Discovery of novel piperonyl derivatives as diapophytoene desaturase inhibitors for the treatment of methicillin-, vancomycin- and linezolid-resistant Staphylococcus aureus infections

European Journal of Medicinal Chemistry 145 (2018) 235e251

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European Journal of Medicinal Chemistry

journal homepage: h ttp://www.elsevier.com/locate/ejmech

Research paper

Discovery of novel piperonyl derivatives as diapophytoene desaturase

inhibitors for the treatment of methicillin-, vancomycin- and

linezolid-resistant Staphylococcus aureus infections

,

Hanwen Wei ^{a 1}, Fei Mao ^{a 1}, Shuaishuai Ni ^{a 1}, Feifei Chen ^b, Baoli Li ^a, Xiaoxia Qiu ^a,

Linghao Hu ^a, Manjiong Wang ^a, Xinyu Zheng ^a, Jin Zhu ^a, Lefu Lan ^b^**, Jian Li ^a

^aShanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University of Science and Technology, 130 Mei Long Road, Shanghai 200237,

,

, *

China

^bState Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China

a r t i c l e i n f o

a b s t r a c t

Article history:

Inhibition of S. aureus diapophytoene desaturase (CrtN) could serve as an alternative approach for

addressing the tricky antibiotic resistance by blocking the biosynthesis of carotenoid pigment which

shields the bacterium from host oxidant killing. In this study, we designed and synthesized 44 derivatives

with piperonyl scaffold targeting CrtN and the structure-activity relationships (SARs) were examined

extensively to bring out the discovery of 21b with potent efﬁcacy and better hERG safety proﬁle

compared to the ﬁrst class CrtN inhibitor benzocycloalkane derivative 2. Except the excellent pigment

inhibitory activity against wild-type S. aureus, 21b also showed excellent pigment inhibition against four

pigmented MRSA strains. In addition, H₂O₂killing and human whole blood killing assays proved 21b

could sensitize S. aureus to be killed under oxidative stress conditions. Notably, the murine study in vivo

validated the efﬁcacy of 21b against pigmented S. aureus Newman, vancomycin-intermediate S. aureus

Mu50 and linezolid-resistant S. aureus NRS271.

Received 12 October 2017

Received in revised form

4 December 2017

Accepted 27 December 2017

Available online 29 December 2017

Keywords:

CrtN inhibitor

STX biosynthesis

Pigment inhibitory activity

Resistant S. aureus

1. Introduction

30% [2]. It's estimated by the U.S. Centers for Disease Control and

Prevention (CDC) that in the U.S. alone, almost half of the total

Staphylococcus aureus (S. aureus) is a pathogen that has been

evolving multi-resistance to many antibiotics and poses a major

menace to human health. The diseases typically present as skin

infections and more serious cases, like sepsis, pneumonia and

bloodstream infections [1]. The mortality related to methicillin-

resistant S. aureus (MRSA) bloodstream infection remains as high as

documented fatalities caused by antibiotic resistant infections was

from MRSA invasive infections [3]. To cope with MRSA infections,

vancomycin and linezolid have been taken as the most reliable

therapeutic agents [4,5]. However, the increasing emergence of

vancomycin-intermediate S. aureus (VISA) and linezolid-resistant

S. aureus (LRSA) makes it a great challenge in response to this

treatment failure [6,7]. These issues emphasize the urgency to ﬁnd

novel structures and mechanism of action to withstand antibiotic

resistant bacteria.

Antivirulence is an alternative approach to revitalize the drug-

development pipeline with new targets and new chemical entities

to treat infections caused by resistant bacteria [8]. Anti-virulence

agents target bacterial products that promote disease by either

damaging the host or evading the host immune system, and allow

bacterial multiplication in the host rather than kill or halt pathogen

growth, thus generating much weaker selection for resistance than

traditional antibiotics [8e11]. As a novel target for antivirulence

therapy against S. aureus, diapophytoene desaturase (CrtN) is an

Abbreviations used: S. aureus, Staphylococcus aureus; MRSA, methicillin-resistant

Staphylococcus aureus; STX, staphyloxanthin; NTF, naftiﬁne hydrochloride; CrtM,

dehydrosqualene synthase; CrtN, diapophytoene desaturases; ROS, reactive oxygen

species; SAR, structure-activity relationship; IC₅₀, half maximal inhibitory concen-

tration; HPLC, high-performance liquid chromatography; MS, mass chromatog-

raphy; CFU, colony-forming unit; PBS, phosphate-buffered saline; DMF, N,N-

dimethylformamide; DMSO, dimethyl sulfoxide; EtOH, ethanol; EtOAc, ethyl ace-

tate; MeOH, methanol; THF, tetrahydrofuran; CH₂Cl₂, dichloromethane; Et₃N,

triethylamine.

* Corresponding author.

** Corresponding author.

E-mail addresses: llan@simm.ac.cn (L. Lan), jianli@ecust.edu.cn (J. Li).

These authors contributed equally to this work.

1

http://dx.doi.org/10.1016/j.ejmech.2017.12.090

236

H. Wei et al. / European Journal of Medicinal Chemistry 145 (2018) 235e251

vancomycin as positive control drugs.

2. Results and discussion

2.1. Chemistry

The general synthetic route of derivatives 11a-h, 12a-c, 13a-e,

14a-g and 15b are outlined in Scheme 1. Commercial acquired

piperonol was oxidized by manganese dioxide to yield piperonyl

aldehyde 3. Compound 4 was prepared by reductive amination of 3

with methylamine. Iodide compounds reacted with acrolein diethyl

acetal through Heck reaction and acidiﬁcation to produce cinnamic

aldehyde derivatives. The latter was subsequently deoxidized by

sodium borohydride and then brominated by phosphorus tri-

bromide to generate compounds 7a-v. 4 reacted with 7a-v via

nucleophilic substitution to afford target derivatives.

Another route in Scheme 2 was applied to prepare cinnamyl

alcohol derivatives and target compounds 12d, 12e, 14h-m, 15a and

15c-e due to the availability of starting materials. Commercial ac-

quired benzaldehyde derivatives reacted with trimethyl phospho-

noacetate to give methyl cinnamate 8a-i via Wittig-Horner

reaction. Cinnamyl alcohol derivatives 9a-i were prepared by ester

reduction with diisobutylaluminum hydride and then brominated

by phosphorus tribromide to generate compounds 10a-i. 4 reacted

with 10a-i via nucleophilic substitution to produce target de-

rivatives 12d, 12e, 14h-m, 15a and 15c-e.

As shown in Scheme 3, to prepare derivatives 16a-c, piper-

onylamine was coupled with intermediate 7v via nucleophilic

substitution to afford derivative 16a, followed by substitution with

an ethyl group or isopropyl group to yield target derivatives 16b

and 16c respectively.

Scheme 4 depicts the synthetic route for derivatives 21a-e. In-

termediate 17 was obtained from 3-ﬂuoro-4-iodobenzotriﬂuoride

via Sonogashira coupling, followed by deprotonation of O-TBDMS

to give key intermediate 19a. Intermediate 5v reacted with triethyl

phosphonoacetate to produce 18a via Wittig-Horner reaction and

then reduced by diisobutylaluminium hydride to yield key

Fig. 1. Structures of Naftiﬁne and benzocycloalkane derivative 2.

established essential enzyme for staphyloxanthin (STX) biosyn-

thesis, which shields golden carotenoid pigmented S. aureus from

host oxidant killing [12e15]. Naftiﬁne (Fig. 1), a US Food and Drug

Administration (FDA)-approved antifungal drug, was proved efﬁ-

cient blocking biosynthesis of carotenoid pigment at nanomolar

concentrations and decreasing bacteria survival rate in murine

abscess formation model without directly killing the pathogen [16].

Based on this, our research group has designed and synthesized a

series of CrtN inhibitors with novel scaffolds as potent drug can-

didates against MRSA infections [17,18]. Benzocycloalkane deriva-

tive 2 (Fig. 1) exhibited potent anti-infectious activity in vitro and in

vivo [18]. However, the further exploration was suspended for the

unsatisfactory hERG safety proﬁle (IC₅₀¼ 3.2

mM) and poor water

solubility (4.24 mg/mL). In an attempt to increase hydrophilicity of

the core ring and gain novel CrtN inhibitors with better hERG safety

proﬁles, we designed and synthesized a collection of piperonyl

derivatives by inserting two oxygen atoms into the cycloalkane ring

and through rational structure modiﬁcation to ﬁnd better CrtN

inhibitors. As an extension to that study, our work was aimed at the

design, synthesis and evaluation of potent CrtN inhibitors with

novel scaffold plus superior physical property and safety proﬁle.

Moreover, the effectiveness of the synthesized compound on

attenuating the virulence of S. aureus strains including vancomycin-

intermediate MRSA Mu50 and linezolid-resistant MRSA NRS271

[19,20] was assessed in a murine abscess formation model from the

aspect of dosage and the mode of administration with linezolid and

Scheme 1. Synthesis of compounds 11a-h, 12a-c, 13a-e, 14a-g and 15b. Reagents and conditions: (a) MnO₂, DCM, r.t., overnight, 95%; (b) methylamine (30%e33% in methanol),

MeOH, r.t., 5 h; then NaBH₄, MeOH, 0 ^ꢀC to r.t., 30 min, 90%, (2 steps); (c) acrolein diethyl acetal, tetrabutylammonium acetate, palladium diacetate, K₂CO₃, KCl, DMF, 90 ^ꢀC, 2 h; then

HCl (33 wt. % in water), 62e85% (2 steps); (d) NaBH₄, MeOH, 0 ^ꢀC to r.t., 30min, 90%; (e) PBr₃, Et₂O, 0 ^ꢀC to r.t., overnight, under N₂, 50e85%; (f) K₂CO₃, DMF, r.t., overnight, 42e65%,

then bubbled into hydrogen chloride gas.

H. Wei et al. / European Journal of Medicinal Chemistry 145 (2018) 235e251

237

Scheme 2. Synthesis of compounds 12d, 12e, 14h-m, 15a and 15c-e. Reagents and conditions: (a) trimethyl phosphonoacetate, NaOMe (28% in methanol), DMF, r.t., 1 h, 95%; (b)

diisobutylaluminum hydride (1.0 M in hexane), DCM, 1.5 h, 0 ^ꢀC to r.t., 65e90%; (c) PBr₃, Et₂O, 0 ^ꢀC to r.t., overnight, under N₂, 50e85%; (d) K₂CO₃, DMF, r.t., overnight, 42e65%, then

bubbled into hydrogen chloride gas.

2.2. Biological activity

2.2.1. In vitro pigment inhibitory activities

To preliminarily assess the activities of synthesized compounds

in vitro, the half maximal inhibitory concentration (IC₅₀) values for

golden carotenoid pigment inhibition of 44 target compounds were

tested against wild-type S. aureus Newman. As shown in Table 1,

compounds bearing unsubstituted phenyl ring or methyl, cyclo-

Scheme 3. Synthesis of compounds 16a-c. Reagents and conditions: (a) K₂CO₃, DMF,

alkyls, heteroaryls (11a-f) showed a loss of the pigment inhibitory

activities. Replacement of phenyl ring at para position with various

substituents, including electron-donating (14f-g) and electron-

withdrawing groups (14a-b, 14d), showed superior activity to their

isomers (12a-e and 13a-e), which demonstrated that the

substituted positions on the phenyl ring substantially affected the

activity. Moreover, there is no obvious relation between pigment

inhibitory activity and the electronic effect of substituents

(comparing the compounds 14a-14g), indicating that both elec-

tron-donating and electron-withdrawing groups were both toler-

able at the phenyl ring. Based on this, more para-substitution

r.t., overnight, 55%; (b) iodoethane or 2-iodopropane, NaH, DMF, 0 ^ꢀC to r.t, overnight,

under N₂, 50%; hydrogen chloride gas.

intermediate 19b. 2-Fluoro-4-(triﬂuoromethyl)benzaldehyde reac-

ted with Wittig salt to give 18b and further reduced as 18a to afford

key intermediate 19c. Intermediate 6v was converted to key in-

termediate 19d and 19e through reduction by hydrogen and car-

bene reaction respectively. The ﬁve key intermediates were

brominated by phosphorus tribromide producing 20a-e and were

ﬁnally reacted with 4 to give the ﬁnal products 21a-e.

Scheme 4. Synthesis of compounds 20a-e. Reagents and conditions: (a) tert-butyldimethyl(2-propynyloxy)silane, Pd(PPh₃)₄, CuI, Et₃N, 0 ^ꢀC to 60 ^ꢀC, 30 min, 89%; (b) TBAF, THF, r.t.,

30 min, 98%; (c) PBr₃, Et₂O, 0 ^ꢀC to r.t., overnight, under N₂, 62%e76%; (d) K₂CO₃, DMF, r.t., overnight, 25e57%; hydrogen chloride gas; (e) triethyl phosphonoacetate, NaOMe (28% in

methanol), DMF, r.t., 1 h, 95%; (f) AlH(Bu-i)₂(1.0 M in hexane), DCM, 1.5 h, 0 ^ꢀC to r.t., 70%; (g) (carbethoxyethylidine)triphenylphosphorane, PhMe, reﬂux, 1 h, 85%; (h) TFA, Et₂Zn

(1.0 M in hexane), CH₂I₂, 0 ^ꢀC to r.t., 2 h; 86% for 19d; (i) Pd/C, MeOH, H₂, r.t., overnight; 94% for 19e.

238

H. Wei et al. / European Journal of Medicinal Chemistry 145 (2018) 235e251

Table 1

Chemical structures and their pigment inhibitory activities against S. aureus Newman for derivatives 11a-h, 12a-e, 13a-e, 14a-m and 15a-e.

Cpd.

R¹

S. aureus Newman IC₅₀(nM)^a

>1000

Cpd.

R¹

S. aureus Newman IC₅₀(nM)^a

>1000

Cpd.

R¹

S. aureus Newman IC₅₀(nM)^a

11a

12e

14g

9.1 0.6

11b

11c

>1000

13a

13b

412.3 12.4

575.5 11.4

14h

14i

12.5 1.8

4.1 0.1

11d

11e

>1000

13c

13d

523.3 136.9

372.4 16.3

14j

7.9 0.5

3.9 0.2

14k

11f

>1000

13e

14a

>1000

14l

5.2 0.4

4.2 0.4

11g

58.4 9.8

14m

11h

12a

4.0 0.1

14b

14c

4.2 0.4

4.6 0.2

15a

15b

17.6 4.1

1.8 0.0

121.2 6.9

12b

12c

>1000

14d

14e

3.5 0.6

15c

15d

57.0 0.5

3.8 0.2

28.8 4.6

82.6 10.5

12d

>1000

14f

15.7 0.3

15e

112.0 20.6

a

All the data represent mean values S.D.

(bromo, nitro, cyan, diﬂuoromethyl, formate, biphenyl, tertiary

butyl and ethyoxyl) and disubstituted compounds (15a-e) were

synthesized and evaluated for obtaining better inhibitors. Com-

parison of IC₅₀values exhibited by 14a-m, the sterically hindered

substituents exerted a beneﬁcial inﬂuence on the activity (14a vs

14d, 14f vs 14l and 14g vs 14m), indicating that the size of sub-

stituents may favor the pigment inhibitory activity. Moreover, the

activity of mono para-substituted derivatives were higher than that

of 3-ﬂuoro-4-disubstitute but lower than that of 2-ﬂuoro-4-dis-

ubstitute derivative (14d vs 15c vs 15b and 14g vs 15e vs 15d).

For further investigating the drug-like properties of these

compounds, two important assays were introduced into the study,

including water solubility and cell toxicity. First, 9 compounds with

IC₅₀values lower than 10 nM were selected to evaluate the cyto-

toxicity against two mammalian cell lines of hepatic carcinoma cell

HepG-2 and normal human embryonic kidney cell HEK-293T by

determining their half cytotoxic concentrations (CC₅₀). Since solu-

bility is a critical property of druggability, UV assay was applied to

determine the water solubility of these compounds in parallel. As

shown in Table 2, all selected compounds displayed good solubility,

and six (14b, 14c, 14d, 14m, 15b and 15d) out of these nine tested

compounds demonstrated excellent water solubility (over 10 mg/

mL). Moreover, compounds 14b, 14m, 15b and 21b exhibited

outstanding cytotoxicity proﬁle against these two cell lines at

comparable levels (CC₅₀> 80 mM). Especially, 15b possessed

balanced cytotoxicity proﬁles against cell lines HepG-2 and HEK-

293T, compared with 14b and 14m. Considering the pigment

inhibitory activity, water solubility and cytotoxicity proﬁle, 15b

thus far emerged as the most attractive candidate for further

structural modiﬁcation.

