Structure-activity relationship of 6-methylidene penems bearing tricyclic heterocycles as broad-spectrum β-lactamase inhibitors: Crystallographic structures show unexpected binding of 1,4-thiazepine intermediates

Article abstract of DOI:10.1021/jm049680x

The design and synthesis of a series of seven tricyclic 6-methylidene penems as novel class A and C serine β-lactamase inhibitors is described. These compounds proved to be very potent inhibitors of the TEM-1 and AmpC β-lactamases and less so against the class B metallo-β-lactamase CcrA. In combination with piperacillin, their in vitro activities enhanced susceptibility of all class C resistant strains from various bacteria. Crystallographic structures of a serine-bound reaction intermediate of 17 with the class A SHV-1 and class C GC1 enzymes have been established to resolutions of 2.0 and 1.4 A?, respectively, and refined to R-factors equal 0.163 and 0.145. In both β-lactamases, a seven-membered 1,4-thiazepine ring has formed. The stereogenic C7 atom in the ring has the R configuration in the SHV-1 intermediate and has both R and S configurations in the GC1 intermediate. Hydrophobic stacking interactions between the tricyclic C7 substituent and a tyrosine side chain, rather than electrostatic or hydrogen bonding by the C3 carboxylic acid group, dominate in both complexes. The formation of the 1,4- thiazepine ring structures is proposed based on a 7-endo-trig cyclization.

Full text of DOI:10.1021/jm049680x

6556
J. Med. Chem. 2004, 47, 6556-6568
Structure-Activity Relationship of 6-Methylidene Penems Bearing Tricyclic
Heterocycles as Broad-Spectrum â-Lactamase Inhibitors: Crystallographic
Structures Show Unexpected Binding of 1,4-Thiazepine Intermediates
Aranapakam M. Venkatesan,† Yansong Gu,† Osvaldo Dos Santos,† Takao Abe,‡ Atul Agarwal,† Youjun Yang,†
Peter J. Petersen,† William J. Weiss,† Tarek S. Mansour,*,† Michiyoshi Nukaga,§ Andrea M. Hujer,|
Robert A. Bonomo,| and James R. Knox*,§
Wyeth Research, 401 N. Middletown Road, Pearl River, New York 10965, Wyeth Lederle Japan, Saitama, Japan, Department
of Molecular and Cell Biology, The University of Connecticut, Storrs, Connecticut 06269-3125, and Research Service,
Department of Veterans Affairs Medical Center, Cleveland, Ohio 44106
Received April 30, 2004
The design and synthesis of a series of seven tricyclic 6-methylidene penems as novel class A
and C serine â-lactamase inhibitors is described. These compounds proved to be very potent
inhibitors of the TEM-1 and AmpC â-lactamases and less so against the class B metallo-â-
lactamase CcrA. In combination with piperacillin, their in vitro activities enhanced susceptibility
of all class C resistant strains from various bacteria. Crystallographic structures of a serine-
bound reaction intermediate of 17 with the class A SHV-1 and class C GC1 enzymes have
been established to resolutions of 2.0 and 1.4 Å, respectively, and refined to R-factors equal
0.163 and 0.145. In both â-lactamases, a seven-membered 1,4-thiazepine ring has formed. The
stereogenic C7 atom in the ring has the R configuration in the SHV-1 intermediate and has
both R and S configurations in the GC1 intermediate. Hydrophobic stacking interactions
between the tricyclic C7 substituent and a tyrosine side chain, rather than electrostatic or
hydrogen bonding by the C3 carboxylic acid group, dominate in both complexes. The formation
of the 1,4- thiazepine ring structures is proposed based on a 7-endo-trig cyclization.
Introduction
based inhibitors of serine-reactive class A and C â-lac-
tamases, whereas others reported transition-state ana-
logue inhibitors.1b,i High-resolution crystallographic
structure determination of an inhibitor with class A and
C â-lactamases describing the mode of action of imidazo-
[2,1-c][1,4]oxazin methylidene penem 7 established the
formation of a novel seven-membered 1,4 thiazepine
intermediate having R stereochemistry at the C7 moi-
ety.12 However, an unexpected feature reported in this
study indicated that the 1,4-thiazepine intermediate is
oriented differently in each complex following the acy-
lation reaction between 7 and active serine residue. This
finding is significant in the context of designing new
analogues with desired potency against both class A and
C enzymes. Accordingly, on the basis of these findings,
we undertook the synthesis and biological evaluation
of novel tricyclic methylidene penems 11-17, followed
by the crystallographic structure determination of reac-
tion intermediates of 17 with the SHV-1 and GC1
â-lactamases, which are well-characterized members of
the class A and C families. It is found that the 1,4-
thiazepine intermediate has distinctive hydrophobic
contacts within the binding site of each â-lactamase.
â-Lactamases are enzymes that hydrolyze â-lactam
rings in all classes of â-lactam antibiotics. The produc-
tion of such enzymes by bacteria is the most common
resistance mechanism against â-lactam antibiotics, a
matter that constitutes a major threat to human health.
â-Lactamases can be either plasmid-mediated, such as
class A TEM enzymes, or chromosomal represented by
class C AmpC enzymes.1 It is estimated that amoxicillin
resistance in Gram-negative Escherichia coli bacteria
amounts to 50% of isolates in hospitals, whereas peni-
cillin resistance to Gram-positive Staphylococcus aureus
bacteria reaches over 90% of clinical isolates.2 Therefore,
the intensive search for a new generation of inhibitors
with broader spectrum of activity than clinically used
clavulanic acid, sulbactam, and tazobactam continues
unabated. This search is especially important given the
fact that these heavily used inhibitors are becoming
ineffective against newly appearing mutant â-lacta-
mases.
Previous work reported on methylidene â-lactam
antibiotics has focused mostly on monocyclic rings
attached to the exocyclic double bond linkage of
penems,3-6 cephems,7 and extended carbon derivatives8
(Figure 1). Investigations in our laboratories have
identified penam sulfones,9 spirocyclopropyl penam
sulfones,10 and methylidene penems11 as mechanism-
Chemistry
The tricyclic 6-methylidene penem carboxylic acid
sodium salts 11-17 were prepared by a two-step
process, starting from (5R,6S)-6-bromo-7-oxo-4-thia-1-
azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid 4-nitroben-
zyl ester 9, which was prepared from the commercially
available 6-aminopenicilanic acid (6-APA) 8 by a mul-
tistep procedure modified13 from the previously avail-
able procedure.14
* To whom correspondence should be addressed. T.S.M.: Tel, (845)
602-3668; Fax, (845) 602-5580; E-mail, mansout@wyeth.com. J.R.K.:
Tel, (860) 486-3133; Fax, (860) 486-4745; E-mail, james.knox@uconn.edu.
† Wyeth Research.
‡ Wyeth Lederle Japan.
§ The University of Connecticut.
| Department of Veterans Affairs Medical Center.
10.1021/jm049680x CCC: $27.50 © 2004 American Chemical Society
Published on Web 11/18/2004

Novel Tricylic 6-Methylidene Penems
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 26 6557
Figure 1. Structure of tazobactam (4) and class A and class C â-lactamase inhibitors.
Scheme 1. General Method To Prepare Compounds 11-17
The first step involved a Lewis acid mediated aldol
condensation between 9 and the appropriately substi-
tuted aldehydes 20a-f and 24 in the presence of
anhydrous MgBr2 and triethylamine (Scheme 1). Previ-
ously, this transformation was carried out using strong
lithium bases such as lithium diisopropylamide, (LDA)
lithium hexamethyldisilazide (LHMDS), or Ph2NLi with
PMB ester group, which led to poor yields and unpre-
dicted side products.15
The aldehydes 20a-f were prepared starting from the
commercially available 2-aminobenzothiazole deriva-
tives 18. The appropriately substituted 2-aminoben-

