β-lactam derivatives as inhibitors of human cytomegalovirus protease

Article abstract of DOI:10.1021/jm980131z

The development of novel monobactam inhibitors of HCMV protease incorporating a carbon side chain at C-4 and a urea function at N-1 is described. Substitution with small groups at the C-3 position of the β- lactam ring gave an increase in enzymatic activity and in stability; however, a lack of selectivity against other serine proteases was noted. The use of both triand tetrasubstituted urea functionalities gave effective inhibitors of HCMV protease. Benzyl substitution of the urea moiety was beneficial, especially when strong electron-withdrawing groups where attached at the para position. Modest antiviral activity was found in a plaque reduction assay.

Full text of DOI:10.1021/jm980131z

2882
J . Med. Chem. 1998, 41, 2882-2891
â-La cta m Der iva tives a s In h ibitor s of Hu m a n Cytom ega lovir u s P r otea se
Christiane Yoakim,* William W. Ogilvie,* Dale R. Cameron, Catherine Chabot, Ingrid Guse, Bruno Hache´,
J ulie Naud, J eff A. O’Meara, Raymond Plante, and Robert De´ziel
Bio-Me´ga Research Division, Boehringer Ingelheim (Canada) Ltd., 2100 Cunard Street, Laval, Que´bec H7S 2G5, Canada
Received March 2, 1998
The development of novel monobactam inhibitors of HCMV protease incorporating a carbon
side chain at C-4 and a urea function at N-1 is described. Substitution with small groups at
the C-3 position of the â-lactam ring gave an increase in enzymatic activity and in stability;
however, a lack of selectivity against other serine proteases was noted. The use of both tri-
and tetrasubstituted urea functionalities gave effective inhibitors of HCMV protease. Benzyl
substitution of the urea moiety was beneficial, especially when strong electron-withdrawing
groups where attached at the para position. Modest antiviral activity was found in a plaque
reduction assay.
Sch em e 1a
Human cytomegalovirus (HCMV), a â-herpesvirus, is
an opportunistic pathogen in immunocompromised in-
dividuals such as AIDS patients and organ transplant
recipients. The current antiviral treatments for HCMV
infection, ganciclovir, cidofovir, and foscarnet, inhibit
the viral DNA polymerase; however, they demonstrate
suboptimal efficacy and safety profiles.1,2 Thus the need
for effective, safe therapeutic agents for HCMV disease
continues to exist. HCMV protease has become a viable
target for antiviral chemotherapy because of its critical
role in capsid assembly and viral maturation.3 The
HCMV UL80 gene encodes an 80-kDa precursor polypro-
tein whose N-terminal domain possesses proteolytic
activity. Autocatalytic cleavage at the R-site (A.A.256)
releases the N-terminal protease domain (No) from the
full-length gene product. Cleavage also occurs near the
C-terminus (A.A.643, M-site) of the polyprotein domain,
a process which is essential for the production of mature
infectious virions. Both No and the full-length protease
precursor are catalytically active.
HCMV protease is a serine protease4 which has little
homology with other serine proteases.5 Biochemical and
mutagenesis studies have identified Ser 132 and His
63 as two residues involved in the catalytic machinery.4
Recent reports of the crystal structure of HCMV pro-
tease6 have identified the third member of the triad as
His 157 and shown that the protease possesses a novel
protein fold.
We have recently reported the development of pepti-
domimetic inhibitors of HCMV protease showing sub-
micromolar potency in an enzymatic assay.7 These
compounds are substrate-based inhibitors incorporating
an activated carbonyl group at the P1 position and are
characterized by having marginal antiviral activity
despite their potency as inhibitors of the enzyme (IC50
< 100 nM). It is reasonable to suggest that this poor
activity could be due to the peptidic nature and large
molecular weight of these inhibitors. This has prompted
us to design small, nonpeptidic inhibitors of HCMV
protease.8 It has previously been reported that â-lac-
a
Reagents and conditions: (a) isobutyl chloroformate, Et3N,
CH2Cl2, CH2N2/Et2O; (b) AgOBz, Et3N, BnOH, THF; (c) H2,
Pd(OH)2/C (20%), MeOH; (d) MsCl, NaHCO3, MeCN, 60 °C; (e)
LiHMDS, THF, BnNCO.
tams are effective inhibitors of classical serine proteases
such as human leukocyte elastase (HLE),9 and we have
recently shown that 4-thioalkyl â-lactams are potent
HCMV protease inhibitors.10 In this paper, we describe
the design and synthesis of novel monobactams which
are selective inhibitors of HCMV protease.
Ch em istr y
The methods used to synthesize the inhibitors are
summarized in Schemes 1-5. Scheme 1 illustrates the
overall procedure for generating the various 4-substi-
tuted â-lactams described herein. N-(Benzyloxycarbo-
nyl)-L-phenylalanine was homologized via formation of
the mixed anhydride, followed by reaction with diazo-
methane to give the corresponding diazomethyl ketone
in 83% yield. A Wolff rearrangement was carried out
using silver benzoate in the presence of benzyl alcohol
to give the ester 1 in 63% yield. Palladium-catalyzed
hydrogenolysis followed by methanesulfonyl chloride-
mediated cyclization11 in the presence of sodium bicar-
bonate gave the key intermediate 2. Ureido formation
was effected via deprotonation with LiHMDS in THF
followed by addition of the appropriate isocyanate to
give substituted ureas such as 3. Other electrophiles
such as phenoxycarbamates12 or carbamoyl chloride
derivatives were also used as needed.
* Corresponding authors. C. Yoakim: e-mail, cyoakim@bio-mega.
boehringer-ingelheim.ca. W. W. Ogilvie: e-mail, wogilvie@bio-mega.
boehringer-ingelheim.ca.
S0022-2623(98)00131-9 CCC: $15.00 © 1998 American Chemical Society
Published on Web 07/01/1998

Inhibitors of Human Cytomegalovirus Protease
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 15 2883
Sch em e 2a
Sch em e 4a
a
Reagents and conditions: (a) TBSCl, DIEA, CH2Cl2; (b) LDA,
THF, MeI or EtI or allyl bromide or O2, (MeO)3P, then CH2N2,
SiO2 or (MeS)2; (c) CsF, MeOH.
a
Reagents and conditions: (a) HCl/Et2O, triphosgene, toluene;
(b) PhOCOCl, Et3N, CH2Cl2.
Sch em e 3a
Sch em e 5a
a
Reagents and conditions: (a) LDA, THF, then 2,6-di-tert-
butylphenol or MeI; (b) CsF, MeOH.
Inhibitors bearing a C-3 substituent can be easily
prepared from intermediate 2 after protection as the
N-tert-butyldimethylsilyl derivative (Scheme 2). Depro-
tonation of 4 with 2 equiv of lithium diisopropylamide
(LDA) followed by alkylation with methyl iodide at -78
°C afforded selectively the 3â-methyl product 5a . This
methodology was also used to generate the correspond-
ing 3â-ethyl and 3â-allyl derivatives (5b,c, respectively).
Methyl ether 5d was obtained by trapping the enolate
with oxygen in the presence of trimethyl phosphite to
provide the corresponding alcohol, which was treated
with diazomethane in the presence of silica gel13 (60%
overall yield). Reaction of the enolate with methyl
disulfide afforded the methylthio ether 5e in 78% yield.
Deprotection was achieved by exposure to cesium fluo-
ride in MeOH to give intermediates 6a -e which were
further elaborated to the ureido derivatives as described
above.
Inversion of the configuration at C-3 of 5a was
achieved via deprotonation (Scheme 3) with lithium
diisopropylamide and quenching the resulting enolate
with 2,6-di-tert-butylphenol14 at -78 °C to give 7a in a
28% yield. Quenching the above enolate with excess
methyl iodide afforded the 3,3-dimethyl compound 7b
in 40% yield. Both compounds were deprotected using
cesium fluoride in MeOH to afford 8a ,b, respectively.
