Substituted benzamide inhibitors of human rhinovirus 3C protease: Structure-based design, synthesis, and biological evaluation

Article abstract of DOI:10.1021/jm9903242

A series of nonpeptide benzamide-containing inhibitors of human rhinovirus (HRV) 3C protease was identified using structure-based design. The design, synthesis, and biological evaluation of these inhibitors are reported. A Michael acceptor was combined with a benzamide core mimicking the P1 recognition element of the natural 3CP substrate, α,β-Unsaturated cinnamate esters irreversibly inhibited the 3CP and displayed antiviral activity (EC₅₀ 0.60/μM, HRV-16 infected H1-HeLa cells). On the basis of cocrystal structure information, a library of substituted benzamide derivatives was prepared using parallel synthesis on solid support. A 1.9 A? cocrystal structure of a benzamide inhibitor in complex with the 3CP revealed a binding mode similar to that initially modeled wherein covalent attachment of the nucleophilic cysteine residue is observed. Unsaturated ketones displayed potent reversible inhibition but were inactive in the cellular antiviral assay and were found to react with nucleophilic thiols such as DTT.

Full text of DOI:10.1021/jm9903242

1670
J . Med. Chem. 2000, 43, 1670-1683
Articles
Su bstitu ted Ben za m id e In h ibitor s of Hu m a n Rh in ovir u s 3C P r otea se:
Str u ctu r e-Ba sed Design , Syn th esis, a n d Biologica l Eva lu a tion
Siegfried H. Reich,* Theodore J ohnson, Michael B. Wallace, Susan E. Kephart, Shella A. Fuhrman,
Stephen T. Worland, David A. Matthews, Thomas F. Hendrickson, Fora Chan, J ames Meador, III,
Rose Ann Ferre, Edward L. Brown, Dorothy M. DeLisle, Amy K. Patick, Susan L. Binford, and Clifford E. Ford
Agouron Pharmaceuticals Inc., 3565 General Atomics Court, San Diego, California 92121
Received J une 24, 1999
A series of nonpeptide benzamide-containing inhibitors of human rhinovirus (HRV) 3C protease
was identified using structure-based design. The design, synthesis, and biological evaluation
of these inhibitors are reported. A Michael acceptor was combined with a benzamide core
mimicking the P1 recognition element of the natural 3CP substrate. R,â-Unsaturated cinnamate
esters irreversibly inhibited the 3CP and displayed antiviral activity (EC50 0.60 µM, HRV-16
infected H1-HeLa cells). On the basis of cocrystal structure information, a library of substituted
benzamide derivatives was prepared using parallel synthesis on solid support. A 1.9 Å cocrystal
structure of a benzamide inhibitor in complex with the 3CP revealed a binding mode similar
to that initially modeled wherein covalent attachment of the nucleophilic cysteine residue is
observed. Unsaturated ketones displayed potent reversible inhibition but were inactive in the
cellular antiviral assay and were found to react with nucleophilic thiols such as DTT.
In tr od u ction
goal was to ultimately develop orally available 3CP
inhibitors; therefore, the design strategy focused on
nonpeptide motifs which would be expected to have
more favorable pharmacokinetic properties. Initial de-
sign was deliberately very simple: positioning an alde-
hyde group so that it could react with the nucleophilic
cysteine and placing a carboxamide group in the S1
recognition pocket. A meta-substituted phenyl ring
positioned these two elements with the appropriate
distance and relative orientation leading to 3-carbamoyl
benzaldehyde as a prototype inhibitor 1a (Figure 1).
This compound was tested and found to be a very weak
inhibitor of the 3CP (Ki ) 104 µM). In addition to the
small size of this inhibitor, we reasoned that its lack of
activity could in part be due to the benzaldehyde
carbonyl being considerably less reactive than its R-ami-
no aldehyde counterpart found in potent peptide-based
aldehyde inhibitors.6b Furthermore, the inherent insta-
bility of aldehydes made this group undesirable for our
purposes. It had been established in our laboratory6c and
others6n that cysteine proteases in general and 3CP in
particular are potently inhibited by Michael acceptors
when incorporated into a peptidic recognition element.
Replacement of the formyl group of inhibitor 1a with
the R,â-unsaturated ethyl ester led to compound 1,
which was found to be an irreversible inhibitor with a
weak inactivation constant7 of 52 s-1 M-1 (Table 1).
Interestingly, compound 1 also showed weak but de-
monstrable antiviral activity (EC50) when tested in a
cytopathic effect assay6 employing H1-HeLa cells in-
fected with HRV-14 (Table 1). Furthermore, compound
1 was nontoxic (CC50) up to 320 µM.
The human rhinoviruses (HRV), which are the pri-
mary cause of the common cold in man, belong to the
Picornovirus family.1 Picornoviruses, such as HRV, have
a single positive-stranded RNA genome,2 which is
translated into a polyprotein of over 2000 amino acids.
The 2A and 3C protease (3CP) process this polyprotein
into its functional viral proteins in HRV.3 For the 3CP,
the consensus cleavage site in the viral polyprotein
substrate is between glutamine (P1) and glycine (P1′)
residues. While the 3CP is a cysteine protease, its
tertiary structure is reminiscent of trypsin-like serine
proteases.4 The requirement for proteolytic processing
of the viral polyprotein, supported by mutagenesis of
the active site residues,5 makes the 3CP a viable target
for antirhinoviral therapy. Solution of the HRV 3CP
crystal structure has facilitated the design of a number
of 3CP inhibitors, which have been previously reported
from our laboratories6a-d and others.6e-n The aim of this
research has been to identify a nonpeptide, low molec-
ular weight inhibitor of 3CP with potent antirhinoviral
activity. In this report we describe the structure-based
design, synthesis, and biological evaluation of a series
of benzamide-containing inhibitors of 3CP.
Design of In h ibitor s
With the aid of the cocrystal structure of a peptide
aldehyde6b bound to the 3CP, the design of a novel
nonpeptide inhibitor was undertaken. Analysis of the
2.3 Å crystal structure of the HRV-14 3CP enzyme
verified that it is structurally related to the trypsin
family of proteases, with a catalytic triad composed of
the residues cysteine, histidine, and glutamic acid.4a Our
With this information in hand, additional esters were
prepared and tested. It was anticipated that by filling
10.1021/jm9903242 CCC: $19.00 © 2000 American Chemical Society
Published on Web 04/01/2000

Human Rhinovirus 3C Protease Inhibitors
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 9 1671
F igu r e 1. Design of benzamide core based on the 3CP cleavage site.
Ta ble 1. Substituted Benzamides
Cmpd No.a
R1
R2
R3
R4
R5
R6
Kobs/[I] (M-1 s-1) [Ki (µM)]b
EC50 (µM)b
CC50 (µM)b
1
2
3
4
5
6
7
8
H
H
H
H
H
H
H
H
H
Me
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
CN
H
H
H
H
H
H
Et
Me
CH2Ph
CH2CH2OH
CH2CH2Ph
CH2(2-pyridyl)
Et
Et
Et
Et
Et
Et
Et
Et
Et
NH2
NH2
NH2
NH2
NH2
NH2
OH
OMe
NH2
NH2
NH2
NH2
NH2
NH2
NH2
52
28
25
42
83
57
NI
NI
[1.5]
10%@25
47
NI
54
15.9
150
5.6
20
100
10
ND
ND
>100
>100
>100
ND
>100
ND
>320
>320
>100
>320
>320
>320
ND
ND
9
>100
>100
>100
ND
>100
ND
10
11
12
13
14
15
OH
OCH2Ph
CH2OH
H
H
OH
OCH2Ph
NI
30
>320
>320
a
b
Elemental analyses (C, H, N) of all compounds agreed to within (0.4% of theoretical values. Serotype 14; see Experimental Section
for assay Method. NI ) no inhibition; ND ) not determined.
the active site in the prime direction (S1′-S2′) additional
Ta ble 2. R,â-Unsaturated Keto Benzamides
affinity might be obtained. The methyl ester 2, while
showing similarly weak inhibition of the enzyme rela-
tive to ethyl ester 1, was surprisingly 10-fold less active
in the antiviral assay. As shown in Table 1, the ester
group, in general, was found to have an unremarkable
effect on the potency, with the benzyl ester 3 showing
the best activity. To determine whether the activity of
the parent compound 1 was due to the inherent reactiv-
ity of the unsaturated ester, alternative substitutions
were examined in the 1-position corresponding to the
S1 recognition pocket. A clear preference was observed
for the primary carboxamide, consistent with recogni-
tion of glutamine in the native substrate peptide.6a
Increased activation of the Michael acceptor toward
nucleophilic addition, by R-cyano substitution, led to a
modest but reversible inhibitor 9. Various unsaturated
imides, also expected to be more reactive than an
unsaturated ester, were poor inhibitors (data not shown).
Exploration of R,â-unsaturated ketones led to some
interesting findings (Table 2). A simple methyl ketone
showed only modest but, again, reversible inhibition.
However, phenyl ketone 17 was considerably more
potent against 3CP. When tested in the antiviral assay,
ketones in general had greater toxicity (lower CC50) and
no measurable antiviral effect. Upon incubation with
DTT, the R,â-unsaturated ketones were completely
inactivated, suggesting that their lack of antiviral
DTT
EC50
CC50
Cmpd No.a
R1
Ki (µM)b inhib.c (µM)b (µM)b
16
17
18
19
20
21
22
Me
Ph
25
0.40
9
1.8
1.8
1.9
0.12
yes
yes
yes
yes
yes
yes
yes
32
28
>15
>22
50
>71
>20
40
>28
>15
>22
>50
>71
>20
Ph(4-NMe2)
Ph(4-OMe)
2-pyridyl
2-furyl
-
a
Elemental analyses (C, H, N) of all compounds agreed to
b
within (0.4% of theoretical values. Serotype 14; see Experimen-
tal Section for assay method. c Indicates loss of 3CP inhibitory
activity after exposure of compound to 5 mM DTT for 2-3 min at
23 °C.
activity may be due to interaction with endogenous
thiols in cells (glutathione).6c Electron-donating sub-
stituents at the 4-position of the phenyl ketone (4-
Me2N-, 4-MeO-), were expected to make the ketones less

