IID572: A New Potentially Best-In-Class β-Lactamase Inhibitor

Article abstract of DOI:10.1021/acsinfecdis.9b00031

Resistance in Gram-negative bacteria to β-lactam drugs is mediated primarily by the expression of β-lactamases, and co-dosing of β-lactams with a β-lactamase inhibitor (BLI) is a clinically proven strategy to address resistance. New β-lactamases that are not impacted by existing BLIs are spreading and creating the need for development of novel broader spectrum BLIs. IID572 is a novel broad spectrum BLI of the diazabicyclooctane (DBO) class that is able to restore the antibacterial activity of piperacillin against piperacillin/tazobactam-resistant clinical isolates. IID572 is differentiated from other DBOs by its broad inhibition of β-lactamases and the lack of intrinsic antibacterial activity.

Full text of DOI:10.1021/acsinfecdis.9b00031

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Featured Article
IID572, A New Potentially Best-In-Class #-Lactamase Inhibitor
Folkert Reck, Alun Bermingham, Johanne Blais, Anthony Casarez, Richard Colvin,
Charles R. Dean, Markus Furegati, Luis Gamboa, Ellena Growcott, Cindy Li, Sara
Lopez, Louis E. Metzger, Sandro Nocito, Flavio Ossola, Kaci Phizackerley, Dita Rasper,
Jacob Shaul, Xiaoyu Shen, Robert Simmons, Dazhi Tang, kyuto tashiro, and Qin Yue
ACS Infect. Dis., Just Accepted Manuscript • DOI: 10.1021/acsinfecdis.9b00031 • Publication Date (Web): 12 Mar 2019
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IID572, A New Potentially Best-In-Class -Lactamase Inhibitor
Folkert Reck¥*, Alun Bermingham¥, Johanne Blais¥, Anthony Casarez¥, Richard Colvin§, Charles R.
Dean¥, Markus Furegati ᴪ, Luis Gamboa¥, Ellena Growcott¥, Cindy Li¥, Sara Lopez¥, Louis Metzger¥,
Sandro Nocito ᴪ, Flavio Ossola ᴪ, Kaci Phizackerley¥, Dita Rasper¥, Jacob Shaul¥, Xiaoyu Shen¥, Robert
L. Simmons¥, Dazhi Tang¥, Kyuto Tashiro¥, Qin Yue¥
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¥ Novartis Institutes for BioMedical Research, 5300 Chiron Way, Emeryville, CA 94608, USA
§ Novartis Institutes for BioMedical Research, 250 Massachusetts Ave, Cambridge, MA 02139, USA
ᴪ Novartis Institutes for BioMedical Research, Synthesis & Technologies Group, Klybeckstrasse 141,
4057 Basel, Switzerland
Resistance in Gram-negative bacteria to -lactam drugs is mediated primarily by the expression of -lactamases and co-dosing of
-lactams with a -lactamase inhibitor (BLI) is a clinically proven strategy to address resistance. New -lactamases that are not
impacted by existing BLIs are spreading and creating the need for development of novel broader spectrum BLIs. IID572 is a novel
broad spectrum BLI of the diazabicyclooctane (DBO) class that is able to restore the antibacterial activity of piperacillin against
piperacillin/tazobactam-resistant clinical isolates. IID572 is differentiated from other DBOs by its broad inhibition of -lactamases,
and the lack of intrinsic antibacterial activity.
Keywords: -lactamase inhibitors, diazabicyclooctane, DBOs, piperacillin, antibiotic.
Multidrug-resistant (MDR) Gram-negative bacteria are
increasing in prevalence in many regions of the world
and present a threat to the health care system, especially
in the hospital. Treatment of infections caused by these
pathogens still relies in large part on the use of -lactam
antibiotics 1. Resistance in Gram-negative bacteria to -
lactam antibiotics is mediated primarily through the
expression of -lactamases, which degrade the -lactam
2. Extended spectrum serine -lactamases (ESBLs) are
spreading and cause resistance to penicillins,
not effectively protect piperacillin against ESBLs and
serine carbapenemases. As a consequence, the
proportion of clinical strains expressing ESBLs or
carbapenemases that are susceptible to TZP is moderate
(60%) and low (0.3%) respectively according to a recent
study 5.
