Design, synthesis of novel 4,5-dihydroisoxazole-containing benzamide derivatives as highly potent FtsZ inhibitors capable of killing a variety of MDR Staphylococcus aureus

B i o o r g an i c &M e d i c i n a l C h e m i s tr y 2 8 (2 020 )1 15729

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Bioorganic & Medicinal Chemistry

journal homepage: www.elsevier.com/locate/bmc

Design, synthesis of novel 4,5-dihydroisoxazole-containing benzamide

derivatives as highly potent FtsZ inhibitors capable of killing a variety of

MDR Staphylococcus aureus

Di Song, Fangchao Bi, Nan Zhang, Yinhui Qin, Xingbang Liu, Yuetai Teng, Shutao Ma^⁎

Department of Medicinal Chemistry, Key Laboratory of Chemical Biology (Ministry of Education), School of Pharmaceutical Sciences, Cheeloo College of Medicine,

Shandong University, 44 West Wenhua Road, Jinan 250012, China

A R T I C L E I N F O

A B S T R A C T

Keywords:

Antibiotic resistance among clinically signiﬁcant bacterial pathogens, such as methicillin-resistant

Staphylococcus aureus (MRSA) and vancomycin-resistant S. aureus (VRSA) is becoming a prevalent threat to

public health, and new antibacterial agents with novel mechanisms of action hence are in an urgent need. As a

part of continuing eﬀort to develop antibacterial agents, we rationally designed and synthesized two series of

4,5-dihydroisoxazol-5-yl and 4,5-dihydroisoxazol-3-yl-containing benzamide derivatives that targeted the bac-

terial cell division protein FtsZ. Evaluation of their activity against a panel of Gram-positive and -negative

pathogens revealed that compound A16 possessing the 4,5-dihydroisoxazol-5-yl group showed outstanding

antibacterial activity (MIC, ≤0.125–0.5 μg/mL) against various testing strains, including methicillin-resistant,

penicillin-resistant and clinical isolated S. aureus strains. Besides, further mouse infection model revealed that

A16 could be eﬀective in vivo and non-toxic to Hela cells. Finally, a detailed discussion of structure-activity

relationships was conducted, referring to the docking results. It is worth noting that substituting a 4,5-dihy-

droisoxazole ring for the isoxazole ring not only broadened the antibacterial spectrum but also resulted in a

signiﬁcant increase in antibacterial activity against S. aureus strains. Taken together, these results suggest a

promising chemotype for the development of new FtsZ-targeting bactericidal agents.

4,5-Dihydroisoxazole-containing benzamide

Design and synthesis

Antibacterial activity

FtsZ inhibitor

Structure-activity relationships

1. Introduction

methoxybenzamide (3-MBA).^10,11Further investigations would be fo-

cused on the optimizing its drug-like properties.^12–15

There is a serious threat to public health due to the increasing of

antimicrobial resistance (AMR),¹and the innovation rate of new anti-

biotics cannot keep up with the emergence of antibiotic-resistant bac-

teria, which deﬁnitely aggravates this problem.²Therefore, to address

the increasing serious antibiotic resistance, new classes of molecules

with novel action mode or newer generations of previously used anti-

biotics should be urgently developed.³

Most benzamide derivatives that have been studied contain parallel-

connected or series-connected heteroaromatic rings which are planar

rigid structures at the 3-O-position, such as PC190723 and compound 1

(Fig. 1). Considering the amino acid residues on the inner surface of the

binding pocket of FtsZ are less likely to shape ﬂat regions, we surmise

that noncoplanar heterocyclic structure could form more suitable in-

teraction with the protein FtsZ though there is an induced ﬁt binding

mode between molecule and target site. According to the above hy-

pothesis and a continuation of eﬀort to further develop benzamide

derivatives as antibacterial agents, we tried to introduce a noncoplanar

heterocyclic structure to increase the ﬂexibility and conformational

diversity of the target molecules, in order to form more changeable

molecular skeleton. We previously reported on some potent anti-

bacterial compounds by introducing an isoxazole ring to connect the

benzamide and the aromatic moieties.¹⁶In this paper, we further de-

Filamentous temperature-sensitive mutant Z (FtsZ) is a key protein

for bacterial cell division and an attractive target for new antibiotics

development.⁴A number of FtsZ-interacting compounds have been

discovered, some of which possess potent antibacterial activity, just

listing several of them being just listed for example in Fig. 1.^5–9Among

these FtsZ-targeting compounds, benzamide derivatives family is an

attractive point and has been extensively investigated by diﬀerent re-

search groups. This continuous research ﬁnally culminated in the de-

velopment of PC190723 which was derived from the weak inhibitor 3-

signed

a series of 4,5-dihydroisoxazol-5-yl-containing benzamide

^⁎Corresponding author.

E-mail address: mashutao@sdu.edu.cn (S. Ma).

https://doi.org/10.1016/j.bmc.2020.115729

Received 3 June 2020; Received in revised form 30 July 2020; Accepted 19 August 2020

A v a i l a b l e o n l i n e 2 7 A u gu s t 2 020

D. Song, et al.

Bioorganic&MedicinalChemistry28(2020)115729

Fig. 1. Chemical structures of some FtsZ inhibitors.

Fig. 2. Brief design process of the 4,5-dihydroisoxazole benzamide derivatives.

derivatives (series A) by substituting a nonplanar 4,5-dihydroisoxazole

ring for the isoxazole ring in the structure. This structural change alters

the planarity of ﬁve-numbered isoxazole heterocycle and can cause a

noncoplanar skeleton and more diverse conformation when the CeO or

CeC bond in the structure twirls. On this basis, we designed another

series of 4,5-dihydroisoxazol-3-yl-containing benzamide derivatives

(series B) by altering the connection of the 4,5-dihydroisoxazole ring in

the structure. The above brief design strategy is illustrated in Fig. 2.

Here we reported the synthesis and in vitro antibacterial activity of the

series A and B. The cell morphology and the FtsZ polymerization as on-

target eﬀect were also performed. The outstanding compounds were

selected to further evaluate their plasma stability, in vivo antimicrobial

activity and cytotoxicity, and ultimately, compound A16 was identiﬁed

to be a novel and potent lead antibacterial agent as an FtsZ inhibitor.

2.2. Synthesis of 4,5-dihydroisoxazol-3-yl-containing benzamide

derivatives (series B)

The synthetic route for series B is listed in Scheme 2. Commercially

available or synthesized benzaldehydes 8 were converted to corre-

sponding styrenes 10 which were then cyclizated with ethyl ni-

troacetate to aﬀord the intermediates 11.¹⁷Reduction with NaBH₄and

then bromination provided the intermediates 12, which were then

condensated with 6 to aﬀord the 4,5-dihydroisoxazol-3-yl-containing

benzamide derivatives B1-B18.

2.3. Analysis and determination of the absolute conﬁguration of

representative compound A16

Representative compound A16 that incorporates a chiral carbon

atom, has two isomers of R- and S-conﬁgurations. The high-perfor-

mance liquid chromatography (HPLC) diagram demonstrated that the

two enantiomers of A16 had the same peak area, and we successfully

resolved the two enantiomers by preparative HPLC. Then, the absolute

conﬁgurations of the two enantiomers were further conﬁrmed by the

agreement exhibiting in experimental and calculated electronic circular

dichroism (ECD) spectra (Fig. 3) (Supplementary data, Figs. S1-S3).

2. Chemistry

2.1. Synthesis of 4,5-dihydroisoxazol-5-yl-containing benzamide

derivatives (series A)

The synthetic route for the series A is shown in Scheme 1. Com-

mercially available 2,4-diﬂuorophenol 3 was protected by benzyl

group, and then treated with dry ice in the presence of n-BuLi, giving

benzoic acid 4. The acid 4 was converted to benzamide 5 using oxalyl

chloride, followed by ammonium carbonate. After deprotection of the

benzyl group, alkylation of 6 with 3-bromopropylene aﬀorded key in-

termediate 7. Finally, cyclization of 7 with various oximes 9 that were

prepared from commercially available or synthesized aldehyde 8 in the

presence of chloramine-T gave the 4,5-dihydroisoxazol-5-yl-containing

benzamide derivatives A1- A27.

3. Results and discussion

3.1. In vitro antibacterial activity of series A and B

The synthesized compounds shown in Scheme 1 and 2 were tested

for their in vitro antibacterial activity through broth microdilution

procedures described in the Clinical and Laboratory Standards Institute

2

D. Song, et al.

Bioorganic&MedicinalChemistry28(2020)115729

Scheme 1. Synthetic route for series A. Reagents and conditions: (i) benzyl chloride, K₂CO₃, NaI, MeCN, 45 °C; (ii) n-BuLi, dry ice, THF, 50 °C to rt; (iii) oxalyl

chloride, DMF, DCM, rt; (iv) ammonium carbonate, DCM, rt; (v) 10% Pd/C, ammonium formate, EtOH, 65 °C; (vi) 3-bromopropyne, K₂CO₃, DMF, 35 °C; (vii) NaOH,

H₂O/EtOH, rt; (viii) chloramine-T, EtOH, rt.

Scheme 2. Synthetic route for series B. Reagents and conditions: (i) methyltriphenylphosphonium bromide, NaH, THF, N₂protection; 0 °C to rt; (ii) ethyl ni-

troacetate, triethylenediamine, EtOH, 78 °C; (iii) NaBH₄, THF/MeOH, rt; (iv) Ph₃P, CBr₄, DCM, rt; (v) K₂CO₃, DMF, 35 °C.

Fig. 3. Experimental ECD (red) and calculated ECD (black) of A16-S.

3

D. Song, et al.

Bioorganic&MedicinalChemistry28(2020)115729

Table 1

The in vitro antibacterial activity of the 4,5-dihydroisoxazol-5-yl-containing benzamide derivatives (series A).

Comp

Minimum inhibitory concentration/MIC (μg/mL)

B. subtilis

B. pumilus

S. aureus

S. aureus CI^cE. coli ATCC25922

P. aeruginosa

ATCC9372

CMCC63202

ATCC25923

ATCC43300^a

PR^b

ATCC27853

A1

A2

16

64

> 64

16

> 64

32

> 64

32

NT

4

> 64

8

> 64

4

16

64

A3

2

8

A4

8

32

A5

4

2

> 64

32

16

32

A6

> 64

64

> 64

64

> 64

64

A7

8

A8

2

4

32

A9

16

64

> 64

32

> 64

16

> 64

64

A10

A11

A12

A13

A14

A15

A16

A17

A18

A19

A20

A21

A22

A23

A24

A25

A26

A27

A16-R

A16-S

PC

32

> 64

8

16

32

> 64

16

0.5

≤0.125

0.25

0.5

> 64

8

2

8

≤0.125

> 64

32

4

16

4

2

4

0.5

0.25

> 64

32

0.5

NT

2

> 64

32

> 64

32

8

32

4

8

4

16

64

> 64

32

4

2

16

4

> 64

64

> 64

64

> 64

64

16

64

> 64

0.5

> 64

4

> 64

2

> 64

1

0.25

≤0.125

0.5

2

≤0.125

0.5

0.25

1

≤0.125

1

0.25

1

≤0.125

1

CIP^d

LIN^e

Ery^f

8

16

> 64

1

16

0.5

≤0.125

1

2

1

> 64

16

> 64

32

≤0.125

> 64

a

S. aureus ATCC43300: methicillin-resistant strain.

S. aureus PR: penicillin-resistant strain.

b

c

d

e

f

S. aureus CI: clinical isolated strain, not characterized.

CIP: ciproﬂoxacin.

LIN: linezolid.

ERY: erythromycin.

(CLSI) guidelines.¹⁸MIC (minimal inhibitory concentration) values for

all the compounds were determined on a panel of sensitive and resistant

bacterial strains. PC (PC190723), CIP (ciproﬂoxacin), LIN (linezolid)

and ERY (erythromycin) were included as references. The MIC results

shown in Table 1 and Table 2 indicates that some of the 4,5-dihy-

droisoxazole-containing benzamide derivatives display superior or

equivalent eﬃcacy to the clinical antibiotics and PC evaluated in this

study.

of A16. There is no doubt that A16 is the best compound in the series A

due to its outstanding activity against all the tested Gram-positive

bacteria strains.

Although A16-R and A16-S are a pair of enantiomers, A16-S dis-

played not only more potent antibacterial activity than A16-R, but also

increased activity compared with A16. In particular, A16-S exhibited

the best antibacterial activity against all the tested Gram-positive pa-

thogens with the MIC values ranging from ≤ 0.125 to 0.25 μg/mL,

better than or equal to PC, CIP, LIN or ERY. For example, A16-S ex-

hibited excellent activity against B. subtilis ATCC9372 and B. pumilus

CMCC63202 with the MIC values of ≤ 0.125 μg/mL, which were equal

to that of ERY. Moreover, the antibacterial activity of A16-S against

three resistant S. aureus strains were stronger than that of the positive

controls. However, A16-S could not inhibit the growth of Gram-nega-

tive pathogens. The above results indicate that A16-S plays a signiﬁcant

role in the antibacterial activity of A16, which is worthy of further

study.

In the series A, A14 demonstrated the best activity against B. subtilis

ATCC9372 with a MIC value ≤ 0.125 μg/mL, comparable to that of CIP

and ERY. Besides, A13, A15, A16 and A17 also showed excellent ac-

tivity against B. subtilis ATCC9372, better than or equal to PC and LIN.

