Synthesis and antiviral activity of monobactams inhibiting the human cytomegalovirus protease

Article abstract of DOI:10.1016/S0968-0896(99)00094-2

A series of monobactam inhibitors of HCMV (N(o)) protease bearing a heterocycle linked by a methylene group at C-4 is described. Inhibitors containing a heterocycle such as a 2-furyl, 2-thiophenyl, 4-methyl-2-tetrazole and 2-benzothiazole were found to be active in a plaque reduction assay. Furthermore, 2-benzothiazole derivatives were shown to inhibit the HCMV protease activity inside cells by using a cell transfection assay, indicating that their antiviral activity in the plaque reduction assay could be attributed to protease inhibition. Copyright (C) 1999 Elsevier Science Ltd.

Full text of DOI:10.1016/S0968-0896(99)00094-2

Bioorganic & Medicinal Chemistry 7 (1999) 1521±1531
Synthesis and Antiviral Activity of Monobactams Inhibiting the
Human Cytomegalovirus Protease
W. W. Ogilvie,* C. Yoakim,* F. Do, B. Hache, L. Lagace, J. Naud, J. A. O'Meara
and R. Deziel
 Â
Boehringer Ingelheim (Canada) Ltd., Bio-Mega Research Division, 2100 Cunard Street, Laval, Quebec, Canada H7S 2G5
Received 23 September 1998; accepted 16 December 1998
AbstractÐA series of monobactam inhibitors of HCMV (No) protease bearing a heterocycle linked by a methylene group at C-4 is
described. Inhibitors containing a heterocycle such as a 2-furyl, 2-thiophenyl, 4-methyl-2-tetrazole and 2-benzothiazole were found
to be active in a plaque reduction assay. Furthermore, 2-benzothiazole derivatives were shown to inhibit the HCMV protease
activity inside cells by using a cell transfection assay, indicating that their antiviral activity in the plaque reduction assay could be
attributed to protease inhibition. # 1999 Elsevier Science Ltd. All rights reserved.
Introduction
within the catalytic domain. Both No and the full-length
protease precursor (UL80 gene product) are catalyti-
cally active.
The human cytomegalovirus (HCMV), a member of the
herpesvirus family, is an opportunistic pathogen in
immunocompromised individuals including AIDS
patients and organ transplant recipients.1,2 An active
infection with HCMV can result in disseminated dis-
ease, pneumonitis, retinitis, oesophagitis and colitis.
Treatments are available, however, the performance of
these drugs is far from ideal. Therefore, the need for
alternative therapies for HCMV infections continues to
exist.
It has been found recently that HCMV protease is
uniquely folded and possesses a novel catalytic triad.8±11
This enzyme has become an attractive target for inhibi-
tion in antiviral chemotherapy.3,4 Strategies that have
been employed include the use of peptidyl-activated
carbonyls,12 aryl tri¯uromethyl ketone derivatives13 and
other small molecule inhibitors.14±17 More recently, we
and others have described the use of monobactam inhi-
bitors.18±21 Mechanistic studies of this class of com-
pounds indicate that inhibition involves processing of
the b-lactam via an acyl enzyme complex.18 While sev-
eral of these monobactams have exhibited sub-
micromolar potency in enzymatic assays,19,21 antiviral
activity in cell culture has so far been modest. In this
paper we describe the preparation of a series of mono-
bactam inhibitors with low micromolar activity in a
plaque reduction assay. Furthermore, by using a cell
based assay, these compounds have been shown to
inhibit protease activity in transfected cells, supporting
the fact that the observed antiviral properties were
e€ected through a decrease in protease activity.
The cytomegalovirus, expresses late in the virus life
cycle, a protease that is essential for viral replication.3,4
The protease is expressed as a precursor protein (UL80
gene product) together with the assembly protein.
The assembly protein substrate is also independently
expressed in approximately 10-fold excess (UL80.5 gene
product). This serine protease5±7 is necessary for the
maturation of the viral capsid. Cleavage occurs to
remove a fragment at the C-terminal end of the UL80
and UL80.5 gene products (M-site, Fig. 1), and also at a
position located near the center of the precursor (R-site)
to excise the catalytic domain (No). In addition to the
M- and R-sites, two more cleavage sites (I and m) are
known for the protease, both of which are located
Chemistry
The synthesis of inhibitors 10±16 and 19±22 proceeded
through common intermediate 4 (Scheme 1) which was
readily prepared on a large scale from the commercially
available serine derivative 1. One carbon extension was
Key words: Human cytomegalovirus; protease inhibitors; b-lactams.
* Corresponding authors. Tel.: +1-450-682-4640; fax: +1-450-682-
4189; e-mail: wogilvie@bio-mega.boehringer-ingelheim.ca, cyoakim
@bio-mega.boehringer-ingelheim.ca
0968-0896/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(99)00094-2

1522
W. W. Ogilvie et al. / Bioorg. Med. Chem. 7 (1999) 1521±1531
achieved by an Arndt±Eistert synthesis using silver
benzoate to catalyze the Wol€ rearrangement. Although
the latter reaction could be done in the presence of
benzyl alcohol to obtain 2 directly, in practice it was
found to be more convenient to prepare the corre-
sponding acid, followed by the esteri®cation. Removal
of the Boc group under acidic conditions and cycliza-
tion using tBuMgCl22,23 a€orded monobactam 3. Pro-
tection using a TIPS group24 followed by removal of the
benzyl ether gave key intermediate 4.
Dess±Martin25 oxidation of 4 to the corresponding
aldehyde was carried out using the modi®cation recently
described by Schreiber.26 Failure to use this modi®ca-
tion led to non-reproducible yields, especially on larger
scales. Exposing the intermediate aldehyde to the
appropriate lithio heterocycle gave the desired alcohols
5a±f as mixtures of epimers. The use of the TIPS func-
tion on the b-lactam nitrogen was found to be necessary
for successful introduction of the heterocycle and sub-
sequent deoxygenation. The corresponding aldehyde
protected at nitrogen by a TBS group gave good yields
only when non-nitrogen containing heterocycles such as
furan were used. The more basic lithio derivative of
benzothiophene a€orded very low yields of the desired
product due to the formation of side products arising
from migration of the silyl group onto oxygen.
The deoxygenation of alcohols 5a±f was accomplished
by treatment with 1,10-(thiocarbonyl)diimidazole fol-
lowed by reduction of the thiocarbamate with Ph3SnH.
The use of Ph3SnH rather than Bu3SnH greatly simpli-
®ed the puri®cation of the desired products 6a±f.
Removal of the TIPS group from 6a±f using CsF in
methanol was followed by treatment with KHMDS and
the appropriate amidyl chloride20,21 to a€ord the
desired protease inhibitors with the general structure 8
(Scheme 2). The tetrazole containing derivatives were
derived from 927,28 in a similar manner. Compounds 15
and 16 were obtained after a ®nal deprotection using
TFA in CH2Cl2.
Figure 1. Genetic organisation of the HCMV protease and assembly
proteins.
Cell based assay
The recombinant vaccinia virus T7 RNA polymerase
transient expression system was used to express the
enzyme and the substrate in COS-7 cells.29 The expres-
sion of the UL80 gene encoding the protease precursor
(Pra) was used to study the e€ect of protease inhibitors
on the processing of the precursor at the M-site giving
rise to the product (Prb) (Fig. 2). The processing of the
assembly protein precursor by the protease was accom-
plished by expressing the catalytic domain of the pro-
tease (No) and the assembly protein precursor (pAP) in
the same cell. The cells were treated with increasing
concentrations of protease inhibitors for a period of 2 h
after which they were metabolically labeled with [35S]-
Met and -Cys for an additional 2 h. The inhibitors were
Scheme 1. Reagents and conditions: (a) IBCF, Et3N, THF, 0ꢀC; (b)
CH2N2, 0ꢀC; (c) AgOBz, Et3N, THF, H2O; (d) BnBr, DBU, CH3CN,
25ꢀC, 65% from 1; (e) 4 N HCl dioxane, 25ꢀC; (f) TMS-Cl, Et3N,
Et2O then tBuMgCl, 0ꢀC to rt, 76% from 2; (g) TIPSOTf, 2,6-lutidine,
CH2Cl2, 0ꢀC; (h) H2, Pd(OH)2, THF, 84% from 3; (i) Dess±Martin
periodinane, CH2Cl2, H2O, 100%; (j) heterocycle, BuLi, 78ꢀC, 52±
76%; (k) Im2CS, DMAP, CH2Cl2; (l) Ph3SnH, AIBN, benzene, ~.
Scheme 2. Reagents and conditions: (a) CsF, CH3OH; (b) KHMDS,
amidyl chloride, THF.

