Design, synthesis and biological evaluation of 1-Phenyl-2-(phenylamino) Ethanone Derivatives as Novel MCR-1 Inhibitors

Article abstract of DOI:10.3390/molecules24152719

Polymyxins are considered to be the last-line antibiotics that are used to treat infections caused by multidrug-resistant (MDR) gram-negative bacteria; however, the plasmid-mediated transferable colistin resistance gene (mcr-1) has rendered polymyxins ine

Full text of DOI:10.3390/molecules24152719

molecules
Article
Design, Synthesis and Biological Evaluation of
1-Phenyl-2-(phenylamino) Ethanone Derivatives as
Novel MCR-1 Inhibitors
Xiu-juan Lan 1,2,3, Hai-tao Yan 3, Feng Lin 4, Shi Hou 1, Chen-chen Li 1, Guang-shu Wang 1,
Wei Sun 1,*, Jun-hai Xiao 2,3,* and Song Li 2,3
* ,
1
School of Pharmaceutical Sciences, Jilin University, Changchun 130021, China
National Engineering Research Center for Strategic Drugs, Beijing Institute of Pharmacology and Toxicology,
Beijing 100850, China
State Key Laboratory of Toxicology and Medical Countermeasures, Beijing Institute of Pharmacology and
Toxicology, Beijing 100850, China
School of Life Sciences, Jilin University, Changchun 130021, China
Correspondence: wgs@jlu.edu.cn (G.-s.W.); wsun@jlu.edu.cn (W.S.); xiaojunhai@139.com (J.-h.X.);
Tel.: +86-10-6693-1642 (J.-h.X.)
2
3
4
*
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 21 June 2019; Accepted: 23 July 2019; Published: 26 July 2019
Abstract: Polymyxins are considered to be the last-line antibiotics that are used to treat infections
caused by multidrug-resistant (MDR) gram-negative bacteria; however, the plasmid-mediated
transferable colistin resistance gene (mcr-1) has rendered polymyxins ineffective. Therefore, the protein
encoded by mcr-1, MCR-1, could be a target for structure-based design of inhibitors to tackle
polymyxins resistance. Here, we identified racemic compound
by virtual screening, and 26 compound derivatives were synthesized and evaluated in vitro
In the cell-based assay, compound 6g 6h 6i 6n 6p 6q, and 6r displayed more potent activity
than compound . Notably, 25 M of compound 6p or 6q combined with 2 mL-1 colistin could
3 as a potential MCR-1 inhibitor
3
.
,
,
,
,
,
3
µ
µg
·
completely inhibit the growth of BL21(DE3) expressing mcr-1, which exhibited the most potent activity.
In the enzymatic assay, we elucidate that 6p and 6q could target the MCR-1 to inhibit the activity of the
protein. Additionally, a molecular docking study showed that 6p and 6q could interact with Glu246
and Thr285 via hydrogen bonds and occupy well the cavity of the MCR-1 protein. These results may
provide a potential avenue to overcome colistin resistance, and provide some valuable information
for further investigation on MCR-1 inhibitors.
Keywords: Colistin resistance; virtual screening; MCR-1 inhibitors; 1-phenyl-2-(phenylamino)
ethanone derivatives
1. Introduction
Nowadays, antibiotic resistance in gram-negative pathogens is recognized as one of the most
serious global threats to public health [
1]. The increase of multidrug-resistant (MDR) organisms results
in more than 700,000 deaths worldwide each year and poses a heavy burden on healthcare costs [
2].
The lack of effective antibiotics to treat infections caused by MDR bacteria is pushing us to the edge of
a postantibiotic era. To address this situation, polymyxins, and old antibiotics, have been reused in
clinics to combat the infections caused by MDR pathogens [
“last line” treatments for MDR pathogens [4].
3], and they are now used as one of the
Polymyxins are cationic antimicrobial peptides (CAMP) that were isolated from Paenibacillus
polymyxa in 1947. There are five types of polymyxins: A, B, C, D and E, and among these, polymyxin B
and polymyxin E (colistin) are used in clinical therapies [
3,
5]. In the past years, the polymyxin
Molecules 2019, 24, 2719; doi:10.3390/molecules24152719
www.mdpi.com/journal/molecules

Molecules 2019, 24, 2719
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resistance mechanisms are mediated chromosomally, such as the two-component regulatory systems
PhoPQ and PmrAB, as well as the inactivation of mgrB, leading to the modification of lipid A
with phosphoethanolamine (PEA) or 4-amino-4-arabinose [6–8]. Recently, in late 2015, the first
plasmid-mediated colistin resistance gene, mcr-1, was reported in China. The mcr-1 gene encodes PEA
transferase, leading to the addition of the PEA moiety, which is from the phosphatidylethanolamine (PE)
donor substrate, to lipid A, decreasing the negative charges of lipid A and impacting the interactions
between colistin and the membrane of pathogens, that is, colistin resistance has occurred [9].
The emergence of mcr-1 is likely to bring on the rapid dissemination of polymyxin resistance,
due to the horizontal transfer of plasmids in various bacteria strains. To date, mcr-1 has been found
in food, animal, and human samples [10]. Additionally, it is worrisome that a growing number of
Enterobacteriaceae strains with mcr-1 coexisting with carbapenem resistance genes, such as NDM-5 and
NDM-9, have been identified [11–13]. Mcr-1 has been isolated from more than 50 countries distributed
on five continents, and there is an increasing risk that pan-drug-resistant bacteria will emerge globally.
Currently, there are few research projects on MCR-1 inhibitors have been reported, such as those
generating ethanolamine (ETA) [14] and pterostilbene [15,16], with the structures shown in Figure 1.
The combination of 10 mM ETA with 4 mL-1 polymyxin B inhibits the growth of Escherichia coli
µg
·
strain Rosetta cells expressing mcr-1; pterostilbene could restore susceptibility to colistin against
mcr-1-positive Escherichia coli strains, and mcr-1-positive K. pneumoniae isolates when it was respectively
32
further clarified. Thus, the development of novel agents, to combat colistin-resistance, was warranted.
We herein report that compound was selected as a potential MCR-1 inhibitor by virtual screening
for the first time, and 26 compound derivatives were designed, synthesized, and evaluated in vitro
µg
·
mL-1 and 16
µg
·mL-1. Both of them displayed low activity, and the mechanism remains to be
3
3
,
and an enzymatic assay was performed to elucidate that active compounds could target the MCR-1
protein. Additionally, structure-activity relationship (SAR) studies and molecular docking analysis were
performed to determine critical structure responsible for their MCR-1 inhibitory activity. The results
might provide some information to address the threats caused by mcr-1.
1
2
Figure 1. Structures of (1) ethanolamine (ETA), and (2) pterostilbene.
2. Results and Discussion
2.1. Finding Potential MCR-1 Inhibitors by Virtual Screening
The full-length MCR-1 protein is composed of an N-terminal transmembrane domain and a
periplasmic C-terminal catalytic domain. However, only the crystal structure of the C-terminal catalytic
domain was reported [17–21]. The active site of the MCR-1 catalytic domain is considered to be
the region of one zinc surrounded by E246, T285, D465, and H466, and T285 was believed to act as
the acceptor for the PEA group during the transfer reaction. However, the current data makes the
role of zinc in the PEA transfer reaction uncertain. In view of the research on the MCR-1 catalytic
domain, we screened over 8000 organic compounds in our in-house compound library based on the
MCR-1 crystal structure (PDB: 5GRR) [19]. The molecules were ranked according to their docking
scores, and the molecules which have shape complementarity and form more hydrogen bonds with
the active site of MCR-1 were selected. Ultimately, a total of 30 compounds were selected as potential
MCR-1 inhibitors.

