Synthesis and biological activity of new salicylanilide N,N-disubstituted carbamates and thiocarbamates

Article abstract of DOI:10.1016/j.bmc.2014.05.064

The development of novel antimicrobial drugs represents a cutting edge research topic. In this study, 20 salicylanilide N,N-disubstituted carbamates and thiocarbamates were designed, synthesised and characterised by IR, ¹H NMR and ¹³C NMR. The compounds were evaluated in vitro as potential antimicrobial agents against Mycobacterium tuberculosis and nontuberculous mycobacteria (Mycobacterium avium and Mycobacterium kansasii) as well as against eight bacterial and fungal strains. Additionally, we investigated the inhibitory effect of these compounds on mycobacterial isocitrate lyase and cellular toxicity. The minimum inhibitory concentrations (MICs) against mycobacteria were from 4 μM for thiocarbamates and from 16 μM for carbamates. Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus, were inhibited with MICs from 0.49 μM by thiocarbamates, whilst Gram-negative bacteria and most of the fungi did not display any significant susceptibility. All (thio)carbamates mildly inhibited isocitrate lyase (up to 22%) at a concentration of 10 μM. The (thio)carbamoylation of the parent salicylanilides led to considerably decreased cytotoxicity and thus improved the selectivity indices (up to 175). These values indicate that some derivatives are attractive candidates for future research.

Full text of DOI:10.1016/j.bmc.2014.05.064

Accepted Manuscript
Synthesis and Biological Activity of New Salicylanilide N,N-Disubstituted Car-
bamates and Thiocarbamates
Martin Krátký, Marie Volková, Eva Novotná, František Trejtnar, Jiřina Stolař
íková, Jarmila Vinšová
PII:
S0968-0896(14)00423-4
DOI:
http://dx.doi.org/10.1016/j.bmc.2014.05.064
BMC 11625
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
22 April 2014
25 May 2014
28 May 2014
Please cite this article as: Krátký, M., Volková, M., Novotná, E., Trejtnar, F., Stolař íková, J., Vinšová, J., Synthesis
and Biological Activity of New Salicylanilide N,N-Disubstituted Carbamates and Thiocarbamates, Bioorganic &
Medicinal Chemistry (2014), doi: http://dx.doi.org/10.1016/j.bmc.2014.05.064
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Synthesis and Biological Activity of New Salicylanilide N,N-Disubstituted
Carbamates and Thiocarbamates
Martin Krátký1, Marie Volková2, Eva Novotná3, František Trejtnar2, Jiřina Stolaříková4 and
Jarmila Vinšová1,*
1
Department of Inorganic and Organic Chemistry, Faculty of Pharmacy, Charles University,
Heyrovského
martin.kratky@faf.cuni.cz (M.K.); jarmila.vinsova@faf.cuni.cz (J.V.)
Department of Pharmacology and Toxicology, Faculty of Pharmacy, Charles University,
Heyrovského 1203, 500 05 Hradec Králové, Czech Republic; E-Mails:
marie.volkova@faf.cuni.cz (M.V.); frantisek.trejtnar@faf.cuni.cz (F.T.)
Department of Biochemical Sciences, Faculty of Pharmacy, Charles University, Heyrovského
1203, 500 05 Hradec Králové, Czech Republic; E-Mail: eva.novotna@faf.cuni.cz
Laboratory for Mycobacterial Diagnostics and Tuberculosis, Regional Institute of Public Health
in Ostrava, Partyzánské náměstí 7, 702 00 Ostrava, Czech Republic; E-Mail:
Jirina.Stolarikova@zu.cz
1203,
500 05
Hradec
Králové,
Czech
Republic;
E-Mails:
2
3
4
* 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
The development of novel antimicrobial drugs represents a cutting edge research topic. In this
study, 20 salicylanilide N,N-disubstituted carbamates and thiocarbamates were designed,
1
synthesised and characterised by IR, H NMR and 13C NMR. The compounds were evaluated in
vitro as potential antimicrobial agents against Mycobacterium tuberculosis and nontuberculous
mycobacteria (M. avium and M. kansasii) as well as against eight bacterial and fungal strains.
Additionally, we investigated the inhibitory effect of these compounds on mycobacterial isocitrate
lyase and cellular toxicity. The minimum inhibitory concentrations (MICs) against mycobacteria
were from 4 µM for thiocarbamates and from 16 µM for carbamates. Gram-positive bacteria,
including methicillin-resistant Staphylococcus aureus, were inhibited with MICs from 0.49 µM by
thiocarbamates, whilst Gram-negative bacteria and most of the fungi did not display any significant
susceptibility. All (thio)carbamates mildly inhibited isocitrate lyase (up to 22%) at a concentration
of 10 µM. The (thio)carbamoylation of the parent salicylanilides led to considerably decreased
cytotoxicity and thus improved the selectivity indices (up to 175). These values indicate that some
derivatives are attractive candidates for future research.
Keywords
antimicrobial activity; antimycobacterial activity; cytotoxicity; isocitrate lyase inhibition;
salicylanilide carbamate; salicylanilide thiocarbamate

1. Introduction
In the 1970s, tuberculosis (TB) was thought to have been nearly eradicated; however, it is currently
one of the most deadly infectious diseases in the world. The main reasons for this unpleasant reality
are co-infection with HIV and the development of drug resistance. It is estimated that 5% of the
more than 9 million people who develop TB annually are infected with multidrug-resistant
tuberculosis (MDR-TB), i.e., a strain that is resistant to at least isoniazid (isonicotinohydrazide,
INH; Fig. 1) and rifampicin (RIF). Other drug-resistant forms of tuberculosis have been described
and defined. Many reasons and risk factors promoting the development of drug resistance have been
proposed and explored. The treatment of resistant TB is more complicated. In 2006, the WHO
declared an ambitious global plan to eradicate TB by the year 2050. This plan should involve
increased research efforts including the development and testing of novel potential
antimycobacterial drugs and/or vaccines.1
Nontuberculous (atypical) mycobacteria are increasingly recognised as causative agents of various
opportunistic human infections. In general, their drug treatment is long and complicated due to
additional factors including the high levels of natural and acquired antibiotic resistance conferred by
a range of mechanisms. The current treatment regimens for diseases caused by nontuberculous
mycobacteria share limited efficacy.2
Similar to mycobacterial species, problems with drug-resistance have been reported for many other
bacterial strains, although it is possible to observe particular amelioration in some cases.
Methicillin-resistant Staphylococcus aureus (MRSA), Streptococcus pneumoniae, enterococci,
Pseudomonas aeruginosa or the family of Enterobacteriaceae that produce extended-spectrum β-
lactamases (ESBL) represent common pathogens with problematic drug susceptibility or
nosocomial strains.3 Mycoses account for the most frequent infections in humans, and they have
become an important public health issue because of the increasing number of immunocompromised
patients. The development of drug resistance also complicates therapy.4
The reported facts justify an intensive future search for antimicrobial chemotherapeutics, especially
those with unique mechanisms of action without any cross-resistance to approved and clinically
established drugs. The development of those antibiotic agents should also overcome organisational,
economic and marketing obstacles.
Some O-aromatic carbamates and thiocarbamates have been reported as potential antimicrobial
agents, in some cases with excellent activity. For example, tolnaftate and tolciclate (Fig. 1), two
thiocarbamate inhibitors of squalene epoxidase, represent clinically used antifungal agents against
dermatophytes.5,6 The analogues of the antituberculosis drug, PA-824, in which the OCH2 linkage is
replaced with carbamate functionality, exhibited more favourable properties7; the introduction of
the N,N-dimethylthiocarbamoyl moiety into the aromatic molecule of aureol substantially increased
its antimycobacterial potency.8 In addition, carbamates and thiocarbamates represent scaffolds that
are often incorporated in the prodrug design including those oriented on antimicrobial drugs.9-13 O-
substituted salicylanilides have exhibited significant antimicrobial activity against mycobacteria,
Gram-positive bacteria and moulds; however, they share pronounced cytotoxicity.14
The main impetus for this study was the work of Férriz et al.,15 which reported promising in vitro
activity of chlorinated salicylanilide N-alkyl carbamates with minimum inhibitory concentrations
(MICs) against both drug-sensitive and MDR M. tuberculosis within the range of 0.5 to 4 µM;
nontuberculous mycobacteria were inhibited within the range of 2 to 32 µM. Moreover, they
displayed acceptable cytotoxicity and stability profiles. It was suggested that the lipophilicity of the
carbamates plays an important role in the biological activity of these compounds.
Various benzanilides, salicylanilides and their esters have been described as being mostly mild
inhibitors of mycobacterial isocitrate lyase (ICL); some of these compounds were comparable or
superior to 3-nitropropionic acid (3-NP), a known ICL inhibitor.16-18 ICL represents one of the
glyoxylate-shunt-pathway enzymes, which is considered to be an attractive drug target especially
for persistent mycobacteria with an assumed potential to shorten and simplify the TB treatment. In
particular, compounds with dual activity against actively growing and non-replicating
mycobacterial subpopulations should be more beneficial.

