Antimicrobial activity of sulfonamides containing 5-chloro-2- hydroxybenzaldehyde and 5-chloro-2-hydroxybenzoic acid scaffold

Article abstract of DOI:10.1016/j.ejmech.2012.01.060

A series of novel sulfonamides containing 5-chloro-2-hydroxybenzaldehyde or 5-chloro-2-hydroxybenzoic acid scaffolds were designed, synthesized and characterized by IR, ¹H NMR and ¹³C NMR. All ten target synthesized derivatives and starting sulfonamides were evaluated in vitro for the activity against Gram-positive and Gram-negative bacteria, fungi, Mycobacterium tuberculosis, Mycobacterium avium and Mycobacterium kansasii. The most active compound against methicillin-sensitive and methicillin-resistant Staphyloccoccus aureus was 5-chloro-N-{4-[N-(4,6-dimethylpyrimidin-2-yl) sulfamoyl]phenyl}-2-hydroxybenzamide with MIC 15.62-31.25 μmol/L. 4-Amino-N-(thiazol-2-yl)benzenesulfonamide and 4-(5-chloro-2- hydroxybenzylideneamino)-N-(thiazol-2-yl)benzenesulfonamide have shown the best activity against M. kansasii at the concentrations of 1-4 μmol/L. The efficacy against other strains was weaker and the studied derivatives exhibited almost none antifungal potency.

Full text of DOI:10.1016/j.ejmech.2012.01.060

European Journal of Medicinal Chemistry 50 (2012) 433e440
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Antimicrobial activity of sulfonamides containing 5-chloro-2-
hydroxybenzaldehyde and 5-chloro-2-hydroxybenzoic acid scaffold
a
a,
b
c
,
d
b
*
ꢀ
ꢀ
Martin Krátký , Jarmila Vinsová , Marie Volková , Vladimír Buchta , Frantisek Trejtnar ,
e
ꢀ
ꢀ
Jirina Stolaríková
a Department of Inorganic and Organic Chemistry, Faculty of Pharmacy, Charles University, Heyrovského 1203, 500 05 Hradec Králové, Czech Republic
b Department of Pharmacology and Toxicology, Faculty of Pharmacy, Charles University, Heyrovského 1203, 500 05 Hradec Králové, Czech Republic
c Department of Clinical Microbiology, Faculty of Medicine and University Hospital, Charles University, Sokolská 581, 500 12 Hradec Králové, Czech Republic
d Department of Biological and Medical Sciences, Faculty of Pharmacy, Charles University, Heyrovského 1203, 500 05 Hradec Králové, Czech Republic
e
ꢀ
Laboratory for Mycobacterial Diagnostics and TB, Regional Institute of Public Health in Ostrava, Partyzánské námestí 7, 702 00 Ostrava, Czech Republic
a r t i c l e i n f o
a b s t r a c t
Article history:
A
series of novel sulfonamides containing 5-chloro-2-hydroxybenzaldehyde or 5-chloro-2-
hydroxybenzoic acid scaffolds were designed, synthesized and characterized by IR, 1H NMR and 13C
NMR. All ten target synthesized derivatives and starting sulfonamides were evaluated in vitro for the
activity against Gram-positive and Gram-negative bacteria, fungi, Mycobacterium tuberculosis, Myco-
bacterium avium and Mycobacterium kansasii. The most active compound against methicillin-sensitive
and methicillin-resistant Staphyloccoccus aureus was 5-chloro-N-{4-[N-(4,6-dimethylpyrimidin-2-yl)
Received 26 September 2011
Received in revised form
26 January 2012
Accepted 31 January 2012
Available online 7 February 2012
sulfamoyl]phenyl}-2-hydroxybenzamide with MIC 15.62e31.25
zenesulfonamide and 4-(5-chloro-2-hydroxybenzylideneamino)-N-(thiazol-2-yl)benzenesulfonamide
have shown the best activity against M. kansasii at the concentrations of 1e4 mol/L. The efficacy against
mmol/L. 4-Amino-N-(thiazol-2-yl)ben-
Keywords:
Antibacterial activity
Antimycobacterial activity
In vitro activity
Salicylanilide
m
other strains was weaker and the studied derivatives exhibited almost none antifungal potency.
Ó 2012 Elsevier Masson SAS. All rights reserved.
Schiff base
Sulfonamide
1. Introduction
Some sulfonamides were the first drugs with antituberculous
effects, but now they are generally considered to have no clinical
Sulfonamides were intensively investigated as the first effective
antibacterial agents. Sulfonamides act generally as structural
analogues of 4-aminobenzoic acid and therefore inhibit dihy-
dropteroate synthase. After the discovery of penicillin followed by
other antibiotics they became less utilized, but later they have
started to attract attention for their synergic activity, e.g. in the
combination with trimethoprim. The antibacterial activity of the
combination of sulfamethoxazole (SMX) with trimethoprim is
more efficient due to the sequential inhibition of the bacterial
synthesis of tetrahydrofolic acid and thereby disrupting nucleic
bases and acids synthesis; it is a drug with a broad spectrum widely
used for the treatment of many infections [1e3]. Unfortunately
some clinical important pathogens are primary or acquired resis-
tant. The increasing rates of resistance among important pathogens
have been reported worldwide [3e5].
usage on Mycobacterium tuberculosis [3,6]; their role is controversial.