6558 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 26
Scheme 2. Synthesis of Aldehydes 20a-f
Venkatesan et al.
Scheme 3. Synthesis of Aldehyde 24
bactam, the new inhibitors exhibited excellent activity
against the enzymes listed here. These compounds were
very potent inhibitors of TEM-1 and AmpC enzymes
with IC50 values in the range of 1-18 and 1-3 nM,
respectively. Furthermore, in comparison to tazobactam,
they exhibited almost 5-100 fold greater potency against
TEM-1 enzyme and were about 28,000-84,000-fold
more potent against AmpC enzymes. Within this series
of new inhibitors, comparison of their potencies reveals
that any substitution on the aromatic ring or any
modification did not alter their potency against the
AmpC enzyme significantly. However, the substitution
on the 7-position (see the numbering in Scheme 1) of
the heterocycle alters the potency against the TEM-1
enzyme slightly. Replacement of hydrogen on the 7-po-
sition of the heterocycle with fluorine (compound 12) or
chlorine (compound 13) does not change their potencies
dramatically. However, the presence of a methyl group
at position 7 (compound 14) increased the potency
against TEM-1 by 2-fold. Moving the methyl from the
7 to the 5 position (compounds 14 and 16) led to a drop
in the potency against TEM-1. This loss in potency was
recovered by changing the aromatic ring of compounds
11-16 to an alicyclic ring (compound 17), which in-
creased the potency against TEM-1 by 10 fold. To
expand further their spectrum of activity, compounds
11-17 were found to be good inhibitors of the class B
metalloenzyme CcrA in comparison to tazobactam (Table
2).
Further in vitro evaluation in cell-based assay (MIC)
reaffirmed the effectiveness of compounds 11-17 as
potent broad spectrum â-lactamase inhibitors. These
data are summarized in Table 3, which also lists the
expressing enzyme and its class. In all these experi-
ments, piperacillin was combined with the newly syn-
thesized inhibitors and tested against various piper-
acillin-resistant pathogens (MIC < 64 µg/mL) expressing
different â-lactamases. The piperacillin + tazobactam
combination was used as a comparator. At the onset, it
is important to note that when tested alone, both
tazobactam and the 6-methylidene penem inhibitors
11-17 did not exhibit any antibacterial activity, which
confirmed the fact that these compounds do not have
any intrinsic antibacterial activity. When combined with
piperacillin, compounds 11 and 17 are as active as
tazobactam against class A producing E. coli pathogens.
In these cases the MIC values of the weaker inhibitor
15 in combination with piperacillin was found to be
higher than that of other inhibitors, which is consistent
with the IC50 values reported against TEM-1. The
permeability in E. coli is evaluated with the permeable
Table 1. In Vitro Activity of Compounds 11-17 against
Different â-Lactamases
IC50, nM
compound
TEM-1
AmpC
tazobactam
100 ( 8
10.0 (2
8.0 ( 1
6.0 ( 1
3.0 ( 1
18.0 ( 2
9.0 ( 2
1.0 ( 0.4
84,000 ( 300
1.0 ( 0.5
2.0 ( 1
11
12
13
14
15
16
17
3.0 ( 1
1.0 ( 0.5
2.0 ( 1
2.0 ( 1
2.0 ( 1
Table 2. In Vitro Activity of Compounds 11-17 against CcrA
compound
IC50, nM
compound
IC50, nM
tazobactam
400,000 ( 200
350 ( 6
14
15
16
17
103 ( 9
250 ( 5
370 ( 8
240 ( 6
11
12
13
370 ( 5
74 ( 3
zothiazole derivatives were reacted with ethyl bromopy-
ruvate in dimethoxyethane. The resulting tricyclic ester
derivatives were reduced to their corresponding alcohols
using LiAlH4 and oxidized to their respective aldehydes
20a-f using active MnO2 in refluxing chloroform for 48
h (Scheme 2). The aldehyde 24 was prepared by a
Hantzch reaction in which R-chlorocyclopentanone was
reacted with thiourea to afford the 2-aminothiazole
derivative 22. This was converted to aldehyde 24 by the
sequence of reactions described for the preparation of
compounds 20a-f and shown in Scheme 3.
Results and Discussion
Structure-Activity Relationship. The in vitro
inhibitory activities (IC50 values in nM) for all the newly
synthesized compounds 11-17 are shown in Tables 1
and 2. As mentioned earlier, all seven compounds were
tested against TEM-1 (class A), CcrA (class B), and
AmpC (class C) â-lactamases. In comparison with tazo-

Novel Tricylic 6-Methylidene Penems
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 26 6559
Table 3. In Vitro Antimicrobial Activity of Inhibitorsa 11-17 in Combination with Piperacillin (Pip) at a Constant 4 µg/mL
Concentration
species & strain
E. coli GC 2844
expression
Tazoa + Pip 11+ Pip 12 + Pip 13 + Pip 14 + Pip 15 + Pip 16 + Pip 17 + Pip
none
2
2
2
2
2
2
16
2
2
E. coli GC 2847
E. coli GC 2920
E. coli GC 2804
E. coli GC 2805
E. coli GC 2252
E. cloacae GC 1477
E. cloacae GC 2071
E. cloacae GC 1475
TEM-1 (class A)
IRT-2 (class A)
Imp
CcrA (class B)
IRT-2 (class A)
AmpC (class C)
Imi+1 + AmpC (class A+C)
P99 (class C)
2
2
8
4
4
8
4
4
0.12
>64
2
2
1
2
2
2
2
<0.06
<0.06
<0.06
<0.06
<0.06
>64
<0.06
<0.06
>64
>64
>64
>64
>64
>64
2
>64
>64
>64
>64
8
16
2
16
32
8
16
16
16
16
8
16
32
8
8
64
16
16
64
16
32
16
16
16
8
16
4
16
4
8
8
8
K. pneumoniae GC 2825 four enzymes
16
32
8
(not characterized)
S. marcescensb GC 1781 Sme-1 + AmpC
P. aeruginosac GC 1764 AmpC
S. marcescens GC 4142 AmpC
E. coli GC 2203
S. aureus GC 2216
1
>64
>64
>64
64
0.50
2
1
16
0.50
2
32
4
1
2
2
2
2
4
4
16
2
1
2
2
2
2
1
1
2
4
2
control
control
1
1
<0.06
<0.06
<0.06
<0.06
<0.06
<0.06
a Tazo ) tazobactam. b S. marcescens ) Serratia marcescens. c P. aeruginosa ) Pseudomonas aeruginosa.
strain 2804, where all inhibitors had potent activity.
Therefore, this aspect should be taken into consideration
in assessing the in vitro activity against strains from
different bacteria.
Figure 2 shows the mixed R-â tertiary structure of
each â-lactamase molecule. Ramachandran analysis of
the SHV-1 and GC1 protein backbone showed 92.0% and
92.3% of residues, respectively, fall in most favored
regions, with none in disallowed regions. Residues 214-
217 in the Ω loop (residues 200-226) of GC1 were not
clear and were not modeled. The difference electron
density of reaction intermediate 27 in each enzyme is
shown in Figure 3.
The absolute configuration at carbon C7 (initially C6′)
is variable in the two complexes. A single conformer 28
with the R configuration is observed in the SHV
complex, whereas in the GC1 complex the conformer
with the R configuration has only 30% occupancy and
coexists with the predominant S conformer (70%) 29.
It is possible that both stereoisomers are formed initially
in each â-lactamase, but the form more stable to
hydrolysis predominates in the crystallographic maps.
Alternatively, stereospecific reaction pathways may
differ in each active site, producing one chiral form in
preference to the other. The C3 carboxylic acid group
of the minor R conformer was not well-defined in the
GC1 map and was not modeled.
It should be noted from Table 3 that even though the
tazobactam + piperacillin combination is potent against
class A producing pathogens, this combination is less
effective against class C producing organisms, such as
Enterobacter cloacae GC 1477, GC 2071, and GC 1475.
However, the addition of the inhibitors 11-17 restored
susceptibility to piperacillin for 100% of the class C
producing strains listed here, including Klebsiella pneu-
moniae GC2825, which expresses four uncharacterized
â-lactamases. However, all inhibitors 11-17 were inef-
fective against E. coli GC 2805 (a class B producing
bacteria) due to their moderate activity against CcrA.
Due to its potent activity, compound 17 was chosen to
elucidate the mechanism of inhibition by X-ray crystal-
lographic techniques using SHV-1 (class A) and the
extended-spectrum GC1 (class C) enzymes. Against
SHV-1 and GC1, compound 17 was determined to have
an IC50 value of 12 and 3.8 nM, respectively.
Structure Determination of the Enzyme Com-
plexes. Rigid-body refinement using the unliganded
SHV-1 (PDB entry 1SHV) and GC1 (PDB entry 1GCE)
â-lactamases was done at 2 Å with CNS.16 R-factors
were ca. 35% for the unsolvated apoprotein models. A
simulated annealing protocol and cross-validation17
were then used in the refinements. XtalView18 was
employed for map displays and for manipulation of the
structures. As the protein models were being optimized,
a serine-bound structure was evident in the difference
density of both complexes. On the basis of previous
crystallographic work on the related penem 7,12 species
28 and 29, the chiral forms of species 27 (Scheme 4),
were added to the model of each complex. Because the
7-atom ring in 27 has three tautomeric forms, only weak
refinement restraints were applied to the ring geometry.
In the case of the GC1 complex, two conformers of the
intermediate were introduced in later refinement steps.
The last stage of the higher resolution refinement of
GC1 was performed with SHELX19 using all data (Table
1, Supporting Information). At 1.4 Å resolution, the
introduction of anisotropic B-factors reduced the R and
Rfree values by 3.5% and 1.1%. Riding hydrogen atoms
were later added in calculated positions.
Inhibitor-Induced Changes in the Enzyme Struc-
tures. When the SHV-1 complex is superposed over the
crystal structure of the uninhibited â-lactamase,20 sev-
eral changes are seen. The side chain of Tyr105 reori-
ents slightly (15°-20°) to sandwich stack with the C7
heterocyclic substituent of the inhibitor (Figure 4). Its
closest contact (ca. 3.5 Å) is with the terminal ring of
the tricyclic substituent. The binding site widens slightly
so that the distance from the 104-106 loop to the B3
â-strand (234-238) increases by nearly 1 Å. Conserved
Glu166 and its hydrogen-bonded water molecule, im-
portant for catalysis, are now closer (2.4 Å) than in the
apoenzyme. This short distance may be an artifact due
to vibration or disorder in either or both groups.
Overlay of the GC1 complex with the uninhibited
enzyme21 shows little indication of a second Ω-loop
pathway seen in other GC1 complexes.12,22 Tyr224 in
the loop is sandwiched with the tricyclic substituent of
the predominant S conformer of the inhibitor intermedi-
ate. The side chain of Gln120 is found in two rotameric
conformations about CR-Câ, one pointing outward and
the other directed inward to hydrogen bond with the N
atom of the imidazoyl ring in the tricyclic substituent.

