Discovery of efficacious pseudomonas aeruginosa-targeted siderophore-conjugated monocarbams by application of a semi-mechanistic pharmacokinetic/pharmacodynamic model

Article abstract of DOI:10.1021/jm501506f

To identify new agents for the treatment of multi-drug-resistant Pseudomonas aeruginosa, we focused on siderophore-conjugated monocarbams. This class of monocyclic β-lactams are stable to metallo-β-lactamases and have excellent P. aeruginosa activities due to their ability to exploit the iron uptake machinery of Gram-negative bacteria. Our medicinal chemistry plan focused on identifying a molecule with optimal potency and physical properties and activity for in vivo efficacy. Modifications to the monocarbam linker, siderophore, and oxime portion of the molecules were examined. Through these efforts, a series of pyrrolidinone-based monocarbams with good P. aeruginosa cellular activity (P. aeruginosa MIC₉₀ = 2 μg/mL), free fraction levels (>20% free), and hydrolytic stability (t_1/2 ≥ 100 h) were identified. To differentiate the lead compounds and enable prioritization for in vivo studies, we applied a semi-mechanistic pharmacokinetic/pharmacodynamic model to enable prediction of in vivo efficacy from in vitro data.

Full text of DOI:10.1021/jm501506f

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Article
Discovery of Efficacious Pseudomonas aeruginosa-Targeted Siderophore-
Conjugated Monocarbams by Application of a Semi-Mechanistic PK/PD Model
Kerry E Murphy-Benenato, Pratik R. Bhagunde, April Chen, Hajnalka E Davis, Thomas
F. Durand-Reville, David E Ehmann, Vince Galullo, Jennifer J. Harris, Holio Hatoum-
Mokdad, Haris Jahic, Aryun Kim, M R Manjunatha, Erika L. Manyak, John Mueller, Sara
Patey, Olga Quiroga, Michael Rooney, Li Sha, Adam B. Shapiro, Mark Sylvester, Beesan
Tan, Andy S Tsai, Maria Uria-Nickelsen, Ye Wu, Mark zambrowski, and Shannon X. Zhao
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm501506f • Publication Date (Web): 06 Feb 2015
Downloaded from http://pubs.acs.org on February 12, 2015
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Discovery of Efficacious Pseudomonas aeruginosa-Targeted Siderophore-Conjugated
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Monocarbams by Application of a Semi-Mechanistic PK/PD Model
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Kerry E. MurphyꢀBenenato,‡a* Pratik R. Bhagunde,‡b April Chen, Hajnalka E. Davis,‡
Thomas F. DurandꢀRéville,‡ David E. Ehmann,‡ Vincent Galullo,‡ Jennifer J. Harris,‡c Holia
HatoumꢀMokdad,‡ Haris Jahić,‡ Aryun Kim,‡* M.R. Manjunatha,† Erika L. Manyak,ǁd John
Mueller,‡ Sara Patey,‡ Olga Quiroga,‡ Michael Rooney,‡ Li Sha,‡ Adam B. Shapiro,‡ Mark
Sylvester,‡ Beesan Tan,‡e Andy S. Tsai,‡f Maria UriaꢀNickelsen,‡e Ye Wu,‡ Mark
Zambrowski,‡g Shannon X. Zhao‡
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‡Infection Innovative Medicines, AstraZeneca R&D Boston, 35 Gatehouse Drive, Waltham,
Massachusetts 02451, USA
†Infection Innovative Medicines, AstraZeneca India Pvt. Ltd., Bellary Road, Bangalore
560024, India
ǁDiscovery Sciences, AstraZeneca R&D Boston, 35 Gatehouse Drive, Waltham,
Massachusetts 02451, USA
Abstract: In order to identify new agents for the treatment of multiꢀdrug resistant
Pseudomonas aeruginosa, we focused on siderophoreꢀconjugated monocarbams. This class
of monocyclic βꢀlactams is stable to metalloꢀβꢀlactamases and have excellent P. aeruginosa
activities due to their ability to exploit the iron uptake machinery of Gramꢀnegative bacteria.
Our medicinal chemistry plan focused on identifying a molecule with optimal potency and
physical properties and activity for in vivo efficacy. Modifications to the monocarbam linker,
siderophore, and oxime portion of the molecules were examined. Through these efforts a
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series of pyrrolidinoneꢀbased monocarbams with good P. aeruginosa cellular activity (P.
aeruginosa MIC90 = 2 ꢀg/ml), free fraction levels (> 20 % free) and hydrolytic stability (t1/2
≥
7
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9
100 h) were identified. In order to differentiate the lead compounds and enable prioritization
for in vivo studies, we applied a mechanistic PK/PD model to enable prediction of in vivo
efficacy from in vitro data.
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Introduction
The need for treatment options for multiꢀdrug resistant Gramꢀnegative bacteria,
especially Pseudomonas aeruginosa (P. aeruginosa) is becoming critical due to the constant
evolution of resistance.1ꢀ3 Strategies to combat this issue are to develop new antiꢀinfectives
that have novel targets, therefore eliminating the concern for crossꢀresistance to other known
agents, or optimize current chemotypes that address the known mechanisms of resistance.
Nevertheless, the clinical pipeline for agents targeting Gramꢀnegative bacteria is thin, with
the carbapenems being the last novel chemotype to be introduced.4, 5
Despite their long history of use, βꢀlactam antibiotics offer a unique opportunity
for the development of new Gramꢀnegative agents.6 They are a clinically validated class of
antibiotics with an improved safety profile as compared to other agents.7 Their targets, the
penicillinꢀbinding proteins (PBPs), are located in the periplasm, eliminating the need to
traverse both cell membranes of the Gramꢀnegative bacterium. Specifically, βꢀlactams’
target the high molecular weight PBPs which are responsible for the transpeptidation reaction
in the synthesis of the bacterium peptidoglycan layer, a key component of all bacterium cell
wall architectures. βꢀLactams work by forming a stable acylꢀenzyme complex with the active
site serine of the PBPs. The target PBPs varies depending on the chemotype.8ꢀ10
Due to the wideꢀspread use of βꢀlactam antibiotics, most modes of resistance to
current therapies are wellꢀunderstood, enabling design efforts to combat them.11 In addition,
for clinically used βꢀlactams there are well established pharmacokinetic/pharmacodynamic
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(PK/PD) relationships.12 Indeed, the successful development of new carbapenems, fourth
generation cephalosporins and novel βꢀlactamase inhibitors leads one to believe there is
potential for success in this area.13ꢀ15
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Two of the largest threats to the effectiveness of P. aeruginosaꢀtargeted βꢀ
lactam antibiotics are inability to access the periplasmic space due to porin mutations and
hydrolysis by βꢀlactamases.10, 16 One of the largest current clinical threats is the rise of panꢀ
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resistant metalloꢀβꢀlactamases in Gramꢀnegative bacteria since there are no known inhibitors
of this class of βꢀlactamases.17ꢀ19 Reports in the literature have highlighted a class of PBP3
selective inhibitors that address these issues due to a novel mode of penetration and a core
structure that is stable to metalloꢀβꢀlactamases: siderophoreꢀconjugated monocarbams (Figure
1).21ꢀ23 Originally reported in the midꢀ1980’s, these compounds, exemplified by 2
(Pirazmonam)21 and 3 (MCꢀ1)23, have two key features which provide them with their unique
activity: a monocyclicꢀβꢀlactam core which renders them stable to metalloꢀβꢀlactamases24, 25
and an ironꢀchelating moiety which is responsible for cellular uptake.26ꢀ28
Figure 1. Monocyclic βꢀlactams
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Bacteria require iron as a nutrient for survival in the host.29 In order to acquire
this essential mineral, pathogens have evolved a number of strategies to sequester iron,
including siderophore production.30 Siderophores are small chelating molecules secreted by
bacteria, for the purpose of acquiring iron from the host and the environment. P. aeruginosa,
like most Gramꢀnegative pathogens, secretes multiple siderophores, each with a dedicated
outer membrane receptor to allow import and eventual intracellular release.31 Compounds 2
and 3 (Figure 1) incorporate a 3ꢀhydroxypyridinone moiety that mimics the bacteria’s natural
siderophores and hijacks its iron uptake machinery for penetration into the periplasm.32, 33
This results in excellent in vitro potency against P. aeruginosa for this class (2 and 4, Table
1) and a clear improvement over the monocyclic βꢀlactam 1 (Aztreonam), (Figure 1).34
Comparison of the acylation rate constants for 1and compounds 2 and 4 for their PBP3 target
reveals the enhanced P. aeruginosa susceptibility for siderophore conjugates is not related to
target affinity and is likely due to better outer membrane permeability.35 Aspects for
optimization of these compounds are the poor hydrolytic stability of 2 (t1/2 < 50 h) and the
low free fraction of 4 (4% free).