Recently avibactam, a novel BLI of the
diazabicyclooctane (DBO) class (Figure 1) was launched
in combination with ceftazidime (CZA), addressing
serine -lactamase mediated resistance in
Enterobacteriaceae and Pseudomononas aeruginosa 6.
However, CZA has a narrower spectrum than TZP,
lacking coverage of methicillin-susceptible
cephalosporins and monobactams. Physicians are
therefore relying increasingly on carbapenems, which
are stable to ESBLs, but this now appears to be driving
the global spread of organisms producing
Staphylococcus aureus and of anaerobes 7, 8. Avibactam
is a broad spectrum BLI, providing coverage of ESBLs,
serine carbapenemases, class C enzymes and some class
D -lactamases. However, emergence of resistance in
the clinic upon treatment of carbapenem-resistant
Enterobacteriaceae (CREs) with CZA has been reported
9, 10, and it is currently not clear what the extend of this
vulnerability is and if other DBO BLIs in the pipeline
will show similar development of resistance upon
treatment 11. In addition to their BLI activity, the DBOs
zidebactam, nacubactam, ETX-2514 and to some extent
avibactam are inhibitors of penicillin binding protein 2
(PBP-2) and exert antibacterial activity against some
Gram-negative organisms 12-15. PBP-2 inhibition by
carbapenemases 3.
-Lactamase-mediated resistance can in principle be
addressed through the development of novel -lactams
with increased stability against -lactamases 4, or by co-
dosing with a -lactamase inhibitor (BLI). The approved
BLIs clavulanic acid, sulbactam and tazobactam have
become limited in their clinical usefulness, because they
are not effective inhibitors of ESBLs or class C serine -
lactamases (cephalosporinases) and are not active
against serine carbapenemases such as Klebsiella
pneumoniae carbapenemases (KPCs). Although
piperacillin/tazobactam (TZP) is still a very important
broad-spectrum hospital drug, the BLI tazobactam does
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of the low overall yield of 12 from 5 of only 0.1%, we
Page 2 of 12
these DBOs acts synergistically with PBP-3-inhibitory
-lactams 16. However, in vitro, reduced susceptibility to
the antibacterial activity of PBP-2 inhibitors, including
DBOs, is selected at high frequency via a multiplicity of
mutations related to the stringent response and it is not
clear at this point if this will translate into rapid
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subsequently designed a more efficient and scalable
route for 12, which we intend to published elsewhere.
HN
O
HN
O
H
N
O
H2N
N
H
H
N
N
N
resistance development in the clinic 12. Another concern
with BLIs that show intrinsic antibacterial activity is that
the use of minimal inhibitory concentrations (MICs) for
the combinations with -lactams as a biomarker to
predict efficacy is not straight-forward, owing to
multiple modes of actions for efficacy and potential
differences in PK/PD for both partners.
N
N
OSO3-Na+
O
OSO3-Na+
O
OSO3-Na+
O
Avibactam, 1
Relebactam, 2
O
IID572, 12
9
O
O
H
O
N
H
HN
N
N
H
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H2N
N
H2N
N
O
N
N
N
N
O
OSO3-Na+
ETX2514
O
OSO3-Na+
O
OSO3-Na+
Zidebactam, 4
Nacubactam, 3
We initiated a program aiming to discover novel DBOs
with improved properties. Our initial goal was to
identify novel DBOs with clinically-relevant intrinsic
antibacterial activity due to inhibition of multiple PBPs
as we hypothesized that inhibition of multiple PBPs
would reduce the frequency of selecting single-step
mutants of the stringent response associated with earlier
DBOs which inhibit PBP-2 only, following the original
vision of chemists at Hoechst Marion Roussel 17.
Fortuitously, we identified a DBO that was a very potent
BLI, providing broad inhibition of serine -lactamases,
but that did not engage PBP-2 and correspondingly
lacked intrinsic antimicrobial activity altogether. Our
vision for this BLI was for it to replace tazobactam in
combination with piperacillin, to restore broad activity
of piperacillin against drug-resistant Enterobacteriaceae.
