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

Article abstract of DOI:10.1021/jm010469k

The structure-based design, chemical synthesis, and biological evaluation of various 2-pyridone-containing human rhinovirus (HRV) 3C protease (3CP) inhibitors are described. These compounds are comprised of a peptidomimetic binding determinant and a Michael acceptor moiety, which forms an irreversible covalent adduct with the active site cysteine residue of the 3C enzyme. The 2-pyridone-containing inhibitors typically display improved 3CP inhibition properties relative to related peptide-derived molecules along with more favorable antiviral properties. The cocrystal structure of one pyridone-derived 3CP inhibitor complexed with HRV-2 3CP is also described along with certain ab initio conformation analyses. Optimization of the 2-pyridone-containing compounds is shown to provide several highly active 3CP inhibitors (k_obs/[I] > 500 000 M^-1 s^-1) that function as potent antirhinoviral agents (EC₅₀ = <0.05 μM) against multiple virus serotypes in cell culture. One 2-pyridone-containing 3CP inhibitor is shown to be bioavailable in the dog after oral dosing (F = 48%).

Full text of DOI:10.1021/jm010469k

J . Med. Chem. 2002, 45, 1607-1623
1607
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. 6. Str u ctu r e-Activity Stu d ies 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, Edward L. Brown, Fausto C. Maldonado, Shella A. Fuhrman,
Leora S. Zalman, Tove Tuntland, Caroline A. Lee, Amy K. Patick, David A. Matthews, Thomas F. Hendrickson,†
Maha B. Kosa, Bo Liu, Minerva R. Batugo, J ean-Paul R. Gleeson, Sylvie K. Sakata, Lijian Chen,
Mark C. Guzman, J ames W. Meador, III, Rose Ann Ferre, and Stephen T. Worland‡
Pfizer Global Research and Development-La J olla/ Agouron Pharmaceuticals, Inc., 10777 Science Center Drive,
San Diego, California 92121-1111
Received October 11, 2001
The structure-based design, chemical synthesis, and biological evaluation of various 2-pyridone-
containing human rhinovirus (HRV) 3C protease (3CP) inhibitors are described. These
compounds are comprised of a peptidomimetic binding determinant and a Michael acceptor
moiety, which forms an irreversible covalent adduct with the active site cysteine residue of
the 3C enzyme. The 2-pyridone-containing inhibitors typically display improved 3CP inhibition
properties relative to related peptide-derived molecules along with more favorable antiviral
properties. The cocrystal structure of one pyridone-derived 3CP inhibitor complexed with HRV-2
3CP is also described along with certain ab initio conformation analyses. Optimization of the
2-pyridone-containing compounds is shown to provide several highly active 3CP inhibitors (kobs
/
[I] > 500 000 M-1 s-1) that function as potent antirhinoviral agents (EC50 ) <0.05 µM) against
multiple virus serotypes in cell culture. One 2-pyridone-containing 3CP inhibitor is shown to
be bioavailable in the dog after oral dosing (F ) 48%).
In tr od u ction
inhibitors might function as broad-spectrum antirhi-
noviral therapeutic agents. Indeed, 3CP has been the
target of several research programs that sought to
identify such antirhinoviral compounds,13 although only
one 3CP inhibitor has currently advanced to the stage
of human clinical trials (see below).
The human rhinoviruses (HRVs) are members of the
picornavirus family and are the single most significant
cause of the common cold in man.1-3 Although consider-
able effort has been expended in the past to identify
clinically efficacious antirhinoviral agents, to date, there
are no marketed antivirals available for the treatment
of this prevalent human pathogen. In addition, the large
number of known HRV serotypes (>100) makes the
development of vaccines seem unlikely.4 Previous at-
tempts at antirhinoviral therapeutic identification in-
clude the use of interferon,2a,c,e the disruption of virus-
receptor (ICAM-1) interactions,5 the examination of
capsid-binding antipicornaviral compounds,6 and the
use of other miscellaneous agents.7,8 In contrast, the
antirhinoviral development efforts at Agouron have
focused on disruption of an early, essential step in the
HRV replication cycle that involves the proteolytic
processing of the large polypeptide produced by cellular
translation of the positive strand viral RNA genome.
The majority of this processing is effected by the HRV
3C protease (3CP),9,10 a cysteine protease possessing
minimal homology with known mammalian enzymes
but which structurally resembles members of the trypsin
protein family.10,11 Because of its importance in the viral
replication cycle and the expected high conservation of
its active site residues among all known HRV sero-
types,12 it is believed that potent and selective 3CP
Previously, we described the design and development
of substrate-derived9,14 peptidyl and peptidomimetic
3CP inhibitors that incorporate C-terminal Michael
acceptor moieties (e.g., compound 1, Figure 1).15-17
These compounds irreversibly inhibit 3CP by forming
a covalent adduct with the active site cysteine residue
of the enzyme and exhibit broad-spectrum antirhinovi-
ral activity in cell culture assays. Our earlier studies
culminated with the selection of one particularly active
peptidomimetic compound, ruprintrivir (USAN, AG7088),
for clinical development as a nasally delivered antirhi-
noviral agent.18-20 This molecule recently demonstrated
encouraging activity in human clinical trials in which
healthy volunteers were deliberately exposed to several
HRVs and is currently the subject of large-scale phase
II studies to examine its effectiveness against naturally
acquired colds.21 Concurrent with these advances, we
also wanted to examine orally bioavailable 3CP inhibi-
tors for potential treatment of the common cold and
other picornaviral infections.22 Unfortunately, potent
3CP inhibitors with sufficient oral bioavailability to
warrant further development were not encountered in
the pool of peptide and peptidomimetic compounds that
led to ruprintrivir (AG7088).23 Therefore, we continued
to seek additional potent, 3CP inhibitors with nonpep-
tidic chemical structures that were distinct from the
molecules that comprised our earlier studies. It was
* To whom correspondence should be addressed. Tel.: (858)622-
7918. Fax: (858)622-7998. E-mail: peter.dragovich@pfizer.com.
† Present address: Schro¨dinger, Inc., 3655 Nobel Drive, Suite 550,
San Diego, CA 92122.
‡ Present address: Anadys Pharmaceuticals, 9050 Camino Santa
Fe, San Diego, CA 92121.
10.1021/jm010469k CCC: $22.00 © 2002 American Chemical Society
Published on Web 03/08/2002

1608 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 8
Dragovich et al.
substituted with a ketomethylene24 or an N-methyl
moiety1 (e.g., compound 2, Figure 1), (iii) the â-sheet
hydrogen-bonding interactions observed between the P3
residue of 1 and the 3CP would be maintained, and (iv)
the previous utilization of related 2-pyridones afforded
potent inhibitors of other trypsinlike proteases that are
structurally similar to 3CP.25,26 The above crystal
structure analysis also suggested that in contrast to
most previous applications of 2-pyridone-containing
enzyme inhibitors, access to the 3CP S2 binding pocket
would best be achieved from a position adjacent to the
P2 amide carbonyl moiety and not from the pyridone
ring itself. In the event, incorporation of an appropriate
P3 3-amino-2-pyridone moiety into the inhibitor design
afforded a compound (3), which displayed greatly im-
proved anti-3CP and antiviral activity relative to the
related peptidyl molecule 1 (Table 1).27 Although the
precise nature of these improvements could not be
identified with certainty,28 they confirmed the above
inhibitor design hypothesis and prompted an extensive
structure-activity study of 2-pyridone-containing 3CP
inhibitors.
Initial 2-pyridone modifications involved variation of
the P2 benzyl moiety of 3 through the introduction of
functional groups that were guided by previous studies
of tripeptidyl 3CP inhibitors.29 Thus, a compound
containing a 4-fluorobenzyl P2 substituent (4) displayed
anti-3CP and antiviral activities nearly equal to that
of 3, while a molecule that incorporated a 3,4-difluo-
robenzyl moiety (5) exhibited significantly improved
inhibition of both the 3C enzyme and the rhinovirus
virulence in cell culture (Table 1). The antiviral activity
trends of compounds 4 and 5 were also observed with
two HRV serotypes other than type 14, suggesting that
they might apply to a large number of rhinoviruses
(Table 1). Somewhat surprisingly, and in contrast to our
earlier studies of tripeptidyl molecules, saturation of the
P2 benzyl moiety resulted in a drastic loss of both 3CP
inhibition activity and antirhinoviral properties (com-
pound 6, Table 1). The origins of the activity reductions
observed with compound 6 have not been rigorously
determined but may result from an unfavorable solution
conformation of the unbound ligand. Importantly, a
2-pyridone-containing compound that lacked the P2
substituent entirely (7) was an extremely poor 3CP
inhibitor and antiviral agent (Table 1). These results
suggested that 2-pyridone 3CP inhibitors required some
type of P2 moiety for effective 3CP recognition but did
not establish a minimum size for such a substituent.
F igu r e 1. Design of 2-pyridone-containing HRV 3CP inhibi-
tors.
envisioned that such compounds would aid in the
identification of orally bioavailable 3CP inhibitors by
further diversifying the physical and biological proper-
ties (e.g., mw, mp, water solubility, metabolic stability,
etc.) of our existing anti-3CP agents. The discovery and
development of one such series of potent, orally bio-
available, 3CP inhibitors are described below.
Having completed the brief exploration of P2 struc-
ture-activity relationships in the 2-pyridone series of
3CP inhibitors described above, we next examined
modification of the N-terminal (P4) substituent. Substi-
tution of the Cbz moiety present in 3 with a simple
acetyl group resulted in drastic loss of both 3CP inhibi-
tory properties and antirhinoviral activity (compare 8
with 3, Table 1). Both of these activities were regained
somewhat by incorporation of a P4 amide derived from
cyclopentanecarboxylic acid into the inhibitor design
(compound 9, Table 1). Interestingly, introduction of a
1,3-dithiolane-2-carboxylic acid-derived N-terminal amide
afforded high levels of 3CP inhibition and antiviral
activity when the resulting compound (10) was tested
against HRV serotype 14. Unfortunately, the molecule
In h ibitor Design a n d In itia l Str u ctu r e-Activity
Stu d ies
Analysis14 of the HRV-2 3CP-1 X-ray crystal struc-
ture15 suggested that the P3 amino acid residue con-
tained in the inhibitor 1 could be replaced with a
3-amino-2-pyridone moiety without significantly affect-
ing key protein-ligand interactions (e.g., compound 3,
Figure 1). Such replacement seemed plausible since (i)
the P3 side chain of 1 did not appreciably contact the
3CP protein, (ii) the P2-P3 amide NH of 1 did not
contribute significantly to 3CP recognition and could be

Human Rhinovirus 3C Protease Inhibitors
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 8 1609
Ta ble 1. 2-Pyridone-Containing 3CP Inhibitors
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 3CP; see ref 15 for assay method and error. Antirhinoviral activity; see ref 15 for assay method
d
and error. e Cytotoxicity; see ref 15 for assay method and error. f 1:1 mixture of diastereomers. ND ) not determined. NA ) not applicable.

1610 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 8
Dragovich et al.
was significantly less potent when examined against
two other HRV serotypes in cell culture, suggesting that
the improvements observed with type 14 HRV would
not be realized against a majority of HRV strains. A
variety of other aliphatic N-terminal amides could be
incorporated into the 2-pyridone-containing inhibitor
series to provide active compounds, although most were
not as potent as the Cbz-containing molecule originally
studied (compare compounds 11 and 12 with 3, Table
1). However, as was observed in our earlier studies of
peptidyl and ketomethylene-containing peptidomimetic
3CP inhibitors, a compound containing an N-terminal
amide derived from 5-methylisoxazole-3-carboxylic acid
(13) displayed improved anti-3CP and in vitro antirhi-
noviral properties relative to the corresponding benzyl
carbamate 3 (Table 1).30 Interestingly, the magnitude
of these improvements was not nearly as great as that
noted in our previous work. Again, the source of the
activity differences between the current series of 2-py-
ridone 3CP inhibitors and the related peptide and
peptidomimetic molecules is not known with certainty.
Utilization of a P4 moiety derived from 5-chloroisox-
azole-3-carboxylic acid also afforded a relatively potent
3CP inhibitor and antirhinoviral agent (compound 14,
Table 1) suggesting that the 5-chloro and 5-methyl
isoxazole substituents could be accommodated nearly
equally in the 3CP active site.
F igu r e 2. Crystal structure of 3 complexed with HRV-2 3CP
(2.3 Å resolution).
In addition to examining modification of the P2 and
P4 substituents contained within the lead compound 3,
we also studied variation of the P1 moiety. Our earlier
studies identified an (S)-γ-lactam fragment that im-
parted high levels of 3CP inhibition activity to peptidyl
and peptidomimetic molecules in which it was incorp-
orated.18a Accordingly, this fragment was introduced
into the 3-amino-2-pyridone-containing 3CP inhibitor
series of the present work. As expected, the resulting
compound displayed improved anti-3CP and antiviral
properties relative to the glutamine-containing molecule
originally studied (compare 15 with 3, Table 1). How-
ever, as noted above for N-terminal isoxazole-containing
2-pyridone 3CP inhibitors, the activity improvements
realized by P1-lactam incorporation were not as great
in the 2-pyridone inhibitor series when compared to
those observed earlier during our studies of peptide and
peptidomimetic compounds. As anticipated from those
earlier studies, introduction of the corresponding P1 (R)-
γ-lactam into the 2-pyridone-containing inhibitor design
resulted in drastic loss of 3CP inhibition activity and
antirhinoviral properties when compared with the mol-
ecule that contained the (S)-lactam isomer (compound
16, Table 1, compare to 15).
F igu r e 3. Schematic diagram of 3 bound in the HRV-2 3CP
active site. Hydrogen bonds are represented as dashed lines,
and the residues that make up the enzyme binding subsites
are depicted.
structures depicted the covalent protein-inhibitor prod-
ucts and not the presumably more relevant transition
states for adduct formation (see below). The specific
details of one particular protein-inhibitor complex are
discussed below.
The 2.3 Å X-ray crystal structure of the covalent
adduct formed between compound 3 and HRV-2 3CP32
is shown in Figure 2, and key protein-inhibitor interac-
tions are illustrated in Figure 3. In general, the inhibitor
bound to the enzyme in a manner similar to that
observed previously for related tripeptidyl molecules15,29
and filled a series of shallow grooves on the protein
surface N-terminal to the scissile amide bond of the
substrate (S1-S4).14 As expected, a covalent bond was
observed between the 3CP active site cysteine residue
(Cys-147) and the â-carbon of the Michael acceptor of
3. As with related peptidyl 3CP inhibitors, a hydrogen
bond between the Michael acceptor ester moiety and the
3CP Cys-147 amide NH was present in the above
complex, but it is uncertain whether this interaction
facilitates addition of the cysteine to 3 or merely arises
after the covalent adduct has been formed.
X-r a y Str u ctu r e An a lysis
The iterative analysis of protein-ligand interactions
by X-ray crystallography is a critical component of any
structure-based inhibitor design program.31 Accordingly,
several crystal structures of 3CP-inhibitor complexes
were obtained during the development of the 2-pyri-
done-containing compounds described above. These
structures confirmed the binding geometries that the
inhibitors adopted when complexed with 3CP and
suggested possibilities for compound modification. As
in our earlier work, the X-ray data were utilized
somewhat cautiously for inhibitor design since the
Several other important protein-ligand interactions
were apparent in addition to those noted in the vicinity
of the active site cysteine, which closely paralleled those
observed previously with related tripeptidyl 3CP inhibi-

