Synthesis and antibacterial evaluation of novel cationic chalcone derivatives possessing broad spectrum antibacterial activity

Article abstract of DOI:10.1016/j.ejmech.2017.12.009

There is an urgent need to identify new antibiotics with novel mechanisms that combat antibiotic resistant bacteria. Herein, a series of chalcone derivatives that mimic the essential properties of cationic antimicrobial peptides were designed and synthesized. Antibacterial activities against drug-sensitive bacteria, including Staphylococcus aureus, Enterococcus faecalis, Escherichia coli and Salmonella enterica, as well as clinical multiple drug resistant isolates of methicillin-resistant S. aureus (MRSA), KPC-2-producing and NDM-1-producing Carbapenem-resistant Enterobacteriaceae were evaluated. Representative compounds 5a (MIC: 1 μg/mL against S. aureus, 0.5 μg/mL against MRSA) and 5g (MIC: 0.5 μg/mL against S. aureus, 0.25 μg/mL against MRSA) showed good bactericidal activity against both Gram-positive and Gram-negative bacteria, including the drug-resistant species MRSA, KPC and NDM. These membrane-active antibacterial compounds were demonstrated to reduce the viable cell counts in bacterial biofilms effectively and do not induce the development of resistance in bacteria. Additionally, these representative molecules exhibited negligible toxicity toward mammalian cells at a suitable concentration. The combined results indicate that this series of cationic chalcone derivatives have potential therapeutic effects against bacterial infections.

Full text of DOI:10.1016/j.ejmech.2017.12.009

Accepted Manuscript
Synthesis and antibacterial evaluation of novel cationic chalcone derivatives
possessing broad spectrum antibacterial activity
Wen-Chao Chu, Peng-Yan Bai, Zhao-Qing Yang, De-Yun Cui, Yong-Gang Hua, Yi
Yang, Qian-Qian Yang, En Zhang, Shangshang Qin
PII:
S0223-5234(17)31015-2
10.1016/j.ejmech.2017.12.009
EJMECH 9987
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 31 October 2017
Revised Date: 2 December 2017
Accepted Date: 2 December 2017
Please cite this article as: W.-C. Chu, P.-Y. Bai, Z.-Q. Yang, D.-Y. Cui, Y.-G. Hua, Y. Yang, Q.-Q.
Yang, E. Zhang, S. Qin, Synthesis and antibacterial evaluation of novel cationic chalcone derivatives
possessing broad spectrum antibacterial activity, European Journal of Medicinal Chemistry (2018), doi:
10.1016/j.ejmech.2017.12.009.
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Graphical abstract

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Synthesis and Antibacterial Evaluation of Novel Cationic Chalcone Derivatives
Possessing Broad Spectrum Antibacterial Activity
#,1
Wen-Chao Chu,#,1 Peng-Yan Bai,
Zhao-Qing Yang,1 De-Yun Cui,1 Yong-Gang
Hua,1 Yi Yang,1 Qian-Qian Yang,1 En Zhang, *1,2 and Shangshang Qin*1,2
1School of Pharmaceutical Sciences; Institute of Drug Discovery and Development;
Key Laboratory of Advanced Pharmaceutical Technology, Ministry of Education of
China; Zhengzhou University, Zhengzhou 450001, PR China.
2Collaborative Innovation Center of New Drug Research and Safety Evaluation,
Henan Province, Zhengzhou 450001, PR China.
E-mail addresses: zhangen@zzu.edu.cn; qinshangshang@126.com

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ABSTRACT
There is an urgent need to identify new antibiotics with novel mechanisms that
combat antibiotic resistant bacteria. Herein, a series of chalcone derivatives that
mimic the essential properties of cationic antimicrobial peptides were designed and
synthesized. Antibacterial activities against drug-sensitive bacteria, including
Staphylococcus aureus, Enterococcus faecalis, Escherichia coli and Salmonella
enterica, as well as clinical multiple drug resistant isolates of methicillin-resistant S.
aureus (MRSA), KPC-2-producing and NDM-1-producing Carbapenem-resistant
Enterobacteriaceae were evaluated. Representative compounds 5a (MIC: 1 µg/mL
against S. aureus, 0.5 µg/mL against MRSA) and 5g (MIC: 0.5 µg/mL against S.
aureus, 0.25 µg/mL against MRSA) showed good bactericidal activity against both
Gram-positive and Gram-negative bacteria, including the drug-resistant species
MRSA, KPC and NDM. These membrane-active antibacterial compounds were
demonstrated to reduce the viable cell counts in bacterial biofilms effectively and do
not induce the development of resistance in bacteria. Additionally, these
representative molecules exhibited negligible toxicity toward mammalian cells at a
suitable concentration. The combined results indicate that this series of cationic
chalcone derivatives have potential therapeutic effects against bacterial infections.
Keywords: Antimicrobial; Cationic chalcone derivatives; Broad spectrum antibacterial
activity; Fluoride atom; Permeabilization; Depolarization

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1. Introduction
The global threat of infectious diseases has been aggravated by the emergence of
bacterial resistance to antibiotics. Such antibiotic resistance has stimulated the urgent
need for the design of new antibiotics with different modes of action.[1-3]
Methicillin-resistant Staphylococcus aureus (MRSA) was initially detected in
Europe,[4] and is now responsible for the majority of community acquired and
nosocomial infections.[5] KPC-2-producing Klebsiella pneumoniae (KPC) was
initially identified in 1996 and has since spread to many countries.[6] New Delhi
metallo-β-lactamase 1 (NDM-1)-producing Enterobacteriaceae hydrolyzes nearly all
known β-lactam antibiotics. Moreover, the NDM-1 gene has quickly disseminated
worldwide and this spread is uncontrollable.[7-9] Unfortunately, the development of
new antibiotics has failed to keep pace with the development of drug-resistance over
the past few decades.[3, 10] Thus, there is a critical global healthcare crisis, which
requires the urgent development of more effective antibiotics.
Figure 1. Three antibacterial peptide mimetic molecules in clinical trials.
Antimicrobial peptides (AMPs) provide broad-spectrum protection against
bacterial infections for most organisms, [11, 12] and have been shown to overcome
bacterial resistance. Cationic and amphiphilic AMPs attach to anionic microbial cell

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surfaces and insert into the microbial membrane bilayers, which then leads to the
disruption of the physical integrity of the cell membrane.[13] Despite the many
advantages, AMPs still have some disadvantages, including easy degradation in vivo,
toxicity in vivo and a high cost of manufacture, which impedes the use of AMPs as
clinical antibacterial agents.[14, 15] Consequently, several groups have used
amphipathic models of AMPs to mimic the action of AMPs and thus produce
membrane-targeting antimicrobial agents.[16, 17] For example, α-peptides,[18]
β-peptides,[19] antimicrobial polymers,[20] oligoacyl lysines,[21] oligoureas,[22]
α-AA peptides[23] and cationic amphiphiles[24, 25] have showed effective activity
against antibiotic-resistant bacteria. Additionally, membrane-active antibacterial
molecules have been synthesized and reported to exhibit good activity and lower
resistance tendencies.[16, 24, 26-28] There are some antibacterial peptide mimetic
molecules including CSA-13[29], LTX-109[30] and PMX 30063[31] (Figure 1) that
are currently undergoing clinical trials.
Figure 2. Representative chalcone compounds have been reported.
Natural products and their derivatives have historically been invaluable as a
source of bioactive lead compounds for drug discovery.[32, 33] Chalcone (1,
3-diphenyl-2-propen-1-ones) is a key substructure of natural compounds that has been
shown to be beneficial to human health.[34, 35] Chalcone and chalcone derivatives
have a wide range of biological activities, including antibacterial,[36] antimalarial,[37,
38] and anti-inflammatory.[39] The unique α,β-unsaturated ketone structure of
chalcone is responsible for the broad-spectrum of biological activities.[40]
Furthermore, their interesting structural scaffolds and significant biological activities

