Combating highly resistant emerging pathogen Mycobacterium abscessus and Mycobacterium tuberculosis with novel salicylanilide esters and carbamates

Article abstract of DOI:10.1016/j.ejmech.2015.07.001

Abstract In the Mycobacterium genus over one hundred species are already described and new ones are periodically reported. Species that form colonies in a week are classified as rapid growers, those requiring longer periods (up to three months) are the mostly pathogenic slow growers. More recently, new emerging species have been identified to lengthen the list, all rapid growers. Of these, Mycobacterium abscessus is also an intracellular pathogen and it is the most chemotherapy-resistant rapid-growing mycobacterium. In addition, the cases of multidrug-resistant Mycobacterium tuberculosis infection are also increasing. Therefore there is an urgent need to find new active molecules against these threatening strains. Based on previous results, a series of salicylanilides, salicylanilide 5-chloropyrazinoates and carbamates was designed, synthesized and characterised. The compounds were evaluated for their in vitro activity on M. abscessus, susceptible M. tuberculosis H₃₇Rv, multidrug-resistant (MDR) M. tuberculosis MDR A8, M. tuberculosis MDR 9449/2006 and on the extremely-resistant Praha 131 (XDR) strains. All derivatives exhibited a significant activity with minimum inhibitory concentrations (MICs) in the low micromolar range. Eight salicylanilide carbamates and two salicylanilide esters exhibited an excellent in vitro activity on M. abscessus with MICs from 0.2 to 2.1 μM, thus being more effective than ciprofloxacin and gentamicin. This finding is potentially promising, particularly, as M. abscessus is a threateningly chemotherapy-resistant species. M. tuberculosis H₃₇Rv was inhibited with MICs from 0.2 μM, and eleven compounds have lower MICs than isoniazid. Salicylanilide esters and carbamates were found that they were effective also on MDR and XDR M. tuberculosis strains with MICs ≥1.0 μM. The in vitro cytotoxicity (IC50) was also determined on human MonoMac-6 cells, and selectivity index (SI) of the compounds was established. In general, salicylanilide derivatives substituted by halogens on both salicyl and aniline rings showed better activity, than 4-benzoylaniline derivatives. The ester or carbamate bond formation of parent salicylanilides mostly retained or improved antimycobacterial potency with moderate selectivity.

Full text of DOI:10.1016/j.ejmech.2015.07.001

Accepted Manuscript
Combating highly resistant emerging pathogen Mycobacterium abscessus and
Mycobacterium tuberculosis with novel salicylanilide esters and carbamates
Zsuzsa Baranyai, Martin Krátký, Jarmila Vinšová, Nóra Szabó, Zsuzsa Senoner, Kata
Horváti, Jiřina Stolaříková, Sándor Dávid, Szilvia Bősze
PII:
S0223-5234(15)30133-1
10.1016/j.ejmech.2015.07.001
EJMECH 7982
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 10 March 2015
Revised Date: 29 June 2015
Accepted Date: 1 July 2015
Please cite this article as: 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, European
Journal of Medicinal Chemistry (2015), doi: 10.1016/j.ejmech.2015.07.001.
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salicylanilides
R1 = Cl, Br
R2 = 4-CF3, 3,4-diCl, 4-CO-Ph
R3 = CH3, Cl, CH2Cl
• Mycobacterium abscessus
• Mycobacterium tuberculosis H37Rv
• multidrug-resistant M. tuberculosis A8 and 9449/2006
• extremely-resistant M. tuberculosis Praha 131
in vitro
antimycobacterial
inhibition
colony forming
minimal inhibitiory
concentration
(Sula)
unit
(LJ)
salicylanilide
5-chloropyrazinoates
salicylanilide carbamates

ACCEPTED MANUSCRIPT
Combating highly resistant emerging pathogen Mycobacterium abscessus and Mycobacterium
tuberculosis with novel salicylanilide esters and carbamates
Zsuzsa Baranyai1, Martin Krátký2, Jarmila Vinšová2, Nóra Szabó3, Zsuzsa Senoner3, Kata Horváti1,
Jiřina Stolaříková4, Sándor Dávid1,3, and Szilvia Bősze1
1
MTA-ELTE Research Group of Peptide Chemistry, Pázmány Péter Sétány 1/A, Budapest, H-1117,
Hungary, P.O. Box 32, 1518 Budapest 112, Hungary; e-mail: bosze@elte.hu (Sz.B.);
baranyaizsuzs@gmail.com (Zs.B.), khorvati@gmail.com (K.H.)
Department of Inorganic and Organic Chemistry, Faculty of Pharmacy, Charles University,
2
Heyrovského 1203, 500 05 Hradec Králové, Czech Republic; e-mail: martin.kratky@faf.cuni.cz
(M.K.); jarmila.vinsova@faf.cuni.cz (J.V.)
Laboratory of Bacteriology, Korányi National Institute for Tuberculosis and Respiratory Medicine,
3
Pihenő út 1, Budapest, H-1122, Hungary; e-mail: nora.szabo@koranyi.hu (N.Sz.)
4 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.
(J. S.)
* Corresponding author: Szilvia Bősze: Pázmány Péter Sétány 1/A, Budapest, H-1117, Hungary, P.O.
Box 32, 1518 Budapest 112, Hungary. e-mail: bosze@elte.hu, tel.: +36-372-2500 ext. 1736, fax:
+36-372-26-20.
Abstract
In the Mycobacterium genus over one hundred species are already described and new ones are
periodically reported. Species that form colonies in a week are classified as rapid growers, those
requiring longer periods (up to three months) are the mostly pathogenic slow growers. More recently,
new emerging species have been identified to lengthen the list, all rapid growers. Of these,
Mycobacterium abscessus is also an intracellular pathogen and it is the most chemotherapy-resistant
rapid-growing mycobacterium. In addition, the cases of multidrug-resistant Mycobacterium
tuberculosis infection are also increasing. Therefore there is an urgent need to find new active
molecules against these threatening strains. Based on previous results, a series of salicylanilides,
salicylanilide 5-chloropyrazinoates and carbamates was designed, synthesized and characterised. The
compounds were evaluated for their in vitro activity on M. abscessus, susceptible M. tuberculosis
H37Rv, multidrug-resistant (MDR) M. tuberculosis MDR A8, M. tuberculosis MDR 9449/2006 and on
the extremely-resistant Praha 131 (XDR) strains. All derivatives exhibited a significant activity with
minimum inhibitory concentrations (MICs) in the low micromolar range. Eight salicylanilide
carbamates and two salicylanilide esters exhibited an excellent in vitro activity on M. abscessus with
MICs from 0.2 to 2.1 ꢀM, thus being more effective than ciprofloxacin and gentamicin. This finding is
potentially promising, particularly, as M. abscessus is a threateningly chemotherapy-resistant species.
