Novel derivatives of nitro-substituted salicylic acids: Synthesis, antimicrobial activity and cytotoxicity

Article abstract of DOI:10.1016/j.bmc.2015.10.029

Inspired by the high antituberculous activity of novel nitro-substituted derivatives and based on promising predicted ADMET properties we have synthesized a series of 33 salicylanilides containing nitro-group in their salicylic part and evaluated them for

Full text of DOI:10.1016/j.bmc.2015.10.029

Bioorganic & Medicinal Chemistry 23 (2015) 7292–7301
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Novel derivatives of nitro-substituted salicylic acids: Synthesis,
antimicrobial activity and cytotoxicity
a
a
b
b
c
´
ˇ
ˇ
Georgios Paraskevopoulos , Martin Krátky , Jana Mandíková , František Trejtnar , Jirina Stolaríková ,
Petr Pávek b, Gurdyal Besra d, Jarmila Vinšová a,
⇑
a Department of Inorganic and Organic Chemistry, Faculty of Pharmacy, Charles University in Prague, Heyrovského 1203, 500 05 Hradec Králové, Czech Republic
b Department of Pharmacology and Toxicology, Faculty of Pharmacy, Charles University in Prague, Heyrovského 1203, 500 05 Hradec Králové, Czech Republic
c
ˇ
Laboratory for Mycobacterial Diagnostics and Tuberculosis, Regional Institute of Public Health in Ostrava, Partyzánské námestí 7, 702 00 Ostrava, Czech Republic
d School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom
a r t i c l e i n f o
a b s t r a c t
Article history:
Inspired by the high antituberculous activity of novel nitro-substituted derivatives and based on promis-
ing predicted ADMET properties we have synthesized a series of 33 salicylanilides containing nitro-group
in their salicylic part and evaluated them for their in vitro antimycobacterial, antimicrobial and antifun-
gal activities. The presence of nitro-group in position 4 of the salicylic acid was found to be beneficial and
Received 21 August 2015
Revised 13 October 2015
Accepted 22 October 2015
Available online 24 October 2015
the resulting molecules exhibited minimum inhibitory concentrations (MICs) ranging from 2 to 32
against Mycobacterium tuberculosis. The best activity was found for 2-hydroxy-4-nitro-N-[4-(trifluo-
romethyl)phenyl]benzamide (MIC = 2 M). 4-Nitrosalicylanilides were also found to be active against
all Staphylococcus species tested while for MRSA strain 2-hydroxy-4-nitro-N-[4-(trifluoromethyl)phe-
nyl]benzamide’s MIC was 0.98 M. None of the nitrosalicylanilides was active against Enterococcus
lM
Keywords:
Salicylanilides
Nitro group
In silico ADMET prediction
In vitro antimycobacterial activity
In vitro antimicrobial activity
Cytotoxicity
l
l
sp. J 14365/08 and no considerable activity was found against Gram-negative bacteria or fungi. The
hepatotoxicity of all nitrosalicylanilides was found to be in the range of their MICs for HepG2 cells.
Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction
need to enrich the pipeline of available drugs against TB, preferably
with candidates offering lower cytotoxicity and with new mode of
According to the World Health Organization, tuberculosis (TB)
is second only to Human Immunodeficiency Virus (HIV) as the
greatest killer worldwide due to a single infectious agent.1 The
numbers reported at the Global Tuberculosis Report for 2014 are
highlighting the severity of the disease. During 2013, 9 million
people fell ill with TB and 1.5 million people died. Whilst TB is
treatable and curable, standard anti-TB drugs, like isoniazid (INH)
and rifampicin (RMP), have been used for decades, and resistance
to the medicines is widespread. Clinical strains that are resistant
to a single anti-TB drug have been documented in every country
surveyed giving rise to multidrug-resistant tuberculosis (MDR-
TB), extensively drug-resistant tuberculosis (XDR-TB) and the
recently reported totally drug-resistant tuberculosis (TDR-TB).2–4
In addition, the standard treatment for adult respiratory TB is a
regimen of drugs developed many years ago and is connected with
several adverse effects like hepatotoxicity, skin reactions, gastroin-
testinal and neurological disorders.5 Among them hepatotoxicity is
the most serious one,6 directing the current research towards the
action in comparison to existing drugs.
On the contrary, various opportunistic human infections are
caused by nontuberculous mycobacteria (NTM) and, according to
the predictions, they will continue to increase their incidence at
least up to year 2050 mainly due to an increasing elderly popula-
tion.7 The treatment of NTM caused infections is complicated and
involves multiple medications due to the high levels of natural
and acquired antibiotic resistance. Furthermore, their current
treatment share limited efficacy,8 and therefore there is a need
for new therapeutic strategies to be developed. Moreover, severe
hospital-acquired infections can be caused due to notable
drug-resistance complications that have been reported for Gram-
positive bacteria, including methicillin-resistant Staphylococcus
aureus (MRSA), Streptococcus pneumoniae and Enterococci.9
Salicylanilides (2-hydroxy-N-phenylbenzamides) have been the
subject of several medicinal chemistry studies due to their signifi-
cant in vitro antimicrobial and antifungal activities.10–12 A number
of salicylanilide derivatives have been developed so far with pro-
ven activity against Mycobacterium tuberculosis (Mtb) and INH-
resistant strains.13–15 However, the precise molecular mode of
action of salicylanilides is not clear. It has been previously reported
⇑
Corresponding author. Tel.: +420 495067343, fax: +420 495067166.
E-mail address: jarmila.vinsova@faf.cuni.cz (J. Vinšová).
http://dx.doi.org/10.1016/j.bmc.2015.10.029
0968-0896/Ó 2015 Elsevier Ltd. All rights reserved.

G. Paraskevopoulos et al. / Bioorg. Med. Chem. 23 (2015) 7292–7301
7293
that a free phenolic hydroxyl on the salicylic acid moiety is
required for activity and suggested that they might function as
proton shuttles that kill bacterial cells by destroying the cellular
proton gradient.16 On the other hand, salicylanilides and their ester
derivatives have been reported to act as moderate inhibitors of
mycobacterial and human methionine aminopeptidases and may
target the function of mycobacterial isocitrate lyase which could
enhance its efficacy due to its particular relevance to mycobacterial
latency.17 These findings have suggested that salicylanilides affect
multiple targets to exert their antimicrobial properties. Further-
more, salicylanilide esters, carbamates and additional derivatives
resulting from their conjugation with existing antimicrobial
agents, have been suggested to work as pro-drugs that are
hydrolyzed in order to express their activity.18
Although salicylanilides and their derivatives (esters,
carbamates etc.) are very potent against Mtb, they often share a
non-preferable cytotoxic profile.16,19 This drawback has limited
their utility as potential therapeutics so far. Recent work has
focused on the discovery of salicylanilide derivatives with
decreased cytotoxicity and, under this concept, novel 2-(phenyl-
carbamoyl)phenyl 4-substituted benzoates were synthesized and
preferable nitro-substitution of the salicylic part of salicylanilides
has not been reported in the literature to date, even if some
nitro-substituted salicylanilides have been previously pre-
sented.16,21,31–34
2. Results and discussion
2.1. ADMET properties prediction
The novel salicylanilides were screened in silico, prior to their
synthesis, for their ADMET (Absorption, Distribution, Metabolism,
Excretion, and Toxicity) properties. The software of choice was
ADMET Predictor (Simulation Plus, Lancaster, CA), one of the
leading computer software for advanced predictive modeling of
ADMET properties.35 The program uses molecular description
values as inputs to independent mathematical models (generally,
nonlinear machine learning technics) in order to generate esti-
mates for each of the ADMET properties.36 ADMET Predictor not
only rapidly estimates a number of vital ADMET properties from
molecular structures, but it is able to validate a series of ‘ADMET
Risk’ or ‘Tox Risk’ parameters. They are parameterized to include
thresholds for a wide range of calculated and predicted properties
that represent potential obstacles to a compound being success-
fully developed as a drug.
found to be active against several mycobacteria (0.125–8
while they exhibited no cytotoxicity at concentrations of up to
50
M against a hepatocyte cell line (HepG2).20 The esterification
lM)
l
products of 5-halogenated salicylanilides with 4-substituted ben-
zoic acids were found to be, not only more potent inhibitors of
Mtb growth but also to share a preferable cytotoxic profile in con-
trast with the parent salicylanilides. The increased lipophilicity of
the afforded derivatives limited their solubility in aqueous media
and hence their drugability.
Our ongoing efforts explore the influence of different substitu-
tions on the parent structure of salicylanilides, in terms of their
activity and cytotoxicity. The introduction of several substituents
is under investigation. In general, it has been previously reported
that an electron withdrawing group is favored in the aniline part
while the influence of the substituents from the acyl moiety is
more complex.21 The best substituents of the parent salicylanilide
structure reported so far is –Br or –Cl atoms in position 5 of the sal-
icylic acid part while a –CF3 group is favoured in position 4 of the
aniline part. More specifically, 5-bromo-2-hydroxy-N-[4-(trifluo-
romethyl)phenyl]benzamide and 5-chloro-2-hydroxy-N-[4-(triflu-
oromethyl)phenyl]benzamide are inhibiting the growth of Mtb at
For the majority of the compounds, the analyses indicated no
potential risk of either ADME/absorption problems related with
physico-chemical properties or any stability risk related to fast
hepatic metabolism. Furthermore, no potential interaction related
to inhibition of drug biotransformation enzymes was indicated.
