4H-1,4-benzothiazine, dihydro-1,4-benzothiazinones and 2-amino-5- fluorobenzenethiol derivatives: Design, synthesis and in vitro antimicrobial screening

Article abstract of DOI:10.1002/ardp.201100309

As part of our studies focused on the design and synthesis of new antimicrobial agents a series of 7-fluoro-3,4-dihydro-2H-1,4-benzothiazine derivatives (4a-4f, 4h) and 7-fluoro-2H-1,4-benzothiazin-3(4H)-one analogues (4j-4o) were synthesized and evaluate

Full text of DOI:10.1002/ardp.201100309

Arch. Pharm. Chem. Life Sci. 2011, 000, 1–10
1
Full Paper
4H-1,4-Benzothiazine, Dihydro-1,4-benzothiazinones and
2-Amino-5-fluorobenzenethiol Derivatives: Design,
Synthesis and in vitro Antimicrobial Screening
Domenico Armenise, Marilena Muraglia, Marco Antonio Florio, Nicolino De Laurentis,
Antonio Rosato, Antonio Carrieri, Filomena Corbo, and Carlo Franchini
Department of Pharmaceutical Chemistry, University of Bari, Bari, Italy
As part of our studies focused on the design and synthesis of new antimicrobial agents a series of 7-
fluoro-3,4-dihydro-2H-1,4-benzothiazine derivatives (4a–4f, 4h) and 7-fluoro-2H-1,4-benzothiazin-3(4H)-
one analogues (4j–4o) were synthesized and evaluated for their in vitro inhibitory activity against a
representative panel of Gram-positive and Gram-negative bacteria strains and also toward selected
fungi species. These compounds were prepared in one step from chloro-substituted-2-amino-5-
fluorobenzenethiol 6a–6c. The biological screening identified in compounds 4a, 4j and 4l the
most promising results of both series showing an interesting antimicrobial activity. Our
antibiotic investigation was also completed by testing the key intermediates 6a–6c. Surprisingly,
6a–6c emerged as the compounds exhibiting the highest antimicrobial activity by possessing a
remarkable antibacterial effect against the Gram-positive strains with MIC (minimal inhibitory
concentration) values between 2 and 8 mg/mL and the fungi panel with MIC values between 2 and
8 mg/mL. These results may prove useful in the design of a novel pool of antimicrobial agents.
Keywords: 4H-1,4-Benzothiazine / Antibacterial activity / Antifungal activity
Received: September 2, 2011; Revised: October 19, 2011; Accepted: October 21, 2011
DOI 10.1002/ardp.201100309
Introduction
of the benzothiazine nucleus, in the recent past, we reported
several examples of benzothiazine derivatives bearing sub-
stituents on the heterocyclic ring able to exhibit both anti-
bacterial and antifungal activity (1, 2, 3, Figure 1) [2–9].
However to our knowledge, few data [2, 9] have been col-
lected to obtain comprehensive SAR indications about the
effects that the substituents on the benzothiazine nucleus
exert on the antibacterial and antifungal biological profile.
In this regard, several questions were raised: (i) how such a
significant effect was produced in the antibacterial activity
by the aromatic substituents (1, Figure 1) and (ii) what sig-
Bacterial resistance to antibiotics has become an increasingly
serious problem in recent years. Drug-resistant pathogens
have evolved the MRSA (methicillin-resistant Staphylococcus
aureus) and the VRSA (vancomycin-resistant Staphylococcus aur-
eus) phenotype. Unfortunately, bacterial resistance has also
emerged against newer agents such as linezolid and dapto-
mycin shortly after their use in clinic [1]. Therefore the
development of new and different antimicrobial drugs is a
very important goal and most of the research program efforts
in this field are directed towards the design of new agents.
It is well documented that the benzothiazine template is
generally recognized as a privileged structure in medicinal
chemistry to investigate both potentially antibacterial [2–9]
and anticancer [10–18] molecules. Looking at the importance
–
nificant effect could a C O moiety exert on the antimicrobial
–
activity in comparison with the R2 and R3 substituents?
(1 vs. 2, Figure 1). In addition, (iii) could dihalogenated deriva-
tives play a role in the antibacterial activity (3, Figure 1)?
In continuation to extend our research on the synthesis
and biological behaviour of 4H-1,4-benzothiazine analogues
as potential drugs for antimicrobial management, here we
have been interested in the preparation of new compounds
containing modified benzothiazine. In detail, in the present
investigation, three new different 4H-1,4-benzothiazine series
Correspondence: Filomena Corbo, Dipartimento Farmaco-Chimico,
`
Facolta di Farmacia, Universita di Bari, via Orabona 4, 70125 Bari, Italy.
E-mail: fcorbo@farmchim.uniba.it
Fax: þ390805442724
`
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D. Armenise et al.
Arch. Pharm. Chem. Life Sci. 2011, 000, 1–10
Figure 1. Initial 3,4-dihydro-2H-1,4-ben-
zothiazines and their derivatives.
(4a–4f, 4h, Table 1, Schemes 1 and 2) and two series of
dihydro-1,4-benzothiazinones (4j–4o, Table 2, Scheme 2) were
synthesized and evaluated. Although compounds 4b, 4c, 4k
and 4l are reported in the literature [2] they have been
included in our research program to screen their antimicro-
bial profile in order to have more data for the SAR proposal.
In relation with the above considerations and because in
the recent literature many benzimidazole, benzoxazole and
benzothiazole derivatives were regarded as promising classes
of bioactive heterocyclic compounds able to exhibit a range
MIC (minimal inhibitory concentration) of the benzothiazine
derivatives (4a–4o) and of compounds 5a–5c and 6a–6c
against different Gram-positive, Gram-negative and fungi
strains belonging to the American Type Culture Collection
(ATCC) was reported (Table 5) and the structure-activity
relationship (SAR) was also discussed. A molecular modeling
study was, at the end, also carried out to update insights into
the molecular determinants most largely affecting the
activity of our newly synthesized antimicrobial agents.
of biological activities [19–22], we recently reported a series of Results and discussion
2-mercapto-1,3-benzothiazole derivatives and their in vitro
antibacterial evaluation (Figure 2). [23] It is disclosed that
the SH moiety at the C2 position of the heterocyclic nucleus
led to a remarkable antibacterial activity.
