Antibacterial activity of indolyl-quinolinium derivatives and study their mode of action

Article abstract of DOI:10.1016/j.bmc.2019.02.024

Filamenting temperature-sensitive mutant Z (FtsZ) is recognized as a promising target for new antibiotics development because of its high conservatism and pivotal role in the bacteria cell division. The aromatic heterocyclic scaffold of indole is known showing merit medical functions in antiviral and antimicrobial. In the present study, a series of 1-methylquinolinium derivatives, which were integrated with an indole fragment at its 2-position and a variety of amino groups (cyclic or linear, mono- or di-amine) at the 4-position were synthesized and their antibacterial activities were evaluated. The results of antibacterial study show that the representative compounds can effectively inhibit the growth of testing strains including MRSA and VRE, with MIC values of 1–4 μg/mL by bactericidal mode. The mode of action assays revealed that c2 can effectively disrupt the rate of GTP hydrolysis and dynamic polymerization of FtsZ, and thus inhibits bacterial cell division and then causes bacterial cell death. In addition, the result of resistance generation experiment reveals that c2 is not likely to induce resistance in S. aureus.

Full text of DOI:10.1016/j.bmc.2019.02.024

Accepted Manuscript
Antibacterial activity of indolyl-quinolinium derivatives and study their mode
of action
Senyuan Cai, Wenchang Yuan, Ying Li, Xuanhe Huang, Qi Guo, Ziwei Tang,
Zhiyuan Fang, Hai Lin, Wing-Leung Wong, Kwok-Yin Wong, Yu-Jing Lu,
Ning Sun
PII:
S0968-0896(18)31971-0
https://doi.org/10.1016/j.bmc.2019.02.024
BMC 14760
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
22 November 2018
9 February 2019
13 February 2019
Please cite this article as: Cai, S., Yuan, W., Li, Y., Huang, X., Guo, Q., Tang, Z., Fang, Z., Lin, H., Wong, W-L.,
Wong, K-Y., Lu, Y-J., Sun, N., Antibacterial activity of indolyl-quinolinium derivatives and study their mode of
action, Bioorganic & Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.bmc.2019.02.024
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Antibacterial activity of indolyl-quinolinium derivatives and
study their mode of action
Senyuan Caia,#, Wenchang Yuanb, #, Ying Lia, Xuanhe Huanga, Qi Guoc, Ziwei Tangd,
Zhiyuan Fangb, Hai Linb, Wing-Leung Wonge, Kwok-Yin Wonge,*, Yu-Jing Lua,*,
Ning Suna,b,e,*
a. School of Chemical Engineering and Light Industry, School of Biomedical and
Pharmaceutical Sciences, Guangdong University of Technology, Guangzhou 510006,
P.R. China; E-mail: luyj@gdut.edu.cn;
b. The Fifth Affiliated Hospital of Guangzhou Medical University, Guangzhou
510700, P.R. China; E-mail: ning.sun@connect.polyu.hk;
c. State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun
Yat-sen University, Guangzhou, P.R. China;
d. School of Pharmaceutical Sciences, Guangzhou Medical University, Guangzhou,
P.R. China;
e. The State Key Laboratory of Chemical Biology and Drug Discovery, Department
of Applied Biology and Chemical Technology, The Hong Kong Polytechnic
University, Hung Hom, Kowloon, Hong Kong, China.
# These authors contributed equally to this paper.
* Corresponding authors
1

Abstract
Filamenting temperature-sensitive mutant Z (FtsZ) is recognized as a promising
target for new antibiotics development because of its high conservatism and pivotal
role in the bacteria cell division. The aromatic heterocyclic scaffold of indole is
known showing merit medical functions in antiviral and antimicrobial. In the present
study, a series of 1-methylquinolinium derivatives, which were integrated with an
indole fragment at its 2-position and a variety of amino groups (cyclic or linear,
mono- or di-amine) at the 4-position were synthesized and their antibacterial activities
were evaluated. The results of antibacterial study show that the representative
compounds can effectively inhibit the growth of testing strains including MRSA and
VRE, with MIC values of 1 to 4 μg/mL by bactericidal mode. The mode of action
assays revealed that c2 can effectively disrupt the rate of GTP hydrolysis and dynamic
polymerization of FtsZ, and thus inhibits bacterial cell division and then causes
bacterial cell death. In addition, the result of resistance generation experiment reveals
that c2 is not likely to induce resistance in S. aureus.
Keywords: Indolyl quinolinium derivatives, antibacterial activity, drug resistant
bacteria, FtsZ, cells division.
1. Introduction
The emergence of multidrug resistance among clinically significant bacterial
pathogens is now being a worldwide public health issue. The high morbidity and
mortality rates could be expected in the future due to lack of effective drugs to treat
with the infections from these super bacteria.1 However, new drugs development
against the multidrug resistant infection is a big challenge. Currently, the first-line
clinical antibiotics display weak or even no activity against resistant bacteria,2 the
development of new types of antibacterial agents that possess a novel mechanism of
action is therefore an urgent need.3
Target selection in new medicines research is critical. Bacterial divisions are
currently believed to be a new area for new generation of antibiotics development
because it has a complex set of biochemical machinery containing many functional
proteins that control cell division process.4 Among these proteins, filamenting
2

temperature-sensitive mutant Z (FtsZ) is highly conserved among bacterial pathogens
but not found in the mammalian cells.5 Therefore, it could be the right target for drug
discovery. As a matter of fact, FtsZ plays a very essential role in the process of
bacterial cell division. In the beginning of bacterial cell division, FtsZ, a GTPase,
assembles into protofilaments and forms the ring-like structure (called the Z-ring) in a
GTP-dependent manner at middle of the cell.6 This ring makes a great contribution to
the cell membrane and serves as a scaffold for the recruitment of many functional
proteins, completing the bacterial cell division.6,7 It is obvious that any factor
disrupting the dynamic assembly or/and GTP hydrolysis of FtsZ could eventually
cause the failure of bacterial cell division and then bacterial cell death. As FtsZ taking
such an essential role in cell division and being an unexploited drug target, it has
become a majority interest in the exploration in recent years.8
Over the past decade, efforts have been continuously devoted to the development
of FtsZ inhibitors and many of them are exhibiting potency of disrupting FtsZ
function.5,8,9 Zantrins (Fig. 1A) and N-methylbenzofuro quinoline derivative were
used to study FtsZ polymerization dynamics and proliferation inhibition effects of
bacterial strains.10,11 A quinoline derivative (Fig. 1B) was also found to inhibit the
mycobacterial FtsZ by disrupting the polymerization of MtbFtsZ.12 Recently, a
quinolinium derivative (Fig. 1C) was reported for its superior antibacterial activity
and disruption of GTPase activity.13 As the molecular scaffold of this quinolinium
derivative has been demonstrated as a very useful pharmacophore, the further
introduction of a suitable hydrophobic side chain or an aromatic heterocyclic moiety
into its framework could be helpful in enhancing their interactions with the
hydrophobic pocket of FtsZ.14 On the other hand, nitrogen-containing compounds
have been widely adopted in molecular design for new drug discovery.15 We speculate
that this rational design may result in a significant improvement in the on-target
activity and the antibacterial activity. In the present study, a series of compounds (Fig.
1D: c1-c15) developed from the molecular fusion of quinolinium-indole-amine was
3