Most importantly, the tested compounds were not found to be
potentially related with toxicity or carcinogenicity as indicated
by parameters of acute toxicity and carcinogenicity in rats
expressed as TD50 and LD50 values, respectively, mutagenic chro-
mosomal aberrations, or maximum recommended therapeutic
dose (MRTD).
More specifically, 17 out of 33 compounds showed a preferable
ADMET Risk (score 0–2). 5 out of 33 compounds presented an
acceptable ADMET Risk (score 3), limited by potential high
lipophilicity and absorption related risk. 11 out of 33 compounds
showed a non-preferable ADMET Risk profile (score 4–5). On the
other hand, 28 out of 33 compounds showed no potential risk of
toxicity (score 0–2) and only 5 out of 33 compounds showed an
acceptable risk of toxicity (score 3). There was no direct correlation
between the position of the nitro group and the potential risk of
toxicity. Furthermore, the substituent on the aniline part is more
likely affecting the potential ADMET properties, as halogens in posi-
tion 3 is preferred while trifluoromethyl group and nitro group are
not preferable. The ADMET Risk, as well as the Tox Risk scores of the
screened compounds are summarized in Table S1. With the encour-
aging prediction results the synthesis of the molecules was decided.
the concentrations of 1 and 2 lM, respectively.
Current research in the field of novel antituberculotics on their
way to the clinic has revealed that many nitro-substituted com-
pounds owe their antimycobacterial activity to the presence of a
nitro group in their scaffold.22,23 For example, the nitro-reduction
of nitroimidazole OPC-67683 (also known as delamanid)24 and
PA-82425 by F420-deazaflavin-dependent nitroreductase (Ddn)
releases nitric oxide, which is thought to inhibit cytochrome
oxidase and other targets. In addition, benzothiazinones (BTZ),
e.g., BTZ-04326 and BTZ-derived inhibitors, e.g., CT13927 may inter-
2.2. Chemistry
act through their nitro-group with
decaprenylphosphoryl-b-
-ribose 20-epimerase (DprE1), either
covalently or not, thus blocking the first step in the epimerization
reaction of decaprenyl-phosphoryl- -ribose (DPR) to decaprenyl-
phosphoryl- -arabinose (DPA) the arabinan donor for arabino-
a cysteine residue of
D
The synthesis of nitro-containing salicylanilides (1–33) is
described in Scheme 1. The final compounds were products of
the conjugation of nitro-salicylic acids with selected anilines in
the presence of PCl3 under microwave irradiation. All compounds
were isolated with good to moderate yields.
D
D
galactan. In a similar manner dinitrobenzamides, e.g., DNB128 are
forming a covalent bond with Cys387 within the active site of
DprE1. More recently, novel dinitrobenzyl-bearing benzazole and
tetrazole derivatives exhibited high and selective antimycobacte-
rial activity.29,30
O
O
R
PCl3, PhCl
MW
R
Herein we describe the influence of the introduction of a nitro-
group at the salicylic part of salicylanilides to their antimycobacte-
rial, antimicrobial and antifungal activities. To the best of our
knowledge, a structure activity relationship (SAR) concerning the
OH
OH
N
H
O2N
O2N
H2N
OH
Scheme 1. Synthesis of nitro-substituted salicylanilides.

7294
G. Paraskevopoulos et al. / Bioorg. Med. Chem. 23 (2015) 7292–7301
2.3. Antimycobacterial activity
(18) (MIC = 2 lM). The favorable substitutions in the aniline part,
regardless of the position of the nitro-group at the salicylic part,
were the mono- or bis-trifluoromethyl-substitution. This is proba-
bly due to the increased lipophilicity (higher calculated logP value)
of the molecule when bearing the trifluoromethyl moiety and is in
accordance with previous results regarding the preferable substi-
tution in the aniline part.21 Furthermore, it is noteworthy that
the presence of a nitro group in the aniline part of the molecule
resulted less active molecules as a result of their decreased
lipophilicity (lower calculated logP values).
The nitro-containing salicylanilides (1–33) were evaluated to
determine their in vitro antimycobacterial activity against
Mycobacterium tuberculosis 331/88 (i.e., H37RV), and the following
nontuberculous mycobacteria: Mycobacterium avium 330/88 and
Mycobacterium kansasii 235/80 and 6509/96 (a clinical isolated
strain). INH, the first-line anti-TB drug, was chosen for the compar-
ison performed in this study. Table 1 reports their minimum
inhibitory concentrations (MICs) as well as their calculated logP
values.
Nitro-substituted salicylanilides did not show any considerable
According to the nitro-substitution of the salicylic part, the
tested compounds could be divided into three groups: Group 1
includes 3-NO2 derivatives (1–11); Group 2 includes 4-NO2 deriva-
tives (12–22); and Group 3 includes 5-NO2 derivatives (23–33). It
was observed that the most preferable position of nitro-group is
position 4 of the salicylic part, followed by the nitro-substitution
in position 5. Nitro-substitution in position 3 of the salicylic part
was the least preferable.
active against M. avium 330/88 (MIC P 32 lM). On the other hand,
all tested molecules were more active than INH against M. kansasii
235/80, while 2-hydroxy-4-nitro-N-[4-(trifluoromethyl)phenyl]
benzamide (18) was the most active (MIC = 8
The same derivative (18) was similarly active with INH against
M. kansasii 6509/96 (MIC = 8 M after 21 days).
lM after 21 days).
l
2.4. Antibacterial and antifungal activity
Some nitrosalicylanilides exhibited good to moderate activity
against Mtb. For the 4-nitro substituted salicylanilides, the activity
Our efforts to further investigate the biological activity of the
nitrosalicylanilides, the derivatives that bear the nitro-group in
positions 4 and 5 of the salicylic part involved testing against eight
ranged between 2 and 32
l
M. The best activity was found
for
2-hydroxy-4-nitro-N-[4-(trifluoromethyl)phenyl]benzamide
Table 1
Antimycobacterial activity of nitrosalicylanilides 1–33
O
R2
N
H
R1
OH
R
R1
ClogP
MIC (lmol/L)
Mtb. 331/88
(H37Rv)
M. avium 330/88
M. kansasii 235/80
M. kansasii 6509/96
14 d
21 d
14 d
21 d
7 d
14 d
21 d
7 d
14 d
21 d
1
2
3
4
5
6
7
8
3-NO2
3-NO2
3-NO2
3-NO2
3-NO2
3-NO2
3-NO2
3-NO2
3-NO2
3-NO2
3-NO2
4-NO2
4-NO2
4-NO2
4-NO2
4-NO2
4-NO2
4-NO2
4-NO2
4-NO2
4-NO2
4-NO2
5-NO2
5-NO2
5-NO2
5-NO2
5-NO2
5-NO2
5-NO2
5-NO2
5-NO2
5-NO2
5-NO2
3-I
3-Br
3-Cl
3-F
3,4-diCl
3-CF3
4-CF3
3-CF3, 4-NO2
3-NO2
4-NO2
3,5-DiCF3
3-I
3-Br
3-Cl
3-F
3,4-DiCl
3-CF3
3.44
3.03
2.76
2.28
3.38
3.12
3.12
3.08
2.1
62.5
62.5
62.5
62.5
16
32
32
16
62.5
32
8
16
16
8
16
4
62.5
62.5
62.5
62.5
32
32
32
16
62.5
32
8
16
16
16
16
8
500
250
500
500
250
500
250
250
>500
125
125
32
32
32
62.5
32
62.5
32
500
250
500
500
250
500
250
500
>500
125
125
62.5
62.5
62.5
125
62.5
62.5
32
62.5
62.5
62.5
62.5
32
32
62.5
32
62.5
32
16
8
8
8
16
4
8
125
125
125
125
62.5
62.5
125
62.5
250
62.5
32
16
16
16
16
8
16
4
32
32
16
16
32
62.5
32
62.5
32
32
16
125
125
250
250
62.5
62.5
125
125
250
125
32
16
16
16
32
8
16
8
32
62.5
32
16
62.5
62.5
62.5
62.5
32
62.5
32
125
125
125
32
125
125
125
125
62.5
125
62.5
32
62.5
62.5
32
16
16
16
16
8
8
4
16
32
32
16
32
32
32
32
32
32
16
62.5
62.5
62.5
16
125
125
125
250
62.5
250
62.5
62.5
250
125
32
16
16
16
32
16
16
8
32
125
125
125
250
62.5
250
125
62.5
250
125
32
16
16
16
32
16
16
8
32
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
2.1
4.1
3.44
3.03
2.76
2.28
3.38
3.12
3.12
3.08
2.1
8
2
16
32
8
8
4
4-CF3
2
3-CF3, 4-NO2
3-NO2
4-NO2
3,5-DiCF3
3-I
3-Br
3-Cl
3-F
3,4-DiCl
3-CF3
16
32
16
8
16
16
16
32
16
16
8
32
62.5
125
16
1
62.5
125
62.5
32
62.5
125
62.5
32
16
32
8
32
32
16
32
32
16
2.1
4.1
4
8
3.44
3.03
2.76
2.28
3.38
3.12
3.12
3.08
2.1
16
16
16
16
8
125
125
125
125
125
125
62.5
125
125
250
32
125
250
125
125
125
125
62.5
125
125
250
32
16
32
16
16
16
16
8
32
16
16
16
>250
62.5
62.5
62.5
62.5
32
62.5
32
62.5
125
125
32
62.5
62.5
62.5
62.5
32
62.5
32
125
125
125
32
8
8
4-CF3
3-CF3, 4-NO2
3-NO2
4-NO2
3,5-DiCF3
32
62.5
62.5
16
0.5
62.5
62.5
62.5
32
2.1
4.1
—
INH
>250
>250
>250
>250
4
8
8
INH = isoniazid. The best values for each strain are provided in bold.

