From 1-acyl-β-lactam human cytomegalovirus protease inhibitors to 1-benzyloxycarbonylazetidines with improved antiviral activity. A straightforward approach to convert covalent to noncovalent inhibitors

Article abstract of DOI:10.1021/jm0492812

Starting from the structure of known β-lactam covalent human cytomegalovirus (HCMV) protease inhibitors and from the knowledge of the residues implicated in the active site of this enzyme, we designed a series of phenylalanine-derived 2-azetidinones beari

Full text of DOI:10.1021/jm0492812

2612
J. Med. Chem. 2005, 48, 2612-2621
From 1-Acyl-â-lactam Human Cytomegalovirus Protease Inhibitors to
1-Benzyloxycarbonylazetidines with Improved Antiviral Activity. A
Straightforward Approach To Convert Covalent to Noncovalent Inhibitors
Guillermo Gerona-Navarro,†,‡ M. Jesu´s Pe´rez de Vega,†,‡ M. Teresa Garc´ıa-Lo´pez,† Graciela Andrei,§
Robert Snoeck,§ Erik De Clercq,§ Jan Balzarini,§ and Rosario Gonza´lez-Mun˜iz*,†
Instituto de Quı´mica Me´dica (CSIC), Juan de la Cierva 3, 28006 Madrid, Spain, and Rega Institute for Medical Research,
Katholieke Universiteit Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium
Received September 2, 2004
Starting from the structure of known â-lactam covalent human cytomegalovirus (HCMV)
protease inhibitors and from the knowledge of the residues implicated in the active site of this
enzyme, we designed a series of phenylalanine-derived 2-azetidinones bearing a 4-carboxylate
moiety that could be apt for additional interactions with the guanidine group of the Arg165/
Arg166 residues of the viral protease. Some compounds within this series showed anti-HCMV
activity at 10-50 µM, but rather high toxicity. The presence of aromatic 1-acyl groups and a
certain hydrophobic character in the region of the 4-carboxylate were stringent requirements
for anti-HCMV activity. To go a step ahead into the search for effective HCMV medicines, we
then envisaged a series of noncovalent inhibitors by simple deletion of the carbonyl group in
the â-lactam derivatives to provide the corresponding azetidines. This led to low micromolar
inhibitors of HCMV replication, with 17 and 27 being particularly promising lead compounds
for further investigation, although their toxicity still needs to be lowered.
Introduction
replication cycle.11-16 Among them, the indolizine-1-
carboxamide MCV423, a highly effective anti-HCMV
agent in vitro, acts on a step of the replicative cycle that
precedes the DNA polymerase step, most likely at the
intermediate early (IE) antigen synthesis.14 The L-ribose
benzimidazole derivative 1263W94 (maribavir), that
appears to be targeted to the UL97 protein kinase, is
at present under clinical trials.15 More recently, a series
of thiourea small molecule derivatives were identified
as the most potent anti-HCMV agents described to date
(nanomolar and subnanomolar inhibitory concentra-
tions).17-19 These compounds act very early in the
replication cycle, inhibiting virion envelope fusion with
the cell plasma membrane.19
A great part of the above-mentioned HCMV inhibitors
were identified through the screening of various classes
of compounds, but some approaches have also been
directed toward the rational design of anti-HCMV
therapies, one of the most relevant being the search for
HCMV protease inhibitors. The HCMV pathogen en-
codes a 780-residue serine protease essential for capsid
assembly and viral maturation.20 The X-ray crystal-
lography revealed that this protein has a backbone fold
unique among serine proteases and an unusual catalytic
triad formed by Ser132, Thr63, and Thr157 residues21-23
that make this protein an attractive target for antiviral
chemotherapy.24-26 Overlays of the catalytic triad of
HCMV protease with that of trypsin suggested that
Arg165 and Arg166 are involved in stabilization of the
oxyanion intermediate formed during the hydrolysis
reaction. The importance of the side chain of these Arg
residues in catalysis has been confirmed by site-directed
mutagenesis studies.27
Human cytomegalovirus (HCMV) is a ubiquitous
member of the â-herpes virus family. Although HCMV
infection of healthy children and adults is usually
asymptomatic, it is a leading cause of birth defects1 and
may result in severe, often life-threatening diseases in
immunocompromised (HIV)2 or immunosuppressed (or-
gan/bone marrow transplant) individuals.3 Thus, an
elevated percentage of kidney, liver, bone marrow, and
heart/lung transplant recipients are affected by HCMV
hepatitis and pneumonia, resulting in decreased graft
and patient survival.3 Although the use of antiretroviral
therapies has significantly diminished the impact of
HCMV diseases in AIDS patients, cessation of treat-
ments in these patients led to recurrence or retinitis,
colitis, and esophagitis, among other HCMV manifesta-
tions.4
Currently, five compounds have been licensed for the
prophylaxis and treatment of HCMV infection: ganci-
clovir (GCV), foscarnet, cidofovir (CDV), valganciclovir,
and fomivirsen.5-7 Despite their antiviral potential, all
these medications suffer from a number of drawbacks,
such as dose-limiting bone marrow and kidney toxicity,
as well as the emergence of single and multiple drug
resistance.8 This has prompted the search for other anti-
HCMV agents that could be potent, safe, and orally
bioavailable, with novel mechanisms of action. Apart
from a few new inhibitors of the viral specific DNA
polymerase,9,10 different nucleoside derivatives as well
as non-nucleoside inhibitors have been discovered, in
recent years, to operate at different states of the
* To whom correspondence should be addressed. Tel: +34-91-
5622900. Fax: +34-91-5644853. E. mail: iqmg313@iqm.csic.es.
† Instituto de Qu´ımica Me´dica.
Since the active site of the HCMV protease has been
identified, the search for inhibitors of this enzyme has
become mainstream. Most of these inhibitors contain
‡ G. G.-N. and M. J. P. V. contributed equally to this work.
§ Rega Institute for Medical Research.
10.1021/jm0492812 CCC: $30.25 © 2005 American Chemical Society
Published on Web 02/23/2005

Anti-HCMV 1-Acyl-â-lactams and 1-Acylazetidines
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 7 2613
Chart 1
Scheme 1a
classical serine protease inhibitor motifs based on an
activated carbonyl group, such as peptidyl keto-
amides,28,29 as well as mechanism-based inhibitors,
such as oxazazinones30,31 and pyrrolidine-5,5-trans-
lactams.32-35 Among the latter, a series of monocyclic
â-lactams 1 (compound 1a being the prototype, Chart
1) has resulted in highly potent derivatives in the
isolated enzyme assay, but their efficacy in cell culture
was quite limited, as for all described inhibitors of this
enzyme.36-40 Despite the vast efforts made in this field,
none of these protease inhibitors has reached the clinic,
most likely due to the fact that they covalently bind to
the enzyme, as probably they also do to other macro-
molecules on their way to the target.
On the basis of the structure of the reported â-lactam
inhibitors 137 and of the residues implicated in the active
site of the HCMV protease,11 our first approach to potent
anti-HCMV agents was directed to increase the contact
points between the protein and the inhibitor. For such
a purpose, a series of phenylalanine-derived 2-azetidi-
nones of general formula A were designed (Chart 1). In
comparison with model compounds 1, these new â-lac-
tams have an additional 4-carboxylate group that could
be suitable for extra interactions with the guanidinium
groups of the Arg165 or Arg166 residue of the viral
protease. Although some compounds in this first series
showed slightly improved anti-HCMV activity when
compared to the leading â-lactam 1a, these compounds
are still less active than the reference compounds
ganciclovir and cidofovir. For the development of thera-
peutically useful serine protease inhibitors with good
pharmacological characteristics, it has generally been
accepted that noncovalent inhibitors are preferred over
covalent ones (even over covalent reversible inhibi-
tors).41,42 With this in mind, we envisaged a series of
noncovalent inhibitors, the azetidine derivatives B,
generated by deletion of the carbonyl lactam group in
compounds A, while enough hydrogen bonds and ionic
and/or van der Waals interactions were maintained with
the biological target. Related serine trap deletion strate-
gies have successfully guided the design of noncovalent
inhibitors of some mammalian serine proteases.41,42
a Reagents and conditions: (a) R2NCO/Cs2CO3/THF. (b) TFA/
DCM. (c) PhCH2OCOCl/DBU/DCM. (d) (Boc)2O/TEA/DMPA/DCM.
This paper deals with the synthesis and evaluation
of the above-mentioned series of compounds, azetidino-
nes A and azetidines B,43 and proposes a general
approach to convert covalent viral protease inhibitors
into noncovalent antiviral agents.
Results and Discussion
1-Acyl-â-lactam Derivatives. The starting N-un-
substituted 2-azetidinones 2ab and 3ab were prepared
from H-Phe-OtBu and H-Phe-OMe, respectively, as
previously described.44 A moderate selectivity during the
cyclization reaction, attributed to the occurrence of a
“memory of chirality” phenomenon,45,46 led to com-
pounds 2 and 3 as mixtures of 4S:4R enantiomers in
about a 2:1 a:b ratio. Taking into account that the
acylation of related â-lactams with isocyanates generally
proceeded in low yield,37 we first investigated the
reaction of azetidinone 2ab with phenyl isocyanate
under different conditions (Scheme 1). Among TEA/
DMAP, DBU, and Cs2CO3, the latter base was found to
be the most effective for the synthesis of compound 4ab

2614 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 7
Gerona-Navarro et al.
Scheme 2a
prepared by treatment of 3 with (S)-1-phenylethyl
isocyanate, followed by chromatographic resolution.