M. tuberculosis H37Rv was inhibited with MICs from 0.2 ꢀM, and eleven compounds have lower MICs
than isoniazid. Salicylanilide esters and carbamates were found that they were effective also on MDR
and XDR M. tuberculosis strains with MICs ≥ 1.0 ꢀM. The in vitro cytotoxicity (IC50) was also
determined on human MonoMac-6 cells, and selectivity index (SI) of the compounds was established.
In general, salicylanilide derivatives substituted by halogens on both salicyl and aniline rings
showed better activity, than 4-benzoylaniline derivatives. The ester or carbamate bond formation of
parent salicylanilides mostly retained or improved antimycobacterial potency with moderate selectivity.
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Keywords
antimycobacterial activity; Mycobacterium abscessus, multidrug-resistant Mycobacterium
tuberculosis; salicylanilide; salicylanilide carbamate; salicylanilide 5-chloropyrazinoate
2

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1. Introduction
The common water contaminant Mycobacterium abscessus is an important human pathogen
responsible for a wide spectrum of infectious diseases, chronic lung disease in patients with cystic
fibrosis, post-traumatic wound infections, soft tissue infections and disseminated cutaneous diseases,
mostly in patients with suppressed immune system. M. abscessus has acquired the reputation of being
the most virulent and chemotherapy-resistant rapidly growing mycobacterial strain. The treatment of
M. abscessus infections is difficult because of the high rate of intrinsic resistance mediated by multiple
mechanisms to both antitubercular drugs and to most of the available antibiotics. Long course of
chemotherapy is required. Antibiotic treatment is guided by in vitro susceptibility testing. Current
guidelines recommend combining a macrolide (clarithromycin or azithromycin) with one
aminoglycoside and one other drug such as moxifloxacin, linezolid, tigecycline, cefoxitin or imipenem.
Clinical efficacy of this multidrug regimen is still controversial. Unfortunately, there is an additional
evidence that none of the commonly administered antimicrobial drugs exerts bactericidal activity in M.
abscessus sensu lato, thus resulting in the poor therapeutic outcome [1,2,3,4].
Tuberculosis (TB) still represents a global and public health problem especially in developing
countries. Appearance of multidrug-resistant (MDR) TB strains resistant at least to isoniazid (INH) and
rifampicin (RIF) and co-infection with HIV makes TB treatment challenging. In 2013 the cases of
MDR-TB were estimated at 480,000 among patients with notified pulmonary TB [5].
The increased number and emergence of resistant mycobacterial strains necessitate the development
of new potent antimycobacterial drugs with enhanced activity with no cross-resistance to clinically
used drugs, fast-acting mechanism, reduced toxicity, enhanced cell penetrating ability and good effect
in the intracellular environment.
Salicylanilides (2-hydroxy-N-phenylbenzamides) have shown a wide variety of interesting
biological activities including antimycobacterial effect. An overview of the previously published most
potent substituted salicylanilide derivatives is summarized in Table 1. They have expressed activity not
only against M. tuberculosis H37Rv, but also against slowly growing mycobacterial strains (M. avium
and M. kansasii), where the standard antituberculotics fail [6]. Their mechanism of action is multiple
and complex, they have displayed the ability to inhibit various bacterial enzymes, e.g., transglycosylase
[7],
D
-alanine-
D
-alanine ligase [8], sortase A [9], mycobacterial isocitrate lyase [10,11,12,13,14],
-alanine dehydrogenase [13], and the two-component regulatory
methionine aminopeptidase [14],
L
systems [15]. Salicylanilides could function as proton shuttles that kill cells by destroying the cellular
proton gradient (uncoupling effect) [16,17].
The temporary masking of hydroxy group of salicylanilides results in improved physicochemical
properties and thus better bioavailability, membrane permeability, higher activity and/or lower toxicity.
In general, salicylanilide esters showed prevailingly improved antimicrobial activity when compared to
parent compounds. They were also active against MDR-TB strains [10,18,19].
Pyrazinamide (PZA), a widely applied first-line anti-TB oral drug, helps to shorten the duration of
the therapy because it kills a population of semi-dormant tuberculosis bacteria in acidic environment
that are not killed by other drugs [20,21]. Although PZA has remarkable in vivo activity, it is not active
against M. tuberculosis under ‘normal’ culture conditions near neutral pH [22]. The acidic pH
requirement for PZA action is the result of increased accumulation of pyrazinoic acid (POA), the active
form of pyrazinamide, in the tubercle bacilli at acidic pH but not at neutral pH [23]. The mechanism of
action of PZA was not clearly elucidated for a long time and more hypotheses about it were proposed
[21,24,25,26]. The newly recognized target of POA was described in 2011: the ribosomal protein S1
(RpsA) which is involved in protein translation and in the ribosome-sparing process of trans-translation
[27].
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The connection of two active molecules across an easily cleavable bridge as a new type of
potentially active molecule is an established approach. Previously described salicylanilide pyrazinoates
[10] can be considered being prodrugs of both salicylanilides and POA, into which these esters are
probably hydrolysed without the presence of pyrazinamidase/nicotinamidase. The most active
pyrazinoates showed MICs for drug-resistant strains in the range of 0.125-2 ꢀM and no cross-resistance
with clinically used drugs. They are the most in vitro efficacious salicylanilide esters so far. This
promising activity is probably due to an additive or synergistic effect of the released POA and
salicylanilides. Selectivity indexes for the most active derivatives ranged up to 64 [10].
5-Chloropyrazinamide, 5-chloropyrazine-2-carboxylic acid as well as its simple esters exhibited
moderate activity against M. tuberculosis including PZA-resistant strains, M. bovis, some strains of
nontuberculous mycobacteria and they are able to inhibit mycobacterial fatty acid synthase I
[28,29,30].
Masking of the phenolic group of salicylanilides by carbamate also protects the molecule against
extensive first-pass metabolism, improves physicochemical properties and broadens its activity profile
[31,32]. A series of salicylanilide N-monoalkyl carbamates exhibited excellent in vitro activity against
M. tuberculosis, atypical mycobacteria and, in particular, drug-resistant strains with MIC values
between 0.5-2 ꢀM. Moreover, they showed decreased cytotoxicity and extended hydrolytic stability
[32]. Salicylanilide N,N-dialkyl/aryl carbamates also inhibited the growth of various mycobacteria,
however they did not exceed the antimycobacterial activity of the parent salicylanilides [11].
Based on the excellent already published in vitro antimycobacterial activity of salicylanilide
scaffold [6] we evaluated four known salicylanilides also against M. abscessus, M. tuberculosis H37Rv
and M. tuberculosis A8 MDR strains. Since they were active, we decided to design and synthesize new
substituted salicylanilides and esters with 5-chloropyrazine-2-carboxylic acid concomitantly with
salicylanilide carbamates. This study can be considered as an additional investigation of previous work
on salicylanilide pyrazinoates [10] and salicylanilide carbamates [11,32] extended by the activity
against the Mycobacterium abscessus which is the most pathogenic and chemotherapy-resistant rapid-
growing mycobacterium.
