Novel benzyl phenyl sulfide derivatives as antibacterial agents against methicillin-resistant Staphylococcus aureus

Article abstract of DOI:10.1038/s41429-019-0257-x

Methicillin-resistant Staphylococcus aureus (MRSA) infection is a major threat to human health due to its resistance to almost all classes of antibiotics. Discovery of novel antibacterial agents with new structures which combat the pathogens responsible f

Full text of DOI:10.1038/s41429-019-0257-x

The Journal of Antibiotics
https://doi.org/10.1038/s41429-019-0257-x
ARTICLE
Novel benzyl phenyl sulfide derivatives as antibacterial agents
against methicillin-resistant Staphylococcus aureus
1
Kuo Lu1 Qi Chen1 Xiao-Fang Xu1 Ying Meng1 Jing Lin1 Wei-Min Chen
●
●
●
●
●
Received: 23 May 2019 / Revised: 22 September 2019 / Accepted: 16 October 2019
© The Author(s), under exclusive licence to the Japan Antibiotics Research Association 2019
Abstract
Methicillin-resistant Staphylococcus aureus (MRSA) infection is a major threat to human health due to its resistance to
almost all classes of antibiotics. Discovery of novel antibacterial agents with new structures which combat the
pathogens responsible for MRSA is urgent. In this study, three series of benzyl phenyl sulfide derivatives were designed
and synthesized, and their antibacterial activity against eleven MRSA strains were evaluated. The results showed that
two series of the synthetic compounds (5a-5l and 12p-12u) exhibit potent antibacterial activity against S. aureus and
MRSA, with minimum inhibitory concentrations of 2–64 μg/mL. The structure-activity relationships are discussed and
the mechanism of the antibacterial activity was shown to involve the destruction of the bacterial cell membrane. Finally,
the MTT assay results suggest that the toxicity of compounds 5f and 5h is selective between bacteria and
mammalian cells.
Introduction
safer antimicrobial agents with novel scaffolds are
urgently needed [4].
Bacterial infection is a serious threat to human health.
Methicillin-resistant Staphylococcus aureus (MRSA) was
recently estimated to be responsible for 60–89% of
nosocomial infections leading to 19,000 deaths and over
$3 billion in health care costs per year in the United States
alone [1–3]. The emergence of increasingly drug-resistant
bacteria leads to the failure of clinical treatment with
frequently-used antibiotics. It is a challenge to develop
effective antibiotics because of the increasing resistance
of antibiotic-resistant microorganisms. More potent and
Sulfides are common in natural materials such as garlic
and chives [5, 6]. It has been reported that synthetic sulfides
showed different pharmacological properties, including
antitumor, antifungal, and antimicrobial activity [6–11]. For
example, benzyl phenyl sulfides have been reported to
inhibit microbial growth [7, 12]. Meanwhile, stilbenoids
have been widely reported to have antibacterial activity
[13–16]. The natural product cajaninstilbene acid (CSA,
Fig. 1) has an antibacterial scaffold and we have identified
several cajaninstilbene acid derivatives with excellent anti-
bacterial activity against gram-positive bacteria, especially
MRSA [15]. Benzyl phenyl sulfides exhibit structural
properties similar to those of stilbenoids (Fig. 1) and show
antibacterial potential. It is a rational approach to replace
the stilbene moiety in cajaninstilbene acid with benzyl
phenyl sulfide.
These authors contributed equally: Kuo Lu, Qi Chen
Supplementary information The online version of this article (https://
doi.org/10.1038/s41429-019-0257-x) contains supplementary
material, which is available to authorized users.
The present study reports the design and synthesis of a
series of novel benzyl phenyl sulfide derivatives, whose
activity against various bacteria including eleven strains of
MRSA was evaluated. The structure-activity relationships
(SAR) and the preliminary antibacterial mechanism have
been examined and are discussed. Among these com-
pounds, two novel compounds (5f and 5h) with minimal
toxicity against mammalian cells and promising activity
against MRSA were identified.
* Jing Lin
linjing_jnu@163.com
* Wei-Min Chen
twmchen@jnu.edu.cn
1
International Cooperative Laboratory of Traditional Chinese
Medicine Modernization and Innovative Drug Development of
Chinese Ministry of Education (MOE), College of Pharmacy,
Jinan University, Guangzhou 510632, PR China

K. Lu et al.
Result and discussion
the acetyl group. Deprotection of 2a-2l with a base gave
compounds 3a-3l. When reacted with isopentenyl bromide
and sodium hydride in toluene, compounds 2a-2l were
converted to 4a-4l. Compounds 4a-4l were hydrolyzed by a
base and then acidified with 10% HCl to give 5a-5l.
