Lead optimization of 2-hydroxymethyl imidazoles as non-hydroxamate LpxC inhibitors: Discovery of TP0586532

Article abstract of DOI:10.1016/j.bmc.2020.115964

Infectious diseases caused by resistant Gram-negative bacteria have become a serious problem, and the development of therapeutic drugs with a novel mechanism of action and that do not exhibit cross-resistance with existing drugs has been earnestly desired. UDP-3-O-acyl-N-acetylglucosamine deacetylase (LpxC) is a drug target that has been studied for a long time. However, no LpxC inhibitors are available on the market at present. In this study, we sought to create a new antibacterial agent without a hydroxamate moiety, which is a common component of the major LpxC inhibitors that have been reported to date and that may cause toxicity. As a result, a development candidate, TP0586532, was created that is effective against carbapenem-resistant Klebsiella pneumoniae and does not pose a cardiovascular risk.

Full text of DOI:10.1016/j.bmc.2020.115964

Bioorg. Med. Chem. 30 (2021) 115964
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Lead optimization of 2-hydroxymethyl imidazoles as non-hydroxamate
LpxC inhibitors: Discovery of TP0586532
Fumihito Ushiyama*, Hajime Takashima, Yohei Matsuda, Yuya Ogata, Naoki Sasamoto,
Risa Kurimoto-Tsuruta, Kaori Ueki, Nozomi Tanaka-Yamamoto, Mayumi Endo, Masashi Mima,
Kiyoko Fujita, Iichiro Takata, Satoshi Tsuji, Haruhiro Yamashita, Hirotoshi Okumura,
Katsumasa Otake, Hiroyuki Sugiyama
Taisho Pharmaceutical Co., Ltd, 1-403 Yoshino-Cho, Kita-Ku, Saitama 331-9530, Japan
A R T I C L E I N F O
A B S T R A C T
Keywords:
Infectious diseases caused by resistant Gram-negative bacteria have become a serious problem, and the devel-
opment of therapeutic drugs with a novel mechanism of action and that do not exhibit cross-resistance with
existing drugs has been earnestly desired. UDP-3-O-acyl-N-acetylglucosamine deacetylase (LpxC) is a drug target
that has been studied for a long time. However, no LpxC inhibitors are available on the market at present. In this
study, we sought to create a new antibacterial agent without a hydroxamate moiety, which is a common
component of the major LpxC inhibitors that have been reported to date and that may cause toxicity. As a result,
a development candidate, TP0586532, was created that is effective against carbapenem-resistant Klebsiella
pneumoniae and does not pose a cardiovascular risk.
LpxC
Non-hydroxamate
Antibacterial agent
Gram-negative bacteria
carbapenem-resistant Enterobacteriaceae
Lead optimization
1. Introduction
dependent deacetylase that catalyzes the essential step in the biosyn-
thesis of lipid A, which is a component of lipopolysaccharide (LPS) in
In recent years, drug-resistant Gram-negative bacteria have been
recognized as a global threat.1,2 The World Health Organization (WHO)
published the Global Action Plan on Antimicrobial Resistance in 2015;
in 2017, they published a list of drug-resistant bacteria that require the
immediate development of new antimicrobial agents.3,4 Until now,
β-lactam, quinolone, and aminoglycoside drugs, etc., have been used as
effective drugs for the treatment of Gram-negative bacterial infections.
However, a large number of Enterobacteriaceae such as Klebsiella
pneumoniae and Escherichia coli resistant to these drugs have been re-
ported in clinical practice.5,6 Among them, carbapenem-resistant
Enterobacteriaceae (CRE) exhibit strong resistance to many drugs
including carbapenem antibiotics, which are regarded as important
agents in the treatment of infectious diseases.7 CRE infection has a high
mortality rate, and its incidence is increasing globally.8 CRE infections
are presently treated with combinations of antimicrobial agents to
which the strain is susceptible, but treatment has been difficult in some
cases.9 Therefore, the development of therapeutic agents with novel
mechanisms of action is desired.10
Gram-negative bacteria.11 LpxC is a well-validated pharmacological
target, and much effort has been devoted to the creation of LpxC in-
hibitors over the past 20 years.12 However, there are still no inhibitors
of LpxC on the market.13 Previous LpxC inhibitors contained
hydroxamic acid at the zinc coordination site.13 However, this structure
often increases the risk of toxicity.14,15 ACHN-975 (Achaogen)16 and
RC-01 (FUJIFILM Toyama Chemical),17 which were leading LpxC in-
hibitors that both contained a hydroxamic acid moiety, reached the
clinical trial stage, but the development of both compounds was
terminated in Phase 1.18,19 (see Fig. 1).
Therefore, we sought to create LpxC inhibitors that do not contain a
hydroxamic acid—and consequently an increased risk of toxicity.14,15
The creation of a novel chemical class of LpxC inhibitors different from
conventional LpxC inhibitors would enable new treatment options. Our
fragment-based drug discovery (FBDD) method showed that 2-(1S-
hydroxyethyl)-imidazole derivatives could be an alternative to com-
pounds containing a hydroxamic acid moiety.20 Among them, com-
pound 1 had an attractive antibacterial activity against E. coli of 0.5 μg/
UDP-3-O-acyl-N-acetylglucosamine deacetylase (LpxC) is a zinc-
mL.20 However, compound 1 also had a high protein binding ability
* Corresponding author.
E-mail address: f-ushiyama@taisho.co.jp (F. Ushiyama).
https://doi.org/10.1016/j.bmc.2020.115964
Received 17 November 2020; Received in revised form 14 December 2020; Accepted 16 December 2020
Available online 29 December 2020
0968-0896/© 2020 Elsevier Ltd. All rights reserved.

F. Ushiyama et al.
Bioorganic & Medicinal Chemistry 30 (2021) 115964
(Table 1) and was not expected to exhibit in vivo efficacy. Therefore, we
sought an approach that would reduce the human protein binding of
compound 1 while maintaining its antibacterial activity against
Enterobacteriaceae. In practice, we explored and optimized the SAR of
the tail site located in the solvent-exposed region, the introduction of a
polar group adjacent to the imidazole moiety, and the hydrophobic
tunnel site. Compounds with reduced protein binding were evaluated in
vivo using efficacy tests and safety pharmacology tests involving the
guinea pig cardiovascular system under anesthesia. Based on the results
of the above examinations, TP0586532, which contains a carboxy group
at the termination of the tail site, was selected and profiled.
inoculation. By contrast, compound 4, in which the positions of the
benzene ring and the acetylene moiety of lead compound 1 were
exchanged, had almost the same antibacterial activity as lead compound
1, while a clear reduction in protein binding was observed. To verify the
effect of the ethynyl-benzene replacement, cocrystals of compound 5 (a
racemate of lead compound 1)20 and compound 4 were compared
(Fig. 3). This examination confirmed that the amino acid residue of
Ile197 was considerably more induced-fit in compound 4 than in com-
pound 5 and that this residue was closer to the hydrophobic tunnel re-
gion. A similar phenomenon has been reported for LpxC inhibitors 23,24
;
this induced-fit increases the spatial tolerance of the hydrophobic tunnel
exit on the tail side, making it easier to deploy a wide range of tail types.
2. Results and discussion
2.3. Second optimization based on compound 4
2.1. Design strategy
Further prioritization was made in consideration of the results shown
in Fig. 3 and the reduction in protein binding enabled by the conversion
of lead compound 1 to compound 4. Namely, we decided to investigate
the derivatives of compound 4, in which the hydrophobic tunnel is
composed of the sequence, beginning from the tail side, “tail-acetylene-
phenyl”. The results of the optimization of the tail and part B (corre-
sponding to the isoxazole site) are shown in Table 2.
In the present study, compound 1, which was discovered in a pre-
viously reported study,20 was used as the lead compound in an effort to
create novel non-hydroxamate LpxC inhibitors. The most important
characteristic of lead compound 1 is that the zinc coordination site is
constructed using 2-(1S-hydroxyethyl)-imidazole. Specifically, com-
pound 1 coordinates with zinc at a secondary hydroxy group and the
nitrogen atom of imidazole. On the other hand, most LpxC inhibitors
reported to date, such as CHIR-09021 (Fig. 2), contain a hydroxamate
moiety at this site, and concerns regarding mutagenicity14,15 exist
because of their distinctive structures.
Further examination of the tail site revealed that the introduction of
the rigid parts tended to favor antibacterial activity over flexible alkyl
chains such as compound 4. In fact, cyclopropylamine 6 showed an
equivalent or better antibacterial activity against E. coli and
K. pneumoniae than compound 4. The serum protein binding to human
and mouse proteins was also reduced to 93.0% and 90.1%, respectively.
Furthermore, in a murine systemic infection model, the survival rate at
7 days was 67%, indicating an in vivo efficacy. Also, compounds 7 and 8,
in which bicyclopentylamine and cyclobutyl alcohol were respectively
introduced into the tail, had stronger antibacterial activities against
E. coli and K. pneumoniae than compound 6. However, a reduction in
serum protein binding was not observed, and no in vivo efficacy was
confirmed. Compound 9, which contained azabicyclohexane at the tail
site, had almost the same antibacterial activity and protein binding
ability as compound 6 but was inferior in terms of in vivo efficacy when
evaluated using a murine systemic infection model.
Similar to previously reported LpxC inhibitors, compound 1 fills the
target hydrophobic tunnel site of LpxC with a linear lipophilic acetylene
and a benzene ring. Also, a space exists in the solvent-exposed area of the
molecule where a tail with adjustable physicochemical properties could
be introduced.
Lead compound 1 exerted antibacterial activities against E. coli ATCC
25922 and K. pneumoniae ATCC 13883 at concentrations of 0.5 μg/mL
and 2 μg/mL, respectively, but the serum protein binding ability for both
human and mouse proteins was >99.5% (Table 1). Thus, we attempted
to create a novel non-hydroxamate LpxC inhibitor with in vivo efficacy
by reducing the protein binding while maintaining or improving the
antibacterial activity.
In addition, derivatives in which the nitrogen and oxygen atoms of
isoxazole were exchanged were also synthesized and evaluated. When
compounds 6 and 10 (NO-replacement form) were compared, com-
pound 10 showed reduced protein binding, but both its enzyme inhib-
itory activity and its antibacterial activity were weakened. The other
NO-replacement compounds (11 and 12) were found to have protein
binding abilities that were lower than or equal to those of compounds 7
and 9, which had the same respective tail types. Compounds 11 and 12
also had improved antibacterial activities and higher protein binding
when compared with compound 10. Although compound 12 had the
strongest antibacterial activity amongst the NO-replacements, its in vivo
efficacy was inferior to that of compound 6 when examined using a
murine systemic infection model.
2.2. First optimization based on compound 1
Based on information for lead compound 1, the tail and the hydro-
phobic tunnel site (part A) were examined (Table 1).
We started replacing the tail part of 1 with hydrophilic part to reduce
protein binding. Compound 2, which contained hydroxypropylazetidine
indicated considerable reduction of the protein binding; however both
the enzyme inhibitory and antibacterial activities against E. coli and
K. pneumoniae were also attenuated, as was the case with other reported
LpxC inhibitors.22 Piperazine 3 had a stronger antibacterial activity
against K. pneumoniae than lead compound 1; a reduction in protein
binding was also confirmed. Even though, in a murine systemic infection
model caused by infection with K. pneumoniae 4102, mice treated with
25 mg/kg of compound 3 only had a 17% survival rate at 7 days after
As described above, compound 6, which was one of the most
promising compounds, had the strongest in vivo efficacy in the murine
HN
O
O
NH2
O
O
H
H
N
N
N
OH
N
OH
H
O
HO
HO
ACHN-975
RC-01
OH
Fig. 1. LpxC inhibitors that advanced to the clinical stage of development.
2

