Design, synthesis and evaluation of novel 9-arylalkyl-10-methylacridinium derivatives as highly potent FtsZ-targeting antibacterial agents

Article abstract of DOI:10.1016/j.ejmech.2021.113480

With the increasing incidence of antibiotic resistance, new antibacterial agents having novel mechanisms of action hence are in an urgent need to combat infectious diseases caused by multidrug-resistant (MDR) pathogens. Four novel series of substituted 9-arylalkyl-10-methylacridinium derivatives as FtsZ inhibitors were designed, synthesized and evaluated for their antibacterial activities against various Gram-positive and Gram-negative bacteria. The results demonstrated that they exhibited broad-spectrum activities with substantial efficacy against MRSA and VRE, which were superior or comparable to the berberine, sanguinarine, linezolid, ciprofloxacin and vancomycin. In particular, the most promising compound 15f showed rapid bactericidal properties, which avoid the emergence of drug resistance. However, 15f showed no inhibitory effect on Gram-negative bacteria but biofilm formation study gave possible answers. Further target identification and mechanistic studies revealed that 15f functioned as an effective FtsZ inhibitor to alter the dynamics of FtsZ self-polymerization, which resulted in termination of the cell division and caused cell death. Further cytotoxicity and animal studies demonstrated that 15f not only displayed efficacy in a murine model of bacteremia in vivo, but also no significant hemolysis to mammalian cells. Overall, this compound with novel skeleton could serve as an antibacterial lead of FtsZ inhibitor for further evaluation of drug-likeness.

Full text of DOI:10.1016/j.ejmech.2021.113480

European Journal of Medicinal Chemistry 221 (2021) 113480
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Design, synthesis and evaluation of novel
9-arylalkyl-10-methylacridinium derivatives as highly potent
FtsZ-targeting antibacterial agents
Di Song, Nan Zhang, Panpan Zhang, Na Zhang, Weijin Chen, Long Zhang, Ting Guo,
Xiaotong Gu, Shutao Ma*
Department of Medicinal Chemistry, Key Laboratory of Chemical Biology (Ministry of Education), School of Pharmaceutical Sciences, Cheeloo College of
Medicine, Shandong University, 44 West Wenhua Road, Jinan, 250012, China
a r t i c l e i n f o
a b s t r a c t
Article history:
With the increasing incidence of antibiotic resistance, new antibacterial agents having novel mechanisms
of action hence are in an urgent need to combat infectious diseases caused by multidrug-resistant (MDR)
pathogens. Four novel series of substituted 9-arylalkyl-10-methylacridinium derivatives as FtsZ in-
hibitors were designed, synthesized and evaluated for their antibacterial activities against various Gram-
positive and Gram-negative bacteria. The results demonstrated that they exhibited broad-spectrum
activities with substantial efficacy against MRSA and VRE, which were superior or comparable to the
berberine, sanguinarine, linezolid, ciprofloxacin and vancomycin. In particular, the most promising
compound 15f showed rapid bactericidal properties, which avoid the emergence of drug resistance.
However, 15f showed no inhibitory effect on Gram-negative bacteria but biofilm formation study gave
possible answers. Further target identification and mechanistic studies revealed that 15f functioned as an
effective FtsZ inhibitor to alter the dynamics of FtsZ self-polymerization, which resulted in termination of
the cell division and caused cell death. Further cytotoxicity and animal studies demonstrated that 15f not
only displayed efficacy in a murine model of bacteremia in vivo, but also no significant hemolysis to
mammalian cells. Overall, this compound with novel skeleton could serve as an antibacterial lead of FtsZ
inhibitor for further evaluation of drug-likeness.
Received 26 March 2021
Received in revised form
13 April 2021
Accepted 15 April 2021
Available online 28 April 2021
Keywords:
9-Arylalkyl-10-methylacridinium
derivatives
Design and synthesis
Antibacterial activity
FtsZ inhibitor
Structure-activity relationships
© 2021 Elsevier Masson SAS. All rights reserved.
1. Introduction
Thus, the discovery of new antibacterial targets and development of
effective antibacterial drugs with new mechanism should be
Infections caused by bacteria have long been common diseases
that even severely threaten human's life. Thanks to the discovery of
penicillin in 1928, and the subsequent application of various anti-
bacterial drugs, millions of lives have been saved. However, the use
of antibacterial drugs inevitably leads to the emergence of bacterial
resistance, and in recent years, especially, the widespread use or
even abuse of antibacterial drugs greatly has accelerated the
prevalence of bacterial resistance, which are undoubtedly exacer-
bating the threat of clinical infections [1]. For example, the
appearance of methicillin-resistant Staphylococcus aureus (MRSA)
and vancomycin-resistant Enterococcus (VRE) contributes to the
enormous difficulty in fighting against bacterial infections [2,3].
accelerated to cope with this issue [4].
The bacterial cell division machinery involves many essential
proteins that are extremely sensitive to perturbation by small
molecules [5]. As a eukaryotic tubulin analogue, the filamentous
temperature-sensitive protein Z (FtsZ) is a key protein involved in
bacterial cell division [6,7]. When bacterial cells divide, FtsZ poly-
merizes into tubulin-like protofilaments at mid-cell that serves as a
highly dynamic cytoskeleton scaffold for the recruitment and or-
ganization of other cell division proteins in the presence of gua-
nosine triphosphate (GTP) to complete the bacterial cell division
[8]. The arrangement of protofilaments is head to tail, which is
followed to assemble into bundles at the site of septum formation.
Intervention of this normal biological function will result in the
inhibition of bacterial division and eventually the death of bacteria
[9]. In addition, FtsZ is highly conservative in the bacterial kingdom
and plays an essential role in prokaryotic cell division. Therefore,
* Corresponding author.
E-mail address: mashutao@sdu.edu.cn (S. Ma).
https://doi.org/10.1016/j.ejmech.2021.113480
0223-5234/© 2021 Elsevier Masson SAS. All rights reserved.

D. Song, N. Zhang, P. Zhang et al.
European Journal of Medicinal Chemistry 221 (2021) 113480
FtsZ has been a promising new target for the development of new
antibacterial drugs. At present, many FtsZ inhibitors have been
reported to exhibit potent antibacterial activity. A few representa-
tive natural and synthesized molecules are shown in Fig. 1 [10e15].
Sanguinarine and berberine with a quaternary pyridinium core,
which are two alkaloids extracted from natural products, have been
identified as FtsZ inhibitors that can alter the Z-ring formation and
disturb the function of FtsZ protein [16,17]. For example, sangui-
narine can inhibit the growth of Escherichia. coli and Bacillus subtilis
by inhibiting Z-ring formation without affecting nucleoid segre-
through structure and activity relationships (SARs) analysis. In
particular, quaternary ammonium nitrogen in structure was found
to be necessary for antibacterial activity. The above findings indi-
cate that it is feasible to simplify the four-membered ring to the
planar structure of the three-membered ring.
Based on this, we obtained the mother molecule of 10-methyl-
acridinium by simplifying the skeleton of sanguinarine and
berberine. 10-methylacridinium is a conjugate rigid plane structure
with polar quaternary ammonium center, which is easily accessible
in 2e3 synthetic steps. We attempted to strengthen the rigidity and
planarity to fit the binding site of FtsZ better and investigate the
effect of side chain on antibacterial activity by introducing various
substituents at the C-9 position of the scaffold. In this research, we
originally linked the aliphatic amine or substituted aromatic amine
to 10-methylacridinium to obtain 10-methyl-9-(alkylamino)acri-
din-10-ium and 10-methyl-9-(arylalkylamino)acridin-10-ium de-
rivatives (Series A), respectively. We expect to form additional
hydrogen bonds of the imine linkers with surrounding amino acid
residues in the narrow cleft of FtsZ to increase antibacterial activity.
And then, we introduced substituted phenyl, styrene and phenyl-
acetylene at the C-9 position of the scaffold to get 10-methyl-9-
arylacridin-10-ium, (E)-10-methyl-9-styrylacridin-10-ium and 10-
methyl-9-(phenylethynyl)acridin-10-ium derivatives (Series B, C
and D), respectively. The linker of Series B is carbon-carbon single
bond possessing enough flexibility, which makes the aromatic ring
at the C-9 position rotate freely in the narrow cleft to form new
binding force. The linker of Series C is carbon-carbon double bond,
which urges the aromatic substituents to extend deep into the
narrow cleft. We expected that the aromatic substituents could
interacted with LEU200, ILE201, VAL203, VAL207, LEU209 in the
narrow cleft through hydrophobic interaction. Besides, double
bonds can change the density of the electron cloud in the mother
molecule, which might affect the antibacterial activity through
changing the capability for cationic ionization. Compared with that
of Series C, the linker of Series D is carbon-carbon triple bond and
the overall structure of this series is linear. This linear skeleton is
beneficial for entering the narrow cleft to interact with the amino
gation. Its IC50 values for E. coli and B. subtilis are 4.6 0.8
mg/mL
and 1.0 0.3 g/mL, respectively. However, sanguinarine may
m
depolymerize microtubules and produce harmful side effects in
mammals, which is a potential drawback in the development of
sanguinarine as antibacterial drug [18]. Berberine, an alkaloid with
similar structure to sanguinarine, can inhibit GTPase activity and
reduce FtsZ polymerization. Although berberine has no inhibitory
effect on microtubules, its antibacterial activity is poor against
Gram-positive bacteria with the minimum inhibitory concentration
values (MICs) in the range of 100e400 mg/mL [19]. Recent litera-
tures have reported that some compounds possessing the quater-
nary or binary pyridinium scaffold with aromatic hydrophobic
groups can significantly enhance antibacterial activity via targeting
FtsZ [20e24]. Our group has been committed to investigating new
FtsZ inhibitors through structural simplification of natural products
such as sanguinarine and berberine, etc. We reported a series of 5-
methyl phenanthridium compounds with moderate activity against
a broad range of Gram-positive bacteria and none of the com-
pounds enhanced polymerization of mammalian tubulin even
though they were at higher concentrations than the positive con-
trol paclitaxel [25]. Further, we accomplished the synthesis of novel
5-methyl-2-phenyl phenanthridium derivatives with an unex-
pected increase in antibacterial activity [26]. Docking results
demonstrated the phenanthridium compound combined in a
relatively narrow cleft delimited by the H7-helix, the T7-loop and
C-terminal subdomain. We found benzodioxolane of sanguinarine
and beberine is not an essential group for antibacterial activity
O
O
O
O
O
Br
HN
H
N
N
O
N
Cl
O
N
O
O
I
synthetic pyridinium derivative
O
berberine, plant natural product
13-substituted cycloberberine (CBBR)
O
F
F
F
N
Br
O
O
O
O
O
N
CH3
N
I
Cl
9-phenoxyalkylberberine
5-methyl-2-phenylphenanthridium 3-phenyl substituted 6,7-dimethoxyisoquinium
Fig. 1. Chemical structures of some FtsZ inhibitors.
2

D. Song, N. Zhang, P. Zhang et al.
European Journal of Medicinal Chemistry 221 (2021) 113480
acid residues in the deep. The above brief design strategy is illus-
trated in Fig. 2.
with various substituted amines in good yields.
The synthetic route for 10-methyl-9-arylacridin-10-ium de-
rivatives (Series B) is outlined in Scheme 2. 9-Bromoacridine 7 was
prepared by the treatment of 3 using phosphorus tribromide as
solvent in a good yield [31]. The 9-arylacridin derivatives were
easily accessible in one-pot in good yields by employing the Suzuki
coupling reaction of 7 with various arylboronic acids in the pres-
ence of Pd [0] as catalyst. 10-methyl-9-arylacridin-10-ium de-
rivatives (9a-9o) were then synthesized via the nucleophilic
substitution reaction of various substituted acridines (8a-8o) with
iodomethane in good yields.
(E)-10-Methyl-9-styrylacridin-10-ium derivatives (Series C) was
synthesized from N,N-diphenylamine as outlined in Scheme 3.
Commercially available 10 was cyclizated with acetic acid to afford
the 9-methylacridine 11 [32]. Bromination of 11 in the presence of
NBS by employing AIBN as initiator of the free radical reaction
furnished the corresponding product 12 in a good yield [33]. The
triethyl phosphite first reacted with the 12 by employing the
Arbuzov reaction to obtain key intermediate 13. Then, 13 generated
a phosphine ylide under the action of a strong base (NaH), under-
going one-pot reaction with various substituted benzaldehydes via
Horner-Emmons reaction to obtain the corresponding products
14a-14m. Methylation of 14a-14m generated the title compounds
(E)-10-methyl-9-styrylacridin-10-ium derivatives (15a-15m) using
iodomethane, following the similar process as described in Scheme
2.
Here, we systematically designed, synthesized and screened a
focused compound library of 61 candidates (Series A, B, C and D) for
their antibacterial activity and identified a new class of FtsZ in-
hibitors. The in vitro antibacterial activity against a panel of Gram-
positive and Gram-negative bacteria strains including MRSA and
VRE was evaluated by testing their MIC values. Bactericidal or
bacteriostatic activity was assessed by testing their minimum
bactericidal concentration (MBC) and MBC/MIC ratio was calcu-
lated to determine their bacteriostatic action. Further, time-kill
kinetics was researched to clarify the sterilization of the promising
compounds in each time period. Besides, we studied the effect on
biofilm formation of Pseudomonas aeruginosa to explain the reason
of weak activity against Gram-negative bacteria and potential
resistance development as an important indicator was evaluated to
observe the occurrence of drug resistance. More importantly, the
cell morphology of Bacillus pumilus, FtsZ polymerization, the as-
sembly and bundling of FtsZ protofilaments as on-target effect were
also investigated. The outstanding compound was selected to
further evaluate its hemolysis and in vivo efficacy. Finally, the
interaction mode between those synthesized molecules with FtsZ
was revealed through the docking analysis with a reported crystal
structure of SaFtsZ (PDB code: 4DXD).
2. Chemistry
As shown in Scheme 4, the chemical synthesis of 10-methyl-9-
(phenylethynyl)acridin-10-ium derivatives (Series D) was initiated
with 9-bromoacridine 7 by employing the Sonogashira coupling
reaction in the presence of Pd [II] and Cu [I] as catalysts in a good
yield [34]. 10-methyl-9-(phenylethynyl)acridin-10-ium derivatives
(17a-17j) were obtained through methylation of 16a-16j, according
to similar procedure as described in Scheme 2.
The synthetic route for 10-methyl-9-(alkylamino)acridin-10-
ium and 10-methyl-9-(arylalkylamino) acridin-10-ium derivatives
(Series A) is illustrated in Scheme 1. Commercially available 2-
bromobenzoic acid reacted with aniline, affording N-phenyl-
anthranilic acid 2 in the presence of excess Cu powder as a catalyst
by employing the Ullmann reaction [27]. The acid was cyclizated
using concentrated sulfuric acid to afford acridone 3 [28]. N-
methylacridone 4 was attained by nucleophilic substitution reac-
tion of 3 with iodomethane [29]. Chlorination of 4 using dichlor-
osulfoxide as solvent furnished key intermediate 5 [30]. 10-methyl-
9-(alkylamino)acridin-10-ium and 10-methyl-9-(arylalkylamino)
acridin-10-ium derivatives (6a-6w) were then synthesized via the
nucleophilic substitution reaction of freshly prepared chloride 5
3. Results and discussions
3.1. In vitro antibacterial activity of 9-arylalkyl-10-
methylacridinium derivatives
The synthesized compounds were initially tested against eight
Fig. 2. Brief design process of the 9-arylalkyl-10-methylacridinium derivatives.
3

D. Song, N. Zhang, P. Zhang et al.
European Journal of Medicinal Chemistry 221 (2021) 113480
Scheme 1. Synthesis of 10-methyl-9-(alkylamino)acridin-10-ium and 10-methyl-9-(arylalkylamino) acridin-10-ium derivatives.
Scheme 2. Synthesis of 10-methyl-9-arylacridin-10-ium derivatives.
susceptible bacteria strains, including B. subtilis ATCC9372,
ATCC43300, S. aureus PR, S. aureus CI, Staphylococcus epidermidis,
S. pyogenes 2, E. faecalis ATCC51299 and E. faecium ATCC51559.
Sanguinarine (San), berberine (Ber), ciprofloxacin (Cip), linezolid
(Lin) and vancomycin (Van) were used as controls to compare the
effect of various linkers and substituents of the synthesized
B. pumilus CMCC63202, S. aureus ATCC25923, Streptococcus pyo-
genes 1, Enterococcus faecalis ATCC29212, Enterococcus faecium
ATCC19434, P. aeruginosa ATCC27853 and Escherichia coli
ATCC25922, and seven resistant bacteria strains, including S. aureus
4