Compounds 12p-12u were synthesized in seven steps, as
shown in Fig. 3. Compound 6 was synthesized from methyl
3-oxobutanoate according to the procedure of Chiarello
[17]. The two free phenolic hydroxyls of compound 6 were
protected by acetylation giving compound 7, which was
then brominated with NBS to give compound 8. Compound
Chemistry
The synthesis of benzyl phenyl sulfides 5a-5l is outlined in
Fig. 2. Briefly, benzyl phenyl sulfides 3a-3l were synthe-
sized in two steps starting from compound 1 which was
reacted with different thiols and then hydrolyzed to remove
R1
R1
COOH
B
B
B
HO
S
A
A
A
R2
R2
9
was obtained by the reaction of
8
with 4-
OCH3
fluorobenzenethiol. Compound 9 was isoprenylated with
isoprenyl bromide to give compound 10. The esters 11p-
11u were obtained by the reaction of compound 10 with
Benzyl phenyl sulfides
Stilbenoids
CSA
Fig. 1 The structures of benzyl phenyl sulfides, stilbenoids, and CSA
COOH
COOCH3
Fig. 2 Synthesis routes of 3a-3l
and 5a-5l. (i) a. RSH, TEA,
DCM; b. K2CO3, MeOH;
(ii) Isoprenyl bromide, NaH,
Toluene; (iii) KOH, MeOH/H2O
HO
R
HO
R
iii
S
S
ii
COOCH3
COOCH3
Br
OMe
OMe
HO
R
OH
5a-5l
4a-4l
i
S
OMe
OMe
COOH
iii
2a-2l
HO
R
1
S
OMe
3a-3l
R=
Cl
H3CO
CH3
OCH3
Cl
Cl
a
b
c
d
e
f
F
F
CF3
OCH3
OCH3
F
OMe
S
N
g
h
i
j
k
l
F
COOCH3
Br
COOCH3
COOCH3
COOCH3
AcO
HO
AcO
HO
O
O
S
i
ii
iii
iv
O
OAc
OH
OAc
OH
6
7
8
9
v
F
F
F
COOH
COOCH3
COOCH3
HO
HO
HO
S
S
S
vii
vi
O
CH3
O
CH3
OH
n
n
n=1, 2, 3, 4, 5, 6
n=1, 2, 3, 4, 5, 6
12p-12u
11p-11u
10
Fig. 3 Synthesis routes of 12p-12u. (i) a. NaH, THF; b. n-BuLi; (ii) Ac2O, TEA, CH2Cl2; (iii) NBS, AIBN, CCl4; (iv) a. 4-Fluorobenzenethiol,
TEA, CH2Cl2; b. MeOH, K2CO3; (v) Isoprenyl bromide, NaH, Toluene; (vi) RX, NaH, DMF or ROH, DIAD, THF; (vii) KOH, MeOH/H2O

Novel benzyl phenyl sulfide derivatives as antibacterial agents against methicillin-resistant. . .
Table 1 In vitro antibacterial activity of 3a-3l
Compounds LogP MIC (μg ml−1
S.aa E.cb P.ac P.vd 43300e 52056f 515992g 513045h 62202i 48706j 49025k 48900l 48966m 49008n 48973o
)
3a
3.89 64
>128 >128 >128 64
64
64
64
128
>128
32
64
64
128
>128
>128
128
128
128
>128
>128
>128
64
64
64
64
3b
3.50 >128 >128 >128 >128 >128
>128
16
>128
64
>128
64
>128
>128
64
>128
>128
64
>128
>128
64
>128
>128
64
>128
>128
128
128
64
3c
3.45 32
4.07 32
4.12 64
4.09 32
3.60 128
3.58 128
3.56 128
4.33 32
>128 >128 >128 64
>128 >128 >128 32
>128 >128 >128 64
>128 >128 >128 32
>128 >128 >128 >128
>128 >128 >128 >128
>128 >128 >128 >128
>128 >128 >128 64
3d
32
32
32
64
3e
32
64
64
128
64
128
64
128
64
64
128
64
3f
32
32
32
64
3g
128
128
>128
64
>128
>128
>128
64
>128
>128
>128
64
>128
>128
>128
128
>128
128
8
>128
128
>128
64
>128
>128
>128
64
>128
>128
>128
64
>128
>128
>128
64
>128
>128
>128
64
3h
3i
3j
3k
3.09 >128 >128 >128 >128 >128
>128
64
>128
128
32
>128
64
>128
64
>128
128
8
>128
128
16
>128
128
8
>128
128
16
>128
128
8
3l
3.64 32
5.37 16
2
>128 >128 >128 >128
>128 >128 >128 16
CSA
16
16
8
Norfloxacin
32
16
16
4
8
16
4
8
4
1
1
1
1
0.5
aStaphylococcus aureus ATCC25923
bEscherichia coli ATCC25922
cPseudomonas aeruginosa ATCC27853
dProteus vulgaris ATCC49101
eMRSA ATCC43300
fMRSA 52056
gMRSA 515992
hMRSA 513045
iMRSA 62202
jMRSA 48706
kMRSA 49025
lMRSA 48900
mMRSA 48966
nMRSA 49008
oMRSA 48973
different halogenated alkanes or alchols, and hydrolysis of
11p-11u gave the target compounds 12p-12u.
