Antibacterial alkoxybenzamide inhibitors of the essential bacterial cell division protein FtsZ

Article abstract of DOI:10.1016/j.bmcl.2008.11.021

3-Methoxybenzamide is a weak inhibitor of the essential bacterial cell division protein FtsZ. Exploration of the structure-activity relationships of 3-methoxybenzamide analogues led to the identification of potent anti-staphylococcal compounds.

Full text of DOI:10.1016/j.bmcl.2008.11.021

Bioorganic & Medicinal Chemistry Letters 19 (2009) 524–527
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Antibacterial alkoxybenzamide inhibitors of the essential bacterial cell
division protein FtsZ
Lloyd G. Czaplewski a, , Ian Collins a, , E. Andrew Boyd b, David Brown a, Stephen P. East b, Mihaly Gardiner b,
Rowena Fletcher a, David J. Haydon a, Vincent Henstock c, Peter Ingram b, Clare Jones b, Caterina Noula c,
Leanne Kennison a, Chris Rockley c, Valerie Rose a, Helena B. Thomaides-Brears a, Rebecca Ure a,
Mark Whittaker b, Neil R. Stokes a,
*
a Prolysis Ltd, Begbroke Science Park, Sandy Lane, Yarnton, Oxfordshire OX5 1PF, UK
b Evotec (UK) Ltd, 114 Milton Park, Abingdon, Oxfordshire OX14 4SA, UK
c Key Organics Ltd, Highfield Industrial Estate, Camelford, Cornwall PL32 9QZ, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
3-Methoxybenzamide is a weak inhibitor of the essential bacterial cell division protein FtsZ. Exploration
of the structure–activity relationships of 3-methoxybenzamide analogues led to the identification of
potent anti-staphylococcal compounds.
Received 2 October 2008
Revised 7 November 2008
Accepted 10 November 2008
Available online 13 November 2008
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Antibacterial
Alkoxybenzamide
FtsZ
3-Methoxybenzamide
Antibacterial efficiency
New antibiotics are urgently needed to treat the increasing
number of life-threatening bacterial infections that are resistant
to current therapies. In particular, the emergence and spread of
drug-resistant staphylococci is of serious concern. Here we report
the discovery and characterization of a novel class of small syn-
thetic antibacterials that have potent activity against
staphylococci.
The essential bacterial cell division protein FtsZ has been recog-
nized as an attractive but as yet underexploited target for antibac-
terial drug discovery.1,2 When bacteria divide, FtsZ undergoes GTP-
dependent polymerization at mid-cell to form the Z-ring and then
recruits other cell division proteins to synthesize the septum that
enables the daughter cells to separate.3 FtsZ is structurally and
functionally homologous to mammalian b-tubulin, which has been
successfully exploited for cancer therapy.4–6 This suggests that FtsZ
may also be amenable to inhibitor development. Several com-
pounds have been reported to block bacterial cell division through
inhibition of FtsZ.7–13 We have explored one of these compounds,
3-methoxybenzamide (3-MBA; Compound 1; Fig. 1)7, leading to
the identification of a potent 3-MBA derivative, PC190723, that
O
F
O
F
F
O
F
NH2
NH2
NH2
O
O
N
O
ALK
1
S
N
Cl
PC190723
Compound 1 3-Methoxybenzamide. B. subtilis MIC 4000 μg.ml-1
Figure 1. Design of 3-MBA analogues leading towards PC190723.
inhibits FtsZ activity and kills staphylococci14 (Fig. 1). Here we
present the early structure–activity relationship (SAR) data leading
to its synthesis.
3-MBA is an attractive fragment-like starting point for FtsZ
inhibitor design. Although it possesses weak on-target antibacte-
rial activity (Bacillus subtilis minimum inhibitory concentration
* Corresponding author. Tel.: +44 01865 854700; fax: +44 01865 854799.
E-mail address: neil.stokes@prolysis.com (N.R. Stokes).
