Synthesis and biological evaluation of penam sulfones as inhibitors of β-lactamases

Article abstract of DOI:10.1016/j.bmc.2005.02.020

The chemical synthesis of a series of new penam sulfone derivatives bearing a 2β-substituted-oxyimino and -hydrazone substituents, their β-lactamase inhibitory properties against selected enzymes representing class A and C β-lactamases are reported. The o

Full text of DOI:10.1016/j.bmc.2005.02.020

Bioorganic & Medicinal Chemistry 13 (2005) 2847–2858
Synthesis and biological evaluation of penam sulfones as
inhibitors of b-lactamases
Oludotun A. Phillips,a,* Andhe V. N. Reddy,b Eduardo L. Setti,b
Paul Spevak,b David P. Czajkowski,b Herninder Atwal,b Sameeh Salama,b
Ronald G. Micetichb and Samarendra N. Maitib
aDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, Kuwait University, PO Box 24923, Safat 13110, Kuwait
bNaeja Pharmaceutical Inc., Edmonton, Alta., Canada T6E 5V2
Received 4 January 2005; revised 10 February 2005; accepted 10 February 2005
Abstract—The chemical synthesis of a series of new penam sulfone derivatives bearing a 2b-substituted-oxyimino and -hydrazone
substituents, their b-lactamase inhibitory properties against selected enzymes representing class A and C b-lactamases are reported.
The oxime containing penam sulfones strongly inhibited the Escherichia coli TEM-1 and Klebsiella pneumoniae cefotaximase (CTX-
1) enzymes, but moderately inhibited the Pseudomonas aeruginosa 46012 cephalosporinase; while the 2b-substituted-hydrazone
derivatives were generally less active against these enzymes. Furthermore, most of the inhibitors enhanced the antibacterial activities
of piperacillin (PIP) and ceftazidime (CAZ) particularly against TEM-1 and CTX-1 producing bacterial strains.
Ó 2005 Elsevier Ltd. All rights reserved.
1. Introduction
b-lactamase-mediated resistance to b-lactam antibiotics
is the use of b-lactam–b-lactamase inhibitor combina-
tions.9–11 Established inhibitors of the serine b-lacta-
mases including clavulanic acid 1, sulbactam 2a, and
tazobactam (TAZ) 2b, (Fig. 1) are useful clinically,11
particularly against mixed infections. These inhibitors
target the most clinically relevant class A b-lactamase
The extensive use of b-lactam antibiotics in hospitals
and in the community has created major resistance
problems world wide, leading to increased morbidity,
mortality, and health-care costs. The most common
form of such resistance is the production of b-lacta-
mases, which hydrolyze the b-lactam ring of b-lactam
antibiotics. Hence, the production of b-lactamases re-
mains the main mechanism for bacterial resistance to
b-lactam antibiotics.1,2 The hydrolytic destruction of
the b-lactam antibiotics may be either serine enzyme
(classes A, C, and D) or metallo-b-lactamases (class B)
catalyzed.3 The classification of the enzymes into these
molecular classes is based on their primary structures
and sequence homology.3–5 Both class A and C enzymes
continue to be of clinical importance because of their
production in strains of Enterobacteriaceae and Pseudo-
monas aeruginosa, selected by the extensive use of the
newer-generation cephalosporins.1,3,6–8 A highly effec-
tive and proven antibacterial strategy for overcoming
producing microorganisms. However, the class
enzymes (AmpC, cephalosporinases) are of profound
C
Keywords: Antibacterial activity; b-Lactamases; b-Lactamase inhibi-
tors; Penam sulfones.
*
Corresponding author. Tel.: +965 531 2300x6070; fax: +965 534
2807; e-mail: dphillips@hsc.edu.kw
Figure 1. Structures of b-lactamase inhibitors.
0968-0896/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.02.020

2848
O. A. Phillips et al. / Bioorg. Med. Chem. 13 (2005) 2847–2858
clinical importance because they are not significantly
inhibited by any of the clinically used b-lactamase inhib-
itors.12,13 Moreover, class C enzymes naturally have
broad-spectrum of action and can hydrolyze Ôb-lacta-
mase resistantÕ b-lactams, such as the third-generation
cephalosporins.11 Furthermore, b-lactam-based inhibi-
tors and newer cephalosporins can upregulate the
expression of class C b-lactamases, thus resulting in
the selection of mutants that hyperproduce chromosom-
ally encoded AmpC b-lactamase through stable dere-
pression.14–16 Both chromosomal and plasmid-borne
class C b-lactamases are reported to be present in 10–
50% of clinical isolates of Citrobacter freundii, Entro-
bacter cloacae, Serratia marcescens, and P. aeruginosa.17
Plasmid-encoded AmpC b-lactamases have also been
described in clinical isolates of Klebsiella spp. and Esch-
erichia coli.18
the corresponding sulfones 9 and 10, respectively.
Column chromatographic purification gave pure benz-
yhydryl-2b-(chloroacetoxy)methyl-6,6-dihydropencilla-
nate-1,1-dioxide, 9. Compound 9 was treated with
thiourea in ethanol to give benzhydryl-2b-hydroxy-
methyl-6,6-dihydropencillanate-1,1-dioxide 11, which
was oxidized with pyridinium chlorochromate (PCC)
in dichloromethane to give the key intermediate benzy-
hydryl-2b-formy1-6,6-dihydropenicillanate-1,1-dioxide
12 in 76% yield. Further chemical transformation by the
reaction of compound 12 with various alkoxylamines
and substituted hydrazines gave the corresponding
oxime 13a–m and hydrazone 14a–f derivatives, respec-
tively (Table 1).
Deprotection of the carboxylic acid protecting group
from the oxime and hydrazone series 13a–m and 14a–f
(Table 1) under catalytic hydrogenation over 10% Pd/
C/hydrogen at 50 psi in ethanol or by the use of trifluo-
roacetic acid gave the corresponding acids 15a–m and
16a–f, respectively (Scheme 1).
Scientists from Hoffmann La-Roche reported the syn-
thesis of a series of 2b-alkenyl penam sulfones that pos-
sess the ability to simultaneously inactivate both class
A pencillinases as well as class C cephalosporinases.19
A compound Ro 48-1220 (3, Fig. 1) from this series
showed best synergy in combination with ceftriaxone
against resistant microorganisms. The increased spec-
trum of activity of Ro 48-1220 was not associated with
increased affinity for, or enhanced reactivity with the
inhibited enzyme, but rather correlated to the slow
deacylation of the inhibited enzyme.19 This was as-
cribed to the presence of the 2b-substituted C@C dou-
ble bond side chain of the penam sulfone 3. The clinical
situation now dictates that second-generation b-lacta-
mase inhibitors capable of encompassing both class A
and class C b-lactamases would combat emerging resis-
tance and provide a vital addition to the antibacterial
armory. We have previously reported 2b-acyl penam
sulfones, exemplified by 4 with extended-spectrum b-
lactamase inhibitory activities.20 In continuation of
our search for new broad-spectrum b-lactamase inhibi-
tors, we reasoned that the introduction of the 2b-
substituted C@N double bond should produce
analogues with strong b-lactamase inhibitory activity.
We hereby report the syntheses, b-lactamase inhibitory
and synergistic activities of a series of 2b-substituted
penam sulfone derivatives 17a–n and 18a–f against
resistant microorganisms in combination with clinically
used b-lactam antibiotics.
Treatment of the crude acids with aqueous NaHCO3
solution followed by purification on a reverse-phase pre-
parative TLC plates and lyophilization gave the corre-
sponding sodium salts 17a–n and 18a–f (Table 2).
Attempts to deprotect 13d under catalytic hydrogena-
tion over 10% P/C resulted in the simultaneous depro-
tection of the diphenylmethyl group and reduction of
the allyl double bond to give the O-propyloxime deriva-
tive. Similarly, hydrogenation of the O-(pyridin-2-
yl)methyloxime derivative 13m over 10% P/C gave a
mixture of the expected compound 17m as a minor com-
ponent in 7% yield and a major component O-(piper-
idin-2-yl)methyloxime derivative 17n in 27% yield.
3. Results and discussion
The inhibitory activities of the new compounds against
E. coli TEM-1, Klebsiella pneumoniae CTX-1 and P.
aeruginosa cephalosporinase b-lactamases were evalu-
ated in comparison with clinically useful inhibitors
namely, clavulanic acid 1, sulbactam 2a, and tazobac-
tam 2b. The 50% inhibitory concentration (IC50) values
against these three b-lactamases are presented in Table
2. In the oxime series, the replacement of the hydroxyl
moiety in 17a with a methoxy 17b, ethoxy 17c, and allyl-
oxy 17d groups resulted in three to four times increased
CTX-1 enzyme inhibitory activities from 0.02 to 0.006,
0.005, and 0.005 lM, respectively. Similarly, increases
in activity were observed against the TEM-1 and cepha-
losporinase. In general, all the oxime derivatives 17a–n
strongly inhibited the TEM-1 enzyme, with 17i and 17j
showing the lowest IC50 of 0.007 and 0.0048 lM, respec-
tively, compared with clavulanic acid (0.065 lM), sul-
bactam (4.5 lM), and tazobactam (0.065 lM). This
strong level of b-lactamases inhibitory activity against
the class A enzymes is typical of penam sulfones.12,13
2. Chemistry
The key intermediate, benzyhydryl-2b-formy1-6,6-
dihydropenicillanate-1,1-dioxide 12 utilized in this work
was synthesized starting from the commercially avail-
able 6-aminopencillanic acid 5, as shown in Scheme 1.
The azetidinone derivative 6 was prepared according
to the literature methods.21,22 Compound 6 was reacted
with chloroacetic acid in the presence of silver acetate to
give an inseparable mixture of benzhydry1-2b-(chloro-
acetoxy)methyl-6,6-dihydropenicillanate 7 and benzhy-
dry1-3-(chloroacetoxy)-3-methyl-6,6-dihydrocephalo-
sporinate 8. The mixture of 7 and 8 was oxidized with
potassium permanganate in glacial acetic acid to give
All the oxime derivatives showed superior inhibitory
activity against the TEM-1 enzyme in comparison to
sulbactam, while the IC50 values for clavulanic acid 1

