Synthesis and on-target antibacterial activity of novel 3-elongated arylalkoxybenzamide derivatives as inhibitors of the bacterial cell division protein FtsZ

Article abstract of DOI:10.1016/j.bmcl.2013.05.056

Novel 3-elongated arylalkoxybenzamide derivatives were designed, synthesized and evaluated for their cell division inhibitory activity and antibacterial activity. Among them, the subseries of 3-alkyloxybenzamide derivatives exhibited greatly improved on-t

Full text of DOI:10.1016/j.bmcl.2013.05.056

Bioorganic & Medicinal Chemistry Letters 23 (2013) 4076–4079
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and on-target antibacterial activity of novel 3-elongated
arylalkoxybenzamide derivatives as inhibitors of the bacterial cell
division protein FtsZ
Siti Ma a, , Chao Cong a, , Xiaohui Meng b, Shasha Cao a, Hongkun Yang a, Yuanyuan Guo a, Xueyi Lu a,
Shutao Ma a,
⇑
a Department of Medicinal Chemistry, Key Laboratory of Chemical Biology (Ministry of Education), School of Pharmaceutical Sciences, Shandong University, 44,
West Culture Road, Jinan 250012, PR China
b Institute of Diagnostics, School of Medicine, Shandong University, 44, West Culture Road, Jinan 250012, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Novel 3-elongated arylalkoxybenzamide derivatives were designed, synthesized and evaluated for their
cell division inhibitory activity and antibacterial activity. Among them, the subseries of 3-alkyloxybenza-
mide derivatives exhibited greatly improved on-target activity against Bacillus subtilis and Staphylococcus
aureus, and remarkably increased antibacterial activity against B. subtilis ATCC9372, penicillin-suscepti-
ble S. aureus ATCC25923, methicillin-resistant S. aureus ATCC29213 (MRSA) and penicillin-resistant
S. aureus PR compared with 3-methoxybenzamide. In contrast, the subseries of 3-phenoxyaklyloxybenza-
mide, 3-heteroarylalkyloxybenzamide and 3-heteroarylthioalkyloxybenzamide derivatives only showed
a significant improvement in on-target activity and antibacterial activity against B. subtilis ATCC9372.
Ó 2013 Elsevier Ltd. All rights reserved.
Received 26 January 2013
Revised 30 April 2013
Accepted 16 May 2013
Available online 24 May 2013
Keywords:
Arylalkoxybenzamide
FtsZ inhibitors
Synthesis
Cell division inhibition activity
Antibacterial activity
The emerging prevalence of methicillin-resistant Staphylococcus
aureus (MRSA), vancomycin-resistant enterococci (VRE) and peni-
cillin-resistant Streptococcus pneumoniae contributes to the enor-
mous difficulty in fighting against bacterial infections.1–3 In
particular, superbugs with New Delhi metallo-b-lactamase-1
(NDM-1) exhibit extensive resistance to almost all the antibiotics
including carbapenems, which have been a serious health con-
cern.4 Consequently, many originally powerful antibiotics in clinic
have showed either weak or no activity against the resistant bacte-
ria. However, the continuous emergence of the bacterial resistance
has also prompted a worldwide effort to develop new alternative
antibacterial agents, especially with novel mechanisms of action.
As a eukaryotic tubulin analogue, filamentous temperature-sen-
sitive protein Z (FtsZ) conserves in almost all bacteria. During bac-
terial cell division, FtsZ offers an essential skeleton for the
formation of Z-ring in the presence of guanosine triphosphate
(GTP).5–7 If FtsZ assembly is restrained, the bacterial cell division
will eventually fail, thereby resulting in bacterial apoptosis.
