Novel salicylanilides from 4,5-dihalogenated salicylic acids: Synthesis, antimicrobial activity and cytotoxicity

Article abstract of DOI:10.1016/j.bmc.2017.01.016

Salicylanilides have proved their activity against tuberculosis (TB). One weak electron-withdrawing substituent is favored at the salicylic part, specially Cl or Br atoms at positions 4 or 5. On the other hand, the antimycobacterial activity of salicylanilides is negatively affected when a strong electron-withdrawing substituent ([sbnd]NO₂) is present at the same positions. Herein we describe the synthesis and characterization of novel salicylanilides possessing two weak electron-withdrawing groups (halogen atoms) at their salicylic part and compare their antitubercular activity with their monohalogenated analogues. All dihalogenated derivatives proved to possess antitubercular activity at a very narrow micromolar range (MIC?=?1–4?μM), similar with their most active monohalogenated analogues. More importantly, the most active final molecules were further screened against multidrug resistant strains and found to inhibit their growth at the range of 0.5–4?μM.

Full text of DOI:10.1016/j.bmc.2017.01.016

Accepted Manuscript
Novel salicylanilides from 4,5-dihalogenated salicylic acids: Synthesis, antimi-
crobial activity and cytotoxicity
Georgios Paraskevopoulos, Sara Monteiro, Rudolf Vosátka, Martin Krátký,
Lucie Navrátilová, František Trejtnar, Jiřina Stolař íková, Jarmila Vinšová
PII:
S0968-0896(16)30882-3
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.01.016
BMC 13495
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
4 October 2016
9 January 2017
12 January 2017
Please cite this article as: Paraskevopoulos, G., Monteiro, S., Vosátka, R., Krátký, M., Navrátilová, L., Trejtnar, F.,
Stolař íková, J., Vinšová, J., Novel salicylanilides from 4,5-dihalogenated salicylic acids: Synthesis, antimicrobial
activity and cytotoxicity, Bioorganic & Medicinal Chemistry (2017), doi: http://dx.doi.org/10.1016/j.bmc.
2017.01.016
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Novel salicylanilides from 4,5-dihalogenated salicylic acids: Synthesis,
antimicrobial activity and cytotoxicity
Georgios Paraskevopoulosa, Sara Monteirob, Rudolf Vosátkac, Martin Krátkýc, Lucie
Navrátilovád, František Trejtnard, Jiřina Stolaříkováe, and Jarmila Vinšovác,
aDepartment of Pharmaceutical Technology, Faculty of Pharmacy, Charles University in
Prague, Heyrovského 1203, 500 05 Hradec Králové, Czech Republic
bInstitute of Organic Chemistry and Technology, Faculty of Chemical Technology, University
of Pardubice, Studentská 573, 532 10 Pardubice, Czech Republic
cDepartment of Inorganic and Organic Chemistry, Faculty of Pharmacy, Charles University in
Prague, Heyrovského 1203, 500 05 Hradec Králové, Czech Republic
dDepartment of Pharmacology and Toxicology, Faculty of Pharmacy, Charles University in
Prague, Heyrovského 1203, 500 05 Hradec Králové, Czech Republic
eLaboratory for Mycobacterial Diagnostics and Tuberculosis, Regional Institute of Public
Health in Ostrava, Partyzánské náměstí 7, 702 00 Ostrava, Czech Republic
* Corresponding author. Heyrovského 1203, 500 05 Hradec Králové, Czech Republic. E-mail
address: jarmila.vinsova@faf.cuni.cz, tel.: +420-495067343, fax: +420-495067166.
Abstract
Salicylanilides have proved their activity against tuberculosis (TB). One weak electron-
withdrawing substituent is favored at the salicylic part, specially Cl or Br atoms at positions 4
or 5. On the other hand, the antimycobacterial activity of salicylanilides is negatively affected
when a strong electron-withdrawing substituent (-NO2) is present at the same positions.
Herein we describe the synthesis and characterization of novel salicylanilides possessing two
weak electron-withdrawing groups (halogen atoms) at their salicylic part and compare their
antitubercular activity with their monohalogenated analogues. All dihalogenated derivatives
proved to possess antitubercular activity at a very narrow micromolar range (MIC= 1-4 μM),
similar with their most active monohalogenated analogues. More importantly, the most active
final molecules were further screened against multidrug resistant strains and found to inhibit
their growth at the range of 0.5-4 μM.
Keywords
Salicylanilides
Double halogenation
Tuberculosis
In vitro antimycobacterial activity
Cytotoxicity
Graphical abstract

1. Introduction
The World Health Organization (WHO) recently reported that around 9.6 million people
were diagnosed with tuberculosis (TB) in 2014, having about 1.5 million of those died from
the disease [1]. According to these data, TB joined Human Immunodeficiency Virus (HIV) on
the top rank of the deadliest infectious diseases, while being responsible for one fourth of
HIV-related deaths. Currently available drugs are not able to eliminate Mycobacterium
tuberculosis (Mtb), the causative agent of TB, while residing in a dormant state after the
initial infection. Apart from that, acquired immunodeficiency, treatments with
immunosuppressive drugs or concomitant diseases may reactivate Mtb [2, 3]. Standard
antimycobaterial agents, like isoniazid (INH) and rifampicin (RIF), have been used for
decades in order to fight the disease. Their overuse or misuse resulted the appearance of
multi-drug resistant (MDR-TB) strains [4-6], extensively drug-resistant TB (XDR-TB) strains
[7] and totally drug-resistant TB (TDR-TB) [8]. The TB drug-development pipeline is
focusing on new regiments to short and improve outcomes of TB treatment and it mainly
includes new and repurposed antimicrobials as well as host directed drugs [9].
2-Hydroxy-N-phenylbenzamides, commonly known as salicylanilides, have been highly
noticed due to their significant in vitro antimicrobial and antifungal activities [10-12]. Many
analogues of salicylanilides have been developed so far with proven activity against Mtb and
INH-resistant nontuberculous mycobacterial strains [13, 14]. In terms of their mode of action,
there is no clear report up to date. The presence of a free phenolic hydroxyl on the salicylic
acid moiety was proved as necessary, and the hypothesis that the molecules function as proton
shuttles that kill bacterial cells by destroying the cellular proton gradient has been suggested
[15]. On the other hand, salicylanilides and their ester derivatives have been reported to
moderate inhibit the 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 [16]. Furthermore, they have been reported to
inhibit the two-component regulatory systems [10] as well as multiple bacterial enzymes, like
transglycosylase [17], sortase A [18] and D-alanine-D-alanine ligase [19]. Finally,
salicylanilide esters, carbamates and additional derivatives resulted from their conjugation
with existing antimicrobial agents, have been suggested to work as pro-drugs that are
hydrolyzed in order to express their activity [20]. Thus, it can be concluded that
salicylanilides affect multiple targets with more than one way in order to exert their
antimicrobial and antimycobacterial properties.
Despite their high anti-tubercular activity, salicylanilides and their derivatives (esters,
carbamates etc.) often share a non-preferable cytotoxic profile [15], which limits their

potential as therapeutics. Recent efforts of our group has been focused on a discovery of
salicylanilide derivatives with decreased cytotoxicity and, under this concept, novel 2-
(phenylcarbamoyl)phenyl 4-substituted benzoates were synthesized and found to be active
against several mycobacteria (0.125-8 μM) while they exhibited no cytotoxicity at
concentrations of up to 50 μM against a hepatocyte cancer cell line (HepG2) [21]. The
esterification products of 5-halosalicylanilides with 4-substituted benzoic acids found to be,
not only more potent inhibitors of Mtb growth but also to share a preferable cytotoxic profile
in contrast with the parent salicylanilides. This way, increased cytotoxicity can be overcome
while the activity of the final molecules remains intact.
The importance of different substituents at the salicylic or aniline part of salicylanilides
(Figure 1) in order to express their antimycobacterial or antimicrobial activities has been
previously highlighted. Up to date, extensive structure-antimycobacterial activity relationship
(SAR) studies at the aniline part (Figure 1, ring B) have been performed and proved that
activity is always favorably influenced by higher lipophilicity and electron-accepting
substituents [15, 22, 23] while trifluoromethyl group at position 4 has been found to be the
best substituent. On the other hand, the influence of the electronic parameters at the acyl part
(Figure 1, ring A) cannot be evaluated in a simple way. To the best of our knowledge, the
preferred substituents for ring A referred up to date is chlorine at position 4 or 5 and bromine
at position 5. More specifically, 5-bromo-2-hydroxy-N-[4-(trifluoromethyl)phenyl]benzamide
and 5-chloro-2-hydroxy-N-[4-(trifluoromethyl)phenyl]benzamide are inhibiting the growth of
Mtb at the concentrations of 1-2 μM respectively and they are the most active salicylanilides
reported so far [22]. The antimycobacterial properties of salicylanilides possessing bromine at
position 4 of the salicylic part have not been previously reported.
Figure 1. General structure of salicylanilides consisting of salicylic part (ring A) and aniline part (ring B)
During our ongoing efforts in order to explore the influence of different substitutions on
the parent structure of salicylanilides, in terms of activity and cytotoxicity, the introduction of
several substituents is under investigation. We have recently reported that the insertion of a
strong electron-withdrawing group, like nitro-group, at the salicylic part of salicylanilides
resulted active molecules with MICs at the range of 2-32 μM against Mycobacterium
tuberculosis, while the nitro group was mostly favored at position 4 [24]. Despite the high
activity of 4-nitrosalicylanilides, the presence of one halogen instead of nitro group was still
more favored (lower MIC).
In our current approach, we examined how antimycobacterial activity is influenced when
two weak electron-withdrawing groups (Cl and Br) are at positions 4 and 5 of the salicylic
part instead of one strong electron-withdrawing during our previous attempt [24].
Furthermore, we compared their antimycobacterial activities and their cytotoxic profile
against HepG2 cells with their monohalogenated analogues.

