Exploitation of Antibiotic Resistance as a Novel Drug Target: Development of a β-Lactamase-Activated Antibacterial Prodrug

Article abstract of DOI:10.1021/acs.jmedchem.8b01923

Expression of β-lactamase is the single most prevalent determinant of antibiotic resistance, rendering bacteria resistant to β-lactam antibiotics. In this article, we describe the development of an antibiotic prodrug that combines ciprofloxacin with a β-lactamase-cleavable motif. The prodrug is only bactericidal after activation by β-lactamase. Bactericidal activity comparable to ciprofloxacin is demonstrated against clinically relevant E. coli isolates expressing diverse β-lactamases; bactericidal activity was not observed in strains without β-lactamase. These findings demonstrate that it is possible to exploit antibiotic resistance to selectively target β-lactamase-producing bacteria using our prodrug approach, without adversely affecting bacteria that do not produce β-lactamase. This paves the way for selective targeting of drug-resistant pathogens without disrupting or selecting for resistance within the microbiota, reducing the rate of secondary infections and subsequent antibiotic use.

Full text of DOI:10.1021/acs.jmedchem.8b01923

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Article
Exploitation of antibiotic resistance as a novel drug target:
development of a #-lactamase-activated antibacterial prodrug
Lindsay E Evans, Aishwarya Krishna, Yajing Ma, Thomas E Webb, Dominic C Marshall,
Catherine L Tooke, James Spencer, Thomas B Clarke, Alan Armstrong, and Andrew Edwards
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01923 • Publication Date (Web): 22 Apr 2019
Downloaded from http://pubs.acs.org on April 22, 2019
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Exploitation of antibiotic resistance as a novel drug
target: development of a β-lactamase-activated
antibacterial prodrug
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Lindsay E. Evans* , Aishwarya Krishna , Yajing Ma , Thomas E. Webb , Dominic C.
§
ǁ
ǁ
†
‡
Marshall , Catherine L. Tooke , James Spencer , Thomas B. Clarke , Alan Armstrong and
Andrew M. Edwards*†
†
MRC Centre for Molecular Bacteriology and Infection, Imperial College London, SW7 2AZ,
United Kingdom.
‡
Department of Chemistry, Molecular Sciences Research Hub, Imperial College London,
W12 0BZ, United Kingdom.
§
Department of Medicine, Imperial College London, SW7 2AZ, United Kingdom.
ǁ
School of Cellular and Molecular Medicine, University of Bristol, Biomedical Sciences Building,
University Walk, Bristol, BS8 1TD, United Kingdom.
ABSTRACT
Expression of β-lactamase is the single most prevalent determinant of antibiotic resistance,
rendering bacteria resistant to β-lactam antibiotics. In this article, we describe the development of
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an antibiotic pro-drug that combines ciprofloxacin with a β-lactamase-cleavable motif. The pro-
drug is only bactericidal after activation by β-lactamase. Bactericidal activity comparable to
ciprofloxacin is demonstrated against clinically-relevant E. coli isolates expressing diverse β-
lactamases; bactericidal activity was not observed in strains without β-lactamase. These findings
demonstrate that it is possible to exploit antibiotic resistance to selectively target β-lactamase-
producing bacteria using our pro-drug approach, without adversely affecting bacteria that do not
produce β-lactamase. This paves the way for selective targeting of drug-resistant pathogens
without disrupting or selecting for resistance within the microbiota, reducing the rate of secondary
infections and subsequent antibiotic use.
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INTRODUCTION
Antimicrobial drug resistance is a global health emergency, threatening advances in many areas
of medicine including surgery, cancer chemotherapy, organ transplantation and survival of pre-
1
,2
term infants. The most prevalent and important resistance determinant is the β-lactamase
enzyme, which hydrolyses members of the β-lactam class of antibiotic (e.g. penicillin,
cephalosporins and carbapenems) and thereby prevents engagement with their therapeutic targets
3,4
the penicillin-binding proteins (PBPs). Of particular concern are the extended-spectrum β-
lactamases (ESBLs) such as the CTX-M class, which are able to cleave a wide range of
clinically-relevant β-lactam antibiotics.5–7
Urinary tract infections (UTIs) are the most prevalent type of bacterial infection globally. These
infections have a high rate of recurrence and can also lead to serious invasive infections such as
8
,9
sepsis, particularly in the elderly. E. coli is the most common causative organism (~75%
cases), of which ~50% are resistant to β-lactam antibiotics due to β-lactamase expression.8,10 As
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a consequence of the high rate of β-lactam resistance in UTI pathogens, second-line, broad-
spectrum antibiotics such as ciprofloxacin are increasingly used therapeutically.11,12
Unfortunately, these broad-spectrum antibiotics are associated with disruption to the beneficial
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bacteria that colonize the gastro-intestinal tract and other surfaces, known as the microbiota.13–17
This disruption can lead to serious secondary infections by antibiotic-resistant bacteria such as
Clostridium difficle or fungi such as Candida albicans, leading to colitis and thrush
respectively.13,18 This is because antibiotics target conserved processes in bacteria such as cell
wall, protein, DNA or RNA biosynthesis, which not only occur in the pathogens that cause
infection but also in the members of the microbiota.19,20
An additional complication associated with some second-line therapeutics such as ciprofloxacin
is host toxicity. Ciprofloxacin holds two black box warnings, one for increased risk of tendonitis
and tendon rupture and one for exacerbation of muscle weakness in myasthenia gravis
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sufferers. Additionally, in 2015, the FDA officially recognized fluoroquinolone-associated
disability (FQAD) as a syndrome. FQAD describes a range of disabling and potentially
permanent side effects including disturbances of tendons, joints, muscles, nerves, the nervous
system and induction of type 2 diabetes.22,23 As a result, strategies with the potential to mitigate
host toxicity by reducing exposure to ciprofloxacin are needed.
Given the drawbacks associated with broad-spectrum antibiotics, efforts have been made to limit
their use.11,23–25 However, these efforts have had limited success with usage rates increasing
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globally, particularly in low- and middle-income countries. In part, this is due to a lack of
access to fast and efficient diagnostic techniques and the need to respond quickly to serious
bacterial infections with effective and cost-efficient treatment regimens that target a wide range
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of different bacterial pathogens. There is, therefore, a pressing need to develop new
therapeutics that kill a broad range of different pathogens without damaging the host microbiota.