F. Ushiyama et al.
Bioorganic & Medicinal Chemistry 30 (2021) 115964
systemic infection model (Table 2). However, the maximum tolerated
dose in mice, as determined using a single intravenous administration
study of compound 6 in mice, was 50 mg/kg, resulting in a narrow
margin of efficacy. Also, convulsive symptoms in mice were confirmed,
and there were concerns about the effects of compound 6 on the central
nervous system. Therefore, studies were conducted to further improve
drug efficacy, reduce central transferability, and minimize off-target
effects. Cocrystal information for compound 4 suggested that a substit-
uent could be introduced into the imidazole-adjacent position. As shown
in Fig. 2, a space where a side chain could be extended is present in lead
compound 1. In contrast, it would be difficult to extend a side chain
while maintaining the existing binding mode on the back side of the
paper. Such side chains would likely form new interactions with amino
acid residues or via water. In addition, the introduction of polar groups
was expected to reduce protein binding. Thus, we next introduced
various side chains to the adjacent position of imidazole, selected the
most promising aminomethyl group, and performed combination syn-
thesis with the attractive tails that had been obtained so far. Further-
more, we actively introduced neutral and acidic components into the tail
site and searched for promising compounds (Table 3).
Fig. 2. Schematic structures of CHIR-090 and lead compound 1.
against K. pneumoniae, from 4 μg/mL to 16 μg/mL. From among the
compounds that had been obtained, compounds that were expected to
have an in vivo efficacy were selected and further narrowed down using a
murine systemic infection model (data not shown). Then, the com-
pounds were evaluated in a murine lung infection model (K. pneumoniae
ATCC BAA-2343, a carbapenem-resistant strain), which is a more clin-
ically oriented in vivo evaluation system. The changes in the number of
viable bacteria in the lung at a dose of 50 mg/kg were determined; as a
result, a decrease of 1 log or more was confirmed for all the evaluated
compounds in Table 3. Furthermore, the murine maximum tolerated
dose (MTD) test was performed using intravenous administrations to
identify compounds with a tolerable dose of 200 mg/kg. From the above
results, four compounds were selected as promising drug candidates:
compounds 15, 17, 18, and 19 (TP0586532).
Compound 13 had a weaker antibacterial activity than compound 6,
which had the same tail, because of a structural conversion introduced
by an aminomethyl group. Compounds 14 and 15 had comparable or
slightly attenuated antibacterial activities compared with compounds 7
and 8 listed in Table 2, but their human protein binding abilities were
improved by 5% or more. Furthermore, compound 17 had a human
protein binding of less than 80% even though it had no basic nitrogen
atom at its tail site. Among other aminomethyl derivatives, a number of
compounds, such as compound 18, with a human protein binding of less
than 80% were obtained. In the hopes of reducing protein binding and
avoiding the toxicity seen in compound 6, a carboxy group, which is an
acidic functional group, was introduced at the tail site in compound 19
(TP0586532), which did not have an aminomethyl group. Compound
19 (TP0586532) had the same antibacterial activity as that of the
compound containing azabicyclohexane, and its human and mouse
protein binding abilities were both less than 80%. We next synthesized
compound 20, which contained an aminomethyl group introduced into
the imidazole adjacent position of compound 19 (TP0586532), but the
antibacterial activity of this compound was attenuated, especially
2.4. Cardiovascular study
Compounds 15, 17, 18, and 19 (TP0586532) were subjected to a
safety pharmacology test using the guinea pig cardiovascular system
under anesthesia to further narrow down the compounds. The admin-
istration of compounds 15, 17, and 18, all of which had an aminomethyl
Table 1
Exploration of tails and part A components.
Cpd
Tail
A
LpxC IC50
0.031
(μ
M)a
Ec MICb
0.5
(μg/mL)
Kpc MIC (
2
μg/mL)
PBd (%) Human Mouse
1 (Lead)
>99.5
>99.5
2
3
4
0.378
0.071
0.051
2
8
73.9
83.5
0.25
1
0.5
2
91.0
91.7
96.9
95.1
a
Fluorescamine method.
b
c
d
E. coli ATCC 25922, MIC: minimum inhibitory concentration.
K. pneumoniae ATCC 13883.
Serum protein binding.
3

F. Ushiyama et al.
Bioorganic & Medicinal Chemistry 30 (2021) 115964
the results of the hERG channel assay, in which compound 19
(TP0586532) showed the weakest activity (IC50 >1000
μM) among
these four compounds. The intravenous infusion of ACHN-975, an LpxC
inhibitor, at a dose of 75 mg/kg in a phase 1 clinical trial reportedly
induced a marked decrease in systolic blood pressure with an increase in
exposure in a cardiovascular safety test using anesthetized rats.27 By
contrast, although a rat model was not used, a decrease in blood pressure
was not observed even after the administration of TP0586532 at a dose
of 150 mg/kg in an anesthetized guinea pig model. These results suggest
that the cardiovascular risk of TP0586532 is significantly less than that
of ACHN-975, although the test conditions differed. Based on the results
that had been obtained, compound 19 (TP0586532) was selected as the
best compound with a low cardiovascular risk.
2.5. Safety profile of TP0586532
Next, genotoxicity tests were performed to further evaluate the
safety of TP0586532. Both a mouse lymphoma assay and an in vivo
micronucleus test were negative. Furthermore, a 4-day repeated dose
study in rats and a 14-day repeated dose study in monkeys showed that
the MTDs were 400 mg/kg (SD rat) and 400 mg/kg or more (cynomolgus
monkey), respectively.
Fig. 3. Structural superposition of 4 (cyan carbons) and 5 (gray carbons) using
MOE software.25 X-ray cocrystal structures of 4 and 5 in Pseudomonas aerugi-
nosa LpxC are shown (PDB ID: 7DEM and 7DEL). The key residue (Ile197) is
represented in stick format. The catalytic zinc is shown as an orange sphere. The
homology of the amino acid residues located in the hydrophobic tunnel region
is highly retained between P. aeruginosa, E. coli, and K. pneumoniae.26.
2.6. X-ray analysis of TP0586532
group adjacent to imidazole, induced QT prolongation and reduced both
the heart rate and the blood pressure of the guinea pigs. The effects were
especially strong for compounds 17 and 18 containing azabicyclohex-
ane. On the other hand, compound 19 (TP0586532), which did not have
an aminomethyl group, produced no remarkable changes in cardiac
parameters (Fig. 4 and Table 4). The effect on QTc was consistent with
A cocrystal of TP0586532 and P. aeruginosa LpxC is shown in Fig. 5.
TP0586532 is chelated with a zinc atom in the same manner as the 2-
(1S-hydroxymethyl)-imidazole derivative that was shown in Fig. 3, and
the nitrogen atom of azabicyclohexane creates
a water-bridged
hydrogen bond with the main chain of Val211. Moreover, the
condensed ring of this azabicyclohexane is located along the wall of the
Table 2
Exploration of tails and part B components.
Cpd
Tail
B
LpxC IC50
0.046e
(
μM)
Eca MIC (
0.5
μ
g/mL)
Kpb MIC (
1
μg/mL)
PBc (%) Human Mouse
Systemic infection (%)d
67
6
93.0
90.1
7
0.049e
0.015f
0.071f
0.25
0.12
0.5
0.5
97.3
96.6
0
8
0.25
0.5
98.3
96.5
0
9g
93.5
91.8
33
10
0.193e
0.065e
0.097f
1
4
88.5
85.0
NTh
NT
17
11
0.5
0.5
2
96.5
93.1
12g
0.5
92.4
92.3
a
E. coli ATCC 25922.
b
c
d
e
f
K. pneumoniae ATCC 13883.
Serum protein binding.
Murine systemic infection model: survival rate at day 7, K. pneumoniae 4102, dose: 25 mg/kg.
LCMS method.
Fluorescamine method.
Mixture of diastereomers at the tail moiety.
Not tested.
g
h
4

F. Ushiyama et al.
Bioorganic & Medicinal Chemistry 30 (2021) 115964
Table 3
Exploration of tails and part R components.
Cpd
13
Tail
R
LpxC IC50
M)
0.282f
Eca MIC (
mL)
μg/
Kpb MIC (
mL)
μg/
PBc (%) Human
Mouse
Lung infection (log10CFU/
lung)d
Mouse MTD (mg/
kg)e
(
μ
2
8
NTh
NT
NT
NT
14
0.279f
0.5
2
89.7
91.6
ꢀ 2.48
100
15
16
<0.037f
0.25
1
1
2
92.6
93.0
ꢀ 1.99
ꢀ 2.84
≥200
0.039 g
90.5
91.0
100
17
NDi
1
1
2
4
72.5
82.0
ꢀ 2.47
ꢀ 1.19
≥200
≥200
18
19
NT
78.8
84.9
0.101g
NT
2
4
4
77.7
75.9
NT
NT
ꢀ 1.63
≥200
(TP0586532)
20
16
NT
NT
a
b
c
E. coli ATCC 25922.
K. pneumoniae ATCC 13883.
Serum protein binding.
d
Murine lung infection model: means of logarithm changes in the bacterial burden in the lung relative to the start of therapy (n = 5), K. pneumoniae ATCC BAA-2343,
dose: 50 mg/kg.
e
Maximum tolerated dose during single intravenous administration, ICR mouse (n = 2).
f
LCMS method.
g
h
Fluorescamine method.
Not tested.
i
Not determined.
tail-side exit of the hydrophobic tunnel, indicating the superiority of this
characteristic tail type.
4. Conclusion
We conducted lead optimization starting from a non-hydroxamate
lead compound 1 with the aim of creating a novel LpxC inhibitor. The
use of protein binding as an index was useful for guiding improvements
in the in vivo efficacy while maintaining the antibacterial activity.
Structural transformation was performed from the tail site to the adja-
cent position of the imidazole, and promising compounds were identi-
fied using cardiovascular studies in anesthetized guinea pigs. These
efforts resulted in the introduction of a carboxy group at the tail site,
rather than the introduction of a primary amine to the adjacent position
of the imidazole. Finally, we identified TP0586532 as a compound with
a low cardiovascular risk that was effective against K. pneumoniae,
including resistant strains. Further detailed evaluations and results will
be reported in due course.
3. Chemistry
The synthesis of compounds 1–20 is depicted in Schemes 1–4. Key
intermediate 21 was coupled to aryl iodide 24 using a Sonogashira
coupling reaction to yield compound 2. Piperazine derivative 3 was
prepared from compound 23 using a palladium-catalyzed Buchwald-
Hartwig amination (Scheme 1).
Isoxazole-phenyl-ethynyl type compounds 4, 6–8, and intermediate
37 were prepared from aryl iodide 25 or 26 using a Suzuki-Miyaura
coupling reaction or Sonogashira coupling. Azabicyclo[3.1.0]hexane
derivative 37 led to the creation of the elongated compounds 9, 16, and
19 according to their respective methods (Scheme 2).
Different isoxazole compounds 10–12 were synthesized from aryl
iodide 40 using procedures similar to those used for compounds 4–9 in
Scheme 2 (Scheme 3).
5. Experimental section
The aminomethyl branch compounds 13–15 were synthesized from
aryl iodide intermediate 45 or 46 containing an N-Boc protected ami-
nomethyl branch. In addition, compounds 17, 18, and 20 were prepared
by procedures similar to those used for compounds 16, 9, and 19 in
Scheme 2, respectively (Scheme 4).
5.1. Chemistry
All reagents and solvents were of commercial quality and were used
without further purification. The progress of the reactions was usually
monitored using thin-layer chromatography (TLC; Merck silica gel 60
F254 plates or Fuji Silysia chromatorex NH plates). Purifications using
silica gel column chromatography were performed using a Biotage
5

F. Ushiyama et al.
Bioorganic & Medicinal Chemistry 30 (2021) 115964
Fig. 4. Safety pharmacology study examining the effects of test compounds on the cardiovascular system in anesthetized guinea pigs. SBP: systolic blood pressure.
DBP: diastolic blood pressure.
performed using a Biotage Initiator+ 60EXP with standard Pyrex vessels
Table 4
(capacity, 2–20 mL). The high-performance liquid chromatography
Toxicokinetic parameters affecting the cardiovascular system in anesthetized
mass spectra (LCMS) and retention times (Rt) were measured under the
guinea pigs.
following conditions (Analytical conditions A–B).
Cpd
15
17
18
19 (TP0586532)
Dose (mg/kg/30 min)
60
180
135
150
Analytical condition A
a,b
fu
0.172
90.7
15.6
0.255
233
0.273
132
0.231
378
C 30min
(
μ
g/mL)c
Measuring instruments: Agilent 1290 Infinity and Agilent 6130 or
6150 of Agilent, with Agilent 385-ELSD being used in combination with
C30min_free
a
(μ
g/mL)d
59.4
36.0
87.3
fu: fraction unbound in plasma, assumed to be equal to the fraction unbound
in serum.
an ELSD detector as an attachment. Column: Acquity CSH C18, 1.7 μm,
2.1 × 50 mm (Waters). Ionization method: ESI. Solvents: Fluid A, 0.1%
formic acid-containing water; Fluid B, 0.1% formic acid containing
acetonitrile. Flow rate: 0.8 mL/min.
b
The protein binding assay was evaluated at a concentration of 500 μg/mL in
guinea pig serum.
c
d
Plasma concentration at the end of infusion.
Detection method: 210 nm and 254 nm. Gradients: 0.0–0.8 min
(Fluid A/Fluid B = 95/5–60/40), 0.8–1.08 min (Fluid A/Fluid B = 60/
40–1/99), 1.08–1.38 min (Fluid A/Fluid B = 1/99).
Unbound plasma concentration at the end of infusion.
Isolera One instrument with Biotage SNAP Ultra Cartridges (particle
size: 25
μm sphere), BÜCHI Reveleris Flash Cartridges (particle size: 40
Analytical condition B
μ
m), or Biotage SNAP Isolute NH2 Cartridges (particle size: 50
μ
m). 1H
NMR spectra were recorded at 400 MHz using a BRUKER AVANCE III
HD 400 spectrometer or 600 MHz using a JEOL ECA600 spectrometer
with tetramethylsilane (TMS) as the internal standard, and proton
chemical shifts were expressed in parts per million (ppm) in the indi-
cated solvent. Multiplicity was defined as s (singlet), d (doublet), t
(triplet), q (quartet), quin (quintet), dd (double doublet), m (multiplet),
or br (broad signal). 13C NMR spectra were recorded at 125 MHz using a
JEOL ECA500 spectrometer with TMS as the internal standard, and
carbon chemical shifts were expressed in parts per million (ppm) in
DMSO‑d6 solvent. High-resolution mass spectrometry (HRMS) findings
were recorded using a Shimadzu LCMS-IT-TOF mass spectrometer with
an electrospray ionization (ESI)/atmospheric pressure chemical ioniza-
tion (APCI) dual source. Microwave irradiation experiments were
The measuring instruments, column, ionization method, solvents,
flow rate, and detection method were the same as those used for
Analytical condition A. Gradients: 0.0–1.2 min (Fluid A/Fluid B = 80/
20–1/99), 1.2–1.4 min (Fluid A/Fluid B = 1/99).
5.1.1. 3-[3-(4-{[3-({2-[(1S)-1-Hydroxyethyl]-1H-imidazol-1-yl}methyl)-
1,2-oxazol-5-yl]ethynyl}phenoxy)azetidin-1-yl]propan-1-ol (2)
A
mixture of 5-ethynyl-3-[(2-{(1S)-1-[(oxan-2-yl)oxy]ethyl}-1H-
imidazol-1-yl)methyl]-1,2-oxazole 21 (59 mg, 0.20 mmol), 1-(3-{[tert-
butyl(dimethyl)silyl]oxy}propyl)-3-(4-iodophenoxy)azetidine 24 (see
supplementary data, 80 mg, 0.18 mmol), Pd(PPh3)2Cl2 (6.3 mg, 0.0089
mmol), CuI (1.0 mg, 0.0054 mmol), and Et3N (0.25 mL, 1.8 mmol) in
6

