Design, synthesis and structure-based optimization of novel isoxazole-containing benzamide derivatives as FtsZ modulators

Article abstract of DOI:10.1016/j.ejmech.2018.09.053

Antibiotic resistance among clinically significant bacterial pathogens is becoming a prevalent threat to public health, and new antibacterial agents with novel mechanisms of action hence are in an urgent need. Utilizing computational docking method and structure-based optimization strategy, we rationally designed and synthesized two series of isoxazol-3-yl- and isoxazol-5-yl-containing benzamide derivatives that targeted the bacterial cell division protein FtsZ. Evaluation of their activity against a panel of Gram-positive and -negative pathogens revealed that compounds B14 and B16 that possessed the isoxazol-5-yl group showed strong antibacterial activity against various testing strains, including methicillin-resistant Staphylococcus aureus and penicillin-resistant S. aureus. Further molecular biological studies and docking analyses proved that the compound functioned as an effective inhibitor to alter the dynamics of FtsZ self-polymerization via a stimulatory mechanism, which finally terminated the cell division and caused cell death. Taken together, these results could suggest a promising chemotype for development of new FtsZ-targeting bactericidal agent.

Full text of DOI:10.1016/j.ejmech.2018.09.053

Accepted Manuscript
Design, synthesis and structure-based optimization of novel isoxazole-containing
benzamide derivatives as FtsZ modulators
Fangchao Bi, Di Song, Nan Zhang, Zhiyang Liu, Xinjie Gu, Chaoyu Hu, Xiaokang Cai,
Henrietta Venter, Shutao Ma
PII:
S0223-5234(18)30831-6
10.1016/j.ejmech.2018.09.053
EJMECH 10764
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 15 July 2018
Revised Date: 19 September 2018
Accepted Date: 19 September 2018
Please cite this article as: F. Bi, D. Song, N. Zhang, Z. Liu, X. Gu, C. Hu, X. Cai, H. Venter, S. Ma,
Design, synthesis and structure-based optimization of novel isoxazole-containing benzamide derivatives
as FtsZ modulators, European Journal of Medicinal Chemistry (2018), doi: https://doi.org/10.1016/
j.ejmech.2018.09.053.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Design, synthesis and structure-based optimization of novel isoxazole-containing
benzamide derivatives as FtsZ modulators
a
Fangchao Bi , Di Song a, Nan Zhang a, Zhiyang Liu a, Xinjie Gu a, Chaoyu Hu a, Xiaokang Cai a,
Henrietta Venter b, 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, China
b School of Pharmacy and Medical Sciences, Sansom Institute for Health Research, University of South
Australia, SA 5000, Australia
Running title: Isoxazole-containing benzamides
* Corresponding author.
E-mail address: mashutao@sdu.edu.cn (S. Ma)
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Abstract
Antibiotic resistance among clinically significant bacterial pathogens is becoming a prevalent threat
to public health, and new antibacterial agents with novel mechanisms of action hence are in an urgent
need. Utilizing computational docking method and structure-based optimization strategy, we rationally
designed and synthesized two series of isoxazol-3-yl- and isoxazol-5-yl-containing benzamide
derivatives that targeted the bacterial cell division protein FtsZ. Evaluation of their activity against a
panel of Gram-positive and -negative pathogens revealed that compounds B14 and B16 that possessed
the isoxazol-5-yl group showed strong antibacterial activity against various testing strains, including
methicillin-resistant Staphylococcus aureus and penicillin-resistant S. aureus. Further molecular
biological studies and docking analyses proved that the compound functioned as an effective inhibitor
to alter the dynamics of FtsZ self-polymerization via a stimulatory mechanism, which finally
terminated the cell division and caused cell death. Taken together, these results could suggest a
promising chemotype for development of new FtsZ-targeting bactericidal agent.
Keywords: Isoxazole-containing benzamide; Antibacterial activity; FtsZ inhibitor; Molecular docking
analysis; Structure-based optimization.
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1. Introduction
At present the widespread bacterial resistance to antibiotics is extremely severe throughout world in
hospital and community settings, which has brought up an urgent need for the discovery and
development of new generation of antibiotics [1, 2]. With respect to the current research, the bacterial
cell division process has been considered as a novel and attractive target for the discovery of new
antibacterial drug [2-5]. During this division process, FtsZ (Filamentous temperature-sensitive mutant
Z) is considered as a fundamental protein forming the division machinery [6-8]. FtsZ is the first protein
to be localized at the incipient division site, and assembles into a highly dynamic cytoskeleton scaffold,
termed Z-ring, by undergoing GTP-dependent polymerization. Subsequently, this cytokinetic structure
recruits other downstream division proteins and contracts enabling septum formation and cell
separation, completing a successful bacterial cell division event [7, 9, 10]. Because of its high
conservation and important function among drug-sensitive and -resistant bacteria, FtsZ has been
confirmed as a promising target for the development of new antibacterial agents by inhibition of the
cell division.
Fig. 1. Chemical structures of some FtsZ inhibitors and their MICs against S. aureus.
By the recent investigations, a number of small molecules have been identified as FtsZ inhibitors, a
few of which just listed (Fig.1). For example, PC190723 is a milestone potent FtsZ inhibitor from
optimization of 3-methoxybenzamide (3-MBA). Despite its initial promise, the poor drug-like and
pharmacokinetic properties have limited its further clinical development. To address this problem,
strategy of prodrug was used and three prodrugs (e.g., TXY436, TXY541 and TXA709) were
developed [11-13], especially TXA709, in which the Cl group on the pyridyl ring has been replaced
with a CF3 functionality resistant to metabolic attack. As a result, the product of TXA709 is associated
with improved pharmacokinetic properties and superior in vivo antistaphylococcal efficacy relative to
PC190723 [13].
The crystal structure of S. aureus FtsZ-PC190723 complex [14] and docking models [15] indicate
that the amide group forms strong hydrogen bonds with surrounding amino acid residues in T7 loop of
FtsZ, which provides the essential binding force between these benzamide compounds and FtsZ.
Besides, the 3-Oside chain stretches into a hydrophobic cleft of FtsZ to form additional interaction.
This explains why 3-MBA, a simple benzamide derivative, can possess FtsZ-targeting effect [16].
Further effective modification is concentrated on the 3-O-side chain. For instance, introduction of the
lipophilic alkyl chain or aromatic ring at the 3-O-position (3 and 4 in Fig.1) significantly enhance
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antibacterial activity but cannot compare favourably with PC190723. This gap indicates that
heteroatom-containing side chain could be more suitable than the pure hydrocarbon side chain for the
hydrophobic cleft of FtsZ. This is because heteroatoms of the 3-O-side chain such as N, O and S may
bring wondrous interactions in this hydrophobic cleft, which differs from the hydrophobic interactions
formed by C atoms of the 3-O-side chain.
In recent years, isoxazole scaffold has attracted more and more attention because of its wide
spectrum of biological activities and therapeutic potential [17]. The structural features of the isoxazole
make it possible for multiple non-covalent interactions, especially hydrogen bonds, pi-pi stack and
hydrophobic interactions. The inclusion of isoxazole may contribute to the increased efficacy,
decreased toxicity, and improved pharmacokinetics profiles [18-20]. Successful applications of
developing isoxazole compounds have resulted in multiple corresponding drugs, including anticancer,
antibacterial, antifungal, antiviral and anti-inflammatory in the market [21].
Fig. 2. Brief design process of isoxazole-benzamide derivatives
Based on the above considerations, we designed the series A of the isoxazol-3-yl-5-benzamide
derivatives through introducing an isoxazol-3-yl group into the 3-O-side chain, and predicted their
potential binding mode through computational docking method. And then, the docking mode was used
to establish their key determinants for affinity to guide the structure-based design, which led to design
of the series B of the isoxazol-5-yl-3-benzamide derivatives. Subsequently, five subordinate analogues
were obtained by further elongating the methylene linkage or introducing Br, Cl or Me into the
3-O-side chain (Fig. 2). From this strategy, we did find some isoxazol-5-yl-3-benzamide derivatives
possessing excellent antibacterial and FtsZ-inhibition activity. In particular, B14 with the isoxazol-5-yl
group displayed about thirty-fold more potent activity against Bacillus subtilis and Bacillus pumilus
(MIC, 0.015 µg/mL) than PC190723, and considerable activity against S. aureus including two
drug-resistant strains (MIC, 2 µg/mL). Here we report the synthesis and antibacterial activity of series
A and B. The cell morphology and the FtsZ polymerization are also performed to indicate their
on-target activity. Their preliminary structure-activity relationships (SARs) and docking study are
described.