W. W. Ogilvie et al. / Bioorg. Med. Chem. 7 (1999) 1521±1531
1523
did not give good activity in the enzymatic assay. Of those
series which were active, compounds possessing a nitro
group on the benzyl urea moiety were two to four times
more potent than the corresponding CF3 analogues, a
result that is in agreement with previous observations.20
The present compounds all showed good selectivity
towards other serine proteases such as human leukocyte
elastase (HLE), porcine pancreatic elastase (PPE) and
the cysteine protease human liver cathepsin B (cat-B). In
all these cases the IC50 values were found to be above the
solubility limit of the assay. Some of these compounds,
however, were less selective towards bovine pancreatic
a-chymotrypsin (BPC). It is interesting to note the e€ect
of di€erent substituents on the observed selectivity. For
example, furan-containing inhibitors (10 and 11) did not
signi®cantly inhibit BPC (IC50 >75 mM), while com-
pounds 12 and 13, bearing a thiophene substituent
showed modest selectivity: three and tenfold, respectively.
The best selectivity was observed with the benzothiazole
derivatives 19 and 20, which were 16- and 80-fold,
respectively, more active against HCMV protease.
Figure 2. HCMV UL80 and UL80.5 gene products used to transfect
COS-7 cells and the gene products analyzed after treatment with inhi-
bitors.
present during the labeling period. The labeled proteins
were immunoprecipitated using a polyclonal antiserum
(K-58) which recognized an amino acid sequence com-
mon to Pra, Prb, pAP and AP as illustrated in Figure 2.
The percent processing of the precursor in each sample
was used to calculate the percent inhibition obtained for
each concentration of inhibitors.
The antiviral activity of these compounds was deter-
mined in a plaque reduction assay. In a few cases, low
solubility prevented an accurate determination of the
antiviral activity and/or the cytotoxicity. However, for
the reported cytotoxicity values, examination of the
curves indicated that the TC50's were all well above the
solubility limit. Five compounds (10, 11, 13, 17, and 18)
had EC50 values below 150 mM, and derivatives 19 and
20 were the most active of the series (EC50=11 and
30 mM, respectively).30
Results and Discussion
The identi®cation of 4-thioalkyl-b-lactams as inhibitors
of HCMV protease has been recently described.19
Mechanistic investigations into the mode of action of
these compounds led to the development of mono-
bactams incorporating a hydrocarbon substituent at the
C-4 position.20 These compounds were e€ective HCMV
protease inhibitors and were more stable in cell culture
media than the 4-thioaryl series. By introducing various
heterocycles linked by a methylthio substituent at the
C-4 position, both enzymatic and cell culture activity
could be improved.21 Although these modi®cations led
to inhibitors with sub-micromolar potency in the enzy-
matic assay and with activities between 50 and 80 mM in
the plaque reduction assay, further improvements in the
potency of this series were dicult to achieve. A possi-
ble explanation for this is that the C-4 heterocycle was
located too distal from and in a non-optimal orientation
relative to the b-lactam nucleus. Employing a one car-
bon tether would modify this while reducing the degrees
of freedom available to the heterocyclic pharmaco-
phore. Compounds 10±22 (Table 1) were, therefore,
prepared and evaluated. Monobactams containing each
heterocyclic group (R) were prepared with an N-methyl-
N-benzyl urea function substituted in the para position
of the phenyl ring by a CF3 or NO2 group (X).20 These
two ureas had been shown previously to impart the
most activity to the monobactam series, and in the case
of 4-methylthioheterocycles, to give the best potency in
the cell culture assay.21 Those inhibitors containing
furan, thiophene, 2-methyltetrazole, thiazole and benzo-
thiazole at the C-4 position of the b-lactam all had
potencies between 0.7 and 7.1 mM. Compounds 14±16,
which incorporated pyridine or benzimidazole residues,
Protease inhibitors 19 and 20 were evaluated for their
ability to inhibit the processing of the protease pre-
cursor as well as the assembly protein precursor in cells.
In order to measure the e€ect on these events indepen-
dently, the recombinant vaccinia virus T7 RNA poly-
merase transient expression system was used to express
the enzyme (No) and the substrates (Pra and pAP) in
COS-7 cells.31,32 Figure 3 illustrates the inhibitory e€ect
of 19 and 20 on the processing of the protease precursor
(Pra). Both compounds exhibited similar e€ects on
processing at the M-site with 70% inhibition at the
maximum concentration tested. Both compounds also
inhibited the processing of the assembly protein pre-
cursor (pAP) by the catalytic domain of the enzyme
(Fig. 4). The e€ect on the processing of the assembly
protein precursor (pAP) occurred at concentrations
similar to that observed for the inhibition of the pro-
tease precursor processing. These data suggest that the
b-lactam CMV protease inhibitors were active against
the precursor as well as the mature form of the protease
in cells. Furthermore, the concentrations at which pro-
tease inhibition was achieved were in the same range as
those observed for the antiviral activities (EC50).
Conclusions
A series of monobactam inhibitors of HCMV protease
bearing a heterocycle linked by a methylene group at

1524
W. W. Ogilvie et al. / Bioorg. Med. Chem. 7 (1999) 1521±1531
Table 1. Biological activities of monobactam HCMV protease inhibitors
IC50 (mM)
c
Compound
R
X
HCMVa
7.1
BPCb
>75
EC50 (mM)
TC50d (mM)
10
CF3
59
>100
11
12
NO2
CF3
3.1
4.5
>75
14
143
>16
>261
>16
13
14
NO2
CF3
1.3
63
15
>300
70
>79
15
CF3
>50
>50
16
17
NO2
CF3
>75
5.7
>75
>300
94
>195
18
19
NO2
CF3
2.7
2.7
27
43
100
11
>133
>25
20
21
NO2
CF3
0.7
4.9
57
12
30
>77
>51
>116
22
NO2
2.5
8
>29
>29
a
HCMV protease inhibition.
Bovine pancreatic a-chymotrypsin inhibition.
b
c
Plaque reduction assay. Ganciclovir was used as a positive control (EC50=1.23 mM). See Experimental.
d
Cytotoxicity as determined by the MTT method.36
Figure 4. Plot showing the inhibition of processing of the assembly
protein precursor (pAP) by the catalytic domain of HMCV protease
(No) in transfected cells. The e€ect of compounds 19 (*) and 20 (&)
at various concentrations is illustrated.
Figure 3. Plot showing the inhibition of autoprocessing of the pro-
tease precursor (Pra) in transfected cells. The e€ect of compounds 19
(*) and 20 (&) at various concentrations is illustrated.
C-4 has been described. Inhibitors containing hetero-
cycles such as a 2-furyl, 2-thiophenyl, 4-methyl-2-tetra-
zole, or 2-benzothiazole were active in a plaque reduction
assay. Furthermore, 2-benzothiazole derivatives (19 and
20) were shown to inhibit the activity of HCMV protease
inside cells by using a novel cell transfection assay, indi-
cating that their antiviral activity in the plaque reduction
assay could be attributed to protease inhibition.

W. W. Ogilvie et al. / Bioorg. Med. Chem. 7 (1999) 1521±1531
1525
Experimental
4-(R)-4-Benzyloxymethyl-azetidin-2-one (3). The Boc
protected amino acid ester 2 (8 g, 20 mmol) was stirred
in 4 N HCl/dioxane (75 mL) for 1 h at which time TLC
indicated that the reaction was complete. The solution
was concentrated and the residue dissolved in a small
volume of water. A 1 M solution of K2CO3 (30 mL) was
then introduced and the mixture was extracted twice
with EtOAc. Drying over MgSO4 followed by removal
of solvent gave the desired amine as yellow oil (5.95 g,
99%). A solution of the amine (5.9 g, 19.7 mmol) in
ether (26 mL) was treated at 0ꢀC with TMS-Cl (2.80 mL,
21.7 mmol) and 10 min later with Et3N (3.3 mL,
23.6 mmol). The resulting suspension was stirred at 0ꢀC
for 1 h. Triethylamine hydrochloride was removed by
®ltration and the ®ltrate was diluted to 0.25 M with
ether and placed in an ice bath. Slow addition of tert-
butylmagnesium chloride (12.8 mL of a 2 M solution in
ether, 25.6 mmol) was followed by stirring at 0ꢀC for 1 h
and overnight at room temperature. The reaction was
quenched by the addition of saturated NH4Cl (5 mL)
and 10% HCl (10 mL) and extracted twice with CH2Cl2.
The combined organic extracts were washed with satu-
rated NaHCO3 and brine and dried over MgSO4. Flash
chromatography (25±60% EtOAc in hexane) a€orded 3
as yellow oil (2.87 g, 76%). [a]2d5 42.9 (c 1.02, CHCl3);
General methods
Unless otherwise noted, materials obtained from com-
mercial sources were used without further puri®cation.
All reactions were carried out under inert atmosphere.
In the last step, the reactions were performed on
approximately 100 mg scale to give the reported yields.
