Synthesis and antibacterial activity of 5-methylphenanthridium derivatives as FtsZ inhibitors

Article abstract of DOI:10.1016/j.bmcl.2017.06.005

5-Methylphenanthridium derivatives were designed, synthesized and evaluated for their in vitro antibacterial activity and cell division inhibitory activity against various Gram-positive and -negative bacteria. Among them, compounds 5A2, 5B1, 5B2, 5B3, 5C1 and 5C2 displayed the best on-target antibacterial activity with an MIC value of 4?μg/mL against B. subtilis ATCC9372 and S. pyogenes PS, showing over 2-fold better activity than sanguinarine. The SARs showed that the 5-methylphenanthridium derivatives with the alkyl side chains at the 2-postion, especially the straight alkyl side chains exerted better on-target antibacterial activity.

Full text of DOI:10.1016/j.bmcl.2017.06.005

Accepted Manuscript
Synthesis and antibacterial activity of 5-methylphenanthridium derivatives as
FtsZ inhibitors
Fang Liu, Henrietta Venter, Fangchao Bi, Susan J. Semple, Jingru Liu, Chaobin
Jin, Shutao Ma
PII:
S0960-894X(17)30607-8
DOI:
http://dx.doi.org/10.1016/j.bmcl.2017.06.005
BMCL 25039
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
16 February 2017
17 May 2017
2 June 2017
Please cite this article as: Liu, F., Venter, H., Bi, F., Semple, S.J., Liu, J., Jin, C., Ma, S., Synthesis and antibacterial
activity of 5-methylphenanthridium derivatives as FtsZ inhibitors, Bioorganic & Medicinal Chemistry Letters
(2017), doi: http://dx.doi.org/10.1016/j.bmcl.2017.06.005
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Synthesis and antibacterial activity of 5-methylphenanthridium derivatives as
FtsZ inhibitors
b
b
a
Fang Liu a, Henrietta Venter Fangchao Bi a, Susan J. Semple , Jingru Liu , Chaobin Jina, Shutao
Ma a,*
a Department of Medicinal Chemistry Key Laboratory of Chemical Biology (Ministry of Education)
School of Pharmaceutical Sciences, Shandong University a. 44 West Culture Road, Jinan 250012
(P.R. China).
b School of Pharmacy & Medical Sciences, Sansom Institute for Health Research, University of South
Australia, GPO Box 2471, Adelaide 5001, Australia
Running title: 5-Methylphenanthridium derivatives
* Corresponding author: Shutao Ma
Department of Medicinal Chemistry
School of Pharmaceutical Sciences, Shandong University
44, West Culture Road, Jinan 250012, P. R. China
Tel/Fax: +86 531 88382009; E-mail: mashutao@sdu.edu.cn
1

Abstract
5-Methylphenanthridium derivatives were designed, synthesized and evaluated for their in vitro
antibacterial activity and cell division inhibitory activity against various Gram-positive and -negative
bacteria. Among them, compounds 5A2, 5B1, 5B2, 5B3, 5C1 and 5C2 displayed the best on-target
antibacterial activity with an MIC value of 4ꢀµg/mL against B. subtilis ATCC9372 and S. pyogenes
PS, showing over 2-fold better activity than sanguinarine. The SARs showed that the
5-methylphenanthridium derivatives with the alkyl side chains at the 2-postion, especially the straight
alkyl side chains exerted better on-target antibacterial activity.
Keywords: Antimicrobials; Biological evaluation; FtsZ; Structure-activity relationships;
5-Methylphenanthridium derivatives.
2