1672 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 9
Reich et al.
F igu r e 2. Stereoview of the 1.9 Å cocrystal structure of benzamide inhibitor 4 in complex with HRV-14 3CP. The catalytic triad
residues (S2 subsite, Glu-71, His-40, and Cys-146) and inhibitor 4 are shown with thicker bonds. The carboxamide group occupies
the S1 specificity pocket. Protein and inhibitor atoms are shown with atoms in green (carbon), blue (nitrogen), red (oxygen), and
yellow (sulfur). Ordered water molecules are represented as red crosses. The protein’s solvent accessible surface is shown in
white.
reactive; however, no effect on their inactivation with
DTT was observed. Similarly, heterocyclic ketones (2-
pyridyl, 2-furyl) maintained their reactivity with DTT.
In an attempt to make the inhibitors more compact, an
indenone scaffold was designed using crystal structure
information. Bicyclic indenone 22 was the most potent
of the ketones; however, it too was inactivated by DTT
and had no antiviral activity. It became clear that while
small nonpeptidic R,â-unsaturated ketones such as 22
afforded potent reversible inhibition of 3CP, this in-
creased reactivity was incompatible with the more
complex cellular milieu present in the cytopathic effect
assay.
One of the more soluble unsaturated ester inhibitors,
hydroxyethyl ester 4, was successfully cocrystallized
with 3CP, and a 1.9 Å crystal structure was solved. The
bound conformation was similar to that of the original
model in terms of the phenyl core and 3-carboxamide
(Figure 2). Interestingly, the orientation of the Michael
ester adduct observed in the crystal structure was
opposite of that predicted based on modeling studies.
That is, the carboethoxy group was rotated away from
the 3-carboxamide group in the crystal structure, whereas
the modeled compound has the carboethoxy group
oriented toward the 3-carboxamide function. This is
consistent with the good hydrogen bond (2.98 Å) ob-
served between the ester carbonyl and Cys147 NH
which may activate the ester toward 1,4-addition in the
transition state and also appears to be a good interaction
in the final complex. In addition, this orientation was
subsequently observed in the peptide-based Michael
acceptors as well.6c Other key interactions between the
protein and inhibitor were also observed. The 3-car-
boxamide is found to make three hydrogen bonds to
His161 and Thr 142. The hydrogen bond from the amide
NH to the Thr142 carbonyl is slightly longer (3.12 Å)
and in a less optimal orientation than that observed
with peptide-based Michael acceptors.6c While the car-
boxamide is 23° out of the aromatic plane, apparently
to secure these hydrogen bonds, further rotation out of
plane would be required to more closely mimic the
hydrogen bonds observed in the P1 glutamine of peptidic
inhibitors, with an associated energetic penalty. This
molecular recognition explains the specificity for the
primary carboxamide over the other groups tested in
this position and is consistent with the binding interac-
tions observed with other inhibitors.6a-c Prior to solution
of the cocrystal structure of the benzamide core, we
began to explore additional substituents on the phenyl
ring to increase affinity. We reasoned that substitution
from the 6-position might allow access to the S2 subsite,
which was unoccupied, and based on other classes of
3CP inhibitors should improve binding significantly.
4-Methyl compound 10, however, proved to be less active
than the parent unsubstituted compound 1. In retro-
spect, the ortho-methyl substitution in 10 would be
expected to prevent the favorable orientation of the
Michael acceptor away from the carboxamide as ob-
served in the cocrystal structure of 1. Substitution of
the Michael acceptor in either the R- or â-position by a
methyl group resulted in complete loss of activity (data
not shown). Finally, substitution at the 6-position also
resulted in loss of activity.
Analysis of the cocrystal structure of 1 indicated that
substitution at the 5-position looked particularly prom-
ising. The aryl-H vector in the 5-position was directed
along the â-sheet toward the S3-S4 pockets, and fur-
thermore, with the appropriate substitution, it appeared
that the S2 pocket might also be accessible (see Figure
2). The phenol 11 had comparable activity to that of 1,
yet the corresponding phenol ethers tested showed a
universal lack of activity (e.g., 12). Hydroxymethylene

Human Rhinovirus 3C Protease Inhibitors
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 9 1673
Ta ble 3. 5-Substituted Benzamides
compound 13, however, was found to retain all of the
potency of the unsubstituted parent 1.
P a r a llel Syn th esis a t th e 5-P osition
With the tolerance for a hydroxymethyl group at the
5-position observed in 13 along with cocrystal structure
information, a parallel synthesis approach was under-
taken to explore substitution at this position. Using the
primary carboxamide group as a handle for attachment
to solid support, it was felt that derivatives could be
readily accessed through nucleophilic substitution of the
corresponding bromomethyl compound (see Chemistry
section). A search of the Available Chemicals Directory
(ACD) for primary amines and mercaptans suitable for
synthesis yielded 3087 possible final compounds.8
A
structure-based computational approach was used to
rank and select a subset of molecules from the total
number that could be synthesized. From the precursor
fragments, a virtual library of 5-substituted benzamides
was created. This library of 3D structures was then run
through a partially fixed docking procedure, where the
benzamide “core” of the molecule was kept fixed to its
position as observed in the cocrystal structure, and the
remaining atoms were adjusted to find their optimal
position in the active site. Once these molecules were
docked, they were analyzed and ranked by low energy
interactions with the protein, number of additional
protein-ligand hydrogen bonds made, and the degree
to which they filled the S2, S3, and/or the S4 pocket.
The best candidate compounds from this screening and
ranking procedure were then selected for synthesis. Of
the 784 compounds prepared and tested, about 30
having greater than 80% inhibition of 3CP at 20 µM9
were selected for resynthesis and full characterization.
As shown in Table 3, a clear preference for branched
aminomethylene groups was observed. The rates of
inactivation (Kobs/I), which are modest, do not correlate
with the antiviral potency. In fact, for the most potent
compound, 30, the antiviral EC50 of 600 nM is excep-
tional given the modest Kobs/I. This lack of a correlation
between the rate of inactivation and the antiviral
activity prompted us to investigate whether the anti-
viral effect of these compounds was in fact due to
inhibition of the 3CP. Examination of proteolytic pro-
cessing by 3CP in the cytopathic effect assay using
polyacrylamide gel electrophoresis with compound 1
clearly showed a dose dependent reduction in proteolytic
fragments consistent with inhibition of 3CP.
a
HRMS, NMR, and HPLC purity were all consistent with the
b
indicated structures; see Experimental Section. Serotype 14; see
Experimental Section for assay method.
fication of 3-formyl benzoic acid followed by Wittig
olefination (method A). A more versatile route was later
devised employing a palladium catalyzed Heck reac-
tion11 between an acrylate ester and iodobenzamide 32
(method B). Preparation of the various cinnamyl ester
derivatives 3-6 utilizes cinnamic acid derivative 33 as
a key intermediate. Scheme 2 details the straightfor-
ward preparation of compounds 7-10 using similar
chemistry. The synthesis of 5-substituted compounds 11
and 12 began with selective reduction of 3,5-dinitroani-
sole and conversion to iodobenzamide 40 followed by
standard Heck coupling as before (Scheme 3). Phenol
14 and phenolic ether 15 were prepared from com-
mercially available 3-cyanoanisaldehyde (Scheme 4).
The synthesis of R,â-unsaturated ketones 16-21
began with the preparation of Weinreb amide 43, again
via a Heck coupling (Scheme 5). Reaction of the required
organolithium species with amide 43 afforded ketones
in good yield. The organolithium reagents were com-
mercial or were obtained through metalation of either
the corresponding bromide or, in the case of furan,12 via
direct metalation of the heterocycle. Finally, indenone
22 was obtained by palladium catalyzed cyanation of
To determine the nature of the binding interactions
with the more highly substituted derivatives, a cocrystal
structure was solved of compound 26 bound to 3CP (data
not shown). In this instance, a significant change in
protein conformation was observed which is very likely
a result of crystal packing forces.10 However, the ben-
zamide core of 26 binds essentially in the same space
and orientation as the unsubstituted compound 4. The
piperizine ring lays directly over the backbone of â-sheet
with the pyridine ring buried deeply into a larger
rearranged P4 subsite (see Figure 2). While the pyridine
ring is largely buried in the protein, the energetic cost
of adopting this new protein conformation is unclear.
Syn th esis
The original synthesis of parent compound 1 is
outlined in Scheme 1 involving functional group modi-

1674 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 9
Reich et al.
Sch em e 1a
P a r a llel Syn th esis
The construction of a library of 5-substituted benza-
mides began with the preparation of the benzylic
bromide intermediate 57 as outlined in Scheme 6.
Protection of commercially available diethyl-5-(hy-
droxymethyl)isophthalate 50 and selective hydrolysis of
one ester gave benzoic acid derivative 52. Selective
reduction of the ester with Li-triethylborohydride af-
forded hydroxymethyl acid 53. Oxidation to the alde-
hyde with TPAP followed by Horner-Emmons conden-
sation and silyl deprotection gave penultimate compound
56 in good yield. Last, conversion of the hydroxymethyl
group to the benzyl bromide with PBr3 produced key
intermediate 57 ready for attachment to solid support.
Monomer 57 was coupled to Rink amide resin (either
free resin or Chiron Crowns) using a standard DIC/
HOBt coupling procedure (Scheme 7). Nucleophilic
displacement of bromide 58 in DMF occurred with no
indication of Michael addition products. TFA deprotec-
tion of coupled products 59 afforded final products of
high purity.
Con clu sion s
The 3CP is a promising and challenging target for the
design of nonpeptide inhibitors. We have identified a
series of nonpeptidic benzamide-containing inhibitors
using the 3CP crystal structure as a guide. Solution of
the cocrystal structure of this new series of inhibitors
in complex with the 3CP confirms that they bind
essentially as modeled. The R,â-unsaturated ester group
suffers irreversible covalent 1,4-addition by the nucleo-
philic catalytic cysteine on the protein, which is con-
firmed in the cocrystal structure. Structural feedback
facilitated the optimization of compounds attempting to
access S2 and S3 subsites of the enzyme. Unfortunately,
access to the S2 subsite was not achieved with this class
of inhibitors. A related series of R,â-unsaturated ketones
display potent reversible inhibition. However, they
suffer from inactivation by free thiols and, presumably
as a result, exhibit no antiviral activity. It has been
shown in previous studies that recognition in the S2
pocket can lead to significant enhancements in binding.
A parallel synthesis effort on solid phase allowed for
the preparation of a large number of benzamide deriva-
tives substituted in the 5-position to access the S3-S4
subsites of the enzyme. These derivatives were con-
firmed to occupy the S3-S4 pockets through crystal-
lographic analysis, yet, only modest improvement in
enzyme inactivation was realized. Despite very modest
inactivation constants (Kobs/I), submicromolar antiviral
activity was observed with compound 30. It appears
clear from our work and that of others that recognition
at S1-S3 subsites and selective covalent binding is
necessary to achieve potent 3CP inactivation and anti-
viral activity.
a
(a) (COCl)2, cat. DMF; (b) NH4OH, THF; (c) Ph3PCH2CO2Et,
PhCH3, reflux; (d) CHdCHCO2Me, Pd(OAc)2, Et3N, 100 °C; (e)
NaOH/MeOH; (f) EDC, DMF, ROH, Et3N, DMAP.
Sch em e 2a
a
(a) CHdCHCO2Et, Pd(OAc)2, Et3N, 100 °C; (b) CH2N2; (c)
NCCH2CO2Et, EtOH, piperidine; (d) (COCl)2, cat. DMF; (e)
NH4OH, THF.
Exp er im en ta l Section
Melting points were determined on a Mel-Temp apparatus
and are uncorrected. The structure of all compounds were
confirmed by proton magnetic resonance spectroscopy, infrared
spectroscopy, and either elemental microanalysis or mass
spectrometry. Proton magnetic resonance spectra were deter-
mined using a General Electric QE-300 spectrometer operating
at a field strength of 300 MHz. Chemical shifts are reported
in parts per million (ppm) and by setting the references such
5-bromoindenone, followed by hydrolysis to the primary
amide. Attempted manipulation of the unprotected
indenone carboxamide 45 led only to decomposition
products. Protection of 45 as the trityl amide,13 however,
permitted R-bromination with NBS followed by elimina-
tion and TFA deprotection to afford the desired inde-
none 22.

