Design, synthesis and structure-activity relationship evaluation of novel LpxC inhibitors as Gram-negative antibacterial agents

Article abstract of DOI:10.1016/j.bmcl.2017.12.005

LpxC inhibitors are new-type antibacterial agents developed in the last twenty years, mainly against Gram-negative bacteria infections. To develop novel LpxC inhibitors with good antibacterial activities and biological metabolism, we summarized the basic skeleton of reported LpxC inhibitors, designed and synthesized several series of compounds and tested their antibacterial activities against Escherichial coli and Pseudomonas aeruginosa in vitro. Structure-activity relationships have been discussed in this article. The metabolism stability of YDL-2, YDL-5, YDL-8, YDL-14, YDL-20–YDL-23 have been evaluated in liver microsomes, which indicated that the 2-amino isopropyl group may be a preferred structure than the 2-hydroxy ethyl group in the design of LpxC inhibitors.

Full text of DOI:10.1016/j.bmcl.2017.12.005

Accepted Manuscript
Design, synthesis and structure-activity relationship evaluation of novel LpxC
inhibitors as Gram-negative antibacterial agents
Shi Ding, Rui-Yang Dai, Wen-Ke Wang, Qiao Cao, Le-Fu Lan, Xian-Li Zhou,
Yu-She Yang
PII:
S0960-894X(17)31165-4
https://doi.org/10.1016/j.bmcl.2017.12.005
BMCL 25461
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
14 September 2017
28 November 2017
4 December 2017
Please cite this article as: Ding, S., Dai, R-Y., Wang, W-K., Cao, Q., Lan, L-F., Zhou, X-L., Yang, Y-S., Design,
synthesis and structure-activity relationship evaluation of novel LpxC inhibitors as Gram-negative antibacterial
agents, Bioorganic & Medicinal Chemistry Letters (2017), doi: https://doi.org/10.1016/j.bmcl.2017.12.005
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Design, synthesis and structure-activity relationship evaluation of novel LpxC
inhibitors as Gram-negative antibacterial agents
b
b
b
Shi Ding a, b, ||, Rui-Yang Dai c, ||, Wen-Ke Wang , Qiao Cao , Le-Fu Lan , Xian-Li Zhou c, *,
Yu-She Yang b,
*
a Key Laboratory of New Drug Research and Development of Liaoning Province, College of
Pharmacy of Liaoning University, Shenyang 10036, Liaoning Province, China
b
State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese
Academy of Sciences, Shanghai 201203, China
c
School of Life Science and Engineering, Southwest Jiaotong University, Chengdu 610031,
Sichuan Province, China
∗Corresponding author: Xian-Li Zhou. Tel: 86-28-8760 3201. Fax: 86-28-8760 3202. E-mail:
zhouxl@swjtu.edu.cn.
∗Corresponding author: Yu-She Yang. Tel: 86-21-5080 6786. Fax: 86-21-5080 6786. E-mail:
ysyang@simm.ac.cn.
||These authors contributed equally to this work.
Abstract
LpxC inhibitors are new-type antibacterial agents developed in the last twenty years, mainly
against Gram-negative bacteria infections. To develop novel LpxC inhibitors with good
antibacterial activities and biological metabolism, we summarized the basic skeleton of reported
LpxC inhibitors, designed and synthesized several series of compounds and tested their
antibacterial activities against Escherichial coli and Pseudomonas aeruginosa in vitro.
Structure-activity relationships have been discussed in this article. The metabolism stability of
YDL-2, YDL-5, YDL-8, YDL-14, YDL-20~YDL-23 have been evaluated in liver microsomes,
which indicated that the 2-amino isopropyl group may be a preferred structure than the 2-hydroxy
ethyl group in the design of LpxC inhibitors.
Keywords: Gram-negative bacteria infection, LpxC inhibitor, Structure-activity relationship, Zinc
binding group, Liver microsomal stability
The growing problems of bacterial resistance gradually evolves into a public health crisis.1 The
emergency of multi-resistant Gram-negative bacteria, including Enterococcus faecium,
Staphylococcus aureus, Klebsiella pheumoniae, Acinetobacter baumanni and Enterobacter
species, account for a large proportion of nosocomial infections.2 Unfortunately, comparing to
rapid spreading of the multi-resistant bacteria, only a few novel antibacterial agents are in the
late-stage clinical development pipeline to treat these intractable infections caused by them.3 Thus,
there is a great need for novel antibacterial agents with new mechanisms of action and chemical
classes.
Different from the membrane structure in the Gram-positive bacteria, the Gram-negative
bacteria possess an additional outer membrane decorated with lipopolysaccharide (LPS),
obstructing the development of new antibiotics severely. LPS is composed of an O-antigen, a
sugar-containing domain and a membrane anchor lipid A. Lipid A is essential for the stability of
the
outer
membrane
of
the
Gram-negative
bacteria.4
The
uridydiphospho-3-O-(R-hydroxydecanoyl)-N-acetylglucosamine deacetylase (LpxC) is a cytosolic
zinc-based deacetylase, which catalyzes the first committed step of the biosynthesis of lipid A in
Gram-negative bacteria and is a validated antibiotic target.5 Thus, the inhibition of LpxC can
obstruct the biosynthesis of lipid A, sequentially destroy the integrity of the outer membrane and
kill most Gram-negative bacteria.