G. Paraskevopoulos et al. / Bioorg. Med. Chem. 23 (2015) 7292–7301
7295
bacterial and eight fungal strains. More specifically, the selected
molecules were tested against Staphylococcus aureus CCM
4516/08, methicillin-resistant Staphylococcus aureus H 5996/08
(MRSA), Staphylococcus epidermis H 6966/08, Enterococcus sp. J
14365/08, Escherichia coli CCM 4517, Klebsiella pneumoniae D
11750/08, extended spectrum b-lactamases producing Klebsiella
pneumoniae J 14368/08, and Pseudomonas aeruginosa CCM 1961.
Furthermore, the selected nitrosalicylanilides were tested against
four Candida strains (Candida albicans ATCC 44859, Candida
tropicalis 156, Candida krusei E28, and Candida glabrata 20/I),
Trichosporon asahii 1188 and three filamentous fungi (Aspergillus
fumigatus 231, Absidia corymbifera 272, and Trichophyton mentagro-
phytes 445). Benzylpenicillin (PNC) and fluconazole (FLU) were
used for comparison during antibacterial and antifungal activity
studies, respectively.
aureus CCM 4516/08 while 4-nitrosalicylanilides were always
more active against MRSA and Staphylococcus epidermis H 6966/08.
None of the tested compounds was found to be active against
any of the tested Gram-negative bacteria (Table S2). In addition,
nitrosalicylanilides were not found to possess any considerable
antifungal activity (Table S3).
2.5. Cytotoxicity evaluation
12 of the synthesized nitrosalicylanilides were evaluated for
their cytotoxic properties using an MTT assay in the HepG2 cell
model. HepG2 cells represent the most likely target tissue of anti-
tuberculotics-related toxicity, which often complicates the treat-
ment of TB.37 The tested compounds were chosen according to
their antimycobacterial activity and the position of the nitro-group
in the salicylic part. More specifically, 4 compounds from each dif-
ferent nitro-substitution of the salicylic part were selected for
cytotoxicity experiments. The substitution for the aniline part
was: 3,4-diCl, 3-CF3, 4-CF3 and 3,5-diCF3. The cytotoxicity results
as well as the selectivity indexes (SI) for the tested compounds
are summarized in Table 3.
Similar to their antimycobacterial activity, 4-nitro substituted
salicylanilides were found to be more active against all different
Staphylococcus species than their 5-nitro substituted analogues
(Table 2). The range of MIC values were between 1–62.5
lM and
3.9–500 M, respectively. The 3,5-bis(trifluoromethyl) moiety
l
was the best substitution for the aniline part for all the different
Staphylococcus species tested. The most active compound against
all different Staphylococcus species was N-[3,5-bis(trifluo-
romethyl)phenyl]-2-hydroxy-4-nitrobenzamide (22), which exhib-
The IC50 values for the tested compounds were in the range of
1.2–13.9
substituted salicylanilides showed consistently the lowest IC50 val-
ues (1.2–4.4 M), while the 3- and 5-nitro-substituted analogues
showed higher IC50 values (3.5–13.9 M). The highest rate of cyto-
lM. According to the position of the nitro group, 4-nitro
ited MIC of 1.95
while for MRSA strain the compound’s MIC was 0.98
l
M for S. aureus CCM 4516/08 and S. epidermis,
M. Once
l
l
l
again, the presence of a nitro-group in the aniline part of the mole-
cule was found to drastically decrease the activity. On the other
hand, no considerable activity was found against Enterococcus sp.
J 14365/08 (MIC P 62.5 lM). In comparison with PNC, none of
the nitrosalicylanilides was more active against Staphylococcus
toxicity was found for 2-hydroxy-4-nitro-N-[4-(trifluoromethyl)
phenyl]benzamide (18). On the other hand, the highest IC50
was found for N-[3,5-bis(trifluoromethyl)phenyl]-2-hydroxy-
5-nitrobenzamide (33). There is no direct correlation between
the substitution at the aniline part and cytotoxicity. SI values for
Table 2
Antibacterial activity of nitrosalicylanilides 12–33 (Gram-positive bacteria)
O
R2
N
H
R1
OH
R1
R2
MIC/IC95
(lmol/L)
SA
MRSA
SE
EF
24 h
48 h
24 h
48 h
24 h
48 h
24 h
48 h
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
PNC
4-NO2
4-NO2
4-NO2
4-NO2
4-NO2
4-NO2
4-NO2
4-NO2
4-NO2
4-NO2
4-NO2
5-NO2
5-NO2
5-NO2
5-NO2
5-NO2
5-NO2
5-NO2
5-NO2
5-NO2
5-NO2
5-NO2
3-I
3-Br
3-Cl
3-F
3,4-DiCl
3-CF3
4-CF3
3-CF3, 4-NO2
3-NO2
4-NO2
3,5-DiCF3
3-I
3-Br
3-Cl
3-F
3,4-DiCl
3-CF3
3.9
3.9
7.81
15.62
3.9
3.9
3.9
15.62
31.25
3.9
3.9
3.9
3.9
3.9
15.62
3.9
15.62
3.9
>500
500
500
500
125
250
250
250
>250
500
62.5
250
500
500
>500
125
>500
>500
>500
>500
>500
500
>500
500
>500
>500
500
500
250
500
>250
>500
125
>500
>500
>500
>500
125
>500
>500
>500
>500
>500
500
7.81
15.62
1.95
1.95
1.95
3.9
15.62
31.25
1.95
1.95
1.95
3.9
7.81
15.62
1.95
1.95
1.95
7.81
31.25
62.5
1.95
31.25
31.25
62.5
125
62.5
31.25
15.62
125
500
500
15.62
31.25
1.95
7.81
7.81
7.81
62.5
62.5
1.95
31.25
250
62.5
250
62.5
31.25
15.62
125
3.9
3.9
7.81
7.81
62.5
62.5
1.95
31.25
31.25
31.25
125
31.25
31.25
31.25
62.5
500
7.81
7.81
62.5
62.5
1.95
31.25
62.5
62.5
125
31.25
31.25
31.25
62.5
500
125
125
31.25
0.98
31.25
31.25
62.5
125
31.25
31.25
31.25
31.25
500
31.25
0.98
31.25
62.5
62.5
125
31.25
31.25
31.25
31.25
500
4-CF3
3-CF3, 4-NO2
3-NO2
4-NO2
3,5-DiCF3
500
500
15.62
250
250
3.9
0.98
250
3.9
0.98
250
7.81
62.5
250
7.81
125
15.62
250
7.81
15.62
SA: Staphylococcus aureus CCM 4516/08, MRSA: Methicillin-resistant Staphylococcus aureus H 5996/08, SE: Staphylococcus epidermidis H 6966/08, EF: Enterococcus sp. J 14365/
08, PNC: benzylpenicillin.

7296
G. Paraskevopoulos et al. / Bioorg. Med. Chem. 23 (2015) 7292–7301
Table 3
proved to retain their activity while possessing a less cytotoxic
profile.20,38 Under this concept, 4-nitrosalicylanilides could be
potential candidates for further development by preparing their
4-(substituted)benzoates. This method was previously reported by
our group for 5-halogenated salicylanilides affording the more
active salicylanilide (4-substituted)benzoates with a preferable
cytotoxic profile than the parent salicylanilides.20 The only limita-
tion observed for the (4-substituted)benzoates of 5-halogenated
salicylanilides was their poor solubility in aqueous media. It is
expected that the presence of a nitro group at the salicylic part,
instead of a halogen, will have a positive impact at the solubility
of 4-(substituted)benzoates of 4-nitrosalicylanilides. The sugges-
tion concerning the solubility is in agreement with the comparison
of the predicted calculated logP values between 4-(substituted)
benzoates of 4-nitrosalicylanilides and 4-(substituted)benzoates
of 5-halogenated salicylanilides (Table S4).
Cytotoxicity of selected nitrosalicylanilides
O
R2
N
H
R1
OH
R1
R2
IC50
(l
M) for
HepG2 cells
Selectivity index
(SI) for Mtb
5
6
7
3-NO2
3-NO2
3-NO2
3-NO2
4-NO2
4-NO2
4-NO2
4-NO2
5-NO2
5-NO2
5-NO2
5-NO2
3,4-DiCl
3-CF3
4-CF3
3,5-DiCF3
3,4-DiCl
3-CF3
5.9
8.9
8.0
7.4
1.4
2.8
1.2
4.4
8.9
7.4
3.5
13.9
0.184–0.369
0.278
0.250
0.925
0.175–0.350
0.350
0.300–0.600
0.550–1.100
0.556–1.113
0.463–0.925
0.438
11
16
17
18
22
27
28
29
33
4-CF3
3,5-DiCF3
3,4-DiCl
3-CF3
4-CF3
3,5-DiCF3
4. Experimental part
4.1. Chemistry
0.869
SI = IC50/MIC100
.
4.1.1. General methods
All of the reagents and solvents were purchased from Sigma–
Aldrich (Darmstadt, Germany) or Penta Chemicals (Prague, Czech
Republic) and used as received. The reactions and the purity of
the products were monitored by thin-layer chromatography using
a mixture of hexane to ethyl acetate of 4:1 or 2:1 as eluent. The
plates were coated with 0.2-mm Merck 60 F254 silica gel and were
visualized by UV irradiation (254 nm). The melting points were
determined on a Büchi Melting Point B-540 apparatus using open
capillaries, and the reported values are uncorrected.
