From phenothiazine to 3-phenyl-1,4-benzothiazine derivatives as inhibitors of the Staphylococcus aureus NorA multidrug efflux pump

Article abstract of DOI:10.1021/jm701623q

Overexpression of efflux pumps is an important mechanism by which bacteria evade effects of substrate antimicrobial agents and inhibition of such pumps is a promising strategy to circumvent this resistance mechanism. NorA is a Staphylococcus aureus multid

Full text of DOI:10.1021/jm701623q

J. Med. Chem. 2008, 51, 4321–4330
4321
From Phenothiazine to 3-Phenyl-1,4-benzothiazine Derivatives as Inhibitors of the
Staphylococcus aureus NorA Multidrug Efflux Pump
Stefano Sabatini,† Glenn W. Kaatz,‡ Gian Maria Rossolini,§ David Brandini,† and Arnaldo Fravolini*,†
Dipartimento di Chimica e Tecnologia del Farmaco, UniVersita` di Perugia, 06123 Perugia, Italy, Department of Internal Medicine, DiVision of
Infectious Diseases, School of Medicine, Wayne State UniVersity and the John D. Dingell Department of Veterans Affairs Medical Center,
Detroit, Michigan 48201, USA, Dipartimento di Biologia Molecolare, Laboratorio di Fisiologia e Biotecnologia dei Microrganismi,
UniVersita` di Siena, 53100 Siena, Italy
ReceiVed December 24, 2007
Overexpression of efflux pumps is an important mechanism by which bacteria evade effects of substrate
antimicrobial agents and inhibition of such pumps is a promising strategy to circumvent this resistance
mechanism. NorA is a Staphylococcus aureus multidrug efflux pump, the activity of which confers decreased
susceptibility to many structurally unrelated agents, including fluoroquinolones, resulting in a multidrug
resistant (MDR) phenotype. In this work, a series of 1,4-benzothiazine derivatives were designed and
synthesized as a minimized structural template of phenothiazine MDR efflux pump inhibitors (EPIs) in an
effort to identify more potent S. aureus NorA EPIs. Almost all derivatives evaluated showed good activity
in combination with ciprofloxacin against S. aureus ATCC 25923; some were capable of completely restoring
ciprofloxacin activity in a norA-overexpressing strain (SA-K2378). Compounds 6k and 7j displayed good
activity against SA-1199B, a strain that also overexpresses norA, in an ethidium bromide (EtBr) efflux
inhibition assay.
Introduction
One strategy used to circumvent efflux-mediated resistance
is the search for new antibiotics that are poor pump substrates
and thus bypass efflux systems.11 A second approach is the
development of efflux pump inhibitors (EPIs) capable of
potentiating the activity of substrate antibiotics. Many inhibitors
may bind to the same site as substrates and inhibit transport by
a competitive or noncompetitive mechanism.12 EPIs used in
combination with antibiotics may not only increase antibacterial
potency13 but also expand the antibacterial spectrum and reduce
the frequency of the emergence of target-based resistance.14
Therefore, efflux pumps are viable antibacterial targets and
identification and development of potent EPIs is a promising
and valid strategy.12
Increasing drug resistance among Gram-positive bacteria is
a significant health problem because these organisms are
responsible for nearly one-third of all community-acquired
infections.15 In particular, methicillin-resistant S. aureus (MRSA)
is one of most frequent nosocomial pathogens in developed
countries.16 For example, in England and Wales, the number
of deaths due to MRSA increased from 51 in 1993 to 1629 in
2005. In 2005, infections caused by MRSA in the U.S. resulted
in 19000 deaths.17 Vancomycin-resistant (VRSA) and -insensi-
tive (VISA) strains of S. aureus have also been identified, further
complicating therapy.17
The most studied chromosomal efflux pump of S. aureus is
NorA, a transporter belonging to the major facilitator super-
family (MFS). As mentioned previously, some MFS pumps,
such as NorA, are capable of extruding multiple structurally
dissimilar substrates.18 Although the NorA binding pocket has
never been studied, sequence homology and the sharing of a
wide range of substrates and/or inhibitors with the Bacillus
subtilis Bmr MDR pump and the plasmid-encoded S. aureus
QacA MDR pump has led to the hypothesis that NorA may
have a large hydrophobic binding site. Such a binding region
would permit substrates to associate with the protein through a
combination of hydrophobic effects and electrostatic attraction
rather than by establishing a precise network of hydrogen bonds.
The treatment of many infectious diseases may be compro-
mised by resistance to antimicrobial agents. The emergence of
resistance among bacteria to a wide variety of structurally
unrelated antibacterial agents such as ꢀ-lactams, macrolides,
tetracyclines, and fluoroquinolones as well as selected dyes and
disinfectants has become a serious public health concern.1
Bacterial drug resistance is achieved by three general mech-
anisms which include enzymatic inactivation,2 modification of
the drug target,3,4 and decreased intracellular drug concentration
either by changes in membrane permeability5 or overexpression
of efflux pumps.6 Efflux is a process by which drug molecules
are removed from the cell by membrane-based proteins, resulting
in sublethal drug concentrations at the active site.7 The natural
role of efflux pumps is to remove toxins that are encountered
in the environment or produced during metabolism. This
protective function enables bacteria to survive in hostile
environments such as in the presence of antibiotics.8 Bacteria
have an array of antiporter-type efflux proteins, energized by
the proton- or sodium-motive force or ATP hydrolysis, that
extrude antibacterial drugs.9 These pumps are capable of
removing specific substrates such as tetracycline (SDR, specific
drug resistance, i.e., TetK) or several structurally dissimilar
substrates such as hydrophilic fluoroquinolones, various bio-
cides, and dyes producing a multidrug resistance (MDR)a
phenotype (i.e., NorA).6,10
* To whom correspondence should be addressed. Phone: +39 075
5855130. Fax: +39 075 5855115. E-mail: fravo@unipg.it.
†
Dipartimento di Chimica e Tecnologia del Farmaco, Universita` di
Perugia.
‡
Department of Internal Medicine, Division of Infectious Diseases,
School of Medicine, Wayne State University and the John D. Dingell
Department of Veterans Affairs Medical Center.
§
Dipartimento di Biologia Molecolare, Laboratorio di Fisiologia e
Biotecnologia dei Microrganismi, Universita` di Siena.
a Abbreviations: EPI, efflux pump inhibitor, MDR, multidrug resistance,
CPX, ciprofloxacin, EtBr, ethidium bromide.
10.1021/jm701623q CCC: $40.75
2008 American Chemical Society
Published on Web 06/25/2008

4322 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 14
Sabatini et al.
