C2 Arylated Benzo[b]thiophene Derivatives as Staphylococcus aureus NorA Efflux Pump Inhibitors

Article abstract of DOI:10.1002/cmdc.201500463

An innovative and straightforward synthesis of second-generation 2-arylbenzo[b]thiophenes as structural analogues of INF55 and the first generation of our laboratory-made molecules was developed. The synthesis of C2-arylated benzo[b]thiophene derivatives

Full text of DOI:10.1002/cmdc.201500463

DOI: 10.1002/cmdc.201500463
Full Papers
C2 Arylated Benzo[b]thiophene Derivatives as
Staphylococcus aureus NorA Efflux Pump Inhibitors
FranÅois Liger,[a] Pascale Bouhours,[b] Carine Ganem-Elbaz,[b] Claude Jolivalt,[b]
StØphane Pellet-Rostaing,[a] Florence Popowycz,*[c] Jean-Marc Paris,*[b] and Marc Lemaire*[a]
An innovative and straightforward synthesis of second-genera-
tion 2-arylbenzo[b]thiophenes as structural analogues of INF55
and the first generation of our laboratory-made molecules was
developed. The synthesis of C2-arylated benzo[b]thiophene de-
rivatives was achieved through a method involving direct ary-
lation, followed by simple structural modifications. Among the
34 compounds tested, two of them were potent NorA pump
inhibitors, which led to a 16-fold decrease in the ciprofloxacin
minimum inhibitory concentration (MIC) against the SA-1199B
strain at concentrations of 0.25 and 0.5 mgmLÀ1 (1 and 1.5 mm,
respectively). This is a promising result relative to that ob-
tained for reserpine (MIC=20 mgmLÀ1), a reference compound
amongst NorA pump inhibitors. These molecules thus repre-
sent promising candidates to be used in combination with ci-
profloxacin against fluoroquinolone-resistant strains.
Introduction
Bacterial infections are becoming more challenging to treat as
a result of the emergence of multidrug resistant (MDR) patho-
genic bacteria, which thus induces serious public health con-
cerns. MDR is increasingly prevalent, especially in Gram-posi-
tive pathogens such as Staphylococcus aureus, Streptococcus
pneumoniae, and Enterococcus spp.,[1] and it is responsible for
many hospital-born infections. Infectious diseases remain the
second major cause of death worldwide.[2] For decades, antibi-
otics have been intensively administrated; this has led to the
intensification of resistance from the bacteria strains, which de-
velop defensive mechanisms, including MDR.[3] As a conse-
quence, the available therapeutic arsenal used with success
from the middle of the 20th Century to fight bacterial infec-
tions has run out. For instance, the emergence of vancomycin
resistance first in enterococci and then in S. aureus has caused
considerable concern, because vancomycin is the drug of last
resort for treatment of methicillin-resistant S. aureus infections.
Several mechanisms are involved in bacterial resistance:[4] en-
zymatic inactivation of the antibiotic, structural modification of
the enzymatic target, substitution of the target by another,
bypass and/or overexpression of the targeted enzyme, and de-
crease in the intramolecular concentration of the antibiotic by
active efflux systems or lack of entry.[5] As an example, resist-
ance mechanisms were observed only a few years after two of
the most recent and sole classes of antibiotics, the oxazolidi-
nones and daptomycin, were brought to the market, launched
in 2000 and 2003, respectively. There is, therefore, an acute
need for new active agents. Important strategies currently
under investigation concern the development of antivirulence
drugs targeting Gram-positive adhesins.[6] Also, the develop-
ment of inhibitors of resistance such as protein-based efflux
pump inhibitors that could restore the activity of the antibiotic
to which the bacteria has become resistant represents a prom-
ising strategy that would allow recycling of known antibacteri-
al agents.[7]
In S. aureus strains, the NorA protein is a drug/proton anti-
porter belonging to the major facilitator family of transporters
and extrudes chemically unrelated molecules such as berber-
ine, ethidium bromide, and ciprofloxacin (Figure 1).[8] A possi-
ble strategy to overcome the resistant bacteria is to combine
the administration of a known antibacterial agent with a NorA
pump inhibitor that could bypass the problem of resistance.[9]
Reserpine is the first inhibitor of the NorA pump that induces
restoration of the antibiotic activities of norfloxaxin and cipro-
floxacin (Figure 2).[10] The restriction associated with the use of
[a] F. Liger, Dr. S. Pellet-Rostaing, Prof. M. Lemaire
Equipe Catalyse Synth›se Environnement, CNRS-UMR 5246
L’Institut de Chimie et Biochimie MolØculaires et SupramolØculaires (ICBMS)
43 Bd. du 11 novembre 1918, 69622 Villeurbanne (France)
E-mail: marc.lemaire@univ-lyon1.fr
[b] P. Bouhours, C. Ganem-Elbaz, Prof. C. Jolivalt, Prof. J.-M. Paris
Laboratoire Charles Friedel, CNRS-UMR 7223, Chimie ParisTech
11 rue Pierre et Marie Curie, 75005 Paris (France)
E-mail: claude.jolivalt@upmc.fr
[c] Prof. F. Popowycz
Institut National des Sciences AppliquØes de Lyon
ICBMS, CNRS-UMR 5246, Equipe Chimie Organique et Bioorganique
20 Avenue Albert Einstein, 69621 Villeurbanne (France)
E-mail: Florence.Popowycz@insa-lyon.fr
Figure 1. Structures of berberine and ciprofloxacin.
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hibit their efflux. Investigation of a broader series of analogues
of these molecules in an effort to improve the efficiency could
therefore be promising.
Results and Discussion
Chemistry
In previous work, we reported the direct bromination of 3-sub-
stituted benzo[b]thiophene[31] with molecular bromine: 6-
bromo-3-cyanobenzo[b]thiophene (1), prepared from commer-
cially available 3-cyanobenzo[b]thiophene, was presented as
a key intermediate in our strategy to access Raloxifen (a drug
used as an oral estrogen receptor modulator) by direct aryla-
tion of 6-methoxy-3-cyanobenzo[b]thiophene (3). To access
a variety of substituted benzo[b]thiophenes at the C6 position
as potential NorA pump inhibitors, palladium-catalyzed cou-
pling reactions on these bromide derivatives was investigated.
As illustrated in Scheme 1, bromide 1 was coupled with 4-me-
Figure 2. NorA pump inhibitors discovered by screening.
this molecule as a therapeutic agent arises from neurotoxic ef-
fects that are observed at the effective antibacterial concentra-
tion of this molecule, so that reserpine was dropped in early
clinical phases.
More recently, several renowned groups from the medicinal
chemistry community have identified a wide range of hetero-
cyclic molecules, quite structurally heterogeneous, displaying
promising biological activities as inhibitors of the NorA pump
bearing the following units: 1,4-benzothiazine,[11] polyphenolic
amide,[12] piperine,[13] piperidine,[14] flavone,[15] indole,[16] quino-
line/quinolone,[17] chalcone,[18] coumarin,[19] 2,1-benzothiazin-4-
one 2,2-dioxide,[20] and other aromatic scaffolds.[11,21] Natural
products such as osthol and curcumin display a four- and
eightfold reduction in the minimum inhibitory concentration
(MIC) of ciprofloxacin,[22] respectively, as do terpenoids, and
citral and citronellal have been used as starting materials for
the preparation of amides that have been identified as potent
efflux pump inhibitors.[23] Boronic acid derivatives were identi-
fied for the first time in 2014 as novel inhibitors of the
S. aureus NorA efflux pump.[7] A high-throughput screening (Di-
verSet chemical library composed of 9600 structurally diverse
compounds) led to the identification of the new NorA pump
inhibitors INF240, INF277, and INF55 (Figure 2) with improved
activities relative to that of reserpine.[24] In particular, 5-nitro-2-
phenylindole INF55 was reported to be eightfold more potent
than the parent compound reserpine. Recently, berberine–
INF55 hybrids were developed, and they have been shown to
enhance the activity of berberine by decreasing its efflux.[25]
Contrary to furan and indole derivatives, benzo[b]thiophene
has received little attention for drug design, although interest-
ing properties in the antipsychotic,[26] anti-inflammatory,[27] anti-
viral,[28] and antithrombotic[29] fields have been described, and
more recently, benzo[b]thiophene was shown to be an efflux
pump inhibitor.[30] In previous work, we showed that several
benzothiophene structures—although they showed no effect
against the susceptible S. aureus strain—exhibited NorA pump
inhibitor properties, and they enhanced the activity of cipro-
floxacin against resistant S. aureus bacteria overexpressing this
efflux pump. Their mode of action as NorA pump inhibitors
was hypothesized because of their ability to enhance the accu-
mulation of ethidium bromide and carbonyl cyanide m-chloro-
phenylhydrazone (two substrates of the NorA efflux pump)
inside a S. aureus NorA overexpressing strain, and they also in-
Scheme 1. Suzuki–Miyaura coupling of 6-bromo-3-cyanobenzo[b]thiophene
(1). Reagents and conditions: a) 4-Methoxyphenylboronic acid, Pd(PPh3)4,
Cs2CO3, anhyd. DME, 958C, 24 h.
thoxyphenylboronic acid (1.5 equiv) in the presence of
Pd(PPh3)4 (10 mol%) as a catalyst and cesium carbonate
(2 equiv) in anhydrous DME at 958C for 24 h. Efforts were de-
voted to optimizing the catalytic system by using a solid min-
eral base instead of classical aqueous sodium hydrogen car-
bonate so that the nitrile functionality could be partially hydro-
lyzed. The optimized conditions afforded 6-substituted ben-
zo[b]thiophene 2 in 91% yield.
