Hybrid molecules between benzenesulfonamides and active antimicrobial benzo[d]isothiazol-3-ones

Article abstract of DOI:10.1016/j.ejmech.2008.07.022

Novel hybrid molecules between benzenesulfonamides and active antimicrobial 2-amino-benzo[d]isothiazol-3-ones were synthesized and characterised and their in vitro antimicrobial activity was evaluated by the minimal inhibitory concentration (MIC). The com

Full text of DOI:10.1016/j.ejmech.2008.07.022

European Journal of Medicinal Chemistry 44 (2009) 2741–2747
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Laboratory note
Hybrid molecules between benzenesulfonamides
and active antimicrobial benzo[d]isothiazol-3-ones
*
Franca Zani , Matteo Incerti, Rocco Ferretti, Paola Vicini
`
Dipartimento Farmaceutico, Universita degli Studi di Parma, Viale G.P. Usberti 27/A, 43100 Parma, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Novel hybrid molecules between benzenesulfonamides and active antimicrobial 2-amino-benzo[d]iso-
thiazol-3-ones were synthesized and characterised and their in vitro antimicrobial activity was evaluated
by the minimal inhibitory concentration (MIC). The compounds exhibit moderate antibacterial properties
Received 5 March 2008
Received in revised form 17 July 2008
Accepted 17 July 2008
against Gram-positive bacteria (MIC 6–100
cocci, including methicillin-resistant Staphylococcus aureus and Staphylococcus epidermidis strains, while
no inhibition of Gram-negative Escherichia coli is detected up to the concentration of 100
g mlꢀ1
m
g mlꢀ1) such as several bacilli, staphylococci and strepto-
Available online 25 July 2008
m
.
Keywords:
Hybrid molecules
Synergistic inhibitory activity occurs when sulfanilamides 4a and 4c are tested in combination with
trimethoprim against S. aureus. Concerning antifungal properties, only compound 4c is able to inhibit the
growth of Saccharomyces cerevisiae and Cryptococcus neoformans yeasts and several dermatophytes.
Structure–activity relationships are discussed.
2-Amino-benzo[d]isothiazol-3-one
derivatives
Benzo[d]isothiazol-3-one
benzenesulfonamides
Antimicrobial activity
Ó 2008 Elsevier Masson SAS. All rights reserved.
1. Introduction
with trimethoprim through a well known synergistic effect. Thus,
sulfamethoxazole is a very active pharmaceutical compound
The emergence of microorganisms resistant to drugs which are
utilised as therapeutic agents stimulates our interest in the search
for novel and more efficient antimicrobial substances.
useful in combination with trimethoprim (Co-trimoxazole) as first
choice treatment of pneumonia and urinary tract bacterial infec-
tions, toxoplasmosis [20] and pneumocystosis in HIV infected
patients [21].
Benzo[d]isothiazol-3-ones are
a
class of heterocyclic
compounds endowed with a broad spectrum of biological activity
[1,2]. They have been reported to possess antiplatelet and spas-
molytic effects [3,4] and they attracted a great deal of interest
particularly on the antimicrobial activity. Their inhibitory prop-
erties as regards several Gram-positive and Gram-negative
bacteria, yeasts and moulds have been extensively demonstrated
[1,5–9]. Recently, in the course of our research on biologically
active benzo[d]isothiazoles, we discovered that 2-amino-benzo[d]
isothiazol-3-one derivatives are wide spectrum antimicrobial
substances exhibiting a good inhibition of the growth of peni-
cillin-resistant staphylococci and, in some cases, possessing
activity equal to or superior to the reference drugs ampicillin and
miconazole [10,11].
Examples of hybrid drugs, incorporating moieties both of
sulfonamide and a different antibacterial agent, i.e. fluo-
roquinolone, are reported in the literature [22–24] as being very
active. Moreover, in our previous paper [25] the antibacterial and
antifungal activities of some N-(benzo[d]isothiazolyl)benzene-
sulfonamides have been described.
According to Wermuth [26], the approach to drug design
based on the preparation of dual- or multiple-ligands, on almost
rational basis, can be expected to yield drugs of superior clinical
value, compared with monotargeted ones. Among antibacterial
agents, ‘‘dual targeting’’ compounds may be a preferred objec-
tive also because they would be less prompted to develop
bacterial resistance. Thus, to prove the impact of the sulfon-
amide group on the biological activity of benzisothiazolones, we
synthesized and evaluated for antibacterial and antifungal
properties a number of unsubstituted and 5-methyl substituted
a
On the other hand, sulfonamides aroused considerable interest
in biology and medicine as antimicrobial antifolic agents [12–19].
Their antibacterial effect is considerably enhanced by combination
2-(benzenesulfonyl)amino-benzo[d]isothiazol-3-ones
(Fig.
1,
compounds 4a–4d) and 2-(bisbenzenesulfonyl)amino-benzo[d]i-
sothiazol-3-ones (Fig. 1, compounds 5a–5d) bearing both the
benzo[d]isothiazol-3-one and the sulfonamido pharmacophoric
moieties.
* Corresponding author. Tel.: þ39 521 905068; fax: þ39 521 905006.
