Discovery of Cyclic Boronic Acid QPX7728, an Ultrabroad-Spectrum Inhibitor of Serine and Metallo-β-lactamases

Article abstract of DOI:10.1021/acs.jmedchem.9b01976

Despite major advances in the β-lactamase inhibitor field, certain enzymes remain refractory to inhibition by agents recently introduced. Most important among these are the class B (metallo) enzyme NDM-1 of Enterobacteriaceae and the class D (OXA) enzymes

Full text of DOI:10.1021/acs.jmedchem.9b01976

Subscriber access provided by Columbia University Libraries
Drug Annotation
Discovery of Cyclic Boronic Acid QPX7728, an Ultra-broad-
spectrum Inhibitor of Serine and Metallo Beta-lactamases
Scott J. Hecker, K. Raja Reddy, Olga Lomovskaya, David C. Griffith, Debora Rubio-Aparicio, Kirk
Nelson, Ruslan Tsivkovski, Dongxu Sun, Mojgan Sabet, Ziad Tarazi, Jonathan Parkinson, Maxim
Totrov, Serge H. Boyer, Tomasz W. Glinka, Orville Antonio Pemberton, Yu Chen, and Michael N. Dudley
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b01976 • Publication Date (Web): 09 Mar 2020
Downloaded from pubs.acs.org on March 10, 2020
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 49
Journal of Medicinal Chemistry
1
2
3
4
5
6
Discovery of Cyclic Boronic Acid QPX7728, an Ultra-broad-spectrum Inhibitor of Serine and
Metallo Beta-lactamases
7
8
9
Scott J. Hecker,*,† K. Raja Reddy,† Olga Lomovskaya,† David C. Griffith,† Debora Rubio-
Aparicio,† Kirk Nelson,† Ruslan Tsivkovski † Dongxu Sun,† Mojgan Sabet,† Ziad Tarazi,†
Jonathan Parkinson,† Maxim Totrov,‡ Serge H. Boyer,† Tomasz W. Glinka,† Orville A.
Pemberton,§ Yu Chen,§ and Michael N. Dudley†
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
†Qpex Biopharma, Inc., 6275 Nancy Ridge Dr., Suite 100, San Diego, CA 92121
‡Molsoft LLC, 11199 Sorrento Valley Rd., San Diego, CA 92121
§Department of Molecular Medicine, University of South Florida Morsani College of Medicine,
12901 Bruce B. Downs Boulevard, Tampa, FL 33612
Abstract: Despite major advances in the beta-lactamase inhibitor field, certain enzymes remain
refractory to inhibition by agents recently introduced. Most important among these are the Class
B (metallo) enzyme NDM-1 of Enterobacteriaceae and the Class D (OXA) enzymes of
Acinetobacter baumannii. Continuing the boronic acid program that led to vaborbactam, efforts
were directed toward expanding the spectrum to allow treatment of a wider range of organisms.
Through key structural modifications of a bicyclic lead, stepwise gains in spectrum of inhibition
were achieved, ultimately resulting in QPX7728 (35). This compound displays a remarkably
broad spectrum of inhibition, including Class B and Class D enzymes, and is little affected by
porin modifications and efflux. Compound 35 is a promising agent for use in combination with a
beta-lactam antibiotic for the treatment of a wide range of multidrug resistant gram-negative
bacterial infections, by both intravenous and oral administration.
-1-
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 2 of 49
1
2
3
4
Introduction
5
6
7
8
9
It has now been several years since the scourge of carbapenem-resistant Enterobacteriaceae was
recognized and the call was sounded for new agents by WHO and other public health
authorities.1 Beta-lactamase inhibitor (BLI) combination products have played a major role in
the response, with FDA approvals awarded for avibactam (with ceftazidime, 2015),2
vaborbactam (with meropenem, 2017)3 and relebactam (with imipenem-cilastatin, 2019)4 (Figure
1). While these agents address the problem of Class A serine carbapenemases such as KPC, they
have no activity against metallo beta-lactamases such as NDM-1, and also have no activity
against the problematic Class D enzymes of Acinetobacter baumannii, such as OXA-23. Hence,
there is an ongoing need for inhibitors showing a broader spectrum that captures these other
types of beta-lactamases. Current work in this area has been reviewed recently.5, 6
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 1. Recently-introduced beta-lactamase inhibitors.
HN
O
O
H
N
H2N
N
H
N
N
S
O
B
CO2H
N
N
O
HO
O
OSO3H
O
OSO3H
relebactam
vaborbactam
avibactam
When our team first began synthesizing boronic acid inhibitors in 2009, many had doubts about
whether it would be possible to achieve potent inhibition of both serine and metallo enzymes
with a single compound. However, recent publications have described boronates with activity
against some enzymes from both classes.7, 8 Herein we describe our own efforts to achieve
broader spectrum, higher potency, and reduced susceptibility to uptake resistance mechanisms
(porin modifications and efflux), resulting in discovery of QPX7728 (35), a remarkably potent
and broad-spectrum inhibitor with potential for both intravenous and oral application.
-2-
ACS Paragon Plus Environment

Page 3 of 49
Journal of Medicinal Chemistry
1
2
3
4
Results and Discussion
5
6
7
Early Evolution of Bicyclic Boronate BLIs
8
9
The benzoxaborinine 1 (Figure 2), first reported by researchers at the University of Toronto,9 has
proven to be an extremely important lead structure in the beta-lactamase inhibitor arena, thanks
to its broad spectrum of inhibition of serine beta-lactamases. Early efforts to develop
compounds included those by Protez Pharmaceuticals10-13 and Rempex Pharmaceuticals,14, 15
with RPX7009 (vaborbactam)16, 17 being the first inhibitor to enter the clinic and achieve
regulatory approvals in the US and EU. Other groups have recently entered other boronate BLIs
into clinical development.18, 19
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 2. The benzoxaborinine beta-lactamase inhibitors lead that started it all.
H
N
O
B
O
HO
CO2H
1
Effect of Substitution ortho to the Carboxylate
Soon after vaborbactam was advanced into development, members of the team that discovered
this agent began investigations into compounds related to benzoxaborinine 1. Work by this
team14, 20 and others21-24 explored a wide variety of acyl groups on the amine alpha to boron. As
was observed in the vaborbactam discovery effort,17 this region is largely insensitive to
modification, as potent, broad-spectrum activity is obtained with a wide variety of substituents.
Conversely, little had been described with respect to substitution on the aromatic ring. Thus, our
initial efforts were directed toward determining the effect of such substituents.
-3-
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 4 of 49
1
2
3
4
5
6
7
8
9
One of the acylamido substituents conferring the best activity in this series was the
methylthioacetamide; therefore, this substituent is selected for purposes of illustration of
synthesis and SAR. Analogs were prepared by analogy to the methods shown in Scheme 1.
Alpha-chloroboronate 2 was obtained by Matteson reaction of its benzylboronate precursor.14
Displacement of the chlorine atom of 2 with inversion is effected with lithium
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
hexamethyldisilazide and is followed by acylation with methylthioacetic acid, giving 3. The
protecting groups are removed by treatment with acid, affording analog 4a.
Scheme 1. Synthesis of benzoxaborinine 4a.
O
SMe
H
Cl
HN
1) LiHMDS, THF
-78 C to RT
N
TFA, Et3SiH
room temp.
O
O
O
O
MeS
B
B
O
B
O
HO
2) MeSCH2CO2H,
EDCI, HOBT,
NMM, DCM
BocO
BocO
CO2H
CO2tBu
CO2tBu
4a
3
2
Evaluation of a range of ortho-substituents against organisms expressing common enzymes of
Classes A and C is shown in Table 1. Potency is expressed as the amount of BLI needed to
reduce the MIC of aztreonam to 4 µg/mL, a concentration that corresponds to a PK-PD
breakpoint based on human dosing. (Aztreonam was selected as the beta-lactam partner because,
unlike carbapenems, it is hydrolyzed by all of these enzymes.) As with the N-acyl substituent,
the effect of substitution ortho to the carboxylate on spectrum and potency of beta-lactamase
inhibition was relatively modest. There is a clear trend toward smaller substituents being
preferred, but all compounds in this set show good potency (≤5 µg/mL) against most strains,
including those with positively (4f) and negatively (4i) charged substituents. A detrimental
effect of substitution is seen with the AmpC enzymes from E. cloacae and P. aeruginosa.
-4-
ACS Paragon Plus Environment

