Discovery of 1,3,4-oxadiazol-2-one-containing benzamide derivatives targeting FtsZ as highly potent agents of killing a variety of MDR bacteria strains

Article abstract of DOI:10.1016/j.bmc.2019.06.010

The spread of infections caused by multidrug-resistant (MDR) pathogens, such as methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant S. aureus (VRSA), has created a need for new antibiotics with novel mechanisms of action. The bacterial division protein FtsZ has been identified as a novel drug target that can be exploited clinically. As part of an ongoing effort to develop FtsZ-targeting antibacterial agents, we describe herein the design, synthesis and bioactivity of six series of novel 1,3,4-oxadiazol-2-one-containing, 1,2,4-triazol-3-one-containing and pyrazolin-5-one-containing benzamide derivatives. Among them, compound A14 was found to be the most potent antibacterial agent, much better than clinical drugs such as ciprofloxacin, linezolid and erythromycin against all the tested gram-positive strains, particularly methicillin-resistant, penicillin-resistant and clinical isolated S. aureus. Subsequent studies on biological activities and docking analyses proved that A14 functioned as an effective compound targeting FtsZ. Preliminary SAR indicated a general direction for further optimization of these novel analogues. Taken together, this research provides a promising chemotype for developing newer FtsZ-targeting bactericidal agents.

Full text of DOI:10.1016/j.bmc.2019.06.010

Accepted Manuscript
Discovery of 1,3,4-oxadiazol-2-one-containing benzamide derivatives targeting
FtsZ as highly potent agents of killing a variety of MDR bacteria strains
Fangchao Bi, Di Song, Yinhui Qin, Xingbang Liu, Yuetai Teng, Na Zhang,
Panpan Zhang, Nan Zhang, Shutao Ma
PII:
S0968-0896(19)30381-5
https://doi.org/10.1016/j.bmc.2019.06.010
BMC 14947
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
6 March 2019
29 May 2019
5 June 2019
Please cite this article as: Bi, F., Song, D., Qin, Y., Liu, X., Teng, Y., Zhang, N., Zhang, P., Zhang, N., Ma, S.,
Discovery of 1,3,4-oxadiazol-2-one-containing benzamide derivatives targeting FtsZ as highly potent agents of
killing a variety of MDR bacteria strains, Bioorganic & Medicinal Chemistry (2019), doi: https://doi.org/10.1016/
j.bmc.2019.06.010
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Discovery of 1,3,4-oxadiazol-2-one-containing benzamide derivatives targeting FtsZ as
highly potent agents of killing a variety of MDR bacteria strains
Fangchao Bi a, Di Song a, Yinhui Qin a, Xingbang Liu a, Yuetai Teng a, Na Zhang a, Panpan Zhang a, Nan
Zhang a, Shutao Ma a, *
a
Department of Medicinal Chemistry, Key Laboratory of Chemical Biology (Ministry of Education),
School of Pharmaceutical Sciences, Shandong University, 44 West Culture Road, Jinan, 250012, China
* Corresponding author
E-mail address: mashutao@sdu.edu.cn
1

Abstract
The spread of infections caused by multidrug-resistant (MDR) pathogens, such as methicillin-resistant
Staphylococcus aureus (MRSA) and vancomycin-resistant S. aureus (VRSA), has created a need for new
antibiotics with novel mechanisms of action. The bacterial division protein FtsZ has been identified as a
novel drug target that can be exploited clinically. As part of an ongoing effort to develop FtsZ-targeting
antibacterial agents, we describe herein the design, synthesis and bioactivity of six series of novel 1,3,4-
oxadiazol-2-one-containing, 1,2,4-triazol-3-one-containing and pyrazolin-5-one-containing benzamide
derivatives. Among them, compound A14 was found to be the most potent antibacterial agent, much
better than clinical drugs such as ciprofloxacin, linezolid and erythromycin against all the tested gram-
positive strains, particularly methicillin-resistant, penicillin-resistant and clinical isolated S. aureus.
Subsequent studies on biological activities and docking analyses proved that A14 functioned as an
effective compound targeting FtsZ. Preliminary SAR indicated a general direction for further
optimization of these novel analogues. Taken together, this research provides a promising chemotype for
developing newer FtsZ-targeting bactericidal agents.
Keywords: 1,3,4-Oxadiazol-2-one-containing benzamides; FtsZ-targeting; Multidrug-resistant bacteria;
Antibacterial activity
2

1. Introduction
The emergence of bacterial drug resistance is rapidly becoming a serious threat to global health, which
has created a critical need for a proactive effort to identify novel therapeutic antibiotics with unique
modes of action [1, 2]. Because efficacious modification of existing antibiotics is becoming increasingly
difficult, targeting new, unexplored essential proteins in bacteria is significant for the discovery of new
class, non-cross resistant antibacterial agents [3-5]. Filamentous temperature-sensitive protein Z (FtsZ)
is a key protein involved in bacterial cell division, which is highly conserved in bacterial pathogens [6-
8]. The essential role in bacterial cell division makes FtsZ a very promising therapeutic target. On the
basis of the FtsZ target, a number of small molecule inhibitors of FtsZ have been developed by some
research groups, such as 3-methoxybenzamide (3-MBA) derivatives [9-11], GTP analogs [12],
guanidinomethyl biaryl derivatives [13], cinnamaldehyde derivatives [14, 15], and pyridinium analogs
[16, 17], etc (Fig.1).
O
NH2
F
O
NH2
F
O
NH2
F
OH
F
N
N
CF3
O
O
O
O
S
Cl
3-MBA
3-MBA derivetive
compound 1
Br
3-MBA derivetive
PC190723
N
CF3
Cl
N
N
N
Cl
O
N
NH2
H
N
O
O
H
H2N
N
S
N
N
N
Cl
H
NH
O
N
pyridinium analogue
cinnamaldehyde derivative
guanidinomethyl biaryl
GTP analogue
Fig. 1. Chemical structures of some FtsZ inhibitors.
Among those compounds, 3-MBA is one of the most attractive starting point for discovering FtsZ-
targeting antibacterial agents. The successive modifications for 3-MBA by 2,6-difluorination, elongation
of alkoxy side chain up to nonyloxy group, and replacement of this 3-alkyloxy side chain with
heteroaryoxy or heteroarymethoxy group finally lead to the discovery of the benzamide derivative
PC190723 with a potent antibacterial activity (MIC, 1 µg/mL against S. aureus) [18, 19]. Further
optimizations for PC190723 has been conducted by replacement of the thiazolopyridine group with a
substituted phenyl-oxazole fragment and introduction of hydroxymethyl substitution at the methylene
linker, as exemplified by compound 1 (Fig. 1) [20], or by improvement of its poor drug-like properties
for clinical utility [11, 21, 22]. The strcuture-activity relationship (SAR) studies indicate that the amide
group is critical for the on-target inhibitory activity and few modifications of the amide are tolerated [23,
24], while the 3-alkyloxy of benzamide group has quite big scope for further optimization, by introducing
long hydrophobic alkyloxy side chain or proper aromatic heterocycle substituent to adapt the
hydrophobic pocket of the binding site.
3

O
NH2
F
Series E
F
N
Br
O
N
O
N
R
change the lactone
to lactam
O
NH2
F
Series B
R
F
O
NH2
F
O
NH2
F
Series F
N
N
O
F
F
introduce a
methylene
O
N
N
and
R
O
O
O
N
N
O
R
Series A
O
change the lactone
to ketone
O
Series C
O
NH2
O
F
F
O
N
change the relative position
of the substitutes
N
O
R
O
NH2
Series D
F
F
N
O
N
R
O
O
Fig. 2. Brief design strategy of 1,3,4-oxadiazol-2-one-containing benzamide derivatives
Based on the above findings, a series of new modifications were considered for the discovery of novel
FtsZ-targting antibacterial agents. In this research, we originally linked the 3-methoxy of 2,6-difluoro-
benzamide and the substituted benzene ring or alky residue using an1,3,4-oxadiazol-2-one moiety to
obtain 1,3,4-oxadiazol-2-one-containing benzamide derivatives (series A), and then introduced a
methylene into the side chain to get five subordinate analogues (series B and C). On this basis, we further
exchanged the relative position of the substitutes on the 1,3,4-oxadiazol-2-one fragment to give the
derivatives of series D, and subsequently changed the lactone to the lactam or ketone to get 1,2,4-triazol-
3-one-containing or pyrazolin-5-one-containing analogues (series E and F) (Fig. 2). The above six series
of target compounds were synthesized and evaluated for their bioactivity.
2. Chemistry
The synthetic route of series A and B is shown in Scheme 1. Commercially available 2,4-
difluorophenol 2 was protected by benzyl group, and then treated with dry ice in the presence of n-BuLi,
giving benzoic acid 3. The benzoic acid was converted to benzamide 4 using the treatment of oxalyl
chloride, and then ammonium carbonate. Deprotection of the benzyl group from 4 produced intermediate
5. Alkylation of 5 with ethyl bromoacetate, followed by ester hydrolysis afforded the acid product 7.
Various substituted hydrazine hydrochloride 10 was prepared from commercially available phenylamine
8 or benzyl halide 9. Amidation followed by cyclization in the presence of triphosgene afforded 1,3,4-
oxadiazol-2-one benzamides A1-21 and B1-3.
4

O
OH
NH2
F
NH2
F
O
O
O
NH2
F
F
F
i, ii
iii, iv
v
vi
F
F
F
F
F
OH
O
OBn
OBn
OH
O
2
4
O
3
5
6
vii
O
NH2
F
F
O
NH2
O
NH2
F
OH
O
F
F
F
xi
O
7
x
H
N
O
N
R
R
N
O
O
N
O
A1-21 and B1-3
H
HCl
O
11
NH2
H
N
viii or ix
Cl (Br)
R2
R1
or
R
NH2
10
R =
9
8
R'
R'
Series A
Series B
Cl
O
O
A12
A11
A5
A8
A10
A9
A6
A7
A4
A1
A2
A3
F
Cl
Cl
Cl
Br
CN
CF3
Cl
Cl
Br
Cl
Cl
A16
A21
B1
B2
A13
A14
A15
A17
A19
A20
B3
A18
Scheme 1. Synthetic route of series A and B. Reagents and conditions: (i) benzyl chloride, K2CO3, NaI, MeCN,
45℃; (ii) n-BuLi, dry ice, THF, -50℃ to r.t.; (iii) oxalyl chloride, DMF, DCM, r.t.; (iv) ammonium carbonate, DCM
r.t.; (v) 10% Pd/C, ammonium formate, EtOH, 65℃; (vi) ethyl bromoacetate, K2CO3, DMF, 35℃; (vii) NaOH
(aq)/MeOH, 60℃; (viii) NaNO2, SnCl2, HCl (aq), -5℃ to r.t.; (ix) hydrazine hydrate, EtOH, concentrated HCl; (x)
EDCI, HOBt, DIPEA, THF/DMF; (xi) triphosgene, THF.
The synthetic route of series C is outlined in Scheme 2. The intermediate 5 was alkylated with 3-
bromo-1-propanol, which was followed by oxidation to carboxylic acid 13. Amidation with two
substituted phenylhydrazines and then cyclization in the presence of triphosgene afforded compounds
C1 and C2.
O
NH2
F
O
NH2
F
O
NH2
F
ii
i
F
F
F
O
O
NH2
F
O
NH2
F
OH
O
OH
O
OH
O
N
13
12
5
F
F
iii
iv
O
O
H
N
R
O
N
N
O
H
HCl
H
R
Series C
14
N
NH2
C1 R = Br
C2 R = t-butyl
R
10
Scheme 2. Synthetic route of series C. Reagents and conditions: (i) 3-bromo-1-propanol, K2CO3, DMF, 35℃; (ii)
Jone‘s reagent, acetone; (iii) EDCI, HOBt, DIPEA, THF/DMF; (iv) THF, triphosgene.
5