E-mail address: franca.zani@unipr.it (F. Zani).
0223-5234/$ – see front matter Ó 2008 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2008.07.022

2742
F. Zani et al. / European Journal of Medicinal Chemistry 44 (2009) 2741–2747
O
O
R
R
R
COCl
SCl
i
ii
N NH2
N NH Boc
S
S
2
1
3a R= H
iii
3b R= CH3
O
N
O
O
S
R
R
O
O
NHR1
NHR1
O
S
N
N
N
S
H
S
NHR1
S
O
O
4a R= R1= H
5a R= R1= H
4b R= H, R1= COCH3
4c R= CH3, R1= H
5b R= H, R1= COCH3
5c R= CH3, R1= H
5d R= CH3, R1= COCH3
4d R= CH3, R1= COCH3
Fig. 1. Scheme of synthesis and structure of compounds 4a–4d and 5a–5d. Reagents and conditions: (i) NH2-NH-Boc (C2H5)2O/pyridine, 60 min 10ꢁC, (ii) TCA/H2O, 150 min, RT,
(iii) 4-COCH3-phenyl-SO2-Cl, pyridine, 120 min, ꢀ10 to ꢀ5 ꢁC; for R1 ¼ H, the following: EtOH/H2O/HCl, 120 min, reflux.
2. Results and discussion
No activity is detected for all of the tested compounds against
Gram-negative Escherichia coli as well as any of the yeasts and
2.1. Chemistry
moulds tested (MIC > 100
a certain antifungal activity inhibiting the growth of Saccharomyces
cerevisiae at the concentration of 100
g mlꢀ1
In all the cases, MIC values are higher than those of the reference
drugs sulfamethoxazole, ampicillin and miconazole and those of
the starting compounds 3a and 3b.
With the aim of gaining deeper insights into the antimicrobial
activity of the new compounds, the minimal bactericidal concen-
trations (MBCs) and the minimal fungicidal concentrations (MFCs)
were determined. As a general trend, these values are higher than
MICs, pointing out that the effectiveness of these substances is
endowed with a bacteriostatic and fungistatic character.
m
g mlꢀ1). Only compound 4c exhibits
The synthetic process for the preparation of the target
compounds is outlined in Fig. 1. Chlorocarbonylphenylsulfenyl-
chloride 1, prepared according to the method previously described
[3], upon cyclization with N-Boc-protected hydrazine afforded the
Boc-protected derivative 2 that, after removal of the N-Boc group by
mild acidic hydrolysis with trichloroacetic acid, gave the key
intermediate 3 [3,27]. The 2-amino-benzo[d]isothiazol-3-ones 3
were then reacted with 4-acetamidobenzene-1-sulfonyl chloride,
in pyridine, in a cooling bath, to afford 2-(benzenesulfonyl)amino-
m
.
benzo[d]isothiazol-3-ones
4 and 2-(bisbenzenesulfonyl)amino-
benzo[d]isothiazol-3-ones 5. Indeed once the sulfonamide 4 is
formed it readily undergoes, by the action of the electrophilic
sulfonyl chloride, a subsequent sulfonylation yielding the disulfonyl
derivative 5. Products 4 and 5 can anyway be simply separated
because of the acidic character of the former.
The structures of the synthesized compounds were supported
by the spectral IR and 1H NMR data and by the results of elemental
analysis which are in agreement with the proposed structures.
The mode of action of the new benzisothiazolone derivatives
here under study was also investigated by detecting the possible
reaction with essential thiol groups of enzymes. In fact, the target of
the class of benzisothiazolones as antimicrobials was identified
with susceptible enzymes bearing accessible thiols at the interac-
tive centres since the growth inhibitory potency of these
compounds was rapidly quenched by the presence of thiol-con-
taining materials such as glutathione or cysteine [28,29]. So the
effect on the growth inhibitory activity of a known concentration of
cysteine, added to the media before inoculation, was estimated. In
all cases MICs increased by a factor two or more (Table 1, values in
italic). That is consistent with the previously cited reports [28,29]
and supports the binding of these compounds to sulphurated
proteins as if they would exert their biological action through
a benzisothiazolone-like mechanism of action.
To clarify the contribution of the sulfonamide moiety to the
antibacterial activity of these compounds, the effect of p-amino-
benzoic acid (PABA) on the MICs was tested by including it in the
medium (Table 1, values underlined). Only for compounds 4a and
4c this supplementation caused a two-fold decrease in the inhibi-
tion of S. aureus growth, while no changes in the MIC values of the
other compounds were observed, in accordance with the sugges-
tion that 4a and 4c against S. aureus behave as dual inhibitors both
by binding to thiol groups and by inhibiting the synthesis of folic
acid.
2.2. Microbiology
The antimicrobial activity of benzo[d]isothiazol-3-one sulfon-
amides 4a–4d and 5a–5d was evaluated by determination of the
minimal inhibitory concentration (MIC) against various bacteria
and fungi, each referred to standard collection strains or fresh
clinical isolates. The results are summarized in Table 1 and are
compared with those of standards ampicillin and sulfamethoxazole
as antibacterial agents and miconazole as an antifungal drug.
