Synthesis and evaluation of sulfonyl piperazine LpxH inhibitors

Article abstract of DOI:10.1016/j.bioorg.2020.104055

The UDP-2,3-diacylglucosamine pyrophosphate hydrolase LpxH is essential in lipid A biosynthesis and has emerged as a promising target for the development of novel antibiotics against multidrug-resistant Gram-negative pathogens. Recently, we reported the c

Full text of DOI:10.1016/j.bioorg.2020.104055

BioorganicChemistry102(2020)104055
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Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Synthesis and evaluation of sulfonyl piperazine LpxH inhibitors
Seung-Hwa Kwaka,1, C. Skyler Cochranea,1, Amanda F. Ennisa, Won Young Lima,
⁎
⁎
Caroline G. Webstera, Jae Chob, Benjamin A. Fentonb, Pei Zhoua,b, , Jiyong Honga,c,
a Department of Chemistry, Duke University, Durham, NC 27708, United States
b Department of Biochemistry, Duke University Medical Center, Durham, NC 27710, United States
c Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, NC, 27710, United States
A R T I C L E I N F O
A B S T R A C T
Keywords:
The UDP-2,3-diacylglucosamine pyrophosphate hydrolase LpxH is essential in lipid A biosynthesis and has
emerged as a promising target for the development of novel antibiotics against multidrug-resistant Gram-ne-
gative pathogens. Recently, we reported the crystal structure of Klebsiella pneumoniae LpxH in complex with 1
(AZ1), a sulfonyl piperazine LpxH inhibitor. The analysis of the LpxH-AZ1 co-crystal structure and ligand dy-
namics led to the design of 2 (JH-LPH-28) and 3 (JH-LPH-33) with enhanced LpxH inhibition. In order to harness
our recent findings, we prepared and evaluated a series of sulfonyl piperazine analogs with modifications in the
phenyl and N-acetyl groups of 3. Herein, we describe the synthesis and structure–activity relationship of sulfonyl
piperazine LpxH inhibitors. We also report the structural analysis of an extended N-acyl chain analog 27b (JH-
LPH-41) in complex with K. pneumoniae LpxH, revealing that 27b reaches an untapped polar pocket near the di-
manganese cluster in the active site of K. pneumoniae LpxH. We expect that our findings will provide designing
principles for new LpxH inhibitors and establish important frameworks for the future development of antibiotics
against multidrug-resistant Gram-negative pathogens.
Antibiotics
Gram-negative bacteria
Lipid A
LpxH
Sulfonyl piperazine
Structure–activity relationship
1. Introduction
Lipid A is also an active component of the bacterial endotoxin re-
sponsible for Gram-negative septic shock during bacterial infection. As
Nosocomial infection by multidrug-resistant Gram-negative patho-
gens is one of the greatest global health challenges [1,2]. However, the
lack of financial return on investment has resulted in pharmaceutical
companies dropping out from the antibacterial field. As a consequence,
no new class of antibiotics for treating Gram-negative bacteria has been
approved since the 1980s [1]. At the same time, widespread antibiotic
resistance has emerged as a major threat to global health. In light of
these issues, the Emerging Infections Network and the WHO cited the
lack of both treatment options for Gram-negative bacteria and devel-
opment pipelines to be the greatest areas of unmet need [1,2]. There-
fore, an urgent need exists for a fundamentally new class of anti-mi-
crobial therapeutics against Gram-negative pathogens, for which there
is no established resistance mechanism.
constitutive biosynthesis of lipid A is required for bacterial viability and
fitness in the human host [3–5], essential lipid A enzymes are excellent
novel antibiotic targets.
Among the nine enzymes involved in lipid A biosynthesis, three
functional orthologs (LpxH in β- and γ-proteobacteria [6], LpxI in α-
proteobacteria [7], and LpxG in Chlamydiae [8]) carry out the cleavage
of the pyrophosphate group of UDP-2,3-diacylglucosamine (UDP-
DAGn) to form lipid X, but they never co-exist. LpxH and LpxG are
unique members of the metal-dependent calcineurin-like phosphoes-
terase (CLP) family, though they share limited sequence similarity [8];
LpxI, on the other hand, is structurally and mechanistically unrelated to
LpxH and LpxG [9,10]. Among these three enzymes, LpxH is most
widespread, functioning in the vast majority of WHO priority Gram-
negative pathogens, including Pseudomonas aeruginosa, Acinetobacter
baumannii, Klebsiella pneumoniae, Escherichia coli, Haemophilius influ-
enzae, and Neisseria gonorrhoeae. Therefore, LpxH inhibitors are ideally
positioned to overcome this emerging public health crisis.
Gram-negative bacteria are characterized by the presence of a un-
ique cell wall component known as lipopolysaccharide (LPS) or lipoo-
ligosaccharide (LOS) in the bacterial outer membrane. Lipid A, a glu-
cosamine-based phospholipid, is the hydrophobic anchor of LPS/LOS.
⁎ Corresponding authors at: Department of Chemistry, Duke University, Durham, NC 27708, United States and Department of Biochemistry, Duke University
Medical Center, Durham, NC 27710, United States.
E-mail addresses: peizhou@biochem.duke.edu (P. Zhou), jiyong.hong@duke.edu (J. Hong).
1 S.-H.K. and C.S.C. contributed equally to this work.
https://doi.org/10.1016/j.bioorg.2020.104055
Received 19 May 2020; Received in revised form 8 June 2020; Accepted 26 June 2020
Availableonline30June2020
0045-2068/©2020ElsevierInc.Allrightsreserved.

S.-H. Kwak, et al.
BioorganicChemistry102(2020)104055
Fig. 1. Sulfonyl piperazine antibiotics inhibit LpxH of the Raetz pathway of lipid A biosynthesis.
A small molecule LpxH inhibitor with a sulfonyl piperazine scaffold
LpxH inhibition. We also envisioned that an extended N-acyl chain of 1
and 3 may reach the di-manganese metal cluster in the active site of
LpxH, leading to LpxH inhibitors with improved potency. We antici-
pated that our effort would help the understanding of the effect of
various substituents on LpxH inhibition. Such information will allow
structural modifications to increase potency and specificity and im-
prove drug performance.
(referred to as AZ1 below; chemical structure shown in Fig. 1) was
discovered to display antibiotic activity against efflux-deficient E. coli
strains [11]. We prepared and biochemically evaluated a series of sul-
fonyl piperazine analogs, and our biochemical characterization estab-
lished a preliminary structure–activity relationship (SAR) and identified
the pharmacophore of this series of LpxH inhibitors [12]. Building upon
this work, we recently reported the first crystal structure of K. pneu-
moniae LpxH in complex with AZ1 (1) [13]. We showed that AZ1 fits
into the L-shaped acyl chain-binding chamber of LpxH with its indoline
ring situated adjacent to the active site, its sulfonyl group adopting a
sharp kink, and its N-CF3-phenyl substituted piperazine group reaching
out to the far side of the LpxH acyl chain-binding chamber. The dis-
covery of two 19F signals of the LpxH-bound AZ1 led us to propose a
model of CF3-phenyl flipping, which resulted in the design of AZ1 de-
rivatives with the phenyl ring doubly substituted with CF3 and chloro
groups, such as JH-LPH-28 and JH-LPH-33 (2 and 3, respectively;
chemical structures shown in Fig. 1) with enhanced potency in enzy-
matic assays [13]. These designed compounds displayed striking im-
provement in antibiotic activity over AZ1 against wild-type K. pneu-
moniae, and co-administration with outer membrane permeability
enhancers significantly sensitizes E. coli to designed LpxH inhibitors.
Encouraged by the promising antibacterial activity of 3, we set off to
further explore the SAR of 3. Herein, we describe our efforts toward the
synthesis and SAR study of second-generation sulfonyl piperazine
analogs with modifications in the head and tail regions in order to
further enhance the antibacterial activity of 3. We also report an LpxH
inhibitor that occupies a polar binding pocket of K. pneumoniae LpxH,
which has never been exploited before.
2.1.1. Analogs with aryl group modifications
To gain insights into the importance of the m-chloro group of 3 in
LpxH inhibition and to evaluate the effect of the size at m-position of
the phenyl ring of 3, we prepared aryl group analogs by replacing the
m-chloro group of 3 with various functional groups, including Br, CH3,
and CF3 (Scheme 1). Our previous synthesis of 3 [13] provided the basis
for the synthesis of m-bromo analog 9a, m-methyl analog 9b, and m-
trifluoromethyl analog 9c (Scheme 1A). Starting from commercially
available 1,3-dibromo-5-(trifluoromethyl)benzene (4a), Pd-mediated
coupling of 4a with 1-Boc-piperazine (5) provided the Boc-protected N-
aryl piperazine 6a. Boc deprotection of 6a by treatment with TFA fol-
lowed by coupling of the resulting piperazine 7a with commercially
available 1-acetyl-5-indolinesulfonyl chloride (8) in the presence of
Et3N proceeded smoothly to afford the desired m-bromo analog 9a in
43% for 2 steps. The m-methyl analog 9b and the m-trifluoromethyl
analog 9c were also prepared in a similar manner starting from the
commercially available 1-bromo-3-methyl-5-(trifluoromethyl)benzene
(4b) and 1,3-bis(trifluoromethyl)-5-bromobenzene (4c), respectively.