Chemistry
The synthetic routes used to synthesize compounds 4a–f, 4h,
and 4j–o are reported in Schemes 1 and 2. Starting materials
6a–6c were prepared by hydrolytic basic cleavage of chloro-6-
fluoro-2-aminobenzothiazoles (5a–5c) commercially available
by following the synthetic pathway described in the litera-
Taking the above points in consideration, to confirm this
preliminary hypothesis we decided to include the isosteric
(NH2 instead of SH) derivatives (5a–5c, Table 3, Scheme 3) and
their reaction products (6a–6c, Table 4, Scheme 3), containing
the SH moiety, in our antibacterial screening.
The structures herein reported are easily prepared,
retained all main characteristics mentioned above and elu-
cidated by spectral data.
In order to enhance selectivity and potency for addressing
the pathological diversity of microbial human disorders, the
Scheme 1. 1,2-Dibromoethane, anhyd. CH3OH or anhyd. EtOH,
0.4 N CH3ONa or 0.65 N CH3ONa, N2, reflux.
Table 1. 7-Fluoro-3,4-dihydro-2H-1,4-benzothiazine derivatives.
F
S
Table 2. 7-Fluoro-2H-1,4-benzothiazin-3(4H)-one derivatives.
N
H
R
Cl
F
S
R
O
N
H
Cl
Compd
5
6
8
R
4a
4b2
4c2
4d
4e
4f
4g
4h
4i
Cl
H
H
H
Ph
Ph
Ph
CH3
CH3
CH3
Cl
Cl
Cl
Compd
5
6
8
R
Cl
Cl
Cl
Cl
Cl
4j
Cl
H
H
H
Ph
Ph
Ph
4k2
4l2
4m
4n
4o
Cl
Cl
Cl
Cl
Cl
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Arch. Pharm. Chem. Life Sci. 2011, 000, 1–10
Antibacterial and Antifungal Activity of 4H-1,4-Benzothiazine
3
Scheme 2. a) Step 1: 2-bromo-1-phenylethanone, anhyd. Et2O, N2, reflux, step 2: acetic acid, CH3OH, NaBH4, 08C; b) step 1:
1-chloroacetone, anhyd. Et2O, N2, reflux, step 2: acetic acid, CH3OH, NaBH4, 08C; c) chloroacetic acid, 2.5 N NaOH, H2O, reflux;
d) bromo(phenyl)acetic acid, 1.87 N EtONa, reflux.
Table 4. Structural formulae for Cl-substituted-2-amino-5-
fluorobenzenethiol.
SH
F
Figure 2. 2-Mercapto-1,3-benzothiazole derivatives [23].
NH
Cl
2
Compd
3
4
6
Table 3. Structural formulae for Cl-substituted-6-fluoro-1,3-
benzothiazol-2-amine.
6a
6b
6c
Cl
Cl
Cl
F
S
NH
2
N
Cl
ture (Scheme 3) [9]. In detail, 4a–4c (Scheme 1) were prepared
according to the method reported in the literature [9] by
using a different starting reagent. In fact, in order to improve
the yield, Cl-substituted-2-amino-5-fluorobenzenethiol 6a–6c
were converted to the corresponding Cl-substituted-7-fluoro-
3,4-dihydro-2H-1,4-benzothiazine 4a–4c starting from 1,2-
dibromoethane rather than 1-bromo-2-chloroethane. As
expected the cyclization reaction afforded heterocyclic
derivatives 4a–4c in higher yields than in the literature [9]
(54% vs. 28% for 4b, 50% vs. 20% for 4c).
Compd
4
5
7
5a
5b
5c
Cl
Cl
Cl
To obtain compounds 4d–4f (Scheme 2), compounds 6a–6c,
in anhyd. Et2O at reflux, were combined with 2-bromo-1-
phenylethanone. After work the crude reaction was first
treated with CH3COOH in MeOH, then with NaBH4 and finally
Scheme 3. Reagents and conditions: 50% NaOH for 24 h; reflux.
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D. Armenise et al.
Arch. Pharm. Chem. Life Sci. 2011, 000, 1–10
Table 5. Antimicrobial activitya) results (MIC mg/mL) of 4H-1,4-benzothiazine (4a–4f, 4h), dihydro-1,4-benzothiazinones (4j–4o) and 2-
amino-5-fluorobenzenethiol derivatives (6a–6c).
Microorganism
Gram positive
Gram negative
E. c. 25922 A. b. 19606
Fungi
C. p. 22019
Compd S. a. 6538P
E. f. 29212
B. s. 6633
C. a. 10231
C. t. 750
C. k. 6258
5a
5b
5c
6a
6b
6c
4a
4b
4c
4d
4e
4f
4h
4j
4k
4l
4m
4n
4o
NRF
OXA
AMB
FLU
256
512
512
8
4
8
32
R
64
R
128
R
125
16
512
250
125
250
500
0.5
0.250
256
256
256
16
4
8
64
R
128
R
256
R
250
32
512
250
64
250
500
4
512
256
256
4
4
2
16
R
64
R
64
R
64
16
R
R
256
512
R
R
R
R
R
128
R
R
R
512
256
512
64
16
R
R
32
R
R
R
n.e.
R
R
256
128
128
8
4
16
32
R
500
R
128
128
n.e.
500
512
250
250
250
R
64
64
64
2
2
4
256
128
128
4
4
8
32
500
500
R
256
128
n.e.
500
512
64
256
128
128
8
16
16
8
32
500
500
R
n.e.
n.e.
n.e.
256
256
n.e.
n.e.
512
n.e.
n.e.
n.e.
n.e.
64
32
n.e.
