Discovery of VNRX-7145 (VNRX-5236 Etzadroxil): An Orally Bioavailable β-Lactamase Inhibitor for Enterobacterales Expressing Ambler Class A, C, and D Enzymes

Article abstract of DOI:10.1021/acs.jmedchem.1c00437

A major antimicrobial resistance mechanism in Gram-negative bacteria is the production of β-lactamase enzymes. The increasing emergence of β-lactamase-producing multi-drug-resistant “superbugs” has resulted in increases in costly hospital Emergency Department (ED) visits and hospitalizations due to the requirement for parenteral antibiotic therapy for infections caused by these difficult-to-treat bacteria. To address the lack of outpatient treatment, we initiated an iterative program combining medicinal chemistry, biochemical testing, microbiological profiling, and evaluation of oral pharmacokinetics. Lead optimization focusing on multiple smaller, more lipophilic active compounds, followed by an exploration of oral bioavailability of a variety of their respective prodrugs, provided 36 (VNRX-7145/VNRX-5236 etzadroxil), the prodrug of the boronic acid-containing β-lactamase inhibitor 5 (VNRX-5236).In vitroandin vivostudies demonstrated that 5 restored the activity of the oral cephalosporin antibiotic ceftibuten against Enterobacterales expressing Ambler class A extended-spectrum β-lactamases, class A carbapenemases, class C cephalosporinases, and class D oxacillinases.

Full text of DOI:10.1021/acs.jmedchem.1c00437

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Article
Discovery of VNRX-7145 (VNRX-5236 Etzadroxil): An Orally
Bioavailable β‑Lactamase Inhibitor for Enterobacterales Expressing
Ambler Class A, C, and D Enzymes
Robert E. Trout,* Allison Zulli, Eugen Mesaros, Randy W. Jackson, Steven Boyd, Bin Liu, Jodie Hamrick,
Denis Daigle, Cassandra L. Chatwin, Kaitlyn John, Lisa McLaughlin, Susan M. Cusick, William J. Weiss,
Mark E. Pulse, Daniel C. Pevear, Greg Moeck, Luigi Xerri, and Christopher J. Burns
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ABSTRACT: A major antimicrobial resistance mechanism in Gram-
negative bacteria is the production of β-lactamase enzymes. The
increasing emergence of β-lactamase-producing multi-drug-resistant
“superbugs” has resulted in increases in costly hospital Emergency
Department (ED) visits and hospitalizations due to the requirement for
parenteral antibiotic therapy for infections caused by these difficult-to-
treat bacteria. To address the lack of outpatient treatment, we initiated
an iterative program combining medicinal chemistry, biochemical
testing, microbiological profiling, and evaluation of oral pharmacokinetics. Lead optimization focusing on multiple smaller, more
lipophilic active compounds, followed by an exploration of oral bioavailability of a variety of their respective prodrugs, provided 36
(VNRX-7145/VNRX-5236 etzadroxil), the prodrug of the boronic acid-containing β-lactamase inhibitor 5 (VNRX-5236). In vitro
and in vivo studies demonstrated that 5 restored the activity of the oral cephalosporin antibiotic ceftibuten against Enterobacterales
expressing Ambler class A extended-spectrum β-lactamases, class A carbapenemases, class C cephalosporinases, and class D
oxacillinases.
infections, such as complicated urinary tract infections
(cUTI) and acute pyelonephritis, new oral treatment options
are urgently needed. An effective oral therapeutic could also
benefit hospitalized patients who are ready for discharge before
completing a full course of IV antibacterial therapy if an
effective IV to oral step-down therapy is available. This has the
potential to significantly reduce hospital stay length, decrease
hospital-related costs, and allow the patient to regain a normal
quality of life sooner.
Currently, there are no approved orally bioavailable β-
lactams or β-lactam/β-lactamase inhibitor (BL/BLI) combi-
nations that cover Enterobacterales expressing key class A or D
carbapenemases or class C cephalosporinases. Resistance and
co-resistance to fluoroquinolones and trimethoprim-sulfame-
thoxazole among ESBL- and carbapenemase-producing Enter-
obacterales further limit oral treatment options.10 The only
approved oral BL/BLI is amoxicillin/clavulanic acid, which
demonstrates limited activity against Gram-negative organisms
INTRODUCTION
■
β-Lactams are the most widely used class of antibiotics in both
the community and hospital setting, representing over 60% of
the total world antibiotic market.1 This preferred, safe, and
efficacious class of antibiotics is under constant threat by
expansion of multi-drug-resistant (MDR) Gram-negative
bacteria including increasing hospital and community preva-
lence of carbapenem-resistant Enterobacterales2 (CRE) and
extended spectrum β-lactamase (ESBL)-producing Enter-
obacterales.3,4 Similarly, the rates of co-resistance to other
classes of antibacterial agents including trimethoprim-sulfame-
thoxazole and fluoroquinolones are higher than recommenda-
tions for use (2010 IDSA Uncomplicated UTI and
Pyelonephritis clinical guidelines) for most of the US and, in
many areas, are increasing steadily.5−7 This growing prevalence
of MDR bacteria in the community has led to an increase in
costly emergency department visits and hospitalizations
required to manage resistant infections by administration of
intravenous (IV) or intramuscular antibiotic therapy, including
carbapenems. As a result, additional economic and resource
burdens are placed on healthcare systems and patient care
while potentially increasing selective pressure for resistance
growth,8 including CRE, and subsequent transmission to other
hospitalized patients.9 To avoid unnecessary institutional
escalations for the treatment of community-associated
Received: March 10, 2021
Published: June 30, 2021
https://doi.org/10.1021/acs.jmedchem.1c00437
© 2021 American Chemical Society
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Figure 1. Boronic acid-based BLIs approved (vaborbactam), in Phase 3 development (taniborbactam), and in Phase 1 development (QPX7728).
expressing Ambler class A ESBLs and lacks activity against
class A carbapenemases, class C cephalosporinases, and class D
oxacillinases. An investigational oral carbapenem (tebipenem
pivoxil hydrobromide)11 and an investigational oral thiopenem
(sulopenem etzadroxil, administered with probenecid)12 are
under development for UTIs due to ESBL-producing Enter-
obacterales; however, their spectrum of activity does not
include carbapenem-resistant organisms.13 Recent studies have
shown that boron-based covalent and slowly reversible
inhibitors, such as vaborbactam (RPX7009),14 taniborbactam
(formerly VNRX-5133),15,16 and QPX772817 (Figure 1),
efficiently inhibit class A and C (vaborbactam) and class A,
B, C, and D enzymes (taniborbactam and QPX7728).
However, the early work on boronic acid-based BLIs by
academic groups18−22 and the pharmaceutical industry focused
on IV products.14−17,23−25 Recognizing the lack of effective
oral agents against the increasingly resistant pathogens
described above, our efforts focused on the development of
an orally bioavailable boronic acid-based β-lactamase inhibitor
capable of restoring antibacterial activity of the third-
generation oral cephalosporin ceftibuten. We present herein
the lead optimization, structure−activity relationship (SAR),
microbiological profiling, and prodrug pharmacokinetics (PK)
that led to the discovery of VNRX-7145 (VNRX-5236
etzadroxil).26,27 This broad-spectrum BLI is currently in
Phase 1 clinical studies (ClinicalTrials.gov Identifier:
NCT04243863).
