5,5,6-Fused tricycles bearing imidazole and pyrazole 6-methylidene penems as broad-spectrum inhibitors of β-lactamases

Article abstract of DOI:10.1016/j.bmc.2007.11.006

β-Lactamases are serine- and metal-dependent hydrolases, produced by the bacteria as defense against β-lactam antibiotics. Commercially available inhibitors such as clavulanic acid, sulbactam, and tazobactam, which are currently used in the hospital setti

Full text of DOI:10.1016/j.bmc.2007.11.006

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 1890–1902
5,5,6-Fused tricycles bearing imidazole and pyrazole 6-methylidene
penems as broad-spectrum inhibitors of b-lactamases
Aranapakam M. Venkatesan,a,* Atul Agarwal,a Takao Abe,b Hideki Ushirogochi,b
Mihira Ado,b Takasaki Tsuyoshi,b Osvaldo Dos Santos,a Zhong Li,a Gerry Francisco,a
Yang I. Lin,a Peter J. Petersen,c Youjun Yang,c William J. Weiss,c
David M. Shlaesc and Tarek S. Mansoura
aWyeth Research, Chemical and Screening Sciences, 401 N. Middletown Road, Pearl River, NY 10965, USA
bWyeth KK, Saitama, Japan
cDepartment of Infectious Disease and Biological Technologies, 401 N. Middletown Road, Pearl River, NY 10965, USA
Received 2 August 2007; revised 25 October 2007; accepted 1 November 2007
Available online 5 November 2007
Abstract—b-Lactamases are serine- and metal-dependent hydrolases, produced by the bacteria as defense against b-lactam antibi-
otics. Commercially available inhibitors such as clavulanic acid, sulbactam, and tazobactam, which are currently used in the hospital
settings, have reduced activity against newly emerging b-lactamases. Bacterial production of diverse b-lactamases including class-A,
class-C, and ESBLs has motivated several research groups to search for inhibitors with a broader spectrum of activity. Previously,
several novel 6-methylidene penems bearing, [5,5] [5,6] and [5,5,5] heterocycles have been synthesized in our laboratory and were
shown to be potent and broad-spectrum b-lactamase inhibitors. As a continuation of our previous work and in order to extend the
structure–activity relationships, in this paper, we describe herein the synthesis and in vitro, in vivo activities of several novel 5,5,6-
fused tricyclic heterocycles attached to the 6-methylidene penem core. The compounds presented in the current paper are potent and
broad-spectrum inhibitors of the TEM-1 and AmpC b-lactamases. In combination with piperacillin, their in vitro activities showed
enhanced susceptibility to class A- and C-resistant strains studied in various bacteria. Some of the newly synthesized compounds
such as 12a–c were shown to have in vivo activity in the acute lethal infection model against TEM-1 producing organisms. The
5,5,6-fused heterocyclic ring cores such as 21, 25, and 35 reported here are hitherto unknown in the literature.
Ó 2007 Elsevier Ltd. All rights reserved.
1. Introduction
bacteria to overcome the effects of the b-lactamase
inhibitors. The currently available inhibitors such as
clavulanic acid 1, sulbactam 2, and tazobactam 3
(Fig. 1) are effective inhibitors against class A producing
organisms; but are not effective against either class C or
class B producing organisms.3a–c Among these classes,
the most problematic b-lactamases are the class C en-
zymes.2 Bacterial genera such as Citrobacter, Enterobac-
ter, Hafnia, Pseudomonas, Morganella, Providencia, and
Serratia are known to express class C enzymes and con-
fer resistance to the above-mentioned commercial prod-
ucts as well as extended-spectrum cephalosporins.4 In
addition to class C producing bacteria, there are strains
which produce multiple enzymes of class A as well as ex-
tended-spectrum b-lactamases (ESBLs),5,6a which are of
great concern in terms of their incidence and treatment
options.6b Thus, the treatment of infection caused by
strains producing ESBLs and or AmpC b-lactamases
in hospital setting is a therapeutic challenge. Therefore,
Development of resistance to b-lactam antibiotics by
various bacteria severely limits the use of penicillins
and cephalosporins in the treatment of bacterial infec-
tion.1 The most significant known mechanism related
to the development of bacterial resistance to the b-lac-
tam antibiotics is the production of class A, class B,
and class C b-lactamases.2 These enzymes hydrolyze
the b-lactam ring of the antibiotics and render them
inactive. To overcome this, co-administering b-lacta-
mase inhibitors with a b-lactam antibiotic is a general
practice in the hospital setting. However, alterations
and mutations within the b-lactamases have allowed
Keyword: Beta-Lactamase Inhibitors.
*
Corresponding author. Tel.: +1 845 602 4023; fax: +1 845 602
5561; e-mail: venkata@wyeth.com
0968-0896/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.11.006

A. M. Venkatesan et al. / Bioorg. Med. Chem. 16 (2008) 1890–1902
1891
O
S
O
O
S
O
OH
O
N
N
N
N
N
N
O
O
O
COONa
COONa
COONa
2
3
1
S
O
R
N
N
N
N
S
S
S
N
O
COO-
O
COO-
O
NH
COONa
NH
O
O
4-6
Ser
Ser
7
8
NH
N
O
S
N
6 = R =
4 = R =
N
5 = R =
N
N
N
Figure 1. Commercial and previously reported b-lactamase inhibitors and 1,4-thiazepine complexes.
a continuous search for novel and broad-spectrum b-
lactamase inhibitors is being actively pursued in several
laboratories.7–15
pared by a novel two-step aldol condensation and reduc-
tive elimination procedures as described previously.20
The pivotal starting material, namely (5R,6S)-6-bro-
mo-7-oxo-4-thia-1-azabicyclo(3.2.0)hept-2-ene-2-car-
boxylic acid-4-nitrobenzyl ester 10, required for this
procedure was prepared from the commercially avail-
able 6-aminopenicilanic acid (6-APA) 9 by using a mod-
ified multi-step procedure.21,22 Compound 10 was
reacted with the appropriately substituted aldehydes
20, 21, 25, 27, and 33–35 in the presence of triethylamine
and anhydrous MgBr2 (Scheme 1).
We have recently shown that several 6-methylidene pen-
ems bearing a monocyclic ring,16a a 6,5-fused bicyclic
heterocycle or 5,5,5-fused tricyclic heterocycle16b are
effective broad-spectrum b-lactamase inhibitors.16c
Compounds such as 4–6 (Fig. 1) are of particular inter-
est because of their broad-spectrum b-lactamase inhibi-
tory activity. These compounds were synthesized on the
basis of both modeling experiments and mechanistic
considerations and were found to be potent both
in vitro and in vivo. High resolution crystallographic
structures of 5 and 6 with SHV-1 (class A) and GC1 en-
zymes revealed the formation of ring expanded seven-
membered dihydro[1,4]thiazepines 7 and 8, respectively.
During these experiments, the stereochemistry of the C-
7 carbon atom was also determined.17,18 Subsequently,
the formation of ring expanded dihydro[1,4]thiazepines
7 and 8 was also supported by electrospray-ionization
mass spectrometry.19 As mentioned above inhibitors
such as 6 displayed good in vitro activities against
ESBLs and class C expressing organisms as well as
in vivo activity.16b This inhibitor 6 bears a 5,5,5-fused
tricyclic system as a part of the 6-methylidene penem
core. Hence, in order to extend the structure–activity
relationships, the terminal cyclopentyl moiety in com-
pound 6 was replaced with more lipophilic ring systems
as in examples 12a, 12d, and 12e and relatively polar
ring system as depicted in example 12b, 12c, 12f, and
12g. The 5,5,6-fused heterocyclic ring cores such as 21,
25, and 35 described here are hitherto unknown in the
literature. The structure–activity relationship in the ser-
ies and the in vitro, in vivo data are presented in the cur-
rent paper.
The intermediate aldol products were trapped as their
respective bromoacetoxy derivatives 11a–g. Reaction
of bromoacetoxy derivatives 11a–g with activated zinc
in phosphate buffer at pH 6.5 resulted in the introduc-
tion of the double bond with the ‘Z’ geometry. During
this process, the carboxyl functionality also underwent
deprotection resulting in the formation of their respec-
tive sodium salts. In the case of compound 11f, about
5% quantity of the ‘E’ isomer 13 was also formed. The
sodium salts of 12a–g and 13 were obtained after purifi-
cation on HP-21 resin (80 mL, Mitsubishi Kasei Co.,
Ltd) by reverse-phase column chromatography.
The aldehydes intermediates 20, 21, 25, 27, and 33–35
required to synthesize all the final compounds were pre-
pared as described in the following section. Aldehydes
20 and 21 required to prepare compounds 12a and
12b, were synthesized starting from the 2-chlorocyclo-
hexanone 14 and 3-chloro tetrahydro-4H-pyran-4-one
15, respectively, by a four-step process as outlined in
Scheme 2. The a-chloro ketone derivatives 14 and 15
were reacted with thiourea to afford the 2-aminothiazole
derivatives 16 and 17. The intermediates 16 and 17 were
reacted with ethyl bromopyruvate in dimethoxyethane.
The resulting tricyclic esters 18 and 19 were reduced to
their corresponding alcohols using lithium aluminum
hydride and then oxidized to their respective aldehydes
20 and 21 using activated manganese dioxide in reflux-
ing chloroform for 48 h (Scheme 2). The aldehyde 25 re-
quired to synthesize compound 12c was prepared by a
2. Chemistry
The tricyclic heterocycle bearing 6-methylidene penem
carboxylic acid sodium salts 12a–g (Scheme 1) were pre-