Based on presented findings, we designed and evaluated a series of salicylanilide N,N-dialkyl/aryl
carbamates and N,N-dimethylthiocarbamates to determine the influence of N,N-disubstitution by
various alkyls/aryls and thionation on antimicrobial activity, cytotoxicity and isocitrate lyase
inhibition.
2. Results and discussion
2.1 Chemistry
Parent salicylanilides (SAL-1-SAL-5) were synthesised from salicylic and 5-chlorosalicylic acids
and appropriate anilines using microwave irradiation and phosphorus trichloride via a previously
described method.19 Based on previously reported structure-activity relationships,14,15,17 we selected
salicylanilides derived from 5-chlorosalicylic acid and 4-substituted anilines; unsubstituted
salicylanilide SAL-5 was used for the comparison.
Salicylanilide carbamates and thiocarbamates (1-5) were synthesised by direct carbamoylation of in
situ generated salicylanilide triethylammonium salts by (thio)carbamoyl chlorides in
dichloromethane (DCM) at rt: 48 h for carbamates and 72 h for thiocarbamates. Yields ranged from
55 to 99%, and thiocarbamates produced generally less yields than corresponding carbamates. The
synthetic plan is depicted in Scheme 1.
When dry acetonitrile (MeCN) was used as the solvent instead of DCM at rt, it was possible to
shorten the reaction time to 18 h for carbamates and 24 h for thiocarbamates. All these reaction
conditions produced desired compounds in similar yields.
We synthesised five parent salicylanilides (SAL-1-SAL-5), fifteen salicylanilide carbamates and
five thiocarbamates. The presented compounds are summarised in Table 1.
2.2 Antimycobacterial activity
Salicylanilides, their carbamates and thiocarbamates were evaluated for their in vitro
antimycobacterial activity against M. tuberculosis 331/88 (H37Rv), Mycobacterium avium 330/88
and two strains of M. kansasii: 235/80 and a clinical isolate, 6509/96. The first-line antituberculosis
drug, isoniazid (INH), and p-aminosalicylic acid (PAS; Fig. 1) as a structurally similar second-line
drug were chosen as the reference compounds.
Salicylanilide N,N-disubstituted carbamates and thiocarbamates (1-5) showed mostly a significant
antimycobacterial activity (MIC ≥ 4 µM; Table 1). One molecule (5b) did not show any activity at a
concentration of 1000 µM and for four derivatives (3d, 4a, 4c, 4d), it was impossible to determine
the exact MIC values because of the precipitation in the testing medium. When parent
salicylanilides were compared, 4-trifluoromethyl group bearing salicylanilide SAL-4 expressed the
lowest MIC, followed by chlorinated SAL-1 and brominated SAL-2 salicylanilides, which is
consistent with previous results. The halogenation of the salicylanilide scaffold enhanced the
antimycobacterial activity (SAL-5 vs. other salicylanilides).
Mycobacterium tuberculosis showed the highest susceptibility (MICs ≥ 4 µM with 4b and 1b
superiority), whereas M. avium with MICs ≥ 16 µM (reached by three thiocarbamates 1b, 2b and
3b) was shown to be the least susceptible; these findings reflect the activity of parent salicylanilides
(SAL-1-SAL-5). The growth of both M. kansasii strains was suppressed most effectively by
thiocarbamate 1b with the lowest MIC values of 8-16 µM. No O-salicylanilide derivative exceeded
the MIC of isoniazid against M. tuberculosis H37Rv and clinically isolated strain of M. kansasii
6509/96, whereas four carbamates (1a, 2a, 3a, 5d) and thiocarbamates (1b, 2b, 3b, 4b) exhibited
clearly lower MICs for M. avium than INH. Thirteen newly synthesised compounds (1a-d, 2a-d,
3a-c, 4b and 5d) inhibited M. kansasii 235/80 at lower concentrations than this substantial first-line
anti-TB drug. All halogenated (thio)carbamates with presented MIC values surpassed p-
aminosalicylic acid, a second-line oral drug with a similar structural fragment, towards M.
tuberculosis and M. kansasii 235/80, but only thiocarbamates produced the same results as well for
M. avium.
Surprisingly, the conversion of salicylanilides to their N,N-disubstituted (thio)carbamates did not
produce noticeable improvement in antimycobacterial activity, as it was reported previously for N-

alkyl carbamates with excellent in vitro activity.15 With one exception (5d), no (thio)carbamate
showed significantly lower MICs than the original molecule; only in some cases, the activity of
thiocarbamates was comparable (i.e., equal or with a difference of one dilution) to parent
salicylanilides (SAL-1-SAL-5) for all or some strains (1b, 2b, 3b). Modification of the most potent
salicylanilide 4 produced carbamates with sharply reduced activity (4a, 4c, 4d). Thiocarbamates of
halogenated salicylanilides exhibited decreased MIC values than the corresponding carbamate
isosters (1a vs. 1b, 2a vs. 2b, 3a vs. 3b, 4a vs. 4b) indicating that the presence of sulphur is
favourable for the antimycobacterial action. The reason may be related to the enhanced lipophilicity
(but it is not a general rule because more lipophilic N,N-diphenylcarbamates do not produce the best
growth inhibition), modified steric parameters or different hydrolytic stability. For M. tuberculosis
and M. avium, N-substitution by two methyl groups represents the most convenient pattern;
substitution of one methyl by phenyl led to the mild reduction of the activity, whereas the presence
of two phenyls on carbamate nitrogen abolished the antimycobacterial properties completely at low
concentrations (1d, 2d, 3d, 4d). It seems that bulkier N-substitution negatively modulates the
described biological activity. For M. kansasii, the activity is not substantially influenced by the N-
substitution and N,N-dimethyl, N-methyl-N-phenyl and N,N-diphenyl carbamates share similar
MICs.
Interestingly, derivatives of unsubstituted salicylanilide 5 expressed a mostly inverse structure-
activity relationship: N,N-dimethylthiocarbamate 5b is less active than analogous N,N-
dimethylcarbamate 5a; N,N-diphenylcarbamate 5d possessed lower MIC values than other
carbamates 5a and 5c.
Based on the comparison of highly antimycobacterial active N-alkylcarbamates (MIC for M.
tuberculosis within the range of 0.5 to 4 µM)15 and less antimycobacterial active salicylanilide N,N-
disubstituted carbamates in this series, we propose three possible hypotheses to explain this
phenomenon. First, the presence of one hydrogen on the carbamate nitrogen is necessary for
effective binding to the cellular target(s), which are currently still not exactly known, most likely
via hydrogen bonding. Second, the longer lipophilic aliphatic tail is more convenient for binding to
the target site, which is the hydrophobic pocket or cavity. The third hypothesis may involve
somewhat different hydrolytic behaviour of various carbamates. Previously, it was demonstrated
that aryl-O-CO-NH-alkyl carbamates show a higher chemical and metabolic lability than
carbamates with a general aryl-O-CO-N(alkyl)2 structure.20-21 Based on this third hypothesis,
carbamate prodrugs serve as temporary salicylanilide depot pool.
2.3 Isocitrate lyase inhibition
Salicylanilide carbamates and thiocarbamates (1-5) were evaluated for their inhibition of
mycobacterial isocitrate lyase (Table 1). The isocitrate lyase (ICL-1) inhibition activity was assayed
via glyoxylate phenyl hydrazone formation at the concentration of investigated compounds being 10
µM. INH was employed as a negative control (inhibition of 0%), when 3-nitropropionic acid (3-NP)
served as a positive control.
(Thio)carbamates expressed consistent but mild inhibition within the range of 3 to 22% at the
concentration of 10 µM. Three carbamates (1d, 3d, 4d) demonstrated ≥ 20% ICL inhibition with
N,N-diphenylcarbamate 3d superiority (22%), thus being slightly less active than 3-NP (25%).
Halogenated salicylanilides and their derivatives seem to be stronger inhibitors than those based on
unsubstituted salicylanilide SAL-5; the trend of the inhibition activity decrease of the parent
compound SAL-5 is observed with O-(thio)carbamoylation, which is in contrast to 4-
trifluoromethylaniline-substituted salicylanilide SAL-4 and its derivatives 4a-4d, generally the
most potent inhibitor scaffold. In the case of 4-monohalogenated salicylanilides SAL-1, SAL-2 and
SAL-3, their dimethyl(thio)carbamates share lower inhibition, while phenylcarbamates enhance this
action when compared to parent compounds. For halogenated salicylanilides, the following is the
inhibitory potency in decreasing order: diphenylcarbamates > (methyl)phenylcarbamates >
dimethylcarbamates ≈ dimethylthiocarbamates; this trend indicates that bulkier and/or aromatic
nitrogen substituents improve enzymatic inhibition. In contrast to the antimicrobial activity,

