Synthesis and biological evaluation of 6-bromo-6-substituted penicillanic acid derivatives as beta-lactamase inhibitors.

Article abstract of DOI:10.1016/S0014-827X(02)01261-2

The synthesis of a selected set of 6-bromopenicillanic acid derivatives with an additional C6 substituent is reported. All these substances were tested as inhibitors of class A and C beta-lactamase enzymes derived from Escherichia coli (TEM-1) and E. cloa

Full text of DOI:10.1016/S0014-827X(02)01261-2

Il Farmaco 57 (2002) 663–669
www.elsevier.com/locate/farmac
Synthesis and biological evaluation of 6-bromo-6-substituted
penicillanic acid derivatives as b-lactamase inhibitors
A. Bedini a,*, C. Balsamini a, B. Di Giacomo a, A. Tontini a, B. Citterio b, L. Giorgi a,
E. Di Modugno c, G. Tarzia a
a Istituto di Chimica Farmaceutica, Uni6ersita` di Urbino, Piazza Rinascimento 6, 61029 Urbino, Italy
b Istituto di Scienze Tossicologiche Igienistiche ed Ambientali, Uni6ersita` di Urbino, Via S. Chiara 27, 61029 Urbino, Italy
c GlaxoSmithKline Medicines Research Centre, Via Fleming 4, 37135 Verona, Italy
Received 7 November 2001; accepted 6 March 2002
Abstract
The synthesis of a selected set of 6-bromopenicillanic acid derivatives with an additional C6 substituent is reported. All these
substances were tested as inhibitors of class A and C b-lactamase enzymes derived from Escherichia coli (TEM-1) and E. cloacae
(P99). As 6-(1-hydroxyethyl) derivatives 4c and 6c were found to be weak b-lactamase inhibitors, they were further investigated
in combination with amoxicillin against a series of b-lactamase-producing bacterial strains. Some structure-activity relationships
´
are discussed. © 2002 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
Keywords: b-Lactams; b-Lactamase inhibitors; Brobactam
class C b-lactamases, there is an urgent need to widen
1. Introduction
the spectrum of the current b-lactamase inhibitors.
6b-Bromopenicillanic acid (brobactam) (Fig. 1) is an
extensively studied b-lactamase inhibitor whose mecha-
nism of action involves the nucleophilic displacement of
bromide to give a dihydrothiazine derivative that is
irreversibly bound to the active site [6,7] (Fig. 2).
Brobactam is as potent as clavulanic acid and inhibits
the same range of b-lactamase enzymes [6].
Herein we report an investigation of some 6-bro-
mopenicillanic acid derivatives (Fig. 3) bearing a second
substituent in C-6 to test if a further substituent in this
position affects the inhibitory activity. The choice of
the second substituent was made on the basis of the
hypothesis that electron-withdrawing substituents could
facilitate the displacement of bromine, which is the
The most common resistance mechanism adopted by
bacteria against b-lactam antibiotics is the production
of b-lactamases [1,2]. These enzymes can be divided
into four classes (A, B, C and D) on the basis of their
primary structure and homology [3] being class A (peni-
cillinases) and C (cephalosporinases) the most clinically
relevant. b-lactamases are able to catalyze the hydroly-
sis of the b-lactam ring, thus leading to inactive com-
pounds [4]. Clavulanic acid, sulbactam and tazobactam
(Fig. 1) are marketed and widely used in association
with piperacillin (tazobactam, Zosyn®, American Home
Products), amoxicillin (clavulanic acid, Augmentin®,
SmithKline Beecham) and ampicillin (sulbactam,
Unasyn®, Pfizer). Although these compounds are effec-
tive inhibitors of class A b-lactamases, they are ineffec-
tive or less effective against class C enzymes [1,5].
Because of the rapid spreading of bacteria that produce
Fig. 1. Structure of representative b-lactam inhibitors of b-lacta-
mases.
* Corresponding author
E-mail address: annalida@uniurb.it (A. Bedini).
´
0014-827X/02/$ - see front matter © 2002 Editions scientifiques et me´dicales Elsevier SAS. All rights reserved.
PII: S0014-827X(02)01261-2

664
A. Bedini et al. / Il Farmaco 57 (2002) 663–669
crucial step in the inhibition of b-lactamase; moreover,
the 6b-(1-hydroxyethyl) group is known to improve the
inhibitory activity of sulbactam against class C b-lacta-
mases [5,8] and to be responsible for the high b-lacta-
mase-inhibitory activity in carbapenems [9].
Fig. 2. Enzyme-bound dihydrothiazine derivative.
2. Chemistry
The synthesis of the 6-bromopenicillanic acid deriva-
tives (3a, 3a*, 3b, 3b*, 4c–d, 6c–d, 8b, 8b*) was per-
formed following the route shown in Fig. 4, 5 and 6
using benzhydryl 6,6-dibromopenicillanate as starting
material [10]. This compound underwent metal–halo-
gen exchange with methylmagnesium bromide in THF
at −78 °C followed by reaction with freshly distilled
benzaldehyde or acetaldehyde, giving isomeric mixtures
of the corresponding bromohydrins (1a [11], 1b).