2. Results and Discussion
2.1. Chemistry
In this study, the synthesis of the selected 4- or 5-monohalogenated salicylanilides at ring
A performed following direct conjugation of the 4- or 5-halogenated salicylic acid with the
corresponding aniline in the presence of PCl3 using chlorobenzene as solvent under
microwave conditions [25]. The aniline part was modified with selected substituents that
proved before to afford the most active analogues [15, 22]. More specifically, the aniline part
modifications were 4-CF3, 3,5-diCF3, 3,4-diCl and 4-Br. The synthesis and mycobacterial
activity for the most 4-chloro-, 5-bromo- and 5-chloro-salicylanilides have been previously
reported by our group and others [15, 21-23, 26] while salicylanilides of 4-bromosalicylic
acid are presented herein for the first time.
The synthesis of 4,5-dihalogenated salicylic acids from 4-halogenated salicylic acids
performed in three steps (Scheme 1).
Scheme 1. Synthesis of 4,5-dihalogehated salicylic acids
Initially, 4-bromo- or 4-chloro-salicylic acid reacted with N-bromosuccinimide (NBS) or
N-chlorosuccinimide (NCS) in the presence of concentrated H2SO4. While repeated attempts
to purify the reaction mixture were unsuccessful, the crude material was subsequently
esterified with MeOH/H2SO4. The new crude material was purified by column
chromatography and the isolated appropriate methyl ester was finally hydrolyzed, resulting
the desired 4,5-dihalogenated salicylic acid. Conjugation of the isolated 4,5-dihalogenated
salicylic acid with substituted aniline afforded the final molecules (Scheme 2).
Scheme 2. Synthesis of final salicylanilides
2.2. Antimycobacterial activity

The synthesized salicylanilides (1a-8d) 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, as a first-line anti-TB drug, and
p-aminosalicylic acid (PAS), a structurally related second-line drug, were chosen for the
comparison performed in this study. Table 1 reports the minimum inhibitory concentrations
(MICs) of salicylanilides containing one halogen at positions 4 or 5 of their salicylic part and
Table 2 reports the MICs of salicylanilides containing two halogens at positions 4 and 5.
Calculated ClogP values are reported as well.
The presence of one halogen at ring A of salicylanilides resulted molecules that are
inhibiting the growth of Mycobacterium tuberculosis at 1-16 μM. In all cases, bromine
derivatives are more active against their chlorine analogues with the same aniline substitution,
regardless the position of the halogen. This is most likely because of the higher calculated
ClogP values of the final salicylanilides when they possess a bromine over a chlorine atom.
Among the tested, the best substitution for the aniline part was the trifluoromethyl group at
position 4. The most active candidates of the series are 5-bromo-2-hydroxy-N-[4-
(trifluoromethyl)phenyl]benzamide
(4c),
4-bromo-N-(3,4-dichlorophenyl)-2-
hydroxybenzamide (2b) and 4-bromo-2-hydroxy-N-[(4-(trifluoromethyl)phenyl)]benzamide
(2c) with MIC=1 μM after 14 days. These molecules are sharing a similar inhibition profile
with INH, a first line anti-TB drug, while all monohalogenated analogues are inhibiting Mtb
more effectively than PAS. The growth of Mycobacterium avium is less effectively inhibited
by monohalogenated analogues, in comparison with Mtb, at 1-32 μM while 5-bromo-2-
hydroxy-N-[4-(trifluoromethyl)phenyl]benzamide (4c) is the most active molecule with
MIC=1μM after 21 days. 5-Chloro-2-hydroxy-N-[4-(trifluoromethyl)phenyl]benzamide (3c)
is the most active inhibitor of Mycobacterium kansasii (235/80) with MIC=1μM after 21 days
while all other candidates of the monohalogenated series had an inhibition range of 2-16 μM
for the specific strain. The growth of the clinical isolated strain of Mycobacterium kansasii
(6509/96) was also inhibited at the range of 2-16 μM while 5-chloro-2-hydroxy-N-[4-
(trifluoromethyl)phenyl]benzamide (3c) was the most active compound.

Table 1: Antimycobacterial activities of salicylanilides possessing one halogen at salicylic part
IC50
(μM)
HepG2
MIC (M)
M. kansasii
SI
Mtb. 331/88
(H37Rv)
M. avium
330/88
M. kansasii
6509/96
ClogP
R1
R2
R3
235/80
14 d
4a
4a
4
4
2
1
1
2
8
21 d
4a
4a
4
4
4
2
2
4
16
8
14 d
21 d
16a
16a
8
16
16
16
8
32
8
16
8
7 d
4
4
2
8
4
4
2
8
4
4
1
8
4
8
2
4
14 d
4
8
4
8
8
4
2
8
4
4
1
16
8
8
2
8
21 d
4
8
4
16
8
4
4
16
4
4
1
16
8
8
4
7 d
4
4
2
8
8
4
2
8
4
8
2
8
8
2
2
4
14 d 21 d
Cl
Cl
Cl
Cl
Br
Br
Br
Br
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
Cl
Cl
Cl
Br
Br
Br
Br
4-Br
3,4-diCl
4-CF3
3,5-diCF3
4-Br
3,4-diCl
4-CF3
3,5-diCF3
4-Br
3,4-diCl
4-CF3
3,5-diCF3
4-Br
3,4-diCl
4-CF3
3,5-diCF3
16a
16a
8
8
4
8
8
3.84
4.12
3.93
4.85
4.11
4.40
4.20
5.12
3.84
4.12
3.93
4.85
4.11
4.40
4.20
5.12
3.24
2.23
2.64
9,55
7.59
17,67
15,04
8,08
3.14
1.99
1.89
10,19
23,60
31,47
1.81
7,80
0.81
0.56
0.66
2.39
1.90
8.84
7.52
2.02
0.20
0.25
0.95
1.27
5.9
1a
1b
1cb
1d
2
4
16
16
16
8
32
8
16
8
32
16d
8d
1a
8
8
8
8
2a
2b
2c
4
8
4
4
16
8
16
8
2d
3ab
3bc
3c
4
2
4
8
8
2
8
2
2
32
16d
8d
16
8
16
8
3d
4a
4b
4c
4d
4d
1a
2
4d
4d
1a
2
H
H
H
2
4
7.87
1.81
3.40
1a
4
4
16
16
8
8
8
4d
0.5
62.5
1
62.5
>250 >250 >250 >250
32 125 125
>250
1000 >1000
4
32
8
125
8
500
INH
PAS
INH: Isoniazid, PAS: p-aminosalicylic acid, aMICs are reported in Ref. [22], bMICs are reported in Ref [26], cMICs are reported in Ref. [21], dMICs are reported
in Ref. [23]

Table 2: Antimycobacterial activities of salicylanilides possessing two halogens at salicylic part
IC50
(μM)
HepG2
MIC (M)
M. kansasii
SI
Mtb. 331/88
(H37Rv)
M. avium
330/88
M. kansasii
6509/96
ClogP
R1
R2
R3
235/80
14 d
2
2
1
2
4
2
2
2
2
2
2
2
2
2
1
2
21 d
2
4
2
2
4
4
2
2
4
2
2
2
4
4
2
4
14 d
21 d
32
125
32
32
32
62.5
62.5
32
7 d
4
8
4
8
4
8
4
4
8
4
4
8
4
8
4
8
14 d
8
8
4
8
8
8
4
8
8
8
8
8
8
8
4
8
21 d
8
8
4
8
8
8
4
8
8
8
8
16
8
8
4
8
7 d
4
8
4
8
8
8
4
8
8
8
4
8
8
8
4
8
14 d 21 d
Br
Br
Br
Br
Br
Br
Br
Br
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Br
Br
Br
Br
Cl
Cl
Cl
Cl
Br
Br
Br
Br
4-Br
3,4-diCl
4-CF3
3,5-diCF3
4-Br
3,4-diCl
4-CF3
3,5-diCF3
4-Br
3,4-diCl
4-CF3
3,5-diCF3
4-Br
3,4-diCl
4-CF3
3,5-diCF3
32
125
32
32
32
62.5
32
16
8
8
8
8
4.67
4.95
4.76
5.68
4.94
5.22
5.03
5.95
4.40
4.68
4.49
5.41
4.67
4.95
4.76
5.68
9.78
11.07
6.52
4.80
17.88
7.96
6.25
3.00
21.41
12.42
9.87
4.86
10.83
7.65
11.03
5.07
4.89
2.76
3.26
2.40
4.47
1.99
3.13
1.50
5.35
6.21
4.94
2.43
2.71
1.91
5.52
1.27
5a
5b
5c
5d
6a
6b
6c
6d
7a
7b
7c
7d
8a
8b
8c
8d
INH
PAS
4
4
8
8
8
8
8
8
4
4
8
8
32
32
8
8
62.5
62.5
32
32
125
32
62.5
62.5
32
8
8
8
8
16
8
16
8
32
125
62.5
32
8
8
4
4
16
8
8
0.5
62.5
1
62.5
>250 >250 >250 >250
32 125 125
>250
4
32
8
8
1000 >1000
125
500
INH: Isoniazid, PAS: p-aminosalicylic acid