This structural peculiarity could explain the broad substrate
specificity of MDR pumps.19
Recently, a structural characterization of NorA was attempted
in silico. The three-dimensional model of NorA consists of 12
transmembrane helices connected by hydrophilic loops of
varying lengths. Two hypothetical binding sites, including a
central hydrophobic pocket and a periplasmic pocket, have been
identified. The maximum docking scores of these sites with
known inhibitors such as reserpine and verapamil provide
supportive evidence for their potential importance.20
Figure 1. Structural modifications of the phenothiazine backbone
leading to the 3-phenyl-2H-1,4-benzothiazines.
correlations have been found only for highly homogeneous sets
of molecules.38 Thus, the rational design of new EPI candidates
is difficult and new entities frequently are discovered seren-
dipitously through testing large libraries of known molecules
or by modifying known EPIs.
In recent years, many EPIs capable of potentiating the activity
of antimicrobial substrates have been identified, although none
has reached the clinic. Early EPIs included the plant alkaloid
reserpine and the synthetic antihypertensive verapamil, but
concentrations required for pump-inhibitory activity are too high
to be clinically relevant.21–23 Several nonantibiotic compounds
such as omeprazole (proton pump inhibitor; antiulcer), parox-
etine (antidepressant), chlorpromazine (neuroleptic), and respec-
tive derivatives have been shown to increase the antibacterial
potency of pump substrates by inhibiting NorA of S. aureus
and other MDR pumps of Gram-positive organisms.23–28 Natural
compounds such as epicatechin-gallate and epigallocatechin-
gallate, and the synthetic acridone derivative GG918 (a P-
glycoprotein inhibitor), have been shown to enhance the activity
of selected antimicrobial agents, including fluoroquinolones,
against efflux-mediated resistant Gram-positive bacteria.29,30
Other natural products such as the porphyrin pheophorbide A
and the flavonolignan 5′-methoxyhydnocarpin (5′-MHC), iso-
lated from Berberis plants, also have been identified as NorA
inhibitors.31 Flavones and synthetic flavolignan analogues of
5′-MHC were evaluated and SAR studies carried out.32 Inhibi-
tion of NorA by synthetic compounds such as phenyl nitroin-
doles and arylthiophenes significantly reduce the frequency at
which ciprofloxacin-resistant S. aureus derivatives emerge.33,34
To date, there are only a few examples of rationally designed
inhibitors and very little work has been done with respect to
the SAR of NorA inhibitors.32,35
Some phenothiazine derivatives, which are known to inhibit
the mammalian MDR pump P-glycoprotein,39 are also inhibitors
of NorA function.27 In some cases, pump-inhibitory activity is
synergized by intrinsic antibacterial activity and in others by a
reduction in the proton motive force, which inhibits pump
function by altering the membrane potential.40,41 Therefore, the
mechanism by which the phenothiazines inhibit efflux is
multifactorial. Unfortunately, phenothiazine derivatives are
recognized for their basic neurotropic activity. Indeed, many
SAR studies have been carried out in an attempt to remove or
decrease this effect.42 Investigations have concentrated on the
substituents in the aromatic ring, on the alkyl chain, the linker
to protonable nitrogen of the same chain, as well as the
substitution of nitrogen at N-10 with vinyl carbon (thioxanthene
derivatives).28
Thinking that the intriguing phenothiazine moiety may be a
valuable template for the synthesis of new and more potent
MDR EPIs, we elected to pursue a structural modification of
this backbone in an attempt to clarify its pharmacophoric moiety
and identify derivatives having improved EPI activity compared
to that of chlorpromazine. Our approach focused on the
elimination of structural features responsible for neuroleptic
activity through a structural minimization with the aim to
identify the minimum pharmacophore entity responsible for EPI
activity. We proceeded with drastic modifications such as the
elimination of one ring of the tricyclic phenothiazine structure,
resulting in a bicyclic benzothiazine scaffold, the elimination
of the chain linked to the N-10 atom, carrying a protonable
tertiary amine essential for the interaction with the dopaminergic
receptor, and the insertion at the C-3 position of a substituted
phenyl ring to ensure a better lipophilic character (Figure 1).
An entry set of benzothiazine derivatives was developed by
synthesizing 3-phenyl-2H-1,4-benzothiazine (6a) as a prototype
The structural heterogeneity of compounds active in reversing
MDR has made it difficult to establish SARs and complicates
the design of new candidates. Indeed, the broad substrate affinity
of MDR pumps suggests that there might be more than a single
binding mechanism within the large binding site.36 The role of
lipophilicity, a characteristic that is generally considered
important for MDR-reversing properties, remains unclear and
no detailed SARs have been done showing any relationship
between partition coefficients and efflux inhibition.37 Successful
Scheme 1a
a (i) PHP, AcOH, 60°C; (ii) Et2O, r.t.; (iii) Et2O, r.t. and then HClg; (iv) PTSA, CH2Cl2, r.t.; (v) K2CO3, DMF, r.t.; (vi) Et3N, EtOH/Et2O, r.t.

Benzothiazines as NorA Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 14 4323
of a new series (see Scheme 1). Analogues substituted at C-4′
with a lipophilic electron donor substituent as a methyl group
(6b), an electron-withdrawing substituent such as a chlorine
atom (6c), and a methoxy group to ensure a different level of
hydrophobicity (6d) also were synthesized. Preliminary screen-
ing of compounds using a wild type S. aureus strain (ATCC
25923) in the presence and absence of ciprofloxacin (CPX)
revealed variable intrinsic and synergistic activity (Table 1).
Compounds 6a and 6d showed no intrinsic antibacterial activity
(MIC > 512 µg/mL), but when the test strain was exposed to
both CPX and 25 µg/mL of either compound, an 8-fold
reduction in the CPX MIC was observed. In a similar manner,
compounds 6b and 6c (MIC 64 µg/mL) reduced the CPX MIC
4-fold.
NorA inhibitory activity was then highlighted by testing this
first set of compounds on modified strains in which the norA
gene was deleted (SA-K1902) or overexpressed from a plasmid
(SA-K2378). As expected, no compound exhibited a significant
effect when coadministered with CPX against SA-K1902. When
compounds 6c, 6b, and 6d were coadministered with CPX
against SA-K2378, MICs were decreased 4-, 8-, and 16-fold,
respectively, with 6d able to completely reverse the effect of
NorA (MIC reduced from 2 to 0.12 µg/mL).
On the basis of these encouraging results, the series of
benzothiazines was enlarged to include new derivatives with
modifications on the phenyl linked to C-3 as a positional shift
of the OMe or its increased presence (see 6e-h), the introduc-
tion of F, NO2, OPr, and Oi-pentyl in the para position (6i-l,
respectively). Other variations included the introduction of CF3
at C-6 for derivatives 7a, 7d, and 7j and introduction of a methyl
group at the N-4 position, resulting in 8k. Finally, sulfur atom
oxidation to sulfone was performed resulting in compounds 12a
and 12c, and hydrogenation of the double bond C-3/N-4 resulted
in 15d and 15m.