Page 5 of 49
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Table 1. Activity of alpha-methylthioacetamides 4a-4l substituted ortho to the carboxylate.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
H
Minimum concentration (µg/mL) of BLI needed to reduce the
N
MeS
MIC of aztreonam (ATM) to 4 µg/mL
O
B
O
R
HO
R:
-H
-F
-OMe
-OH
-SMe
CO2H
ATM
alone
Organism
Strain
Enzyme Class
4a
4b
4c
4d
4e
4f
K. pneumoniae KP1009 CTX-M-15
A
A
A
A
C
C
>128
>128
128
>128
64
0.6
≤0.3
5
≤0.3
≤0.3
1.25
0.6
≤0.3
≤0.3
≤0.3
≤0.3
0.6
1.25
≤0.3
2.5
1.25
2.5
5
0.6
0.04
1.25
0.6
0.6
0.6
5
E. coli
E. coli
EC308
EC302
SHV-5
TEM-10
KPC-2
K. pneumoniae KP1004
E. cloacae
P. aeruginosa PAM 2035 AmpC
0.6
0.6
2.5
1.25
10
ECL1002 chAmpC
2.5
1.25
20
64
10
20
20
OH
O
R:
O
ATM
alone
Organism
Strain
BL
Class
4g
4h
4i
4j
4k
4l
K. pneumoniae KP1009 CTX-M-15
A
A
A
A
C
C
>128
>128
128
>128
64
0.6
0.16
1.25
2.5
1.25
0.04
1.25
2.5
2.5
0.08
5
1.25
≤0.3
2.5
2.5
≤0.3
2.5
5
2.5
≤0.3
2.5
5
E. coli
E. coli
EC308
EC302
SHV-5
TEM-10
KPC-2
K. pneumoniae KP1004
E. cloacae
5
1.25
5
ECL1002 chAmpC
2.5
5
5
5
2.5
>20
P. aeruginosa PAM 2035 AmpC
64
>20
>20
>20
>20
>20
Abbreviations: ATM, aztreonam
One of the issues that we had encountered with earlier unsubstituted analogs in this series was a
propensity to form oligomers in solution. This phenomenon was observed by HPLC as longer
retention time peaks whose mass corresponded to dimers, trimers, etc. The extent of formation
was pH- and concentration-dependent. Such species were not observed with monocyclic BLIs
such as vaborbactam. Although the structures were never formally characterized, we believed
these to be of the type shown in Figure 3, as boronic acids are known to complex with
salicylates.25 Representative chromatograms, LC-MS data and tentative structural assignments
are provided in the Supporting Information. While we believed these issues were manageable,
-5-
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 6 of 49
1
2
3
4
5
6
we envisioned that the formation of such species in an intravenous dosage form would
complicate the toxicological qualification of impurities to enable human administration.
7
8
9
Figure 3. Putative structures of dimer 5a and trimer 5b.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
OH
H
R
O
N
CO2H
O
O
B
OH
O
O
H
N
H
N
R
CO2H
R
O
O
B
O
B
O
O
O
(HO)2B
(HO)2B
O
O
R
NH
O
R
NH
5b
5a
Upon synthesis of ortho-substituted analogs, we quickly learned that the presence of an ortho-
substituent markedly reduces the extent of oligomer formation. The extent of oligomerization
was evaluated by HPLC-UV at various concentrations at pH 7.8 and is expressed as area-% of
monomer (the desired form). As shown in Table 2, compounds lacking an ortho substituent
exhibited a concentration-dependent decrease in monomer content, whereas compounds with an
ortho substituent had a much-reduced tendency to undergo oligomerization, even at higher
concentrations.
Table 2: Monomer content at pH 7.8 +/- 0.2 at 1, 10 and 100 mg/mL concentration
H
H
N
N
MeS
O
B
S
O
B
O
R
O
R
HO
HO
CO2H
CO2H
4a-4c
4m-4o
Compound
R
% Monomer (HPLC, 220 nm)
1 mg/mL
10 mg/mL
100 mg/mL
4a
4b
4c
H
F
72.1
98.0
98.2
43.0
97.5
95.5
14.9
96.4
96.5
OMe
-6-
ACS Paragon Plus Environment

Page 7 of 49
Journal of Medicinal Chemistry
1
2
3
4
5
6
4m
4n
4o
H
F
80.0
94.6
99.4
37.0
8.3
93.5
98.6
87.6
67.0
OMe
7
8
9
Another benefit of substitution ortho to the carboxylate concerned the extent to which the MIC in
P. aeruginosa was affected by the MexAB-OprM efflux pump. To evaluate the impact of
substitution in this region on efflux, the plasmid expressing the gene encoding the KPC-2
carbapenemase was introduced into two isogenic strains of P. aeruginosa, PAM1032 and
PAM1154 that overexpressed or lacked MexAB-OprM, respectively. Biapenem is not affected
by efflux in P. aeruginosa, and both strains had the same biapenem MIC of 32 µg/ml due to the
expression of KPC-2. The potency of BLIs in potentiation of biapenem in these strains was
determined. Potency was defined as the minimum concentration of BLI needed to decrease the
MIC of biapenem 64-fold, from 32 µg/ml to 0.5 µg/ml, or minimum potentiating concentration
(MPC64). The Efflux Index (EI) for each BLI was defined as the ratio of MPC64 values in
PAM1032/KPC-2 (efflux proficient) and PAM1154/KPC-2 (efflux deficient).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 3 shows MPC64 and EIs values for selected analogs. A modest but consistent trend is
observed wherein the impact of efflux is reduced by the presence of a methoxy or fluoro
substituent. Interestingly, a hydroxy substituent (4d) does not offer this benefit. It is also
noteworthy that incorporation of a basic group on the acylamido substituent (4p-4r) completely
removes the impact of efflux, even when no ortho substituent is present.
-7-
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 8 of 49
1
2
3
4
5
6
7
8
9
Table 3. MexAB-OprM Dependent Efflux of BLIs from P. aeruginosa
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
HN
H
H
H
N
N
N
MeS
S
O
B
S
O
B
O
B
O
R
O
R
O
R
HO
HO
HO
CO2H
CO2H
CO2H
4a-4d
4m-4o
4p-4r
PAM1032/
KPC-2
PAM1154/
KPC-2
Biapenem
MPC64 (µg/mL)
Biapenem
MPC64 (µg/mL)
Entry
R
EI
4a
4b
4c
4d
4m
4n
4o
4p
4q
4r
H
F
80
20
10
40
80
40
40
2.5
5
5
5
8
4
OMe
OH
H
5
2
2.5
2.5
10
5
16
32
4
F
OMe
H
8
2.5
5
1
F
1
OMe
5
5
1
Alpha-thio analogs with much improved metallo activity.
While in this compound series the potent inhibition of a broad spectrum of serine beta-
lactamases was well-established, activity against metallo enzymes (Class B) had not been
reported at that time. Our modeling studies suggested that members of this series would be
capable of binding in the active site of metallo beta-lactamases such as NDM-1. Compounds
were therefore evaluated in biochemical and microbiological assays to determine the extent of
inhibition of this enzyme. Indeed, compound 4s, in addition to being a potent inhibitor of the
serine carbapenemases KPC-2 (Class A) and OXA-48 (Class D), demonstrated modest inhibition
-8-
ACS Paragon Plus Environment