Elemental analysis (C, H and N) was performed on an automatic
microanalyser CHNS-O CE instrument (FISONS EA 1110, Milano,
Italy). Infrared spectra (ATR) were recorded on a FT-IR spectrome-
ter (Nicolet 6700 FT-IR) in the range of 400–4000 cmꢀ1. The NMR
spectra were measured in DMSO at ambient temperature using a
Varian V NMR S500 instrument (500 MHz for 1H and 125 MHz
for 13C, Varian Comp., Palo Alto, CA, USA) or a Varian Mercury-Vxbb
300 (300 MHz for 1H and 75.5 MHz for 13C, Varian, Inc., Palo Alto,
CA, USA). The chemical shifts, d, are given in ppm with respect to
tetramethylsilane, which was used as an internal standard. The
coupling constants (J) are reported in Hz.
the tested compounds was always 61.113 indicating that antimy-
cobacterial activity trends with cytotoxicity.
3. Conclusions
In order to investigate the influence of the introduction of a
nitro group at the salicylic part of salicylanilides on their antimy-
cobacterial, antimicrobial and antifungal activities, a series of novel
salicylanilides, containing nitro group in their salicylic part, were
designed and synthesized. All compounds were screened in silico
with ADMET Predictor prior to their synthesis, in order to obtain
an estimated prediction about their potential ADME properties
and toxicity risk. The prediction revealed no potential risk of either
ADME/absorption problems related with physico-chemical proper-
ties or any stability risk related to fast hepatic metabolism for the
majority of the screened compounds. In addition the nitrosalicy-
lanilides showed minimum to acceptable risk of toxicity as
indicated from their Tox Risk score.
The synthesized compounds were evaluated for their antimy-
cobacterial activity against Mtb and found to possess activity in
the low micromolar range, comparable with the 5-halogenated sal-
icylanilides which are the most active salicylanilides presented to
date. The most preferable position of nitro-group was found to
be position 4 of the salicylic part, followed by the nitro substitution
in position 5. Nitro substitution in position 3 of the salicylic part
was the least preferable. Although nitro-substituted salicylanilides
did not show any considerable activity against M. avium, they were
more active than INH against M. kansasii 235/80. 2-hydroxy-4-
nitro-N-[4-(trifluoromethyl)phenyl]benzamide (18) was found to
possess similar activity with INH against M. kansasii 6509/96. Fur-
thermore, 4-nitro substituted salicylanilides were found to be
more active against all Staphylococcus species tested than their 5-
nitro substituted analogues while none of the nitrosalicylanilides
The calculated logP values (ClogP), which are the logarithms of
the partition coefficients for octan-1-ol/water, were determined
using the CS ChemOffice Ultra program (version 12.0, Cambridge-
Soft, Cambridge, MA, USA).
4.1.2. Synthesis
All salicylanilides (1–33) were synthesized via a previously
described method39 with yields of 60–85%. This microwave
assisted syntheses were performed in
a microwave reactor
(530 W, 600 rpm, MicroSYNTH Milestone) for 30–60 min to reflux.
All compounds were purified by recrystallization from ethanol or
acetone or mixture of ethanol/acetone.
4.1.2.1. 2-Hydroxy-N-(3-iodophenyl)-3-nitrobenzamide (1).
Yellow solid; yield 74%; mp 188.5–189.5 °C. IR (ATR): 3376, 3083,
1676 (CO). 1H NMR (500 MHz, DMSO): d 10.73 (1H, s, NH), 8.17
(1H, s, H20), 8.16 (1H, dd, J = 8.0 Hz, J = 1.0 Hz, H4), 8.13 (1H, dd,
J = 8.0 Hz, J = 1.0 Hz, H6), 7.70 (1H, d, J = 8.5 Hz, H40), 7.53 (1H, d,
J = 7.5 Hz, H60), 7.20 (1H, t, J = 8.0 Hz, H50), 7.15 (1H, t, J = 8.0 Hz,
presented considerable activity against Enterococcus sp.
J
14365/08. In addition, the synthesized compounds were found to
be inactive against all tested Gram-negative bacteria and fungi.
Nitro substituted salicylanilides were found to share a non-prefer-
able cytotoxic profile as their IC50 values against HepG2 cells were
found to be in the range of their MIC values.
H5). 13C NMR (125 MHz, DMSO):
d 166.35, 152.94, 138.38,
138.23, 134.08, 133.30, 130.97, 129.37, 128.75, 122.11, 120.43,
118.73, 94.59. Anal. Calcd for C13H9IN2O4 (384.13): C, 40.65; H,
2.36; N, 7.29. Found: C, 40.60; H, 2.38; N, 7.28.
The presence of a free phenolic hydroxyl at the salicylanilide
scaffold has been suggested to be responsible for the cytotoxicity
of these molecules.16 Several derivatives of salicylanilides have been

G. Paraskevopoulos et al. / Bioorg. Med. Chem. 23 (2015) 7292–7301
7297
4.1.2.2. 2-Hydroxy-N-(3-bromophenyl)-3-nitrobenzamide (2).
Yellow solid; yield 80%; mp 160 °C. IR (ATR): 3375, 3084, 1677
(CO). 1H NMR (300 MHz, DMSO): d 10.79 (1H, s, NH), 8.16 (1H,
dd, J = 8.1 Hz, J = 1.5 Hz, H4), 8.13 (1H, dd, J = 7.2 Hz, J = 1.8 Hz,
H6), 8.03 (1H, s, H20), 7.67–7.65 (1H, m, H50), 7.37–7.35 (2H, m,
H40, H60), 7.15 (1H, t, J = 8.1 Hz, H5). 13C NMR (75 MHz, DMSO): d
166.20, 152.64, 139.45, 138.03, 133.99, 130.80, 128.57, 127.20,
123.32, 122.20, 121.46, 119.76, 118.64. Anal. Calcd for
J = 270.0 Hz), 122.87, 120.74, 118.70. Anal. Calcd for C14H9F3N2O4
(326.23): C, 51.54; H, 2.78; N, 8.59. Found: C, 51.10; H, 2.98; N,
8.37.
4.1.2.8. 2-Hydroxy-3-nitro-N-(4-nitro-3-[trifluoromethyl)phe-
nyl]benzamide (8). Yellow solid; yield 78%; mp 161–162 °C. IR
(ATR): 3331, 3096, 1686 (CO). 1H NMR (500 MHz, DMSO): d
11.38 (1H, s, NH), 8.39 (1H, d, J = 2.0 Hz, H20), 8.26 (1H, d,
J = 8.9 Hz, H50), 8.21 (1H, dd, J = 9.0 Hz, J = 2.1 Hz, H60), 8.14 (1H,
dd, J = 8.2 Hz, J = 1.7 Hz, H4), 8.08 (1H, dd, J = 7.7 Hz, J = 1.6 Hz,
H6), 7.16 (1H, t, J = 7.9 Hz, H5). 13C NMR (125 MHz, DMSO): d
166.12, 151.80, 142.97, 142.05, 138.00, 134.51, 128.49, 127.63,
123.90, 123.67, 122.84 (q, J = 33.3 Hz), 122.02 (q, J = 271 Hz),
C13H9BrN2O4 (337.13): C, 46.32; H, 2.69; N, 8.31. Found: C, 45.83;
H, 2.90; N, 8.02.
4.1.2.3. 2-Hydroxy-N-(3-chlorophenyl)-3-nitrobenzamide (3).
Yellow solid; yield 85%; mp 153–154 °C. IR (ATR): 3365, 3091,
1667 (CO). 1H NMR (300 MHz, DMSO): d 10.81 (1H, s, NH), 8.16
(1H, dd, J = 7.8 Hz, J = 1.2 Hz, H4), 8.13 (1H, dd, J = 8.4 Hz,
J = 1.5 Hz, H6), 7.89 (1H, t, J = 1.8 Hz, H20), 7.62 (1H, dd, J = 8.1 Hz,
J = 0.9 Hz, H60), 7.15 (1H, t, J = 7.8 Hz, H50), 7.25 (1H, dd,
J = 7.8 Hz, J = 1.5 Hz, H40), 7.15 (1H, t, J = 8.1 Hz, H5). 13C NMR
118.85, 118.54 (q, J = 6.0 Hz). Anal. Calcd for
C14H8F3N3O6
(371.23): C, 45.30; H, 2.17; N, 11.32. Found: C, 44.65; H, 2.61; N,
10.87.
4.1.2.9. 2-Hydroxy-3-nitro-N-(3-nitrophenyl)benzamide (9).
Yellow solid; yield 66%; mp 204–205 °C. IR (ATR): 3350, 3112,
1670 (CO). 1H NMR (500 MHz, DMSO): d 11.06 (1H, s, NH), 8.71
(1H, t, J = 2.1 Hz, H20), 8.16 (1H, dd, J = 5.7 Hz, J = 1.6 Hz, H60),
8.14 (1H, dd, J = 6.2 Hz, J = 1.7 Hz, H40), 8.08 (1H, dd, J = 8.2 Hz,
J = 1.3 Hz, H4), 8.01 (1H, dd, J = 8.1 Hz, J = 2.3 Hz, H6), 7.68 (1H, t,
J = 8.2 Hz, H50), 7.16 (1H, t, J = 8.1 Hz, H5). 13C NMR (125 MHz,
DMSO): d 166.32, 152.39, 147.90, 139.17, 138.02, 134.14, 130.26,
128.59, 126.72, 122.62, 118.97, 118.77, 114.93. Anal. Calcd for
(75 MHz, DMSO):
d 166.21, 152.63, 139.33, 138.02, 134.00,
133.06, 130.51, 128.56, 124.31, 122.27, 120.47, 119.37, 118.65.