The development of LpxC inhibitors have been more than two decades since the discovery of
L-573,655 in 1996.6 However, despite extensive researches have been conducted in these years,
few inhibitors have reached the clinic and none of them have reached the market. The most
well-studied LpxC inhibitor is CHIR-090, which displayed high affinity to both E. coli and P.
aeruginosa LpxC and remarkable antibacterial activity against a wide range of Gram-negative
pathogens.7 Later, Lee et al. and Liang et al. synthesized and evaluated a series of compounds
with linear diphenylacetylene scaffold. Some of these compounds, especially LPC-058, exhibited
excellent therapeutic potential against multi-drug resistant (MDR)/extensively drug resistant
(XDR) strains in vitro.8 In 2012, Pfizer reported a series of novel LpxC inhibitors with
methylsulfone, exemplified by 4 and 5.9 ACHN-975, developed by Achaogen, is the only LpxC
inhibitor accessed the Phase I clinical research, but there is no further progress revealed by now10
(Fig. 1).
Figure 1. The structures of classic LpxC inhibitors.
To have a better understand of the general mechanism of LpxC inhibitors, we analyzed the
X-ray crystal structure of CHIR-090 and LpxC from Aquifex aeolicus (aaLpxC) intensively.11 The
structure information reveals that LpxC is mainly composed of a catalytic zinc ion and a
hydrophobic tunnel accommodating a myristate fatty acid side chain. Thus, most of LpxC
inhibitors contain a zinc-binding group and a lipophilic tail to occupy the hydrophobic tunnel. The
lipophilic tails of the LpxC inhibitors make important van der Waals interactions with the LpxC in
the hydrophobic tunnel, which is also critical for the inhibition activity.12
P2
P1: The zinc-binding group
P3
HO
P2: The substitute group
O
H
N
P3: Linker, conecting the lipophilic
OH
N
tail and the zinc-binding group
H
P4
O
P1
P4: The lipophilic tail
O
P5: The terminal group
P5
N
Figure 2. The general structure of LpxC inhibitors exampled by CHIR-090.
As discussed above, we divided the structure of LpxC inhibitor into 5 parts for analysis,
exemplified by CHIR-090 (Fig. 2). P1 site is the zinc-binding group, which is designed to bind the
catalytic zinc ion in LpxC. P2 site is the substitute group, various kinds of substitute groups
generate different effect in the antibacterial activity. P3 site is the linker proportion, aims to
connect the lipophilic tail and the zinc-binding group. P4 site is the lipophilic tail with different
length, occupying the hydrophobic tunnel in LpxC. P5 site is the terminal group, various kinds of
groups have been introduced to this position to improve the antibacterial activity and biological
metabolism of the LpxC inhibitors. Thus, we designed and synthesized several series of