1b was found to be a diastereomeric mixture of three
different 6-(1-hydroxyethyl) derivatives and it was par-
tially separated by chromatography, affording 1c as a
single diastereoisomer and 1d as a mixture of two
diastereoisomers (ratio 1c/1d 2:1).
Fig. 3. New 6-bromopenicillanic acid derivatives studied.
The configurations of the hydroxyethyl group and of
the bromine atom were assigned according to previous
1
crystallographic and H NMR studies carried out on
benzyl ester analogues [12].
1c and 1d were separately oxidized with pyridinium
dichromate in CH2Cl2 to the corresponding acetyl
derivatives 2b and 2b*, which were then deprotected
using trifluoroacetic acid–anisole [13] for 2b or m-
cresol [14] for 2b* to obtain their corresponding acids
3b and 3b* (Fig. 4).
Hydroxybenzyl adducts (1a [11], diastereomeric ratio
not determined) were directly oxidized with pyridinium
dichromate in CH2Cl2 to the corresponding benzoyl
derivatives 2a and 2a* which, after separation by flash-
chromatography, were deprotected with m-cresol [14]
to their corresponding acids 3a and 3a* (Fig. 4). The
configuration of the 6-benzoyl derivatives was assigned
as 6b for 2a and 6a for 2a* on the basis of the observed
Fig. 4. Reagents and conditions: (i) (a) CH3MgBr, THF, −78 °C,
(b) benzaldehyde or acetaldehyde; (ii) pyridinium dichromate,
CH2Cl2, r.t.; (iii) m-cresol; 50 °C for 3a, 3a*, 3b*; TFA, anisole for
3b.
1
chemical shift of the proton in position C-5 in the H
NMR spectrum compared with the shift of the same
proton in the 6-acyl derivatives 2b and 2b*.
1c and 1d were deprotected with trifluoroacetic acid–
anisole [13], affording 4c and 4d, respectively, or oxi-
dized to their corresponding sulfones 5c and 5d using
m-chloroperoxybenzoic acid and then deprotected to 6c
and 6d (Fig. 5).
Attempts to oxidize the benzhydryl 6-acyl sulfide
derivatives to the corresponding sulfones by standard
oxidation procedures (m-CPBA or KMnO4) lead to
extensive decomposition and the same result was ob-
tained in attempts to oxidize 5c or 5d with pyridinium
dichromate to obtain 6-bromo-6-acetyl derivatives.
Fig. 5. Reagents and conditions: (i) m-chloroperoxybenzoic acid,
CH2Cl2; (ii) TFA, anisole.

A. Bedini et al. / Il Farmaco 57 (2002) 663–669
665
treatment with methoxylamine hydrochloride and pyri-
dine; intermediates 7b and 7b* were subjected to depro-
tection by means of trifluoroacetic acid–anisole (Fig.
6).
3. Results and discussion
The b-lactamase-inhibitory activity of the 6-bro-
mopenicillanic acid derivatives studied as inhibitors of
P99 and TEM-1 b-lactamase derived from E. cloacae
and Escherichia coli, respectively, is reported in Table 1.
Compounds 4c, 6c and 6d were found to be weak
inhibitors of TEM-1 b-lactamase, and 6c was able to
inhibit the class-C b-lactamase P99.
Fig. 6. Reagents and conditions: (i) methoxylamine hydrochloride,
pyridine, Na2SO4, r.t.; (ii) TFA, anisole.
Table 1
b-Lactamase-inhibitory activity of 6-bromopenicillanic acid deriva-
tives against isolated TEM-1 and P99 enzymes, IC50 (mg ml−1
)
Table 2 reports the in vitro activity of 4c and 6c both
alone and in 1:2 or 1:1 combinations with the known
antibiotic amoxicillin. A similar combination of clavu-
lanic acid and amoxicillin was used as a reference
standard. These data show that neither 4c nor 6c
possess antibacterial activity against the selected bacte-
rial strains. These combinations with amoxicillin did
not provide the synergic effect against class A b-lacta-
mase-producing bacteria that was observed with amoxi-
cillin–clavulanic acid, nor did they give a synergic
effect against class C b-lactamase-producing strains.
These results show that the presence of a carbonyl or
methoxyimino moiety in position C-6 in addition to the
bromine is detrimental for b-lactamase inhibitory acti-
vity regardless of their configuration.
Comp.
TEM-1 (E. coli )
P99 (E. cloacae)
Clav. acid
3a
3a*
3b
3b*
4c
4d
6c
6d
8b
0.7 a
N I b
N I b
N I b
N I b
825
\4000 a
N I b
N I b
N I b
N I b
N I b
N I b
61
N I b
760
1000
N I b
N I b
N I b
N I b
N I b
8b*
a See ref. [6,15].
b N I: non-inhibiting.