Since β-lactamase enzymes are not found in mammalian cells, we hypothesized that we could
exploit this enzyme as a novel anti-bacterial target. Furthermore, β-lactamase expression is
prevalent amongst UTI pathogens, which can both colonize the gut and cause infection of the
GU tract.8,10 Consequently, this represents an opportunity to selectively target disease-causing
bacteria without causing significant disruption to the microbiota or select for drug resistance as
has been reported for broad-spectrum antibiotics such as ciprofloxacin.28–30 Therefore, the aim of
this work was to develop a small molecule antibacterial agent that is selectively active against
bacteria that express β-lactamase. To do this, we employed a pro-drug strategy that utilized a β-
lactam cleavable motif linked to the broad-spectrum antibiotic ciprofloxacin.
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In support of our approach, the use of β-lactams as prodrug modifiers in antibody-directed
enzyme prodrug therapy approaches has been explored in disease areas such as cancer (1 – 3,
Fig 1).31–35 Additionally, β-lactam-fluoroquinolone conjugates have been proposed as a co-drug
strategy to treat bacterial infections (4 and 5, Fig 1).36–39 However, our approach is different in
that it is designed to selectively deliver a broad-spectrum, bactericidal antibiotic to only bacteria
that express β-lactamase, whilst having minimal effect on bacteria which do not express the
resistance determinant. By contrast, previous dual activity co-drug approaches were designed to
have broad-spectrum activity against both drug-sensitive bacteria and those that express β-
lactamase.
Herein we describe the design and development of the pro-drug, including optimization of the β-
lactam motif to reduce the antibacterial activity of the intact molecule and increase the efficiency
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of β-lactamase mediated ciprofloxacin release. This is, to our knowledge, the first example of a
β-lactam-fluoroquinolone pro-drug with selective activity against drug-resistant bacteria.
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NHBz
OH
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H N
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Ro 23-9424 (5)
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Figure 1. Selected representative examples of cephalosporin pro-drugs (PROTAX 1, 2, and
3
5
38
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BMY-46633 3 ) and codrugs (MCO 4 and Ro 23-9424 5 ).
RESULTS AND DISCUSSION
Prodrug design
In order to create a pro-drug molecule that is selectively activated in β-lactamase producing
bacteria it was important to select a β-lactamase cleavable motif, linkage strategy and active
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antibiotic that gave a stable non-bactericidal intact molecule and enabled the rapid and efficient
release of the antibiotic upon activation by β-lactamase. The success of this strategy required a
pro-drug motif that would enable efficient substrate turnover rather than inhibition of the β-
lactamase enzyme. The cephalosporin class of β-lactams are efficiently hydrolysed by β-
lactamases and have been previously employed as a prodrug motif as cleavage of the β-lactam
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ring is associated with the loss of the functional group at the 3’-position (Fig 2a). In addition,
the chemistry associated with changing the 3’-substituent of cephalosporins is well-established
and a wide variety of substituents at the C-3’ position are tolerated by β-lactamases.37,41,42
Consequently, a cephalosporin core was selected as the β-lactam component. To achieve the
desired selectivity profile, ciprofloxacin was attached via the carboxylic acid to give the 3’-
cephem ester 6 (Fig 2b). Derivatization of the carboxylic acid group of fluoroquinolone
antibiotics is associated with a significant decrease in antibacterial activity due to a decreased
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ability to bind to bacterial DNA-enzyme complexes. Whilst this choice of attachment site was
selected to remove the ciprofloxacin activity from the intact prodrug, it remained likely that the
pro-drug molecule would retain antibacterial activity as a result of the ability of the cephem
portion of the molecule to interact with PBPs. Therefore, to further increase selectivity it was
essential to undertake a program of optimization of the β-lactam motif to reduce PBP activity
and increase or maintain β-lactamase activity. Initial optimization was performed on the
cephalosporin portion of the prodrug to enable the rapid generation of analogues and evaluation
of biological activity. The cephalosporin analogues with the most desirable activity profile were
then selected for preparation as the full pro-drug.
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Figure 2. (A) Mechanism of β-lactamase triggered cephalothin hydrolysis; (B) General structure
of proposed cephalosporin-ciprofloxacin pro-drug 6.
β-Lactam Analogue Preparation and Biological Evaluation
Analysis of the literature identified the amide functionality at C-7 of the cephem ring as central
to PBP and β-lactamase activity44–51 and therefore structural changes at this position provided the
initial focus of investigation. Using cephalothin 7 (Table 1) as the starting point, analogues were
prepared to explore bioisosteric replacement,52,53 functionalities present in early generation β-
lactam antibiotics, and to probe steric and electronic tolerance.54,55 All compounds were
synthesized according to the previously reported methods (Fig S1).55,56 Antibacterial activity was
assessed by determining the minimal concentration required to inhibit bacterial growth, known
as the minimal inhibitory concentration (MIC), against the E.coli strain DH5α ± expression of
57
the ESBL TEM-116. The susceptibility to β-lactamase mediated hydrolysis was assessed by
determining the physiological efficiency (k /K ) of hydrolysis by recombinant AmpC
cat
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protein.3,58,59
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For all compounds (Table 1), a higher MIC value was determined for the E. coli strain
expressing TEM-116 than the strain not expressing β-lactamase, indicating hydrolytic activity by
the β-lactamase. Introduction of a substituent to the thiophene ring (8) or switching from a C-2 to
a C-3 substitution (9) gave a modest increase in MIC values and a small decrease in k /K
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compared to cephalothin 7. Although no measurable MIC value could be determined for any of
the phenyl analogues (20-23), this was accompanied by a >3-fold decrease in k /K . In general,
cat
m
a quaternary carbon (20-24) or tertiary carbon (10 and 25) at the α-position relative to the amide
carbonyl was not well tolerated by AmpC. This finding is consistent with prior reports and has
previously been exploited to reduce β-lactamase activity in the development of later-generation
β-lactams. Compounds containing straight-chain aliphatic groups (26-28) retained some
antibacterial activity; an increase in k /K was observed with increasing chain length.
cat
m
Table 1. Antibacterial activities (MIC) and AmpC hydrolytic efficiency of synthesized
compounds and reference compound cephalothin (ceph) 7.
