Phenolic N-monosubstituted carbamates: Antitubercular and toxicity evaluation of multi-targeting compounds

Article abstract of DOI:10.1016/j.ejmech.2019.111578

The research of novel antimycobacterial drugs represents a cutting-edge topic. Thirty phenolic N-monosubstituted carbamates, derivatives of salicylanilides and 4-chlorophenol, were investigated against Mycobacterium tuberculosis H₃₇Ra, H₃₇Rv including multidrug- and extensively drug-resistant strains, Mycobacterium avium, Mycobacterium kansasii, Mycobacterium aurum and Mycobacterium smegmatis as representatives of nontuberculous mycobacteria (NTM) and for their cytotoxic and cytostatic properties in HepG2 cells. Since salicylanilides are multi-targeting compounds, we determined also inhibition of mycobacterial isocitrate lyase, an enzyme involved in the maintenance of persistent tuberculous infection. The minimum inhibitory concentrations were from ≤0.5 μM for both drug-susceptible and resistant M. tuberculosis and from ≤0.79 μM for NTM with no cross-resistance to established drugs. The presence of halogenated salicylanilide scaffold results into an improved activity. We have verified that isocitrate lyase is not a key target, presented carbamates showed only moderate inhibitory activity (up to 18% at a concentration of 10 μM). Most of the compounds showed no cytotoxicity for HepG2 cells and some of them were without cytostatic activity. Cytotoxicity-based selectivity indexes of several carbamates for M. tuberculosis, including resistant strains, were higher than 125, thus favouring some derivatives as promising features for future development.

Full text of DOI:10.1016/j.ejmech.2019.111578

Journal Pre-proof
Phenolic N-Monosubstituted carbamates: Antitubercular and toxicity evaluation of
multi-targeting compounds
Martin Krátký, Ondřej Janďourek, Zsuzsa Baranyai, Eva Novotná, Jiřina Stolaříková,
Szilvia Bősze, Jarmila Vinšová
PII:
S0223-5234(19)30712-3
DOI:
https://doi.org/10.1016/j.ejmech.2019.111578
EJMECH 111578
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 19 December 2018
Revised Date: 29 July 2019
Accepted Date: 30 July 2019
Please cite this article as: M. Krátký, Ondř. Janďourek, Z. Baranyai, E. Novotná, Jiř. Stolaříková, S.
Bősze, J. Vinšová, Phenolic N-Monosubstituted carbamates: Antitubercular and toxicity evaluation
of multi-targeting compounds, European Journal of Medicinal Chemistry (2019), doi: https://
doi.org/10.1016/j.ejmech.2019.111578.
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Phenolic N-Monosubstituted Carbamates: Antitubercular and Toxicity
Evaluation of Multi-targeting Compounds
Martin Krátký1,*, Ondřej Janďourek2, Zsuzsa Baranyai3, Eva Novotná4, Jiřina Stolaříková5, Szilvia
Bősze3 and Jarmila Vinšová1
1
Department of Organic and Bioorganic Chemistry, Faculty of Pharmacy in Hradec Králové,
Charles University, Akademika Heyrovského 1203, 500 05 Hradec Králové, Czech Republic
Department of Biological and Medical Sciences, Faculty of Pharmacy in Hradec Králové,
2
Charles University, Akademika Heyrovského 1203, 500 05 Hradec Králové, Czech Republic
MTA-ELTE Research Group of Peptide Chemistry, Eötvös Loránd University, Pázmány Péter
3
Sétány 1/A, Budapest, H-1117, P.O. Box 32, 1518 Budapest 112, Hungary
Department of Biochemical Sciences, Faculty of Pharmacy in Hradec Králové, Charles
4
University, Akademika Heyrovského 1203, 500 05 Hradec Králové, Czech Republic
Laboratory for Mycobacterial Diagnostics and Tuberculosis, Regional Institute of Public Health
5
in Ostrava, Partyzánské náměstí 7, 702 00 Ostrava, Czech Republic
* Corresponding author. Akademika Heyrovského 1203, 500 05 Hradec Králové, Czech Republic.
E-mail address: martin.kratky@faf.cuni.cz, tel.: +420-495067343, fax: +420-495067166.
Abstract
The research of novel antimycobacterial drugs represents a cutting-edge topic. Thirty phenolic N-
monosubstituted carbamates, derivatives of salicylanilides and 4-chlorophenol, were investigated
against Mycobacterium tuberculosis H37Ra, H37Rv including multidrug- and extensively drug-
resistant strains, Mycobacterium avium, Mycobacterium kansasii, Mycobacterium aurum and
Mycobacterium smegmatis as representatives of nontuberculous mycobacteria (NTM) and for their
cytotoxic and cytostatic properties in HepG2 cells. Since salicylanilides are multi-targeting
compounds, we determined also inhibition of mycobacterial isocitrate lyase, an enzyme involved in
the maintenance of persistent tuberculous infection. The minimum inhibitory concentrations were
from ≤0.5 µM for both drug-susceptible and resistant M. tuberculosis and from ≤0.79 µM for NTM
with no cross-resistance to established drugs. The presence of halogenated salicylanilide scaffold
results into an improved activity. We have verified that isocitrate lyase is not a key target, presented
carbamates showed only moderate inhibitory activity (up to 18% at a concentration of 10 ꢀM).
Most of the compounds showed no cytotoxicity for HepG2 cells and some of them were without
cytostatic activity. Cytotoxicity-based selectivity indexes of several carbamates for M. tuberculosis,
including resistant strains, were higher than 125, thus favouring some derivatives as promising
features for future development.
Keywords
antimycobacterial activity; carbamate; cytotoxicity; multi-targeting; Mycobacterium tuberculosis;
salicylanilide
1

1. Introduction
The development of novel antimicrobial agents represents an essential public health topic.
Tuberculosis (TB), a severe infectious contagious disease caused by Mycobacterium tuberculosis
(Mtb.) complex is one of the leading causes of morbidity and mortality worldwide. According to the
World Health Organization, TB remains a global emergency. In 2017, an estimated 10.3 million
people fell ill with TB, and approximately 1.3 million HIV-negative people died from this disease.
HIV and TB co-infection as well as development of drug-resistant TB forms have brought
additional problems. There were an estimated 558,000 new cases of rifampicin (RIF)-resistant and
multidrug-resistant tuberculosis (MDR-TB). MDR-TB consists of in vitro resistance to both
isoniazid (INH) and rifampicin, the two most effective first-line oral drugs. XDR-TB was defined as
an MDR-TB plus resistance to at least one drug in both essential classes of medicines used in an
MDR-TB regimen: fluoroquinolones and second-line injectable drugs (amikacin, capreomycin, and
kanamycin).1 Their treatment is more challenging with limited therapeutic possibilities.
Ubiquitous nontuberculous (also called atypical) mycobacteria (NTM) are recognised more
frequently as causative agents of serious opportunistic human infections in both immunocompetent
and immunocompromised patients. They affect various tissues including respiratory tract, skin,
joints, bones and soft tissues. Their incidence is expected to continue to increase at least up to 2050.
There are multiple reasons for this fact, including heightened awareness, better diagnostics, an
increased number of vulnerable hosts and increasing medical tourism. Their very long treatment
requires administration of multiple drugs in combination, but there is a high level of both intrinsic
and acquired resistance. That is why the current treatment regimens have only a limited efficacy and
the outcome is poor. Unfortunately, many NTM cases are misdiagnosed as TB, however the
treatment of NTM caused diseases is not like that of TB.2,3,4
To achieve a global control against epidemics of resistant mycobacterial strains, there is an urgent
need for development of novel drugs. Ideally, they should avoid any cross-resistance to current
therapeutic options.
Carbamate bond has been highlighted in medicinal chemistry as both drugs and prodrugs. This
structure motif has been incorporated in many molecules of various indication classes in clinical
practice, illustratively antiviral, antiparasitic or central nervous system targeting drugs. Generally,
carbamate functional group provides sufficient chemical stability and capability to increase
permeability across biological membranes.5 Carbamates have been involved in prodrug design of
phenolic compounds frequently. They are more stable towards enzymatic hydrolysis than
corresponding esters of carboxylic acids.5,6
Diverse carbamates have been proposed and evaluated as potential antimycobacterial agents active
against not only Mtb.7,8,9,10,11,12 but also NTM like highly chemoresistant Mycobacterium
abscessus8, Mycobacterium avium and Mycobacterium kansasii7,10; for some of them truly
promising results were reported. They have been able to inhibit also MDR- and XDR-TB strains.7,9
Carbamates obtained from salicylanilides (2-hydroxy-N-phenylbenzamides) have shown, e.g.,
significant antimycobacterial properties7,9,10, activity against Gram-positive bacteria including
methicillin-resistant Staphylococcus aureus10,13, yeasts and fungi10,13, or inhibition of
acetylcholinesterase and butyrylcholinesterase.14,15 Importantly, they provided a better in vitro
antitubercular activity concomitantly with a reduced cytotoxicity and improved physicochemical
properties (e.g., low lipophilicity required for passive diffusion via cellular membranes), i.e., the
main disadvantages of their parent salicylanilide compounds.16
Salicylanilide-based molecules have been reported as inhibitors of many bacterial and/or
mycobacterial enzymes targeting various metabolic pathways and physiological functions:
D
-Ala-
D-
Ala ligase17, transglycosylase18,19, isocitrate lyase and methionine aminopeptidase20,
L-alanine
dehydrogenase, lysine ꢀ-aminotransferase, chorismate mutase, pantothenate synthetase21 or they are
able to disrupt proton gradient as uncouplers.22 However, there is no one “leading” target identified
as crucial for anti-TB activity and, likely, their antimycobacterial properties are a result of
interaction with multiple cellular proteins and structures. This “multi-targeting” nature may provide
a benefit of more difficult development of an acquired resistance.
2