1-phenylethanones 14d44 and 14m, which then were reduced
with LiAlH4 to afford, after spontaneous cyclization, the
expected 3-phenyl-3,4-dihydro-2H-1,4-benzothiazines 15d and
15m in good yields (Scheme 3).
Results and Discussion
In this study, a series of 1,4-benzothiazine derivatives was
designed and synthesized as possible minimizing structures of
phenothiazine MDR EPIs and developed as a template to obtain
more effective S. aureus NorA EPIs. A microbiological ap-
proach was used to assess EPI function by determining the
intrinsic antimicrobial activity of synthesized compounds against
wild-type S. aureus ATCC 25923 and the effect of combining
these compounds with CPX on the same strain. Successive steps
involved an evaluation of synergistic antimicrobial activity
against SA-K1902 (norA-) and SA-K2378, which overex-
presses norA from a multicopy plasmid. To confirm the
inhibitory mechanism an ethidium bromide (EtBr), efflux
inhibition assay was carried out using SA-1199B, a well-
characterized strain that overexpresses norA.52
The modification of the 3-phenyl ring on the 6a prototype
involved the shifting of a methoxy group to the para, meta, and
ortho positions, giving rise to compounds 6d, 6f, and 6e,
respectively. When combined with CPX at a concentration of
25 µg/mL, MIC reductions of 4- to 8-fold versus S. aureus
ATCC 25923 and 8- to 16-fold versus SA-K2378 were observed
(Table 1). Compounds 6g (dimethoxy) and 6h (trimethoxy)
potentiated the activity of CPX by 4- to 16-fold versus ATCC
25923 and SA-K2378, respectively. Elongation of the ethereal
alkyl chain at C-4′ of 6d to n-propyloxy resulted in the same
activity for 6k (CPX MIC decreased g8-fold) against S. aureus
ATCC 25923 but a lesser effect was observed against SA-K2378
(4-fold). Introduction of an i-pentyl chain 6l had a detrimental
effect with a reduction of activity against both S. aureus ATCC
25923 and SA-K2378 observed. Substitution at C-4′ with an
electron withdrawing group give different results: NO2 6j
resulted in diminished synergistic activity when combined with
CPX, while fluorine 6i potentiated activity by 16-fold against
SA-K2378. A hydrophobic electron-donor methyl group 6b also
was quite beneficial, resulting in a 4- to 8-fold potentiation of
CPX activity versus the ATCC strain and SA-K2378, respectively.
Further modifications were carried out on the benzothiazine
nucleus. The introduction at C-6 of CF3, a hydrophobic electron-
withdrawing group that is also present both in phenothiazine
and thioxanthene EPI derivatives (i.e., fluphenazine and flu-
pentixol, respectively) gave rise to compounds 7a, 7d, and 7j.
These compounds had variable intrinsic activities (7a ) 7j >
7d), and when combined with CPX at 25 µg/mL, 7j potentiated
the activity of CPX by g8- and g32-fold against S. aureus
ATCC 25923 and SA-K2378, respectively. The 4′-unsubstituted
7a and 4′-methoxy 7d derivatives exhibited a low activity on
the wild-type strain; however, against SA-K2378 the activity
for 7a was very strong with a MIC reduction of 16-fold at both
25 and 12.5 µg/mL.
Chemistry
The synthetic route giving rise to the 3-phenyl-1,4-benzothi-
azine derivatives 6a-l, 7a,d,j, and 8k entailed the reaction of
appropriate 2-aminothiophenol or its sodium salt 1,43 2, and
344 with a substituted R-bromo acetophenone 5a-f, 5g,45 and
5h46-l (Scheme 1). R-Bromo acetophenones 5k,l were obtained
by bromuration with pyridinium perbromide hydrobromide
(PHP) of correspondent acetophenones 4k47 and 4l.47
Compounds 6a,48 6d,44 6j44-l, 7a, and 7j were obtained in
low yields (32-43%) because they were accompanied by
dimerization compounds,48 a byproduct of spontaneous self-
oxidation at the C-2 position in polar solvents. A slight
modification of the classical procedure resulted in compounds
6b,49 6c,49 6e-i, and 7d, which were obtained as hydrochlorides
in better yields. The N-4 methyl derivative 8k was prepared in
the same manner by reacting 2-methylamino thiophenol sodium
salt 344 with R-bromo-4′-propoxyacetophenone 5k.
The direct oxidation of the benzothiazine sulfur atom of 6d44
to the corresponding sulfoxide and/or sulfone derivative was
prevented by the high reactivity of the C-2 position, which
resulted in the corresponding 3-[4-(methyloxy)phenyl]-2H-1,4-
benzothiazin-2-one 9d.44 3-Phenyl-1,4-benzothiazine-1,1-dioxide
derivatives 12a50and 12c were obtained, as illustrated in
(Scheme 2) by reacting the sodium salt of 2-nitro-benzensulfinic
acid 1051 with substituted R-bromo acetophenones 5a,c to obtain
the nitrosulphones 11a50 and 11c that were then reduced with
H2/Raney-Ni to afford, after spontaneous cyclization, the
expected sulfones 12a50 and 12c in high yields.
To obtain further information on the SAR of this new class
of EPIs, other modifications were carried out. Methylation at
N-4, with consequent stabilization of the endocyclic double bond
in the C-2/C-3 positions, gave derivative 8k that caused a slight
reduction of CPX MIC (4-fold) only for SA-K2378. The
3-phenyl benzothiazine-1,1-dioxide 12a and its 4′-chloro deriva-
tive 12c, obtained by S-1 oxidation, had minimal potentiation
activity. Reduction of the endocyclic double bond at the C-3/
N-4 positions resulted in the 3,4-dihydro-2H-1,4-benzothiazine
15d, which had half the potentiation activity observed with 6d
Reacting the 2-nitrobenzenesulfenyl chloride 13 with substi-
tuted acetophenones 4d,m resulted in the 2-[(2-nitrophenyl)thio]-

4324 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 14
Table 1. Evaluation of Antibacterial Activity
Sabatini et al.
a n-Fold reduction of CPX MIC.

Benzothiazines as NorA Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 14 4325
Table 2. Inhibition of EtBr Efflux by SA-1199Ba
Scheme 2a
b
b
compd
%
compd
%
6a
6d
6e
6g
6h
6i
0.0
19.5
0.0
22.8
23.6
0.0
6l
7a
7j
8k
15d
15m
reserpine
16.9
8.4
69.7
0.0
10.4
28.2
81.6
81.0
6j
6k
19.6
84.7
chlorpromazine
a All compounds were tested at a concentration of 50 µM. b Percent
efflux inhibition.
a (i) DMF, r.t.; (ii) H2, Raney-Ni, r.t.
Scheme 3a
Figure 2. Effect of compounds 6k (0), 7j ()), reserpine (∆), and
chlorpromazine (×) on EtBr efflux of SA-1199B.