Human Rhinovirus 3C Protease Inhibitors
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 9 1675
Sch em e 3a
a
(a) NaSH, MeOH; (b) NaNO2, HCl, KI; (c) Fe3(CO)12, EtOH, reflux; (d) Zn(CN)2, Pd(PPh3)4, DMF; (e) H2O2, KOH, H2O; (f) BBr3,
CH2Cl2; (g) CHdCHCO2Et, Pd(OAc)2, Et3N, 100 °C; (h) BzBr, K2CO3, DMF.
Sch em e 4a
a
(a) H2SO4, 100 °C; (b) BBr3, CH2Cl2; (c) Ph3PdCHCO2Et, DMF; (d) BzBr, K2CO3, DMF.
that, in CDCl3, the CHCl3 peak is at 7.26 ppm and, in DMSO-
d6, the DMSO peak is at 2.49 ppm, and, in acetone-d6, the
acetone peak is at 2.04 ppm. Standard and peak multiplicities
are designated as follows: s, singlet; d, doublet; dd, doublet of
doublets; t, triplet; brs, broad singlet; brd, broad doublet; br,
broad signal; and m, multiplet. Mass spectra were determined
at Scripps Research Institute, San Diego, CA, Mass Spectro-
metric Facilities. Infrared absorption spectra were taken on a
Perkin-Elmer 457 spectrometer or a MIDAK high resolution
FT IR, and values are reported in cm-1. Elemental mi-
croanalyses were performed by Atlantic Microlabs Inc., Nor-
cross, GA, and gave results for the elements stated within
(0.4% of the theoretical values. N,N-Dimethylformamide and
N,N-dimethylacetamide were used as is from Aldrich. Tet-
rahydrofuran (THF) was distilled from sodium benzophenone
ketyl or CaH2 under nitrogen. Flash chromatography was
performed using silica gel 60 (Merck Art 9385), unless stated
otherwise. Thin layer chromatographs (TLC) were performed
on precoated sheets of silica 60 F254 (Merck Art 5719).
Abbreviations: NMO, 4-methylmorpholine N-oxide; TPAP,
tetrapropylammonium perruthenate; TBAF, tetrabutylammo-
nium fluoride; HATU, O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-
tetramethyluronium; FMOC, 9-fluorenylmethoxycarbonyl;
DIEA, diisopropylethylamine; HOBT, 1-hydroxybenzotriazole
hydrate; DIC, 1,3-diisopropylcarbodiimide.
3CP In h ibition Assa ys a n d An tivir a l Assa ys. 1. En -
zym e In h ibition Assa ys. The general conditions of the
fluorescence resonance energy transfer assay utilized to assess
3CP activity and procedures for reversible inhibitor Ki deter-
minations are described in ref 6b. Continuous fluorometric

1676 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 9
Reich et al.
Sch em e 5a
a
(a) CHdCHCON(OMe)Me, Pd(OAc)2, Et3N, 100 °C; (b) RLi, -78 °C; (c) Zn(CN)2, Pd(PPh3)4, DMF; (d) H2O2, KOH, H2O; (e) Ph3COH,
Ac2O, AcOH, cat. H2SO4; (f) NBS, hν; then Et3N; (g) TFA, CH2Cl2.
Sch em e 6. Preparation of Monomer 57 for Parallel Synthesisa
a
(a) TBDPS-Cl, imidazole, DMF; (b) MeOH, 1 equiv NaOH; (c) LiHB(Et3); (d) TPAP; (e) EtO2CCH2P(O)(OEt)2, NaH; (f) TBAF;
(g) PBr3; (h) RINK-NH2, HATU, DMF; (i) TBAF, THF; (j) TFA-H2O.
enzyme assays were conducted using a Perkin-Elmer LS50B
spectrofluorimeter with a four place motorized cuvette holder.
The Kobs/[I] values for the irreversible inhibitors were obtained
from reactions initiated by addition of 50 nM 3CP, containing
1 µM substrate, and varying inhibitor concentrations. Typi-
cally, three to five concentrations were examined, and three
related methods were utilized for subsequent data analysis.
For reactions in which less than 20% of the substrate was
processed when 100% inactivation was observed, data from
the continuous assay were directly analyzed with the nonlinear
regression analysis program ENZFITTER to obtain first-order
rate constants for enzyme inactivation at each inhibitor
concentration. The slope of a graph of Kobs/(I) vs [I] was
calculated using ENZFITTER and reported as kobs/[I]. The
error associated with this determination is less than 10% of a
given value and is often less than 5%. For very slow irrevers-
ible inhibitors (kobs/[I] < 1000 M-1 s-1) and/or when >20%
consumption of substrate was observed at 100% 3CP inactiva-
tion, the primary data were adjusted to account for expected
changes in velocity due to changes in substrate concentration.
The transformed data were used to calculate kobs/[I] values
and the kobs/[I] figure was determined utilizing the slope
method described above.
2. An tivir a l Assa ys. All strains of human rhinovirus (HRV)
were purchased from American Type Culture Collection
(ATCC). HRV stocks were propagated, and antiviral assays
were performed in H1-HeLa cells (ATCC). Cells were grown
in Minimal Essential Medium with 10% fetal bovine serum.
The ability of compounds to protect cells against HRV infection
was measured by the XTT dye reduction method. Briefly, H1-
HeLa cells were infected with HRV-14 at a multiplicity of
infection (moi) of 0.08 or mock-infected with medium only.
Infected or uninfected cells were resuspended at 8 × 105 cells
per milliliter and incubated with appropriate concentrations
of drug. Two days later, XTT/PMS was added to the test plates,
and the amount of formazan produced was quantified spec-

Human Rhinovirus 3C Protease Inhibitors
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 9 1677
core. Three-dimensional coordinates of the library were gener-
ated with CORINA Ver. 1.7.15 The structures were then energy
minimized in the Batchmin module of MacroModel16 Ver. 5.5
using the AMBER* force field (atomic charges were the default
from the force field).
Sch em e 7. Parallel Synthesis of 5-Substituted
Benzamides on Solid Supporta
Compounds with favorable predicted binding modes to
either the P2 or P4 pocket were selected by a partially fixed
docking routine as implemented in AGDOCK.17 Each com-
pound was run through the docking process eight times against
the target protein active site from the cocrystal structure of
compound 1, with the benzamide core and protein atoms fixed
at the coordinates of the cocrystal structure.18 All torsions of
the 5-position benzamide substituents were flexible during
docking. To remove docked structures with highly unfavorable
contacts with the target, the fixed ligand atom constraints were
removed, and the ligand structures were energy minimized
in the context of the rigid protein. Only those structures which
after minimization in the protein maintained the coordinates
of the benzamide core close to the cocrystal structure (RMS
deviation e 1.0 for heavy atoms) were kept for further analysis.
The docked molecules were then scored and ranked using
HTS,19 a program developed in-house for rapidly estimating
the free energy of protein-ligand association. Structures with
poor HTS scores were discarded; and among the multiple
docked conformations of the same compound, the one with the
best HTS score was retained. Final ranking was made on the
basis of which compounds satisfied at least one specific
hydrogen bond donor or acceptor interaction with protein
atoms which are known to form â-strand type interactions with
the native peptide substrate and which fit well into the P2 or
P4 pockets, as judged by ligand-protein atom distance criteria
to selected protein atoms in the subsite.
Ch em istr y. 3-Ca r ba m oyl-ben za ld eh yd e (1a ). Oxalyl
chloride (13.3 mL, 152 mmol) and DMF (50 µL) were added to
a suspension of 3-carboxybenzaldehyde (11.4 g, 76.2 mmol) in
200 mL of CH2Cl2 and stirred at 23 °C for 18 h. The resulting
clear solution was concentrated, dissolved in 100 mL of CH2-
Cl2, and concentrated again. The crude acid chloride was
dissolved in 20 mL of THF, poured into a mixture of concen-
trated NH4OH (26 mL, 381 mmol) with 100 mL of crushed
ice, and allowed to warm to 23 °C with stirring. The mixture
was acidified with concentrated HCl to pH ∼3, then concen-
trated to remove THF. The resulting aqueous suspension was
filtered and the white solid product dried under vacuum to
give 7.4 g (65%) of 3-carbamoyl-benzaldehyde 1a : 1H NMR
(DMSO-d6) δ 10.06 (1 H, s), 8.40 (1 H, s), 8.19 (1 H, s), 8.17 (1
H, d, J ) 6.6 Hz), 8.04 (1 H, d, J ) 7.7 Hz), 7.69 (1 H, t, J )
7.7 Hz), 7.56 (1 H, s); IR (KBr pellet) 3391, 3205, 1711, 1694,
1664, 1385, 1217. Anal. (C8H7NO2‚0.1H2O) C, H, N.
a
(a) 57, DIC, HOBt; (b) RR1NH or RSH, DIEA, DMF, 70 °C;
(c) TFA.
trophotometrically at 450/650 nm. The EC50 was calculated
as the concentration of drug that increased the percentage of
formazan production in drug-treated, virus-infected cells to
50% of that by drug-free, uninfected cells. The reported values
were obtained from either a single antiviral determination or
the mean of two or more experiments. To avoid false positives
due to toxicity, only compounds displaying CC50’s greater than
10 times the observed EC50’s were considered to be truly active
antirhinoviral agents. Using the above method, the EC50 of
the known antirhinoviral agent Pirodavir was determined to
be 0.02 ( 0.01 µM (range 0.18-1 µM), comparable to the 0.03
µM minimal inhibitory concentration value previously re-
ported.
P r otein Cr ysta lla gr a p h y. Serotype 2 human rhinovirus
3C protease was incubated with a 3-fold molar excess of
compound 4 in the presence of 2% DMSO for 24 h at 4 °C.
The complex was concentrated to 10 mg/mL and then passed
through a 0.22 µm cellulose-acetate filter. Crystals were grown
at 13 °C using a hanging drop vapor diffusion method in which
equal volumes (3 µL) of the protein/ligand complex and
reservoir solution were mixed on plastic coverslips and sealed
over individual wells filled with 1 mL of reservoir solution
containing 1.2 M ammonium sulfate, 0.325 M sodium phos-
phate, 0.325 M potassium phosphate, 0.1 M ADA pH 6.6, and
2.5% (v/v) 1,4-dioxane. A single crystal measuring 0.6 × 0.4 ×
0.2 mm (space group P21212; a ) 61.22, b ) 77.71, c ) 34.35
Å) was prepared for low temperature data collection by a 2
min immersion in an artificial mother liquor solution consist-
ing of 400 µL of the reservoir solution mixed with 125 µL of
glycerol, followed by flash freezing in a stream of N2 gas at
-170 °C. X-ray diffraction data were collected using a MAR
imaging plate and processed with DENZO. Diffraction data
were 74% complete to a resolution of 1.85 Å with R(sym) )
1.9%. Protein atomic coordinates from the cocrystal structure
determination (ref 6c) were used to initiate rigid body refine-
ment in X-PLOR followed by simulated annealing and conju-
gate gradient minimization protocols. Placement of the inhibi-
tor, addition of ordered solvent, and further refinement
proceeded as described in ref 6c. The final R factor was 20.8%
(10 680 reflections with F > 2_(F)). The root-mean-square
deviations from ideal bond lengths and angles were 0.013 Å
and 2.6°, respectively. The final model consisted of all atoms
for residues 1-180 (excluding the side chain of residue 21)
plus 104 water molecules.
3-(3-Ca r ba m oylp h en yl)-a cr ylic Acid E t h yl E st er (1).
Method A: A solution of 3-carbamoyl-benzaldehyde 1a (7.38
g, 49.5 mmol) and (carbethoxymethylene)triphenylphosphorane
(17.23 g, 49.5 mmol) in 200 mL of toluene was heated to reflux
for 24 h. After cooling to room temperature, the reaction
mixture was partitioned between 750 mL of CH2Cl2 and a
mixture of 200 mL of water with 200 mL of brine. The organic
layer was washed again with a mixture of 100 mL of water
with 100 mL of brine, then dried over MgSO4, filtered, and
concentrated to a crude yellow oil which crystallized on
standing. The combined aqueous layers were filtered, and the
insoluble material was combined with the crude solid obtained
from the organic layer. The crude product was purified by
recrystallization from methanol. A 5.25 g (48%) yield of 1 was
1
isolated in two crops as yellow needles: mp 168-170 °C; H
NMR (CDCl3) δ 7.99(1 H, s), 7.80 (1 H, d, J ) 7.7 Hz), 7.71 (1
H, d, J ) 11.8 Hz), 7.68 (1 H, s), 7.49 (1 H, t, J ) 7.7 Hz), 6.52
(1 H, d, J ) 16.2 Hz), 6.10 (1 H, br s, NH), 5.75 (1 H, br s,
NH), 4.28 (2 H, q, J ) 7.0 Hz), 1.35 (3 H, t, J ) 7.0 Hz); IR
(neat film) 3414, 3177, 1695, 1682, 1639, 1400, 1313, 1215,
1192. Anal. (C12H13NO3) C, H, N.
Ca lcu la tion s Used in Vir tu a l Libr a r y An a lysis. We
selected 3087 primary amines and mercaptans from the ACD.14
The library was built with PEONY (an in-house program
which synthesizes the 2D virtual library from its fragments)
attaching the compounds at the 5-position of the benzamide
Compound 1 was also prepared in 78% yield as a white solid
using ethyl acrylate in the same procedure (Method B) for the
preparation of 2 described below.
3-Iod o-ben za m id e (32). 3-Iodo-benzamide was prepared