1892
A. M. Venkatesan et al. / Bioorg. Med. Chem. 16 (2008) 1890–1902
H2N
O
Br
S
S
PNB =
N
N
O
COOH
COOPNB
NO2
9
10
6-APA
RCHO/ a,b
O
R
(a) MgBr2/ Et3N/ THF:Acetonitrile
(b) Acetic anhydride/ 0o C- -20oC
(c) Activated Zinc/ 6.5 pH Phosphate
buffer/ THF: Acetonitrile/ RT/ 0.1NaOH
R
O
O
O
Br
c
S
S
N
N
COONa
N
COOPNB
12a-g
11a-g
S
S
N
S
N
N
N
O
N
N
N
R =
N
N
11b,12b
11c,12c
11a,12a
6
O
N
O
N
O
O
N
N
O
O
N
N
N
N
N
N
E and Z isomers
11g,12g
11e, 12e
11d, 12d
11f,12f
S
O
N
N
N
O
COONa
13
O
Scheme 1. General method to prepare compounds 12a–g.
CHO
COOEt
c,d
N
O
N
N
a
N
b
N
NH2
X
X
X
X
S
S
S
Cl
X = CH2 , 14
X = O, 15
X = CH2 , 16
X = O, 17
X = CH2 , 18
X = O, 19
X = CH2 , 20
X = O, 21
Scheme 2. General method to prepare aldehydes 20 and 21. Reagents and conditions: (a) Thiourea, EtOH, THF, reflux; (b) BrCH2COCOOEt,
DME, reflux; (c) LiAlH4, THF, 0 °C; (e) MnO2, CHCl3, reflux.
similar sequence of reactions starting from compound
23 and ethyl bromopyruvate (Scheme 3). Compound
23 required for this transformation was prepared by
the hydrogenation of 3-amino-1,2,4-triazolopyridine
2223a with Pd/C at room temperature at 40 psi pressure
(Scheme 3). The tricyclic aldehyde 27 was synthesized
starting from 1-methyl-2-amino benzimidazole 26 as
indicated in Scheme 4. The tricyclic aldehydes 33–35
were synthesized by reacting ethyl-5-hydroxy-1H-pyra-
zole-3-carboxylate 2823b and corresponding a-chloro
cyclic ketones 14, 15, and 29 in the presence of K2CO3
in boiling acetonitrile as indicated in Scheme 5.
ability.24 b-Lactamase inhibitory activities were deter-
mined spectrophotometrically as described by Bush
et al.24b 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 antibiotic (piperacillin)
and antibiotic plus inhibitors (compounds 12a–g and 13)
were determined by the broth microdilution method as
recommended by the Clinical Laboratory and Standards
Institute (CLSI)25 using Mueller–Hinton II broth
(MHBII, BBL Cockeysville, MD). Microtiter plates
containing serial dilutions of each antimicrobial agent
or antimicrobial agent plus inhibitor were inoculated
with each organism to yield the appropriate density
(105 CFU/ml) in a 100-ll final volume. The plates were
3. Biology
All the compounds synthesized were tested in vitro
against TEM-1 and Amp-C enzymes for their inhibitory

A. M. Venkatesan et al. / Bioorg. Med. Chem. 16 (2008) 1890–1902
1893
N
N
N
N
N
N
b
a
COOEt
N
NH2
NH2
N
N
N
23
22
24
c,d
N
N
CHO
N
N
25
Scheme 3. Synthesis of aldehyde 25. Reagents and conditions: (a) H2, Pd, C, 40 psi, RT; (b) BrCH2COCOOEt, DME, sealed tube, reflux ; (c)
DIBAL, THF, ꢀ78 to 0 °C; (e) MnO2, CHCl3, reflux.
Table 1. In vitro activity of compounds 6, 13, and 12a–g against
different b-lactamases, IC50 (nM)
N
N
N
N
a,b,c
NH2
N
Compound
Class-A (IC50) nM
Class-C (IC50) nM
Amp-C
CHO
TEM-1
Imi-1
26
27
Tazobactam
6
12a
100
1.4
1.9
2.8
2.4
24
8
NA
72
84,000 300
Scheme 4. Synthesis of aldehyde 27. Reagents and conditions: (a)
BrCH2COCOOEt, DME, reflux ; (b) LiBH4, THF, MeOH, reflux; (c)
MnO2, CHCl3, reflux.
2.1
0.62
1.5
1.4
16
5.5
4
33
12b
12c
100
14
12d
12e
50
incubated for 18–24 h at 35 °C in ambient air. The min-
imum inhibitory concentration (MIC) for all isolates
was defined as the lowest concentration of antimicrobial
agent that completely inhibited the growth of the organ-
isms as detected by the unaided eye. The MIC values
presented in this article were determined for piperacillin
(PIP) alone, the inhibitor alone, and the inhibitor in
combination with piperacillin at a constant concentra-
tion of 4 lg/mL of the inhibitor. The potent compounds
in this assay were taken to an in vivo acute lethal infec-
tion 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
intraperitoneally with a lethal dose (10–100 LD50) of
the pathogen. The same group was injected intrave-
nously with piperacillin alone or piperacillin combined
with inhibitor (compounds 12a–g and 13) at a ratio of
4:1 (Pip:Inhibitor). The seven days survival ratios from
three separate tests were pooled for determination of
median effective dose (ED50) by probit analysis.
4
160
140
2900
98
(Z)-12f
(E)-13
12g
4.3
150
1.2
53
4
zymes and their IC50 values are enlisted in Table 1. In all
our experiments, tazobactam was used as the standard
and the comparator. As can be seen from Table 1, all
the newly synthesized inhibitors 12a–g with ‘Z’ olefin
geometry are potent inhibitors of both TEM-1 and
AmpC enzymes with IC50 values of 1.2–24 and 0.62–
16 nM, respectively. As compared to tazobactam, all
the newly synthesized inhibitors were about 4- to 90-fold
more potent against TEM-1 and about 5000- to 84,000-
fold more active against AmpC enzymes. All the inhib-
itors are modestly potent against Imi. Among the seven
6-methylidene penems reported here, compounds 12a–d
represent the ‘imidazole’-fused tricyclic compounds and
the remaining compounds bear the pyrazole ring system
as a part of their tricyclic appendage. It was shown ear-
lier16b that compound 6, which bears the imidazole ring,
has broad spectrum of activities. Hence, in order to ex-
tend the structure-activity relationships the cyclopentyl
moiety in compound 6 was replaced with more lipophilic
cyclohexyl (example 12a) and relatively polar pyran ring
system (example 12b). These modifications did not alter
the in vitro activity against TEM-1. However, the cyclo-
hexyl ring containing compound 12a is about 3- to 4-
fold more potent than 6 against class C enzyme. Com-
4. Structure–activity relationship
All the newly synthesized 6-methylidene penem inhibi-
tors bearing tricyclic heterocycles (6, 12a–g, and 13)
are listed in Table 1.
The in vitro enzyme inhibition studies were carried out
against TEM-1, Imi-1 (class A), and AmpC (class C) en-
O
O
O
COOEt
a
b, c
COOEt
CHO
N
X
N
X
X
+
N
N
N
Cl
HO
N
H
X = CH2, 14
X = O, 15
X = NCOOEt, 29
X = CH2, 30
X = O, 31
X = NCOOEt, 32
X = CH2, 33
X = O, 34
X = NCOOEt, 35
28
Scheme 5. General method to prepare compounds 33–35. Reagents and conditions: (a) K2CO3, CH3CN, reflux; (b) LiBH4, THF, 0 °C; (c) MnO2,
CHCl3, reflux.

