Synthesis and biological evaluation of pyridinium-functionalized carbazole derivatives as promising antibacterial agents

Article abstract of DOI:10.1016/j.bmcl.2017.08.040

Various pyridinium-functionalized carbazole derivatives were constructed by coupling the key fragments of carbazole skeleton and pyridinium nucleus in a single molecular architecture. Antibacterial bioassays revealed that some of the title compounds displ

Full text of DOI:10.1016/j.bmcl.2017.08.040

Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of pyridinium-functionalized
carbazole derivatives as promising antibacterial agents
a
a
⇑
⇑
Pei-Yi Wang , He-Shu Fang , Wu-Bin Shao, Jian Zhou, Zhuo Chen, Bao-An Song , Song Yang
State Key Laboratory Breeding Base of Green Pesticide and Agricultural Bioengineering, Key Laboratory of Green Pesticide and Agricultural Bioengineering, Ministry of
Education, Center for R&D of Fine Chemicals of Guizhou University, Guiyang 550025, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Various pyridinium-functionalized carbazole derivatives were constructed by coupling the key fragments
of carbazole skeleton and pyridinium nucleus in a single molecular architecture. Antibacterial bioassays
revealed that some of the title compounds displayed impressive bioactivities against plant pathogens
such as Xanthomonas oryzae pv. oryzae, Ralstonia solanacearum, and Xanthomonas axonopodis pv. citri with
minimal EC50 values of up to 0.4, 0.3, and 0.3 mg/L, respectively. These bioactivities were achieved by sys-
tematically tuning and optimizing bridging linker, alkyl length of the tailor, and substituents on the car-
bazole scaffold. Compared with the bioactivity of the lead compound (AP-10), antibacterial efficacy
dramatically increased by approximately 13-, 104- and 21-fold. This finding suggested that these com-
pounds can serve as new lead compounds in research on antibacterial chemotherapy.
Received 5 June 2017
Revised 15 August 2017
Accepted 17 August 2017
Available online xxxx
Keywords:
Pyridinium
Carbazole
Synthesis
Optimization
Antibacterial
Ó 2017 Elsevier Ltd. All rights reserved.
Disease-causing bacteria are gaining considerable attention
over the last decade due to the significant threats they impose
on agricultural products and their remarkable ability in acquiring
additional resistance mechanisms.1–3 Xanthomonas oryzae pv. ory-
zae (Xoo), Ralstonia solanacearum (R. solanacearum) and Xan-
thomonas axonopodis pv. citri (Xac) are three widely distributed
Gram-negative opportunistic pathogens that can infect an array
of individual species including rice, tomato, potato, tobacco and
citrus.4–6 Infection by these bacteria can present necrotic lesions
on leaves, stems and/or fruits, which consequently result in a seri-
ous loss in agricultural output.7,8 In addition, the emergence and
worldwide spread of multidrug resistant pathogens has exacer-
bated the management of these persistent plant bacterial diseases.
Although a few of the commercial drugs have been used to combat
these diseases such as bismerthiazol (BT) and thiodiazole copper
(TC), they failed to effectively treat the infected plants under field
conditions considering their poor efficiency, high phytotoxicity
and/or bacterial resistance.9,10 Therefore, exploring and developing
new anti-bacterial drugs is imperative with novel chemical motifs
preferably owning unique modes of action rather than analogues of
the existing ones.
Carbazole skeleton, owning impressive electronic and charge-
transport properties, is a crucial type of nitrogen-containing aro-
matic heterocyclic scaffold, present in many naturally occurring
products and biologically active substances.11,12 In addition, this
privileged building block can be easily tuned and modified with
various functional groups, which endow carbazole-based deriva-
tives and analogues with an admirable array of pharmacological
activities such as anticancer, anti-inflammatory, antiviral, antifun-
gal and antioxidant activities.13–16 In particular, the antimicrobial
activity has been widely investigated for their extensively poten-
tial applications in the pharmaceutical industry.17,18 For example,
Bremner and co-workers reported several carbazole-linked cyclic
and acyclic peptoids as growth inhibitors of Staphylococcus aureus
with a minimum inhibitory concentration (MIC) of 15 mg/mL.19 Gu
and co-workers had synthesized and evaluated the antimicrobial
activity of a series of new carbazole derivatives of ursolic acid
and found that some compounds exhibited significant antibacterial
activities against both Gram-positive and Gram-negative bacteria
with MIC values ranging from 3.9 mg/mL to 15.6 mg/mL.20 Thus,
the fusion of a carbazole motif with the target molecule might
probably lead to improved biological activity owing to the syner-
gistic effect of these valuable moieties.