compounds mainly with the modifications in P1 site to P4 site to study the structure-activity
relationship, which would provide useful information for further study of the novel LpxC
inhibitors.
The synthetic route of compound YDL-1 is described in Scheme 1. Intermediate I-02 was
obtained by the nucleophilic reaction of sodium methanesulphinate and material I-01 in acetone,
then reacted with 4-bromophenethyl bromide and got intermediate I-03. The hydrolyzation of
intermediate I-03 was carried under alkaline condition, followed by the Suzuki coupling reaction
catalyzed by Pd(PPh3)4 to afford acid I-05. Finally, a condensation reaction was performed
between acid I-05 and o-phenylenediamine to yield compound YDL-1.
Scheme 1. Reagents and conditions: (a) sodium methanesulphinate, acetone, 70 ; (b)
4-bromophenethyl bromide, Cs2CO3, DMF, 50 ; (c) LiOH (1 N in water), EtOH，rt; (d)
phenylboronic acid, Na2CO3, Pd(PPh3)4, H2O/isopropanol, 70 ; (e) o-phenylenediamine, HATU,
Et3N, DMF, rt.
The preparation of compound YDL-4 is outlined in Scheme 2. An addition-elimination reaction
was carried between material II-01 and L-Threonine methyl ester hydrochloride under alkaline
condition and got sulfamide II-02, followed by the Sonogashira cross-coupling reaction with
ethynyltrimethylsilane catalyzed by Pd(PPh3)2Cl2 and deprotection process under alkaline
condition to get terminal alkyne II-04. Intermediate II-05 was obtained by the Eglinton reaction
with terminal alkyne II-04 and ethynylcyclopropane catalyzed by Cu(OAc)2, then reacted with the
aqueous solution of NH2OH to yield hydroxamic acid YDL-4.
Scheme 2. Reagents and conditions: (a) L-Threonine methyl ester hydrochloride, TEA, DCM,
0
to rt; (b) ethynyltrimethylsilane, Pd(PPh3)2Cl2, CuI, THF, 60 ; (c) K2CO3，MeOH, rt; (d)
ethynylcyclopropane, Cu(OAc)2, MeOH/Pydine, rt; (e) NH2OH (50% in water), EtOH, 40 .
Key intermediates III-08~III-09 were afforded according the procedure illustrated in Scheme 3
and Scheme 4. Chiral acid III-04 with a single configuration was afforded in one-pot procedure of
three components (III-01, III-02 and III-03) under alkaline condition, then transferred to ester
III-05 by the methylation reaction using CH3I. A hydrogenation reaction was adopted with the
existence of Zn powder and NH4Cl to yield amine III-06, followed by N-Boc protection reaction
with Boc2O and gave intermediate III-07. Finally, deprotection reaction of 2-phenethyl group was
carried using hydrogen under pressure (3 KG) to yield key intermediate III-08. Intermediate
IV-02 was obtained by the protection of hydroxy group of material IV-01 with TBSCl, then the
reduction reaction was carried with the existence of LiBH4 to afford hydroxyl intermediate IV-03.
Aldehyde IV-04 was afforded by oxidation of hydroxy with TPAP and then reacted with NH2-OBn
to yield Schiff base IV-05, followed by the reduction reaction of imine with NaBH3CN to yield
intermediate IV-06. A condensation reaction was carried between intermediate IV-06 and HCOOH
using CDI, then deprotected the O-TBS group in TBAF and the N-Boc group in trifluoroacetic
acid separately to yield key intermediate IV-09.