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A. M. Venkatesan et al. / Bioorg. Med. Chem. 16 (2008) 1890–1902
pound 12a is 2-fold more potent against Imi when com-
pared to the lead compound 6. When the thiazole ring in
examples 6, 12a, and 12b was replaced with 1,2,4-tria-
zole ring system as in example 12c, the potency remained
almost identical to 12a and 12b against TEM-1 and
AmpC, and exhibited excellent potency against Imi-1.
The potency decreased as the terminal saturated rings
of 6 and 12a–12c were replaced with a phenyl. Thus,
compound 12d is almost 10-fold less potent against
TEM-1 than compounds 6 and 12a–c. 6-Methylidene
penem derivatives 12e–g bearing a pyrazole ring dis-
played good potency against TEM-1 and AmpC en-
zymes. The imidazole fused tricyclics 6 and 12a–c are
more potent inhibitors of TEM-1 and AmpC as com-
pared to the pyrazole fused tricyclic bearing penem
inhibitors 12e–g. During the preparation of example
12f, a small quantity of the corresponding ‘E’ isomer
13 was isolated and tested for in vitro activity against
all of the four enzymes. As can be seen in Table 1, the
potency of 13 is >50-fold weaker against TEM-1, as
compared to its corresponding ‘Z’ isomer 12f. This de-
crease in the potency can be explained by modeling of
12f and 13 at the TEM-1 binding site26 (Fig. 2).
is important to note that, when tested alone, both the
tazobactam and the newly synthesized inhibitors (6,
12a–g, and 13) did not exhibit any antibacterial activity
(data not shown) against the isolates used in this study,
which established the lack of inherent antibacterial
activity. The newly synthesized compounds 6, 12a–g,
and 13, and tazobactam, when combined with piperacil-
lin at a constant 4 lg/mL concentration, reduced the
MIC values (except 12d and 13) of piperacillin
(>64 lg/mL) against TEM-1 producing isolates. How-
ever, it is evident from Table 2 that even though the
tazobactam plus piperacillin combination has synergy
against class A producing isolates, this combination is
less effective against most class C b-lactamase producing
organisms (S. marcescus is an exception). The addition
of the newly synthesized inhibitors such as 6, 12a and
12b effectively reduced the MIC value and restored the
susceptibility to piperacillin for the class C producing
organisms. Among the newly synthesized inhibitors,
the imidazole-fused tricyclic derivatives attached to the
6-methylidene penem moiety 12a–c are more effective
than the pyrazole ring-containing compounds such as
12e–g. Compound 12g, in spite of its low IC50 values,
exhibited poor whole cell activity. It is noteworthy that
all the newly synthesized compounds reported here
exhibited good aqueous solution state stability in the
pH range of 7–8.5.
Our optimized protein:ligand complex models (TEM-
1:12f, TEM-1:13, AmpC:12f, and AmpC:13) indicated
a preference for the Z isomer over E isomer (Fig. 2).
b-Lactamase inhibition involves positioning of the lac-
tam oxygen in the oxyanion hole, and subsequently, its
acylation. b-Lactam and the tricyclic head groups are
co-planar in the predicted bioactive conformations of
12f and 13. The shape of the Z isomeric form allows
the tricyclic head group to be directed toward the open-
ing of the binding site and still makes good H-bonding
and vdW contacts with the binding site amino acids.
This orientation of the tricyclic head group allows the
lactam oxygen to be positioned deep into the oxyanion
hole and facilitates nucleophilic attack by the active ser-
ine. In contrast, as shown in Figure 2B the E isomer
places the tricyclic head group deeper into the binding
site, and, thereby, pulls the lactam oxygen away from
the oxyanion hole, making the acylation more difficult
and resulting in weaker enzyme inhibition. It is also
noteworthy to see from Figure 2B that the E isomer is
less firmly embedded in the oxyanion hole and makes
fewer contacts with the binding pocket residues as com-
pared to the corresponding Z isomer (12f). These molec-
ular modeling observations are consistent for the AmpC
binding site as well.
6. In vivo efficacy
The in vivo efficacy data (ED50 values) of piperacillin
and the most potent inhibitors 12a–c (4:1 ratio) are
listed in Table 3. These data were generated in a murine
acute lethal infection model with Escherichia coli LSU
80-8 (a TEM-1 producing organism).16b In all these
experiments, tazobactam was used for comparison.
When piperacillin was administered alone (IV), it did
not have efficacy (ED50 > 128 mg/kg) against E. coli
LS80-8. However, when piperacillin was combined with
the newly synthesized inhibitors 6 and 12a–c, reduced
ED50 values in the range of 19–32 mg/kg were observed.
In the same experiment, tazobactam demonstrated sim-
ilar effectiveness against E. coli LSU 80-8.
7. Conclusions
Guided by the mechanistic understanding, computa-
tional chemistry and as an extension to our previous
work, several novel tricyclic heterocycle-substituted 6-
methylidene penem molecules were synthesized. Many
of these molecules are potent and broad-spectrum b-lac-
tamase inhibitors. During the course of this investiga-
tion, several novel tricyclic aldehydes 20, 21, 25, 27,
and 33–35 were synthesized. The newly synthesized 6-
methylidene penem derivatives 6, 12a–c, and 12e–g have
a broader spectrum of activity than any of the currently
available inhibitors on the market. They were shown to
have good in vitro activity against both class A and class
C enzymes. In vitro antimicrobial susceptibility testing
studies demonstrated that, the newly synthesized com-
pounds 12a–c, 12e, and 12f reduced the MIC values of
5. In vitro antibacterial activity
The in vitro effectiveness of the newly synthesized com-
pounds was evaluated in the cell-based assay by measur-
ing their minimum inhibitory concentration (MIC).
These data are summarized in Table 2, which also lists
the expressing enzyme and its class. In all these
experiments, piperacillin was combined with the newly
synthesized inhibitors and tested against different piper-
acillin-resistant bacteria (MIC > 64 lg/mL) expressing
various b-lactamases. The piperacillin + tazobactam
combination was used for comparison. At the onset, it

A. M. Venkatesan et al. / Bioorg. Med. Chem. 16 (2008) 1890–1902
1895
Figure 2. Nitrogen, oxygen, carbon, sulfur and hydrogen atoms are shown with blue, red, green, yellow and white colors, respectively. Carbons of
compound 13 are colored magenta. The inter-molecular hydrogen bonds are shown with dotted yellow lines. There is a salt bridge interaction
between carboxylate function of the ligand and lysine sidechain. (A) 12f docked in TEM-1 binding site. (B) Compound 13 docked in TEM-1 binding
site. This E isomer is less firmly embedded in the oxyanion hole and makes fewer contacts with the binding pocket residues as compared to the
corresponding Z isomer (12f). (C) Overlay of predicted bioactive conformations of 12f and 13 demonstrating how the tricyclic head groups are
oriented differently when superimposed by the b-lactam ring. (D) Compound 12f docked in AmpC binding site.
piperacillin to the susceptible range for the class A and
some of the class C producing organisms. In particular,
compounds, 12a–c, which bear an imidazole ring in the
tricyclic ring skeleton, when combined with piperacillin
rendered the organisms listed here susceptible. The same
compounds 6 and 12a–c in vivo enhanced the activity of
piperacillin against E. coli LSU 80–8, a TEM-1 produc-
ing organism. Further investigations in various other

1896
A. M. Venkatesan et al. / Bioorg. Med. Chem. 16 (2008) 1890–1902
Table 2. In Vitro Antimicrobial activity of Piperacillin (PIP) and the combined newly synthesized inhibitors (12a–g, 13, 6) or Tazobactam at a
constant 4 lg/mL concentration
Organisms and their enzyme expression
Tazo
6
12a
12b
12c
12d
12e
12f
13
12g
E. coli GC 2844
2
2
2
2
2
4
2
2
2
2
E. coli GC 2847 (TEM-1)
E. coli GC 2920 (IRT-2)
E. coli GC 2883 (OXA-10 + PSE-2)
E. coli GC 2894 (AmpC)
E. coli GC 2905 (P99)
2
4
8
4
8
>64
4
16
4
32
2
>64
8
32
4
4
2
2
2
2
1
32
2
2
4
2
64
32
8
8
16
32
8
2
4
2
2
32
32
4
16
16
8
64
32
64
<0.06
>64
16
>64
64
64
64
2
64
2
4
4
8
2
32
16
32
64
E. coli GC 2906 (Imi-1)
E. coli GC 2804 (imp)
E. coli GC 2805 (CcrA)
2
<0.06
2
2
64
<0.06
>64
16
>64
8
<0.06
>64
2
<0.06
>64
16
32
8
<0.06
>64
16
16
8
<0.06
>64
32
<0.06
>64
16
64
32
32
32
2
<0.06
>64
16
64
32
16
32
2
0.12
>64
32
>64
8
E. coli GC 2253 (IRT-2)
E. cloacae GC 1477 (AmpC)
E. cloacae GC 4142 (AmpC)
E. cloacae GC 6991 (AmpC)
E. cloacae GC 1475 (P99)
S. marcescens GC 1781 (Sme-1 + AmpC)
P. aeruginosa GC 1764 (AmpC)
S. maltophilia GC 1712 (L1)
S. marcescens GC 4132 (AmpC)
E. coli GC 2203
>64
64
16
4
>64
>64
32
>64
>64
32
>64
64
16
4
16
8
16
8
16
2
64
32
0.50
>64
>64
64
1
1
1
0.50
32
>64
1
2
64
4
64
2
2
4
64
>64
8
32
>64
8
>64
>64
8
>64
2
>64
2
>64
2
>64
16
>64
32
1
0.50
2
2
<0.06
4
1
0.25
2
64
2
16
4
16
2
64
2
16
S. aureus GC 2216
2
2
Table 3. In vivo efficacy (IV)a of select compounds in murine acute
lethal infection model with E. coli LSU 80-8, a TEM-1 producing
organism
(Fuji Silysia). Thin-layer chromatography (TLC) was
performed on Merck PLC prescored plates 60F254. 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 commercial sources and were used without further
purification.
Compound
ED50 (mg/kg)
Tazobactam
20
12a
12b
12c
6
23.3
25.3
32.5
19
a Piperacillin: inhibitor = 4:1; ED50 value of piperacillin alone
>128 mg/kg.
9. Molecular modeling
The lowest energy conformations of compounds 11f and
12f were manually docked in TEM-1 (RCSB PDB code:
1BTL) and AmpC (RCSB PDB code: 1FSW) as de-
scribed previously.16a,26 Hydrogen atoms were added
and the four models were soaked in a box of water
(TIP3P) applying periodic boundary conditions using
MOE.26 The complexes were optimized by energy mini-
mizing at a constant dielectric of 1 with MMFF94 force-
field in MOE.
organisms of related compounds are in progress to
determine their potential as clinically viable therapeutic
agents.
8. Experimental
8.1. General methods
Melting points were determined in open capillary tubes
on a Meltemp melting point apparatus and are uncor-
rected. 1H NMR spectra were determined with a Bruker
DPX-400 spectrometer at 400 MHz. Chemical shifts d
are reported in parts per million (d) relative to residual
chloroform (7.26 ppm), TMS (0 ppm), or dimethylsulf-
oxide (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. Electron impact
and high-resolution mass spectra were obtained on a
Finnigan MAT-90 spectrometer. Combustion analysis
was obtained using Perkin–Elmer Series II 2400
CHNS/O analyzer. Chromatographic purifications were
performed by open chromatography using IBW-127ZH
10. Compound 12a
10.1. Preparation of (5R),(6Z)-7-oxo-6-(5,6,7,8-tetrahy-
droimidazo[2,1-b][1,3]benzothiazol-2-ylmethylene)-4-thia-
1-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid (12a)
10.1.1. Step 1: Preparation of 2-Carbethoxy-{5,6,7,8-
tetrahydro}-imidazo[2,1-b][1,3]benzothiazole (18). A mix-
ture of 2-chlorocyclohexanone (13.2 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
(12.0 g separated). This was dissolved in anhydrous eth-
anol (100 ml) and sodium methoxide (3.3 g, 63 mmol).
To this ethyl bromopyruvate (15.0 g, 76 mmol) was
added and stirred at room temperature for 2 h. It was