Human Rhinovirus 3C Protease Inhibitors
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 8 1611
Ta ble 2. 4-Methyl-2-Pyridone-Containing 3CP Inhibitors
tors. For example, the glutamine amide of 3 formed
hydrogen bonds with the His-161 side chain and both
the Thr-142 side chain and the amide carbonyl in the
bottom and on the edge of the 3CP S1 binding pocket,
respectively. In addition, the inhibitor phenylalanine
residue bound to 3CP in a canyon formed by the side
chains of Leu-127, Ser-128, and Asn-130 on one side and
His-40 on the other. As expected, the greatest differ-
ences between the 3CP binding of the 2-pyridone-
containing inhibitor 3 and the related tripeptidyl mol-
ecules were noted in the vicinity of the pyridone ring
itself. The conformationally mobile serine residue that
forms a hydrogen bond with the P2-P3 amide NH of
most peptidyl 3CP inhibitors (Ser-128) was positioned
within van der Waals contact distance adjacent to the
pyridone ring (Figure 3). As was noted previously during
studies of peptidyl 3CP inhibitors, the N-terminal Cbz
moiety of 3 was situated in a hydrophobic pocket on the
surface of the protein formed by the side chains of Tyr-
122, Ile-125, and Phe-170. Also as expected, an anti-
parallel â-sheet hydrogen-bonding interaction was noted
between the pyridone moiety and the Gly-164 (Figure
3), which mimicked that crystallographically observed
between the 3CP and the P3 amino acid residue of
related tripeptidyl 3CP inhibitors. The other backbone
amide NH present in 3 formed a hydrogen bond with
3CP Val-162, while an additional hydrogen bond be-
tween the inhibitor Cbz carbonyl moiety and the 3CP
Ser-128 amide NH was also evident (Figure 3). Thus, a
total of eight hydrogen bonds involving both the inhibi-
tor amino acid residues and the 2-pyridone moiety were
observed in the complex between compound 3 and
HRV-2 3CP.
a
b
Method of preparation: see Scheme 1. Elemental analyses
(C, H, N) of all compounds agreed to within (0.4% of theoretical
values. c Inhibition activity against HRV-14 3CP; see ref 15 for
assay method and error.
not well understood but may involve uncertainties
associated with modeling efforts conducted with ir-
reversible Michael reaction products rather than the
transition states of such transformations.
In h ibitor Op tim iza tion
On the basis of the experimental results described
above, additional inhibitor optimization efforts were
conducted utilizing 3-amino-2-pyridones that lacked
substituents at the pyridone 4-position. Accordingly,
various beneficial functional groups identified during
the initial phase of structure-activity relationship
studies described above (Table 1) were combined to form
new 3CP inhibitors. Thus, a 2-pyridone containing a P1
(S)-γ-lactam moiety, a P2 benzyl substituent, and an
N-terminal (P4) isoxazole fragment displayed very good
levels of 3CP inhibition activity as well as extremely
potent antirhinoviral properties in cell culture (com-
pound 19, Table 3). This molecule also exhibited potent
antiviral activity against two HRV strains other than
type 14. However, the compound was extensively me-
tabolized when incubated with human liver microsomes
(Table 3), and additional efforts were therefore made
to improve upon the biological stability of the molecule.
Analysis of the incubation mixtures of 19 with human
liver microsomes identified several metabolites includ-
ing compounds derived from oxidation of the 4-fluo-
rophenyl aromatic ring and hydrolysis of the carboxylic
acid ethyl ester.33 In an attempt to reduce the formation
of metabolites resulting from the former process, an
additional fluorine atom was introduced at the 3-posi-
tion of the P2 benzyl substituent present in 19. The
resulting difluorinated compound (20) displayed im-
proved anti-3CP and antirhinoviral properties relative
to the monofluorinated molecule (Table 3, compare 19
with 20), and this result was consistent with similar
improvements noted previously for other “unoptimized”
2-pyridone 3CP inhibitors (see Table 1, compounds 4
Analysis of the above complex suggested several
possibilities for beneficial modification of 3CP inhibitors
that contained the 2-pyridone fragment. In particular,
the crystal structure indicated that the pyridone-P4
carbamate moiety was significantly twisted out of the
plane defined by the pyridone ring. However, molecular
mechanics calculations (MM2) conducted with uncom-
plexed 3-carbamoyl-2-pyridones predicted a coplanar
relationship between the carbamate and the heterocyclic
ring. Together, these observations suggested that alkyl
substitution of the pyridone ring adjacent to the car-
bamate moiety might minimize the conformational
differences between the 3CP-complexed and the uncom-
plexed structures. This suggestion was supported by ab
initio studies conducted at the HF 6-31G* level of
theory. The crystal structure analysis also indicated that
introduction of such an alkyl moiety should not steri-
cally interfere with the binding of such compounds to
the 3CP enzyme. In the event, however, a 3-carbamoyl-
4-methyl-2-pyridone-containing compound displayed sig-
nificantly reduced 3CP inhibition activity when com-
pared to a related nonmethylated molecule (compare 17
with 3, Table 2). An additional comparison was made
between two amide-containing molecules (compounds 13
and 18, Table 2). Again, the methylated pyridone 18
displayed reduced anti-3CP activity when compared to
its nonmethylated analogue 13, although the difference
in activity between the two amide-containing com-
pounds was not as dramatic as that noted above for the
benzyl carbamates. The failure of the 4-methyl pyri-
dones to improve 3CP inhibition properties is currently

1612 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 8
Ta ble 3. Optimized 2-Pyridone-Containing 3CP Inhibitors
Dragovich et al.
kobs/[I]
EC50
CC50
HLM
plasma
c
compd no.
R
X
H
prepa
B
formulab
serotype
(M-1 s-1
)
(µM)d
(µM)e
(% met)f
95
T1/2 (h)g
19
CH2CH3
C30H32FN5O7‚0.50H2O
14
1A
10
14
1A
10
14
1A
10
2
1 250 000
ND
ND
1 800 000
ND
ND
548 000
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
0.002
0.015
0.004
0.001
0.006
0.005
0.003
0.132
0.085
0.048
0.033
0.109
0.036
0.100
0.044
0.044
0.012
0.003
0.009
0.010
0.003
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
ND
1.4
9.7
20
21
CH2CH3
F
F
B
C
C30H31F2N5O7
80
55
CH(CH3)2
C31H33F2N5O7‚0.75H2O
3
9
16
25
39
13
78
11
19
23
Hanks
ND
ND
a
b
Method of preparation: see Schemes 2 and 3. Elemental analyses (C, H, N) of all compounds agreed to within (0.4% of theoretical
values. c Inhibition activity against HRV 3CP; see ref 15 for assay method and error. Antirhinoviral activity; see ref 15 for assay method
d
and error. e Cytotoxicity; see ref 15 for assay method and error. f Loss of compound (% metabolized) after 30 min exposure to human liver
g
microsomes; see Experimental Section for additional details. Half-life of compound upon exposure to human plasma; see Experimental
Section for additional details.
and 5). In addition, the difluorinated compound 20
displayed improved stability toward human liver mi-
crosomes as compared with 19, although the metabolism
of 20 was still somewhat facile in the in vitro assay
(Table 3). Unfortunately, and somewhat surprisingly
based on our earlier studies of peptidyl and peptidomi-
metic 3CP inhibitors,18a compound 20 exhibited a short
half-life when exposed to human plasma (Table 3), and
efforts were accordingly focused on improving this
important biological stability parameter.
Or a l Bioa va ila bility Stu d ies
The R,â-unsaturated esters contained within tripep-
tidyl molecules related to the 2-pyridones of the present
study were previously shown to be quite unstable
toward a variety of rodent plasmas (e.g., compound 1,
t1/2 ) 10 min in rat plasma).15 This rodent plasma
instability precluded the use of rats and/or mice for oral
bioavailability testing of the compounds of the present
study.34 Accordingly, the dog was selected as an ap-
propriate species in which to conduct the desired
analysis. In the event, inhibitor 21 displayed good
bioavailability and pharmacokinetic properties after oral
administration in the dog (Figure 4, Table 4). Encourag-
ingly, plasma levels of the molecule were observed after
initial oral dosing, which substantially exceeded the
compound’s previously determined average in vitro EC50
antirhinoviral potency (0.045 µM, 0.028 µg/mL, 15 HRV
serotypes), and these levels remained above this average
potency for more than 8 h. Because no animal model
predictive of rhinovirus-associated human symptoma-
tology has been identified to date, the ultimate signifi-
cance of the above pharmacokinetic profile cannot be
determined with certainty. However, the PK results do
demonstrate that 2-pyridone-containing, peptidomi-
metic 3CP inhibitors can display good oral bioavailabil-
ity properties in the dog and suggest that additional
study of such compounds for use as orally bioavailable
HRV 3CP inhibitors is warranted.
We anticipated that the instability of compound 20
toward human plasma was primarily due to enzyme-
mediated hydrolysis of the ethyl ester contained in the
molecule. Accordingly, the ethyl ester was replaced with
a sterically more demanding isopropyl ester (compound
21). This alteration afforded improved performance in
the human liver microsomal stability assay and dra-
matically improved the half-life of the compound in the
presence of human plasma (Table 3). These benefits,
however, were realized with a simultaneous worsening
of anti-3CP and antiviral potency of 21 relative to the
ethyl ester-containing molecule 20. Despite these reduc-
tions, compound 21 displayed good absolute levels of
antirhinoviral potency when tested against 15 HRV
serotypes in cell culture (Table 3). Because this molecule
combined good levels of antiviral potency with relatively
good biological stability, it was therefore chosen for
subsequent oral bioavailability studies.

Human Rhinovirus 3C Protease Inhibitors
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 8 1613
done ring that was encountered when more direct
synthetic methods (e.g., P4-P3-P2 + P1 coupling) were
explored.
F igu r e 4. Pharmacokinetic profile of 21 in dogs after IV and
oral administration (n ) 3). Vehicle ) 80:20 propylene glycol:
H2O. The average antirhinoviral potency of 21 as determined
against 15 HRV serotypes in cell culture is indicated by the
arrow. An additional sample was collected 24 h after both IV
and PO dosing, but the concentration of 21 was below the limit
of detection (5 ng/mL) in each.
The silyl protecting group present in 27 was removed,
and the resulting alcohol (28) was subjected to an
oxidation/olefination sequence to provide intermediate
29 in good overall yield. As was encountered during
previous syntheses of peptidyl and peptidomimetic 3CP
inhibitors,15,18a the above olefination process afforded
the desired trans isomer with <5% of the corresponding
Syn th esis
The 2-pyridone-containing 3CP inhibitors described
in this study were prepared by three related synthetic
methods (A, B, and C). The particular method employed
to synthesize a given compound is indicated in Tables
1-3, and representative examples of each are given
below. These syntheses differ primarily in the nature
of the P1 fragment utilized to begin each sequence and
employ several synthetic transformations and interme-
diates from previously reported preparations of related
tripeptidyl 3CP inhibitors.15,18a The first method (method
A) is illustrated in Scheme 1 with the preparation of
compound 13 and involves alkylation of a Boc-protected
pyridone entity (P3 fragment) with an activated P2-P1
moiety. Thus, commercially available N-R-Cbz-γ-trityl-
L-glutamine was reduced to primary alcohol 22 by a two
step process, and this entity was subsequently converted
to the corresponding tert-butyldimethylsilyl ether 23 in
good yield. Deprotection of the Cbz moiety present in
23 followed by coupling of the resulting amine (not
shown) with commercially available (2R)-2-hydroxy-3-
phenylpropionic acid afforded intermediate 24 in good
yield. The secondary alcohol moiety present in 24 was
then converted to the corresponding mesylate (25), and
the crude material thus obtained was condensed with
the sodium salt of (2-hydroxypyridin-3-yl)carbamic acid
tert-butyl ester (26, see Experimental Section) at el-
evated temperature to afford product 27 as a single
diastereomer in moderate yield. Attempts to form the
triflate corresponding to 25 utilizing trifluoromethane-
sulfonic anhydride resulted in the unwanted removal
of both the primary silyl ether and the trityl protecting
group present in 24. These side reactions were presum-
ably caused by the formation of trifluoromethanesulfonic
acid in the reaction medium and occurred even in the
presence of a large excess of various amine bases (e.g.,
2,6-lutidine).35 The synthesis of intermediate 27 pre-
dominantly provided the N-alkylated pyridone product
as evidenced by thin-layer chromatography (TLC) analy-
sis of the reaction mixture and 1H nuclear magnetic
resonance (NMR) analysis of the pyridone product.36,37
The described preparation of intermediate 27 minimized
racemization of the stereocenter adjacent to the pyri-
1
cis isomer as determined by H NMR analysis of the
crude reaction mixture. Selective removal of the Boc
protecting group present in 29 proved to be surprisingly
difficult but was eventually accomplished through ther-
mal deprotection. The resulting amine (not shown) was
condensed with 5-methylisoxazole-3-carbonyl chloride
to provide product 30 in good yield. Subsequent removal
of the trityl moiety contained within 30 afforded the
desired 3CP inhibitor 13 in good yield after purification
by silica gel chromatography.
The majority of the other 2-pyridone-containing 3CP
inhibitors described in this work could be prepared by
variation of synthetic method A described above. Uti-
lization of (2-hydroxypyridin-3-yl)carbamic acid benzyl
ester 3138 in lieu of 26 allowed for the synthesis of
compound 3 (Table 1) without additional manipulation
of the 3-amino-pyridone substituent following alkylation
of the pyridone moiety. Similarly, incorporation of either
Boc- or Cbz-protected 3-amino-2-hydroxy-4-methylpy-
ridine (prepared in analogy to 26 and 31) into Scheme
1 above (replacing 26) allowed for the synthesis of
inhibitors 17 and 18. Alternatively, substitution of
appropriately functionalized 2-hydroxyacetic acids in
place of the (2R)-2-hydroxy-3-phenylpropionic acid em-
ployed in Scheme 1 above enabled the synthesis of
compounds 4-7 (Cbz-protected pyridone 31 was also
employed). The hydroxy acid required for the prepara-
tion of compound 7 was commercially available. The
remainder was prepared from the corresponding (com-
mercially available) Boc-protected D-amino acids by a
two step process analogous to that described for the
synthesis of intermediate 34 below. Finally, replacement
of 5-methylisoxazole-3-carbonyl chloride in the Scheme
1 sequence with other carboxylic acids and/or acid
chlorides allowed for the preparation of compounds
8-14 in addition to inhibitor 13 described above.39
The synthesis of 2-pyridone-containing 3CP inhibitors
that incorporate P1-γ-lactam moieties is illustrated in
Scheme 2 with the preparation of compound 20 (syn-
thetic method B). Thus, the known18a γ-lactam-contain-
ing alcohol 32 was converted to the corresponding tert-
butyldiphenylsilyl ether 33 in good yield. Deprotection