Previously reported data concerning the parent 2-amino-
benzisothiazolones 3a and 3b have also been included in the table
in order to provide structure–activity relations.
Apart from a few exceptions, the new compounds exhibit
moderate antibacterial activity against representative Gram-posi-
tive bacteria Bacillus subtilis and Staphylococcus aureus with MICs
ranging from 12 to 100
shows the highest inhibition of the above bacteria (MIC 12
and 50
g mlꢀ1, respectively). Compound 4b and compounds 4d
m
g mlꢀ1. Among the active compounds, 5c
g mlꢀ1
Compounds 4a, 4c and 5a–5d, demonstrated to be the most
active against Gram-positive bacteria, were subjected to
a screening against a wide spectrum of bacteria such as Sarcina
lutea, several bacilli, staphylococci and streptococci, including
m
m
and 5d have no effect up to the dose of 100
bacteria and S. aureus only, respectively.
m
g mlꢀ1 against both

F. Zani et al. / European Journal of Medicinal Chemistry 44 (2009) 2741–2747
2743
Table 1
Antimicrobial activity, expressed as MIC (
m
g mlꢀ1) and, in brackets, as MBC and MFC (
m
g mlꢀ1
)
Compounds
Bacteriaa
Fungib
SC
BS
SA
EC
CT
AN
3ac
3bc
4a
0.7 (1.5)
0.7 (1.5)
6 (12)
6 (25)
50 (>100)
>100
6 (50)
25 (100)
>100
6 (25)
6 (12)
>100
6 (25)
12 (25)
>100
100 (>100)
50 (>100)
>100
50 (>100)
100d
50e
100
4b
4c
>100
25 (100)
50
>100
50 (>100)
100
>100
>100
>100
100 (>100)
>100
>100
>100
>100
>100
25
100
4d
5a
5b
5c
5d
100 (>100)
>100
100
25 (50)
>100
25
50 (>100)
>100
50
12 (100)
>100
12
50 (>100)
>100
50
6 (25)
0.007 (0.15)
–
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
50 (>100)
>100
50
100 (>100)
>100
100
50 (100)
>100
50
>100
Sulfamethoxazole
Ampicillin
Miconazole
25 (100)
0.07 (1.5)
–
>100
3 (25)
–
>100
–
6 (25)
>100
–
3 (12)
f
–
12 (25)
a
Gram-positive bacteria: Bacillus subtilis ATCC 6633 (BS) and Staphylococcus aureus ATCC 25923 (SA); Gram-negative bacteria: Escherichia coli SPA 27 (EC).
b
c
d
e
f
Yeasts: Saccharomyces cerevisiae ATCC 9763 (SC) and Candida tropicalis ATCC 1369 (CT); mould: Aspergillus niger ATCC 6275 (AN).
Values reported in a previous paper [10].
MIC obtained in the presence of cysteine.
MIC obtained in the presence of PABA.
Not tested.
methicillin-resistant S. aureus and Staphylococcus epidermidis
Table 3 presents the antifungal effectiveness of compound 4c
strains (Table 2). Comparison tests were performed by using sul-
famethoxazole as standard drug. The compounds exert a moderate
inhibition of all the tested microorganisms, methicillin-resistant
bacteria included. S. lutea appears to be the most sensitive strain. In
against various yeasts and moulds. This compound acts towards
Cryptococcus neoformans at 100
(MFC > 100
g mlꢀ1) and shows significant inhibition of mould
Madurella mycetomatis at 12
g mlꢀ1 and dermatophytes Epi-
m
g mlꢀ1 in a fungistatic manner
m
m
general, MIC values range from 6 to 100
m
g mlꢀ1 and in the case of
dermothyton floccosum, Microsporum gypseum, Trichophyton men-
tagrophytes, Trichophyton rubrum and Trichophyton soudanense at
both methicillin-susceptible and methicillin-resistant S. epi-
dermidis, Streptococcus faecalis and Streptococcus faecium benziso-
thiazolone sulfonamides are either more active or equipotent to
sulfamethoxazole. MBCs (Table 2) increase by a factor of two or
more when compared with MICs, confirming the bacteriostatic
effect of the tested compounds.
concentrations of 6–50 m
g mlꢀ1. However, its antifungal activity is
less potent than that of miconazole standard drug.
The analysis of the results reported in Table 1 shows that the
activity of the chemicals tested can be correlated to the substituents
both in the sulfonamide and in the heterocyclic nucleus.