Figure 1. Selected BLIs of the DBO type.
Since 12 showed an interesting profile as a -lactamase
inhibitor (discussed in the next section), we prepared a
number of analogs, including those with substitutions on
the amide, as well as the corresponding six-membered
lactam, the synthesis of which is described in the patent
21. These analogs were synthetically more complex than
12, with similar properties to 12 in early profiling and
are therefore not discussed in this report.
Hydrolytic stability. The chemical hydrolytic stability
of 12 was determined in phosphate buffer pH 7.4 at 37
oC and was found to be similar to other DBOs (t1/2 = 55
h, Table 1). As a point of reference, the carbapenem
imipenem and the monobactam aztreonam show a t1/2 of
46 h and >2000 h respectively in this assay 4. Since
DBOs are hydrolytically labile, similar to carbapenems,
RESULTS AND DISCUSSION
care must be taken to ensure sufficient chemical solution
stability of analogs for development and for biological
assays.
Chemistry. The preparation of DBOs is challenging,
due to long and linear synthetic routes and the high ring
strain of the diazabicyclooctane scaffold 18, 19 leading to
instability towards nucleophiles. Therefore, we chose to
access novel analogs of DBOs through late stage
modification, which has the advantage of divergent
synthetic routes for more efficient analoging. Our initial
strategy was to utilize the alpha/beta unsaturated ester 6
(Scheme 1) for accessing 3-substituted analogs, which
had not been explored much in the literature, in part
probably due to the synthetic difficulties. Conventional
Michael additions to 6 failed to give the desired
products, resulting instead in opening of the cyclic urea
moiety. However, radical Michael additions via visible
light-mediated photo-redox catalysis 20 proved
successful, leading to the diastereomeric methylene
amino-substituted analogs 7-10 in 79% overall yield
(Scheme 1). Boc-deprotection of the amino group with
TFA in 7 resulted in cyclization, providing after
Intrinsic antibacterial activity for 12 and reference
DBOs. Antibacterial activity was determined by broth
microdilution assay following the recommended CLSI
methodology 22 against quality control strains of E. coli,
K. pneumoniae and P. aeruginosa (Table 1). Avibactam
showed weak antibacterial activity against E. coli and K.
pneumoniae, whereas nacubactam and zidebactam
showed stronger antibacterial activity against E. coli and
for the latter also against P. aeruginosa, in agreement
with reports in the literature 23-25. As discussed above,
the antibacterial activity observed for some DBOs is a
result of selective PBP-2 inhibition, a feature that selects
for high frequencies of resistance in vitro. Compound 12
did not show measurable antibacterial activity against
any of the isolates tested (MICs >64 g/mL), similar to
relebactam 26.
neutralization the lactam 11. No lactam formation was
observed for isomers 8-10 following similar treatment.
Formation of the lactam was initially not intended,
however, we were interested to profile a final compound
with this novel structural feature. The benzyl group in 11
was removed by hydrogenation and a sulfonyl group
was introduced using SO3/pyridine to give 12. Because
Potentiation of piperacillin by compound 12 against
strains expressing -lactamases. Potentiation of
piperacillin by 12 in the presence of a wide range of
serine -lactamases was determined using isogenic
strains of E. coli expressing individual -lactamases
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from a multi-copy plasmid 4. Comparative data were
the presence of metallo -lactamases, as expected (Table
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generated for the BLIs tazobactam, avibactam,
relebactam and vaborbactam 27. MICs for piperacillin/
BLI combinations were determined with a fixed
concentration of BLI at 4 g/mL. Vaborbactam was
tested at 8 g/mL, relebactam at 2 g/mL, tentatively
reflecting the relative high and low human doses and
exposures for vaborbactam 28 and relebactam 29
respectively (Tables 2 and S1).
S1).