To evaluate the effect of symmetrical substituents, we prepared the
m-difluoro analog 13a and the m-dichloro analog 13b (Scheme 1B).
Commercially available 1-bromo-3,5-difluorobenzene (10) was coupled
to 1-Boc-piperazine (5) to afford the N-aryl piperazine 11. Boc depro-
tection of 11 followed by coupling of the resulting piperazine 12a to 1-
acetyl-5-indolinesulfonyl chloride (8) completed the synthesis of the m-
difluoro analog 13a. Similarly, the m-dichloro analog 13b was prepared
by coupling commercially available 1-(3–5-dichlorophenyl)piperazine
(12b) with 8.
2. Results and discussion
2.1. Chemistry
AZ1 (1) and JH-LPH-33 (3) consist of three parts (Fig. 1): the sub-
stituted phenyl group, sulfonyl piperazine linker, and N-acetyl indoline
group. Following our previous report that the replacement of the m-
hydrogen of the phenyl group of 1 with a m-chloro substituent sig-
nificantly enhanced the LpxH inhibition and antibacterial activity of 1
[13], we set out to systematically derivatize the substituents of the
phenyl group in order to identify the optimal substituent pattern for
2.1.2. Analogs with indoline modifications
We also synthesized an indole analog 17 and an N-methanesulfonyl
group analog 19 by replacing the indoline and N-acetyl group of 3 with
an indole group and a methanesulfonyl group, respectively. 1-(3-
Chloro-5-(trifluoromethyl)phenyl)piperazine (14) [13] was coupled to
2

S.-H. Kwak, et al.
BioorganicChemistry102(2020)104055
Scheme 2. Synthesis of analogs with indoline modifications.
carboxylic acid 22 and then coupled 22 with NH2OTBS. The final TBS
deprotection of 23 by TFA successfully afforded the extended N-acyl
chain hydroxamic acid analog 24. To gain insights into the effect of
chain length on LpxH inhibition, we further elongated the acyl chain by
replacing methyl 4-aminobutanoate with methyl 5-aminopentanoate
and prepared the analogs 27a and 27b (Scheme 3B).
2.2. Analysis of structure–activity relationships
2.2.1. Substituted phenyl and indoline analogs
Scheme 1. Synthesis of analogs with aryl group modifications.
After the completion of analog synthesis, we biochemically char-
acterized the K. pneumoniae LpxH inhibition by these analogs at 0.1 μM
using the nonradioactive, colorimetric malachite green assay that we
had previously reported [12]. Among the tested analogs, nearly all of
the compounds show comparable or better activity than 1 (AZ1, 22%
inhibition; Table 1). The extended N-acyl chain analog 27b (JH-LPH-
41) showed the strongest inhibition of LpxH activity and inhibited
~64% of LpxH activity. Such an activity is only slightly worse than 3
(JH-LPH-33, 79% inhibition), but it is better than 2 (JH-LPH-28, 48%
inhibition). Several other analogs, including 9a (57% inhibition), 9b
(50% inhibition), 13b (59% inhibition), and 17 (48% inhibition), also
showed noticeable LpxH inhibition, whereas 9c (16% inhibition), 19
(12% inhibition), and 24 (15% inhibition) showed significantly lower
activity than 3, but their activities were comparable to 1 (Table 1).
The LpxH activity assay data provided several valuable insights into
the SAR of sulfonyl piperazine LpxH inhibitors (Fig. 2). Among the
phenyl group analogs of 3 with a second m-substituent of the m-tri-
fluoromethyl substituted phenyl ring (e.g., m-hydrogen substituted
analog 1, m-fluoro substituted analog 2, m-methyl substituted analog
9b, and m-chloro substituted analog 3), there is a general trend of in-
creasing potency following the increase in the volume of substituents
(H < F < CH3 < Cl), except for m-bromo substituted analog 9a and
m-trifluoromethyl substituted analog 9c, which may have become too
bulky for the buried acyl chain chamber to tolerate [13].
1H-indole-5-sulfonyl chloride (15) to give 16 (Scheme 2A). N-Acet-
ylation of the indole ring of 16 by treatment with Ac2O completed the
synthesis of the indole analog 17. Compound 3 was converted to the N-
methanesulfonyl group analog 19 by Ac deprotection under acidic
conditions followed by N-methanesulfonylation of the resulting indo-
line.
2.1.3. Analogs with extended N-acyl chains
In order to explore the role of the N-acetyl group of 3 in LpxH in-
hibition, we prepared several sulfonyl piperazine compounds with ex-
tended N-acyl chains. Since LpxH contains two manganese metals in the
active site, we hypothesized that a sulfonyl piperazine LpxH inhibitor
that exploits the chelation to the di-manganese metal cluster would be
more potent than the parent compounds, 1 or 3. Therefore, we capped
the N-acyl group with a hydroxamic acid since hydroxamic acid is a
well-characterized manganese-chelating group [14]. We envisioned the
hydroxamic acid group would tightly bind to the manganese metals in
the LpxH active site and improve the binding affinity of LpxH in-
hibitors. Since the N-acetylsulfanilyl analog of 1 was active in our
previous study [12], we modeled our new extended N-acyl chain ana-
logs based on a sulfanilamide scaffold. Starting from the known 4-((4-
(3-(trifluoromethyl)phenyl)piperazin-1-yl)sulfonyl)aniline (20a) [12],
we successfully introduced a urea linkage with methyl 4-aminobu-
tanoate in the presence of CDI (Scheme 3A). To install a hydroxamic
acid group, we hydrolyzed the methyl ester 21 to the corresponding
A similar trend was also observed when both m-positions are sub-
stituted with fluoro (analog 13a), chloro (analog 13b), and tri-
fluoromethyl (analog 9c) groups, with the dichloro substituted analog
13b displaying better activity (59% inhibition) than the difluoro
3

S.-H. Kwak, et al.
BioorganicChemistry102(2020)104055
When the N-acetyl group of the indoline ring of 3 was replaced with a
methanesulfonyl group (analog 19), the activity dropped significantly
(12% inhibition), indicating that the N-acetyl group of 3 is critical to
the LpxH inhibition.
2.2.2. Extended N-acyl chain analogs
As our previous structural analysis of LpxH in complex with 1 and 3
has shown that the active site of LpxH was not occupied by sulfonyl
piperazine LpxH inhibitors such as 1 or 3 [13], we synthesized analogs
with extended acyl chains to test the feasibility of expanding the
compound-LpxH interaction to the active site. For proof-of-concept
studies, we selected the sulfanilamide scaffold and attached an ex-
tended acyl chain to the aniline nitrogen. Three compounds (24, 27a,
and 27b) were synthesized containing different lengths of acyl chains
with a terminal hydroxamate group designed to chelate the di-manga-
nese cluster in the active site. The shorter N-acyl chain analog 24
showed slightly lower activity (15% inhibition) than 1 (22% inhibi-
tion), whereas extending the acyl chain of 24 by one methylene group
(analog 27a, 30% inhibition) improved the potency over 1. Considering
that aryl replacement of the indoline ring always results in reduced
activity [12], attaching an acyl chain generally improves the potency of
the compound. Combining the long acyl chain with the phenyl ring
doubly substituted with trifluoromethyl and chloro groups yielded the
most active compound of this series 27b (JH-LPH-41) that inhibited
64% activity of KpLpxH at 0.1 μM compound concentration. The ac-
tivity of 27b is only slightly worse than 3 (79% inhibition). Despite the
excellent in vitro activity of 27b, its long acyl chain with many rotatable
bonds negatively impacted the antibiotic activity of 27b, yielding an
unimpressive MIC of 32 μg/mL against K. pneumoniae. The MIC value
27b was still an improvement over 1 (MIC > 64 μg/mL), but is sig-
nificantly worse than 3 (1.6 μg/mL) [13]. Such a result was attributed
to the poor membrane permeability of 27b due to the highly flexible
and hydrophobic nature of the extended acyl chain of 27b.