The three substances active as weak inhibitors of
isolated b-lactamase enzymes (Table 1) each bear a
hydroxyethyl group in C-6, giving further confirmation
Methoxyimino derivatives 8b and 8b* were synthe-
sized from 6-bromo-6-acetyl derivatives 2b and 2b* by
Table 2
In vitro synergy study of compounds 4c and 6c in combination with amoxicillin
Comp.
E. coli,
E. coli,
1850E,
W.T. b
E. coli,
18520E,
P.M. c
E. coli, 1919E,
P.M. c,
E. cloacae,
1051E,
E. cloacae,
1321E,
P99−
S. aureus,
S. aureus,
853E,
ATTC, 35218,
ATTC, 43387,
b-lact.+ a
TEM-1 d
P99+ e
b-lact.+ a
PC1+ f
Clavulanic acid
Amoxicillin
Amox.+clav. 2:1
Amox.+clav. 1:1
4c
Amox.+4c 2:1
Amox.+4c 1:1
6c
32
\256
8
64
8
8
8
\256
16
8
\256
16
8
64
8
4
4
\256
8
8
\256
4
4
64
\256
16
64
\256
128
64
4
4
4
\256
4
4
\256
4
4
32
256
B=2
B=2
\256
128
128
\256
128
32
128
B=2
B=2
\256
128
128
\256
128
8
8
64
\256
\256
\256
\256
\256
\256
\256
\256
\256
\256
\256
\256
\256
\256
\256
\256
\256
\256
Amox.+6c 2:1
Amox.+6c 1:1
128
128
The MIC is expressed as mg ml−1
a b-Lactamase-producing strain.
b W.T.=wild type.
.
c P.M.=permeable membrane.
d TEM-1-producing strain.
e P99-producing strain.
f PC1-producing strain.

666
A. Bedini et al. / Il Farmaco 57 (2002) 663–669
of the importance of this moiety in the inhibitory
activity [16]. In the case of 6c, the presence of the
sulfone extends this activity to class C enzymes. The
6b-configuration of the hydroxyethyl group in deriva-
tive 6c is in accordance with the configuration of class
C inhibitor derivatives lacking the bromine atom [16].
In any case, the reduced activity of our compounds
with respect to that of their debrominated analogues
shows that the double substitution we attempted is
detrimental for b-lactamase inhibitory activity.
4.1.2. 6h-Bromo-6i-(1-hydroxy-ethyl)-3,3-dimethyl-7-
oxo-4-thia-1-aza-bicyclo[3.2.0]heptane-2-carboxylic
acid benzhydryl ester (1c) and 6i-bromo-6h-(1-hydroxy-
ethyl)-3,3-dimethyl-7-oxo-4-thia-1-aza-bicyclo[3.2.0]-
heptane-2-carboxylic acid benzhydryl ester (1d)
Benzhydryl 6,6-dibromo penicillanate [10] (800 mg,
1.52 mmol) in dry THF (14 ml) at −78 °C under
nitrogen was treated, dropwise, with a solution of
methylmagnesium bromide in diethyl ether (3 M; 0.64
ml, 1.92 mmol) and the resulting solution was allowed
to react for 20 min under stirring. Freshly distilled
acetaldehyde (0.6 ml, 10.7 mmol) in dry THF (2.5 ml)
was then added. After 20 min saturated aqueous am-
monium chloride was added and the reaction mixture
extracted with EtOAc. The organic phases were com-
bined, washed with water and brine, dried on Na2SO4
and the solvent was evaporated to yield a crude mate-
rial which was subjected to flash-chromatography on
silica gel (cyclohexane–EtOAc 7:3).
4. Experimental
4.1. Chemistry
Melting points were taken on a Buchi SMP-510
capillary apparatus and are uncorrected. Infrared spec-
tra (IR) were recorded on a Bruker FT-48 spectrometer
and absorbances are reported in w (cm−1). Elemental
analyses were performed on a Carlo Erba analyzer and
were within 90.4% of the theoretical values (when
solvent is reported in the analytical formula, it has been
detected even by 1H NMR analysis). EI-MS spectra
(70 eV) were taken on a Fison-Trio 1000 instrument.
1H NMR spectra were recorded on a Bruker 200 spec-
trometer, chemical shifts are reported in ppm (l scale).
Column chromatography purifications were performed
under ‘flash’ conditions using Merck 230–400 mesh
silica gel. Analytical thin-layer chromatography (TLC)
was carried out on Merck silica gel 60 F254 plates.
Compound 1c: 68% yield; amorphous solid. IR
1
(KBr): 3474, 1781, 1745 cm−1. H NMR (CDCl3) l:
1.27 (s, 3H); 1.28 (d, 3H, J=5.96 Hz); 1.43 (s, 3H);
4.22 (m, 1H), 4.61 (s, 1H); 5.63 (s, 1H); 6.94 (s, 1H);
7.38–7.35 (m, 10H). MS (m/z): 225–223, 167 (100%),
152, 114.