1614 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 8
Dragovich et al.
Ta ble 4. Pharmacokinetic Parameters for 21 in Dogs after IV and Oral Administration (n ) 3)
Cmax (µg/mL)
max (min)
AUC (0f∞)
(µg‚min/mL)
CL (mL/
min/kg)
V
(L/kg)
T1/2
(min)
F
(%)
dose
T
15 mg/kg iv
30 mg/kg po
10.6 at 4.0
3.5 at 70
1028
980
14.7
33
4.2
9.6
194
204
48
Sch em e 1a
a
t
Synthetic method A. Reagents and conditions (Tr ) CHPh3, TBS ) Si(CH3)2 Bu): (a) 1.0 equiv ethyl chloroformate, 1.0 equiv NMM,
THF, -10 °C, 20 min, then 2.25 equiv NaBH4, H2O, 23 °C, 5 h; (b) 2.3 equiv imidazole, 1.1 equiv TBSCl, DMF, 23 °C, 12 h, 71%; (c)
H2/Pd/C, EtOH, 23 °C, 12 h; (d) 1.0 equiv (2R)-2-hydroxy-3-phenylpropionic acid, 2.0 equiv Pr2Net, 1.0 equiv HATU, DMF, 0f23 °C, 1.5
h, 62%; (e) 1.4 equiv Pr2Net, 1.2 equiv CH3SO2Cl, CH2Cl2, -10 °C, 30 min; (f) 2.5 equiv 26, 2.2 equiv NaH, THF, 0 °C, 20 min, then 1.0
i
i
equiv 25, reflux, 61 h, 77%; (g) Et3N‚3HF, 1:1 CH3CN:H2O, 23 °C, 12 h; (h) 1.1 equiv Dess-Martin periodinane, CH2Cl2, 23 °C, 2 h; (i)
1.4 equiv Ph3PdCHCO2Et, THF, reflux 1 h, then 23 °C, 12 h, 77%; (j) 190-200 °C, 1 h; (k) 2.0 equiv 5-methylisoxazole-3-carbonyl chloride,
i
2.0 equiv NMM, CH3CN, 0f23 °C, 1 h, 55%; (l) 3.0 equiv Pr3SiH, 2:3 TFA:CH2Cl2, 23 °C, 30 min, 85%.
of the Boc moiety present in 33 under mildly acidic
conditions followed by coupling of the resulting amine
salt (not shown) with (2R)-3-(3′,4′-difluorophenyl)-2-
hydroxypropionic acid 34 afforded intermediate 35 in
good yield. Carboxylic acid 34 was prepared from Boc-
D-3,4-difluorophenylalanine by a two step process analo-
gous to that generally utilized for the conversion of
R-amino acids to the corresponding R-hydroxy acids (see
Experimental Section).40 Intermediate 35 was converted
to the corresponding mesylate (compound 36), and this
material was condensed in crude form with the sodium
salt of pyridone 37 (see Experimental Section) to provide
product 38 in good yield. As was observed in the
preparation of pyridone 27 in Scheme 1 above, the
synthesis of intermediate 38 predominantly provided
the N-alkylated pyridone product as a single diastere-
omer.36,37
Removal of the silyl protecting group present in 38,
oxidation of the resulting alcohol (39), and subsequent
olefination of the corresponding aldehyde (not shown)
afforded intermediate 40 in moderate yield. The 2,4-
dimethoxybenzyl moiety present in 40 was removed by
exposure to 2,3-dichloro-5,6-dicyano-1,4-benzoquinone
(DDQ) at elevated temperature to give compound 20.41
This deprotection could also be accomplished by expo-
sure of 40 to ceric ammonium nitrate (CAN),42 although
this latter method was somewhat lower yielding than
the DDQ deprotection mentioned above. Compounds 15
and 19 were prepared in a manner analogous to that
described above for 20 utilizing (2R)-2-hydroxy-3-phe-
nylpropionic acid (commercially available) and (2R)-3-
(4′-fluorophenyl)-2-hydroxypropionic acid (prepared from
the corresponding D-amino acid), respectively, in place
of intermediate 34. Similarly, compound 16 was syn-
thesized in a manner analogous to that employed for
inhibitor 15 above, beginning with the appropriate
R-lactam isomer of γ-lactam-containing alcohol 32.18a
An alternate method for preparing γ-lactam-contain-
ing 3CP inhibitors is illustrated in Scheme 3 (method
C) and employs an unprotected P1 moiety (45)43 in place
of the dimethoxybenzyl-containing fragment (32) uti-
lized in method B above. Method C avoids the need for
lactam deprotection in the final step of inhibitor syn-
thesis and therefore enabled more rapid variation of the
Michael acceptor moiety incorporated in the compounds
under investigation. Surprisingly, several early syn-
thetic intermediates analogous to those depicted in
Scheme 2 that lacked the dimethoxybenzyl group proved
troublesome to manipulate due to drastically different
physical properties. An alternate method for coupling
the various inhibitor components together was therefore
explored.

Human Rhinovirus 3C Protease Inhibitors
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 8 1615
Sch em e 2a
a
Synthetic method B. Reagents and conditions (DMB ) 2,4-dimethoxybenzyl, TBDPS ) SiPh2tBu): (a) 5.0 equiv Et3N, 2.0 equiv
TBDPSCl, 0.25 equiv DMAP, CH2Cl2, 0f23 °C, 2 h, 50%; (b) HCl, 1,4-dioxane, 23 °C, 1 h; (c) 1.5 equiv 34, 5.0 equiv NMM, 1.5 equiv
i
HATU, CH3CN, 0f23 °C, 2.5 h, 54%; (d) 1.4 equiv Pr2Net, 1.25 equiv CH3SO2Cl, CH2Cl2, -10 °C, 30 min; (e) 2.0 equiv 37, 1.8 equiv
NaH, THF, 23 °C, 20 min, then 1.0 equiv 36, THF, reflux, 16 h, 74%; (f) HF, 30:1 CH3CN:H2O, 23 °C, 20 min, 90%; (g) 1.2 equiv Dess-
Martin periodinane, CH2Cl2, 23 °C, 2 h; (h) 1.3 equiv Ph3PdCHCO2Et, THF, reflux 1.7 h, 63%; (i) 4.2 equiv DDQ, 10:1 CHCl3:H2O, 60 °C,
4 h, 72%.
Sch em e 3a
a
Synthetic method C. Reagents and conditions: (a) HCl, CH3OH, 23 °C, 20 h; (b) 1.7 equiv 2,6-lutidine, 1.6 equiv Tf2O, 0 °C, 30 min;
(c) 1.1 equiv 37, 1.0 equiv NaH, THF, 23 °C, 30 min, then 1.0 equiv 42, THF, 23 °C, 2 h, 81%; (d) 2.5 equiv LiI, pyridine, reflux, 30 min,
96%; (e) (from 45) 2:3 TFA:CH2Cl2, 23 °C, 1 h, then 0.95 equiv 44, 7.0 equiv NMM, 1.1 equiv HATU, DMF, -10f23 °C, 3 h, 48%; (f) 1.2
i
equiv Dess-Martin periodinane, CH2Cl2, 23 °C,1 h; (g) 1.2 equiv Ph3PdCHCO2 Pr, THF, reflux 30 min, 46%.
Thus, esterification of (2R)-3-(3′,4′-difluorophenyl)-2-
hydroxypropionic acid (34) under acidic conditions af-
forded intermediate 41 in moderate yield (Scheme 3).
This material was converted to the corresponding tri-
flate 42 and condensed with the sodium salt of pyridone
37 to give intermediate 43 in good yield after purifica-
tion on silica gel. The ester present in 43 was converted
to the corresponding carboxylic acid (44) under near-
neutral conditions, and the crude material thus obtained
was coupled with γ-lactam 4543 to give alcohol 46 in
moderate overall yield. Oxidation of alcohol 46 and
olefination of the resulting aldehyde (not shown) using
(triphenyl-λ5-phosphanylidene)acetic acid isopropyl es-
ter44 afforded compound 21 in reasonable yield.
Con clu sion s
The studies presented above demonstrate that pep-
tidomimetic, 2-pyridone-containing irreversible inhibi-
tors of the HRV 3CP can function as potent, broad-
spectrum, orally bioavailable antirhinoviral agents.
Such 2-pyridone-containing molecules typically display
improved HRV 3CP inhibition activities and antiviral