D. Song, N. Zhang, P. Zhang et al.
European Journal of Medicinal Chemistry 221 (2021) 113480
Scheme 3. Synthesis of (E)-10-methyl-9-styrylacridin-10-ium derivatives.
Scheme 4. Synthesis of 10-methyl-9-(phenylethynyl)acridin-10-ium derivatives.
compounds on antibacterial activity. Their MIC values were listed in
Tables 1 and 2.
CI with the MIC values of 4 and 8
7-carbon alkyl chain not only performed moderate activity against
S. aureus ATCC25923 and S. pyogenes 1 with an MIC value of 16 g/
mg/mL, respectively. 6f possessing
m
mL, but also offered the activity against resistant bacterial strains of
S. aureus PR and S. aureus CI. Besides, 6s with pyrrole ring and 6w
with pyrimidine ring showed weak activity against some Gram-
positive bacterial strains.
Based on the above MIC data, the structure-activity relation-
ships (SARs) of Series A were summarized as follows. Introduction
of a long unbranched alkyl chain contributed to the increased
antibacterial activity (see 6a-6g). The antibacterial activity of 6g (8-
carbon alkyl chain) was stronger than that of 6f (7-carbon alkyl
chain), which was induced by the lengthening of the side chain. We
3.1.1. In vitro antibacterial activity of Series A analogues
The target compounds of Series A were evaluated for in vitro
antibacterial activity (Tables 1 and 2). The results demonstrated
that only 6f, 6g, 6s and 6w showed inhibitory effect on the tested
bacterial strains. Among them, 6g with 8-carbon alkyl chain
exhibited the moderate activity against the four tested susceptible
bacterial strains of B. subtilis ATCC9372, B. pumilus CMCC63202,
S. aureus ATCC25923 and S. pyogenes 1 with the MIC values of 16,16,
8 and 16
mg/mL, respectively. On the other hand, 6g also inhibited
the growth of resistant bacterial strains of S. aureus PR and S. aureus
5

D. Song, N. Zhang, P. Zhang et al.
European Journal of Medicinal Chemistry 221 (2021) 113480
Table 1
In vitro antibacterial activity against susceptible-bacteria of Series A, B, C and D (6a-6w, 9a-9o, 15a-15m and 17a-17j).
Comp
Minimum Inhibitory Concentration/MIC (mg/mL)
B. subtilis
B. pumilus
S. aureus
S.pyogenes
1d
E. faecalis
E. faecium
P. aeruginosa
E. coli
ATCC9372a
CMCC63202b
ATCC25923c
ATCC29212e
ATCC19434f
ATCC27853g
ATCC25922h
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j-6r
6s
6t
6u
6v
6w
9a
9b
9c
9d
9e
9f
9g
9h
9i
9j
9k
9l
64
>64
>64
64
64
32
16
>64
32
>64
64
>64
64
>64
16
16
32
8
16
16
64
4
2
1
0.5
8
1
4
4
>64
1
1
1
1
1
0.25
2
8
1
2
2
1
0.5
2
8
8
2
2
>64
>64
>64
>64
64
32
16
>64
>64
>64
64
>64
>64
>64
64
32
>64
16
32
32
>64
8
4
2
1
16
1
16
8
>64
2
2
0.5
1
1
0.5
1
16
1
2
1
1
2
2
8
8
2
2
2
4
4
4
>64
>64
>64
>64
64
16
8
>64
>64
>64
32
>64
>64
>64
16
2
8
1
4
8
16
1
0.25
0.25
0.03
4
64
32
>64
16
32
16
16
>64
>64
>64
16
>64
>64
>64
>64
8
64
4
8
4
16
4
2
1
1
8
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
4
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
4
>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
>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
>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
>64
64
NT
4
NT
4
>64
64
0.125
1
0.5
1
1
9m
9n
9o
NT
NT
NT
NT
NT
NT
NT
NT
2
NT
NT
NT
NT
NT
NT
2
NT
NT
NT
NT
NT
NT
NT
NT
4
NT
NT
NT
NT
NT
NT
4
>64
>64
>64
>64
>64
64
0.5
64
0.5
0.25
0.5
0.5
1
0.5
0.5
4
0.5
1
1
0.5
0.5
2
4
8
2
2
4
4
4
4
64
0.25
0.25
0.25
0.5
0.5
<0.125
<0.125
2
0.5
0.5
1
0.25
0.25
4
8
16
4
4
8
16
16
16
16
>64
2
>64
8
1
15a
15b
15c
15d
15e
15f
15g
15h
15i
15j
15k
15l
15m
17a
17b
17c
17d
17e
17f
17g
17h
17i
17j
AD
Sani
Berj
Cipk
Linl
Vanm
64
>64
>64
>64
>64
32
>64
>64
64
32
NT
NT
NT
4
NT
NT
NT
8
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
32
2
4
4
4
4
4
8
>64
4
>64
2
0.5
0.5
NT
NT
NT
NT
NT
>64
16
>64
32
4
NT
NT
NT
NT
NT
>64
64
>64
>64
8
8
>64
4
>64
16
2
8
>64
4
64
4
2
>64
8
>64
>64
>64
0.25
>64
>64
2
1
0.25
4
2
a
B. subtilis ATCC9372: Bacillus subtilis ATCC9372, penicillin-susceptible strain.
B. pumilus CMCC63202: Bacillus pumilus CMCC63202, penicillin-susceptible strain.
S. aureus ATCC25923: Staphylococcus aureus ATCC25923, erythromycin-susceptible strain.
S. pyogenes 1: Streptococcus pyogenes 1, erythromycin-susceptible strain isolated clinically.
E. faecalis ATCC29212: Enterococcus faecalis ATCC29212, vancomycin-susceptible strain.
E. faecium ATCC19434: Enterococcus faecium ATCC19434, vancomycin-susceptible strain.
P. aeruginosa ATCC27853: Pseudomonas aeruginosa ATCC27853, penicillin-susceptible strain, not characterized.
E. coli ATCC25922: Escherichia coli ATCC25922, penicillin-susceptible strain.
San: Sanguinarine.
Ber: Berberine.
Cip: Ciprofloxacin.
Lin: Linezolid.
b
c
d
e
f
g
h
i
j
k
l
m
Van: Vancomycin.
6

D. Song, N. Zhang, P. Zhang et al.
European Journal of Medicinal Chemistry 221 (2021) 113480
inferred that their lipid/water partition coefficients (logP) as one of
the most important factors could affected the activity [35]. There-
fore, we theoretically calculated the logP values (ClogP) by the
commercial ChemBioOffice 2014 (Cambridge Soft, USA) to further
confirm their influence on antibacterial activity. Consequently, the
ClogP value of 6g with 8-carbon alkyl chain was the highest, which
suggested the ability of penetrating the lipid bilayer contributed
significantly to antibacterial activity. The above results confirmed
that the presence of an alkyl group could produce moderate ac-
tivity. Other compounds with stronger electronegativity state could
lead to the loss of antibacterial activity as a result of their poor cell
penetration. Besides, we inferred that aniline as substituent in
structure could rotate freely in space, which reduced the planarity
and made it difficult to enter into a narrow hydrophobic cavity of
FtsZ. These findings help us to conduct further structural
optimization.
the MIC values of 0.25, 0.5, 0.5, <0.125, 2 and 4 mg/mL, respectively.
Besides, 15f displayed strong activity against MRSA, S. epidermidis,
S. pyogenes 2 and VRE, better than or equal to control drugs.
Similarly, 15m not only exerted the excellent activity against
B. subtilis ATCC9372 and S. aureus ATCC25923 with a low MIC value
of 0.5
mg/mL but also effectively inhibited the growth of three
resistant S. aureus strains and VRE.
The SARs of Series C were summarized below. The antibacterial
activity of compounds with C-2 and C-4 position substituents were
similar to that of 15a (no substituent on the phenyl ring), which
indicated that the extension direction of the substituents in space
had little effect on antibacterial activity. In particular,15f containing
a tert-butyl side chain demonstrated the best antibacterial activity,
which suggested that the appropriate length and flexibility more
easily generated hydrophobic interaction with the binding site of
FtsZ. In addition,15f had a higher ClogP value (3.087) than the other
compounds, which was also beneficial for the compound to pene-
trate the bacterial cell membrane. In contrast, decreasing the chain
lipophilicity by introducing an oxygen atom (15g and 15h) on the
phenyl ring or introducing the electron-donating substituents (15j
and 15k) led to decreased antibacterial activity.
3.1.2. In vitro antibacterial activity of Series B analogues
The MIC data of Series B analogues were exhibited in Tables 1
and 2. The results showed that 9j possessed equivalent antibacte-
rial activity (0.5
mg/mL) against B. subtilis ATCC9372 to that of
linezolid and vancomycin, and 9j and 9l demonstrated stronger
activity against B. pumilus CMCC63202 than the controls. Moreover,
9h, 9i, 9j, 9l and 9n exhibited 8e133 fold increase in activity against
S. aureus ATCC25923 compared with the lead compound. The ac-
tivity of 9i, 9j, 9l, 9m and 9n against S. pyogenes 1 were comparable
3.1.4. In vitro antibacterial activity of Series D analogues
The antibacterial activity of Series D was showed in Tables 1 and
2 In Series D, 17a, 17d and 17e showed inhibitory effect on the four
tested susceptible bacterial strains of B. subtilis ATCC9372,
B. pumilus CMCC63202, S. aureus ATCC25923 and S. pyogenes 1 with
to that of linezolid (1
(0.25 g/mL). As for susceptible enterococcus strains, 9j and 9l
showed equivalent antibacterial activity (4 g/mL) to that of van-
mg/mL) but not better than that of vancomycin
m
the MIC values of 2, 2, 2 and 4
mentioning that 17e exhibited remarkable activity against
E. faecalis ATCC29212 with a MIC value of 2 g/mL, which was 2-fold
mg/mL, respectively. It was worth
m
comycin. Furthermore, 9j and 9l as the promising compounds in
this series could inhibit the growth of all the tested resistant bac-
teria strains including MRSA and VRE with the MIC values ranging
m
more potent than that of vancomycin. Especially, 17d exhibited the
best activity against all the tested resistant bacteria, with the MIC
from <0.125 to 8
mg/mL, which were comparable or superior to that
values ranging from 2 to 8 mg/mL, better than or equal to sangui-
of the controls.
narine, ciprofloxacin, linezolid and vancomycin.
The SARs of Series B were summarized as follows. The MIC re-
sults showed that the antibacterial activity of compound without
substituents on the phenyl group (9a) was stronger than that of the
compounds with C-2 position substituents. Compounds with
electron-donating substituents of C-3 position on the phenyl ring
(9c and 9d) were only marginally more active than 9a, whereas
electron-withdrawing substituent of C-3 position (9f) led to a loss
of activity. Surprisingly, compounds with substituents at the C-4
position of the phenyl ring displayed significantly improved activ-
ity. For example, 9j with 4-carbon alkyl chain offered the most
potent antibacterial activity. Furthermore, with the extension of
alkyl chain, the activity also increased. The above analysis indicated
that when there was the same substituent on the phenyl group, its
extension direction in space had great effect on the antibacterial
activity. Besides, the antibacterial activity of 9l with biphenyl group
was second only to 9j, which emphasized the importance in
planarity of scaffold. In view of this, we inferred that the bulky
substituent (9l and 9j) could extend into the binding cavity of FtsZ
to form new binding force. Thus, it was necessary to lengthen the
linker to two carbons, and keep the mother nucleus and sub-
stituents on the same plane.
The SARs of Series D indicated that the C-3 and C-4 position
substitutions on the phenyl ring demonstrated improved antibac-
terial activity over a C-2 substituted scaffold. Besides, introducing
an oxygen atom (17g, 17h and 17i) on the phenyl ring led to
decreased antibacterial activity compared with introducing an alkyl
chain (17e and 17f).
In general, Series C (substituted with alkene) as the most
outstanding series demonstrated excellent antibacterial activity
against all the susceptible and resistant Gram-positive bacterial
strains, some of which were better than control drugs. Among
them, 15f showed a strong inhibitory effect on the susceptible and
resistant bacterial strains including MRSA and VRE with the MIC
values ranging from <0.125 to 4 mg/mL. All the compounds in Series
D (substituted with alkyne) performed good antibacterial activity
against all the tested Gram-positive bacterial strains, but on the
whole they were not as good as those in Series C. In addition, 15a
(no substituent on the phenyl ring) in Series C and 17a (no sub-
stituent on the phenyl ring) in Series D had satisfactory activity
with the MIC values ranging from 0.25 to 8 mg/mL, which indicated
the introduction of substituents had little effect on the antibacterial
activity. The compounds in Series B (substituted with phenyl) made
a big difference in antibacterial activity and the introduction of
substituent on the phenyl ring affected the activity significantly.
Among them, 9j with 4-carbon alkyl chain could exert excellent
3.1.3. In vitro antibacterial activity of Series C analogues
The antibacterial activity of Series C was showed in Tables 1 and
2. All the test compounds except for 15h exhibited excellent anti-
bacterial activity against Gram-positive bacteria, some of which
were better than control drugs in antibacterial activity. Among
them, 15f as the most promising compound showed excellent ac-
tivity against the six tested susceptible bacterial strains of B. subtilis
ATCC9372, B. pumilus CMCC63202, S. aureus ATCC25923,
S. pyogenes 1, E. faecalis ATCC29212 and E. faecium ATCC19434 with
antibacterial activity with the MIC values ranging from 0.03 to 8 mg/
mL. The antibacterial activity of Series B to susceptible bacteria is
good, but barely satisfactory to resistant bacteria. In Series A, 6f, 6g,
6s and 6w showed inhibitory effect on the tested bacterial strains.
Among them, 6f with 7-carbon alkyl chain and 6g with 8-carbon
alkyl chain displayed moderate antibacterial activity against the
four tested susceptible bacterial strains, which confirmed that the
7

D. Song, N. Zhang, P. Zhang et al.
European Journal of Medicinal Chemistry 221 (2021) 113480
Table 2
In vitro antibacterial activity against resistant-bacteria of Series A, B, C and D (6a-6w, 9a-9o, 15a-15m and 17a-17j).
Comp
Minimum Inhibitory Concentration/MIC (
m
g/mL)
S. aureus ATCC43300a
S. aureus PRb
S. aureus CIc
S. epidermidisd
S. pyogenes2e
E. faecalis ATCC51299f
E. faecium ATCC51559g
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j-6r
6s
6t
6u
6v
6w
9a
9b
9c
9d
9e
9f
9g
9h
9i
9j
9k
9l
>64
>64
>64
>64
64
32
16
>64
>64
>64
>64
>64
>64
>64
32
>64
>64
64
>64
>64
64
64
16
4
2
>64
2
16
32
>64
4
4
4
2
8
1
2
16
2
8
2
2
1
2
4
8
2
2
4
8
4
4
>64
>64
>64
64
64
16
4
>64
>64
>64
>64
64
16
8
>64
>64
>64
64
>64
>64
>64
32
8
>64
64
>64
64
64
32
16
>64
>64
>64
64
>64
>64
>64
>64
>64
>64
64
>64
>64
>64
64
32
8
2
>64
2
32
32
>64
8
8
2
4
2
1
4
32
2
8
2
2
2
4
16
16
4
8
8
16
8
8
8
>64
8
64
64
>64
32
32
32
16
64
64
>64
64
>64
>64
>64
>64
32
>64
16
32
16
64
16
4
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
4
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
NT
2
>64
>64
>64
64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
64
16
8
>64
8
64
64
>64
8
16
8
8
8
2
8
64
4
16
4
4
2
4
8
16
2
4
8
64
4
8
16
32
4
1
0.25
<0.125
8
0.25
2
2
>64
2
1
1
1
2
0.5
1
8
1
2
2
1
1
2
2
4
2
2
4
4
4
4
16
>64
8
>64
16
2
4
2
16
1
2
NT
4
NT
2
9m
9n
9o
NT
NT
NT
NT
NT
NT
NT
NT
4
NT
NT
NT
NT
NT
NT
8
NT
NT
NT
NT
NT
NT
NT
NT
2
NT
NT
NT
NT
NT
NT
4
4
>64
0.5
0.5
0.5
1
0.5
0.25
0.5
4
0.5
0.5
1
0.5
0.5
8
16
16
4
4
8
16
8
16
16
>64
2
64
>64
1
15a
15b
15c
15d
15e
15f
15g
15h
15i
15j
15k
15l
15m
17a
17b
17c
17d
17e
17f
17g
17h
17i
17j
AD
San
Ber
Cip
Lin
Van
NT
NT
NT
8
NT
NT
NT
4
8
4
NT
NT
NT
NT
NT
>64
32
>64
>64
4
NT
NT
NT
NT
NT
>64
32
>64
>64
4
16
8
16
16
>64
16
>64
>64
4
16
>64
8
>64
16
2
>64
>64
4
4
2
NT
4
1
>64
>64
a
S. aureus ATCC43300: Staphylococcus aureus ATCC43300, methicillin-resistant strain.
S. aureus PR: Staphylococcus aureus PR, penicillin-resistant strain.
S. aureus CI: Staphylococcus aureus, clinical isolated strain, not characterized.
S. epidermidis: Staphylococcus epidermidis, penicillin-resistant strain isolated clinically, not characterized.
S. pyogenes 2: Streptococcus pyogenes 2, erythromycin-resistant strain isolated clinically.
E. faecalis ATCC51299: Enterococcus faecalis ATCC51299, vancomycin-resistant strain.
E. faecium ATCC51559: Enterococcus faecium ATCC51559, vancomycin-resistant strain.
b
c
d
e
f
g
presence of an alkyl group gave moderate activity. The order of
antibacterial activity of the four series is Series C > Series D > Series
B > Series A. Series A containing substituted amines could rotate
flexibly in space, whereas the narrow cleft binding site required
some degree of planarity in order to fit better. Increasing the
scaffold rigidity by the introduction of alkene (Series C) and alkyne
8