Keeping in mind especially a breakthrough study of Férriz et al.7 reporting salicylanilide N-n-alkyl
carbamates highly active against MDR-TB strains with minimum inhibitory concentrations (MIC)
from 0.5 µM, we completed synthetically the series of our phenolic N-
cycloalkyl/phenyl/phenylalkyl carbamates13,15 and investigated them against various mycobacterial
strains and HepG2 cell line. We have identified salicylanilide derivatives as inhibitors of isocitrate
lyase; that is why we also evaluated their in vitro potency to inhibit this mycobacterial enzyme
involved in the maintenance of latency.10,20,23,24,25
2. Results and discussion
2.1 Chemistry
The synthetic overview is depicted in Scheme 1. First, salicylanilides were obtained by treatment of
salicylic or 5-chlorosalicylic acid with appropriate anilines in the presence of phosphorus trichloride
under microwave irradiation.26
5-Chloro-2-hydroxy-N-[4-(trifluoromethyl)phenyl]benzamide was converted to novel carbamates 5
by a reaction with commercially available isocyanates (1.1 of equivalents) in the presence of 1 eq.
of triethylamine. Dry acetonitrile was employed as a solvent. The reaction time was 2 hours at rt,
only the preparation of adamantan-1-yl isocyanate 4e required 8 h under reflux conditions. Yields
ranged from 70 to 94%, symmetrical 1,3-disubstituted ureas resulting from isocyanates13 were
observed as side products in the case of phenylalkyl isocyanates and especially phenyl isocyanate,
but not for cyclohexyl isocyanate. Salicylanilide bearing CF3 group was chosen since it and also its
derivatives have shown the most potent anti-TB activity in our previous studies.9,10,20,21,23,24,28 The
synthesis and characterization of the carbamates 1-3, 5 and 6 were reported previously by our
group.13,15 To evaluate an influence of halogenation on biological activity and also the contribution
of 2-[N-(substituted phenyl)carbamoyl] moiety, we also included non-halogenated salicylanilide
carbamates 5 and corresponding “simplified” 4-chlorophenyl carbamates 6, respectively. The parent
salicylanilides of here presented derivatives are identical to salicylanilides involved in our previous
study dealing with N,N-disubstituted carbamates to allow the direct comparison of these both
groups.10
Scheme 1. Synthesis of salicylanilides, salicylanilide carbamates 1-5 and 4-chlorophenyl carbamates
6 [R1 = H, Cl; R2 = H, Cl, Br, F, CF3; R3 = cyclohexyl a, phenyl b, benzyl c, phenethyl d,
adamantan-1-yl e; MW: microwave irradiation (530 W, 600 rpm, 22 min); PhCl: chlorobenzene;
MeCN: acetonitrile; Et3N: triethylamine].
The design of the carbamic part of these molecules is outlined in Scheme 2. Férriz et al.7 identified
salicylanilide N-hexylcarbamates as the most active against Mtb. Thus, we can obtain by an
imaginary cyclization of this substituent N-cyclohexylcarbamates a. Their dehydrogenation
provides phenylcarbamates b, which can also be homologues of highly antitubercular active
3

salicylanilide benzoates27,28 derived via an incorporation of -NH- group between ester and phenyl
moiety. Benzyl- and phenethylcarbamates c and d are higher homologues of phenylcarbamates
obtained by an elongation of the linker connecting planar aromatic portion with a more hydrophilic
carbamate functional group. Adamantane molecule (carbamates e) represents one of the frequent
and proved scaffolds for anti-TB drugs used for a sufficient lipophilicity control.29
The new compounds were characterized by melting points, IR and NMR spectra; the purity was
checked by the thin-layer chromatography and elemental analysis. In the IR spectra, sharp and
strong bands appear at around 1710–1722 cm-1 (carbamic C=O) and 1659–1668 (amide I). Amide II
bands were present at 1522–1536 cm-1. Most of the derivatives displayed also two clearly visible
N–H stretch bands (3261–3336 cm-1).
In some NMR spectra, we observed the existence of two rotamers (syn/anti) due to a hindered
rotation of C-N “pseudo-double” bond. For the carbamates, this phenomenon is well known.
Secondary carbamates usually prefer anti-conformation.5 The existence of two distinct rotamers
was the most pronounced in the spectra of the phenethylcarbamate 5d; the ratio of the dominant and
minority conformer was approx. 100:18.
All of the derivatives investigated in this study are summarized in Table 1.
Scheme 2. Design of antimycobacterial salicylanilide 1-5 and phenolic carbamates 6 (structural
modifications-new fragments are highlighted using blue colour)
2.2 Antimycobacterial activity
Initially, salicylanilide and 4-chlorophenol-based carbamates 1-6 were investigated in vitro against
mycobacteria actively growing in culture: Mtb. 331/88 (i.e., H37Rv), three NTM strains –
Mycobacterium avium 330/88, M. kansasii 235/80 and M. kansasii 6509/96 (a clinical isolate). 4-
Chlorophenol, isoniazid (INH), the first-line anti-TB drug, and p-aminosalicylic acid (PAS) as a
structurally similar compound were involved as the reference compounds for comparison.
Presented N-cycloalkyl/phenyl/phenylalkyl carbamates 1-6 (Table 1) did not exhibit a uniform
behaviour. This group should be divided into two: highly active halogenated salicylanilide
derivatives 1-4 (MIC values of ≤0.5 to 62.5 µM) and, on the other hand, non-halogenated
salicylanilide carbamates 5 together with slightly less potent 4-chlorophenyl carbamates 6 (MIC of
62.5 to 1000 µM).
In general, Mtb. is more susceptible than NTM. It was inhibited by the carbamates 1-4 with MIC up
to 16 µM with trifluoromethyl derivatives (4a, 4b, 4d and 4e) superiority (≤1 µM). This activity is
comparable to INH. Non-halogenated salicylanilide carbamates 5 led to a single MIC of 125 µM,
while 4-chlorophenyl derivatives inhibited this strain at 62.5-500 µM with 6e superiority. MIC for
4

atypical mycobacteria of the first group of derivatives ranged from 2 to 62.5 µM, for the second one
of 125-1000 µM. The highest activity was found for four carbamates again (4a, 4b, 4d, 4e; 2-8
µM), followed by brominated derivatives 2. The activities against M. avium and two strains of M.
kansasii were comparable (±one dilution). Usually, salicylanilide derivatives have supressed M.
kansasii stronger (e.g.,28) than M. avium. All of the salicylanilide carbamates 1-5 and some of the
carbamates 6 exhibited lower MIC than INH against INH-resistant strains of M. avium and M.
kansasii 235/80. A major part of phenolic derivatives (1a-2e, 3b, 3d-4e) showed an activity
comparable to INH against the clinical isolate of M. kansasii (6509/96). All halogenated
compounds 1-4 exhibited a significantly better activity against Mtb. than PAS (up to >125 times),
whereas other investigated derivatives were usually comparable (5, adamantane-based molecule 6e)
to this second-line anti-TB drug. The carbamates 1-4 showed also identical (3c) or the remaining
ones even substantially lower MIC values for M. avium, especially after 3 weeks of incubation.
Illustratively, the derivative 4e more than 31 times more potent than PAS. Additionally, both strains
of M. kansasii were dramatically more susceptible to halosalicylanilide carbamates 1-4 than to PAS
(up to >250 times). With one exception, also non-halogenated salicylic 5 and 4-chlorophenyl
carbamates 6 produced a better growth inhibition of M. kansasii 235/80 after 2 and 3 weeks of
incubation; the activities against M. kansasii 6509/96 are comparable mutually (i.e., equal or ±one
dilution). Surprisingly, the modification of 4-chlorophenol with mild intrinsic antimycobacterial
properties (MIC ≥125 µM) to form more lipophilic carbamates 6 did not result in any significant
improvement in activity.
Focusing on structure-activity relationships, following issues were identified:
1) The presence of the salicylanilide scaffold is essential for the low MIC values. The removal of 2-
[N-(substituted phenyl)carbamoyl] fragment to provide less lipophilic carbamates of 4-chlorophenol
(derivatives 6) led to significantly lower antimycobacterial properties (up to two orders of
magnitude).
2) The halogenation of the salicylanilide core and corresponding increased lipophilicity are required
for the outstanding activity (compounds 1-4 vs. 5) with CF3 group superiority (4) for both Mtb.
H37Rv and NTM. Among particular halogens, fluorine atom is the best one for Mtb. but not NTM,
where brominated derivatives led to the most potent growth inhibition.
3) Carbamates 1, 2 and 5 obtained from 5-chloro-N-(4-chlorophenyl)-2-hydroxybenzamide, N-(4-
bromophenyl)-5-chloro-2-hydroxybenzamide and unsubstituted salicylanilide, respectively,
exhibited equal MIC values (±one dilution) despite carbamic N-substituent. On the other hand,
among fluorine derivatives 3 and 4, N-benzyl compounds c showed a decreased activity when
compared to other carbamates. N-Adamantan-1-yl group (6e) represents the most convenient moiety
for 4-chlorophenyl carbamates.
Table 1. Antimycobacterial activity of the carbamates 1-6
MIC [µM]
Mtb.
331/88
R1
R2
R3
M. avium
M. kansasii 235/80
M. kansasii 6509/96
logP
14 d 21 d
14 d
21 d
16
16
16
8
7 d
14 d
32
32
8
8
16
21 d
7 d
8
8
4
4
14 d
8
16
8
8
16
21 d
8
16
8
8
16
1a Cl
1b Cl
1c Cl
1d Cl
1e Cl
Cl
Cl
Cl
Cl
Cl
Cyclohexyl
Phenyl
Benzyl
Phenethyl
Adamantan-1-yl
16
16
16
8
16
16
16
16
16
16
16
8
8
16
16
16
8
8
8
32
32
16
16
16
5.0
5.11
5.18
5.46
5.28
16
16
8
5