studied for the first time to explore their antimicrobial activities and the mode of
action targeting FtsZ protein.
Fig. 1. Structures of zantrin Z3 (A), quinoline derivative (B), quinolinium derivative (C), and
indolyl quinolinium derivatives (D).
2. Results and discussion
2.1. Synthesis of quinolinium-indole-amine fused derivatives
The target compounds c1 – c15 were prepared in good yields by the integration
of 1-methylquinolinium and an indolyl-vinyl fragment through a straightforward
two-step synthesis process16 (Scheme 1). The intermediates (1-methylquinolinium
iodide or chloro-methylquinolinium iodide) were obtained by the methylation reaction
of 2-methylquinoline, and 4-chloro-2-methylquinoline with iodomethane in the
presence of sulfolane. Further condensation reaction of the intermediate with suitable
indolyl carbaldehyde and different amides in one-pot can attain the target compounds.
All the compounds reported herein were characterized by 1H NMR, 13C NMR and
HRMS. Among these compounds, c1, c5 and c7 – c8 were reported as bio-molecular
sensing probe by our group.16-18
4

Scheme 1. Synthetic routes of indolyl quinolinium derivatives (c1–c15). Reagents and
conditions: (i) iodomethane, sulfolane, 60 oC for 8 h; (ii)
1H-indole-2-carbaldehyde/1H-indole-3-carbaldehyde, amines, anhydrous ethanol 70 oC for 6
h.
2.2. Antibacterial susceptibility assays
The minimum inhibitory concentrations (MICs) of the quinolinium derivatives
were tested against Gram-positive (Table 1) and Gram-negative (Table 2) bacteria by
broth microdilution procedures described in the Clinical and Laboratory Standards
Institute (CLSI) guidelines.19 The determined MICs values were compared with
penicillin, ciprofloxacin, and vancomycin under the same assay conditions for
antimicrobial susceptibility assessment. The results showed that the inhibition effect
of these compounds on gram-positive bacteria was generally better than that of
gram-negative bacteria, indicating that these compounds had good potential to
develop further into effective antibacterial agents against gram-positive bacteria. In
addition, for certain potent compounds, they can inhibit the growth of the tested
gram-positive strains, including drug-resistant strains, with MIC values lower than 10
μg/mL (Table 1). It is interesting to observe that these most active compounds (c2, c9)
possess a common piperidine group at the 4-position of the 1-methylquinolinium core,
indicating that the integration of these substituent is the key contribution to excellent
potency of antibacterial activity. The results show that the compounds containing
5

amino substituents including piperidine (c2/c9), 4-methylpiperidine (c3/c10),
pyrrolidine (c4/c11) at the 4-position of the 1-methylquinolinium core, have a better
antibacterial activity than that have 2-diethylaminoethylamine (c6/c13),
2-piperidinoethylamine (c7), 2-pyrrolidinoethylamine (c14), 2-morpholinoethylamine
(c15). Among all these compounds, c2 and c9 are showing the best inhibition ability
against S. aureus ATCC 29213 with the MIC values of 1 μg/mL, which are
comparable or even superior to the three antibiotic references. From Table 2, some of
the compounds also showed moderate to good inhibitory activity against
Gram-negative bacteria. It seems that compounds c2/c9 and c3/c10 possess higher
potency against E. coli ATCC 25922 and ATCC BAA-2469 than other derivatives
probably due to the common-shared piperidine or 4-methylpiperidine substituent
group at the 4-position of the 1-methylquinolinium core. Interestingly, this
observation in inhibition activity is similar to that found in the treatment with
Gram-positive bacteria shown in Table 1. Therefore, the inclusion of piperidine and
indole scaffolds could be somehow important for the consideration in new molecular
drug design against Gram-positive bacteria targeting at the inhibition of FtsZ protein
activity.
Table 1 Minimum inhibitory concentrations (MICs) of compounds against Gram-positive
bacteria strains (μg/mL).
Bacterial strains
S. aureus S. aureus S. aureus E. faecium E. faecium
S. aureus
ATCC
E. faecalis
ATCC
B. subtilis
ATCC
ATCC
ATCC
ATCC
ATCC
49624
29213b BAA-41c 43300c 700221d
168
25923
29212
Pena
Cipa
Vana
c1
32
2
<0.125
>64
2
>64
>64
2
>64
2
>64
>64
>64
64
4
0.25
32
0.5
32
2
0.5
4
<0.125
0.25
32
4
2
32
1
2
2
2
32
1
32
2
64
2
32
4
c2
8
4
4
8
8
8
4
8
c3
8
4
2
8
8
16
32
64
32
8
16
64
64
64
c4
8
16
16
16
16
16
16
16
32
32
16
64
32
16
32
16
c5
16
16
c6
c7
6

32
4
32
2
32
1
32
2
32
4
64
4
32
2
32
4
c8
c9
4
2
4
4
4
4
2
4
c10
c11
c12
c13
c14
c15
8
4
2
4
16
32
32
64
64
8
4
16
16
64
64
64
16
16
32
64
16
16
16
64
16
16
16
32
32
32
64
64
32
64
64
64
16
32
32
32
a. Pen: penicillin; Cip: ciprofloxacin; Van: vancomycin; b penicillin-resistant strain; c
methicillin-resistant strain; d vancomycin-resistant strain; Other strains are drug susceptible
strains.
Table 2 Minimum inhibitory concentrations (MICs) of compounds against Gram-negative
bacteria strains (μg/mL).
Bacterial strains
A.
E. coli
E. coli
K. pneumoniae P. aeruginosa
E. cloacae
ATCC
BAA-1143d
baumannii
ATCC ATCC
25922 BAA-2469b
ATCC
ATCC
ATCC
BAA-2470b
BAA-2108c
19606c
64
Pena
Cipa
Vana
c1
1
<0.125
>64
64
>64
>64
>64
64
>64
64
>64
64
>64
0.5
2
64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
32
16
16
c2
32
32
>64
64
c3
32
32
c4
64
64
>64
>64
64
c5
64
64
c6
32
64
c7
7

>64
8
>64
16
>64
16
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
c8
c9
16
32
64
64
64
>64
32
32
c10
c11
c12
c13
c14
c15
32
64
>64
64
>64
>64
>64
>64
64
>64
a. Pen: penicillin; Cip: ciprofloxacin; Van: vancomycin; b expressing NDM-1 beta-lactamase; c
multidrug-resistant strain; d expressing AmpC beta-lactamase; Other strains are drug
susceptible strains.
2.3. Bactericidal or bacteriostatic assay
Based on the MICs results, we further assessed the minimum bactericidal
concentrations (MBCs) of the selected compounds against methicillin susceptible S.
aureus ATCC 29213 and MRSA ATCC BAA-41. Vancomycin was used as a control
for the tests. According to the CLSI standards, an MBC/MIC ratio of 1-2 is
considered as indicative of bactericidal behavior. By contrast, for the MBC/MIC ratio
≧ 8 is considered indicative of bacteriostatic behavior.20 The results listed in Table 3
showed that vancomycin was observed as bactericidal agents with the MBC/MIC
ratios of 1 against two S. aureus strains (The results of time-killing kinetics assay can
be referred to ESI Fig. S47). For our compounds, c2 and c9 also exhibited a
bactericidal mode against both methicillin susceptible and resistant S. aureus with the
MBC/MIC ratio of 2.
Table 3 Comparison of MIC and MBC Values for c2 and c9 against two S. aureus strains.
Comp
MBC(μg/mL)
MIC(μg/mL)
MBC/MIC
S. aureus ATCC 29213
2
2
2
1
1
2
2
2
1
c2
c9
vancomycin
S. aureus ATCC BAA-41
8