Sulfonamides are not recommended for a routine susceptibility
testing of M. tuberculosis isolates [7]. Recently, several sulfonamides
revealed the inhibition of M. tuberculosis b-carbonic anhydrase at low
concentrations. Such an action can be relevant for the design of
antituberculosis drugs with an innovative mechanism of action [8,9].
Simultaneous testing of the M. tuberculosis susceptibility to various
sulfonamides showed some activity, e.g. minimum inhibitory
concentration (MIC) of the most active sulfathiazole was 10
of sulfadiazine slightly over 50 g/mL; for others were higher. SMX
exhibited MIC against M. tuberculosis of 1000 g/mL [6], whereas
g/
mg/mL or
m
m
other studies revealed good susceptibility at concentrations ꢀ38
m
mL. Moreover, these values are withinreadilyachievable serum levels
of this drug [10]. Sulfathiazole causes higher than 80% growth inhi-
bition of M. tuberculosis H37Rv with IC50 ꢁ5
mM [11].
Sulfamethoxazole was shown to inhibit dihydropteroate syn-
thase of Mycobacterium avium [12] and it affected the growth of
three strains of M. avium complex with MIC50/80 about 30 mg/mL,
but not all strains were susceptible [13e16]. Also other sulfon-
amides were active against M. avium complex [13,15,17].
* Corresponding author. Tel.: þ420 495067343; fax: þ420 495067166.
ꢀ
E-mail address: jarmila.vinsova@faf.cuni.cz (J. Vinsová).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2012.01.060

434
M. Krátký et al. / European Journal of Medicinal Chemistry 50 (2012) 433e440
SMX was tested against Mycobacterium kansasii with majority
g/mL, including strains resistant to isoniazid (INH),
ethambutol or rifampicin. SMX was also useful in patient studies
[8,16].
aromatic imines (Scheme 1) and amides (Scheme 2) were synthe-
MIC ꢀ4
m
sized. The yields of Schiff bases ranged from 81 to 96%, of amides
44e69% due to repeated purifications. An overview of the
compounds brings Table 1.
For slowly growing mycobacteria, SMX is acceptable for routine
susceptibility testing of M. kansasii and Mycobacterium marinum.
2.2. Pharmacology
Strains with MIC80 ꢀ32
mg/mL are considered to be susceptible [7].
Some members of the series of 5-chloro-2-hydroxybenzaldehyde
(5-chlorosalicylaldehyde) Schiff bases were touched to be antibac-
terial active (e.g. Escherichia coli, Pseudomonas fluorescens and
Staphylococcus aureus), whereas fungi including Candida albicans
were in general resistant [18].
2.2.1. Antimycobacterial activity
All the prepared compounds were tested for their in vitro anti-
mycobacterial activity against M. tuberculosis 331/88, M. avium
(330/88) and two strains of M. kansasii (235/80 and 6509/96) as
well as four parent sulfonamides and isoniazid as a reference
compound (see Table 2).
Sulfonamides are still modified to found derivatives with more
convenient antimicrobial properties (e.g. [19]). A large range of
sulfonamide Schiff bases having a wide spectrum of biological
activities is known. The Schiff bases with substituted salicylalde-
hydes (in a free or a complex form with inorganic ions) were shown
to be able to stop the growth of bacterial species like E. coli,
S. aureus, Pseudomonas aeruginosa, Shigella flexneri, Klebsiella
pneumoniae, Salmonella typhi in the wobble ranges of MIC. Data
about their antifungal activity differ; some derivatives were highly
active, some moderate active and others inactive [20e26].
Salicylanilide derivatives expressed obvious and in some
cases excellent antibacterial activity (including drug-sensitive and
multidrug-resistant Mycobacterium sp., methicillin-resistant
S. aureus) and antifungal activity (e.g. [27e30]), 5-chloro-2-
hydroxybenzoic (5-chlorosalicylic) acid derivatives were the most
active.
These findings led us to combine sulfonamides with 5-
chlorosalicyladehyde to form Schiff bases and prepare amides
with 5-chlorosalicylic acid (Fig. 1) as potential antimicrobial agents.
Additionally, we wanted to compare the antimicrobial activity of
sulfonamide’s salicylic amides (Fig. 2) and Schiff bases mutually as
well as the evaluation of the convenience of sulfonamide substi-
tution of salicylanilide molecules. This study contributes to the
description of the antimicrobial activity of sulfonamide and sali-
cylic derivatives.
Sulfonamides were found being more efficient against myco-
bacteria than towards other bacteria. M. tuberculosis was inhibited
at the concentrations of 32e125
superiority (32 mol/L; the concentration achievable by standard
clinical dosing is 32 g/mL, i.e. approximately 126 mol/L, and it is
mmol/L with sulfamethoxazole 1a
m
m
m
considered to be a break point [10]). Almost at the same concen-
tration SMX 1a inhibits the growth of M. avium (MIC
32e62.5
m
mol/L). M. kansasii, both collection and clinical isolated
mol/L). Sulfathia-
types, is the most susceptible species (MIC ꢁ1
m
zole 3a blocked its growth unambiguously more potently than
others. All sulfonamides revealed markedly lower activity against
M. tuberculosis than a standard drug isoniazid. On the other side,
they expressed significantly lower MIC against M. avium and
M. kansasii 235/80 and except sulfamethazine derivatives (4a-c)
they are slightly more effective than INH against M. kansasii 6509/
96. This activity against atypical strains is notable and hopeful.
When expertising the structureeactivity relationships of
synthesized derivatives, we shall distinguish two subgroups e
activity against M. tuberculosis and M. avium on one side, and
activity against M. kansasii on the other side. Tuberculous strain
was at least sensitive (MIC 32e500
modification resulted mostly in twice or more increased MIC. The
most active derivative is Schiff base 3b (32e62.5 mol/L). Activities
of sulfamethoxazole, sulfadiazine and their modifications (1b,c,
2b,c) resemble themselves.