1H NMR spectra were obtained on a Bruker AMX 400
spectrometer with tetramethylsilane as internal standard
(d scale) and in the solvent given. FAB mass spectra
were recorded on an Autospec VG spectrometer. ES
mass spectra were recorded on a Quattro II spectro-
meter. Column chromatography was performed either
on silica gel (10±40 mm or 230±400 mesh ASTM, E.
Merck) or by preparative HPLC using a Partisil 10
ODS-3, C18 preparative column (50 cmÂ22 mm). Ana-
lytical 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% tri¯uoro-
acetic acid (TFA) in water/0.06% TFA; System B:
Vydac C18, 10 mm analytical column (24 cmÂ4.6 mm);
mobile phase, acetonitrile in 20 mM aqueous Na2HPO4
at pH 8.2.
1
3(R)-4-Benzyloxy-3-tert-butoxycarbonylamino-butyric
acid benzyl ester (2). To a solution of 1 (18.59 g,
63.16 mmol) and Et3N (9.2 mL, 66.3 mmol) in THF
(126 mL) at 0ꢀC was added dropwise isobutyl chlor-
oformate (9.0 mL, 69.5 mmol). After stirring for 40 min,
an excess of ethereal CH2N2 was added and stirring was
continued for an additional 1.5 h. Sucient glacial ace-
tic acid was then introduced to destroy excess CH2N2,
the solution was ®ltered and the ®ltrate washed with
saturated NaHCO3 and brine and dried over MgSO4.
Removal of solvent under reduced pressure gave the
IR (neat) 3262, 1759 cm 1; H NMR (CDCl3) d 7.39±
7.30 (m, 5H), 6.03 (br, 1H), 4.56 (s, 2H), 3.86±3.81 (m,
1H), 3.67 (dd, J=10.4, 4.6 Hz, 1H), 3.48 (dd, J=9.5,
7.6 Hz, 1H), 3.04 (ddd, J=14.9, 5.4, 1.9 Hz, 1H), 2.65
(ddd, J=14.6, 2.2, 1.6 Hz, 1H); MS (ES) 192 (M+H);
214 (M+23); HRMS calcd for C11H14NO2 (M+H)
192.1025, found 192.1028.
4-(R)-4-Hydroxymethyl-1-triisopropylsilanyl-azetidin-2-one
(4). Triisopropylsilyl tri¯uromethanesulfonate (1.07 mL,
3.98 mmol) was added via syringe to a solution of
1
desired diazoketone (20.17 g, 85%). H NMR (CDCl3)
3 (585 mg, 3.06 mmol) and 2,6-lutidine (0.89 mL,
d 7.38±7.29 (m, 5H), 5.56 (br, 1H), 5.41 (br, 1H), 4.53 (s,
2H), 4.33 (br, 1H), 3.87 (dd, J=9.2, 3.2 Hz, 1H), 3.62
(dd, J=9.5, 5.1 Hz, 1H), 1.46 (s, 9H). This material
(5.12 g, 16.0 mmol) was dissolved in THF (32 mL) and
water (5.8 mL). Silver benzoate (367 mg, 1.6 mmol) in
Et3N (3.4 mL) was then added slowly (caution: nitro-
gen gas evolution). After stirring at room temperature
for 2 h the solution was diluted with ether and washed
twice with 10% HCl, once with brine and dried over
MgSO4. Evaporation of the solvent gave the crude acid
which was dissolved in CH3CN (32 mL) containing
benzyl bromide (2.1 mL, 17.6 mmol) and DBU (2.88 mL,
19.2 mmol). After stirring for 2 h at room temperature,
the mixture was diluted with ether washed twice with
10% citric acid, twice with NaHCO3, once with brine
and dried over MgSO4. Removal of solvent under
reduced pressure gave the crude ester which was puri®ed
by ¯ash chromatography (10% EtOAc in hexanes) to
7.65 mmol) in CH2Cl2 (6 mL) at 0ꢀC. After stirring for
3 h, the solution was diluted with EtOAc and washed
with water, twice with 10% citric acid, twice with
NaHCO3 once with brine and dried over MgSO4. Eva-
poration of the solvent and ¯ash chromatography (10%
EtOAc in hexanes) gave the desired silyl amide as a
colorless oil (927 mg, 87%). [a]2d5 +33.1 (c 1.08, CHCl3);
IR (neat) 1744 cm 1; 1H NMR (CDCl3) d 7.33±7.22 (m,
5H), 4.48 (s, 2H), 3.74 (ddd, J=10.8, 5.7, 2.9 Hz, 1H),
3.59 (dd, J=9.9, 5.1 Hz, 1H), 3.47 (dd, J=9.9, 5.7 Hz,
1H), 3.12 (dd, J=15.3, 5.7 Hz, 1H), 2.76 (dd, J=15.3,
2.5 Hz, 1H), 1.31 (sept, J=2.6 Hz, 3H), 1.06 (d, J=
7.3 Hz, 9H), 1.02 (d, J=7.3 Hz, 9H); 13C NMR (CDCl3)
d 173.4, 137.9, 128.6, 128.0, 127.9, 73.6, 73.3, 48.8, 42.2,
18.4, 18.2, 12.0; MS (ES) 348 (M+H); 370 (M+23);
HRMS calcd for C20H34NO2Si (M+H) 348.2359,
found 348.2371. A mixture of the benzyl ether so
obtained (3.80 g, 10.9 mmol) and 10% Pd(OH)2/C
(300 mg) in THF (100 mL) was stirred under an atmo-
sphere of hydrogen for 5 h. Filtration through Celite1
and removal of the solvent gave pure 4 (3.38 g, 96%)
deliver the desired ester 2 (12.4 g, 77%). [a]2d5+5.0 (c
1
1.11, CHCl3); IR (neat) 3364, 1713 cm
;
1H NMR
(CDCl3) d 7.39±7.28 (m, 10H), 5.16 (br, 1H), 5.10 (s,
2H), 4.51 (d, J=12.1 Hz, 1H), 4.47 (d, J=12.4 Hz, 1H),
4.20 (br, 1H), 3.58 (dd, J=9.2, 3.8 Hz, 1H), 3.52 (dd,
J=9.5, 4.1 Hz, 1H), 2.75±2.65 (m, 2H), 1.45 (s, 9H); MS
(ES) 400 (M+H); 422 (M+23); HRMS calcd for
C23H30NO5 (M+H) 400.2124, found 400.2113.
after ¯ash chromatography (25% EtOAc in hexanes).
1
[a]2d5+41.5 (c 1.06, CHCl3); IR (KBr) 3380, 1707 cm
;
1H NMR (CDCl3) d 3.88±3.82 (m, 1H), 3.77±3.69 (m,
2H), 3.15 (dd, J=15.3, 5.4 Hz, 1H), 2.88 (dd, J=15.3,
2.2 Hz, 1H), 1.83 (dd, J=7.0, 4.5 Hz, 1H), 1.37 (sept,

1526
W. W. Ogilvie et al. / Bioorg. Med. Chem. 7 (1999) 1521±1531
J=7.6 Hz, 3H), 1.14 (d, J=7.3 Hz, 9H), 1.11 (d, J=
7.3 Hz, 9H); 13C NMR (CDCl3) d 173.6, 65.2, 50.3, 41.5,
18.4, 18.3, 12.0; MS (ES) 258 (M+H); 280 (M +23).