F. Ushiyama et al.
Bioorganic & Medicinal Chemistry 30 (2021) 115964
Fig. 5. (a) Overlap of TP0586532 (cyan carbons, PDB ID: 7DEN) and ACHN-975 (gray carbons, PDB ID: 6MOO) bound to P. aeruginosa LpxC. The residues on the
surface are colored as follows: lipophilic reagions are in light green, hydrophilic in pink, and exposed in red. The catalytic zinc is displayed as an orange sphere. (b)
View of TP0586532 (cyan carbons) from the tail side. Val211 is represented in stick format. A water molecule is displayed as a red ball. The relevant interactions are
indicated by the dashed lines.
N
O
N
O
OH
OTHP
a, b
N
N
HO
N
N
2
: R =
: R =
N
∗
O
R
∗
21
N
3
N
MeO
c, d
f, g
N
O
N
O
OTBS
N
OTBS
e
N
N
N
I
22
23
Scheme 1. Synthesis of isoxazole-ethynyl-phenyl type compounds 2 and 3. Reagents and conditions: (a) 1-(3-{[tert-butyl(dimethyl)silyl]oxy}propyl)-3-(4-
iodophenoxy)azetidine 24 (see supplementary data), Pd(PPh3)2Cl2, CuI, Et3N, DMF, 60 ◦C, 1 h; (b) TsOH⋅H2O, MeOH, rt, 16 h, 40% over two steps; (c) TsOH⋅H2O,
MeOH, rt, 2 h; (d) TBSCl, imidazole, DMF, rt, 3 h, 80% over two steps; (e) 1,4-diiodobenzene, Pd(PPh3)4, CuI, Et3N, MeCN, rt, 1 h, 61%; (f) 1-(2-methoxyethyl)
piperazine, Pd2(dba)3, 2-dicyclohexylphosphino-2′-(N,N-dimethylamino)biphenyl, tert-BuONa, toluene, 60 ◦C, 5.5 h, 76%; (g) 1 mol/L TBAF in THF, THF, rt, 0.5
h, 80%.
DMF (0.51 mL) was stirred at 60 ◦C for 1 h. The mixture was diluted with
CHCl3. The insoluble matter was removed by filtration, and the filtrate
was concentrated. The residue was dissolved in MeOH followed by the
addition of TsOH⋅H2O (136 mg, 0.72 mmol). After stirring for 16 h, the
reaction mixture was poured into aqueous saturated NaHCO3, and the
mixture was extracted twice with 5% MeOH in CHCl3. The combined
organic layer was dried over MgSO4, filtered, and concentrated. The
residue was purified using column chromatography on NH silica gel and
eluted with MeOH/CHCl3 to obtain 2 as a colorless solid (30 mg, 40%
over two steps). 1H NMR (600 MHz, DMSO‑d6) δ 7.59–7.55 (m, 2H),
7.16 (d, J = 1.2 Hz, 1H), 6.95–6.91 (m, 2H), 6.82 (s, 1H), 6.80 (s, 1H),
5.46–5.39 (m, 3H), 4.87–4.82 (m, 2H), 4.39 (t, J = 5.0 Hz, 1H),
3.75–3.69 (m, 2H), 3.44–3.38 (m, 2H), 2.97–2.90 (m, 2H), 2.48–2.44
(m, 2H), 1.47–1.41 (m, 5H). 13C NMR (125 MHz, DMSO‑d6) δ 161.6,
158.4, 152.9, 149.7, 133.7, 126.5, 120.9, 115.3, 111.8, 107.6, 98.8,
74.2, 66.6, 61.4, 60.5, 58.9, 56.3, 40.5, 30.6, 21.8. LCMS (ESI) m/z 423
[M+H]+. Rt 0.527 min (Analytical condition A). HRMS (ESI/APCI dual)
m/z calcd for C23H26N4O4 [M+H]+ 423.2027, found 423.2020.
imidazol-1-yl}methyl)-5-[(4-iodophenyl)ethynyl]-1,2-oxazole 23 (50
mg, 0.094 mmol), 1-(2-methoxyethyl)piperazine (27 mg, 0.19 mmol),
Pd2(dba)3 (4.3 mg, 0.0047 mmol), and 2-dicyclohexylphosphino-2′-(N,
N-dimethylamino)biphenyl (3.7 mg, 0.0094 mmol) in toluene (1 mL).
The mixture was stirred at 60 ◦C for 5.5 h. After cooling, the mixture
was diluted with aqueous NaHCO3 and extracted with CHCl3. The
organic layer was dried over MgSO4 and concentrated. The residue was
purified using column chromatography on NH silica gel and eluted
with EtOAc/n-hexane to obtain 1-(4-{[3-({2-[(1S)-1-{[tert-butyl
(dimethyl)silyl]oxy}ethyl]-1H-imidazol-1-yl}methyl)-1,2-oxazol-5-yl]
ethynyl}phenyl)-4-(2-methoxyethyl)piperazine as a pale yellow amor-
phous substance (39 mg, 76%). LCMS (ESI) m/z 550 [M+H]+. Rt 0.597
min (Analytical condition B).
In step 2, 1 mol/L TBAF in THF (0.089 mL, 0.089 mmol) was added
to a solution of the above-described amorphous substance (38 mg,
0.069 mmol) in THF (0.7 mL). The mixture was stirred at room tem-
perature for 0.5 h, quenched with 20% aqueous potassium carbonate,
and extracted with 10% MeOH in CHCl3. The organic extract was passed
through a phase separator and concentrated. The residue was purified
using column chromatography on NH silica gel and eluted with EtOAc/
n-hexane and MeOH/CHCl3 to obtain 3 as a pale yellow solid (24 mg,
80%). 1H NMR (400 MHz, DMSO‑d6) δ 7.44 (d, J = 8.7 Hz, 2H), 7.16 (d,
J = 0.8 Hz, 1H), 6.97 (d, J = 8.7 Hz, 2H), 6.81 (d, J = 0.8 Hz, 1H), 6.72
5.1.2. (1S)-1-(1-{[5-({4-[4-(2-Methoxyethyl)piperazin-1-yl]phenyl}
ethynyl)-1,2-oxazol-3-yl]methyl}-1H-imidazol-2-yl)ethan-1-ol (3)
In step 1, sodium tert-butoxide (18 mg, 0.19 mmol) was added to a
solution of 3-({2-[(1S)-1-{[tert-butyl(dimethyl)silyl]oxy}ethyl]-1H-
7

F. Ushiyama et al.
Bioorganic & Medicinal Chemistry 30 (2021) 115964
O
B
O
N
O
TBSO
OTHP
∗
32
: R1 =
TBSO
OTHP
N
I
27
N
O
N
N
a
25
N
N
N
O
R1
OTHP
28 / 29 / 30 / 31
R1
32-36
CF3OCHN
BocHN
∗
N
b
28, 33
: R1 =
I
26
∗
29, 34
30, 35
: R1 =
∗
: R1 =
TBSO
H
N
O
OH
∗
c
N
31, 36
: R1 =
N
BocN
H
R2
∗
HO
∗
4
: R2 =
: R2 =
: R2 =
OH
d
e
8
: R =
9
: Z = MeO
HO
Z
2
H2N
∗
6
7
H
∗
∗
16
: Z = -CHO
N
H
37
: R =
H2N
2
f, g
Z
= H
19:
Z = -(CH2)3CO2H
Scheme 2. Synthesis of isoxazole-phenyl-ethynyl type compounds 4, 6–9, 16, and 19. Reagents and conditions: (a) 27 (see supplementary data), Pd(PPh3)4,
Na2CO3, toluene, EtOH, 80 ◦C, 4 h; (b) 28, 29, 30 (see supplementary data) or 31 (see supplementary data), Superstable Pd(0), CuI, Et3N, MeCN, rt, 1–2.5 h, 81%–
97%; (c) 4: 32, TsOH⋅H2O, MeOH, rt, 16 h, 19% over two steps; 6: i) 33, K2CO3, MeOH, rt, 8 h, 95%; ii) TsOH⋅H2O, MeOH, rt, 2 h, 99%; 7: 34, TFA, CHCl3, 0 ◦C, 4 h,
87%; 8: 35, 4 mol/L HCl in 1,4-dioxane, 1,4-dioxane, rt, 2 h, 72%; 37: 36, 4 mol/L HCl in 1,4-dioxane, 1,4-dioxane, MeOH, rt, 100 min, 60%; (d) 37, 2-
(methoxymethyl)oxirane, MeOH, 50 ◦C, 1.5 h, microwave, 82%; (e) N-formylsaccharin, THF, DMF, rt, 1 h, 70%; (f) ethyl 4-bromobutanoate, K2CO3, NaI, DMF, 80 ◦C,
80 min, 75%; (g) 2 mol/L NaOH, MeOH, THF, rt, 2 h, 77%. Superstable Pd = tris{tris[3,5-bis(trifluoromethyl)phenyl]phosphine}palladium.
(s, 1H), 5.45–5.35 (m, 3H), 4.90–4.80 (m, 1H), 3.46 (t, J = 5.7 Hz, 2H),
3.29–3.21 (m, 6H), 2.57–2.49 (m, 7H), 1.46 (d, J = 6.5 Hz, 3H). 13C
NMR (125 MHz, DMSO‑d6) δ 161.5, 153.3, 151.8, 149.7, 133.0, 126.5,
120.9, 114.2, 107.5, 106.9, 100.2, 73.8, 70.0, 61.4, 58.0, 56.9, 52.8,
46.7, 40.5, 21.8. LCMS (ESI) m/z 436 [M+H]+. Rt 0.570 min (Analytical
condition A). HRMS (ESI/APCI dual) m/z calcd for C24H29N5O3 [M+H]+
436.2343, found 436.2326.
on silica gel and eluted with MeOH/CHCl3 to obtain the crude product
of 5-[4-(5-{[tert-butyl(dimethyl)silyl]oxy}pent-1-yn-1-yl)phenyl]-3-[(2-
{(1S)-1-[(oxan-2-yl)oxy]ethyl}-1H-imidazol-1-yl)methyl]-1,2-oxazole
32, which was dissolved in MeOH (1.1 mL); TsOH⋅H2O (106 mg, 0.56
mmol) was then added. The mixture was stirred for 16 h. The mixture
was poured into saturated aqueous NaHCO3 and extracted with 5%
MeOH in CHCl3 twice. The combined organic extracts were dried over
MgSO4 and concentrated. The residue was purified using column
chromatography on NH silica gel and eluted with MeOH/EtOAc to
obtain 4 as a pale yellow oil (15 mg, 19% over two steps). 1H NMR
(600 MHz, CHLOROFORM-d) δ 7.65 (d, J = 8.3 Hz, 2H), 7.46 (d, J =
8.3 Hz, 2H), 7.02 (d, J = 1.2 Hz, 1H), 6.97 (d, J = 1.2 Hz, 1H), 6.35 (s,
1H), 5.28–5.41 (dd, J = 15.7, 15.7, 2H), 5.06–5.00 (m, 1H), 3.85–3.80
(m, 2H), 2.57 (t, J = 6.6 Hz, 2H), 2.37 (d, J = 6.6 Hz, 1H), 1.88 (quin,
J = 6.6 Hz, 2H), 1.70 (d, J = 6.6 Hz, 3H). 13C NMR (125 MHz,
DMSO‑d6) δ 168.8, 161.7, 149.7, 132.0, 126.2, 125.7, 125.6, 125.3,
121.0, 100.1, 93.2, 79.9, 61.3, 59.4, 40.7, 31.4, 21.8, 15.4. LCMS (ESI)
m/z 352 [M+H]+. Rt 0.830 min (Analytical condition A). HRMS (ESI/
APCI dual) m/z calcd for C20H21N3O3 [M+H]+ 352.1656, found
352.1650.
5.1.3. 5-{4-[3-({2-[(1S)-1-Hydroxyethyl]-1H-imidazol-1-yl}methyl)-1,2-
oxazol-5-yl]phenyl}pent-4-yn-1-ol (4)
A mixture of 5-iodo-3-[(2-{(1S)-1-[(oxan-2-yl)oxy]ethyl}-1H-imi-
dazol-1-yl)methyl]-1,2-oxazole 25 (90 mg, 0.22 mmol), tert-butyl
(dimethyl)({5-[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]
pent-4-yn-1-yl}oxy)silane 27 (107 mg, 0.27 mmol), Pd(PPh3)4 (26 mg,
0.022 mmol), and 2 mol/L aqueous Na2CO3 (0.22 mL, 0.44 mmol) in
toluene (0.45 mL) and EtOH (0.22 mL) was stirred at 80 ◦C for 2 h,
followed by the addition of Pd(PPh3)4 (26 mg, 0.022 mmol) and
additional stirring at 80 ◦C for 2 h. After cooling, the mixture was
poured into ice water and extracted with EtOAc twice. The combined
organic extracts were washed with brine, dried over MgSO4 and
concentrated. The residue was purified using column chromatography
8