1678 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 9
Reich et al.
using a modification of the procedure by Remsen.20 3-Iodo-
benzoic acid (28.68 g, 116 mmol) was stirred in CH2Cl2 (200
mL) at 23 °C under argon. Oxalyl chloride (30.2 mL, 349 mmol)
was added slowly, and slow gas evolution was observed. DMF
(0.1 mL) was then added, accelerating gas evolution consider-
ably, and the reaction was stirred for 2 h. Solvent was
removed, and the brown oily residue was dissolved in THF
(50 mL) and added to a solution of 18% aqueous NH4OH (260
mL) at 0 °C. After the mixture was stirred for 15 min, the
liquid was decanted off, and the remaining sludge was acidified
with 1 N HCl. The white solid was collected by filtration,
washed with water, and dried under vacuum to give 25.68 g
(90%) of 3-iodo-benzamide 32: 1H NMR (DMSO-d6) δ 8.21 (1
H, t, J ) 1.5 Hz), 8.05 (1 H, s), 7.87 (2 H, dd, J ) 8.1, 1.5 Hz),
7.47 (1 H, s), 7.25 (1 H, t, J ) 7.8 Hz); IR (KBr) 3343, 3164,
1661, 1628, 1561, 1424, 1389, 1125. Anal. (C7H6INO) C, H,
N.
3410, 3165, 1705, 1678, 1630, 1576, 1393, 1373, 1283, 1261.
Anal. (C18H17NO3) C, H, N.
3-(3-Car bam oyl-ph en yl)-acr ylic Acid P yr idin -3-yl Meth -
yl Ester (6). This compound was prepared in 68% yield as a
white solid using 3-pyridyl carbinol in the same procedure for
the preparation of 3 described above: 1H NMR (CDCl3) δ 8.68
(1 H, s), 8.60 (1 H, d, J ) 4.8 Hz), 7.99 (1 H, s), 7.66-7.81 (4
H, m), 7.48 (1 H, t, J ) 7.5 Hz), 7.26-7.35 (1 H, m), 6.55 (1 H,
d, J ) 16.2 Hz), 6.16 (1 H, br s), 5.79 (1 H, br s), 5.28 (2 H, s);
IR (KBr) 3418, 3160, 1700, 1676, 1630, 1576, 1397, 1285, 1259.
Anal. (C16H14N2O3) C, H, N.
3-(2-Eth oxyca r bon yl-vin yl)-ben zoic Acid (7). Using pro-
cedure B described above, compound 7 was prepared in 72%
yield as a white solid: 1H NMR (CDCl3) δ 11.22 (1 H, s), 8.29
(1 H, s), 8.13 (1 H, d, J ) 7.8 Hz), 7.71-7.78 (2 H, m), 7.52 (1
H, t, J ) 7.5 Hz), 6.54 (1 H, d, J ) 16.2 Hz), 4.29 (2 H, q, J )
7.2 Hz), 1.36 (3 H, t, J ) 7.2 Hz); IR (KBr) 2984, 2672, 2564,
1725, 1642, 1445, 1302, 1209, 1177 cm-1. Anal. (C12H12O4‚
0.2H2O) C, H.
3-(2-Eth oxyca r bon yl-vin yl)-ben zoic Acid Meth yl Ester
(8). Compound 7 (225 mg, 1.02 mmol) was dissolved in a
mixture of dichloromethane (2 mL) and methanol (2 mL).
(Trimethylsilyl)diazomethane (2 M solution in hexanes, ∼0.8
mL, ∼1.6 mmol) was added dropwise until gas evolution ceased
and a faint yellow color remained for 10 min. The reaction was
concentrated and purified by flash chromatography (1% MeOH/
CHCl3) to give 196 mg (82%) of 8 as a white solid: 1H NMR
(CDCl3) δ 8.21 (1 H, s), 8.05 (1 H, d, J ) 7.8 Hz), 7.71 (1 H, d,
J ) 16.2 Hz), 7.70 (1 H, d, J ) 7.8 Hz), 7.47 (1 H, t, J ) 7.8
Hz), 6.50 (1 H, d, J ) 16.3), 4.28 (2 H, q, J ) 7.2 Hz), 3.94 (s,
3H), 1.35 (3 H, t, J ) 7.2 Hz); IR (KBr) 3399, 3094, 3065, 3034,
2980, 2907, 1717, 1638, 1447 cm-1. Anal. (C13H14O4) C, H.
3-(3-Ca r ba m oyl-p h en yl)-a cr ylic Acid Meth yl Ester (2).
Method B: 3-Iodo-benzamide 32 (1.16 g, 4.7 mmol), methyl
acrylate (530 µL, 5.87 mmol), palladium(II) acetate (16 mg,
0.071 mmol), and triethylamine (820 µL, 5.87 mmol) were
stirred in 10 mL of acetonitrile under argon in a sealed tube
at 100 °C for 5 h. The reaction was cooled to 0 °C, and the
gray precipitate was collected. Recrystallization from methanol
gave 425 mg (44%) 2 as a white solid: 1H NMR (DMSO-d6) δ
8.20 (1 H, s), 8.04 (1 H, s), 7.87 (2 H, dd, J ) 7.8, 18 Hz), 7.69
(1 H, d, J ) 16.2 Hz), 7.50 (1 H, t, J ) 7.8 Hz), 7.47 (1 H, s),
6.73 (1 H, d, J ) 15.9 Hz), 3.73 (3 H, s); IR (KBr) 3424, 3366,
3173, 2957, 1728, 1659, 1578, 1431, 1399, 1317. Anal. (C11H11
-
NO3‚0.2H2O) C, H, N.
3-(3-Ca r ba m oyl-p h en yl)-a cr ylic Acid (33). Compound 1
(1.75 g, 7.99 mmol) was hydrolyzed in 2:1 0.8 N aqueous
NaOH/methanol (48 mL), stirring at 23 °C for 5 h. The solution
was then concentrated to ∼20 mL volume, cooled to 0 °C, and
acidified to pH ) 4 with 1 N HCl. The resulting crystals were
collected, washed with 5 mL of cold H2O, and dried under
vacuum to give 1.47 g (96%) of 3-(3-carbamoyl-phenyl)-acrylic
acid 33 as a white solid: 1H NMR (DMSO-d6) δ 12.50 (1 H, br
s), 8.17 (1 H, s), 8.05 (1 H, s), 7.85 (2 H, dd, J ) 23.7, 7.8 Hz),
7.61 (1 H, d, J ) 16.2 Hz), 7.49 (1 H, t, J ) 7.5 Hz), 7.47 (1 H,
s), 6.62 (1 H, d, J ) 15.6 Hz); IR (KBr) 3451, 3202, 2924, 1690,
1640, 1443, 1395, 1316, 1219. Anal. (C10H9NO3‚0.25H2O) C,
H, N.
3-(3-Ca r ba m oyl-p h en yl)-a cr ylic Acid Ben zyl Ester (3).
3-(3-Carbamoyl-phenyl)-acrylic acid (212 mg, 1.11 mmol) and
benzyl alcohol (172 µL, 1.66 mmol) were stirred in DMF (3
mL) at 0 °C. EDC (318 mg, 1.66 mmol), triethylamine (170
µL, 1.22 mmol), and DMAP (14 mg, 0.11 mmol) were added,
and the reaction was allowed to stir for 3 h while warming to
23 °C. The solution was concentrated, and the residue was
purified by flash chromatography (3% EtOH/CHCl3) to give
90 mg (29%) of 3 as a white solid: 1H NMR (DMSO-d6) δ 8.22
(1 H, s), 8.04 (1 H, s), 7.94 (2 H, dd, J ) 12.0, 7.8 Hz), 7.61 (1
H, d, J ) 16.2 Hz), 7.34-7.50 (7 H, m), 6.79 (1 H, d, J ) 16.2
Hz), 5.23 (2 H, s); IR (KBr) 3420, 3316, 3150, 1711, 1665, 1630,
1402, 1308, 1167. Anal. (C17H15NO3) C, H, N.
3-(3-Ca r ba m oyl-p h en yl)-a cr ylic Acid 2-Hyd r oxyeth yl
Ester (4). This compound was prepared in 33% yield as a
white solid using ethylene glycol in the same procedure for
the preparation of 3 described above: 1H NMR (DMSO-d6) δ
8.21 (1 H, s), 8.05 (1 H, br s), 7.90 (1 H, d, J ) 7.8 Hz), 7.86 (1
H, d, J ) 7.8 Hz), 7.71 (1 H, d, J ) 16.2 Hz), 7.51 (1 H, t, J )
7.8 Hz), 7.47 (1 H, br s), 6.74 (1 H, d, J ) 16.2 Hz), 4.86 (1 H,
t, J ) 5.1 Hz), 4.17 (2 H, t, J ) 5.1 Hz), 3.64 (2 H, q, J ) 5.1
Hz); IR (KBr) 3418, 3212, 1705, 1676, 1626, 1574, 1399, 1280.
Anal. (C12H13NO4) C, H, N.
3-(3-Ca r ba m oyl-p h en yl)-a cr ylic Acid P h en eth yl Ester
(5). This compound was prepared in 49% yield as a white solid
using phenethyl alcohol in the same procedure for the prepa-
ration of 3 described above: 1H NMR (CDCl3) δ 7.98 (1 H, s),
7.79 (1 H, d, J ) 7.8 Hz), 7.68 (1 H, d, J ) 16.2 Hz), 7.66 (1 H,
d, J ) 7.8 Hz), 7.48 (1 H, t, J ) 7.5 Hz), 7.23-7.36 (5 H, m),
6.50 (1 H, d, J ) 16.2 Hz), 6.14 (1 H, br s), 5.83 (1 H, br s),
4.44 (2 H, t, J ) 6.9 Hz), 3.02 (2 H, t, J ) 6.9 Hz); IR (KBr)
(E)-3-(3-Ca r ba m oyl-p h en yl)-2-cya n o-a cr ylic Acid Eth -
yl Ester (9). Piperidine (150 µL, 1.52 mmol) was added to a
solution of 3-formyl benzamide 30 (111 mg, 0.74 mmol) and
ethyl cyanoacetate (79 µL, 0.74 mmol) in ethanol (2 mL) at 0
°C. After the mixture was stirred for 5 h at 23 °C, solvent was
removed. The residue was dissolved in CH2Cl2 and washed
with 0.1 N HCl, H2O, and then brine. Organics were dried (Na2-
SO4) and concentrated. Purification by flash chromatography
(3% MeOH/CHCl3) gave 86 mg (47%) of 9 as a white solid: 1H
NMR (CDCl3) δ 8.37 (1 H, s), 8.31 (1 H, s), 8.18 (1 H, d, J )
7.8 Hz), 8.04 (1 H, d, J ) 7.8 Hz), 7.63 (1 H, t, J ) 7.8 Hz),
6.09 (1 H, br s), 5.71 (1 H, br s), 4.41 (2 H, q, J ) 7.2 Hz), 1.42
(3 H, t, J ) 7.2 Hz); IR (KBr) 3435, 3351, 3306, 3167, 2224,
1724, 1701, 1626, 1601, 1578, 1433, 1383, 1275, 1211 cm-1
.
Anal. (C13H12N2O3‚0.5H2O) C, H, N.
6-Meth yl-3-(3-car bam oylph en yl)-acr ylic Acid Eth yl Es-
ter (10). 3-Iodo-4-methyl benzoic acid was converted to the
corresponding benzamide according to the procedure described
for compound 1a above ((COCl)2, NH4OH), to provide 3-iodo-
4-methyl benzamide in 93% yield. Further conversion accord-
ing to procedure B described above provided compound 10 in
48% yield: 1H NMR (CDCl3) δ 8.02 (1 H, d, J ) 1.6 Hz), 7.95
(1 H, d, J ) 15.9 Hz), 7.68 (1 H, d, J ) 7.9 Hz), 7.29 (1 H, d,
J ) 7.9 Hz), 6.46 (1 H, d, J ) 15.9 Hz), 6.10 (1 H, br s), 5.75
(1 H, br s), 4.08 (2 H, q, J ) 7.1 Hz), 2.48 (3 H, s), 1.42 (3 H,
t, J ) 7.1 Hz). Anal. (C13H15N2O3‚0.2H2O) C, H, N.
3-Meth oxy-5-n itr o-p h en yla m in e (34). Sodium bicarbon-
ate (6.07 g, 72.3 mmol) was added to sodium sulfide nonahy-
drate (18.2 g, 75.9 mmol) in deionized water (50 mL). When
the sodium bicarbonate was completely dissolved, methanol
(50 mL) was added, and the solution cooled to 0 °C. A
precipitate formed, which was removed by filtration through
a Celite pad; the filtered solution was added to 3,5-dinitroani-
sole (8.02 g, 40.5 mmol) in methanol (50 mL). After heating
at reflux for 30 min, the solution was concentrated in vacuo
to remove methanol. The aqueous residue was poured into 200
mL of ice water, and the resulting orange precipitate was
collected by suction filtration. Chromatography (1:2 EtOAc/
hexanes) of the crude solid yielded unreacted 3,5-dinitroanisole
(0.98 g, 12%) and aniline product 34 (4.96 g, 73%; 83% based
on recovered 3,5-dinitroanisole) as an orange solid: mp ) 117-
1
119 °C; H NMR (CDCl3) δ 7.12 (s, 2H), 6.48 (s, 1H), 3.98 (br