introduction of isoprenyl group into compounds 3a-3l
greatly enhances their antibacterial activity and electron-
withdrawing substituents in the B ring contribute to the
antibacterial activity. With electron-withdrawing sub-
stituents such as chlorine, fluorine, and trifluoromethyl in
ring B, compounds 5d-5j have significant antibacterial
activity and 5d, with an o-chlorophenyl B ring and 5h, with
an m-fluorophenyl B ring, proved to be the most potent.
Electron-donating substituents in the B ring are evidently
not conducive to antibacterial activity. For example, 5k,
which has m-methoxy and p-methoxy substituents in ring B,
shows only weak antibacterial activity. Compounds 5a-5c,
with electron-donating substituents such as methyl and
methoxy in ring B show moderate activity. Compound 5l in
which ring B has been replaced by a heterocycle were also
synthesized. Its activities against bacteria were evaluated
and are shown in Table 2. Substitution with a heterocyclic
ring leads to no obvious improvement in the antibacterial
activity; the MIC values of 5l, containing a heterocyclic
MIC determination and SAR study
The minimum inhibitory concentration (MIC) of all syn-
thetic compounds was evaluated in vitro by a serial dilution
method [18]. The MIC is defined as the lowest concentration
of antibacterial agent at which no growth of the strains is
observed [19]. CSA and norfloxacin were used as positive
controls. MIC values were determined with eleven strains of
MRSA and one strain of methicillin-sensitive S. aureus, as
well as three strains of gram-negative bacteria (Escherichia
coli, Proteus vulgaris, Pseudomonas aeruginosa).
As shown in Table 1, 3a-3l displayed little or no activity
against most bacteria, indicating the importance of isoprenyl
group. The MIC of 5a-5l was determined and values are
summarized in Table 2. Compared with 3a-3l, the anti-
bacterial activity of 5a-5l against MRSA bacteria is greatly
improved (2–16 folds change). This indicated that the
ring, are in the range of 4–32 μg ml−1
.

K. Lu et al.
Table 2 In vitro antibacterial activity of 5a-5l
Compounds LogP MIC (μg ml−1
S.aa E.cb P.ac P.vd 43300e 52056f 515992g 513045h 62202i 48706j 49025k 48900l 48966m 49008n 48973o
)
5a
5.46 16
5.07 16
5.02 32
>128 >128 >128 32
>128 >128 >128 64
>128 >128 >128 64
8
8
32
32
64
4
8
16
32
64
8
16
32
64
8
16
32
64
8
16
32
64
16
8
16
32
64
16
16
8
16
32
64
8
5b
16
16
4
128
64
4
16
32
8
5c
5d
5.64
5.69
5.67
4
4
4
>128 >128 >128
>128 >128 >128
>128 >128 >128
4
8
8
5e
4
4
8
8
8
8
8
16
8
5f
2
4
4
8
8
8
8
8
5g
5.18 16
5.15
5.13 16
5.91
>128 >128 >128 16
8
8
16
4
64
4
16
8
16
8
16
16
16
8
16
16
16
8
16
16
16
8
16
8
5h
4
>128 >128 >128
>128 >128 >128
>128 >128 >128
4
8
8
4
4
5i
8
8
8
32
8
16
8
16
8
16
8
5j
4
4
8
8
5k
4.66 64
5.21 16
5.37 16
2
>128 >128 >128 128
>128 >128 >128
>128 >128 >128 16
32 16 16
32
4
128
16
32
16
>128
32
16
4
>128
16
8
128
8
128
8
128
8
128
8
128
8
128
8
5l
8
CSA
16
8
8
8
16
1
8
16
1
8
Norfloxacin
4
8
4
1
1
0.5
aStaphylococcus aureus ATCC25923
bEscherichia coli ATCC25922
cPseudomonas aeruginosa ATCC27853
dProteus vulgaris ATCC49101
eMRSA ATCC43300
fMRSA 52056
gMRSA 515992
hMRSA 513045
iMRSA 62202
jMRSA 48706
kMRSA 49025
lMRSA 48900
mMRSA 48966
nMRSA 49008
oMRSA 48973
Compounds 2a-2l and 4a-4l showed no activities against
all the test strains at the concentration of 128 μg ml−1
(data not shown). This suggests that a free carboxyl group
is essential to the antibacterial activity and indeed, ester-
ification of the carboxyl leads to loss of antibacterial
activity.