2a Cl
2b Cl
2c Cl
2d Cl
2e Cl
3a Cl
3b Cl
3c Cl
3d Cl
3e Cl
Br
Br
Br
Br
Br
F
F
F
F
F
Cyclohexyl
Phenyl
Benzyl
Phenethyl
Adamantan-1-yl
Cyclohexyl
Phenyl
8
8
16
8
8
4
4
8
4
4
16
16
16
8
8
8
8
16
4
4
1
8
8
8
8
8
32
32
62.5
32
32
16
16
16
8
8
32
32
62.5
32
32
4
4
4
4
4
16
8
16
8
8
4
8
8
8
8
8
32
16
32
16
8
4
4
16
4
4
250
250
250
250
125*
500
250
250
250
125*
>250
>250
1000
8
8
8
8
8
32
32
32
16
16
8
4
16
8
4
250
250
250
250
125*
1000
500
500
500
125*
>250
>250
>1000
4
4
4
4
4
16
8
16
8
8
2
8
8
8
8
8
16
16
32
16
8
4
4
16
4
4
250
250
250
250
250
250
250
250
250
125*
250
8
8
8
8
8
8
32
16
32
16
16
4
4
16
4
4
250
250
250
250
250
250
500
500
500
125*
>250
8
5.27
5.38
5.45
5.73
5.55
4.60
4.71
4.78
5.06
4.88
5.36
5.48
5.54
5.82
5.64
3.88
4.0
4.07
4.35
4.16
3.63
3.75
3.82
4.10
3.91
2.20
-0.60
0.88
Benzyl
Phenethyl
Adamantan-1-yl
Cyclohexyl
Phenyl
Benzyl
Phenethyl
Adamantan-1-yl ≤0.5
Cyclohexyl
Phenyl
4a Cl CF3
4b Cl CF3
4c Cl CF3
4d Cl CF3
4e Cl CF3
1
≤0.5
2
8
8
32
8
8
8
32
8
1
4
1
1
4
8
4
4
2
8
2
4
≤0.5
4
4
5a
5b
5c
5d
5e
6a
6b
6c
6d
6e
H
H
H
H
H
-
-
-
-
-
H
H
H
H
H
-
-
-
-
-
125 125
125 125
125 125
125 125
125 125
500 500
250 500
125 250
250 250
125
125
125
125
125
500
500
500
250
125
>250
>250
32
125
125
125
125
125*
1000
1000
500
500
250
>250
>250
125
125
125
125
125
125
250
125
125
125
125
250
>250
125
125
125
125
125
125
125
125
125
125
125
125
4
Benzyl
Phenethyl
Adamantan-1-yl
Cyclohexyl
Phenyl
Benzyl
Phenethyl
Adamantan-1-yl 62.5 125
125 250
4-Cl-phenol
INH
0.5
62.5 62.5
1
PAS
32
125
500
INH: isoniazid; PAS: p-aminosalicylic acid; * at this concentration, the growth of the strain was observed; at duplex concentration, a precipitate
and/or a turbidity was present, therefore it was not possible to determine the exact MIC value.
Based on these promising data, we decided to extend the commonly used panel of mycobacteria to
confirm an excellent wide-spectrum antimycobacterial activity against actively growing
mycobacteria. We involved Mycobacterium tuberculosis H37Ra ITM-M006710 and two NTM:
Mycobacterium aurum DSM 43999 and Mycobacterium smegmatis DSM 43465. Avirulent strain of
Mtb. is commonly used in screening showing comparable results to virulent strain with benefit
shown in lower risk of infection. Other two strains are counted between fast growing mycobacteria
and their benefit is in generation time that is much shorter than Mtb. Three first-line oral anti-TB
drugs, INH, rifampicin (RIF) and ethambutol (EMB) were used as the standards. The results are
summarized in Table 2.
Table 2. Activity of the carbamates 1-6 against additional mycobacterial strains
MIC [µg/mL] (µM)
R1
R2
R3
Mtb. H37Ra
M. aurum
M. smegmatis
48 h
3.125
1.56
1.56
1.56
3.125
1.56
1.56
72 h
1.56
1.56
1.56
1.56
3.91
1.56
1.56
1.98
1.56
1.56
1.56
1.98
3.125
3.125
120 h
1.56
1.56
1.56
1.56
3.91
1.56
1.56
1.98
3.125
3.125
3.91
1.98
3.125
3.125
1a Cl
1b Cl
1c Cl
1d Cl
1e Cl
2a Cl
2b Cl
2c Cl
2d Cl
2e Cl
3a Cl
3b Cl
3c Cl
3d Cl
Cl
Cl
Cl
Cl
Cl
Br
Br
Br
Br
Br
F
Cyclohexyl
Phenyl
Benzyl
Phenethyl
Adamantan-1-yl
Cyclohexyl
Phenyl
Benzyl
3.91
0.78 (1.65)
1.56
Phenethyl
Adamantan-1-yl
Cyclohexyl
Phenyl
3.125
3.125
3.125
3.125
F
F
F
Benzyl
Phenethyl
6

3e Cl
F
Adamantan-1-yl
Cyclohexyl
Phenyl
0.99 (2.24)
≤0.39 (≤0.88)
≤0.39 (≤0.90)
≤0.39 (≤0.87)
0.39-0.78
(0.84-1.69)
0.99 (2.00)
15.625
1.98
1.98
4a Cl CF3
4b Cl CF3
4c Cl CF3
≤0.39 (≤0.88)
≤0.39 (≤0.90)
≤0.39 (≤0.87)
≤0.39 (≤0.88)
≤0.39 (≤0.90)
0.78 (1.74)
Benzyl
4d Cl CF3
4e Cl CF3
Phenethyl
≤0.39 (≤0.84)
≤0.39 (≤0.84)
Adamantan-1-yl
Cyclohexyl
Phenyl
≤0.39 (≤0.79)
15.625
31.25
31.25
31.25
≥250
1.98 (4.00)
15.625
31.25
31.25
31.25
250
≥500
15.625
125
5a
5b
5c
5d
5e
6a
6b
6c
6d
6e
H
H
H
H
H
-
H
H
H
H
H
-
-
-
-
-
31.25
15.625
15.625
250
500
7.81
31.25
31.25
Benzyl
Phenethyl
Adamantan-1-yl
Cyclohexyl
Phenyl
Benzyl
Phenethyl
Adamantan-1-yl
≥500
15.625
125
125
≥125
-
-
-
-
62.5
≥125
62.5
0.125-0.25
(0.91-1.82)
0.0039-0.0078
(0.005-0.009)
0.5 (1.80)
INH
3.91
15.625
RIF
0.39 (0.47)
0.25 (0.90)
12.5-25
EMB
0.5 (1.80)
INH: isoniazid; RIF: rifampicin; EMB: ethambutol. For the most active compounds (MIC lower than 1 µg/mL) we calculated also MIC in µM for
direct comparison with values reported in Table 1.
Obviously, the carbamates 1-5 were also able to inhibit more mycobacterial strains effectively: Mtb.
H37Ra, M. smegmatis and M. aurum. We confirmed the excellence of the compounds substituted by
CF3 group (derivatives 4) as well as significantly higher MIC values of non-halogenated
salicylanilide carbamates 5 and 4-chlorophenyl carbamates 6.
The most active derivatives are fully comparable or superior to clinically used drugs INH and EMB
at low micromolar or even submicromolar concentrations. In contrast to INH and RIF, there were
no differences between MIC values of the carbamates 1-6 against Mtb. and NTM strains
predominantly. Thus, we confirmed the broad-spectrum antimycobacterial action of the investigated
carbamates. Interestingly, generally fewer active compounds 5-6 inhibited the additional
mycobacterial strains in this follow-up assay. Other important finding is that the activity our
compounds does not depend on the medium used for the determination of MIC; in this assay,
Middlebrook 7H9 medium was used. Comparison of results between virulent and avirulent strain
also showed that values of MIC are comparable and reliable. The advantage of using Mtb. H37Ra is
also connected with a shorter time of screening (results are read after 120 hours compared to 14 or
21 days).
2.2.1 Activity against drug-resistant TB strains
The carbamates with the highest in vitro efficacy against drug-susceptible Mtb. H37Rv (i.e., 4a, 4b,
4d, and 4e with MIC of ≤1 ꢀM, all being the derivatives of 5-chloro-2-hydroxy-N-[4-
(trifluoromethyl)phenyl]benzamide) were evaluated also against MDR- and XDR-TB additionally.
They exhibited also an interesting activity against six multidrug-resistant Mtb. strains and one
XDR-TB strain within the range of 0.5-2 ꢀM, independently of the resistance patterns (Table 3). N-
Phenethylcarbamate 4d showed only a negligibly lower in vitro efficacy. Importantly, these values
are comparable or identical to those obtained for Mtb. H37Rv indicating that there is no cross-
resistance with currently used drugs for the treatment of both drug-susceptible and resistant TB
(INH, RIF, rifabutin, ethambutol, streptomycin, fluoroquinolones represented by ofloxacin,
clofazimine, and aminoglycosides). Regarding these facts, the salicylanilide carbamates seem to be
promising wide-spectrum anti-TB and antimycobacterial agents against drug-susceptible, drug-
resistant and NTM mycobacterial strains.
Table 3. MIC of salicylanilide carbamates 4 for drug-resistant Mtb. strains
7