Utilizing the structure-based design work by Ness et al.32
and the extensive SAR studies and microbiological profiling by
Burns et al.25 and Liu et al.,15 the authors determined that the
need to balance the attributes that allow for Gram-negative
bacterial penetration with the attributes necessary for oral
absorption was the key hurdle to overcome. To protect β-
lactam activity against the targeted clinically relevant β-
lactamase-producing bacteria, the partner BLI must cross the
outer membrane of diverse Gram-negative pathogens. O’Shea
and Moser33 observed that high-molecular-weight compounds
with clog P < 1 and a topological polar surface area (tPSA) >
150 A2 have preferred access into Gram-negative bacteria.
However, it has been well established that lower-molecular-
weight compounds with higher clog P and tPSA < 140 A2 have
an increased oral absorption.34,35
Keeping these conflicting attributes in mind, our design
strategy was to maintain the cyclic boronate scaffold that
interacts covalently with the active-site serine residue with the
plan to create a prodrug of the carboxylic acid group to
increase lipophilicity and aid oral absorption (Figure 2). The
amide side of the molecule would focus on small lipophilic
groups that maintained BLI activity but limited the molecular
weight and the tPSA.
RESULTS AND DISCUSSION
■
Design of BLIs to Protect Oral Cephalosporins
against Serine-β-Lactamases. Identification of an oral
cephalosporin partner was key in the development of an orally
bioavailable BLI. For cUTI and uUTI, approved oral
cephalosporin candidates, such as cefpodoxime proxetil and
ceftibuten, were once reliable treatments, but their effective-
ness has been eroded by the spread of β-lactamase-mediated
resistance in Gram-negative organisms. Comparative suscepti-
bilities of clinical isolates producing extended spectrum β-
lactamases to ceftibuten relative to other oral β-lactams
indicate that ceftibuten is far less susceptible to hydrolysis by
ESBLs, while maintaining significant affinity to penicillin
binding proteins to provide intrinsic potency.28−30 These
features facilitate restoration of antibacterial activity of the β-
lactam by a BLI, and combined with its high absorption and
favorable PK profile, ceftibuten is the optimal choice for
combination with an oral BLI.31
Figure 2. Strategy for designing a cyclic boronate-based BLI with
potential for oral absorption.
Synthesis of the Cyclic Boronate-Based BLIs. The
cyclic boronate-based BLIs were prepared using modifications
of previously reported procedures (Scheme 1).15,25 Compound
1, isolated from two consecutive homologation reactions
following Matteson’s protocol,36 was treated with lithium
bis(trimethylsilyl)amide at −20 °C to afford the desired
stereoisomer intermediate 2. The α-silylaminoboronate inter-
mediate was treated in situ with HATU and N-methylmorpho-
line and coupled with the desired carboxylic acid. The
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lactamases, all the analogues displayed similar potent inhibition
of the class A SHV-5 ESBL and KPC-2 carbapenemase and the
class C AmpC cephalosporinase (Table 1). Potency against the
Scheme 1. Generalized Synthesis of Cyclic Boronate-Based
BLIs
Table 1. Biochemical Activity of BLIs 5−14 against Purified
a
β-Lactamases
IC50 (μM)
SHV-5
KPC-2
(class A)
AmpC
(class C)
OXA-48
(class D)
entry clog P tPSA (class A)
5
6
7
8
0.48
1.01
0.72
0.07
0.23
−0.64
−0.26
0.53
−0.92
0.68
96
96
96
120
113
139
142
96
0.126
0.036
0.008
0.012
0.025
0.514
0.012
0.017
0.008
0.006
0.080
0.093
0.043
0.033
0.116
0.069
0.028
0.031
0.035
0.041
0.014
0.009
0.009
0.014
0.008
0.006
0.005
0.006
0.003
0.007
0.317
0.426
1.62
0.687
1.45
1.79
4.67
0.627
8.55
2.39
9
10
11
12
13
14
carboxylic acids (RCO2H) used for the SAR studies were
either commercially available or readily synthesized from
available starting materials. The resultant amides 3 were
deprotected and cyclized by treatment with BCl3 (1 M in
DCM) at −78 °C to afford the crude boronates 4 which were
purified by reverse-phase high-performance liquid chromatog-
raphy (HPLC) and then lyophilized to dryness.
156
96
a
IC50 values reported as the mean from duplicate measurements on
separate days with automatic repeat if data differed by more than 20%
from the previous result.
SAR: β-Lactamase Inhibitor. A set of cyclic boronates
(Figure 3) was evaluated for both inhibition of purified β-
lactamase enzymes and in vitro rescue of ceftibuten activity
against selected ceftibuten-resistant Enterobacterales strains.
Our primary in-house biochemical screening panel was
composed of four β-lactamases belonging to Ambler class A
(SHV-5, KPC-2), class C (AmpC), and class D (OXA-48).
Antibacterial activity assays were performed using ceftibuten-
resistant Klebsiella pneumoniae and Escherichia coli isolates; the
minimum concentration of each BLI necessary to restore the
antibacterial activity of ceftibuten (fixed at 1 μg/mL) against
clinical isolates of E. coli and K. pneumoniae expressing various
β-lactamases, by enzyme category, was determined to rank-
order the level of BLI potentiation of ceftibuten activity. This
concentration of ceftibuten is clinically relevant as it
corresponds to the EUCAST Susceptible breakpoint for
treatment of infections originating in the urinary tract
(EUCAST 2021).37
class D OXA-48 carbapenemase was more varied, with IC50’s
ranging from 0.32 to 8.55 μM. The BLIs generally exhibited
comparable potency (≤8-fold variation across BLIs, by strain)
in rescuing ceftibuten fixed at 1 μg/mL against individual
representative ceftibuten-resistant strains of K. pneumoniae and
E. coli expressing ESBL, KPC, class C, and OXA-48 β-
lactamases, with certain exceptions (Table 2). Some increased
variation in potency among the BLIs against strains expressing
class C and OXA-48 β-lactamases was observed. In particular,
compounds 7, 12, and 14 exhibited reduced activity that could
be attributed to their higher clog P and lower tPSA values. The
remaining BLIs possessed the requisite biochemical and
microbiological activity to be progressed to the next stage of
SAR development.
Synthesis of the Cyclic Boronate-Based BLI Prodrugs.
With the focus of the program on discovering an orally
bioavailable BLI, comparison of prodrugs of the active BLIs
was initiated to differentiate between BLIs with similar
biochemical and antibacterial activity. A pair of carboxylic
acid ester prodrugs of each BLI were prepared. Two types of
ester prodrugs were utilized: alkyl ester (ethyl) and acyloxy
ester (pivoxyl). Ethyl esters 15 were synthesized from the
carboxylic acids by addition of 4 N HCl in dioxane in the
Keeping the emphasis on balancing the clog P and tPSA to
maximize Gram-negative bacterial penetration with oral
absorption of the prodrug, our efforts focused on small
amide side chains with limited functionality and moderate-to-
high lipophilicity. When evaluated against a primary panel of β-
Figure 3. Chemical structures of screened BLIs.
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presence of ethanol (Scheme 2). For pivoxyl esters 18,
intermediate 3 was treated with trifluoroacetic acid in
dichloromethane (DCM) or 4 N HCl in dioxane to provide
acid 16. Reaction of 16 with sodium carbonate, sodium iodide,
and chloromethyl pivalate in N,N-dimethylformamide afforded
ester 17. The pinanediol and methyl ether were selectively
removed by treatment with aluminum chloride in DCM to
provide the desired pivoxyl esters 18 (Scheme 2).