1616 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 8
Dragovich et al.
i
equiv), Pr2NEt (4.25 mL, 24.4 mmol, 2.0 equiv), and HATU
(4.64 g, 12.2 mmol, 1.0 equiv) in DMF (35 mL) at 0 °C. After
it was stirred for 1 h, the reaction mixture was allowed to
warm to 23 °C and stirred 20 min more. Then, 5% KHSO4 (80
mL) and MTBE (600 mL) were added, and the phases were
separated. The organic phase was washed with H2O (80 mL)
and brine (70 mL), dried over Na2SO4, and evaporated. The
residue was purified by flash column chromatography (50%
EtOAc in hexanes) to provide 24 as a white foam (4.85 g, 62%).
IR (cm-1): 3394, 3295, 1666, 1649, 1519, 1255, 1114, 1085.
1H NMR (CDCl3): δ -0.03-0.02 (m, 6H), 0.85 (s, 9H), 1.70-
1.89 (m, 2H), 2.18-2.42 (m, 3H), 2.83 (dd, 1H, J ) 13.8, 8.1),
3.15 (dd, 1H, J ) 13.8, 4.0), 3.40 (dd, 1H, J ) 10.0, 4.7), 3.51
(dd, 1H, J ) 10.0, 3.1), 3.83-3.94 (m, 1H), 4.17-4.23 (m, 1H),
6.79 (d, 1H, J ) 8.7), 7.09 (s, 1H), 7.17-7.32 (m, 20H). Anal.
(C39H48N2O4Si‚0.30H2O) C, H, N.
properties when compared with related tripeptidyl 3CP
inhibitors. One particular 2-pyridone-containing com-
pound was shown to be orally bioavailable in the dog
(F ) 48%). Importantly, the plasma concentrations of
this molecule in the dog exceeded its average antirhi-
noviral activity as determined by cell culture assays
(EC50, 15 HRV serotypes) for more than 8 h postadmin-
istration. Collectively, these results suggest that ad-
ditional studies of 2-pyridone-containing HRV 3CP
inhibitors are warranted and may lead to the identifica-
tion of such molecules suitable for clinical development
as orally delivered agents.
Exp er im en ta l Section
(1′S,2R)-Meth a n esu lfon ic Acid 1-[1′-(ter t-Bu tyld im eth -
ylsila n yloxym eth yl)-3′-(tr itylca r ba m oyl)p r op ylca r ba m -
oyl]-2-p h en yleth yl Ester (25). Intermediate 24 (1.96 g, 3.08
General descriptions of experimental procedures, reagent
purifications, and instrumentation along with conditions and
uncertainties for enzyme and antiviral assays are provided
elsewhere.15 1H NMR chemical shifts are reported in parts per
million (δ) downfield relative to internal tetramethylsilane,
and coupling constants are given in Hertz. Abbreviations also
apply as follows: HATU [O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-
tetramethyluronium hexafluorophosphate], HOBt (1-hydroxy-
benzotriazole hydrate), EDC [1-(3-dimethylaminopropyl)-3-
ethylcarbodiimide hydrochloride], CDI (1,1′-carbonyldiimidazole),
MTBE (tert-butyl methyl ether), and TFA (trifluoroacetic acid).
Pirodavir49 was kindly provided by J anssen Pharmaceuticals.
Pleconaril was prepared as described in the literature.6b
Rep r esen ta tive Exa m p le of P r ep a r a tion Meth od A.
Syn th esis of tr a n s-(2′S,4S)-6-Ca r ba m oyl-4-(2′-{3′′-[(5′′′-
m eth ylisoxazole-3′′′-car bon yl)am in o]-2′′-oxo-2′′H-pyr idin -
1′′-yl}-3′-p h en ylp r op ion yla m in o)h ex-2-en oic Acid Eth yl
E st er (13). (1S)-[1-Hyd r oxym et h yl-3-(t r it ylca r b a m oyl)-
p r op yl]ca r ba m ic Acid Ben zyl Ester (22). 4-Methylmor-
pholine (1.89 mL, 17.2 mmol, 1.0 equiv) and ethyl chlorofor-
mate (1.65 mL, 17.3 mmol, 1.0 equiv) were added to a mixture
of commercially available N-R-Cbz-γ-trityl-L-glutamine (9.00
g, 17.2 mmol, 1.0 equiv) in tetrahydrofuran (THF; 23 mL) at
-10 °C. After the mixture was stirred for 20 min, the reaction
mixture was filtered and the filtrate was added dropwise to a
suspension of NaBH4 (1.47 g, 38.9 mmol, 2.25 equiv) in H2O
(10 mL) at 0 °C. The resulting mixture was allowed to warm
to 23 °C and stirred for 5 h. It was then cooled again to 0 °C,
was quenched by the careful addition of 1 N HCl (30 mL), and
then was partitioned between MTBE (500 mL) and brine (2 ×
100 mL). The organic phase was dried over Na2SO4 and
evaporated to provide intermediate 22, which was used
without further purification.
(1S)-[1-(ter t-Bu tyld im eth ylsila n yloxym eth yl)-3-(tr ityl-
ca r ba m oyl)p r op yl]ca r ba m ic Acid Ben zyl Ester (23). In-
termediate 22, prepared above, was dissolved in dimethylfor-
mamide (DMF; 10 mL). Imidazole (2.69 g, 39.5 mmol, 2.3
equiv) and tert-butyldimethylsilyl chloride (2.86 g, 19.0 mmol,
1.1 equiv) were added. The reaction mixture was stirred
overnight, then diluted with MTBE (500 mL), and washed
sequentially with 2.5% KHSO4, H2O, NaHCO3, H2O, and brine
(100 mL each). The organic phase was dried over MgSO4 and
evaporated. The residue was purified by flash column chro-
matography (gradient elution, 25f40% EtOAc in hexanes) to
provide 23 (7.6 g, 71%) as a white amorphous solid. IR (cm-1):
3307, 1708, 1660, 1496, 1249. 1H NMR (CDCl3): δ -0.01-
0.05 (m, 6H), 0.89 (s, 9H), 1.76-1.93 (m, 2H), 2.29-2.40 (m,
2H), 3.56-3.77 (m, 3H), 5.03-5.16 (m, 3H), 7.00 (s, 1H), 7.18-
7.39 (m, 20H). Anal. (C38H46N2O4Si) C, H, N.
i
mmol, 1.0 equiv) and Pr2NEt (0.752 mL, 4.32 mmol, 1.4 equiv)
were dissolved in CH2Cl2 (30 mL) and cooled to -10 °C.
Methanesulfonyl chloride (0.286 mL, 3.70 mmol, 1.2 equiv) was
added slowly (dropwise) with vigorous stirring. After 30 min,
the reaction mixture was diluted with CH2Cl2 (200 mL),
washed with brine (50 mL), dried over Na2SO4, and concen-
trated to provide intermediate 25, which was used without
further purification.
(2-Hyd r oxyp yr id in -3-yl)ca r ba m ic Acid ter t-Bu tyl Es-
ter (26). A suspension of 10% palladium on carbon (0.35 g)
and 2-hydroxy-3-nitropyridine (5.00 g, 35.7 mmol, 1.0 equiv)
in EtOH (170 mL) was subjected to 1 atm of hydrogen for 16
h. After the reaction vessel was purged with argon, the mixture
was filtered through Whatman no. 3 paper and the filtrate
was evaporated to give 2-hydroxy-3-aminopyridine, which was
used without further purification. This crude material thus
obtained was dissolved in THF (100 mL) at 23 °C. Di-tert-butyl
dicarbonate (7.79 g, 35.7 mmol, 1.0 equiv) was added, and the
reaction mixture was heated to reflux for 4 h. Additional di-
tert-butyl dicarbonate (6.0 g, 27 mmol, 0.8 equiv) was then
added, and the reaction mixture was refluxed overnight. The
solvent was evaporated, and the residue was purified by flash
column chromatography (gradient elution, 50f60% EtOAc in
hexanes) to provide 26 as a white solid (6.48 g, 83%). IR (cm-1):
3225, 1725, 1649, 1514. 1H NMR (CDCl3): δ 1.52 (s, 9H), 6.33
(dd, 1H, J ) 7.4, 6.6), 7.01 (dd, 1H, J ) 6.6, 1.8), 7.56 (s, 1H),
8.11 (d, 1H, J ) 7.1), 12.61 (s, 1H). Anal. (C10H14N2O3) C, H,
N.
(1′′S,2′S)-(1-{1′-[1′′-(ter t-Bu tyldim eth ylsilan yloxym eth yl)-
3′′-(tr itylca r ba m oyl)p r op ylca r ba m oyl]-2′-p h en yleth yl}-
2-oxo-1,2-d ih yd r op yr id in -3-yl)ca r ba m ic Acid ter t-Bu tyl
Ester (27). Hydroxypyridine 26 from above (0.838 g, 3.99
mmol, 1.3 equiv) was stirred in THF (20 mL). Sodium hydride
(60% dispersion in mineral oil, 0.148 g, 3.70 mmol, 1.2 equiv)
was added. After the mixture was stirred for 20 min, a solution
of mesylate 25 (1.0 equiv based on 26) in THF (15 mL) was
added. The resulting mixture was refluxed for 40 h, whereupon
TLC showed the reaction to be only 50% complete. In a
separate flask, additional sodium hydride (60% dispersion in
mineral oil, 0.111 g, 2.78 mmol, 0.9 equiv) was added to a
suspension of 26 (0.647 g, 3.08 mmol, 1.0 equiv) in THF (10
mL). After this was stirred for 20 min, this mixture was added
to the original reaction vessel and the resulting mixture was
refluxed for an additional 21 h and then stirred at 23 °C for
48 h. The crude reaction mixture was diluted with MTBE (600
mL), washed with a mixture of brine and 10% KHSO4 (3:1, 80
mL) and brine (80 mL), then was dried over Na2SO4, and
concentrated. The residue was purified by flash column
chromatography (gradient elution, 30f35% EtOAc in hexanes)
to afford 27 as a white foam (1.98 g, 77%). IR (cm-1): 3389,
(2′R,4S)-5-(ter t-Bu tyldim eth ylsilan yloxy)-4-(2′-h ydr oxy-
3′-p h en ylp r op ion yl-a m in o)p en ta n oic Acid Tr ityla m id e
(24). Intermediate 23 (7.6 g, 12 mmol, 1 equiv) and 10%
palladium on carbon (0.45 g) were suspended in EtOH (140
mL) at 23 °C and hydrogenated at 50 psi overnight. The
reaction mixture was filtered through Whatman no. 3 paper,
the paper was washed with EtOH (120 mL), and the combined
filtrates were evaporated. The residue was combined with (2R)-
2-hydroxy-3-phenylpropionic acid (1.42 g, 12.2 mmol, 1.0
3307, 1725, 1678, 1649, 1590, 1502. 1H NMR (CDCl3):
δ
-0.02-0.04 (m, 6H), 0.86 (s, 9H), 1.52 (s, 9H), 1.55-1.88 (m,
2H), 2.08-2.14 (m, 2H), 3.19 (dd, 1H, J ) 13.7, 8.1), 3.39-
3.51 (m, 2H), 3.53 (dd, 1H, J ) 14.2, 7.8), 3.82-3.93 (m, 1H),
5.60-5.67 (m, 1H), 6.17 (t, 1H, J ) 7.3), 6.44 (d, 1H, J ) 8.3),

Human Rhinovirus 3C Protease Inhibitors
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 8 1617
7.04 (s, 1H), 7.12-7.36 (m, 21H), 7.59 (s, 1H), 7.94 (d, 1H, J )
4.43-4.54 (m, 1H), 5.68 (dd, 1H, J ) 15.7, 1.3), 5.74-5.82 (m,
1H), 6.00 (s, 1H), 6.19 (s, 1H), 6.32 (t, 1H, J ) 7.3), 6.46 (s,
1H), 6.69 (dd, 1H, J ) 15.7, 5.5), 7.13-7.30 (m, 5H), 7.48 (dd,
1H, J ) 7.3, 1.6), 7.62 (d, 1H, J ) 7.6), 8.39 (dd, 1H, J ) 7.3,
1.6), 9.46 (s, 1H). Anal. (C28H31N5O7‚0.50H2O) C, H, N.
7.1). Anal. (C49H60N4O6Si) C, H, N.
(1′′S,2′S)-(1-{1′-[1′′-Hydr oxym eth yl-3′′-(tr itylcar bam oyl)-
p r op ylca r ba m oyl]-2′-p h en yleth yl}-2-oxo-1,2-d ih yd r op yr -
id in -3-yl)ca r ba m ic Acid ter t-Bu tyl Ester (28). Intermedi-
ate 27 (0.96 g, 1.16 mmol, 1.0 equiv) was dissolved in a mixture
of CH3CN (15 mL) and H2O (1.5 mL) in a plastic tube.
Triethylamine trihydrofluoride (11 drops) was added, and the
reaction solution was stirred overnight at 23 °C. The mixture
was then diluted with EtOAc (400 mL), washed with brine (3
× 40 mL), dried over MgSO4, and concentrated to provide
intermediate 28, which was used without further purification.
tr a n s-(2′S,4S)-4-[2′-(3′′-ter t-Bu t oxyca r b on yla m in o-2′′-
oxo-2′′H-p yr id in -1′′-yl)-3′-p h en ylp r op ion yla m in o]-6-(tr i-
tylca r ba m oyl)h ex-2-en oic Acid Eth yl Ester (29). Crude
alcohol 28 from above was dissolved in CH2Cl2 (10 mL) at 23
°C, and Dess-Martin periodinane45 (Lancaster, 0.545 g, 1.28
mmol, 1.1 equiv) was added. After the mixture was stirred for
2 h, the solvent was evaporated and the residue was repeatedly
suspended in toluene and concentrated (40 mL, then 2 × 10
mL) to give a yellow foam. This material was dissolved in THF
(17 mL). (Carbethoxymethylene)triphenylphosphorane (0.563
g, 1.62 mmol, 1.4 equiv) was added, and the reaction mixture
was heated to reflux for 1 h and then stirred at 23 °C
overnight. After the solvent was evaporated, the residue was
purified by flash column chromatography (gradient elution,
40f50% EtOAc in hexanes) to provide 29 (0.710 g, 77%). IR
(cm-1): 3378, 3284, 1719, 1649, 1596, 1508, 1267. 1H NMR
(CDCl3): δ 1.28 (t, 3H, J ) 7.1), 1.47 (s, 9H), 1.54-1.69 (m,
1H), 1.87-2.02 (m, 1H), 2.09-2.22 (m, 2H), 3.12 (dd, 1H, J )
13.7, 7.7), 3.47 (dd, 1H, J ) 13.7, 8.1), 4.17 (q, 2H, J ) 7.1),
4.43-4.54 (m, 1H), 5.51-5.58 (m, 1H), 5.64 (dd, 1H, J ) 15.7,
1.6), 6.12 (t, 1H, J ) 7.2), 6.60-6.68 (m, 3H), 7.08-7.31 (m,
21H), 7.51 (s, 1H), 7.90 (d, 1H, J ) 7.1). Anal. (C47H50N4O7‚
0.50H2O) C, H, N.
Rep r esen ta tive Exa m p le of P r ep a r a tion Meth od B.
Syn th esis of tr a n s-(2′S,3′′′′′S,4S)-4-[3′-(3′′,4′′-Diflu or op h e-
n yl)-2′-(3′′′-{[1′′′-(5′′′′-m et h ylisoxa zol-3′′′′-yl)m et h a n oyl]-
a m in o}-2′′′-oxo-2′′′H -p yr id in -1′′′-yl)p r op a n oyla m in o]-5-
(2′′′′′-oxop yr r olid in -3′′′′′-yl)p en t-2-en oic Acid Eth yl Ester
(20). (1S,3′S)-{2-ter t-Bu tyld iph en ylsila n yloxy)-1-[1′-(2′′,4′′-
d im eth oxyben zyl)-2′-oxo-p yr r olid in -3′-ylm eth yl]eth yl}-
ca r ba m ic Acid ter t-Bu tyl Ester (33). Triethylamine (7.52
mL, 54.0 mmol, 5.0 equiv), tert-butylchlorodiphenylsilane (5.53
mL, 21.6 mmol, 2.0 equiv), and 4-(dimethylamino)pyridine
(0.330 g, 2.70 mmol, 0.25 equiv) were added successively to a
0 °C solution of (1S,3′S)-{1-[1′-(2′′,4′′-dimethoxybenzyl)-2′-
oxopyrrolidin-3′-ylmethyl]-2-hydroxyethyl}carbamic acid tert-
butyl ester 3218a (4.41 g, 10.8 mmol, 1 equiv) in CH2Cl2 (50
mL). The reaction mixture was allowed to warm to 23 °C and
stirred for 2 h. It was then diluted with MTBE (400 mL),
washed with brine (2 × 100 mL), dried over MgSO4, and
evaporated. The residue was purified by flash column chro-
matography (gradient elution, 30f40% EtOAc in hexanes) to
give 33 (3.51 g, 50%) as a white foam; Rf ) 0.40 (40% EtOAc
in hexanes). IR (cm-1): 3319, 1678, 1508. 1H NMR (CDCl3): δ
1.05 (s, 9H), 1.42 (s, 9H), 1.44-1.65 (m, 2H), 2.05-2.17 (m,
1H), 2.23-2.35 (m, 1H), 2.44-2.56 (m, 1H), 3.14-3.21 (m, 2H),
3.55-3.68 (m, 2H), 3.69-3.81 (m, 1H), 3.79 (s, 3H), 3.79 (s,
3H), 4.42 (s, 2H), 4.77 (d, 1H, J ) 9.3), 6.41-6.46 (m, 2H),
7.09-7.13 (m, 1H), 7.34-7.46 (m, 6H), 7.61-7.67 (m, 4H).
Anal. (C37H50N2O6Si) C, H, N.
(2R)-3-(3′,4′-Diflu or op h en yl)-2-h yd r oxyp r op ion ic Acid
(34). Boc-D-3,4-difluorophenylalanine (5.00 g, 16.6 mmol, 1.0
equiv) was stirred for 4 h in a solution of HCl in 1,4-dioxane
(4.0 M, 25 mL). Hexanes (25 mL) were added, and the mixture
was stirred an additional 20 min. The resulting solid was
collected by filtration, air-dried, dissolved in 1 M H2SO4 (70
mL), and cooled to -2 °C. A solution of sodium nitrite (40%
aq, 8.25 mL) was then added dropwise, keeping the temper-
ature 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 stir for 16 h more. The reaction mixture was
extracted with MTBE (3 × 70 mL), and the organic phases
were dried over MgSO4 and concentrated. The residue was
subjected to flash column chromatography (3% CH3OH in CH2-
Cl2 with 0.2% AcOH) to provide 34 (1.36 g) as an oil, which
still contained some minor, unidentified impurities.
tra ns-(2′S,4S)-4-(2′-{3′′-[(5′′′-Methylisoxazole-3′′′-carbonyl)-
a m in o]-2′′-oxo-2′′H -p yr id in -1′′-yl}-3′-p h en ylp r op ion yl-
a m in o)-6-(tr itylca r ba m oyl)h ex-2-en oic
Acid
Eth yl
Ester (30). Intermediate 29 from above (0.088 g, 0.11 mmol,
1.0 equiv) was heated (neat) to between 190 and 200 °C for 65
min and then cooled to 23 °C. The resulting amine, obtained
as a dark residue, was dissolved in CH3CN (2 mL) and cooled
to 0 °C. 5-Methylisoxazole-3-carbonyl chloride (0.033 g, 0.23
mmol, 2.0 equiv) and 4-methylmorpholine (0.025 mL, 0.23
mmol, 2.0 equiv) were added, and the reaction mixture was
allowed to warm to 23 °C. After the mixture was stirred for
40 min, a mixture of 10% KHSO4 and brine (1:1, 15 mL) and
EtOAc (70 mL) was added. The phases were separated, and
the organic phase was washed with brine, dried over Na2SO4,
and evaporated. The residue was purified by flash column
chromatography (50% EtOAc in hexanes) to provide 30 (0.049
g, 55%). IR (cm-1): 3331, 1678 (br), 1590, 1525. 1H NMR
(CDCl3): δ 1.28 (t, 3H, J ) 7.1), 1.58-1.72 (m, 1H), 1.87-
2.03 (m, 1H), 2.10-2.26 (m, 2H), 2.48 (s, 3H), 3.15 (dd, 1H, J
) 13.7, 7.8), 3.47 (dd, 1H, J ) 13.7, 8.1), 4.17 (q, 2H, J ) 7.1),
4.43-4.55 (m, 1H), 5.54-5.61 (m, 1H), 5.65 (dd, 1H, J ) 15.8,
1.5), 6.17 (t, 1H, J ) 7.3), 6.45 (s, 1H), 6.65 (dd, 1H, J ) 15.8,
5.4), 6.72 (s, 1H), 6.84 (d, 1H, J ) 8.0), 7.08-7.32 (m, 22 H),
8.35 (dd, 1H, J ) 7.3, 1.5), 9.49 (s, 1H). Anal. (C47H46N5O7‚
0.25H2O) C, H, N.
(1S,2′′′R,3′S)-N-{2-(ter t-Bu tyld ip h en ylsila n yloxy)-1-[1′-
(2′′,4′′-d im et h oxyb en zyl)-2′-oxop yr r olid in -3′-ylm et h yl]-
eth yl}-3′′′-(3′′′′,4′′′′-d iflu or op h en yl)-2′′′-h yd r oxyp r op ion a -
m id e (35). Carbamate 33 (2.49 g, 3.85 mmol, 1.0 equiv) was
dissolved in 1,4-dioxane (16 mL) at 23 °C. A solution of HCl
in the same solvent (4.0 M, 16 mL) was added. After the
solution was stirred for 75 min at 23 °C, the volatiles were
evaporated under reduced pressure. The resulting residue was
dissolved in CH3CN (20 mL) and cooled to 0 °C. Hydroxyacid
34 (1.17 g, 5.79 mmol, 1.5 equiv), 4-methylmorpholine (2.12
mL, 19.3 mmol, 5.0 equiv), and HATU (2.20 g, 5.79 mmol, 1.5
equiv) were added sequentially, and the reaction mixture was
allowed to warm to 23 °C and then stirred for 2.5 h. It was
then partitioned between EtOAc (500 mL) and brine (2 × 125
mL), and the organic layer was dried over MgSO4 and
concentrated. The residue was purified by flash column
chromatography (gradient elution, 2f2.5% CH3OH in CH2-
Cl2) to provide 35 (1.07 g, 54%) as a white foam; Rf ) 0.21
(50% EtOAc in hexanes). IR (cm-1): 3389, 3319, 1660, 1514.
1H NMR (CDCl3): δ 1.05 (s, 9H), 1.54-1.67 (m, 2H), 2.01-
2.12 (m, 1H), 2.20-2.40 (m, 2H), 2.78 (dd, 1H, J ) 14.1, 7.9),
3.08 (dd, 1H, J ) 14.1, 3.7), 3.14-3.21 (m, 2H), 3.50 (dd, 1H,
J ) 10.1, 5.7), 3.62 (dd, 1H, J ) 10.1, 3.7), 3.77 (s, 3H), 3.78
(s, 3H), 3.89 (d, 1H, J ) 4.6), 3.96-4.10 (m, 1H), 4.21-4.28
(m, 1H), 4.36 (s, 2H), 6.37-6.44 (m, 2H), 6.89-6.95 (m, 2H),
tr a n s-(2′S,4S)-6-Ca r b a m oyl-4-(2′-{3′′-[(5′′′-m et h ylisox-
a zole-3′′′-ca r b on yl)a m in o]-2′′-oxo-2′′H -p yr id in -1′′-yl}-3′-
p h en ylp r op ion yla m in o)h ex-2-en oic Acid E t h yl E st er
(13). Intermediate 30 (0.047 g, 0.059 mmol, 1.0 equiv) was
dissolved in CH2Cl2 (3 mL). Triisopropylsilane (0.036 mL, 0.176
mmol, 3.0 equiv) and TFA (2 mL) were added. The bright
yellow solution was stirred for 25 min and then diluted with
CCl4 (3 mL), and all of the volatiles were evaporated. The
residue was purified by flash column chromatography (3% CH3-
OH in CH2Cl2) to afford 13 (0.028 g, 85%) as an amorphous
1
foam. IR (cm-1): 3342, 1666 (br), 1590, 1531, 1455. H NMR
(CDCl3): δ 1.30 (t, 3H, J ) 7.1), 1.70-1.84 (m, 1H), 1.85-
1.99 (m, 1H), 2.17-2.24 (m, 2H), 2.48 (s, 3H), 3.18 (dd, 1H, J
) 13.7, 7.8), 3.50 (dd, 1H, J ) 13.7, 8.1), 4.19 (q, 2H, J ) 7.1),