Using Buchwald–Hartwig conditions, amine derivatives 5–7
were also prepared by using aniline, 2-aminopyridine, and
morpholine in a Pd(OAc)2/2,2’-bis(diphenylphosphino)-1,1’-bi-
naphthyl (BINAP) catalytic system in the presence of cesium
carbonate (or K3PO4) in toluene [or 1,2-dimethoxyethane
(DME)] at 1008C. Morpholine was also introduced to 4-bromo-
benzo[b]thiophene carbonitrile (4)[31] to generate regioisomer 8
in moderate yield (Scheme 2).
For substrates 2–8, direct arylation was applied to build
aryl–aryl bonds in a cleaner chemical transformation.[32] Six dif-
ferent molecules were arylated according this method by
using bromobenzene (2 equiv) in the presence of Pd(OAc)2/
PPh3/K2CO3 in DMF at 1208C. Depending on the substituents
on the phenyl group of benzo[b]thiophene, (step 1, Scheme 3)
a longer reaction time (from 20 to 120 h) and a higher loading
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Scheme 2. Overview of the Buchwald–Hartwig reactions on bromide starting
materials. Reagents and conditions: a) Amine, Pd(OAc)2, BINAP, Cs2CO3, tolue-
ne,1008C, 30 h; b) amine, Pd(OAc)2, BINAP, K3PO4, DME, 958C, 18 h.
of the catalyst (from 5 to 20 mol% of palladium acetate) were
required to achieve arylation at the C2 position. In each case,
a double amount of triphenylphosphine was used relative to
the amount of the source of metal. Highly functionalized mole-
cules 9–14 were obtained in moderate to good yields (37–
86%). The nitrile moiety was subsequently hydrolyzed under
basic conditions to provide carboxylic acids and intermediate
amides 15–21 (partial hydrolysis was observed for compounds
15 and 21). These 13 newly synthesized molecules were inte-
grated in the biological assays (see below). Reduction of the
carboxylic acid moieties by using an excess amount of BH3·THF
(3 to 4 equiv) in THF at reflux provided corresponding alcohols
22–25 (Scheme 4).
Scheme 4. Access to oxime derivatives 29–31. Reagents and conditions:
a) BH3·THF, THF, reflux, 18 h; b) MnO2 or DMP, CH2Cl2, RT, 24 h; c) NH2OH·HCl,
pyridine, EtOH, 908C, 24 h.
provided 32 in 71% yield. Subsequent hydrolysis of the nitrile
group of 32 in basic medium failed. Carboxylic derivative 33
was alternatively obtained by demethylation of 17. Despite nu-
The choice of either MnO2 or Dess–Martin periodinane
(DMP)[33] for selective oxidation was strongly dependent on the
degradation of compounds in the reaction mixture. Com-
pounds 26 and 28 were obtained by using MnO2, whereas
compound 27 was prepared by using DMP. Compounds 22–25
were all successfully and selectively oxidized into aldehydes,
except compound 23, which decomposed. Oxime synthesis
from benzo[b]thiophene-3-carboxaldehyde was performed in
the presence of hydroxylamine chlorohydrate in a 9:1 mixture
of pyridine/ethanol at reflux.[34] Desired compounds 29–31
were isolated in yields ranging from 67 to 91% (Scheme 4).
Additional phenol derivatives were prepared (Scheme 5). De-
methylation of 10 with boron tribromide at room temperature
Scheme 5. Preparation of 6-hydroxybenzo[b]thiophene derivatives. Reagents
and conditions: a) 1m BBr3 in THF, CH2Cl2, RT, 18 h; b) BH3·THF, THF, reflux,
18 h.
Scheme 3. Synthesis of C2-arylated benzo[b]thiophenes followed by hydrolysis. Reagents and conditions: a) Bromobenzene, Pd(OAc)2, PPh3, K2CO3, DMF,
1208C, 20–120 h; b) 3m NaOH(aq), dioxane or ethylene glycol, 4 days, reflux.
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merous attempts to purify compound 33, the chemical purity
was incompatible with biological tests. Compound 33 was re-
duced to alcohol 34 by using an excess amount of borane so-
lution in THF in a global yield of 50% (two steps).
If the MIC was found to be superior to 128 mgmLÀ1, the
compound showed no intrinsic antibacterial activity and could
unambiguously be further investigated for biological activity as
an efflux pump inhibitor. In that case, the compound was
tested in the presence of a sub-inhibitory concentration of ci-
profloxacin against SA-1199B (MIC=16 mgLÀ1). From Table 1, it
can be seen that most of the tested compounds showed no in-
trinsic activity against sensitive or resistant S. aureus strains.
Only compounds 16, 18, and 34 displayed activities on both
strains; the measured MICs range from 64 to 128 mgmLÀ1,
which is indicative of significant, but low, intrinsic antibacterial
activity. All other compounds displayed no intrinsic antibacteri-
al properties against any strain. Their screening as NorA efflux
pump inhibitors was thus undertaken against resistant-strain
Previous studies on the bromination of 3-substituted ben-
zo[b]thiophenes[27] have shown that depending on the reaction
conditions (e.g., solvent, number of equivalents of bromine
source), the 3-benzo[b]thiophene carboxylic acid could be
halogenated simultaneously at the C5 and C6 positions. So-
formed compound 35 was modified in the framework of this
project to enlarge the set of tested compounds. Ullman con-
densation by using 2m sodium methoxide in methanol with
10% DMF at 1208C led to complete conversion (Scheme 6).
SA-1199B in combination with ciprofloxacin (CIP) at 4 mgmLÀ1
,
that is, a concentration fourfold lower than the MIC of CIP
alone. Biological results pointed out that six molecules dis-
Table 1. Minimum inhibitory concentrations of benzothiophene deriva-
tives alone or in combination with a sub-inhibitory concentration of ci-
profloxacin.
Compd
MIC[a] [mgmLÀ1
]
ATCC 25923
SA-1199B
SA-1199B+CIP4
CIP
2
3
5
6
0.25
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
64
>128
32–64
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
64
16
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
64
–
>128
32
Scheme 6. Preparation of benzo[b]thiophenes highly functionalized in the
C2, C3, C5, and C6 positions. Reagents and conditions: a) MeONa, MeOH,
DMF, CuBr, Cu, 1208C, 9 days; b) MeOH, H2SO4, 708C, 18 h; c) bromoben-
zene, Pd(OAc)2, PPh3, K2CO3, DMF, 1208C, 20 days; d) LiOH(aq), THF, 658C,
4 days.
4
4
8
9
>128
>128
>128
1–2
>128
>128
32
>128
16
>128
8
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
34
35
36
37
38
Classical esterification with MeOH in the presence of an acid
catalyst furnished 36 in 43% yield over two steps. Arylation
under the previously described conditions only allowed the
formation of compound 37 in low yield. The ester function
seems to be less suitable than the nitrile moiety for the palladi-
um-catalyzed coupling reaction. Subsequent hydrolysis with
lithium hydroxide provided carboxylic acid 38 in quantitative
yield.
>128
64
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
64
64
64
64
>128
0.5
Biological evaluation
8
The antibacterial activity of the library of benzo[b]thiophene
derivatives as well as the synthetic intermediates (altogether
34 molecules) was evaluated on sensitive ATCC 25923 and re-
sistant SA-1199B S. aureus strains (Table 2). S. aureus SA-1199B
is a ciprofloxacin-resistant [thanks to NorA pump overexpres-
sion and a mutation on the topoisomerase-IV (A116E GrlA), the
fluoroquinolone target (QRDR) in S. aureus] strain derived from
the clinical isolate S. aureus SA-1199 and recovered from the
vegetation of a rabbit treated with this drug.[35]
>128
>128
16–32
>128
>128
>128
>128
0.25
16
>128
>128
>128
>128
>128
>128
>128
>128
>128
64
Determination of the MIC, defined as the minimum concen-
tration of the tested compound required to inhibit growth of
the bacteria, was performed for all compounds by optical den-
4
64
[a] Variance of MIC data can be estimated to a twofold difference as
a result of the microdilution method used. MIC values were determined
against sensitive ATCC 25923 and resistant SA-1199B S. aureus strains in
the absence or presence of ciprofloxacin at 4 mgmLÀ1 (CIP4).
sity measurements.
A MIC value inferior or equal to
128 mgmLÀ1 indicated intrinsic antibacterial activity of the
compound.
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played promising results with MICꢀ8 mgmLÀ1. For comparison,
previous studies[1] showed that the 2-phenylbenzo[b]thiophene
derivative has a MIC of 12.5 mgmLÀ1. The general tendency of
structural modification to improve biological activity is quite
difficult to define on the basis of the obtained results. The
presence of a bulky group at either the C2 or C6 position
seems to be a beneficial parameter. The crucial role of C2Àaryl
bond formation was established by Samosorn et al.[16d] with
indole derivatives tested as NorA pump inhibitors. No activity
was detected for C5,C6-disubstituted compounds; this led to
the suggestion of steric hindrance, which hampers interaction
with the NorA pump.
also at 20 mgmLÀ1, as its MIC against SA-1199B is >32 mgmLÀ1
The effects of the benzo[b]thiophene derivatives on the EtBr
accumulation of SA-1199B, taking reserpine as a reference, are
shown in Figure 3. Compounds 6, 23, and 32 all allowed a sig-
A sublibrary representative of the most active compounds
was further tested with lower CIP sub-inhibitory concentra-
tions. The biological results (Table 2) revealed three com-
Table 2. Minimum inhibitory concentration of selected compounds in
the presence of sub-inhibitory low concentrations of ciprofloxacin.