2.3. Structure of K. Pneumoniae LpxH in complex with 27b (JH-LPH-41)
In order to gain a better understanding of the nature of the inter-
action of 27b with K. pneumoniae LpxH, we determined the co-crystal
structure of the K. pneumoniae LpxH/27b complex at 1.85 Å (Fig. 3,
Figure S1, and Table S1). We found that, similar to previously reported
sulfonyl piperazine compound structures, [13] 27b also occupies the
hydrophobic substrate-binding chamber between the calcineurin-like
phosphatase (CLP) domain and the insertion cap domain (Fig. 3A).
However, its N-acyl chain snakes into the active site as designed: the
acyl chain picks up additional hydrophobic interactions with Y125 of
the insertion cap and I171 on the loop connecting the cap back to the
CLP core domain (Fig. 3B). Most unexpectedly, the hydroxamate group
did not chelate the di-manganese cluster as we designed; instead, its
carbonyl group and N-hydroxyl group form two hydrogen bonds with
the backbone amide and carbonyl group of M172 of the same loop as
I171 (Fig. 3B). Although these hydroxamate-containing acyl chains did
not reach the metal cluster as we designed, they demonstrate the fea-
sibility to expand molecular interactions into the active site of LpxH.
3. Conclusion
We recently reported the first crystal structure of K. pneumoniae
LpxH in complex with AZ1 (1), a sulfonyl piperazine LpxH inhibitor,
and the identification of a more potent LpxH inhibitor, JH-LPH-33 (3).
In order to further elaborate the SAR of these compounds, we prepared
and biochemically characterized a series of sulfonyl piperazine LpxH
inhibitors. Our SAR study revealed an important correlation between
the compound activity and the volume of the functional groups at the
meta-position of the trifluoromethyl substituted distal phenyl ring, with
the compound potency increasing from H, F, CH3 and achieving max-
imal activity at Cl substitution. Further increases in volume with Br and
Scheme 3. Synthesis of analogs with extended acyl chains.
substituted analog 13a (39% inhibition) and the difluoromethyl sub-
stituted analog 9c (16% inhibition).
Replacement of the indoline of 3 with indole (analog 17) slightly
decreased the potency (48% inhibition for 17 vs 79% inhibition for 3).
4

S.-H. Kwak, et al.
BioorganicChemistry102(2020)104055
Table 1
Specific activity K. pneumoniae LpxH in the presence of sulfonyl piperazine LpxH inhibitors.
Compounds
Structure
0.1 μM compound
Activity (μmol/min/mg)a
Percentage Activity
Percentage Inhibition
DMSO
298.7
9.0
100
78b
3
0
1 (AZ1)
22
2 (JH-LPH-28)
52b
21b
43
50
84
61
41
52
88
85
70
36
48
79
57
50
16
39
59
48
12
15
30
64
3 (JH-LPH-33)
9a
128.3
148.8
252.1
182.4
121.7
156.1
262.7
253.3
210.0
107.6
12
4
3
9b
9.3
9c
38.9
16.7
25.1
15.2
34.7
20.0
26.3
15.6
13
6
13a
13b
8
17
5
19
12
7
24
27a
9
27b (JH-LPH-41)
5
a
b
Data are mean values of three independent experiments. Errors represent standard error.
Values calculated from previously reported IC50 curves [13] obtained under identical assay conditions.
5

S.-H. Kwak, et al.
BioorganicChemistry102(2020)104055
equivalent resonances, br = broad). Electrospray ionization (ESI) mass
spectrometry (MS) was recorded with an Agilent 1100 series (LC/MSD
trap) spectrometer in order to obtain the molecular masses of com-
pounds. The purity of final compounds used in bioassays was de-
termined by NMR and was found to be > 95%.
tert-Butyl 4-(3-bromo-5-(trifluoromethyl)phenyl)piperazine-1-car-
boxylate (6a). Anhydrous toluene (4.8 mL) was added to a mixture of
1,3-dibromo-5-(trifluoromethyl)benzene (4a, 500 mg, 1.6 mmol), 1-
Boc-piperazine (5, 596 mg, 3.2 mmol), NaOt-Bu (307 mg,
3.2 mmol), JohnPhos (71.6 mg, 0.24 mmol), and Pd2(dba)3 (68.6 mg,
0.08 mmol). Argon (Ar) was bubbled through the reaction mixture for
15 min before the reaction mixture was heated to reflux for 14 h. The
reaction mixture was concentrated in vacuo, dissolved in CH2Cl2/MeOH
(1/1), and filtered through a pad of Celite. The filtrate was concentrated
in vacuo and purified by column chromatography (silica gel, hexanes/
EtOAc, 5/1) to afford 6a (100 mg, 14%) as a yellow solid: 1H NMR
(400 MHz, CDCl3) δ 7.20 (s, 1H), 7.15 (s, 1H), 7.01 (s, 1H), 3.61–3.53
(m, 4H), 3.23–3.15 (m, 4H), 1.48 (s, 9H); HRMS (ESI) m/z 431.0545
[(M + Na)+ calcd for C16H20BrF3N2O2 431.0553].
Fig. 2. SAR of phenyl and indoline substitutions.
CF3 substitutions resulted in reduced activity. A similar trend was ob-
served with the symmetrical substitutions at the meta-positions of the
distal phenyl ring. Moreover, our efforts resulted in the identification of
27b (JH-LPH-41) that occupies a previously untapped pocket near the
di-manganese cluster in the LpxH active site. Despite having a proximal
phenyl group instead of an indoline group, the acyl chain extension of
27b into the active site nearly restored the in vitro activity of 27b to that
of 3 with an indoline group. These findings establish a clear SAR of the
m-substituted distal phenyl ring and demonstrate the feasibility to ex-
pand drug interactions to the active site of LpxH. We anticipate our
study will ultimately contribute to developing more potent and selec-
tive LpxH inhibitors for multidrug-resistant Gram-negative pathogens.
1-(3-Bromo-5-(trifluoromethyl)phenyl)piperazine (7a). To a cooled
(0 °C) solution of 6a (100 mg, 0.22 mmol) in anhydrous CH2Cl2 (1 mL)
was added dropwise TFA (0.5 mL). After stirring at 25 °C for 2.5 h, the
solvents were removed under reduced pressure to give 7a (100 mg) as
an orange solid. Compound 7a was used in the following step without
further purification: 1H NMR (400 MHz, CDCl3) δ 9.35 (br s, 1H), 7.34
(s, 1H), 7.21 (s, 1H), 7.05 (s, 1H), 3.50 (t, J = 5.1 Hz, 4H), 3.45–3.36
(m, 4H); HRMS (ESI) m/z 309.0216 [(M + H)+ calcd for C11H12BrF3N2
309.0209].
4. Materials and methods
1-(5-((4-(3-Bromo-5-(trifluoromethyl)phenyl)piperazin-1-yl)sul-
4.1. Synthesis of sulfonyl piperazine LpxH inhibitors
fonyl)indolin-1-yl)ethan-1-one (9a).
A solution of 7a (100 mg,
0.32 mmol) and Et3N (50 μL, 0.38 mmol) in anhydrous 1,4-dioxane
(0.22 M, 1.5 mL) was heated to 60 °C. 1-Acetylindoline-5-sulfonyl
chloride (8, 41.5 mg, 0.16 mmol) in anhydrous 1,4-dioxane (1 mL) was
added to the reaction mixture. After stirring at 60 °C for 3 h, the re-
action mixture was cooled to 25 °C. The reaction was quenched by an
addition of H2O and the resulting mixture was extracted with EtOAc.
The combined organic layers were washed with brine, dried over an-
hydrous Na2SO4, and concentrated in vacuo. The residue was purified
by column chromatography (silica gel, hexanes/EtOAc, 1/1) to afford
9a (37 mg, 43% for 2 steps) as a white solid: 1H NMR (400 MHz, CDCl3)
δ 8.34 (d, J = 8.5 Hz, 1H), 7.63 (d, J = 8.4 Hz, 1H), 7.57 (s, 1H), 7.21
(s, 1H), 7.09 (s, 1H), 6.95 (s, 1H), 4.16 (t, J = 8.6 Hz, 2H), 3.34–3.24
(m, 6H), 3.17–3.11 (m, 4H), 2.27 (s, 3H); HRMS (ESI) m/z 532.0517
[(M + H)+ calcd for C21H21BrF3N3O3S 532.0512].