Anal. Calcd for C13H9ClN2O4 (292.68): C, 53.35; H, 3.10; N, 9.57.
Found: C, 52.80; H, 3.51; N, 9.32.
4.1.2.4. 2-Hydroxy-N-(3-fluorophenyl)-3-nitrobenzamide (4).
Yellow solid; yield 76%; mp 111–112 °C. IR (ATR): 3366, 3088,
1662 (CO). 1H NMR (500 MHz, DMSO): d 10.81 (1H, s, NH), 8.16
(1H, dd, J = 7.8 Hz, J = 1.4 Hz, H4), 8.13 (1H, dd, J = 8.3 Hz,
J = 1.5 Hz, H6), 7.67 (1H, dt, J = 11.5 Hz, J = 2.1 Hz, H20), 7.48 (1H,
d, J = 8.3 Hz, H60), 7.43 (1H, dt, J = 10.0 Hz, J = 7.4 Hz, H50) 7.15
(1H, t, J = 7.9 Hz, H5), 7.00 (1H, td, J = 8.5 Hz, J = 2.2 Hz, H40). 13C
C13H9N3O6 (303.23): C, 51.49; H, 2.99; N, 13.96. Found: C, 50.68;
H, 3.24; N, 13.47.
4.1.2.10. 2-Hydroxy-3-nitro-N-(4-nitrophenyl)benzamide (10).
Yellow solid; yield 60%; mp 221–222 °C. IR (ATR): 3355, 3100,
1677 (CO). 1H NMR (300 MHz, DMSO): d 11.74 (1H, s, NH), 8.27
(2H, d, J = 9.1 Hz, H30, H50), 8.09 (2H, d, J = 7.8 Hz, H4, H6), 7.97
(2H, d, J = 9.1 Hz, H20, H60), 7.03 (1H, t, J = 7.9 Hz, H5). 13C NMR
NMR (125 MHz, DMSO):
d 166.17, 162.00 (d, J = 242.2 Hz),
152.60, 139.57 (d, J = 11.1 Hz), 137.98, 134.00, 130.43 (d,
J = 9.4 Hz), 128.51, 122.30, 118.64, 111.08 (d, J = 21.0 Hz), 107.77
(d, J = 26.3 Hz). Anal. Calcd for C13H9FN2O4 (276.22): C, 56.53; H,
3.28; N, 10.14. Found: C, 55.94; H, 3.67; N, 9.92.
(75 MHz, DMSO):
134.68, 128.59, 124.93, 123.83, 120.09, 117.29. Anal. Calcd for
13H9N3O6 (303.23): C, 51.49; H, 2.99; N, 13.96. Found: C, 50.72;
d 165.99, 153.86, 144.69, 142.73, 138.30,
4.1.2.5. 2-Hydroxy-N-(3,4-dichlorophenyl)-3-nitrobenzamide(5).
Yellow solid; yield 73%; mp 153–154 °C. IR (ATR) 3365, 3107, 1673
(CO). 1H NMR (300 MHz, DMSO): d 10.88 (1H, s, NH), 8.13 (2H, d,
J = 8.1 Hz, H4, H6), 8.07 (1H, s, H20), 7.65 (2H, s, H50, H60), 7.15 (1H,
t, J = 8.1 Hz, H5). 13C NMR (75 MHz, DMSO): d 166.10, 152.42,
138.09, 138.01, 134.08, 131.03, 130.74, 128.56, 126.09, 122.55,
122.07, 120.83, 118.72. Anal. Calcd for C13H8Cl2N2O4 (327.12): C,
47.73; H, 2.47; N, 8.56. Found: C, 47.02; H, 2.78; N, 8.09.
C
H, 3.37; N, 13.40.
4.1.2.11. N-[3,5-Bis(trifluoromethyl)phenyl]-2-hydroxy-3-nitro-
benzamide (11).
Yellow solid; yield 71%; mp 183–184 °C. IR
(ATR): 3355, 3109, 1671 (CO). 1H NMR (300 MHz, DMSO): d
11.21 (1H, s, NH), 8.41 (2H, s, H20, H60), 8.14 (1H, dd, J = 8.0 Hz,
J = 1.5 Hz, H4), 8.12 (1H, dd, J = 8.0 Hz, J = 1.5 Hz, H6), 7.17 (1H, t,
J = 8.1 Hz, H5). 13C NMR (75 MHz, DMSO): d 166.28, 152.09,
140.09, 138.10, 134.20, 130.79 (q, J = 33.0 Hz), 128.60, 123.17 (q,
J = 271.0 Hz), 122.95, 120.40, 118.81, 117.18 (q, J = 3.7 Hz). Anal.
Calcd for C15H8F6N2O4 (394.23): C, 45.70; H, 2.05; N, 7.11. Found:
C, 45.08; H, 2.31; N, 6.89.
4.1.2.6. 2-Hydroxy-N-[3-(trifluoromethyl)phenyl]-3-nitrobenza-
mide (6). Yellow solid; yield 76%; mp 142–143.5 °C. IR (ATR):
3364, 3089, 1665 (CO). 1H NMR (500 MHz, DMSO): d 10.94 (1H,
s, NH), 8.17 (1H, dd, J = 7.8 Hz, J = 1.4 Hz, H4), 8.16 (1H, d,
J = 1.5 Hz, H20), 8.14 (1H, dd, J = 8.3 Hz, J = 1.5 Hz, H6), 7.95 (1H,
d, J = 8.3 Hz, H40), 7.64 (1H, t, J = 8.0 Hz, H50), 7.52 (1H, d,
J = 7.8 Hz, H60), 7.16 (1H, t, J = 7.8 Hz, H5). 13C NMR (125 MHz,
DMSO): d 160.30, 152.54, 138.72, 138.05, 134.02, 130.07, 129.48
(q, J = 31.8 Hz), 128.55, 124.51, 124.03 (q, J = 270.00 Hz), 122.35,
120.87 (q, J = 3.8 Hz), 118.66, 117.06 (q, J = 4.00 Hz). Anal. Calcd
for C14H9F3N2O4 (326.23): C, 51.54; H, 2.78; N, 8.59. Found: C,
51.03; H, 2.94; N, 8.22.
4.1.2.12. 2-Hydroxy-N-(3-iodophenyl)-4-nitrobenzamide (12).
Yellow solid; yield 77%; mp 243–245 °C. IR (ATR): 3337, 3125,
1640 (CO). 1H NMR (500 MHz, DMSO): d 11.75 (1H, br s, OH),
10.49 (1H, s, NH), 8.23 (1H, s, H20), 7.92 (1H, d, J = 9.2 Hz, H6),
7.78–7.74 (2H, m, H3, H5), 7.68 (1H, d, J = 8.1 Hz, H40), 7.50 (1H,
d, J = 9.2 Hz, H60), 7.17 (1H, t, J = 8.0 Hz, H50). 13C NMR (125 MHz,
DMSO): d 164.01, 156.57, 149.49, 139.74, 132.66, 130.83, 130.81,
128.30, 127.10, 119.44, 113.66, 111.16, 94.55. Anal. Calcd for C13
-
4.1.2.7. 2-Hydroxy-N-[4-(trifluoromethyl)phenyl]-3-nitrobenza-
mide (7). Yellow solid; yield 69%; mp 156–157 °C. IR (ATR): 3362,
3093, 1678 (CO). 1H NMR (500 MHz, DMSO): d 10.96 (1H, s, NH),
8.16 (1H, dd, J = 7.8 Hz, J = 1.4 Hz, H4), 8.14 (1H, dd, J = 8.3 Hz,
J = 1.5 Hz, H6), 7.94 (2H, d, J = 8.5 Hz, H30, H50), 7.76 (2H, d,
J = 8.5 Hz, H20, H40), 7.16 (1H, t, J = 8.0 Hz, H5). 13C NMR
(125 MHz, DMSO): d 166.22, 152.41, 141.63, 137.95, 134.21,
128.47, 126.05 (q, J = 3.8 Hz), 124.42 (q, J = 32.5 Hz), 124.27 (q,
H9IN2O4 (384.13): C, 40.65; H, 2.36; N, 7.29. Found: C, 40.48; H,
2.59; N, 7.01.
4.1.2.13. N-(3-Bromophenyl)-2-hydroxy-4-nitrobenzamide (13).
Yellow solid; yield 68%; mp 220–221.5 °C. IR (ATR): 3313, 3085,
1639 (CO). 1H NMR (500 MHz, DMSO): d 11.79 (1H, br s, OH),
10.49 (1H, s, NH), 8.08 (1H, s, H20), 7.92 (1H, d, J = 9.2 Hz, H6),
7.78–7.74 (2H, m, H3, H5), 7.67–7.65 (1H, m, H60), 7.35–7.30 (2H,

7298
G. Paraskevopoulos et al. / Bioorg. Med. Chem. 23 (2015) 7292–7301
m, H40, H50). 13C NMR (125 MHz, DMSO): d 164.06, 156.56, 149.50,
139.93, 130.84, 130.77, 127.07, 126.72, 122.46, 121.54, 118.98,
113.65, 111.17. Anal. Calcd for C13H9BrN2O4 (337.13): C, 46.32;
H, 2.69; N, 8.31. Found: C, 45.91; H, 3.01; N, 7.96.