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have prompted many groups to isolate and modify chalcone for the development of
new drug candidates, and there are some existing drugs including metochalcone and
antipeptic ulcer mucoprotective sofalcone.[41, 42] Natural chalcone derivatives such
as xanthohumol[43] and licochalcone E[44] (Figure 2) show good antibacterial
activity.
In our continuous endeavor for new structures with good biological activity
against drug-resistant bacteria,[45, 46] antibacterial small molecular peptidomimetics
were developed by taking advantage of the hydrophobic scaffold of chalcone.[45]
These molecules contained cationic residues, which are positioned on the scaffold to
recognize the outer membrane. Some compounds of this series exhibited potent
antimicrobial properties against Gram-positive bacteria and Gram-negative bacteria,
with rapid killing rates.. Importantly, the antibacterial mechanisms of our compounds
involved depolarization and permeabilization of the bacterial cell membrane. These
results provide new insights to the potential use of chalcone peptidomimetics as novel
and efficient antimicrobial agents.
2.Results and discussion
2.1. Synthesis and characterization

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a): NaOH, EtOH, H2O, r. t., overnight. b): Chloroacetyl chloride, K2CO3, acetone,
r. t., 30 min. c): N,N-dimethyl alkylamine, acetonitrile, 85 °C, hydrothermal reaction
vessel, 24 h.
Scheme 1. Synthesis of cationic chalcone derivatives
Twenty-nine cationic chalcone analogs (5a-5ac), which differed in the length of
the N,N-dimethyl alkylamine and had various substituted aryl rings, were designed
and synthesized. The molecules were synthesized using a three-step method. The
synthetic route is shown in Scheme 1. Intermediate compounds 4-aminochalcone 3
were synthesized via the aldol condensation reaction from various substituted
aromatic formaldehydes and 4-aminoacetophenone (or 3-aminoacetophenone).[47]
Compounds 4 were then obtained by the acylation of 3 using chloroacetyl chloride.
Finally, cationic chalcone analogs 5 were obtained from N,N-dimethyl alkylamine and
intermediates 4.[24] The target molecules 5a-5ac were characterized by 1H NMR, 13C
NMR and high-resolution mass spectrometry (HRMS) as described for compound 5g
(Figure 3).

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Scheme 2. The accidental synthesis of 5n'
Figure 3. 1H NMR and 13C NMR chemical shifts of compound 5g.
During the synthesis of compound 5n', unexpectedly 3n was obtained rather than
the production of 3n' (Scheme 2). From the NMR and HRMS data of 3n, we
presumed that the fluorine of 2-fluoro-5-nitrobenzaldehyde (2n) or intermediate 3n'
was replaced by an ethoxyl during the aldol reaction in ethanol.[48] Compound 5n
was obtained from this accidental compound 3n according the same method (Scheme
1).

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Table 1. Minimal Inhibitory Concentration (MIC) and Hemolytic Activity (HC50) of
the Final Products
MIC(µg/mL)
Compd
HC50(µg/mL)
S. aureus
E. faecalis
E. coli
S. enterica
ATCC29213 ATCC29212 ATCC25922 ATCC13076
1
2
1
2
4
8
228
＞500
186
83
5a
5b
2
4
4
2
1
4
5c
2
2
2
4
5d
32
16
0.5
4
64
＞128
64
2
＞128
128
4
68
5e
16
262
137
293
192
444
56
5f
1
5g
8
16
＞128
＞128
2
32
5h
8
＞128
＞128
＞128
8
5i
＞128
0.5
2
＞128
5j
2
2
5k
4
4
54
5l
32
8
32
2
128
＞128
16
8
128
＞128
16
50
5m
5n
102
284
136
153
124
374
＞500
200
127
187
363
83
2
4
5o
2
4
16
5p
1
1
4
8
5q
1
2
4
4
5r
2
2
8
8
5s
128
4
128
8
>128
16
16
16
16
8
>128
32
5t
5u
4
8
32
5v
4
8
32
5w
5x
4
8
32
2
8
16
5y
1
4
4
16
137
133
360
249
5z
0.5
32
2
1
4
8
5ab
5aa
5ac
VANa
MEMb
32
4
64
16
128
16
c
c
c
c
2
-
-
-
-
c
c
c
c
-
-
<0.125
-
-
aVAN (vancomycin), bMEM (meropenem), cnot determined.

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2.2. Antibacterial activity
All compounds were initially screened for antibacterial activity against
Gram-positive bacteria Staphylococcus aureus (S. aureus) and Enterococcus faecalis
(E. faecalis), and against Gram-negative bacteria Escherichia coli (E. coli) and
Salmonella enterica (S. enterica), and resistant bacteria including MRSA, KPC and
NDM. The antibacterial efficacy was expressed as the minimum inhibitory
concentration (MIC) that is required to inhibit the growth of the bacteria.[49] MIC
values of the tested compounds are listed in Table 1.
In general, all compounds except 5e, 5i, 5j, 5n and 5t showed good activity
against all the bacteria tested. The cationic molecules were more effective toward
Gram-positive bacteria than toward Gram-negative bacteria. For example, the range
of MIC values for compounds 5a-5d were 1–2 µg/mL against Gram-positive strains,
whereas the range of MIC values were 2–8 µg/mL against Gram-negative strains.
However, the antibacterial activities of the cationic molecules were found to vary
significantly because of the different hydrophobicity of the alkyl chain. For example,
compounds 5f-5j with different alkyl chain lengths exhibited noticeably different
antibacterial activity, with 5g (medium hydrophobicity) showing the highest
antibacterial activity. The antibacterial activity of compounds 5g-5j decreased with
increasing spacer length. This decrease in the antibacterial activity may be caused by
the aggregation tendency because of the long hydrophobic chain.[50] This finding
indicates the importance of an amphiphilic balance.
2.2.1. Effect of phenyl group and the fluoride atom on antibacterial activity
Different aryl groups of chalcone showed distinct activities. Compounds 5a-5s
with different substituted phenyl groups and compounds 5z-5ac with thienyl or
furanyl groups showed good activity, whereas compounds 5t-5y with pyridyl groups
showed poorer activity when compared with the activity of the other compounds. The
MICs of mono-substituted phenyl compounds 5d, 5e, 5q, 5r and 5s against
Gram-positive bacteria ranged from 1 to 2 µg/mL, whereas the range of MIC values
against Gram-negative bacteria were 4–8 µg/mL. P-methyl (5q) and p-methoxyl (5r)