MIC [µM]
Mtb.
Mtb.
Mtb.
Mtb.
Mtb.
Mtb.
Mtb.
7357/1998
9449/2006
8666/2010
234/2005
Praha 1
Praha 4
Praha 131
14 d
21 d 14 d 21 d 14 d 21 d
14 d 21 d 14 d 21 d 14 d 21 d 14 d
21 d
4a
4b
4d
4e
0.5
1
1
2
1
0.5
1
1
1
2
1
0.5
0.5
2
1
1
2
1
0.5
1
1
1
2
1
0.5
0.5
1
1
1
1
1
0.5
0.5
2
1
1
2
1
0.5
1
1
1
2
0.5
2
2
2
1
0.5
1
0.5
1
0.5
0.5
1
1
MIC values equal or lower than 1 ꢀM are given in bold.
MDR-TB strains: 7357/1998 resistant to INH, RIF, rifabutin, streptomycin, ethambutol and ofloxacin; 9449/2006 resistant to INH, RIF, rifabutin and
streptomycin; 234/2005 resistant to INH, RIF, rifabutin, streptomycin and ethambutol; 8666/2010 resistant to INH, RIF, rifabutin, streptomycin,
ethambutol, ofloxacin and clofazimine; Praha 1 resistant to INH, RIF, rifabutin, streptomycin, ethambutol and clofazimine; Praha 4 resistant to INH,
RIF, rifabutin, streptomycin, ethambutol, ofloxacin and clofazimine. XDR-TB strain: Praha 131 resistant to INH, RIF, rifabutin, streptomycin,
ethambutol, ofloxacin, gentamicin and amikacin.
Comparing to previous salicylanilide carbamates, the most active derivatives 1-4 are comparable to
salicylanilide N-n-alkylcarbamates and chloroalkycarbamates7,9, and significantly superior to N,N-
disubstituted carbamates.10
2.3 Evaluation of isocitrate lyase inhibition
Since salicylanilide derivatives are multi-targeting agents, we investigated the carbamates against
one of the targets described earlier. Previous works have reported salicylanilides10,20, their esters
with various carboxylic acids20,23,24, benzenesulfonic acid20, N,N-disubstituted (thio)carbamates10
and benzamides25 as in vitro inhibitors of mycobacterial isocitrate lyase (ICL1), but to the best of
our knowledge, inhibition activity of phenolic N-monosubstituted carbamates has not been reported
to date.
ICL1, one of two enzymes involved in glyoxylate cycle, a metabolic pathway replenishing
tricarboxylic acid cycle intermediates, and methylcitrate cycle represents an attractive target for
combating especially persistent intracellular mycobacterial subpopulation during chronic infection.
However, inhibition of ICL has no significant observable effect against actively growing
mycobacteria25 (here reported in Tables 1-3). It was presumed that the ICL inhibitors may result in
the shortening of TB therapy. Especially dual activity against both actively growing and non-
replicating mycobacteria should provide an extra benefit.25,30 Moreover, this enzyme has been
proposed to be linked to endogenous resistance or a higher tolerance to anti-TB drugs.31
ICL inhibition assay was selected to extend the potential application to latent mycobacteria as an
adjunctive target illustrating the multi-targeting nature of the carbamates 1-6. We determined their
activity at a single concentration of 10 µM using glyoxylate phenyl hydrazone formation assay
(Table 4). INH and PAS were employed as negative controls; 3-nitropropionic acid, a known
substrate-analogue inhibitor25, served as a positive control. We also intended to determine IC50 for
the most potent inhibitors (i.e., inhibition rate comparable to 3-nitropropionic acid: 25%). However,
all of the derivatives produced only a mild inhibition of the enzyme within the range of 5-18% with
2e and 3a superiority, even though the differences are rather marginal.
The highest activity was observed for the brominated and monofluorinated derivatives 2 and 3,
which are superior to dichlorocarbamates 1. In contrast to whole-cell antimycobacterial activity, the
presence of CF3 group (4a-4e) does not offer any significant advantage. The halogenation improved
the inhibition potency substantially (5 vs. 1 and especially 2-4 vs. 5). Comparing salicylanilide
molecules 1-5 and 4-chlorophenyl carbamates 6, the (4-halogenophenyl)carbamoyl fragment is not
required for an effective ICL inhibition. Due to insignificant distinctions, it is also unfeasible to
identify the most convenient carbamic N-substituent.
In accordance to some previous reports regarding salicylanilide carboxylates, benzenesulfonates
esters and N,N-disubstituted (thio)carbamates10,20,23,24, here presented phenolic carbamates were
8

found as mild ICL1 inhibitors comparable with the majority of them and, importantly, superior to
parent salicylanilides with free phenolic group.
As expected25,30, we did not identify a clear relationship of in vitro MIC against actively growing
mycobacteria (low micromolar or submicromolar values; Tables 1, 2 and 3) and ICL inhibition
(only partial inhibition at 10 µM; Table 4). Moreover, there should not be a direct correlation
between strong anti-TB properties under our experimental conditions and ICL suppression, since
both tests are focused on different stages of mycobacterial life cycle. Since ICL is indispensable
only in non-replicating mycobacteria, its inhibition has shown partial potential against persistent or
latent mycobacteria during a long-term use in the TB therapy, but not in short-term assay
determining suppression of actively growing extracellular bacteria used in this study. Using in
combination with other drugs, it may help to increase susceptibility to them. Thus, the consideration
of ICL as a key target of the carbamates 1-6 is inappropriate and it is in accordance with the known
assumptions. Their mechanism of action against actively growing mycobacteria remains to be
elucidated in depth and it is not related to described mild ICL inhibition. Analogous phenomenon
was described for methyl 4-(4-methoxyphenyl)-4-oxobut-2-enoate. This ester inhibited ICL1 (IC50
= 30.9 µM) and showed an activity in model of intracellularly persistent mycobacteria, but it was
also active against various mycobacterial strains in log-phase in culture (MIC90 0.25-1 µg/mL). The
explanation of this excellent activity cannot be attributed to moderate ICL inhibition suggesting
likely an additional unknown actual target remaining to be confirmed.32.
Giving together, the here reported ICL inhibition of the salicylanilide carbamates brings probably
only a certain minor benefit concerning nonreplicating mycobacteria and latent TB. Actual and key
anti-TB target of the carbamates 1-6 remains to be confirmed.
Table 4. Inhibition of isocitrate lyase
Inhibition rate
Code
R1
R2
R3
[%] at 10 µM
13 ± 1.56
10 ± 1.56
11 ± 1.58
10 ± 1.21
11 ± 1.58
16 ± 3.70
17 ± 1.42
17 ± 2.94
17 ± 1.15
18 ± 2.61
18 ± 0.75
16 ± 4.54
15 ± 2.71
15 ± 2.49
14 ± 3.93
14 ± 3.66
13 ± 2.47
14 ± 3.00
11 ± 4.72
15 ± 1.33
8 ± 2.77
5 ± 1.56
8 ± 1.35
6 ± 1.99
9 ± 1.58
16 ± 2.27
14 ± 0.83
14 ± 2.38
12 ± 1.10
13 ± 2.88
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
H
Cl
Cl
Cl
Cl
Cl
Br
Br
Br
Br
Br
F
F
F
F
F
CF3
CF3
CF3
CF3
CF3
H
Cyclohexyl
Phenyl
Benzyl
Phenethyl
Adamantan-1-yl
Cyclohexyl
Phenyl
1a
1b
1c
1d
1e
2a
2b
2c
2d
2e
3a
3b
3c
3d
3e
4a
4b
4c
4d
4e
5a
5b
5c
5d
5e
6a
6b
6c
6d
6e
Benzyl
Phenethyl
Adamantan-1-yl
Cyclohexyl
Phenyl
Benzyl
Phenethyl
Adamantan-1-yl
Cyclohexyl
Phenyl
Benzyl
Phenethyl
Adamantan-1-yl
Cyclohexyl
Phenyl
H
H
H
H
-
-
-
-
H
H
H
H
-
-
-
-
Benzyl
Phenethyl
Adamantan-1-yl
Cyclohexyl
Phenyl
Benzyl
Phenethyl
Adamantan-1-yl
-
-
9