thiocarbamates did not produce better results (i.e., higher inhibition rates) than corresponding
carbamates.
We tried to determine IC50 for the most potent inhibitors (1d, 2d, 3d and 4d), but the sharply
increased lipophilicity of these molecules (ClogP values of 6.61-7.37) caused problems with the
enzymatic assessment. Diphenylcarbamates precipitated after a very short period in the testing
medium at the concentrations higher than 50 µM; therefore it was not possible to find exact IC50
values.
Similar to other salicylanilide esters,16-18 presented (thio)carbamates act as moderate ICL inhibitors
with no superiority to 3-NP. Thus, this inhibition represents probably only a minor benefit in
addition to the action against actively growing mycobacteria. As expected and observed previously
for other benzanilides, there is not a clear relationship of in vitro MICs and ICL inhibition. Here
reported MIC values were obtained for actively growing mycobacteria, while ICL inhibition has
displayed the potential against persistent or non-growing mycobacterial subpopulations.
2.4 Antibacterial and antifungal activity evaluation
All of the salicylanilide carbamates and thiocarbamates (1-5) were assayed in vitro against eight
bacterial strains; four of them were Gram-positive [Staphylococcus aureus, methicillin-resistant
Staphylococcus aureus (MRSA), Staphylococcus epidermidis, Enterococcus sp.], and four were
Gram-negative (Escherichia coli, Klebsiella pneumoniae, ESBL-positive Klebsiella pneumoniae
and Pseudomonas aeruginosa). Benzylpenicillin (PNC) and bacitracin (BAC) were used as
comparative drugs.
None of the salicylanilide derivatives inhibited the growth of Gram-negative bacteria at a
concentration of 125 µM or lower, whereas Gram-positive cocci were inhibited by some derivatives
at 0.49-125 µM. Compounds, which are not reported in Table 2, were found sharing all MIC values
≥ 125 µM; compound 4a precipitated in the testing medium. O-{2-[(4-Bromophenyl)carbamoyl]-4-
chlorophenyl} dimethylcarbamothioate 2b showed best MICs (0.49 µM) against Staphyloccus sp.
including MRSA, superior to benzylpenicillin). Three thiocarbamates (1b, 2b, 4b) expressed mostly
lower MIC values than bacitracin, an established antibacterial drug used particularly in the topical
treatment of infections caused by Gram-positive bacteria. Despite the presence of drug-resistance,
the most susceptible strain was S. aureus.
It is obvious that halogenation of the salicylanilide core improves the antibacterial activity when
compared to unsubstituted salicylanilide (5 vs. 1a, 1b, 2b, 3a, 3b, 4b) with bromine as the most
superior (2b), followed by chlorine (1b) as aniline ring substitution patterns in accordance with
increasing lipophilicity. The thiocarbamates possess a stronger in vitro antibacterial activity than
corresponding carbamates (1a vs. 1b, 2a vs. 2b, 3a vs. 3b). The possible explanation may consist
in significantly increased lipophilicity or changed steric parameters. The substitution of carbamate
nitrogen by small alkyls is a necessary requirement for this biological activity, while the
replacement of one or both methyls by phenyl(s) results in its complete abolition.
The antifungal properties of investigated compounds were evaluated in vitro against eight species:
Candida albicans, C. tropicalis, C. krusei, C. glabrata, Trichosporon asahii, Aspergillus fumigatus,
Absidia corymbifera and Trichophyton mentagrophytes. Fluconazole (FLU) and amphotericin B
(AMB) were used as reference drugs.
Among all strains, only T. mentagrophytes was inhibited by carbamates and thiocarbamates at the
concentration of 125 µM and lower; MIC values range of 1.95 to 500 µM. MICs of derivatives not
involved in Table 2 exceeded 125 µM; compound 4a precipitated in the testing medium. Under our
conditions, two thiocarbamates 1b and 2b share the identical in vitro antifungal activity as an
established and highly effective drug amphotericin B and superior to fluconazole.
The structure-activity relationship, although based on limited data, revealed similar results as for
antibacterial activity: thiocarbamates expressed more potent MICs than corresponding carbamates
(1a vs. 1b, 2a vs. 2b, 3a vs. 3b), the monohalogen substitution of aniline ring improves the activity
(with chlorine 1b and bromine 2b superiority) and the presence of any phenyl on carbamic nitrogen
prevents in vitro antifungal properties at low concentrations.

2.5 Cytotoxicity evaluation
Parent salicylanilides (SAL) and their derivatives (1-5) underwent cytotoxicity determination in
HepG2 cell model using a standard colorimetric method measuring a tetrazolium salt reduction.
Their cytotoxicity is expressed as IC50, i.e., concentration, which reduces the viability of the cells to
50% of the maximal viability.
IC50 of parent salicylanilides (SAL-1-SAL-5) ranged between 0.36 and 89.8 µM, and the
halogenation decreased the values to 0.36 µM for trifluoromethyl derivative 4. All carbamates
showed an IC50 that was higher than 188 µM and thiocarbamates ≥ 67.3 µM. Six compounds (1d,
2d, 3d, 4c, 4d and 5b) started to crystallise from water-containing testing medium just in non-
cytotoxic concentrations; therefore, it was not possible to determine the exact IC50 values, although
the highest non-cytotoxic and fully soluble concentration is reported (Table 3). The less toxic
carbamates were dimethyl derivatives 4a and 5a.
Importantly, carbamoylation and thiocarbamoylation of parent salicylanilides sharply decreases
their substantial cytotoxicity – e.g., regarding 4 and 4a, the cytotoxicity was decreased more than
18,077-fold. Thiocarbamates of halogenated salicylanilides showed a higher cytotoxicity (≥ 67.3
µM) than corresponding carbamates (≥ 188.7 µM); the replacement of oxygen by sulphur leads to
2.3-97.6 times higher cytotoxicity. For halogenated salicylanilides (SAL-1-SAL-4), the following is
the cytotoxicity reduction in decreasing order: (methyl)phenylcarbamate > dimethylcarbamate >
diphenylcarbamate (limited validity due to not exact data) > dimethylthiocarbamate. Interestingly,
the order is reversed for unsubstituted salicylanilide 5 derivatives.
While the O-modification of parent phenolic salicylanilides did not markedly increase the
antimycobacterial activity, it mitigated the cytotoxicity in the HepG2 model, thus improving the
selectivity indices (SI). SI is calculated as a ratio of IC50/MIC, and its values higher than 10 indicate
rather acceptable toxicity (based on the analogy of the therapeutic index). The SI of the parent
salicylanilides, SAL-1-SAL-5, ranged from 0.05 to 5.4 for mycobacteria and 0.05 to 16.0 for T.
mentagrophytes. The SI of (thio)carbamates was up to 29.5 for mycobacteria, up to 174.8 for S.
aureus including MRSA and up to 43.9 for T. mentagrophytes. Seven molecules, carbamates 1a,
2a, 2c, 3a and thiocarbamates 1b, 2b, 4b, showed both SI values higher than 10 for M. tuberculosis
with 2c superiority (29.5); two derivatives (1b, 2c) exhibited at least one value exceeding this
threshold for nontuberculous mycobacteria. Two thiocarbamates with a significant in vitro
antibacterial activity share SI values for Staphylococci over 10: 1b and especially 2b (174.8), which
selectivity index is superior to bacitracin. Three thiocarbamates (1b, 2b, 3b) also exhibited a
satisfactory ratio in the case of T. mentagrophytes, even in comparison with fluconazole and
especially amphotericin B.
In general, derivatives of 4-Cl and 4-bromoanilines demonstrated more convenient activity/toxicity
data than other salicylanilide-based compounds.
3. Conclusions
In this study, we synthesised twenty salicylanilide carbamates and thiocarbamates from
(thio)carbamoyl chlorides and salicylanilide triethylammonium salts. New compounds were
characterised, and all of them underwent a set of biological tests as potential antimicrobial agents
against mycobacteria, other bacterial and fungal strains, regarding their cytotoxicity and inhibition
of mycobacterial isocitrate lyase.
The presented derivatives inhibited the growth of both tuberculous and nontuberculous
mycobacteria in the micromolar concentration range; however, they did not exceed
antimycobacterial activity of parent salicylanilides. On the other hand, the modification of the
salicylic phenol group by (thio)carbamoylation markedly alleviated the substantial toxicity of
salicylanilides against HepG2 cells, thus improving the selectivity indices and the toxicity profile.
Several derivatives exhibited low micromolar MICs for Staphylococci including MRSA and T.
mentagrophytes. Some structure-activity relationships have been identified; for example, N,N-
dimethylthiocarbamates share more enhanced antimicrobial and cytotoxic properties than