mmol/L) and any sulfonamide
m
MIC of sulfonamide derivatives for M. avium varied from 32 up
2. Results and discussion
to 125
mmol/L. 3-Chlorobenzaldehyde Schiff base 1d and amide 4c
had maximal inhibitory activity (32e62.5
mmol/L). For M. avium is
2.1. Chemistry
the attenuation of the antimycobacterial acting by derivatization
not so sharp, three structures exhibited the same in vitro effectivity
as parent sulfonamides (1d, 2c, 4c).
Ten sulfonamide derivatives (1bee, 2b,c, 3b,c, 4b,c) based on
the modification of primary aromatic amine group by forming
The introduction of either imine or amide is generally disad-
vantageous in the case of M. tuberculosis and M. avium. However, for
sulfadiazine 2a and sulfamethazine 4a are preferred amides, for
sulfathiazole and sulfamethoxazole derivatives (1d vs. 1e) Schiff
Fig. 1. General structure of synthesized compounds.
Scheme 1. Synthesis of sulfonamide Schiff bases (R1 ¼ H, OH).
Fig. 2. “Salicylanilide core” in the molecules of sulfonamide amides.
Scheme 2. Synthesis of sulfonamide amides (R1 ¼ H, OH).

M. Krátký et al. / European Journal of Medicinal Chemistry 50 (2012) 433e440
435
Table 1
Overview of evaluated compounds.
Structure
Sulfonamide
Schiff base
Amide
1a
R1 ¼ R2 ¼ H
sulfamethoxazole
2a
R1 ¼ R2 ¼ H
sulfadiazine
3a
R1 ¼ R2 ¼ H
sulfathiazole
4a
R1 ¼ R2 ¼ H
sulfamethazine
Table 2
Antimycobacterial activity of sulfonamide derivatives.
MIC [mmol/L]
M. tuberculosis
M. avium
M. kansasii
M. kansasii
331/88
330/88
235/80
6509/96
14 d
21 d
14 d
21 d
7 d
14 d
21 d
7 d
14 d
21 d
1a
1b
1c
1d
1e
2a
2b
2c
3a
3b
3c
4a
4b
4c
INH
32
32
32
62.5
125
125
62.5
500a
62.5
125
8
4
8
4
2
16
8
4
2
16
8
16
8
16
16
16
8
4
4
8
4
8
8
16
4
1
2
8
16
125
16
4
4
4
8
4
8
8
32
8
2
2
8
16
125
62.5
4e8
4
8
8
4
8
8
32
8
2
125
125
62.5
500a
32
125
125
32
250
250
125
500a
62.5
250
250
62.5
62.5
250a
125
500
125
1
62.5
62.5
32
500a
62.5
125
62.5
62.5
62.5
250a
32
8
8
16
16
8
4
4
16
32
125
32
>250
32
32
16
4
62.5
62.5
125
32
2
8
4
2
8
250a
62.5
250
125
0.5e1
250a
62.5
125
16
125
250
62.5
>250
16
62.5
16
>250
32
250
62.5
8e16
62.5
32
>250
62.5
>250
INH e isoniazid; one or two lowest MIC values for each strain are bolded.
a
At presented concentration the grow of strain was observed, at duplex concentration there was present precipitate and/or turbidity, therefore it was not possible to
determine exact MIC value.

436
M. Krátký et al. / European Journal of Medicinal Chemistry 50 (2012) 433e440
Table 3
In vitro antibacterial activity.
31.25 up to 500
m
mol/L, whereas MIC of sulfadiazine 2a and sulfa-
methazine 4a were ꢁ500
mmol/L. No significant difference between
drug-sensitive and MRSA strain was observed. S. epidermidis and
enterococci are in vitro resistant, only SMX 1a partly inhibited first
MIC/IC90
SA
[m
mol/L]
MRSA
24 h
SE
named strain at 250e500
(E. coli, two strains of K. pneumoniae and P. aeruginosa) showed
mol/L). Paradoxically, sulfon-
mmol/L. Four Gram-negative strains
24 h
48 h
48 h
24 h
48 h
1a
1b
1c
1d
1e
2a
2b
2c
3a
3b
3c
4a
4b
4c
250
31.25
125
31.25
>500
500
250
125
31.25
31.25
>125
500
500
31.25
125
62.5
250
250
500
completely insensitivity (MIC ꢁ500
m
15.62
15.62
31.25
>500
500
125
31.25
62.5
31.25
>125
500
125
15.62
15.62
31.25
31.25
62.5
>500
>500
125
31.25
125
31.25
>125
>500
250
>500
>500
>500
>500
>500
>500
>500
>500
>500
>125
>500
>500
31.25
15.62
>500
>500
>500
>500
>500
>500
>500
>500
>500
>125
>500
>500
250
amides have been recommended largely for the treatment of Gram-
negative infections. These result supports the facts about problems
with the resistance to alone sulfonamides [5].