Table 2
Antibacterial activity, expressed as MIC (
m
g mlꢀ1) and, in brackets, as MBC (
Compounds
m
g mlꢀ1), against Gram-positive bacteria
Microorganisms
4a
4c
5a
100 (>100)
50 (>100)
50 (>100)
50 (100)
25 (50)
50 (>100)
25 (50)
5b
>100
25 (>100)
100 (>100)
50 (100)
100 (>100)
>100
100 (>100)
100 (>100)
100 (>100)
>100
5c
5d
SMZa
Bacillus cereus ATCC 11966
100 (>100)
50 (>100)
25 (>100)
50 (>100)
50 (>100)
50 (>100)
6 (12)
100 (>100)
100 (>100)
100 (>100)
50 (>100)
50 (>100)
100 (>100)
50 (>100)
50 (100)
50 (>100)
25 (100)
25 (50)
50 (>100)
50 (100)
25 (>100)
12 (50)
50 (>100)
50 (>100)
50 (>100)
50 (>100)
25 (>100)
50 (>100)
25 (>100)
25 (50)
50 (>100)
25 (50)
25 (50)
25 (50)
25 (50)
>100
12 (50)
1.5 (12)
1.5 (6)
1.5 (12)
1.5 (12)
1.5 (12)
0.7 (3)
12 (50)
50 (>100)
>100
25 (>100)
6 (50)
>100
>100
3 (25)
Bacillus circulans BGSC 16A1
Bacillus megaterium BGSC 7A2
Bacillus pumilus BGSC 8E2
100 (>100)
50 (100)
100 (>100)
100 (>100)
>100
50 (>100)
100 (>100)
>100
>100
>100
100 (>100)
100 (>100)
>100
Bacillus subtilis var. sotto BGSC 27A1
Bacillus thuringiensis var. kurstak, BGSC 4D1
Sarcina lutea ATCC 9341
50 (>100)
25 (50)
Staphylococcus aureus methicillin-resistantb
Staphylococcus epidermidisb
50 (>100)
25 (100)
25 (>100)
25 (>100)
25 (>100)
25 (>100)
25 (>100)
50 (>100)
50 (>100)
25 (100)
Staphylococcus epidermidis methicillin-resistantb
Staphylococcus haemolyticusb
Streptococcus agalactiaeb
25 (>100)
50 (>100)
50 (>100)
50 (>100)
50 (>100)
50 (100)
>100
100 (>100)
100 (>100)
100 (>100)
50 (100)
Streptococcus faecalisb
Streptococcus faeciumb
Streptococcus pyogenesb
50 (>100)
a
Sulfamethoxazole.
Isolate of clinical origin.
b

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F. Zani et al. / European Journal of Medicinal Chemistry 44 (2009) 2741–2747
Table 3
B. subtilis and S. aureus. The interactions were detected in
comparison with sulfamethoxazole–trimethoprim mixture and are
summarized by the isobolograms of Figs. 2 and 3. With regard to B.
subtilis, the graphical representation (Fig. 2) displays straight lines
which connect the individual fractional inhibitory concentrations
of the drugs and the fractional inhibitory concentration indices
range from 1 (for compounds 4a, 4c, 4d, 5a and 5c) to 1.5 (for
compounds 5b and 5d). It suggests that only additivity or indif-
ference occurs from the combined action of trimethoprim with
compounds 4a, 4c, 4d, 5a and 5c and with compounds 5b and 5d,
respectively. On the contrary, subinhibitory concentrations of
trimethoprim potentiates the antibacterial activity of mono-sulfa-
nilamides 4a and 4c against S. aureus cells by reducing their MIC
values. Fig. 3A, that refers to the latter compounds, exhibits concave
isobols and fractional inhibitory concentration indices lower than
0.5. This synergistic effect is comparable, even if less marked, to
that produced by the reference sulfamethoxazole–trimethoprim
mixture and supports a sulfa-like mechanism of action. On the
contrary, combinations of bis-sulfonamides 5a–5c have shown to
be additive (Fig. 3B), FIC indices being >0.5 and <1. Thus, in
accordance with the observations done when the compounds were
tested in the presence of PABA, it can be pointed out that only 4a
and 4c act as ‘‘dual targeting’’ compounds, both by binding to thiol
groups and by inhibiting the synthesis of folic acid. Nevertheless,
their antibacterial efficacy against S. aureus is not higher than that
of the corresponding bis-sulfanilamides 5a and 5c, which probably
owe their quantitatively similar activity to the favourable increase
of lipophilicity with respect to 4a and 4c.
Antifungal activity of compound 4c against yeasts and moulds (MIC and, in brackets,
MFC expressed in m )
g mlꢀ1
Fungi
4c
Miconazole
Yeasts
Candida albicans ATCC 10231
Candida guilliermondiia
Candida parapsilosisa
Cryptococcus neoformansa
>100
>100
>100
6
0.15
3
100 (>100)
0.3
Mouldsa
Epidermophyton floccosum
Madurella mycetomatis
Microsporum gypseum
Pseudoallescheria boydii
Sporothrix schenckii
Trichophyton interdigitalis
Trichophyton mentagrophytes
Trichophyton rubrum
12
12
50
0.01
0.007
12
100
50
1
1
1
0.1
100
>100
>100
50
6
12
Trichophyton soudanense
a
Clinical isolates.
Compounds 4a, 4c, 5a and 5c, having a free amino group at the 4
position of the sulfonamide moiety, are more potent than their
corresponding acetylamino parent compounds.
Moreover, the inhibition activity seems to be driven to a certain
degree by the lipophilicity, because the 5-methyl substituted ben-
zothiazolones 4c, 4d, 5c and 5d are more active than their 5-
unsubstituted analogs 4a, 4b, 5a and 5b. Similarly, 2-(bisbenzene-
sulfonyl)amino-benzo[d]isothiazol-3-ones exhibit, in general,
higher inhibitory activity than the corresponding mono-
benzenesulfonyl derivatives. This trend supports the hypothesis
that these compounds can penetrate the microbial membranes of
sensitive Gram-positive bacteria and interact with their cellular
targets at a higher extent.