The synthetic route of series D is outlined in Scheme 3. Corresponding substituted benzoic acid 15
was converted to benzoyl hydrazine 17 through esterification and hydrazinolysis, then condensation of
17 with triphosgene yielded intermediate 1,3,4-oxadiazol-2(3H)-one 18. Hydroxy-methylation of 18 with
formaldehyde followed by bromination afforded compound 20, which was coupled with 5 to form
compounds D1-3.
O
N
O
O
O
O
OH
O
O
ii
i
iii
NH2
NH
iv
OH
O
N
N
O
N
H
R
R
R
19
R
R
17
16
18
15
v
O
N
Br
N
O
NH2
F
20
R
F
D1 R = methyl
D2 R = t-butyl
D3 R = Cl
vi
O
NH2
F
N
R
O
N
F
O
O
Series D
OH
5
Scheme 3. Synthetic route of series D. Reagents and conditions: (i) MeOH, SOCl2, 0℃ to 45℃; (ii) hydrazine
hydrate, MeOH, 45℃; (iii) triphosgene, DIPEA, DCM; (iv) formaldehyde, TEA, DMF; (v) CBr4, PPh3, DCM; (vi)
K2CO3, DMF, 35℃.
The synthetic route of series E is outlined in Scheme 4. The compound A14 was treated with
ammonium hydroxide or hydrazine hydrate to get compounds E1 and E2. E2 was then dimethylated to
afford compound E3. 4-Bromoaniline was reacted with ethyl 2-chloroacetoacetate to provide the
intermediate 23. Amination of 23 using ammonium hydroxide and subsequently cyclization using
triphosgene to give 1,2,4-triazol-3-one intermediate 25. Alkylation of 25 with corresponding alkyl halide
produced 26, which then was subjected to reduction and bromination to generate intermediate 28.
Reaction of 28 with 5 to get the desired compounds E4-11.
6

O
NH2
F
O
NH2
F
O
NH2
F
O
NH2
F
F
F
F
F
iii
N
i or ii
Br
N
N
O
Br
N
or
Br
O
Br
N
O
O
O
N
O
N
O
N
O
O
HN
N
N
A14
E1
H2N
N
E2
E3
O
NH2
O
NH
Cl
O
H
H
O
O
N
O
NH2
iv
vi
N
O
v
N
N
N
N
O
+
O
O
Br
Br
Br
Br
25
vii
Cl
24
23
21
22
R
N
R
N
R
N
O
O
O
O
ix
viii
N
N
N
N
N
O
NH2
F
N
OH
O
Br
E4 R = methyl
E5 R = ethyl
Br
Br
F
26
Br
F
27
28
E6 R = propyl
E7 R = butyl
E8 R = isobutyl
E9 R = isoamyl
E10 R = octyl
E11 R = benzyl
x
N
Br
O
N
O
O
NH2
F
N
R
E4-11
OH
5
Scheme 4. Synthetic route of series E. Reagents and conditions: (i) ammonium hydroxide, EtOH, sealed tube, 78℃;
(ii) hydrazine hydrate, EtOH, reflux; (iii) K2CO3, CH3I, DMF, sealed tube, 80℃; (iv) EtOH/H2O, concentrated HCl,
NaNO2, AcONa; -5℃; (v) ammonium hydroxide, 1,4-dioxane; (vi) triphosgene, DCM; (vii) K2CO3, corresponding
alkyl halides, DMF; (viii) NaBH4, THF/MeOH; (ix) CBr4, PPh3, DCM; (x) K2CO3, DMF, 35℃.
The synthetic route of series F is outlined in Scheme 5. The intermediate 5 was alkylated with ethyl
4-chloroacetoacetate to get 29, which was followed by cyclization with substituted phenylhydrazines to
produce the desired compounds F1-4.
O
NH2
F
O
NH2
F
F
i
O
F
Cl
+
F1 R = Cl
F2 R = Br
F3 R = t-butyl
F4 R = n-butyl
O
O
O
OH
O
O
NH2
F
5
O
O
29
ii
F
N
HCl
R
O
H
N
O
N
NH2
Series F
R
10
Scheme 5. Synthetic route of series F. Reagents and conditions: (i) KOH, DMSO, r.t.; (ii) EtOH, 75℃.
3. Results and discussion
3.1. In vitro antibacterial activity
The synthesized compounds showed in Scheme 1-4 were tested for their in vitro antibacterial activity
through broth microdilution procedures described in the Clinical and Laboratory Standards Institute
(CLSI) guidelines [25]. MIC values (minimal inhibitory concentration) for all the compounds were
7

determined on a panel of sensitive and resistant bacterial strains, PC (PC190723), CIP (ciprofloxacin),
LIN (linezolid) and ERY (erythromycin) were included as comparison.
The MIC results listed in Table 1 showed that some of the 1,3,4-oxadiazol-2-one-containing
benzamide derivatives displayed superior or comparable efficacy, compared with the clinical antibiotics
and PC evaluated in this study. Among series A, A12 and A14 were the most effective compounds. For
example, A12 and A14 displayed arrestive activity against susceptible and resistant S. aureus strains with
MIC range of 0.5–2 μg/mL, which was much better than CIP (2, 16, >64, 32 μg/mL) and ERY
(4, >64, >64, >64 μg/mL). Especially, A14 possessing MIC values of 0.5–1 μg/mL against one
susceptible and three resistant S. aureus strains was comparable to PC, and superior to CIP, LIN and
ERY. Note that there was no significant difference among the MIC values of these outstanding
compounds against susceptible and resistant S. aureus strains, which underlined the practicability of FtsZ
as a promising antibacterial target in developing new vigorous agents against a variety of MDR bacteria
strains.
Based on the preliminary results of series A, increasing the carbon number of the linkage (series B and
C) or exchanging the substitute’s relative position in the 1,3,4-oxadiazol-2-one ring (series D), all
decreased their antibacterial activity, especially for three S. aureus strains. Comparison of A12 and D3
indicated that the configuration of 1,3,4-oxadiazol-2-one ring was crucial for the antibacterial activity.
Besides, the position of the methylene in the side chain had considerable influence on the antibacterial
activity. In series B, for instance, the introduced-methylene definitely increased the molecular length (B2)
that could not be accommodated by the binding pocket of FtsZ, while in series C, the molecular length
(C1) could be partly compressed by the flexibility of three-atom chain.
Table 1
Series A, B, C, D analogues with their in vitro antibacterial activity.
Minimum inhibitory concentration/MIC (μg/mL)
B. subtilis
B. pumilus
S. aureus
S. aureus
S. aureus S. aureus
E. coli
P. aeruginosa
Comp
ATCC937
CMCC63202
ATCC25923
ATCC43300a
PRb
CIc
ATCC25922
ATCC27853
2
4
A1
A2
64
8
16
4
16
4
32
4
16
4
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
0.5
16
A3
64
16
4
64
>64
2
64
64
2
>64
>64
4
64
>64
2
A4
16
A5
0.5
1
A6
2
16
8
16
8
32
16
>64
64
4
16
16
2
A7
1
2
A8
0.015
2
0.5
2
>64
32
2
64
16
4
A9
32
4
A10
A11
A12
A13
A14
A15
A16
1
8
16
>64
2
64
1
>64
2
>64
2
64
2
0.25
8
>64
1
>64
1
>64
1
>64
1
>64
0.5
2
0.125
2
8
16
4
8
16
4
0.125
2
2
4
8

A17
A18
A19
A20
A21
B1
2
4
4
4
4
>64
4
4
64
2
8
>64
4
16
64
4
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
≤0.125
>64
16
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
≤0.125
>64
32
1
0.5
0.5
32
0.5
4
2
4
4
>64
32
>64
>64
>64
>64
4
>64
64
64
>64
2
>64
>64
>64
>64
4
16
>64
64
>64
4
8
B2
8
32
B3
32
32
C1
1
≤0.125
≤0.125
>64
32
C2
0.25
>64
8
4
1
4
2
D1
>64
>64
>64
1
>64
>64
>64
1
>64
>64
>64
1
>64
>64
>64
0.5
0.5
1
D2
D3
>64
0.5
≤0.125
0.5
≤0.125
>64
0.5
PC
CIPd
LINe
ERYf
0.25
2
0.25
2
0.25
2
32
2
≤0.125
≤0.125
>64
>64
>64
a S. aureus ATCC43300: methicillin-resistant strain.
b S. aureus PR: penicillin-resistant strain.
c S. aureus CI: clinical isolated strain, not characterized.
d CIP: ciprofloxacin; e LIN: linezolid; f ERY: erythromycin.
The MIC results of series E and F are shown in Table 2. This two series of compounds were designed
and synthesized on the consideration of increasing hydrolytic stability of the five membered lactone ring,
which was addressed by replacing the ester bond with amide bond or ketone. However, this replacement
resulted in a dramatic loss of antibacterial activity, which might result from the decrease of the molecular
lipophilicity (the calculated Log P of A14, E1 and F2 are 3.41, 2.74 and 2.69, respectively) or the increase
of the steric effect. This helps us recognize that the FtsZ binding site for the 3-O-substituted side chain
may prefer lipophilic groups to hydrophilic groups.
Table 2
Series E and F analogues with their in vitro antibacterial activity.
Minimum inhibitory concentration/MIC (μg/mL)
B. subtilis
B. pumilus
S. aureus
S. aureus
S. aureus
E. coli
P.
aeruginosa
ATCC27853
>64
Comp
ATCC9372
CMCC63202
ATCC25923
ATCC43300a
PRb
ATCC25922
E1
E2
E3
E4
E5
E6
E7
E8
>64
16
>64
64
>64
>64
32
>64
>64
16
>64
>64
32
>64
>64
>64
>64
>64
>64
>64
>64
>64
32
32
>64
64
1
>64
>64
>64
>64
>64
>64
>64
32
>64
>64
>64
>64
>64
>64
>64
32
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
9

E9
E10
E11
F1
>64
>64
>64
>64
>64
>64
>64
>64
>64
0.5
>64
>64
>64
>64
>64
>64
>64
1
>64
>64
>64
>64
>64
>64
>64
1
>64
>64
>64
>64
>64
64
>64
>64
>64
>64
>64
>64
>64
>64
≤0.125
>64
16
>64
>64
>64
>64
>64
>64
>64
>64
≤0.125
>64
32
>64
>64
F2
>64
F3
>64
F4
>64
64
PC
0.5
1
CIPc
LINd
ERYe
≤0.125
0.5
0.25
2
0.25
2
0.25
2
32
2
≤0.125
≤0.125
≤0.125
>64
>64
a S. aureus ATCC43300: methicillin-resistant strain.
b S. aureus PR: penicillin-resistant strain.
c CIP: ciprofloxacin; d LIN: linezolid; e ERY: erythromycin.
3.2. Bactericidal or bacteriostatic assay
The primary in vitro antibacterial activity of A14 highlighted by the results in Table 1 prompted us to
further investigate whether its activity is bactericidal or bacteriostatic. Therefore, the MBC values
(minimal bactericidal concentration) of A14 against susceptible (ATCC25923), methicillin-resistant
(ATCC43300) and penicillin-resistant S. aureus stains were tested and compared with its corresponding
MIC values. PC190723 and clinical drugs linezolid and vancomycin were included as comparator
controls in determinations. The results listed in Table 3 indicated that the MBC/MIC ratios of linezolid
was ≥32 which was observed as bacteriostatic activity. Significantly, A14 exhibited equivalent MBC
values with MIC (MBC/MIC ratio is 1) for all the tested S. aureus which was the same as PC and
vancomycin, indicating a bactericidal mode clearly.
Table 3
Comparison of MIC and MBC Values for A14 in three S. aureus.
Comp
MBC (μg/mL)
MIC (μg/mL)
MBC/MIC
susceptible S. aureus (ATCC25923)
A14
1
1
1
1
2
2
1
1
PC190723
vancomycin
linezolid
2
1
>64
>32
methicillin-resistant S. aureus (ATCC43300)
A14
1
1
1
1
1
2
1
1
PC190723
vancomycin
linezolid
1
1
>64
>32
penicillin-resistant S. aureus
A14
1
1
1
1
1
1
1
1
1
PC190723
vancomycin
10