62.5
>500
>500
250
125
62.5
31.25
>125
>500
500
15.62
31.25
In general, the introduction of both 5-chlorosalicylaldehyde and
5-chlorosalicylic acid scaffolds resulted in the increased antibacte-
rial activity. For staphylococci, sulfamethoxazole derivatives 1b and
1c are up to eight times more active than parent compound 1a (250
vs. 31.25 mmol/L). Schiff base 1d without salicylic hydroxyl expressed
almost the same activityas its “hydroxylated counterpart” 1b. On the
other side, the replacement of 2-hydroxy group from the salicyla-
mide 1c resulted in total loss of the antibacterial activity (1e).
The sulfonamide Schiff bases expressed all lower MIC than
parent compounds e MIC against S. aureus were 31.25e500
and for MRSA 15.62e250 mol/L, when salicylamides exhibited
else better efficacy in the range from 15.62 up to 125 mol/L. The
250
15.62
15.62
31.25
31.25
BAC
31.25
BAC: bacitracin; SA: Staphylococcus aureus CCM 4516/08; MRSA: methicillin-
resistant Staphylococcus aureus 5996/08; SE: Staphylococcus epidermidis
H 6966/08. One or two lowest MIC values for each strain are bolded.
mmol/L
H
m
m
bases. It appears that the presence of 2-hydroxy group on phenyl
ring is necessary for the activity against M. tuberculosis and
M. avium only for amides 1e, not for Schiff base 1d, which exhibited
aboveeaverage activity.
most effective compounds in vitro are imines 1b, 3b and amide 4c,
all comparable to bacitracin, a topical antimicrobial agent, used for
the treatment of Gram-positive infections [31]. Interestingly, the
activities of derivatives on MRSA strain are slightly better than on
methicillin-sensitive one. However, the role of sulfonamides in the
treatment of MRSA infections is controversial [32,33]. Based on MIC
values, the activity against S. aureus seems to be bactericidal.
S. epidermidis and Enterococcus are more impervious, only 4c
Similarly to starting sulfonamides, M. kansasii was affected by
the lowest concentration levels (ꢁ2
mmol/L); 1d, 1e and 3b dis-
played the highest rate of both strains suppression. The presence of
phenolic group is not demanded for a good activity e both 1d and 1e
(MIC 2e8
The order of growth inhibition potency for different sulfonamide
derivatives is: sulfathiazoles (2e4 mol/L) > sulfamethoxazoles
(2e16 mol/L) > sulfadiazines (4e32 mol/L) > sulfamethazines
(16e250 mol/L). These facts correspond to the basal effectiveness
of alone sulfonamides. Their derivatization led to structures with
improved activity (e.g. 1a vs. 1d, 2a vs. 2c), the same or nearly same
MIC (3a vs. 3b) or with a decreased potency (e.g. 4a vs. 4b, 3a vs. 3c).
Sulfonamides containing salicylamide fragment are less potent
against M. tuberculosis than salicylanilides substituted at the para-
position by moieties with negative mesomeric effect like nitro,
alkyloxycarbonyl or cyano groups; however, the growth inhibiting
activity of newly prepared amides against M. kansasii is similar or
superior when compared with these salicylanilides [27].
mmol/L) are the most active sulfamethoxazole derivatives.
inhibited their growth significantly (MIC 31.25e250
S. epidermidis and 125e500 mol/L for Enterococcus).
mmol/L for
m
m
Amides seem to be more beneficial than Schiff bases for sulfadi-
azine 2a and sulfamethazine 4a. Reverse relationship was observed
for sulfamethoxazole 1a and sulfathiazole 3a. The formation of the
Schiff base of the very low active sulfamethazine 4a brought deriv-
ative 4bwith onlya moderate improvementof MIC, but amide 4c was
described being the most antibacterial active molecule in this series.
In vitro efficacy of new derivatives against Gram-negative
bacteria was much lower than against Gram-positive,
correspondingly to unsubstituted sulfonamides. E. coli was the
m
m
m
most susceptible species, affected by 1b (MIC of 125e500
1c (250e500 mol/L), 3b, 4b (500 mol/L) and above all 2b
(62.5e250 mol/L). Both strains of K. pneumoniae were affected only
mmol/L),
m
m
m
by a high concentration of Schiff bases 1b, 2b and 3b (500
P. aeruginosa was completely insusceptible (MIC ꢁ500
m
m
mol/L).
mol/L).
2.2.2. Antibacterial activity (Table 3)
The in vitro antibacterial activity was assayed against
Gram-positive and Gram-negative strains: S. aureus, methicillin-
resistant S. aureus (MRSA), Staphylococcus epidermidis, Entero-
coccus sp., E. coli, K. pneumoniae, ESBL-positive K. pneumoniae and
P. aeruginosa. Zinc salt of bacitracin (BAC) and starting sulfonamides
were used as comparative drugs.
However, the activity is still only mild, but favourable over starting
sulfonamides. The presence of o-hydroxy group related to the azo-
methine bridge seems to be necessary for the activity.
It is obvious, that both modification of sulfonamide’s primary
amino group brings promising significant improvement of anti-
bacterial activity.
Sulfonamides exhibited only moderate activity against Gram-
positive bacterial strains. Two strains of S. aureus were affected by
sulfamethoxazole 1a and sulfathiazole 3a with MIC ranging from
2.2.3. Antifungal activity
Antifungal activity was determined in vitro against four yeast
strains (C. albicans, Candida tropicalis, Candida krusei, Candida
glabrata) and four moulds e Trichosporon asahii, Aspergillus fumi-
gatus, Absidia corymbifera and Trichophyton mentagrophytes.
As expected, sulfonamides expressed almost lack of antifungal
Table 4
IC50 and selectivity indexes for selected sulfonamide derivatives.