K2378. In addition to NorA, characterized S. aureus MDR efflux
pumps that do transport CPX include NorB, NorC, and MepA.53
Gene expression analyses would be required to determine if
any of these are at play in ATCC 25923, but is beyond the
scope of this study.
a (i) CH3CN dry, reflux; (ii) LiAlH4, THF dry, r.t.
Several derivatives (6a,d,e,g-l, 7a,j, 8k, and 15d,m) were
evaluated for their abilities to interfere with EtBr efflux from
SA-1199B, employing a 50 µM concentration. None of these
compounds had any intrinsic antibacterial activity against this
strain (data not shown). 6k and 7j showed good efflux inhibitory
activity; 6k was equipotent to reserpine and chlorpromazine
(84.7, 81.6, and 81.0% inhibition of efflux, respectively) (Table
2). A dose-response efflux inhibition curve was generated for
6k and 7j, the most active compounds. Reserpine and chlor-
promazine were used as comparators, and the data are presented
in Figure 2. Compound 6k reduced NorA-mediated efflux as
well as reserpine at all tested concentrations.
Isobolograms demonstrating the effect of combining 6k, 7j,
reserpine, and chlorpromazine on CPX MICs against SA-K1902
and SA-K2378 are shown in Figure 3. It can be seen that the
CPX MICs for SA-K1902 (norA-) were constant at all the
concentrations of 6k, indicating no significant activity against
non-NorA pumps that this strain may express. With respect to
7j and chlorpromazine, CPX MICs decreased 4-fold at con-
centrations greater than 6.25 and 12.5 µg/mL, respectively
(Figure 3a). Compound 7j and chlorpromazine have weak
antibacterial activity against this strain (MIC 25 µg/mL), and it
is possible that as this concentration is approached, the
antimicrobial effect produces the MIC changes observed. The
small MIC reduction produced by 3.13 µg/mL of reserpine,
which is devoid of any antimicrobial activity against this strain,
is probably the result of inhibition of one or more non-NorA
efflux pumps that have CPX as a substrate.
against SA-K2378, and its 4′-thio analogue 15m, which was
equipotent to compound 6d against both strains.
Many of the tested compounds, such as 6b, 6d-i, 6k, 7a,
7d, 7j, 15d, and 15m, displayed the same or better activity than
the reference compounds reserpine and chlorpromazine against
S. aureus ATCC 25923 and SA-K2378 when coadministered
with CPX. In some cases (see 6b, 6e, 6f, 6h, 6i, 7a, 7d, and 7j
in Table 1), the strong activity results, at least for the wild-type
strain, from NorA pump inhibition and from their weak intrinsic
antibacterial activity that can give a synergistic interaction with
CPX. Compounds 6d, 6g, and 15m, which are devoid of any
antibacterial activity, are able to restore CPX susceptibility in
SA-K2378 to the same level as that observed for the SA-K1902
(norA-) by complete inhibition of NorA.
The 2-fold reductions in CPX MICs observed for compounds
6 g-i and 7j against SA-K1902 (norA-deleted strain) are within
the accepted variance of microdilution susceptibility testing and
are thus not significant. Interestingly, some compounds dem-
onstrated greater activity against ATCC 25923 than against the
norA-overexpressing strain SA-K2378 (see 6a and 6k data in
Table 1), and some had the opposite effect (see 6 g-i). In the
latter case, the explanation is most likely related to inhibition
of NorA. In cases where the activity of a compound was greater
against the ATCC strain than against the norA-overexpressing
strain, the explanation may be as simple as the fact that these
are two very different genetic backgrounds. The S. aureus
genome encodes many putative pumps and most have not been
characterized.53 It is feasible that baseline expression of a known
or undefined pump(s) capable of CPX transport susceptible to
the activity of 6a or 6k in ATCC 25923 is greater than that of
either SA-K1902 or its norA-overexpressing derivative SA-
For SA-K2378, which overexpresses norA from a multicopy
plasmid, there is a gradual reduction of CPX MICs across all
tested concentrations of both 6k and 7j (Figure 3b). The CPX
MIC is reduced to a value identical to that of SA-K1902 at a

4326 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 14
Sabatini et al.
reference compounds reserpine and chlorpromazine. The first
group, consisting of 6a, 6e, and 6i, did not show any EtBr efflux
inhibition. The second group, including 6d and 15m, displayed
moderate EtBr efflux inhibitory activity (20-30% inhibition),
and the third group, including 6k, 7j, reserpine, and chlorpro-
mazine, exhibited high activity (more than 50% efflux inhibi-
tion). Several in silico parameters were calculated for these
groups, and some of these molecular descriptors seem to
correlate with efflux inhibition (Table 3).
The physicochemical data reported in Table 3 suggest that
the more active compounds share with reserpine and chlorpro-
mazine some properties that may contribute to confer inhibitory
activity of EtBr efflux by NorA pump. Relatively to EPIs
compounds, the NorA inhibitory activity seems to be related
both to hydrophobic properties (Log P > 3.9, W1 > 1050,
SOLY < –4, and %FU4 < 57.6) and molecular dimension (MW
> 280, V > 600, and G > 1.35). To better understand the
relative weight of each physicochemical property on NorA pump
inhibition, a PLS model was built taking into account the X
descriptor matrix (Table 3) and the percent of EtBr efflux on
SA-1199B. The model confirms that Log P is directly correlated
with efflux inhibition. Furthermore, water solubility (SOLY)
as well as percentage of uncharged molecules at pH 4 (%FU4)
were strongly but inversely correlated to NorA inhibition.
Polarity (PSA), molecular dimension (MW, V, and G), and
hydrophilic accessible volume (W1) are less correlated with
inhibition of EtBr efflux. The model therefore could suggest
that H-bond pattern is more relevant than molecular size and
shape in respect to efflux.
In conclusion, the 3-phenyl-1,4-benzothiazine derivatives,
designed by structural minimization of phenothiazine NorA
pump inhibitors, show modest or no intrinsic antistaphylococcal
activity and restore, in a concentration-dependent manner, the
antibacterial activity of CPX against norA-overexpressing S.
aureus strains. The EPI activity of some of these benzothiazine
derivatives was superior to that shown for the progenitor
compound chlorpromazine and the reference compound reser-
pine both against S. aureus ATCC 25923 and SA-K2378.
Figure 3. Effect of combining 6k (0), 7j ()), reserpine (∆), and
chlorpromazine (×) on ciprofloxacin MICs against SA-K1902 (a) and
SA-K2378 (b).
concentration of 25 µg/mL for compound 6k and 12.5 µg/mL
for 7j. 6k is devoid of any antimicrobial activity against this
strain (MIC >100 µg/mL), and 7j has weak activity (MIC 50
µg/mL). The fact that a concentration of no more than one-
fourth the MIC of compound 7j reduces the ciprofloxacin MIC
to that of the norA-null parent strain strongly suggests that the
mechanism of the effect is the complete inhibition of NorA
activity.