H
H
R
N
S
O
N
OAc
O
CO H
2
Compound
R group
MIC value (μM)
AmpC-catalysed
hydrolytic efficiency
DH5α +
TEM-116
DH5α
-1
k /K (μM/S )
cat
m
S
S
Ceph 7
12.5
100
2.86 ± 0.33
2.50 ± 0.28
2.36 ± 0.10
8
9
50
50
200
200
Br
S
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Me
1
0
400
>800
0.87 ± 0.15
11
50
100
400
200
400
800
1.86 ± 0.09
1.77 ± 0.19
1.34 ± 0.20
4.78 ± 0.13
5.06 ± 0.32
1
1
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0
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8
9
0
1
1
1
1
2
3
4
5
100
100
100
200
Me
F
Cl
Br
1
6
>800
>800
3.99 ± 0.88
1
1
1
2
7
8
9
0
>800
200
>800
400
7.33 ± 1.72
9.13 ± 1.66
31.14 ± 2.67
0.52 ± 0.18
O
O
>800
>400
>800
>400
2
1
>400
>800
>400
>800
0.26 ± 0.10
0.31 ± 0.38
0.88 ± 0.15
ND
Me
22
OMe
2
2
3
4
>400
>400
>400
>400
NO2
O
2
2
5
6
>400
200
>400
400
0.17 ± 0.09
0.53 ± 0.16
Me
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100
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>800
0.64 ± 0.04
0.81 ± 0.09
0
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9
0
1
2
3
4
5
6
7
8
9
0
Examination of the tested analogues (Table 1) immediately revealed the importance of bulky
benzylic substituents (11-19). Thiophene rings are frequently used as a bioisosteric replacement
for a phenyl groups and it is therefore perhaps unsurprising that there was only a modest 4-fold
increase in MIC value against E. coli DH5α and a slight decrease in k /K for 11 compared to
cat
m
cephalothin.52,53 However, introduction of substituents at the para-position (12-17) gave a
further 2- to 4-fold increase in MIC against E. coli DH5α compared to unsubstituted benzyl 11.
Substitution at the para-position also affected hydrolysis by AmpC with the following order of
activity observed F < Me = H < Cl = Br. Movement of the methyl substituent from the para- (12)
to the meta-position (18) gave a 5-fold increase in k /K , and a 3-fold increase compared to
cat
m
cephalothin. High k /K values were determined for bisaryl 16 and the para- and meta-
cat
m
substituted biphenyl ethers 17 and 19 (3.99 ± 0.88, 7.33 ± 1.72 and 31.14 ± 2.67, respectively).
In addition, no measurable MIC values could be determined for 16, 17 or 19. This led us to
question if the results were indicative of no antibacterial activity, or simply a result of increased
efflux activity out of, or a lack of permeability in to, the bacterial cell.
β-Lactamase hydrolytic activity in whole-cell NMR assay
To address the question of compound permeability/efflux, a whole-cell β-lactamase hydrolysis
assay was used to detect the penetration of compounds into the periplasm.60,61 Hydrolytic
1
decomposition of β-lactam rings is associated with changes in H NMR signals, which can be
1
detected using whole bacterial cells in real time by H NMR spectroscopy (Fig S2). As
hydrolysis occurred within the bacterial periplasm, only compounds with sufficient intracellular
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accumulation were hydrolysed. Compound 16 and 17 were selected as representative examples
of a high lipophilicity compounds with no measurable antibacterial activity and moderate in vitro
β-lactamase hydrolysis. We evaluated the hydrolysis of bisaryl 16, biaryl ether 17 and
cephalothin 7 in DH5α ± TEM-116 (Table 2). After 90 minutes, 16 was 69% hydrolyzed in
DH5α + TEM-116 compared to 14% hydrolyzed in DH5α - TEM-116 and 17 was 53%
hydrolyzed in DH5α + TEM-116 compared to 13% hydrolyzed in DH5α - TEM-116. These
results indicated a high degree of in vivo β-lactamase mediated hydrolysis and that 16 and 17
accumulated in the bacterial cell. We therefore concluded that the lack of antibacterial activity of
this compound against E. coli DH5α without β-lactamase was due to an absence of PBP
engagement and not due poor permeability or efflux activity.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 2. Percentage hydrolysis of cephalothin (ceph) 7 and compounds 16 and 17 by DH5α cells
±
β-lactamase in whole-cell NMR hydrolysis assay.
Conc.
Incubation
time (mins)
Strain
% Hydrolysis by NMR
Compound
(μM)
- βla
-
+ βla
61
Ceph 7
50
60
90
90
60
60
60
1
1
6
7
100
100
100
100
100
DH5a ± TEM-116
DH5a ± CTX-M-1
14
13
0
0
0
69
53
100
100
95
Ceph 7
1
1
6
7
Biological evaluation in uropathogenic E. coli
Initial assessment of compound activity was performed in the laboratory E. coli strain DH5α. To
assess the activity of the β-lactams against a clinically-relevant pathogenic strain of E. coli we
selected the uropathogenic strain CFT073. This bacterium was isolated from the blood of a
patient with acute pyelonephritis, is devoid of all virulence plasmids commonly associated with
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uropathogenic strains and proved tractable for genetic manipulation.62,63 The plasmid pSU18,
either without the coding sequence for β-lactamase (referred to here as pEMP) or encoding for
the β-lactamase CTX-M-1 was introduced into CFT073, enabling comparison of compound
activity in CFT073 and CFT073 + pSU18 ± β-lactamase. The primary β-lactamase used in this
work was CTX-M-1 because CTX-M enzymes are the most prevalent β-lactamases amongst
Enterobacteria such as E. coli. As part of a class of extended-spectrum β-lactamases (ESBL) it
confers resistance to most β-lactam antibiotics, with the exception of carbapenems.6
0
1
2
3
4
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6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
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5
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7
8
9
0
1
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8
9
0
1
2
3
4
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7
8
9
0
In the first instance, MIC values were determined for selected compounds against CFT073 +
pSU18 ± CTX-M-1 (Table 3). For all the compounds tested, the MIC values for CFT073 +
pSU18 were within 2-fold of those determined against DH5α. Next the hydrolytic activity of
these compounds was assessed in the whole cell NMR assay (Table 3). For the majority of the
compounds tested, a low level of hydrolysis, <20% after 60 minutes, was detected. However,
levels of hydrolysis comparable to that observed for cephalothin (68%) were observed only for
2
4 and 26 (64% and 67%, respectively).
Table 3. Antibacterial activities (MIC) against CFT073 ± CTX-M-1 and percentage hydrolysis
in CFT073 + CTX-M-1 cells for selected compounds and reference compound cephalothin
(ceph) 7.