IC50
[m
mol/L]
SI for M. kansasii
SI for S. aureus
activity, only SMX 1a inhibited A. fumigatus at 250
base 2b, as the most efficacious compound in vitro, was found to
have moderate activity against C. albicans, C. glabrata (125 mol/L)
mol/L). Amide 4c affected T. asahii and T.
mol/L. Other derivatives were completely
inactive at the concentrations higher than 125 mmol/L (1b, 3b, 3c,
mmol/L. Schiff
1a
1b
3a
3b
4c
2622.0
193.2
4161.0
312.9
337.0
163.9e655.5
12.1e48.3
1040.3e4161.0
78.2e156.5
5.4e21.1
5.2e42.0
6.1e12.4
33.2e133.2
10.0
m
and T. asahii (62.5e125
mentagrophytes at 125
m
m
10.8e21.6
SI ¼ IC50/MIC100 (M. kansasii) or IC50/MIC90 (both strains of S. aureus).

M. Krátký et al. / European Journal of Medicinal Chemistry 50 (2012) 433e440
437
4b) or 500
m
mol/L (1c,1d,1e). The introduction of salicylic fragment
a Melting Point machine B-540 (Büchi) apparatus using open
capillaries and the reported values are uncorrected.
into sulfonamide molecules brings only a negligible benefit despite
of the kind of connection (amide/azomethine).
Infrared spectra (ATR) were recorded on FT-IR spectrometer
Nicolet 6700 FT-IR in the range of 400e4000 cmꢂ1. The NMR
spectra were recorded on a Varian VNMR S500 (500 MHz for 1H and
125 MHz for 13C; Varian, Inc., Palo Alto, USA) at ambient temper-
ature using deuterated dimethyl sulfoxide (DMSO-d6) solutions of
2.2.4. In vitro cytotoxicity
Cytotoxicity (Hep G2 cells, a method measuring a tetrazolium
salt reduction) was determined for the most active derivatives 1b,
3b, 4c and sulfonamides 1a and 3a (Table 4). The cytotoxicity is
expressed as IC50, i.e. concentration, at which was the viability of
the cells decreased to 50% from the maximal viability.
Although synthesized derivatives (1b, 3b, 4c) did not exhibit
the samples. The chemical shifts d are given in ppm with respect to
tetramethylsilane as an internal standard. The coupling constants
(J) are reported in Hz. Elemental analysis (C, H, N) were performed
on an automatic microanalyser CHNSeO CE instrument (FISONS EA
1110, Italy).
sharp cytotoxicity (IC50 192.2e337.0 mmol/L), the introduction of 2-
hydroxyphenyl ring into sulfonamide molecules resulted in the
increased cytotoxicity (1a vs. 1b and 3a vs. 3b) more than ten times.
Based on the comparison of MIC and IC50, the antimicrobial activity
of presented compounds is not caused by general cytotoxic impact,
but they have additional specific effect(s).
4.1.2. Synthesis of Schiff bases (Scheme 1)
An appropriate sulfonamide (1.5 mmol) was dissolved in 7 mL of
96% ethanol, one equivalent of 5-chlorosalicylaldehyde or 3-
chlorobenzaldehyde (in the case of 1d) was added in one portion
by micropipette. The solution was refluxed with stirring for 3 h and
after this time let stand at the room temperature and then for 12 h
at þ4 ꢃC. The precipitate was filtered off and repeatedly washed
with 5 mL of cold 96% ethanol. The product was recrystallized from
boiling ethanol, if necessary.
We calculated selectivity indexes for two the most susceptible
bacteria, M. kansasii (both strains) and S. aureus including MRSA.
Both sulfamethoxazole 1a and sulfathiazole 3a exhibit very high SI
against M. kansasii (ꢁ163.9), whereas sulfamethoxazole Schiff base
1b, sulfathiazole Schiff base 3b and sulfamethazine amide 4c showed
clearly lower values (5.1e156.5). The SI for 1b and 3b are all still
satisfactory (SI values higher than 10 could be considered being
sufficient, others are poor). This decreasing of SI is conditioned by the
increased cytotoxicity and not significantly improved anti-
mycobacterial activity. However, it is not definite and clarified, that
these salicyl derivatives act by the same way as parent sulfonamides.
For S. aureus, the favourable molecule is sulfathiazole 3a due to
very low cytotoxicity. Its Schiff base 3b and sulfamethazine amide
4c exhibited sufficient SI ꢁ10, whereas SMX 1a and its Schiff base
1b revealed for some concentrations poor SI <10 and for others
higher ones.
4.1.3. Synthesis of amides (Scheme 2)
5-Chlorosalicylic acid or 3-chlorobenzoic acid (in the case of 1e)
and appropriate sulfonamide (both 1.5 mmol) were suspended in
15 mL of the mixture of toluene and chlorobenzene (1:1), PCl3
(0.75 mmol) was added in one portion by micropipette. The reac-
tion was carried out with vigorous stirring in a microwave reactor
(550 W) for 30 min to reflux. Then the reaction mixture was filtered
while hot, let stand at room temperature and then at þ4 ꢃC for 12 h.
The crude product was filtered off and recrystallized from boiling
ethanol to obtain a pure product.
Although the activity of synthesized derivatives against staph-
ylococci was improved, concomitantly the cytotoxicity was higher
and SI are diminished. However, the selectivity indexes especially
of Schiff bases 1b and 3b towards M. kansasii and Schiff base 3b and
amide 4c against S. aureus are satisfactory.