4
4
2
2
2
2
2
2
1
c2
c9
vancomycin
2.4. Time-killing kinetics determinations
In order to further study the antibacterial activity of compounds, time-kill
kinetics assays monitored by the viable counts method were used to confirm the
bactericidal property of c2 against S. aureus ATCC BAA41. As shown in Fig. 2A, the
bacterial cells grew rapidly in the first 8 h without c2. After 8 h incubation, the growth
of S. aureus went into the stationary phase. On the other hand, 2 × MIC and 4 × MIC
of c2 can rapidly reduce the viable counts (103 CFU mL−1) of S. aureus after
incubation for 4 h. The growth inhibition of different concentration gradient revealed
that the rate at which c2 killed S. aureus was dependent on the compound
concentration. The higher the concentration of the compound applied, the greater the
rates of killing resulted. Moreover, c2 at the 1 ×, 2 ×, 4 × MIC concentrations caused
a similar reduction of nearly 1 × 103 CFU mL−1 for S. aureus in 24 h. The results
indicated that c2 inhibited the growth of S. aureus in a bactericidal manner.
2.5. Drug resistance study
To further evaluate the drug resistance of these compounds, we selected two
commercial antibiotics (Rifampin and Norfloxacin) as the positive control against S.
aureus ATCC29213. As shown in Fig. 2B, comparing with the positive control, the
MIC values for c2 and c9 increased only 4-fold, while for rifampin and norfloxacin
increased over 2000 and 500-fold of MIC values after 15 passages. The results
showed that c2 and c9 exhibit potent antibacterial activity and may delay the
development of drug resistance.
9

Fig. 2. (A) Time-killing Kinetics determinations for c2. At time zero, samples of a growing
culture of S. aureus ATCC BAA41 were incubated with concentrations of c2 equivalent to 1×
(red), 2× (blue), or 4× (pink) the MIC. Vehicle (1% DMSO, black) was included. Samples
were removed at the time intervals indicated for the determination of viable cell counts. (B)
Bacterial resistance studies of rifampin, norfloxacin, c2 and c9 against S. aureus ATCC
29213.
2.6. Morphology and membrane integrity of B. subtilis cells
One characteristic of bacterial cell division inhibition observation is the cell
elongation of an enlarged phenotype, which caused by disrupting the cell division
function of FtsZ protein. Apart from this, the perturbation of membrane integrity may
cause bacterial cell lysis and death.21 For these purpose, we investigated the bacterial
cell morphology and membrane integrity by using a red fluorescent dye FM4-64 in the
absence and in the presence of MIC concentration of the screened compounds from
the antimicrobial susceptibility assessment. The observations were recorded by using
a super high-resolution laser confocal microscopy. As shown in Fig. 3, c2 remarkably
increased the cell length of B. subtilis 168 from the range of 5-13 μm to 70-130 μm.
Other compounds, c9 and c10 exhibited moderate effect on B. subtilis 168 (Fig. S1).
Moreover, c2 did not show any detectable impairment of cell membrane integrity
(Fig. 4B) compared with the untreated bacterial cells (Fig. 4A). The results strongly
indicated that c2 inhibits the bacterial proliferation by disrupting the cell division
function of FtsZ protein without perturbing the membrane integrity.
10

Fig. 3. The morphology of B. subtilis. Cells of B. subtilis 168 were grown in the absence (A)
and presence of MIC concentration of c2 (B). Scale bar = 20 μm.
Fig. 4. The membrane integrity of B. subtilis. Cells of B. subtilis 168 were stained by red
fluorescent dye FM4-64 in the absence (A) and presence of MIC concentration of c2 (B).
Scale bar = 20 μm.
2.7. Effects of indolyl quinolinium derivatives on the GTPase activity of FtsZ
A recent report showed that the dynamic polymerization of cell division protein
FtsZ is dependent on the rate of GTP hydrolysis.22 The possible mechanism of
antibacterial activity inhibition was studied with two selected compounds (c2 and c9),
which possess better antibacterial activity. The effects of indolyl quinolinium
derivatives on the GTPase activity were carried out. The interferential effects on the
GTPase activity of FtsZ were shown in Fig. 5A and Table S1. The results show that
11

both c2 and c9 inhibited GTP hydrolysis of FtsZ in a concentration-dependent
manner. The GTPase activity of negative control (no compounds) is 19.8 units/L (ESI
Table S1). The GTPase inhibitory effect of c2 was found approximately 50% at the
concentration of 32 μg/mL.
2.8. Light scattering assay
In the attempt to further investigate the mechanism of antibacterial activities with
indolyl quinolinium derivatives, we used a light scattering assay to assess the effect of
c2 on the dynamic polymerization of FtsZ. Fig. 5B shows a time-dependent manner
polymerization profiles of FtsZ in the absence and in the presence of c2 at the
concentration ranged from 0.4 to 1.2 μg/mL. From the results, the compound
exhibited an obvious enhancement effect on FtsZ polymerization at the concentration
of 0.4 μg/mL compared with a negative control using DMSO conducted under the
same conditions in the assay. The results indicate that the compound shows strong
efficacy in the disruption of the SaFtsZ polymerization with a dose-dependent
manner.
Fig. 5. (A) Inhibition of GTPase activity of FtsZ by compounds c2 and c9. (B) Effect of
indolyl quinolinium derivative (c2) on the polymerization of FtsZ.
2.9. Antibacterial activity of c2 and c9 FtsZ over-expression strain
To further confirm the in vivo on-target effect of the indolyl quinolinium
derivatives, c2 and c9 were selected to test their antibacterial activity against a FtsZ
over-expression strain (E. coli BL21). The over expression of FtsZ can be induced by
IPTG. The MIC values of c2 and c9 were 8 μg/mL against this strain under
non-over-expression condition. It is interesting to note that the MIC values of c2 and
12

c9 increased to 32 and 16 μg/mL respectively, when FtsZ was over expressed (Table
4). The decline of antibacterial activity suggests that the over-expression FtsZ calls for
more compounds to inhibit the function of FtsZ. The results suggest that c2 and c9
may interact with FtsZ in vivo.
Table 4 MIC of c2 and c9 against E. coli BL21 strain (μg/mL).
Compounds
E. coli BL21
E. coli BL21+IPTG
8
8
32
16
c2
c9
2.10. Predicted binding mode of c2 in FtsZ
From the above biological assays, c2 is ascertained as a potent FtsZ-targeting
inhibitor. Molecular modeling study was used to further identify the potential binding
site of the FtsZ protein with the compound. The optimal docking pose suggest that c2
probably binds to a hydrophobic and narrow cleft constituted by the H7-helix,
T7-loop and a four-stranded β-sheet (Fig. 6A). A 2D ligand interaction diagram (Fig.
6B) shows the predicted interactions between c2 and FtsZ residues. The dominant
binding of c2 with Gln192, Gly196, Leu200, Val203, Leu209, Met226, Gly227, Ile
228, and Val297 of FtsZ protein are through hydrophobic interactions. In addition, an
electrostatic interaction can be found between the positively charged amine and
Asp199. Two carbon hydrogen bonds were found between the piperidine group of c2
and Leu200/Val203. Moreover, a lot of amino acids in this pocket (such as Gly205,
Val310, Ile311, etc.) can form van der waals force with c2.
13