linezolid
>64
2
>32
a PC190723, vancomycin and linezolid are included as control agents.
3.3. Kinetics of the bactericidal activity
With the bactericidal nature of A14, we next investigated the time-kill curves on methicillin-resistant
S. aureus ATCC43300. As shown in Fig. 3, the growth trend of bacteria was suppressed obviously when
incubated with (1, 2, 4 and 8) × MIC of A14 compared with none drug treated curve. At those
concentrations, A14 decreased the bacteria counts by four logs within 24 h, which were similar with
PC190723 and clinical drug vancomycin at 2 μg/mL. The growth inhibition at different concentration
gradient of A14 reveal that, the kill rate for S. aureus is dependent on the exposure time, with ≥MIC
concentrations resulting in similar rates of kill at equal time point. Note that the kill rate under 4 μg/mL
of linezolid also decreased the bacteria counts by about one log within 24 h.
Fig. 3. Time-kill curves for S. aureus ATCC43300 (MRSA). Each data point reflects the average of two
independent measurements.
3.4. Effects on the morphology of B. pumilus cells
We have known that FtsZ-target compounds such as PC190723 could inhibit cell division and lead to
bacterial cell elongation or enlargement [26]. So we next explored the effect of A14 on the bacterial cell
morphology through microscopic observation, in order to confirm whether its mode of action was
associated with alteration of FtsZ. As shown in Fig. 4, compared with untreated bacterial cells, A14
increased the cell length of B. pumilus significantly at 0.5 × MIC concentration, which are consistent
with other reported 3-substituted benzamide derivatives [9, 27]. This result indicates that A14 interferes
with normal function of FtsZ, then leads to improper cell division and causes bacterial cell death.
11

Fig. 4. Effect of compound A14 on the cell morphology (400×). B. pumilus cells were observed in the absence (A)
or in the presence of 0.06 μg/mL A14 (B).
3.5. Stimulation of FtsZ polymerization dynamics
Deep studies on the dynamic of FtsZ protein have shown that some FtsZ-targeting agents could
stimulate FtsZ polymerization dynamics and stabilize FtsZ polymeric structures which contribute to the
antibacterial activity of FtsZ inhibitors [28, 29]. Therefore, the polymerization assays of B. subtilis FtsZ
(BsFtsZ) under different concentrations of A14 were performed utilizing a microplate reader at 340 nm
(A340) [13]. The time-dependent A340 profiles illustrated in Fig. 5A indicated that A14 increases the
kinetics of BsFtsZ polymerization, with magnitude of stimulatory effect in a concentration-dependent
manner. As expected, both linezolid and DMSO vehicle had a negligible effect on the polymerization of
BsFtsZ. In addition, D3, E1, E5 and F2 at 5 μg/mL was find to display negligible stimulatory effect
compared with A14 at lower concentrations, as shown in Fig. 5B. This different stimulatory impact on
FtsZ polymerization indicates that they have no interaction with the FtsZ target, which tallies with the
result of their weak antibacterial activity.
Fig. 5. Impact of A14 on the polymerization of BsFtsZ in a concentration-dependent manner. Polymerization profiles
were acquired in the presence of DMSO vehicle or the indicated concentrations of compounds. Linezolid was
included as a negative (non-FtsZ-targeting) control.
3.6. Compound’s stability studies in mouse plasma
As a consideration in the potential biological stability issue of A14, we designed and synthesized series
E and F through changing the lactone to the lactam or ketone. However, this series of compounds lost
12

their FtsZ-targeting and antibacterial activities. For all that, we also should validate our original intention.
Thus, we next sought to evaluate the time-dependent stability of A14 and E1 in the mouse plasma at 37℃
in vitro. Fig. 6 shows HPLC chromatograms of A14 and E1 at 25 µg/mL in freshly prepared mouse
plasma over a period of 24 h. Inspection of those chromatograms reveals that the degradation of A14 in
the mouse plasma is almost completed in 12 h with a half-life of approximately 2.5 h, considering the
more-hostile environment in vivo, the biodegradation rate of A14 would be more rapid. By contrast, E1
remains essentially undegraded in the mouse plasma over 24 h, which displays a much more chemical
stability than that of A14 in in vitro plasma.
Fig. 6. Time-dependence of integrated HPLC peak areas for A14 and E1 after incubation at 37℃ in mouse plasma
in vitro. AU denotes arbitrary units.
3.7. Compound’s stability study in liver microsome
After the plasma stability, the in vitro metabolic stabilities of analogs A14 and E1 were examined by
measuring their half-life upon incubation with mouse liver microsome in the presence of NADPH and
testosterone was used as a positive control. The result showed a clearance rate of 24.3% at 20 min. As
shown in Fig. 7, the two compounds possessed good stability profile in mouse microsome with a half-
life of more than 90 minutes. The improved metabolic stability of A14 in liver microsome compared to
plasma could be caused by the different enzyme system between plasma and liver microsome.
Fig. 7. In vitro microsomal stabilities of compounds A14 and E1.
13

3.8. A14 is minimally toxic to mammalian cells
To evaluate any potential toxicity against mammalian cell, a 48 h MTT-based assay was performed to
assess the cytotoxicity of A14 to Hela cells [11]. This 1,3,4-oxadiazol-2-one-containing benzamide
derivative was proved to be minimally toxic against Hela cells, with a 50% inhibitory concentration (IC50)
of >64 µg/mL which is much higher than the antibacterial MIC values. Albeit testing a single mammalian
cell line, this initial result suggests that A14 could be associated with a significant therapeutic window.
3.9. Computational docking studies of A14-FtsZ bingding mode
To further explore the interaction mode between those synthesized molecules with the target protein,
the most potent compound A14 was chosen as a representative for the docking analysis with a reported
crystal structure of SaFtsZ (PDB code: 3vob) [24] utilizing AutoDock program [30]. The docking mode
shown in Fig. 8 indicates that A14 binds into the pocket formed by T7 loop, H7 helix and C-terminal
sheet, which is same as PC190723 [24]. Its amide group and ether oxygen atom form major hydrogen
bonds with surrounding amino acid residues, and the conjugated aromatic rings extend into a
hydrophobic channel (Fig. 8A). Besides, what attracts our attention is that the lactone bond of the1,3,4-
oxadiazol-2-one ring orientates the pocket wall, as shown in Fig. 8B and 8C. This boundary effect could
collide with the large substitute on the 1-O-position of the five-membered heterocyclic ring, which might
result in the loss of antibacterial activity of series D and E.
Fig. 8. The molecular docking model of SaFtsZ (PDB code: 3vob) in complex with A14. (A) The hydrogen bonds
are shown as yellow dot lines; (B and C) The ginger arrows indicate boundary facing to the lactone bond.
4. Conclusion
Based on the rational design strategy, we designed and synthesized six series of novel 1,3,4-oxadiazol-
2-one-containing, 1,2,4-triazol-3-one-containing and pyrazolin-5-one-containing benzamide derivatives,
and tested for their in vitro biological activities. The result brings two outstanding compounds, such as
A14 and A12, with significant antibacterial activity against six Gram-positive bacterial strains including
14

three drug-resistant S. aureus strains. Their mechanism of action was also confirmed as targeting FtsZ
through morphological observation and protein dynamic study. It is noteworthy that A14 shows similar
MIC values (0.5–1 µg/mL) against three resistant and one sensitive strains of S. aureus, which are the
same as that of PC190723, much better than commercial drugs such as CIP, LIN and ERY according to
their bactericidal or bacteriostatic efficacy. In addition, difference of the activity among these series
compounds draw that the substitution on the five-membered aromatic ring should have a volume limit
according to the binding pocket of FtsZ, which could guide further to develop FtsZ-targeting agents.
Further study on the pharmacokinetics of A14 and more efforts on improving its biostability is still under
exploration and research.
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 indicated
1
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. Melting points were determined using a RY-1
melting point apparatus without corrected.
All the 1H NMR, 13C NMR, MS spectra of those compounds and intermediates can be referenced in
the supporting information.
5.2. Typical procedure for the synthesis of series A, B and C
5.2.1. 3-(Benzyloxy)-2,6-difluorobenzoic acid (3)
To a solution of 2,4-difluorophenol 2 (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 2, and
brine, subsequently. The organic layer was dried and concentrated to give the benzyl-protected 2 (31.8
g, 99%) as an off-white crystalline solid. this intermediate was used in the next step without additional
purification. 1H NMR (600 MHz, DMSO-d6) δ 7.46 – 7.43 (m, 2H), 7.42 – 7.38 (m, 2H), 7.37 – 7.33 (m,
1H), 7.31 – 7.24 (m, 2H), 7.03 – 6.98 (m, 1H), 5.16 (s, 2H).
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, 150 mL), 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 3 (11.4 g, 95%) was used directly without
additional purification. 1H NMR (600 MHz, DMSO-d6) δ 13.97 (s, 1H), 7.47 – 7.43 (m, 2H), 7.43 – 7.37
15

(m, 3H), 7.37 – 7.33 (m, 1H), 7.12 (td, J = 9.2, 1.9 Hz, 1H), 5.20 (s, 2H). ESI-MS calcd for C14H9F2O3
[M - H]-, 263.2, found 263.2.
5.2.2. 2,6-Difluoro-3-hydroxybenzamide (5)
To a solution of 3 (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 6 h 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 4 (9.6 g, 97%). The residue was used directly without
additional purification.
To a solution of 4 (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 5 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.
5.2.3. 2-(3-Carbamoyl-2,4-difluorophenoxy)acetic acid (7)
A mixture of 5 (2.5 g, 14.5 mmol), ethyl bromoacetate (3.4 g, 20 mmol) and K2CO3 (4.1 g, 30 mmol)
in DMF (18 mL) was stirred at 35℃ overnight. 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 6
as a white solid (3.2 g, 85%). 1H NMR (600 MHz, DMSO-d6) δ 8.15 (s, 1H), 7.85 (s, 1H), 7.17 (td, J =
9.4, 5.1 Hz, 1H), 7.05 (td, J = 8.9, 1.8 Hz, 1H), 4.89 (s, 2H), 4.17 (q, J = 7.1 Hz, 2H), 1.21 (t, J = 7.1 Hz,
3H).
To a mixture solution of 15 mL MeOH and 15 mL NaOH aq (2 M) was added 6 (3 g, 11.6 mmol) and
heated at 50℃ for 4 h. Upon completion, the mixture was concentrated to remove most of the MeOH,
the residual aqueous solution was treated with concentrated HCl in ice-bath. The precipitated solid was
filtered out and dried, getting the acid intermediate 7 as a white solid (2.4 g, 90%). 1H NMR (600 MHz,
DMSO-d6) δ 13.13 (s, 1H), 8.15 (s, 1H), 7.85 (s, 1H), 7.14 (td, J = 9.3, 5.1 Hz, 1H), 7.04 (td, J = 9.0, 1.7
Hz, 1H), 4.79 (s, 2H).
5.2.4. 3-(3-carbamoyl-2,4-difluorophenoxy)propanoic acid (13)
A mixture of 5 (2 g, 11.6 mmol), 3-bromo-1-propanol (2.1 g, 15.1 mmol) and K2CO3 (3.2 g, 23.2
mmol) in DMF (16 mL) was stirred at 35℃ for 7 h. The reaction mixture was then diluted with EtOAc
(80 mL), washed with NH4Cl (aq) and brine, dried over Na2SO4, concentrated, and purified on silica gel.
16