Oral Bioavailability of β-Lactamase Inhibitors. Ethyl
and pivoxyl prodrugs of the remaining BLIs were prepared and
dosed via oral gavage to rats. The oral bioavailability data
demonstrated a clear effect of clog P and tPSA on the oral
absorption of the BLI prodrugs tested (Table 3). Prodrug
analogues with clog P < 1.00 and/or tPSA > 120 exhibited
poor bioavailability. The BLI analogues whose prodrugs
demonstrated reasonable to good (F ∼ 20−99%) absorption
included compounds 5, 6, 8, and 9. The pivoxyl prodrugs
increased the clog P of the BLIs above 1.00, which appeared to
maximize absorption when compared with the ethyl versions.
One exception to this was the ethyl prodrug of 5 which had a
higher clog P (1.23) but had lower bioavailability (F < 5%) due
to lack of hydrolysis of the ethyl ester (F ∼ 20−30% of 19 was
observed).
Based on the high oral bioavailability data of pivoxyl
prodrugs 20 and 26, studies were undertaken to select the
most promising BLI. To further differentiate between BLIs 5
and 9, the compounds were tested against a challenge set of
clinical isolates of E. coli and K. pneumoniae expressing ESBL (n
= 5), KPC (n = 11), class C (n = 4), and OXA-48 (n = 5) β-
lactamases. Against this set of 25 strains, with ceftibuten fixed
at 1 μg/mL, 5 rescued ceftibuten with a minimum inhibitory
concentration required to inhibit the growth of 90% of
organisms tested (MIC90) = 1 μg/mL, while 9 had an MIC90
=
4 μg/mL (Table 4). As a result, 5 was selected as the active
BLI to further evaluate and maximize its oral absorption.
Optimization of Oral Bioavailability of Compound 5.
A series of acyloxy and alkyl carbonate prodrugs were prepared
following the synthesis presented in Scheme 2 (Figure 4).
Following a similar protocol, all compounds were dosed via
oral gavage in rats. The majority of prodrugs exhibited
reasonable oral bioavailability (Table 5). Despite clog P values
ranging from 1.09 to 2.15, the various alkyl carbonate prodrugs
tested showed similar results with bioavailability (F) values
from 23 to 33%. Among the acyloxy prodrugs tested, the
pivoxyl- (20) and 3-pentylacyloxy- (36) prodrugs demon-
strated the highest oral bioavailability and were tested in
additional species.
Upon dosing the pivoxyl- (20) and 3-pentylacyloxy- (36)
prodrugs in mice, dogs, and monkeys, 36 demonstrated the
most consistent oral bioavailability across species (Table 6).
While 20 exhibited excellent oral bioavailability in rats (F =
99%), bioavailability was considerably lower in the other three
tested species. In addition, prodrugs that liberate pivalate
(trimethylacetic acid) upon hydrolysis have resulted in the
generation of pivaloylcarnitine which, upon elimination in the
urine, leads to depletion of the limited carnitine pool in the
body.38,39 While the potential toxicity resulting from carnitine
depletion is dependent on the amount of pivalate dosed/
generated, 36 does not have this liability and as a result, based
on these data and its consistent and high oral bioavailability
data across species, was selected as the candidate for further
development.
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Scheme 2. Generalized Synthesis of Cyclic Boronate-Based BLI Ethyl and Pivoxyl Prodrugs
that cytochrome P450 enzymes do not play a role in the
hydrolysis. The half-life in human plasma was short at about 11
min, which was closer to what was observed in the rodent
species compared to the longer half-lives in dogs (43.9 min)
and monkeys (22.0 min). In addition, 5 demonstrated long
half-lives (>120 min) in intestinal S9, liver S9, and plasma
across all species (data not shown). Overall, 36 was nearly
completely hydrolyzed to 5 in all tested matrixes from all
species.
Compound 36 Yielded Only One Metabolite, 5, in
Hepatocytes across Species. An in vitro study to investigate
the biotransformation of 36 in cryopreserved hepatocytes from
mice, rats, rabbits, beagle dogs, cynomolgus monkeys, and
humans was performed.40 After incubation, one metabolite was
detected resulting from hydrolysis of 36. The metabolism was
extensive in all species with 0−5% 36 remaining. The structure
of the metabolite was positively identified as 5 by comparing
the observed accurate mass of the metabolite with the observed
accurate mass of the authentic chemical standard for 5. No
other metabolites were detected in any species tested.
Compound 36 Shows High Permeability through
Caco-2 Monolayers. In an effort to gain insight into the
absorption potential of 36 in humans, a Caco-2 cell
permeability study was undertaken.40
The apparent permeability (Papp) for 36 averaged to 9.22 ×
10−6 cm/s, which classifies it as having a high absorption
potential in humans (Table 8).41 The active BLI (5)
demonstrated poor absorption potential, which further
emphasized the need for the prodrug approach for oral
delivery.
Combination of Ceftibuten and 5 Showed In Vivo
Efficacy. In vivo efficacy was demonstrated in a lethal murine
septicemia model42 by dosing 5 (subcutaneously) and 36
(orally) with ceftibuten (dosed both subcutaneously with 5
and orally with 36). A comparison of ceftibuten/5 dosed
subcutaneously and ceftibuten/36 dosed orally demonstrated
similar activity with ED50 (median effective dose) values of
13.5 and 12.9 mg/kg, respectively.43 Additionally, the ability to
demonstrate in vivo efficacy in a UTI model which more
Table 3. Oral Bioavailability of BLI Esters in Rats
a
entry
ester
clog P
tPSA
dose (mg/kg)
F (%)
b
5
19 (ethyl)
20 (pivoxyl)
21 (ethyl)
22 (pivoxyl)
23 (ethyl)
24 (pivoxyl)
25 (ethyl)
26 (pivoxyl)
27 (ethyl)
28 (pivoxyl)
29 (ethyl)
30 (pivoxyl)
31 (ethyl)
32 (pivoxyl)
1.23
1.54
1.76
2.07
0.69
1.00
0.98
1.29
0.12
0.43
0.50
0.81
−0.16
0.15
85
111
85
3
<5
99
NT
31
<5
20
<5
95
<5
<5
NT
<5
<5
NT
10
NT
b
6
111
109
135
102
128
128
154
131
157
145
171
3
5
8
10
5
10
b
9
10
11
13
3
3
b
NT
b
3
3
b
NT
a
F (%) = percent absolute oral bioavailability corrected for MW
b
difference between the prodrug and parent. Dosed as a cassette with
another ester; NTnot tested.
Compound 36 Demonstrated Nearly Complete
Hydrolysis to 5 in Various Matrixes across Species.
With the goal of creating a prodrug that allowed for complete
biological conversion upon absorption to the active BLI, the
metabolic stability of 36 was assessed in vitro in intestinal S9,
liver S9, and plasma from CD-1 mice, Sprague-Dawley rats,
beagle dogs, cynomolgus monkeys, and humans.40
In intestinal S9, 36 was rapidly cleaved with short half-lives
in all species with the exception of beagle dogs (Table 7). Very
short half-lives were also observed in liver S9 across all species.