Carbamylation and decarbamylation kinetics against
CTX-M-15 and KPC-2 -lactamases. DBOs cause
carbamylation of the active site serine of -lactamases,
with reversible but slow decarbamylation 33-35. The
kinetic constants for carbamylation of the enzymes
CTX-M-15 and KPC-2 by compound 12 and avibactam
were determined using a stopped-flow
9
Piperacillin is a substrate for all four classes of -
lactamases, the serine -lactamases classes A, C and D,
as well as the metallo -lactamases (class B, Table S1),
as apparent from the large shift of the MIC against
strains expressing -lactamases relative to the parental
strain.
spectrophotometric assay following nitrocefin (NCF)
substrate hydrolysis (see supplementary methods). The
values for the carbamylation efficiencies k2/Ki of
avibactam for CTX-M-15 and KPC-2 were 5.9±2.6 x
105 M-1s-1 and 7.2±3.6 x 104 M-1s-1 respectively (Table
3), similar to the values reported by Ehmann et al 36 (1.3
x 105 M-1s-1 and 1.3 x 104 M-1s-1 respectively).
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Tazobactam did not effectively potentiate piperacillin in
the presence of KPCs, TEM-1, BEL-1 and OXA-type
enzymes, apparent from the elevated MICs for strains
expressing these enzymes. A smaller MIC shift is
observed for tazobactam and the class C enzymes AmpC
and P99, indicating sub-optimal inhibition by
tazobactam. This is also reflected in the clinic, where
TEM-1 hyper production, as well as AmpC upregulation
and OXA enzymes have been associated with elevated
MICs for TZP against clinical strains 30, which often
express multiple of these -lactamases. Tazobactam’s
inability to inhibit KPC-2 is probably at least in part due
to tazobactam being a substrate for KPC-2 31.
Compound 12 showed ~ 32- and 7- fold higher
carbamylation efficiencies than avibactam with CTX-M-
15 and KPC-2 respectively. It is possible that the fused
five-membered lactam ring in compound 12 exerts
additional ring strain on the cyclic urea warhead in 12,
leading to a higher reactivity of the cyclic urea moiety
towards the active site serine of these -lactamases.
Both avibactam and especially 12 appear to carbamylate
CTX-M-15 faster than KPC-2. The residence times of
binding for compound 12 to CTX-M-15 and KPC-2
were 12 and 36 minutes respectively, shorter than those
observed for avibactam (33 and 152 minutes). It is
possible that the strain through the lactam moiety in the
adduct of 12 to the -lactamase is leading to higher off-
rates. However, we have not yet confirmed the structure
of the adduct between 12 and CTX-M-15 and KPC-2, so
it is also conceivable that 12 may react with the active
site serine of -lactamases through ring opening of the
five membered lactam. We have not confirmed
reversibility for the inhibition of -lactamases by 12, so
it is also possible that 12 is not re-generated upon
cleavage from the -lactamases, but is hydrolyzed.
Further studies are needed to elucidate the mechanism of
inhibition of -lactamases by 12, and to understand the
impact of mechanism and kinetics on human dose
selection.
Piperacillin/ relebactam showed elevated MICs for
strains expressing CTX-M-15, TEM-10, SHV-12 and all
OXAs, indicating the limitations for relebactam to
inhibit class A enzymes other than KPCs and its lack of
activity against class D enzymes. A similar profile of
narrow spectrum inhibition of class A and lack of
activity against class D enzymes was also observed for
vaborbactam when combined with piperacillin.
Relebactam and vaborbactam are intended for use in
combination with carbapenems, which are relatively
stable to most serine -lactamases. However, the lack of
activity against OXA carbapenemases like OXA-48,
limits the utility of these combinations to
carbapenemases of the class A type (mainly KPCs). Our
data also show that these BLIs are not able to effectively
potentiate a -lactam that is more labile to serine -
lactamases than a carbapenem, like piperacillin.
Potentiation of piperacillin by compound 12 against
clinical strains. In vitro activity of piperacillin and
compound 12 against 190 TZP-non-susceptible clinical
isolates of Enterobacteriaceae was determined by broth
microdilution, with 12 tested at a fixed concentration of
4 g/mL (Tables 4 and S2). Annotated mechanisms of
resistance for the strains included de-repressed class C
enzymes, KPCs, upregulated TEM-1, ESBLs like SHV-
5 and -7, as well as OXA-48-like enzymes. This panel
did not include metallo–-lactamase producing strains.