Clearly, the insertion of the sulfonamido group plays a negative
role on the antimicrobial activity of the 2-aminobenzisothiazolones
3a and 3b as it produces a remarkable decrease of the inhibitory
potency towards all the tested microorganisms. This suggests that
a sulfa-like mode of action would make no contribution to the in
vitro activity of the compounds under study and that the steric
hindrance due to the sulfonamido group partially prevents the
interaction of the benzisothiazolone derivatives with their target
thiol groups.
3. Conclusion
Unsubstituted and 5-methyl substituted 2-(benzenesulfonyl)a-
mino-benzo[d]isothiazol-3-ones and 2-(bisbenzenesulfonyl)a-
mino-benzo[d]isothiazol-3-ones exhibit moderate antimicrobial
properties against Gram-positive bacteria, including methicillin-
resistant strains. The inhibition activity seems to be controlled to
a certain degree by the facility of entering the bacterial membrane
and blocking the essential enzymes involved in the cell metabo-
lism. Although the newly synthesized compounds, carrying the
1
SMZ
4a, 4c, 4d, 5a, 5c
In addition, to investigate if the antibacterial effectiveness of the
new sulfonamides increases in the presence of trimethoprim,
synergism studies were carried out. So, the inhibition profile of the
sulfonamides 4a, 4c, 4d and 5a–5d active against B. subtilis and of
the sulfonamides 4a, 4c and 5a–5c active against S. aureus was
evaluated in combination with trimethoprim against the respective
sensible strains. The results were, then, compared with the anti-
bacterial activity of the substances used individually. Sulfame-
thoxazole–trimethoprim mixture was taken as positive control.
At first the paper strip diffusion determinations on agar medium
were performed. The antibacterial activity of the sulfanilamides 4a
and 4c was potentiated by trimethoprim when S. aureus was
assayed. In this case the size of inhibition zones was greatest where
the two drugs overlapped (data not shown), as observed for
trimethoprim and sulfamethoxazole. On the contrary, no syner-
gistic action was presented by combination of trimethoprim and
compounds 5a–5c for the same strain and by the interaction of
trimethoprim and all the sulfonamides against B. subtilis.
5b, 5d
0.8
0.6
0.4
0.2
0
0
0.2
0.4
0.6
0.8
1
Since the paper strip test has a qualitative character, the anti-
microbial synergism was then quantitatively assessed by the two-
fold dilution method in the checkerboard titration. Sulfonamides
and trimethoprim were tested together in a large number of dilu-
tions to determine the concentrations of each drug in combination
with trimethoprim that produce an endpoint of no growth towards
FIC Trimethoprim
Fig. 2. Assessment of combinations of sulfamethoxazole (SMZ) and sulfonamides 4a,
4c, 4d, 5a–5d with trimethoprim against Bacillus subtilis ATCC 6633 in the checker-
board broth dilution method. The concentrations of the drugs are expressed as frac-
tions of the minimal inhibitory concentrations (FIC, fractional inhibitory
concentration).

F. Zani et al. / European Journal of Medicinal Chemistry 44 (2009) 2741–2747
2745
in the analytical laboratory of Dipartimento Farmaceutico, Uni-
1
0.8
0.6
0.4
0.2
0
A
SMZ
4a
`
versita di Parma, on a ThermoQuest (Italia) FlashEA 1112 Elemental
Analyzer, for C, H, N and S. The found values for C, H, N, S were
always ꢂ0.4% of the theoretical ones. IR spectra were recorded, as
KBr pellets, on a Jasco FT-IR 300E spectrophotometer (Jasco Ltd.,
Tokyo, Japan) and the reported wavenumbers are given in cmꢀ1. 1H
NMR spectra, in DMSO-d6 solutions, were recorded on a Bruker AC
4c
300 instrument at 298 K. Chemical shifts are reported as
d (ppm)
relative to TMS as internal standard. The progress of the reactions
was monitored by thin layer chromatography with F254 silica-gel
precoated sheets (Merck, Darmstadt, Germany) and UV light was
used for detection.
4.1.1. General procedure for synthesis of
2-(40-acetylaminobenzenesulfonyl)amino-benzo[d]isothiazol-3-
ones (4b, 4d) and of 2-(bis-40-acetylaminobenzenesulfonyl)
amino-benzo[d]isothiazol-3-ones (5b, 5d)
Small portions of 4-acetylaminobenzene-1-sulfonyl chloride
(10 mmol) were added to a stirred mixture of the suitable 2-amino-
benzo[d]isothiazol-3-one 3 (9 mmol) in pyridine (9 ml) at 0–5 ꢁC
for 1 h and then kept at room temperature for 2 h. The reaction
mixture was poured into water and crushed ice (20 ml/20–30 g),
acidified with conc. HCl and stirred at room temperature overnight.