No effect on the half-life of 36 was observed when including or
excluding NADPH in either intestinal S9 or liver S9, indicating
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Table 4. Concentration of BLIs 5 and 9 to Restore the Antibacterial Activity of Ceftibuten (Fixed at 1 μg/mL) against Clinical
Isolates of E. coli and K. pneumoniae Expressing Various β-Lactamases by Enzyme Category
BLI concentration (μg/mL)
category
ESBL
strain
E. coli
K. pneumoniae
E. coli
designation
β-lactamases
CTX-M-15, TEM-1
SHV-12, TEM-1
CTX-M-2
TEM-1, SHV-12
SHV-12, TEM-1
SHV-5, KPC-2, TEM-1
AmpC SHV-12, CTX-M-15, TEM-1
SHV-1, TEM-1, KPC-2
SHV-11, KPC-3, TEM-1
SHV-12, KPC-2, AmpC, TEM-1
SHV-11, TEM-1, KPC-2
KPC-3, TEM-1, AmpC
KPC
5
9
ESBL 5
ESBL 10
SI LP377
3327
304487
UMM
0.12
0.06
0.004
0.03
0.016
≤0.002
0.016
1
0.06
0.25
0.5
0.5
0.5
≤0.002
2
≤0.002
0.03
0.5
0.016
0.12
0.016
0.03
1
0.03
0.03
1
0.12
0.06
≤0.002
0.016
0.016
≤0.002
0.016
4
E. coli
K. pneumoniae
K. pneumoniae
E. coli
K. pneumoniae
K. pneumoniae
K. pneumoniae
K. pneumoniae
K. pneumoniae
E. coli
KPC
233
136928
137052
155140
156309
166964 #1
786978
849121
845661
845670
J53
SI-P026TC
196477
217917
DOV
SI-C05
SI-C17
664507
664520
0.25
0.5
2
0.5
1
E. coli
KPC
KPC
KPC
≤0.002
4
≤0.002
0.03
0.5
0.06
0.12
0.06
0.06
4
K. pneumoniae
K. pneumoniae
E. coli
class C
SHV-5, AmpC, TEM-1
CMY-2, TEM-1
AmpC, CMY-2, TEM-1, CTX-M-15
CMY-2, TEM-1
OXA-48
OXA-48
OXA-48
OXA-48
OXA-48
E. coli
K. pneumoniae
K. pneumoniae
K. pneumoniae
K. pneumoniae
K. pneumoniae
E. coli
OXA-48
0.06
0.5
E. coli
BLI concentration to rescue activity of ceftibuten (fixed at 1 μg/mL) against >90% of tested strains
4
Table 5. Oral Bioavailability of Compound 5 Prodrugs in
Rats
a
entry
clog P
tPSA
dose (mg/kg)
F (%)
20
33
34
35
36
37
38
39
40
41
1.54
2.19
0.31
1.14
2.20
1.09
1.09
2.15
1.78
1.72
111
111
111
111
111
120
120
120
111
120
10
5
10
10
5
10
10
10
10
10
99
29
10
38
82
23
28
33
19
24
a
F (%) = percent absolute oral bioavailability corrected for MW
difference between the prodrug and parent.
respectively). These results would allow for more control
over the exposure of 5 in the efficacy study.
Figure 4. Chemical structures of compound 5 prodrugs.
Once the route of administration was selected, the in vivo
efficacy study of ceftibuten with and without co-administration
of 5 was conducted in the murine ascending the UTI model
with three strains of E. coli expressing TEM-1 + CTX-M-15,
CTX-M-15, or KPC-2 + SHV-12.44 To establish the ascending
UTI model, groups of five female C3H/HeJ mice were placed
on 5% glucose water for 6 days and then transurethrally
infected with approximately 9 log10 colony forming units (cfu)
of each bacterial isolate. Treatments of ceftibuten alone (1−
300 mg/kg), ceftibuten with compound 5 1:1 (1−300 mg/kg),
and a comparator agent (amoxicillin-clavulanate, 2:1, 10−300
mg/kg) were initiated 4 days post infection and administered
closely mimics the target cUTI indication in humans was
explored. A PK study was performed to guide selection of
dosing regimens for the cUTI efficacy study. Oral dosing of 10
and 90 mg/kg of 36 in mice resulted in F = 72% and F = 38%
of 5, respectively. The lack of dose proportionality of 5 by oral
dosing prompted a transition to subcutaneous dosing for the in
vivo efficacy study. A PK study utilizing subcutaneous
injections of 1.2, 12, and 38 mg/kg of 5 showed dose
proportionality of AUC (7.8, 76, and 250 mg∗h/L,
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Table 6. Oral Bioavailability of 20 and 36 across Species
rat
dose (mg/kg)
mouse
dose (mg/kg)
dog
monkey
dose (mg/kg)
a
a
a
a
entry
F (%)
F (%)
dose (mg/kg)
F (%)
F (%)
20
36
10
5
99
82
5
10
42
72
10
5
42
62
10
5
52
61
a
F (%) = percent absolute oral bioavailability corrected for MW difference between the prodrug and parent.
a
Table 7. Metabolic Stability of 36 across Multiple Species
intestinal S9 T1/2 (min)
liver S9 T1/2 (min)
species
+NADPH
−NADPH
+NADPH
−NADPH
plasma T1/2 (min)
CD-1 mouse
Sprague-Dawley rat
beagle dog
cynomolgus monkey
human
1.1
3.9
49.0
11.2
2.9
1.1
4.0
119
10.1
3.3
1.2
2.3
0.5
0.8
1.0
1.3
2.8
0.7
0.9
1.1
4.6
1.6
43.9
22.0
10.7
a
Abbreviations: NADPH = nicotinamide adenine dinucleotide phosphate.
Table 8. Caco-2 Permeability Results for 36 and 5
Papp (10−6 cm/s)
a
b
c
test article
direction
recovery (%)
R1
R2
avg
efflux Ratio
absorption potential classification
significant efflux
36
A-to-B
B-to-A
A-to-B
B-to-A
90
98
103
8.25
28.3
0.10
0.15
10.2
30.5
0.11
9.22
29.4
0.11
0.15
3.2
high
yes
5
1.4
low
no
106
0.14
a
b
c
Efflux ratio (ER) is Papp (B-to-A)/Papp (A-to-B). Papp (A-to-B) < 1.0 × 10−6 cm/s: low, Papp (A-to-B) ≥ 1.0 × 10−6 cm/s: high. ER ≥ 2.0 and Papp
(B-to-A) ≥ 1.0 × 10−6 cm/s.
subcutaneously every 12 h for 3 days. At day 7 post infection,
mean bacterial titers for the three bacterial strains were 6.7
log10 cfu in the kidney, 5.8 log10 cfu in the bladder, and 6.7
log10 cfu/mL in urine for the untreated controls. Admin-
istration of ceftibuten alone resulted in minimal cfu reductions
in the kidneys and cfu reductions in the bladder and urine that
did not exceed 2 log10 at ceftibuten doses of 100 and 300 mg/
kg. The addition of 5 to ceftibuten in a 1:1 ratio resulted in
increased efficacy, with bacterial titers that were up to 2.0 log10
cfu lower in kidneys, up to 3.2 log10 cfu lower in bladders, and
up to 4.0 log10 cfu/mL lower in urine than the corresponding
ceftibuten alone doses. The combination of ceftibuten and 5
also showed improved efficacy compared to amoxicillin-
clavulanate (2:1) with bacterial titers that were up to 2.3
log10 cfu lower in kidneys, 2.4 log10 cfu lower in bladders, and
3.7 log10 cfu/mL lower in urine. The UTI model demonstrated
that 5 rescues ceftibuten activity against uropathogenic strains
of E. coli expressing ESBLs or a combination of ESBLs and a
KPC carbapenemase.
ceftibuten activity in a mouse model of UTI due to ESBL- and
KPC-carbapenemase-producing strains of E. coli and K.
pneumoniae. Compound 36 is in Phase 1 clinical studies
(ClinicalTrials.gov Identifier: NCT04243863).