H
N
H
R
S
O
N
OAc
O
CO H
2
MIC value (μM)
%
hydrolysis in
CFT073 +
pSU18 +
CTX-M-1
Compound
R group
CFT073 +
pSU18
CFT073 + pSU18
a
+
CTX-M-1
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S
Ceph 7
25
>400
68
ND
S
8
50
>400
Br
S
1
1
1
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4
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4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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7
8
9
0
1
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8
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0
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9
0
1
2
3
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7
8
9
0
9
50
>400
>400
32
0
Me
1
0
>400
1
1
1
1
1
1
2
3
4
5
50
>400
>400
>400
>400
>400
17
7
100
100
100
100
Me
F
18
14
9
Cl
Br
16
>400
>400
4
1
1
1
2
7
8
9
0
>400
200
>400
>400
>400
>400
4
16
0
O
O
≥400
>400
26
2
1
>400
>400
>400
>400
3
3
Me
22
OMe
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9
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6
O
2
2
4
5
>400
ND
>400
ND
64
0
0
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0
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9
0
2
2
2
6
7
8
400
200
100
>400
>400
>400
67
25
7
Me
a
Percentage hydrolysis determined for 100 μM compound at 1 hour by whole-cell NMR assay.
ND = Not done.
A clear feature of the SAR was that CTX-M-1 mediated hydrolytic activity in whole CFT073
cells correlated with lipophilicity. Plotting the calculated LogP (cLogP) values for compounds
against the log of percentage hydrolysis revealed that moderate-high levels of hydrolysis (>30%)
were only observed for compound with cLogP values below 0.1 (Fig 3). Linear regression
2
analysis revealed moderate correlation (R = 0.60), despite the degree of hydrolysis reflecting
both cellular penetration and β-lactamase activity, which are both sensitive to compound
lipophilicity.
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Figure 3. Plot of log percentage hydrolysis in CFT073 + CTX-M-1 cells against calculated logP
(clogP) for synthesized compounds (filled circle) and cephalothin 7 (open circle). Linear
2
regression (dashed line), R = 0.60 (GraphPad prism 7).
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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0
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8
9
0
Interestingly, compounds 16 and 17 were hydrolyzed rapidly (69% and 54% after 90 minutes,
respectively) in DH5α expressing the TEM-116 β-lactamase (Table 2) but only 4% hydrolyzed
after 60 minutes in CFT073 expressing the CTX-M-1 β-lactamase. We hypothesized that the low
level of hydrolytic activity observed for many of the compounds could be a result of poor
intracellular accumulation in CFT073 E. coli since clinical isolates often have reduced
6
4
permeability to antibiotics. To test this hypothesis, hydrolysis in DH5α expressing CTX-M-1
was determined for cepaholothin 7, 16 and 17 (Table 2). After 60 minutes complete hydrolysis
for cepaholothin 7 and 16 and 94% hydrolysis for 17 was observed, suggesting that the low level
of hydrolysis observed in CFT073 + CTX-M-1 was not due to the inability of CTX-M-1 to
hydrolyze this chemotype. Instead it is likely that either due to poor membrane permeability or
increased efflux activity, lipophilic analogues were unable to engage with CTX-M-1 in CFT073.
For compounds with low hydrolytic activity in the whole cell NMR assay with CFT073 we were
unable to discern if a high MIC value in the absence of CTX-M-1 truly reflected a lack of
antibacterial activity or a lack of permeability/high efflux activity. Therefore, compound 26, with
its high MIC value in both CFT073 ± CTX-M-1 (≥400 μM) and high hydrolytic activity in whole
CFT073 cells (67% after 60 minutes), was selected for incorporation into the full prodrug
molecule. Compound 24, which also possessed a high MIC value in both CFT073 ± CTX-M-1
(≥400 μM) and high hydrolytic activity in whole CFT073 cells (64% after 60 minutes) was not
progressed at this time as we wished to avoid the potential for toxicity problems arising from the
furan ring, which has been identified as common toxicophore due to metabolic instability.65
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6
Pro-drug preparation
Preparation of the pro-drug derived from compound 26 required coupling of an activated 26-
derivative, iodo-cephalosporin 30, to ciprofloxacin-derivative 33 (Scheme 1). The iodo-
cephalosporin 30 was prepared in three steps from commercially available 7-
0
1
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8
9
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8
9
0
1
2
3
4
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7
8
9
0
aminocephalosporanic acid (7-ACA). First, 7-ACA was reacted with acetic anhydride to give N-
5
5
acetyl 26. Protection of the carboxylic acid as the tert-butyl ester was then performed using
tert-butyl 2,2,2-trichloroacetimidate (TBTA) enabling formation of the tert-butyl ester 29 in the
6
6
absence of base, which has previously been reported to be associated with isomerization from
3
2
67–69
the ∆ - to the biologically inactive ∆ -cephem.
Iodination at the 3’-position with TMSI gave
3
6
the activated iodo-cephalosporin 30 ready for coupling. The ciprofloxacin component was
prepared by BOC protection of the piperazine NH to give 32 and subsequent conversion of the
6
9
carboxylic acid to the sodium salt 33. Coupling of compounds 30 and 33 was performed in 3:1
1,4-dioxane/DMF to give the protected cephalosporin-ciprofloxacin conjugate 34.36,67 Finally,
global de-protection with TFA to remove the BOC and tert-butyl ester afforded the final pro-
7
1
drug 35. Synthesis of 35 was achieved in 7-steps from commercially-available materials
without the requirement for toxic metal reagents.
Scheme 1. Synthesis of pro-drug 35.a
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3
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0
1
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8
9
0
1
2
3
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8
9
0
1
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7
8
9
0
a
o
Reagents and conditions: (i) Acetic anhydride, NaHCO , H O, acetone, 0 C, 30 min; (ii)
3
2
o
TBTA, DCM, 60 C, 16 hr; (iii) TMSI, DCM, rt, 2 hr; (iv) Boc O, 1 M NaOH, THF, rt, 16 hr;
2
(v) 0.1 M NaOH, MeOH, rt, 30 min; (vi) 3:1 1,4-dioxane/DMF, rt, 4 hr; (vii) 1:1 TFA/DCM,
o
anisole, 0 C - rt.