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achieved similar activity as compound 5a against S. aureus. The presence of a
fluoride atom was observed to be important for antibacterial activity. For example,
compound 5g with a fluoride atom substituted phenyl ring displayed the highest
antibacterial activity. In addition, the position of the fluoride atom was found to affect
the antibacterial activity. The different activity of 5d, 5e and 5g suggests that the
position of the fluoride atom affects the antibacterial activity of these compounds. The
compounds 5k-5m with different positions of the second fluoride atom exhibited
varying antibacterial activity. Compound 5k displayed similar activity to that of 5g
(MICs.aureus = 0.5 µg/mL), indicating the position of the fluoride atom modulates the
antibacterial efficacy of the compounds.
2.2.2. Activity against drug-resistant bacteria
Table 2. MIC of Cationic Small Molecules against Drug-Resistant MRSA
MRSA
Compd
M-1
1
M-2
0.5
1
M-3
1
M-4
8
M-5
4
M-6
0.5
2
M-7
1
M-8
4
M-9
2
5a
5s
1
1
16
4
8
2
32
1
2
1
0.5
0.5
0.5
0.5
1
1
2
0.5
0.5
0.5
0.25
0.5
2
2
2
5b
5c
1
0.5
0.5
0.5
0.5
0.5
0.5
0.5
2
8
2
1
4
1
1
8
2
1
4
2
5d
5g
5l
1
8
2
1
4
1
1
8
4
1
4
2
1
0.5
0.5
1
16
4
4
0.5
1
1
1
5p
5q
5r
1
2
0.5
0.5
2
4
2
1
8
4
1
1
1
4
2
32
16
8
4
16
1
2
5z
1
1
0.5
4
1
2
2
5ab
The activity of compounds 5a-5d, 5g, 5l, 5p-5s, 5z and 5ab against
drug-resistant bacteria MRSA (9 clinical isolated strains) was further tested. All
selected molecules had MIC values in the range of 0.25–32 µg/mL (Table 2).
Compound 5g exhibited the highest activity with an MIC of 0.25 µg/mL against
MRSA-6, and the MICs of the other compounds were primarily 0.5–8 µg/mL.

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Table 3. MIC of Cationic Small Molecules against Drug-Resistant KPC
KPC
Compd
K-1 K-2
K-3
16
8
K-4
K-5
32
8
K-6
16
8
K-7
＞128
16
K-8
8
K-9
16
8
K-10
＞128
32
K-11
K-12
＞128
16
MIC90
＞128
16
16
8
16
8
＞128
＞128
5s
5b
5c
16
8
8
8
4
4
4
8
4
4
8
4
4
8
8
8
4
4
8
4
4
4
16
2
4
8
8
8
8
5d
5g
5l
4
4
8
8
4
4
16
4
4
16
8
16
16
4
8
8
8
4
4
16
8
4
8
8
8
8
8
8
8
＞128
8
4
＞128
16
4
8
16
8
16
＞128
16
5p
5q
5r
4
4
8
8
8
4
8
4
4
16
4
16
8
8
8
4
8
2
8
8
16
16
32
16
16
16
16
8
32
8
32
8
32
8
16
8
16
8
2
8
16
8
32
32
32
5z
2
8
16
16
16
5ab
Antibacterial activity of selected compounds 5a-5d, 5g, 5l, 5p-5s, 5z and 5ab
against the drug-resistant bacteria KPC (12 clinical isolated strains) was investigated.
The MIC of compound 5c ranged from 4 to 8 µg/mL (Table 3). Compounds 5r, 5z and
5ab exhibited good activity against KPC-7 (MIC: 2 µg/mL).
Table 4. MIC of Cationic Small Molecules against Drug-Resistant NDM-1
NDM-1
Compd
N-1
16
16
4
N-2
16
4
N-3
2
N-4
8
N-5
1
N-6
16
8
N-7
8
N-8
8
N-9
16
8
N-10
128
16
16
16
16
16
128
1
N-11
16
8
N-12
1
MIC90
16
16
16
16
16
8
5s
5b
5c
4
8
16
4
4
4
8
4
1
4
4
4
4
4
8
16
16
4
4
4
1
4
16
16
8
4
4
16
4
8
4
5d
5g
5l
4
4
1
4
16
4
4
4
4
4
4
2
4
4
4
4
4
2
8
8
1
8
16
8
8
8
4
8
4
4
16
8
5p
5q
5r
4
4
2
8
4
4
4
8
4
32
32
64
8
4
8
1
8
4
8
8
8
8
16
32
4
8
16
32
8
16
4
16
8
4
8
32
4
16
8
16
8
16
8
16
2
16
8
5z
2
8
5ab
The activity of compounds 5a-5d, 5g, 5l, 5p-5s, 5z and 5ab against the
drug-resistant bacteria NDM (12 clinical isolated strains) was tested. The range of
MIC values against NDM were primarily between 1 and 16 µg/mL (Table 4).
Compound 5ab showed the highest activity (MIC: 2–8 µg/mL). In addition,
antibacterial activity toward NDM was better than the activity measured against KPC.

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Figure 4. Hemolytic activity (HC50) of part compounds.
2.3. Hemolytic activity
The ability of compounds to lyse red blood cells (RBCs), which were isolated
from sheep blood, was used as a measure of the toxicity of the compounds toward
mammalian cells. The hemolytic activity of all cationic molecules was represented as
HC50 values (the concentration at which 50% hemolysis occurs). HC50 values of the
cationic molecules ranged from 50 µg/mL to >500 µg/mL (Table 1). The HC50 values
correlated with the presence or absence of a fluoride atom(s) and the length of the
alkyl chain. The number of fluoride atoms on the phenyl group influenced the
hemolytic activity. Here, the HC50 values of compounds 5d, 5e and 5g with one
fluorine atom were 83, 68 and 137 µg/mL, respectively, whereas the HC50 values of
compounds 5k, 5l and 5m that have two fluoride atoms were 56, 54 and 50 µg/mL,
respectively. The HC50 value of 5g was 2-fold higher than that of compound 5k
(Figure 4). The above results revealed that 5d, 5e and 5g were more selective than
compounds 5k, 5l and 5m toward bacteria over mammalian cells. Similarly, the HC50
value of 5w was 2-fold higher than that of compound 5y. In addition, the selectivity
values (HC50/MIC) of the potential compounds 5a and 5g were 228 and 274. The
HC50 values of compounds 5f-5j did not systematically increase with an increase in
the length of the alkyl chain. Thus, these results indicate that hydrophobicity did not

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clearly affect hemolytic activity.
Figure 5. Biofilm disruption of chalcone derivatives at different concentrations. (a)
Cell viability of S. aureus in biofilms treated with compound 5g. (b) Cell viability of
E. coli in biofilms treated with compound 5a.
2.4. Disruption activity
The action of antimicrobial peptides involves the cationic moieties of the peptide
interacting with the anionic moieties displayed on the surface of bacteria membranes.
Subsequently, the hydrophobic moiety of the AMP disperses the established biofilms,
leading to cell death.[51] The antibiofilm activity of the most promising cationic
small molecules, 5g and 5a, was tested against established S. aureus and E. coli
biofilms, respectively (Figure 5). S. aureus biofilms were cultured for 24 h and then
treated with compound 5g at different concentrations. As shown in Figure 5a, S.
aureus cell viability in the biofilms decreased with increasing concentration of
compound 5g. The starting number of S. aureus in the biofilms was 1015.96 CFU/mL,
which was used as a reference point. After 24 h, the cell viabilities in biofilms
decreased to 1013.93, 1012.93, 1011.696, 1010.95, 109.59, 109.41, 108.95 CFU/mL at 5g
concentrations of 2, 4, 8, 16, 32, 64 and 128 µg/mL, respectively. In contrast, the cell
viability in not-treated biofilms increased to 1018.93 CFU/mL. The above results
indicate that the cationic small molecule 5g reduced bacteria viability in biofilms.
In addition, E. coli cell viability in biofilms was also observed to decrease with
increasing concentrations of 5a (Figure 5b). E. coli biofilms were cultured for 72 h
and then treated with compound 5a at different concentrations. The starting cell
number of E. coli in the biofilms was 1014.51 CFU/mL. After 24 h, the cell viabilities