Experimental Section
Bacterial Strains. The strains of S. aureus employed were ATCC
25923 (wild-type), SA-1902 (norA-deleted), and SA-1199B (over-
expressing norA and also possesses an A116E GrlA substitution).52,55
In addition SA-K2378, which overexpresses norA from a multicopy
plasmid, also was used. This strain was produced by cloning norA
and its promoter into plasmid pCU1 and then introducing the
construct into SA-K1902.56
Microbiologic Procedures. All microbiologic procedures em-
ployed S. aureus ATCC 29523, SA-K1902, and SA-K2378. MICs
were determined in duplicate by microdilution techniques according
to CLSI guidelines.57 The effect of combining reserpine and
chlorpromazine or scalar dilutions of freshly prepared solutions of
each synthesized compound on the MICs of CPX also was
determined. Checkerboard combination studies using CPX and 6k
or 7j were performed as described previously.58
EtBr Efflux. The loss of EtBr from S. aureus SA-1199B was
determined fluorometrically as previously described.59 Experiments
were performed in duplicate, and the results were expressed as the
mean total efflux over a 5 min time course. The effect of increasing
concentrations of reserpine, 6k, and 7j on the EtBr efflux of SA-
1199B also was determined. EtBr efflux of SA-1199B in the
presence of test compounds was compared to that determined in
their absence and percent reduction in efflux was calculated.
Synthesis. All reactions were routinely checked by thin-layer
chromatography (TLC) on silica gel 60F254 (Merck) and visualized
using UV illumination. Flash column chromatography was per-
formed on Merck silica gel 60 (mesh 230-400) using the indicated
Data collected in this study highlight that 3-phenyl-1,4-
benzothiazine derivatives obtained by structural minimization
of phenothiazine EPIs can be effective inhibitors of NorA. These
results indicate that the benzothiazine moiety is the key feature
for activity of the two different series of derivatives. The SAR
for this new class of benzothiazine EPIs has shown that
substituents on the C-3 phenyl ring play an important role and
in particular 4′-methoxy and 4′-thiomethyl groups give potent
compounds (6d and 15m) against wild-type and norA-overex-
pressing strain SA-K2378. Propyloxy compound 6k displays
good activity against both a wild-type strain and in the EtBr
efflux inhibition assay against the norA-overexpressing strain
SA-1199B. A nitro group in the C-4′ position, when coupled
with the presence of a trifluoromethyl group in the C-6 position
of the benzothiazine nucleus, gives the potent compound 7j
characterized by good efflux inhibitory activity and in combina-
tion susceptibility testing with CPX. Oxidation of the S-1
position or N-4 methylation gave less active compounds, which
were probably the result of an increased polarity and/or planarity
of the benzothiazine nucleus.
To investigate if there is a correlation between NorA
inhibitory activity and the physicochemical parameters calcu-
lated in silico,54 seven compounds were selected based on their
EtBr efflux inhibitory activity and were compared with the

Benzothiazines as NorA Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 14 4327
Table 3. Physicochemical Properties of Selected Compounds
compd
activitya
MW
Log P
Vb
Gc
W1d
SOLYe
PSAf
%FU4g
6a
6e
6i
6d
15m
6k
7j
0.0
0.0
0.0
19.5
28.2
84.7
69.7
81.6
81.0
225.31
255.34
243.30
255.34
273.42
283.34
338.30
608.68
318.86
3.0
2.9
3.2
3.1
4.5
4.0
3.9
4.0
5.2
531.4
584.4
534.5
590.2
623.5
669.6
644.8
1275.0
719.5
1.26
1.30
1.27
1.32
1.33
1.39
1.40
1.79
1.37
890.9
956.1
-3.66
-3.60
-3.45
-3.68
-4.46
-4.43
-4.19
-6.05
-4.25
39.4
50.8
39.4
50.8
65.6
50.8
76.6
126.1
33.0
92.0
78.3
87.0
23.5
18.2
23.5
57.6
0.2
1031.1
1017.8
1034.6
1086.5
1303.0
1784.2
1067.0
reserpine
chlorpromazine
<0.1
a Percent inhibition of EtBr efflux from SA-1199B. b V: molecular volume. c G: globularity. d W1: hydrophilic accessible volume. e SOLY: intrinsic
solubility in water. f PSA: polar surface area. g %FU4: % fraction of uncharged molecules at pH 4.
solvents. Yields were of purified product and were not optimized.
Melting points were determined in capillary tubes (Electrothermal
model 9100) and are uncorrected. Elemental analyses were per-
formed on a Carlo Erba elemental analyzer (model 1106), and the
data for C, H, and N are within 0.4% of the theoretical values. 1H
NMR spectra were recorded at 200 MHz (Bruker Avance AC 200)
or 400 MHz (Bruker Advance-DRX 400), with CDCl3 as solvent,
unless otherwise indicated, and with Me4Si as the internal standard.
The chemical shift (δ) values are reported in ppm, and the coupling
constants (J) are given in Hz. The abbreviations used are as follows:
s, singlet; bs, broad singlet; d, doublet; dd, double doublet; t, triplet;
m, multiplet. The spectral data are consistent with the assigned
structures. Reagents and solvents were purchased from common
commercial suppliers and were used as received. For routine
aqueous workup, the reaction mixture was extracted with CH2Cl2
or EtOAc. The organic layer was washed with brine, dried over
anhydrous Na2SO4, and concentrated with a Bu¨chi rotary evaporator
at low pressure. All starting materials were commercially available,
unless otherwise indicated.
2-Bromo-1-(4-propoxyphenyl)ethanone (5k). Pyridinium per-
bromide hydrobromide (3.50 g, 11.0 mmol) was added to a solution
of 1-(4-propoxyphenyl)ethanone 4k47 (1.30 g, 7.3 mmol) in acetic
acid (15 mL) and warmed to 55-60 °C for 30 min under magnetic
stirring. After cooling, the mixture was poured in water, neutralized
with NaHCO3, and extracted with EtOAc (3 × 50 mL). The organic
extracts were dried with Na2SO4, and the solvent was removed
under reduced pressure to obtain a residue that was purified by
flash column chromatography with a mixture of EP:Et2O 95:5 to
obtain 0.90 g (yield 48%) of pure 2-bromo-1-(4-propoxyphenyl)e-
thanone 5k as colorless oil. 1H NMR (CDCl3): δ 1.05 (3H, t, J )
7.45 Hz, OCH2CH2CH3), 1.75-1.95 (2H, m, OCH2CH2CH3), 4.00
(2H, t, J ) 6.55 Hz, OCH2CH2CH3), 4.40 (2H, s, CH2Br), 6.95
(2H, d, J ) 9.02 Hz, H-2, H-6), 7.95 (2H, d, J ) 9.02 Hz H-3,
H-5).