General chemistry procedures. All reactions were conducted in oven-
dried glassware under nitrogen or argon. Unless otherwise stated all
reagents were purchased from commercial suppliers and used without
further purification. All solvents were American Chemical Society
(ACS) grade or better and used without further purification except
tetrahydrofuran (THF), which was freshly distilled from sodium/ben-
zophenone each time before use. Analytical thin layer chromatography
(TLC) was performed with glass backed silica gel (60 Å) plates with
fluorescent indication (Whatman). Visualization was accomplished by
UV irradiation at 254 nm and/or by staining with p-anisaldehyde so-
lution. Flash column chromatography was performed by using silica gel
(particle size 230–400 mesh, 60 Å). All 1H spectra were recorded with a
Varian 400 spectrometer. All 1H NMR δ values are given in parts per
million (ppm) and are referenced to the residual solvent signals (CDCl3:
δ = 7.26 ppm, CD3OD: δ = 3.31 ppm, CD3COCD3: δ = 2.05 ppm).
Coupling constants (J) are given in Hertz (Hz) and multiplicities are
tert-Butyl 4-(3-methyl-5-(trifluoromethyl)phenyl)piperazine-1-car-
boxylate (6b). To a solution of 1-bromo-3-methyl-5-(trifluoromethyl)
benzene (4b, 50 mg, 0.21 mmol), 1-Boc-piperazine (5, 51 mg,
0.27 mmol), and NaOt-Bu (30 mg, 0.31 mmol) in toluene (0.63 mL) was
indicated using the conventional abbreviation (s
=
singlet,
d = doublet, t = triplet, q = quartet, m = multiplet or overlap of non-
Fig. 3. Expansion of the LpxH inhibitor interaction into the active site of LpxH. (A) Side view of the KpLpxH/27b (JH-LPH-41) complex. LpxH is shown in the cartoon
model, the catalytic di-manganese cluster is shown in the sphere model, and 27b is shown in the stick model. Location of the cap domain and the CLP core domain is
labeled. (B) Top view of the KpLpxH/27b complex. Hydrophobic residues of LpxH (Y125 and I171) interacting with the N-acyl chain of 27b are labeled. The
hydrogen bonds between the hydroxamate group of 27b and the backbone of M172 of LpxH are indicated by dashed lines.
6

S.-H. Kwak, et al.
BioorganicChemistry102(2020)104055
added JohnPhos (6.3 mg, 0.02 mmol, 10 mol%), and Pd2(dba)3
(9.2 mg, 0.01 mmol, 5 mol%). Argon (Ar) was bubbled through the
reaction mixture for 15 min and then the reaction was heated to reflux
for 15 h. The reaction mixture was concentrated in vacuo, dissolved in
CH2Cl2/MeOH (1/1), and filtered through a pad of Celite. The residue
was purified by column chromatography (silica gel, hexanes/EtOAc,
20/1) to afford 6b (58 mg, 81%): 1H NMR (400 MHz, CDCl3) δ 6.92 (s,
2H), 6.87 (s, 1H), 3.62–3.51 (m, 4H), 3.21–3.08 (m, 4H), 2.35 (s, 3H),
1.47 (s, 9H).
J = 8.5 Hz, 2H), 3.36–3.34 (m, 4H), 3.28 (t, J = 8.8 Hz, 2H), 3.16–3.15
(m, 4H), 2.27 (s, 3H); HRMS (ESI): m/z 522.1281 [(M + H)+ calcd for
C
22H21F6N3O3S 522.1289].
tert-Butyl 4-(3,5-difluorophenyl)piperazine-1-carboxylate (11).
Toluene (4 mL) was added to a mixture of 1-bromo-3,5-difluorobenzene
(10, 250 mg, 1.3 mmol), 1-Boc-piperazine (5, 315 mg, 1.69 mmol),
NaOt-Bu (187 mg, 1.95 mmol), JohnPhos (39 mg, 10 mol%), and
Pd2(dba)3 (60 mg, 5 mol%). Argon (Ar) was bubbled through the re-
action mixture for 30 min, and the reaction mixture was refluxed for
15 h. The reaction mixture was concentrated in vacuo and the residue
was dissolved in CH2Cl2/MeOH (1/1), filtered through a pad of Celite,
and concentrated in vacuo. The residue was purified by column chro-
matography (silica gel, hexanes/EtOAc, 20/1) to afford 11 (436 mg,
quantitative) as a solid: 1H NMR (400 MHz, CDCl3) δ 6.36 (d,
J = 8.8 Hz, 2H), 6.27 (t, J = 8.8 Hz, 1H), 3.60–3.52 (m, 4H),
3.20–3.11(m, 4H), 1.48 (s, 9H).
1-(3-Methyl-5-(trifluoromethyl)phenyl)piperazine (7b). To a solu-
tion of 6b (58 mg, 0.17 mmol) in CH2Cl2 (0.85 mL) was added TFA
(0.33 mL). The reaction mixture was stirred at 25 °C for 2 h and then
was concentrated in vacuo to afford 7b (41 mg). Compound 7b was used
in the following step without further purification: 1H NMR (400 MHz,
CDCl3) δ 7.03 (s, 1H), 6.93 (s, 1H), 6.88 (s, 1H), 3.60–3.46 (m, 4H),
2.58–2.40 (m, 4H), 2.37 (s, 3H).
1-(5-((4-(3-Methyl-5-(trifluoromethyl)phenyl)piperazin-1-yl)sul-
1-(3,5-Difluorophenyl)piperazine (12a). TFA (1.4 mL) was added to
a solution of 11 (207 mg, 0.69 mmol) in CH2Cl2 (3.5 mL), and the
resulting mixture was stirred at 25 °C for 1 h. The reaction mixture was
concentrated in vacuo to afford 12a. Compound 12a was used in the
following step without further purification: 1H NMR (400 MHz, CD3OD)
δ 6.61 (d, J = 10.0 Hz, 2H), 6.42 (t, J = 9.0 Hz, 1H), 3.48–3.45 (m,
4H), 3.36–3.33 (m, 4H); HRMS (ESI) m/z 199.1041 [(M + H)+ calcd
for C10H12F2N2 199.1041].
fonyl)indolin-1-yl)ethan-1-one (9b).
A solution of 7b (27 mg,
0.11 mmol) and Et3N (17 μL, 0.13 mmol) in 1,4-dioxane (1.63 mL) was
heated to 60 °C. To this solution, 1-acetylindoline-5-sulfonyl chloride
(8, 29 mg, 0.11 mmol) was added and the resulting mixture was stirred
at 60 °C for 3 h followed by at 25 °C for 14 h. The reaction was
quenched by an addition of H2O and the aqueous layer was extracted
with EtOAc. The combined organic layers were dried over anhydrous
Na2SO4 and concentrated in vacuo. The crude product was purified by
column chromatography (silica gel, hexanes/EtOAc, 2/1) to afford 9b
(26.3 mg, 51% for 2 steps): 1H NMR (400 MHz, CDCl3) δ 8.33 (d,
J = 7.9 Hz, 1H), 7.62 (d, J = 7.9 Hz, 1H), 7.56 (s, 1H), 6.93 (s, 1H),
6.85 (s, 1H), 6.82 (s, 1H), 4.14 (t, J = 8.6 Hz, 2H), 3.31–3.22 (m, 6H),
3.18–3.10 (m, 4H), 2.32 (s, 3H), 2.25 (s, 3H); HRMS (ESI) m/z
468.1554 [(M + H)+ calcd for C22H24F3N3O3S 468.1563].
1-(5-((4-(3,5-Difluorophenyl)piperazin-1-yl)sulfonyl)indolin-1-yl)
ethan-1-one (13a). A solution of 12a (50 mg, 0.25 mmol) and Et3N
(40 μL, 0.3 mmol) in 1,4-dioxane (3.7 mL) was heated to 60 °C and 1-
acetyl-5-indolinesulfonyl chloride (8, 65 mg, 0.25 mmol) was added.
After stirring at 60 °C for 3 h, the reaction mixture was cooled to 25 °C
and stirred for additional 14 h. The reaction was quenched by an ad-
dition of H2O. The reaction mixture was extracted with EtOAc. The
combined organic layers were dried over anhydrous Na2SO4 and con-
centrated in vacuo. The residue was purified by column chromato-
graphy (silica gel, hexanes/EtOAc, 1/1) to afford 13a (48 mg, 46% for 2
steps) as a white solid: 1H NMR (400 MHz, CDCl3) δ 8.34 (d,
J = 8.2 Hz, 1H), 7.62 (d, J = 8.2 Hz, 1H), 7.56 (s, 1H), 6.31–6.25 (m,
3H), 4.16 (t, J = 8.5 Hz, 2H), 3.30–3.26 (m, 6H), 3.12–3.11 (m, 4H),
2.26 (s, 3H); HRMS (ESI) m/z 422.1349 [(M + H)+ calcd for
C20H21F2N3O3S 422.1345].
tert-Butyl
4-(3,5-bis(trifluoromethyl)phenyl)piperazine-1-carbox-
ylate (6c). Toluene (1.6 mL) was added to a mixture of 1,3-bis(tri-
fluoromethyl)-5-bromobenzene (4c, 150 mg, 0.51 mmol), 1-Boc-pi-
perazine (5, 123 mg, 0.66 mmol), NaOt-Bu (74 mg, 0.77 mmol),
JohnPhos (15 mg, 10 mol%), and Pd2(dba)3 (28 mg, 5 mol%). Argon
(Ar) was bubbled through the reaction mixture for 30 min, and the
reaction mixture was refluxed for 18 h. The reaction mixture was
concentrated in vacuo and the residue was dissolved in CH2Cl2/MeOH
(1/1), filtered through a pad of Celite, and concentrated in vacuo. The
residue was purified by column chromatography (silica gel, hexanes/
EtOAc, 20/1) to afford 6c (203 mg, quantitative): 1H NMR (400 MHz,
CDCl3) δ 7.30 (s, 1H), 7.25 (s, 2H), 3.65–3.59 (m, 4H), 3.27–3.24 (m,
4H), 1.48 (s, 9H).