These authors contributed equally to this work.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.11.021

L. G. Czaplewski et al. / Bioorg. Med. Chem. Lett. 19 (2009) 524–527
525
(MIC) 4000
l
g mlꢀ1), because of its low molecular weight it has a
Our objective was to increase the potency of 3-MBA against
bacterial cells while retaining the on-target inhibition of cell divi-
sion. The biological activities of the compounds were characterized
by measuring their MICs against B. subtilis 168 and by morphomet-
ric analysis to determine on-target activity as expressed by fila-
mentation of the bacilli due to continued short-term growth in
the absence of cell division (Fig. 2).11
high ligand efficiency.15 It is able to penetrate bacterial cells, which
is often a barrier to novel antibacterial discovery, and it has proven
to be an effective starting point for, or has been a key feature in, the
discovery of inhibitors in other therapeutic areas.16–18
Selected compounds were also tested against Staphylococcus
aureus ATCC 29213 to determine both potency and on-target activ-
ity, expressed by ballooning of the cocci in this species (Fig. 2).14,19
Genetic studies were used to confirm that selected compounds in
the series inhibited cell division through their interaction with
FtsZ.7,14
The preliminary SAR exploration of compound 1 started with
purchasable close analogues and extended to those that could be
synthesized from commercial building blocks in one to four steps
as illustrated in Scheme 1. Conversion of the carboxylic acids to
the corresponding primary amides was achieved via the acyl chlo-
rides and subsequent reaction with aqueous ammonia. In the case
of compounds 5 and 7a, methylamine and O-benzyl-hydroxyl-
amine were used, respectively, while catalytic hydrogenation of
7a afforded compound 7. Thiobenzamide 6 was prepared from 1
in one-step using Lawesson’s reagent. The deprotection of the
methoxy group in 1 was performed with boron tribromide to pro-
vide 9. Compounds 29–43 were synthesized by alkylation of the
phenol with alkyl halides in the presence of catalytic sodium iodide
under modified Finkelstein conditions. The chemical structures of
Figure 2. Cells of B. subtilis 168 (A and B) or S. aureus ATCC 29213 (C and D) were
cultured (4 h) in the absence (A and C) or presence of 8 l
g mlꢀ1 compound 32 (B
and D) and analysed by phase-contrast microscopy. B. subtilis filaments and S.
aureus balloons in response to contact with cell division inhibitors.
O
S
O
5; R8=NHMe
iv)
R8
NHOH
NH2
7a; R8=NHOBn
7
6
O
O
O
iii)
3; R4,5,6,7=H
vii)
1; R4,5,6,7=H
16; R5=MeO
18; R7=MeO
19; R4=NO2
R7
R7
R7
O
O
O
R6
R5
R6
R5
R6
R5
i)
ii)
OH
R4
Cl
R4
NH2
20; R5=NO2
21; R7=NO2
R4
O
O
O
22; R4=Me
23; R4=F
24; R5=F
25; R7=F
1, 16-25
v)
v)
R4,7=H
R7
R7
R7
29; R9=n-propyl
30; R9=n-butyl
31; R9=n-pentyl
32; R9=n-hexyl
33; R9=n-heptyl
34; R9=n-octyl
35; R9=n-nonyl
36; R9=n-decyl
O
O
O
i) ii)
OH
R4
NH2
R4
NH2
R4
vi)
OH
OH
O
R9
9; R4,7=H
29-43
37; R9=n-undecyl
38; R9=n-dodecyl
O
O
R9=n-nonyl
39; R4=F, R7=H
40; R4=H, R7=F
41; R4=F, R7=F
42; R4=F, R7=Cl
43; R4=Cl, R7=F
OH
NH2
i) ii)
10; R2=SMe
11; R2=NHMe
13; R2=OCF3
R2
R2
Scheme 1. Reagents and conditions: (i) SOCl2, toluene, reflux; (ii) THF, aq NH3; (iii) MeNH2 or BnONH2; (iv) H2 10% Pd/C; (v) BBr3, CH2Cl2, rt; (vi) RBr or RCl, K2CO3, NaI, DMF,
60 °C; (vii) Lawesson’s reagent. Compounds 2, 4, 8, 12, 14, 15, 17, 26, 27, 28 are not shown on the scheme and were purchased.