Isocyanates which were not commercially available
were prepared as described in Scheme 4. 1(R)-Phenyl-
propyl isocyanate (10) was prepared from the com-
mercially available amine 9 by reaction with triphos-
gene. Alternatively, preactivation of amine 11 could be
achieved using phenyl chloroformate12 in the presence
of triethylamine to give the corresponding phenoxycar-
bamate 12 in 42% yield after recrystallization.
Noncommercial secondary benzylic amines could be
readily prepared from the corresponding substituted
benzyl bromides as exemplified in Scheme 5. The
appropriate benzyl bromide 13 reacted with methyl-
amine in ethanol to afford the corresponding secondary
amine 14, which was isolated as the hydrochloride salt.
a
Reagents and conditions: (a) MeNH2, EtOH, HCl/Et2O; (b)
phosgene, DIEA, CH2Cl2.
Further reaction with phosgene and diisopropylethyl-
amine (DIEA) in dichloromethane gave the desired
amidoyl chloride.
Resu lts a n d Discu ssion
We recently described the development of 4-thioalkyl
â-lactams as HCMV protease inhibitors.10 Preliminary
investigations15 of their mode of action suggested that
these compounds did not inhibit HCMV protease via a
“double hit” mechanism.16 Mechanistic investigations
of related HLE inhibitors had demonstrated that those
compounds also did not inhibit by a “double hit”.17
These observations suggested to us that it might be
possible that monobactams possessing a carbon append-
age at C-4 would be effective HCMV protease inhibitors.
This approach could be valuable since compounds
incorporating such a structure would be less likely to
cause irreversible inhibition of endogenous proteases.
Compounds 17 and 18 were therefore prepared and
tested for activity against HCMV protease. As shown
in Table 1, replacement of the C-4 thiophenyl group10
with a benzyl side chain resulted in only a 2-fold loss
in potency (entries 1 and 2).18 Compound 18, in which
the configuration at C-4 is R, was not an inhibitor of
HCMV protease. These results prompted us to try to
improve activity via modifications of the C-3 position
of the â-lactam and to the urea moiety.
The structure-activity relationship (SAR) based on
compound 17 began with an investigation of the effect
of the nature of the urea moiety (Table 2). Removal of
the alkyl group from the benzylic position of the urea
gave a 2-fold drop in inhibitor activity (entries 1 and

2884 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 15
Ta ble 1. Monobactams with Various Substituents at C-4
Yoakim et al.
Ta ble 3. Various Urea Substitutions
Ta ble 2. Modifications to the Benzylic Position of the Urea
Ta ble 4. Effect of Substitutions at the C-3 Position
2). A 10-fold loss in activity was observed with inversion
of the configuration at the benzylic position of the urea
substituent (entries 1 and 3). Extension of the chiral
alkyl side chain of urea 17 resulted in an increase in
inhibitory activity relative to compound 17 (entry 4).
Further expansion of this group did not significantly
improve potency (entry 5).
Other modifications to the urea function were also
examined in an effort to establish the scope of the
substituents which were tolerated at this position (Table
3). The use of a 2-phenylethyl group gave a 3-fold
potency loss, whereas a simple phenyl led to almost a
4-fold increase in activity (entries 2 and 3). This
increase in potency was accompanied, unfortunately, by
a significant drop in the stability in cell culture media
of compound 23 relative to compound 3.19 The use of
ureas based on para-substituted anilines did not give
an improvement in either activity or stability relative
to compound 23 (entries 4-6). The incorporation of a
cyclohexyl group gave an inhibitor with potency com-
parable to that of 3 (entry 7) accompanied by a con-
comitant drop in solubility (data not shown). The use
of pyridyl-substituted ureas did not give an increase in
activity, but there was an improvement in inhibitor
a
Compound 38 has an R configuration at C-4 of the â-lactam.
solubility relative to compound 3 (data not shown).
Although aniline derivatives (23-26) were somewhat
more active against HCMV protease than 3, the fact
that these compounds were less stable than 3 led us to
continue further SAR studies on the benzylic series.
Previously it had been reported that the addition of
substituents to the C-3 position of other monobactam
inhibitors gave improvements in inhibitory activity,
stability, and selectivity.10,20 Substitutions at this posi-
tion were therefore explored with the aim of improving
both the potency and the stability profile of our com-
pounds (Table 4). The addition of a methyl group cis
to the C-4 benzyl group (31) resulted in a 6-fold drop in

Inhibitors of Human Cytomegalovirus Protease
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 15 2885
Ta ble 6. Incorporation of Tetrasubstituted Ureas
Ta ble 5. Selectivity Data for Various Monobactam Inhibitors
IC50 (µM)
Entry
Compound
PPE
HLE
Chymotrypsin
1
2
3
4
5
6
7
8
30
31
32
33
34
35
36
37
>75
19
53
45
1.7
4.7
0.3
<0.2
>75
36
25
>75
16
>75
>75
19
6
38
2.2
3
1.2
0.3
0.9
<0.2
inhibitor activity, while the corresponding trans isomer
32 was equipotent with compound 30 (entries 1-3).
Compounds 31 and 32 each demonstrated marginal
improvements in stability. Disubstitution at the C-3
position gave a dramatic stability improvement (entry
4) which was incurred at the expense of inhibitory
activity. Alkyl substituents other than methyl at C-3
led to a decrease in activity while maintaining stability
in cell culture media (entries 5 and 6). The introduction
of methoxy or methylthio moieties gave significant
improvements in inhibitory activity, but these were
accompanied by large losses in stability (entries 7 and
8). Previous results from the Merck group21 had shown
that disubstitution at the C-3 position of monobactams
may lead to a shift in binding mode in which the
opposite configuration at C-4 is favored. We therefore
tested compound 38 to be sure that the inactivity of 33
was not due to a similar change. As shown in entry 9
however, activity could not be recovered in this manner.
Some of these compounds also displayed unsatisfac-
tory selectivity profiles against other common serine
proteases. The activity of the monobactam compounds
was measured against porcine pancreatic elastase (PPE),
human leukocyte elastase (HLE), bovine pancreatic
R-chymotrypsin (BPC), and the cysteine protease human
liver cathepsin B.22 As shown in Table 5,23 monobactam
30, which is unsubstituted at C-3, was not an inhibitor
of the other serine proteases tested. The introduction
of methyl groups onto C-3 in either the R or â configu-
ration or disubstitution at C-3 gave compounds which
interacted with PPE and HLE (entries 2-4) and, in the
case of 31, with R-chymotrypsin. Substituents larger
than methyl gave further decreases in selectivity (en-
tries 5-8) particularly in the case of compound 37 which
was a more potent inhibitor of PPE and HLE than of
HCMV protease. For this reason the remaining SAR
was performed without substituents at C-3.
Ta ble 7. Various Tetrasubstituted Urea Derivatives
As shown in Table 6, the addition of an N-methyl
group to 3 gave a compound (39) with comparable
activity toward HCMV protease. The use of alkyl
substituents such as ethyl or cyclopropyl (entries 2 and
3) gave losses in activity. Although the use of alkoxy-
substituted ureas gave small increases in inhibitor
activity, this was accompanied by a loss of compound
stability (entries 5 and 6).