4.1.4. 4-(5-Chloro-2-hydroxybenzylideneamino)-N-(5-
methylisoxazol-3-yl)benzenesulfonamide (1b)
Red solid; yield 84%; mp 226.5e228.5 ꢃC. IR: 3098, 2997, 2912,
2814, 1619 (azomethine, eHC]Ne), 1588, 1563, 1475, 1412, 1351,
1271, 1168, 1092, 1045, 939, 917, 889, 821, 780, 746, 662. 1H NMR
(500 MHz, DMSO):
d 12.30 (1H, s, OH), 11.49 (1H, s, SO2NH), 8.90
3. Conclusion
(1H, s, azomethine), 7.91 (2H, d, J ¼ 8.6 Hz, H20), 7.75 (1H, d,
J ¼ 2.7 Hz, H6), 7.53 (1H, d, J ¼ 8.6 Hz, H4), 7.46 (2H, dd, J ¼ 8.8,
J ¼ 2.7 Hz, H30), 7.00 (1H, d, J ¼ 8.8 Hz, H3), 6.16 (1H, s, isoxazole),
We designed and synthesized ten derivatives of four sulfon-
amides based on the formation of Schiff bases derived from 5-
chlorosalicylaldehyde (5-chloro-2-hydroxybenzaldehyde) and of
amides from 5-chlorosalicylic acid (5-chloro-2-hydroxybenzoic
acid). Sulfonamides and these derivatives were evaluated in vitro
against eight bacterial and fungal strains and four mycobacterial
strains. The best activity was found against M. kansasii and S. aureus
including MRSA strain. Fungi were the less susceptible group. In
general, selectivity indexes of sulfamethoxazole and sulfathiazole
5-chloro-2-hydroxybenzaldehyde-derived Schiff bases and sulfa-
methazine 5-chloro-2-hydroxybenzoic acid-derived amide against
two named pathogens exceed 10.
2.29 (3H, s, CH3). 13C NMR (125 MHz, DMSO):
157.7, 153.0, 137.3, 133.7, 130.8, 128.5, 123.0, 122.4, 121.0, 119.0, 95.6,
12.3. Anal. Calcd. for C17H14ClN3O4S (391.83): C, 52.11; H, 3.60; N,
10.72. Found: C, 52.30; H, 3.71; N, 10.63.
d 170.6, 163.8, 159.0,
4.1.5. 5-Chloro-2-hydroxy-N-(4-(N-(5-methylisoxazol-3-yl)
sulfamoyl)phenyl)benzamide (1c)
White solid; yield 50%; mp 266e268 ꢃC. IR: 3069, 2835, 1661,
1614 (C]O),1593,1552,1511,1498,1471,1422,1331,1227,1166,1093,
884, 825, 796, 685, 657. 1H NMR (500 MHz, DMSO): 11.39 (1H, s,
SO2NH),
d 10.97 (1H, s, OH), 10.70 (1H, s, CONH), 7.91 (2H, d,
J ¼ 8.9 Hz, H20), 7.84 (1H, d, J ¼ 2.7 Hz, H6), 7.49 (1H, d, J ¼ 8.8 Hz, H4),
7.45 (2H, dd, J ¼ 8.8 Hz, J ¼ 2.7 Hz, H30), 7.05 (1H, d, J ¼ 8.8 Hz, H3),
6.08 (1H, s, isoxazole), 2.27 (3H, s, CH3). 13C NMR (125 MHz, DMSO):
4. Experimental protocols
4.1. Chemistry
d 170.5, 165.1, 158.1, 156.3, 142.8, 134.1, 133.2, 129.0, 128.2, 123.0,
120.5,119.2,113.7, 95.6,12.3. Anal. Calcd. for C17H14ClN3O5S (407.83):
C, 50.07; H, 3.46; N, 10.30. Found: C, 50.34; H, 3.67; N, 10.62.
4.1.1. General methods
All the reagents and solvents were purchased from
SigmaeAldrich or Penta Chemicals and they were used as received.
Reactions were monitored by thin layer chromatography (plates
coated with 0.2 mm Merck 60 F254 silica gel) and were visualized
by UV irradiation (254 nm). Melting points were determined on
4.1.6. 4-(3-Chlorobenzylideneamino)-N-(5-methylisoxazol-3-yl)
benzenesulfonamide (1d)
White solid; yield 81%; mp 85e88 ꢃC. IR: 3085, 2992, 2894, 1616
(azomethine, eHC]Ne), 1594, 1507, 1470, 1393, 1329, 1270, 1158,

438
M. Krátký et al. / European Journal of Medicinal Chemistry 50 (2012) 433e440
1092, 1036, 921, 826, 789, 689. 1H NMR (500 MHz, DMSO):
d
11.44
J ¼ 8.5 Hz, H4), 7.45 (2H, dd, J ¼ 8.8, J ¼ 2.7 Hz, H30), 7.00 (1H, d,
J ¼ 8.8 Hz, H3), 6.75 (1H, s, pyrimidine), 2.25 (6H, s, CH3). 13C NMR
(1H, s, SO2NH), 8.62 (1H, s, azomethine), 7.86 (2H, d, J ¼ 8.5 Hz, H20),
7.64e7.39 (6H, m, H2, H4, H5, H6, H30), 6.15 (1H, s, isoxazole), 2.26
(125 MHz, DMSO):
d 170.4, 163.5, 159.0, 156.2, 151.9, 139.2, 133.5,
(3H, s, CH3). 13C NMR (125 MHz, DMSO):
d
170.2, 162.4, 158.1, 153.5,
130.9, 129.7, 123.0, 121.5, 120.9, 118.9, 111.9, 18.8. Anal. Calcd. for
C19H17ClN4O3S (416.88): C, 54.74; H, 4.11; N, 13.44. Found: C, 54.99;
H, 3.92; N, 13.51.