A14, A15, A16 and A17 demonstrated potent activity against B. pumilus

CMCC63202 with the MIC values ≤ 0.125 μg/mL, better than PC

(0.5 μg/mL), CIP (8 μg/mL) and LIN (1 μg/mL). In particular, A16 not

only displayed the best antibacterial activity against S. aureus

ATCC25923 with a low MIC value of 0.5 μg/mL but also eﬀectively

inhibited the growth of three resistant S. aureus strains. For example,

the antibacterial activity of A16 against S. aureus ATCC43300 was 4-,

64- and 8-fold more active than that of the positive controls PC (1 μg/

mL), CIP (16 μg/mL) and LIN (2 μg/mL). Moreover, A16 showed re-

markable activity against S. aureus PR with a MIC value of 0.5 μg/mL,

which was 2- and 2-fold more potent than that of PC and LIN. Notably,

although the clinical isolated S. aureus (CI) pathogen had a deﬁnite

resistance to CIP and ERY, it could not escape suppression at 0.5 μg/mL

In the series B, B13 derived from exchanging the N and O atom’s

relative position in the 4,5-dihydroisoxazole ring was found to display

excellent activity against B. subtilis ATCC9372 with a MIC value

of ≤ 0.125 μg/mL, comparable to ERY. B3, B4, B10, B11 and B12

showed moderate antibacterial activity against B. subtilis ATCC9372

with the MIC values of 2–4 μg/mL. Notably, B4 and B13 exerted

stronger activity against B. pumilus CMCC63202 than the others with

the MIC values 0.5 μg/mL, better than or equal to that of LIN (1 μg/mL)

4

D. Song, et al.

Bioorganic&MedicinalChemistry28(2020)115729

Table 2

The in vitro antibacterial activity of the 4,5-dihydroisoxazol-3-yl-containing benzamide derivatives (series B).

Comp

Minimum inhibitory concentration/MIC (μg/mL)

B. subtilis

B. pumilus

S. aureus

S. aureus ATCC43300^a

S. aureus

S. aureus CI^cE. coli ATCC25922

P. aeruginosa

ATCC9372

CMCC63202

ATCC25923

PR^b

ATCC27853

B1

B2

16

32

> 64

16

> 64

64

> 64

32

> 64

64

NT

16

> 64

8

> 64

4

64

B3

4

B4

2

0.5

32

16

32

B5

16

> 64

64

> 64

64

> 64

32

> 64

32

NT

1

B6

16

B7

8

64

32

B8

> 64

64

> 64

64

B9

16

B10

B11

B12

B13

B14

B15

B16

B17

B18

PC

4

16

4

8

64

2

4

64

32

≤0.125

> 64

64

0.5

8

4

> 64

64

> 64

1

> 64

64

> 64

1

16

8

32

64

0.5

2

0.5

1

CIP^d

LIN^e

Ery^f

8

16

> 64

1

16

0.5

≤0.125

1

2

1

> 64

16

> 64

32

≤0.125

> 64

a

S. aureus ATCC43300: methicillin-resistant strain.

S. aureus PR: penicillin-resistant strain.

b

c

d

e

f

S. aureus CI: clinical isolated strain, not characterized.

CIP: ciproﬂoxacin;

LIN: linezolid;

ERY: erythromycin.

corresponding MIC values. Clinical drug LIN and clarithromycin (CLA)

were used as controls in the S. aureus determinations. The results shown

in Table 3 indicate that the MBC/MIC ratios of LIN and CLA are ≥ 32,

which was observed as bacteriostatic activity. Signiﬁcantly, A16 ex-

hibited almost equivalent MBC and MIC values (i.e., MBC/MIC ratio of

1 and 2) for both susceptible and resistant S. aureus, which indicates its

bactericidal mode clearly.

Table 3

Comparison of MIC and MBC Values for A16 in four strains of S. aureus.

Comp

MBC (μg/mL)

MIC (μg/mL)

MBC/MIC

Susceptible S. aureus (ATCC25923)

A16

LIN

0.5

> 64

4

0.5

2

1

> 32

32

CLA

0.125

Methicillin-resistant S. aureus (ATCC43300)

3.3. Kinetics of the bactericidal activity

A16

LIN

0.5

0.25

2

> 64

N/A

> 32

N/A

CLA

> 64

In view of its bactericidal nature, we next investigated the time-kill

curves of A16 on a clinical isolated S. aureus strain. As shown in Fig. 4,

the growth trend of the bacterial strain was suppressed obviously when

incubated with 0.5 × MIC of A16 compared with no drug treated

curve. At the concentration of 1 × MIC, 2 × MIC and 4 × MIC, A16

could reduce the viable counts below the lowest detectable limit (10³

CFU/mL) after incubation for 24, 12 and 9 h, respectively. The time-kill

curves of diﬀerent concentration gradient indicate that the speed at

which A16 killed S. aureus is positively correlated with its concentra-

tion. The higher concentration resulted in the faster rate of killing.

Penicillin-resistant S. aureus

A16

LIN

1

0.5

1

2

> 64

N/A

CLA

N/A

> 64

Clinical isolated S. aureus

A16

LIN

0.5

64

0.5

1

64

CLA

N/A

> 64

N/A

^aLIN and CLA are included as control agents.

^bN/A, not applicable.

3.4. Eﬀects on the morphology of B. subtilis cells and S. aureus cells

and PC (0.5 μg/mL). Besides, B13 demonstrated moderate antibacterial

activity against three S. aureus strains with the MIC values of 4–8 μg/

mL, which is slightly lower than that of PC and LIN.

We have known that FtsZ-targeting compounds such as PC190723

can inhibit bacterial cell division and result in bacterial cell elongation

or enlargement.¹⁹Thus we next explored the eﬀect of A16 on the

bacterial cell morphology through microscopic observation to conﬁrm

whether its mode of action was associated with alteration of FtsZ. As

shown in Fig. 5, compared with untreated bacterial cells, A16 elongates

the cell length of B. subtilis and enlarges the cell size of S. aureus ATCC

CI signiﬁcantly at 0.5 × MIC concentration, which is consistent with

other reported 3-substituted benzamide derivatives.^11,20Fig. 5 (A) in-

dicates that the normal cells of B. subtilis have characteristic short rod

morphology with the average length of 2.28 μm (n = 15, Standard

3.2. Bactericidal or bacteriostatic assay

The primary in vitro antibacterial activity of A16 highlighted by the

results in Table 1 prompted us to further investigate whether its activity

was bactericidal or bacteriostatic. Therefore, the MBC (minimal

bactericidal concentration) values of A16 against susceptible

(ATCC25923), methicillin-resistant (ATCC43300), penicillin-resistant

and clinical isolated S. aureus stains were tested and compared with its

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D. Song, et al.

Bioorganic&MedicinalChemistry28(2020)115729

Fig. 4. Time-kill curves for clinical isolated S. aureus at diﬀerent concentrations of A16.

Deviation = 0.29). After treatment with A16 (0.25 μg/mL), almost all

the bacteria showed iconic elongated phenotype in rod-shaped B. sub-

tilis cells with the average length of 29.33 μm (n = 15, SD = 4.56)

(Fig. 5 (B) and 5 (C)). Fig. 5 (D) points out that the normal cell of S.

aureus ATCC CI is spherical in shape with the average diameter of

0.79 μm (n = 15, SD = 0.05). After treatment with A16 (0.25 μg/mL),

S. aureus ATCC CI cells became large with the average diameter of

2.41 μm (n = 15, SD = 0.49) (Fig. 5 (E) and 5 (F)). The above results

conﬁrm that A16 interferes with the normal function of FtsZ, further

leads to improper cell division and ﬁnally causes bacterial cell death.

assay of B. subtilis FtsZ (BsFtsZ) at diﬀerent concentrations of A16 were

performed utilizing a microplate reader at 340 nm (A₃₄₀). The time-

dependent A₃₄₀proﬁles illustrated in Fig. 6(A) indicates that A16 in-

creases the kinetics of BsFtsZ polymerization, with the magnitude of

stimulatory eﬀect in a concentration-dependent manner. As expected,

neither CIP, LIN nor vehicle impacted the polymerization of BsFtsZ to

any signiﬁcant degree. Fig. 6(B) shows that A16-S possesses a stronger

impact than A16-R and A16 on the FtsZ polymerization, which is

correlated with its antibacterial activity. In contrast, A26_predisplays a

much weaker stimulatory eﬀect than A16 at the same concentration,

which tallies with its antibacterial activity as well.

3.5. Stimulation of FtsZ polymerization dynamics

3.6. Stability of A16 in mouse plasma in vitro

In-depth studies on the dynamic of FtsZ protein have shown that

some FtsZ-targeting agents can stimulate FtsZ polymerization dynamics

and stabilize FtsZ polymeric structures, which contribute to the anti-

bacterial activity of FtsZ inhibitors.^21,22Therefore, the polymerization

To evaluate the primary pharmacokinetic proﬁle of A16, the time-

dependent stability of A16 in the mouse plasma at 37 °C was assessed in

vitro. The data of liquid chromatography-tandem mass spectrometry

Fig. 5. Eﬀects of compound A16 on the bacterial cell morphology. B. subtilis cells were grown in the absence (A) or in the presence (B and C) of A16 at 0.25 μg/mL. S.

aureus CI cells were grown in the absence (D) or in the presence (E and F) of A16 at 0.25 μg/mL. Scale Bar = 10 μm.

6

D. Song, et al.

Bioorganic&MedicinalChemistry28(2020)115729

Fig. 6. The impact of A16 on the polymerization of BsFtsZ. (A) Eﬀect on the polymerization of FtsZ in the absence or presence of A16 at the concentration of 2.5, 5

and 10 μg/mL. CIP and LIN were included as negative (non-FtsZ-targeting) controls. (B) Eﬀect on the polymerization of FtsZ in the absence or in the presence of 5 μg/

mL of compound A26_pre, A16, A16-R and A16-S.

cells, with a 50% inhibitory concentration (IC₅₀) of > 64 μg/mL which

is much higher than its antibacterial MIC values. Albeit testing a single

mammalian cell line, based on this initial result obtained we found that

A16 was associated with a signiﬁcant therapeutic window. Further

studies on additional mammalian and human cell lines will be directed

toward assessing the general nature of this result.

3.9. The docking analysis of A16, B13 and A26_prewith FtsZ

Docking study was undertaken to rationalize the observed anti-

bacterial activity and investigate the interactions of the representative

compounds with the FtsZ binding site. The docking analysis of A16,

B13 and A26_prewere explored and contrasted utilizing a reported

crystal structure of SaFtsZ (PDB code: 3vob)²⁴and Ledock. The docking

results are shown in Fig. 8. Both A16, B13 and A26_prewere well

Fig. 7. Eﬃcacy of A16 in mouse model of blood infection with S. aureus. The

combined with FtsZ protein. The molecular conformations of A16,

CFU numbers of 0.1 mL blood was recorded and LIN was included as a positive

A26_preand PC190723 almost overlapped with each other (Fig. 8A),

control.

which indicate that the binding site of A16 is in good agreement with

PC190723. Their three pharmacophores of A16 are connected by the

(LC-MS) demonstrated an almost constant peak area, which corre-

sponded with the molecular mass of A16. The above results revealed

that A16 did not get oxidized to the corresponding isoxazole product

and remained basically undegraded in the mouse plasma over 24 h,

which indicates its excellent chemical stability in plasma

(Supplementary data, Figs. S4-S13).

middle OeC bond, and the whole molecule presents an M−shaped

conformation (Fig. 8A).¹⁵The benzamide moiety of the above three

compounds forms the same hydrogen bonds and ion–dipole forces with

the surrounding amino acid residues Asn263, Thr296, Asn208 and

Leu209, as well as Ca⁺, which are critical forces to grasp the FtsZ

protein. The series-connected heterocycle on the right extends into a

hydrophobic cavity. Comparing the docking conformations of A16 and

B13 (Fig. 8C and 8D), a conventional hydrogen bonding interaction was

predicted between the oxygen atom in the 4,5-dihydroisoxazole of A16

and Gly196. Besides, A16 could also form hydrogen-bonding interac-

tion with a water molecule, while B13 lacked the above two interac-

tions. Consequently, this little conformational change caused an addi-

tional aﬃnity of A16 for FtsZ protein, and further increased stimulatory

eﬀect on FtsZ polymerization, thereby leading to the enhanced anti-

bacterial activity.

3.7. In vivo eﬃcacy against S. aureus (MDRS) in mouse model

Three groups of mice were inoculated with 8.5 × 10⁸CFU clinical

isolated S. aureus which is resistant to CIP and ERY through tail vein

injection. Then the infected mice were administered with vehicle, LIN

and A16, respectively, in a dose of 10 mg/kg each time and three times

a day through tail vein injection. After two days, the bacterial CFU

(colony-forming units) of blood collected from retro-orbital plexus was

recorded.^12,23Fig. 7 displays that LIN and A16 at same doses both can

signiﬁcantly decrease the bacterial burden in blood. Note that, even

having superior eﬃcacy in vitro and time-kill kinetics, A16 still did not

work better in vivo than LIN, which could be due to its inadequate

pharmacokinetic properties.

3.10. Discussion of structure-activity relationships (SARs)

Detailed SARs analysis on series A is summarized to reveal diﬀerent

substituents that are crucial to maintaining the antibacterial activity.

Firstly, A3-A8 with halogen substitutions exhibited stronger activity

than A1 without substitution on the benzene ring, exhibiting a 2–8 fold

increase in antibacterial activity against B. subtilis ATCC9372. Similarly,

they also showed increased activity against B. pumilus CMCC63202 with

the MIC values of 2–8 μg/mL, which was 8–32 fold more potent than of

that of A1. It is suggested that the introduction of halogen atoms on the

3.8. Cytotoxicity of A16 to mammalian cells

To evaluate any potential toxicity against mammalian cell, a 48 h

MTT-based assay was performed to assess the cytotoxicity of A16 to

Hela cells.¹⁵This compound was proved to be minimally toxic to Hela

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D. Song, et al.