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2.2. Evaluation of the Activity of Potential MCR-1 Inhibitors
We here evaluated the activities of the above 30 compounds by applying the broth microdilution
method [22], and the strain of E. coli BL21(DE3) was used to perform the cell-inhibitory assay. In this
assay, BL21(DE3) and BL21(DE3) transformed with only the null pET-28a(+) plasmid were used
as a negative control (NC), and BL21(DE3) transformed with the pET-28a(+) plasmid carrying the
full-length mcr-1 gene was used as the wild type (WT) control. The Clinical and Laboratory Standards
Institute (CLSI) defines colistin resistance as
The MICs of colistin against BL21(DE3), BL21(DE3)+pET-28a(+), and BL21(DE3)+pET-28a(+)+mcr-1
≥
8
µ
g
·
mL−1 and colistin susceptibility as
≤
2
µg .
mL−1
·
are shown in Table 1. As can be seen, the MICs of colistin against the negative control bacterias
are 2
µ
g
·
mL−1, and the MIC of colistin against BL21(DE3) transformed with the mcr-1 gene is
8
µg
·
mL−1, the results indicated that BL21(DE3) expressing mcr-1 confers colistin resistance. Therefore,
the MIC assay using the BL21(DE3)+pET-28a(+)+mcr-1 was performed to screen the efficacies of the
30 compounds. First, we tested whether the compounds alone could influence the growth of the
above strain at a concentration of 100
results showed that none of the compounds influenced the growth of the strain. Then, we tested
which compound could decrease the colistin resistance of 8
mL−1 to colistin susceptibility of
mL−1. The concentrations of the compounds used were 50
M and 100 M. Among the 30
screened compounds, 100 M compound combined with 2
mL−1 colistin could completely inhibit
the growth of BL21(DE3)+pET-28a(+)+mcr-1, and all of the results suggested that compound may
is
µM. The wells of the microwell plates were turbid, and the
µg
·
2
µg
·
µ
µ
µ
3
µg
·
3
inhibit the activity of MCR-1 and restore the susceptibility of colistin. Therefore, compound
3
identified as a potential MCR-1 inhibitor. The structure of compound 3 is shown in Figure 2a.
Table 1. The colistin MIC of Escherichia coli BL21(DE3) strains that carry the mcr-1 gene.
E. coli BL21(DE3) Strains
Colistin MIC (µg·mL−1
)
BL21(DE3)
pET28a
pET28a-mcr-1
2
2
8
a
b
c
Figure 2.
(a) Structure of racemic compound
3. (b) Docking pattern of the interactions between
compound
3
and MCR-1 catalytic domain with H-bonds shown by red lines. (c) The core structure of
1-phenyl-2-(phenylamino) ethanone.
2.3. Compound Design
The active site of 5GRR occupies a shallow depression on a relatively flat face of the protein.
However, the zinc was so far away from the shallow depression that molecules could not coordinate
with zinc, while molecules could interact with key amino acids. The docking results between MCR-1
catalytic domain and compound 3 are presented in Figure 2b. The results showed that compound 3
occupies well the shallow depression of MCR-1 catalytic domain, and the carboxyl group interacted
with Glu246 and Thr285 by hydrogen bonding (H-bond). The above observations indicated that

Molecules 2019, 24, 2719
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1-phenyl-2-(phenylamino) ethanone which mainly contributed to occupying the cavity and the carboxyl
group which forms H-bonds with essential amino acids might be important for the activity against
MCR-1, therefore, the structure of 1-phenyl-2-(phenylamino) ethanone was chosen as the core structure
(Figure 2c), and the effects of the carboxyl group (R), R0 and R00 substituents were investigated on SARs.
Recently, the modelled structure of the full-length MCR-1 through structural modelling with the
Neisseria meningitidis EptA [23] (NmEptA, PDB: 5FGN) as a template was reported, and the study
suggested that the trans-membrane domain was essential for MCR-1 activity and there is a cavity
for PE substrate entry into MCR-1. The cavity which is composed of a catalytic site and a PE lipid
substrate-binding site was considered to be the active site of MCR-1 [24]. Based on the above study,
we attempted to synthesize a series of compounds with a linear alkyl chain at R0, which were similar
to the fatty chain of PE substrate, and investigated its effect on activity.
2.4. Chemistry
The synthetic routes of all of the new compounds in this study are shown in Schemes 1–3.
In Scheme 1, commercially available acetophenone with various substituent groups
selenium dioxide, yielding corresponding phenylglyoxals , and then, was reacted with primary
amines to yield 6a 6r. In Scheme 2, the synthesis of 8a 8d also started from various acetophenones
by adding copper (II) bromide, and the bromation was accomplished to give . Then, 4-aminobenzoic
acid was added to an ethanolic solution of , yielding 8a 8d. In Scheme 3, the acetophenones
with various substituent groups were oxidized by selenium dioxide, yielding the corresponding
phenylglyoxals . Then, 4-aminobenzoic acid reacted with in the presence of selenium dioxide and
pyridine, an oxidation reaction occurs, and 9a–9d were obtained.
4 was oxidized by
5
5
–
–
4,
7
7
–
4
5
5
Scheme 1. The synthetic routes of 6a–6r. Reagents and conditions: (a) SeO2, 1,4-dioxane, H2O, reflux;
(b) ROH (CH3OH, EtOH, (CH3)2CHOH), r.t.
Scheme 2. The synthetic routes of 8a–8d. Reagents and conditions: (a) CuBr2, EtOAc, reflux; (b) EtOH, r.t.
Scheme 3. The synthetic routes of 9a
(b) SeO2, Py, MeCN, reflux.
–9d. Reagents and conditions: (a) SeO2, 1,4-dioxane, H2O, reflux;

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2.5. Activity Evaluation and SAR Study
As mentioned in Section 2.2, first, we tested whether the synthesized compounds alone could
influence the growth of the strain, the wells of the microwell plates were turbid when the concentration
of the compounds used was 100
influenced the growth of the strain. After that, the activities of the compounds in combination with
mL−1 colistin were tested. The concentrations of the compounds used were 12.5
M, 25 M,
M, and 100 M. The results of the activity evaluation of the synthesized compounds are listed
in Tables 2 and 3. As can be seen, seven compounds (6g 6h 6i 6n 6p 6q, and 6r) displayed more
potent activity than compound . Among them, 25 M of compound 6p and 6q in combination with
mL−1 colistin could completely inhibit the growth of BL21(DE3) expressing mcr-1, which exhibited
the most potent activity.
For the first series of compounds 6a
µM, and the results showed that none of the novel compounds
2
50
µ
g
·
µ
µ
µ
µ
,
,
,
,
,
3
µ
2
µg
·
–
6i, shortening the length of the R1 substituent was investigated,
and the benzyl was replaced by smaller fluorine, hydroxyl, piperidine, and cyclohexyl groups;
meanwhile, the R4 position was substituted with carboxyl, ester, or nitro group. As shown in Table 2,
the introduction of fluorine, hydroxyl, or piperidine at R1 (6a–6d) led to a complete loss of activity.
As for the cyclohexyl substituent (6e–6i), the esterification or the introduction of nitro group at R4
resulted in the loss of activity, and retaining the carboxyl of the R4 group (6g
, 6h, and 6i) could increase
the MCR-1 inhibitory activity.
Then, the compounds 6j–6r, which switched the benzyl group of R1 to a linear alkane chain,
were synthesized based on the study of the modelled structure of the full-length MCR-1 [24]. As shown
in Table 2, compounds 6j–6l, which introduced n-propyl at R1, lost potency for MCR-1. When R1
was substituted by n-hexyl or n-octyl, the compounds (6m–6r) which keep the carboxyl group at R4
exhibited MCR-1 inhibitory activity, especially 6p and 6q, which displayed the most potent activity.
Surprisingly, compared to 6n, the introduction of methyl at R2 (6p) resulted in a significant increase
in the inhibitory activity. However, the introduction of a methyl group at R3 (6o and 6r) led to a
decreased activity. Additionally, from 6j, 6n, and 6q, we found that the extension of carbon chains
is more conducive to MCR-1 inhibitory activity with n-octyl group > n-hexyl > n-propyl. Therefore,
the SARs existing in these compounds listed in Table 2 indicated that the R1 and R4 substituents play
important roles in the MCR-1 inhibitory activity, and the compounds in which R1 was substituted by
alaphatic group (with a cyclohexyl, n-hexyl or n-octyl group) and the carboxyl was kept constant at R4
displayed inhibitory activity against MCR-1. Additionally, the length of the chain substitution had a
significant influence on the MCR-1 inhibitory activity, the longer the substitution was, the better the
activity. Moreover, the activity of 6o or 6r substituted with –CH3 at R3 was lower than 6p substituted
with –CH3 at R2.
Having produced optimized substituents at positions R1 and R4, we turned our attention to the
R5 substituent. Based on the compounds 6g
, 6h, and 6i, which displayed the same activities with
the only difference in R5, it was reasonable to assume that the removal of the R5 substituent will still
result in the maintenance of some activity. Thus, the compounds 8a–8d and 9a–9d that removed the
alkoxy substituents were synthesized. The results listed in Table 3 showed that the removal of the
R5 substituent resulted in activity losses, and the results indicated that the R5 substituents are also
essential to the MCR-1 inhibitory activity.
Taking into account the above SAR studies, we could conclude that the combination of R1, R4,
and R5 substituents is very important for MCR-1 inhibitory activity, and the SARs of these compound
3
derivatives was that compounds substituted with a lipophilic R1 (cyclohexyl, n-hexyl or n-octyl group)
and the maintenance of the carboxyl and EtO at R4 and R5 displayed better MCR-1 inhibitory activity.
Additionally, the activity of the compound substituted with –CH3 at R2 was superior to that of the
compound substituted with –CH3 at R3.