Human Rhinovirus 3C Protease Inhibitors
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 9 1679
s, 2H), 3.83 (s, 3H); IR (KBr pellet) 3447, 3364, 1637, 1523,
1344 cm-1; Anal. (C7H8N2O3) C, H, N.
3-Ben zyloxy-5-iod o-ben za m id e (49). A solution of benzyl
bromide (128 mg, 0.75 mmol), phenol 40 (131.5 mg, 0.50
mmol), and potassium carbonate (138 mg, 1.00 mmol) in DMF
(3.0 mL) was heated to 60 °C for 1.5 h. After cooling to 23 °C,
the solution was filtered, concentrated in vacuo, and purified
by silica gel chromatography (1:1 EtOAc/hexanes) to give
benzyl ether 49 (132.5 mg, 75%) as a white solid: mp ) 124-
1-Iod o-3-m eth oxy-5-n itr o-ben zen e (35). Concentrated
HCl (15 mL) was added to a solution of aniline 34 (5.25 g, 31.2
mmol) in water (15 mL) at 0 °C. To this was added a chilled
solution of sodium nitrite (3.88 g, 56.2 mmol) in water (20 mL),
dropwise, with vigorous mechanical stirring. Stirring was
continued at 0 °C for 15 min after the addition was complete,
and then a solution of potassium iodide (10.37 g, 62.4 mmol)
in water (20 mL) was added carefully. The cooling bath was
removed, and the reaction heated to boiling. When the produc-
tion of purple vapor ceased, the mixture was cooled to 23 °C
and extracted with CH2Cl2 (3 × 200 mL). The combined organic
extracts were dried (MgSO4), filtered, and concentrated in
vacuo. Purification by silica gel chromatography (1:9 EtOAc/
hexanes) gave pure iodide 35 (7.35 g, 84%) as a colorless
1
125 °C; H NMR (CDCl3) δ 7.67 (s, 1H), 7.49 (s, 1H), 7.40 (m,
6H), 6.0-5.8 (2 br s, 2H), 5.08 (s, 2H); IR (KBr pellet) 3369,
3192, 1660, 1564 cm-1; Anal. (C14H12INO2) C, H, N.
3-(3-Ben zyloxy-5-ca r ba m oyl-p h en yl)-a cr ylic Acid Eth -
yl Ester (12). Iodide 49 (51.6 mg, 0.146 mmol) was coupled
with ethyl acrylate (36.6 mg, 0.365 mmol) under the standard
conditions to give 12 (18.5 mg, 39%) as an off-white solid: mp
) 193-194 °C; 1H NMR (DMSO-d6) δ 8.01 (s, 1H), 7.81 (s,
1H), 7.63 (d, J ) 15.8 Hz, 1H), 7.54 (d, J ) 4.8 Hz, 2H), 7.45
(m, 6H), 6.75 (d, J ) 15.8 Hz, 1H), 5.18 (s, 2H), 4.19 (q, J )
7.0 Hz, 2H), 1.26 (t, J ) 7.0 Hz, 3H); IR (KBr pellet) 3418,
3173, 1705, 1670, 1589, 1288 cm-1; Anal. (C19H19NO4‚0.2H2O)
C, H, N.
1
solid: mp ) 81-82 °C; H NMR (CDCl3) δ 8.15 (s, 1H), 7.70
(s, 1H), 7.56 (s, 1H), 3.88 (s, 3H); IR (KBr pellet) 1527, 1342
cm-1; Anal. (C7H6INO3) C, H, N.
3-Iod o-5-m eth oxy-p h en yla m in e (36). A mixture of tri-
irondodecacarbonyl (16.24 g, 32.2 mmol), 35 (7.50 g, 26.9
mmol), methanol (15 mL), and toluene (200 mL) was heated
at reflux for 3.5 h. After cooling, filtration, concentration in
vacuo, and silica gel chromatography (1:4 EtOAc/hexanes),
aniline 36 (6.11 g, 91%) was obtained as a yellow oil which
crystallized on standing: mp ) 84-86 °C; 1H NMR (CDCl3) δ
6.64 (s, 2H), 6.15 (s, 1H), 3.71 (s, 3H), 3.66 (br s, 2H); IR (KBr
pellet) 3414, 3308, 3208, 1572 cm-1; Anal. (C7H8INO) C, H,
N.
3-Ca r ba m oyl-4-m eth oxy-ben za ld eh yd e (41). 3-Bromo-
anisaldehyde (8.0 g, 37.2 mmol) and copper cyanide (4.0 g,
44.67 mmol) were stirred in DMF (100 mL) at 150 °C for 16
h. To this was added an iron nitrate solution (20 g of iron(III)
nitrate, 6 mL of concetrated HCl, 40 mL of H2O), and the
mixture was stirred 10 min before it was allowed to cool to 23
°C. The reaction was then diluted with H2O (200 mL) and
extracted with CHCl3 (3 × 80 mL). Organic layers were
combined and solvents were removed, taking care to pump off
the residual DMF. The brown/green residue was once again
dissolved in CHCl3 (100 mL) and washed with 1 N HCl (50
mL) and brine (50 mL). The material was dried (Na2SO4), and
solvent was removed to give the crude nitrile as a tan solid.
The nitrile was stirred in concentrated H2SO4 (60 mL) at 100
°C for 1 h. The solution was cooled, poured into H2O (250 mL),
and extracted with CHCl3 (8 × 50 mL). Organics were dried
(Na2SO4) and concentrated. The resulting solid was recrystal-
lized from methanol to give 2.98 g (45%) of 3-carbamoyl-4-
methoxy-benzaldehyde 41 as a white solid: 1H NMR (CDCl3)
δ 9.92 (1 H, s), 8.28 (1 H, s), 8.00 (1 H, d, J ) 9.0 Hz), 7.74 (1
H, br s), 7.69 (1 H, br s), 7.33 (1 H, d, J ) 9.0 Hz), 3.98 (3 H,
3-Am in o-5-m et h oxy-b en zon it r ile (37). By the same
method used to make compound 44, iodide 36 (2.51 g, 10.1
mmol) was converted to nitrile 37 (1.24 g, 83%), a yellow
1
solid: mp ) 81-85 °C; H NMR (CDCl3) δ 6.53 (m, 2H), 6.39
(t, J ) 2.0 Hz, 1H), 3.87 (br s, 2H), 3.77 (s, 3H); IR (neat film)
3408, 3333, 3221, 2228, 1597 cm-1; Anal. (C8H8N2O) C, H, N.
3-Iod o-5-m eth oxy-ben zon itr ile (38). By the same method
used to prepare 35, nitrile 37 (1.12 g, 7.6 mmol) was converted
to iodide 38 (1.25 g, 64%): 1H NMR (CDCl3) δ 7.55 (s, 1H),
7.47 (s, 1H), 7.12 (s, 1H), 3.82 (s, 3H); IR (neat film) 2231,
1587, 1284 cm-1; Anal. (C8H6INO) C, H, N.
3-Iod o-5-m eth oxy-ben za m id e (39). By the same method
used to prepare amide 45, nitrile 38 (1.245 g, 4.81 mmol) was
converted to amide 39 (1.039 g, 78%), a white solid: mp )
175-176 °C; 1H NMR (CDCl3) δ 7.65 (s, 1H), 7.40 (s, 1H), 7.34
(s, 1H), 6.0-5.8 (2 br S, 2H), 3.83 (s, 3H); IR (KBr pellet) 3389,
3194, 1658, 1568, 1390 cm-1; Anal. (C8H8INO2) C, H, N.
3-Hyd r oxy-5-iod o-ben za m id e (40). Amide 39 (1.032 g,
3.72 mmol) suspended in CH2Cl2 (100 mL) was cooled to -78
°C. Boron tribromide solution (1.0 M in CH2Cl2, 7.45 mL, 7.45
mmol) was added. After being stirred at -78 °C for 30 min,
the solution was heated to reflux for 4 h. Another equivalent
of boron tribromide (3.7 mL) was added, and the solution was
stirred at 23 °C for 16 h. The reaction mixture was quenched
with water (50 mL), causing a white precipitate to form. Ether
(50 mL) was added to dissolve the precipitate, and the layers
separated. The aqueous layer was discarded, and the ether
layer was washed with 2 N NaOH (2 × 100 mL). The combined
basic washes were treated with 6 N HCl until pH ∼4, then
extracted with ether (2 × 150 mL). These ether extracts were
dried (MgSO4), filtered, and concentrated in vacuo to give
phenol 40 (803.3 mg, 82%) as a white solid: mp ) 192-195
s); IR (KBr) 3399, 3183, 1676, 1589, 1433, 1262, 1204 cm-1
Anal. (C9H9NO3) C, H, N.
.
5-F or m yl-2-h yd r oxy-b en za m id e (42). 3-Carbamoyl-4-
methoxy-benzaldehyde 41 (1.065 g, 5.95 mmol) was stirred in
dry CH2Cl2 (120 mL) at -78 °C under argon. Boron tribromide
(10.71 mL, 1.0 M, 10.71 mmol) was added, and the reaction
stirred 18 h while warming to 23 °C. The reaction was
quenched with 0.05 N HCl (80 mL) and was allowed to stir
for 15 min. The organic layer was collected, and the aqueous
layer was further extracted with EtOAc (2 × 50 mL). Organics
were combined, dried (Na2SO4), and concentrated. Purification
by flash chromatography (60% EtOAc/CHCl3) gave 540 mg
(55%) of 5-formyl-2-hydroxy-benzamide 42 as a white solid:
1H NMR (DMSO-d6) δ 14.00 (1 H, s), 9.88 (1 H, s), 8.73 (1 H,
br s), 8.55 (1 H, d, J ) 1.5 Hz), 8.21 (1 H, br s), 8.00 (1 H, dd,
J ) 8.7, 1.5 Hz), 7.13 (1 H, d, J ) 8.7 Hz); IR (KBr) 3420,
3237, 1686, 1618, 1493, 1375, 1279, 1196 cm-1. Anal. (C8H7-
NO3) C, H, N.
3-(3-Ca r ba m oyl-4-h yd r oxy-p h en yl)-a cr ylic Acid Eth yl
Ester (14). 5-Formyl-2-hydroxy-benzamide 42 (65 mg, 0.40
mmol) and (carbethoxymethylene)triphenylphosphorane (281
mg, 0.81 mmol) were stirred in DMF (3 mL) at 23 °C for 2 h.
Solvent was removed, and the residue was purified by flash
chromatography (2% MeOH/CHCl3) to give 44 mg (48%) of 14
as a white solid: 1H NMR (DMSO-d6) δ 13.50 (1 H, s), 8.52 (1
H, br s), 8.29 (1 H, s), 8.07 (1 H, br s), 7.77 (1 H, d, J ) 8.7
Hz), 7.56 (1 H, d, J ) 15.6 Hz), 6.92 (1 H, d, J ) 8.7 Hz), 6.57
(1 H, d, J ) 15.6 Hz), 4.18 (2 H, q, J ) 7.2 Hz), 1.26 (3 H, t, J
) 7.2); IR (KBr) 3387, 3198, 2988, 2359, 1688, 1620, 1491,
1441, 1372, 1279 cm-1. Anal. (C12H13NO4) C, H, N.
1
°C; H NMR (DMSO-d6) δ 9.99 (s, 1H), 7.93 (s, 1H), 7.61 (s,
1H), 7.37 (s, 1H), 7.24 (s, 2H); IR (KBr pellet) 3483, 3396, 3232,
1680, 1649, 1433 cm-1; Anal. (C7H6INO2) C, H, N.
3-(3-Ca r ba m oyl-5-h yd r oxy-p h en yl)-a cr ylic Acid Eth yl
Ester (11). Iodide 40 (24.6 mg, 0.093 mmol) was coupled with
ethyl acrylate (23.4 mg, 0.234 mmol) under the standard
conditions described above to give 11 (17.3 mg, 79%) as a white
1
solid: mp ) 200-202 °C; H NMR (DMSO-d6) δ 9.86 (s, 1H),
7.92 (s, 1H), 7.67 (s, 1H), 7.57 (d, J ) 16.8 Hz, 1H), 7.36 (s,
1H), 7.31 (s, 1H), 7.15 (s, 1H), 6.61 (d, J ) 16.2 Hz, 1H), 4.18
(q, J ) 7.0 Hz, 2H), 1.25 (t, J ) 7.0 Hz, 3H); IR (KBr pellet)
3402, 3209, 1680, 1593, 1296 cm-1; Anal. (C12H13NO4‚0.4H2O)
C, H, N.
2-Ben zyloxy-5-for m yl-ben za m id e (42a ). Compound 42
(105 mg, 0.64 mmol) and benzyl bromide (114 µL, 0.95 mmol)
were stirred in DMF (3 mL) with K2CO3 (176 mg, 1.27 mmol)