corresponding N,N-dimethylcarbamates. Together, some carbamates and thiocarbamates may
represent attractive molecules for future investigation, especially with respect to M. tuberculosis,
Gram-positive cocci and Trichophyton mentagrophytes fungus.
4. Experimental part
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. Reactions and the
purity of the products were monitored by thin layer chromatography with a toluene/ethyl acetate 4:1
or toluene/methanol 9:1 mixture as eluent; plates were coated with 0.2 mm Merck 60 F254 silica
gel and were visualised by UV irradiation (254 nm). Melting points were determined on a Büchi
Melting Point machine B-540 apparatus 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 FT-IR
spectrometer Nicolet 6700 FT-IR in the range of 400 to 4,000 cm-1. The NMR spectra were
1
measured in CDCl3 at ambient temperature on a Varian VNMR S500 instrument (500 MHz for H
and 125 MHz for 13C; Varian Comp. Palo Alto, CA, USA). The chemical shifts, δ, are given in
ppm, with respect to tetramethylsilane as an internal standard. The coupling constants (J) are
reported in Hz.
The calculated logP values (ClogP), that are the logarithms of the partition coefficients for octan-1-
ol/water, were determined using the program CS ChemOffice Ultra version 12.0 (CambridgeSoft,
Cambridge, MA, USA).
4.1.2 Synthesis
Parent salicylanilides (SAL-1-SAL-5) were synthesised via a previously described method.19 This
microwave-assisted synthesis was carried out in a microwave reactor (530 W, 600 rpm;
MicroSYNTH Milestone) for 22 min to refluxing.
An equivalent of appropriate salicylanilide (1 mmol) was suspended under vigorous stirring in dry
dichloromethane (DCM; 10 mL), and then triethylamine (1.5 of equivalents) was added in one
portion. The mixture was stirred for 5 minutes to allow complete dissolution of the salicylanilide
due to formation of its triethylammonium salt. Then, appropriate (thio)carbamoyl chloride (1.5 of
equivalents) was added in one portion, and the mixture was stirred at room temperature for 48 h
(carbamates) or 72 h (thiocarbamates). The reaction was monitored using TLC. Then, the solution
was evaporated till dryness, ethyl acetate was added and the suspension was stored at 4 °C for
approximately 30 min. Then, the precipitate was removed by filtration, the filtrate was collected,
partly evaporated and then n-hexane was added to initiate crystallisation. After 24 hours at 4 °C, the
precipitate was filtered to give resulting (thio)carbamates 1-5. Products were recrystallised from
ethyl acetate, if necessary.
When dry acetonitrile (MeCN) was used as the solvent at rt, the reaction time was shortened to 18 h
for carbamates and 24 h for thiocarbamates. Under refluxing in acetonitrile, the reaction time of 2.5
h was sufficient for carbamates and 5 h for thiocarbamates. However, these methods produced
similar yields and purity of the product as the described general method performed in
dichloromethane at rt.
4-Chloro-2-[(4-chlorophenyl)carbamoyl]phenyl dimethylcarbamate (1a). White solid; yield 94%;
mp 171.5-173.5 °C. IR (ATR): 3301 (NH), 1710 (O-CO-N), 1676 (CO-NH), 1598, 1534, 1492,
1400, 1388, 1318, 1216, 1174, 1143, 1103, 1016, 862, 824, 809, 801, 737, 665 cm-1. 1H NMR (500
MHz, CDCl3): δ 8.68 (1H, bs, NH), 7.70 (1H, d, J = 2.6 Hz, H3), 7.56-7.50 (2H, m, H2´, H6´), 7.40
(1H, dd, J = 2.6 Hz, J = 8.7 Hz, H5), 7.32-7.28 (2H, m, H3′, H5′), 7.06 (1H, d, J = 8.7 Hz, H6), 3.09
(3H, s, CH3), 3.01 (3H, s, CH3). 13C NMR (125 MHz, CDCl3): 162.9, 154.9, 146.6, 136.4, 131.8,

131.7, 131.5, 129.8, 129.5, 129.0, 124.7, 120.9, 37.0, 36.7. Anal. Calcd. for C16H14Cl2N2O3
(353.20): C, 54.41; H, 4.00; N, 7.93. Found: C, 54.23; H, 3.86; N, 8.12.
O-{4-Chloro-2-[(4-chlorophenyl)carbamoyl]phenyl} dimethylcarbamothioate (1b). Yellowish
solid; yield 55%; mp 144-145 °C. IR (ATR): 3272 (NH), 1645 (CO-NH), 1599, 1541 (O-CS-N),
1
1534, 1492, 1477, 1403, 1389, 1313, 1287, 1205, 1123, 1097, 1012, 822, 814, 739 cm-1. H NMR
(500 MHz, CDCl3): δ 9.13 (1H, bs, NH), 7.68 (1H, d, J = 2.6 Hz, H3), 7.16-7.55 (2H, m, H2´, H6´),
7.45 (1H, dd, J = 2.7 Hz, J = 8.7 Hz, H5), 7.32-7.26 (2H, m, H3′, H5′), 6.97 (1H, d, J = 8.7 Hz, H6),
3.44 (3H, s, CH3), 3.29 (3H, s, CH3). 13C NMR (125 MHz, CDCl3): 186.9, 162.8, 148.8, 136.6,
132.4, 132.0, 131.7, 129.8, 129.4, 129.0, 125.3, 120.6, 43.5, 39.2. Anal. Calcd. for C16H14Cl2N2O2S
(369.27): C, 52.04; H, 3.82; N, 7.59. Found: C, 52.23; H, 3.76; N, 7.40.
4-Chloro-2-[(4-chlorophenyl)carbamoyl]phenyl methyl(phenyl)carbamate (1c). White solid; yield
93%; mp 126.5-127.5 °C. IR (ATR): 3316 (NH), 1698 (O-CO-N), 1676 (CO-NH), 1597, 1531,
1
1493, 1400, 1373, 1312, 1223, 1140, 1103, 1014, 971, 917, 839, 818, 797, 768, 730, 699 cm-1. H
NMR (500 MHz, CDCl3): δ 8.20 (1H, bs, NH), 7.76 (1H, d, J = 2.6 Hz, H3), 7.42-7.23 (10H, m,
H5, H2´, H6´, H3´, H5´, H2´´, H6´´, H3´´, H5´´, H4´´), 7.12 (1H, d, J = 8.7 Hz, H6), 3.37 (3H, s,
13
CH3). C NMR (125 MHz, CDCl3): 162.3, 153.2, 146.6, 141.9, 136.1, 131.8, 131.6, 130.1, 129.6,
129.5, 128.9, 127.5, 126.1, 124.5, 121.4, 38.8. Anal. Calcd. for C21H16Cl2N2O3 (415.27): C, 60.74;
H, 3.88; N, 6.75. Found: C, 60.97; H, 3.89; N, 6.92.
4-Chloro-2-[(4-chlorophenyl)carbamoyl]phenyl diphenylcarbamate (1d). White solid; yield 91%;
mp 141-143 °C. IR (ATR): 3342 (NH), 1699 (O-CO-N), 1666 (CO-NH), 1596, 1528, 1492, 1400,
1355, 1313, 1239, 1210, 1104, 1015, 825, 762, 729, 698 cm-1. 1H NMR (500 MHz, CDCl3): δ 8.32
(1H, bs, NH), 7.66 (1H, d, J = 2.6 Hz, H3), 7.39-7.23 (13H, m, H5, H2´, H6´, H2´´, H6´´, H3´´,
H5´´, H4´´, H2´´´, H6´´´, H3´´´, H5´´´, H4´´´), 7.16-7.12 (3H, m, H6, H3′, H5′). 13C NMR (125
MHz, CDCl3): 162.7, 152.5, 146.5, 141.6, 136.1, 131.7, 131.4, 130.0, 129.6, 129.5, 129.3, 128.7,
127.2, 127.0, 124.2, 121.4. Anal. Calcd. for C26H18Cl2N2O3 (477.34): C, 65.42; H, 3.80; N, 5.87.
Found: C, 65.19; H, 3.98; N, 5.55.
2-[(4-Bromophenyl)carbamoyl]-4-chlorophenyl dimethylcarbamate (2a). White solid; yield 89%;
mp 176-177 °C. IR (ATR): 3305 (NH), 1709 (O-CO-N), 1676 (CO-NH), 1593, 1533, 1488, 1475,
1387, 1316, 1252, 1215, 1173, 1142, 1103, 1070, 1012, 862, 822, 808, 800, 736, 663 cm-1. 1H NMR
(500 MHz, CDCl3): δ 8.70 (1H, bs, NH), 7.69 (1H, d, J = 2.6 Hz, H3), 7.48-7.42 (4H, m, H2´, H6´,
H3′, H5′), 7.39 (1H, dd, J = 2.6 Hz, J = 8.7 Hz, H5), 7.05 (1H, d, J = 8.7 Hz, H6), 3.09 (3H, s,
CH3), 3.00 (3H, s, CH3). 13C NMR (125 MHz, CDCl3): 162.9, 154.9, 146.6, 136.9, 132.0, 131.8,
131.4, 129.7, 124.7, 121.3, 121.2, 117.1, 37.0, 36.7. Anal. Calcd. for C16H14BrClN2O3 (397.65): C,
48.33; H, 3.55; N, 7.04. Found: C, 48.54; H, 3.80; N, 7.01.
O-{2-[(4-Bromophenyl)carbamoyl]-4-chlorophenyl} dimethylcarbamothioate (2b). Yellowish
solid; yield 81%; mp 146-148.5 °C. IR (ATR): 3292 (NH), 1679 (CO-NH), 1590, 1538 (O-CS-N),
1514, 1489, 1479, 1396, 1388, 1310, 1285, 1220, 1151, 1135, 1102, 1070, 1013, 890, 816, 746 cm-
1
1. H NMR (500 MHz, CDCl3): δ 9.12 (1H, bs, NH), 7.68 (1H, d, J = 2.7 Hz, H3), 7.55-7.51 (2H,
m, H2´, H6´), 7.46-7.43 (3H, m, H5, H3′, H5′), 6.97 (1H, d, J = 8.7 Hz, H6), 3.44 (3H, s, CH3), 3.29
(3H, s, CH3). C NMR (125 MHz, CDCl3): 186.9, 162.8, 148.8, 137.1, 132.4, 132.0, 131.9, 131.7,
13
129.8, 125.3, 121.0, 117.0, 43.6, 39.2. Anal. Calcd. for C16H14BrClN2O2S (413.72): C, 46.45; H,
3.41; N, 6.77. Found: C, 46.26; H, 3.50; N, 6.51.
2-[(4-Bromophenyl)carbamoyl]-4-chlorophenyl methyl(phenyl)carbamate (2c). White solid; yield
89%; mp 154.5-155.5 °C. IR (ATR): 3316 (NH), 1698 (O-CO-N), 1680 (CO-NH), 1597, 1531,
1489, 1395, 1372, 1313, 1208, 1136, 1104, 1071, 1010, 816, 765, 746, 729, 698, 668 cm-1. 1H NMR
(500 MHz, CDCl3): δ 8.20 (1H, bs, NH), 7.76 (1H, d, J = 2.7 Hz, H3), 7.39 (1H, dd, J = 2.7 Hz, J =

