Novel imidazole substituted 6-methylidene-penems as broad-spectrum β-lactamase inhibitors

Article abstract of DOI:10.1016/j.bmc.2004.08.039

A series of novel imidazole substituted 6-methylidene-penems has been synthesized (R = heterocycle) and was shown to be potent, broad-spectrum β-lactamase inhibitors against class-A and class-C enzymes. The present paper deals with the design, synthesis, and structure-activity relationships (SAR) of compounds 11-15. β-Lactamases are serine and metallo-dependent enzymes produced by the bacteria in defense against β-lactam antibiotics. Production of class-A, class-B, and class-C enzymes by the bacteria make the use of β-lactam antibiotics ineffective in certain cases. To overcome resistance to β-lactam antibiotics, several β-lactamase inhibitors such as clavulanic acid, sulbactam, and tazobactam are widely used in the clinic in combination with β-lactam antibiotics. However, single point mutations within these enzymes have allowed bacteria to overcome the inhibitory effect of the commercially approved β-lactamase inhibitors. Although the commercially available β-lactamase inhibitor/β-lactam antibiotic combinations are effective against class-A producing bacteria and many extended spectrum β-lactamase (ESBL's) producing bacteria they are less effective against class-C enzymes expressing bacteria. To circumvent this problem, based on modeling studies several novel imidazole substituted 6-methylidene-penem derivatives were synthesized and tested against various β-lactamase producing isolates. The present paper deals with the synthesis and structure-activity relationships (SAR) of these compounds.

Full text of DOI:10.1016/j.bmc.2004.08.039

Bioorganic & Medicinal Chemistry 12 (2004) 5807–5817
Novel imidazole substituted 6-methylidene-penems as
broad-spectrum b-lactamase inhibitors
Aranapakam M. Venkatesan,a,* Atul Agarwal,a Takao Abe,b Hideki Ushirogochi,b
Itsuki Yamamura,b Toshio Kumagai,b Peter J. Petersen,a William J. Weiss,a Eileen Lenoy,a
Youjun Yang,a David M. Shlaes,a John L. Ryanc and Tarek S. Mansoura
aWyeth Research, 401 N. Middletown Road, Pearl River, NY 10965, USA
bWyeth Lederle Japan, Saitama, 353-8511, Japan
cWyeth Research, 87 Cambridge Park Drive, Cambridge, MA02140, USA
Received 28 June 2004; revised 20 August 2004; accepted 26 August 2004
Available online 21 September 2004
Abstract—b-Lactamases are serine and metallo-dependent enzymes produced by the bacteria in defense against b-lactam antibiotics.
Production of class-A, class-B, and class-C enzymes by the bacteria make the use of b-lactam antibiotics ineffective in certain cases.
To overcome resistance to b-lactam antibiotics, several b-lactamase inhibitors such as clavulanic acid, sulbactam, and tazobactam
are widely used in the clinic in combination with b-lactam antibiotics. However, single point mutations within these enzymes have
allowed bacteria to overcome the inhibitory effect of the commercially approved b-lactamase inhibitors. Although the commercially
available b-lactamase inhibitor/b-lactam antibiotic combinations are effective against class-A producing bacteria and many extended
spectrum b-lactamase (ESBLꢀs) producing bacteria they are less effective against class-C enzymes expressing bacteria. To circumvent
this problem, based on modeling studies several novel imidazole substituted 6-methylidene-penem derivatives were synthesized and
tested against various b-lactamase producing isolates. The present paper deals with the synthesis and structure–activity relationships
(SAR) of these compounds.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
with amoxicillin or ticarcillin), sulbactam3b 2 (given in
combination with ampicillin), and tazobactam3c 3 (given
in combination with piperacillin) are effective class-A
enzyme inhibitors. However, these inhibitors/antibiotics
combinations are the least effective against class-C and
class-B producing bacteria including some extended
spectrum b-lactamases (ESBL) producing bacteria.
b-Lactamases are serine-dependent enzymes produced
by the bacteria in defense against all classes of b-lactam
antibiotics, such as penicillins, cephalosporins, carba-
penems, and monobactams. In order to overcome this
resistance several b-lactamase inhibitor/b-lactam antibi-
otic combinations are currently being used in the clinical
setting. However, single point mutations within these
enzymes have rendered b-lactamases relatively resistant
to inactivation.1 Based upon the amino acid sequence
variations, the b-lactamase enzymes have been divided
into class-A, class-B, class-C, and class-D b-lacta-
mases.1d,2 Currently, only three b-lactamase inhibitors
are used in the clinic in combination with b-lactam anti-
biotics (Fig. 1). Clinically employed b-lactamase inhibi-
tors such as clavulanic acid3a 1 (given in combination
During the last decade, several groups have focused on
mechanistic considerations in designing new broad-spec-
trum inhibitors.4,5 In this regard, boronates6 have been
reported as transition state inhibitors and acyl phospho-
nates and phosphonamidates7 as active serine phos-
phorylating agents. In seeking insights into designing
new 6-methylidene-penem inhibitors, we demonstrated
that electrospray ionization mass spectrometry tech-
niques provides a powerful method for characterizing
transformations that occur upon the interaction of
inhibitors with TEM-1, SHV-1, and AmpC b-lacta-
mases.8 High-resolution crystallographic structures of
two novel penems carrying bicyclic or tricyclic hetero-
cycles at the 6-methylidene moiety indicated the
Keyword: b-lactamase inhibitors.
Corresponding author. Tel.: +1 845 602 4023; fax: +1 845 602
5561; e-mail: venkata@wyeth.com
*
0968-0896/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.08.039

5808
A. M. Venkatesan et al. / Bioorg. Med. Chem. 12 (2004) 5807–5817
O
O
O
O
S
S
O
N
N
OH
N
N
N
N
O
O
O
COONa
COONa
COONa
3
2
1
N N
N
N
N
N
N
N
S
S
S
N
N
N
O
O
O
COONa
COONa
COONa
4
5
6
SYN-1012
BRL-42715
SB-206999Z
Figure 1. Currently used commercial b-lactamase inhibitors and penem inhibitors 4–6.
dominance of hydrophobic stacking interactions between
the heterocycle and amino acid residues in SHV-1 and
GC1 enzymes.9 The formation of 1,4-thiazepine ring
structures10 in both the enzymes was established with
different orientation of the thiazepine ring and the
heterocyclic moieties.9 These findings for penems bear-
ing bicyclic and tricyclic heterocyles at the methylidene
moiety observed by crystallography9 are different from
modeling studies of a related monocyclic penem.5,10,11
In the earlier modeling studies5 the importance of
hydrophobic interactions, the orientation of the thiaze-
pine ring and the stereochemistry at the carbon bearing
the heterocycle were not predicted. With these consider-
ations in mind, we undertook the design and synthesis of
penem inhibitors bearing a 6-methylidene monocyclic
heterocyle. Earlier work has demonstrated that several
penems containing furyl, oxazolyl, isoxazolyl, thiadiazo-
lyl, and triazolyl rings are broad-spectrum inhibitors of
class A and C enzymes.12 However, 6-methylidene-pen-
ems bearing an imidazole nucleus have never been re-
ported in the literature. Hence, it is the objective of
this article to synthesize and elucidate the structure–
activity relationships of several novel 6-methylidene-
penems bearing imidazole derivatives against several
bacteria expressing different b-lactamases. Unlike the
other b-lactamase inhibitors,3 the 6-methylidene struc-
tural feature of 4 imparts a unique mechanism of action
for the b-lactamase inhibition.5,8,11
penem moiety of the inhibitor present comparable inter-
action opportunities in TEM-1 and AmpC enzymes.