Bioorganic&MedicinalChemistry28(2020)115729

Fig. 8. Predicted binding modes of SaFtsZ (PDB ID: 3vob) in complex with A16, B13 and 26_pre. (A) The predicted binding models of A16 (yellow structure), A26_pre

(green structure) and PC190723 (cyan structure); (B) The predicted binding models of A26_pre; (C) The predicted binding model of A16; (D) The predicted binding

models of B13 (brown structure).

benzene ring is beneﬁcial to enhance the antibacterial activity of

compounds. We infer that the conjugation of halogen atoms may in-

crease the lipid solubility of compounds and facilitate their penetration

through bacterial cell membranes, thereby enhancing their anti-

bacterial activity. Secondly, the antibacterial activity of the most

compounds in A11-A26 was signiﬁcantly better than that of A1 when

tested against B. subtilis ATCC9372 and B. pumilus CMCC63202. A14-

A17 generated potent activity, which was superior or equivalent to

controls (PC, CIP, LIN and ERY). The tested results also show that the

antibacterial activity of the compounds is enhanced when the electron-

donating groups are attached to the benzene ring. We speculate that the

electron-rich substituted benzene rings may produce π-π stacking and

hydrophobic interactions with the surrounding amino acid residues in

the hydrophobic cavity of FtsZ. Besides, the length of alkyl substitution

on the benzene ring has a signiﬁcant inﬂuence on the antibacterial

activity as well. Increasing the length of the alkyl group on the benzene

ring (A11 to A17) leads to enhanced antibacterial activity. When pos-

sessing the n-butyl group (A16), the activity reaches a peak value, while

the further elongation of the alkyl side chain (A17) decreases the ac-

tivity dramatically. This may be well explained by the fact that the

binding pocket of FtsZ for these compounds has a volume restriction.

For A19-A25, their antibacterial activity gradually increased with the

extension of the alkoxy side chain, which demonstrates the same trend

as A11-A17 in antibacterial activity. Thirdly, the position of substituent

on the phenyl ring plays a crucial role in the antibacterial potency. For

example, varying the position of chlorine atom from para‐position (A4)

to ortho-position (A2) sharply decrease the antibacterial activity.

Replacing the para-methoxy group (A21) by an ortho-methoxy group

(A19) directly leads to the loss of antibacterial activity. It is possible

that para-substituent with appropriate length and ﬂexibility is more

suitable for the narrow hydrophobic slit of FtsZ. Taken together, when

aliphatic alkane was introduced to the para-position of the benzene

ring, the contribution of antibacterial activity was signiﬁcantly stronger

than that of other substituents. The representative compound A16 of-

fered the most potent and balanced activity against all the tested Gram-

positive bacteria (MIC = 0.125–4 μg/mL).

In series B, B1-B7 had diﬀerent halogen atoms on the benzene ring.

Among them, the antibacterial activity of B3 and B4 was stronger than

that of B1-B2 and B5-B7, which can be inferred that the contribution of

halogen atom for activity is Cl > Br > F, and chlorine atom at the C-2

and C-4 position of the phenyl ring oﬀered the most potent antibacterial

activity. By comparing the structures of B8-B13, increasing the length

of the alkyl side chain was found to improve the antibacterial activity

greatly. This may be explained that the long-chain aliphatic alkanes

have ﬂexibility, which are allowed to enter the hydrophobic cavity and

bind with amino acid residues. As for B14-B18 that possess diﬀerent

length alkoxy groups on the benzene ring, shortening the length of the

alkoxy group signiﬁcantly weakened their antibacterial activity, which

8

D. Song, et al.

Bioorganic&MedicinalChemistry28(2020)115729

Fig. 9. The structures of the selected compounds used for comparison from our present and previous studies.

are similar to that of B8-B13. Besides, altering the position of chlorine

atom from para-position (B3) to ortho-position (B1) or meta-position

(B2) sharply decreased the antibacterial activity. Similarly, replacing

the para-ethoxy group (B17) with an ortho-ethoxy group (B15) or meta-

ethoxy substitution (B16) also weakened the antibacterial activity.

Thus, we can conclude that the extension direction of the side chain on

the benzene ring in space has a signiﬁcant eﬀect on the antibacterial

activity. In short, aliphatic alkanes on para-position of the benzene rings

exert more potent antibacterial activity than those on other positions.

The representative compound B13 with the n-butyl group exhibited

excellent antibacterial activity against B. subtilis ATCC9372 and B. pu-

milus CMCC63202 with the MIC values of ≤ 0.125 and 0.5 μg/mL,

respectively.

dihydroisoxazol-containing benzamide derivatives also show broad

antibacterial spectrum, and are more active against S. aureus strain. For

example, the antibacterial activity of A14, A15, A16, A17 and A24

against B. pumilus CMCC63202 were at least 2-fold more active than

A24_pre, A25_pre, A26_pre, A27_preand A18_pre(Fig. 9), respectively. In

particular, A16 has not only signiﬁcantly improved activity against S.

aureus ATCC25923, which is 128-fold more active than A26_pre, but also

remarkably inhibits the growth of S. aureus PR, which is a 128-fold

improvement compared with A26_pre. It is unexpected that A14 and A15

exhibit 2- to 16-fold better activity against S. aureus than A24_preand

A25_pre. Besides, A3, A8, A24 and A25 exhibited moderate antibacterial

activity against the tested Gram-positive bacteria with the MIC values

of 2–32 μg/mL. In contrast, the corresponding A6_pre, A3_pre, A18_preand

A17_prewere inactive against the above tested strains except for B.

subtilis ATCC9372. As for series B, B3, B17 and B18 exert better activity

against B. subtilis ATCC9372 and B. pumilus CMCC63202, while the

corresponding B2_pre, B8_preand B10_pre(Fig. 9) have no obvious anti-

bacterial activity. Moreover, B4 and B13 show a broad spectrum ac-

tivity, but the corresponding B5_preand B15_pre(Fig. 9) barely inhibit the

growth of B. subtilis ATCC9372.

In general, the structures of the series A and series B almost overlap

with each other, and their structural diﬀerence lies in the 4,5-dihy-

droisoxazole. From the MIC results, A11-A16 have much better anti-

bacterial activity than B8-B13, but these compounds possess the same

warhead of 2,6-diﬂuorobenzamide and the same tail of the substituted

benzene ring. The only diﬀerence in their structure is connections of the

4,5-dihydroisoxazole ring, which interfere with the potency of their

antibacterial activity. Also, A19-A25 with alkoxy side chains on the

benzene rings are structurally similar to B14-B18 but exhibit stronger

antibacterial activity. Thus, it is concluded that the 4,5-dihydroisox-

azole ring in the structure has a positive eﬀect on the antibacterial

activity and the relative position of heteroatoms in the 4,5-dihy-

droisoxazole ring may have a direct inﬂuence on the interaction with

FtsZ binding pocket, which was conﬁrmed by the docking results. The

oxygen atom on the 4,5-dihydroisoxazole of the series A is adjacent to

Gly196 and a water molecule, tending to interact with each other. The

subtle diﬀerences in heteroatom positions on the 4,5-dihydroisoxazole

alter the binding aﬃnity of compounds to FtsZ target, which explain the

signiﬁcant diﬀerences in antibacterial activity between series A and

series B.

Although there is little change in their structure, the antibacterial

activity is greatly improved, and the antibacterial spectrum has been

broadened. Thus, we can conclude that the replacement of the isoxazole

ring with a 4,5-dihydroisoxazole ring produces a positive inﬂuence on

antibacterial activity. Based on stereochemistry of A16 and A26_pre, the

4,5-dihydroisoxazole ring in molecular structure has some ﬂexibility

and can be twisted to suit the hydrophobic cavity of FtsZ. In contrast,

the isoxazole ring in molecular structure is rigid and limits its distortion

to accommodate hydrophobic cavity, which was conﬁrmed by the

docking results (Fig. 8B and 8C). Also, lipid/water partition coeﬃcients

(logP) as one of the most important factors have been extensively em-

ployed to predict the bioactivity of target molecules.²⁵Therefore, we

theoretically calculated the logP values (ClogP) of A16 and A26_pre

molecules by the commercial ChemBioOﬃce 2010 (Cambridge Soft,

USA) to further conﬁrm their inﬂuence on antibacterial activity. Results

showed that the ClogP values of A16 and A26_preare 4.18 and 4.19,

respectively, which don’t aﬀect their antibacterial activity. Taken to-

gether, the replacement of the isoxazole ring in molecular structure

with a 4,5-dihydroisoxazole ring leads to the change of spatial conﬁg-

uration of the target compound. It suits the hydrophobic cavity of FtsZ,

thereby strengthening aﬃnity for FtsZ, which is consistent with our

design strategy.

A16 has the most promising antibacterial activity among all tested

compounds, while B14_pre(Fig. 9) is the best compound that was

screened from our previous work.¹⁶For all that, A16 still displays 4-, 8-

and 8-fold more potent antibacterial activity against S. aureus

ATCC25923, S. aureus ATCC43300 and S. aureus PR than B14_pre

.

Moreover, this compound also exhibits excellent activity against B.

pumilus CMCC63202, comparable to B14_pre. This indicates that 4,5-

dihydroisoxazol-containing benzamide derivatives demonstrate in-

creased antibacterial activity, compared with our reported 4,5-isoxazol-

containing benzamide derivatives.¹⁶Not only that, 4,5-

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D. Song, et al.

Bioorganic&MedicinalChemistry28(2020)115729

4. Conclusion

(m, 1H), 7.11 (td, J = 9.1, 5.1 Hz, 1H), 6.90 (td, J = 9.1, 2.0 Hz, 1H),

5.94 (d, J = 44.1 Hz, 2H), 5.16–5.10 (m, 1H), 4.24–4.17 (m, 2H),

3.74–3.52 (m, 2H). ¹³C NMR (150 MHz, DMSO‑d₆) δ 161.77, 156.37,

152.51 (dd, J_F-C= 241.6, 6.7 Hz), 148.38 (dd, J_F-C= 248.7, 8.6 Hz),

143.38 (dd, J_F-C= 10.6, 3.3 Hz), 132.11, 131.78, 131.22, 130.96,

129.04, 127.94, 117.11 (dd, J_F-C= 25.0, 20.3 Hz), 116.20 (dd, J_F-

_C= 9.3, 2.3 Hz), 111.43 (dd, J_F-C= 23.1, 3.8 Hz), 79.30, 71.16, 39.45.

ESI-MS calcd for C₁₇H₁₄ClF₂N₂O₃[M+H]⁺, 367.1, found 367.2.

Using the structure-based optimization strategy, two series of 4,5-

dihydroisoxazol-5-yl- and 4,5-dihydroisoxazol-3-yl-containing benza-

mide derivatives were designed, synthesized, and tested for their in vitro

and in vivo biological activities. They were pharmacologically evaluated

as FtsZ-targeting antibacterial agents as well. The results indicated that

several compounds exhibited signiﬁcant antibacterial activity against

most of the tested strains, including the multidrug-resistant strains. It is

noteworthy that A16 display signiﬁcantly activity against three resistant

strains of methicillin-resistant, penicillin-resistant and clinical isolated S.

aureus, and a sensitive strain of S. aureus with the MIC values ranging

from 0.25 to 0.5 μg/mL, which are comparable to those observed with

PC190723 and LIN, and are much better than that of CLA. It is worth

noting that substituting a 4,5-dihydroisoxazole ring for the isoxazole ring

resulted in a great increase in antibacterial activity and the broadening of

antibacterial spectrum. Regarded as a whole, the research ﬁndings de-

scribed herein highlight A16 as a lead compound with potent bacter-

icidal activity and reinforce the importance of FtsZ as a promising anti-

bacterial target. Further optimization of the 4,5-dihydroisoxazol-5-yl-

containing benzamide derivatives is ongoing in our lab.

5.2.3. 2,6-Diﬂuoro-3-((3-(3-chlorophenyl)-4,5-dihydroisoxazol-5-yl)

methoxy)benzamide (A3)

White solid, Mp: 144–146 °C; ¹H NMR (400 MHz, DMSO‑d₆) δ 8.11

(s, 1H), 7.83 (s, 1H), 7.74–7.63 (m, 2H), 7.58–7.46 (m, 2H), 7.25 (td,

J = 9.3, 5.2 Hz, 1H), 7.08 (td, J = 9.0, 1.9 Hz, 1H), 5.16–5.06 (m, 1H),

4.27–4.14 (m, 2H), 3.61 (dd, J = 17.2, 11.1 Hz, 1H), 3.39–3.32 (m,

1H). ¹³C NMR (150 MHz, DMSO‑d₆) δ 161.73, 156.28, 152.52 (dd, J_F-

_C= 241.3, 6.6 Hz), 148.39 (dd, J_F-C= 248.7, 8.4 Hz), 143.35 (dd, J_F-

= 11.0, 3.2 Hz), 134.04, 131.80, 131.22, 130.36, 126.67, 125.72,

C

117.09 (dd, J_F-C= 24.9, 20.4 Hz), 116.32 (dd, J_F-C= 9.4, 2.3 Hz),

111.44 (dd, J_F-C= 23.1, 3.9 Hz), 79.53, 71.30, 36.77. ESI-MS calcd for

C

₁₇H₁₄ClF₂N₂O₃[M+H]⁺, 367.1, found 367.2.

5. Experimental section

5.2.4. 2,6-Diﬂuoro-3-((3-(4-chlorophenyl)-4,5-dihydroisoxazol-5-yl)

methoxy)benzamide (A4)

5.1. General experimental protocol

White solid, Mp: 146–147 °C; ¹H NMR (600 MHz, DMSO‑d₆) δ 8.11

(s, 1H), 7.82 (s, 1H), 7.71 (d, J = 8.6 Hz, 2H), 7.54 (d, J = 8.6 Hz, 2H),

7.25 (td, J = 9.3, 5.2 Hz, 1H), 7.07 (td, J = 8.8, 1.8 Hz, 1H), 5.13–5.06

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 solvents using silica gel 200–300 mesh

size. ¹H NMR spectra were recorded at 400 or 600 MHz, and ¹³C NMR

spectra were recorded at 150 MHz 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.

ECD spectra were performed on a Chirascan spectropolarimeter.

Analytical HPLC was carried out on an Agilent 1200 Series equipment

coupled to ultraviolet–visible detector (UVD) at 254 nm with a

Diamonil C18 reversed phase column (150 × 4.6 mm, 5 μm) at ambient

temperature. The eﬀective chromatogram was obtained using the mo-

bile phase of methanol: water (60 : 40, vol./vol.) at the ﬂow rate of

1.0 mL/min. The purity of the tested compounds as determined by

analytical HPLC was more than 95%. Compounds 4, 5, 6 and 9 were

synthesized according to our previous reports.¹⁶The detailed synthetic

methods of series A and B refer to the Supplementary data.