500
512
64
250
250
R
R
125
R
R
250
128
250
500
0.250
0.250
125
250
250
500
0.03
R
32
250
250
500
4
250
250
R
16
512
0.5
0.5
1
1
1
1
a)
Antimicrobial activity was estimated by using NCCLS assay [24]. Abbreviations: S. a., S. aureus; E. f., E. faecalis; B. s., B. subtilis; E. c., E. coli;
A. b., A. baumannii; C. a., C. albicans; C. p., C. parapsilosis; C. t., C. tropicalis; C. k., C. krusei; NRF, norfloxacin; OXA, oxacillin; AMB, amphotericin
B; FLU, fluconazole; R, resistant; n.e., not evaluated.
basified with NaOH pellets to yield the desired compounds
4d–4f in good overall yields. We next attempted to transfer
the synthetic route described above to obtain the 4g–4i
(Scheme 2) series using 1-chloroacetone as starting material.
This reaction pathway gave only the 4h derivative in very low
yield (11%). 7-Fluoro-2H-1,4-benzothiazin-3(4H)-one analogues
4j–4l and 4m–4o were respectively synthesized in good yields
by reaction of the respective chloro-substituted-2-amino-5-
fluorobenzenethiol 6a–6c with chloroacetic acid in NaOH
or with chloro(phenyl)acetic acid in EtONa (Scheme 2).
The structures of the obtained compounds were fully sup-
ported through their spectroscopic data (IR, 1H NMR, and
MS). Further details about the synthetic procedures are
reported in the Experimental section.
the selected molecules were also tested against a panel of
fungi strains (Candida albicans 10231, Candida parapsilosis
22019, Candida tropicalis 750 and Candida krusei 6258).
Norfloxacin, oxacillin, amphothericin B and fluconazole
were used as references drugs. The results expressed as
MIC (mg/mL) are listed in Table 5.
The combined data showed that the screened compounds
exerted inhibitory activity against the tested bacterial strains
with MIC values between 2 and 512 mg/mL. The obtained
results generally indicate that the majority of these series
of compounds are more active against Gram-positive than
Gram-negative bacteria. It is worth noting that among the
selected fungi, Candida parapsilosis growth seems to be especi-
ally affected by the tested molecules, ranging from 2 to
512 mg/mL.
Biological evaluation
Among both mentioned series 4a–4o, the most promising
results were recorded for 4a, 4j and 4l derivatives as showed
in Table 5.
According to NCCLS guidelines [24] these compounds were
tested against an assortment of Gram-positive and Gram-
negative bacteria belonging to the ATCC collection
(Staphylococcus aureus 6538P, Enterococcus faecalis 29212,
Bacillus subtilis 6633, Escherichia coli 25922, Acinetobacter bauman-
nii 19606) to evaluate their antibacterial profile. In addition,
In detail, among 7-fluoro-3,4-dihydro-2H-1,4-benzothiazine
derivatives (4a–4f, 4h), 4a resulted in a significant potency
against both Gram-positive and fungi species. In particular,
4a revealed its best antimicrobial activity against
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Arch. Pharm. Chem. Life Sci. 2011, 000, 1–10
Antibacterial and Antifungal Activity of 4H-1,4-Benzothiazine
5
Staphylococcus aureus (MIC: 32 mg/mL), Bacillus subtilis (MIC:
16 mg/mL) and Candida parapsilosis (MIC: 8 mg/mL).
Interestingly, the 4b analogue did not demonstrate an anti-
microbial profile, while isomer 4c only slightly affected
Acinetobacter baumannii growth (MIC: 32 mg/mL). The above
results suggest that a chlorine atom at the C5 position of
the 7-fluoro-3,4-dihydro-2H-1,4-benzothiazine nucleus could
influence the antimicrobial activity. A comparison of 7-flu-
oro-3,4-dihydro-2H-1,4-benzothiazine derivatives 4d–4f and
4h with the 4a–4c series revealed that the introduction of
a methyl or a phenyl group at the C3 position of the hetero-
cycle results in a significant decrease of the antimicrobial
activity.
possessing a remarkable antibacterial effect against Gram-
positive strains with MIC values between 2 and 8 mg/mL and
against the fungi panel with MIC values between 2 and
8 mg/mL. It must be underlined that we found significant
antibacterial activity of 6c against Bacillus subtilis (MIC:
2 mg/mL) and Gram-negative Acinetobacter baumannii (MIC:
16 mg/mL). Moreover, the broad-spectrum antifungal activity
registered for 6b with MIC values between 2 and 16 mg/mL is
noteworthy.
In summary, comparing the overall antimicrobial
trend of the 6a–6c series with the 4a–4o series it could be
hypothesized that the lesser steric hindrance associated to
the SH moiety could be more favourable for inducing both
antibacterial and antifungal activity.
Next, antimicrobial profile was measured for 7-fluoro-2H-
1,4-benzothiazin-3(4H)-one analogues (4j–4o). Among this
series, 4j, bearing a chlorine atom at the C5 position of
the heteronucleus, exhibited a good antibacterial activity
against Staphylococcus aureus (MIC: 16 mg/mL), and Bacillus sub-
tilis (MIC: 16 mg/mL). Moreover, compound 4l, having a
chlorine atom at the C8 position of the heterocycle displayed
a moderate activity against Gram-negative Acinetobacter bau-
mannii (MIC: 32 mg/mL) and the fungi Candida parapsilosis and
Candida tropicalis with MIC values of 64 mg/mL. Interestingly,
introduction of a phenyl group at the C2 position of the 7-
fluoro-2H-1,4-benzothiazin-3(4H)-one analogues (4m–4o) led
to a decrease of the antimicrobial profile.
Molecular modelling
As a final task of this study, a molecular modelling study was
carried out to better perceive and evaluate the biological
profile of most interesting compounds emerged from the
experimental assays. In detail, the remarkable antifungal
activity of 4a, but especially 6b, was interpreted through
docking experiments carried out on an assumed target for
our antifungal agents represented by the cytochrome P450-
dependent lanosterol 14a-demethylase (CYP51) of Candida
albicans.