A. M. Venkatesan et al. / Bioorg. Med. Chem. 16 (2008) 1890–1902
1897
then refluxed for 48 h. At the end, reaction mixture was
cooled to room temperature and concentrated. The res-
idue was extracted with chloroform and washed well
with water. The product was purified by silica-gel col-
umn chromatography by eluting it with 50% ethyl ace-
tate:hexane. Red semi-solid; Yield: 6.2 g (39%); (M+H)
251.
duced pressure at room temperature. The yellow solid
was washed with acetone, filtered, and dried. Yield:
120 mg, 20%; yellow solid; mp 250 °C (dec);
1
(M+H+Na) 382. H NMR (DMSO-d6) d 1.9 (m,2H),
2.5 (m, 2H), 3.2–3.4 (m, 4H), 6.6 (s, 1H), 7.1 (s, 1H),
7.5 (s, 1H), 8.1 (s, 1H).
C16H12N3NaO3S2.
The ester was reduced with LiBH4 and the resultant
alcohol was oxidized with active MnO2. The aldehyde,
namely 5,6,7,8-tetrahydroimidazo[2,1-b][1,3]benzothia-
zole-2-carbaldehyde 20, obtained was taken to next step.
Calcd C 50.39%, H 3.17%, N 11.02%; found: C 50.10%,
H 3.42, N 10.99.
10.1.2. Step 2: Preparation of 4-nitrobenzyl (5R)-6-[(acet-
yloxy)(5,6,7,8-tetrahydroimidazo[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 (11a). To a stirred solution of 5,6,7,8-
tetrahydroimidazo[2,1-b][1,3]benzothiazole-2-carbaldehyde
20 (412 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-ni-
tro-benzyl ester 10 (770 mg, 2 mmol) in dry THF
(10 mL)/acetonitrile (15 mL), anhydrous MgBr2:O(Et)2
(1.2 g, 3.0 mmol) was added under an argon atmosphere
at room temperature. After cooling to ꢀ20 °C, Et3N
(2.0 mL) was added and the reaction vessel was covered
with foil to exclude light. The reaction mixture was stirred
for 4 h at ꢀ20 °C and treated with acetic anhydride
(1.04 mL) in one portion. The reaction mixture was
warmed to 0 °C and stirred for 15 h at 0 °C. The mixture
was diluted with ethyl acetate and washed with 5% citric
acid aqueous solution, saturated sodium hydrogen car-
bonate, and brine. The organic layer was dried (MgSO4)
and filtered through a pad of Celite. The pad was washed
with ethyl acetate. The filtrate was concentrated under
reduced pressure. The residue was applied to silica gel col-
umn chromatography and eluted with ethyl acetate:hex-
ane (1:1). Collected fractions were concentrated under
reduced pressure and the mixture of diastereomers was
taken to the next step. Pale yellow amorphous solid;
Yield: 980 mg, 77%; (M+H) 634.
11. Compound 12b
11.1. Preparation of (5R),(6Z)-6-(5,8-dihydro-6H-imidazo
[2,1-b]pyrano[4,3-d][1,3]thiazol-2-ylmethylene)-7-oxo-4-
thia-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid (12b)
11.1.1. Step 1: Preparation of ethyl 5,8-dihydro-6H-
imidazo[2.1-b]pyrano[4,3-d][1,3]thiazole-2-carboxylate
(19). To a mixture of tetrahydro-4H-pyran-4-one (5.0 g,
50 mmol) in CCl4 (100 ml) at 0 °C, SO2Cl2 (7.4 g,
55 mmol) was slowly added. After the addition, reaction
mixture was stirred at room temperature for 4 h and care-
fully quenched with ice-cold water. Reaction mixture was
washed well and dried over anhydrous MgSO4. The or-
ganic layer was filtered and concentrated. The colorless
oil obtained was dissolved in THF/EtOH (2:1) containing
thiourea (4.0 g, 52 mmol) and refluxed for 8 h. At the end,
reaction mixture was cooled to room temperature and the
separated, 6,7-dihydro-4H-pyrano[4,3-d][1,3]thiazol-2-
amine hydrochloride 17 white solid was filtered. Yield
4.5 g (47%); mp 115 °C, (M+H) 157.
To a stirred solution of, 6,7-dihydro-4H-pyrano[4,3-
d][1,3]thiazol-2-amine hydrochloride (4.0 g, 20.8 mmol)
in anhydrous ethanol (100 ml), sodium methoxide
(1.1 g, 21 mmol) was added. This was stirred at room
temperature for 30 min and to this ethyl bromopyruvate
(10.0 g, 51 mmol) was added and stirred at room tem-
perature for 2 h. Then it was refluxed for 48 h. At the
end reaction mixture was cooled to room temperature
and concentrated. The residue was basified with 2 M
NaHCO3 solution. extracted with chloroform, and
washed well with water. The product was purified by sil-
ica-gel column chromatography by eluting it with 50%
ethyl acetate:hexane. Red semi-solid; yield: 3.1 g
(59%), (M+H) 253.
10.1.3. Step 3: Preparation of (5R),(6Z)-7-oxo-6-(5,6,7,8-
tetrahydroimidazo[2,1-b][1,3]benzothiazol-2-ylmethyl-
ene)-4-thia-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid
sodium salt (12a). 4-Nitrobenzyl (5R)-6-[(acetyloxy)(5,
6,7,8-tetrahydroimidazo[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 11a (980 mg, 1.55 mmol) was dis-
solved in THF (20 mL) and acetonitrile (10 mL).
Freshly activated Zn dust (5.2 g) was rapidly added
along with 0.5 M phosphate buffer (pH 6.5, 28 mL).
The reaction vessel was covered with foil to exclude light
and the reaction mixture was vigorously stirred for 2 h
at room temperature. The reaction mixture was filtered,
cooled to 3 °C, and 0.1 N NaOH was added to adjust
the pH to 8.5. The filtrate was washed with ethyl acetate
and the aqueous layer was separated. The aqueous layer
was concentrated under high vacuum at 35 °C to give a
yellow precipitate. The product was purified by HP21
resin reverse phase column chromatography. Initially
the column was eluted with deionized water (2 L) and la-
ter with 10% acetonitrile:water. The fractions containing
the product were collected and concentrated under re-
The ester 19 was reduced with LiBH4 and the resultant
alcohol was oxidized with active MnO2. The aldehyde
obtained was taken to next step.
11.1.2. Step 2: Preparation of 4-nitrobenzyl (5R)-6-
[(acetyloxy)(5,8-dihydro-6H-imidazo[2,1-b][1,3]pyrano-
[4,3-d][1,3]thiazol-2-yl)-6-bromo-7-oxo-4-thia-1-azabicyclo-
[3.2.0]hept-2-ene-2-carboxylate (11b). Starting from 2-
formyl-5,8-dihydro-6H-imidazo[2.1-b]pyrano[4,3-d][1,3]-
thiazole (208 mg, 1.0 mmol), (5R,6S)-6-bromo-7-oxo-4-
thia-1-aza-bicyclo[3.2.0]hept-2-ene-2-carboxylic acid 4-
nitro-benzyl ester (400 mg, 1.3 mmol), and anhydrous
MgBr2:O(Et)2 (1.2 g, 3.0 mmol) and following the proce-