2. Chemistry
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Scheme 1. Synthetic route for series A. Regents and conditions: (i) benzyl chloride, K2CO3, NaI, MeCN, 45 ; (ii)
n-BuLi, dry ice, THF, -50 to rt; (iii) oxalyl chloride, DMF, DCM, r.t.; (iv) ammonium carbonate, DCM rt; (v) 10%
Pd/C, ammonium formate, EtOH, 65 ; (vi) 3-bromopropyne, K2CO3, DMF, 35 ; (vii) NaOH, H2O/EtOH, rt;
(viii) chloramine-T, EtOH, rt.
The synthetic route for series A is shown in Scheme 1. Commercially available 2,4-difluorophenol 6
was protected by benzyl group, and then treated with dry ice in the presence of n-BuLi, giving the
benzoic acid 7. The acid was converted to benzamide 8 using oxalyl chloride, followed by ammonium
carbonate. After deprotection of the benzyl group, alkylation of 9 with 3-bromopropyne afforded
intermediate 10. Various oximes 12 were prepared from commercially available or synthesized
aldehyde 11, which were then cyclized with 10 to the isoxazol-3-yl-5-benzamides A1-A35 in the
presence of chloramine-T.
Scheme 2. Synthetic route for series B. Regents and conditions: (i) CH3MgBr, THF, 0
to rt; (ii) MnO2,
acetonitrile, 70 ; (iii) NaH, diethyl oxalate, toluene, 90 ; (iv) NH2OH-HCl, EtOH, reflux; (v) NaBH4,
EtOH/H2O, rt; (vi) SOCl2, 1H-benzotriazole, DCM, rt; or CBr4, PPh3, DCM, 0 to rt; (vii) K2CO3, DMF, 35
(viii) Acetyl chloride, AlCl3, 1,2-Dichloroethane, 0 to rt.
;
The synthetic route for series B is outlined in Scheme 2. Commercially available or synthetic
acetophenone 13 was converted to 14 in the presence of NaH and diethyl oxalate, which was then
condensed with hydroxylamine hydrochloride to yield intermediate isoxazole 15. Reduction by NaBH4
followed by chlorination or bromination afforded key intermidiate 16, which was coupled with 9
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formed the isoxazol-5-yl-3-benzamides B1-B16.
Scheme 3. Synthetic route for subordinate series copounds B14-2, B14-Br, Cl, Me and B2-Br. Regents and
conditions: (i) NaH, THF, n-BuLi, -10 to 0 ; (ii) 2-Iodoxybenzoic acid, MeCN, 0 to reflux; (iii) NH2OH-HCl,
EtOH, 80 ; (iv) NaBH4, EtOH/H2O, rt; (v) TsCl, TEA, DMAP, DCM, 0 to rt; (vi) intermediate 9, K2CO3, DMF,
rt; (vii) CH3I, NaH, DMF, rt; (viii) NH2OH-HCl, EtOH, 80 ; (ix) NaBH4, EtOH/H2O, rt; (x) CBr4, PPh3, DCM, 0
to rt; (xi) intermediate 9, K2CO3, DMF, rt; (xii) NBC or NCS, AcOH, reflux.
Subordinate compound B14-2 derived from B14 was synthesized as shown in Scheme 3.
Commercially available 4-tert-butylbenzaldehyde was condensed with ethyl acetoacetate to provide
intermediate 17. Oxidation of 17 using 2-iodoxybenzoic acid followed by cyclization with
hydroxylamine hydrochloride produced 19. Reduction of 19 using NaBH4 followed by sulfonylation in
the presence of TsCl in DCM furnished 20. This intermediate was then treated with 9 to give the
desired B14-2.
Four subordinate compounds B14-Br, B14-Cl, B14-Me and B2-Br were synthesized as shown in
Scheme 3. Bromination or chlorination of B2 and B14 in the presence of NBS or NCS in acetic acid
furnished the corresponding B2-Br, B14-Br and B14-Cl in a moderate yield. Methylation of
intermediate 14-B14 using CH3I followed by the similar process as described in Scheme 2 provided the
bromomethyl intermediate 22. Nucleophilic substitution of 22 with intermediate 9 gave the desired
compound B14-Me.
3. Results and discussion
3.1. In vitro antibacterial activity of series A analogues
The target compounds listed in Scheme 1, 2 and 3 were evaluated for their in vitro antibacterial
activity by broth microdilution procedures described in the method recommended by the Clinical and
Laboratory Standards Institute (CLSI) guidelines [22]. Minimal inhibitory concentration (MIC) values
for all the target compounds were determined in comparison with PC190723 (PC), ciprofloxacin (CIP),
linezolid (LIN) against a panel of sensitive and resistant strains.
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The MIC results shown in Table 1 indicated that some of the isoxazol-3-yl-containing compounds in
the series A displayed superior or comparable efficacy to the clinical antibiotics and PC190723
evaluated in this study. In particular, compounds A25 and A26 exhibited over 16-fold potent activity
(MIC, 0.03 µg/mL) against B. subtilis than PC and other two reference antibiotics, and A25 displayed
moderate activity against three susceptible and resistant S. aureus with MIC values of 4–8 µg/mL,
which was much better than ciprofloxacin, which was slightly inferior to PC190723.
Table 1
Series A analogues with their in vitro antibacterial activity.
Minimum inhibitory concentration/MIC (µg/mL)
Compd
B. subtilis
B. pumilus
CMCC63202
>64
S. aureus
ATCC25923
>64
>64
>64
>64
>64
>64
8
S. aureus
ATCC43300a
>64
>64
>64
>64
>64
>64
4
S. aureus
PRb
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
8
E. coli
ATCC25922
>64
P. aeruginosa
ATCC27853
>64
ATCC9372
64
4
A1
A2
>64
>64
>64
1
>64
>64
>64
A3
4
>64
>64
>64
A4
8
16
>64
>64
A5
8
>64
>64
>64
A6
0.5
64
3
0.5
>64
>64
A7
>64
>64
64
>64
4
>64
>64
A8
>64
>64
>64
A9
64
>64
>64
4
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
8
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
4
>64
>64
A10
A11
A12
A13
A14
A15
A16
A17
A18
A19
A20
A21
A22
A23
A24
A25
A26
A27
A28
A29
A30
A31
A32
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
1
>64
>64
>64
>64
>64
>64
2
16
>64
>64
16
64
>64
64
>64
64
8
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
2
32
>64
>64
0.03
0.03
1
0.25
0.25
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
2
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
64
32
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
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32
8
>64
>64
>64
0.5
8
>64
>64
>64
1
>64
>64
>64
1
>64
>64
>64
1
>64
>64
>64
>64
8
>64
>64
>64
>64
4
A33
A34
A35
PC
CIPc
LINd
32
0.5
2
8
16
>64
1
1
2
1
2
>64
>64
a S. aureus ATCC43300: methicillin-resistant strain. b S. aureus PR: penicillin-resistant strain isolated clinically,
not characterized. c CIP: Ciprofloxacin. d LIN: Linezolid.
3.2. Structure-based rational optimization leading to series B analogues
Given that A25 with the isoxazol-3-yl group was screened as a promising potent compound, and this
could offer an attractive starting point for structure-based optimization. Subsequently, we investigated
the potential binding mode of A25 using a reported crystal structure of S. aureus FtsZ (PDB ID: 3vob)
[14]. The docking orientation from the computational analysis is shown in Fig. 3. Binding of A25 into
a relatively narrow cleft delimited by H7-helix, T7-loop and C-terminal β-sheet, was the same as the
binding site of PC190723 (Fig. S2) [11]. The amide group and ether oxygen atom formed some
important hydrogen bonds with surrounding amino acid residues, as key force to grasp the FtsZ protein,
and the 4-tert-butylphenyl group extended to a hydrophobic region. What draw more of our attention
was that there was Asp199 residue near the isoxazol-3-yl group in the 3-O-side chain. The distances
between the carboxyl group of Asp199 and the N, O atoms of the isoxazol-3-yl group were 4.1 and 3.4
Å, respectively. If the position of the N and O atoms of the isoxazol-3-yl group exchanged, the
isoxazol-3-yl group was converted to the isoxazol-5-yl group in the 3-O-side chain. The N atom of the
isoxazol-5-yl group was 3.4 Å away from the carboxyl group of Asp199. In the physiological pH
environment, the carboxyl group existed in the form of anion. The N atom of the isoxazol-5-yl group
could form moderate ion-dipole interaction with the carboxyl anion. Thus, our next strategy was to
exchange the N and O atom’s relative position in the isoxazole ring, in order to form a direct ion-dipole
interaction with Asp199. To this end, the series B of the isoxazol-5-yl-3-benzamide derivatives
analogues were designed, as shown in Fig. 2.