Introduction of a second halogen at the ring A of salicylanilides afforded molecules that
inhibit the growth of Mtb at a narrow micromolar range (MICs=1-4 μM). When these results
compared with ring A nitro substituted salicylanilides [24], it is obvious that two weak
electron-withdrawing groups are preferred at the salicylic part instead of one strong electron-
withdrawing group. On the other hand, the dihalogenated products are similarly active with
their most active monohalogenated analogues. Even if the presence of the second halogen
affects the ClogP values of the final molecules, no direct correlation between activities and
ClogP values was noticed. The best substitution for the aniline part is trifluoromethyl group at
position 4, like for the monohalogenated analogues. The most active inhibitors of the series
are 4-bromo-5-chloro-2-hydroxy-N-[4-(trifluoromethyl)phenyl]benzamide (5c) and 5-bromo-
4-chloro-2-hydroxy-N-[4-(trifluoromethyl)phenyl]benzamide (8c) with MIC=1 μM after 14
days. The growth of Mycobacterium avium is less effectively inhibited by dihalogenated
analogues, in comparison with Mtb as well as in comparison with the monohalogenated
analogues, at 16-62.5 μM. The most active compounds against Mycobacterium avium were N-
[3,5-bis(trifluoromethyl)phenyl]-4,5-dibromo-2-hydroxybenzamide
(6d)
and
N-[3,5-
bis(trifluoromethyl)phenyl]-5-bromo-4-chloro-2-hydroxybenzamide (8d) with MIC=32 μM
after 21 days, proving the superiority of 3,5-bis(trifluoromethyl) substitution on ring B.
Mycobacterium kansasii (235/80) was inhibited in the range of 4-16 μM from ring A
dihalogenated analogues, without important differences between the different substitutions.
The growth of Mycobacterium kansassi (6509/96) was inhibited at the same range (4-16 μM)
while
the
highest
activity
observed
for
4,5-dibromo-2-hydroxy-N-[4-
(trifluoromethyl)phenyl]benzamide (6c) and was 4 μM after 21 days.
The most active salicylanilides against Mtb among all monohaloganated and dihalogenated
ring A salicylanilides were those possessing a trifluoromethyl group at position 4 of the
aniline part. These molecules were selected for further screening against five MDR-TB strains
with various resistance patterns (234/2005, 9449/2007, 242/2015, Praha 1 and Praha 4) and
one XDR-TB strain (Praha 131). The results are summarized in Table 3.

Table 3: Antimycobacterial activities of selected salicylanilides against MDR-TB and XDR-TB strains.
MIC [M
M. tuberculosis
M. tuberculosis
M. tuberculosis
242/2015
M. tuberculosis
M. tuberculosis
M. tuberculosis
Code
234/2005
9449/2007
Praha 1
Praha 4
Praha 131
14 d
2
1
21 d
2
2
14 d
2
1
14 d
1
1
21 d
1
1
14 d
1
1
14 d
1
1
21 d
1
1
14 d
1
1
21 d
2
2
14 d
2
1
21 d
2
1
1c
2c
0.5
2
0.5
4
0.5
2
0.5
1
0.5
1
0.5
2
0.5
1
0.5
1
0.5
2
0.5
4
0.5
2
0.5
4
3c
4c
2
2
2
2
2
2
2
2
2
4
2
2
5c
2
2
2
1
1
2
1
1
2
2
2
2
6c
1
2
2
2
2
2
1
2
1
2
1
2
1
2
1
2
1
2
2
2
1
2
2
2
7c
8c
16
32
16
16
16
16
16
16
16
16
16
16
INH
INH: Isoniazid

All tested salicylanilides exhibited excellent activities against MDR and XDR strains
ranging between 0.5-4 μM which are similar or even lower than the MICs observed for drug-
sensitive strains. The insertion of a second halogen at ring A doesn’t seem to dramatically
affect the antimycobacterial activity of the final molecules against these strains even if the
ring A dihalogenated compounds are slightly less active than their monohalogenated
analogues. The most active ring A monohalogenated derivative was 5-chloro-2-hydroxy-N-[4-
(trifluoromethyl)phenyl]benzamide (3c) with MIC=0.5 μM against all tested strains while the
most
active
dihalogenated
derivatives
were
and
4,5-dibromo-2-hydroxy-N-[4-
4,5-dichloro-2-hydroxy-N-[4-
(trifluoromethyl)phenyl]benzamide
(6c)
(trifluoromethyl)phenyl]benzamide (7c) with MIC=1-2 μM against all resistant strains.
2.3. Cytotoxicity evaluation
Since hepatotoxic effects are a very common and serious problem in patients who are
undergoing treatment for tuberculosis [27], all synthesized salicylanilides were evaluated for
their in vitro cytotoxic properties using an MTT assay in the human liver model of HepG2
cell line. The cytotoxicity results as well as the selectivity indexes (SI= IC50/MIC against Mtb
in 21 days) for the tested compounds are summarized in Table 1 and Table 2 together with
their antimycobacterial activities.
The IC50 values for the salicylanilides bearing one halogen at ring A were in the range of
1.81 to 31.47 μM. The majority of the compounds of this group possess a non-preferable
cytotoxic profile, since their IC50 values are in the range of their MICs, thus having SI values
around 1. The non-preferable cytotoxic profile is expected for these molecules, since the
presence of a free phenolic hydrogen is closely connected, not only with the activity of
salicylanilides but also with their cytotoxicity [15]. Interestingly, two of the most active
salicylanilides of 4-bromosalicylic acid, which are presented here for the first time, are having
a comparatively high value of SI. More specifically, 4-bromo-N-(3,4-dichlorophenyl)-2-
hydroxybenzamide (2b) and 4-bromo-2-hydroxy-N-[4-(trifluoromethyl)phenyl]benzamide
(2c) are having SI values of 8.84 and 7.52 respectively.
The insertion of a second halogen on the ring A afforded molecules characterized by a
generally more preferable cytotoxic profile in comparison with their monohalogenated
analogues, since SI values are in the range of 1.27-6.21. It is noteworthy, that the presence of
two trifluoromethyl groups at the aniline part is always negatively affecting the cytotoxicity of
the final salicylanilides (lower SI values). In terms of SI, the best compounds of the series are
4,5-dichloro-N-(3,4-dichlorophenyl)-2-hydroxybenzamide
(7b),
5-bromo-4-chloro-2-
hydroxy-N-[4-(trifluoromethyl)phenyl]benzamide (8c) and N-(4-bromophenyl)-4,5-dichloro-
2-hydroxybenzamide (7a) with SI=6.21, 5.52 and 5.35 respectively, while their activities
against Mtb are 2, 2 and 4 μM after 21 days.
3. Conclusions
Herein, the effect on antimycobacterial activity of salicylanilides when two weak electron-
withdrawing groups (bromine and chlorine atoms) are present at the positions 4 and 5 of their
salicylic part has been examined. Initially, salicylanilides possessing bromine at position 4 of
salicylanilides were synthesized in order to complete the series of 4 and 5 monohalogenated
salicylanilides. To the best of our knowledge, 4-bromosalicylanilides, together with their
antimycobacterial activities, are presented herein for the first time. 4,5-Dihalogenated
salicylic acids synthesized from 4-halogenated salicylic acids and used for the synthesis of
novel salicylanilides possessing two halogens at positions 4 and 5 of their salicylic part. All

new compounds were screened for their antimycobacterial activities against Mtb 331/88 (i.e.,
H37RV) and three strains of nontuberculous mycobacteria.
Introduction of a second halogen at ring A resulted salicylanilides sharing a high
antimycobacterial activity, similar with the most active monohalogenated salicylic acid
derivatives. The growth of Mtb is inhibited at a very narrow micromolar range (MICs=1-4
μM). Furthermore, as a continuation of our previous findings [24], the insertion of a second
weak electron-withdrawing substituent at ring A is much preferable over the presence of one
single strong electron-withdrawing substituent. The cytotoxicity of the monohalogenated
salicylic acid derivatives, confirmed once more that the parent structure of salicylanilides is
having a non-preferable cytotoxic profile, since IC50 values were close to the range of their
MICs. Small exception noticed for two 4-bromosalicylic acid derivatives which exhibited
SI>7 against Mycobacterium tuberculosis. The insertion of a second halogen, did not prove to
dramatically affect the overall cytotoxic profile of the final salicylanilides, even if afforded
molecules with generally more acceptable selectivity indexes (all values were >1). Finally, the
synthesized compounds were proved to be very active against all multidrug-resistant and one
extensively drug-resistant mycobacterial strains (MICs = 0.5-4 μM). Our results suggest that
multiple halogen substitution has a positive effect to the overall antimycobacterial activity of
the final molecules and that multiple substitution at ring A is a promising method for the
discovery of novel molecules with increased activity over Mtb.
4. Experimental part
4.1. Chemistry
4.1.1.General methods
All 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 (TLC). 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 Bűchi 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 spectrometer (Nicolet 6700 FT-IR) in the range of 400-4000 cm-1. The NMR spectra
were measured in deuterated solvents at ambient temperature using a Varian V NMR S500
1
13
instrument (500 MHz for H and 125 MHz for C, Varian Comp., Palo Alto, CA, USA) or a
1
13
Varian Mercury-Vxbb 300 (300 MHz for H and 75.5 MHz for C, Varian, Inc., Palo Alto,
CA, USA). The chemical shifts, δ, are given in ppm with respect to tetramethylsilane, which
was used as an internal standard. The coupling constants (J) are reported in Hz.
The ClogP values, which are the logarithms of the partition coefficients for octan-1-
ol/water, were determined using the CS ChemOffice Ultra program (version 15.0, Cambridge-
Soft, Cambridge, MA, USA).
4.1.2.Synthesis

