Synthesis and evaluation of peptidyl Michael acceptors that inactivate human rhinovirus 3C protease and inhibit virus replication

Article abstract of DOI:10.1021/jm980114+

Human rhinovirus, the chief cause of the common cold, contains a positive-sense strand of RNA which is translated into a large polyprotein in infected cells. Cleavage of the latter to produce the mature viral proteins required for replication is catalyzed in large part by a virally encoded cysteine proteinase (3C(pro)) which is highly selective for -Q~GP- cleavage sites. We synthesized peptidyl derivatives of vinylogous glutamine or methionine sulfone esters (e.g., Boc-Val-Leu-Phe-vGln-OR: R = Me, 1; R = Et, 2) and evaluated them as inhibitors of HRV-14 3C protease (3C(pro)). Compounds 1 and 2 and several related tetra- and pentapeptide analogues rapidly inactivated 3C(pro) with submicromolar IC₅₀ values. Electrospray mass spectrometry confirmed the expected 1:1 stoichiometry of 3C(pro) inactivation by 1, 2, and several other analogues. Compound 2 also proved to be useful for active site titration of 3C(pro) which has not been possible heretofore because of the lack of a suitable reagent. In contrast to 1, 2, and congeners, peptidyl Michael acceptors lacking a P₄ residue have greatly reduced or negligible activity against 3C(pro), consistent with previously established structure-activity relationships for 3C(pro) substrates. Hydrolysis of the P₁ vinylogous glutamine ester to a carboxylic acid also decreased inhibitory activity considerably, consistent with the decreased reactivity of acrylic acids vs acrylic esters as Michael acceptors. Incorporating a vinylogous methionine sulfone ester in place of the corresponding glutamine derivative in 1 also reduced activity substantially. Compounds 1 and 2 and several of their analogues inhibited HRV replication in cell culture by 50% at low micromolar concentrations while showing little or no evidence of cytotoxicity at 10-fold higher concentrations. Peptidyl Michael acceptors and their analogues may prove useful as therapeutic agents for pathologies involving cysteine proteinase enzymes.

Full text of DOI:10.1021/jm980114+

J . Med. Chem. 1998, 41, 2579-2587
2579
Syn th esis a n d Eva lu a tion of P ep tid yl Mich a el Accep tor s Th a t In a ctiva te Hu m a n
Rh in ovir u s 3C P r otea se a n d In h ibit Vir u s Rep lica tion
J ian-she Kong,† Shankar Venkatraman,† Kelly Furness,† Sanjay Nimkar,† Timothy A. Shepherd,‡ Q. May Wang,‡
J effrey Aube´,† and Robert P. Hanzlik*,†
Department of Medicinal Chemistry, University of Kansas, Lawrence, Kansas 66045-2506, and Lilly Research Laboratories,
Eli Lilly and Company, Indianapolis, Indiana 46285
Received February 20, 1998
Human rhinovirus, the chief cause of the common cold, contains a positive-sense strand of
RNA which is translated into a large polyprotein in infected cells. Cleavage of the latter to
produce the mature viral proteins required for replication is catalyzed in large part by a virally
encoded cysteine proteinase (3Cpro) which is highly selective for -Q∼GP- cleavage sites. We
synthesized peptidyl derivatives of vinylogous glutamine or methionine sulfone esters (e.g.,
Boc-Val-Leu-Phe-vGln-OR: R ) Me, 1; R ) Et, 2) and evaluated them as inhibitors of HRV-14
3C protease (3Cpro). Compounds 1 and 2 and several related tetra- and pentapeptide analogues
rapidly inactivated 3Cpro with submicromolar IC50 values. Electrospray mass spectrometry
confirmed the expected 1:1 stoichiometry of 3Cpro inactivation by 1, 2, and several other
analogues. Compound 2 also proved to be useful for active site titration of 3Cpro, which has
not been possible heretofore because of the lack of a suitable reagent. In contrast to 1, 2, and
congeners, peptidyl Michael acceptors lacking a P4 residue have greatly reduced or negligible
activity against 3Cpro, consistent with previously established structure-activity relationships
for 3Cpro substrates. Hydrolysis of the P1 vinylogous glutamine ester to a carboxylic acid also
decreased inhibitory activity considerably, consistent with the decreased reactivity of acrylic
acids vs acrylic esters as Michael acceptors. Incorporating a vinylogous methionine sulfone
ester in place of the corresponding glutamine derivative in 1 also reduced activity substantially.
Compounds 1 and 2 and several of their analogues inhibited HRV replication in cell culture
by 50% at low micromolar concentrations while showing little or no evidence of cytotoxicity at
10-fold higher concentrations. Peptidyl Michael acceptors and their analogues may prove useful
as therapeutic agents for pathologies involving cysteine proteinase enzymes.
In tr od u ction
tein in vitro,4 their inhibition by small-molecule inhibi-
tors should stop viral replication in vivo. Thus these
proteinases are attractive targets for development of
new approaches to antiviral chemotherapy.
Many plant and animal viruses depend on virus-
encoded proteinases for cleaving virus-specified polypep-
tides to generate proteins related to viral replication.1
One of the largest families of human pathogenic RNA
viruses is the Picornaviridae, which includes poliovirus,
hepatitis A virus, rhinovirus, and others. Rhinovirus,
the chief cause of the common cold, occurs in over 100
different serotypes; this makes immunization impracti-
cal as an approach to prevention, and it invites small-
molecule chemotherapeutic approaches to treatment.
Like other picornaviruses, rhinoviruses contain a posi-
tive-sense strand of RNA, translation of which in
infected cells yields a large polyprotein. This polypro-
tein is cleaved co- and posttranslationally by virally
encoded proteinases to produce mature viral enzymes
and structural proteins. These cleavages are catalyzed
in an organized cascade involving the viral cysteine
proteinases 2Apro, 3Cpro, and 3CDpro. These enzymes are
highly substrate-selective, highly specific in their choice
of cleavage sites, and absent from normal (noninfected)
host cells.2,3 Since mutagenesis of their active site
residues renders them incapable of processing polypro-
The 2A and 3C proteinases are susceptible to inhibi-
tion in vitro by a number of nonspecific thiol-proteinase
inhibitors as well as by more specific agents such as
elastatinal, leupeptin, and methoxysuccinyl-Ala-Ala-
Pro-Val-chloromethyl ketone.5,6 The latter also reduces
the yield of poliovirus type 1, coxsackievirus A21, and
human rhinovirus 2 in infected HeLa cells.5 Similarly,
the peptidyl bromomethyl ketone (Cbz-Phe-azaglutamine-
CH2Br) is a moderately potent time-dependent inacti-
vator of HRV 3Cpro (k2/Ki > 2500 M-1 s-1).7 Interest-
ingly the epoxysuccinyl derivative E-64, which is a
potent active site-directed irreversible inhibitor and
active site titrant toward cysteine proteinases of the
papain family, does not inhibit 3C proteinases from
HRV-146 or poliovirus.8,9 Small molecules containing
reactive carbonyl groups can be potent inhibitors of
cysteine proteinases via reversible formation of covalent
hemithioacetal or -ketal adducts with the active site
thiol (cf. elastatinal and leupeptin above).10-12 Peptide
aldehydes tailored to resemble substrates for 2A and
3C proteinases have also provided quite potent in vitro
inhibition13-15 and have inspired the development of
nonpeptidic carbonyl compounds including â-lactams,16
isatins,17 and homophthalimides18 as HRV 3Cpro inhibi-
* Address correspondence to: Dr. Robert P. Hanzlik at University
of Kansas. Tel: 785-864-3750. Fax: 785-864-5326. E-mail: rhanzlik@
rx.pharm.ukans.edu.
† University of Kansas.
‡ Eli Lilly and Co.
S0022-2623(98)00114-9 CCC: $15.00 © 1998 American Chemical Society
Published on Web 06/16/1998

2580 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 14
Kong et al.
Sch em e 1
Sch em e 2. Synthesis of Boc-VLF-OHa
a
(a) DCC, NMM, NHS, CH2Cl2; (b) H2, Pd/C, EtOH.
Sch em e 3a
tors. Thus far, however, only the isatins have provided
potent in vitro inhibitors, and results in cell culture have
been disappointing.
a
(a) DCC, NHS, THF, DMF; (b) NaBH4, 90% EtOH, HCl.
Because peptidyl Michael acceptors based on vinylo-
gous amino acids19 inactivate cysteine proteinases se-
lectively and specifically,20-22 we became interested in
for P1 glutamine is rationalized on the basis that its
γ-carbonyl oxygen accepts two important hydrogen
bonds from T141 and H160 of the enzyme. Thus
asparagine, aspartate, and glutamate are poor substi-
tutes for glutamine, although a dipeptide aldehyde with
methionine sulfone at the P1 position is a good competi-
tive inhibitor or HRV-14 3Cpro (Ki ) 0.47 µM),15 and in
the case of the related hepatitis A 3Cpro, N,N-dimeth-
ylglutamine derivatives are accepted.13 The above
structural requirements for substrates were translated
into the design of the peptidyl Michael acceptors shown
in Table 1.
exploring this approach to inhibition of the HRV 3Cpro
.