3-(2-Methoxyphenyl)-2H-1,4-benzothiazine Hydrochloride (6e).
Obtained from 2-bromo-1-[2-(methyloxy)phenyl]ethanone 5e as
brown solid in 62% yield after crystallization with cycloesane/Et2O;
1
mp 137.5-138 °C. H NMR (CDCl3): δ 3.95 (5H, s, OCH3 and
CH2), 7.04 (1H, d, J ) 8.43 Hz, H-3′), 7.20 (1H, t, J ) 7.19 Hz,
H-5′), 7.30-7.45 (3H, m, H-7, H-4′ and H-6′), 7.66 (1H, t, J )
7.35 Hz, H-6), 8.10 (1H, d, J ) 7.59 Hz, H-5), 8.45-8.55 (1H, m,
H-8). Anal. (C15H14ClNOS) C, H, N.
3-(3-Methoxyphenyl)-2H-1,4-benzothiazine Hydrochloride (6f).
Obtained from 2-bromo-1-[3-(methyloxy)phenyl]ethanone 5f as
brown solid in 55% yield after crystallization with cycloesane/Et2O;
mp 159-160 °C. 1H NMR (CDCl3): δ 3.75 (2H, s, CH2), 3.95
(3H, s, OCH3), 7.10-7.25 (2H, m, H-2′ and H-5′), 7.30-7.45 (3H,
m, H-5, H-7 and H-4′), 7.55-7.65 (1H, m, H-6′), 7.80-7.95 (2H,
m, H-6 and H-8). Anal. (C15H14ClNOS) C, H, N.
3-(3,4-Dimethoxyphenyl)-2H-1,4-benzothiazine Hydrochloride
(6g). Obtained from 1-[3,4-bis(methyloxy)phenyl]-2-bromoethanone
5g45 as pale-brown solid in 47% yield after crystallization with
cycloesane/Et2O; mp 198-198.5 °C. 1H NMR (CDCl3): δ 4.10 (5H,
s, OCH3 and CH2), 4.30 (3H, s, OCH3), 7.10 (1H, d, J ) 8.85 Hz,
H-5′), 7.35-7.55 (3H, m, H-6, H-7 and H-6′), 7.83 (1H, d, J )
6.64 Hz, H-5), 8.64 (1H, s, H-2′), 8.82 (1H, d, J ) 7.7 Hz, H-8).
Anal. (C16H16ClNO2S) C, H, N.
3-(3,4,5-Trimethoxyphenyl)-2H-1,4-benzothiazine Hydrochlo-
ride (6h). Obtained from 2-bromo-1-[3,4,5-tris(methyloxy)phe-
nyl]ethanone 5h46 as brown solid in 41% yield after crystallization
with cycloesane/Et2O; mp 157-158 °C. 1H NMR (CDCl3): δ 3.93
(2H, s, CH2), 4.03 (3H, s, OCH3), 4.08 (6H, s, OCH3), 7.35-7.45
(3H, m, H-5, H-6 and H-7), 7.65 (2H, s, H-2′ and H-6′), 8.55 (1H,
d, J ) 7.7 Hz, H-8). Anal. (C17H18ClNO3S) C, H, N.
3-(4-Fluorophenyl)-2H-1,4-benzothiazine Hydrochloride (6i).
Obtained from 2-bromo-1-(4-fluorophenyl)ethanone 5i as yellow
solid in 53% yield after crystallization with cycloesane/Et2O; mp
1
2-Bromo-1-[4-(isopentyloxy)phenyl]ethanone (5l). Obtained by
the previous procedure, from 4-(isopentyloxyphenyl)ethanone 4l,47
as a yellow oil in 44% yield, after purification by column
187-188 °C. H NMR (CDCl3): δ 4.05 (2H, s, CH2), 7.35-7.55
(5H, m, H-6, H-2′, H-3′, H-5′ and H-6′), 8.53-8.78 (3H, m, H-5,
H-6 and H-8). Anal. (C14H11ClFNS) C, H, N.
1
chromatography (EP/Et2O 95:5). H NMR (CDCl3): δ 1.00 (6H,
3-(4-Propoxyphenyl)-2H-1,4-benzothiazine (6k). Obtained from
2-bromo-1-(4-propoxyphenyl)ethanone 5k as yellow solid in 32%
yield after purification by flash column chromatography (CH2Cl2/
d, J ) 6.40 Hz, OCH2CH2CH(CH3)2), 1.55-1.95 (3H, m,
OCH2CH2CH(CH3)2), 4.05 (2H, t, J ) 6.55 Hz, OCH2CH2CH-
(CH3)2), 4.40 (2H, s, CH2Br), 6.95 (2H, d, J ) 9.00 Hz, H-2, H-6),
7.95 (2H, d, J ) 9.00 Hz, H-3, H-5).
1
EP 60:40 f 40:60); mp 115-117 °C. H NMR (CDCl3): δ 1.05
(3H, t, J ) 7.35 Hz, OCH2CH2CH3), 1,75-1,95 (2H, m,
OCH2CH2CH3), 3.90 (2H, s, CH2), 4.10 (2H, t, J ) 6.50 Hz,
OCH2CH2CH3), 6.98 (2H, d, J ) 8.64 Hz, H-3′, H5′), 7.10 (1H,
dt, J ) 7.53 and 1.49 Hz, H-7), 7.23 (1H, dt, J ) 7.77 and 1.67
Hz, H-6), 7.33 (1H, dd, J ) 7.56 and 1.63 Hz, H-5), 7.47 (1H, dd,
J ) 7.81 and 1.56 Hz, H-7), 7.98 (2H, d, J ) 8.64, H-2′,H-6′).
Anal. (C17H17NOS) C, H, N.
Procedure for the Synthesis of Substituted 3-Phenyl-2H-1,4-
benzothiazines 6a-l and 8k. To a suspension of sodium 2-ami-
nothiophenate 143 (1.5 equiv) in dry Et2O (30 mL) was added
dropwise to a solution of appropriate 2-bromo-1-phenylethanone
(1.0 equiv) in the same solvent. The reaction mixture was
maintained at room temperature under nitrogen atmosphere and
magnetic stirring until 2-bromo-1-phenylethanone disappeared
(TLC). Then, for compounds 6a,48 6d,44 6j44-l, and 8k, the mixture
was filtered to eliminate NaBr and the organic solvent was removed
under reduced pressure to obtain a residue that was purified by
flash column chromatography to give the target compounds. For
compounds 6b,49 6c,49 and 6e-i, after filtration, the reaction
mixture was bubbled with HClg until the target compound was
completely precipitated as hydrochloride.