Accordingly, FtsZ is considered as an attractive target to develop
antibacterial agents with selective toxicity to bacterial pathogens
due to its essential role in prokaryotic cell division, widespread
conservation in the bacterial kingdom, absence in the mitochon-
dria of higher eukaryotes and evolutionary distance from tubulin.8
Nevertheless, FtsZ remains largely unexploited as a new target for
antibacterial agents, which has aroused the great interest of many
researchers to develop novel FtsZ inhibitors. To date, a certain
number of FtsZ inhibitors such as totarol, cinnamaldehyde, berber-
ine and 3-methoxybenzamide (3-MBA) (Fig. 1), which disturb
either the polymerization or the GTPase activity of FtsZ or both
of them, have been screened from natural sources or small synthe-
sized molecules.9–13
Although 3-MBA shows weak on-target antibacterial activity
against Bacillus subtilis with minimum inhibitory concentration
(MIC) values of 4096 lg/mL, it is able to penetrate bacterial cell
and bind to FtsZ at high ligand efficiency, which makes 3-MBA an
attractive starting point for the discovery of potent FtsZ inhibi-
tors.14 PC190723 (Fig. 1) as a potent derivative of 3-MBA is charac-
terized by substituted benzamide and thiazolopyridine moieties
joined together via an ether linkage.15 It not only inhibits FtsZ
GTPase activity in vitro and FtsZ localization in vivo, but also
exhibits potent antibacterial activity with MIC values in the range
of 0.5–1.0 lg/mL against B. subtilis and various staphylococci includ-
ing MRSA. More importantly, PC190723 has no inhibitory activity
against yeasts or human hepatocytes. Another derivative of
3-MBA obtained by replacing the thiazolopyridine of PC190723
⇑
Corresponding author. Tel.: +86 531 88382009; fax: +86 531 88382548.
E-mail address: mashutao@sdu.edu.cn (S. Ma).
These authors contributed equally to the work.
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.05.056

S. Ma et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4076–4079
4077
The 3-elongated arylalkoxybenzamide derivatives (4–31) were
synthesized from 3-hydroxybenzoic acid (1) as outlined in
Scheme 1. The chlorination of 1 with thionyl chloride was followed
by reaction with aqueous ammonia to afford 3-hydroxybenzamide
(3) in 71% yield. The alkylation of 3 with corresponding alkyl ha-
lides or in the presence of potassium carbonate gave the 3-alkyl-
oxybenzamide derivatives (4–15) in yields ranging from 55% to
74%. Further alkylation of the 4-substituted phenol with 9 or 10
provided the 3-phenoxyaklyloxybenzamide derivatives (16–23)
in yields within the range of 61–73%. Similarly, diazole or triazole
derivatives were subjected to the alkylation with 9 or 10 to give
3-heteroarylalkyloxybenzamide derivatives (24–27) in 71–78%
yields. The condensation of heteroarylthiol with 8, 9 or 10, respec-
tively, was catalyzed by potassium carbonate to afford the 3-het-
eroarylthioalkyloxybenzamide derivatives (28–31) in yields
within the range of 58–66%.
Figure 1. Structures of compounds inhibiting FtsZ polymerization and GTPase
activity.
The 3-elongated arylalkoxybenzamide derivatives were deter-
mined for cell division inhibitory activity and in vitro antibacterial
activity, respectively. The cell division inhibitory activity of the
tested compounds against B. subtilis and S. aureus was measured
by assessing cell morphology using phase-contrast light micros-
copy.21 The lowest concentration at which filamentation of B. sub-
tilis or ballooning of S. aureus appeared was recorded as cell
division inhibitory activity indicating on-target activity. The MIC
value of the tested compounds as in vitro antibacterial activity
was determined using a standard broth microdilution method rec-
ommended by NCCLS.22 The tested bacteria were four penicillin-
susceptible strains of B. subtilis ATCC9372, S. aureus ATCC25923,
S. pneumonia ATCC49619 and Streptococcus pyogenes PS, a methicil-
lin-resistant strain of S. aureus ATCC29213 (MRSA), and a penicil-
lin-resistant strain of S. aureus PR. The results of cell division
with a benzothiazole group displays more potent activity with an
MIC value of 0.25 l
g/mL against S. aureus than PC190723.16,17 on
the other hand, the structure–activity relationships (SARs) indicate
that the amide and 3-methoxy group of 3-MBA appear to be critical
for cell division inhibition while the replacement of the 3-methoxy
group with various moieties such as alkyl, aryl or heteroaryl groups
results in drug-like 3-MBA derivatives with on-target antibacterial
activity.18,19 Furthermore, all of the active derivatives of 3-MBA
convincingly dock into a cleft (a hydrophobic channel) between he-
lix 7 and the C-terminal domain of FtsZ in S. aureus.20 The docking
model also predicts a long space equivalent in length to at least
9–10 carbons for substitutions off the benzamide group. Accord-
ingly, a reasonably long hydrophobic side chain and a preferred ter-
minal group will be helpful for the interaction with the
hydrophobic cleft, thereby resulting in a remarkable improvement
in on-target activity and antibacterial activity.