In this paper we report our results on the design,
synthesis, and evaluation of peptidyl Michael acceptors
as inhibitors of HRV 3Cpro and as inhibitors of viral
replication in cell culture in vitro. We show that with
appropriate peptidyl carrier moieties, vinylogous
glutamine esters are extremely potent inactivators of
HRV 3Cpro in vitro; in fact they can actually be used as
active site titrants for this enzyme, for which no other
titrant has been described. In addition, some of the
compounds are also highly effective at inhibiting viral
replication in cell culture while showing very low
cytotoxicity.
Syn th esis
The peptide Boc-V-L-F-OH, a common component of
many of the inhibitors in Table 1, was synthesized using
standard peptide-coupling chemistry as indicated in
Scheme 2. The vinylogous glutamine derivatives were
synthesized from Boc-L-glutamine by a sequence of
cyclization to Boc-aminoglutarimide and reduction to
Boc-glutaminal, which exists primarily as a cyclic hemi-
aminal (Scheme 3). The latter was converted to the
vinylogous glutamine esters by Horner-Emmons chem-
istry20-22 and on to the various peptidyl Michael accep-
tors as shown in Scheme 4. A similar approach starting
with Boc-L-methionine was used for the synthesis of the
vinylogous methionine sulfone derivatives 7 and 9
(Scheme 5).
Since the racemization of peptide aldehydes can occur
relatively easily, their potential epimerization during
the Horner-Emmons olefination was of some concern.
The fact that products 13 and 14, which have a single
chiral center, show appreciable optical activity indicates
that complete racemization did not occur. However,
other observations suggest that some epimerization
In h ibitor Design
The 3C proteinase of HRV-14 has been cloned, ex-
pressed, and purified in a number of laboratories (see
Birch et al.23 and references therein). Its substrate
requirements have been explored using synthetic pep-
tides related to viral polyprotein cleavage sites and a
consensus substrate has been defined.6,24-26 A crystal
structure of HRV-14 3Cpro and a hypothetical substrate
docking model27 rationalize the enzyme’s observed
kinetic specificity and highlight the following main
points. The minimum consensus substrate which is
efficiently cleaved is P5-P4-P3-P2-Q∼G-P, where Q∼G
represents the scissile (P1-P1′) bond (Scheme 1). Posi-
tions P5 and P3 are quite tolerant to substitution, and
the docking model suggests these side chains project out
into solution. On the other hand, kinetic studies
indicate that P4 should be small and nonpolar while P2
may be large and hydrophobic, and the crystal structure
of the enzyme reveals the existence of pockets comple-
mentary to such side chains. The stringent requirement

Synthesis, Evaluation of Peptidyl Michael Acceptors
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 14 2581
Sch em e 4a
a
(a) (MeO)2P(O)CH2CO2Me, NaH, THF; (b) (EtO)2P(O)CH2CO2Et, NaH, THF; (c) (EtO)2P(O)CH2SO2Ph, NaH, THF; (d) TFA, CH2Cl2;
(e) PhCH2OCOCl, Et3N, CH2Cl2; (f) DECP, Et3N, THF; (g) Boc-F-OH; (h) Boc-VLF-OH; (i) Cbz-F-OSu, Et3N; (j) aq NaOH, THF; (k) Cbz-
F-OH; (l) Boc-E(tBu)-OH.
Sch em e 5a
tive results are shown in Table 1. A considerable range
of inhibitory activities was observed, with compounds
1-4 giving complete inhibition even at the lowest
concentration tested, while others gave only 18-40%
inhibition at the highest concentration tested. For the
more active compounds we next determined IC50 values
using more narrowly spaced concentrations of inhibitor
(chosen in light of the preliminary results) and recording
progress curves for chromophore generation in each
reaction. Progress curves obtained from the less active
compounds showed curvature indicative of progressive
enzyme inactivation during the observation period (data
not shown). For the more active compounds the inac-
tivation was so fast that it could not be observed in this
way; instead linear progress curves were observed in
all reactions in which enzyme inactivation was not
complete. In addition, the IC50 values of the more active
compounds were all very similar and approximately
equal to one-half of the enzyme concentration used in
the assays.
a
(a) CDI, THF, then MeONHCH3; (b) LiAlH4, Et2O; (c)
(MeO)2P(O)CH2CO2Me, NaH, THF; (d) Oxone, MeOH, H2O; (e)
TFA, CH2Cl2; (f) DECP, Et3N, DMF; (g) Boc-VLF-OH; (h) Cbz-F-
OH, EDC, HOBT, NMM.
The above observations are consistent with a very
rapid, stoichiometric, covalent inactivation of the en-
zyme by the Michael acceptors. We therefore used
electrospray mass spectrometry to examine the enzyme
after treatment with several peptidyl Michael acceptors.
In each case the only protein species observed had the
mass expected for a 1:1 protein:ligand adduct (Table 2).
Moreover, these complexes were unaffected by dialysis
for 4 h at 4 °C. In parallel with these experiments a
control experiment was performed using the dipeptide
aldehyde 3Cpro inhibitor LY338387 (Cbz-FM(O2)-H).15
In this case the undialyzed protein showed a MW of
20 446.4 ( 2.5 Da, as expected for a 1:1 hemithioacetal
adduct, but after dialysis only the ligand-free native
protein was observed. These results indicate that the
inhibition of HRV-14 3Cpro by the Michael acceptors is
irreversible and thus covalent as well as stoichiometric.
probably did occur. This was most notable in the case
of compound 7, for which both HPLC and 1H NMR
clearly show the presence of two diastereomers in a 3:5
ratio. In the glutamine series (e.g., compounds 1, 2, 4,
10, and 11) diastereomeric purity tended to be signifi-
cantly higher, typically 85-95% as determined by HPLC
and/or 1H NMR, and this was deemed adequate for
initial evaluation of the compounds for enzyme inhibi-
tion and antiviral activity. That such epimerizations
were not encountered during the synthesis of simple
peptide derivatives (e.g., compounds 16-19) further
implicates the Horner-Emmons reaction as the source
of epimerization, but efforts to minimize or control it
were not undertaken.
Resu lts a n d Discu ssion
The fact that the most potent inhibitors appeared to
be literally “titrating” the enzyme suggested that they
might actually be exploited for this purpose. This would
be significant since E-64, the epoxysuccinyl derivative
En zym e In h ibition . For evaluation as inhibitors of
HRV-14 3Cpro all compounds were tested initially at
concentrations of 1, 5, 25, and 100 µg/mL. Representa-

2582 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 14
Kong et al.
Ta ble 1. Structures, Numbering, Enzyme Inhibition, and Antiviral Activity of Peptidyl Michael Acceptors
enzyme inhbition
rangefinding
plaque reduction
b
compd
structure
µg/mL
% inhiba
IC50 (µM)
IC50 (µg/mL)
1
2
Boc-V-L-F-vQ-OMe
Boc-V-L-F-vQ-OEt
Cbz-V-L-F-vQ-OMe
Boc-E(tBu)-V-L-F-vQ-OMe
Boc-V-L-F-vQ-OH
E-V-L-F-vQ-OMe
Boc-V-L-F-vM(O2)-OMe
Cbz-F-vQ-OMe
1
1
100
100
100
100
43
0.25 ( 0.02
0.13 ( 0.01
0.17 ( 0.02
0.2 ( 0.01
17.7 ( 2.1
0.49 ( 0.03
13.6 ( 2.5
9.5 ( 1.9
ndc
0.74
0.41
0.93
4.5
>10
>10
>10
3.2
>10
>10
>10
>10
ntd
3
1
4
1
25
1
5
5
25
100
100
100
100
100
100
5
6
100
48
7
8
51
9
Cbz-F-vM(O2)-OMe
Boc-F-vQ-OMe
27
10
11
12
13
14
15
42
nd
Boc-F-vQ-OEt
39
nd
Cbz-vQ-OEt
18
nd
Boc-vQ-OMe
18
nd
Boc-vQ-OEt
27
nd
>10
>10
(see Scheme 4)
42
nd
a
b
d
Average of duplicates which agreed within (10%. See the Experimental Section for details. c nd ) not determined. nt ) not tested.