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Table 2. The activities of 6a–6r in combination with 2
expressing mcr-1.
µ
g
mL−1 colistin against the growth of BL21(DE3)
·
Comp.
3
R1
R2
H
H
H
H
H
H
H
H
H
R3
H
H
H
R4
R5
Conc. (µM)
100
6a
N.I.A.
N.I.A.
N.I.A.
N.I.A.
N.I.A.
N.I.A.
50
6b
6c
6d
6e
H
H
H
H
H
6f
6g
6h
50
6i
H
H
50
6j
6k
6l
H
H
H
H
H
H
H
H
H
H
H
N.I.A.
N.I.A.
N.I.A.
N.I.A.
50
6m
6n
6o
6p
6q
6r
100
H
H
25
H
H
25
50
N.I.A. = No Inhibitory activity.
2.6. Inhibitory Effects Against MCR-1
In order to elucidate that the synthesized compounds could inhibit the activity of MCR-1 protein,
we performed an enzymatic assay to evaluate whether synthesized compounds could target the
MCR-1. In this assay, the MCR-1 protein was purified as Xu and coauthors described [24], and the PEA
donor substrate with a fluorescent label (NBD-glycerol-3-PEA) was used [23]. As shown in Figure 3,
the MCR-1 protein could remove the PEA moiety from the substrate NBD-glycerol-3-PEA and give a
fluorescent product NBD-glycerol. The substrate NBD-glycerol-3-PEA and the product NBD-glycerol
could exhibit different Rf value by thin layer chromatography (TLC) using blue light (455–485 nm),
therefore, the TLC was used to separate the mixture in the enzymatic reaction [23]. If the synthesized
compounds could inhibit the reaction of PEA transfer catalyzed by MCR-1, and the TLC will mainly
show the substrate NBD-glycerol-3-PEA band. Thus, this system might be used to test the inhibitory
effect against MCR-1 protein.

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Table 3. The activities of 8a–8d and 9a–9d in combination with 2
of BL21(DE3) expressing mcr-1.
µg
mL−1 colistin against the growth
·
8a, 9a: R1=
8
9
8b, 9b: R1=
8c, 9c: R1=
8d, 9d: R1=
Comp.
Conc. (µM)
Comp.
Conc. (µM)
8a
8b
8c
N.I.A.
N.I.A.
N.I.A.
N.I.A.
9a
9b
9c
N.I.A.
N.I.A.
N.I.A.
N.I.A.
8d
9d
N.I.A.= No Inhibitory activity.
In this assay, 0.2 mM substrate NBD-glycerol-3-PE was used, and the results are shown in Figure 4,
the substrate NBD-glycerol-3-PEA was the lower spot, and the control 1 showed that the upper spot
was the product NBD-glycerol [24]. The control 2 showed that compound 3, 6p, 6q or 6m (6m was
one of the inactive compounds) could not influence the spot of the substrate. Therefore, this system
was used to test whether these selected compounds could target the MCR-1 protein. For compound
3
, the TLC exhibited one spot of the substrate when it was 10 mM, and the results indicated that
compound could inhibit the reaction of PEA transfer catalyzed by MCR-1. For 6p and 6q, the TLC
3
exhibited one spot of the substrate when they were respectively 1.6 mM and 0.8 mM, and the results
showed that compound 6p and 6q also could inhibit the reaction of PEA transfer catalyzed by MCR-1,
and compound 6q showed more potent inhibition than 6p. Obviously, both of them exhibited more
potent inhibition against MCR-1 protein than compound 3. However, compound 6m still clearly
exhibited two spots of the substrate and the product when it was 10 mM, and the results indicated
that 6m could not inhibit the reaction of PEA transfer catalyzed by MCR-1, which was consistent
with the loss of inhibitory activity in the cell-based assay. The above results suggested that the active
compounds 6p and 6q could interact with MCR-1 and inhibit the activity of the protein.
Figure 3. The scheme for the enzymatic reaction catalyzed by the MCR-1 protein.

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Compound 3
NBD-glycerol
Control 1
Substrate only
Control 2
10 mM
5 mM
2 mM
Compound 6p
Compound 6q
Control 1 Substrate only Control 2 1.6 mM
0.4 mM
0.1 mM Control 2
0.8 mM
0.4 mM
0.2mM
Compound 6m
Control 1 Substrate only Control 2 10 mM
5 mM
1 mM
Figure 4. The bands of compounds 3, 6p, 6q, and 6m interacted with MCR-1 protein for inhibiting
the reaction of phosphoethanolamine (PEA) transfer by thin layer chromatography (TLC); Control 1:
The bands of substrate interacted with MCR-1 protein; Control 2: The bands of tested compounds
interacted with the substrate.
2.7. Molecular Docking
After the previous test of biological activity, in order to further obtain the relationship between
the structure of the active compounds and the inhibition of MCR-1 protein, several compounds were
molecularly docked with the full-length MCR-1 modelled by Swiss-Model. The molecular docking
simulation results are presented in Figure 5. Since the studies have suggested that Thr285 was
believed to act as the acceptor for the PEA group during the transfer reaction, therefore, the interaction
with Thr285 is crucial to MCR-1 inhibitory activity. As shown in Figure 5a–c, the carboxyl group
of the active compound 6q, 6n or 6p bonds to Glu246 and Thr285 via hydrogen bonds, and the
fatty chain of compounds occupies well the cavity of MCR-1 protein. Comparing the docking result
of 6p, compound 6o lost the hydrogen bond interaction with the Thr285 (Figure 5d), therefore,
the decrease in the anti-MCR-1 activity of 6o might be due to the loss of the H-bond with Thr285.
Additionally, molecular docking was also performed to study the binding mode of compound 8d
and 9d (the compounds of series 2 and series 3), and the docking results are shown in Figure 5e,f.
As can be seen, both of them lost the H-bonds with Glu246 and Thr285, which played an important
role in anti-MCR-1 activity. This result might explain why 8d and 9d lost their activity against MCR-1
protein, and the results also elucidate the SARs that the R5 substituents are also essential to the MCR-1
inhibitory activity.
Additionally, another configuration of these compounds was also molecularly docked with the
full-length MCR-1. The docking results showed that the two configurations of the molecules did not
display an obvious difference in the docking modes, and the cavity of MCR-1 have a good match with
the two configurations. Therefore, on the basis of the docking results, the configuration of compounds
may not contribute to the MCR-1 inhibitory activity. The docking modes of another configuration are
provided in the Supplementary Data.