Elution with DCM/MeOH (70:1) afforded the according primary alcohol intermediate 12 as white solid.
ESI-MS calcd for C10H12F2NO3 [M + H]+, 232.2, found 232.2.
The above alcohol intermediate 12 was dissolved in acetone, freshly prepared Jones reagent (3.5 eq,
calculated by CrO3) was dropwise added at room temperature. The resulting mixture was further stirred
for 3 h, upon completion, excessive isopropanol was added. Insoluble materials was filtered off and
washed with MeOH, the filtrate was collected, concentrated and purified on silica gel, Elution with
DCM/MeOH (30:1) afforded the acid intermediate 13 as white solid (2.0 g, 75% for two steps). 1H NMR
(600 MHz, DMSO-d6) δ 12.41 (s, 1H), 8.11 (s, 1H), 7.82 (s, 1H), 7.24 (td, J = 9.3, 5.2 Hz, 1H), 7.06 (td,
J = 8.9, 1.8 Hz, 1H), 4.23 (t, J = 6.0 Hz, 2H), 2.71 (t, J = 5.9 Hz, 2H). ESI-MS calcd for C10H8F2NO4 [M
- H]-, 244.2, found 244.3.
5.2.5. Typical procedure for the synthesis of hydrazine hydrochloride intermediates (10)
Dissolve the substituted anline (3 mmol) in HCl aq (6 M) at -5℃, and slowly added an aqueous solution
of NaNO2 (0.23 g, 3.3 mmol). The mixture was stirred for further 0.5 h, a solution of SnCl2 (1.7 g, 7.5
mmol) dissolved in concentrated HCl was added dropwise, then the resulting mixture was moved to room
temperature and stirred for further 1 h. The muddy mixture was filtered out, washed with a small amount
of HCl solution and dried in vacuum. This desired phenylhydrazine hydrochloride intermediate was used
directly without additional purification.
To a solution of substituted benzyl halide 3 mmol in EtOH was added the hydrazine hydrate 30 mmol,
and the mixture was stirred at room temperature overnight till the reaction was completed monitored by
TLC. The reaction mixture was concentrated to dryness, and re-dissolved in a little EtOH, then added an
aqueous HCl (12 M) and stirred for awhile at 0℃. The resulting precipitate was collected by filtration
and dried. This desired benzylhydrazine hydrochloride intermediate was used directly without additional
purification.
5.2.6. Typical procedure for the synthesis of 1,3,4-oxadiazol-2-one-benzamide derivatives (series A, B
and C)
A 10 mL round-bottom flask equipped with a magnetic stirrer was charged with 7 or 13 (0.26 mmol),
EDCI (55 mg, 0.29 mmol), HOBt (39 mg, 0.29 mmol) and THF (5 mL), DMF (1 mL), this mixture was
stirred at room temperature for 3 h. Then the intermediate 10 (0.4 mmol) and followed DIPEA (155 mg,
1.2 mmol) were added to the reaction, and stirred overnight. The mixture was then diluted with EtOAc
(30 mL), washed with NH4Cl (aq) and brine, dried over Na2SO4, concentrated in a rotovapor, and purified
on silica gel. Elution with DCM/MeOH (30:1) afforded the according products 11 or 14 in good yield.
1
Example 11-A12, H NMR (600 MHz, DMSO-d6) δ 10.04 (s, 1H), 8.14 (s, 1H), 8.00 (s, 1H), 7.86 (s,
1H), 7.21 – 7.14 (m, 3H), 7.13 – 7.08 (m, 1H), 6.71 (d, J = 6.8 Hz, 2H), 4.75 (s, 2H). ESI-MS calcd for
C15H13ClF2N3O3 [M + H]+, 356.1, found 356.3.
The above acylhydrazine intermediate 11 or 14 was dissolved in THF (5 mL), and triphosgene (2 eq)
was added, the mixture was stirred at room temperature for 1h. Upon completion, the reaction mixture
was then diluted with EtOAc (25 mL), washed with brine, dried over Na2SO4, concentrated in vacuum,
and purified on silica gel. Elution with petroleum ether/EtOAc (2:1 to 1:1) afforded the according
products in good yield.
5.2.6.1. 2,6-Difluoro-3-((4-phenyl-5-oxo-1,3,4-oxadiazol-2-yl)methoxy)benzamide. (A1). White solid,
yield 52% for two steps, Mp:161–162℃; 1H NMR (600 MHz, DMSO-d6) δ 8.16 (s, 1H), 7.88 (s, 1H),
17

7.74 (d, J = 7.7 Hz, 2H), 7.55 – 7.50 (m, 2H), 7.45 (td, J = 9.3, 5.1 Hz, 1H), 7.34 (t, J = 7.4 Hz, 1H), 7.15
(td, J = 9.0, 1.8 Hz, 1H), 5.30 (s, 2H). 13C NMR (151 MHz, DMSO-d6) δ 161.52, 154.04 (d, JF-C = 6.0
Hz), 152.44, 150.75, 148.63 (dd, JF-C = 250.7, 8.3 Hz), 142.14 (dd, JF-C = 10.6, 3.0 Hz), 135.93, 129.90,
126.86, 118.83, 117.49 (d, JF-C = 15.1 Hz), 117.12 (d, JF-C = 21.1 Hz) 111.62 (dd, JF-C = 22.7, 7.6 Hz),
62.60. ESI-MS calcd for C16H12F2N3O4 [M + H]+, 348.3, found 348.4.
5.2.6.2. 2,6-Difluoro-3-((4-(p-tolyl)-5-oxo-1,3,4-oxadiazol-2-yl)methoxy)benzamide. (A2). White solid,
yield 54% for two steps, Mp:165–167℃; 1H NMR (600 MHz, Chloroform-d) δ 7.68 (d, J = 8.6 Hz, 2H),
7.25 (d, J = 8.2 Hz, 2H), 7.21 (td, J = 9.0, 5.1 Hz, 1H), 6.93 (td, J = 9.0, 2.0 Hz, 1H), 5.96 (d, J = 35.7
Hz, 2H), 5.05 (s, 2H), 2.37 (s, 3H). ESI-MS calcd for C17H14F2N3O4 [M + H]+, 362.3, found 362.3.
5.2.6.3. 2,6-Difluoro-3-((4-(m-tolyl)-5-oxo-1,3,4-oxadiazol-2-yl)methoxy)benzamide. (A3). White solid,
Mp:155–157℃; 1H NMR (600 MHz, DMSO-d6) δ 8.16 (s, 1H), 7.88 (s, 1H), 7.57 – 7.52 (m, 2H), 7.47
– 7.38 (m, 2H), 7.18 – 7.12 (m, 2H), 5.29 (s, 2H), 2.37 (s, 3H). ESI-MS calcd for C17H14F2N3O4 [M +
H]+, 362.3, found 362.4.
5.2.6.4. 2,6-Difluoro-3-((4-(o-tolyl)-5-oxo-1,3,4-oxadiazol-2-yl)methoxy)benzamide. (A4). White solid,
Mp:180–182℃; 1H NMR (600 MHz, DMSO-d6) δ 8.16 (s, 1H), 7.88 (s, 1H), 7.46 – 7.39 (m, 4H), 7.37
– 7.33 (m, 1H), 7.15 (td, J = 8.9, 1.8 Hz, 1H), 5.27 (s, 2H), 2.22 (s, 3H). ESI-MS calcd for C17H14F2N3O4
[M + H]+, 362.3, found 362.4.
5.2.6.5. 2,6-Difluoro-3-((4-(4-ethylphenyl)-5-oxo-1,3,4-oxadiazol-2-yl)methoxy)benzamide. (A5). White
1
solid, Mp:177–178℃; H NMR (600 MHz, Chloroform-d) δ 7.70 (d, J = 8.6 Hz, 2H), 7.27 (d, J = 8.9
Hz, 2H), 7.20 (td, J = 9.0, 5.1 Hz, 1H), 6.93 (td, J = 9.0, 2.0 Hz, 1H), 6.01 (s, 2H), 5.06 (s, 2H), 2.67 (q,
J = 7.6 Hz, 2H), 1.24 (t, J = 7.6 Hz, 3H). ESI-MS calcd for C18H16F2N3O4 [M + H]+, 376.3, found 376.4.
5.2.6.6. 2,6-Difluoro-3-((4-(4-isopropylphenyl)-5-oxo-1,3,4-oxadiazol-2-yl)methoxy)benzamide. (A6).
White solid, Mp:189–191℃; 1H NMR (600 MHz, Chloroform-d) δ 7.70 (d, J = 8.6 Hz, 2H), 7.30 (d, J =
8.6 Hz, 2H), 7.21 (td, J = 9.0, 5.1 Hz, 1H), 6.93 (td, J = 9.0, 2.0 Hz, 1H), 5.95 (d, J = 45.5 Hz, 2H), 5.06
(s, 2H), 2.94 (p, J = 7.0 Hz, 1H), 1.26 (d, J = 6.9 Hz, 6H). ESI-MS calcd for C19H18F2N3O4 [M + H]+,
390.4, found 390.4.
5.2.6.7. 2,6-Difluoro-3-((4-(4-(tert-butyl)phenyl)-5-oxo-1,3,4-oxadiazol-2-yl)methoxy)benzamide. (A7).
White solid, Mp:171–173℃; 1H NMR (400 MHz, DMSO-d6) δ 8.17 (s, 1H), 7.90 (s, 1H), 7.64 (d, J =
8.8 Hz, 2H), 7.54 (d, J = 8.9 Hz, 2H), 7.44 (td, J = 9.4, 5.2 Hz, 1H), 7.15 (td, J = 8.9, 1.8 Hz, 1H), 5.30
(s, 2H), 1.29 (s, 9H). ESI-MS calcd for C20H20F2N3O4 [M + H]+, 404.4, found 404.4.
5.2.6.8. 2,6-Difluoro-3-((4-(4-butylphenyl)-5-oxo-1,3,4-oxadiazol-2-yl)methoxy)benzamide. (A8). White
solid, Mp:159–161℃; 1H NMR (400 MHz, DMSO-d6) δ 8.17 (s, 1H), 7.90 (s, 1H), 7.62 (d, J = 8.5 Hz,
2H), 7.44 (td, J = 9.4, 5.2 Hz, 1H), 7.33 (d, J = 8.4 Hz, 2H), 7.15 (td, J = 9.0, 1.8 Hz, 1H), 5.29 (s, 2H),
2.60 (t, J = 7.6 Hz, 2H), 1.56 (p, J = 7.5 Hz, 2H), 1.30 (h, J = 7.4 Hz, 2H), 0.89 (t, J = 7.3 Hz, 3H).
ESI-MS calcd for C20H23F2N4O4 [M + NH4]+, 421.4, found 421.4.
5.2.6.9. 2,6-Difluoro-3-((4-(2,4-dimethylphenyl)-5-oxo-1,3,4-oxadiazol-2-yl)methoxy)benzamide. (A9).
18