3-[4-(Isopentyloxy)phenyl]-2H-1,4-benzothiazine (6l). Obtained
from 2-bromo-1-[4-(isopentyloxy)phenyl]ethanone 5l as yellow
semisolid in 39% yield after purification by flash column chroma-
1
tography (CH2Cl2/EP 60:40 f 40:60). H NMR (CDCl3): δ 1.00
(6H, d, J ) 6.42 Hz, OCH2CH2CH(CH3)2), 1.60-1.95 (3H, m,
OCH2CH2CH(CH3)2), 2,45 (1H, bs, NH), 4.10 (2H, t, J ) 6.50
Hz, OCH2CH2CH(CH3)2), 5.95 (1H, s, H-2), 7.00 (2H, d, J ) 8.98,
H-3′, H5′), 7.20-7.50 (3H, m, H-5, H-6, H-7), 7.60 (1H, dd, J )

4328 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 14
Sabatini et al.
8.95 and J ) 2.05, H-8), 8.05 (2H, d, J ) 8.98, H-2′,H-6′). Anal.
(C19H21NOS) C, H, N.
1-(4-Chlorophenyl)-2-[(2-nitrophenyl)sulfonyl]ethanone (11c).
To a solution of [(2-nitrophenyl)sulfonyl]sodium salt 1051 (1.00 g,
4.78 mmol) in DMF dry (8 mL) was added dropwise a solution of
2-bromo-1-(4-chlorophenyl)ethanone 5c (1.12 g, 4.78 mmol) in the
same solvent (7 mL) and the mixture was maintained at room
temperature under nitrogen atmosphere and magnetic stirring for
6 h. The mixture was then poured in ice/water to obtain a yellow
solid that was filtered and purified by flash column chromatography
(cyclohexane:Et2O 80:20) to give 0.91 g (yield 56%) of pure 1-(4-
chlorophenyl)-2-[(2-nitrophenyl)sulfonyl]ethanone 11c as pale-
4-Methyl-3-(4-propoxyphenyl)-4H-1,4-benzothiazine (8k). Ob-
tained from 2-(methylamino)benzenethiol sodium salt 344 and
2-bromo-1-(4-propoxyphenyl)ethanone 5k as yellow solid in 35%
yield after purification by flash column chromatography (CH2Cl2/
EP 60:40 f 40:60). mp 91-93 °C. 1H NMR (CDCl3): δ 1.05 (3H,
t, J ) 7.45 Hz, OCH2CH2CH3), 1.80-1.95 (2H, m, OCH2CH2CH3),
3.65 (3H, s, NCH3), 4.00 (2H, t, J ) 6.55 Hz, OCH2CH2CH3),
6.30 (1H, s, H-2), 7.00 (2H, d, J ) 8.70 Hz, H-3′, H-5′), 7.32 (2H,
d, J ) 8.70 Hz, H-2′, H-6′), 7.40 (1H, t, J ) 7.12 Hz, H-6), 7.55
(1H, d, J ) 8.55 Hz, H-8), 7.65 (1H, t, J ) 7.01 Hz, H-7), 8.50
(1H, d, J ) 8.04 Hz, H-5). Anal. (C18H19NOS) C, H, N.
3-(4-Methoxyphenyl)-2H-1,4-benzothiazin-2-one (9d44). Solid
m-chloroperbenzoic acid (0.20 g, 1.17 mmol) was added to a
solution of 3-[4-(methyloxy)phenyl]-2H-1,4-benzothiazine 6d44
(0.10 g, 0.39 mmol) in CH2Cl2 (10 mL) and maintained under
magnetic stirring for 30 min at room temperature. The mixture then
was poured in water, neutralized with NaHCO3, and extracted with
CH2Cl2 (3 × 50 mL). The organic extracts were dried with Na2SO4,
and the solvent was removed under reduced pressure to obtain a
residue that was purified by flash column chromatography with
CHCl3 to obtain 0.078 g (yield 74%) of pure 3-(4-methoxyphenyl)-
2H-1,4-benzothiazin-2-one 9d44 as yellow solid; mp 111-113 °C.
1H NMR (CDCl3): δ 3.80 (3H, s, OCH3), 6.95 (2H, d, J ) 8.95
Hz, H-3′, H-5′), 7.30-7.55 (3H, m, H-5, H-6, H-7), 7.90 (1H, d,
J ) 9.35 Hz, H-8), 8.20 (2H, d, J ) 8.95 Hz, H-2′, H-6′). Anal.
(C15H11NO2S) C, H, N.
1
yellow solid; mp 153-154 °C. H NMR (CDCl3): δ 5.30 (2H, s,
CH2), 7.53 (2H, d, J ) 8.88 Hz, H-3′, H-5′), 7.80-8.00 (5H, m,
Ar-H), 8.17-8.24 (1H, m, H-5).
3-(4-Chlorophenyl)-4H-1,4-benzothiazine 1,1-Dioxide (12c). A
stirred solution of 1-(4-chlorophenyl)-2-[(2-nitrophenyl)sulfonyl]e-
thanone 11c (0.18 g, 0.54 mmol) in a mixture of EtOH:DMF (1:2)
(25 mL) was hydrogenated over catalytic amount of Raney-Ni and
under H2 atmosphere at room temperature for 2 h. The mixture
was then filtered over celite, and the filtrate was evaporated to
dryness. The solid residue was crystallized by EtOH to give 3-(4-
chlorophenyl)-4H-1,4-benzothiazine 1,1-dioxide 12c (0.07 g, 44%)
as white solid; mp 153-153.5 °C. 1H NMR (CDCl3): δ 6.32 (1H,
s, H-2), 7.30 (1H, t, J ) 7.97 Hz, H-6), 7.50-7.70 (4H, m, H-5,
H-7, H-3′, H-5′), 7.77 (2H, d, J ) 8.67 Hz, H-2′, H-6′), 7.83 (1H,
dd, J ) 8.02 and 1.18 Hz, H-8), 10.90 (1H, bs, NH). Anal.
(C14H10ClNO2S) C, H, N.
1-[4-(Methylthio)phenyl]-2-[(2-nitrophenyl)thio]ethanone (14m).
To a solution of 2-nitrobenzenesulfenyl chloride 13 (2.00 g, 10.55
mmol) in CH3CN dry (20 mL) was added dropwise a solution of
1-[4-(methylthio)phenyl]ethanone 4m (1.16 g, 7.04 mmol) in the
same solvent (5 mL), and the mixture was maintained at reflux
under nitrogen atmosphere and magnetic stirring for 90 min. After
this time, a yellow solid was separated from the mixture by filtration
and purified by flash column chromatography (CHCl3:MeOH 95:
5) to give 1.50 g (yield 67%) of pure 1-[4-(methylthio)phenyl]-2-
[(2-nitrophenyl)thio]ethanone 14m as pale-yellow solid; mp
113.5-114.5 °C. 1H NMR (CDCl3): δ 2.5 (3H, s, SCH3), 4.30 (2H,
s, CH2), 7.53 (2H, d, J ) 8.88 Hz, H-3′, H-5′), 7.80-8.00 (5H, m,
Ar-H), 8.17-8.24 (1H, m, H-5).