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2. Results and Discussion
2.1. Chemistry
5-Chloropyrazine-2-carboxylic acid 1 was synthesized from 5-hydroxypyrazine-2-carboxylic acid
and thionyl chloride. The reaction was promoted by addition of a catalytic amount of N,N-
dimethylformamide (DMF) and then the intermediate was hydrolyzed according to previously
described procedure [33] (Scheme 1).
The starting salicylanilides 2 were selected according to their previously described high in vitro
antimycobacterial activity (2a-d) (Table 1) [6]. Two new salicylanilides (2e, 2f) were synthesized.
Carbonyl group was introduced as a benzoyl moiety to the salicylanilide scaffold in order to form
oxime or hydrazone bond with carrier molecules in future work. Salicylanilides 2 were prepared by the
reaction of the appropriate substituted salicylic acids and the substituted anilines in the presence of
phosphorus trichloride (0.5 eq.) in chlorobenzene (Scheme 2). The reaction was carried out in a
microwave reactor for 22 min to reflux. This procedure led to an increase of the yield and shortening
the reaction time from several hours to minutes [19]. The yields ranged from 26-93% and
salicylanilides substituted by benzoyl group (2e, 2f) produced significantly lower yields than those
substituted only by halogens (2a-d).
Salicylanilide esters 3 with 5-chloropyrazine-2-carboxylic acid 1 were obtained by the previously
described activation with N,N’-dicyclohexylcarbodiimide (DCC) in DMF (Scheme 3) [19] with yields
within 29-60%.
For the synthesis of carbamates 4, a suspension of the appropriate salicylanilide 1 in acetonitrile
(MeCN) was treated with one equivalent of triethylamine (TEA), followed by addition of the
corresponding isocyanate (1 eq.; Scheme 4) [32]. Chloroalkyl moiety was introduced in order to form
covalent bond with cysteine-containing carriers in future work. This reaction was performed at room
temperature due to thermal instability of the products with yields from 20 to 64%. The prepared
carbamates 4 belong into three series according to the substituent R3: 2-chloroethyl (R3 = Cl)
carbamates, 3-chloropropyl carbamates (R3 = CH2Cl) and propyl (R3 = CH3) carbamates.
In Table 2 the physicochemical properties (molecular weight, calculated log P and solubility) of the
compounds are summarized. Increased lipophilicity of esters 3 (log P: 4.45 - 4.92) in comparison with
both free acid 1 (log P: 0.24) and salicylanilides 2 (log P: 3.93 - 4.49) may facilitate transport through
cell walls and biomembranes.
Compounds that lack aqueous solubility could cause difficulties in in vitro studies (<10 mg/mL low,
10–60 mg/mL moderate, >60 mg/mL high solubility), but all of the compounds presented here,
however, showed at least moderate solubility, up to 100 ꢀM, in aqueous media as Sula and RPMI-1640
used for in vitro mycobacterial and cytotoxicity testing (Table 2).
The stability of the active ester and carbamate type compounds was determined in culture medium
sampling over 24 h, modelling the in vitro conditions. The selected representative results are
summarised in the Supplementary Information (SFigure 1-9). The salicylanilide esters were more
stable, their half-time was significantly higher (> 3 h) than the half-time of carbamates. The release of
the parent salicylanilide and 5-chloropirazin-2-carboxylic acid was observed (SFigure 4-6). The half-
time of the carbamates was markedly low (< 1h, SFigure 7-9), parent salicylanilide was also detected as
a released compound. According to the stability of salicylanilide 5-chloropyrazinoates, similarly as
authors published earlier for salicylanilide pyrazinoates [10], we can also hypothesize that 5-
chloropyrazinoates 3 might be considered as prodrugs with a prolonged liberation.
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2.2. In Vitro Antimycobacterial Activity
5-Chloropyrazine-2-carboxylic acid 1, salicylanilides 2, esters 3 and carbamates 4 were evaluated
for their in vitro antimycobacterial activity against a fast growing mycobacterium – Mycobacterium
abscessus, and two strains of slowly growing Mycobacterium tuberculosis: H37Rv and MDR strain A8
(resistant to both INH and RIF, two first-line antituberculars). INH, ciprofloxacin (CIPX) and
gentamicin (GEN) as antimycobacterial active drugs were involved for comparison. Table 3 reports
obtained minimum inhibitory concentrations (MICs).
In our assay, 5-chloropyrazine-2-carboxylic acid 1 itself had no in vitro activity at low
concentrations on the extracellular bacteria. This molecule is effective on the intracellular bacteria at
acidic conditions [21].
The in vitro activity of the compounds was studied on the M. abscessus (Table 3), which is a
common environment contaminant human pathogen responsible for a wide spectrum of infectious
diseases. M. abscessus is a chemotherapy-resistant strain (resistant to INH, RIF, polypeptides,
quinolones, tetracyclines, p-aminosalicylic acid, thioamides and most of the aminoglycosides) [34].
The MIC values of the control drugs INH, CIPX and GEN were 40 ꢀg/mL (291.8 ꢀM), 1 ꢀg/mL (3.0
ꢀM) and 5 ꢀg/mL (10.5 ꢀM) respectively.
Fortunately, our salicylanilide derivatives have uniquely good inhibitory effect (0.1-20 ꢀg/mL, i.e.,
0.2-43.6 ꢀM; Table 3) on M. abscessus. In summary, derivatives of 5-chlorosalicylic acid showed
better activity than those derived from 5-bromosalicylic acid, among the esters 3,4-dichloroaniline
fragment improved the activity in comparison with 4-CF3-aniline. Both halogenated anilines showed
sharp superiority to 4-benzoylaniline derivatives (MICs from 22.9 ꢀM). When focused on derivatives
of salicylanilides 2a-d, the presence of carbamate linkage (4a-i) confers the highest activity with MICs
of 0.1-2 ꢀg/mL (0.2-4.3 ꢀM), followed by esters 3a-d (1.0-5.5 ꢀM) and then by parent salicylanilides
2a-d (3.2-13.9 ꢀM). The esterification by 5-chloropyrazine-2-carboxylic acid represents an
advantageous approach for this strain.
Among the carbamates 4a-e, 4g and 4h exhibited the highest activity (0.2-1.3 ꢀM) and the MIC
values of 4b, 4g and 4h are 15-times lower than MIC of ciprofloxacin (3.0 ꢀM) and 53-times lower
than those obtained for gentamicin (10.5 ꢀM). These drugs may be used in the treatment of M.
abscessus infections, but acquired resistance has hampered their wide use. One additional carbamate
(4i) and two salicylanilide esters (3b, 3d) share lower MICs than CIPX, and two salicylanilides 2a-b
showed comparable growth inhibition. With the exception of N-(4-benzoylphenyl)-2-
hydroxybenzamide derivatives and salicylanilide 2c, the remaining compounds were more active in
vitro than GEN. INH was practically inactive as well as 5-chloropyrazine-2-carboxylic acid 1.
This is first evidence that salicylanilide-based molecules are able to inhibit M. abscessus, even at
low micromolar concentrations. With respect to the lack of highly effective drugs without any
resistance and poor therapeutic outcomes, our findings are potentially very promising.