It has been reported that the introduction of hydrophobic
chains into compounds is beneficial to antibacterial activity
[15]. Thus, in order to get more potent antibacterial agents
and to comprehensively illuminate the structure-activity
relationship of benzyl phenyl sulfides with antibacterial
activity, hydrocarbon chains with 2–7 carbons were intro-
duced at the p-position of the carboxyl group in ring A.
Considering the starting material (4-fluorophenyl)metha-
nethiol is commercially available, the p-fluoro substituent in
B ring was retained for its contribution to the antibacterial
activity. The activity of compounds 12p-12u against dif-
ferent bacterial strains was evaluated (Table 3) and a
slight, 2–4 folds increase in the activity against bacteria
was observed. With an n-heptyl at the p-position to the
carboxyl group, 12u proved to be the most potent com-
pound. However, as the number of carbon atoms was
increased, the logP of the compounds increased and the
solubility of the compounds decreased sharply. 12u proved
to be poorly soluble in aqueous media. Lacking the free
carboxyl, compounds 11p-11u show no antibacterial effect
at a concentration of 128 μg ml−1 (data not shown), proving
that the carboxyl group is essential for the antibacterial
activity.
MTT assay
The effects of 5f and 5h on of cell viabilities were deter-
mined using mouse macrophage cell lines (RAW 264.7)
and IC50 values were shown in Table 4. Compared with the
nature product CSA, 5f, and 5h exhibited similar effects on
cell viabilities, while their antibacterial ability improved
2–16 folds. Compared IC50 values to MIC values, 5f and 5h
show moderate selectivity between bacterial and mamma-
lian cells.

Novel benzyl phenyl sulfide derivatives as antibacterial agents against methicillin-resistant. . .
Table 3 In vitro antibacterial activity of 12p-12u
Compounds
n
LogP MIC (μg/mL)
S.aa E.cb P.ac P.vd 43300e 52056f 515992g 513045h 62202i 48706j 49025k 48900l 48966m 49008n 48973o
12p
1
2
3
4
5
6
5.55 16 >128 >128 >128
8
16
4
16
8
16
4
16
8
16
8
8
8
8
4
4
4
8
1
16
8
8
8
8
4
4
4
8
1
16
8
8
12q
6.06
6.62
7.12
7.63
8.12
4
8
8
4
4
>128 >128 >128 64
>128 >128 >128
8
12r
4
4
4
8
4
8
8
8
8
12s
>128 >128 >128 16
>128 >128 >128 64
>128 >128 >128 32
4
8
8
8
4
4
4
4
12t
2
4
4
4
4
4
4
4
12u
2
4
4
4
4
2
4
2
CSA
Norfloxacin
5.37 16 >128 >128 >128 16
32 16 16
16
8
32
16
16
4
8
8
16
1
16
1
8
2
4
8
4
0.5
aStaphylococcus aureus ATCC25923
bEscherichia coli ATCC25922
cPseudomonas aeruginosa ATCC27853
dProteus vulgaris ATCC49101
eMRSA ATCC43300
fMRSA 52056
gMRSA 515992
hMRSA 513045
iMRSA 62202
jMRSA 48706
kMRSA 49025
lMRSA 48900
mMRSA 48966
nMRSA 49008
oMRSA 48973
Table 4 the MIC and IC50 values of 5f, 5h, and CSA
Bacterial membrane permeability
Compounds
MIC (μg ml−1
)
IC50 (μM)
The cell membrane of bacteria is one of the main targets
of antibiotics. Drugs that target the bacterial cell membrane
often are effective against drug-resistant bacteria [20–22].
Propidium iodide (PI) is a dye that can pass through the
membrane of compromised bacterial cells and which fluoresces
upon binding to DNA, indicating membrane damage [23–25].
The ability of compounds 5 f and 5 h with good antibacterial
activity against MRSA to penetrate the bacterial cell membrane
was studied using a PI assay [23]. As can be seen from Fig. 4,
both compounds were efficient in penetrating the membranes
of S. aureus at concentrations of 4 μg ml−1 and 8 μg ml−1
and are more effective than the positive control melittin, a
peptide which is a cytoplasmic membrane pore-forming toxin
[26, 27]. The compounds tested exhibit a concentration-
dependent effect on the penetration of the bacterial cell mem-
brane. The results suggest that this class of compounds is likely
to kill bacteria by damaging its cytoplasmic membrane.
5f
2–8
79.01 1.13
86.37 1.87
84.34 7.18
5h
4–16
8–32
CSA
clinical isolated MRSA strains were also determined. The
synthesized derivatives were potent against S. aureus and,
especially against MRSA. The MIC values against MRSA of
5d, 5e, 5f, 5h, and 5j are in the range of 2–16 μg ml−1. More
importantly, 5h and 5j exhibit good selectivity for bacterial
over mammalian cells. The preliminary antibacterial
mechanism of benzyl phenyl sulfides derivatives was shown
to involve destruction of the bacterial cell membrane. The
structure–activity relationships of this class of derivatives
were summarized, and is expected to be useful in the future
design and modification of new benzyl phenyl sulfide deri-
vatives as candidate antibacterial agents.