F. Ushiyama et al.
Bioorganic & Medicinal Chemistry 30 (2021) 115964
O
N
O
O
R1
N
N
OTHP
N
28 / 29 / 31
OTHP
a
N
OH
N
b
N
I
I
R1
39
40
41-43
CF3OCHN
∗
H
28, 41
: R1 =
∗
31, 43
: R1 =
∗
BocN
29, 42
: R1 =
H
O
BocHN
N
OH
H
c
N
N
R2
H
H2N
H2N
∗
∗
∗
10
: R2 =
d
12
: R2 =
OH
44
: R2 =
∗
MeO
N
HN
H
H
11
: R2 =
Scheme 3. Synthesis of isoxazole-phenyl-ethynyl type compounds 10–12. Reagents and conditions: (a) 2-{(1S)-1-[(oxan-2-yl)oxy]ethyl}-1H-imidazole, TMAD,
Bu3P, THF, rt, 22 h, 65%; (b) 41, 42: 28 or 29, Pd(PPh3)4, CuI, Et3N, DMF, 65 ◦C, 0.5–1 h; 43: 31, Superstable Pd(0), CuI, Et3N, MeCN, rt–55 ◦C, 1.5 h, 92%; (c) 10: i)
41, 2 mol/L HCl in MeOH, MeOH, 0 ◦C–rt, 1.5 h; ii) 1 mol/L NaOH, MeOH, rt, 16 h, 81% over three steps; 11: 42, TFA, CHCl3, 0 ◦C–rt, 2 h, 88% over two steps; 12:
43, 4 mol/L HCl in 1,4-dioxane, rt, 3.5 h, 99%; (d) 2-(methoxymethyl)oxirane, EtOH, MeOH, 60 ◦C, 1 h, 75%.
5.1.4. (1S)-1-{1-[(5-{4-[(1-Aminocyclopropyl)ethynyl]phenyl}-1,2-
oxazol-3-yl)methyl]-1H-imidazol-2-yl}ethan-1-ol (6)
5.1.5. (1S)-1-{1-[(5-{4-[(3-Aminobicyclo[1.1.1]pentan-1-yl)ethynyl]
phenyl}-1,2-oxazol-3-yl)methyl]-1H-imidazol-2-yl}ethan-1-ol (7)
In step 1, a 20% aqueous K2CO3 (5.4 g) solution was added to a
solution of 2,2,2-trifluoro-N-{1-[(4-{3-[(2-{(1S)-1-[(oxan-2-yl)oxy]
ethyl}-1H-imidazol-1-yl)methyl]-1,2-oxazol-5-yl}phenyl)ethynyl]
cyclopropyl}acetamide 33 (683 mg, 1.29 mmol) in MeOH (25 mL),
and the mixture was stirred at room temperature for 4 h. Additional
20% aqueous K2CO3 (5.4 g) solution was then added at that temper-
ature. After stirring at the same temperature for 4 h, water was added
to the mixture, which was then extracted with CHCl3. The organic
extract was passed through a phase separator and concentrated. The
residue was purified using column chromatography on NH silica gel
and eluted with MeOH/CHCl3 to obtain 1-[(4-{3-[(2-{(1S)-1-[(oxan-
2-yl)oxy]ethyl}-1H-imidazol-1-yl)methyl]-1,2-oxazol-5-yl}phenyl)
ethynyl]cyclopropan-1-amine as a pale yellow syrup (529 mg, 95%).
LCMS (ESI) m/z 433 [M+H]+. Rt 0.686 min (Analytical condition A).
In step 2, TsOH⋅H2O (647 mg, 3.40 mmol) was added to a solution of
the above-described product (368 mg, 0.85 mmol) in MeOH (8.5 mL).
After stirring at room temperature for 2 h, 20% aqueous K2CO3 solution
was added to the mixture, which was extracted with CHCl3. The organic
extract was passed through a phase separator and concentrated. The
residue was purified using column chromatography on NH silica gel and
eluted with MeOH/CHCl3 to obtain 6 as a colorless amorphous sub-
stance (292 mg, 99%). 1H NMR (600 MHz, CHLOROFORM-d) δ 7.65 (d,
J = 8.3 Hz, 2H), 7.44 (d, J = 8.3 Hz, 2H), 7.01 (d, J = 1.2 Hz, 1H), 6.96
(d, J = 1.2 Hz, 1H), 6.36 (s, 1H), 5.41–5.37 (m, 1H), 5.33–5.28 (m, 1H),
5.06–4.99 (m, 1H), 1.69 (d, J = 6.6 Hz, 3H), 1.08–1.00 (m, 4H). 13C
NMR (125 MHz, DMSO‑d6) δ 168.8, 161.8, 149.7, 131.8, 126.5, 125.7,
125.5, 125.2, 120.9, 100.1, 98.9, 77.1, 61.4, 40.7, 24.5, 21.8, 18.1.
LCMS (ESI) m/z 349 [M+H]+. Rt 0.484 min (Analytical condition A).
HRMS (ESI/APCI dual) m/z calcd for C20H20N4O2 [M+H]+ 349.1659,
found 349.1671.
TFA (0.096 mL, 1.3 mmol) was added to a solution of tert-butyl {3-
[(4-{3-[(2-{(1S)-1-[(oxan-2-yl)oxy]ethyl}-1H-imidazol-1-yl)methyl]-
1,2-oxazol-5-yl}phenyl)ethynyl]bicyclo[1.1.1]pentan-1-yl}carbamate
34 (70 mg, 0.13 mmol) in CHCl3 (0.42 mL) at 0 ◦C. After stirring for 2
h, additional TFA (0.096 mL, 1.3 mmol) was added. The mixture was
stirred at 0 ◦C for another 2 h and quenched by adding MeOH, then
concentrated. The residue was partitioned between 10% MeOH in
CHCl3 and 2 mol/L Na2CO3. The aqueous layer was extracted three
times with 10% MeOH in CHCl3. The combined organic extracts were
dried over MgSO4 and concentrated. The residue was purified using
column chromatography on NH silica gel and eluted with MeOH/CHCl3
to obtain 7 as a colorless amorphous substance (41 mg, 87%). 1H NMR
(400 MHz, METHANOL-d4) δ 7.74 (d, J = 8.4 Hz, 2H), 7.46 (d, J = 8.4
Hz, 2H), 7.15 (d, J = 1.1 Hz, 1H), 6.92 (d, J = 1.1 Hz, 1H), 6.72 (s, 1H),
5.58–5.41 (m, 2H), 5.04 (q, J = 6.6 Hz, 1H), 2.12 (s, 6H), 1.59 (d, J =
6.6 Hz, 3H). 13C NMR (125 MHz, DMSO‑d6) δ 168.7, 161.8, 149.7,
132.1, 126.5, 125.8, 125.7, 124.6, 120.9, 100.2, 91.3, 80.1, 61.4, 57.6,
51.6, 40.7, 23.6, 21.8. LCMS (ESI) m/z 375 [M+H]+. Rt 0.442 min
(Analytical condition A). HRMS (ESI/APCI dual) m/z calcd for
C
22H22N4O2 [M+H]+ 375.1816, found 375.1798.
5.1.6. (1S,3r)-3-({4-[3-({2-[(1S)-1-Hydroxyethyl]-1H-imidazol-1-yl}
methyl)-1,2-oxazol-5-yl]phenyl}ethynyl)cyclobutan-1-ol (8)
Four mol/L HCl in 1,4-dioxane (2.0 mL) was added to a solution of
5-(4-{[(1r,3S)-3-{[tert-butyl(dimethyl)silyl]oxy}cyclobutyl]ethynyl}
phenyl)-3-[(2-{(1S)-1-[(oxan-2-yl)oxy]ethyl}-1H-imidazol-1-yl)
methyl]-1,2-oxazole 35 (47 mg, 0.084 mmol) in 1,4-dioxane (2.0 mL).
The mixture was stirred at room temperature for 2 h and concen-
trated. The residue was basified with saturated aqueous NaHCO3 and
extracted with CHCl3. The organic extract was passed through a phase
separator and concentrated. The residue was purified using column
chromatography on NH silica gel and eluted with MeOH/CHCl3 to
obtain 8 as a colorless solid (22 mg, 72%). 1H NMR (400 MHz,
9