H. Wei et al. / European Journal of Medicinal Chemistry 145 (2018) 235e251

239

Table 2

Pigment (S. aureus Newman, IC₅₀), cytotoxicity (CC₅₀) and water solubility results of representative compounds.

Cpd.

S. aureus Newman IC₅₀(nM)^a

Cytotoxicity CC₅₀

HepG2

(

m

M)^a

Water Solubility (mg/mL)^b

HEK293T

11h

14b

14c

14d

14i

14k

14m

15b

4.02 0.13

4.24 0.38

4.55 0.19

3.51 0.61

4.10 0.12

3.87 0.24

4.22 0.38

1.81 0.04

3.84 0.21

4.02 0.23

e

60.48 2.04

140.51 10.98

104.40 8.96

69.23 5.52

137.82 8.84

53.55 1.63

82.61 5.07

94.67 3.85

120.45 7.42

90.33 4.73

28.62 4.22

48.32 0.96

83.83 6.01

68.33 4.87

133.10 15.90

67.99 6.06

59.12 5.32

122.45 12.45

111.88 9.58

76.83 3.98

92.64 6.68

36.49 5.08

4.56

18.71

17.46

13.50

9.03

5.02

24.25

14.03

19.22

8.69

15d

21b

Amphotericin B

e

a

All the data represent mean values S.D.

The values given are the solubility in water.

b

Further structural modiﬁcation was carried out on 15b. Firstly,

inhibition test was performed to evaluate the safety proﬁle of 15b

and 21b (Table 4). As shown in Table 2, both 15b and 21b exhibited

good water solubility, along with lower cytotoxicity than Ampho-

tericin B. Moreover, ﬁnding compound with better hERG safety

proﬁle is one major task of our modiﬁcation. Considering the su-

perior hERG safety proﬁle of 21b (~3-fold higher than 15b), we

ﬁnally chose 21b for further in vitro/in vivo assessments.

To assess the pigment inhibitory activities against antibiotic

resistant S. aureus, 21b together with 15b was incubated with two

community-acquired MRSAs (USA300 LAC and USA400 MW)

[21,22] and two hospital-acquired MRSAs (vancomycin-interme-

diate MRSA Mu50 and linezolid-resistant MRSA NRS271) to

examine the pigment inhibitory activity against multidrug-resis-

tant strains. The IC₅₀values are presented in Table 4, indicating that

15b and 21b are efﬁcient to a variety of pigmented MRSA strains.

N-methyl group was replaced by hydrogen, ethyl and isopropyl

(16a, 16b and 16c) to conﬁrm the steric interference of N-sub-

stituents matters. And comparing with 15b, the N-methyl substit-

uent was necessary for pigment inhibitory activity. Then, various

linker substituents were synthesized and tested, and only the diene

linker derivative displayed the single-digit nanomolar pigment

inhibitory activity.

The analysis of the data shown in Tables 1 and 3 revealed some

noteworthy observations for the SAR study: (1) In the studied set of

R¹group (Table 1), removing the substituents from the phenyl ring

or replacing with methyl, cycloalkyls or heteroaryls resulted in the

loss of potency. Para-position of phenyl ring is the best location of

substituents compared with ortho- or meta-position. The electronic

effect of the substituents for the potency was ﬁnite, and both

electron-donating and electron-withdrawing groups were toler-

able at the phenyl ring. Moreover, various types of substituted

group on the phenyl could remarkably affect the pigment inhibitory

activities, and the structure and activity relationships were un-

ambiguous. (2) N-methyl fragment was essential for the potency

(Table 3), for N-substituents variation resulted in the elimination of

potency. (3) For the allyl linker (Table 3), inserting a vinyl retained

its potency, whereas the allyl linker was irreplaceable. (4) Intro-

duction of oxygen atoms to the benzocycloalkane was tolerable for

achieving high potency.

2.2.2. Determination of the target enzyme

To verify whether our derivatives targeted the same enzyme as

NTF (the ﬁrst class CrtN inhibitor [16]), an HPLC experiment was

performed at 286 nm to analysis the 4,4⁰-diapophytoene, which is

the product of CrtM and the substrate of CrtN. As shown in Fig. 2,

the expression of CrtM in Escherichia coli displayed the peak of 4,4⁰-

diapophytoene (Fig. 2B, arrow character) similar to that of wild-

type S. aureus Newman (Fig. 2C), including retention time and UV

absorption spectra in the HPLC chromatogram. Subsequently, this

peak disappeared in the carotenoid extract of CrtM mutant (Fig. 2D)

and augmented in CrtN mutant (Fig. 2E), which further proved that

this peak belonged to 4,4⁰-diapophytoene. Lastly, either NTF-

treated or 21b-treated Newman (Fig. 2G) both had the HPLC peak

similar to that of the CrtN mutant, which led to the accumulation of

4,4⁰-diapophytoene in the S. aureus Newman. These results indi-

cated that CrtN is the enzyme target of 21b.

Before choosing the ﬁnal compound, which would be subjected

to further biological tests, the water solubility and cytotoxicity of

21b were tested (Table 2). Besides, hERG potassium channel

Table 3

Chemical structures and their pigment inhibitory activities against S. aureus New-

man for derivatives 16a-c and 21a-e.

S. aureus Newman IC₅₀(nM)^a

Cpd.

S. aureus Newman IC₅₀(nM)^a

To further prove the piperonyl derivatives targeting CrtN and

measure the efﬁcacy of enzymatic inhibition directly, CrtN enzy-

matic inhibitory activities in vitro of 21b and 15b were tested using

the previous protocol [9]. The results exhibited in Table 4 demon-

strated that 21b and 15b could inhibit the CrtN enzymatic activity

with IC₅₀values of 407.8 nM and 430.0 nM, respectively.

Cpd.

16a

16b

16c

21a

>1000

17.9 2.3

21b

21c

21d

21e

4.0 0.2

>1000

a

All the data represent mean values S.D.

Table 4

Pigment inhibition (S. aureus Newman, Mu50, NRS271, USA400 MW2 and USA300 LAC, IC₅₀), enzyme (CrtN IC₅₀), and hERG results of 15b and 21b.

Cpd.

Mu50

NRS271

USA400 MW2

IC₅₀(nM)^a

USA300 LAC

IC₅₀(nM)^a

CrtN

hERG

IC₅₀(mM)

IC₅₀(nM)^a

15b

21b

2.2 0.2

2.5 0.1

2.0 0.2

4.1 0.3

6.1 0.3

7.6 0.1

11.0 0.8

12.9 1.4

430.0 32.0

407.8 40.3

4.22

14.00

a

The data represent mean values S.D.

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H. Wei et al. / European Journal of Medicinal Chemistry 145 (2018) 235e251

Fig. 2. 21b treatment resulted in the accumulation of 4,4⁰-diapophytoene. (AeG) HPLC chromatograms (absorption at 286 nm) of the carotenoid extracts from E. coli (A), E. coli

expressing S. aureus crtM (B), wild-type S. aureus Newman (C), CrtM mutant (D), CrtN mutant (E), NTF-treated wild-type S. aureus Newman (F), 21b-treated wild-type S. aureus

Newman strains (G). Insets on the right show the absorbance spectra of the indicated HPLC peaks. mAu, milli-absorbance units. Absorbance (Abs) represents the amount of light

absorbed by the sample.

In addition, compound 21b did not affect the growth of S. aureus

strains (Newman strain and three MRSA strains) at 0.2 mM, indi-

cating that 21b did not function as traditional antibiotic (Fig. S1,

Supporting Information).

incubation with 21b also sensitized Mu50, USA300 LAC, and

USA400 MW2 to killing by H₂O₂(2.00% vs 23.33%, 2.67% vs 26.33%,

2.33% vs 29.67%, respectively), and the addition of NAC promoted

the survival rates of all three MRSA strains. The results above

illustrated that the oxidizability of H₂O₂could reduce the survival

of S. aureus, and the pigment served as protective antioxidant.

Fig. 3E indicates that 21b-treated S. aureus Newman dramatically

reduced bacteria survival in fresh human whole blood compared to

vehicle control (1.83% vs 20.0%, reduced by a factor of ~10.9).

Similarly, the other three 21b-treated MRSA strains (and Mu50,

USA300 LAC, and USA400 MW2) also presented less survivors than

untreated S. aureus strains (0.28% vs 8.66%%, 1.57% vs 23.33%, 0.93%

vs 14.33, respectively). These results demonstrated that through

inhibiting protective pigment biosynthesis, 21b could render

S. aureus to be more susceptible to immune clearance and lower the

survival rate in vitro.

2.2.3. In vitro oxidative killing assay

Two independent experiments of hydrogen peroxide killing and

human whole blood killing assays were carried out to assess the

effects of the most promising compound 21b on sensitizing

S. aureus Newman and three MRSA strains (Mu50, USA300 LAC, and

USA400 MW2) to immune clearance. The results were shown in

Fig. 3. In hydrogen peroxide killing assay, the survival rate of 21b-

treated S. aureus Newman was reduced signiﬁcantly than that of

untreated S. aureus Newman by a factor of ~11.7 (survival, 2.33% vs

26.67%), which survived worse than the antioxidant Nacetylcys-

teine (NAC)-treated S. aureus Newman cells (survival, 26.67% vs

57.33%) for NAC served as an oxidant scavenger. Moreover,

H. Wei et al. / European Journal of Medicinal Chemistry 145 (2018) 235e251

241

Fig. 3. Effects of 21b on susceptibility of S. aureus to killing by hydrogen peroxide and human whole blood; * p < 0.05, ** p < 0.01, *** p < 0.001via a two-tailed t-test (n ¼ three

biological replicates, each with two technical replicates).

2.2.4. In vivo effects of 21b on attenuating the virulence of

Newman, Mu50 and NRS271

showed that the dose-dependence effect of the 21b was not

obvious and the effects of treatment mode was limited.

In the Mu50 model, all the four treatment groups decreased

bacterial loads in both the kidneys and the livers with signiﬁcance

level of P value less than 0.001 (Fig. 4CeD). Especially in the liver, all

the treatment groups except low-dosage with post-treatment

group showed comparative clearance relative to that of linezolid-

treatment group.

In the NRS271 model, bacterial populations were counted in the

livers (Fig. 4E). The therapeutic effect of two pre-treatment groups

were comparable with that of linezolid-treatment, besides, the

post-treatment group showed lower staphylococcal loads than

linezolid-treatment group with statistical signiﬁcance. However,

the four treatment groups displayed no statistical signiﬁcance. In

this regard, 21b have an advantage over linezolid.

Since derivative 21b possessed potent pigment inhibition ac-

tivity in vitro and superior hERG safety proﬁle, the in vivo efﬁcacy of

21b against wild-type S. aureus Newman, MRSA Mu50 and LRSA

NRS271 was evaluated in a murine abscess formation model where

four regimens were established to systemically assess the impacts

of dosage as well as the mode of administration. The four regimens

included two dose levels (0.4 mg/bid/4.5 d and 0.1 mg/bid/4.5 d)

twice per day for two different administration modes of pre-

infection and post-infection administration. The pre-treatment and

post-treatment groups were injected intraperitoneally with deriv-

ative 21b 24 h before and 6 h after retro-orbital inoculation with

S. aureus strains, respectively. The two positive control groups were

vancomycin and linezolid at a dosage of 0.4 mg/bid/4.5 d via pre-

infection administration. The results are summarized in Fig. 4.

In the Newman model, the mice were inoculated with

2.2 ꢁ 10⁷colony-forming units (CFU) of S. aureus Newman bacteria.

Upon euthanatizing the mice at 108 h, the bacterial survivals in the

livers and kidneys were counted. In the kidneys (Fig. 4A), staphy-

lococcal loads of the treatment groups (high dosage with pre-

treatment, high dosage with post-treatment, low dosage with pre-

treatment and low dosage with post-treatment) were signiﬁcantly

lower than those of the vehical control group, with statistical sig-

niﬁcance (P ¼ 0 .0017, 0.0006, 0.0010, 0.0014, respectively). There

was no signiﬁcant statistical difference with respect to either

dosage or treatment mode.

3. Conclusions

In summary, 44 piperonyl derivatives were synthesized and

evaluated to obtain potent CrtN inhibitors with excellent pigment

inhibition against S. aureus Newman. Through structure optimiza-

tion, we found that the diene linker possessed better hERG cardiac

safety proﬁle, coupled with fair aqueous solubility. Taken together,

safety and efﬁcacy as well as physical property allowed 21b to be

more appealing than other derivatives. The screened compounds

were then evaluated for in vitro pigment inhibitory against four

MRSA strains. The HPLC experiment and the enzymatic inhibitory

activity conﬁrmed that 21b targets the enzyme CrtN. In the

oxidative killing assay, 21b was proved capable of sensitizing

S. aureus strains to be killed by H₂O₂or human whole blood in vitro.

To evaluated the efﬁcacy in vivo as well as systemically examine the

suitable dosage as well as the mode of administration, four treat-

ment regimen and two positive control were set in a murine ab-

scess formation model. In this model, compound 21b validated its

therapeutic efﬁcacy against wild-type S. aureus Newman, Mu50

and NRS271, which could signiﬁcantly alleviate the staphylococcal

loads in host organs. The study in vivo also indicated that the

In the livers (Fig. 4B), the high-dose for pre-treatment group and

low-dose for post-treatment group signiﬁcantly decreased the

bacterial survival by 1.90 log₁₀CFU and 1.53 log₁₀CFU, respectively

(equal to 98.74% and 97.06% decrease in surviving bacteria), slightly

higher than linezolid-treatment group, which led to a reduction by

1.41 log₁₀CFU, but there was no signiﬁcant difference, so the same

for vancomycin-treatment group (2.13 log₁₀CFU reduction) which

also demonstrated no signiﬁcant difference. On the whole, all of the

four regimens reduced the Newman staphylococcal loads in the

livers by at least 95% decrease in surviving bacteria. The results

242

H. Wei et al. / European Journal of Medicinal Chemistry 145 (2018) 235e251

Fig. 4. In vivo effect of 21b on reducing bacteria survival. Bacteria survival in the kidneys and livers of mice challenged with Newman bacteria (A and B). Bacteria survival in the

kidneys and livers of mice challenged with Mu50 bacteria (C and D). Bacteria survival in the livers of mice challenged with NRS271 bacteria (E). P ¼ pretreatment, drugs or

compounds were intraperitoneally administered 24 h before infection, compared with the post-treatment (compound was administered 6 h after infection). Statistical signiﬁcance

was determined by the Mann-Whitney test (two-tailed): * p < 0.05, ** p < 0.01, *** p < 0.001, n.s. indicates no signiﬁcant difference. Each symbol represents the value for an

individual mouse. Horizontal bars indicate the observation means, and dashed lines mark the limits of detection.

H. Wei et al. / European Journal of Medicinal Chemistry 145 (2018) 235e251

243

administration of 21b after occurrence of infection could achieve

comparative effects with prophylactical treatment, and moreover,

21b may possess considerably broad therapeutic window concen-

tration against pigmented S. aureus strains.

water. The mixture was extracted with EtOAc for three times. The

combined extracts were washed with brine, dried over anhydrous

MgSO₄, ﬁltered and condensed. The residue was then puriﬁed by

silica gel column chromatography (eluent, EtOAc/petroleum ether,

1/15, V/V) to afford 5a as a yellowish oil.

4. Experimental section

4.1.4. Synthesis of (E)-3-(furan-2-yl)prop-2-en-1-ol (6a)

4.1. General chemistry

To a solution of 5a (244.0 mg, 2.0 mmol) in methanol (10 mL)

was added NaBH₄(76 mg, 2.0 mmol) at 0 ^ꢀC. The reaction mixture

was then stirred at room temperature for 30 min and concentrated.

The residue was poured into water and extracted with EtOAc. The

combined extracts were washed with brine, dried over anhydrous

Na₂SO₄, ﬁltered, and condensed to afford 6a as a colorless oil. The

crude was used for the next step without further puriﬁcation.