Fig. 6. (A) 1,2,4-trimethylquinolin-1-ium iodide derivatives (c2) was predicted to bind into
the C-terminal interdomain cleft of FtsZ; (B) predicted interactions between c2 and the amino
acids of FtsZ.
3. Conclusions
In conclusion, we demonstrated a series of molecules fused with
quinolinium-indole-amine in the FtsZ-targeted antibacterial applications for the first
time. The class of compounds exhibit significant antibacterial activity against several
clinically representative strains including some drug resistant bacterial strains. Some
of the compounds displayed superior or comparable antibacterial activity to penicillin,
ciprofloxacin, and vancomycin. Two compounds (c2 and c9) were identified with
strong antibacterial activity against MRSA with MIC values from 1 to 4 µg/ml
respectively. Based on the morphology of B. subtilis cell, c2 can remarkably increase
the cell length of B. subtilis 168 by inhibiting bacterial cell division without
perturbing the membrane integrity. The results of biochemical assays revealed that c2
can effectively disrupt the GTPase activity and polymerization of FtsZ in a
dose-dependent manner and it does not trigger the development of bacterial
resistance. Based on these studies, these indolyl-quinolinium derivatives may be
worth for further structural modification and conducting in-depth antibacterial
research.
4. Materials and methods
4.1. Chemistry
All reagents and chemicals were purchased from commercial unless otherwise
specified. All the solvents were of analytical reagent grade and used without further
purification. Melting point measurement was carried out using a SRS Opti Mel
14

automated melting point instrument without correction. High resolution mass spectra
(HRMS) were recorded with Shi-madzu LCMS-IT-TOF. 1H and 13C NMR spectra
were recorded in DMSO using TMS as the reference with a Bruker Bio Spin Gmb
Hspectrometer at 400 MHz and 100 MHz, respectively. Reactions progress was
monitored by thin-layer chromatography (TLC) on 0.25-mm pre-coated silica GF254
plates.
4.2. General synthetic procedure for the intermediates b1-b2
Intermediates b1–b2 were prepared according to similar previously reported
protocol.23 To the solution of 2-methylquinoline and 4-chloro-2-methylquinoline (0.2
g) in sulfolane (10 mL) were added iodomethane (0.42 mL, 6.74 mmol). The reaction
mixture was stirred at 50 °C for 18 h, cooled and anhydrous ether is added after the
shock, suction filtration, the solid was washed with anhydrous diethyl ether, and dried
in vacuum to give of intermediates b1-b2 in >80% yield.
4.3. General synthetic procedure for indolyl quinolinium derivatives (c1 - c15).
A solution of intermediates b (0.5 mmol), 1H-indole-2-carbaldehyde or
1H-indole-3-carbaldehyde, amides (3.6 mmol) in a closed pressure reactor with 10
mL anhydrous ethanol were heated to 70 oC for reaction for 6 h. When the reaction
mixture was cooled to room temperature, the mixture was poured into a beaker with
50 mL ether. Then the solution was filtered to remove solid and the filtrate was
extracted with ether for three times (3 × 10 mL). The ether layers were combined and
then were removed under reduced pressure. The obtained solid which is target
compounds was dried in vacuum.
4.3.1. (E)-2-(2-(1H-indol-2-yl)vinyl)-1-methylquinolin-1-ium iodide (c1)
Purple solid, yield 76%; Melting point: 256–258 °C; 1H NMR (400 MHz,
DMSO-d6) δ 11.94 (s, 1H), 9.29 (d, J = 6.5 Hz, 1H), 8.90 (d, J = 8.4 Hz, 1H), 8.49 (d,
J = 6.5 Hz, 1H), 8.45 (d, J = 8.9 Hz, 1H), 8.35 – 8.22 (m, 3H), 8.12 (t, J = 7.7 Hz, 1H),
7.66 (d, J = 7.9 Hz, 1H), 7.50 (d, J = 8.2 Hz, 1H), 7.29 (t, J = 7.6 Hz, 1H), 7.08 (dd, J
= 14.8, 7.1 Hz, 2H), 4.52 (s, 3H). 13C NMR (100 MHz, DMSO) δ 154.0 (s), 147.0 (s),
139.2 (s), 139.0 (s), 138.0 – 138.1 (m), 135.0 (s), 133.5 (s), 129.0 (s), 126.6 (s), 125.9
(s), 125.8 (s), 123.6 (s), 122.0 (s), 120.8 (s), 119.4 (s), 115.1 (s), 113.8 (s), 113.2 (s),
113.1 (s), 44.4 (s). HRMS m/z: calcd. for C20H17N2 [M − I] + 285.1386, found
285.1378.
4.3.2. (E)-2-(2-(1H-indol-2-yl)vinyl)-1-methyl-4-(piperidin-1-yl)quinolin-1-ium iodide
(c2)
15