All of the evaluated compounds 2-4 showed significant activity against drug-susceptible M.
tuberculosis H37Rv (Table 3). Polyhalogenated salicylanilides 2a-d exhibited MICs of 0.5 ꢀg/mL (i.e.,
1.4-1.6 ꢀM), thus being approximately ten-fold more active than derivatives of 4-benzoylaniline 2e and
2f with MIC of 5 ꢀg/mL (12.7-14.2 ꢀM). Four esters 3 share low MIC values up to 1 ꢀg/mL (≤2.0 ꢀM).
Carbamates derived from salicylanilides 2a-d exhibited excellent in vitro antitubercular activity with
MICs of 0.1-1 ꢀg/mL (0.2-2.3 ꢀM), while salicylanilide carbamates where R2= 4-CO-Ph showed
slightly higher MIC values of 4 ꢀg/mL (8.0-9.2 ꢀM). The most active molecules are 4-chloro-2-[(3,4-
dichlorophenyl)carbamoyl]phenyl
dichlorophenyl)carbamoyl]phenyl
propylcarbamate
(2-chloroethyl)carbamate
4e,
4-bromo-2-[(3,4-
4-bromo-2-{[4-
4h,
(trifluoromethyl)phenyl]carbamoyl}phenyl (2-chloroethyl)carbamate 4f and 4-bromo-2-[(3,4-
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dichlorophenyl)carbamoyl]phenyl (3-chloropropyl)carbamate 4i with MICs of 0.2, 0.2, 0.4 and 0.4 ꢀM,
respectively. These four compounds exhibited also significantly higher activity than isoniazid
(1.17 ꢀM). On the other side, seven derivatives produced comparatively higher MICs than this essential
drug (2e, 2f, 4j, 4k, 4l, 4m, 4n).
In case of the in vitro activity on the M. tuberculosis H37Rv the esterification or carbamoylation of
parent salicylanilides mostly retained or improved antimycobacterial activity, i.e., more than seven
times (2d vs. 4h, or 2b vs. 4e). For the high in vitro efficacy of O-modified salicylanilide derivatives,
4-Br and 4-Cl at the salicylic ring are both interchangeable as well as the 3,4-diCl and 4-CF3
substituents of the aniline ring. The antimycobacterial action of carbamates 4 is not sharply influenced
by the N-substituting moiety: 2-chloroethyl, 3-chloropropyl and propyl carbamates produced
comparable results.
The multidrug-resistant strain A8 was affected at similar or only slightly higher concentrations than
the H37Rv strain (0.5-10 ꢀg/mL, i.e. 1.0-21.3 ꢀM; Table 3). Similarly to the drug-susceptible H37Rv
strain, derivatives with incorporated 3,4-dichloro- or 4-trifluoromethylaniline moieties share escalated
anti-MDR action within a narrow concentration range of 1.0-4.4 ꢀM. O-Substituted salicylanilide
derivatives showed mostly decreased MICs when compared to parent salicylanilides. Carbamates 4g
and 4i showed the lowest MIC of 1.0 ꢀM. Only six compounds (2e, 2f, 4j-4m) showed higher MICs
than INH (> 7.3 ꢀM); all of them are derivatives of 4-benzoylaniline.
Ten compounds among the salicyilanilde esters and carbamates with the lowest MIC (≤1.1 ꢀM)
against the M. tuberculosis H37Rv (3a, 3b, 3c, 4a, 4b, 4e, 4f, 4g, 4h and 4i) were evaluated against two
different drug-resistant strains with a different method. The M. tuberculosis 9449/2006 is an MDR
strain (resistant to INH, RIF, rifabutine and streptomycin) and the M. tuberculosis Praha 131 strain is
an extremely-resistant (XDR) strain (resistant to INH, RIF, rifabutine, streptomycin, ethambutol,
ofloxacin, gentamicin and amikacin). Table 4 reports the obtained MICs of the ten compounds. Both
the evaluated esters and the carbamates revealed excellent activity on the MDR and XDR strain within
the concentration range of 0.5-2 ꢀM. The MDR and XDR strains showed similar susceptibility despite
their different resistance pattern. The lowest MIC after 21 days of incubation against MDR-TB
9449/2006 was found for two carbamates, 4a and 4e (with MICs of 1 ꢀM). Against the XDR-TB
salicylanilide ester 3a and two carbamates 4e and 4i (with MICs 1 ꢀM) showed the highest activity
after 21 days of incubation.
In comparison with the previously described salicylanilide pyrazinoates [10], here reported
salicylanilide 5-chloropyrazinoates 3a-c displayed comparable activity against the multidrug-resistant
M. tuberculosis 9449/2006 and Praha 131 (XDR) strains (see Table 4). The salicylanilide pyrazinoate
analogs of the salicylanilid 5-chloropyrazinoates showed slightly lower MICs for these two strains, this
finding suggest that the presence of the chlorine on the pyrazine ring is not necessary.
The low MIC values against the M. tuberculosis 9449/2006 and Praha 131 indicate that the selected
salicylanilide esters and carbamates could be used as potential agents for combating MDR and XDR-
TB.
The influence of the lipophilicity (calculated logP) on the activity of the compounds against bacteria
is not direct. 5-chloropyrazine-2-carboxylic acid has the lowest logP value (0.24) and it was not
effective on the tested bacteria under the given circumstances as mentioned before. The salicylanilide
5-chloropyrazionates have higher logP values than the 5-chloropyrazine-2-carboxylic acid and the
parent salicylanilides itself. This finding results in slight improvement in the activity of the esters
compared to the parent salicylanilides in general. The carbamates also have higher lipophilicity then
their parent salicylanilides. The most lipophilic derivatives according to the logP values are
salicylanilide carbamates 4i (5.09), 4m (5.08) and 4n (5.19). Moreover 4i has excellent activity against
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the tested mycobacteria, especially on the MDR and XDR TB strains. This compound has moderate
cytotoxicity, the selectivity indexes of 4i range between 4.5 and 23.8 (Table 6).
2.2. In Vitro Cytotoxic Activity
Determination of the in vitro cytotoxic effect of the compounds was carried out on human
monocytic cell line MonoMac6 using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide) assay. The IC50 values (the concentration which decreases the viability of the cells to 50%
from the maximal viability) were determined from the dose-response curves (Table 5). The MonoMac6
cells are considered as an in vitro model of the host cell macrophages. The selectivity indexes (SI)
related to each bacterial strain were determined as a ratio of the IC50 value for cytotoxic activity to the
MIC value (Table 5).