Compounds 5a, 5b, 6a, and 6b were hydrolyzed to the
corresponding free carboxylic acids 8a, 8b, 9a, and 9b
by treatment with (1:4) TFA/CH2Cl2. The absolute
configuration at C4 in the main diastereoisomers a was
assigned as S, since major enantiomers in 2 and 3 have
S configuration, as previously established.47 Neverthe-
less, to corroborate that the aliphatic-aromatic dipep-
tide rule48,49 is also valid for these 1-acyl-â-lactams,
compounds 8a and 8b were transformed into the cor-
responding Ala-dipeptide derivatives 12a and 12b. As
expected, the signal corresponding to the â-H protons
of Ala residue is more shielded in the R,S-dipeptide 12b
than in the S,S-analogue 12a (Scheme 2),48 while in
HPLC the heterochiral derivative 12b is more retained
than the homochiral one.49
Finally, the 1-urethane-substituted â-lactams 10 and
11 were prepared as 2:1 S:R enantiomeric mixtures by
treatment of azetidinone 2 with benzyl chloroformate
and tert-butyl dicarbonate, respectively, as previously
described.50
The target 1-acyl-â-lactams 4-11 were evaluated for
their ability to inhibit the replication of HCMV in
vitro,14 and the results were compared to those obtained
for the reference compounds ganciclovir and cidofovir
(Table 1). For comparative purposes, the reported anti-
HCMV activity of the model â-lactam 1a was also
included in the table. Compounds 4-11 did not interfere
a (a) H-Ala-OMe/BOP/TEA/THF.
(82%). Using these optimized conditions, the reaction
of compound 2 with (R)-1- and (S)-1-phenylethyl iso-
cyanate afforded compounds 5ab and 6ab, respectively.
These diastereoisomeric mixtures, which were easily
separated by column chromatography, were obtained in
approximately 1.5:1 a:b ratio, indicating some kinetic
resolution during the acylation reaction. The methyl
ester-containing derivatives 7a (62%) and 7b (29%) were
Table 1. Activity of 1-Acyl-â-lactam Derivatives against Human Cytomegalovirus (HCMV) and Varicella-zoster (VZV) Virus in HEL
Cell Cultures
EC50 (µM)
anti-VZV activityb
anti-HCMV activitya
cytotoxicity (µM)
OKA
(TK+)
07/1
R1
R2
AD-169
Davis
(TK-)
MCCc
CC50
d
compd
4ab
tBu
tBu
tBu
tBu
tBu
Me
Me
H
Ph
50
50
33
13
200
118
70
>200
184
164
>50
>50
>200
>200
187
>200
>200
36
5a
CH(R-CH3)Ph
CH(R-CH3)Ph
CH(S-CH3)Ph
CH(S-CH3)Ph
CH(S-CH3)Ph
CH(S-CH3)Ph
CH(R-CH3)Ph
CH(R-CH3)Ph
CH(S-CH3)Ph
CH(S-CH3)Ph
tBu
32
33
>50
>50
37
23
200
5b
100
35
32
107
35
>50
20
14
>200
200
200
>50
50
>200
200
200
6a
6b
33
>50
>50
>50
>200
>200
>200
>200
>200
12
7a
>50
>50
>200
>50
>50
>200
>200
11
-
>50
>50
151
>200
110
111
>200
8
7b
-
8a
>200
>50
>50
>200
>200
13
8b
H
9a
H
9b
H
>200
>200
50
11ab
10ab
1ae
tBu
tBu
CH2Ph
53
-
-
-
-
>250
>150
>150
511
ganciclovir
cidofovir
acyclovir
brivudin
1.5
1.5
1.0
20
-
-
>1500
>1400
>1200
1201
0.6
-
-
17
7.1
111
103
>100
>100
0.03
249
a Effective concentration required to inhibit by 50% the HCMV-induced cytopathicity in human embryonic lung (HEL) fibroblast cell
cultures at 7 days postinfection, as described in ref 8c. Virus input was 100 plaque-forming units (PFU). b Effective concentration required
to reduce VZV plaque formation after 5 days in HEL cell cultures by 50%, as compared to untreated controls. c Compound concentration
required to cause a microscopically visible alteration of normal cell morphology. d Cytotoxic concentration required to reduce cell growth
by 50%. e From ref 17b.

Anti-HCMV 1-Acyl-â-lactams and 1-Acylazetidines
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 7 2615
Scheme 3a
with the replication of a broad range of DNA or RNA
viruses (data not shown), but some compounds showed
moderate activity against VZV reference strains (Table
1).
As shown in Table 1, the phenylurea-substituted
â-lactams 4 weakly blocked HCMV plaque formation
with an EC50 value of 50 µM, fully comparable to that
reported for the prototype 2-azetidinone 1a. A slight
increase in the antiviral activity was observed for
compounds 5 and 6, incorporating chiral R-methylbenzyl
urea substituents at position 1. Evaluation of the anti-
HCMV activity of 1′R and 1′S diastereoisomers 5a and
6a indicated no appreciable influence of the absolute
configuration at the R-methylbenzyl moiety on the
inhibition of viral replication. In contrast, a 4S config-
uration seems to be preferred, at least in the case of
compounds bearing an R-R-methylbenzyl moiety (com-
pare 4S-5a with 4R-5b). Although intriguing, the activ-
ity-stereochemistry relationships in our â-lactam series
is quite similar to those found for model compounds 1.37
Thus, in compound 1b the change of the C-4 configu-
ration (S by R) provided an almost inactive compound,
while inversion at the R-methylbenzyl chiral carbon led
to a minor potency loss. The lack of activity of the
methyl ester derivatives 7a and 7b, as well as of the
free carboxylic acid analogues 8a, 8b, 9a and 9b, might
suggest a role for the tert-butyl ester moiety, either as
a bulky substituent for hydrophobic interaction with the
viral target or by increasing cellular and/or nuclear
membrane penetration. Nevertheless, since tert-butyl
ester-hydrolyzing enzymes are uncommon, a higher
enzymatic hydrolysis of methyl versus tert-butyl esters,
to provide the inactive carboxylic derivatives, could also
explain the inactivity of compounds 7. Replacing the
urea moieties in 4-6 by a benzyloxycarbonyl group to
give 10 had a significant effect on the antiviral potency.
Compound 10 showed the highest anti-HCMV activity
within the â-lactam series, but its cell toxicity value was
close to the EC50 data for viral inhibition. Since com-
pound 10 is highly reactive toward O- and N-nucleo-
philes,50 it could bind to many nucleophiles en route to
the intended target, most likely resulting in toxic side
effects.41,42 Finally, the presence of an aromatic group
at the 1-acyl substituent seems important for the
antiviral activity, since the tert-butoxycarbonyl-substi-
tuted derivative 11 was completely inactive.
Almost in parallel to the inhibition of the HCMV
replication, we also noted a modest anti-VZV activity
for compounds 4ab, 5a, 6a, 6b, and 10ab. Dual anti-
VZV and anti-HCMV activity has also been described
for a series of bicyclic furanopyrimidine deoxynucleo-
sides.51 Interestingly, compound 10ab, which is the most
active against HCMV, also proved to be the most active
against VZV.
The precise mechanism of action of these 1-acyl-â-
lactams against HCMV remains unclear. However,
considering their structural analogy with model com-
pounds 1, we presumed that compounds 4-6 and 10
could inhibit HCMV replication in an irreversible man-
ner through acylation of the virus serine protease. As
other irreversible enzyme inhibitors, compound 10 is
highly reactive toward nucleophiles,50 and this reactivity
could be masking, at least to some extent, its real
antiviral effects. In fact, in the series of the potent 5,5-
a (a) R2NCO/Cs2CO3/THF. (b) PhCH2OCOCl/DCM. (c) TFA/
DCM. (d) R2NH2/BOP/TEA/THF. (e) H-Ala-R3/BOP/TEA/THF. (f)
NH3/MeOH.
trans-lactam serine protease inhibitors, a combination
of low nanomolar potency against HCMV protease and
sufficient plasma stability were strictly required to get
compounds with low micromolar activity against the
virus in the intact cells.35
1-Acylazetidine Derivatives. Following the idea
that conversion of the 1-acyl-â-lactams into the corre-
sponding azetidines could lead to more active and safe
HCMV inhibitors, we first focused on removing the
carbonyl lactam group from the hypothetically covalent
inhibitors 6 and 10 to obtain the corresponding azetidine
derivatives 15 and 17 (Scheme 3). After a positive proof
of concept, some other azetidine derivatives were pre-
pared to set up structure-activity relationships. Thus,
the methyl ester derivatives 16 were designed as
negative controls, since their 1-acylazetidinone coun-
terparts 7a and 7b were inactive. On the other hand,
the substitution of the OtBu group in 10 by NHtBu and
NHChx to provide compounds 20 and 21 could serve to
restrict the conformational flexibility, through the pos-
sibility of intramolecular hydrogen bonds, while a bulky
hydrophobic group was kept at this point. The knowl-
edge that the substrate cleavage site across all the
herpesvirus proteases are highly conserved (Ala-Ser),
led us to design azetidine derivatives 22-27, which
incorporate differently substituted Ala residues to allow
access to the S1 subsite of the enzyme.52
The common starting azetidines 13ab and 14ab were
prepared, as enantiomeric mixtures of approximately
2.5:1 a:b ratio, from suitable â-lactam precursors,
following a chemoselective reduction method previously
described.53 As shown in Scheme 3, the treatment of
azetidine derivatives 13 and 14 with (S)-1-phenylethyl
isocyanate afforded the diastereoisomeric mixtures 15ab

2616 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 7
Gerona-Navarro et al.
Table 2. Chemical Shifts and Temperature Coefficients of Amide Protons in Compounds 20, 21, and 27
δ 2-CONH
DMSOa
δ NHMe
DMSOa
a
a
compd
CDCl3
∆δ
∆δ/∆Tb
CDCl3
∆δ
∆δ/∆Tb
20
7.81
7.86
8.18
8.13
7.72
7.85
8.23
8.11
0.09
-2.7
-3.0
-2.8
-2.9
21
0.01
27ac
27bc
-0.05
0.02
6.34
6.34
7.81
7.90
1.47
1.56
-5.4
-5.0
a Measured for the major trans rotamer (ppm). b Determined by least-squares linear regression analysis from measurements over the
range 30-60 °C (seven points), in DMSO-d6 (ppb/K). c Measured in approximately 4:1 enriched mixtures of each diastereoisomer.
and 16ab in moderate to good yield. In this case, only
compound 16ab could be efficiently resolved into its
corresponding diastereoisomers. In a similar way, acy-
lation of 13 and 14 with benzyl chloroformate resulted
in the corresponding Z-substituted derivatives 17ab and
18ab, as enantiomeric mixtures. Carboxamide deriva-
tives 20ab and 21ab were prepared by reaction of the
carboxylic derivative 19ab with tert-butyl- and cyclo-
hexylamine, respectively, using BOP as the coupling
agent. A similar coupling reaction was conducted to
prepare Ala dipeptide derivatives 22ab and 23ab, two
diastereoisomeric mixtures that could not be chromato-
graphically resolved. Compound 23 was then trans-
formed into the corresponding free carboxylic and amide
derivatives 24 and 25 by saponification and ammonoly-
sis with NH3/MeOH, respectively. It is interesting to
note that, in the last reaction, the expected compound
25 was obtained as a single diastereoisomer, 25a, along
with a 1:1.1 diastereoisomeric mixture of compound
26ab, resulting from the methanolysis of the Z group.