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in the biofilms decreased to 1013.20, 1012.41, 1012.10, 1010.00, 108.10, 107.10, 106.35
CFU/mL at 5a concentrations of 2, 4, 8, 16, 32, 64 and 128 µg/mL, respectively. The
cell viability in not-treated biofilms increased to 1016.1 CFU/mL. Thus, compound 5a
was able to reduce the number of viable bacteria even in mature (72 h) E. coli
biofilms. These results are attributed to the properties of the compounds to eradicate
preformed biofilms. Biofilm disruption activity of compounds 5g and 5a was visually
observed by crystal violet staining (Figure S1).
Figure 6. Propensity of the development of bacterial resistance against compound 5g.
(a) For S. aureus, where antibiotic norfloxacin was used as the control. (b) For E. coli,
where the lipopeptide colistin was used as the control.
2.5. Propensity to induce bacterial resistance
Bacterial resistance against most antibiotics presents a serious problem.[52] Thus,
the propensity of compounds to inhibit bacterial resistance is an important property.
The ability of 5g to suppress the development of resistance was tested. Norfloxacin
and colistin were selected as positive controls for S. aureus and E. coli, respectively.
Resistance is usually defined as a > 4-fold increase in the original MIC.[53] The
results showed no change in the MIC of compound 5g, indicating that exposure of the
bacteria to this compound did not lead to the development of bacterial resistance over
20 generations. In contrast, norfloxacin and colistin showed 256-fold and 32-fold
increases in the MIC values (Figure 6). The above results of the resistance study
revealed that resistance by the tested bacteria toward 5g is not easily developed.

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Figure 7. Plasma stability and bactericidal activity in complex mammalian fluids. (a)
Compound 5g against S. aureus in plasma treated at different times. (b) Compounds
5a and 5g against S. aureus in different culture mediums (50% serum, 50% plasma
and 50% blood) against MRSA.
2.6. Plasma stability
Enzymes and proteins present in plasma will contribute to the degradation of
natural antibacterial peptides. Plasma stability is an important factor that limits the
application of antibacterial peptides.[21] The active compound 5g was dissolved in 50%
sterile Milli-Q water and 50% plasma. Three parallel tests were pre-incubated at
37 °C in 50% plasma for 0, 3 and 6 h. Bactericidal efficacy expressed as the minimum
bactericidal concentration (MBC), which is the minimum drug concentration required
to kill 99.9% of a tested microorganism, was examined. After the treatment, the MBC
values of 5g dissolved in 100% media as the positive control was 8 µg/mL. The MBC
values of 5g increased to 16 µg/mL after treatment in 50% plasma for 0, 3 and 6 h
against S. aureus (Figure 7a). There was no influence of the incubation time on the
observed activity. The above results indicate that compound 5g is stable in plasma,
even after a 6 h treatment.
2.7. Bactericidal activity in complex mammalian fluids
One of the major limitations of natural antimicrobial peptides is the loss of
bactericidal efficacy in the presence of mammalian fluids due to enzymatic hydrolysis.

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Activity in different culture mediums (50% serum, 50% plasma and 50% blood) was
also determined to establish the efficacy of the compounds further. Bactericidal
efficacy in different fluids was expressed as the MBC. It is interesting that 5a and 5g
showed the same activity in different culture mediums. The MBC values of 5a and 5g
in the 50% plasma were 16 µg/mL (Figure 7b). The MBC values of 5a and 5g in the
50% serum were 32 µg/mL, which is 2-fold higher than in 50% plasma. The MBC
value was 128 µg/mL in 50% blood. These results indicate that compounds 5a and 5g
were active in 50% plasma and 50% serum, whereas in 50% blood the compounds
were not effective.
Figure 8. Time-dependent killing of pathogens by compound 5a. (a) S. aureus was
challenged with compound 5a (at 6 × MIC and 8 × MIC) and vancomycin. (b) E. coli
was challenged with compound 5a (at 6 × MIC and 8 × MIC) and moxalactam. (c)
Images show S. aureus after treatment for 6 h. (d) Images show E. coli after treatment
for 6 h. The control was only treated with sterile water. Data are representative of 3
independent experiments.
2.8. Bactericidal time-kill kinetics
To study the bactericidal activity of the highest compound 5a, a time-kill assay
was carried out against S. aureus and E. coli at different concentrations (6 × MIC and

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8 × MIC). Compound 5a showed rapid bactericidal activity. The initial count of S.
aureus was 106.1 CFU/mL. The number of viable bacteria dropped to 100 CFU/mL
after 3 h at a concentration of 6 × MIC. In contrast, vancomycin (6 × MIC) did not
kill the bacteria after 6 h. It is encouraging that compound 5a also displayed the same
rapid bactericidal activity against E. coli as observed for S. aureus (Figure 8). These
results clearly demonstrate the superiority of compound 5a over commonly used
antibiotics vancomycin and moxalactam in killing both Gram-positive bacteria and
Gram-negative bacteria.
Figure 9. Mechanism of antibacterial action of potential cationic chalcone derivatives.
(a) and (b) cytoplasmic membrane depolarization of S. aureus and E. coli, respectively,
at 10 µg/mL. (c) and (d) inner membrane permeabilization of S. aureus and E. coli,
respectively, at 10 µg/mL.
2.9. Mechanism of antibacterial action
To confirm the mechanism of action, 11 selected compounds (5a, 5s, 5b, 5c, 5d,
5g, 5k, 5l, 5p, 5q, 5r) with good antibacterial activity were used in the cytoplasmic

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membrane depolarization assay and the inner membrane permeabilization assay.
2.9.1. Cytoplasmic membrane depolarization
In the cytoplasmic membrane depolarization assay, the membrane
potential-sensitive cyanine dye DiSC35 was used indirectly to reflect the integrity of
the cytoplasmic membrane. If depolarization of the cell membrane is enhanced, the
dye embeds into the hydrophobic environment of the bilayer of the membrane and the
fluorescence intensity enhances significantly.[54] Depolarization of the cytoplasmic
membrane was determined using S. aureus and E. coli. The results are shown in
Figure 9a, 9b, which showed that all compounds were able to depolarize the
membranes of S. aureus and E. coli. However, the membrane depolarization ability by
the compounds varied. Compounds 5c, 5g and 5r showed the highest depolarization
activity of the cytoplasmic membrane of S. aureus, and compounds 5k, 5a and 5g
showed the highest depolarization activity of the cytoplasmic membrane of E. coli.
The other compounds showed lower activity against both S. aureus and E. coli. The
above results agree with the MIC data.
2.9.2. Inner membrane permeabilization
Permeabilization of the cytoplasmic inner membranes of bacteria S. aureus and E.
coli was studied using propidium iodide (PI). The PI dye fluoresces red after
embedding in double-stranded DNA. Although PI cannot pass through live cell
membranes, it can traverse damaged cell membranes and stain the nucleus. Thus,
using the unique attributes of PI, the permeabilization of the inner membrane was
monitored. The results are shown in Figure 9c, 9d. Fluorescence intensity
enhancement was observed following treatment of the cells with the aforementioned
compounds. Thus, the chalcone derivatives were efficient in permeabilizing the inner
membranes of both S. aureus and E. coli. Nonetheless, there were differences in the
ability of the compounds to permeabilize the inner membranes. Compounds 5a, 5k
and 5l showed the highest permeabilization activity towards the membrane of S.
aureus, and compounds 5l, 5b and 5c showed the highest permeabilization activity

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towards the membrane of E. coli. The above results agree with the MIC data.
Figure 10. Electron scanning microscopy images of HeLa cells after treated with
small molecule 5a for 24 h. (a) Non-treated cells (negative control). (b) Cells treated
with 5a (2 × MIC). (c) Cells treated with 5a (4 × MIC) and (d) cells treated with 0.1%
Triton X (positive control). Scale bar is 10 µm.