D. Song, N. Zhang, P. Zhang et al.
European Journal of Medicinal Chemistry 221 (2021) 113480
(Series D) strengthened antibacterial activity, which contributed to
penetrating into the hydrophobic cavity of FtsZ forming hydrogen
negative bacteria.
bond interaction,
p
-p
stacking interaction, electrostatic interaction
3.5. Evaluation of potential resistance development
and hydrophobic interaction. Besides, prolonging the linker to two
carbons contributed to the increased activity. This might be because
compounds suit the hydrophobic cavity of FtsZ better, thereby
strengthening affinity for FtsZ. In summary, lengthening the linker,
and keeping the mother nucleus and substituents on the same
plane were crucial to antibacterial activity. It was worth mentioning
that all the series except for Series A demonstrated increased
antibacterial activity, as compared to the mother molecule (AD),
which was corresponding to our optimization strategy. In the next
assays, 15f as the most potent compound was selected for further
evaluation.
Bacterial resistance to the currently used antimicrobials is a
major cause of ineffective treatment of infectious diseases [37,38].
Thus, the evaluation of potential resistance development is neces-
sary in developing new antimicrobials [39,40]. The propensity of
bacterial resistance development for 15f was performed using a
multipassage resistance selection assay. We passaged S. aureus
ATCC25923 at subinhibitory concentration (0.5 ꢂ MIC) of 15f and
ciprofloxacin for 18 cycles. As shown in Fig. 5, no greater than a 2-
fold increase in the MIC values was observed for 15f after 18 pas-
sages, while the MIC value of ciprofloxacin was increased by 64-fold
after 9 passages. These results demonstrated that 15f could avoid
inducing bacterial resistance. We inferred that new antibacterial
mechanism and the rapid bactericidal pattern of 15f played an
important role in overcoming the emergence of bacterial
resistance.
3.2. Bactericidal or bacteriostatic activity
To further investigate whether its antibacterial activity was
bactericidal or bacteriostatic, we determined the MBC of 15f against
three susceptible bacteria strains (S. aureus ATCC25923, B. subtilis
ATCC9372 and B. pumilus CMCC63202) and three resistant bacteria
strains (S. aureus ATCC43300, S. aureus PR and penicillin-resistant
S. epidermidis). Sanguinarine and linezolid were used as controls in
the MBC determinations. According to the Clinical and Laboratory
Standards Institute (CLSI) standards, an MBC/MIC ratio ꢀ 4 is
regarded as a bactericidal behavior. The results shown in Table 3
indicate that the MBC/MIC ratios of sanguinarine and linezolid are
ꢁ8, which were observed as bacteriostatic activity. By contrast, 15f
was observed as a bactericidal agent with the MBC/MIC ratios ꢀ4.
Significantly, 15f exhibited almost identical MBC and MIC values
(i.e. MBC/MIC ratio ¼ 1 or 2) for both susceptible and resistant
bacterial strains, indicating its bactericidal mode clearly.
3.6. Effects on the morphology of B. pumilus cells
Berberine and benzamide FtsZ inhibitors is known to disrupt
FtsZ activity and then cause the morphology of bacteria to grow
larger or longer [22,41,42]. Because the combination of those
compounds with FtsZ inhibits the normal function of FtsZ, and then
hinders bacterial division process. Increasing or prolonged bacterial
cell volume eventually leads to bacterial lysis and death. Thus, we
used an optical microscope to observe the bacterial morphology of
B. pumilus cultured with or without 15f. Cell morphological changes
are shown in Fig. 6. The result showed that 15f significantly
increased the length of B. pumilus compared to the control. For
example, the normal cell length of B. pumilus cells is around
3.3. Kinetics of the bactericidal activity
1.2e3
mm (Fig. 6A), while the cell length after incubated with
0.5 ꢂ MIC concentration (0.25
m
g/mL) was longer than 20 mm
The time-kill kinetic approach against S. aureus ATCC25923 was
used to investigate the kinetics of the bactericidal activity. As
shown in Fig. 3, the bacteria increased sharply in the counts of CFU
without 15f or in the presences of 0.5 ꢂ MIC of 15f. By contrast, 15f
achieved a 2.2 log reduction in initial bacterial load (more than
99.99% of bacteria were killed) in 3 h at 4 ꢂ MIC, and a 2.43 log CFU
reduction in 24 h at 2 ꢂ MIC. It was noteworthy that the com-
mercial antibiotic linezolid at 16 ꢂ MIC achieved approximately
same killing efficacy to 15f at 1 ꢂ MIC. Fast bactericidal perfor-
mance would shorten the treatment time, which could reduce the
probability of bacterial resistance. These findings revealed that 15f
could serve as an excellent bactericidal agent in a concentration-
dependent manner.
(Fig. 6B). These cell elongation phenomena were similar to that of
other known FtsZ inhibitors, indicating preliminarily its inhibitory
effect on FtsZ.
3.7. Stimulation of FtsZ polymerization dynamics
Research on the dynamic of FtsZ has demonstrated that some
FtsZ-targeting inhibitors can stimulate the polymerization of FtsZ
and stabilize the structure of FtsZ polymer, which is beneficial for
improving the antibacterial activity of FtsZ inhibitors [43,44]. The
antibacterial activity of sanguinarine and berberine might owe to
their inhibitory effect on the polymerization of FtsZ [16,17,45]. Thus,
we selected 15f for testing on the polymerization of purified
B. subtilis FtsZ (BsFtsZ) in vitro. For this assay, we utilized a light
scattering approach to detect the effect of 15f on the FtsZ poly-
merization. Fig. 7 showed the time-dependent polymerization
profiles of BsFtsZ in the absence and in the presence of 15f at
3.4. Effect on biofilm formation of P. aeruginosa
The antibacterial activity of the test compounds against Gram-
negative bacteria was much weaker than that against Gram-posi-
tive bacteria. One potential explanation is that the lipopolysac-
charide (LPS) layer outside the cell membrane of Gram-negative
bacteria could prevent the entry of the compounds and result in the
very low permeability of membrane. Moreover, biofilm plays an
essential role in drug resistance as well. Thus, 15f was selected to
measure its effect on P. aeruginosa ATCC27853 biofilm formation
using the crystal violet assay [36]. As shown in Fig. 4, the inhibition
rate of the P. aeruginosa ATCC27853 biofilm formation was very
similar to the negative control even though the concentration of 15f
different concentrations ranged from 2.5 to 10 mg/mL. Moreover,
the time-dependent light scattering intensity illustrated in Fig. 7
revealed that 15f stimulated the kinetics of BsFtsZ polymerization
in a concentration-dependent manner. As expected, both linezolid
(10 mg/mL) and DMSO vehicle had a negligible effect on the poly-
merization of BsFtsZ. This different stimulatory impact on FtsZ
polymerization indicated that 15f exerted antibacterial activity
through interacting with the FtsZ.
3.8. Promoting the assembly and bundling of FtsZ protofilaments
reached to 64 mg/mL. This result showed that 15f was impossible to
inhibit the biofilm formation of P. aeruginosa ATCC27853, thereby
Apart from the light scattering assay, the effect of 15f on the
demonstrating that it had no antibacterial activity against Gram-
polymerization of FtsZ were further analyzed by transmission
9

D. Song, N. Zhang, P. Zhang et al.
European Journal of Medicinal Chemistry 221 (2021) 113480
electron microscopy (TEM) to investigate the morphological change
of FtsZ filaments. As shown in Fig. 8, short, thin and single strand
FtsZ filaments were observed in the absence of the test compound.
After treated with 10 mg/mL of 15f, the size and thickness of the FtsZ
polymers substantially increased, implying that this compound
stimulated the formation of bundling of the FtsZ protofilaments in a
longitudinal and lateral manner. As expected, high efficacy of pro-
moting both the assembly of BsFtsZ and bunding of BsFtsZ polymers
were observed for 15f, which was in accordance with its result of
light scattering assay.
3.9. Evaluation of potential hemolysis
15f containing quaternary ammonium nitrogen might disrupt
mammalian cell membrane. In order to determine whether it was
cytotoxic to mammalian cells, the hemolysis assay was performed
with mouse red blood cells. 2% Triton X-100 was selected as a
positive control and the results were displayed in Fig. 9. When
tested at the concentration of 2 ꢂ MIC, 15f barely exhibited he-
molysis (0.71%). With the increase of the tested concentration,
however, its potential hemolysis also heightened. Nevertheless, 15f
only displayed 9.89% hemolysis even though its concentration
reached to 16 ꢂ MIC. All hemolysis levels were below 10%, which
indicated that 15f was not significantly hemolytic within the range
of its antibacterial concentration. These findings encouraged us to
explore its in vivo efficacy.
Fig. 3. Time-kill curves of compound 15f against S. aureus ATCC25923. Each data point
reflects the average of two independent measurements, with the error bars reflecting
the standard deviation from the mean.
3.10. In vivo efficacy against murine bacterial infection model
In view of its excellent in vitro antibacterial activity, 15f was
further assessed for in vivo efficacy using murine bacteremia model,
which are commonly used to evaluate the systemic effects of an-
tibiotics [46]. Thus, murine bacteremia model was established by
intraperitoneal injection with about 107 CFU/mL of the S. aureus
ATCC25923 solution, and 0.9% saline was injected as a negative
control group. The treatment was performed after the 1-h injection,
which is sufficient for the bacteria to spread throughout the mouse
[47] and then, mice were intraperitoneally treated with different
doses of 15f and linezolid dissolved in vehicle. As shown in Fig. 10,
Table 3
Fig. 4. 18 h biofilm biomass was measured by the crystal violet assay of P. aeruginosa
ATCC27853 bacterial culture and different concentrations of the test compound 15f
(Values are presented as the mean SD, n ¼ 3).
Comparison of MIC and MBC values for 15f in six Gram-positive bacteria strains.
Comp
MBC (
mg/mL)
MIC (
mg/mL)
MBC/MIC
Susceptible S. aureus (ATCC25923)
15f
San
Lin
2
>64
4
0.5
2
0.125
4
>32
32
15f at 25 mg/kg significantly reduced the bacterial load compared
with the no drug control group, and the difference was statistically
significant (P < 0.001). Contrarily, 15f at 1 mg/kg could not inhibit
the growth of bacteria. We inferred that this was related to its
metabolism in vivo. Furthermore, 15f could reduce the number of
viable pathogens of infected mice at medium dosage (5 mg/kg), and
its efficacy was as good as that of linezolid at the same concen-
tration. Although 15f showed better efficacy at high dosage, line-
zolid demonstrated better in vivo antibacterial activity than 15f.
These results suggested that 15f possessed therapeutic potential as
a novel antibacterial agent.
Methicillin-resistant S. aureus (ATCC43300)
15f
San
Lin
4
>64
NT
1
2
>64
4
>32
NT
Penicillin-resistant S. aureus PR
4
128
16
15f
San
Lin
2
16
4
2
8
4
Susceptible B. subtilis (ATCC9372)
15f
San
Lin
0.5
64
64
0.25
4
0.5
2
16
128
Susceptible B. pumilus (CMCC63202)
15f
San
Lin
2
>64
NT
0.5
1
>64
4
>64
NT
3.11. Computational studies of the binding mode
To gain more insights into the interaction mode of 15f with FtsZ,
computational docking studies were conducted using the reported
crystal structure of S. aureus FtsZ (PDB ID: 4DXD). The docking re-
sults demonstrated that 15f was inclined to bind near the T7-loop
Penicillin-resistant S. epidermidis
1
64
NT
15f
San
Lin
1
1
>64
1
64
NT
10

D. Song, N. Zhang, P. Zhang et al.
European Journal of Medicinal Chemistry 221 (2021) 113480
demonstrated moderate antibacterial activity, which compelled us
to further design and synthesize Series B, C and D based on the
analysis of the SARs to discover better antibacterial agents. As ex-
pected, compounds of Series B, C and D exhibited significant anti-
bacterial activity against most of the tested strains, including the
multidrug-resistant strains, compared with those of Series A. In
addition, some of the above compounds could inhibit the growth of
VRE effectively. It is noteworthy that the MIC values of 15f against
susceptible and resistant Gram-positive bacteria ranged from
<0.125 to 4
mg/mL, which were much lower than those of lead
compound berberine and less than or equal to those observed with
antibiotics commonly used in clinical as well. Particularly, 15f had
an MIC value of 4
mg/mL against VRE, which was much lower than
that observed for vancomycin (MIC > 64
mg/mL). Considering the
fact that 15f was inactive to Gram-negative bacteria, we further
confirmed that 15f could not inhibit the biofilm formation of
P. aeruginosa ATCC27853. In bactericidal kinetic assay, 15f was
found to have rapid bactericidal properties, reaching 2.2 log
reduction for S. aureus ATCC25923 (more than 99.99% of bacteria
were killed) within 3 h at 4 ꢂ MIC, which demonstrated that 15f
was an excellent bactericidal agent. Its rapid bactericidal activity
contributed to overcoming the emergence of drug resistance.
Further mechanistic studies by employing various biochemical
Fig. 5. Bacterial resistance studies of compound 15f and ciprofloxacin against S. aureus
ATCC25923.
(Fig. 11A). This binding site is a narrow hydrophobic cavity sur-
rounded by H7-helix, T7-loop, and a four-stranded b-sheet. This
narrow hydrophobic cavity required a certain degree of planarity,
which was in good agreement with configuration of 15f through
hydrophobic interactions. The side chain of 15f extended into the
depth of cavity forming hydrophobic interaction with LEU200,
VAL203, VAL207 and ILE201. Furthermore, quaternary ammonium
center of 15f was hydrophilic, which interacted with the sur-
rounding amino acid residues through an electrostatic interaction
or van der waals force with GLN192, GLY193 and GLY227. (Fig. 11B).
The docking result revealed 15f suited the hydrophobic cavity of
FtsZ perfectly, which was consistent with our design strategy.
4. Conclusion
In summary, four series of novel substituted 10-methyl-
acridinium derivatives were designed and synthesized for evalua-
tion of their in vitro and in vivo antibacterial activity. These
compounds were easily accessible in 2e5 synthetic steps with good
yields by employing some common intermediates, which con-
structed the compound library rapidly for the study of antibacterial
activity. The results suggested that some compounds of Series A
Fig. 7. Time-dependent polymerization profiles of BsFtsZ in the absence and presence
of compound 15f at a concentration range from 2.5 to 10 mg/mL.
Fig. 6. Effects of compound 15f on the cell morphology of B. pumilus cells in the absence (A) and in the presence (B) of 15f at 0.25 mg/mL.
11