Compound 24 was finally converted into the N-methyl-
amide derivatives 27ab by treatment with methylamine
in the presence of BOP.
Compounds 15-17 and 20-27 were evaluated as
inhibitors of the AD-169 and Davis HCMV strains, as
previously described (Table 3).14 As noted in Table 3,
the 2-tert-butoxycarbonylazetidine derivative 15ab re-
tains the potency of its â-lactam equivalents 6a and 6b,
but with a slightly increased cytostatic activity, while
the methyl ester analogues 16a and 16b were inactive,
as in the â-lactam series. Strikingly, compound 17
showed a considerable improvement in the antiviral
activity with respect to 10 and all other â-lactamic
inhibitors. Although certain toxicity persisted, the thera-
peutic index (ratio CC50/EC50 g 50) was clearly improved
with respect to the â-lactam analogue 10 (therapeutic
index ≈ 3). Therefore, its antiviral selectivity was at
least 10-fold increased. The EC50 value obtained for this
azetidine derivative was similar to that of the standard
reference compound cidofovir. Further modification of
the structure of 17 by transformation to 2-carboxamide
analogues 20 and 21 resulted in a 5-8-fold decrease in
antiviral potency, probably due to the lower conforma-
tional flexibility of these latter compounds. The impor-
tance of a hydrophobic environment by the carboxylate
in position 2 was suggested by the anti-HCMV activity
of compound 22 and the inactivity of its analogues 23
and 24, with a small methyl group and a free carboxylic
acid, respectively. However, a high susceptibility toward
enzymatic hydrolysis of the methyl ester derivative 23
could also take account for its inactivity. Contrastingly,
the antiviral activity was restored with the primary
2-carboxamide derivative 25, indicating that the NH2
protons could be implicated in direct interactions with
the biological target. Again, an increase in the hydro-
phobic character at this part of the structure, as in the
NHMe derivative 27, is followed by an enhancement of
the activity and no signs of cytotoxicity at 50 µM. As
previously noted for the parent â-lactams, the antiviral
activity in the homologous azetidine series is dependent
on the presence of an aromatic moiety within the
substituent at position 1, as deduced from the lack of
activity of compound 26.
1
The examination of the H NMR spectra of all these
1-acylazetidines evidenced a different conformational
behavior among compounds with 2-carboxylic esters and
2-carboxamide derivatives. Thus, compounds 15-18
showed in CDCl3 nearly equimolecular extent of cis and
trans rotamers around the N1-CO bond (38-50% cis).
In contrast, the percentage of cis rotamer was lower
than 15% for all the carboxamide derivatives (20-27)
in the same solvent. We found that, even in a highly
coordinating solvent, such as DMSO, the ratio of cis
rotamer for the latter compounds did not exceed 25%,
indicating a lower conformational flexibility of these
derivatives compared with the carboxylic ester ana-
logues. To ascertain if the restricted rotation around the
N
1-CO bond could be due to the stabilization of folded
conformations, we selected 20, 21, and 27 as model
compounds to evaluate the differences in the amide NH
protons chemical shifts between CDCl3 and DMSO, as
well as the temperature coefficients for these protons
in DMSO, two parameters that could be related to the
existence of intramolecular hydrogen bonds.54 As shown
in Table 2, the chemical values for the 2-CONH protons
in CDCl3 were in all cases >7 ppm; these values suffer
an imperceptible variation in DMSO, and the temper-
ature dependence of the chemical shifts are smaller
than, or equal to, 3 ppb/K, as an absolute value. These
observations are consistent with the participation of the
2-CONH proton of compounds 20, 21, and 27 in a stable
intramolecular hydrogen bond, fixing a γ-turn confor-
mation. In contrast, the NHMe amide protons in 27a
and 27b showed chemical shift values and temperature
coefficients that suggested non-hydrogen-bonded states.
All compounds within this series, except for 15 and
21, did not show notable inhibition of the cytopathicity
of a broad panel of viruses, pointing to their selectivity
as anti-HCMV agents.
Conclusions
Starting from a series of described covalent inhibitors
of the HCMV serine protease, we describe a two-step
approach to develop more effective inhibitors of HCMV
replication. First, we tried to introduce additional
protein-inhibitor close contacts by incorporating car-
boxylate-related groups at position 4 of the model
â-lactams and by fine-tuning the structure through
other minor modifications. The second more challenging
step was directed to remove the â-lactam ring carbonyl

Anti-HCMV 1-Acyl-â-lactams and 1-Acylazetidines
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 7 2617
Table 3. Activity of 1-Acylazetidine Derivatives against Human Cytomegalovirus (HCMV) and Varicella-zoster Virus (VZV) in HEL
Cell Cultures
EC50 (µM)
anti-VZV activityb
anti-HCMV activitya
AD-169 Davis
cytotoxicity (µM)
MCCc
CC50
OKA
(TK+)
07/1
d
compd
R1
(TK-)
15ab
OtBu
40
33
36
25
400
57
16a
OMe
>20
>20
0.7
-
50
50
>50
>50
5
>50
>50
34
16b
OMe
-
>50
>2
>3.2
7.8
>50
>2
>16
5.1
>16
>80
>80
>80
346
>50
-
17ab
OtBu
0.6
4.4
3.2
4.4
>80
>80
32
20ab
NHtBu
NHChx
Ala-OtBu
Ala-OMe
Ala-OH
Ala-NH2
Ala-NH2
Ala-NH-Me
5.5
g16
g16
80
36
21ab
5.9
37
22ab
16
>16
>80
>80
>80
>400
>50
-
33
23ab
>80
>400
39
400
50
24ab
g400
g400
>400
>50
>1500
>1400
>1200
1201
124
50
25a
26ab
>400
7.4
>400
5.0
1.5
1.0
20
>200
>50
27ab
ganciclovir
cidofovir
acyclovir
brivudin
1.5
>150
>150
511
0.6
-
-
111
103
17
7.1
>100
>100
0.03
249
a Effective concentration required to inhibit by 50% the HCMV-induced cytopathicity in human embryonic lung (HEL) fibroblast cell
cultures at 7 days postinfection, as described in ref 8c. Virus input was 100 plaque-forming units (PFU). b Effective concentration required
to reduce VZV plaque formation after 5 days in HEL cell cultures by 50%, as compared to untreated controls. c Compound concentration
required to cause a microscopically visible alteration of normal cell morphology. d Cytotoxic concentration required to reduce cell growth
by 50%.
Gemini 200 or a Varian XL-300 operating at 50 and 75 MHz,
respectively. Elemental analyses were obtained on a CH-O-
RAPID apparatus. Optical rotations were determined with a
Perkin-Elmer 141 polarimeter. Analytical HPLC was per-
formed on a Waters Nova-pak C18 column (3.9 × 150 mm, 4
µm) with a flow rate of 1 mL/min and using a tunable UV
detector set at 214 nm. Mixtures of CH3CN (solvent A) and
0.05% TFA in H2O (solvent B) were used as mobile phase.
Compounds 2, 3, 10, 11, 13, and 14 were prepared as
previously described.44,50,53
General Procedure for the Reaction of N-Unsubsti-
tuted â-Lactams with Isocyanates. To a solution of the
corresponding azetidinone (2.54 mmol) in dry THF (4 mL) was
added Cs2CO3 (0.83 g, 2.54 mmol), followed by the correspond-
ing isocyanate (3.05 mmol). After stirring at room temperature
for 10-20 min, the solvent was eliminated, and the resulting
residue was dissolved in EtOAc and washed with brine. The
organic layer was separated, dried over Na2SO4, and evapo-
rated to dryness. The obtained residue was purified on a silica
gel column or on preparative plates, using the solvent system
indicated in each case.
group, responsible for the covalent acylation of the
enzyme, in an attempt to convert covalent inhibitors to
noncovalent ones. Following this strategy, we obtained
compounds 17 and 27, which showed activity at 0.6-
7.4 µM against both strains of HCMV. These promising
leads could represent the first class of noncovalent
inhibitors for HCMV protease reported to date, although
their exact mechanism of action remains to be deter-
mined. The high throughput screening of various librar-
ies, followed by optimization, has allowed the discovery
of a series of competitive, noncovalent HCMV protease
inhibitors, but their ability to reduce HCMV replication
in cell assays was not disclosed.55 The rationale here
used could be applied to other families of HCMV
protease covalent inhibitors to explore its utility as a
general approach for transforming covalent to non-
covalent inhibitors.
Experimental Section
(4R,S)-4-Benzyl-4-tert-butoxycarbonyl-1-phenylami-
nocarbonyl-2-azetidinone (4ab). Flash chromatography:
EtOAc/hexane (1:8). Yield: 82% (from 2ab and phenyl iso-
Chemistry. All reagents were of commercial quality. Sol-
vents were dried and purified by standard methods. Amino
acid derivatives were obtained from Bachem AG. Analytical
TLC was performed on aluminum sheets coated with a 0.2-
mm layer of silica gel 60 F254 (Merck), and preparative TLC
was done on 20 × 20 glass plates coated with a 2 mm layer of
silica gel PF254 (Merck). Silica gel 60 (230-400 mesh) (Merck)
was used for flash chromatography. Melting points were taken
on a micro hot stage apparatus and are uncorrected. 1H NMR
spectra were recorded with a Varian Gemini 200 or a Varian
XL-300, operating at 200 and 300 MHz, respectively, using
TMS as reference. Temperature coefficients were obtained
from least-squares fits to data of 30, 35, 40, 45, 50, 55, and 60
°C in DMSO-d6. 13C NMR spectra were recorded with a Varian
cyanate), solid. Mp: 88-90 °C (EtOAc/hexane). HPLC: tR
)
16.6 min (A:B ) 50:50). ESI-MS: 403.2 (M + Na)+. Anal.