1618 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 8
Dragovich et al.
7.02-7.09 (m, 2H), 7.31-7.45 (m, 7H), 7.60-7.65 (m, 4H).
(cm-1): 3366 br, 3331, 1672 br, 1596, 1531 br. 1H NMR
(CDCl3): δ 1.48-1.62 (m, 2H), 1.91-2.19 (m, 2H), 2.30-2.40
(m, 1H), 2.49 (s, 3H), 3.10-3.21 (m, 3H), 3.45-3.56 (m, 3H),
3.75 (s, 3H), 3.78 (s, 3H), 3.89-4.05 (m, 2H), 4.17 (d, 1H, J )
14.4), 4.38 (m, 1H, J ) 14.4), 5.59-5.68 (m, 1H), 6.20-6.27
(m, 1H), 6.38-6.45 (m, 3H), 6.81-6.87 (m, 1H), 6.91-7.02 (m,
3H), 7.24-7.29 (m, 1H), 8.19-8.25 (m, 1H), 8.36-8.41 (m, 1H),
9.52 (s, 1H). Anal. (C35H37F2N5O8) C, H, N.
Anal. (C41H48F2N2O6Si) C, H, N.
5-Meth ylisoxa zole-3-ca r boxylic Acid (2′-Hyd r oxyp yr i-
d in -3′-yl)a m id e (37). Palladium, 10% on activated carbon
(0.45 g), and 2-hydroxy-3-nitropyridine (7.00 g, 50.0 mmol, 1.0
equiv) in EtOH (210 mL) were subjected to 1 atm of hydrogen
for 16 h. After the reaction vessel was purged with argon, the
mixture was filtered through Whatman no. 3 paper and the
filtrate was evaporated to give 2-hydroxy-3-aminopyridine,
which was used without further purification. This crude
material was suspended in CH3CN (170 mL) and cooled to 0
°C. 5-Methylisoxazole-3-carbonyl chloride (8.00 g, 55.0 mmol,
1.0 equiv) was added in one portion. After 25 min at 0 °C, the
reaction mixture was allowed to warm to 23 °C and stirred
for an additional 75 min. The thick mixture was then poured
into dilute HCl (0.02 M, 150 mL) and mixed thoroughly. The
undissolved solid was collected by filtration, washed with H2O
(2 × 20 mL), and then dried under vacuum overnight to
provide 37 (7.1 g, 65%) as a white solid; mp 270-272 °C, dec;
Rf ) 0.38 (10% CH3OH in CHCl3). IR (cm-1): 3460, 3331, 1637,
tr a n s-(2′S,3′′′′′S,4S)-4-(3′-(3′′,4′′-Diflu or op h en yl)-2′-{3′′′-
[(5′′′′-m eth ylisoxa zole-3′′′′-ca r bon yl)-a m in o]-2′′′-oxo-2′′′H-
p yr id in -1′′′-yl}p r op ion yla m in o)-5-[1′′′′′-(2′′′′′′,4′′′′′′-d im eth -
oxyben zyl)-2′′′′′-oxop yr r olid in -3′′′′′-yl]p en t-2-en oic Acid
Eth yl Ester (40). Dess-Martin periodinane45 (Lancaster,
0.390 g, 0.913 mmol, 1.2 equiv) and 39 (0.528 g, 0.761 mmol,
1.0 equiv) were combined in CH2Cl2 (8 mL) and stirred for 2 h
at 23 °C. The volatiles were then removed under reduced
pressure. The residue was suspended in toluene and concen-
trated to dryness (2 × 10 mL), and the resulting residue was
dissolved in THF (16 mL). (Carbethoxymethylene)tri-
phenylphosphorane (0.345 g, 0.990 mmol, 1.3 equiv) was
added, and the reaction mixture was refluxed for 1.75 h. It
was allowed to cool to 23 °C and then was concentrated. The
residue was purified by flash column chromatography (2% CH3-
OH in CH2Cl2) to provide 40 (0.363 g, 63%) as a foam; Rf )
0.36 (5% CH3OH in CH2Cl2). IR (cm-1): 3331, 3284, 1713,
1666, 1649, 1596. 1H NMR (CDCl3): δ 1.27 (t, 3H, J ) 7.1),
1.46-1.65 (m, 2H), 1.85-1.97 (m, 1H), 2.10-2.21 (m, 1H),
2.35-2.47 (m, 1H), 2.50 (s, 3H), 3.10-3.22 (m, 3H), 3.50 (dd,
1H, J ) 13.9, 7.5), 3.75 (s, 3H), 3.77 (s, 3H), 4.12-4.21 (m,
3H), 4.38 (d, 1H, J ) 14.5), 4.44-4.55 (m, 1H), 5.56-5.63 (m,
1H), 5.73 (dd, 1H, J ) 15.6, 1.5), 6.21-6.27 (m, 1H), 6.38-
6.47 (m, 3H), 6.73 (dd, 1H, J ) 15.6, 5.6), 6.84-6.90 (m, 1H),
6.92-7.07 (m, 3H), 7.22-7.26 (m, 1H), 8.37-8.41 (m, 1H), 8.69
(d, 1H, J ) 6.4), 9.55 (s, 1H). Anal. (C39H41F2N5O9‚0.75H2O)
C, H, N.
tr a n s-(2′S,3′′′′′S,4S)-4-[3′-(3′′,4′′-Diflu or op h en yl)-2′-(3′′′-
{[1′′′-(5′′′′-m et h ylisoxa zol-3′′′′-yl)m et h a n oyl]a m in o}-2′′′-
oxo-2′′′H -p yr id in -1′′′-yl)p r op a n oyla m in o]-5-(2′′′′′-oxop y-
r r olid in -3′′′′′-yl)p en t-2-en oic Acid Eth yl Ester (20). DDQ
(0.130 g, 0.573 mmol, 1.4 equiv) was added to a solution of 40
(0.312 g, 0.410 mmol, 1.0 equiv) in a mixture of CHCl3 and
H2O (10:1, 11 mL) at 23 °C. The reaction vessel was placed in
an oil bath and was heated to 60 °C. After the mixture was
stirred for 1.5 h, more DDQ (0.130 g, 0.573 mmol, 1.4 equiv)
was added. After an additional 1.5 h, more DDQ (0.130 g, 0.573
mmol, 1.4 equiv) was added. After 4 h total time, the reaction
mixture was allowed to cool to 23 °C, was diluted with EtOAc
(300 mL), and was washed sequentially with a mixture of 10%
KHSO4 and brine (1:1, 100 mL) and a mixture of saturated
NaHCO3 and brine (1:1, 100 mL). The organic layer was dried
over MgSO4 and concentrated. The residue was purified by
flash column chromatography (2% CH3OH in CH2Cl2) to
provide 20 (0.181 g, 72%) as a white foam; Rf ) 0.43 (10% CH3-
OH in CHCl3). IR (cm-1): 3331, 1690, 1649, 1596, 1531, 1455,
1278. 1H NMR (CDCl3): δ 1.30 (t, 3H, J ) 7.1), 1.44-1.54 (m,
1H), 1.63-1.78 (m, 1H), 2.08-2.29 (m, 3H), 2.49 (d, 3H, J )
0.9), 3.05 (dd, 1H, J ) 13.6, 7.5), 3.20-3.42 (m, 3H), 4.19 (dq,
2H, J ) 7.1, 1.7), 4.34-4.45 (m, 1H), 5.64 (dd, 1H, J ) 15.7,
1.4), 6.00 (t, 1H, J ) 7.8), 6.32 (t, 1H, J ) 7.3), 6.45 (s, 1H),
6.71 (dd, 1H, J ) 15.7, 5.6), 6.86-6.91 (m, 1H), 6.98-7.08 (m,
2H), 7.15 (s, 1H), 7.68 (dd, 1H, J ) 7.3, 1.7), 8.39 (dd, 1H, J )
7.3, 1.7), 8.65 (d, 1H, J ) 6.8), 9.46 (s, 1H). Anal. (C30H31F2N5O7)
C, H, N.
1
1543. H NMR (DMSO-d6): δ 2.48 (s, 3H), 6.29 (dd, 1H, J )
7.2, 6.6), 6.69 (s, 1H), 7.19 (dd, 1H, J ) 6.6, 1.8), 8.26 (dd, 1H,
J ) 7.2, 1.8), 9.43 (s, 1H), 12.20 (s, 1H). Anal. (C10H9N3O3) C,
H, N.
(1′′′S,2′′R,3′′′′S)-5-Meth ylisoxa zole-3-ca r boxylic Acid
{1′-[1′′-{2′′′-(ter t-Bu tyldiph en ylsilan yloxy)-1′′′-[1′′′′-(2′′′′′,4′′′′′-
d im eth oxyben zyl)-2′′′′-oxop yr r olid in -3′′′′-ylm eth yl]eth yl-
ca r b a m oyl}-2′′-(3′′′′′′,4′′′′′′-d iflu or op h en yl)et h yl]-2′-oxo-
1′,2′-d ih yd r op yr id in -3′-yl}a m id e (38). N,N-Diisopropyl-
ethylamine (0.357 mL, 2.05 mmol, 1.4 equiv) and 35 (1.07 g,
1.46 mmol, 1.0 equiv) were combined in CH2Cl2 (20 mL) and
cooled to -10 °C. Methanesulfonyl chloride (0.142 mL, 1.83
mmol, 1.25 equiv) was added slowly (dropwise) with vigorous
stirring. After 30 min, the reaction mixture was diluted with
MTBE (250 mL), washed sequentially with a mixture of brine
and 10% KHSO4 (1:1, 60 mL) and brine (60 mL), dried over
Na2SO4, and concentrated to give the crude mesylate 36, which
was used without further purification.
Sodium hydride (60% dispersion in mineral oil, 0.105 g, 2.62
mmol, 1.8 equiv) was added to a solution of 37 (0.642 g, 2.93
mmol, 2.0 equiv) in THF (7 mL) at 23 °C. After the mixture
was stirred for 20 min, a solution of the crude mesylate 36
(prepared above) in THF (7 mL) was added. The resulting
mixture was refluxed for 16 h, cooled to 23 °C, diluted with
MTBE (20 mL), and washed sequentially with a mixture of
brine and 10% KHSO4 (3:1, 30 mL) and brine (30 mL). The
organic layer was dried over Na2SO4 and was concentrated.
The resulting residue was purified by flash column chroma-
tography (gradient elution, 2f2.5% CH3OH in CH2Cl2) to
provide 38 (1.00 g, 74%) as a white foam; Rf ) 0.62 (5% CH3-
OH in CH2Cl2). IR (cm-1): 3331, 3295, 1666, 1649, 1596, 1525.
1H NMR (CDCl3): δ 1.03 (s, 9H), 1.43-1.54 (m, 1H), 1.55-
1.67 (m, 1H), 1.90-2.02 (m, 1H), 2.05-2.26 (m, 2H), 2.50 (s,
3H), 3.02-3.17 (m, 3H), 3.43 (dd, 1H, J ) 14.1, 7.3), 3.51 (dd,
1H, J ) 10.1, 5.8), 3.70 (dd, 1H, J ) 10.1, 3.7), 3.76 (s, 3H),
3.78 (s, 3H), 3.95-4.07 (m, 1H), 4.25 (d, 1H, J ) 14.6), 4.36
(d, 1H, J ) 14.6), 5.50-5.58 (m, 1H), 6.19-6.26 (m, 1H), 6.39-
6.47 (m, 3H), 6.78-6.84 (m, 1H), 6.86-7.05 (m, 3H), 7.12-
7.17 (m, 1H), 7.32-7.45 (m, 6H), 7.54-7.64 (m, 5H), 8.36-
8.40 (m, 1H), 9.56 (s, 1H). Anal. (C51H55 F2N5O8Si) C, H, N.
(1′′S,1′′′S,3′′′′S)-5-Meth ylisoxa zole-3-ca r boxylic Acid [1′-
(2′′-(3′′′,4′′′-Diflu or oph en yl)-1′′-{1′′′-[1′′′′-(2′′′′′,4′′′′′-dim eth oxy-
b e n zyl)-2′′′′-oxop yr r olid in -3′′′′-ylm e t h yl]-2′′′-h yd r oxy-
e t h ylca r b a m oyl}e t h yl)-2′-oxo-1′,2′-d ih yd r op yr id in -3′-
yl]a m id e (39). Hydrofluoric acid (48%, 2 mL) was added
dropwise to a stirred solution of 38 (0.929 g, 0.997 mmol, 1.0
equiv) in a mixture of CH3CN (14 mL) and H2O (0.45 mL) in
a plastic tube at 23 °C. After 1.5 h, more hydrofluoric acid (2
mL) was added. After 3 h, the reaction mixture was poured
into saturated aqueous NaHCO3 (125 mL), extracted with CH2-
Cl2 (3 × 200 mL), dried over Na2SO4, and evaporated. The
residue was purified by flash column chromatography (gradi-
ent elution, 2f3% CH3OH in CH2Cl2) to provide 39 (0.621 g,
90%) as a white foam; Rf ) 0.25 (5% CH3OH in CH2Cl2). IR
P r epar ation Meth od C. Syn th esis of tr a n s-(2′S,3′′′′′S,4S)-
4-[3′-(3′′,4′′-Diflu or op h en yl)-2′-(3′′′-{[1′′′′-(5′′′′-m eth ylisox-
a zol-3′′′′-yl)m et h a n oyl]a m in o}-2′′′-oxo-2′′′H -p yr id in -1′′′-
yl)p r op a n oyla m in o]-5-(2′′′′′-oxop yr r olid in -3′′′′′-yl)-p en t -
2-en oic Acid Isop r op yl Ester (21). (2R)-3-(3′,4′-Diflu or o-
p h en yl)-2-h yd r oxyp r op ion ic Acid Meth yl Ester (41). A
solution of HCl in 1,4-dioxane (0.100 mL) and 34 (0.170 g,
0.841 mmol, 1 equiv) were combined in CH3OH (5 mL) at 23
°C and maintained for 20 h at that temperature. The reaction
mixture was then concentrated under reduced pressure, and
the resulting residue was purified by flash column chroma-