O. A. Phillips et al. / Bioorg. Med. Chem. 13 (2005) 2847–2858
2849
Scheme 1. Synthesis of 2b-substituted-oxyimino and -hydrazone penam sulfones. Reagents and conditions: (i) ClCH2CO2H, AgOAc; (ii) KMnO4/
AcOH/acetone; (iii) thiourea/ethanol; (iv) pyridinium chlorochromate, DCM; (v) R0ONH2 or R0R00NNH2/ethanol–DCM; (vi) TFA, anisole, 0 °C,
NaHCO3 or 10% Pd/C–ethanol, (vii) NaHCO3 aqueous.
and tazobactam 2b were about 14 times greater than
that of 17j. Unfortunately, this strong in vitro inhibitory
activity did not translate into synergistic activity in com-
bination with PIP; hence, compound 17j and others
with the exception of compounds 17g, 17i, and 17k were
devoid of synergistic activity against E. coli TEM-1
strain. Therefore, only compounds 17g, 17i, and 17k en-
hanced the antibacterial activity of PIP against E. coli
TEM-1 strain with MICs of 0.78, 0.39, and 0.78 lg/
mL, respectively. The lack of synergy could be due to
a variety of factors including poor uptake through the
bacterial cell wall,7 or the inhibitor could be over-
whelmed by the hyperproduction of b-lactamases.23,24
However, most of the compounds demonstrated syner-
gistic activity comparable to that of tazobactam in com-
bination with PIP against E. coli TEM-3 and TEM-7
strains (Table 3).
TEM-7 with >4-fold reduction in MIC values compared
with CAZ alone.
K. pneumoniae CTX-1 enzyme was strongly inhibited by
all the oxyimino penam sulfone derivatives with the IC50
values ranging from 0.0025 to 0.5 lM for 17j and 17i,
respectively. In addition, both compounds 17j and 17k
displayed the strongest inhibitory activity against the
CTX-1 enzyme with IC50 values of 0.0025 and
0.0028 lM, respectively. Removal of the tert-butyl pro-
tecting group of 17k to give a more water soluble
disodium salt 17j did not significantly affect the b-
lactamases inhibitory activity (Table 2). All the oxime
derivatives demonstrated stronger synergistic activity
against K. pneumoniae CTX-1 in combination with
CAZ than with PIP. On other hand, TAZ did not pro-
tect CAZ against hydrolysis by the K. pneumoniae
CTX-1 enzyme, despite the strong in vitro b-lactamase
inhibitory activity demonstrated by TAZ, but only mod-
erately protected PIP.
Furthermore, 17j showed strong synergy with PIP
against E. coli TEM-3, TEM-7, and OXA-1 strains by
exhibiting >4-fold reduction in MIC values over PIP
alone (Table 3). Similarly, in combination with CAZ,
17j showed strong synergy against E. coli TEM-3, and
The P. aeruginosa cephalosporinase was only moder-
ately inhibited by the oxime derivatives with IC50 values

2850
O. A. Phillips et al. / Bioorg. Med. Chem. 13 (2005) 2847–2858
Table 1. Structures of 2b-substituted-oxyimino and -hydrazone penam
sulfone esters
coli TEM-3, TEM-7, and K. pneumoniae CTX-1 these
three compounds showed strong synergy in combination
with CAZ. Furthermore, the hydrazone compounds
18a, 18b, and 18c protected PIP against the E. coli
TEM-3 and TEM-7 enzymes, but did not offer protec-
tion against K. pneumoniae CTX-1.
O
O
S
N
R'
N
O
CO2 CH(Ph)2
In conclusion, a series of 2b-oxyimino and -hydrazone
penam sulfones have been synthesized and evaluated
for their activity as b-lactamase inhibitors and as poten-
tial partners to protect b-lactamase labile b-lactam anti-
biotics. Most of the compounds had broad-spectrum
inhibitory activity against the b-lactamases including
the class A enzymes, normally sensitive to such com-
pounds10,19,25 and the class C enzymes; although activity
against class C enzymes were markedly reduced com-
pared to those against TEM-1 and CTX-1. This could
be explained based on the understanding that high affin-
ity with class C enzymes is dependent on C-6 substitu-
tion at the penam ring as noted for the penicillins.
However, these 2b-substituted penam sulfones were
stronger inhibitors of the serine b-lactamases, particu-
larly the TEM-1 enzyme in comparison to Ro 48-1220
(IC50, 1.1 lM).17 The 2b-oxyimino penam sulfones dem-
onstrated stronger inhibitory activity against all the
three serine b-lactamases compared to the hydrazone
derivatives. Overall, compounds 17b (R0 = OCH3) and
18a (R0 = NHCOCH3) were identified as the most active
representative compounds from the oxyimino and
hydrazone series, respectively. From a reversibility of
b-lactamase inhibition study carried out on 17b and
18a it was show that very little enzyme activity was
recovered after about 21 h of incubation of these com-
pounds with TEM-1, CTX-1, and cephalosporinase en-
zymes (data not shown). This may be an indication that
the 2b-substituted penam derivatives reported in this
study will irreversibly inhibit these b-lactamases, with
possibly a slow deacylation of the inhibited enzyme as
reported for Ro 48-1220.
Compounds
R0
13a
13b
13c
13d
13e
13f
13g
13h
–OH
–OCH3
–OCH2CH3
–OCH2CH@CH2
–OCH2CH2OH
–OCH2OCH3
–OC(CH3)3
–OCH2CONH2
13i
-O
13j
13k
–OCH2CO2CHPh2
–OCH2CO2C(CH3)3
N
N
13l
N
-O
13m
14a
-O
N
–NHCOCH3
NH
N
14b
O
–NHCOPh
14c
14d
14e
14f
–NHCSNH2
–NHCONH2
–NHCOOCH2Ph
ranging from 0.45 to >10.0 lM for compounds 17k and
17m, respectively. A significant reduction in the cephalo-
sporinase inhibition was observed by the substitution of
the six-member heterocycles pyridine and piperidine as
found in 17m and 17n, respectively. This moderate ceph-
alosporinase inhibitory activity (Table 2) is a strong
reflection of the observed weak to lack of synergistic
activity in combination with either PIP or CAZ against
cephalosporinase producing strains of E. cloacae P99
and P. aeruginosa (Tables 3 and 4). However, against
E. coli strains producing other TEM (TEM-3 and
TEM-7) and OXA-1 enzymes, the oxime series showed
significant synergistic activity in combination with either
PIP or CAZ.
4. Experimental
All column chromatographic purifications were accom-
plished on silica gel 60 (E. Merck, 230–400 mesh) using
the appropriate solvent gradients. TLC was done on
commercial reverse-phase silica gel plates (Analtech)
containing calcium sulfate binder and fluorescent indica-
1
tor. H NMR spectra were determined with a Bruker
AC-200-F (200 MHz) spectrometer in appropriate deu-
terated solvents and are expressed in ppm downfield
from TMS as internal standard. Elemental analyses
were performed on a Carlo Erba Ea 11108 analyzer
and results were within 0.4% of theoretical values for
C, H, and N.
In comparison to the oxime derivatives, the hydrazone
penam sulfones were generally much weaker inhibitors
of all the three b-lactamases. However, compound
(18a, R0 = NHCOCH3) showed the strongest TEM-1
enzyme inhibitory activity that is about seven and five
times the activities of 17i and 17j, respectively.
Although, compounds 18a, 18b, and 18c did not show
synergistic activity against E. coli TEM-1 in combina-
tion with PIP; they demonstrated strong synergy against
E. coli TEM-3, TEM-7, and S. aureus strains. Against E.
4.1. Diphenylmethyl (2S,3R,5R)-3-formyl-3-methyl-7-
oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate-4,4-
dioxide (12)19
To a stirred solution of diphenylmethyl (2S,3R,5R)-3-
hydroxymethyl-3-methyl-7-oxo-4-thia-1-azabicyclo[3.2.0]-
heptane-2-carboxylate-4,4-dioxide22 (11, 6.84 g, 16.46