Potentiation of piperacillin by 12 was observed for the
majority of the strains, with MIC50/90 values of 8/ 16
g/mL respectively for the combination, representing
Avibactam has been reported to have a broader spectrum
of -lactamase inhibitory activity, resulting in better
protection against class A, with some coverage of class
D enzymes, especially OXA-48 32, and our data are in
agreement with these findings. Compound 12 provided
the broadest protection of piperacillin from this set of
BLIs, including better activity against the OXA-type
enzymes than avibactam and good coverage of class A
and C enzymes, including KPC-2. None of the serine -
lactamase inhibitors studied potentiated piperacillin in
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broad susceptibility of most of the TZP-resistant strains,
Page 4 of 12
in rat and dog. Plasma protein binding of  was low to
moderate for all preclinical species and 23% for human.
The clearance of 12 in rat was somewhat lower than
clearance determined for avibactam (9.7 vs 18
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6
7
8
based on the approved breakpoint for TZP of 16 g/mL.
Exceptions, aside from metallo--lactamase expressing
strains which were not investigated here, were several
OXA-181 or CTX-M-15/CMY-42 expressing strains of
E. coli from Turkey, Thailand and the Philippines
(supplementary Table S3). According to our isogenic
panel studies, compound 12 inhibits these β-lactamases,
suggesting that there may be additional β-lactamases
that are not inhibited by compound 12, or that non-β-
lactamase mediated mechanisms that decrease
mL/min/kg respectively), however the doses for 12 and
avibactam were different and the values close, so the
difference may not be significant. Clearance for
avibactam in rat has been reported in the literature as 24
mL/min/kg 42. Clearance for 12 in dog was lower than
the reported value for avibactam 43 (2.3 vs 6.1
mL/min/kg respectively). The half-life for 12 in rat was
longer than for typical -lactams (6.2 h), due to a long
t1/2 . If the relative low clearance observed for 12 in rat
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susceptibility to PIP (or compound 12) are present in
these isolates. Consistent with the latter, we found that
these isolates harbored an alteration of the ftsI gene
encoding a YRIN insertion in the target of PIP, (PBP-3
37) that was previously shown to impact susceptibility to
aztreonam 38 and LYS228 39. We showed that an E. coli
ATCC25922 derivative harboring the YRIN insertion
(or a previously described YRIK insertion 38 was also 4-
fold less susceptible to PIP than ATCC 25922 (MIC
shifting from 4 µg/mL to 16 µg/mL, supplementary
Table S4). Therefore these insertions directly reduce
susceptibility to PIP via a target-based mechanism that
would not be overcome by the BLI activity of compound
12. It is possible that other non-β-lactamase mechanisms
of resistance are also be present in these isolates. The
impact of PBP-3 alteration on susceptibility to PIP/12
found here is reminiscent of a recent report implicating a
TIPY insertion in PBP-3 in reducing susceptibility of a
carbapenem resistant E. coli clinical isolate to
and dog is also observed in man, then this may lower the
dose requirement for 12, without risk of accumulation in
q4 dosing typically used for piperacillin.
Efficacy of PIP/12 in the murine thigh infection
model. The combination of compound 12 with
piperacillin was profiled for efficacy in the neutropenic
mouse thigh infection model (Table 7). We chose the
same ratio for PIP:BLI as for TZP (8:1) for this study.
However the human dose prediction has not been
performed yet and may indicate a different ratio for
optimal efficacy. We chose a relatively resistant strain of
K. pneumoniae expressing KPC-11 and SHV-12 for this
experiment. The MIC for TZP against this strain was
>64 g/mL, higher than the clinical breakpoint for TZP
of 16 g/mL. At the doses administered, TZP did not
achieve efficacy against this strain in this model,
consistent with the elevated MIC and the presence of a
KPC -lactamase, which is not inhibited by tazobactam.
PIP/12 was efficacious against this strain, consistent
with the lower MIC of 16 g/mL reflecting inhibition of
class A -lactamases including KPCs by 12.
ceftazidime, ceftaroline or aztreonam in combination
with avibactam 40.