However, there is significant difference between the
remainder of the binding sites of TEM-1 and AmpC en-
zymes, which makes the design of broad-spectrum inhib-
itors challenging. Hypotheses for the atomic interactions
were derived from several crystal structures available in
the public domain with inhibitor fragments bound to the
b-lactamase enzymes.13c,d In the present work these
crystal structures were13c,d used to design new inhibitors.
A binding mode for the penem moiety was predicted
that was consistent with the derived hypotheses. Our
molecular modeling work predicted higher potency for
the Z configuration at the C6–C7 bond in the penem nu-
cleus, which is also an observed fact with other 6-substi-
tuted methyledine penem derivatives. Subsequently, the
triazole containing penem, BRL-42715, was manually
docked in the TEM-1 (RCSB PDB code: 1BTL) and
AmpC (RCSB PDB code: 1FSW) binding sites and a
bioactive conformation was predicted. Keeping the pe-
nem moiety constant, novel methylidene substituents
were designed that will be able to interact with amino
acids of both, TEM-1 and AmpC binding sites.13c,d
Due to sequence heterogeneity within each class, the de-
signed inhibitors were refined against several experimen-
tally determined and modeled mutant b-lactamase 3D
structures. A hydrophobic substituent at the 2-position
of the imidazole was designed that was predicted to en-
hance interaction with Glu 104 and Tyr 105 (in TEM-1)
or Leu 119 and Gln 120 (in AmpC) residues. This
hypothesis is reasonable since SYN-1012, compound 6,
demonstrated broad-spectrum activity and good syn-
ergy with many b-lactam antibiotics.14 SYN-1012 is a
penem with a methyltriazole substitution at the C6 posi-
tion that differs from 4 by having an extended linker.
Based on the molecular modeling studies, and consider-
ations for hydrophobicity, several novel 6-methylidene-
penems bearing 2-substituted imidazole derivatives
11–15 were designed and synthesized. The inhibitor bind-
ing site is known in class-A and class-C b-lactamase en-
zymes.13 However, due to the fast acylation of the
active serine residues followed by ring opening, it has
not been possible to use X-ray crystallography to cap-
ture the intact inhibitor structure with the enzyme as is
also confirmed by mass spectrometric studies.4,8 At the
onset, a salt bridge between the inhibitor carboxylate
and binding site lysine 234 in class A and 315 in class
C, Amp-C helps in positioning the inhibitor b-lactam
ring in the oxyanion hole.13c,d The residues around the
2. Chemistry
The 6-methylidene-penem carboxylic acid sodium salts
11–15 were prepared by a two step process (Scheme 1)
starting from (5R,6S)-6-bromo-7-oxo-4-thia-1-azabicy-
clo[3.2.0]hept-2-ene-2-carboxylic
acid-4-nitro-benzyl

A. M. Venkatesan et al. / Bioorg. Med. Chem. 12 (2004) 5807–5817
5809
H2N
O
Br
S
S
PNB =
N
N
O
COOH
COOPNB
NO2
6-APA 7
8
RCHO/ a,b
9a-e
O
R
(a) MgBr2/ Et3N/ THF:Acetonitrile
(b) Acetic anhydride/ 0oC-20oC
(c) Activated Zinc/ 6.5 pH Phosphate
buffer/ THF: Acetonitrile/ RT
R
O
O
O
Br
c
S
N
S
N
COONa
COOPNB
11-15
10 a-e
9a,10a , 11 = R
9b,10b, 12 = R
=
=
N
N
CHO
9a
HN
N
R'
N
N
9b
9c
CHO
CHO
HN
N
N
R'
N
N
N
9c, 10c, 13 = R
9d,10d, 14 = R
=
N
N
N
S
S
S
HN
N
R'
9a-c = R' = PNZ
=
O
N
PNZ =
O
N
N
9e ,10f, 15 = R
=
NO2
N
Scheme 1. General method to prepare compounds 11–15.
ester 8 and the appropriately substituted aldehydes 9a–e.
The bromopenem derivative 8 required for this opera-
tion was prepared from the commercially available
6-amino penicillanic acid 7 (6-APA) by a modified multi-
step procedure as reported previously.15 The first step
involved a Lewis acid mediated (anhydrous MgBr2)
aldol condensation reaction between the appropriately
substituted aldehyde 9a–e and compound 8 in the pres-
ence of triethylamine as the base. The resulting diaste-
reomeric mixture of bromohydrins was trapped as
their respective acetylated derivatives 10a–e. In the
past,15,16 this transformation was achieved by using
strong bases such as LDA, LiHMDS, or Ph2NLi. The
use of the above mentioned strong bases led to poor
yields and unpredicted side products. However, the use
of a mild base in conjunction of a Lewis acid gave a bet-
ter and reproducible yield without the formation of any
side product. This is the first report, where MgBr2/Et3N
mediated procedure has been applied to 6-bromopenem
derivative 8 with monocyclic aldehydes to form C6–C7
carbon bond. After standardizing this novel aldol con-
densation procedure, we next turned our attention to
the reductive elimination procedure to introduce a Z-
double bond between the C6 and C7, carbon atoms on
the acetoxy bromohydrins 10a–e. The resulting 6-methyl-
idene-penem molecules 11–15 were prone to decompo-
sition but stable at pH in the range of 5–9. Therefore,
a neutral procedure was devised17 using freshly prepared
activated zinc and 0.5M (pH6.5) phosphate buffer. Zinc
dust was activated using 0.1N HCl for 15min and fil-
tered. It was washed well with deionized water and used
as such. The amount of zinc required for this transfor-
mation was found to be four times the weight of the sub-
strate bromohydrins. After several attempts with
different solvent systems to carry out this transforma-
tion at room temperature, acetonitrile–THF (1:2)
emerged as the most suitable combination.
The resultant crude products were purified by Diaion
HP-21 resin (80mL, Mitsubishi Kasei Co. Ltd) column

5810
A. M. Venkatesan et al. / Bioorg. Med. Chem. 12 (2004) 5807–5817
chromatography. After adsorbing, the column was
eluted with water and then 5% acetonitrile aqueous solu-
tion. It is important to mention that at this juncture the
use of activated zinc/phosphate buffer not only intro-
duced the double bond between the C6 and C7 carbon
atoms with the exclusive formation of Z isomer but also
resulted in deprotection of the carboxyl functionality,
thus avoiding an extra deprotection step. The reason
for the exclusive formation of Z isomer is unknown
and rather surprising. It might be due to the effect of
the imidazole ring, which can chelate with the acetoxy-
zinc enolate intermediate, which is formed during this
reaction.15,18 Osborne et al.15 carried out this transfor-
mation in the preparation of 4 using Zn/NH4Cl/TME-
DAÆ2HCl in DMF at room temperature with a E:Z in
the ratio of 1:13, respectively. Additionally, unlike the
MeAlCl2 procedure15 (which was used previously to
deprotect the carboxy functionality) the mild nature of
this transformation avoided racemization at the C5
position. The aldehydes 9a–e were prepared by the
methods already reported in the literature.19
inhibitor alone and in combination with piperacillin
(Pip) at a constant concentration of 4lg/mL of the
inhibitor. The potent compounds in this assay were
taken to an in vivo acute lethal infection model. In this
model, five dose levels per compound with five female
CD-1 (Charles River Laboratories) mice at each dose
level were used. Each group was injected intraperitone-
ally with a lethal dose (10–100 LD50) of the pathogen.
The same group was injected intravenously with pipera-
cillin alone or piperacillin combined with inhibitor
(compounds 11–15) at a ratio of 4:1 (Pip:inhibitor).