(C22H24N2O4) C, H, N.
(4S,1′R)-1-[(1′-Phenyl)ethyl]aminocarbonyl-4-benzyl-4-
tert-butoxycarbonyl -2-azetidinone (5a). Preparative
TLC: EtOAc/hexane (1:8). Yield: 50% (from 2ab and R-(+)-
(1-phenyl)ethyl isocyanate), as a solid. Mp: 44 °C (EtOAc/
hexane). [R]D ) +123.37 (c ) 1.01, CHCl3). HPLC: tR ) 24.10
1
min (A:B ) 45:55). H NMR (300 MHz, CDCl3): δ 7.36-7.2
(m, 10H, C6H5), 6.78 (d, 1H, NH, J ) 8.05), 5.09 (m, 1H, 1′-H),
3.67 (d, 1H, 3-H, J ) 14.6), 3.28 (d, 1H, 3-H, J ) 14.6), 2.90
(s, 2H, 4-CH2), 1.57 (d, 3H, 2′-H, J ) 6.9), 1.36 (s, 9H, CH3,

2618 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 7
Gerona-Navarro et al.
tBu). 13C NMR (75 MHz, CDCl3): δ 169.47 (COO), 165.33 (2-
General Procedure for the Preparation of Azetidi-
none-Containing Dipeptides. A solution of the correspond-
ing 4-carboxy-2-azetidinone 8 (0.021 g, 0.06 mmol) in THF (1
mL) was successively treated with H-Ala-OMe‚HCl (0.012 g,
0.085 mmol), BOP (0.037 g, 0.085 mmol), and TEA (0.024 mL,
0.17 mmol) and stirred overnight at room temperature. After
evaporation, the residue was dissolved in EtOAc and washed
with citric acid (10%), NaHCO3 (10%), H2O, and brine. The
organic layer was dried over Na2SO4 and evaporated to leave
a residue that was purified on a silica gel column, using EtOAc:
hexane (1:5) as eluent.
(4S,1′R,1′′S)-1-[(1′-Phenyl)ethyl]aminocarbonyl-4-[(1′′-
methoxycarbonyl)ethyl]aminocarbonyl-4-benzyl-2-aze-
tidinone (12a). Yield: 81% (from 8a), as a white solid. Mp:
72-74 °C (EtOAc/hexane). [R]D ) -83.6 (c ) 0.64, CHCl3).
HPLC: tR ) 7.32 min (A:B ) 45:55). 1H NMR (200 MHz,
CDCl3): δ 8.72 (d, 1H, 4-CONH, J ) 7.2), 7.46-6.94 (m, 11H,
C6H5 and CONH), 5.11 (m, 1H, 1′-H), 4.58 (m, 1H, 1′′-H), 3.78
(s, 3H, OMe), 3.59 (d, 1H, 3-H, J ) 14.2), 3.43 (d, 1H, 4-CH2,
J ) 16.5), 3.11 (d, 1H, 3-H, J ) 14.2), 2.92 (d, 1H, 4-CH2, J )
16.5), 1.55 (d, 3H, 2′-H, J ) 7.0), 1.49 (d, 3H, 2′′-H, J ) 7.3).
ESI-MS: 438.2 (M + 1)+. Anal. (C24H27N3O5) C, H, N.
(4R,1′R,1′′S)-1-[(1′-Phenyl)ethyl]aminocarbonyl-4-[(1′′-
methoxycarbonyl)ethyl]aminocarbonyl-4-benzyl-2-aze-
tidinone (12b). Yield: 80% (from 8b), as a white solid. Mp:
123-124 °C (EtOAc/hexane). [R]D ) +37.5 (c ) 0.45, CHCl3).
HPLC: tR ) 8.40 min (A:B ) 45:55). 1H NMR (200 MHz,
CDCl3): δ 8.72 (d, 1H, 4-CONH, J ) 6.9), 7.40-7.05 (m, 11H,
C6H5 and 1-CONH), 5.13 (m, 1H, 1′-H), 4.51 (m, 1H, 1′′-H),
3.76 (s, 3H, OMe), 3.72 (d, 1H, 3-H, J ) 14.3), 3.42 (d, 1H,
4-CH2, J ) 16.5), 3.24 (d, 1H, 3-H, J ) 14.3), 2.96 (d, 1H,
4-CH2, J ) 16.5), 1.59 (d, 3H, 2′-H, J ) 6.9), 1.36 (d, 3H, 2′′-
H, J ) 7.3). ESI-MS: 438.2 (M + 1)+. Anal. (C24H27N3O5) C,
H, N.
C), 149.20 (NCON), 142.93, 134.47, 130.49, 128.52, 128.41,
t
127.23, 127.18, 125.94 (Ar), 82.88 (C, Bu), 60.77 (4-C), 49.36
t
(1′-C), 43.80 (3-C), 35.95 (4-CH2), 27.52 (CH3, Bu), 22.29 (2′-
C). ESI-MS: 409.2 (M + 1)+. Anal. (C24H28N2O4) C, H, N.
(4R,1′R)-1-[(1′-Phenyl)ethyl]aminocarbonyl-4-benzyl-
4-tert-butoxycarbonyl-2-azetidinone (5b). Preparative
TLC: EtOAc/hexane (1:8). Yield: 33% (from 2ab and R-(+)-
(1-phenyl)ethyl isocyanate), as a foam. [R]D ) -145.93 (c )
1
1.01, CHCl3). HPLC: tR ) 23.10 min (A:B ) 45:55). H NMR
(300 MHz, CDCl3): δ 7.43-7.01 (m, 10H, C6H5), 6.78 (d, 1H,
NH, J ) 7.8), 5.08 (m, 1H, 1′-H), 3.64 (d, 1H, 3-H, J ) 14.5),
3.21 (d, 1H, 3-H, J ) 14.5), 2.91 (m, 2H, 4-CH2), 1.54 (s, 9H,
t
CH3, Bu), 1.53 (d, 3H, 2′-H, J ) 6.9). 13C NMR (75 MHz,
CDCl3): δ 169.66 (COO), 165.13 (2-C), 149.22 (NCON), 143.23,
134.14, 130.55, 128.61, 128.38, 127.34, 127.18, 125.90 (Ar),
t
83.07 (C, Bu), 60.97 (4-C), 49.44 (1′-C), 43.87 (3-C), 35.84 (4-
CH2), 27.82 (CH3, tBu), 22.53 (2′-C). ESI-MS: 409.2 (M + 1)+.
Anal. (C24H28N2O4) C, H, N.
(4S,1′S)-1-[(1′-Phenyl)ethyl]aminocarbonyl-4-benzyl-4-
tert-butoxycarbonyl-2-azetidinone (6a). Preparative
TLC: EtOAc/hexane (1:5). Yield: 44% (from 2ab and S-(-)-
(1-phenyl)ethyl isocyanate), as a foam. [R]D ) +150.43 (c )
1.14, CHCl3). HPLC: tR ) 23.10 min (A:B ) 45:55). ESI-MS:
409.2 (M + 1)+. Anal. (C24H28N2O4) C, H, N. 1H and 13C NMR
data identical to that of its enantiomer 5b.
(4R,1′S)-1-[(1′-Phenyl)ethyl]aminocarbonyl-4-benzyl-4-
tert-butoxycarbonyl-2-azetidinone (6b). Preparative
TLC: EtOAc/hexane (1:5). Yield: 30% (from 2ab and S-(-)-
(1-phenyl)ethyl isocyanate), as a solid. Mp: 43 °C (EtOAc/
hexane). [R]D ) -126.03 (c ) 0.92, CHCl3). HPLC: tR ) 24.10
min (A:B ) 45:55). ESI-MS: 409.2 (M + 1)+. Anal. (C24H28N2O4)
C, H, N. 1H and 13C NMR data identical to that of its
enantiomer 5a.
General Procedure for the Reaction of N-Unsubsti-
tuted Azetidines with Isocyanates. A solution of the
corresponding azetidine 13ab or 14ab (0.62 mmol) in dry CH2-
Cl2 (2 mL) was treated with TEA (0.086 mL, 0.62 mmol) and
S-(-)-(1-phenyl)ethyl isocyanate (0.74 mmol) and stirred at
room temperature for 1 h. After evaporation of the solvent,
the resulting residue was purified on a silica gel column using
the eluent system indicated in each case.
(2S,R,1′S)-1-[(1′-Phenyl)ethyl]aminocarbonyl-2-benzyl-
2-tert-butoxycarbonylazetidine (15ab). Eluent: CH2Cl2:
EtOAc (15:1). Yield: 73% (from 13ab), as a white foam. ESI-
MS: 395.2 (M + 1)+. Anal. (C24H30N2O3) C, H, N.
(4S,1′S)-1-[(1′-Phenyl)ethyl]aminocarbonyl-4-benzyl-4-
methoxycarbonyl-2-azetidinone (7a). Flash chromatogra-
phy: EtOAc/hexane (1:5). Yield: 62% (from 3ab and R-(-)-
(1-phenyl)ethyl isocyanate), as a syrup. [R]D ) +161.66 (c )
0.54, CHCl3). HPLC: tR ) 12.84 min (A:B ) 40:60). ESI-MS:
367.1 (M + 1)+. Anal. (C21H22N2O4) C, H, N.
(4R,1′S)-1-[(1′-Phenyl)ethyl]aminocarbonyl-4-benzyl-4-
methoxycarbonyl-2-azetidinone (7b). Flash chromatogra-
phy: EtOAc/hexane (1:5). Yield: 29% (from 3ab, and R-(-)-
(1-phenyl)ethyl isocyanate), as a syrup. [R]D ) -96.41 (c )
0.94, CHCl3). HPLC: tR ) 14.56 min (A:B ) 40:60). ESI-MS:
367.1 (M + 1)+. Anal. (C21H22N2O4) C, H, N.
General Procedure for the Hydrolysis of OtBu Esters.