526
L. G. Czaplewski et al. / Bioorg. Med. Chem. Lett. 19 (2009) 524–527
the 3-MBA derivatives obtained were confirmed by 1H NMR and
mass spectrometry and the purity was demonstrated by HPLC
analysis.
The amide and 3-ether substituents of compound 1 appeared to
be critical for inhibition of cell division (Table 1). Only 3-ethoxy-
benzamide 12 improved antibacterial activity.
Table 2
B. subtilis MIC and cell division inhibitory activity for compounds 15–28
R7
O
R6
NH2
Preliminary SAR indicated that few substitutions of the benz-
amide ring were tolerated (Table 2). R4 and R7 substitution with
small halogens was preferred leading to compounds 23 and 26,
which demonstrated greater potency and on-target activity than
compound 1.
R4
R5
O
Compound R4
R5
R6
R7
B. subtilis MIC
Cell division
(l
g mlꢀ1
)
inhibitiona g mlꢀ1
(l )
As options for optimization of the benzamide to improve po-
tency appeared to be limited, and encouraged by the biological
activity of compound 12 we focused on the modification of the
3-methoxy substituent of compound 1 as shown in Table 3.
Extension of the 3-alkyloxy substituent resulted in a substantial
improvement in antibacterial activity and led to the identification
of compound 35 with on-target activity and MICs of 0.5 and
15
16
17
18
19
20
21
22
23
24
25
26
27
28
MeO
H
H
H
NO2
H
H
Me
F
H
H
MeO
H
H
H
NO2
H
H
H
F
H
H
MeO
H
H
H
H
H
H
H
H
H
H
H
H
H
>4000
>4000
>4000
W.T.
W.T.
W.T.
W.T.
W.T.
W.T.
W.T.
W.T.
128
W.T.
W.T.
256
256
2048
MeO >4000
H
H
NO2
H
H
H
F
F
Cl
F
>4000
>4000
>4000
>4000
512
>4000
800
512
2
l
g mlꢀ1 against B. subtilis and S. aureus, respectively. The SAR
indicates that extension beyond the optimal nonyl alkyl chain re-
sults in a reduction of activity.
H
F
F
Cl
H
H
H
H
The effect of the active halogen substitutions (compounds 23–
28) in combination with the 3-nonyloxy substituent of 35 was ex-
plored (Table 4). Halogenation at R4 was associated with further
improvements in antibacterial activity. Compounds 39 and 41 are
>10,000 times more potent than 1 and retain on-target inhibition
of cell division in bacterial cells. We believe these compounds
are the most potent antibacterial cell division inhibitors described
to date.
Genetic studies confirmed that selected compounds exerted
their cell division inhibitory activity through their interaction with
FtsZ. Spontaneous compound 32 resistant mutants of S. aureus
ATCC 601055 were isolated at a frequency of 4 ꢁ 10ꢀ7 at 4ꢁ MIC.
The FtsZ genes were sequenced and point mutations M218I and
P300R were identified (Table 5). The FtsZ mutations conferred
resistance to compounds 1, 32 and 35. Furthermore, in B. subtilis
1024
>4000
H
a
Lowest concentration at which filamentation of B. subtilis is observed indicating
on-target activity. W.T. No effect on morphology.