Free 5-chloropyrazin 2-carboxylic acid 1 did not show cytotoxic effect at the measured
concentration range (up to 250 ꢀM). The parent salicylanilides, salicylanilide 5-chloropyrazinoates and
carbamates showed high to moderate cytotoxic activity on the MonoMac 6 cells. Two carbamates, 4j
and 4m expressed the highest IC50 of 37.8±4.3 and 24.9±4.5 ꢀM, respectively. On the other hand two
esters 3a and 3c showed the lowest values of 1.1±1.6 and 1.1±0.5 ꢀM, respectively. Based on these
findings it seems that both antimycobacterial and cytotoxic properties are mostly associated with the
salicylanilide core. Salicylanilide derivatives substituted by benzoyl group on the aniline ring 2d, 2e,
4j-e, showed lower cytotoxicity (with IC50 values of 12.6-48.0 ꢀM) than salicylanilide derivatives
substituted by halogens on the aniline ring (with IC50 values of 1.1-10.1 ꢀM). However these 4-
benzoylaniline derivatives possessed significantly higher MIC values which means that their selectivity
is also low.
Selectivity indexes ranged from 0.2 up to 44.0 for M. abscessus; from 1.0 up to 35.0 for M.
tuberculosis H37Rv; from 0.6 up to 9.5 for M. tuberculosis A8 MDR; from 0.6 up to 7.6 for M.
tuberculosis 9449/2006 MDR; and from 0.6 up to 9.5 for M. tuberculosis Praha 131 XDR.
SI values higher than 10 indicate rather acceptable toxicity and selectivity for bacteria. In this aspect
salicylanilide carbamate 4b exhibited the most favourable SI values for most of the mycobacterium
strains (SI from 4.4 up to 44.0), making it the most attractive drug-candidate from the tested
salicylanilide derivatives, especially in combination with its very low MIC values on the different
mycobacteria strains (0.2-2 ꢀM).
Contrary to our expectations the salicylanilide esters did not show lower cytotoxicity than the parent
salicylanilides [10]. In our case masking of the phenolic group of the salicylanilides with ester or
carbamate bond did not result in significant decrease of cytotoxicity.
8

ACCEPTED MANUSCRIPT
3. Conclusions
In this study, we synthesized and evaluated novel salicylanilides, salicylanilide 5-
chloropyrazinoates and N-alkyl (or N-chloroalkyl) carbamates as potential antimycobacterial agents.
All of these derivatives exhibited notable in vitro micromolar or submicromolar activity in spite of the
presence of multidrug-resistance. This is the first and unique evidence that salicylanilide-based
compounds (parent amides, their esters and carbamates) are able to inhibit a chemotherapy-resistant
species of Mycobacterium abscessus at very low concentrations similar to those obtained for both drug-
susceptible, MDR and XDR M. tuberculosis. The esterification or carbamoylation of parent
salicylanilides mostly improved antimycobacterial activity. Especially the carbamoylation of
polyhalogenated salicylanilides provided promising derivatives with excellent activity against both M.
abscessus and M. tuberculosis (MICs ≥ 0.2 µM). Unfortunately, in many cases the outstanding
antimycobacterial activity of the salicylanilide derivatives is overshadowed by their high or moderate
cytotoxicity to mammalian cells, however compounds with acceptable selectivity have been found.
Future works need focusing on increasing the selectivity.
4. Experimental Section
4.1. Chemistry
4.1.1. General Methods
All reagents and solvents were purchased from Sigma-Aldrich and Merck. Commercial grade
reagents were used without a further purification. Reactions were monitored by thin layer
chromatography plates coated with 0.2 mm silica gel 60 F254 (Merck), which were visualized by UV
irradiation (254 nm). All melting points were determined on a Melting Point machine B-540 (Büchi)
apparatus using open capillaries and they are uncorrected. Infrared spectra (KBr pellets) were recorded
on FT-IR spectrometer Nicolet 6700 FT-IR (Thermo Fisher Scientific, Waltham, MA, USA) in the
range of 400-4000 cm-1 using the ATR technique. The NMR spectra were measured at ambient
1
13
temperature on a Varian Mercury-Vxbb 300 (300 MHz for H and 75.5 MHz for C; Varian Comp.
1
13
Palo Alto, USA) or with a Varian VNMR S500 instrument (500 MHz for H and 126 MHz for C;
Varian Comp. Palo Alto, CA, USA) using deuterated dimethyl sulfoxide (DMSO-d6; for
salicylanilides, 5-chloropyrazine-2-carboxylic acid and carbamates) or CDCl3 (for esters) solutions of
the samples. The chemical shifts, δ, are given in ppm, related to tetramethylsilane as an internal
standard. The coupling constants (J) are reported in Hz. Elemental analysis (C, H, N) was performed on
an automatic microanalyser CHNS-O CE instrument (FISONS EA 1110, Milano, Italy). Measured
monoisotopic molecular mass was acquired by Bruker Esquire 3000+ ESI-MS. Samples were dissolved
in a mixture of acetonitrile/water = 1/1 (v/v) containing 0.1% acetic acid and introduced by a syringe
pump with a flow rate of 10 ꢀL/min. The log P values (the calculated logarithm of the partition
coefficient for n-octanol/water) were calculated using the program CS ChemOffice Ultra ver. 11.0
(CambridgeSoft, Cambridge, MA, USA).
4.1.2. Synthesis of 5-chloropyrazine-2-carboxylic acid 1
5-Hydroxypyrazine-2-carboxylic acid (2 mmol) was suspended in 6 mL of thionyl chloride and 200
ꢀL of N,N-dimethylformamide (DMF) was added. The mixture was heated at 75 °C for 4 hours. After
this period, excess of thionyl chloride was distilled off to get crude compound and the reaction
intermediate was hydrolysed according to previously described procedure [33] to obtain 5-
chloropyrazine-2-carboxylic acid 1.
9

ACCEPTED MANUSCRIPT
5-Chloropyrazine-2-carboxylic acid (1) [33]
Brown solid; yield: 75%; mp: decomposes above 150 °C. IR (ATR): 3408, 1708 (C=O), 1524, 1322,
1298, 1271, 1167, 1122, 1026, 920, 884, 800, 740, 720, 652. 1H NMR (300 MHz, DMSO-d6): δ 13.87
(1H, s, OH), 9.00 (1H, d, J = 1.3 Hz, H3), 8.90 (1H, d, J = 1.4 Hz, H6). 13C NMR (75 MHz, DMSO-
d6): δ 164.54, 151.30, 145.64, 144.61, 142.66. MSmonoizotopic (calc/meas): 158.0 / 158.0. Anal. Calcd. for
C5H3ClN2O2 (158.54): C, 37.88; H, 1.91; N, 17.67; Found: C, 38.02; H, 1.88; N, 17.54.
4.1.3. Synthesis of salicylanilides 2
Salicylanilides 2 were prepared by the reaction of the appropriate substituted salicylic acids (5
mmol) and the substituted anilines (5 mmol) in the presence of phosphorus trichloride (2.5 mmol) in
chlorobenzene [19]. The reaction was carried out with vigorously stirring in a microwave reactor
(MicroSYNTH MLS ETHOS 1600 URM) for 22 min to reflux. The reaction mixture was filtered while
hot, let stand at 20 °C and then at 4 °C for 24 h. The crude product was filtered off and once or more
times recrystallized from aqueous ethanol to obtain the pure product. The mother liquor was partly
evaporated to the crystallization to obtain the second portion of crude product.
