Structure-based design, synthesis, and biological evaluation of irreversible human rhinovirus 3C protease inhibitors. 4. Incorporation of P1 lactam moieties as L-glutamine replacements

Article abstract of DOI:10.1021/jm9805384

The structure-based design, chemical synthesis, and biological evaluation of various human rhinovirus (HRV) 3C protease (3CP) inhibitors which incorporate P₁ lactam moieties in lieu of an L-glutamine residue are described. These compounds are comprised of a tripeptidyl or peptidomimetic binding determinant and an ethyl propenoate Michael acceptor moiety which forms an irreversible covalent adduct with the active site cysteine residue of the 3C enzyme. The P₁-lactam-containing inhibitors display significantly increased 3CP inhibition activity along with improved antirhinoviral properties relative to corresponding L-glutamine-derived molecules. In addition, several lactam-containing compounds exhibit excellent selectivity for HRV 3CP over several other serine and cysteine proteases and are not appreciably degraded by a variety of biological agents. One of the most potent inhibitors (AG7088, mean antirhinoviral EC₉₀ ? 0.10 μM, n = 46 serotypes) is shown to warrant additional preclinical development to explore its potential for use as an antirhinoviral agent.

Full text of DOI:10.1021/jm9805384

J . Med. Chem. 1999, 42, 1213-1224
1213
Str u ctu r e-Ba sed Design , Syn th esis, a n d Biologica l Eva lu a tion of Ir r ever sible
Hu m a n Rh in ovir u s 3C P r otea se In h ibitor s. 4. In cor p or a tion of P 1 La cta m
Moieties a s L-Glu ta m in e Rep la cem en ts
Peter S. Dragovich,* Thomas J . Prins, Ru Zhou, Stephen E. Webber, J oseph T. Marakovits, Shella A. Fuhrman,
Amy K. Patick, David A. Matthews, Caroline A. Lee, Clifford E. Ford, Benjamin J . Burke, Paul A. Rejto,
Thomas F. Hendrickson, Tove Tuntland, Edward L. Brown, J ames W. Meador III, Rose Ann Ferre,
J ames E. V. Harr, Maha B. Kosa, and Stephen T. Worland
Agouron Pharmaceuticals, Inc., 3565 General Atomics Court, San Diego, California 92121
Received September 25, 1998
The structure-based design, chemical synthesis, and biological evaluation of various human
rhinovirus (HRV) 3C protease (3CP) inhibitors which incorporate P1 lactam moieties in lieu of
an L-glutamine residue are described. These compounds are comprised of a tripeptidyl or
peptidomimetic binding determinant and an ethyl propenoate Michael acceptor moiety which
forms an irreversible covalent adduct with the active site cysteine residue of the 3C enzyme.
The P1-lactam-containing inhibitors display significantly increased 3CP inhibition activity along
with improved antirhinoviral properties relative to corresponding L-glutamine-derived mol-
ecules. In addition, several lactam-containing compounds exhibit excellent selectivity for HRV
3CP over several other serine and cysteine proteases and are not appreciably degraded by a
variety of biological agents. One of the most potent inhibitors (AG7088, mean antirhinoviral
EC90 ≈ 0.10 µM, n ) 46 serotypes) is shown to warrant additional preclinical development to
explore its potential for use as an antirhinoviral agent.
In tr od u ction
The human rhinoviruses (HRVs) are members of the
picornavirus family and are the single most significant
cause of the common cold.1,2 We recently described the
design and development of substrate-derived3,4 tripep-
tidyl HRV 3C protease (3CP) inhibitors which incorpo-
rate C-terminal Michael acceptor moieties (e.g., com-
pound 1, Figure 1, Table 1).5-7 These compounds
irreversibly inhibit 3CP by forming a covalent adduct
with the active site cysteine residue of the enzyme and
exhibit antirhinoviral activity in cell culture. In addi-
tion, the preceding paper describes the modification of
such molecules by the introduction of a P2-P3 keto-
methylene dipeptide isostere into the inhibitor design.8
The resulting peptidomimetic compounds display sub-
stantially improved in vitro antiviral properties relative
to the corresponding peptide-derived molecules. In an
effort to further modify the structure of the initially
studied inhibitor series, we sought to alter amide
moieties other than that linking the P2-P3 amino acid
residues of 1 without significantly interfering with 3CP
recognition. The results of our efforts to derivatize the
primary amide present in the P1 Gln9 side chain of 1
are described below.
F igu r e 1. Design of HRV 3CP inhibitors.
Ta ble 1
compd
no.
sero-
kobs/[I]
EC50 CC50
c
R1
R2
formulaa
typeb (M-1 s-1
)
(µM)c (µM)c
1
H
H
C32H42N4O7
25 000
0.54 >320
1.6
2
2 000
750
60
2
3
CH3
H
C33H44N4O7
5.6
4.0
>100
>100
In h ibitor Design a n d Str u ctu r e-Activity
Stu d ies
CH3 CH3 C34H46N4O7
a
Elemental analyses (C, H, N) of all compounds agreed to
Analysis of the HRV-2 3CP-1 X-ray crystal structure5
indicated that only the trans P1 Gln amide hydrogen
atom interacted with the protease while the cis NH was
solvent-exposed (Figure 2). Thus, selective alkylation of
the latter was envisioned to allow for amide modification
without adversely affecting 3CP inhibition properties.
However, a molecule containing a monomethylated P1
b
within (0.4% of theoretical values. Serotype-14 unless otherwise
noted. c See ref 5 for assay method and error.
glutamine moiety (2)6 displayed drastically reduced 3CP
inhibition activity relative to the nonalkylated inhibitor
1 (Figure 1, Table 1).10 A comparison of 1, 2, and the
dimethylated analogue 36 (Figure 1, Table 1) suggested
10.1021/jm9805384 CCC: $18.00 © 1999 American Chemical Society
Published on Web 03/19/1999

1214 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 7
Dragovich et al.
Accordingly, the P1 lactam moiety was incorporated
into tripeptidyl molecules containing a P3 Val amino
acid residue and an N-terminal amide derived from
5-methylisoxazole-3-carboxylic acid (9 and 10, Table
3).15 The resulting compounds rapidly and irreversibly
inhibited HRV-14 3CP and displayed very potent anti-
viral properties in cell culture. Inclusion of a Val-Phe
ketomethylene dipeptide isostere8 in the inhibitor design
resulted in slightly diminished anti-3CP activity but
further improved antirhinoviral properties in cell cul-
ture (compare 9 with 11 and 10 with 12, Table 3).
Similar activity differences between ketomethylene- and
peptide-derived 3CP inhibitors were previously observed
during the examination of non-P1-lactam-containing
molecules.8 In addition to the compounds described
above, a tripeptide-derived inhibitor which contained a
P3 tBuGly residue also exhibited impressive anti-3CP
and antiviral properties (compound 13, Table 3).
F igu r e 2. Schematic diagram of 1 bound in the HRV-2 3CP
active site.5 Hydrogen bonds are represented as dashed lines,
and the residues which make up the enzyme binding subsites
are depicted.
Two of the most active antiviral agents were also
examined against multiple rhinovirus serotypes in cell
culture (Table 4). The tripeptide-derived inhibitor 9
exhibited potent EC90 antiviral activity against all
serotypes tested.16 As anticipated from previous stud-
ies,8 the ketomethylene-containing molecule 11 dis-
played more potent EC90 antiviral properties than the
peptide 9. This improvement, however, was not nearly
as dramatic as that observed when examining P1
glutamine-derived peptidyl and peptidomimetic 3CP
inhibitors.8 A comparison of the lactam-containing
molecule 11 with its glutamine analogue 148 indicated
that incorporation of the P1 lactam moiety into the
inhibitor design improved EC90 antiviral activity by a
factor of between 8 and 16 (depending on HRV serotype).
Similar improvements in antiviral activity were noted
when comparing other lactam- and glutamine-contain-
ing molecules (data not shown). Compounds 9 and 11
rank among the most potent known antirhinoviral
agents, and they compare favorably with the capsid-
binding molecules Pirodavir17 and Pleconaril18 (Table
4).
that the poor anti-3CP activity exhibited by 2 was
primarily attributable to an unfavorable equilibrium in
which the trans methyl amide isomer predominated
over the desired cis isomer (Figure 3).11 We therefore
sought to enforce the cis amide geometry by the incor-
poration of a P1 lactam moiety into the inhibitor design.
Modeling studies (see below) utilizing the HRV-2 3CP-1
crystal structure5 suggested that such a lactam moiety
could be accommodated in the 3CP S1 binding pocket
and predicted the (S)-stereochemistry at the lactam
R-carbon to be the most favorable for effective 3CP
recognition (Figure 3).
In the event, the (S)-γ-lactam-containing compound
4 displayed dramatically improved inhibitory activity
against 3CPs from several rhinovirus serotypes relative
to the corresponding glutamine-derived molecule 1
(Tables 1 and 2). Equally important, inhibitor 4 also
exhibited improved antiviral properties relative to 1 and
was not cytotoxic in cell culture to the limits of its
solubility.13 The corresponding (S)-δ-lactam-containing
inhibitor 5 also displayed significantly improved anti-
3CP and antiviral activity relative to 1, while a molecule
incorporating an (R)-γ-lactam (6) exhibited reduced 3CP
inhibition properties in accordance with computational
predictions (see below, Table 2). A similar reduction in
anti-3CP activity was obtained by incorporation of a
cyclic urea moiety into the inhibitor design (7). The poor
inhibition properties of 7 relative to 4 paralleled reduc-
tions in anti-3CP activity observed previously with
inhibitors containing acyclic P1 urea functionalities.6
The promising antirhinoviral activity exhibited by the
(S)-γ- and (S)-δ-lactam-containing Cbz-Leu-Phe-derived
compounds prompted the incorporation of these lactam
entities into other 3CP inhibitors. Thus, substitution of
a P3 Val amino acid residue in place of Leu (8, Table 2)
significantly improved anti-3CP activity in direct anal-
ogy to structure-activity relationships observed for
related glutamine-containing 3CP inhibitors.6 This re-
sult indicated that the P1 lactam-containing compounds
bound to 3CP in a manner similar to that observed for
the corresponding glutamine-derived molecules and
suggested that other beneficial modifications of the
latter series might improve the former as well.14
Due to the encouraging antiviral activity exhibited by
the compounds depicted in Table 3, several of their other
biological properties were examined in anticipation of
possible preclinical development efforts. One such prop-
erty was the ability of the molecules to selectively inhibit
HRV 3CP without affecting other proteolytic enzymes.
None of the Michael acceptor-containing compounds
examined in this study appreciably inhibited a variety
of serine proteases when incubated at relatively high
concentrations for 10-15 min (Table 5). However,
measurable inhibition of the cysteine protease cathepsin
B by the tripeptidyl molecule 1 was observed under the
above assay conditions. In contrast, the more potent
antirhinoviral agents described in this study (9 and 11)
did not affect any of the nonrhinoviral cysteine proteases
examined, suggesting that optimization of their 3CP
inhibition properties also led to improvements in en-
zyme selectivity.
In addition to the selectivity studies described above,
the stability of many 3CP inhibitors toward several
biologically relevant agents was also examined. As was
the case with previously examined irreversible 3CP
inhibitors,5 the ethyl propenoate Michael acceptor was
employed extensively in the current work due to its ease

P1 Lactam Moieties as L-Glutamine Replacements
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 7 1215
F igu r e 3. Design of P1-lactam-containing HRV 3CP inhibitors.