The seven days survival ratio from three separate tests
were pooled for determination of median effective dose
(ED50).
4. Structure–activity relationships
The inhibitory activities (IC50 values in nanomolar) of
the five methylidene-penem derivatives 11–15 and tazo-
bactam against TEM-1, Imi-1 (class-A), CcrA (class-
B), and AmpC (class-C) are listed in Table 1. All the
newly synthesized molecules were found to be potent
inhibitors of both class-A and class-C enzymes except
for compound 15, which was found to be less active
against Imi-1. All the new analogs were more potent
than tazobactam. Against TEM-1, all the new com-
pounds synthesized were 25–150-fold more potent than
tazobactam. Tazobactam is a very weak inhibitor of
AmpC enzyme and was 12,000–84,000-fold less active
than all the newly synthesized inhibitors. But among
these newly synthesized derivatives 11–15, the N-methyl
imidazolyl-2-thiazole derivative 13 and the correspond-
ing desmethyl compound 14 were found to be about
10-fold less active than 12 against TEM-1. The 2-phenyl
substituted imidazole derivative 11 was found to be less
potent than the one carbon extended 2-benzyl substi-
tuted imidazole derivative 12 by about 10-fold. Molecu-
lar modeling suggested that the benzyl substituent in
compound 12 forms a p–p stacking interaction with
Tyr 105 in the TEM-1 active site (Fig. 2). In the case
of phenyl analog 11 this interaction is predicted to be
weaker, which might explain the differences in the poten-
cies against TEM-1. Replacing the phenyl with 2-thiaz-
ole moiety (compound 13) and subsequently
methylating the imidazole nitrogen (compound 14) did
not alter the potency against TEM-1. However,
replacing the 2-thiazole moiety in compound 14 with
the N-methylimidazole group (compound 15) increased
the potency against TEM-1 and AmpC enzymes. But
as mentioned before the inhibitory activity of compound
3. Biology
All the compounds synthesized were tested in vitro
against TEM-1 and AmpC enzymes for their inhibitory
ability.20 b-Lactamase inhibitory activities were deter-
mined spectrophotometrically as described by Bush
et al.20 using nitrocefin as substrate. IC50 values were
calculated using WinNonlin (Pharsight Corp., Moun-
tainview, CA). In each experiment, tazobactam 3 was
used as a standard. The potent compounds from the
above mentioned enzyme inhibition in vitro assay, were
tested in the antimicrobial susceptibility assay. In this
assay, the in vitro activities of the antibiotics (piperacil-
lin) and antibiotics+inhibitors (compound 11–15) were
determined by the broth microdilution method as rec-
ommended by the National Committee for Clinical Lab-
oratory Standards (NCCLS) using Mueller-Hinton II
broth (MHBII, BBL Cockeysville, MD). Microtiter
plates containing serial dilutions of each antimicrobial
agents or antimicrobial agents+inhibitor were inocu-
lated with each organism to yield the appropriate den-
sity (105 CFU/mL) in a 100lL final volume. The plates
were incubated for 18–24h at 35ꢁC in ambient air.
The minimum inhibitory concentration (MIC) for all
isolates was defined as the lowest concentration of anti-
microbial agent that completely inhibited the growth of
the organisms as detected by the unaided eye. The MIC
values presented in this article were determined for the
Table 1. In vitro activity of compounds 11–15 against different b-lactamases, IC50 (nM)
Compound
TEM-1 (IC50, nM)
Imi-1 (IC50, nM)
CcrA (IC50, nM)
400,000 200
AmpC (IC50, nM)
84,000 300
Tazobactam
100
4.0
8
1
NA
11
12
13
14
15
6
6
2
3
4
250
230
260
220
190
5
8
5
9
7
2.0
2.0
6.0
1
1
1
0.4 0.2
6.0
5.0
1
1
68
11 50
1300
3.0 0.2
1.0
1.0 0.3
1
1
NA: Not available.

A. M. Venkatesan et al. / Bioorg. Med. Chem. 12 (2004) 5807–5817
5811
Figure 2. A and B: Compound 11 docked to Amp-C active site (class-C); C and D: compound 11 docked to TEM-1 active site (class-A).
15 against Imi-1, a carbapenem hydrolyzing enzyme21 is
less than the other molecules listed in Table 1. As can be
seen from Table 1, all the newly synthesized compounds
showed only a modest in vitro inhibitory activity against
class-B enzymes. However, compounds 11–15 were
found to be more potent than tazobactam against
class-B enzyme.
isolates. It should be noted from Table 2 that even
though the tazobactam+piperacillin combination has
synergy against class-A producing organisms, this com-
bination is less effective against class-C producing
organisms (E. cloacae GC 2071, E. cloacae GC1475, P.
aeruginosa GC 1764, and S. marcescens). However, the
addition of the newly synthesized inhibitors 11–15 re-
stored susceptibility to piperacillin for 50–90% of the
strains. Especially the combination of compound 12
and piperacillin considerably reduced the MIC values
of piperacillin against both class-A and class-C produc-
ing organisms rendering the susceptibility range to 90%
of the organisms. This combination is very effective
against P. aeruginosa GC 1764 as well.
5. In vitro antibacterial activity
The above mentioned in vitro potent compounds in the
enzyme assay 11–15 were tested for their in vitro
antibacterial activity alone and in combination with
piperacillin against various bacteria expressing different
b-lactamases. These data are summarized in Table 2. In
all these experiments, piperacillin+tazobactam were
used at a constant 4lg/mL for comparison purpose.
All the isolates enlisted in Table 2 were resistant to pip-
eracillin alone (>64lg/mL). When tested alone, neither
tazobactam nor any of the newly synthesized penem
inhibitors 11–15 exhibited antibacterial activity against
the isolates used in this study, which established the lack
of inherent antibacterial activity (except for E. coli GC
2804).
6. In vivo efficacy
The in vivo data (ED50 values) of piperacillin and inhib-
itors (4:1 ratio) in a murine acute lethal infection model
with E. coli LSU 80-8 (a TEM-1 producing organism)
are listed in Table 3. When administered alone, pipera-
cillin had no efficacy (ED50 > 128mg/kg). When pipera-
cillin was combined with tazobactam or compounds 11,
13–15 the ED50 values were greater than 64mg/kg. How-
ever, compound 12, when co-administered intrave-
nously, reduced the ED50 value of piperacillin to
43mg/kg. It should be stressed here the in vivo data
(ED50 values) of piperacillin reported here were only
performed against a TEM-1 producing b-lactamase.