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A
B
C
E
D
F
Figure 5. Docking patterns of the interaction between compounds
A: 6q; B: 6n; C: 6p; D: 6o; E: 8d;
F: 9d and the full-length MCR-1 protein with H-bonds shown by red lines.
3. Materials and Methods
3.1. Structure-Based Virtual Screening
The MCR-1 crystal structure (PDB: 5GRR) was downloaded from the Protein Data Bank (PDB),
and the molecular docking was carried out using Gold (The Cambridge Crystallographic Data Centre).
Before docking calculations, the Zn601 which surrounded by E246, T285, D465, and H466 was kept,
the other zinc and the water molecules of the crystal structure were deleted, and hydrogen atoms
were added. The structures of 8141 compounds in our in-house compound library were transferred
from two-dimensional to three-dimensional by concord4.0 with energy minimization automatically
for the next docking simulations. During docking calculations, zinc was selected as the active site,
and the cavity was set to 10 Å; template was set to chemscore_kinase; number of GA Runs were set to
30; scoring function was set to GoldScore; GA search options was set to slow. After the docking process
was finished, the docking scores were in order from high scores to low scores, firstly, the compounds
which scored more than 50 were selected, and 286 compounds were obtained, and then, the molecules
which have shape complementarity and form more hydrogen bonds with the active site of MCR-1
were used for the analysis of docking and the interactions.
3.2. Chemistry
Solvents and reagents used in the synthesis were obtained from Beijing Innochem Science
and Technology Co. Ltd. and J&K Scientific Ltd. Chemical reactions were monitored by thin layer
chromatography (TLC). The structures of the products were identified by 1H and 13C-NMR spectroscopy
(Bruker Advance III 400 MHz spectrometer), with DMSO-d6 used as the solvent. The molecular weights
of the products were measured by high resolution mass spectrometry (HRMS) with electrospray

Molecules 2019, 24, 2719
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ionization (ESI) as the ionization mode (Agilent 1260-G6230A). The synthesis of the compounds,
NMR and mass spectra are provided in the Supplementary Materials.
Methyl 4-((1-Ethoxy-2-(4-fluorophenyl)-2-oxoethyl)amino)benzoate (6a) 1H-NMR (400 MHz, DMSO-d6)
δ
8.17 (dd, J = 8.9, 5.6 Hz, 2H), 7.76 (d, J = 8.8 Hz, 2H), 7.46–7.38 (m, 3H), 7.02 (d, J = 8.9 Hz, 2H),
6.29 (d, J = 8.6 Hz, 1H), 3.77 (s, 3H), 3.56–3.49 (m, 1H), 3.44–3.37 (m, 1H), 1.03 (t, J = 7.0 Hz, 3H). 13C-NMR
(101 MHz, DMSO-d6)
δ 192.94, 166.73, 150.90, 132.63, 131.32, 118.97, 116.45, 116.23, 113.50, 81.86, 60.81,
51.96, 15.76. HRMS(ESI) (m/z): Calad. for C18H18FNO4 331.3382; found 286.0875 [M − C2H5O]+.
Methyl 4-((1-Ethoxy-2-(4-hydroxyphenyl)-2-oxoethyl)amino)benzoate (6b
DMSO-d6) 10.55 (s, 1H), 8.00 (d, J = 8.8 Hz, 2H), 7.76 (d, J = 8.8 Hz, 2H), 7.35 (d, J = 8.4 Hz,
1H), 7.02 (d, J = 8.9 Hz, 2H), 6.88 (d, J = 8.8 Hz, 2H), 6.9 (d, J = 8.4 Hz, 1H), 3.77 (s, 3H), 3.53–3.45
(m, 1H), 3.41–3.33 (m, 1H), 1.02 (t, J = 7.0 Hz, 3H). 13C-NMR (101 MHz, DMSO-d6)
192.15, 166.76,
)
1H-NMR (400 MHz,
δ
δ
163.29, 151.00, 132.33, 131.30, 126.10, 118.77, 115.87, 113.47, 81.29, 60.38, 51.95, 15.80. HRMS(ESI) (m/z):
Calad. for C18H19NO5 329.3472; found 284.0919 [M-C2H5O]+.
4-((1-Ethoxy-2-oxo-2-(4-(piperidin-1-yl)phenyl)ethyl)amino)-2-methylbenzoic Acid (6c
(400 MHz, DMSO-d6) 12.05 (s, 1H), 7.93 (d, J = 9.1 Hz, 2H), 7.72 (d, J = 9.3 Hz, 1H), 7.05 (d, J = 8.5 Hz, 1H),
)
1H-NMR
δ
6.96 (d, J = 9.1 Hz, 2H), 6.82 (d, J = 6.7 Hz, 2H), 6.13 (d, J = 8.5 Hz, 1H), 3.51–3.44 (m, 1H), 3.41 (brs, 4H),
3.37–3.32 (m, 1H), 2.47 (s, 3H), 1.58 (brs, 6H), 1.02 (t, J = 7.0 Hz, 3H). 13C-NMR (101 MHz, DMSO-d6)
δ
191.21, 168.73, 154.60, 149.81, 142.15, 133.15, 131.84,122.84, 118.86, 116.33, 113.14, 111.02, 81.01, 60.09,
48.08, 25.40, 24.54, 22.89, 15.87. HRMS(ESI) (m/z): Calad. for C23H28N2O4 396.4794; found 351.1708
[M-C2H5O]+.
1
2-Ethoxy-2-((4-nitrophenyl)amino-1-(4-(piperidin-1-yl)phenyl)ethanone (6d) H-NMR (400 MHz,
DMSO-d6)
2H), 6.97 (d, J = 8.2 Hz, 2H), 6.22 (d, J = 7.8 Hz, 1H), 3.52–3.48 (m, 1H), 3.42 (brs, 4H), 3.40–3.32 (m, 1H),
1.58 (brs, 6H), 1.03 (t, J = 6.9 Hz, 3H). 13C-NMR (101 MHz, DMSO-d6)
190.68, 154.68, 153.13, 138.19,
δ 8.06 (d, J = 8.9 Hz, 2H), 7.95 (d, J = 8.6 Hz, 2H), 7.88 (d, J = 7.9 Hz, 1H), 7.10 (d, J = 8.9 Hz,
δ
131.94, 126.30, 122.58, 113.48, 113.11, 80.94, 60.45, 48.05, 25.42, 24.53, 15.79. HRMS(ESI) (m/z): Calad. for
C21H25N3O4 383.4409; found 384.1918 [M + H]+.
Methyl 4-((2-(4-CYCLOHEXYLPHENYl)-1-ethoxy-2-oxoethyl)amino)benzoate (6e) 1H-NMR (400 MHz,
DMSO-d6)
δ 8.02 (d, J = 8.3 Hz, 2H), 7.77 (d, J = 8.8 Hz, 2H), 7.44 (d, J = 8.6 Hz, 1H), 7.40 (d, J = 8.4 Hz,
2H), 7.02 (d, J = 8.8 Hz, 2H), 6.26 (d, J = 8.6 Hz, 1H), 3.77 (s, 3H), 3.56–3.48 (m, 1H), 3.45–3.37 (m, 1H),
2.62–2.55 (m, 1H), 1.80–1.69 (m, 5H), 1.47–1.22 (m, 5H), 1.04 (t, J = 7.0 Hz, 3H). 13C-NMR (101 MHz,
DMSO-d6)
δ 193.63, 166.74, 154.50, 150.97, 132.61, 131.32, 129.81, 127.59, 118.87, 113.46, 81.68, 60.73,
51.95, 44.41, 33.98, 26.71, 26.00, 15.80. HRMS(ESI) (m/z): Calad. for C24H29NO4 395.4914; found
350.1751 [M − C2H5O]+.
1-(4-Cyclohexylphenyl)-2-ethoxy-2-((4-nitrophenyl)amino)ethanone (6f) 1H-NMR (400 MHz, DMSO-d6)
δ
8.08 (d, J = 9.2 Hz, 2H), 8.04–8.01 (m, 3H), 7.42 (d, J = 8.3 Hz, 2H), 7.10 (d, J = 9.3 Hz, 2H),
6.35 (d, J = 8.3 Hz, 1H), 3.59–3.51 (m, 1H), 3.46–3.39 (m, 1H), 2.63–2.57 (m, 1H), 1.81–1.69 (m, 5H),
1.48–1.23 (m, 5H), 1.05 (t, J = 7.0 Hz, 3H). 13C-NMR (101 MHz, DMSO-d6)
193.27, 154.66, 153.02,
δ
138.36, 132.45, 129.88, 127.62, 126.32, 113.49, 81.55, 61.05, 44.42, 33.99, 26.70, 26.00, 15.75. HRMS(ESI)
(m/z): Calad. for C22H26N2O4 382.4528; found 337.1546 [M − C2H5O]+.
4-((2-(4-Cyclohexylphenyl)-1-ethoxy-2-oxoethyl)amino)benzoic Acid (6g) 1H-NMR (400 MHz, DMSO-d6)
δ
12.26 (s, 1H), 8.01 (d, J = 8.4 Hz, 2H), 7.73 (d, J = 8.8 Hz, 2H), 7.41 (d, J = 8.4 Hz, 2H), 7.35 (d, J = 8.7 Hz,
1H), 6.99 (d, J = 8.9 Hz, 2H), 6.24 (d, J = 8.6 Hz, 1H), 3.55–3.48 (m, 1H), 3.45–3.37 (m, 1H), 2.62–2.57 (m,
1H), 1.80–1.69 (m, 5H), 1.48–1.22 (m, 5H), 1.04 (t, J = 7.0 Hz, 3H). 13C-NMR (101 MHz, DMSO-d6)
δ
193.68, 167.88, 154.48, 150.62, 132.63, 131.47, 129.79, 127.60, 120.01, 113.31, 81.73, 60.73, 44.40, 33.99,
26.71, 26.00, 15.82. HRMS(ESI) (m/z): Calad. for C23H27NO4 381.4648; found 336.1594 [M − C2H5O]+.