Scheme 3. Reagents and conditions: (a) KOH，H2O, 45 ; (b) CH3I, Cs2CO3，DMF, rt; (c) Zn
powder, NH4Cl, AcOH, THF, rt; (d) Boc2O, TEA, THF, rt; (e) H2 (3 KG), Pd(OH)2, THF, 40 .
Scheme 4. Reagents and conditions: (a) TBSCl, imidazole, DMF, rt; (b) LiBH4, ethanol, 55 ; (c)
NMO, TPAP, 4Å-MS, CH2Cl2, rt; (d) NH2-OBn·HCl, pyridine, methanol, rt; (e) NaBH3CN,
CH3COOH, rt; (f) CDI, HCOOH, CH2Cl2, rt; (g) 1N TBAF in THF, rt; (h) TFA, CH2Cl2, rt.
The convergent synthetic strategy toward compounds YDL-2, YDL-3, YDL-5~YDL-14,
YDL-19~YDL-23 is summarized in Scheme 5. Protected alkynes V-13~V-24 were obtained by
the Sonogashira cross-coupling reactions with bromides V-01~V-12 and ethynyltrimethylsilane
catalyzed by Pd(PPh3)2Cl2, then their O-TMS protections were removed under alkaline condition
to get corresponding terminal alkynes V-25~V-36. The Eglinton reactions catalyzed by Cu(OAc)2
were carried between terminal alkynes V-25~V-36 and ethynylcyclopropane to afford
corresponding intermediates V-37~V-48, then hydrolyzed under alkaline condition to yield vital
acids V-49~V-60. There were three pathways leading to the final productions. 1) Amides
V-61~V-64 were obtained by condensation reactions between acids V-49, V-50, V-53, V-59 and
key intermediate III-08 separately, then deprotected the N-Boc groups under acid condition to get
corresponding amines V-65~V-68. Finally, these amines were reacted with the aqueous solution of
NH2OH to afford hydroxamic acids YDL-20~YDL-23. 2) A condensation reaction was carried
between acid V-49 and key intermediate IV-09 to afford intermediate V-69, then the deprotection
process was performed with the existence of BCl3 to get reversed hydroxamic acid YDL-3. 3)
Threonine derivatives V-70~V-81 were obtained by the dehydration process between
corresponding acids V-49~V-60 and L-Threonine methyl ester hydrochloride under alkaline
condition separately, which were then converted to hydroxamic acids YDL-2, YDL-5~YDL-14,
YDL-19 using the aqueous solution of NH2OH.