Ta ble 2
compd
no.
kobs/[I]
EC50
CC50
c
R
X
n
formulaa
serotypeb
2
(M-1 s-1
)
(µM)c
(µM)c
4
CH2CH(CH3)2
(S)-CH
1
C34H44N4O7‚0.50H2O
257 000
31 000
239 000
18 000
10 900
500 000
0.10
ND
0.03
1.6
0.60
0.03
>100
5
6
7
8
CH2CH(CH3)2
CH2CH(CH3)2
CH2CH(CH3)2
CH(CH3)2
(S)-CH
(R)-CH
N
2
1
1
1
C35H46N4O7‚0.50H2O
C34H44N4O7
C33H43N5O7
>100
>100
>100
>100
(S)-CH
C33H42N4O7
a
b
Elemental analyses (C, H, N) of all compounds agreed to within (0.4% of theoretical values. Serotype-14 unless otherwise noted.
c See ref 5 for assay method and error. ND ) not determined.
Ta ble 3
compd
no.
kobs/[I]
EC50
CC50
b,c
R
X
n
formulaa
(M-1 s-1
)
(µM)b,c
(µM)c
9
10
11
12
13
CH(CH3)2
CH(CH3)2
CH(CH3)2
CH(CH3)2
C(CH3)3
NH
NH
CH2
CH2
NH
1
2
1
2
1
C30H38FN5O7
C31H40FN5O7
C31H39FN4O7
C32H41FN4O7
C31H40FN5O7
1 500 000
900 000
1 090 000
500 000
980 000
0.01
0.02
0.005
0.001
0.01
>100
>100
>100
>100
>100
a
b
Elemental analyses (C, H, N) of all compounds agreed to within (0.4% of theoretical values. Serotype-14. c See ref 5 for assay
method and error.
of preparation. However, a concern existed that this
moiety might undergo facile in vivo metabolism, thereby
compromising the usefulness of molecules in which it
was incorporated.19 As expected based on previous
studies,5 inhibitor 11 was rapidly degraded in rat
plasma, presumably due to hydrolysis of the R,â-
unsaturated ethyl ester present in the molecule (Table
6).20 However, the compound was quite stable toward
human and dog plasmas and exhibited negligible deg-
radation in the presence of hydrolytic enzymes such as
chymotrypsin and peptidase. In addition, the molecule
was unaffected by prolonged exposure to high concen-
trations of dithiothreitol (5 mM), suggesting that the
inhibitor did not react readily with nonenzymatic thiols.
Similar stability toward the various biological agents
listed (and instability toward rat plasma) was noted for
many other 3CP inhibitors related to the molecules
described in this study (data not shown). Although the
studies described above do not address every aspect of
in vivo metabolism, they do examine some of the more
obvious degradation events that might affect the com-
pounds detailed in this work.
The extremely potent antirhinoviral activity displayed
by compound 11, along with its excellent protease
selectivity, low cytotoxicity in cell culture, and stability
toward various biological agents prompted its selection

1216 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 7
Dragovich et al.
Ta ble 4. Antirhinoviral Activity of Selected 3CP Inhibitors against Several HRV Serotypes
a
b
See ref 5 for assay method and error. Provided by J anssen Pharmaceuticals. c Prepared as described in ref 18.
Ta ble 5. Inhibition of Serine and Cysteine Proteases by
Ta ble 7. Computational Studies of P1-Lactam-Containing HRV
Selected Antirhinoviral Agents
3CP Inhibitors
% activity remaining after 10-15 mina
predicted predicted
measured
affinitya
affinity
affinity
compd
chymo-
cathepsin
B
compd
no.
kobs/[I]
relative
rate
(no. 1)a,b
(no. 2)a,c
no.b thrombin trypsin trypsin elastase
calpain
(M-1 s-1
)
(kcal/mol) (kcal/mol) (kcal/mol)
1c
9
11
13
101
103
100
101
100
96
101
100
97
98
100
100
95
92
100
99
39
96
98
100
100
100
100
100
1
4
5
6
25 000
257 000
239 000
18 000
1
10
9.6
0.72
0.0
-1.4
-1.3
0.0
-2.0
-1.7
0.7
0.0
-1.4
-1.0
5.9
96
81
0.20
DMSO
100
100
100
100
a
Relative values; assumes changes in kobs/[I] values are repre-
a
Each enzyme was present in the appropriate assay at ∼10 nM.
Tested at 10 µM unless otherwise noted. c Tested at 2 µM.
b
sentative of changes in ligand-3CP affinity. Calculated using the
b
covalent protein-ligand complex. c Calculated using the nonco-
valent protein-ligand encounter complex. See the Experimental
Section for additional details.
Ta ble 6. Stability of Compound 11 toward Various Biological
Agents
biological agenta
half-life (min)
The 3CP affinity of compound 1 was calculated in both
analyses to serve as a reference, and the latter calcula-
tions also included an entropic correction to account for
the expected differences in flexibility between lactam-
and glutamine-containing molecules.21 As seen in Table
7, both methods qualitatively predicted the improved
anti-3CP activity of (S)-lactam containing molecules 4
and 5 relative to glutamine-derived inhibitors such as
1. Both methods also qualitatively anticipated the
reduced 3CP affinity exhibited by the (R)-lactam con-
taining compound 6. Additional details of these molec-
ular modeling studies are included in the Experimental
Section.
rat plasma
dog plasma
human plasma
chymotrypsin
peptidase
<2
>60
>60
>60
>60
>30
dithiothreitol (5 mM)
a
See the Experimental Section for assay conditions and details.
as a candidate for further development. This molecule
(AG7088) is currently undergoing testing to evaluate its
possible clinical use as an antirhinoviral agent, and
additional data concerning these studies will be dis-
closed in due course.
Syn th esis
Molecu la r Mod elin g Stu d ies
The lactam-containing compounds utilized in this
study were prepared by a variety of methods which
incorporate several elements from previous syntheses
of related 3CP inhibitors. The preparation of a tripep-
tidyl (S)-γ-lactam-containing inhibitor is illustrated in
Scheme 1 by the synthesis of compound 4. The known
methyl ester 1522 (derived from L-glutamic acid in four
steps) was hydrolyzed under basic conditions to provide
the corresponding carboxylic acid (16) in quantitative
yield. This material was not purified, but was instead
coupled with (S)-(-)-4-benzyl-2-oxazolidinone to give
Two related computational approaches were utilized
to estimate the binding affinities of various lactam-
containing molecules to HRV 3CP. The results of both
methods are depicted in Table 7 and are compared with
experimental values. Each analysis employed lactam
model compounds constructed from the previously de-
scribed HRV-2 3CP-1 complex.5 In the first approach,
the expected covalent protein-inhibitor complex was
examined, while in the second the noncovalent encoun-
ter complex between enzyme and ligand was studied.

P1 Lactam Moieties as L-Glutamine Replacements
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 7 1217
Sch em e 1a
a
DMB ) 2,4-dimethoxybenzyl. Reagents and conditions: (a) 3.0 equiv of NaOH, CH3OH, 23 °C, 3.5 h, 100%; (b) 3.0 equiv of Et3N, 1.0
equiv of t-BuCOCl, 1.1 equiv of LiCl, 0.95 equiv of (S)-(-)-4-benzyl-2-oxazolidinone, THF, 0f23 °C, 19 h, 83%; (c) 1.0 equiv of NaN(TMS)2,
3.0 equiv of allyl iodide, THF, -78f-45 °C, 2 h, 55%; (d) O3, 2.0 equiv of CH3OH, CH2Cl2, -78 °C, then 10 equiv of Me2S, 0 °C, 1 h; (e)
4.0 equiv of HCl‚H2N-DMB, 4.0 equiv of NaOAc, 2.0 equiv of NaBH3CN, 2:1 THF:EtOH, 23 °C, 18 h; (f) 0.20 equiv of TsOH‚H2O, CH3OH,
50 °C, 2.5 h, 44% from 18; (g) 3.0 equiv of DMSO, 1.5 equiv of oxalyl chloride, 6.0 equiv of Et3N, CH2Cl2, -78 °C, 1.5 h, 100%; (h) 1.0 equiv
of NaN(TMS)2, 1.0 equiv of (EtO)2P(O)CH2CO2Et, THF, -78f0 °C, 45 min, 61%; (i) HCl, 1,4-dioxane, 23 °C, 1.5 h; (j) 1.3 equiv of Cbz-
L-Leu-L-Phe-OH, 4.0 equiv of NMM, 1.7 equiv of HOBt, 1.7 equiv of EDC, CH2Cl2, 23 °C, 20 h, 70%; (k) 4.9 equiv of DDQ, 10:1 CHCl3:
H2O, 50 °C, 8 h, 55%.
acyl oxazolidinone 17 in good yield.23 Low-temperature
alkylation of the sodium enolate derived from 17 with
allyl iodide then provided the allyl derivative 18 after
purification on silica gel.24,25 This material was con-
verted to lactam 19 by ozonolysis and subsequent
reductive amination of the resulting aldehyde (not
shown) with 2,4-dimethoxybenzylamine. Lactam 19 was
difficult to separate completely from contaminating (S)-
(-)-4-benzyl-2-oxazolidinone, and the mixture of the two
was typically hydrolyzed under mildly acidic conditions
to provide the more easily purified amino alcohol 20.
The conversion of 20 into inhibitor 4 then paralleled
several previous syntheses of tripeptide-derived Michael
acceptors quite closely.5,6 Thus, Swern oxidation26 of 20
followed by olefination of the resulting aldehyde (21)
provided the trans R,â-unsaturated ethyl ester 22 in
moderate yield. As was observed previously,5 only minor
amounts (<5%) of the corresponding cis olefin isomer
were formed during the conversion of 21 to 22. Acidic
removal (HCl) of the Boc protecting group from 22 and
carbodiimide-mediated coupling of the resulting amine
hydrochloride salt with commercially available Cbz-L-
Leu-L-Phe-OH afforded the tripeptidyl molecule 23 in
good overall yield. The 2,4-dimethoxybenzyl moi-
ety was removed from 23 by treatment with 2,3-
dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) at ele-
vated temperature to give γ-lactam-containing inhibitor
4 in moderate yield.27 Alternatively, this deprotection
could be accomplished by exposure of 23 to ceric am-
monium nitrate (CAN).28 In both cases, flash column
chromatography was necessary to remove impurities
generated during the deprotection reaction from the
desired inhibitor. The (R)-γ-lactam-containing com-
pound 6 was prepared in a manner that was completely
analogous to the synthesis of 4 described above, with
the exception that (R)-(+)-4-benzyl-2-oxazolidinone was
employed for coupling with carboxylic acid 16 (data not
shown).29
The preparation of an intermediate required for the
synthesis of inhibitors incorporating a P1 (S)-δ-lactam
moiety is illustrated in Scheme 2. Hydroboration of the
allyl derivative 18 described in Scheme 1 afforded
alcohol 24 in good yield after oxidative workup and
chromatographic purification. This material was treated
with SO3‚pyridine, and the resulting aldehyde (not
shown) was condensed with 2,4-dimethoxybenzylamine
utilizing the reductive amination conditions described
above for the preparation of compound 19 to give
intermediate 25. As before, 25 was difficult to separate
from the (S)-(-)-4-benzyl-2-oxazolidinone produced dur-
ing lactam formation. Impure 25 was therefore hydro-
lyzed under acidic conditions to give alcohol 26 which
could be satisfactorily purified by flash column chro-
matography. Swern oxidation26 of 26 and subsequent
olefination of the resulting aldehyde (27) then afforded
R,â-unsaturated ester 28. This intermediate was con-
verted into inhibitors 5, 10, and 12 by methods that
were completely analogous to other syntheses reported
in this work (cf., Schemes 1, 4, and 5).