inhibitory activity and antibacterial activity in units of
shown in Table 1.
lg/mL are
Therefore, on the basis of the consideration detailed above, we
designed novel 3-elongated arylalkoxybenzamide derivatives pos-
sessing the 3-elongated sided chains with 2–9 atoms from 3-oxy-
gen atom to the terminal groups such as halogen, aryl and
heteroaryl groups. Our objective was to further probe the effect
of the lengths of 3-elongated sided chains and various terminal
groups on on-target antibacterial activity.
3-Alkyloxybenzamide derivatives 4–15 with 2–9 atoms from
the 3-oxygen atom to the terminal halogen (or carbon) atom on
their 3-elongated side chains displayed significantly improved
on-target activity against B. subtilis and S. aureus, and remarkably
increased antibacterial activity against B. subtilis and three tested
strains of S. aureus, but failed to show improved antibacterial activ-
ity against S. pneumonia and S. pyogenes in comparison with 3-MBA
Scheme 1. Reagents and conditions: (a) SOCl2, toluene, reflux, 4.5 h; (b) aqueous ammonia, 70.8% for two-steps; (c) R1Cl2 or R1Cl, K2CO3, NaI, DMF (or R1Br2, K2CO3, CH3CN),
60 °C, 16–20 h, 55.4–73.6%; (d) 9 (or 10), 4-substituted phenol, K2CO3, DMF, 65–70 °C, 14–18 h, 61.4–72.6%; (e) 9 (or 10), diazole or triazole derivatives, K2CO3, DMF, 65–70 °C,
14–18 h, 71.9–77.2%; (f) 8 (or 9, 10), heteroaryl thiol, K2CO3, DMF, 65–70 °C, 58.6–65.2%.

4078
S. Ma et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4076–4079
and their precursor 3. In the subseries of the 3-chloroalkoxy deriv-
atives, 3-chlorohexyloxy derivative 7 was found to have the most
greatly improved on-target activity and antibacterial activity
against B. subtilis ATCC937, showing 128-fold and 256-fold better
than 3-MBA, respectively, while 3-chlorobutoxy derivative 5 dem-
onstrated the most remarkably improved on-target activity and
antibacterial activity against S. aureus, exhibiting 256-, 256- and
512-fold higher antibacterial activity against S. aureus ATCC25923,
S. aureus ATCC29213 and S. aureus PR than 3-MBA, respectively. In
the subseries of the 3-bromoalkoxy derivatives, 3-bromobutoxy
derivative 10 was the most effective in on-target activity and anti-
bacterial activity against B. subtilis and S. aureus, showing 256-,
256-, 256- and 512-fold better antibacterial activity against B. sub-
tilis ATCC9372, S. aureus ATCC25923, S. aureus ATCC29213 and S.