Compd
MIC[a] [mgmLÀ1
]
SA-1199B+CIP1
SA-1199B+CIP2
Figure 3. Accumulation of ethidium bromide in S. aureus SA-1199B overex-
pressing NorA. Uptake of ethidium bromide (5 mgLÀ1) in the presence of
6
16–32
>128
2
>32
>64
2
>128
>128
>128
>128
8
>128
1
8–16
>64
1
>128
>128
16
11
23
24
27
32
35
36
37
38
*
2.6% DMSO with no addition ( ) as negative control or with the addition of
~
~
&
*
reserpine ( ), compound 6 ( ), compound 23 ( ), and compound 32 ( ).
Reserpine and tested benzothiophene derivatives were added after 10 min
incubation of the S. aureus strain with EtBr (indicated by an arrow) at
20 mgmLÀ1
.
>128
nificant EtBr fluorescence increase relative to that shown by
the negative control. Compound 6 was the least efficient mole-
cule, but its accumulation level was very similar to that of re-
serpine, the positive control. Compounds 32 and 23 showed
better capability in EtBr accumulation than reserpine, of which
compound 23 was the most effective, and this correlates well
with antibacterial potency of these compounds deduced from
their MIC measurements and corroborates the hypothesis that
the mode of action of these antibacterial agents could be by
blocking the NorA pump in S. aureus.
[a] Variance of MIC data can be estimated to a twofold difference as
a result of the microdilution method used. MIC values were determined
against the S. aureus SA-1199B strain in the presence of ciprofloxacin at
1 mgmLÀ1 (CIP1) or 2 mgmLÀ1 (CIP2).
pounds exhibiting significant activity against the resistant
S. aureus SA-1199B strain, and compounds 23 and 32 were es-
pecially promising with MICs of 2 mgmLÀ1 in the presence of
only 1 mgmLÀ1 ciprofloxacin, which thus decreases the CIP MIC
of the strain by a factor of 16. The resistance of the strain was
thus significantly decreased in the presence of these benzo[b]-
thiophene derivatives, and they were able to reach the value
of the parent sensitive S. aureus strain SA-1199, that is,
Conclusions
In the present work, we developed an innovative and straight-
forward synthesis of a second generation of 2-arylbenzo[b]thio-
phenes as structural analogues of INF55 and the first genera-
tion of molecules developed in our laboratory.[30] This work led
to the design of 34 new molecules, the biological evaluation
of which as NorA pump inhibitors was assayed. The screened
benzo[b]thiophene derivatives were synthesized by direct ary-
lation of benzo[b]thiophene in the C2 position. The synthetic
method based on regioselective bromination and direct aryla-
tion at the C2 position on the same scaffold afforded a signifi-
cant series of original molecules substituted at both the C2
and C6 positions. Two hit compounds emerged from this li-
brary with more than a 10-fold increase in potency relative to
that of benzo[b]thiophene derivatives with no substitution at
the C6 position previously assayed in our group. The two most
1 mgmLÀ1 [31]
.
To obtain further insight into the mode of action of these
active compounds and to check their ability to act as efflux
pump inhibitors (EPIs), we evaluated their ability to interfere
with ethidium bromide (EtBr) accumulation in SA-1199B. All
tests were performed by using the synthesized compounds at
20 mgmLÀ1, that is, a concentration lower than their MICs
alone against SA-1199B. These conditions excluded the possi-
bility that any observed EtBr fluorescence increase could be re-
lated to cellular death and subsequent collapse of the NorA
pump proton-driven activity rather than to active EtBr efflux in-
hibition. Reserpine, a well-known NorA pump inhibitor widely
used in EtBr accumulation tests, was used as a positive control,
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tween H2O (50 mL) and CH2Cl2 (50 mL). The organic layer was sepa-
rated, and the aqueous layer was extracted with CH2Cl2 (2”30 mL).
The combined extract was dried (MgSO4), filtered, and concentrat-
ed under reduced pressure. The resulting residue was purified by
flash chromatography (SiO2, cyclohexane/EtOAc 80:20) to afford
the pure desired compound.
promising molecules as NorA pump inhibitors are substituted
at the C2 position with a phenyl group and at the C6 position
with either an aniline or a hydroxy group, both of which are
nucleophiles and able to form hydrogen bonds. The main con-
straint to the efficient design of NorA pump inhibitors remains
the mechanism of inhibition, which still remains unsolved.
These compounds represent promising candidates to be used
in association with ciprofloxacin against fluoroquinolone-resist-
ant strains.
General procedure C for total hydrolysis of the nitrile: 3m NaOH
in H2O (1 mL) was added to a stirred solution of the nitrile deriva-
tive (0.29 mmol) in ethylene glycol (10 mL), and the mixture was
heated at reflux for 4 days. After cooling, water (30 mL) was added
to the mixture, and the resulting solution was acidified with 1 n
HCl(aq) until pH 3. After extraction with CH2Cl2 (3”10 mL), the or-
ganic layer was dried (MgSO4) and concentrated to give the car-
boxylic acid.
Experimental Section
General methods
General procedure D for the BH3 reduction of the carboxylic
acid: Under an argon atmosphere, 1m BH3 in THF (0.88 mmol) was
added to a stirred solution of the carboxylic acid (0.22 mmol) in an-
hydrous THF (4.4 mL). The mixture was heated at reflux for 18 h
and, after cooling, was partitioned between H2O (20 mL) and
CH2Cl2 (20 mL). The organic layer was separated, and the aqueous
layer was extracted with CH2Cl2 (2”20 mL). The combined extract
was dried (MgSO4), filtered, and concentrated under reduced pres-
sure. The resulting residue was purified by flash chromatography
(SiO2, cyclohexane/EtOAc 70:30).
Reagents and solvents were supplied by Aldrich, Acros, Lancaster,
Alfa Aesar, or Fluka; they were purchased at the highest commer-
cial quality and were used without further purification. All reactions
were performed under an argon atmosphere with anhydrous sol-
vents. NMR spectra were recorded with a Bruker DPX-300 (1H:
300 MHz; 13C: 75 MHz) spectrometer by using CDCl3 and DMSO as
solvents. The chemical shifts and coupling constants are reported
in the standard fashion. The following abbreviations are used to
explain the multiplicities: s=singlet, d=doublet, t=triplet, q=
quartet, m=multiplet, br=broad. Electrospray ionization (ESI)
mass spectrometry (MS) experiments were performed with
a Thermo Finnigan LCQ Advantage mass. High-resolution mass
spectrometry (HRMS) was performed with a Finnigan Mat 95xL
mass spectrometer by using chemical ionization. Analytical thin-
layer chromatography was performed on silica gel Merck 60 D254
(0.25 mm). Flash chromatography was performed on Merck Si 60
silica gel (40–63 mm) or Merck aluminum oxide 90 active neutral
(63–200 mm).
General procedure E for MnO2 oxidation: MnO2 (4.6 mmol) was
added to a stirred solution of the alcohol (0.115 mmol) in CH2Cl2
(11.5 mL). The mixture was stirred vigorously for 24 h and then fil-
tered over Celite. The filtrate was concentrated under reduced
pressure and purified by flash chromatography (SiO2, cyclohexane/
EtOAc 90:10).
General procedure F for oxime synthesis: Hydroxylamine hydro-
chloride (0.259 mmol) and pyridine (0.291 mmol) were added to
a stirred solution of the aldehyde (0.064 mmol) in ethanol (1.5 mL).
The mixture was heated at reflux for 18 h and, after cooling, was
partitioned between H2O (20 mL) and CH2Cl2 (20 mL). The organic
layer was separated, and the aqueous layer was extracted with
CH2Cl2 (2”20 mL). The combined extract was dried (MgSO4), fil-
tered, and concentrated under reduced pressure. The resulting res-
idue was purified by flash chromatography (SiO2, cyclohexane/
EtOAc 70:30).
Chemistry
General procedure A for direct arylation: A Schlenk tube was
charged with bromobenzo[b]thiophene-3-carbonitrile
1 or 4
(0.5 mmol), Pd(OAc)2 (3 mol%), racemic BINAP (4.5 mol%), and
Cs2CO3 (0.6 mmol) under an argon atmosphere and was sealed
with a rubber septum. Then, anhydrous toluene (1 mL) and amine
(0.65 mmol) were added. The mixture was heated at 1008C for 18
to 30 h and, after cooling, was partitioned between H2O (25 mL)
and CH2Cl2 (25 mL). The organic layer was separated, and the aque-
ous layer was extracted with CH2Cl2 (2”20 mL). The combined or-
ganic extract was dried (MgSO4), filtered, and concentrated under
reduced pressure. The resulting residue was purified by flash chro-
matography.
6-(4-Methoxyphenyl)benzo[b]thiophene-3-carbonitrile (2):
A
Schlenk tube was charged with 6-bromobenzo[b]thiophene-3-car-
bonitrile (1; 95 mg, 0.4 mmol), 4-methoxyphenylboronic acid
(91 mg, 0.6 mmol), Pd(PPh3)4 (46 mg, 0.06 mmol), Cs2CO3 (261 mg,
0.8 mmol), and anhydrous DME (0.8 mL) under an argon atmos-
phere. The mixture was heated at 958C for 24 h and, after cooling,
was partitioned between H2O (25 mL) and CH2Cl2 (25 mL). The or-
ganic layer was separated, and the aqueous layer was extracted
with CH2Cl2 (2”20 mL). The combined extract was dried (MgSO4),
filtered, and concentrated under reduced pressure. The resulting
residue was purified by flash chromatography (SiO2, cyclohexane/
EtOAc 95:5) to give 2 (97 mg, 91%) as a white solid; mp: 117–
General procedure A1 for direct arylation: Slight changes were
made to general procedure A in the choice of the base (K3PO4), sol-
vent (DME), and temperature (908C).