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McPherson et. al. demonstrated that monocarbam 3, enters P. aeruginosa via
the catechol siderophore receptors PiuA and PirA.36 Furthermore, they reported 3 is stable to
a broad spectrum of serine and metalloꢀβꢀlactamases, and is not affected by porin deletions or
overꢀexpression of efflux pumps. They also concluded the first step resistance mutation to
monocarbam 3, knockout of the siderophore receptor PiuA, is not relevant in vivo. However,
a recent report by Tomaras et. al. on a related siderophoreꢀconjugated monobactam MBꢀ1
described adaptive resistance.37 In this report they described an in vitro assay which was
predictive of the observed in vivo results however this assay has only been shown to be
predictive for a single compound and no data to support its applicability to the monocarbam
class has been reported. Therefore additional in vivo experiments will be required to
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establish a strong in vitro to in vivo correlation aimed at enabling the development of
siderophore containing βꢀlactams.
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Recognizing successful progression for this project would require extensive in
vivo experimentation, we anticipated that informed decisions on the selection of compounds
for in vivo experiments would require quantitative PK/PD modeling. Thus, our program had
two main goals: (1) identify monocarbam compounds with optimal physical properties (i.e.,
high free fraction and good hydrolytic stability) for in vivo efficacy, and (2) develop a semiꢀ
mechanistic PK/PD model to enable prediction of in vivo efficacy.38 The PK/PD relationship
of βꢀlactam antibiotics are well described, and free time above MIC has been shown to be the
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driver for all classes of βꢀlactams.39 The previously mentioned lack of correlation between
P. aeruginosa PBP target affinity and cellular activity led us to take a nonꢀstructure based
drug design approach. Instead, we focused on chemical modifications aimed at optimizing
the plasma protein binding (PPB) and clearance (CL) since free drug exposure is the key
factor for efficacy for this class of compounds.40 Through our medicinal chemistry
optimization we identified a series of pyrrolidinoneꢀbased monocarbams which demonstrated
an unprecedented balance in their P. aeruginosa activity, hydrolytic stability, PPB and CL.
Through our studies we developed the structure activity relationships (SAR) around
permeation, efflux and target engagement. After establishing the in vitro activity of the
series, we then applied a mechanistic PK/PD model which allows prediction of in vivo
efficacy from in vitro kill kinetics and therefore prioritization of in vivo experiments aimed at
identifying potential clinical candidates.
Chemistry
The synthesis of the siderophoreꢀconjugated monocarbams utilized one of two
general synthetic schemes: (1) monocarbam formation with desired heterocycle followed by
introduction of the siderophore piece (Scheme 1), or (2) coupling of the siderophore to the
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desired heterocycle, followed by monocarbam formation (Scheme 2). Two representative
examples are depicted here.
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As can be seen in Scheme 1, coupling of known aminothiazole βꢀlactam 541, 42
with unfunctionalized heterocycles (e.g., 6) under traditional monocarbam formation
conditions,22, 23 followed by azide reduction provided coupling partner 7. Amide formation
and twoꢀstep deprotection afforded the target compound 9. Alternatively, coupling of a
protected siderophore (e.g., 8)27 to the target heterocycle (10), followed by monocarbam
formation and deprotection steps, provided fully elaborated monocarbams 13. The key
challenge in accessing the monocarbams was purification and isolation of intermediates and
final compounds due to their instability and polar nature. Synthetic details for all
monocarbams can be found in the supporting information.
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Scheme 1. Example of general route 1 for the synthesis of siderophoreꢀconjugated
monocarbams
(a) (i) MSTFA, THF; (ii) CSI, DCM, 0 °C; (iii) THF, 0 °C – rt, 66%; (b) H2, 10 mol % Pd/C, EtOH; (c) HATU, iꢀPr2EtN,
DMF; (d) H2, Pd black, MeOH; (e) TFA, DCM, 0 °C, 17% over four steps
Scheme 2. Example of general route 2 for the synthesis of siderophoreꢀconjugated
monocarbams
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(a) 8, EDCI, HOBT, DMF, 89%; (b) (i) MSTFA, THF; (ii) 5, CSI, DCM, 0 °C; (iii) THF/DCM, 0 °C ꢀ rt; (c) H2, Pd black,
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MeOH; (d) TFA, DCM, 33% over three steps
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Results and Discussion
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Structure activity relationships of the monocarbam scaffold. From the outset, our goal
was to identify potent inhibitors of P. aeruginosa which addressed the hydrolytic stability and
physical property concerns of the previously reported monocarbams.23 The initial designs
focused on introduction of a saturated linker, analogous to compound 2,21 in order to reduce
the planarity of the molecule and therefore increase free fraction.40 A variety of saturated
heterocycles (9, 13ꢀ16) were synthesized in addition to compounds with shortened linkers
(17, 18) (Table 1). To evaluate these compounds, P. aeruginosa whole cell activity (MICs),
P. aeruginosa PBP3 acylation rate constants,35 human PPB, rat CL, and hydrolytic stability
were analyzed.
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Except for dihydrotriazinone 14 and aniline 18, all compounds demonstrated
excellent activity against the multiple efflux pump knockꢀout strain P. aeruginosa ARC546
and good to moderate activity against wild type P. aeruginosa (ARC545, PAO1). In
addition, all compounds displayed PBP3 acylation rate constants of >1 × 103 Mꢀ1sꢀ1. Overall,
these compounds are effective inhibitors of P. aeruginosa PBP3 and are able to permeate the
outer membrane of the Gramꢀnegative bacteria.