Fig. 3. The Molecular docking model of SaFtsZ (PDB ID: 3vob) in complex with A25. The hydrogen bonds were
shown as cyan dot lines; the distances of Asp199 and N, O atoms of the isoxazol-3-yl group were shown as green
dot lines.
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3.3. In vitro antibacterial activity of series B analogues
The MIC results of series B analogues were exhibited in Table 2. In the series B, B14 with the
isoxazol-5-yl group was found to have more potent antibacterial activity than A25 with the
isoxazol-3-yl group, both of which were isomers with the different connection of the isoxazole group in
the 3-O-side chain. . For example, B14 displayed the MIC values of 0.015–0.03 µg/mL against B.
subtilis and B. pumilus stains, respectively, which was about 32-fold better than PC190723. It was also
worth mentioning that B14 and B16 inhibited the growth of one susceptible and two resistant
(methicillin- and penicillin-resistant) S. aureus strains with the MIC values of 1–4 µg/mL, respectively,
which were much stronger than ciprofloxacin (MIC, 8, 16 and >64 µg/mL), and comparable to
linezolid and PC190723. The nearly indiscriminate MIC values of these compounds against susceptible
and resistant S. aureus strains also confirmed the important role of FtsZ as an antibacterial target in
discovering new antibiotics.
Based on the structure of the most effective compound B14, elongating the methylene linkage
(compound B14-2) or introducing halogen or alkyl group in the isoxazol-5-yl group (compounds
B2-Br, B14-Br, B14-Cl and B14-Me) all decreased their antibacterial activity, especially for three S.
aureus strains (MIC, >64 µg/mL). According to the PC190723-FtsZ 3D structure (PDB code: 3vob)
and the docking models (Fig. S1), we reasoned that the binding pocket could just hold ligands of
specific length, and compounds which are longer than B14 could not enter into the limited domain. The
low activity caused by the redundant atoms introduced to B14 might be resulted by the space restricted
binding pocket of FtsZ. Furthermore, the increased activity from A25 to B14 revealed that the relative
position of heteroatoms in isoxazole ring could have direct influence on the interaction with FtsZ
binding pocket, which was corresponding to our computational deduction.
Table 2
Series B and four subordinate analogues with their in vitro antibacterial activity.
Minimum inhibitory concentration/MIC (µg/mL)
Compd
B. subtilis
B. pumilus
CMCC63202
>64
S. aureus
ATCC25923
>64
S. aureus
ATCC43300a
>64
S. aureus
PRb
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
4
E. coli
ATCC25922
>64
P. aeruginosa
ATCC27853
>64
ATCC9372
64
64
B1
B2
>64
>64
>64
>64
>64
8
>64
>64
>64
>64
>64
B3
>64
0.5
64
>64
>64
>64
>64
>64
B4
8
>64
>64
>64
>64
B5
>64
>64
>64
>64
>64
B6
64
>64
>64
>64
>64
>64
B7
>64
32
>64
>64
>64
>64
>64
B8
>64
>64
>64
>64
>64
B9
>64
4
>64
>64
>64
>64
>64
B10
B11
B12
B13
B14
B15
>64
>64
>64
>64
>64
64
>64
>64
>64
>64
>64
32
>64
>64
>64
>64
>64
0.015
0.06
0.03
2
2
>64
>64
>64
>64
>64
>64
>64
>64
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>64
>64
>64
>64
1
0.06
>64
>64
>64
1
1
2
2
>64
>64
>64
>64
>64
>64
>64
8
>64
>64
>64
>64
>64
>64
>64
4
B16
B14-2
B14-3
B2-Br
B14-Br
B14-Me
PC
>64
>64
>64
>64
>64
1
>64
>64
>64
>64
>64
1
>64
>64
>64
>64
>64
1
0.5
0.5
2
1
0.5
8
CIPc
LINd
8
16
>64
2
1
1
2
1
>64
>64
a
b
S. aureus ATCC43300: methicillin-resistant strain. S. aureus PR: penicillin-resistant strain isolated clinically,
not characterized. c CIP: Ciprofloxacin. d LIN: Linezolid.
3.4. Bactericidal or bacteriostatic assay
The antibacterial activity of B14 highlighted by the results in Tables 1 and 2 prompted further
investigation as to whether or not its activity was bactericidal or bacteriostatic in nature. Therefore, the
minimal bactericidal concentration (MBC) values of B14 against susceptible (ATCC25923),
methicillin-resistant (ATCC43300) and penicillin-resistant S. aureus stains were determined and
subsequently compared with corresponding MIC values. Clinical drug linezolid and clarithromycin
were used as controls in the S. aureus determinations. According to the CLSI standards, an MBC/MIC
ratio of 1 to 2 is viewed as being indicative of bactericidal behavior. By contrast, an MBC/MIC ratio of
≥8 is considered indicative of bacteriostatic behavior. The test results shown in Table 3 indicated that
linezolid and clarithromycin were observed as bacteriostatic agents with the MBC/MIC ratios of 16 and
32, respectively. Significantly, B14 exhibited almost identical MBC and MIC values (i.e., MBC/MIC
ratio of 1 and 2) for both susceptible and resistant S. aureus, indicating a bactericidal mode against the
above bacterial species.
Table 3
Comparison of MIC and MBC Values for B14 in three S. aureus.
Compd
MBC (µg/mL)
MIC (µg/mL)
MBC/MIC
susceptible S. aureus (ATCC25923)
4
32
4
2
2
B14
linezolid
2
16
32
clarithromycin
0.125
methicillin-resistant S. aureus (ATCC43300)
2
2
1
1
B14
linezolid
32
32
penicillin-resistant S. aureusb
8
4
2
2
B14
linezolid
32
16
a linezolid and clarithromycin are included as control agents.
b penicillin-resistant strain isolated clinically.
3.5. Kinetics of the bactericidal activity
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After the bactericidal nature of B14 was established, the kinetics of killing would be next
investigated in methicillin-resistant S. aureus ATCC43300. Time-kill curves are shown in Fig. 4. The
growth of bacteria was obvious when it was incubated without B14 or with 0.5 × MIC of B14. At the
concentration of 1 × MIC and 2 × MIC, B14 could reduce the viable counts below the lowest
detectable limit (103 CFU/mL) after incubation for 12 h. And B14 at the concentration of 4 × MIC
could rapidly reduce the viable counts after incubation for 6 h. The growth inhibition of different
concentration gradient revealed that, the rate at which B14 killed S. aureus strain was dependent on the
compound concentration, and the higher concentrations resulted in the greater rates of killing. It was
noteworthy that the killing rate of B14 under 8 µg/mL was much greater than that of linezolid with an
equivalent concentration.
Fig. 4. Time−kill curves for S. aureus ATCC43300 (MRSA). Each data point reflects the average of two
independent measurements, with the error bars reflecting the standard deviation from the mean.
3.6. Effects on the morphology of B. pumilus cells
To confirm whether the antibacterial mechanism of B14 was associated with alteration of FtsZ
function, we explored the bacterial cell morphology through microscopic observations. As shown in
Fig. 5, B14 significantly increased the cell length of B. pumilus CMCC63202 at 0.5 × MIC
concentration, as compared with untreated cells. Such cell filamentation was thought to be a hallmark
of FtsZ-directed inhibitors such as PC190723 [23]. These results, which were consistent with other
reported 3-substituted benzamides [16, 24], suggested that B14 could interfere with proper FtsZ
function, which led to the abnormal bacterial cell division and then cause cell death.
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Fig. 5. Effects of compound B14 on the cell morphology. B. pumilus cells were grown in the absence (A) or in the
presence (B) of B14 at 0.015 µg/mL.
3.7. Stimulation of FtsZ polymerization dynamics
Recent studies showed that the stimulation of FtsZ polymerization dynamics and stabilization of
FtsZ polymerization were thought to underlie the antibacterial activity of FtsZ-targeting agents [25, 26].