General procedure B for direct CÀH arylation: A Schlenk tube
was charged with the substituted benzo[b]thiophene (1 mmol),
Pd(OAc)2 (11 mg, 0.05 mmol), PPh3 (26 mg, 0.10 mmol), and K2CO3
(415 mg, 3 mmol) under an argon atmosphere and was sealed
with a rubber septum. Then, anhydrous DMF (1 mL) and the aryl
bromide (2 mmol) were added, and the mixture was heated at
1208C for 48 h. If the reaction was not complete (as observed by
1
119 8C (CH2Cl2/pentane); H NMR (300 MHz, CDCl3): d=3.88 (s, 3H,
OMe), 7.02 (d, J=8.8 Hz, 2H), 7.59 (d, J=8.8 Hz, 2H), 7.74 (dd, J=
8.5, 1.2 Hz, 1H), 8.02 (d, J=8.5 Hz, 1H), 8.05 (d, J=1.2 Hz, 1H),
8.10 ppm (s, 1H); 13C NMR (75 MHz, CDCl3): d=55.5 (CH3), 106.9
(C), 114.5 (2CH), 114.6 (CH), 120.4 (CH), 122.7 (CH), 125.5 (CH),
128.6 (2CH), 132.5 (C), 135.9 (C), 137.4 (CH), 139.4 (2C), 159.7 ppm
(C).
1
TLC or H NMR spectroscopy), additional aliquots of Pd(OAc)2 and
PPh3 were added (0.05 equiv per 0.05 equiv Pd(OAc)2; 0.10 equiv
per 0.10 equiv PPh3) until total consumption of the starting materi-
al was observed. After cooling, the mixture was partitioned be-
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6-Phenylaminobenzo[b]thiophene-3-carbonitrile (5): Procedure A
starting from 1 (119 mg, 0.5 mmol) and aniline; flash chromatogra-
phy (SiO2, cyclohexane/EtOAc/Et3N 90:10:1). Yield: 84 mg (67%);
gray solid; mp: 93–958C (MeOH); 1H NMR (300 MHz, CDCl3): d=
5.98 (brs, 1H, NH), 7.05 (t, J=7.4 Hz, 1H), 7.13–7.21 (m, 3H), 7.30–
7.38 (m, 2H), 7.53 (d, J=1.7 Hz, 1H), 7.81 (d, J=8.6 Hz, 1H),
7.86 ppm (s, 1H); 13C NMR (75 MHz, CDCl3): d=106.8 (C), 108.3
(CH), 114.7 (C), 117.9 (CH), 119.3 (2CH), 122.5 (CH), 123.2 (CH),
129.7 (2CH), 131.1 (C), 134.4 (CH), 140.5 (C), 142.0 (C), 142.8 ppm
(C); HRMS (ESI): m/z: calcd for C15H11N2S: 251.0637 [M+H+], found:
251.0636.
CDCl3): d=55.9 (CH3), 101.7 (C), 105.0 (CH), 115.5 (C), 116.2 (CH),
123.4 (CH), 128.1 (2CH), 129.4 (2CH), 130.2 (CH), 131.8 (C), 133.2
(C), 139.1 (C), 152.5 (C), 158.8 ppm (C); HRMS (ESI): m/z: calcd for
C16H12NOS: 266.0640 [M+H+], found: 266.0638.
2-Phenyl-6-phenylaminobenzo[b]thiophene-3-carbonitrile (11):
Procedure B starting from 5, Pd(OAc)2 (20 mol%), and PPh3
(40 mol%); 20 h. Yield: 76%; yellow solid; mp: 142–1448C (CH2Cl2/
1
pentane); H NMR (300 MHz, CDCl3): d=5.93 (brs, 1H), 7.04 (t, J=
7.4 Hz, 1H), 7.15–7.20 (m, 3H), 7.31–7.37 (m, 2H), 7.43–7.54 (m,
4H), 7.81 (d, J=8.7 Hz, 1H), 7.83–7.88 ppm (m, 2H); 13C NMR
(75 MHz, CDCl3): d=101.8 (C), 108.1 (CH), 115.5 (C), 118.0 (CH),
119.3 (2CH), 122.5 (CH), 123.5 (CH), 128.1 (2CH), 129.4 (2CH), 129.7
(2CH), 130.2 (CH), 131.9 (C), 133.2 (C), 139.4 (C), 142.1 (C), 142.7 (C),
151.8 ppm (C); HRMS (ESI): m/z: calcd for C21H15N2S: 327.0950 [M+
H+], found: 327.0944.
6-(Pyridin-2-ylamino)benzo[b]thiophene-3-carbonitrile (6): Proce-
dure A starting from 1 (119 mg, 0.5 mmol), pyridin-2-yl amine,
Pd(OAc)2 (5 mol%), and racemic BINAP (10 mol%); 30 h; flash chro-
matography (SiO2, cyclohexane/EtOAc/Et3N 90:10:1). Yield: 55 mg
(47%); yellow solid; mp: 143–1458C (CH2Cl2/pentane); 1H NMR
(300 MHz, CDCl3): d=6.63 (brs, 1H), 6.80–6.86 (m, 2H), 7.36 (dd,
J=2.0, 8.8 Hz, 1H), 7.53–7.59 (dt, J=1.9, 7.5 Hz, 1H), 7.89 (d, J=
8.8 Hz, 1H), 7.96 (s, 1H), 8.28 (dd, J=1.1, 5.1 Hz, 1H), 8.33 ppm (d,
J=2.0 Hz, 1H); 13C NMR (75 MHz, CDCl3): d=106.7 (C), 109.8 (CH),
111.6 (CH), 114.7 (C), 115.9 (CH), 119.4 (CH), 122.8 (CH), 132.1 (C),
135.5 (CH), 137.9 (CH), 139.6 (C), 140.2 (C), 148.2 (CH), 155.4 ppm
(C); HRMS (ESI): m/z: calcd for C14H10N3S: 252.0595 [M+H+], found:
342.0594.
2-Phenyl-6-(pyridin-2-ylamino)benzo[b]thiophene-3-carbonitrile
(12): Procedure B starting from 6, Pd(OAc)2 (10 mol%), and PPh3
(20 mol%); 120 h. Yield: 37%; yellow solid; mp: 157–1598C
(CH2Cl2/cyclohexane); 1H NMR (300 MHz, CDCl3): d=6.79–6.85 (m,
2H), 6.99 (brs, 1H), 7.34 (dd, J=1.9, 8.6 Hz, 1H), 7.43–7.59 (m, 4H),
7.82–7.94 (m, 3H), 8.22 (d, J=1.5 Hz, 1H), 8.28 ppm (d, J=4.5 Hz,
1H); 13C NMR (75 MHz, CDCl3): d=101.7 (C), 109.8 (CH), 111.3 (CH),
115.5 (C), 115.9 (CH), 119.4 (CH), 123.0 (CH), 128.1 (2CH), 129.4
(2CH), 130.3 (CH), 131.8 (C), 134.1 (C), 138.0 (CH), 139.0 (C), 139.5
(C), 148.3 (CH), 152.9 (C), 155.4 ppm (C); HRMS (ESI): m/z: calcd for
C20H14N3S: 328.0903 [M+H+], found: 328.0914.
6-Morpholin-4-ylbenzo[b]thiophene-3-carbonitrile (7): Procedur-
e A1 starting from 1 (238 mg, 1.0 mmol), morpholine, Pd(OAc)2
(5 mol%), and racemic BINAP (7.5 mol%); 18 h; flash chromatogra-
phy (SiO2, cyclohexane/EtOAc/Et3N 85:15:1). Yield: 174 mg (71%);
6-Morpholin-4-yl-2-phenylbenzo[b]thiophene-3-carbonitrile (13):
Procedure B starting from 7, Pd(OAc)2 (10 mol%), PPh3 (20 mol%);
72 h. Yield: 68%; yellow solid; mp: 139–1418C (MeOH); 1H NMR
(300 MHz, CDCl3): d=3.23 (t, J=4.6 Hz, 4H), 3.89 (t, J=4.6 Hz, 4H),
7.15 (dd, J=1.9, 8.9 Hz, 1H), 7.23 (d, J=1.3 Hz, 1H), 7.42–7.53 (m,
3H), 7.79 (d, J=8.9 Hz, 1H), 7.83–7.87 ppm (m, 2H); 13C NMR
(75 MHz, CDCl3): d=49.6 (2CH2), 66.8 (2CH2), 101.6 (C), 107.2 (CH),
115.5 (C), 116.7 (CH), 123.0 (CH), 128.0 (2CH), 129.3 (2CH), 130.1
(CH), 131.9 (C), 132.5 (C), 139.4 (C), 150.3 (C), 151.8 ppm (C); HRMS
(ESI): m/z: calcd for C19H17N2OS: 321.1056 [M+H+], found:
321.1055.
1
white solid; mp: 145–1478C (MeOH); H NMR (300 MHz, CDCl3): d=
3.25 (t, J=4.9 Hz, 4H), 3.92 (t, J=4.9 Hz, 4H), 7.21 (dd, J=8.9,
1.7 Hz, 1H), 7.34 (d, J=1.7 Hz, 1H), 7.85 (d, J=8.9 Hz, 1H),
7.90 ppm (s, 1H); 13C NMR (75 MHz, CDCl3): d=49.6 (2CH2), 66.7
(2CH2), 106.6 (C), 107.6 (C), 114.6 (C), 116.8 (CH), 122.8 (CH), 130.6
(C), 134.6 (CH), 140.5 (CH), 150.1 ppm (C); HRMS (ESI): m/z: calcd
for C13H13N2OS: 245.0749 [M+H+], found: 245.0748.