1898
A. M. Venkatesan et al. / Bioorg. Med. Chem. 16 (2008) 1890–1902
dure outlined for 12a, (Step 2) 400 mg, (yield, 62%) of the
titled product was isolated. Mp 78 °C; (M+H) 636.
with ethyl acetate–methanol (1/1). The title compound
24 was obtained as a pale yellow solid (7.56 g, 77%).
1H NMR (400 MHz, CDCl3) d 1.42 (t, 3H, J = 7.1 Hz),
2.14–2.25 (m, 4H), 3.11 (t, 2H, J = 6.1 Hz), 4.37 (t,
2H, J = 5.7 Hz), 4.41 (q, 2H, J = 7.1 Hz), 7.57 (s, 1H);
(M+H) 235.
11.1.3. Step 3: Preparation of (5R),(6Z)-6-(5,8-dihydro-
6H-imidazo[2,1-b]pyrano[4,3-d][1,3]thiazol-2-ylmethylene)-
7-oxo-4-thia-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylic
acid (12b). Starting from 4-nitrobenzyl (5R)-6-[(acetyl-
oxy)(5,8-dihydro-6H-imidazo[2,1-b][1,3]pyrano[4,3-d][1,3]-
thiazol-2-yl)-6-bromo-7-oxo-4-thia-1-azabicyclo[3.2.0]-
hept-2-ene-2-carboxylate (500 mg, 0.79 mmol) and fol-
lowing the procedure outlined for 12a (Step 3) the titled
compound was isolated. The yellow solid was washed
with acetone, filtered, and dried. Yield: 85 mg, 30%; as
12.1.3. Step 3: 4,5,6,7-Tetrahydro-1,3a,3b,8-tetraaza-
cyclopenta[a]indene-2-carbaldehyde (25). Diisobutylalu-
minum hydride (1.01 M) in toluene (1.06 mL) was added
dropwise to the solution of 4,5,6,7-tetrahydro-1,3a,3b,8-
tetraaza-cyclopenta[a]indene-2-carboxylic acid ethyl
ester (100 mg) in dry THF (5 mL) at ꢀ78 °C under a
nitrogen atmosphere. The reaction mixture was stirred
for 30 min at ꢀ78 °C and treated with ethanol (ca
1 mL). The mixture was warmed to 0 °C and stirred
for 1 h at that temperature. The reaction solution was
diluted with ethyl acetate (20 mL), treated with 0.5 mL
saturated ammonium chloride solution, and sonicated
for ca. 5 min (until a precipitate was deposited enough).
The reaction mixture was filtered and the organic layer
was dried (Na2SO4) and filtered through a pad of Celite.
The filtrate was concentrated under reduced pressure.
The residue was crystallized from dichloromethane
and diethyl ether to give the title compound (47.4 mg,
58%). 1H NMR (400 MHz, CDCl3) d 2.16–2.27 (m,
4H), 3.14 (t, 2H, J = 6.1 Hz), 4.39 (t, 2H, J = 5.7 Hz),
7.53 (s, 1H), 10.01 (s, 1H), (M+H) 191.
1
yellow crystals; mp 205 °C; (M+H+Na) 383. H NMR
(DMSO-d6) d 2.8 (m, 2H), 4.0 (m,2H), 4.6 (s,2H), 6.4
(s,1H), 6.5 (s,1H), 7.0 (s,1H), 8.1 (s,1H).
C15H10N3NaO4S2.
Calcd C 46.99%, H 2.63%, N 10.96%; found: C 46.91%,
H 2.68, N 10.91.
12. Compound 12c
12.1. Preparation of (5R),(6Z)-6-(4,5,6,7-tetrahydro-1,3a,
3b,8-tetraaza-cyclopenta[a]indene-2-ylmethylene)-7-oxo-4-
thia-1-aza-bicyclo[3.2.0]hept-2-ene-2-carboxylic acid
sodium salt (12c)
12.1.4. Step 4: (5R,6RS)-6-{(RS)-Acetoxy-[4,5,6,7-tetra-
hydro-1,3a,3b,8-tetraaza-cyclopenta[a]indene-2-yl]-methyl}-
6-bromo-7-oxo-4-thia-1-aza-bicyclo[3.2.0]hept-2-ene-2-
carboxylic acid 4-nitro-benzyl ester (11c). Starting
from 4,5,6,7-Tetrahydro-1,3a,3b,8-tetraaza-cyclopenta[a]-
indene-2-carbaldehyde (2.97 g, 5 mmol), (5R,6S)-6-
bromo-7-oxo-4-thia-1-aza-bicyclo[3.2.0]hept-2-ene-2-car-
boxylic acid 4-nitro-benzyl ester (10) (2.97 g, 7.7 mmol),
and anhydrous MgBr2 and following the procedure as
outlined for 12a (Step 2) the title compound was ob-
tained as a brown amorphous solid. Yield: 651.6 mg,
13%. 1H NMR (400 MHz, CDCl3) d 2.10–2.24 (m,
4H), 2.29 (s, 3H), 3.04–3.07 (m, 2H), 4.28–4.32 (m,
2H), 5.27 (d, 1H, J = 13.7 Hz), 5.43 (d, 1H,
J = 13.7 Hz), 6.19 (s, 1H), 6.91 (s, 1H), 7.01 (s, 1H),
7.49 (s, 1H), 7.59–7.62 (m, 2H), 8.23–8.25 (m, 2H);
(M+H) 619.
12.1.1. Step 1: 5,6,7,8-Tetrahydro-[1,2,4]triazolo[1,5-
a]pyridin-2-ylamine (23). The 12.7% solution of HCl in
ethanol (5.35 mL) and 10% Pd–C (50% wet) (2.5 g) were
added to the mixture of [1,2,4]triazolo[1,5-a]pyridin-2-
ylamine 22 (2.5 g) in ethanol (72 mL). The reaction mix-
ture was hydrogenated at 40 psi of H2 for 3 days at
room temperature. The mixture was filtered and concen-
trated under reduced pressure. The residue was treated
with saturated potassium carbonate solution and ex-
tracted with chloroform. The organic layer was dried
(Na2SO4) and concentrated under reduced pressure.
The title compound 23 was obtained as a pale yellow so-
1
lid (2.31 g, 90%). H NMR (400 MHz, CDCl3) d 1.88–
1.94 (m, 2H), 1.98–2.05 (m, 2H), 2.77 (t, 2H,
J = 6.2 Hz), 3.95 (t, 2H, J = 6.2 Hz), 4.09 (br s, 2H).
12.1.2. Step 2: 4,5,6,7-Tetrahydro-1,3a,3b,8-tetraaza-
cyclopenta[a]indene-2-carboxylic acid ethyl ester (24).
Ethyl bromopyruvate (10.23 g) was added to the mix-
ture of 5,6,7,8-tetrahydro-[1,2,4]triazolo[1,5-a]pyridin-
2-ylamine (5.8 g) in 1,2-dimethoxyethane (320 mL).
The reaction mixture was stirred for 5 h at room temper-
ature and concentrated to 100 mL under reduced pres-
sure. To the stirred solution, diethyl ether (200 mL)
was added and the separated solid was filtered. The pre-
cipitate was dissolved in ethanol (175 mL) and stirred
for 20 h at 110 °C in sealed tube. The reaction mixture
was cooled to room temperature and concentrated un-
der reduced pressure. The residue was treated with satu-
rated potassium carbonate solution and extracted with
chloroform. The organic layer was dried (Na2SO4) and
concentrated under reduced pressure. The residue was
applied to silica gel column chromatography and eluted
12.1.5. Step 5: (5R),(6Z)-6-(4,5,6,7-tetrahydro-1,3a,3b,8-
tetraaza-cyclopenta[a]indene-2-ylmethylene)-7-oxo-4-thia-
1-aza-bicyclo[3.2.0]hept-2-ene-2-carboxylic acid sodium
salt (12c). Starting from (5R, 6RS)-6-{(RS)-Acetoxy-
[4,5,6,7-tetrahydro-1,3a,3b,8-tetraaza-cyclopenta[a]indene-
2-yl]-methyl}-6-bromo-7-oxo-4-thia-1-aza-bicyclo[3.2.0]-
hept-2-ene-2-carboxylic acid 4-nitro-benzyl ester
(643.6 mg, 1.03 mmol) and following the procedure out-
lined for 12a (Step 3) the title compound was obtained
as a yellow amorphous solid (68 mg, 18%, pH 7.4).
Mp 175 °C (dec); (M+H+Na) 366; 1H NMR
(400 MHz, D2O) d 1.85–2.03 (m, 4H), 2.85–2.99 (m,
2H), 4.07–4.14 (m, 2H), 6.34 (s, 1H), 6.74 (s, 1H), 6.76
(s, 1H), 7.28 (s, 1H).
C15H12N5NaO3S2.

A. M. Venkatesan et al. / Bioorg. Med. Chem. 16 (2008) 1890–1902
1899
Calcd 49.31%, H 3.31%, N 19.17%; found: C 49.01%, H
3.40, N 19.08.
14. Compound 12e
14.1. Preparation of (5R,6Z)-7-oxo-6-(5,6,7,8-tetrahyd-
ropyrazolo[5,1-b][1,3]benzoxazol-2-ylmethylene)-4-thia-1-
azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid sodium salt
(12e)
13. Compound 12d
13.1. Preparation of (5R),(6Z)-8-[(9-methyl-9H-imidazo-
[1,2-a]benzimidazol-2-yl)methylene]-7-oxo-4-thia-1-azabi-
cyclo[3.2.0]hept-2-ene-2-carboxylic acid (12d)
14.1.1. Step 1: Preparation of ethyl-5-[(2-oxocyclo-
hexyl)oxy]-1H-pyrazole-3-carboxylate. To the stirred
suspension of ethyl 5-hydroxy-1H-pyrazole-3-carboxyl-
ate 28 (6.25 g, 40 mmol) and potassium carbonate
(22.1 g, excess) in 500 ml of acetonitrile was added 2-
chlorocyclohexanone 14 (6.35 g, 48 mmol) and refluxed
for 16 h. The reaction mixture was allowed to cool to
room temperature and filtered. The residue was washed
with acetonitrile and the filtrate was concentrated to an
oil. The oil was dissolved in ethyl acetate and extracted
with water. The organic phase was dried over MgSO4
and evaporated to dryness. The product was purified
by silica-gel column chromatography by eluting it with
1:1 ethyl acetate:hexane. The desired product (4.92 g
(49%)) was obtained as a white solid. Mp 122–124 °C;
(M+H) 253.
13.1.1. Step 1: Preparation of 9-methyl-9H-imidazo[1,2-
a]benzimidazole-2-carbaldehyde (27). To a stirred solu-
tion of LiBH4 (1.79 g, 82 mmol) in THF at 0 °C, ethyl
9-methyl-9H-imidazo[1,2-a]benzimidazole-2-carboxyl-
ate (2.5 g, 10.3 mmol) was added dropwise. The reaction
mixture was refluxed for 2 h and cooled to room temper-
ature and carefully quenched with ice-cold water and
acidified with concd HCl to pH 4. The reaction mixture
was stirred at room temperature for 1 h and basified
with K2CO3. The residue was extracted with chloro-
form;methanol (3:1) and dried over anhydrous MgSO4.
It was filtered and concentrated to yield: 1.3 g of its cor-
responding alcohol.
The residue (1.3 g, 6.4 mmol) was oxidized with MnO2
(5.0 g) in CHCl3 under refluxing condition. After the
completion, reaction mixture was filtered and concen-
trated. It was purified by SiO2 column chromatography
by eluting it with 1:1 ethyl acetate:hexane. Brown solid,
yield: 330 mg (25%); (M+H) 200.
14.1.2. Step 2: Preparation of ethyl 5,6,7,8-tetrahydro-
A
pyrazolo[5,1-b][1,3]benzoxazole-2-carboxylate (30).
mixture of ethyl-5-[(2-oxocyclohexyl)oxy]-1H-pyrazole-
3-carboxylate (127.6 mg, 0.5 mmol), methane sulfonic
acid (95 mg), and acetic acid 9.5 mL in toluene (50 ml)
was refluxed for 18 h using a Dean–Stark trap to remove
water. The reaction mixture was allowed to cool to
room temperature and it was filtered. The filtrate was
concentrated to an oil. The residue was dissolved in
ethyl acetate and aqueous bicarbonate solution. The or-
ganic layer was washed with water and dried over
MgSO4. After removal of the ethyl acetate, the residue
was purified by silica gel chromatography eluting with
1:1 ethyl acetate/hexane to give 69.7 mg (59%) of the de-
sired product 30 as white solid. Mp 55–57 °C; (M+H)
235.0.
13.1.2. Step 2: Preparation of 4-nitrobenzyl(5R)-6-
[(acetyloxy)(9-methyl-9H-imidazo[1,2-a]benzimidazole-2-)-
methyl]-6-bromo-7-oxo-4-thia-1-azabicyclo[3.2.0]hept-2-
ene-2-carboxylate (11d). Starting from 9-methyl-9H-imi-
dazo[1,2-a]benzimidazole-2-carbaldehyde 27 (330 mg,
1.65 mmol), (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 (770 mg, 2 mmol), and anhydrous MgBr2:O(Et)2
(1.2 g, 3.0 mmol) and following the procedure as out-
lined for compound 12a (Step 2) the titled compound
(as mixture of diastereomers) was isolated as pale
yellow amorphous solid; yield: 330 mg, 31%; (M+H)
628.
14.1.3. Step 3: Preparation of 5,6,7,8-tetrahydropyrazol-
o[5,1-b][1,3]benzoxazol-2-ylmethanol. To the stirred
solution of ethyl 5,6,7,8-tetrahydropyrazolo[5,1-b][1,3]-
benzoxazole-2-carboxylate 30 (3.84 g, 16.4 mmol) in
100 ml of THF were added 3.05 g of LiBH4 and 3 ml
of methanol. The solution was heated at 40 °C for
2.5 h. The reaction was quenched with 1 N HCl, the
pH was adjusted to 1.0, and stirred at room temperature
for 1 h. The reaction mixture pH was again adjusted to 8
with K2CO3 and extracted with ethyl acetate. The or-
ganic layer was dried over MgSO4 and concentrated to
an oil. The product was purified by SiO2 column chro-
matographed to give 2.62 g of the desired product
(83%). Mp 82–84 °C; (M+H) 193.
13.1.3. Step 3: Preparation of (5R),(6Z)-8-[(9-methyl-9H-
imidazo[1,2-a]benzimidazol-2-yl)methylene]-7-oxo-4-thia-
1-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid sodium
salt (12d). Starting from 4-nitrobenzyl(5R)-6-[(acetyl-
oxy)(9-methyl-9H-imidazo[1,2-a]benzimidazole-2-)
methyl]-6-bromo-7-oxo-4-thia-1-azabicyclo[3.2.0]hept-
2-ene-2-carboxylate 11d (1 g, 1.6 mmol) and following
the procedure as outlined for 12a (Step 3) the titled com-
pound was separated as a yellow solid. Yield: 140 mg,
23%; as yellow solid; mp 220 °C (Dec); (M+H+Na)
1
375. H NMR (DMSO-d6) d 3.4 (s,3H), 6.54 (s, 1H),
6.56 (s, 1H), 7.01 (s, 1H), 7.21 (t, 1H), 7.3 (t, 1H), 7.56
(d, 1H), 7.85 (d,1H), 8.1 (s,1H).
14.1.4. Step 4: Preparation of 5,6,7,8-tetrahydropyrazol-
o[5,1-b][1,3]benzoxazole-2-carbaldehyde (33). To the stir-
red solution of 5,6,7,8-tetrahydropyrazolo[5,1-b][1,3]-
benzoxazol-2-ylmethanol (2.30 g, 11.97 mmol) in 60 ml
of CHCl3 was added 10 g of MnO2. The suspension
was refluxed for 1.5 h under a nitrogen atmosphere.
The reaction mixture was filtered through a pad of Cel-
C17H11N4NaO3S.
Calcd 54.54%, H 2.96%, N 14.97%; found: C 54.51%, H
2.91, N 14.86.