Ta ble 2. Apparent Masses of HRV-14 3C Protease after
Inactivation with Various Michael Acceptors
these compounds. In view of the extremely high reac-
tivity of these inhibitors and the modest sensitivity of
the photometric assay available to us, we did not
attempt to determine apparent second-order rate con-
stants (i.e., k2/Ki) for the inactivation of HRV-14 3Cpro
by any of our compounds. Nevertheless the inhibition
data presented in Table 1 allow some conclusions to be
drawn about the effects of structure on the relative
efficacy of the compounds. All of the potent compounds
are tetra- or pentapeptides having a glutamine-like side
chain at P1 and a hydrophobic side chain at P4; smaller
compounds lacking the P4 residue are very much less
active (viz. compounds 10-14). These features are
consistent with the kinetic specificity of HRV-14 3Cpro
as delineated using peptide substrates comprised of only
standard proteinogenic amino acids.6,24-26 Although
HRV-14 3Cpro is highly selective for cleaving -Q∼GP-
sites in protein substrates, it evidently tolerates re-
placement of the P1′-P2′ Gly-Pro leaving group with
nonphysiological groups including p-nitroaniline28 or the
acrylic ester moiety of the peptidyl Michael acceptors.
As Michael acceptors, acrylate esters are much more
reactive than the corresponding acrylic acids which are
negatively charged at physiological pH values.22,29,30
This may explain why compound 5 is much less effective
as an inactivator of HRV-14 3Cpro than its ester ana-
logues 1 and 2. At the P5 position the enzyme accepts
the lipophilic Boc or Cbz groups in 1-3, the blocked
glutamate derivative Boc-E(tBu) in 4, or the unblocked
zwitterionic glutamyl residue in 6. However, replace-
ment of the P1 glutamine side chain (-CH2CH2CONH2)
by a methionine sulfone side chain (-CH2CH2SO2CH3)
in 7 leads to substantial loss of activity. This is in
contrast to the significant potency of the methional
sulfone derivative Cbz-FM(O2)-H as a competitive in-
MW
ligand, structure
ligand
adducta
∆MW
none
0
645
659
831
674
679
468
446
19 998.4
20 644.4
20 657.8
20 831.0
20 674.8
20 465.2
20 465.2
20 446.4
1, Boc-V-L-F-vQ-OMe
2, Boc-V-L-F-vQ-OEt
4, Boc-E(OtBu)-V-L-F-vQ-OMe
6, E-V-L-F-vQ-OMe
19 998.6
19 998.8
20 000.0
20 000.8
19 997.2
19 997.2
20 000.0
3, Cbz-V-L-F-vQ-OMe
8, Cbz-F-vQ-OMe
LY338387, Cbz-FM(O2)-H
a
b
Observed MW of protein ((2.5 Da). The calculated mass of
the native enzyme is 19 997 (ref 23).
F igu r e 1. Active site titration of HRV 3C protease with Boc-
V-L-F-vQ-OEt (compound 2).
widely used for titrating many cysteine proteinases in
the papain clan, does not inhibit HRV-14 3Cpro 6
As
.
shown in Figure 1, incremental addition of compound
2 to aliquots of HRV 3Cpro leads to proportional de-
creases in enzyme activity with complete loss of activity
being reached once 0.4 nmol of 2 had been added. These
results, coupled with the observation of 1:1 adducts by
mass spectrometry, indicate that the enzyme prepara-
tion used actually contained only 47% active 3C pro-
tease, a result which agrees with estimates made by
isoelectric focusing gel analysis of the purified recom-
binant protein followed by kinetic assays for 3Cpro
activity (data not shown). Thus Michael acceptors such
as 2 can indeed be used to determine the operational
normality of solutions of HRV 3C protease.
hibitor of HRV-14 3Cpro 15
Thus the ability of the
.
sulfone group to function as a H-bond-accepting17,27
surrogate for the amide side chain of glutamine evi-
dently depends at least in part on the nature of other
enzyme-ligand interactions and the degree to which
these individual interactions may be cooperative.
An tivir a l Activity a n d Cytotoxicity. Active 3Cpro
is essential for viral replication, but for compounds
which inactivate this enzyme in cell-free systems to
exhibit antiviral activity in cells, they must be capable
Because the IC50 values for compounds 1-4, and
perhaps 6, are very close to one-half of the enzyme
concentration used in the assays, the IC50 data do not
reveal potential differences in kinetic specificity among

Synthesis, Evaluation of Peptidyl Michael Acceptors
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 14 2583
from KOH pellets. Melting points were determined in capil-
lary tubes using a Thomas-Hoover apparatus and are uncor-
rected. NMR spectra were recorded using a GE QE-Plus (300-
MHz) or a Bruker DRX-400 (400-MHz) spectometer. Fast
atom bombardment mass spectra were obtained using an
AUTOSPEC-Q tandem hybrid mass spectrometer or a ZAB-
HS mass spectrometer. Spectra were recorded from samples
in matrixes of glycerol, glycerol/thioglycerol, or m-nitrobenzyl
alcohol saturated with LiOAc. A Nermag R10-10b mass
spectrometer was used for electron impact and desorption-
chemical ionization mass spectra, the latter with ammonia as
the reagent gas. Combustion analyses were performed using
a Perkin-Elmer model 2400-CHN elemental analyzer. Glass
plates coated with silica gel GLHF (250 µm, Analtech) were
used for analytical thin-layer chromatography. HPLC analy-
ses were performed using a Shimadzu gradient HPLC system
with a Vydac C-18 column (218TP54, 5 µm, 4.6 × 250 mm).
Depending on the compound being analyzed, the mobile phase
contained either MeOH (40-60%, v/v) or MeCN (10-40%, v/v)
in water containing 0.1% (v/v) TFA, but elution was always
isocratic at a flow rate of 1.0 mL/min; peak detection was at
214 nm.
Syn th esis. Boc-V-L-OBn (16). Boc-valine (1.0 g, 4.6
mmol), leucine benzyl ester p-toluenesulfonate salt (1.81 g, 4.6
mmol), CH2Cl2 (50 mL), NMM (1.52, mL, 13.8 mmol), and NHS
(0.58 g, 5.06 mmol) were combined in a flask and cooled to 0
°C. DCC (1.04 g, 5.06 mmol) was added, and the reaction
mixture was stirred for 3 h at 0 °C and for 18 h at room
temperature. The solvent was evaporated and the residue
dissolved in ethyl acetate (250 mL). The solution was washed
successively with 1 M HCl, water, saturated NaHCO3 solution,
and brine (40 mL each), dried over MgSO4, and filtered.
Evaporation of the filtrate yielded 4.81 g of a white solid which
was chromatographed over silica gel (CH2Cl2/MeOH, 19:1) to
give compound 16 as a white solid (3.5 g, 70%). Mp: 88-89
°C. 1H NMR (400 MHz, CDCl3): δ 0.86-0.94 (m, 12H), 1.44
(s, 9H), 1.53-1.69 (m, 3H), 2.08 (m, 1H), 3.87 (dd, J ) 6.4, 8.8
Hz, 1H), 4.68 (m, 1H), 5.04 (br d, J ) 8 Hz, 1H), 5.15 (ABq, J
) 12.3 Hz, ∆ν ) 9.0 Hz, 2H), 6.22 (br s, 1H), 7.30-7.40 (m,
5H).
Boc-V-L-OH (17). Compound 16 (3.46 g, 8.23 mmol) was
dissolved in ethanol (100 mL) and hydrogenated over Pd/C (200
mg) at 30 psi H2 for 12 h at room temperature. The catalyst
was removed by filtration and the solvent evaporated yielding
17 as a white solid in quantitative yield. 1H NMR (400 MHz,
CDCl3): δ 0.79-0.91 (m, 12H), 1.37 (s, 9H), 1.52 (m, 2H), 1.62
(m, 1H), 1.92 (m, 1H), 3.78 (m, 1H), 4.26 (m, 1H), 6.65 (d, J )
9.1 Hz, 1H), 7.96 (d, J ) 7.9 Hz, 1H).
Boc-V-L-F -OBn (18). Compound 17 (2.58 g, 8.23 mmol),
phenylalanine benzyl ester (2.10 g, 8.23 mmol), HOBt (1.22 g,
9.05 mmol), NMM (2.3 mL, 24.7 mmol), and 50 mL of CH2Cl2
were combined in a flask and cooled to 0 °C. EDC (1.85 g, 9.7
mmol) was added, and the mixture was stirred for 3 h at 0 °C
and for 18 h at room temperature. The solvent was evaporated
and the residue dissolved ethyl acetate (250 mL). The solution
was washed successively with 1 M HCl, water, saturated
NaHCO3 solution, and brine (40 mL each), dried over MgSO4,
and filtered. Evaporation of the filtrate yielded 3.11 g of a
white solid which was chromatographed over silica gel (CH2-
Cl2/MeOH, 19:1) to give compound 18 (3.11 g, 55%). Mp: 151-
152 °C. 1H NMR (400 MHz, CDCl3): δ 0.89-0.95 (m, 12H),
1.43 (s, 9H), 1.47-1.53 (m, 1H), 1.55-1.65 (m, 2H), 1.89-1.97
(m, 1H), 2.05-2.15 (m, 1H), 3.09 (d, J ) 6 Hz, 2H), 3.83-3.89
(m, 1H), 4.37-4.46 (m, 1H), 4.83-4.90 (m, 1H), 5.11 (ABq, J
) 12.1 Hz, ∆ν ) 21.4 Hz, 2H), 7.00 (m, 2H), 7.21 (m, 3H), 7.35
(m, 2H), 7.75 (m, 3H). FABMS (NBA/LiOAc): m/z 574 (M +
Li), 474 (M - Boc + Li). HRMS: calcd for C32H46N3O6,
568.3387; found (FAB, glycerol), 568.3375.