4-Morpholin-4-ylbenzo[b]thiophene-3-carbonitrile (8): Procedur-
e A1 starting from 4 (238 mg, 1.0 mmol), morpholine, Pd(OAc)2
(5 mol%), and racemic BINAP (7.5 mol%); 18 h; flash chromatogra-
phy (SiO2, cyclohexane/EtOAc/Et3N 85:15:1). Yield: 150 mg (61%);
4-Morpholin-4-yl-2-phenylbenzo[b]thiophene-3-carbonitrile (14):
Procedure B starting from 8; 48 h. Yield: 86%; white solid; mp:
160–1628C (CH2Cl2/pentane); 1H NMR (300 MHz, CDCl3): d=3.14
(brs, 4H), 4.01 (brs, 2H), 4.10 (brs, 2H), 7.18 (d, J=7.8 Hz, 1H),
7.41 (t, J=7.8 Hz, 1H), 7.48–7.55 (m, 3H), 7.58 (d, J=7.8 Hz, 1H),
7.83–7.86 ppm (m, 2H); 13C NMR (75 MHz, CDCl3): d=53.9 (2CH2),
66.2 (2CH2), 101.1 (C), 116.3 (CH), 117.0 (C), 118.1 (CH), 126.8 (CH),
129.0 (2CH), 129.3 (2CH), 130.4 (CH), 131.7 (C), 133.5 (C), 139.3 (C),
149.3 (C), 156.3 ppm (C); HRMS (ESI): m/z: calcd for C19H17N2OS:
321.1056 [M+H+], found: 321.1055.
1
white solid; mp: 149–1518C (MeOH); H NMR (300 MHz, CDCl3): d=
3.10 (brs, 4H), 5.03 (brs, 4H), 7.16 (d, J=7.5 Hz, 1H), 7.42 (dd, J=
7.5, 7.9 Hz, 1H), 7.62 (d, J=7.9 Hz, 1H), 8.12 ppm (s, 1H); 13C NMR
(75 MHz, CDCl3): d=53.8 (2CH2), 66.3 (2CH2), 105.9 (C), 116.0 (CH),
116.4 (C), 118.6 (CH), 127.1 (CH), 131.8 (C), 139.0 (CH), 140.5 (C),
149.3 ppm (C); HRMS (ESI): m/z: calcd for C13H13N2OS: 245.0749
[M+H+], found: 245.0748.
6-(4-Methoxyphenyl)-2-phenylbenzo[b]thiophene-3-carbonitrile
(9): Procedure B starting from 2; 48 h. Yield: 71%; yellow solid;
mp: 178–1808C (CH2Cl2/pentane); 1H NMR (300 MHz, CDCl3): d=
3.87 (s, 3H, OMe), 7.01 (d, J=8.5 Hz, 2H), 7.51–7.61 (m, 4H), 7.71
(d, J=8.5 Hz, 2H), 7.89–7.99 ppm (m, 4H); 13C NMR (75 MHz,
CDCl3): d=55.5 (CH3), 101.9 (C), 114.6 (2CH), 115.3 (C), 120.0 (CH),
122.8 (CH), 125.6 (CH), 128.3 (2CH), 128.5 (2CH), 129.5 (2CH), 130.5
(CH), 131.7 (C), 132.7 (C), 138.0 (C), 138.3 (C), 139.4 (C), 154.8 (C),
159.7 ppm (C); HRMS (ESI): m/z: calcd for C22H16NOS: 342.0947
[M+H+], found: 342.0948.
6-(4-Methoxyphenyl)-2-phenylbenzo[b]thiophene-3-carboxamide
(15): 3m NaOH(aq) (2 mL) was added to a stirred solution of 9
(210 mg, 0.61 mmol) in dioxane (20 mL), and the mixture was
heated at reflux for 4 days. After cooling, water (40 mL) was added
to the mixture, and the resulting solution was neutralized with 1 n
HCl(aq). After extraction with CH2Cl2 (3”20 mL), the organic layer
was dried (MgSO4) and concentrated to dryness to give 15
(195 mg, 89%) as a white solid; mp: >2108C (CH2Cl2); 1H NMR
(300 MHz, [D6]DMSO): d=3.81 (s, 3H, OMe), 7.06 (d, J=8.7 Hz, 2H),
7.41–7.53 (m, 3H), 7.67–7.76 (m, 5H), 7.82 (d, J=8.5 Hz, 1H), 7.95
(brs, 2H), 8.29 ppm (d, J=1.1 Hz, 1H); 13C NMR (75 MHz,
[D6]DMSO): d=55.2 (CH3), 114.5 (2CH), 119.3 (CH), 123.3 (CH), 123.7
(CH), 128.0 (2CH), 128.2 (2CH), 128.8 (CH), 128.9 (2CH), 130.2 (C),
131.9 (C), 133.1 (C), 136.8 (C), 137.5 (C), 138.8 (C), 140.1 (C), 159.1
6-Methoxy-2-phenylbenzo[b]thiophene-3-carbonitrile (10): Pro-
cedure B starting from 3; 48 h. Yield: 84%; white solid; mp: 131–
1
1338C (CH2Cl2/pentane); H NMR (300 MHz, CDCl3): d=3.91 (s, 3H,
OMe), 7.14 (dd, J=2.3 and 8.9 Hz, 1H), 7.31 (d, J=2.3 Hz, 1H),
7.47–7.55 (m, 3H), 7.83–7.88 ppm (m, 3H); 13C NMR (75 MHz,
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(C), 166.9 ppm (C); HRMS (ESI): m/z: calcd for C22H18NO2S: 360.1058
[M+H+], found: 360.1058.
138.1 (C), 139.2 (C), 149.5 (C), 168.4 ppm (C); HRMS (ESI): m/z: calcd
for C19H19N2O2S: 339.1167 [M+H+], found: 339.1169.
6-(4-Methoxyphenyl)-2-phenylbenzo[b]thiophene-3-carboxylic
acid (16): Procedure C starting from 9. Yield: 95%; yellow solid;
mp: 183–1858C (CH2Cl2); H NMR (300 MHz, [D6]DMSO): d=3.81 (s,
{6-(4-Methoxyphenyl)-2-phenylbenzo[b]thiophen-3-yl}methanol
(22): Procedure D starting from 16. Yield: 79%; white solid; mp:
154–1568C (CH2Cl2/pentane); H NMR (300 MHz, CDCl3): d=3.87 (s,
1
1
3H, OMe), 7.06 (d, J=8.7 Hz, 1H), 7.46–7.53 (m, 3H), 7.56–7.60 (m,
2H), 7.74 (d, J=8.7 Hz, 2H), 7.79 (dd, J=1.7, 8.7 Hz, 1H), 8.28 (d,
J=8.7 Hz, 1H), 8.31 (d, J=1.3 Hz, 1H), 13.02 ppm (brs, 1H);
13C NMR (75 MHz, [D6]DMSO): d=55.3 (CH3), 114.6 (2CH), 119.4
(CH), 124.0 (C), 124.2 (CH), 124.6 (CH), 128.3 (C), 128.5 (2CH), 129.1
(CH), 129.3 (2CH), 131.9 (C), 133.6 (C), 136.9 (C), 137.2 (C), 138.9 (C),
149.3 (C), 159.2 (C), 165.0 ppm (C); HRMS (ESI): m/z: calcd for
C22H15O3S: 359.0742 [MÀHÀ], found: 359.0743.
3H, OMe), 4.90 (s, 2H), 7.01 (d, J=8.3 Hz, 2H), 7.42–7.51 (m, 3H),
7.58–7.63 (m, 5H), 7.94 (d, J=8.1 Hz, 1H), 8.00 ppm (s, 1H);
13C NMR (75 MHz, CDCl3): d=55.5 (CH3), 57.1 (CH2), 114.4 (2CH),
120.0 (CH), 122.6 (CH), 124.1 (CH), 128.4 (2CH), 128.6 (CH), 128.9
(2CH), 129.8 (2CH), 130.2 (C), 133.5 (C), 133.8 (C), 137.7 (C), 138.8
(C), 140.0 (C), 143.1 (C), 159.3 ppm (C); HRMS (ESI): m/z: calcd for
C22H18NaO2S: 369.0920 [M+Na+], found: 369.0917.
(2-Phenyl-6-phenylaminobenzo[b]thiophen-3-yl)methanol (23):
Procedure D starting from 18. Yield: 63%; gray solid; mp: 98–
6-Methoxy-2-phenylbenzo[b]thiophene-3-carboxylic acid (17):
Procedure C starting from 10. Yield: 100%; pale brown solid; mp:
1
1008C (CH2Cl2/pentane); H NMR (300 MHz, CDCl3): d=4.62 (d, J=
1
187–1898C (CH2Cl2); H NMR (300 MHz, [D6]DMSO): d=3.84 (s, 3H,
5.1 Hz, 2H), 5.26 (t, J=5.1 Hz, 1H), 6.85 (t, J=7.4 Hz, 1H), 7.11–7.15
(m, 2H), 7.18 (dd, J=2.1, 8.6 Hz, 1H), 7.23–7.29 (m, 2H), 7.39–7.43
(m, 1H), 7.48–7.53 (m, 2H), 7.60–7.65 (m, 3H), 7.83 (d, J=8.7 Hz,
1H), 8.38 ppm (brs, 1H); 13C NMR (75 MHz, CDCl3): d=55.1 (CH2),
116.4 (CH), 116.8 (2CH), 119.9 (CH), 123.7 (CH), 128.1 (CH), 128.9
(2CH), 129.1 (2CH), 129.3 (2CH), 131.7 (C), 133.8 (C), 134.0 (C),
136.9 (C), 139.6 (C), 141.0 (C), 143.4 ppm (C); HRMS (ESI): m/z: calcd
for C22H18NOS: 332.1104 [M+H+], found: 332.1105.