Human Rhinovirus 3C Protease Inhibitors
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 8 1619
tography (25% EtOAc in hexanes) to provide 41 (0.104 g, 57%)
as a white solid; mp 49-50 °C; Rf ) 0.40 (40% EtOAc in
6.92-7.02 (m, 2H), 7.44-7.49 (m, 1H), 8.17 (d, 1H, J ) 7.9),
8.33-8.40 (m, 1H), 9.44 (s, 1H). Anal. (C26H27F2N5O6‚1.0H2O)
C, H, N.
1
hexanes). IR (cm-1): 3465, 1737. H NMR (CDCl3): δ 2.83 (d,
1H, J ) 5.5), 2.91 (dd, 1H, J ) 14.0, 6.6), 3.09 (dd, 1H, J )
14.0, 4.2), 3.79 (s, 3H), 4.40-4.47 (m, 1H), 6.90-6.96 (m, 1H),
7.02-7.13 (m, 2H). Anal. (C10H10F2O3) C, H, N.
tr a n s-(2′S,3′′′′′S,4S)-4-[3′-(3′′,4′′-Diflu or op h en yl)-2′-(3′′′-
{[1′′′′-(5′′′′-m et h ylisoxa zol-3′′′′-yl)m et h a n oyl]a m in o}-2′′′-
oxo-2′′′H -p yr id in -1′′′-yl)p r op a n oyla m in o]-5-(2′′′′′-oxop y-
r r olid in -3′′′′′-yl)-p en t-2-en oic Acid Isop r op yl Ester (21).
Dess-Martin periodinane45 (Lancaster, 0.267 g, 0.625 mmol,
1.15 equiv) was added to a 0 °C solution of 46 (0.295 g, 0.543
mmol, 1 equiv) in CH2Cl2 (10 mL). The reaction vessel was
then warmed to 23 °C and stirred for 45 min. The volatiles
were evaporated, and the residue was repeatedly concentrated
from toluene (3 × 5 mL). The residue was then dissolved in
THF (10 mL). (Triphenyl-λ5-phosphanylidene)acetic acid iso-
propyl ester44 (0.226 g, 0.624 mmol, 1.15 equiv) was added,
and the reaction mixture was refluxed for 30 min, then cooled
to 23 °C, and concentrated under reduced pressure. The
resulting residue was purified by flash column chromatogra-
phy (2% CH3OH in CHCl3) to afford 21 contaminated with
several impurities. This material was dissolved in CHCl3 (100
mL), washed sequentially with a mixture of saturated NaHCO3
and brine (1:1, 40 mL) and brine (20 mL). The organic layer
was dried over MgSO4 and concentrated to give pure 21 (0.155
g, 46%) as a pale yellow foam; Rf ) 0.29 (5% CH3OH in CH2-
(2R)-3-(3′,4′-Diflu or op h en yl)-2-tr iflu or om eth a n esu lfo-
n yloxyp r op ion ic Acid Meth yl Ester (42). A solution of 41
(0.091 g, 0.421 mmol, 1.0 equiv) and 2,6-lutidine (0.083 mL,
0.713 mmol, 1.7 equiv) was cooled to 0 °C. Trifluoromethane-
sulfonic anhydride (0.113 mL, 0.672 mmol, 1.6 equiv) was
added dropwise. The reaction mixture was stirred 30 min at
0 °C, then was diluted with MTBE (90 mL), and washed
sequentially with 1 M HCl (20 mL) and brine (2 × 20 mL).
The organic layer was dried over MgSO4 and concentrated to
provide crude 42, which was used without further purification.
(2S)-3-(3′,4′-Diflu or oph en yl)-2-{3′′-[(5′′′-m eth ylisoxazole-
3′′′-c a r b o n y l)a m in o ]-2′′-o x o -2′′H -p y r id in -1′′-y l}p r o -
p ion ic Acid Meth yl Ester (43). A solution of 37 (0.102 g,
0.465 mmol, 1.1 equiv) in THF was treated with sodium
hydride (60% suspension in mineral oil, 0.017 g, 0.425 mmol,
1.0 equiv) at 23 °C. The reaction mixture was stirred for 30
min at that temperature while gas evolution ceased, and then,
a THF solution (4 mL) of crude 42 prepared above was added
rapidly over 2 min. The resulting reaction mixture was stirred
for 2 h at 23 °C, then was partitioned between MTBE (100
mL) and brine (2 × 20 mL). The organic layer was dried over
MgSO4 and was concentrated. The residue was purified by
flash column chromatography (40% EtOAc in hexanes) to
provide 43 (0.142 g, 81%) as a white foam; Rf ) 0.29 (40%
EtOAc in hexanes). IR (cm-1): 3342, 1743, 1690, 1649, 1602.
1H NMR (CDCl3): δ 2.50 (s, 3H), 3.33 (dd, 1H, J ) 14.4, 9.9),
3.52 (dd, 1H, J ) 14.4, 5.5), 3.77 (s, 3H), 5.32 (dd, 1H, J ) 9.9,
5.5), 6.17-6.23 (m, 1H), 6.47-6.49 (m, 1H), 6.66-6.82 (m, 2H),
6.89-7.07 (m, 2H), 8.40-8.44 (m, 1H), 9.56 (s, 1H). Anal.
(C20H17F2N3O5) C, H, N.
1
Cl2). IR (cm-1): 3507, 3331, 3284, 1690, 1649, 1596, 1525. H
NMR (CDCl3): δ 1.27 (d, 3H, J ) 6.2), 1.28 (d, 3H, J ) 6.2),
1.45-1.56 (m, 1H), 1.62-1.78 (m, 1H), 2.08-2.29 (m, 3H), 2.49
(s, 3H), 3.06 (dd, 1H, J ) 13.8, 7.8), 3.21-3.44 (m, 3H), 4.36-
4.47 (m, 1H), 4.99-5.12 (m, 1H), 5.66 (dd, 1H, J ) 15.6, 1.5),
5.96-6.04 (m, 1H), 6.32 (t, 1H, J ) 7.3), 6.45 (s, 1H), 6.71 (dd,
1H, J ) 15.6, 5.6), 6.84-6.91 (m, 1H), 6.98-7.17 (m, 3H), 7.66
(dd, 1H, J ) 7.3, 1.6), 8.39 (dd, 1H, J ) 7.3, 1.6), 8.64 (d, 1H,
J ) 6.6), 9.45 (s, 1H). Anal. (C31H33F2N5O7‚0.75H2O) C, H, N.
The following compounds were prepared using modifications
of either general synthetic method A, B, or C as indicated in
Tables 1-3.
tr a n s-(2′S,4S)-4-[2′-(3′′-Ben zyloxyca r b on yla m in o-2′′-
oxo-2′′H-p yr id in -1′′-yl)-3′-p h en ylp r op ion yla m in o]-6-ca r -
ba m oylh ex-2-en oic Acid Eth yl Ester (3). Rf ) 0.28 (5%
CH3OH in CH2Cl2). IR (cm-1): 3298, 1713, 1655, 1590, 1508,
1196. 1H NMR (CDCl3): δ 1.30 (t, 3H, J ) 7.1), 1.65-1.96 (m,
2H), 2.02-2.19 (m, 2H), 3.15 (dd, 1H, J ) 13.8, 7.6), 3.49 (dd,
1H, J ) 13.8, 8.3), 4.18 (q, 2H, J ) 7.1), 4.42-4.53 (m, 1H),
5.17 (s, 2H), 5.62-5.81 (m, 4H), 6.26-6.52 (m, 1H), 6.66 (dd,
1H, J ) 15.6, 5.4), 7.12-7.40 (m, 12H), 7.81 (s, 1H), 7.97-
8.04 (m, 1H). Anal. (C31H34N4O7‚0.25H2O) C, H, N.
(2S)-3-(3′,4′-Diflu or oph en yl)-2-{3′′-[(5′′′-m eth ylisoxazole-
3′′′-c a r b o n y l)a m in o ]-2′′-o x o -2′′H -p y r id in -1′′-y l}p r o -
p ion ic Acid (44). Lithium iodide (1.15 g, 8.59 mmol, 2.5 equiv)
and 43 (1.43 g, 3.43 mmol, 1.0 equiv) were combined in
pyridine (6 mL) and refluxed for 30 min. After the mixture
was cooled to 23 °C, the reaction mixture was partitioned
between 1 M HCl (150 mL) and CH2Cl2 (3 × 150 mL). The
combined organic phases were washed with a mixture of brine
and 1 M HCl (10:1, 3 × 25 mL), then dried over MgSO4, and
concentrated to provide the crude carboxylic acid 44 (1.33 g,
96%), which was used without further purification.
tr a n s-(2′S,4S)-4-[2′-(3′′-Ben zyloxyca r b on yla m in o-2′′-
oxo-2′′H-pyr idin -1′′-yl)-3′-(4′′′-flu or oph en yl)pr opion ylam i-
n o]-6-ca r ba m oylh ex-2-en oic Acid Eth yl Ester (4). mp 99-
101 °C; Rf ) 0.66 (10% CH3OH in CH2Cl2). IR (cm-1): 3308,
(1′′S,1′′′′S,3′′′′′S)-5-Meth ylisoxa zole-3-ca r boxylic Acid
(1′-{2′′-(3′′′,4′′′-Diflu or op h en yl)-1′′-[2′′′′-h yd r oxy-1′′′′-(2′′′′′-
oxop yr r olid in -3′′′′′-ylm et h yl)et h ylca r b a m oyl]et h yl}-2′-
oxo-1′,2′-d ih yd r op yr id in -3′-yl)a m id e (46). TFA (4 mL) and
4543 (0.591 g, 2.29 mmol, 1.0 equiv) were combined in CH2Cl2
(6 mL) and stirred for 50 min. The volatiles were then removed
under reduced pressure, and the residue was evaporated
repeatedly from CCl4 (3 × 25 mL). The resulting residue and
crude 44 (0.877 g, 2.17 mmol, 0.95 equiv) were combined in
DMF (10 mL) and cooled to -10 °C. 4-Methylmorpholine (1.76
mL, 16.0 mmol, 7.0 equiv) and HATU (0.957 g, 2.52 mmol,
1.1 equiv) were added. After the mixture was stirred for 2 h
at -10 °C, the vessel was allowed to warm to 23 °C and stirred
an additional 1 h. 10% KHSO4 (30 mL), brine (50 mL), and
EtOAc (650 mL) were added. The phases were separated, and
the organic phase was washed sequentially with brine (30 mL),
a mixture of saturated NaHCO3 and brine (1:1, 50 mL), and
brine (25 mL), then dried over MgSO4, and concentrated. The
residue was purified by flash column chromatography (gradi-
ent elution, 3f5% CH3OH in CHCl3) to provide 46 (0.566 g,
48%) as a white foam; Rf ) 0.38 (10% CH3OH in CHCl3). IR
1
1714, 1650, 1511, 1199. H NMR (CDCl3): δ 1.33 (t, 3H, J )
6.9), 1.72-1.82 (m, 1H), 1.86-1.96 (m, 1H), 2.10-2.18 (m, 2H),
3.10-3.17 (m, 2H), 3.10-3.17 (m, 2H), 3.46-3.53 (m, 1H),
3.63-3.74 (m, 2H), 4.22 (q, 2H, J ) 6.9), 4.48-4.57 (m, 1H),
5.21 (s, 2H), 5.58-5.65 (m, 2H), 5.91-5.97 (m, 1H), 6.19-6.22
(m, 1H), 6.35 (t, 1H, J ) 7.2), 6.69 (dd, 1H, J ) 15.6, 5.1), 6.98
(t, 2H, J ) 8.7), 7.13-7.18 (m, 2H), 7.36-7.40 (m, 5H), 7.85
(s, br. 1H), 8.06-8.09 (m, 1H). Anal. (C33H33N4O7‚1.25 H2O)
C, H, N.
tr a n s-(2′S,4S)-4-[2′-(3′′-Ben zyloxyca r b on yla m in o-2′′-
oxo-2′′H-p yr id in -1′′-yl)-3′-(3′′′,4′′′-d iflu or op h en yl)p r op io-
n yla m in o]-6-ca r ba m oylh ex-2-en oic Acid Eth yl Ester (5).
mp 175-178 °C; Rf ) 0.43 (10% CH3OH in CH2Cl2). IR (cm-1):
1
3298, 1661, 1516, 1266. H NMR (CDCl3): δ 1.32 (t, 3H, J )
7.2), 1.74-1.95 (m, 2H), 2.12-2.20 (m, 2H), 3.05-3.12 (m, 1H),
3.42-3.50 (m, 1H), 4.21 (q, 2H, J ) 7.2), 4.45-4.56 (m, 1H),
5.21 (s, 2H), 5.58-5.71 (m, 3H), 6.07-6.10 (m, 1H), 6.32-6.34
(m, 1H), 6.71 (dd, 1H, J ) 15.6, 5.1), 6.88-6.91 (m, 1H), 6.99-
7.11 (m, 2H), 7.30-7.33 (m, 1H), 7.36-7.39 (m, 5H), 7.53 (s,
br. 1H), 7.86 (s, br. 1H), 8.04 (s, br. 1H). Anal. (C31H32N4O7‚
0.50H2O) C, H, N.
1
(cm-1): 3331, 1678 br, 1590, 1531. H NMR (CDCl3): δ 1.42-
1.53 (m, 1H), 1.64-1.80 (m, 1H), 1.98-2.10 (m, 1H), 2.19-
2.32 (m, 2H), 2.48 (s, 3H), 3.12 (dd, 1H, J ) 14.0, 8.2), 3.19-
3.33 (m, 2H), 3.43 (dd, 1H, J ) 14.0, 7.4), 3.52 (s, 2H), 3.80-
3.88 (m, 1H), 3.92-4.04 (m, 1H), 5.78-5.85 (m, 1H), 6.25-
6.32 (m, 1H), 6.44 (s, 1H), 6.72 (s, 1H), 6.80-6.88 (m, 1H),
tr a n s-(2′S,4S)-4-[2′-(3′′-Ben zyloxyca r b on yla m in o-2′′-
oxo-2′′H-p yr id in -1′′-yl)-3′-cycloh exylp r op ion yla m in o]-6-
ca r ba m oylh ex-2-en oic Acid Eth yl Ester (6). mp 64-66 °C;