(m, 1H), 4.26–4.16 (m, 2H), 3.64–3.56 (m, 1H), 3.31–3.26 (m, 1H). ¹³

C

NMR (150 MHz, DMSO‑d₆) δ 161.71, 156.29, 152.51 (dd, J_F-C= 240.1,

9.8 Hz), 148.38 (dd, J_F-C= 247.6, 11.3 Hz), 143.31 (dd, J_F-C= 13.6,

3.8 Hz), 135.16, 129.40, 128.85, 128.61, 117.08 (dd, J_F-C= 25.7,

19.6 Hz), 116.36 (d, J_F-C= 15.1 Hz), 111.51 (dd, J_F-C= 22.7, 5.3 Hz),

79.42, 71.31, 36.87. ESI-MS calcd for C₁₇H₁₄ClF₂N₂O₃[M+H]⁺,

367.1, found 367.3.

5.2.5. 2,6-Diﬂuoro-3-((3-(2,4-dichlorophenyl)-4,5-dihydroisoxazol-5-yl)

methoxy)benzamide (A5)

White solid, Mp: 103–104 °C; ¹H NMR (600 MHz, DMSO‑d₆) δ 8.12

(s, 1H), 7.83 (s, 1H), 7.77 (d, J = 2.1 Hz, 1H), 7.66 (d, J = 8.4 Hz, 1H),

7.55 (dd, J = 8.4, 2.1 Hz, 1H), 7.26 (td, J = 9.3, 5.2 Hz, 1H), 7.08 (td,

J = 9.0, 1.8 Hz, 1H), 5.16–5.09 (m, 1H), 4.27–4.16 (m, 2H), 3.68 (dd,

J = 17.2, 11.0 Hz, 1H), 3.38 (dd, J = 17.3, 7.0 Hz, 1H). ¹³C NMR

(150 MHz, DMSO‑d₆) δ 161.73, 155.63, 152.50 (dd, J_F-C= 241.5,

6.7 Hz), 148.36 (dd, J_F-C= 248.8, 8.3 Hz), 143.35 (dd, J_F-C= 10.8,

3.1 Hz), 135.55, 133.14, 132.44, 130.53, 128.20, 128.02, 117.11 (dd,

J_F-C= 25.0, 20.5 Hz), 116.21 (d, J_F-C= 9.5 Hz), 111.43 (dd, J_F-

=

22.8, 3.8 Hz), 79.49, 71.12, 39.21. ESI-MS calcd for

C

5.2. Typical compounds of the series A

₁₇H₁₆Cl₂F₂N₃O₃[M+NH₄]⁺, 418.1, found 418.5.

5.2.1. 2,6-Diﬂuoro-3-((3-phenyl-4,5-dihydroisoxazol-5-yl)methoxy)

benzamide (A1)

5.2.6. 2,6-Diﬂuoro-3-((3-(3,4-dichlorophenyl)-4,5-dihydroisoxazol-5-yl)

methoxy)benzamide (A6)

White solid, Mp: 148–150 °C; ¹H NMR (400 MHz, DMSO‑d₆) δ 8.11

(s, 1H), 7.83 (s, 1H), 7.73–7.66 (m, 2H), 7.51 – 7.44 (m, 3H), 7.26 (td,

J = 9.3, 5.2 Hz, 1H), 7.07 (td, J = 8.9, 1.9 Hz, 1H), 5.13–5.03 (m, 1H),

4.28–4.14 (m, 2H), 3.61 (dd, 1H), 3.32–3.25 (m, 1H). ¹³C NMR

(150 MHz, DMSO‑d₆) δ 161.77, 157.03, 152.51 (dd, J_F-C= 241.2,

6.6 Hz), 148.39 (dd, J_F-C= 248.7, 8.4 Hz), 143.38 (dd, J_F-C= 11.0,

3.2 Hz), 130.60, 129.70, 129.30, 127.09, 117.08 (dd, J_F-C= 24.9,

20.4 Hz), 116.32 (dd, J_F-C= 9.4, 2.2 Hz), 111.44 (dd, J_F-C= 23.0,

3.8 Hz), 79.09, 71.37, 37.02. HRMS (ESI) calcd for C₁₇H₁₅F₂N₂O₃[M

+H]⁺, 333.1045, found 333.1044.

White solid, Mp: 181–182 °C; ¹H NMR (400 MHz, DMSO‑d₆) δ 8.12

(s, 1H), 7.90 (d, J = 1.8 Hz, 1H), 7.84 (s, 1H), 7.75 (d, J = 8.4 Hz, 1H),

7.69 (dd, J = 8.5, 1.9 Hz, 1H), 7.25 (td, J = 9.3, 5.2 Hz, 1H), 7.08 (td,

J = 9.0, 1.9 Hz, 1H), 5.18–5.08 (m, 1H), 4.26–4.15 (m, 2H), 3.66–3.56

(m, 1H), 3.38–3.34 (m, 1H). ¹³C NMR (150 MHz, DMSO‑d₆) δ 161.70,

155.72, 152.52 (dd, J_F-C= 241.6, 6.6 Hz), 148.38 (dd, J_F-C= 248.6,

8.4 Hz), 143.33 (dd, J_F-C= 10.9, 3.1 Hz), 133.04, 132.16, 131.57,

130.38, 128.83, 127.10, 117.10 (dd, J_F-C= 25.0, 20.4 Hz), 116.33 (d,

J

_F-C= 9.4 Hz), 111.44 (dd, J_F-C= 22.7, 3.8 Hz), 79.80, 71.25, 36.67.

ESI-MS calcd for C₁₇H₁₃Cl₂F₂N₂O₃[M+H]⁺, 401.0, found 401.2.

5.2.2. 2,6-Diﬂuoro-3-((3-(2-chlorophenyl)-4,5-dihydroisoxazol-5-yl)

methoxy)benzamide (A2)

5.2.7. 2,6-Diﬂuoro-3-((3-(4-ﬂuorophenyl)-4,5-dihydroisoxazol-5-yl)

methoxy)benzamide (A7)

White solid, Mp: 130–132 °C; ¹H NMR (600 MHz, Chloroform-d) δ

7.66 – 7.61 (m, 1H), 7.46–7.42 (m, 1H), 7.39–7.35 (m, 1H), 7.34–7.30

White solid, Mp: 155–156 °C; ¹H NMR (600 MHz, Chloroform-d) δ

7.71–7.66 (m, 2H), 7.14–7.07 (m, 3H), 6.89 (td, J = 9.1, 2.0 Hz, 1H),

10

D. Song, et al.

Bioorganic&MedicinalChemistry28(2020)115729

5.91 (d, J = 52.1 Hz, 2H), 5.13 – 5.07 (m, 1H), 4.26–4.13 (m, 2H),

3.56–3.38 (m, 2H). ¹³C NMR (150 MHz, DMSO‑d₆) δ 164.34, 162.70,

161.74, 156.20, 152.51 (dd, J_F-C= 241.5, 6.6 Hz), 148.38 (dd, J_F-

6.88 (td, J = 9.1, 2.0 Hz, 1H), 5.92 (d, J = 45.6 Hz, 2H), 5.08 (dq,

J = 11.1, 5.1 Hz, 1H), 4.26 – 4.09 (m, 2H), 3.57–3.35 (m, 2H), 2.39 (s,

3H). ¹³C NMR (150 MHz, DMSO‑d₆) δ 161.75, 156.86, 152.50 (dd, J_F-

_C= 241.6, 6.6 Hz), 148.39 (dd, J_F-C= 248.7, 8.4 Hz), 143.38 (dd, J_F-

= 248.5, 8.4 Hz), 143.36 (dd, J_F-C= 10.9, 3.2 Hz), 129.41 (d, J_F-

C

= 8.6 Hz), 126.33 (d, J_F-C= 3.2 Hz), 117.09 (dd, J_F-C= 25.0,

= 11.0, 3.2 Hz) 140.35, 129.85, 127.03, 126.92, 117.09 (dd, J_F-

C

20.4 Hz), 116.35 (d, J_F-C= 21.9 Hz), 111.43 (dd, J_F-C= 23.1, 3.8 Hz),

79.22, 71.33, 37.11. ESI-MS calcd for C₁₇H₁₄F₃N₂O₃[M+H]⁺, 351.3,

found 351.4.

= 25.0, 20.4 Hz), 116.29 (dd, J_F-C= 9.2, 2.4 Hz), 111.42 (dd, J_F-

C

= 22.7, 3.8 Hz), 78.89, 71.38, 37.12, 21.44. ESI-MS calcd for

C

₁₈H₁₇F₂N₂O₃[M+H]⁺, 347.3, found 347.3.

5.2.8. 2,6-Diﬂuoro-3-((3-(4-bromophenyl)-4,5-dihydroisoxazol-5-yl)

methoxy)benzamide (A8)

5.2.13. 2,6-Diﬂuoro-3-((3-(4-ethylphenyl)-4,5-dihydroisoxazol-5-yl)

methoxy)benzamide (A13)

1

White solid, Mp: 144–146 °C; ¹H NMR (600 MHz, Chloroform-d) δ

7.55 (s, 4H), 7.09 (td, J = 9.1, 5.1 Hz, 1H), 6.89 (td, J = 9.1, 2.0 Hz,

1H), 5.92 (d, 2H), 5.14–5.08 (m, 1H), 4.24–4.13 (m, 2H), 3.54–3.37 (m,

2H). ¹³C NMR (150 MHz, DMSO‑d₆) δ 161.72, 156.40, 152.51 (dd, J_F-

_C= 241.4, 6.7 Hz), 148.38 (dd, J_F-C= 248.7, 8.5 Hz), 143.35 (dd, J_F-

White solid, Mp: 132–133 °C; H NMR (600 MHz, Chloroform-d) δ

7.60 (d, J = 8.2 Hz, 2H), 7.24 (d, J = 8.2 Hz, 2H), 7.09 (td, J = 9.1,

5.1 Hz, 1H), 6.88 (td, J = 9.1, 2.0 Hz, 1H), 5.91 (d, J = 47.4 Hz, 2H),

5.11–5.04 (m, 1H), 4.25–4.10 (m, 2H), 3.57–3.36 (m, 2H), 2.68 (q,

J = 7.6 Hz, 2H), 1.25 (t, J = 7.6 Hz, 3H). ¹³C NMR (150 MHz, DMSO‑d₆)

δ 161.75, 156.88, 152.51 (dd, J_F-C= 241.5, 6.6 Hz), 148.39 (dd, J_F-

= 10.7, 3.2 Hz), 132.30, 129.05, 123.91, 117.09 (dd, J_F-C= 24.9,

C

20.4 Hz), 116.31 (dd, J_F-C= 9.8, 2.3 Hz), 111.43 (dd, J_F-C= 22.8,

3.9 Hz), 79.44, 71.31, 36.81. ESI-MS calcd for C₁₇H₁₄BrF₂N₂O₃[M

+H]⁺, 411.0, found 411.2.

= 248.6, 8.5 Hz), 146.58, 143.38 (dd, J_F-C= 10.7, 3.2 Hz), 128.68,

C

127.14, 117.09 (dd, J_F-C= 24.9, 20.4 Hz), 116.30 (dd, J_F-C= 9.3,

2.3 Hz), 111.42 (dd, J_F-C= 23.1, 3.8 Hz), 78.89, 71.38, 37.14, 28.52,

15.85. ESI-MS calcd for C₁₉H₁₉F₂N₂O₃[M+H]⁺, 361.4, found 361.3.

5.2.9. 2,6-Diﬂuoro-3-((3-(4-(dimethylamino)phenyl)-4,5-

dihydroisoxazol-5-yl)methoxy)benzamide (A9)

5.2.14. 2,6-Diﬂuoro-3-((3-(4-isopropylphenyl)-4,5-dihydroisoxazol-5-yl)

methoxy)benzamide (A14)

White solid, Mp: 217–219 °C; ¹H NMR (600 MHz, DMSO‑d₆) δ 8.12

(s, 1H), 7.84 (s, 1H), 7.50 (d, J = 8.9 Hz, 2H), 7.26 (td, J = 9.3, 5.1 Hz,

1H), 7.08 (td, J = 9.0, 1.8 Hz, 1H), 6.75 (d, J = 8.8 Hz, 2H), 5.02–4.94

(m, 1H), 4.23–4.12 (m, 2H), 3.52 (dd, J = 16.8, 10.8 Hz, 1H), 3.24 (dd,

J = 16.9, 7.3 Hz, 1H), 2.96 (s, 6H). ¹³C NMR (150 MHz, DMSO‑d₆) δ

161.74, 156.66, 153.26 (d, J_F-C= 6.3 Hz), 151.77, 151.66 (d, J_F-

_C= 6.7 Hz), 148.36 (dd, J_F-C= 248.7, 8.2 Hz), 143.41 (dd, J_F-C= 11.4,

2.6 Hz), 128.21, 117.08 (dd, J_F-C= 24.8, 20.5 Hz), 116.74, 116.29 (d,

White solid, Mp: 132–133 °C; ¹H NMR (600 MHz, Chloroform-d) δ

7.62 (d, J = 8.2 Hz, 2H), 7.28 (d, J = 8.4 Hz, 2H), 7.09 (td, J = 9.1,

5.1 Hz, 1H), 6.88 (td, J = 9.1, 2.0 Hz, 1H), 5.91 (d, J = 49.5 Hz, 2H),

5.12–5.04 (m, 1H), 4.24–4.10 (m, 2H), 3.55–3.38 (m, 2H), 2.98–2.90

(m, 1H), 1.26 (d, J = 6.9 Hz, 6H). ¹³C NMR (150 MHz, DMSO‑d₆) δ

161.75, 156.86, 152.51 (dd, J_F-C= 241.5, 6.6 Hz), 151.14, 148.39 (dd,

J

_F-C= 248.6, 8.6 Hz), 143.39 (dd, J_F-C= 10.8, 3.1 Hz), 127.32, 127.20

J

_F-C= 9.1 Hz), 112.15, 111.42 (dd, J_F-C= 23.2, 3.7 Hz), 78.21, 71.43,

(d, J_F-C= 6.7 Hz), 117.09 (dd, J_F-C= 24.9, 20.4 Hz), 116.31 (dd, J_F-

_C= 9.4, 2.2 Hz), 111.43 (dd, J_F-C= 22.8, 3.8 Hz), 78.88, 71.37, 37.15,

33.82, 24.11. ESI-MS calcd for C₂₀H₂₁F₂N₂O₃[M+H]⁺, 375.4, found

375.4.