White solid, Mp:180–182℃; 1H NMR (600 MHz, DMSO-d6) δ 8.16 (s, 1H), 7.88 (s, 1H), 7.43 (td, J =
9.3, 5.1 Hz, 1H), 7.30 (d, J = 8.0 Hz, 1H), 7.21 (s, 1H), 7.17 – 7.12 (m, 2H), 5.26 (s, 2H), 2.33 (s, 3H),
2.16 (s, 3H). ESI-MS calcd for C18H19F2N4O4 [M + NH4]+, 393.4, found 393.3.
5.2.6.10. 2,6-Difluoro-3-((4-(4-methoxyphenyl)-5-oxo-1,3,4-oxadiazol-2-yl)methoxy)benzamide. (A10).
1
White solid, Mp:150–152℃; H NMR (600 MHz, DMSO-d6) δ 8.16 (s, 1H), 7.88 (s, 1H), 7.65 – 7.58
(m, 2H), 7.44 (td, J = 9.3, 5.1 Hz, 1H), 7.14 (td, J = 9.0, 1.8 Hz, 1H), 7.10 – 7.04 (m, 2H), 5.28 (s, 2H),
3.79 (s, 3H). ESI-MS calcd for C17H17F2N4O5 [M + NH4]+, 395.3, found 395.3.
5.2.6.11. 2,6-Difluoro-3-((4-(3-methoxyphenyl)-5-oxo-1,3,4-oxadiazol-2-yl)methoxy)benzamide. (A11).
1
White solid, Mp:144–145℃; H NMR (600 MHz, DMSO-d6) δ 8.16 (s, 1H), 7.88 (s, 1H), 7.47 – 7.41
(m, 2H), 7.35 – 7.31 (m, 1H), 7.29 (t, J = 2.2 Hz, 1H), 7.15 (td, J = 8.9, 1.8 Hz, 1H), 6.94 – 6.90 (m, 1H),
5.30 (s, 2H), 3.79 (s, 3H). ESI-MS calcd for C17H17F2N4O5 [M + NH4]+, 395.3, found 395.3.
5.2.6.12. 2,6-Difluoro-3-((4-(4-chlorophenyl)-5-oxo-1,3,4-oxadiazol-2-yl)methoxy)benzamide. (A12).
White solid, Mp:148–149℃; 1H NMR (600 MHz, DMSO-d6) δ 8.16 (s, 1H), 7.88 (s, 1H), 7.75 (d, J =
9.0 Hz, 2H), 7.60 (d, J = 9.0 Hz, 2H), 7.44 (td, J = 9.3, 5.2 Hz, 1H), 7.14 (td, J = 8.9, 1.8 Hz, 1H), 5.30
(s, 2H). 13C NMR (151 MHz, DMSO-d6) δ 161.51, 153.26 (dd, JF-C = 243.1, 7.6 Hz), 152.59, 150.62,
148.64 (dd, JF-C = 249.2, 8.3 Hz), 142.14 (dd, JF-C = 10.6, 3.0 Hz), 134.86, 130.81, 129.88, 120.37, 117.43
(dd, JF-C = 7.6, 3.0 Hz), 117.11 (d, JF-C = 21.1 Hz), 111.59 (dd, JF-C = 22.7, 5.3 Hz), 62.56. ESI-MS calcd
for C16H14ClF2N4O4 [M + NH4]+, 399.1, found 399.3.
5.2.6.13. 2,6-Difluoro-3-((4-(3-chlorophenyl)-5-oxo-1,3,4-oxadiazol-2-yl)methoxy)benzamide. (A13).
White solid, Mp:138–139℃; 1H NMR (600 MHz, Chloroform-d) δ 7.87 (s, 1H), 7.77 (ddd, J = 8.3, 2.2,
0.9 Hz, 1H), 7.39 (t, J = 8.1 Hz, 1H), 7.27 (dd, J = 2.1, 1.0 Hz, 1H), 7.20 (td, J = 9.1, 5.1 Hz, 1H), 6.94
(td, J = 9.0, 2.0 Hz, 1H), 5.97 (d, J = 28.4 Hz, 2H), 5.07 (s, 2H). ESI-MS calcd for C16H14ClF2N4O4 [M
+ NH4]+, 399.1, found 399.3.
5.2.6.14. 2,6-Difluoro-3-((4-(4-bromophenyl)-5-oxo-1,3,4-oxadiazol-2-yl)methoxy)benzamide. (A14).
White solid, Mp:104–106℃; 1H NMR (600 MHz, DMSO-d6) δ 8.16 (s, 1H), 7.89 (s, 1H), 7.73 (d, J =
9.2 Hz, 2H), 7.69 (d, J = 9.2 Hz, 2H), 7.44 (td, J = 9.3, 5.2 Hz, 1H), 7.14 (td, J = 8.9, 1.7 Hz, 1H), 5.30
(s, 2H). 13C NMR (101 MHz, DMSO-d6) δ 161.49, 153.29 (dd, JF-C = 244.4, 7.1 Hz), 152.62, 150.57,
148.71 (dd, JF-C = 250.5, 9.1 Hz), 142.22 (dd, JF-C = 12.1, 3.0 Hz), 135.31, 132.80, 120.64, 119.01, 117.52
(d, JF-C = 9.1 Hz), 117.26, 111.76 (dd, JF-C = 23.2, 3.0 Hz), 62.61. ESI-MS calcd for C16H11BrF2N3O4 [M
+ H]+, 426.0, found 426.2.
5.2.6.15. 2,6-Difluoro-3-((4-(4-fluorophenyl)-5-oxo-1,3,4-oxadiazol-2-yl)methoxy)benzamide. (A15).
1
White solid, Mp:128–130℃; H NMR (600 MHz, DMSO-d6) δ 8.16 (s, 1H), 7.89 (s, 1H), 7.77 – 7.72
(m, 2H), 7.44 (td, J = 9.3, 5.2 Hz, 1H), 7.40 – 7.35 (m, 2H), 7.15 (td, J = 8.9, 1.8 Hz, 1H), 5.30 (s, 2H).
ESI-MS calcd for C16H11F3N3O4 [M + H]+, 366.3, found 366.3.
5.2.6.16.
2,6-Difluoro-3-((4-(4-(trifluoromethyl)phenyl)-5-oxo-1,3,4-oxadiazol-2-
1
yl)methoxy)benzamide. (A16). White solid, Mp:127–129℃; H NMR (600 MHz, DMSO-d6) δ 8.17 (s,
1H), 7.97 (d, J = 8.7 Hz, 2H), 7.91 (d, J = 8.8 Hz, 2H), 7.89 (s, 1H), 7.45 (td, J = 9.3, 5.1 Hz, 1H), 7.15
19

(td, J = 9.0, 1.8 Hz, 1H), 5.33 (s, 2H). ESI-MS calcd for C17H11F5N3O4 [M + H]+, 416.3, found 416.4.
5.2.6.17. 2,6-Difluoro-3-((4-(4-cyanophenyl)-5-oxo-1,3,4-oxadiazol-2-yl)methoxy)benzamide. (A17).
White solid, Mp:168–170℃; 1H NMR (600 MHz, DMSO-d6) δ 8.17 (s, 1H), 8.01 (d, J = 8.9 Hz, 2H),
7.93 (d, J = 8.9 Hz, 2H), 7.89 (s, 1H), 7.45 (td, J = 9.3, 5.1 Hz, 1H), 7.15 (td, J = 8.9, 1.7 Hz, 1H), 5.33
(s, 2H). ESI-MS calcd for C17H14F2N5O4 [M + NH4]+, 390.3, found 390.3.
5.2.6.18.
2,6-Difluoro-3-((4-(3,4-dichlorophenyl)-5-oxo-1,3,4-oxadiazol-2-yl)methoxy)benzamide.
(A18). White solid, Mp:170–172℃; 1H NMR (600 MHz, DMSO-d6) δ 8.16 (s, 1H), 7.95 (d, J = 2.5 Hz,
1H), 7.89 (s, 1H), 7.81 (d, J = 8.9 Hz, 1H), 7.72 (dd, J = 8.9, 2.5 Hz, 1H), 7.44 (td, J = 9.3, 5.1 Hz, 1H),
7.15 (td, J = 8.9, 1.7 Hz, 1H), 5.31 (s, 2H). ESI-MS calcd for C16H13Cl2F2N4O4 [M + NH4]+, 433.0, found
433.3.
5.2.6.19.
2,6-Difluoro-3-((4-(2,4-dichlorophenyl)-5-oxo-1,3,4-oxadiazol-2-yl)methoxy)benzamide.
(A19). White solid, Mp:154–156℃; 1H NMR (600 MHz, DMSO-d6) δ 8.16 (s, 1H), 7.94 (d, J = 2.3 Hz,
1H), 7.89 (s, 1H), 7.75 (d, J = 8.6 Hz, 1H), 7.66 (dd, J = 8.6, 2.3 Hz, 1H), 7.42 (td, J = 9.3, 5.1 Hz, 1H),
7.15 (td, J = 8.9, 1.8 Hz, 1H), 5.30 (s, 2H). ESI-MS calcd for C16H13Cl2F2N4O4 [M + NH4]+, 433.0, found
433.4.
5.2.6.20. 2,6-Difluoro-3-((4-(4-chloro-2-methylphenyl)-5-oxo-1,3,4-oxadiazol-2-yl)methoxy)benzamide.
1
(A20). White solid, Mp:144–146℃; H NMR (600 MHz, DMSO-d6) δ 8.16 (s, 1H), 7.89 (s, 1H), 7.53
(d, J = 2.2 Hz, 1H), 7.48 (d, J = 8.5 Hz, 1H), 7.46 – 7.41 (m, 2H), 7.14 (td, J = 8.9, 1.7 Hz, 1H), 5.27 (s,
2H), 2.22 (s, 3H). ESI-MS calcd for C17H16ClF2N4O4 [M + NH4]+, 413.1, found 413.4.
5.2.6.21. 2,6-Difluoro-3-((4-(tert-butyl)-5-oxo-1,3,4-oxadiazol-2-yl)methoxy)benzamide. (A21). White
solid, Mp:123–125℃; 1H NMR (600 MHz, Chloroform-d) δ 7.16 (td, J = 9.1, 5.1 Hz, 1H), 6.92 (td, J =
9.0, 2.0 Hz, 1H), 5.98 (d, J = 27.5 Hz, 2H), 4.91 (s, 2H), 1.53 (s, 9H). ESI-MS calcd for C14H19F2N4O4
[M + NH4]+, 345.3, found 345.3.
5.2.6.22. 2,6-Difluoro-3-((4-(4-chlorobenzyl)-5-oxo-1,3,4-oxadiazol-2-yl)methoxy)benzamide. (B1).
1
White solid, yield 48% for two steps, Mp:132–133℃; H NMR (600 MHz, DMSO-d6) δ 8.15 (s, 1H),
7.88 (s, 1H), 7.44 (d, J = 8.5 Hz, 2H), 7.38 – 7.31 (m, 3H), 7.12 (td, J = 9.0, 1.8 Hz, 1H), 5.17 (s, 2H),
4.91 (s, 2H). 13C NMR (151 MHz, DMSO-d6) δ 161.49, 153.41, 153.23 (dd, JF-C = 243.1, 7.6 Hz), 151.83,
148.69 (dd, JF-C = 250.7, 8.3 Hz), 142.01 (dd, JF-C = 10.6, 3.0 Hz), 134.75, 133.19, 130.17, 129.15, 117.45
(dd, JF-C = 13.6, 5.3 Hz), 117.17 (d, JF-C = 21.1 Hz), 111.57 (dd, JF-C = 22.7, 8.3 Hz), 62.61, 48.49. ESI-
MS calcd for C17H13ClF2N3O4 [M + H]+, 396.1, found 396.2.
5.2.6.23. 2,6-Difluoro-3-((4-(4-bromobenzyl)-5-oxo-1,3,4-oxadiazol-2-yl)methoxy)benzamide. (B2).
White solid, Mp:107–108℃; 1H NMR (600 MHz, DMSO-d6) δ 8.15 (s, 1H), 7.88 (s, 1H), 7.58 (d, J =
8.4 Hz, 2H), 7.35 (td, J = 9.3, 5.1 Hz, 1H), 7.26 (d, J = 8.4 Hz, 2H), 7.12 (td, J = 8.9, 1.8 Hz, 1H), 5.17
(s, 2H), 4.89 (s, 2H). ESI-MS calcd for C17H16BrF2N4O4 [M + NH4]+, 457.0, found 457.3.
5.2.6.24. 2,6-Difluoro-3-((4-(4-(tert-butyl)benzyl)-5-oxo-1,3,4-oxadiazol-2-yl)methoxy)benzamide. (B3).
White solid, Mp:120–122℃; 1H NMR (600 MHz, Chloroform-d) δ 7.38 (d, J = 8.3 Hz, 2H), 7.26 (d, J =
20