F. Ushiyama et al.
Bioorganic & Medicinal Chemistry 30 (2021) 115964
O
B
BocHN
N
O
O
OTHP
N
I
47 48
/
BocHN
N
N
R1
O
45
a
OR2
N
BocHN
N
N
30 / 49 / 50
51-54, 56
O
R1
R1
OTBS
b
N
H
∗
N
∗
I
BocHN
∗
46
R1 =
47, 51
48, 52
: R1 =
: R1 =
N
H
BocHN
Z
R2 = THP
R2 =THP
49, 54
: Z = -COCF3
R2 = TBS
55
: Z = H
∗
c, d
30, 53
: R1 =
R2 = H
TBSO
R2 = TBS
50, 56
: Z = -(CH2)3CO2Et
R2 = TBS
: Z = -(CH2)3CO2Et
R2 = H
d
57
H
∗
H2N
∗
H2N
13
: R3 =
R3 =
N
H
Z
e
N
O
51-53
55
∗
O
OH
∗
17
18
20
: Z =
f, g / h, i
j, k
N
14
15
: R3 =
N
H2N
HO
∗
: Z = HO
57
∗
R3
: R3 =
O
∗
: Z =
HO
Scheme 4. Synthesis of aminomethyl branch compounds 13–15, 17, 18, and 20. Reagents and conditions: (a) 51: 47, Pd-PEPPSI-IPent catalyst, 2 mol/L Na2CO3,
toluene, EtOH, 100 ◦C, 2 h; 52: 48, Pd-PEPPSI-IPent catalyst, 2 mol/L Na2CO3, toluene, EtOH, 80 ◦C, 1 h; (b) 53: 30, Superstable Pd(0), CuI, Et3N, MeCN, 60 ◦C, 10
min, 99%; 54: 49, Pd(PPh3)4, CuI, Et3N, DMF, 65 ◦C, 0.5 h; 56: 50, Superstable Pd(0), CuI, Et3N, MeCN, rt, 2 h; 90%; (c) 1 mol/L NaOH, THF, MeOH, rt, 20 min, 77%
over two steps; (d) 1 mol/L TBAF in THF, THF, rt, 20 min–1 h, 85%–95%; (e) 13: 51, TFA, CHCl3, rt, 2 h, 20% over two steps; 14: 52, TFA, CHCl3, MeOH, rt, 1.25 h,
31% over two steps; 15: 53, 4 mol/L HCl in 1,4-dioxane, MeOH, rt, 5 h, 94%; (f) 55, N-formylsaccharin, THF, rt, 40 min, 89%; (g) 4 mol/L HCl in 1,4-dioxane, MeOH,
rt, 1 h, 85%; (h) 55, (2S)-2-methyloxirane, MeOH, 50 ◦C, 2.5 h, microwave; (i) 4 mol/L HCl in 1,4-dioxane, MeOH, rt, 1 h, quant over two steps; (j) 57, 4 mol/L HCl in
1,4-dioxane, MeOH, rt, 1 h; (k) 1 mol/L NaOH, THF, rt, 2 h, 34% over two steps. Pd-PEPPSI-IPent = [1,3-bis(2,6-di-3-pentylphenyl)imidazol-2-ylidene](3-chlor-
opyridyl)dichloropalladium(II).
CHLOROFORM-d) δ 7.65 (d, J = 8.1 Hz, 2H), 7.46 (d, J = 8.1 Hz, 2H),
7.02 (br s, 1H), 6.97 (br s, 1H), 6.36 (s, 1H), 5.44–5.26 (m, 2H),
5.09–4.97 (m, 1H), 4.66 (quin, J = 6.7 Hz, 1H), 3.28–3.18 (m, 1H),
stirred at 50 ◦C for 0.5 h under microwave irradiation. Additional 2-
(methoxymethyl)oxirane (35.3 mg, 0.40 mmol) was then added, and
the mixture was stirred at 50 ◦C for 1 h under microwave irradiation
and concentrated. The residue was purified using column chroma-
tography on NH silica gel and eluted with MeOH/CHCl3 to obtain 9 as
a pale yellow amorphous substance (30 mg, 82%). 1H NMR (400 MHz,
METHANOL-d4) δ 7.72 (d, J = 8.2 Hz, 2H), 7.42 (d, J = 8.2 Hz, 2H),
7.15 (s, 1H), 6.92 (s, 1H), 6.70 (s, 1H), 5.56–5.41 (m, 2H), 5.04 (q, J
= 6.6 Hz, 1H), 3.88–3.70 (m, 1H), 3.47–3.24 (m, 5H), 3.20–3.08 (m,
2H), 2.57–2.38 (m, 4H), 1.95–1.86 (m, 1H), 1.86–1.76 (m, 2H), 1.59
(d, J = 6.6 Hz, 3H). 13C NMR (125 MHz, DMSO‑d6) δ 168.8, 161.7,
149.7, 132.0, 126.5, 125.6, 125.5, 125.3, 120.9, 100.0, 94.8, 76.5,
75.3, 67.9, 61.4, 58.3, 57.3, 54.3, 54.1, 40.7, 26.1, 25.9, 21.8, 7.8.
LCMS (ESI) m/z 463 [M+H]+. Rt 0.565 min (Analytical condition A).
HRMS (ESI/APCI dual) m/z calcd for C26H30N4O4 [M+H]+ 463.2340,
found 463.2345.
2.57–2.46 (m, 2H), 2.38–2.26 (m, 2H), 1.69 (d, J = 6.4 Hz, 3H). 13
C
NMR (125 MHz, DMSO‑d6) δ 168.8, 161.8, 149.7, 132.0, 126.5,
125.7, 125.6, 125.2, 120.9, 100.1, 97.4, 80.0, 64.4, 61.4, 40.9, 40.7,
21.8, 17.4. LCMS (ESI) m/z 364 [M+H]+. Rt 0.460 min (Analytical
condition B). HRMS (ESI/APCI dual) m/z calcd for C21H21N3O3
[M+H]+ 364.1656, found 364.1642.
5.1.7. 1-[(1R,5S,6s)-6-({4-[3-({2-[(1S)-1-Hydroxyethyl]-1H-imidazol-1-
yl}methyl)-1,2-oxazol-5-yl]phenyl}ethynyl)-3-azabicyclo[3.1.0]hexan-3-
yl]-3-methoxypropan-2-ol (9)
2-(Methoxymethyl)oxirane (35.3 mg, 0.40 mmol) was added to a
solution of (1S)-1-(1-{[5-(4-{[(1R,5S,6s)-3-azabicyclo[3.1.0]hexan-6-
yl]ethynyl}phenyl)-1,2-oxazol-3-yl]methyl}-1H-imidazol-2-yl)ethan-
1-ol 37 (30 mg, 0.080 mmol) in MeOH (1 mL). The mixture was
10

F. Ushiyama et al.
Bioorganic & Medicinal Chemistry 30 (2021) 115964
5.1.8. (1S)-1-{1-[(3-{4-[(1-Aminocyclopropyl)ethynyl]phenyl}-1,2-
1H), 6.73 (s, 1H), 5.61 (dd, J = 15.7, 15.7 Hz, 2H), 5.03 (q, J = 6.5 Hz,
1H), 2.15 (s, 6H), 1.59 (d, J = 6.5 Hz, 3H). 13C NMR (125 MHz,
DMSO‑d6) δ 169.3, 161.4, 149.6, 132.0, 127.8, 126.8, 126.5, 124.4,
121.1, 101.1, 90.4, 80.2, 61.5, 57.4, 50.8, 40.7, 23.8, 21.8. LCMS (ESI)
m/z 375 [M+H]+. Rt 0.426 min (Analytical condition A). HRMS (ESI/
APCI dual) m/z calcd for C22H22N4O2 [M+H]+ 375.1816, found
375.1798.
oxazol-5-yl)methyl]-1H-imidazol-2-yl}ethan-1-ol (10)
A
mixture of 3-(4-iodophenyl)-5-[(2-{(1S)-1-[(oxan-2-yl)oxy]
ethyl}-1H-imidazol-1-yl)methyl]-1,2-oxazole 40 (70 mg, 0.15 mmol),
CuI (1.4 mg, 0.0073 mmol), Pd(PPh3)4 (17 mg, 0.015 mmol), Et3N
(0.20 mL, 1.5 mmol), and DMF (0.29 mL) was heated at 65 ◦C for 5
min, and
a
solution of N-(1-ethynylcyclopropyl)-2,2,2-tri-
fluoroacetamide 28 (34 mg, 0.19 mmol) in DMF (0.38 mL) was then
added in a dropwise manner. After stirring for 0.5 h, the mixture was
diluted with EtOAc and filtered through a short pad of NH silica gel.
The filtrate was concentrated, and the residue was purified using
column chromatography on NH silica gel and eluted with EtOAc/n-
hexane to obtain 2,2,2-trifluoro-N-{1-[(4-{5-[(2-{(1S)-1-[(oxan-2-yl)
oxy]ethyl}-1H-imidazol-1-yl)methyl]-1,2-oxazol-3-yl}phenyl)ethy-
nyl]cyclopropyl}acetamide 41 as a crude product, which was dis-
solved in MeOH (1.5 mL); then, 2 mol/L HCl in MeOH (1.2 mL, 2.4
mmol) was added to the solution at 0 ◦C. After stirring at the same
temperature for 0.5 h, the mixture was stirred at room temperature
for 1 h and concentrated to obtain 2,2,2-trifluoro-N-[1-({4-[5-({2-
[(1S)-1-hydroxyethyl]-1H-imidazol-1-yl}methyl)-1,2-oxazol-3-yl]
5.1.10. 1-[(1R,5S,6s)-6-({4-[5-({2-[(1S)-1-Hydroxyethyl]-1H-imidazol-
1-yl}methyl)-1,2-oxazol-3-yl]phenyl}ethynyl)-3-azabicyclo[3.1.0]hexan-
3-yl]-3-methoxypropan-2-ol (12)
2-(Methoxymethyl)oxirane (34 mg, 0.39 mmol) was added to a
suspension of (1S)-1-(1-{[3-(4-{[(1R,5S,6s)-3-azabicyclo[3.1.0]hexan-
6-yl]ethynyl}phenyl)-1,2-oxazol-5-yl]methyl}-1H-imidazol-2-yl)ethan-
1-ol 44 (29 mg, 0.077 mmol) in EtOH (1 mL). After stirring at room
temperature overnight, MeOH (1 mL) and 2-(methoxymethyl)oxirane
(15 mg, 0.17 mmol) were added to the mixture, which was stirred at
60 ◦C for 1 h and concentrated. The residue was purified using column
chromatography on NH silica gel and eluted with MeOH/CHCl3 to
obtain 12 as a light yellow amorphous substance (27 mg, 75%). 1H
NMR (400 MHz, METHANOL-d4) δ 7.73 (d, J = 8.1 Hz, 2H), 7.41 (d, J
= 8.1 Hz, 2H), 7.19 (s, 1H), 6.94 (s, 1H), 6.72 (s, 1H), 5.68–5.53 (m,
2H), 5.03 (q, J = 6.6 Hz, 1H), 3.83–3.74 (m, 1H), 3.42–3.36 (m, 2H),
3.35 (s, 3H), 3.15 (br dd, J = 9.0, 5.2 Hz, 2H), 2.54–2.38 (m, 4H),
1.92–1.87 (m, 1H), 1.83–1.78 (m, 2H), 1.59 (d, J = 6.6 Hz, 3H). 13C
NMR (125 MHz, DMSO‑d6) δ 169.2, 161.4, 149.6, 131.9, 127.3, 126.7,
126.5, 125.1, 121.0, 101.1, 94.2, 76.5, 75.3, 67.9, 61.5, 58.3, 57.3,
54.2, 54.1, 40.7, 26.1, 25.8, 21.8, 7.7. LCMS (ESI) m/z 463 [M+H]+. Rt
0.560 min (Analytical condition A). HRMS (ESI/APCI dual) m/z calcd
for C26H30N4O4 [M+H]+ 463.2340, found 463.2326.
phenyl}ethynyl)cyclopropyl]acetamide hydrochloride as
a crude
product, which was used in the next step without further purification.
The crude product was dissolved in MeOH (1.5 mL) followed by the
addition of 1 mol/L NaOH solution (1.5 mL, 1.5 mmol) at room
temperature. After stirring for 16 h, the mixture was concentrated; the
residue was then partitioned between 10% MeOH in CHCl3 and 2
mol/L aqueous Na2CO3 solution. The aqueous layer was extracted
with 10% MeOH in CHCl3, and the combined organic extracts were
dried over MgSO4 and concentrated. The residue was then purified
using preparative TLC on silica gel and eluted with CHCl3/MeOH/
NH4OH to obtain 10 as a colorless amorphous substance (41 mg, 81%
over three steps). 1H NMR (400 MHz, METHANOL-d4) δ 7.76 (d, J =
8.7 Hz, 2H), 7.45 (d, J = 8.7 Hz, 2H), 7.20 (s, 1H), 6.94 (s, 1H), 6.73
(s, 1H), 5.61 (dd, J = 14.9, 14.9 Hz, 2H), 5.03 (q, J = 6.4 Hz, 1H),
1.59 (d, J = 6.4 Hz, 3H), 1.08–0.93 (m, 4H). 13C NMR (125 MHz,
DMSO‑d6) δ 169.3, 161.4, 149.6, 131.7, 127.3, 126.7, 126.5, 125.0,
121.1, 101.1, 98.3, 77.1, 61.5, 40.8, 24.5, 21.8, 18.0. LCMS (ESI) m/z
349 [M+H]+. Rt 0.393 min (Analytical condition A). HRMS (ESI/APCI
dual) m/z calcd for C20H20N4O2 [M+H]+ 349.1659, found 349.1667.
5.1.11. (1S)-1-{1-[(1R)-2-Amino-1-(5-{4-[(1-aminocyclopropyl)ethynyl]
phenyl}-1,2-oxazol-3-yl)ethyl]-1H-imidazol-2-yl}ethan-1-ol (13)
Two mol/L Na2CO3 (0.26 mL, 0.53 mmol) was added to a mixture
of tert-butyl [(2R)-2-(5-iodo-1,2-oxazol-3-yl)-2-(2-{(1S)-1-[(oxan-2-yl)
oxy]ethyl}-1H-imidazol-1-yl)ethyl]carbamate 45 (70 mg, 0.13 mmol),
tert-butyl (1-{[4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]
ethynyl}cyclopropyl)carbamate 47 (76 mg, 0.20 mmol), and Pd-
PEPPSI-IPent catalyst (10 mg, 0.013 mmol) in toluene (0.53 mL) and
EtOH (0.53 mL). After stirring at 100 ◦C for 2 h, EtOAc and saturated
aqueous NaHCO3 were added to the mixture, which was extracted with
EtOAc. The separated organic extract was dried over MgSO4 and
concentrated. The residue was purified using column chromatography
on silica gel and eluted with MeOH/CHCl3 to obtain tert-butyl [(2R)-2-
{5-[4-({1-[(tert-butoxycarbonyl)amino]cyclopropyl}ethynyl)phenyl]-
1,2-oxazol-3-yl}-2-(2-{(1S)-1-[(oxan-2-yl)oxy]ethyl}-1H-imidazol-1-yl)
ethyl]carbamate 51 as a crude product [LCMS (ESI) m/z 662 [M+H]+.
Rt 0.932 min (Analytical condition B)], which was taken up in CHCl3
(2.2 mL). TFA (0.42 mL, 5.4 mmol) was added, and the mixture was
stirred at room temperature for 2 h, then basified with saturated
aqueous K2CO3 at 0 ◦C and extracted with 10% MeOH in CHCl3 ten
times. The combined organic extracts were passed through a phase
separator and concentrated. The residue was purified using column
chromatography on silica gel and eluted with CHCl3/MeOH/NH4OH,
then purified again using column chromatography on NH silica gel and
eluted with MeOH/CHCl3 to obtain 13 as a pale yellow amorphous
substance (10 mg, 20% over two steps). 1H NMR (600 MHz, METH-
ANOL-d4) δ 7.76 (d, J = 8.3 Hz, 2H), 7.47 (d, J = 8.3 Hz, 2H), 7.24 (d,
J = 1.2 Hz, 1H), 6.97 (d, J = 1.2 Hz, 1H), 6.78 (s, 1H), 5.93–5.88 (m,
1H), 5.09 (q, J = 6.6 Hz, 1H), 3.52 (dd, J = 13.6, 6.2 Hz, 1H), 3.39 (dd,
J = 13.6, 8.3 Hz, 1H), 1.66 (d, J = 6.6 Hz, 3H), 1.06–1.02 (m, 2H),
1.00–0.96 (m, 2H). 13C NMR (125 MHz, DMSO‑d6) δ 168.7, 163.5,
150.1, 131.8, 126.9, 125.6, 125.6, 125.2, 117.6, 100.0, 98.9, 77.1,
60.8, 54.1, 44.8, 24.5, 21.5, 18.1. LCMS (ESI) m/z 189 [M + 2H]2+. Rt
0.201 min (Analytical condition A). HRMS (ESI/APCI dual) m/z calcd
for C21H23N5O2 [M+H]+ 378.1925, found 378.1910.
5.1.9. (1S)-1-{1-[(3-{4-[(3-Aminobicyclo[1.1.1]pentan-1-yl)ethynyl]
phenyl}-1,2-oxazol-5-yl)methyl]-1H-imidazol-2-yl}ethan-1-ol (11)
A
mixture of 3-(4-iodophenyl)-5-[(2-{(1S)-1-[(oxan-2-yl)oxy]
ethyl}-1H-imidazol-1-yl)methyl]-1,2-oxazole 40 (70 mg, 0.15 mmol),
CuI (1.4 mg, 0.0073 mmol), Pd(PPh3)4 (17 mg, 0.015 mmol), Et3N
(0.20 mL, 1.5 mmol), and DMF (0.29 mL) was heated at 65 ◦C for 5
min, and then a solution of tert-butyl (3-ethynylbicyclo[1.1.1]pentan-1-
yl)carbamate 29 (39 mg, 0.19 mmol) in DMF (0.38 mL) was added in a
dropwise manner. After stirring for 1 h, the mixture was diluted with
EtOAc and filtered through a short pad of NH silica gel. The filtrate was
concentrated, and the residue was purified using column chromatog-
raphy on NH silica gel and eluted with EtOAc/n-hexane to obtain tert-
butyl {3-[(4-{5-[(2-{(1S)-1-[(oxan-2-yl)oxy]ethyl}-1H-imidazol-1-yl)
methyl]-1,2-oxazol-3-yl}phenyl)ethynyl]bicyclo[1.1.1]pentan-1-yl}
carbamate 42 as a crude product. This product was then dissolved in
CHCl3 (0.42 mL), and TFA (0.34 mL) was added to the solution at 0 ◦C.
After stirring at the same temperature for 1 h, the mixture was stirred
at room temperature for another 1 h. The mixture was concentrated,
and the residue was dissolved in 10% MeOH in CHCl3 (3 mL) and
basified with 2 mol/L Na2CO3 (3 mL). The phases were separated, and
the aqueous layer was extracted with 10% MeOH in CHCl3. The com-
bined organic layer was dried over MgSO4 and concentrated. The res-
idue was purified using preparative TLC on silica gel and eluted with
CHCl3/MeOH/NH4OH to obtain 11 as a colorless amorphous substance
(48 mg, 88% over two steps). 1H NMR (400 MHz, METHANOL-d4) δ
7.75 (d, J = 8.3 Hz, 2H), 7.45 (d, J = 8.3 Hz, 2H), 7.20 (s, 1H), 6.94 (s,
11