37.49, 26.82, 25.85. ESI-MS calcd for C₁₉H₂₀F₂N₃O₃[M+H]⁺, 376.4,

found 376.4.

5.2.10. 2,6-Diﬂuoro-3-((3-(4-cyanophenyl)-4,5-dihydroisoxazol-5-yl)

methoxy)benzamide (A10)

5.2.15. 2,6-Diﬂuoro-3-((3-(4-(tert-butyl)phenyl)-4,5-dihydroisoxazol-5-

yl)methoxy)benzamide (A15)

White solid, Mp: 156–158 °C; ¹H NMR (600 MHz, Chloroform-d) δ

7.79 (d, J = 8.5 Hz, 2H), 7.71 (d, J = 8.6 Hz, 2H), 7.09 (td, J = 9.0,

5.1 Hz, 1H), 6.89 (td, J = 9.1, 2.0 Hz, 1H), 5.90 (d, J = 52.2 Hz, 2H),

5.20 – 5.13 (m, 1H), 4.26–4.18 (m, 2H), 3.56–3.42 (m, 2H). ¹³C NMR

(150 MHz, DMSO‑d₆) δ 161.69, 156.41, 152.50 (dd, J_F-C= 241.5,

6.6 Hz), 148.36 (dd, J_F-C= 248.7, 8.4 Hz), 143.31 (dd, J_F-C= 11.2,

3.0 Hz), 134.06, 133.23, 127.79, 118.95, 117.08 (dd, J_F-C= 25.2,

20.3 Hz), 116.31 (d, J_F-C= 8.9 Hz), 112.74, 111.43 (dd, J_F-C= 23.1,

3.8 Hz), 79.99, 71.26, 36.45. ESI-MS calcd for C₁₈H₁₄F₂N₃O₃[M+H]⁺,

358.3, found 358.2.

White solid, Mp: 157–159 °C; ¹H NMR (600 MHz, Chloroform-d) δ

7.62 (d, J = 8.4 Hz, 2H), 7.44 (d, J = 8.4 Hz, 2H), 7.10 (td, J = 9.1,

5.0 Hz, 1H), 6.88 (td, J = 9.1, 2.0 Hz, 1H), 5.90 (d, J = 59.7 Hz, 2H),

5.08 (ddt, J = 10.4, 6.8, 5.0 Hz, 1H), 4.17 (ddd, J = 47.7, 10.3, 5.0 Hz,

2H), 3.56–3.37 (m, 2H), 1.34 (s, 9H). ¹³C NMR (150 MHz, DMSO‑d₆) δ

161.73, 156.79, 153.33, 153.28, 151.70 (d, J_F-C= 6.6 Hz), 148.39 (dd,

J

_F-C= 248.5, 8.5 Hz), 143.38 (dd, J_F-C= 11.0, 3.2 Hz), 126.95, 126.92,

126.07, 117.09 (dd, J_F-C= 24.9, 20.4 Hz), 116.36, 116.34, 116.29,

111.43 (dd, J_F-C= 23.1, 4.0 Hz), 78.87, 71.36, 37.14, 35.05, 31.39.

ESI-MS calcd for C₂₁H₂₃F₂N₂O₃[M+H]⁺, 389.4, found 389.3.

5.2.11. 2,6-Diﬂuoro-3-((3-(m-tolyl)-4,5-dihydroisoxazol-5-yl)methoxy)

benzamide (A11)

5.2.16. 2,6-Diﬂuoro-3-((3-(4-butylphenyl)-4,5-dihydroisoxazol-5-yl)

methoxy)benzamide (A16)

White solid, Mp: 108–109 °C; ¹H NMR (600 MHz, Chloroform-d) δ

7.52 (s, 1H), 7.47 (d, J = 7.7 Hz, 1H), 7.30 (t, J = 7.6 Hz, 1H), 7.24 (d,

J = 7.6 Hz, 1H), 7.10 (td, J = 9.1, 5.1 Hz, 1H), 6.88 (td, J = 9.2,

2.0 Hz, 1H), 5.92 (d, J = 49.5 Hz, 2H), 5.12–5.06 (m, 1H), 4.24–4.12

(m, 2H), 3.56–3.39 (m, 2H), 2.39 (s, 3H). ¹³C NMR (150 MHz,

DMSO‑d₆) δ 161.76, 157.05, 152.51 (dd, J_F-C= 241.3, 6.6 Hz), 148.39

(dd, J_F-C= 248.6, 8.5 Hz), 143.38 (dd, J_F-C= 10.9, 3.2 Hz), 138.55,

131.24, 129.63, 129.18, 127.55, 124.28, 117.08 (dd, J_F-C= 25.1,

20.4 Hz), 116.31 (dd, J_F-C= 9.4, 2.4 Hz), 111.43 (dd, J_F-C= 23.1,

3.8 Hz), 78.99, 71.35, 37.07, 21.36. ESI-MS calcd for C₁₈H₁₇F₂N₂O₃[M

+H]⁺, 347.3, found 347.3.

White solid, Mp: 143–144 °C; ¹H NMR (600 MHz, Chloroform-d) δ

7.59 (d, J = 8.1 Hz, 2H), 7.23 (d, J = 8.1 Hz, 2H), 7.09 (td, J = 9.1,

5.1 Hz, 1H), 6.88 (td, J = 9.1, 2.0 Hz, 1H), 5.93 (d, J = 34.7 Hz, 2H),

5.11 – 5.05 (m, 1H), 4.24–4.10 (m, 2H), 3.55–3.37 (m, 2H), 2.64 (t,

2H), 1.64–1.58 (m, 2H), 1.36 (h, J = 7.4 Hz, 2H), 0.93 (t, J = 7.3 Hz,

3H). ¹³C NMR (150 MHz, DMSO‑d₆) δ 161.72, 156.88, 152.53 (dd, J_F-

_C= 242.4, 7.1 Hz), 148.42 (dd, J_F-C= 249.5, 9.1 Hz), 145.17, 143.42

(dd, J_F-C= 11.1, 3.5 Hz), 129.19, 127.17, 127.07, 128.61, 117.12 (dd,

J_F-C= 25.3, 20.2 Hz), 116.43 (d, J_F-C= 10.1 Hz), 111.42 (dd, J_F-

_C= 22.2, 4.5 Hz), 78.89, 71.40, 37.15, 35.11, 33.35, 22.15, 14.21. ESI-

MS calcd for C₂₁H₂₃F₂N₂O₃[M+H]⁺, 389.4, found 389.3.

5.2.12. 2,6-Diﬂuoro-3-((3-(p-tolyl)-4,5-dihydroisoxazol-5-yl)methoxy)

benzamide (A12)

5.2.17. 2,6-Diﬂuoro-3-((3-(4-pentylphenyl)-4,5-dihydroisoxazol-5-yl)

methoxy)benzamide (A17)

White solid, Mp: 135–137 °C; ¹H NMR (600 MHz, Chloroform-d) δ

7.58 (s, 2H), 7.22 (d, J = 7.9 Hz, 2H), 7.09 (td, J = 9.0, 5.3 Hz, 1H),

White solid, Mp: 148–149 °C; ¹H NMR (600 MHz, DMSO‑d₆) δ 8.12

11

D. Song, et al.

Bioorganic&MedicinalChemistry28(2020)115729

(s, 1H), 7.83 (s, 1H), 7.59 (d, J = 8.2 Hz, 2H), 7.28 (d, J = 8.3 Hz, 2H),

7.27 – 7.23 (m, 1H), 7.07 (td, J = 9.0, 1.8 Hz, 1H), 5.08–5.02 (m, 1H),

4.24–4.15 (m, 2H), 3.58 (dd, J = 17.0, 11.0 Hz, 1H), 3.29 (dd,

J = 17.0, 7.2 Hz, 1H), 2.61 (t, J = 7.6 Hz, 2H), 1.58 (p, J = 7.6 Hz,

2H), 1.34–1.23 (m, 4H), 0.86 (t, J = 7.1 Hz, 3H). ¹³C NMR (150 MHz,

DMSO‑d₆) δ 161.73, 156.89, 152.50 (dd, J_F-C= 241.5, 6.7 Hz), 148.39

(dd, J_F-C= 248.8, 8.4 Hz), 145.19, 143.38 (dd, J_F-C= 10.9, 3.2 Hz),

129.20, 127.17, 127.07, 117.09 (dd, J_F-C= 25.0, 20.4 Hz), 116.35,

116.33, 116.28, 111.42 (dd, J_F-C= 22.8, 3.8 Hz), 78.88, 71.36, 37.14,

35.39, 31.28, 30.88, 22.40, 14.38. ESI-MS calcd for C₂₂H₂₅F₂N₂O₃[M

+H]⁺, 403.4, found 403.5.

5.2.22. 2,6-Diﬂuoro-3-((3-(3-ethoxyphenyl)-4,5-dihydroisoxazol-5-yl)

methoxy)benzamide (A22)

White solid, Mp: 152–153 °C; ¹H NMR (400 MHz, DMSO‑d₆) δ 8.11

(s, 1H), 7.83 (s, 1H), 7.41–7.33 (m, 1H), 7.30–7.16 (m, 3H), 7.12–6.99

(m, 2H), 5.13–5.02 (m, 1H), 4.28–4.13 (m, 2H), 4.06 (q, J = 7.0 Hz,

2H), 3.59 (dd, J = 17.1, 11.0 Hz, 1H), 3.3–3.23 (m, 1H), 1.34 (t,

J = 7.0 Hz, 3H). ¹³C NMR (150 MHz, DMSO‑d₆) δ 161.72, 159.08,

157.01, 151.72, 147.53, 143.41, 130.98, 130.44, 119.45, 117.09 (dd,

J_F-C= 25.1, 20.1 Hz), 116.85, 116.33 (d, J_F-C= 8.2 Hz), 112.59,

111.44 (dd, J_F-C= 23.1, 3.6 Hz), 79.13, 71.35, 63.64, 37.08, 15.06.

ESI-MS calcd for C₁₉H₁₉F₂N₂O₄[M+H]⁺, 377.4, found 377.5.

5.2.23. 2,6-Diﬂuoro-3-((3-(4-ethoxyphenyl)-4,5-dihydroisoxazol-5-yl)

methoxy)benzamide (A23)

5.2.18. 2,6-Diﬂuoro-3-((3-(4-cyclohexylphenyl)-4,5-dihydroisoxazol-5-yl)

methoxy)benzamide (A18)

White solid, Mp: 132–134 °C; ¹H NMR (600 MHz, DMSO‑d₆) δ 8.11

(s, 1H), 7.83 (s, 1H), 7.61 (d, J = 8.9 Hz, 2H), 7.25 (td, J = 9.3, 5.2 Hz,

1H), 7.07 (td, J = 8.9, 1.8 Hz, 1H), 6.99 (d, J = 8.9 Hz, 2H), 5.03 (dd,

J = 10.8, 6.0 Hz, 1H), 4.23–4.14 (m, 2H), 4.07 (q, J = 7.0 Hz, 2H),

3.56 (dd, J = 17.0, 10.9 Hz, 1H), 3.27 (dd, J = 17.0, 7.3 Hz, 1H), 1.34

(t, J = 7.0 Hz, 3H). ¹³C NMR (150 MHz, DMSO‑d₆) δ 161.74, 160.42,

156.49, 152.49 (dd, J_F-C= 241.5, 6.6 Hz), 148.38 (dd, J_F-C= 248.7,

8.4 Hz), 143.39 (dd, J_F-C= 11.0, 3.2 Hz), 128.68, 121.97, 117.09 (dd,

White solid, Mp: 178–180 °C; ¹H NMR (600 MHz, DMSO‑d₆) δ 8.11

(s, 1H), 7.83 (s, 1H), 7.60 (d, J = 8.3 Hz, 2H), 7.31 (d, J = 8.3 Hz, 2H),

7.25 (td, J = 9.3, 5.2 Hz, 1H), 7.07 (td, J = 9.0, 1.8 Hz, 1H), 5.09–5.01

(m, 1H), 4.23–4.15 (m, 2H), 3.57 (dd, J = 17.0, 11.0 Hz, 1H), 3.28 (dd,

J = 17.1, 7.2 Hz, 1H), 2.58–2.52 (m, 1H), 1.82–1.76 (m, 4H), 1.71 (d,

J = 12.7 Hz, 1H), 1.45–1.33 (m, 4H), 1.28–1.20 (m, 1H). ¹³C NMR

(150 MHz, DMSO‑d₆) δ 161.73, 156.87, 152.51 (dd, J_F-C= 241.2,

6.7 Hz), 150.28, 148.40 (dd, J_F-C= 248.8, 8.3 Hz), 143.38 (dd, J_F-

J

_F-C= 25.0, 20.4 Hz), 116.34, 116.33, 116.27, 115.11, 111.42 (dd, J_F-

= 10.9, 3.2 Hz), 127.58, 127.33, 127.15, 117.09 (dd, J_F-C= 25.0,

C

_C= 22.8, 3.8 Hz), 78.71, 71.38, 63.72, 37.30, 15.01. ESI-MS calcd for

20.4 Hz), 116.32 (dd, J_F-C= 9.3, 2.3 Hz), 111.42 (dd, J_F-C= 22.8,

3.8 Hz), 78.86, 71.36, 44.11, 37.14, 34.19, 26.73, 26.00. ESI-MS calcd

for C₂₃H₂₅F₂N₂O₃[M+H]⁺, 415.5, found 415.4.