1d: 31% yield (diastereomeric mixture 3:1 ratio from
1H NMR integration); amorphous solid. IR (KBr):
1
3423, 1765, 1736 cm−1. H NMR (CDCl3) l: 1.26 (s);
1.44 (d); 1.66 (s); 4.16 (m, major isomer); 4.27 (m,
minor isomer); 4.61 (s); 5. 51 (s); 6.94 (s); 7.36–7.34
(m). MS (m/z): 410, 167 (100%), 152, 114.
4.1.3. 6h-Bromo-6i-(1-hydroxy-ethyl)-3,3-dimethyl-
4,4,7-trioxo-4u6-thia-1-aza-bicyclo[3.2.0]heptane-2-car-
boxylic acid benzhydryl ester (5c) and 6i-bromo-6h-
(1-hydroxy-ethyl)-3,3-dimethyl-4,4,7-trioxo-4u6-
thia-1-aza-bicyclo[3.2.0]heptane-2-carboxylic acid benz-
hydryl ester (5d)
Hydroxy sulfides 1c and 1d were separately oxidized
to sulfones. A solution of 57–86% m-chloroperoxyben-
zoic acid (376 mg) in 6 ml of CH2Cl2 was added
dropwise to a solution of 1c (or 1d; 150 mg, 0.3 mmol)
in 6 ml of CH2Cl2 at 0 °C. The mixture was stirred for
20 h at r.t., then washed with 3% Na2SO3 solution in
saturated aqueous NaHCO3, then with brine. The or-
ganic layer was dried and the solvent evaporated to give
a residue which was purified by flash-chromatography
on silica gel (cyclohexane–EtOAc 8:2).
4.1.1. 6i-Benzoyl-6h-bromo-3,3-dimethyl-7-oxo-4-thia-
1-aza-bicyclo[3.2.0]heptane-2-carboxylic acid benzhydryl
ester (2a) and 6h-benzoyl-6i-bromo-3,3-dimethyl-7-
oxo-4-thia-1-aza-bicyclo[3.2.0]heptane-2-carboxylic
acid benzhydryl ester (2a*)
Pyridinium dichromate (306 mg, 0.81 mmol) was
added to a solution of 1a [10] (150 mg, 0.27 mmol) in 2
ml of dry CH2Cl2 at 0 °C. The reaction mixture was
stirred for 18 h at r.t., filtered on Celite, washed with
hot EtOAc and then evaporated to give a crude residue.
The two isomers were separated by flash-chromatogra-
phy on silica gel (cyclohexane-EtOAc 9:1).
Compound 2a: 26% yield; amorphous solid. IR (Nu-
1
jol): 1799, 1734, 1683 cm−1. H NMR (CDCl3) l: 1.20
(s, 3H); 1.51 (s, 3H); 4.64 (s, 1H); 5.88 (s, 1H); 6.97 (s,
1H); 7.39–7.32 (m, 10H), 7.63–7.47 (m, 3H); 7.99–7.97
(m, 2H). MS (m/z): 224–226, 167 (100%), 152, 105.
Compound 2a*: 54% yield; m.p. 145 °C (dec.)
Compound 5c: 69% yield; m.p. 140 °C (Et2O–hexa-
1
ne). IR (KBr): 3536, 1808, 1734, 1325 cm−1. H NMR
(CDCl3) l: 1.14 (s, 3H); 1.37 (d, 3H, J=6.2 Hz), 1.58
(s, 3H); 4.61 (s, 1H); 4.81 (m, 1H), 4.81 (s, 1H); 6.97 (s,
1H); 7.40–7.32 (m, 10H). MS (m/z): 167 (100%), 152.
5d: 76% yield (diastereomeric mixture 2:1 ratio from
1H NMR integration); amorphous solid. IR (KBr):
(EtOAc–hexane). IR (Nujol): 1785, 1734, 1669 cm−1
.
1H NMR (CDCl3) l: 1.31 (s, 3H); 1.72 (s, 3H); 4.69 (s,
1H); 6.15 (s, 1H); 6.92 (s, 1H); 7.35–7.33 (m, 10H),
7.63–7.48 (m, 3H); 8.32–8.28 (m, 2H). MS (m/z): 470,
167 (100%), 152, 105.
3487, 1805, 1749, 1334 cm−1 1H NMR (CDCl3) l:
.

A. Bedini et al. / Il Farmaco 57 (2002) 663–669
667
1.12 (s); 1.46 (d, minor isomer, J=5.98 Hz); 1.50 (d,
4.1.6. General procedure for the deprotection of
major isomer, J=6.4 Hz); 1.60 (s); 4.11 (m, minor
isomer); 4.27 (m, major isomer); 4.59 (s); 4.71 (s); 6.94
(s, major isomer); 6.96 (s, minor isomer); 7.36–7.32
(m). MS (m/z): 167 (100%), 152.
benzhydryl esters to acids
Method A was used to deprotect 2a, 2a* and 2b* and
method B to deprotect all the other benzhydryl esters.
4.1.6.1. Method A. Suitable ester (0.18 mmol) were
dissolved in 1 ml of m-cresol and the solution was
stirred at 50 °C for 6 h. The reaction mixture was
extracted 2× with NaHCO3 solution; the water phases
were combined and washed 2× with EtOAc. The
aqueous phase at 0 °C was acidified to pH 2/3 with 2 N
HCl and then extracted with EtOAc. The organic
phases were combined, dried on Na2SO4 and evapo-
rated to give the desired compound.