Brown solid, yield 50%; Melting point: 178 - 186°C; 1H NMR (400 MHz,
DMSO) δ 11.82 (s, 1H), 8.27 (d, J = 8.8 Hz, 1H), 8.11 (d, J = 8.3 Hz, 1H), 8.06 – 7.98
(m, 2H), 7.74 (t, J = 7.59 Hz, 1H), 7.63 (dd, J = 11.8, 9.1 Hz, 2H), 7.49 – 7.35 (m,
2H), 7.25 (t, J = 7.6 Hz, 1H), 7.06 (t, J = 7.5 Hz, 1H), 7.01 (s, 1H), 4.23 (s, 3H), 3.76
(s, 4H), 1.84 (s, 4H), 1.78 (s, 2H). 13C NMR (100 MHz, DMSO) δ 160.0 (s), 153.6
(s), 141.4 (s), 138.9 (s), 136.0 (s), 134.1 (s), 133.5 (s), 128.4 (s), 127.0 (s), 126.4 (s),
124.9 (s), 121.8 (s), 120.5 (s), 120.2 (s), 119.6 (s), 117.6 (s), 112.0 (s), 109.8 (s),
104.4 (s), 56.5 (s), 53.3 (s), 40.6 (s), 40.4 (s), 40.2 (s), 40.0 (s), 39.8 (s), 39.6 (s), 39.4
(s), 38.4 (s), 25.9 (s), 24.0 (s), 19.0 (s). HRMS m/z: calcd. for C25H26N3 [M - I]+
368.2118, found 368.2114.
4.3.3.
(E)-2-(2-(1H-indol-2-yl)vinyl)-1-methyl-4-(4-methylpiperidin-1-yl)quinolin-1-ium
iodide (c3)
Brown solid, yield 72%; Melting point: 266–269 °C; 1H NMR (400 MHz,
DMSO) δ 11.99 (s, 1H), 8.24 (t, J = 12.9 Hz, 2H), 8.16 (d, J = 6.2 Hz, 2H), 8.09 (d, J
= 8.3 Hz, 1H), 7.99 (t, J = 7.9 Hz, 1H), 7.71 (t, J = 7.6 Hz, 1H), 7.57 – 7.51 (m, 1H),
7.49 (s, 1H), 7.40 (d, J = 15.7 Hz, 1H), 7.36 – 7.21 (m, 2H), 4.23 (s, 3H), 4.08 (d, J =
12.8 Hz, 2H), 1.93 (dd, J = 39.1, 10.6 Hz, 2H), 1.83 – 1.69 (m, 1H), 1.50 (dd, J =
22.3, 10.4 Hz, 2H), 1.04 (d, J = 6.4 Hz, 3H). 13C NMR (100 MHz, DMSO) δ 159.8
(s), 153.6 (s), 141.4 (s), 138.9 (s), 135.6 (s), 133.8 (d, J = 56.9 Hz), 133.8 (d, J = 56.9
Hz), 128.4 (s), 127.1 (s), 126.4 (s), 124.9 (s), 121.8 (s), 120.3 (d, J = 27.3 Hz), 119.6
(s), 118.2 – 117.8 (m), 112.0 (s), 109.8 (s), 104.4 (s), 60.8 (s), 52.6 (s), 35.2 (s), 34.1
(s), 19.1 (s). HRMS m/z: calcd. for C26H28N3 [M − I] + 382.2278, found 382.2275.
4.3.4. (E)-2-(2-(1H-indol-2-yl)vinyl)-1-methyl-4-(pyrrolidin-1-yl)quinolin-1-ium
iodide (c4)
Yellow solid, yield 43%; Melting point: 206 - 213°C; 1H NMR (400 MHz,
DMSO) δ 11.77 (s, 1H), 8.51 (d, J = 8.39 Hz, 1H), 8.16 (d, J = 8.8 Hz, 1H), 8.00 (t, J
= 7.8 Hz, 1H), 7.88 (d, J = 15.8 Hz, 1H), 7.67 (t, J = 7.7 Hz, 1H), 7.61 (d, J = 8.0 Hz,
1H), 7.55 (d, J = 15.8 Hz, 1H), 7.44 (d, J = 8.19 Hz, 1H), 7.22 (t, J = 7.39 Hz, 1H),
7.05 (t, J = 7.2 Hz, 2H), 6.89 (s, 1H), 4.12 (s, 3H), 4.02 (s, 4H), 2.06 (s, 4H). 13C
NMR (100 MHz, DMSO) δ 155.4 (s), 154.7 (s), 152.1 (s), 151.8 (s), 141.3 (s), 138.6
(s), 137.4 (s), 135.6 (s), 133.8 (s), 133.7 (s), 132.9 (s), 132.0 (s), 128.4 (s), 128.2 (s),
127.9 (s), 127.6 (s), 125.5 (s), 125.3 (s), 124.6 (s), 123.8 (s), 121.7 (s), 121.2 (s),
120.6 (s), 120.4 (s), 120.2 (s), 119.0 (s), 118.7 (s), 118.6 (s), 118.2 (s), 111.9 (s),
108.9 (s), 106.5 (s), 103.2 (s), 100.0 (s), 56.5 (s), 53.9 (s), 38.1 (s), 25.7 (s), 19.0 (s).
HRMS m/z: calcd. for C24H24N3 [M - I]+ 354.1965, found 354.1958.
4.3.5. (E)-2-(2-(1H-indol-2-yl)vinyl)-1-methyl-4-morpholinoquinolin-1-iumiodide (c5)
16

Brown solid, yield 57%; Melting point: 248 - 256°C; 1H NMR (400 MHz,
DMSO) δ 11.79 (s, 1H), 8.31 (d, J = 8.9 Hz, 1H), 8.19 (d, J = 7.9 Hz, 1H), 8.06 (dd, J
= 14.4, 10.9 Hz, 2H), 7.75 (t, J = 7.59 Hz, 1H), 7.66 (d, J = 3.3 Hz, 1H), 7.63 (d, J =
4.3 Hz, 1H), 7.57 (s, 1H), 7.47 (d, J = 8.19 Hz, 1H), 7.31 (t, J = 7.3 Hz, 1H), 7.07 (t, J
= 7.4 Hz, 1H), 7.11 (s, 1H), 4.27 (s, 3H), 3.91 (d, J = 4.4 Hz, 4H), 3.80 (d, J = 4.1 Hz,
4H). 13C NMR (100 MHz, DMSO) δ 159.8 (s), 154.1 (s), 153.8 (s), 141.3 (s), 138.9
(s), 135.5 (s), 134.3 (s), 134.0 (s), 128.4 (s), 127.0 (s), 126.8 (s), 125.1 (s), 121.9 (s),
120.6 (s), 120.2 (s), 119.7 (s), 117.4 (s), 112.0 (s), 110.2 (s), 104.9 (s), 66.3 (s), 52.5
(s), 38.5 (s). HRMS m/z: calcd. for C24H24N3O [M - I]+ 371.1928, found 371.1924.
4.3.6.
(E)-2-(2-(1H-indol-2-yl)vinyl)-4-((2-(diethylamino)ethyl)amino)-1-methylquinolin-1-i
um iodide (c6)
Brown solid, yield 49%; Melting point: 213 - 219°C; 1H NMR (400 MHz,
DMSO) δ 11.79 (s, 1H), 8.48 (d, J = 8.3 Hz, 1H), 8.19 (d, J = 9.0 Hz, 1H), 8.03 (t, J =
7.6 Hz, 1H), 7.88 (d, J = 15.8 Hz, 1H), 7.76 (t, J = 7.6 Hz, 1H), 7.61 (dd, J = 16.1,
11.9 Hz, 2H), 7.46 (d, J = 8.19 Hz, 1H), 7.19 (t, J = 7.6 Hz, 1H), 7.08 (s, 1H), 7.06 (t,
J = 7.5 Hz, 1H), 6.93 (s, 1H), 4.15 (s, 3H), 3.74 (t, J = 6.3 Hz, 2H), 2.78 (t, J = 6.3 Hz,
2H), 2.58 (dd, J = 14.1, 7.0 Hz, 4H), 0.96 (t, J = 7.0 Hz, 6H). 13C NMR (100 MHz,
DMSO) δ 154.2 (s), 154.0 (s), 140.0 (s), 138.7 (s), 135.5 (s), 134.2 (s), 132.5 (s),
128.4 (s), 126.7 (s), 124.7 (s), 123.6 (s), 121.7 (s), 120.4 (s), 119.4 (s), 118.3 (s),
117.8 (s), 111.9 (s), 109.1 (s), 97.0 (s), 51.4 (s), 47.1 (s), 46.9 (s), 42.1 (s), 37.8 (s),
12.3 (s). HRMS m/z: calcd. for C26H31N4 [M - I]+ 399.2543, found 399.2539.
4.3.7.
(E)-2-(2-(1H-indol-2-yl)vinyl)-1-methyl-4-((2-(piperidin-1-yl)ethyl)amino)quinolin-1-
ium iodide (c7)
Dark brown solid, yield 65%; Melting point: 190 - 195°C; 1H NMR (400 MHz,
DMSO) δ 11.82 (s, 1H), 8.27 (d, J = 8.8 Hz, 1H), 8.11 (d, J = 8.3 Hz, 1H), 8.06 – 7.98
(m, 2H), 7.74 (t, J = 7.59 Hz, 1H), 7.63 (dd, J = 11.8, 9.1 Hz, 2H), 7.48 – 7.39 (m,
2H), 7.19 (t, J = 7.6 Hz, 1H), 7.06 (t, J = 7.5 Hz, 1H), 6.98 (s, 1H), 4.23 (s, 3H), 3.76
(s, 4H), 1.84 (s, 4H), 1.78 (s, 2H). 13C NMR (100 MHz, DMSO) δ 160.0 (s), 153.6
(s), 141.4 (s), 138.9 (s), 135.6 (s), 134.1 (s), 133.5 (s), 129.3 (s), 128.4 (s), 127.0 (s),
126.4 (s), 124.9 (s), 121.8 (s), 120.5 (s), 120.2 (s), 119.6 (s), 117.6 (s), 112.0 (s),
109.8 (s), 104.4 (s), 100.1 (s), 56.5 (s), 53.3 (s), 38.4 (s), 25.9 (s), 24.0 (s), 19.0 (s).
HRMS m/z: calcd. for C27H30N4 [M - I]+ 411.2528, found 411.2532.
4.3.8. (E)-2-(2-(1H-indol-3-yl)vinyl)-1-methylquinolin-1-ium iodide (c8)
Purple solid, yield 78%; Melting point: 253–254 °C; 1H NMR (400 MHz,
DMSO-d6) δ 12.31 (s, 1H), 8.81 (d, J = 9.1 Hz, 1H), 8.64 (d, J = 15.5 Hz, 1H), 8.60
17