6560 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 26
Venkatesan et al.
Scheme 4. Mechanism of Inhibition of SHV-1 and GC1 â-Lactamases by 17a
a Species 27 is observed in the crystal structures of both enzymes.
chains of Leu119 and Leu296 shift toward the inter-
mediate.
Mechanism of Inhibition. A proposed pathway for
the conversion of penem 17 to the intermediate observed
by crystallography is given in Scheme 4. Acylation of
the reactive serine (Ser70 or Ser64) of the SHV-1 class
A or GC1 class C â-lactamase produces species 25,
which may be very susceptible to hydrolysis.4,31 Rotation
about the C5-C6 bond and ring opening produces a
reactive thiolate species 26. Thiolate 26 undergoes a
7-endo-trig cyclization in preference to a 6-exo-trig
process to produce the seven-membered thiazepine ring.
The thiolate attack on the C6-C6′ double bond can
occur from either side to produce chirality at carbon 7
(initially C6′). The dihydrothiazepine 27 is drawn as the
ion pair of three tautomeric forms 27a-c. The experi-
mental electron density at C6 is almost planar, so we
believe the amount of tautomer 27c is small.
Orientation and Interactions of the Intermedi-
ate in Each Binding Site. In both class A and class C
binding sites the serine-bound intermediate has its
â-lactamyl carbonyl group hydrogen bonded to main
chain amide groups of the reactive serine and â-strand
B3. The positions of the 7-atom rings are generally
equivalent in the two binding sites, with the exception
that the ring of the minor R conformer in the class C
complex is rotated ca. 180° about the bond to the serine
ester.
Figure 2. Tertiary structure of (a) the class A SHV-1
â-lactamase complex and (b) the class C GC1 complex. The
thiazepine intermediate 27 is bound to the reactive serines
70 and 64, respectively. Four residues in the Ω loop at the
bottom of the GC1 enzyme were not seen. Drawn by MOL-
SCRIPT.37
Asn152 also rotates slightly to bind with this N atom.
Other changes involve the polypeptide backbone. The
segment from 118 to 123 on the left of the binding site
moves about 0.5 Å, and the 293-298 segment above the
binding site moves about 1.4 Å, so the hydrophobic side

Novel Tricylic 6-Methylidene Penems
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 26 6561
Figure 3. Stereoview of the Fo - Fc electron density of the intermediate 27 bonded to the reactive serine in (a) the SHV-1
â-lactamase and (b) the GC1 â-lactamase. The density contour level is 3σ. The figure was made by XtalView18 and Raster3D.38
In both complexes the C3 carboxylic acid group of the
thiazepine ring is solvated and has no interactions with
the enzyme. In the class A â-lactamase, the N4 atom in
the thiazepine ring is 3.1 Å from, but poorly oriented
to, the main chain carbonyl oxygen atom of residue 237
on the B3 â-strand. The N4 atom of the main conformer
of the class C complex has no protein interactions,
though in the minor conformer it is 3.2 Å from the
Asn152 amide group.
The rotameric conformation of the tricyclic substitu-
ent about the C7-C heterocycle bond differs from one
enzyme to the other. In the SHV-1 class A binding site,
the ring edge containing the S and N atoms is directed
inward toward the hydrophobic Val216 at the top of the
binding site. In both conformers of the GC1 class C
complex, the S-N edge of the substituent is turned
outward. In the major conformer the imidazole N atom
has hydrogen bonds with the amide groups of Gln120
(2.8 Å) and Asn152 (2.9 Å), while in the minor conformer
the N atom has no interactions with protein.
It would be useful to determine the generality of this
stacking interaction in other complexes of each enzyme.
Hydrolysis of the Acyl Intermediate. Neither the
heterocylic group nor the thiazepine ring is able to block
the approach of a water molecule to the acyl ester bond,
as seen in some â-lactamase complexes.22-24 In the
SHV-1 class A binding site, a water molecule activated
by Glu166 is thought to attack from the relatively buried
side of the ester bond25 (Figure 3). We observe this water
molecule, but the short length of its hydrogen bond
indicates the presence of some disorder, which may
impair efficient deacylation. In class C enzymes, the
water is believed to attack from the more exposed top
side of the ester. Normally, conserved Tyr150, and
possibly the â-lactamyl intermediate itself,25-27 may
activate the hydrolytic water molecule.28,29 In GC1 a
molecule of water is found above but rather far (3.6 Å)
from the ester bond, and the water is uninvolved with
the Tyr150 hydroxyl group (4.2 Å). Substrate assistance
in activating this water, via the intermediate’s C3
carboxylate group or N4 nitrogen atom, is not possible
here. Thus, the stability of the acyl intermediate is more
readily accounted for in the GC1 class C case than in
class A. Structural reasons for the disorder (SHV-1) and
poor activation (GC1) of the water remain unclear.
Perhaps more important, in both cases, is the large
contribution to stability provided by the conjugation of
the acyl ester with the large dihydrothiazepine ring
Of possibly greater significance in the binding is a
sandwich stacking of the large tricyclic ring with a
tyrosine ring in each binding site. Tyr105 in the class
A SHV-1 enzyme and Tyr224 in GC1 are each about
3.5 Å from the tricyclic ring. These tyrosines are almost
conserved in class A and class C â-lactamases (Tyr224
is conventionally numbered 221 in wild-type sequences)
but are in very different regions of the two binding sites.

6562 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 26
Venkatesan et al.
Figure 4. Stereoview of the overlay of the intermediates of 17 (red) and 7 (light blue) in (a) the class A SHV-1 â-lactamase and
(b) the class C GC1 â-lactamase. Tyrosines Tyr105 or Tyr224 stack with the heterocyclic group on C7. Only the major S conformer
of 17 is shown for the GC1 enzyme. The minor R conformer is near the position of 7.
system, particularly in the vinylogous urethane tauto-
meric structures 27a and 27b.
than the tricyclic system in 17. The more conjugated
17 easily stacks with Tyr224, 7 less so.
Comparison of the Binding by Tricyclic 17 and
Bicyclic 7. Our crystal structures show that both
penems react to form the same thiazepine intermediate,
the 7-atom rings of which overlay very closely (Figure
4). The stereoconfiguration at C7 in the complexes of
these two enzymes with 7 is exclusively R,12 whereas
here in the complexes with 17 the R form is found
exclusively only in the SHV-1 class A enzyme. The
heterocycle of both inhibitors is found in the upper
region of the SHV-1 class A binding site but in the
bottom of the GC1 class C binding site. In all cases the
heterocyclic group lies on the side of the thiazepine ring
opposite the expected position of the hydrolytic water
molecule. Thus, the rather large heterocycles do not
block an attack by water on the acyl ester bond.
In the case of 17, the conformer engaged in tyrosine
stacking has higher crystallographic occupancy than the
unstacked conformer. One explanation, applicable to
both enzymes, is that the stacked conformer is more
resistant to hydrolysis by virtue of this anchoring and
fixed position. An unstacked conformer may have more
degrees of freedom, some of which might better position
the intermediate for hydrolysis. But this argument
appears to fail in the case of 7, where the stacked
conformer is found only in the SHV-1 class A enzyme.12
However, with 7, the Tyr224 of the GC1 class C enzyme
had two main-chain positions in the crystal structure,
so that full-time stacking was not possible. Further, the
bicyclic ring system in 7 is relatively less conjugated
Generally, binding by these thiazepine intermediates
appears to be only marginally dependent on hydrogen
bonding and electrostatics. For example, one of the
expected functionalities,4 the C3 carboxylic acid group,
is solvated in most complexes and has few significant
protein interactions. Rather, the hydrophobicity of the
multicyclic substituent at C7 and, possibly, its degree
of conjugation appear to have greater influence on the
positioning of the intermediate. Optimization of the
design of the C7 substituent to exploit this stacking
interaction could prove beneficial.
Prior Penem Studies. A comparison can be made
with earlier chemical and modeling studies of the
related monocyclic penem 14,5,31 All these workers
predicted the expanded ring structure now observed by
crystallography after the reaction of 7 or 17 with both
â-lactamases. Major differences are found, however, in
the predicted C7 stereochemistry of the intermediate
and in its interactions with the â-lactamases. In an
energy minimization study,4 the thiazepine intermedi-
ate of 1 was docked in the binding sites of class A and
class C â-lactamases. The modeling indicated that only
the C7(S) conformer could be accommodated in each
enzyme, and it predicted specific hydrogen-bonding
interactions of the C3 carboxylic acid group, none of
which are observed in our four crystal structures (ref
12 and present work). It is notable too that the tyrosine
stacking interaction with the C7 heterocycle was not
predicted, possibly because the methyltriazolyl group