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4-((2-(4-Cyclohexylphenyl)-1-methoxy-2-oxoethyl)amino)benzoic Acid (6h
)
1H-NMR (400 MHz,
DMSO-d6)
2H), 7.37 (d, J = 8.6 Hz, 1H), 7.02 (d, J = 8.8 Hz, 2H), 6.25 (d, J = 8.5 Hz, 1H), 3.17 (s, 3H), 2.62–2.56
(m 1H), 1.80–1.69 (m, 5H), 1.47–1.22 (m, 5H). 13C-NMR (101 MHz, DMSO-d6)
193.46, 167.88, 154.57,
δ 12.34 (s, 1H), 8.02 (d, J = 8.3 Hz, 2H), 7.76 (d, J = 8.7 Hz, 2H), 7.40 (d, J = 8.3 Hz,
δ
150.47, 132.60, 131.46, 129.82, 127.62, 120.18, 113.44, 82.22, 52.33, 44.42, 33.99, 26.71, 26.00. HRMS(ESI)
(m/z): Calad. for C22H25NO4 367.4382; found 336.1594 [M − CH3O]+.
4-((2-(4-Cyclohexylphenyl)-1-isopropoxy-2-oxoethyl)amino)benzoic Acid (6i) H-NMR (400 MHz,
1
DMSO-d6)
δ 12.26 (s, 1H), 8.01 (d, J = 8.4 Hz, 2H), 7.73 (d, J = 8.8 Hz, 2H), 7.40 (d, J = 8.3 Hz,
2H), 7.34 (d, J = 8.8 Hz, 1H), 6.96 (d, J = 8.9 Hz, 2H), 6.22 (d, J = 8.7 Hz, 1H), 3.87–3.81 (m, 1H),
2.61–2.56 (m, 1H), 1.80–1.69 (m, 5H), 1.47–1.22 (m, 5H), 1.06 (d, J = 6.1 Hz, 3H), 1.03 (d, J = 6.1 Hz, 3H).
13C-NMR (101 MHz, DMSO-d6)
δ 194.13, 167.87, 154.33, 150.67, 132.75, 131.46, 129.80, 127.54, 119.90,
113.27, 80.89, 67.92, 44.38, 33.99, 26.71, 26.00, 23.43, 23.29. HRMS(ESI) (m/z): Calad. for C24H29NO4
395.4914; found 336.1594 [M − C3H7O]+.
4-((1-Ethoxy-2-oxo-2-(4-propylphenyl)ethyl)amino)benzoic Acid (6j) 1H-NMR (400 MHz, DMSO-d6)
12.26 (s, 1H), 8.01 (d, J = 8.3 Hz, 2H), 7.74 (d, J = 8.8 Hz, 2H), 7.38 (d, J = 8.4 Hz, 2H), 7.35 (d, J = 8.8 Hz,
1H), 7.00 (d, J = 8.8 Hz, 2H), 6.25 (d, J = 8.6 Hz, 1H), 3.56–3.48 (m, 1H), 3.45–3.37 (m, 1H), 2.63 (t, J = 7.2 Hz
2H), 1.66–1.57 (m, 2H), 1.04 (t, J = 7.0 Hz, 3H), 0.90 (t, J = 7.3 Hz, 3H). 13C-NMR (101 MHz, DMSO-d6)
δ
,
δ
193.76, 167.89, 150.63, 149.38, 132.52, 131.47, 129.68, 129.22, 120.01, 113.32, 81.68, 60.71, 37.75, 24.26, 15.81,
14.16. HRMS(ESI) (m/z): Calad. for C20H23NO4 341.4009; found 296.1282 [M − C2H5O]+.
Methyl 4-((1-Ethoxy-2-oxo-2-(4-propylphenyl)ethyl)amino)benzoate (6k) methyl 4-aminobenzoate
(0.50 g, 3.31 mmol) was added to a solution of 2-oxo-2-(4-propylphenyl)acetaldehyde (0.58 g, 3.31 mmol)
in EtOH (20 mL), and the solution was stirred at room temperature overnight. The solvent was
evaporated under reduced pressure, the crude residue was purified using silica gel chromatography
1
and was eluted with PE/CH3COCH3 (v:v=8:1) to give 6k as white solid in a yield of 22%. H-NMR
(400 MHz, DMSO-d6)
7.37 (d, J = 8.4 Hz, 2H), 7.04 (d, J = 8.9 Hz, 2H), 6.28 (d, J = 8.6 Hz, 1H), 3.78 (s, 3H), 3.57–3.49 (m, 1H),
3.45–3.38 (m, 1H), 2.63 (t, J = 7.2 Hz, 2H), 1.66–1.57 (m, 2H), 1.04 (t, J = 7.0 Hz, 3H), 0.90 (t, J = 7.3 Hz, 3H)
13C-NMR (101 MHz, DMSO-d6)
193.71, 166.74, 150.97, 149.40, 132.49, 131.31, 129.69, 129.21, 118.86,
δ 8.03 (d, J = 8.4 Hz, 2H), 7.78 (d, J = 8.8 Hz, 2H), 7.44 (d, J = 8.6 Hz, 1H),
.
δ
113.46, 81.62, 60.70, 51.95, 37.75, 24.26, 15.79, 14.15. HRMS(ESI) (m/z): Calad. for C21H25NO4 355.4275;
found 310.1438 [M − C2H5O]+.
2-Ethoxy-2-((4-nitrophenyl)amino)-1-(4-propylphenyl)ethanone (6l) 1H-NMR (400 MHz, DMSO-d6)
δ
8.08 (d, J = 9.3 Hz, 2H), 8.04 (d, J = 8.4 Hz, 2H), 8.01 (s, 1H), 7.39 (d, J = 8.4 Hz, 2H), 7.11 (d, J = 9.3 Hz,
2H), 6.36 (d, J = 8.3 Hz, 1H), 3.59–3.52 (m, 1H), 3.46–3.39 (m, 1H), 2.64 (t, J = 7.2 Hz, 2H), 1.67–1.58
(m, 2H), 1.05 (t, J = 7.0 Hz, 3H), 0.90 (t, J = 7.3 Hz, 3H). 13C-NMR (101 MHz, DMSO-d6)
δ 193.36, 153.02,
149.56, 138.35, 132.34, 129.76, 129.24, 126.32, 113.49, 81.51, 61.04, 37.76, 24.25, 15.75, 14.14. HRMS(ESI)
(m/z): Calad. for C19H22N2O4 342.3890; found 297.1234 [M − C2H5O]+.
2-Ethoxy-1-(4-hexylphenyl)-2-((4-nitrophenyl)amino)ethanone (6m) 1H-NMR (400 MHz, DMSO-d6)
δ
8.07 (d, J = 8.8 Hz, 2H), 8.02 (d, J = 7.3 Hz, 2H), 7.99 (s, 1H), 7.38 (d, J = 7.7 Hz, 2H), 7.10 (d, J = 9.3 Hz,
2H), 6.35 (d, J = 8.3 Hz, 1H), 3.58–3.50 (m, 1H), 3.45–3.37 (m, 1H), 2.65 (t, J = 7.6 Hz, 2H), 1.58 (brs, 2H),
1.26 (brs, 6H), 1.04 (t, J = 7.0 Hz, 3H), 0.84 (brs, 3H). 13C-NMR (101 MHz, DMSO-d6)
δ 193.39, 153.04,
149.82, 138.35, 132.31, 129.79, 129.21, 126.33, 113.50, 81.52, 61.06, 35.72, 31.59, 31.0, 28.85, 22.58, 15.75,
14.48. HRMS(ESI) (m/z): Calad. for C22H28N2O4 384.4687; found 339.1704 [M − C2H5O]+.
4-((1-Ethoxy-2-(4-hexylphenyl)-2-oxoethyl)amino)benzoic Acid (6n) 1H-NMR (400 MHz, DMSO-d6)
δ
12.29 (s, 1H), 8.01 (d, J = 8.4 Hz, 2H), 7.74 (d, J = 8.8 Hz, 2H), 7.38–7.34 (m, 3H), 7.00 (d, J = 8.9 Hz, 2H),
6.25 (d, J = 8.6 Hz, 1H), 3.55–3.48 (m, 1H), 3.44–3.39 (m, 1H), 2.65 (t, J = 7.2 Hz, 2H), 1.60–1.56 (m, 2H),
1.27 (brs, 6H), 1.03 (t, J = 7.0 Hz, 3H), 0.85 (t, J = 7.2 Hz, 3H). 13C-NMR (101 MHz, DMSO-d6)
δ 193.76,