In vitro DNA gyrase activity
Members of the fluoroquinolone antibiotic family, including Ciprofloxacin 31, target the type II
topoisomerase enzymes, DNA gyrase and topoisomerase IV. Inhibition of these enzymes results
in the arrest of DNA replication and transcription preventing bacterial cell growth.43,72 Having
successfully prepared pro-drug 35, we moved to testing our hypothesis that the intact prodrug
would not inhibit DNA gyrase or topoisomerase IV but β-lactamase triggered hydrolysis would
result in the release of free ciprofloxacin capable of engaging these targets. To test this
hypothesis, we evaluated the ability of pro-drug 35 and ciprofloxacin to inhibit recombinant
DNA gyrase enzyme activity both in the absence and presence of the purified recombinant β-
lactamase CTX-M-15 (Fig 4). Compounds were incubated with relaxed pBR322 plasmid DNA
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with and without recombinant CTX-M-15 and recombinant DNA gyrase. As predicted,
inhibition of DNA gyrase by pro-drug 35 was not observed in the absence of CTX-M-15.
However, in the presence of CTX-M-15, 1 μM 35 was capable of reducing DNA gyrase activity
by >50%. Ciprofloxacin 31 activity was not affected by CTX-M-15. These results confirmed that
β-lactamase-specific hydrolysis of 35 results in liberation of active antibiotic capable of DNA
gyrase inhibition in vitro.
0
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8
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1
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3
4
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8
9
0
Figure 4. Activity of pro-drug 35 and ciprofloxacin 31 against recombinant DNA gyrase ± CTX-
M-15. A) DNA was separated by agrose gel electrophoresis with 2-log DNA ladder. oc, open
circle DNA; rel, relaxed DNA; sc, supercoiled DNA. B) Quantification of gel bands
corresponding to supercoiled DNA and normalized to no gyrase and gyrase only activity (ImageJ
1.52a). Gyr, DNA gyrase; cip, ciprofloxacin 31; PD, pro-drug 35; CTX, CTX-M-15. Error bars
represent SEM (n = 4); prodrug vs prodrug + CTX-M-15 was analyzed by unpaired t-test, p =
0
.0004 (GraphPad Prism 7.03).
Selective pro-drug activity against uropathogenic E. coli expressing β-lactamase
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Next, the activity of pro-drug 35 was evaluated using whole bacterial cells. MIC values for 35
and ciprofloxacin 31 were determined in E. coli CFT073 expressing the disease-relevant β-
lactamases CTX-M-1, New Delhi metallo-β-lactamase 1 (NDM-1) and Klebsiella pneumoniae
carbapenemase (KPC-3) β-lactamase (Fig 5). NDM-1 is an example of an increasingly prevalent
β-lactamase that is capable of hydrolyzing carbapenems, usually considered the last line of
1
1
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0
1
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1
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0
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9
0
7
3
defence against β-lactamase expressing bacteria , whilst KPC enzymes are class A β-lactamases
and the most common carbapenemases globally.74,75
Figure 5. Antibacterial activities for pro-drug 35 (blue) and ciprofloxacin 31 (red) against
CFT073 E.coli cells WT and expressing empty plasmid (pEMP), CTX-M-15 (pCTX), NDM1
(pNMD1) and KPC (pKPC). (A) Dose-response curves, each point represents the mean ± SEM,
n = 3; (B) Summary of MIC values.
As expected, the MIC value determined for ciprofloxacin was consistent across all tested strains
at 31 nM. The MIC determined for pro-drug 35 in E. coli CFT073 WT and expressing empty
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plasmid (pEMP) was 310 nM, representing a 10-fold decrease in activity compared to
ciprofloxacin 31 in the absence of β-lactamase. By contrast to bacteria without β-lactamase, the
MIC value for 35 was 63 nM for E. coli CFT073 strains expressing CTX-M-1, NDM1 or KPC,
only 2-fold higher than ciprofloxacin 31. These data demonstrate efficient and selective β-
lactamase mediated pro-drug cleavage and active antibiotic release, resulting in arrest of
bacterial cell growth at comparable concentrations to that with free ciprofloxacin.
0
1
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0
The activity of pro-drug 35 compared to ciprofloxacin was profiled further in six independently-
isolated uropathogenic E. coli clinical isolates expressing the CTX-M-15 β-lactamase, which
were obtained from Charing Cross Hospital, Imperial College NHS Trust. Three of the strains
were ciprofloxacin sensitive (EC11, EC16 and EC17) and three were ciprofloxacin resistant
(EC12, EC13 and EC19) as determined by diagnostic susceptibility testing. Activity of pro-drug
3
5 was confirmed against the three ciprofloxacin sensitive bacterial strains, whilst no arrest in
bacterial growth was observed for either ciprofloxacin 31 or pro-drug 35 for strains EC12, EC13
or EC19 (Fig 6 and Table S1). These results demonstrate that the antibacterial activity of 35
observed in β-lactamase expressing strains is mediated through liberated ciprofloxacin and
provide evidence for the clinical utility of 35. The gut microbiota includes both Gram-negative
bacteria such as E. coli and Gram-positive organisms such as E. faecalis. Since we had shown
that 35 was inactive against E. coli we decided to further examine the potential clinical value of
the pro-drug by testing its activity against two representative E. faecalis strains, that did not
express β-lactamase. Pro-drug 35 showed reduced activity compared to ciprofloxacin, indicating
that our approach could minimize undesirable damage to the microbiota caused by
fluoroquinolones (Fig S3). We also assessed the activity of 35 against CFT073 pEMP or pCTX-
M-1 in the presence of human serum, which can modulate drug activity via protein-binding and
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also contains esterases that have the potential to activate the pro-drug by cleaving the ester
linkage. However, data from MIC assays performed in the presence of human serum (Fig S4),
were equivalent to those obtained in the absence of serum (Fig 5). Combined, these findings
provided further confidence in the selectivity of prodrug 35 and its stability in the host
environment.
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Figure 6. Effect of pro-drug 35 (blue) or ciprofloxacin 31 (red) against six uropathogenic E.coli
clinical isolates. Each point represents mean ± SEM, n = 3.
Selective bactericidal activity against β-lactamase expressing bacteria
Finally, the ability of pro-drug 35 to kill bacteria, rather than arrest growth was evaluated.