The activity of PIP/12 against clinical strains of the
anaerobic species Bacteroides fragilis group,
methicillin-susceptible Staphylococcus aureus (MSSA),
Pseudomonas aeruginosa and Haemophilus. influenzae
isolates was determined as well (Table 5). PIP/12 was
equipotent compared to TZP against B. fragilis group, P.
aeruginosa and H. influenzae isolates, and 2-fold more
potent than TZP against MSSA (piperacillin is not active
against methicillin-resistant S. aureus (MRSA), due to
changes in the target PBP2a 41, which cannot be rescued
by addition of a BLI). Thus the combination of PIP/12
has the potential for empiric broad coverage, including
the treatment of drug-resistant Enterobacteriaceae.
Conclusions. Compound 12 is a potent and broad
spectrum -lactamase inhibitor that does not inhibit
PBP-2 and lacks measureable intrinsic antibacterial
activity. This may remove potential development and
therapeutic liabilities attributed to selective PBP-2
inhibition. Compound 12 effectively protects piperacillin
against degradation by serine -lactamase-expressing
drug-resistant Enterobacteriaceae and other organisms,
restoring activity against the majority of TZP-resistant
strains tested. The pharmacokinetic parameters for 12
were evaluated in rodents and dog and found to be
similar to avibactam, except for 12 having a longer half
life, especially in dog. PIP/ 12 but not TZP showed
efficacy against a KPC-expressing strain of K.
Pharmacokinetics of compound 12 in preclinical
species. Pharmacokinetic parameters for 12 were
determined in mouse, rat and dog. Data in rat were also
generated for avibactam for comparison (Table 6). The
profile of 12 was similar to other DBOs and -lactams in
all species, showing low clearance with a significant
portion of the dose being recovered unchanged in urine
pneumoniae in the murine thigh infection model.
Table 1. Antibacterial activity and hydrolytic stability of compound 12 and reference DBOs
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Hydrolytic stability
t1/2 [h]a
MIC [g/mL]
1
2
3
4
5
6
7
8
9
10
DBO
E. coli
K. pneumoniae
P. aeruginosa
ATCC 25922
ATCC 43816
ATCC 27853
1 (Avibactam)
2 (Relebactam)
3 (Nacubactam)
4 (Zidebactam)
12
16
>64
2
32
>64
>64
>64
2
61
60
32
53
55
>64
>64
>64
>64
0.125
>64
>64
aHydrolytic chemical stability in phosphate buffer pH7.4 at 37°C.
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Table 2. In vitro activity of piperacillin in combination with -lactamase inhibitors against isogenic strains of E. coli expressing
individual serine -lactamases
Piperacillin MIC [g/mL], when tested alone or in combination with BLIa
Ambler classb
No BLI
Strain
(-lactamase)
12
TAZ
AVI
REL
VAB
parent
CTX-M-15
KPC-2
N/A
A
A
A
A
A
A
C
4
2
4
4
4
4
4
2
4
2
4
4
4
8
4
16
4
4
1
2
4
16
4
2
4
>64
>64
>64
>64
>64
32
>64
32
4
2
4
TEM-1
2
8
>32
>32
>32
4
TEM-10
SHV-12
PER-1
2
>32
>32
4
4
2
4
2
AmpC
>64
32
8
2
4
8
CMY-2
DHA-1
C
2
2
4
4
C
64
4
2
2
4
OXA-10
D
D
D
D
D
>64
>64
>64
>64
>64
32
64
>64
>64
>64
16
8
>32
>32
>32
>32
>32
>32
>32
>32
>32
>32
OXA-23
OXA-40
OXA-48
OXA-146
16
2
32
a 12, TAZ and AVI tested at 4 g /mL, vaborbactam at 8 g /mL, relebactam at 2 g /mL; TAZ: tazobactam, AVI: avibactam, REL:
relebactam, VAB: vaborbactam. b Ambler -lactamase class 44-46
Table 3. Kinetic values for carbamylation and decarbamylation of compound 12 against CTX-M-15 and KPC-2 in comparison
to avibactama
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1
2
3
4
5
6
7
8
Parameter
CTX-M-15
1.9±0.8 x 107
5.9±2.6 x 105
0.0014
KPC-2
5.6±2.8 x 105
7.2±3.6 x 104
0.0005
12
Carbamylation k2/Ki (M-1s-1)
Avibactam
12
Decarbamylation Koff (s-1)
Avibactam
12
0.0003
0.0001
12
36
Residence time (min)
9
Avibactam
48
219
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
15±7
29±4
Km nitrocefin (M)
a The values represent an average of 4 or 5 replicates for the carbamylation and 3 replicates for decarbamylation experiments.