from 4 (160 mg, 0.62 mmol) and thiophene (0.07 mL,
0.94 mmol) in 70% yield (147 mg) using a procedure
similar to that described for compound 5f. HPLC (A)
1
100%, (B) 97%; mp 112±114ꢀC; IR (neat) 1711 cm
;
4-(R)-4-(Hydroxy-thiazol-2-yl-methyl)-1-triisopropylsil-
anyl-azetidin-2-one (5f). To a solution of b-lactam alco-
hol 4 (1.08 g, 4.2 mmol) and Dess±Martin periodinane
(2.68 g, 6.3 mmol) in dry CH2Cl2 (50 mL) was added
dropwise reagent grade CH2Cl2 (30 mL). After stirring
for 1 h, 50 mL of 10% Na2S2O3 and 50 mL of saturated
NaHCO3 was added and the resulting mixture stirred
vigorously until both layers were clear. The mixture was
diluted with ether and the combined organic phases
washed with satd NaHCO3, brine and dried over
MgSO4. Removal of solvent under reduced pressure
gave the desired aldehyde as a white solid (1.08 g,
100%). Thiazole (0.45 mL, 6.3 mmol) was dissolved in
THF (40 mL) and the resulting solution cooled to
50ꢀC. nBuLi (4.9 mL of a 1.2 M solution in hexanes,
5.88 mmol) was added and stirring was continued for
30 min. The solution was then cooled further to 78ꢀC
and the previously prepared aldehyde was slowly intro-
duced as a solution in THF (8 mL). TLC analysis after
20 min indicated that the reaction was complete. The
reaction was quenched by the addition of acetic acid
(0.4 mL) in THF (3 mL), saturated NH4Cl was added
and the mixture extracted with ether. The combined
ethereal extracts were washed with brine and dried over
MgSO4. Puri®cation by ¯ash chromatography (10%
EtOAc in benzene) gave the desired alcohol 5f (1.08 g,
76%) as a mixture of isomers. HPLC (A) 96%, (B)
97%; mp 79±81ꢀC; IR (KBr) 1738, 1719, 1682 cm 1; 1H
NMR (CDCl3) d isomer A: 7.77 (d, J=3.2 Hz, 1H), 7.34
(d, J=3.2 Hz, 1H), 5.32 (dd, J=3.5, 2.2 Hz, 1H), 4.23±
4.20 (m, 1H), 3.07 (dd, Jab=15.3, Jax=2.7 Hz, 1H),
2.94 (d, J=3.2 Hz, 1H), 2.86 (dd, Jab=15.3, Jbx=
5.5 Hz, 2H), 1.46±1.37 (m, 3H), 1.20±1.15 (m, 18H);
isomer B: 7.76 (d, J=3.2 Hz, 1H), 7.36 (d, J=3.2 Hz,
1H), 4.97 (dd, J=7.6, 5.1 Hz, 1H), 3.97±3.93 (m, 1H),
1H NMR (CDCl3) d 7.30±7.28 (m, 1H), 7.05±6.98 (m,
2H), 4.86±4.84 (m, 1H), 3.89±3.85 (m, 1H), 3.06±3.00 (m,
1H), 2.55 (dd, J=15.6, 2.7 Hz, 1H), 2.15 (s, 1H), 1.62±
1.37 (m, 3H), 1.25±1.13 (m, 18H); MS (ES ) 338 (M
H); HRMS calcd for C17H30NO2SSi (M+H), 340.1767,
found 340.1773.
-
4-(R)-4-(Hydroxy-pyridin-2-yl-methyl)-1-triisopropylsil-
anyl-azetidin-2-one (5c). This compound was prepared
from 4 (200 mg, 0.78 mmol) and 2-bromopyridine
(185 mg, 1.17 mmol) using a procedure similar to that
described for compound 5f. The reaction was carried
out in CH2Cl2 at 78ꢀC.33 HPLC (B) 96%; IR (neat)
1719 cm 1; 1H NMR (CDCl3) d 8.59±8.54 (m, 1H), 7.68
(m, 1H), 7.32±7.23 (m, 4H), 5.06±5.04 and 4.67 (2Âm,
1H), 4.11 and 4.07 (2Âd, J=7.0 and 4.5 Hz, 1H), 4.01±
3.98 and 3.85±3.81 (2 m, 1H), 2.98±2.88 (m, 1H), 2.66±
2.59 (m, 1H), 1.57±1.42 (m, 3H), 1.23±1.13 (m, 18H);
MS (ES+) 335 (M+H); HRMS calcd for C18H31
N2O2Si (M+H), 335.2155, found 335.2163.
4-(R)-4-{Hydroxy-[1-(2-trimethylsilanyl-ethoxymethyl)-
1H-benzoimidazol-2-yl]-methyl}-1-triisopropylsilanyl-aze-
tidin-2-one (5d). This compound was prepared from 4
(1.03 g, 4.03 mmol) and 1-[[2-(trimethylsilyl)ethoxy]-
methyl]-1H-benzimidazole34 (1.56 g, 6.05 mmol) in 52%
yield (1.05 g) using a procedure similar to that described
1
for compound 5f. IR (neat) 3226, 1734 cm 1; H NMR
(CDCl3) d 7.77 (dd, J=6.4, 1.6 Hz, 0.3H), 7.74 (dd,
J=7.6, 1.9 Hz, 0.7H), 7.46 (dd, J=6.7, 1.6 Hz, 0.3H),
7.41 (dd, J=6.4, 0.9 Hz, 0.7H), 7.36±7.29 (m, 2H), 5.70
(d, J=11.4 Hz, 0.7H), 5.68 (d, J=11.1 Hz, 0.3H), 5.56
(d, J=11.1 Hz, 0.7H), 5.54 (d, J=11.1 Hz, 0.3H), 5.32
(t, J=8.6 Hz, 0.3H), 4.92 (dd, J=8.6, 5.4 Hz, 0.7H),
4.42 (ddd, J=8.9, 6.0, 2.9 Hz, 0.7H), 4.32 (dt, J=5.1,
2.5 Hz, 0.3H), 4.00±3.97 (m, 0.3H), 3.93 (s, 0.7H), 3.64±
3.57 (m, 2H), 3.31 (dd, J=15.6, 2.5 Hz, 0.3H), 3.17 (dd,
J=15.6, 6.0 Hz, 0.7H), 3.11 (dd, J=15.6, 5.4 Hz, 0.3H),
2.59 (dd, J=15.6, 2.9 Hz, 0.7H), 1.62 (septet, J=7.3 Hz,
2H), 1.44 (septet, J=7.6 Hz, 1H), 1.17±1.10 (m, 18H),
0.96±0.90 (m, 2H), 0.02 (s, 0.7H), 0.03 (s, 0.3H); MS
(ES+) 504 (M+H); HRMS calcd for C26H46N3O3Si2,
504.3078, found 504.3004; Anal. calcd for C26H45
N3O3Si2: C, 61.98; H, 9.00; N, 8.34. Found: C, 61.56; H,
9.32; N, 8.24.
3.57 (d, J=5.1 Hz, 1H), 3.09 (dd, Jab=15.6, Jax
=
5.7 Hz, 1H), 2.81 (dd, Jab=15.6, Jbx=7.8 Hz, 2H),
1.57±1.46 (m, 3H), 1.17±1.12 (m, 18H); MS (ES+) 341
(M+H), 363 (M +23); HRMS calcd for C16H29
N2O2SSi, 341.1719, found 341.1722.
4-(R)-4-(Furan-2-yl-hydroxy-methyl)-1-triisopropylsilanyl-
azetidin-2-one (5a). This compound was prepared from
4 (1.15 g, 4.47 mmol) and furan (0.65 mL, 8.94 mmol)
in 53% yield (760 mg) using a procedure similar to
that described for compound 5f except that the con-
densation with furan was done in ether at 20ꢀC.
4-(R)-4-Thiazol-2-yl-methyl-1-triisopropylsilanyl-azetidin-
2-one (6f). Alcohol 5f (1 g, 2.94 mmol) and 1,10-(thio-
carbonyl)diimidazole (1.56 g, 8.82 mmol) were stirred
together with DMAP (357 mg, 2.94 mmol) in CH2Cl2
(30 mL) for 18 h. Removal of the solvent and ¯ash
chromatography (50% EtOAc in hexanes) gave the
desired thiocarbamate (1.26 g, 95%) which was imme-
diately dissolved in benzene (56 mL, 0.05 M) containing
Ph3SnH (1.47 g, 4.2 mmol) and AIBN (92 mg,
0.56mmol). The solution was degassed (two freeze±thaw
cycles) and re¯uxed for 45 min. Removal of solvent and
¯ash chromatography (10% EtOAc in hexanes the 30%
EtOAc in hexanes) gave 6f as a colorless oil (793 mg,
1
HPLC (A) 100%, (B) 98%; IR (neat) 1736 cm 1; H
NMR (CDCl3) d 7.39 and 7.25 (2Âm, 1H), 6.37±6.28
(m, 2H), 5.01 and 4.67 (2Âm, 1H), 3.96 (m, 1H), 3.25
and 2.65 (2Âm, 1H), 3.11±2.97 (m, 1H), 2.11 (m, 1H),
1.55±1.32 (m, 3H), 1.18-1.10 (m, 18H); MS (ES+) 324
(M+H); HRMS calcd for C17H30NO3Si (M+H),
324.1995, found 324.2004; Anal. calcd for C17H29NO3Si:
C, 63.12; H, 9.04; N, 4.33. Found: C, 63.34; H, 9.42; N,
4.50.