F. Ushiyama et al.
Bioorganic & Medicinal Chemistry 30 (2021) 115964
5.1.12. (1S)-1-{1-[(1R)-2-Amino-1-(5-{4-[(3-aminobicyclo[1.1.1]
pentan-1-yl)ethynyl]phenyl}-1,2-oxazol-3-yl)ethyl]-1H-imidazol-2-yl}
ethan-1-ol (14)
ethynyl}phenyl)-1,2-oxazol-3-yl]methyl}-1H-imidazol-2-yl)ethan-1-ol
37 (20 mg, 0.053 mmol) in THF (1 mL) and DMF (0.5 mL). After
stirring at room temperature for 1 h, the mixture was diluted with
CHCl3 and saturated aqueous NaHCO3, then extracted with a com-
bined solvent of CHCl3 and MeOH. The organic extract was concen-
trated, and the residue was purified using column chromatography on
NH silica gel and eluted with MeOH/EtOAc to obtain 16 as a colorless
solid (15 mg, 70%). 1H NMR (400 MHz, METHANOL-d4) δ 8.10 (s,
1H), 7.73 (d, J = 8.2 Hz, 2H), 7.45 (d, J = 8.2 Hz, 2H), 7.15 (s, 1H),
6.92 (s, 1H), 6.71 (s, 1H), 5.56–5.41 (m, 2H), 5.04 (br q, J = 6.6 Hz,
1H), 3.95–3.79 (m, 2H), 3.72–3.64 (m, 1H), 3.39–3.23 (m, 1H),
2.09–1.97 (m, 2H), 1.59 (d, J = 6.6 Hz, 3H), 1.39–1.33 (m, 1H). 13C
NMR (125 MHz, DMSO‑d6) δ 168.7, 161.8, 161.7, 149.7, 132.1,
126.5, 125.7, 124.9, 120.9, 100.1, 92.9, 77.3, 61.4, 47.2, 44.4, 40.6,
24.8, 24.7, 21.8, 9.7. LCMS (ESI) m/z 403 [M+H]+. Rt 0.838 min
(Analytical condition A). HRMS (ESI/APCI dual) m/z calcd for
C23H22N4O3 [M+H]+ 403.1765, found 403.1747.
A mixture of intermediate tert-butyl [(2R)-2-(5-iodo-1,2-oxazol-3-
yl)-2-(2-{(1S)-1-[(oxan-2-yl)oxy]ethyl}-1H-imidazol-1-yl)ethyl]carba-
mate 45 (170 mg, 0.32 mmol), tert-butyl (3-{[4-(4,4,5,5-tetramethyl-
1,3,2-dioxaborolan-2-yl)phenyl]ethynyl}bicyclo[1.1.1]pentan-1-yl)
carbamate 48 (170 mg, 0.42 mmol), Pd-PEPPSI-IPent catalyst (25 mg,
0.032 mmol), and 2 mol/L Na2CO3 (0.64 mL, 1.3 mmol) in toluene
(1.6 mL) and EtOH (1.6 mL) was stirred at 80 ◦C for 1 h. The mixture
was poured into 2 mol/L Na2CO3 and extracted with EtOAc three times.
The combined organic extracts were washed with brine, dried over
MgSO4 and concentrated. The residue was purified using column
chromatography on NH silica gel and eluted with EtOAc/n-hexane to
obtain tert-butyl [(2R)-2-{5-[4-({3-[(tert-butoxycarbonyl)amino]bicy-
clo[1.1.1]pentan-1-yl}ethynyl)phenyl]-1,2-oxazol-3-yl}-2-(2-{(1S)-1-
[(oxan-2-yl)oxy]ethyl}-1H-imidazol-1-yl)ethyl]carbamate 52 as
a
crude product, which was used in the next step without further
purification.
5.1.15. (1R,5S,6s)-6-[(4-{3-[(1R)-2-Amino-1-{2-[(1S)-1-hydroxyethyl]-
1H-imidazol-1-yl}ethyl]-1,2-oxazol-5-yl}phenyl)ethynyl]-3-azabicyclo
[3.1.0]hexane-3-carbaldehyde (17)
TFA (0.81 mL, 11 mmol) was added to a solution of 52 in CHCl3 (1.4
mL) at room temperature. After stirring for 45 min, MeOH (1.5 mL) was
added, and the resulting mixture was stirred at that temperature for
another 0.5 h and concentrated. The residue was partitioned between 2
mol/L Na2CO3 and 10% MeOH in CHCl3. The aqueous layer was
extracted four times with 10% MeOH in CHCl3. The combined organic
extracts were dried over MgSO4 and concentrated. The residue was
purified using preparative TLC on silica gel and eluted with CHCl3/
MeOH/NH4OH to obtain 14 as a pale yellow amorphous substance (40
mg, 31% over two steps). 1H NMR (600 MHz, METHANOL-d4) δ 7.75 (d,
J = 8.3 Hz, 2H), 7.46 (d, J = 8.3 Hz, 2H), 7.24 (s, 1H), 6.97 (s, 1H), 6.79
(s, 1H), 5.90 (dd, J = 8.1, 6.2 Hz, 1H), 5.09 (q, J = 6.3 Hz, 1H), 3.52 (dd,
J = 13.6, 6.2 Hz, 1H), 3.39 (dd, J = 13.6, 8.1 Hz, 1H), 2.12 (s, 6H), 1.66
(d, J = 6.3 Hz, 3H). 13C NMR (125 MHz, DMSO‑d6) δ 168.6, 163.5,
150.1, 132.1, 126.9, 125.9, 125.7, 124.6, 117.6, 100.1, 91.3, 80.1, 60.8,
57.6, 54.1, 51.6, 44.8, 23.6, 21.5. LCMS (ESI) m/z 404 [M+H]+. Rt
0.221 min (Analytical condition A). HRMS (ESI/APCI dual) m/z calcd
for C23H25N5O2 [M+H]+ 404.2081, found 404.2076.
In step 1, N-formylsaccharin (189 mg, 0.90 mmol) was added to a
solution of tert-butyl [(2R)-2-[5-(4-{[(1R,5S,6s)-3-azabicyclo[3.1.0]
hexan-6-yl]ethynyl}phenyl)-1,2-oxazol-3-yl]-2-{2-[(1S)-1-hydrox-
yethyl]-1H-imidazol-1-yl}ethyl]carbamate 55 (410 mg, 0.81 mmol) in
THF (8.1 mL). After stirring at room temperature for 40 min, saturated
aqueous NaHCO3 was added to the mixture at 0 ◦C and extracted with
CHCl3. The organic extract was passed through a phase separator and
concentrated. The residue was purified using column chromatography
on NH silica gel and eluted with MeOH/EtOAc to obtain tert-butyl [(2R)-
2-[5-(4-{[(1R,5S,6s)-3-formyl-3-azabicyclo[3.1.0]hexan-6-yl]ethynyl}
phenyl)-1,2-oxazol-3-yl]-2-{2-[(1S)-1-hydroxyethyl]-1H-imidazol-1-yl}
ethyl]carbamate as a colorless solid (386 mg, 89%). LCMS (ESI) m/z 532
[M+H]+. Rt 0.659 min (Analytical condition B).
In step 2, 4 mol/L HCl in 1,4-dioxane (9.0 mL, 36 mmol) was added
to a solution of the above-described solid (384 mg, 0.72 mmol) in MeOH
(7.2 mL) at 0 ◦C. The mixture was stirred at room temperature for 1 h
and concentrated. The residue was basified with 20% aqueous K2CO3
and a combined solvent of CHCl3 and MeOH. The organic extract was
passed through a phase separator and concentrated. The residue was
purified using column chromatography on silica gel and eluted with
MeOH containing 28% aqueous ammonia/CHCl3 to obtain 17 as a
colorless solid (264 mg, 85%). 1H NMR (400 MHz, METHANOL-d4) δ
8.10 (s, 1H), 7.74 (d, J = 7.8 Hz, 2H), 7.45 (d, J = 7.8 Hz, 2H), 7.24 (s,
1H), 6.97 (s, 1H), 6.77 (s, 1H), 5.94–5.85 (m, 1H), 5.14–5.03 (m, 1H),
3.96–3.78 (m, 2H), 3.75–3.61 (m, 1H), 3.59–3.46 (m, 1H), 3.45–3.16
(m, 2H), 2.09–1.96 (m, 2H), 1.66 (d, J = 6.4 Hz, 3H), 1.39–1.33 (m, 1H).
13C NMR (125 MHz, DMSO‑d6) δ 168.6, 163.5, 161.7, 150.1, 132.1,
126.9, 125.7, 125.6, 124.9, 117.6, 100.0, 92.9, 77.3, 60.8, 54.1, 47.2,
44.9, 44.4, 24.8, 24.7, 21.5, 9.6. LCMS (ESI) m/z 432 [M+H]+. Rt 0.652
min (Analytical condition A). HRMS (ESI/APCI dual) m/z calcd for
5.1.13. (1R,3r)-3-[(4-{3-[(1R)-2-Amino-1-{2-[(1S)-1-hydroxyethyl]-1H-
imidazol-1-yl}ethyl]-1,2-oxazol-5-yl}phenyl)ethynyl]cyclobutan-1-ol (15)
Four mol/L HCl in 1,4-dioxane (10 mL) was added to a solution of
tert-butyl
[(2R)-2-[5-(4-{[(1r,3R)-3-{[tert-butyl(dimethyl)silyl]oxy}
cyclobutyl]ethynyl}phenyl)-1,2-oxazol-3-yl]-2-{2-[(1S)-1-{[tert-butyl
(dimethyl)silyl]oxy}ethyl]-1H-imidazol-1-yl}ethyl]carbamate
53
(0.535 mg, 0.74 mmol) in MeOH (1 mL). The mixture was stirred at
room temperature for 5 h and concentrated. The residue was basified
with 20% aqueous K2CO3 and extracted with a combined solvent of
CHCl3 and MeOH. The separated organic extract was passed through a
phase separator and concentrated. The residue was purified using col-
umn chromatography on NH silica gel and eluted with MeOH/CHCl3 to
obtain 15 as a colorless amorphous substance (274 mg, 94%). 1H NMR
(400 MHz, CHLOROFORM-d) δ 7.66 (d, J = 8.1 Hz, 2H), 7.47 (d, J = 8.1
Hz, 2H), 7.09 (s, 1H), 6.96 (s, 1H), 6.30 (s, 1H), 5.65 (br dd, J = 9.4, 3.8
Hz, 1H), 5.05 (q, J = 6.4 Hz, 1H), 4.72–4.60 (m, 1H), 3.63 (br dd, J =
13.4, 3.8 Hz, 1H), 3.52–3.39 (m, 1H), 3.31–3.18 (m, 1H), 2.58–2.46 (m,
2H), 2.40–2.24 (m, 2H), 1.77 (d, J = 6.4 Hz, 3H). 13C NMR (125 MHz,
DMSO‑d6) δ 168.7, 163.5, 150.1, 132.0, 126.9, 125.6, 125.2, 117.6,
100.0, 97.4, 80.0, 64.4, 60.8, 54.1, 44.9, 21.5, 17.4. LCMS (ESI) m/z 393
[M+H]+. Rt 0.710 min (Analytical condition A). HRMS (ESI/APCI dual)
m/z calcd for C22H24N4O3 [M+H]+ 393.1921, found 393.1902.
C
24H25N5O3 [M+H]+ 432.2030, found 432.2025.
5.1.16. (S)-1-((1R,5S,6R)-6-((4-(3-((R)-2-Amino-1-(2-((S)-1-
hydroxyethyl)-1H-imidazol-1-yl)ethyl)isoxazol-5-yl)phenyl)ethynyl)-3-
azabicyclo[3.1.0]hexan-3-yl)propan-2-ol (18)
(2S)-2-Methyloxirane (0.042 mL, 0.60 mmol) was added to a solu-
tion of tert-butyl [(2R)-2-[5-(4-{[(1R,5S,6s)-3-azabicyclo[3.1.0]hexan-
6-yl]ethynyl}phenyl)-1,2-oxazol-3-yl]-2-{2-[(1S)-1-hydroxyethyl]-1H-
imidazol-1-yl}ethyl]carbamate 55 (20 mg, 0.040 mmol) in MeOH (1
mL). The mixture was stirred at 50 ◦C for 1.5 h under microwave irra-
diation. Additional (2S)-2-methyloxirane (0.042 mL, 0.60 mmol) was
added, and the mixture was stirred at 50 ◦C for 1 h under microwave
irradiation and concentrated. The residue was purified using column
chromatography on NH silica gel and eluted with MeOH/EtOAc to
obtain tert-butyl [(2R)-2-{2-[(1S)-1-hydroxyethyl]-1H-imidazol-1-yl}-2-
5.1.14. (1R,5S,6s)-6-({4-[3-({2-[(1S)-1-Hydroxyethyl]-1H-imidazol-1-
yl}methyl)-1,2-oxazol-5-yl]phenyl}ethynyl)-3-azabicyclo[3.1.0]hexane-3-
carbaldehyde (16)
N-Formylsaccharin (12 mg, 0.057 mmol) was added to a solution
of
(1S)-1-(1-{[5-(4-{[(1R,5S,6s)-3-azabicyclo[3.1.0]hexan-6-yl]
12

