Synthesis and antibacterial activity of 3-benzylamide derivatives as FtsZ inhibitors

Article abstract of DOI:10.1016/j.bmcl.2017.02.032

The emergence and spread of multidrug-resistant strains of the human pathological bacteria are generating a threat to public health worldwide. In the current study, a series of PC190723 derivatives was synthesized and investigated for their antimicrobial activity. The compounds exhibited good activity against several Gram-positive bacteria as determined by comparison of diameters of the zone of inhibition of test compounds and standard antibiotics. Compound 9 with a fluorine substitution on the phenyl ring showed the best antibacterial activity in the series against M. smegmatis with the zone ratio of 0.62, and against S. aureus with the zone ratio of 0.44. The results from this study indicate that based on the unique 3-methoxybenzamide pharmacophore, compound 9 may represent a promising lead candidate against Gram-positive bacteria that are worthy of further investigation

Full text of DOI:10.1016/j.bmcl.2017.02.032

Accepted Manuscript
Synthesis and antibacterial activity of 3-benzylamide derivatives as FtsZ inhib-
itors
Zhongping Hu, Shasha Zhang, Weicheng Zhou, Xiang Ma, Guangya Xiang
PII:
S0960-894X(17)30166-X
http://dx.doi.org/10.1016/j.bmcl.2017.02.032
BMCL 24704
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
25 November 2016
5 February 2017
15 February 2017
Please cite this article as: Hu, Z., Zhang, S., Zhou, W., Ma, X., Xiang, G., Synthesis and antibacterial activity of 3-
benzylamide derivatives as FtsZ inhibitors, Bioorganic & Medicinal Chemistry Letters (2017), doi: http://dx.doi.org/
1
0.1016/j.bmcl.2017.02.032
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Synthesis and antibacterial activity of 3-benzylamide derivatives as FtsZ
inhibitors
a
a
b
a,*
a,*
Zhongping Hu , Shasha Zhang , Weicheng Zhou , Xiang Ma , Guangya Xiang
a
School of Pharmacy, Tongji Medical College, Huazhong University of Science and
Technology, Wuhan, 430030, Hubei, China.
b
State Key Lab of New Drug & Pharmaceutical Process, Shanghai Key Lab of
Anti-Infectives, Shanghai Institute of Pharmaceutical Industry, State Institute of
Pharmaceutical Industry, Shanghai 200437, China
*
Authors to whom correspondence should be addressed; E-Mails:
xiangma@hust.edu.cn (X.M.); gyxiang1968@hotmail.com (G.X.); Tel.:

+86-27-8369-2793 (X.M. & G.X.); Fax: +86-27-8369-2762 (X.M. & G.X.)
Abstract
The emergence and spread of multidrug-resistant strains of the human pathological
bacteria are generating a threat to public health worldwide. In the current study, a
series of PC190723 derivatives were synthesized and investigated for their
antimicrobial activity. The compounds exhibited good activity against several
Gram-positive bacteria as determined by comparison of diameters of the zone of
inhibition of test compounds and standard antibiotics. Compound 9 with a fluorine
substituted on the phenyl ring showed best antibacterial activity in the series against
M. smegmatis with the zone ratio of 0.62, and against S. aureus with the zone ration
of 0.44. The results from this study indicate that based on the unique
3
-methoxybenzamide pharmacophore, compound 9 may represent a promising lead
candidate against Gram-positive bacteria that are worthy of further investigation
Key Words: antibacterial agents; 3-methoxybenzamide; FtsZ; Mycobacterium