The preparation of the cyclic-urea-containing inhibitor
7 is depicted in Scheme 3. Attempts to synthesize the
P1 intermediate corresponding to 22 and 28 (Schemes
1 and 2) were unsuccessful in the case of the cyclic urea,
and an alternate approach to the target compound was
therefore utilized. Accordingly, olefination of the known
tripeptide 2930 uneventfully provided the R,â-unsatu-
rated ester 30. Removal of the Boc protecting group from
30 and coupling of the resulting amine salt with N-Boc-

1218 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 7
Dragovich et al.
Sch em e 2a
Sch em e 4a
a
DMB ) 2,4-dimethoxybenzyl. Reagents and conditions: (a) 1.0
equiv of BH3‚THF, THF,
0 °C, 30 min, then 1.0 equiv of
a
DMB ) 2,4-dimethoxybenzyl. Reagents and conditions: (a)
HCl, 1,4-dioxane, 23 °C, 1.5 h; (b) 1.2 equiv of Boc-L-(4-F-Phe)-
OH, 3.0 equiv of (i-Pr)2NEt, 1.1 equiv of HATU, DMF, 0f23 °C,
1.5 h, 77%; (c) HCl, 1,4-dioxane, 23 °C, 1.5 h; (d) 1.2 equiv of Boc-
L-Val-OH, 4.0 equiv of NMM, 1.7 equiv of HOBt, 1.7 equiv of EDC,
CH2Cl2, 23 °C, 18 h, 88%; (e) HCl, 1,4-dioxane, 23 °C, 1.5 h; (f) 2.2
equiv of NMM, 1.1 equiv of 5-methylisoxazole-3-carbonyl chloride,
CH2Cl2, 0f23 °C, 1 h, 69%; (g) 1.3 equiv of DDQ, 10:1 CHCl3:
H2O, reflux, 9 h, 56%.
NaBO3‚4H2O, 25 °C, 1 h, 72%; (b) 4.0 equiv of SO3‚pyridine, 3.6
equiv of Et3N, DMSO, 0f23 °C, 3 h; (c) 4.0 equiv of HCl‚H2N-
DMB, 4.0 equiv of NaOAc, 2.0 equiv of NaBH3CN, 2:1 THF:EtOH,
23 °C, 20 h; (d) 0.20 equiv of TsOH‚H2O, CH3OH, 50 °C, 2.5 h,
44% from 24; (e) 4.0 equiv of DMSO, 3.0 equiv of oxalyl chloride,
5.0 equiv of Et3N, CH2Cl2, -78 °C, 1.5 h; (f) 1.0 equiv of
NaN(TMS)2, 1.0 equiv of (EtO)2P(O)CH2CO2Et, THF, -78f0 °C,
45 min, 48% from 26.
Sch em e 3a
vided the corresponding tripeptide 33 (not shown). This
material was also deprotected under acidic conditions
(HCl), and the amine hydrochloride thus produced was
derivatized with 5-methylisoxazole-3-carbonyl chloride
to give intermediate 34 in moderate overall yield. As
described for the preparation of 4 above, treatment of
34 with DDQ at elevated temperature provided the
tripeptidyl inhibitor 9 after purification on silica gel.
Similarly, coupling of the amine hydrochloride salt
derived from HCl deprotection of 22 with the keto-
methylene dipeptide isostere 358 (used in crude form)
gave intermediate 36 in moderate overall yield (Scheme
5). This material was transformed into inhibitor 11 in
a manner analogous to that used for the conversion of
intermediate 33 to inhibitor 9 described above.
a
Reagents and conditions: (a) 1.2 equiv of Ph3PdCHCO2Et,
THF, 23 °C, 18 h; (b) HCl, 1,4-dioxane, 23 °C, 18 h; (c) 1.1 equiv
of BocNHCH2CHO, 1.0 equiv of NaBH3CN, CH3OH, 23 °C, 18 h,
44%; (d) TFA, CH2Cl2, 23 °C, 2 h, 92%; (e) 0.92 equiv of CDI, THF,
23 °C, 3.5 h, 54%.
2-aminoethanal under reductive amination conditions
provided the protected diamine compound 31 in moder-
ate yield. Boc deprotection (TFA), neutralization of the
amine salt thus obtained, and subsequent exposure of
the free diamine to 1,1′-carbonyldiimidazole afforded the
cyclic-urea-containing inhibitor 7 in good yield.
Inhibitors 9 and 11 were prepared by derivatization
of intermediate 22 with amino acid residues or a
ketomethylene dipeptide isostere, respectively. Thus,
acidic deprotection (HCl) of 22 and carbodiimide-medi-
ated coupling of the resulting amine salt with Boc-L-
Phe(4-F)-OH afforded the dipeptide product 32 in
moderate yield (Scheme 4). A second HCl deprotection
and subsequent coupling with Boc-L-Val-OH then pro-
Con clu sion s
The studies presented above illustrate how the in-
corporation of P1 lactam glutamine replacements into
substrate-derived, irreversible human rhinovirus 3C
protease (3CP) inhibitors substantially improves the
anti-3CP and antiviral activity of such compounds. Both
peptidyl and peptidomimetic molecules benefit from
such incorporation, and the resulting optimized inhibi-
tors rank among the most potent antirhinoviral agents
described in the literature to date. In addition, most of
the compounds presented in this work display excellent
selectivity for HRV 3CP over other serine and cysteine

P1 Lactam Moieties as L-Glutamine Replacements
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 7 1219
Sch em e 5a
over MgSO4 and were gravity-filtered. The filtrate was con-
centrated under reduced pressure and the residue dried under
vacuum to afford 16 (10.0 g, 100% crude yield) as a colorless
oil. This material was used without further purification: 1H
NMR (CDCl3, mixture of isomers) δ 1.49 (s), 1.57 (s), 1.60 (s),
1.84-2.05 (m), 2.39-2.41 (m), 3.71-3.74 (m), 3.91-4.05 (m).
(4S,4′′S)-4-[3′-(4′′-Ben zyl-2′′-oxo-oxazolidin -3′′-yl)-3′-oxo-
p r op yl]-2,2-d im eth yloxa zolid in e-3-ca r boxylic Acid ter t-
Bu t yl E st er (17). Triethylamine (8.87 mL, 63.6 mmol, 3.0
equiv) and pivaloyl chloride (2.61 mL, 21.2 mmol, 1.0 equiv)
were added sequentially to a solution of 16 (5.80 g, 21.2 mmol,
1 equiv) in THF (450 mL) at 0 °C. The cloudy reaction mixture
was stirred at 0 °C for 3.5 h, and then lithium chloride (0.988
g, 23.3 mmol, 1.1 equiv) and (S)-(-)-4-benzyl-2-oxazolidinone
(3.57 g, 20.1 mmol, 0.95 equiv) were added sequentially. After
warming to 23 °C and being stirred for 19 h, the reaction
mixture was partitioned between 0.5 M HCl (150 mL) and
EtOAc (2 × 150 mL). The combined organic layers were
washed with half-saturated Na2CO3 (150 mL), dried over
MgSO4, and gravity-filtered. The filtrate was concentrated
under reduced pressure, and the residue was purified by flash
column chromatography (30% EtOAc in hexanes) to give 17
(7.17 g, 83%) as a colorless oil which slowly crystallized over
7 days: mp ) 60-64 °C; Rf ) 0.69 (50% EtOAc in hexanes);
IR (cm-1) 2978, 1783, 1694; 1H NMR (CDCl3, mixture of
isomers) δ 1.49 (s), 1.59 (s), 1.63 (s), 2.01-2.10 (m), 2.76 (dd,
J ) 13.5, 9.8), 2.82-3.13 (m), 3.30-3.41 (m), 3.76-3.82 (m),
3.90 (s, br), 3.97 (dd, J ) 9.0, 5.6), 4.10-4.19 (m), 4.63-4.71
(m), 7.22-7.36 (m). Anal. (C23H32N2O6) C, H, N.
a
DMB ) 2,4-dimethoxybenzyl. Reagents and conditions: (a)
HCl, 1,4-dioxane, 23 °C, 1.5 h; (b) 1.4 equiv of 35,8 4.0 equiv of
NMM, 1.7 equiv of HOBt, 1.7 equiv of EDC, CH2Cl2, 23 °C, 18 h,
63%; (c) HCl, 1,4-dioxane, 23 °C, 1.5 h; (d) 2.2 equiv of NMM, 1.1
equiv of 5-methylisoxazole-3-carbonyl chloride, CH2Cl2, 0f23 °C,
1 h, 85%; (e) 1.4 equiv of DDQ, 10:1 CHCl3:H2O, reflux, 20 h, 39%.
(2′S,4S,4′′S)-4-[2′-(4′′-Ben zyl-2′′-oxo-oxa zolid in e-3′′-ca r -
b on yl)-p en t -4′-en yl]-2,2-d im et h yloxa zolid in e-3-ca r b ox-
ylic Acid ter t-Bu tyl Ester (18). A solution of 17 (7.17 g, 16.6
mmol, 1 equiv) in THF (50 mL) was added to a solution of
sodium bis(trimethylsilyl)amide (16.6 mL of a 1.0 M solution
in THF, 16.6 mmol, 1.0 equiv) in the same solvent (150 mL)
at -78 °C. The reaction mixture was stirred for 20 min at -78
°C, and then allyl iodide (4.55 mL, 49.8 mmol, 3.0 equiv) was
added. After being stirred an additional 3 h at -78 °C, the
reaction mixture was maintained at -45 °C for 2 h and then
was partitioned between a 2:1 mixture of half-saturated NH4Cl
and 5% Na2S2O3 (300 mL) and a 1:1 mixture of EtOAc and
hexanes (2 × 200 mL). The combined organic layers were
washed with H2O (200 mL), dried over MgSO4, and gravity-
filtered. The filtrate was concentrated under reduced pressure,
and the residue was purified by flash column chromatography
(15% EtOAc in hexanes) to provide 18 (4.29 g, 55%) as a
proteases and exhibit good stability toward a variety of
biological agents. The above studies led to the selection
of one inhibitor (compound 11, AG7088) as a develop-
ment candidate, and the molecule is currently undergo-
ing testing to evaluate its potential clinical use as an
antirhinoviral agent.
Exp er im en ta l Section
General descriptions of experimental procedures, reagent
purifications, and instrumentation along with conditions and
uncertainties for enzyme and antiviral assays are provided
elsewhere.5 1H NMR chemical shifts are reported in ppm (δ)
downfield relative to internal tetramethylsilane, and coupling
constants are given in hertz. A simplified naming system
employing amino acid abbreviations is used to identify some
intermediates and final products. When utilizing this naming
system, italicized amino acid abbreviations represent modifi-
cations at the C-terminus of that residue where acrylic acid
esters are reported as “E” (trans) propenoates. In addition, the
terminology “AA1Ψ[COCH2]-AA2” indicates that, for any pep-
tide sequence, two amino acids (AA1 and AA2) usually linked
by an amide bond are replaced by a ketomethylene dipeptide
isostere. The following abbreviations also apply: HATU [O-(7-
azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluo-
rophosphate], HOBt [1-hydroxybenzotriazole hydrate], EDC
[1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochlo-
ride], CDI [1,1′-carbonyldiimidazole], MTBE [tert-butyl methyl
ether], DDQ [2,3-dichloro-5,6-dicyano-1,4-benzoquinone], (S)-
Pyrrol-Ala [(2S,3′S)-2-amino-3-(2′-oxopyrrolidin-3′-yl)propionic
acid], (S)-Piper-Ala [(2S,3′S)-2-amino-3-(2′-oxopiperidin-3′-yl)-
propionic acid]. Pirodavir was kindly provided by J anssen
Pharmaceuticals. Pleconaril was prepared as described in the
literature.18
(4S)-4-(2′-Car boxyeth yl)-2,2-dim eth yloxazolidin e-3-car -
boxylic Acid ter t-Bu tyl Ester (16). Sodium hydroxide (27
mL of a 4.0 M solution in H2O, 108 mmol, 3.0 equiv) was added
to a solution of (4S)-4-(2-methoxycarbonylethyl)-2,2-dimethyl-
oxazolidine-3-carboxylic acid tert-butyl ester 1522 (10.5 g, 36.5
mmol, 1 equiv) in CH3OH (150 mL), and the resulting cloudy
reaction mixture was stirred at 23 °C for 3.5 h. The mixture
was concentrated under reduced pressure to ∼30 mL volume
and then was partitioned between 0.5 M HCl (150 mL) and
EtOAc (2 × 150 mL). The combined organic layers were dried
colorless oil: Rf ) 0.44 (30% EtOAc in hexanes); IR (cm-1
)
2978, 1780, 1695; 1H NMR (CDCl3, mixture of isomers) δ 1.45
(s), 1.49 (s), 1.68-1.80 (m), 2.13-2.47 (m), 2.49-2.67 (m), 3.32
(dd, J ) 13.4, 3.1), 3.69-3.97 (m), 4.11-4.21 (m), 4.66-4.74
(m), 5.06-5.13 (m), 5.74-5.88 (m), 7.20-7.36 (m). Anal.