8.8 Hz, H5), 7.37-7.22 (9H, m, H2´, H6´, H3´, H5´, H2´´, H6´´, H3´´, H5´´, H4´´), 7.12 (1H, d, J =
8.7 Hz, H6), 3.37 (3H, s, CH3). 13C NMR (125 MHz, CDCl3): 162.2, 152.9, 146.6, 141.8, 136.6,
131.8, 131.6, 130.1, 129.7, 129.5, 127.5, 126.1, 124.5, 121.7, 117.2, 38.8. Anal. Calcd. for
C21H16BrClN2O3 (459.72): C, 54.86; H, 3.51; N, 6.09. Found: C, 55.07; H, 3.68; N, 6.21.
2-[(4-Bromophenyl)carbamoyl]-4-chlorophenyl diphenylcarbamate (2d). White solid; yield 98%;
mp 175.5-177.5 °C. IR (ATR): 3341 (NH), 1699 (O-CO-N), 1669 (CO-NH), 1593, 1529, 1490,
1
1393, 1355, 1312, 1238, 1211, 1104, 1072, 1039, 1015, 822, 762, 729, 698 cm-1. H NMR (500
MHz, CDCl3): δ 8.26 (1H, bs, NH), 7.68 (1H, d, J = 2.6 Hz, H3), 7.38 (1H, dd, J = 2.7 Hz, J = 8.8
Hz, H5), 7.36-7.24 (12H, m, H2´, H6´, H2´´, H6´´, H3´´, H5´´, H4´´, H2´´´, H6´´´, H3´´´, H5´´´,
H4´´´), 7.22-7.18 (2H, m, H3′, H5′), 7.15 (1H, d, J = 8.7 Hz, H6). 13C NMR (125 MHz, CDCl3):
162.6, 152.5, 146.4, 141.6, 136.6, 131.8, 131.7, 131.5, 130.1, 129.7, 129.3, 127.2, 124.3, 121.7,
121.6, 117.3. Anal. Calcd. for C26H18BrClN2O3 (521.79): C, 59.85; H, 3.48; N, 5.37. Found: C,
59.70; H, 3.63; N, 5.59.
4-Chloro-2-[(4-fluorophenyl)carbamoyl]phenyl dimethylcarbamate (3a). White solid; yield 87%;
mp 148.5-149.5 °C. IR (ATR): 3296 (NH), 1702 (O-CO-N), 1665 (CO-NH), 1613, 1536, 1510,
1408, 1390, 1317, 1215, 1177, 1103, 1017, 893, 859, 828, 810, 747, 734 cm-1. 1H NMR (500 MHz,
CDCl3): δ 8.70 (1H, bs, NH), 7.67 (1H, d, J = 2.6 Hz, H3), 7.52-7.48 (2H, m, H2´, H6´), 7.34 (1H,
dd, J = 2.6 Hz, J = 8.7 Hz, H5), 7.04–6.99 (3H, m, H6, H3′, H5′), 3.09 (3H, s, CH3), 3.00 (3H, s,
CH3). 13C NMR (125 MHz, CDCl3): 162.9, 159.4 (d, J = 243.9 Hz), 154.8, 146.7, 133.8 (d, J = 2.8
Hz), 131.6, 131.5, 131.2, 129.6, 124.6, 121.6 (d, J = 7.9 Hz), 115.6 (d, J = 22.5 Hz), 36.9, 36.7.
Anal. Calcd. for C16H14ClFN2O3 (336.75): C, 57.07; H, 4.19; N, 8.32. Found: C, 56.88; H, 3.99; N,
8.39.
O-{4-Chloro-2-[(4-fluorophenyl)carbamoyl]phenyl} dimethylcarbamothioate (3b). Yellowish solid;
yield 56%; mp 164-165.5 °C. IR (ATR): 3291 (NH), 1643 (CO-NH), 1541 (O-CS-N), 1509, 1473,
1
1412, 1390, 1288, 1236, 1204, 1159, 1143, 1124, 1097, 1055, 826, 814, 791, 773, 714 cm-1. H
NMR (500 MHz, CDCl3): δ 9.05 (1H, bs, NH), 7.69 (1H, d, J = 2.6 Hz, H3), 7.61-7.57 (2H, m, H2´,
H6´), 7.45 (1H, dd, J = 2.7 Hz, J = 8.8 Hz, H5), 7.03 (2H, dt, J = 2.1 Hz, J = 8.7 Hz, H3′, H5′), 6.97
(1H, d, J = 8.6 Hz, H6), 3.45 (3H, s, CH3), 3.30 (3H, s, CH3). 13C NMR (125 MHz, CDCl3): 187.0,
162.7, 159.4 (d, J = 243.7 Hz), 148.8, 134.1 (d, J = 2.9 Hz), 132.4, 132.1, 131.6, 129.9, 125.3,
121.1 (d, J = 7.8 Hz), 115.7 (d, J = 22.6 Hz), 43.5, 39.2. Anal. Calcd. for C16H14ClFN2O2S
(352.81): C, 54.47; H, 4.00; N, 7.94. Found: C, 54.69; H, 3.80; N, 7.79.
4-Chloro-2-[(4-fluorophenyl)carbamoyl]phenyl methyl(phenyl)carbamate (3c). White solid; yield
99%; mp 150.5-151 °C. IR (ATR): 3321 (NH), 1687 (O-CO-N), 1672 (CO-NH), 1599, 1532, 1509,
1501, 1408, 1375, 1282, 1215, 1138, 1102, 822, 767, 730, 701 cm-1. 1H NMR (500 MHz, CDCl3): δ
8.15 (1H, bs, NH), 7.78 (1H, d, J = 2.6 Hz, H3), 7.39 (1H, dd, J = 2.6 Hz, J = 8.6 Hz, H5), 7.37-
7.24 (7H, m, H2´, H6´, H2´´, H6´´, H3´´, H5´´, H4´´), 7.13 (1H, d, J = 8.6 Hz, H6), 6.99 (2H, t, J =
8.5 Hz, H3′, H5′), 3.37 (3H, s, CH3). 13C NMR (125 MHz, CDCl3): 162.2, 159.4 (d, J = 244.3 Hz),
153.0, 146.4, 141.9, 133.5 (d, J = 2.8 Hz), 131.7, 131.6, 130.1, 129.5, 127.5, 126.1, 124.5, 122.1 (d,
J = 7.8 Hz), 115.6 (d, J = 22.5 Hz), 38.8. Anal. Calcd. for C21H16ClFN2O3 (398.81): C, 63.24; H,
4.04; N, 7.02. Found: C, 63.50; H, 3.90; N, 6.85.
4-Chloro-2-[(4-fluorophenyl)carbamoyl]phenyl diphenylcarbamate (3d). White solid; yield 93%;
mp 171.5-172.5 °C. IR (ATR): 3330 (NH), 1703 (O-CO-N), 1669 (CO-NH), 1530, 1509, 1493,
1406, 1358, 1319, 1260, 1213, 1105, 1013, 845, 833, 758, 706, 693, 666 cm-1. 1H NMR (500 MHz,
CDCl3): δ 8.20 (1H, bs, NH), 7.69 (1H, d, J = 2.6 Hz, H3), 7.39-7.24 (13H, m, H5, H2´, H6´, H2´´,
H6´´, H3´´, H5´´, H4´´, H2´´´, H6´´´, H3´´´, H5´´´, H4´´´), 7.15 (1H, d, J = 8.5 Hz, H6), 6.99 (2H, t,
J = 8.2 Hz, H3′, H5′). 13C NMR (125 MHz, CDCl3): 162.6, 159.5 (d, J = 243.9 Hz), 152.5, 146.5,
141.6, 133.5 (d, J = 2.6 Hz), 131.7, 131.5, 130.1, 129.7, 129.3, 127.1, 124.3, 122.1 (d, J = 8.1 Hz),