tuberculosis; Staphylococcus aureus
An increase of multidrug resistance to antibiotics among pathogenic strains of
1
-3
bacteria significantly threatens to public health . In particular, the emergence and
4
spread of drug-resistant staphylococci aureus is of serious concern . New approaches
to combat them are sorely needed. Recent efforts have included finding new ways to
modify the structures of existing antibiotics as well as discovering and developing
5
, 6
new leads and new molecular targets . Cell division has been of considerable
1
, 2, 7
interest to the antibacterial drug discovery as a promising target
. The bacterial cell
division process encodes essential proteins forming the divisome, which are
extremely sensitive to the inhibition. Among those proteins, the essential cytoskeletal
cell division protein FtsZ（Filamenting temperature-sensitive mutant Z）is the most
important and highly conserved protein in bacteria and archaea cell division
8
-10
machinery . During the cell division, FtsZ undergoes GTP dependent
polymerization to form a Z-ring, which allows the constriction to give rise to two
equal daughter cells. If FtsZ assembly is restrained, the bacterial cell division would
eventually fail, thereby resulting in bacterial apoptosis. FtsZ is structurally and
functionally homologous to mammalian β-tubulin, which has been successfully
11
exploited for cancer therapy . It suggests that FtsZ may also be amenable to the
inhibitor discovery for antibacterial agents. Furthermore, the validation of FtsZ as a
novel antibacterial drug target has been confirmed by the work of various research
groups, and the recent advances in the development of small molecules that target
1
, 3, 11-13
FtsZ has been the subject of some recently reviews
.
3
-Methoxybenzamide (3-MBA) (Fig. 1), which has been identified as a small

molecule that stabilizes the polymerization or disturbs the GTPase activity of FtsZ or
1
4
both of them, was a promising lead for the development of new antibacterial agents .
Optimization of this lead with the aim to improving the antibacterial potency and drug
1
5, 16
like properties, a potent derivative PC190723 was discovered
. PC190723 has
potent and selective in vitro bactericidal activity against staphylococci, including
methicillin- and multi-drug-resistant Staphylococci aureus, and kills staphylococci in
1
6
vivo . Later modification of PC190723 led to its prodrugs TXY541, TXY436 and
TXA709 with superior antibacterial activity and improved drug-like
1
7-20
properties
(Fig. 1). Further modifications were also explored by replacing the
methyloxy linked thiazolo[5,4-b]pyridine on PC190723 with other heteroaryloxy or
aryloxy systems as exemplified by compounds I and II (Fig.1)with enhanced
2
1, 22
pharmacokinetic and improved in vivo efficacy
.

Fig.
1. Chemical Structures of some 3-MBA based FtsZ specific inhibitors
The crystal structure of the S. aureus FtsZ-PC190723 complex has demonstrated
the mechanism of action of the cell division inhibit and identified the binding site
2
3
located between the two domains of FtsZ . Several SAR studies demonstrated that
benzamide group is critical for the inhibitory activity with little space for the
modification, while many reasonably hydrophobic alkyloxy substituents or
equivalents linkage on the 3-position of benzamide is helpful for the interaction on the
1
5, 21
binding site resulting the improvement on the antibacterial activity
. On the basis
of medicinal chemistry principles and SAR studies of the above findings, a series of
new modifications based on PC190723 was considered for the discovery of novel
antibacterial agents: a) the amide function as in PC190723 was replaced by the
bioisosteric sulphonamide functional group, which is a typical functional group in the

antibacterial sulfa drug used for conditions such as acne and urinary tract infections
Compounds 1 and 2); b) phenyl functions were introduced at the end of the chain
(
with different oxygen or nitrogen containing linker (Compounds 3-22).
Fig. 2. Structures of 3-MBA and PC190723 analogues
Therefore, in an attempt to design and develop new potential antibacterial agents
we report the synthesis and antibacterial activity of compounds 1-22. Our objective
was to further explore the effect of the lengths of 3-elongated side chain and various
linker group and its bioisosteric version of PC190723 on its antibacterial activity.
They were evaluated as antimicrobial agents on the standard bacterial strains using
PC190723 as a reference compound to compare their activity with the standard
antibacterial drugs.
The synthesis of the bioisosteric version of PC190723 was based on the condensation
of 3-hydroxybenzlysuphonamide with 2-halomethyl-thiazolopyridine. This procedure
2
4
was originally described by Haydon, D. J. et al and further expanded by Sorto, N. A.
2
5
26, 27
and our group
. Considering these results, our attention was turned to develop a