Page 9 of 49
Journal of Medicinal Chemistry
1
2
3
4
5
6
of NDM-1 (Class B) with a Ki of 7.4 µM (Table 4). Whole-cell activity against NDM-1
producing strains, however, was lacking.
7
8
9
In parallel with pursuit of the above series, one of our chemists prepared sulfur-linked compound
6a, in part because it was easily accessible from the alpha-chloroboronate intermediate being
used to make amide-linked analogs. These efforts resulted in a 300-fold improvement in potency
against NDM-1 (Ki = 0.024 µM). Importantly, in whole cells this compound at a concentration
of 0.6 µg/mL restored susceptibility to biapenem of an NDM-1 producing strain of K.
pneumoniae (Table 4). The potency of compound 6a was not affected by increasing
concentrations of zinc (up to 0.6 mM), suggesting a mechanism other than zinc chelation or
competition with zinc for binding to the protein. Importantly, this compound did not inhibit any
members of a panel of human metallo-proteases. Recently, other reports have appeared on
observations of metallo activity with certain acylamido analogs.7, 8
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 4. Biochemical and whole-cell activity of compounds 4s and 6a.
N N
H
S
N
S
H2N
S
S
O
B
N N
B
O
HO
O
HO
CO2H
CO2H
4s
6a
Ki (µM) of inhibition of purified enzyme Concentration of BLI (µg/ml) to decrease
with indicated substrates MIC to biapenem to 1 µg/ml
BLI
KPC-2
OXA-48
nitrocefin
NDM-1
imipenem
KPC-2
OXA-48
KP1086
NDM-1
KP1081
nitrocefin
0.011
0.021
0.15
KP1004
≤0.04
0.08
Avibactam
0.027
0.0005
0.0014
>80
1.25
2.5
5
>20
4s
6a
7.4
>20
0.024
0.16
0.6
Based on the observations above, an optimization program was initiated around sulfur- and
oxygen-linked analogs. Initial efforts in this series were conducted in the series with no
-9-
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 10 of 49
1
2
3
4
5
6
7
8
9
substituent ortho to the carboxylate, with the intention of optimizing that substituent later.
Compounds 6a-r were obtained by reaction of chloroboronate 2 with the appropriate
nucleophile, followed by deprotection (Scheme 2). Displacement by sulfur nucleophiles was
straightforward, whereas displacement by oxygen nucleophiles failed in most cases; thus, only
two examples are shown.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Scheme 2. Synthesis of Compounds 6a-r
Cl
R-SH or R-OH
O
O
R
TFA, Et3SiH
room temp.
B
B
TEA/DCM
room temp.
O
BocO
HO
CO2H
CO2tBu
6a-r
2
In Table 5, activity is expressed as the minimum concentration of BLI needed to reduce the MIC
of a specified beta-lactam (β-L) to the concentration that corresponds to the PK-PD breakpoint
for that beta-lactam based on human dosing (Potentiation Target Value, PTV). Since aztreonam
is stable to metallo beta-lactamases, biapenem was selected as the partner beta-lactam for
evaluation of inhibition of NDM-1 and VIM-1. Analog selection was influenced by a desire to
maintain small size and incorporate non-charged polarity, as these features were generally
associated with better uptake (as measured by the ratio of MIC to IC50; data not shown). Simple
substituents like methylthio (6b) and methoxy (6c) were among the first to be made and have
some of the best potency. These compounds had good activity against carbapenemases (KPC,
NDM and VIM) but had poor activity against important cephalosporinases (CTX-M, SHV,
TEM). Simple uncharged substituents (6d-6e) had a similar profile to 6b and 6c, whereas basic
groups were variable (6f vs. 6g). Among nitrogen-containing heterocyclic analogs, activity
varied widely depending on the location of the nitrogen atoms. Most analogs retained activity
against the metallo enzymes (NDM, VIM) with the exception of imidazole-2-ylthio 6l and pyrid-
-10-
ACS Paragon Plus Environment

Page 11 of 49
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
2-ylthio 6m; perhaps this is due to the interaction of a nucleophilic nitrogen with the boron atom.
In this heteroarylthio series, activity against KPC was only observed with N-aminotriazolylthio
analog 6k and aminothiadiazolylthio 6q.
10
11
12
13
14
15
16
17
Table 5. Activity of 6a-r with Oxygen- and Sulfur-linked Substituents Alpha to Boron.
R
Minimum concentration (µg/mL) of BLI needed to
reduce the MIC of β-L to PTV (µg/mL)
B
O
HO
R:
MeS-
MeO-
CO2H
Organism
K. pneumoniae KP1009 CTX-M-15
E. coli
E. coli
K. pneumoniae KP1004
E. cloacae
K. pneumoniae KP1081
K. pneumoniae KP1054
Strain
BL
Class
β-L
ATM
ATM
ATM
ATM
ATM
BP
β-L alone PTV
6a
>20
2.5
20
10
20
6b
>20
>20
>20
2.5
>20
0.6
≤0.3
6c
10
2.5
20
0.3
2.5
0.6
0.16
6d
>10
>10
>10
5
>10
0.6
2.5
6e
>20
>20
>20
5
>20
1.25
1.25
6f
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
A
A
A
A
C
B
B
>128
>128
128
>128
64
4
4
4
4
4
1
1
>20
>20
>20
>20
>20
>20
>20
KP308
EC302
SHV-5
TEM-10
KPC-2
ECL1002 chAmpC
NDM-1
VIM-1
16
0.6
2.5
BP
16
R:
Organism
K. pneumoniae KP1009 CTX-M-15
E. coli
E. coli
K. pneumoniae KP1004
E. cloacae
K. pneumoniae KP1081
K. pneumoniae KP1054
Strain
BL
Class
β-L
ATM
ATM
ATM
ATM
ATM
BP
β-L alone PTV
6g
>20
>20
>20
>20
2.5
6h
5
6i
6j
6k
10
10
>20
5
5
6l
A
A
A
A
C
B
B
>128
>128
128
>128
64
4
4
4
4
4
1
1
>20
>20
>20
>20
>20
≤0.3
≤0.3
>20
>20
>20
>20
>20
≤0.3
≤0.3
>10
>10
>10
>10
>10
>20
>20
KP308
EC302
SHV-5
TEM-10
KPC-2
>40
>40
>40
>40
≤0.3
2.5
ECL1002 chAmpC
NDM-1
VIM-1
16
0.6
0.6
0.16
0.3
BP
16
R:
Organism
K. pneumoniae KP1009 CTX-M-15
E. coli
E. coli
K. pneumoniae KP1004
E. cloacae
K. pneumoniae KP1081
K. pneumoniae KP1054
Strain
BL
Class
β-L
ATM
ATM
ATM
ATM
ATM
BP
β-L alone PTV
6m
>20
>20
>20
>20
>20
10
6n
6o
>20
>20
>20
>20
>20
2.5
6p
>20
10
>20
>20
>20
0.3
2.5
6q
>10
5
>10
10
>10
0.3
0.16
6r
>20
5
>20
>20
>20
0.3
0.6
A
A
A
A
C
B
B
>128
>128
128
>128
64
4
4
4
4
4
1
1
>10
>10
>10
>10
>10
>20
>20
KP308
EC302
SHV-5
TEM-10
KPC-2
ECL1002 chAmpC
NDM-1
VIM-1
16
BP
16
20
1.25
Abbreviations: β-L, beta-lactam; PTV, potentiation target value; ATM, aztreonam; BP, biapenem
Ortho-substitution and Oral Bioavailability.
In addition to potent, broad-spectrum activity, one of our objectives was to achieve suitable
properties for oral administration. Of the analogs in Table 5, we selected thiadiazole 6a as the
-11-
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 12 of 49
1
2
3
4
5
6
7
8
9
basis for further optimization of the substituent ortho to the carboxylate, since it displayed a
balanced spectrum and did not contain an additional H-bond donor (which would likely hamper
oral absorption). Table 6 shows the results of a series of small ortho-substituents 7a-k. These
compounds were prepared in a manner analogous to that used for compounds 6, starting with the
appropriate substituted benzoic acid.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
As was seen in the amide series (Table 1), the best activity was observed with the small
substituents methoxy (7a) and fluoro (7b). The benefit of this substituent (relative to
unsubstituted analog 6a) was apparent in all strains tested. With larger substituents, the benefit
was greatly reduced or absent, particularly with respect to Class A serine enzymes. Interestingly,
activity against the metallo enzymes (Class B) was less affected by the size of this substituent,
and in fact activity against IMP-26 was significantly improved with the larger substituents in 7g-
7k.
Table 6. Activity of Thiadiazolylthio analogs 7.
S
Minimum concentration (µg/mL) of BLI needed to reduce
the MIC of β-L to PTV (µg/mL)
S
N N
B
HO
O
R
R:
CO2H
Organism
K. pneumoniae KP1009 CTX-M-15
E. coli
E. coli
K. pneumoniae KP1004
E. cloacae
P. aeruginosa PAM 2035 AmpC
K. pneumoniae KP1160
K. pneumoniae KP1081
K. pneumoniae KP1054
Strain
BL
Class β-L β-L alone PTV
6a
>20
2.5
20
10
20
ND
40
0.6
2.5
7a
2.5
0.3
2.5
1.25
10
10
5
0.3
0.3
7b
10
0.3
5
2.5
5
20
20
0.6
1.25
7c
20
2.5
20
5
>20
20
20
1.25
1.25
7d
>20
10
20
20
>20
>20
>20
2.5
2.5
7e
20
5
20
5
A
A
A
A
C
C
B
B
B
ATM
ATM
ATM
ATM
ATM
ATM
MP
>128
>128
128
>128
64
64
16
16
16
4
4
4
4
4
4
1
1
1
KP308
EC302
SHV-5
TEM-10
KPC-2
ECL1002 chAmpC
>20
20
2.5
0.6
0.16
IMP-26
NDM-1
VIM-1
BP
BP
R:
Organism
K. pneumoniae KP1009 CTX-M-15
E. coli
E. coli
Strain
BL
Class β-L β-L alone PTV
7f
7g
>20
>20
>20
10
7h
>20
10
20
>20
7i
20
10
>20
20
7j
>20
10
>20
20
7k
20
5
5
20
A
A
A
A
ATM
ATM
ATM
ATM
>128
>128
128
4
4
4
4
>20
>20
>20
>20
KP308
EC302
SHV-5
TEM-10
KPC-2
K. pneumoniae KP1004
>128
-12-
ACS Paragon Plus Environment