of diffusion and/or transport into infected cells. Once
inside they must not be inactivated (e.g., by conjugation
with glutathione or hydrolysis of ester or peptide bonds
required for enzyme inhibition) or transported back out
of the cells before they have had a chance to react
irreversibly with the viral 3Cpro. Compounds 1-3 have
very significant antiviral activity in cell culture assay,
inhibiting viral replication and plaque formation by 50%
at submicromolar concentrations (Table 1). Clearly they
fulfill all the criteria for activity, cellular uptake, and
metabolic stability stated above. The reduced activity
of compound 6, despite its potency as a 3Cpro inactivator
in cell-free systems, is probably attributable to difficulty
in entering cells, although the possibility that it is
rapidly hydrolyzed intracellularly (e.g., by an aminopep-
tidase) cannot be ruled out. With the possible exception
of compound 8, the other Michael acceptor compounds
showed only weak antiviral activity at best, consistent
with their relatively low activity as enzyme inactivators.
To ensure that the observed antiviral activity was not
due to a general cytotoxic effect, all compounds were
evaluated for toxicity in uninfected cells using the XTT
reduction assay. In this assay a tetrazolium dye (XTT)
is metabolically reduced to form a colored product by
metabolically viable cells. At the highest concentration
of test compound examined, 10 µg/mL, compound 1
caused a 16% reduction in metabolic activity while
compound 2 caused an 8% reduction; all other com-
pounds showed no difference from controls, suggesting
a very low level of cytotoxicity for these peptidyl Michael
acceptors.
Con clu sion s
Peptidyl Michael acceptors comprising an appropriate
recognition peptide and a vinylogous amino acid ester
are extremely active as affinity-labeling inactivators of
human rhinovirus 3C proteinase in vitro. They form
1:1 covalent adducts with the active site cysteine and
can be used for active site titration of HRV-14 3Cpro. In
cell cultures the more active inhibitors show excellent
antiviral activity at concentrations far below those
causing cytotoxicity. Peptidyl Michael acceptors and
their analogues warrant further investigation as poten-
tial therapeutic agents for pathologies involving cysteine
proteinase enzymes.
Exp er im en ta l Section
The abbreviations which are used are as follows: Cbz,
benzyloxycarbonyl; CDI, 1,1-carbonyldiimidazole; CI, chemical
ionization; DCC, N,N′-dicyclohexylcarbodiimide; DECP, diethyl
cyanophosphonate; DMF, N,N-dimethylformamide; EDC, 1-[3-
(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride; EI,
electron ionization; FAB, fast atom bombardment; FD, field
desorption; G/TG, glycerol/thioglycerol; HOBt, 1-hydroxyben-
zotriazole; HPLC, high-performance liquid chromatography;
HR, high resolution; MS, mass spectrometry; NBA, m-ni-
trobenzyl alcohol; NHS, N-hydroxysuccinimide; NMM, N-
methylmorpholine; OSu, N-succinimidinyloxy; TFA, trifluoro-
acetic acid; THF, tetrahydrofuran.
In str u m en ts a n d Ma ter ia ls. Chemicals, solvents, and
reagents were obtained from commercial sources. The peptide
p-nitroanilide EALFQ-pNA used for photometric assays was
custom synthesized by American Peptide Co. (CA) and was
characterized by amino acid analysis and mass spectrometry.
THF was distilled from sodium and benzophenone, DMF was
distilled from phthalic anhydride at reduced pressure, aceto-
nitrile was distilled from CaH2, and triethylamine was distilled
Boc-V-L-F -OH (19). Compound 18 was hydrogenated as
described above for Boc-V-L-OBn giving Boc-V-L-F-OH as a
white solid in quantitative yield. Mp: 124-125 °C. 1H NMR
(400 MHz, CDCl3): δ 0.82-0.92 (m, 12H), 1.43, (s, 9H), 1.53-
1.62 (m, 2H), 1.71 (m, 1H), 1.92 (m, 1H), 2.05 (m, 1H), 3.03
(m, 1H), 3.17 (m, 1H), 3.89 (m, 1H), 4.58 (m, 1H), 4.77 (m,

2584 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 14
Kong et al.
1H), 5.20 (br s, 1H), 7.02 (br s, 2H), 7.13-7.28 (m, 5H).
HRMS: calcd for C25H40N3O6, 478.2917; found (FAB, G/TG),
478.2900.
NaHCO3 solution, the organic phase was dried over MgSO4,
filtered, and concentrated to yield compound 12 as a white
solid (500 mg, 70%). Mp: 72-76 °C. [R]D -58 (c ) 1.0,
MeOH). 1H NMR (400 MHz, CDCl3): δ 1.28 (t, 3H), 1.94 (m,
2H), 1.98 (m, 1H), 2.28 (m, 2H), 4.19 (q, 2H), 4.39 (m, 1H),
5.12 (m, 2H), 5.41 (br s, 1H), 5.54 (br s, 1H), 5.74 (br s, 1H),
5.94 (d, J ) 16 Hz, 1H), 6.82 (dd, J ) 6, 16 Hz, 1H), 7.32 (m,
5H). HPLC (MeOH/H2O/TFA, 50:50:0.1),tR ) 5.60 min. Anal.
(C17H22N2O5) C, H, N.
Boc-F -vQ-OEt (11). Boc-vQ-OEt (300 mg, 1.0 mmol) was
stirred in 2 mL of TFA/CH2Cl2 (1:1) for 30 min at room
temperature. The solution was evaporated to dryness, and the
residue was combined with DMF (15 mL), Boc-F-OH (0.4 g,
1.5 mmol), Et3N (0.5 mL), and DECP (0.25 mL, 1.5 mmol).
After the mixture stirred for 44 h, EtOAc (250 mL) was added,
and the solution was extracted with saturated NaHCO3
solution, 0.1 N citric acid, and brine (40 mL each). The organic
extract was dried over MgSO4, filtered, and concentrated to
give 600 mg of a white paste. The latter was chromatographed
over silica gel (CH2Cl2/MeOH, 20:1) which provided ester 11
as a white solid. Mp: 86-89 °C, 70% yield. FABMS (G/TG):
m/z 448 (MH), 348 (M - Boc + H). Anal. (C23H33N3O6) C, H,
N. HPLC (MeOH/H2O/TFA, 50:50:0.1) shows this material to
Boc-L-2-a m in oglu ta r im id e (20). Boc-L-glutamine (2.46 g,
10.0 mmol) and N-hydroxysuccinimide (1.5 g, 10 mmol) were
dissolved in a mixture of DMF (2.25 mL) and THF (15 mL)
and cooled to 0 °C. DCC (20.8 g, 10.0 mmol) was added, and
the mixture was stirred at room temperature overnight and
then filtered. The filtrate was concentrated in vacuo, and the
residue was extracted with hot CHCl3 several times. The
extract was concentrated, and the residue was taken up in
EtOAc. The latter was washed three times with water, dried
over MgSO4, filtered, and concentrated in vacuo. Chromatog-
raphy of the solid residue over silica gel (EtOAc/hexanes, 1:1)
provided Boc-L-2-aminoglutarimide (20) (752 mg, 34%) as a
white solid. Mp: 153-155 °C. [R]D -58.8 (c ) 1.125, MeOH).
1H NMR (300 MHz, CDCl3): δ 1.5 (s, 9H), 1.8-2.0 (m, 1H),
2.4-2.6 (m, 1H), 2.6-2.9 (m, 2H), 4.3 (m, 1H), 5.35 (br s, 1H),
8.2 (br s, 1H). CIMS: m/z 246 (M + NH4), 229 (MH). IR
(KBr): 3350 (sh), 1720, 1690 cm-1. Anal. (C10H16N2O4) C, H,
N.