We probed for the propensity of B14 to exert such kind of effect on the polymerization of purified B.
subtilis FtsZ (BsFtsZ) in vitro. For this assay, we utilized a microliter plate-based spectrophotometric
approach to detect FtsZ polymerization in a time-dependent increase in solution absorbance at 340 nm
(A340). The time-dependent A340 profiles of BsFtsZ in the absence or presence of B14 at the
concentrations ranging from 2.5 to 10 µg/mL is illustrated in Fig. 6A. Plainly, B14 increased the
kinetics of BsFtsZ polymerization, with the magnitude of these stimulatory effects increasing
dependent on compound concentration. Ciprofloxacin was included as a non-FtsZ-targeting control
drug in this assay. As expected, neither ciprofloxacin nor vehicle impacted the polymerization of
BsFtsZ to any significant degree. It should be noted that A25 at 10 µg/mL displayed much weaker
stimulatory effect than B14 at same concentration, as shown in Fig. 6B. This different stimulatory
impact on FtsZ polymerization tallied with their different antibacterial activity.
Fig. 6. The impact of B14 on the polymerization of BsFtsZ in a concentration-dependent manner. (A)
Polymerization profiles were acquired in the presence of DMSO vehicle or the indicated concentrations of B14.
Ciprofloxacin was included as a negative (non-FtsZ-targeting) control. (B) Polymerization profiles in the presence
of B14 and A25 at 10 µg/mL, respectively.
3.8. Promotion of assembly of FtsZ and bunding of FtsZ polymers
Apart from the light scattering assay, the effects of B14 on the polymerization of FtsZ were also
visualized using transmission electron microscopy (TME). Negatively stained electron micrographs of
B14-induced polymers of BsFtsZ are shown in Fig. 7. It was found that, in the presence of B14 at 10
µg/mL, the size and thickness of the FtsZ polymers and the bundling of the FtsZ protofilaments
drastically increased (Fig. 7A), however, under the experimental conditions employed, negligible
BsFtsZ polymers was observed in the absence of B14 (Fig. 7B), which was corresponding with other
empirical results reported previously [27-30]. Thick filaments of bundled FtsZ polymers would scatter
light more effectively than unpolymerized FtsZ [27]. Viewed as a whole, the TME assay suggested that
12

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the B14 promoted both the assembly of BsFtsZ and bunding of BsFtsZ polymers, which accounted for
the increasing magnitudes of the light-scattering signals (A340) as shown in Fig. 6.
Fig. 7. Transmission electron micrographs of BsFtsZ polymers in the presence of B14 (A) at 10 µg/mL and in the
presence of DMSO vehicle (B).
3.9. Computational studies of the binding mode
The computational analyses of B14, A25 and PC190723 were also explored and contrasted utilizing
a reported crystal structure of SaFtsZ (PDB code: 3vob) and Autodock. The binding modes of B14 and
PC190723 in Fig. 8A showed that the amide group and ether oxygen atom formed strong hydrogen
bonds with surrounding amino acid residues as key force to grasp the FtsZ protein, and the
4-tert-butylphenyl group and the 6-chloro-thiazolopyridine group extended to a hydrophobic region to
produce additional van der Waals interaction. Comparing the docking conformations of B14 and A25 in
Fig. 8B, we found that even both conformations were nearly overlap with each other, and the nitrogen
atom in the isoxazol-3-yl group of B14 could form unique ion-dipole interaction with the ionized
carboxyl group of Asp199 and hydrogen-bonding interaction with a water molecule, while the nitrogen
atom in the isoxazol-5-yl group of A 25 lacked the above interactions. Thus, this docking result was
consistent with our computer-assisted optimization strategy mentioned above, and could partly explain
the enhanced antibacterial activity and increased stimulatory effect on FtsZ polymerization of B14.
Fig. 8. Predicted binding modes of SaFtsZ (PDB ID: 3vob) in complex with B14 and A25. (A) The hydrogen
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bonds of PC190723 (The conformation of PC190723 was not docking result, and it was the actual binding model
in the X-ray diffraction.) and B14 were shown as pink and yellow dot lines, respectively. (B) The hydrogen bonds
of B14 and A25 were shown as yellow and cyan dot lines, respectively.
3.10. In vivo efficacy against mice MRSA infection
To evaluate the in vivo antistaphylococcal efficacy of B14, we initially used a mouse peritonitis
model of systemic infection with S. aureus ATCC43300 (MRSA). But most of the mice were still alive
after inoculated intraperitoneally with about 6.6 × 109 CFU of bacteria, due to the low virulent of
MRSA ATCC43300 [31]. Considering too much bacterial count could influence the effective dose of
the test agents, we next characterized the efficacy of B14 in a mouse blood model of infection with S.
aureus ATCC43300 [32-34]. B14 and linezolid (or the vehicle control) were administered
intraperitoneally at the doses and times indicated, after 5.4 × 107 CFU of S. aureus was injected
intravenously. The numbers of bacterial CFU recovered from the blood after 24 h are shown in Fig. 9.
Five i.p. doses of 30 mg/kg of B14, administered 0.1, 3, 6, 9 and 12 h postinfection, were sufficient to
cause significant decreases in bacterial burden after 24 h compared with results from the untreated
control group. Two i.p. doses of 30 mg/kg of linezolid, administered 0.1, 6 h postinfection, were
adopted as a positive control. Note that none of the mice, including the untreated group, died within
one week after the intravenous administration of bacteria.
Fig. 9. Efficacy of B14 in a mouse blood model of infection with MRSA ATCC43300. The numbers of CFU
recovered from the blood after 24 h are shown. *, all CFU reductions were significant (P < 0.01) relative to those
of the untreated control (vehicle).
3.11. Cytotoxic evaluation on mammalian cells
To probe for any potential mammalian cytotoxicity, we used a 48 h tetrazole (MTT)-based assay to
assess the cytotoxicity of A25, B14 and B16 against human cervical cancer (Hela) cells [13]. These
isoxazole-containing derivatives were found to be minimally toxic to Hela cells, with 50% inhibitory
concentrations (IC50) of >128 µg/mL. Significantly, this IC50 value is much higher than the
antistaphyloccal MIC values of B14. Albeit derived from a single mammalian cell line, this initial
result suggests that B14 is associated with a significant therapeutic window. Future studies with
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additional mammalian and human cell lines will be directed toward assessing the general nature of this
result.
4. Conclusion
Using computational docking results and structure-based optimization, two series of isoxazol-3-yl-
and isoxazol-5-yl-containing benzamide derivatives were designed, synthesized, and tested for their in
vitro biological activities. They were pharmacologically evaluated as FtsZ-targeting antibacterial agents
as well. The results indicate that several compounds exhibit significant antibacterial activity against
most of the tested strains, including the drug-resistant strains. It is noteworthy that the antibacterial
activity of B14 against B. subtilis and B. pumilus is about 32-fold more active than those of PC190723.
Particularly, the MIC values of B14 and B16 against two resistant and one sensitive S. aureus strains
range between 1 and 4 µg/mL, which are comparable to those observed with PC190723 and linezolid,
and are much better than those of ciprofloxacin. Furthermore, the relative position of N and O atoms in
the isoxazole ring of the 3-O-side chain can really influence the antibacterial activity, as illustrated in
the computational docking studies. Regarded as a whole, the studies described herein highlight B14 as
a lead compound with potent bactericidal activity and reinforce the importance of FtsZ as a promising
antibacterial target. Further optimization of the isoxazol-3-yl-containing benzamide derivatives and in
vivo studies with lead compound B14 are ongoing in our lab.
5. Experimental section
5.1. General experimental protocol
All commercially available chemicals and reagents were used without any further purification unless
otherwise indicated. Reactions progress was monitored by thin-layer chromatography (TLC) on
0.25-mm pre-coated silica GF254 plates. Flash column chromatography was carried out with the
1
indicated solvents using silica gel 200–300 mesh size. H NMR spectra were recorded at 400 or 600
MHz, 13C NMR spectra were recorded at 151 MHz, and reported in ppm using appropriate deuterated
solvents. Mass spectra were obtained on the API 4000 instrument. HRMS were measured using an
Orbitrap analyzer. Melting points were determined using a RY-1 melting point apparatus without
corrected.