To a solution of the corresponding tert-butoxycarbonyl-2-
azetidinone (0.26 mmol) in dry CH2Cl2 (1 mL) was added TFA
(0.2 mL, 2.6 mmol). After a 2-h reaction, the solution was
evaporated to dryness, coevaporating several times with CH2-
Cl2. The resulting residue was purified on a silica gel column
using CH2Cl2:MeOH (30:1) as eluent.
(4S,1′R)-1-[(1′-Phenyl)ethyl]aminocarbonyl-4-benzyl-4-
carboxy-2-azetidinone (8a). Yield: 90% (from 5a), as a solid.
Mp: 165-167 °C (EtOAc/hexane). [R]D ) +95.31 (c ) 0.38,
CHCl3). HPLC: tR ) 6.80 min (A:B ) 40:60). ESI-MS: 353.1
(M + 1)+. Anal. (C24H28N2O4) C, H, N.
(2R,1′S)-1-[(1′-Phenyl)ethyl]aminocarbonyl-2-benzyl-2-
methoxycarbonylazetidine (16a). Eluent: EtOAc:hexane
(1:3). Yield: 53% (from 14ab). Gummy solid. HPLC: tR ) 7.63
1
min (A:B ) 40:60). H NMR (300 MHz, CDCl3): δ 7.33-7.06
(m, 11H, C6H5 and NH), 4.96 (m, 1H, 1′-H), 3.65 (s, 3H, OMe),
3.54 (m, 1H, 4-H), 3.29 (d, 1H, 4-CH2, J ) 13.7), 3.26 (m, 1H,
2-H), 3.04 (d, 1H, 4-CH2, J ) 13.7), 2.30 (m, 1H, 3-H), 2.08
(m, 1H, 3-H), 1.43 (d, 3H, 2′-H, J ) 6.9). 13C NMR (75 MHz,
CDCl3): δ 174.27 (COO), 157.11 (NCON), 144.38, 134.94,
130.26, 128.34, 128.19, 126.88, 126.79, 125.99 (Ar), 71.39 (2-
C), 52.38 (OMe), 49.18 (1′-C), 43.75 (4-C), 39.33 (2-CH2), 23.29
(3-C), 22.58 (2′-C). ESI-MS: 353.2 (M + 1)+. Anal. (C21H24N2O3)
C, H, N.
(4R,1′R)-1-[(1′-Phenyl)ethyl]aminocarbonyl-4-benzyl-
4-carboxy-2-azetidinone (8b). Yield: 80% (from 5b), as a
solid. Mp: 170-172 °C (EtOAc/hexane). [R]D ) -120.33 (c )
0.38, CHCl3). HPLC: tR ) 6.42 min (A:B ) 40:60). ESI-MS:
353.1 (M + 1)+. Anal. (C24H28N2O4) C, H, N.
(4S,1′S)-1-[(1′-Phenyl)ethyl]aminocarbonyl-4-benzyl-4-
carboxy-2-azetidinone (9a). Yield: 98% (from 6a), as a solid.
Mp: 171-173 °C (EtOAc/hexane). [R]D ) +119.54 (c ) 0.81,
CHCl3). HPLC: tR ) 6.40 min (A:B ) 40:60). ESI-MS: 353.1
(M + 1)+. Anal. (C24H28N2O4) C, H, N.
(4R,1′S)-1-[(1′-Phenyl)ethyl]aminocarbonyl-4-benzyl-4-
carboxy-2-azetidinone (9b). Yield: 72% (from 6b), as a solid.
Mp: 161-163 °C (EtOAc/hexane). [R]D ) -99.19 (c ) 0.81,
CHCl3). HPLC: tR ) 6.80 min (A:B ) 40:60). ESI-MS: 353.1
(M + 1)+. Anal. (C24H28N2O4) C, H, N.
(2S,1′S)-1-[(1′-Phenyl)ethyl]aminocarbonyl-2-benzyl-2-
methoxycarbonylazetidine (16b). Eluent: EtOAc:hexane
(1:3). Yield: 23% (from 14ab), as a foam. [R]D ) -41.1 (c )
1
0.43, CHCl3). HPLC: tR ) 7.07 min (A:B ) 40:60). H NMR
(300 MHz, CDCl3): δ 7.31-7.11 (m, 11H, C6H5 and NH), 4.95
(m, 1H, 1′-H), 3.64 (s, 3H, OMe), 3.54 (m, 1H, 2-H), 3.39 (d,
1H, 4-CH2, J ) 13.8), 3.23 (m, 1H, 2-H), 3.07 (d, 1H, 4-CH2, J
) 13.8), 2.31 (m, 1H, 3-H), 2.09 (m, 1H, 3-H), 1.39 (d, 3H, 2′-
H, J ) 6.9). 13C NMR (75 MHz, CDCl3): δ 174.04 (COO),
156.85 (NCON), 144.51, 135.16, 130.47, 128.43, 128.31, 126.98,
126.90, 125.92 (Ar), 71.34 (2-C), 52.49 (OMe), 49.22 (1′-C),
43.87 (4-C), 39.38 (2-CH2), 23.69 (3-C), 22.90 (2′-C). ESI-MS:
353.2 (M + 1)+. Anal. (C21H24N2O3) C, H, N.
General Procedure for the Synthesis of N-Benzyloxy-
carbonylazetidines. A solution of the corresponding azeti-

Anti-HCMV 1-Acyl-â-lactams and 1-Acylazetidines
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 7 2619
dine (0.56 mmol) in dry CH2Cl2 (3 mL), cooled at 0 °C, was
treated with propylene oxide (0.595 mL, 8.4 mmol) and benzyl
chloroformate (0.158 mL, 1.12 mmol). After reaction overnight
at room temperature, the solvent was evaporated and the
resulting residue was purified on a silica gel column as
specified in each case.
(2R,S)-2-Benzyl-1-benzyloxycarbonyl-2-tert-butoxycar-
bonylazetidine (17ab). Eluent: EtOAc:hexane (1:4). Yield:
82% (from 13ab), as a syrup. HPLC: tR ) 14.24 min (A:B )
50:50). ESI-MS: 382.2 (M + 1)+. Anal. (C23H27NO4) C, H, N.
(2R,S)-2-Benzyl-1-benzyloxycarbonyl-2-metoxy-
carbonylazetidine (18ab). Eluent: EtOAc:hexane (1:6).
Yield: 77% (from 14ab), as a syrup. HPLC: tR ) 5.38 min
(A:B ) 50:50). ESI-MS: 340.1 (M + 1)+. Anal. (C20H21NO4) C,
H, N.
(2R,S)-2-Benzyl-1-benzyloxycarbonyl-2-carboxylazeti-
dine (19ab). A solution of azetidine 18ab (0.4 g, 1.18 mmol)
in MeOH (8 mL) was treated with 2 N NaOH (1.32 mL, 2.36
mmol) and stirred at room temperature overnight. After
evaporation, the residue was dissolved in H2O, acidified with
1 M HCl to pH ) 3, and extracted with EtOAc. The organic
layer was dried over Na2SO4 and evaporated to leave a residue
that was used without further purification (0.35 g, 92%, foam).
HPLC: tR ) 4.21 min (A:B ) 40:60).
times with CH2Cl2, to afford 51 mg (99%) of the title compound
as a syrup. a:b ratio ) 2.4:1. ESI-MS: 397.2 (M + 1)+. Anal.
(C22H24N2O5‚H2O) C, H, N.
(2R,1′S)-2-Benzyl-1-benzyloxycarbonyl-2-[1′-(car-
bamoyl)ethyl]carbamoylazetidine (25a). Compound 23ab
(30 mg, 0.07 mmol) was treated with a saturated solution of
NH3 in MeOH (5 mL), stirred at room temperature for 48 h,
and evaporated. The reaction was repeated twice. The solution
was evaporated to dryness, and the resulting residue was
purified on a silica gel column using a gradient from 1 to 5%
MeOH in CH2Cl2, to afford 7 mg (24%) of the title compound
as a white solid. A second, more retained, component was
identified as the diastereoisomeric mixture of compounds 26ab
(4 mg, 17%), as a white foam.
Compound 25a: HPLC: tR ) 3.74 (A:B ) 40:60). ESI-MS:
396.3 (M + 1)+. Anal. (C22H25N3O4) C, H, N.
(2S,R,1′S)-2-Benzyl-1-methoxycarbonyl-2-[1′-(car-
bamoyl)ethyl]carbamoylazetidine (26ab). HPLC: tR
)
1.76 min (A:B ) 40:60). a:b ratio ) 1:1.1. ESI-MS: 320.2 (M
+ 1)+. Anal. (C16H21N3O4) C, H, N.
(2S,R,1′S)-2-Benzyl-1-benzyloxycarbonyl-2-[1′-(methyl-
carbamoyl)ethyl]carbamoylazetidine (27ab). To a solution
of compound 24ab (31 mg, 0.095 mmol) in dry CH2Cl2 (1 mL)
was successively added methylamine hydrochloride (13 mg,
0.19 mmol), BOP (0.127 g, 0.285 mmol), and TEA (0.04 mL,
0.285 mmol). The mixture was stirred at room temperature
overnight and evaporated. The resulting residue was dissolved
in EtOAc and washed with citric acid (10%), NaHCO3 (10%),
H2O, and brine. The organic layer was separated, dried over
Na2SO4, and evaporated. The residue was purified on a silica
gel column using EtOAc:hexane (1:4) to yield 19.2 mg (60%)
of the title compounds as a white solid. HPLC: tR ) 17.56 and
19.93 min (A:B ) 30:70). a:b ratio ) 4:1. Major isomer
(2R,1′S): 1H NMR (300 MHz, CDCl3): δ 8.18 (d, 1H, 1′-NH, J
) 7.2), 7.32 (m, 5H, C6H5), 7.05 (m, 5H, C6H5), 6.34 (br s, 1H,
CONH), 5.26 (d, 1H, OCH2, J ) 12.3), 4.96 (d, 1H, OCH2, J )
12.3), 4.37 (m, 1H, 1′-H), 3.44 (d, 1H, 4-CH2, J ) 14.2), 3.42
(m, 1H, 4-H), 2.86 (d, 1H, 4-CH2, J ) 14.2), 2.77 (m, 1H, 4-H),
2.74 (d, 3H, N-CH3, J ) 4.9), 2.50 (m, 1H, 3-H), 2.11 (m, 1H,
3-H), 1.33 (d, 3H, 1′-CH3, J ) 7.1). 13C NMR (50 MHz,
CDCl3): δ 173.84 and 172.41 (NCO), 156.30 (NCOO), 136.05,
134.64, 130.34, 128.54, 128.35, 127.10 (Ar), 73.33 (2-C), 67.08
(OCH2), 49.06 (1′-C), 44.84 (4-C), 39.01 (2-CH2), 26.28 (N-
CH3), 23.56 (3-C), 17.34 (2′-C). Minor isomer (2S,1′S): 1H NMR
(300 MHz, CDCl3): δ 8.13 (d, 1H, 1′-NH, J ) 7.2), 7.32 (m.