Table 3
B. subtilis and S. aureus MICs and cell division inhibitory activity for compounds 29–38
Compound
R2 (Table 1)
MIC (
l
g mlꢀ1
)
B. subtilis cell division
inhibitiona g mlꢀ1
(l )
B. subtilis
S. aureus
29
30
31
32
33
34
35
36
37
38
Propyloxy
Butyloxy
Pentyloxy
Hexyloxy
Heptyloxy
Octyloxy
Nonyloxy
Decyloxy
Undecyloxy
Dodecyloxy
500
128
32
16
4
256
128
32
16
8
4
2
64
>256
>256
375
24
24
8
1.5
0.37
0.18
0.5
1
1
0.5
1
4
Table 1
>256
>128
B. subtilis MIC and cell division inhibitory activity for compounds 1–14
a
Lowest concentration at which filamentation of B. subtilis is observed indicating
O
R1
O
on-target activity.
NH2
NH2
Table 4
R3
B. subtilis and S. aureus MICs and cell division inhibitory activity for halogenated 3-
nonyloxybenzamide compounds 39–43
O
R2
8-13
14
1-7
Compound R4 R7
MIC (
l
g mlꢀ1
)
Cell division inhibitiona g mlꢀ1
(l )
B. subtilis S. aureus B. subtilis
S. aureus
Compound R-group
B. subtilis MIC
g mlꢀ1
Cell division inhibitiona
(
l
)
(l )
g mlꢀ1
39
40
41
42
43
F
H
F
F
Cl
H
F
F
Cl
F
0.125
1
0.5
8
0.125
1
0.5
2
0.25
0.5
1
1
2
3
4
–CONH2
–CH2NH2
–COOH
–
4000
250
>4000
>4000
500
0.125
0.5
0.5
0.5
1
2
0.125
0.25
0.5
O.T.
W.T.
W.T.
a
CH2CONH2
–CONHMe
–CSNH2
–CONHOH
–CH3
Lowest concentration at which filamentation of B. subtilis or ballooning of S.
aureus is observed indicating on-target activity.
5
6
7
>4000
2000
4000
>2000
>4000
>4000
>4000
2000
W.T.
W.T.
W.T.
W.T.
3000
W.T.
W.T.
500
8
a site-directed FtsZ mutation V307R14 increased the MIC of com-
pound 32 from 16 to >128 l .
g mlꢀ1
9
–OH
10
11
12
13
14
–SMe
–NHMe
–OEt
–OCF3
–OCH3
Compounds 1 and 32 were effective against four methicillin-
resistant S. aureus (MRSA), three methicillin-sensitive S. aureus
(MSSA) strains and one strain of Staphylococcus epidermidis (Table
6). There was no evidence that methicillin-resistance altered sensi-
tivity to the alkyloxybenzamide cell division inhibitors.
>4000
>4000
W.T.
W.T.
a
Lowest concentration at which filamentation of B. subtilis is observed indicating
An apocrystal X-ray structure of the B. subtilis FtsZ protein
(2vxy) has been used to develop a docking model that is consistent
on-target activity. W.T. No effect on morphology. O.T. Off-target activity observed,
for example cell death without filamentation.

L. G. Czaplewski et al. / Bioorg. Med. Chem. Lett. 19 (2009) 524–527
527
Table 5
Table 7
Potency of compounds 1, 32 and 35 against wild-type S. aureus ATCC 601055 and
Antibacterial efficiency of selected compounds
strains with spontaneous FtsZ point mutations M218I and P300R
Compound
Mol
wt
Number of
non-hydrogen MIC
atoms
S. aureus
Antibacterial efficiency
Compound
MIC (l
g mlꢀ1
)
(mgꢀm1 lꢀ1 Nnon-hydrogen
(Da)a
(
l
g mlꢀ1
)
)
atom
S. aureus
FtsZ
FtsZ
ATCC 601055
M218I
P300R
1
12
23
26
29
30
31
32
33
34
35
39
151
165
169
187
179
193
207
221
235
249
263
281
299
331
11
12
12
13
13
14
15
16
17
18
19
20
21
24
20
24
35
37
2048
1024
512
512
256
128
32
16
8
4
2
0.5
0.5
0.5
4.0
2.0
0.12
0.06
ꢀ0.065
ꢀ0.002
0.056
0.051
0.105
0.147
0.229
0.258
0.284
0.307
0.327
0.380
0.362
0.317
0.276
0.258
0.257
0.261
1
32
35
4000
32
>8000
>128
>128
>8000
>128
>128
2
Table 6
Potency of compounds 1 and 32 against isolates of S. aureus and S. epidermidis
Strain
MIC (l )
g mlꢀ1
Compound 1
Compound 32
41
S. aureus ATCC 601055 (MSSA)
S. aureus ATCC 607004 (MRSA)
S. aureus ATCC 700698 (MRSA)
S. aureus ATCC 25923 (MSSA)
S. aureus ATCC 29213 (MRSA)
S. aureus ATCC 43300 (MRSA)
S. aureus ATCC 19636 (MSSA)
S. epidermidis ATCC 12228
4096
4096
4096
4096
2048
4096
2048
4096
32
32
32
16
16
16
16
32
Ciprofloxacin
Chloramphenicol 323
Linezolid
Mupirocin
Fusidic acid
337
500
516
a
Rounded to nearest whole Da.