OMe), 7.11 (dd, J=2.2, 9.1 Hz, 1H), 7.42–7.50 (m, 3H), 7.51–7.56 (d,
J=2.2 Hz, 1H), 8.11 (d, J=9.1 Hz, 1H), 12.98 ppm (brs, 1H);
13C NMR (75 MHz, [D6]DMSO): d=55.5 (CH3), 104.7 (CH), 115.4 (CH),
123.7 (C), 125.0 (CH), 128.4 (2CH), 128.7 (CH), 129.2 (2CH), 132.2
(C), 133.7 (C), 139.4 (C), 146.5 (C), 157.4 (C), 165.0 ppm (C); HRMS
(ESI): m/z: calcd for C16H13O3S: 285.0580 [M+H+], found: 285.0583.
2-Phenyl-6-phenylaminobenzo[b]thiophene-3-carboxylic
acid
(18): Procedure C starting from 11. Yield: 85%; brown solid; mp:
>2108C (CH2Cl2); 1H NMR (300 MHz, [D6]DMSO): d=6.88 (t, J=
7.4 Hz, 1H), 7.14–7.30 (m, 5H), 7.43–7.53 (m, 5H), 7.64 (s, 1H), 8.06
(d, J=8.9 Hz, 1H), 8.45 (brs, 1H), 12.91 ppm (brs, 1H); 13C NMR
(75 MHz, [D6]DMSO): d=107.0 (CH), 117.1 (CH), 117.3 (2CH), 120.3
(CH), 123.9 (C), 124.8 (CH), 128.4 (2CH), 128.6 (CH), 129.1 (2CH),
129.3 (2CH), 131.6 (C), 133.8 (C), 139.5 (C), 141.4 (C), 142.9 (C),
145.1 (C), 165.1 ppm (C); HRMS (ESI): m/z: calcd for C21H14NO2S:
344.0745 [MÀHÀ], found: 344.0740.
(6-Morpholin-4-yl-2-phenylbenzo[b]thiophen-3-yl)methanol (24):
Procedure D starting from 20. Yield: 81%; white solid; mp: 153–
1558C (CH2Cl2/pentane); 1H NMR (300 MHz, [D6]acetone): d=3.22
(t, J=4.9 Hz, 4H), 3.82 (t, J=4.9 Hz, 4H), 4.23 (t, J=5.2 Hz, 1H),
4.78 (d, J=5.2 Hz, 2H), 7.19 (dd, J=2.3, 8.9 Hz, 1H), 7.38–7.44 (m,
2H), 7.46–7.52 (m, 2H), 7.65–7.69 (m, 2H), 7.88 ppm (d, J=8.9 Hz,
1H); 13C NMR (75 MHz, [D6]acetone): d=50.6 (2CH2), 56.7 (CH2),
67.4 (2CH2), 108.0 (CH), 116.1 (CH), 124.2 (CH), 128.9 (CH), 129.6
(2CH), 130.3 (CH), 132.4 (C), 135.0 (C), 135.3 (C), 139.0 (C), 141.4 (C),
150.5 ppm (C); HRMS (ESI): m/z: calcd for C19H20NO2S: 326.1209
[M+H+], found: 326.1211.
2-Phenyl-6-(pyridin-2-ylamino)benzo[b]thiophene-3-carboxylic
acid (19): Procedure C starting from 12. Yield: 88%; brown solid;
1
mp: 120–1228C (CH2Cl2); H NMR (300 MHz, [D6]DMSO): d=6.80 (t,
(6-Methoxy-2-phenylbenzo[b]thiophen-3-yl)methanol (25): Pro-
cedure D starting from 17. Yield: 75%; white solid; mp: 139–1418C
J=6.3 Hz, 1H), 6.91 (d, J=8.3 Hz, 1H), 7.44–7.63 (m, 7H), 8.09 (d,
J=8.9 Hz, 1H), 8.23 (d, J=4.0 Hz, 1H), 8.72 (s, 1H), 9.35 (brs, 1H),
12.94 ppm (brs, 1H); 13C NMR (75 MHz, [D6]DMSO): d=108.8 (CH),
111.2 (CH), 114.7 (CH), 117.6 (CH), 123.9 (C), 124.2 (CH), 128.4 (2CH),
128.6 (CH), 129.2 (2CH), 131.9 (C), 133.8 (C), 137.4 (CH), 139.0 (C),
139.5 (C), 145.6 (C), 147.2 (CH), 155.6 (C), 165.1 ppm (C); HRMS
(ESI): m/z: calcd for C20H15N2O2S: 347.0849 [M+H+], found:
347.0840.
1
(CH2Cl2/pentane); H NMR (300 MHz, CDCl3): d=3.89 (s, 3H, OMe),
4.85 (s, 2H), 7.05 (dd, J=2.3, 8.9 Hz, 1H), 7.32 (d, J=2.3 Hz, 1H),
7.37–7.49 (m, 3H), 7.56–7.60 (m, 2H), 7.81 ppm (d, J=8.9 Hz, 1H);
13C NMR (75 MHz, CDCl3): d=55.8 (CH3), 57.2 (CH2), 105.0 (CH),
114.6 (CH), 123.2 (CH), 128.4 (CH), 128.9 (2CH), 129.7 (2CH), 130.0
(C), 133.9 (C), 134.1 (C), 140.5 (C), 140.7 (C), 157.7 ppm (C); HRMS
(ESI): m/z: calcd for C16H14NaO2S: 293.0607 [M+Na+], found:
293.0613.
6-Morpholin-4-yl-2-phenylbenzo[b]thiophene-3-carboxylic acid
(20): Procedure C starting from 13. Yield: 98%; brown solid; mp:
6-(4-Methoxyphenyl)-2-phenylbenzo[b]thiophene-3-carbalde-
hyde (26): Procedure E starting from 22; flash chromatography
(SiO2, cyclohexane/EtOAc 90:10). Yield: 75%; yellow solid; mp:
1
184–1868C (CH2Cl2); H NMR (300 MHz, [D6]DMSO): d=3.20 (t, J=
4.7 Hz, 4H), 3.77 (t, J=4.7 Hz, 4H), 7.23 (dd, J=2.3, 9.1 Hz, 1H),
7.40–7.54 (m, 6H), 8.06 (d, J=9.1 Hz, 1H), 12.86 ppm (brs, 1H);
13C NMR (75 MHz, [D6]DMSO): d=48.7 (2CH2), 66.0 (2CH2), 106.4
(CH), 115.5 (CH), 123.8 (C), 124.5 (CH), 128.3 (2CH), 128.6 (CH),
129.1 (2CH), 131.2 (C), 133.8 (C), 139.6 (C), 145.4 (C), 149.0 (C),
165.1 ppm (C); HRMS (ESI): m/z: calcd for C19H18NO3S: 340.1007
[M+H+], found: 340.1008.
1
192–1948C (CH2Cl2/pentane); H NMR (300 MHz, CDCl3): d=3.87 (s,
3H), 7.01 (d, J=8.9 Hz, 2H), 7.52–754 (m, 3H), 7.57–7.63 (m, 4H),
7.71 (dd, J=1.6, 8.5 Hz, 1H), 7.99 (d, J=1.6 Hz, 1H), 8.79 (d, J=
8.5 Hz, 1H), 10.07 ppm (s, 1H); 13C NMR (75 MHz, CDCl3): d=55.5
(CH3), 114.5 (2CH), 119.3 (CH), 125.4 (CH), 125.7 (CH), 128.5 (2CH),
129.1 (2CH), 130.1 (C), 130.1 (CH), 130.7 (2CH), 131.8 (C), 133.1 (C),
135.8 (C), 138.9 (C), 159.6 (C), 159.6 (C), 160.7 (C), 186.9 ppm (CHO);
HRMS (ESI): m/z: calcd for C22H17O2S: 345.0949 [M+H+], found:
345.0948.