synthetic approach for the 2,6-difluoro-3-hydroxybenzenesulfonamide (30). Since the
sulphonamide functional group was on the meta position of the hydroxyl group in the
2
,4-difluorophenol, the classical methods involving the direct sulphonation would be
ineffective. We resorted to the 3-trimethylsilylated intermediate, which has been
successfully used in the functionalisation of the 2 position of 1,3-disubstituted
2
8
benzenes by para-directing groups . In the synthesis of 3-trimethylsilylated derivative,
commercial available 2,4-difluoro-1-methoxybenzene (25) was smoothly
deprotonated with sec-butyllithium at the position flanked by the two fluoro atoms
(
Scheme 1). The deprotonated compound then react with chlorotrimethylsilane to
form compound 26 in total of 87.4% yield. The 3-trimethylsilylated compound 26 was
then reacted with the freshly prepared trimethylsilyl chlorosulfate to form sulphonic
acid 28 after a hydrolysis work-up with sodium bicarbonate in 85.3% yield. The
sulphonic acid 28 was further reacted with PCl
subsequently the concentrated ammonia water was added in the system to form the
sulphonamide 29. Treatment of the compound 29 with BBr effected the
5
/POCl
3
for the chlorination, and
3
demethylation to provide the fragment 30 in a high yield. The synthesis of the
fragment 6-chloro-2-(chloromethyl)-thiazolo[5,4-b]pyridine (31) was completed
2
6,
27
according to previously described procedures
.
And the fragment
5
2
-chloro-2-(chloromethyl)benzo[d]thiazole (33) was obtained starting from
2
9
-amino-4-chlorobenzenethiol (32) . The simultaneously acylation and cyclization
between 32 and chloroacetyl chloride under the optimized condition allowed the
compound 33 to be efficiently prepared in 51.5 % yield. By using K CO as a base in
2
3

CH
catalyzed by the 0.2 eq. of NaI resulted in the formation of 1 or 2 in good yields
Scheme 1).
3
CN at room temperature, the reaction of the fragment 30 and fragment 31 or 33
(
o
o
Scheme 1. Reagents and conditions: (a) sec-BuLi, -78 C, 1h, Me
ClSO SiMe , CCl , reflux; (c) NaHCO , H O, HCl; (d) POCl , PCl
ClCOCH Cl, Et N, toluene; (g) NaI, K CO , CH CN.
3
SiCl, -78 C, 1h, dry THF; (b)
, NH OH; (e) BBr , CH Cl ; (f)
3
3
4
3
2
3
5
4
3
2
2
2
3
2
3
3
The synthesis of compounds 3-22 was based on the connection from the
,6-difluoro-3-hydroxybenzamide fragment 34. As reported in our or others literatures,
2
the 2,6-difluoro-3-hydroxybenzamide (34) could be introduced as aldehyde, ester, or
2
5, 26, 30
carboxylic acid
for the preparation of amide, we began to use the commercially available
,4-difluoro-phenol (35) as the starting material to introduce cyanide functional group
. Since the cyanide functional group was also a common source
2
for the attempt (Scheme 2). Protection of 2,4-difluorophenol with chloromethyl
methyl ether followed by regioselective lithiation with s-BuLi, the corresponding
aromatic nitrile 37 was then formed by treatment with DMF and POCl
3
, followed by
3
1, 32
the reaction with molecular iodine in aq NH
3
. The benzonitrile 37 was converted
to amide 38 in aqueous alkaline medium accelerating by hydrogen peroxide.
Treatment of compound 38 with methanolic HCl effected deprotection to provide