4.1.2.1. Synthesis of methyl esters of dihalogenated salicylic acids (III-VI), general
procedure A:
4-Chloro or 4-bromosalicylic acid (1.0 eq) was dissolved in concentrated H2SO4 (10 mmol
salicylic acid/30 mL H2SO4). N-Chlorosuccinimide or N-bromosuccinimide (1.3 eq) were
o
slowly added in small portions while keeping the temperature between 0-5 C during the
addition. The reaction mixture was stirred at r.t. and, after complete consumption of starting
material, water was carefully added. The formed precipitate was filtered and washed with cold
water. The crude product was dissolved in MeOH and concentrated H2SO4 was added to the
solution (1 mL H2SO4/10 mL MeOH). The reaction mixture was heated at boiling temperature
for 8-16 hours. After cooling at r.t., AcOEt was added (equal amount with MeOH) followed
by extensive washing with saturated solution of NaHCO3. The organic phase was dried over
anhydrous Na2SO4, filtrated and evaporated to dryness. The crude material was purified by
column chromatography with hexane/AcOEt as an eluent in order to afford the pure product.
Yields were 35-42% after the described two-step procedure, calculated from the initial
amount of 4-monohalogenated salicylic acid.
4.1.2.2. Synthesis of 4,5-dihalogenated salicylic acids (VII-X), general procedure B:
The methyl ester of 4,5-dihalogenated salicylic acid (1 eq) was dissolved in a mixture of
THF:MeOH:H2O (4:1:1, 0.15 mmol acid/1 mL solution). LiOH was added (4 eq) to the
solution and the reaction mixture stirred at r.t until complete consumption of starting material
(1-2 hours). Solvents were removed under reduced pressure and the resulting solid dissolved
in water. The solution was acidified with 2M HCl until pH=3 (precipitate formed) and washed
with AcOEt (3 x equal volume). The combined organic phases were dried (Na2SO4), filtrated
and evaporated to dryness to afford the desired 4,5-dihalogenated salicylic acid with yields of
94-97 %.
4.1.2.3. Synthesis of salicylanilides, general procedure C:
All salicylanilides (1a-8d) were synthesized via a previously described microwave assisted
(MicroSYNTH Milestone) method [25]. Briefly, the appropriately substituted salicylic acid (1
eq) and the substituted aniline (1 eq) were suspended or dissolved in chlorobenzene
(1mL/0.15 mmol of acid). Phosphorus trichloride (PCl3) was added (0.5 eq) and the mixture
was stirred (600 rpm) and radiated (530 W) for 30-60 min. The mixture was filtrated while
hot and was let cool, initially at r.t. and overnight at 4 oC. The formed precipitate was filtered
and recrystallized with ethanol or mixture of hexane/AcOEt in order to afford the final
compounds with yields of 41-83%.
4.1.3. Compounds’ characterization
4.1.3.1.
Methyl 4-bromo-5-chloro-2-hydroxybenzoate (III)
Prepared by general procedure A using 4-bromosalicylic acid and N-chlorosuccinimide.
Purified by column chromatography, eluent hexane/AcOEt:20/1. White solid; yield 42% in
two steps; mp 120.5-122.0 oC. IR (ATR): 3152, 1668, 1599, 1455, 1438, 1200, 1100, 788. 1H
13
NMR (500 MHz, CDCl3): δ 10.66 (s, 1H), 7.89 (s, 1H), 7.31 (s, 1H), 3.96 (s, 3H). C NMR
(125 MHz, CDCl3): δ 169.2, 159.9, 130.3, 130.0, 124.7, 122.9, 112.6, 77.2, 52.8. Anal. Calcd
for C8H6BrClO3 (265.49): C, 36.19; H, 2.28. Found: C, 36.16; H, 2.26.

4.1.3.2.
Methyl 4,5-dibromo-2-hydroxybenzoate (IV): Prepared by general
procedure A using 4-bromosalicylic acid and N-bromosuccinimide. Purified by column
chromatography, eluent Hexane/AcOEt:15/1. White solid; yield 38% in two steps; mp 107.0-
o
1
108.0 C. IR (ATR): 3152, 1669, 1597, 1451, 1439, 1193, 1100, 902, 787. H NMR (300
MHz, CDCl3): δ 10.67 (s, 1H), 8,04 (s, 1H), 7.31 (s, 1H), 3.96 (s, 3H). 13C NMR (75 MHz,
CDCl3): δ 169.6, 161.0, 134.3, 132.8, 123.5, 114.3, 113.6, 53.4. Anal. Calcd for C8H6Br2O3
(309.94): C, 31.00; H, 1.95. Found: C, 30.97; H, 1.96.
4.1.3.3.
Methyl 4,5-dichloro-2-hydroxybenzoate (V): Prepared by general procedure
A using 4-chlorosalicylic acid and N-chlorosuccinimide. Purified by column chromatography,
o
eluent hexane/AcOEt:50/1. White solid; yield 35% in two steps; mp 100.0-101.0 C. IR
1
(ATR): 3093, 1669, 1606, 1463, 1449, 1342, 1248, 1210, 1103, 935, 794. H NMR (500
13
MHz, CDCl3): δ 10.69 (s, 1H), 7.90 (s, 2H), 7.12 (s, 2H), 3.96 (s, 3H). C NMR (125 MHz,
CDCl3): δ 169.5, 160.5, 140.0, 131.1, 123.2, 119.9, 112.5, 53.2. Anal. Calcd for C8H6Cl2O3
(221.03): C, 43.47; H, 2.74. Found: C, 43.44; H, 2.72.
4.1.3.4.
Methyl 5-bromo-4-chloro-2-hydroxybenzoate (VI): Prepared by general
procedure A using 4-chlorosalicylic acid and N-bromosuccinimide. Purified by column
chromatography, eluent hexane/AcOEt (50:1). White solid; yield 38% in two steps; mp 93.5-
o
1
94.5 C. IR (ATR): 3097, 1672, 1601, 1493, 1439, 1323, 1242, 1207, 1104, 917, 788. H
NMR (300 MHz, CDCl3): δ 10.71 (s, 1H), 8.06 (s, 2H), 7.13 (s, 1H), 3.96 (s, 3H). 13C NMR
(75 MHz, CDCl3): δ 169.1, 160.9, 141.6, 134.2, 119.6, 112.7, 111.6, 52.9. Anal. Calcd for
C8H6BrClO3 (265.49): C, 36.19; H, 2.28. Found: C, 36.18; H, 2.27
4.1.3.5.
4-Bromo-5-chloro-2-hydroxybenzoic acid (VII): Prepared by general
procedure B from methyl 4-bromo-5-chloro-2-hydroxybenzoate. White solid; yield 96%; mp
222.5-223.5 oC. IR (ATR): 3051, 2851, 1657, 1599, 1461, 1439, 1313, 1237, 1199, 887, 863.
13
1H NMR (300 MHz, DMSO-d6): δ 11.59 (bs, 1H), 7.86 (s, 2H), 7.41 (s, 1H). C NMR (75
MHz, DMSO-d6): δ 169.9, 159.5, 130.7, 128.1, 123.0, 122.4, 114.6. Anal. Calcd for
C7H4BrClO3 (251.46): C, 33.44; H, 1.60. Found: C, 33.42; H, 1.59
4.1.3.6.
4,5-Dibromo-2-hydroxybenzoic acid (VIII): Prepared by general procedure
o
B from methyl 4,5-dibromo-2-hydroxybenzoate. White solid; yield 94%; mp 226.0-228.0 C.
IR (ATR): 2922, 2851, 1659, 1596, 1460, 1439, 1311, 1236, 1198, 882, 863. H NMR (300
MHz, DMSO-d6): δ 11.08 (bs, 1H), 7.98 (s, 1H), 7.41 (s, 1H). C NMR (75 MHz, DMSO-
1
13
d6): δ 169.8, 160.0, 134.0, 130.4, 122.4, 115.0, 112.5. Anal. Calcd for C7H4Br2O3 (295.91): C,
28.41; H, 1.36. Found: C, 28.40; H, 1.35
4.1.3.7.
4,5-Dichloro-2-hydroxybenzoic acid (IX): Prepared by general procedure B
o
from methyl 4,5-dichloro-2-hydroxybenzoate. White solid; yield 97%; mp 208.0-209.0 C.
[lit[28]: mp 210-211 C]. All spectroscopic characterization was in accordance with
o
previously reported values.
4.1.3.8.
5-Bromo-4-chloro-2-hydroxybenzoic acid (X): Prepared by general
procedure B from methyl 5-bromo-4-chloro-2-hydroxybenzoate. White solid; yield 95%; mp