1-(5-((4-(3,5-Dichlorophenyl)piperazin-1-yl)sulfonyl)indolin-1-yl)
ethan-1-one (13b). To a solution of commercially available 1-(3–5-di-
chlorophenyl)piperazine (12a, 50 mg, 0.22 mmol) in 1,4-dioxane
(3.0 mL) was added Et3N (34 μL, 0.26 mmol) at 25 °C. The reaction
mixture was treated with 1-acetyl-5-indoline sulfonyl chloride (8,
57 mg, 0.22 mmol) and stirred under N2 at 60 °C for 3 h. The reaction
mixture was cooled to 25 °C and then stirred for an additional 20 h. The
reaction mixture was diluted with EtOAc. The layers were separated,
and the aqueous layer was extracted with EtOAc. The combined organic
layers were washed with brine, dried over anhydrous Na2SO4, and
concentrated in vacuo. The residue was purified by column chromato-
graphy (silica gel, hexanes/EtOAc, 4/1) to afford 13b as a white solid
(46 mg, 47%): 1H NMR (400 MHz, CDCl3) δ 8.34 (d, J = 8.5 Hz, 1H),
7.60 (dd, J = 8.6, 1.9 Hz, 1H), 7.55 (s, 1H), 6.83 (t, J = 1.7 Hz, 1H),
6.69 (d, J = 1.8 Hz, 2H), 4.15 (t, J = 8.6 Hz, 2H), 3.27–3.24 (m, 6H),
3.13–3.10 (m, 4H), 2.26 (s, 3H); HRMS (ESI): m/z 454.0753 [(M + H)+
calcd for C20H21Cl2N3O3S 454.0759].
1-(3,5-Bis(trifluoromethyl)phenyl)piperazine (7c). To a solution of
6c (200 mg, 0.5 mmol) in CH2Cl2 (3 mL) was added TFA (1.3 mL) at
0 °C. The reaction mixture was stirred under N2 at 0 °C for 10 min and
then warmed to 25 °C. The reaction mixture was stirred under N2 at
25 °C for 3.5 h. The reaction mixture was concentrated in vacuo to give
7c as an orange solid. Compound 7c was used in the following step
without further purification: 1H NMR (400 MHz, CDCl3) δ 7.45 (s, 1H),
7.30 (s, 2H), 3.95 (br s, 1H), 3.59 (m, 4H), 3.46 (m, 4H).
1-(5-((4-(3,5-Bis(trifluoromethyl)phenyl)piperazin-1-yl)sulfonyl)in-
dolin-1-yl)ethan-1-one (9c). To a solution of 7c (50 mg, 0.17 mmol) in
1,4-dioxane (3 mL) was added Et3N (32 μL, 0.24 mmol) at 25 °C. 1-
Acetyl-5-indoline sulfonyl chloride (8, 44 mg, 0.17 mmol) was added to
the reaction mixture. The resulting mixture was stirred under N2 at
60 °C for 3 h. The reaction mixture was cooled to 25 °C and kept at the
same temperature for 10 h. The reaction mixture was diluted with
EtOAc. The layers were separated, and the aqueous layer was extracted
with EtOAc. The combined organic layers were washed with brine,
dried over anhydrous Na2SO4, and concentrated under reduced pres-
sure. The residue was purified by column chromatography (silica gel,
hexanes/EtOAc, 4/1) to afford 9c as a white solid (35 mg, 40% for 2
steps): 1H NMR (400 MHz, CDCl3) δ 8.34 (d, J = 8.6 Hz, 1H), 7.62 (dd,
J = 8.5, 1.9 Hz, 1H), 7.57 (s, 1H), 7.31 (s, 1H), 7.19 (s, 2H), 4.17 (t,
5-((4-(3-Chloro-5-(trifluoromethyl)phenyl)piperazin-1-yl)sulfonyl)-
1H-indole (16). To a solution (60 °C) of 1H-indole-5-sulfonyl chloride
(15) (50 mg, 0.23 mmol) and Et3N (72 μL, 0.55 mmol) in anhydrous
1,4-dioxane (0.22 M, 1 mL) was added 1-(3-chloro-5-(trifluoromethyl)
phenyl)piperazine [13] (14, 121 mg, 0.46 mmol) in anhydrous 1,4-di-
oxane (0.5 mL). After stirring at 60 °C for 2 h, the reaction mixture was
cooled to 25 °C and stirred for an additional 18 h. The reaction was
quenched by an addition of H2O and the resulting mixture was diluted
with EtOAc. The combined organic layers were washed with brine,
dried over anhydrous Na2SO4, and concentrated in vacuo. The residue
7

S.-H. Kwak, et al.
BioorganicChemistry102(2020)104055
was purified by column chromatography (silica gel, hexanes/EtOAc, 3/
1) to afford 16 (32 mg, 32%) as a white solid: 1H NMR (400 MHz,
CDCl3) δ 8.58 (br s, 1H), 8.15 (s, 1H), 7.60 (d, J = 8.5 Hz, 1H), 7.52 (d,
J = 8.3 Hz, 1H), 7.39–7.35 (m, 1H), 7.04 (s, 1H), 6.91 (s, 1H), 6.89 (s,
1H), 6.70 (s, 1H), 3.34–3.26 (m, 4H), 3.20–3.13 (m, 4H); HRMS (ESI)
m/z 444.0760 [(M + H)+ calcd for C19H17ClF3N3O2S 444.0755].
1-(5-((4-(3-Chloro-5-(trifluoromethyl)phenyl)piperazin-1-yl)sul-
The layers were separated, and the aqueous layer was extracted with
CH2Cl2. The combined organic layers were dried over anhydrous
Na2SO4 and concentrated in vacuo. The residue was purified by column
chromatography (silica gel, CH2Cl2/MeOH (10/1) to 100% MeCN) to
afford 22 (90 mg, 97%) as a colorless oil: 1H NMR (400 MHz, CD3OD) δ
7.68 (d, J = 8.8 Hz, 2H), 7.62 (d, J = 8.7 Hz, 2H), 7.37 (dd, J = 7.8,
7.9 Hz, 1H), 7.15 (d, J = 8.6 Hz, 1H), 7.14 (s, 1H), 7.08 (d, J = 7.6 Hz,
1H), 3.28–3.22 (m, 6H), 3.14–3.09 (m, 4H), 2.35 (t, J = 7.3 Hz, 2H),
1.84–1.82 (m, 2H); HRMS (ESI) m/z 515.1570 [(M + H)+ calcd for
fonyl)-1H-indol-1-yl)ethan-1-one (17). To a solution of 16 (15 mg,
0.03 mmol), Et3N (12 μL, 0.09 mmol), and N,N-dimethyl-4-aminopyr-
idine (1.4 mg, 0.012 mmol) in anhydrous 1,2-dichloroethane (1 mL)
was added Ac2O (11.2 μL, 0.12 mmol). The resulting mixture was
stirred at 80 °C for 24 h, the reaction was quenched by an addition of
H2O. The mixture was extracted with EtOAc. The combined organic
layers were dried over anhydrous Na2SO4 and concentrated in vacuo.
The residue was purified by column chromatography (silica gel, hex-
anes/EtOAc, 3/1) to afford 17 (14 mg, 96%) as a white solid: 1H NMR
(400 MHz, CDCl3) δ 8.63 (d, J = 8.8 Hz, 1H), 8.05 (s, 1H), 7.75 (d,
J = 9.1 Hz, 1H), 7.58 (d, J = 3.7 Hz, 1H), 7.04 (s, 1H), 6.92 (s, 1H),
6.89 (s, 1H), 6.77 (d, J = 3.7 Hz, 1H), 3.37–3.25 (m, 4H), 3.24–3.12
(m, 4H), 2.69 (s, 3H); HRMS (ESI) m/z 486.0867 [(M + H)+ calcd for
C22H25F3N4O5S 515.1571].