Parent salicylanilides 2a, 2b, 2c and 2d were reported previously [6].
N-(4-Benzoylphenyl)-5-chloro-2-hydroxybenzamide (2e)
White solid; yield: 44%; mp: 218.0-218.8 °C. IR (ATR): 3314 (NH), 1644, 1621 (CONH-I), 1599
1
(CONH-II), 1222 (C-O). H NMR (300 MHz, DMSO): δ 11.63 (1H, s, OH), 10.67 (1H, bs, NH),7.94-
7.88 (3H, m, H6, H3´, H5´), 7.81-7.76 (2H, m, H2´, H6´), 7.75-7.70 (2H, m, H2´´, H6´´), 7.65 (1H, tt, J
= 1.9 Hz, J = 7.4 Hz, H4´´), 7.59-7.52 (2H, m, H3´´, H5´´), 7.46 (1H, dd, J = 2.7 Hz, J = 8.8 Hz, H4),
13
7.03 (1H, d, J = 8.8 Hz, H3). C NMR (75 MHz, DMSO): δ 194.78, 165.14, 156.43, 142.52, 137.61,
133.23, 132.53, 132.35, 131.21, 129.61, 128.86, 128.69, 123.01, 120.56, 119.92, 119.18. MSmonoizotopic
(calc/meas): 351.1 / 351.3. Anal. Calcd. for C20H14ClNO3 (351.07): C, 68.28; H, 4.01; N, 3.98; Found:
C, 68.23; H, 3.88; N, 3.56.
N-(4-Benzoylphenyl)-5-bromo-2-hydroxybenzamide (2f)
White solid; yield: 26%; mp: 229.7-231.1 °C. IR (ATR): 3314 (NH), 1644, 1620 (CONH-I), 1599
1
(CONH-II), 1221 (C-O). H NMR (500 MHz, DMSO): δ 11.64 (1H, s, OH), 10.66 (1H, bs, NH), 8.01
(1H, d, J = 2.6 Hz, H6), 7.92-7.88 (2H, m, H3´, H5´), 7.80-7.76 (2H, m, H2´, H6´), 7.74-7.71 (2H, m,
H2´´, H6´´), 7.66 (1H, tt, J = 1.9 Hz, J = 7.4 Hz, H4´´), 7.59-7.53 (3H, m, H4, H3´´, H5´´), 6.97 (1H, d,
J = 8.8 Hz, H3). 13C NMR (126 MHz, DMSO): δ 194.76, 165.07, 156.86, 142.49, 137.60, 136.03,
132.50, 132.35, 131.69, 131.18, 129.58, 128.67, 121.07, 119.93, 119.60, 110.41. MSmonoizotopic
(calc/meas): 395.0 / 395.2. Anal. Calcd. for C20H14BrNO3 (396.23): C, 60.62; H, 3.56; N, 3.53; Found:
C, 60.34; H, 3.54; N, 3.46.
4.1.4. Synthesis of salicylanilide esters 3
The corresponding substituted salicylanilide 2 (1 mmol) and 5-chloropyrazine-2-carboxylic acid 1
(1 mmol) were dissolved in dry DMF (8 mL), the solution was cooled to -10 °C and DCC (1.2 mmol)
was added in three portions during 1 h. The mixture was stirred for additional 3 hours at the same
temperature and then stored at +4 °C for 20 h. The precipitate of N,N´-dicyclohexylurea was removed
by filtration and the solvent was evaporated in vacuo till dryness. The crude products 3 were purified
by one or repeated crystallization from ethyl acetate-hexane mixture.
4-Chloro-2-{[4-(trifluoromethyl)phenyl]carbamoyl}phenyl 5-chloropyrazine-2-carboxylate (3a)
White solid; yield: 35%; mp: 163.4-165.0 °C. IR (ATR): 3372 (NH), 3081, 2934, 2857, 1751 (C=O
ester), 1663, 1648, 1633, 1600, 1530, 1520, 1477, 1411, 1323, 1297, 1274, 1259, 1190, 1163, 1125,
10

ACCEPTED MANUSCRIPT
1104, 1066, 1020, 879, 839, 826, 786, 715, 702, 668. 1H NMR (500 MHz, DMSO-d6): δ 9.40 (1H, bs,
NH), 9.23 (1H, d, J = 1.4 Hz, H3´´), 8.69 (1H, d, J = 1.4 Hz, H5´´), 8.13 (1H, d, J = 2.6 Hz, H3), 7.78
(2H, d, J = 8.4 Hz, H3´, H5´), 7.61 (2H, d, J = 8.3 Hz, H2´, H6´), 7.57 (1H, dd, J = 2.5 Hz, J = 8.6 Hz,
13
H5), 7.52 (1H, d, J = 8.7 Hz, H6); C NMR (126 MHz, CDCl3): δ 161.43, 160.35, 154.25, 146.70,
146.04, 144.31, 140.76, 139.71, 132.72, 131.40, 127.46, 126.66 (q, J = 32.8 Hz), 126.19 (q, J = 3.8
Hz), 123.93 (q, J = 272.6 Hz), 123.92, 120.45. MSmonoizotopic (calc/meas): 455.0 / 455.2; Anal. Calcd.
for C19H10ClF3N3O3 (456.20): C, 50.02; H, 2.21; N, 9.21; Found: C, 50.34; H, 2.45; N, 9.43.
4-Chloro-2-[(3,4-dichlorophenyl)carbamoyl]phenyl 5-chloropyrazine-2-carboxylate (3b)
White solid; yield: 60%; mp: 172.5-174.8 °C. IR (ATR): 3365 (NH), 3093, 2931, 2860, 1757 (C=O
ester), 1702, 1668, 1645, 1589, 1519, 1475, 1397, 1375, 1322, 1298, 1270, 1237, 1192, 1132, 1108,
1023, 911, 875, 860, 822, 709, 679, 663. 1H NMR (500 MHz, CDCl3): δ 9.33 (1H, bs, NH), 9.23 (1H,
d, J = 1.4 Hz, H3´´), 8.72 (1H, d, J = 1.4 Hz, H5´´), 8.16 (1H, d, J = 2.5 Hz, H2´), 7.80 (1H, d, J = 2.4
Hz, H3), 7.64 (1H, dd, J = 2.5 Hz, J = 8.8 Hz, H5), 7.57-7.55 (2H, m, H6, H6´), 7.41 (1H, d, J = 8.8
Hz, H5´). 13C NMR (126 MHz, DMSO): δ 161.07, 160.22, 154.48, 146.75, 146.20, 144.32, 139.87,
137.45, 132.88, 132.68, 131.75, 130.65, 130.63, 128.20, 127.28, 123.77, 122.54, 120.07. MSmonoizotopic
(calc/meas): 454.9 / 455.0. Anal. Calcd. for C18H9Cl4N3O3 (457.09): C, 47.30; H, 1.98; N, 9.19; Found:
C, 47.07; H, 2.04; N, 9.03.