4-(R)-4-(Hydroxy-thiophen-2-yl-methyl)-1-triisopropylsil-
anyl-azetidin-2-one (5b). This compound was prepared

W. W. Ogilvie et al. / Bioorg. Med. Chem. 7 (1999) 1521±1531
1527
83%). HPLC (A) 97%, (B) 97%; [a]d+85ꢀ (c 0.55,
4-(R)-4-Benzothiazol-2-yl-methyl-1-triisopropylsilanyl-aze-
tidin-2-one (6e). This compound was prepared from 5e
in 71% yield using a similar procedure to that described
1
CHCl3); IR (CHCl3) 1730 cm 1; H NMR (CDCl3) d
7.73 (d, J=3.5 Hz, 1H), 7.25 (d, J=3.5 Hz, 1H), 4.08±
4.02 (m, 1H), 3.64 (dd, Jab=14.5, Jax=3.6 Hz, 1H),
3.23 (dd, Jab=15.6, Jax=5.4 Hz, 1H), 3.11 (dd, Jab=
above for 6f. HPLC (A) 100%, (B) 100%; [a]d+81ꢀ (c
1
0.61, CHCl3); IR (CHCl3) 1745 cm
;
1H NMR
14.5, Jax=10.7 Hz, 1H), 2.85 (dd, Jab=15.6, Jax
=
(CDCl3) d 8.00 (d, J=7.6 Hz, 1H), 7.87 (d, J=7.9 Hz,
1H), 7.51±7.46 (m, 1H), 7.41±7.37 (m, 1H), 4.19±4.13
(m, 1H), 3.73 (dd, Jab=14.6, Jax=3.8 Hz, 1H), 3.19
(dd, Jab=14.6, Jax=10.5 Hz, 1H), 3.28 (dd, Jab=15.6,
Jax=5.4 Hz, 1H), 2.91 (dd, Jab=15.6, Jbx=2.5 Hz, 1H);
MS (ES+) 375 (M+H), 397 (M+23); HRMS calcd for
C20H31N2OSSi, 375.1926, found 375.1939; Anal. calcd
for C20H30N2OSSi: C, 64.12; H, 8.07; N, 7.46. Found:
C, 64.19; H, 8.13; N, 7.40.
2.5 Hz, 1H), 1.41±1.34 (m, 3H), 1.18±1.13 (m, 18H); MS
(ES+) 325 (M+H); HRMS calcd for C16H29N2OSSi,
325.1770, found 325.1764; Anal. calcd for C16H28
N2OSSi: C, 59.21; H, 8.70; N, 8.63. Found: C, 59.20; H,
8.98; N, 8.66.
4-(S)-4-Furan-2-yl-methyl-1-triisopropylsilanyl-azetidin-2-
one (6a). This compound was prepared from 5a in 71%
yield using a similar procedure to that described above
for 6f. [a]2d5+70ꢀ (c 0.95, CH2Cl2); IR (neat) 1745 cm
;
4-(R)-4-Thiazol-2-yl-methyl-azetidin-2-one (7f). Lactam
6f (743 mg, 2.29 mmol) was stirred in methanol (20 mL)
containing CsF (418 mg, 2.75 mmol) at room tempera-
ture for 1 h. The methanol was then removed, the resi-
due suspended in EtOAc and washed twice with brine.
Drying over MgSO4 and ¯ash chromatography
(EtOAc) gave 7f (269 mg, 70%) as a colorless oil which
solidi®ed upon standing. Mp 56ꢀC; [a]d 52ꢀ (c 0.51,
1
1H NMR (CDCl3) d 7.32 (m, 1H), 6.29 (m, 1H), 6.06
(m, 1H),3.86 (m, 1H), 3.20 (m, 2H), 2.73 (m, 2H), 1.41±
1.33 (m, 3H), 1.16±1.11 (m, 18H); MS (ES+) 308
(M+H); HRMS calcd for C17H30NO2Si (M+H),
308.2046, found 308.2056.
4-(R)-4-Thiophen-2-yl-methyl-1-triisopropylsilanyl-azetidin-
2-one (6b). This compound was prepared from 5b in
67% yield using a similar procedure to that described
above for 6f. HPLC (A) 100%; [a]2d5+50ꢀ (c 0.80,
CH2Cl2); IR (neat) 1744 cm 1; 1H NMR (CDCl3) d 7.17
(d, J=5.1 Hz, 1H), 6.95 (dd, J=5.1, 3.2 Hz, 1H), 6.83
(d, J=3.2 Hz, 1H), 3.83±3.79 (m, 1H), 3.45 (dd, J=
14.3, 3.5 Hz, 1H), 3.16 (dd, J=15.5, 5.4 Hz, 1H), 2.87
(dd, J=14.3, 11.1 Hz, 1H), 2.76 (dd, J=5.6, 3.0 Hz, 1H),
1.38 (m, 3H), 1.15 (m, 18H); MS (ES+) 324 (M+H);
HRMS calcd for C17H30NOSSi (M+H), 324.1817,
found 324.1808.
1
CHCl3); IR (KBr) 1739 cm 1; H NMR (CDCl3) d 7.73
(d, J=3.5 Hz, 1H), 7.26 (d, J=3.5 Hz, 1H), 6.13 (br,
1H), 4.12±4.07 (m, 1H), 3.39 (dd, Jab=15.3, Jab=5.0Hz,
1H), 3.27 (dd, Jab=15.3, Jax=8.3 Hz, 1H), 3.21±3.16
(m, 1H), 2.80±2.76 (m, 1H); MS (ES+) 169 (M+H), 191
(M+23); HRMS calcd for C7H10N2OS, 169.0436,
found 169.0440; Anal. calcd For C7H9N2OS: C, 49.98;
H, 4.79; N, 16.65. Found: C, 49.75; H, 4.74; N, 16.43.
4-(S)-4-Furan-2-yl-methyl-azetidin-2-one (7a). Lactam 6a
(270 mg, 0.88 mmol) was treated with CsF as described
above to give 7a (120 mg, 90%) as a white solid. HPLC
(A) 99%, (B) 99%; [a]2d5 118ꢀ (c 0.28, CH2Cl2); IR
4-(S)-4-Pyridin-2-yl-methyl-1-triisopropylsilanyl-azetidin-
2-one (6c). This compound was prepared from 5c in
91% yield using a similar procedure to that described
above for 6f. HPLC (B) 99%; [a]2d5+249ꢀ (c 0.57,
CH2Cl2); IR (neat) 1740 cm 1; 1H NMR (CDCl3) d 8.54
(d, J=4.8 Hz, 1H), 7.62 (dt, J=7.7, 2.0 Hz, 1H), 7.17±
7.11 (m, 2H), 4.09±4.03 (m, 1H), 3.49 (dd, J=14.6,
5.2 Hz, 2H), 3.08 (dd, J=15.5, 5.3 Hz, 1H), 2.84±2.74
(m, 1H), 1.46±1.35 (m, 3H), 1.22±1.14 (m, 18H); MS
(ES+) 319 (M+H); HRMS calcd for C18H31N2OSi
(M+H), 319.2206, found 319.2223.
1
(neat) 1742 cm 1; H NMR (CDCl3) d 7.34 (dd, J=1.9,
0.6 Hz, 1H), 6.31 (dd, J=3.2, 1.0 Hz, 1H), 6.08 (dd,
J=3.2, 1.0 Hz, 1H), 5.89 (br, 1H), 3.90 (m, 1H), 3.13
(ddd, J=15.0, 4.8, 1.9 Hz, 1H), 3.00 (dd, J=14.9,
5.4 Hz, 1H), 2.91 (dd, J=14.9, 5.4 Hz, 1H), 2.72 (ddd,
J=15.0, 2.5, 1.6 Hz, 1H); MS (ES+) 152 (M+H);
HRMS calcd for C8H9NO2, 155.0633, found 155.0634.
4-(R)-4-Thiophen-2-yl-methyl-azetidin-2-one (7b). Lactam
6b (260 mg, 0.81 mmol) was treated with CsF as descri-
bed above to give 7b (120 mg, 92%) as a white solid.
4-(R)-1-Triisopropylsilanyl-4-[1-(2-trimethylsilanyl-ethoxy-
methyl)-1H-benzoimidazol-2-yl-methyl]-azetidin-2-one (6d).
This compound was prepared from 5d in 82% yield
HPLC (A) 99%, (B) 95%; [a]2d5 14ꢀ (c 0.85, CH2Cl2);
1
IR (neat) 1764 cm
;
1H NMR (CDCl3) d 7.18 (dd,
J=5.4, 1.3 Hz, 1H), 6.96 (dd, J=5.1, 3.5 Hz, 1H), 6.84
(m, 1H), 5.98 (br, 1H), 3.89±3.84 (m, 1H), 3.18 (dd,
J=14.9, 5.7 Hz, 1H), 3.14±3.06 (m, 2H), 2.73±2.68 (m,
1H); MS (ES+) 168 (M+H); HRMS calcd for
C8H10NOS, 168.0483, found 168.0489.
using a similar procedure to that described above for 6f.
;
1H
[a]2d5+78ꢀ (c 0.61, CHCl3); IR (neat) 1740 cm
1
NMR (CDCl3) d 7.76±7.74 (m, 1H), 7.69±7.67 (m, 1H),
7.49±7.40 (m, 2H), 5.48 (s, 2H), 4.41±4.36 (m, 1H),
3.58±3.53 (m, 3H), 3.40 (dd, J=15.9, 5.4 Hz, 1H), 3.04
(dd, J=15.6, 11.1 Hz, 1H), 2.85 (dd, J=15.6, 2.5 Hz,
1H), 1.42 (septet, J=7.3 Hz, 3H), 1.20 and 1.17 (2Âd,
J=7.3 Hz, 18H), 0.93 (t, J=7.9, 2H), 0.02 (s, 9H); 13C
NMR (CDCl3) d 173.3, 151.5, 136.3, 135.3, 130.6, 129.3,
123.4, 122.9, 119.8, 109.4, 72.5, 67.0, 47.6, 45.6, 34.9,
18.5, 18.4, 18.0, 12.0, 1.3; MS (ES+) 488 (M+H), 510
(M+23); HRMS calcd for C26H46N3O2Si2 (M+H),
488.3129, found 488.3145.