Novel Tricylic 6-Methylidene Penems
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 26 6563
Reaction with Penem Inhibitor. A crystal of SHV-1 was
soaked at 21 °C for 3 h in a 30% PEG holding solution (pH 7)
containing 10 mM of 17. The inhibitor solution was refreshed
15 min before crystal cooling and X-ray data collection. A crys-
tal of the GC1 enzyme was soaked with 2 mM 17 for 70 min.
in 1 is smaller than the bi- and tricyclic groups studied
here. Nevertheless, our results emphasize that modeling
the binding of acyl intermediates in these enzymes can
be misleading, and they demonstrate the limitation of
some docking algorithms in quantitating the weak van
der Waals interactions that are often critical in com-
plexation.
X-ray Data Collection. For cryoprotection, the inhibitor-
soaked SHV-1 and GC1 crystals were immersed for 10-15 s
in a solution containing 25% MPD or 25% glycerol, respec-
tively, in the PEG holding solution. Crystals were flash-cooled
at 100 K with a nitrogen gas stream. For SHV-1, data were
collected on a Bruker HISTAR multiwire area detector on a
Rigaku RU-200 X-ray generator (λ ) 1.542 Å) with double-
mirror Franks focusing. Frames were counted for 2 min
through an ω range of 0.2° and were processed with XGEN
(Molecular Simulations, Inc.). For GC1, 1° oscillation images
were collected on a Q210 CCD detector (Area Detector Systems
Corp.) at station A1 of the Cornell High Energy Synchrotron
Source (MacCHESS). The HKL programs36 were used to
process GC1 intensities. Results are given in Table 2 of the
Supporting Information. Atomic coordinates and structure
factors have been deposited in the Protein Data Bank at the
Research Collaboratory for Structural Bioinformatics at Rut-
gers University (entries 1Q2P and 1Q2Q for the SHV and GC1
complexes, respectively).
Conclusion
Novel 6-methylidene penems bearing tricyclic hetero-
cycles were synthesized from bromopenem 9 by employ-
ing a unique aldol reaction mediated by MgBr2/trieth-
ylamine followed by reductive elimination. The new
penems proved to be potent inhibitors of serine-reactive
class A and C â-lactamases and had excellent MIC
values in combination with piperacillin. The inhibitory
reaction of 17 with the SHV-1 class A and GC1 class C
â-lactamases as monitored by X-ray crystallography
revealed the formation of a 1,4-thiazepine intermediate
with unexpected modes of binding. The significance of
hydrophobic stacking of the C7 heterocycle with Tyr105
and Tyr224 in SHV-1 and GC1, respectively, and its
possible role in protecting the intermediate from hy-
drolytic attack are presented. Both chiral forms of the
intermediate were found, but with differing degrees of
crystallographic occupancy in each enzyme. While C7-
(R) is seen in both enzymes to a different extent, C7(S)
is seen only in GC1. It is unknown whether both chiral
forms are made initially in equal proportion, or stereo-
selectively, as one chiral form may have hydrolyzed
more readily than the other. The present work together
with that reported earlier12 on a bicyclic penem discloses
new insights and considerations into â-lactamase inhi-
bition that impacts the design of new inhibitors.
General Methods. Melting points were determined in open
capillary tubes on a Meltemp melting point apparatus and are
uncorrected. 1H NMR spectra were determined with a Bruker
DPX-400 spectrometer at 400 MHz. Chemical shifts δ are
reported in parts per million (δ) relative to residual chloroform
(7.26 ppm), TMS (0 ppm), or dimethyl sulfoxide (2.49 ppm) as
an internal reference with coupling constants (J) reported in
hertz (Hz). The peak shapes are denoted 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. Elec-
tron impact and high-resolution mass spectra were obtained
on a Finnigen MAT-90 spectrometer. Combustion analysis was
obtained using a Perkin-Elmer Series II 2400 CHNS/O ana-
lyzer. Chromatographic purifications were performed by open
chromatography using IBW-127ZH (Fuji Silysia). Thin-layer
chromatography (TLC) was performed on Merck PLC pres-
Experimental Section
cored plates 60F254
.
Biological Testing. All the compounds prepared here were
tested in vitro against TEM-1 (class A), CcrA (class B), and
Amp-C (class C) â-lactamases for their inhibitory activity by
the spectrometric method as described by Bush et al. using
nitrocefin as the substrate.32 Homogeneously purified â-lac-
tamases were prepared from E. coli (TEM-1, SHV-1), E. cloacae
(Imi-1, AmpC, GC1), and Bacteroides fragilis (CcrA). The
enzyme concentrations were 4.4 nM (TEM-1), 31 nM (SHV-
1), 1.2 nM (CcrA), 2.1 nM (AmpC), and 5.7 nM (GC1),
respectively. The maximal residual velocity was then mea-
sured.
After testing these compounds for the enzyme inhibition
assay, the test compounds were subjected to the whole cell in
vitro susceptibility testing assay. The in vitro activities, as
minimum inhibitory concentrations (MIC’s), were obtained by
the microbath dilution method.33 MIC values were determined
for the combination of inhibitor with piperacillin (Pip) at a
constant concentration of 4 µg/mL of the inhibitor.
Subcloning and Protein Purification. The SHV-1 â-lac-
tamase gene (blaSHV-1, GenBank AF124984) was directionally
subcloned into pBCSK from a clinical strain of K. pneumoniae
15571.34 E. coli DH10B was used to harvest the SHV-1
enzyme.20 The E. cloacae GC1 enzyme, a natural extended-
spectrum variant of the E. cloacae wild-type P99 enzyme, was
obtained from the GC1 â-lactamase gene on the pCS101
plasmid in E. coli AS226-51, as previously described.21,35
Crystallization of the Apo â-Lactamases. SHV-1 crystals
were grown from PEG (Mr ) 6000) as previously described.20
The 28.9 kDa SHV-1 enzyme crystallizes in P212121 with one
molecule in the asymmetric unit. The â-lactamase from E.
cloacae GC1 was crystallized in PEG (Mr ) 8000) at pH 7.21
The GC1 crystals have space group P21212 with one molecule
of 39.4 kDa in the asymmetric unit.
The terms “concentrated” and “evaporated” refer to removal
of solvents using a rotary evaporator at water aspirator
pressure with a bath temperature equal to or less than 40 °C.
Unless otherwise noted, reagents were obtained from com-
mercial sources and were used without further purification.
All the substituted and unsubstituted ethylimidazo[2,1-b]-
benzothiazole-2-carboxylates were prepared by the known
literature procedure.39 The esters were reduced to alcohols and
the crude products (after characterizing them using mass-
spectra) were taken to the next step to prepare novel aldehydes
20a-f and 24.
Preparation of (5R,6Z)-6-(Imidazo[2,1-b][1,3]benzothia-
zol-2-ylmethylene)-7-oxo-4-thia-1-azabicyclo[3.2.0]hept-2-
ene-2-carboxylic Acid Sodium Salt (11). Step 1: Ethyl
Imidazo[2,1-b]benzthiazole-2-carboxylate. Ethyl bromopy-
ruvate (9.8 g, 50 mmol) was added dropwise to a stirred
solution of 2-aminobenzothiazole (7.5 g, 50 mmol) in dimethoxy-
ethane (100 mL) at room temperature. After the addition, the
reaction mixture was heated to reflux for 6 h. The reaction
mixture was cooled to room temperature and quenched with
ice cold water. The aqueous layer was neutralized with NH4-
OH and the separated solid was filtered. It was washed well
with water and dried. The crude product was purified by silica
gel column chromatography by eluting it with 75% ethyl
acetate:hexane: brown solid; yield 10 g, 81%; mp 97 °C; MS
(M + H)+ 248.
Step 2: Imidazo[2,1-b]benzthiazole-2-methanol. To a
stirred slurry of LiAlH4 (2.0 g, excess) in dry THF was slowly
added ethyl imidazo[2,1-b]benzthiazole-2-carboxylate (4.9 g,
20 mmol) in THF (100 mL) at 0 °C. After the addition, the
reaction mixture was stirred at room temperature for 1 h and
quenched with saturated NH4Cl/NH4OH. The separated solid