5.2. Typical procedure for the synthesis of series A and B
5.2.1. 3-(Benzyloxy)-2,6-difluorobenzoic acid (7)
To a solution of 2,4-difluorophenol 6 (20.0 g, 154 mmol) in acetonitrile (180 mL) was added
benzyl chloride (18.5 g, 146 mmol) , K2CO3 (32.0 g, 231 mmol) and catalytic amount of NaI. The
mixture was stirred overnight at 70 . Upon completion, the mixture was diluted with petroleum ether,
insoluble salts was filtered off through diatomite and washed with petroleum ether. The filtrate was
transferred into separating funnel, further washed with water, 10% sodium hydroxide to remove the
unreacted 6, and brine, subsequently. The organic layer was dried and concentrated to give the
benzyl-protected 6 (31.8 g, 99%) as an off-white crystalline solid. this intermediate was used in the
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next step without additional purification.
To the above intermediate (10.0 g, 45.5 mmol) in THF (70 mL) under a nitrogen atmosphere at -50
was added dropwise n-BuLi (2.5 M in hexane, 20 mL, 50 mmol). The reaction solution was stirred for
2 h and poured onto freshly crushed dry ice, and the resulting mixture was allowed to warm to room
temperature overnight. To the mixture was added NaOH solution (1.5 M, 150mL), then washed with
petroleum ether to remove the unreacted reagent. The remaining alkaline aqueous solution was
acidified using concentrated hydrochloric acid in ice bath, the precipitated solid was collected through
negative-pressure filtration, and vacuum drying. This acid intermediate 7 (11.4 g, 95%) was used
directly without additional purification.
5.2.2. 3-(Benzyloxy)-2,6-difluorobenzamide (8)
To a solution of 7 (10 g, 37.9 mmol) in DCM (100 mL) was added a drop of DMF for catalyst, and
followed added oxalyl chloride (3.8 mL, 45.5 mmol). Then stirred the mixture for 6h at room
temperature until completion. The organic solvent was evaporated in vacuo to dry, then the resulting
mixture was diluted with DCM (100 mL), and added crushed ammonium carbonate (8.0 g, NH3 ≥
40%,).The reaction mixture was sealed with a balloon, and stirred overnight at room temperature. Upon
completion, 6 mL MeOH was added to the reaction solution to dissolve the product, and then filtered
out the undissolvable salt, and further washed the filter cake with DCM/MeOH (20:1), the resulting
filtrate was collected and evaporated to dryness getting amide intermediate 8 (9.6 g, 97%). The residue
was used directly without additional purification.
5.2.3. 2,6-difluoro-3-hydroxybenzamide (9)
To a solution of 8 (9 g, 34.2 mmol) in EtOH (100 mL) were added 10% Pd/C (0.9 g) and ammonium
formate (13 g, 205 mmol). The reaction mixture was sealed with a balloon, and stirred overnight at
65 . Upon completion, the mixture was concentrated, and the getting residue was redissolved with
DCM/MeOH (10/1), then filtered off the insoluble salts. The filtrate was evaporated, and the crude
product was purified by column chromatography (DCM/MeOH, 20/1) yielding intermediate 9 as a
white solid (5.7 g, 96%). Mp: 118–120 ; 1H NMR (600 MHz, DMSO-d6) δ 9.94 (s, 1H), 8.06 (s, 1H),
7.77 (s, 1H), 6.99 – 6.88 (m, 2H). 13C NMR (151 MHz, DMSO-d6) δ 162.24, 151.08 (dd, J = 238.3, 6.2
Hz), 147.38 (dd, J = 253.5, 8.7 Hz), 141.95 (dd, J = 12.2, 3.2 Hz), 117.85 (dd, J = 8.9, 3.8 Hz), 116.91
(dd, J = 24.5, 20.1 Hz), 111.40 (dd, J = 23.0, 4.1 Hz). ESI-MS calcd for C7H6F2NO2 [M + H]+, 174.1,
found 174.2.
5.2.4. 2,6-difluoro-3-(prop-2-yn-1-yloxy)benzamide (10)
A mixture of 9 (2 g, 11.6 mmol), 3-bromopropyne (1.8 g, 15.1 mmol) and K2CO3 (3.2 g, 23.2 mmol)
in DMF (16 mL) was stirred at 35 for 7h. The reaction mixture was then diluted with EtOAc (80
mL), washed with water and brine, dried over Na2SO4, concentrated, and crystallized with petroleum
ether and ethyl acetate. The precipitated solid was filtered out and dried, getting the key intermediate
10 as a white solid (2 g, 82%). Mp: 138–140 ; 1H NMR (600 MHz, DMSO-d6) δ 8.16 (s, 1H), 7.88 (s,
1H), 7.28 (td, J = 9.4, 5.2 Hz, 1H), 7.12 (td, J = 8.9, 1.8 Hz, 1H), 4.91 (d, J = 2.5 Hz, 2H), 3.65 (t, J =
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2.4 Hz, 1H). ESI-MS calcd for C10H8F2NO2 [M + H]+, 212.2, found 212.1.
5.2.5. Typical procedure for the synthesis of oxime intermediates (12)
Dissolve hydroxylamine hydrochloride (0.21 g, 3 mmol) in water (6 mL) and neutralized with
aqueous sodium hydroxide solution (10%). A solution of according aldehyde 11 (2.8 mmol) in ethanol
(2 mL) was slowly added to the above mixture with stirring at room temperature, and stirred for about
3 h. upon the completion of reaction, the residue was diluted with ethyl acetate (30 mL), and then
washed with brine. The organic layer was dried over anhydrous Na2SO4 and the solvent was evaporated
in vacuum. The oxime was used without further purification.
5.2.6 Typical procedure for the synthesis of the isoxazol-3-yl-5-benzamide derivatives (series A)
A 10-mL round-bottom flask equipped with a magnetic stirrer was charged with 10 (50 mg, 0.24
mmol), 12 (0.5 mmol) and ethanol (2.5 mL). A solution of chloramine-T (160 mg, 0.7 mmol) in water
(1 mL) was added dropwise to the reaction mixture, and stirred at room temperature overnight. The
reaction mixture was then diluted with EtOAc (25 mL), washed with brine, dried over Na2SO4,
concentrated in a rotovapor, and purified on silica gel. Elution with petroleum ether/EtOAc (2:1)
afforded the according products in good yield.
5.2.6.1. 3-((3-Phenylisoxazol-5-yl)methoxy)-2,6-difluorobenzamide. (A1): white solid, yield 62%; Mp:
170–171 ; 1H NMR (600 MHz, DMSO-d6) δ 8.16 (s, 1H), 7.86 – 7.91 (m, 3H), 7.50 – 7.54 (m, 3H),
7.41 (td, J = 9.3, 5.1 Hz, 1H), 7.22 (s, 1H), 7.13 (td, J = 8.9, 1.7 Hz, 1H), 5.44 (s, 2H). HRMS (ESI)
calcd for C17H13F2N2O3 [M + H]+, 331.0889, found 331.0892.
5.2.6.2. 3-((3-(4-Chlorophenyl)isoxazol-5-yl)methoxy)-2,6-difluorobenzamide. (A2): white solid, yield
64%; Mp: 173–174 ; 1H NMR (600 MHz, DMSO-d6) δ 8.15 (s, 1H), 7.93 (d, J = 8.5 Hz, 2H), 7.87 (s,
1H), 7.61 (d, J = 8.6 Hz, 2H), 7.41 (td, J = 9.3, 5.1 Hz, 1H), 7.25 (s, 1H), 7.13 (td, J = 8.9, 1.7 Hz, 1H),
5.45 (s, 2H). 13C NMR (151 MHz, DMSO-d6) δ 168.54, 161.71, 161.59, 152.87 (dd, J = 242.4, 6.5 Hz),
148.52 (dd, J = 249.3, 8.4 Hz), 142.36 (dd, J = 10.9, 2.9 Hz), 135.60, 129.77, 128.95, 127.45, 117.16
(dd, J = 24.9, 20.5 Hz), 116.74 (d, J = 9.1 Hz), 113.58 (dd, J = 23.3, 3.8 Hz), 103.35, 62.21. ESI-MS
calcd for C17H12ClF2N2O3 [M + H]+, 365.7, found 365.3.
Physical characteristics, 1H NMR, 13C NMR, MS and HRMS for other target compounds of series A,
were reported in the supporting information.