1620 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 8
Dragovich et al.
Rf ) 0.47 (10% CH3OH in CH2Cl2). IR (cm-1): 3302, 2925,
1721, 1651, 1197. 1H NMR (CDCl3): δ 0.93-1.05 (m, 2H),
1.18-1.22 (m, 4H), 1.32 (t, 3H, J ) 7.2), 1.66-1.75 (m, 4H),
1.82-1.91 (m, 2H), 1.94-2.06 (m, 2H), 2.10-2.17 (m, 2H),
2.90-2.92 (m, 2H), 4.23 (q, 2H, J ) 7.2), 4.52-4.60 (m, 1H),
5.23 (s, 2H), 5.65 (t, 1H, J ) 8.1), 5.82-5.86 (m, 1H), 5.62 (dd,
1H, J ) 15.9, 1.8), 6.01-6.05 (m, 1H), 6.37 (t, 1H, J ) 7.2),
6.85 (dd, 1H, J ) 15.9, 5.7), 7.25-7.29 (m, 1H), 7.36-7.43 (m,
5H), 7.88 (s, br. 1H), 8.09 (d, 1H, J ) 6.9). Anal. (C31H40N4O7‚
1.0H2O) C, H, N.
7.39 (m, 7H), 8.33-8.38 (m, 1H), 8.59 (s, 1H). Anal. (C28H36N4O6‚
0.50H2O) C, H, N.
tr a n s-(2′S,4S)-6-Ca r b a m oyl-4-(2′-{3′′-[(5′′′-ch lor oisox-
a zole-3′′′-ca r b on yl)a m in o]-2′′-oxo-2′′H -p yr id in -1′′-yl}-3′-
p h en ylp r op ion yla m in o)h ex-2-en oic Acid E t h yl E st er
(14). mp 163-165 °C; Rf ) 0.49 (10% CH3OH in CH2Cl2). IR
1
(cm-1): 3335, 1648, 1533, 1179. H NMR (CDCl3): δ 0.34 (t,
3H, J ) 7.2), 1.76-1.86 (m, 1H), 1.94-2.00 (m, 1H), 2.23 (t,
2H, J ) 6.9), 3.20-3.27 (m, 1H), 3.35-3.42 (m, 2H), 3.51-
3.58 (m, 1H), 4.22 (q, 2H, J ) 7.2), 4.52-4.57 (m, 1H), 5.69
(dd, 1H, J ) 15.6, 1.5), 6.70 (s, br. 1H), 6.14 (s, br. 1H), 6.32
(s, br. 1H), 6.39 (t, 1H, J ) 7.2), 6.69 (dd, 1H, J ) 15.6, 5.4),
7.21-7.33 (m, 4H), 7.47 (d, 1H, J ) 7.2), 8.44 (d, 1H, J ) 7.5),
9.47 (s, br. 1H). Anal. (C27H28ClN5O7‚0.60H2O) C, H, N.
tr a n s-(4S)-4-[2′-(3′′-Ben zyloxyca r b on yla m in o-2′′-oxo-
2′′H-p yr id in -1′′-yl)-a cetyla m in o]-6-ca r ba m oylh ex-2-en o-
ic Acid Eth yl Ester (7). mp 169-174 °C; Rf ) 0.24 (10%
CH3OH in CH2Cl2). IR (cm-1): 3273, 1719, 1649. 1H NMR
(DMSO-d6): δ 1.19 (t, 3H, J ) 7.1), 1.64-1.85 (m, 2H), 2.10
(t, 2H, J ) 7.6), 4.11 (q, 2H, J ) 7.1), 4.38-4.41 (m, 1H), 4.60
(d, 1H, J ) 15.5), 4.67 (d, 1H, J ) 15.5), 5.15 (s, 2H), 5.92 (dd,
1H, J ) 15.8, 1.3), 6.26 (t, 1H, J ) 7.1), 6.76-6.83 (m, 2H),
7.09-7.42 (m, 7H), 7.84 (d, 1H, J ) 7.3), 8.41-8.44 (m, 2H).
Anal. (C24H28N4O7‚0.50H2O) C, H, N.
tr a n s-(2′S,4S)-4-[2′-(3′′-Acetylam in o-2′′-oxo-2′′H-pyr idin -
1′′-yl)-3′-p h en yp r op ion yla m in o]-6-ca r ba m oylh ex-2-en o-
ic Acid Eth yl Ester (8). Rf ) 0.12 (5% CH3OH in CH2Cl2).
IR (cm-1): 3307, 1708, 1666, 1643, 1590, 1519. 1H NMR
(CDCl3): δ 1.30 (t, 3H, J ) 7.1), 1.70-1.86 (m, 2H), 2.05-
2.24 (m, 5H), 3.15 (dd, 1H, J ) 13.7, 8.1), 3.50 (dd, 1H, J )
13.7, 7.8), 4.19 (q, 2H, J ) 7.1), 4.45-4.56 (m, 1H), 5.66-5.77
(m, 2H), 5.82 (s, 1H), 5.94 (s, 1H), 6.28 (t, 1H, J ) 7.2), 6.69
(dd, 1H, J ) 15.7, 5.6), 7.10-7.29 (m, 5H), 7.32-7.45 (m, 2H),
8.28-8.36 (m, 2H). Anal. (C25H30N4O6‚0.50H2O) C, H, N.
tr a n s-(2′S,4S)-6-Ca r ba m oyl-4-{2′-[3′′-cyclop en ta n eca r -
b on yla m in o-2′′-oxo-2′′H -p yr id in -1′′-yl]-3′-p h e n ylp r o-
p ion yla m in o}h ex-2-en oic Acid Eth yl Ester (9). Rf ) 0.27
(5% CH3OH in CH2Cl2). IR (cm-1): 3319, 1713, 1666, 1590,
1514. 1H NMR (CDCl3): δ 1.31 (t, 3H, J ) 7.1), 1.55-2.02 (m,
10H), 2.04-2.22 (m, 2H), 2.68-2.80 (m, 1H), 3.16 (dd, 1H, J
) 13.7, 7.7), 3.51 (dd, J ) 13.7, 8.1), 4.19 (q, 2H, J ) 7.1),
4.45-4.56 (m, 1H), 5.57-5.74 (m, 4H), 6.29 (t, 1H, J ) 7.4),
6.68 (dd, 1H, J ) 15.8, 5.5), 7.10-7.32 (m, 7H), 8.30 (s, 1H),
8.35 (dd, 1H, J ) 7.4, 1.7). Anal. (C29H36N4O6‚0.50H2O) C, H,
N.
tr a n s-(2′S,3′′′S,4S)-4-[2′-(3′′-Ben zyloxyca r bon yla m in o-
2′′-oxo-2′′H -p yr id in -1′′-yl)-3′-p h en ylp r op ion yla m in o]-5-
(2′′′-oxop yr r olid in -3′′′-yl)p en t-2-en oic Acid Eth yl Ester
(15). Rf ) 0.28 (5% CH3OH in CH2Cl2). IR (cm-1): 3272, 1684
1
(br), 1590, 1514, 1273, 1196. H NMR (CDCl3): δ 1.30 (t, 3H,
J ) 7.1), 1.42-1.55 (m, 1H), 1.59-1.75 (m, 1H), 2.00-2.27 (m,
3H), 3.07-3.28 (m, 3H), 3.43 (dd, 1H, J ) 13.7, 7.3), 4.19 (q,
2H, J ) 7.1), 4.36-4.47 (m, 1H), 5.12-5.21 (m, 2H), 5.75 (dd,
1H, J ) 15.6, 1.2), 5.85-5.94 (m, 1H), 6.26 (t, 1H, J ) 7.2),
6.58 (s, 1H), 6.70 (dd, 1H, J ) 15.6, 5.7), 7.10-7.41 (m, 10H),
7.44-7.50 (m, 1H), 7.71 (s, 1H), 7.97 (d, 1H J ) 6.2), 8.28 (d,
1H, J ) 6.8). Anal. (C33H36N4O7‚0.25H2O) C, H, N.
tr a n s-(2′S,3′′′R,4S)-4-[2′-(3′′-Ben zyloxyca r bon yla m in o-
2′′-oxo-2′′H -p yr id in -1′′-yl)-3′-p h en ylp r op ion yla m in o]-5-
(2′′′-oxop yr r olid in -3′′′-yl)p en t-2-en oic Acid Eth yl Ester
(16). Rf ) 0.27 (5% CH3OH in CH2Cl2). IR (cm-1): 3272, 1684
(br), 1514, 1267, 1196. 1H NMR (CDCl3): δ 1.31 (t, 3H, J )
7.1), 1.48-1.79 (m, 2H), 2.02-2.24 (m, 2H), 2.27-2.39 (m, 1H),
3.12 (dd, 1H, J ) 13.7, 8.2), 3.19-3.34 (m, 2H), 3.48 (dd, 1H,
J ) 13.7, 7.8), 4.19 (q, 2H, J ) 7.1), 4.43-4.53 (m, 1H), 5.17
(s, 2H), 5.73 (dd, 1H, J ) 15.6, 1.3), 5.90-5.98 (m, 1H), 6.27
(t, 1H, J ) 7.1), 6.63 (dd, 1H, J ) 15.6, 6.0), 6.65-6.71 (m,
1H), 7.13-7.27 (m, 6H), 7.31-7.40 (m, 4H), 7.50 (dd, 1H, J )
7.1, 1.6), 7.75 (s, 1H), 7.97 (d, 1H, J ) 6.6), 8.69 (d, 1H, J )
7.0). Anal. (C33H36N4O7‚0.50H2O) C, H, N.
tr a n s-(2′S,4S)-4-[2′-(3′′-Ben zyloxyca r b on yla m in o-4′′-
m eth yl-2′′-oxo-2′′H-p yr id in -1′′-yl)-3′-p h en ylp r op ion yla m i-
n o]-6-ca r ba m oylh ex-2-en oic Acid Eth yl Ester (17). Rf )
0.23 (5% CH3OH in CH2Cl2). IR (cm-1): 3284, 1684 (br), 1596,
tr a n s-(2S,4′S)-6-Car bam oyl-4-(2′-{3′′-[([1′′′,3′′′]dith iolan e-
2′′′-ca r bon yl)a m in o-2′′-oxo-2′′H-p yr id in -1′′-yl}-3′-p h en yl-
p r op ion yla m in o)h ex-2-en oic Acid Eth yl Ester (10). Rf )
0.28 (5% CH3OH in CH2Cl2). IR (cm-1): 3295, 1672 (br), 1590,
1519 (br), 1273. 1H NMR (CDCl3): δ 1.30 (t, 3H, J ) 7.1), 1.66-
2.22 (m, 4H), 3.17 (dd, 1H, J ) 13.7, 7.8), 3.28-3.45 (m, 4H),
3.52 (dd, 1H, J ) 13.7, 8.1), 4.19 (q, 2H, J ) 7.1), 4.43-4.55
(m, 1H), 5.01 (s, 1H), 5.66 (dd, 1H, J ) 15.8, 1.5), 5.67 (s, 1H),
5.86 (s, 2H), 6.29 (t, 1H, J ) 7.3), 6.67 (dd, 1H, J ) 15.8, 5.5),
7.12-7.40 (m, 7H), 8.31 (dd, 1H, J ) 7.3, 1.6), 9.57 (s, 1H).
Anal. (C27H32N4O6S2‚0.50H2O) C, H, N.
t r a n s-(2′S ,4S )-6-Ca r b a m oyl-4-(2′-{2′′-oxo-3′′-[(t e t r a -
hydrofuran-2′′′-carbonyl)amino]-2′′H-pyridin-1′′-yl}-3′-phenyl-
p r op ion yla m in o)h ex-2-en oic Acid Eth yl Ester (11). mp
64-67 °C; Rf ) 0.28 (10% CH3OH in CH2Cl2). IR (cm-1): 3344,
1646, 1519, 1178. 1H NMR (CDCl3): δ 1.34 (t, 3H, J ) 7.2),
1.72-1.82 (m, 1H), 1.95-2.04 (m, 2H), 2.16-2.23 (m, 2H),
2.32-2.43 (m, 1H), 3.18-3.27 (m, 1H), 3.51-3.60 (m, 5H),
3.93-4.00 (m, 1H), 4.05-4.12 (m, 1H), 4.22 (q, 2H, J ) 7.2),
4.46-4.55 (m, 2H), 5.54-5.69 (m, 2H), 6.34-6.41 (m, 2H), 6.68
(dd, 1H, J ) 15.6, 5.4), 6.86-6.93 (m, 1H), 7.17-7.41 (m, 5H),
8.42-8.45 (m, 1H), 9.37 (d, 1H, J ) 10.2). Anal. (C28H34N4O7‚
1.5TFA) C, H, N.
1
1327, (br), 1308, 1267, 1237. H NMR (CDCl3): δ 1.29 (t, 3H,
J ) 7.1), 1.55-1.68 (m, 1H), 1.82-2.07 (m, 4H), 2.14 (s, 3H),
3.08 (dd, 1H, J ) 13.6, 7.1), 3.47 (dd, J ) 13.6, 8.8), 4.17 (q,
2H, J ) 7.1), 4.41-4.52 (m, 1H), 5.06 (d, 1H, J ) 12.3), 5.12
(d, 1H, J ) 12.3), 5.59 (dd, 1H, J ) 15.7, 1.6), 5.67-5.74 (m,
1H), 5.90 (s, 1H), 6.02 (s, 1H), 6.17 (d, 1H, J ) 7.3), 6.68 (dd,
1H, J ) 15.7, 5.1), 7.01 (s, 1H), 7.13-7.38 (m, 10H), 7.48 (d,
1H, J ) 7.3). Anal. (C32H36N4O7‚0.75H2O) C, H, N.
tr a n s-(2′S,4S)-6-Ca r b a m oyl-4-(2′-{4′′-m et h yl-3′′-[(5′′′-
m eth ylisoxazole-3′′′-car bon yl)am in o]-2′′-oxo-2′′H-pyr idin -
1′′-yl}-3′-p h en ylp r op ion yla m in o)h ex-2-en oic Acid Eth yl
Ester (18). mp 138-141 °C; Rf ) 0.52 (10% CH3OH in CH2-
1
Cl2). IR (cm-1): 3289, 1663, 1542, 1203. H NMR (CDCl3): δ
1.22 (t, 3H, J ) 7.2), 1.62-1.89 (m, 2H), 1.99 (s, 3H), 2.02-
2.07 (m, 1H), 2.47 (s, 3H), 3.03-3.39 (m, 5H), 4.12 (q, 2H, J )
7.2), 4.32-4.41 (m, 1H), 5.76 (dd, 1H, J ) 15.6, 1.5), 5.80-
5.83 (m, 1H), 6.13 (d, 1H, J ) 7.5), 6.60 (s, br. 1H), 6.75 (dd,
1H, J ) 15.6, 5.4), 7.15-7.24 (m, 5H), 7.76 (d, 1H, J ) 7.2),
8.65 (d, 1H, J ) 7.8), 9.59 (s, br. 1H). Anal. (C29H33N5O7‚
1.5TFA) C, H, N.
tr a n s-(2′S,3′′′′′S,4S)-4-(3′-(4′′-F lu or op h en yl)-2′-{3′′′-[(5′′′′-
m eth ylisoxa zole-3′′′′-ca r bon yl)a m in o]-2′′′-oxo-2′′′H-p yr i-
d in -1′′′-yl}p r op ion yla m in o)-5-(2′′′′′-oxop yr r olid in -3′′′′′-yl)-
p en t-2-en oic Acid Eth yl Ester (19). Rf ) 0.24 (5% CH3OH
in CH2Cl2). IR (cm-1): 3331, 1690, 1590, 1531, 1455. 1H NMR
(CDCl3): δ 1.30 (t, 3H, J ) 7.0), 1.45-1.55 (m, 1H), 1.64-
1.75 (m, 1H), 2.03-2.31 (m, 3H), 2.49 (s, 3H), 3.10 (dd, 1H, J
) 13.7, 7.9), 3.20-3.46 (m, 3H), 4.20 (q, 2H, J ) 7.0), 4.36-
4.47 (m, 1H), 5.67 (dd, 1H, J ) 15.7, 1.4), 5.85-5.92 (m, 1H),
6.29 (t, 1H, J ) 7.2), 6.45 (s, 1H), 6.70 (dd, 1H, J ) 15.7, 5.7),
6.86 (s, 1H), 6.90-6.97 (m, 2H), 7.10-7.16 (m, 2H), 7.60 (dd,
tr a n s-(2′S,4S)-6-Ca r ba m oyl-4-{2′-[3′′-(2′′′,2′′′-d im eth yl-
p r op ion yla m in o)-2′′-oxo-2′′H -p yr id in -1′′-yl]-3′-p h en yl-
p r op ion yla m in o}h ex-2-en oic Acid Eth yl Ester (12). Rf )
0.38 (5% CH3OH in CH2Cl2). IR (cm-1): 3378, 3307, 3213,
1713, 1666, 1643, 1590, 1514, 1273. 1H NMR (CDCl3): δ 1.18-
1.37 (m, 12H), 1.67-1.98 (m, 2H), 2.05-2.20 (m, 2H), 3.17 (dd,
1H, J ) 13.6, 7.7), 3.50 (dd, 1H, J ) 13.6, 8.2), 4.19 (q, 2H, J
) 7.1), 4.43-4.54 (m, 1H), 5.62-5.72 (m, 2H), 5.81-5.92 (m,
2H), 6.29 (t, 1H, J ) 7.2), 6.66 (dd, 1H, J ) 15.8, 5.7), 7.13-