Scheme 5. Reagents and conditions: (a) ethynyltrimethylsilane, Pd(PPh3)2Cl2, CuI, TEA, THF,
60oC; (b) K2CO3，MeOH, rt; (c) ethynylcyclopropane, Cu(OAc)2, MeOH/Pydine, rt; (d) LiOH (1
N in water), EtOH; (e) III-08, EDCI, HOBt, DIEA, DMF, rt; (f) TFA, DCM, rt. (g) NH2OH (50%
in water), DCM/MeOH, rt; (h) IV-09, EDCI, HOBt, DIEA, DMF, rt; (i) BCl3, CH2Cl2, -78~0 . (j)
L-Threonine methyl ester hydrochloride, EDCI, HOBt, DIEA, DMF, rt;
The target compounds YDL-15~YDL-18 were afforded according to the procedure illustrated
in Scheme 6. The ring-open reaction was adopted between commercially available materials
VI-01 and VI-02 of different chiral configurations and 4-bromoaniline yielded dihydroxyl
intermediates VI-03~VI-04, which were further cyclized using diethyl carbonate under alkaline
condition to give oxazolidinones VI-05~VI-06. The oxidation reactions were carried to transfer
hydroxyl oxazolidinones VI-05~VI-06 to corresponding acids VI-07~VI-08, then the methylation
process was performed to afford corresponding esters VI-09~VI-10. Intermediates VI-11~VI-14
were obtained by Suzuki coupling reactions catalyzed by Pd(PPh3)4 or Sonogashira cross-coupling
reactions catalyzed by Pd(PPh3)2Cl2 from esters VI-09~VI-10, then hydrolyzed to corresponding
acids VI-15~VI-18. The hydroxy of material VI-19 was protected by PMBCl, then ammonolysis
reaction was proceeded using hydrazine hydrate in ethanol and afforded amine VI-21.
Intermediate VI-22 was obtained by the condensation process between amine VI-21 and
N-Boc-threonine, followed by deprotection with BiCl3, gave the key amine VI-23. The
condensation reactions were carried between acids VI-15~VI-18 and amine VI-23 separately to
get corresponding intermediates VI-24~VI-27, which were reacted with trifluoroacetic acid to
deprotect the O-PMB group and yielded compounds YDL-15~YDL-18.
Scheme 6. Reagents and conditions: (a) 4-bromoaniline, EtOH, 50 ; (b) diethyl carbonate,
MeOH/MeONa, toluene, 105 ; (c) TEMPO, NaClO, NaClO2, CH3CN/H2O, 80 ; (d) CH3I,
Cs2CO3 ， DMF, rt; (e) phenylboronic acid, Pd(PPh3)4, K2CO3, EtOH/H2O, 85 ; (f)
phenylacetylene, Pd(PPh3)2Cl2, CuI, TEA, THF, 60 ; (g) LiOH (1 N in water), EtOH; (h) PMBCl,
K2CO3, DMSO, rt; (i) hydrazine hydrate, EtOH, 50 ; (j) N-Boc-threonine，EDCI, HOBt, DIEA,
DMF, rt; (k) BiCl3, acetonitrile /H2O, 50 ; (l) L-Threonine methyl ester hydrochloride, EDCI,
HOBt, DIEA, DMF, rt; (m) NH2OH (50% in water), DCM/MeOH, rt.
Minimum inhibitory concentration (MIC) of each compound was tested by the modified
National Committee for Clinical Laboratory Standards, which is adapted to 96-well plates and LB
media in the presence of 5% DMSO.13 The detail of the testing method can be found in supporting
information. Tested bacterial strains were classic Gram-negative pathogens, including P.
aeruginosa (PAO1, PA14) and E. coli (AB1157, DH5α).
Our effects began with the replacement of the P1 site. Hydroxamic acid is the most commonly
used zinc-binding group of the LpxC inhibitors, and exhibit remarkable zinc-binding capacity.

However, the weakness of metabolism is also existed in hydroxamic acid, which would be turned
into toxic hydroxylamine in vivo.14 The acyl o-phenylenediamine and reversed hydroxamate acid
were also proved as excellent zinc-binding groups in the previous literature.15 Thus, we attempted
to replace the hydroxylamine of hydroxamic acid to o-phenylenediamine and reverse the
hydroxamate acid, expecting to get novel LpxC inhibitors with both remarkable antibacterial
activity and excellent biological metabolism. We chose the racemates of compounds 4 and YDL-2
as leading compounds, replaced the zinc-binding group and tested their antibacterial activities.
The MIC values of all tested compounds were listed in Table 1. Unfortunately, YDL-1 and
YDL-3 lost their antibacterial activities totally, which implied that the hydroxamic acid is
essential for the inhibition activity and hard to be replaced.
Table 1.
In vitro antibacterial activity of compounds with the modification of the hydroxamate acid group.
MIC (µg/mL)
Compound
Structure
P. aeruginosa P. aeruginosa E. coli
E. coli
PAO1
PA14
DH5α AB1157
O
S
O
H
N
OH
0.31
0.31
0.4
>256
0.024
>256
1
4 (racemate)
O
>256
0.19
>256
0.19
>256
0.024
>256
YDL-1
YDL-2
YDL-3
HO
O
H
N
N
OH
H
O
>256
>256
P3 site is the linker position, connecting the lipophilic tail and the zinc-binding group. Early
literatures show that there is some synergistic effect between the sulfamide group and hydroxamic
acid, enhancing the combining capacity of the inhibitors to metalloenzyme.16 Thus, we attempted
to introduce the sulfamide group into the P3 site and get compound YDL-4. The results show that
the antibacterial activities against both E. coli and P. aeruginosa have dramatically decreased after
the replacement (Table 2).
Table 2.
In vitro antibacterial activity of compounds with the modification of amide group.
MIC (µg/mL)
Compound
Structure
P. aeruginosa P. aeruginosa E. coli E. coli
PAO1 PA14 DH5α AB1157