As can be seen in Table 1, the monocarbams with saturated linkers afforded free
fraction values similar to 2, a 4 to 10ꢀfold increase over triazolone 4. In addition, the rat PK
of some of the more potent analogs (9 and 13) showed reasonable clearance levels. In
general, this set of compounds had good hydrolytic stability, demonstrating excellent
stabilities of >100 h. Examination of the hydrolytic stability reaction profiles indicated the
major degradation products of resulted from decomposition of the sideꢀchains and not βꢀ
lactam hydrolysis.
Table 1. Antibacterial activity and physiochemical properties of novel monocarbams
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P. aeruginosa MIC
P. aeruginosa PBP3
acylation constant
kinact (Mꢀ1sꢀ1)
Human
PPBc
Hydrolytic
Stabilityd
Rat CLe
(ꢀg/mL)
Compound
ARC546a
ARC545b
(% free)
t
(h)
(mL/min/kg)
½
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4
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13
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18
0.25
0.06
<0.06
<0.06
<0.06
2
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<0.06
<0.06
1
4
0.25
0.25
1
1
16
8
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16
2.6 × 105
1.4 × 105
7.2 × 104
1.5 × 105
2.6 × 104
3.8 × 103
2.4 × 104
4.6 × 104
2.5 × 104
62f
>150f
41 ± 9.9
138 ± 17
>150g
21
NT
9
16
12
NT
NT
NT
NT
NT
29 ± 4.8
4 ± 0.90
24 ± 0.95
14 ± 0.30
16 ± 0.30
26 ± 2.3
33f
38 ± 7.5
40 ± 4.7
>150g
>150g
61 ± 1.2
23 ± 1.8
>150g
1.3 × 104
103 ± 6.4
aP. aeruginosa ARC546 = PAO1 ∆MexABCDXY; P. aeruginosa ARC545 = PAO1; cn = 3, standard deviation; dpH = 7.4,
b
37 °C; eHan Wistar Rat (male), iv infusion, 1 mg/kg, (n=2); NT not tested; fn = 1; gNot determined
The promising results of the initial designs prompted elaboration of the SAR
around the linker between the siderophore moiety and the heterocycle in order to address the
stability issue and increase the P. aeruginosa antimicrobial activity. Being the most potent,
compounds 9, 13, and 16 were all candidates for elaboration. Attempts to introduce
functionalization to triazinone 13 were unsuccessful. The excellent PBP3 acylation rate
constant of 9 (as compared to pyrrolidine 16) indicated the carbonyl of the pyrrolidinone may
be part of a favorable interaction with the PBP3 enzyme, which has been observed
crystallographically with triazolone based compounds.43 Hypothesizing this improved
acylation rate constant could be beneficial to achieving improved P. aeruginosa MICs, we
chose to pursue scaffold 9 and focus on improving the MICs and hydrolytic stability.
The compounds synthesized had modified linkages of the siderophore to the
heterocycle (Table 2). As previously observed, the compounds all show good to excellent
activity against the P. aeruginosa pump knockꢀout strain albeit diminished as compared to
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the previous set (Table 1). The P. aeruginosa PBP3 acylation rate constants indicate that
reduced target engagement is not the cause of reduced activity. This suggests there may be a
relationship between the siderophore linker and the ability of the molecule to penetrate the
outer membrane of P. aeruginosa. We also observed a dramatic change in activity against
wildꢀtype P. aeruginosa. Where the enantiomers of the pyrrolidinone (9 and 19)
demonstrated equivalent MIC values and PPB, amine 20, urea 21 and triazole 22 showed 8ꢀ
32ꢀfold reduction in activity against P. aeruginosa PAO1. Comparing the amino acidꢀ
derived linkers 23ꢀ27 a range of activities (PAO1 MIC = 1 – 8 ꢀg/ml and PPB = 14 – 38%
free) was observed, with glycine containing analogue 23 and aspartic acid containing
analogue 24 showing the most potent P. aeruginosa MICs. Comparing the linkages, the
amides were generally more stable than the amino (20), urea (21) or triazine (22) linkage.
Analysis of the hydrolytic stability profiles indicated the major products were not due to βꢀ
lactam ringꢀopening. Instead the major cause of instability may be due to the propensity of
the sideꢀchain to βꢀeliminate (e.g. 20, 22) or hydrolyze (e.g. 21). It was hypothesized the
instability of compound 25 is caused by the additional basic amine.
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Table 2. Antibacterial activity and physiochemical properties of modified siderophoreꢀ
conjugated monocarbams
P. aeruginosa MIC
(ꢀg/mL)
P. aeruginosa PBP3
acylation rate constant
kinact (Mꢀ1sꢀ1)
Human
PPBc
(% free)
Hydrolytic
Stabilityd
Compound
ARC546a
ARC545b
t
(h)
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<0.06
<0.06
0.5
1
0.5
8
16
4
2
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4
4
1.5 × 105
3.5 × 105
5.7 × 104
2.4 × 104
2.3 × 105
2.4 × 104
3.8 × 104
9.4 × 103
1.8 × 104
1.4 × 104
24 ± 0.95
25 ± 3.5
40 ± 8.2
26 ± 3.8
31 ± 10
38 ± 0.56
14 ± 1.4
18 ± 2.4
21 ± 3.1
28 ± 1.9
>150e
>150e
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22
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27
48 ± 3.8
30 ± 5.7
117 ± 12
>150e
1
<0.06
0.25
0.5
0.5
0.25
0.5
>150e
51 ± 5.3
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>150e
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114 ± 30
aP. aeruginosa ARC546 = PAO1 ∆MexABCDXY; P. aeruginosa ARC545 = PAO1; cn = 3, standard deviation; dpH = 7.4,
b
37 °C; eNot determined.
To date, there has been limited SAR generated on the siderophore moiety in the
monocarbam series.44 SAR from other classes of siderophoreꢀcontaining monocyclic βꢀ
lactams indicate some differences in activity can be observed between a 3ꢀhydroxypyridinone
and a 1,3ꢀdihydroxypyridinone,45 however this has never been explored in the monocarbam
class. To investigate this feature 1,3ꢀdihydroxypyridinone derivatives of compounds with
differing activities were synthesized. The compounds and data for both the parent 3ꢀ
hydroxypyridinone and 1,3ꢀdihydroxypyridinone derivatives are highlighted in Table 3.
Comparison of the modified siderophore compounds with the parent compounds presented a
few trends as well as some unexpected effects. The P. aeruginosa MIC value of compound
28 was similar to 9, however the free fraction was reduced (24 to 8% free). Comparison of
ureas 21 and 29 showed a marked improvement in potency (PAO1 MIC = 0.5 ꢀg/ml versus
16 ꢀg/ml) and hydrolytic stability (87 h versus 30 h). The amino acid derivatives 30 and 31
provided decreased MICs, and increased acylation rate constants and free fraction compared
to the parent compounds (23 and 24, respectively). In general, the 1,3ꢀdihydroxypyridinone
analogs afforded compounds with improved potency and hydrolytic stability compared to the
corresponding 3ꢀhydroxypyridinones while maintaining good free fractions. Attempts to
correlate the changes in PPB and hydrolytic stability to changes in the pKa of the siderophore
did not reveal any information. It was concluded the changes we observed are due to
unpredicted effects caused by the modified 3ꢀdimensional structures of the compounds.