1680 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 9
Reich et al.
at 60 °C for 1 h. Solvent was removed, and the residue was
purified by flash chromatography (2% MeOH/CHCl3) to give
135 mg (83%) of 42a as a white solid: 1H NMR (DMSO-d6) δ
9.91 (1 H, s), 8.24 (1 H, s), 7.98 (1 H, d, J ) 8.7 Hz), 7.67 (2 H,
br s), 7.51 (2 H, d, J ) 7.2 Hz), 7.41-7.34 (4 H, m), 5.36 (2 H,
s); IR (KBr) 3387, 3192, 2849, 1690, 1649, 1599, 1437, 1389,
1265, 1206 cm-1. Anal. (C15H13NO3‚0.2H2O) C, H, N.
3-(4-Ben zyloxy-3-ca r ba m oyl-p h en yl)-a cr ylic Acid Eth -
yl Ester (15). 2-Benzyloxy-5-formyl-benzamide 42a (105 mg,
0.412 mmol) and (carbethoxymethylene)triphenylphosphorane
(287 mg, 0.824 mmol) were stirred in DMF (3 mL) at 40 °C
for 2 h. Solvent was removed, and the residue was purified by
flash chromatography (1% MeOH/CHCl3) to give 95.8 mg (72%)
of 15 as a white solid: 1H NMR (CDCl3) δ 8.45 (1 H, d, J ) 1.8
Hz), 7.70-7.60 (3 H, m), 7.43 (5 H, br s), 7.08 (1 H, d, J ) 8.7
Hz), 6.43 (1 H, d, J ) 15.6 Hz), 5.75 (1 H, br s), 5.23 (2 H, s),
4.25 (2 H, q, J ) 7.2 Hz), 1.33 (3 H, t, J ) 7.2 Hz); IR (KBr)
3441, 3154, 1689, 1676, 1593, 1500, 1431, 1371 cm-1. Anal.
(C19H19NO4) C, H, N.
3-[3-(4-Meth oxy-ph en yl)-oxo-pr open yl]-ben zam ide (19).
This compound was prepared in 47% yield as a white solid
using 4-bromoanisole in the same procedure for the prepara-
tion of 18 described above: 1H NMR (CDCl3) δ 8.15 (1 H, s),
8.06 (2 H, d, J ) 8.7 Hz), 7.84-7.75 (3 H, m), 7.63 (1 H, d, J
) 16.2 Hz), 7.51 (1 H, t, J ) 7.8 Hz), 6.99 (2 H, d, J ) 9.0 Hz),
6.18 (1 H, br s), 5.73 (1 H, br s), 3.90 (3 H, s); IR (KBr) 3376,
3192, 1659, 1607, 1439, 1227 cm-1. Anal. (C17H15NO3‚0.2H2O)
C, H, N.
3-(3-Oxo-3-p yr id in -2-yl-p r op en yl)-ben za m id e (20). This
compound was prepared in 17% yield as a white solid using
2-bromopyridine in the same procedure for the preparation of
18 described above: 1H NMR (CDCl3) δ 8.76 (1 H, d, J ) 4.8
Hz), 8.39 (1 H, d, J ) 16.2 Hz), 8.19 (2 H, t, J ) 1.5 Hz), 7.95
(1 H, d, J ) 16.2 Hz), 7.91-7.82 (3 H, m), 7.52 (2 H, t, J ) 7.8
Hz), 6.26 (1 H, br s), 5.91 (1 H, br s); IR (KBr) 3416, 3207,
3055, 1672, 1607, 1580, 1391,1332, 1221 cm-1. Anal. (C15H12
-
N2O2‚0.2H2O) C, H, N.
3-(3-F u r a n -2-yl-3-oxo-p r op en yl)-ben za m id e (21). To a
solution of freshly distilled furan (425 µL, 5.85 mmol) in dry
THF (8 mL) at -10 °C under argon was added n-butyllithium
(1.56 mL, 2.5 M in hexanes, 3.9 mmol).13 After being stirred
for 2 h at 0 °C, the solution was cooled to -50 °C, and a
solution of 3-[2-(methoxy-methyl-carbamoyl)-vinyl]benzamide
(114 mg, 0.487 mmol) in THF (1 mL) was added slowly. The
reaction was stirred 1 h while warming to 0 °C and was then
poured over saturated NH4Cl and extracted with CHCl3.
Organics were washed with brine, dried (Na2SO4), and con-
centrated. Purification by flash chromatography (2 to 6%
MeOH/CHCl3) gave 49 mg (42%) of 21 as a white solid: 1H
NMR (CDCl3) δ 8.16 (1 H, s), 7.89 (1 H, d, J ) 15.6 Hz), 7.83-
7.76 (2 H, m), 7.68 (1 H, t, J ) 0.9 Hz), 7.57-7.48 (2 H, m),
7.37 (1 H, d, J ) 3.6 Hz), 6.62 (1 H, dd, J ) 2.1, 0.9 Hz), 6.15
(1 H, br s), 5.69 (1 H, br s); IR (KBr) 3474, 3354, 3191, 1668,
1605, 1466, 1393, 1325 cm-1. Anal. (C14H11NO3‚0.2H2O) C, H,
N.
1-Oxo-in d a n -5-ca r bon itr ile (44). A solution of 5-bromo-
1-indanone (5.28 g, 25 mmol, zinc cyanide (1.76 g, 15 mmol),
and tetrakis(triphenylphosphine) palladium(0) (1.15 g, 1.0
mmol) in DMF (25 mL) was heated to 80 °C for 2 h. After
cooling to 23 °C, the solution was diluted with toluene (50 mL),
washed with 2 N NH4OH (2 × 50 mL) and brine (50 mL), dried
over MgSO4, filtered, and concentrated in vacuuo. Chroma-
tography of the residue (1:1 EtOAc/hexanes) yielded 3.33 g
(85%) of 44 as a yellow solid: 1H NMR (CDCl3) δ 7.85 (d, J )
7.7 Hz, 1H), 7.82 (s, 1H), 7.67 (d, J ) 7.7 Hz, 1H), 3.21 (t, J )
5.9 Hz, 2H), 2.77 (dd, J ) 6.3, 5.9 Hz, 2H); IR (KBr pellet)
2226, 1715 cm-1. Anal. (C10H7NO‚0.1H2O) C, H, N.
3-[2-(Meth oxy-m eth yl-car bam oyl)-vin yl]ben zam ide (43).
3-Iodo-benzamide 32 (3.0 g, 12.1 mmol), N-methoxy-N-methy-
lacrylamide21 (1.8 g, 15 mmol), palladium(II) acetate (40 mg,
0.18 mmol), and triethylamine (2.1 mL, 15 mmol) were stirred
in acetonitrile (12 mL) under argon in a sealed tube at 100 °C
for 2.5 h. The reaction was allowed to cool, and solvent was
removed. The residue was dissolved in CH2Cl2, washed with
0.1 N HCl and then with brine, dried (MgSO4), and concen-
trated. Recrystallization twice from methanol gave 1.64 g
(58%) of 3-[2-(methoxy-methyl-carbamoyl)-vinyl]benzamide 43
as a white solid: 1H NMR (CDCl3) δ 8.06 (1 H, s), 7.78 (1 H,
d, J ) 7.8 Hz), 7.73 (1 H, d, J ) 16.2 Hz), 7.69 (1 H, d, J ) 7.8
Hz), 7.46 (1 H, t, J ) 7.8 Hz), 7.10 (1 H, d, J ) 16.2 Hz), 6.33
(1 H, br s), 5.97 (1 H, br s), 3.77 (3 H, s), 3.31 (3 H, s); IR (Kbr)
3385, 3173, 1680, 1649, 1613, 1582, 1476, 1433, 1397, 1182,
1105 cm-1. Anal. (C12H14N2O3‚0.33H2O) C, H, N.
3-(3-Oxo-bu t-1-en yl)-ben za m id e (16). Method C: 3-[2-
(Methoxy-methyl-carbamoyl)-vinyl]benzamide 43 (100 mg,
0.427 mmol) was stirred in dry THF (4 mL) at 0 °C under
argon. Methyllithium (1.6 mL, 1.5 M in ether, 2.4 mmol) was
added, and the reaction stirred for 1.5 h. The reaction was
poured over 0.1 N HCl and extracted with CH2Cl2. Organics
were dried (MgSO4) and concentrated. Purification by flash
chromatography (2 to 5% EtOH/CH2Cl2) gave 54 mg (67%) of
16 as a white solid: 1H NMR (CDCl3) δ 8.03 (1 H, s), 7.80 (1
H, d, J ) 7.8 Hz), 7.70 (1 H, d, J ) 7.8 Hz), 7.54 (1 H, d, J )
16.2 Hz), 7.50 (1 H, t, J ) 7.8 Hz), 6.80 (1 H, d, J ) 16.2 Hz),
6.14 (1 H, br s), 5.82 (1 H, br s), 2.39 (3 H, s); IR (KBr) 3345,
3160, 2363, 1667, 1400, 1264 cm-1. Anal. (C11H11NO2 ) C, H,
N.
1-Oxo-in d a n -5-ca r boxylic Acid Am id e (45). A solution
of nitrile 44 (3.02 g, 20.4 mmol) in 3% aqueous H2O2 (110 mL)
was heated at 50 °C for 4 h. The mixture was then cooled to
0 °C for 1 h, and the resulting precipitate was collected by
suction filtration and dried under vacuum to give 2.40 g (67%)
of 45 as a yellow solid. The aqueous mother liquor was
concentrated to dryness and triturated with hot methanol. The
methanol solubles were concentrated and purified by silica gel
chromatography (5% MeOH in CH2Cl2) to give 184.5 mg (5%)
3-(3-Oxo-3-p h en yl-p r op -1-en yl)-ben za m id e (17). Com-
pound 17 was prepared in 20% yield as a white solid using
phenyllithium according to method C described above: 1H
NMR (CDCl3) δ 8.15 (1 H, s), 8.03 (2 H, d, J ) 7.2 Hz), 7.76-
7.84 (3 H, m), 7.48-7.64 (5 H, m), 6.26 (1 H, br s), 5.85 (1 H,
br s); IR (KBr) 3383, 3192, 3057, 2361, 1653, 1609, 1580, 1447
cm-1. Anal. (C16H13NO2) C, H, N.
3-[3-(4-Dim eth yla m in o-p h en yl)-oxo-p r op en yl]-ben za -
m id e (18). 4-Bromo-N,N-dimethylaniline (770 mg, 3.85 mmol)
was stirred in dry THF (6 mL) at -40 °C under argon.
n-Butyllithium (1.5 mL, 2.5 M in hexanes, 3.75 mmol) was
added dropwise, and the solution was stirred for 15 min. A
solution of 3-[2-(methoxy-methyl-carbamoyl)-vinyl]benzamide
43 (150 mg, 0.64 mmol) in THF (3 mL) was added slowly, and
the reaction was stirred for 1 h while warming to 23 °C. The
reaction was poured over saturated NH4Cl and was then
extracted with CHCl3. Organics were washed with brine, dried
(Na2SO4), and concentrated. Purification by flash chromatog-
raphy (1 to 4% MeOH/CHCl3) gave 140 mg of 18 (74%) as a
bright orange solid: 1H NMR (CDCl3) δ 8.14 (1 H, s), 8.02 (2
H, d, J ) 9.0 Hz), 7.83-7.75 (3 H, m), 7.67 (1 H, d, J ) 15.6
Hz), 7.50 (1 H, t, J ) 7.8 Hz), 6.71 (2 H, d, J ) 9.0 Hz), 6.13
(1 H, br s), 5.66 (1 H, br s), 3.10 (6 H, s); IR (KBr) 3372, 3192,
1671, 1609, 1578, 1377, 1188 cm-1. Anal. (C18H18N2O2‚0.1H2O)
C, H, N.
1
more 45 as a white solid: mp ) 207 °C; H NMR (DMSO-d6)
δ 8.14 (s, 1H), 8.02 (s, 1H), 7.85 (d, J ) 7.7 Hz, 1H), 7.67 (d, J
) 7.7 Hz, 1H), 7.57 (s, 1H), 3.13 (t, J ) 5.7 Hz, 2H), 2.68 (m,
2H); IR (KBr pellet) 1697, 1660, 1622 cm-1; Anal. (C10H9NO2)
C, H, N.
1-Oxo-in d a n -5-ca r boxylic Acid Tr ityl-a m id e (46). Acetic
anhydride (1.05 mL, 11.1 mmol) and concentrated H2SO4 (0.01
mL) were added to a solution of amide 45 (620.5 mg, 3.54
mmol) and triphenylmethyl alcohol (615 mg, 2.36 mmol) in
glacial acetic acid (12 mL).12 The solution was heated at 40 °C
for 3.5 h, cooled to 23 °C, and concentrated in vacuuo. The
residue was purified by silica gel chromatography (1:1 EtOAc/
hexanes to 10:1 EtOAc/hexanes) to give 432 mg (44% based
on trityl alcohol) of 46 as a yellow solid: 1H NMR (CDCl3) δ
7.92 (s, 1H), 7.78 (q, J ) 8.5 Hz, 2H), 7.30 (m, 16H), 3.18 (t, J
) 5.9 Hz, 2H), 2.74 (m, 2H). Anal. (C29H23NO2‚0.25H2O) C, H,
N.