(3S)-3-[(ter t-Bu toxyca r bon yl)a m in o]-2-h yd r oxyp ip er i-
d in -6-on e (21). By analogy to reductions of peptidyl gluta-
timide derivatives reported by Kaldor et al.,14 compound 20
(1.5 g, 6.6 mmol) was dissolved in 60 mL of EtOH/H2O (9:1)
and cooled to -15 °C. Sodium borohydride (965 mg, 25.4
mmol) was added in one portion followed by 3 drops of 2 N
HCl. The solution was stirred at -10 °C while 2 drops of 2 N
HCl was added every 15 min for 90 min. The mixture was
poured into brine (50 mL) and extracted with CHCl3. Removal
of solvent and chromatography of the residue using EtOAc/
MeOH (15:1) yielded 0.98 g (42.5%) of compound 21, a mixture
of C-2 epimers, as white crystals. Mp: 113-115 °C. [R]D -6.8
(c ) 0.5, MeOH). 1H NMR (300 MHz, CDCl3): δ 1.40 (s, 9H),
1.7-1.9 (m, 2H), 2.20 (m, 1H), 2.45 (m, 1H), 3.77 (m, 1H), 4.7
(br s, 1H), 4.85 (m, 1H), 4.95 (s, 1H), 6.7 (br s, 1H). MS (EI):
m/z 212 (M - H2O). IR (KBr): 3300 (br), 1670 (br) cm-1. This
material was used in subsequent reactions without further
characterization.
Meth yl (2E,4S)-7-Am in o-4-[(ter t-bu toxyca r bon yl)a m i-
n o]-7-oxo-2-h ep ten oa te (Boc-vQ-OMe, 13). A solution of
trimethyl phosphonoacetate (1.82 g, 10 mmol) in 25 mL of THF
was added dropwise to a stirred suspension of NaH (330 mg,
13.75 mmol) in anhydrous THF (75 mL). After 1 h at room
temperature a solution of compound 21 (1.15 g, 5 mmol) in 25
mL of THF was added. The mixture was stirred for 2.5 h,
quenched with water (10 mL), and concentrated under reduced
pressure. The residue was dissolved in CHCl3, washed suc-
cessively with 2% HCl, water, and saturated NaHCO3 solution,
and dried over MgSO4. After concentration the crude product
was triturated with Et2O leaving a white crystalline product
(1.19 g, 83%). Mp: 116-117 °C. [R]D -27 (c ) 0.4, MeOH).
1H NMR (300 MHz, CDCl3): δ 1.42 (s, 9H), 1.80 (m, 1H), 2.00
(m, 1H), 2.30 (m, 2H), 3.75 (s, 3H), 4.35 (br s, 1H), 4.90 (br d,
1H), 5.45 (br s, 1H), 5.95 (d, J ) 16 Hz, 1H), 6.85 (dd, J ) 16,
5 Hz, 1H). CIMS: m/z 287 (MH)+. Anal. (C13H22N2O5) C, H,
N.
Boc-vQ-OEt (14). This compound was prepared as de-
scribed for compound 13 using triethyl phosphonoacetate in
place of trimethyl phosphonoacetate. Yield: 81%. Mp: 123-
124.5 °C. [R]D -42 (c ) 1.0, MeOH). 1H NMR (400 MHz,
CDCl3): δ 1.30 (t, 3H), 1.45 (s, 9H), 1.82 (m, 1H), 1.97 (m, 1H),
2.31 (m, 2H), 4.17 (q, 2H), 4.35 (br s, 1H), 4.96 (m, 1H), 5.78
(br s, 1H), 5.94 (d, J ) 16 Hz, 1H), 6.10 (br s, 1H), 6.83 (dd, J
) 16, 6 Hz, 1H). HRMS: calcd for C14H25N2O5, 301.1763;
found (FABMS, G/TG), 301.1777. HPLC (MeOH/H2O/TFA, 50:
50:0.1) tR ) 5.91 min.
be a mixture of two components, a minor one (20%) with tR
)
8.16 min and a major one (80%) with tR ) 15.8 min. In
addition, in the 1H NMR spectrum of this product several of
the characteristically patterned peaks are accompanied by
small peaks showing the same pattern but with a slight offset
in chemical shift in a fashion suggestive of a diastereomeric
impurity. However, preparative separation of the diastereo-
mers was not pursued, and the spectrum reported below is
that for the major diastereomer. 1H NMR (400 MHz, CDCl3):
δ 1.28 (t, 3H), 1.40 (s, 9H), 1.75 (m, 1H), 1.95 (m, 1H), 2.20
(m, 2H), 3.06 (d, 2H), 4.17 (q, 2H), 4.57 (m, 1H), 5.10 (br s,
1H), 5.45 (br s, 1H), 5.62 (d, J ) 16 Hz, 1H), 5.97 (br s, 1H),
6.35 (br s, 1H), 6.65 (dd, J ) 16, 6 Hz, 1H), 7.25 (m, 5H).
Boc-V-L-F -vQ-OMe (1). Boc-vQ-OMe (70 mg, 0.24 mmol)
was dissolved in CH2Cl2 (1 mL), trifluoroacetic acid (1 mL) was
added, and the solution was stirred for 2 h at room tempera-
ture and then concentrated in vacuo. The crude product was
dissolved in DMF (5 mL), and Boc-V-L-F-OH (105 mg, 0.22
mmol), triethylamine (204 µL, 1.3 mmol), and diethyl cyano-
phosphonate (108 µL, 0.66 mmol) were added. After the
mixture stirred for 44 h, ethyl acetate (250 mL) was added,
and the solution was washed with saturated NaHCO3 solution,
0.1 N citric acid, and brine (25 mL each). The organic layer
was dried over MgSO4 and filtered. After concentration, HPLC
(MeOH/HOH/TFA, 60:40:0.1) showed two peaks at tR ) 10.3
and 11.2 min. Trituration of the residue with small portions
of EtOAc selectively removed the latter and left behind white
crystals of compound 1 (105 mg, 74%). Mp: 222 °C dec. 1H
NMR (300 MHz, DMSO-d6): δ 0.85 (m, 12H), 1.40 (s, 9H), 1.56
(m, 1H), 1.67 (m, 1H), 1.72 (m, 1H), 1.90 (m, 1H), 2.06 (m,
2H), 2.86 (m, 1H), 2.96 (m, 1H), 3.56 (s, 3H), 3.75 (m, 1H),
4.35 (m, 2H), 4.46 (m, 1H), 5.65 (d, J ) 15 Hz, 1H), 6.67 (dd,
J ) 15, 6 Hz, 1H), 6.72 (m, 1H), 7.20 (m, 6H), 7.81 (m, 1H),
8.00 (m, 1H), 8.08 (m, 1H). EIMS: m/z 645 (M+). FABMS
(G/TG): m/z 646 (MH), 546 (M - Boc + H).
Boc-V-L-F -vQ-OEt (2). This compound was prepared as
described for 1 using Boc-vQ-OEt in place of Boc-vQ-OMe.
After trituration of the initial solid product with EtOAc, the
yield of product was 50%. Mp: 142-146 °C. HPLC (MeCN/
H2O/TFA, 40:60:0.1) tR ) 9.3 min. 1H NMR (300 MHz, DMSO-
d6): δ 0.80 (m, 12H), 1.25 (m, 3H), 1.40 (m, 9H), 1.55 (m, 1H),
1.66 (m, 1H), 1.75 (m, 1H), 1.90 (m, 1H), 2.07 (m, 2H), 2.88
(m, 1H), 2.95 (m, 1H), 3.75 (m, 1H), 4.12 (q, 2H), 4.35 (m, 2H),
4.49 (m, 1H), 5.62 (d, J ) 6 Hz, 1H), 6.70 (m, 2H), 7.20 (m,
5H), 7 0.85 (m, 1H), 8.04 (m, 1H), 8.10 (m, 1H). FABMS
(NBA/LiOAc): m/z 666 (M + Li), 566 (M - Boc + Li). HRMS:
calcd for C34H53N5O8Li, 666.4037; found (FAB, NBA/LiOAc),
666.4054.
Cbz-vQ-OEt (12). Boc-vQ-OEt (500 mg, 1.74 mmol) was
stirred in 2 mL of TFA/CH2Cl2 (1:1) for 30 min at room
temperature. The mixture was evaporated to dryness and the
residue dissolved in CH2Cl2 (10 mL) and Et3N (0.5 mL) and
cooled to 0 °C. Benzyl chloroformate (0.3 mL, 1.6 mmol) was
added, and the mixture was stirred for 3 h and then diluted
into CH2Cl2 (50 mL). After washing with water and saturated
Boc-E(tBu )-V-L-F -vQ-OMe (4). Boc-VLFvQ-OMe (100
mg, 0.15 mmol) was stirred with 36 µL of TFA and 20 µL of
CH2Cl2 for 30 min at room temperature, and the mixture was

Synthesis, Evaluation of Peptidyl Michael Acceptors
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 14 2585
evaporated to dryness. DMF (5 mL) was added, followed by
Boc-E(tBu)-OH (100 mg, 0.3 mmol), Et3N (66 µL, 0.45 mmol),
and DECP (75 µL, 0.45 mmol), and the mixture was stirred
for 24 h at room temperature. EtOAc (25 mL) was added, and
the solution was filtered and evaporated. The residue was
triturated with THF (5 mL) and then with ether (20 mL) and
dried under vacuum leaving 50 mg (33% yield) of a white solid.