4-(S)-4-Pyridin-2-yl-methyl-azetidin-2-one (7c). Lactam
6c (550 mg, 1.73 mmol) was treated with CsF as descri-
bed above to give 7c (230 mg, 82%) as a white solid.
HPLC (B) 99%; mp 89-90ꢀC; [a]d25 204ꢀ (c 0.60,
1
CH2Cl2); IR (neat) 3167, 1739 cm 1; H NMR (CDCl3)
d 8.54 (d, J=4.7 Hz, 1H), 7.63 (dt, J=7.6, 1.6 Hz, 1H),
7.17 (d, J=6.4 Hz, 1H), 7.16 (t, J=7.6 Hz, 1H), 6.08
(br, 1H), 4.13-4.05 (m, 1H), 3.17±3.11 (m, 2H), 3.04 (dd,

1528
W. W. Ogilvie et al. / Bioorg. Med. Chem. 7 (1999) 1521±1531
J=14.6, 8.4 Hz, 1H), 2.77±2.73 (m, 1H); MS (ES+) 163
(M+H); HRMS calcd for C9H11N2O, 163.0871, found
163.0875; Anal. calcd for C9H10N2O: C, 66.65; H, 6.21;
N, 17.27. Found: C, 66.68; H, 6.10; N, 16.93.
was prepared from 7a (46 mg, 0.31 mmol) using a pro-
cedure similar to that described above for compound
10. Compound 11 was obtained as an oil (60 mg, 57%)
after ¯ash chromatography (50% EtOAc in hexanes).
HPLC (A) 99%, (B) 99%; [a]2d5 126ꢀ (c 0.27, CH2Cl2);
IR (neat) 1777, 1668 cm 1; 1H NMR (CDCl3) d 8.21 (d,
J=8.9 Hz, 2H), 7.43 (d, J=8.9 Hz, 2H), 7.30 (d,
J=0.7 Hz, 1H), 6.30 (dd, J=2.9, 2.0 Hz, 1H), 6.10 (dd,
J=2.9, 0.7 Hz, 1H), 4.71±4.61 (m, 2H), 4.45 (m, 1H),
3.21±2.82 (m, 4H), 2.97 (s, 3H); MS (ES+) 344 (M+H);
HRMS calcd for C17H18N3O5, 344.1246, found
344.1251; Anal. calcd for C17H17N3O5: C, 59.47; H,
4.99; N, 12.24. Found: C, 59.80; H, 5.06; N, 11.97.
4-(R)-4-[1-(2-Trimethylsilanyl-ethoxymethyl)-1H-benzo-
imidazol-2-yl-methyl]-azetidin-2-one (7d). Lactam 6d
(445 mg, 0.91 mmol) was treated with CsF as described
above to give 7d (240 mg, 79%) as a white solid. Mp
68ꢀC; [a]d25 74ꢀ (c 0.67, CHCl3); IR (KBr) 3177,
1
1745 cm
;
1H NMR (CDCl3) d 7.73±7.69 (m, 1H),
7.42±7.39 (m, 1H), 7.37±7.26 (m, 2H), 6.51 (s, 1H), 5.48
(s, 2H), 4.34±4.28 (m, 1H), 3.56 (dd, J=8.3, 8.3 Hz,
2H), 3.35 (dd, J=16.2, 4.1 Hz, 1H), 3.28 (ddd, J=14.9,
5.1, 1.9 Hz, 1H), 3.12 (dd, J=15.9, 9.2 Hz, 1H), 2.82
(ddd, J=14.9, 2.2, 1.3 Hz, 1H), 0.91 (dd, J=8.0, 8.0 Hz,
2H), 0.04 (s, 9H); MS (ES+) 332 (M+H), 354 (M
+23); HRMS calcd for C17H26N3O2Si, 332.1794, found
332.1806; Anal. calcd for C17H25N3O2Si: C, 61.60; H,
7.60; N, 12.68. Found: C, 61.48; H, 7.79; N, 12.46.
4-(R)-4-Thiophen-2-yl-methyl-2-oxo-azetidine-1-carboxylic
acid methyl-(4-tri¯uoromethyl-benzyl)-amide (12). This
compound was prepared from 7b (30 mg, 0.18 mmol)
using a procedure similar to that described above for
compound 10. Compound 12 was obtained as an oil
(40 mg, 50%) after ¯ash chromatography (50% EtOAc
in hexanes). HPLC (A) 97%, (B) 95%; [a]2d5+249ꢀ (c
1
4-(R)-4-Benzothiazol-2-yl-methyl-azetidin-2-one (7e).
Lactam 6e (819 mg, 2.19 mmol) was treated with CsF
as described above to give 7e (330 mg, 69%) as a white
0.25, CH2Cl2); IR (neat) 1779, 1668 cm
;
1H NMR
(CDCl3) d 7.61 (d, J=7.9 Hz, 2H), 7.38 (d, J=7.9 Hz,
2H), 7.18 (dd, J=5.4, 1.3 Hz, 1H), 6.95 (dd, J=5.4,
3.2 Hz, 1H), 6.84 (dd, J=3.2, 1.3 Hz, 1H), 4.78±4.60 (m,
2H), 4.49±4.44 (m, 1H), 3.42 (dd, J=14.9, 3.8 Hz, 1H),
3.19 (m, 1H) 2.99 (s, 3H), 2.98 (dd, J=15.9, 6.1 Hz, 1H),
2.76 (dd, J=15.9, 3.8 Hz, 1H); MS (ES+) 383 (M+H);
HRMS calcd for C18H18F3N2O2S (M+H), 383.1041,
found 383.1052.
solid. mp 115ꢀC; HPLC (A) 100%, (B) 100%; [a]d 66ꢀ
1
(c 0.41, CHCl3); IR (CHCl3) 1768 cm
;
1H NMR
(CDCl3) d 7.98 (d, J=7.9 Hz, 1H), 7.87 (d, J=7.9 Hz,
1H), 7.51±7.47 (m, 1H), 7.42±7.38 (m, 1H), 6.20 (br, 1H),
4.23±4.18 (m, 1H), 3.48 (dd, Jab=15.5, Jax=4.6 Hz,
1H), 3.36 (dd, Jab=15.5, Jax=8.4 Hz, 1H), 3.24 (ddd,
J=14.9, 5.1, 2.2 Hz, 1H), 2.85±2.81 (m, 1H); MS (ES+)
219 (M+H); HRMS calcd for C11H11N2OS, 219.0592,
found 219.0599; Anal. calcd for C11H10N2OS: C, 60.53;
H, 4.62; N, 12.83. Found: C, 60.82; H, 4.33; N, 12.86.
4-(R)-2-Oxo-4-thiophen-2-yl-methyl-2-oxo-azetidine-1-
carboxylic acid methyl-(4-nitro-benzyl)-amide (13). This
compound was prepared from 7b (30 mg, 0.18 mmol)
using a procedure similar to that described above for
compound 10. Compound 13 was obtained as a pale-
yellow syrup (32 mg, 50%) after ¯ash chromatography
4-(S)-4-Furan-2-yl-methyl-2-oxo-azetidine-1-carboxylic
acid methyl-(4-tri¯uoromethyl-benzyl)-amide (10). Lac-
tam 7a (50 mg, 0.33 mmol) was dissolved in THF (5 mL)
and cooled to 50ꢀC. A 0.5 M solution of KHMDS in
toluene (0.73 mL, 0.36 mmol) was added and the
resulting solution was stirred at 50ꢀC for 20 min. This
solution was then added rapidly via cannula to a THF
solution of amidyl chloride (250 mg, 1 mmol) at 50ꢀC.
The resulting pale-yellow mixture was stirred for 10 min
and then quenched by the addition of saturated NH4Cl.