Page 13 of 49
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
E. cloacae
P. aeruginosa PAM 2035 AmpC
K. pneumoniae KP1160
K. pneumoniae KP1081
K. pneumoniae KP1054
ECL1002 chAmpC
C
C
B
B
B
ATM
ATM
MP
BP
BP
64
64
16
16
16
4
4
1
1
1
>20
>20
>20
>20
20
>20
20
1.25
1.25
0.3
>20
20
0.3
1.25
0.16
>20
>20
1.25
0.6
>20
10
0.6
2.5
1.25
>20
>20
2.5
2.5
0.3
IMP-26
NDM-1
VIM-1
0.16
Abbreviations: β-L, beta-lactam; PTV, potentiation target value; ATM, aztreonam; MP, meropenem; BP, biapenem
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Of the various substituted analogs 7, ortho-methoxy analog 7a displayed the best overall activity
against Enterobacteriaceae; however, its oral bioavailability was poor (< 5%). Given the
presence of a carboxylic acid handle, prodrugs were investigated. In practice, it proved to be
straightforward to obtain compounds displaying high oral bioavailability in the rat. These
prodrugs were prepared in a single step by alkylation of the carboxylic acid under basic
conditions. Table 7 shows the results of five prodrugs; double esters 8b and 8c, as well as
carbonate 8d, gave essentially quantitative oral bioavailability.
Table 7. Oral bioavailability of prodrugs of 7a in the rat.
S
S
S
S
N N
B
N N
B
HO
O
OMe
HO
O
OMe
OH
O
O
O
8a-e
7a
Compound
Prodrug
Oral Bioavailability*
~3%
7a
--
O
8a
8b
8c
8d
8e
52%
100%
100%
94%
O
O
O
O
O
O
O
O
O
64%
O
O
* Based on AUC relative to i.v. dosing of 7a; 50-75 mg/kg dose
-13-
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 14 of 49
1
2
3
4
Removal of the substituent next to boron.
5
6
7
8
9
To further broaden the scope of our findings, we sought to replace the methoxy-bearing carbon
atom of 7a with a nitrogen atom. As the synthesis of this target (9) was expected to be
challenging, we began with a model system lacking the substituent next to boron. The synthesis
of this compound (16) is shown in Scheme 3. Saponification of methyl 3-benzyloxy-4-bromo-2-
pyridinecarboxylate (10) followed by re-esterification affords tert-butyl ester 12. Palladium-
catalyzed installation of the vinyl group using potassium vinyltrifluoroborate affords 13, which is
subjected to iridium-catalyzed hydroboration to give boronate 14. Hydrogenolysis of the benzyl
group gives ester 15; the tert-butyl ester was then hydrolyzed using TFA in triethylsilane. The
crude product 16 was purified first as the free acid, and then was converted to the sodium salt for
repurification.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Scheme 3. Synthesis of Compound 16.
BF3- K+
Br
Br
Br
Boc2O
LiOH, THF
N
N
BnO
N
N
t-BuOH
BnO
TEA, PdCl2(dppf)
MeOH, H2O
BnO
BnO
CO2t-Bu
CO2t-Bu
CO2H
CO2Me
13
10
12
11
O
Pd/C, H2
MeOH
dppe, DCM
B
1) Et3SiH, TFA
2) 0.1N NaOH
Na+
O
B-
N
B
N
HO
HO
O
HO
O
N
PinBH, [IrCl(COD)]2
BnO
CO2- Na+
CO2t-Bu
CO2t-Bu
16
14
15
Upon successful synthesis of 16, we observed far better activity than was expected, given the
lack of a substituent thought to be required for potent inhibition. We immediately sought to
capitalize on our previous observations that the fluoro and methoxy substituents generally
afforded the best activity in the benzo series. The syntheses of these compounds are provided in
the Supporting Information.
-14-
ACS Paragon Plus Environment