TLC was performed on HSGF 254 (150e200 mm thickness; Yantai

Huiyou Co., China). UV light and also, I₂and DNP were used to

monitor synthetic progress. Column chromatography was per-

formed on silica gel (200e300 mesh) and eluted with ethyl acetate

and petroleum ether. NMR spectra data were recorded on a Bruker

AMX-400 NMR. Chemical shifts were reported in parts per million

(ppm). NMR signals were described from the aspect of multiplicity

(s ¼ singlet, d ¼ doublet, t ¼ triplet, q ¼ quartet, q ¼ quartet,

m ¼ multiplet and br ¼ broad), coupling constant (Hz) and inte-

grated value. Infrared spectra were recorded in potassium bromide

disks on a Nicolet FT-IR 6700 spectrometer, and n_maxare partially

reported in cm^ꢂ1. The mass spectral (MS) data were acquired with

electron spray ionization (ESI) produced by a Finnigan MAT-95 and

LCQ-DECA spectrometer. Each reported compound had >95% pu-

rity, as determined by HPLC analysis on an Agilent 1100 with a

quaternary pump and diode-array detector (DAD) (Supporting In-

formation). Melting points of each compound were determined on

an SGW X-4 melting point apparatus. All starting reagents and

solutions were commercially available and were used without

further puriﬁcation.

4.1.5. Synthesis of (E)-2-(3-bromoprop-1-en-1-yl)furan (7a)

To a solution of 6a (248 mg, 2.0 mmol) in anhydrous ether

(10 mL) was added phosphorus tribromide (75 m

L, 0.8 mmol) at 0 ^ꢀC

under a nitrogen atmosphere. The reaction mixture was stirred at

room temperature overnight and poured into ice water containing

saturated sodium bicarbonate. The mixture was then extracted

with EtOAc. The combined extracts were washed with brine, dried

over anhydrous Na₂SO₄, ﬁltered, and condensed at 30 ^ꢀC to afford

7a as a yellow oil. The crude was used for the next step without

further puriﬁcation.

4.1.6. Synthesis of methyl (E)-4-(o-tolyl)but-3-enoate (8a)

Sodium methoxide (28% in MeOH, 386 mg, 2.0 mmol) was

added to a stirred solution of 2-methylbenzaldehyde (253 mg,

2.1 mmol) and trimethyl phosphonoacetate (364 mg, 2.0 mmol) in

dimethylformamide (10 mL) at 0 ^ꢀC. After the mixture was stirred

for 2 h, the reaction mixture was poured into water to give crystals.

Then the crystals were ﬁltered to give 8a as colorless needles. The

crude was used for the next step without further puriﬁcation. ¹H

4.1.1. Synthesis of benzo[d](1,3)dioxole-5-carbaldehyde (3)

To a stirred solution of 1-hydroxymethyl-3,4-methylenediox-

ybenzene (304 mg, 2.0 mmol) in DCM (25 mL) was added MnO₂

(20 mmol, 1.74 g), and the reaction mixture was stirred at room

temperature overnight. Subsequently, the mixture was ﬁltrate over

Celite, and the organic solvent was evaporated under reduced

pressure to produce 3 as a colorless oil without further puriﬁcation.

NMR (400 MHz, DMSO-d₆)

J ¼ 7.6 Hz, 1H), 7.32 (d, J ¼ 6.8 Hz, 1H), 7.29e7.24 (m, 2H), 6.53 (d,

J ¼ 15.9 Hz, 1H), 3.74 (s, 3H), 2.40 (s, 3H).

d

7.88 (d, J ¼ 15.9 Hz, 1H), 7.73 (d,

¹H NMR (400 MHz, DMSO-d₆)

7.33 (s, 1H), 7.15 (d, J ¼ 8.0 Hz, 1H), 6.18 (s, 2H).

d

9.81 (s, 1H), 7.55 (d, J ¼ 8.0 Hz, 1H),

4.1.7. Synthesis of (E)-3-(o-tolyl)prop-2-en-1-ol (9a)

Diisobutylaluminum hydride (1.0 M solution in hexane, 4 mL,

4.0 mmol) was added dropwise to a stirred solution of 8a (296 mg,

2.0 mmol) in dichloromethane (10 mL) at 0 ^ꢀC. After the mixture

was stirred at room temperature for 1.5 h, methyl (0.4 mL) and

water (0.4 mL) were added to the mixture with cooling. The reac-

tion mixture was acidiﬁed with concentrated hydrochloric acid and

extracted with dichloromethane. The extract was washed with

brine, dried over MgSO₄, and condensed to give the 9a as a colorless

oil. The crude was used for the next step without further puriﬁca-

4.1.2. Synthesis of 1-(benzo[d](1,3)dioxol-5-yl)-N-

methylmethanamine (4)

To a stirred solution of 3 (300 mg, 2.0 mmol) in anhydrous

methanol (10 mL) was added methylamine (601 mg, 33% in meth-

anol) at room temperature. After stirring for 5 h, the solvent was

evaporated under reduced pressure to remove the excessive

methylamine and the residue was re-dissolved in methanol (15 mL)

with stirring at 0 ^ꢀC. NaBH₄(76 mg, 2.0 mmol) was added after-

wards and then the reaction was transferred to r.t for 30 min. The

solvent was removed under reduced pressure and extracted with

EtOAc and water. The combined organic layers were washed with

brine, dried over anhydrous Na₂SO₄, ﬁltered and condensed under

reduced pressure. The residue was then puriﬁed by silica gel col-

umn chromatography (eluent, EtOAc/petroleum ether, 1/1, V/V) to

tion. ¹H NMR (400 MHz, DMSO-d₆)

d

7.47 (d, J ¼ 8.7 Hz, 1H),

7.19e7.12 (m, 3H), 6.76 (d, J ¼ 15.8 Hz, 1H), 6.24 (dt, J ¼ 15.8, 5.0 Hz,

1H), 4.90 (t, J ¼ 5.5 Hz, 1H), 4.14 (td, J ¼ 5.3, 1.5 Hz, 2H), 2.29 (s, 3H).

4.1.8. Synthesis of (E)-N-(benzo[d][1,3]dioxol-5-ylmethyl)-N-

methyl-3-phenylprop-2-en-1-amine hydrochloride (11a)

afford 4 as a colorless oil. ¹H NMR (400 MHz, DMSO-d₆)

1H), 6.78e6.74 (m, 2H), 5.95 (s, 2H), 4.06 (s,1H), 3.65 (s, 2H), 2.43 (s,

3H).

d

6.83 (s,

A mixture of intermediate 4 (182 mg, 1.1 mmol), (E)-(3-bromo-

prop-1-en-1-yl)benzene (197 mg, 1.0 mmol) and K₂CO₃(276 mg,

2.0 mmol) in DMF (10 mL) was stirred at room temperature over-

night. The reaction mixture was then poured into water and

extracted with ethyl acetate. The combined organic layers were

washed with brine, dried over anhydrous Na₂SO₄, and then ﬁltered

and condensed. The residue was then puriﬁed by silica gel column

chromatography (eluent, EtOAc/petroleum ether, 1/5, V/V) to give

the free base of 11a as a yellow oil. Subsequently, the free base was

dissolved in EtOAc (10 mL) and inﬂated with hydrogen chloride gas

until the solids stopped showing. Then the solvent was evaporated

4.1.3. Synthesis of (E)-3-(furan-2-yl)acrylaldehyde (5a)

A mixture of 2-iodofuran (388 mg, 2.0 mmol), acrolein diethyl

acetal (729 mg, 5.6 mmol), tetrabutylammonium acetate (1.20 g,

4.0 mmol), palladium diacetate (14 mg, 0.06 mmol), K₂CO₃(553 mg,

4.0 mmol), and KCl (209 mg, 2.8 mmol) in anhydrous DMF (10 mL)

was stirred for 2 h at 90 ^ꢀC. The resulting dark brown mixture was

quenched with 3% (w/w) hydrochloric acid, and then poured into

244

H. Wei et al. / European Journal of Medicinal Chemistry 145 (2018) 235e251

in vacuo and the residue was suspended in a mixture of ethyl ac-

etate and petroleum ether under ultrasonic. The precipitate was

ﬁltrated and washed with ethyl ether to afford the ﬁnal compound

11a in the form of hydrochloride as a white solid. Spectroscopic

data given below are in their hydrochloride form. Yield: 59%; m.p.:

4.1.13. Synthesis of (E)-N-(benzo[d][1,3]dioxol-5-ylmethyl)-3-

cyclohexyl-N-methylprop-2-en-1-amine hydrochloride (11f)

Compound 11f was prepared by the experimental procedure

depicted for compound 11a as a white solid. Yield: 52%; m.p.:

147e150 ^ꢀC. ¹H NMR (400 MHz, CD₃OD)

d 7.00e6.88 (m, 3H),

139e141 ^ꢀC. ¹H NMR (400 MHz, CD₃OD)

d

7.52 (d, J ¼ 7.1 Hz, 2H),

6.06e5.95 (m, 3H), 5.55 (dt, J ¼ 14.8, 7.4 Hz, 1H), 4.30 (d, J ¼ 13.1 Hz,

1H), 4.09 (d, J ¼ 13.1 Hz, 1H), 3.79 (dd, J ¼ 13.1, 6.7 Hz, 1H), 3.61 (dd,

J ¼ 13.0, 7.9 Hz, 1H), 2.69 (s, 3H), 2.10 (s, 1H), 1.83e1.64 (m, 5H),

7.42e7.30 (m, 3H), 7.06e6.99 (m, 2H), 6.94 (s, 1H), 6.91 (d,

J ¼ 9.4 Hz, 1H), 6.35 (dt, J ¼ 15.2, 7.5 Hz, 1H), 6.02 (d, J ¼ 7.6 Hz, 2H),

4.40 (d, J ¼ 13.0 Hz, 1H), 4.18 (d, J ¼ 13.1 Hz, 1H), 4.03 (dd, J ¼ 13.2,

7.1 Hz, 1H), 3.86 (dd, J ¼ 13.2, 7.9 Hz, 1H), 2.79 (s, 3H). ¹³C NMR

1.41e1.08 (m, 5H). ¹³C NMR (100 MHz, DMSO-d₆)

d 148.52, 147.89,

147.05, 125.78, 124.16, 116.98, 111.57, 108.82, 101.90, 57.67, 56.60,

(100 MHz, DMSO-d₆)

d

148.54, 147.90, 138.79, 135.98, 129.21 (2C),

38.02, 32.33 (2C), 26.02, 25.85, 25.75 (2C); HRMS (ESI) m/z calcd for

129.06, 127.27 (2C), 125.85, 124.24, 118.73, 111.64, 108.83, 101.90,

57.80, 56.57, 38.26; HRMS (ESI) m/z calcd for C₁₈H₂₀NO₂[MþH]^þ

282.1494, found 282.1492.

C

₁₈H₂₆NO₂[MþH]^þ288.1964, found 288.1963.

4.1.14. Synthesis of (E)-N-(benzo[d][1,3]dioxol-5-ylmethyl)-N-

methyl-3-(naphthalen-1-yl)prop-2-en-1-amine hydrochloride (11g)

Compound 11g was prepared by the experimental procedure

depicted for compound 11a as a white solid. Yield: 58%; m.p.:

4.1.9. Synthesis of (E)-N-(benzo[d][1,3]dioxol-5-ylmethyl)-3-

(furan-2-yl)-N-methylprop-2-en-1-amine hydrochloride (11b)

Compound 11b was prepared by the experimental procedure

depicted for compound 11a as a white solid. Yield: 44%; m.p.:

173e176 ^ꢀC. ¹H NMR (400 MHz, CD₃OD)

d

8.17 (d, J ¼ 8.3 Hz, 1H),

7.90 (dd, J ¼ 7.6, 5.5 Hz, 2H), 7.78 (d, J ¼ 7.5 Hz, 1H), 7.75 (s, 1H),

7.61e7.47 (m, 3H), 7.05 (d, J ¼ 8.2 Hz, 2H), 6.95 (d, J ¼ 7.7 Hz, 1H),

6.39 (dt, J ¼ 15.2, 7.4 Hz, 1H), 6.02 (s, 2H), 4.46 (d, J ¼ 13.1 Hz, 1H),

4.25 (d, J ¼ 13.1 Hz, 1H), 4.17 (dd, J ¼ 13.2, 7.1 Hz, 1H), 4.00 (dd,

J ¼ 13.1, 8.0 Hz, 1H), 2.86 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆)

114e115 ^ꢀC. ¹H NMR (400 MHz, CD₃OD)

d 7.53 (s, 1H), 7.05e6.87 (m,

3H), 6.75 (d, J ¼ 15.7 Hz, 1H), 6.57e6.43 (m, 2H), 6.15 (dt, J ¼ 15.4,

7.6 Hz, 1H), 6.02 (s, 2H), 4.37 (d, J ¼ 13.1 Hz, 1H), 4.14 (d, J ¼ 13.1 Hz,

1H), 4.00 (dd, J ¼ 13.2, 7.1 Hz,1H), 3.81 (dd, J ¼ 13.0, 8.3 Hz,1H), 2.76

(s, 3H). ¹³C NMR (100 MHz, DMSO-d₆)

d 151.31, 148.54, 147.89,

d

148.57, 147.92, 136.26, 133.67, 133.43, 130.98, 129.20, 128.97,

126.99, 126.56, 126.18, 125.92, 124.60, 124.20, 124.05, 121.56, 111.71,

144.18, 127.04, 125.85, 124.16, 116.57, 112.36, 111.63, 110.85, 108.83,

101.90, 57.86, 56.51, 38.06; HRMS (ESI) m/z calcd for C₁₆H₁₈NO₃

[MþH]^þ272.1287, found 272.1289.

108.85, 101.91, 57.89, 56.64, 38.34; HRMS (ESI) m/z calcd for

C

₂₂H₂₂NO₂[MþH]^þ332.1651, found 332.1649.

4.1.15. Synthesis of (E)-N-(benzo[d][1,3]dioxol-5-ylmethyl)-N-

methyl-3-(naphthalen-2-yl)prop-2-en-1-amine hydrochloride

(11h)

4.1.10. Synthesis of (E)-N-(benzo[d][1,3]dioxol-5-ylmethyl)-N-

methyl-3-(thiophen-2-yl)prop-2-en-1-amine hydrochloride (11c)

Compound 11c was prepared by the experimental procedure

depicted for compound 11a as a white solid. Yield: 42%; m.p.:

Compound 11h was prepared by the experimental procedure

depicted for compound 11a as a white solid. Yield: 56%; m.p.:

113e115 ^ꢀC. ¹H NMR (400 MHz, CD₃OD)

d

7.40 (d, J ¼ 5.0 Hz, 1H),

174e176 ^ꢀC. ¹H NMR (400 MHz, CD₃OD)

d 7.90 (s, 1H), 7.89e7.82 (m,

7.18 (d, J ¼ 3.3 Hz, 1H), 7.10e6.87 (m, 5H), 6.08 (dt, J ¼ 15.5, 7.6 Hz,

1H), 6.01 (s, 2H), 4.38 (d, J ¼ 13.0 Hz, 1H), 4.16 (d, J ¼ 13.0 Hz, 1H),

4.00 (dd, J ¼ 13.3, 7.1 Hz, 1H), 3.82 (dd, J ¼ 13.2, 8.1 Hz, 1H), 2.77 (s,

3H), 7.73 (d, J ¼ 8.6 Hz, 1H), 7.56e7.44 (m, 2H), 7.08 (d, J ¼ 15.8 Hz,

1H), 7.03 (d, J ¼ 7.5 Hz, 2H), 6.94 (d, J ¼ 8.2 Hz, 1H), 6.53e6.39 (m,

1H), 6.02 (s, 2H), 4.43 (d, J ¼ 13.1 Hz, 1H), 4.21 (d, J ¼ 13.1 Hz, 1H),

4.13e4.02 (m, 1H), 3.96e3.86 (m, 1H), 2.82 (s, 3H). ¹³C NMR

3H). ¹³C NMR (100 MHz, DMSO-d₆)

d 148.56, 147.91, 140.55, 132.14,

128.37, 128.34, 127.07, 125.84, 124.19, 117.69, 111.60, 108.85, 101.91,

57.95, 56.62, 38.20; HRMS (ESI) m/z calcd for C₁₆H₁₈NO₂S [MþH]^þ

288.1058, found 288.1057.

(100 MHz, DMSO-d₆)

d 148.56, 147.92, 138.85, 133.56, 133.48,

133.38, 128.78, 128.52, 128.10, 127.54, 127.08, 126.96, 125.87, 124.27,

124.05, 119.20, 111.65, 108.85, 101.91, 57.82, 56.65, 38.32; HRMS

(ESI) m/z calcd for C₂₂H₂₂NO₂[MþH]^þ332.1651, found 332.1651.