Conclusion
Experimental
A series of benzyl phenyl sulfides were designed and syn-
thesized. Their MICs against one methicillin-sensitive S.
aureus, three-gram-negative bacteria strains, as well as eleven
All reagents were used as purchased without further pur-
ification. All solvents were dried before use according to

K. Lu et al.
Fig. 4 Membrane permeabilization against MRSA 43300. a Treatment with 5f. b Treatment with 5h. Mellitin was used at the concentration of
8 μg/mL and act as positive control. 0.1% DMSO (vol/vol) was used as negative control
standard methods. Thin-layer chromatography (TLC) was
performed on F254 precoated plates silica gel from Qingdao
Haiyang Inc. and observed under 254 nm UV light. All
compounds were purified by flash chromatography with
General procedure for synthesis of compounds 3a-3l
Solid KOH (5 equiv) was added to a solution of 2a-2l (200
mg) in a mixed MeOH and H2O solvent (10 ml, MeOH:
H2O, 5:1). The reaction mixture was refluxed for 6 h in an
oil bath. After the reaction was complete, the mixture was
quenched with ice water and acidified with 10% HCl. The
precipitate was filtered and recrystallized from petroleum
ether/EtOAc to obtain pure 3a-3l.
1
200–300 mesh silica gel. H (300 MHz) and 13C NMR (75
MHz) spectra were measured on a Bruker Magnetic System
1
300 MHz spectrometer and the H NMR (400 MHz) and
13C NMR (100 MHz) spectra were collected on Bruker
Magnetic System 400 MHz spectrometer. High-resolution
mass spectra were recorded by Agilent 6210 LC/MSD TOF
or Waters Q-TOF spectrometers. Characterization data for
all compounds are included in the Supplementary Infor-
mation (SI). LogP values were calculated at the web site of
Molinspiration Cheminformatics https://www.molinspira
tion.com/.
General procedure for synthesis of compounds 4a-4l
Compounds 2a-2l (3 mmol) were dissolved in dry toluene
(20 ml). NaH (144 mg, 3.6 mmol, 60% in mineral oil) was
added to the solution at 0 °C. The reaction mixture was
stirred at 50 °C for 0.5 h, and then cooled to r.t. when iso-
prenyl bromide (745 mg, 4.5 mmol, 90%) was added
dropwise. The reaction was stirred at 78 °C for a further 2 h,
then it was quenched by addition of ice water (20 ml). The
mixture was extracted with EtOAc (2 × 10 ml) and the
combined organic layer was washed with brine (2 × 10 ml),
dried over anhydrous sodium sulfate and filtered. The fil-
trate was concentrated under reduced pressure. The crude
products were purified by silica-gel column to give com-
pounds 4a-4l.
General procedure for synthesis of compounds 2a-2l
Compound 1 (1.00 g, 3.10 mmol) and an aromatic thiol
(3.70 mmol, 1.2 equiv) was dissolved in dry CH2Cl2 (10 ml)
and Et3N (0.88 ml, 6.33 mmol) was added to the solution.
The reaction mixture was stirred at r.t. for 1 h with the
reaction monitored by TLC. After the reaction was com-
plete, the mixture was filtered and the filtrate was con-
centrated under reduced pressure. The crude compound was
then dissolved in MeOH (10 ml), and K2CO3 (1.14 g, 1.5
equiv) was added to the solution. The suspension was
stirred at r.t. for 30 min and then the solvent was removed
under reduced pressure. The residue was dissolved in H2O
(10 ml), neutralized with 2 N HCl and extracted with
CH2Cl2 (3 × 10 ml). The combined organic extracts were
washed with H2O (2 × 10 ml) and brine (2 × 10 ml), then
dried over anhydrous sodium sulfate and filtered. The fil-
trate was concentrated under reduced pressure to give a
crude product which was purified on a silica-gel column to
obtain compounds 2a-2l.
General produce for synthesis of compounds 5a-5l
Solid KOH (560 mg 10 mmol) was added to a solution of
4a-4l (2 mmol) in a mixture of MeOH and H2O (MeOH 8
ml, H2O 2 ml). The reaction mixture was refluxed for 6 h in
an oil bath. After the reaction completed, the mixture was
quenched by ice water and acidified with 10% HCl. The
precipitate was filtered and recrystallized from petroleum
ether/EtOAc to get pure 5a-5l.

Novel benzyl phenyl sulfide derivatives as antibacterial agents against methicillin-resistant. . .