The sterols-biosynthesis pathway, and therefore the
enzymes specifically involved, are indeed already known to
be targeted by compounds such as azoles (i.e. fluconazole and
miconazole), but also by other drugs characterized by a differ-
ent molecular scaffold (i.e. benzothiazines and benzoxazine)
[25–27], responsible for the cell growth inhibition determined
by the depletion of ergosterol due to CYP51 inhibition.
In order to accomplish our task, 4a and 6b were docked into
the catalytic site of a homology based model of Candida albicans
CYP51. The choice of this theoretical structure was supported by
the evidence that the same has been proved to be a robust tool
for understanding the molecular basis for antifungal activity of
a previously published series of 1,4-benzothiazines [6–7].
The binding poses achieved throughout the docking simu-
lations highlighted some interesting features that might be
in charge of the antifungal activity of our newly synthesized
compounds: as it might be perceived from Figure 3, both 4a
and 6b are capable to accommodate the active site of CA-
CYP51 mainly anchoring to the heme group with the fluorine
atom. For both the examined compounds, the same atom is
On the basis of the biological data until here discussed, due
both to the small numbers of evaluated series and to the low
chemical diversity, an attempt to analyse the structure
activity relationships does not seem obvious. Anyway, several
comparison on the results led us to hypothesize that the
insertion of a steric bulk as methyl or phenyl group on
the heteronucleus could be detrimental to antimicrobial
activity. On the contrary, the chlorine atom could play a role
in the antimicrobial activity even if its effect does not depend
on the relative position on the heterocyclic ring. Generally
among the analyzed series, compared with 7-fluoro-2H-1,4-
benzothiazin-3(4H)-one analogues (4j–4o, Table 2), 7-fluoro-
3,4-dihydro-2H-1,4-benzothiazine derivatives (4a–4f, 4h,
Table 1) showed a higher antimicrobial profile suggesting
–
that a C O moiety exerts a low ability to inhibit bacterial and
–
fungal growth.
Then, these preliminary results led us to include com-
pounds 5a–5c and intermediates 6a–6c in our biological
screening with the aim to generate a new pool of antimicro-
bial molecules. Interesting, the 2-mercapto-1,3-benzothiazole
derivatives (Figure 2) isosters 5a–5c essentially lost antimi-
crobial activity. These data are consistent with our previous
findings [23] that the SH moiety is clearly essential for the
antibacterial profile.
˚
placed at 1.9 A distance from the iron atom generating a
highly favorable coordination bond (ꢀ27.59 and ꢀ26.97 kcal/mol
respectively), with the rest of the molecular skeleton being
almost perpendicular to the plane defined by the heme
group. It has to be pointed out that SAR already outlined
the value of this kind of substitution, since the unsubstituted
derivatives showed a very low antifungal profile [3], moreover
Surprisingly, as shown in Table 5, 6a–6c emerged as the
compounds exhibiting the highest antimicrobial activity by
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Arch. Pharm. Chem. Life Sci. 2011, 000, 1–10
should be basically referred to its lipophilicity, and therefore
to the capability of passing the cell membrane, as suggested
by the calculated partition coefficient values. In fact, corre-
spondingly CLogP for 4a and 6b are 2.41 and 2.85, while the
deschloro analogues data are well below an optimal value of
2.0, especially for the latter compounds.
Then, from this modeling study it emerged a plausible
mechanism of inhibition of CA-CYP51 elicited by our com-
pounds, suggesting a mandatory role of a fluorine properly
placed on the aromatic ring of the molecule, but at the same
time showing the need of additional functional groups for a
better antifungal activity. In this context, the synthesis of novel
derivatives with a correct volume and hindrance required to
fulfill the binding site cavity is our ongoing project.
Conclusions
In summary in this study we report on the synthesis and
antimicrobial activity of 7-fluoro-3,4-dihydro-2H-1,4-benzo-
thiazine derivatives (4a–4f, 4h, Table 1) and 7-fluoro-2H-1,4-
benzothiazin-3(4H)-one analogues (4j–4o, Table 2). Additional
investigation was realized on Cl-substituted-6-fluoro-1,3-ben-
zothiazol-2-amine (5a–5c) and Cl-substituted-2-amino-5-fluo-
robenzenethiol (6a–6c).
Among the 7-fluoro-3,4-dihydro-2H-1,4-benzothiazine
derivatives (4a–4f, 4h), 4a exhibited highly potent antimicro-
bial activity against Gram-positive bacteria and fungi strains.
Its MIC values toward Bacillus subtilis (MIC: 16 mg/mL) and
Candida parapsilosis (MIC: 8 mg/mL) are very interesting.
Derivatives 4d–f and 4h containing the sterically bulky
phenyl and methyl, respectively, at the C3 position of the
heterocyclic ring showed weak antimicrobial activity com-
pared to the 4a–4c analogues.
Among series 4j–4o, the most significant data were
obtained for 4j and 4l. Significant antibacterial activity
against Bacillus subtilis (MIC: 16 mg/mL) was recorded for com-
pound 4j. 4l showed an interesting activity against Gram-
negative Acinetobacter baumannii (MIC: 32 mg/mL). Compounds
4m–4o, bearing a phenyl at the C2 position of the hetero-
cycle, are only weakly active against the bacteria and fungi
panels.
Figure 3. Docking poses for 4a (top) and 6b (bottom) in the active
site of CA-CYP51. Ligands and iron atom are displayed as ball and
stick to help interpretation.
a similar interaction pattern has been also experimentally
observed in the X-ray structure of fluconazole with CYP51 of
Mycobacterium tuberculosis (PDB code: 1EA1, 1E9X) selected as
template for the comparative building of CA-CYP51 [28].