5.2.7. General procedure for the synthesis of ketone 13
To a solution of aldehyde 11 (4 mmol) in THF (12 mL) at 0 under nitrogen, was dropwise added
methylmagnesium bromide (1.0 M in THF, 5 mmol). After stirring for 10 min the reaction mixture was
allowed to warm to room temperature and was stirred for 3 h. Upon completion, the reaction was
quenched with saturated NH4Cl solution and extracted with EtOAc. The combined organic layer was
washed with brine, dried over Na2SO4, and evaporated under reduced pressure. The crude residue was
used directly.
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The above secondary alcohol was dissolved in acetonitrile (30 mL). Molecular sieve (1.5 g),
silica gel (1.5 g) and activated MnO2 (3.5 g, 40 mmol) were added, and the mixture was stirred at 70
for 8 h. upon completion of the oxidation, the resulting suspension was filtered through diatomite, and
the filtrate was concentrated by rotary evaporation. Purification of the residue by column
chromatography (petroleum ethrt/EtOAc, 15:1) afforded ketone 13.
For the compound B-16, we used the friedel-crafts acylation to synthesize the ketone intermediate
13-B16. A solution of the 5-tert-butyl-m-xylene (3 mmol) dissolved in 1, 2-dichloroethane (2 mL) was
added dropwise to a cold (0 ) solution of anhydrous aluminium chloride (3.9 mmol) and acid chloride
(3.3 mmol) in 1,2-dichloroethane (8 mL). After complete addition, the mixture was warmed to room
temperature and stirred for 5 h. The solution was pouted over ice/water (40 g) which was then extracted
with DCM. The organic layer was dried over Na2SO4, filtered and the solvent was removed to give the
crude product. The product was purified by column chromatography (petroleum ether/EtOAc) to afford
ketone 13-B16 as colorless oil. Yield 81%. 1H NMR (600 MHz, Chloroform-d) δ 7.02 (s, 2H), 2.47 (s,
3H), 2.25 (s, 6H), 1.29 (s, 9H).
5.2.8. General procedure for the synthesis of intermediates 14
A round-bottom flask equipped with a magnetic stirrer was charged with alkanone 13 (3.5 mmol),
diethyl oxalate (0.73 g, 5 mmol) and toluene (15 mL), and a suspension of NaH (80%, 0.21 g, 7 mmol)
was added. The reaction mixture was stirred at 90
for 2 h. The reaction mixture was poured into
ice-water (30 mL), acidified with aqueous HCl to pH 2~3 and extracted with EtOAc. The combined
organic layer was dried over Na2SO4 and evaporated under reduced pressure. The resulting residue was
then purified by flash column chromatography on silica using 10% ethyl acetate in petroleum ether.
Yield 75–85%. Example 14-B14, name: ethyl 4-(4-(tert-butyl)phenyl)-2,4-dioxobutanoate, yellow oil,
1
yield 78%; H NMR (600 MHz, Chloroform-d) δ 15.39 (s, 1H), 7.94 (d, J = 8.6 Hz, 2H), 7.52 (d, J =
8.5 Hz, 2H), 7.07 (s, 1H), 4.40 (q, J = 7.2 Hz, 2H), 1.42 (t, J = 7.2 Hz, 3H), 1.36 (s, 9H). MS calcd for
C16H21O4 [M + H]+, 277.3, found 277.4.
5.2.9. General procedure for the synthesis of intermediates 15
To a solution of 14 (2.5 mmol) in EtOH (15 mL) was added hydroxylamine hydrochloride (0.52 g,
7.5 mmol). The mixture was heated to reflux for 2 h. upon completion, the reaction mixture was
concentrated, and the residue was partitioned between EtOAc and brine, dried over Na2SO4,
concentrated in a rotovapor, and purified on silica gel [35, 36]. Elution with 5% EtOAc/petroleum ether
afforded
the
intermediate
15.
Example
15-B14,
name:
ethyl
5-(4-(tert-butyl)phenyl)isoxazole-3-carboxylate, yield 81 %; MS calcd for C16H20NO3 [M + H]+, 274.3,
found 274.3.
5.2.10. General procedure for the synthesis of intermediates 16
The ethyl ester intermediate 15 (2 mmol) was dissolved in ethanol/water (3:1, 10 mL), and NaBH4
(0.38 g, 10 mmol) was added. The reaction mixture was sealed with a balloon and stirred overnight at
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room temperature. Upon the completion of reduction, the solution was partitioned between EtOAc (40
mL) and 1N HCl (25 mL). The organic layer was washed with saturated NaHCO3 and brine, dried over
Na2SO4, concentrated in a rotovapor. The alcohol was used directly for next step without purification.
The chloride intermediate 16 was prepared as followed, the above alcohol intermediate was
dissolved in DCM (10 mL), 1H-benzotriazole (0.48 g, 4 mmol) and SOCl2 (0.33 mL, 3 mmol) were
added to this mixture successively. The resulting reaction mixture was stirred overnight at room
temperature. The reaction mixture was then diluted with DCM, washed with saturated NaHCO3 and
brine, dried over Na2SO4, concentrated, and purified on silica gel with petroleum ether/EtOAc (50:1)
getting the chloride intermediate 16 in good yield. Example 16-B14, name:
5-(4-(tert-butyl)phenyl)-3-(chloromethyl)isoxazole, yellow solid, yield 87%; 1H NMR (600 MHz,
Chloroform-d) δ 7.71 (d, J = 8.5 Hz, 2H), 7.49 (d, J = 8.5 Hz, 2H), 6.58 (s, 1H), 4.63 (s, 2H), 1.35 (s,
9H). MS calcd for C14H17ClNO [M + H]+, 322.1, found 322.4.
The bromide intermediate 16 was prepared from alcohol intermediate using the procedure as
followed. To the alcohol dissolved in DCM at 0 , was added CBr4 (1.5eq) and PPh3 (3eq)
successively. The mixture was stirred at room temperature overnight. Upon completion, purification
was carried out using silica gel column with appropriate eluent. Example 16-B16, name:
1
3-(bromomethyl)-5-(4-(tert-butyl)-2,6-dimethylphenyl)isoxazole. H NMR (600 MHz, Chloroform-d)
δ 7.14 (s, 2H), 6.32 (s, 1H), 4.50 (s, 2H), 2.22 (s, 6H), 1.33 (s, 9H). MS calcd for C16H21BrNO [M +
H]+, 250.7, found 250.3.
5.2.11. General procedure for the synthesis of the isoxazol-5-yl-3-benzamide derivatives (series B)
A round-bottom flask equipped with a magnetic stirrer was charged with intermediate 9 (50 mg, 0.29
mmol), chloride 16 (0.35 mmol), K2CO3 (83 mg, 0.6 mmol) and DMF (3 mL). The reaction mixture
was stirred at 35 for 5–8 h. Upon completion, the reaction solution was diluted with EtOAc (30 mL),
washed with water (20 mL × 2) and brine (15 mL). The organic layer was dried over Na2SO4,
concentrated, and purified on silica gel using petroleum ether/EtOAc (2:1) offered the corresponding
compound in good yield.
5.2.11.1. 3-((5-Phenylisoxazol-3-yl)methoxy)-2,6-difluorobenzamide. (B1): white solid, yield 84%; Mp:
183–184 ; 1H NMR (600 MHz, Chloroform-d) δ 7.77 – 7.81 (m, 2H), 7.51 – 7.44 (m, 3H), 7.16 (td, J
= 9.1, 5.1 Hz, 1H), 6.89 (td, J = 9.1, 2.0 Hz, 1H), 6.67 (s, 1H), 5.94 (d, 2H), 5.26 (s, 2H). HRMS (ESI)
calcd for C17H13F2N2O3 [M + H]+, 331.0889, found 331.0889.
5.2.11.2. 3-((5-(4-Chlorophenyl)isoxazol-3-yl)methoxy)-2,6-difluorobenzamide. (B2): white solid, yield
87%; Mp: 201–203 ; 1H NMR (600 MHz, DMSO-d6) δ 8.16 (s, 1H), 7.94 (d, J = 8.6 Hz, 2H), 7.88 (s,
1H), 7.62 (d, J = 8.6 Hz, 2H), 7.37 (td, J = 9.3, 5.1 Hz, 1H), 7.24 (s, 1H), 7.11 (td, J = 8.9, 1.7 Hz, 1H),
5.35 (s, 2H). 13C NMR (151 MHz, DMSO-d6) δ 169.07, 161.64, 161.35, 152.77, 148.55, 142.59,
135.75, 129.88, 127.97, 125.85, 117.39, 116.62, 111.41, 101.02, 63.24. HRMS (ESI) calcd for
C17H12ClF2N2O3 [M + H]+, 365.0499, found 365.0506.