The results of the ED50 values could be different if
used against other hyperproducing extended spectrum
The newly synthesized compounds when combined with
piperacillin enhanced its activity against piperacillin
resistant b-lactamase producing organisms. The MIC
values for piperacillin were reduced to 2–64lg/mL in
the presence of a constant 4lg/mL of each of the inhib-
itors, including tazobactam against class-A producing

5812
A. M. Venkatesan et al. / Bioorg. Med. Chem. 12 (2004) 5807–5817
Table 2. In vitro antimicrobial activity of inhibitorsa in combination with piperacillin (Pip) at a constant 4lg/mL concentration
Species and strain
Expression
Tazo+Pip
Pip+11
Pip+12
Pip+13
Pip+14
Pip+15
E. coli GC 2844
E. coli GC 2847
None
TEM-1
IRT-2
2
2
2
16
2
2
2
64
2
2
32
1
2
64
2
E. coli GC 2920
E. coli GC 2883
4
2
0.5
16
OXA+10+PSE-2
AmpC
P99
2
4
2
8
8
E. coli GC 2894
E. coli GC 2905
2
8
2
32
1
2
32
2
4
1
4
2
64
E. coli GC 2906
E. coli GC 2804
Imi-1
Imp
2
4
2
<0.06
>64
16
32
4
64
<0.06
>64
32
>64
32
32
>64
1
4
0.12
>64
2
<0.06
>64
16
32
64
8
<0.06
>64
16
32
>64
4
<0.06
>64
>64
>64
>64
>64
>64
8
E. coli GC 2805
E. coli GC 2252
CcrA
IRT-2
E. cloacae GC 1477
E. cloacae GC 2071
E. cloacae GC 1475
K. pneumoniae GC 2825
S. marcescens GC 1781
P. aeruginosa GC 1764
S. maltophilia GC 1712
S. marcescens GC 4142
E. coli GC 2203
AmpC
Imi+1+AmpC
P99
>64
>64
>64
>64
1
4
4 blaꢀs
64
1
8
>64
1
Sme-1+AmpC
AmpC
L1
1
>64
>64
>64
>64
64
32
>64
1
4
16
>64
4
8
64
>64
2
>64
2
>64
8
AmpC
Control
Control
2
<0.06
1
<0.06
2
1
<0.06
1
0.5
S. aureus GC 2216
1
a MIC values of piperacillin and the inhibitors when tested alone were found to be >64lg/mL.
Table 3. In vivo efficacy (IV)a in a murine acute lethal infection model
with E. coli LSU 80-8, a TEM-1 producing organism
lin, the MICꢀs values for piperacillin were reduced to
susceptible range for most of the organisms enlisted in
Table 1. Especially the 2-benzyl substituted imidazole
derivative 12, when combined with piperacillin, rendered
90% of the organisms listed here susceptible. The same
compound 12, in vivo enhanced the activity of piperacil-
lin against E. coli LSU 80-8, a TEM-1 producing organ-
ism. Further investigations in various organisms are in
progress to determine their potential as therapeutic
agents.
Compound
ED50 (mg/kg)
Tazobactam
>64
>64
11
12
13
14
15
43
5
>64
>64
>64
a Piperacillin; inhibitors = 4:1: ED50 value of piperacillin alone >
128mg/kg.
b-lactamase strains or an inhibitor resistance b-lacta-
mase strain. The in vivo efficacy of compound 12 in
other acute lethal infection models against various bac-
teria, producing different classes of b-lactamases are un-
der investigation.
8. Experimental section
8.1. General methods
Melting points were determined in an open capillary
tube on a Meltemp melting point apparatus (Laboratory
Devices, Cambridge, MA) and are uncorrected. 1H
NMR spectra were determined with Bruker DPX-400
spectrometer at 400MHz. Chemical shifts d are reported
in parts per million (d) relative to residual chloroform
(7.26ppm), TMS (0ppm), or dimethylsulfoxide
(2.49ppm) as an internal reference with coupling con-
stants (J) reported in hertz (Hz). The peak shapes are de-
noted as follows: s, singlet; d, doublet; t, triplet; q,
quartet; m, multiplet; br, broad. Electrospray (ES) mass
spectra were recorded in positive or negative mode on a
Micromass Platform spectrometer. Electron impact and
high-resolution mass spectra were obtained on a Finni-
gen MAT-90 spectrometer. Combustion analysis were
obtained using Perkin–Elmer Series II 2400 CHNS/O
analyzer (Robertson Microlit, Maddison, NJ). Chroma-
tographic purifications were performed by open
chromatography using IBW-127ZH (Fuji Silysia).
Thin-layer chromatography (TLC) was performed on
Merck PLC prescored plates 60F254. The terms ꢁconcen-
tratedꢀ and ꢁevaporatedꢀ refer to the removal of solvents
using a rotary evaporator at water aspirator pressure with
7. Conclusion
In this paper, based on the modeling experiments and
mechanistic understanding several novel imidazole sub-
stituted 6-methylidene-penem molecules were designed
and synthesized as a potent b-lactamase inhibitors.
Preparation of these compounds 11–15 was synthetically
challenging and therefore a novel Lewis acid mediated
aldol condensation reaction was standardized. This no-
vel C6–C7 bond formation reaction was shown to be
rather mild, efficient, and a useful variation of the aldol
reaction. In this paper, we have also described a mild
and efficient reductive elimination procedure that can
be applied to other pH sensitive molecules such as pen-
ems. The newly synthesized compounds have a broader
spectrum of activity than any of the currently available
inhibitors in the market and were shown to have excel-
lent in vitro activity against both class-A and class-C en-
zymes. In vitro antimicrobial susceptibility testings,
when compounds 11–15 were combined with piperacil-

A. M. Venkatesan et al. / Bioorg. Med. Chem. 12 (2004) 5807–5817
5813
a bath temperature equal to or less than 40ꢁC. Unless
otherwise noted, reagents were obtained from commer-
cial sources and were used without further purification.
ꢀ20ꢁC and treated with acetic anhydride (0.277mL) in
one portion and warmed to 0ꢁC and stirred at that tem-
perature for 27h. The mixture was diluted with ethyl
acetate and washed with 5% citric acid aqueous solu-
tion, saturated sodium hydrogen carbonate, and brine.
The organic layer was dried (MgSO4) and filtered
through a pad of Celite. The pad was washed with ethyl
acetate and the filtrate was concentrated under reduced
pressure. The residue was applied to silica gel column
chromatography and eluted with ethyl acetate–hexane
(2/3–1/1). The title compound was obtained as three dia-
stereomixture (6/4/1.5, a pale yellow amorphous solid,
8.2. Materials and methods
8.2.1. Molecular modeling. Hydrogen atoms were added
to the pdb files, 1BTL (TEM-1) and 1FSW (Amp-C),
and energy minimized in Quanta/CHARMM29 (Accel-
rys Inc., San Diego, CA). BRL-42715 was modeled
using Quanta and energy minimized with Gaussian 98
using 6-31G* basis set. CHARMM29 was parameter-
ized to reproduce the inhibitor geometry obtained from
quantum mechanics energy minimization. Inhibitor con-
formations that were up to 5kcal/mol above the global
minima were manually docked in the b-lactamase inhib-
itor binding pockets of TEM-1 and AmpC proteins. The
inhibitor was docked placing the penem carboxylate
oxygen 2.4A away from the Lys 234 (TEM-1)/Lys 315
(AmpC) Nz. Assuming a short distance required for
the nucleophillic attack, the lactam oxygen was placed
2.2A away from the Ser 70 (TEM-1)/Ser 64 (AmpC).
The bioactive inhibitor conformation was chosen based
upon lowest interaction energy (calculated using
CHARMM29) with the binding pocket amino acids.
Inhibitors with the lowest interaction energies were rec-
ommended for synthesis.
1
986mg, 86%). H NMR (d, CDCl3) 2.04 (s, 0.35 · 3H),
2.10 (s, 0.13 · 3H), 2.30 (s, 0.52 · 3H), 5.25–5.43 (m,
4H), 6.09 (s, 0.52 · 1H), 6.22 (s, 0.13 · 1H), 6.31 (s,
0.35 · 1H), 6.33 (s, 0.35 · 1H), 6.87 (s, 0.13 · 1H), 6.92
(d, 0.52 · 1H, J = 1.4Hz), 7.31–7.76 (m, 11H), 8.17–
8.25 (m, 4H).
8.3.3. Step 3: Preparation of (5R),(6Z)-6-(2-phenyl-
1H-imidazol-4-ylmethylene)-7-oxo-4-thia-1-aza-bicy-
clo[3.2.0]hept-2-ene-2-carboxylic acid sodium salt (11).
