The synthesis and SAR of rhodanines as novel class C β-lactamase inhibitors

Article abstract of DOI:10.1016/S0960-894X(00)00444-3

β-Lactam antibiotics such as the cephalosporins and penicillins have diminished clinical effectiveness due to the hydrolytic activity of diverse β-lactamases, especially those in molecular classes A and C. A structure-activity relationship (SAR) study of

Full text of DOI:10.1016/S0960-894X(00)00444-3

Bioorganic & Medicinal Chemistry Letters 10 (2000) 2179±2182
The Synthesis and SAR of Rhodanines as Novel Class C
ꢀ-Lactamase Inhibitors
Eugene B. Grant,* Deodialsingh Guiadeen, Ellen Z. Baum, Barbara D. Foleno,
Haiyong Jin, Deborah A. Montenegro, Erin A. Nelson, Karen Bush and Dennis J. Hlasta
Antimicrobial Agents Research, The R.W. Johnson Pharmaceutical Research Institute, Route 202, PO Box 300, Raritan, NJ 08869, USA
Received 27 March 2000; accepted 24 July 2000
AbstractÐb-Lactam antibiotics such as the cephalosporins and penicillins have diminished clinical e€ectiveness due to the hydro-
lytic activity of diverse b-lactamases, especially those in molecular classes A and C. A structure±activity relationship (SAR) study of
a high-throughput screening lead resulted in the discovery of a potent and selective non-b-lactam inhibitor of class C b-lactamases.
#
2000 Elsevier Science Ltd. All rights reserved.
Bacterial resistance to b-lactam antibiotics can result
from their overuse in treating mild infections and from
patient failure in following a prescribed dosing regimen.
The surviving bacteria often become resistant due to the
1
b-lactam hydrolyzing enzymes known as b-lactamases.
To circumvent this resistance, bacterial infections have
been treated by administering a b-lactamase inhibitor
with the b-lactam antibiotic.
b-Lactamases are divided into four classes based on their
molecular structure. Class C and class A b-lactamases,
with an active site serine, represent the majority of all
2
b-lactamases. Commercially available inhibitors such as
clavulanic acid (1), tazobactam (2, TZB), and sulbactam
(3) inactivate most common class A b-lactamases, but
are ine€ective clinically against class C b-lac-tamases
3
(
Fig. 1). Chromosomally-encoded class C b-lactamases
rapidly degrade cephalosporins, and they are generally
not inhibited by the existing b-lactamase inhibitors. The
most active broad-spectrum b-lactamase inhibitor that
is e€ective against class A and C b-lactamases is BRL
Figure 1.
4
2715 (4). However, this compound was not developed
4
as a clinical agent.
The class C cephalosporin-hydrolyzing b-lactamase
class is an attractive target for drug discovery. As a
dominant resistance mechanism in Gram-negative bac-
teria, it is expected to become more widespread in the
absence of a commercially available inhibitor.
High-throughput screening of our chemical library
identi®ed rhodanine 5 as a promising lead inhibitor
candidate (Fig. 2). P99, isolated from Enterobacter
cloacae, and TEM-1, isolated from Escherichia coli,
were used in these studies as prototypical class C and
5
class A b-lactamases. Rhodanine 5 is a non-b-lactam
with an IC50 of 2.6 mM against the class C enzyme P99
and an IC50 of 8.7 mM against the class A enzyme
TEM-1. This paper describes our e€orts to discover a
*
8
Corresponding author. Tel.: +1-908-218-6589; fax: +1-908-203-
109; e-mail: egrant@prius.jnj.com
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00444-3