Additional non-bonded interactions between the cyclic
moieties of our antifungal agents and a cluster of nonpolar
residues, comprising Tyr118, Leu121, Phe228 and Leu376,
defining a hydrophobic dome placed above the heme group,
are also detected, nonetheless with relatively low contri-
bution to the binding energy (ꢀ16.51 and ꢀ13.60 kcal/mol
of 4a and 6b respectively) while no significant weight can be
ascribed to the chlorine atoms, since in both dockings no
direct involvement was observed. On the other hand, it might
be postulated that the valence of this molecular element
Rather surprisingly, Cl-substituted-2-amino-5-fluorobenze-
nethiol derivatives (6a–6c) are the most promising com-
pounds of all series described in this study, having a
strong activity against both bacteria and fungi strains (MIC
values between 2 and 16 mg/mL). The high antibacterial effect
of 6c toward Bacillus subtilis (MIC: 2 mg/mL) and the antifungal
activity of 6b against Candida parapsilosis (MIC: 2 mg/mL) are
worthy to note.
Then, taking into account that this preliminary study does
not produce conclusive evidences about a structure-antibac-
terial relationship the results presented in this work suggest
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Arch. Pharm. Chem. Life Sci. 2011, 000, 1–10
Antibacterial and Antifungal Activity of 4H-1,4-Benzothiazine
7
that a) the 7-fluoro-3,4-dihydro-2H-1,4-benzothiazine nucleus
rather than 7-fluoro-2H-1,4-benzothiazin-3(4H)-one, b) the SH
moiety and c) the chlorine atom on the aromatic ring may
positively favor an antimicrobial profile. On the contrary, an
increase of the steric hindrance on the heterocycle probably
could lead to a loss of antimicrobial activity.
layer was separated, dried over anhyd. Na2SO4 and evapor-
ated under reduced pressure. The residue was purified by
silica gel column-chromatography with petroleum ether/
EtOAc (8:2 v/v) to provide compound 4a as a brown oil.
Yield: 56%; 1H NMR (300 MHz, CDCl3): d 3.03–3.08 (m, 2H,
SCH2), 3.65–3.68 (m, 2H, NCH2), 3.9 (s, br, 1H, NH), 6.68 and
6.70 (dd, 1H, J ¼ 3.2 Hz, H6), 6.78 and 6.80 (dd, 1H,
J ¼ 3.2 Hz, H8). MS (70 eV) m/z (%) 203 (Mþ), 188 (100).
In light of the data herein discussed we focussed our
attention on the most promising series 6a–6c. Then, in our
ongoing research program to the synthesis of new antimi-
crobial agents, several synthetic routes are in progress to
acquire more data for guiding the further rational design
of 2-amino-5-fluorobenzenethiol analogues to serve as a new
cluster for the antibacterial and antifungal investigation.
Synthesis of 6-chloro-7-fluoro-3,4-dihydro-2H-1,4-
benzothiazine (4b) [2]
2-Amino-4-chloro-5-fluorobenzenethiol (6b) (1 mmol) in etha-
nol (10 mL) and 1,2-dibromoethane (4.3 mmol) was heated at
reflux under nitrogen, for 1.5 h. Then 0.65 N CH3ONa (10 mL)
was added and heating was continued for 2 h. After cooling
to room temperature, the reaction mixture was diluted with
2 N NaHCO3 (10 mL) and extracted with CH2Cl2. The organic
layer was separated, dried over anhyd. Na2SO4 and then
evaporated under reduced pressure. The crude product was
separated by column-chromatography on silica gel by using
light petroleum ether and EtOAc (7:3 v/v) as eluent to obtain
the pure compound 4b as a grey solid.
Experimental section
Chemistry
Melting points were recorded on a Gallenkamp melting point
apparatus in open glass capillary tubes. The IR spectra were
recorded on a Perkin-Elmer Spectrum One FT spectropho-
tometer and band positions were given in reciprocal centi-
meters (cmꢀ1). 1H NMR spectra were recorded on a FT Bruker
Aspect 3000 spectrometer using CDCl3 as the solvent, unless
otherwise indicated. Chemical shifts were reported in part
per million (ppm) relative to solvent resonance: CDCl3, d 7.26
(1H NMR). Amino proton assignments were confirmed
by D2O exchange. J values are given in Hz. EIMS spectra were
Mp is in agreement with the published data [2].
Yield: 54%; IR (nujol mull): 3410 cmꢀ1; 1H NMR (300 MHz,
CDCl3): d 3.00–3.06 (m, 2H, SCH2), 3.55–3.61 (m, 2H, NCH2),
3.92 (s, br, 1H, NH), 6.46 (d, 1H, J ¼ 6.3 Hz, H5), 6.77 (d, 1H,
J ¼ 9.0 Hz, H8); MS (70 eV) m/z (%) 203 (Mþ), 188 (100), 135,
113.
recorded with
a Hewlett-Packard 6890-5973 MSD gas
chromatograph/mass spectrometer at low resolution. Silica
gel chromatographic separations were performed by chroma-
tography with silica gel (Kieselgel 60, 40–63 mm, Merck)
packed in glass columns, using the technique described in
the literature [29]. The weight of the silica gel was approxi-
mately 100 times that of the substance. The eluting solvent
indicated in parentheses for each purification was deter-
mined by TLC, which was performed on precoated silica
gel on aluminum sheets (Kieselgel 60, F254, Merck). TLC plates
were visualized with UV light and/or in an iodine chamber.
All chemicals were purchased from Aldrich Chemical Co.
in the highest quality commercially available. The structures
of the compounds were confirmed by routine spectrometric
and spectroscopic analyses. Only spectra for compounds not
previously described are given.
Synthesis of 8-chloro-7-fluoro-3,4-dihydro-2H-1,4-
benzothiazine (4c) [2]
The title compound was prepared starting from 6-amino-2-
chloro-3-fluorobenzenethiol (6c) according to the synthetic
protocol described for 4b. After standard workup, the desired
product was isolated as a brown oil.