(5R,6RS)-6-{(RS)-Acetoxy-[1-(4-nitro-benzyloxycarbo-
nyl)-2-phenyl-1H-imidazol-4-yl]-methyl}-6-bromo-7-
oxo-4-thia-1-aza-bicyclo[3.2.0]hept-2-ene-2-carboxylic
acid 4-nitro-benzyl ester (10a) (1.15g) was dissolved in
THF (16.1mL) and acetonitrile (7.5mL). Freshly acti-
vated Zn dust [a mixture of Zn dust (15g) and 0.1N
HCl was stirred at room temperature for 15min and fil-
tered. Washed well with water and 0.5M phosphate buf-
fer and used as such] (4.6g) and 0.5M phosphate buffer
(pH6.4, 23.6mL) were added to the mixture. The reac-
tion vessel was covered with a foil to exclude light and
vigorously stirred for 3h at room temperature. At the
end, reaction solution was cooled to 10ꢁC and 1M
NaOH solution was added to adjust the pH to 7.5.
Reaction mixture was mixed with ethyl acetate and fil-
tered through a pad of Celite. The pad was washed with
water and the aqueous layer was separated. The aqueous
layer was concentrated under high vacuum at 35ꢁC and
applied to Diaion HP-21 (100mL, Mitsubishi Kasei Co.
Ltd) resin column chromatography. After adsorbing,
the column was eluted with water and then with 5–
10% acetonitrile–water. The combined fractions were
concentrated under high vacuum at 35ꢁC and lyophi-
lized to give the title compound as a yellow amorphous
solid (322mg, 63%). Mp 281ꢁC (dec); 1H NMR (d,
D2O) 6.32 (s, 1H), 6.76 (s, 1H), 6.79 (s, 1H), 7.22 (s,
1H), 7.24–7.33 (m, 3H), 7.60–7.63 (m, 2H).
8.3. Preparation of (5R),(6Z)-6-(2-phenyl-1H-imidazol-4-
ylmethylene)-7-oxo-4-thia-1-aza-bicyclo[3.2.0]hept-2-ene-
2-carboxylic acid sodium salt (11)
8.3.1. Step 1: Preparation of 4-formyl-2-phenyl-imidaz-
ole-1-carboxylic acid 4-nitro-benzyl ester (9a). To a stir-
red mixture of 4-formyl-2-phenylimidazole (624mg)
and sodium hydrogen carbonate (791mg) in dioxane
(3.6mL), THF (3.6mL), and water (7.2mL), 48.7% solu-
tion of p-nitrobenzyl chloroformate (PNZCl) in dioxane
(2.08mL) was added at room temperature and stirred
for 2.5h. The reaction mixture was diluted with ethyl
acetate and washed with brine. The organic layer was
dried (MgSO4) and concentrated under reduced pres-
sure. The residue was crystallized from ethyl acetate
and n-hexane to give the title compound (956mg,
1
75%). H NMR (d, CDCl3) 5.41 (s, 2H), 7.32 (d, 2H,
J = 8.6Hz), 7.40–7.51 (m, 3H), 7.56–7.58 (m, 2H),
8.17–8.20 (m, 2H), 8.22 (s, 1H), 9.97 (s, 1H).
8.3.2. Step 2: Preparation of (5R,6RS)-6-{(RS)-acetoxy-
[1-(4-nitro-benzyloxycarbonyl)-2-phenyl-1H-imidazol-4-
yl]-methyl}-6-bromo-7-oxo-4-thia-1-aza-bicyclo[3.2.0]-
hept-2-ene-2-carboxylic acid 4-nitro-benzyl ester (10a).
4-Formyl-2-phenyl-imidazole-1-carboxylic acid 4-nitro-
benzyl ester (568mg) and the dry THF solution
(15mL) of (5R,6S)-6-bromo-7-oxo-4-thia-1-aza-bicy-
clo[3.2.0]hept-2-ene-2-carboxylic acid 4-nitro-benzyl es-
ter (587mg) were added successively to the dry
acetonitrile (15mL) solution of anhydrous MgBr2
(622.4mg) under a nitrogen atmosphere at room tem-
perature. After cooling the reaction mixture to ꢀ20ꢁC,
triethylamine (0.494mL) was added in one portion.
The reaction vessel was covered with a foil to exclude
light. The reaction mixture was stirred for 7h at
8.4. Preparation of (5R),(6Z)-6-(2-benzyl-1H-imidazol-4-
ylmethylene)-7-oxo-4-thia-1-aza-bicyclo[3.2.0]hept-2-ene-
2-carboxylic acid sodium salt (12)
8.4.1. Step 1: Preparation of 2-benzyl-1H-imidazole-4-
carbaldehyde. 2-Phenyl-acetoamidine (2g) was added to
the solution of 2-bromo-3-isopropoxy-propenal (2.88g)
in CHCl3 (30mL) and stirred for 1h at room tempera-
ture. Triethylamine (2.09mL) was added to the mixture
and heated to reflux for 6h. The mixture was cooled to
room temperature, diluted with CHCl3, and washed with
10% potassium hydrogen carbonate. The organic layer
was dried (MgSO4) and concentrated under reduced

5814
A. M. Venkatesan et al. / Bioorg. Med. Chem. 12 (2004) 5807–5817
pressure. The residue was purified by silica gel column
chromatography and eluted with ethyl acetate–hexane
(3/1). The crude compound was crystallized from ethyl
acetate and n-hexane to give the title compound (1.10g,
40%). H NMR (d, CDCl3) 4.17 (s, 2H), 7.24–7.36 (m,
5H), 7.72 (br s, 1H), 9.60 (br s, 1H), 10.28 (br s, 1H).
4-nitro-benzyl ester (10b) (2.5g) was dissolved in THF
(35mL) and acetonitrile (16.3mL). Freshly activated
Zn dust (10g) and 0.5M phosphate buffer (pH6.4,
51.3mL) were added to the mixture. The reaction vessel
was covered with a foil to exclude light and stirred
vigorously for 2h at room temperature. The mixture
was cooled to 10ꢁC, and 1 M NaOH aqueous solu-
tion was added to adjust pH to 7.5. The reaction
solution was mixed with ethyl acetate and filtered
through a pad of Celite. The pad was washed with water
and the aqueous layer was separated. The aqueous layer
was concentrated under high vacuum at 35ꢁC and
applied to Diaion HP-21 (250mL, Mitsubishi Kasei
Co. Ltd) resin column chromatography. After adsorb-
ing, the column was eluted with water and then with
5–10% acetonitrile–water. The combined fractions were
concentrated under high vacuum at 35ꢁC and lyophi-
lized to give the title compound as a yellow amorphous
solid (780mg, 67%). Mp 146ꢁC (dec); 1H NMR (d, D2O)
3.92 (s, 2H), 6.39 (d, 1H, J = 0.8Hz), 6.74 (s, 1H), 6.89
(s, 1H), 7.13–7.16 (m, 3H), 7.21–7.25 (m, 3H).