General procedure for synthesis of compounds 12p-
12u
General procedure for preparation of 11p, 11r, 11s, 11t:
NaH (50.6 mg, 1.26 mmol) was added to a solution of
compound 10 (300 mg, 0.97 mmol) in DMF (5 ml), and the
mixture was stirred for 1 h. Then alkyl halide (1.45 mmol)
was added to the mixture dropwise, the reaction was stirred
for 3 h and was then quenched with H2O. The mixture was
extracted with EtOAc (3 × 10 ml). The combined organic
layer was dried over Na2SO4 and concentrated in vacuo.
The residue was purified by flash chromatography giving
compounds 11p, 11r, 11s and 11t.
General procedure for preparation of compounds 11q
and 11u: A mixture of compound 10 (300 mg, 0.78 mmol),
PPh3 (354 mg, 1.17 mmol) and alkyl alcohol (0.78 mmol)
was dissolved in dry THF (3 ml). DIAD (236 mg,
1.17 mmol) was added to the solution dropwise, the reaction
was stirred for 16 h and then quenched with H2O. The
mixture was extracted with EtOAc (3 × 10 ml). The com-
bined organic layer was dried over Na2SO4, concentrated in
vacuo. The residue was purified by flash chromatography to
afford compounds 11q and 11u.
General procedure for preparation of 12p-12u: KOH
(80 mg, 2 mmol) was added to a solution of compounds
11p-11u (0.5 mmol) in MeOH (5 ml) and H2O (2 ml). The
reaction mixture was refluxed overnight and then quenched
with ice water and acidified with aqueous HCl (2 M). The
mixture was extracted with EtOAc (3 × 10 ml). The com-
bined organic layer was dried over Na2SO4 and concentrated
in vacuo. The residues were purified by crystallized from
PE/EtOAc. The products were obtained as white solids.
Preparation of compound 6 [17]. Methyl acetoacetate
(20.0 g, 172.2 mmol) was added dropwise to a suspension of
NaH (6.2 g, 258.4 mmol) in THF (100 ml) at 0 °C. The
solution was cooled to −78 °C then a 2.5 M solution of
n-BuLi (65.5 ml, 163.6 mmol) was added dropwise. The
reaction mixture was warmed to ambient temperature and
stirring was continued overnight. Then the mixture was
acidified to pH 2 with 6 N HCl at 0 °C and extracted with
EtOAc (3 × 200 ml). The combined organic layer was dried
over Na2SO4 and concentrated in vacuo, and the residue was
purified by flash chromatography to afford compound 6.
Preparation of compound 7: Ac2O (18 ml, 176 mmol)
and TEA (24 ml, 176 mmol) were added to a mixture
of compound 6 (8 g, 44 mmol) and DMAP (200 mg,
1.76 mmol) in CH2Cl2 (100 ml). The reaction was stirred 1 h
before being quenched with H2O. The mixture was then
extracted with CH2Cl2 (3 × 100 ml) and the combined
organic layer was dried over Na2SO4 and concentrated in
vacuo. The residue was purified by flash chromatography to
afford compound 7.
Preparation of compound 8: NBS (1.8 g, 10 mmol)
and BPO (20 mg) were added to a solution of compound
7 (4.4 g, 16.4 mmol) in CCl4. The reaction was refluxed for
3 h and then more NBS (1.8 g, 10 mmol) and BPO
(20 mg) were added. The reaction was refluxed for another 3
h. The mixture was filtered after cooling to ambient tem-
perature. The filtrate was concentrated in vacuo. The residue
was purified by flash chromatography to afford compound 8.
Preparation of compound 9: 4-Fluorothiophenol
(445 mg, 3.4 mmol) and TEA (0.8 ml, 5.8 mmol) were
added to a solution of compound 8 (1.00 g, 2.9 mmol) in
CH2Cl2 (10 ml). The reaction was stirred for 2 h. Then the
mixture was concentrated in vacuo and the residue was
dissolved in MeOH (10 ml). K2CO3 (1.20 g, 8.7 mmol) was
added to the solution and the mixture was stirred overnight
before being quenched with aqueous HCl (1 M), and then
extracted with EtOAc (3 × 10 ml). The combined organic
layer was dried over Na2SO4 and concentrated in vacuo.
The residue was purified by flash chromatography to give
compound 9.
Bacterial strains and growth conditions
Bacterial strains S. aureus ATCC25923 E. coli
ATCC25922 P. aeruginosa ATCC27853 P. vulgaris
ATCC49101, MRSA ATCC43300, were purchased from
the American Type Culture Collection. MRSA 52056,
MRSA 515992, MRSA 513045, and MRSA 62202 were
isolated from clinical patients by the Guangzhou Military
Region Guangzhou General Hospital. MRSA 48760,
MRSA 49025, MRSA 48900, MRSA 48966, MRSA
49008, and MRSA 48973 were isolated from clinical
patients by the Second Clinical College of Jinan University.