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Figure 11. Fluorescence microscopy images of HeLa cells after treatment with 5a for
24 h and staining with calcein AM and propidium iodide (PI). (a - c) Non-treated cells
(negative control). (d - f) Cells treated with 5a (2 × MIC). (g - i) Cells treated with 5a
(4 × MIC), and (j-l) cells treated with 0.1% Triton-X (positive control). Scale bar is
200 nm.
2.10. Electron scanning and fluorescence microscopy
The toxicity of compound 5a against HeLa cells was studied. Cells were seeded
into the wells and then treated with compound 5a at different concentrations (2 × MIC
and 4 × MIC). The treated cell lines were imaged by optical microscopy to visualize
the cell morphology (Figure 10). The images presented in Figure 10b, 10c show that
the treated cells had spindle shapes with intact morphology at 5a concentrations of 2

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× MIC and 4 × MIC, and the results were similar to the untreated cell line (Figure
10a). In contrast, cells treated with Triton-X had shapes that indicated extensive cell
damage (Figure 10d). Using the different attributes of the dyes calcein-AM and PI,
fluorescent microscopic studies were carried out to visualize live and dead cells. Cells
treated with compound 5a fluoresced green even at 2 × MIC and 4 × MIC
concentrations (Figure 11d-f, g-i) and were similar to untreated cells (Figure 11a-c).
In contrast, cells treated with 0.1% Triton-X fluoresced red (Figure 11j-l). These
results thus indicate that compound 5a is non-toxic toward mammalian cells at 4 ×
MIC value.
3. Conclusions
A new series of cationic molecules with the chalcone skeleton and a hydrophobic
alkyl chain were designed and synthesized. These compounds displayed potent
broad-spectrum and membrane-active antibacterial activity against various
drug-sensitive and drug-resistant bacteria. Molecules with different substituted aryl
rings showed different antibacterial and hemolytic activities. Moreover, a fluoride
atom was found to influence positively the activities of the compounds. In addition,
the cationic chalcone derivatives were shown to be stable under plasma conditions
and even in complex mammalian fluids such as serum and plasma. The potential
compounds can depolarize and permeabilize bacterial membranes, leading to the rapid
death of bacteria. Importantly, the compounds did not allow bacteria to develop
resistance and exhibited negligible toxicity toward mammalian cells at a given
concentration. The results indicate that these cationic chalcone derivatives could be
potential therapeutic agents to treat bacterial infections in the future.
4. Experimental section
4.1. Materials and instrumentation: Reagents and solvents were purchased from
commercial sources (shanghai darui finechemical, Energy Chemical, Aladdin,
Macklin) and were used without further purification. All the solvents were of reagent
grade. All the chemicals were used as supplied. Drug-sensitive bacterial strains, E.
coli, S. aureus, E. faecalis, S. enterica were obtained from American type culture

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collection. Drug-resistant bacteria, Methicillin-resistant S. aureus (MRSA),
Carbapenemase-producing K. pneumonia (KPC) and NDM-1-producing
Enterobacteriaceae (NDM-1) were collected from a teaching hospital located in
Zhengzhou, Henan Province. All isolates were identified by VITEK2 compact
(bioMerieux, France) and 16S rRNA gene sequencing. PCR and nucleotide
sequencing were employed to screen for the presence of blaNDM gene, the primers and
PCR program conditions were described previously [55]. Sheep RBCs were used for
hemolytic assay.
1H NMR and 13C NMR spectra were recorded on a Bruker 400 MHz and 101 MHz
spectrometer, respectively, and TMS as internal standard reference. Coupling constants
(J) are given in hertz (Hz). High resolution mass spectra (HRMS) were recorded on a
Waters Micromass Q-T of Micromass spectrometer. Analytical thin layer
chromatography (TLC) was performed on glass plates pre-coated with silica gel,
(Qingdao Marine Chemical Factory (China)) to monitor the reactions. Visualization
was accomplished using UV light. Column chromatography was performed on silica
gel (300-400 meshes). For optical density and fluorescence measurements, Tecan
Infinite Pro series M200 Microplate Reader was used. The biological experiments were
performed with a 1300 series A2 Biological Safety Cabinet. TDL-5M Desktop
Low-speed Refrigerated Centrifuge was used in antibacterial studies.
4.2. General procedure for the synthesis of Chalcone analogs (3a-3w)
NaOH (9.25 mmol, 1.25 eq.) in H2O (50 mL) was stirred at room temperature.
Compound 1-(4-aminophenyl) ethan-1-one or 1-(3-aminophenyl) ethan-1-one (7.40
mmol, 1.00 eq.) and aromatic aldehyde (7.58 mmol, 1.02 eq.) were dissolved in ethanol
(50 mL). Then ethanol solution was added into H2O solution. The reaction mixture was
stirred at room temperature. After the reaction was completed (monitored by TLC light
petroleum / EtOAc, 2/1, v/v), the mixture was poured into ice water (50 mL). The
precipitate formed was filtered and washed with water. The products were dried under
vacuum and purified via silica gel column chromatography (light petroleum /EtOAc,
3/1, v/v) to obtain intermediate products 3a-3w in 10%-86% yield. The purified
1
compounds were subsequently characterized using H NMR, 13C NMR. The NMR

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data of compounds (3a-3w) were listed in supplementary material.
4.3. General procedure for the synthesis of amide coupling compounds (4a-4w)
Chloroacetyl chloride (0.6 mmol, 1.20 eq.) was dropped to the mixture of
intermediates 3a-3w (0.5 mmol, 1.00 eq.) and K2CO3 (0.6 mmol, 1.20 eq.) in acetone
(1.5 mL). The reaction mixture was stirred at room temperature for 0.5-1 h. After the
reaction finished (monitored by TLC), the mixture was poured into ice water (3 mL).
The precipitate formed was filtered and washed with water. The products were dried
under vacuum to obtain target compounds 4a-4w without any further purification. The
purified compounds were in 41%-91% yield and subsequently characterized using 1H
NMR, 13C NMR. The NMR data of compounds (4a-4w) were listed in supplementary
material.
4.4. General procedure for the synthesis of amphiphilic small molecules (5a-5ac)
Compounds (4a-4w) (528.73 µmol, 1.0eq.) were reacted with N, N- dimethyl
tertiary amine (1.58 mmol, 3 eq.) in anhydrous acetonitrile (6 mL) in a screw-top
pressure tube at 85 °C for about 24 h. After the reaction, the solvent was evaporated
and the products were precipitated using excess ether. Finally, the products were
filtered and washed with ether for 3-5 times. The products were dried under vacuum
to obtain target compounds 5a-5ac without any further purification. The purified
1
compounds were in 50%-95% yield and subsequently characterized using H NMR,
13C NMR, IR and HR-MS.
4.4.1. (E)N-(2-((4-cinnamoylphenyl)amino)-2-oxoethyl)-N,N-dimethyloctan-1-ami
nium chloride (5a). Light yellow solid, yield: 78%. 1H NMR (400 MHz, DMSO-d6) δ
12.21 (s, 1H), 8.24 (d, J = 8.0 Hz, 2H), 8.04 – 7.88 (m, 5H), 7.77 (d, J = 15.5 Hz, 1H),
7.48 (s, 3H), 4.64 (s, 2H), 3.58 (s, 2H), 3.34 (s, 6H), 1.79 (s, 2H), 1.24-1.23 (m, 6H),
1.23-1.13 (m, 4H), 0.83 (d, J = 6.1 Hz, 3H). 13C NMR (101 MHz, DMSO-d6) δ
187.61, 162.71, 143.60, 142.37, 134.70, 133.16, 130.54, 129.84, 128.88, 121.89,
119.09, 64.88, 62.32, 51.24, 31.12, 28.37, 25.67, 22.00, 21.83, 13.91. IR ʋ (cm −1):
764, 975, 1447, 1601, 1661, 1696, 3025, 3166, 3245; HR-MS (ESI) Calculated for
C27H37ClN2O2 [M-Cl] +: 421.2850, found: 421.2856.