2
,6-difluoro-3-hydroxybenzamide fragment 34 in a high yield.
Scheme 2. Reagents and conditions: (a) ClCH
s-BuLi, anhy THF, -78℃, 2h; ⅱ) DMF, 0℃, 2h; ⅲ) NH
10%), H (30%), rt, 3h. (d) CH OH-6 M HCl, rt, 2h.
2
OCH
3
, DIPEA, CH
2
Cl
2
, rt, 2h. (b) ⅰ)
3
(28%), I
2
, rt, 3h. (c) NaOH
(
2
O
2
3
Compounds 3-13 were then obtained by the O-alkylation of 34 with prepared
substituted phenyl 2-chloroacetate，2-substitutedphenoxyethyl 2-bromoacetate, phenyl
3
2
-chloropropanoate, or benzyl bromoacetate (Scheme 3). Substituted phenyl
-chloroacetates were synthesized from acylation on various phenols with
chloroacetyl chloride. The above used intermediates 2-substitutedphenoxyethyl
2
2
-bromoacetates were synthesized from phenols, which were elongated with
-bromoethanol in aqueous medium to alcoholic derivatives, and then acylated with
bromoacetyl bromide to the bromoacetate intermediates. The above used phenyl
3
3
-chloropropanoate was synthesized from the acylation with phenol using
-chloropropionyl chloride. The followed alkylation with 34 gave compound 12,
while the O-alkylation of 34 with commercial available benzyl bromoacetate formed
compound 13 (Scheme 3).

Scheme 3. Reagents and conditions: (a) NaI, K
2-24h. (c) K CO , DMF, rt, 12h
2
CO
3
, DMF, rt, 24h. (b) K
2
CO , rt,
3
1
2
3
Aniline 39 was acylated with chloroacetyl chloride and was followed by the
reaction with 2,6-difluoro-3-hydroxybenzamide (34), providing the nitrogen
containing compound 14. Compounds 15 and 16 were prepared starting from
potassium benzoate 41, which was reacted with dibromoalkane (1,2-dibromoethane
and 1,3-dibromopropane, respectively) to give mono-bromoalkane 42-43. The
subsequent O-alkylation was achieved through the reaction between the 41 or 42 with
of 2,6-difluoro-3-hydroxybenzamide (34) to form the title compounds 15 and 16
(
Scheme 4).

Scheme 4. Reagents and conditions: (a) chloroacetyl chloride, Et
4, NaI, K CO , DMF, rt, 12h; (c) 1,2-dibromoethane or 1,3-dibromopropane, CH
reflux, 12h. (d) 34, K CO , DMF, rt, 24h.
3
N, CH
2
Cl
2
, rt, 1h. (b)
3
2
3
3
CN,
2
3
The bicyclic aromatic tailing derivatives 17-22 were synthesized from
-hydroxyquinoline derivatives or 1- or 2-naphthol as outlined in Scheme 5. The
8
substituted 8-hydroxyquinoline or 1- or 2-naphthol 44a-44f were alkylated with
,3-dibromopropane in the presence of sodium hydroxide aqueous solution and
1
tetrabutylammonium iodide (TBAI) in dichloromethane to afford mono-halogenated
intermediates 45a-45f. Then the target compounds 17-22 were obtained via the
alkylation of compound 34 with the corresponding alkyl bromides.
Scheme 5. Reagents and conditions: (a) NaOH, TBAI, CH
2
Cl
2
, rt, 24h; (b) 34, K
2
CO ,
3
DMF, rt, 24h.
Compound 1-22 were screened at the loading of 100 µg per disc against a panel of

bacteria (Staphylococcus aureus ATCC 6538P, Pseudomonas aeruginosa ATCC 9027,
Mycobacterium smegmatis ATCC 607, Bacillus subtilis ATCC 6633, Klebsiella
aerogenes ATCC 9621, Escherichia coli ATCC 25922) and two fungus (Candida
albicans ATCC 10231 and Aspergillus niger ATCC 16404) using paper disc diffusion
3
3
method , and compared with PC190723 tested in the same in vitro model. The zone
ratios which were used to express the comparative efficiency to the standard drugs are
summarized in the Table 1. In general, no inhibitory activity against gram negative
bacteria and fungus was observed in the current study. Only the gram positive bacteria
S. aureus, B.subtilis, M. smegmatis were sensitive to most of the tested compounds.
Compounds demonstrated relatively weaker inhibitory activity against S. aureus and
B.subtilis than positive control cephaloridine with the zone ratio < 1，and none of
these compounds showed superiority to PC190728 which demonstrated about equal
potency to the cephaloridine (zone ratio = 1.2). These result was consistency with
other reports on the PC190723 and 3-MBA analogs, and PC190723 was report to only
show the significant efficacy on Staphylococcus aureus, including MRSA and
1
5, 24
MDRSA, but no efficacy on Escherichia coli
.
Table 1. Antimicrobial activity of compounds presented as ratios of zone of inhibition for the test
compounds
a,c
Zone Ratio (100µg)
Compd
S. aureus
B.subtilis
M.smegmatis
d
1
2
3
4
5
6
7
8
-
-
-
-
-
-
0.07
0.25
-
0.19
0.50
0.07
0.19
0.36
-
0.3
0.15
0.18
0.25
0.51
0.18
0.10
-
0.37