(d, J = 9.2 Hz, 1H), 8.44 (d, J = 8.9 Hz, 1H), 8.40 (d, J = 2.8 Hz, 1H), 8.31 – 8.17 (m,
2H), 8.08 (t, J = 7.7 Hz, 1H), 7.84 (t, J = 7.5 Hz, 1H), 7.64 – 7.48 (m, 2H), 7.31 (dd, J
= 5.9, 3.0 Hz, 2H), 4.46 (s, 3H). 13C NMR (100 MHz, DMSO) δ 156.1 (s), 156.1 (s),
143.7 (s), 139.6 (d, J = 16.5 Hz), 137.3 (s), 135.5 (s), 135.2 (s), 130.5 (s), 129.2 (s),
128.5 (s), 128.0 (s), 126.0 (s), 122.3 (s), 120.8 (d, J = 5.4 Hz), 119.6 (s), 116.6 (s),
112.6 (s), 112.2 (s). HRMS m/z: calcd. for C20H17N2 [M − I] + 285.1386, found
285.1383.
4.3.9. (E)-2-(2-(1H-indol-3-yl)vinyl)-1-methyl-4-(piperidin-1-yl)quinolin-1-ium iodide
(c9)
Yellow solid, yield 56%; Melting point: 175 - 185°C; 1H NMR (400 MHz,
DMSO) δ 11.99 (s, 1H), 8.24 (dd, J = 16.4, 12.3 Hz, 2H), 8.16 (dd, J = 6.6, 2.6 Hz,
2H), 8.09 (d, J = 8.2 Hz, 1H), 8.00 (t, J = 7.9 Hz, 1H), 7.69 (t, J = 7.6 Hz, 1H), 7.52
(dd, J = 6.3, 2.4 Hz, 1H), 7.50 (s, 1H), 7.41 (d, J = 15.7 Hz, 1H), 7.31 – 7.23 (m, 2H),
4.23 (s, 3H), 3.69 (s, 3H), 1.85 (s, 3H). 13C NMR (100 MHz, DMSO) δ 141.3 (s),
141.2 (s), 137.7 (s), 136.6 (s), 136.6 (s), 133.7 (s), 133.5 (s), 131.7 (s), 131.4 (s),
128.7 (s), 127.7 (s), 127.5 (s), 125.8 (s), 125.5 (s), 125.4 (s), 125.1 (s), 123.2 (s),
122.5 (s), 121.4 (s), 120.6 (s), 120.4 (s), 119.7 (s), 118.7 (s), 118.6 (s), 116.4 (s),
114.1 (s), 113.7 (s), 112.9 (s), 112.6 (s), 110.8 (s), 103.2 (s), 99.4 (s), 53.8 (s), 53.4
(s), 25.7 (s), 25.4 (s). HRMS m/z: calcd. for C25H26N3 [M - I]+ 368.2119, found
368.2124.
4.3.10.
(E)-2-(2-(1H-indol-3-yl)vinyl)-1-methyl-4-(4-methylpiperidin-1-yl)quinolin-1-ium
iodide (c10)
Brown solid, yield 78%; Melting point: 266–269 °C; 1H NMR (400 MHz,
DMSO) δ 12.12 (s, 1H), 8.28 – 8.18 (m, 2H), 8.16 (d, J = 2.5 Hz, 2H), 8.07 (d, J = 8.2
Hz, 1H), 7.98 (t, J = 7.8 Hz, 1H), 7.71 (t, J = 7.6 Hz, 1H), 7.57 – 7.50 (m, 1H), 7.48
(s, 1H), 7.39 (d, J = 15.7 Hz, 1H), 7.26 (p, J = 7.7 Hz, 2H), 4.22 (s, 3H), 4.08 (d, J =
12.8 Hz, 2H), 1.87 (d, J = 12.6 Hz, 2H), 1.79 (d, J = 6.1 Hz, 1H), 1.49 (dd, J = 22.0,
10.7 Hz, 2H), 1.03 (d, J = 6.3 Hz, 3H). 13C NMR (100 MHz, DMSO) δ 159.6 (s),
155.5 (s), 141.3 (s), 138.8 (s), 137.8 (s), 133.8 (s), 132.8 (s), 126.9 (s), 126.3 (s),
125.5 (s), 123.4 (s), 121.7 (s), 120.8 (s), 120.4 (s), 119.5 (s), 114.0 – 113.8 (m), 113.2
(s), 113.1 (s), 104.4 (s), 52.5 (s), 38.3 (s), 34.0 (s), 30.5 (s), 22.0 (s). HRMS m/z:
calcd. for C26H28N3 [M − I] +382.2278, found 382.2276.
4.3.11. (E)-2-(2-(1H-indol-3-yl)vinyl)-1-methyl-4-(pyrrolidin-1-yl)quinolin-1-ium
iodide (c11)
Brown solid, yield 54%; Melting point: 205 -215°C; 1H NMR (400 MHz,
DMSO) δ 12.05 (s, 1H), 8.50 (s, 1H), 8.17 – 8.10 (m, 2H), 8.01 (dt, J = 15.8, 5.3 Hz,
18