o
o
213.0-214.5 C. [lit[28]: mp 208-213 C]. All spectroscopic characterization was in
accordance with previously reported values.
4.1.3.9.
N-(4-Bromophenyl)-4-chloro-2-hydroxybenzamide (1a): Prepared by
o
general procedure C. White solid; yield 73%; mp 220.0-221.0 C. [lit[22]: mp 220.0-222.0
oC]. All spectroscopic characterization was in accordance with previously reported values.
4.1.3.10. 4-Chloro-N-(3,4-dichlorophenyl)-2-hydroxybenzamide (1b): Prepared by
o
general procedure C. White solid; yield 77%; mp 222.0-223.0 C. [lit[22]: mp 221.0-223.0
oC]. All spectroscopic characterization was in accordance with previously reported values.
4.1.3.11. 4-Chloro-2-hydroxy-N-(4-(trifluoromethyl)phenyl)benzamide
(1c):
o
Prepared by general procedure C. White solid; yield 71%; mp 212.0-213.5 C. [lit[22]: mp
o
213.0-216.0 C]. All spectroscopic characterization was in accordance with previously
reported values.
4.1.3.12. N-(3,5-Bis(trifluoromethyl)phenyl)-4-chloro-2-hydroxybenzamide
(1d):
o
Prepared by general procedure C. White solid; yield 82%; mp 203.0-204.5 C. [lit[29]: mp
o
204.0-205.0 C]. All spectroscopic characterization was in accordance with previously
reported values.
4.1.3.13. 4-Bromo-N-(4-bromophenyl)-2-hydroxybenzamide (2a): Prepared by
o
general procedure C. White solid; yield 63%; mp 229.0-230.0 C. IR (ATR): 3322, 3087,
1
1602, 1590, 1550, 1448, 1225, 1076, 827, 810. H NMR (500 MHz, DMSO-d6): δ 11.89 (brs,
1H), 10.42 (s, 1H), 7.82 (d, J = 8.5 Hz, 1H), 7.69 (d, J = 9.0 Hz, 2H), 7.55 (d, J = 8.5 Hz, 2H),
13
7.19 (d, J = 2.0 Hz, 1H), 7.16 (dd, J = 8.3, 2.0 Hz, 1H). C NMR (125 MHz, DMSO-d6): δ
165.4, 158.5, 137.6, 131.6, 131.0, 126.1, 122.6, 122.1, 119.6, 118.0, 115.9. Anal. Calcd for
C13H9Br2NO2 (371.03): C, 42.08; H, 2.44; N, 3.78. Found: C, 42.11; H, 2.45; N, 3.76.
4.1.3.14. 4-Bromo-N-(3,4-dichlorophenyl)-2-hydroxybenzamide (2b): Prepared by
o
general procedure C. Light purple solid; yield 54%; mp 234.5-235.5 C. IR (ATR): 3304,
1
3161, 1631, 1597, 1586, 1442, 1223, 1136, 899, 819. H NMR (500 MHz, DMSO-d6): δ
11.75 (brs, 1H), 10.50 (s, 1H), 8.10 (d, J = 2.5 Hz), 7.77 (d, J = 8.5 Hz, 1H), 7.66 (dd, J = 8.5,
2.5 Hz, 1H), 7.61 (d, J = 9.0 Hz, 1H), 7.20 (d, J = 2.0 Hz, 1H), 7.18 (dd, J = 8.3, 2.0 Hz, 1H).
13C NMR (125 MHz, DMSO-d6): δ 165.4, 158.1, 138.4, 131.1, 130.9. 130.6, 126.1, 125.5,
122.2, 121.7, 120.5, 119.6, 118.2. Anal. Calcd for C13H8BrCl2NO2 (371.03): C, 42.25; H,
2.23; N, 3.88. Found: C, 42.28; H, 2.22; N, 3.89.
4.1.3.15. 4-Bromo-2-hydroxy-N-(4-(trifluoromethyl)phenyl)benzamide
(2c):
o
Prepared by general procedure C. White solid; yield 44%; mp 224.0-225.5 C. IR (ATR):
1
3314, 2934, 1609, 1591, 1572, 1421, 1227, 1107, 1070, 914, 840. H NMR (500 MHz,
DMSO-d6): δ 11.79 (brs, 1H), 10.58 (s, 1H), 7.94 (d, J = 8.5 Hz, 2H), 7.81 (d, J = 8.5 Hz,
13
1H), 7.73 (d, J = 8.5 Hz, 2H), 7.21 (d, J = 2.0 Hz, 1H), 7.17 (dd, J = 8.5, 2.0 Hz, 1H). C
NMR (125 MHz, DMSO-d6): δ 165.7, 158.3, 142.1, 131.4, 126.2 (q, J = 3.63 Hz), 126.3,

124.5 (q, J = 264.3 Hz), 124.1 (q, J = 44.4 Hz), 122.4, 120.6, 119.8, 118.6. Anal. Calcd for
C14H9BrF3NO2 (360.13): C, 46.69; H, 2.52; N, 3.89. Found: C, 46.70; H, 2.50; N, 3.86.
4.1.3.16. N-(3,5-Bis(trifluoromethyl)phenyl)-4-bromo-2-hydroxybenzamide
(2d):
o
Prepared by general procedure C. White solid; yield 47%; mp 218.0-219.0 C. IR (ATR):
1
3316, 3169, 1642, 1381, 1275, 1184, 1130, 906, 693. H NMR (300 MHz, DMSO-d6): δ
11.62 (brs, 1H), 10.79 (s, 1H), 8.43 (s, 2H), 7.81 (s, 1H), 7.76 (d, J = 8.1 Hz, 1H), 7.21 (d, J =
1,5 Hz, 1H), 7.17 (dd, J = 8.4 Hz, 1,8 Hz, 1H). 13C NMR (75 MHz, DMSO-d6): δ 65.9, 158.1,
140.3, 131.2, 130.7 (q, J = 33.0 Hz), 126.7, 123.1 (q, J = 294.3 Hz), 122.2, 120.3 (q, J = 3.8
Hz), 119.6, 118.3, 116.8 (q, J = 3.8 Hz). Anal. Calcd for C15H8BrF6NO2 (428.12): C, 42.08;
H, 1.88; N, 3.27. Found: C, 42.04; H, 2.00; N, 3.85.
4.1.3.17. N-(4-Bromophenyl)-5-chloro-2-hydroxybenzamide (3a): Prepared by
o
o
general procedure C. White solid; yield 56%; mp 240.0-241.5 C. [lit[23]: mp 240-241 C].
All spectroscopic characterization was in accordance with previously reported values.
4.1.3.18. 5-Chloro-N-(3,4-dichlorophenyl)-2-hydroxybenzamide (3b): Prepared by
o
general procedure C. Light purple solid; yield 59%; mp 246.0-248.0 C. [lit[23]: mp 248.0-
o
250.0 C]. All spectroscopic characterization was in accordance with previously reported
values.
4.1.3.19. 5-Chloro-2-hydroxy-N-[4-(trifluoromethyl)phenyl]benzamide
(3c):
o
Prepared by general procedure C. White solid; yield 54%; mp 218.0-219.0 C. [lit[22]: mp
o
217.0-218.0 C]. All spectroscopic characterization was in accordance with previously
reported values.
4.1.3.20. N-[3,5-Bis(trifluoromethyl)phenyl]-5-chloro-2-hydroxybenzamide
(3d):
o
Prepared by general procedure C. White solid; yield 62%; mp 169.0-170.0 C. [lit[15]: mp
o
172.0-173.0 C]. All spectroscopic characterization was in accordance with previously
reported values.
4.1.3.21. 5-Bromo-N-(4-bromophenyl)-2-hydroxybenzamide (4a): Prepared by
o
general procedure C. White solid; yield 67%; mp 240.0-241.5 C. [lit[23]: mp 240.0-241.0
oC]. All spectroscopic characterization was in accordance with previously reported values.
4.1.3.22. 5-Bromo-N-(3,4-dichlorophenyl)-2-hydroxybenzamide (4b): Prepared by
o
general procedure C. White solid; yield 63%; mp 235.0-236.0 C. [lit[23]: mp 234.0-236.0
oC]. All spectroscopic characterization was in accordance with previously reported values.
4.1.3.23. 5-Bromo-2-hydroxy-N-[4-(trifluoromethyl)]benzamide (4c): Prepared by
o
general procedure C. White solid; yield 61%; mp 202.0-203.0 C. [lit[22]: mp 202.0-203.0
oC]. All spectroscopic characterization was in accordance with previously reported values.