Table 4. In vitro activity of piperacillin/ compound 12 against 190 piperacillin/tazobactam-nonsusceptible Enterobacteriaceae
isolatesa
MIC (g/mL)
Antibiotic
Susceptiblea (%)
Range
MIC50
8
MIC90
16
Piperacillin/ 12b
TZPb
0.5 - >64
32 - >64
64 - >64
≤0.06 - 16
≤0.06 - >64
NAc
0
>64
>64
1
>64
>64
4
Piperacillin
CZAb
0
99.5
37.4
Meropenem
8
>64
a Susceptibility as defined by CLSI (CLSI M100-S28). b BLIs were tested at a fixed concentration of 4 g/mL. c Not applicable, breakpoint
not defined. TZP: Piperacillin/ Tazobactam; CZA: Ceftazidime/ Avibactam. Strains tested include: Citrobacter freudii (18), Enterobacter
aerogenes (3), E. cloacae (22), E. hormaechei (1), E. coli (28), Klebsiella oxytoca (2), K. pneumoniae (99), Salmonella spp. (5), Serratia
marcescens (12). For the MIC distribution, MIC50 and MIC90 values of PIP/12 against the various species tested, see Table S2.
Table 5. In vitro activity of piperacillin/ compound 12 and reference compounds against Bacteroides fragilis group, methicillin-
susceptible Staphylococcus aureus (MSSA) and Pseudomonas aeruginosaa
MIC (g/mL)
Range
Micro organism
(no. of isolates)
Antibiotic
Susceptiblea (%)
MIC50
MIC90
32
32
>64
1
B. fragilis groupb (27)
Piperacillin/ 12c
TZPc
0.25 - 64
≤0.06 - >64
2 - >64
4
NAd
93
4
Piperacillin
Meropenem
Piperacillin/ 12c
TZPc
32
0.5
1
59
0.125 - >16
0.5 - 1
96
MSSA (40)
1
NAd
100
NAd
100
0.5 - 8
1
2
Piperacillin
Vancomycin
1 - >64
4
>64
1
0.5 - 1
0.5
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P. aeruginosa (124)
H. influenzae (25)
Piperacillin/ 12c
TZPc
≤0.06 - 64
8
16
NAd
90
1
2
3
4
5
6
7
8
0.25 - 64
8
16
Piperacillin
Meropenem
Piperacillin/ 12c
TZPc
0.25 - >64
≤0.06 - 32
≤0.06 – 0.5
0.25 – 0.5
0.25 - >64
0.25 - >32
8
32
77
1
8
82
≤0.06
≤0.06
≤0.06
4
≤0.06
≤0.06
64
NAd
100
NAd
52
9
10
11
12
Piperacillin
Azithromycin
>32
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
a Susceptibility as defined by CLSI (CLSI M100-S28). For susceptibility of MSSA to piperacillin/ TAZ, the USA FDA breakpoint was
applied (ZOSYN® package insert). b B. fragilis (9), B. ovatus (6), B. thetaiotaomicron (7) and B. vulgatus (5). c BLIs were tested at a fixed
concentration of 4 g/mL. d Not applicable.
Table 6. Pharmacokinetics in mouse, rat and dog and plasma protein bindinga
Clearance
[mL/min/kg]
Volume
[L/kg]
% dose in
urine
PPBb
[% bound]
Compound
Half-life [h]
AUC [M h]
Mouse
Rat
12
12
9.2
9.7
18
0.2
0.5
0.4
0.2
1.4
6.2
1.5
2.5
60
187
18c
80
NDd
45
20
61
Avibactam
12
30
NDd
24
Dog
2.3
88
a Pharmacokinetic parameters following administration of 10 mg/kg (mouse), 30 mg/kg and 5 mg/kg for 12 and avibactam respectively
(rat) or 3 mg/kg (dog), by iv infusion. b Plasma protein binding. c Different dose for 12 and avibactam, see a. d Not determined.