H20), 8.24 (1H, d, J = 9.0 Hz, H50), 8.20 (1H, dd, J = 9.0 Hz,
J = 1.8 Hz, H60), 7.90 (1H, d, J = 8.2 Hz, H6), 7.79–7.74 (2H, m, H3,
H5). 13C NMR (125 MHz, DMSO):
d 164.79, 156.73, 149.74,
143.17, 141.87, 131.04, 127.66, 127.10, 123.29, 122.94 (q,
J = 33.0 Hz), 122.05 (q, J = 271.5 Hz), 118.16 (q, J = 6.0 Hz), 113.48,
111.26. Anal. Calcd for C13H8F3N3O6 (371.23): C, 45.30; H, 2.17;
N, 11.32. Found: C, 44.72; H, 2.63; N, 10.92.
4.1.2.14. N-(3-Chlorophenyl)-2-hydroxy-4-nitrobenzamide (14).
Yellow solid; yield 71%; mp 227–228 °C. IR (ATR): 3329, 3080,
1637 (CO). 1H NMR (500 MHz, DMSO): d 11.80 (1H, br s, OH),
10.57 (1H, s, NH), 7.95–7.91 (2H, m, H6, H20), 7.78–7.74 (2H, m,
H3, H5), 7.61 (1H, dd, J = 8.2 Hz, J = 1.0 Hz, H40), 7.39 (1H, t,
J = 8.1 Hz, H50), 7.19 (1H, dd, J = 7.8 Hz, J = 1.8 Hz, H60). 13C NMR
(125 MHz, DMSO): d 164.06, 156.56, 149.50, 139.80, 133.11,
130.85, 130.47, 127.06, 123.82, 119.62, 118.59, 113.65, 111.17.
Anal. Calcd for C13H9ClN2O4 (292.68): C, 53.35; H, 3.10; N, 9.57.
Found: C, 52.98; H, 3.41; N, 9.30.
4.1.2.20. 2-Hydroxy-4-nitro-N-(3-nitrophenyl)benzamide (20).
Yellow solid; yield 63%; mp 221–222 °C. IR (ATR): 3400, 3091,
1655 (CO). 1H NMR (500 MHz, DMSO): d 11.77 (1H, br s, OH),
10.85 (1H, s, NH), 8.77 (1H, s, H20), 8.04 (1H, dd, J = 7.8 Hz,
J = 1.3 Hz, H40), 7.98 (1H, dd, J = 8.2 Hz, J = 2.2 Hz, H60), 7.93 (1H,
d, J = 9.3 Hz, H6), 7.80–7.73 (2H, m, H3, H5), 7.65 (1H, t,
J = 8.2 Hz, H50). 13C NMR (125 MHz, DMSO): d 164.42, 156.58,
149.58, 147.95, 139.55, 130.91, 130.22, 127.09, 126.13, 118.60,
114.23, 113.67, 111.20. Anal. Calcd for C13H9N3O6 (303.23): C,
51.49; H, 2.99; N, 13.86. Found: C, 50.94; H, 3.24; N, 13.52.
4.1.2.15. N-(3-Fluorophenyl)-2-hydroxy-4-nitrobenzamide (15).
Yellow solid; yield 75%; mp 238–239 °C. IR (ATR): 3393, 3110,
1684 (CO). 1H NMR (500 MHz, DMSO): d 11.80 (1H, br s, OH),
10.59 (1H, s, NH), 7,93 (1H, d, J = 8.2 Hz, H6), 7.78–7.74 (2H, m,
H3, H5), 7.72 (1H, d, J = 11.6 Hz, H20), 7.47 (1H, d, J = 8.3 Hz, H60),
7.43–7.37 (1H, m, H50), 7.19 (1H, td, J = 8.4 Hz, J = 2.3 Hz, H40).
13C NMR (125 MHz, DMSO): d 164.02, 162.26 (d, J = 241.6 Hz),
156.52, 149.49, 140.07 (d, J = 11.0 Hz), 130.87, 130.44 (d,
J = 9.5 Hz), 127.13, 115.91, 113.66, 111.16, 110.58 (d, J = 21.1 Hz),
106.93 (d, J = 26.3 Hz). Anal. Calcd for C13H9FN2O4 (276.22): C,
56.53; H, 3.28; N, 10.14. Found: C, 55.87; H, 3.65; N, 9.98.
4.1.2.21. 2-Hydroxy-4-nitro-N-(4-nitrophenyl)benzamide (21).
Yellow solid; yield 61%; mp 253–255 °C. IR (ATR): 3392, 3090,
1655 (CO). 1H NMR (500 MHz, DMSO): d 10.90 (1H, s, NH), 8.30–
8.21 (2H, m, H30, H50), 8.02–7.96 (2H, m, H20, H60), 7.94 (1H, d,
J = 8.4 Hz, H6), 7.80–7.73 (2H, m, H3, H5). 13C NMR (125 MHz,
DMSO): d 164.23, 156.47, 149.60, 144.30, 142.75, 130.79, 126.86,
124.60, 119.74, 113.38, 111.11. Anal. Calcd for
C13H9N3O6
(303.23): C, 51.49; H, 2.99; N, 13.86. Found: C, 50.98; H, 3.18; N,
13.61.
4.1.2.16. N-(3,4-Dichlorophenyl)-2-hydroxy-4-nitrobenzamide
(16). Yellow solid; yield 83%; mp 230–232 °C. IR (ATR): 3325,
3115, 1638 (CO). 1H NMR (500 MHz, DMSO): d 11.79 (1H, br s,
OH), 10.67 (1H, s, NH), 8.12 (1H, d, J = 2.0 Hz, H20), 7.91 (1H, d,
J = 8.2 Hz, H6), 7.78–7.74 (2H, m, H3, H5), 7.66 (1H, dd, J = 8.8 Hz,
J = 2.2 Hz, H60), 7.61 (1H, d, J = 8.8 Hz, H50). 13C NMR (125 MHz,
DMSO): d 164.13, 156.53, 149.54, 138.51, 131.06, 130.90, 130.73,
127.10, 125.57, 121.31, 120.18, 113.64, 111.18. Anal. Calcd for
4.1.2.22. N-[3,5-Bis(trifluoromethyl)phenyl]-2-hydroxy-4-nitro-
benzamide (22). Yellow solid; yield 74%; mp 253–255 °C. IR
(ATR): 3340, 3116, 1640 (CO). 1H NMR (300 MHz, DMSO): d
11.72 (1H, br s, OH), 10.98 (1H, s, NH), 8.43 (2H, s, H20, H60), 7.92
(1H, d, J = 8.0 Hz, H6), 7.83 (1H, s, H40), 7.80–7.73 (2H, m, H3,
H5). 13C NMR (75 MHz, DMSO): d 164.69, 156.48, 149.69, 140.36,
130.97, 130.81 (q, J = 32.3 Hz), 126.98, 123.20 (q, J = 271.5 Hz),
119.91 (q, J = 3.75 Hz), 116.90 (q, J = 3.8 Hz), 113.71, 111.19. Anal.
Calcd for C13H8F6N2O4 (394.23): C, 45.70; H, 2.05; N, 7.11. Found:
C, 45.24; H, 2.39; N, 8.89.
C13H8Cl2N2O4 (327.12): C, 47.73; H, 2.47; N, 8.56. Found: C,
47.24; H, 2.71; N, 8.29.
4.1.2.17. 2-Hydroxy-4-nitro-N-[3-(trifluoromethyl)phenyl]ben-
zamide (17). Yellow solid; yield 65%; mp 215–216 °C. IR (ATR):
3316, 3085, 1639 (CO). 1H NMR (500 MHz, DMSO): d 11.80 (1H,
br s, OH), 10.73 (1H, s, NH), 8.23 (1H, s, H20), 7.96–7.91 (2H, m,
H6, H40), 7.78–7.74 (2H, m, H3, H5), 7.61 (1H, t, J = 8.0 Hz, H50),
7.48 (1H, d, J = 7.7 Hz, H60). 13C NMR (125 MHz, DMSO): d 164.50,
156.84, 149.73, 139.35, 131.03, 130.21, 129.71 (q, J = 31.6 Hz),
127.23, 124.26 (q, J = 271.3 Hz), 123.96, 120.61 (q, J = 3.7 Hz),
116.44 (q, J = 4.1 Hz), 113.80, 111.39. Anal. Calcd for C13H9F3N2O4
(326.23): C, 51.54; H, 2.78; N, 8.59. Found: C, 51.11; H, 2.96; N,
8.20.
4.1.2.23. 2-Hydroxy-N-(3-iodophenyl)-5-nitrobenzamide (23).
White solid; yield 80%; mp 239–240 °C. IR (ATR): 3332, 3129,
1637 (CO). 1H NMR (500 MHz, DMSO): d 10.58 (1H, s, NH), 8.72
(1H, d, J = 2.8 Hz, H6), 8.28 (1H, dd, J = 9.1 Hz, J = 2.8 Hz, H4), 8.20
(1H, s, H20), 7.69 (1H, d, J = 8.2 Hz, H60), 7.51 (1H, d, J = 7.8 Hz,
H40), 7.18 (1H, t, J = 10.0 Hz, H50), 7.16 (1H, d, J = 10.0 Hz, H3). 13C
NMR (125 MHz, DMSO): d 164.15, 163.07, 139.45, 139.33, 132.85,
130.79, 128.76, 128.40, 125.78, 119.88, 119.72, 117.96, 94.49. Anal.
Calcd for C13H9IN2O4 (384.13): C, 40.65; H, 2.36; N, 7.29. Found: C,
40.11; H, 2.71; N, 6.94.