O. A. Phillips et al. / Bioorg. Med. Chem. 13 (2005) 2847–2858
Table 2. 50% Inhibitory concentration (IC50, lM) values of penam sulfone derivatives
2851
O
O
S
N
R'
N
O
-
+
Na
CO2
Compounds
R0
IC50, (lM) values against
TEM-1 (E. coli)
CTX-1 (K. pneumoniae)
Cephalosporinase (P. aeruginosa 46012)
17a
17b
17c
17d
17e
17f
17g
17h
–OH
–OCH3
0.58
0.3
0.02
7.0
1.9
1.0
2.0
1.4
1.0
4.0
3.2
0.006
0.005
0.005
0.007
0.0036
0.45
–OCH2CH3
–OCH2CH@CH2
–OCH2CH2OH
–OCH2OCH3
–OC(CH3)3
–OCH2CONH2
0.5
0.1
0.5
0.1
1.00
0.01
0.008
17i
0.007
0.5
1.4
-OH2C
17j
17k
–OCH2COꢀ2 Naþ
–OCH2CO2C(CH3)3
0.0048
0.031
0.0025
0.0028
1.0
0.45
N
17l
0.7
0.006
0.01
2.6
N
N
O
17m
0.08
>10.0
N
-OH2C
-OH2C
17n
18a
0.4
0.01
0.1
10
N
H
–NHCOCH3
0.0014
0.6
NH
N
18b
n/t
n/t
n/t
O
18c
18d
–NHCOPh
n/t
n/t
n/t
–NHCSNH2
–NHCONH2
–NHCOOCH2Ph
>10.0
2.5
3.0
ꢁ 1.0
0.1
0.1
>10.0
1.0
1.0
>50
20
18e
18f
Clavulanic acid
Sulbactam
Tazobactam (TAZ)
0.065
4.5
0.065
0.0025
0.05
0.005
3
n/t = not tested.
mmol) in dry methylene chloride (340 mL), was added
pyridinium chlorochromate (7.1 g) in one portion. The
mixture was stirred at room temperature for 18 h, and
an additional amount of pyridinium chlorochromate
(1.78 g) was added, and the mixture was stirred for
additional 5 h. The reaction mixture was diluted with
methylene chloride, treated with charcoal, and filtered
through a bed of Celite. The filtrate was washed
with water, brine, dried (Na2SO4), and evaporated to
give 12, after flash column chromatography as a
foam 5.14 g (76%). 1H NMR (DMSO-d6): d 9.85 (s,
1H), 7.23–7.38 (m, 10H), 6.85 (s, 1H), 5.82 (s, 1H),
5.25 (dd, 1H, J = 1.2 Hz, J = 4.43 Hz), 3.74 (dd,
1H, J = 4.43 Hz, J = 16.7 Hz), 3.30 (dd, 1H, J =
1.2 Hz, J = 16.7 Hz), 1.26 (s, 3H). Anal. (C21H19NO6S)
C, H, N.
4.2. Diphenylmethyl (2S,3R,5R)-3-formyl-3-methyl-7-
oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate-31-
(hydroxyloxime)-4,4-dixoide (13a)
To a solution of intermediate aldehyde 12 (2.5 g,
6.05 mmol) in a mixture of methylene chloride (30 mL)
and ethanol (60 mL) was added hydroxylamine hydro-
chloride (504 mg, 7.26 mmol) followed by pyridine (430
mg, 440 lL) and the mixture was stirred at room tem-
perature for 5.5 h. The mixture was concentrated to dry-
ness and the residue was taken up in ethyl acetate,
washed with water, brine, dried (Na2SO4), and concen-
trated to give the crude product. Purification of the
crude product over a silica gel column using benzene–
ethyl acetate (10:1) gave 900 mg (35%) of 13a as a foam.
1H NMR (DMSO-d6): d 12.0 (s, 1H), 7.81 (s, 1H),

Table 3. Synergistic activity of penam sulfones 17a–m and 18a–c with PIP
Test organisms
MIC (lg/mL) of
PIP alone
+TAZ
+17a
+17b
0.78
+17c
0.39
+17e
0.39
+17g
+17h
0.39
0.78
50
+17i
+17j
0.39
0.39
50
+17k
0.78
60.2
0.78
+17l
0.39
+17m
1.56
0.78
12.5
+18a
0.39
+18b
0.78
0.78
50
1.56
12.5
+18c
0.39
S. aureus 54K
S. aureus 80K
12.5
12.5
>50
>50
>50
25
0.78
0.39
0.39
1.56
0.78
3.13
12.5
1.56
1.56
0.78
50
60.2
60.2
12.5
1.56
0.78
0.78
1.56
0.39
0.78
12.5
0.78
1.25
60.2
50
60.2
60.2
0.39
0.78
60.2
1.56
12.5
0.78
6.25
1.56
0.78
0.78
6.25
60.2
12.5
60.2
25
60.2
25
60.2
25
60.2
25
E. coli TEM-1
E. coli TEM-3
1.56
1.56
60.2
1.56
25
0.78
0.78
60.2
1.56
12.5
1.56
3.13
6.25
6.25
1.56
0.39
6.25
0.78
0.39
0.78
1.56
1.56
0.39
1.56
1.56
0.78
12.5
1.56
3.13
25
>50
12.5
E. coli TEM-7
E. coli OXA-1
6.25
6.25
0.78
6.25
25
25
K. pneumoniae CTX-1
S. marcescens 200L
P. vulgaris CT106
C. freundii 2046E
E. cloacae P99
>40
>50
>50
>50
>50
>50
50
12.5
12.5
>50
1.56
>50
12.5
>50
6.25
12.5
3.13
25
1.56
>50
50
3.13
12.4
>50
12.5
25
12.5
<0.2
0.39
3.13
0.78
0.78
3.13
1.56
0.78
3.13
12.5
1.56
0.39
6.25
0.39
1.25
0.78
>50
3.13
>50
>50
25
60.2
50
25
60.2
>50
12.5
0.78
60.2
>50
25
60.2
>50
50
12.5
25
>50
25
>50
50
>50
25
0.39
25
25
>50
50
12.5
25
50
12.5
1.56
P. aeruginosa L. 46007
M. morganii 36014
50
<0.2
25
60.2
50
12.5
60.2
60.2
6.25
0.39
25
1.56
60.2
1.56
Table 4. Synergistic activity of penam sulfones 17a–m and 18a–c with CAZ
Test organisms
MIC (lg/mL) of
CAZ alone
+TAZ
+17a
+17b
+17c
+17e
0.39
60.2
1.56
0.78
12.5
12.5
6.25
60.2
+17g
+17h
3.13
6.25
25
12.5
>50
+17i
+17j
0.78
+17k
+17l
1.56
3.13
12.5
0.39
>50
12.5
+17m
0.39
60.2
1.56
1.56
>50
12.5
12.5
1.56
+18a
+18b
+18c
E. coli TEM-3
E. coli TEM-7
25
25
0.39
12.9
0.78
12.5
1.56
3.13
50
0.39
0.39
1.56
0.39
12.5
12.5
3.13
60.2
0.39
0.39
1.56
0.78
0.39
0.39
1.56
0.78
0.39
0.78
1.56
0.39
25
60.2
0.39
0.78
0.39
12.5
25
0.78
1.56
1.56
6.25
6.25
0.78
3.13
1.56
12.5
6.25
25
0.78
3.13
6.25
6.25
6.25
12.5
6.25
1.56
0.78
3.13
1.56
K. pneumoniae CTX-1
P. vulgaris CT-106
E. cloacae P99
>50
12.5
>50
25
>50
12.5
12.5
25
25
25
6.25
12.5
60.2
>50
12.5
12.5
3.13
P. aeruginosa 46220 DR-2
E. aerogens 41004
M. morganii 36014
1.56
12.5
<0.20
25
12.5
3.13
12.5
25
12.5
12.5
3.13
12.5
6.25
60.2
25
25
12.5
25
25
25
3.13
1.56
0.78
6.25
0.78