4.1.4. 6i-Acetyl-6h-bromo-3,3-dimethyl-7-oxo-4-
thia-1-aza-bicyclo[3.2.0]heptane-2-carboxylic acid
benzhydryl ester (2b) and 6h-acetyl-6i-bromo-3,3-
dimethyl-7-oxo-4-thia-1-aza-bicyclo[3.2.0]heptane-2-
carboxylic acid benzhydryl ester (2b*)
Activated molecular sieves and pyridinium dichro-
mate (288 mg, 0.76 mmol) were added to a solution of
the appropriate alcohol (1c or 1d; 125 mg, 0.225 mmol)
in 2 ml of dry CH2Cl2 at 0 °C. The mixture was stirred
for 18 h at r.t., filtered on celite, washed with hot
EtOAc and then evaporated to give a residue which was
purified by flash-chromatography on silica gel (cyclo-
hexane–EtOAc 6:4).
4.1.6.2. Method B. TFA (0.17 ml; 2.2 mmol) was added
to a solution of 0.146 mmol of the suitable ester in 0.17
ml of anisole at 0 °C. The mixture was stirred at r.t. for
3 h.
Compound 2b: 74% yield; m.p. 121 °C (CH2Cl2–
hexane). 1H NMR (CDCl3) l: 1.25 (s, 3H); 1.54 (s, 3H);
2.42 (s, 3H); 4.57 (s, 1H); 5.57 (s, 1H); 6.94 (s, 1H);
7.38–7.30 (m, 10H). MS (m/z): 487–489 (M+), 320–
322, 167 (100%), 152, 114.
4.1.7. 6i-Benzoyl-6h-bromo-3,3-dimethyl-7-oxo-4-
thia-1-aza-bicyclo[3.2.0]heptane-2-carboxylic acid (3a)
1
Yield 45%; amorphous solid. H NMR (CDCl3) l:
Compound 2b*: 95% yield; m.p. 150 °C (CH2Cl2–
1.53 (s, 3H); 1.56 (s, 3H); 4.55 (s, 1H); 5.83 (s, 1H);
1
hexane). IR (Nujol): 1785, 1733, 1716 cm−1. H NMR
7.64–7.48 (m, 3H), 8.04–7.99 (m, 2H). MS (m/z):
(CDCl3) l: 1.27 (s, 3H); 1.66 (s, 3H); 2.52 (s, 3H); 4.61
(s, 1H); 5.89 (s, 1H); 6.93 (s, 1H); 7.39–7.30 (m, 10H).
MS (m/z): 487–489 (M+), 320–322, 167 (100%), 152,
114.
383–385
(M+);
105
(100%),
77.
Anal.
(C15H14BrNO4S·0.5EtOAc) C, H, N.
4.1.8. 6h-Benzoyl-6i-bromo-3,3-dimethyl-7-oxo-4-
thia-1-aza-bicyclo[3.2.0]heptane-2-carboxylic acid (3a*)
Yield 72%; amorphous solid. IR (KBr): 3381, 1731,
4.1.5. 6h-Bromo-6i-(1-methoxyimino-ethyl)-3,3-
dimethyl-7-oxo-4-thia-1-aza-bicyclo[3.2.0]heptane-2-
carboxylic acid benzhydryl ester (7b) and 6i-bromo-
6h-(1-methoxyimino-ethyl)-3,3-dimethyl-7-oxo-4-
thia-1-aza-bicyclo[3.2.0]heptane-2-carboxylic acid
benzhydryl ester (7b*)
1
1720, 1676 cm−1. H NMR (CDCl3) l: 1.60 (s, 3H);
1.75 (s, 3H); 4.60 (s, 1H); 6.07 (s, 1H); 7.66–7.43 (m,
3H), 8.31–8.27 (m, 2H). MS (m/z): 304, 105 (100%),
77. Anal. (C15H14BrNO4S·0.5EtOAc) C, H, N.
Na2SO4 (250 mg), pyridine (0.24 ml) and methoxyl-
amino hydrochloride (200 mg, 2.4 mmol) were added to
a solution of the 6-acetyl derivative (2b or 2b*; 195 mg,
0.4 mmol) in 5 ml of dry CH2Cl2. The mixture was
stirred 5 days at r.t. then added of water, extracted with
CH2Cl2 and dried on Na2SO4. The solvent was evapo-
rated to dryness and the residue purified by flash-chro-
matography on silica gel (cyclohexane–EtOAc 9:1).
Compound 7b: 42% yield; m.p. 145 °C (hexane). IR
4.1.9. 6i-Acetyl-6h-bromo-3,3-dimethyl-7-oxo-4-
thia-1-aza-bicyclo[3.2.0]heptane-2-carboxylic acid (3b)
The product crystallized spontaneously after concen-
tration of the reaction mixture and addition of hexane.