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Initial attempts to predict the 3ꢀdimensional shapes and create computational models to
corroborate these hypotheses were not successful and continued efforts were out of scope of
the presented project. However a deeper understanding of how the identity of the
siderophore and its linker to the scaffold would provide useful incite for future design.
Table 3. Comparison of the antibacterial activity and physiochemical properties of 3ꢀ
hydroxypyridinone and 1,3ꢀhydroxypyridinone siderophoreꢀconjugated monocarbams
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P. aeruginosa MIC
(ꢀg/mL)
P. aeruginosa PBP3
acylation rate constant
kinact (Mꢀ1sꢀ1)
Human
PPBc
(% free)
24 ± 0.95
8 ± 0.18
26 ± 3.8
26 ± 6.9
38 ± 0.56
45e
Hydrolytic
Stabilityd
Compound
ARC546a
ARC545b
t
(h)
½
9
<0.06
<0.06
1
<0.06
0.25
<0.06
0.5
1
0.5
16
0.5
2
1.5 × 105
3.0 × 105
2.4 × 104
5.9 × 104
2.4 × 104
1.0 × 105
3.8 × 104
2.0 × 105
>150f
28
21
29
23
30
24
31
>150f
30 ± 5.7
87 ± 10
>150f
0.5
1
>150f
14 ± 1.4
>150f
<0.06
0.25
43e
>150f
aP. aeruginosa ARC546 = PAO1 ∆MexABCDXY; P. aeruginosa ARC545 = PAO1; cn = 3, standard deviation; dpH = 7.4,
b
37 °C; en = 1; fNot determined
To expand the SAR we next examined introduction and removal of a methyl
substituent on several portions of the scaffold as can be seen in Table 4. These changes had
large effects on chemical stability and potency. Methylation of the urea nitrogens had little
effect on potency or PPB, however compound 32 had a significant reduction in stability
presumably due to the increased basicity of the nitrogen and therefore favoring elimination.
Removal of the gemꢀdimethyl substituent from the aminoꢀthiazole oxime (34) resulted in a
loss of antimicrobial activity against P. aeruginosa and 10ꢀfold reduction in acylation
efficiency. Based on reported crystal structures of aminoꢀthiazole oxime containing βꢀ
lactams, this reduction in target engagement is due to loss of hydrophobic interactions in that
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portion of the P. aeruginosa PBP3 pocket, however the efflux mutant P. aeruginosa MIC
(ARC546 MIC = 0.13 ꢀg/ml) indicates a high rate constant is not critical for cellular
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activity.43 Introduction of an additional methyl to the βꢀlactam core (35) resulted in a marked
increase in MIC and a large decrease in acylation rate constant. It has been shown that this
substitution is tolerated on monobactams and monosulfactams. However with the larger
activating group of a monocarbam the additional substituent on the βꢀlactam core causes
unfavorable clashes with the enzyme.43, 46 These data indicate that modification of the parts
of the inhibitors that interact with the PBP3 enzyme has a direct effect on acylation rate
constants but this does not always correlate with antimicrobial activity.
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Table 4. Antibacterial activity and physiochemical properties of monocarbam analogs
P. aeruginosa
P. aeruginosa MIC
(ꢀg/mL)
ARC546a ARC545b
Human
PPBc
Hydrolytic
Stabilityd
PBP3 acylation
rate constant
kinact (Mꢀ1sꢀ1)
5.9 × 104
Compound
R1
Me
Me
Me
H
R2
H
H
H
H
Me
R3
H
Me
H
H
H
R4
H
H
Me
H
(% free)
26 ± 6.9
27 ± 2.4
26 ± 5.5
26 ± 0.34
23 ± 1.1
t
(h)
½
29
32
33
34
35
<0.06
0.25
0.25
0.13
1
0.5
0.5
1
4
8
87 ± 10
19 ± 1.9
>150e
3.1 × 104
1.9 × 104
3.4 × 103
87 ± 20
Me
H
8.1 × 102
>150e
aP. aeruginosa ARC546 = PAO1 ∆MexABCDXY; P. aeruginosa ARC545 = PAO1; cn = 3, standard deviation; dpH = 7.4,
b
37 °C; dNot determined
Population MICs and PK properties. After identification of a number of compounds with
favorable potency and properties (P. aeruginosa PAO1 MICs ≤ 1, free fractions >20%, and
stabilities >100 h) we profiled them further for differentiation in terms of susceptibility or
PK. Data for select compounds can be seen in Table 5. These compounds, though not as
potent as triazolone 4, are effective against a panel of clinical isolates of P. aeruginosa,
including strains containing metalloꢀβꢀlactamases and P. aeruginosa specific extended
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spectrum βꢀlactamases (ESBLs),47 with MIC90 values of ≤ 2 ꢀg/ml (details of the strains
included in the MIC90 panels can be found in the supporting information). Against other
Gramꢀnegative pathogens, they were less effective agents (Table 5). Reasons for the elevated
MIC90 values against the enterobacteriacea are due to the susceptibility of these compounds to
certain classes of ESBLs (e.g., TEM, CTXꢀM, SHV) which are prevalent in this class of
bacteria. Reduced susceptibility to Acinetobacter baumannii did not correlate with the βꢀ
lactamase content suggesting the elevated MICs are due to poor penetration.
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The new pyrrolidinoneꢀbased monocarbams consistently demonstrate
acceptable levels of free drug. The rat free fraction, showing similar trends to the human free
fraction, is increased compared to compound 4 while low clearance is observed. Overall the
monocarbams have PK properties consistent with other βꢀlactams (e.g.1, Table 5).48
Table 5. Population MICs and PK properties of monocarbam analogs
MIC90 (ꢀg/mL)a
(range)
1
4
9
13
29
1024
(4 ꢀ >1024)
>512
(0.03 ꢀ >512)
>512
(0.25 ꢀ >512)
>512
(16 ꢀ >512)
0.25
(0.03 ꢀ 2)
16
(0.016 ꢀ 128)
8
(0.03 ꢀ 32)
>128
(1 ꢀ >128)
2
(0.13 ꢀ 2)
32
(0.03 ꢀ >128)
32
(0.03 ꢀ 64)
>64
(2 ꢀ >64)
2
(0.13 ꢀ 8)
64
(0.25 ꢀ >128)
64
(0.03 ꢀ >32)
>128
(8 ꢀ >128)
2
(0.13 ꢀ 2)
16
(0.06 ꢀ >128)
32
(0.03 ꢀ >128)
>128
(1 ꢀ >128)
P. aeruginosa
E. colib
K. pneumoniaeb
A. baumanniib
PK Propertiesc
Rat PPBd (% free)
Rat CL (mL/min/kg)
Rat t1/2 (h)
60 ± 0.10
21
4d
9
0.39
0.28
6 ± 0.94
16
0.34
15 ± 3.4
12
0.37
29 ± 1.2
9
0.49
0.29
0.30
0.44
Rat Vss (L/kg)
0.44
0.42
an = 22 for E. coli and n = 20 for P. aeruginosa, K. pneumonia, A. baumannii. b E. coli = Escherichia coli; K. pneumoniae =
Klebsiella pneumoniae; A. baumannii = Acinetobacter baumannii. Han Wistar Rat (male), iv infusion, 1 mg/kg, n = 2; dn =
c
3, standard deviation; eNot determined
PK/PD modeling to predict in vivo efficacy from in vitro killing kinetics. As can be seen
from the MIC90 data in Table 5, the compounds were effective antiꢀP. aeruginosa agents.