4.1.2.18. 2-Hydroxy-4-nitro-N-[4-(trifluoromethyl)phenyl]ben-
zamide (18)31. Yellow solid; yield 69%; mp 215–216 °C. IR
(ATR): 3401, 3109, 1665 (CO). 1H NMR (500 MHz, DMSO): d
11.80 (1H, br s, OH), 10.74 (1H, s, NH), 8.23 (1H, s, H20), 7.97–
7.91 (3H, m, H6, H20, H60), 7.78–7.75 (2H, m, H3, H5), 7.73 (2H, d,
4.1.2.24. N-(3-Bromophenyl)-2-hydroxy-5-nitrobenzamide (24).
White solid; yield 76%; mp 231–232 °C. IR (ATR): 3333, 3077, 1617
(CO). 1H NMR (300 MHz, DMSO): d 10.64 (1H, s, NH), 8.70 (1H, d,
J = 2.9 Hz, H6), 8.28 (1H, dd, J = 9.2 Hz, J = 2.9 Hz, H4), 8.06 (1H, s,
H20), 7.65 (1H, m, H50), 7.35 (2H, m, H40, H60), 7.16 (1H, d,
J = 9.2 Hz, H3). 13C NMR (75 MHz, DMSO): d 164.16, 162.98,
139.70, 139.36, 130.78, 128.42, 126.93, 125.85, 122.91, 121.52,
119.89, 119.40, 117.95. Anal. Calcd for C13H9BrN2O4 (337.13): C,
46.32; H, 2.69; N, 8.31. Found: C, 45.91; H, 2.97; N, 8.04.
J = 8.7 Hz, H30, H50). 13C NMR (125 MHz, DMSO):
d 164.25,
156.49, 149.53, 141.99, 130.94, 127.29, 126.09 (q, J = 3.7 Hz),
124.31 (q, J = 270.0 Hz), 124.05 (q, J = 32.1 Hz), 120.03, 113.67,
111.16. Anal. Calcd for C13H9F3N2O4 (326.23): C, 51.54; H, 2.78;
N, 8.59. Found: C, 51.08; H, 2.99; N, 8.14.
4.1.2.25. N-(3-Chlorophenyl)-2-hydroxy-5-nitrobenzamide (25)32
.
4.1.2.19. 2-Hydroxy-4-nitro-N-[4-nitro-3-(trifluoromethyl)phe-
nyl]benzamide (19). Yellow solid; yield 69%; mp 194–195 °C. IR
(ATR): 3406, 3120, 1667 (CO). 1H NMR (500 MHz, DMSO): d
11.68 (1H, br s, OH), 11.16 (1H, s, NH), 8.42 (1H, d, J = 1.1 Hz,
White solid; yield 76%; mp 227.5–228.5 °C. IR (ATR): 3331, 3085,
1630 (CO). 1H NMR (500 MHz, DMSO): d 10.64 (1H, s, NH), 8.71
(1H, d, J = 2.9 Hz, H6), 8.28 (1H, dd, J = 9.0 Hz, J = 2.9 Hz, H4), 7.92
(1H, s, H20), 7.62 (1H, m, H60), 7.40 (1H, t, J = 8.1 Hz, H50), 7.20 (1H,

G. Paraskevopoulos et al. / Bioorg. Med. Chem. 23 (2015) 7292–7301
7299
d, J = 8.2 Hz, H40), 7.16 (1H, d, J = 9.1 Hz, H3). 13C NMR (125 MHz,
4.1.2.31. 2-Hydroxy-5-nitro-N-(3-nitrophenyl)benzamide (31)32,34
.
DMSO): d 164.14, 162.95, 139.54, 139.36, 133.08, 130.45, 128.39,
125.84, 124.01, 120.07, 119.86, 119.01, 117.94. Anal. Calcd for C13H9-
ClN2O4 (392.68): C, 53.35; H, 3.10; N, 9.57. Found: C, 52.89; H, 3.52;
N, 9.10.
White solid; yield 66%; mp 235–236 °C. IR (ATR): 3342, 3091, 1626
(CO). 1H NMR (500 MHz, DMSO): d 10.89 (1H, s, NH), 8.74 (1H, t,
J = 1.9 Hz, H20), 8.70 (1H, d, J = 2.8 Hz, H6), 8.28 (1H, dd, J = 9.1 Hz,
J = 2.8 Hz, H4), 8.06 (1H, d, J = 8.1 Hz, H40), 7.99 (1H, dd, J = 8.1 Hz,
J = 2.0 Hz, H60), 7.66 (1H, d, J = 8.2 Hz, H50), 7.17 (1H, d, J = 9.1 Hz,
H3). 13C NMR (125 MHz, DMSO): d 164.44, 162.95, 147.91, 139.32,
139.31, 130.17, 128.49, 126.50, 125.90, 119.87, 118.73, 117.95,
114.63. Anal. Calcd for C13H9N3O6 (303.23): C, 51.49; H, 2.99; N,
13.86. Found: C, 51.12; H, 3.14; N, 13.55.
4.1.2.26.
N-(3-Fluorophenyl)-2-hydroxy-5-nitrobenzamide
(26)32,33. White solid; yield 73%; mp 218–219 °C. IR (ATR): 3333,
3085, 1612 (CO). 1H NMR (500 MHz, DMSO): d 10.66 (1H, s, NH),
8.70 (1H, d, J = 2.8 Hz, H6), 8.28 (1H, dd, J = 9.1 Hz, J = 2.8 Hz, H4),
7.71 (1H, d, J = 11.5 Hz, H20), 7.47 (1H, d, J = 8.2 Hz, H60), 7.41
(1H, dd, J = 15.0 Hz, J = 8.0 Hz, H50), 7.16 (1H, d, J = 9.1 Hz, H3),
6.98 (1H, td, J = 8.5 Hz, J = 2.4 Hz, H40). 13C NMR (125 MHz, DMSO):
d 164.07, 162.88, 162.07 (d, J = 240.0 Hz), 139.81 (d, J = 11.0 Hz),
139.38, 130.41 (d, J = 9.4 Hz), 128.36, 125.86, 119.97, 117.92,
116.32 (d, J = 2.7 Hz), 110.78 (d, J = 21.1 Hz), 107.37 (d,
J = 26.2 Hz). Anal. Calcd for C13H9FN2O4 (276.22): C, 56.53; H,
3.28; N, 10.14. Found: C, 56.29; H, 3.61; N, 9.85.
4.1.2.32. 2-Hydroxy-5-nitro-N-(4-nitrophenyl)benzamide (32).
White solid; yield 67%; mp 208–210 °C. IR (ATR): 3375, 3088,
1660 (CO). 1H NMR (300 MHz, DMSO): d 11.04 (1H, s, NH), 8.63
(1H, d, J = 2.7 Hz, H6), 8.27 (3H, m, H4, H30, H50), 7.98 (2H, d,
J = 9.1 Hz, H20, H60), 7.16 (1H, d, J = 9.1 Hz, H3). 13C NMR
(75 MHz, DMSO):
130.79, 126.86, 124.60, 119.74, 113.38, 111.12. Anal. Calcd for
13H9N3O6 (303.23): C, 51.49; H, 2.99; N, 13.86. Found: C, 51.21;
d 164.23, 156.48, 149.61, 144.31, 142.76,
C
4.1.2.27. N-(3,4-Dichlorophenyl)-2-hydroxy-5-nitrobenzamide
(27)32,33. White solid; yield 84%; mp 273–274 °C. IR (ATR): 3391,
3097, 1655 (CO). 1H NMR (300 MHz, DMSO): d 10.70 (1H, s, NH),
8.67 (1H, d, J = 2.8 Hz, H6), 8.27 (1H, dd, J = 9.2 Hz, J = 2.9 Hz, H4),
8.09 (1H, d, J = 2.1 Hz, H20), 7.67 (1H, dd, J = 8.8 Hz, J = 2.2 Hz,
H60), 7.61 (1H, d, J = 8.8 Hz, H50), 7.15 (1H, d, J = 9.0 Hz, H3). 13C
NMR (75 MHz, DMSO): d 164.13, 162.87, 139.34, 138.26, 131.04,
130.68, 128.46, 125.91, 125.80, 121.75, 120.56, 119.92, 117.94.
Anal. Calcd for C13H8Cl2N2O4 (327.12): C, 47.73; H, 2.47; N, 8.56.
Found: C, 47.21; H, 2.78; N, 8.12.
H, 3.21; N, 13.47.
4.1.2.33. N-[3,5-Bis(trifluoromethyl)phenyl]-2-hydroxy-5-nitro-
benzamide (33)16. White solid; yield 69%; mp 223–224 °C. IR
(ATR): 3387, 3097, 1645 (CO). 1H NMR (300 MHz, DMSO): d
11.02 (1H, s, NH), 8.69 (1H, d, J = 2.9 Hz, H6), 8.42 (2H, s, H20,
H60), 8.28 (1H, dd, J = 9.1 Hz, J = 2.9 Hz, H4), 7.82 (1H, s, H40), 7.16
(1H, d, J = 9.1 Hz, H3). 13C NMR (75 MHz, DMSO): d 164.64,
162.90, 140.15, 139.29, 130.73 (q, J = 32.9 Hz), 128.58, 125.97,
125.90, 123.19 (q, J = 272.8 Hz), 120.32, 120.29, 119.78, 117.98,
117.96, 116.96 (q, J = 3.7 Hz). Anal. Calcd for
C15H8F6N2O4
4.1.2.28. 2-Hydroxy-5-nitro-N-[3-(trifluoromethyl)phenyl]ben-
zamide (28)33. White solid; yield 71%; mp 181–183 °C. IR (ATR):
3342, 3199, 1632 (CO). 1H NMR (500 MHz, DMSO): d 10.79 (1H,
s, NH), 8.73 (1H, d, J = 2.8 Hz, H6), 8.29 (1H, dd, J = 9.1 Hz,
J = 2.9 Hz, H4), 8.21 (1H, s, H20), 7.94 (1H, d, J = 8.1 Hz, H40), 7.62
(1H, t, J = 8.0 Hz, H50), 7.50 (1H, d, J = 7.7 Hz, H60), 7.17 (1H, d,
J = 9.0 Hz, H3). 13C NMR (125 MHz, DMSO): d 164.40, 163.03,
139.34, 138.90, 130.00, 129.49 (q, J = 31.6 Hz), 128.42, 125.83,
124.06 (q, J = 271.0 Hz), 124.21, 120.61 (q, J = 3.7 Hz), 119.82,
(394.23): C, 45.70; H, 2.05; N, 7.11. Found: C, 45.24; H, 2.57; N,
6.87.