Human Rhinovirus 3C Protease Inhibitors
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 9 1681
1-Oxo-1H-in d en e-5-ca r boxylic Acid Tr ityl-a m id e (47).
A solution of N-Bromosuccinimide (67.3 mg, 0.38 mmol) and
amide 46 (157.8 mg, 0.38 mmol) in CCl4 (10 mL) was heated
to reflux for 2 h while irradiated with a 200 W lamp. After
the solution cooled to 23 °C, the succinimide precipitate was
filtered off. The clear solution was cooled to 0 °C and treated
with triethylamine (0.055 mL, 0.40 mmol) for 2 h, then
concentrated in vacuuo. Chromatography of the residue (1:1
EtOAc/hexanes) afforded 73.6 mg (47%) of enone 47 as a yellow
solid: 1H NMR (CDCl3) δ 7.66 (d, J ) 7.3 Hz, 1H), 7.61 (d, J
) 5.9 Hz, 1H), 7.51 (s, 1H), 7.47 (d, J ) 7.3 Hz, 1H), 7.28 (m,
16H), 5.98 (d, J ) 5.9 Hz, 1H); IR (neat film) 1711, 1670, 1493
(1H, s), 8.09 (1H, s), 7.73-7.66 (4H, m), 7.50 (1H, br s), 7.44-
7.36 (6H, m), 4.87 (2H, s), 1.13 (9H, s).
A solution of triethyl phosphonoacetate (58.0 mL, 295.0
mmol) in DMF (300 mL) was cooled to 0 °C and treated with
sodium hydride (11.8 g of 60% in mineral oil, 295 mmol). This
mixture is held at 0 °C for 30 min, then treated with a solution
of 54 (21.0 g, 50.2 mmol) in DMF (300 mL) at 0 °C. This
mixture was allowed to warm to 23 °C over a period of 3 h,
then held at 23 °C overnight. The mixture was then acidified
with saturated aqueous citric acid (500 mL), then extracted
with EtOAc (1 L). The organic layer was washed with brine
(3 × 150 mL), then evaporated to yield 54.0 g of a dark oil.
Purification by silica gel chromatography (EtOAc-hexanes
elutant) yielded 14.8 g (51% overall) of a colorless oil that
solidified upon standing at 23 °C: 1H NMR (CDCl3) δ 8.17 (1H,
s), 8.06 (1H, s), 7.74-7.67 (6H, m), 7.50 (1H, br s), 7.45-7.39
(6H, m), 6.50 (1 H, d, J ) 16.2), 4.82 (2H, s), 4.29 (2H, q, J )
7.0), 1.36 (3H, t, J ) 7.0), 1.12 (9H, s).
3-Br om om eth yl-5-(2-eth oxycar bon yl-vin yl)-ben zoic Acid
(57). A solution of 55 (5.0 g, 10.2 mmol) in THF (34 mL) was
treated with TBAF (15.3 mL of a 1 M solution in THF, 15.3
mmol) and allowed to stand at 23 °C overnight. The solution
was then treated with a saturated aqueous sodium bicarbonate
solution and extracted with diethyl ether (2 × 20 mL). The
aqueous layer was acidified with a saturated citric acid
solution (50 mL), then extracted with EtOAc (2 × 50 mL).
Evaporation of the organics yielded 2.6 g of a white powder
56: 1H NMR (CDCl3) δ 8.18 (1H, s), 8.10 (1H, s), 7.79 (1H, s),
7.72 (1H, d, J ) 16.0), 7.50 (1H, br s), 6.55 (1H, d, J ) 16.0),
4.81 (2H, s), 4.28 (2H, q, J ) 7.0), 1.35 (3H, t, J ) 7.0), 1.20
(1H, br s).
This material (56) was dissolved in methylene chloride (50
mL), treated with phosphorus tribromide (2.91 mL, 30.6
mmol), and held at 23 °C overnight. The solution was then
treated with saturated aqueous sodium bicarbonate (50 mL)
and extracted with diethyl ether (2 × 30 mL). The aqueous
layer was acidified with saturated aqueous citric acid (50 mL),
then extracted with citric acid (3 × 30 mL). Evaporation of
the organic layer yielded 2.2 g (70% overall) of 57 as a white
powder: 1H NMR (CDCl3) δ 8.20 (1H, s), 8.13 (1H, s), 7.77
(1H, s), 7.71 (1H, d, J ) 16.2), 7.50 (1H, br s), 6.56 (1H, d, J
) 16.2), 4.54 (2H, s), 4.29 (2H, q, J ) 7.0), 1.35 (3H, t, J )
7.0).
3-(3-Ca r ba m oyl-5-h yd r oxym eth yl-p h en yl)-a cr ylic Acid
Eth yl Ester (13). FMOC-Rink polystyrene resin (0.50 g, 0.16
mmol) in a shaking vessel was treated with a 1:1 mixture of
piperidine and DMF (15 mL) and shaken for 30 min. The resin
was washed with DMF (3 × 15 mL) and CH2Cl2 (3 × 15 mL).
Acid 55 (122 mg, 0.25 mmol) in DMF (10 mL) was added to
the resin, followed by DIEA (0.09 mL, 0.50 mmol) and HATU
(95 mg, 0.25 mmol). The mixture was shaken 1 h, then drained
and washed with DMF (3 × 15 mL) and CH2Cl2 (3 × 15 mL).
TBAF (0.8 mL of 1 M solution in THF, 0.80 mmol) and THF
(10 mL) were added to the resin and shaken 4 h. The vessel
was drained and washed with THF (3 × 15 mL), MeOH (3 ×
15 mL), H2O (3 × 15 mL), MeOH (3 × 15 mL), and CH2Cl2 (3
× 15 mL). The linker was cleaved with 95:5 TFA-H2O (20
mL). Evaporation of the solvent, followed by purification of
the residue on silica gel (EtOAC elutant) yielded 37 mg (90%)
of 13: 1H NMR (CD3OD) δ (1H, s), 7.94 (1H, s), 7.80 (1H, s),
7.77 (1H, d, J ) 16.2), 6.66 (1H, d, J ) 16.1), 4.72 (2H, s), 4.29
(2H, q, J ) 7.0) 1.36 (3H, t, J ) 7.0); MS (FAB) 250 (MH+),
272 (MNa+).
cm-1
.
1-Oxo-1H-in d en e-5-ca r boxylic Acid Am id e (22). Trif-
luoroacetic acid (3 mL) was added to a solution of 47 (68.3
mg, 0.16 mmol) in CH2Cl2 (3.0 mL). After 30 min at 23 °C, the
solution was concentrated in vacuuo and purified by silica gel
chromatography (5% MeOH in CH2Cl2) to give 16.6 mg (60%)
1
of 22 as a yellow solid: mp ) 275 °C (decomposes); H NMR
(DMSO-d6) δ 8.08 (s, 1H), 7.95 (d, J ) 5.9 Hz, 1H), 7.78 (d, J
) 7.4 Hz, 1H), 7.66 (s, 1H), 7.53 (s, 1H), 7.44 (d, J ) 7.4 Hz,
1H), 6.08 (d, J ) 5.9 Hz, 1H); IR (KBr pellet) 3383, 1705, 1658
cm-1; HRMS (M + H+) Calcd. for C10H8NO2: 174.0555; Found
174.0559. Anal. (C10H7NO2‚0.1H2O) C, H, N.
5-(ter t-Bu t yl)-d ip h en yl-sila n yloxym et h yl)-isop h t h a l-
ic Acid Dieth yl Ester (51). A solution of 5-hydroxymethyl-
isophthalic acid diethyl ester 50 (20.0 g, 79.4 mmol) and
imidazole (10.81 g, 158.8 mmol) in DMF (265 mL) was treated
with tert-butylchlorodiphenyl silane (19.6 mL, 75.4 mmol) at
23 °C. This solution was held at 23 °C overnight, then
saturated aqueous ammonium chloride (200 mL) and EtOAc
(200 mL) are added. The EtOAc layer was washed with
additional ammonium chloride solution (100 mL) and then
washed with brine (100 mL). Evaporation of the organics
yielded 39.9 g of product as colorless oil: 1H NMR (CDCl3) δ
8.57 (1H, s), 8.22 (1H, s), 7.70-7.67 (5H, m), 7.46-7.35 (6H,
m), 4.84 (2H, s), 4.40 (4H, q, J ) 7.1), 1.42 (6H, t, J ) 7.1),
1.12 (9H, s).
5-(ter t-Bu tyl-d ip h en yl-sila n yloxym eth yl)-isop h th a lic
Acid Mon om eth yl Ester (52). A solution of 51 (39.9 g, 81.3
mmol) in MeOH (1.2 L) was treated with 0.95 N aqueous
NaOH (83.6 mL, 79.4 mmol) at 23 °C. The resulting solution
was held at 23 °C for 3 d, then acidified with saturated aqueous
citric acid. MeOH was removed by evaporation, and the
remaining aqueous solution was cooled to 5 °C and held
overnight, upon which precipitation occurred. The precipitate
was collected by filtration, washed with ice water (3 × 50 mL),
then dried to yield 31.0 g (87% overall) of 52 as a white
powder: 1H NMR (CDCl3) δ (1H, s), 8.28 (1H, s), 8.25 (1H, s),
7.70-7.67 (4H, m), 7.45-7.36 (6H, m), 4.84 (2H, s), 3.96 (3H,
s), 1.12 (9H, s).
3-(ter t-Bu tyl-d ip h en yl-sila n yloxym eth yl)-5-h yd r oxym -
eth yl-ben zoic Acid (53). A solution of 52 (27.2 g, 60.6 mmol)
in THF (400 mL) was treated with lithium triethylborohydride
(212.0 mL of a 1 M solution in THF, 212.0 mmol) at 23 °C.
The resulting solution was held overnight at 23 °C and then
quenched with a saturated aqueous citric acid solution (200
mL). This mixture was evaporated to dryness, then redissolved
in EtOAc (500 mL), and washed with brine (3 × 100 mL).
Evaporation of the organic layer yielded 26.8 g of alcohol as a
white powder: 1H NMR (CDCl3) δ 8.01 (2H, s), 7.74-7.68 (4H,
m), 7.59 (1H, s), 7.50 (1H, br s), 7.44-7.36 (6H, m), 4.82 (2H,
s), 4.77 (2H, s), 3.50 (1H, br s), 1.12 (9H, s); MS (FAB) 553
(MCs+).
3-(ter t-Bu tyl-d ip h en yl-sila n yloxym eth yl)-5-(2-eth oxy-
ca r bon yl-vin yl)-ben zoic Acid (55). A mixture of 53 (24.8
g, 59.0 mmol,), NMO (10.4 g, 88.5 mmol), and powdered 3 Å
molecular sieves (5 g) in methylene chloride (118 mL) was
treated with TPAP (1.04 g, 2.95 mmol), then stirred vigorously
for 2 h at 23 °C. The mixture was then treated with saturated
aqueous citric acid (100 mL) and EtOAc (500 mL). The organic
layer was washed with brine (200 mL) then evaporated to yield
3-(tert-butyl-diphenyl-silanyloxymethyl)-5-formyl-benzoic acid
54 (21.0 g): 1H NMR (CDCl3) δ 10.08 (1H, s), 8.49 (1H, s), 8.33
Resin 58 (F u n ction a lized w ith 3-Br om om eth yl-5-(2-
eth oxycar bon yl-vin yl)-ben zoic Acid 57). FMOC-Rink amide
polystyrene resin (3.00 g, 1.97 mmol) was treated with a 1:1
mixture of piperidine and DMF (30 mL) and shaken 30 min.
The vessel was drained, and the resin was washed with DMF
(3 × 25 mL) and then CH2Cl2 (3 × 25 mL). In another flask,
57 (0.93 g, 2.96 mmol) and HOBT (0.40 g, 2.96 mmol) in CH2-
Cl2 (30 mL) were treated with DIC (0.93 mL, 5.91 mmol) and
held at 23 °C for 45 min. This solution was then added to the
resin and shaken for 6 h. The vessel was drained, and the resin
was washed with CH2Cl2 (3 × 25 mL), MeOH (3 × 25 mL),