4-Bromo-2-{[4-(trifluoromethyl)phenyl]carbamoyl}phenyl 5-chloropyrazine-2-carboxylate (3c)
White solid; yield: 29%; mp: 159.7-161.3 °C. IR (ATR): 3356 (NH), 3076, 2937, 2858, 1759 (C=O
ester), 1661, 1647, 1600, 1531, 1521, 1473, 1413, 1321, 1294, 1269, 1196, 1165, 1140, 1115, 1102,
1
1093, 1093, 1068, 1023, 874, 839, 820, 785, 711, 687, 653. H NMR (300 MHz, DMSO-d6): δ 9.36
(1H, bs, NH), 9.24 (1H, d, J = 1.4 Hz, H3´´), 8.70 (1H, d, J = 1.3 Hz, H5´´), 8.30 (1H, d, J = 2.5 Hz,
H3), 7.79 (2H, d, J = 8.4 Hz, H3´, H5´), 7.73 (1H, dd, J = 2.5 Hz, J = 8.6 Hz, H5), 7.62 (2H, d, J = 8.5
13
Hz, H2´, H6´), 7.46 (1H, d, J = 8.6 Hz, H6). C NMR (75 MHz, CDCl3): δ 161.26, 160.27, 154.29,
146.73, 146.59, 144.32, 140.76, 139.69, 135.73, 134.41, 127.67, 126.65 (q, J = 32.8 Hz), 126.21 (q, J =
3.7 Hz), 124.19, 123.93 (q, J = 269.3 Hz), 120.41. MSmonoizotopic (calc/meas): 499.0 / 499.1. Anal.
Calcd. for C19H10BrClF3N3O3 (500.65): C, 45.58; H, 2.01; N, 8.39; Found: C, 45.48; H, 2.04; N, 8.42.
4-Bromo-2-[(3,4-dichlorophenyl)carbamoyl]phenyl 5-chloropyrazine-2-carboxylate (3d)
White solid; yield: 53%; mp: 176.9-178.6 °C. IR (ATR): 3364 (NH), 3097, 2935, 2859, 1756 (C=O
ester), 1702, 1667, 1645, 1587, 1519, 1475, 1394, 1374, 1321, 1298, 1269, 1235, 1190, 1133, 1096,
1023, 860, 827, 818, 709, 676. 1H NMR (500 MHz, CDCl3): δ 9.43 (1H, bs, NH), 9.25 (1H, d, J = 1.3
Hz, H3´´), 8.80 (1H, d, J = 1.4 Hz, H5´´), 8.32 (1H, d, J = 2.5 Hz, H3), 7.82 (1H, d, J = 2.5 Hz, H2´),
7.73 (1H, dd, J = 2.6 Hz, J = 8.7 Hz, H5), 7.66 (1H, dd, J = 2.5 Hz, J = 8.7 Hz, H6´), 7.50 (1H, d, J =
13
8.7 Hz, H6), 7.42 (1H, d, J = 8.7 Hz, H5´). C NMR (126 MHz, DMSO): δ 160.92, 160.05, 154.44,
146.80, 146.57, 144.30, 142.17, 139.65, 137.31, 135.74, 134.66, 132.57, 130.61, 127.27, 124.07,
122.39, 120.34, 119.94. MSmonoizotopic (calc/meas): 499.0 / 499.0. Anal. Calcd. for C18H9BrCl3N3O3
(501.55): C, 43.11; H, 1.81; N, 8.38; Found: C, 452.98; H, 1.74; N, 8.33.
4.1.5. Synthesis of salicylanilide carbamates 4
A suspension of the appropriate salicylanilide 2 (1 mmol) in acetonitrile (6 mL) was treated with 1
mmol of triethylamine for 5 minutes, followed by addition of the corresponding isocyanate (1 mmol)
[32]. The mixture was stirred for 3 hours at room temperature. After this time, resulting carbamates 4
were separated by filtration and the crystals were washed twice with a small amount of cold methanol.
The products were crystallized from ethyl-acetate/hexane, if necessary.
4-Chloro-2-{[4-(trifluoromethyl)phenyl]carbamoyl}phenyl (2-chloroethyl)carbamate (4a)
White solid; yield: 52%; mp: decomposes above 191.3 °C; IR (ATR): 3337 (NH), 3277 (NH), 1715 (O-
CONH-), 1661 (CONH-I) 1219 (C-O). 1H NMR (300 MHz, DMSO): δ 10.71 (1H, bs, NH amide), 8.11
11

ACCEPTED MANUSCRIPT
(1H, t, J = 5.7 Hz, NH carbamate), 7.93-7.87 (2H, m, H3´, H5´), 7.74-7.67 (3H, m, H3, H2´, H6´), 7.60
(1H, dd, J = 2.6 Hz, J = 8.6 Hz, H5), 7.29 (1H, d, J = 8.7 Hz, H6), 3.53 (2H, t, J = 6.1 Hz, CH2-Cl),
3.30 (2H, q, J = 6.0 Hz, N-CH2). 13C NMR (75 MHz, DMSO): δ 163.57, 153.86, 147.19, 131.66,
131.25, 129.33, 128.59, 126.14 (q, J = 3.9 Hz), 125.36, 124.53 (q, J = 271.3 Hz), 123.92 (q, J = 31.9
Hz), 120.61, 119.83, 43.11, 42.76. MSmonoizotopic (calc/meas): 420.0 / 420.2. Anal. Calcd. for
C17H13Cl2F3N2O3 (421.20): C, 48.48; H, 3.11; N, 6.65; Found: C, 48.07; H, 3.14; N, 6.47.
4-Chloro-2-{[4-(trifluoromethyl)phenyl]carbamoyl}phenyl (3-chloropropyl)carbamate (4b)
White solid; yield: 54%; mp: 197.0-220.1 °C. IR (ATR): 3335 (NH), 3277 (NH), 1716 (O-CONH-),
1659 (CONH-I), 1217 (C-O). 1H NMR (500 MHz, DMSO-d6): δ 10.70 (1H, bs, NH amide), 7.95-7.87
(3H, m, NH carbamate, H3´, H5´), 7.75-7.67 (3H, m, H3, H2´, H6´), 7.59 (1H, dd, J = 2.7 Hz, J = 8.6
Hz, H5), 7.28 (1H, d, J = 8.7 Hz, H6), 3.55 (2H, t, J = 6.6 Hz, CH2-Cl), 3.10 (2H, q, J = 6.3 Hz, N-
13
CH2), 1.79 (2H, p, J = 6.6 Hz, C-CH2-C). C NMR (126 MHz, DMSO): δ 163.67, 153.78, 147.36,
131.66, 131.25, 129.20, 128.56, 126.13 (q, J = 4.1 Hz), 125.39, 124.52 (q, J = 271.5 Hz), 123.90 (q, J =
32.0 Hz), 120.59, 119.76, 42.70, 38.00, 32.29. MSmonoizotopic (calc/meas): 434.0 / 434.2. Anal. Calcd. for
C18H15Cl2F3N2O3 (435.22): C, 49.67; H, 3.47; N, 6.44; Found: C, 49.54; H, 3.38; N, 6.21.