137.7, 133.9, 132.0, 131.1, 129.2, 128.7, 127.8, 121.9, 112.8, 95.5, 12.3.
Anal. Calcd. for C17H14ClN3O3S (375.83): C, 54.33; H, 3.75; N, 11.18.
Found: C, 54.52; H, 3.64; N, 11.33.
4.1.13. 5-Chloro-N-(4-(N-(4,6-dimethylpyrimidin-2-yl)sulfamoyl)
phenyl)-2-hydroxybenzamide (4c)
4.1.7. 3-Chloro-N-(4-(N-(5-methylisoxazol-3-yl)sulfamoyl)phenyl)
benzamide (1e)
Slightly yellow solid; yield 69%; mp 121.5e123.5 ꢃC. IR: 2838,
1623 (C]O),1595,1531,1495,1416,1382,1302,1163,1143,1081, 970,
917, 839, 778, 705, 660. 1H NMR (500 MHz, DMSO): 11.01 (1H, s,
Slightly yellow solid; yield 44%; mp 227e228.5 ꢃC. IR: 3321,
3247, 1669, 1615 (C]O), 1589, 1518, 1472, 1402, 1389, 1315, 1285,
1260, 1166, 1094, 930, 903, 835, 800, 737, 696, 676. 1H NMR
SO2NH),
d 10.94 (1H, s, OH), 10.67 (1H, s, CONH), 7.97 (2H, d,
(500 MHz, DMSO):
d
11.37 (1H, s, SO2NH), 10.71 (1H, s, CONH),
J ¼ 8.8 Hz, H20), 7.84 (1H, d, J ¼ 2.8 Hz, H6), 7.53 (1H, d, J ¼ 8.9 Hz, H4),
7.44 (2H, dd, J ¼ 8.8 Hz, J ¼ 2.7 Hz, H30), 6.95 (1H, d, J ¼ 8.1 Hz, H3),
6.73 (1H, s, pyrimidine), 2.23 (6H, s, CH3). 13C NMR (125 MHz,
8.00e7.83 (6H, m, H2, H6, H20, H30), 7.68 (1H, d, J ¼ 8.1 Hz, H4),
7.59e7.55 (1H, m, H5), 6.14 (1H, s, isoxazole), 2.29 (3H, s, CH3). 13
C
NMR (125 MHz, DMSO):
d
170.5, 164.9, 157.7, 143.4, 136.5, 133.9,
DMSO): d 170.7, 167.5, 159.9, 156.5, 142.1, 135.4, 133.2, 130.5, 130.2,
133.5, 132.0, 130.7, 128.1, 127.8, 126.9, 120.3, 95.6, 12.3. Anal. Calcd.
for C17H14ClN3O4S (391.83): C, 52.11; H, 3.60; N, 10.72. Found: C,
51.90; H, 3.78; N, 10.54.
129.4, 123.0, 119.7, 119.5, 113.6, 23.1. Anal. Calcd. for C19H17ClN4O4S
(432.88): C, 52.72; H, 3.96; N,12.94. Found: C, 52.97; H, 3.64; N,13.22.
4.2. Pharmacology
4.1.8. 4-(5-Chloro-2-hydroxybenzylideneamino)-N-(pyrimidin-2-
yl)benzenesulfonamide (2b) [34]
4.2.1. Antimycobacterial susceptibility
Slightly orange solid; yield 88%; mp 260.5e263 ꢃC (241 ꢃC
(acetone) [34]); IR: 1624 (azomethine, eHC]Ne).
All the prepared compounds were tested for their in vitro anti-
mycobacterial activity against M. tuberculosis 331/88 (H37Rv)
(dilution of the strain was 10ꢂ3) and moreover for some non-
tuberculous INH-resistant strains M. avium (330/88, resistant to
INH, rifampicin, ofloxacin, and ethambutol; dilution 10ꢂ5) and
M. kansasii (235/80, dilution 10ꢂ4). One strain was clinically isolated
M. kansasii 6509/96 (dilution 10ꢂ5); other strains were obtained
from the Czech National Collection of Type Cultures (CNCTC). The
micromethod for the determination of MIC was used. Anti-
4.1.9. 5-Chloro-2-hydroxy-N-(4-(N-pyrimidin-2-ylsulfamoyl)
phenyl)benzamide (2c)
White solid; yield 63%; mp 223.5e226 ꢃC. IR: 3423, 3355, 3076,
2939, 2872, 1622 (C]O), 1594, 1580, 1492, 1441, 1409, 1326, 1153,
1092, 942, 843, 824, 797, 719, 681, 667. 1H NMR (500 MHz, DMSO):
d
11.57 (1H, s, SO2NH), 11.15 (1H, s, OH), 10.66 (1H, s, CONH), 8.47
(2H, d, J ¼ 4.8 Hz, pyrimidine C400 and C600), 7.89 (2H, d, J ¼ 8.9 Hz,
H20), 7.84 (1H, d, J ¼ 2.9 Hz, H6), 7.62 (1H, d, J ¼ 8.8 Hz, H4), 7.44
(2H, dd, J ¼ 8.8 Hz, J ¼ 2.7 Hz, H30), 7.00 (1H, d, J ¼ 8.8 Hz, H3), 6.59
(1H, t, J ¼ 4.4 Hz, pyrimidine H500). 13C NMR (125 MHz, DMSO):
mycobacterial activities were determined in the Sula semisynthetic
ꢀ
medium [35] (SEVAC, the Czech Republic). The tested compounds
were added to the medium as solutions in dimethyl sulfoxide
(DMSO). The following concentrations were used: 1000, 500, 250,
d
170.9, 158.5, 157.4, 152.6, 142.7, 135.4, 133.5, 130.2, 130.0, 128.6,
125, 62, 32, 16, 8, 4, 2, and 1
after incubation at 37 ꢃC for 14 and 21 days, for M. kansasii addi-
tionally for 7 days. MIC ( mol/L) was the lowest concentration at
mmol/L. The MICs were determined
125.6, 120.0, 115.7, 112.8. Anal. Calcd. for C17H13ClN4O4S (404.83): C,
50.44; H, 3.24; N, 13.84. Found: C, 50.78; H, 3.47; N, 13.54.