N-((tert-Butyldimethylsilyl)oxy)-4-(3-(4-((4-(3-(trifluoromethyl)
phenyl)piperazin-1-yl)sulfonyl)phenyl)ureido)butanamide (23). To a
solution of 22 (22 mg, 0.04 mmol) in anhydrous THF (2.86 mL) were
added ethyl chloroformate (8 µL, 0.08 mmol) and Et3N (11 µL,
0.08 mmol). After stirring at 25 °C for 1 h, NH2OTBS (12.5 mg,
0.08 mmol) in anhydrous MeOH (0.66 mL) was added to the reaction
mixture. After stirring at 25 °C for 1 h, the reaction mixture was con-
centrated in vacuo. The residue was purified by column chromato-
graphy (silica gel, CH2Cl2/MeOH, 50/1 to 10/1) to afford 23 (23 mg,
85%) as a white solid: 1H NMR (400 MHz, CD3OD) δ 7.68 (d,
J = 9.3 Hz, 2H), 7.62 (d, J = 9.1 Hz, 2H), 7.37 (dd, J = 7.9, 8.0 Hz,
1H), 7.14 (d, J = 7.6 Hz, 1H), 7.13 (s, 1H), 7.08 (d, J = 7.7 Hz, 1H),
6.35–6.30 (m, 1H), 3.32–3.21 (m, 6H), 3.13–3.09 (m, 4H), 2.17 (t,
J = 7.4 Hz, 2H), 1.87–1.78 (m, 2H), 0.95 (s, 9H), 0.16 (s, 6H); HRMS
(ESI) m/z 644.2542 [(M + H)+ calcd for C28H40F3N5O5SSi 644.2544].
N-Hydroxy-4-(3-(4-((4-(3-(trifluoromethyl)phenyl)piperazin-1-yl)
sulfonyl)phenyl)ureido)butanamide (24). To a cooled (0 °C) solution of
23 (7.7 mg, 0.01 mmol) in anhydrous CH2Cl2 (1.7 mL) was added
dropwise TFA (0.5 mL). After stirring at 25 °C for 25 min, the reaction
mixture was concentrated in vacuo. The residue was purified by column
chromatography (silica gel, CH2Cl2/MeOH, 10/1) to afford 24 as a
white solid (7 mg, quantitative): 1H NMR (400 MHz, CD3OD) δ 7.68 (d,
J = 8.7 Hz, 2H), 7.62 (d, J = 8.4 Hz, 2H), 7.38 (dd, J = 7.8, 7.8 Hz,
1H), 7.16 (d, J = 8.1 Hz, 1H), 7.14 (s, 1H), 7.09 (d, J = 7.8 Hz, 1H),
3.29–3.19 (m, 6H), 3.15–3.07 (m, 4H), 2.15 (t, J = 7.5 Hz, 2H),
1.87–1.78 (m, 2H); HRMS (ESI) m/z 530.1680 [(M + H)+ calcd for
C
21H19ClF3N3O3S 486.0872].
5-((4-(3-Chloro-5-(trifluoromethyl)phenyl)piperazin-1-yl)sulfonyl)
indoline (18). To a cooled (0 °C) solution of 3 [13] (89 mg, 0.18 mmol)
in EtOH (0.41 mL) was added c-HCl (0.2 mL). The resulting mixture was
refluxed for 2 h and then ice water was added followed by 35% NH4OH.
The reaction was extracted with EtOAc. The combined organic layers
were dried over anhydrous Na2SO4 and concentrated in vacuo to afford
18 (80 mg). Compound 18 was used in the following step without
further purification: 1H NMR (400 MHz, CDCl3) δ 7.42 (d, J = 6.2 Hz,
1H), 7.41 (s, 1H), 7.03 (s, 1H), 6.93 (s, 1H), 6.90 (s, 1H), 6.57 (d,
J = 8.8 Hz, 1H), 3.68 (t, J = 8.4 Hz, 2H), 3.32–3.25 (m, 4H), 3.15–3.09
(m, 4H), 3.08 (t, J = 8.5 Hz, 2H).
5-((4-(3-Chloro-5-(trifluoromethyl)phenyl)piperazin-1-yl)sulfonyl)-
1-(methylsulfonyl)indoline (19). To
a solution of 18 (36 mg,
0.08 mmol) in pyridine (3.5 mL) was added methanesulfonyl chloride
(0.03 mL, 0.39 mmol). The reaction mixture was stirred at 25 °C for
18 h. The reaction was quenched by an addition of saturated NH4Cl
solution and the organic layer was extracted with CH2Cl2. The com-
bined organic layers were dried over anhydrous Na2SO4 and con-
centrated in vacuo. The residue was purified by column chromato-
graphy (silica gel, hexanes/EtOAc, 2/1) to afford 19 as a white solid
(29 mg, 67% for 2 steps): 1H NMR (400 MHz, CDCl3) δ 7.62 (d,
J = 8.5 Hz, 1H), 7.59 (s, 1H), 7.49 (d, J = 7.5 Hz, 1H), 7.06 (s, 1H),
6.94 (s, 1H), 6.91 (s, 1H), 4.08 (t, J = 8.6 Hz, 2H), 3.33–3.27 (m, 4H),
3.23 (t, J = 8.6 Hz, 2H), 3.17–3.12 (m, 4H), 2.95 (s, 3H); HRMS (ESI)
m/z 524.0689 [(M + H)+ calcd for C20H21ClF3N3O4S2 524.0687].
Methyl 4-(3-(4-((4-(3-(trifluoromethyl)phenyl)piperazin-1-yl)sul-
fonyl)phenyl)ureido)butanoate (21). To a solution of 4-((4-(3-(tri-
C
22H26F3N5O5S 530.1680].
Methyl 5-(3-(4-((4-(3-(trifluoromethyl)phenyl)piperazin-1-yl)sul-
fonyl)phenyl)ureido)pentanoate (25a). To a solution of 20a (88 mg,
0.23 mmol) and CDI (185 mg, 1.14 mmol) in THF (1.14 mL) was added
DIPEA (0.2 mL, 1.14 mmol). After stirring at 25 °C for 2 h, the reaction
mixture was treated with methyl 5-aminopentanoate [15] (150 mg,
1.14 mmol) and allowed to stir for 1 h. The reaction mixture was
concentrated in vacuo and the residue was purified by column chro-
matography (silica gel, hexanes/EtOAc, 1/1) to afford 25a (75 mg,
60%) as a colorless oil: 1H NMR (400 MHz, CDCl3) δ 7.73 (s, 1H), 7.62
(d, J = 8.9 Hz, 2H), 7.53 (d, J = 8.7 Hz, 2H), 7.33 (dd, J = 8.2, 8.2 Hz,
1H), 7.10 (d, J = 7.7 Hz, 1H), 7.05 (s, 1H), 7.00 (d, J = 8.2 Hz, 1H),
5.60 (t, J = 5.5 Hz, 1H), 3.64 (s, 3H), 3.27–3.22 (m, 6H), 3.13–3.11 (m,
4H), 2.33 (t, J = 7.1 Hz, 2H), 1.70–1.62 (m, 2H), 1.58–1.51 (m, 2H).