4.1.3.24. N-[3,5-Bis(trifluoromethyl)phenyl]-5-bromo-2-hydroxybenzamide
(4d):
o
Prepared by general procedure C. White solid; yield 83%; mp 201.0-202.0 C. [lit[15]: mp
o
194.0-195.0 C]. All spectroscopic characterization was in accordance with previously
reported values.
4.1.3.25. 4-Bromo-N-(4-bromophenyl)-5-chloro-2-hydroxybenzamide (5a): Prepared
o
by general procedure C. White solid; yield 45%; mp 262.0-263.0 C. IR (ATR): 3322, 3086,
1605, 1549, 1488, 1402, 1237, 1078, 820. 1H NMR (500 MHz, DMSO-d6): δ 11.90 (brs, 1H),
10.44 (s, 1H), 8.03 (s, 1H), 7.68 (d, J = 8.5 Hz, 2H), 7.55 (d, J = 8.5 Hz, 2H), 7.37 (s, 1H). 13C
NMR (125 MHz, DMSO-d6): δ 164.1, 156.5, 137.4, 131.6, 130.1, 125.5, 123.0, 122.5, 121.8,
120.2, 116.0. Anal. Calcd for C13H8Br2ClNO2 (405.47): C, 38.51; H, 1.99; N, 3.45. Found: C,
38.50; H, 2.01; N, 3.44.
4.1.3.26. 4-Bromo-5-chloro-N-(3,4-dichlorophenyl)-2-hydroxybenzamide
(5b):
o
Prepared by general procedure C. Light purple solid; yield 47%; mp 264.0-265.5 C. IR
(ATR): 3321, 3089, 1626, 1599, 1541, 1476, 1402, 1237, 1135, 858, 818, 671. 1H NMR (500
MHz, DMSO-d6): δ 11.79 (brs, 1H), 10.52 (s, 1H), 8.08 (d, J = 2.0 Hz, 1H), 7.98 (s, 1H), 7.65
13
(dd, J = 9.0, 2.0 Hz, 1H), 7.61 (d, J = 9.0 Hz, 1H), 7.36 (s, 1H, H3). C NMR (125 MHz,
DMSO-d6): δ 164.3, 155.4, 138.4, 131.2, 130.8, 130.3, 125.9, 125.7, 123.3, 122.0, 121.9,
120.7, 120.5. Anal. Calcd for C13H7BrCl3NO2 (395.46): C, 39.48; H, 1.78; N, 3.54. Found: C,
39.44; H, 1.81; N, 3.41.
4.1.3.27. 4-Bromo-5-chloro-2-hydroxy-N-[4-(trifluoromethyl)phenyl]benzamide
o
(5c): Prepared by general procedure C. White solid; yield 43%; mp 236.0-238.0 C. IR
(ATR): 3320, 3087, 1609, 1414, 1324, 1232, 1117, 1070, 841, 708, 673. 1H NMR (500 MHz,
DMSO-d6): δ 11.81 (brs, 1H), 10.62 (s, 1H), 8.01 (s, 1H), 7.92 (d, J = 8.5 Hz, 2H), 7.73 (d, J
13
= 9.0 Hz, 2H), 7.37 (s, 1H). C NMR (125 MHz, DMSO-d6): δ 164.2, 156.2, 141.8, 130.2,
126.1 (q, J = 3.5 Hz), 125.5, 124.0 (q, J = 270.0 Hz), 124.3 (q, J = 32.0 Hz), 123.1, 121.8,
121.8, 120.7, 120.4. Anal. Calcd for C14H8BrClF3NO2 (394.57): C, 42.62; H, 2.04; N, 3.55.
Found: C, 42.59; H, 2.06; N, 3.51.
4.1.3.28. N-[3,5-Bis(trifluoromethyl)phenyl]-4-bromo-5-chloro-2-
hydroxybenzamide (5d): Prepared by general procedure C. White solid; yield 46%; mp
o
1
208.5-209.5 C. IR (ATR): 3322, 3297, 1639, 1379, 1275, 1188, 1177, 1127, 896, 702. H
NMR (500 MHz, DMSO-d6): δ 11.67 (brs, 1H), 10.82 (s, 1H), 8.41 (s, 2H), 7.99 (s, 1H), 7.82
(s, 1H), 7.38 (s, 1H). 13C NMR (125 MHz, DMSO-d6): δ 164.6, 156.2, 140.1, 133.4, 130.7 (q,
J = 32.5 Hz), 130.2, 125.8, 123.3 (q, J = 234.5 Hz), 123.1, 121.8, 120.3, 120.2 (q, J = 3.8 Hz),
119.9, 116.9 (q, J = 3.3 Hz). Anal. Calcd for C15H7BrClF6NO2 (462.57): C, 38.95; H, 1.53; N,
3.03. Found: C, 38.92; H, 1.55; N, 3.00.
4.1.3.29. 4,5-Dibromo-N-(4-bromophenyl)-2-hydroxybenzamide (6a): Prepared by
o
general procedure C. White solid; yield 53%; mp 262.0-263.0 C. IR (ATR): 3323, 3086,
1604, 1548, 1488, 1402, 1237, 820. 1H NMR (500 MHz, DMSO-d6): δ 11.91 (brs, 1H), 10.44
13
(s, 1H), 8.14 (s, 1H), 7.68 (d, J = 8.6 Hz, 2H), 7.55 (d, J = 8.6 Hz, 2H), 7.37 (s, 1H). C
NMR (125 MHz, DMSO-d6): δ 164.0, 157.0, 137.4, 133.3, 131.6, 127.9, 122.5, 121.9, 120.4,

116.0, 112.6. Anal. Calcd for C13H8Br3NO2 (405.47): C, 34.70; H, 1.79; N, 3.11. Found: C,
34.65; H, 1.82; N, 3.09.
4.1.3.30. 4,5-Dibromo-N-(3,4-dichlorophenyl)-2-hydroxybenzamide (6b): Prepared
o
by general procedure C. White solid; yield 52%; mp 263.0-264.0 C. IR (ATR): 3324, 3087,
1
1597, 1540, 1477, 1398, 1236, 858, 818, 680. H NMR (500 MHz, DMSO-d6): δ 11.80 (brs,
1H), 10.53 (s, 1H), 8.10 (s, 1H), 8.08 (d, J = 2.1 Hz, 1H), 7.65 (dd, J = 8.8, 2.1 Hz, 1H), 7.62
13
(d, J = 8.8 Hz, 1H), 7.37 (s, 1H). C NMR (125 MHz, DMSO-d6): δ 164.1, 156.8, 138.2,
133.3, 131.0, 130.6, 128.0, 125.7, 121.8, 121.7, 120.6, 120.5, 112.6. Anal. Calcd for
C13H7Br2Cl2NO2 (439.91): C, 35.49; H, 1.60; N, 3.18. Found: C, 35.48; H, 1.60; N, 3.17.
4.1.3.31. 4,5-Dibromo-2-hydroxy-N-[4-(trifluoromethyl)phenyl]benzamide
(6c):
o
Prepared by general procedure C. White solid; yield 48%; mp 240.5-241.5 C. IR (ATR):
3319, 3084, 1631, 1589, 1414, 1323, 1116, 1069, 841, 702. 1H NMR (500 MHz, DMSO-d6):
δ 11.83 (brs, 1H), 10.62 (s, 1H), 8.12 (s, 1H), 7.93 (d, J = 8.5 Hz, 2H), 7.74 (d, J = 8.5 Hz,
13
2H), 7.38 (s, 1H). C NMR (125 MHz, DMSO-d6): δ 164.2, 156.7, 141.8, 127.9, 126.0 (q, J
= 3.7 Hz), 124.3 (q, J = 268.8 Hz), 124.1 (q, J = 31.9 Hz), 121.8, 120.9, 120.4. Anal. Calcd
for C14H8Br2F3NO2 (439.02): C, 38.30; H, 1.84; N, 3.19. Found: C, 38.26; H, 1.86; N, 3.18.
4.1.3.32. N-[3,5-Bis(trifluoromethyl)phenyl]-4,5-dibromo-2-hydroxybenzamide
o
(6d): Prepared by general procedure C. White solid; yield 53%; mp 206.0-207.0 C. IR
1
(ATR): 3224, 1642, 1379, 1275, 1173, 1127, 896, 701. H NMR (500 MHz, DMSO-d6): δ
16.42 (s, 0H), 15.59 (s, 1H), 13.18 (s, 2H), 12.87 (s, 1H), 12.59 (s, 1H), 12.14 (s, 1H). 13C
NMR (125 MHz, DMSO-d6): δ 164.6, 156.8, 140.1, 133.4, 130.7 (q, J = 32.9 Hz), 128.2,
123.2 (q, J = 272.8 Hz), 121.9, 120.6, 120.3 (d, J = 3.8 Hz), 117.1 – 116.9 (d, J = 3.8 Hz),
112.6. Anal. Calcd for C15H7Br2F6NO2 (50.02): C, 35.53; H, 1.39; N, 2.76. Found: C, 35.50;
H, 1.41; N, 2.75.
4.1.3.33. N-(4-Bromophenyl)-4,5-dichloro-2-hydroxybenzamide (7a): Prepared by
o
general procedure C. White solid; yield 59%; mp 266.0-267.0 C. IR (ATR): 3327, 3091,
1606, 1548, 1489, 1404, 1241, 1099, 820. 1H NMR (500 MHz, DMSO-d6): δ 11.96 (brs, 1H),
10.45 (s, 1H), 8.06 (s, 1H), 7.68 (d, J = 9.0 Hz, 2H), 7.55 (d, J = 8.5 Hz, 2H), 7.23 (s, 1H). 13C
NMR (125 MHz, DMSO-d6): δ 164.0, 156.7, 137.4, 134.9, 131.6, 130.4, 122.5, 121.0, 119.6,
118.7, 116.1. Anal. Calcd for C13H8BrCl2NO2 (361.02): C, 43.25; H, 2.23; N, 3.88. Found: C,
43.22; H, 2.23; N, 3.86.
4.1.3.34. 4,5-Dichloro-N-(3,4-dichlorophenyl)-2-hydroxybenzamide (7b): Prepared
o
by general procedure C. White solid; yield 67%; mp 262.0-263.5 C. IR (ATR): 3321, 3089,
1
1658, 1625, 1413, 1324, 1233, 1121, 1105, 842, 710, 682. H NMR (500 MHz, DMSO-d6): δ
11.83 (brs, 1H), 10.53 (s, 1H), 8.08 (d, J = 2 Hz, 1H), 8.01 (s, 1H), 7.62 (dd, J = 9.0, 2.0 Hz,
13
1H), 7.61 (d, J = 9.0 Hz, 1H), 7.23 (s, 1H). C NMR (125 MHz, DMSO-d6): δ 164.1, 156.5,
138.2, 135.0, 131.0, 130.7, 130.5, 125.7, 121.7, 121.0, 120.5, 119.8, 118.6. Anal. Calcd for
C13H7Cl4NO2 (351.01): C, 44.48; H, 2.01; N, 3.99. Found: C, 44.41; H, 2.04; N, 3.95.

