Design, synthesis and biological activity evaluation of novel 2,6-difluorobenzamide derivatives through FtsZ inhibition

Article abstract of DOI:10.1016/j.bmcl.2016.12.081

Novel series of 3-substituted 2,6-difluorobenzamide derivatives as FtsZ inhibitors were designed, synthesized and evaluated for their in vitro antibacterial activity against various phenotype of Gram-positive and Gram-negative bacteria, and their cell division inhibitory activity against three representative strains. As a result, 3-chloroalkoxy derivative 7, 3-bromoalkoxy derivative 12 and 3-alkyloxy derivative 17 were found to exhibit the best antibacterial activity against Bacillus subtilis with MICs of 0.25–1 μg/mL, and good activity (MIC < 10 μg/mL) against both susceptible and resistant Staphylococcus aureus. Additionally, all the three compounds displayed potent cell division inhibitory activity with MIC values of below 1 μg/mL against Bacillus subtilis and Staphylococcus aureus.

Full text of DOI:10.1016/j.bmcl.2016.12.081

Accepted Manuscript
Design, synthesis and biological activity evaluation of novel 2,6-difluoroben-
zamide derivatives through FtsZ inhibition
Fangchao Bi, Liwei Guo, Yinhu Wang, Henrietta Venter, Susan J. Semple, Fang
Liu, Shutao Ma
PII:
S0960-894X(16)31360-9
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.12.081
BMCL 24570
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
5 October 2016
19 December 2016
29 December 2016
Please cite this article as: Bi, F., Guo, L., Wang, Y., Venter, H., Semple, S.J., Liu, F., Ma, S., Design, synthesis and
biological activity evaluation of novel 2,6-difluorobenzamide derivatives through FtsZ inhibition, Bioorganic &
Medicinal Chemistry Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl.2016.12.081
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Design,
synthesis
and
biological
activity
evaluation
of
novel
2,6-difluorobenzamide derivatives through FtsZ inhibition
Fangchao Bia, Liwei Guo a, Yinhu Wang a, Henrietta Venter b, Susan J. Semple b, Fang Liu a, Shutao
Ma a,*
aDepartment 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).
bSchool of Pharmacy & Medical Sciences, Sansom Institute for Health Research, University of South
Australia, GPO Box 2471, Adelaide 5001, Australia
Running title: 2,6-Difluorobenzamide 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
The authors “Fangchao Bi” and “Liwei Guo” contributed equally to the work.
1

Abstract
Novel series of 3-substituted 2,6-difluorobenzamide derivatives as FtsZ inhibitors were designed,
synthesized and evaluated for their in vitro antibacterial activity against various phenotype of
Gram-positive and Gram-negative bacteria, and their cell division inhibitory activity against three
representative strains. As a result, 3-chloroalkoxy derivative 7, 3-bromoalkoxy derivative 12 and
3-alkyloxy derivative 17 were found to exhibit the best antibacterial activity against Bacillus subtilis with
MICs of 0.25-1 μg/mL, and good activity (MIC<10 μg/mL) against both susceptible and resistant
Staphylococcus aureus. Additionally, all the three compounds displayed potent cell division inhibitory
activity with MIC values of below 1 μg/mL against Bacillus subtilis and Staphylococcus aureus.
Keywords: benzamide derivatives; design and synthesis; FtsZ inhibition; biological activity.
2

Bacterial resistance to current antibiotic therapies is a major global public health concern.1, 2 The
problem of antibiotic resistance is magnified by the lack of new therapeutic agents and the reduced
investment of the pharmaceutical industry.3, 4 There are two main strategies to overcome this problem.
One is to extensively modify existing antibacterial drugs in order to maintain activity against their targets
and the other is to develop novel antibacterial agents that can work by unique mode of action on known
targets or by interacting with novel targets.
Filamentous temperature-sensitive protein Z (FtsZ), which is an essential cell division protein of
bacteria, has been recently considered as an attractive target for the development of new antibacterial
drugs.5 When bacteria divide, FtsZ with bound GTP polymerizes into tubulin-like protofilaments at
mid-cell that serves as a scaffold for the recruitment and organization of other cell division proteins,
causing assembly of the septal ring.6-8 The septal ring then constricts in concert with septal progression,
enabling the daughter cells to separate.9 The appeal of FtsZ as an antibacterial target lies in its essential
role for bacterial viability, its highly conservation among important bacterial pathogens and its absence
in higher eukaryotes.7, 10 Accordingly, disrupting proper FtsZ assembly could result in bacterial growth
inhibition and cell death.11 To this end, various chemical classes of FtsZ inhibitors have been identified
by different investigation approaches, such as 3-methoxybenzamide (3-MBA)12, cinnamaldehyde13,
curcumin14, benzimidazole15, etc (Fig. 1).
Fig. 1. Structures of some mentioned compounds inhibiting FtsZ
In particular, 3-MBA is one of the most attractive starting point for discovering FtsZ-targeting
antibacterial compounds. For example, PC190723, a synthetic analogues of 3-MBA, has demonstrated
potent activity against B. subtilis and various resistant S. aureus by disruption of FtsZ function.16-18 The
3