F. Ushiyama et al.
Bioorganic & Medicinal Chemistry 30 (2021) 115964
{5-[4-({(1R,5S,6R)-3-[(2S)-2-hydroxypropyl]-3-azabicyclo[3.1.0]
which was used in the next step without further purification. LCMS
(ESI) m/z 518 [M+H]+. Rt 0.592 min (Analytical condition A).
In step 2, the above-described product was taken up in THF (2 mL).
To this suspension, 1 mol/L aqueous NaOH (2 mL, 4 mmol) was added.
The mixture was stirred at room temperature for 2 h, neutralized with 1
mol/L hydrochloric acid, and washed with EtOAc. The separated
aqueous layer was purified using Diaion HP-20 to obtain 20 as a pale
yellow solid (19 mg, 34% over two steps). 1H NMR (400 MHz, METH-
ANOL-d4) δ 7.74 (d, J = 8.2 Hz, 2H), 7.44 (d, J = 8.2 Hz, 2H), 7.24 (s,
1H), 6.98 (s, 1H), 6.77 (s, 1H), 5.92 (br t, J = 7.2 Hz, 1H), 5.09 (q, J =
6.5 Hz, 1H), 3.58–3.34 (m, 4H), 2.91–2.80 (m, 4H), 2.34 (br t, J = 6.5
Hz, 2H), 2.06–2.00 (m, 2H), 1.88–1.75 (m, 3H), 1.66 (d, J = 6.5 Hz, 3H).
13C NMR (125 MHz, DMSO‑d6) δ 174.8, 168.7, 163.5, 150.1, 132.0,
126.9, 125.6, 125.5, 125.3, 117.6, 99.9, 94.8, 76.4, 60.8, 54.1, 53.6,
53.5, 44.9, 32.7, 26.0, 24.0, 21.5, 7.9. LCMS (ESI) m/z 490 [M+H]+. Rt
0.514 min (Analytical condition A). HRMS (ESI/APCI dual) m/z calcd
for C27H31N5O4 [M+H]+ 490.2449, found 490.2442.
hexan-6-yl}ethynyl)phenyl]-1,2-oxazol-3-yl}ethyl]carbamate
as
a
crude product, which was dissolved in MeOH (5 mL). Then, 4 mol/L HCl
in 1,4-dioxane (1.0 mL, 4.0 mmol) was added to the solution. The
mixture was stirred at room temperature for 1 h and concentrated. The
residue was basified with 20% aqueous K2CO3 and extracted with a
combined solvent of CHCl3 and MeOH. The organic extract was passed
through a phase separator and concentrated. The residue was purified
using column chromatography on NH silica gel and eluted with MeOH/
CHCl3 to obtain 18 as a pale yellow solid (19 mg, quant over two steps).
1H NMR (400 MHz, METHANOL-d4) δ 7.73 (d, J = 8.0 Hz, 2H), 7.42 (d,
J = 8.0 Hz, 2H), 7.24 (s, 1H), 6.97 (s, 1H), 6.75 (s, 1H), 5.90 (t, J = 6.9
Hz, 1H), 5.14–5.03 (m, 1H), 3.88–3.05 (m, 5H), 2.51–2.26 (m, 4H),
1.97–1.90 (m, 1H), 1.85–1.77 (m, 2H), 1.66 (d, J = 6.2 Hz, 3H), 1.11 (d,
J = 5.6 Hz, 3H). 13C NMR (125 MHz, DMSO‑d6) δ 168.7, 163.5, 150.1,
132.0, 126.9, 125.6, 125.5, 125.3, 117.6, 99.9, 94.9, 76.4, 64.5, 61.9,
60.8, 54.1, 54.1, 54.0, 44.9, 26.1, 25.9, 21.5, 21.4, 7.7. LCMS (ESI) m/z
462 [M+H]+. Rt 0.211 min (Analytical condition A). HRMS (ESI/APCI
dual) m/z calcd for C26H31N5O3 [M+H]+ 462.2500, found 462.2488.
5.2. Evaluation of the inhibitory activity on E. coli LpxC
5.1.17. 4-[(1R,5S,6s)-6-({4-[3-({2-[(1S)-1-Hydroxyethyl]-1H-imidazol-
1-yl}methyl)-1,2-oxazol-5-yl]phenyl}ethynyl)-3-azabicyclo[3.1.0]hexan-
3-yl]butanoic acid (19)
The IC50 of E. coli LpxC was determined using the synthetic lipid
substrate UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine, as
described previously with slight modifications.28,29 E. coli LpxC purified
from E. coli cells was used for the assay. An LpxC enzyme assay was
performed with reaction mixtures containing 40 mM MES (pH6.5),
0.02v/v% Brij35, 80 µM dithiothreitol, 25 nM ZnCl2, 3.1 nM E. coli LpxC
and 20 µM UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine. The
reaction mixtures were incubated for 120 min at room temperature. For
the fluorescamine assay, the reaction was terminated by the addition of
0.2 M sodium phosphate buffer and 1.0 mg/mL fluorescamine in a 1:1
vol mixture of N,N-dimethylformamide and acetonitrile. Fluorescence
(390 nm excitation/495 nm emission) was detected using an EnSpire
instrument (PerkinElmer Japan Co., Ltd., Tokyo, Japan). For the LCMS
assay, the reaction terminated by the addition of acetonitrile containing
In step 1, K2CO3 (332 mg, 2.40 mmol) and NaI (120 mg, 0.80 mmol)
were added to a mixture of (1S)-1-(1-{[5-(4-{[(1R,5S,6s)-3-azabicyclo
[3.1.0]hexan-6-yl]ethynyl}phenyl)-1,2-oxazol-3-yl]methyl}-1H-imida-
zol-2-yl)ethan-1-ol 37 (300 mg, 0.80 mmol) and ethyl 4-bromobuta-
noate (172 mg, 0.88 mmol) in DMF (7.5 mL). After stirring at 80 ◦C
for 80 min, 20% aqueous K2CO3 was added to the mixture, which was
extracted with EtOAc. The separated organic extract was washed twice
with 20% aqueous K2CO3 and concentrated. The residue was purified
using column chromatography on NH silica gel and eluted with EtOAc/
n-hexane and MeOH/CHCl3 to obtain ethyl 4-[(1R,5S,6s)-6-({4-[3-({2-
[(1S)-1-hydroxyethyl]-1H-imidazol-1-yl}methyl)-1,2-oxazol-5-yl]
phenyl}ethynyl)-3-azabicyclo[3.1.0]hexan-3-yl]butanoate as
a
pale
25
μM UDP-GlcNAc was used as an internal standard. The mixture was
yellow amorphous substance (293 mg, 75%). LCMS (ESI) m/z 245 [M +
centrifuged, and the supernatant was injected into
a
liquid
2H]2+. Rt 0.834 min (Analytical condition A).
chromatography-tandem mass spectrometry (LC-MS/MS) instrument.
The samples were separated using an Inertsil Amide column (3.0 µm, 50
mm × 2.1 mm I.D.; GLScience, Japan) using the Shimadzu HPLC system.
The mobile phase was 8 mM ammonium acetate containing 72%
acetonitrile, and the flow rate was 0.2 mL/min. MS/MS detection of
each component was performed using a TSQ Quantum system (Thermo
Fisher Scientific, USA) or a QTRAP-5500 system (SCIEX, USA) with an
electrospray interface in negative ion detection mode. The inhibition
values were calculated from the product signals of the control DMSO
samples containing no compound (0% inhibition) and samples con-
taining no substrate (100% inhibition).
In step 2, 2 mol/L NaOH solution (3.0 mL, 6.0 mmol) was added to a
solution of the above-described substance (291 mg, 0.60 mmol) in
MeOH (3 mL) and THF (3 mL). After stirring at room temperature for 2
h, the mixture was neutralized with 1 mol/L hydrochloric acid at 0 ◦C
and concentrated. The residue was purified using Diaion HP-20 to obtain
19 as a pale yellow amorphous substance (212 mg, 77%). 1H NMR (400
MHz, METHANOL-d4) δ 7.73 (d, J = 8.2 Hz, 2H), 7.44 (d, J = 8.2 Hz,
2H), 7.15 (s, 1H), 6.93 (s, 1H), 6.71 (s, 1H), 5.56–5.41 (m, 2H), 5.04 (q,
J = 6.6 Hz, 1H), 3.62 (d, J = 11.0 Hz, 2H), 3.09 (br d, J = 10.6 Hz, 2H),
3.03 (br t, J = 6.0 Hz, 2H), 2.47–2.39 (m, 2H), 2.15–2.10 (m, 2H),
1.88–1.79 (m, 3H), 1.59 (d, J = 6.6 Hz, 3H). 13C NMR (125 MHz,
DMSO‑d6) δ 174.4, 168.8, 161.8, 149.7, 132.0, 126.5, 125.7, 125.5,
125.3, 120.9, 100.1, 94.7, 76.5, 61.4, 53.5, 53.3, 40.7, 31.7, 26.0, 23.4,
21.8, 7.9. LCMS (ESI) m/z 461 [M+H]+. Rt 0.641 min (Analytical con-
dition A). HRMS (ESI/APCI dual) m/z calcd for C26H28N4O4 [M+H]+
461.2183, found 461.2169.
5.3. Evaluation of antibacterial activity
The MIC was determined using the Clinical and Laboratory Stan-
dards Institute (CLSI) broth micro-dilution method.30
5.4. In vivo models
5.1.18. 4-{(1R,5S,6s)-6-[(4-{3-[(1R)-2-Amino-1-{2-[(1S)-1-
hydroxyethyl]-1H-imidazol-1-yl}ethyl]-1,2-oxazol-5-yl}phenyl)ethynyl]-3-
azabicyclo[3.1.0]hexan-3-yl}butanoic acid (20)
All in vivo experimental protocols used in this study were approved
by the Taisho Pharmaceutical Animal Care Committee and were in
accordance with the Guidelines for Proper Conduct of Animal Experi-
ments (Science Council of Japan, 2006). Animals were maintained
under controlled temperature (23 ± 3 ◦C) and humidity (50 ± 20%)
conditions and a 12-h light/dark cycle (lights on at 07:00 h).
In step 1, 4 mol/L HCl in 1,4-dioxane (2.0 mL, 8.0 mmol) was added
to
a solution of ethyl 4-{(1R,5S,6s)-6-[(4-{3-[(1R)-2-[(tert-butox-
ycarbonyl)amino]-1-{2-[(1S)-1-hydroxyethyl]-1H-imidazol-1-yl}
ethyl]-1,2-oxazol-5-yl}phenyl)ethynyl]-3-azabicyclo[3.1.0]hexan-3-yl}
butanoate 57 (73 mg, 0.12 mmol) in MeOH (0.1 mL). The mixture was
stirred at room temperature for 1 h and concentrated to obtain ethyl 4-
{(1R,5S,6s)-6-[(4-{3-[(1R)-2-amino-1-{2-[(1S)-1-hydroxyethyl]-1H-
imidazol-1-yl}ethyl]-1,2-oxazol-5-yl}phenyl)ethynyl]-3-azabicyclo
[3.1.0]hexan-3-yl}butanoate hydrogen chloride as a crude product,
5.5. Efficacy studies
5.5.1. Murine systemic infection model
Four-week-old male ICR mice (Japan SLC, Inc., Shizuoka, Japan)
13