6564 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 26
Venkatesan et al.
was extracted with chloroform:MeOH (3:1) and filtered through
a pad of Celite. The organic layer was washed once with
saturated NaCl and dried over anhydrous MgSO4. It was
filtered and concentrated. The brown solid obtained was taken
to next step without purification: yield 3.8 g, 93%; mp 131
°C; MS (M + H)+ 205.
benzthiazole-2-carboxylate (2.64 g, 0.01 mol) and LiBH4 (50
mg) in THF at refluxing temperature for 2 h. The reaction
mixture was quenched with ice-cold water and acidified with
10 N HCl. The reaction mixture was stirred for 1 h and
neutralized with K2CO3. The separated residue was extracted
with chloroform:methanol (3:1), dried over anhydrous MgSO4,
filtered, and concentrated. The crude reaction mixture was
found to be pure and taken to the next step with out any
purification: semisolid; yield 1.5 g 68%; MS (M + H)+ 223.
Step 3: 2-Formylimidazo[2,1-b]benzthiazole (20a). To
a stirred solution of imidazo[2,1-b]benzthiazole-2-methanol
(2.04 g, 10 mmol) in chloroform (200 mL) was added activated
MnO2 (15 g, excess). The reaction mixture was refluxed for 24
h and filtered through a pad of Celite. The reaction mixture
was concentrated and the product was purified by silica gel
column chromatography by eluting it with 75% ethyl acetate:
Step 3: 2-Formyl-7-fluoro-imidazo[2,1-b]benzthiazole
(20b) was prepared according to the procedure outlined for
compound 11 (step 3). Starting from 7-fluoroimidazo[2,1-b]-
benzthiazole-2-methanol (1.5 g 6.7 mmol) and active MnO2 (12
g, excess), in boiling chlorofom (300 mL) 1.1 g (78% yield) of
the aldehyde derivative was isolated as brown solid. The
product was purified by silica gel column chromatography by
1
hexane: brown solid; yield 800 mg, 40%; H NMR (CDCl3) δ
9.97 (s, 1H), 8.61 (s, 1H), 7.61 (m, 1H), 7.57 (m, 1H), 7.31-
7.13 (m, 2H); MS (M + H)+ 203.
1
Step 4: 4-Nitrobenzyl 6-[(Acetyloxy)(imidazo[2,1-b][1,3]-
benzothiazol-2-yl)methyl]-6-bromo-7-oxo-4-thia-1-azabi-
cyclo[3.2.0]hept-2-ene-2-carboxylate (10a). To a stirred
solution of 2-formylimidazo[2,1-b]benzthiazole (20a) (444 mg,
2.2 mmol) in dry THF-acetonitrile solution (20 mL and 15
mL) was added anhydrous MgBr2:etherate (619 mg, 2.4 mmol)
at room temperature. The reaction mixture was stirred for 30
min and cooled to -20 °C followed by the addition of (5R,6S)-
6-bromo-7-oxo-4-thia-1-azabicyclo[3.2.0]hept-2-ene-2-carbox-
ylic acid 4-nitrobenzyl ester (9) (772 mg, 2 mmol) and Et3N
(2.0 mL) under an argon atmosphere. The reaction vessel was
covered with foil to exclude light and stirred for 16 h at -20
°C. At the end, the reaction mixture was treated with acetic
anhydride (1.04 mL) in one portion. The reaction mixture was
warmed to 0 °C and stirred for 15 h at that temperature. The
mixture was diluted with ethyl acetate 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 purified by a silica gel column by eluting it with ethyl
acetate:hexane (1:1). Collected fractions were concentrated
under reduced pressure, and the mixture of diastereoisomers
was taken to the next step: pale yellow amorphous solid; yield
850 mg, 67%; mp 69 °C; MS (M + H)+ 630.
eluting it with 3:1 ethyl acetate:hexane. H NMR (CDCl3) δ
9.8 (s, 1H), 9.1 (s, 1H), 8.2 (dd, 1H, J ) 4 Hz), 8.0 (dd, 1H, J
) 4 Hz), 7.5 (m, 1H); MS (M + H)+ 221.
Step 4: 4-Nitrobenzyl-6-[(acetyloxy)(7-fluoroimidazo-
[2,1-b][1,3]benzothiazol-2-yl)methyl]-6-bromo-7-oxo-4-
thia-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylate (10b) was
prepared according to the procedure outlined for the prepara-
tion of compound 11 (step 4). Starting from 2-formyl-7-
fluoroimidazo[2,1-b]benzthiazole (20b) (500 mg, 2.3 mmol) and
(5R,6S)-6-bromo-7-oxo-4-thia-1-azabicyclo[3.2.0]hept-2-ene-2-
carboxylic acid 4-nitro-benzyl ester (9) (875 mg, 2.3 mmol),
compound 10b was isolated as a mixture of diastereoisomers,
which were taken to the next step. Prior to that, the compound
was purified by silica gel column chromatography by eluting
it with 1:1 ethyl acetate:hexane: pale yellow amorphous solid;
yield 330 mg, 22%; MS (M + H)+ 649.
Step 5: (5R,6Z)-6-[(7-Fluoroimidazo[1,2-b][1,3]benzo-
thiazol-2-ylmethylene)]-7-oxo-4-thia-1-azabicyclo[3.2.0]-
hept-2-ene-2-carboxylic acid sodium salt (12) was pre-
pared according to the procedure outlined for the preparation
of compound 11 (step 5). Starting from 4-nitrobenzyl-6-
[(acetyloxy)(7-fluoroimidazo[2,1-b][1,3]benzothiazol-2-yl)methyl]-
6-bromo-7-oxo-4-thia-1-azabicyclo[3.2.0]hept-2-ene-2-carboxy-
late (10b) (710 mg, 1.07 mmol), compound 12 was isolated as
a yellow amorphous solid after purification using an HP-21
reverse phase column: yellow crystals; yield 80 mg, 19%; mp
Step 5: (5R,6Z)-6-(Imidazo[1,2-b][1,3]benzothiazol-2-yl-
methylene)-7-oxo-4-thia-1-azabicyclo[3.2.0]hept-2-ene-2-car-
boxylic Acid Sodium Salt (11). 4-Nitrobenzyl-6-[(acetyloxy)-
(imidazo[2,1-b][1,3]benzothiazol-2-yl)methyl]-6-bromo-7-oxo-4-
thia-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylate (10a) (500 mg,
0.79 mmol) was dissolved in THF (17 mL) and acetonitrile (34
mL), and freshly activated Zn dust (5.2 g) was added rapidly
along with 0.5 M phosphate buffer (pH 6.5, 28 mL). The
reaction vessel was covered with foil to exclude light and
vigorously stirred for 2 h at room temperature. The reaction
mixture was filtered and cooled to 3 °C. The pH of the filtrate
was adjusted to 8.5, and the filtrate was washed with ethyl
acetate. The ethyl acetate layer was separated and the aqueous
layer was concentrated under high vacuum at 35 °C to give a
yellow precipitate. The precipitate was dissolved in acetoni-
trile/water and loaded on a HP-21 reverse phase column. It
was eluted with deionized water (2 L) and latter eluted with
10% acetonitrile:water: yellow crystals; yield 105 mg, 35%;
1
200 °C (dec); H NMR (DMSO-d6) δ 6.53 (s, 1H), 6.63 (s, 1H),
7.1 (s, 1H), 7.45 (t, 1H), 8.04 (m, 1H), 8,13-8.10 (m, 1H), 8.61
(s, 1H); MS (M + Na)+ 396. Anal. (C16H7FN3O3S2Na) C, H, N.
Preparation of (5R,6Z)-6-[(7-Chloroimidazo[2,1-b][1,3]-
benzothiazol-2-ylmethylene)-7-oxo-4-thia-1-azabicyclo-
[3.2.0]hept-2-ene-2-carboxylic Acid Sodium Salt (13).
Step 1: Ethyl 7-chloroimidazo[2,1-b]benzthiazole-2-car-
boxylate was prepared according to the procedure outlined
for the preparation of compound 11 (step 1). Starting from
6-chloro-2-aminobenzothiazole (9.2 g, 50 mmol) and ethyl
bromopyruvate (11.6 g, 60 mmol), 8.5 g (60% yield) of ethyl
7-chloroimidazo[2,1-b]benzthiazole-2-carboxylate was isolated
as a brown solid. The product was purified using silica gel
column chromatography by eluting with 1:1 ethyl acetate:
hexane; MS (M + H)+ 281.
1
mp 233 °C; H NMR (DMSO-d6) δ 6.51 (s, 1H), 6.53 (s, 1H),
7.09 (s, 1H), 7.47 (t, 1H, J ) 7.5 Hz), 7.54 (t, 1H, J ) 7.5 Hz),
8,06 (t, 1H), 8.62 (s, 1H); MS (M + H)+ 356, (M + Na)+ 378.
Anal. (C16H8N3O3S2Na) C, H, N.
Step 2: 7-Chloroimidazo[2,1-b]benzthiazole-2-metha-
nol was prepared according to the procedure outlined for the
preparation of compound 11 (step 2). Starting from ethyl
7-chloroimidazo[2,1-b]benzthiazole-2-carboxylate (9.0 g, 32.1
mmol) and LiAlH4 (4.0 g, excess), 5.5 g (72% yield) of the
alcohol derivative was isolated as a brown solid. The product
was found to be pure enough and taken to the next step
without purification: mp 166 °C; MS (M + H)+ 239.
Step 3: 2-Formyl-7-chloroimidazo[2,1-b]benzthiazole
(20c) was prepared according to the procedure outlined for
the preparation of compound 11 (step 3). Starting from
7-chloroimidazo[2,1-b]benzthiazole-2-methanol (4.0 g 16.8 mmol)
in refluxing chloroform:methanol (300:50 mL) and active MnO2
(20 g, excess), 2.2 g (55% yield) of the aldehyde derivative was
isolated as a brown solid. Product was purified by silica gel
column chromatography by eluting it with 1:1 ethyl acetate:
Preparation of (5R,6Z)-6-[(7-Fluoroimidazo[2,1-b][1,3]-
benzothiazol-2-ylmethylene)-7-oxo-4-thia-1-azabicyclo-
[3.2.0]hept-2-ene-2-carboxylic Acid Sodium Salt (12).
Step 1: Ethyl 7-fluoroimidazo[2,1-b]benzthiazole-2-car-
boxylate was prepared according to the procedure outlined
for compound 11 (step 1). Starting from 6-fluoro-2-aminoben-
zothiazole (10.0 g, 59.5 m.mol) and ethyl bromopyruvate (17.4
g, 89.2 mmol), 3.0 g (19% yield) of ethyl 7-fluoro-imidazo[2,1-
b]benzthiazole-2-carboxylate was isolated as a brown semisolid
after purifying the reaction mixture using silica gel column
chromatography; MS (M + H)+ 265.
Step 2: 7-Fluoroimidazo[2,1-b]benzthiazole-2-metha-
nol was prepared starting from ethyl 7-fluoroimidazo[2,1-b]-