1
8.4.2. Step 2: Preparation of 2-benzyl-4-formyl-imidaz-
ole-1-carboxylic acid 4-nitro-benzyl ester (9b). To a stir-
red solution of 2-benzyl-1H-imidazole-4-carbaldehyde
(1g) and sodium hydrogen carbonate (1.13g) in diox-
ane–water (1:1, 60mL), 48.7% solution of p-nitrobenzyl
chloroformate (PNZCl) in dioxane (2.62g) was added at
0ꢁC and stirred for 2.5h. The reaction mixture was di-
luted with ethyl acetate and washed with saturated so-
dium hydrogen carbonate and brine. The organic layer
was dried (MgSO4) and concentrated under reduced
pressure. The residue was applied to silica gel column
chromatography and eluted with chloroform–acetone
(9/1). The title compound was obtained as pale brown
oil (1.97g, 100%). 1H NMR (d, CDCl3) 4.46 (s,
2H), 5.42 (s, 2H), 7.19–7.30 (m, 5H), 7.44–7.47 (m,
2H), 8.06 (s, 1H), 8.21–8.25 (m, 2H), 9.91 (s, 1H).
8.5. Preparation of (5R),(6Z)-6-(2-thiazol-2-yl-1H-imid-
azol-4-ylmethylene)-7-oxo-4-thia-1-aza-bicyclo[3.2.0]hept-
2-ene-2-carboxylic acid sodium salt (13)
8.4.3. Step 3: Preparation of (5R,6RS)-6-{(RS)-acetoxy-
[2-benzyl-1-(4-nitro-benzyloxycarbonyl)-1H-imidazol-
4-yl]-methyl}-6-bromo-7-oxo-4-thia-1-aza-bicyclo[3.2.0]-
hept-2-ene-2-carboxylic acid 4-nitro-benzyl ester (10b).
To a stirred solution of 2-benzyl-4-formyl-imidazole-1-
carboxylic acid 4-nitro-benzyl ester (1.9g) in dry aceto-
nitrile (10mL) was added a dry acetonitrile (50mL)
solution of anhydrous MgBr2 (2.41g) under a nitrogen
atmosphere at room temperature. The dry THF solution
(60mL) of (5R,6S)-6-bromo-7-oxo-4-thia-1-aza-bicy-
clo[3.2.0]hept-2-ene-2-carboxylic acid 4-nitro-benzyl es-
ter (2g) was added to the mixture, cooled to ꢀ20ꢁC,
and triethylamine (1.8mL) was added in one portion.
The reaction vessel was covered with a foil to exclude
light. The reaction mixture was stirred for 6h at
ꢀ20ꢁC and treated with 4-dimethylaminopyridine
(127mg) and acetic anhydride (0.98mL) in one portion.
The reaction mixture was warmed to 0ꢁC and stirred for
16h at 0ꢁC. The mixture was diluted with ethyl acetate
and washed with H2O, saturated sodium hydrogen car-
bonate, and brine. The organic layer was dried (MgSO4)
and filtered through a pad of Celite and washed
with ethyl acetate. The filtrate was concentrated under
reduced pressure. The residue was applied to silica gel
column chromatography, then eluted with ethyl ace-
tate–hexane (2/3–1/1). The title compound was obtained
as two diastereomixture (3/2, a pale yellow amorphous
solid, 2.8g, 70%). 1H NMR (d, CDCl3) 2.00 (s,
0.6 · 3H), 2.26 (s, 0.4 · 3H), 4.34–4.37 (m, 2H), 5.25–
5.47 (m, 4H), 6.02 (s, 0.4 · 1H), 6.22 (s, 0.6 · 1H), 6.25
(s, 0.6 · 1H), 6.85 (d, 0.4 · 1H, J = 1.5Hz), 7.14–7.62
(m, 11H), 8.21–8.25 (m, 4H).
8.5.1. Step 1: Preparation of 4-formyl-2-thiazol-2-yl-
imidazol-1-carboxylic acid 4-nitro-benzyl ester (9c). To
a stirred solution of 2-thiazol-2-yl-1H-imidazol-4-carb-
aldehyde (570mg in dry CH2Cl2 (35mL), N,N0-diisopro-
pylethylamine and 48.7% solution of p-nitrobenzyl
chloroformate (PNZCl) in dioxane (1.69mL) were
added slowly at 0ꢁC. The reaction mixture was stirred
for 2h and diluted with CHCl3. The organic layer was
washed with saturated sodium hydrogen carbonate in
brine and dried over MgSO4. It was concentrated under
reduced pressure and the residue was crystallized from
ethyl acetate and n-hexane to give the title compound
1
(1.05g, 92%). H NMR (d, CDCl3) 5.49 (s, 2H), 7.50
(d, 2H, J = 8.8Hz), 7.55 (d, 1H, J = 3.4Hz), 7.89 (d,
1H, J = 3.1Hz), 8.19 (s, 1H), 8.24 (dq, 2H, J = 8.8,
2.0Hz), 9.97 (s, 1H).
8.5.2. Step 2: Preparation of (5R,6RS)-6-{(RS)-acetoxy-
[1-(4-nitro-benzyloxycarbonyl)-2-thiazol-2-yl-1H-imid-
azol-4-yl]-methyl}-6-bromo-7-oxo-4-thia-1-aza-bicyclo
[3.2.0]hept-2-ene-2-carboxylic acid 4-nitro-benzyl ester
(10c).
4-Formyl-2-thiazol-2-yl-imidazol-1-carboxylic
acid 4-nitro-benzyl ester (940mg) was added to the dry
acetonitrile (35mL) solution of anhydrous MgBr2
(1.26g) under a nitrogen atmosphere at room tempera-
ture. A dry THF solution (28mL) of (5R,6S)-6-bromo-
7-oxo-4-thia-1-aza-bicyclo[3.2.0]hept-2-ene-2-carboxylic
acid 4-nitro-benzyl ester (1.01g) was added to the mix-
ture, cooled to ꢀ20ꢁC, and triethylamine (0.942mL)
was added in one portion. The reaction vessel was cov-
ered with a foil to exclude light and stirred for 4h at
ꢀ20ꢁC and treated with 4-dimethylaminopyridine
(64mg) and acetic anhydride (0.495mL) in one portion.
The reaction mixture was warmed to 0ꢁC and stirred for
20h at 0ꢁC. The mixture was diluted with ethyl acetate
and washed with 0.1M phosphate buffer (pH7), satu-
rated sodium hydrogen carbonate, and brine. The
8.4.4. Step 4: Preparation of (5R),(6Z)-6-(2-benzyl-
1H-imidazol-4-ylmethylene)-7-oxo-4-thia-1-aza-bicy-
clo[3.2.0]hept-2-ene-2-carboxylic acid sodium salt (12).
(5R,6RS)-6-{(RS)-Acetoxy-[2-benzyl-1-(4-nitro-benzyl-
oxycarbonyl)-1H-imidazol-4-yl]-methyl}-6-bromo-7-oxo-
4-thia-1-aza-bicyclo[3.2.0]hept-2-ene-2-carboxylic acid

A. M. Venkatesan et al. / Bioorg. Med. Chem. 12 (2004) 5807–5817
5815
organic layer was dried over anhydrous MgSO4 and fil-
tered through a pad of Celite and washed with ethyl ace-
tate. The filtrate was concentrated under reduced
pressure and the residue was purified by silica gel col-
umn chromatography (ethyl acetate–hexane = 1/1
ꢁethyl acetate only) and (5R,6RS)-6-(RS)-acetoxy-[1-
(4-nitro-benzyloxycarbonyl)-2-thiazol-2-yl-1H-imidazol-
4-yl]-methyl-6-bromo-7-oxo-4-thia-1-aza-bicyclo[3.2.0]-
hept-2-ene-2-carboxylic acid 4-nitro-benzyl ester (860
mg) was obtained as a spongy solid.
J = 3.2Hz), 7.68 (s, 1H), 7.89 (d, 1H, J = 3.2Hz),
9.91 (s, 1H).