Although the great development of antimicrobials in the past century has gave rise to the
tremendous advances for human beings, making the rate of mortality from bacterial infections
decrease remarkably and presenting a perception of the final triumph to the public in the battle
against bacteria.1 However, bacterial infections remain to be one of the exceedingly serious threats to
the human society, impacting the lives of people all over world and the global economy
significantly.2 On the one hand, the profligate use of antibacterial agents has accelerated the
evolution of bacterial resistance and even the epidemiological landscape of the Gram-positive and
-negative pathogens.3 On the other hand, the past almost three-decade-long drug discovery in the
field of antibacterial agents has avoided any new classes of antimicrobial drugs.4 In the current status,
except for the restrictive use of antibacterial agents which provides a solution to the rapidly
increasing bacterial resistance, it is becoming ever more urgent to discover new antimicrobial agents
with novel or so-far-underexplored targets to address the recent antibiotic crisis.
Filamentous temperature-sensitive protein Z (FtsZ), as a crucial bacterial cell division protein,
has been identified as an appealing target for the development of novel antimicrobials in the recent
years, because of its high conservatism among bacterial pathogens and absence in mammalian cells.5,
6
It plays a pivotal role in the bacteria cell division. When the bacterial division starts, FtsZ protein
self-polymerizes into protofilaments in a GTP-dependent fashion, and then forms the so-called Z-ring
at the center of the cell.7, 8 The resulting Z-ring serves as a scaffold to recruit other cell division
proteins.9, 10 After the recruitment, Z-ring contracts, enabling the septum formation and the final cell
separation. Therefore, any factor disturbing the function of FtsZ protein will eventually result in the
failure of cell division and then the bacterial death.
Sanguinarine (Fig. 1), a kind of benzophenanthridine alkaloid, has been identified as a small
molecule inhibiting FtsZ with antibacterial activity.11 It can strongly induce bacterial filaments and
alter Z-ring formation. Some in vitro experiments showed that sanguinarine inhibits FtsZ induced
polymerization and hinders lateral interaction between the raw silks. Therefore, the number of FtsZ
bundles is reduced, thereby inhibiting the formation of Z rings and reducing the frequency of their
occurrence. However, it can also inhibit tubulin polymerization into microtubules, producing toxic
side effects on mammalian cells.12 Chelerythrine (Fig. 1), similar to sanguinarine in the structure,
showed similar activity against S. aureus and B. subtilis. By modifying their structures and reducing
their toxic effects on the human body, it is possible to develop new antibacterial drugs.
3

Fig. 1. The structures of sanguinarine, chelerythrine and 5-methylphenanthridium.
The general synthetic methodology employed for the synthesis of 5-methylphenanthridium
derivatives is shown in Scheme 1. O-Bromobenzoic acid 1 was converted to the acid chloride in situ
using SOCl2, and this acid chloride was then reacted with the anilines to obtain the benzamides 2A-C
in high yields. After that, by treating the benzamides with NaH and CH3I in DMF to give the
corresponding N-methylbenzamides 3A-C. The N-methylbenzamides were then cyclized to the
phenanthridinones 4A-C via an intramolecular Heck reaction using catalytic Pd(OAc)2, PPh3 and
Na3CO3 in good yields. The phenanthridinones were reduced by DIBAL as the reducing agent and
subsequent treatment with dilute HCl produced the phenanthridinium salts 5A-C in high yields. The
structures of the target compounds were characterized by MS, 1H-NMR, and all the spectral data
were in agreement with the proposed structures.
4

Scheme 1. Reagents and conditions: (a) i) SOCl2, reflux, 2 h; ii) phenyl anilines, TEA, DCM, rt, 3 h
78.6~89.3%; (b) NaH, DMF, 0 °C~rt, 70.1~75.6%; (c) Pd(OAc)2, PPh3, Na2CO3, dry DMF, reflux, 5
h, 76.8~83.2%; (d) i) DIBAL, -10 °C~rt; ii) 2 M HCl, 20 min, 15.2~29.0%.
The 5-methylphenanthridium derivatives (5A1-5C1) were determined for their in
vitro antibacterial activity and cell division inhibitory activity, respectively. The in vitro antibacterial
activity was tested using tube dilution method recommended by NCCLS and the cell division
inhibitory activity was measured by assessing cell morphology using phase-contrast light
microscopy. The tested strains included S. aureus ATCC25923, S. aureus ATCC29213 (MRSA), S.
epidermidis, S. pyogenes PS, E. Coli ATCC25922, B. subtilis ATCC9372 and P. Aeruginosa
ATCC27853. S.
aureus ATCC25923, B.
subtilis ATCC9372, E.
coli ATCC25922
and P.
aeruginosa ATCC27853 were used to determine the cell division inhibitory activity of the tartget
compounds. The results in the unit of µg/mL are shown in Table1, Table 2 and Fig. 2.
The 5-methylphenanthridium derivatives universally exhibited better antibacterial activity against
B. subtilis ATCC9372, S. aureus PR, S. pyogenes PS and S. pyogenes PR than the other tested
strains. Among them, many derivatives also showed superior or comparable activity against B.
subtilis ATCC9372 and S. pyogenes PS to sanguinarine and ciprofloxacin. Particularly, compounds
5A2, 5B1, 5B2, 5B3, 5C1 and 5C2 displayed the best antibacterial activity with an MIC value of
4ꢀµg/mL against B. subtilis ATCC9372, showing over 8, 2, 2, and 2-fold better activity than
curcumin, sanguinarine, ciprofloxaxin and oxacillin sodium, respectively. In the inhibition of S.
pyogenes PS, the most active compounds5A2, 5B1, 5B2, 5B3, 5C1 and 5C2 exhibited the same MIC
value of 4ꢀµg/mL, being 2, 16, and 4-fold better than sanguinarine and oxacillin sodium, respectively.
Furthermore, compound 5B5 also showed the strongest activity with an MIC value of 16ꢀµg/mL
against S. aureus ATCC29213 in all of the tested compounds, being 8-fold and 8-fold more potent
than curcumin, and oxacillin sodium, but 2- and weaker 4-fold weaker than sanguinarine and
ciprofloxacin. Compounds 5A2, 5B1, 5C1 and 5C2 exhibited the same activity against S. aureus PR
to sanguinarine. Besides, all of the compounds showed basically the same activity against S.
pyogenes PS and S. pyogenes PR.
5