EXPERIMENTAL SECTION
■
General Methods. All solvents and reagents were purchased from
commercial vendors and were used without further purification unless
otherwise mentioned. Column chromatography was conducted using
prepacked silica gel cartridges (Biotage) on a Biotage Isolera Prime
system. LC−MS was conducted on an Agilent 1100 series HPLC and
6120 Quadrupole LC/MS in the API-ES mode using a Waters
Xbridge C18 column (4.6 mm × 50 mm, 3.5 μm). Mobile phase A
was 0.01% trifluoroacetic acid in water, and mobile phase B was 0.01%
1
trifluoroacetic acid in acetonitrile. H NMR spectra were recorded
using a Varian Mercury 300 MHz spectrometer. The final products
were purified by preparative HPLC on a Waters XBridge C18 column
(19 mm × 100 mm, 5 μm). The purity of all tested compounds was
≥95%, as determined by HPLC with UV detection at 220 nm and 1H
NMR analyses.
Calculations of clog P and tPSA were performed using ChemDraw
(Pro, Version 19.0). For clog P, ChemDraw uses the calculator from
Biobyte Corp, Claremont, CA (www.biobyte.com).45 For tPSA,
calculations were based on fragment contributions using the SMILES
Daylight Toolkit module.46
All animal experiments performed in the article were conducted in
compliance with institutional guidelines as defined by the Institutional
Animal Care and Use Committee (IACUC).
General Method for the Preparation of 3. To a solution of
tert-butyl 3-((S)-2-chloro-2-((3aS,4S,6S,7aR)-3a,5,5-trimethylhexahy-
dro-4,6-methanobenzo[d][1,3,2]dioxaborol-2-yl)ethyl)-2-methoxy-
benzoate (1)15,24 (1.35 g, 3 mmol) in THF (9 mL) at −78 °C was
added dropwise a solution of lithium bis(trimethylsilyl)amide (3.0
mL, 1 M in THF, 3 mmol). Following addition, the bath was removed
and stirring was continued for 17 h. The resulting solution, containing
CONCLUSIONS
■
Starting from a cyclic boronate template, we discovered highly
potent inhibitors of Ambler class A, C, and D β-lactamase
enzymes that rescued the activity of an oral cephalosporin,
ceftibuten, against ceftibuten-resistant E. coli and K. pneumo-
niae, two frequently isolated species of Enterobacterales
causing clinical infections. The synthesis of prodrugs of these
active BLIs, followed by comparisons of oral bioavailability in
rats, led to the selection of 36. Compound 36 demonstrated
excellent oral bioavailability in rats, dogs, and monkeys.
Hydrolysis of the prodrug ester to release the active BLI, 5,
was demonstrated in multiple species. Compound 5 restored
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approximately 0.25 M of intermediate 2 in THF, was used without
further purification.
7.83 (d, 1H), 7.30 (d, 1H), 6.96 (t, 1H), 4.85 (m, 1H), 2.92 (m, 2H),
2.50 (m, 2H), 2.05 (m, 2H), 1.43 (t, 3H). ESI-MS m/z: 328 (M +
H)+.
To a mixture of carboxylic acid (1 mmol) and HATU (1.1 mmol)
was added dimethylacetamide (DMA) (3 mL), followed by N-methyl-
morpholine (1.1 mmol). The resulting solution was stirred for 90 min.
To this solution was added a solution of 2 (1 mmol). The resulting
mixture was stirred for 2.5 h, diluted with ethyl acetate (EtOAc),
washed with water and brine, dried over anhydrous Na2SO4, filtered,
and concentrated. The residue was purified by silica gel chromatog-
raphy (10 g of silica; eluted with 20−100% EtOAc/hexanes) to
provide amide 3 (50−70% yield).
General Method for the Preparation of 15. To a solution of
carboxylic acid 4 (1.28 mmol) in ethanol (30 mL) was added 4 N
HCl in dioxane (6 mL). The solution was stirred at 40 °C for 18 h,
concentrated in vacuo, purified by reverse-phase flash chromatog-
raphy, and dried using lyophilization (20−40% yield).
Ethyl (R)-2-Hydroxy-3-propionamido-3,4-dihydro-2H-benzo[e]-
[1,2]oxaborinine-8-carboxylate (19). Prepared from the general
1
method for 15. H NMR (CD3OD): δ 7.35 (d, 1H), 7.19 (d, 1H),
General Method for the Preparation of 4. To a solution of
amide 3 (0.30 mmol) in anhydrous DCM (5 mL) at −78 °C was
added BCl3 (1 M in DCM, 2.1 mmol). After 1 h, the reaction mixture
was warmed to 0 °C. The reaction mixture was stirred for 1 h and
quenched by addition of water (5 mL) at 0 °C. The aqueous phase
was washed with DCM and then purified by reverse-phase preparative
HPLC. The product-containing fractions were combined and
lyophilized to dryness to give carboxylic acids 5−14 (40−70% yield).
(R)-2-Hydroxy-3-propionamido-3,4-dihydro-2H-benzo[e][1,2]-
oxaborinine-8-carboxylic Acid (5). Prepared from the general
6.85 (t, 1H), 4.05 (m, 2H), 3.45 (m, 1H), 2.58 (m, 2H), 2.03−2.45
(m, 2H), 1.10 (m, 3H), 0.92 (m, 3H). ESI-MS m/z: 292 (M + H)+.
Ethyl (R)-3-Butyramido-2-hydroxy-3,4-dihydro-2H-benzo[e][1,2]-
oxaborinine-8-carboxylate (21). Prepared from the general method
1
for 15. H NMR (CD3OD): δ 9.05 (s, 1H), 7.45 (d, 1H), 7.12 (d,
1H), 6.78 (t, 1H), 4.30 (m, 2H), 3.05 (m, 1H), 2.79 (m, 2H), 2.20
(m, 2H), 1.38 (m, 5H), 0.50 (m, 3H). ESI-MS m/z: 306 (M + H)+.
Ethyl (R)-3-(3-Cyanopropanamido)-2-hydroxy-3,4-dihydro-2H-
benzo[e][1,2]oxaborinine-8-carboxylate (23). Prepared from the
general method for 15. 1H NMR (CD3OD): δ 10.15 (s, 1H), 7.48 (d,
1H), 7.17 (d, 1H), 6.80 (t, 1H), 4.30 (q, 2H), 3.10 (m, 1H), 2.83 (m,
2H), 2.62 (m, 4H), 1.38 (t, 3H). ESI-MS m/z: 317 (M + H)+.
Ethyl (R)-2-Hydroxy-3-(4-oxopentanamido)-3,4-dihydro-2H-
benzo[e][1,2]oxaborinine-8-carboxylate (25). Prepared from the
general method for 15. 1H NMR (CD3OD): δ 9.85 (s, 1H), 7.45 (d,
1H), 7.18 (d, 1H), 6.81 (t, 1H), 4.32 (q, 2H), 3.05 (m, 1H), 2.82 (m,
2H), 2.62 (m, 2H), 2.42 (m, 2H), 1.98 (s, 3H), 1.38 (t, 3H). ESI-MS
m/z: 334 (M + H)+.
Ethyl (R)-3-(4-Amino-4-oxobutanamido)-2-hydroxy-3,4-dihydro-
2H-benzo[e][1,2]oxaborinine-8-carboxylate (27). Prepared from the
general method for 15. 1H NMR (CD3OD): δ 9.90 (s, 1H), 7.45 (d,
1H), 7.18 (d, 1H), 6.80 (t, 1H), 4.32 (q, 2H), 3.05 (m, 1H), 2.80 (m,
2H), 2.48 (m, 2H), 2.30 (m, 2H), 1.38 (t, 3H). ESI-MS m/z: 335 (M
+ H)+.
1
methods for 3 and 4. H NMR (CD3OD): δ 10.19 (s, 1H), 7.82
(d, 1H, J = 7.8 Hz), 7.25 (d, 1H, J = 7.1 Hz), 6.98 (t, 1H, J = 7.5 Hz),
3.20 (s, 1H), 2.90 (m, 2H), 2.20 (m, 2H), 0.90 (t, 3H, J = 7.5 Hz).