Yield 70%; m.p. 125 °C (dec.). IR (KBr): 3141, 1774,
1
1735, 1710 cm−1. H NMR (CDCl3) l: 1.56 (s, 3H);
1.60 (s, 3H); 2.43 (s, 3H); 4.49 (s, 1H); 5.53 (s, 1H). MS
(m/z): 321–323 (M+), 114, 100 (100%). Anal.
(C10H12BrNO4S) C, H, N.
1
(KBr): 1786, 1744 cm−1. H NMR (CDCl3) l: 1.21 (s,
3H); 1.53 (s, 3H); 2.12 (s, 3H); 3.92 (s, 3H); 4.59 (s,
1H); 5.77 (s, 1H); 6.94 (s, 1H); 7.37–7.33 (m, 10H). MS
(m/z): 349–351, 208–210, 167 (100%), 152, 114.
4.1.10. 6h-Acetyl-6i-bromo-3,3-dimethyl-7-oxo-4-
thia-1-aza-bicyclo[3.2.0]heptane-2-carboxylic acid (3b*)
Yield 39%; amorphous solid. IR (KBr): 3243, 1792,
1
Compound 7b*: 72% yield; oil. H NMR (CDCl3) l:
1
1.27 (s, 3H); 1.66 (s, 3H); 2.11 (s, 3H); 3.89 (s, 3H); 4.60
(s, 1H); 5.59 (s, 1H); 6.92 (s, 1H); 7.36–7.33 (m, 10H).
MS (m/z): 349–350, 208–210, 167 (100%), 152,
114.
1764, 1721 cm−1. H NMR (CDCl3) l: 1.58 (s, 3H);
1.70 (s, 3H); 2.54 (s, 3H); 4.55 (s, 1H); 5.83 (s, 1H). MS
(m/z): 321–323 (M+), 114, 100 (100%). Anal.
(C10H12BrNO4S·0.1EtOAc) C, H, N.

668
A. Bedini et al. / Il Farmaco 57 (2002) 663–669
4.1.11. 6h-Bromo-6i-(1-hydroxy-ethyl)-3,3-dimethyl-7-
oxo-4-thia-1-aza-bicyclo[3.2.0]heptane-2-carboxylic
acid (4c)
quently recrystallized. Yield 74%; m.p. 141 °C (dec.)
(Et2O–hexane). IR (KBr): 3168, 1762, 1746, 1734
1
cm−1. H NMR (CDCl3) l: 1.54 (s, 3H); 1.58 (s, 3H);
The product crystallized after concentration of the
reaction mixture and addition of light petroleum. Yield
75%; m.p. 148 °C (dec.). IR (KBr): 3392, 1772, 1748
2.12 (s, 3H); 3.93 (s, 3H); 4.53 (s, 1H); 5.74 (s, 1H). MS
(m/z): 350–352 (M+), 208–210, 191–193, 160, 84
(100%). Anal. (C11H15BrN2O4S·0.15Et2O) C, H, N.
1
cm−1. H NMR (CDCl3) l: 1.28 (d, 3H, J=6.12 Hz);
1.58 (s, 3H); 1.68 (s, 3H); 4.24 (m, 1H); 4.55 (s, 1H);
5.57 (s, 1H). MS (m/z): 160 (100%), 114. Anal.
(C10H14BrNO4S) C, H, N.
4.1.16. 6i-Bromo-6h-(1-methoxyimino-ethyl)-3,3-
dimethyl-7-oxo-4-thia-1-aza-bicyclo[3.2.0]heptane-2-
carboxylic acid (8b*)
The reaction mixture was concentrated and then
subjected to flash chromatography on silica gel
(EtOAc). Yield 52%; m.p. 124 °C (dec.) (CH2Cl2-light
pet.). IR (KBr): 3306, 1784, 1751, 1718 cm−1. 1H NMR
(CDCl3) l: 1.58 (s, 3H); 1.72 (s, 3H); 2.05 (s, 3H); 3.94
(s, 3H); 4.57 (s, 1H); 5.88 (s, 1H). MS (m/z): 350–352
(M+), 208–210, 178–180, 84 (100%). Anal.
(C11H15BrN2O4S) C, H, N.
4.1.12. 6i-Bromo-6h-(1-hydroxy-ethyl)-3,3-dimethyl-7-
oxo-4-thia-1-aza-bicyclo[3.2.0]heptane-2-carboxylic
acid (4d)
The product crystallized after concentration of the
reaction mixture and addition of light petroleum then it
was recrystallized. Yield 86% (diastereomeric mixture
2:1 ratio from 1H NMR integration); m.p. 134 °C
(dec.) (CH2Cl2-light pet.). IR (KBr): 3295, 1780, 1760,
1
1737 cm−1. H NMR (CDCl3) l: 1.45 (d, minor iso-
4.2. Biology
mer, J=5.48 Hz); 1.48 (d, major isomer, J=6.1 Hz);
1.56 (s); 1.70 (s); 4.22 (m, minor isomer); 4.28 (m, major
isomer); 4.53 (s); 5.47 (s, major isomer); 5.49 (s, minor
isomer). MS (m/z): 323–325, 160 (100%), 114. Anal.