(C26H36N2O6) C, H, N.
(1S,3′S)-{2-[1′-(2′′,4′′-Dim eth oxyben zyl)-2′-oxo-p yr r oli-
d in -3′-yl]-1-h yd r oxym eth yleth yl}-ca r ba m ic Acid ter t-Bu -
tyl Ester (20). Ozone was bubbled through a solution of 18
(4.29 g, 9.08 mmol, 1 equiv) in CH2Cl2 (200 mL) containing
CH3OH (0.735 mL, 18.1 mmol, 2.0 equiv) at -78 °C until a
blue color persisted. The reaction mixture was then purged
with argon until it became colorless. Methyl sulfide (6.67 mL,
90.8 mmol, 10 equiv) was added, and the mixture was stirred
at -78 °C for 3.5 h and then was maintained at 0 °C for an
additional 1 h. After partitioning the reaction mixture between
H2O (200 mL) and a 1:1 mixture of EtOAc and hexanes (2 ×
200 mL), the combined organic layers were dried over MgSO4
and were gravity-filtered. The filtrate was concentrated under
reduced pressure, and the residue was immediately utilized
without further purification.
The above residue was dissolved in a 2:1 mixture of THF
and EtOH (240 mL) at 23 °C, and 2,4-dimethoxybenzylamine
hydrochloride (7.40 g, 36.3 mmol, 4.0 equiv), sodium acetate
(2.98 g, 36.2 mmol, 4.0 equiv), and sodium cyanoborohydride
(1.14 g, 18.1 mmol, 2.0 equiv) were added sequentially. The
resulting suspension was stirred for 18 h at 23 °C and then
was partitioned between 0.5 M HCl (400 mL) and EtOAc (2 ×

1220 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 7
Dragovich et al.
200 mL). The combined organic layers were washed with half-
saturated NaHCO3 (300 mL), dried over Na2SO4, and concen-
trated under reduced pressure. The residue was passed
through a short silica gel column (eluting with 50% EtOAc in
hexanes) to give 19 contaminated with (S)-(-)-4-benzyl-2-
oxazolidinone.
The combined organic layers were dried over Na2SO4 and were
concentrated. Purification of the residue by flash column
chromatography (60% EtOAc in hexanes) provided 23 (0.158
g, 70%) as a tan foam: Rf ) 0.30 (5% CH3OH in CH2Cl2); IR
(cm-1) 3289, 1713, 1655; 1H NMR (CDCl3) δ 0.87-0.92 (m, 6H),
1.28 (t, 3H, J ) 7.2), 1.46-1.68 (m, 5H), 1.74-1.86 (m, 1H),
1.97-2.19 (m, 2H), 3.02 (dd, 1H, J ) 13.7, 5.6), 3.11-3.24 (m,
3H), 3.78 (s, 3H), 3.79 (s, 3H), 4.17 (q, 2H, J ) 7.2), 4.20-4.30
(m, 2H), 4.35-4.45 (m, 2H), 4.82-4.90 (m, 1H), 5.07 (d, 1H, J
) 12.3), 5.13 (d, 1H, J ) 12.3), 5.36 (d, 1H, J ) 7.8), 5.82 (dd,
1H, J ) 15.6, 1.2), 6.42-6.46 (m, 2H), 6.72 (dd, 1H, J ) 15.6,
5.3), 6.88 (d, 1H, J ) 8.7), 7.09 (d, 1H, J ) 9.0), 7.13-7.20 (m,
5H), 7.29-7.37 (m, 5H), 8.09 (d, 1H, J ) 6.5). Anal. (C43H54N4O9‚
0.5H2O) C, H, N.
This material was dissolved in CH3OH (100 mL), and TsOH‚
H2O (0.345 g, 1.81 mmol, 0.20 equiv) was added. The reaction
mixture was heated to 50 °C and was maintained at that
temperature for 2.5 h. After cooling to 23 °C, the reaction
mixture was concentrated under reduced pressure to ∼20 mL
volume and was partitioned between half-saturated NaHCO3
(150 mL) and a 9:1 mixture of CH2Cl2 and CH3OH (2 × 150
mL). The combined organic layers were dried over Na2SO4 and
were concentrated under reduced pressure. Purification of the
residue by flash column chromatography (3% CH3OH in
CH2Cl2) afforded 20 (1.62 g, 44% from 18) as a foam: Rf )
0.03 (50% EtOAc in hexanes); IR (cm-1) 3328, 1669; 1H NMR
(CDCl3) δ 1.44 (s, 9H), 1.50-1.75 (m, 2H), 1.90-2.00 (m, 1H),
2.17-2.27 (m, 1H), 2.52-2.62 (m, 1H), 3.14-3.24 (m, 2H),
3.51-3.65 (m, 3H), 3.70-3.78 (m, 1H), 3.80 (s, 6H), 4.35 (d,
1H, J ) 14.3), 4.48 (d, 1H, J ) 14.3), 5.51-5.54 (m, 1H), 6.42-
6.46 (m, 2H), 7.09-7.12 (m, 1H). Anal. (C21H32N2O6) C, H, N.
E t h yl-3-{Boc-L-[(N-2,4-d im et h oxyb en zyl)-(S)-P yr r ol-
Ala ]}-E-p r op en oa te (22). DMSO (0.270 mL, 3.80 mmol, 3.0
equiv) was added dropwise to a -78 °C solution of oxalyl
chloride (0.166 mL, 1.90 mmol, 1.5 equiv) in CH2Cl2 (14 mL).
The reaction mixture was stirred 20 min at -78 °C and then
a solution of 20 (0.518 g, 1.27 mmol, 1 equiv) in CH2Cl2 (13
mL) was added via cannula along the side of the reaction
vessel. After an additional 20 min, triethylamine (1.06 mL,
7.60 mmol, 6.0 equiv) was added dropwise, and the reaction
mixture was stirred for 1.5 h at -78 °C. Acetic acid (0.479 mL,
8.37 mmol, 6.6 equiv) was then added, and the reaction
mixture was warmed to 0 °C for 5 min, then diluted with
MTBE (200 mL), and washed with water (25 mL), saturated
NaHCO3 (25 mL), and brine (25 mL). The organic phase was
dried over Na2SO4 and was concentrated to provide 21 as a
white foam (0.516 g, 100%). This material was used without
further purification.
Eth yl-3-{Cbz-L-Leu -L-P h e-L-[(S)-P yr r ol-Ala ]}-E-p r op en -
oa te (4). DDQ (0.066 g, 0.29 mmol, 1 equiv) and 23 (0.225 g,
0.291 mmol, 1.0 equiv) were combined in a mixture of CHCl3
(4 mL) and water (0.4 mL). After the mixture was stirred for
2 h in a 50 °C oil bath, additional DDQ (0.095 g, 0.42 mmol,
1.4 equiv) was added. After 2 h more at 50 °C, a third portion
of DDQ (0.099 g, 0.44 mmol, 1.5 equiv) was added. The reaction
mixture was stirred for 2 h at 50 °C, and then DDQ (0.065 g,
0.29 mmol, 1.0 equiv) was added for the fourth time. After
stirring for 2 h more at 50 °C, the reaction mixture was allowed
to cool overnight, then was diluted with EtOAc (250 mL), and
washed with saturated NaHCO3 (40 mL) and brine (25 mL).
The organic phase was dried over Na2SO4 and was concen-
trated. Purification of the residue by flash column chroma-
tography (2.4% CH3OH in CH2Cl2) provided 4 (0.100 g, 55%)
as a white foam: Rf ) 0.26 (5% CH3OH in CH2Cl2); IR (cm-1
)
3278, 1690, 1637; 1H NMR (CDCl3) δ 0.85-0.92 (m, 6H), 1.28
(t, 3H, J ) 7.2), 1.39-1.65 (m, 4H), 1.68-1.93 (m, 2H), 2.08-
2.20 (m, 1H), 2.27-2.38 (m, 1H), 3.02-3.13 (m, 2H), 3.24-
3.32 (m, 2H), 4.11-4.20 (m, 1H), 4.18 (q, 2H, J ) 7.2), 4.47-
4.58 (m, 1H), 4.81-4.89 (m, 1H), 5.05 (d, 1H, J ) 12.1), 5.12
(d, 1H, J ) 12.1), 5.26 (d, 1H, J ) 8.1), 5.78 (dd, 1H, J ) 15.7,
1.2), 6.23 (s, 1H), 6.72 (dd, 1H, J ) 15.7, 5.3), 7.13-7.25 (m,
6H), 7.30-7.37 (m, 5H), 7.54 (d, 1H, J ) 7.2). Anal. (C34H44N4O7‚
0.5H2O) C, H, N.
(2′S,4S,4′′S)-4-[2′-(4′′-Ben zyl-2′′-oxo-oxa zolid in e-3′′-ca r -
bon yl)-5′-h yd r oxyp en tyl]-2,2-d im eth yloxa zolid in e-3-ca r -
boxylic Acid ter t-Bu tyl Ester (24). A solution of borane-
tetrahydrofuran complex (0.96 mL of a 1.0 M solution in THF,
0.96 mmol, 1.0 equiv) was added to a 0 °C solution of 18 (0.455
g, 0.963 mmol, 1 equiv) in THF (3 mL). After the mixture was
stirred for 30 min, water (3 mL) and sodium perborate
tetrahydrate (0.148 g, 0.962 mmol, 1.0 equiv) were added and
the ice bath was removed. After an additional hour, the
reaction mixture was diluted with MTBE (125 mL) and was
washed with water (15 mL) and brine (2 × 15 mL). The
combined organic layers were dried over Na2SO4 and were
concentrated. Purification of the residue by flash column
chromatography (50% EtOAc in hexanes) provided 24 (0.339
g, 72%) as a colorless glass: Rf ) 0.41 (50% EtOAc in hexanes);
IR (cm-1) 3486, 1780, 1693; 1H NMR (CDCl3, mixture of
isomers) δ 1.42-1.85 (m), 2.13-2.24 (m), 2.70 (dd, J ) 13.1,
10.0), 3.29-3.38 (m), 3.61-4.22 (m), 4.63-4.76 (m), 7.19-7.38
(m). Anal. (C26H38N2O7‚0.50H2O) C, H, N.