ESI-MS m/z: 264 (M + H)+.
(R)-3-Butyramido-2-hydroxy-3,4-dihydro-2H-benzo[e][1,2]-
oxaborinine-8-carboxylic Acid (6). Prepared from the general
1
methods for 3 and 4. H NMR (CD3OD): δ 10.22 (s, 1H), 7.85
(d, 1H), 7.32 (d, 1H), 6.90 (t, 1H), 3.27 (s, 1H), 2.94 (m, 2H), 2.31
(m, 1H), 2.22 (m, 1H), 1.43 (q, 2H), 0.53 (t, 3H). ESI-MS m/z: 278
(M + H)+.
(R)-2-Hydroxy-3-(4,4,4-trifluorobutanamido)-3,4-dihydro-2H-
benzo[e][1,2]oxaborinine-8-carboxylic Acid (7). Prepared from the
1
general methods for 3 and 4. H NMR (CD3OD): δ 10.45 (s, 1H),
7.85 (d, 1H), 7.33 (d, 1H), 6.97 (t, 1H), 4.80 (m, 1H), 2.96 (m, 2H),
2.69 (m, 2H), 2.64 (m, 2H). ESI-MS m/z: 332 (M + H)+.
Ethyl (R)-2-Hydroxy-3-(3-(methylsulfonamido)propanamido)-
(R)-3-(3-Cyanopropanamido)-2-hydroxy-3,4-dihydro-2H-benzo-
[e][1,2]oxaborinine-8-carboxylic Acid (8). Prepared from the general
3,4-dihydro-2H-benzo[e][1,2]oxaborinine-8-carboxylate (29). Pre-
1
1
pared from the general method for 15. H NMR (CD3OD): δ 10.02
methods for 3 and 4. H NMR (CD3OD): δ 10.6 (s, 1H), 7.38 (d,
(s, 1H), 7.45 (d, 1H), 7.18 (d, 1H), 6.81 (t, 1H), 4.34 (q, 2H), 3.18
(m, 2H), 3.05 (m, 1H), 2.82 (m, 2H), 2.80 (s, 3H), 2.50 (m, 2H),
1.40 (t, 3H). ESI-MS m/z: 385 (M + H)+.
1H), 7.32 (d, 1H), 6.97 (t, 1H), 4.90 (m, 1H), 2.94 (m, 2H), 2.62
(m, 2H), 2.40 (m, 2H). ESI-MS m/z: 289 (M + H)+.
(R)-2-Hydroxy-3-(4-oxopentanamido)-3,4-dihydro-2H-benzo[e]-
[1,2]oxaborinine-8-carboxylic Acid (9). Prepared from the general
Ethyl (R)-2-Hydroxy-3-(3-sulfamoylpropanamido)-3,4-dihydro-
1
2H-benzo[e][1,2]oxaborinine-8-carboxylate (31). Prepared from
methods for 3 and 4. H NMR (CD3OD): δ 10.2 (s, 1H), 7.74 (d,
1
the general method for 15. H NMR (CD3OD): δ 10.05 (s, 1H),
1H), 7.24 (d, 1H), 6.90 (t, 1H), 4.78 (m, 1H), 3.22 (m, 1H), 2.62
(m, 2H), 2.38 (m, 2H), 1.91 (s, 3H). ESI-MS m/z: 306 (M + H)+.
(R)-3-(4-Amino-4-oxobutanamido)-2-hydroxy-3,4-dihydro-2H-
benzo[e][1,2]oxaborinine-8-carboxylic Acid (10). Prepared from the
7.42 (d, 1H), 7.12 (d, 1H), 6.78 (t, 1H), 4.25 (q, 2H), 3.18 (m, 2H),
3.03 (m, 1H), 2.85 (m, 2H), 2.65 (m, 2H), 1.36 (t, 3H). ESI-MS m/
z: 371 (M + H)+.
General Methods for the Preparation of 16. Method A: A
solution of benzoic acid tert-butyl ester 3 (2.35 mmol) and 4 N HCl
in dioxane (18 mL) was stirred at room temperature for 7 h. The
reaction mixture was concentrated in vacuo and azeotroped two times
with toluene. The crude product was carried forward without
purification.
Method B: Trifluoroacetic acid (1.6 mL) was added to a solution of
benzoic acid tert-butyl ester 3 (0.71 mmol) in DCM (8 mL), and the
reaction mixture was stirred at room temperature for 30 min. The
reaction mixture was concentrated, and the product was azeotroped
with toluene and carried forward without further purification.
General Method for the Preparation of 17. To a solution of
16 (0.53 mmol) in DMF (2.5 mL) under argon was added sodium
carbonate (1.07 mmol), and the reaction mixture was stirred at room
temperature for 5 min. Chloromethyl pivalate (0.902 mmol) in DMF
(0.4 mL) was added, followed by sodium iodide (0.280 mmol), and
the reaction mixture was stirred at room temperature for 18 h. The
reaction mixture was quenched with water and extracted three times
with EtOAc. The combined organic layers were washed with brine,
dried over Na2SO4, filtered, and concentrated. The crude product was
purified by flash chromatography on silica gel (10−100% EtOAc/
hexanes) (50−70% yield).
1
general methods for 3 and 4. H NMR (CD3OD): δ 7.85 (d, 1H),
7.33 (d, 1H), 6.98 (t, 1H), 4.85 (m, 1H), 3.92 (m, 2H), 2.35−2.60
(m, 4H). ESI-MS m/z: 307 (M + H)+.
(R)-2-Hydroxy-3-(3-(methylsulfonamido)propanamido)-3,4-di-
hydro-2H-benzo[e][1,2]oxaborinine-8-carboxylic Acid (11). Pre-
1
pared from the general methods for 3 and 4. H NMR (CD3OD):
δ 7.83 (d, 1H), 7.32 (d, 1H), 6.97 (t, 1H), 4.90 (m, 1H), 3.18−3.26
(m, 2H), 2.95 (m, 2H), 2.85 (s, 3H), 2.55 (m, 2H). ESI-MS m/z: 357
(M + H)+.
(R)-2-Hydroxy-3-(pent-4-ynamido)-3,4-dihydro-2H-benzo[e]-
[1,2]oxaborinine-8-carboxylic Acid (12). Prepared from the general
1
methods for 3 and 4. H NMR (CD3OD): δ 10.4 (s, 1H), 7.83 (d,
1H), 7.32 (d, 1H), 6.98 (t, 1H), 4.95 (m, 1H), 2.95 (m, 2H), 2.45−
2.80 (m, 2H), 2.40 (m, 2H), 1.95 (s, 1H). ESI-MS m/z: 288 (M +
H)+.
(R)-2-Hydroxy-3-(3-sulfamoylpropanamido)-3,4-dihydro-2H-
benzo[e][1,2]oxaborinine-8-carboxylic Acid (13). Prepared from the
1
general methods for 3 and 4. H NMR (CD3OD): δ 7.83 (d, 1H),
7.34 (d, 1H), 6.98 (t, 1H), 4.95 (m, 1H), 3.05−3.25 (m, 2H), 2.95
(m, 2H), 2.80 (m, 2H). ESI-MS m/z: 343 (M + H)+.