Mp: 189-194 °C. HPLC in two solvent systems showed a
single peak (MeOH/H2O/TFA, 50:50:0.1) tR ) 8.64 min; (MeCN/
H2O/TFA, 50:50:0.1) tR ) 8.06 min. Likewise the 1H NMR
spectrum (400 MHz, CDCl3) showed no indication of diaster-
eomeric impurities: δ 0.80 (m, 12H), 1.36 (s, 18H), 1.50 (m,
1H), 1.68 (m, 2H), 1.80 (m, 1H), 1.88 (m, 1H), 2.05 (t, 1H),
2.19 (t, 1H), 2.48 (s, 3H), 2.82 (dd, 1H, J ) 8.82, 11.9 Hz), 2.92
(dd, 1H, J ) 6.1, 11.9 Hz), 3.32 (s, 3H), 3.64 (s, 2H), 3.95 (m,
1H), 4.04 (m, 1H), 4.28 (m, 1H), 4.34 (m, 1H), 4.46 (m, 1H),
5.62 (d, J ) 16 Hz, 1H), 6.66 (dd, J ) 16, 6 Hz, 1H), 6.75 (s,
1H), 7.03 (d, 1H), 7.17 (m, 5H), 6.71 (d, 1H), 7.89 (d, 1H), 8.05
(d, 1H). HRMS: calcd for C42H67N6O11, 831.4868; found (FAB,
glycerol), 831.4885.
Cbz-V-L-F -vQ-OMe (3). This compound was synthesized
by removing the Boc protecting group of 1 (viz. 4 above)
followed by introduction of the Cbz protecting group (viz. 12
above). The product, mp 228-242 °C dec, appeared as a single
peak upon HPLC (MeCN/H2O/TFA, 35:65:0.1) tR ) 20.3 min;
(MeOH/H2O/TFA, 60:40:0.1) tR ) 11.5 min. 1H NMR (400
MHz, DMSO-d6): δ 0.74-0.87 (m, 12H), 1.36 (m, 2H), 1.54
(m, 1H), 1.65 (m, 1H), 1.70 (m, 1H), 2.04 (t, 2H), 2.90 (dq, 2H),
3.64 (s, 2H), 3.82 (m, 1H), 4.30 (m, 2H), 4.65 (m, 1H), 5.02 (s,
2H), 5.62 (d, J ) 15 Hz, 1H), 6.67 (dd, J ) 15, 6 Hz, 1H), 6.75
(br s, 1H), 7.20 (m, 5H), 7.33 (m, 5H), 7.93 (d, 1H), 7.98 (d,
1H), 8.05 (d, 1H). HRMS: calcd for C36H49N5O8Li, 686.3741;
found (FAB, NBA/LiOAc), 686.3740.
E-V-L-F -vQ-OMe‚TF A (6). This compound was prepared
by stirring Boc-E(tBu)-V-L-F-vQ-OMe (100 mg) with 50 µL of
TFA for 30 min at room temperature and evaporating to
dryness under vacuum. The resulting solid was used without
further purification. FABMS (glycerol): m/z 673 (M - H)-.
HRMS: calcd for C33H51N6O9, 675.3718; found (FAB, glycerol),
675.3705. 1H NMR (300 MHz, DMSO-d6): δ 0.82-0.95 (m,
12H), 1.35-1.80 (m, 8H), 2.05 (t, 1H), 2.30 (t, 1H), 2.88 (dq,
2H), 3.60 (m, 1H), 3.88 (m, 1H), 4.14 (m, 1H), 4.34 (m, 1H),
4.48 (m, 1H), 5.62 (d, J ) 16 Hz, 1H), 6.70 (dd, J ) 6, 16 Hz,
1H), 6.78 (br s, 1H), 7.20 (m, 5H), 7.95 (d, 1H), 8.07 (m, 4H),
8.45 (d, 1H).
Cbz-F -vQ-OMe (8). Boc-vQ-OMe (49 mg, 0.17 mmol) was
dissolved in 2 mL of CH2Cl2, and 2 mL of TFA was added.
After the mixture stirred for 1 h at room temperature, CH2-
Cl2 was added, and the mixture was concentrated to a viscous
oil. The latter was dissolved in DMF (3 mL) and triethylamine
(102 µL, 0.73 mmol), Cbz-F-OSu (68 mg, 0.17 mmol) was
added, and the mixture stirred for 2.5 h. Ethyl acetate was
added, and the mixture was extracted with 10% aqueous
NaHCO3, 1% citric acid, and brine and dried over MgSO4. After
concentration the residue was fractionated by chromatotron
(MeOH/CH2Cl2, 4:96) to give 34 mg (43%) of a white solid.
FDMS: 468. 1H NMR (300 MHz, CDCl3/MeOH-d4, 9:1): δ 1.52
(m, 1H), 2.00 (m, 2H), 2.80 (m, 2H), 3.58 (s, 3H), 4.15 (m, 1H),
4.30 (m, 1H), 4.84 (s, 2H), 5.42 (d, J ) 16 Hz, 1H), 6.46 (dd, J
) 16, 6 Hz, 1H), 7.20 (m, 5H), 7.62 (d, 1H). Anal. (C25H29N3O6),
C, H, N.
h at room temperature, the pH was brought to 3.0 using 1 M
H2SO4, whereupon a white precipitate formed. The solid was
collected by filtration, washed with ether, and dried under
vacuum. Yield: 0.42 g (85%). Mp: 171-175 °C. HPLC
(MeCN/H2O, 40:60) tR ) 4.6 min; (MeOH/H2O/TFA, 50:50:0.1)
tR ) 29.0 min. 1H NMR (400 MHz, DMSO-d6): δ 0.80 (m,
12H), 1.37 (s, 9H), 1.57 (m, 1H), 1.70 (m, 2H), 1.90 (m, 1H),
2.05 (t, 2H), 2.85 (dd, 1H, J ) 8.40, 13.6 Hz), 2.93 (dd, 1H, J
) 5.7, 13.6 Hz), 3.75 (m, 1H), 4.35 (m, 2H), 4.48 (m, 1H), 5.61
(d, J ) 15 Hz, 1H), 6.61 (dd, J ) 15, 6 Hz, 1H), 6.76 (br s, 1H),
7.19 (m, 5H), 7.84 (d, 1H), 8.00 (d, 1H), 8.08 (d, 1H). HRMS:
calcd for C32H50N5O8, 632.3659; found (FAB, glycerol), 632.3636.
ter t-Bu tyl N-[(E)-1-(3-Am in o-3-oxop r op yl)-3-(p h en yl-
su lfon yl)-2-p r op en yl]ca r ba m a te (23). A solution of diethyl
(phenylsulfonyl)methylphosphonate (254 mg, 0.87 mmol) in
THF was added to a suspension of NaH (35 mg, 0.87 mmol)
in THF. After the mixture stirred for 30 min at room
temperature, a THF solution of Boc-L-glutarimide (200 mg,
0.87 mmol) was added, and stirring was continued for 2.5 h.
A saturated solution of NH4Cl was added, and the mixture
was extracted with ethyl acetate. The extract was dried over
MgSO4 giving an oil which was fractionated by chromatotron
(EtOAc/MeOH, 90:10) to give 100 mg (31%) of a white solid.
1H NMR (300 MHz, CDCl3): δ 1.38 (s, 9H), 1.90 (m, 2H), 2.30
(m, 2H), 4.38 (m, 1H), 5.38 (d, J ) 8 Hz, 1H), 5.82 (br s, 1H),
6.12 (br s, 1H), 6.45 (d, J ) 16 Hz, 1H), 6.83 (dd, J ) 15, 5 Hz,
1H), 7.5-7.6 (m, 3H), 7.8-8.0 (m, 2H). Anal. (C17H24N2O5)
C, H, N.
Ben zyl N-((1S)-2-{[(1S,2E)-1-(3-Am in o-3-oxop r op yl)-3-
(p h en ylsu lfon yl)-2-p r op en yl]a m in o}-1-ben zyl-2-oxoeth -
yl)ca r ba m a te (15). Compound 23 (90 mg, 0.24 mmol) was
dissolved in 2 mL of CH2Cl2, and 2 mL of TFA was added.