C

₁₉H₁₉F₂N₂O₄[M+H]⁺, 377.4, found 377.5.

5.2.24. 2,6-Diﬂuoro-3-((3-(3-propoxyphenyl)-4,5-dihydroisoxazol-5-yl)

methoxy)benzamide. (A24)

5.2.19. 2,6-Diﬂuoro-3-((3-(2-methoxyphenyl)-4,5-dihydroisoxazol-5-yl)

methoxy)benzamide (A19)

White solid, Mp: 145–146 °C; ¹H NMR (600 MHz, Chloroform-d) δ

7.31 (t, J = 8.0 Hz, 1H), 7.27 (d, J = 2.3 Hz, 1H), 7.20 (d, J = 7.7 Hz,

1H), 7.10 (td, J = 9.1, 5.1 Hz, 1H), 6.99 – 6.94 (m, 1H), 6.88 (td,

J = 9.1, 2.0 Hz, 1H), 5.92 (d, J = 46.0 Hz, 2H), 5.13–5.06 (m, 1H),

4.24–4.12 (m, 2H), 3.95 (t, J = 6.6 Hz, 2H), 3.55–3.38 (m, 2H), 1.82

(h, J = 7.3 Hz, 2H), 1.04 (t, J = 7.4 Hz, 3H). ¹³C NMR (150 MHz,

DMSO‑d₆) δ 161.73, 159.25, 157.02, 152.51 (dd, J_F-C= 241.5, 6.6 Hz),

148.39 (dd, J_F-C= 248.7, 8.4 Hz), 143.37 (dd, J_F-C= 10.9, 3.2 Hz),

130.98, 130.43, 119.44, 117.09 (dd, J_F-C= 25.0, 20.3 Hz), 116.87,

116.33 (d, J_F-C= 9.3 Hz), 112.65, 111.43 (dd, J_F-C= 23.0, 3.9 Hz),

79.14, 71.36, 69.53, 37.08, 22.47, 10.85. ESI-MS calcd for

White solid, Mp: 108–109 °C; ¹H NMR (600 MHz, Chloroform-d) δ

7.74–7.70 (m, 1H), 7.42–7.37 (m, 1H), 7.10 (td, J = 9.1, 5.1 Hz, 1H),

6.98 (t, J = 7.5 Hz, 1H), 6.95 (d, J = 8.4 Hz, 1H), 6.88 (td, J = 9.1,

2.0 Hz, 1H), 5.93 (d, J = 55.4 Hz, 2H), 5.07–5.01 (m, 1H), 4.22–4.10

(m, 2H), 3.87 (s, 3H), 3.69–3.45 (m, 2H). ¹³C NMR (150 MHz,

DMSO‑d₆) δ 161.78, 157.75, 156.11, 152.48 (dd, J_F-C= 241.3, 6.6 Hz),

148.37 (dd, J_F-C= 248.7, 8.4 Hz), 143.42 (dd, J_F-C= 11.0, 3.2 Hz),

131.98, 129.37, 121.07, 118.66, 117.09 (dd, J_F-C= 25.0, 20.4 Hz),

116.22 (dd, J_F-C= 9.4, 2.3 Hz), 112.68, 111.41 (dd, J_F-C= 22.8,

3.9 Hz), 78.79, 71.33, 56.16, 26.81. ESI-MS calcd for C₁₈H₁₇F₂N₂O₄[M

+H]⁺, 363.3, found 363.3.

C

₂₀H₂₁F₂N₂O₄[M+H]⁺, 391.4, found 391.3.

5.2.25. 2,6-Diﬂuoro-3-((3-(4-propoxyphenyl)-4,5-dihydroisoxazol-5-yl)

methoxy)benzamide (A25)

5.2.20. 2,6-Diﬂuoro-3-((3-(3-methoxyphenyl)-4,5-dihydroisoxazol-5-yl)

methoxy)benzamide (A20)

White solid, Mp: 162–163 °C; ¹H NMR (600 MHz, Chloroform-d) δ

7.61 (d, J = 8.9 Hz, 2H), 7.10 (td, J = 9.1, 5.1 Hz, 1H), 6.92 (d,

J = 8.9 Hz, 2H), 6.88 (td, J = 9.1, 1.9 Hz, 1H), 5.91 (d, J = 61.0 Hz,

2H), 5.06 (ddt, J = 10.5, 6.9, 5.1 Hz, 1H), 4.17 (ddd, J = 48.3, 10.2,

5.1 Hz, 2H), 3.96 (t, J = 6.5 Hz, 2H), 3.54–3.35 (m, 2H), 1.87–1.79 (m,

2H), 1.05 (t, J = 7.4 Hz, 3H). ¹³C NMR (150 MHz, DMSO‑d₆) δ 161.73,

160.57, 156.48, 152.48 (dd, J_F-C= 241.6, 6.6 Hz), 148.37 (dd, J_F-

_C= 248.7, 8.4 Hz), 143.38 (dd, J_F-C= 10.8, 3.1 Hz), 128.66, 121.97,

117.08 (dd, J_F-C= 24.9, 20.4 Hz), 116.33, 115.15, 111.41 (dd, J_F-

White solid, Mp: 114–116 °C; ¹H NMR (600 MHz, DMSO‑d₆) δ 8.11

(s, 1H), 7.83 (s, 1H), 7.38 (t, J = 8.0 Hz, 1H), 7.30–7.20 (m, 3H),

7.11–7.02 (m, 2H), 5.12–5.04 (m, 1H), 4.26–4.15 (m, 2H), 3.80 (s, 3H),

3.59 (dd, J = 17.1, 11.0 Hz, 1H), 3.30–3.27 (m, 1H). ¹³C NMR

(150 MHz, DMSO‑d₆) δ 161.72, 159.81, 157.00, 152.50 (dd, J_F-

_C= 241.6, 6.7 Hz), 148.38 (dd, J_F-C= 248.6, 8.5 Hz), 143.37 (dd, J_F-

= 10.9, 3.2 Hz), 131.00, 130.45, 119.60, 117.09 (dd, J_F-C= 24.9,

C

20.5 Hz), 116.47, 116.33 (d, J_F-C= 8.4 Hz), 112.02, 111.44 (dd, J_F-

= 23.0, 4.1 Hz), 79.16, 71.35, 55.69, 37.09. ESI-MS calcd for

C

= 22.8, 3.8 Hz), 78.70, 71.38, 69.56, 37.30, 22.42, 10.81. ESI-MS

C

₁₈H₁₇F₂N₂O₄[M+H]⁺, 363.3, found 363.3.

calcd for C₂₀H₂₁F₂N₂O₄[M+H]⁺, 391.4, found 391.3.

5.2.21. 2,6-Diﬂuoro-3-((3-(4-methoxyphenyl)-4,5-dihydroisoxazol-5-yl)

methoxy)benzamide (A21)

5.2.26. 2,6-Diﬂuoro-3-((3-(benzo[d][1,3]dioxol-5-yl)-4,5-

dihydroisoxazol-5-yl)methoxy)benzamide (A26)

White solid, Mp: 175–176 °C; ¹H NMR (600 MHz, Chloroform-d) δ

7.63 (d, J = 8.5 Hz, 2H), 7.14–7.05 (m, 1H), 6.98–6.84 (m, 3H), 5.93

(d, J = 50.0 Hz, 2H), 5.11–5.02 (m, 1H), 4.25–4.08 (m, 2H), 3.85 (s,

3H), 3.55–3.34 (m, 2H). ¹³C NMR (150 MHz, DMSO‑d₆) δ 161.74,

161.13, 156.49, 152.49 (dd, J_F-C= 241.5, 6.7 Hz), 148.38 (dd, J_F-

_C= 248.8, 8.4 Hz), 143.39 (dd, J_F-C= 10.8, 3.1 Hz), 128.68, 122.13,

117.08 (dd, J_F-C= 24.9, 20.4 Hz), 116.29 (dd, J_F-C= 9.5, 2.3 Hz),

114.71, 111.42 (dd, J_F-C= 23.1, 3.8 Hz), 78.73, 71.38, 55.76, 37.31.

ESI-MS calcd for C₁₈H₁₇F₂N₂O₄[M+H]⁺, 363.3, found 363.3.

White solid, Mp: 150–152 °C; ¹H NMR (600 MHz, Chloroform-d) δ

7.29 (d, J = 1.6 Hz, 1H), 7.12–7.04 (m, 2H), 6.89 (td, J = 9.1, 2.0 Hz,

1H), 6.83 (d, J = 8.1 Hz, 1H), 6.01 (s, 2H), 5.91 (d, J = 59.7 Hz, 2H),

5.10–5.03 (m, 1H), 4.24–4.10 (m, 2H), 3.52–3.33 (m, 2H). ¹³C NMR

(150 MHz, DMSO‑d₆) δ 161.75, 156.61, 152.50 (dd, J_F-C= 241.5,

6.6 Hz), 149.37, 148.19, 147.56 (d, J_F-C= 8.5 Hz), 143.37 (dd, J_F-

_C= 11.0, 3.2 Hz), 123.68, 122.12, 117.08 (dd, J_F-C= 24.9, 20.4 Hz),

116.31 (dd, J_F-C= 9.4, 2.2 Hz), 111.42 (dd, J_F-C= 23.1, 3.8 Hz),

108.89, 106.44, 102.03, 78.96, 71.35, 37.29. ESI-MS calcd for

12

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Bioorganic&MedicinalChemistry28(2020)115729

5.3.5. 2,6-Diﬂuoro-3-((5-(3,4-dichlorophenyl)-4,5-dihydroisoxazol-3-yl)

C₁₈H₁₅F₂N₂O₅[M+H]⁺, 377.3, found 377.3.

methoxy)benzamide (B5)

White solid, Mp: 114–119 °C; ¹H NMR (600 MHz, Chloroform-d) δ

7.44–7.43 (d, J = 8.3 Hz, 1H), 7.38 (d, J = 2.1 Hz, 1H), 7.13–7.09 (m,

2H), 6.92–6.89 (td, J = 9.1, 2.0 Hz, 1H), 5.97–5.93 (d, J = 22.8 Hz,

2H), 5.64–5.60 (dd, J = 11.1, 7.6 Hz, 1H), 4.92 (s, 2H), 3.60–3.55 (dd,

J = 17.4, 11.1 Hz, 1H), 3.06–3.02 (dd, J = 17.4, 7.6 Hz, 1H). ¹³C NMR

(150 MHz, DMSO‑d₆) δ 161.63, 155.76, 152.88 (dd, J_F-C= 242.2,

6.6 Hz), 148.64 (dd, J_F-C= 249.0, 8.5 Hz), 142.59 (d, J_F-C= 3.3 Hz),

142.53, 131.75, 131.36, 131.12, 128.53, 126.75, 117.19 (dd, J_F-

_C= 25.0, 20.4 Hz), 116.97 (d, J_F-C= 9.4 Hz), 111.50 (dd, J_F-C= 22.9,

3.9 Hz), 80.44, 64.64, 42.98. ESI-MS calcd for C₁₇H₁₂Cl₂F₂N₂O₃[M

+H]⁺, 401.1, [M+NH₄]⁺, 418.1, found 401.2, 418.3.

5.2.27. 2,6-Diﬂuoro-3-((3-(furan-2-yl)-4,5-dihydroisoxazol-5-yl)

methoxy)benzamide (A27)

White solid, Mp: 168–169 °C; ¹H NMR (600 MHz, Chloroform-d) δ

7.53 (d, J = 1.8 Hz, 1H), 7.09 (td, J = 9.1, 5.1 Hz, 1H), 6.89 (td,

J = 9.2, 2.0 Hz, 1H), 6.76 (d, J = 3.5 Hz, 1H), 6.51 (dd, J = 3.5,

1.8 Hz, 1H), 5.93 (d, J = 53.6 Hz, 2H), 5.10–5.02 (m, 1H), 4.24–4.10

(m, 2H), 3.54–3.36 (m, 2H). ESI-MS calcd for C₁₅H₁₃F₂N₂O₄[M+H]⁺,

323.3, found 323.3.

5.3. Typical compounds of the series B

5.3.6. 2,6-Diﬂuoro-3-((5-(4-ﬂurophenyl)-4,5-dihydroisoxazol-3-yl)

methoxy)benzamide (B6)

5.3.1. 2,6-Diﬂuoro-3-((5-(2-chlorophenyl)-4,5-dihydroisoxazol-3-yl)

methoxy)benzamide (B1)

Waxy solid, Mp: 99–103 °C; ¹H NMR (600 MHz, Chloroform-d) δ

7.28–7.27 (m, 1H), 7.26 (s, 1H), 7.14–7.10 (td, J = 9.1, 5.1 Hz, 1H),

7.06–7.03 (t, J = 8.7 Hz, 2H), 6.92–6.88 (td, J = 9.1, 2.0 Hz, 1H), 5.96

(s, 2H), 5.66–5.63 (dd, J = 11.0, 8.3 Hz, 1H), 4.95–4.90 (d, J = 2.9 Hz,

2H), 3.57–3.52 (dd, J = 17.4, 11.1 Hz, 1H), 3.09–3.04 (dd, J = 17.4,

8.3 Hz, 1H). ¹³C NMR (150 MHz, DMSO‑d₆) δ 163.15, 161.69, 161.53,

155.58, 152.90 (dd, J_F-C= 242.2, 6.6 Hz), 148.70 (dd, J_F-C= 248.9,

8.4 Hz), 142.60 (dd, J_F-C= 10.7, 3.3 Hz), 137.52 (d, J_F-C= 3.1 Hz),

128.69 (d, J_F-C= 8.3 Hz), 117.20 (d, J_F-C= 4.6 Hz), 117.07 (d, J_F-

White solid, Mp: 106–109 °C; ¹H NMR (600 MHz, Chloroform-d) δ

7.43–7.41 (dd, J = 6.9, 2.5 Hz, 1H), 7.39–7.37 (m, 1H), 7.28–7.27 (m,

1H), 7.26–7.24 (m, 1H), 7.11–7.07 (td, J = 9.1, 5.0 Hz, 1H), 6.89–6.86

(td, J = 9.1, 2.0 Hz, 1H), 5.96–5.85 (m, 3H), 4.93–4.87 (m, 2H),

3.73–3.68 (dd, J = 17.6, 11.3 Hz, 1H), 3.00–2.96 (dd, J = 17.6, 7.2 Hz,

1H). ESI-MS calcd for C₁₇H₁₃ClF₂N₂O₃[M+H]⁺, 367.1, [M+NH₄]⁺,

384.1, found 367.2, 384.2.