Survival of E. coli CFT073 pEMP or pCTX-M-1 with no treatment or exposed to ciprofloxacin
3
1 or pro-drug 35 were determined over time by CFU counts (Fig 7). After 6 hours incubation
with pro-drug 35 there was >100-fold greater killing of E. coli expressing CTX-M-1, compared
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with bacteria that did not express the enzyme. Killing activity of 35 in E. coli expressing CTX-
M-1 was almost identical to free ciprofloxacin, whilst growth comparable to no treatment
controls was detected for CFT073 expressing empty plasmid incubated with 35. These findings
demonstrate that it is possible to selectively kill β-lactamase-producing bacteria using our pro-
drug approach, without adversely affecting bacteria that do not produce β-lactamase.
0
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Figure 7. Survival of CFT073 pEMP (open circle) and pCTX (filled circle) with no treatment
(green), ciprofloxacin 31 (78 nM) (red) or pro-drug 35 (78 nM) (blue). Cipro, ciprofloxacin; PD,
pro-drug 35.
CONCLUSIONS
A novel cephalosporin-fluoroquinolone antibiotic pro-drug has been designed, synthesized and
evaluated for biological activity. A program of optimization was successfully undertaken to
reduce the antibacterial activity of the intact pro-drug though modification to the cephalosporin
component. Pro-drug 35 exhibits similar growth inhibitory activity to ciprofloxacin against
uropathogenic E. coli expressing the diverse ESBLs CTX-M-1, NDM-1 or KPC, but little
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activity against strains that did not express β-lactamases. The selectively observed for
bactericidal activity was even greater, with pro-drug 35 killing β-lactamase expressing bacteria at
the same rate as free ciprofloxacin, whilst not affecting the growth of bacteria that did not
express β-lactamases.
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Overall, the activity of pro-drug 35 is consistent with: 1) permeability to pathogenic gram-
negative bacteria; 2) a low-level of antibacterial activity for the intact pro-drug; 3) β-lactamase
mediated intracellular release of ciprofloxacin upon cleavage of the cephalosporin; 4) activation
of the pro-drug by a broad range of β-lactamases.
Together, these studies demonstrate that our pro-drug approach can harness resistance as a
therapeutic opportunity to selectively kill antibiotic-resistant bacteria. Since fluoroquinolones are
a clinically useful, broad-spectrum antibiotic we envisage that increasing the selectivity profile
will have two major advantages. Firstly, increased selectivity of fluoroquinolones will enable
maintenance of the microbiota leading to reduced secondary infection rate and subsequent
antibiotic use. Secondly, a decreased side-effect profile due to minimized exposure of host cells
to fluoroquinolone antibiotic.
The focus of this work was uropathogenic E. coli (UPEC), which are a major cause of UTIs and
frequently expresses β-lactamase. Our approach is expected to result in high concentrations of
active ciprofloxacin at the site of infection (bladder and kidneys), without causing disruption to
the host microbiota. However, it is important to consider that the primary reservoir of UPEC is
the gut, and it is envisaged that our pro-drug approach would also enable the selective
decolonization of ESBL-expressing E. coli from the GI tract of people who suffer from recurrent
UTI. An additional use could be the treatment of lung infections caused by P. aeruginosa in
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patients with cystic fibrosis, for which fluoroquinolones are the only available oral
antibiotics.76,77 Since >65% P. aeruginosa isolates express the AmpC β-lactamase, it is possible
that our pro-drug approach could be used treat the infection without the associated damage to the
microbiota.
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In summary, this study paves the way for the development and use of small molecule
therapeutics that selectively target drug-resistant pathogens using broad-spectrum antibiotics,
whilst minimizing selection for resistance and without collateral damage to the microbiota. This
compliments on-going efforts to alter the spectrum of activity of existing antibiotics to enable
7
9
them to be used in new ways. For example, recent work from Liu and co-workers described an
approach to broaden the spectrum of activity of the otherwise Gram-positive restricted
oxazolidinone antibiotics to confer activity against Gram-negative bacteria. By contrast, our
approach restricts the activity of the normally broad-spectrum agent ciprofloxacin to only those
bacteria that express the β-lactamase resistance determinant. We anticipate that the modification
of existing antibiotics will prolong and expand their clinical utility, whilst efforts to discover new
antibiotic classes are underway.
EXPERIMENTAL SECTION
Experimental Procedures (Chemistry). Unless otherwise stated, reactions were conducted in
oven-dried glassware under an atmosphere of argon using anhydrous solvents. All commercially
obtained reagents and solvents were used as received. TLC analysis was performed on precoated
aluminum sheets of silica (60 F254 nm, Merck) and visualized using short-wave UV light.
Column chromatography was also performed on an IsoleraTM Spektra Four purification system
1
using Biotage Flash silica cartridges (SNAP KPSil, SNAP Ultra or SNAP KP-C18-HS). H
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NMR spectra were recorded on Bruker Av-400 spectrometers at 400 MHz using an internal
deuterium lock. Chemical shifts are quoted in parts per million (ppm) using the following
internal references: CDCl (δ 7.26), D O (δ 4.79), and DMSO-d (δ 2.50). Signal
3
H
2
H
6
H
1
1
1
1
1
1
1
1
1
1
2
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0
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9
0
multiplicities are recorded as singlet (s), doublet (d), triplet (t), quartet (q), multiplet (m), doublet
of doublets (dd), triplet of triplets (tt), apparent (app), broad (br), or obscured (obs). Coupling
1
3
constants, J, were measured to the nearest 0.1 Hz. C NMR spectra were recorded on Bruker
Av-400 spectrometers at 101 MHz using an internal deuterium lock. Chemical shifts are quoted
to 0.1 ppm, unless greater accuracy was required, using the following internal references: CDCl3
(
δ 77.0) and DMSO-d (δ 39.5). High resolution mass spectra were recorded on a Waters LCT
C 6 C
with a Waters Aquilty UPLC I-class system operating in ES+ or ES- mode. Analytical separation
was performed using a Waters BEH Acquity C18, 50mm x 2.1mm column using a flow rate of
0
.5ml/min in a 4 minute gradient elution at 40 °C. The mobile phase was a mixture of 99.9%
Water and 0.1% Formic Acid (solvent A), and 99.9% Acetonitrile and 0.1% Formic Acid
solvent B). Gradient elution was as follows: 95:5 (A/B) to 5:95 (A/B) over 3.2 min, and then
(
reversion back to 95:5 (A/B) over 0.3 min, finally 95:5 (A/B) for 0.5 min. For accurate mass
determination samples were referenced against Leucine Enkaphalin or Sulfadimethoxine. All
tested compounds were ≥95% purity by LCMS analysis. All final compounds were screened
through computational PAINS and aggregator filters and gave no structural alerts as potential
assay interference compounds or aggregators.80,81 All compounds were soluble at the
concentrations used for biological evaluation.