1682 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 9
Reich et al.
then CH2Cl2 (3 × 25 mL). The resin 58 was dried under
vacuum and stored in a desiccator.
Ack n ow led gm en t . The authors thank the mem-
bers of the RVP Project Team for their support and
Takeo Yuno of J apan Tobacco for preparation of com-
pound 1a . We also thank Dr. Robert Love for help with
Figure 2.
3-[3-Ca r ba m oyl-5-(5-p yr id in -2-yl-[1,3,4]oxa ld ia zol-2-yl-
su lfa n ylm eth yl-p h en yl]-a cr ylic Acid Eth yl Ester (25).
Resin 58 (100 mg, 0.063 mmol) in DMF (1 mL) and DIEA (0.11
mL, 0.63 mmol) in a screw-top vial was treated with 2-(2-
pyridyl)-5-thiol-1,3,4-oxadiazole (50 mg, 0.28 mmol) and heated
at 70 °C overnight. The resin was then transferred to a fritted
vessel and washed with DMF (3 × 10 mL), MeOH (3 × 10
mL), and CH2Cl2 (3 × 10 mL). The resin was treated with 95:5
TFA-CH2Cl2 (10 mL), shaken 1 h, and filtered and the filtrate
evaporated. The residue was treated with 10% Et3N-MeOH
(3 mL), then evaporated again. The resulting material was
purified by silica gel chromatography (EtOAc elutant) to yield
10 mg (38%) of 25: 1H NMR (CDCl3) δ 8.75 (1H, d, J ) 4.0),
8.18 (1H, d, J ) 7.7), 8.04-7.88 (3H, m), 7.80 (1H, s), 7.65
(1H, d, J ) 16.2), 7.51-7.47 (1H, m), 6.51 (1H, d, J ) 16.2),
6.40 (1H, br s), 5.70 (1H, br s), 4.54 (2H, s), 4.25 (2H, q,
J ) 7.0), 1.32 (3H, t, J ) 7.0); MS (FAB) 411 (MH+), 433
(MNa+).
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rystal Structure Solution of P1 Glutamine Isosteric Replace-
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Structure-Based Design, Synthesis, and Biological Evaluation
3-[3-Ca r ba m oyl-5-(4-p yr id in -2-yl-p ip er izin -1-m eth yl)-
p h en yl]-a cr ylic Acid Eth yl Ester (26). This was prepared
with 1-(2-pyridyl)piperazine using conditions described for the
synthesis of 25 to yield 12 mg (48%) of 26: 1H NMR (CDCl3)
δ (1H, m), 7.91 (1H, s), 7.79 (1H, s), 7.64 (1H, d, J ) 16.0),
7.62 (1H, s), 7.47-7.41 (1H, m), 6.62-6.57 (2H, m), 6.48 (1H,
d, J ) 16.0), 4.21 (2H, q, J ) 7.0), 3.55 (2H, s), 3.49-3.45 (4H,
m), 2.55-2.51 (4H, m), 1.32 (3H, t, J ) 7.0); MS (FAB) 395
(MH+), 417 (MNa+).
3-(3-{[Ben zyl-(2-et h oxyca r b on yl-et h yl)-a m in o]-m et h -
yl}-5-ca r ba m oyl-p h en yl)-a cr ylic Acid Eth yl Ester (23).
This was prepared with N-benzyl-3-aminopropionic acid ethyl
ester using conditions described for the synthesis of 25 to yield
18 mg (67%) of 23: 1H NMR (CDCl3) δ 7.95 (1H, s), 7.85 (1H,
s), 7.70 (1H, d, J ) 16.0), 7.50 (1H, s), 7.38-7.20 (5H, m), 6.95
(1H, s), 6.60 (1H, d, J ) 16.0), 5.70 (1H, s), 4.25 (2H, q, J )
7.0), 4.15 (2H, q, J ) 7.0), 3.75 (2H, s), 3.63 (2H, s), 2.82 (2H,
t, J ) 5.0), 2.55 (2H, t, J ) 5.0), 1.36 (3H, t, J ) 7.0); MS (ES)
439 (MH+), 461 (MNa+).
3-{3-Ca r ba m oyl-5-[(et h yl-p yr id in -4-ylm et h yl-a m in o)-
m eth yl]-p h en yl}-a cr ylic Acid Eth yl Ester (27). This was
prepared with 4-(ethylaminomethyl)pyridine using conditions
described for the synthesis of 25 to yield 10 mg (43%) of 27:
1H NMR (CDCl3) δ 8.75 (2H, d, J ) 4.0), 8.05 (1H, s), 7.95
(1H, s), 7.75-7.55 (4H, m), 6.50 (1H, d, J ) 16.0), 4.30 (2H, q,
J ) 7.0), 4.18 (2H, q, J ) 8.0), 3.95 (2H, s), 3.90 (2H, s), 1.40-
1.20 (6H, m); MS (ES) 368 (MH+), 390 (MNa+).
4-[3-Ca r b a m oyl-5-(2-et h oxyca r b on yl-vin yl)-b en zyl]-
p ip er a zin e-1-ca r boxylic Acid Eth yl Ester (28). This was
prepared with ethyl-1-piperazine carboxylate using conditions
described for the synthesis of 25 to yield 15 mg (63%) of 28:
1H NMR (CDCl3) δ 8.07 (1H, s), 7.90 (1H, s), 7.66 (1H, s), 4.19
(2H, q, J ) 7.4), 4.14 (2H, s), 4.07 (2H, q, J ) 7.4), 3.68 (4H,
m), 3.55 (4H, m), 1.26 (3H, t, J ) 7.4), 1.19 (3H, t, J ) 7.4).
MS (ES) 390 (MH+), 412 (MNa+).
3-{3-Ca r ba m oyl-5-(4-p yr im id in -2-yl-p ip er a zin -1-ylm e-
th yl)-p h en yl]-a cr ylic Acid Eth yl Ester (29). This was
prepared with 1-(2-pyrimidyl)piperazine using conditions de-
scribed for the synthesis of 25 to yield 15 mg (60%) of 29: 1H
NMR (CDCl3) δ (2H, d, J ) 4.8), 7.88 (1H, s), 7.80 (1H, s),
7.71 (1H, d, J ) 16.2), 7.70 (1H, s), 6.53 (1H, d, J ) 16.2), 6.48
(1H, t, J ) 4.8), 4.27 (2H, q, J ) 7.0), 3.84 (4H, t, J ) 5.2),
3.59 (2H, s), 2.51 (4H, t, J ) 4.8), 1.34 (3H, t, J ) 7.0); MS
(FAB) 396 (MH+), 418 (MNa+).
3-{3-Ca r ba m oyl-5-[4-(2-cya n o-p h en yl)-p ip er a zin -1-yl-
m eth yl]-p h en yl}-a cr ylic Acid Eth yl Ester (30). This was
prepared with 1-(2-cyanophenyl)-piperazine using conditions
described for the synthesis of 25 to yield 19 mg (73%) of 30:
1H NMR (CDCl3) δ 7.90 (1H, s), 7.81 (1H, s), 7.70 (1H, d, J )
15.8), 7.69 (1H, s), 7.57-7.54 (1H, m), 7.51-7.45 (1H, m),
7.03-6.98 (2H, m), 6.53 (1H, d, J ) 15.8), 4.27 (2H, q, J )
7.4), 3.63 (2H, s), 3.24 (4H, q, J ) 4.8), 2.68 (4H, q, J ) 4.8),
1.34 (3H, t, J ) 7.0); MS (FAB) 419 (MH+), 441 (MNa+).

Human Rhinovirus 3C Protease Inhibitors
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