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167.88, 150.62, 149.63, 132.49, 131.46, 129.70, 129.16, 120.01, 113.33, 81.68, 60.71, 35.71, 31.59, 31.05, 28.86,
22.58, 15.80, 14.48. HRMS(ESI) (m/z): Calad. for C23H29NO4 383.4807; found 338.1749 [M − C2H5O]+.
1
4-((1-Ethoxy-2-(4-hexylphenyl)-2-oxoethyl)amino)-2-methylbenzoic Acid (6o) H-NMR (400 MHz,
DMSO-d6)
δ 12.09 (s, 1H), 8.01 (d, J = 7.7 Hz, 2H), 7.73 (d, J = 8.4 Hz, 1H), 7.37 (d, J = 7.2 Hz,
2H), 7.18 (d, J = 8.7 Hz, 1H), 6.83 (s, 2H), 6.25 (d, J = 8.8 Hz, 1H), 3.56–3.48 (m, 1H), 3.45–3.37
(m, 1H), 2.65 (t, J = 7.4 Hz, 2H), 2.48 (s, 3H), 1.59–1.56 (m, 2H), 1.27 (brs, 6H), 1.04 (t, J = 7.0 Hz, 3H),
0.85 (t, J = 5.7 Hz, 3H). δ 193.77, 168.68, 149.73, 149.59, 142.18, 133.17,
13C-NMR (101 MHz, DMSO-d6)
132.49, 129.69, 129.15, 119.04, 116.33, 111.02, 81.57, 60.68, 35.71, 31.59, 31.06, 28.86, 22.87, 22.58, 15.82,
14.48. HRMS(ESI) (m/z): Calad. for C24H31NO4 397.5072; found 352.1909 [M − C2H5O]+.
1
4-((1-Ethoxy-2-(4-hexylphenyl)-2-oxoethyl)amino)-3-methylbenzoic Acid (6p) H-NMR (400 MHz,
DMSO-d6)
7.06 (d, J = 8.6 Hz, 1H), 6.28 (d, J = 6.8 Hz, 2H), 3.57–3.50 (m, 1H), 3.39–3.36 (m, 1H), 2.65 (t, J = 7.6 Hz
2H), 2.20 (s, 3H), 1.60–1.57 (m, 2H), 1.27 (brs, 6H), 1.02 (t, J = 7.0 Hz, 3H), 0.85 (t, J = 6.8 Hz, 3H).
13C-NMR (101 MHz, DMSO-d6)
.194.10, 167.97, 149.72, 147.90, 132.30, 132.01, 129.83, 129.46, 129.12,
δ 12.27 (s, 1H), 8.04 (d, J = 8.2 Hz, 2H), 7.70–7.66 (m, 2H), 7.37 (d, J = 8.3 Hz, 2H),
,
δ
122.55, 120.18, 111.67, 81.79, 60.14, 35.72, 31.58, 31.02, 28.85, 22.58, 17.85, 15.75, 14.48. HRMS(ESI) (m/z):
Calad. for C24H31NO4 397.5072; found 352.1909 [M − C2H5O]+.
4-((1-Ethoxy-2-(4-octylphenyl)-2-oxoethyl)amino)benzoic Acid (6q) 1H-NMR (400 MHz, DMSO-d6)
δ
12.26 (s, 1H), 8.01 (d, J = 8.3 Hz, 2H), 7.74 (d, J = 8.8 Hz, 2H), 7.38–7.33 (m, 3H), 7.00 (d, J = 8.9 Hz,
2H), 6.25 (d, J = 8.6 Hz, 1H), 3.55–3.48 (m, 1H), 3.43–3.37 (m, 1H), 2.65 (t, J = 7.6 Hz, 2H), 1.60–1.56
(m, 2H), 1.27–1.23 (m, 10H), 1.03 (t, J = 7.0 Hz, 3H), 0.85 (t, J = 6.8 Hz, 3H). 13C-NMR (101 MHz,
DMSO-d6)
δ 193.75, 167.88, 150.62, 149.62, 132.48, 131.46, 129.69, 129.16, 120.01, 113.32, 81.67, 60.70,
35.71, 31.80, 31.09, 29.33, 29.19, 22.62, 15.80, 14.50. HRMS(ESI) (m/z): Calad. for C25H33NO4 411.5338;
found 366.2067 [M − C2H5O]+.
4-((1-Ethoxy-2-(4-octylphenyl)-2-oxoethyl)amino)-2-methylbenzoic Acid (6r) H-NMR (400 MHz,
1
DMSO-d6)
δ 12.08 (s, 1H), 8.00 (d, J = 8.3 Hz, 2H), 7.73 (d, J = 9.2 Hz, 1H), 7.37 (d, J = 8.2 Hz, 2H),
7.17 (d, J = 8.7 Hz, 1H), 6.83–6.82 (m, 2H), 6.24 (d, J = 8.7 Hz, 1H), 3.56–3.48 (m, 1H), 3.45–3.37 (m, 1H),
2.65 (t, J = 7.6 Hz, 2H), 2.47 (s, 3H), 1.60–1.56 (m, 2H), 1.27–1.23 (m, 10H), 1.03 (t, J = 7.0 Hz, 3H),
0.85 (t, J = 6.8 Hz, 2H). 13C-NMR (101 MHz, DMSO-d6)
δ 193.76, 168.68, 149.73, 149.59, 142.18,
133.17,132.49, 129.69, 129.14, 119.04, 116.34, 111.02, 81.57, 60.67, 35.71, 31.81, 31.10, 29.34, 29.20, 22.87,
22.63, 15.82, 14.50. HRMS(ESI) (m/z): Calad. for C26H35NO4 425.5604; found 380.2222 [M − C2H5O]+.
4-((2-(4-Cyclohexylphenyl)-2-oxoethyl)amino)benzoic Acid (8a) 1H-NMR (400 MHz, DMSO-d6)
(s, 1H), 8.00 (d, J = 8.4 Hz, 2H), 7.67 (d, J = 8.9 Hz, 2H), 7.41 (d, J = 8.4 Hz, 2H), 6.70 (d, J = 8.9 Hz, 2H),
4.75 (s, 2H), 2.64–2.58 (m, 1H), 1.81–1.70 (m, 5H), 1.49–1.20 (m, 5H). 13C-NMR (101 MHz, DMSO-d6)
δ 12.10
δ
195.96, 168.03, 154.25, 152.61, 133.39, 131.51, 128.70, 127.67, 117.97, 112.01, 49.78, 44.41, 34.07, 26.73,
26.01. HRMS(ESI) (m/z): Calad. for C21H23NO3 337.4122; found 338.1749 [M+H]+.
4-((2-Oxo-2-(4-propylphenyl)ethyl)amino)benzoic Acid (8b) 1H-NMR (400 MHz, DMSO-d6)
δ 12.07 (s,
1H), 8.00 (d, J = 8.2 Hz, 2H), 7.68 (d, J = 8.8 Hz, 2H), 7.39 (d, J = 8.2 Hz, 2H), 6.71–6.67 (m, 3H), 4.75 (s,
2H), 2.65 (t, J = 7.6 Hz, 2H), 1.67–1.58 (m, 2H), 0.90 (t, J = 7.3 Hz, 3H). 13C-NMR (101 MHz, DMSO-d6)
δ
195.97, 168.05, 152.61, 149.11, 133.25, 131.52, 129.30, 128.59, 118.00, 112.02, 49.79, 37.73, 24.35, 14.14.
HRMS(ESI) (m/z): Calad. for C18H19NO3 297.3484; found 298.1438 [M + H]+.
1
4-((2-(4-Hexylphenyl)-2-oxoethyl)amino)benzoic Acid (8c) H-NMR (400 MHz, DMSO-d6)
δ 12.07
(s, 1H), 8.00 (d, J = 8.2 Hz, 2H), 7.67 (d, J = 8.6 Hz, 2H), 7.38 (d, J = 8.2 Hz, 2H), 6.70 (d, J = 8.8 Hz, 2H),
6.67 (t, J = 5.2 Hz, 1H), 4.74 (d, J = 5.2 Hz, 2H), 2.66 (t, J = 7.6 Hz, 2H), 1.63–1.55 (m, 2H), 1.28 (brs, 6H),
0.86 (t, J = 6.8 Hz, 3H). 13C-NMR (101 MHz, DMSO-d6)
δ 195.98, 168.03, 152.62, 149.35, 133.23, 131.51,
129.24, 128.61, 118.00, 112.02, 49.79, 35.68, 31.61, 31.14, 28.84, 22.60, 14.50. HRMS(ESI) (m/z): Calad.
for C21H25NO3 339.4281; found 340.1907 [M + H]+.