1900
A. M. Venkatesan et al. / Bioorg. Med. Chem. 16 (2008) 1890–1902
ite and concentrated to give yellow solid. The product
was purified by SiO2 column chromatography to give
the titled aldehyde. Yield: 1.95 g 85.5%, (M+H) 191.
and methane sulfonic acid (192 mg) in acetic acid
(7 ml) and toluene (50 ml) was refluxed for 18 h using
a Dean–Stark trap to remove water. The reaction mix-
ture was allowed to cool to room temperature. The reac-
tion mixture was filtered. The filtrate was concentrated
to an oil. The residue was dissolved in ethyl acetate
aqueous bicarbonate solution. The organic layer was
washed with water and dried over MgSO4. After re-
moval of the ethyl acetate, the residue was purified by
silica gel chromatography eluting with ethyl acetate/hex-
ane to give 120 mg (51%) of the desired product as white
solid. Mp 116–118 °C; (M+H) 237.0
14.1.5. Step 5: 4-Nitrobenzy (5R)-6-[(acetyloxy)(5,67,8-
tetrahydropyrazolo[5,1-b][1,3]benzoxazol-2-yl)methyl-6-
bromo-7-oxo-4-thia-1-azabicyclo[3.2.0]hept-2-ene-2-car-
boxylate (11e). Starting from 5,6,7,8-tetrahydropyrazol-
o[5,1-b][1,3]benzoxazole-2-carbaldehyde 33 (589 mg,
3.1 mmol), (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 10 (1.54 g, 4.6 mmol) and anhydrous MgBr2:O(Et)2
(2.21 g, 8.5 mmol) and following the procedure as out-
lined for 12a (Step 2) the titled compound was isolated
as the mixture of diastereo isomers. Pale yellow amor-
phous solid. Yield: 792 mg, 42%; mp 160–162 °C;
(M+H) 618.
15.1.3. Step 3: Preparation of 7,8-dihydro-5H-pyrano[4,3-
d]pyrazolo[5,1-b][1,3]oxazol-2-ylmethanol. To the stirred
solution of 7,8-dihydro-5H-pyrano[4,3-d]pyrazolo[5,1-
b][1,3]oxazole-2-carboxylate 31 (1.5 g, 6.3 mmol) in
100 ml of THF were added LiBH4 (1.05 g) and methanol
(2 ml). The solution was heated at 40 °C for 2.5 h. The
reaction mixture was quenched by 1 N HCl, and the
mixture adjusted to pH 1.3 and stirred at room temper-
ature for 1 h. The reaction mixture was adjusted to pH 8
with K2CO3 and was extracted with ethyl acetate. The
organic layer was dried over MgSO4, concentrated to
an oil, and column chromatographed to give the desired
product. Yield 740 mg, 60%; (M+H) 196.
14.1.6. Step 6: Preparation of (5R,6Z)-7-oxo-6-(5,6,7,8-
tetrahydropyrazolo[5,1-b][1,3]benzoxazol-2-ylmethylene)-
4-thia-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylic
acid
sodium salt (12e). Starting from 4-Nitrobenzy (5R)-
6-[(acetyloxy)(5,67,8-tetrahydropyrazolo[5,1-b][1,3]ben-
zoxazol-2-yl)methyl-6-bromo-7-oxo-4-thia-1-azabicyclo-
[3.2.0]hept-2-ene-2-carboxylate 11e (318 mg, 0.5 mmol),
and following the procedure as outlined for 12a (Step
3) the titled product was obtained as a yellow solid.
Yield 150 mg, (76%); Yellow solid; (M+H+Na) 365.2.
H NMR (D2O): d 6.92 (1H, s), 6.91 (1H, s), 6.32 (1H,
s), 5.85 (1H, s), 2.59 (4H, m), 1.80 (4H, m).
15.1.4. Step 4: Preparation of 7,8-dihydro-5H-pyrano[4,3-
d]pyrazolo[5,1-b][1,3]oxazol-2-carbaldehyde (34). To the
stirred solution of 7,8-dihydro-5H-pyrano[4,3-d]pyraz-
olo[5,1-b][1,3]oxazol-2-ylmethanol (1.0 g, 5.1 mmol) in
60 ml of CHCl3 was added 8 g of activated MnO2. The
suspension was refluxed for 1.5 h under a nitrogen
atmosphere. The reaction mixture was filtered through
a pad of Celite. The filtrate was concentrated to give yel-
low oil. The product was purified by chromatography.
Yield 790 mg, 80 (M+H) 193.
C16H12N3NaO4S2.
Calcd 52.60%, H 3.31%, N 11.50%; found: C 52.48%, H
3.29, N 11.46.
15. Compound 12f
15.1.5. Step 5: 4-Nitrobenzy (5R)-6-[(acetyloxy)(7,8-
dihydro-5H-pyrano[4,3]pyrazolo[5,1-b][1,3]oxazol-2-yl)-
methyl]-6-bromo-7-oxo-4-thia-1-azabicyclo[3.2.0]hept-2-
ene-2-carboxylate (11f). Starting from 7,8-dihydro-5H-
pyrano[4,3-d]pyrazolo[5,1-b][1,3]oxazol-2-carbaldehyde
34 (600 mg, 3.1 mmol), (5R, 6S)-6-bromo-7-oxo-4-thia-
1-aza-bicyclo[3.2.0]hept-2-ene-2-carboxylic acid 4-nitro-
benzyl ester 10 (1.54 g, 4.6 mmol) and anhydrous
MgBr2:O(Et)2 (2.21 g, 8.5 mmol), and following the pro-
cedure as outlined for 12a (Step 2) the titled compound
was isolated as the mixture of diastereo isomers. Pale
yellow amorphous solid; Yield: 1.35 g, 70%; (M+H) 619.
15.1. Preparation of (5R,6Z)-6-(7,8-dihydro-5H-pyr-
ano[4,3-d]pyrazolo[5,1-b][1,3]oxazol-2-ylmethylene)7-
oxo-4-thia-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylic
acid sodium salt (12f)
15.1.1. Step 1: Preparation of ethyl-5-[(4-oxotetrahydro-
2H-pyran-3-yl)oxy]-1H-pyrazole-3-carboxylate. To the
stirred suspension of ethyl 5-hydroxy-1H-pyrazole-3-
carboxylate 28 (7.0 g, 45 mmol) and potassium carbon-
ate (24.0 g excess) in 500 ml of acetonitrile was added
3-bromo-tetrahydro-pyran-4-one (8.0 g, 45 mmol) 15,
and refluxed for 16 h. The reaction mixture was allowed
to cool to room temperature and filtered. The residue
was washed with acetonitrile and the filtrate was concen-
trated to an oil. The residue was dissolved in ethyl ace-
tate and washed with water. The organic phase was
dried over MgSO4 and evaporated to dryness. The de-
sired product (9.0 g, 78%) was obtained as a white solid.
Mp 121–123 °C; (M+H) 255.
15.1.6. Step 6: Preparation of (5R,6Z)-6-(7,8-dihydro-5H-
pyrano[4,3-d]pyrazolo[5,1-b][1,3]oxazol-2-methylene)7-
oxo-4-thia-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylic acid
sodium salt (12f) and (5R,6E)-6-(7,8-dihydro-5H-
pyrano[4,3-d]pyrazolo[5,1-b][1,3]oxazol-2-ylmethylene)7-
oxo-4-thia-1-azabicyclo[3.2.0]hept-2-ene-2-carboxylic
acid sodium salt (13). Starting from 4-Nitrobenzy(5R)-6-
[(acetyloxy)(7,8-dihydro-5H-pyrano[4,3]pyrazolo[5,1-b]-
[1,3]oxazol-2-yl)methyl]-6-bromo-7-oxo-4-thia-1-azabi-
cyclo[3.2.0]hept-2-ene-2-carboxylate (1.2 g, 1.9 mmol)
and following the procedure as outlined for 12a (Step
3) the titled compounds were isolated as a yellow solid.
15.1.2. Step 2: Preparation of ethyl 7,8-dihydro-5H-
pyrano[4,3-d]pyrazolo[5,1-b][1,3]oxazole-2-carboxylate
(31). A mixture of ethyl-5-[(4-oxotetrahydro-2H-pyran-
3-yl)oxy]-1H-pyrazole-3-carboxylate (254 mg, 1 mmol)