(1S,3′S)-{2-[1′-(2′′,4′′-Dim eth oxyben zyl)-2′-oxo-piper idin -
3′-yl]-1-h yd r oxym eth yleth yl}-ca r ba m ic Acid ter t-Bu tyl
Ester (26). A solution of sulfur trioxide-pyridine complex
(5.04 g, 31.67 mmol, 4.0 equiv) in DMSO (150 mL) was added
to an ice-cooled solution of 24 (3.88 g, 7.92 mmol, 1 equiv) in
triethylamine (2.75 mL, 28.51 mmol, 3.6 equiv) at a rate which
maintained the reaction temperature between 8 and 17 °C.
The resulting mixture was then stirred at 23 °C for 3 h, cooled
to 0 °C, and was quenched by the addition of H2O (150 mL).
The quenched reaction mixture was extracted with EtOAc (2
× 150 mL), and the combined organic layers were washed with
5% citric acid (100 mL) and brine (100 mL), then were dried
over Na2SO4, and concentrated. The residue was dried further
under vacuum to give a white foam (3.53 g).
Sodium bis(trimethylsilyl)amide (1.23 mL of a 1.0 M solution
in THF, 1.23 mmol, 1.0 equiv) was added to a solution of
triethyl phosphonoacetate (0.244 mL, 1.23 mmol, 1.0 equiv)
in THF (15 mL) at -78 °C, and the resulting mixture was
stirred for 20 min at that temperature. A solution of crude 21
(prepared above, 0.500 g, 1.23 mmol, 1 equiv) in THF (13 mL)
was added via cannula along the side of the reaction vessel,
and the resulting mixture was stirred for 45 min at -78 °C,
warmed to 0 °C for 7 min, then was partitioned between 0.5
M HCl (20 mL) and MTBE (2 × 50 mL). The combined organic
layers were dried over MgSO4 and were concentrated. Puri-
fication of the residue by flash column chromatography (60%
EtOAc in hexanes) provided 22 (0.356 g, 61%) as a white
foam: Rf ) 0.43 (60% EtOAc in hexanes); IR (cm-1) 3307, 1708,
1
1678; H NMR (CDCl3) δ 1.28 (t, 3H, J ) 7.2), 1.43 (s, 9H),
1.52-1.70 (m, 2H), 1.98-2.09 (m, 1H), 2.21-2.34 (m, 1H),
2.48-2.59 (m, 1H), 3.16-3.24 (m, 2H), 3.80 (s, 6H), 4.18 (q,
2H, J ) 7.2), 4.27-4.40 (m, 1H), 4.41 (s, 2H), 5.40 (d, 1H, J )
8.1), 5.95 (dd, 1H, J ) 15.6, 1.6), 6.41-6.48 (m, 2H), 6.86 (dd,
1H, J ) 15.6, 5.3), 7.08-7.13 (m, 1H). Anal. (C25H36N2O7‚
0.25H2O) C, H, N.
Eth yl-3-{Cbz-L-Leu -L-P h e-L-[(N-2,4-d im eth oxyben zyl)-
(S)-P yr r ol-Ala ]}-E-p r op en oa te (23). A solution of HCl in 1,4-
dioxane (4.0 M, 4 mL) was added to a solution of 22 (0.139 g,
0.292 mmol, 1 equiv) in the same solvent (4 mL) at 23 °C. After
the mixture was stirred for 1.5 h at that temperature, the
volatiles were evaporated to give the crude amine hydrochlo-
ride salt. This material was dissolved in CH2Cl2 (7 mL), and
Cbz-L-Leu-L-Phe-OH (0.156 g, 0.378 mmol, 1.3 equiv), 4-meth-
ylmorpholine (0.128 mL, 1.16 mmol, 4.0 equiv), HOBt (0.067
g, 0.50 mmol, 1.7 equiv) and EDC (0.095 g, 0.50 mmol, 1.7
equiv), were added sequentially. After being stirred for 20 h
at 23 °C, the reaction mixture was partitioned between brine
(15 mL) and a 1:9 mixture of CH3OH and CH2Cl2 (3 × 25 mL).
To a solution of this material (3.53 g, 7.22 mmol, 1 equiv)
in a 2:1 mixture of THF and EtOH (120 mL) were added 2,4-

P1 Lactam Moieties as L-Glutamine Replacements
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 7 1221
dimethoxybenzylamine hydrochloride (5.88 g, 28.89 mmol, 4.0
equiv), NaOAc (2.37 g, 28.89 mmol, 4.0 equiv), and NaBH3-
CN (0.908 g, 14.45 mmol, 2.0 equiv) at 23 °C. The reaction
mixture was stirred 20 h at 23 °C and then was diluted with
MTBE (200 mL). The organic layer was washed with 10%
KHSO4 (100 mL), saturated NaHCO3 (100 mL), and brine (100
mL), dried over Na2SO4, and concentrated to give 25 as a pale
yellow foam. This material was utilized without further
purification.
p-Toluenesulfonic acid monohydrate (0.275 g, 1.44 mmol,
0.20 equiv) was added to a solution of crude 25 (3.34 g, 7.22
mmol, 1 equiv) in CH3OH (50 mL) at 23 °C. The reaction
mixture was maintained at 50 °C for 2.5 h and then was
partitioned between CH2Cl2 (100 mL) and saturated NaHCO3
(100 mL). The organic layer was dried over Na2SO4 and was
concentrated. Purification of the residue by flash column
chromatography (3% CH3OH in CH2Cl2) afforded 26 (1.33 g,
44% from 24) as a white foam: 1H NMR (CDCl3) δ 1.44 (s,
9H), 1.71-1.85 (m, 2H), 1.92-1.98 (m, 2H), 2.40-2.48 (m, 1H),
2.71-2.78 (m, 1H), 3.19-3.32 (m, 2H), 3.45-3.69 (m, 4H),
4.11-4.20 (m, 2H), 4.68 (m, 1H), 5.47 (m, 1H), 6.44 (s, 1H),
7.20-7.33 (m, 2H).
E t h yl-3-{Boc-L-[(N -2,4-d im e t h oxyb e n zyl)-(S)-P ip er -
Ala ]}-E-p r op en oa te (28). DMSO (0.133 mL, 1.87 mmol, 4.0
equiv) was added dropwise to a -78 °C solution of oxalyl
chloride (0.123 mL, 1.41 mmol, 3.0 equiv) in CH2Cl2 (7 mL).
The reaction mixture was stirred 20 min at -78 °C, and then
a solution of 26 (0.198 g, 0.468 mmol, 1 equiv) in CH2Cl2 (5
mL) was added via cannula along the side of the reaction
vessel. After an additional 20 min, triethylamine (0.327 mL,
2.34 mmol, 5.0 equiv) was added dropwise, and the reaction
mixture was maintained at -78 °C for 1.5 h. Acetic acid (0.148
mL, 2.59 mmol, 5.5 equiv) was added, and the reaction mixture
was warmed to 0 °C for 5 min, then was diluted with MTBE
(50 mL), and was washed with water (15 mL), saturated
NaHCO3 (15 mL), and brine (15 mL). The organic phase was
dried over Na2SO4 and was concentrated to provide 27 as a
foam which was used without further purification.
Sodium bis(trimethylsilyl)amide (0.468 mL of a 1.0 M
solution in THF, 0.468 mmol, 1.0 equiv) was added to a
solution of triethyl phosphonoacetate (0.093 mL, 0.47 mmol,
1.0 equiv) in THF (5 mL) at -78 °C, and the resulting solution
was stirred for 20 min at that temperature. A solution of 27
(0.468 mmol, 1 equiv) in THF (4 mL) was added via cannula
along the side of the reaction vessel, and the reaction mixture
was stirred for 45 min at -78 °C, then warmed to 0 °C for 7
min, and was partitioned between 0.5 M HCl (20 mL) and
MTBE (2 × 50 mL). The combined organic layers were dried
over MgSO4 and were concentrated. Purification of the residue
by flash column chromatography (40% EtOAc in hexanes)
provided 28 (0.110 g, 48%) as a white foam: Rf ) 0.29 (40%
EtOAc in hexanes); IR (cm-1) 3296, 1716, 1616; 1H NMR
(CDCl3) δ 1.28 (t, 3H, J ) 7.2), 1.42-1.87 (m, 4H), 1.44 (s,
9H), 2.03-2.24 (m, 2H), 2.36-2.46 (m, 1H), 3.16-3.30 (m, 2H),
3.79 (s, 6H), 4.18 (q, 2H, J ) 7.2), 4.26-4.38 (m, 1H), 4.53 (s,
2H), 5.50 (d, 1H, J ) 7.8), 5.95 (dd, 1H, J ) 15.6, 1.6), 6.42-
6.48 (m, 2H), 6.86 (dd, 1H, J ) 15.6, 5.1), 7.08-7.15 (m, 1H).
Anal. (C26H38N2O7‚0.25H2O) C, H, N.
the mixture was stirred overnight, the volatiles were removed
under reduced pressure and the residue was triturated with
Et2O (20 mL). The resulting solids were collected by vacuum
filtration and were washed with Et2O (3 × 10 mL) to give the
crude amine salt (0.63 g 73%, 1.05 mmol).
This material was dissolved in CH3OH (10 mL) at 23 °C,
and N-Boc-2-aminoethanal (Aldrich, 0.19 g, 1.16 mmol, 1.1
equiv) and NaBH3CN (0.069 g, 1.05 mmol, 1.0 equiv) were
added sequentially. The reaction mixture was stirred at 23
°C overnight and then was concentrated. The residue was
dissolved in EtOAc (25 mL), and the organic layer was washed
with water (25 mL) and brine (25 mL), dried over MgSO4, and
concentrated. The residue was purified by flash column
chromatography (gradient elution, 0f3% CH3OH in CH2Cl2)
to provide 31 (0.32 g, 44%) as a white amorphous solid: Rf )
0.20 (5% CH3OH in CHCl3); IR (cm-1) 1712, 1649, 1537, 1252,
1175; 1H NMR (DMSO-d6) δ 0.79 (d, 3H, J ) 6.6), 0.82 (d, 3H,
J ) 6.6), 1.21 (t, 3H, J ) 7.0), 1.26-1.37 (m, 13H), 1.46-1.54
(m, 1H), 2.56-2.60 (m, 2H), 2.82-2.97 (m, 4H), 3.98-4.04 (m,
1H), 4.10 (q, 2H, J ) 7.0), 4.42-4.49 (m, 2H), 4.98 (d, 1H, J )
12.5), 5.04 (d, 1H, J ) 12.9), 5.59 (d, 1H, J ) 15.8), 6.73-6.75
(m, 1H), 6.77 (dd, 1H, J ) 15.8, 4.8), 7.20-7.34 (m, 10H), 7.41
(d, 1H, J ) 8.1), 7.97 (d, 1H, J ) 7.0), 8.07 (d, 1H, J ) 7.0).
Anal. (C37H53N5O8‚0.50H2O) C, H, N.
Eth yl-3-[Cbz-L-Leu -L-P h e-L-1-(2-im id a zolid on e)Ala ]-E-
p r op en oa te (7). TFA (0.8 mL) was added to a solution of 31
(0.29 g, 0.42 mmol, 1 equiv) in CH2Cl2, (8 mL) and the reaction
mixture was stirred at 23 °C for 2 h. The volatiles were then
removed under reduced pressure, and the residue was dis-
solved in EtOAc (25 mL) and washed with saturated NaHCO3
solution (25 mL), water (25 mL), and brine (25 mL). The
organic layer was dried over MgSO4 and was concentrated to
give the crude diamine (0.23 g, 92%, 0.39 mmol) as a tan
amorphous solid.
This material was dissolved in THF (4 mL) at 23 °C, and
carbonyldiimidazole (0.06 g, 0.36 mmol, 0.92 equiv) was added.