General Synthetic Procedures. Method A. 7-Aminocephalosporanic acid (1 equiv) was
dissolved in sat. NaHCO (aq) and acetone added, followed by acid chloride (1.2 or 2 equiv).
3
The reaction was stirred at room temperature for 30 min then washed with EtOAc. The aqueous
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55
layer was acidified to pH 2 with 1 M HCl and extracted with DCM (x3). The organic extracts
were combined, dried over Na SO , evaporated and the resulting solid triturated with ice-cold
2
4
Et O (unless otherwise stated) to afford the product.
2
0
1
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Method B. 7-Aminocephalosporanic acid (1 equiv) and acid chloride (2 equiv) were dissolved
in EtOAc and heated to reflux for 30 min. After cooling to room temperature, aniline (1.3 –
3
equiv) was added and stirred for 1 h before the reaction mixture was diluted with 3% NaHCO3
(
aq). The aqueous layer was separated and the organic layer washed with 3% NaHCO (aq) (x2).
3
The aqueous layers were combined, washed with EtOAc and acidified to pH 2 with 1 M HCl.55
The desired product was isolated as described.
Method C. Carboxylic acid (1 equiv) was dissolved in DCM and oxalyl chloride (1.2 equiv)
56
added followed by DMF (1 drop) and the reaction stirred for 16 h. The solvent was removed
under reduced pressure to afford the acyl chloride, which was used without further purification.
Preparation of compounds
6
R,7R)-3-(Acetoxymethyl)-7-(2-(4-bromothiophen-2-yl)acetamido)-8-oxo-5-thia-1
azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (8). 2-(4-Bromothiophen-2-yl)acetic acid
203 mg, 0.92 mmol), N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (193 mg,
.01 mmol) and 7-aminocephalosporanic acid (250 mg, 0.92 mmol) were suspended in DMF (8
(
1
8
2
ml) and stirred at room temperature for 48 h. The resulting mixture was filtered and the filtrate
diluted with H O and extracted with EtOAc (x3). The organic extracts were combined, washed
2
with 1 M LiCl (aq) and brine, and dried over Na SO . Solvent was removed under reduced
2
4
pressure and the resulting oil triturated with Et O. The precipitate was collected by vacuum
2
filtration and washed with DCM to afford the product as beige amorphous solid (36 mg, 8%). IR
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-
1 1
(
solid): max 3273, 3101, 2837, 1774, 1748, 1707, 1662, 1539, 1223 cm . H NMR (400 MHz,
DMSO-d ) δ 9.15 (d, J = 8.1 Hz, 1H), 7.51 (d, J = 1.2 Hz, 1H), 6.93 (s, 1H), 5.68 – 5.59 (m, 1H),
6
5.06 (d, J = 4.8 Hz, 1H), 5.00 (d, J = 12.6 Hz, 1H), 4.70 (d, J = 12.6 Hz, 1H), 3.78 (d, J = 2.6 Hz,
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
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3
3
3
3
4
4
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4
4
4
4
4
4
4
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5
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5
5
5
5
5
5
5
6
0
1
2
3
4
5
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7
8
9
0
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2
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0
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0
13
2
H), 3.58 (d, J = 18.0 Hz, 1H), 3.42 (d, J = 18.7 Hz, 1H), 2.02 (s, 3H). C NMR (101 MHz,
DMSO) δ 170.3, 169.4, 162.8, 139.1, 128.5, 122.8, 107.7, 59.0, 57.2, 35.6, 25.4, 20.6. HRMS
+
+
(ESI ): calcd for C H BrN O S (M + H) 496.9453, found 496.9479.
1
6
17
2
6 2
(
6R,7R)-3-(Acetoxymethyl)-8-oxo-7-(2-(thiophen-3-yl)acetamido)-5-thia-1-
azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (9). 3-Thiopheneacetic acid (104 mg, 0.74 mmol),
oxalyl chloride (76 μL, 0.88 mmol) and DMF (1 drop) were reaction in DCM (3 mL) according
to method C. The resulting acid chloride and 7-aminocephalosporanic acid (100 mg, 0.37 mmol)
were reacted in EtOAc (5 mL) prior to the addition of aniline (100 μL) according to method B.
The aqueous layer was extracted with DCM (x3) and the organic layers were combined, dried
over Na SO and evaporated. The resulting solid was triturated with ice-cold DCM to afford the
2
4
product as an off-white amorphous solid (48 mg, 33%). IR (solid): max 3284, 1751, 1730, 1651,
-1 1
1
621, 1536, 1241 cm . H NMR (400 MHz, DMSO-d ) δ 8.95 (d, J = 8.3 Hz, 1H), 7.46 (dd, J =
6
4.9, 3.0 Hz, 1H), 7.26 (dd, J = 2.9, 1.0 Hz, 1H), 7.03 (dd, J = 4.9, 1.2 Hz, 1H), 5.46 (dd, J = 8.3,
4
3
.8 Hz, 1H), 4.99 (d, J = 11.9 Hz, 1H), 4.93 (d, J = 4.8 Hz, 1H), 4.73 (d, J = 11.9 Hz, 1H), 3.61 –
1
3
.49 (m, 2H), 3.45 (d, J = 17.2 Hz, 1H), 3.19 (d, J = 17.3 Hz, 1H), 2.00 (s, 3H). C NMR (101
MHz, DMSO-d ) δ 170.6, 170.5, 163.3, 162.8, 135.8, 135.6, 128.6, 125.7, 122.3, 64.7, 58.5,
6
+
+
5
3
7.2, 36.4, 25.1, 20.8. HRMS (ESI ): calcd for C H N O S (M + H) 397.0528, found
16 16 2 6 2
97.0540.