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4.4.2. (E)-N-(2-((4-(3-(3-chlorophenyl)acryloyl)phenyl)amino)-2-oxoethyl)-N,N
1
dimethyloctan-1-aminium chloride (5b). White solid, yield: 93%. H NMR (400
MHz, DMSO-d6) δ 11.87 (s, 1H), 8.19 (s, 2H), 7.90 (d, J = 18.9 Hz, 5H), 7.71 (d, J =
14.1 Hz, 1H), 7.52 (s, 2H), 4.50 (s, 2H), 3.51 (s, 2H), 3.26 (s, 6H), 1.74 (s, 2H),
13
1.42-0.98 (m, 10H), 0.82 (s, 3H). C NMR (101 MHz, DMSO-d6) δ 187.51, 162.69,
142.37, 142.12, 135.00, 133.69, 133.10, 130.52, 129.89, 128.92, 122.65, 119.12,
−1
64.93, 62.33, 51.26, 31.11, 28.35, 25.66, 21.98, 21.83, 13.89. IR ʋ (cm ): 723, 984,
1465, 1596, 1658, 1698, 3085, 3170, 3245; HR-MS (ESI) Calculated for
C27H36Cl2N2O2 [M-Cl] +: 455.2460, found: 455.2464.
4.4.3. (E)-N-(2-((4-(3-(3-chlorophenyl)acryloyl)phenyl)amino)-2-oxoethyl)-N,N-dim
1
ethyloctan-1-aminium chloride (5c). Light yellow solid, yield: 67%. H NMR (400
MHz, DMSO-d6) δ 11.99 (s, 1H), 8.24 (d, J = 8.6 Hz, 2H), 8.11 – 8.02 (m, 2H), 7.92
(d, J = 8.6 Hz, 2H), 7.84 (d, J = 6.1 Hz, 1H), 7.72 (d, J = 15.6 Hz, 1H), 7.53 – 7.46 (m,
2H), 4.56 (s, 2H), 3.60 – 3.50 (m, 2H), 3.30 (s, 6H), 1.78 (s, 2H), 1.31-1.25 (m, 6H),
1.25-1.18 (m, 4H), 0.84 (t, J = 6.6 Hz, 3H). 13C NMR (101 MHz, DMSO-d6) δ 187.94,
163.23, 143.00, 142.37, 137.47, 134.27, 133.46, 131.16, 130.48, 128.38, 123.89,
119.56, 65.38, 62.80, 51.73, 31.62, 28.87, 26.17, 22.50, 22.33, 14.41. IR ʋ (cm −1):
727, 971, 1447, 1600, 1665, 1699, 3027, 3163, 3245; HR-MS (ESI) Calculated for
C27H36Cl2N2O2 [M-Cl] +: 455.2460, found: 455.2463.
4.4.4. (E)-N-(2-((4-(3-(4-fluorophenyl)acryloyl)phenyl)amino)-2-oxoethyl)-N,N-dim
1
ethyloctan-1-aminium chloride (5d). White solid, yield: 83%. H NMR (400 MHz,
DMSO-d6) δ 12.04 (s, 1H), 8.22 (d, J = 8.5 Hz, 2H), 8.05 – 7.88 (m, 5H), 7.75 (d, J =
15.6 Hz, 1H), 7.32 (t, J = 8.6 Hz, 2H), 4.57 (s, 2H), 3.65 – 3.48 (m, 2H), 3.31 (s, 6H),
1.78 (s, 2H), 1.31-1.25 (m, 6H), 1.25-1.18 (m, 4H), 0.84 (t, J = 6.4 Hz, 3H). 13C NMR
(101 MHz, DMSO-d6) δ 187.50, 163.13 (d, J = 250.07 Hz), 162.70, 142.36, 133.13,
131.39 (d, J = 3.0 Hz), 131.19 (d, J = 8.6 Hz), 129.84, 121.78, 119.07, 115.89 (d, J =
21.7 Hz), 64.88, 62.31, 51.23, 31.11, 28.36, 25.66, 22.00, 21.83, 13.90. IR ʋ (cm −1):
739, 983, 1465, 1597, 1656, 1697, 3085, 3172, 3241; HR-MS (ESI) Calculated for
C27H36ClFN2O2 [M-Cl] +: 439.2755, found: 439.2762.
4.4.5. (E)-N-(2-((4-(3-(3-fluorophenyl)acryloyl)phenyl)amino)-2-oxoethyl)-N,N-dim

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1
ethyloctan-1-aminium chloride (5e). Light yellow solid, yield: 58%. H NMR (400
MHz, DMSO-d6) δ 12.09 (s, 1H), 8.24 (d, J = 8.7 Hz, 2H), 8.05 (d, J = 15.6 Hz, 1H),
7.91 (dd, J = 23.1, 9.5 Hz, 3H), 7.80 – 7.65 (m, 2H), 7.52 (dd, J = 14.2, 7.9 Hz, 1H),
7.30 (td, J = 8.6, 2.3 Hz, 1H), 4.59 (s, 2H), 3.56 (dd, J = 10.2, 6.5 Hz, 2H), 3.31 (s,
6H), 1.79 (s, 2H), 1.31-1.25 (m, 6H), 1.25-1.18 (m, 4H), 0.84 (t, J = 6.8 Hz, 3H). 13C
NMR (101 MHz, DMSO-d6) δ 187.49, 162.73, 162.56 (d, J = 244.62), 142.51, 142.09
(d, J = 2.7 Hz), 137.29 (d, J = 8.1 Hz), 132.97, 130.80 (d, J = 8.4 Hz), 129.94, 125.53
(d, J = 2.4 Hz), 123.33, 119.07, 117.16 (d, J = 21.4 Hz), 114.62 (d, J = 22.0 Hz),
64.87, 62.32, 51.23, 31.11, 28.36, 25.67, 22.00, 21.84, 13.89. IR ʋ (cm −1): 733, 971,
1446, 1601, 1665, 1697, 3028, 3165, 3239; HR-MS (ESI) Calculated for
C27H36ClFN2O2 [M-Cl] +: 439.2755, found: 439.2763.
4.4.6. (E)-N-(2-((4-(3-(2-fluorophenyl)acryloyl)phenyl)amino)-2-oxoethyl)-N,N-dim
1
ethylbutan-1-aminium chloride (5f). Light yellow solid, yield: 76%. H NMR (400
MHz, DMSO-d6) δ 12.06 (s, 1H), 8.32 – 8.09 (m, 3H), 8.02 (d, J = 15.7 Hz, 1H), 7.93
(d, J = 8.5 Hz, 2H), 7.84 (d, J = 15.7 Hz, 1H), 7.54 (dd, J = 13.2, 6.7 Hz, 1H), 7.34
(dd, J = 13.0, 6.2 Hz, 2H), 4.58 (s, 2H), 3.66 – 3.50 (m, 2H), 3.31 (s, 6H), 1.77 (d, J =
7.4 Hz, 2H), 1.33 (dd, J = 14.4, 7.2 Hz, 2H), 0.94 (t, J = 7.3 Hz, 3H). 13C NMR (101
MHz, DMSO-d6) δ 187.44, 162.73, 160.87 (d, J = 251.3 Hz), 142.53, 134.71 (d, J =
4.2 Hz), 132.89, 132.59 (d, J = 8.6 Hz), 129.92, 129.09, 124.93 (d, J = 3.4 Hz),
124.01 (d, J = 4.0 Hz), 122.32 (d, J = 11.2 Hz), 119.14, 116.05 (d, J = 21.6 Hz), 64.77,
62.35, 51.18, 23.87, 19.13, 13.43. IR ʋ (cm −1): 743, 963, 1456, 1598, 1659, 1691,
+
3034, 3163, 3235; HR-MS (ESI) Calculated for C23H28ClFN2O2 [M-Cl] : 383.2129,
found: 383.2135.
4.4.7. (E)-N-(2-((4-(3-(2-fluorophenyl)acryloyl)phenyl)amino)-2-oxoethyl)-N,N-dim
1
ethyloctan-1-aminium chloride (5g). Light yellow solid, yield: 84%. H NMR (400
MHz, CDCl3) δ 12.03 (s, 1H), 7.97 (dd, J = 19.1, 8.6 Hz, 4H), 7.89 (d, J = 15.9 Hz,
1H), 7.66 (d, J = 7.4 Hz, 1H), 7.62 (d, J = 15.7 Hz, 1H), 7.44 – 7.32 (m, 1H), 7.20 (t,
J = 7.5 Hz, 1H), 7.16 – 7.05 (m, 1H), 4.95 (s, 2H), 3.75 – 3.57 (m, 2H), 3.44 (s, , 6H),
1.83 (s, 2H), 1.46-1.12 (m, 10H), 0.86 (t, J = 6.4 Hz, 3H). 13C NMR (101 MHz,
CDCl3) δ 188.99, 161.73 (d, J = 255 Hz), 161.71, 141.79, 137.19, 134.27, 131.77 (d, J