Page 15 of 49
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Results of microbiological testing of these compounds (17b-c), as well as the ortho-unsubstituted
analog (17a), are shown in Table 8, in which the best performing compounds from earlier series
(4b and 7a) are included for comparison. The benefit of the ortho substituent (17b-c vs. 17a) is
readily apparent in many (but not all) isolates. Compounds 17b and 17c display excellent
potency against the Class A and Class C enzymes, and in addition these analogs show
remarkable activity against the difficult Class D (OXA-23, OXA-72) enzymes of Acinetobacter
baumannii. Further, 17b retains excellent activity against the Class B (metallo) enzymes NDM-
1 and VIM-2, with the only remaining deficiency being the metallo enzyme IMP-26.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 8. Activity of compounds unsubstituted next to boron.
H
S
S
S
N
S
MeS
N N
N N
B
N
O
B
B
B
N
B
O
O
F
HO
O
OMe
CO2H
HO
O
HO
O
R
HO
HO
CO2H
CO2H
CO2H
CO2H
4b
7a
16
17a: R = H
17b: R = OMe
17c: R = F
9
(not made)
Minimum concentration (µg/mL) of BLI needed to reduce
the MIC of β-lactam (β-L) to PTV
β-L PTV
alone
Organism
Strain
BL
Class β-L
4b
7a
16
17a
17b
17c
K. pneumoniae KP1009 CTX-M-15
E. coli ECM6625 SHV-5
E. coli ECM6619 TEM-10
K. pneumoniae KP1004 KPC-2
E. cloacae ECL1002 chAmpC
P. aeruginosa PAM 2035
A
ATM >128
ATM >128
4
4
4
4
4
4
8
8
1
1
1
0.3
0.15
1.25
1.25
2.5
2.5
0.3
2.5
1.25
10
2.5
5
20
20
0.6
0.6
0.6
0.6
0.08
0.16
0.16
0.6
2.5
2.5
>20
0.6
5
A
A
A
C
C
D
D
B
B
B
0.08
0.16
1.25
0.3
10
0.08
0.16
0.3
ATM 128
ATM >128
0.6
5
ATM
ATM
MP
MP
MP
BP
64
64
64
64
16
16
16
0.6
>20
20
1.25
0.6
AmpC
OXA-72
OXA-23
IMP-26
NDM-1
VIM-1
10
10
1.25
2.5
A. baumannii
A. baumannii
AB1053
AB1054
>20
>20
>20
>20
>20
>20
>20
5
0.6
>20
1.25
20
>20
1.25
10
>20
0.6
K. pneumoniae KP1160
K. pneumoniae KP1081
K. pneumoniae KP1054
0.3
0.3
0.6
BP
Abbreviations: β-L, beta-lactam; PTV, potentiation target value; ATM, aztreonam; MP, meropenem; BP, biapenem
Compound 17b was advanced to further evaluation, and displayed an excellent PK profile in the
rat, including 50% oral bioavailability. Further evaluation in the dog and monkey, however,
-15-
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 16 of 49
1
2
3
4
5
6
7
8
9
revealed significant oxidative deboronation to afford the long-lived metabolite 18 (Figure 4). The
extremely slow clearance of 18 was attributed to high serum protein binding (approaching 100%).
The generation of a substantial levels of a long-lived metabolite was viewed as a development risk,
and further efforts to advance 17b into development were suspended.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 4. Oxidative deboronation of 17b.
HO
[O]
B
HO
OMe
CO2H
HO
O
OMe
CO2H
18
17b
SAR of Analogs Designed to Address the Metabolic Liability of 17b
In order to block the oxidative deboronation of 17b, we sought to introduce steric congestion or
alter the electronic environment close to the boronic acid moiety. The results of testing several
analogs are shown in Table 9. Unfortunately, in most cases modification resulted in a significant
loss of potency and/or spectrum of inhibition. However, fused-cyclopropyl analog 25 (racemic)
represented a notable exception, retaining essentially all of the activity of 17b. This analog was
immediately tested for metabolite formation, and it was shown to be far more stable to metabolic
oxidation.
Table 9. Analogs of 17b designed to block oxidative deboronation.
F
HO
B
B
B
B
B
HO
O
HO
O
OMe
CO2H
HO
O
HO
O
OMe
CO2H
HO
O
OMe
CO2H
CO2H
CO2H
23
22
19
20
21
HO
B
B
B
B
B
HO
O
OMe
CO2H
HO
O
OMe
CO2H
HO
O
OMe
CO2H
HO
O
OMe
CO2H
HO
O
OMe
CO2H
27
28
26
25
24
-16-
ACS Paragon Plus Environment

Page 17 of 49
Journal of Medicinal Chemistry
1
2
3
4
Minimum concentration (µg/mL) of BLI needed to
reduce the MIC of β-L to PTV
5
6
β-L
alone
Organism
Strain
BL
Class β-L
PTV
19
20
21
22
23
7
8
9
K. pneumoniae KP1009 CTX-M-15
A
A
A
A
C
C
D
D
B
B
B
ATM >128
ATM >128
4
4
4
4
4
4
8
8
1
1
1
10
20
1.25
1.25
1.25
0.16
0.3
5
>20
20
>20
>20
>20
5
>20
>20
>20
10
E. coli
E. coli
KP308
EC302
SHV-5
TEM-10
KPC-2
ATM
128
20
>20
1.25
20
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
K. pneumoniae KP1004
E. cloacae
P. aeruginosa PAM 2035 AmpC
ATM >128
2.5
10
ECL1002 chAmpC
ATM
ATM
MP
MP
MP
BP
64
64
64
64
16
16
16
>20
1.25
>20
>20
>20
10
20
10
1.25
>20
>20
>20
1.25
5
>20
>20
>20
>20
>20
>20
A. baumannii
A. baumannii
AB1053 OXA-72
AB1054 OXA-23
20
2.5
5
>20
>20
>20
>20
K. pneumoniae KP1160
K. pneumoniae KP1081
K. pneumoniae KP1054
IMP-26
NDM-1
VIM-1
2.5
5
BP
>20
20
β-L
alone
Organism
Strain
BL
Class β-L
PTV
24
25
26
27
28
K. pneumoniae KP1009 CTX-M-15
A
A
A
A
C
C
D
D
B
B
B
ATM >128
ATM >128
4
4
4
4
4
4
8
8
1
1
1
20
20
0.3
0.3
0.3
0.16
0.3
5
>20
>20
>20
5
2.5
1.25
5
10
10
10
5
E. coli
E. coli
KP308
EC302
SHV-5
TEM-10
KPC-2
ATM
128
>20
5
K. pneumoniae KP1004
E. cloacae
P. aeruginosa PAM 2035 AmpC
ATM >128
1.25
2.5
10
ECL1002 chAmpC
ATM
ATM
MP
MP
MP
BP
64
64
64
64
16
16
16
10
10
10
10
5
20
10
A. baumannii
A. baumannii
AB1053 OXA-72
AB1054 OXA-23
10
0.6
1.25
5
>20
>20
>20
10
1.25
2.5
>20
2.5
20
10
10
20
10
5
K. pneumoniae KP1160
K. pneumoniae KP1081
K. pneumoniae KP1054
IMP-26
NDM-1
VIM-1
>20
2.5
2.5
0.6
0.08
BP
5
Abbreviations: β-L, beta-lactam; PTV, potentiation target value; ATM, aztreonam; MP, meropenem; BP, biapenem
Optimization of Cyclopropyl Analogs
Buoyed by these findings, we sought to prepare several close-in cyclopropyl analogs; the results
are shown in Table 10. Of the two enantiomers 29 and 30 of compound 25, the one with the
cyclopropyl group “down” as drawn (30) was consistently more potent. The same preference was
observed with other substituents ortho to the carboxylate, including H (32 vs. 31), F (35 vs. 34)
and replacement of the benzene ring by pyridine (37 vs. 36). Overall, the best overall activity was
observed with compounds 30 and 35. Of the two, 35 (QPX7728) was preferred due to having
lower human protein binding (89% vs. 96%).
-17-
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 18 of 49
1
2
3
4
5
6
7
8
9
Table 10. Optimization of cyclopropyl analogs.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
F
B
F
B
B
B
B
HO
O
HO
O
HO
O
OMe
CO2H
HO
O
OMe
CO2H
HO
O
OMe
CO2H
CO2H
CO2H
31
32
29
30
33
HO
HO
B
N
B
N
B
B
B
HO
O
HO
O
O
OMe
CO2H
HO
O
F
HO
O
F
CO2H
CO2H
CO2H
CO2H
36
37
38
34
35
Minimum concentration (µg/mL) of BLI needed to
reduce the MIC of β-lactam (β-L) to Potentiation
Target Value (PTV)
β-L
alone
Organism
Strain
BL
Class β-L
PTV
29
30
31
32
33
K. pneumoniae KP1009 CTX-M-15
A
A
A
A
C
C
D
D
B
B
B
ATM >128
ATM >128
ATM 128
ATM >128
ATM 64
ATM 64
4
4
4
4
4
4
8
8
1
1
1
0.6
0.3
0.3
0.08
0.08
0.16
0.3
10
5
0.6
0.3
0.16
0.16
0.3
0.6
5
1.25
1.25
5
E. coli
E. coli
KP308
EC302
SHV-5
TEM-10
KPC-2
0.6
5
K. pneumoniae KP1004
E. cloacae
P. aeruginosa PAM 2035 AmpC
0.16
1.25
2.5
0.16
10
1.25
10
ECL1002 chAmpC
2.5
2.5
20
10
A. baumannii
A. baumannii
AB1053 OXA-72
AB1054 OXA-23
MP
MP
MP
BP
BP
64
64
16
16
16
0.6
0.6
5
1.25
20
1.25
5
20
10
10
K. pneumoniae KP1160
K. pneumoniae KP1081
K. pneumoniae KP1054
IMP-26
NDM-1
VIM-1
>20
1.25
10
20
>20
2.5
10
0.6
0.3
0.6
0.16
0.3
0.04
β-L
Organism
Strain
BL
Class β-L
PTV
34
35
36
37
38
alone
K. pneumoniae KP1009 CTX-M-15
A
A
A
A
C
C
D
D
B
B
ATM >128
ATM >128
ATM 128
ATM >128
ATM 64
ATM 64
4
4
4
4
4
4
8
8
1
1
1.25
1.25
1.25
0.3
2.5
5
0.3
0.3
0.16
0.3
0.6
5
20
10
5
2.5
0.6
0.3
0.6
5
10
5
E. coli
E. coli
KP308
EC302
SHV-5
TEM-10
KPC-2
5
K. pneumoniae KP1004
E. cloacae
P. aeruginosa PAM 2035 AmpC
1.25
20
10
5
1.25
2.5
20
ECL1002 chAmpC
2.5
0.3
1.25
>20
2.5
A. baumannii
A. baumannii
AB1053 OXA-72
AB1054 OXA-23
MP
MP
MP
BP
64
64
16
16
5
0.6
1.25
2.5
0.6
2.5
10
10
10
>20
20
K. pneumoniae KP1160
K. pneumoniae KP1081
IMP-26
NDM-1
>20
5
2.5
0.6
-18-
ACS Paragon Plus Environment