preliminary structure-activity relationship (SAR) indicates that the amide functional group is critical for
inhibition of cell division, and 2,6-difluoro groups on the benzamide can substantially enhance inhibitory
activity, while the 3-alkyloxy side chain could continue to be investigated for further improvement in the
on-target activity and the antibacterial activity.19-21 Accordingly, leaving the amino function unchanged
and introducing a reasonably long hydrophobic side chain or a preferred aromatic heterocycle on the
3-oxygen, will coordinate with the hydrophobic pocket and enhance their interaction. We speculate that
this rational design may result in a significant improvement in the on-target activity and the antibacterial
activity. Actually, in our previous work, compound 6c that just held the two simple pharmacophores
exhibited good antibacterial activity against B. subtilis ATCC9372 and S. aureus ATCC29213 with MICs
of 8 and 4 μg/mL. Furthermore, in silico docking model performed by AutoDock has further indicated
that compound 6c bound into SaFtsZ (3vob.pdb) in the same pocket as PC190723.22 Its amide group and
ether oxygen atom formed strong hydrogen bonds with the surrounding amino acid residues in the pocket,
and its flexible ether side chain extended into a hydrophobic channel formed by the H7 helix and the C
terminal sheet of the SaFtsZ (Fig. 2).
Fig. 2. Model of compound 6c docked into SaFtsZ (PDB code, 3vob). In the model, the predicted
hydrogen bonds between compound 6c and amino acid residues are indicated by dotted lines.
On the basis of the consideration detailed above, we designed a structural series of novel
3-O-arylalkyl-2,6-difluorobenzamide derivatives by introducing fluorine atoms at the 2,6 positions of the
benzene ring, the 3-O-arylalkyl side chain of which possessed various groups such as alkyl halide,
4

branched alkyl, ester, and heterocyclic groups. It’s hoped that the 2,6-difluorobenzamide derivatives
could exhibit more potent anti-FtsZ effect and higher antibacterial activity against various
drug-susceptible and -resistant bacterial strains.
The synthesis of compounds 4‒21 and 25‒36 is shown in Schemes 1 and 2, respectively
(Supplementary data, Chemical Synthesis). The commercially available 2,4-difluorophenol 1 was
etherified with chloromethyl ethyl ether in the presence of DIPEA (N,N-diisopropylethylamine) and the
resulting product was poured onto crushed carbon dioxide in the presence of n-BuLi (n-butyl lithium) to
give protected 2,6-difluorobenzoic acid 2. The acid was converted into amide in the presence of aqueous
ammonia,
DIPEA and
ethyl
chloroformate,
and
then
deprotected
to
provide
3-hydroxy-2,6-difluorobenzamide 3. The alkylation of 3 with different alkyl chloride or bromide in the
presence of potassium carbonate afforded 3-O-alkyl-2,6-difluorobenzamide derivatives 4‒21. Similarly,
the alkylation of 3 with 2-chloromethyl benzimidazole 23 gave 2-benzoimidazol-2,6-difluorobenzamide
derivatives 25 and 26. The nucleophilic substitution of the intermediates 8‒12 with benzimidazole 24
provided 1-benzoimidazol-2,6-difluorobenzamide derivatives 27‒36.
Scheme 1. Reagents and conditions: (a) chloromethyl ethyl ether, DIPEA, CH2Cl2, 0 °C, 2 h, 91.1%; (b)
n-BuLi, CO2, -78 °C, 2 h, 93.4%; (c) aqueous ammonia, DIPEA, ethyl chloroformate, rt, 6 h, 71.6%; (d)
6M HCl, CH3OH (1:1), rt, 2 h, 70.1%; (e) alkyl chloride or alkyl bromide, K2CO3, DMF, 25–45 °C,
12–14 h, 48.2–67.0%.
5