100
50
20
40
YDL-4
YDL-2
HO
O
H
N
N
OH
H
O
0.19
0.19
0.024
0.024
P4 site is the lipophilic tail of LpxC inhibitors, designed to occupy the hydrophobic tunnel in
LpxC. The majority of the LpxC inhibitors (CHIR-090, LPC-058, ACHN-975, et al.) preferred the
unsubstituted benzene ring as the “head” of the lipophilic tail adjacent to the P3 site, probably
concerning the space of the hydrophobic tunnel. In 2016, a series of novel LpxC inhibitors with
the framework of oxazolidone were synthesized by Kurasaki et al.17 They introduced kinds of
substitute groups on the benzene ring in the P4 site to discuss their influence and found that the
compounds with β-substitution showed higher antibacterial activity to the ones with no
substitution. But the P2 site and P3 site of these LpxC inhibitors had been incorporated into one
site (oxazolidone) in their discussions, the SARs summarized by them may not be the same as the
ones with separated P2 and P3 sites (CHIR-090, LPC-058, ACHN-975, et al.). Thus, we
introduced kinds of small-size substitute groups to the benzene ring of compound YDL-2 with
separated P2 and P3 sites, to evaluate their influence on the antibacterial activities of this kind of
LpxC inhibitors. Compounds YDL-5 ~YDL-14 have been synthesized and their MICs are listed in
table 3. The antibacterial activities of these compounds are related to the sizes of the substitute
groups, which will decrease more with larger substitutions (YDL-9, YDL-10, YDL-12 and
YDL-13) than the ones with smaller substitutions (YDL-5, YDL-6, YDL-7, YDL-8 and YDL-11).
Unexpectedly, the compounds with hydrophilic groups (YDL-8 and YDL-11) almost maintain
their antibacterial activities against both E. coli and P. aeruginosa, which is contradicted against
the common sense of the hydrophobic tunnel of LpxC. As shown in Figure 3, we speculated that
intramolecular hydrogen bonds may exist between the hydrophilic substitutions (hydroxy in
YDL-8 and amino in YDL-11) in the P4 site and the carboxide group in the P3 site, which may
influence the spatial configuration of the molecules and favor their combination to LpxC. This
effect would reduce the decrease of antibacterial activities induced by the introduction of
hydrophilic substitute group in the hydrophobic tunnel. The position of the substitutions is also
important, compounds with β-substitution are more favorable to the ones with α-substitution for
the performance of their antibacterial activities (YDL-5 vs. YDL-14), and both of them are weaker
than the unsubstituted one (YDL-2).
Table 3.
In vitro antibacterial activity of compounds with various substitutions in the benzene ring.
HO
R1
O
H
R2
N
N
OH
H
O

MIC (µg/mL)
Compound
R1
R2
P. aeruginosa P. aeruginosa
E. coli
DH5α
0.31
E. coli
AB1157
0.31
PAO1
2.5
PA14
1.25
F
Cl
H
H
H
H
H
H
H
H
H
F
YDL-5
YDL-6
6.25
5
6.25
5
0.048
0.156
0.156
2.5
0.192
0.156
0.078
1.25
0.156
0.04
0.6
CH3
OH
OCH3
OCHF2
NH2
NO2
CN
H
YDL-7
YDL-8
0.625
10
0.625
10
YDL-9
10
10
0.31
0.04
0.4
YDL-10
YDL-11
YDL-12
YDL-13
YDL-14
YDL-2
0.625
5
0.625
5
40
40
40
80
0.63
0.19
2.5
0.63
0.19
2.5
0.31
0.024
0.156
0.4
H
H
/
0.024
0.156
CHIR-090
/
Figure 3. The intramolecular hydrogen-bond interaction in compounds YDL-8 and YDL-11.
In 2011, Mansoor et al. designed and synthesized two series of novel urea derivatives, the
benzene rings (adjacent to the P3 site) of which were replaced by aliphatic heterocycles.18 These
compounds remained the inhibitory activities to Gram-negative bacteria and were worthy of study.
To have a further exploration of the structure-activity relationships in the P4 site, we attempted to
introduce the oxazolidinone ring and the pyridine ring to replace the benzene ring adjacent to the
P3 site and synthesized compounds YDL-15 to YDL-19. The results are shown in table 4.
However, all the compounds containing oxazolidinone lose their antibacterial activities totally
(YDL-15~YDL-18). The introduction of pyridine ring is also unfavorable for the antibacterial
activity (YDL-19 vs. YDL-2).
Table 4.
In vitro antibacterial activity of compounds with replacements of the benzene ring to the
oxazolidinone ring and the pyridine ring.
HO
O
H
N
N
OH
R1
H
O
R2