Molecules 2019, 24, 2719
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1
4-((2-(4-Octylphenyl)-2-oxoethyl)amino)benzoic Acid (8d) H-NMR (400 MHz, DMSO-d6)
δ 12.06
(s, 1H), 7.99 (d, J = 8.3 Hz, 2H), 7.67 (d, J = 8.9 Hz, 2H), 7.38 (d, J = 8.2 Hz, 2H), 6.70 (d, J = 8.8 Hz, 2H),
6.66 (t, J = 5.2 Hz, 1H), 4.74 (d, J = 5.4 Hz, 2H), 2.66 (t, J = 7.2 Hz, 2H), 1.63–1.55 (m, 2H), 1.29–1.24
(m, 10H), 0.85 (t, J = 7.2 Hz, 3H). 13C-NMR (101 MHz, DMSO-d6)
δ 195.97, 168.02, 152.61, 149.35,
133.22, 131.50, 129.24, 128.60, 117.99, 112.01, 49.79, 35.68, 31.82, 31.18, 29.35, 29.21, 29.18, 22.63, 14.51.
HRMS(ESI) (m/z): Calad. for C23H29NO3 367.4813; found 368.2221 [M + H]+.
4-(2-(4-Cyclohexylphenyl)-2-oxoeacetamido)benzoic Acid (9a) 1H-NMR (400 MHz, DMSO-d6)
(s, 1H), 11.23 (s, 1H), 7.97 (d, J = 8.7 Hz, 4H), 7.86 (d, J = 8.7 Hz, 2H), 7.48 (d, J = 8.2 Hz, 2H), 2.66–2.60
(m, 1H), 1.82–1.70 (m, 5H), 1.49–1.23 (m, 5H). 13C-NMR (101 MHz, DMSO-d6)
189.22, 167.35, 164.40,
δ 12.88
δ
155.85, 142.25, 131.03, 130.83, 130.81, 128.08, 126.96, 120.01, 44.51, 33.95, 26.67, 25.98. HRMS(ESI) (m/z):
Calad. for C21H21NO4 351.3957; found 352.1542 [M + H]+.
1
4-(2-Oxo-2-(4-propylphenyl)acetamido)benzoic Acid (9b) H-NMR (400 MHz, DMSO-d6)
δ 12.89
(s, 1H), 11.25 (s, 1H), 7.99–7.97 (m, 4H), 7.86 (d, J = 8.9 Hz, 2H), 7.44 (d, J = 8.3 Hz, 2H), 2.67 (t, J = 7.2 Hz
,
2H), 1.68–1.58 (m, 2H), 0.90 (t, J = 7.3 Hz, 3H). 13C-NMR (101 MHz, DMSO-d6)
δ
189.24, 167.35, 164.41,
150.80, 142.25, 131.03, 130.70, 129.70, 126.96, 120.01, 37.83, 24.24, 14.10. HRMS(ESI) (m/z): Calad. for
C18H17NO4 311.3319; found 312.1230 [M + H]+.
4-(2-(4-Hexylphenyl)-2-oxoacetamido)benzoic Acid (9c) 1H-NMR (400 MHz, DMSO-d6) δδ 12.89 (s, 1H),
11.24 (s, 1H), 7.98–7.96 (m, 4H), 7.86 (d, J = 8.9 Hz, 2H), 7.44 (d, J = 8.3 Hz, 2H), 2.69 (t, J = 7.2 Hz,
2H), 1.63–1.56 (m, 2H), 1.28 (brs, 6H), 0.85 (t, J = 6.8 Hz, 3H). 13C-NMR (101 MHz, DMSO-d6)
δ 189.23,
167.36, 164.42, 151.06, 142.24, 131.02, 130.71, 130.65, 129.64, 126.98, 120.00, 35.81, 31.58, 31.02, 28.81,
22.58, 14.49. HRMS(ESI) (m/z): Calad. for C21H23NO4 353.4116; found 354.1700 [M + H]+.
4-(2-(4-Octylphenyl)-2-oxoacetamido)benzoic Acid (9d) 1H-NMR (400 MHz, DMSO-d6)
11.24 (s, 1H), 7.98–7.96 (m, 4H), 7.86 (d, J = 8.8 Hz, 2H), 7.44 (d, J = 8.2 Hz, 2H), 2.68 (t, J = 8.0 Hz, 2H),
1.61–1.58 (m, 2H), 1.27–1.24 (m, 10H), 0.85 (t, J = 6.8 Hz, 3H). 13C-NMR (101 MHz, DMSO-d6)
189.23,
δ 12.89 (s, 1H),
δ
167.36, 164.41, 151.06, 142.24, 131.02, 130.72, 130.65, 129.64, 126.99, 120.01, 35.81, 31.80, 31.06, 29.33,
29.18, 29.16, 22.62, 14.50. HRMS(ESI) (m/z): Calad. for C23H27NO4 381.4648; found 382.2011 [M + H]+.
3.3. Biological Assay
3.3.1. Determination of the MIC of Colistin
The MIC of colistin was determined using the broth microdilution method. E. coli BL21(DE3) and
pET-28a(+) were obtained from commercial sources (YouBio). The full length of the colistin-resistance
gene mcr-1 was synthesized by the Sangon Biotech (Shanghai) Co., Ltd. Antibiotics, culture media,
and other reagents used were purchased commercially. Bacterial colonies were selected from a LB agar
and transferred to a cation-adjusted Mueller-Hinton broth (CAMHB) tube, and the broth was shaken at
37 ◦C. After adjusting the turbidity to OD600 0.08-0.1 (1
diluted (1:100) to obtain an inoculum of 1
106 cfu mL−1. The working concentrations of colistin
(ranging from 0.0625 L of
mL−1 to 64 mL−1 in 2-fold dilutions) were prepared in CAMHB. 50
each intermediate concentration of colistin solution was dispensed into the wells of the microwell
plates, and 50 L of the bacterial suspension was added to the 96-well microplates. The final bacterial
concentration is 5
105 cfu mL−1. The plates were incubated in an ambient air incubator at 37 ◦C for
×
108 cfu mL−1), the suspensions were further
·
×
·
µg
·
µg
·
µ
µ
×
·
16 h. The MIC was determined as the lowest concentration that completely inhibits bacterial growth in
the wells, and the data was obtained in at least two independent experiments.
3.3.2. The Susceptibility Testing of Colistin Combined with Compounds
The broth microdilution method was applied to identify potential inhibitors of MCR-1 in the
presence of 2
µ
g
·
mL−1 colistin in CAMHB. Dimethyl sulfoxide (DMSO) was added with a final
concentration of 0.1%. As described above, bacterial colonies were selected from a LB agar and