Extensive substitutions to 39 were carried out in
order to try to increase the activity of this compound
(Table 7). Nitro substituents were introduced onto the
various positions of the urea benzyl function (entries
2-4). Addition of substituents onto the ortho and meta
positions did not improve the potency; however, a 7-fold
increase in potency was noted for the para-substituted
nitro function (entry 4). Alkyl substituents induced a
small improvement in inhibitory activity as did a chloro
substituent (entries 5-7). Methoxy and cyano substitu-
tions did not induce significant improvements in potency
(entries 8 and 9), and other groups such as an acetamide
or amine led to losses in activity (entries 10-11). Strong
electron-withdrawing groups such as nitro or CF3 (entry
12) produced the best results.
The apparent correlation between IC50 and the elec-
tron-withdrawing power of the para substituents sug-
gested to us the possibility of observing a quantitative

2886 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 15
Yoakim et al.
activity (EC50 ) 60 µM); however, the window between
the activity and the cytotoxicity was smaller (TC50
)
178 µM). This compound was also somewhat unstable
in the cell culture media (Table 6, entry 5), and so the
EC50 value should be treated with caution. Compounds
46, 51, and 56 each gave results near 100 µM in the
plaque reduction assay27 and low-micromolar IC50 val-
ues. This suggests that improved cell culture activity
could be realized by further improving the potency of
these compounds, studies of which will be reported in
due course.
Su m m a r y
The development of a new series of monobactam
inhibitors of HCMV protease incorporating a benzyl side
chain at C-4 has been described. Substitution at the
C-3 position was tolerated and gave small increases in
stability and enzymatic activity. These compounds were
much less selective, however, than the corresponding
inhibitors which were unsubstituted at C-3 and so the
remaining SAR was performed with azetidinones which
did not incorporate substituents at C-3. Substitution
of the urea moiety suggested that benzyl groups were
the best choice at this position. Both tri- and tetra-
substituted ureas were effective with tetra substitution
giving a slight stability advantage. Modification of the
benzyl function indicated that strong electron with-
drawing groups at the para position had the best
activity. Preliminary mechanistic investigations indi-
cate that these compounds are reversible and competi-
tive inhibitors of HCMV protease and that inhibition
involves the formation of an acyl-enzyme species.15
Details of these studies will be published separately.
Modest antiviral activity in a plaque reduction assay
suggested that these compounds could potentially be
useful antiviral agents. Studies are ongoing to improve
the activity of the present series of inhibitors.
F igu r e 1. Plot of log IC50 (µM) vs σp for compounds 39, 46,
47, 49-51, 53, and 54 (r2 ) 0.74).
F igu r e 2. Plot of log IC50 (µM) vs ESP (kcal/mol) for
compounds 39, 46, 47, 49-51, 53, and 54 (r2 ) 0.92).
structure-activity relationship for substitutions at this
position.24 Shown in Figure 1 is a Hammett plot using
readily available σp values.25 Figure 2 shows a similar
graph using electrostatic potential (ESP) values calcu-
lated at the electronic surface of the centroid of the
ring26 (see the Experimental Section). Both plots show
modest correlations (r2 ) 0.74 and 0.92, respectively)
suggestive of an electronic interaction between the
enzyme and the aromatic ring. This correlation
prompted us to try other strong electron-withdrawing
Exp er im en ta l Section
Ch em istr y. Ma ter ia ls a n d Meth od s. Unless otherwise
noted, materials obtained from commercial sources were used
without further purification. In the last step, the reactions
were done on an approximately 100-mg scale to give the
inhibitor with the reported yield. 1H NMR spectra were
obtained on a Bruker AMX 400 spectrometer with tetrameth-
ylsilane as internal standard (δ scale) and in the given solvent.
FAB mass spectra were recorded on an Autospec VG spec-
trometer. Column chromatography was performed either on
silica gel (10-40 µm or 230-400 mesh ASTM, E. Merck) or
by preparative HPLC using a Partisil 10 ODS-3, C18 prepara-
tive column (50 cm × 22 mm). Analytical HPLC was carried
out in the following systems: system A, Vydac C18, 10-mm
analytical column (24 cm × 4.6 mm), mobile phase acetonitrile/
0.06% trifluoroacetic acid (TFA) in water/0.06% TFA; system
B, Vydac C18, 10-mm analytical column (24 cm × 4.6 mm),
mobile phase acetonitrile in 20 mM Na2HPO4 at pH 8.0.
Analytical data for final compounds are given in Table 8.
3(S)-{{(Ben zyloxy)ca r b on yl}a m in o}-4-p h en ylb u t yr ic
Acid Ben zyl Ester (1). To a cooled solution (-10 °C) of
N-(benzyloxycarbonyl)-L-phenylalanine (19 g, 62 mmol) in THF
(300 mL) was added Et3N (9.5 mL, 68 mmol). Isobutyl
chloroformate (11 mL, 81 mmol) was added dropwise over 10
min. After 30 min at -10 °C and 30 min at room temperature,
a solution of diazomethane in Et2O (0.3-0.5 M, 500 mL) was
added. The reaction mixture was stirred for 10 min and then
purged with nitrogen for 2 h. The resulting white precipitate
was removed by filtration, and the filtrate was concentrated
under reduced pressure. The residue was purified by flash
25
groups which were chosen based upon their σp and
ESP values (entries 13-15). Unfortunately a large
increase in potency was not realized, although the
activity of the additional compounds examined did
correlate approximately with the electron-withdrawing
strength of the appendage.
The activity in cell culture of those compounds with
IC50 values less than 10 µM was determined in a plaque
reduction assay.7 With the exception of compounds 27,
42, and 48, all of the compounds tested were not
cytotoxic, giving TC50 values greater than 250 µM. In
general the EC50 values were greater than 100 µM,
despite the fact that many compounds were very effec-
tive in the enzymatic assay. Five compounds showed
more interesting activity in the plaque reduction assay.
Compound 3 gave an EC50 of 53 µM with a toxicity
window of at least 5-fold. Compound 42 showed similar

Inhibitors of Human Cytomegalovirus Protease
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 15 2887
Ta ble 8. Physical Data
Compound
HPLC A
HPLC B
Formula
Anal.
Compound
HPLC A
HPLC B
Formula
Anal.
3
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
99
100
100
100
99
96
100
98
100
98
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
18H18N2O2
19H20N2O2
19H20N2O2
19H20N2O2
20H22N2O2
21H24N2O2
19H20N2O2
17H16N2O2
18H18N2O2
18H18N2O3
17H15FN2O2
17H22N2O2
17H17N3O2
17H17N3O2
17H17N3O2
18H19N3O2
18H19N3O2
19H21N3O2
19H21N3O2
20H21N3O2
18H19N3O2
HRMS
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
HRMS
HRMS
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
HRMS
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
99
100
100
95
99
100
97
96
96
99
96
99
100
98
99
94
98
99
99
99
98
100
95
96
98
99
91
96
97
99
95
98
99
98
100
99
95
97
C18H19N3O2S
C19H20N2O2
C19H20N2O2
C20H22N2O2
C21H22N2O2
C19H20N2O3
C25H24N2O3
C19H19N3O4
C19H19N3O4
C19H19N3O4
C20H22N2O2
C22H26N2O2
C19H19ClN2O2
C20H22N2O3
C20H19N3O2
C21H23N3O3
C19H21N3O2
C20H19F3N2O2
C20H19F3N2O2S
C20H19F3N2O3S
C20H19F3N2O4S
HRMS
C,H,N
C,H,N
C,H,N
C,H,N
C,H,N
HRMS
HRMS
C,H,N
HRMS
C,H,N
C,H,N
C,H,N
HRMS
HRMS
HRMS
HRMS
C,H,N
C,H,N
HRMS
HRMS
100
99
99
99
100
100
100
100
98
100
98
100
100
99
99
99
99
100
95
97
100
100
100
96
99
100
98
99
96
99
98
98
100
100
98
97
chromatography (20% EtOAc in hexane) to give 15.9 g (83%
yield) of the desired diazo ketone as a yellow solid.