Table 1. Antibacterial activity of 5-methylphenanthridium derivatives (µg/mL)
S. aureus
B. subtilis
ATCC9372 a)
E. coli
ATCC25922 b)
P. aeruginosa
ATCC27853 c)
S. aureus
ATCC25923 d)
S. aureus
PR f)
S. epidermidis S. pyogenes S. pyogenes
PR i)
Compounds
ATCC29213
e)
g)
PS h)
5A1
5A2
5A3
5A4
5A5
5A6
5A7
5A8
5A9
5A10
5B1
16
4
>128
>128
>128
>128
>128
>128
>128
>128
>128
128
128
>128
>128
>128
>128
>128
>128
>128
128
>128
64
>128
128
>128
>128
>128
128
>128
>128
>128
>128
>128
64
32
4
>128
128
128
>128
128
64
32
4
32
8
8
64
16
8
16
8
16
8
4
64
128
8
128
>128
128
128
>128
>128
128
8
128
8
128
8
128
16
128
32
32
32
8
128
8
128
32
32
32
4
128
>128
>128
>128
128
64
128
32
32
32
4
16
16
4
>128
>128
>128
>128
>128
>128
>128
>128
>128
8
>128
>128
>128
>128
>128
>128
>128
>128
8
5B2
4
16
8
4
8
5B3
4
16
64
32
4
8
5B4
8
>128
16
>128
16
16
8
128
>128
128
64
16
8
16
8
5B5
8
5C1
4
64
32
4
4
8
5C2
Sang j)
Cipro k)
4
128
8
32
4
4
8
8
8
4
64
8
8
8
32
4
16
>128
>128
8
16
>128
2
OS l)
8
128
128
8
128
1
a)
b)
c)
B. subtilis ATCC9372: penicillin-susceptible strain; E. coli ATCC25922: penicillin-susceptible strain; P. aeruginosa ATCC27853: penicillin-susceptible strain; S. aureus ATCC25923:
d)
e)
f)
S. aureus ATCC29213: methicillin-resistant strain; S. aureus PR: penicillin-resistant strain isolated clinically, not characterized;
g)
penicillin-susceptible strain;
S. epidermidis:
h)
penicillin-resistant strain isolated clinically, not characterized; S. pyogenes PS: penicillin-susceptible strain; S. pyogenes PR: penicillin-resistant strain; Sang: Sanguinarine Cipro:
i)
j)
k)
ciprofloxacin; l) OS: oxacillin sodium.
6

All the above results indicated that 5-methylphenanthridium and its derivatives were the
effective simplified forms of sanguinarine and chelerythrine. The study of SARs showed that
the introduction of the alkyl side chains at the 2-position of the 5-methylphenanthridium (such
as compounds 5A2, 5A3 and 5A4) could improve the antibacterial activity. Furthermore, the
straight alkyl side chain was found to be better than the branched alkyl side chain in the
antibacterial activity. By comparison, introducing halogen group into the 2-position (such as
compounds 5A9 and 5A10) slightly decrease the antibacterial activity while introducing
methoxyl and methylthio groups at the 2-position retained the antibacterial activity. However,
the substituent group at the 4-position was less rigorous and regular. Nonetheless, introduction
of the alkyl side chain (such as compounds 5B1, 5B2 and 5B3) into the 4-position could
enhance the antibacterial activity as well. Additionally, introduction of the side chains at the 2-
and 4-positions simultaneously (such as compounds 5C1 and 5C2) could increase the
antibacterial activity.
As seen from Table 2 and Fig. 3, basically the cell division inhibitory activity of all the target
compounds fitted their in vitro antibacterial activity against S. aureus ATCC25923, B.
subtilis ATCC9372, E. coli ATCC25922 and P. aeruginosa ATCC27853. Therefore, the above
results indicated that the 5-methylphenanthridium derivatives were promising FtsZ inhibitors with
on-target antibacterial activity.
7