0
0
INH
PAS
25 ± 4.1
3-nitropropionic acid
INH: isoniazid; PAS: p-aminosalicylic acid.
2.4. Cytotoxic and cytostatic properties evaluation
All of the carbamates 1-6 underwent determination of their in vitro cytotoxic and cytostatic action
on hepatocellular carcinoma cells (HepG2) using MTT assay. The HepG2 cell line serves as an in
vitro model for the hepatotoxicity for early drug screen. Liver tissue represents the most likely
target for the chronic toxicity of antitubercular drugs, which often complicates the six months long
therapy of TB.33 That is why we have chosen this cell line for selectivity indexes (SI) determination.
The same approach was used for our previously reported salicylanilide derivatives10,20,23,24,28 and
allows a direct comparison of different series.
In general, the cytotoxicity of salicylanilide-based molecules has been reported as an unwanted
effect of these potential antimicrobial agents.16 The evaluation of cytotoxicity allows calculating the
selectivity indexes, which are defined as a ratio of IC50 and MIC (cytotoxicity on HepG2 cells).
Analogously to the therapeutic index, SI values higher than 10 suggest an acceptable toxicity. There
were solubility problems with several derivatives preventing determination of IC50 values at higher
concentrations (especially 3b). Hence, the highest non-cytotoxic and concentrations with no
precipitation observed are reported.
The evaluated derivatives can be divided into several groups regarding their toxicities (Table 5).
First, the carbamates that are neither cytotoxic nor cytostatic at the concentration of 125 µM or
higher (all 4-chlorophenyl carbamates 6 and two adamantyl compounds 3e and 5e). Second, the
largest group is comprised of the derivatives with no cytotoxicity but with cytostatic properties (1a-
1c, 2a-2d, 3b, 3c, 4a, 4c-4e, 5c and 5d). It means that they do not kill the cell directly, although
they arrest cell growth, multiplication and proliferation. This cytostatic action together with no
cytotoxicity was reported for salicylanilide 4-substituted benzoates.28 The remaining molecules
exhibited both cytotoxic and cytostatic action for HepG2 cells, although at variable concentrations
(1d, 1e, 2e, 3a, 3d, 4b, 5a and 5b).
The most cytotoxic compound was the adamantyl derivative 2e (IC50 = 4.20 µM), on the other
hand, the less toxic carbamates shared IC50 higher than 250 µM (1a-1c, 5e). The majority of
halogenated salicylanilide carbamates caused a significant cytostasis in HepG2 cells (i.e., IC50 ≤10
µM; 1b-3b, 3d, 4a-4e), three of them with IC50 values of ≤1 µM (1b, 4a, 4d). The presence of
halogenated salicylanilide scaffold is translated mostly into enhanced cytostatic properties but not
cytotoxicity. Focusing on N-carbamic substitution, there is no clear structure-activity relationship.
From an alternative point of view, especially 2-[(4-bromophenyl)carbamoyl]-4-chlorophenyl
adamantan-1-ylcarbamate 2e may be considered as a potent cytotoxic and cytostatic agent with a
concomitant antibacterial, antifungal13 and antimycobacterial properties.
In term of SI, ten derivatives provided a sufficient selectivity for Mtb. (1a-1c, 3d-4e), even up to
>125 with 4a, 4d and 4e superiority. The adamantyl carbamate 4e was found as the most selective
one for NTM (SI >31.3), followed by 1c, 4a and 4d (SI >15.6), whereas dichlorocarbamates 1a and
1b were selective certainly only for M. avium. Moreover, these most selective derivatives were
comparably selective or even better than INH and PAS.
Table 5. Cytotoxic and cytostatic action of phenolic carbamates
IC50 [µM]
cytotoxicity
>250
IC50 [µM]
cytostasis
33.60±8.66
0.90±0.00
6.40±1.56
5.10±3.39
8.15±1.06
9.40±0.71
SI for Mtb.
SI for NTM
Code
R1
R2
R3
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Br
Cyclohexyl
Phenyl
Benzyl
Phenethyl
Adamantan-1-yl
Cyclohexyl
>7.8 (Mk), >15.6 (Ma)
>7.8 (Mk), >15.6 (Ma)
1a
1b
1c
1d
1e
2a
>15.6
>15.6
>15.6
1.9
1.5
>6.3
>250
>250
15.00
24.00
>15.6
>0.9
1.5-3
>3.1
>50
10

Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
H
H
H
H
H
-
Br
Br
Br
Br
F
F
F
F
F
CF3
CF3
CF3
CF3
CF3
H
H
H
H
H
Phenyl
Benzyl
Phenethyl
Adamantan-1-yl
Cyclohexyl
Phenyl
>50
>50
>50
5.45±0.07
1.65±0.64
2.20±1.27
4.35±0.07
8.60±1.84
3.25±1.34
34.15±12.37
7.60±0.85
>125
0.75±0.35
2.45±1.77
3.00±0.85
1.00±0.42
4.85±2.47
51.50
>6.3
>3.1
>6.3
0.5
>3.1
>3.1
>6.3
2b
2c
2d
2e
3a
3b
3c
3d
3e
4a
4b
4c
4d
4e
5a
5b
5c
5d
5e
4.20±0.99
21.20±4.67
>20
>125
41.50±4.95
>125
>125
9.80±3.54
>125
>125
>125
0.5-1
2.7-5.3
>2.5
>7.8
10.4
31.3
>125
9.8-19.6
>31.3
>125
>125
0.4
0.5
>1
>1
>2
>0.3
>0.3
>0.5
>0.5
>1
0.7-1.3
0.6-2.5
>2
1.3-5.2
>3.9
>15.6
1.2-9.8
>3.9
>15.6
>31.3
0.2-0.4
0.2-0.5
>0.5
>0.5
>0.5
>0.1
>0.1
>0.3
>0.3
>0.5
Benzyl
Phenethyl
Adamantan-1-yl
Cyclohexyl
Phenyl
Benzyl
Phenethyl
Adamantan-1-yl
Cyclohexyl
Phenyl
49.00±8.91
57.65±9.40
>125
26.80
54.30
55.60
Benzyl
Phenethyl
Adamantan-1-yl
Cyclohexyl
Phenyl
>125
>250
>125
>125
>125
>125
>125
>250
233.00±24.04
>125
-
-
-
-
-
-
-
6a
6b
6c
6d
6e
-
-
-
-
-
-
>125
>125
>250
>250
ND
ND
Benzyl
Phenethyl
Adamantan-1-yl
-
≥31.3 (Mk 6509/96)
<2.2-70
INH
PAS
>250
35.8
-
2,240
INH: isoniazid; PAS: p-aminosalicylic acid. SI = IC50 for cytotoxicity/MIC. SI values higher than 10 are given in bold. Mk = M. kansasii, Ma = M.
avium.
3. Conclusions
In this work, we completed the series of salicylanilide N-cycloalkyl/phenyl/phenylalkyl carbamates
and together with analogous 4-chlorophenyl carbamates, they were screened for their biological
activity. We assessed them against various mycobacterial strains (Mtb. including MDR- and XDR-
TB, broad-spectrum of NTM), for their cytotoxicity and cytostatic properties in liver cells model
and also inhibition of isocitrate lyase, one of the mycobacterial enzymes responsible for the
maintenance of persistent TB infection.
Minimum inhibitory concentrations for both tuberculous and nontuberculous mycobacteria were in
the micromolar concentration range and some of them were even submicromolar including activity
against drug-resistant Mtb. strains. Importantly, the carbamic acid derivatives do not share any
cross-resistance to clinically used anti-TB drugs. Their mechanism of action is multiple affecting
many cellular targets. Moreover, all the compounds inhibited isocitrate lyase mildly and the
majority of the carbamates were not cytotoxic, several derivatives also non-cytostatic. In sum, some
here presented carbamates may represent attractive antimycobacterial molecules for future
investigation, especially with respect to drug-resistant Mtb. and nontuberculous isoniazid-resistant
species, thus confirming carbamates as an auspicious scaffold in medicinal chemistry for combating
mycobacteria. Key mycobacterial target of the carbamates remains to be elucidated.
4. Material and Methods
4.1 Chemistry
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 they were used as received. The reactions and the
purity of the products were monitored by the thin layer-chromatography using a mixture with a ratio
of toluene to ethyl acetate 4:1 (v/v) as the eluent. Plates were coated with 0.2 mm Merck 60 F254
silica gel (Merck Millipore, Darmstadt, Germany) and were visualised by UV irradiation (254 nm).
11