Novel Tricylic 6-Methylidene Penems
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 26 6565
hexane: 1H NMR (CDCl3) δ 9.9 (s, 1H), 8.3 (s, 1H), 7.7 (s, 1H),
7.6 (d, 1H, J ) 8 Hz), 7.5 (m, 1H); MS (M + H)+ 236.
bromo-7-oxo-4-thia-1-azabicyclo[3.2.0]hept-2-ene-2-carboxy-
late (10d) (350 mg, 0.54 mmol) compound 14 was isolated as
yellow amorphous solid after the purification using an HP-21
reverse phase column. The yellow solid was washed with
acetone, filtered, and dried: yellow crystals; yield 110 mg, 55%;
Step 4: 4-Nitrobenzyl-6-[(acetyloxy)(7-chloroimidazo-
[2,1-b][1,3]benzothiazol-2-yl)methyl]-6-bromo-7-oxo-4-
thia-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylate (10c) was
prepared according to the procedure outlined for the prepara-
tion of compound 11 (step 4). Starting from 2-formyl-7-
chloroimidazo[2,1-b]benzthiazole (20c) (270 mg, 1.14 mmol)
and (5R,6S)-6-bromo-7-oxo-4-thia-1-azabicyclo[3.2.0]hept-2-
ene-2-carboxylic acid 4-nitrobenzyl ester (9) (500 mg, 1.14
mmol), compound 10c was isolated as a mixture of diastereo-
isomers, which were taken to the next step. Prior to that, the
compound was purified by silica gel column chromatography
by eluting with 1:1 ethyl acetate:hexane: pale yellow amor-
phous solid; yield 495 mg, 65%; MS (M + H)+ 665.
Step 5: (5R,6Z)-6-[(7-Chloroimidazo[1,2-b][1,3]benzo-
thiazol-2-ylmethylene)]-7-oxo-4-thia-1-azabicyclo[3.2.0]-
hept-2-ene-2-carboxylic acid sodium salt (13) was pre-
pared according to the procedure outlined for the preparation
of compound 11 (step 5). Starting from 4-nitrobenzyl-6-
[(acetyloxy)(7-chloroimidazo[2,1-b][1,3]benzothiazol-2-yl)methyl]-
6-bromo-7-oxo-4-thia-1-azabicyclo[3.2.0]hept-2-ene-2-carboxy-
late (10c), compound 13 was isolated as a yellow amorphous
solid after purification using an HP-21 reverse phase column.
The yellow solid was washed with acetone, filtered, and
dried: yellow crystals; yield 80 mg, 18%; mp 240 °C; 1H NMR
(DMSO-d6) δ 6.6 (s, 2H), 7.1 (s, 1H), 7.62 (dd, 1H), 8.11 (d,
1H), 8.2 (s, 1H), 8.6 (s, 1H); MS (M + H + Na)+ 412. Anal.
(C16H7ClN3O3S2Na) C, H, N.
Preparation of (5R,6Z)-6-[(7-methylimidazo[2,1-b][1,3]-
benzothiazol-2-ylmethylene)-7-oxo-4-thia-1-azabicyclo-
[3.2.0]hept-2-ene-2-carboxylic Acid Sodium Salt (14).
Step 1: Ethyl 7-methylimidazo[2,1-b]benzthiazole-2-car-
boxylate was prepared according to the procedure outlined
for the preparation of compound 11 (step 1). Starting from
6-methyl-2-aminobenzothiazole (3.2 g, 20 mmol) and ethyl
bromopyruvate (4.0 g, 20.4 mmol), 3.0 g (57% yield) of ethyl
7-methylimidazo[2,1-b]benzthiazole-2-carboxylate was isolated
as a brown solid. The product was purified by silica gel column
chromatography by eluting with 1:1 ethyl acetate:hexane: MS
(M + H)+ 261.
1
mp 178 °C (dec); H NMR (DMSO-d6) δ 8.56 (s, 1H), 7.93 (d,
1H), 7.83 (s, 1H), 7.38 (d, 1H), 7.07 (s, 1H), 6.51 (s, 2H), 2.42
(s, 3H); MS (M + H + Na)+ 392. Anal. (C17H10N3O3S2Na) C,
H, N.
Step 4: (5R,6Z)-6-[(7-Methylimidazo[1,2-b][1,3]benzo-
thiazol-2-ylmethylene)]-7-oxo-4-thia-1-azabicyclo [3.2.0]-
hept-2-ene-2-carboxylic Acid Sodium Salt (14) (Proce-
dure B). 4-Nitrobenzyl 6-[(acetyloxy)(7-methylimidazo[2,1-b]-
[1,3]benzothiazol-2-yl)methyl]-6-bromo-7-oxo-4-thia-1-azabicyclo-
[3.2.0]hept-2-ene-2-carboxylate (350 mg, 0.54 mmol) was
dissolved in THF (40 mL) and 6.5 pH phosphate buffer (40
mL) and hydrogenated over Pd/C (10%, 200 mg) at 40 psi
pressure for 3 h at room temperature. The reaction mixture
was filtered through a pad of Celite and washed with aceto-
nitrile. The reaction mixture was concentrated to 40 mL and
cooled to 0 °C, and the pH was adjusted to 8.5 by adding 1 N
NaOH. The product was directly loaded over an HP21 resin
reverse phase column. Initially, the column was eluted with
deionized water (2 L) and later with 10% acetonitrile:water.
The fractions were concentrated and the yellow solid was
washed with acetone, filtered, and dried: yield 110 mg, 55%.
Preparation of (5R,6Z)-6-[(7-Methoxyimidazo[2,1-b][1,3]-
benzothiazol-2-ylmethylene)-7-oxo-4-thia-1-azabicyclo[3.2.0]-
hept-2-ene-2-carboxylic Acid Sodium Salt (15). Step 1:
Ethyl 7-methoxyimidazo[2,1-b]benzthiazole-2-carboxy-
late was prepared according to the procedure outlined for
compound 11 (step 1). Starting from 6-methoxy-2-amino
benzothiazole (27 g, 0.15 mol) and ethyl bromopyruvate (39.9
g, 0.2 mol), 24 g (43% yield) of ethyl 7-methoxyimidazo[2,1-b]-
benzthiazole-2-carboxylate was isolated as a brown solid. The
product was purified by silica gel column chromatography by
eluting with 1:1 ethyl acetate:hexane: MS (M + H)+ 277.
Step 2: 7-Methoxyimidazo[2,1-b]benzthiazole-2-metha-
nol was prepared according to the procedure outlined for
compound 11 (step 2). Starting from ethyl 7-methoxyimidazo-
[2,1-b]benzthiazole-2-carboxylate (12.5 g, 43.5 mmol) and
LiAlH4 solution (43.5 mL, 0.5 M solution in THF), 4.0 g (40%
yield) of the alcohol derivative was isolated as a brown solid:
MS (M + H)+ 235.
Step 3: 2-Formyl-7-methoxyimidazo[2,1-b]benzthia-
zole (20e) was prepared according to the procedure outlined
for compound 11 (step 4). Starting from 7-methoxyimidazo-
[2,1-b]benzthiazole-2-methanol (4.0 g 17 mmol) in refluxing
chloroform (300 mL) and active MnO2 (12 g, excess), 822 mg
(21% yield) of the aldehyde derivative was isolated as brown
solid: 1H NMR (CDCl3) δ 9.9 (s, 1H), 8.3 (s, 1H), 7.6 (d, 1H, J
) 9 Hz), 7.24 (d, 1H, J ) 2.4 Hz), 7.05 (m, 1H), 3.9 (s, 3H);
MS (M + H)+ 233.
Step 4: 4-Nitrobenzyl 6-[(acetyloxy)(7-methoxyimid-
azo[2,1-b][1,3]benzothiazol-2-yl)methyl]-6-bromo-7-oxo-4-thia-
1-azabicyclo[3.2.0]hept-2-ene-2-carboxylate (10e) was pre-
pared according to the procedure outlined for the preparation
of compound 11 (step 4). Starting from 2-formyl-7-methoxy-
imidazo[2,1-b]benzthiazole (20e) (822 mg, 3.5 mmol) and
(5R,6S)-6-bromo-7-oxo-4-thia-1-aza-bicyclo[3.2.0]hept-2-ene-2-
carboxylic acid 4-nitrobenzyl ester (9) (1.364, 3.54 mmol),
compound 10e was isolated as a mixture of diastereoisomers,
which were taken to next step. Prior to that, the compound
was purified by silica gel column chromatography by eluting
with 1:1 ethyl acetate:hexane: pale yellow amorphous solid;
yield 2.24 g, 95%; MS (M + H)+ 660.
Step 5: (5R,6Z)-6-[(7-methoxyimidazo[1,2-b][1,3]benzo-
thiazol-2-ylmethylene)]-7-oxo-4-thia-1-azabicyclo[3.2.0]-
hept-2-ene-2-carboxylic acid sodium salt (15) was pre-
pared according to the procedure outlined for the preparation
of compound 11 (step 5). Starting from 4-nitrobenzyl-6-[(acetyl-
oxy)(7-methoxyimidazo[2,1-b][1,3]benzothiazol-2-yl)methyl]-6-bro-
mo-7-oxo-4-thia-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylate (10e)
Step 2: 2-Formyl-7-methylimidazo[2,1-b]benzthiazole
(20d). To a stirred solution of ethyl 7-methylimidazo[2,1-b]-
benzthiazole-2-carboxylate (4.0 g, 15.38 mmol) in dry THF at
-78 °C was added DIBAL (1 M solution in toluene) (16.0 mL,
16 mmol). The reaction mixture was stirred at -78 °C and
slowly elevated to room temperature. The reaction mixture was
stirred at room temperature for 30 min and quenched with
saturated NH4Cl. The reaction mixture was extracted with
chloroform and washed well with water. The organic layer was
dried over anhydrous MgSO4, filtered, and concentrated. The
residue was purified by SiO2 column chromatography by
eluting with chloroform:methanol (20:1): brown solid; yield 800
1
mg, 24%; H NMR (CDCl3) δ 9.3 (s, 1H), 8.3 (s, 1H), 7.6-7.5
(m, 2H), 7.36 (s, 1H), 2.4 (s, 3H); MS (M + H)+ 217.
Step 3: 4-Nitrobenzyl 6-[(acetyloxy) (7-methylimidazo-
[2,1-b][1,3]benzothiazol-2-yl)methyl]-6-bromo-7-oxo-4-
thia-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylate (10d) was
prepared according to the procedure outlined for the prepara-
tion of compound 11 (step 4). Starting from 2-formyl-7-
methylimidazo[2,1-b]benzthiazole (20d) (432 mg, 2.0 mmol)
and (5R,6S)-6-bromo-7-oxo-4-thia-1-azabicyclo[3.2.0]hept-2-
ene-2-carboxylic acid 4-nitro-benzyl ester (9) (772 mg, 2.0
mmol), compound 10d was isolated as a mixture of diastereo-
isomers, which were taken to the next step. Prior to that, the
compound was purified by silica gel column chromatography
by eluting with 1:1 ethyl acetate:hexane: pale yellow amor-
phous solid; yield 400 mg, 31%; MS (M + H)+ 645.
Step 4: (5R,6Z)-6-[(7-Methylimidazo[1,2-b][1,3]benzo-
thiazol-2-ylmethylene)]-7-oxo-4-thia-1-azabicyclo[3.2.0]-
hept-2-ene-2-carboxylic acid sodium salt (14) was pre-
pared according to the procedure outlined for the preparation
of compound 11 (step 5). Starting from 4-nitrobenzyl 6-[(acetyl-
oxy)(7-methylimidazo[2,1-b][1,3]benzothiazol-2-yl)methyl]-6-