Bacteria were cultured in Mueller-Hinton Broth (MHB) or
lysogeny broth (LB broth) at 37 °C.
Preparation of compound 10: NaH (260 mg, 6.49 mmol)
was added to the suspension of compound 9 (2.00 g,
6.49 mmol) in toluene (60 ml) and the resulting mixture was
stirred at 50 °C for 4 h. The mixture was cooled to 35 °C
prior to the addition of isoprenyl bromide (1.32 g,
8.437 mmol) and then stirred overnight. The reaction was
quenched with H2O and extracted with EtOAc (3 × 50 ml).
The combined organic layer was dried over Na2SO4 and
concentrated in vacuo. The residue was purified by flash
chromatography to obtain compound 10.
MIC determination
The MICs were determined using a slightly modified broth
micro-dilution method by Wiegand et al. [18]. Stock solu-
tions of test compounds were prepared in DMSO. Various
concentrations of test compounds were serially diluted in
sterile MHB to final volumes of 100 μl in 96-well plates.
Each well was then inoculated with 100 μl of the test

K. Lu et al.
organism in MHB at a final concentration of 5 × 105 CFU
ml. The MICs were defined as the lowest concentration of
test compounds at which growth was inhibited after 16 h of
incubation at 37 °C [28].
Publisher’s note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
References
1. Guidos RJ, et al. Combating antimicrobial resistance: policy
recommendations to save lives. Clin Infect Dis. 2011;52:
S397–S428.
MTT assay
2. Klevens RM, et al. Invasive methicillin-resistant Staphylococcus
aureus infections in the United States. JAMA-J Am Med Assoc.
2007;298:1763–71.
3. Talbot GH, et al. Bad bugs need drugs: an update on the devel-
opment pipeline from the antimicrobial availability task force of
the infectious diseases society of America. Clin Infect Dis.
2006;42:657–68.
4. Lin Z, et al. Total synthesis and antimicrobial evaluation of natural
albomycins against clinical pathogens. Nat Commun.
2018;9:3445.
5. Wu CC, Chung JG, Tsai SJ, Yang JH, Sheen LY. Differential
effects of allyl sulfides from garlic essential oil on cell cycle
regulation in human liver tumor cells. Food Chem Toxicol.
2004;42:1937–47.
6. Rattanachaikunsopon P, Phumkhachorn P. Diallyl sulfide content
and antimicrobial activity against food-borne pathogenic bacteria
of chives (Allium schoenoprasum). Biosci Biotechnol Biochem.
2008;72:2987–91.
7. Hatano A, Nishimura M, Souta I. Impact of unnatural nucleosides
on the control of microbial growth. Biocontrol Sci.
2009;14:55–60.
The MTT assay were performed as described before [29].
Briefly, the RAW264.7 cells were cultured in DMED medium
which contains 10% fetal bovine serum and 2 mM L-glutamine
and 100 units/ml penicillin/streptomycin. The cultures were
maintained in an atmosphere of 5% CO2 at 37 °C. Cells were
passaged at preconfluent densities using a solution containing
0.25% trypsin and 0.5 mM EDTA. Cells were seeded at a
density of 5000 cells/well in a 96-well plate for 24 h. The test
compounds were stored in DMSO and diluted in the medium
before use. For each group, the final DMSO concentration was
less than 0.1%. Cell viability was assessed on the basis of
cellular conversion of MTT into a formazan product with
DMSO after 24 h exposure to the test compounds. Absorbance
was detected by a Gen5 Reader at 570 nm. The experiments
were performed in five replicate wells for each compound or
concentration with at least three experimental runs.
8. Mostofi M, Mohammadi Ziarani G, Lashgari N. Design, synthesis
and biological evaluation of benzofuran appended benzothiaze-
pine derivatives as inhibitors of butyrylcholinesterase and anti-
microbial agents. Bioorg Med Chem. 2018;26:3076–95.
9. Lee MK, Senius JD, Grossman MJ. Sulfur-specific microbial
desulfurization of sterically hindered analogs of dibenzothio-
phene. Appl Environ Microbiol. 1995;61:4362–6.
10. Wang N, Saidhareddy P, Jiang X. Construction of sulfur-
containing moieties in the total synthesis of natural products.
Nat Prod Rep. 2019; https://doi.org/10.1039/c8np00093j.
11. Feng M, Tang B, Liang SH, Jiang X. Sulfur containing scaffolds
in drugs: synthesis and application in medicinal chemistry. Curr
Top Med Chem. 2016;16:1200–16.