D. Song, N. Zhang, P. Zhang et al.
European Journal of Medicinal Chemistry 221 (2021) 113480
assays, microscopic observation and computational docking
methods revealed that 15f functioned as an effective FtsZ inhibitor
to stimulate FtsZ polymerization through binding into the inter-
domain cleft of FtsZ. In a murine infection model induced by
S. aureus ATCC25923, 15f showed an excellent antibacterial efficacy
and no significant hemolysis to mammalian cells. Therefore, 15f can
be used as an effective candidate drug for further development to
combat drug-resistant pathogen infections.
5. Experimental sections
5.1. General experimental protocol
All commercially available reagents and solvents were used
without further purification. Analytical thin-layer chromatography
(TLC) was performed on 0.25-mm pre-coated silica GF254 plates
and visualized under UV light. Column chromatography of silica gel
(200e300 mesh) was used to separate and purify intermediates
and compounds. High resolution mass spectra (HRMS) were per-
formed at the Analytical Center for Structural Constituent and
Physical Property of Shandong University, Jinan, China. 1H NMR and
13C NMR spectra were recorded on Bruker Avance DRX 400/600
spectrometer (Bruker, Switzerlands) at ambient temperature in
CDCl3, DMSO‑d6 or CD3OD solution. Tetramethylsilane (TMS) was
employed as an internal standard of chemical shifts. Melting points
were measured using an uncorrected RY-1 melting point apparatus.
The high-performance liquid chromatography (HPLC) method was
established using an Agilent 1200 Series equipment coupled to
ultravioletevisible detector (UVD) at 254 nm with a Diamonil C18
Fig. 9. The hemolysis rate of mice red blood cell induced by positive control (Triton X-
100), negative control (PBS) and compound 15f at 1 ꢂ MIC, 2 ꢂ MIC, 4 ꢂ MIC, 8 ꢂ MIC
and 16 ꢂ MIC, respectively. Bars represent the standard deviation (n ¼ 3).
the filtrate was acidified with diluted HCl to pH 5e6. The precipi-
tate was purified using recrystallization from ethanol to obtain pure
product as a grey solid (1.65g, 78%). 1H NMR (400 MHz, DMSO‑d6)
d
13.06 (s, 1H), 9.63 (s, 1H), 7.90 (dd, J ¼ 8.0, 1.5 Hz, 1H), 7.37 (ddd,
reversed phase column (150 ꢂ 4.6 mm, 5
m
m), injection volume of
J ¼ 12.8, 9.7, 7.3 Hz, 3H), 7.23 (t, J ¼ 7.5 Hz, 3H), 7.07 (t, J ¼ 7.3 Hz,
1H), 6.78 (t, J ¼ 7.5 Hz, 1H). MS (ESI) m/z calcd for C13H11NO2
[M ꢃ H]-, 212.1, found: 212.20.
5
m
L, and it was maintained at ambient temperature. The effective
chromatogram was obtained using the mobile phase of acetonitrile:
0.025 mol/L potassium dihydrogen phosphate buffer (60 : 40, vol/
vol) and the pH value was adjusted to 3 with phosphoric acid at the
flow rate of 1.0 mL/min. The purity of the test compounds as
determined by analytical HPLC was more than 95%.
5.2.2. 9(10H)-Acridinone (3)
To a 100 mL round bottom flask containing 2 (2.0 g, 9.4 mmol),
was added concentrated H2SO4 (10 mL). The mixture was stirred at
100 ꢄC under N2 atmosphere for 6 h, monitored by using TLC. After
cooling down, the reaction mixture was poured into ice water,
stirred and filtered. The obtained solid was washed with saturated
NaHCO3 solution, and dried to give intermediate 3 as a yellow solid
5.2. Typical procedure for the synthesis of Series A, B, C and D
5.2.1. 2-(Phenylamino)benzoic acid (2)
A solution of 2-bromobenzoic acid (2.0 g, 10 mmol), aniline
(1.85 g, 20 mmol), Cu powder (0.2e0.3
mm, 4.97 mmol), and K2CO3
(1.5g, 82%). 1H NMR (400 MHz, DMSO‑d6)
d 11.73 (s, 1H), 8.23 (d,
(1.37g, 10 mmol) in EtOH (80 ml) was heated under reflux for 12 h,
monitored by using TLC. The cooled reaction mixture was poured
into hot water. The mixture was filtrated through Celite, and then
J ¼ 7.8 Hz, 2H), 7.75e7.71 (m, 2H), 7.54 (d, J ¼ 8.3 Hz, 2H), 7.25 (t,
J ¼ 7.2 Hz, 2H). MS (ESI) m/z calcd for C13H9NO [M þ H]þ, 196.1,
found: 391.3.
Fig. 8. Transmission electron micrographs of BsFtsZ polymers in the absence (A) and in the presence (B) of 15f at 10
12
mg/mL.

D. Song, N. Zhang, P. Zhang et al.
European Journal of Medicinal Chemistry 221 (2021) 113480
(4 mL) and the mixture was refluxed under nitrogen atmosphere
for 2 h. After the completion of the reaction, intermediate 5 was
obtained as a light yellow solid after concentration under reduced
pressure at 50 ꢄC to remove the excess of SOCl2. This intermediate
could be used in the next step without purification.
5.2.5. Typical procedure for the synthesis of Series A
A solution of 5 (1.0 eqiv) and substituted amines (1.1eqiv) in
anhydrous acetonitrile (7 mL) was heated at 60 ꢄC for 5 h, moni-
tored by using TLC. The reaction mixture was concentrated under
reduced pressure and purified on silica gel with DCM/MeOH (20/1)
to give the compounds of Series A in good yields.
5.2.5.1. 10-Methyl-9-(ethylamino)acridin-10-ium chloride. (6a).
Yellow solid, yield 82%, m.p.: 240e243 ꢄC; 1H NMR (600 MHz,
Chloroform-d)
d 12.16 (s, 1H), 9.45 (s, 1H), 8.18 (s, 1H), 7.87 (d,
J ¼ 29.9 Hz, 2H), 7.60 (dd, J ¼ 97.0, 77.2 Hz, 4H), 4.05 (s, 3H), 3.48 (s,
2H), 1.73 (t, J ¼ 7.1 Hz, 3H). 13C NMR (150 MHz, CDCl3)
d 159.01,
142.48, 139.65, 135.35, 134.93, 127.52, 127.12, 124.07, 121.98, 116.07,
114.98, 112.09, 44.26, 35.80, 15.20. HRMS (ESI) m/z calcd for
C
16H17ClN2 [M ꢃ Cl]þ, 237.1386, found: 237.1395.
Fig. 10. Efficacy of 15f in the bacteremia model infected with S. aureus ATCC25923. The
infected mice were treated with 15f (1, 5 and 25 mg/kg) or linezolid (1, 5 and 25 mg/
kg) via intraperitoneal injection (n ¼ 6). Each point represents the determination of a
single animal, and the line shows the mean value. *P < 0.05, **P < 0.01, ***P < 0.001,
compared with untreated control group.
5.2.5.2. 10-Methyl-9-(propylamino)acridin-10-ium chloride. (6b).
Yellow solid, yield 78%, m.p.: 200e202 ꢄC; 1H NMR (600 MHz,
Chloroform-d)
d 12.13 (s, 1H), 9.49 (s, 1H), 8.15 (s, 1H), 7.87 (d,
J ¼ 23.7 Hz, 2H), 7.73e7.44 (m, 4H), 4.10e4.07 (m, 2H), 4.05 (s, 3H),
2.25e2.19 (m, 2H),1.05 (t, J ¼ 7.3 Hz, 3H). 13C NMR (150 MHz, CDCl3)
d
158.77, 135.09, 134.74, 127.13, 126.81, 123.58, 121.61, 115.97, 114.98,
5.2.3. 10-Methylacridin-9(10H)-one (4)
50.64, 35.69, 22.73, 10.89. HRMS (ESI) m/z calcd for C17H19ClN2
Intermediate 3 (1.5 g, 7.7 mmol) was dissolved in DMF (40 mL)
and NaH (65% oil dispersion, 460 mg, 19.2 mmol) was added at 0 ꢄC.
The suspension was stirred at 0 ꢄC for 30 min and at 60 ꢄC for
30 min, and then CH3I (2.7g, 19.2 mmol) was added. The resultant
solution was continuously stirred at 60 ꢄC for 18 h. After cooling to
room temperature, the reaction solution was quenched with water.
The resulting solid was filtrated and dried in vacuo to afford the title
compound as a yellow solid (1.4g, 87%). 1H NMR (400 MHz,
[M ꢃ Cl]þ, 251.1543, found: 251.1550.
5.2.5.3. 10-Methyl-9-(isopropylamino)acridin-10-ium chloride. (6c).
Yellow solid, yield 75%, m.p.: 218e220 ꢄC; 1H NMR (600 MHz,
Chloroform-d)
d 11.94 (s, 1H), 9.80 (s, 1H), 8.10 (s, 1H), 7.86 (s, 2H),
7.59 (d, J ¼ 83.5 Hz, 4H), 4.67e4.62 (m, 1H), 4.04 (s, 3H), 1.83 (d,
J ¼ 6.4 Hz, 6H). 13C NMR (150 MHz, CDCl3)
d 158.80, 135.06, 127.50,
123.98, 121.97, 115.96, 114.89, 52.99, 35.75, 22.77. HRMS (ESI) m/z
DMSO‑d6)
d
8.36e8.33 (m, 2H), 7.88e7.81 (m, 4H), 7.34 (ddd, J ¼ 7.9,
calcd for C17H19ClN2 [M ꢃ Cl]þ, 251.1543, found: 251.1559.
6.4, 1.4 Hz, 2H), 3.95 (s, 3H). MS (ESI) m/z calcd for C14H11NO [M þ
H]þ, 210.1, found: 210.3.
5.2.5.4. 10-Methyl-9-(pentylamino)acridin-10-ium chloride. (6d).
Yellow solid, yield 72%, m.p.: 194e197 ꢄC; 1H NMR (600 MHz,
5.2.4. 9-Chloro-10-methylacridin-10-ium chloride (5)
Chloroform-d)
d 12.12 (s, 1H), 9.45 (s, 1H), 8.16 (s, 1H), 7.86 (d,
Intermediate 4 (150 mg, 0.57 mmol) was dissolved in SOCl2
J ¼ 22.6 Hz, 2H), 7.67 (d, J ¼ 72.4 Hz, 3H), 7.44 (s, 1H), 4.12e4.08 (m,
Fig. 11. Predicted binding mode of 10-methylacridinium derivatives. (A) 15f bound to the C-terminal interdomain cleft of SaFtsZ (PDB: 4DXD); (B) Predicted interaction between 15f
and amino acid residues of SaFtsZ.
13

D. Song, N. Zhang, P. Zhang et al.
European Journal of Medicinal Chemistry 221 (2021) 113480
2H), 4.04 (s, 3H), 2.20e2.15 (m, 2H), 1.44e1.38 (m, 4H), 0.91 (t,
(600 MHz, Chloroform-d) d 13.49 (s, 1H), 8.76 (s, 2H), 7.83 (d,
J ¼ 7.0 Hz, 3H). 13C NMR (150 MHz, CDCl3)
d
158.94, 142.47, 139.60,
J ¼ 62.0 Hz, 3H), 7.66 (d, J ¼ 7.7 Hz,1H), 7.40e7.32 (m, 5H), 7.28e7.27
135.30, 134.84, 127.42, 127.10, 123.94, 121.84, 116.08, 114.99, 112.05,
(m, 1H), 4.22 (s, 3H), 2.97e2.94 (m, 2H), 1.36 (t, J ¼ 7.5 Hz, 3H). 13
C
49.27, 35.79, 29.24, 28.77, 21.98, 13.77. HRMS (ESI) m/z calcd for
NMR (150 MHz, CDCl3) d 156.88, 141.04, 139.27, 137.57, 137.28,
C
19H23ClN2 [M ꢃ Cl]þ, 279.1856, found: 279.1850.
135.87, 129.35,128.75,128.64,128.56,128.42, 127.30,127.20,126.44,
126.38, 123.38, 123.14, 116.83, 114.04, 24.20, 23.40, 13.59, 13.23.
HRMS (ESI) m/z calcd for C22H21ClN2 [M ꢃ Cl]þ, 313.1699, found:
313.1707.
5.2.5.5. 10-Methyl-9-(hexylamino)acridin-10-ium chloride. (6e).
Yellow solid, yield 74%, m.p.: 188e190 ꢄC; 1H NMR (600 MHz,
Chloroform-d)
d 12.16 (s, 1H), 9.46 (s, 1H), 8.15 (s, 1H), 7.86 (d,
J ¼ 33.1 Hz, 2H), 7.74e7.44 (m, 4H), 4.12e4.08 (m, 2H), 4.04 (s, 3H),
5.2.5.12. 10-Methyl-9-((2-methoxyphenyl)amino)acridin-10-ium
2.18e2.16 (m, 2H), 1.45e1.42 (m, 2H), 1.34e1.31 (m, 4H), 0.86 (t,
chloride. (6l). Yellow solid, yield 83%, m.p.: 124e128 ꢄC; 1H NMR
J ¼ 7.0 Hz, 3H). 13C NMR (150 MHz, CDCl3)
d
158.91, 142.39, 139.58,
(600 MHz, Chloroform-d) d 13.38 (s, 1H), 8.90 (s, 2H), 7.83 (d,
135.31, 134.81, 127.38, 127.01, 123.89, 121.82, 116.07, 115.02, 112.03,
49.28, 35.83, 35.80, 30.98, 29.46, 26.30, 22.25, 13.72. HRMS (ESI) m/
z calcd for C20H25ClN2 [M ꢃ Cl]þ, 293.2012, found: 293.2002.
J ¼ 37.1 Hz, 4H), 7.37 (s, 2H), 7.22 (s, 2H), 6.86 (d, J ¼ 37.4 Hz, 2H),
4.24 (s, 3H), 3.82 (s, 3H). HRMS (ESI) m/z calcd for C21H19ClN2
[M ꢃ Cl]þ, 315.1492, found: 315.1498.
5.2.5.6. 10-Methyl-9-(heptylamino)acridin-10-ium chloride. (6f).
Yellow solid, yield 79%, m.p.: 180e183 ꢄC; 1H NMR (600 MHz,
5.2.5.13. 10-Methyl-9-((2-(trifluoromethyl)phenyl)amino)acridin-10-
ium chloride. (6m). Yellow solid, yield 79%, m.p.: 199e202 ꢄC; 1H
Chloroform-d)
d
12.10 (s, 1H), 9.42 (s, 1H), 8.16 (s, 1H), 7.86 (d,
NMR (600 MHz, Chloroform-d) d 13.68 (s, 1H), 8.19 (s, 2H), 7.73 (d,
J ¼ 22.1 Hz, 2H), 7.72e7.43 (m, 4H), 4.10 (d, J ¼ 8.3 Hz, 2H), 4.04 (s,
3H), 2.18e2.15 (m, 2H), 1.44e1.42 (m, 2H), 1.37e1.34 (m, 2H),
1.28e1.27 (m, 4H), 0.86 (t, J ¼ 6.8 Hz, 3H). 13C NMR (150 MHz,
J ¼ 35.6 Hz, 4H), 7.56 (d, J ¼ 8.6 Hz, 3H), 7.34 (s,1H), 7.19 (s, 2H), 4.02
(s, 3H). HRMS (ESI) m/z calcd for C21H16ClF3N2 [M ꢃ Cl]þ, 353.1260,
found: 353.1266.
CDCl3)
d 159.08, 142.52, 139.68, 135.28, 134.93, 127.61, 127.28,
124.13, 121.86, 116.04, 114.90, 112.22, 49.40, 35.76, 31.53, 29.62,
28.63, 26.73, 22.38, 13.90. HRMS (ESI) m/z calcd for C21H27ClN2
[M ꢃ Cl]þ, 307.2169, found: 307.2174.
5.2.5.14. 10-Methyl-9-([1,10-biphenyl]-4-ylamino)acridin-10-ium
chloride. (6n). Yellow solid, yield 81%, m.p.: 167e170 ꢄC; 1H NMR
(600 MHz, Chloroform-d)
d
7.64 (d, J ¼ 7.4 Hz, 2H), 7.58 (d,
J ¼ 8.3 Hz, 3H), 7.53 (d, J ¼ 7.3 Hz, 1H), 7.45e7.38 (m, 6H), 7.33e7.27
5.2.5.7. 10-Methyl-9-(octylamino)acridin-10-ium chloride. (6g).
Yellow solid, yield 80%, m.p.: 186e188 ꢄC; 1H NMR (600 MHz,
(m, 2H), 7.06 (s, 3H), 3.83 (s, 3H). HRMS (ESI) m/z calcd for
C
26H21ClN2 [M ꢃ Cl]þ, 361.1699, found: 361.1694.
Chloroform-d)
d 12.18 (s, 1H), 9.47 (s, 1H), 8.16 (s, 1H), 7.87 (d,
J ¼ 23.9 Hz, 2H), 7.73e7.44 (m, 4H), 4.11 (s, 2H), 4.05 (s, 3H), 2.18 (s,
5.2.5.15. 10-Methyl-9-((2,4-difluorophenyl)amino)acridin-10-ium
chloride. (6o). Yellow solid, yield 84%, m.p.: 254e258 ꢄC; 1H NMR
2H), 1.44 (t, J ¼ 7.3 Hz, 2H), 1.36e1.34 (m, 2H), 1.29e1.23 (m, 6H),
0.86 (t, J ¼ 7.0 Hz, 3H). 13C NMR (150 MHz, CDCl3)
d
158.86, 142.35,
(600 MHz, DMSO‑d6)
(d,
126.6 Hz, 5H), 4.23 (s, 3H). 13C NMR (150 MHz,
CD3OD þ CDCl3) 157.85, 142.95, 142.27, 137.20, 134.79, 129.34 (d,
d
11.65 (s, 1H), 8.10 (d, J ¼ 115.6 Hz, 6H), 7.38
139.53, 135.29, 134.78, 127.33, 126.92, 123.84, 121.80, 116.09, 115.04,
111.97, 49.26, 35.84, 31.40, 29.48, 28.85, 28.77, 26.61, 22.27, 13.80.
HRMS (ESI) m/z calcd for C22H29ClN2 [M ꢃ Cl]þ, 321.2325, found:
321.2335.
J
¼
d
JF-C ¼ 8.6 Hz), 127.02, 125.72, 124.92, 121.79, 118.40, 116.07, 115.16,
113.52 (d, JF-C ¼ 24.0 Hz), 105.83 (d, JF-C ¼ 24.0 Hz), 37.57. HRMS
(ESI) m/z calcd for C20H15ClF2N2 [M ꢃ Cl]þ, 321.1198, found:
321.1207.
5.2.5.8. 10-Methyl-9-(cyclopropylamino)acridin-10-ium
(6h). Yellow solid, yield 78%, m.p.: 220e223 ꢄC; 1H NMR (600 MHz,
Chloroform-d)
chloride.
d
12.19 (s, 1H), 8.93 (s, 1H), 7.88 (d, J ¼ 20.4 Hz, 2H),
5.2.5.16. 10-Methyl-9-((2,4-dichlorophenyl)amino)acridin-10-ium
7.74e7.45 (m, 5H), 4.08 (s, 3H), 3.44e3.40 (m, 1H), 1.57e1.54 (m,
2H), 1.21e1.17 (m, 2H). HRMS (ESI) m/z calcd for C17H17ClN2
[M ꢃ Cl]þ, 249.1386, found: 249.1395.
chloride. (6p). Yellow solid, yield 78%, m.p.: 249e254 ꢄC; 1H NMR
(600 MHz, DMSO‑d6)
d
7.77 (d, J ¼ 37.6 Hz, 7H), 7.35 (d, J ¼ 91.4 Hz,
4H), 4.00 (s, 3H). HRMS (ESI) m/z calcd for C20H15Cl3N2 [M ꢃ Cl]þ,
353.0607, found: 353.0601.
5.2.5.9. 10-Methyl-9-(cyclohexylamino)acridin-10-ium chloride. (6i).
Yellow solid, yield 72%, m.p.: 215e219 ꢄC; 1H NMR (600 MHz,
5.2.5.17. 10-Methyl-9-((2,4-dimethylphenyl)amino)acridin-10-ium
Chloroform-d)
d
11.75 (s,1H), 9.77 (s, 1H), 7.86 (s, 2H), 7.65e7.50 (m,
chloride. (6q). Yellow solid, yield 82%, m.p.: 189e192 ꢄC; 1H NMR
5H), 4.19 (d, J ¼ 9.1 Hz, 1H), 4.03 (s, 3H), 2.73 (d, J ¼ 14.2 Hz, 2H),
2.02 (d, J ¼ 12.8 Hz, 2H), 1.94 (d, J ¼ 13.5 Hz, 2H), 1.66 (d, J ¼ 13.3 Hz,
1H), 1.49e1.46 (m, 1H), 1.27 (d, J ¼ 13.3 Hz, 2H). 13C NMR (150 MHz,
(600 MHz, Chloroform-d)
d
7.83 (d, J ¼ 64.0 Hz, 3H), 7.47 (d,
J ¼ 8.0 Hz, 2H), 7.39 (s, 1H), 7.12 (s, 1H), 7.07 (s, 2H), 7.03 (d,
J ¼ 7.9 Hz, 2H), 4.21 (s, 3H), 2.52 (s, 6H). HRMS (ESI) m/z calcd for
CDCl3)
d
158.97, 135.06, 133.73, 127.58, 121.11, 114.69, 60.47, 54.44,
C
22H21ClN2 [M ꢃ Cl]þ, 313.1699, found: 313.1689.
35.67, 32.40, 25.18, 24.28. HRMS (ESI) m/z calcd for C20H23ClN2
[M ꢃ Cl]þ, 291.1856, found: 291.1856.
5.2.5.18. 10-Methyl-9-((2,4-dimethoxyphenyl)amino)acridin-10-ium
chloride. (6r). Yellow solid, yield 79%, m.p.: 146e148 ꢄC; 1H NMR
5.2.5.10. 10-Methyl-9-(phenylamino)acridin-10-ium chloride. (6j).
Yellow solid, yield 78%, m.p.: 231e235 ꢄC; 1H NMR (600 MHz,
(600 MHz, Chloroform-d) d 7.46 (s, 3H), 7.28 (s, 2H), 7.26 (s, 1H),
6.78e6.77 (m, 2H), 6.51e6.46 (m, 3H), 3.82 (s, 3H), 3.71 (s, 3H), 3.62
Chloroform-d)
d
13.57 (s, 1H), 8.76 (s, 3H), 7.73 (d, J ¼ 67.8 Hz, 4H),
(s, 3H). 13C NMR (150 MHz, CDCl3 þ CD3OD)
d 155.88, 154.72,
7.26 (s, 6H), 4.12 (s, 3H). 13C NMR (150 MHz, CDCl3 þ CD3OD)
149.72, 134.80, 130.53, 126.80, 119.96, 113.49, 104.58, 99.78, 55.09,
55.04, 33.55. HRMS (ESI) m/z calcd for C22H21ClN2O2 [M ꢃ Cl]þ,
345.1598, found: 345.1600.
d
155.31, 143.53, 141.44, 134.39, 129.45, 127.05, 125.87, 122.47,
122.39, 116.20, 115.58, 35.33. HRMS (ESI) m/z calcd for C20H17ClN2
[M ꢃ Cl]þ, 285.1386, found: 285.1396.
5.2.5.19. 10-Methyl-9-(1H-pyrrol-1-yl)acridin-10-ium chloride. (6s).
Brown solid, yield 68%, m.p.: 199e203 ꢄC; 1H NMR (600 MHz,
5.2.5.11. 10-Methyl-9-((2-ethylphenyl)amino)acridin-10-ium chlo-
ride. (6k). Yellow solid, yield 82%, m.p.: 187e191 ꢄC; 1H NMR
DMSO‑d6)
d
8.58 (d, J ¼ 5.0 Hz, 1H), 8.52 (d, J ¼ 5.2 Hz, 2H), 8.34 (dd,
14