residue was purified by flash chromatography (30% EtOAc in
hexane) to give 3 (56 mg, 15% yield) as a colorless oil: 1H NMR
(CDCl3) δ 7.46 (t, J ) 6.4 Hz, 1H), 7.36-7.21 (m, 10H), 4.36
(d, J ) 6.4 Hz, 2H), 4.21 (m, 1H), 3.27 (dd, J ) 11.8, 3.8 Hz,
1H), 3.06 (dd, J ) 15.8, 5.6 Hz, 1H), 2.93 (dd, J ) 11.8, 8.9
Hz, 1H), 2.73 (dd, J ) 15.8, 3.0 Hz, 1H); IR (CDCl3) υ 1769,
1700 cm-1; FAB MS m/z 295.2 (MH+); HRMS calcd for
The diazo ketone (13 g, 43 mmol) and benzyl alcohol (4.7
mL, 45 mmol) were dissolved in THF (150 mL). Silver
benzoate (977 mg, 4.29 mmol) in Et3N (8.9 mL, 64 mmol) was
added portionwise (vigorous gas evolution). After 30 min at
room temperature, the reaction mixture was concentrated
under reduced pressure. The residue was dissolved in EtOAc,
and the solution was washed with H2O and brine, dried
(MgSO4), and concentrated. The residue was purified by flash
chromatography (15% EtOAc in hexane) to give 1 (11 g, 63%
yield) as a white solid: 1H NMR (CDCl3) δ 7.40-7.10 (m, 15H),
5.27 (brd, J ) 8.0 Hz, 1H), 5.14 (d, J ) 12.2 Hz, 1H), 5.10 (d,
J ) 12.2 Hz, 1H), 5.06 (s, 2H), 4.30-4.20 (m, 1H), 2.93 (dd, J
) 13.3, 6.5 Hz, 1H), 2.82 (dd, J ) 13.3, 7.6 Hz, 1H), 2.57 (dd,
J ) 16, 5.5 Hz, 1H), 2.50 (dd, J ) 16, 5.0 Hz, 1H); IR (KBr) υ
3323, 1711, 1691 cm-1; FAB MS m/z 404.2 (MH+), 426.2 (M +
Na+).
4(S)-Ben zyla zet id in -2-on e (2). The 3(S)-{{(benzyloxy)-
carbonyl}amino}-4-phenylbutyric acid benzyl ester (1) (10.97
g, 27.2 mmol) in MeOH (1 L) was stirred at room temperature
for 7 h under hydrogen (1 atm) in the presence of 20%
Pd(OH)2/C (50 mg). The catalyst was removed by filtration
through diatomaceous earth. The filtrate was concentrated
under reduced pressure to give 3(S)-amino-4-phenylbutyric
acid (4.53 g, 93% yield) as a white solid.
C
18H19N2O2 295.1447 (MH+), found 295.1452.
4(S)-Ben zyl-1-(ter t-b u t yld im et h ylsilyl)a zet id in -2-on e
(4). To a solution of 4(S)-benzylazetidin-2-one (400 mg, 2.48
mmol) in CH2Cl2 (8 mL) was added DIEA (648 µL, 3.72 mmol),
followed by tert-butyldimethylsilyl chloride (411 mg, 2.73
mmol). The reaction mixture was stirred for 16 h at room
temperature. The solvent was evaporated, and the residue
was purified by flash chromatography (12% EtOAc in hexane)
to give 4 (647 mg, 95% yield) as a white solid: 1H NMR (CDCl3)
δ 7.34-7.15 (m, 5H), 3.77-3.70 (m, 1H), 3.25 (dd, J ) 13.5,
3.5 Hz, 1H), 2.99 (dd, J ) 15.5, 5 Hz, 1H), 2.70 (dd, J ) 15.5,
2.5 Hz, 1H), 2.59 (dd, J ) 13.5, 11 Hz, 1H), 1.01 (s, 9H), 0.31
(s, 3H), 0.29 (s, 3H); IR (KBr) υ 1725 cm-1; FAB MS m/z 276.2
(MH+) 298.2 (M + Na+). Anal. (C16H25NOSi) C, H, N.
Gen er a l P r oced u r e for â-La cta m Alk yla tion : 4(S)-
Ben zyl-1-(ter t-bu tyld im eth ylsilyl)-3(S)-m eth yla zetid in -
2-on e (5a ). To a solution of diisopropylamine (705 µL, 5.03
mmol) in anhydrous THF (12 mL) at -20 °C was added
n-butyllithium (2.9 mL, 4.6 mmol, 1.6 M in hexane). After
the reaction mixture was cooled to -78 °C, a solution of 4(S)-
benzyl-1-(tert-butyldimethylsilyl)azetidin-2-one (4) (640 mg,
2.32 mmol) in THF (4 mL) was introduced, and the mixture
was stirred at -78 °C for 15 min followed by addition of methyl
iodide (488 mg, 214 µL, 3.44 mmol). After 10 min, the reaction
mixture was poured into EtOAc (125 mL). The organic phase
was washed with aqueous NaHSO4 (1 M) and brine, dried
(MgSO4), filtered, and concentrated. The residual oil was
purified by flash chromatography (6% EtOAc in hexane) to give
5a (557 mg, 83% yield) as a pale-yellow solid: 1H NMR (CDCl3)
δ 7.33-7.15 (m, 5H), 3.35 (ddd, J ) 10.8, 3.8, 2.5 Hz, 1H),
3.21 (dd, J ) 13.4, 3.8 Hz, 1H), 2.88 (qd, J ) 7.5, 2.5 Hz, 1H),
2.60 (dd, J ) 13.4, 10.8 Hz, 1H), 1.02 (d, J ) 7.5 Hz, 3H), 1.00
(s, 9H), 0.31 (s, 3H), 0.27 (s, 3H); IR (CHCl3) υ 1723, 1512,
A suspension of NaHCO3 (12.7 g, 152 mmol) in MeCN (1.55
L) was stirred and heated to gentle reflux. Freshly distilled
methanesulfonyl chloride (2.15 mL, 27.8 mmol) was added,
followed by the portionwise addition of the above acid (4.53 g,
25.3 mmol) over 5 h. After 16 h under reflux, the solid was
removed by filtration at 60 °C, and the filtrate was concen-
trated under reduced pressure. The residual solid was tritu-
rated with EtOAc and filtered. The filtrate was concentrated
and the residue purified by flash chromatography (40% EtOAc
in hexane) to give 2 (2.20 g, 54% yield) as a white solid: 1H
NMR (CDCl3) δ 7.35-7.17 (m, 5H), 5.83 (br s, 1H), 3.88-3.82
(m, 1H), 3.08 (ddd, J ) 14.8, 5.0, 2.2 Hz, 1H), 2.98 (dd, J )
13.7, 5.7 Hz, 1H), 2.84 (dd, J ) 13.7, 7.9 Hz, 1H), 2.70 (ddd, J
) 14.9, 2.0, 1.3 Hz, 1H); IR (KBr) υ 3215, 3171, 1732 cm-1
FAB MS m/z 323.1 (MH+). Anal. (C10H11NO) C, H, N.
;
1429 cm-1; FAB MS m/z 290 (MH+); HRMS calcd for C17H28
-
NOSi 290.1940 (MH+), found 290.1932.