After the mixture stirred for 1 h, CH2Cl2 was added, and the
mixture was concentrated to a viscous oil which was used
immediately. DMF (3 mL) and triethylamine (102 µL, 0.73
mmol) were added, followed by Cbz-F-OSu (97 mg, 0.24 mmol),
and the mixture was stirred for 2 h. Ethyl acetate was added,
and the mixture was extracted with 10% aqueous NaHCO3,
1% citric acid, and brine and dried over MgSO4. After
concentration the white solid residue was fractionated by
chromatotron (MeOH/CH2Cl2, 4:96) to give 28 mg (21%) of a
white solid. 1H NMR (300 MHz, CDCl3/MeOH-d4, 9:1): δ 1.58
(m, 1H), 2.03 (m, 2H), 2.81 (m, 2H), 4.15 (t, J ) 7 Hz, 1H),
4.42 (m, 1H), 4.91 (s, 2H), 5.82 (d, J ) 16 Hz, 1H), 6.60 (dd, J
) 16, 5 Hz, 1H), 7.0-7.2 (m, 5H), 7.4-7.8 (m, 5H). Anal.
(C25H29N3O6‚0.5H2O) C, H, N.
Boc-M-N(CH3)OCH3 (24). Boc-L-methionine (5.0 g, 20
mmol) was dissolved in THF (30 mL), 1,1-carbonyldiimidazole
(3.25 g, 20 mmol) was added, and the solution was stirred for
2 h at room temperature. N,O-Dimethylhydroxylamine hy-
drochloride (5.85 g, 60 mmol) was introduced, and the reaction
was stirred for 18 h at room temperature and concentrated.
The residue was dissolved in 250 mL of EtOAc and the latter
extracted with 50-mL portions of 1 M HCl, water, 1 M
NaHCO3, and brine, dried over MgSO4, and concentrated. The
residue was chromatographed over silica gel (EtOAc/hexanes,
1:1) to give 4.4 g (75%) of amide 24 as a white solid. TLC: Rf
) 0.6 (CH2Cl2/MeOH, 20:1). 1H NMR (300 MHz, CDCl3): δ
1.41 (s, 9H), 1.80 (m, 2H), 2.12 (s, 3H), 2.54 (m, 2H), 3.10 (s,
3H), 3.78 (s, 3H), 4.78 (m, 1H), 5.71 (br d, 1H). This material
was used without further purification.
Boc-F -vQ-OMe (10). This compound was prepared as
described for Boc-F-vQ-OEt but using Boc-vQ-OMe in place
of Boc-vQ-OEt. Yield: 50%. Mp: 74-77 °C. HPLC (MeCN/
H2O/TFA, 40:60:0.1) showed two peaks at tR ) 5.3 min (14%)
and 6.65 min (86%). 1H NMR (300 MHz, DMSO-d6): δ 1.30
(m, 9H), 1.70 (m, 1H), 1.77 (m, 1H), 2.10 (m, 2H), 2.75 (m,
1H), 2.92 (m, 1H), 3.65 (s, 1H), 4.15 (m, 1H), 4.40 (m, 1H),
5.70 (d, J ) 15 Hz, 1H), 7.75 (m, 2H), 7.91 (m, 1H), 7.20 (m,
6H), 8.07 (m, 1H). FABMS (NBA/LiOAc): m/z 440 (M + Li),
340 (M - Boc + Li). Anal. (C22H31N3O6) C, H, N.
Boc-m eth ion a l (25). Amide 24 (4.4 g, 15 mmol) was
dissolved in ether (100 mL) and cooled in an ice bath. LiAlH4
(0.72 g, 18.8 mmol) was added cautiously, and the mixture was
stirred and monitored by TLC. After 35 min the reaction was
quenched by cautious addition of 0.5 M KHSO4 solution (40
mL) while maintaining vigorous stirring. Ether (100 mL) was
added, and the aqueous phase was separated and extracted
with ether (5 × 50 mL). The ether phases were combined,
extracted with 3 M HCl (3 × 30 mL), saturated NaHCO3
solution (3 × 20 mL), and brine (3 × 20 mL), dried over MgSO4,
and concentrated to give Boc-methional as a translucent white
oil (3.1 g, 88%). TLC: Rf ) 0.37 (hexanes/EtOAc, 3:1). 1H
NMR (300 MHz, CDCl3): δ 1.42 (s, 9H), 2.05 (s, 3H), 2.08 (m,
Boc-V-L-F -vQ-OH (5). Boc-V-L-F-vQ-OMe (0.5 g, 0.77
mmol) was dissolved in THF/H2O (1:1, 4 mL), and 1.4 mL of 1
N NaOH solution was added. After the mixture stirred for 5

2586 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 14
Kong et al.
2H), 2.54 (m, 2H), 4.30 (m, 1H), 5.21 (m, 1H), 9.52 (s, 1H).
This material was used immediately for the next reaction.
MeOH/CH2Cl2, 5:95), plus an additional 110 mg of 9 contami-
nated with a small amount of sulfoxide. FDMS: 502. 1H NMR
(300 MHz, CDCl3/MeOH-d4, 9:1): δ 1.81 (m, 1H), 2.00 (m, 1H),
2.78 (s, 3H), 2.8-3.0 (m, 4H), 3.75 (s, 3H), 4.20 (m, 1H), 4.52
(m, 1H), 4.98 (s, 2H), 5,43 (d, J ) 16 Hz, 1H), 6.18 (br d, J )
8 Hz, 1 H), 6.52 (dd, J ) 5, 16 Hz, 1H), 7.0-7.3 (m, 10H), 7.65
(br d, J ) 8 Hz, 1H). Anal. (C25H30N2O7S) C, H, N.
Boc-vM-OMe (26). Trimethyl phosphonoacetate (5.40 mL,
33.4 mmol) was dissolved in THF (20 mL) and added to a
stirred suspension of NaH (1.11 g, 27.85 mmol) in THF (20
mL). After 30 min at room temperature a solution of Boc-
methional (2.60 g, 11.1 mmol) in THF (25 mL) was added, and
stirring continued for 1 h. Water (5 mL) was cautiously added
dropwise with stirring, the mixture concentrated, and the
residue partitioned between CHCl3 (200 mL) and water (20
mL). The CHCl3 layer was extracted with dilute HCl, NaHCO3
solution, and brine, dried over MgSO4, and concentrated to
give an off-white solid. Chromatography of the latter over
silica gel (EtOAc) gave Boc-vM-OMe (2.22 g, 70%). 1H NMR
(300 MHz, CDCl3): δ 1.41 (s, 9H), 2.05 (s, 3H), 2.08 (m, 2H),
2.47 (m, 2H), 3.58 (s, 3H), 4.38 (m, 1H), 4.61 (br d, 1H), 5.88
(d, J ) 15 Hz, 1H), 6.68 (dd, J ) 6, 15 Hz, 1H).
Boc-vM(O2)-OMe (27). Compound 26 (1.5 g, 5.35 mmol)
was dissolved in MeOH (15 mL) and water (5 mL) and cooled
in an ice bath, and a cold solution of Oxone (9.21 g, 14.98
mmol) in water (10 mL) was added. The solution was allowed
to come to room temperature and stirred for 3 h while
monitoring by TLC (EtOAc). Chloroform (25 mL) was added
and the aqueous phase removed. The chloroform was dried
over MgSO4 and concentrated to yield a transparent oil (1.72
g). Chromatography of the latter over silica gel (EtOAc/
hexanes, 10:1) afforded Boc-vM-OMe (0.61 g, 32%) as a white
solid. Mp: 125-131 °C dec. [R]D 3.2 (c ) 1.1, MeOH). 1H
NMR (400 MHz, CDCl3): δ 1.43 (s, 9H), 2.05 (m, 1H), 2.20
(m, 1H), 2.94 (s, 3H), 3.08 (s, 3H), 3.75 (s, 3H), 4.43 (m, 1H),
4.67 (br d, 1H), 5.97 (d, J ) 5 Hz, 1H), 6.84 (dd, J ) 5, 15 Hz,
1H). FABMS (NBA/LiOAc): m/z 328 (M + Li).
Boc-V-L-F -vM(O2)-OMe (7). Boc-vM(O2)-OMe was depro-
tected and coupled to Boc-V-L-F-OH as described for compound
1 above. The product, a white solid (mp 180 °C), was evidently
a mixture of two diastereomers whose preparative separation
was not attempted. HPLC (MeCN/H2O/TFA, 40:60:0.1) showed
two peaks in a 3:5 ratio, tR ) 24.9 and 26.3 min, respectively.