O. A. Phillips et al. / Bioorg. Med. Chem. 13 (2005) 2847–2858
2853
7.29–7.46 (m, 10H), 6.92 (s, 1H), 5.40 (s, 1H), 5.30 (br d,
J = 2.9 Hz), 3.74 (dd, 1H, J = 4.5 Hz, J = 16.6 Hz), 3.32
(dd, 1H, J = 1.2 Hz, J = 16.6 Hz), 1.36 (s, 3H). Anal.
(C21H20N2O6S) C, H, N.
solid (229 mg), which was purified by reverse-phase pre-
parative TLC using acetonitrile–water (7:1.5) as the
developing solvent, to afford 44 mg (22%) of 17b as a
white fluffy solid. 1H NMR (DMSO-d6): d 7.55 (s,
1H), 5.03 (br d, 1H, J = 2.9 Hz), 4.32 (s, 1H), 3.83 (s,
3H), 3.55 (dd, 1H, J = 4.2 Hz, J = 16.1 Hz), 3.12 (dd,
1H, J = 1.3 Hz, J = 16.1 Hz), 1.52 (s, 3H). Anal.
(C9H11N2NaO6S) C, H, N.
4.3. Sodium (2S,3R,5R)-3-formyl-3-methyl-7-oxo-4-thia-
1-azabicyclo[3.2.0]heptane-2-carboxylate-31-(hydroxyl-
oxime)-4,4-dioxide (17a)
A mixture of compound 13a (200 mg, 0.47 mmol) in
ethanol (20 mL) and 10% Pd/C (200 mg) was hydroge-
nated at 50 psi for 5.5 h. The reaction mixture was fil-
tered over a bed of Celite and the filtrate was
concentrated under reduced pressure. The residue was
triturated with a mixture of ether–hexane to give the
crude as a white solid, which was collected by filtration
(68 mg). The acid was dissolved in a solution of NaH-
CO3 (24 mg) in water (10 mL), stirred at room temper-
ature for 10 min, and freeze-dried to give a white solid
(75 mg). The product was purified by reverse-phase
preparative TLC using acetonitrile–water (7:1.5) as
developing solvent, to give 30 mg (23%) of 17a as
white fluffy solid. 1H NMR (DMSO-d6): d 11.56 (s,
1H), 7.42 (s, 1H), 5.00 (br d, 1H, J = 2.9 Hz), 4.27 (s,
1H), 3.54 (dd, 1H, J = 4.6 Hz, J = 16.6 Hz), 3.12 (dd,
1H, J = 1.2 Hz, J = 16.5 Hz), 1.53 (s, 3H). Anal.
(C8H9N2NaO6S) C, H, N.
4.6. Diphenylmethyl (2S,3R,5R)-3-formyl-3-methyl-7-
oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate-31-
(O-ethyloxime)-4,4-dioxide (13c)
Compound 13c was synthesized from intermediate 12
(1.83 g, 4.43 mmol) and ethyloxyamine hydrochloride
(0.494 g, 4.43 mmol) as described for 13a, to give
428 mg (23%) of 13c as a foam after silica gel column
purification using benzene–ethyl acetate (30:1). 1H
NMR (DMSO-d6): d 7.93 (s, 1H), 7.25–7.50 (m, 10H),
6.92 (s, 1H), 5.45 (s, 1H), 5.32 (dd, 1H, J = 1.2 Hz,
J = 4.5 Hz), 4.13 (q, 2H, J = 7.0 Hz), 3.75 (dd, 1H,
J = 4.5 Hz, J = 16.6 Hz), 3.33 (dd, 1H, J = 1.2 Hz,
J = 16.6 Hz), 1.36 (s, 3H), 1.21 (t, 3H, J = 7.0 Hz). Anal.
(C23H24N2O6S) C, H, N.
4.7. Sodium (2S,3R,5R)-3-formyl-3-methyl-7-oxo-4-thia-
1-azabicyclo[3.2.0]heptane-2-carboxylate-31-(O-ethyl-
oxime)-4,4-dioxide (17c)
4.4. Diphenylmethyl (2S,3R,5R)-3-formyl-3-methyl-7-
oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate-31-
(O-methyloxime)-4,4-dioxide (13b)
Compound 17c was prepared from compound 13c
(0.298 g, 0.653 mmol) by hydrogenation over 10% Pd/
C (300 mg) at 50 psi for 2.5 h as described for 17a;
and purified by reverse-phase preparative TLC plates
A suspension of the aldehyde 12 (1.0 g, 2.42 mmol) in
95% ethanol (50 mL) was treated with methoxylamine
hydrochloride (242 mg, 2.903 mmol) followed by
pyridine (230 mg, 2.903 mmol) and the mixture was
stirred at room temperature for 5 h and concentrated
under reduced pressure. The residue was dissolved in
methylene chloride, washed with 5% HCl, water and
brine. After drying (Na2SO4), the solvent was removed
under reduced pressure to give 1.0 g of crude,
which was purified over a silica gel column using hex-
ane–ethyl acetate as eluent to give 467 mg (44%) of 13b
1
to give 89 mg, (59%) of 17c as a yellow fluffy solid. H
NMR (DMSO-d6): d 7.53 (s, 1H), 5.02 (br s, 1H), 4.34
(s, 1H), 4.09 (q, 2H, J = 7.04 Hz), 3.56 (dd, 1H,
J = 4.3 Hz, J = 16.1 Hz), 3.13 (dd, 1H, J = 1.1 Hz,
J = 16.0 Hz), 1.53 (s, 3H), 1.20 (t, 3H, J = 7.04 Hz).
Anal. (C10H13N2NaO6S) C, H, N.
4.8. Diphenylmethyl (2S,3R,5R)-3-formyl-3-methyl-7-
oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate-31-
(O-allyloxime)-4,4-dioxide (13d)
1
as a foam. H NMR (DMSO-d6): d 7.89 (s, 1H); 7.22–
7.40 (m, 10H), 6.86 (s, 1H), 5.39 (s, 1H), 5.26 (dd, 1H,
J = 1.4 Hz, J = 4.4 Hz), 3.82 (s, 3H), 3.68 (dd, 1H,
J = 4.6 Hz, J = 16.7 Hz), 3.26 (dd, 1H, J = 1.4 Hz,
J = 16.7 Hz), 1.28 (s, 3H). Anal. (C22H22N2O6S) C,
H, N.
Compound 13d was synthesized from the intermediate
aldehyde 12 (2.23 g, 5.39 mmol) and allyloxyamine
hydrochloride (709 mg, 6.47 mmol) as described for
13a to give 800 mg (32%) of 13d as a foam, after silica
gel column purification using benzene–ethyl acetate
1
(30:1). H NMR (DMSO-d6): d 8.01 (s, 1H), 7.30–7.46
4.5. Sodium (2S,3R,5R)-3-formyl-3-methyl-7-oxo-4-thia-
1-azabicyclo[3.2.0]heptane-2-carboxylate-31-(O-methyl-
oxime)-4,4-dioxide (17b)
(m, 10H), 6.93 (s, 1H), 5.87–6.08 (m, 1H), 5.47 (s, 1H),
5.18–5.40 (m, 3H), 4.62 (d, 2H, J = 5.6 Hz), 3.76 (dd,
1H, J = 4.5 Hz, J = 16.7 Hz), 3.34 (dd, 1H, J = 1.5 Hz,
J = 16.7 Hz), 1.35 (s, 3H). Anal. (C24H24N2O6S) C, H,
N.
To a solution of compound 13b (294 mg, 0.664 mmol) in
dry anisole (768 lL) at ꢀ5 °C under N2, was added TFA
(758 mg, 512 lL) and stirred at 0 °C for 3.5 h. The mix-
ture was concentrated to dryness under reduced pressure
and the residue was triturated with a mixture of ether–
hexane, to the crude acid, which was collected by filtra-
tion (186 mg). The acid was treated with a solution of
NaHCO3 (113 mg) in water (10 mL), stirred at room
temperature for 10 min, and freeze-dried to give a fluffy
4.9. Sodium (2S,3R,5R)-3-formyl-3-methyl-7-oxo-4-thia-
1-azabicyclo[3.2.0]heptane-2-carboxylate-31-(O-allyl-
oxime)-4,4-dioxide (17d)
Compound 17d was prepared from 13d (300 mg,
0.64 mmol) and anisole/TFA (768 lL/493 lL) as de-
scribed for 17b to give 15 mg (7%) of 17d as a light