Table 7. In vivo efficacy of piperacillin/ compound 12 and piperacillin/ TAZ against an SHV-12 and KPC-11 expressing strain
of K. pneumoniae in the neutropenic mouse thigh infection modela
Maximum Efficacy
Highest dose tested
PIP:BLI [mg/kg/day]
Static dose [mg/kg/day]
MIC (g/mL)
[ Log 10 CFU/thigh]
Piperacillin/12
16
1695
- 0.84 ± 0.09
5120:640
5120:640
TZP
>64
Stasis not achieved
Not efficacious
a Animals were inoculated with approximately 1x106 CFU/thigh. After treatment with vehicle for 24 hrs, bacterial burden increased by
2.34 ± 0.61 Log10 CFU compared to static levels in the thigh (0 hr, 5.96 ± 0.24 Log10 CFU/thigh). Maximum efficacy was benchmarked
against the bacterial load in the thigh at the time treatment began (stasis). Compounds were dosed subcutaneously every 3 hrs. PIP and
BLIs were dosed in a ratio of 8:1. TZP: Piperacillin/ Tazobactam.
Scheme 1. Synthesis of compound 12 by photoredox couplinga
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H
H
N
H
N
H
N
CO2H
N
Boc
Boc
Boc
Boc
1
2
3
4
5
6
7
O
HN
O
O
O
O
O
H
N
H
H
N
H
N
H
N
Boc
HO
O
O
O
O
O
a-c
d
N
N
N
N
N
N
N
N
OBn
OBn
OBn
OBn
O
O
O
O
OBn
OBn
O
O
7
8
9
10
5
6
(10%)
(15%)
(41%)
(13%)
(21%)
8
9
e
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
HN
HN
H
N
H
O
f-g
O
H
H
N
N
N
OSO3Na
O
OBn
O
12
11
(40-63%)
aReagents and conditions: (a) MeOH, DMAP, DCC, 0 oC  rt; (b) NH(ipr)2, n-BuLi, PhSeCl, -78 oC  -10 oC; (c) H2O2/AcOH, K2SO3,
0 oC; (d) Boc-Gly-OH, Ir[df(CF3)ppy2(dtbpy]PF6, K2HPO3, 8W UVA fluorescence tube; (e) TFA, rt, then N(Et)3, 0 oC  rt; (f) Pd-C, MeOH;
(g) SO3py
Supporting Information
The Supporting information is available free of charge on the ACS Publications website at DOI:
Supplementary methods
Supplemental Table S1. MICs for piperacillin +/- BLI against isogenic strains of E. coli expressing individual -
lactamases
Supplementary Table S2. MIC distribution of PIP/12 against 190 Enterobacteriaceae isolates
Supplemental Table S3. Presence of ftsI mutations in E. coli clinical isolates that are not highly susceptible to
PIP/12
Supplementary Table S4. Impact of YRIN and YRIK insertions in PBP-3 on susceptibility of E. coli to β-lactam
antibiotics
Supplementary references
Author Information
Corresponding Author
*E-mail: Folkert.Reck@Novartis.com
Conflict of interest
The authors declare no competing financial interests.
Acknowledgements
The authors thank JMI Laboratories, Inc. and International Health Management Associates (IHMA) for clinical isolates
and Jennifer Leeds for critical review of the manuscript.
Abbreviations
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BLI, -lactamase inhibitor; DBO, diazabicyclooctane; MDR, multidrug-resistant; ESBL, extended spectrum -
lactamase; KPC, Klebsiella pneumoniae carbapenemase; TZP, piperacillin/tazobactam; CZA, ceftazidime/avibactam;
CRE, carbapenem-resistant Enterobacteriaceae; PBP, penicillin binding protein; MIC, minimal inhibitory
concentration; PIP, piperacillin; REL, relebactam; VAB, vaborbactam; AVI, avibactam; TAZ, tazobactam.
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