The melting points were determined on a Büchi Melting Point B-540 apparatus (BÜCHI, Flawil,
Switzerland) using open capillaries, and the reported values are uncorrected. Elemental analysis (C,
H, 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
(ThermoFisher Scientific, Waltham, MA, USA) in the range of 650-4000 cm-1. The NMR spectra
were measured in DMSO-d6 or THF-d8 at ambient temperature using a Varian V NMR S500
1
13
instrument (500 MHz for H and 126 MHz for C; Varian Comp. Palo Alto, CA, USA). The
chemical shifts, δ, are given in ppm with respect to tetramethylsilane used as an internal standard.
The coupling constants (J) are reported in Hz.
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 18.0 (Cambridge-
Soft, Cambridge, MA, USA).
4.1.2 Synthesis
Salicylanilides were prepared via a previously reported microwave-assisted method.26 The synthesis
of the dihalogenated carbamates 1-413 and 4-chlorophenyl carbamates 615 was also already
published by our group.
The carbamates 5 were synthesised according to the ref.13 5-Chloro-2-hydroxy-N-[4-
(trifluoromethyl)phenyl]benzamide (1 mmol, 315.7 mg) was suspended under vigorous stirring in
dry acetonitrile (MeCN; 8 mL), and then triethylamine (1 equivalent, 139.4 ꢀL) was added in one
portion. The mixture was stirred to allow complete dissolution of the salicylanilide. Then,
appropriate isocyanate (1.1 of equivalents) was added when divided into two portions, second
portion after 15 minutes, and the mixture was stirred at room temperature for additional 2 h
(cyclohexyl, phenyl, benzyl and phenethyl isocyanates) or it was refluxed for 8 h (adamantan-1-yl
isocyanate). After this time, the resulting precipitate was collected by filtration, washed with a small
volume of cold methanol and dried. The products were recrystallized from ethyl acetate.
4-Chloro-2-{[4-(trifluoromethyl)phenyl]carbamoyl}phenyl cyclohexylcarbamate (4a)
White solid; yield 94%; mp 215-217 °C. IR (ATR): 3261 (N-H), 2930, 2857, 1710 (C=O
carbamate), 1659 (amide I), 1602, 1552, 1529 (amide II), 1479, 1410, 1395, 1318, 1270, 1222,
1
1178, 1166, 1116, 1106, 1065, 1017, 883, 836, 743, 728, 699, 666 cm-1. H NMR (500 MHz,
DMSO): δ 10.63 (1H, bs, amide NH), 7.95-7.87 (3H, m, carbamate NH, H2´, H6´), 7.77-7.66 (3H,
m, H3, H3´, H5´), 7.58 (1H, dd, J = 8.7 Hz, J = 2.7 Hz, H5), 7.25 (1H, d, J = 8.7 Hz, H6), 3.24-3.14
13
(1H, m, CH), 1.74-1.45 (5H, m, cyclohexyl), 1.28-0.97 (5H, m, cyclohexyl). C NMR (126 MHz,
DMSO): δ 165.28, 152.87, 147.49, 142.81, 131.84, 131.21, 129.07, 128.81, 126.16 (q, J = 3.8 Hz),
125.34, 124.04 (q, J = 271.7 Hz), 123.86 (q, J = 32.8 Hz), 120.53, 119.75, 50.06, 32.42, 24.84,
24.61. Anal. Calcd. for C21H20ClF3N2O3 (440.84): C, 57.21; H, 4.57; N, 6.35. Found: C, 57.13; H,
4.80; N, 6.27.
4-Chloro-2-{[4-(trifluoromethyl)phenyl]carbamoyl}phenyl phenylcarbamate (4b)
White solid; yield 70%; mp 202.5-205 °C. IR (ATR): 3313 (N-H), 3281 (N-H), 1722 (C=O
carbamate), 1663 (amide I), 1602, 1536 (amide II), 1511, 1478, 1446, 1405, 1330, 1264, 1224,
1
1207, 1184, 1160, 1116, 1102, 1066, 1015, 840, 833, 763, 745, 708, 691, 664 cm-1. H NMR (500
MHz, DMSO): δ 10.82 (1H, bs, amide NH), 10.28 (1H, bs, carbamate NH), 7.89 (2H, d, J = 8.5 Hz,
H2´, H6´), 7.80 (1H, d, J = 2.6 Hz, H3), 7.72-7.63 (3H, m, H5, H3´, H5´), 7.47-7.39 (3H, m, H6,
13
H2´´, H6´´), 7.27 (2H, t, J = 7.9 Hz, H3´´, H5´´), 7.01 (1H, t, J = 7.3 Hz, H4´´). C NMR (126
MHz, DMSO): δ 165.28, 163.61, 151.16, 146.99, 139.91, 133.24, 131.49, 129.74, 129.02, 128.84,
128.77, 126.20 (q, J = 3.7 Hz), 124.02 (d, J = 32.0 Hz), 124.10 (q, J = 271.4 Hz), 121.99, 120.60,
118.37. Anal. Calcd. for C21H14ClF3N2O3 (434.80): C, 58.01; H, 3.25; N, 6.44. Found: C, 58.12; H,
3.14; N, 6.39.
4-Chloro-2-{[4-(trifluoromethyl)phenyl]carbamoyl}phenyl benzylcarbamate (4c)
12

White solid; yield 72%; mp 188.5-190 °C. IR (ATR): 3321 (N-H), 3032, 2926, 2874, 1713 (C=O
carbamate), 1665 (amide I), 1626, 1610, 1573, 1541, 1525 (amide II), 1474, 1454, 1338, 1327,
1257, 1219, 1115, 1070, 1020, 837, 752, 730, 697, 659 cm-1. 1H NMR (500 MHz, DMSO): δ 10.74
(1H, bs, amide NH), 8.37 (1H, t, J = 6.2 Hz, carbamate NH), 7.92 (2H, d, J = 8.5 Hz, H2´, H6´),
7.76-7.69 (3H, m, H3, H3´, H5´), 7.61 (1H, dd, J = 8.7 Hz, J = 2.7 Hz, H5), 7.31 (1H, d, J = 8.7 Hz,
13
H6), 7.25-7.15 (5H, m, H2´´, H3´´, H4´´, H5´´, H6´´), 4.21 (2H, d, J = 6.2 Hz, N-CH2). C NMR
(126 MHz, DMSO): δ 163.67, 154.15, 147.37, 142.77, 139.14, 131.91, 131.28, 129.34, 128.59,
128.28, 126.95, 126.88, 126.14 (q, J = 3.9 Hz), 125.49, 124.56 (q, J = 271.2 Hz), 123.89 (q, J =
32.0 Hz), 119.86, 44.04. Anal. Calcd. for C22H16ClF3N2O3 (448.82): C, 58.87; H, 3.59; N, 6.24.
Found: C, 58.95; H, 3.47; N, 6.30.
4-Chloro-2-{[4-(trifluoromethyl)phenyl]carbamoyl}phenyl phenethylcarbamate (4d)
White solid; yield 90%; mp 160.5-162 °C. IR (ATR): 3336 (N-H), 3280 (N-H), 2929, 1717 (C=O
carbamate), 1662 (amide I), 1603, 1573, 1537, 1524 (amide II), 1513, 1476, 1409, 1327, 1265,
1
1215, 1165, 1123, 1067, 1021, 926, 836, 748, 738, 700, 662 cm-1. H NMR (500 MHz, DMSO): δ
10.70 (1H, bs, amide NH), 7.96-7.90 (3H, m, NH carbamate, H2´, H6´), 7.73-7.68 (3H, m, H3, H3´,
H5´), 7.59 (1H, dd, J = 8.7 Hz, J = 2.7 Hz, H5), 7.27-7.10 (6H, m, H6, H2´´, H3´´, H4´´, H5´´,
13
H6´´), 3.20 (2H, dt, J = 7.9 Hz, J = 6.2 Hz, N-CH2), 2.66 (2H, t, J = 7.4 Hz, CH2-Ph). C NMR
(126 MHz, DMSO): δ 163.65, 153.66, 147.30, 142.77, 139.26, 131.80, 131.15, 129.17, 128.77,
128.51, 128.40, 126.21, 126.15 (q, J = 3.6 Hz), 125.42, 124.53 (q, J = 271.2 Hz), 123.91 (q, J =
32.0 Hz), 119.83, 42.23, 35.23. Anal. Calcd. for C23H18ClF3N2O3 (462.85): C, 59.68; H, 3.92; N,
5.68. Found: C, 59.90; H, 3.87; N, 5.77.
4-Chloro-2-{[4-(trifluoromethyl)phenyl]carbamoyl}phenyl adamantan-1-ylcarbamate (4e)
White solid; yield 71%; mp 242-244.5 °C (decomp.). IR (ATR): 3303 (N-H), 2911, 2853, 1717
(C=O carbamate), 1668 (amide I), 1603, 1540, 1522 (amide II), 1508, 1475, 1457, 1381, 1356,
1343, 1319, 1214, 1188, 1163, 1107, 1065, 1017, 923, 854, 837, 750, 702, 665 cm-1. 1H NMR (500
MHz, THF): δ 10.40 (1H, bs, NH), 7.99 (1H, d, J = 2.7 Hz, H3), 7.94 (2H, d, J = 8.5 Hz, H2´, H6´),
7.69-7.65 (3H, m, NH carbamate, H2´, H6´), 7.40 (1H, dd, J = 8.7 Hz, J = 2.6 Hz, H5), 6.95 (1H, d,
13
J = 8.9 Hz, H6), 1.91-1.89 (3H, m, CH), 1.74 (6H, s, C-CH2), 1.70-1.63 (6H, m, CH-CH2). C
NMR (126 MHz, DMSO): δ 165.26, 152.56, 147.05, 142.64, 131.81, 131.15, 129.01, 128.83,
126.23 (q, J = 3.8 Hz), 125.37, 124.48 (q, J = 271.4 Hz), 123.92 (q, J = 32.4 Hz), 120.49, 119.64,
50.43, 41.00, 36.05, 28.92. Anal. Calcd. for C25H24ClF3N2O3 (492.92): C, 60.92; H, 4.91; N, 5.68.
Found: C, 61.01; H, 4.98; N, 5.33.
4.2 Biological activity
4.2.1 Antimycobacterial activity evaluation
4.2.1.1 Activity against M. tuberculosis H37Rv, M. avium and M. kansasii
The in vitro antimycobacterial activity of the carbamates 1-6 against Mycobacterium tuberculosis
331/88 (H37Rv) and three strains of nontuberculous mycobacteria (Mycobacterium avium 330/88
[resistant to isoniazid, rifamycines, ofloxacin, and ethambutol], Mycobacterium kansasii 235/80
from the Czech National Collection of Type Cultures, and a clinical isolate of M. kansasii 6509/96)
was evaluated using a previously reported method.23 The micromethod for the determination of
MIC was used involving the Šula’s semisynthetic medium (SEVAC, Prague, Czech Republic). The
investigated compounds were added to the medium as solutions in DMSO; the final volume
contained 1.0 % DMSO (v/v). The following concentrations were used: 1000, 500, 250, 125, 62.5,
32, 16, 8, 4, 2, 1 and 0.5 ꢀM. The MIC were determined after incubation at 37 °C for 14 and 21
days, for M. kansasii additionally for 7 days. MIC (in ꢀM) was the lowest concentration at which
the complete inhibition of mycobacterial growth occurred. Two oral antitubercular drugs, isoniazid
(INH) and p-aminosalicylic acid (PAS), were involved as reference drugs.
The most active carbamates 4 (4a, 4b, 4d, 4e) were evaluated against seven drug-resistant TB
strains (dilution of the strains was 10-3) with different resistance patterns. All of the strains are
13