A. M. Venkatesan et al. / Bioorg. Med. Chem. 16 (2008) 1890–1902
1901
In this reaction both E and Z isomers were formed and
they were separated by prep. HPLC.
by eluting with 1:1 ethyl acetate/hexane to give the de-
sired product as an yellow oil; yield: 175 mg, 57%;
(M+H) 308.0.
(5R,6Z)-6-(7,8-Dihydro-5H-pyrano[4,3-d]pyrazolo[5,1-b]-
[1,3]oxazol-2-ylmethylene)7-oxo-4-thia-1-azabicyclo[3.2.0]-
hept-2-ene-2-carboxylic acid sodium salt 12f; yield
87 mg, (25%); yellow solid; (M+H+Na) 368.2.
16.1.3. Step 3: Preparation of ethyl 2-(hydroxymethyl)-
7,8-dihydropyrazolo [50,10:2,3][1,3]oxazolo[5,4-c]pyridine-
6(5H)-carboxylate. To the stirred solution of diethyl 7,8-
tetrahydropyrazolo[50,10:2,3][1,3]oxazolo[5,4-c]pyridine-
2,6(5H)-dicarboxylate (307 mg, mmol) of 40 ml of THF
were added LiBH4 (305 mg, excess) and 1 ml of metha-
nol. The solution was heated at 40 °C for 2.5 h. The
reaction mixture was quenched by 1 N HCl, and ad-
justed to pH 1.3 and stirred at room temperature for
1 h. The reaction mixture was again adjusted pH to 8
with K2CO3 and extracted with ethyl acetate. The or-
ganic layer was dried over MgSO4, concentrated to an
oil, and column chromatographed to give the desired
product. Yield: 172 mg, 65%; (M+H) 266.
H NMR (D2O): 7.04 (1H, s), 7.01 (1H, s), 6.45 (1H, s),
6.09 (1H, s), 4.76 (2H, m), 4.12 (2H, m), 2.96 (2H, m).
(5R,6E)-6-(7,8-dihydro-5H-pyrano[4,3-d]pyrazolo[5,1-b]-
[1,3]oxazol-2-ylmethylene)7-oxo-4-thia-1-azabicyclo[3.2.0]-
hept-2-ene-2-carboxylic acid sodium salt 13; yield
75 mg, (21%); yellow solid; (M+H+Na) 368.2.
H NMR (D2O): 7.08 (1H, s), 6.81 (1H, s), 6.71 (1H, s),
6.40 (1H, s), 4.68 (2H, m), 4.03 (2H, m), 2.87 (2H, m).
Compound 12f: C15H10N3NaO5S.
16.1.4. Step 4: Preparation of ethyl 2-formyl-7,8-dihyd-
ropyrazolo [50,10:2,3][1,3]oxazolo[5,4-c]pyridine-6(5H)-
carboxylate (35). To the stirred solution of ethyl 2-
(hydroxymethyl)-7,8-dihydropyrazolo [50,10:2,3][1,3]oxazolo-
[5,4-c]pyridine-6(5H)-carboxylate (1.76 g, 6.6 mmol) in
CHCl3 (60 ml) was added activated MnO2 (10 g, excess).
The suspension was refluxed for 1.5 h under a nitrogen
atmosphere. The reaction mixture was filtered through
a pad of Celite and the filtrate was concentrated to give
a yellow solid. The product was purified by chromatog-
raphy. 1.43 g of the product was obtained (82%); Mp
97–99 °C, (M+H) 264.
Calcd 49.05%, H 2.74%, N 11.44%; found: C 48.98%, H
2.72, N 11.48.
16. Compound 12g
16.1. Preparation of (5R,6Z)-6-{[6-(ethoxycarbonyl)-5,6,7,
8-tetrahydropyrazolo[50,10:2,3][1,3]oxazolo[5,4-c]pyridin-2-
yl]methylene}-7-oxo-4-thia-1-azabicyclo[3.2.0]hept-2-ene-
2-carboxylic acid sodium salt (12g)
16.1.1. Step 1: Preparation of ethyl 3-{[(3-ethoxycar-
bonyl)-1H-pyrazol-5-yl]oxy}-4-oxopiperidine-1-carboxyl-
ate. To the stirred suspension of ethyl 5-hydroxy-1H-
pyrazole-3-carboxylate 28 (19.5 g, 127 mmol) and potas-
sium carbonate (50.0 g, excess) in 500 ml of acetonitrile
was added 3-bromo-4-oxo-piperidine-1-carboxylic acid
ethyl ester 29 (37.45 g, 149 mmol), and refluxed for
16 h. The reaction mixture was allowed to cool to room
temperature, and filtered. The residue was washed with
acetonitrile and the filtrate was concentrated to an oil.
The residue was dissolved in ethyl acetate and washed
with water. The organic phase was dried over MgSO4
and evaporated to dryness. The product was purified
by silica-gel column chromatography by eluting it with
1:1 ethyl acetate:hexane. 8.5 g (19%) of the desired prod-
uct was obtained as a yellow oil; (M+H) 326.
16.1.5. Step 5: Preparation of ethyl 2-[(acetyloxy)(5R)-6-
bromo-2-Z{[(4-nitrobenzyl)oxy]carbonyl}-7-oxo-4-thia-1-
azabicyclo[3.2.0]hept-2-en-6-yl)methyl]-7,8-dihydropyraz-
olo[50,10:2,3][1,3]oxazolo[5,4-c]pyridine-6(5H)-carboxyl-
ate (11g). Starting from ethyl 2-formyl-7,8-dihydro-
pyrazolo
[50,10:2,3][1,3]oxazolo[5,4-c]pyridine-6(5H)-
carboxylate 35 (790 mg, 3. mmol), (5R,6S)-6-bromo-7-
oxo-4-thia-1-aza-bicyclo[3.2.0]hept-2-ene-2-carboxylic
acid 4-nitro-benzyl ester 10 (1.54 g, 4.6 mmol), and
anhydrous MgBr2: O(Et)2 (2.21 g, 8.5 mmol) and fol-
lowing the procedure as outlined for 12a (Step 2) the
titled compound was separated as the mixture of diaste-
reo isomers. Pale yellow amorphous solid; yield: 1.67 g,
81%; (M+H) 690.
16.1.6. Step 6: Preparation of (5R,6Z)-6-{[6-(ethoxycar-
bonyl)-5,6,7,8-tetrahydropyrazolo[50,10:2,3][1,3]oxazolo-
[5,4-c]pyridin-2-yl]methylene}-7-oxo-4-thia-1-azabicyclo-
[3.2.0]hept-2-ene-2-carboxylic acid sodium salt (12g).
Ethyl 2-[(acetyloxy)(5R)-6-bromo-2-Z{[(4-nitrobenzyl)-
oxy]carbonyl} -7-oxo-4-thia-1-azabicyclo[3.2.0]hept-2-
en-6-yl)methyl)]-7,8-dihydropyrazolo[50,10:2,3][1,3]-
oxazolo[5,4-c]pyridine-6(5H)-carboxylate 11g (828 mg,
0.5 mmol) was dissolved in THF (20 mL), acetonitrile
(10 mL) and 0.5 M phosphate buffer (pH 6.5, 28 mL)
and hydrogenated over 10% Pd/C (200 mg) at 40 psi
pressure. After 4 h the reaction mixture was filtered,
cooled to 3 °C, and 0.1 M NaOH was added to adjust
pH to 8.5. The filtrate was washed with ethyl acetate
and the aqueous layer was separated. The aqueous layer
was concentrated under high vacuum at 35 °C to give
16.1.2. Step 2: Preparation of diethyl 7,8-tetrahydropy-
razolo[50,10:2,3][1,3]oxazolo[5,4-c]pyridine-2,6(5H)-dicar-
boxylate (32).
A mixture of ethyl 3-{[(3-ethoxy-
carbonyl)-1H-pyrazol-5-yl]oxy}-4-oxopiperidine-1-car-
boxylate (325 mg, 1 mmol) and methane sulfonic acid
(95 mg) in acetic acid (5 ml) and toluene (50 ml) was re-
fluxed for 18 h using a Dean–Stark trap to remove
water. The reaction mixture was allowed to cool to
room temperature and the reaction mixture was filtered.
The filtrate was concentrated to an oil. The oil was dis-
solved in ethyl acetate and aqueous bicarbonate solu-
tion. The organic layer was washed with water and
dried over MgSO4. After removal of the ethyl acetate,
the residue was purified by silica gel chromatography