2854
O. A. Phillips et al. / Bioorg. Med. Chem. 13 (2005) 2847–2858
1
yellow fluffy solid after purification. H NMR (DMSO-
d6): d 7.59 (s, 1H), 5.90–6.05 (m, 1H), 5.20–5.40 (m, 2H),
5.02 (br d, 1H, J = 2.9 Hz), 4.58 (d, 2H, J = 5.6 Hz), 4.32
(s, 1H), 3.54 (dd, 1H, J = 4.1 Hz, J = 16.0 Hz), 3.14 (dd,
1H, J = 1.2 Hz, J = 16.0 Hz), 1.52 (s, 3H). Anal.
(C11H13N2NaO6S) C, H, N.
4.14. Diphenylmethyl (2S,3R,5R)-3-formyl-3-methy1-7-
oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate-31-
(O-t-butyloxime)-4,4-dioxide (13g)
A solution of compound 12 (1.81 g, 4.39 mmol) in a
mixture of ethanol (98%, 20 mL) and methylene chloride
(15 mL) was treated with t-butoxylamine hydrochloride
(0.5 g, 3.98 mmol) and triethylamine (0.282 g, 2.79
mmol). The mixture was stirred at room temperature
overnight, and worked up as described for 13a. Purifica-
tion of the crude by silica gel column chromatography
using hexane–ethyl acetate (3:2) gave 350 mg (16.5%)
4.10. Diphenylmethyl (2S,3R,5R)-3-formyl-3-methyl-7-
oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate-31-
[O-(2-hydroxyethyl)oxime]-4,4-dioxide (13e)
Compound 13e was synthesized from intermediate 12
(1.5 g, 3.52 mmol) and 2-hydroxy-ethyloxyamine hydro-
chloride (0.955 g, 3.519 mmol) as described for 13a to
afford 202 mg (12%) of 13e as a yellow gum, after silica
1
of 13g. H NMR (DMSO-d6): d 7.82 (s, 1H), 7.29–7.43
(m, 10H), 6.92 (s, 1H), 5.44 (s, 1H), 5.30 (dd, 1H,
J = 1.2 Hz, J = 4.5 Hz), 3.74 (dd, 1H, J = 4.5 Hz,
J = 16.6 Hz), 3.30 (dd, 1H, J = 1.2 Hz, J = 16.6 Hz),
1.38 (s, 3H), 1.24 (s, 9H). Anal. (C25H28N2O6S) C,
H, N.
1
gel column purification (benzene–ethyl acetate, 3:1). H
NMR (DMSO-d6): d 7.95 (s, 1H), 7.29–7.45 (m, 10H),
6.93 (s, 1H), 5.46 (s, 1H), 5.31 (br d, 1H, J = 2.5 Hz),
4.80 (t, 1H, J = 5.4 Hz), 4.12 (t, 2H, J = 5.0 Hz), 3.75
(dd, 1H, J = 4.2 Hz, J = 16.0 Hz), 3.60–3.72 (m, 2H),
3.35 (dd, 1H, J = 1.2 Hz, J = 16.0 Hz), 1.34 (s, 3H).
Anal. (C23H24N2O7S) C, H, N.
4.15. Sodium (2S,3R,5R)-3-formyl-3-methyl-7-oxo-4-
thia-1-azabicyclo[3.2.0]heptane-2-carboxylate-31-(O-t-
butyloxime)-4,4-dioxide (17g)
4.11. Sodium (2S,3R,5R)-3-formyl-3-methyl-7-oxo-4-
thia-1-azabicyclo[3.2.0]heptane-2-carboxylate-31-[O-(2-
hydroxyethyl)oxime]-4,4-dioxide (17e)
A solution of compound 13g (315 mg, 0.65 mmol) in a
mixture of ethyl acetate (6 mL) and ethanol (20 mL)
was hydrogenated over 10% Pd/C (310 mg) at 50 psi
for 8 h, and worked up as described for 17a. The crude
acid was treated with a solution of NaHCO3 (55 mg) in
water and freeze-dried to afford the crude product
(200 mg), which was purified by reverse-phase prepara-
tive TLC (acetonitrile–water, 8:1) to yield 66 mg (30%)
Compound 17e was prepared from 13e (188 mg,
0.398 mmol) by hydrogenation over 10% Pd/C
(0.190 g) at 50 psi for 2.5 h, as described for 17a to
give 84 mg (58%) of 17e as a pale yellow solid after
purification. 1H NMR (DMSO-d6): d 7.55 (s, 1H),
5.03 (br d, 1H, J = 2.5 Hz), 4.78 (br s, 1H), 4.34
(s, 1H), 4.07 (t, 2H, J = 4.9 Hz), 3.50–3.68 (m, 3H),
3.10 and 3.18 (2s, 1H), 1.53 (s, 3H). Anal.
(C10H13N2NaO7S) C, H, N.
1
of 17g. H NMR (DMSO-d6): d 7.44 (s, 1H), 4.98 (dd,
1H, J = 1.1 Hz, J = 4.0 Hz), 4.33 (s, 1H), 3.53 (dd, 1H,
J = 4.3 Hz, J = 16.0 Hz), 3.12 (dd, 1H, J = 1.1 Hz,
J = 16.0 Hz), 1.54 (s, 3H), 1.25 (s, 9H). Anal.
(C12H17N2NaO6S) C, H, N.
4.12. Diphenylmethyl (2S,3R,5R)-3-formyl-3-methyl-7-
oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate-31-
[O-(methoxymethyl)oxime]-4,4-dioxide (13f)
4.16. Diphenylmethyl (2S,3R,5R)-3-formyl-3-methyl-7-
oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate-31-
[O-(carboxamidomethyl)oxime]-4,4-dioxide (13h)
Compound 13f was prepared in the same manner as de-
scribed for compound 13b; from intermediate 12 and
methoxymethyloxyamine hydrochloride and purified
by silica gel column chromatography to give 33% of
Compound 13h was prepared from intermediate 12
(2.55 g, 6.163 mmol), carboxamidomethyloxy amine
hydrochloride (0.65 g, 5.136 mmol), and pyridine
(0.284 g), for 10 h as described for 13a. Purification by
silica gel column chromatography using hexane–ethyl
acetate (1:1) gave 472 mg (16%) of 13h. 1H NMR
(DMSO-d6): d 8.05 (s, 1H), 7.29–7.47 (m, 12H), 6.93
(s, 1H), 5.44 (s, 1H), 5.32 (d, 1H, J = 3.0 Hz), 4.49 (s,
2H), 3.76 (dd, 1H, J = 3.5 Hz, J = 16.0 Hz), 3.34 (dd,
1H, J = 1.1 Hz, J = 16.0 Hz), 1.32 (s, 3H). Anal.
(C23H23N3O7S) C, H, N.
1
13f. H NMR (CDCl3): d 7.64 (s, 1H), 7.28–7.40 (m,
10H), 6.93 (s, 1H), 5.17 (q, 2H, J = 7.3 Hz), 5.09 (s,
1H), 4.64 (br t, 1H), 3.50 (t, 2H), 3.40 (s, 3H), 1.34 (s,
3H). Anal. (C23H24N2O7S) C, H, N.
4.13. Sodium (2S,3R,5R)-3-formyl-3-methyl-7-oxo-4-
thia-1-azabicyclo[3.2.0]heptane-2-carboxylate-31-[O-
(methoxymethyl)oxime]-4,4-dioxide (17f)
4.17. Sodium (2S,3R,5R)-3-formyl-3-methyl-7-oxo-
4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate-31-[O-
(carboxamidomethyl)oxime]-4,4-dioxide (17h)
Compound 17f was prepared from 13f (370 mg,
0.783 mmol) and 10% Pd/C (370 mg) at 50 psi for 5 h
as described for 17a after treating with a solution of
NaHCO3 (70 mg) in water, and purified to give 180 mg
Compound 17h was prepared from 13h (380 mg,
0.783 mmol), 10% Pd/C (380 mg), and NaHCO3
(66 mg) as described for 17g, to give 190 mg (71%) of
1
(70%) of 17f. H NMR (D2O): d 7.81 (s, 1H), 5.17 (s,
2H), 5.09 (dd, 1H, J = 1.6 Hz, J = 4.2 Hz), 4.78 (s,
1H), 3.68 (dd, 1H, J = 4.3 Hz, J = 16.8 Hz), 3.48
(s, 3H), 3.40 (dd, 1H, J = 1.6 Hz, J = 16.8 Hz), 1.65 (s,
3H). Anal. (C10H13N2NaO7S) C, H, N.
1
17h after purification. H NMR (DMSO-d6): d 7.69 (s,
1H), 7.28 (d, 2H, J = 19.6 Hz), 5.03 (br d, 1H), 4.43 (s,
2H), 4.31 (s, 1H), 3.55 (dd, 1H, J = 4.0 Hz,