resistant to INH, RIF, rifabutin, and streptomycin, and resistance to other drugs was observed in
some cases: strain 7357/1998 was resistant additionally to ethambutol and ofloxacin, strain
234/2005 to ethambutol, strain 8666/2010 resistant to ethambutol, ofloxacin and clofazimine, strain
Praha 1 exhibited an additional resistance to ethambutol and clofazimine; Praha 4 to ethambutol,
ofloxacin and clofazimine (all MDR-TB strains); and Praha 131 was resistant to INH, rifamycines,
streptomycin, ethambutol, ofloxacin, gentamicin and amikacin (i.e., XDR-TB strain). The following
concentrations were used for drug-resistant strains: 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.125, 0.064, and
0.032 ꢀM.
4.2.1.2 Activity against M. tuberculosis H37Ra, M. aurum and M. smegmatis
Antimycobacterial assay was performed with fast growing Mycobacterium smegmatis DSM 43465
(ATCC 607), Mycobacterium aurum DSM 43999 (ATCC 23366) from German Collection of
Microorganisms and Cell Cultures (Braunschweig, Germany) and with avirulent strain of
Mycobacterium tuberculosis H37Ra ITM-M006710 (ATCC 9431) from Belgian Co-ordinated
Collections of Micro-organisms. The technique used for activity determination was microdilution
broth panel method using 96-well microtitration plates. Culturing medium was Middlebrook 7H9
broth (Sigma-Aldrich, Steinheim, Germany) enriched with 0.4% of glycerol (Sigma-Aldrich,
Steinheim, Germany) and 10% of Middlebrook OADC growth supplement (Himedia, Mumbai,
India).
Mycobacterial strains were cultured on Middlebrook 7H9 agar and suspensions were prepared in
Middlebrook 7H9 broth. Final density was adjusted to value 1.0 according to McFarland scale and
diluted in ratio 1:20 (for fast growing mycobacteria) or 1:10 (for M. tuberculosis) with broth.
Tested compounds were dissolved in DMSO (Sigma-Aldrich, Steinheim, Germany) then MB broth
was added to obtain concentration 2000 µg/mL. Standards used for activity determination were
isoniazid (INH), rifampicin (RIF) and ethambutol (EMB) (Sigma-Aldrich, Steinheim, Germany).
Final concentrations were reached by binary dilution and addition of mycobacterial suspension and
were set at 500, 250, 125, 62.5, 31.25, 15.625, 7.81 and 3.91 µg/mL or 50, 25, 12.5, 6.25, 3.13,
1.56, 0.78 and 0.39 µg/mL according to final MIC. Isoniazid was diluted in range 500-3.91 µg/mL
for screening against fast growing mycobacteria and in range 1-0.0078 µg/mL for screening against
M. tuberculosis. Rifampicin final concentrations ranged from 50 to 0.39 µg/mL for fast growing
mycobacteria and from 1 to 0.0039 µg/mL for M. tuberculosis. Ethambutol was used for screening
antimycobacterial activity with the final concentrations 2, 1, 0.5, 0.25, 0.125, 0.0625, 0.0313,
0.0156 µg/mL. The final concentration of DMSO did not exceeded 2.5% (v/v) and did not affect the
growth of M. smegmatis, M. aurum nor M. tuberculosis. Positive (broth, DMSO, bacteria) and
negative (broth, DMSO) controls were included.
Plates were sealed with polyester adhesive film and incubated in dark at 37 °C without agitation.
The addition of 0.01% solution of resazurin sodium salt followed after 48 hours of incubation for
M. smegmatis, 72 hours of incubation for M. aurum and after 120 hours of incubation for M.
tuberculosis, respectively. Stain was prepared by dissolving resazurin sodium salt (Sigma-Aldrich,
Steinheim, Germany) in deionised water to get 0.02% solution. Then 10% aqueous solution of
Tween 80 (Sigma-Aldrich, Steinheim, Germany) was prepared. Both liquids were mixed up making
use of the same volumes and filtered through syringe membrane filter. Microtitration panels were
then incubated for further 2.5 hours for determination of activity against M. smegmatis, 4 hours for
M. aurum and 24 hours for M. tuberculosis, respectively.
Antimycobacterial activity was expressed as minimal inhibition concentration (MIC) and the value
was read on the basis of stain colour change (blue colour – active compound; pink colour – not
active compound). MIC values for standards were 15.625 µg/mL for INH, 12.5-25 µg/mL for RIF
and 0.5 µg/mL for EMB against M. smegmatis, and 3.91 µg/mL for INH, 0.39 µg/mL for RIF and
0.25 µg/mL for EMB against M. aurum, and 0.125-0.25 µg/mL for INH, 0.0039-0.0078 µg/mL for
RIF and 0.5 µg/mL for EMB against M. tuberculosis, respectively. All experiments were conducted
in duplicate.
14

4.2.2 Isocitrate lyase inhibition determination
The isocitrate lyase (ICL1) inhibition activity was assayed according to Dixon and Kornberg
procedure (glyoxylate phenyl hydrazone formation) using a single concentration of the carbamates
1-6 of 10 µM.23 Results are expressed as inhibition rates [%] and the data represent an average of
triplicate experiments ± standard deviation (SD). Isoniazid and p-aminosalicylic acid were
employed as negative controls (inhibition of 0%), while 3-nitropropionic acid served as a positive
control.
4.2.3 Cytostatic and cytotoxic activity determination
HepG2, human hepatocellular carcinoma cells (ATCC HB-8065) were cultured in RPMI-1640
medium containing 10% fetal calf serum (FCS), 2 mM L-glutamine, 160 µg/mL gentamicin at
37 °C, 5% CO2 in water-saturated atmosphere and grown to confluence and were plated into 96-
well plate with initial cell number of 5.0-10.0 × 103 per well. After 24 h incubation at 37 °C, cells
were treated with the compounds in 100 ꢀL final volume containing 2.0 % DMSO (v/v). Cells were
incubated with the compounds at 0.0032-250 µM concentration range for overnight. Control cells
were treated with serum free medium (RPMI-1640) only or with DMSO (c = 1.0 v/v %) at 37 °C
for overnight. After incubation, the cells were washed twice with serum free medium (RPMI-1640).
To determine the in vitro cytostatic effect, cells were cultured for a further 72 h in medium
containing serum. To measure the in vitro cytotoxicity of the compounds, MTT-assay was carried
out immediately after the washing step following the overnight treatment.
The cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) assay. The solution of MTT (45 ꢀL, 2 mg/mL) was added to each well, which was reduced
to insoluble violet formazan dye crystals within the living cells. After 3.5 h of incubation at 37 °C
the cells were centrifuged for 5 min (2000 rpm) and supernatant was removed. The obtained
formazan crystals were dissolved in 100 ꢀL of DMSO and the optical density (OD) of the samples
was measured at λ = 540 and 620 nm, respectively, employing ELISA Reader instrument (iEMS
Reader, Labsystems, Finland). OD620 values were subtracted from OD540 values and the percent of
cytostasis or cytotoxicity was calculated using equation “Cytostatic effect/Cytotoxicity (%) = [1 −
(ODtreated/ODcontrol)] × 100” (where ODtreated and ODcontrol correspond to the optical densities of the
treated and the control cells, respectively). For each compound, at least two independent
experiments were carried out with four parallel measurements.
The 50% inhibitory concentration (IC50) values were determined from the dose-response curves.
The curves were defined using MicrocalTM Origin1 (version 7.6) software: cytostasis (%) or
cytotoxicity (%) was plotted as a function of concentration, fitted to a sigmoidal curve and, based
on this curve, the half maximal inhibitory concentration (IC50) value was determined representing
the concentration of a compound required for 50% inhibition in vitro.
Acknowledgements
This work was supported by the Czech Science Foundation [grant number 17-27514Y]; by the
project EFSA-CDN (No. CZ.02.1.01/0.0/0.0/16_019/0000841) co-funded by ERDF; the COST
action [CA15135 (Multitarget Paradigm for Innovative Ligand Identification in the Drug Discovery
Process MuTaLig)] and the Hungarian Research Fund [grant number OTKA K-104275, Ministry of
Human Capacities (1783-3/2018/FEKUTSRAT)].
We would like to thank the staff of the Department of Organic and Bioorganic Chemistry, Faculty
of Pharmacy in Hradec Králové for the technical assistance; Pavel Mašek and Jaroslava Urbanová
for the language assistance provided.
Conflict of Interest
The authors declare no conflict of interest.
References
15