1-Methyl-2-thiazol-2-yl-1H-imidazol-5-carbaldehyde:
1H NMR (d, CDCl3) 4.48 (s, 3H), 7.51 (d, 1H,
J = 3.1Hz), 7.82 (s, 1H), 7.97 (d, 1H, J = 3.1Hz),
9.82 (s, 1H).
8.6.2. Step 2: Preparation of (5R,6RS)-6-[(RS)-acetoxy-
(1-methyl-2-thiazol-2-yl-1H-imidazol-4-yl)-methyl]-6-bromo-
7-oxo-4-thia-1-aza-bicyclo[3.2.0]hept-2-ene-2-carboxylic
acid 4-nitro-benzyl ester (10d). 1-Methyl-2-thiazol-2-yl-
1H-imidazol-4-carbaldehyde (380mg) was added to the
dry acetonitrile (28mL) solution of anhydrous MgBr2
(1.03g) under a nitrogen atmosphere at room tempera-
ture. A colorless powder was deposited over a period
of 10min. To this suspension a dry THF solution
(28mL) of (5R,6S)-6-bromo-7-oxo-4-thia-1-aza-bicy-
clo[3.2.0]hept-2-ene-2-carboxylic acid 4-nitro-benzyl es-
ter (760mg) was added and cooled to ꢀ20ꢁC. To this
stirred solution triethylamine (0.768mL) was added in
one portion. The reaction vessel was covered with a foil
to exclude light and was stirred for 5h at ꢀ20ꢁC and
treated with 4-dimethylaminopyridine (48mg) and acetic
anhydride (0.371mL) in one portion. The reaction mix-
ture was warmed to 0ꢁC and stirred for 22h at this tem-
perature. At the end, the mixture was diluted with ethyl
acetate and washed with 5% citric acid aqueous solu-
tion, saturated sodium hydrogen carbonate, and brine.
The organic layer was dried (MgSO4) and filtered
through a pad of Celite and the pad was washed with
ethyl acetate. The filtrate was concentrated under re-
duced pressure. The residue was purified by silica gel
column chromatography and eluted with ethyl acetate–
hexane (3/2). The title compound was obtained as two
diastereomeric mixture (1/1, a pale yellow amorphous
solid, 673.4mg, 52%). 1H NMR (d, CDCl3) 2.00 (s,
0.5 · 3H), 2.28 (s, 0.5 · 3H), 4.12 (s, 3H), 5.28 (d,
0.5 · 1H, J = 13.5Hz), 5.29 (d, 0.5 · 1H, J = 13.5Hz),
5.44 (d, 0.5 · 1H, J = 13.5Hz), 5.47 (d, 0.5 · 1H,
J = 13.5Hz), 6.16 (s, 0.5 · 1H), 6.30 (s, 0.5 · 1H), 6.42
(s, 0.5 · 1H), 6.87 (d, 0.5 · 1H, J = 0.8Hz), 6.92 (d,
0.5 · 1H, J = 0.8Hz), 7.19 (s, 0.5 · 1H), 7.34–7.36 (m,
1H), 7.47 (s, 0.5 · 1H), 7.50 (s, 0.5 · 1H), 7.60–7.64
(m, 2H), 7.83–7.85 (m, 1H), 8.23–8.26 (m, 1H).
8.5.3. Step 3: Preparation of (5R),(6Z)-6-(2-thiazol-2-yl-
1H-imidazol-4-ylmethylene)-7-oxo-4-thia-1-aza-bicy-
clo[3.2.0]hept-2-ene-2-carboxylic acid sodium salt (13).
To a stirred solution of (5R,6RS)-6-{(RS)-acetoxy-[1-(4-
nitro-benzyloxycarbonyl)-2-thiazol-2-yl-1H-imidazol-4-
yl]-methyl}-6-bromo-7-oxo-4-thia-1-aza-bicyclo[3.2.0]-
hept-2- ene-2-carboxylic acid 4-nitro-benzyl ester
(860mg) in THF (12mL) and acetonitrile (5.6mL)
freshly activated Zn dust (3.44g) and 0.5M phosphate
buffer (pH6.4, 17.6mL) were added. The reaction vessel
was covered with a foil to exclude light and stirred vig-
orously for 2h at room temperature. At the end, reac-
tion mixture was cooled to 10ꢁC and 1M NaOH
solution was added to adjust the pH to 7.5. The reaction
mixture was mixed with ethyl acetate and filtered
through a pad of Celite. The pad was washed with water
and the aqueous layer was separated. The aqueous layer
was concentrated under high vacuum at 35ꢁC and ap-
plied to Diaion HP-21 (90mL, Mitsubishi Kasei Co.
Ltd) resin column chromatography. After adsorbing,
the column was eluted with water and then with 2.5–
5% acetonitrile–water. The combined fractions were
concentrated under high vacuum at 35ꢁC and lyophi-
lized to give the title compound as a yellow amorphous
solid (126mg, 11%). Mp 145ꢁC (dec); 1H NMR (d,
D2O) 6.40 (d, 1H, J = 0.7Hz), 6.81 (s, 1H), 6.89 (s,
1H), 7.38 (s, 1H), 7.48 (d, 1H, J = 3.1Hz), 7.71 (d, 1H,
J = 3.3Hz).
8.6. Preparation of (5R),(6Z)-6-(1-methyl-2-thiazol-2-yl-
1H-imidazol-4-ylmethylene)-7-oxo-4-thia-1-aza-bicy-
clo[3.2.0]hept-2-ene-2-carboxylic acid sodium salt (14)
8.6.1. Step 1: Preparation of 1-methyl-2-thiazol-2-yl-1H-
imidazol-4-carbaldehyde (9d). To a stirred solution of
potassium tert-butoxide (494mg) in dry THF–DMF
(3:1, 40mL) 2-thiazol-2-yl-1H-imidazol-4-carbaldehyde
(717mg) and 18-crown-6 (106mg) was added at room
temperature. The reaction mixture was stirred for
10min and treated with methyl iodide (0.274mL). After
stirring for 17.5h at room temperature, it was concen-
trated under reduced pressure. The residue was dis-
solved with ethyl acetate and filtered and the filtrate
was evaporated under reduced pressure. The crude
material was purified with silica gel column chromato-
graphy (ethyl acetate–hexane=1/1). The title compound
(410mg, 53%) and its regio-isomer 1-methyl-2-thiazol-2-
yl-1H-imidazol-5-carbaldehyde (240mg, 31%) were
obtained as a white solid.
8.6.3. Step 3: Preparation of (5R),(6Z)-6-(1-methyl-2-
thiazol-2-yl-1H-imidazol-4-ylmethylene)-7-oxo-4-thia-1-
aza-bicyclo[3.2.0]hept-2-ene-2-carboxylic acid sodium salt
(14). To a stirred solution of (5R,6RS)-6-[(RS)-acetoxy-
(1-methyl-2-thiazol-2-yl-1H-imidazol-4-yl)-methyl]-6-
bromo-7-oxo-4-thia-1-aza-bicyclo[3.2.0]hept-2-ene-2-
carboxylic acid 4-nitro-benzyl ester (10d) (604mg) in
THF (8.5mL)–acetonitrile (3.9mL) freshly activated
Zn dust (2.41g) and 0.5M phosphate buffer (pH6.4,
23.6mL) were added. The reaction vessel was covered
with a foil to exclude light and vigorously stirred for
3h at room temperature. At the end, reaction mixture
was cooled to 10ꢁC and 1M NaOH solution was added
to adjust the pH to 7.5. The reaction solution was mixed
with ethyl acetate and filtered through a pad of Celite.