Page 19 of 49
Journal of Medicinal Chemistry
1
2
3
4
5
K. pneumoniae KP1054
VIM-1
B
BP
16
1
>20
0.16
20
0.3
0.16
Abbreviations: β-L, beta-lactam; PTV, potentiation target value; ATM, aztreonam; MP, meropenem; BP, biapenem
6
7
8
9
The synthesis of compound 35 is shown in Scheme 4. 2-Bromo-5-fluorophenol (39) is converted
to tert-butylcarbonate 40; treatment with LDA effects deprotonation and acyl transfer, after which
the resulting phenol is re-protected, giving 41. The two protecting groups are replaced with a
single acetonide in 42, which is then subjected to a Heck reaction to introduce the acrylate side-
chain, giving 43. Bromination is followed by decarboxylative elimination to afford cis-vinyl
bromide 44. Palladium-mediated borylation with the (+)-pinanediol ester of tetrahydroxydiboron
affords cis-vinylboronate 45, which is subjected to palladium-catalyzed cyclopropanation with
diazomethane to afford 46. Separation of the two diastereomers by HPLC is followed by
sequential hydrolysis of the acetonide and pinanediol protecting groups. Single-crystal X-ray
structure determination of the diastereomer of 46 leading to the more active final product allowed
assignment of the cyclopropane stereochemistry as that shown in 35.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Scheme 4. Synthesis of Compound 35.
Br
Br
1) TFA
1) LDA, THF
Br
Boc2O
Br
O
F
2) acetone, TFA
TFAA, 65 °C
BocO
F
2) Boc2O, DMAP,
DCM
DMAP, DCM
CO2tBu
O
O
HO
F
BocO
F
39
40
41
42
B2((+)pinanediol)2,
COOH
HOOC
PdCl2(dppf), KOAc
Pd(OAc)2, P(o-toly)3
Et3N, DMF, 100 °C
1) Br2, CHCl3, 0 °C
Br
O
F
dioxane, 60 °C
O
F
2) Et3N, DMF, 0 °C
O
O
O
O
44
43
H
1) Column chromatography
2) 3N NaOH, dioxane, r.t.
H
O
O
CH2N2, Pd(OAc)2
THF, -10 °C to r.t.
B
B
B
O
F
O
F
HO
O
F
O
O
3) TFA, TES, i-BuB(OH)2
O
O
O
O
O
OH
45
46
35
-19-
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 20 of 49
1
2
3
4
5
6
7
Structural Biology of Compound 35
8
9
The complex crystal structures of 35 with KPC-2, NDM-1, OXA-48, and VIM-2 were obtained
through soaking and co-crystallization experiments (see Experimental Section). In the serine
enzymes KPC-2 and OXA-48, the catalytic serine residue is covalently bound to the boron atom
of the inhibitor. In the metallo enzymes NDM-1 and VIM-2, the catalytic water molecule is
covalently bound to the boron atom. The conformation of 35 is nearly identical in the various
structures, with the exception of the carboxylate moiety which is nearly co-planar in the metallo
enzymes and closer to orthogonal in the serine enzymes. While the polar interactions vary from
one structure another, in all cases the hydrophobic region of 35 is packed against lipophilic
(predominantly aromatic) sidechains.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 5. High resolution structures of 35 with multiple enzymes
KPC-2 (1.30 Å)
OXA-48 (1.80 Å)
NDM-1 (1.05 Å)
VIM-2 (1.20 Å)
-20-
ACS Paragon Plus Environment

Page 21 of 49
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Hydrogen atoms were added using ICM-Pro (Molsoft, San Diego).
Accession codes: 6V1J (KPC-2: 35), 6V1M (NDM-1: 35), 6V1O (OXA-48: 35), and 6V1P (VIM-2:
35)
Biochemical evaluation. QPX7728 displays “slow-binding” kinetics with serine beta-
lactamases, whereas it exhibits “fast on – fast off” kinetics with metallo enzymes (data not
shown; to be described in a future publication from these laboratories). Inhibition constants (Ki)
for various beta-lactamases purified from overexpressing recombinant E. coli strains, using
nitrocefin (NCF) or imipenem (IMI) as substrate, are shown in Table 11. Ki values were <100
nM for all enzymes except IMP-1. Compound 35 was clearly superior to the comparators
vaborbactam and avibactam against the Class D enzymes OXA-48 and OXA-23 of
Acinetobacter baumannii, and against the Class B metalloenzymes NDM-1, VIM-1 and IMP-1.
Table 11. Ki values (in µM) of beta-lactamase inhibition by 35, vaborbactam and avibactam
Enzyme
Class
35
Vaborbactam
Avibactam
CTX-M-14
TEM-10
SHV-12
KPC-2
A
A
A
A
C
D
D
B
B
B
0.00029 ± 0.00006
0.00066 ±0.00023
0.00074 ± 0.00021
0.0019 ± 0.0010
0.0085 ± 0.0024
0.00028 ± 0.00015
0.00074 ± 0.00028
0.0080 ± 0.0035
0.032 ± 0.014
0.033 ± 0.013
0.14 ± 0.014
0.021 ± 0.004
0.056 ± 0.015
0.035 ± 0.015
14 ± 5
0.00041 ± 0.00013
0.0014 ± 0.0004
0.00027 ± 0.00011
0.011 ± 0.003
0.010 ± 0.002
0.027 ± 0.015
1.7 ± 0.4
AmpC
OXA-48
OXA-23
VIM-1
>40
>40
>40
NDM-1*
IMP-1*
>40
>40
0.22 ± 0.05
>40
>40
-21-
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 22 of 49
1
2
3
4
Ki values were measured with imipenem as substrate.
5
6
7
8
Microbiology. Compound 35 (at a fixed concentration of 8 µg/mL) was evaluated in
combination with cefepime, ceftolozane and meropenem against panels of Enterobacterales,
including Klebsiella pneumoniae (n = 511), E. coli (n = 297), Enterobacter cloacae complex (n
= 88), and other organisms (n = 119); of these, 507 isolates expressed ESBL enzymes and 508
were carbapenem-resistant isolates. Compound 35 was also tested against carbapenem-resistant
Acinetobacter baumannii (n = 503) and Pseudomonas aeruginosa (n = 500). The MIC50/MIC90
values are shown in Table 12. Comparative data with the marketed agents ceftazidime-
avibactam and meropenem-vaborbactam are also shown. All 3 combinations with 35 were
highly effective against Enterobacterales producing ESBLs as well as strains expressing the KPC
and OXA-48 carbapenemases. In strains expressing metallo enzymes (NDM and VIM),
cefepime and meropenem potencies were enhanced to MIC90s of 1 and 2 µg/mL, respectively,
whereas ceftolozane achieved little benefit. This is attributed to a high ceftolozane hydrolyzing
activity of MBLs combined with a lower (compared to serine enzymes) inhibition of MBLs by
35. Against carbapenem-resistant A. baumannii, only meropenem potency was adequately
enhanced, whereas against P. aeruginosa, all 3 combinations achieved an MIC90 ≤8 µg/mL.
Studies in strains overexpressing and/or deficient in efflux pumps and porin modifications
showed that 35 is largely unaffected by these common resistance mechanisms (data not shown).
Interestingly, 35 displays weak antibacterial activity of its own, with a modal MIC of 16 µg/mL
against Enterobacterales (range <1 to ≥32 µg/mL); in general this activity is at much higher
concentration than the potentiated MICs, and therefore is unlikely to be a major contributor to
the activity of the combinations. Overall, the results of these studies demonstrate the broad
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
-22-
ACS Paragon Plus Environment