O. A. Phillips et al. / Bioorg. Med. Chem. 13 (2005) 2847–2858
2855
J = 16.0 Hz), 3.14 (dd, 1H, J = 1.0 Hz, J = 16.0 Hz),
1.51 (s, 3H). Anal. (C10H12N3NaO7S) C, H, N.
CO3 (124 mg, 1.47 mmol) in water as described for
17g, followed by usual purification and freeze-drying
to give 33 mg (12%) of 17j. H NMR (D2O): d 7.80 (s,
1
4.18. Diphenylmethyl (2S,3R,5R)-3-formyl-3-methyl-7-
oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate-31-
[O-(cyclopropylmethyl)oxime]-4,4-dioxide (13i)
1H), 5.13 (dd, 1H, J = 1.8 Hz, J = 2.5 Hz), 4.82 (s,
1H), 4.59 (s, 2H), 3.73 (dd, 1H, J = 4.3 Hz,
J = 16.8 Hz), 3.48 (dd, 1H, J = 1.8 Hz, J = 16.8 Hz),
1.68 (s, 3H). Anal. (C10H10N2Na2O8S) C, H, N.
Compound 13i was prepared from intermediate 12
(2.17 g,
hydrochloride (0.54 g, 4.37 mmol), and pyridine
5.24 mmol),
cyclopropyl-methyloxyamine
4.22. Diphenylmethyl (2S,3R,5R)-3-formyl-3-methyl-7-
oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate-31-
[O-(t-butyloxycarbonylmethyl)oxime]-4,4-dioxide (13k)
(283 lL) as described for 13a to give 13i 590 mg (23%)
as a white foam after column purification. H NMR
1
(DMSO-d6): d 7.93 (s, 1H), 7.25–7.50 (m, 10H), 6.92
(s, 1H), 5.43 (s, 1H), 5.32 (dd, 1H, J = 1.5 Hz,
J = 3.0 Hz), 3.92 (d, 2H, J = 7.1 Hz), 3.75 (dd, 1H,
J = 4.5 Hz, J = 16.6 Hz), 3.33 (dd, 1H, J = 1.2 Hz,
J = 16.6 Hz), 1.36 (s, 3H), 1.00–1.20 (m, 1H), 0.44–
0.55 (m, 2H), 0.23–0.30 (m, 2H). Anal. (C25H26N2O6S)
C, H, N.
Compound 13k was synthesized from the aldehyde 12
(326 mg, 0.79 mmol), t-butyloxycarbonylmethyloxy-
amine (116 mg, 0.79 mmol), and a drop of 6 M HCl,
under similar conditions described for 13a. Purified by
silica gel column chromatography (hexane–ethyl ace-
tate, 3:2) to give 131 mg (30.6%) of 13k. 1H NMR
(DMSO-d6): d 8.08 (s, 1H), 7.29–7.48 (m, 10H), 6.92
(s, 1H), 5.48 (s, 1H), 5.33 (br d, 1H, J = 3.0 Hz), 4.60
(ABq, 2H, J = 12.0 Hz), 3.75 (dd, 1H, J = 3.0 Hz,
J = 16.0 Hz), 3.34 (dd, 1H, J = 1.1 Hz, J = 16.0 Hz),
1.42 (s, 9H), 1.31 (s, 3H). Anal. (C27H30N2O8S) C, H, N.
4.19. Sodium (2S,3R,5R)-3-formyl-3-methyl-7-oxo-4-
thia-1-azabicyclo[3.2.0]heptane-2-carboxylate-31-[O-
(cyclopropylmethyl)oxime]-4,4-dioxide (17i)
Compound 17i was prepared from compound 13i
(350 mg, 0.73 mmol), after hydrogenation over 10%
Pd/C (450 mg) and treated with a solution of NaHCO3
(61 mg) in water as described for 17g, and purified to
4.23. Sodium (2S,3R,5R)-3-formyl-3-methyl-7-oxo-4-
thia-1-azabicyclo[3.2.0]heptane-2-carboxylate-31-[O-(t-
butyloxycarbonylmethyl)oxime]-4,4-dioxide (17k)
1
give 54 mg (21%) of 17i. H NMR (DMSO-d6): d 7.54
Compound 17k was obtained from 13k (317 mg,
0.584 mmol) after hydrogenation over 10% Pd/C
(300 mg) as described for 17g. Treatment with a solution
of NaHCO3 (26 mg) in water, and purification gave 17k
45 mg (36%). 1H NMR (DMSO-d6): d 7.66 (s, 1H), 5.02
(br d, 1H, J = 2.9 Hz), 4.55 (br d, 2H), 4.34 (s, 1H), 3.55
(dd, 1H, J = 4.1 Hz, J = 16.0 Hz), 3.15 (dd, 1H,
J = 1.0 Hz, J = 16.0 Hz), 1.51 (s, 3H), 1.43 (s, 9H). Anal.
(C14H19N2NaO8S) C, H, N.
(s, 1H), 5.01 (br d, 1H, J = 3.0 Hz), 4.30 (s, 1H), 3.90
(d, 2H, J = 7.2 Hz), 3.53 (dd, 1H, J = 4.2 Hz,
J = 16.2 Hz), 3.13 (dd, 1H, J = 1.1 Hz, J = 16.1 Hz),
1.52 (s, 3H), 1.03–1.20 (m, 1H), 0.47–0.57 (m, 2H),
0.22–0.30 (m, 2H). Anal. (C12H15N2NaO6S) C, H, N.
4.20. Diphenylmethyl (2S,3R,5R)-3-formyl-3-methyl-7-
oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate-31-
[O-(diphenylmethyloxycarbonylmethyl)oxime]-4,4-diox-
ide (13j)
4.24. Diphenylmethyl (2S,3R,5R)-3-formyl-3-methyl-7-
oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate-31-
[O-[2-(1H-1,2,3-triazol-1-yl)ethyl]oxime]-4,4-dioxide
(13l)
To a solution of the aldehyde 12 (1.2 g, 2.90 mmol) in a
mixture of ethanol (15 mL) and methylene chloride
(15 mL), was added diphenylmethyloxycarbonyl meth-
yloxyamine (2.90 mmol, generated in situ from diphe-
nylmethyloxycarbonyl methyloxy phthalimido by
treatment with hydrazine), and treated with two drops
of concd HCl. The mixture was stirred for 14 h at room
temp; and worked up as described for 13a. Purification
by column chromatography (hexane–ethyl acetate,
3:2), gave 13j 645 mg (34%) as a foam. 1H NMR
(DMSO-d6): d 8.10 (s, 1H), 7.12–7.42 (m, 20H), 6.82
and 6.85 (2s, 2H), 5.46 (s, 1H), 5.30 (br d, 1H), 4.83
(ABq, 2H, J = 16.0 Hz), 3.69 (dd, 1H, J = 3.0 Hz,
J = 16.0 Hz), 3.25 (dd, 1H, J = 1.0 Hz, J = 16.0 Hz),
1.25 (s, 3H). Anal. (C36H32N2O8S) C, H, N.
Compound 13l was synthesized from the penam alde-
hyde 12 (3.3 g, 7.98 mmol), 1,2,3-triazol-1-ylethyloxy-
amine hydrochloride (0.87 g, 7.98 mmol) and pyridine
(0.5 g) as described for 13a, followed by purification
by silica gel column chromatography (hexane–ethyl ace-
1
tate) to give 900 mg (22%) of 13l as foam. H NMR
(DMSO-d6): d 7.98 and 8.00 (2s, 2H), 7.70 (s, 1H),
7.33–7.40 (m, 10H), 6.92 (s, 1H), 5.46 (s, 1H), 5.33 (br
d, 1H, J = 3.0 Hz), 4.65 (br d, 2H), 4.47 (br t, 2H),
3.75 (dd, 1H, J = 4.0 Hz, J = 15.1 Hz), 3.32 (dd, 1H,
J = 1.0 Hz, J = 15.0 Hz), 1.33 (s, 3H). Anal.
(C25H25N5O6S) C, H, N.
4.21. (2S,3R,5R)-3-Formyl-3-methyl-7-oxo-4-thia-1-aza-
bicyclo[3.2.0]heptane-2-carboxylic acid-31-[O-(carb-
oxymethyl)oxime]disodium salt 4,4-dioxide (17j)
4.25. Sodium (2S,3R,5R)-3-formyl-3-methyl-7-oxo-4-
thia-1-azabicyclo[3.2.0]heptane-2-carboxylate-31-[O-(2-
(1H-1,2,3-triazol-1-yl)ethyl)oxime]-4,4-dioxide (17l)
Compound 17j was synthesized from compound 13j
(480 mg, 0.74 mmol), after hydrogenation over 10%
Pd/C (500 mg) and treatment with a solution of NaH-
Compound 17l was prepared from compound 13l
(400 mg, 0.764 mmol) after hydrogenation over 10%
Pd/C (420 mg) as described for 17h, and purified to give