After being stirred for 3.5 h at 23 °C, the reaction mixture
was concentrated under reduced pressure, and the residue was
purified by flash column chromatography (gradient elution,
0f2% CH3OH in CH2Cl2) to give 7 (0.12 g, 54%) as a white
amorphous solid: mp ) 161-164 °C; Rf ) 0.21 (5% CH3OH in
1
CHCl3); IR (cm-1) 1701, 1647, 1535, 1277; H NMR (DMSO-
d6) δ 0.79 (d, 3H, J ) 6.6), 0.82 (d, 3H, J ) 6.6), 1.21 (t, 3H, J
) 7.0), 1.27-1.35 (m, 2H), 1.48-1.52 (m, 1H), 2.79-2.86 (m,
1H), 2.92-3.05 (m, 3H), 3.14-3.19 (m, 2H), 3.25-3.30 (m, 2H),
3.98-4.03 (m, 1H), 4.10 (q, 2H, J ) 7.0), 4.47-4.49 (m, 1H),
4.59-4.63 (m, 1H), 4.97-5.02 (m, 2H), 5.72 (d, 1H, J ) 15.8),
6.37 (s, 1H), 6.71 (dd, 1H, J ) 15.8, 5.5), 7.15-7.39 (m, 10H),
7.42 (d, 1H, J ) 8.1), 8.00 (d, 1H, J ) 8.1), 8.18 (d, 1H, J )
8.1). Anal. (C33H43N5O7) C, H, N.
E t h yl-3-{Boc-L-P h e(4-F )-L-[(N-2,4-d im et h oxyb en zyl)-
(S)-P yr r ol-Ala ]}-E-p r op en oa te (32). A solution of HCl in 1,4-
dioxane (4.0 M, 12 mL) was added to a solution of 22 (0.432 g,
0.906 mmol, 1 equiv) in the same solvent (12 mL) at 23 °C.
After the mixture was stirred for 1.5 h at 23 °C, the solvent
was evaporated to give the crude amine salt which was
dissolved in DMF (7 mL) and cooled to 0 °C. Boc-L-Phe(4-F)-
OH (0.308 g, 1.09 mmol, 1.2 equiv), N,N-diisopropylethylamine
(0.474 mL, 2.72 mmol, 3.0 equiv), and HATU (0.379 g, 0.997
mmol, 1.1 equiv) were added sequentially, and the reaction
mixture was allowed to warm to 23 °C. After 1.5 h, the mixture
was diluted with MTBE (200 mL), washed with 5% KHSO4
(20 mL) and brine (20 mL), dried over MgSO4, and concen-
trated. The residue was purified by flash column chromatog-
raphy (60% EtOAc in hexanes) to provide 32 (0.447 g, 77%)
Eth yl-3-[Cbz-L-Leu -L-P h e-L-N-Boc-a min oAla ]-E-pr open -
oa te (30). (Carbethoxymethylene)triphenylphosphorane (1.20
g, 3.28 mmol, 1.2 equiv) was added to a solution of 2930 (1.60
g, 2.73 mmol, 1 equiv) in THF (55 mL), and the reaction
mixture was stirred at 23 °C overnight. The volatiles were then
removed under reduced pressure, and the residue was purified
by flash column chromatography (gradient elution, 0f1.5%
CH3OH in CH2Cl2) to give 30 (0.968 g, contaminated with
triphenylphosphine oxide). This material was used without
further purification.
E t h yl-3-[Cb z-L-Leu -L-P h e-L-(2-Boc-2-a m in oeth yl)a m i-
n oAla ]-E-p r op en oa te (31). A solution of HCl in 1,4-dioxane
(4.0 M, 10 mL) was added to a solution of 30 (0.95 g, 1.46 mmol,
1 equiv) in the same solvent (20 mL) at 23 °C. The reaction
mixture was stirred at that temperature for 1.5 h, and then
additional HCl in 1,4-dioxane (4.0 M, 10 mL) was added. After
as a white foam: Rf ) 0.34 (60% EtOAc in hexanes); IR (cm-1
)
3258, 1705, 1666; 1H NMR (CDCl3) δ 1.28 (t, 3H, J ) 7.2),
1.45 (s, 9H), 1.51-1.66 (m, 2H), 1.78-1.90 (m, 1H), 2.06-2.23
(m, 2H), 2.99 (dd, 1H, J ) 13.7, 6.2), 3.11 (dd, 1H, J ) 13.7,
5.3), 3.17-3.23 (m, 2H), 3.80 (s, 3H), 3.81 (s, 3H), 4.18 (q, 2H,
J ) 7.2), 4.35 (s, 2H), 4.38-4.51 (m, 2H), 5.29-5.37 (m, 1H),
5.76 (d, 1H, J ) 15.8), 6.43-6.47 (m, 2H), 6.72 (dd, 1H, J )
15.8, 5.3), 6.83-6.91 (m, 2H), 7.09-7.17 (m, 3H), 7.92 (br, 1H).
Anal. (C34H44FN3O8) C, H, N.

1222 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 7
Dragovich et al.
Eth yl-3-{Boc-L-Va l-L-P h e(4-F )-L-[(N-2,4-d im eth oxyben -
zyl)-(S)-P yr r ol-Ala ]}-E-p r op en oa te (33). A solution of HCl
in 1,4-dioxane (4.0 M, 10 mL) was added to a solution of 32
(0.425 g, 0.662 mmol, 1 equiv) in the same solvent (10 mL) at
23 °C. After the mixture was stirred for 1.5 h at 23 °C, the
solvent was evaporated to give the crude amine salt. This
material was combined with Boc-L-Val-OH (0.173 g, 0.796
mmol, 1.2 equiv) and dissolved in CH2Cl2 (20 mL) at 23 °C.
HOBt (0.152 g, 1.12 mmol, 1.7 equiv), 4-methylmorpholine
(0.291 mL, 2.65 mmol, 4.0 equiv), and EDC (0.216 g, 1.13
mmol, 1.7 equiv) were then added sequentially. After being
stirred overnight at 23 °C, the reaction mixture was diluted
with MTBE (200 mL) and washed with 5% KHSO4 (20 mL),
saturated NaHCO3 (20 mL), and brine (20 mL). The organic
layer was dried over Na2SO4 and was concentrated. The
residue was purified by flash column chromatography (gradi-
ent elution, 60f100% EtOAc in hexanes) to provide 33 (0.430
g, 88%) as a white foam: Rf ) 0.24 (60% EtOAc in hexanes);
(dd, 1H, J ) 15.6, 5.4), 6.86-6.94 (m, 2H), 7.01 (d, 1H, J )
9.0), 7.11-7.18 (m, 2H), 7.21 (d, 1H, J ) 8.7), 7.76 (d, 1H, J )
7.2). Anal. (C30H38FN5O7) C, H, N.
E t h yl-3-{Boc -L -Va lΨ[COCH 2]-L -P h e (4-F )-L -[(N -2,4-
d im eth oxyben zyl)-(S)-P yr r ol-Ala ]}-E-p r op en oa te (36). A
solution of HCl in 1,4-dioxane (4.0 M, 10 mL) was added to a
solution of 22 (0.342 g, 0.718 mmol, 1 equiv) in the same
solvent (10 mL) at 23 °C. After the mixture was stirred for
1.5 h at that temperature, the volatiles were removed under
reduced pressure to give the crude amine salt. This material
was combined with crude 358 (1.00 mmol, 1.4 equiv) and
dissolved in CH2Cl2 (17 mL) at 23 °C. HOBt (0.165 g, 1.22
mmol, 1.7 equiv), 4-methylmorpholine (0.316 mL, 2.87 mmol,
4.0 equiv) and EDC (0.234 g, 1.22 mmol, 1.7 equiv) were added
sequentially. After stirring overnight at 23 °C, the reaction
mixture was partitioned between brine (30 mL) and a 1:9
mixture of CH3OH and CH2Cl2 (3 × 70 mL). The organic layers
were dried over Na2SO4 and were concentrated. The residue
was purified by flash column chromatography (55% EtOAc in
hexanes) to provide 36 (0.337 g, 63%) as a white foam: Rf )
0.24 (60% EtOAc in hexanes); IR (cm-1) 3293, 1717, 1668; 1H
NMR (CDCl3) δ 0.82 (d, 3H, J ) 6.8), 1.01 (d, 3H, J ) 6.8),
1.30 (t, 3H, J ) 7.2), 1.51-1.65 (m, 2H), 1.84-1.96 (m, 1H),
2.16-2.37 (m, 2H), 2.47 (s, 3H), 2.49-2.55 (m, 3H), 2.85-3.01
(m, 2H), 3.12-3.25 (m, 3H), 3.77 (s, 3H), 3.78 (s, 3H), 4.18 (q,
2H, J ) 7.2), 4.31-4.49 (m, 3H), 4.65-4.70 (m, 1H), 5.53 (dd,
1H, J ) 15.7, 1.4), 6.39-6.44 (m, 3H), 6.63 (dd, 1H, J ) 15.7,
5.4), 6.93-7.01 (m, 2H), 7.05-7.10 (m, 1H), 7.12-7.18 (m, 2H),
7.24 (d, 1H, J ) 8.7), 7.47 (d, 1H, J ) 6.5). Anal. (C40H54FN3O9‚
0.50H2O) C, H, N.
1
IR (cm-1) 3284, 1713, 1678 br, 1643; H NMR (CDCl3) δ 0.91
(d, 3H, J ) 6.8), 0.97 (d, 3H, J ) 6.8), 1.28 (t, 3H, J ) 7.2),
1.45 (s, 9H), 1.50-1.62 (m, 2H), 1.66-1.82 (m, 1H), 1.90-2.02
(m, 1H), 2.08-2.21 (m, 2H), 2.94 (dd, 1H, J ) 13.5, 5.8), 3.17-
3.27 (m, 3H), 3.80 (s, 3H), 3.82 (s, 3H), 3.97-4.05 (m, 1H), 4.17
(q, 2H, J ) 7.2), 4.27 (d, 1H, J ) 14.3), 4.29-4.38 (m, 1H),
4.40 (d, 1H, J ) 14.3), 4.86-4.93 (m, 1H), 5.10 (d, 1H, J )
8.7), 5.76 (dd, 1H, J ) 15.6, 1.2), 6.45-6.52 (m, 2H), 6.70 (dd,
1H, J ) 15.6, 5.4), 6.79-6.88 (m, 3H), 7.12-7.22 (m, 3H), 8.30
(d, 1H, J ) 5.9). Anal. (C39H53FN4O9) C, H, N.
Eth yl-3-{(5′-m eth ylisoxa zole-3′-ca r bon yl)-L-Va l-L-P h e-
(4-F )-L-[(N-2,4-d im eth oxyben zyl)-(S)-P yr r ol-Ala ]}-E-p r o-
p en oa te (34). A solution of HCl in 1,4-dioxane (4.0 M, 10 mL)
was added to a solution of 33 (0.410 g, 0.553 mmol, 1 equiv)
in the same solvent (10 mL) at 23 °C. After the mixture was
stirred for 1.5 h at 23 °C, the solvent was evaporated to give
the crude amine salt which was dissolved in CH2Cl2 (20 mL)
and cooled to 0 °C. 4-Methylmorpholine (0.134 mL, 1.22 mmol,
2.2 equiv) and 5-methylisoxazole-3-carbonyl chloride (0.089 g,
0.61 mmol, 1.1 equiv) were added sequentially, and the ice
bath was removed. After being stirred for 1 h at 23 °C, the
reaction mixture was diluted with MTBE (200 mL), washed
with 5% KHSO4 (20 mL), saturated NaHCO3 (20 mL), and
brine (20 mL), dried over Na2SO4, and concentrated. The
residue was purified by flash column chromatography (2%
CH3OH in CH2Cl2) to provide 34 (0.288 g, 69%) as a white
foam: Rf ) 0.36 (5% CH3OH in CH2Cl2); IR (cm-1) 3284, 1717,
E t h y l-3-{(5′-m e t h y lis o x a zo le -3′-c a r b o n y l)-L -Va lΨ-
[COCH2]-L-P h e(4-F )-L-[(N-2,4-d im eth oxyben zyl)-(S)-P yr -
r ol-Ala ]}-E-p r op en oa te (37). A solution of HCl in 1,4-dioxane
(4.0 M, 5 mL) was added to a solution of 36 (0.309 g, 0.418
mmol, 1 equiv) in the same solvent (5 mL) at 23 °C. After the
mixture was stirred for 1.5 h at 23 °C, the volatiles were
removed under reduced pressure to give the crude amine salt.