However the in vitro MICs did not differentiate between a variety of chemotypes. To address
this concern, we sought to utilize an in vitro tool that would enable us to differentiate and
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therefore prioritize compounds for in vivo efficacy studies. Two compounds with similar in
vitro profiles but different structural motifs, triazinone 13 and pyrrolidinone 29, were selected
for PK/PD modeling and simulation. Monocarbam 4 was also evaluated for validation
purposes since it had been shown to be efficacious in vivo against a strain of P. aeruginosa.36
For all PK/PD modeling work, metalloꢀβꢀlactamase producing P. aeruginosa ARC3502
(VIMꢀ1) was utilized as the study strain.
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For each compound, the relationship between free drug concentration and
bacterial response was evaluated as a function of time by generating in vitro kill kinetics
(fixed static concentrations over time) and in vivo kill kinetics (dynamic concentrationꢀtime
profiles following single dose) in an immunoꢀcompromised mouse thigh infection model.49
A full doseꢀranging efficacy experiment was additionally generated for compound 4 to verify
predictions of in vivo response at the 24 h endpoint following multiple doses every 3 h. A
PK/PD model previously validated to effectively describe the in vivo response of P.
aeruginosa to βꢀlactams50ꢀ53 was used to fit the in vitro kill kinetics for compound 4 against
P. aeruginosa ARC3502. As seen in Figures 2a and 2b, the model fit the in vitro data for
monocarbam 4 reasonably well (r2 = 0.90). Next, the estimated model (bacterial kill rate, Kk
= 1.2 hꢀ1; halfꢀmaximal effective concentration, EC50 = 0.01 mg/ml) was used to simulate the
in vivo response. Figures 2c and 2d, respectively, show the estimated model generated from
in vitro data reasonably predicted the in vivo response for the singleꢀdose kill kinetics and for
the doseꢀranging efficacy experiment. Model validation was established with compound 4, as
the simulated data correctly predicted net bacterial stasis at 24 h with 50 mg/kg every 3 h and
2ꢀlog kill with the highest dosing regimen, 200 mg/kg every 3 h (Figure 2d).
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Figure 2. PK/PD modeling for compound 4 against P. aeruginosa ARC3502. (a) Model fit of in vitro kill kinetics at fixed
static concentrations (Kk = 1.2 hꢀ1, EC50 = 0.01 mg/mL). Symbols represent observations; solid lines are model
fit/predictions. (b) Observations versus model predictions for in vitro data fit. (c) Prediction of in vivo kill kinetics
following single doses by estimated model. Symbols represent observations; solid lines are model fit/predictions. (d)
Prediction of in vivo efficacy following multiple doses in a doseꢀranging experiment. Symbols represent observations at 24
h time point (6 h for vehicle); solid lines are model fit/predictions.
The modeling of in vitro kill kinetics and subsequent prediction of in vivo
response was repeated for compounds 13 and 29. Again we fit the in vitro data to the model
and then employed estimated parameters (13, Kk = 4.3 hꢀ1, EC50 = 2.1 mg/ml; 29, Kk = 5.2 hꢀ
1, EC50 = 1.4 mg/ml) to simulate the in vivo response. Figures 3a and 4a show the model fit
the in vitro kill kinetics for 13 and 29, respectively, with both compounds demonstrating a
similar in vitro profile. The in vitro observed bacterial burdens were in line with the model
predictions, with an r2 of 0.95 and 0.91 for 13 (Figure 3b) and 29 (Figure 4b), respectively.
Once more, the model was efficient in predicting the in vivo kill kinetics for both compounds
(Figure 3c for 13, Figure 4c for 29) and correctly predicted the higher rate of bacterial kill in
vivo for pyrrolidinone 29 irrespective of the similar in vitro profiles. This is because when
predicting in vivo response the model combines the growth and kill parameters with the in
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vivo pharmacokinetics, which is significantly different between 13 and 29 for the same dose
levels. For example, the unbound AUC of 29 is approximately 2ꢀfold higher as compared to
the unbound AUC of 13 (PK parameters are reported in the supporting information). The
result is a meaningful difference in in vivo response between 13 and 29.
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Taking it one step further, we utilized the model to simulate a full doseꢀranging
efficacy experiment (multiple doses every 3 h) for 13 and 29 in a similar fashion as
monocarbam 4. As seen in Figures 3d and 4d, differences between these two compounds are
highlighted by our PK/PD modeling, with 29 predicted to be more efficacious in vivo than 13
against this strain of P. aeruginosa. Similar to compound 4, pyrrolidinone 29 was predicted
to achieve stasis with the 50 mg/kg every 3 h regimen and 2ꢀlog kill with 200 mg/kg every 3
h.
Figure 3. PK/PD modeling for compound 13 against P. aeruginosa ARC3502. (a) Model fit of in vitro kill kinetics at
fixed static concentrations (Kk = 4.3 hꢀ1, EC50 = 2.1 mg/mL). Symbols represent observations; solid lines are model
fit/predictions. (b) Observations versus model predictions for in vitro data fit. (c) Prediction of in vivo kill kinetics
following single doses by estimated model. Symbols represent observations; solid lines are model predictions. (d)
Prediction of in vivo efficacy following multiple doses in a doseꢀranging experiment. Solid lines are model predictions.
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Figure 4. PK/PD modeling for compound 29 against P. aeruginosa ARC3502. (a) Model fit of in vitro kill kinetics at
fixed static concentrations (Kk = 5.2 hꢀ1, EC50 = 1.4 mg/mL). Symbols represent observations; solid lines are model
fit/predictions. (b) Observations versus model predictions for in vitro data fit. (c) Prediction of in vivo kill kinetics
following single doses by estimated model. Symbols represent observations; solid lines are model predictions. (d)
Prediction of in vivo efficacy following multiple doses in a doseꢀranging experiment. Solid lines are model predictions.
It should be noted that despite a similar in vitro killing profile for the three
compounds (Figures 2a, 3a, 4a), the estimated kill rate of compound 4 (Kk = 1.2 hꢀ1) is
approximately 2ꢀ3 fold lower than the estimated kill rates for compounds 13 and 29 (Kk = 4.3
hꢀ1 and Kk = 5.2 hꢀ1 respectively). This is because the in vitro kill kinetics study for
compound 4 was performed in isosensitest media, where Mueller Hinton Broth (MHB) media
was used for compounds 13 and 29. Due to the different media, the in vitro growth rate of
the bacterium was 2ꢀ3ꢀfold lower for compound 4 as compared to the growth rates in the
experiments with compounds 13 and 29. Since bacterial population dynamics are a
combination of growth and kill rates, the estimated kill rate for compound 4 was lower than
for compounds 13 and 29. Therefore, with media adjusted growth and kill rates, the model is
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able to predict the in vivo response for all three compounds reasonably well, demonstrating
the robustness of the modeling approach.