(R)-3-(4,4-Difluoropentanamido)-2-hydroxy-3,4-dihydro-2H-
benzo[e][1,2]oxaborinine-8-carboxylic Acid (14). Prepared from the
1
general methods for 3 and 4. H NMR (CD3OD): δ 10.38 (s, 1H),
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General Method for the Preparation of 18. Aluminum
chloride (3.31 mmol) was added to a solution of 17 (0.41 mmol)
in DCM (10 mL), and the reaction mixture was stirred at room
temperature for 18 h. The reaction mixture was quenched with water
and methanol, concentrated, purified by reverse-phase HPLC, and
dried using lyophilization to provide the desired prodrugs (20−40%
yield).
((2-Ethylbutanoyl)oxy)methyl (R)-2-Hydroxy-3-propionamido-
3,4-dihydro-2H-benzo[e][1,2]oxaborinine-8-carboxylate (36). Pre-
pared from the general methods for 16−18 utilizing chloromethyl 2-
ethylbutanoate in place of chloromethyl pivalate in the preparation of
1
17. H NMR (CD3OD): δ 9.82 (s, 1H), 7.60 (d, 1H, J = 7.8 Hz),
7.21 (d, 1H, J = 7.1 Hz), 6.80 (t, 1H, J = 7.5 Hz), 5.98 (s, 2H), 3.12
(s, 1H), 2.82 (m, 2H), 2.20 (m, 3H), 1.60 (m, 4H), 0.91 (m, 9H).
ESI-MS m/z: 414 (M + Na)+.
(Pivaloyloxy)methyl (R)-2-Hydroxy-3-propionamido-3,4-dihy-
((Isopropoxycarbonyl)oxy)methyl (R)-2-Hydroxy-3-propionami-
do-3,4-dihydro-2H-benzo[e][1,2]oxaborinine-8-carboxylate (37).
Prepared from general methods for 16−18 utilizing chloromethyl
isopropyl carbonate in place of chloromethyl pivalate in the
preparation of 17. 1H NMR (CD3OD): δ 9.03 (s, 1H), 7.59 (d,
1H), 7.21 (d, 1H), 6.81 (t, 1H), 5.95 (m, 2H), 4.85 (m, 1H), 3.04 (s,
1H), 2.83 (m, 2H), 2.22 (m, 2H), 1.28 (m, 6H), 0.92 (t, 3H). ESI-
MS m/z: 402 (M + Na)+.
((2-Ethoxy-2-methylpropanoyl)oxy)methyl (R)-2-Hydroxy-3-pro-
pionamido-3,4-dihydro-2H-benzo[e][1,2]oxaborinine-8-carboxy-
late (38). Prepared from general methods for 16−18 utilizing
chloromethyl 2-ethoxy-2-methylpropanoate in place of chloromethyl
pivalate in the preparation of 17. 1H NMR (CD3OD): δ 9.02 (s, 1H),
7.59 (d, 1H), 7.21 (d, 1H), 6.81 (t, 1H), 5.99 (m, 2H), 3.45 (m, 2H),
3.04 (s, 1H), 2.82 (m, 2H), 2.21 (m, 2H), 1.40 (s, 6H), 1.12 (t, 3H),
0.89 (t, 3H). ESI-MS m/z: 430 (M + Na)+.
dro-2H-benzo[e][1,2]oxaborinine-8-carboxylate (20). Prepared
1
from general methods for 16−18. H NMR (CD3OD): δ 9.80 (s,
1H), 7.58 (d, 1H, J = 7.8 Hz), 7.20 (d, 1H, J = 7.1 Hz), 6.80 (t, 1H, J
= 7.5 Hz), 5.98 (s, 2H), 3.05 (s, 1H), 2.82 (m, 2H), 2.20 (m, 2H),
1.20 (s, 9H), 0.90 (t, 3H, J = 7.5 Hz). ESI-MS m/z: 400 (M + Na)+.
(Pivaloyloxy)methyl (R)-3-Butyramido-2-hydroxy-3,4-dihydro-
2H-benzo[e][1,2]oxaborinine-8-carboxylate (22). Prepared from
1
general methods for 16−18. H NMR (CD3OD): δ 10.18 (s, 1H),
7.85 (d, 1H), 7.50 (d, 1H), 7.12 (t, 1H), 6.23 (s, 2H), 3.38 (m, 1H),
3.15 (m, 2H), 2.50 (m, 2H), 1.68 (m, 2H), 1.52 (s, 9H), 0.82 (t, 3H).
ESI-MS m/z: 414 (M + Na)+.
(Pivaloyloxy)methyl (R)-3-(3-Cyanopropanamido)-2-hydroxy-
3,4-dihydro-2H-benzo[e][1,2]oxaborinine-8-carboxylate (24). Pre-
1
pared from general methods for 16−18. H NMR (CD3OD): δ 10.0
(s, 1H), 7.53 (d, 1H), 7.20 (d, 1H), 6.81 (t, 1H), 5.94 (s, 2H), 3.12
(m, 1H), 2.83 (m, 2H), 2.61 (m, 2H), 1.23 (s, 9H). ESI-MS m/z: 424
(M + Na)+.
(((Pentan-3-yloxy)carbonyl)oxy)methyl (R)-2-Hydroxy-3-propio-
namido-3,4-dihydro-2H-benzo[e][1,2]oxaborinine-8-carboxylate
(39). Prepared from general methods for 16−18 utilizing
chloromethyl pentan-3-yl carbonate in place of chloromethyl pivalate
(Pivaloyloxy)methyl (R)-2-Hydroxy-3-(4-oxopentanamido)-3,4-
dihydro-2H-benzo[e][1,2]oxaborinine-8-carboxylate (26). Prepared
1
1
from general methods for 16−18. H NMR (CD3OD): δ 9.90 (s,
in the preparation of 17. H NMR (CD3OD): δ 9.02 (s, 1H), 7.59
1H), 7.53 (d, 1H), 7.20 (d, 1H), 6.82 (t, 1H), 5.93 (s, 2H), 3.03 (m,
1H), 2.82 (m, 2H), 2.62 (m, 2H), 2.41 (m, 2H), 1.98 (s, 3H), 1.21 (s,
9H). ESI-MS m/z: 442 (M + Na)+.
(m, 1H), 7.21 (m, 1H), 6.81 (m, 1H), 5.94 (m, 2H), 4.60 (m, 1H),
3.04 (s, 1H), 2.82 (m, 2H), 2.20 (m, 2H), 1.62 (m, 4H), 0.90 (m,
9H). ESI-MS m/z: 430 (M + Na)+.
(Pivaloyloxy)methyl (R)-3-(4-Amino-4-oxobutanamido)-2-hy-
droxy-3,4-dihydro-2H-benzo[e][1,2]oxaborinine-8-carboxylate
(28). Prepared from general methods for 16−18. 1H NMR
(CD3OD): δ 9.92 (s, 1H), 7.53 (d, 1H), 7.20 (d, 1H), 6.80 (t,
1H), 5.98 (d, 2H), 3.05 (m, 1H), 2.85 (m, 2H), 2.62 (m, 2H), 2.43
(m, 2H), 1.21 (s, 9H). ESI-MS m/z: 443 (M + Na)+.
((Cyclopentanecarbonyl)oxy)methyl (R)-2-Hydroxy-3-propiona-
mido-3,4-dihydro-2H-benzo[e][1,2]oxaborinine-8-carboxylate
(40). Prepared from general methods for 16−18 utilizing
chloromethyl cyclopentanecarboxylate in place of chloromethyl
1
pivalate in the preparation of 17. H NMR (CD3OD): δ 9.02 (s,
1H), 7.58 (d, 1H), 7.20 (d, 1H), 6.81 (t, 1H), 5.93 (m, 2H), 3.04 (s,
1H), 2.82 (m, 2H), 2.21 (m, 2H), 1.55−1.98 (m, 9H), 0.90 (t, 3H).