(
6R,7R)-3-(Acetoxymethyl)-8-oxo-7-((R)-2-phenylpropanamido)-5-thia-1-
azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (10). (R)-(−)-2-Phenylpropionic acid (101 μL,
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1
1
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2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
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5
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0
.74 mmol), oxalyl chloride (76 μL, 0.88 mmol) and DMF (1 drop) were reaction in DCM
(3 mL) according to method C. The resulting acid chloride and 7-aminocephalosporanic acid
(100 mg, 0.37 mmol) were reacted in EtOAc (5 mL) prior to the addition of aniline (100 μL)
according to method B. The aqueous layer was extracted with DCM (x3) and the organic layers
were combined, dried over Na SO and evaporated. The resulting solid was triturated with ice-
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
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9
0
1
2
3
4
5
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7
8
9
0
1
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8
9
0
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2
3
4
5
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9
0
2
4
cold DCM to afford the product as a white amorphous solid (42 mg, 28%). IR (solid): max 3571,
-1
1
3
317, 1785, 1759, 1718, 1662, 1517, 1238 cm . H NMR (400 MHz, CDCl ) δ 8.94 (d, J = 8.4
3
Hz, 1H), 7.34 – 7.26 (m, 4H), 7.23 – 7.18 (m, 1H), 5.57 (dd, J = 8.4, 4.8 Hz, 1H), 4.99 – 4.91 (m,
2H), 4.68 (d, J = 12.3 Hz, 1H), 3.82 (q, J = 7.1 Hz, 1H), 3.44 (d, J = 17.6 Hz, 1H), 3.24 (d, J =
13
1
1
7.7 Hz, 1H), 1.99 (s, 3H), 1.34 (d, J = 7.0 Hz, 3H). C NMR (101 MHz, DMSO-d ) δ 174.1,
6
70.3, 163.7, 163.1, 141.6, 128.1, 127.2, 126.6, 63.7, 58.5, 57.4, 44.2, 25.2, 20.7, 17.8. HRMS
+
+
(ESI ): calcd for C H N O S (M + H) 405.1120, found 405.1131.
24 24 2 6
(6R,7R)-3-(Acetoxymethyl)-8-oxo-7-(2-phenylacetamido)-5-thia-1-azabicyclo[4.2.0]oct-2-
ene-2-carboxylic acid (11). 7-Aminocephalosporanic acid (100 mg, 0.35 mmol) and
phenylacetyl chloride (97 μL, 0.73 mmol) were reacted in satd NaHCO (aq) (10 mL) and
3
acetone (5 mL) according to method A to afford the product as a white amorphous solid (45 mg,
-1
1
3
1%). IR (solid): max 3254, 3034, 1778, 1737, 1707, 1654, 1536, 1223 cm . H NMR (400
MHz, DMSO-d ) δ 13.71 (br s, 1H), 9.10 (d, J = 8.3 Hz, 1H), 7.35 – 7.18 (m, 5H), 5.68 (dd, J =
6
8.3, 4.8 Hz, 1H), 5.08 (d, J = 4.8 Hz, 1H), 5.00 (d, J = 12.8 Hz, 1H), 4.68 (d, J = 12.8 Hz, 1H),
13
3
1
.66 – 3.47 (m, 4H), 2.03 (s, 3H). C NMR (101 MHz, DMSO) δ 170.9, 170.2, 164.7, 162.8,
35.8, 129.0, 128.2, 126.54, 126.48, 123.1, 62.7, 59.1, 57.4, 41.6, 25.5, 20.6. HRMS (ESI+):
+
calcd for C H N O S (M + H) 391.0964, found 391.0972.
18 19
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(
6R,7R)-3-(Acetoxymethyl)-8-oxo-7-(2-(p-tolyl)acetamido)-5-thia-1-azabicyclo[4.2.0]oct-2-
ene-2-carboxylic acid (12). 4-Methylphenylacetic acid (111 mg, 0.74 mmol), oxalyl chloride
(76 μL, 0.88 mmol) and DMF (1 drop) were reaction in DCM (3 mL) according to method C.
The resulting acid chloride and 7-Aminocephalosporanic acid (100 mg, 0.37 mmol) were reacted
in EtOAc (5 mL) prior to the addition of aniline (100 μL) according to method B. The resulting
precipitate was collected by vacuum filtration and washed with DCM to afford the product as a
white amorphous solid (63 mg, 42%). IR (solid): max 3261, 3045, 1778, 1752, 1707, 1655, 1536,
1
1
1
1
1
1
1
1
1
1
2
2
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2
2
2
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1
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4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
-1 1
1
223 cm . H NMR (400 MHz, DMSO-d ) δ 13.66 (br s, 1H), 9.04 (d, J = 8.3 Hz, 1H), 7.15 (d,
6
J = 8.1 Hz, 2H), 7.10 (d, J = 8.0 Hz, 2H), 5.67 (dd, J = 8.3, 4.8 Hz, 1H), 5.08 (d, J = 4.8 Hz, 1H),
5
3
1
.00 (d, J = 12.8 Hz, 1H), 4.69 (d, J = 12.8 Hz, 1H), 3.62 (d, J = 18.1 Hz, 1H), 3.54 – 3.41 (m,
1
3
H), 2.26 (s, 3H), 2.03 (s, 3H). C NMR (101 MHz, DMSO) δ 171.1, 170.2, 164.8, 162.8,
+
35.5, 132.7, 128.9, 128.8, 62.7, 59.1, 57.4, 41.2, 25.5, 20.63, 20.57. HRMS (ESI ): calcd for
+
C H N O S (M + H) 405.1120, found 405.1119.
1
9
21
2
6
(
6R,7R)-3-(Acetoxymethyl)-7-(2-(4-fluorophenyl)acetamido)-8-oxo-5-thia-1-
azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid (13). 4-Fluorophenylacetyl chloride (98 μL,
.74 mmol) and 7-aminocephalosporanic acid (100 mg, 0.37 mmol) were reacted in EtOAc
0
(5 mL) prior to the addition of aniline (100 μL) according to method B. The resulting mixture
o
was cooled to 4 C and the precipitate collected by vacuum filtration and washed with ice-cold
DCM to afford the product as a white amorphous solid (61 mg, 41%). IR (solid): max 3273,
-1
1
1
763, 1736, 1659, 1532, 1215 cm . H NMR (400 MHz, DMSO-d ) δ 13.67 (br s, 1H), 9.10 (d, J
6
=
8.2 Hz, 1H), 7.30 (dd, J = 8.4, 5.6 Hz, 2H), 7.12 (app t, J = 8.8 Hz, 2H), 5.67 (dd, J = 8.1, 4.8
Hz, 1H), 5.08 (d, J = 4.8 Hz, 1H), 5.00 (d, J = 12.8 Hz, 1H), 4.69 (d, J = 12.8 Hz, 1H), 3.65 –
1
3
3.46 (m, 4H), 2.03 (s, 3H). C NMR (101 MHz, DMSO-d ) δ 170.8, 170.1, 164.6, 162.8, 131.9,
6
ACS Paragon Plus Environment
2
9