5H, C6H5), 7.05 (m, 5H, C6H5), 6.34 (br s, 1H, CONH), 5.22 (d,
1H, OCH2, J ) 12.2), 5.02 (d, 1H, OCH2, J ) 12.2), 4.37 (m,
1H, 1′-H), 3.42 (d, 1H, 4-CH2, J ) 14.0), 3.42 (m, 1H, 4-H),
2.94 (d, 1H, 4-CH2, J ) 14.0), 2.77 (m, 1H, 4-H), 2.72 (d, 1H,
N-CH3, J ) 5.2), 2.50 (m, 1H, 3-H), 2.11 (m, 1H, 3-H), 1.38
(d, 3H, 1′-CH3, J ) 7.1). 13C NMR (75 MHz, CDCl3): δ 174.28
and 172.43 (NCO), 156.40 (NCOO), 136.07, 134.66, 130.40,
130.35, 128.57, 128.39, 127.12 (Ar), 73.40 (2-C), 67.04 (OCH2),
49.12 (1′-C), 44.80 (4-C), 39.00 (2-CH2), 26.30 (N-CH3), 23.48
(3-C), 17.31 (2′-C). ESI-MS: 410.2 (M + 1)+, 432.1 (M + Na)+.
Anal. (C23H27N3O4) C, H, N.
General Procedure for Coupling 2-Carboxyazetidine
19 with Amines and Amino Acids. A solution of compound
19ab (0.124 g, 0.38 mmol) in dry CH2Cl2 (2 mL) was succes-
sively treated with the corresponding amine or R-amino ester
(0.57 mmol), BOP (0.219 g, 0.49 mmol), and TEA (0.069 mL,
0.49 mmol, or 0.12 mL, 0.87 mmol, if the amine was in the
hydrochloride form). After stirring overnight at room temper-
ature, the solvent was eliminated. The residue was dissolved
in EtOAc and washed with citric acid (10%), NaHCO3 (10%),
H2O, and brine. The organic layer was separated, dried over
Na2SO4, and evaporated to dryness. The obtained residue was
purified on a silica gel column as indicated in each case.
(2S,R)-2-Benzyl-1-benzyloxycarbonyl-2-(tert-butyl)car-
bamoylazetidine (20ab). Eluent: gradient from 12 to 60%
EtOAc in hexane. Yield: 67% (from 19ab and tert-butylamine),
as a white solid. HPLC: tR ) 25.23 min (A:B ) 40:60). 1H RMN
(200 MHz, CDCl3): δ 7.80 (br s, 1H, NH), 7.30 (m. 5H, C6H5),
7.16 (m, 5H, C6H5), 5.22 (d, 1H, OCH2, J ) 12.2), 4.99 (d, 1H,
OCH2, J ) 12.2), 3.42 (d, 1H, 4-CH2, J ) 13.8), 3.38 (m, 1H,
4-H), 2.83 (d, 1H, 4-CH2, J ) 13.8), 2.72 (m, 1H, 4-H), 2.51
t
(m, 1H, 3-H), 2.01 (m, 1H, 3-H), 1.32 (s, 9H, Bu). ESI-MS:
381.2 (M + 1)+, 403.3 (M + Na)+. Anal. (C23H28N2O3) C, H, N.
(2S,R)-2-Benzyl-1-benzyloxycarbonyl-2-(cyclohexyl)-
carbamoylazetidine (21ab). Eluent: gradient from 10 to
60% EtOAc in hexane. Yield: 50% (from 19ab and cyclohex-
ylamine), as a syrup. HPLC: tR ) 8.51 min (A:B ) 50:50). 1H
RMN (200 MHz, CDCl3): δ 8.86 (d, 1H, NH, J ) 7.8), 7.32 (m.
5H, C6H5), 7.25 (m, 5H, C6H5), 5.21 (d, 1H, OCH2, J ) 12.2),
5.01 (d, 1H, OCH2, J ) 12.2), 3.72 (m, 1H, 1-Chx), 3.41 (d,
1H, 4-CH2, J ) 14.0), 3.39 (m, 1H, 4-H), 2.85 (d, 1H, 4-CH2, J
) 14.0), 2.72 (m, 1H, 4-H), 2.51 (m, 1H, 3-H), 2.05 (m, 1H,
3-H), 1.82-1.14 (m, 10H, Chx). ESI-MS: 407.3 (M + 1)+, 429.3
(M + Na)+. Anal. (C25H30N2O3) C, H, N.
Antiviral Assays. HCMV. Confluent human embryonic
lung (HEL) fibroblasts were grown in 96-well microtiter plates
and infected with the human cytomegalovirus (HCMV) strains
Davis and AD-169 at 100 PFU per well. After a 2-h incubation
period, residual virus was removed and the infected cells were
further incubated with medium containing different concen-
trations of the test compounds (in duplicate). After incubation
for 7 days at 37 °C, virus-induced cytopathogenicity was
monitored microscopically after ethanol fixation and staining
with Giemsa. Antiviral activity was expressed as the EC50 or
concentration required to reduce virus-induced cytopathoge-
nicity by 50%. EC50 values were calculated from graphic plots
of the percentage of cytopathogenicity as a function of con-
centration of the compounds.
(2S,R,1′S)-2-Benzyl-1-benzyloxycarbonyl-2-[1′-(tert-
butoxycarbonyl)ethyl]carbamoylazetidine (22ab). Elu-
ent: EtOAc:hexane (1:4). Yield: 53% (from 19ab and HCl‚H-
Ala-OtBu), as a syrup. HPLC: tR ) 34.69 and 30.61 min (A:B
) 40:60). a:b ratio ) 2.5:1. ESI-MS: 453.3 (M + 1)+. Anal.
(C26H32N2O5) C, H, N.
(2S,R,1′S)-2-Benzyl-1-benzyloxycarbonyl-2-[1′-(meth-
oxycarbonyl)ethyl]carbamoylazetidine (23ab). Eluent:
gradient from 2 to 20% EtOAc in CH2Cl2. Yield: 53% (from
19ab and HCl‚H-Ala-OMe), as a syrup. HPLC: tR ) 9.23 and
9.95 min (A:B ) 40:60). a:b ratio ) 2:1. ESI-MS: 411.2 (M +
1)+. Anal. (C23H26N2O5) C, H, N.
(2S,R,1′S)-2-Benzyl-1-benzyloxycarbonyl-2-[1′-(carboxy)-
ethyl]carbamoylazetidine (24ab). To a solution of com-
pound 22ab (50 mg, 0.11 mmol) in dry CH2Cl2 (1 mL) was
added TFA (0.2 mL, 2.6 mmol). After a 2-h reaction, the
solution was evaporated to dryness, coevaporating several
VZV. The laboratory wild-type VZV strain Oka and the
thymidine kinase-deficient VZV strain 07/1 were used. Con-
fluent HEL cells grown in 96-well microtiter plates were
inoculated with VZV at an input of 20 PFU per well. After a

2620 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 7
Gerona-Navarro et al.
(11) Martinez, A.; Gil, C.; Perez, C.; Castro, A.; Prieto, C.; Otero, J.;
Andrei, G.; Snoeck, R.; Balzarini, J.; De Clercq, E. Nonnucleoside
human cytomegalovirus inhibitors: Synthesis and antiviral
evaluation of (chlorophenylmethyl)benzothiadiazine dioxide
derivatives J. Med. Chem. 2000, 43, 3267-3273.
(12) Slater, M. J.; Baxter, R.; Bonser, R. W.; Cockerill, S.; Gohil, K.;
Parry, N.; Robinson, E.; Randall, R.; Yeates, C.; Snowden, W.;
Walters, A. Synthesis of N-alkyl substituted indolocarbazoles
as potent inhibitors of human cytomegalovirus replication.
Bioorg. Med. Chem. Lett. 2001, 11, 1993-1995.
(13) Reefschlaeger, J.; Bender, W.; Hallenberger, S.; Weber, O.;
Eckenberg, P.; Goldman, S.; Haerter, M.; Buerger, I.; Trappe,
J.; Herrington, J. A.; Haebich, D.; Ruebsamen-Waigmann, H.
Novel nonnucleoside inhibitors of cytomegaloviruses (BAY 38-
4766): In vitro and in vivo antiviral activity and mechanism of
action. J. Antimicrob. Chemother. 2001, 48, 757-767.
(14) Snoeck, R.; Andrei, G.; Bodaghi, B.; Lagneaux, L.; Daelemans,
D.; De Clercq, E.; Neytes, J.; Schols, D.; Naesens, L.; Michelson,
S.; Bron, D.; Otto, M. J.; Bousseau, A.; Nemecek, C.; Roy, C.
2-Chloro-3-pyridin-3-yl-5,6,7,8-tetrahydroindolizine-1-carboxa-
mide (CMV423), a new lead compound for the treatment of
human cytomegalovirus infections. Antiviral Res. 2002, 55, 413-
424.
(15) Biron, K. K.; Harvey, R. J.; Chamberlain, S. C.; Good, S. S.;
Smith, A. A., III; Davis, M. G.; Talarico, C. L.; Miller, W. H.;
Ferris, R.; Dornsife, R. E.; Stanat, S. C.; Drach, J. C.; Townsend,
L. B.; Koszalka, G. W. Potent and selective inhibition of human
cytomegalovirus replication by 1263W94, a benzimidazole L-
riboside with a unique mode of action. Antimicrob. Agents
Chemother. 2002, 46, 2365-2372.
2-h incubation period, residual virus was removed and varying
concentrations of the test compounds were added (in dupli-
cate). Antiviral activity was expressed as EC50, the compound
concentration required to reduce viral plaque formation after
5 days by 50% as compared with untreated controls.