As an important functional fragment, pyridinium nucleus exists
extensively in various kinds of pharmaceutical agents.21,22 Nor-
mally, compounds featuring this scaffold always acquire various
physicochemical properties and subsequently resulted in reformed
⇑
Corresponding author.
E-mail addresses: songbaoan22@yahoo.com (B.-A. Song), jhzx.msm@gmail.com
(S. Yang).
a
The two authors contribute equally to this work.
http://dx.doi.org/10.1016/j.bmcl.2017.08.040
0960-894X/Ó 2017 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Wang P.-Y., et al. Bioorg. Med. Chem. Lett. (2017), http://dx.doi.org/10.1016/j.bmcl.2017.08.040

2
P.-Y. Wang et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
Fig. 1. Design strategy for the target compounds.
has desirable electron characteristics, is probably responsible for
better binding with the enzymes or the receptors of bacteria; pyri-
dinium, which has a positive charge, is employed to possibly inter-
act with anionic cell components and increase the water solubility
as well as membrane permeability; bridging linker and alkyl chain
length of the tailor are used to tune the balance of hydrophobicity/
hydrophilicity of target compounds. Better bioactive structures
would be fabricated by utilizing the synergistic effect of these priv-
ileged moieties. The antibacterial activities of all the title com-
pounds were tested against plant pathogens such as Xoo, R.
solanacearum and Xac.
To investigate the antibacterial effect after replacing the anthra-
cene ring of AP-10 into carbazole skeleton, compound 1 was firstly
designed and synthesized. As indicated in Scheme 1, intermediate
2-(9H-carbazol-9-yl)ethanol was obtained by adding 2-bro-
moethanol into a mixture of carbazole and NaH in dry THF, fol-
lowed by treating it by two-step consecutive reactions with 11-
bromoundecanoyl chloride and pyridine to provide the title mole-
cule 1. The structure was confirmed by 1H NMR, 13C NMR and MS
experiments (detailed information see Supplementary data). The
antibacterial bioassays against Xoo, R. solanacearum and Xac were
performed as previously described,27,28 and the commercial
antibacterial agents (BT and TC) were co-assayed as positive con-
trols under similar conditions. The inhibitory effect of 1 was indeed
improved by introducing carbazole moiety in the target molecule
(Table 1). In particular, anti-R. solanacearum activity was signifi-
cantly increased with EC50 values from 31.3 mg/L to 1.6 mg/L,
approximately 19-fold enhancement in the antibacterial efficacy,
which validated the author’s assumption.
Further, we wonder if the removed ester group within the alkyl
chain would reform the bioactive efficacy, because compound 1
exhibited good antimicrobial potentials. Thereby, compound 2
was primarily constructed by sequential substitution reactions of
carbazole with 1,12-dibromododecane to produce bromide-tai-
lored carbazole, which was then treated with pyridine at 50 °C
(Scheme 2). The structure was also characterized by 1H NMR, 13C
NMR and MS experiments (detailed information see Supplemen-
tary data). The bioassay result is shown in Table 1. A dramatically
Scheme 1. Synthesis of compound 1.
or enhanced biological activities, because the positive charge can
strengthen their specificity for the target species.23 Moreover, pyri-
dinium-tailored amphiphiles are considered one of the powerful
molecular templates to create biologically active compounds, espe-
cially in the field of antimicrobial drug discovery.24,25 For example,
Eren et al. investigated the antibacterial activity of some pyri-
dinium functionalized polynorbornenes and found that com-
pounds bearing octyl tails on the pyridine rings showed potent
inhibitory effects against Escherichia coli and Bacillus subtilis.26
Apparently, these repeatedly numerous studies on this functional
scaffold opened a new avenue for the discovery and development
of novel high-efficient bioactive molecules.