The reaction mixture was diluted with ether, washed
twice with water, once with brine and dried over
MgSO4. The crude residue was puri®ed by ¯ash chro-
matography (25% EtOAc in hexanes to 50% EtOAc in
hexanes) to provide compound 10 (86 mg, 71%) as a
pale-yellow gum. HPLC (A) 100%, (B) 100%; [a]2d5 39ꢀ
(50% EtOAc in hexanes). HPLC (A) 99%, (B) 99%;
1
[a]2d5 141ꢀ (c 0.26, CH2Cl2); IR (neat) 1661, 1607 cm
;
1H NMR (CDCl3) d 8.21 (d, J=8.5 Hz, 2H), 7.43 (d,
J=8.5 Hz, 2H), 7.18 (dd, J=5.1, 1.0 Hz, 1H), 6.96 (dd,
J=5.1, 3.5 Hz, 1H), 6.85 (dd, J=3.5, 1.0 Hz, 1H), 4.74±
4.64 (m, 2H), 4.47 (m, 1H), 3.41 (dd, J=14.9, 3.8 Hz,
1H), 3.23 (dd, J=14.9, 7.0 Hz, 1H), 3.02 (s, 3H), 3.00
(dd, J=17.1, 6.1 Hz, 1H), 2.79 (dd, J=15.9, 3.8 Hz,
1H); MS (ES+) 360 (M+H); HRMS calcd for C17H18
N3O4S (M+H), 360.1018, found 360.1010.
4-(S)-4-Pyridin-2-yl-methyl-2-oxo-azetidine-1-carboxylic
acid methyl-(4-tri¯uoromethyl-benzyl)-amide (14). This
compound was prepared from 7c (50 mg, 0.31 mmol)
using a procedure similar to that described above for
compound 10. Compound 14 was obtained as a pale-
yellow syrup (70 mg, 61%) after ¯ash chromatography
(90% EtOAc in hexanes). HPLC (A) 100%, (B)
1
(c 0.31, CH2Cl2); IR (neat) 1778, 1669 cm 1; H NMR
(CDCl3) d 7.61 (d, J=8.3 Hz, 2H), 7.38 (d, J=8.3 Hz,
2H), 7.29 (d, J=1.0 Hz, 1H), 6.30 (dd, J=3.2, 1.9 Hz,
1H), 6.10 (dd, J=3.2, 1.0 Hz, 1H), 4.62±4.58 (m, 2H),
4.47±4.42 (m, 1H), 3.21±2.82 (m, 4H), 2.96 (s, 3H); MS
(ES+) 367 (M+H); HRMS calcd for C18H18F3N2O3
(M+H), 367.1269, found 367.1263; Anal. calcd For
C18H17F3N2O3: C, 59.02; H, 4.68; N, 7.65. Found: C,
59.16; H, 4.74; N, 7.45.
100%; [a]2d5+167ꢀ (c 0.24, CH2Cl2); IR (neat) 1777,
1
1668 cm
;
1H NMR (CDCl3) d 8.51 (dd, J=4.5,
1.3 Hz, 1H), 7.62±7.51 (m, 3H), 7.39 (d, J=8.0 Hz, 2H),
7.17±7.14 (m, 2H), 4.61 (br, 1H), 4.61±4.56 (m, 2H),
3.35 (dd, J=13.7, 4.2 Hz, 1H), 3.15 (br, 1H), 3.01±2.99
(m, 2H), 2.94 (s, 3H); MS (ES+) 378 (M+H); HRMS
calcd for C19H19F3N3O2(M+H), 378.1429, found
378.1420.
4-(S)-4-Furan-2-yl-methyl-2-oxo-azetidine-1-carboxylic
acid methyl-(4-nitro-benzyl)-amide (11). This compound

W. W. Ogilvie et al. / Bioorg. Med. Chem. 7 (1999) 1521±1531
1529
1
4-(R)-4-(1H-Benzoimidazol-2-yl-methyl)-2-oxo-azetidine-1-
carboxylic acid methyl-(4-tri¯uoromethyl-benzyl)-amide
(15). This compound was prepared from 7d using a
procedure similar to that described above for compound
10. Final deprotection was accomplished by stirring in
2/1 TFA/CH2Cl2 for 6 h. Removal of solvent followed
by dissolution in EtOAc, washing with NaHCO3 and
brine and drying over MgSO4 gave 15 (12.7 mg, 40%) as
a white solid after ¯ash chromatography (10% acetone
in EtOAc). Mp 232ꢀC (dec); HPLC (A) 100%, (B)
(neat) 1779, 1669 cm 1; H NMR (CDCl3) d 8.22 (d,
J=8.6 Hz, 2H), 7.47 (d, J=8.9 Hz, 2H), 4.73±4.56 (m,
3H), 4.29 (s, 3H), 3.43 (dd, J=15.0, 4.5 Hz, 1H), 3.36
(dd, J=15.0, 6.4 Hz, 1H), 3.09 (dd, J=15.9, 6.0 Hz,
1H), 2.99 (s, 3H), 2.88 (dd, J=16.2, 3.5 Hz, 1H); MS
(FAB) 360 (M+H); HRMS calcd for C15H18N7O4,
360.1420, found 360.1414.
4-(R)-4-Benzothiazol-2-yl-methyl-2-oxo-azetidine-1-car-
boxylic acid methyl-(4-tri¯uoromethyl-benzyl)-amide (19).
This compound was prepared from 7e (141 mg,
0.65 mmol) using a procedure similar to that described
above for compound 10. Compound 19 was obtained as
a pale-yellow syrup (153 mg, 55%) after ¯ash chroma-
tography (40% EtOAc in hexanes). HPLC (A) 100%,
92%; [a]d +22ꢀ (c 0.46, CH3CN); IR (CHCl3) 1729,
1
1655 cm
;
1H NMR (CDCl3) d 8.23±8.21 (m, 1H),
7.72±7.69 (m, 1H), 7.54 (d, J=8.0 Hz, 2H), 7.41±7.38
(m, 2H), 7.29 (d, J=8.3 Hz, 2H), 4.71±4.64 (m, 1H),
4.51 (s, 2H), 4.46 (d, J=6.4 Hz, 1H), 3.47 (dd, Jab=16.8,
Jax=4.4 Hz, 1H), 3.33 (dd, Jab=16.8, Jbx=6.4, 1H),
(B) 100%; [a]d+91ꢀ (c 0.49, CHCl3); IR (CHCl3) 1782,
1
3.15 (dd, Jab=17.3, Jax=3.7 Hz, 1H), 3.11 (dd, Jab
=
1669 cm
;
1H NMR (CDCl3) d 7.95 (d, J=8.3 Hz,
17.3, Jbx=6.4, 1H), 2.81 (s, 3H); MS (ES+) 417
(M+H), 439 (M+23); HRMS calcd for C21H20F3N4O2
(M+H), 417.1538, found 417.1521.
1H), 7.84 (d, J=7.9 Hz, 1H), 7.59 (d, J=8.3 Hz, 2H),
7.49±7.45 (m, 1H), 7.41±7.37 (m, 3H), 4.72±4.61 (m,
3H), 3.69 (dd, Jab=15.1, Jax=4.1 Hz, 1H), 3.52 (dd,
Jab=15.1, Jax=7.3 Hz, 1H), 3.12 (dd, Jab=16.2,
Jax=6.3 Hz, 1H), 3.06 (dd, Jab=16.2, Jax=3.6 Hz,
1H), 2.98 (s, 3H); MS (ES+) 434 (M+H); HRMS calcd
for C21H19F3N3O2S, 434.1150, found 434.1164.
4-(R)-4-(1H-Benzoimidazol-2-yl-methyl)-2-oxo-azetidine-
1-carboxylic acid methyl-(4-nitro-benzyl)-amide (16).
This compound was prepared from 7d (79 mg,
0.24 mmol) using a procedure similar to that described
above for compound 10. Final deprotection was
accomplished by stirring in 2/1 TFA/CH2Cl2 for 6 h.
Removal of solvent followed by dissolution in EtOAc,
washing with NaHCO3 and brine and drying over
MgSO4 gave 16 (18 mg, 19%) as a white solid after ¯ash
chromatography (10% acetone in EtOAc). Mp 194ꢀC;
HPLC (A) 100%, (B) 95%; [a]d +32ꢀ (c 0.45, CH3CN);
IR (CHCl3) 1729, 1657 cm 1; 1H NMR (CDCl3) d 8.21±
8.19 (m, 1H), 8.13±8.11 (m, 1H), 7.71±7.69 (m, 1H),
7.40±7.37 (m, 2H), 7.33 (d, J=8.9 Hz, 2H), 4.71±4.64
(m, 1H), 4.55 (s, 2H), 4.45 (d, J=6.0 Hz, 1H), 3.46 (dd,
Jab=16.9, Jax=4.2 Hz, 1H), 3.35 (dd, Jab=16.9,
Jbx=6.0, 1H), 3.14 (d, J=5.1 Hz, 2H), 2.84 (s, 3H); MS
(ES+) 394 (M+H), 416 (M+23); HRMS calcd for
C20H20N5O4, 394.1515, found 394.1528.
4-(R)-4-Benzothiazol-2-yl-methyl-2-oxo-azetidine-1-car-
boxylic acid methyl-(4-nitro-benzyl)-amide (20). This
compound was prepared from 7e (70 mg, 0.32 mmol)
using a procedure similar to that described above for
compound 10. Compound 20 was obtained as a pale-
yellow syrup (44 mg, 33%) after ¯ash chromatography
using TLC grade silica gel (60% EtOAc in hexanes).