3-(4-Methoxyphenyl)-3,4-dihydro-2H-1,4-benzothiazine (15d).
To a suspension of LiAlH4 (0.03 g, 0.83 mmol) in THF dry (10
mL) was added dropwise a solution of 1-(4-methoxyphenyl)-2-[(2-
nitrophenyl)thio]ethanone 14d44 (0.10 g, 0.33 mmol) in the same
solvent (30 mL). The reaction mixture was maintained at room
temperature under nitrogen atmosphere and magnetic stirring for
40 min. Then the mixture was added of EtOAc (10 mL), poured in
water, and extracted with EtOAc (3 × 30 mL). The organic extracts
were dried with Na2SO4, and the solvent was removed under
reduced pressure to obtain a residue that was purified by flash
column chromatography (EP:Et2O 90:10 f 80:20) to give 0.04 g
(yield 47%) of pure 3-(4-methoxyphenyl)-3,4-dihydro-2H-1,4-
benzothiazine 15d as colorless oil. 1H NMR (CDCl3): δ 2.48 (1H,
d, J ) 3.00 Hz, N-H), 3.26 (2H, d, J ) 5.70 Hz, CH2), 3.80 (3H,
s, OCH3), 4.85-5.00 (1H, m, CH), 6.90 (2H, d, J ) 8.78 Hz, H-3′,
H-5′), 7.25-7.30 (1H, m, H-6), 7.35 (2H, d, J ) 8.78 Hz, H-2′,
H-6′), 7.45-7.60 (2H, m, H-5, H-7), 8.20 (1H, dd, J ) 8.18 and
1.34 Hz, H-8). Anal. (C15H15NOS) C, H, N.
3-[4-(Methylthio)phenyl]-3,4-dihydro-2H-1,4-benzothiazine (15m).
Obtained by the procedure described for compound 15d, from 1-[4-
(methylthio)phenyl]-2-[(2-nitrophenyl)thio]ethanone 14m as a col-
orless oil in 57% yield. 1H NMR (CDCl3): δ 2.39 (1H, bs, N-H),
2.52 (3H, s, SCH3), 3.25-3.35 (2H, m, CH2), 4.85-5.00 (1H, m,
CH), 7.20-7.35 (5H, m, H-5, H-2′, H-3′, H-5′, H-6′), 7.45-7.65
(2H, m, H-6, H-7), 8.20 (1H, d, J ) 8.18 Hz, H-8). Anal.
(C15H15ClNS2) C, H, N.
Procedure for the Synthesis of Substituted 3-Phenyl-6-(triflu-
oromethyl)-2H-1,4-benzothiazines 7a,j. A suspension of 2-amino-
4-(trifluoromethyl)benzenethiol hydrochloride 2 (1.2 equiv) and
K2CO3 (3.0 equiv) in dry DMF (20 mL) was maintained at room
temperature under nitrogen atmosphere and magnetic stirring for
45 min, then a solution of appropriate 2-bromo-1-phenylethanone
(1.0 equiv) in the same solvent was added dropwise. When
2-bromo-1-phenylethanone disappeared (TLC), the mixture was
poured in water and extracted with EtOAc (3 × 50 mL). The
organic extracts were dried with Na2SO4, and the solvent was
removed under reduced pressure to obtain a residue that was purified
by flash column chromatography (EP:Et2O 95:5 f 50:50) to give
the target compounds.
3-Phenyl-6-(trifluoromethyl)-2H-1,4-benzothiazine (7a). Ob-
tained from 2-bromo-1-phenylethanone 5a as yellow solid in 43%
1
yield; mp 83.5-84 °C. H NMR (CDCl3): δ 6.04 (1H, s, H-2),
7.40-7.60 (6H, m, Ar-H and N-H), 7.92-7.98 (1H, m, H-5),
8.05-8.15 (2H, m, H-7 and H-8). Anal. (C15H10F3NS) C, H, N.
3-(4-Nitrophenyl)-6-(trifluoromethyl)-2H-1,4-benzothiazine (7j).
Obtained from 2-bromo-1-(4-nitrophenyl)ethanone 5j as orange
1
solid in 52% yield; mp 138.5-139 °C. H NMR (CDCl3): δ 3.75
(2H, s, CH2), 7.40-7.55 (2H, m, H-7 and H-8), 7.78-7.82 (1H,
m, H-5), 8.23 (2H, d, J ) 6.94 Hz, H-3′, H-5′), 8.38 (2H, d, J )
6.92 Hz, H-2′, H-6′). Anal. (C15H9F3N2O2S) C, H, N.
3-(4-Methoxyphenyl)-6-(trifluoromethyl)-2H-1,4-benzothiaz-
ine Hydrochloride (7d). A suspension of 2-amino-4-(trifluorom-
ethyl)benzenethiol hydrochloride 2 (0.37 g, 1.63 mmol) and
triethylamine (0.45 mL, 3.27 mmol) in Et2O/EtOH 50:50 (50 mL)
was maintained at room temperature under nitrogen atmosphere
and magnetic stirring for 15 min, then a solution of 2-bromo-1-
[4-(methyloxy)phenyl]ethanone 5d (0.25 g, 1.09 mmol) in the same
solvent was added dropwise. After 90 min, the mixture was filtrated
and organic solution was bubbled with HClg until the target
compound was completely precipitated as hydrochloride. The solid
was collected after crystallization with cycloesane/Et2O to give 3-(4-
methoxyphenyl)-6-(trifluoromethyl)-2H-1,4-benzothiazine hydro-
chloride 7d (0.28 g) as a yellow solid in 79% yield; mp 171-172.5
Supporting Information Available: Elemental analysis data for
final compounds and figure of PLS loading plot for properties
reported in Table 3 and percent of inhibition of EtBr efflux against
SA1199B (Activity). This material is available free of charge via
the Internet at http://pubs.acs.org.
1
°C. H NMR (CDCl3): δ 3.98 (3H, s, OCH3), 4.05 (2H, s, CH2),
7.17 (2H, d, J ) 8.93 Hz, H-2′, H-6′), 7.50-7.55 (2H, m, H-7,
H-8), 8.53 (2H, d, J ) 8.93 Hz, H-3′, H-5′), 8.82 (1H, s, H-5).
Anal. (C16H13ClF3NOS) C, H, N.

Benzothiazines as NorA Inhibitors
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 14 4329
(24) Vidaillac, C.; Guillon, J.; Arpin, C.; Forfar-Bares, I.; Ba, B. B.; Grellet,
J.; Moreau, S.; Caignard, D-H.; Jarry, C.; Quentin, C. Syntesis of
omeprazole analogues and evaluation of these as potential inhibitors
of the multidrug efflux pump NorA of Staphylococcus aureus.
Antimicrob. Agents Chemother. 2007, 51, 831–838.
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