D. Song, N. Zhang, P. Zhang et al.
European Journal of Medicinal Chemistry 221 (2021) 113480
J ¼ 8.0, 1.3 Hz, 2H), 7.87 (d, J ¼ 8.4 Hz, 2H), 7.85e7.82 (m, 2H),
7.36e7.33 (m, 2H), 6.88 (t, J ¼ 5.2 Hz, 1H), 3.95 (s, 3H). 13C NMR
ether/EtOAc (8/1) to give titled compound (0.13 g) as a white solid
in 89% yield. 1H NMR (400 MHz, Chloroform-d)
d
8.31 (d, J ¼ 8.2 Hz,
(150 MHz, CD3OD)
d
152.88, 142.40, 138.37, 131.48, 127.71, 126.58,
2H), 7.77 (dd, J ¼ 18.2, 8.0 Hz, 4H), 7.54e7.50 (m, 1H), 7.47e7.43 (m,
2H), 7.13 (dd, J ¼ 8.2, 2.2 Hz, 1H), 7.03 (d, J ¼ 7.5 Hz, 1H), 6.99 (d,
J ¼ 2.0 Hz, 1H), 3.87 (s, 3H). MS (ESI) m/z calcd for C20H15NO [M þ
H]þ, 287.1, found: 287.6.
125.70, 123.92, 120.33, 120.30, 118.70, 111.97, 38.40. HRMS (ESI) m/z
calcd for C18H15ClN2 [M ꢃ Cl]þ, 259.1230, found: 259.1228.
5.2.5.20. 10-Methyl-9-(thiazol-2-ylamino)acridin-10-ium chloride.
(6t). Dark red solid, yield 69%, m.p.: 137e141 ꢄC; 1H NMR
5.2.8. Typical procedure for the synthesis of Series B
(600 MHz, Chloroform-d)
d
8.14e8.13 (m, 2H), 7.64 (t, J ¼ 7.5 Hz,
Intermediate 8d (100 mg, 0.39 mmol) was dissolved in an excess
of CH3I (0.25 mL, 3.9 mmol) within a sealed tube. The mixture was
heated at 85 ꢄC for 24 h and then isopropyl ether (50 mL) was added
to separate out a dark red solid. The crude product was purified via
column chromatography (DCM/MeOH 10/1) to yield the corre-
sponding product as a red solid (80 mg, 76%).
2H), 7.47 (d, J ¼ 8.2 Hz, 3H), 7.14 (t, J ¼ 7.5 Hz, 2H), 6.90e6.89 (m,
1H), 3.89 (s, 3H), 3.49 (s, 1H). 13C NMR (150 MHz, CDCl3 þ CD3OD)
d
158.09, 141.55, 133.68, 132.43, 127.85, 127.17, 121.03, 120.86, 119.16,
114.60, 114.48, 34.14. HRMS (ESI) m/z calcd for C17H14ClN3S
[M ꢃ Cl]þ, 292.0903, found: 292.0893.
5.2.5.21. 10-Methyl-9-(pyridin-2-ylamino)acridin-10-ium chloride.
(6u). Yellow solid, yield 72%, m.p.: 188e191 ꢄC; 1H NMR (600 MHz,
5.2.8.1. 10-Methyl-9-phenylacridin-10-ium iodide. (9a). Red solid,
yield 76%, m.p.: 236e240 ꢄC; 1H NMR (600 MHz, Chloroform-d)
Chloroform-d)
d
8.57 (dd, J ¼ 8.0, 1.7 Hz, 2H), 7.85 (d, J ¼ 5.4 Hz, 1H),
d
8.94 (d, J ¼ 9.2 Hz, 2H), 8.42 (ddd, J ¼ 9.0, 6.7, 1.3 Hz, 2H), 8.01 (dd,
J ¼ 8.7, 1.1 Hz, 2H), 7.81 (dd, J ¼ 8.5, 6.8 Hz, 2H), 7.75e7.71 (m, 3H),
7.50e7.49 (m, 2H), 5.28 (s, 3H). 13C NMR (150 MHz, CDCl3)
160.78,
7.74 (ddd, J ¼ 8.7, 7.0, 1.7 Hz, 2H), 7.61e7.58 (m, 2H), 7.55 (d,
J ¼ 8.7 Hz, 2H), 7.31 (t, J ¼ 7.5 Hz, 2H), 6.85 (d, J ¼ 8.7 Hz,1H), 6.70 (t,
J ¼ 6.4 Hz, 1H), 3.92 (s, 3H). 13C NMR (150 MHz, CDCl3 þ CD3OD)
d
140.86, 138.94, 132.26, 130.02, 129.63, 129.23, 128.54, 127.58, 125.51,
118.92, 40.80. HRMS (ESI) m/z calcd for C20H16IN [M ꢃ I]þ, 270.1277,
found: 270.1279.
d
177.94, 155.94, 142.03, 140.60, 139.77, 133.61, 132.27, 127.53,
126.93, 121.70, 120.94, 114.57, 112.37, 111.52, 33.23. HRMS (ESI) m/z
calcd for C19H16ClN3 [M ꢃ Cl]þ, 286.1339, found: 286.1338.
5.2.5.22. 10-Methyl-9-(pyrimidin-2-ylamino)acridin-10-ium
ride. (6v). Orange solid, yield 71%, m.p.: 179e183 ꢄC; 1H NMR
(600 MHz, DMSO‑d6)
(dd, J ¼ 8.7, 1.1 Hz, 2H), 8.40e8.37 (m, 2H), 7.96 (dd, J ¼ 8.3, 7.1 Hz,
2H), 7.60e7.59 (m, 1H), 6.96 (t, J ¼ 3.6 Hz, 1H), 6.66e6.49 (m, 1H),
4.79 (s, 3H). HRMS (ESI) m/z calcd for C18H15ClN4 [M ꢃ Cl]þ,
287.1291, found: 287.1280.
chlo-
5.2.8.2. 10-Methyl-9-(o-tolyl)acridin-10-ium iodide. (9b). Red solid,
yield 72%, m.p.: 230e233 ꢄC; 1H NMR (600 MHz, Chloroform-d)
d
12.42 (s, 1H), 8.74 (d, J ¼ 9.1 Hz, 2H), 8.51
d
8.97 (d, J ¼ 9.2 Hz, 2H), 8.44e8.41 (m, 2H), 7.88e7.86 (m, 2H), 7.80
(dd, J ¼ 8.5, 6.7 Hz, 2H), 7.65e7.62 (m, 1H), 7.55e7.50 (m, 2H), 7.27
(s, 1H), 5.30 (s, 1H), 1.93 (s, 1H). 13C NMR (150 MHz, CDCl3)
161.44,
d
141.16, 139.35, 135.88, 132.30, 130.70, 130.35, 129.36, 129.10, 128.10,
126.18, 125.75, 119.57, 41.22, 19.80. HRMS (ESI) m/z calcd for
C
21H18IN [M ꢃ I]þ, 284.1434, found: 284.1428.
5.2.5.23. 10-Methyl-9-(pyrimidin-4-ylamino)acridin-10-ium
ride. (6w). Yellow solid, yield 70%, m.p.: 191e194 ꢄC; 1H NMR
(600 MHz, Chloroform-d)
chlo-
5.2.8.3. 10-Methyl-9-(m-tolyl)acridin-10-ium iodide. (9c). Red solid,
yield 69%, m.p.: 226e228 ꢄC; 1H NMR (600 MHz, Chloroform-d)
d
8.82 (s, 1H), 8.36 (d, J ¼ 5.6 Hz, 1H), 8.03
(d, J ¼ 7.3 Hz, 2H), 7.68 (t, J ¼ 7.6 Hz, 2H), 7.52 (d, J ¼ 8.4 Hz, 2H), 7.18
d
8.95 (d, J ¼ 9.2 Hz, 2H), 8.42 (ddd, J ¼ 9.0, 6.7, 1.3 Hz, 2H), 8.02 (dd,
(t, J ¼ 7.3 Hz, 2H), 6.71 (s, 1H), 3.93 (s, 3H). 13C NMR (150 MHz,
J ¼ 8.7, 1.1 Hz, 2H), 7.81 (dd, J ¼ 8.5, 6.8 Hz, 2H), 7.59 (t, J ¼ 7.9 Hz,
CDCl3)
d 159.42, 156.43, 141.75, 132.44, 128.30, 121.03, 119.42,
1H), 7.53 (d, J ¼ 7.6 Hz, 1H), 7.27 (d, J ¼ 7.1 Hz, 2H), 5.27 (s, 3H), 2.53
114.50, 111.10, 34.24. HRMS (ESI) m/z calcd for C18H15ClN4 [M ꢃ Cl]þ,
(s, 3H). 13C NMR (150 MHz, CDCl3)
d 161.42, 141.14, 139.14, 138.74,
287.1291, found: 287.1289.
132.56,130.99,130.04,129.92,128.65,127.73,126.69, 125.80, 119.20,
41.11, 21.29. HRMS (ESI) m/z calcd for C21H18IN [M ꢃ I]þ, 284.1434,
found: 284.1442.
5.2.6. 9-Bromoacridine (7)
PBr3 (19.5 mL, 205 mmol) was added dropwise to a 250 mL
round bottom flask containing 3 (4.0 g, 20.5 mmol) at 0 ꢄC under N2
atmosphere. After stirring at 110 ꢄC for 24 h, the resultant solution
was quenched by slow addition of H2O and an aqueous NaOH so-
lution was added to the resultant mixture until pH ¼ 14. The crude
product was extracted with CH2Cl2 (50 mL ꢂ 3). The combined
organic extracts were washed with brine, dried over Na2SO4, fil-
trated, and concentrated under reduced pressure to obtain the title
compound as a yellow solid (3.81 g, 72%). 1H NMR (400 MHz,
5.2.8.4. 10-Methyl-9-(3-methoxyphenyl)acridin-10-ium iodide. (9d).
Red solid, yield 70%, m.p.: 227e229 ꢄC; 1H NMR (600 MHz, Chlo-
roform-d)
d
8.94 (dd, J ¼ 9.2, 3.6 Hz, 2H), 8.43e8.40 (m, 2H),
8.05e8.03 (m, 2H), 7.82e7.80 (m, 2H), 7.63e7.60 (m, 1H), 7.25e7.23
(m, 1H), 7.05 (dd, J ¼ 7.4, 1.3 Hz, 1H), 7.02e7.01 (m, 1H), 5.26 (s, 3H),
3.89 (s, 3H). 13C NMR (150 MHz, CDCl3)
d 160.62, 159.24, 140.99,
139.08, 133.64, 129.95, 129.79, 127.70, 125.59, 121.67, 119.05, 115.35,
115.20, 55.38, 40.96. HRMS (ESI) m/z calcd for C21H18INO [M ꢃ I]þ,
300.1383, found: 300.1373.
Chloroform-d)
d
8.45 (d, J ¼ 8.8 Hz, 2H), 8.27 (d, J ¼ 8.6 Hz, 2H),
7.85e7.81 (m, 2H), 7.69e7.65 (m, 2H). MS (ESI) m/z calcd for
C
13H8BrN [M þ H]þ, 258.0, found: 260.2.
5.2.8.5. 10-Methyl-9-(3-fluorophenyl)acridin-10-ium iodide. (9e).
Red solid, yield 68%, m.p.: 230e232 ꢄC; 1H NMR (600 MHz, Chlo-
5.2.7. 9-(3-Methoxyphenyl)acridine (8d)
To a solution of 7 (150 mg, 0.58 mmol) in dioxane and water
(5:1), aryl boronic acid (0.11 g, 0.87 mmol), and K2CO3 (0.24 g,
17.4 mmol) was added under N2 atmosphere. After Pd(PPh3)4
(0.005 mmol) was added, the resulting solution was heated to
100 ꢄC for 8 h. After completion of the reaction, the aqueous layer
was extracted with CH2Cl2 (30 mL ꢂ 3). The combined organic layer
was dried over Na2SO4, filtrated, concentrated under reduced
pressure and the residue was purified on silica gel with petroleum
roform-d)
d
8.95 (d, J ¼ 9.2 Hz, 2H), 8.43 (ddd, J ¼ 8.9, 6.7, 1.5 Hz,
2H), 7.98 (d, J ¼ 8.6 Hz, 2H), 7.85e7.83 (m, 2H), 7.74e7.70 (m, 1H),
7.47e7.44 (m,1H), 7.32 (d, J ¼ 7.5 Hz,1H), 7.24 (t, J ¼ 2.1 Hz,1H), 5.28
(s, 3H). 13C NMR (150 MHz, CDCl3)
d 163.19, 161.52, 158.85 (d, JF-
¼ 2.0 Hz), 141.34, 139.31, 134.62 (d, JF-C ¼ 7.7 Hz), 131.04 (d, JF-
C
C
¼ 8.3 Hz), 129.49, 128.16, 125.78, 125.56 (d, JF-C ¼ 3.3 Hz), 119.59,
117.45 (d, JF-C ¼ 20.9 Hz),116.88 (d, JF-C ¼ 23.2 Hz), 41.45. HRMS (ESI)
m/z calcd for C20H15FIN [M ꢃ I]þ, 288.1183, found: 288.1196.
15