HPLC (A) 100%, (B) 99%; [a]d +96ꢀ (c 0.60, CHCl3);
IR (CHCl3) 1782, 1670 cm 1; H NMR (CDCl3) d 8.18
1
(d, J=8.9 Hz, 2H), 7.94 (d, J=8.3 Hz, 1H), 7.85 (d,
J=7.6 Hz, 1H), 7.50±7.38 (m, 4H), 4.75±4.64 (m, 3H),
3.67 (dd, Jab=15.0, Jax=4.0 Hz, 1H), 3.56 (dd, Jab=
15.0, Jax=6.8 Hz, 1H), 3.17±3.05 (m, 2H), 3.01 (s, 3H);
MS (ES+) 411 (M+H); HRMS calcd for C20H19
N4O4S, 411.1127, found 411.1117; Anal. calcd for
C20H18N4O4S: C, 58.53; H, 4.42; N, 13.65. Found: C,
58.29; H, 4.40; N, 13.45.
4-(R)-4-(2-Methyl-2H-tetrazol-5-yl-methyl)-2-oxo-azetidine-
1-carboxylic acid methyl-(4-tri¯uoromethyl-benzyl)-amide
(17). This compound was prepared from 8 (40 mg,
0.24 mmol) using a procedure similar to that described
above for compound 10. Compound 17 was obtained as
a pale-yellow oil (49 mg, 55%) after ¯ash chromato-
graphy (50% EtOAc in hexanes). HPLC (A) 100%, (B)
4-(R)-4-Thiazol-2-yl-methyl-2-oxo-azetidine-1-carboxylic
acid methyl-(4-tri¯uoromethyl-benzyl)-amide (21). This
compound was prepared from 7f (53 mg, 0.32 mmol)
using a procedure similar to that described above for
compound 10. Compound 21 was obtained as a pale-
yellow oil (64 mg, 53%) after ¯ash chromatography
using TLC grade silica gel (50% EtOAc in hexanes).
HPLC (A) 100%, (B) 100%; [a]d +72ꢀ (c 0.55, CHCl3);
1
99%; IR (neat) 1779, 1669 cm 1; H NMR (CDCl3) d
7.56 (d, J=8.0 Hz, 2H), 7.35 (d, J=8.0 Hz, 2H), 4.70±
4.49 (m, 3H), 4.21 (s, 3H), 3.40 (dd, J=15.0, 4.1 Hz,
1H), 3.28 (dd, J=15.0, 6.5 Hz, 1H), 3.03 (dd, J=16.0,
6.0 Hz, 1H), 2.90 (s, 3H), 2.83 (dd, J=16.0, 3.7 Hz, 1H);
MS (FAB) 383 (M+H); HRMS calcd for C16H18
F3N6O6, 383.1444, found 383.1452.
IR (CHCl3) 1781, 1668 cm 1; H NMR (CDCl3) d 7.72
1
(d, J=3.2 Hz, 1H), 7.62 (d, J=8.3 Hz, 2H), 7.40 (d,
J=7.9 Hz, 2H), 7.25 (d, J=3.5 Hz, 1H), 4.75±4.55 (m,
3H), 3.59 (dd, Jab=14.9, Jax=4.2 Hz, 1H), 3.45 (dd,
Jab=14.9, Jax=6.9 Hz, 1H), 3.10±3.01 (m, 2H), 2.97 (s,
3H); MS (ES+) 389 (M+H), 406 (M+23); HRMS
calcd for C17H17F3N3O2S, 384.0994, found 384.0987;
Anal. calcd For C17H16F3N3O2S: C, 53.26; H, 4.21; N,
10.96. Found: C, 53.08; H, 4.29; N, 10.88.
4-(R)-4-(2-Methyl-2H-tetrazol-5-yl-methyl)-2-oxo-azetidine-
1-carboxylic acid methyl-(4-nitro-benzyl)-amide (18). This
compound was prepared from 8 (50 mg, 0.29 mmol)
using a procedure similar to that described above for
compound 10. Compound 18 was obtained as a pale-
yellow syrup (54 mg, 53%) after ¯ash chromatography
(50% EtOAc in hexanes). HPLC (A) 96%, (B) 96%; IR
4-(R)-4-Thiazol-2-yl-methyl-2-oxo-azetidine-1-carboxylic
acid methyl-(4-nitro-benzyl)-amide (22). This compound

1530
W. W. Ogilvie et al. / Bioorg. Med. Chem. 7 (1999) 1521±1531
was prepared from 7f (50 mg, 0.30 mmol) using a pro-
cedure similar to that described above for compound
10. Compound 21 was obtained as a pale-yellow oil
(40 mg, 37%) after ¯ash chromatography using TLC
grade silica gel (60% EtOAc in hexanes). HPLC (A)
incubated in cysteine and methionine de®cient DMEM
for 20 min and labeled for 2 h with Easy Tag Express
(100 mCi/mL), NEN. Cells were lysed in ice-cold RIPA
bu€er containing protease inhibitors. The samples were
centrifuged and the supernatant subjected to immuno-
precipitation. Total [35S] incorporation into protein was
determined using TCA precipitation on Whatman
chromatography paper (Et-31). Aliquots representing
the same number of counts were incubated with 10 mL
of rabbit K-58 polyclonal antibody coupled to anti
rabbit IgG-conjugated magnetic beads (Dynal). K-58
serum was produced using a peptide representing amino
acids 196-213 of HCMV assembly protein. The immu-
noprecipitated proteins were solubilized in SDS sample
bu€er and denatured by heating at 95ꢀC for 5 min.
Samples were electrophoresed on SDS-polyacrylamide
gels. The gels were dried and exposed to phosphor-
autoradiography. The quantitation of the radioactivity
contained in the precursor and the product was done
using a phosphoimager (Molecular Dynamics).
100%, (B) 99%; [a]d +66ꢀ (c 0.62, CHCl3); IR (CHCl3)
1
1781, 1670 cm
;
1H NMR (CDCl3) d 8.23±8.20 (m,
2H), 7.72 (d, J=3.2 Hz, 1H), 7.46 (d, J=8.6 Hz, 2H),
7.26 (d, J=3.2 Hz, 1H), 4.68±4.57 (m, 3H), 3.58 (dd,
Jab=14.9, Jax=4.1 Hz, 1H), 3.47 (dd, Jab=14.9,
Jax=6.8 Hz, 1H), 3.11±3.03 (m, 2H), 2.99 (s, 3H); MS
(ES+) 361 (M+H), 383 (M+23); HRMS calcd for
C16H17 N4O4S, 361.0971, found 361.0958; Anal. calcd
for C16H16N4O4S: C, 53.32; H, 4.47; N, 15.55. Found:
C, 53.06; H, 4.43; N, 15.35.
Enzymatic and plaque reduction assays
IC50 and EC50 values were determined by previously
published methods.12,20,35 Each reported IC50 value
represents the mean of at least 3 determinations. The
reproducibility of the enzymatic assay was gauged using
Ac-Gly-Tbg-Val-Asn-Ala(CF3) as a standard. This
compound gave an IC50 of 8.28‹0.27 (95% con®dence
limit based on 2sm) on 302 determinations. Ganciclovir
was used as a positive control in the plaque reduction
assay and gave an EC50 of 1.23‹0.07 mM (95% con-
®dence limit based on 2sm) on 82 determinations. The
cytotoxicity of drugs (TC50) was determined with the
tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-
nyl tetrazolium bromide (MTT)36 under the same cell
culture assay conditions used in the plaque reduction
assay.
Acknowledgements
S. Valois, C. Boucher, S. Bordeleau, and M. Little are
gratefully acknowledged for excellent analytical sup-
port. P. Bonneau, C. Plou€e and H. Montpetit per-
formed the enzymatic assay. Thanks to D. Wernic for
the synthesis of the substrates used in the enzymatic
assay. The plaque reduction assay was performed by N.
Dansereau, M. Liuzzi and A.-M. Bonneau. Thanks to J.
Kelland for assistance during the preparation of this
manuscript.
Cell based assay
The coding sequences for the HCMV UL80 (protease
precursor) and UL80.5 (assembly protein precursor)
genes were ampli®ed from HCMV genomic DNA by
PCR and cloned into pCR3 plasmid vector (Invitrogen)
which can direct expression of protein under the control
of the T7 promoter in mammalian cells.29 For expres-
sion of the catalytic domain of the protease, the DNA
sequence encoding amino acid 1-256 of the UL80 gene
was ampli®ed by PCR using HCMV genomic DNA as a
template and cloned in the pCR3 vector (Invitrogen).
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in Dulbecco's modi®ed Eagle's medium (DMEM) con-
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