Membrane permeabilization assay
A single S. aureus colony was inoculated into LB broth and
cultured overnight. The culture was diluted into fresh LB
broth with a dilution of 1:100 and allowed to continue
culturing for 6 h to reach the exponential phase. The cells
were harvested by centrifuged (3500 rpm, 5 min, 4 °C). The
template was washed and resuspended in HEPES buffer (5
mM glucose, 5 mM HEPES, pH 7.2) in 1:1 ratio. PI was
added to the culture to a final concentration of 10 μg ml−1
.
Different concentrations of the test compounds (5h and 5f)
were then added into the culture containing PI. 0.1% DMSO
(vol/vol) was used as negative control. Fluorescence was
monitored at an excitation wavelength of 530 nm and an
emission wavelength of 590 nm. The uptake of PI was
monitored by the increase in fluorescence for 10 min as an
index of inner membrane permeabilization.
12. Hatano A, et al. Inhibitory effects on microbial growth using the
derivatives of benzyl phenyl sulfide. Biocontrol Sci.
2011;16:63–7.
13. Asgharpour Z, Farzaneh F, Ghiasi M, Azarkish M. Synthesis,
characterization, density functional theory studies and anti-
bacterial activity of a new Schiff base dioxomolybdenum(VI)
complex with tryptophan as epoxidation catalyst. Appl Organomet
Chem. 2017;31 (e3782).
14. Wan MS, et al. Synthesis and properties of novel stilbene-twelve
alkyl quaternary ammonium salts as antibacterial optical whiten-
ing agents. Cellulose. 2017;24:3209–18.
Acknowledgements We sincerely thank the National Natural Science
Foundation of China (81673336), Pearl River S&T Nova Program of
Guangzhou (201806010116), and the Fundamental Research Funds
for the Central University (21617478) for financial support of this
study. We sincerely thank Lingwei Wang and Yuemei Lu from the
Second Clinical Medical College of Jinan University for their donation
of clinical isolated MRSA strains.
15. Geng Z-Z, et al. Novel cajaninstilbene acid derivatives as anti-
bacterial agents. Eur J Med Chem. 2015;100:235–45.
16. Huang MY, et al. Anti-inflammatory effects of cajaninstilbene
acid and its derivatives. J Agric Food Chem. 2016;64:2893–900.
17. Wilson ZE, Brimble MA. A flexible asymmetric synthesis of the
tetracyclic core of berkelic acid using a Horner-Wadsworth-
Emmons/oxa-Michael cascade. Org Biomol Chem. 2010;
8:1284–6.
Compliance with ethical standards
18. Wiegand I, Hilpert K, Hancock RE. Agar and broth dilution
methods to determine the minimal inhibitory concentration (MIC)
of antimicrobial substances. Nat Protoc. 2008;3:163–75.
Conflict of interest The authors declare that they have no conflict of
interest.

Novel benzyl phenyl sulfide derivatives as antibacterial agents against methicillin-resistant. . .
19. Koh J-J, et al. Rapid bactericidal action of alpha-mangostin
against MRSA as an outcome of membrane targeting. Biochim
Biophys Acta Biomembr. 2013;1828:834–44.
25. Yarlagadda V, Akkapeddi P, Manjunath GB, Haldar J. Membrane
active vancomycin analogues: a strategy to combat bacterial
resistance. J Med Chem. 2014;57:4558–68.
20. Fauvart M, Van den Bergh B, Michiels J. Stabbed while sleeping:
synthetic retinoid antibiotics kill bacterial persister cells. Mol Cell.
2018;70:763–64.
26. Koh JJ, et al. Nonpeptidic amphiphilic xanthone derivatives:
structure-activity relationship and membrane-targeting properties.
J Med Chem. 2016;59:171–93.
21. Kim W, et al. NH125 kills methicillin-resistant Staphylococcus
aureus persisters by lipid bilayer disruption. Future Med Chem.
2016;8:257–69.
27. Seydlova G, et al. Lipophosphonoxins II: design, synthesis, and
properties of novel broad spectrum antibacterial agents. J Med
Chem. 2017;60:6098–118.
22. Kim W, et al. A new class of synthetic retinoid antibiotics
effective against bacterial persisters. Nature. 2018;556:103–7.
23. Konai MM, Ghosh C, Yarlagadda V, Samaddar S, Haldar J.
Membrane active phenylalanine conjugated lipophilic norspermi-
dine derivatives with selective antibacterial activity. J Med Chem.
2014;57:9409–23.
28. Nobmann P, Bourke P, Dunne J, Henehan G. In vitro anti-
microbial activity and mechanism of action of novel carbohydrate
fatty acid derivatives against Staphylococcus aureus and MRSA. J
Appl Microbiol. 2010;108:2152–61.
29. Huang M-Y, et al. Design, synthesis and anti-inflammatory effects
of novel 9-O-substituted-berberine derivatives. MedChemComm.
2016;7:658–66.
24. Ghosh C, et al. Small molecular antibacterial peptoid mimics: the
simpler the better! J Med Chem. 2014;57:1428–36.