Yield: 49%; IR (nujol mull): 3377 cmꢀ1; 1H NMR (300 MHz,
CDCl3): d 3.08–3.15 (m, 2H, SCH2), 3.52–3.60 (m, 2H, NCH2),
3.70 (s, br, 1H, NH), 6.34–6.42 (m, 1H, H5), 6.72 (t, 1H,
J ¼ 8.2 Hz, H6); MS (70 eV) m/z (%) 203 (Mþ), 188 (100).
General procedure for the synthesis of 7-fluoro-3,4-
dihydro-2H-1,4-benzothiazine derivatives (4d–f)
The preparation of 5-chloro-7-fluoro-3-phenyl-3,4-dihydro-2H-
1,4-benzothiazine (4d) can be taken as reference for the
synthesis of 7-fluoro-3,4-dihydro-2H-1,4-benzothiazine deriva-
tives (4d–f).
Synthesis of 5-chloro-7-fluoro-3,4-dihydro-2H-1,4-
benzothiazine (4a)
2-Amino-3-chloro-5-fluorobenzenethiol (6a) (1 mmol) in
methanol (10 mL) and 1,2-dibromoethane (8.8 mmol) were
stirred at reflux under N2 atmosphere for 15 min. Then 0.4 N
CH3ONa (20 mL) was added dropwise over 10 min. After cool-
ing to room temperature, the reaction mixture was quenched
with 2 N NaHCO3 and extracted with EtOAc. The organic
A mixture of 6a (1 mmol) and 2-bromo-1-phenylethanone
(1 mmol) in anhydrous Et2O (30 mL) was heated at reflux
under N2 atmosphere for 30 min. After cooling to room
temperature, 2 N NaHCO3 (10 mL) was added in one portion.
Then the reaction mixture was extracted with CH2Cl2. The
obtained organic layer was separated, dried over anhyd.
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8
D. Armenise et al.
Arch. Pharm. Chem. Life Sci. 2011, 000, 1–10
Na2SO4 and concentrated under reduced pressure. A mixture
of CH3COOH and methanol (30 mL, 1:1) at 08C was carefully
added to the crude residue. Subsequently, NaBH4 (4.84 mmol)
was added portionwise under stirring. The mixture was
diluted with H2O and then basified with NaOH pellets
(pH ffi 11). The basic layer was extracted with CHCl3
(3 Â 30 mL). Organic layers were dried over anhyd. Na2SO4
and concentrated in vacuum. The residue was purified by
silica gel column-chromatography (petroleum ether/EtOAc,
8:2) to afford the desired compound 4d as a brown oil.
Yield: 10%; 1H NMR (300 MHz, CDCl3): d 3.12 (d, 2H,
J ¼ 4.7 Hz, SCH2), 4.35 (s, l, 1H, NH), 4.63 (s, 1H, CH-Ph),
6.75 and 6.95 (dd, 1H, J ¼ 8.3 Hz, H6), 6.84 and 6.87 (dd,
1H, J ¼ 10.7 Hz, H8), 7.35–7.44 (m, 5H, H-Arom); MS
(70 eV) m/z (%) 279 (Mþ), 264, 211, 188 (100).
(3 Â 30 mL). Organic layers were dried over anhyd. Na2SO4
and concentrated in vacuum. The residue was purified by
silica gel column-chromatography (petroleum ether/EtOAc,
7:3) to provide compound 4h as an orange solid.
Yield: 12%; mp 212–2148C (EtOAc); IR (nujol mull): 3351
cmꢀ1
;
1H NMR (300 MHz, CDCl3): d 1.30 (d, 3H, J ¼ 6.2 Hz,
CH3), 2.7–2.9 (m, 2H, CH2), 3.6–3.7 (m, 2H, CH), 3.77 (s, br, 1H,
NH), 6.45 (d, 1H, J ¼ 7.1 Hz, H5), 6.81 (d, 1H, J ¼ 9.4 Hz, H8).
MS (70 eV) m/z (%) 217 (Mþ), 202 (100).
General procedure for the synthesis of 7-fluoro-2H-1,4-
benzothiazin-3(4H)-one derivatives (4j–l)
The preparation of 5-chloro-7-fluoro-2H-1,4-benzothiazin-
3(4H)-one (4j) can be taken as reference for the synthesis of
7-fluoro-2H-1,4-benzothiazin-3(4H)-one derivatives (4j–l).
2-Amino-3-chloro-5-fluorobenzenethiol (6a) (1 mmol) in
2.5 N NaOH (15 mL) was heated under reflux for 30 min.
6-Chloro-7-fluoro-3-phenyl-3,4-dihydro-2H-1,4-
benzothiazine (4e)
Then
a
solution of chloroacetic acid (2 mmol) in
The title compound was prepared by using the corresponding
benzenethiol derivative 6b as starting material. The synthetic
procedure provided 4e as a brown oil.
H2O (3.5 mL) was added dropwise. The reflux was continued
for 1.5 h. The reaction mixture was then cooled at room
temperature and extracted with EtOAc. The organic layer
was dried over anhyd. Na2SO4 and concentrated under
reduced pressure.
Yield: 17%; IR (nujol mull): 3379, 1645 cmꢀ1 1H NMR
;
(300 MHz, DMSO-d6): d 2.9–3.3 (m, 2H, CH2), 4.0–4.2 (m, 1H,
CH2–CH), 4.07 (s, br, 1H, NH), 6.53 (d, 1H, J ¼ 6.0 Hz, H5), 6.86
(d, 1H, J ¼ 8.8 Hz, H8), 7.4–7.6 (m, 5H, H-Arom); MS (70 eV) m/z
(%) 279 (Mþ), 264, 211, 188 (100).
The crude residue was purified by silica gel column-
chromatography (petroleum ether/EtOAc, 7:3) to afford 4j
as a yellow solid.
8-Chloro-7-fluoro-3-phenyl-3,4-dihydro-2H-1,4-
benzothiazine (4f)
5-Chloro-7-fluoro-2H-1,4-benzothiazin-3(4H)-one (4j)
Yield: 25%; mp 209–2128C (EtOAc); IR (nujol mull): 3183, 1666
The title compound was prepared by using the corresponding
benzenethiol derivative 6c as starting material. The synthetic
procedure provided 4f as a brown oil.