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O. A. Phillips et al. / Bioorg. Med. Chem. 13 (2005) 2847–2858
170 mg (59%) of 17l as a fluffy solid. 1H NMR (DMSO-
d6): d 8.16 (s, 1H), 7.70 (s, 1H), 7.58 (s, 1H), 5.05 (br d,
1H), 4.60–4.72 (m, 2H), 4.38–4.49 (m, 2H), 4.35 (s, 1H),
3.54 (dd, 1H, J = 3.0 Hz, J = 16.0 Hz), 3.14 (dd, 1H,
J = 0.9 Hz, J = 16.0 Hz), 1.55 (s, 3H). Anal.
(C12H14N5NaO6S) C, H, N.
(Na2SO4). Purification by silica gel column chromato-
graphy (hexane–ethyl acetate) gave 400 mg (29%) of
14a. H NMR (DMSO-d6): d 11.69 (s, 1H, exchanged
with D2O), 7.56 (s, 1H), 7.30–7.50 (m, 10H), 6.90 (s,
1H), 5.77 (s, 1H), 5.34 (br d, 1H), 3.75 (dd, 1H,
J = 3.0 Hz, J = 16.1 Hz), 3.50 (dd, 1H, J = 1.2 Hz,
J = 16.0 Hz), 2.00 (s, 3H), 1.39 (s, 3H). Anal.
(C23H23N3O6S) C, H, N.
1
4.26. Diphenylmethyl (2S,3R,5R)-3-formyl-3-methyl-7-
oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate-31-
[O-[(pyridin-2-yl)methyl]oxime]-4,4-dioxide (13m)
4.30. Sodium (2S,3R,5R)-3-formyl-3-methyl-7-oxo-4-
thia-1-azabicyclo[3.2.0]heptane-2-carboxylate-31-(acetyl-
hydrazone)-4,4-dioxide (18a)
Compound 13m was prepared from the penam aldehyde
12 (3.5 g, 8.47 mmol), pyridin-2-ylmethyloxyamine
hydrochloride (1.23 g) and pyridine (0.964 g) as de-
scribed for 13a, and purified by silica gel column chro-
matography (hexane–ethyl acetate) to give 700 mg
A solution of the 14a (320 mg, 0.68 mmol) in a mixture
of ethanol (12 mL) and ethyl acetate (18 mL), was
hydrogenated over 10% Pd/C (350 mg) at 50 psi for
8 h; and worked up as described for 17a to give the
crude acid. The crude acid was treated with a solution
NaHCO3 (57 mg) in water (10 mL) and the solution
was freeze-dried to give a solid, which was purified by
reverse-phase preparative TLC (acetonitrile–water,
1
(16%) of 13m. H NMR (DMSO-d6): d 8.54–8.57 (m,
1H), 8.13 (s, 1H), 7.66–7.74 (m, 1H), 7.30–7.45 (m,
12H), 6.91 (s, 1H), 5.49 (s, 1H), 5.32 (dd, 1H,
J = 1.0 Hz, J = 3.0 Hz), 5.24 (s, 2H), 3.75 (dd, 1H,
J = 4.5 Hz, J = 16.6 Hz), 3.34 (dd, 1H, J = 1.0 Hz,
J = 16.6 Hz), 1.31 (s, 3H). Anal. (C27H25N3O6S) C, H,
N.
1
7:1), to give 50 mg (23%) of 18a. H NMR (D2O): d
7.69 (s, 1H), 5.13 (br d, 1H), 4.88 (s, 1H), 3.73 (dd,
1H, J = 3.0 Hz, J = 16.0 Hz), 3.40 (dd, 1H, J = 1.0 Hz,
J = 16.0 Hz), 2.12 (s, 3H), 1.71 (s, 3H). Anal.
(C10H12N3NaO6S) C, H, N.
4.27. Sodium (2S,3R,5R)-3-formyl-3-methyl-7-oxo-4-
thia-1-azabicyclo[3.2.0]heptane-2-carboxylate-31-[O-
[(pyridin-2-yl)methyl]oxime]-4,4-dioxide (17m)
4.31. Diphenylmethyl (2S,3R,5R)-3-methyl-7-oxo-3-[N-
(2-oxo-1-imidazolidinyl)formimidoyl]-4-thia-1-azabicy-
clo[3.2.0]heptane-2-carboxylate-4,4-dioxide (14b)
This compound was prepared from 13m (410 mg,
0.789 mmol) hydrogenated over 10% Pd/C (410 mg) as
described for 17l, and purified to give 20 mg (7%) of
1
17m as the minor component. H NMR (DMSO-d6): d
Compound 14b was prepared from the aldehyde 12
(600 mg, 1.21 mmol) and 1-amino-2-oxo-imidazolidine
(122 mg, 1.21 mmol) as described for 14a. Purification
by silica gel column chromatography (hexane–ethyl ace-
tate, 4:1) gave 190 mg (26%) of 14b as a foam. 1H NMR
(CDCl3): d 7.23–7.40 (m, 10H), 6.95 (s, 1H), 6.89 (s, 1H),
5.40–5.62 (br m, 1H, exchanged with D2O), 5.05 (s, 1H),
4.69 (br t, 1H), 3.55–3.80 (m, 4H), 3.42–3.55 (m, 2H),
1.52 (s, 3H). Anal. (C24H24N4O6S) C, H, N.
8.56 (d, 1H), 7.78–7.86 (m, 1H), 7.72 (s, 1H), 7.34–
7.43 (m, 2H), 5.20 (s, 2H), 5.04 (br d, 1H), 4.36 (s,
1H), 3.56 (dd, 1H, J = 4.0 Hz, J = 16.0 Hz), 3.15 (dd,
1H, J = 1.0 Hz, J = 16.1 Hz), 1.52 (s, 3H). Anal.
(C14H14N3NaO6S) C, H, N.
4.28. Sodium (2S,3R,5R)-3-formyl-3-methyl-7-oxo-4-
thia-1-azabicyclo[3.2.0]heptane-2-carboxylate-31-[O-
[(piperidin-2-yl)methyl]oxime]-4,4-dioxide (17n)
4.32. Sodium (2S,3R,5R)-3-methyl-7-oxo-3-[N-(2-oxo-1-
imidazolidinyl)formimidoyl]-4-thia-1-azabicyclo-
[3.2.0]heptane-2-carboxylate-4,4-dioxide (18b)
Compound 17n was obtained as the major component
as a fluffy solid 80 mg (27%). H NMR (D2O): d 7.76
(d, 1H, J = 2.2 Hz), 5.11 (br d, 1H), 4.79 (s, 1H), 4.18–
1
4.45 (m, 2H), 3.49 (dd, 1H, J = 4.2 Hz, J = 16.8 Hz),
d, 3H,
J = 16.8 Hz), 2.99 (t, 1H, J = 9.0 Hz), 1.46–1.89 (m,
overlapped with a s, 9H). Anal. (C14H20N3NaO6S) C,
H, N.
A solution of 14b (190 mg, 0.383 mmol) in ethanol
(20 mL) was hydrogenated over 10% Pd/C (200 mg) at
50 psi for 2.5 h; and worked up as described for 17a.
The crude acid was treated with a solution of NaHCO3
(40 mg) in water (10 mL), and freeze-dried to give a
solid, which was purified by reverse-phase preparative
3.38–3.61 (br m, overlapped with
a
1
4.29. Diphenylmethyl (2S,3R,5R)-3-formyl-3-methyl-7-
oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate-31-
(acetylhydrazone)-4,4-dioxide (14a)
TLC to give 17 mg (12%) of 18b. H NMR (D2O): d
6.97 (s, 1H), 5.10 (dd, 1H, J = 1.6 Hz, J = 4.0 Hz),
4.77 (s, 1H), 3.50–3.88 (m, 5H), 3.40 (dd, 1H,
J = 1.6 Hz, J = 16.6 Hz), 1.69 (s, 3H). Anal. (C11H13-
N4NaO6S) C, H, N.
To a stirred solution of penam aldehyde 12 (1.2 g,
2.9 mmol) in a mixture of ethanol (15 mL) and methyl-
ene chloride (15 mL), was added acetic hydrazide
(221 mg, 2.9 mmol) followed by one drop of concd
HCl. The mixture was stirred at room temperature for
14 h; the solvent was removed under reduced pressure
and the residue was taken up in methylene chloride
(100 mL), washed with water, brine, and dried
4.33. Diphenylmethyl (2S,3R,5R)-3-formyl-3-methyl-7-
oxo-4-thia-1-azabicylco[3.2.0]heptane-2-carboxylate-31-
(benzoylhydrazone)-4,4-dioxide (14c)
Compound 14c was prepared from the aldehyde 12
(0.8 g, 1.94 mmol) and benzoic hydrazide (0.263 g,