Scheme 2. Reagents and conditions: (a) chloroacetic acid, 4M HCl, reflux, 4 h, 79.4%; (b) K2CO3, DMF,
60–65 °C, 12–14 h, 55.1–62.2%; (c) formic acid, 4M HCl, reflux, 4 h, 78.1%.
All the synthesized compounds 4‒21 and 25‒36 were determined for their biological activity,
including the in vitro antibacterial activity and the cell division inhibitory activity (Supplementary data,
Antibacterial Testing). The MIC value of in vitro antibacterial activity was determined with the
application of the standard broth microdilution method recommended by NCCLS23. Curcumin and the
key intermediate 3 were used as the FtsZ-targeted references. The tested strains included six
Gram-positive strains of S. aureus ATCC 25923, S. aureus ATCC 29213, S. aureus PR, B. subtilis ATCC
9372, S. pneumoniae ATCC 49619 and S. pyogenes PS, two Gram-negative strains of E. coli ATCC
25922 and P. aeruginosa ATCC27853. The results of minimum inhibitory activity in units of μg/mL are
shown in Table 1.
The cell division inhibitory activity of the synthesized compounds against three representative strains
including B. subtilis ATCC9372, S. aureus ATCC25923 and E. coli ATCC25922 was assessed by
analyzing the cell morphology with the application of phase-contrast light microscopy24. Lowest drug
concentration at which filamentation of B. subtilis and E. coli or ballooning of S. aureus appeared was
recorded as cell division inhibitory concentration indicating on-target activity. The results of cell division
inhibitory activity in units of μg/mL are shown in Table 2.
Table 1
3-O-Arylalkyl-2,6-difluorobenzamide derivatives with their antibacterial activity.
Minimum inhibitory concentration/ MIC (µg/mL)
Compd
B. subtilis
S. aureus
S. aureus
ATCC29213c
4096
512
32
S. aureus
PRd
S. pyogenes
PSe
S. pneumoniae E.coli
P. aeruginosa
ATCC27853h
4096
512
128
ATCC9372a ATCC25923b
ATCC49619f
>4096
ATCC25922g
curcumin
3
4
32
1024
32
2048
1024
16
4096
1024
32
>4096
>4096
128
512
512
128
4096
>128
6

5
6
7
8
4
4
0.5
16
32
4
16
1
8
16
2
16
16
4
8
1
256
64
32
64
1
256
128
128
64
1024
512
1024
256
1024
1024
1024
1024
512
512
512
1024
8
32
16
64
32
32
16
2
256
128
128
16
8
16
8
32
32
8
16
2
256
128
64
16
8
512
256
256
256
1024
512
1024
1024
1024
1024
1024
512
512
2048
1024
1024
64
64
>128
64
64
128
128
64
128
128
128
>128
>128
>128
128
>128
128
128
128
128
64
128
128
128
128
256
256
512
256
512
512
512
128
512
512
256
256
128
128
256
128
128
128
256
128
256
128
256
128
256
128
128
128
256
256
512
512
256
512
1024
1024
512
1024
512
512
512
128
128
256
128
128
128
256
128
128
128
128
128
128
128
128
128
128
256
256
1024
512
512
512
512
512
512
512
512
512
9
10
11
12
13
14
15
16
17
18
19
20
21
25
26
27
28
29
30
31
32
33
34
35
36
256
128
64
>128
>128
>128
>128
>128
>128
>128
>128
>128
1024
1024
1024
512
1024
512
1024
256
1024
1024
512
8
0.25
512
64
64
64
1024
64
1024
64
1024
256
512
64
512
512
256
1024
8
256
256
256
128
512
256
512
512
512
512
512
256
512
1024
256
512
512
a B. subtilis ATCC9372: standard susceptible strain.
b S. aureus ATCC25923: standard susceptible strain.
c S. aureus ATCC29213: methicillin-resistant strain.
d S. aureus PR: penicillin-resistant strain isolated clinically, not characterized.
e S. pyogenes PS: penicillin-susceptible strain.
f S. pneumoniae ATCC49619: standard susceptible strain.
g E.coli ATCC25922: standard susceptible strain.
h P. aeruginosa ATCC27853: standard susceptible strain.
Table 2
3-O-Arylalkyl-2,6-difluorobenzamide derivatives with their cell division inhibitory activity.
Cell division inhibitiona (µg/mL)
Compd
B. subtilis ATCC937
S. aureus ATCC25923
E.coli ATCC25922
curcumin
3
4
5
16
1024
16
2
1024
2048
32
4
256
512
64
64
6
7
8
9
10
11
12
13
2
0.25
2
16
1
2
16
0.5
16
16
4
4
1
128
64
128
32
64
64
64
64
64
0.5
128
7