Yield: 25%; 8C; IR (nujol mull): 3414, 1598 cmꢀ1; H NMR
cmꢀ1 1H NMR (300 MHz, CDCl3): d 3.42 (m, 2H, CH2), 6.92
;
(d, 1H, JH-F ¼ 6.11 Hz, H5), 7.12 (d, 1H, JH-F ¼ 8.33 Hz, H8), 8.72
(s, br, 1H, NH); MS (70 eV) m/z (%) 217 (Mþ, 100), 188, 172, 113.
1
(300 MHz, DMSO-d6): d 3.16 (d, 2H, J ¼ 8.6 Hz, CH2), 4.10 (s, br,
1H, NH), 4.52 and 4.56 (dd, 1H, J ¼ 2.4 Hz, CH2–CH), 6.38–6.42
(d, 1H, J ¼ 5.0 Hz, H5), 6.75 (t, 1H, J ¼ 8.8 Hz, H6), 7.3–7.4 (m,
5H, H-Arom); MS (70 eV) m/z (%) 279 (Mþ), 264, 211, 188 (100).
6-Chloro-7-fluoro-2H-1,4-benzothiazin-3(4H)-one (4k) [2]
The title compound was prepared by using the corresponding
benzenethiol derivative 6b as starting material. The synthetic
procedure provided 4k as a yellow solid.
Mp is in agreement with the published data [2].
Yield: 25%; IR (nujol mull): 3184, 1667 cmꢀ1 1H NMR
;
6-Chloro-7-fluoro-3-methyl-3,4-dihydro-2H-1,4-
benzothiazine (4h)
(300 MHz, CDCl3): d 3.48 (m, 2H, CH2), 6.73 and 6.77 (dd,
1H, J ¼ 6.10 Hz, H6), 6.98 (t, 1H, J ¼ 8.30 Hz, H5), 8.60 (s,
br, 1H, NH); MS (70 eV) m/z (%) 217 (Mþ, 100), 188, 172, 113.
A
mixture of 6b (1 mmol) and 1-chloropropan-2-one
(1.5 mmol) in anhydrous Et2O (30 mL) were heated under
reflux in an N2 atmosphere for 30 min. After cooling to room
temperature, 2 N NaHCO3 (10 mL) were added in one portion.
Then the reaction mixture was extracted with CH2Cl2. The
obtained organic layer was separated, dried over anhyd.
Na2SO4 and concentrated under reduced pressure. A mixture
of CH3COOH and methanol (30 mL, 1:1) at 08C was carefully
added to the crude residue. Subsequently, NaBH4 (4.84 mmol)
was added portionwise under stirring. The mixture was
diluted with H2O and then basified with NaOH pellets
(pH ffi 11). The basic layer was extracted with CHCl3
8-Chloro-7-fluoro-2H-1,4-benzothiazin-3(4H)-one (4l) [2]
The title compound was prepared by using the corresponding
benzenethiol derivative 6c as starting material. The synthetic
procedure provided 4l as a green solid.
Mp is in agreement with the published data [2].
Yield: 32%; IR (nujol mull): 3140, 1678 cmꢀ1 1H NMR
;
(300 MHz, CDCl3):
d 3.42 (m, 2H, CH2), 6.90 (d, 1H,
J ¼ 7.0 Hz, H8), 7.12 (d, 1H, J ¼ 8.8 Hz, H5), 8.15 (s, br, 1H,
NH); MS (70 eV) m/z (%) 217 (Mþ, 100), 188, 172, 113.
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Arch. Pharm. Chem. Life Sci. 2011, 000, 1–10
Antibacterial and Antifungal Activity of 4H-1,4-Benzothiazine
9
General procedure for the synthesis of 7-fluoro-2H-1,4-
benzothiazin-3(4H)-one derivatives (4m–o)
Stock solutions of the tested compounds were obtained in
DMSO. Stock solutions of lower concentrations were pre-
pared for those substances which did not dissolve well.
Then two-fold serial dilutions in the suitable test medium
between 512 and 0.5 mg/mL were plated. To be sure that the
solvent had no adverse effect on bacterial growth, a control
test was carried out by using ethanol at its maximum con-
centration along with the medium.
The preparation of 5-chloro-7-fluoro-2-phenyl-2H-1,4-benzo-
thiazin-3(4H)-one (4m) can be taken as reference for the syn-
thesis of 7-fluoro-2H-1,4-benzothiazin-3(4H)-one derivatives
(4m–o). 2-Amino-3-chloro-5-fluorobenzenethiol (6a) (1 mmol)
in 1.87 N EtONa (5 mL) was heated under reflux for 30 min.
Then, a solution of bromo(phenyl)acetic acid (1.5 mmol) was
added dropwise over 10 min. The reaction mixture was
stirred for 24 h at reflux and subsequently quenched
with H2O (5 mL) and then basified with NaOH pellets. The
aqueous phase was extracted with CHCl3 (3 Â 25 mL). The
combined organic layers were dried over anhyd. Na2SO4 and
concentrated in vacuum. Purification of the crude product by
silica gel chromatography (petroleum ether/EtOAc, 1:1) led to
the pure compound 4m as a yellow solid.
Bacteria strains available as freeze-dried discs, belonging to
the ATCC collection, were used: Gram-positive strains such as
Staphylococcus aureus 6538P, Enterococcus faecalis 29212, Bacillus
subtilis 6633, and Gram-negative ones such as Escherichia coli
25922, Acinetobacter baumannii 19606.
To preserve the purity of cultures and to allow the repro-
ducibility, a series of cryovials of all microbial strains in
glycerolic medium was set up and stored at ꢀ808C.
Pre-cultures of each bacterial strain were prepared in
Cation Adjusted Mueller-Hinton broth (CAMHB) and incu-
bated at 378C until the growth ceased. The turbidity of
bacterial cell suspension was calibrated to 0.5 McFarland
Standard by spectrophotometric method (625 nm, range
0.08–0.10), and further the standardized suspension was
diluted 1:100 with CAMHB to have 1–2 Â 106 CFU/mL (rif. a).