115.4 (d, J = 22.4 Hz). Anal. Calcd. for C26H18ClFN2O3 (460.88): C, 67.76; H, 3.94; N, 6.08.
Found: C, 67.54; H, 4.12; N, 6.25.
4-Chloro-2-{[4-(trifluoromethyl)phenyl]carbamoyl}phenyl dimethylcarbamate (4a). White solid;
yield 94%; mp 183.5-185 °C. IR (ATR): 3286 (NH), 1694 (O-CO-N), 1676 (CO-NH), 1604, 1532,
1
1405, 1390, 1317, 1257, 1221, 1178, 1167, 1110, 1064, 1015, 885, 847, 836, 728 cm-1. H NMR
(500 MHz, CDCl3): δ 8.98 (1H, bs, NH), 7.68-7.65 (3H, m, H3, H2´, H6´), 7.55 (2H, d, J = 8.6 Hz,
H3′, H5′), 7.36 (1H, dd, J = 2.6 Hz, J = 8.7 Hz, H5), 7.04 (1H, d, J = 8.6 Hz, H6), 3.11 (3H, s,
CH3), 3.02 (3H, s, CH3). 13C NMR (125 MHz, CDCl3): 163.2, 154.9, 146.7, 140.9, 131.9, 131.6,
131.1, 129.6, 126.2 (q, J = 32.8 Hz), 126.1 (q, J = 3.8 Hz), 124.6, 124.0 (q, J = 271.5 Hz), 119.4,
36.9, 36.7. Anal. Calcd. for C17H14ClF3N2O3 (386.75): C, 52.79; H, 3.65; N, 7.24. Found: C, 52.98;
H, 3.42; N, 7.37.
O-(4-Chloro-2-{[4-(trifluoromethyl)phenyl]carbamoyl}phenyl)
dimethylcarbamothioate
(4b).
Yellowish solid; yield 91%; mp 138.5-139.5 °C. IR (ATR): 3391 (NH), 1666 (CO-NH), 1599, 1536
(O-CS-N), 1473, 1411, 1392, 1332, 1283, 1255, 1196, 1166, 1090, 1064, 1015, 850, 821, 721 cm-1.
1H NMR (500 MHz, CDCl3): δ 9.36 (1H, bs, NH), 7.75 (2H, d, J = 8.5 Hz, H2´, H6´), 7.69 (1H, d, J
= 2.6 Hz, H3), 7.59 (2H, d, J = 8.5 Hz, H3′, H5′), 7.46 (1H, dd, J = 2.6 Hz, J = 8.6 Hz, H5), 6.98
(1H, d, J = 8.5 Hz, H6), 3.45 (3H, s, CH3), 3.30 (3H, s, CH3). 13C NMR (125 MHz, CDCl3): 186.9,
163.1, 148.8, 141.0, 132.5, 131.9, 131.8, 129.9, 126.3 (q, J = 4.0 Hz), 126.1 (q, J = 31.8 Hz), 125.3,
124.0 (q, J = 271.5 Hz), 119.1, 43.6, 39.2. Anal. Calcd. for C17H14ClF3N2O2S (402.81): C, 50.69; H,
3.50; N, 6.95. Found: C, 50.90; H, 3.72; N, 6.78.
4-Chloro-2-{[4-(trifluoromethyl)phenyl]carbamoyl}phenyl methyl(phenyl)carbamate (4c). White
solid; yield 84%; mp 129-130 °C. IR (ATR): 3334 (NH), 1704 (O-CO-N), 1676 (CO-NH), 1606,
1540, 1409, 1373, 1336, 1323, 1265, 1214, 1165, 1148, 1138, 1105, 1067, 1016, 841, 767, 731,
1
695, 675 cm-1. H NMR (500 MHz, CDCl3): δ 8.46 (1H, bs, NH), 7.77 (1H, d, J = 2.6 Hz, H3),
7.58-7.25 (10H, m, H5, H2´, H6´, H3′, H5′, H2´´, H6´´, H3´´, H5´´, H4´´), 7.13 (1H, d, J = 8.7 Hz,
H6), 3.44 (3H, s, CH3). 13C NMR (125 MHz, CDCl3): 162.5, 153.0, 146.7, 141.9, 140.6, 132.0,
131.6, 130.1, 129.5, 127.5, 126.3-125.8 (m), 126.0, 124.5, 124.0 (q, J = 271.5 Hz), 119.8, 38.8.
Anal. Calcd. for C22H16ClF3N2O3 (448.81): C, 58.78; H, 3.59; N, 6.24. Found: C, 59.01; H, 3.41; N,
6.52.
4-Chloro-2-{[4-(trifluoromethyl)phenyl]carbamoyl}phenyl diphenylcarbamate (4d). White solid;
yield 95%; mp 155.5-157 °C. IR (ATR): 3397 (NH), 1707 (O-CO-N), 1675 (CO-NH), 1605, 1536,
1493, 1411, 1345, 1322, 1257, 1214, 1189, 1172, 1152, 1112, 1067, 1016, 996, 838, 758, 701, 694
1
cm-1. H NMR (500 MHz, CDCl3): δ 8.62 (1H, bs, NH), 7.53 (1H, d, J = 2.6 Hz, H3), 7.38-7.22
(15H, m, H5, H2´, H6´, H3´, H5´, H2´´, H6´´, H3´´, H5´´, H4´´, H2´´´, H6´´´, H3´´´, H5´´´, H4´´´),
7.06 (1H, d, J = 8.6 Hz, H6). 13C NMR (125 MHz, CDCl3): δ 163.1, 152.6, 146.7, 141.6, 140.6,
131.8, 131.0, 129.6, 129.3, 129.2, 127.2, 126.3 (q, J = 32.7 Hz), 125.7 (q, J = 3.9 Hz), 125.6, 123.9
(q, J = 271.0 Hz), 123.4, 119.6. Anal. Calcd. for C27H18ClF3N2O3 (510.88): C, 63.48; H, 3.55; N,
5.48. Found: C, 63.19; H, 3.26; N, 5.44.
2-(Phenylcarbamoyl)phenyl dimethylcarbamate (5a)22. White solid; yield 93%; mp 115.5-117 °C.
IR (ATR): 3280 (NH), 1721 (O-CO-N), 1652 (CO-NH).
O-[2-(Phenylcarbamoyl)phenyl] dimethylcarbamothioate (5b). Yellowish solid; yield 77%; mp
136.5-137.5 °C. IR (ATR): 3291 (NH), 1676 (CO-NH), 1594, 1545 (O-CS-N), 1509, 1493, 1439,
1400, 1318, 1286, 1253, 1212, 1173, 1146, 778, 768, 759, 697 cm-1. 1H NMR (300 MHz, CDCl3): δ
9.06 (1H, bs, NH), 7.74 (1H, dd, J = 1.7 Hz, J = 7.6 Hz, H3), 7.65 (2H, d, J = 7.8 Hz, H2´, H6´),
7.53-7.47 (1H, m, H4), 7.41-7.30 (3H, m, H5, H3´, H5´), 7.15-7.09 (1H, m, H4´), 7.04 (1H, d, J =
8.1 Hz, H6), 3.45 (3H, s, CH3), 3.29 (3H, s, CH3). 13C NMR (75 MHz, CDCl3): δ 187.3, 164.2,