Cytotoxicity Assays. Cytotoxicity measurements were
based on the inhibition of HEL cell growth. HEL cells were
seeded at a rate of 5 × 103 cells/well into 96-well microtiter
plates and allowed to proliferate for 24 h. Then, medium
containing different concentrations of the test compounds was
added. After 3 days of incubation at 37 °C, the cell number
was determined with a Coulter counter. The cytostatic con-
centration was calculated as the CC50, the compound concen-
tration required to reduce growth by 50% relative to the
number of cells in the untreated controls. CC50 values were
estimated from graphic plots of the number of cells (percentage
of control) as a function of the concentration of the test
compounds. Cytotoxicity was expressed as minimum cytotoxic
concentration (MCC) or the compound concentration that
causes a microscopically detectable alteration of cell morphol-
ogy.
Acknowledgment. This work was supported by
CICYT (SAF2003-07207-C02) and Geconcerteerde Onder-
zoeksacties-Vlaanderen (GOA-00/12). G.G.-N. acknowl-
edges a predoctoral fellowship from the Spanish Min-
istry of Science and Technology (1998-2002).
(16) McGuigan, C.; Pathirana, R. N.; Snoeck, R.; Andrei, G.; De
Clercq, E.; Balzarini, J. Discovery of a new family of inhibitors
of human cytomegalovirus (HCMV) based upon lipophilic alkyl
furano pyrimidine dideoxy nucleosides: Action via a novel
nonnucleoside mechanism. J. Med. Chem. 2004, 47, 1847-1851.
(17) (a) Bloom, J. D.; DiGrandi, M. J.; Dushin, R. G.; Curran, K. J.;
Ross, A. A.; Norton, E. B.; Terefenko, E.; Jones, T. R.; Feld, B.;
Lang, S. A. Thiourea inhibitors of herpes viruses. Part 1: Bis-
(aryl)thiourea inhibitors of CMV. Bioorg. Med. Chem. Lett. 2003,
13, 2929-2932.
Supporting Information Available: 1H and 13C NMR
spectra of compounds 4ab, 5a, 7b, 8a, 8b, 9a, 9b, 17a, 18ab,
19ab, 22ab, 23ab, 24ab, 25a, and 26ab and elemental
analysis for all new compounds. This material is available free
of charge via the Internet at http://pubs.acs.org.
(18) Bloom, J. D.; Dushin, R. G.; Curran, K. J.; Donahue, F.; Norton,
E. B.; Terefenko, E.; Jones, T. R.; Ross, A. A.; Feld, B.; Lang, S.
A.; DiGrandi, M. J. Thiourea inhibitors of herpes viruses. Part
2: N-Benzyl-N′-arylthiourea inhibitors of CMV. Bioorg. Med.
Chem. Lett. 2004, 14, 3401-3406.
(19) Jones, T. R.; Lee, S. W.; Johann, S. V.; Razinkov, V.; Visalli, R.
J.; Feld, B.; Bloom, J. D.; O’Connell, J. Specific inhibition of
human cytomegalovirus glycoprotein B-mediated fusion by a
novel thiourea small molecule. J. Virol. 2004, 78, 1289-1300.
(20) Welch, A. R.; Woods, A. S.; McNally, L. M.; Cotter, R. J.; Gibson,
W. A. A herpesvirus maturational proteinase assembling: Iden-
tification of its gene, putative active site domain and cleavage
site. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 10792-10796.
(21) Tong, L.; Quian, C. G.; Massariol, M. J.; Bonneau, P. R.;
Cordingley, M. G.; Lagace, L. A new serine-protease fold revealed
by the crystal structure of human cytomegalovirus protease.
Nature 1996, 383, 272-275.
(22) Shieh, H.-S.; Kurumbail, R. G.; Stevens, A. M.; Stegeman, R.
A.; Sturman, E. J.; Pak, J. Y.; Wittwer, A. J.; Palmier, M. O.;
Wiegand, R. C.; Holwerda, B. C.; Stallings, W. C. Three-
dimensional structure of human cytomegalovirus protease.
Nature 1996, 383, 279-282.
(23) Shieh, H.-S.; Stallings, W. C.; Wittwer, A. J.; Hippenmeyer, P.;
Kurumbail, R. G.; Holwerda, B. C. Crystal structure of human
cytomegalovirus protease delineate a new target for antiherpes
drug design. Int. Antiviral News 1997, 5, 71-73.
(24) Holwerda, B. C. Herpesvirus proteases: Targets for novel
antiviral drugs. Antiviral Res. 1997, 35, 1.
References
(1) Arvin, A. M.; Alford, C. A. Chronic intrauterine and perinatal
infections. In Antiviral Agents and Viral Diseases of Man;
Galasso, G. J., Whitley, R. J., Merigan, T. C., Eds.; Raven
Press: New York, 1990; pp 497-580.
(2) Falagas, M. E.; Paya, C.; Ruthazer, R.; Bradley, A.; Wiesner,
R.; Griffith, J.; Freeman, R.; Rohrer, R.; Werner, B. G.; Snydman,
D. R.; Wiesner, R.; Griffith, J.; Freeman, R.; Rohrer, R.; Werner,
B. G.; Snydman, D. R. Significance of cytomegalovirus for long-
term survival after orthotopic liver transplantationsA prospec-
tive derivation and validation cohort analysis. Transplantation
1998, 66, 1020-1028.
(3) Casado, J. L.; Pe´rez-El´ıas, M. J.; Mart´ı-Belda, P.; Antela, A.;
Sua´rez, M.; Ciancas, E.; Frutos, B.; Pe´rez, M. D.; Guerrero, A.
Improved outcome of cytomegalovirus retinitis in AIDS patients
after introduction of protease inhibitors. J. Acq. Immune Def.
Synd. 1998, 19, 130-134.
(4) Torriani, F. J.; Freeman, W. R.; Macdonald, J. C.; Karavellas,
M. P.; Duran, D. M.; Jeffrey, D. D.; Meyland, P. R. A.; Schrier,
R. D. CMV retinitis recurs after stopping treatment in virological
and immunological failures of potent antiretroviral therapy.
AIDS 2000, 14, 173-180.
(5) Field, A. K. Human Cytomegalovirus: Challenges opportunities
and new drug development. Antiviral Chem. Chemother. 1999,
10, 219-232.
(6) Mart´ınez, A.; Castro, C.; Gil, C.; Pe´rez, C. Recent strategies in
the development of new human cytomegalovirus inhibitors. Med.
Chem. Rev. 2001, 21, 227-244.
(7) De Clercq, E. New inhibitors of human cytomegalovirus (HCMV)
on the horizon. J. Antimicrob. Chemother. 2003, 51, 1079-1083.
(8) Jabs, D. A.; Enger, C.; Dunn, J. P.; Forman, M. Cytomegalovirus
retinitis and viral resistance: Ganciclovir resistance. J. Infect.
Dis. 1998, 177, 770-773.
(9) Vaillancourt, V. A.; Cudahy, M. M.; Staley, S. A.; Brideau, R.
J.; Conrad, S. J.; Knechtel, M. L.; Oien, N. L.; Wieber, J. L.;
Yoshihiko, Y.; Watchen, M. W. Naphthalene carboxamides as
inhibitors of human cytomegalovirus DNA polymerase. Bioorg.
Med. Chem. Lett. 2000, 10, 2079-2081.
(25) Waxman, L.; Darke, P. L. Recent advances in antiviral re-
search: Identification of inhibitors of the herpesvirus proteases.
Antiviral Chem. Chemother. 2000, 11, 1-21.
(26) Ogilvie, W. W. Nonnucleoside inhibitors of the human cyto-
megalovirus. Curr. Med. Chem. Anti-Infective Agents 2002, 1,
177-191.
(27) Liang, P.-H.; Brun, K. A.; Field, J. A.; O’Donnell, K.; Doyle, M.
L.; Green, S. M.; Baker, A. E.; Blackburn, M. N.; Abdel-Meguid,
S. S. Site-directed mutagenesis probing the catalytic role of
arginines 165 and 166 of human cytomegalovirus protease.
Biochemistry 1998, 37, 5923-5929.
(28) Ogilvie, W.; Bailey, M.; Poupart, M.-A.; Abraham, A.; Bhavsar,
A.; Bonneau, P.; Bordeleau, J.; Bousquet, Y.; Chabot, C.; Du-
ceppe, J.-S.; Fazal, G.; Goulet, S.; Grand-Maitre, C.; Guse, I.;
Halmos, T.; Lavalle´e, P.; Leach, M.; Malenfant, E.; O’Meara, J.;
Plante, R.; Plouffe, C.; Poirier, M.; Soucy, F.; Yoakim, C.; De´ziel,
R. Peptidomimetic inhibitors of the human cytomegalovirus
protease. J. Med. Chem. 1997, 40, 4113-4135.
(10) Oien, N. L.; Brideau, R. J.; Hopkins, T. A.; Wieber, J. L.;
Knechtel, M. L.; Shelly, J. A.; Anstadt, R. A.; Wells, P. A.;
Poorman, R. A.; Huang, A.; Vaillancourt, V. A.; Clayton, T. L.;
Tucker, J. A.; Wathen, M. W. Broad-spectrum anti-herpes
activities of 4-hydroxyquinoline carboxamides, a novel class of
herpesvirus polymerase inhibitors. Antimicrob. Agents Chemo-
ther. 2002, 46, 724-730.

Anti-HCMV 1-Acyl-â-lactams and 1-Acylazetidines
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 7 2621
(29) LaPlante, S. R.; Bonneau, P.; Aubry, N.; Cameron, D. R.; De´ziel,
R.; Grand-Maitre, C.; Plouffe, C.; Tong, L.; Kawai, S. Charac-
terization of the human cytomegalovirus protease as an induced-
fit serine protease and the implications to the design of mech-
anism-based inhibitors. J. Am. Chem. Soc. 1999, 121, 2974-
2978.