2
180
E. B. Grant et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2179±2182
rhodanine b-lactamase inhibitor which is selective for
P99 over TEM-1.
Finally, we decided to examine heterocyclic sidechains.
Substitution of an electron withdrawing triazole for the
double bond yielded a moderately active analogue, 20,
while the imidazole analogue 21 was inactive. Replace-
ment of the phenyl group with a furan to give analogue
2
3 further increased the activity. The most active ana-
logue in this study was the 5-nitrofuran analogue 24.
Rhodanine 24 was 200-fold more selective for P99 than
TEM-1, as compared to our original rhodanine 5 which
was only threefold more selective.
We next examined 24 for synergy with the b-lactam
piperacillin in a six-organism panel using TZB as a
positive control (Table 3). This panel includes bacterial
strains producing class A enzymes and isogenic strains
of S. marcescens and P. aeruginosa producing class C
b-lactamases in an inducible or constitutive state. As
demonstrated in this panel, piperacillin MICs were
lowered against S. marcescens and P. aeruginosa strains
Figure 2.
The simplest modi®cation of 5 was to excise the pyrroli-
done ring and to construct analogues by condensation of
substituted aromatic aldehydes with 2-thio-thiazolidi-4-
none (Fig. 3). In general, synthesis of these alkylidene
rhodanines was facile, and only the Z isomer was isolated.6
Table 1. Inhibitory activity of substituted rhodanines7 against P99
and TEM-18
IC50 (mM)
Compound
R1
R2
P99
TEM-1
6
7
Phenyl
Phenyl
H
Ethyl
455
240
>700
432
8
9
Phenyl
Phenyl
>700
246
>700
138
Figure 3.
10
Phenyl
435
>700
A wide variety of rhodanines was prepared and tested as
inhibitors of P99 and TEM-1 b-lactamases. Examining
the activity of selected rhodanines, two general trends
emerged. First, the most selective compounds for P99
lacked a substituent at the ring nitrogen (Table 1). Sec-
ond, the analogues with a nitro group on the benzyli-
dene phenyl ring had superior activity.
1
1
2
H
H
335
>700
>700
1
>700
1
3
H
H
H
290
43
>700
>700
>700
The relatively weak but selective activity of these ana-
logues encouraged us to extend the sidechain with a
two-carbon spacer in order to mimic the cephalosporin
substrate of class C b-lactamases. The addition of the
two-carbon spacer led to improved IC 's, while main-
14
5
0
taining essentially the same trends observed for the
simpler analogues (Table 2). Interestingly, the activity
of the saturated analogue 19 was superior to unsatu-
rated 17, however the selectivity for P99 was dimin-
ished. Preparation of the E isomer at C3 of 17 was
attempted, but was unsuccessful.
1
1
5
6
500
H
>700
190

E. B. Grant et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2179±2182
Table 2. Inhibitory activity of substituted rhodanines against P99
Table 4. Antimicrobial activity of rhodanines 24 and 25a
MIC (mg/mL)
2181
and TEM-1
Bacteria
Enzyme
24
25
E. coli OC 4075
TEM-1
P99
E2 (ind)
2
2
32
32
32
32
1
0.5
4
4
32
32
E. cloacae OC 4080
E. cloacae OC 4092
E. cloacae OC 4081
P. aeruginosa OC 4270
P. aeruginosa OC 4290
E2 (const)
AmpC (ind)
AmpC (const)
IC50 (mM)
TEM-1
Compound
X
S
R1
P99
455
aSee Table 3 footnotes for abbreviations.
6
>700
>700
100
17
18
19
S
S
S
76
4.2
with inducible or constitutive production of class C
cephalosporinases. Because of the inherent anti-
biotic activity of 24, it was impossible to evaluate the
synergistic potential at concentrations greater than its
MIC.10
9.7
32
2
0
1
S
S
83
>700
>700
The nitrofuran moiety of 24 was suspected to be
associated with the antibacterial activity since 2-nitro-
furans, such as nitrofurantoin, are known anti-
bacterials.12 To test this hypothesis, thiazolidinedione
2
>700
2
(
5 was prepared and tested in the biological assays
Table 4). Thiazolidinedione 25 had better MICs
against E. cloacae isolates as compared to 24, but
5 lacked inhibitory activity at the concentrations
2
2
2
2
3
4
5
S
S
8
>100
100
2
tested (IC50 >5 0 mM). Therefore, it is likely that
the nitrofuran was not responsible for inhibition of
P99 in 24, but is responsible for the antibacterial activity
of 24.
62
S
0.45
100
In conclusion, the rhodanines represent a novel class of
b-lactamase inhibitors that can be modi®ed to show
selectivity for class C b-lactamases. The rhodanines
di€er chemically from traditional b-lactamase inhibitors
such as clavulanic acid and tazobactam, but do exhibit
inhibitory activity against both class A and class C
b-lactamases.
2
O
>5 0
>5 0
7.7a
2
TZB
0.06
aAverage IC50 of tazobactam in 10 experiments.
Table 3. Susceptibility testing of 24 in combination with piperacillin
a
MIC (mg/mL)
Bacteria
Enzyme
Molecular class
Piperacillin
PIP/TZBb
24
PIP/24 (1)c
PIP/24 (4)c
E. coli OC 4075
TEM-1
P99
AmpC (ind)
A
C
C
C
C
C
>64
>64
8
>64
8
8
64
4
32
8
2
2
8
8
32
32
>64
>64
2
32
4
16
NAd
NA
2
E. cloacae OC 4080
S. marcescens OC 4101
S. marcescens OC 4294
P. aeruginosa OC 4270
P. aeruginosa OC 4290
e
f
f
AmpC (const)
8
4
8
e
AmpC (ind)
AmpC (const)
64
32
a
MICs were determined using standard methods according to NCCLS.11
PIP, piperacillin; TZB, tazobactam at 4 mg/mL.
b
c
Inhibitor concentration in mg/mL shown in parentheses.
d
e
NA, not applicable.
Inducible b-lactamase production.
Constitutive b-lactamase production.
f

2
182
E. B. Grant et al. / Bioorg. Med. Chem. Lett. 10 (2000) 2179±2182
1
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8
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