150.3, 138.3, 131.5, 130.9, 130.0, 129.0, 126.7, 124.2, 123.8, 119.4, 43.4, 39.1. Anal. Calcd. for
C16H16N2O2S (300.38): C, 63.98; H, 5.37; N, 9.33. Found: C, 64.13; H, 5.55; N, 9.20.
2-(Phenylcarbamoyl)phenyl methyl(phenyl)carbamate (5c). White solid; yield 93%; mp 141.5-
142.5 °C. IR (ATR): 3318 (NH), 1714 (O-CO-N), 1682 (CO-NH), 1598, 1536, 1496, 1453, 1442,
1
1378, 1318, 1311, 1201, 1139, 969, 775, 744, 703, 692, 669, 658 cm-1. H NMR (500 MHz,
CDCl3): δ 8.16 (1H, bs, NH), 7.82 (1H, d, J = 7.9 Hz, H3), 7.50-7.39 (3H, m, H4, H2´, H6´), 7.36-
7.23 (8H, m, H5, H3´, H5´, H2´´, H6´´, H3´´, H5´´, H4´´), 7.19 (1H, d, J = 7.9 Hz, H6), 7.15 (1H, t,
J = 7.3 Hz, H4´), 3.38 (3H, s, CH3). 13C NMR (125 MHz, CDCl3): δ 163.7, 153.5, 148.0, 142.1,
137.8, 131.8, 130.3, 129.3, 129.0, 128.9, 127.3, 126.2, 124.4, 123.1, 120.1, 38.6. Anal. Calcd. for
C21H18N2O3 (346.38): C, 72.82; H, 5.24; N, 8.09. Found: C, 72.57; H, 5.03; N, 8.24.
2-(Phenylcarbamoyl)phenyl diphenylcarbamate (5d). White solid; yield 91%; mp 170-172 °C. IR
(ATR): 3373 (NH), 1702 (O-CO-N), 1676 (CO-NH), 1601, 1542, 1491, 1442, 1349, 1328, 1202,
1
1191, 1012, 772, 775, 745, 702, 692, 680, 653 cm-1. H NMR (300 MHz, CDCl3): δ 8.10 (1H, bs,
NH), 7.82 (1H, dd, J = 1.9 Hz, J = 7.9 Hz, H3), 7.53-7.43 (3H, m, H4, H2´, H6´), 7.37-7.21 (14H,
m, H5, H6, H3´, H5´, H2´´, H6´´, H3´´, H5´´, H4´´, H2´´´, H6´´´, H3´´´, H5´´´, H4´´´), 7.15 (1H, t, J
= 7.4 Hz, H4´). 13C NMR (75 MHz, CDCl3) δ 163.8, 152.9, 147.8, 141.7, 137.7, 131.8, 130.1,
129.3, 129.2, 128.9, 126.9, 126.3, 124.5, 123.1, 120.2. Anal. Calcd. for C26H20ClF3N2O3 (408.45):
C, 76.45; H, 4.94; N, 6.86. Found: C, 76.19; H, 4.75; N, 6.84.
4.2 Biological activity
4.2.1 In vitro antimycobacterial susceptibility determination
Salicylanilide carbamates and thiocarbamates were evaluated for their in vitro antimycobacterial
activity against M. tuberculosis 331/88 (H37Rv; dilution of this strain was 10-3), Mycobacterium
avium 330/88 (resistant to INH, RIF, ofloxacin and ethambutol; dilution 10-5) and two strains of M.
kansasii: 235/80 (dilution 10-4) and the clinically isolated strain 6509/96 (dilution 10-5). The used
method is described in ref.17. The following concentrations were used: 1000, 500, 250, 125, 62.5,
32, 16, 8, 4, 2, 1, 0.5, 0.25, and 0.125 µM. MIC (reported in µM) was the lowest concentration at
which the complete inhibition of mycobacterial growth occurred. Isoniazid (INH) and p-
aminosalicylic acid (4-amino-2-hydroxybenzoic acid, PAS) as a structurally similar second-line
drug were chosen as the reference compounds. For each compound, MICs were determined in
quadruplicate and repeated twice.
4.2.2 In vitro antibacterial activity determination
The in vitro antibacterial activity was assayed against eight Gram-positive and Gram-negative
strains: Staphylococcus aureus CCM 4516/08, methicillin-resistant Staphylococcus aureus H
5996/08 (MRSA), Staphylococcus epidermidis H 6966/08, Enterococcus sp. J 14365/08;
Escherichia coli CCM 4517, Klebsiella pneumoniae D 11750/08, ESBL-positive Klebsiella
pneumoniae J 14368/08, and Pseudomonas aeruginosa CCM 1961.
The microdilution broth method in Mueller-Hinton broth was used. The tested compounds were
dissolved in DMSO to the final concentrations ranging from 500 to 0.49 µM. Benzylpenicillin
(penicillin G; PNC) and bacitracin (BAC) were used as the comparative drugs. The minimum
inhibitory concentrations were assayed as 95% (IC95) or higher reduction of growth compared to the
control. For each compound, MIC determination was performed twice. The used method is
described in ref.17.
4.2.3 In vitro antifungal activity determination
The antifungal properties of all synthesised compounds were evaluated in vitro against four
Candida strains (Candida albicans ATCC 44859, Candida tropicalis 156, Candida krusei E28, and
Candida glabrata 20/I), Trichosporon asahii 1188 and three filamentous fungi (Aspergillus
fumigatus 231, Absidia corymbifera 272, and Trichophyton mentagrophytes 445). The

microdilution broth method was used in RPMI 1640 with glutamine. Fluconazole (FLU) and
amphotericin B (AMB) were used as the reference drugs. The MICs were assayed as an 80% (IC80)
or higher reduction of growth in comparison to the control; for filamentous fungi, MICs are
expressed as IC50 values. For each compound, MIC determination was performed twice. The used
method is described in ref.19.
4.2.4 Isocitrate lyase inhibition assay (ICL1)
The isocitrate lyase (ICL) activity was assayed according to the protocol reported by Dixon and
Kornberg (glyoxylate phenyl hydrazone formation)23 at the investigated compounds concentration
of 10 µM. IC50 data represent an average of triplicate experiments SD. Isoniazid was employed as
a negative control (inhibition of 0%), and 3-nitropropionic acid (3-NP) served as a positive
control.17
4.2.5 Cytotoxicity evaluation (HepG2 cells)
All compounds were tested for their cytotoxicity in the human hepatocellular liver carcinoma cell
line HepG2 (passage 3–4; ECACC, Salisbury, UK) using a standard colorimetric method that
involves measuring a tetrazolium salt reduction (CellTiter(R) 96 AQueous One Solution Assay,
Promega G3580, Madison, WI, USA).
The cells were routinely cultured in Eagle’s minimum essential media supplemented with 10%
foetal bovine serum, 1% L-glutamine solution, and a non-essential amino acid solution. The
investigated compounds were dissolved in a very small amount of DMSO, and a small volume was
added to the cell culture. The tested compounds were prepared in triplicate at eight incubation
concentrations. The following types of controls were included: determination of 100% viability and
0% viability (the cells treated by 10% DMSO), no cell control, control for the determination of
possible interaction of tested compounds with reagents, control of the setting of incubation medium
and the control of the toxicity of DMSO.
The results are expressed as the inhibitory concentration that reduces cell viability to 50% of the
maximal (control) viability (IC50). IC50 was calculated in each of the tested substances using
GraphPad Prism software (version 5.02; GraphPad Software Inc., San Diego, CA, USA) and
Microsoft Excel 2010.
This method is described in depth in ref.17.
Acknowledgements
This publication is a result of the project implementation: „Support of establishment, development,
and mobility of quality research teams at the Charles University“, project number
CZ.1.07/2.3.00/30.0022, supported by The Education for Competitiveness Operational Programme
(ECOP) and co-financed by the European Social Fund and the state budget of the Czech Republic.
This work was also financially supported by IGA NT 13346 (2012).
We want to thank to Ida Dufková for the excellent performance of antibiotic susceptibility tests, the
staff of the Department of Inorganic and Organic Chemistry, Faculty of Pharmacy, for the technical
assistance and J. Urbanová, M.A., for the language help.
This manuscript was checked and edited by Elsevier Language Editing Services.
Conflict of Interest
The authors declare no conflict of interest.
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N
O
CH3
N
CH3
N
OH
OH
-aminosalicylic acid
O
CH3
O
CH3
H2N
S
O
NHNH2
S
isoniazid
p
tolnaftate
tolciclate
Figure 1. Structures of some antimycobacterial and thiocarbamate-based antimycotic drugs

Scheme 1. Synthesis of salicylanilides (SAL-1-SAL-5) and their carbamates and thiocarbamates (1-
5). [R1 = H, Cl; R2 = H, Cl, Br, F, CF3; R3 and R4 = CH3, phenyl; X = O (carbamates), S
(thiocarbamates); MW: microwave irradiation (530 W, 600 rpm, 22 min); PhCl: chlorobenzene;
DCM: dichloromethane; MeCN: acetonitrile; Et3N: triethylamine].