4-Morpholin-4-yl-2-phenylbenzo[b]thiophene-3-carboxamide
(21): Procedure C starting from 14. Yield: 94%; pale brown solid;
1
mp: >2108C (CH2Cl2); H NMR (300 MHz, [D6]DMSO): d=2.90 (brs,
4H), 3.75 (brs, 4H), 7.28 (d, J=7.5 Hz, 1H), 7.36–7.50 (m, 4H), 7.70
(brs, 1H), 7.74–7.78 ppm (m, 3H); 13C NMR (75 MHz, [D6]DMSO):
d=53.5 (2CH2), 65.9 (2CH2), 117.8 (CH), 118.8 (CH), 125.7 (CH),
128.4 (2CH), 128.5 (CH), 128.8 (2CH), 130.9 (C), 133.4 (C), 135.3 (C),
6-Morpholin-4-yl-2-phenylbenzo[b]thiophene-3-carbaldehyde
(27): 15 wt.% Dess–Martin periodinane solution in CH2Cl2 (600 mL,
0.291 mmol) was added to a stirred solution of 24 (62 mg,
0.191 mmol) in CH2Cl2 (7 mL). The mixture was stirred at room tem-
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perature for 6 h and then partitioned between H2O (20 mL) and
CH2Cl2 (15 mL). Then, a saturated aqueous solution of Na2S2O3
(5 mL) and a saturated aqueous solution of NaHCO3 (1 mL) were
successively added. The organic layer was separated, and the aque-
ous layer was extracted with CH2Cl2 (2”15 mL). The combined ex-
tract was dried (MgSO4), filtered, and concentrated under reduced
pressure. The resulting residue was purified by flash chromatogra-
phy (SiO2, cyclohexane/EtOAc 80:20) to give 27 (27 mg, 44%) as
hydrous CH2Cl2 (3.8 mL). The mixture was stirred for 4 h at room
temperature and then partitioned between H2O (20 mL) and
CH2Cl2 (20 mL). The organic layer was separated, and the aqueous
layer was extracted with CH2Cl2 (2”20 mL). The combined extract
was dried (MgSO4), filtered, and concentrated under reduced pres-
sure. The resulting residue was purified by flash chromatography
(SiO2, cyclohexane/EtOAc 70:30) to give 32 (67 mg, 71%) as a pale
yellow solid; mp: 175–1778C (CH2Cl2); 1H NMR (300 MHz,
[D6]acetone): d=7.16 (dd, J=2.1, 8.7 Hz, 1H), 7.46 (d, J=1.9 Hz,
1H), 7.49–7.60 (m, 3H), 7.75 (d, J=8.7 Hz, 1H), 7.85 (d, J=6.6 Hz,
2H), 9.03 ppm (brs, 1H); 13C NMR (75 MHz, [D6]acetone): d=102.4
(C), 108.5 (CH), 115.6 (C), 117.3 (CH), 123.9 (CH), 128.7 (2CH), 130.2
(2CH), 131.0 (CH), 132.6 (C), 132.8 (C), 140.0 (C), 152.1 (C),
157.6 ppm (C); HRMS (ESI): m/z: calcd for C15H9NNaOS: 274.0297
[M+Na+], found: 274.098.
a
yellow solid; mp: 134–1368C (CH2Cl2/pentane); 1H NMR
(300 MHz, CDCl3): d=3.25 (t, J=4.8 Hz, 4H), 3.91 (t, J=4.8 Hz, 4H),
7.16 (dd, J=2.3, 9.0 Hz, 1H), 7.27 (d, J=2.3 Hz, 1H), 7.46–7.53 (m,
3H), 7.54–7.59 (m, 2H), 8.62 (d, J=8.9 Hz, 1H), 10.00 ppm (s, 1H);
13C NMR (75 MHz, CDCl3): d=49.8 (2CH2), 67.0 (2CH2), 106.7 (CH),
116.8 (CH), 125.6 (CH), 129.0 (2CH), 129.9 (CH), 130.1 (C), 130.5
(CH), 130.6 (2CH), 131.9 (C), 139.9 (C), 150.0 (C), 158.2 (C),
187.0 ppm (CHO); HRMS (ESI): m/z: calcd for C19H18NO2S: 324.1058
[M+H+], found: 324.1060.
(6-Hydroxy-2-phenylbenzo[b]thiophen-3-yl)methanol (34): Under
an argon atmosphere, pure BBr3 (125 mL, 1.33 mmol) was slowly
added to a stirred solution of 16 (188 mg, 0.66 mmol) in anhydrous
CH2Cl2 (5 mL). The mixture was stirred at room temperature for
18 h and was then partitioned between H2O (25 mL) and CH2Cl2
(25 mL). The organic layer was separated, and the aqueous layer
was extracted with CH2Cl2 (2”20 mL). The combined extract was
dried (MgSO4), filtered, and concentrated under reduced pressure.
The crude product (128 mg, 0.473 mmol) was dissolved in anhy-
drous THF (4.8 mL) and then 1m BH3 in THF (950 mL, 0.95 mmol)
was added under an argon atmosphere. After heating at reflux for
18 h, the mixture was cooled and partitioned between H2O (25 mL)
and CH2Cl2 (25 mL). The organic layer was separated, and the aque-
ous layer was extracted with CH2Cl2 (2”20 mL). The combined ex-
tract was dried (MgSO4), filtered, and concentrated under reduced
pressure. The resulting residue was purified by flash chromatogra-
phy (SiO2, cyclohexane/EtOAc 70:30) to give 34 (70 mg, 50% over
two steps) as a white solid; mp: 166–1688C (CH2Cl2/pentane);
1H NMR (300 MHz, [D4]MeOH): d=4.90 (s, 2H), 6.97 (dd, J=2.3,
8.7 Hz, 1H), 7.24 (d, J=2.1 Hz, 1H), 7.38–7.50 (m, 3H), 7.60–7.64
(m, 2H), 7.81 ppm (d, J=8.7 Hz, 1H); 13C NMR (75 MHz, [D4]MeOH):
d=56.9 (CH2), 107.9 (CH), 115.4 (CH), 124.5 (CH), 129.2 (CH), 129.7
(2CH), 130.6 (2CH), 131.5 (C), 135.0 (C), 135.5 (C), 140.2 (C), 141.8
(C), 156.5 ppm (C).
6-Methoxy-2-phenylbenzo[b]thiophene-3-carbaldehyde (28): Pro-
cedure E starting from 25; flash chromatography (SiO2, cyclohex-
ane/EtOAc 90/10). Yield: 91%; white solid; mp: 121–1238C
1
(MeOH); H NMR (300 MHz, CDCl3): d=3.91 (s, 3H, OMe), 7.13 (dd,
J=2.4, 9.0 Hz, 1H), 7.31 (d, J=2.3 Hz, 1H), 7.49–7.54 (m, 3H), 7.55–
7.61 (m, 2H), 8.66 (d, J=8.9 Hz, 1H), 10.02 ppm (s, 1H); 13C NMR
(75 MHz, CDCl3): d=55.7 (CH3), 104.4 (CH), 115.8 (CH), 126.0 (CH),
129.0 (2CH), 129.9 (CH), 130.0 (C), 130.6 (2CH), 131.1 (C), 131.8 (C),
139.6 (C), 158.4 (C), 158.5 (C), 186.9 ppm (CHO); HRMS (ESI): m/z:
calcd for C16H13O2S: 269.0631 [M+H+], found: 269.0632.
trans-6-(4-Methoxyphenyl)-2-phenylbenzo[b]thiophene-3-carbal-
dehyde oxime (29): Procedure G starting from 26. Yield: 91%; pale
yellow solid; mp: 194–1968C (CH2Cl2); 1H NMR (400 MHz,
[D6]DMSO): d=3.82 (s, 3H, OMe), 7.06 (d, J=6.4 Hz, 2H), 7.51–7.58
(m, 5H), 7.75 (d, J=6.4 Hz, 2H), 7.80 (dd, J=1.1, 6.4 Hz, 1H), 8.2 (s,
1H), 8.31 (d, J=1.1 Hz, 1H), 8.60 (d, J=6.4 Hz, 1H), 11.40 ppm (s,
1H); 13C NMR (100 MHz, [D6]DMSO): d=55.2 (CH3), 114.5 (2CH),
119.4 (CH), 123.9 (C), 124.0 (CH), 125.5 (CH), 128.0 (2CH), 129.1
(2CH), 129.2 (CH), 129.8 (2CH), 131.9 (C), 132.6 (C), 135.7 (C), 137.0
(C), 138.9 (C), 144.0 (CH), 145.1 (C), 159.1 ppm (C); HRMS (ESI): m/z:
calcd for C22H18NO2S: 360.1053 [M+H+], found: 360.1052.
trans-6-Morpholin-4-yl-2-phenylbenzo[b]thiophene-3-carbalde-
hyde oxime (30): Procedure G starting from 27. Yield: 67%; pale
brown solid; mp: 186–1888C (MeOH); 1H NMR (300 MHz,
[D6]acetone): d=3.25 (t, J=4.9 Hz, 4H), 3.82 (t, J=4.9 Hz, 4H), 7.20
(dd, J=2.2, 9.1 Hz, 1H), 7.44 (d, J=2.2 Hz, 1H), 7.47–7.55 (m, 5H),
8.26 (s, 1H), 8.48 (d, J=9.1 Hz, 1H), 10.35 ppm (s, 1H); 13C NMR
(75 MHz, [D6]acetone): d=50.2 (2CH2), 67.4 (2CH2), 107.6 (CH),
116.3 (CH), 125.1 (C), 126.9 (CH), 129.6 (CH), 129.8 (2CH), 130.8
(2CH), 131.4 (C), 134.3 (C), 141.3 (C), 143.7 (C), 145.7 (CH),
150.7 ppm (C).
5,6-Dibromobenzo[b]thiophene-3-carboxylic acid methyl ester
(35): N-Bromosuccinimide (979 mg, 5.5 mmol) was added to
a stirred solution of benzo[b]thiophene-3-carboxylic acid methyl
ester (435 mg, 2.5 mmol) in CH2Cl2 (25 mL); this was followed by
the addition of trifluoroacetic acid (2.5 mL) and concentrated
H2SO4 (0.25 mL). The resulting mixture was stirred at room temper-
ature for 24 h and was then partitioned between H2O (50 mL) and
CH2Cl2 (50 mL). The organic layer was separated, and the aqueous
layer was extracted with CH2Cl2 (2”25 mL). The combined organic
extract was dried (MgSO4), filtered, and concentrated under re-
duced pressure. The resulting residue was purified by flash chro-
matography (SiO2, cyclohexane/EtOAc 93:7) to give 35 (350 mg,
40%) as a white solid; mp: 130–1328C (MeOH).1H NMR (300 MHz,
CDCl3): d=3.94 (s, 3H, OMe), 7.97 (s, 1H), 8.33 (s, 1H), 8.82 ppm (s,
1H); 13C NMR (75 MHz, CDCl3): d=52.14 (CH3), 121.52 (C), 122.47
(C), 126.23 (C), 126.74 (CH), 129.19 (CH), 137.11 (C), 138.24 (CH),
139.87 (C), 162.58 ppm (C).
trans-6-Methoxy-2-phenylbenzo[b]thiophene-3-carbaldehyde
oxime (31): Procedure G starting from 28. Yield: 79%; pale brown
1
solid; mp: 175–1778C (MeOH); H NMR (300 MHz, CDCl3): d=3.90
(s, 3H, OMe), 7.07 (dd, J=2.3, 8.9 Hz, 1H), 7.30 (d, J=2.3 Hz, 1H),
7.42–7.50 (m, 5H), 7.52 (brs, 1H), 8.33 (s, 1H), 8.49 ppm (d, J=
9.0 Hz, 1H); 13C NMR (75 MHz, CDCl3): d=55.8 (CH3), 104.6 (CH),
115.0 (CH), 123.4 (C), 126.5 (CH), 128.9 (2CH), 128.9 (C), 130.3
(2CH), 131.5 (C), 133.2 (C), 140.3 (C), 145.4 (C), 147.1 (CH),
157.9 ppm (C); HRMS (ESI): m/z: calcd for C16H14NO2S: 284.0745
[M+H+], found: 284.0744.