5.3.2. 2,6-Diﬂuoro-3-((5-(3-chlorophenyl)-4,5-dihydroisoxazol-3-yl)

= 7.6 Hz), 115.90 (d, J_F-C= 21.5 Hz), 111.53 (dd, J_F-C= 23.2,

C

3.9 Hz), 81.38, 64.77, 42.87. ESI-MS calcd for C₁₇H₁₃F₃N₂O₃[M+H]⁺,

351.1, [M+NH₄]⁺, 368.1, found 351.3, 368.3.

methoxy)benzamide (B2)

1

Waxy solid, Mp: 108–112 °C; H NMR (600 MHz, Chloroform-d) δ

7.30–7.27 (m, 3H), 7.17–7.09 (m, 2H), 6.92–6.88 (td, J = 9.1, 1.9 Hz,

1H), 5.97–5.94 (d, J = 13.8 Hz, 2H), 5.65–5.62 (dd, J = 11.1, 7.8 Hz,

1H), 4.93 (s, 2H), 3.59–3.55 (dd, J = 17.4, 11.2 Hz, 1H), 3.08–3.04

(dd, J = 17.4, 7.8 Hz, 1H). ¹³C NMR (150 MHz, DMSO‑d₆) δ 161.65,

155.67, 152.87 (dd, J_F-C= 242.1, 6.7 Hz), 148.63 (dd, J_F-C= 249.0,

8.4 Hz), 143.92, 142.59 (dd, J_F-C= 11.0, 3.2 Hz), 133.77, 131.06,

128.50, 126.31, 125.12, 117.19 (dd, J_F-C= 25.0, 20.4 Hz), 116.92 (d,

5.3.7. 2,6-Diﬂuoro-3-((5-(4-bromophenyl)-4,5-dihydroisoxazol-3-yl)

methoxy)benzamide (B7)

White solid, Mp: 122–124 °C; ¹H NMR (600 MHz, Chloroform-d) δ

7.50–7.48 (d, J = 8.4 Hz, 2H), 7.17–7.16 (d, J = 8.4 Hz, 2H),

7.13–7.09 (td, J = 9.1, 5.1 Hz, 1H), 6.91–6.88 (td, J = 9.1, 2.0 Hz,

1H), 5.95 (s, 2H), 5.64–5.61 (dd, J = 11.1, 8.1 Hz, 1H), 4.94–4.89 (m,

2H), 3.58–3.53 (dd, J = 17.4, 11.1 Hz, 1H), 3.07–3.02 (dd, J = 17.4,

8.0 Hz, 1H). ¹³C NMR (150 MHz, DMSO‑d₆) δ 161.65, 155.59, 152.87

(dd, J_F-C= 242.2, 6.7 Hz), 148.65 (dd, J_F-C= 249.0, 8.5 Hz), 142.56

(dd, J_F-C= 10.7, 3.2 Hz), 140.80, 132.01, 128.70, 121.69, 117.18 (d,

J_F-C= 4.5 Hz), 117.06 (d, J_F-C= 8.6 Hz), 111.53 (dd, J_F-C= 23.2,

3.8 Hz), 81.23, 64.71, 42.90. ESI-MS calcd for C₁₇H₁₃BrF₂N₂O₃[M

+H]⁺, 411.1, [M+NH₄]⁺, 428.1, found 411.2, 430.3.

J

_F-C= 9.3 Hz), 111.50 (dd, J_F-C= 23.1, 3.8 Hz), 81.07, 64.66, 42.99.

ESI-MS calcd for C₁₇H₁₃ClF₂N₂O₃[M+H]⁺, 367.1, [M+NH₄]⁺, 384.1,

found 367.2, 384.3.

5.3.3. 2,6-Diﬂuoro-3-((5-(4-chlorophenyl)-4,5-dihydroisoxazol-3-yl)

methoxy)benzamide (B3)

White solid, Mp: 127–129 °C; ¹H NMR (600 MHz, DMSO‑d₆) δ 8.14

(s, 1H), 7.87 (s, 1H), 7.45 (d, J = 8.5 Hz, 2H), 7.35–7.29 (m, 3H), 7.10

(td, J = 8.9, 1.7 Hz, 1H), 5.67 (dd, J = 11.1, 8.0 Hz, 1H), 5.01 (s, 2H),

5.3.8. 2,6-Diﬂuoro-3-((5-(3-methylphenyl)-4,5-dihydroisoxazol-3-yl)

methoxy)benzamide (B8)

3.58 (dd, J = 17.5, 11.1 Hz, 1H), 3.03 (dd, J = 17.6, 8.0 Hz, 1H). ¹³

C

Waxy solid, Mp: 78–80 °C; ¹H NMR (600 MHz, Chloroform-d) δ

7.26–7.23 (t, J = 7.6 Hz, 1H), 7.15–7.06 (m, 4H), 6.91–6.88 (td,

J = 9.1, 2.1 Hz, 1H), 5.97 (s, 2H), 5.64–5.61 (dd, J = 11.1, 8.3 Hz,

1H), 4.95–4.90 (m, 2H), 3.55–3.51 (dd, J = 17.4, 11.1 Hz, 1H),

3.11–3.06 (dd, J = 17.4, 8.3 Hz, 1H), 2.34 (s, 3H). ¹³C NMR (150 MHz,

DMSO‑d₆) δ 161.65, 155.45, 152.85 (dd, J_F-C= 242.1, 6.6 Hz), 148.64

(dd, J_F-C= 248.9, 8.5 Hz), 142.61 (dd, J_F-C= 11.0, 3.2 Hz), 141.29,

138.32, 129.21, 128.98, 127.00, 123.65, 117.20 (dd, J_F-C= 24.9,

20.4 Hz), 116.92 (dd, J_F-C= 9.4, 2.2 Hz), 111.51 (dd, J_F-C= 23.2,

3.8 Hz), 82.07, 64.72, 42.86, 21.45. ESI-MS calcd for C₁₈H₁₆F₂N₂O₃[M

+H]⁺, 347.1, [M+NH₄]⁺, 364.1, found 347.3, 364.3.

NMR (150 MHz, DMSO‑d₆) δ 160.55, 154.51, 151.74 (dd, J_F-C= 244.6,

6.8 Hz), 147.48 (dd, J_F-C= 249.2, 9.1 Hz), 141.44 (dd, J_F-C= 12.1,

4.5 Hz), 139.30, 132.04, 128.01, 127.32, 116.19 (d, J_F-C= 19.6 Hz),

115.90 (d, J_F-C= 16.6 Hz), 110.51 (dd, J_F-C= 22.7, 6.8 Hz), 80.09,

63.59, 41.85. ESI-MS calcd for C₁₇H₁₄ClF₂N₂O₃[M+H]⁺, 367.1, found

367.2.

5.3.4. 2,6-Diﬂuoro-3-((5-(2,4-dichlorophenyl)-4,5-dihydroisoxazol-3-yl)

methoxy)benzamide (B4)

White solid, Mp: 108–112 °C; ¹H NMR (600 MHz, Chloroform-d) δ

7.41–7.40 (d, J = 2.0 Hz, 1H), 7.37–7.36 (dd, J = 8.4, 0.7 Hz, 1H),

7.26–7.25 (m, 1H), 7.11–7.07 (td, J = 9.1, 5.1 Hz, 1H), 6.90–6.87 (td,

J = 9.1, 2.1 Hz, 1H), 5.95–5.87 (m, 3H), 4.92–4.86 (m, 2H), 3.73–3.68

5.3.9. 2,6-Diﬂuoro-3-((5-(4-methylphenyl)-4,5-dihydroisoxazol-3-yl)

methoxy)benzamide (B9)

(dd, J = 17.6, 11.3 Hz, 1H), 2.97–2.93 (dd, J = 17.6, 7.1 Hz, 1H). ¹³

C

White solid, Mp: 106–110 °C; ¹H NMR (600 MHz, Chloroform-d) δ

7.26 (s, 1H), 7.18–7.11 (m, 4H), 6.91–6.88 (td, J = 9.1, 2.1 Hz, 1H),

5.95 (s, 2H), 5.64–5.61 (dd, J = 11.1, 8.4 Hz, 1H), 4.95–4.90 (m, 2H),

3.54–3.49 (dd, J = 17.4, 11.1 Hz, 1H), 3.10–3.05 (dd, J = 17.4, 8.4 Hz,

1H), 2.35 (s, 3H). ¹³C NMR (150 MHz, DMSO‑d₆) δ 161.65, 155.46,

152.85 (dd, J_F-C= 241.9, 6.6 Hz), 148.65 (dd, J_F-C= 248.8, 8.4 Hz),

142.62 (dd, J_F-C= 10.8, 3.3 Hz), 138.28, 137.91, 129.61, 126.53,

NMR (150 MHz, DMSO‑d₆) δ 161.62, 155.74, 152.89 (dd, J_F-C= 242.2,

6.6 Hz), 148.64 (dd, J_F-C= 248.9, 8.4 Hz), 142.53 (dd, J_F-C= 11.0,

3.3 Hz), 137.92, 133.82, 132.25, 129.62, 128.71, 128.17, 117.31,

117.03 (dd, J_F-C= 8.4, 2.6 Hz), 111.49 (dd, J_F-C= 23.3, 3.7 Hz), 78.64,

64.58, 42.46. ESI-MS calcd for C₁₇H₁₂Cl₂F₂N₂O₃[M+H]⁺, 401.1, [M

+NH₄]⁺, 418.1, found 401.2, 418.3.

13

D. Song, et al.

Bioorganic&MedicinalChemistry28(2020)115729

117.20 (d, J_F-C= 4.7 Hz), 116.98 (dd, J_F-C= 9.7, 2.2 Hz), 111.51 (dd,

J_F-C= 23.2, 3.8 Hz), 82.02, 64.79, 42.74, 21.18. ESI-MS calcd for

1H). ¹³C NMR (150 MHz, DMSO‑d₆) δ 161.68, 159.90, 155.52, 152.87

(dd, J_F-C= 242.1, 6.6 Hz), 148.65 (dd, J_F-C= 248.8, 8.4 Hz), 142.97,

142.64 (dd, J_F-C= 11.0, 3.2 Hz), 130.27, 118.51, 117.19 (dd, J_F-

C

₁₈H₁₆F₂N₂O₃[M+H]⁺, 347.1, [M+NH₄]⁺, 364.1, found 347.3,

364.3.

= 25.0, 20.4 Hz), 116.91 (dd, J_F-C= 9.3, 2.2 Hz), 114.02, 111.98,

C

111.49 (dd, J_F-C= 23.1, 3.8 Hz), 81.91, 64.73, 55.50, 42.94. ESI-MS

calcd for C₁₈H₁₆F₂N₂O₄[M+H]⁺, 363.1, [M+NH₄]⁺, 380.1, found

363.3, 380.4.

5.3.10. 2,6-Diﬂuoro-3-((5-(4-ethylphenyl)-4,5-dihydroisoxazol-3-yl)

methoxy)benzamide (B10)

Waxy solid, Mp: 86–88 °C; ¹H NMR (600 MHz, Chloroform-d) δ

7.21–7.18 (m, 4H), 7.15–7.11 (td, J = 9.1, 5.1 Hz, 1H), 6.91–6.88 (td,

J = 9.1, 2.0 Hz, 1H), 5.95–5.92 (d, J = 18.1 Hz, 2H), 5.65–5.62 (dd,

J = 11.1, 8.5 Hz, 1H), 4.95–4.90 (m, 2H), 3.54–3.49 (dd, J = 17.4,

11.1 Hz, 1H), 3.11–3.07 (dd, J = 17.4, 8.5 Hz, 1H), 2.6 –2.63 (q,

J = 7.6 Hz, 2H), 1.24–1.22 (t, J = 7.6 Hz, 3H). ¹³C NMR (150 MHz,

DMSO‑d₆) δ 161.66, 155.46, 152.86 (dd, J_F-C= 242.2, 6.7 Hz), 148.66

(dd, J_F-C= 248.8, 8.3 Hz), 144.26, 142.62 (dd, J_F-C= 11.0, 3.3 Hz),

138.52, 128.45, 126.60, 117.19 (d, J_F-C= 4.7 Hz), 116.98 (d, J_F-

5.3.15. 2,6-Diﬂuoro-3-((5-(2-ethoxyphenyl)-4,5-dihydroisoxazol-3-yl)

methoxy)benzamide (B15)

White solid, Mp: 124–128 °C; ¹H NMR (600 MHz, Chloroform-d) δ

7.31–7.29 (dd, J = 7.5, 1.7 Hz, 1H), 7.26–7.24 (dd, J = 7.8, 1.8 Hz,

1H), 7.13–7.09 (td, J = 9.1, 5.1 Hz, 1H), 6.93–6.85 (m, 3H), 5.94–5.86

(m, 3H), 4.93–4.86 (m, 2H), 4.10–4.02 (m, 2H), 3.57–3.52 (dd,

J = 17.5, 11.3 Hz, 1H), 3.01–2.97 (dd, J = 17.5, 7.8 Hz, 1H),

1.42–1.36 (t, J = 7.0 Hz, 3H). ¹³C NMR (150 MHz, DMSO‑d₆) δ 161.60,

155.92, 155.39, 152.76 (dd, J_F-C= 242.1, 6.7 Hz), 148.54 (dd, J_F-

_C= 248.8, 8.4 Hz), 142.64 (dd, J_F-C= 11.0, 3.2 Hz), 129.70, 129.01,

126.78, 120.63, 117.14 (dd, J_F-C= 25.1, 20.4 Hz), 116.81 (d, J_F-

= 9.3 Hz), 111.51 (dd, J_F-C= 23.2, 3.8 Hz), 82.04, 64.78, 42.72,

C

28.33, 16.04. ESI-MS calcd for C₁₉H₁₈F₂N₂O₃[M+H]⁺, 361.1, [M

+NH₄]⁺, 378.1, found 361.3, 378.4.