5-(3-(4-((4-(3-(Trifluoromethyl)phenyl)piperazin-1-yl)sulfonyl)
fluoromethyl)phenyl)piperazin-1-yl)sulfonyl)aniline
[12]
(20a,
103 mg, 0.26 mmol) and CDI (216 mg, 1.33 mmol) in anhydrous THF
(1.3 mL) was added DIPEA (0.23 mL, 1.33 mmol). After stirring at 25 °C
for 2 h, methyl 4-aminobutanoate (204 mg, 1.33 mmol) was added to
the reaction mixture. After stirring at 25 °C for 1.5 h, the reaction
mixture was concentrated under reduced pressure. The residue was
purified by column chromatography (silica gel, hexanes/EtOAc, 1/1) to
afford 21 (100 mg, 71%) as a colorless oil: 1H NMR (400 MHz, CDCl3) δ
7.64 (d, J = 8.5 Hz, 2H), 7.56 (d, J = 8.7 Hz, 2H), 7.37–7.29 (m, 1H),
7.11 (d, J = 7.6 Hz, 1H), 7.06 (s, 1H), 7.01 (d, J = 8.6 Hz, 1H), 5.55 (br
s, 1H), 3.68 (s, 3H), 3.34–3.23 (m, 6H), 3.16–3.08 (m, 4H), 2.41 (t,
J = 6.8 Hz, 2H), 1.91–1.83 (m, 2H); HRMS (ESI) m/z 529.1726
[(M + H)+ calcd for C23H27F3N4O5S 529.1727].
phenyl)ureido)pentanoic acid (26a). 1 N LiOH (0.25 mL) was added to
25a (34 mg, 0.063 mmol) in THF/H2O (2/1, 0.63 mL) at 25 °C. After
stirring at 25 °C for 17 h, the reaction mixture was cooled to 0 °C and
acidified with 1 N HCl. After an addition of H2O, the resulting mixture
was extracted with CH2Cl2. The organic layers were combined, dried
over anhydrous Na2SO4, and concentrated in vacuo. The residue was
purified by column chromatography (silica gel, CH2Cl2/MeOH, 10/1) to
afford 26a (18 mg, 54%) as a white solid: 1H NMR (400 MHz, CD3OD) δ
7.67 (d, J = 9.0 Hz, 2H), 7.62 (d, J = 9.0 Hz, 2H), 7.37 (dd, J = 8.3,
8.3 Hz, 1H), 7.15 (d, J = 7.4 Hz, 1H), 7.14 (s, 1H), 7.08 (d, J = 7.6 Hz,
1H), 3.29–3.26 (m, 4H), 3.22 (t, J = 6.7 Hz, 2H), 3.11–3.10 (m, 4H),
2.34 (t, J = 8.0 Hz, 2H), 1.70–1.63 (m, 2H), 1.60–1.53 (m, 2H); HRMS
(ESI) m/z 529.1733 [(M + H)+ calcd for C23H27F3N4O5S 529.1727].
N-Hydroxy-5-(3-(4-((4-(3-(trifluoromethyl)phenyl)piperazin-1-yl)
sulfonyl)phenyl)ureido)pentanamide (27a). Ethyl chloroformate
4-(3-(4-((4-(3-(Trifluoromethyl)phenyl)piperazin-1-yl)sulfonyl)
phenyl)ureido)butanoic acid (22). To a solution of 21 (100 mg,
0.18 mmol) in THF/H2O (2/1, 1.8 mL) was added 1 N LiOH (0.37 mL)
at 25 °C. After stirring for 18 h, the reaction was quenched by an ad-
dition of 1 N HCl, and the resulting mixture was diluted with CH2Cl2.
8

S.-H. Kwak, et al.
BioorganicChemistry102(2020)104055
(6.2 mg, 0.057 mmol) and Et3N (8 μL) were added to a solution of 26a
(15 mg, 0.028 mmol) in THF (2 mL). After stirring for 1 h, the reaction
mixture was treated with NH2OTBS (8.4 mg, 0.057 mmol) in MeOH
(0.44 mL). After an additional 2 h, NH2OTBS (8.4 mg, 0.057 mmol) in
MeOH (0.44 mL) was added and the resulting mixture was left to stir for
additional 2 h. The reaction mixture was concentrated in vacuo, dis-
solved in CH2Cl2 (2 mL), and treated with TFA (0.17 mL). The resulting
mixture was stirred at 0 °C for 1 h and then concentrated in vacuo to
afford 27a (8.1 mg, 53% for 2 steps) as a solid: 1H NMR (400 MHz,
CD3OD) δ 7.69 (d, J = 8.9 Hz, 2H), 7.62 (d, J = 8.9 Hz, 2H), 7.39 (dd,
J = 7.9, 7.9 Hz, 1H), 7.19 (d, J = 8.3 Hz, 1H), 7.18 (s, 1H), 7.11 (d,
J = 7.5 Hz, 1H), 3.32–3.30 (m, 4H), 3.22 (t, J = 6.7 Hz, 2H), 3.14–3.11
(m, 4H), 2.14 (t, J = 7.3 Hz, 2H), 1.72–1.63 (m, 2H), 1.61–1.50 (m,
2H); HRMS (ESI) m/z 544.1840 [(M + H)+calcd for C23H28F3N5O5S
544.1836].
10.04 (br s, 1H), 8.09 (br s, 1H), 7.74 (br s, 2H), 7.66 (br s, 2H), 7.21 (s,
1H), 7.18 (s, 1H), 7.06 (s, 1H), 3.49–3.37 (m, 4H), 3.26–3.18 (m, 2H),
3.12–3.10 (m, 4H), 2.19–2.11 (m, 2H), 1.70–1.59 (m, 2H), 1.56–1.48
(m, 2H); HRMS (ESI) m/z 578.1441 [(M
+
H)+ calcd for
C23H27ClF3N5O5S 578.1446].
4.2. Cloning and purification of K. Pneumoniae LpxH
Cloning and purification of K. pneumoniae LpxH for crystallography
studies were carried out as previously reported [13]. Briefly, K. pneu-
moniae LpxH was cloned into a modified pET21b (Novagen/Millipore
Sigma) vector, yielding the LpxH fusion protein with a C-terminal TEV
protease site (ENLYFQGS) and His10 tag. Vector-transformed BL21
STAR (DE3) E. coli cells (Thermo Fisher Scientific) were grown in M9
minimal medium to an OD600 of 0.5 at 37 °C, prior to being induced
with 1 mM IPTG at 30 °C. After 5 h, the cells were then harvested by
centrifugation. Protein purification was carried out at 4 °C. Cell pellets
were lysed in a buffer containing 50 mM phosphate-citrate, 20 mM MES
(pH 6.0), 600 mM NaCl, 10% sucrose, 5 mM 2-mercaptoethanol, 10 mM
imidazole, and 0.1% Triton X-100 using a French press. After removing
cell debris by centrifugation, a HisPur Ni-NTA column (Thermo Fisher
Scientific) was pre-equilibrated with the lysis buffer, and the super-
natant was loaded. Following extensive washes using a purification
buffer containing 20 mM phosphate-citrate, 20 mM MES (pH 6.0),
300 mM NaCl, 5% glycerol, 5 mM 2-mercaptoethanol, and 40 mM
imidazole, LpxH was eluted from the column by increasing the imida-
zole concentration stepwise from 40 to 400 mM. The protein sample
was concentrated and further purified with size-exclusion chromato-
graphy (Superdex 200; GE Healthcare Life Sciences) in the FPLC buffer
containing 20 mM MES (pH 6.0), 800 mM NaCl, 1 mM DTT, and 5%
glycerol.
Methyl 5-(3-(4-((4-(3-chloro-5-(trifluoromethyl)phenyl)piperazin-1-
yl)sulfonyl)phenyl)ureido)pentanoate (25b). To a solution of 20b
(290 mg, 0.69 mmol) and CDI (561 mg, 3.46 mmol) in anhydrous THF
(3.45 mL) was added DIPEA (0.6 mL, 3.46 mmol). After stirring at 25 °C
for 2 h, the reaction mixture was transferred to methyl 5-amino-
pentanoate (114 mg, 0.87 mmol). After stirring at 25 °C for 1.5 h, the
reaction mixture was concentrated under reduced pressure and the
residue was purified by column chromatography (silica gel, hexanes/
EtOAc, 1/1) to afford 25b (350 mg, 87%) as a white sticky solid: 1H
NMR (400 MHz, CDCl3) δ 7.63 (s, 1H), 7.59 (d, J = 8.5 Hz, 2H), 7.49
(d, J = 8.2 Hz, 2H), 7.05 (s, 1H), 6.95 (s, 1H), 6.93 (s, 1H), 5.56 (br s,
1H), 3.65 (s, 3H), 3.34–3.20 (m, 6H), 3.15–3.06 (m, 4H), 2.33 (t,
J = 7.2 Hz, 2H), 1.69–1.59 (m, 2H), 1.58–1.50 (m, 2H).
5-(3-(4-((4-(3-Chloro-5-(trifluoromethyl)phenyl)piperazin-1-yl)sul-
fonyl)phenyl)ureido)pentanoic acid (26b). To a solution of 25b (34 mg,
0.05 mmol) in THF/H2O (2/1, 0.5 mL) was added 1 N LiOH (0.13 mL)
at 25 °C. After stirring for 18 h, the reaction was quenched by an ad-
dition of 1 N HCl, and the resulting mixture was diluted with CH2Cl2.