9
0.44
0.18
0.15
0.11
0.3
0.18
0.10
-
0.62
1
0
1
2
3
4
5
6
7
8
9
0
1
2
0.22
-
-
1
1
1
1
1
1
1
1
1
2
2
2
-
0.41
-
0.10
-
-
-
-
-
-
-
0.07
-
-
-
-
-
0.25
0.18
-
0.1
0.1
-
-
0.2
-
-
-
-
PC190723
1.2
-
-
inhibitionzone of test compound
inhibitionzone of standard
a
zone ratio 
b
standard drugs for the calculation: S.aureus: cephaloridine disk, B.subtilis:
Tetracycline, M.smegmatis: Streptomycin disk;
c
The results are averages of three separate experiments
d
‘-’= not active,
In compounds 1 and 2, the crucial amide functional group in PC190723 was
replaced with the bioisosteric sulphonamide functional group, and the rest of the main
structure feature was maintained. However, the results show that such a modification
is not tolerated. Replacement of thiazolo[5,4-b]pyridine functional with phenyl ring
and using an ester linger to connect both ring system derived compounds 3-13.
Compounds 3-5, which have the ester group directly connected with phenyl ring,
showed weak activities on S. aureus and B.subtilis. Introducing an ethylene linker
between the phenyl ring and ester group as in compounds 6-11 only marginally
increased the antibacterial activity. Other variations including inserting a methylene
either between the ester and phenyl ring or between the ester and alkyloxy group as
exampled by compounds 12 and 13, or replacing the oxygen with amine as in

compound 14, were deleterious on the antibacterial activity. Ketone analogues
showed no activity as evidenced by compounds 15 and 16. Similarly, replacement
with the benzo-condensed bicyclic ring system was unproductive as evidenced by
compounds 17-22. Compounds 17-20 showed slightly activities against a wider
spectrum of microorganism, like gram-positive, gram-negative bacterium, even
fungus, although the activity was inferior to the standard drugs. Compounds 21-22
showed no efficacy on the screening. It indicates that nitrogen in the ring system may
increase polarity and water-solubility of the compounds, and then is beneficial for the
antibacterial activity. Among all the tested compounds, compound 9 with a fluorine
substituted on the phenyl ring, showed best antibacterial activity in the series against
M. smegmatis with the zone ratio of 0.62, and against S. aureus with the zone ration
of 0.44.
A series compounds bearing phenyl or quinolyl side chain of 3-alkyloxy-2,6-
difluorobenzamide were designed and synthesised in order to identify new potent
bactericidal agents. Most of tested compounds showed activities on Gram-positive
bacterium, Compound 9 apparently have better inhibitory activity against S. aureus
and M. smegmatis. Herein these results suggest that based on the unique 3-MBA
pharmacophore, compound 9 may represent promising lead candidates against gram
positive bacteria that are worthy of further investigation. Halogen atom and polarity
of the side chain may improve the antibacterial activity and broad-spectrum
properties.

Acknowledgment
This work was supported by the Fundamental Research Fund for the Central
Universities of Huazhong University of Science and Technology (HUST)
(
2016YXMS140, 2016YXMS147, 2016JCTD109, and 2014TS090) and Natural
Science Foundation of Hubei Province (2016CFB379)
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Graphical Abstract
Synthesis and antibacterial activity of 3-benzylamide derivatives as FtsZ
inhibitors
a
a
b
a,*
a,*
Zhongping Hu , Shasha Zhang , Weicheng Zhou , Xiang Ma , Guangya Xiang