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= 8.8 Hz), 129.74, 124. 43 (d, J = 6.9 Hz), 123.08 (d, J = 11.2 Hz), 119.84, 116.27 (d,
J = 21.9 Hz), 66.50, 63.90, 52.10, 31.54, 28.96, 26.17, 22.91, 22.52, 14.01. IR ʋ (cm
−1): 723, 980, 1444, 1600, 1662, 1696, 3026, 3165, 3235; HR-MS (ESI) Calculated
for C27H36ClFN2O2 [M-Cl] +: 439.2755, found: 439.2764.
4.4.8. (E)-N-(2-((4-(3-(2-fluorophenyl)acryloyl)phenyl)amino)-2-oxoethyl)-N,N-dim
1
ethyldodecan-1-aminium chloride (5h). Light yellow solid, yield: 77%. H NMR
(400 MHz, DMSO-d6) δ 12.05 (d, J = 21.5 Hz, 1H), 8.20 (d, J = 8.6 Hz, 2H), 8.15 (t,
J = 7.7 Hz, 1H), 8.01 (d, J = 15.7 Hz, 1H), 7.93 (d, J = 8.0 Hz, 2H), 7.84 (d, J = 15.7
Hz, 1H), 7.53 (dd, J = 13.5, 6.9 Hz, 1H), 7.34 (dd, J = 13.0, 5.5 Hz, 2H), 4.57 (s, 2H),
3.66 – 3.48 (m, 2H), 3.30 (s, 6H), 1.78 (s, 2H), 1.30 – 1.17 (m, 18H), 0.83 (t, J = 6.4
Hz, 3H). 13C NMR (101 MHz, DMSO-d6) δ 187.29, 162.73, 160.87 (d, J = 251.4 Hz),
142.58, 134.63 (d, J = 4.1 Hz), 132.86, 132.46 (d, J = 8.7 Hz), 129.81, 129.06, 124.83
(d, J = 3.1 Hz), 123.86 (d, J = 3.9 Hz), 122.32 (d, J = 11.2 Hz), 119.09, 115.95 (d, J =
21.7 Hz), 64.72, 62.33, 51.29, 31.27, 29.03, 28.94, 28.76, 28.72, 28.39, 25.68, 22.05,
21.86, 13.83. IR ʋ (cm −1): 753, 981, 1467, 1601, 1659, 1704, 3091, 3175, 3246;
HR-MS (ESI) Calculated for C31H44ClFN2O2 [M-Cl] +: 495.3381, found: 495.3388.
4.4.9. (E)-N-(2-((4-(3-(2-fluorophenyl)acryloyl)phenyl)amino)-2-oxoethyl)-N,N-dim
1
ethyltetradecan-1-aminium chloride (5i). Light yellow solid, yield: 79%. H NMR
(400 MHz, DMSO-d6) δ 11.85 (s, 1H), 8.19 (d, J = 8.8 Hz, 2H), 8.13 (t, J = 7.5 Hz,
1H), 8.00 (d, J = 15.7 Hz, 1H), 7.89 (d, J = 8.7 Hz, 2H), 7.83 (d, J = 15.7 Hz, 1H),
7.53 (dd, J = 13.1, 6.4 Hz, 1H), 7.33 (dd, J = 13.2, 5.7 Hz, 2H), 4.51 (s, 2H), 3.53 (dd,
J = 10.1, 6.6 Hz, 2H), 3.28 (s, 6H), 1.76 (s, 2H), 1.29 – 1.16 (m, 22H), 0.83 (t, J = 6.8
Hz, 3H). 13C NMR (101 MHz, DMSO-d6) δ 187.24, 162.72, 160.88 (d, J = 251.5 Hz),
142.57, 134.63 (d, J = 4.1 Hz), 132.86, 132.42 (d, J = 8.7 Hz), 129.80, 129.03, 124.80
(d, J = 3.1 Hz), 123.82 (d, J = 3.8 Hz), 122.33 (d, J = 11.2 Hz), 119.07, 115.92 (d, J =
21.7 Hz), 64.67, 62.31, 51.30, 31.28, 29.10, 29.05, 28.96, 28.78, 28.73, 28.40, 25.68,
22.06, 21.86, 13.82. IR ʋ (cm −1): 758, 985, 1468, 1597, 1661, 1696, 3084, 3161,
+
3245; HR-MS (ESI) Calculated for C33H48ClFN2O2 [M-Cl] : 523.3694, found:
523.3697.
4.4.10. (E)-N-(2-((4-(3-(2-fluorophenyl)acryloyl)phenyl)amino)-2-oxoethyl)-N,N-di