14
15
16
17
18
19
20
21
25
26
27
28
29
30
31
32
33
34
35
128
32
4
0.125
256
64
32
32
OTb
64
32
16
4
0.25
256
128
64
128
512
128
OT
256
256
1024
OT
512
OT
OT
128
OT
64
64
64
64
64
64
64
64
128
128
256
128
512
512
512
256
512
256
256
256
OT
32
512
128
OT
OT
OT
32
128
64
36
a
Lowest concentration at which filamentation of B. subtilis and E.coli or ballooning of S. aureus is observed
indicating cell division inhibitory activity.
b off-target activity: no effect on morphology at 2048-4096 μg/mL.
All the synthesized 3-alkyloxy derivatives 4‒21 displayed significantly improved antibacterial activity
against all the tested strains in comparison with curcumine and their precursor 3. Especially for B.
subtilis ATCC9372 and three S. aureus strains of S. aureus ATCC25923, S. aureus ATCC29213 and S.
aureus PR, the most potent compounds exhibited 4096-, 1024-, 256- and 512-fold higher efficacy against
those four strains than the precursor 3 respectively, indicating a successful optimization in this program.
In the subseries of the 3-chloroalkoxy derivatives 4‒7 and 3-bromoalkoxy derivatives 8‒12, there was
a clear trend that the longer carbon chain from the 3-oxygen atom to the terminal halogen resulted in the
better antibacterial activity. For instance, compounds 7 and 12 possessing a 6 carbon atoms chain
exhibited much stronger antibacterial activity against B. subtilis (MICs, 0.5 and 1 μg/mL) than their
analogs 4 and 9 with a 3 carbon atoms chain (MICs, 32 and 32 μg/mL). In the subseries of the branched
alkyl substituted derivatives 13‒19, those compounds also showed an improved trend in the antibacterial
activity with elongation of their 3-alkoxy side chain. Among them, compound 17 with 3-O-(2-ethyl)
hexyl group exhibited the most potent activity with MIC values of 0.25, 1, 8 and 8 μg/mL against above
four B. subtilis and S. aureus strains, respectively. For the cycloalkyl substituted derivatives 18 and 19,
however, the improvement of their antibacterial activity was not obvious. This could be because the
hydrophobic channel tolerating the 3-substituents was volume-limited. When the elongated 3-O-alkyl
side chain was changed to 3-O-ester side chain containing five or seven atoms length, the antibacterial
8

activity was decreased. For example, compounds 20 and 21 showed relatively weak activity against B.
subtilis with same MIC values of 64 μg/mL, indicating that the interaction sites could not prefer the
ester chains to the alkyl chains.
Fig. 3. Structures of compounds 6g, 25 and 27
By contrast, the benzimidazolyl substituted derivatives 25‒36 just showed slightly improved
antibacterial activity against those tested strains compared to curcumine and their precursor 3. In
previous studies of Haydon, D.J, compound 6g containing a benzothiazole (Fig. 3) as a key precursor
leading to the discovery of PC190723, displayed potent antibacterial and cell division inhibitory activity
against S. aureus with levels of 2 and 4 μg/mL, respectively.25 Benzimidazole and benzothiazole share
similar molecular skeleton, which are widely applied as versatile and important pharmacophores in
medicinal chemistry due to their good antitumor, antiviral and antimicrobial activities.26-28 Given that, we
replaced the benzothiazole with benzimidazole to obtain compound 25 (Fig. 3). However, this
modification did not achieve a desired result. We suspected that the polar secondary amine of
benzimidazole was not suitable for interaction with the hydrophobic channel. In order to reduce the
hydrophilicity of benzimidazole, different length side chains were introduced into the polar
secondary amine, providing compounds 27‒36. But none of this subseries could showed comparable
antibacterial activity to compound 6g. We attributed this contrast to speculation that the stereostructure of
this hydrophobic binding site might be very strict with the 3-substituted groups, especially with the rigid
heterocyclic moieties while the flexible side chains could fit comfortably into the critical hydrophobic
pocket. This was consistent with the results that the 3-O-alkyl substituted compounds 4‒17 are generally
much better activity than the 3-O-heterocyclic substituted compounds 25‒36.
9