Gen er a l P r oced u r e for Ur eid o F or m a tion . 4(S)-Ben -
zyl-2-oxoa zetid in e-1-ca r boxylic Acid Ben zyla m id e (3). To
a solution of 4(S)-benzylazetidin-2-one (2) (200 mg, 1.24 mmol)
in THF (4 mL) at -78 °C was added lithium bis(trimethylsilyl)-
amide (1.36 mL, 1.36 mmol, 1 M in THF). After 10 min, benzyl
isocyanate (180 mL, 1.49 mmol) was added. Stirring was
continued at -78 °C for 45 min. The reaction mixture was
diluted with EtOAc (50 mL), washed with aqueous NaHSO4
(1 M) and brine, dried (MgSO4), filtered, and concentrated. The
4(S)-Ben zyl-1-(ter t-bu tyld im eth ylsilyl)-3(S)-eth yla ze-
tid in -2-on e (5b). Following the same alkylation procedure
but replacing methyl iodide with ethyl iodide, 5b was obtained
in 82% yield: 1H NMR (CDCl3) δ 7.32-7.15 (m, 5H), 3.38 (ddd,
J ) 10.8, 4.0, 2.2 Hz, 1H), 3.22 (dd, J ) 13.0, 3.8 Hz, 1H),
2.79 (ddd, J ) 8.6, 5.7, 2.5 Hz, 1H), 2.57 (dd, J ) 13.0, 10.8
Hz, 1H), 1.60-1.50 (m, 1H), 1.41-1.30 (m, 1H), 1.00 (s, 9H),
0.52 (t, J ) 7.3 Hz, 3H), 0.32 (s, 3H), 0.27 (s, 3H); IR (CHCl3)

2888 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 15
Yoakim et al.
υ 1723, 1472, 1255 cm-1; FAB MS m/z 304 (MH+); HRMS calcd
in 88% yield: 1H NMR (CDCl3) δ 7.34-7.17 (m, 5H), 5.75 (br
s, 1H), 3.53-3.50 (m, 1H), 2.94 (dd, J ) 13.7, 6.0 Hz, 1H), 2.87
(dd, J ) 13.5, 7.6 Hz, 1H), 2.86-2.81 (m, 1H), 1.82-1.71 (m,
1H), 1.66-1.56 (m, 1H), 0.87 (t, J ) 7.6 Hz, 3H); IR (CHCl3) ν
1753, 1216 cm-1; FAB MS m/z 190.1 (MH+); HRMS calcd for
for C18H30NOSi 304.2097 (MH+), found 304.2083.
4(S)-Ben zyl-1-(ter t-bu tyld im eth ylsilyl)-3(S)-a llyla zeti-
d in -2-on e (5c). Following the same alkylation procedure but
replacing methyl iodide with allyl bromide, 5c was obtained
in 89% yield: 1H NMR (CDCl3) δ 7.25-7.13 (m, 5H), 5.34-
5.25 (m, 1H), 4.88-4.75 (m, 2H), 3.44 (ddd, J ) 10.6, 3.8, 2.5
Hz, 1H), 3.21 (dd, J ) 13.2, 3.8 Hz, 1H), 2.91 (ddd, J ) 7.8,
5.7, 2.2 Hz, 1H), 2.58 (dd, J ) 13.2, 10.5 Hz, 1H), 2.21-2.09
(m, 2H), 1.01 (s, 9H), 0.31 (s, 3H), 0.27 (s, 3H); IR (CHCl3) υ
1725, 1471, 1255 cm-1; FAB MS m/z 316 (MH+); HRMS calcd
for C19H30NOSi 316.2097 (MH+), found 316.2091.
4(S)-Ben zyl-1-(ter t-bu tyldim eth ylsilyl)-3(S)-m eth oxyaze-
tid in -2-on e (5d ). To a solution of diisopropylamine (800 µL,
5.7 mmol) in anhydrous THF (40 mL) at -20 °C was added
n-butyllithium (3.6 mL, 5.7 mmol, 1.6 M in hexane). After 15
min, the reaction was cooled to -78 °C, and freshly distilled
trimethyl phosphite (1.1 mL, 7.6 mmol) was added followed
C
12H16NO 190.1232 (MH+), found 190.1238.
4(S)-Ben zyl-3(S)-a llyla zetid in -2-on e (6c). Following the
same deprotection procedure, compound 6c was obtained in
100% yield: 1H NMR (CDCl3) δ 7.30-7.15 (m, 5H), 5.79 (s,
1H), 5.74-5.64 (m, 1H), 5.10-5.00 (m, 2H), 3.55 (ddd, J ) 8.3,
5.7, 1.9 Hz, 1H), 2.99-2.93 (m, 2H), 2.83 (dd, J ) 13.7, 8.3
Hz, 1H), 2.51-2.45 (m, 1H), 2.38-2.30 (m, 1H); IR (CHCl3) ν
1755, 1496, 1371 cm-1; FAB MS m/z 202 (MH+); HRMS calcd
for C13H16NO 202.1232 (MH+), found 202.1225.
4(S)-Ben zyl-3(S)-m eth oxya zetid in -2-on e (6d ). Follow-
ing the same deprotection procedure, compound 6d was
obtained in 100% yield: 1H NMR (CDCl3) δ 7.36-7.18 (m, 5H),
6.00 (br s, 1H), 4.26 (t, J ) 1.9 Hz, 1H), 3.80 (ddd, J ) 7.8,
6.2, 1.6 Hz, 1H), 3.34 (s, 3H), 2.98 (dd, J ) 14, 6.2 Hz, 1H),
2.88 (dd, J ) 14, 7.8 Hz, 1H).
4(S)-Ben zyl-3(S)-(m eth ylth io)a zetid in -2-on e (6e). Fol-
lowing the same deprotection procedure, compound 6e was
obtained in 97% yield: 1H NMR (CDCl3) δ 7.73-7.20 (m, 5H),
5.82 (br s, 1H), 3.78-3.74 (m, 2H), 3.09 (dd, J ) 14.0, 5.4 Hz,
1H), 2.89 (dd, J ) 14.0, 8.0 Hz, 1H), 2.09 (s, 3H); IR (CDCl3)
ν 1733 cm-1; FAB MS m/z 208 (MH+).
4(S)-Ben zyl-1-(ter t-bu tyldim eth ylsilyl)-3(R)-m eth ylaze-
tid in -2-on e (7a ). Using the same procedure for enolate
formation as in 5a followed by addition of 2,6-di-tert-butylphe-
nol gave 7a in 28% yield: 1H NMR (CDCl3) δ 7.32-7.17 (m,
5H), 4.07-4.03 (m, 1H), 3.40-3.33 (m,1H), 3.12 (dd, J ) 14.9,
3.5 Hz, 1H), 2.86 (dd, J ) 14.9, 11.1 Hz, 1H), 1.15 (d, J ) 7.6
Hz, 3H), 1.00 (s, 9H), 0.27 (s, 3H), 0.25 (s, 3H); IR (CHCl3) ν
2944, 1725 cm-1; HRMS calcd for C17H28NOSi 290.1940 (MH+),
found 290.1944.
by a solution of 4 (1.05 g, 3.8 mmol) in THF (10 mL).
A
constant stream of oxygen was introduced, and the mixture
was stirred at -78 °C for 3 h. The reaction was quenched
with saturated aqueous NH4Cl and extracted with EtOAc (120
mL). The organic layer was washed with brine, dried (MgSO4),
filtered, and concentrated. The residue was purified by flash
chromatography (10% EtOAc in hexane to 30% EtOAc in
hexane) to give 4(S)-benzyl-1-(tert-butyldimethylsilyl)-3(S)-
hydroxyazetidin-2-one (671 mg, 60% yield) as a white solid:
1H NMR (CDCl3) δ 7.25-7.21 (m, 5H), 4.51 (d, J ) 2 Hz, 1H),
3.75-3.71 (m, 1H), 3.26 (br s, 1H), 3.20 (dd, J ) 14, 3.8 Hz,
1H), 2.66 (dd, J ) 14, 11.1 Hz, 1H), 1.00 (s, 9H), 0.31 (s, 3H),
0.29 (s, 3H).