D. Song, N. Zhang, P. Zhang et al.
European Journal of Medicinal Chemistry 221 (2021) 113480
5.2.8.6. 10-Methyl-9-(3-nitrophenyl)acridin-10-ium iodide. (9f).
Red solid, yield 72%, m.p.: 232e236 ꢄC; 1H NMR (600 MHz, Chlo-
126.55, 125.17, 118.93. HRMS (ESI) m/z calcd for C26H20IN [M ꢃ I]þ,
346.1590, found: 346.1583.
roform-d)
(ddd, J ¼ 8.5, 6.1, 1.7 Hz, 2H), 8.39 (s, 1H), 8.01e7.97 (m, 2H),
7.89e7.86 (m, 4H), 5.27 (s, 3H). 13C NMR (150 MHz, DMSO)
157.35,
d
8.92 (d, J ¼ 9.2 Hz, 2H), 8.62 (d, J ¼ 7.9 Hz, 1H), 8.44
5.2.8.13. 10-Methyl-9-(4-(trifluoromethyl)phenyl)acridin-10-ium io-
dide. (9m). Red solid, yield 71%, m.p.: 241e246 ꢄC; 1H NMR
d
147.64, 140.82, 138.26, 135.81, 134.25, 130.38, 129.12, 127.86, 125.29,
124.57, 124.44, 118.93. HRMS (ESI) m/z calcd for C20H15IN2O2
[M ꢃ I]þ, 315.1128, found: 315.1138.
(600 MHz, Chloroform-d)
d
8.97 (d, J ¼ 9.2 Hz, 2H), 8.44 (ddd,
J ¼ 9.0, 6.7, 1.4 Hz, 2H), 8.00 (d, J ¼ 8.0 Hz, 2H), 7.90e7.89 (m, 2H),
7.85e7.82 (m, 2H), 7.70 (d, J ¼ 7.9 Hz, 2H), 5.28 (s, 3H). 13C NMR
(150 MHz, CDCl3)
d 158.53, 141.13, 139.20, 136.37, 132.04 (q, JF-
5.2.8.7. 10-Methyl-9-(p-tolyl)acridin-10-ium iodide. (9g). Red solid,
yield 71%, m.p.: 240e242 ꢄC; 1H NMR (600 MHz, Chloroform-d)
¼ 33.1 Hz), 130.02, 129.15, 128.13, 125.72 (q, JF-C ¼ 3.7 Hz), 125.59,
C
124.13, 122.32, 119.40, 41.21. HRMS (ESI) m/z calcd for C21H15F3IN
[M ꢃ I]þ, 338.1151, found: 338.1163.
d
8.93 (d, J ¼ 9.1 Hz, 2H), 8.42e8.40 (m, 2H), 8.05 (d, J ¼ 8.6 Hz, 2H),
7.81e7.79 (m, 2H), 7.52 (d, J ¼ 7.7 Hz, 2H), 7.38 (d, J ¼ 7.8 Hz, 2H),
5.26 (s, 3H), 2.58 (s, 3H). 13C NMR (150 MHz, CDCl3)
161.51, 141.05,
d
5.2.8.14. 10-Methyl-9-(4-(trifluoromethoxy)phenyl)acridin-10-ium
iodide. (9n). Red solid, yield 67%, m.p.: 204e208 ꢄC; 1H NMR
140.59, 139.04, 129.97, 129.53, 129.46, 129.37, 127.62, 125.80, 119.08,
40.97, 21.24. HRMS (ESI) m/z calcd for C21H18IN [M ꢃ I]þ, 284.1434,
found: 284.1449.
(600 MHz, Chloroform-d)
J ¼ 9.0, 6.7, 1.3 Hz, 2H), 8.00e7.95 (m, 2H), 7.85e7.83 (m, 2H),
7.61e7.57 (m, 4H), 5.27 (s, 3H). 13C NMR (150 MHz, CDCl3)
158.98,
d
8.95 (d, J ¼ 9.2 Hz, 2H), 8.43 (ddd,
d
5.2.8.8. 10-Methyl-9-(4-ethylphenyl)acridin-10-ium iodide. (9h).
Red solid, yield 66%, m.p.: 240e242 ꢄC; 1H NMR (600 MHz, Chlo-
150.23 (d, JF-C ¼ 2.1 Hz), 141.12, 139.13, 131.34, 130.98, 129.33,
128.02, 125.78, 120.98, 119.33, 41.15. HRMS (ESI) m/z calcd for
roform-d)
d
8.94 (d, J ¼ 9.2 Hz, 2H), 8.43e8.40 (m, 2H), 8.05 (dd,
C
21H15F3INO [M ꢃ I]þ, 354.1100, found: 354.1117.
J ¼ 8.7, 1.1 Hz, 2H), 7.80 (dd, J ¼ 8.4, 6.9 Hz, 2H), 7.54 (d, J ¼ 7.9 Hz,
2H), 7.40 (d, J ¼ 8.0 Hz, 2H), 5.26 (s, 3H), 2.90e2.86 (m, 2H), 1.41 (t,
5.2.8.15. 10-Methyl-9-(4-formylphenyl)acridin-10-ium iodide. (9o).
Red solid, yield 69%, m.p.: 246e248 ꢄC; 1H NMR (600 MHz, Chlo-
J ¼ 7.6 Hz, 3H). 13C NMR (150 MHz, CDCl3)
d 161.42, 146.61, 140.94,
138.96, 129.91, 129.57, 129.55, 128.08, 127.54, 125.70, 118.97, 40.86,
28.36, 14.94. HRMS (ESI) m/z calcd for C22H20IN [M ꢃ I]þ, 298.1590,
found: 298.1590.
roform-d)
d
10.26 (s, 1H), 8.95 (d, J ¼ 9.2 Hz, 2H), 8.45e8.42 (m, 2H),
8.25 (d, J ¼ 7.8 Hz, 2H), 7.91 (d, J ¼ 8.4 Hz, 2H), 7.83 (dd, J ¼ 8.7,
6.6 Hz, 2H), 7.74 (d, J ¼ 7.9 Hz, 2H), 5.28 (s, 3H). 13C NMR (150 MHz,
DMSO þ CD3OD)
d 192.68, 158.82, 141.08, 138.78, 138.37, 136.88,
5.2.8.9. 10-Methyl-9-(4-propylphenyl)acridin-10-ium iodide. (9i).
Red solid, yield 74%, m.p.: 230e232 ꢄC; 1H NMR (600 MHz, Chlo-
130.53, 129.55, 129.10, 127.94, 125.16, 119.13. HRMS (ESI) m/z calcd
for C21H16INO [M ꢃ I]þ, 298.1226, found: 298.1229.
roform-d)
d
8.94 (d, J ¼ 9.2 Hz, 2H), 8.42 (ddd, J ¼ 9.0, 6.7, 1.3 Hz,
2H), 8.05 (dd, J ¼ 8.7, 1.1 Hz, 2H), 7.80 (dd, J ¼ 8.5, 6.8 Hz, 2H), 7.52
(d, J ¼ 7.9 Hz, 2H), 7.39 (d, J ¼ 8.0 Hz, 2H), 5.26 (s, 3H), 2.82e2.79 (m,
2H), 1.84e1.78 (m, 2H), 1.07 (t, J ¼ 7.3 Hz, 3H). 13C NMR (150 MHz,
5.2.9. 9-Methylacridine (11)
A mixture of N,N-diphenylamine (3.0g, 17.7 mmol), AcOH (3.2 g,
53.2 mmol), and ZnCl2 (12.0 g, 88.6 mmol) was heated with effi-
cient stirring. When the temperature reached to 180 ꢄC, the excess
AcOH was removed from the reaction mixture by distillation. Then,
the reaction mixture was heated at 220 ꢄC for additional 5 h under
stirring. After the addition of aqueous ammonia solution, the yel-
low precipitates were obtained by filtration and subsequently dis-
solved in CH2Cl2. The CH2Cl2 solution was neutralized by washing
with aqueous NaHCO3 and brine, dried over Na2SO4, filtrated,
concentrated under reduced pressure. The residue was purified on
silica gel with petroleum ether/EtOAc (5/1), yielding the pale yel-
CDCl3) d 161.51,145.26,141.06,139.04,129.99,129.71,129.57,128.73,
127.62, 125.80, 119.10, 41.00, 37.55, 24.05, 13.63. HRMS (ESI) m/z
calcd for C23H22IN [M ꢃ I]þ, 312.1747, found: 312.1751.
5.2.8.10. 10-Methyl-9-(4-butylphenyl)acridin-10-ium iodide. (9j).
Red solid, yield 73%, m.p.: 190e194 ꢄC; 1H NMR (600 MHz, Chlo-
roform-d)
d
8.94 (d, J ¼ 9.2 Hz, 2H), 8.42 (ddd, J ¼ 9.0, 6.7, 1.3 Hz,
2H), 8.06e8.04 (m, 2H), 7.80 (dd, J ¼ 8.4, 6.9 Hz, 2H), 7.52 (d,
J ¼ 8.0 Hz, 2H), 7.39 (d, J ¼ 8.0 Hz, 2H), 5.26 (s, 3H), 2.84e2.81 (m,
2H), 1.79e1.74 (m, 2H), 1.52e1.45 (m, 2H), 1.02 (t, J ¼ 7.4 Hz, 3H). 13
C
low solid (2.77 g, 81%). 1H NMR (400 MHz, Chloroform-d)
d 8.24
NMR (150 MHz, CDCl3)
d
161.36, 145.31, 140.88, 138.91, 129.84,
(dd, J ¼ 12.0, 8.9 Hz, 4H), 7.79e7.75 (m, 2H), 7.58e7.54 (m, 2H), 3.13
129.50, 129.44, 128.52, 127.50, 125.63, 118.91, 40.79, 35.06, 32.91,
21.96, 13.54. HRMS (ESI) m/z calcd for C24H24IN [M ꢃ I]þ, 326.1903,
found: 326.1913.
(s, 3H). MS (ESI) m/z calcd for C14H11N [M þ H]þ, 194.1, found: 194.4.
5.2.10. 9-(Bromomethyl)acridine (12)
To a solution of 11 (2.0 g, 10.4 mmol) in CCl4 (100 mL) was added
AIBN (0.17 g, 1.0 mmol) and heated to 60 ꢄC under stirring for
30 min. Then NBS (0.17 g, 11.4 mmol) was added and the reaction
mixture was heated under refluxing conditions for 4 h. After
completion of the reaction, the reaction solution was diluted with
CH2Cl2. The organic layer was separated, dried over Na2SO4, fil-
trated, and concentrated under reduced pressure. The residue was
purified by silica gel using petroleum ether/EtOAc (5/1) as eluent to
afford intermediate 12 (2.45 g) in 87% yield. 1H NMR (400 MHz,
5.2.8.11. 10-Methyl-9-(4-methoxyphenyl)acridin-10-ium iodide. (9k).
Red solid, yield 69%, m.p.: 248e250 ꢄC; 1H NMR (600 MHz, Chlo-
roform-d)
d
8.91 (d, J ¼ 9.2 Hz, 2H), 8.41 (ddd, J ¼ 9.0, 6.7, 1.4 Hz,
2H), 8.11e8.09 (m, 2H), 7.82e7.80 (m, 2H), 7.45e7.43 (m, 2H), 7.23
(d, J ¼ 8.7 Hz, 2H), 5.23 (s, 3H), 3.99 (s, 3H). 13C NMR (150 MHz,
CDCl3)
d 161.29, 160.99, 141.00, 138.90, 131.40, 130.00, 127.54,
125.84, 124.19, 118.95, 114.26, 55.47, 40.80. HRMS (ESI) m/z calcd for
C
21H18INO [M ꢃ I]þ, 300.1383, found: 300.1382.
DMSO‑d6)
d
8.85 (d, J ¼ 8.8 Hz, 1H), 8.29 (d, J ¼ 8.6 Hz, 4H),
5.2.8.12. 10-Methyl-9-([1,10-biphenyl]-4-yl)acridin-10-ium
(9l). Red solid, yield 77%, m.p.: 230e232 ꢄC; 1H NMR (600 MHz,
Chloroform-d)
iodide.
7.98e7.94 (m, 2H), 7.80 (d, J ¼ 7.7 Hz, 1H), 5.89 (s, 2H). MS (ESI) m/z
calcd for C14H10BrN [M þ H]þ, 272.0, found: 274.3.
d
8.95 (d, J ¼ 9.2 Hz, 2H), 8.43 (ddd, J ¼ 9.0, 6.7,
1.3 Hz, 2H), 8.13e8.11 (m, 2H), 7.94 (d, J ¼ 8.1 Hz, 2H), 7.85e7.82 (m,
5.2.11. Diethyl (acridin-9-ylmethyl)phosphonate (13)
2H), 7.76e7.75 (m, 2H), 7.60e7.55 (m, 4H), 7.48 (t, J ¼ 7.4 Hz, 1H),
The mixture of 12 (1.0 g, 3.7 mmol) and triethyl phosphite (4 mL)
was heated to reflux for 4 h. After cooling down to room temper-
ature, the excess triethyl phosphite was removed under reduced
5.28 (s, 3H). 13C NMR (150 MHz, DMSO)
d 159.82, 141.29, 140.79,
138.57, 138.07, 131.74, 130.29, 129.21, 128.80, 127.84, 127.60, 126.65,
16