Fig. 4. Compound 17 inhibits cell division in S. aureus ATCC 25923 (A and B) and B. subtilis
ATCC9372 (C and D) were grown in the absence (A and C) or presence (B and D) of 0.25μg/mL of 17
and these phenotypes were analyzed by phase-contrast light microscopy at a magnification of ×400.
In the cell division inhibitory assay, the same trend as the in vitro antibacterial activity was observed.
Most of the 3-alkyloxy derivatives 4‒21 showed improved cell division inhibitory activity against the
three tested strains compared to the curcumine and their precursor 3. The most active compounds were 7,
12 and 17 with effective concentrations of 0.25, 0.5 and 0.125 μg/mL against B. subtilis ATCC9372,
respectively (Fig. 4). And they were also the three best compounds standing out from the antibacterial
assay. In the benzimidazolyl substituted derivatives 25‒36, many of them showed off-target effects,
which suggested that the large groups could hinder the interaction with target FtsZ, thereby resulting in
poor antibacterial activity. Generally, the cell inhibitory activity was accordant with the in vitro
antibacterial activity, indicating that these compounds inhibited the proliferation of bacteria via
influencing the function of FtsZ.
In summary, a series of novel 3-O-arylalkyl-2,6-difluorobenzamide derivatives were designed,
synthesized and evaluated for their antibacterial activity and cell division inhibitory activity against
various pathogen strains. In all the tested compounds, the 3-O-alkyl derivatives showed much more
potent cell division inhibitory and antibacterial activity than the 3-O-heteroaromatic derivatives. In
particular, compounds 7, 12 and 17 that possessed 3-O-flexible side chains displayed the best
antibacterial activity (MICs, 0.25‒8 μg/mL) against both susceptible and resistant strains including B.
subtilis ATCC9372, S. aureus ATCC25923, S. aureus ATCC29213, S. aureus PR. For the 3-O-rigid
heteroaryl substituted derivatives, however, their improved activity was not outstanding, although some
derivatives had very similar structure to previously discovered compound 6g. Further optimization of
2,6-difluorobenzamide derivatives wiht potent FtsZ-targeted antibacterial activity should continue to be
10

investigated.
Conflict of interest
The authors declare that this study was carried out only with public funding. There is no funding or no
agreement with commercial for profit firms.
Acknowledgements
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) and Major Project of Science and
Technology of Shandong Province (2015ZDJS04001).
A. Supplementary data
Supplementary data associated with this article can be found, in the online version,
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12

Graphical abstract:
Design, synthesis and biological activity evaluation of novel 2,6-difluorobenzamide derivatives
through FtsZ inhibition
Fangchao Bi a, Liwei Guo a, Yinhu Wang a, Henrietta Venter b, Susan J. Semple b, Fang Liu a, Shutao Ma
a,
*
The 2,6-difluorobenzamide derivative (17) bearing 3-O-flexible alkyl side chains exhibited modest in
vitro antibacterial and cell division inhibitory activities, while the the 3-O-rigid heteroaryl substituted
derivative (25) showed weak antibacterial activity although it had very similar structure to previously
discovered compound 6g.
13

Research Highlights
> Novel 2,6-difluorobenzamide derivatives were synthesized and evaluated. > Some compounds
showed potent activity against both susceptible and resistant strains. > They exerted their effects by
inhibition of bacterial cell division protein FtsZ. > Replacing the benzothiazole with benzimidazole lost
its antibacterial activity.
14