1902
A. M. Venkatesan et al. / Bioorg. Med. Chem. 16 (2008) 1890–1902
L.; Anderson, M.; Manousos, M.; Barrett, J. F. J.
Antibiotics 1987, 40, 803.
12. Buynak, J. D.; Wu, K.; Bachmann, B.; Khasnis, D.; Hua,
L.; Wguyen, H. K.; Carver, C. L. J. Med. Chem. 1995, 38,
1022.
13. Philips, O. A.; Czajkowski, D. P.; Spevak, P.; Singh, M.
P.; Hanehara-Kunugita, C.; Hyodo, A.; Micetich, R. G.;
Maiti, S. N. J. Antibiotics 1997, 50, 350.
14. (a) Sandanayaka, V. P.; Yang, Y. Org. Lett. 2000, 2, 3087;
(b) Sandanayaka, V. P. Bioorg. Med. Chem. Lett. 2001, 11,
997; (c) Bitha, P.; Zhong, L.; Francisco, G. D.; Rasmus-
sen, B.; Lin, Y. I. Bioorg. Med. Chem. Lett. 1999, 9, 991;
(d) Bitha, P.; Zhong, L.; Francisco, G. D.; Yang, Y.;
Petersen, P. J. B.; Lin, Y. I. Bioorg. Med. Chem. Lett.
1999, 9, 997.
yellow precipitate. The product was purified by HP21
resin reverse phase column chromatography. Initially
the column was eluted with deionized water (2 L) and la-
ter with 10% acetonitrile:water. The fractions containing
the product were collected and concentrated at reduced
pressure at room temperature. The yellow solid was
washed with acetone and filtered. Yield 375 mg (71%);
yellow solid; (M+H+Na) 438.4.
H NMR (D2O): d 6.96 (1H, s), 6.94 (1H, s), 6.41 (1H, s),
6.00 (1H, s), 4.53 (2H, m), 4.13 (2H,q), 3.78 (2H,m), 2.78
(2H, m), 1.21 (3H, t).
C15H15N4NaO6S.
15. Sandanayaka, V. P.; Prashad, A. S.; Yang, Y.; William-
son, T.; Lin, Y. I.; Mansour, T. S. J. Med. Chem. 2003, 46,
2569.
Calcd 49.32%, H 3.45%, N 12.78%; found: C 49.30%, H
3.41, N 12.67.
16. (a) Venkatesan, A. M.; Agarwal, A.; Abe, T.; Ushirogo-
chi, H.; Yamamura, I.; Kumagai, T.; Petersen, P. J.;
Weiss, W. J.; Lenoy, E.; Yang, Y.; Shlaes, D. M.; Ryan, J.
L.; Mansour, T. S. Bioorg. Med. Chem. 2004, 12, 5807; (b)
Weiss, W. J.; Petersen, P. J.; Murphy, T. M.; Tardio, L.
A.; Yang, Y.; Bradford, P. A.; Venkatesan, A. M.; Abe,
T.; Isoda, T.; Mihira, A.; Ushirogochi, H.; Takasake, T.;
Projan, S.; O’Connell, J.; Mansour, T. S. Antimicrob.
Agents Chemother. 2004, 48, 4589; (c) Venkatesan, A. M.;
Agarwal, A.; Abe, T.; Ushirogochi, H.; Yamamura, I.;
Ado, M.; Tsuyoshi, T.; Dos Santos, O.; Gu, Y.; Sum, F.
W.; Li, Z.; Francisco, G.; Lin, Y. I.; Petersen, P. J.; Yang,
Y.; Kumagai, T.; Weiss, W. J.; Shlaes, D. M.; Knox, J. R.;
Mansour, T. S. J. Med. Chem. 2006, 49, 4623.
17. Venkatesan, A. M.; Gu, Y.; Dos Santos, O.; Abe, T.;
Agarwal, A.; Yang, Y.; Petersen, P. J.; Weiss, W. J.;
Mansour, T. S.; Michiyoshi, N.; Hujer, A. M.; Bonomo,
R. A.; Knox, J. R. J. Med. Chem. 2004, 47, 6556.
18. Nukaga, M.; Abe, T.; Venkatesan, A. M.; Mansour, T. S.;
Bonomo, R. A.; Knox, J. R. Biochemistry 2003, 42, 13152.
19. Tabei, K.; Feng, X.; Venkatesan, A. M.; Abe, T.; Hideki,
U.; Mansour, T. S.; Siegel, M. M. J. Med. Chem. 2004, 47,
3674.
Acknowledgment
The authors thank the members of the Wyeth Chemical
Technologies group for analytical and spectral
determination.
References and notes
1. (a) Sandanayaka, V. P.; Prashad, A. S. Curr. Med. Chem
2002, 9, 1145; (b) Micetich, R. G.; Salama, S. M.; Maiti, S.
N.; Reddy, A. V. N.; Singh, R. Curr. Med. Chem 2002, 1,
193; (c) Bush, K. Curr. Pharm. Des. 1999, 5, 839.
2. Bush, K.; Jocoby, G. A.; Medeiros, A. A. Antimicrob.
Agents Chemother. 1995, 39, 1211.
3. (a) Reading, C.; Farmer, T. Biochem. J. 1981, 199, 779;
(b) English, A. R.; Retsema, J. A.; Girard, A. E.;
Lynch, J. E.; Barth, W. E. Antimicrob. Agents Chemo-
ther. 1978, 14, 414; (c) Micetich, R. G.; Maiti, S. N.;
Spevak, P.; Hall, T. W.; Yamabe, S.; Ishida, N.;
Tanaka, M.; Yamazaki, T.; Nakai, A.; Ogawa, K. J.
Med. Chem. 1987, 30, 1469.
20. Abe, T.; Sato, C.; Ushirogochi, H.; Sato, K.; Takasaki, T.;
Isoda, T.; Mihira, A.; Shiro, M.; Venkatesan, A. M.;
Mansour, T. S. J. Org. Chem. 2004, 69, 5850.
21. Abe, T.; Matsunaga, H.; Mihira, A.; Sato, C.; Ushirogo-
chi, H.; Takasaki, T.; Venkatesan, A. M.; Mansour, T. S.;
2003, WO 2003093277.
4. (a) Bush, K. Curr. Opin. Investig. Drugs 2002, 3, 1284; (b)
Miller, L. A.; Ratnam, K.; Payne, D. J. Curr. Opin.
Pharmacol. 2001, 1, 451.
22. Osborne, N. F.; Atkins, R. J.; Broom, N. J. P.; Coulton,
S.; Harbridge, J. B.; Harris, M. A.; Francois, I. S. J. Chem.
Soc. Perkin Trans. 1 1994, 179.
23. (a) Potts, K. T.; Burton, H. R.; Bhattacharya, J. J. Org.
Chem. 1966, 31, 260; (b) Zauhan, J.; Ladouceur, B. F.
Can. J. Chem 1976, 54, 488.
5. Nukaga, M.; KiKuo, T.; Yamaguchi, H.; Sawai, T.
Antimicrob. Agents Chemother. 1994, 38, 1374.
6. (a) Yang, Y.; Bhachech, N.; Bradford, P. A.; Jett, B. D.;
Sahm, D. F.; Bush, K. Antimicrob. Agents Chemother.
1998, 42, 1671; (b) Bradford, P. A. Clin. Microbiol. Rev.
2001, 14, 933.
24. (a) Yang, Y.; Testa, R. T.; Bhachech, N.; Rasmussen, B.
A.; Bush, K. Antimicrob. Agents Chemother. 1999, 43,
2904; (b) Bush, K.; Macalintal, C.; Rasmussen, B. A.; Lee,
V.; Yang, Y. Antimicrob. Agents Chemother. 1993, 37, 851.
25. (a) CLSI, Methods for dilution antimicrobial susceptibil-
ity tests for bacteria that grow aerobically. Approved
standards, 2006, 26, Clinical Laboratory Standard Insti-
tute, Wayne, PA; (b) CLSI, Performance standareds for
antimicrobial susceptibility testing: M100-S16, 2006, 26,
Clinical Laboratory Standard Institute, Wayne, PA.
26. MOE software (Version 2006.08) available from Chemical
Computing Group Inc., 1010 Sherbrooke St. West,
Montreal, Quebec, Canada; www.chemcomp.com.
7. Buynak, J. D. Curr. Med. Chem. 2004, 11, 1951.
8. (a) Bennett, I. S.; Broom, N. J. P.; Bruton, G.; Calvert, S.
A.; Clarke, B. P.; Coleman, K.; Edmondson, R.; Edwards,
P.; Jones, D.; Osborne, N. F.; Walker, G. J. Antibiotics
1991, 44, 331; (b) Bennett, I. S.; Brooks, G.; Brown, N. J.
P.; Calvert, S. H.; Coleman, K.; Francois, I. J. Antibiotics
1991, 44, 969.
9. Bulychev, A.; Massova, I.; Lerner, S. A.; Mobashery, S. J.
Am. Chem. Soc. 1995, 117, 4797.
10. Broom, N. J. P.; Farmer, T. H.; Osborne, N. F.; Tyler, J.
W. J. Chem. Soc. Chem. Commun. 1992, 1663.
11. Chen, L. Y.; Chang, C.-W.; Hedberg, K.; Guarineo, K.;
Welch, W. M.; Kiessling, L.; Retsema, J. A.; Haskell, S.