D. Song, N. Zhang, P. Zhang et al.
European Journal of Medicinal Chemistry 221 (2021) 113480
pressure to afford intermediate 13 (1.1 g) in a 92% yield.
2H),1.69e1.64 (m, 2H),1.42e1.39 (m, 2H), 0.97 (t, J ¼ 7.4 Hz, 3H). 13
C
NMR (150 MHz, CDCl3)
d 157.94, 146.71, 146.33, 140.81, 138.79,
5.2.12. (E)-9-styrylacridine (14a)
132.18, 129.18, 128.86, 128.04, 127.49, 124.63, 119.06, 118.56, 40.73,
35.39, 33.16, 22.06, 13.72. HRMS (ESI) m/z calcd for C26H26IN
[M ꢃ I]þ, 352.2060, found: 352.2072.
To a solution of 13 (0.15 g, 0.45 mmol) and benzaldehyde (0.05 g,
0.45 mmol) in dry THF (25 mL), NaH (65% oil dispersion, 33.5 mg,
0.9 mmol) was added carefully in an ice-water bath. After the
completion of the addition, the solution mixture was warmed to
room temperature and stirred overnight. After quenching by water,
the resulting mixture was extracted with EtOAc (50 mL ꢂ 3). The
combined organic layer was washed with brine, dried over Na2SO4,
filtrated, and concentrated under reduced pressure. The resulting
crude product was purified by silica gel using DCM/MeOH (60/1) to
afford 14a (85 mg) in a 67% yield.
5.2.13.6. (E)-10-methyl-9-(4-(tert-butyl)styryl)acridin-10-ium
dide. (15f). Brown solid, yield 69%, m.p.: 218e220 ꢄC; 1H NMR
(600 MHz, Chloroform-d)
io-
d
8.83 (d, J ¼ 9.2 Hz, 2H), 8.69 (d,
J ¼ 8.6 Hz, 2H), 8.41e8.38 (m, 2H), 8.10 (d, J ¼ 16.3 Hz, 1H),
7.90e7.87 (m, 2H), 7.73 (d, J ¼ 8.3 Hz, 2H), 7.57 (d, J ¼ 8.3 Hz, 2H),
7.23 (d, J ¼ 16.3 Hz, 1H), 5.13 (s, 3H), 1.40 (s, 9H). 13C NMR (150 MHz,
CDCl3) d 157.96,154.45,146.51,140.81,138.80,131.96,128.85,127.90,
127.49, 126.08, 124.63, 119.07, 118.78, 40.72, 34.82, 30.93. HRMS
5.2.13. Typical procedure for the synthesis of Series C
The titled compound 15a (66 mg, 74%) were prepared from 14a
(85 mg, 0.3 mmol) and CH3I (0.43 g, 3.0 mmol) according to the
preparation procedure of 9d described above.
(ESI) m/z calcd for C26H26IN [M ꢃ I]þ, 352.2060, found: 352.2062.
5.2.13.7. (E)-10-methyl-9-(4-ethoxystyryl)acridin-10-ium
(15g). Brown solid, yield 67%, m.p.: 186e189 ꢄC; 1H NMR
(600 MHz, Chloroform-d)
iodide.
d
8.83 (d, J ¼ 9.1 Hz, 2H), 8.69 (d,
5.2.13.1. (E)-10-methyl-9-styrylacridin-10-ium
Dark red solid, yield 76%, m.p.: 200e204 ꢄC; 1H NMR (600 MHz,
Chloroform-d)
iodide.
(15a).
J ¼ 8.6 Hz, 2H), 8.42e8.39 (m, 2H), 8.11 (d, J ¼ 16.3 Hz, 1H),
7.92e7.89 (m, 2H), 7.43 (t, J ¼ 7.9 Hz,1H), 7.33 (d, J ¼ 7.6 Hz,1H), 7.29
(s, 1H), 7.18 (d, J ¼ 16.3 Hz, 1H), 7.04 (dd, J ¼ 8.2, 2.1 Hz, 1H), 5.13 (s,
3H), 4.18e4.14 (m, 2H), 1.48 (t, J ¼ 7.0 Hz, 3H). 13C NMR (150 MHz,
d
8.84 (d, J ¼ 9.2 Hz, 2H), 8.70 (d, J ¼ 7.8 Hz, 2H), 8.41
(ddd, J ¼ 9.2, 6.6, 1.4 Hz, 2H), 8.12 (d, J ¼ 16.4 Hz, 1H), 7.92e7.89 (m,
2H), 7.79e7.77 (m, 2H), 7.56e7.51 (m, 3H), 7.23 (d, J ¼ 16.4 Hz, 1H),
CDCl3) d 159.46,157.75,146.11,141.03, 138.95, 136.02, 130.19, 128.94,
5.14 (s, 3H). 13C NMR (150 MHz, CDCl3)
d
157.78, 146.11, 140.88,
127.70, 124.89, 120.47, 119.87, 119.30, 116.94, 113.39, 63.85, 40.94,
14.70. HRMS (ESI) m/z calcd for C23H20INO [M ꢃ I]þ, 340.1696,
found: 340.1706.
138.91, 134.60, 130.59, 129.10, 128.91, 127.95, 127.66, 124.75, 119.69,
119.15, 40.80. HRMS (ESI) m/z calcd for C22H18IN [M ꢃ I]þ, 296.1434,
found: 296.1438.
5.2.13.8. (E)-10-methyl-9-(3,4-dimethoxystyryl)acridin-10-ium io-
5.2.13.2. (E)-10-methyl-9-(2-methoxystyryl)acridin-10-ium iodide.
dide. (15h). Brown solid, yield 63%, m.p.: 224e227 ꢄC; 1H NMR
(15b). Dark red solid, yield 69%, m.p.: 184e187 ꢄC; 1H NMR
(600 MHz, Chloroform-d)
d
8.77 (d, J ¼ 8.5 Hz, 2H), 8.70 (d,
(600 MHz, Chloroform-d)
d
8.84 (s, 2H), 8.71 (d, J ¼ 8.2 Hz, 2H), 8.40
J ¼ 9.0 Hz, 2H), 8.36e8.33 (m, 2H), 8.18 (d, J ¼ 16.0 Hz, 1H),
7.89e7.86 (m, 2H), 7.41 (s, 1H), 7.33 (d, J ¼ 8.1 Hz, 1H), 7.20 (d,
J ¼ 16.1 Hz, 1H), 6.97 (d, J ¼ 8.2 Hz, 1H), 5.02 (s, 3H), 4.06 (s, 3H),
(s, 2H), 8.26 (d, J ¼ 16.1 Hz, 1H), 7.89 (s, 2H), 7.78 (d, J ¼ 7.2 Hz, 1H),
7.51e7.47 (m, 2H), 7.13 (t, J ¼ 7.3 Hz, 1H), 7.05 (d, J ¼ 8.4 Hz, 1H), 5.14
(s, 3H), 3.96 (s, 3H). 13C NMR (150 MHz, DMSO þ CDCl3)
d
157.63,
3.98 (s, 3H). 13C NMR (150 MHz, DMSO þ CDCl3)
d 157.67, 150.83,
157.22, 140.48, 140.35, 137.63, 131.58, 128.52, 127.81, 126.90, 123.85,
123.42, 120.75, 120.37, 118.64, 111.35, 55.40. HRMS (ESI) m/z calcd
for C23H20INO [M ꢃ I]þ, 326.1539, found: 326.1532.
148.70, 147.01, 140.46, 137.55, 128.73, 128.20, 126.74, 123.75, 123.32,
118.57, 118.20, 111.12, 109.89, 55.48, 55.33. HRMS (ESI) m/z calcd for
C
24H22INO2 [M ꢃ I]þ, 356.1645, found: 356.1641.
5.2.13.3. (E)-10-methyl-9-(4-methylstyryl)acridin-10-ium
(15c). Brown solid, yield 77%, m.p.: 250e253 ꢄC; 1H NMR
(600 MHz, DMSO‑d6)
iodide.
5.2.13.9. (E)-10-methyl-9-(2-([1,10-biphenyl]-4-yl)vinyl)acridin-10-
ium iodide. (15i). Dark red solid, yield 71%, m.p.: 209e211 ꢄC; 1H
d
8.85 (d, J ¼ 9.6 Hz, 2H), 8.75 (d, J ¼ 9.2 Hz,
NMR (600 MHz, Chloroform-d)
d
8.83 (d, J ¼ 9.1 Hz, 2H), 8.73 (d,
2H), 8.55 (d, J ¼ 16.4 Hz, 1H), 8.44e8.42 (m, 2H), 8.00e7.98 (m, 2H),
J ¼ 8.5 Hz, 2H), 8.42e8.39 (m, 2H), 8.18 (d, J ¼ 16.3 Hz, 1H), 7.91 (dd,
J ¼ 8.4, 6.9 Hz, 2H), 7.86 (d, J ¼ 8.2 Hz, 2H), 7.78 (d, J ¼ 8.2 Hz, 2H),
7.68 (d, J ¼ 7.1 Hz, 2H), 7.51 (t, J ¼ 7.6 Hz, 2H), 7.43 (t, J ¼ 7.4 Hz, 1H),
7.28 (d, J ¼ 16.4 Hz, 1H), 5.14 (s, 3H). 13C NMR (150 MHz, DMSO)
7.90 (d, J ¼ 8.1 Hz, 2H), 7.37 (d, J ¼ 7.9 Hz, 2H), 7.31 (d, J ¼ 16.3 Hz,
1H), 4.81 (s, 3H), 2.41 (s, 3H). 13C NMR (150 MHz, DMSO)
d 157.52,
145.78, 140.53, 140.01, 137.72, 132.55, 129.20, 128.73, 128.07, 126.96,
123.92, 119.65, 118.69, 54.57, 48.21, 20.76. HRMS (ESI) m/z calcd for
d 157.20, 145.08, 141.27, 140.44, 138.72, 137.72, 134.27, 128.71,
C
23H20IN [M ꢃ I]þ, 310.1590, found: 310.1604.
128.65, 127.63, 126.99, 126.64, 126.31, 123.88, 120.65, 118.67. HRMS
(ESI) m/z calcd for C28H22IN [M ꢃ I]þ, 372.1747, found: 372.1737.
5.2.13.4. (E)-10-methyl-9-(4-ethylstyryl)acridin-10-ium
(15d). Brown solid, yield 72%, m.p.: 222e226 ꢄC; 1H NMR
(600 MHz, Chloroform-d)
iodide.
5.2.13.10. (E)-10-methyl-9-(4-fluorostyryl)acridin-10-ium
(15j). Brown solid, yield 69%, m.p.: 225e228 ꢄC; 1H NMR (600 MHz,
DMSO‑d6)
iodide.
d
8.82 (d, J ¼ 8.5 Hz, 2H), 8.69 (d,
J ¼ 8.5 Hz, 2H), 8.39 (t, J ¼ 7.1 Hz, 2H), 8.10 (d, J ¼ 16.2 Hz, 1H),
7.90e7.87 (m, 2H), 7.71 (d, J ¼ 7.9 Hz, 2H), 7.37 (d, J ¼ 7.7 Hz, 2H),
7.22 (d, J ¼ 16.2 Hz, 1H), 5.11 (s, 3H), 2.77e2.73 (m, 2H), 1.30 (t,
d
8.86 (d, J ¼ 8.6 Hz, 2H), 8.77 (d, J ¼ 9.2 Hz, 2H), 8.55 (d,
J ¼ 16.3 Hz, 1H), 8.45e8.42 (m, 2H), 8.09e8.06 (m, 2H), 8.01e7.99
(m, 2H), 7.40 (t, J ¼ 8.6 Hz, 2H), 7.34 (d, J ¼ 16.3 Hz, 1H), 4.82 (s, 3H).
J ¼ 7.6 Hz, 3H). 13C NMR (150 MHz, CDCl3)
d
157.97, 147.69, 146.69,
13C NMR (150 MHz, DMSO)
d 163.59, 161.94, 157.16, 144.01, 140.47,
140.89, 138.83, 132.25, 128.88, 128.70, 128.14, 127.54, 124.70, 119.14,
118.60, 40.79, 28.72, 15.26. HRMS (ESI) m/z calcd for C24H22IN
[M ꢃ I]þ, 324.1747, found: 324.1756.
137.76, 131.80 (d, JF-C ¼ 3.2 Hz), 130.31 (d, JF-C ¼ 8.5 Hz), 128.71,
127.02, 123.89, 120.51 (d, JF-C ¼ 2.5 Hz), 118.70, 115.55 (d, JF-
¼ 21.7 Hz), 48.20. MS (ESI) m/z calcd for C22H17FIN [M ꢃ I]þ,
C
314.13, found: 314.46.
5.2.13.5. (E)-10-methyl-9-(4-butylstyryl)acridin-10-ium
(15e). Brown solid, yield 78%, m.p.: 186e188 ꢄC; 1H NMR (600 MHz,
Chloroform-d)
iodide.
5.2.13.11. (E)-10-methyl-9-(4-chlorostyryl)acridin-10-ium
(15k). Brown solid, yield 70%, m.p.: 169e172 ꢄC; 1H NMR
(600 MHz, Chloroform-d)
8.84e8.81 (m, 2H), 8.75 (d, J ¼ 8.9 Hz,
1H), 8.28 (d, J ¼ 8.4 Hz, 2H), 7.96 (d, J ¼ 8.2 Hz, 1H), 7.78e7.72 (m,
iodide.
d
8.83 (s, 2H), 8.70 (d, J ¼ 8.0 Hz, 2H), 8.40 (s, 2H),
8.09 (d, J ¼ 15.7 Hz, 1H), 7.89 (s, 2H), 7.69 (d, J ¼ 7.7 Hz, 2H), 7.35 (d,
d
J ¼ 7.4 Hz, 2H), 7.23 (d, J ¼ 15.9 Hz, 1H), 5.14 (s, 3H), 2.73e2.70 (m,
17