All wells were seeded with 100 mL of inoculums.
5-Chloro-7-fluoro-2-phenyl-2H-1,4-benzothiazin-3(4H)-
one (4m)
Yield: 60%; mp 210–2128C (EtOAc); IR (nujol mull): 3188, 1668
1
cmꢀ1; H NMR (300 MHz, CDCl3): d 4.25 (s, 1H, CH), 7.23 (d,
1H, JH-F ¼ 8.65 Hz, H6), 7.32 (d, 1H, JH-F ¼ 12 Hz, H8),
7.15–7.20 (m, 5H, H–Arom.), 9.2 (s, br, 1H, NH); MS
(70 eV) m/z (%) 293 (Mþ, 100), 264, 188, 172.
A number of wells containing only inoculated broth as
control growth were prepared.
6-Chloro-7-fluoro-2-phenyl-2H-1,4-benzothiazin-3(4H)-
one (4n)
The plates were incubated at 378C for 24 h, and the MIC
values were recorded as the last well containing no bacterial
growth. The MIC were determined by using an antibacterial
assayed repeated twice in triplicate.
The title compound was prepared by using the corresponding
benzenethiol derivative 6b as starting material. The synthetic
procedure provided 4n as a yellow solid.
Yield: 79%; mp 210–2158C (EtOAc); IR (nujol mull): 3130,
Norfloxacin and oxacillin were used as standard drugs.
1668 cmꢀ1 1H NMR (300 MHz, CDCl3): d 4.72 (s, 1H, CH),
;
6.68–6.75 (m, 1H, H5), 6.97 (t, 1H, JH-F ¼ 8.7 Hz, H8), 7.28–7.40
(m, 5H, H–Arom.), 8.2 (s, br, 1H, NH); MS (70 eV) m/z (%) 293
(Mþ, 100), 264, 188, 172.
Antifungal activity
Four yeast strains (ATCC) were used: Candida albicans 10231,
Candida parapsilosis 22019, Candida tropicalis 750 and Candida
krusei 6258.
8-Chloro-7-fluoro-2-phenyl-2H-1,4-benzothiazin-3(4H)-
one (4o)
Pre-cultures of each yeast strain were prepared in
Sabouraud broth 2% glucose (SAB), and incubated at 358C
until the growth ceased. The turbidity of yeast stock suspen-
sion was calibrated to 0.5 McFarland Standard by spectropho-
tometric method (530 nm, range 0.12–0.15), and further the
standardized suspension was diluted first 1:50 with SAB ad
then 1:20 in the same medium to have 1–5 Â 106 CFU/mL
(rif. b).
The title compound was prepared by using the corresponding
benzenethiol derivative 6c as starting material. The synthetic
procedure provided 4o as a yellow solid.
Yield: 40%; mp 189–2018C (EtOAc); IR (nujol mull): 3140,
1670 cmꢀ1 1H NMR (300 MHz, CDCl3): d 4.72 (s, 1H, CH),
;
6.7–6.9 (m, 5H, H–Arom.), 7.2–7.4 (m, 2H, H5 þ H6), 9.4 (s, br,
1H, NH); MS (70 eV) m/z (%) 293 (Mþ, 100), 264, 188, 172.
All wells were seeded with 100 mL of inoculums.
A number of wells containing only inoculated broth as
control growth were prepared.
Biology
The in-vitro minimum inhibitory concentrations (MICs,
mg/mL) of the prepared compounds were assessed by the
broth microdilution method, using 96-well plates, according
to the National Committee for Clinical Laboratory Standard
(CLSI) formerly NCCLS.
The plates were incubated at 358C for 24–48 h, and the MIC
values were recorded as the last well containing no fungal
growth.
The MIC were determined by using an antifungal assay
repeated twice in triplicates.
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D. Armenise et al.
Arch. Pharm. Chem. Life Sci. 2011, 000, 1–10
[9] D. Armenise, N. De Laurentis, A. Rosato, F. Morlacchi,
J. Heterocyclic Chem. 2006, 43, 1371–1378.
Amphotericin B and fluconazole were used as standard
drugs.
[10] R. R. Gupta, R. Kumar, R. K. Gutam, Journal of Fluorine
Chemistry 1985, 28, 381–385.
Molecular modeling
[11] R. R. Gupta, R. Kumar, Journal of Fluorine Chemistry 1986, 31,
19–24.
Dockings were carried out by means of the Glue model
implemented in the Grid software [30]. The binding site
was defined as 10 Â 10 Â 10 cubic box centering on the iron
atom of the heme group. Molecular interaction maps were
calculated using a set of properly selected probes reproduc-
ing the molecular fragments of the examined compounds,
namely CL, F, S1, STH, N1, N2 and DRY according to the Grid
atoms code. The maximum number of sites and interactions
was set to 10 000, while the minimum energy change was
0.001 kcal/mol. Electrostatic contribution was considered,
and the pose scored with the best binding affinity was
selected. CLogP were calculated with ACD/ChemSketch
v.12.01.
[12] R. R. Gupta, A. Thomas, H. K. Gautam, V. Gupta, Journal of
Fluorine Chemistry 1989, 44, 1–14.
[13] V. Gupta, R. R. Gupta, Journal fuer Praktische Chemie 1991, 333,
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This work was accomplished thanks to the financial support of the
Ministero dell’Istruzione, dell’Universita` e della Ricerca (MIUR).
[18] Park Chang Ha, Yous Said, Nativelle Serpentini Celine,
Seralini Gilles Eric, Chang Soon Jae, Lesieur Daniel (Yang
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Patent No. FR 2860235, Application: FR 2003-11397.
The authors kindly acknowledge Prof. Antonio Macchiarulo for supplying
the CA-CYP51 structure.
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The authors have declared no conflict of interest.
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ß 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com