MIC (µg/mL)
Compound
R1
R2
P. aeruginosa P. aeruginosa E. coli E. coli
PAO1
PA14
DH5α AB1157
>256
>256
>256
>256
>256
>256
0.625
0.024
>256
>256
>256
>256
1.25
YDL-15
YDL-16
YDL-17
YDL-18
YDL-19
YDL-2
>256
>256
>256
20
>256
>256
>256
10
0.19
0.19
0.024
P2 site is the substituted group, various kinds of substitutions have been introduced in this site
of LpxC inhibitors. The 2-hydroxy ethyl group and the 2-amino isopropyl group are proved and
most widely used substitute groups in the P2 site. But there are few literatures to compare these
groups directly. We selected four compounds (YDL-2, YDL-5, YDL-8 and YDL-14) as leading
compounds. Compounds YDL-20~YDL-23 were synthesized and their antibacterial activities
were evaluated to recognize the better substitute group for LpxC inhibitors. Their MIC values are
listed in table 5. The results display that the 2-amino isopropyl group is better than the 2-hydroxy
ethyl group in antibacterial aspect generally (YDL-20 vs. YDL-2, YDL-21 vs. YDL-5, YDL-22
vs. YDL-8, YDL-23 vs. YDL-14).
Table 5.
In vitro antibacterial activity of compounds with the 2-hydroxy ethyl group and the 2-amino
isopropyl group.
MIC (µg/mL)
Compound
R1
R2
R3
P. aeruginosa P. aeruginosa
E. coli
E. coli
PAO1
PA14
DH5α
AB1157
H
H
F
H
H
H
0.156
0.078
0.012
0.024
0.04
0.039
0.024
0.078
YDL-20
YDL-2
0.19
0.19
0.31
0.625
YDL-21

F
OH
OH
H
H
H
H
F
2.5
1.25
0.625
0.625
0.31
0.31
0.078
0.156
0.019
0.31
0.31
0.078
0.078
0.019
0.4
YDL-5
YDL-22
YDL-8
0.625
0.625
0.31
YDL-23
YDL-14
H
F
0.63
0.63
To get deep understanding of the structure-activity relationship, we selected the most potent
compound YDL-20 to investigate its binding mode in the binding site of LpxC. The cocrystal
structure of CHIR-090 bound to Pseudomonas aeruginosa LpxC (PDB entry code 5U39) was
prepared with Schrodinger software to be the enzyme model, and Glide XP was utilized for
docking study by constraint with the Metal Coordination function implemented in the program.
The detail of the method can be found in supporting information. As shown in Figure 4, the
predicted binding conformation of YDL-20 is very similar to CHIR-090, which demonstrated that
the hydroxamic acid formed strong interactions with the zinc ion and amine group of YDL-20 is
located at similar position of hydroxyl group. As CHIR-090, the benzene group of YDL-20
located in a hydrophobic subpocket, and interacted with several residues including Leu200,
Ala206 and Ala214, which indicated that it only can accommodate small substituents at the
benzene group. And large groups (YDL-9, YDL-10, YDL-12 and YDL-13) will crash with nearby
protein atoms, therefore decrease the binding affinity. The alkynes of YDL-20 stretched out the
binding channel and the cyclopropane group just situated at the entrance of the binding site, which
is still in the hydrophobic environment. Meanwhile, the benzene ring in the P4 site and carboxide
are nearly on the same plane. The intramolecular hydrogen bonds between the hydrophilic
substitutions (hydroxy in YDL-8 and amino in YDL-11) on the benzene ring and the carboxide
group may enhance the plane configuration of these molecules and favor their combination to
LpxC, which would reduce the decrease of antibacterial activities induced by the introduction of
hydrophilic substitute group in the hydrophobic tunnel.