4.1.3.35. 4,5-Dichloro-2-hydroxy-N-(4-(trifluoromethyl)phenyl)benzamide
(7c):
o
Prepared by general procedure C. White solid; yield 58%; mp 238.0-240.0 C. IR (ATR):
3318, 2922, 1613, 1414, 1324, 1232, 1120, 1071, 842, 710. 1H NMR (500 MHz, DMSO-d6):
δ 11.88 (brs, 1H), 10.63 (s, 1H), 8.04 (s, 1H), 7.93 (d, J = 8.5 Hz,2H), 7.73 (d, J = 8.5 Hz,
2H), 7.24 (s, 1H). 13C NMR (125 MHz, DMSO-d6): δ 154.2, 146.5, 131.7, 124.9, 120.6, 116.0
(q, J = 3.6 Hz), 114.14 (q, J = 32.1 Hz), 114.06 (q, J = 277.2 Hz), 111.1, 110.4, 110.1, 108.6.
Anal. Calcd for C14H8Cl2F3NO2 (350.12): C, 48.03; H, 2.30; N, 4.00. Found: C, 47.85; H,
2.36; N, 3.81.
4.1.3.36. N-[3,5-Bis(trifluoromethyl)phenyl]-4,5-dichloro-2-hydroxybenzamide
o
(7d): Prepared by general procedure C. White solid; yield 57%; mp 216.0-217.0 C. IR
1
(ATR): 3334, 3161, 1641, 1379, 1276, 1190, 1180, 1128, 895, 703. H NMR (500 MHz,
DMSO-d6): δ 11.72 (brs, 1H), 10.82 (s, 1H), 8.42 (s, 2H), 8.02 (s, 1H), 7.83 (s, 1H), 7.24 (s,
13
1H). C NMR (125 MHz, DMSO-d6): δ 164.7, 156.7, 140.3, 135.4, 130.9 (q, J = 32.5Hz),
130.5, 123.3 (q, J = 271.6 Hz), 121.2, 120.4 (q, J = 3.5 Hz), 119.9, 118.9, 117.1 (q, J = 3.5
Hz). Anal. Calcd for C15H7Cl2F6NO2 (418.12): C, 43.09; H, 1.69; N, 3.35. Found: C, 32.98;
H, 1.72; N, 3.31.
4.1.3.37. 5-Bromo-N-(4-bromophenyl)-4-chloro-2-hydroxybenzamide (8a): Prepared
o
by general procedure C. White solid; yield 41%; mp 256.0-258.0 C. IR (ATR): 3326, 2930,
1605, 1548, 1488, 1402, 1240, 1098, 820. 1H NMR (500 MHz, DMSO-d6): δ 11.98 (brs, 1H),
10.44 (s, 1H), 8.18 (s, 1H), 7.67 (d, J = 8.5 Hz, 2H), 7.55 (d, J = 9.0 Hz, 2H), 7.23 (s, 1H). 13C
NMR (125 MHz, DMSO-d6): δ 164.0, 157.4, 137.4, 136.9, 133.5, 131.6, 122.5, 119.8, 118.6,
116.1, 110.2. Anal. Calcd for C13H8Br2ClNO2 (405.47): C, 38.51; H, 1.99; N, 3.45. Found: C,
38.46; H, 2.03; N, 3.41.
4.1.3.38. 5-Bromo-4-chloro-N-(3,4-dichlorophenyl)-2-hydroxybenzamide
(8b):
o
Prepared by general procedure C. White solid; yield 47%; mp 261.0-263.0 C. IR (ATR):
1
3326, 3090, 1625, 1601, 1541, 1476, 1401, 1238, 1136, 859, 818, 670. H NMR (500 MHz,
DMSO-d6): δ 11.85 (brs, 1H), 10.53 (s, 1H), 8.13 (s, 1H), 8.07 (s, 1H), 7.65 (d, J = 8.5 Hz,
13
1H), 7.61 (d, J = 8.5 Hz, 1H), 7.22 (s, 1H). C NMR (125 MHz, DMSO-d6): δ 164.0, 157.1,
138.2, 137.0, 133.6, 131.0, 130.6, 125.7, 121.7, 120.5, 120.0, 118.6, 110.2. Anal. Calcd for
C13H7BrCl3NO2 (395.46): C, 39.48; H, 1.78; N, 3.54. Found: C, 39.43; H, 1.80; N, 3.51.
4.1.3.39. 5-Bromo-4-chloro-2-hydroxy-N-[4-(trifluoromethyl)phenyl]benzamide
o
(8c): Prepared by general procedure C. White solid; yield 43%; mp 243.5-244.5 C. IR
(ATR): 3320, 3083, 1633, 1608, 1413, 1324, 1238, 1111, 1069, 840, 712, 658. 1H NMR (500
MHz, DMSO-d6): δ 11.89 (brs, 1H), 10.62 (s, 1H), 8.16 (s, 1H), 7.92 (d, J = 8.5 Hz, 2H), 7.73
(d, J = 9.0 Hz, 2H), 7.23 (s, 1H). 13C NMR (125 MHz, DMSO-d6): δ 164.1, 157.1, 141.7,
136.9, 133.7, 124.2 (q, J = 262.5 Hz), 124.3 (q, J = 31.3 Hz), 126.0 (q, J = 3.63 Hz), 120.4,
120.3, 118.6, 110.2. Anal. Calcd for C14H8BrClF3NO2 (394.57): C, 42.62; H, 2.04; N, 3.55.
Found: C, 42.57; H, 2.08; N, 3.50.
4.1.3.40. N-[3,5-Bis(trifluoromethyl)phenyl]-5-bromo-4-chloro-2-
hydroxybenzamide (8d): Prepared by general procedure C. White solid; yield 49%; mp

o
1
228.5-229.5 C. IR (ATR): 3312, 3092, 1637, 1379, 1275, 1186, 1133, 902, 704. H NMR
(500 MHz, DMSO-d6): δ 11.70 (s, 1H), 10.83 (s, 1H), 8.41 (s, 2H), 8.13 (s, 1H), 7.83 (s, 1H),
7.24 (s, 1H). 13C NMR (125 MHz, DMSO-d6): δ 164.5, 157.1, 140.1, 137.2, 133.7, 130.7 (q, J
= 32.8 Hz), 123.2 (q, J = 272.8 Hz), 120.3 (q, J = 3.4 Hz), 120.2, 118.7, 117.0 (q, J = 3.4 Hz)
110.2. Anal. Calcd for C15H7BrClF6NO2 (462.57): C, 38.95; H, 1.53; N, 3.03. Found: C,
38.89; H, 1.57; N, 2.98.
4.2. Biology
4.2.1. In vitro antimycobacterial evaluation
The in vitro antimycobacterial activity of the synthesized compounds was determined
against Mtb My 331/88 (H37RV; dilution of strain 10-3), M. avium My 330/80 (dilution of
strain 10-5), M. kansasii 235/80 (dilution of strain 10-4) and M. kansasii 6509/96 (dilution of
strain 10-4). A selected set of compounds was further tested against five MDR-TB strains
(dilution of all MDR-TB strains was 10-3 while 234/2005 is resistant to INH, RIF, rifabutin,
streptomycin, ethambutol and ofloxacin; 9449/2007 is resistant to INH, RIF, rifabutin, and
streptomycin; 242/2015 is resistant to INH, RIF, rifabutin, streptomycin and ethambutol;
Praha 1 is resistant to INH, (RIF), rifabutin and streptomycin and Praha 4 is resistant to INH,
RIF, rifabutin, streptomycin, ethambutol, ofloxacin and clofazimine) and one XDR-TB strain
(Praha 131 which is resistant to INH, RIF, rifabutin, streptomycin, ethambutol, ofloxacin,
gentamicin and amikacin). 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. The antimycobacterial activity of the compounds was determined in a
Šula’s semisynthetic medium (SEVAC, Prague, Czech Republic) via the micromethod for the
o
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 [30]. 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 0.5 μM. INH and PAS were chosen as reference drugs.
4.2.2. Cytotoxicity
All salicylanilides were tested for cytotoxicity against the human hepatocellular liver
carcinoma cell line HepG2 (passages 33-37; ECACC, Salisbury, UK) using a standard
colorimetric method measuring a tetrazolium salt reduction (CellTiter(R) 96 AQueous One
Solution Assay, Promega G3580, Madison, WI, USA).
The cells were routinely cultured in Minimum Essentials Eagle Medium (Sigma-Aldrich,
Darmstadt, Germany) supplemented with 10% fetal bovine serum (PAA, Biotech, Prague,
Czech Republic), 1% of L-glutamine solution (Sigma-Aldrich), and non-essential amino acids
o
solution (Sigma-Aldrich) in a humidified atmosphere containing 5% CO2 at 37 C. For
subculturing, the cells were harvested after trypsin/EDTA (Sigma-Aldrich) treatment at 37 oC.
The cells treated with the tested substances were used as experimental groups. Untreated
HepG2 cells were used as control groups.
The cells were seeded in density 1 x 104 cells per well in a 96-well plate with microscopic
control. Next day, the cells were treated with each of the tested substances. All tested
substances were prepared at concentrations 0.1-100 μM and tested in triplicates. The