To a solution of 4(S)-benzyl-1-(tert-butyldimethylsilyl)-3(S)-
hydroxyazetidin-2-one (150 mg, 0.51 mmol) in Et2O (70 mL)
at 0 °C was added silica gel (40-60 µm, 9 g). The vigorously
stirred mixture was treated with diazomethane in Et2O (50
mL, 0.3-0.5 M solution). Once the yellow color had almost
disappeared after about 15 min, additional diazomethane
solution (20 mL) was added. This procedure was repeated
several times until no more starting material could be detected
by TLC (about 1.5 h). The reaction mixture was stirred for
an additional hour at room temperature and then filtered and
concentrated to give 5d (157 mg, 99% yield) as a white solid
which was used without purification: 1H NMR (CDCl3) δ 7.27-
7.20 (m, 5H), 4.16 (d, J ) 1.9 Hz, 1H), 3.67 (ddd, J ) 11.1,
3.8, 1.9 Hz, 1H), 3.23 (dd, J ) 13.5, 3.8 Hz, 1H), 2.94 (s, 3H),
2.57 (dd, J ) 13.4, 11.1 Hz, 1H), 1.01 (s, 9H), 0.33 (s, 3H),
0.31 (s, 6H).
4(S)-Ben zyl-1-(ter t-b u t yld im et h ylsilyl)-3(S)-(m et h yl-
th io)a zetid in -2-on e (5e). Following the same alkylation
procedure but replacing methyl iodide with dimethyl disulfide,
compound 5e was obtained in 78% yield: 1H NMR (CDCl3) δ
7.36-7.20 (m, 5H), 3.75 (d, J ) 2.6 Hz, 1H), 3.66 (ddd, J )
10.5, 3.8, 2.2 Hz, 1H), 3.29 (dd, J ) 13.7, 3.8, 1H), 2.67 (dd, J
) 13.7, 10.5 Hz, 1H), 1.82 (s, 3H), 1.03 (s, 9H), 0.34 (s, 3H),
0.31 (s, 3H); IR (KBr) ν 1733 cm-1; FAB MS m/z 322.2 (MH+),
344.2 (M + Na+). Anal. (C17H27NOSSi) C, H, N.
Gen er a l P r oced u r e for N-Silyl Clea va ge: 4(S)-Ben zyl-
3(S)-m eth yla zetid in -2-on e (6a ). To a solution of 4(S)-
benzyl-1-(tert-butyldimethylsilyl)-3(S)-methylazetidin-2-one (557
mg, 1.92 mmol) in MeOH (25 mL) at 0 °C was added cesium
fluoride (439 mg, 2.89 mmol). After 1 h the solvent was
evaporated under reduced pressure, and the residue was
dissolved in EtOAc. The organic phase was washed with H2O
and brine, dried (MgSO4), filtered, and concentrated. The
residue was purified by flash chromatography (50% EtOAc in
hexane) to give 6a (239 mg, 71% yield) as a white solid: 1H
NMR (CDCl3) δ 7.35-7.17 (m, 5H), 5.78 (br s, 1H), 3.46 (ddd,
J ) 8.0, 5.9, 2.0 Hz, 1H), 2.98 (dd, J ) 13.5, 5.9 Hz, 1H), 2.91
(qd, J ) 7.3, 2.0 Hz, 1H), 2.84 (dd, J ) 13.5, 8.0 Hz, 1H), 1.26
(d, J ) 7.3 Hz, 3H); IR (CHCl3) ν 1751, 1601, 1514 cm-1; FAB
MS m/z 176 (MH+); HRMS calcd for C11H14NO 176.1075
(MH+), found 176.1079.
4(S)-Ben zyl-1-(ter t-b u t yld im et h ylsilyl)-3,3-d im et h yl-
a zetid in -2-on e (7b). Using the same procedure for enolate
formation as in 5a followed by addition of excess MeI gave 7b
in 40% yield: 1H NMR (CDCl3) δ 7.33-7.15 (m, 5H), 3.74 (dd,
J ) 11.1, 3.8 Hz, 1H), 3.15 (dd, J ) 14.8, 3.8 Hz, 1H), 2.82
(dd, J ) 14.8, 11.1 Hz, 1H), 1.21 (s, 3H), 1.10 (s, 3H), 1.01 (s,
9H), 0.27 (s, 3H), 0.24 (s, 3H); IR (CHCl3) ν 1723, 1259, 1178
cm-1; FAB MS m/z 304 (MH+); HRMS calcd for C18H30NOSi
304.2097 (MH+), found 304.2090.
4(S)-Ben zyl-3(R)-m eth yla zetid in -2-on e (8a ). Following
the same deprotection procedure, compound 8a was obtained
in 98% yield: 1H NMR (CDCl3) δ 7.35-7.17 (m, 5H), 5.76 (br
s, 1H), 3.94-3.90 (m, 1H), 3.41-3.34 (m, 1H), 2.93 (dd, J )
13.7, 4.1 Hz, 1H), 2.73 (dd, J ) 13.8, 10.2 Hz, 1H), 1.30 (d, J
) 7.6 Hz, 3H); IR (CHCl3) ν 2900, 1753 cm-1; HRMS calcd for
C
11H14NO 176.1075 (MH+), found 176.1066.
4(S)-Ben zyl-3,3-d im eth yla zetid in -2-on e (8b). Following
the same deprotection procedure, compound 8b was obtained
in 31% yield: 1H NMR (CDCl3) δ 7.34-7.17 (m, 5H), 5.60 (br
s, 1H), 3.57 (dd, J ) 10.0, 4.1 Hz, 1H), 2.94 (dd, J ) 13.8, 4.4
Hz, 1H), 2.72 (dd, J ) 13.7, 9.8 Hz, 1H), 1.35 (s, 3H), 1.27 (s,
3H); IR (CHCl3) ν 1757, 1428 cm-1; FAB MS m/z 190 (MH+);
HRMS calcd for C12H16NO 190.1232 (MH+), found 190.1226.
Gen er a l P r oced u r e for Isocya n a te F or m a tion : 1(R)-
P h en ylp r op yl Isocya n a te (10).9 To a solution of 1(R)-
phenylpropylamine (9) (14.3 g, 106 mmol) in Et2O (102 mL)
was added a 1.0 M solution of HCl/Et2O (212 mL, 212 mmol).
The resulting solution was stirred for 30 min and then
evaporated to dryness on a rotary evaporator. The resulting
white hydrochloride salt was suspended in toluene (200 mL).
Triphosgene was added (11.7 g, 39.3 mmol), and the resulting
suspension was stirred at reflux for 3 h and at room temper-
ature for 18 h. The reaction mixture was concentrated and
the final volume adjusted to 200 mL with toluene giving a final
concentration of 0.53 M. The resulting isocyanate solution was
used as such. An aliquot (170 mL) was concentrated to give a
colorless oil: 1H NMR (CDCl3) δ 7.36-7.22 (m, 5H), 4.50 (t, J
) 6.7 Hz, 1H), 1.82 (q, J ) 7.3 Hz, 2H), 0.94 (t, J ) 7.3 Hz,
3H).