Human Rhinovirus 3C Protease Inhibitors
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 8 1621
1H, J ) 7.2, 1.6), 8.37 (dd, 1H, J ) 7.2, 1.6), 8.51 (d, 1H, J )
6.6), 9.47 (s, 1H). Anal. (C30H32FN5O7‚0.50H2O) C, H, N.
Or a l Bioa va ila b ilit y of Com p ou n d 21 in t h e Bea gle
Dog. The oral bioavailability of compound 21 was determined
after intravenous (15 mg/kg, n ) 3) and oral (30 mg/kg, n ) 3)
single agent dosing of the molecule to male beagle dogs (BW
a total run time of 25 min. The retention times for compounds
20 and 21 were 12.7 and 13.4 min, respectively. All molecules
were monitored at 215 nm.
P r otein -Liga n d Cr ysta l Str u ctu r e Deter m in a tion .
HRV 3CP of serotype 2 was incubated with a 3-fold molar
excess of compound 3 in the presence of 2% dimethyl sulfoxide
for 24 h at 4 °C. The resulting complex was concentrated to 7
mg/mL and then passed through a 0.45 µM cellulose-acetate
filter. Crystals were grown at 13 °C using the hanging drop
vapor diffusion method in which equal volumes (4 µL) of the
protein/ligand complex and reservoir solution were layered on
plastic coverslips and sealed over wells filled with 1 mL of
reservoir solution containing 0.9 M sodium phosphate, 0.9 M
potassium phosphate, 0.2 M ammonium sulfate, 0.1 M Hepes
at pH 7.5 (HCl titrated), and 20 mM dithiothreitol. A rectan-
gular blocklike crystal of approximate dimensions 0.2 mm ×
0.07 mm × 0.07 mm (space group P212 12; a ) 61.41, b ) 77.74,
c ) 33.97 Å) was prepared for low-temperature data collection
by transfer to an artificial mother liquor solution consisting
of 400 µL of the reservoir solution mixed with 125 µL of
glycerol and then flash frozen in a stream of N2 gas at -170
°C. X-ray diffraction data were collected with a MAR Research
345 mm imaging plate and processed with DENZO.46 Diffrac-
tion data were 96.8% complete to a resolution of 2.3 Å with
Rsym ) 7.4%. Protein atomic coordinates from a previously
described isomorphous cocrystal structure of type 2 3CP15 were
used to initiate rigid-body refinement in XPLOR47 followed by
simulated annealing and conjugate gradient protocols. Place-
ment of the inhibitor, addition of ordered solvent, and further
refinement were conducted as described previously.48 The final
R factor was 18.4% [(6279 reflections with F > 2σ(F)]. The root-
mean-square deviations from ideal bond lengths and angles
were 0.019 Å and 3.2 deg, respectively. The final model
consisted of all atoms for residues 1-180 (excluding side chains
of residues 12, 45, 55, and 65) plus 141 water molecules.
∼9.3 kg). The compound was administered in
a vehicle
consisting of 80% propylene glycol and 20% sterile water at a
concentration of 15.0 mg/mL. Blood samples were collected
periodically for up to 24 h postdosing. The plasma concentra-
tions of 21 were determined by liquid chromatography mass
spectrometry (limit of detection equal to 5 ng/mL). Pharma-
cokinetic parameters 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 (20 and 21) 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 high-perfor-
mance liquid chromatography (HPLC) with a Primesphere
reversed phase column (5 µm, 4.6 mm × 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 to 40
µg/mL. The half-life of a given compound’s metabolic conver-
sion rate was determined by linear regression (WinNonlin
Professional, Pharsight, Mountainview, CA) of the mean
plasma concentration-time data obtained from the incubation
studies.
Micr osom e Sta bility Stu d ies. Human pooled liver mi-
crosomes (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 (19, 20, or 21) and proceeded for 30 min followed
by the addition of 500 µL of 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 2500g
for 20 min. The supernatant was directly injected onto an
HPLC system and analyzed. For compound 19, chromato-
graphic separation was achieved using a Hewlett-Packard
1050 HPLC with a Primesphere C18 HC reversed phase column
(5 µm, 4.6 mm × 150 mm). Analyte elution was conducted
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 a total run time of 24 min. The retention
time of compound 19 was 15.1 min under these conditions.
For compounds 20 and 21, chromatographic separation was
achieved using a Zorbax Eclipse XDB C18 column (5 µm, 4.6
mm × 150 mm). These compounds 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
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 Dr. Steven Bender. We also
thank Dr. Xinjun Hou for assistance with the prepara-
tion of Figure 2.
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(22) Ruprintrivir (AG7088) has exhibited antiviral activity against
several nonrhinoviral picornaviruses.19a Treatment of many such
picornaviral-mediated diseases would presumably be best ef-
(13) (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

Human Rhinovirus 3C Protease Inhibitors
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 8 1623
fected with an orally bioavailable therapeutic agent. For ad-
ditional details, see Rotbart, H. A. Antiviral Therapy for
Enteroviruses and Rhinoviruses. Antiviral Chem. Chemother.
2000, 11, 261-271.
(32) In the discussion of the X-ray crystal structure, the notation
“3CP” indicates 3C protease derived from HRV-2. This protein
provided diffraction quality crystals more routinely than the
serotype 14-derived protease. The inhibitor-binding regions of
the two enzymes do not differ significantly.
(23) A detailed account of these studies will be disclosed in the future.
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