Table 2. Cell division inhibitory activity of 5-methylphenanthridium derivatives (µg/mL)
B. subtilis ATCC9372 a)
E. coli ATCC25922 b)
P. aeruginosa ATCC27853 c)
S. aureus ATCC25923 d)
Compounds
5A1
5A2
5A3
5A4
5A5
5A6
5A7
5A8
5A9
5A10
5B1
5B2
5B3
5B4
5B5
5C1
5C2
Sang e)
16
4
>128
>128
128
128
>128
>128
>128
>128
>128
>128
>128
128
>128
32
8
64
4
128
64
64
8
>128
>128
>128
128
128
>128
128
128
>128
>128
128
8
64
8
16
16
4
>128
128
>128
>128
>128
>128
>128
>128
128
>128
>128
>128
>128
128
4
4
16
8
>128
16
8
2
>128
>128
>128
64
2
128
128
8
8
>128
a) B. subtilis ATCC9372: penicillin-susceptible strain; b) E. coli ATCC25922: penicillin-susceptible strain; c) P. aeruginosa ATCC27853: penicillin-susceptible strain; d) S. aureus ATCC25923:
penicillin-susceptible strain; e) Sang:Sanguinarine
8

Fig. 2. B. subtilis ATCC9372 without (A) and with (B) compound 5B3 at 128 µg/mL; S. aureus
ATCC25923 without (C) and with (D) compound 5B3 at 128 µg/mL.
Besides, the 5-methylphenanthridium derivatives were also determined using a commercial
tubulin assay kit for their effect on the polymerisation of mammalian tubulin. The results showed
that none of the compounds enhanced polymerisation of mammalian tubulin even though they
were added at concentrations much higher than that of the positive control paclitaxel (Figure 3A
on the page 6 in supplementary material). A marginal inhibitory effect was observed on tubulin
polymerisation in the presence of compounds 5A5 and 5B2 (Figure 3B on the page 6 in
supplementary material). These compounds were tested at relatively high concentrations though
(32 µg/mL and 256 µg/ml for 5B2 and 5A7 respectively). The effect of compound 5A5 on
mammalian tubulin polymerisation could not be determined as the compound interfered with the
fluorescence of the reporter used in the assay kit. However, the MIC values of this compound
were at least 128 µg/mL for all the bacterial strains tested. Hence, this derivative would not be
selected for further optimisation anyway.
9

In conclusion, a series of 5-methylphenanthridium derivatives were designed, synthesized and
evaluated for their in vitro antibacterial activity and cell division inhibitory activity against various
Gram-positive and Gram-negative bacteria. Besides, the activity against polymerisation of mammalian
tubulin was determined. Among them, compounds 5A2, 5B1, 5B2, 5B3, 5C1 and 5C2 displayed the
best on-target antibacterial activity with an MIC value of 4ꢀµg/mL against B. subtilis ATCC9372 and S.
pyogenes PS, showing over 2-fold better activity than sanguinarine. The SARs showed that the
introduction of the alkyl side chains at the 2-position of the 5-methylphenanthridium could improve its
antibacterial activity, especially the straight alkyl side chain being better than the branched alkyl side
chain in the antibacterial activity while introducing halogen group into the 2-position could cause
slightly reduced antibacterial activity.
Acknowledgments
This research was supported financially by the National Natural Science Foundation of China
(81673284), the Natural Science Foundation of Shandong (ZR2015BM019), Major Project of Research
and development of Shandong Province (2016GSF201202), Major Project of Science and Technology
of Shandong Province (2015ZDJS04001) and the Project-sponsored by SRF for ROCS, SEM.
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10

Highlights
˃ 5-Methylphenanthridium derivatives were designed and synthesized. ˃ They were tested for their
antibacterial and cell division inhibitory activities. ˃ Compounds 5A2, 5B1, 5B2, 5B3, 5C1 and 5C2
exerted the best on-target activity.
11