with the SAR of this alkyloxybenzamide series of FtsZ inhibitors.14
The series did not convincingly dock into the GTPase site of FtsZ
but did dock into an adjacent cleft. The SAR of the series correlates
well with the model, which predicts an optimal hydrophobic alkyl
substituent equivalent in length to 9–10 carbons with little space
for substitutions off the benzamide group. This is in agreement
with our experimental findings (Tables 2 and 3).
Acknowledgments
We thank various colleagues including Mike Marriott, Geoff
Lawton, Ian Skidmore, Jeff Errington and Steve Ruston for assis-
tance, advice and support. This work was funded by investments
from East Hill Management (Boston, USA), a LINK grant in Applied
Genomics (AppGen55) from the UK Biotechnology and Biological
Sciences Research Council and the UK Department of Trade and
Industry. Compounds are the subject of patent application PCT/
GB2007/001012. The Prolysis authors declare financial interests.
The concept and use of ligand efficiency15 provides a means of
normalizing the potency and molecular weight of a compound to
enable useful comparison of compounds within a series. Due to dif-
ficulties associated with biochemical analysis of FtsZ we have not
been able to measure the ligand efficiencies of these compounds
directly. Instead we propose to use antibacterial efficiency to pro-
vide a means to do this. We define antibacterial efficiency in Eq. 1,
as a logarithmic function of the MIC in mg mlꢀ1 per non-hydrogen
atom (Table 7). This metric describes the ability of the compound
to penetrate the cell and to interact with its target to kill the cell
as a function of molecular weight.
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Antibacterial efficiency ¼ ꢀ ln MICðmg mlꢀ1 =Nnon-hydrogen atoms
ð1Þ
Þ
The antibacterial efficiencies of clinically approved lower
molecular weight compounds as opposed to higher molecular
weight natural products, are in the 0.25 to 0.32 mg mlꢀ1 per non-
hydrogen atom range (Table 7). Compound 1 was much less effi-
cient at ꢀ0.065 but extension of the alkoxy substitution signifi-
cantly improved antibacterial efficiency to >0.3, comparable with
marketed drugs. The fluorinated benzamide analogues demon-
strated an improvement in antibacterial efficiency compared to
non-fluorinated compounds.
Antibacterial efficiency may be a useful tool to direct fragment-
based antibacterial optimization. It reduces reliance on the MIC
and enables decision making to focus on the efficiency of the li-
gand. This may lead to lower molecular weight Leads and Drug
Candidates.
Our exploration of 3-MBA (1) SAR has led to the identification of
potent inhibitors of FtsZ that could form the basis of a new tar-
geted treatment for staphylococcal infection. Further exploration
and optimization of the compound series, particularly with regard
to replacing the long alkyl substituent with more drug-like alterna-
tives, while retaining antibacterial efficiency, is ongoing and will be
reported in due course.
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