6566 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 26
Venkatesan et al.
(659 mg, 1.0 mmol), compound 15 was isolated as a yellow
amorphous solid after purification using an HP-21 reverse
phase column. The precipitate was filtered and washed with
H2O, MeCN, and acetone to give the title compound: yellow
and sodium methoxide (2.7 g, 51 mmol). To this was added
ethyl bromopyruvate (10 0.0 g) and the mixture stirred at room
temperature for 2 h. Then it was refluxed for 48 h. The reaction
mixture was cooled to room temperature and concentrated. The
residue was extracted with chloroform and washed well with
water. The product was purified by silica gel column chroma-
tography by eluting with 50% ethyl acetate:hexane: red
semisolid; yield 3.0 g 13%; MS (M + H)+ 237.
Step 2: 6,7-Dihydro-5H-cyclopenta[d]imidazo[2,1-b]-
[1,3]thiazole-2-methanol was prepared according to the
procedure outlined for the preparation of compound 11 (step
2). Starting from ethyl 6,7-dihydro-5H-cyclopenta[d]imidazo-
[2,1-b][1,3]thiazole-2-carboxylate (4.7 g, 20 mmol) and LiAlH4
solution (22 mL, 0.5 M solution in THF), 2.5 g (62% yield) of
the alcohol derivative was isolated as a brown solid; MS (M
+ H)+ 195.
Step 3: 2-Formyl-6,7-dihydro-5H-cyclopenta[d]imidazo-
[2,1-b][1,3]thiazole (24) was prepared according to the
procedure outlined for the preparation of compound 11 (step
3). Starting from 6,7-dihydro-5H-cyclopenta[d]imidazo[2,1-b]-
[1,3]thiazole-2-methanol (2.0 g, 10.3 mmol) in refluxing chlo-
roform:methanol (300:50 mL) and active excess MnO2 (12 g,),
900 mg (45%) of the aldehyde derivative was isolated as a
brown solid. 1H NMR (CDCl3) δ 9.9 (s, 1H), 7.9 (s, 1H), 2.97-
2.95 (m, 4H), 2.59-2.55 (m, 2H); MS (M + H)+ 193.
Step 4: 4-Nitrobenzyl (5R)-6-[(acetyloxy)(6,7-dihydro-
5H-cyclopenta[d]imidazo[2,1-b][1,3]thiazol-2-yl)-6-bromo-
7-oxo-4-thia-1-azabicyclo[3.2.0]hept-2-ene-2-carboxy-
late (10g) was prepared according to the procedure outlined
for the preparation of compound 11 (step 4). Starting from
2-formyl-6,7-dihydro-5H-cyclopenta[d]imidazo[2,1-b][1,3]-
thiazole (24) (600 mg, 3.1 mmol) and (5R,6S)-6-bromo-7-oxo-
4-thia-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid 4-ni-
trobenzyl ester (9) (1.2 g, 3 mmol), compound 10g was isolated
as a mixture of diastereoisomers, which were taken to the next
step. Prior to that, the compound was purified by silica gel
column chromatography by eluting with 1:1 ethyl acetate:
hexane: pale yellow amorphous solid; yield 850 mg, 45%; MS
(M + H)+ 620.
1
crystals; yield: 68 mg, 23%; mp 284 °C; H NMR (DMSO-d6)
δ 3.89 (s, 3H), 6.58 (s, 1H), 6.64 (s, 1H), 7.14 (s, 1H), 7.2 (dd,
1H, J ) 6.0 Hz), 7.75 (d, 1H, J ) 3.0 Hz), 8.03 (d, J ) 6.0 Hz,
1H), 8.62 (s, 1H); MS (M + H)+ 386, (M + Na)+ 407. Anal.
(C17H10N3O4S2Na) C, H, N.
Preparation of (5R,6Z)-6-[(5-Methylimidazo[2,1-b][1,3]-
benzothiazol-2-ylmethylene)-7-oxo-4-thia-1-azabicyclo-
[3.2.0]hept-2-ene-2-carboxylic acid sodium salt (16). Step
1: Ethyl 5-methylimidazo[2,1-b]benzthiazole-2-carboxy-
late was prepared according to the procedure outlined for
compound 11 (step 1). Starting from 4-methyl-2-aminoben-
zothiazole (8.0 g, 48.7 mmol) and ethyl bromopyruvate (14.0
g, 71.7 mmol), 6.0 g (45% yield) of ethyl 5-methylimidazo[2,1-
b]benzthiazole-2-carboxylate was isolated as a brown solid. The
product was purified by silica gel column chromatography by
eluting it with 1:1 ethyl acetate:hexane. MS (M + H)+ 261.
Step 2: 5-Methylimidazo[2,1-b]benzthiazole-2-metha-
nol was prepared according to the procedure outlined for the
preparation of compound 11 (step 2). Starting from ethyl
5-methylimidazo[2,1-b]benzthiazole-2-carboxylate (5.2 g, 20
mmol) and LiAlH4 solution (22 mL, 0.5 M solution in THF), 3
g (69% yield) of the alcohol derivative was isolated as a brown
solid; MS (M + H)+ 219.
Step 3: 2-Formyl-5-methylimidazo[2,1-b]benzthiazole
(20f) was prepared according to the procedure outlined for the
preparation of compound 11 (step 3), starting from 5-meth-
ylimidazo[2,1-b]benzthiazole-2-methanol (2.0 g, 9.1 mmol) in
refluxing chloroform:methanol (300:50 mL) and active MnO2
(12 g, excess): brown solid; yield 700 mg 35%; 1H NMR (CDCl3)
δ 9.9 (s, 1H), 8.5 (s, 1H), 7.5 (d, 1H, J ) 8 Hz), 7.3-7.1 (m,
2H), 2.7 (s, 3H); MS (M + H)+ 217.
Step 4: 4-Nitrobenzyl 6-[(acetyloxy) (5-methylimidazo-
[2,1-b][1,3]benzothiazol-2-yl)methyl]-6-bromo-7-oxo-4-
thia-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylate (10f) was
prepared according to the procedure outlined for the prepara-
tion of compound 11 (step 4). Starting from 2-formyl-5-
methylimidazo[2,1-b]benzthiazole (20f) (432 mg, 2.0 mmol)
and (5R,6S)-6-bromo-7-oxo-4-thia-1-aza-bicyclo[3.2.0]hept-2-
ene-2-carboxylic acid 4-nitrobenzyl ester (9) (770 mg, 2 mmol),
compound 10f was isolated as a mixture of diastereoisomers,
which were taken to the next step. Prior to that, compound
was purified by silica gel column chromatography by eluting
with 1:1 ethyl acetate:hexane: pale yellow amorphous solid;
yield 270 mg, 20%; MS (M + H)+ 644.
Step 5: (5R,6Z)-6-(6,7-Dihydro-5H-cyclopenta[d]imi-
dazo[2,1-b][1,3]thiazol-2-ylmethylene)-7-oxo-4-thia-1-
azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid sodium salt
(17) was prepared according to the procedure outlined for the
preparation of compound 11 (step 5). Starting from 4-nitroben-
zyl (5R)-6-[(acetyloxy)(6,7-dihydro-5H-cyclopenta[d]imidazo-
[2,1-b][1,3]thiazol-2-yl)-6-bromo-7-oxo-4-thia-1-azabicyclo[3.2.0]-
hept-2-ene-2-carboxylate (850 mg, 1.37 mmol), compound 17
was isolated as a yellow amorphous solid after purification
using an HP-21 reverse phase column. The yellow solid was
washed with acetone, filtered, and dried: yellow crystals; yield
Step 5: (5R,6Z)-6-[(5-Methylimidazo[1,2-b][1,3]benzo-
thiazol-2-ylmethylene)]-7-oxo-4-thia-1-azabicyclo[3.2.0]-
hept-2-ene-2-carboxylic acid sodium salt (16) was pre-
pared according to the procedure outlined for the preparation
of compound 11 (step 5). Starting from 4-nitrobenzyl-6-
[(acetyloxy)(5-methylimidazo[2,1-b][1,3]benzothiazol-2-yl)-
methyl]-6-bromo-7-oxo-4-thia-1-azabicyclo[3.2.0]hept-2-ene-2-car-
boxylate (10f) (400 mg, 0.62 mmol) compound 16 was isolated
as yellow an amorphous solid after purification using an HP-
21 reverse phase column. The precipitate was filtered and
washed with H2O, MeCN, and acetone to give the title
1
138 mg, 29%; mp 192°C; H NMR (DMSO-d6) δ 2.51 (m, 4H),
3.01 (m, 2H), 8.2 (s, 1H), 7.1 (s, 1H), 6.55 (s, 1H), 6.4 (s, 1H);
MS (M + H + Na)+ 367. Anal. (C15H10N3O3S2Na) C, H, N.
Acknowledgment. X-ray data for the SHV-1 com-
plex were collected at the Institute of Materials Science
of the University of Connecticut. Data for GC1 were
obtained at the Cornell High Energy Synchrotron
Source (CHESS, supported by NSF Award DMR-
9311772), using the Macromolecular Diffraction facility
(MacCHESS, supported by NIH Award RR-01646).
Work in Cleveland was supported by a Department of
Veterans Affairs Merit Review Award to R.A.B. The
authors thank the members of Wyeth’s Chemical Tech-
nology Department for analytical and spectral deter-
mination and Drs. David Shlaes and Beth Rasmussen
for stimulating discussions and encouragement.
1
compound: yellow crystals; yield 60 mg, 24%; mp 192 °C; H
NMR (DMSO-d6) δ 2.1 (s, 3H), 6.53 (s, 2H), 7.1 (s, 1H), 7.34-
7.36 (m, 2H), 7.85 (m, 1H), 8.58 (s, 1H); MS (M + Na)+ 392.
Anal. (C17H10N3O3S2Na) C, H, N.
Preparation of (5R,6Z)-6-(6,7-Dihydro-5H-cyclopenta-
[d]imidazo[2,1-b][1,3]thiazol-2-ylmethylene)-7-oxo-4-thia-
1-azabicyclo[3.2.0]hept-2-ene-2-carboxylic Acid Sodium
Salt (17). Step 1: Preparation of Ethyl 6,7-Dihydro-5H-
cyclopenta[d]imidazo[2,1-b][1,3]thiazole-2-carboxy-
late. A mixture of 2-chlorocyclopentanone (11.8 g, 100 mmol)
and thiourea (8.0 g 101 mmol) was refluxed in ethanol:THF
(1:2) for 16 h. The reaction mixture was cooled to room
temperature and the separated white solid was filtered (9.0 g
separated). This was dissolved in anhydrous ethanol (100 mL)
Appendix
Abbreviations. Cymal-6, cyclohexyl-(n-hexyl)-â-D-
maltoside; HEPES, N-(2-hydroxyethyl)piperazine-N′-(2-
ethanesulfonic acid); IC50, concentration of a drug that