Table 1. Antimycobacterial activity of salicylanilide carbamates and thiocarbamates (1-5)
MIC [µM]
% ICL
inhibition at
10 µM
M. tuberculosis
331/88
14 d
Code
R1
R2
R3
R4
X
M. avium 330/88
M. kansasii 235/80
M. kansasii 6509/96
ClogP
( SD)
21 d
4
14 d
8
21 d
16
7 d
14 d
8
21 d
8
7 d
14 d
8
21 d
SAL-1
1a
1b
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
parent salicylanilide
4
16
4
62.5
8
4
62.5
16
8
125
16
13 2.90
3.57
3.69
4.67
5.35
7.01
3.84
3.96
4.94
5.62
7.28
3.17
3.29
4.27
4.95
6.61
3.93
4.05
5.03
5.71
7.37
3.29
2.57
3.55
4.23
5.9
CH3
CH3
CH3
Ph
CH3
CH3
Ph
O
S
16
62.5
16
62.5
16
62.5
16
62.5
16
125
16
6
6
1.36
1.92
4
32
8
32
1c
1d
O
O
250*
250*
8
250*
250*
16
62.5
32
125*
125*
8
125*
125*
8
32
32
62.5
62.5*
8
125
62.5*
8
17 1.63
20 1.08
13 2.04
Ph
250*
4
250*
4
SAL-2
2a
2b
parent salicylanilide
4
4
CH3
CH3
CH3
Ph
CH3
CH3
Ph
O
S
16
16
62.5
16
62.5
16
62.5
16
125*
16
125*
16
62.5
16
62.5
32
62.5
32
9
1.43
8
32
8
32
11 0.50
14 2.70
19 0.32
14 3.01
2c
2d
O
O
250*
250*
16
250*
250*
32
32
32
125*
125*
16
125*
125*
16
32
32
62.5
62.5*
16
125
62.5*
16
Ph
250*
8
250*
8
SAL-3
3a
3b
parent salicylanilide
8
8
F
CH3
CH3
CH3
Ph
CH3
CH3
Ph
O
S
32
32
16
125
32
125
32
62.5
16
125
32
125
32
62.5
16
125
32
125
32
8
7
1.64
0.33
F
8
32
3c
3d
F
O
O
62.5
250*
2
250*
250*
4
250*
250*
8
62.5
62.5*
2
125
62.5*
4
125*
62.5*
4
62.5
125*
2
125
125*
4
125*
125*
4
14 1.09
22 2.02
F
Ph
250*
1
SAL-4
4a
CF3
CF3
CF3
CF3
CF3
H
parent salicylanilide
6
1.9
11 0.50
12 1.97
16 2.17
21 1.12
CH3
CH3
CH3
Ph
CH3
CH3
Ph
O
S
250*
16
250*
16
250*
16
250*
32
250*
32
250*
16
250*
32
250*
32
>1000
4
>1000
4
4b
4c
O
O
125*
125*
32
125*
125*
125
1000
250*
250*
125
1000
250*
250*
250
1000
125*
125*
125
1000
125*
125*
250
1000
125*
125*
250
1000
125*
125*
125
1000
125*
125*
250
1000
125*
125*
250
1000
4d
Ph
SAL-5
5a
5b
parent salicylanilide
9 3.8
10 1.12
H
H
CH3
CH3
CH3
Ph
CH3
CH3
Ph
O
S
1000
6
3
8
1.53
0.58
0.74
H
H
>
1000
250
125
0.5
>
1000
>1000
>1000
>
1000
250
>
1000
500
>1000
>
1000
250
125
2
>
1000
500
125
4
>1000
5c
H
H
O
O
250
125
1
500
125
1000
125
>1000
>1000
5d
H
H
Ph
62.5
250
62.5
250
62.5
125
0
-0.6
INH
4 or 8
>
>
>250
>250
>250

PAS
3-NP
62.5
1000
62.5
1000
32
125
125
1000
1000
1000
>1000
1000
32
125
500
1000
10 1.60
25 4.1
0.88
-0.4
>
>
>
1000
>1000
>
>
>
>
1000
>1000
>
Ph: phenyl; INH: isoniazid; PAS: p-aminosalicylic acid; 3-NP = 3-nitropropionic acid. NT – not tested. * at the specified concentration, the growth of the tested strain was observed; at duplex concentration, precipitate and/or
turbidity was present, therefore it was not possible to determine the exact MIC value.
The best values for each strain are provided in bold.

Table 2. Antibacterial and antifungal activity of salicylanilide carbamates and thiocarbamates (1-5)
MIC/IC95/IC50[µM]
SE
SA
MRSA
EF
TM
72 h 120 h
31.25 31.25
Code
R2
X
24 h
62.5
3.9
48 h
125
24 h
48 h
125
24 h
125
48 h
125
24 h
125
48 h
>125
>125
>500
>125
>500
>500
15.62
62.5
-
1a
1b
2b
3a
3b
4b
Cl
Cl
Br
F
O
S
S
O
S
S
62.5
3.9
1.95
1.95
125
7.81
500
-
1.95
1.95
125
7.81
500
-
7.81
0.49
>125
7.81
0.49
7.81
0.49
>125
31.25
0.49
>125
125
0.49
125
0.49
>500
125
>125 >125
F
15.62 31.25 31.25 31.25 31.25 31.25
>500
>500
7.81
CF3
7.81
0.98
7.81
-
7.81
0.98
7.81
62.5
7.81
125
31.25 31.25
250 250
PNC
BAC
FLU
AMB
15.62 15.62 15.62 15.62 31.25 15.62
-
-
-
-
-
-
-
-
7.81
1.95
125
1.95
-
-
-
-
-
-
-
-
SA: Staphylococcus aureus CCM 4516/08; MRSA: methicillin-resistant Staphylococcus aureus H 5996/08; SE: Staphylococcus epidermidis H
6966/08; EF: Enterococcus sp. J 14365/08. TM: Trichophyton mentagrophytes 445. PNC: benzylpenicillin; BAC: bacitracin, FLU: fluconazole,
AMB: amphotericin B.
The best values for each strain are provided in bold.

Table 3. Cytotoxicity and selectivity indices of parent salicylanilides (SAL-1-SAL-5), carbamates
and thiocarbamates (1-5)
SI for
SI for M.
tuberculosis
SI for S.
aureus
Code
R1
R2
R3
R4
X
IC50 [µM]
atypical
mycobacteria
1.4-5.4
1.5-3.0
5.2-10.4
≤9.0
SI for TM
SAL-1
1a
1b
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Br
Br
Br
Br
Br
F
parent salicylanilide
21.71
188.7
83.51
289.0
5.4
11.8
-
1.5-3.0
11.1
6.0
CH3
CH3
CH3
CH3
O
S
10.4-20.9
9.0
10.7-21.4
-
42.8
-
1c
CH3
phenyl
phenyl
O
O
-
-
-
-
1d
phenyl
>100
15.72
342.5
85.64
945.1
SAL-2
2a
2b
parent salicylanilide
3.9
1.0-3.9
≤5.5
-
16.0
-
-
CH3
CH3
CH3
CH3
O
S
21.4
10.7
2.7-5.4
≤29.5
-
174.8
-
43.9
-
2c
CH3
phenyl
phenyl
O
O
29.5
-
-
-
2d
phenyl
>100
26.06
444.5
116.8
512.0
SAL-3
3a
3b
parent salicylanilide
3.3
0.8-3.3
3.6-7.1
3.7-7.3
≤8.2
-
3.3
F
CH3
CH3
CH3
CH3
O
S
14.0
7.3-14.6
8.2-16
3.6
≤3.6
3.7-7.5
F
15.0
-
-
-
3c
F
CH3
phenyl
phenyl
O
O
-
-
-
3d
F
phenyl
>100
0.36
SAL-4
4a
4b
Cl CF3
Cl CF3
Cl CF3
Cl CF3
parent salicylanilide
0.18-0.36
0.05-0.18
-
-
0.05-0.09
CH3
CH3
CH3
CH3
CH3
O
S
6508.0
67.29
>100
-
16.8
-
-
<1
-
2.1-4.2
-
8.6
-
-
4c
phenyl
phenyl
O
O
-
0.7-2.08
6.0
-
-
-
4d
SAL-5
5a
Cl CF3 phenyl
>100
H
H
H
H
H
H
H
H
H
H
parent salicylanilide
89.80
5985.9
>500
0.36-0.72
6.0
-
-
CH3
CH3
CH3
CH3
O
S
-
-
-
-
-
-
5b
279.9
211.1
-
-
5c
CH3
phenyl
phenyl
O
O
1.1
≤1.1
1.7-3.4
5d
phenyl
1.7
-
-
≥31.3 (only
Mk)
-
-
INH
>250
>250
PAS
3-NP
PNC
BAC
FLU
AMB
2,240
692.5
>10,000
254.6
>300
35.8
<0.7
<2.2-70
<0.7
-
-
-
-
-
-
-
-
-
-
-
-
-
>40->10,204
8.1-32.6
-
-
>2.4->38.4
1.3
2.5
-
SI = IC50/MIC. SI values higher than 10 are given in bold. Mk = M. kansasii 6509/96. MICs of salicylanilides 1, 2, 3 and 4 against T. mentagrophytes
were reported previously 19
.

Graphical abstract