m
which the complete inhibition of mycobacterial growth occurred.
As the reference compounds were chosen starting unmodified
sulfonamides e sulfamethoxazole, sulfadiazine, sulfathiazole, sul-
famethazine (well-known and formerly widely clinically used) and
a standard antituberculotic drug isoniazid.
4.1.10. 4-(5-Chloro-2-hydroxybenzylideneamino)-N-(thiazol-2-yl)
benzenesulfonamide (3b) [34]
Yellow solid; yield 92%; mp 234e236 ꢃC (224 ꢃC (acetone) [34]);
IR: 1620 (azomethine, eHC]Ne).
4.1.11. 5-Chloro-2-hydroxy-N-(4-(N-thiazol-2-ylsulfamoyl)phenyl)
benzamide (3c)
4.2.2. Antibacterial evaluation
The in vitro antibacterial activity was assayed against Gram-
positive and Gram-negative strains: S. aureus CCM 4516/08,
methicillin-resistant S. aureus H 5996/08 (MRSA), S. epidermidis H
White solid; yield 44%; mp 276e278 ꢃC. IR: 3114, 1645 (C]O),
1588, 1570, 1533, 1497, 1417, 1328, 1281, 1146, 1091, 936, 851, 832,
814, 732, 686, 661. 1H NMR (500 MHz, DMSO):
d
12.72 (1H, s,
6966/08, Enterococcus sp. J 14365/08; E. coli CCM 4517,
SO2NH),11.61 (1H, s, OH),10.61 (1H, s, CONH), 7.87e7.77 (4H, m, H4,
H6, H20), 7.46 (2H, dd, J ¼ 8.9 Hz, J ¼ 2.6 Hz, H30), 7.25 (1H, d,
J ¼ 4.5 Hz, thiazole]CHeN), 7.02 (1H, d, J ¼ 8.8 Hz, H3), 6.82 (1H, d,
K. pneumoniae D 11750/08, ESBL-positive K. pneumoniae J 14368/08,
and P. aeruginosa CCM 1961.
The microdilution broth method modified according to standard
M07-A07 [36] in Mueller-Hinton broth (HiMedia Laboratories,
India) adjusted to pH 7.4 (ꢄ0.2) was used. The investigated
compounds were dissolved in DMSO to the final concentrations
J ¼ 4.5 Hz, thiazole]CHeS). 13C NMR (125 MHz, DMSO):
d 169.0,
165.1, 156.4, 141.6, 137.4, 133.2, 128.8, 127.9, 127.1, 124.7, 123.0, 120.3,
119.2, 108.4. Anal. Calcd. for C16H12ClN3O4S2 (409.87): C, 46.89; H,
2.95; N, 10.25. Found: C, 46.58; H, 2.89; N, 10.34.
ranging from 500 to 0.49 mmol/L. Zinc salt of bacitracin and starting
sulfonamides were used as comparative drugs. Bacterial inoculum
in sterile water was prepared to match 0.5 McFarland scale
(1.5 ꢅ 108 CFU/mL). The minimum inhibitory concentrations (MIC)
were assayed as 90% (IC90) or higher reduction of growth when
compared to the control. The determination of results was per-
formed visually and spectrophotometrically (at 540 nm). The
values of MICs were determined after 24 and 48 h of incubation in
the darkness at 35 ꢃC (ꢄ0.1) in a humid atmosphere.
4.1.12. 4-(5-Chloro-2-hydroxybenzylideneamino)-N-(4,6-
dimethylpyrimidin-2-yl)benzenesulfonamide (4b)
Orange solid; yield 96%; mp 132.5e134.5 ꢃC. IR: 3073, 1621
(azomethine, eHC]Ne), 1595, 1564, 1479, 1434, 1384, 1353, 1268,
1157, 1078, 977, 867, 840, 788, 692, 659. 1H NMR (500 MHz, DMSO):
d
12.45 (1H, s, OH), 11.83 (1H, s, SO2NH), 8.92 (1H, s, azomethine),
8.04 (2H, d, J ¼ 8.5 Hz, H20), 7.76 (1H, d, J ¼ 2.7 Hz, H6), 7.50 (1H, d,

M. Krátký et al. / European Journal of Medicinal Chemistry 50 (2012) 433e440
439
4.2.3. Antifungal evaluation
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kit (with contained MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-
carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) was
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