Figure 4. The predicted binding mode of compound YDL-20 in LpxC from the Glide docking
study. The protein as well as CHIR-090 was colored in cyan, while YDL-20 was colored in yellow.
Water molecules were shown as red sphere and Zinc ion was shown as gray sphere.
In addition, we evaluated the metabolic stability of compounds YDL-2, YDL-5, YDL-8,
YDL-14, YDL-20~YDL-23 in liver microsomes (human and mouse). The detail of the testing
method can be found in supporting information. As shown in Table 6, compounds
YDL-20~YDL-23 exhibited better metabolic stability than YDL-2, YDL-5, YDL-8, YDL-14
respectively in the liver microsomes of human, which indicated that the 2-amino isopropyl group
may be a preferred structure than the 2-hydroxy ethyl group in the design of LpxC inhibitors.
Table 6.
Half-lives and intrinsic clearances of YDL-2, YDL-5, YDL-8, YDL-14, YDL-20~YDL-23 in
liver microsomes a
Compound Species Microsomal t1/2 b (min)
Clint c (mL⋅min/g/protein)
Human
Mouse
Human
Mouse
Human
Mouse
Human
Mouse
Human
Mouse
Human
Mouse
Human
Mouse
Human
Mouse
633
190
3415
206
200
85
3
11
1
YDL-2
YDL-20
YDL-5
10
11
25
0
--d
YDL-21
YDL-8
240
273
233
--d
9
8
9
0
YDL-22
YDL-14
YDL-23
95
22
1
2664
143
--d
15
0
135
16

a0.33 mg/mL microsomal protein, NADP+-regenerating system, [inhibitor], 0.1 µmol/L, incubation
at 37 , samples taken at 0, 7, 17, 30, and 60 min, determination of parent compound by MS. b t1/2:
c
d
elimination half-life in rat liver microsomes. Clint: intrinsic body clearance. t1/2 was not
calculated, because compound YDL-21~YDL-23 were not metabolized at 60 min.
In conclusion, several series of novel LpxC inhibitors were synthesized and their
structure-activity relationships had been evaluated by the MIC values against E. coli and P.
aeruginosa in vitro. In the P1 site, the hydroxylamine of hydroxamic acid had been replaced with
o-phenylenediamine and reversed, accompanying with the degression of their antibacterial
activities. In the P2 site, we proved that the 2-amino isopropyl group is better than the 2-hydroxy
ethyl group in the aspects of antibacterial activity in vitro and metabolism stability in human liver
microsomes. In the P3 site, the introduction of the sulfamide group did not generate the synergistic
effect with hydroxamic acid and decrease the antibacterial activities deeply. In the P4 site, the
introduction of substitute groups to the benzene ring or the replacement of the benzene ring with
oxazolidinone ring or pyridine ring would decrease the antibacterial activities in different degrees.
We hope that these structure-activity relationships would be very useful for further development of
the LpxC inhibitors.
Acknowledgment
The authors thank Professor Xiong Bing from Shanghai Institute of Materia Medica, for
molecular docking experiments. This work was supported by Key New Drug Creation and
Manufacturing Program of China (No. 2014ZX09507009-016) and Youth Project of Education
Department of Liaoning Province (No. LQN201709).
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Graphical abstract