4.1.11. Synthesis of (E)-N-(benzo[d][1,3]dioxol-5-ylmethyl)-N-

methylbut-2-en-1-amine hydrochloride (11d)

4.1.16. Synthesis of (E)-N-(benzo[d][1,3]dioxol-5-ylmethyl)-3-(2-

ﬂuorophenyl)-N-methylprop-2-en-1-amine hydrochloride (12a)

Compound 12a was prepared by the experimental procedure

depicted for compound 11a as a white solid. Yield: 55%; m.p.:

Compound 11d was prepared by the experimental procedure

depicted for compound 11a as a yellow oil. Yield: 45%. ¹H NMR

(400 MHz, CD₃OD)

d

6.97 (d, J ¼ 8.4 Hz, 2H), 6.92 (d, J ¼ 7.7 Hz, 1H),

152e154 ^ꢀC. ¹H NMR (400 MHz, CD₃OD)

d

7.65 (t, J ¼ 7.6 Hz, 1H),

6.17e5.96 (m, 3H), 5.63 (dt, J ¼ 15.1, 7.4 Hz, 1H), 4.13 (s, 2H), 3.68 (s,

2H), 2.71 (s, 3H), 1.79 (d, J ¼ 6.4 Hz, 3H). ¹³C NMR (100 MHz, DMSO-

7.39 (dd, J ¼ 13.7, 6.9 Hz, 1H), 7.23 (t, J ¼ 7.6 Hz, 1H), 7.20e7.13 (m,

1H), 7.07 (d, J ¼ 16.0 Hz, 1H), 7.03 (d, J ¼ 7.8 Hz, 2H), 6.95 (d,

J ¼ 7.7 Hz, 1H), 6.51e6.41 (m, 1H), 6.04 (s, 2H), 4.39 (s, 1H), 4.24 (s,

1H), 4.08 (s, 1H), 3.94 (s, 1H), 2.82 (s, 3H). ¹³C NMR (100 MHz,

d₆)

d 148.50, 147.92, 136.58, 125.78, 124.18, 119.93, 111.57, 108.82,

101.90, 57.67, 56.33, 38.00, 18.33; HRMS (ESI) m/z calcd for

C

₁₃H₁₈NO₂[MþH]^þ220.1338, found 220.1337.

DMSO-d₆)

d

160.09 (d, J_C-F¼ 248.5 Hz), 148.54, 147.89, 130.91 (d, J_C-

4.1.12. Synthesis of (E)-N-(benzo[d][1,3]dioxol-5-ylmethyl)-3-

cyclopentyl-N-methylprop-2-en-1-amine hydrochloride (11e)

Compound 11e was prepared by the experimental procedure

depicted for compound 11a as a white solid. Yield: 48%; m.p.:

¼ 10.0 Hz), 130.66, 128.45, 125.86, 125.22, 124.21, 123.56 (d, J_C-

¼ 11.8 Hz), 121.90, 116.30 (d, J_C-F¼ 21.9 Hz), 111.65, 108.80, 101.89,

F

57.88, 56.66, 38.30; HRMS (ESI) m/z calcd for C₁₈H₁₉NO₂F [MþH]^þ

300.1400, found 300.1400.

97e98 ^ꢀC. ¹H NMR (400 MHz, CD₃OD)

d 7.00e6.89 (m, 3H),

6.09e5.98 (m, 3H), 5.66e5.51 (m, 1H), 4.28 (s, 1H), 4.09 (s, 1H), 3.78

(s, 1H), 3.63 (s, 1H), 2.70 (s, 3H), 2.62e2.51 (m, 1H), 1.92e1.77 (m,

2H), 1.75e1.57 (m, 4H), 1.43e1.28 (m, 2H). ¹³C NMR (100 MHz,

4.1.17. Synthesis of (E)-N-(benzo[d][1,3]dioxol-5-ylmethyl)-3-(2-

chlorophenyl)-N-methylprop-2-en-1-amine hydrochloride (12b)

Compound 12b was prepared by the experimental procedure

depicted for compound 11a as a white solid. Yield: 47%; m.p.:

DMSO-d₆)

d 148.50, 147.88, 145.99, 125.78, 124.18, 117.39, 111.58,

108.80, 101.89, 57.67, 56.50, 42.89, 38.00, 32.65 (2C), 25.12 (2C);

HRMS (ESI) m/z calcd for C₁₇H₂₄NO₂[MþH]^þ274.1807, found

274.1808.

162e165 ^ꢀC. ¹H NMR (400 MHz, CD₃OD)

d 7.72e7.67 (m, 1H), 7.41

(dd, J ¼ 6.5, 3.0 Hz, 1H), 7.32 (dd, J ¼ 4.8, 3.8 Hz, 2H), 7.27 (d,

J ¼ 15.8 Hz, 1H), 7.02e6.96 (m, 2H), 6.91 (d, J ¼ 7.8 Hz, 1H),

H. Wei et al. / European Journal of Medicinal Chemistry 145 (2018) 235e251

245

6.40e6.30 (m, 1H), 5.99 (s, 2H), 4.38 (d, J ¼ 13.0 Hz, 1H), 4.18 (d,

J ¼ 13.1 Hz, 1H), 4.05 (dd, J ¼ 13.0, 7.0 Hz, 1H), 3.90 (dd, J ¼ 13.2,

7.9 Hz, 1H), 2.79 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆)

148.56,

m/z calcd for C₁₈H₁₉NO₂F [MþH]^þ300.1400, found 300.1403.

d

4.1.22. Synthesis of (E)-N-(benzo[d][1,3]dioxol-5-ylmethyl)-3-(3-

147.90, 134.13, 133.92, 132.58, 130.60, 130.12, 128.12, 128.05, 125.87,

124.17, 122.51, 111.65, 108.82, 101.90, 57.87, 56.33, 38.39; HRMS (ESI)

m/z calcd for C₁₈H₁₉NO₂Cl [MþH]^þ316.1104, found 316.1104.

chlorophenyl)-N-methylprop-2-en-1-amine hydrochloride (13b)

Compound 13b was prepared by the experimental procedure

depicted for compound 11a as a white solid. Yield: 48%; m.p.:

123e124 ^ꢀC. ¹H NMR (400 MHz, CD₃OD)

d 7.56 (s, 1H), 7.43 (d,

4.1.18. Synthesis of (E)-N-(benzo[d][1,3]dioxol-5-ylmethyl)-N-

methyl-3-(2-(triﬂuoromethyl)phenyl)prop-2-en-1-amine

hydrochloride (12c)

J ¼ 6.8 Hz, 1H), 7.41e7.31 (m, 2H), 7.09e6.94 (m, 2H), 6.95e6.85 (m,

2H), 6.44e6.33 (m, 1H), 6.02 (s, 2H), 4.37 (s, 1H), 4.20 (s,1H), 4.00 (s,

1H), 3.87 (s, 1H), 2.78 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆)

Compound 12c was prepared by the experimental procedure

depicted for compound 11a as a white solid. Yield: 49%; m.p.:

d 148.54,147.90, 138.27, 137.14, 134.01,131.05,128.72, 126.72, 126.12,

125.86, 124.23, 120.65, 111.64, 108.83, 101.90, 57.75, 56.25, 38.38;

HRMS (ESI) m/z calcd for C₁₈H₁₉NO₂Cl [MþH]^þ316.1104, found

316.1104.

159e161 ^ꢀC. ¹H NMR (400 MHz, CD₃OD)

d

7.81 (d, J ¼ 7.9 Hz, 1H),

7.73 (d, J ¼ 7.7 Hz, 1H), 7.66 (t, J ¼ 7.6 Hz, 1H), 7.53 (t, J ¼ 7.6 Hz, 1H),

7.27 (d, J ¼ 15.0 Hz, 1H), 7.05e6.89 (m, 3H), 6.34 (dt, J ¼ 15.4, 7.6 Hz,

1H), 6.02 (s, 2H), 4.38 (s, 1H), 4.21 (s, 1H), 4.08 (s, 1H), 3.95 (s, 1H),

4.1.23. Synthesis of (E)-N-(benzo[d][1,3]dioxol-5-ylmethyl)-N-

methyl-3-(3-(triﬂuoromethyl)phenyl)prop-2-en-1-amine

hydrochloride (13c)

2.74 (s, 3H). ¹³C NMR (100 MHz, DMSO-d6)

d 148.58, 147.92, 134.79

(q, J_C-F¼ 1.6 Hz), 133.89, 133.39, 129.26, 128.71, 126.40 (q, J_C-

¼ 28.4 Hz), 126.23 (q, J_C-F¼ 5.8 Hz), 125.87, 124.68 (q, J_C-

Compound 13c was prepared by the experimental procedure

depicted for compound 11a as a white solid. Yield: 61%; m.p.:

F

¼ 272.1 Hz), 124.37, 124.12, 111.64, 108.82, 101.91, 57.92, 56.23,

F

38.28; HRMS (ESI) m/z calcd for C₁₉H₁₉NO₂F₃[MþH]^þ350.1368,

149e151 ^ꢀC. ¹H NMR (400 MHz, CD₃OD)

d 7.82 (s, 1H), 7.78 (d,

found 350.1370.

J ¼ 7.5 Hz, 1H), 7.67e7.56 (m, 2H), 7.01 (d, J ¼ 7.1 Hz, 2H), 6.94 (t,

J ¼ 8.1 Hz, 2H), 6.52e6.41 (m, 1H), 6.02 (s, 2H), 4.37 (s, 1H), 4.22 (s,

1H), 4.02 (s, 1H), 3.91 (s, 1H), 2.80 (s, 3H). ¹³C NMR (100 MHz,

4.1.19. Synthesis of (E)-N-(benzo[d][1,3]dioxol-5-ylmethyl)-N-

methyl-3-(o-tolyl)prop-2-en-1-amine hydrochloride (12d)

Compound 12d was prepared by the experimental procedure

depicted for compound 11a as a white solid. Yield: 44%; m.p.:

DMSO-d₆)

d

148.63, 147.95, 137.47(q, J_C-F¼ 3.9 Hz), 137.00, 131.22,

130.35, 130.03 (q, J_C-F¼ 31.7 Hz), 125.87, 125.49 (q, J_C-

¼ 2.4 Hz),124.55 (q, J_C-F¼ 270.6), 124.03, 123.71 (q, J_C-F¼ 3.8 Hz),

F

179e181 ^ꢀC. ¹H NMR (400 MHz, CD₃OD)

d

7.33 (s, 1H), 7.30 (d,

120.76, 111.46, 108.94, 101.93, 58.24, 56.67, 38.84; HRMS (ESI) m/z

calcd for C₁₉H₁₉NO₂F₃[MþH]^þ350.1368, found 350.1369.

J ¼ 7.8 Hz, 1H), 7.25 (t, J ¼ 7.5 Hz, 1H), 7.16 (d, J ¼ 7.2 Hz, 1H),

7.05e6.99 (m, 2H), 6.93 (dd, J ¼ 7.6, 0.8 Hz, 1H), 6.87 (d, J ¼ 15.7 Hz,

1H), 6.37e6.25 (m, 1H), 6.01 (s, 2H), 4.37 (d, J ¼ 12.9 Hz, 1H), 4.17 (d,

J ¼ 13.1 Hz, 1H), 4.01 (dd, J ¼ 12.9, 6.8 Hz, 1H), 3.85 (dd, J ¼ 13.3,

8.1 Hz, 1H), 2.78 (s, 3H), 2.35 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆)

4.1.24. Synthesis of (E)-N-(benzo[d][1,3]dioxol-5-ylmethyl)-N-

methyl-3-(m-tolyl)prop-2-en-1-amine hydrochloride (13d)

Compound 13d was prepared by the experimental procedure

depicted for compound 11a as a white solid. Yield: 46%; m.p.:

d

148.55, 147.90, 136.92, 136.04, 135.05, 130.81, 128.86, 126.65,

126.32, 125.87, 124.19, 119.84, 111.66, 108.83, 101.90, 57.80, 56.69,

38.23,19.80; HRMS (ESI) m/z calcd for C₁₉H₂₂NO₂[MþH]^þ296.1651,

found 296.1648.

136e137 ^ꢀC. ¹H NMR (400 MHz, CD₃OD)

d 7.57e7.52 (m, 1H),

7.29e7.11 (m, 4H), 7.06e6.99 (m, 2H), 6.94 (d, J ¼ 8.0 Hz, 1H), 6.20

(dt, J ¼ 15.3, 7.4 Hz, 1H), 6.01 (s, 2H), 4.40 (d, J ¼ 13.1 Hz, 1H), 4.18 (d,

J ¼ 13.0 Hz, 1H), 4.05 (dd, J ¼ 13.3, 7.1 Hz, 1H), 3.88 (dd, J ¼ 13.2,

7.9 Hz, 1H), 2.80 (s, 3H), 2.38 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆)

4.1.20. Synthesis of (E)-N-(benzo[d][1,3]dioxol-5-ylmethyl)-3-(2-

methoxyphenyl)-N-methylprop-2-en-1-amine hydrochloride (12e)

Compound 12e was prepared by the experimental procedure

depicted for compound 11a as a white solid. Yield: 42%; m.p.:

d

148.53, 147.90, 138.92, 138.33, 135.92, 129.73, 129.09, 127.72,

125.85, 124.56, 124.25, 118.47, 111.64, 108.83, 101.90, 57.76, 56.60,

38.22, 21.41; HRMS (ESI) m/z calcd for C₁₉H₂₂NO₂[MþH]^þ296.1651,

found 296.1652.

149e151 ^ꢀC. ¹H NMR (400 MHz, CD₃OD)

d

7.51 (dd, J ¼ 7.7, 1.6 Hz,

1H), 7.38e7.29 (m, 1H), 7.17 (d, J ¼ 15.9 Hz, 1H), 7.05e6.98 (m, 3H),

6.98e6.90 (m, 2H), 6.40e6.28 (m, 1H), 6.01 (s, 2H), 4.38 (d,

J ¼ 12.9 Hz, 1H), 4.17 (d, J ¼ 13.2 Hz, 1H), 4.01 (d, J ¼ 13.1 Hz, 1H),

3.87 (s, 3H), 3.83 (d, J ¼ 12.7 Hz, 1H), 2.77 (s, 3H). ¹³C NMR

4.1.25. Synthesis of (E)-N-(benzo[d][1,3]dioxol-5-ylmethyl)-3-(3-

methoxyphenyl)-N-methylprop-2-en-1-amine hydrochloride (13e)

Compound 13e was prepared by the experimental procedure

depicted for compound 11a as a white solid. Yield: 45%; m.p.:

(100 MHz, DMSO-d₆) d 156.97, 148.53,147.90, 133.48, 130.44, 127.50,

125.83, 124.39, 124.25, 121.04, 119.09, 111.95, 111.62, 108.82, 101.90,

57.84, 57.06, 55.97, 38.26; HRMS (ESI) m/z calcd for C₁₉H₂₂NO₃

[MþH]^þ312.1600, found 312.1601.

150e152 ^ꢀC. ¹H NMR (400 MHz, CD₃OD)

d

7.28 (t, J ¼ 7.9 Hz, 1H),

7.08 (d, J ¼ 7.6 Hz,1H), 7.06 (s, 1H), 7.01 (d, J ¼ 7.6 Hz, 2H), 6.96e6.84

(m, 3H), 6.39e6.26 (m, 1H), 6.03 (s, 2H), 4.39 (d, J ¼ 12.9 Hz, 1H),

4.17 (d, J ¼ 13.1 Hz, 1H), 4.01 (dd, J ¼ 13.0, 7.0 Hz, 1H), 3.91e3.74 (m,

4.1.21. Synthesis of (E)-N-(benzo[d][1,3]dioxol-5-ylmethyl)-3-(3-

ﬂuorophenyl)-N-methylprop-2-en-1-amine hydrochloride (13a)

Compound 13a was prepared by the experimental procedure

depicted for compound 11a as a white solid. Yield: 50%; m.p.:

4H), 2.79 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆)

d 159.99, 148.54,

147.90, 138.70, 137.42, 130.26, 125.85, 124.25, 119.75, 119.05, 114.63,

112.59, 111.63, 108.83, 101.90, 57.80, 56.50, 55.61, 38.30; HRMS (ESI)

m/z calcd for C₁₉H₂₂NO₃[MþH]^þ312.1600, found 312.1602.