Molecules 2019, 24, 2719
14 of 16
transferred to a CAMHB tube, and the broth was shaken at 37 ◦C. After adjusting the turbidity to
OD600 0.08–0.1 (1
108 cfu mL−1), the suspensions were further diluted (1:100) to obtain an inoculum
of 1 M to 400 M in
106 cfu mL−1. The working concentrations of compounds (ranging from 50
2-fold dilutions) were prepared in CAMHB. 50 L of the bacterial suspension was added to the 96-well
microplates, and 25 L of each intermediate concentration of the compound solution was dispensed
into the wells of the microwell plates. Then, 25 L of the prepared colistin solution was added into the
microwell plates. The final concentrations of compounds were 12.5 M, 25 M, 50 M, and 100 M.
mL−1. The plates were incubated in an ambient air
×
·
×
·
µ
µ
µ
µ
µ
µ
µ
µ
µ
The final bacterial concentration is 5
×
105 cfu
·
◦
incubator at 37 C for 16 h. The activity was evaluated by recording the lowest concentrations of
compounds in combination with 2
bacteria, and the data was obtained in at least two independent experiments.
µg
mL−1 colistin that completely inhibited the visible growth of the
·
3.3.3. The Assay for MCR-1 Enzymatic Activity
In this biochemical assay, the fluorescently-labeled substrate (NBD-PEA) is 1-acyl-2-{12-[(7-nitro-
2-1,3-benzoxadiazol-4-yl) amino] dodecanoyl}-sn-glycero-3-phosphoethanol amine (Avanti Lipids,
USA). The reaction system (10
µL in total) was kept for 18 h at room temperature and contained 50 mM
HEPES (pH 7.50), 100 mM NaCl, 0.03% of DDM, 0.2 mM NBD-PEA, 4
µM MCR-1 and compounds
(compound 3 is 2 mM, 5 mM, 10 mM; compound 6p is 0.1 mM, 0.4 mM, 1.6 mM; compound 6q is
0.2 mM, 0.4mM, 0.8 mM; compound 6m is 1 mM, 5 mM, 10 mM). The reaction products were subjected
to TLC in a mobile phase consisting of ethyl acetate: methanol: water (v:v:v = 7:2:1). The fluorescent
signal on the TLC plate was detected under Epi blue light (455–485 nm) with a gel imaging system
(Bio-Rad), and the data was obtained in at least two independent experiments.
3.4. Molecular Docking
The full-length structure of MCR-1 was modelled by the software Swiss-Model using the structure
of Neisseria meningitidis EptA (PDB: 5FGN) as a template, and the molecular docking was described as
Section 3.1. After the docking process was finished, the best pose of each molecule was used to analyze
the interactions and study the relationship between the compounds and the MCR-1 protein.
4. Conclusions
This study shows that a combination of colistin and MCR-1 inhibitors could be an alternative
option for reverting colistin resistance, and this strategy may not place selective pressure on bacterium.
The present research on MCR-1 inhibitors (such as ETA and pterostilbene) exhibited low activity,
and the mechanism remains to be further clarified. To solve the issue of polymyxins resistance caused
by mcr-1, it is important to develop new agents for high-efficiency against MCR-1. In this work,
we identified racemic compound
for the first time, and 26 novel compounds were designed and synthesized based on the structure of
1-phenyl-2-(phenylamino) ethanone. In the evaluation of cell-based assay, seven compounds (6g 6h
6i 6n 6p 6q, and 6r) displayed more potent activity than compound . Among them, 25 M of
compounds 6p or 6q in combination with 2
mL−1 colistin could completely inhibit the growth of
3 as a potential MCR-1 inhibitor by structure-based virtual screening
,
,
,
,
,
3
µ
µg
·
BL21(DE3) expressing mcr-1, which exhibited the most potent activity. Moreover, in the enzymatic
assay, we elucidate that compound 6p and 6q could target the MCR-1 and inhibit the activity of the
protein. Additionally, molecular docking studies on several compounds were carried out to elucidate
the SARs, and the docking results exhibited that the active compounds could interact with Glu246 and
Thr285 via hydrogen bonds, and they occupy well the cavity of MCR-1 protein. Therefore, we conclude
that the essential hydrogen bonding and appropriate substitution size could promote the inhibitory
activity against MCR-1 protein. We are doing research on further structural optimization, and we will
look for valuable information to develop potential MCR-1 inhibitors.

Molecules 2019, 24, 2719
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Supplementary Materials: The following are available online.
Author Contributions: Conceptualization, J.-h.X. and S.L.; methodology, X.-j.L., H.-t.Y., F.L. and J.-h.X.; software,
X.-j.L., S.H. and C.-c.L.; validation, X.-j.L. and J.-h.X.; formal analysis, J.-h.X.; investigation, X.-j.L.; resources,
J.-h.X. and S.L.; data curation, X.-j.L. and J.-h.X.; writing—original draft preparation, X.-j.L.; writing—review
and editing, W.S. and G.-s.W.; visualization, F.L.; supervision, J.-h.X.; project administration, G.-s.W. and J.-h.X.;
funding acquisition, J.-h.X. and S.L.
Funding: This research was funded by the National Science and Technology Major Project of China
(2018zx09711003-003).
Conflicts of Interest: The authors declare no conflict of interest.
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2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
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(CC BY) license (http://creativecommons.org/licenses/by/4.0/).