8.2 Hz, 2H), 7.11 (td, J = 9.1, 5.1 Hz, 1H), 6.87 (td, J = 9.0, 2.0 Hz, 1H), 5.92 (d, J = 29.2 Hz, 2H), 4.91
(s, 2H), 4.84 (s, 2H), 1.31 (s, 9H). ESI-MS calcd for C21H25F2N4O4 [M + NH4]+, 435.4, found 435.4.
5.2.6.25. 2,6-Difluoro-3-(2-(4-(4-bromophenyl)-5-oxo-1,3,4-oxadiazol-2-yl)ethoxy)benzamide. (C1).
White solid, Mp:107–109℃; 1H NMR (600 MHz, DMSO-d6) δ 8.11 (s, 1H), 7.84 (s, 1H), 7.71 (s, 4H),
7.30 (td, J = 9.3, 5.2 Hz, 1H), 7.09 (td, J = 8.9, 1.8 Hz, 1H), 4.41 (t, J = 6.1 Hz, 2H), 3.20 (t, J = 6.1 Hz,
2H).13C NMR (151 MHz, DMSO-d6) δ 161.68, 155.25, 152.67 (dd, JF-C = 243.1, 6.8 Hz), 150.83, 148.51
(dd, JF-C = 247.6, 8.3 Hz), 142.92 (d, JF-C = 12.1 Hz), 135.45, 132.76, 120.23, 118.58, 117.19 (dd, JF-C
=
28.7, 21.1 Hz), 116.62 (d, JF-C = 15.1 Hz), 111.59 (dd, JF-C = 24.2, 7.6 Hz), 65.59, 26.90. ESI-MS calcd
for C17H13BrF2N4O4 [M + H]+, 440.0, found 440.3.
5.2.6.26.
2,6-Difluoro-3-(2-(4-(4-(tert-butyl)phenyl)-5-oxo-1,3,4-oxadiazol-2-yl)ethoxy)benzamide.
(C2). White solid, Mp:112–114℃; 1H NMR (600 MHz, DMSO-d6) δ 8.10 (s, 1H), 7.84 (s, 1H), 7.65 (d,
J = 8.7 Hz, 2H), 7.52 (d, J = 8.7 Hz, 2H), 7.30 (td, J = 9.3, 5.1 Hz, 1H), 7.09 (td, J = 8.9, 1.7 Hz, 1H),
4.40 (t, J = 6.1 Hz, 2H), 3.20 (t, J = 6.1 Hz, 2H), 1.29 (s, 9H). ESI-MS calcd for C21H22F2N3O4 [M + H]+,
418.4, found 418.4.
5.3. Typical procedure for the synthesis of series D
5.3.1. Typical procedure for the synthesis of 1,3,4-oxadiazol-2(3H)-one intermediates (18)
The substituted benzoic acid 15 (6 mmol) was dissolved in 20 mL MeOH, SOCl2 (1.4 g, 12 mmol)
was added slowly in ice-bath, then the mixture was heated at 45℃ for 5 h. Upon completion, the mixture
was concentrated, the residue was re-dissolved in EtOAc and washed with NaHCO3 and brine. The
organic layer was dried with Na2SO4 and concentrated to give the desired ester intermediate 16, which
was used in the next step without further purification.
The above ester 16 was dissolved in 15 mL MeOH, hydrazine hydrate (2 mL, 32 mmol), then the
mixture was heated at 45℃ for 5 h. Upon completion, the mixture was concentrated, the residue was re-
dissolved in EtOAc and washed with brine. The organic layer was concentrated and purified by silica gel
affording the desired acylhydrazine intermediate 17 as a white solid in good yield.
Add benzhydrazide 17 (2 mmol), CH2Cl2 (20 mL) and DIPEA (0.52 g, 4 mmol) to a round bottomed
flask under N2 protection. Triphosgene (0.3 g, 1 mmol) dissolved in DCM (4 mL) Using a syringe, the
triphosgene/DCM solution was added dropwise to the stirred solution of hydrazide, and stirred at room
temperature for 1 h. The reaction mixture was concentrated by rotary evaporation, the crude product was
purified by chromatography on silica (DCM/MeOH = 100:1) affording the desired products 18 as white
solid (about 70% yield for three steps). Example 18-D3, 1H NMR (600 MHz, DMSO-d6) δ 12.66 (s, 1H),
7.80 (d, J = 8.6 Hz, 2H), 7.62 (d, J = 8.6 Hz, 2H). ESI-MS calcd for C8H4ClN2O2 [M - H]-, 195.0, found
195.2.
5.3.2. Typical procedure for the synthesis of 3-(hydroxymethyl)-1,3,4-oxadiazol-2(3H)-one intermediates
(19)
The above intermediate 18 (1.5 mmol) was dissolved in DMF, then added TEA (0.3 g,3 mmol) and
formaldehyde solution (0.6 mL, 7.5 mmol). The mixture was stirred at room temperature for 6 h. The
residue was diluted with EtOAc and washed with NH4Cl and brine, the organic layer was concentrated
and purified by silica gel. Elution with petroleum ether/EtOAc (10:1) afforded the according products 19
21

as white solid (yield about 60%). Example 19-D3, 1H NMR (600 MHz, DMSO-d6) δ 7.83 (d, J = 8.6 Hz,
2H), 7.65 (d, J = 8.6 Hz, 2H), 6.92 (t, J = 7.5 Hz, 1H), 5.09 (d, J = 7.2 Hz, 2H).
5.3.3. Typical procedure for the synthesis of 3-(bromomethyl)-1,3,4-oxadiazol-2(3H)-one intermediates
(20)
To the alcohol intermediate 19 dissolved in DCM at 0℃, was added CBr4 (1.5 eq) and PPh3 (3 eq)
successively. The mixture was stirred at room temperature for 6 h. Upon completion, purification was
carried out using silica gel column with petroleum ether/EtOAc (50:1). Example 20-D3, 1H NMR (600
MHz, Chloroform-d) δ 7.82 (d, J = 8.7 Hz, 2H), 7.48 (d, J = 8.6 Hz, 2H), 5.61 (s, 2H).
5.3.4. Typical procedure for the synthesis of 1,3,4-oxadiazol-2-one-benzamide derivatives (series D)
A round-bottom flask equipped with a magnetic stirrer was charged with intermediate 5 (50 mg, 0.29
mmol), bromide 20 (0.3 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) afforded the corresponding
compound in good yield.
5.3.4.1. 2,6‐Difluoro‐3‐((5‐(4‐methylphenyl)‐2‐oxo‐1,3,4‐oxadiazol‐3‐yl)methoxy)benzamide. (D1).
White solid, yield 84%; Mp:179–181℃; 1H NMR (600 MHz, Chloroform-d) δ 7.74 (d, J = 8.3 Hz, 2H),
7.33 – 7.28 (m, 3H), 6.91 (td, J = 9.0, 2.0 Hz, 1H), 5.99 (d, J = 61.9 Hz, 2H), 5.70 (s, 2H), 2.42 (s, 3H).
ESI-MS calcd for C17H14F2N3O4 [M + H]+, 362.3, found 362.3.
5.3.4.2. 2,6‐Difluoro‐3‐((5‐(4-(tert-butyl)phenyl)‐2‐oxo‐1,3,4‐oxadiazol‐3‐yl)methoxy)benzamide. (D2).
1
White solid, yield 86%; Mp:158–160℃; H NMR (600 MHz, DMSO-d6) δ 8.14 (s, 1H), 7.87 (s, 1H),
7.76 (d, J = 8.6 Hz, 2H), 7.60 (d, J = 8.5 Hz, 2H), 7.49 (td, J = 9.3, 5.3 Hz, 1H), 7.15 (td, J = 9.0, 1.8 Hz,
1H), 5.80 (s, 2H), 1.31 (s, 9H). ESI-MS calcd for C20H23F2N4O4 [M + NH4]+, 421.4, found 421.5.
5.3.4.3. 2,6‐Difluoro‐3‐((5‐(4-chlorophenyl)‐2‐oxo‐1,3,4‐oxadiazol‐3‐yl)methoxy)benzamide. (D3).
1
White solid, yield 83%; Mp:186–188℃; H NMR (600 MHz, DMSO-d6) δ 8.14 (s, 1H), 7.86 (s, 1H),
7.84 (d, J = 8.7 Hz, 2H), 7.65 (d, J = 8.7 Hz, 2H), 7.49 (td, J = 9.3, 5.3 Hz, 1H), 7.15 (td, J = 8.9, 1.7 Hz,
1H), 5.80 (s, 2H). 13C NMR (151 MHz, DMSO-d6) δ 161.44, 153.87 (dd, JF-C = 244.6, 6.8 Hz), 153.01,
152.88, 149.51 (dd, JF-C = 249.2, 9.1 Hz), 140.67 (dd, JF-C = 12.1, 3.0 Hz), 137.37, 130.03, 127.98, 122.41,
119.64 (d, JF-C = 9.1 Hz), 117.27 (dd, JF-C = 25.7, 18.1 Hz), 111.83 (dd, JF-C = 22.7, 7.6 Hz), 74.76. ESI-
MS calcd for C16H14ClF2N4O4 [M + NH4]+, 399.1, found 399.2.
5.4. Typical procedure for the synthesis of series E
5.4.1. 2,6‐Difluoro-3‐((1‐(4‐bromophenyl)‐5‐oxo‐4H‐1,2,4‐triazol‐3‐yl)methoxy)benzamide. (E1).
The compound A14 (50 mg, 0.12 mmol), ammonium hydroxide (0.3 mL, 2.1 mmol) and EtOH (3 mL)
was added to a sealed tube, and the mixture was stirred at 80℃ for 9 h. Upon completion monitored by
TLC, the reaction solution was concentrated by rotary evaporation, the residue was redissolved in EtOAc
and washed with brine. The organic layer was concentrated and purified on silica gel using DCM/MeOH
(50:1) afforded the compound E1. White solid, yield 64%; Mp: > 260℃; 1H NMR (600 MHz, DMSO-
22