(C10H14BrNO4S) C, H, N.
4.2.1. i-Lactamase inhibition assay
The activity of compounds 3a, 3a*, 3b, 3b*, 4c–d,
6c–d, 8b, 8b* as b-lactamase inhibitors was assessed by
determining the concentrations needed to obtain a 50%
inhibition of the hydrolysis of nitrocefin, which was
used as the reporter substrate in competition experi-
ments. The tests were performed on TEM-1 and P99 as
representative enzymes for class A and class C b-lacta-
mases, respectively. Nitrocefin was used at final concen-
trations of 180 mM for TEM-1 and of 250 mM for P99.
The compounds were dissolved in 50 mM sodium
phosphate buffer, pH 7.0, at a final concentration of 2.5
mM. 1.5 ml of 1:10 diluted enzyme (in 50 mM sodium
phosphate buffer, pH 7.0) were added to the reaction
mixture (100 ml final volume: 50 ml substrate+50 ml
compound) and nitrocefin hydrolysis was recorded at
482 nm for 3 min at 30 °C, using a Spectramax spec-
trophotometer. Compounds were assayed at 14 diffe-
rent concentrations ranging from 0.015 to 1.25 mM.
The rates of hydrolysis at each inhibitory concentration
were calculated and the IC50 values (expressed as mg
ml−1) were determined by plotting the percentage of
inhibition against the inhibitor concentration.
4.1.13. 6h-Bromo-6i-(1-hydroxy-ethyl)-3,3-dimethyl-
4,4,7-trioxo-4u6-thia-1-aza-bicyclo[3.2.0]heptane-2-
carboxylic acid (6c)
The product crystallized after concentration of the
reaction mixture and addition of CH2Cl2-light pet.
Yield 66%; m.p. 170 °C (dec.). IR (KBr): 3247, 1801,
1
1774, 1325 cm−1. H NMR (CDCl3) l: 1.38 (d, 3H,
J=6.04 Hz); 1.52 (s, 3H); 1.65 (s, 3H); 4.55 (s, 1H);
4.83 (m, 1H); 4.85 (s, 1H). Anal. (C10H14BrNO6S) C, H,
N.
4.1.14. 6i-Bromo-6h-(1-hydroxy-ethyl)-3,3-dimethyl-
4,4,7-trioxo-4u6-thia-1-aza-bicyclo[3.2.0]heptane-2-
carboxylic acid (6d)
The product crystallized after concentration of the
reaction mixture and addition of light pet. Yield 34%
1
(diastereomeric mixture 5:1 ratio from H NMR inte-
1
gration). IR (KBr): 3420, 1799, 1756, 1327 cm−1. H
NMR (CDCl3–DMSO-d6) l: 1.25 (d, minor isomer,
J=6.14 Hz); 1.33 (d, major isomer, J=6.14 Hz); 1.36
(s); 1.49 (s); 3.97 (m, minor isomer); 4.08 (m, major
isomer); 4.29 (s); 4.96 (s, major isomer); 5.00 (s, minor
isomer). MS (m/z): 312–314, 202–204, 148–150
(100%). Anal. (C10H14BrNO6S·0.1C6H12) C, H, N.
4.2.2. Bacterial strains and culture conditions
Eight reference strains were used in these studies: E.
coli ATTC 35218, E. coli 1850E (wild type=W.T.),
E.coli 1852E (permeable membrane=P.M.), E. coli
1919E (TEM-1), E. cloacae 1051E (P99 enzyme pro-
ducer=P99+), E. cloacae 1321E (P99 enzyme non-
producer=P99−) and S. aureus 853E (penicillinase
producer=PC1).
E. coli and S. aureus were grown overnight at 37 °C
on Triptycase Soya Agar and on Baird–Parker Agar
Base, respectively. For in vitro studies of pharmacody-
4.1.15. 6h-Bromo-6i-(1-methoxyimino-ethyl)-3,3-
dimethyl-7-oxo-4-thia-1-aza-bicyclo[3.2.0]heptane-2-
carboxylic acid (8b)
The product crystallized after concentration of the
reaction mixture and addition of hexane, and subse-

A. Bedini et al. / Il Farmaco 57 (2002) 663–669
669
inhibitors of Class A and Class C b-Lactamases II, Biorg. Med.
Chem. Lett. 9 (1999) 997–1002.
[6] S. Coulton, I. Francois, b-Lactamases: targets for drug design,
Prog. Med. Chem. 31 (1994) 297–349.
[7] T.R. Aplin, C.V. Robinson, C.J. Schofield, S.G. Waley, Use of
electrospray mass spectrometry to investigate the inhibition of
b-lactamases by 6-halogenopenicillanic acids, J. Chem. Soc.,
Chem. Commun. (1993) 121–123.
namics, logarithmic growth-phase cultures were pre-
pared by inoculating colonies into Mueller Hinton
Broth (Oxoid), to obtain a final inoculum of approxi-
mately 105 CFU ml−1
.