170e172 ^ꢀC. ¹H NMR (400 MHz, CD₃OD)

d

7.40 (dd, J ¼ 8.0, 5.9 Hz,

1H), 7.36e7.27 (m, 2H), 7.13e6.99 (m, 3H), 6.92 (dd, J ¼ 11.7, 9.2 Hz,

2H), 6.45e6.35 (m, 1H), 6.02 (s, 2H), 4.40 (d, J ¼ 13.1 Hz, 1H), 4.19 (d,

J ¼ 13.0 Hz, 1H), 4.03 (dd, J ¼ 13.3, 7.1 Hz, 1H), 3.87 (dd, J ¼ 13.3,

4.1.26. Synthesis of (E)-N-(benzo[d][1,3]dioxol-5-ylmethyl)-3-(4-

ﬂuorophenyl)-N-methylprop-2-en-1-amine hydrochloride (14a)

Compound 14a was prepared by the experimental procedure

depicted for compound 11a as a white solid. Yield: 60%; m.p.:

7.9 Hz, 1H), 2.80 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆)

d 162.93 (d,

J_C-F¼ 243.3 Hz), 148.55, 147.90, 138.61 (d, J_C-F¼ 7.9 Hz), 137.44 (d, J_C-

158e161 ^ꢀC. ¹H NMR (400 MHz, CD₃OD)

d 7.60e7.49 (m, 2H),

¼ 2.1 Hz), 131.17 (d, J_C-F¼ 8.5 Hz), 125.86, 124.22, 123.71 (d, J_C-

7.17e7.07 (m, 2H), 7.04e6.86 (m, 4H), 6.28 (dt, J ¼ 15.2, 7.5 Hz, 1H),

6.02 (s, 2H), 4.39 (d, J ¼ 12.9 Hz, 1H), 4.17 (d, J ¼ 13.0 Hz, 1H),

4.08e3.91 (m, 1H), 3.90e3.77 (m, 1H), 2.73 (s, 3H). ¹³C NMR

F

¼ 2.4 Hz), 120.49, 115.72 (d, J_C-F¼ 21.2 Hz), 113.49 (d, J_C-

F

¼ 21.9 Hz), 111.63, 108.83, 101.90, 57.77, 56.22, 38.37; HRMS (ESI)

F

246

H. Wei et al. / European Journal of Medicinal Chemistry 145 (2018) 235e251

(100 MHz, DMSO-d₆)

d

162.64 (d, J_C-F¼ 245.8 Hz), 148.55, 147.91,

4.1.31. Synthesis of (E)-N-(benzo[d][1,3]dioxol-5-ylmethyl)-N-

methyl-3-(p-tolyl)prop-2-en-1-amine hydrochloride (14f)

137.62, 132.60 (d, J_C-F¼ 3.1 Hz), 129.33 (d, J_C-F¼ 8.3 Hz, 2C), 125.84,

124.22, 118.57, 116.11 (d, J_C-F¼ 21.6 Hz, 2C), 111.62, 108.84, 101.91,

57.79, 56.48, 38.29; HRMS (ESI) m/z calcd for C₁₈H₁₉NO₂F [MþH]^þ

300.1400, found 300.1399.

Compound 14f was prepared by the experimental procedure

depicted for compound 11a as a white solid. Yield: 48%; m.p.:

169e171 ^ꢀC. ¹H NMR (400 MHz, CD₃OD)

7.19 (d, J ¼ 7.9 Hz, 2H), 7.04e6.98 (m, 2H), 6.93 (d, J ¼ 7.7 Hz, 1H),

6.87 (d, J ¼ 15.6 Hz, 1H), 6.32e6.23 (m, 1H), 6.03 (s, 2H), 4.27 (s, 2H),

3.91 (s, 2H), 2.77 (s, 3H), 2.34 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆)

d

7.40 (d, J ¼ 8.0 Hz, 2H),

4.1.27. Synthesis of (E)-N-(benzo[d][1,3]dioxol-5-ylmethyl)-3-(4-

chlorophenyl)-N-methylprop-2-en-1-amine hydrochloride (14b)

Compound 14b was prepared by the experimental procedure

depicted for compound 11a as a white solid. Yield: 58%; m.p.:

d

148.50, 147.89, 138.72, 138.55, 133.24, 129.76 (2C), 127.21 (2C),

125.78, 124.37, 117.63, 111.58, 108.82, 101.89, 57.84, 56.72, 38.28,

21.32; HRMS (ESI) m/z calcd for C₁₉H₂₂NO₂[MþH]^þ296.1651, found

296.1648.

160e162 ^ꢀC. ¹H NMR (400 MHz, CD₃OD)

d

7.51 (d, J ¼ 8.5 Hz, 2H),

7.38 (d, J ¼ 8.4 Hz, 2H), 7.05e6.99 (m, 2H), 6.93 (d, J ¼ 7.8 Hz, 1H),

6.89 (d, J ¼ 16.0 Hz, 1H), 6.42e6.29 (m, 1H), 6.01 (s, 2H), 4.39 (d,

J ¼ 13.0 Hz, 1H), 4.18 (d, J ¼ 12.9 Hz, 1H), 4.02 (dd, J ¼ 13.3, 7.0 Hz,

1H), 3.86 (dd, J ¼ 13.3, 8.0 Hz, 1H), 2.79 (s, 3H). ¹³C NMR (100 MHz,

4.1.32. Synthesis of (E)-N-(benzo[d][1,3]dioxol-5-ylmethyl)-3-(4-

methoxyphenyl)-N-methylprop-2-en-1-amine hydrochloride (14g)

Compound 14g was prepared by the experimental procedure

depicted for compound 11a as a white solid. Yield: 43%; m.p.:

DMSO-d₆)

d 148.55, 147.90, 137.42, 134.94, 133.45, 129.21 (2C),

122e124 ^ꢀC. ¹H NMR (400 MHz, CD₃OD)

d

7.6 (d, J ¼ 8.2 Hz, 2H),

129.01 (2C), 125.85, 124.20, 119.71, 111.62, 108.84, 101.91, 57.82,

56.42, 38.34; HRMS (ESI) m/z calcd for C₁₈H₁₉NO₂Cl [MþH]^þ

316.1104, found 316.1104.

7.07e6.97 (m, 2H), 6.94e6.90 (m, 3H), 6.84 (d, J ¼ 15.7 Hz, 1H),

6.22e6.12 (m, 1H), 6.02 (s, 2H), 4.38 (d, J ¼ 13.1 Hz, 1H), 4.16 (d,

J ¼ 13.1 Hz, 1H), 3.99 (dd, J ¼ 12.9, 6.8 Hz, 1H), 3.89e3.74 (m, 4H),

2.77 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆)

d 160.08, 148.54, 147.91,

4.1.28. Synthesis of (E)-N-(benzo[d][1,3]dioxol-5-ylmethyl)-3-(4-

bromophenyl)-N-methylprop-2-en-1-amine hydrochloride (14c)

Compound 14c was prepared by the experimental procedure

depicted for compound 11a as a white solid. Yield: 58%; m.p.:

138.59, 128.69 (2C), 128.59, 125.82, 124.26, 115.93, 114.60 (2C),

111.60, 108.84, 101.90, 57.75, 56.78, 55.68, 38.19; HRMS (ESI) m/z

calcd for C₁₉H₂₂NO₃[MþH]^þ312.1600, found 312.1603.

162e164 ^ꢀC. ¹H NMR (400 MHz, CD₃OD)

d

7.54 (d, J ¼ 8.5 Hz, 2H),

4.1.33. Synthesis of (E)-4-(3-((benzo[d][1,3]dioxol-5-

ylmethyl)(methyl)amino)prop-1-en-1-yl)benzonitrile hydrochloride

(14h)

Compound 14h was prepared by the experimental procedure

depicted for compound 11a as a white solid. Yield: 55%; m.p.:

7.48e7.36 (m, 2H), 7.01 (d, J ¼ 8.9 Hz, 2H), 6.93 (d, J ¼ 7.7 Hz, 1H),

6.88 (d, J ¼ 15.8 Hz, 1H), 6.43e6.31 (m, 1H), 6.02 (s, 2H), 4.39 (d,

J ¼ 13.1 Hz, 1H), 4.18 (d, J ¼ 13.0 Hz, 1H), 4.01 (dd, J ¼ 13.1, 6.9 Hz,

1H), 3.85 (dd, J ¼ 13.0, 7.8 Hz, 1H), 2.78 (s, 3H). ¹³C NMR (100 MHz,

DMSO-d₆)

d 148.55, 147.91, 137.48, 135.28, 132.13 (2C), 129.29 (2C),

194e196 ^ꢀC. ¹H NMR (400 MHz, CD₃OD)

d

7.75 (d, J ¼ 8.4 Hz, 2H),

125.84, 124.20, 122.11, 119.79, 111.62, 108.84, 101.91, 57.83, 56.43,

38.35; HRMS (ESI) m/z calcd for C₁₈H₁₉NO₂Br [MþH]^þ360.0599,

found 360.0601.

7.70 (d, J ¼ 8.4 Hz, 2H), 7.06e7.01 (m, 2H), 6.98 (d, J ¼ 15.0 Hz, 1H),

6.93 (d, J ¼ 7.8 Hz, 1H), 6.59e6.47 (m, 1H), 6.03 (s, 2H), 4.41 (d,

J ¼ 13.0 Hz, 1H), 4.21 (d, J ¼ 13.0 Hz, 1H), 4.07 (dd, J ¼ 13.4, 7.0 Hz,

1H), 3.91 (dd, J ¼ 13.3, 7.7 Hz, 1H), 2.81 (s, 3H). ¹³C NMR (100 MHz,

DMSO-d₆)

d 148.56, 147.90, 140.62, 136.91, 133.15 (2C), 128.04 (2C),

4.1.29. Synthesis of (E)-N-(benzo[d][1,3]dioxol-5-ylmethyl)-N-

methyl-3-(4-(triﬂuoromethyl)phenyl)prop-2-en-1-amine

hydrochloride (14d)

125.88, 124.18, 122.98, 119.25, 111.64, 111.10, 108.83, 101.91, 57.88,

56.20, 38.48; HRMS (ESI) m/z calcd for C₁₉H₁₉N₂O₂[MþH]^þ

307.1447, found 307.1445.

Compound 14d was prepared by the experimental procedure

depicted for compound 11a as a white solid. Yield: 65%; m.p.:

160e162 ^ꢀC. ¹H NMR (400 MHz, CD₃OD)

d

7.72 (d, J ¼ 8.7 Hz, 2H),

4.1.34. Synthesis of (E)-N-(benzo[d][1,3]dioxol-5-ylmethyl)-3-(4-

(diﬂuoromethyl)phenyl)-N-methylprop-2-en-1-amine

hydrochloride (14i)

7.69 (d, J ¼ 8.7 Hz, 2H), 7.07e6.96 (m, 3H), 6.94 (d, J ¼ 7.8 Hz, 1H),

6.57e6.46 (m, 1H), 6.02 (s, 2H), 4.42 (d, J ¼ 13.0 Hz, 1H), 4.21 (d,

J ¼ 13.1 Hz, 1H), 4.07 (dd, J ¼ 13.3, 6.9 Hz, 1H), 3.91 (dd, J ¼ 13.2,

Compound 14i was prepared by the experimental procedure

depicted for compound 11a as a white solid. Yield: 52%; m.p.:

7.8 Hz, 1H), 2.81 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆)

d 148.56,

154e157 ^ꢀC. ¹H NMR (400 MHz, CD₃OD)

d

7.64 (d, J ¼ 8.0 Hz, 2H),

147.91, 140.05, 137.08, 128.97 (d, J_C-F¼ 31.6 Hz), 127.93 (2C), 126.09

(q, J_C-F¼ 3.6 Hz, 2C), 125.10 (q, J_C-F¼ 272.4 Hz) 124.18, 123.30,

122.07, 111.64, 108.83, 101.91, 57.88, 56.30, 38.42; HRMS (ESI) m/z

calcd for C₁₉H₁₉NO₂F₃[MþH]^þ350.1368, found 350.1368.

7.56 (d, J ¼ 8.1 Hz, 2H), 7.05e6.92 (m, 4H), 6.77 (t, J ¼ 56.2 Hz, 1H),

6.49e6.36 (m, 1H), 6.02 (s, 2H), 4.38 (s, 1H), 4.21 (s, 1H), 4.01 (s, 1H),

3.91 (s, 1H), 2.74 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆)

d 148.57,

147.92, 138.50, 137.74, 134.35 (t, J_C-F¼ 22.2 Hz), 127.63 (2C), 126.60

(t, J_C-F¼ 6.0 Hz, 2C), 125.85, 124.19, 120.75, 115.22 (t, J_C-

4.1.30. Synthesis of (E)-N-(benzo[d][1,3]dioxol-5-ylmethyl)-N-

methyl-3-(4-nitrophenyl)prop-2-en-1-amine hydrochloride (14e)

Compound 11c was prepared by the experimental procedure

depicted for compound 11a as a brown solid. Yield: 57%; m.p.:

¼ 235.7 Hz), 111.62, 108.85, 101.91, 57.89, 56.42, 38.40; HRMS (ESI)

F

m/z calcd for C₁₉H₂₀NO₂F₂[MþH]^þ332.1462, found 332.1461.

4.1.35. Synthesis of (E)-methyl 4-(3-((benzo[d][1,3]dioxol-5-

ylmethyl)(methyl)amino)prop-1-en-1-yl)benzoate hydrochloride

(14j)

200e203 ^ꢀC. ¹H NMR (400 MHz, CD₃OD)

d

8.24 (d, J ¼ 8.6 Hz, 2H),

7.75 (d, J ¼ 8.7 Hz, 2H), 7.02 (d, J ¼ 8.1 Hz, 2H), 7.00 (d, J ¼ 3.7 Hz,

1H), 6.92 (d, J ¼ 7.8 Hz, 1H), 6.62e6.53 (m, 1H), 6.01 (s, 2H), 4.41 (d,

J ¼ 13.0 Hz, 1H), 4.21 (d, J ¼ 13.1 Hz, 1H), 4.07 (dd, J ¼ 13.4, 6.9 Hz,

1H), 3.92 (dd, J ¼ 13.4, 7.7 Hz, 1H), 2.81 (s, 3H). ¹³C NMR (100 MHz,

Compound 14j was prepared by the experimental procedure

depicted for compound 11a as a white solid. Yield: 62%; m.p.:

167e170 ^ꢀC. ¹H NMR (400 MHz, CD₃OD)

d

8.02 (d, J ¼ 8.4 Hz, 2H),

DMSO-d₆)

d

148.57, 147.91, 147.45, 142.59, 136.43, 128.34 (2C),

7.63 (d, J ¼ 8.3 Hz, 2H), 7.02 (s, 1H), 7.01e6.92 (m, 3H), 6.54e6.42

125.89, 124.46 (2C), 124.16, 123.96, 111.65, 108.84, 101.91, 57.93,

56.23, 38.51; HRMS (ESI) m/z calcd for C₁₈H₁₉N₂O₄[MþH]^þ

327.1345, found 327.1348.