(30) (a) Pinto, I. L.; Jarvest, R. L.; Clarke, B.; Dabrowski, C. E.;
Fenwick, A.; Gorczyca, M. M.; Jennings, L. J.; Lavery, P.;
Sternberg, E. J.; Tew, D. G.; West, A. Inhibition of human
cytomegalovirus protease by enedione derivatives of thieno[2,3-
d]oxazinones through a novel dual acylation/alkylation mecha-
nism. Bioorg. Med. Chem. Lett. 1999, 9, 449-452.
(31) Smith, D. G.; Gribble, A. D.; Haigh, D.; Ife, R. J.; Lavery, P.;
Skett, P.; Slingsby, B. P.; Stacey, R.; Ward, R. W.; West, A. The
inhibition of human cytomegalovirus (hCMV) protease by hy-
droxylamine derivatives. Bioorg. Med. Chem. Lett. 1999, 9,
3137-3142.
(32) Borthwick, A. D.; Angier, S. J.; Crame, A. J.; Exall, A. M.; Haley,
T. M.; Hart, G. J.; Mason, A. M.; Pennell, A. M. K.; Weingarten,
G. G. Design and synthesis of pyrrolidine-5,5-trans-lactams (5-
oxo-hexahydro-pyrrolo[3,2-b]pyrroles) as novel mechanism-based
inhibitors of human cytomegalovirus protease. 1. The R-methyl-
trans-lactam template. J. Med. Chem. 2000, 43, 4452-4464.
(33) Borthwick, A. D.; Crame, A. J.; Erlt, P. F.; Exall, A. M.; Haley,
T. M.; Hart, G. J.; Mason, A. M.; Pennell, A. M. K.; Sing, O. M.
P.; Weingarten, G. G.; Woolven, J. M. Design and synthesis of
pyrrolidine-5,5-trans-lactams (5-oxo-hexahydro-pyrrolo[3,2-b]-
pyrroles) as novel mechanism-based inhibitors of human cy-
tomegalovirus protease. 2. Potency and chirality. J. Med. Chem.
2002, 45, 1-18.
(34) Borthwick, A. D.; Exall, A. M.; Haley, T. M.; Jackson, D. L.;
Mason, A. M.; Weingarten, G. G. Pyrrolidine-5,5-trans-lactams
as novel mechanism-based inhibitors of human cytomegalovirus
protease. Part 3. Potency and plasma stability. Bioorg. Med.
Chem. Lett. 2002, 12, 1719-1722.
(35) Borthwick, A. D.; Davies, D. E.; Erlt, P. F.; Exall, A. M.; Haley,
T. M.; Hart, G. J.; Jackson, D. L.; Parry, N. R.; Patikis, A.;
Trivedi, N.; Weingarten, G. G.; Woolven, J. M. Design and
synthesis of pyrrolidine-5,5-trans-lactams (5-oxo-hexahydro-
pyrrolo[3,2-b]pyrroles) as novel mechanism-based inhibitors of
human cytomegalovirus protease. 4. Antiviral activity and
plasma stability. J. Med. Chem. 2003, 46, 4428-4449.
(36) De´ziel, R.; Malenfant, E. Inhibition of human cytomegalovirus
protease N0 with monocyclic â-lactams. Bioorg. Med. Chem. Lett.
1998, 8, 1437-1442.
(43) Gerona-Navarro, G.; Pe´rez de Vega, M. J.; Garc´ıa-Lo´pez, M. T.;
Andrei, G.; Snoeck, R.; De Clercq, E.; Balzarini, J.; Gonza´lez-
Mun˜iz, R. Synthesis and anti-HCMV activity of 1-acyl-â-lactams
and 1-acylazetidines derived from phenylalanine. Bioorg. Med.
Chem. Lett. 2004, 14, 2253-2256.
(44) Gerona-Navarro, G.; Bonache, M. A.; Herranz, R.; Garcı´a-Lo´pez,
M. T.; Gonza´lez-Mun˜iz, R. Entry to new conformationally
constrained amino acids. First synthesis of 3-unsubstituted
4-alkyl-4-carboxy-2-azetidinone derivatives via an intramolecu-
lar NR-CR-cyclization strategy. J. Org. Chem. 2001, 43, 3538-
3547.
(45) Bonache, M. A.; Gerona-Navarro, G.; Mart´ın-Mart´ınez, M.;
Garc´ıa-Lo´pez, M. T.; Lo´pez, P.; Cativiela, C.; Gonza´lez-Mun˜iz,
R. Memory of chirality in the stereoselective synthesis of
â-lactams derived from amino acids. Influence of the reaction
conditions. Synlett 2003, 1007-1011.
(46) Bonache, M. A.; Gerona-Navarro, G.; Garc´ıa-Aparicio, C.; Al´ıas,
M.; Mart´ın-Mart´ınez, M.; Garc´ıa-Lo´pez, M. T.; Lo´pez, P.;
Cativiela, C.; Gonza´lez-Mun˜iz, R. Memory of chirality in the
stereoselective synthesis of â-lactams: Importance of the start-
ing amino acid derivative. Tetrahedron: Asymmetry 2003, 14,
2161-2169.
(47) Gerona-Navarro, G.; Garc´ıa-Lo´pez, M. T.; Gonzalez-Mun˜iz, R.
General approach for the stereocontrolled construction of the
â-lactam ring in amino acid-derived 4-alkyl-4-carboxy-2-azeti-
dinones. J. Org. Chem. 2002, 67, 3953-3956.
(48) Gonza´lez-Mun˜iz, R.; Cornille, F.; Bergeron, F.; Ficheux, D.;
Pothier, J.; Durieux, C.; Roques, B. P. Solid-phase synthesis of
a fully active analogue of cholecystokinin using the acid-stable
Boc-Phe(p-CH2)SO3H as a substitute for Boc-Tyr(SO3H) in CCK8.
Int. J. Pept. Protein Res. 1991, 37, 331-340.
(49) Fournie´-Zaluski, M. C.; Lucas-Soroca, E.; Devin, J.; Roques, B.
P. Proton NMR configurational correlation for retro-inverso
dipeptides: Application to the determination of the absolute
configuration of enkephalinase inhibitors. Relationships between
stereochemistry and enzyme recognition. J. Med. Chem. 1986,
29, 751-757.
(50) Gerona-Navarro, G.; Garc´ıa-Lo´pez, M. T.; Gonzalez-Mun˜iz, R.
Easy access to orthogonally protected R-alkyl aspartic acid and
R-alkyl asparagine derivatives by controlled opening of â-lac-
tams. Tetrahedron Lett. 2003, 44, 6145-6148.
(51) McGuigan, C.; Brancale, A.; Andrei, G.; Snoeck, R.; De Clercq,
E.; Balzarini, J. Novel bicyclic furanopyrimidines with dual anti-
VZV and -HCMV activity. Bioorg. Med. Chem. Lett. 2003, 13,
4511-4513.
(52) Borthwick, A. D.; Weingarten, G.; Haley, T. M.; Tomaszewski,
M.; Wang, W.; Hu, Z.; Bedard, J.; Jin, H.; Yuen, L.; Mansour, T.
S. Design and synthesis of monocyclic â-lactams as mechanism-
based inhibitors of human cytomegalovirus protease. Bioorg.
Med. Chem. Lett. 1998, 8, 365-370.
(53) Gerona-Navarro, G.; Bonache, M. A.; Al´ıas, M.; Pe´rez de Vega,
M. J.; Garc´ıa-Lo´pez, M. T.; Lo´pez, P.; Cativiela, C.; Gonza´lez-
Mun˜iz, R. Simple access to novel azetidine-containing confor-
mationally restricted amino acids by chemoselective reduction
of â-lactams. Tetrahedron Lett. 2004, 45, 2193-2196.
(54) Belvisi, L.; Gennari, C.; Mielgo, A.; Potenza, D.; Scolastico, C.
Conformational preferences of peptides containing reverse-turn
mimetic bicyclic lactams: Inverse γ-turn versus type-II′ â-turns.
Insights into â-hairpin stability. Eur. J. Org. Chem. 1999, 389-
400.
(55) Gopalsamy, A.; Lim, K.; Ellingboe, J. W.; Mitsner, B.; Nikitenko,
A.; Upeslacis, J.; Mansour, T. S.; Olson, M. W.; Bebernitz, G.
A.; Grinberg, D.; Feld, B.; Moy, F. J.; O’Connell, J. Design and
synthesis of 1,6-naphthalene derivatives as selective HCMV
protease inhibitors. J. Med. Chem. 2004, 47, 1893-1899.
(37) Yoakim, C.; Ogilvie, W.; Cameron, D. R.; Chabot, C.; Guse, I.;
Hache´, B.; Naud, J.; O’Meara, J. A.; Plante, R.; De´ziel, R.
â-Lactam derivatives as inhibitors of human cytomegalovirus
protease. J. Med. Chem. 1998, 41, 2882-2891.
(38) Yoakim, C.; Ogilvie, W. W.; Cameron, D. R.; Chabot, C.; Grand-
Maitre, C.; Guse, I.; Hache´, B.; Kawai, S.; Naud, J.; O’Meara, J.
A.; Plante, R.; De´ziel, R. Potent â-lactmam inhibitors of human
cytomegalovirus protease. Antiviral Chem. Chemother. 1998, 9,
379-387.
(39) Ogilvie, W.; Yoakim, C.; Do, F.; Hache´, B.; Lagace´, L.; Naud, J.;
O’Meara, J. A.; De´ziel, R. Synthesis and antiviral activity of
monobactams inhibiting the human cytomegalovirus protease.
Bioorg. Med. Chem. 1999, 7, 1521-1531.
(40) Bonneau, P.; Hasani, F.; Plouffe, C.; Malenfant, E.; Laplante,
S. R.; Guse, I.; Ogilvie, W. W.; Plante, R.; Davidson, W. C.;
Hopkins, J. L.; Morelock, M. M.; Cordingley, M. G.; De´ziel, R.
Inhibition of human cytomegalovirus protease by monocyclic
â-lactam derivatives: Kinetic characterization using a fluores-
cent probe. J Am. Chem. Soc. 1999, 121, 2965-2973.
(41) Sanderson, P. E. J. Small, noncovalent serine protease inhibitors.
Med. Res. Rev. 1999, 19, 179-197.
(42) Leung, D.; Abbenante, G.; Failie, D. P. Protease inhibitors:
Current status and future prospect. J. Med. Chem. 2000, 43,
305-441.
JM0492812