aureus PR than 3-MBA, respectively. In the subseries of the 3-alk-
oxypentyloxy derivatives, 3-butoxypentyloxy derivative 15 was
the most effective in on-target activity and antibacterial activity
against B. subtilis and S. aureus, showing 512-, 256-, 512- and
512-fold better antibacterial activity against B. subtilis ATCC9372,
S. aureus ATCC25923, S. aureus ATCC29213 and S. aureus PR than
3-MBA, respectively. The above results suggest that the extension
of the 3-alkoxy groups with two to nine atoms from the 3-oxygen
atom to the terminal halogen (or carbon) atom leads to a substan-
tial improvement in on-target activity and antibacterial activity
against B. subtilis and S. aureus. In particular, the introduction of
3-halogenoalkoxy group with 4 atoms or 3-etheroxy group with
9 atoms from the 3-oxygen atom to the terminal halogen (or
carbon) atom shows the most greatly improved antibacterial activ-
ity against S. aureus.
3-Phenoxyaklyloxybenzamide derivatives 16–23, which have
the 3-elongated side chain with 4–5 atoms from the 3-oxygen
atom to the terminal benzene ring, exhibited remarkably improved
on-target activity and antibacterial activity, especially against
B. subtilis compared to 3-MBA and their precursor 3. Among them,
4-chlorophenyl derivative 22 was found to have the most potent
on-target activity and antibacterial activity (0.25 and 0.5 lg/mL)
against B. subtilis ATCC9372. However, this series did not show
notably improved antibacterial activity against S. aureus,
S. pneumonia and S. pyogenes. Those indicate that the introduction
of 3-phenoxyaklyloxy groups with 4–5 atoms from the 3-oxygen
atom to the terminal benzene ring can enhance on-target activity
and antibacterial activity against B. subtilis.
3-Heteroarylalkyloxybenzamide derivatives 24–27 possessing
the 4–5 atom carbon chain from the 3-oxygen atom to the terminal
heteroaryl ring showed slightly improved antibacterial activity
against B. subtilis and S. aureus compared with 3-MBA. Only triazole
derivative 24 had remarkably improved on-target activity and
antibacterial activity against B. subtilis. Similarly, 3-hetero-
arylthioalkyloxybenzamide derivatives 28–31 containing the
thioether chain with 3–5 atoms from the 3-oxygen atom to the ter-
minal heteroaryl ring displayed improved on-target activity and
antibacterial activity against B. subtilis and S. aureus in comparison
with the references. In particular, benzoheterocyclic derivatives 28
and 30 showed greatly improved on-target activity and antibacte-
Table 1
Cell division inhibitory activity and antibacterial activity for the 3-elongated arylalkoxybenzamide derivatives
Compound
Cell division inhibitiona
(l
g/mL)
Minimum inhibitory concentration /MIC(lg/mL)
B. subtilis
S. aureus
B. subtilis
ATCC9372
S. aureus
ATCC25923
S. aureus
S. aureus
PRe
S. pneumoniae
S. pyogenes
PSg
ATCC9372b
ATCC25923c
ATCC29213d
ATCC49619f
3-MBA
3
4
5
6
7
8
9
512
4096
64
16
32
4
64
128
8
32
16
64
8
8
8
1024
4
4
32
32
0.25
32
16
WT
128
WT
16
512
4
WTh
4096
64
32
32
32
16
32
8
64
64
128
32
16
1024
512
512
512
512
WT
256
512
1024
1024
128
WT
1024
256
512
512
4096
512
128
32
64
16
128
128
16
64
32
64
32
8
32
1024
16
4
64
2048
4096
64
8
32
32
64
64
8
32
64
64
16
2048
4096
64
8
32
64
128
64
8
16
4096
4096
64
8
32
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
32
128
128
8
32
64
64
32
8
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
64
64
32
4
8
1024
1024
1024
1024
1024
4096
1024
1024
2048
1024
256
1024
2048
512
1024
1024
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
1024
0.5
32
32
1024
128
1024
32
512
16
128
128
a
b
c
Lowest concentration at which filamentation of B. subtilis or ballooning of S. aureus is observed indicating on-target activity.