Novel Tricylic 6-Methylidene Penems
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 26 6567
Derivatives as Inhibitors of Class-A and Class-C â-Lactamase
I. Bioorg. Med. Chem. Lett. 1999, 9, 991-996. (d) Bitha, P.;
Zhong, L.; Francisco, G. D.; Yang, Y.; Petersen, P. J. B.; Lin, Y.
I. 6-(1-Hydroxalkyl) Penam Sulfone Derivatives as Inhibitors of
Class-A and Class-C â-Lactamase II. Bioorg. Med. Chem. Lett.
1999, 9, 997-1002.
will result in 50% inhibition of the target enzyme; LDA,
lithium diisopropylamide; LiHMDS, lithium hexameth-
yldisilazide; MIC, concentration of the lowest dilution
of the drug in which bacterial growth is absent as
detected by the unaided eye; MPD, 2-methyl-2,4-pen-
tanediol; PEG, poly(ethyleneglycol); rmsd, root-mean-
square deviation.
(10) Sandanayaka, V. P.; Prashad, A. S.; Yang, Y.; Williamson, T.;
Lin, Y. I.; Mansour, T. S. Spirocyclopropyl â-Lactams as Mech-
anism Based Inhibitors of Serine â-Lactamases. Synthesis by
Rhodium-Catalyzed Cyclopropanation of 6-Diazo Penicillanate
Sulfones. J. Med. Chem. 2003, 46, 2569-2571.
(11) (a) Abe, T.; Ushirogochi, H.; Yamamura, I.; Kumagai, T.;
Mansour, T. S.; Venkatesan, A. M.; Agarwal, A.; Petersen, P.
J.; Weiss, W. J.; Lenoy, E.; Yang, Y.; Shlaes, D. Novel Penem
Bearing a Methylidene Linkage as a Broad Spectrum â-Lacta-
mase Inhibitors. Poster presented at 43rd ICCAC, Chicago, IL,
2003, no. 538. (b) Tabei, K.; Feng, X.; Venkatesan, A. M.; Abe,
T.; Hideki, U.; Mansour, T. S.; Siegel, M. M., Mechanism of
Inactivation of â-Lactamases by Novel 6-Methylidene Penems
Elucidated Using Electrospray Ionization Mass Spectrometry.
J. Med. Chem. 2004, 47, 3674-3688.
(12) Nukaga, M.; Abe, T.; Venkatesan, A. M.; Mansour, T. S.; Bonomo,
R. A.; Knox, J. R. Inhibition of Class-A and Class-C â-Lacta-
mases by Penems: Crystallographic Structures of Novel 1,4-
Thiazepine Intermediate. Biochemistry 2003, 42, 13152-13159.
(13) Abe, T.; Matsunaga, H.; Mihira, A.; Sato, C.; Ushirogochi, H.;
Takasaki, T.; Venkatesan, A. M.; Mansour, T. S. Preparation of
Heteroaryl-6-alkylidene-penems as â-Lactamase Inhibitors for
Use as Antibacterial Agents, 2003, WO-2003093277.
(14) (a) Osborne, N. F.; Atkins, R. J.; Broom, N. J. P.; Coulton, S.;
Harbridge, J. B.; Harris, M. A.; Francois, I. S.; Walker, Synthesis
of (5R)-(Z)-6-(1-Methyl-1,2,3-triazol-4-ylmethylene) Penem-3-
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from 6-Aminopenicillanic Acid G. J. Chem. Soc Perkin Trans.
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Supporting Information Available: Crystallographic
refinement data (Table 1), X-ray data collection (Table 2), and
elemental analysis data for compounds 11-17. This mate-
rial is available free of charge via the Internet at http://
pubs.acs.org.
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