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Overall we have shown that a range of parameters are essential to predicting in
vivo response. There is a complex interplay between the various parameters, which
combined with the inherent model nonlinearity provided in vivo predictions. With these
results we were confident we had a predictive tool to enable compound prioritization for
further in vivo testing. The results from the further in vivo pharmacology will be reported
elsewhere.54
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Conclusion
In summary, we have identified a new series of siderophoreꢀconjugated
monocarbams which display excellent biochemical and cellular P. aeruginosa activity, good
hydrolytic stability, and favorable PK parameters. We developed this series with the aim of
addressing the known physical property and stability issues of monocarbams. By
introduction of a saturated linker we succeeded in significantly increasing free fraction
compared to triazolones. Modifying the siderophore to a 1,3ꢀdihydroxypyridinone provided
compounds with improved potencies and physical properties. We observed these compounds
were not prone to hydrolysis of the βꢀlactam, but instead suffered from decomposition of the
sidechains. Throughout our optimization we were able to maintain low clearance values
while increasing the human free fraction and therefore free drug concentrations. We applied
a semiꢀmechanistic PK/PD model which enables differentiation of compounds and prediction
of in vivo efficacy from in vitro kill kinetics. This enabled us, with minimal compound
requirements (<5 mg), to screen compounds for predicted efficacy and make timely project
decisions.
Experimental Procedures:
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General Considerations: All solvents and reagents used were obtained commercially and
1
used as such unless noted otherwise. H NMR spectra were recorded in CDCl3, D2O or
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DMSOꢀd6 solutions at 300 K using a Brucker Ultrashield 300 MHz instrument or a Brucker
Ultrashield 400 MHz instrument. 13C NMR spectra were recorded in DMSOꢀd6 solutions at
300 K and 126 MHz using a Brucker DRXꢀ500 500 MHz instrument with a QNP cryoprobe
or at 75.5 MHz using a Brucker Ultrashield 300 MHz instrument. Chemical shifts are
reported as parts per million relative to TMS (0.00) for 1H and 13C NMR. Highꢀresolution
mass spectra (HRMS) were obtained using a hybrid quadrupole timeꢀofꢀflight mass
spectrometer (microTOFq II, Bruker Daltonics) in ESI+ mode. Silica gel chromatographies
were performed on an ISCO Combiflash Companion Instruments using ISCO RediSep Flash
Cartridges (particle size: 35ꢀ70 microns) or Silicycle SiliaSep Flash Cartridges (particle size:
40ꢀ63 microns). Reverse phase chromatographies were performed on an ISCO Combiflash
Companion Instruments using RediSep High Performance Gold C18 columns. When not
indicated, compound intermediates and reagents were purchased from chemical supply
houses. All final compounds were determined to be greater than 95% pure via analysis by
reversed phase UPLCꢀMS (retention times, RT, in minutes) with a Waters Acquity UPLC
instrument with DAD and ELSD and a UPLC HSS T3, 2.1 x 30 mm, 1.8 um column and a
gradient of 2 to 98% acetonitrile in water with 0.1% formic acid over 2.0 minutes at 1
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mL/min. Injection volume was 1 µL and the column temperature was 30 °C. Detection was
based on electrospray ionization (ESI) in positive and negative polarity using Waters ZQ
mass spectrometer (Milford, MA, USA), diodeꢀarray UV detector from 210 to 400 nm, and
evaporative light scattering detector (Sedex 75, Sedere, Alfortville Cedex, France). 1
(Aztreonam) was purchased from MP Biomedicals Santa Anna, California, compounds 2 and
4 were synthesized according to published procedures.23, 55
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(S)-4-Azidopyrrolidin-2-one (6): In a 50 ml round bottom flask was combined (R)ꢀ4ꢀ
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hydroxypyrrolidinꢀ2ꢀone (0.35 g, 3.5 mmol) and DCM (15 ml). The reaction was cooled to 0
°C in an ice bath and 4ꢀDMAP (0.85 g, 6.9 mmol) was added followed by toluenesulfonyl
chloride (0.69 g, 3.6 mmol). The reaction was allowed to warm to room temperature and stir
for 16h. The reaction was then washed with 1M HCl (2 × 15 ml). The organics were
separated and concentrated to yield a brown oil which was diluted in THF (10 mL) and
cooled to 0 ºC. Tetrabutylammonium azide (1.1 g, 3.8 mmol) was added and the reaction
was allowed to warm to room temperature. After 3h, the reaction was concentrated and
purified by SiO2 column chromatography (0ꢀ10% MeOH in DCM) to yield the product as a
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1
white solid (175 mg, 40%). H NMR (300 MHz, CHLOROFORMꢀd) δ ppm 5.79 (br. s., 1
H) 4.36 (tt, J=6.9, 3.6 Hz, 1 H) 3.65 ꢀ 3.80 (m, 1 H) 3.29 ꢀ 3.49 (m, 1 H) 2.69 (dd, J=17.2, 7.4
Hz, 1 H) 2.42 (dd, J=17.3, 4.0 Hz, 1 H). ESIꢀMS m/z: 126.9 [M + H]+.
tert-Butyl
2-((Z)-2-((2S,3S)-1-((S)-4-azido-2-oxopyrrolidin-1-ylsulfonylcarbamoyl)-2-
methyl-4-oxoazetidin-3-ylamino)-1-(2-(tert-butoxycarbonylamino)thiazol-4-yl)-2-
oxoethylideneaminooxy)-2-methylpropanoate: In a round bottom flask was combined (S)ꢀ
4ꢀazidopyrrolidinꢀ2ꢀone (100 mg, 0.79 mmol), THF (4 mL), and NꢀmethylꢀNꢀ(trimethylsilyl)
trifluoroacetamide (MSTFA) (0.44 mL, 2.4 mmol). The solution was stirred at room
temperature for 2h, after which time the reaction was concentrated in vacuo for 2h, and then
reꢀdissolved in THF (4 mL). In a separate round bottom flask containing tertꢀbutyl 2ꢀ((E)ꢀ1ꢀ
(2ꢀ(tertꢀbutoxycarbonylamino)thiazolꢀ5ꢀyl)ꢀ2ꢀ((2S,3S)ꢀ2ꢀmethylꢀ4ꢀoxoazetidinꢀ3ꢀylamino)ꢀ2ꢀ
oxoethylideneaminooxy)ꢀ2ꢀmethylpropanoate (5)23 (345 mg, 0.67 mmol) and DCM (3 mL) at
0 ºC was added CSI (58 ꢀl, 0.67 mmol) dropwise. This solution was allowed to stir at the
same temperature for 1h before dropwise addition of the amide solution. The reaction was
allowed to warm to room temperature and stir for 16h. The reaction was then concentrated
and the crude material directly purified by SiO2 column chromatography (10:1 DCM:MeOH)
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to yield the product as a white solid (330 mg, 66%). H NMR (300 MHz, DMSOꢀd6) δ ppm
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11.72 (s, 1 H) 8.97 (d, J=8.1 Hz, 1 H) 7.18 (s, 1 H) 4.50 (dd, J=8.1, 3.0 Hz, 1 H) 4.38 (t,
J=6.2 Hz, 1 H) 4.08 (dd, J=10.8, 5.7 Hz, 1 H) 3.82 ꢀ 3.94 (m, 1 H) 3.59 ꢀ 3.71 (m, 1 H) 2.75
(dd, J=17.5, 7.0 Hz, 1 H) 2.19 ꢀ 2.33 (m, 1 H) 1.13 ꢀ 1.58 (m, 27 H). ESIꢀMS m/z: 743.1 [M
+ H]+.