The solid obtained was filtered and washed thoroughly with water
(3 ꢃ10 ml), then poured again into water (200 ml), added with 10%
Na2CO3 (5 ml) and kept under stirring for 4 h. The residue (product
5) was filtered off and the filtrate, treated with conc. HCl, afforded
compound 4 that was collected by filtration and purified by column
chromatography (CH2Cl2/EtOH, 95:5). Compounds 4 and 5 were
then recrystallised from suitable solvents.
0
0.2
0.4
0.6
0.8
1
FIC Trimethoprim
1
B
SMZ
5a
5b
5c
0.8
0.6
0.4
0.2
0
4.1.1.1. 2-(40-Acetylaminobenzenesulfonyl)amino-benzo[d]isothiazol-
3-one (4b). Yield: 35%; mp 189–190 ꢁC (ethanol). IR (KBr): 3379,
3163 (NH), 1691 (CO), 1336, 1157 (SO2) cmꢀ1; 1H NMR (DMSO-d6,
d,
ppm): 11.10 (s, 1H, NHSO2); 10.36 (s, 1H, NHCO); 7.88–7.78 (m, 6H,
H-4, H-7, H-20, H-30, H-50, H-60); 7.69 (t, 1H, J ¼ 7.2, H-5); 7.40 (t, 1H,
J ¼ 7.2, H-6); 2.10 (s, 3H, CH3). Anal. Calcd. for C15H13N3O4S2
(363.41): C, 49.57; H, 3.61; N, 11.56; S, 17.65. Found: C, 49.52; H,
3.66; N, 11.44; S, 17.53.
4.1.1.2. 2-(40-Acetylaminobenzenesulfonyl)amino-5-methyl-benzo[d]
isothiazol-3-one (4d). Yield: 32%; mp 175–177 ꢁC (dichloro-
methane). IR (KBr): 3373, 3170 (NH), 1687 (CO), 1338, 1157 (SO2)
0
0.2
0.4
0.6
0.8
1
cmꢀ1 1H NMR (DMSO-d6,
; d, ppm): 11.11 (s, 1H, NHSO2); 10.37 (s,
FIC Trimethoprim
1H, NHCO); 7.77–7.74 (m, 5H, H-7, H-20, H-30, H-50, H-60); 7.63 (s,1H,
H-4); 7.52 (dd, 1H, J ¼ 8.4, J ¼ 1.8, H-6); 2.37 (s, 3H, CH3); 2.10 (s, 3H,
COCH3). Anal. Calcd. for C16H15N3O4S2 (377.44): C, 50.91; H, 4.01; N,
11.13; S, 16.99. Found: C, 50.73; H, 4.06; N, 10.93; S, 16.79.
Fig. 3. Assessment of combinations of sulfamethoxazole (SMZ) and sulfonamides 4a
(A), 4c (A), 5a–5c (B) with trimethoprim against Staphylococcus aureus ATCC 25923 in
the checkerboard broth dilution method. The concentrations of the drugs are
expressed as fractions of the minimal inhibitory concentrations (FIC, fractional inhib-
itory concentration).
4.1.1.3. 2-(Bis-40-acetylaminobenzenesulfonyl)amino-benzo[d]iso-
thiazol-3-one (5b). Yield: 36%; mp 216–218 ꢁC (ethyl acetate/
petroleum ether). IR (KBr): 3316 (NH), 1680 (CO), 1375, 1169 (SO2)
benzisothiazolone moiety and the sulfonamide portion, could be
considered hybrid drugs from a structural point of view, notwith-
standing two of them act also by a sulfa-like mechanism, it can be
stated the sulfa-like mode of action would make no or little
contribution to the detected antimicrobial activity.
cmꢀ1 1H NMR (DMSO-d6,
; d, ppm): 10.53 (s, 2H, NH); 7.96 (d, 1H,
J ¼ 8.1, H-4); 7.92–7.86 (m, 9H, H-20, H-30, H-50, H-60, H-200, H-300, H-
500, H-600, H-7); 7.78 (t, 1H, J ¼ 7.2, H-5); 7.47 (t, 1H, J ¼ 7.2, H-6); 2.13
(s, 6H, CH3). Anal. Calcd. for C23H20N4O7S3 (560.63): C, 49.27; H,
3.60; N, 9.99; S, 17.16. Found: C, 49.48; H, 3.93; N, 9.66; S, 16.81.
4. Experimental protocols
4.1.1.4. 2-(Bis-40-acetylaminobenzenesulfonyl)amino-5-methyl-ben-
zo[d]isothiazol-3-one (5d). Yield: 62%; mp 175–178 ꢁC (ethyl
acetate/petroleum ether). IR (KBr): 3322 (NH), 1685 (CO), 1371, 1165
4.1. Chemistry
Synthetic starting material, reagents and solvents were
purchased from Aldrich or Fluka. Solvents, unless otherwise spec-
ified were reagent grade or of the highest quality commercially
available. Melting points (ꢁC) were determined with a Buchi 512
apparatus and are uncorrected. Elemental analysis were performed
(SO2) cmꢀ1 1H NMR (DMSO-d6,
; d, ppm): 10.55 (s, 2H, NH); 7.88–
7.81 (m, 9H, H-20, H-30, H-50, H-60, H-200, H-300, H-500, H-600, H-7); 7.71
(s, 1H, H-4); 7.61 (dd, 1H, J ¼ 8.3, J ¼ 1.35, H-6); 2.40 (s, 3H, CH3);
2.13 (s, 6H, CH3). Anal. Calcd. for C24H22N4O7S3 (574.65): C, 50.16; H,
3.86; N, 9.75; S, 16.74. Found: C, 50.09; H, 4.13; N, 9.34; S, 16.57.