F. Ushiyama et al.
Bioorganic & Medicinal Chemistry 30 (2021) 115964
were inoculated intraperitoneally with 0.5 mL of bacterial suspension
including 3w/v% mucin (Sigma-Aldrich). The challenge doses of
K. pneumoniae 4102 were 3.8–5.5 × 106 CFU/mouse. The test com-
pounds were subcutaneously administered at 1 h after inoculation (n =
6). The survival rates of each group were evaluated for 7 days. The test
compounds were dissolved in 10w/v% hydroxypropyl-β-cyclodextrin
(HP-β-CD; NIHON SHOKUHIN KAKO CO., LTD., Tokyo, Japan). All the
vehicle-treated mice (10w/v% HP-β-CD) died after inoculation.
cannulated into the femoral vein. The infusion rate for pentobarbital was
8 mg/kg/h. Another polyethylene tube filled with saline was cannulated
into the jugular vein and was used for the administration of the vehicle
or test compounds. A measurement cannula filled with heparinized sa-
line connected to a pressure transducer was cannulated into the carotid
artery to measure blood pressure using a blood pressure amplifier.
Electrocardiography was performed using an electrocardiogram ampli-
fier. The anesthetized guinea pigs were intravenously administered in-
fusions of the vehicle or test compounds for 30 min. The QTc was
calculated by correcting the QT interval in Bazett’s formula. Heart rate,
QTc, systolic blood pressure, and diastolic blood pressure were recorded
as the change from the baseline value.
5.5.2. Murine lung infection model
Five-week-old female ICR mice (Japan SLC, Inc.) were used in this
study. Neutropenia was induced with the intraperitoneal administration
of 150 and 100 mg/kg of cyclophosphamide (SHIONOGI & CO., LTD.,
Osaka, Japan) at 4 days and 1 day prior to infection, respectively. Mice
were inoculated intranasally with 0.05 mL of bacterial suspension
Blood was collected at the end of the infusion period and was
centrifuged to obtain the plasma for the exposure assessment. The
plasma concentration at the end of infusion (C30min) was determined
using a LC-MS/MS system (AB SCIEX, Framingham, MA). The unbound
plasma concentration at the end of infusion (C30min, free) was calculated
by multiplying the C30min value and the unbound fraction in plasma,
which was assumed to be equal to the unbound fraction in serum.
(1.5–1.9
×
106 CFU/mouse) of K. pneumoniae ATCC BAA-2343
(blaKPC+). The test compounds were subcutaneously administered at 2
h after inoculation (n = 5). Viable cells in the lungs were counted at 2
(control) and 26 h after inoculation. The test compounds were dissolved
in 10w/v% HP-β-CD.
5.8. MTD and repeated dose studies
5.6. Protein binding assay
Exploratory MTD studies were conducted using male ICR mice
(Charles River Laboratories Japan, Inc., Kanagawa, Japan) at dose levels
of up to 200 mg/kg using a single intravenous injection via the tail vein.
For the repeated dose studies, TP0586532 was intravenously
administered to 6-week-old male and female Sprague Dawley (SD) rats
(Charles River Laboratories Japan, Inc., Kanagawa, Japan) for 4 days
(bolus, via tail vein) and to 3-year-old female cynomolgus monkeys
(Vanny Bio-Research [Cambodia] Corporation Ltd., Cambodia) for 14
days (infusion for 30 min, via saphenous vein) to assess the MTDs using
repeated dosing.
The protein binding assay was conducted using a 96-well equilib-
rium dialysis method. The pooled serum of male ICR mice was obtained
from Charles River Laboratories Japan, Inc. Guinea pigs serum was
obtained from male Hartley guinea pigs (Japan SLC, Inc.) by venous
puncture and were collected without the use of an anticoagulant.
Human serum was obtained from healthy male and female volunteers by
venous puncture and were collected without the use of an anticoagulant.
The compounds were diluted in serum to yield final concentrations of 1
μ
g/mL (human and mouse) and 500 μg/mL (guinea pig). Serum samples
were dialyzed using a dialysis membrane (molecular weight cut-off:
12000–14000 Da) against 50 mM sodium phosphate buffer at 37 ◦C
for 4 h in a 5% CO2-incubator. The concentration of the test compound
on each side of the membrane was determined using LC-MS/MS. The
serum protein binding ability was calculated using the following
equation:
5.9. hERG channel assay
An automated patch clamp method (Q-PatchHTX) (Sophion Biosci-
ence, Ballerup, Denmark) was used to evaluate the effects of 8-(meth-
ylamino)-2-oxo-1,2-dihydroquinoline derivatives on the hERG
potassium channel. Q cell (CHO hERG Duo optimized for the Q-patch)
was purchased from Sophion Bioscience, and the experiment was con-
ducted in accordance with their protocol. The IC50 values of compounds
15, 17, 18, and 19 (TP0586532) and of quinidine (positive control) on
whole-cell hERG currents were determined.
Serum protein binding (%) = (Cs ꢀ Cb)/Cs × 100,
where Cs and Cb represent the concentration of the test compound in the
serum and buffer, respectively.
The unbound fraction in guinea pig serum was calculated using the
following equation:
5.10. Protein production
Fraction unbound in serum = 1 ꢀ (Serum protein binding)/100.
P. aeruginosa LpxC(1–299) carrying the C40S mutation was
expressed and purified as reported previously.20
5.7. Cardiovascular study
5.11. Crystallization, data collection and processing
The maintenance and experimental protocols conformed to the
Guide for the Care and Use of Laboratory Animals of Taisho Pharma-
ceutical Co., Ltd.
Complexes with inhibitors were crystallized using the sitting drop
vapor diffusion method at 293 K. Crystals were flash-frozen and were
measured at a temperature of 100 K. Diffraction data for the cocrystals
was collected at either the beamline BL45XU of SPring-8 (Japan) or in-
house using a MicroMax 007 HF Generator (Rigaku) with a PILATUS
200 K (Rigaku). Data processing was performed using CrysAlisPro
(Rigaku). The structure was determined using molecular replacement
with MolRep and the structure of the PDB code of 3UHM as the search
model. The structure was refined with Refmac5. The data collection and
refinement statistics are summarized in the Supplementary Data
(Table S1).
Two cardiovascular studies were conducted to assess the effects of
the test compounds. Compounds 15 and 19 (TP0586532) were assessed
in the first study, and compounds 17 and 18 were assessed in the second.
Male Hartley guinea pigs were obtained from Japan SLC, Inc. (Shizuoka,
Japan). An aqueous solution (concentration, 11w/v%; pH4) of sulfo-
butyl ether-β-cyclodextrin (SBE-β-CD; Cyclolab Cyclodextrin Research
and Development Laboratory Ltd., Budapest, Hungary) was used in the
vehicle control group in both studies.
Six- to seven-week-old guinea pigs were anesthetized with pento-
barbital (60 mg/kg, double dilution with saline, intraperitoneal
administration) and placed in a supine position on a thermal heated
plate during surgery. A tube was cannulated into the trachea and con-
nected to a ventilator. The artificial respiration conditions were 15 mL/
kg and 60 times/min. A polyethylene tube filled with pentobarbital was
Declaration of Competing Interest
The authors declare that they have no known competing financial
14

F. Ushiyama et al.
Bioorganic & Medicinal Chemistry 30 (2021) 115964
12 Supuran CT, Carta F, Scozzafava A. Metalloenzyme inhibitors for the treatment of
Gram-negative bacterial infections: a patent review (2009–2012). Expert Opin Ther
Pat. 2013;23:777–788. https://doi.org/10.1517/13543776.2013.777042.
13 Kalinin DV, Holl R. LpxC inhibitors: a patent review (2010–2016). Expert Opin Ther
Pat. 2017;27:1227–1250. https://doi.org/10.1080/13543776.2017.1360282.
14 Shen S, Kozikowski AP. Why hydroxamates may not be the best histone deacetylase
inhibitors—what some may have forgotten or would rather forget? ChemMedChem.
2016;11:15–21. https://doi.org/10.1002/cmdc.201500486.
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgments
The authors thank Dr. Takashi Yoshizumi for his contributions to the
project, Maho Honda-Yamagishi and Toru Yamaguchi-Sasaki for the
preparation of the compounds, Reiko Wada for performing the hERG
channel assay, Dr. Atsushi Okada for recording the nuclear magnetic
resonance spectra, and Akira Higuchi for recording the high-resolution
mass spectra.
15 Smith GF. Designing drugs to avoid toxicity. Prog Med Chem. 2011;50:1–47. https://
doi.org/10.1016/B978-0-12-381290-2.00001-X.
16 Serio AW, Kubo A, Lopez S, et al. Structure, Potency AND Bactericidal Activity
of ACHN-975, a First-In-Class LpxC Inhibitor. In 53rd Interscience Conference on
Antimicrobial Agents and Chemotherapy; Achaogen; 2013 [poster #F-1226].
17 Shoji M, Suzumura Y, Fujiwara M, Kurosaki C, Nozaki Y, Mizunaga S. T-1228 (RC-
01), A New LpxC Inhibitor Exhibiting Potent Activity against Multidrug-Resistant
Gram-Negative Pathogens: Design, Synthesis and Structure-Activity Relationship.
Presented at ASM Microbe, June 20ꢀ 24, San Francisco, CA, USA, Sunday-AAR-740;
2019.
Appendix A. Supplementary material
18 US National Library of Medicine. ClinicalTrials.gov, https://clinicaltrials.gov/ct2/sh
ow/NCT01870245; 2013.
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bmc.2020.115964.
19 US National Library of Medicine. ClinicalTrials.gov, https://clinicaltrials.gov/ct2/sh
ow/NCT03832517; 2019.
20 Yamada Y, Takashima H, Walmsley DL, et al. Fragment-based discovery of novel
non-hydroxamate LpxC inhibitors with antibacterial activity. J Med Chem. 2020;63:
14805–14820. https://doi.org/10.1021/acs.jmedchem.0c01215.
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