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1
methyloctadecan-1-aminium chloride (5j). Light yellow solid, yield: 78%. H NMR
(400 MHz, DMSO-d6) δ 11.81 (s, 1H), 8.19 (d, J = 8.8 Hz, 2H), 8.13 (t, J = 7.5 Hz,
1H), 8.00 (d, J = 15.7 Hz, 1H), 7.89 (d, J = 8.7 Hz, 2H), 7.84 (d, J = 15.8 Hz, 1H),
7.53 (dd, J = 13.1, 6.5 Hz, 1H), 7.33 (dd, J = 13.2, 5.5 Hz, 2H), 4.51 (s, 2H), 3.53 (dd,
J = 10.1, 6.6 Hz, 2H), 3.29 (s, 6H), 1.76 (s, 2H), 1.35 – 1.12 (m, 32H), 0.84 (t, J = 6.8
Hz, 3H). 13C NMR (101 MHz, DMSO-d6) δ 186.21, 161.60, 159.75 (d, J = 251.3 Hz),
141.38, 133.57 (d, J = 4.2 Hz), 131.76, 131.43 (d, J = 8.8 Hz), 128.76, 127.94, 123.77
(d, J = 3.2 Hz), 122.79 (d, J = 3.8 Hz), 121.19 (d, J = 11.2 Hz), 117.97, 114.90 (d, J =
21.5 Hz), 63.53, 61.10, 50.23, 30.13, 27.92, 27.90, 27.85, 27.80, 27.55, 27.19, 24.46,
20.93, 20.63, 12.77. IR ʋ (cm −1): 757, 985, 1467, 1597, 1661, 1696, 3084, 3161,
+
3249; HR-MS (ESI) Calculated for C37H56ClFN2O2 [M-Cl] : 579.4320, found:
579.4323.
4.4.11. (E)-N-(2-((4-(3-(2,3-difluorophenyl)acryloyl)phenyl)amino)-2-oxoethyl)-N,N
1
-dimethyloctan-1-aminium chloride (5k). White solid, yield: 73%. H NMR (400
MHz, DMSO-d6) δ 12.10 (s, 1H), 8.21 (d, J = 8.8 Hz, 2H), 8.05 (d, J = 15.7 Hz, 1H),
7.98 (d, J = 7.6 Hz, 1H), 7.94 (d, J = 8.8 Hz, 2H), 7.80 (d, J = 15.7 Hz, 1H), 7.54 (dd,
J = 17.2, 8.3 Hz, 1H), 7.33 (dd, J = 12.8, 7.7 Hz, 1H), 4.59 (s, 2H), 3.56 (dd, J = 10.2,
6.5 Hz, 2H), 3.31 (s, 6H), 1.79 (s, 2H), 1.31-1.25 (m, 6H), 1.25-1.20 (m, 4H), 0.84 (t,
J = 6.8 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ 192.52, 168.00, 155.21 (dd, J = 246.9,
J = 12.7 Hz), 153.79 (dd, J = 253.6, 13.4 Hz), 147.92, 138.64 (t, J = 3.6 Hz), 137.95,
135.21, 130.64 (d, J = 4.1 Hz), 130.36 (d, J = 5.3 Hz), 130.30 (d, J = 3.0 Hz), 129.93
(d, J = 8.2 Hz), 129.49 (d, J = 2.6 Hz), 124.39, 124.19, 70.17, 67.61, 56.49, 36.36,
33.60, 30.93, 27.24, 27.10, 19.12. IR ʋ (cm −1): 743, 963, 1467, 1602, 1661, 1700,
+
3081, 3165, 3250; HR-MS (ESI) Calculated for C27H35ClF2N2O2 [M-Cl] : 457.2661,
found: 457.2666.
4.4.12. (E)-N-(2-((4-(3-(2,4-difluorophenyl)acryloyl)phenyl)amino)-2-oxoethyl)-N,N
1
-dimethyloctan-1-aminium chloride (5l). Light yellow solid, yield: 75%. H NMR
(400 MHz, DMSO-d6) δ 12.11 (s, 1H), 8.38 – 8.10 (m, 3H), 8.01 (d, J = 15.7 Hz, 1H),
7.93 (d, J = 8.4 Hz, 2H), 7.78 (d, J = 15.7 Hz, 1H), 7.33 (dt, J = 16.5, 9.1 Hz, 2H),
4.59 (s, 2H), 3.66 – 3.51 (m, 2H), 3.31 (s, 6H), 1.79 (s, 2H), 1.31-1.25 (m, 6H),

ACCEPTED MANUSCRIPT
1.25-1.16 (m, 4H), 0.83 (d, J = 6.7 Hz, 3H). 13C NMR (101 MHz, DMSO-d6) δ
187.34, 163.51 (dd, J = 252.4, 12.7 Hz), 162.73, 161.25 (dd, J = 254.9, 12.7 Hz),
142.54, 133.75 (d, J = 1.9 Hz), 132.87, 130.71 (dd, J = 10.0, 3.6 Hz), 129.87, 123.76,
119.26 (d, J = 3.8 Hz), 119.13, 112.58 (d, J = 3.4 Hz), 112.37 (d, J = 3.5 Hz), 104.55
(t, J = 26.1 Hz), 64.90, 62.35, 51.25, 31.10, 28.35 (d, J = 1.9 Hz), 25.67, 21.98, 21.84,
13.87. IR ʋ (cm −1): 749, 974, 1466, 1600, 1661, 1695, 3093, 3173, 3240; HR-MS
(ESI) Calculated for C27H35ClF2N2O2 [M-Cl] +: 457.2661, found: 457.2668.
4.4.13. (E)-N-(2-((4-(3-(2,6-difluorophenyl)acryloyl)phenyl)amino)-2-oxoethyl)-N,N
1
-dimethyloctan-1-aminium chloride (5m). Brown solid, yield: 95%. H NMR (400
MHz, DMSO-d6) δ 11.98 (s, 1H), 8.09 (d, J = 8.7 Hz, 2H), 7.90 (dd, J = 17.4, 12.4 Hz,
3H), 7.69 (d, J = 16.0 Hz, 1H), 7.63 – 7.50 (m, 1H), 7.27 (t, J = 8.8 Hz, 2H), 4.55 (s,
2H), 3.60 – 3.49 (m, 2H), 3.30 (s, 6H), 1.78 (s, 2H), 1.31-1.25 (m, 6H), 1.25-1.19 (m,
4H), 0.84 (t, J = 6.7 Hz, 3H). 13C NMR (101 MHz, DMSO-d6) δ 187.53, 162.76,
160.77 (dd, J = 254.6, 6.8 Hz), 142.62, 132.63, 132.47 (d, J = 11.1 Hz), 129.80,
128.91, 127.31 (t, J = 7.5 Hz), 119.25, 112.47 (d, J = 5.0 Hz), 112.20 (d, J = 9.4 Hz),
111.93 (d, J = 15.3 Hz), 64.87, 62.27, 51.24, 31.12, 28.36, 25.66, 22.00, 21.83, 13.89.
IR ʋ (cm −1): 749, 962, 1469, 1597, 1661, 1696, 3084, 3161, 3249; HR-MS (ESI)
Calculated for C27H35ClF2N2O2 [M-Cl] +: 457.2661, found: 457.2668.
4.4.14. (E)-N-(2-((4-(3-(2-ethoxy-5-nitrophenyl)acryloyl)phenyl)amino)-2-oxoeth
1
yl)-N,N-dimethyloctan-1-aminium chloride (5n). Light yellow solid, yield: 58%. H
NMR (400 MHz, DMSO-d6) δ 11.98 (s, 1H), 8.82 (s, 1H), 8.29 (d, J = 7.3 Hz, 1H),
8.21 (d, J = 8.3 Hz, 2H), 8.13 (d, J = 15.8 Hz, 1H), 7.94 (dd, J = 23.0, 12.0 Hz, 3H),
7.32 (d, J = 9.2 Hz, 1H), 4.56 (s, 2H), 3.55 (s, 2H), 3.30 (s, 6H), 1.78 (s, 2H),
1.31-1.26 (m, 6H), 1.26-1.18 (m, 4H), 0.84 (d, J = 6.6 Hz, 3H). 13C NMR (101 MHz,
DMSO-d6) δ 187.48, 162.73, 162.04, 142.49, 140.85, 135.86, 132.99, 129.92, 127.17,
124.31, 123.97, 123.73, 119.10, 113.01, 65.26, 64.91, 62.33, 51.25, 31.11, 28.36,
25.67, 21.99, 21.83, 14.31, 13.89. IR ʋ (cm −1): 747, 956, 1467, 1589, 1660, 1699,
+
3045, 3191, 3249; HR-MS (ESI) Calculated for C29H40ClN3O5 [M-Cl] : 510.2962,
found: 510.2963.
4.4.15. (E)-N-(2-((4-(3-(4-(tert-butyl)phenyl)acryloyl)phenyl)amino)-2-oxoethyl)-N,