(m, 1H), 6.02 (s, 2H), 4.38 (s, 1H), 4.21 (s, 1H), 4.03 (s, 1H), 3.91 (s,

4H), 2.80 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆)

d

166.34, 148.54,

147.90, 140.61, 137.46, 130.06 (2C), 129.67, 127.51 (2C), 125.86,

H. Wei et al. / European Journal of Medicinal Chemistry 145 (2018) 235e251

247

124.22, 121.93, 111.64, 108.83, 101.90, 57.88, 56.39, 52.65, 38.39;

HRMS (ESI) m/z calcd for C₂₀H₂₂NO₄[MþH]^þ340.1549, found

340.1548.

depicted for compound 11a as a white solid. Yield: 54%; m.p.:

165e166 ^ꢀC. FTIR: 3422.6, 3027.5, 2905.6, 1627.2, 1609.1, 1334.8,

1251.2, 1170.4, 1128.7, 1040.9, 910.6, 878.7 cm-1¹H NMR (400 MHz,

CD₃OD)

J ¼ 16.0 Hz, 1H), 7.05e6.82 (m, 3H), 6.67e6.49 (m, 1H), 6.02 (s, 2H),

4.39 (s, 1H), 4.22 (s, 1H), 4.08 (s, 1H), 3.94 (s, 1H), 2.77 (s, 3H). ¹³

NMR (100 MHz, DMSO-d₆)

d

7.85 (t, J ¼ 7.7 Hz, 1H), 7.65e7.42 (m, 2H), 7.09 (d,

4.1.36. Synthesis of (E)-3-([1,1⁰-biphenyl]-4-yl)-N-(benzo[d][1,3]

dioxol-5-ylmethyl)-N-methylprop-2-en-1-amine hydrochloride

(14k)

C

d

159.60 (d, J_C-F¼ 250.9 Hz), 148.57,

Compound 14k was prepared by the experimental procedure

depicted for compound 11a as a white solid. Yield: 53%; m.p.:

147.91, 130.56 (qd, J_C-F¼ 32.8, 8.6 Hz), 129.66 (d, J_C-F¼ 3.6 Hz),

129.21 (d, J_C-F¼ 2.1 Hz), 127.81 (d, J_C-F¼ 11.7 Hz), 125.88, 125.08 (q,

J_C-F¼ 271.8 Hz), 124.13, 124.07 (d, J_C-F¼ 2.7 Hz), 122.06 (p, J_C-

182e185 ^ꢀC. ¹H NMR (400 MHz, CD₃OD)

d 7.73e7.52 (m, 6H), 7.44 (t,

J ¼ 7.6 Hz, 2H), 7.35 (t, J ¼ 7.3 Hz, 1H), 7.07e6.89 (m, 4H), 6.49e6.31

(m,1H), 6.02 (s, 2H), 4.41 (d, J ¼ 13.0 Hz,1H), 4.19 (d, J ¼ 13.1 Hz,1H),

4.05 (dd, J ¼ 13.2, 7.1 Hz, 1H), 3.88 (dd, J ¼ 13.2, 7.9 Hz, 1H), 2.80 (s,

¼ 3.7 Hz), 113.91 (dq, J_C-F¼ 21.8, 4.1 Hz), 111.64, 108.82, 101.90,

F

57.99, 56.42, 38.49.¹⁹

F NMR (376 MHz, DMSO-d₆/CF₃COOH)

d

ꢂ61.17 (s, 3F), ꢂ115.52(dd, J ¼ 9.4, 8.0 Hz, 1F); HRMS (ESI) m/z

3H). ¹³C NMR (100 MHz, DMSO-d₆)

d

148.55, 147.91, 140.61, 139.90,

calcd for C₁₉H₁₈NO₂F₄[MþH]^þ368.1274, found 368.1272.

138.33, 135.12, 129.47 (2C), 128.15, 127.91 (2C), 127.40 (2C), 127.03

(2C), 125.87, 124.23, 118.82, 111.64, 108.85, 101.91, 57.84, 56.65,

38.30; HRMS (ESI) m/z calcd for C₂₄H₂₄NO₂[MþH]^þ358.1807,

found 358.1806.

4.1.41. Synthesis of (E)-N-(benzo[d][1,3]dioxol-5-ylmethyl)-3-(3-

ﬂuoro-4-(triﬂuoromethyl)phenyl)-N-methylprop-2-en-1-amine

hydrochloride (15c)

Compound 15c was prepared by the experimental procedure

depicted for compound 11a as a white solid. Yield: 51%; m.p.:

4.1.37. Synthesis of (E)-N-(benzo[d][1,3]dioxol-5-ylmethyl)-3-(4-

(tert-butyl)phenyl)-N-methylprop-2-en-1-amine hydrochloride

(14l)

167e170 ^ꢀC. ¹H NMR (400 MHz, CD₃OD)

d

7.70 (t, J ¼ 7.7 Hz, 1H),

7.54 (d, J ¼ 11.8 Hz, 1H), 7.48 (d, J ¼ 7.8 Hz, 1H), 7.02 (s, 1H),

7.01e6.87 (m, 3H), 6.60e6.47 (m, 1H), 6.02 (s, 2H), 4.37 (s, 1H), 4.22

(s, 1H), 4.05 (s, 1H), 3.92 (s, 1H), 2.74 (s, 3H). ¹³C NMR (100 MHz,

Compound 14l was prepared by the experimental procedure

depicted for compound 11a as a white solid. Yield: 50%; m.p.:

220e222 ^ꢀC. ¹H NMR (400 MHz, CD₃OD)

d

7.49e7.36 (m, 4H),

DMSO-d₆)

d

159.62 (dq, J_C-F¼ 252.8, 3.7 Hz), 148.58, 147.92, 143.23

7.04e6.98 (m, 2H), 6.94 (d, J ¼ 8.0 Hz, 1H), 6.88 (d, J ¼ 15.8 Hz, 1H),

6.29 (dt, J ¼ 15.2, 7.5 Hz, 1H), 6.01 (s, 2H), 4.37 (d, J ¼ 13.0 Hz, 1H),

4.17 (d, J ¼ 13.1 Hz, 1H), 4.01 (dd, J ¼ 13.6, 6.8 Hz, 1H), 3.84 (dd,

J ¼ 13.2, 8.0 Hz, 1H), 2.78 (s, 3H), 1.32 (s, 9H). ¹³C NMR (100 MHz,

(d, J_C-F¼ 7.7 Hz), 136.01e135.85 (m), 128.18 (q, J_C-F¼ 3.8 Hz), 125.87,

124.16, 123.94 (d, J_C-F¼ 2.5 Hz), 123.78 (d, J_C-F¼ 1.4 Hz), 123.09 (q,

J_C-F¼ 270.3 Hz), 116.64e115.94 (m), 115.15 (d, J_C-F¼ 21.1 Hz), 111.62,

108.85, 101.91, 57.85, 55.95, 38.55.¹⁹F NMR (376 MHz, DMSO-d₆/

DMSO-d₆)

d

151.72, 148.55, 147.91, 138.73, 133.25, 127.05 (2C),

CF₃COOH)

d

ꢂ59.84 (d, J ¼ 12.0 Hz, 3F), ꢂ115.86 to ꢂ116.16 (m, 1F);

125.95 (2C), 125.83, 124.23, 117.76, 111.62, 108.84, 101.90, 57.79,

56.68, 38.22, 34.86, 31.47 (3C); HRMS (ESI) m/z calcd for C₂₂H₂₈NO₂

[MþH]^þ338.2120, found 338.2119.

HRMS (ESI) m/z calcd for C₁₉H₁₈NO₂F₄[MþH]^þ368.1274, found

368.1276.

4.1.42. Synthesis of (E)-N-(benzo[d][1,3]dioxol-5-ylmethyl)-3-(2-

ﬂuoro-4-methoxyphenyl)-N-methylprop-2-en-1-amine

hydrochloride (15d)

Compound 15d was prepared by the experimental procedure

depicted for compound 11a as a white solid. Yield: 49%; m.p.:

4.1.38. Synthesis of (E)-N-(benzo[d][1,3]dioxol-5-ylmethyl)-3-(4-

ethoxyphenyl)-N-methylprop-2-en-1-amine hydrochloride (14m)

Compound 14m was prepared by the experimental procedure

depicted for compound 11a as a white solid. Yield: 49%; m.p.:

121e123 ^ꢀC. ¹H NMR (400 MHz, CD₃OD)

d

7.44 (d, J ¼ 8.7 Hz, 2H),

132e134 ^ꢀC. ¹H NMR (400 MHz, CD₃OD)

d

7.53 (t, J ¼ 8.8 Hz, 1H),

7.00 (d, J ¼ 7.6 Hz, 2H), 7.04e6.90 (m, 3H), 6.84 (d, J ¼ 15.7 Hz, 1H),

6.23e6.09 (m, 1H), 6.03 (s, 2H), 4.38 (d, J ¼ 12.5 Hz, 1H), 4.16 (d,

J ¼ 11.6 Hz, 1H), 4.05 (q, J ¼ 7.0 Hz, 2H), 3.97 (d, J ¼ 6.4 Hz, 1H), 3.83

(d, J ¼ 7.5 Hz, 1H), 2.77 (s, 3H), 1.38 (t, J ¼ 7.0 Hz, 3H). ¹³C NMR

7.07e6.88 (m, 4H), 6.78 (d, J ¼ 8.8 Hz, 1H), 6.74 (dd, J ¼ 12.9, 2.1 Hz,

1H), 6.34e6.21 (m, 1H), 6.02 (s, 2H), 4.38 (d, J ¼ 12.4 Hz,1H), 4.16 (d,

J ¼ 12.6 Hz, 1H), 4.00 (d, J ¼ 6.1 Hz, 1H), 3.91e3.73 (m, 4H), 2.73 (s,

3H). ¹³C NMR (100 MHz, DMSO-d₆)

d

161.32 (d, J_C-F¼ 11.4 Hz),

(100 MHz, DMSO-d₆)

d

159.35, 148.53, 147.90, 138.60, 128.69 (2C),

160.93 (d, J_C-F¼ 247.0 Hz), 148.54, 147.90, 130.75, 129.14 (d, J_C-

128.45, 125.81, 124.27, 115.84, 115.03 (2C), 111.60, 108.84, 101.90,

¼ 5.1 Hz), 125.83, 124.23, 118.88, 115.91 (d, J_C-F¼ 12.3 Hz), 111.61,

F

63.58, 57.74, 56.79, 38.18, 15.06; HRMS (ESI) m/z calcd for

111.58, 108.82, 101.98 (d, J_C-F¼ 26.0 Hz), 101.90, 57.82, 56.89, 56.25,

C

₂₀H₂₄NO₃[MþH]^þ326.1756, found 326.1757.

38.22.¹⁹F NMR (376 MHz, DMSO-d₆/CF₃COOH)

d

ꢂ115.65 (dd,

J ¼ 12.5, 9.1 Hz, 1F); HRMS (ESI) m/z calcd for C₁₉H₂₁NO₃F [MþH]^þ

4.1.39. Synthesis of (E)-N-(benzo[d][1,3]dioxol-5-ylmethyl)-3-(2,4-

dichlorophenyl)-N-methylprop-2-en-1-amine hydrochloride (15a)

Compound 15a was prepared by the experimental procedure

depicted for compound 11a as a white solid. Yield: 60%; m.p.:

330.1505, found 330.1504.

4.1.43. Synthesis of (E)-N-(benzo[d][1,3]dioxol-5-ylmethyl)-3-(3-

ﬂuoro-4-methoxyphenyl)-N-methylprop-2-en-1-amine

hydrochloride (15e)

188e190 ^ꢀC. ¹H NMR (400 MHz, CD₃OD)

d

7.72 (d, J ¼ 8.5 Hz, 1H),

7.53 (s, 1H), 7.38 (d, J ¼ 8.5 Hz, 1H), 7.23 (d, J ¼ 15.8 Hz, 1H), 7.01 (d,

J ¼ 7.9 Hz, 2H), 6.93 (d, J ¼ 7.7 Hz, 1H), 6.44e6.33 (m, 1H), 6.03 (s,

2H), 4.37 (s, 1H), 4.21 (s, 1H), 4.04 (s, 1H), 3.94 (s, 1H), 2.81 (s, 3H).

Compound 15e was prepared by the experimental procedure

depicted for compound 11a as a white solid. Yield: 48%; m.p.:

152e154 ^ꢀC. ¹H NMR (400 MHz, CD₃OD)

d

7.34 (dd, J ¼ 12.5, 2.1 Hz,

¹³C NMR (100 MHz, DMSO-d₆)

d

148.56, 147.90, 134.11, 133.35,

1H), 7.24 (d, J ¼ 8.5 Hz, 1H), 7.09 (t, J ¼ 8.6 Hz, 1H), 7.04e6.98 (m,

2H), 6.93 (d, J ¼ 7.8 Hz, 1H), 6.82 (d, J ¼ 15.7 Hz, 1H), 6.27e6.15 (m,

1H), 6.02 (s, 2H), 4.32 (s, 1H), 4.21 (s, 1H), 3.93 (s, 1H), 3.89 (s, 4H),

132.99, 130.60, 129.54, 129.38, 128.28, 125.87, 124.15, 123.34, 111.64,

108.83, 101.91, 57.87, 56.20, 38.44; HRMS (ESI) m/z calcd for

C

₁₈H₁₈Cl₂NO₂[MþH]^þ350.0715, found 350.0714.

2.77 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆)

d

152.02 (d, J_C-

¼ 243.8 Hz), 148.52, 147.89, 147.80, 137.50, 129.32 (d, J_C-F¼ 6.6 Hz),

F

4.1.40. Synthesis of (E)-N-(benzo[d][1,3]dioxol-5-ylmethyl)-3-(2-

ﬂuoro-4-(triﬂuoromethyl)phenyl)-N-methylprop-2-en-1-amine

hydrochloride (15b)

125.82, 124.42 (d, J_C-F¼ 3.0 Hz), 124.28, 117.63, 114.22, 113.98 (d, J_C-

¼ 18.5 Hz), 111.62, 108.82, 101.89, 57.66, 56.52, 56.36, 38.26.¹⁹

F

NMR (376 MHz, DMSO-d₆/CF₃COOH)

d

ꢂ135.30 (dd, J ¼ 12.6, 9.0 Hz,

Compound 15b was prepared by the experimental procedure

1F); HRMS (ESI) m/z calcd for C₁₉H₂₁NO₃F [MþH]^þ330.1505, found

248

H. Wei et al. / European Journal of Medicinal Chemistry 145 (2018) 235e251

330.1507.

J ¼ 14.0, 7.4 Hz, 1H), 3.80e3.71 (m, 1H), 1.49 (d, J ¼ 6.7 Hz, 3H), 1.44

(d, J ¼ 6.7 Hz, 3H). ¹³C NMR (100 MHz, DMSO-d₆)

d 159.51 (d, J_C-

4.1.44. Synthesis of (E)-N-(benzo[d][1,3]dioxol-5-ylmethyl)-3-(2-

ﬂuoro-4-(triﬂuoromethyl)phenyl)prop-2-en-1-amine hydrochloride

(16a)

A solution of intermediate 7v (1.42 g, 5.0 mmol) was added

slowly to

(831 mg, 5.5 mmol) and K₂CO₃(1.38 g, 10.0 mmol) in DMF (10 mL)

with stirring at room temperature overnight. The reaction mixture

was then poured into water and extracted with ethyl acetate. The

combined organic layers were washed with brine, dried over

anhydrous Na₂SO₄, and then ﬁltered and condensed. The residue

was then puriﬁed by silica gel column chromatography (eluent,

EtOAc/petroleum ether, 1/5, V/V) to give the free base of 16a as a

yellow oil. The ﬁnal compound 16a in the form of hydrochloride

was prepared by the saliﬁcation procedure described for compound

11a as a white solid. Yield: 60%; m.p.: 104e105 ^ꢀC. ¹H NMR

¼ 250.6 Hz), 148.52, 147.93, 130.56 (qd, J_C-F¼ 32.8, 8.4 Hz), 129.65

F

(d, J_C-F¼ 3.6 Hz), 128.54 (d, J_C-F¼ 3.2 Hz), 127.90 (d, J_C-F¼ 12.0 Hz),

125.83, 124.92 (d, J_C-F¼ 4.2 Hz), 124.15, 123.52 (qd, J_C-F¼ 271.8,

2.4 Hz), 122.07 (p, J_C-F¼ 3.4 Hz), 113.86 (dq, J_C-F¼ 25.6, 3.7 Hz),

111.62, 108.84, 101.92, 52.45, 51.22, 49.84, 15.47 (2C); HRMS (ESI) m/

z calcd for C₂₁H₂₂F₄NO₂[MþH]^þ396.1587, found 396.1586.

a mixture of benzo[d][1,3]dioxol-5-ylmethanamine

4.1.47. Synthesis of tert-butyl((3-(2-ﬂuoro-4-(triﬂuoromethyl)

phenyl)prop-2-yn-1-yl)oxy)dimethylsilane (17)