4.2. Biology
4.2.1. In vitro antimycobacterial evaluation
The in vitro antimycobacterial activity of the synthesized com-
pounds was determined against Mycobacterium tuberculosis My
331/88 (H37RV), M. avium My 330/80, M. kansasii 235/80 and M.
kansasii 6509/96. All strains were obtained from the Czech
National Collection of Type Cultures (CNCTC, Brno, Czech Republic)
except of M. kansasii 6509/96, which was clinically isolated. Basic
suspensions of the mycobacterial strains were prepared according
to a 1.0 McFarland standard. From the basic suspension, subse-
117.96, 116.73 (q, J = 4.1 Hz). Anal. Calcd for
C14H9F3N2O4
(326.23): C, 51.54; H, 2.78; N, 8.59. Found: C, 51.08; H, 3.01; N,
8.10.
4.1.2.29. 2-Hydroxy-5-nitro-N-[4-(trifluoromethyl)phenyl]ben-
zamide (29)31. White solid; yield 76%; mp 255–256 °C. IR (ATR):
3391, 3097, 1632 (CO). 1H NMR (300 MHz, DMSO): d 10.79 (1H,
s, NH), 8.69 (1H, d, J = 2.9 Hz, H6), 8.28 (1H, dd, J = 9.2 Hz,
J = 2.9 Hz, H4), 7.94 (2H, d, J = 8.6 Hz, H30, H50), 7.73 (2H, d,
J = 8.7 Hz, H20, H60), 7.17 (1H, d, J = 9.2 Hz, H3). 13C NMR
quent dilutions of each strain were made: M. tuberculosis 10ꢀ3
,
M. avium 10ꢀ5 and M. kansasii 10ꢀ4. The antimycobacterial activity
of the compounds was determined in a Šula’s semisynthetic med-
ium (SEVAC, Prague, Czech Republic) via the micromethod for the
determination of the minimum inhibitory concentration (MIC) at
37 °C after 14 and 21 days and after 7, 14 and 21 days for M.
kansasii.40 The tested compounds were added to the medium as
DMSO solutions while INH was used as a standard in a sterile
water solution. The concentrations of the tested compounds were
used as following: 500, 250, 125, 62.5, 32, 16, 8, 4, 2, 1 and
(75 MHz, DMSO):
d 164.21, 162.78, 141.80, 141.79, 139.38,
128.40, 126.07 (q, J = 3.75 Hz), 124.12 (q, J = 31.5 Hz), 122.51,
120.41, 120.26, 117.91. Anal. Calcd for C14H9F3N2O4 (326.23): C,
51.54; H, 2.78; N, 8.59. Found: C, 51.12; H, 3.12; N, 8.03.
4.1.2.30. 2-Hydroxy-5-nitro-N-[4-nitro-3-(trifluoromethyl)phe-
nyl]benzamide (30). White solid; yield 62%; mp 255–256 °C. IR
(ATR): 3358, 3094, 1665 (CO). 1H NMR (500 MHz, DMSO): d
11.15 (1H, s, NH), 8.63 (1H, d, J = 2.9 Hz, H6), 8.42 (1H, s, H20),
8.59 (1H, dd, J = 9.1 Hz, J = 2.9 Hz, H4), 8.24 (1H, d, J = 9.1 Hz,
H50), 8.22 (1H, dd, J = 9.0 Hz, J = 1.9 Hz, H60), 7.17 (1H, d,
J = 9.2 Hz, H3). 13C NMR (125 MHz, DMSO): d 164.70, 162.76,
143.15, 142.14, 139.45, 128.74, 127.72, 126.27, 123.77, 122.73 (q,
J = 31.3 Hz), 122.26 (q, J = 273 Hz), 120.63, 118.66 (q, J = 6.25 Hz),
118.05. Anal. Calcd for C14H9F3N2O4 (371.23): C, 45.30; H, 2.17;
N, 11.32. Found: C, 44.88; H, 2.46; N, 11.01.
0.5
250
l
l
M. The same concentrations within the range from 0.5 to
M were used for INH.
4.2.2. In vitro antibacterial evaluation
The in vitro antibacterial activity was assayed against eight
Gram-positive and Gram-negative strains: Staphylococcus aureus
CCM 4516/08, methicillin-resistant Staphylococcus aureus
H
5996/08 (MRSA), Staphylococcus epidermis H 6966/08, Enterococcus
aureus H 6966/08, Enterococcus sp. J 14365/08, Escherichia coli CCM
4517, Klebsiella pneumoniae D 11750/08, ESBL-positive Klebsiella
pneumoniae J 14368/08, and Pseudomonas aeruginosa CCM 1961.

7300
G. Paraskevopoulos et al. / Bioorg. Med. Chem. 23 (2015) 7292–7301
The microdilution broth method modified according to stan-
The treated cells were incubated together with controls at 37 °C
for 24 h in 5% CO2 atmosphere. After this time reagent from the
kit CellTiter 96 AQueous One Solution Cell Proliferation
Assay was added. The CellTiter 96 assay is based on the
reduction of tetrazolium salt MTS [3-(4,5-dimethylthiazol-2-yl)-5-
(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] to
water-soluble formazan dye by metabolically active cells. The
reduction of the reagent is attributed to availability of NADH or
NADPH. The decline in levels of these metabolically important com-
pounds in the cell causes that the production of formazan is
reduced. The tested plate was incubated for 2 h at 37 °C and 5%
CO2 and after this time, the absorbance was recorded at 490 nm
using a 96-well plate reader (TECAN, Infinite M200, Grödig, Austria).
The results were expressed as inhibitory concentration which is
necessary to inhibit cell viability to 50% from the maximal (control)
viability (IC50). A standard toxicological parameter IC50 was calcu-
lated in each of the tested substances using GraphPad Prism soft-
ware (version 6; GraphPad Software Inc. San Diego, CA, USA).
dard M07-A07 in Mueller–Hinton broth (HiMedia Laboratories,
Mumbai, India) adjusted to pH 7.4 ( 0.2) was used. The tested
compounds were dissolved in DMSO to the final concentrations
ranging from 500 to 0.49 lmol/L. Benzylpenicillin (penicillin G)
was used as a comparative drug. Bacterial inoculum in sterile
water was prepared to match 0.5 McFarland scale (1.5 108 CFU/
mL). The minimum inhibitory concentrations were assayed as
95% (IC95) or higher reduction of growth when compared to the
control. The determination of results was performed visually and
spectrophotometrically (at 540 nm). The values of MICs were
determined after 24 and 48 h of incubation in the darkness at
35 °C ( 0.1) in a humid atmosphere.
4.2.3. In vitro antifungal evaluation
The antifungal properties were evaluated in vitro against four
Candida strains (Candida albicans ATCC 44859, Candida tropicalis
156, Candida krusei E28, and Candida glabrata 20/I), Trichosporon
asahii 1188 and three filamentous fungi (Aspergillus fumigatus
231, Absidia corymbifera 272, and Trichophyton mentagrophytes
445). The microdilution broth method was used according to the
CLSI M27-A3 and M38-A2 guidelines in RPMI 1640 with glutamine
(KlinLab, Prague, Czech Republic) buffered to pH 7.0 with 0.165 mol
of 3-morpholino-propane-1-sulfonic acid (Sigma–Aldrich, Darm-
stadt, Germany). DMSO served as a diluent for all of the compounds.
In yeast, the final size of the inoculum was 5 ꢁ 103 0.2 CFU/mL. In
the case of the molds Aspergillus, Trichophyton and Absidia corymb-
ifera, the spores were harvested after cultivation of a given fungal
strain grown on Sabouraud agar from 3 to 10 days. The final size
of the inoculum was 0.5–5 ꢁ 104 CFU/mL. The sizes of the fungal
inocula were checked using a Bürker chamber. Fluconazole was
used as a reference drug. The MIC values for yeasts and filamentous
fungi were assayed as a reduction of growth of at least 80% (IC80) or
of at least 50% (IC50) compared with the control, respectively. The
results were analyzed visually and spectrophotometrically (at
540 nm). The MIC values were determined after 24 and 48 h of
incubation in the dark at 35 °C ( 0.1) in a humid atmosphere, but
for T. mentagrophytes, the final MIC values were determined after
72 and 120 h of incubation.
Acknowledgments
The study was supported by the European Social Fund
and the state budget of the Czech Republic. Project no.
CZ.1.07/2.3.00/30.0061 and by the Research project IGA NT
13346 (2012).
The authors would like to thank the staff of the Department of
Inorganic and Organic Chemistry, Faculty of Pharmacy, for the
technical assistance as well as Mrs. I. Dufková from the Depart-
ment of Biological and Medical Sciences, for providing the antibi-
otic susceptibility tests.
Supplementary data
Supplementary data (Tables S1, S2, S3 and S4) associated with
this article can be found, in the online version, at http://dx.doi.
org/10.1016/j.bmc.2015.10.029.
References and notes
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