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tert-Butyl 2-((Z)-2-((2S,3S)-1-((S)-4-amino-2-oxopyrrolidin-1-ylsulfonylcarbamoyl)-2-
methyl-4-oxoazetidin-3-ylamino)-1-(2-(tert-butoxycarbonylamino)thiazol-4-yl)-2-
oxoethylideneaminooxy)-2-methylpropanoate (7): tertꢀButyl 2ꢀ((Z)ꢀ2ꢀ((2S,3S)ꢀ1ꢀ((S)ꢀ4ꢀ
azidoꢀ2ꢀoxopyrrolidinꢀ1ꢀylsulfonylcarbamoyl)ꢀ2ꢀmethylꢀ4ꢀoxoazetidinꢀ3ꢀylamino)ꢀ1ꢀ(2ꢀ(tertꢀ
butoxycarbonylamino)thiazolꢀ4ꢀyl)ꢀ2ꢀoxoethylideneaminooxy)ꢀ2ꢀmethylpropanoate (0.3 g,
0.44 mmol) was dissolved in ethanol (5 mL) and 10% Pd/C (30 mg) was added. The reaction
was allowed to stir under 1 atm H2 for 3h. The reaction was then filtered through celite,
washed with ethanol, and concentrated to yield the product as a grey solid which was used
1
directly in subsequent reactions. H NMR (300 MHz, DMSOꢀd6) δ ppm 11.72 (br. s., 1 H)
8.95 (d, J=8.1 Hz, 1 H) 7.18 (s, 1 H) 4.41 (dd, J=8.1, 3.0 Hz, 1 H) 4.08 (dd, J=11.1, 6.4 Hz, 1
H) 3.65 ꢀ 3.93 (m, 3 H) 2.63 ꢀ 2.85 (m, 1 H) 2.31 (dd, J=17.4, 3.3 Hz, 1 H) 1.18 ꢀ 1.52 (m, 27
H). ESIꢀMS m/z: 717.1 [M + H]+.
2-((((Z)-1-(2-Aminothiazol-4-yl)-2-(((2S,3S)-1-((((S)-4-(5-hydroxy-4-oxo-1,4-
dihydropyridine-2-carboxamido)-2-oxopyrrolidin-1-yl)sulfonyl)carbamoyl)-2-methyl-4-
oxoazetidin-3-yl)amino)-2-oxoethylidene)amino)oxy)-2-methylpropanoic acid (9): In a
25 ml flask was combined 4,5ꢀbis(benzyloxy)picolinic acid27 (100 mg, 0.30 mmol), tertꢀbutyl
2ꢀ((Z)ꢀ2ꢀ((2S,3S)ꢀ1ꢀ((S)ꢀ4ꢀaminoꢀ2ꢀoxopyrrolidinꢀ1ꢀylsulfonylcarbamoyl)ꢀ2ꢀmethylꢀ4ꢀ
oxoazetidinꢀ3ꢀylamino)ꢀ1ꢀ(2ꢀ(tertꢀbutoxycarbonylamino)thiazolꢀ4ꢀyl)ꢀ2ꢀ
oxoethylideneaminooxy)ꢀ2ꢀmethylpropanoate (7) (220 mg, 0.30 mmol), HATU (133 mg,
0.35 mmol), DIPEA (55 ꢁL, 0.4 mmol), and DMF (3 mL). The reaction was allowed to stir
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at room temperature for 16h after which time it was diluted with EtOAc. The organics were
washed three times with 50% brine, and then concentrated. The crude material was eluted
through a plug of SiO2 with 10:1 DCM:MeOH. The pooled fractions were concentrated and
dissolved in MeOH (3 mL). Pd black (50 mg) was added and the suspension was allowed to
stir under 1 atm H2 for 3h until complete debenzylation was observed. The reaction was then
filtered and the filtrate was concentrated and reꢀdissolved in DCM (2 mL). The reaction was
cooled to 0 ˚C and TFA (1 mL) was added. The reaction was allowed to warm to room
temperature and stir for 3h. The reaction was concentrated, purified by C18 reverse phase
chromatography (0ꢀ100% MeCN/H2O with 0.1% formic acid) and lyophilized to yield the
product as a white solid (35 mg, 17%). UPLC RT = 0.46 min, MS (ES) MH+: 698 for
C24H27N9O12S2; 1H NMR (300 MHz, DMSOꢀd6) δ ppm 9.14 (d, J=6.78 Hz, 1H), 9.03 (d,
J=8.10 Hz, 1H), 7.96 (s, 1H), 7.57 (s, 1H), 6.75 (s, 1H), 4.52 (dd, J=8.10, 3.01 Hz, 2H), 4.13
(dd, J=10.08, 6.69 Hz, 1H) 3.83 ꢀ3.99 (m, 1H), 3.68ꢀ3.91 (m, 2H), 2.65 ꢀ 2.85 (m, 1H), 2.53
(d, J=4.90, 1H), 1.32 ꢀ 1.57 (m, 9H). 13C NMR (126 MHz, 60 °C, DMSOꢀd6) δ ppm 176.5,
172.5, 171.1, 166.7, 164.4, 164.1, 158.8, 152.9, 150.7, 148.2, 142.7, 142.3, 134.3, 112.8,
112.4, 83.4, 63.4, 57.6, 55.9, 45.5, 40.6, 26.0, 19.6. HRMS: (ES+) Calcd. for C24H28N9O12 S2
[M + H]+: 698.1293; Found 698.1303.
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2-Methyl-1,2,4-triazinan-3-one (10): To a 200 ml flask was added 2ꢀmethylꢀ4,5ꢀdihydroꢀ
1,2,4ꢀtriazinꢀ3(2H)ꢀone56 (2.1 g, 19 mmol) and MeOH (120 mL) under nitrogen. The
reaction was cooled to 0 °C and treated with sodium cyanoborohydride (2.4 g, 38 mmol).
Hydrochloric acid (4.0 mL, 49 mmol) was then added dropwise (a white suspension began to
form after the addition of HCl) and the reaction was allowed to warm to room temperature
and was stirred for 3h. The reaction was treated with 1N NaOH dropwise until the solid
dissolved and the pH was between 7 and 8. The reaction was extracted three times with
EtOAc. The combined organics were concentrated and purified by SiO2 column
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chromatography (2ꢀ20% MeOH in EtOAc) to afford the desired product as a white solid (2.0
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g, 93%). H NMR (300 MHz, DMSOꢀd6) δ ppm 6.36 (br. s., 1 H) 5.11 (t, J=7.16 Hz, 1 H)
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3.05 ꢀ 3.23 (m, 2 H) 2.75 ꢀ 2.95 (m, 5 H).
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1-(4,5-Bis(benzyloxy)picolinoyl)-2-methyl-1,2,4-triazinan-3-one (11): 4,5ꢀ
Bis(benzyloxy)picolinic acid (5.8 g, 17 mmol), 2ꢀmethylꢀ1,2,4ꢀtriazinanꢀ3ꢀone (2.0 g, 17
mmol), EDC (5.0 g, 26 mmol), and HOBT (1.3 g, 8.7 mmol) were suspended in DMF (55
mL) and allowed to stir at room temperature under nitrogen overnight. The reaction was
diluted with water and allowed to stir for 1 hr. The solid formed was filtered and dried. The
solid was then reꢀsuspended in Et2O and filtered to afford the desired product (6.7 g, 89%).