This material was dissolved in CH2Cl2 (15 mL) and cooled to
0 °C. 4-Methylmorpholine (0.101 mL, 0.919 mmol, 2.2 equiv)
and 5-methylisoxazole-3-carbonyl chloride (0.067 g, 0.46 mmol,
1.1 equiv) were added sequentially, and the ice bath was
removed. After being stirred for 1 h at 23 °C, the reaction
mixture was partitioned between brine (15 mL) and a 1:9
mixture of CH3OH and CH2Cl2 (3 × 30 mL). The organic layers
were dried over Na2SO4 and were concentrated. The residue
was purified by flash column chromatography (70% EtOAc in
hexanes) to provide 37 (0.266 g, 85%) as a white foam: Rf )
0.24 (60% EtOAc in hexanes); IR (cm-1) 3293, 1717, 1668; 1H
NMR (CDCl3) δ 0.82 (d, 3H, J ) 6.8), 1.01 (d, 3H, J ) 6.8),
1.30 (t, 3H, J ) 7.2), 1.51-1.65 (m, 2H), 1.84-1.96 (m, 1H),
2.16-2.37 (m, 2H), 2.47 (s, 3H), 2.49-2.55 (m, 3H), 2.85-3.01
(m, 2H), 3.12-3.25 (m, 3H), 3.77 (s, 3H), 3.78 (s, 3H), 4.18 (q,
2H, J ) 7.2), 4.31-4.49 (m, 3H), 4.65-4.70 (m, 1H), 5.53 (dd,
1H, J ) 15.7, 1.4), 6.39-6.44 (m, 3H), 6.63 (dd, 1H, J ) 15.7,
5.4), 6.93-7.01 (m, 2H), 7.05-7.10 (m, 1H), 7.12-7.18 (m, 2H),
7.24 (d, 1H, J ) 8.7), 7.47 (d, 1H, J ) 6.5). Anal. (C40H49FN4O9‚
0.5H2O) C, H, N.
1
1650; H NMR (CDCl3) δ 0.97 (d, 3H, J ) 6.8), 1.01 (d, 3H, J
) 6.8), 1.28 (t, 3H, J ) 7.2), 1.51-1.64 (m, 2H), 1.72-1.84 (m,
1H), 1.95-2.05 (m, 1H), 2.11-2.33 (m, 2H), 2.48 (s, 3H), 2.98
(dd, 1H, J ) 13.7, 5.6), 3.16-3.24 (m, 3H), 3.80 (s, 3H), 3.81
(s, 3H), 4.17 (q, 2H, J ) 7.2), 4.23 (d, 1H, J ) 14.3), 4.31-4.42
(m, 1H), 4.40 (d, 1H, J ) 14.3), 4.44-4.50 (m, 1H), 4.88-4.96
(m, 1H), 5.79 (dd, 1H, J ) 15.6, 1.4), 6.43-6.49 (m, 3H), 6.71
(dd, 1H, J ) 15.6, 5.3), 6.80-6.88 (m, 2H), 6.94 (d, 1H, J )
9.3), 7.11-7.17 (m, 3H), 7.29 (d, 1H, J ) 8.7), 8.33 (d, 1H, J )
6.2). Anal. (C39H48FN5O9‚0.50H2O) C, H, N.
Eth yl-3-{(5′-m eth ylisoxa zole-3′-ca r bon yl)-L-Va l-L-P h e-
(4-F )-L-[(S)-P yr r ol-Ala ]}-E-p r op en oa te (9). A suspension of
34 (0.263 g, 0.351 mmol, 1 equiv), water (2 drops), and DDQ
(0.104 g, 0.458 mmol, 1.3 equiv) in CHCl3 (10 mL) was refluxed
for 9 h and then allowed to cool to room temperature over 8 h.
The reaction mixture was diluted with CH2Cl2 (200 mL) and
washed with a 2:1 mixture of saturated NaHCO3 and 1 M
NaOH (20 mL). The organic phase was dried over MgSO4 and
was concentrated. Purification of the residue by flash column
chromatography (gradient elution, 2f3% CH3OH in CH2Cl2)
gave 9 (0.117 g, 56%) as a white solid: mp ) 219-220 °C; Rf
) 0.23 (5% CH3OH in CH2Cl2); IR (cm-1) 3401 br, 3295, 1655
E t h y l-3-{(5′-m e t h y lis o x a zo le -3′-c a r b o n y l)-L -Va lΨ-
[COCH2]-L-P h e(4-F)-L-[(S)-P yr r ol-Ala ]}-E-pr open oate (11).
A suspension of DDQ (0.102 g, 0.449 mmol, 1.4 equiv), 37
(0.240 g, 0.320 mmol, 1 equiv), and water (2 drops) in CHCl3
(10 mL) was refluxed for 20 h and then allowed to cool. The
reaction mixture was diluted with CH2Cl2 (200 mL) and
washed with a 2:1 mixture of saturated NaHCO3 and 1 M
NaOH (20 mL). The organic phase was dried over Na2SO4 and
was concentrated. Purification of the residue by flash column
chromatography (2% CH3OH in CH2Cl2) gave 11 (0.074 g, 39%)
as a faintly yellow solid: mp ) 178-181 °C; Rf ) 0.49 (10%
1
br; H NMR (CDCl3) δ 0.94 (d, 3H, J ) 6.8), 0.97 (d, 3H, J )
1
6.5), 1.29 (t, 3H, J ) 7.2), 1.54-1.65 (m, 1H), 1.72-1.91 (m,
2H), 2.07-2.26 (m, 2H), 2.28-2.39 (m, 1H), 2.49 (d, 3H, J )
0.9), 3.01 (dd, 1H, J ) 13.8, 6.1), 3.12 (dd, 1H, J ) 13.8, 6.4),
3.26-3.38 (m, 2H), 4.18 (q, 2H, J ) 7.2), 4.34 (dd, 1H, J )
8.7, 7.2), 4.43-4.54 (m, 1H), 4.90 (dt, 1H, J ) 9.0, 6.2), 5.76
(dd, 1H, J ) 15.6, 1.6), 6.00 (s, 1H), 6.42 (q, 1H, J ) 0.9), 6.72
CH3OH in CHCl3); IR (cm-1) 3295, 1678 br; H NMR (CDCl3)
δ 0.85 (d, 3H, J ) 6.8), 1.03 (d, 3H, J ) 6.5), 1.30 (t, 3H, J )
7.2), 1.51-1.62 (m, 1H), 1.71-1.93 (m, 2H), 2.27-2.40 (m, 2H),
2.47 (s, 3H), 2.51-2.75 (m, 3H), 2.82-2.98 (m, 2H), 3.11-3.24
(m, 1H), 3.26-3.42 (m, 2H), 4.18 (q, 2H, J ) 7.2), 4.41-4.53
(m, 1H), 4.63-4.72 (m, 1H), 5.50 (d, 1H, J ) 15.4), 5.88 (s,

P1 Lactam Moieties as L-Glutamine Replacements
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 7 1223
1H), 6.39 (s, 1H), 6.63 (dd, 1H, J ) 15.4, 5.3), 6.92-7.03 (m,
2H), 7.08-7.31 (m, 4H). Anal. (C31H39FN4O7) C, H, N.
wavelength detector. The standard curve for compound 11
ranged from 0.05 µg/mL to 40.0 µg/mL. Using the above HPLC
conditions, the retention time for compound 11 was ap-
proximately 13 min. The hydrolysis of 1-naphthyl butyrate
served as a positive control for carboxyl esterase activity in
human plasma.
Molecu la r Mod elin g Stu d ies. A simple computational
approach was employed to estimate the binding free energies
of complexes formed between the HRV 3C protein (P) and
various ligands (L). This approach assumes that a single
protein-ligand conformation predominates in the bound com-
plex while another dominates in solution. The protein-ligand
association was expressed as a two-step process involving
hypothetical bound conformations of the ligand and protein
in solution, where the binding energy is given by the sum of
an interaction term and two strain energies (eq 1). Unlike free
P r otea se Selectivity Assa ys. Assays were performed
using commercially available proteases (at ∼10 nM concentra-
tions) essentially as described by the supplier. Human liver
cathepsin B, porcine erythrocyte calpain I, and human neu-
trophil elastase were purchased from Calbiochem. Bovine
chymotrypsin and human thrombin were purchased from
Boehringer Mannheim. Bovine trypsin was purchased from
Sigma.
Degr a d a tion Stu d ies of Com p ou n d 11. Compound 11
(25 µM) and 100 mM potassium phosphate buffer (pH 7.4) were
preincubated in test tubes for 5 min at 37 °C in a shaking
water bath. Chymotrypsin (Sigma, 5 units/mL) or porcine
intestinal peptidase (Sigma, 0.1 units/mL) was preincubated
at 37 °C and then added to the appropriate test tube containing
11 to initiate the reaction. After 0 and 60 min, a 200 µL aliquot
of each incubation mixture was transferred to a microcentri-
fuge tube containing 200 µL of CH3CN. These tubes were
capped, vortexed, and centrifuged in an Eppendorf (Hamburg)
5415C centrifuge at 14 000 rpm for 10 min. A 15 µL portion of
each supernatant was then injected onto a Hewlett-Packard
1100 HPLC equipped with a Zorbax RX C8 (2.1 m × 150 mm)
column (DuPont, Wilmington, DE). The aqueous component
of the mobile phase was 0.1% Et3N in water adjusted to pH
3.5 with approximately 0.05% trifluoroacetic acid. Compound
11 was eluted using a solvent gradient beginning with 55:25:
20 buffer:CH3CN:CH3OH, increasing to 20:60:20 buffer:CH3CN:
CH3OH over 10 min and held for 10 min before returning to
the original conditions at a flow rate of 0.3 mL/min. Compound
11 was detected by UV at 220 nm. The hydrolysis of 1-naphthyl
butyrate served as a positive control for the esterase activity
of the peptidase under study.
P la sm a Sta bility Stu d ies of Com p ou n d 11. Human
plasma anticoagulated with CPDA (citrate phosphate dextrose
adenine) was obtained from the San Diego Blood Bank, San
Diego, CA. Heparinized plasma from several animal species
was purchased from Covance Research, Denver, PA. The
plasma was distributed into 5 mL cryotubes and frozen at -70
°C until analysis. In preparation for the experiment, 1990 µL
of plasma (n ) 3) or 1990 µL of 100 mM potassium phosphate
pH 7.4 buffer (n ) 3) were placed into separate test tubes.
Because of enhanced plasma esterase activity relative to that
of other species, rat plasma was diluted 1:1 with 100 mM
potassium phosphate pH 7.4 buffer (n ) 3) prior to the
experiments. All tubes were preincubated in a shaking water
bath for a few minutes until the solutions had reached 37 °C.