d6) δ 12.53 (s, 1H), 8.14 (s, 1H), 7.87 (s, 1H), 7.84 (d, J = 9.0 Hz, 2H), 7.64 (d, J = 9.0 Hz, 2H), 7.38 (td,
J = 9.3, 5.1 Hz, 1H), 7.12 (td, J = 8.9, 1.8 Hz, 1H), 5.11 (s, 2H). 13C NMR (101 MHz, DMSO-d6) δ
169.69, 161.58, 153.05 (dd, JF-C = 243.4, 7.1 Hz), 152.89, 148.74 (dd, JF-C = 250.5, 9.1 Hz), 144.08,
142.43 (dd, JF-C = 11.1, 3.0 Hz), 137.31, 132.42, 120.30, 117.61, 117.22 (d, JF-C = 10.1 Hz), 111.49 (dd,
JF-C = 23.2, 4.0 Hz), 63.65. ESI-MS calcd for C16H12BrF2N4O3 [M + H]+, 425.0, found 425.2.
5.4.2. 2,6‐Difluoro-3‐((4‐amino‐1‐(4‐bromophenyl)‐5‐oxo‐1,2,4‐triazol‐3‐yl)methoxy)benzamide. (E2)
The compound A14 (50 mg, 0.12 mmol), hydrazine hydrate (0.1 mL, 1.6 mmol) and EtOH (5 mL)
was added to a round-bottom flask, and the mixture was refluxed for 6 h. Upon completion monitored
by TLC, the reaction solution was concentrated by rotary evaporation, the residue was redissolved in
EtOAc and washed with brine. The organic layer was concentrated and purified on silica gel using
DCM/MeOH (25:1) afforded the compound E2. White solid, yield 71%; Mp: 174–175℃; 1H NMR (600
MHz, DMSO-d6) δ 8.14 (s, 1H), 7.90 – 7.83 (m, 3H), 7.70 – 7.66 (m, 2H), 7.43 (td, J = 9.3, 5.0 Hz, 1H),
7.12 (td, J = 8.9, 1.8 Hz, 1H), 5.57 (s, 2H), 5.16 (s, 2H). ESI-MS calcd for C16H16BrF2N6O3 [M + NH4]+,
457.0, found 457.2.
5.4.3.
2,6‐Difluoro-
3‐((1‐(4‐bromophenyl)‐4‐(dimethylamino)‐5‐oxo‐1,2,4‐triazol‐3‐yl)methoxy)benzamide. (E3)
The compound E2 (30 mg, 0.07 mmol), iodomethane (200 mg, 1.4 mmol) K2CO3 (100 mg, 0.7 mmol)
and DMF (2 mL) was added to a sealed tube, and the mixture was heated at 85℃ for 36 h. The resulting
solution was diluted with EtOAc and washed with NH4Cl aq and brine, the organic layer was
concentrated and purified by silica gel using DCM/MeOH (50:1) afforded the compound E3. White solid,
Mp: 167–169℃; 1H NMR (600 MHz, DMSO-d6) δ 8.14 (s, 1H), 7.87 (s, 1H), 7.82 (d, J = 9.0 Hz, 2H),
7.67 (d, J = 9.0 Hz, 2H), 7.44 (td, J = 9.3, 5.1 Hz, 1H), 7.13 (td, J = 8.9, 1.7 Hz, 1H), 5.14 (s, 2H), 2.95
(s, 6H). ESI-MS calcd for C18H20BrF2N6O3 [M + NH4]+, 485.1, found 485.3.
5.4.4. (Z)-ethyl 2-amino-2-(2-(4-bromophenyl)hydrazono)acetate (24)
Add 4-bromoaniline (3.3 g, 19.2 mmol) to a solution of concentrated HCl (6.5 mL), EtOH (10 mL)
and water (3.5 mL). A solution of NaNO2 in water was added dropwise to the mixture at -5℃ and stirred
for further 30 min. A cold solution of 2-chloroacetoacetate (3.16 g, 19.2 mmol), sodium acetate (4.72 g,
57.6 mmol) in EtOH (20 mL) and water (15 mL) was added, and the reaction was stirred at -5℃ for 4 h.
The reaction was quenched with water (100 mL) and stirred overnight. The solid was collected by
filtration and dried in vacuum to furnish intermedia 23 as light yellow solid (4.3 g, 74%), which was used
in the next step without further purification.
The above intermediate 23 (3 g, 9.9 mmol) was dissolved in dioxane (40 mL), then ammonia (6 mL,
40 mmol) was added to the reaction. The mixture was stirred at room temperature for 6 h. Upon
completion, the solution was concentrated and the residue was redissolved in EtOAc, washed with brine.
The organic layer was dried with Na2SO4, concentrated and purified by silica gel using petroleum
ether/EtOAc (4:1) afforded the product 24 as yellow solid (2.56 g, 91%). ESI-MS calcd for
C10H13BrN3O2 [M + H]+, 286.0, found 286.2.
5.4.5. Ethyl 1-(4-bromophenyl)-5-oxo-4,5-dihydro-1H-1,2,4-triazole-3-carboxylate (25)
A solution of triphosgene (2.4 g, 8.1 mmol) in anhydrous THF (10 mL) was added dropwise to a stirred
solution of intermediate 24 (2.3 g, 8.1 mmol) in anhydrous THF (20 mL) at 0°C. After the addition was
23

completed, the mixture was stirred 3 h at room temperature. Then most of the solvent was removed at
reduced pressure, the residue was diluted with EtOAc, and washed with brine. The organic layer was
concentrated, and purified on silica gel. Elution with petroleum ether/EtOAc (2:1) afforded the desired
product 25 as white solid (2.1 g, 83%). ESI-MS calcd for C11H11BrN3O3 [M + H]+, 313.1, found 313.2.
5.4.6. Typical procedure for the synthesis of intermediates (28)
A mixture of intermediate 25 (0.15 g, 0.48 mmol), various alkyl halides (0.7 mmol) and K2CO3 (138
mg, 1 mmol) in DMF (2 mL) was stirred at 35℃ for 6 h. The reaction mixture was then diluted with
EtOAc (20 mL), washed with water and brine, concentrated in rotary evaporator, and purified on silica
gel getting the corresponding intermediates 26 in good yield. Example 26-E5, ESI-MS calcd for
C13H15BrN3O3 [M + H]+, 340.0, found 340.3.
NaBH4 (1.5 mmol) was added to a solution of intermediate 26 (0.4 mmol) dissolved in THF (4 mL)
and MeOH (1 mL), the mixture was sealed with a balloon, and stirred at room temperature overnight.
Upon the completion of reduction, most of the solution was evaporated in vacuum, and the residue 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 and purified by silica gel with DCM/MeOH (60:1)
getting the alcohol intermediate 27. Example 27-E5, 1H NMR (600 MHz, DMSO-d6) δ 7.88 (d, J = 9.0
Hz, 2H), 7.65 (d, J = 9.0 Hz, 2H), 5.76 (t, J = 5.9 Hz, 1H), 4.48 (d, J = 5.9 Hz, 2H), 3.77 (q, J = 7.2 Hz,
2H), 1.26 (t, J = 7.2 Hz, 3H).
To the alcohol intermediate 27 dissolved in DCM at 0℃, was added CBr4 (1.5eq) and PPh3 (3 eq)
successively. The mixture was stirred at room temperature for 6 h. Upon completion, purification was
carried out using silica gel column with appropriate eluent affording the corresponding intermediates 28
in good yield. Example 28-E5, ESI-MS calcd for C11H12Br2N3O [M + H]+, 362.0, found 362.1.
5.4.7. General procedure for the synthesis of E4–E11
A round-bottom flask equipped with a magnetic stirrer was charged with intermediate 5 (50 mg, 0.29
mmol), bromide 28 (0.25 mmol), K2CO3 (138 mg, 1 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) afforded the corresponding
compound in good yield.
5.4.7.1.
2,6‐Difluoro-3‐((1‐(4‐bromophenyl)‐4‐methyl‐5‐oxo‐1,2,4‐triazol‐3‐yl)methoxy)benzamide.
(E4). White solid, Mp: 204–206℃; 1H NMR (600 MHz, DMSO-d6) δ 8.14 (s, 1H), 7.88 (s, 1H), 7.85 (d,
J = 9.0 Hz, 2H), 7.67 (d, J = 9.0 Hz, 2H), 7.46 (td, J = 9.3, 8.5, 4.4 Hz, 1H), 7.14 (td, J = 9.1, 1.6 Hz,
1H), 5.28 (s, 2H), 3.33 (s, 3H). ESI-MS calcd for C17H17BrF2N5O3 [M + NH4]+, 456.0, found 456.2.
5.4.7.2. 2,6‐Difluoro-3‐((1‐(4‐bromophenyl)‐4‐ethyl‐5‐oxo‐1,2,4‐triazol‐3‐yl)methoxy)benzamide. (E5).
White solid, Mp: 181–182℃; 1H NMR (600 MHz, DMSO-d6) δ 8.14 (s, 1H), 7.90 – 7.84 (m, 3H), 7.67
(d, J = 9.0 Hz, 2H), 7.46 (td, J = 9.3, 5.1 Hz, 1H), 7.15 (td, J = 9.0, 1.8 Hz, 1H), 5.29 (s, 2H), 3.81 (q, J
= 7.2 Hz, 2H), 1.29 (t, J = 7.2 Hz, 3H). 13C NMR (151 MHz, DMSO-d6) δ 161.55, 153.06 (dd, JF-C
=
243.1, 7.6 Hz), 151.78, 148.65 (dd, JF-C = 249.2, 9.1 Hz), 143.85, 142.09 (dd, JF-C = 10.6, 3.0 Hz), 137.13,
132.54, 120.38, 117.99, 117.35 (d, JF-C = 19.6 Hz), 117.15 (d, JF-C = 10.6 Hz), 111.55 (dd, JF-C = 22.7,
6.0 Hz), 62.85, 37.34, 14.33. ESI-MS calcd for C18H19BrF2N5O3 [M + NH4]+, 470.1, found 470.2.
24

5.4.7.3. 2,6‐difluoro-3‐((1‐(4‐bromophenyl)‐4‐propyl‐5‐oxo‐1,2,4‐triazol‐3‐yl)methoxy)benzamide. (E6).
White solid, Mp: 108–109℃; 1H NMR (600 MHz, DMSO-d6) δ 8.14 (s, 1H), 7.90 – 7.83 (m, 3H), 7.67
(d, J = 9.0 Hz, 2H), 7.46 (td, J = 9.2, 5.1 Hz, 1H), 7.15 (td, J = 8.9, 1.8 Hz, 1H), 5.28 (s, 2H), 3.73 (t,
2H), 1.74 (h, J = 7.4 Hz, 2H), 0.90 (t, J = 7.4 Hz, 3H). ESI-MS calcd for C19H21BrF2N5O3 [M + NH4]+,
484.1, found 484.3.
5.4.7.4. 2,6‐difluoro-3‐((1‐(4‐bromophenyl)‐4‐butyl‐5‐oxo‐1,2,4‐triazol‐3‐yl)methoxy)benzamide. (E7).
White solid, Mp: 126–128℃; 1H NMR (600 MHz, DMSO-d6) δ 8.14 (s, 1H), 7.90 – 7.83 (m, 3H), 7.67
(d, J = 9.0 Hz, 2H), 7.45 (td, J = 9.2, 5.2 Hz, 1H), 7.15 (td, J = 8.9, 1.8 Hz, 1H), 5.28 (s, 2H), 3.76 (t, J
= 7.6 Hz, 2H), 1.69 (p, J = 7.6 Hz, 2H), 1.32 (h, J = 7.4 Hz, 2H), 0.89 (t, J = 7.4 Hz, 3H). ESI-MS calcd
for C20H20BrF2N4O3 [M + H]+, 481.1, found 481.2.
5.4.7.5.
2,6‐difluoro-
3‐((1‐(4‐bromophenyl)‐4‐(2‐methylpropyl)‐5‐oxo‐1,2,4‐triazol‐3‐yl)methoxy)benzamide. (E8). White
solid, Mp: 157–128℃; 1H NMR (600 MHz, DMSO-d6) δ 8.14 (s, 1H), 7.90 – 7.84 (m, 3H), 7.67 (d, J =
9.0 Hz, 2H), 7.46 (td, J = 9.2, 5.1 Hz, 1H), 7.15 (td, J = 8.9, 1.7 Hz, 1H), 5.26 (s, 2H), 3.61 (d, J = 7.7
Hz, 2H), 2.23 – 2.15 (m, 1H), 0.90 (d, J = 6.7 Hz, 6H). ESI-MS calcd for C20H23BrF2N5O3 [M + NH4]+,
498.1, found 498.2.
5.4.7.6.
2,6‐difluoro-
3‐((1‐(4‐bromophenyl)‐4‐(3‐methylbutyl)‐5‐oxo‐1,2,4‐triazol‐3‐yl)methoxy)benzamide. (E9). White
solid, Mp: 150–151℃; 1H NMR (600 MHz, DMSO-d6) δ 8.14 (s, 1H), 7.89 – 7.84 (m, 3H), 7.67 (d, J =
9.0 Hz, 2H), 7.45 (td, J = 9.4, 5.3 Hz, 1H), 7.15 (td, J = 8.8, 1.8 Hz, 1H), 5.28 (s, 2H), 3.77 (t, 2H), 1.64
– 1.56 (m, 3H), 0.90 (d, J = 6.2 Hz, 6H). ESI-MS calcd for C21H25BrF2N5O3 [M + NH4]+, 512.1, found
512.3.
5.4.7.7. 2,6‐difluoro-3‐((1‐(4‐bromophenyl)‐4‐octyl‐5‐oxo‐1,2,4‐triazol‐3‐yl)methoxy)benzamide. (E10).
White solid, Mp: 153–154℃; 1H NMR (600 MHz, DMSO-d6) δ 8.12 (s, 1H), 7.89 – 7.83 (m, 3H), 7.67
(d, J = 9.0 Hz, 2H), 7.45 (td, J = 9.3, 5.0 Hz, 1H), 7.15 (td, J = 9.0, 1.8 Hz, 1H), 5.27 (s, 2H), 3.75 (t, J
= 7.6 Hz, 2H), 1.70 (p, J = 7.7 Hz, 2H), 1.30 – 1.19 (m, 10H), 0.84 (t, J = 7.0 Hz, 3H). ESI-MS calcd for
C24H31BrF2N5O3 [M + NH4]+, 554.2, found 554.3.
5.4.7.8.
2,6‐difluoro-3‐((1‐(4‐bromophenyl)‐4‐benzyl‐5‐oxo‐1,2,4‐triazol‐3‐yl)methoxy)benzamide.
(E11). White solid, Mp: 160–161℃; 1H NMR (600 MHz, DMSO-d6) δ 8.11 (s, 1H), 7.91 – 7.84 (m, 3H),
7.70 – 7.66 (m, 1H), 7.51 – 7.46 (m, 1H), 7.35 – 7.27 (m, 6H), 7.13 – 7.08 (m, 1H), 5.20 (s, 2H), 5.02 (s,
2H). ESI-MS calcd for C23H21BrF2N5O3 [M + NH4]+, 532.1, found 532.4.
5.5. Typical procedure for the synthesis of series F
5.5.1. Ethyl 4-(3-carbamoyl-2,4-difluorophenoxy)-3-oxobutanoate (29)
To a mixture of potassium hydroxide (224 mg, 4.0 mmol) in 3 mL of DMSO was dropwise added the
25