2746
F. Zani et al. / European Journal of Medicinal Chemistry 44 (2009) 2741–2747
4.1.2. General procedure for synthesis of
2-(40-aminobenzenesulfonyl)amino-benzo[d]isothiazol-3-ones
(4a, 4c) and of 2-(bis-40-aminobenzenesulfonyl)
millilitre of medium was added. After an incubation period of
24 h at 37 ꢁC (bacteria) and of 48 h at 30 ꢁC (fungi), the minimum
inhibitory concentrations (MIC,
m
g mlꢀ1) were detected as the
amino-benzo[d]isothiazol-3-ones (5a, 5c)
lowest concentrations of compound that resulted in complete
inhibition of the microbial growth.
To detect the mechanism of action of the tested compounds,
the experiments described above were repeated by assaying the
antimicrobial properties of the active chemicals in the presence
A suspension of the appropriate 40-acetylaminobenzenesulfo-
nylamino-benzo[d]isothiazol-3-one 4b, 4d or 5b, 5d (5 mmol) in
ethanol (75 ml) and 1.2 N HCl (20 ml) were refluxed for 120 min.
The solvent was evaporated under reduced pressure. The residue
was added with water (100 ml) and sodium acetate to pH 5–6. The
precipitate was filtered, washed with water, dried and crystallized
from suitable solvent.
of
L-cysteine hydrochloride. Thus, cysteine hydrochloride was
dissolved in water at the concentration of 3.5 mg mlꢀ1 and ster-
ilized by filtration. Fifty microlitres of the obtained solution were
added to each millilitre of the testing suspensions before incu-
bation. In addition, Mueller Hinton broth containing PABA at
4.1.2.1. 2-(4-Aminobenzenesulfonyl)amino-benzo[d]isothiazol-3-one
(4a). Yield: 70%; mp 172–174 ꢁC (ethanol). IR (KBr): 3473, 3345
10
MICs.
The minimum bactericidal concentrations (MBC) and the
minimum fungicidal concentrations (MFC), both expressed in
g mlꢀ1, were determined by subculturing on fresh medium
100 l of liquid from each suspension remaining clear and incu-
m
g mlꢀ1 was used to test the effect of this substance on the
(NH2), 3180 (NH), 1673 (CO), 1348, 1163 (SO2) cmꢀ1 1H NMR
;
(DMSO-d6, d, ppm): 11.12 (s, 1H, NHSO2); 7.88–7.81 (m, 2H, H-4, H-
7); 7.68 (t, 1H, J ¼ 7.2, H-5); 7.47–7.38 (m, 3H, H-6, H-20, H-60); 6.60
(d, 2H, J ¼ 9, H-30, H-50); 6.14 (s, 2H, NH2). Anal. Calcd. for
C13H11N3O3S2 (321.38): C, 48.59; H, 3.45; N, 13.08; S, 19.05. Found:
C, 48.81; H, 3.77; N, 12.78; S, 18.96.
m
m
bating the samples obtained at 37 ꢁC for 24 h (bacteria) and at
30 ꢁC for 48 h (fungi). MBC and MFC values represent the lowest
concentration of drug needed for the reduction of the initial
inoculum of 99.9%.
4.1.2.2. 2-(4-Aminobenzenesulfonyl)amino-5-methyl-benzo[d]iso-
thiazol-3-one (4c). Yield: 73%; mp 187–190 ꢁC (ethyl acetate/
petroleum ether). IR (KBr): 3471, 3389 (NH2), 3037 (NH), 1670 (CO),
Compound 4c, the only one active against fungi, was screened
for its antifungal activity by mean of serial plate dilution method
towards several pathogenic moulds (Table 3) freshly isolated from
pathological material. A suspension of each fungal strain in physi-
ologic saline was streaked onto the surface of Sabouraud Dextrose
1345, 1155 (SO2) cmꢀ1 1H NMR (DMSO-d6,
; d, ppm): 10.58 (s, 1H,
NHSO2); 7.74 (d, 1H, J ¼ 8.3, H-7); 7.64 (s, 1H, H-4); 7.51 (dd, 1H,
J ¼ 8.3, J ¼ 1.5, H-6); 7.41 (d, 2H, J ¼ 8.5, H-20, H-60); 6.59 (d, 2H,
J ¼ 8.5, H-30, H-50); 6.14 (s, 2H, NH2); 2.38 (s, 3H, CH3). Anal. Calcd.
for C14H13N3O3S2 (335.41): C, 50.13; H, 3.91; N, 12.53; S, 19.12.
Found: C, 50.00; H, 4.09; N, 12.22; S, 18.82.
Agar plates containing 0.001–100 m
g mlꢀ1 of compound and the
plates were incubated at 25 ꢁC for 14 days. MICs were determined
and the antifungal activity was compared with that of miconazole
used as standard drug.