Page 23 of 49
Journal of Medicinal Chemistry
1
2
3
4
utility of 35 and that there may be benefit to more than one combination being available to the
5
6
clinician.
7
8
9
10
11
12
13
14
Table 12. Minimum inhibitory concentrations of beta-lactam antibiotics alone and in the
presence of Compound 35 (8 µg/mL).
MIC50/90 (µg/ml)
No. of
strains
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Organism
MP-VAB
CPM
CPM-35
CTLZN CTLZN-35
MP
MP-35
CTZDM-AVI
All Enterobacterales
1015 32/>32 ≤0.06/0.25
32/>32
0.125/8
1/>32
≤0.015/0.125 0.5/>32
0.06/>32
0.03/0.06
0.06/2
ESBLs (No CRE)* 507
CRE KPC 157
16/32 ≤0.015/0.0.03 8/>32
≤0.06/0.25 0.03/0.125 0.015/0.03
0.125/0.5
1/4
32/>32 ≤0.06/0.25
>32/>32
>32/>32
0.125/0.5
0.25/0.5
32/>32
32/>32
≤0.06/0.125
≤0.06/0.125
CRE OXA-48 150 >32/>32 0.125/0.5
Non-CP CRE* 51
0.5/1
1/2
16/>32
1/4
>32/>32 0.125/0.5
>32/>32
>32/>32
>32/>32
0.5/4
0.125/1
16/>32
8/32
8/16
32/>32
>32/>32
0.5/16
0.125/0.25
≤0.015/2
0.5/4
CRE MBL (NDM, VIM) 150 >32/>32
≤0.06/1
16/32
2/8
>32/>32
32/>32
2/4
>32/>32
32/>32
0.5/16
Carbapenem-res.
503 >32/>32
A. baumannii
P. aeruginosa
500
4/32
0.5/1
0.25/8
Abbreviations: CPM, cefepime; CTLZN, ceftolozane; MP, meropenem; CTZDM-AVI, ceftazidime-avibactam; MP-
VAB, meropenem-vaborbactam.
* Strains that do not produce carbapenem hydrolyzing enzymes
Selectivity of Protease Inhibition. Compound 35 was evaluated against a panel of common
mammalian serine and metallo proteases at concentrations up to 1000 µM. As shown in Table
13, only one enzyme had an IC50 below 100 µM, Cathepsin A. The significance of the activity
of 35 on this target has yet to be determined.
Table 13. IC50 Values (µM) for Inhibition of Mammalian Proteases by 35.
Protease
Type
IC50, µM
>1000
>100
Chymotrypsin
Thrombin
Serine
Serine
Serine
Serine
Serine
Serine
Serine
Neutrophil elastase
Trypsin
132
>1000
>1000
101
Plasmin
Elastase
Urokinase
>1000
-23-
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 24 of 49
1
2
3
4
5
6
7
8
9
Chymase
Serine
Serine
Serine
Serine
Serine
Serine
Metallo
Metallo
Metallo
Metallo
>1000
DPP7
>1000
>1000
>1000
666
DPP8
DPP9
Cathepsin G
Cathepsin A
ECE-1
1.1
431
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ACE-2
>1000
>1000
>1000
MMP-1
MMP-9
Pharmacokinetics. The pharmacokinetics of compound 35 by intravenous (IV) and oral (PO)
administration were evaluated in the rat at multiple doses (Table 14). The systemic exposure by
IV administration, as shown by the Cmax and AUC values, increased in a dose proportional
manner. Overall the parameters are similar to those of most β-lactam antibiotics, evidencing
high Cmax and AUC, short half-life and low volume of distribution. Plasma protein binding in
the rat is 85%.
Compound 35 also displays oral bioavailability (F%) in fasted rats, with values ranging between
43-53% at doses of 30-100 mg/kg, whereas it declined to 24-28% at higher doses. Given its
polarity (logD7.4 -2.85), we speculate that active transport may be involved in oral uptake, which
may be saturated at higher doses. The compound was well tolerated at all doses.
Table 14. Pharmacokinetics of 35 in rats following intravenous and oral administration.
Dose
(mg/kg)
AUC
(mg*hr/L)
CL
(L/hr/kg)
Cmax
(mg/L)
Half-Life
(hr)
F%
Route
IV
IV
IV
PO
PO
PO
PO
10
100
300
30
100
300
1000
35.2 ± 8.2
221.0± 46.7
608.1 ± 15.3
34.0± 7.3
89.7 ± 10.4
176.8 ± 28.0
504.2 ± 141.7
0.29 ± 0.06
0.47 ± 0.16
0.49 ± 0.01
0.91 ± 0.18
1.11 ± 0.13
1.71 ± 0.28
2.05 ± 0.54
26.1 ± 3.5
208.3 ± 6.3
543.8 ± 22.3
8.6 ± 1.5
1.25 ± 0.36
1.46 ± 0.72
1.18 ± 0.41
4.40 ± 1.23
3.23 ± 0.61
3.65 ± 0.25
4.00 ± 1.33
--
--
--
53.2 ± 3.7
42.6 ± 4.8
28.0 ± 4.4
24.3 ± 7.5
28.8 ± 2.5
52.0 ± 5.0
90.4 ± 8.7
-24-
ACS Paragon Plus Environment

Page 25 of 49
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Efficacy in infection models. Compound 35 was evaluated in a 24 h neutropenic mouse thigh
infection model against Klebsiella pneumoniae strain KP1244 (Figure 6). This strain expresses
KPC-3, SHV-11 and SHV-12, is resistant to carbapenems, with an MIC to meropenem of >64
µg/mL; in the presence of 8 µg/mL of 35, the MIC to meropenem is 0.25 µg/mL. The doses
shown of both meropenem and QPX7728 were administered every 2 hours for 24 hours (12
doses of the designated amount per day). In this study, meropenem at a dose of 300 mg/kg given
every 2 hours fails to control the infection, whereas the co-administration of 0.5-1 mg/kg of 35
affords a static effect, and 10 mg/kg achieves a >1 log reduction in CFU/thigh (relative to the
start of treatment).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 6. Activity of meropenem alone or in combination with Compound 35 against K.
pneumoniae KP1244 in a 24 h neutropenic mouse thigh infection model.
1.0
0.5
0.62
-0.03
0.0
-0.42
-0.88
-0.5
-1.0
-1.5
-2.0
-2.5
-1.15
-1.66
-2.26
Meropenem
300 mg/kg
Meropenem
Meropenem
Meropenem
Meropenem
Meropenem
Meropenem
300 mg/kg + 300 mg/kg + 300 mg/kg + 300 mg/kg + 300 mg/kg + 300 mg/kg +
Compound 35 Compound 35 Compound 35 Compound 35 Compound 35 Compound 35
0.5 mg/kg
1.0 mg/kg
5.0 mg/kg
10 mg/kg
25 mg/kg
50 mg/kg
-25-
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 26 of 49
1
2
3
4
5
6
7
8
9
Safety. Compound 35 was studied in a 7-day pilot toxicology study at daily doses of 30, 100 and
300 mg/kg in rats (5 male, 5 female per dose level) administered by IV infusion. No changes
were observed (tissue histology and clinical chemistry).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Conclusions
This Drug Annotation describes the path from the narrow-spectrum first-generation boronic acid
BLI vaborbactam to a remarkably potent and broad-spectrum agent 35 (Figure 7). The amide
series represented by 4b displays potent inhibition of serine enzymes of Classes A and C, but has
only weak activity against Class B (metallo) enzymes. Replacement of the amide linkage by a
thio linkage, as exemplified in 7a, achieves much-improved potency against Class B enzymes.
As a model system for introduction of a heteroatom into the aryl ring, compounds unsubstituted
next to the boron atom were prepared. Compound 17b represented a breakthrough in terms of
potency and spectrum. Susceptibility of 17b to metabolism precipitated work to identify
substituents that would block metabolism while retaining all other desirable properties,
culminating in 35 (QPX7728).
Figure 7. Evolution of beta-lactamase inhibitors leading to compound 35.
H
H
S
N
S
N
MeS
O
B
N
N
B
B
B
S
O
HO
B
CO2H
O
F
O
OMe HO
CO2H
O
OMe
CO2H
17b
HO
O
F
HO
HO
O
CO2H
CO2H
4b
7a
35
vaborbactam
Compound 35 displays potent activity against the vast majority of beta-lactamase enzymes,
including not only the serine enzymes of Classes A and C, but also metallo enzymes such as
NDM-1 and the difficult Class D (OXA) enzymes of Acinetobacter baumannii. In vivo, 35
displays pharmacokinetics in the rat similar to beta-lactam antibiotics and has good oral
-26-
ACS Paragon Plus Environment