In our previous work, 1-[11-(9-anthracenyl methoxy)-11-
oxoundecyl] pyridinium bromide (AP-10) exhibited good antibac-
terial activities towards plant pathogens.27 Encouraged by the
aforementioned facts and in a continuing search for high-efficient
antibacterial agents, we report herein the design and synthesis of
a series of pyridinium-tailored carbazole derivatives by coupling
key fragments of carbazole skeleton and pyridinium nucleus in a
single molecular architecture (Fig. 1). The carbazole group, which
Table 1
Antibacterial activities of target compounds 1–5 against plant pathogen Xoo, R. solanacearum and Xac in vitro.
No.
Xoo
R. solanacearum
Xac
Regression equationa
r
EC50 (mg/L)
Regression equation
r
EC50 (mg/L)
Regression equation
r
EC50 (mg/L)
AP-10
1
2
3
4
5
BT
TC
y = 3.29x + 2.60
y = 3.54x + 3.36
y = 16.27x + 10.85
y = 9.67x + 5.07
y = 14.16x À 2.52
y = 3.91x + 0.26
y = 1.50x + 2.05
/
0.93
0.98
0.98
0.98
0.96
0.95
0.98
/
5.3 0.7
2.9 0.3
0.4 0.1
1.0 0.1
3.4 0.1
16.4 2.4
92.6 + 2.2
/
y = 1.01x + 3.50
y = 12.73x + 2.39
y = 16.91x + 15.26
y = 9.55x + 8.89
y = 4.73x + 4.89
y = 3.85x + 2.07
/
0.97
0.98
0.96
1.00
0.98
0.99
/
31.3 8.2
1.6 0.2
0.3 0.1
0.4 0.1
1.1 0.1
5.8 0.5
/
y = 2.74x + 2.80
y = 12.87x À 2.71
y = 3.59x + 6.60
y = 2.03x + 4.57
y = 5.18x + 4.38
y = 2.12x + 4.24
/
0.96
0.94
0.95
1.00
0.95
1.00
/
6.3 0.5
4.0 0.2
0.3 0.1
1.6 0.4
1.3 0.1
2.3 0.2
/
y = 1.03x + 2.94
0.99
99.1 5.1
y = 2.15x + 0.94
0.98
77.0 2.0
a
Five different concentrations (such as 80, 40, 20, 10, 5 mg/L, depending on the bioactivity of different compounds, the concentrations were chosen in two times decline
trend to make sure the EC50 values are inside the concentration ranges tested) of the test compounds and positive control were selected to test the corresponding inhibition
rates. By using the SPSS 17.0 software and the obtained inhibition rates at different concentrations, a related regression equation was provided to calculate the related EC50
values.10,27,28
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P.-Y. Wang et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
3
improved antibacterial potency was observed against Xoo, R. sola-
nacearum and Xac, and their EC50 values varied from 2.9 mg/L to
0.4 mg/L, 1.6 mg/L to 0.3 mg/L and 4.0 mg/L to 0.3 mg/L, respec-
tively, suggesting that the ester group might serve as a disadvanta-
geous factor to block any further interactions between compound 1
and bacterial receptors.
Comparing the bioactivity of the lead compound (AP-10), the
antibacterial efficacy was strongly enhanced by approximately
13-, 104- and 21-fold, respectively, indicating that a better bioac-
tive structure was achieved. To tune the balance of hydrophobic-
ity/hydrophilicity of target molecules, pyridinium-tailored
carbazoles (3, 4, 5) bearing alkyls of different lengths were fabri-
cated following the synthetic protocols of compound 2. EC50 values
provided an enhancive tendency with the decrease in the alkyl
chain length, demonstrating that the decrease in molecular
lipophilicity was unfavourable to the bioactivity.
To study the effect of the substitution on the carbazole ring
towards bioactivity, title compounds 6–21 possessing different
substitutional units were designed and synthesized from Scheme 3.
In general, the intermediates containing diacetyl or di-(p-chlorl-
benzoyl) groups at the 3, 6-positions of the carbazole skeleton
were provided through the Friedel Crafts reaction with acetyl chlo-
ride or 4-chlorobenzoyl chloride,29,30 which subsequently reacted
with lithium aluminum hydride to produce the other two crucial
intermediates bearing diethyl or di-(p-chlorobenzyl) groups.