Physical characteristics, 1H NMR, 13C NMR, MS and HRMS for other target compounds of series B,
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were reported in the supporting information.
5.2.12. Typical procedure for the synthesis of subordinate B14-2
To a suspension of NaH (80%, 0.15 g, 5 mmol) in dry THF (10 mL) was added acetoacetate
(4 mmol) under nitrogen, stirred for 30 min at room temperature, then cold to −10 °C. To this reaction
mixture was added n-BuLi (in hexane 1.5 M, 3.3 mL, 5 mmol) and stirred for further 30 min. Then
4-tert-butybenzaldehyde (0.5 g, 3 mmol) was added, and stirred for further 1–2 h at 0 °C. Upon
completion, saturated NH4Cl (30 mL) was added and the aqueous was extracted with ethyl acetate. The
combined extract was dried and evaporated in vacuum. The residue was purified on silica gel to afford
the secondary alcohol as light yellow oil.
The above alcohol intermediate 17 was dissolved in acetonitrile and added 2-Iodoxybenzoic acid
(0.84 g, 3 mmol) at 0 °C, then heated the reaction to reflux for 3 h. The resulting mixture was filtered
through diatomite and the filtrate was collected and concentrated. The residue was purified on silica gel
getting the intermediate 18 as yellow oil.
The cyclization of 18 to 19 using hydroxylamine hydrochloride was carried out as described above
1
for the synthesis of series B [37]. Intermediate 19, H NMR (600 MHz, Chloroform-d) δ 7.71 (d, J =
8.5 Hz, 2H), 7.48 (d, J = 8.5 Hz, 2H), 6.56 (s, 1H), 4.22 (q, J = 7.1 Hz, 2H), 3.77 (s, 2H), 1.35 (s, 9H),
1.30 (t, J = 7.1 Hz, 3H).
The reduction of ethyl ester 19 to alcohol using NaBH4 was carried out as described above for the
synthesis of series B. The according primary alcohol (100 mg, 0.4 mmol) was dissolved in DCM (8
mL), to this solution was added TEA (121 mg, 1.2 mmol), DMAP (catalytic amount) and TsCl (114 mg,
0.6 mmol). The mixture was stirred overnight at room temperature with N2 protection. The resulting
solution was diluted with DCM (20 mL), washed with saturated NaHCO3 (15 mL) and brine (15 mL).
The organic layer was dried over Na2SO4, concentrated in vacuo, purified by
silica gel column chromatography eluting with petroleum ether/EtOAc. The product 20 was isolated as
a white solid in 90% yield. 1H NMR (600 MHz, Chloroform-d) δ 7.75 (d, J = 8.4 Hz, 2H), 7.65 (d, J =
8.4 Hz, 2H), 7.48 (d, J = 8.5 Hz, 2H), 7.28 (d, J = 8.1 Hz, 2H), 6.30 (s, 1H), 4.35 (t, J = 6.6 Hz, 2H),
3.08 (t, J = 6.5 Hz, 2H), 2.36 (s, 3H), 1.35 (s, 9H). MS calcd for C22H26NO4S [M + H]+, 400.5, found
400.5.
To a round-bottom flask equipped with a magnetic stirrer was added intermediate 9 (45 mg, 0.26
mmol), 20 (100 mg, 0.25 mmol), K2CO3 (70 mg, 0.5 mmol) and DMF (2 mL). The suspension was
stirred overnight at room temperature, and then diluted with EtOAc (25 ML) washed with water (10
mL × 2) and brine (10 mL). The organic layer was dried, concentrated and purified on silica gel using
DCM/MeOH (100:1) to afford the desired product B14-2.
5.2.12.1. 3-(2-(5-(4-(Tert-butyl)phenyl)isoxazol-3-yl)ethoxy)-2,6-difluorobenzamide. (B14-2): white
solid, yield 20%; Mp: 157–158 ; 1H NMR (600 MHz, DMSO-d6) δ 8.10 (s, 1H), 7.83 (s, 1H), 7.75 (d,
J = 8.5 Hz, 2H), 7.55 (d, J = 8.5 Hz, 2H), 7.28 (td, J = 9.3, 5.2 Hz, 1H), 7.08 (td, J = 9.0, 1.8 Hz, 1H),
6.96 (s, 1H), 4.40 (t, J = 6.4 Hz, 2H), 3.15 (t, J = 6.4 Hz, 2H), 1.31 (s, 9H). 13C NMR (151 MHz,
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DMSO-d6) δ 169.35, 162.04, 161.80, 153.60, 152.40, 148.35, 143.21, 126.56, 125.78, 124.70, 117.08,
116.09, 111.52, 100.41, 67.82, 35.12, 31.34, 26.31. MS calcd for C22H23F2N2O3 [M + H]+, 401.4, found
401.4.
5.2.13. Typical procedure for the synthesis of subordinate compounds B2-Br, B14-Br and B14-Cl
A round-bottom flask equipped with a magnetic stirrer was charged with B2 or B14 (20 mg) and
acetic acid (2 mL), to this mixture was added NBS or NCS (1.1 eq), and heated to reflux for 2 h. upon
completion, the reaction mixture was diluted with EtOAc (20 mL), and washed with water (10 mL × 2),
saturated NaHCO3 (10 mL) and brine (10 mL). The organic layer was dried over Na2SO4, concentrated
and purified on silica gel with DCM/MeOH (100:1), afforded the desired compounds in good yield.
5.2.13.1. 3-((5-(4-Chlorophenyl)-4-bromoisoxazol-3-yl)methoxy)-2,6-difluorobenzamide. (B2-Br):
1
white solid, yield 75%; Mp: 179–181 ; H NMR (600 MHz, DMSO-d6) δ 8.15 (s, 1H), 8.03 (d, J =
8.7 Hz, 2H), 7.87 (s, 1H), 7.71 (d, J = 8.6 Hz, 2H), 7.43 (td, J = 9.3, 5.2 Hz, 1H), 7.13 (td, J = 9.0, 1.8
Hz, 1H), 5.35 (s, 2H). HRMS (ESI) calcd for C17H11BrClF2N2O3 [M + H]+, 442.9604, found 442.9597.
5.2.13.2. 3-((5-(4-(Tert-butyl)phenyl)-4-bromoisoxazol-3-yl)methoxy)-2,6-difluorobenzamide. (B14-Br):
1
white solid, yield 77%; Mp: 122–123 ; H NMR (600 MHz, DMSO-d6) δ 8.15 (s, 1H), 7.94 (d, J =
8.5 Hz, 2H), 7.89 – 7.84 (m, 1H), 7.65 (d, J = 8.6 Hz, 2H), 7.43 (td, J = 9.3, 5.2 Hz, 1H), 7.13 (td, J =
9.0, 1.7 Hz, 1H), 5.33 (s, 2H), 1.33 (s, 9H). MS calcd for C21H23BrF2N2O3 [M + NH4]+, 482.1, found
482.3.
5.2.13.3. 3-((5-(4-(tert-butyl)phenyl)-4-chloroisoxazol-3-yl)methoxy)-2,6-difluorobenzamide. (B14-Cl):
1
white solid, yield 73%; Mp: 99–100 ; H NMR (600 MHz, Methanol-d4) δ 8.15 (s, 1H), 7.92 (d, J =
8.6 Hz, 2H), 7.87 (s, 1H), 7.65 (d, J = 8.6 Hz, 2H), 7.43 (td, J = 9.2, 5.1 Hz, 1H), 7.14 (td, J = 8.9, 1.7
Hz, 1H), 5.37 (s, 2H), 1.33 (s, 9H). HRMS (ESI) calcd for C21H19ClF2N2NaO3 [M + Na]+, 443.0944,
found 443.0944.
5.2.14. Typical procedure for the synthesis of subordinate compound B14-Me
The intermediate 14-B14 (150 mg, 0.54 mmol) prepared in series B, NaH (80%, 18 mg, 0.6 mmol)
and DMF (2 mL) was added to a round-bottom flask under nitrogen protection, this resulting
suspension was stirred at room temperature for 30 min. Then iodomethane was added to this mixture
and stirred overnight. The mixture was diluted with EtOAc (25 mL) and washed with water (10 mL ×
2), brine (10 mL). The organic layer was dried, concentrated and purified on silica gel getting the
methylated intermediate 21. Yield 45%.