3H), 7.65 (t, J = 7.7 Hz, 1H), 7.51 (d, J = 7.7 Hz, 1H), 7.47 (d, J = 15.7 Hz, 1H),
7.28-7.19 (m, 2H), 7.00 (s, 1H), 4.11 (s, 3H), 4.01 (s, 4H), 2.06 (s, 4H). 13C NMR
(100 MHz, DMSO) δ 159.8 (s), 155.6 (s), 141.3 (s), 138.8 (s), 137.8 (s), 133.8 (s),
132.8 (s), 126.8 (s), 126.3 (s), 125.5 (s), 123.4 (s), 121.7 (s), 120.7 (s), 120.4 (s),
119.5 (s), 114.1 (s), 113.3 (s), 113.1 (s), 104.4 (s), 53.2 (s), 38.3 (s), 25.9 (s), 24.0 (s).
HRMS m/z: calcd. for C24H24N3 [M - I]+ 354.1949, found 354.1955.
4.3.12. (E)-2-(2-(1H-indol-3-yl)vinyl)-1-methyl-4-morpholinoquinolin-1-ium iodide
(c12)
Yellow solid, yield 53%; Melting point: 248 - 255°C; 1H NMR (400 MHz,
DMSO) δ 12.03 (s, 1H), 8.30 (dd, J = 12.3, 3.2 Hz, 2H), 8.18 (t, J = 5.2 Hz, 3H), 7.99
(t, J = 7.5 Hz, 1H), 7.73 (t, J = 7.6 Hz, 1H), 7.58 – 7.51 (m, 2H), 7.43 (d, J = 15.7 Hz,
1H), 7.32 – 7.24 (m, 2H), 4.27 (s, 3H), 3.97 – 3.89 (m, 4H), 3.72 (d, J = 4.2 Hz, 4H).
13C NMR (100 MHz, DMSO) δ 159.8 (s), 155.6 (s), 141.3 (s), 138.8 (s), 137.8 (s),
133.8 (s), 132.8 (s), 126.8 (s), 126.3 (s), 125.5 (s), 123.4 (s), 121.7 (s), 120.7 (s),
120.4 (s), 119.5 (s), 114.1 (s), 113.3 (s), 113.1 (s), 104.4 (s), 53.2 (s), 40.6 (s), 40.4
(s), 40.2 (s), 40.0 (s), 39.8 (s), 39.6 (s), 39.4 (s), 38.3 (s), 25.9 (s), 24.1 (s). HRMS
m/z: calcd. for C24H24N3O[M - I]+ 371.1928, found 371.1932.
4.3.13.
(E)-2-(2-(1H-indol-3-yl)vinyl)-4-((2-(diethylamino)ethyl)amino)-1-methylquinolin-1-i
um iodide (c13)
Yellow-brown solid, yield 49%; Melting point: 213 - 217°C; 1H NMR (400
MHz, DMSO) δ 11.90 (s, 1H), 8.45 (d, J = 8.3 Hz, 1H), 8.18 (d, J = 8.8 Hz, 1H), 8.11
(d, J = 8.19Hz, 1H), 8.06 (d, J = 16.2 Hz, 2H), 8.02 – 7.97 (m, 1H), 7.69 (t, J = 7.59
Hz, 1H), 7.59 (d, J = 6.4 Hz, 1H), 7.38 (d, J = 15.8 Hz, 1H), 7.29 – 7.22 (m, 2H), 7.16
(s, 1H), 4.15 (s, 3H), 3.73 (t, J = 6.4 Hz, 2H), 2.81 (t, J = 6.4 Hz, 2H), 2.61 (q, J = 7.1
Hz, 4H), 1.07 (t, J = 7.1 Hz, 6H). 13C NMR (100 MHz, DMSO) δ 155.7 (s), 153.7 (s),
140.0 (s), 137.8 (s), 137.1 (s), 134.0 (s), 131.7 (s), 126.5 (s), 126.3 (s), 125.5 (s),
123.5 (s), 123.2 (s), 121.4 (s), 120.5 (s), 119.3 (s), 119.0 (s), 117.7 (s), 114.1 (s),
113.8 (s), 113.0 (s), 96.3 (s), 51.4 (s), 47.1 (s), 37.8 (s), 22.8 (s), 12.4 (s). HRMS m/z:
calcd. for C26H31N4 [M - I]+ 399.2539, found 399.2534.
4.3.14.
(E)-2-(2-(1H-indol-3-yl)vinyl)-1-methyl-4-((2-(pyrrolidin-1-yl)ethyl)amino)quinolin-1
-ium iodide (c14)
Yellow brown solid, yield 74%; Melting point: 205 - 210°C; 1H NMR (400
MHz, DMSO) δ 11.90 (s, 1H), 8.51 (d, J = 8.1 Hz, 1H), 8.21 (d, J = 8.8 Hz, 1H), 8.09
(dd, J = 19.0, 11.8 Hz, 3H), 8.02 – 7.97 (m, 1H), 7.73 (t, J = 7.6 Hz, 1H), 7.53 (d, J =
7.1 Hz, 1H), 7.38 (d, J = 15.8 Hz, 1H), 7.29 – 7.19 (m, 2H), 7.14 (s, 1H), 4.15 (s, 3H),
19

3.79 (t, J = 6.3 Hz, 2H), 2.86 (s, 2H), 2.61 (s, 4H), 1.71 (s, 4H). 13C NMR (100 MHz,
DMSO) δ 155.8 (s), 153.7 (s), 140.0 (s), 137.8 (s), 137.3 (s), 134.0 (s), 131.8 (s),
126.3 (s), 125.5 (s), 123.9 (s), 123.2 (s), 121.4 (s), 120.5 (s), 119.2 (s), 117.8 (s),
114.0 (s), 113.8 (s), 113.0 (s), 96.3 (s), 79.8 (s), 79.5 (s), 79.1 (s), 66.7 (s), 56.8 (s),
53.9 (s), 37.8 (s). HRMS m/z: calcd. for C26H29N4 [M - I]+ 397.2381, found 397.2376.
4.3.15.
(E)-2-(2-(1H-indol-3-yl)vinyl)-1-methyl-4-(3-morpholinopropyl)quinolin-1-ium iodide
(c15)
Yellow brown solid, yield 45%; Melting point: 145 - 149°C; 1H NMR (400
MHz, DMSO) δ 11.97 (s, 1H), 8.89 (s, 1H), 8.57 (d, J = 8.03 Hz, 1H), 8.20 (d, J =
8.79 Hz, 1H), 8.20 – 8.02 (m, 3H), 8.01 – 7.85 (m, 1H), 7.72 (t, J = 7.6 Hz, 1H), 7.49
(d, J = 8.2 Hz, 1H), 7.4 (d, J = 15.8 Hz, 1H), 7.25 (t, J = 5.6 Hz, 2H), 7.16 (s, 1H),
4.15 (s, 3H), 3.80 (d, J = 4.8 Hz, 2H), 3.58 (s, 4H), 2.73 (s, 2H), 2.53 (d, J = 8.9 Hz,
4H). 13C NMR (100 MHz, DMSO) δ 155.8 (s), 153.7 (s), 140.0 (s), 137.8 (s), 137.3
(s), 134.0 (s), 131.8 (s), 126.3 (s), 125.5 (s), 123.9 (s), 123.2 (s), 121.4 (s), 120.5 (s),
119.2 (s), 117.8 (s), 114.0 (s), 113.8 (s), 113.0 (s), 96.3 (s), 79.8 (s), 79.5 (s), 79.1 (s),
66.7 (s), 56.8 (s), 53.9 (s), 37.8 (s). HRMS m/z: calcd. for C27H30N3O[M - I]+
413.2329, found 413.2331.
4.4. Antibacterial susceptibility assays
The minimum inhibitory concentrations (MICs) of the test compounds were
determined using the broth micro-dilution procedure described in the Clinical and
Laboratory Standards Institute (CLSI) guidelines.19 Two or three single colonies of
the testing bacterial strain on a TSB agar plate were inoculated in 5 mL of MH or
CA-MH broth and then grown at 37 oC overnight with shaking. The bacterial culture
was transferred to fresh MH or CA-MH broth and diluted to a final concentration of
approximately 5× 105 CFU/mL in a 96-well microtiter plate. Then, a series of
concentrations of the test compounds were added. After 18 h of incubation at 37 oC,
The MIC value is defined as the lowest concentration of test compounds which
prevents visible growth of bacterium.
4.5. Minimum bactericidal concentration (MBC) assay
MBC assay was conducted using the broth microdilution assay described in the
above section. The MBC was determined by plating 10 μL of culture volume from the
MIC assay onto TSB agar plate and colony formation was examined after 24 hours at
20