To a mixture of the 2-ﬂuoro-1-iodo-4-(triﬂuoromethyl)benzene

(580 mg,

2.0 mmol),

tetrakis(triphenylphosphine)palladium

(231 mg, 0.2 mmol), and CuI (381 mg, 2.0 mmol) in triethylamine at

0 ^ꢀC was added tert-butyldimethyl(2-propynyloxy)silane (341 mg,

2.0 mmol). The reaction mixture was heated to 60 ^ꢀC for 30 min and

then ﬁltrated through a short pad of silica gel. The residue was

washed with dichloromethane and the ﬁltrate concentrated. The

crude product was then puriﬁed by silica gel column chromatog-

raphy (eluent, petroleum ether) to afford 17 as a brown oil. ¹H NMR

(400 MHz, CD₃OD)

d

7.80 (t, J ¼ 7.8 Hz, 1H), 7.51 (t, J ¼ 7.9 Hz, 2H),

7.04 (d, J ¼ 16.1 Hz, 1H), 7.01e6.97 (m, 2H), 6.90 (dd, J ¼ 7.7, 0.5 Hz,

1H), 6.55 (dt, J ¼ 16.0, 7.1 Hz, 1H), 6.00 (s, 2H), 4.17 (s, 2H), 3.89 (d,

J ¼ 7.1 Hz, 2H). ¹³C NMR (100 MHz, DMSO-d₆)

d 159.58 (d, J_C-

(400 MHz, CDCl₃)

2H), 4.58 (s, 2H), 0.92 (d, J ¼ 9.8 Hz, 9H), 0.17 (s, 6H).

d

7.54 (t, J ¼ 7.4 Hz, 1H), 7.35 (dd, J ¼ 11.3, 8.9 Hz,

¼ 250.7 Hz), 148.12, 147.75, 130.36 (qd, J_C-F¼ 32.9, 8.5 Hz), 129.52

F

(d, J_C-F¼ 3.7 Hz), 127.94 (d, J_C-F¼ 12.0 Hz), 127.41 (d, J_C-F¼ 2.6 Hz),

126.97 (d, J_C-F¼ 4.9 Hz), 125.91, 123.72 (qd, J_C-F¼ 270.6, 2.6 Hz),

124.67, 122.09 (p, J_C-F¼ 3.6 Hz), 113.90 (dq, J_C-F¼ 25.6, 3.8 Hz),

110.87, 108.70, 101.75, 49.55, 48.03; HRMS (ESI) m/z calcd for

4.1.48. Synthesis of ethyl (2E,4E)-5-(2-ﬂuoro-4-(triﬂuoromethyl)

phenyl)penta-2,4-dienoate (18a)

Sodium methoxide (28% in MeOH, 386 mg, 2.0 mmol) was

added to a stirred solution of intermediate 5v (253 mg, 2.1 mmol)

and triethyl phosphonoacetate (448 mg, 2.0 mmol) in dime-

thylformamide (10 mL) at 0 ^ꢀC. After the mixture was stirred for 2 h,

the reaction mixture was poured into water to give crystals. Then

the crystals were ﬁltered to give 18a as colorless needles. The crude

was used for the next step without further puriﬁcation. ¹H NMR

C

₁₈H₁₆F₄NO₂[MþH]^þ354.1117, found 354.1118.

4.1.45. Synthesis of (E)-N-(benzo[d][1,3]dioxol-5-ylmethyl)-N-

ethyl-3-(2-ﬂuoro-4-(triﬂuoromethyl)phenyl)prop-2-en-1-amine

hydrochloride (16b)

To a solution of 16a (424 mg, 1.2 mmol) in DMF (10 mL) was

added sodium hydride (48 mg, 1.2 mmol) at 0 ^ꢀC under a nitrogen

atmosphere. The reaction mixture was stirred for 15 min, and

iodoethane (0.2 mL, 2.4 mmol) was added into the solution. The

mixture was stirred at room temperature overnight, poured into

water, and extracted with EtOAc. The combined organic layers were

washed with brine, dried over anhydrous Na₂SO₄, ﬁltered, and

condensed. The residue was then puriﬁed by silica gel column

chromatography (eluent, EtOAc/petroleum ether, 1/5, V/V) to give

the free base of 16b as a yellowish oil. The ﬁnal compound 16b in

the form of hydrochloride was prepared by the saliﬁcation pro-

cedure described for compound 11a as a white solid. Yield: 55%;

(400 MHz, DMSO-d₆)

d

7.95 (t, J ¼ 7.7 Hz, 1H), 7.74 (d, J ¼ 10.5 Hz,

1H), 7.62 (d, J ¼ 8.2 Hz, 1H), 7.51 (dd, J ¼ 15.2, 10.5 Hz, 1H), 7.34 (dd,

J ¼ 15.5, 10.7 Hz, 1H), 7.25 (d, J ¼ 15.7 Hz, 1H), 6.24 (d, J ¼ 15.2 Hz,

1H), 3.98 (q, J ¼ 8.0 Hz, 2H), 1.20 (t, J ¼ 8.0 Hz, 3H).

4.1.49. Synthesis of ethyl (E)-3-(2-ﬂuoro-4-(triﬂuoromethyl)

phenyl)-2-methylacrylate (18b)

To

a solution of 2-ﬂuoro-4-(triﬂuoromethyl)benzaldehyde

(384 mg, 2.0 mmol) in toluene (60 mL) was added (carbethox-

yethylidine)triphenylphosphorane (1.05 g, 2.9 mmol) in one

portion. The reaction mixture was heated to 100 ^ꢀC for 1 h before

being cooled to room temperature. The solvent was removed under

reduced pressure and petroleum ether was added to the residue.

The precipitated triphenylphosphine oxide was removed via

ﬁltration and the ﬁltrate concentrated. Puriﬁcation of the crude

material by silica gel column chromatography (eluent, EtOAc/pe-

troleum ether, 1/20, V/V) to afford 18b as a colorless oil. ¹H NMR

m.p.: 150e152 ^ꢀC. ¹H NMR (400 MHz, CD₃OD)

d

7.85 (t, J ¼ 7.8 Hz,

1H), 7.52 (t, J ¼ 8.0 Hz, 2H), 7.07 (d, J ¼ 16.4 Hz, 1H), 7.03 (d,

J ¼ 8.2 Hz, 2H), 6.93 (d, J ¼ 8.1 Hz, 1H), 6.62e6.51 (m, 1H), 6.02 (d,

J ¼ 2.6 Hz, 2H), 4.33 (d, J ¼ 3.2 Hz, 2H), 4.01 (dd, J ¼ 6.7, 4.3 Hz, 2H),

3.26 (dt, J ¼ 7.4, 5.9 Hz, 2H), 1.40 (t, J ¼ 7.3 Hz, 3H). ¹³C NMR

(100 MHz, DMSO-d₆)

d

159.55 (d, J_C-F¼ 250.8 Hz), 148.50, 147.94,

130.56 (qd, J_C-F¼ 32.9, 8.4 Hz), 129.61 (d, J_C-F¼ 3.6 Hz), 128.81 (d, J_C-

(400 MHz, CDCl₃)

d 7.58 (s, 1H), 7.42e7.34 (m, 2H), 7.31 (d,

¼ 3.1 Hz), 127.87 (d, J_C-F¼ 11.3 Hz), 125.81, 124.91 (d, J_C-F¼ 4.2 Hz),

F

J ¼ 10.0 Hz,1H), 4.23 (q, J ¼ 7.1 Hz, 2H), 1.96 (t, J ¼ 1.3 Hz, 3H),1.29 (t,

124.15, 123.74 (qd, J_C-F¼ 273.0, 2.4 Hz), 122.03 (p, J_C-F¼ 3.6 Hz),

113.88 (dq, J_C-F¼ 25.4, 3.8 Hz), 111.63, 108.82, 101.89, 55.54, 52.92,

46.65, 8.93; HRMS (ESI) m/z calcd for C₂₀H₂₀F₄NO₂[MþH]^þ

382.1430, found 382.1431.

J ¼ 7.1 Hz, 3H).

4.1.50. Synthesis of 3-(2-ﬂuoro-4-(triﬂuoromethyl)phenyl)prop-2-

yn-1-ol (19a)

4.1.46. Synthesis of (E)-N-(benzo[d][1,3]dioxol-5-ylmethyl)-3-(2-

ﬂuoro-4-(triﬂuoromethyl)phenyl)-N-isopropylprop-2-en-1-amine

hydrochloride (16c)

To a solution of 17 (332 mg, 1,0 mmol) in tetrahydrofuran

(10 mL) was added tetrabutylammonium ﬂuoride (2.6 g, 10 mmol)

at 0 ^ꢀC for 30 min. The solvent was then evaporated in vacuo. The

residue was extracted with water and EtOAc. The combined organic

layers were washed with brine, dried over anhydrous Na₂SO₄,

ﬁltered and condensed. The residue was then puriﬁed by silica gel

column chromatography to afford 19a as a colorless oil. ¹H NMR

Compound 16c was prepared by the experimental procedure

depicted for compound 16b as a white solid. Yield: 30%; m.p.:

155e156 ^ꢀC. ¹H NMR (400 MHz, CD₃OD)

d

7.74 (t, J ¼ 7.8 Hz, 1H),

7.55e7.47 (m, 2H), 7.04e6.96 (m, 3H), 6.91 (d, J ¼ 8.2 Hz, 1H),

6.46e6.36 (m, 1H), 5.99 (d, J ¼ 3.7 Hz, 2H), 4.43 (d, J ¼ 13.1 Hz, 1H),

4.18 (d, J ¼ 13.2 Hz, 1H), 4.07 (dd, J ¼ 14.5, 7.6 Hz, 1H), 3.95 (dd,

(400 MHz, DMSO-d₆)

d

7.81 (d, J ¼ 9.6 Hz,1H), 7.75 (t, J ¼ 7.6 Hz,1H),

7.61 (d, J ¼ 8.1 Hz, 1H), 5.51 (t, J ¼ 6.0 Hz, 1H), 4.38 (d, J ¼ 6.0 Hz, 2H).

H. Wei et al. / European Journal of Medicinal Chemistry 145 (2018) 235e251

249

4.1.51. Synthesis of (2E,4E)-5-(2-ﬂuoro-4-(triﬂuoromethyl)phenyl)

penta-2,4-dien-1-ol (19b)

7.65 (d, J ¼ 9.4 Hz, 1H), 7.61 (d, J ¼ 8.1 Hz, 1H), 7.05 (s, 2H), 6.96 (d,

J ¼ 8.4 Hz, 1H), 6.05 (s, 2H), 4.39 (s, 2H), 4.35 (s, 2H), 2.99 (s, 3H). ¹³

C

To a solution of 18a (288 mg, 1.0 mmol) in dichloromethane

(10 mL) was added diisobutyl aluminium hydride (1.0 M in hexane,

2 mL, 2.0 mmol) dropwise at 0 ^ꢀC under nitrogen atmosphere. The

reaction solution was stirred at room temperature for 1.5 h and

quenched with methyl (0.2 mL) and water (0.2 mL) with cooling.

The reaction mixture was acidiﬁed with concentrated hydrochloric

acid and extracted with dichloromethane. The combined organic

layers were washed with brine, dried over anhydrous Na₂SO₄,

ﬁltered and condensed to generate 19b as a colorless oil without

NMR (100 MHz, DMSO-d₆)

d

162.44 (d, J_C-F¼ 252.7 Hz), 148.71,

147.91, 135.57, 131.89 (qd, J_C-F¼ 33.1, 8.1 Hz), 125.93, 123.76, 123.43

(qd, J_C-F¼ 273.6, 3.4 Hz), 122.34e122.08 (m), 114.25 (d, J_C-

¼ 15.8 Hz), 113.91 (dq, J_C-F¼ 24.4, 3.0 Hz), 111.64, 108.86, 101.94,

F

87.32, 81.55, 57.69, 44.13, 39.06; HRMS (ESI) m/z calcd for

C

₁₉H₁₆F₄NO₂[MþH]^þ366.1117, found 366.1118.

4.1.56. Synthesis of (2E,4E)-N-(benzo[d][1,3]dioxol-5-ylmethyl)-5-

(2-ﬂuoro-4-(triﬂuoromethyl)phenyl)-N-methylpenta-2,4-dien-1-

amine hydrochloride (21b)

Compound 21b was prepared by the experimental procedure

depicted for compound 11a as a white solid. Yield: 45%; m.p.:

168e170 ^ꢀC. FTIR: 3421.7, 3015.5, 2904.1, 1622.1, 1494.3, 1374.1,

1331.8, 1260.3, 1164.7, 1128.0, 1040.7, 912.1, 883.1 cm-1¹H NMR

further puriﬁcation. ¹H NMR (400 MHz, DMSO-d₆)

d 7.88 (dd,

J ¼ 16.2, 8.3 Hz, 1H), 7.66 (d, J ¼ 10.8 Hz, 1H), 7.55 (d, J ¼ 8.2 Hz, 1H),

7.17 (dd, J ¼ 15.8, 10.6 Hz, 1H), 6.69 (d, J ¼ 15.6 Hz, 1H), 6.46 (dd,

J ¼ 15.2, 10.7 Hz, 1H), 6.12 (dt, J ¼ 15.2, 5.0 Hz, 1H), 4.91 (t, J ¼ 5.4 Hz,

1H), 4.08 (t, J ¼ 4.9 Hz, 2H).

4.1.52. Synthesis of ((1R,2R)-2-(2-ﬂuoro-4-(triﬂuoromethyl)phenyl)

cyclopropyl)methanol (19d)

(400 MHz, CD₃OD)

d

7.85 (t, J ¼ 7.8 Hz, 1H), 7.50 (t, J ¼ 9.3 Hz, 2H),

7.20 (dd, J ¼ 15.8, 10.5 Hz, 1H), 7.08e6.99 (m, 2H), 6.99e6.89 (m,

A solution of diethyl zinc (1.1 M, 2.72 mL, 3.0 mmol) in hexanes

was added to 5 mL of dichloromethane under nitrogen. The

resulting solution was cooled to 0 ^ꢀC. Triﬂuoroacetic acid (0.23 mL,

3.0 mmol) was added slowly to the cooled diethylzinc solution.

After the completion of addition, the resulting mixture was stirred

for 20 min. A solution of diiodomethane, (0.24 mL, 3.0 mmol) in

5 mL of dichloromethane was added to the mixture. Subsequently,

a solution of intermediate 6v (330 mg, 1.5 mmol) in 5 mL of

dichloromethane was added. After completing addition, the reac-

tion mixture was warmed to room temperature and stirred for 2 h.

Excess reagent was quenched by slow addition of 5 mL of 1 M hy-

drochloric acid. The resulting solution was partitioned with EtOAc

and water. The combined organic extracts were washed with

saturated ammonium chloride, dried over MgSO₄, ﬁltered and

concentrated in vacuo. The crude was puriﬁed by silica gel column

chromatography (eluent, EtOAc/petroleum ether, 1/5, V/V) to afford

2H), 6.79 (dd, J ¼ 15.0, 10.6 Hz, 1H), 6.16e5.97 (m, 3H), 4.28 (s, 2H),

3.92 (s, 2H), 2.78 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆)

d 159.58 (d,

J_C-F¼ 250.6 Hz), 148.55, 147.89, 138.79, 133.39 (d, J_C-F¼ 4.5 Hz),

129.85 (qd, J_C-F¼ 32.8, 8.7 Hz), 129.05 (d, J_C-F¼ 3.5 Hz), 128.73 (d, J_C-

¼ 11.8 Hz), 125.85, 125.60, 125.00, 124.17, 123.80 (qd, J_C-F¼ 270.3,

F

2.7 Hz), 122.10e121.86 (m), 113.82 (dq, J_C-F¼ 25.5, 3.8 Hz), 111.64,

108.80, 101.90, 57.88, 56.34, 38.20.¹⁹F NMR (376 MHz, DMSO-d₆/

CF₃COOH)

d

ꢂ61.13 (s, 3F), ꢂ115.82 (dd, J ¼ 11.3, 8.0 Hz, 1F); HRMS

(ESI) m/z calcd for C₂₁H₂₀F₄NO₂[MþH]^þ394.1430, found 394.1431.

4.1.57. Synthesis of (E)-N-(benzo[d][1,3]dioxol-5-ylmethyl)-3-(2-

ﬂuoro-4-(triﬂuoromethyl)phenyl)-N,2-dimethylprop-2-en-1-amine

hydrochloride (21c)

Compound 21c was prepared by the experimental procedure

depicted for compound 11a as a white solid. Yield: 55%; m.p.:

19d as a colorless oil. ¹H NMR (400 MHz, DMSO-d₆)

d 7.66 (d,

155e158 ^ꢀC. ¹H NMR (400 MHz, CD₃OD)

d 7.63e7.57 (m, 1H), 7.53 (t,

J ¼ 10.8 Hz,1H), 7.46 (d, J ¼ 7.9 Hz,1H), 7.22 (t, J ¼ 7.9 Hz,1H), 4.70 (t,

J ¼ 5.6 Hz, 1H), 3.52 (dt, J ¼ 11.1, 5.5 Hz, 1H), 3.41 (dt, J ¼ 11.4, 5.8 Hz,

1H), 2.02 (dd, J ¼ 8.9, 4.6 Hz, 1H), 1.51e1.39 (m, 1H), 1.08e0.95 (m,

2H).