The 1H NMR spectrum (400 MHz, DMSO-d6) also indicated
the presence of two diastereomers in a 3:5 ratio. Major
isomer: δ 2.94 (s), 3.64 (s), 3.75 (m), 5.62 (d, J ) 6 Hz), 6.78
(dd, J ) 6, 16 Hz). Minor isomer: δ 2.90 (s), 3.62 (s), 4.00
(m), 5.87 (d, J ) 6 Hz), 6.67 (dd, J ) 6, 16 Hz). In addition
the following common resonances were observed: (δ) 0.75-
0.85 (m, 12H), 1.06-2.00 (m, 8H), 2.80-3.10 (m, 3-4H), 4.25-
4.55 (m, 3H), 5.05 (d, 1H), 7.15-7.28 (m, 5H), 7.72 (t, 1H),
8.08-8.20 (m, 2H). HRMS: calcd for C33H52N4O9SLi, 687.345;
found (FABMS, NBA/LiOAc), 687.3638.
Cbz-F -vM-OMe (28). Compound 26 (432 mg, 1.5 mmol)
was dissolved in 3 mL of CH2Cl2 and cooled to 0 °C. TFA (3
mL) was added, the ice bath was removed, and the reaction
was stirred for 3 h and concentrated. The residue was
dissolved in THF (8 mL) and cooled to 0 °C. HOBt (201 mg,
1.5 mmol), EDC (286 mg, 1.5 mmol), Cbz-F-OH (447 mg, 1.5
mmol), and NMM (0.5 mL, 4.5 mmol) were added, the ice bath
was removed, and the mixture was stirred for 18 h at room
temperature. EtOAc (200 mL) was added, and the solution
was extracted with dilute HCl, NaHCO3 solution, and brine
and dried over MgSO4. After evaporation the residue was
fractionated by chromatotron (EtOAc) and concentrated to give
compound 28 as a white solid (360 mg, 51%). 1H NMR (300
MHz, CDCl3): δ 1.63-1.88 (m, 2H), 2.03 (s, 3H), 2.40 (t, J )
5 Hz, 2H), 3.01 (dd, J ) 5, 7 Hz, 1H), 5.11 (q, J ) 8 Hz, 2H),
5.26 (m, 1H), 5.71 (d, J ) 16 Hz, 1H), 5.95 (br d, J ) 8 Hz,
1H), 6.65 (dd, J ) 5, 16 Hz, 1H), 7.2-7.4 (m, 10 H). Anal.
(C25H30N2O5S) C, H, N.
En zym e P u r ifica tion a n d Assa y. Recombinant HRV-14
3Cpro was expressed and purified as described previously.23 The
chromogenic substrate EALFQ-pNA was used to measure its
activity.28 A typical assay (200 µL) contained 50 mM Hepes,
pH 7.5, 150 mM NaCl, 1 mM EDTA, 250 µM substrate
(EALFQ-pNA), and 3Cpro (ca. 0.4 µM). Assay substrate and
inhibitors were added as DMSO solutions, keeping the total
DMSO concentration in each incubation constant at 10% (v/
v). Control assays were identical but with pure DMSO added
in place of inhibitor solution. Assays were run in duplicate
at 30 °C; A405 readings were taken every 2 min for 20 min,
and reaction rates were calculated from ∆A/∆t using ∆ꢀ )
10 300 M-1 cm-1. All reactions were performed in microtiter
plate wells and monitored using a temperature-controlled
microplate reader (Molecular Devices). Rates were propor-
tional to substrate concentration over the range 0-250 µM (the
upper limit of solubility), and the apparent second-order
28
catalytic constant (kcat/Km) was 840 ( 11 M-1 s-1
.
For initial evaluation, each inhibitor was tested at concen-
trations of 1, 5, 25, and 100 µg/mL. Substrate solution, buffer,
and inhibitor solution were combined, and reactions were
initiated by adding an aliquot of 3Cpro enzyme solution. A405
readings were recorded every 2 min to generate progress
curves for each reaction. The percent inhibition was deter-
mined after 20 min by comparing A405 for the test case to that
of the uninhibited control reaction. For the more active
compounds IC50 values were determined using a total of eight
concentrations of inhibitor (controls and assay conditions as
described above). A405 readings were taken after 20 min of
reaction, and IC50 values were determined from plots of percent
inhibition vs inhibitor concentration.
Active Site Titr ation of HRV 3C P r otease with Mich ael
Accep t or 2. To each of seven polystyrene microcuvettes
(1-mL capacity, 1-cm path length) was added, in order, assay
buffer (525 µL), enzyme solution (32.5 µL, 0.52 mg of protein/
mL), DMSO (13, 18, 20, 23, 27, 30, or 33 µL), and a solution of
Michael acceptor 2 in DMSO (30 µM; 20, 15, 13, 10, 6, 3, or 0
µL, respectively). After the last addition each cuvette was
inverted several times for mixing and allowed to stand at room
temperature for 60 min. The amount of enzyme activity was
then determined by adding a solution of assay substrate
(EALFQ-pNA, 5.11 mM in DMSO, 32.5 µL) to each cuvette
and recording ∆A/∆t as described above.
P r otein Ma ss Sp ectr om etr y. Purified HRV-14 3Cpro (0.8
nmol) was incubated with a given Michael acceptor (10 nmol)
in a volume of 0.4 mL for 60 min at room temperature, with
other conditions as described for enzyme assays. Samples
were then subjected to electrospray ionization mass spectrom-
etry analysis using a PESciex API III triple stage quadrupole
mass spectrometer. Sample was continuously infused into the
interface at a rate of 10-20 µL/min. The instrument was
operated in the positive ion detection mode with an ion spray
voltage of 3500 V and an inlet orifice potential of 55 V. Mass
spectra were collected over a mass range of 300-2000 U at
0.1 U intervals with a dwell time of 1 ms/interval. The final
spectrum was obtained by averaging a total of 5-10 scans.
P la qu e Red u ction Assa y for HRV-14. Confluent mono-
layers of HeLa cells were infected using 100 plaque-forming
units/plate. Controls consisted of mock-infected plates. After
an adsoprtion period of 30 min the innocula were replaced by
1.5 mL of a maintenance medium overlay containing 0.5%
agarose and supplemented with various concentrations of test
compound. The plates were incubated at 35 °C for 48 h, and
the infected monolayers were then fixed with buffered 10%
formalin and stained with crystal violet after removal of the
overlay. The mean plaque number was calculated from a
duplicate series of counts, converted to a percentage of
Cbz-F -vM(O2)-OMe (9). Compound 28 (190 mg, 0.4 mmol)
was dissolved in MeOH (3 mL) and cooled to 0 °C. A solution
of Oxone (339 mg, 1.2 mmol) in H2O (3 mL) was added, and
the mixture was stirred vigorously for 3 h at room tempera-
ture. The mixture was extracted with CHCl3 (2 × 100 mL)
and the latter dried over MgSO4 and concentrated giving a
white solid which was fractionated by chromatotron (MeOH/
CH2Cl2, 4:96) yielding 45 mg (22%) of pure 9 (TLC Rf ) 0.45;

Synthesis, Evaluation of Peptidyl Michael Acceptors
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untreated controls, and plotted against the log concentration
of test compound to determine the 50% inhibitory concentra-
tion (IC50). Each experiment was repeated two or three times
with results agreeing within (25%. For each compound the
value reported in Table 1 is the highest of the interpolated
IC50 values, which thus gives the most conservative estimate
of antiviral potency. As a control on consistency of the plaque
reduction assay, the antirhinoviral compound enviroxime31 was
routinely included among the other test compounds assayed.
In the three series of assays which affoded the data in Table
1, enviroxime showed IC50 concentrations of 0.045, 0.054, and
0.047 µg/mL.
Cyt ot oxicit y Assa y. HeLa cells were cultured without
virus infection but with the test compounds as described above,
and cell viability was determined by the metabolism of the
tetrazolium dye XTT.32 In this assay metabolically active cells
transform XTT to a soluble, colored formazan product which
is measured spectrophotometrically. Microtiter plates (96-
well) were seeded with 104 cells/well in medium 199 containing
Earl’s balanced salt solution, 1% fetal bovine serum, penicillin
(100 units/mL), streptomycin (100 µg/mL), and varying con-
centrations of test compound. Plates were incubated under
5% CO2 at 37 °C for 3 days. Each well received 0.05 mL of
XTT solution (1 mg/mL in phosphate-buffered saline contain-
ing 0.025 mM phenazine methosulfate). Plates were then
incubated for an additional 4 h at 37 °C, and A450 was
measured. The absorption was proportional to the number of
viable cells in the well. The concentration of compound which
produced a 50% reduction in the number of viable cells was
designated the TC50 concentration.
Ack n ow led gm en t. We thank Dr. Elcira Villareal
for antiviral activity testing, Mark Wakulchik for
performing cytotoxicity testing, J ohn Richardson for
mass spectral analyses, and Drs. Carlos Lopez, Steven
Kaldor, and Louis J ungheim for their advice and inter-
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