4(S)-Ben zyl-3(S)-eth yla zetid in -2-on e (6b). Following
the same deprotection procedure, compound 6b was obtained

Inhibitors of Human Cytomegalovirus Protease
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 15 2889
Gen er a l P r oced u r e for th e P r ep a r a tion of P h en oxy-
ca r ba m a te Der iva tives: 4-{{(P h en oxyca r bon yl)a m in o}-
m eth yl}p yr id in e (12). To a solution of 4-(aminomethyl)-
pyridine (11) (10.7 g, 98.5 mmol) in CH2Cl2 (245 mL) at 0 °C
was added Et3N (14.2 mL, 19.9 g, 197 mmol), followed by a
dropwise addition of phenyl chloroformate (14.8 mL, 18.5 g,
118 mmol). After stirring for 1 h, the resulting mixture was
diluted with EtOAc (1.5 L). The organic phase was washed
twice with water and then brine, dried (MgSO4), and concen-
trated under reduced pressure. Purification of the residue by
chromatography (gradient EtOAc to 10% MeOH/CHCl3) gave
a yellow solid which was recrystallized from EtOAc/hexane (2:
1) to yield the desired compound 12 (9.55 g, 42% yield) which
was used immediately without further purification: 1H NMR
(CDCl3) δ 8.61 (d, J ) 5.7 Hz, 2H), 7.40-7.15 (m, 7H), 5.61
(br s, 1H), 4.50 (d, J ) 6.4 Hz, 2H).
Gen er a l P r oced u r e for th e P r ep a r a tion of Ca r ba m oyl
Ch lor id es: N-Met h yl-N-{{4-(t r iflu or om et h yl)p h en yl}-
m eth yl}ca r ba m oyl Ch lor id e (15n ). To a solution of {4-
(trifluoromethyl)phenyl}methyl bromide (13n ) (20.0 g, 83.7
mmol) in EtOH was added methylamine (100 mL of 40%
aqueous solution, 1.3 mol). After 2 h, the reaction was
concentrated under reduced pressure. The aqueous phase was
extracted with EtOAc (2 × 100 mL). The combined organic
phases were washed with 5% aqueous NaHCO3 solution and
then brine, dried (MgSO4), filtered, and evaporated to dryness.
The resulting residue was dissolved in HCl/dioxane (4 N, 100
mL). The solvent was removed under reduced pressure. The
resulting solid was triturated with Et2O and collected by
suction filtration to provide hydrochloride salt 14n (17.0 g, 90%
yield) as a white solid. The salt was suspended in CH2Cl2 (150
mL), and the suspension was cooled to 0 °C. DIEA (30.2 mL,
173 mmol) was added, followed by the addition of a phosgene
solution in toluene (1.93 M, 55 mL, 105.7 mmol). After 2 h at
0 °C, the reaction mixture was concentrated, and the resulting
thick gum was extracted with Et2O. Evaporation of the Et2O
extract gave a light-yellow oil which was purified by flash
chromatography (10% EtOAc in hexane) to give 15n as a pale-
yellow oil (16.0 g, 84% yield) which was used immediately
without further purification: 1H NMR (CDCl3) δ 7.59 (m, 2H),
7.33 (m, 2H), 4.72 and 4.58 (2s, 2H), 3.04 and 2.97 (2s, 3H).
4(S)-Ben zyl-2-oxoa zetid in e-1-ca r boxylic Acid (4-P yr i-
d in ylm eth yl)a m id e (30). Following the same procedure as
for compound 3 but using 12 as the reactant, compound 30
was obtained in 81% yield as a white solid: 1H NMR (400 MHz,
CDCl3) δ 8.55 (m, 2H), 7.38-7.12 (m, 7H), 7.01 (m, 1H), 4.47
(m, 2H), 4.29 (m, 1H), 3.41 (dd, J ) 14.0, 3.0 Hz, 1H), 2.99
(dd, J ) 16.2, 5.8 Hz, 1H), 2.93 (dd, J ) 14.0, 8.4 Hz, 1H),
2.73 (dd, J ) 16.2, 2.9 Hz, 1H); IR (CDCl3) υ 3357, 1764, 1694
cm-1; FAB MS m/z 296.1 (MH+); HRMS calcd for C17H18N3O2
296.1399, found 296.1408.
4(S)-Ben zyl-2-oxoa zetid in e-1-ca r boxylic Acid N-Meth -
yl-N-{{4-(tr iflu or om eth yl)p h en yl}m eth yl}a m id e (54). To
a solution of 4(S)-benzylazetidin-2-one (2) (110 mg, 0.68 mmol)
in THF (6 mL) at -5 °C was added potassium bis(trimethyl-
silyl)amide (1.43 mL, 0.717 mmol, 0.5 M in toluene). After 20
min the reaction mixture was added via cannula to a solution
of 15n (860 mg, 3.4 mmol) in THF (6 mL) at -78 °C. The
reaction mixture was stirred for 2 h during which time the
temperature rose to -20 °C. The reaction was then quenched
with brine, and the mixture was diluted with EtOAc. The
aqueous phase was extracted twice with EtOAc, and the
combined organic layers were washed with brine, dried
(MgSO4), filtered, and concentrated. The residue was purified
by flash chromatography (20% EtOAc in hexane) to give
compound 54 (102 mg, 40% yield) as a colorless oil: 1H NMR
(400 MHz, CDCl3) δ 7.58 (d, J ) 7.9 Hz, 2H), 7.31 (d, J ) 7.9
Hz, 2H), 7.26-7.10 (m, 5H), 4.80-4.53 (m, 2H), 4.41 (m, 1H),
3.15 (dd, J ) 14.8, 3.8 Hz, 1H), 2.85 (s, 3H), 2.84 (m, 2H), 2.65
(dd, J ) 14.8, 3.5 Hz, 1H); IR (neat) υ 1778, 1665 cm-1; FAB
MS m/z 377 (MH+); HRMS calcd for C20H20F3N2O2 377.1477,
found 377.1488.
(59 mg, 0.17 mmol) in EtOH (5 mL) was stirred under 1 atm
of hydrogen for 2 h in the presence of 10% Pd/C (30 mg). The
catalyst was removed by filtration through microfiber. The
filtrate was concentrated under reduced pressure to give a
crude material which was purified by preparative HPLC. The
resulting TFA salt was suspended in EtOAc and washed with
saturated NaHCO3 to give, after drying over MgSO4 and
removal of solvent, 53 as a yellow foam (45.4 mg, 84%): 1H
NMR (CDCl3) δ 7.30-7.23 (m, 3H), 7,18-7.14 (m, 2H), 7.07
(d, J ) 7.9 Hz, 2H), 6.67 (d, J ) 7.9 Hz, 2H) 4.50-4.40 (m,
3H), 3.22 (dd, J ) 15.9, 3.5 Hz, 1H), 2.93-2.84 (m, 5H), 2.67
(dd, J ) 15.9, 3.8 Hz, 1H); IR (neat) υ 3456, 3360, 1773, 1662
cm-1; FAB MS m/z 324 (MH+), 346 (M + 23); HRMS calcd for
C
18H22N3O2 324.1712 (MH+), found 324.1700.
4(S)-Ben zyl-2-oxoa zetid in e-1-ca r boxylic Acid N-Meth -
yl-N-{(4-a ceta m id op h en yl)m eth yl}a m id e (52). A solution
of azetidinone 46 (26 mg, 0.07 mmol) and excess acetic
anhydride in THF (5 mL) was stirred under 1 atm of hydrogen
for 24 h in the presence of 10% Pd/C (10 mg). The catalyst
was removed by filtration through microfiber. The filtrate was
concentrated under reduced pressure to give a colorless syrup
which was purified by preparative TLC (EtOAc) to afford 52
1
as a colorless gel (15.9 mg, 60%): H NMR (CDCl3) δ 7.49 (d,
J ) 8.3 Hz, 2H), 7.32-7.20 (m, 4H), 7.19-7.14 (m, 3H), 4.57-
4.42 (m, 3H), 3.21 (dd, J ) 13.7, 3.2 Hz, 1H), 2.95-2.85 (m,
5H), 2.69 (dd, J ) 15.9, 3.5 Hz, 1H), 2.19 (s, 3H); IR (neat) υ
3316, 1776, 1666 cm-1; FAB MS m/z 366 (MH+), 388 (M + 23);
HRMS calcd for C21H24N3O3 366.1819 (MH+), found 366.1826.