ESI-MS m/z: 412 (M + Na)+.
(Pivaloyloxy)methyl (R)-2-Hydroxy-3-(3-(methylsulfonamido)-
propanamido)-3,4-dihydro-2H-benzo[e][1,2]oxaborinine-8-car-
1
boxylate (30). Prepared from general methods for 16−18. H NMR
(((Cyclopentyloxy)carbonyl)oxy)methyl (R)-2-Hydroxy-3-propio-
namido-3,4-dihydro-2H-benzo[e][1,2]oxaborinine-8-carboxylate
(41). Prepared from general methods for 16−18 utilizing
chloromethyl cyclopentyl carbonate in place of chloromethyl pivalate
in the preparation of 17. 1H NMR (CD3OD): δ 9.02 (s, 1H), 7.58 (d,
1H), 7.21 (d, 1H), 6.81 (t, 1H), 5.93 (m, 2H), 5.10 (m, 1H), 3.04 (s,
1H), 2.82 (m, 2H), 2.22 (m, 2H), 1.58−1.95 (m, 8H), 0.93 (t, 3H).
ESI-MS m/z: 428 (M + Na)+.
(CD3OD): δ 7.68 (d, 1H), 7.47 (m, 1H), 7.20 (m, 1H), 5.98 (s, 2H),
3.38 (m, 1H), 3.15 (m, 2H), 2.97 (s, 3H), 2.80 (m, 2H), 2.63 (m,
2H), 1.21 (s, 9H). ESI-MS m/z: 493 (M + Na)+.
(Pivaloyloxy)methyl (R)-2-Hydroxy-3-(3-sulfamoylpropanami-
do)-3,4-dihydro-2H-benzo[e][1,2]oxaborinine-8-carboxylate (32).
1
Prepared from general methods for 16−18. H NMR (CD3OD): δ
7.65 (d, 1H), 7.52 (m, 1H), 7.18 (m, 1H), 5.95 (s, 2H), 3.79 (m,
2H), 3.19 (m, 1H), 2.88 (m, 2H), 2.75 (m, 2H), 1.20 (s, 9H). ESI-
MS m/z: 479 (M + Na)+.
ASSOCIATED CONTENT
■
(Benzoyloxy)methyl (R)-2-Hydroxy-3-propionamido-3,4-dihy-
dro-2H-benzo[e][1,2]oxaborinine-8-carboxylate (33). Prepared
from general methods for 16−18 utilizing chloromethyl benzoate in
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00437.
1
place of chloromethyl pivalate in the preparation of 17. H NMR
(CD3OD): δ 9.02 (s, 1H), 8.08 (d, 2H), 7.62 (m, 2H), 7.53 (m, 2H),
7.20 (d, 1H), 6.81 (t, 1H), 6.22 (dd, 2H), 3.05 (s, 1H), 2.83 (m, 2H),
2.18 (q, 2H), 0.88 (t, 3H). ESI-MS m/z: 420 (M + Na)+.
Acetoxymethyl (R)-2-Hydroxy-3-propionamido-3,4-dihydro-2H-
benzo[e][1,2]oxaborinine-8-carboxylate (34). Prepared from general
methods for 16−18 utilizing chloromethyl acetate in place of
chloromethyl pivalate in the preparation of 17. 1H NMR
(CD3OD): δ 7.59 (m, 1H), 7.21 (m, 1H), 6.82 (m, 1H), 5.97 (m,
2H), 3.06 (s, 1H), 2.80 (m, 2H), 2.22 (m, 2H), 2.12 (s, 3H), 0.90 (m,
3H). ESI-MS m/z: 336 (M + H)+.
Inhibition assays, antimicrobial susceptibility testing, PK
screening studies, mouse PK studies, metabolic stability,
metabolite profiling, bidirectional permeability through
Caco-2 monolayers, mouse efficacy study, HPLC
1
method and HPLC of compounds 5 and 36, and H
NMR of compounds 5 and 36 (PDF)
Molecular formula strings (CSV)
(Isobutyryloxy)methyl (R)-2-Hydroxy-3-propionamido-3,4-dihy-
dro-2H-benzo[e][1,2]oxaborinine-8-carboxylate (35). Prepared
from general methods for 16−18 utilizing chloromethyl isobutyrate
AUTHOR INFORMATION
■
Corresponding Author
1
in place of chloromethyl pivalate in the preparation of 17. H NMR
Robert E. Trout − Venatorx Pharmaceuticals, Inc., Malvern,
Pennsylvania 19355, United States; orcid.org/0000-
0001-6649-0493; Email: trout@venatorx.com
(CD3OD): δ 9.02 (s, 1H), 7.58 (d, 1H), 7.21 (d, 1H), 6.81 (t, 1H),
5.98 (m, 2H), 3.04 (s, 1H), 2.83 (m, 2H), 2.23 (m, 3H), 1.60 (m,
6H), 0.92 (t, 3H). ESI-MS m/z: 386 (M + Na)+.
10163
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Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
Authors
absolute oral bioavailability; HATU, 1-[bis(dimethylamino)-
methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium-3-oxide hexa-
fluorophosphate; HR-MS/MS, hi-res tandem mass spectrom-
etry; KPC, Klebsiella pneumoniae carbapenemase; MBL,
metallo-β-lactamase; MDR, multi-drug-resistant; MIC, mini-
mum inhibitory concentration; NADPH, nicotinamide adenine
dinucleotide phosphate; PK, pharmacokinetics; SAR, struc-
ture−activity relationship; SBL, serine-β-lactamase; UPLC,
ultra-performance liquid chromatography; UTI, urinary tract
infection
Allison Zulli − Venatorx Pharmaceuticals, Inc., Malvern,
Pennsylvania 19355, United States
Eugen Mesaros − Venatorx Pharmaceuticals, Inc., Malvern,
Pennsylvania 19355, United States
Randy W. Jackson − Venatorx Pharmaceuticals, Inc., Malvern,
Pennsylvania 19355, United States
Steven Boyd − Venatorx Pharmaceuticals, Inc., Malvern,
Pennsylvania 19355, United States
Bin Liu − Venatorx Pharmaceuticals, Inc., Malvern,
Pennsylvania 19355, United States; orcid.org/0000-
0002-2538-312X
REFERENCES
■
Jodie Hamrick − Venatorx Pharmaceuticals, Inc., Malvern,
Pennsylvania 19355, United States
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Christopher J. Burns − Venatorx Pharmaceuticals, Inc.,
Malvern, Pennsylvania 19355, United States
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.1c00437
Notes
The authors declare the following competing financial
interest(s): R.T., A.Z., E.M., R.J., S.B., B.L., J.H., D.D., C.C.,
K.J., L.M., S.C., D.P., G.M., L.X., C.B. are employees of and/or
shareholders in Venatorx Pharmaceuticals.
ACKNOWLEDGMENTS
■
This project has been funded in whole or in part from the
National Institute for Allergy and Infectious Diseases, National
Institutes of Health, Department of Health and Human
Services, under Contract no. HHSN272201600029C and grant
nos. R44AI109879, R01AI089512, R01AI111539, and
R43AI109879.
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ABBREVIATIONS
■
AUC, area under the concentration−time curve; BL, β-lactam
antibiotic; BLI, β-lactamase inhibitor; cfu, colony-forming unit;
CRE, carbapenem-resistant Enterobacteriaceae spp.; DCM,
dichloromethane; DMA, dimethyl acetamide; DMF, N,N-
dimethylformamide; DMSO, dimethyl sulfoxide; ESBL,
extended spectrum β-lactamase; EtOAc, ethyl acetate; F,
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