5,6-Dimethoxybenzo[b]thiophene-3-carboxylic acid methyl ester
(36): A test tube was charged with 35 (326 mg, 0.93 mmol) and
CuBr (36 mg, 0.22 mmol). Then, a freshly prepared solution of 2m
NaOMe in MeOH (10 mL, 20 mmol), Cu (118 mg, 1.86 mmol) and
DMF (2 mL) were added. The tube was sealed and heated at 1208C
for 9 days. After cooling, H2O (40 mL) was added, and the mixture
6-Hydroxy-2-phenylbenzo[b]thiophene-3-carbonitrile (32): Under
an argon atmosphere, 1m BBr3 in CH2Cl2 (760 mL, 0.76 mmol) was
slowly added to a stirred solution of 10 (100 mg, 0.38 mmol) in an-
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was acidified to pH 2 with 1m HCl(aq) solution. The reaction mixture
was extracted with CH2Cl2 (2”40 mL), and the combined organic
extracts were dried (MgSO4), filtrated, and concentrated. A solution
of the crude product in anhydrous MeOH (10 mL), concentrated
H2SO4 (100 mL) was heated at reflux for 24 h. After cooling, the mix-
ture was partitioned between H2O (20 mL) and CH2Cl2 (20 mL), the
organic layer was separated, and the aqueous layer was extracted
with CH2Cl2 (2”20 mL). The combined organic layers were dried
(MgSO4), filtered, and concentrated under reduced pressure. The
resulting residue was purified by flash chromatography (SiO2, cy-
clohexane/EtOAc 95:5) to give 36 (100 mg, 43%) as a white solid;
(100 mL) were dispensed in a 96-well microplate after twofold serial
dilutions in MH by using a Biomek 2000 Workstation (Beckman
Coulter, Villepinte, France). The bacterial inoculum (100 mL, pre-
pared as describe above) was then added in each well. Growth of
bacterial strains was measured by using a DTX 880 microplate
reader (Beckman Coulter, Villepinte, France) by monitoring absorp-
tion at l=620 nm after 0, 2, 4, 6, and 24 h incubation at 378C. All
tested compounds were dissolved in DMSO at a concentration of
10 mgmLÀ1 before dilution in MH for use in MIC determination.
MIC was defined as the minimal concentration of the tested com-
pound that completely inhibited cell growth during 24 h incuba-
tion at 378C. Benzothiophene compounds were first tested at final
concentrations ranging from 0.125 to 128 mgLÀ1. The highest final
DMSO concentration used (2.56% v/v) induced no significant bac-
terial growth inhibition. MIC determination of ciprofloxacin
(16 mgmLÀ1 for SA-1199B and 0.25 mgmLÀ1 for SA-ATCC 25923)
was performed as a control on each microplate. The accepted var-
iance on the MIC value was estimated to a twofold difference
owing to the microdilution method used.
1
mp: 118–1208C (MeOH); H NMR (300 MHz, CDCl3): d=3.92 (s, 3H,
OMe), 3.94 (s, 3H, OMe), 3.99 (s, 3H, OMe), 7.24 (s, 1H), 8.06 (s,
1H), 8.17 ppm (s, 1H); 13C NMR (75 MHz, CDCl3): d=51.7 (CH3), 56.1
(CH3), 56.2 (CH3), 103.6 (CH), 105.6 (CH), 126.3 (C), 130.9 (C), 132.7
(C), 134.2 (CH), 148.7 (C), 149.0 (C), 163.5 ppm (C); HRMS (ESI): m/z:
calcd for C12H12NaO4S: 275.0349 [M+Na+], found: 275.0352.
5,6-Dimethoxy-2-phenylbenzo[b]thiophene-3-carboxylic
acid
methyl ester (37): Procedure B starting from 36, Pd(OAc)2
(10 mol%), and PPh3 (20 mol%); 20 days. Yield: 33%; colorless
oil.1H NMR (300 MHz, CDCl3): d=3.72 (s, 3H, OMe), 3.96 (s, 3H,
OMe), 4.00 (s, 3H, OMe), 7.23 (s, 1H), 7.39–7.43 (m, 3H), 7.45–7.50
(m, 2H), 7.87 ppm (s, 1H); 13C NMR (75 MHz, CDCl3): d=51.5 (CH3),
56.2 (2CH3), 103.0 (CH), 105.9 (CH), 122.2 (C), 128.2 (2CH), 128.7
(CH), 129.6 (2CH), 131.5 (C), 132.6 (C), 134.4 (C), 148.7 (C), 148.9 (C),
150.2 (C), 164.7 ppm (C); HRMS (ESI): m/z: calcd for C18H16NaO4S:
351.0662 [M+Na+], found: 351.0659.
Ethidium bromide accumulation assays: The accumulation of ethid-
ium bromide by SA cells with or without inhibitors was determined
by fluorimetry. SA strains were grown overnight in MH broth, dilut-
ed 20-fold in fresh, pre-warmed MH broth, and were grown in
a shaking water bath at 378C under agitation (130 rpm) over 3.5 h,
which corresponds to the mid-log phase of growth. Samples
(10 mL) were aseptically withdrawn from the culture, bacteria were
harvested by centrifugation (4000 g, 10 min), the supernatant was
thrown, and bacterial cells were resuspended in fresh sterile pre-
warmed MH broth containing EtBr (20 mL, final concentration,
5 mgLÀ1) and incubated again in a shaking water bath at 378C
under agitation (130 rpm) for 30 min. Aliquots (1 mL) were taken at
fixed intervals (0, 5, 10, 15, 20, and 25 min) for fluorescence mea-
surement (lex =530 nm, lem =600 nm). After 10 min, tested com-
pounds were added (final concentration 20 mgLÀ1) and fluores-
cence was measured for an additional 15 min. Experiments were
performed in duplicate. The fluorescence intensity difference be-
tween the compound containing assay and the fluorescence base-
line before addition was indicative of the ability of the compound
to inhibit the efflux of EtBr. Reserpine was used as a positive con-
trol.
5,6-Dimethoxy-2-phenylbenzo[b]thiophene-3-carboxylic
acid
(38): 3m LiOH in H2O (0.3 mL) was added to a stirred solution of
37 (30 mg, 0.09 mmol) in THF (1 mL), and the mixture was heated
at reflux for 4 days. After cooling, water (10 mL) was added to the
mixture, and the resulting solution was acidified with 1 n HCl(aq)
until pH 3. After extraction with CH2Cl2 (3”10 mL), the organic
layer was dried (MgSO4) and concentrated to dryness to give 38
(29 mg, 100%) as a pale brown solid; mp: 203–2058C (CH2Cl2);
1H NMR (400 MHz, [D6]DMSO): d=3.83 (s, 3H, OMe), 3.85 (s, 3H,
OMe), 7.40–7.48 (m, 3H), 7.50–7.54 (m, 2H), 7.60 (s, 1H), 7.74 (s,
1H), 12.82 ppm (brs, 1H); 13C NMR (100 MHz, [D6]DMSO): d=55.5
(CH3), 55.8 (CH3), 104.0 (CH), 105.5 (CH), 123.4 (C), 128.3 (2CH),
128.6 (CH), 129.2 (2CH), 130.7 (C), 131.9 (C), 133.9 (C), 147.3 (C),
148.4 (C), 148.4 (C), 165.0 ppm (C); HRMS (ESI): m/z: calcd for
C17H15O4S: 315.0691 [M+H+], found: 315.0692.
Acknowledgements
SA-1199 and SA-1199B were kindly provided by Glenn W. Kaatz
(John D. Dingell Department of Veterans Affairs, Medical Center,
Detroit MI, USA).
Biology
Bacterial strains: S. aureus ATCC 25923 was purchased from the In-
stitut Pasteur (CRBIP, Paris, France). The wild-type overproducing
NorA mutant clinical isolate S. aureus SA-1199B was generously
provided by G. W. Kaatz.
Keywords: antibiotics · arylation · efflux pump inhibitors ·
inhibitors · sulfur heterocycles
Media, antibiotics, and culture conditions: Mueller–Hinton broth
(MH, Bio Rad) was used for all bacteria overnight cultures at 378C,
susceptibility testing, and ethidium bromide accumulation experi-
ments. Ciprofloxacin was obtained from Sigma–Aldrich (Saint
Quentin Fallavier, France). Reserpine was purchased from Alfa
Aesar (Schiltigheim, France). Stock solutions (ciprofloxacin and
EtBr) were prepared in sterile water except for reserpine, which
was dissolved in DMSO.
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Received: October 8, 2015
Published online on January 6, 2016
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