The cyclization of 21 and followed reduction to alcohol were carried out as described above for the
synthesis of series B. The according primary alcohol (60 mg, 0.24 mmol) was then dissolved in DCM
(5 mL) at 0 , to this solution was added CBr4 (122 mg, 0.37 mmol) and PPh3 (188 mg, 0.72 mmol)
successively. After 10 min, the mixture was warmed to room temperature and stirred for 5 h. The
resulting solution was concentrated in vacuo, purified by silica gel column chromatography eluting
1
with petroleum ether/EtOAc (50:1), afforded the bromide intermediate 22, yield 87%. H NMR (600
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MHz, Chloroform-d) δ 7.65 (d, J = 8.5 Hz, 2H), 7.51 (d, J = 8.5 Hz, 2H), 4.46 (s, 2H), 2.27 (s, 3H),
1.36 (s, 9H). MS calcd for C15H19BrNO [M + H]+, 308.1, found 308.1.
To a round-bottom flask equipped with a magnetic stirrer was added intermediate 9 (40 mg, 0.23
mmol), 22 (65 mg, 0.21 mmol), K2CO3 (70 mg, 0.5 mmol) and DMF (2 mL). The suspension was
stirred overnight at room temperature, and then diluted with EtOAc (25 ML) washed with water (10
mL × 2) and brine (10 mL). The organic layer was dried, concentrated and purified on silica gel using
DCM/MeOH (100:1) to afford the desired product B14-Me.
5.2.14.1.
3-((5-(4-(Tert-butyl)phenyl)-4-methylisoxazol-3-yl)methoxy)-2,6-difluorobenzamide.
1
(B14-Me): white solid, yield 92%; Mp: 152–154 ; H NMR (600 MHz, DMSO-d6) δ 8.15 (s, 1H),
7.87 (s, 1H), 7.70 (d, J = 8.5 Hz, 2H), 7.60 (d, J = 8.5 Hz, 2H), 7.43 (td, J = 9.3, 5.1 Hz, 1H), 7.14 (td,
J = 9.0, 1.7 Hz, 1H), 5.34 (s, 2H), 2.25 (s, 3H), 1.33 (s, 9H). MS calcd for C22H23F2N2O3 [M + H]+,
401.4, found 401.4.
5.3. In vitro antibacterial assay
The minimum inhibitory concentration (MIC) of the synthesized compounds was determined in a
96-well microplate using the broth microdilution method recommended in the Clinical and Laboratory
Standards Institute (CLSI) guidelines [22]. Log-phase bacteria were added to 96-well microtiter plates
containing 2-fold serial dilutions of compound or comparator drug in cation-adjusted Mueller-Hinton
(CAMH) broth at concentrations ranging from 64 to 0.125 µg/mL. The final volume in each well was
0.1 mL, and the microtiter plates were incubated aerobically for 24 h at 37 °C. Turbidity was observed,
the last well with no growth of bacterial was recorded to represent the MIC value.
5.4. Minimum bactericidal concentration (MBC) assay
MBC assay was conducted using the broth microdilution assay described in the preceding section.
After the 24 h incubation period, aliquots from the microtiter wells were plated onto tryptic soy agar
(TSA). The colonies that grew after 24 h of incubation were counted, with MBC being defined as the
lowest compound concentration resulting in a ≥3-log reduction in the number of colony forming units
(CFU).
5.5. Time-kill curve assay
A growing culture of S. aureus ATCC 43300 was diluted to approximately 106 CFU/mL in volumes
of CAMH broth, containing various concentrations of B14 or comparator drug. Cultures were
incubated at 37 with shaking. At appropriate time intervals, 100 µL samples were removed for serial
dilution in 900 µL volumes of CAMH broth and 100 µL volumes from three dilutions were spread on
to TSA. All TSA plates were incubated at 37 °C, and the CFU/mL at each time point was determined
by counting colonies after 24 h.
5.6. Visualization of bacterial morphology
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The B. pumilus strain CMCC63202 cells were grown in LB medium. The cultures at an A600 of 0.01
from an overnight culture were inoculated in the same medium containing absence or 0.5 × MIC
concentration of the test compound and grown at 37 for 18 h. The cells for morphology studies were
harvested and resuspended in 0.5 mL of PBS buffer. 10 µL of the suspension mixture were then placed
on a microscopic slide pretreated with 0.1% (w/v) poly-L-lysine and the morphology of the bacterial
cells was observed under a phase-contrast optical microscope at 40 magnification. The images were
captured using an Olympus CKX41 microscope.
5.7. FtsZ polymerization assay
B. subtilis FtsZ was cloned, overexpressed, and purified as described previously [23]. The
polymerization of BsFtsZ protein was monitored using a microtiter plate-based light-scattering assay in
which changes in light scattering are reflected by corresponding changes in absorbance at 340 nm (A340
[27]. Test compound or comparator drug (at concentrations ranging from 0 to 10 µg/mL) were
combined with 1 mM GTP and 5 µM FtsZ in 100 µL of reaction solution, which contained 50 mM
MES buffer , 50 mM KCl, 2 mM magnesium acetate. Reactions were assembled in half-volume,
flat-bottom microtiter plates, and polymerization was continuously monitored at 25 °C by measuring
)
A
340 in a Thermo Multiskan FC plate reader over a time period of 30 min.
5.8. Transmission electron microscopy (TME)
At room temperature, BsFtsZ was incubated in the absence and in the presence of the test compound
in polymerization reaction solution described above. After 20 min, 25-µL drops of the resulting
dilutions were placed on glow-discharged, copper, 400 mesh, Formvar/carbon-coated grids. Excess
solution was wicked away with filter paper. The grids were negatively stained with a solution of 1%
phosphotungstic acid for 1 min and blotted dry. The grids were then digitally imaged at 100 kV on a
JEM-1011 transmission microscope interfaced with an Olympus camera.
5.9. Computational study
Docking simulations were performed using Autodock (version 4.2) docking package [38]. The
crystal structure of FtsZ (PDB ID: 3vob) was downloaded from the Protein Data Bank (PDB). The
ligand molecule was built using the Chembio3D software. The refined ligands and protein receptor
model were converted to pdbqt format using Autodock Tools. Automated docking studies were carried
out using Autodock as implemented through the graphical interface MGLTools protocol. The docking
results were visually inspected and analyzed using Pymol (version 1.5).
5.10. Efficacy modles of infection
Antistaphylococcal efficacy in vivo was assessed in a mouse blood model of infection with S. aureus
ATCC43300 (MRSA). All experimental procedures conformed to the animal experiment guidelines of
the Animal Care and Welfare Committee of Shandong University. Groups of KM mice with an average
weight of 20 g were infected intravenously with an inoculum of MRSA (0.1 mL of saline containing
5.4 × 107 CFU). Compounds (or the vehicle control) were administered i.p. at the doses and times
23

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indicated. The vehicle contained 1% DMAC, 30% TEG, and 7% HPβCD in water (final). After 24 h,
the animals were anaesthetized, and 0.2 mL blood was drawn from heart and then were spread on to
TSA plates for CFU enumeration. The numbers of CFU were determined form each plate following
overnight incubation.
5.11. MTT cytotoxicity assay
The cytotoxicity of compounds was assessed in human cervical cancer (Hela) cells using a 48 h
continuous 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay as described
previously [39]. Cells were plated at 104 cell/well in a 96-well plate for overnight incubation, and then,
100 µl of each compound were added. After 36-h incubation, 20 µl of MTT solution (5 mg/mL) was
added and incubated for 4 h at 37 . The MTT solution was then removed, substituted with 150 µl
DMSO for 10 min, and the absorbance was measured at 570 nm.
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), Major Project of Research and Development of Shandong Province (2016GSF201202),
Key Research and Development Project of Shandong Province (2017CXGC1401), Major Project of
Science and Technology of Shandong Province (2015ZDJS04001).
Appendix A. Supplementary data
Supplementary data related to this article can be found at
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Research Highlights
˃ Two novel series of isoxazole-containing benzamide derivatives were designed and synthesized. ˃
They were evaluated for their biological activities as FtsZ-targeting antibacterial agents. ˃
Computational docking program was used to analyze for the structure-based rational optimization. ˃
This optimization strategy indeed led to a potent compound B14 with better activity. ˃ In-depth study
on the mechanism confirmed this compound targeted FtsZ protein.