Finally, a series of target compounds simultaneously owning dif-
ferent alkyl chain lengths (n = 6, 8, 10, 12) were obtained through
the synthetic protocols of compound 2.
Preliminary antibacterial bioassays revealed that these com-
pounds exhibited poor to good activities against the three tested
strains. As noted in Table 2, the substituents had a considerable
influence towards bioactivity. Compounds 6–9 and 14–17 contain-
ing diacetyl or diethyl groups can effectively inhibit the growth of
Xoo at 80 mg/L, even lowered the concentration to 40 mg/L. By
contrast, the anti-Xoo capacity was significantly decreased after
placing the sterically hindered groups (p-chlorlbenzoyl or p-
chlorobenzyl) at the 3, 6-positions of the carbazole ring. A phe-
nomenon for EC50 values of compounds 6–9 against Xoo was firstly
decreased and then increased with improvement in the length of
alkyl tailors (Table 3). Thereby, compound 8 produced the minimal
EC50 value of up to 1.4 mg/L, suggesting that even slight changes in
the ratio of hydrophobicity/hydrophilicity can affect their bioactiv-
ities. However, the EC50 values of compounds 14–17 followed the
order of 14 (0.9 mg/L) < 15 (1.1 mg/L) < 16 (10.3 mg/L) < 17
(10.9 mg/L), revealing that the enhancement of molecular
lipophilicity might result in negative effects to the anti-Xoo activ-
ity. The electronic effect of substituents on the carbazole scaffold
also had a significant effect on the anti-Xoo activity, illuminated
by comparing the EC50 values of 6 (15.4 mg/L) and 14 (0.9 mg/L),
7 (5.3 mg/L) and 15 (1.1 mg/L), 8 (1.4 mg/L) and 16 (10.3 mg/L), 9
(3.3 mg/L) and 17 (10.9 mg/L) with the former bearing electron-
withdrawing groups (CH3CO-) and the latter containing electron-
donating groups (CH3CH2–). The antibacterial efficacy towards R.
solanacearum was dramatically reduced after introducing small
or large sterically hindered fragments, though compound 14
showed an inhibition rate of 92.7% at a dosage of 80 mg/L. For
Scheme 2. Synthesis of compounds 2, 3, 4, and 5.
Scheme 3. Synthesis of compounds 6–21.
Table 2
Inhibitory effect of target compounds 6–21 against plant pathogen Xoo, R. solanacearum and Xac in vitro.
No.
Inhibition (%)
Xoo
R. solanacearum
Xac
80 mg/L
40 mg/L
80 mg/L
40 mg/L
80 mg/L
40 mg/L
6
7
8
100
100
100
85.6 4.8
98.4 0.3
100
22.5 9.4
41.8 4.3
34.3 4.0
11.6 2.3
17.9 0.7
27.5 6.9
96.5 2.6
100
100
62.6 3.7
100
100
9
100
100
5.9 3.1
4.5 3.8
100
100
10
11
12
13
14
15
16
17
18
19
20
21
BT
TC
61.4 1.5
66.4 2.0
33.5 9.5
55.8 7.4
100
100
100
100
53.3 5.1
38.8 2.1
26.9 9.4
4.1 5.8
45.1 5.2
/
58.3 3.7
58.3 8.0
18.6 8.7
25.1 4.6
100
100
100
95.5 2.8
26.7 2.6
18.8 3.6
0
0
0
0
0
0
0
0
0
36.8 5.4
21.2 2.1
22.7 1.7
24.6 6.3
100
28.4 1.1
17.1 1.4
18.3 9.9
16.5 0.7
100
92.7 3.2
20.4 7.4
0
0
0
0
0
0
66.7 6.0
0
0
0
0
0
0
0
100
100
100
100
49.4 6.9
40.8 7.0
10.0 3.5
17.3 4.6
18.9 2.7
/
27.7 8.9
30.2 5.4
8.9 2.3
11.0 2.5
13.8 4.6
/
0
35.3 3.4
/
/
/
43.2 1.6
12.1 2.3
51.2 3.2
32.4 2.8
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P.-Y. Wang et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
Table 3
Antibacterial activities of target compounds (6–11, 13–18) against pathogen Xoo, R. solanacearum and Xac in vitro.