= 9.3 Hz), 112.46, 111.45 (dd, J_F-C= 23.1, 3.9 Hz), 78.17, 64.74,

C

5.3.11. 2,6-Diﬂuoro-3-((5-(4-isopropylphenyl)-4,5-dihydroisoxazol-3-yl)

methoxy)benzamide (B11)

63.84, 42.03, 15.00. ESI-MS calcd for C₁₉H₁₈F₂N₂O₄[M+H]⁺, 377.1,

[M+NH₄]⁺, 394.1, found 377.3, 394.2.

White solid, Mp: 88–90 °C; ¹H NMR (600 MHz, Chloroform-d) δ 7.26

(s, 1H), 7.24–7.22 (m, 1H), 7.21–7.17 (m, 2H), 7.15–7.11 (td, J = 9.1,

5.1 Hz, 1H), 6.91–6.88 (td, J = 9.1, 2.0 Hz, 1H), 5.97–5.93 (d,

J = 22.8 Hz, 2H), 5.65–5.61 (dd, J = 11.1, 8.5 Hz, 1H), 4.959–4.90 (m,

2H), 3.54–3.49 (dd, J = 17.4, 11.1 Hz, 1H), 3.12–3.08 (dd, J = 17.4,

8.5 Hz, 1H), 2.93–2.87 (h, J = 6.9 Hz, 1H), 1.25–1.24 (d, J = 6.9 Hz,

6H). ¹³C NMR (150 MHz, DMSO‑d₆) δ 161.69, 155.46, 152.89 (dd, J_F-

= 242.2, 6.6 Hz), 149.53 (d, J_F-C= 8.4 Hz), 148.89, 147.87 (d, J_F-

^C_C= 8.5 Hz), 142.64 (dd, J_F-C= 11.0, 3.3 Hz), 138.63, 126.99, 126.62,

117.21 (d, J_F-C= 4.6 Hz), 116.99 (d, J_F-C= 9.0 Hz), 111.50 (dd, J_F-

_C= 22.8, 3.9 Hz), 82.07, 64.80, 42.68, 33.65, 24.27. ESI-MS calcd for

5.3.16. 2,6-Diﬂuoro-3-((5-(3-ethoxyphenyl)-4,5-dihydroisoxazol-3-yl)

methoxy)benzamide (B16).

1

Waxy solid, Mp: 106–110 °C; H NMR (600 MHz, Chloroform-d) δ

7.26–7.24 (d, J = 7.9 Hz, 1H), 7.13–7.10 (td, J = 9.1, 5.1 Hz, 1H),

6.90–6.78 (m, 4H), 6.01–5.95 (d, J = 35.2 Hz, 2H), 5.64–5.61 (dd,

J = 11.1, 8.0 Hz, 1H), 4.92 (s, 2H), 4.05–3.98 (qd, J = 7.0, 3.7 Hz, 2H),

3.55–3.51 (dd, J = 17.4, 11.2 Hz, 1H), 3.10–3.05 (dd, J = 17.4, 8.0 Hz,

1H), 1.42–1.39 (t, J = 7.0 Hz, 3H). ¹³C NMR (151 MHz, DMSO‑d₆) δ

161.68, 159.18, 155.49, 152.86 (dd, J_F-C= 242.3, 6.7 Hz), 148.65 (dd,

C

₂₀H₂₀F₂N₂O₃[M+H]⁺, 375.2, [M+NH₄]⁺, 392.2, found 375.4,

J

_F-C= 248.8, 8.4 Hz), 142.96, 142.64 (dd, J_F-C= 11.0, 3.3 Hz), 130.24,

392.3.

118.43, 117.20 (dd, J_F-C= 24.9, 20.4 Hz), 116.87 (d, J_F-C= 9.2 Hz),

114.45, 112.46, 111.49 (dd, J_F-C= 23.1, 3.8 Hz), 81.92, 64.71, 63.45,

42.93, 15.06. ESI-MS calcd for C₁₉H₁₈F₂N₂O₄[M+H]⁺, 377.1, [M

+NH₄]⁺, 394.1, found 377.4, 394.3.

5.3.12. 2,6-Diﬂuoro-3-((5-(4-(tert-butyl)phenyl)-4,5-dihydroisoxazol-3-

yl)methoxy)benzamide (B12)

1

Waxy solid; H NMR (600 MHz, DMSO‑d₆) δ 8.14 (s, 1H), 7.87 (s,

1H), 7.39 (d, J = 8.4 Hz, 2H), 7.33 (td, J = 9.3, 5.2 Hz, 1H), 7.23 (d,

J = 8.2 Hz, 2H), 7.11 (td, J = 8.9, 1.8 Hz, 1H), 5.60 (dd, J = 11.0,

8.3 Hz, 1H), 5.01 (s, 2H), 3.54 (dd, J = 17.5, 11.2 Hz, 1H), 3.04 (dd,

J = 17.5, 8.4 Hz, 1H), 1.27 (s, 9H). ¹³C NMR (150 MHz, DMSO‑d₆) δ

161.63, 155.47, 152.84 (dd, J_F-C= 242.1, 6.7 Hz), 151.08, 148.64 (dd,

5.3.17. 2,6-Diﬂuoro-3-((5-(4-ethoxyphenyl)-4,5-dihydroisoxazol-3-yl)

methoxy)benzamide (B17).

White solid, Mp: 106–109 °C; ¹H NMR (600 MHz, Chloroform-d) δ

7.19–7.18 (m, 2H), 7.15–7.11 (td, J = 9.1, 5.1 Hz, 1H), 6.91–6.84 (m,

3H), 5.97–5.93 (d, J = 23.2 Hz, 2H), 5.62–5.59 (dd, J = 10.9, 8.6 Hz,

1H), 4.93 (s, 2H), 4.05–4.01 (q, J = 7.0 Hz, 2H), 3.51–3.46 (dd,

J = 17.4, 11.0 Hz, 1H), 3.09–3.05 (dd, J = 17.4, 8.5 Hz, 1H),

1.42–1.40 (t, J = 7.0 Hz, 3H). ¹³C NMR (150 MHz, DMSO‑d₆) δ 161.66,

158.91, 155.48, 152.86 (dd, J_F-C= 242.1, 6.7 Hz), 148.67 (dd, J_F-

_C= 248.8, 8.4 Hz), 142.62 (dd, J_F-C= 10.8, 3.2 Hz), 132.88, 128.08,

117.20 (d, J_F-C= 4.5 Hz), 117.02 (d, J_F-C= 8.3 Hz), 114.89, 111.52

(dd, J_F-C= 23.2, 3.9 Hz), 81.99, 64.83, 63.51, 42.49, 15.07. ESI-MS

calcd for C₁₉H₁₈F₂N₂O₄[M+H]⁺, 377.1, [M+NH₄]⁺, 394.1, found

377.4, 394.3.

J

_F-C= 248.7, 8.6 Hz), 142.61 (dd, J_F-C= 11.0, 3.3 Hz), 138.26, 126.32,

125.83, 117.21 (d, J_F-C= 4.5 Hz), 116.99 (d, J_F-C= 9.5 Hz), 111.51

(dd, J_F-C= 22.8, 3.9 Hz), 81.92, 64.77, 42.65, 34.76, 31.56. ESI-MS

calcd for C₂₁H₂₃F₂N₂O₃[M+H]⁺, 389.4, found 389.3.

5.3.13. 2,6-Diﬂuoro-3-((5-(4-butylphenyl)-4,5-dihydroisoxazol-3-yl)

methoxy)benzamide (B13)

White solid, Mp: 101–102 °C; ¹H NMR (600 MHz, Chloroform-d) δ

7.21–7.16 (m, 4H), 7.13 (td, J = 9.1, 5.1 Hz, 1H), 6.90 (td, J = 9.1,

2.0 Hz, 1H), 5.93 (d, J = 30.7 Hz, 2H), 5.63 (dd, J = 11.0, 8.5 Hz, 1H),

4.97–4.89 (m, 2H), 3.51 (dd, J = 17.4, 11.1 Hz, 1H), 3.09 (dd,

J = 17.4, 8.4 Hz, 1H), 2.60 (t, 2H), 1.61–1.58 (m, 2H), 1.39–1.31 (m,

2H), 0.92 (t, J = 7.4 Hz, 3H). HRMS (ESI) calcd for C₂₁H₂₃F₂N₂O₃[M

+H]⁺, 389.1671, found 389.1673.

5.3.18. 2,6-Diﬂuoro-3-((5-(4-propoxyphenyl)-4,5-dihydroisoxazol-3-yl)

methoxy)benzamide (B18).

White solid, Mp: 76–78 °C; ¹H NMR (600 MHz, Chloroform-d) δ

7.19–7.18 (m, 2H), 7.15–7.11 (td, J = 9.1, 5.1 Hz, 1H), 6.91–6.86 (m,

3H), 5.97–5.96 (d, J = 9.8 Hz, 2H), 5.62–5.59 (dd, J = 10.9, 8.6 Hz,

1H), 4.93 (s, 2H), 3.92–3.90 (t, J = 6.6 Hz, 2H), 3.51–3.46 (dd,

J = 17.4, 11.0 Hz, 1H), 3.09–3.05 (dd, J = 17.4, 8.5 Hz, 1H),

1.83–1.77 (h, J = 7.3 Hz, 2H), 1.04–1.02 (t, J = 7.4 Hz, 3H). ESI-MS

calcd for C₂₀H₂₀F₂N₂O₄[M+H]⁺, 391.1, [M+NH₄]⁺, 408.1, found

391.3, 408.4.

5.3.14. 2,6-Diﬂuoro-3-((5-(3-methoxyphenyl)-4,5-dihydroisoxazol-3-yl)

methoxy)benzamide (B14)

White solid, Mp: 116–120 °C; ¹H NMR (600 MHz, Chloroform-d) δ

7.28–7.26 (m, 2H), 7.13–7.09 (td, J = 9.1, 5.1 Hz, 1H), 6.90–6.84 (m,

3H), 6.80–6.79 (t, J = 2.1 Hz, 1H), 6.00–5.96 (d, J = 27.5 Hz, 2H),

5.65–5.62 (dd, J = 11.1, 8.0 Hz, 1H), 4.93 (s, 2H), 3.80 (s, 3H),

3.56–3.51 (dd, J = 17.4, 11.1 Hz, 1H), 3.10–3.06 (dd, J = 17.4, 8.0 Hz,

14

D. Song, et al.

Bioorganic&MedicinalChemistry28(2020)115729

5.4. In vitro antibacterial assay

were determined form each plate following overnight incubation.

The MIC values of the synthesized compounds was determined in a

96-well microplate using the broth microdilution method re-

commended in the Clinical and Laboratory Standards Institute (CLSI)

guidelines.¹⁸The measurements were performed as previously de-

scribed.¹⁶

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 previously de-

scribed ²⁷. The experimental method was referred to our previous

study.¹⁶

5.5. Minimum bactericidal concentration (MBC) assay

The measurements were performed as previously described.¹⁶

5.6. Time-kill curve assay

5.12. Computational analysis

The molecular docking study was performed using Le-Dock.

Molecular ligand A16, B13 and A26_prewere constructed and saved as

Mol2 ﬁles by Chembio3D software. The FtsZ crystal structure (PDB

code: 3vob) was obtained from Protein Data Bank (PDB) and then

prepared for docking by using Le-Dock. The docking results were vi-

sually inspected and analyzed using Pymol (version 1.5).

The measurements were performed as previously described at var-

ious concentrations of A16 or comparator drug.¹⁶

5.7. Visualization of bacterial morphology

B. subtilis ATCC9372 and S. aureus ATCC CI were selected as tested

baterial strains, and the measurements were performed as previously

described.¹⁶

Declaration of Competing Interest

The authors declare that they have no known competing ﬁnancial

interests or personal relationships that could have appeared to inﬂu-

ence the work reported in this paper.

5.8. FtsZ polymerization assay

B. subtilis FtsZ was cloned, overexpressed and puriﬁed as previously

described.¹⁹The 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

Acknowledgements

This research was supported ﬁnancially by the National Natural

Science Foundation of China (81973179 and 81673284), Key Research

and Development Project of Shandong Province (2017CXGC1401) and

the China-Australia Centre for Health Sciences Research (CACHSR no.

2019GJ05).

340 nm (A₃₄₀

)

²⁶. The experimental method was referred to our previous

study.¹⁶

5.9. Compound’s stability study in vitro mouse plasma

Appendix A. Supplementary material

The fresh mice plasma was prepared by drawing blood, antic-

oagulation, and centrifugation. The experimental solution of the tested

compound at concentrations of 25 μg/mL was prepared from 2.56 mg/

mL DMSO stock solutions in freshly prepared mouse plasma and then

incubated at 37 °C. 20 μL of the solution was added to 60 μL of me-

thanol at a speciﬁc time point throughout 24 h. The obtained samples

were centrifuged at 12000 rpm for 4 min, and then the supernatants of

each sample were assessed using LC-MS.

Supplementary data to this article can be found online at https://

doi.org/10.1016/j.bmc.2020.115729.

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16

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Design, synthesis of novel 4,5-dihydroisoxazole-containing benzamide derivatives as highly potent FtsZ inhibitors capable of killing a variety of MDR Staphylococcus aureus

DOI: 10.1016/j.bmc.2020.115729

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