4.1.2.3. 2-(Bis-40-aminobenzenesulfonyl)amino-benzo[d]isothiazol-
3-one (5a). Yield: 70%; mp 203–205 ꢁC (ethyl acetate/petroleum
ether). IR (KBr): 3494, 3394 (NH2), 1693 (CO), 1375, 1159 (SO2)
All the experiments were performed in triplicate and the
reported results were obtained from three independent
measurements.
cmꢀ1 1H NMR (DMSO-d6,
; d, ppm): 7.94–7.87 (m, 2H, H-4, H-7);
7.75 (t, 1H, J ¼ 7.2, H-5); 7.55–7.41 (m, 5H, H-6, H-20, H-60, H-200, H-
600); 6.62 (d, 4H, J ¼ 8.4, H-30, H-50, H-300, H-500); 6.45 (br s, 4H, NH2).
Anal. Calcd. for C19H16N4O5S3 (476.55): C, 47.89; H, 3.38; N, 11.76; S,
20.19. Found: C, 48.09; H, 3.69; N, 11.38; S, 19.87.
4.2.2. Synergism studies
The effects of combinations of new sulfonamides with
trimethoprim (Sigma, Milano, Italy) against B. subtilis ATCC 6633
and S. aureus ATCC 25923 was examined in vitro according to two
procedures: the paper strip diffusion method and the checker-
board titration technique [25,31]. The antimicrobial combination
of sulfamethoxazole with trimethoprim was used as positive
standard control. The solutions of the standard drugs were
prepared in water by adding the minimal amount of 2.5 M NaOH
(for sulfamethoxazole) or 0.05 M HCl (for trimethoprim) to
dissolve them.
4.1.2.4. 2-(Bis-40-aminobenzenesulfonyl)amino-5-methyl-benzo[d]
isothiazol-3-one (5c). Yield: 60%; mp 204–206 ꢁC (ethanol/water).
IR (KBr): 3480, 3379 (NH2), 1691 (CO), 1369, 1159 (SO2) cmꢀ1 1H
;
NMR (DMSO-d6,
d
, ppm): 7.8 (d, 1H, J ¼ 8.4, H-7); 7.69 (s, 1H, H-4);
7.58 (dd, 1H, J ¼ 8.5, J ¼ 1.5, H-6); 7.47 (d, 4H, J ¼ 9, H-20, H-60, H-200,
H-600); 6.61 (d, 4H, J ¼ 9, H-30, H-50, H-300, H-500); 6.48 (s, 4H, NH2);
2.39 (s, 3H, CH3). Anal. Calcd. for C20H18N4O5S3 (490.58): C, 48.97;
H, 3.70; N, 11.42; S, 19.61. Found: C, 48.55; H, 3.80; N, 11.81; S,
19.31.
As regards the checkerboard titration technique, sulfonamides
were tested at concentrations ranging from 100 to 0.15
trimethoprim from 0.0003 to 3
m
g mlꢀ1 and
m
g mlꢀ1. The effect of the studied
combinations was expressed by the fractional inhibitory concen-
tration (FIC) of each tested compound (S, sulfonamides; T,
trimethoprim):
4.2. Microbiology
4.2.1. Antimicrobial activity evaluation
The new compounds were tested in vitro for their antimicrobial
properties against a wide spectrum of microorganisms (Tables 1–3)
using the serial double dilution method [30].
FICS ¼ MIC of each sulfonamide in combination/MIC of each
sulfonamide alone
FICT ¼ MIC of trimethoprim in combination/MIC of trimethoprim
The compounds under investigation were dissolved in
dimethyl sulfoxide and diluted in the media (Tryptose Phosphate
Broth for Streptococcus pyogenes, Mueller Hinton broth for other
bacteria and Sabouraud liquid medium for fungi) so as to achieve
alone.
The FIC indices (FICI) for the most effective combinations were
then calculated by summing the separate FIC values of each
sulfonamide and trimethoprim:
the concentration range of 0.001–100 m
g mlꢀ1. To ensure that
dimethyl sulfoxide had no effect on microbial growth, a control
test with media supplemented with the solvent was performed.
Ampicillin, sulfamethoxazole and miconazole were used as
reference antibacterial and antifungal agents, respectively. An
inoculum of 5 ꢃ104 CFU for bacteria and 1 ꢃ103 CFU for fungi per
FICI ¼ FICS þ FICT
Drug interactions are defined as synergistic when the FIC index
is ꢄ0.5, additive when the FIC index is >0.5 and ꢄ1, while values >1

F. Zani et al. / European Journal of Medicinal Chemistry 44 (2009) 2741–2747
2747
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[32].
These results were summarized graphically on the isobolograms
constructed taking along the x-axis the FIC of trimethoprim and
along the y-axis the corresponding FIC values of each sulfonamide.
The isobolograms were constructed by connecting with a line
(isobol) the series of points generated for each drug combination.
Synergistic interaction is diagrammed as concave isobol, additive
effect as straight line and antagonism as a convex isobol.
All tests were performed in triplicate and the results were
confirmed by three separate experiments.
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This study was supported by the grant FIL 2007 of the University
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