37°C. MBC is defined as the lowest compound concentration resulting in a ≧ 3-log
reduction in the number of colony forming units (CFU) 24.
4.6. Time-killing kinetics assay
A growing culture of S. aureus ATCC BAA41 was diluted to approximately 106
CFU. mL-1 in volumes of Mueller Hinton broth. The bacterial culture was grown in
the presence of various concentrations (0×, 1×, 2×, 4×MIC) of compounds and
incubated at 37 oC with shaking. At the appropriate time intervals (0, 1, 2, 4, 8, 24h),
100 μL of culture were removed for serial dilutions in appropriate times, and 30 µL
was spread on MH agar plates. The cell counts (CFU.mL-1) were recorded after
incubating the plates at 37 oC for 24 h.
4.7. Drug resistance study
The drug resistance study was based on the minimum inhibitory concentrations
(MICs) tests. The initial MIC values of two compounds and two control antibiotics
(rifampin and norfloxacin) against S. aureus ATCC 29213 were determined as
described above. The bacterial culture from 96-well microtiter plate at a concentration
of 0.5× MIC was continuously used to prepare the bacterial dilution (approximately 5 ×
105 CFU/mL) for the next MIC assay. Then these bacterial suspensions were seeded
into another new 96-well microtiter plate and incubated with various concentrations of
two compounds and the two control antibiotics. After incubation for 24 h at 37 °C, the
fold change of MIC values was determined. This process was repeated for about 17
passages.
4.8. Visualization of bacterial morphology and membrane staining
The B. subtilis 168, stored at -80 oC, was transferred into LB medium and grown at
37oC with shaking overnight. The cultures were then diluted to approximately 1×105
CFU/mL and inoculated in the same medium containing absence or MIC concentration
of the test compounds. After grown at 37 oC for 4 h, the cells for bacterial morphology
were harvested by centrifugation and then resuspended in 100 microliter phosphate
buffered saline buffer (PBS, pH 7.4). For membrane staining, the cells were stained by
a red fluorescent dye FM4-64 (1.6 μM, Invitrogen, Eugene, OR, USA) for 15 min at
21

37 °C without shaking. Then the procedure of cell harvest was performed as previously
described. Five microliter of sample mixtures were placed on a microscopic slide
pretreated with 0.1% (w/v) poly-L-lysine. The bacterial cell morphology was
visualized using microscopy with a 40× magnification. The membrane staining of cells
was observed using fluorescence microscopy with a 40× magnification. The images
were captured using a super high-resolution laser confocal microscopy (Carl Zeiss,
LSM 800 with Airyscan). The scale bar is 20 μm. Each assay was performed in
triplicates.
4.9. GTPase activity assay
GTPase Activity of SaFtsZ was determined in 96- well micro plates using the
ATPase/GTPase Activity Assay Kit (SIGMA-ALDRICH, MAK113) according to an
optimized protocol and the manufacturer’s instructions.25 In this assay, 10 μL SaFtsZ
(4.6 μM) protein was pre-incubated with 10 μL vehicle (1% DMSO) or serial
dilutions of the test compounds in assay buffer (40 mM Tris, 80 mM NaCl, 8 mM
MgAc2, 1 mM EDTA, pH 7.5, containing 0.01% Triton X-100 to avoid compound
aggregation) for 10 min at room temperature. Then 30μL GTP (0.4 mM) was added
into the reaction mix and incubated at 37oC about 30 min. Then 50 μL of reactions
system were quenched by adding 200 μL of reagent for 30 min at 37oC to terminate
the enzyme reaction. The concentration of inorganic phosphate was measured by
reading the absorbance at 620 nm with a microplate reader.
4.10. Light scattering assay
The overexpression and purification of S. aureus FtsZ were carried out according
to the method as described in this literature.26 Light scattering assay was performed
following the procedure as described in this work.27 Briefly, the polymerization and
depolymerization of recombinant S. aureus FtsZ was measured by using a
thermostatically (37 oC) fluorescence spectrometer (Perkin Elmer, USA) in a model of
90° light scattering. Both excitation and emission wavelengths were set to 600 nm
with a slit width of 2.5 nm. S. aureus FtsZ (12 μM) in 50 mM of MOPS buffer (pH
6.5) was incubated with vehicle (1% DMSO) or different concentrations of the test
compounds. 50 mM KCl and 10 mM MgCl2 were then added to establish a baseline.
A final concentration of 1 mM GTP was added after that. The variation in light
scattering was measured for an additional 2000s. All experimental data have been
subtracted appropriate blanks. Each assay was performed in triplicate.
22

4.11. Antibacterial activity against FtsZ over-expression strain.
The strain was constructed following previous study.28 The FtsZ was over
expressed by incubating with 0.4 mM IPTG. The general procedure of antibacterial
activity assay was mentioned in section 4.4.
4.12. Molecular docking study
The molecular modeling procedures were performed using Discovery Studio
2016. The X-ray crystal structure of SaFtsZ downloaded from the PDB database
(PDB entry: 4DXD; resolution: 2.0 Å).29 Water molecules and co-crystal ligands were
removed from the structure and the protein was prepared for docking using an
automated procedure of Discovery Studio (DS). The ligand c2 were sketched and
prepared for docking using the tools of small molecules of DS. Automated docking
studies were carried out using DS-CDocker protocol of DS. The top-scoring poses
were visually inspected.
Acknowledgements
We acknowledge the support from the National Natural Science
Foundation of China (81703333 and 81473082), Natural Science Foundation of
Guangdong Province, China (2017A030313078), and Science and Technology
Program of Guangdong Province (2016A020209009, 2016B020211002 and
510093637143), and Department of Agriculture and Rural Affairs of
Guangdong Province, China (2018LM2175). The authors are also grateful to
the support from Nanshan Scholar Program of Guangzhou Medical University
(B185006006008) and Innovation and Technology Commission of Hong Kong,
The Hong Kong Polytechnic University.
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Highlights
 Novel indolyl-quinolinium derivatives were synthesized.
 The antibacterial activity of quinolinium derivatives were evaluated.
 c2 and c9 possess potent antibacterial activity against MRSA and VRE.
 Biological assays suggest antibacterial activity of c2 may associated with FtsZ.
A new series of indolyl-quinolinium derivatives perturb the polymerization of FtsZ with
strong antibacterial activities.
26