At the start of the incubation, 10 µL of a freshly made 5 mM
solution of compound 11 in CH3CN was pipetted into a test
tube containing plasma or buffer to achieve a final concentra-
tion of 25 µM. After vortexing the tube, a 200 µL sample was
collected, and the tube was returned to the water bath. The
test tubes were then incubated at 37 °C and 200 µL samples
were collected periodically for 1 h. These samples were
immediately transferred to borosilicate tubes prefilled with 2.0
mL of CH3CN, and the mixtures were vortexed to ensure
protein precipitation and termination of metabolic transforma-
tions. After subsequent centrifugation for 10 min at 4000 rpm
at 10 °C in a Sorvall RT 7 centrifuge, the clear supernatant
was decanted into a new set of borosilicate tubes and the
volatiles were removed under a gentle stream of nitrogen using
a Dri-Block sample concentrator (Techne, Princeton, NJ ) at
40 °C. The samples were reconstituted in 250 µL of mobile
phase (60% 25 mM NH4H2PO4 at pH ) 5.1 and 40% CH3CN)
and subjected to HPLC analysis.
bound
LP
∆G ) E
- EuLnbound - EPunbound
bound
LP
) [E
- EbLound - EbPound] + [ELbound - ELunbound] +
[EbPound - EPunbound
]
interaction
LP
) ∆E
+ ∆EsLtrain + ∆EPstrain
(1)
energy perturbation methods, this approach neglects entropic
contributions to binding affinity and represents a zero-tem-
perature enthalpic estimate for the binding free energy. A
simple entropic correction was therefore applied to the calcu-
lated binding affinities of the noncovalent encounter complexes
to account for this fact.21
The binding energy of the complex was obtained by local
minimization using the Amber* force field and GB/SA solva-
tion correction contained in the Macromodel (v5.5) software
package.31 The ligand was assigned partial charges and
minimized in an enzyme active site that was reduced to 631
atoms to allow truncated-Newton conjugate gradient (TNCG)
minimization. Constraints to the protein were applied in
concentric spheres with atoms far from the ligand fixed rigidly
in their crystallographic positions. Atoms near the ligand were
constrained with increasing flexibility, utilizing a harmonic
force constant of 400 kJ /Å at the outer edge of the site, 200
kJ /Å between 10 and 15 Å from the ligand, 10 kJ /Å between
5 and 10 Å from the ligand, and no constraints on atoms less
than 5 Å from the ligand. The strain energies were obtained
in an analogous manner.
Meth od No. 1. A minimized model of the covalent adduct
between the glutamine-derived compound 1 and HRV 3CP was
prepared using the HRV-2 3CP-1 cocrystal structure.5 Covalent
models of the lactam-containing molecules 4, 5, and 6 were
similarly constructed. Nonbonded protein-ligand interactions
were assumed to determine binding trends in the series. No
additional constraints were applied to the covalent connection
between the protein and the ligand since bond-making and
bond-breaking terms were assumed to cancel for each ligand.
Meth od No. 2. Noncovalent encounter complexes between
HRV 3CP and compounds 1, 4, 5, and 6 were constructed from
the HRV-2 3CP-1 co-crystal structure5 and were investigated
as described above. For these complexes, an entropic correc-
tion21 was applied to the calculated binding affinities.
Ack n ow led gm en t. We are grateful for many helpful
discussions throughout the course of this work with
Prof. Larry Overman and Dr. Steven Bender. We also
thank Dr. Kim Albizati, Dr. Srinivasan Babu, Terence
Moran, Ray Dagnino, and Miguel Pagan for the large-
scale preparation of several intermediates.
Chromatographic analyses were performed using a 1100
Hewlett-Packard HPLC with a Primesphere reversed phase
column (5 µm, 4.6 m × 150 mm, Phenomenex, Torrance, CA)
at flow rate 1 mL/min and a gradient elution of NH4H2PO4 25
mM buffer, pH ) 5.1/CH3CN/CH3OH (95/3/2 to 70/28/2 over 5
min, changing to 50/48/2 over the next 5 min, and reverting
to 95/3/2 for the final 5 min). A sample volume of 100 µL was
injected onto the column, and compound 11 was detected by
UV absorption at 212 nm utilizing a Hewlett-Packard multi-
Refer en ces
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(15) Dragovich, P. S.; Zhou, R.; Skalitzky, D. J .; Fuhrman, S. A.;
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Phase Synthesis of Irreversible Human Rhinovirus 3C Protease
Inhibitors. 1. Optimization of Tripeptides Incorporating N-
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culture. In general, the activity observed for a given compound
vs serotypes 1A, 2, and 10 was representative of its broad-
spectrum antiviral properties. Serotype 14 nearly always proved
to be the most susceptible to the antiviral agents under study.
Compound 11 (AG7088) has been tested against 46 HRV
serotypes to date and is active against all with a mean EC90 of
approximately 0.10 µM. Full details of these experiments will
be presented elsewhere (Patick, A. K. Manuscript in prepara-
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(17) Andries, K.; Dewindt, B.; Snoeks, J .; Willebrords, R.; Van
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(19) The R,â-unsaturated carboxylic acids corresponding to the
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(20) The assumption that rat plasma degradation of 11 primarily
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(21) Mammen, M.; Shakhnovich, E. I.; Whitesides, G. M. Using a
Convenient, Quantitative Model for Torsional Entropy To Es-
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Assignment of Stereochemistry in the Oligomycin/Rutamycin/
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(25) The diastereomeric excess obtained in the preparation of 18 was
not rigorously determined, but it is inferred to be high based on
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diastereomers in the 1H NMR spectra of 18 and subsequent
synthetic intermediates.
(26) (a) Mancuso, A. J .; Swern, D. Activated Dimethyl Sulfoxide:
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Tidwell, T. T. Oxidation of Alcohols by Activated Dimethyl
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(27) Mori, S.; Iwakura, H.; Takechi, S. A New Amidoalkynylation
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5394 and references therein.
(28) Overman, L. E.; Osawa, T. A Convenient Synthesis of 4-Unsub-
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(29) During the preparation of 6, the diastereomeric excess of the
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rigorously determined. See ref 25 above.
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Substrates by the Human Rhinovirus 14 3C Proteinase, Cloned
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(4) The nomenclature used for describing the individual amino acid
residues of a peptide substrate (P2, P1, P1′, P2′, etc.) and the
corresponding enzyme subsites (S2, S1, S1′, S2′, etc.) is described
in the following: Schechter, I.; Berger, A. On the Size of the
Active Site in Proteases. I. Papain. Biochem. Biophys. Res.
Commun. 1967, 27, 157-162.
(5) Dragovich, P. S.; Webber, S. E.; Babine, R. E.; Fuhrman, S. A.;
Patick, A. K.; Matthews, D. A.; Lee, C. A.; Reich, S. R.; Prins, T.
J .; Marakovits, J . T.; Littlefield, E. S.; Zhou, R.; Tikhe, J .; Ford,
C. E.; Wallace, M. B.; Meador, J . W., III; Ferre, R. A.; Brown, E.
L.; Binford, S. L.; Harr, J . E. V.; DeLisle, D. M.; Worland, S. T.
Structure-Based Design, Synthesis, and Biological Evaluation
of Irreversible Human Rhinovirus 3C Protease Inhibitors. 1.
Michael Acceptor Structure-Activity Studies. J . Med. Chem.
1998, 41, 2806-2818.
(6) Dragovich, P. S.; Webber, S. E.; Babine, R. E.; Fuhrman, S. A.;
Patick, A. K.; Matthews, D. A.; Reich, S. H.; Marakovits, J . T.;
Prins, T. J .; Zhou, R.; Tikhe, J .; Littlefield, E. S.; Bleckman, T.
M.; Wallace, M. B.; Little, T. L.; Ford, C. E.; Meador, J . W., III;
Ferre, R. A.; Brown, E. L.; Binford, S. L.; DeLisle, D. M.;
Worland, S. T. Structure-Based Design, Synthesis, and Biological
Evaluation of Irreversible Human Rhinovirus 3C Protease
Inhibitors. 2. Peptide Structure-Activity Studies. J . Med. Chem.
1998, 41, 2819-2834.
(7) A similar series of HRV 3CP inhibitors has also recently been
described. See: Kong, J .-s.; Venkatraman, S.; Furness, K.;
Nimkar, S.; Shepherd, T. A.; Wang, Q. M.; Aube´, J .; Hanzlik, R.
P. Synthesis and Evaluation of Peptidyl Michael Acceptors That
Inactivate Human Rhinovirus 3C Protease and Inhibit Virus
Replication. J . Med. Chem. 1998, 41, 2579-2587.
(8) Preceding paper in this issue. Dragovich, P. S.; Prins, T. J .; Zhou,
R.; Fuhrman, S. A.; Patick, A. K.; Matthews, D. A.; Ford, C. E.;
Meador, J . W., III; Ferre, R. A.; Worland, S. T. Structure-Based
Design, Synthesis, and Biological Evaluation of Irreversible
Human Rhinovirus 3C Protease Inhibitors. 3. Structure-Activ-
ity Studies of Ketomethylene-Containing Peptidomimetics. J .
Med. Chem. 1999, 42, 1203-1212.
(9) All amino acids described in the text are L-isomers unless
otherwise noted.
(10) Note that the reduction in the antiviral potency of 2 (or 3)
relative to 1 was not nearly as great as the loss in 3CP inhibition
activity. This observation suggested that Gln amide N-alkylation
may result in improved cell permeability properties.
(11) The loss in anti-3CP activity of 2 relative to 1 was approximately
equal to the expected energetic difference between the cis and
trans amide isomers of 2 (2.2 kcal/mol extrapolated from
measured kobs/[I] values vs 2.6 kcal/mol reported for simple model
systems).12 This analysis assumes that changes in kobs/[I] values
approximate changes in ligand-3CP binding affinity and that
the anti-3CP activities of cis- and trans-2 are similar to those
displayed by compounds 1 and 3, respectively.
(30) Webber, S. E.; Okano, K.; Little, T. L.; Reich, S. H.; Xin, Y.;
Fuhrman, S. A.; Matthews, D. A.; Hendrickson, T. F.; Love, R.
A.; Patick, A. K.; Meador, J . W., III; Ferre, R. A.; Brown, E. L.;
Ford, C. E.; Binford, S. L.; Worland, S. T. Tripeptide Aldehyde
Inhibitors of Human Rhinovirus 3C Protease: The Design,
Synthesis, Biological Evaluation and Cocrystal Structure Solu-
tion of P1 Glutamine Isosteric Replacements. J . Med. Chem.
1998, 41, 2786-2805.
(31) Mohamadi, F.; Richards, N. G. J .; Guida, W. C.; Liskamp, R.;
Lipton, M.; Caufield, C.; Chang, G.; Hendrickson, T.; Still, W.
C. MacroModel-An Integrated Software System for Modeling
Organic and Bioorganic Molecules Using Molecular Mechanics.
J . Comput. Chem. 1990, 11, 440-467.
(12) J orgensen, W. L.; Gao, J . Cis-Trans Energy Difference for the
Peptide Bond in the Gas Phase and in Aqueous Solution. J . Am.
Chem. Soc. 1988, 110, 4212-4216 and references therein.
(13) In several cases, the solubility of a given inhibitor was deter-
mined to be less than the reported CC50 value. However, the
most active antiviral agents were clearly soluble and nontoxic
to more than 10× the reported EC50 values.
(14) Analysis of the HRV-2 3CP-8 X-ray crystal structure showed that
the lactam-containing inhibitor 8 bound to 3CP in a manner
nearly identical to that observed previously5 for the glutamine-
derived molecule 1 (Matthews, D. A. Unpublished results).
J M9805384