The pad was washed with water and the aqueous layer
was separated and concentrated under high vacuum at
1-Methyl-2-thiazol-2-yl-1H-imidazol-4-carbaldehyde:
1H NMR (d, CDCl3) 4.21 (s, 3H), 7.44 (d, 1H,

5816
A. M. Venkatesan et al. / Bioorg. Med. Chem. 12 (2004) 5807–5817
35ꢁC. The concentrate was applied to Diaion HP-21
(60mL, Mitsubishi Kasei Co. Ltd) resin column chro-
matography. After adsorbing, the column was eluted
with water and then with 5% acetonitrile–water and
10% acetonitrile–water. The combined fractions were
concentrated under high vacuum at 35ꢁC and lyophi-
lized to give the title compound as a yellow amorphous
solid (231mg, 64%). Mp 214ꢁC (dec); 1H NMR (d, D2O)
3.91 (s, 3H), 6.44 (s, 1H), 6.84 (s, 1H), 6.91 (s, 1H), 7.43
(s, 1H), 7.60 (d, 1H, J = 3.3Hz), 7.84 (d, 1H, J = 3.3Hz).
at that temperature. The mixture was diluted with ethyl
acetate and washed with 5% citric acid aqueous solu-
tion, saturated sodium hydrogen carbonate, and brine.
The organic layer was dried (MgSO4) and filtered and
the filtrate was concentrated under reduced pressure.
The residue was purified by silica gel column chroma-
tography and eluted with chloroform–methanol (9/1).
The title compound was obtained as a spongy yellow so-
lid. Yield: 507.6mg, 91.4%. 1H NMR (d, CDCl3) 2.19 (s,
3H), 3.75 (s, 3H), 4.03 (s, 3H), 5.29 (d, 1H, J = 13.5Hz),
5.42 (d, 1H, J = 13.5Hz), 6.03 (s, 1H), 6.65 (s, 1H), 7.34
(s, 1H), 7.48 (d, 1H, J = 1.2Hz), 7.55 (d, 1H, J = 1.2Hz),
7.59–7.61 (m, 3H), 8.23–8.26 (m, 2H).
8.7. Preparation of (5R),(6Z)-6-(3,10-dimethyl-3H,10H-
[2,40]biimidazolyl-4-ylmethylene)-7-oxo-4-thia-1-aza-
bicyclo[3.2.0]hept-2-ene-2-carboxylic acid sodium salt
(15)
8.7.3. Step 3: Preparation of (5R),(6Z)-6-(3,10-dimethyl-
3H,10H-[2,40]biimidazolyl-4-ylmethylene)-7-oxo-4-thia-1-
aza-bicyclo[3.2.0]hept-2-ene-2-carboxylic acid sodium salt
(15). To a stirred solution of (5R,6RS)-6-[(S)-acetoxy-
(3,10-dimethyl-3H,10H-[2,40]biimidazolyl-4-yl)-methyl]-
6-bromo-7-oxo-4-thia-1-aza-bicyclo[3.2.0]hept-2-ene-2-
carboxylic acid 4-nitro-benzyl ester (10e) (596mg) in
THF (8.3mL) and acetonitrile (3.9mL), freshly acti-
vated Zn dust (2.38g) and 0.5M phosphate buffer
(pH6.5, 12.2mL) were added. The reaction vessel was
covered with a foil to exclude light and stirred vigor-
ously for 2h at room temperature. At the end, reaction
mixture was cooled to 10ꢁC and 1M NaOH solution
was added to adjust the pH to 7.5. The reaction solution
was mixed with ethyl acetate and filtered through a pad
of Celite. The pad was washed with water and the aque-
ous layer was separated and concentrated under high
vacuum at 35ꢁC. The concentrate was applied to Diaion
HP-21 (60mL, Mitsubishi Kasei Co. Ltd) resin column
chromatography. After adsorbing, the column was
eluted with water and then with 5–10% acetonitrile–
water. The combined fractions were concentrated under
high vacuum at 35ꢁC and lyophilized to give the title
compound as a yellow amorphous solid (66.3mg,
19%). Mp 113ꢁC (dec); 1H NMR (d, D2O) 3.31 (s,
3H), 3.52 (s, 3H), 5.74 (s, 1H), 6.43 (s, 2H), 6.70 (s,
1H), 7.27 (s, 1H), 7.46 (s 1H).
8.7.1. Step 1: Preparation of 3,10-dimethyl-3H,10H-
[2,40]biimidazolyl-4-carbaldehyde (9e). To
a stirred
solution of potassium tert-butoxide (3.23g) in dry
DMF (140mL),1H,10H-[2,40]biimidazolyl-4-carbalde-
hyde (1.95g) and 18-crown-6 (634mg) were added at
0ꢁC. The reaction mixture was stirred for 10min and
treated with methyl iodide (1.85mL). After stirring for
30min at 0ꢁC the reaction mixture was stirred at
room temperature for 17h. The reaction mixture was
concentrated under reduced pressure and the residue
was extracted with chloroform, washed well with water
and dried over anhydrous MgSO4. The organic layer
was filtered and evaporated under reduced pressure.
The crude material was purified by silica gel column
chromatography (chloroform–methanol = 9/1). The title
compound (887mg, 39%) and its regio-isomer 1,10-di-
methyl-1H,10H-[2,40]biimidazolyl-4-carbaldehyde (296
mg, 13%) were obtained as a pale pink solid.
3,10-Dimethyl-3H,10H-[2,40]biimidazolyl-4-carbaldehyde:
1H NMR (d, CDCl3) 3.78 (s, 3H), 4.37 (s, 3H), 7.51 (d,
1H, J = 1.0Hz), 7.62 (d, 1H, J = 1.0Hz), 7.76 (s, 1H),
9.71 (s, 1H).
1,10-Dimethyl-1H,10H-[2,40]biimidazolyl-4-carbaldehyde:
1H NMR (d, CDCl3) 3.76 (s, 3H), 4.10 (s, 3H), 7.47 (d,
1H, J = 0.9Hz), 7.57 (s, 1H), 7.59 (d, 1H J = 0.9Hz),
9.85(s, 1H).
Acknowledgements
8.7.2. Step 2: Preparation of (5R,6RS)-6-[(RS)-acetoxy-
(3,10-dimethyl-3H,10H-[2,40]biimidazolyl-4-yl)-methyl]-6-
bromo-7-oxo-4-thia-1-aza-bicyclo[3.2.0]hept-2-ene-2-
carboxylic acid 4-nitro-benzyl ester (10e). To a stirred
solution of 3,10-dimethyl-3H,10H-[2,40]biimidazolyl-4-
carbaldehyde (171mg) in dry acetonitrile (20mL) a solu-
tion of anhydrous MgBr2 (800mg) under a nitrogen
atmosphere at room temperature was added. It was
stirred at room temperature for 10min and a dry THF
solution (20mL) of (5R,6S)-6-bromo-7-oxo-4-thia-1-
aza-bicyclo[3.2.0]hept-2-ene-2-carboxylic acid 4-nitro-
benzyl ester (346mg) was added to the mixture, cooled
to ꢀ20ꢁC, and triethylamine (0.768mL) was added in
one portion. The reaction vessel was covered with a foil
to exclude light. The reaction mixture was stirred for 6h
at ꢀ20ꢁC and treated with 4-dimethylaminopyridine
(22mg) and acetic anhydride (0.17mL) in one portion
and subsequently warmed to 0ꢁC and stirred for 17h
The authors thank the members of the Wyeth Chemical
Technologies group for analytical and spectral determi-
nation, Drs. Frank Hollinger for early contributions and
Drs. Ray Testa, Patricia Bradford, and Beth Rasmussen
for useful discussions.
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