B. subtilis ATCC9372: penicillin-susceptible strain.
S. aureus ATCC25923: penicillin-susceptible strain.
S. aureus ATCC29213: methicillin-resistant strain.
S. aureus PR: penicillin-resistant strain isolated clinically, not characterized.
S. pneumoniae ATCC49619: penicillin-susceptible strain.
d
e
f
g
h
S. pyogenes PS: penicillin-susceptible strain.
No effect on morphology at 2048–4096 lg/mL.

S. Ma et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4076–4079
4079
rial activity against B. subtilis. Among the heteroaryl derivatives
Acknowledgments
24–31, compound 30 exhibited the most significantly improved
on-target activity and antibacterial activity against B. subtilis,
showing 128- and 256-fold activity than 3-MBA, respectively.
On the whole, the 3-elongated arylalkoxybenzamide derivatives
had greatly improved on-target activity and antibacterial activity
against B. subtilis compared to the references. Especially, the
3-alkyloxybenzamide derivatives showed obviously increased
activity against three phenotypes of S. aureus in addition to
B. subtilis. Among all of the tested compounds, 4-chlorophenyl
derivative 22 had the most potent antibacterial activity against
B. subtilis while 3-chlorobutoxy derivative 5, 3-bromobutoxy deriv-
ative 10, and 3-butoxypentyloxy derivative 15 displayed the most
significantly improved antibacterial activity against S. aureus
ATCC25923 and S. aureus PR, respectively. Notably, 3-butoxypen-
tyloxy derivative 15 presented the best antibacterial activity
against resistant S. aureus ATCC29213. These results described
above led us to conclude that: (i) the extension of 3-alkoxy chains
with small terminal groups would be beneficial for increasing
on-target activity and antibacterial activity against B. subtilis and
S. aureus; (ii) the replacement of the small terminal groups of the
3-elongated alkoxy chains with large heteroaryl groups would
increase on-target activity and antibacterial activity against
B. subtilis. In addition, the preliminary SARs indicated that the
small terminal group might be a suitable for interaction with
the hydrophobic cleft in FtsZ of both B. subtilis and S. aureus, while
the large terminal group might only be beneficial for affinity to the
hydrophobic cleft in FtsZ of B. subtilis.
In conclusion, novel 3-elongated arylalkoxybenzamide deriva-
tives were designed, synthesized and evaluated for their on-target
activity and antibacterial activity. The 3-alkyloxybenzamide deriv-
atives showed greatly improved on-target antibacterial activity
against both B. subtilis and three phenotypes of S. aureus compared
with 3-MBA. In contrast, 3-phenoxyaklyloxybenzamide, 3-hetero-
arylalkyloxybenzamide and 3-heteroarylthioalkyloxybenzamide
derivatives only had a significant improvement in on-target activ-
ity and antibacterial activity against B. subtilis. Among all of the
tested compounds, 4-chlorophenyl derivative 22 had the most po-
tent antibacterial activity against B. subtilis and 3-butoxypentyloxy
derivative 15 presented the best antibacterial activity against resis-
tant S. aureus ATCC29213. Additionally, 3-chlorobutoxy derivative
5, 3-bromobutoxy derivative 10, and 3-butoxypentyloxy derivative
15 displayed the most significantly improved antibacterial activity
against S. aureus ATCC25923 and S. aureus PR, respectively. It is
noteworthy that the extension of 3-alkoxy chains with the small
terminal groups may enhance on-target antibacterial activity
against B. subtilis and S. aureus while the replacement of the small
terminal groups of the 3-elongated alkoxy chains with large het-
eroaryl groups only increases on-target antibacterial activity
against B. subtilis.
This research was financially supported by National Natural Sci-
ence Foundation of China (20872081and 21072114), Natural Sci-
ence Foundation of Shandong (ZR2010HM092) and the Project-
sponsored by SRF for ROCS, SEM.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.05.
056.
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