No.
Xoo
R. solanacearum
Xac
Regression equation
r
EC50 (mg/L)
Regression equation
r
EC50 (mg/L)
Regression equation
r
EC50 (mg/L)
6
7
8
9
y = 1.29x + 3.47
y = 2.73x + 3.02
y = 2.83x + 4.57
y = 3.25x + 3.32
y = 0.77x + 3.85
y = 0.99x + 3.56
y = 1.92x + 1.41
y = 2.94x + 5.16
y = 1.11x + 4.95
y = 1.35x + 3.63
y = 2.07x + 2.85
y = 1.74x + 1.71
y = 1.50x + 2.05
/
0.94
0.97
0.94
0.93
0.95
0.99
0.97
0.99
0.94
0.96
0.96
0.98
0.98
/
15.4 1.2
5.3 0.4
1.4 0.1
3.3 0.1
31.2 0.5
28.1 5.4
73.9 3.0
0.9 0.1
1.1 0.1
10.3 1.3
10.9 0.7
77.6 5.2
92.6 + 2.2
/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
>80
>80
>80
>80
>80
>80
>80
19.1 4.4
>80
>80
>80
>80
/
y = 2.49x + 1.67
y = 2.18x + 3.15
y = 2.73x + 3.40
y = 0.83x + 5.22
/
/
/
y = 4.45x + 1.83
y = 2.25x + 3.12
y = 1.18x + 3.48
/
/
/
0.91
0.94
0.99
0.91
/
21.8 0.3
7.1 1.4
3.8 0.5
0.5 0.1
>80
>80
>80
5.1 0.7
6.8 0.2
19.4 5.3
>80
10
11
13
14
15
16
17
18
BT
TC
/
/
y = 1.95x + 2.51
/
/
/
/
/
0.92
/
/
/
/
/
1.00
1.00
0.98
/
/
/
>80
/
77.0 2.0
y = 1.03x + 2.94
0.99
99.1 5.1
y = 2.15x + 0.94
0.98
anti-Xac bioassays, compounds 7–9 and 14–16 exerted potent
growth suppression effects, even at 40 mg/L, whereas compounds
10–13 and 18–21 did not produce significant activity even at a
high concentration of 80 mg/L, further demonstrating that large
groups on the carbazole ring would block the bioactivity of title
compounds. Compounds 6–9 displayed enhanced anti-Xac activity
with increasing alkyl chain length, and their EC50 values reached a
minimum of 0.5 mg/L. This fact might be ascribed to the enhanced
molecular lipophilicity. In comparison, compounds 14–17 pro-
vided reduced anti-Xac ability with the increase in the alkyl tailors.
In view of the above study findings, the antibacterial activity can
be affected by a variety of factors including alkyl chain length of
the tailor, bridging linker, electronic properties and steric hin-
drance of substituents on the carbazole scaffold, which reminded
us to carefully optimize the molecular structures in the exploration
of novel, high-efficient bioactive substances.
In conclusion, a series of pyridinium-tailored carbazole com-
pounds were designed and synthesized by rationally tuning and
optimizing bridging linker, alkyl length of the tailor, and sub-
stituents on the carbazole skeleton. Antibacterial evaluation
results revealed that some of the target compounds exhibited
promising bioactivities against plant pathogen Xoo, R. solana-
cearum and Xac, with minimal EC50 values of 0.4, 0.3 and 0.3 mg/
L, respectively. Considering their simple structures and conve-
niently synthetic protocols, these kinds of compounds can be fur-
ther studied as new lead compounds in the field of antibacterial
chemotherapy.
A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2017.08.
040.
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We acknowledge the financial support of the Key Technologies
R&D Program (2014BAD23B01), National Natural Science Founda-
tion of China (21372052, 21662009), the Research Project of Min-
istry of Education of China (NCET-13-0748, 213033A), and Guizhou
Provincial S&T Program ([2012]6012, [2016]1029, 201341).
Please cite this article in press as: Wang P.-Y., et al. Bioorg. Med. Chem. Lett. (2017), http://dx.doi.org/10.1016/j.bmcl.2017.08.040