4(S)-Ben zyl-2-oxoa zetid in e-1-ca r boxylic Acid N-Meth -
y l-N -{{4-(t r iflu o r o m e t h y ls u lfin y l)p h e n y l}m e t h y l}-
a m id e (56) a n d 4(S)-Ben zyl-2-oxoa zetid in e-1-ca r boxylic
Acid N-Meth yl-N-{{4-(tr iflu or om eth ylsu lfon yl)p h en yl}-
m eth yl}a m id e (57). To a solution of azetidinone 55 (130 mg,
0.32 mmol) in MeOH/water/acetone (1:3:2) (6 mL) was added
Oxone (1.27 g, 2.08 mmol). After stirring at room temperature
overnight, the methanol and acetone were removed by rotary
evaporation. The residue was diluted with EtOAc and filtered.
The filtrate was washed with saturated NaHCO3 and brine
and dried over MgSO4. Flash chromatography (30% EtOAc
in hexanes) afforded 122 mg of a mixture of the desired
compounds which were separated by preparative HPLC (56,
39.4 mg, 29%; 57, 43.9 mg, 31%). 56: 1H NMR (CDCl3) δ 7.73
(d, J ) 8.3 Hz, 2H), 7.44 (d, J ) 8.3 Hz, 2H), 7.25-7.18 (m,
3H), 7.12 (m, 2H), 4.73-4.49 (m, 2H), 4.44-4.38 (m, 1H), 3.14
(dd, J ) 13.7, 3.8 Hz, 1H), 2.94 (s, 3H), 2.89 (br m, 1H), 2.88
(dd, J ) 16.2, 6.0 Hz, 1H), 2.64 (dd, J ) 16.2, 3.8 Hz, 1H); IR
(neat) υ 1776, 1668 cm-1; FAB MS m/z 425 (MH+); HRMS calcd
for C20H19F3N2O3S 425.1147 (MH+), found 425.1133. 57: 1H
NMR (CDCl3) δ 7.96 (d, J ) 8.3 Hz, 2H), 7.48 (d, J ) 8.3 Hz,
2H), 7.26-7.19 (m, 3H), 7.12-7.10 (m, 2H), 4.76-4.57 (br m,
2H), 4.43-4.39 (m, 1H), 3.14 (dd, J ) 13.7, 4.2 Hz, 1H), 2.96
(s, 3H), 2.90 (dd, J ) 15.9, 6.0 Hz, 1H), 2.95-2.82 (brm, 1H),
2.67 (dd, J ) 15.9, 3.5 Hz, 1H); IR (neat) υ 1778, 1670 cm-1
;
FAB MS m/z 441 (MH+); HRMS calcd for C20H19F3N2O4S
441.1096 (MH+), found 441.1080.
Electr osta tic P oten tia ls. The urea benzyl portion of 39
was modeled as toluene, and compounds 46-57 were modeled
as the corresponding para-substituted toluenes. Equilibrium
geometries were calculated using the AM1 semiempirical
Hamiltonian28 as implemented in Spartan 4.1.129 on an SGI
Challenge L. The electron density and electrostatic potential
were calculated on grids of 0.5-Å resolution. The total electron
density was plotted as an isosurface of value 0.002 e/au3
representing the molecular surface, and the electrostatic
potential was mapped onto that surface as a property. Elec-
trostatic potentials (in kcal/mol) were then measured as the
minimum potential on the surface at the center of the phenyl
ring.26
Bioch em istr y. Ma ter ia l a n d Meth od s: Fluorescence
measurements were recorded on a Perkin-Elmer LS-50B
spectrofluorimeter equipped with a plate reader accessory. UV
measurements were recorded on a Thermomax microplate
reader from Molecular Devices Corp., Menlo Park, CA.
4(S)-Ben zyl-2-oxoa zetid in e-1-ca r boxylic Acid N-Meth -
yl-N-{(4-a m in op h en yl)m eth yl}a m id e (53). Azetidinone 46

2890 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 15
Yoakim et al.
HCMV No P r otea se Assa y: HCMV No protease30 was
assayed with an internally quenched fluorogenic substrate
based on the maturation cleavage site (Abz-VVNASSRLY(3-
NO2)R-OH, kcat/KM ) 260 M-1 s-1). The fluorescence increase
upon cleavage of the Ala-Ser amide bond was monitored using
excitation λ ) 312 nm (slit 2.5 nm) and emission λ ) 415 nm
(slit 5 nm). A protocol adaptable to a 96-well plate format was
designed for the determination of IC50 values of inhibitors.
Briefly, HCMV No was incubated for 2.5 h at 30 °C in the
presence of the substrate with a range of sequentially diluted
inhibitor concentrations (300-0.06 µM depending on the
potency of each compound). After this period, enzymatic
hydrolysis of the fluorogenic substrate in the absence of
inhibitor led to about a 30% conversion. Quenching was not
required before fluorescence measurement since the total
scanning time by the plate reader accessory was brief relative
to the duration of the reaction. The aqueous incubation buffer
contained 50 mM tris(hydroxymethyl)aminomethane‚HCl, pH
8, 0.5 M Na2SO4, 50 mM NaCl, 0.1 mM EDTA, 1 mM tris(2-
carboxyethyl)phosphine‚HCl, 3% v/v DMSO, and 0.05% w/v
casein. The final concentrations of HCMV No protease (ex-
pressed in terms of total monomer concentration) and sub-
strate were 100 nM and 5 µM, respectively. IC50 values were
obtained through fitting of the inhibition curve to a competitive
inhibition model using SAS NLIN procedure. The mode of
inhibition was determined by measurements of the initial rates
(in cuvettes) at various substrate concentrations in the buffer
as described above. The IC50 values listed were obtained
according to this assay.
P la qu e Red u ction Assa y: Hs-68 cells (ATCC # CRL 1635)
were seeded in 12-well plates at 83 000 cells/well in 1 mL of
DMEM medium (Gibco Canada Inc.) supplemented with 10%
fetal bovine serum (FBS; Gibco Canada Inc.). The plates were
incubated for 3 days at 37 °C to allow the cells to reach 80-
90% confluency prior to the assay. The medium was removed
from the cells by aspiration. The cells were then infected with
approximately 50 PFU of HCMV (strain AD169, ATCC VR-
538) in DMEM medium (commercially available) supple-
mented with 5% inactivated FBS (assay medium). The virus
was allowed to adsorb to cells for 2 h at 37 °C. Following viral
adsorption, the medium was removed from the wells by
aspiration. The cells were then incubated with or without 1
mL of appropriate concentrations of test reagent in assay
medium. Occasionally, test compounds were added 24 h
postinfection. After 4 days of incubation at 37 °C, the medium
was exchanged with fresh medium containing test compound,
and 4 days later the cells were fixed with 1% aqueous
formaldehyde and stained with a 2% violet solution in 20%
ethanol in water. Microscopic plaques were counted using a
stereomicroscope. Drug effects were calculated as a percent
reduction in the number of plaques in the presence of each
drug concentration compared to the number observed in the
absence of drug. Ganciclovir was used as a positive control in
all experiments.31
described in the Experimental Section (11 pages). Ordering
information is given on any current masthead page.
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and B. Simoneau for assistance during the preparation
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Su p p or tin g In for m a tion Ava ila ble: Full tabulation of
1H NMR, FAB MS, IR, and HRMS for all inhibitors not

Inhibitors of Human Cytomegalovirus Protease
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 15 2891
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> 250 µM. 56: EC50 ) 110 µM, TC50 > 250 µM.
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J M980131Z