The layers were separated, and the aqueous layer was extracted with
CH2Cl2. The combined organic layers were dried over anhydrous
Na2SO4 and concentrated in vacuo. The residue was purified by column
chromatography (silica gel, CH2Cl2/MeOH (30/1) to 100% MeCN) to
afford 26b (15 mg, 45%) as a white solid: 1H NMR (400 MHz,
CD3COCD3) δ 8.60 (s, 1H), 7.75 (d, J = 8.6 Hz, 2H), 7.65 (d,
J = 8.6 Hz, 2H), 7.20 (s, 1H), 7.17 (s, 1H), 7.06 (s, 1H), 6.18 (br s, 1H),
3.50–3.41 (m, 4H), 3.27–3.24 (m, 2H), 3.15–3.07 (m, 4H), 2.33 (t,
J = 7.2 Hz, 2H), 1.67–1.64 (m, 2H), 1.59–1.56 (m, 2H); HRMS (ESI) m/
z 563.1340 [(M + H)+ calcd for C23H26ClF3N4O5S 563.1337].
N-Hydroxy-5-(3-(4-((4-(3-chloro-5-(trifluoromethyl)phenyl)piper-
4.3. Co-crystallization of K. Pneumoniae LpxH with a sulfonyl piperazine
antibiotic 27b (JH-LPH-41)
Peak fractions containing K. pneumoniae LpxH were buffer-ex-
changed into a buffer containing 20 mM MES (pH 6.0), 200 mM NaCl,
1 mM DTT, and 5% glycerol. During buffer exchange, concentrated 27b
solution in DMSO was added to the protein solution in a 1:1 M ratio.
The solution was then concentrated to 8 mg/mL for crystallization and
additional concentrated 27b solution in DMSO was added to the protein
solution in a 1:1 M ratio (final ratio = 2:1 drug: protein, final 27b
concentration = 0.54 mM, DMSO = 2%).
Protein crystals were grown using the sitting-drop vapor diffusion
method at 20 °C. Each drop was prepared by mixing 1 μL of the protein
solution with 1 μL of the reservoir solution (200 mM KCl, 100 mM
sodium citrate, 37% pentaerythritol propoxylate (5/4 PO/OH), pH 5.5).
The final drop solution contained 4 mg/mL of LpxH with 0.27 mM 27b,
10 mM MES (pH 6.0), 100 mM NaCl, 100 mM KCl, 50 mM sodium
citrate (pH 5.5), 18.5% pentaerythritol propoxylate (5/4 PO/OH),
0.5 mM DTT, 1% DMSO, and 2.5% glycerol. Diffraction quality protein
crystals were harvested after 2 weeks and soaked with the reservoir
solution additionally containing 20% glycerol, 100 μM MnCl2, 0.27 mM
27b, and 2% DMSO for cryoprotection.
azin-1-yl)sulfonyl)phenyl)ureido)-pentanamide
(27b).
[Coupling
Reaction] To a solution of 26b (13 mg, 0.02 mmol) in anhydrous THF
(1.69 mL) were added ethyl chloroformate (4.3 µL, 0.04 mmol) and
Et3N (6 µL, 0.04 mmol). After stirring at 25 °C for 1 h, NH2OTBS (6 mg,
0.04 mmol) in anhydrous MeOH (0.39 mL) was added to the reaction
mixture. After stirring at 25 °C for 1 h, the reaction mixture was con-
centrated in vacuo and purified by column chromatography (silica gel,
CH2Cl2/MeOH, 30/1) to afford N-((tert-butyldimethylsilyl)oxy)-5-(3-(4-
((4-(3-chloro-5-(trifluoromethyl)phenyl)piperazin-1-yl)sulfonyl)
phenyl)ureido)pentanamide (13 mg) as
a
white solid: 1H NMR
(400 MHz, CD3COCD3) δ 9.78 (br s, 1H), 8.46 (s, 1H), 7.74 (d,
J = 9.2 Hz, 2H), 7.66 (d, J = 8.9 Hz, 2H), 7.20 (s, 1H), 7.17 (s, 1H),
7.06 (s, 1H), 6.04 (t, J = 5.5 Hz, 1H), 3.50–3.42 (m, 4H), 3.26–3.19 (m,
2H), 3.15–3.07 (m, 4H), 2.15–2.13 (m, 2H), 1.69–1.60 (m, 2H),
1.56–1.53 (m, 2H), 0.94 (s, 9H), 0.15 (s, 6H); HRMS (ESI) m/z
692.2313 [(M + H)+ calcd for C29H41ClF3N5O5SSi 692.2311]; [TBS
Deprotection] To a cooled (0 °C) solution of the TBS protected hydro-
xamic acid (13 mg, 0.01 mmol) in anhydrous CH2Cl2 (2.8 mL) was
added dropwise TFA (240 µL). After stirring for 25 min at 25 °C, the
reaction mixture was concentrated in vacuo and purified by column
chromatography (silica gel, CH2Cl2/MeOH, 10/1) to afford 27b (7 mg,
55% for 2 steps) as a white solid: 1H NMR (400 MHz, CD3COCD3) δ
4.4. Structural analysis of K. Pneumoniae LpxH complexes with 27b (JH-
LPH-41)
The X-ray diffraction data of the K. pneumoniae LpxH complex with
27b were collected at the Northeastern Collaborative Access Team
(NECAT) 24-ID-C beamline at the Advanced Photon Source at Argonne
National Laboratory. The X-ray diffraction data was processed using
XDS [16]. The phase information of the crystal structures of the K.
pneumoniae LpxH complex was obtained by molecular replacement with
the PHASER module in the PHENIX suite [17] using the PDB entry 6PJ3
as the search model. Restraints of the inhibitors were generated by
9

S.-H. Kwak, et al.
BioorganicChemistry102(2020)104055
using eLBOW [18] and edited manually. Iterative model building and
refinement was carried out using COOT [19] and PHENIX [17]. The
2mFo-DFc omit maps were generated using PHENIX [17].
GM007105). The authors would like to thank Dr. Jinshi Zhao for in-
sightful discussions about the LpxH inhibition assay.
Appendix A. Supplementary material
4.5. Enzymatic assay for LpxH inhibition
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bioorg.2020.104055.
The LpxE-coupled LpxH activity assay [12] was conducted as de-
scribed previously using the GB1-K. pneumoniae LpxH-His10 fusion
protein [13]. Briefly, two reaction mixtures were prepared that contain
20 mM Tris-HCl (pH 8.0), 0.5 mg/mL BSA, 0.02% Triton X-100, 1 mM
MnCl2, 1 mM DTT, and 10% DMSO, with one additionally containing
200 μM substrate (UDP-DAGn) and the other containing both LpxH
(20 ng/mL) and 0.2 μM inhibitor. The reaction mixtures were pre-in-
cubated at 37 °C for 10 min before an equal volume of the LpxH mixture
was added to the substrate mixture to initiate the reaction at 37 °C. The
final reaction solution contains 100 μM substrate, 10 ng/mL enzyme,
and 0.1 μM inhibitor. At the desired reaction time points, an aliquot of
20 μL reaction mixture was removed and added to a well in 96-well
half-area plate containing 5 mM EDTA (final concentration) to quench
the LpxH reaction. Purified Aquifex aeolicus LpxE was then added to a
final concentration of 5 μg/mL. The plate was incubated at 37 °C for
30 min followed by addition of formic acid to a final concentration of
3.75 M to quench the LpxE reaction. The malachite green reagent
(Sigma Aldrich, catalog MAK307) was added with a 5-fold dilution, and
the solution was incubated for 30 min at room temperature before the
absorbance at 620 nm was measured. All measurements were done in
triplicates, and standard error was calculated. Percentage LpxH activ-
ities for 1, 2, and 3 at 0.1 μM were calculated from previously reported
IC50 values [13], which were extracted from fitting of the dose–r-
esponse curve of vi/v0 = 1/(1 + [I]/IC50) assayed under identical
conditions.
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Author contributions
S.-H.K. and C.S.C. contributed equally; P.Z. and J.H. conceived the
project, designed the overall experimental strategy, and analyzed and
discussed the results; S.-H.K., A.F.E., W.Y.L., and C.G.W. synthesized
the sulfonyl piperazine LpxH inhibitors used in this study; C.S.C. pur-
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Declaration of Competing Interest
The authors declared that there is no conflict of interest.
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Acknowledgments
This work was supported in part by the grants from the National
Institute of Allergy and Infectious Diseases (AI139216) and National
Institute of General Medical Sciences (GM115355). X-ray diffraction
data were collected at the Northeastern Collaborative Access Team
beam lines (24-ID-C), which is funded by the National Institute of
General Medical Sciences from the National Institutes of Health (P30
GM124165). The Pilatus 6 M detector on 24-ID-C beam line is funded
by a NIH-ORIP HEI grant (S10 RR029205). This research used resources
of the Advanced Photon Source, Department of Energy Office of
Science, United States User Facility operated for the Department of
Energy Office of Science by Argonne National Laboratory under
Contract No. DE-AC02-06CH11357. C.G.W. and A.F.E were supported
by the NIGMS Pharmacological Sciences Training Grant (NIH
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