D. Song, N. Zhang, P. Zhang et al.
European Journal of Medicinal Chemistry 221 (2021) 113480
2H), 7.61 (d, J ¼ 8.3 Hz, 2H), 7.54 (d, J ¼ 8.3 Hz, 2H), 7.28e7.27 (m,
1H), 7.22e7.19 (m, 1H), 5.10 (s, 3H). HRMS (ESI) m/z calcd for
C
4.82 (s, 3H), 4.13 (s, 3H). 13C NMR (150 MHz, DMSO)
d 140.36,137.77,
134.06, 133.99, 128.47, 127.99, 125.41, 120.54, 118.85, 71.65, 59.63,
55.89, 47.98. HRMS (ESI) m/z calcd for C23H18INO [M ꢃ I]þ,
324.1383, found: 324.1386.
22H17ClIN [M ꢃ I]þ, 330.1044, found: 330.1046.
5.2.13.12. (E)-10-methyl-9-(4-bromostyryl)acridin-10-ium
(15l). Brown solid, yield 71%, m.p.: 179e181 ꢄC; 1H NMR (600 MHz,
DMSO‑d6)
iodide.
5.2.15.4. 10-Methyl-9-(m-tolylethynyl)acridin-10-ium iodide. (17d).
Brown solid, yield 62%, m.p.: 203e205 ꢄC; 1H NMR (600 MHz,
d
8.84 (d, J ¼ 8.6 Hz, 2H), 8.77 (d, J ¼ 9.2 Hz, 2H), 8.62 (d,
J ¼ 16.4 Hz, 1H), 8.45e8.43 (m, 2H), 8.02e7.99 (m, 2H), 7.96 (d,
DMSO‑d6)
d
9.05e9.04 (m, 2H), 8.82 (d, J ¼ 9.2 Hz, 2H), 8.49e8.46
J ¼ 8.4 Hz, 2H), 7.76 (d, J ¼ 8.4 Hz, 2H), 7.31 (d, J ¼ 16.4 Hz, 1H), 4.83
(m, 2H), 8.13e8.11 (m, 2H), 8.02 (s, 1H), 7.98 (d, J ¼ 6.7 Hz, 1H),
7.57e7.53 (m, 2H), 4.85 (s, 3H), 2.46 (s, 3H). HRMS (ESI) m/z calcd
for C23H18IN [M ꢃ I]þ, 308.1434, found: 308.1444.
(s, 3H). 13C NMR (150 MHz, DMSO)
d 157.09, 143.72, 140.58, 137.83,
134.43, 131.53, 129.89, 128.77, 127.11, 123.99, 123.18, 121.64, 118.76,
48.21. HRMS (ESI) m/z calcd for C22H17BrIN [M ꢃ I]þ, 374.0539,
found: 374.0542.
5.2.15.5. 10-Methyl-9-((4-ethylphenyl)ethynyl)acridin-10-ium
dide. (17e). Brown solid, yield 66%, m.p.: 182e186 ꢄC; 1H NMR
(600 MHz, DMSO‑d6)
io-
5.2.13.13. (E)-10-methyl-9-(2,4-dichlorostyryl)acridin-10-ium iodide.
d
9.05e9.04 (m, 2H), 8.81 (d, J ¼ 9.2 Hz, 2H),
(15m). Dark red solid, yield 73%, m.p.: 212e215 ꢄC; 1H NMR
8.49e8.46 (m, 2H), 8.13e8.10 (m, 4H), 7.52 (d, J ¼ 8.3 Hz, 2H), 4.84
(s, 3H), 2.79e2.75 (m, 2H), 1.26 (t, J ¼ 7.6 Hz, 3H). HRMS (ESI) m/z
calcd for C24H20IN [M ꢃ I]þ, 322.1590, found: 322.1601.
(600 MHz, DMSO‑d6)
d
8.85e8.84 (m, 2H), 8.79 (d, J ¼ 9.2 Hz, 2H),
8.62 (d, J ¼ 16.3 Hz, 1H), 8.47e8.44 (m, 3H), 8.03e8.00 (m, 2H), 7.82
(d, J ¼ 2.1 Hz, 1H), 7.70 (dd, J ¼ 8.6, 2.1 Hz, 1H), 7.42 (d, J ¼ 16.4 Hz,
1H), 4.85 (s, 3H). 13C NMR (150 MHz, DMSO)
d
156.54, 140.67,
5.2.15.6. 10-Methyl-9-((4-butylphenyl)ethynyl)acridin-10-ium
dide. (17f). Brown solid, yield 67%, m.p.: 192e194 ꢄC; 1H NMR
(600 MHz, DMSO‑d6)
io-
138.30, 137.92, 134.72, 133.85, 131.96, 129.41, 128.98, 128.64, 127.66,
127.24, 124.74, 124.15, 118.84, 48.21. HRMS (ESI) m/z calcd for
d
9.05e9.03 (m, 2H), 8.80 (d, J ¼ 9.2 Hz, 2H),
C
22H16Cl2IN [M ꢃ I]þ, 364.0654, found: 364.0650.
8.48e8.45 (m, 2H), 8.12e8.09 (m, 4H), 7.50 (d, J ¼ 8.2 Hz, 2H), 4.84
(s, 3H), 2.75e2.72 (m, 2H), 1.65e1.62 (m, 2H), 1.46e1.44 (m, 2H),
0.95e0.92 (m, 3H). HRMS (ESI) m/z calcd for C26H24IN [M ꢃ I]þ,
350.1903, found: 350.1906.
5.2.14. 9-(o-Tolylethynyl)acridine (16b)
To a 100 mL round bottom flask containing 7 (0.15 g, 0.58 mmol),
was added Pd (PPh3)2Cl2 (8.1 mg, 0.01 mmol) and CuBr2 (3.3 mg,
0.02 mmol). Et3N (10 mL) was added as solvent and the suspension
was stirred for 10 min at room temperature under N2 atmosphere.
After 10 min under stirring, phenylacetylene (65 mg, 0.64 mmol)
was added and the solution was refluxed at 85 ꢄC for 24 h. When
cooled to room temperature, the reaction solution was filtered
through Celite. The solvent was removed under reduced pressure
and the residue was purified via silica gel using petroleum ether/
EtOAc (3/1) as eluent to yield the desired product as a yellow solid
5.2.15.7. 10-Methyl-9-((4-methoxyphenyl)ethynyl)acridin-10-ium
iodide. (17g). Brown solid, yield 59%, m.p.: 216e221 ꢄC; 1H NMR
(600 MHz, DMSO‑d6)
d
9.03 (d, J ¼ 8.4 Hz, 2H), 8.78 (d, J ¼ 9.1 Hz,
2H), 8.46e8.44 (m, 2H), 8.17 (d, J ¼ 8.6 Hz, 2H), 8.10e8.08 (m, 2H),
7.23 (d, J ¼ 8.6 Hz, 2H), 4.81 (s, 3H), 3.93 (s, 3H). HRMS (ESI) m/z
calcd for C23H18INO [M ꢃ I]þ, 324.1383, found: 324.1384.
5.2.15.8. 10-Methyl-9-((4-ethoxyphenyl)ethynyl)acridin-10-ium io-
(76%, 0.12 g). 1H NMR (400 MHz, Chloroform-d)
2H), 8.29 (d, J ¼ 4.4 Hz, 2H), 7.85e7.81 (m, 2H), 7.75 (dd, J ¼ 7.5,
1.4 Hz, 1H), 7.67e7.63 (m, 2H), 7.48e7.44 (m, 1H), 7.09e7.02 (m,
2H), 4.07 (s, 3H).
d
8.70 (d, J ¼ 8.5 Hz,
dide. (17h). Brown solid, yield 65%, m.p.: 179e183 ꢄC; 1H NMR
(600 MHz, DMSO‑d6)
d
9.04e9.02 (m, 2H), 8.77 (d, J ¼ 9.2 Hz, 2H),
8.46e8.43 (m, 2H), 8.15 (d, J ¼ 8.8 Hz, 2H), 8.10e8.07 (m, 2H), 7.21
(d, J ¼ 8.8 Hz, 2H), 4.81 (s, 3H), 4.23e4.19 (m, 2H), 1.40 (t, J ¼ 7.0 Hz,
3H). 13C NMR (150 MHz, DMSO)
d 140.26, 137.68, 135.30, 128.73,
5.2.15. Typical procedure for the synthesis of Series D
Compound 17b (71.6 mg, 68%) were prepared from 16b (0.1 g,
0.36 mmol) and CH3I (0.51 g, 3.6 mmol) according to the prepara-
tion procedure of 9d described above.
127.80, 125.28, 118.73, 114.85, 63.27, 13.85. HRMS (ESI) m/z calcd for
C
24H20INO [M ꢃ I]þ, 338.1539, found: 338.1530.
5.2.15.9. 10-Methyl-9-((4-propoxyphenyl)ethynyl)acridin-10-ium io-
dide. (17i). Brown solid, yield 63%, m.p.: 194e198 ꢄC; 1H NMR
5.2.15.1. 10-Methyl-9-(phenylethynyl)acridin-10-ium iodide. (17a).
Brown solid, yield 61%, m.p.: 205e209 ꢄC; 1H NMR (600 MHz,
(600 MHz, DMSO‑d6)
d
9.04e9.02 (m, 2H), 8.78 (d, J ¼ 9.2 Hz, 2H),
8.46e8.43 (m, 2H), 8.16 (d, J ¼ 8.8 Hz, 2H), 8.10e8.07 (m, 2H), 7.22
(d, J ¼ 8.8 Hz, 2H), 4.81 (s, 3H), 4.11 (t, J ¼ 6.5 Hz, 2H), 1.82e1.78 (m,
2H), 1.02 (t, J ¼ 7.4 Hz, 3H). HRMS (ESI) m/z calcd for C25H22INO
[M ꢃ I]þ, 352.1696, found: 352.1693.
DMSO‑d6)
d
9.05 (d, J ¼ 8.5 Hz, 2H), 8.82 (d, J ¼ 9.2 Hz, 2H),
8.49e8.47 (m, 2H), 8.18 (d, J ¼ 7.3 Hz, 2H), 8.14e8.11 (m, 2H),
7.73e7.66 (m, 3H), 4.85 (s, 3H). HRMS (ESI) m/z calcd for C22H16IN
[M ꢃ I]þ, 294.1277, found: 294.1288.
5.2.15.10. 10-Methyl-9-([1,10-biphenyl]-4-ylethynyl)acridin-10-ium
5.2.15.2. 10-Methyl-9-(o-tolylethynyl)acridin-10-ium iodide. (17b).
Brown solid, yield 63%, m.p.: 228e231 ꢄC; 1H NMR (600 MHz,
iodide. (17j). Brown solid, yield 67%, m.p.: 199e204 ꢄC; 1H NMR
(600 MHz, DMSO‑d6)
d
9.10e9.09 (m, 2H), 8.82 (d, J ¼ 9.2 Hz, 2H),
DMSO‑d6)
d
9.05 (d, J ¼ 8.4 Hz, 2H), 8.80 (d, J ¼ 9.2 Hz, 2H),
8.50e8.47 (m, 2H), 8.29 (d, J ¼ 8.4 Hz, 2H), 8.14e8.12 (m, 3H), 8.00
(d, J ¼ 8.4 Hz, 2H), 7.86 (d, J ¼ 7.3 Hz, 2H), 7.56 (t, J ¼ 7.6 Hz, 2H), 4.86
(s, 3H). HRMS (ESI) m/z calcd for C28H20IN [M ꢃ I]þ, 370.1590,
found: 370.1574.
8.48e8.46 (m, 2H), 8.16e8.14 (m, 2H), 8.08 (d, J ¼ 7.3 Hz,1H), 7.71 (t,
J ¼ 8.2 Hz, 1H), 7.34 (d, J ¼ 8.4 Hz, 1H), 7.21 (t, J ¼ 7.4 Hz, 1H), 4.83 (s,
3H), 4.14 (s, 3H). HRMS (ESI) m/z calcd for C23H18IN [M ꢃ I]þ,
308.1434, found: 308.1435.
5.3. In vitro antibacterial assay
5.2.15.3. 10-Methyl-9-((2-methoxyphenyl)ethynyl)acridin-10-ium
iodide. (17c). Brown solid, yield 59%, m.p.: 218e222 ꢄC; 1H NMR
The MIC of the prepared compounds was determined using the
standard broth microdilution procedures recommended by CLSI
[48,49]. Bacterial strains were incubated on Mueller Hinton Agar
(MHA) medium at 37 ꢄC for 24 h and then log-phase bacteria were
(600 MHz, DMSO‑d6)
2H), 8.48e8.45 (m, 2H), 8.16e8.13 (m, 2H), 8.07 (d, J ¼ 7.5 Hz, 1H),
d
9.03 (d, J ¼ 8.5 Hz, 2H), 8.79 (d, J ¼ 9.2 Hz,
7.72e7.69 (m, 1H), 7.33 (d, J ¼ 8.4 Hz, 1H), 7.20 (t, J ¼ 7.4 Hz, 1H),
18

D. Song, N. Zhang, P. Zhang et al.
European Journal of Medicinal Chemistry 221 (2021) 113480
suspended in 10 mL sterile water to make a 0.5 McFarland standard
inoculum. The standard inoculum was diluted 10 times for further
use. A series of gradient solutions of the prepared compounds and
the control drugs were prepared in sterile Mueller Hinton (MH)
broth by using a 2-fold dilution method with concentrations
a 96-well plate at a concentration of 0.5 ꢂ MIC were used to prepare
the bacterial suspension (~107 CFU/mL) for the next MIC mea-
surement. Ciprofloxacin and rifampicin were used as comparisons
to investigate the resistance development. After incubation with
sample at 37 ꢄC for 24 h, the new MICs were determined. The
experiment process was lasted for 18 passages.
ranging from 0.125 to 64
mg/mL. Then 5 mL of diluted standard
inoculum (1.5 ꢂ 107 cfu/mL) was added to the above solutions and
96-well plates were incubated aerobically for 24 h at 37 ꢄC. After
24 h, turbidity was observed and the last tube with no growth of
5.8. Visualization of bacterial morphology
bacteria was recorded to represent the MIC value, expressed in
mL.
m
g/
The cells of B. pumilus CMCC63202 was incubated in MH broth at
37 ꢄC and then the bacterial suspension was diluted with MH broth
to an OD620 of 0.01. The prepared bacterial solutions contain
0.5 ꢂ MIC concentration of the test compound and grow at 37 ꢄC for
18 h. The cells for morphology studies were collected and resus-
5.4. Minimum bactericidal concentration (MBC) assay
MBC assay was conducted using the broth microdilution assay
described in the preceding section [50]. After 24 h of incubation,
5 mL of the clear solutions from the microtiter wells were plated
onto tryptic soy agar (TSA). The TSA plates were incubated aero-
bically for 24 h at 37 ꢄC. After 24 h, the colonies were observed on
TSA plates. If there were no colonies, the corresponding minimum
concentration was the MBC value.
pended in 0.5 mL of PBS buffer and 10 mL of the suspension mixture
was transferred on a microscopic slide pretreated with 0.1% (w/v)
poly-L-lysine. Phase-contrast light microscope (the Olympus
CKX41) was used to capture cell morphology.
5.9. FtsZ polymerization assay
The polymerization of BsFtsZ was monitored using the light
scattering assay adapted from the literature [52]. The polymeriza-
tion was measured using Hitachi fluorescence spectrophotometer
(model F-2500) and the excitation and emission wavelengths were
set at 411 and 430 nm, respectively. The PMT Voltage was set at
5.5. Time-kill curve assay
The log-phase S. aureus ATCC 25923 was diluted to approxi-
mately 106 CFU/mL in volumes of MH broth containing a series of
concentrations (1/2, 1, 2 and 4 ꢂ MIC) of the test compound. The
700 V, and the slit width was 5 nm. BsFtsZ (5 mM) in 20 mM of Tris
resultant mixtures were incubated at 37 ꢄC with shaking. 20
m
L
buffer (pH 7.4, containing 20 mM KCl, 5 mM MgCl2 and 0.01% Triton
X-100 to avoid compound aggregation) was placed in a fluorometer
cuvette, and then was incubated with vehicle (2% DMSO) or
different concentrations of the test compound. A final concentra-
tion of 2 mM GTP was added at the last fraction and the increase in
light scattering was measured for an additional 800s. Moreover,
samples were removed for serial dilution in 180
broth at 0, 3, 6, 9, 12 and 24 h, respectively. Then, 10
m
L volumes of MH
m
L of the above
diluted samples were plated onto TSA plates with a smear loop. All
TSA plates were incubated at 37 ꢄC for 24 h and the CFU/mL at each
time point was obtained by counting colonies. Then, we draw a
time-kill curve with data of different concentrations and different
time points.
negative control experiments with 10
formed in the same way.
mg/mL linezolid were per-
5.6. Biofilm formation quantification assay
5.10. Transmission electron microscopy (TEM)
Biofilm inhibition was performed using the crystal violet assay
as previously described [51]. Bacterial strains were grown over-
night in MH medium, washed in fresh media and diluted to an
OD620 of 0.05. This culture was grown for 5e6 h to mid-log phase
(OD620 of 0.3e0.5) and then diluted with MH media to an OD620 of
BsFtsZ (5
of the test compound in polymerization reaction solution described
above. After 20 min, 10 L of the resulting dilutions were placed on
glow-discharged Formvar carbon-coated copper grids (400 mesh).
After 10 min, the grids were negatively stained with a solution of 1%
phosphotungstic acid (PTA) for 1 min and excess PTA was wicked
away with filter paper, air-dried. The specimen was then digitally
imaged on a transmission electron microscope (HITACHI, HT-7700)
interfaced with a CCD camera.
mM) was incubated in the absence and in the presence
m
0.05. 100
amounts of the test compounds into the 96-well plate at a final
concentration of 64, 32, 16, 8 and 4 g/mL, set 3 parallel wells for
each group. The control group was added to 100 L of bacterial
culture per well and the blank group was added to 100 L of fresh
mL of bacterial suspension was aliquoted with certain
m
m
m
MH medium. Then the culture was incubated statically for 18 h at
37 ꢄC. After that, the bacterial culture was removed and the 96-well
plate washed three times with PBS and dried (pH ¼ 7.2e7.4). The
remaining biofilms were fixed with anhydrous methanol for 15 min
before dried. Then biofilms were stained with 0.1% (w/v) crystal
violet (CV) solution for 30 min. After removing CV, each well was
washed three times with distilled water, dried out and solubilized
5.11. Hemolytic activity assay
The final concentrations ranged from 1 ꢂ MIC to 16 ꢂ MIC
mg/mL
and 50 L of each was added to 96-well plates. Fresh red blood cells
m
of mice were collected by centrifugation at a speed of 1500 rpm for
10 min. Following that, PBS was used to wash the red blood cells
with the same method three times. After addition of 20-fold vol-
in 120
mL 1% sodium dodecyl sulphate. Biofilm inhibition was
ume PBS to washed red blood cells, 50 mL of the resulting solution
determined by measuring the absorbance at 540 nm by a microtiter
plate reader. Each assay was performed in triplicate.
was added to each vial containing the serially diluted compound
and incubated at 37 ꢄC for 1 h. 96-well plates were centrifuged at
1500 rpm for 10 min, and then 30
mL of the suspension was taken
5.7. Resistance development study
out and transferred to another 96-well plate containing 70
mL of
PBS in each vial. The absorbance of the mixture was observed at
450 nm. The negative control was the PBS buffer not treated with
the drug, and the positive control was the PBS buffer treated with
2% Triton. The experiment was performed in triplicate and the
The propensity of bacterial resistance development for test
compound was assessed using a laboratory resistance simulation
assay. The initial MIC of the test compound against S. aureus
ATCC25923 was obtained from the above method. Bacterial cells in
hemolysis activity was calculated by the formula
%
19

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European Journal of Medicinal Chemistry 221 (2021) 113480
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PBS)] ꢂ 100.
¼
[(Abs sample e Abs PBS)/(Abs Triton e Abs
5.12. In vivo efficacy
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The mid-log phase cultures of S. aureus ATCC25923 were
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5.13. Computational study
Molecular docking was performed using Discovery Studio. The
X-ray crystal structure of FtsZ (PDB ID: 4DXD; resolution: 2.0 Å) was
downloaded from the Protein Data Bank (PDB) database. Water
molecules and co-crystal ligands were removed from the structure
and the SaFtsZ protein was prepared for further molecular docking.
The ligand molecule was built using the Chembio3D software and
minimized using the Discovery Studio molecule preparation tools.
Through the graphical user interface DS CDocker protocol, auto-
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Declaration of competing interest
[22] N. Sun, F.Y. Chan, Y.J. Lu, M.A.C. Neves, H.K. Lui, Y. Wang, K.Y. Chow, K.F. Chan,
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The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
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Acknowledgements
This work was supported financially by National Natural Science
Foundation of China (81973179 and 81673284) and Key Project of
Major Project of New and Old Kinetic Energy Conversion of Shan-
dong Province in 2020.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2021.113480.
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