following types of controls were included: determination of 100% viability and 0% viability
(the cells treated by 10% DMSO), no cell control and vehiculum controls. These checking
samples were prepared in triplicates.
o
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 compounds in the cell causes that the production of
o
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 calculated in each of the tested substances using GraphPad Prism software
(version 6; GraphPad Software Inc. San Diego, CA, USA).
Acknowledgments
The authors would like to thank the staff of the Department of Inorganic and Organic
Chemistry, Faculty of Pharmacy, for the technical assistance.
References
[1] Global tuberculosis report 2015,
http://apps.who.int/iris/bitstream/10665/191102/1/9789241565059_eng.pdf?ua=1, last accessed at
3.10.2016
[2] C. Dye, Global epidemiology of tuberculosis, Lancet, 367 (2006) 938-940.
[3] O.M. Jolobe, Anti-TNFalpha treatment and reactivation of latent tuberculosis, Lancet, 370 (2007)
27-28.
[4] P. Onyebujoh, A. Zumla, I. Ribeiro, R. Rustomjee, P. Mwaba, M. Gomes, J.M. Grange, Treatment of
tuberculosis: present status and future prospects, Bull. World. Health. Organ., 83 (2005) 857-865.
[5] M.H. Cynamon, Y. Zhang, T. Harpster, S. Cheng, M.S. DeStefano, High-dose isoniazid therapy for
isoniazid-resistant murine Mycobacterium tuberculosis infection, Antimicrob. Agents Chemother., 43
(1999) 2922-2924.
[6] P. Bemer-Melchior, A. Bryskier, H.B. Drugeon, Comparison of the in vitro activities of rifapentine
and rifampicin against Mycobacterium tuberculosis complex, J. Antimicrob. Chemother., 46 (2000)
571-576.
[7] A. Jain, R. Mondal, Extensively drug-resistant tuberculosis: current challenges and threats, FEMS
Immunol. Med. Microbiol., 53 (2008) 145-150.
[8] S.K. Parida, R. Axelsson-Robertson, M.V. Rao, N. Singh, I. Master, A. Lutckii, S. Keshavjee, J.
Andersson, A. Zumla, M. Maeurer, Totally drug-resistant tuberculosis and adjunct therapies, J. Intern.
Med., 277 (2015) 388-405.
[9] R.S. Wallis, M. Maeurer, P. Mwaba, J. Chakaya, R. Rustomjee, G.B. Migliori, B. Marais, M. Schito,
G. Churchyard, S. Swaminathan, M. Hoelscher, A. Zumla, Tuberculosis--advances in development of
new drugs, treatment regimens, host-directed therapies, and biomarkers, Lancet Infect. Dis., 16
(2016) e34-46.
[10] M.J. Macielag, J.P. Demers, S.A. Fraga-Spano, D.J. Hlasta, S.G. Johnson, R.M. Kanojia, R.K. Russell,
Z. Sui, M.A. Weidner-Wells, H. Werblood, B.D. Foleno, R.M. Goldschmidt, M.J. Loeloff, G.C. Webb, J.F.

Barrett, Substituted salicylanilides as inhibitors of two-component regulatory systems in bacteria, J.
Med. Chem., 41 (1998) 2939-2945.
[11] R. De La Fuente, N.D. Sonawane, D. Arumainayagam, A.S. Verkman, Small molecules with
antimicrobial activity against E. coli and P. aeruginosa identified by high-throughput screening, Br. J.
Pharmacol., 149 (2006) 551-559.
[12] A. Imramovský, J. Vínšová, J.M. Férriz, V. Buchta, J. Jampílek, Salicylanilide esters of N-protected
amino acids as novel antimicrobial agents, Bioorg. Med. Chem. Lett., 19 (2009) 348-351.
[13] J. Vinšová, J. Kozic, M. Krátký, J. Stolaříková, J. Mandíková, F. Trejtnar, V. Buchta, Salicylanilide
diethyl phosphates: synthesis, antimicrobial activity and cytotoxicity, Bioorg. Med. Chem., 22 (2014)
728-737.
[14] K. Pauk, I. Zadražilová, A. Imramovský, J. Vinšová, M. Pokorná, M. Masaříková, A. Čížek, J.
Jampílek, New derivatives of salicylamides: Preparation and antimicrobial activity against various
bacterial species, Bioorg. Med. Chem., 21 (2013) 6574-6581.
[15] I.Y. Lee, T.D. Gruber, A. Samuels, M. Yun, B. Nam, M. Kang, K. Crowley, B. Winterroth, H.I.
Boshoff, C.E. Barry, 3rd, Structure-activity relationships of antitubercular salicylanilides consistent
with disruption of the proton gradient via proton shuttling, Bioorg. Med. Chem., 21 (2013) 114-126.
[16] M. Krátký, J. Vinšová, E. Novotná, J. Mandíková, V. Wsól, F. Trejtnar, V. Ulmann, J. Stolaříková, S.
Fernandes, S. Bhat, J.O. Liu, Salicylanilide derivatives block Mycobacterium tuberculosis through
inhibition of isocitrate lyase and methionine aminopeptidase, Tuberculosis (Edinb), 92 (2012) 434-
439.
[17] T.J. Cheng, Y.T. Wu, S.T. Yang, K.H. Lo, S.K. Chen, Y.H. Chen, W.I. Huang, C.H. Yuan, C.W. Guo, L.Y.
Huang, K.T. Chen, H.W. Shih, Y.S. Cheng, W.C. Cheng, C.H. Wong, High-throughput identification of
antibacterials against methicillin-resistant Staphylococcus aureus (MRSA) and the transglycosylase,
Bioorg. Med. Chem., 18 (2010) 8512-8529.
[18] B.C. Chenna, B.A. Shinkre, J.R. King, A.L. Lucius, S.V. Narayana, S.E. Velu, Identification of novel
inhibitors of bacterial surface enzyme Staphylococcus aureus Sortase A, Bioorg. Med. Chem. Lett., 18
(2008) 380-385.
[19] G. Triola, S. Wetzel, B. Ellinger, M.A. Koch, K. Hubel, D. Rauh, H. Waldmann, ATP competitive
inhibitors of D-alanine-D-alanine ligase based on protein kinase inhibitor scaffolds, Bioorg. Med.
Chem., 17 (2009) 1079-1087.
[20] M. Krátký, J. Vinšová, Salicylanilide ester prodrugs as potential antimicrobial agents--a review,
Curr. Pharm. Des., 17 (2011) 3494-3505.
[21] M. Krátký, S. Bősze, Z. Baranyai, I. Szabó, J. Stolaříková, G. Paraskevopoulos, J. Vinšová, Synthesis
and in vitro biological evaluation of 2-(phenylcarbamoyl)phenyl 4-substituted benzoates, Bioorg.
Med. Chem., 23 (2015) 868-875.
[22] K. Waisser, O. Bureš, P. Holý, J. Kuneš, R. Oswald, L. Jirásková, M. Pour, V. Klimešová, L.
Kubicová, J. Kaustová, Relationship between the structure and antimycobacterial activity of
substituted salicylanilides, Arch. Pharm. (Weinheim), 336 (2003) 53-71.
[23] K. Waisser, J. Hladůvková, J. Kuneš, L. Kubicová, V. Klimešová, P. Karajannis, J. Kaustová,
Synthesis and antimycobacterial activity of salicylanilides substituted in position 5, Chem. Pap., 55
(2001) 121-129.
[24] G. Paraskevopoulos, M. Krátký, J. Mandíková, F. Trejtnar, J. Stolaříková, P. Pávek, G. Besra, J.
Vinšová, Novel derivatives of nitro-substituted salicylic acids: Synthesis, antimicrobial activity and
cytotoxicity, Bioorg. Med. Chem., 23 (2015) 7292-7301.
[25] M. Krátký, J. Vinšová, V. Buchta, K. Horvati, S. Bősze, J. Stolaříková, New amino acid esters of
salicylanilides active against MDR-TB and other microbes, Eur. J. Med. Chem., 45 (2010) 6106-6113.
[26] M. Krátký, J. Vinšová, E. Novotná, J. Stolaříková, Salicylanilide pyrazinoates inhibit in vitro
multidrug-resistant Mycobacterium tuberculosis strains, atypical mycobacteria and isocitrate lyase,
Eur. J. Pharm. Sci., 53 (2014) 1-9.
[27] B.E. Senousy, S.I. Belal, P.V. Draganov, Hepatotoxic effects of therapies for tuberculosis, Nat.
Rev. Gastroenterol. Hepatol., 7 (2010) 543-556.

[28] M. Tashiro, H. Tsuzuki, J.-i. Matsumoto, S. Mataka, K. Nakayama, et al., Reductive Dechlorination
of Chloro-phenols and -benzoic acids by Raney Co-Al Alloy in an Alkaline Deuterium Oxide Solution
and Preparation of Deuterated Salicylic Acids, J. Chem. Res. (M), (1989) 2826 - 2851.
[29] S. Kang, H.-J. Min, M.-S. Kang, M.-G. Jung, S. Kim, Discovery of novel 2-hydroxydiarylamide
derivatives as TMPRSS4 inhibitors, Bioorg. Med. Chem. Lett., 23 (2013) 1748-1751.
[30] J. Kaustová, Quantitative Micromethod for Susceptibility Testing of Mycobacteria in Šula's
Medium, J. Klin. Microbiol. Inf. Lék., 3 (1997) 115-124.