J ¼ 9.1 Hz, 2H), 7.07e7.00 (m, 2H), 6.95 (d, J ¼ 8.2 Hz, 1H), 6.81 (s,

1H), 6.04 (s, 2H), 4.40 (d, J ¼ 13.1 Hz, 1H), 4.24 (d, J ¼ 13.0 Hz, 1H),

4.03 (d, J ¼ 13.0 Hz, 1H), 3.87 (d, J ¼ 12.9 Hz, 1H), 2.83 (s, 3H), 1.93 (t,

J ¼ 1.2 Hz, 3H). ¹³C NMR (100 MHz, DMSO-d₆)

d 159.61 (d, J_C-

¼ 250.1 Hz), 148.56, 147.90, 138.85 (d, J_C-F¼ 2.6 Hz), 130.50 (qd, J_C-

F

4.1.53. Synthesis of 3-(2-ﬂuoro-4-(triﬂuoromethyl)phenyl)propan-

1-ol (19e)

¼ 32.0, 8.8 Hz), 129.20 (d, J_C-F¼ 2.4 Hz), 127.77 (d, J_C-F¼ 12.5 Hz),

F

125.86, 124.85 (d, J_C-F¼ 3.6 Hz), 123.91 (q, J_C-F¼ 270.5 Hz), 124.16,

122.98e122.43 (m), 113.95 (dq, J_C-F¼ 22.4, 3.6 Hz), 111.60, 108.81,

101.90, 58.89, 54.62, 38.99, 15.42; HRMS (ESI) m/z calcd for

A solution of intermediate 6v (330 mg, 1.5 mmol) in methanol

was subjected to hydrogenation at atmospheric pressure in the

presence of 10% Pd/C (33 mg, 0.3 mmol) at ambient temperature

overnight. The reaction solution was ﬁltered and the ﬁltrate was

concentrated as a colorless oil to afford 19e without further puri-

C

₂₀H₂₀F₄NO₂[MþH]^þ382.1430, found 382.1431.

ﬁcation. ¹H NMR (400 MHz, DMSO-d₆)

7.57e7.47 (m, 2H), 4.56 (t, J ¼ 5.1 Hz, 1H), 3.42 (dd, J ¼ 11.6, 6.2 Hz,

2H), 2.72 (t, J ¼ 7.6 Hz, 2H), 1.70 (tt, J ¼ 16.2, 8.1 Hz, 2H).

d

7.59 (d, J ¼ 10.1 Hz, 1H),

4.1.58. Synthesis of 1-(benzo[d][1,3]dioxol-5-yl)-N-(((1R,2R)-2-(2-

ﬂuoro-4-(triﬂuoromethyl)phenyl)cyclopropyl)methyl)-N-

methylmethanamine hydrochloride (21d)

4.1.54. Synthesis of 1-(3-bromoprop-1-yn-1-yl)-2-ﬂuoro-4-

(triﬂuoromethyl)benzene (20a)

Compound 21d was prepared by the experimental procedure

depicted for compound 11a as a white solid. Yield: 25%; m.p.:

Intermediate 20a was prepared by the experimental procedure

160e162 ^ꢀC. ¹H NMR (400 MHz, CD₃OD)

d 7.49e7.40 (m, 2H), 7.27 (s,

depicted for intermediate 7a as a yellow oil. ¹H NMR (400 MHz,

1H), 7.00 (d, J ¼ 8.7 Hz, 2H), 6.93 (d, J ¼ 7.7 Hz, 1H), 6.03 (s, 2H), 4.45

(d, J ¼ 11.6 Hz, 1H), 4.19 (d, J ¼ 12.8 Hz, 1H), 3.50e3.35 (m, 1H),

3.28e3.10 (m, 1H), 2.86 (s, 3H), 2.31 (s, 1H), 1.63 (s, 1H), 1.38 (s, 1H),

CDCl₃)

d

7.57 (t, J ¼ 7.4 Hz, 1H), 7.39 (d, J ¼ 8.3 Hz, 1H), 7.35 (d,

J ¼ 9.2 Hz, 1H), 4.18 (s, 2H).

1.23 (dd, J ¼ 14.1, 6.9 Hz, 1H). ¹³C NMR (100 MHz, DMSO-d₆)

d 161.08

4.1.55. Synthesis of N-(benzo[d][1,3]dioxol-5-ylmethyl)-3-(2-

ﬂuoro-4-(triﬂuoromethyl)phenyl)-N-methylprop-2-yn-1-amine

hydrochloride (21a)

(d, J_C-F¼ 245.7 Hz), 148.53, 147.88, 133.81 (qd, J_C-F¼ 34.4, 3.5 Hz),

128.88 (d, J_C-F¼ 12.1 Hz), 128.31 (d, J_C-F¼ 3.7 Hz), 125.83, 124.05,

123.93 (qd, J_C-F¼ 270.1, 2.4 Hz), 121.98e121.75 (m), 112.85 (dq, J_C-

Compound 21a was prepared by the experimental procedure

depicted for compound 11a as a white solid. Yield: 57%; m.p.:

¼ 25.9, 3.8 Hz), 111.62, 108.80, 101.89, 58.48, 57.80, 38.45, 17.04,

F

16.42, 14.19; HRMS (ESI) m/z calcd for C₂₀H₂₀F₄NO₂[MþH]^þ

138e140 ^ꢀC. ¹H NMR (400 MHz, CD₃OD)

d

7.83 (t, J ¼ 7.2 Hz, 1H),

382.1430, found 382.1431.

250

H. Wei et al. / European Journal of Medicinal Chemistry 145 (2018) 235e251

4.1.59. Synthesis of N-(benzo[d][1,3]dioxol-5-ylmethyl)-3-(2-

ﬂuoro-4-(triﬂuoromethyl)phenyl)-N-methylpropan-1-amine

hydrochloride (21e)

Compound 21e was prepared by the experimental procedure

depicted for compound 11a as a white solid. Yield: 33%; m.p.:

different concentrations of test compounds (15b and 21b) or mock

(corresponding amount of ddH₂O), 3.5 L FAD stock solution

(10 mM), and 300 L CrtN lysate (~1.41 mg CrtN, as estimated by

western blot using a known concentration of the puriﬁed 6His-crtN

protein), then 0.02 M HEPES buffer (pH 7.5) to 700 L. The tests

were proceeded under anaerobic atmosphere by adding a ﬁnal

concentration of 20 U/mL glucose oxidase (Sigma-Aldrich, G2133),

20000 U/mL catalase (Sigma-Aldrich, C1345), and 2 mM glucose as

an oxygen-trapping system. The reaction mixture was started by

adding the lysate and incubating overnight at 37 ^ꢀC and then

stopped by methanol. The pigments were extracted twice against

m

188e190 ^ꢀC. ¹H NMR (400 MHz, CD₃OD)

d

7.30 (d, J ¼ 9.6 Hz, 1H),

7.01e6.89 (m, 2H), 6.67e6.47 (m, 3H), 5.95 (s, 2H), 4.33 (s, 2H), 3.15

(t, J ¼ 8.6 Hz 2H), 2.70e2.54 (m, 5H), 2.22e2.01 (m, 2H). ¹³C NMR

(100 MHz, DMSO-d₆)

d

161.59 (d, J_C-F¼ 250.7 Hz), 148.55, 147.87,

133.50 (qd, J_C-F¼ 32.6, 8.2 Hz), 132.43, 131.43 (d, J_C-F¼ 12.8 Hz),

125.84, 124.07, 123.90 (qd, J_C-F¼ 270.0, 2.6 Hz), 121.96e121.73 (m),

112.82 (dq, J_C-F¼ 21.8, 3.8 Hz), 111.58, 108.79, 101.91, 58.37, 53.78,

38.95, 25.61, 23.66; HRMS (ESI) m/z calcd for C₁₉H₂₀F₄NO₂[MþH]^þ

370.1430, found 370.1431.

700

m

L chloroform. The organic phase was combined, concentrated,

L chloroform and OD₄₅₀was recorded. The

and redissolved in 200

m

IC₅₀values were obtained by ﬁtting the OD data to a normal dose-

response curve using Graphpad Prism 5.0 software.

4.2. Biological methods

4.2.4. Bacterial growth assays of S. aureus Newman and MRSA

strains

4.2.1. Pigment inhibition assay

S. aureus Newman bacteria were cultured in TSB (4 mL) medium

21b was dissolved in DMSO to 20 mM as a stock solution and

diluted with fresh TSB medium to produce a ﬁnal concentration of

at 37 ^ꢀC for 48 h with 20

mL synthesized compounds dissolved in

DMSO and diluted to a set of concentrations, in duplicate. The

vehicle control was prepared by adding equal amount of DMSO. The

positive control was using nonpigmented CtrN mutant of S. aureus

Newman instead of Newman, with no compounds or DMSO added.

3 mL bacteria cultures were centrifuged and washed twice with

0.01 M phosphate-buffered saline (PBS) and resuspended in

methanol to extract pigment. The absorbance value was deter-

mined at 450 nm on a NanoDrop 2000c (Thermo scientiﬁc) spec-

trophotometer. IC₅₀values were calculated by Graphpad Prism 5.0

software. The IC₅₀values of the MRSA strains NRS271, USA300 LAC,

USA400 MW and Mu 50 were determined in the same way.

0.2 mM and 0.05 mM 100

well plates, together with the growth controls (containing equal

amount of DMSO). 60 L parafﬁn wax was covered onto the di-

lutions to prevent the medium from evaporating. The dilutions

were placed at 37 ^ꢀC for 4 h to sufﬁciently dissolve the test com-

pounds. Overnight cultured S. aureus strains were washed twice

with PBS and diluted with fresh medium to obtain an optical

density at 600 nm (OD₆₀₀) of ~1.0. Test and growth control wells

mL of each dilution was distributed in 96-

m

were inoculated with

5 mL of a bacterial suspension (ﬁnal

OD₆₀₀z 0.05). The 96-well plates were incubated at 37 ^ꢀC over-

night, and the OD₆₀₀was recorded every half an hour with a Syn-

ergy

2 (Biotek) plate reader following the manufacturer's

4.2.2. Cytotoxicity assay

instructions.

The cytotoxic activity against HepG 2 and HEK-293T cell lines in

vitro was determined using the CCK-8 assay. The cells were plated

in 96-well plates at density of 5000 cells per well and incubated at

37 ^ꢀC in 5% CO₂atmosphere for 24 h. The tested compounds were

dissolved in DMSO and diluted with culture medium (DMSO ﬁnal

concentration < 0.4%). The vehicle control was prepared by mixing

the culture medium with the corresponding concentration of

DMSO. Amphotericin B was set as reference drug. The various

concentration of tested compounds and controls were treated with

the cells for 72 h at 37 ^ꢀC in a 5% CO₂incubator. Then the super-

4.2.5. Hydrogen peroxide killing and human whole blood killing

assays

For H₂O₂killing assay, four strains, including Newman, USA300

LAC, USA400 WM2 and Mu50, were cultured in TSB and grown at

37 ^ꢀC for 24 h with 1

mM compound 21b (40 mL in DMSO) or 40 mL

DMSO as vehicle control or equal amount of N-Acetyl-Cysteine

(NAC). The bacteria were washed twice in PBS and then diluted to a

concentration of 4 ꢁ 10⁶CFU per 250

mL reaction mixture in a 2-mL

Eppendorf tube. After H₂O₂was added to a ﬁnal concentration of

1.5%, the tubes were incubated for 30 min at 37 ^ꢀC with shaking at

250 r.p.m. The reaction was terminated by the addition of 1000 U/

mL exogenous catalase (Sigma-Aldrich). Bacterial survival was

assessed by serial dilutions on TSA plates for enumeration of CFU.

For human whole blood killing assay, overnight cultured strains

were centrifuged and suspended in sterile PBS to generate a sus-

natant liquor was removed and 100 mL of new media diluted CCK-8

solution (10% CCK-8) was added to each well with 1 h incubating.

The cell survival was determined by measuring the absorbance at

450 nm. The assay was measured in triplicate.

4.2.3. CrtN enzyme inhibition assay

Diapophytoene was puriﬁed from diapophytoene-producing

E. coli BL21 (DE3)/pET28a:crtM extracting with acetone. 8 mg dia-

pophytoene was mixed with 24 mg of phosphatidylcholine (Sigma-

pension of 1 ꢁ 10⁷CFU/mL. Whole blood (360

mL) from healthy

human volunteer was collected using a BD VACUTAINER PT tube

and then mixed with 40 mL bacterial sample, which resulted in a

Aldrich) in 200

mL CHCl₃to prepare diapophytoene emulsion. The

concentration of 1 ꢁ 10⁶CFU/mL. The tubes were incubated at 37 ^ꢀC

for 6 h, and then the dilutions were plated on TSA agar plates for

enumeration of the surviving CFUs.

mixture was spun-dried and incubated with 2 mL 0.02 M HEPES

buffer (pH 7.5) followed by sonicating in ice water to obtain the

homogeneous emulsion. For the preparation of CrtN lysate, E. coli

BL21 (DE3)/pET28a:crtN was sub-cultured into 1000 mL of LB broth

4.2.6. S. aureus systemic infection models

supplemented with 50

~0.1 and grown to an OD₆₀₀of ~0.5. The expression of 6His-CrtN

m

g/mL kanamycin to achieve an OD₆₀₀of

6-8-week-old female BALB/c mice were obtained from JSJ Lab

Animal, Ltd. and housed under speciﬁed pathogen-free conditions.

The pre-treatment groups received intraperitoneal injections of

21b at a total dose of 50 mg/kg or 200 mg/kg (in 12 intervals for

108 h) twelve hours before infection. Similarily, vancomycin, line-

zolid and ddH₂O were given as the pre-treatment group at a total

dose of 200 mg/kg. The post-treatment groups received intraperi-

toneal injections at a total dose of 39 mg/kg or 156 mg/kg (in 12

protein was induced with 0.5 mM isopropyl-b-D-thiogalactoside

(IPTG) at 16 ^ꢀC overnight. The cells were harvested, and the pellets

were suspended in 30 mL HEPES buffer and lysed at 4 ^ꢀC by

sonication.

The enzyme activity was determined in triplicate, with a total of

700 mL of the following: 50 mL diapophytoene emulsion, 70 mL

H. Wei et al. / European Journal of Medicinal Chemistry 145 (2018) 235e251

426e433.

251

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All the compounds were dissolved in sterile ddH₂O. For the mouse

model of abscess formation, the mice were challenged with 100 mL

of a bacterial suspension of either 2.2 ꢁ 10⁷CFU of S. aureus New-

man, 1.1 ꢁ 10⁹CFU of S. aureus Mu50, or 1.8 ꢁ 10⁸CFU of S. aureus

NRS271 via retro-orbital injection. Animals were euthanized 90 h

after infection. Kidneys, and livers were isolated and homogenized

in 1 mL PBS plus 0.01% Triton X-100 to obtain single-cell suspen-

sions, and serial dilutions of each organ were plated on TSA (Difco)

plates for the enumeration of CFUs. The statistical signiﬁcance was

determined by the Mann-Whitney Test (two-tailed).

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Financial support for this research provided by the National

Natural Science Foundation of China (Grant 21672064), the Na-

tional Key R&D Program of China (Grant 2017YFB0202600), the

“Shu Guang” project supported by the Shanghai Municipal Educa-

tion Commission and Shanghai Education Development Founda-

tion (Grant 14SG28), Shanghai Sailing Program (Grants

17YF1403600), and the Fundamental Research Funds for the Cen-

tral Universities are gratefully acknowledged.

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HPLC analysis data of derivatives 11a-h, 12a-e, 13a-e, 14a-m,

15a-e, 16a-c and 21a-e; bacterial growth assays of S. aureus New-

man and MRSA strains, the hERG inhibition assay and MIC values of

derivatives against MRSA strains.

Supplementary data related to this article can be found at

https://doi.org/10.1016/j.ejmech.2017.12.090.

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Article Doi

Discovery of novel piperonyl derivatives as diapophytoene desaturase inhibitors for the treatment of methicillin-, vancomycin- and linezolid-resistant Staphylococcus aureus infections

DOI: 10.1016/j.ejmech.2017.12.090

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