O. A. Phillips et al. / Bioorg. Med. Chem. 13 (2005) 2847–2858
2857
1.94 mmol) as described for 14a. Purification by silica
gel column chromatography (hexane–ethyl acetate) gave
530 mg (51%) of 14c as a foam. 1H NMR (DMSO-d6): d
8.07 (s, 1H), 7.28–8.04 (m, 16H), 6.88 (s, 1H), 5.40 (br s,
1H), 5.31 (s, 1H), 3.79 (dd, 1H, J = 4.0 Hz, J = 16.0 Hz),
3.36 (dd, 1H, J = 1.0 Hz, J = 16.0 Hz), 1.47 (s, 3H).
Anal. (C28H25N3O6S) C, H, N.
scribed for 14a to give the crude foam (2.4 g). Purifica-
tion by silica gel column chromatography (hexane–
1
ethyl acetate, 1:1) gave 480 mg (18%) of 14e. H NMR
(DMSO-d6): d 10.67 (s, 1H), 7.25–7.48 (m, 10H), 6.92
(s, 1H), 6.61 (br s, 2H), 5.39 (s, 1H), 5.33 (br d, 1H,
J = 2.90 Hz), 3.73 (dd, 1H, J = 4.5 Hz, J = 16.5 Hz),
3.34 (dd, 1H, J = 1.0 Hz, J = 16.5 Hz), 1.43 (s, 3H).
Anal. (C22H22N4O6S) C, H, N, S.
4.34. Sodium (2S,3R,5R)-3-formyl-3-methyl-7-oxo-4-
thia-1-azabicyclo[3.2.0]heptane-2-carboxylate-31-(ben-
zoylhydrazone)-4,4-dioxide (18c)
4.38. Sodium (2S,3R,5R)-3-formyl-3-methyl-7-oxo-4-
thia-1-azabicyclo[3.2.0]heptane-2-carboxylate-31-semi-
carbazone-4,4-dioxide (18e)
Compound 18c was prepared from the ester 14c
(310 mg, 0.58 mmol), hydrogenated over 10% Pd/C
(350 mg) as described for 18a, treated with a solution
of NaHCO3 (41 mg) in water, and purified on reverse-
phase preparative TLC to give 100 mg (59%) of
This compound was prepared as described for com-
pound 18a, from compound 14e (380 mg, 0.81 mmol),
by hydrogenation over 10% Pd/C at 50 psi for 24 h
and treatment with a solution of NaHCO3 (68 mg) in
water. Purification on reverse-phase preparative TLC
gave 18e as a fluffy solid 126 mg (62%). 1H NMR
(DMSO-d6): d 10.42 (br s, 1H), 7.22 (s, 1H), 6.33 (br s,
H), 4.97 (br s, 1H), 4.19 (s, 1H), 3.53 (dd, 1H,
J = 4.0 Hz, J = 16.0 Hz), 3.10 (dd, 1H, J = 1.0 Hz,
J = 15.9 Hz), 1.52 (s, 3H). Anal. (C9H11N4NaO6S) C,
H, N.
1
18c. H NMR (DMSO-d6): d 7.80–8.05 (m, 3H), 7.40–
7.60 (m, 4H), 5.07 (br s, 1H), 4.30 (br s, 1H), 3.58 (dd,
1H, J = 3.0 Hz, J = 16.0 Hz), 3.17 (dd, 1H, J = 1.0 Hz,
J = 16.1 Hz), 1.19 (s, 3H). Anal. (C15H14N3NaO6S) C,
H, N.
4.35. Diphenylmethyl (2S,3R,5R)-3-formyl-3-methyl-7-
oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate-31-
thiosemicarbazone-4,4-dioxide (14d)
4.39. Diphenylmethyl (2S,3R,5R)-3-formyl-3-methyl-7-
oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate-31-
(benzyloxycarbonyl-hydrazone)-4,4-dioxide (14f)
Compound 14d was prepared from the penam aldehyde
12 (1.7 g, 4.11 mmol) and thiosemicarbazide (0.375 g,
4.11 mmol), as described for 14a and purified by silica
gel column chromatography (hexane–ethyl acetate) to
give 900 mg (45%) of 14d. 1H NMR (DMSO-d6): d
11.75 (br s, 1H, exchanged with D2O), 8.41 (br s, 1H, ex-
changed with D2O), 8.06 (br s, 1H, exchanged with
D2O), 7.46 (s, 1H), 7.20–7.40 (m, 10H), 6.80 (s, 1H),
5.45 (s, 1H), 5.25 (br d, 1H, J = 2.5 Hz), 3.65 (dd, 1H,
J = 4.4 Hz, J = 16.1 Hz), 3.22 (dd, 1H, J = 1.0 Hz,
J = 16.0 Hz), 1.35 (s, 3H). Anal. (C22H22N4O5S2) C,
H, N, S.
Compound 14f was prepared from the penam aldehyde
12 (2.0 g, 4.84 mmol), N-benzyloxycarbonyl hydrazine
hydrochloride (891 mg, 4.3978 mmol), and pyridine
(313 mg, 320 lL) as described for 14e. Purification by
silica gel column chromatography (hexane–ethyl ace-
1
tate) gave 745 mg (27%) of 14f as a foam. H NMR
(DMSO-d6): d 11.75 (br s, 1H), 7.61 (s, 1H), 7.22–7.41
(m, 15H), 6.87 (s, 1H), 5.20–5.40 (m, 4H), 3.75 (dd,
1H, J = 4.7 Hz, J = 16.6 Hz), 3.32 (dd, 1H, J = 1.0 Hz,
J = 16.6 Hz), 1.38 (s, 3H). Anal. (C29H27N3O7S) C, H,
N, S.
4.36. Sodium (2S,3R,5R)-3-formyl-3-methyl-7-oxo-4-
thia-1-azabicyclo[3.2.0]heptane-2-carboxylate-31-thio-
semicarbazone-4,4-dioxide (18d)
4.40. Sodium (2S,3R,5R)-3-formyl-3-methyl-7-oxo-4-
thia-1-azabicyclo[3.2.0]heptane-2-carboxylate-31-(benzyl-
oxycarbonyl-hydrazone)-4,4-dioxide (18f)
Compound 18d was prepared from 14d (300 mg,
0.617 mmol) and 10% Pd/C (600 mg) and H2, as de-
scribed for 18a. Treatment with a solution of NaHCO3
(47 mg), and purification on reverse-phase preparative
Compound 18f was prepared by catalytic hydrogenation
of 14f (292 mg, 0.520 mmol), over 10% Pd/C at 50 psi
for 24 h as described for 18a. Treatment with a solution
of NaHCO3 in water, followed by purification on re-
verse-phase TLC gave 26 mg (12%) of 18f as a white
1
TLC gave 38 mg (18%) of 18d. H NMR (DMSO-d6):
d 11.05 (br s, 1H), 7.93 (br s, 1H), 7.61 (s, 1H), 7.44 (br
s, 1H), 6.51 (s, 1H), 4.20 (br d, 1H), 3.10 (br d, 1H,
J = 14.9 Hz), 2.80 (dd, 1H, J = 3.2 Hz, J = 15.0 Hz),
1.90 (s, 3H). Anal. (C9H11N4NaO5S2) C, H, N.
1
fluffy solid. H NMR (DMSO-d6): d 11.34 (br s, 1H),
7.23–7.45 (m, 5H), 5.11 (s, 2H), 4.97 (br d, 1H,
J = 2.9 Hz), 4.15 (s, 1H), 3.52 (dd, 1H, J = 3.9 Hz,
J = 16.0 Hz), 3.10 (dd, 1H, J = 1.0 Hz, J = 16.0 Hz),
1.50 (s, 3H). Anal. (C16H16N3NaO7S) C, H, N.
4.37. Diphenylmethyl (2S,3R,5R)-3-formyl-3-methyl-7-
oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate-31-
semicarbazone-4,4-dioxide (14e)
5. Assay of b-lactamase activity and b-lactamase
inhibitory activity
To a stirred solution of the penam aldehyde 12 (2.3 g,
5.6 mmol) in a mixture of ethanol (10 mL) and methyl-
ene chloride (10 mL) was treated with semicarbazide
hydrochloride (0.477 g, 4.3 mmol) and pyridine
(0.204 g, 2.58 mmol). The reaction was worked up as de-
The b-lactamase enzymes were obtained as crude cell ex-
tracts (from E. coli TEM-1, K. pneumoniae CTX-1, and
P. aeruginosa 46012 cephalosporinase) prepared by

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O. A. Phillips et al. / Bioorg. Med. Chem. 13 (2005) 2847–2858
penicillin as b-lactamase inhibitors IL Farmaco 2002, 57,
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the UV method.26 The amount of protein was measured
by the Bio-Rad Protein Assay. The b-lactamase inhibi-
tory activity was performed by UV spectrophotometry
using freshly prepared antibiotic solutions in 50 mM
phosphate buffer at pH 7.4. The assay was run at
30 °C, by monitoring the hydrolysis of the substrates
at the following wavelengths; 235 nm for ampicillin
(100 lM) and 260 nm for cephaloridine (100 lM) as
substrates. The substrates were added to the reaction
mixture after pre-incubation of the enzyme with b-lacta-
mase inhibitor for 5 min. Clavulanic acid, sulbactam
and tazobactam were used as reference drugs in the
determination of 50% inhibitory concentration (IC50,
lM) values.
8. Li, C.; Nicolau, D. P.; Lister, P. D.; Quintiliani, R.;
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6. In vitro susceptibility testing
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The in vitro synergistic antibacterial activities were
determined by the agar dilution method. A total of 106
cells of bacterial suspension per mL were spotted on
the Mueller Hinton agar containing the antibiotics
(piperacillin, PIP or ceftazidime, CAZ) alone or in com-
bination with 5 lg/mL of the inhibitor. The minimum
inhibitory concentration (MIC, lg/mL) values defined
as the lowest antibiotic concentration that prevented vis-
ible bacterial growth were recorded after incubation
overnight at 37 °C. Tazobactam was synthesized in our
laboratories, while ceftazidime, clavulanic acid, pipera-
cillin, and sulbactam were obtained as commercial
preparations.
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The authors thank NAEJA Pharmaceuticals Inc. and
Kuwait University for their support during the course
of this project and in the preparation of the manuscript.
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