4.2.3. MIC determination
Compounds 4c and 6c were tested alone or in combi-
nation with amoxicillin (ratio 1:1 and 1:2) against a
selected series of b-lactamase-producing strains. Amoxi-
cillin and clavulanic acid alone and in combination
were also tested as references. The MICs were deter-
mined by a standard broth microdilution method (NC-
CLS) [17]. The final compound concentrations ranged
from 0.25 to 128 mg ml−1. The MIC was defined as the
lowest drug concentration, which inhibited visible
growth of micro-organisms after 24 h of incubation at
37 °C.
[8] T. Nagase, D. Golemi, A. Ishiwata, S. Mobashery, Inhibition of
beta-lactamases by 6,6-bis(hydroxylmethyl)penicillanate, Bioorg.
Chem. (2001) 140–145.
[9] Y.-I. Lin, P. Bitha, S.M. Sakya, T.W. Strohmeyer, Z. Li, V.J.
Lee, S.A. Lang Jr., Y. Yang, N. Bhachech, W.J. Weiss, P.J.
Petersen, N.V. Jacobus, K. Bush, R.T. Testa, F.P. Tally, Pep-
tidic prodrugs of novel aminomethyl-THF 1b-methylcarbapen-
ems, Bioorg. Med. Chem. Lett. 7 (1997) 1665–1670.
[10] R.A. Volkmann, R.D. Carroll, R.B. Drolet, M.L. Elliott, B.S.
Moore, Efficient preparation of 6,6-dihalopenicillanic acid. Syn-
thesis of penicillanic acid S,S-dioxide (sulbactam), J. Org. Chem.
47 (1982) 3344–3345.
[11] C.D. Foulds, M. Kosmirak, P.G. Sammes, Substituted penicil-
lanic acid 1,1-dioxides as b-lactam inhibitors: studies on 6-ben-
zylidene- and hydroxybenzylpenam sulfones, J. Chem. Soc.,
Perkin Trans. I (1985) 963–968.
Acknowledgements
[12] F. DiNinno, T.R. Beattie, B.G. Christensen, Aldol condensa-
tions of regiospecific penicillanate and cephalosporanate eno-
lates. Hydroxyethylation at C-6 and C-7, J. Org. Chem. 42
(1977) 2960–2965.
[13] E.L. Setti, O.A. Phillips, A.V.N. Reddy, C. Kunugita, A. Hy-
odo, R.G. Micetich, S.N. Maiti, Synthesis and biological evalua-
tion of sodium 2b-[(2,5-dihydro-6-hydroxy-2-methyl-5-oxo-
1,2,4-triazin-3-yl)sulfonylmethyl]-2a-methylpenam-3a-carboxyl-
ate-1,1-dioxide, Heterocycles 42 (1996) 645–651.
The authors wish to thank Dr. Antonio Felici,
GlaxoSmithKline Medicines Research Centre, for
doing microbiological determinations.
References
[14] H.G.F. Richter, P. Angehrn, C. Hubschwerlen, M. Kania, G.P.
Page, J.-L. Specklin, F. Winkler, Design, synthesis and evalua-
tion of 2b-alkenyl penam sulfone acids as inhibitors of b-lacta-
mases, J. Med. Chem. 39 (1996) 3712–3722.
[1] O.A. Mascaretti, O.D. Danelon, E.L. Setti, M. Laborde, E.G.
Mata, Recent advances in the chemistry of b-lactam compounds
as selected active-site b-lactamase inhibitors, Curr. Pharm. Des.
5 (1999) 939–953.
[15] C. Reading, M. Cole, Structure activity relationships amongst
b-lactamase inhibitors, J. Enzyme Inhib. 1 (1986) 83–104.
[16] P. Bitha, Z. Li, G.D. Francisco, B.A. Rasmussen, Y.-I. Lin,
6-(1-Hydroxyalkyl)penam sulfone derivatives as inhibitors of
Class A and Class C b-Lactamases I, Bioorg. Med. Chem. Lett.
9 (1999) 991–996.
[17] National Committee for Clinical Laboratory Standards. Meth-
ods for dilution antimicrobial susceptibility tests for bacteria that
grow aerobically. Approved standard, NCCLS publication M7-
A4, 10, N 8, Villanova, PA, USA, 1998.
[2] D.J. Payne, W. Du, J.H. Bateson, b-Lactamase epidemiology
and the utility of established and novel b-lactamase inhibitors,
Expert Opin. Invest. Drugs 9 (2000) 247–261.
[3] R.P. Ambler, The structure of b-lactamases, Philos. Trans. R.
Soc. London B 289 (1980) 321–331.
[4] M.I. Page, The reactivity of b-lactams, the mechanism of cataly-
sis and the inhibition of b-lactamases, Curr. Pharm. Des. 5
(1999) 895–913.
[5] P. Bitha, Z. Li, G.D. Francisco, Y. Yang, P.J. Petersen, E.
Lenoy, Y. Lin, 6-(1-Hydroxyalkyl)penam sulfone derivatives as