intermediate 5 (346 mg, 2.0 mmol). The mixture was stirred at room temperature for 30 min and then
ethyl 4-chloroacetoacetate (329 mg, 2.0 mmol) was added. The mixture was stirred at room temperature
overnight and then acidified by addition of hydrochloric acid (1 M). The mixture was extracted with
EtOAc and the organic layer was washed with water and brine, dried over Na2SO4, then concentrated in
vacuum evaporator. The crude product was purified by silica gel chromatography with DCM/MeOH
(80:1) getting the intermediate 29 (287 mg, 48%). White solid, 1H NMR (600 MHz, DMSO-d6) δ 8.18
(s, 1H), 7.88 (s, 1H), 7.11 (td, J = 9.2, 5.2 Hz, 1H), 7.06 (td, J = 8.9, 1.5 Hz, 1H), 5.04 (s, 2H), 4.11 (q,
J = 7.1 Hz, 2H), 3.70 (s, 2H), 1.19 (t, J = 7.1 Hz, 3H). ESI-MS calcd for C13H14F2NO5 [M + H]+, 302.3,
found 302.4.
5.5.2. General procedure for the synthesis of F1–F4
A solution of intermediate 29 (50 mg, 0.166 mmol) in 3 mL of EtOH was added the corresponding
substituted phenylhydrazine hydrochloride (0.166 mmol), and the mixture was heated at 75℃ for 5 h.
the resulting solution was concentrated and purified by silica gel with DCM/MeOH (gradient from 150:1
to 60:1) getting the desired compounds in a yield about 30%.
5.5.2.1. 2,6-Difluoro-3-((1-(4-chlorophenyl)-5-oxo-4,5-dihydro-1H-pyrazol-3-yl)methoxy)benzamide.
(F1). Off-white solid, Mp: 178–179℃; 1H NMR (600 MHz, DMSO-d6) δ 8.15 (s, 1H), 7.87 (s, 1H), 7.77
(d, J = 8.9 Hz, 2H), 7.52 (d, J = 8.9 Hz, 2H), 7.36 (td, J = 9.3, 5.2 Hz, 1H), 7.08 (td, J = 9.0, 1.8 Hz, 1H),
5.65 (s, 1H), 5.05 (s, 2H). 13C NMR (151 MHz, DMSO-d6) δ 161.80, 154.12, 152.36 (dd, JF-C = 241.6,
7.6 Hz), 148.46 (dd, JF-C = 249.2, 9.1 Hz), 148.21, 143.13 (dd, JF-C = 10.6, 3.0 Hz), 137.94, 130.30,
129.39, 122.89, 117.06 (dd, JF-C = 27.2, 19.6 Hz), 116.28 (d, JF-C = 13.6 Hz), 111.24 (dd, JF-C = 24.2,
7.6 Hz), 88.06, 66.29. ESI-MS calcd for C17H13ClF2N3O3 [M + H]+, 380.1, found 380.3.
5.5.2.2. 2,6-Difluoro-3-((1-(4-bromophenyl)-5-oxo-4,5-dihydro-1H-pyrazol-3-yl)methoxy)benzamide.
(F2). Off-white solid, Mp: 112–114℃; 1H NMR (600 MHz, DMSO-d6) δ 8.16 (s, 1H), 7.88 (s, 1H), 7.71
(d, J = 8.9 Hz, 2H), 7.65 (d, J = 8.9 Hz, 2H), 7.36 (td, J = 9.3, 5.1 Hz, 1H), 7.08 (td, J = 8.9, 1.7 Hz, 1H),
5.65 (s, 1H), 5.05 (s, 2H). 13C NMR (101 MHz, DMSO-d6) δ 161.79, 154.05, 152.39 (dd, JF-C = 242.4,
7.1 Hz), 148.50 (dd, JF-C = 249.5, 9.1 Hz), 148.29, 143.21 (dd, JF-C = 10.1, 3.0 Hz), 138.37, 132.33,
123.22, 118.59, 117.12 (dd, JF-C = 25.3, 20.2 Hz), 116.45 (d, JF-C = 9.1 Hz), 111.41 (dd, JF-C = 23.2, 3.0
Hz), 88.04, 66.34. ESI-MS calcd for C17H13BrF2N3O3 [M + H]+, 424.0, found 424.2.
5.2.2.3.
2,6-Difluoro-3-((1-(4-(tert-butyl)phenyl)-5-oxo-4,5-dihydro-1H-pyrazol-3-
yl)methoxy)benzamide. (F3). Off-white solid, Mp: 94–96℃; 1H NMR (600 MHz, DMSO-d6) δ 11.68 (s,
1H), 8.15 (s, 1H), 7.87 (s, 1H), 7.61 (d, J = 8.8 Hz, 2H), 7.47 (d, J = 8.8 Hz, 2H), 7.37 (td, J = 9.3, 5.2
Hz, 1H), 7.08 (td, J = 9.0, 1.7 Hz, 1H), 5.62 (s, 1H), 5.04 (s, 2H), 1.30 (s, 9H). ESI-MS calcd for
C21H22F2N3O3 [M + H]+, 402.4, found 402.4.
5.5.2.4.
2,6-Difluoro-3-((1-(4-butylphenyl)-5-oxo-4,5-dihydro-1H-pyrazol-3-yl)methoxy)benzamide.
1
(F4). Off-white solid, Mp: 185–186℃; H NMR (600 MHz, DMSO-d6) δ 11.71 (s, 1H), 8.15 (s, 1H),
7.87 (s, 1H), 7.59 (d, J = 8.4 Hz, 2H), 7.37 (td, J = 9.4, 5.2 Hz, 1H), 7.27 (d, J = 8.5 Hz, 2H), 7.08 (td, J
= 9.0, 1.8 Hz, 1H), 5.62 (s, 1H), 5.04 (s, 2H), 2.60 (t, J = 7.7 Hz, 2H), 1.56 (p, J = 7.5 Hz, 2H), 1.31 (h,
J = 7.4 Hz, 2H), 0.90 (t, J = 7.4 Hz, 3H). ESI-MS calcd for C21H22F2N3O3 [M + H]+, 402.4, found 402.4.
26

5.6. 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. 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℃. Turbidity was observed,
the last well with no growth of bacterial was recorded to represent the MIC value.
5.7. 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).
5.8. 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 A14 or comparator drug. Cultures were incubated
at 37℃ with shaking. At appropriate time intervals, 10 µL samples were removed for serial dilution in
90 µL volumes of CAMH broth and 10 µL volumes from three dilutions were spread on to TSA. All
TSA plates were incubated at 37℃, and the CFU/mL at each time point was determined by counting
colonies after 24 h.
5.9. Visualization of bacterial morphology
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.
5.10. FtsZ polymerization assay
B. subtilis FtsZ was cloned, overexpressed, and purified as described previously [26]. 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
)
[13]. Test compound or comparator drug (at concentrations ranging from 0 to 2.5 µg/mL) were combined
27

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, flatbottom microtiter
plates, and polymerization was continuously monitored at 25℃ by measuring A340 in a Thermo Multiskan
FC plate reader over a time period of 30 min.
5.11. Compound’s stability studies in vitro mouse plasma
Experimental solutions of A14 or E1 at concentrations of 25 µg/mL were prepared from 2.56 mg/mL
DMSO stock solutions in freshly prepared mouse plasma, and then incubated at 37℃. At different time
point, 20 µL of solution was added to 60 µL of methanol, and centrifuged at 4000 rpm for 15 min. The
supernatants of each sample was then assessed using reverse-phase HPLC as described below. Mouse
plasma was obtained by drawing blood, anticoagulation and centrifugation.
5.12. Compound’s stability studies in vitro mouse liver microsome
For each compound, the experimental solution was prepared by adding 1.5 µL of compound solution
(100 µm in DMSO) to 148.5 µL of 0.5 mg/mL liver microsome. 5 µL of NADPH agent (10 mM) was
added to initiate the reaction, and the solution was incubated at 37℃. At different time point, 20 µL of
solution was added to 40 µL acetonitrile, and centrifuged at 10000 rpm for 10 min. The supernatants of
each sample was then assessed using reverse-phase HPLC.
5.12. Reverse-phase HPLC
For the HPLC measurement, a reverse-phase Diamonsil C18 (2) column was used on an Agilent 1200
series liquid chromatograph equipped with a G1315C DAD detector (set at 254 nm). The column size
was 150 × 4.6 mm, with the particle and pore sizes being 5 µm and 100 Å, respectively. Column
temperature was not controlled under room temperature. A 20 µL sample of each experimental solution
was injected and a flow rate of 1 mL/min was applied, along with a gradient of 10–90% acetonitrile
[containing 0.1% (v/v) trifluoroacetic acid (TFA)] and water in the mobile phase running for 20 min. The
retention time of A14 and E1 was about 14.5min and 12.2min, respectively. Peak areas were determined
using the ChemStation software package.
5.13. 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 [31]. Cells were plated at 104 cell/well in a 96-well plate for overnight incubation, and then,
gradient volumes of compound were added. After 48-h incubation, 20 µL of MTT solution (5 mg/mL)
was added and incubated for 3 h at 37℃. The MTT solution was then removed, substituted with 150 µL
DMSO for 10 min, and the absorbance was measured at 490 nm in a Thermo Multiskan FC plate reader.
5.14. Computational study
Docking simulations were performed using Autodock (version 4.2) docking package [30]. The crystal
28

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).
Conflicts 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), Key Research and Development Project of Shandong Province (2017CXGC1401).
Appendix A. Supplementary data
Supplementary data to this article can be found online at
29