1
Global
Tuberculosis
Report
2018.
http://apps.who.int/iris/bitstream/handle/10665/274453/9789241565646-eng.pdf?ua=1 (accessed 14
November 2018).
2
J. van Ingen, M.J. Boeree, D. van Soolingen, J.W. Mouton, Resistance mechanisms and drug
susceptibility testing of nontuberculous mycobacteria, Drug Resist. Update 15 (2012) 149–161.
3
M.J. Nasiri, M. Haeili, M. Ghazi, H. Goudarzi, A. Pormohammad, A.A.I. Fooladi, M.M.
Feizabadi, New Insights in to the Intrinsic and Acquired Drug Resistance Mechanisms in
Mycobacteria, Front. Microbiol. 8 (2017) 681.
4 G. Sood, N. Parrish, Outbreaks of nontuberculous mycobacteria, Curr. Opin. Infect. Dis. 30 (2017)
404–409.
5
A.K. Ghosh, M. Brindisi, Organic Carbamates in Drug Design and Medicinal Chemistry, J. Med.
Chem. 58 (2015) 2895–2940.
6
J.M. Férriz, J. Vinšová, Prodrug Design of Phenolic Drugs, Curr. Pharm. Des. 16 (2010) 2033–
2052.
7
J.M. Férriz, K. Vávrová, F. Kunc, A. Imramovský, J. Stolaříková, E. Vavříková, J. Vinšová,
Salicylanilide carbamates: Antitubercular agents active against multidrug-resistant Mycobacterium
tuberculosis strains, Bioorg. Med. Chem. 18 (2010) 1054–1061.
8
C. Gomez, P. Ponien, N. Serradji, A. Lamouri, A. Pantel, E. Capton, V. Jarlier, G. Anquetin, A.
Aubry, Synthesis of gatifloxacin derivatives and their biological activities against Mycobacterium
leprae and Mycobacterium tuberculosis, Bioorg. Med. Chem. 21 (2013) 948–956.
9 Z. Baranyai, M. Krátký, J. Vinšová, N. Szabó, Z. Senoner, K. Horváti, J. Stolaříková, S. Dávid, S.
Bősze, Combating highly resistant emerging pathogen Mycobacterium abscessus and
Mycobacterium tuberculosis with novel salicylanilide esters and carbamates, Eur. J. Med. Chem.
101 (2015) 692–704.
10
M. Krátký, M. Volková, E. Novotná, F. Trejtnar, J. Stolaříková, J. Vinšová, Synthesis and
biological activity of new salicylanilide N,N-disubstituted carbamates and thiocarbamates, Bioorg.
Med. Chem. 22 (2014) 4073–4082.
11 P.P. de Castro, D.L. Campos, F.R. Pavan, G.W. Amarante, Dual-protected amino acid derivatives
as new antitubercular agents, Chem. Biol. Drug Des. 92 (2018) 1576–1580.
12
Y.G. Kang, C.Y. Park, H. Shin, R. Singh, G. Arora, C. Yu, I.Y. Lee, Synthesis and anti-
tubercular activity of 2-nitroimidazooxazines with modification at the C-7 position as PA-824
analogs, Bioorg. Med. Chem. Lett. 25 (2015) 3650–3653.
13
M. Krátký, J. Vinšová, Salicylanilide N-monosubstituted carbamates: Synthesis and in vitro
antimicrobial activity, Bioorg. Med. Chem. 24 (2016) 1322–1330.
14
M. Krátký, Š. Štěpánková, K. Vorčáková, M. Švarcová, J. Vinšová, Novel Cholinesterase
Inhibitors Based on O-Aromatic N,N-Disubstituted Carbamates and Thiocarbamates, Molecules 21
(2016) 191.
15
M. Krátký, Š. Štěpánková, K. Vorčáková, J. Vinšová, Investigation of salicylanilide and 4-
chlorophenol-based N-monosubstituted carbamates as potential inhibitors of acetyl- and
butyrylcholinesterase, Bioorg. Chem. 80 (2018) 668–673.
16
M. Krátký, J. Vinšová, Salicylanilide Ester Prodrugs as Potential Antimicrobial Agents – a
Review, Curr. Pharm. Des. 17 (2011) 3494–3505.
17
G. Triola, S. Wetzel, B. Ellinger, M.A. Koch, K. Hübel, 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.
18
T.J.R. 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.E. 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.
19
W.S. Wu, W.C. Cheng, T.J.R. Cheng, C.H. Wong, Affinity-Based Screen for Inhibitors of
Bacterial Transglycosylase, J. Am. Chem. Soc. 140 (2018) 2752–2755.
16

20
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 92
(2012) 434–439.
21
M. Krátký, E. Novotná, S. Saxena, P. Yogeeswari, D. Sriram, M. Švarcová, J. Vinšová,
Salicylanilide Diethyl Phosphates as Potential Inhibitors of Some Mycobacterial Enzymes, Sci.
World J. 2014 (2014) 703053.
22
I.Y. Lee, T.D. Gruber, A. Samuels, M. Yun, B. Nam, M. Kang, K. Crowley, B. Winterroth, H.I.
Boshoff, C.E. Barry, Structure–activity relationships of antitubercular salicylanilides consistent
with disruption of the proton gradient via proton shuttling, Bioorg. Med. Chem. 21 (2013) 114–126.
23
M. Krátký, J. Vinšová, E. Novotná, J. Mandíková, F. Trejtnar, J. Stolaříková, Antibacterial
Activity of Salicylanilide 4-(Trifluoromethyl)benzoates, Molecules 18 (2013) 3674–3688.
24
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.
25
M. Krátký, J. Vinšová, Advances in Mycobacterial Isocitrate Lyase Targeting and Inhibitors,
Curr. Med. Chem. 19 (2012) 6126–6137.
26
M. Krátký, J. Vinšová, Antifungal Activity of Salicylanilides and Their Esters with 4-
(Trifluoromethyl)benzoic Acid, Molecules 17 (2012) 9426–9442.
27
M. Krátký, J. Vinšová, J. Stolaříková, Antimycobacterial assessment of salicylanilide benzoates
including multidrug-resistant tuberculosis strains, Molecules 17 (2012) 12812–12820.
28
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. 24 (2015) 868–875.
29
D. Addla, A. Jallapally, D. Gurram, P. Yogeeswari, D. Sriram, S. Kantevari, Design, synthesis
and evaluation of 1,2,3-triazole-adamantylacetamide hybrids as potent inhibitors of Mycobacterium
tuberculosis, Bioorg. Med. Chem. Lett. 24 (2014) 1974–1979.
30
R.P. Bhusal, G. Bashiri, B.X.C. Kwai, J. Sperry, I.K.H. Leung, Targeting isocitrate lyase for the
treatment of latent tuberculosis, Drug Discov. Today 22 (2017) 1008–1016.
31
M. Nandakumar, C. Nathan, K.Y. Rhee, Isocitrate lyase mediates broad antibiotic tolerance in
Mycobacterium tuberculosis, Nat. Commun. 5 (2014) 4306.
32
Y. Liu, S. Zhou, Q. Deng, X. Li, J. Meng, Y. Guan, C. Li, C. Xiao, Identification of a novel
inhibitor of isocitrate lyase as a potent antitubercular agent against both active and non-replicating
Mycobacterium tuberculosis, Tuberculosis 97 (2016) 38–46.
33
B.E. Senousy, S.I. Belal, P.V. Draganov, Hepatotoxic effects of therapies for tuberculosis, Nat.
Rev. Gastroenterol. Hepatol. 7 (2010) 543-556.
17

Highlights
•Salicylanilide N-monosubstituted carbamates are highly active against mycobacteria.
•MIC against multidrug-resistant mycobacteria are from ≤0.5 ꢀM.
•Salicylanilide carbamates are mild inhibitors of mycobacterial isocitrate lyase.
•The majority of carbamates are non-cytotoxic and selective for mycobacteria.