Page 27 of 49
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
bioavailability without the need for a prodrug. Efficacy in a mouse infection model in
combination with meropenem against a strain of Klebsiella pneumoniae expressing multiple
beta-lactamase enzymes was established. Selectivity for the beta-lactamase target was
established by screening against a panel of mammalian proteases. Preliminary toxicology
studies in rats showed no adverse findings. Compound 35 has been advanced to late-stage pre-
clinical development to support IND filing.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
EXPERIMENTAL SECTION
General methods. Unless otherwise noted, solvents and reagents were obtained from commercial
suppliers and were used without further purification. All reactions involving air- or moisture-
sensitive reagents were performed under an argon atmosphere. Thin-layer chromatography was
performed on Merck silica gel 60 plates coated with 0.25 mm layer with fluorescent indicator.
Components were visualized by UV light (λ = 254 and 366 nm) and iodine vapors. Column
chromatography was conducted either under medium pressure on silica gel (Merck silica gel 40–
63 μm) or on prepacked silica gel cartridges (Isco) on an Isco system. Purity and low-resolution
mass spectrometry (MS) data were determined on a ThermoFinnigan Surveyor HPLC and
ThermoFinnigan LCQ Advantage instruments equipped with ESI, using an Agilent Eclipse Plus
C18 (4.6 mm × 150 mm, 5 μm) column. Mobile phase A was 0.1% trifluoroacetic acid in water
and mobile phase B was 0.1% trifluoroacetic acid in acetonitrile. The following conditions were
used: a gradient from 10% to 90% B in 6 min and held at 90% B for 1.5 min; UV detection at 220
and 254 nm; a flow rate of 2 mL/min; an injection volume of 10μL; full scan, mass range from
100 to 1000 amu. Masses are given as an m/z ratio. 1H NMR spectra were acquired in the indicated
-27-
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 28 of 49
1
2
3
4
5
6
7
8
9
solvents on a Bruker NMR spectrometer (Avance 400 MHz for 1H). Data are reported as following:
chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, bs = broad singlet, dd
= doublet of doublet, td = triplet of doublet, m = multiplet), coupling constants, and number of
protons. The purity of all compounds by HPLC was ≥95% except where noted in the Supporting
Information.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
All experiments utilizing animals were conducted under protocols approved by the Qpex
Institutional Animal Care and Use Committee (IACUC).
Disodium salt of 4,4-Dihydroxy-5-oxa-8-aza-4-boranuidabicyclo[4.4.0]deca-1(6),7,9-triene-
7-carboxylate (16)
3-(Benzyloxy)-4-bromopicolinic acid (11):
To a solution of compound 1026 (7 g, 21.73 mmol, 1.0 eq) in THF:MeOH:H2O (2:2:1, 100
mL) was added LiOH (1.37 g, 32.6 mmol, 1.5 eq) at room temperature. The mixture was stirred at
room temperature for 1.5 hours. The solvent was removed under reduced pressure. The residue
was re-dissolved in water, adjusted pH to 1 with 1N HCl, and extracted with ethyl acetate. The
organics were dried over Na2SO4, and concentrated to yield residue, which was purified by column
1
chromatography (DCM:MeOH = 10:1) to give compound 11 (4.85 g, 72%). H NMR (CD3OD,
300 MHz): δ 8.21 (d, J = 4.8 Hz, 1H), 7.88 (d, J = 4.8 Hz, 1H), 7.55-7.36 (m, 5H), 5.15 (s, 2H).
tert-Butyl 3-(benzyloxy)-4-bromopicolinate (12):
To a solution of compound 11 (4.8 g, 15.58 mmol, 1.0 eq) in tBuOH (150 mL) was added
DMAP (195 mg, 1.596 mmol, 0.1 eq) and Boc2O (10.2 g, 46.74 mmol, 3 eq). The mixture was
-28-
ACS Paragon Plus Environment

Page 29 of 49
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
o
stirred overnight at 60 C. The solvent was removed under reduced pressure, and the resulting
residue was purified by column chromatography (ethyl acetate:hexanes = 1:3) to give compound
12 (4.9 g, 86%). 1H NMR (CDCl3, 300 MHz): δ 8.23 (d, J = 4.8 Hz, 1H), 7.63 (d, J = 5.1 Hz, 1H),
7.55-7.38 (m, 5H), 5.13 (s, 2H), 1.58-1.57 (m, 9H).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
tert-Butyl 3-(benzyloxy)-4-vinylpicolinate (13):
To a solution of compound 12 (4.9 g, 13.45 mmol, 1.0 eq) in isopropyl alcohol (250 mL)
was added PdCl2(dppf) (878 mg, 1.08 mmol, 0.08 eq), potassium vinyltrifluroborate (4.5 g, 33.59
mmol, 2.5 eq), and triethylamine (2.04 g, 20.17 mmol, 1.5 eq). The mixture was purged with N2
and stirred at 86 oC for 3 hours. The solvent was removed under reduced pressure, and the resulting
residue was purified by column chromatography (ethyl acetate:hexanes = 1:3) to give compound
13 (3.77 g, 90%). 1H NMR(CDCl3,300 MHz): δ 8.38 (d, 1H), 7.52-7.28 (m, 6H), 6.95 (dd, 1H), 5.
95 (dd, 1H), 5.56 (dd, 1H), 5.01 (s, 2H), 1.6-1.58 (m, 9H).
tert-Butyl 3-(benzyloxy)-4-(2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)ethyl)picolinate (14):
To a solution of pinacolborane (1.86 g, 14.54 mmol, 1.2 eq), [IrCl(COD)]2 (163 mg, 0.263
mmol, 0.02 eq) and dppe (191 mg, 0.479 mmol, 0.04 eq) in DCM (100 mL) was added compound
13 (3.77 g, 12.11 mmol, 1.0 eq) in DCM (50 mL). The mixture was purged with N2 and stirred
overnight at room temperature. After addition of MeOH (10 mL), the solvent was removed under
reduced pressure, and the resulting residue was purified by column chromatography (ethyl
1
acetate:hexanes = 1:3) to give compound 14 (2.58 g, 48%). H NMR (CDCl3,300 MHz): δ 8.36
(d, 1H), 7.51-7.29 (m, 6H), 5.03 (s, 2H), 2.80 (t, 2H), 1.59 (s, 9H). 1.29-1.18 (m, 14H).
-29-
ACS Paragon Plus Environment