1H NMR (300 MHz, DMSOꢀd6) δ ppm 8.27 (s, 1 H) 7.18 ꢀ 7.73 (m, 11 H) 6.95 (br. s., 1 H)
5.14 ꢀ 5.44 (m, 4 H) 3.94 (br. s., 2 H 2.75 ꢀ 3.25 (m, 5 H). ESIꢀMS m/z: 433.6 [M + H]+.
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2-((Z)-1-(2-Aminothiazol-4-yl)-2-((2S,3S)-1-(1-(5-hydroxy-4-oxo-1,4-dihydropyridine-2-
carbonyl)-2-methyl-3-oxo-1,2,4-triazinan-4-ylsulfonylcarbamoyl)-2-methyl-4-
oxoazetidin-3-ylamino)-2-oxoethylideneaminooxy)-2-methylpropanoic acid (13): The
same procedure as described for tertꢀButyl 2ꢀ((Z)ꢀ2ꢀ((2S,3S)ꢀ1ꢀ((S)ꢀ4ꢀazidoꢀ2ꢀoxopyrrolidinꢀ
1ꢀylsulfonylcarbamoyl)ꢀ2ꢀmethylꢀ4ꢀoxoazetidinꢀ3ꢀylamino)ꢀ1ꢀ(2ꢀ(tertꢀ
butoxycarbonylamino)thiazolꢀ4ꢀyl)ꢀ2ꢀoxoethylideneaminooxy)ꢀ2ꢀmethylpropanoate (7) was
followed, using tertꢀbutyl 2ꢀ((E)ꢀ1ꢀ(2ꢀ(tertꢀbutoxycarbonylamino)thiazolꢀ5ꢀyl)ꢀ2ꢀ((2S,3S)ꢀ2ꢀ
methylꢀ4ꢀoxoazetidinꢀ3ꢀylamino)ꢀ2ꢀoxoethylideneaminooxy)ꢀ2ꢀmethylpropanoate (5) (200
mg, 0.28 mmol) and 1ꢀ(4,5ꢀbis(benzyloxy)picolinoyl)ꢀ2ꢀmethylꢀ1,2,4ꢀtriazinanꢀ3ꢀone (137
mg, 0.33 mmol). The reaction was concentrated and filtered through a plug of SiO2 eluting
with 100:10:1 DCM:MeOH:NH4OH. The filtrate was dissolved in MeOH (2 mL) and Pd
black (50 mg) was added. The suspension was allowed to stir under 1 atm of H2 for 3h until
complete debenzylation was observed. The reaction was filtered and concentrated. The
crude was dissolved in DCM (2 mL) and TFA (1 mL) was added at 0 ºC. After 2h at 0 ºC,
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the reaction was concentrated, purified by C18 reverse phase chromatography (0ꢀ100%
MeCN/H2O with 0.1% formic acid) and lyophilized to yield the product as a white solid (70
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8
1
mg, 33%). UPLC RT = 0.28 min, MS (ES) MH+: 713.1 for C24H28N10O12S2. H NMR (300
9
MHz, DMSOꢀd6) δ ppm 9.12 (d, J=7.9 Hz, 1 H) 8.04 (s, 1 H) 7.33 (s, 1 H) 6.81 (s, 1 H) 4.64
(d, J=4.9 Hz, 1 H) 4.50ꢀ3.95 (m, 4 H) 3.85 (d, J=6.4 Hz, 1H) 3.07 (br. s., 3 H) 1.36 ꢀ 1.56 (m,
9 H). 13C NMR (75 MHz, DMSOꢀd6) δ ppm 174.3, 169.2, 165.2, 164.3, 161.8, 159.1, 158.6,
158.1, 157.6, 147.5, 145.5, 138.9, 117.3, 113.5, 112.4, 110.5, 81.8, 61.6, 55.7, 23.8, 17.0.
HRMS (ES+) Calcd for C24H29N10O12S2 [M + H]+ 713.1402; Found 713.1401.
ASSOCIATED CONTENT
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Supporting Information
Synthesis and characterization details for compounds 14ꢀ35; Methods for hydrolytic stability
assay, susceptibility testing, in vitro and in vivo pharmacokinetics, in vivo efficacy and
mathematical PK/PD modelling; PK details for 4, 13 and 29. This material is available free
of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Authors
* Kerry E. MurphyꢀBenenato, kerry.benenato@modernatx.com; Aryun Kim,
aryun.kim@astrazeneca.com
Present Addresses
a Moderna Therapeutics, Inc., 200 Technology Square, Cambridge, MA, 02139, United States
b Merck & Co., Inc., 90 E Scott Avenue, Rahway, New Jersey 07065, United States
c Incyte Corporation, Route 141 & Henry Clay Road, Wilmington, Delaware 19880, United
States
d Agios Pharmaceuticals, 38 Sidney Street, Cambridge, MA 02139, United States
e Pfizer Inc., 610 Main Street, Cambridge, Massachusetts 02139, United States
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f Pfizer Inc., Eastern Point Road, Groton, Connecticut 06340, United States
g Novartis Institutes for BioMedical Research, Inc., 250 Massachusetts Avenue, Cambridge,
Massachusetts 02139, United States
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Notes
The authors declare the following competing financial interest(s): We are or have been
employed by AstraZeneca Pharmaceuticals and may possess company stock.
ACKNOWLEDGMENT
We would like to thank Camil Joubran, Sharon Tentarelli, and Nancy DeGrace for generating
characterization data; Robert Giacobbe, Linda Otterson and Marie Potter for generating MIC
data; Sandeep Ghorpade for helpful discussions; Baolei Zheng and his colleagues at
Pharmaron for synthesis of intermediates and final compounds.
ABBREVIATIONS
MIC, minimum inhibitory concentration; PBP, penicillin binding protein; PPB, plasma
protein binding; CL, clearance; Vss, volume of distribution steady state; VIMꢀ1, Verona
integronꢀencoded metalloꢀβꢀlactamase; CFU, colony forming units; AUC, area under the
curve; MSTFA, NꢀMethylꢀNꢀ(trimethylsilyl)trifluoroacetamide; THF, Tetrahydrofuran; CSI,
Chlorosulfonyl isocyanate; DCM, Dichloromethane; HATU, Nꢀ[(Dimethylamino)ꢀ1Hꢀ1,2,3ꢀ
triazoloꢀ[4,5ꢀb]pyridinꢀ1ꢀylmethylene]ꢀNꢀmethylmethanaminium
hexafluorophosphate Nꢀ
oxide; DMF, N,NꢀDimethylformamide; TFA, Trifluoroacetic acid; EDCI, Nꢀ(3ꢀ
Dimethylaminopropyl)ꢀN′ꢀethylcarbodiimide hydrochloride; HOBT, 1ꢀHydroxybenzotriazole
hydrate; 4ꢀDMAP, 4ꢀDimethylaminopyridine; DMSO, Dimethyl sulfoxide; DIPEA, N,Nꢀ
Diisopropylethylamine; EtOAc, Ethyl acetate; MeCN, Acetonitrile; UPLC, Ultra
Performance Liquid Chromatography.
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