4-Chloro-2-[(3,4-dichlorophenyl)carbamoyl]phenyl (2-chloroethyl)carbamate (4c)
White solid; yield: 30%; mp: 247.5-248.5°C. IR (ATR): 3334 (NH), 3257 (NH), 1717 (O-CONH-),
1655 (CONH-I), 1218 (C-O). 1H NMR (300 MHz, DMSO): δ 10.63 (1H, bs, NH amide), 8.11 (1H, t, J
= 5.8 Hz, NH carbamate), 8.06 (1H, d, J = 2.0 Hz, H2´), 7.70 (1H, d, J = 2.6 Hz, H3), 7.62-7.57 (3H,
m, H5, H6, H6´), 7.26 (1H, d, J = 8.7 Hz, H5´), 3.55 (2H, t, J = 6.1 Hz, CH2-Cl), 3.32 (2H, q, J = 5.9
13
Hz, N-CH2). C NMR (75 MHz, DMSO): δ 163.41, 153.83, 147.19, 139.20, 131.51, 131.32, 131.10,
130.83, 130.78, 129.35, 128.54, 125.39, 121.14, 119.98, 43.18, 42.78. MSmonoizotopic (calc/meas): 420.0 /
420.1. Anal. Calcd. for C16H12Cl4N2O3 (422.09): C, 45.43; H, 2.87; N, 6.64; Found: C, 45.15; H, 2.62;
N, 6.62.
4-Chloro-2-[(3,4-dichlorophenyl)carbamoyl]phenyl (3-chloropropyl)carbamate (4d)
White solid; yield: 61%; mp: 245.0-247.2 °C. IR (ATR): 3334 (NH), 3262 (NH), 1715 (O-CONH-),
1659 (CONH-I), 1215 (C-O). 1H NMR (300 MHz, DMSO): δ 10.63 (1H, bs, NH amide), 8.06 (1H, d, J
= 2.0 Hz, H2´), 7.92 (1H, t, J = 5.8 Hz, NH carbamate), 7.69 (1H, d, J = 2.7 Hz, H3), 7.61-7.56 (3H, m,
H5, H6, H6´), 7.24 (1H, d, J = 8.7 Hz, H5´), 3.56 (2H, t, J = 6.3 Hz, CH2-Cl), 3.10 (2H, q, J = 6.1 Hz,
13
N-CH2), 1.80 (2H, p, J = 6.5 Hz, C-CH2-C). C NMR (75 MHz, DMSO): δ 163.56, 153.79, 147.41,
139.24, 131.52, 131.37, 131.15, 130.82, 129.25, 128.56, 125.45, 125.40, 121.08, 119.92, 42.69, 38.01,
32.34. MSmonoizotopic (calc/meas): 434.0 / 434.1. Anal. Calcd. for C17H14Cl4N2O3 (436.12): C, 46.82; H,
3.24; N, 6.42; Found: C, 46.85; H, 3.18; N, 6.54.
4-Chloro-2-[(3,4-dichlorophenyl)carbamoyl]phenyl propylcarbamate (4e)
White solid; yield: 20%; mp: 247.6-248.9 °C. IR (ATR): 3330 (NH), 3268 (NH), 1714 (O-CONH-),
1
1662 (CONH-I), 1219 (C-O). H NMR (300 MHz, DMSO): δ 10.60 (1H, bs, NH amide), 8.06 (1H, s,
H2´), 7.81 (1H, t, J = 5.9 Hz, NH carbamate), 7.68 (1H, d, J = 2.7 Hz, H3), 7.61-7.56 (3H, m, H5, H6,
H6´), 7.26 (1H, d, J = 8.6 Hz, H5´), 2.92 (2H, q, J = 6.5 Hz, N-CH2), 1.36 (2H, h, J = 7.0 Hz, C-CH2-
C), 0.77 (3H, t, J = 6.5 Hz, CH3). 13C NMR (75 MHz, DMSO): δ 163.51, 153.68, 147.42, 139.26,
131.66, 131.26, 131.06, 130.77, 129.12, 128.46, 125.42, 125.31, 121.08, 119.94, 42.38, 22.51, 11.23.
MSmonoizotopic (calc/meas): 400.0 / 400.2. Anal. Calcd. for C17H15Cl3N2O3 (401.67): C, 50.83; H, 3.76;
N, 6.97; Found: C, 50.58; H, 3.55; N, 6.86.
4-Bromo-2-{[4-(trifluoromethyl)phenyl]carbamoyl}phenyl (2-chloroethyl)carbamate (4f)
White solid; yield: 38%; mp: 187.5-215.0 °C. IR (ATR): 3339 (NH), 3276 (NH), 1715 (O-CONH-),
1659 (CONH-I), 1219 (C-O). 1H NMR (500 MHz, DMSO): δ 10.70 (1H, bs, NH amide), 8.10 (1H, t, J
= 5.8 Hz, NH carbamate), 7.90 (2H, d, J = 8.5 Hz, H3´, H5´),7.82 (1H, d, J = 2.6 Hz, H3), 7.75-7.67
(3H, m, H5, H2´, H6´), 7.23 (1H, d, J = 8.7 Hz, H6), 3.53 (2H, t, J = 6.1 Hz, CH2-Cl), 3.30 (2H, q, J =
12

ACCEPTED MANUSCRIPT
6.2 Hz, N-CH2). 13C NMR (126 MHz, DMSO): δ 163.44, 153.76, 147.65, 142.69, 134.19, 132.01,
131.37, 126.10 (q, J = 3.8 Hz), 125.67, 124.51 (q, J = 271.2 Hz), 123.90 (q, J = 32.0 Hz), 119.83,
117.30, 43.08, 42.75. MSmonoizotopic (calc/meas): 464.0 / 464.1. Anal. Calcd. for C17H13BrClF3N2O3
(465.65): C, 43.85; H, 2.81; N, 6.02; Found: C, 43.66; H, 3.01; N, 5.78.
4-Bromo-2-{[4-(trifluoromethyl)phenyl]carbamoyl}phenyl (3-chloropropyl)carbamate (4g)
White solid; yield: 43%; mp: 181.0-219.0 °C. IR (ATR): 3334 (NH), 3276 (NH), 1715 (O-CONH-),
1657 (CONH-I), 1218 (C-O). 1H NMR (500 MHz, DMSO): δ 10.69 (1H, bs, NH amide), 8.10 (1H, t, J
= 5.8 Hz,), 7.92-7.86 (3H, m, NH carbamate, H3´, H5´), 7.80 (1H, d, J = 2.5 Hz, H3), 7.74-7.65 (3H,
m, H5, H2´, H6´), 7.21 (1H, d, J = 8.7 Hz, H6), 3.54 (2H, t, J = 6.6 Hz, CH2-Cl), 3.08 (2H, q, J = 6.3
Hz, N-CH2), 1.78 (2H, p, J = 6.6 Hz, C-CH2-C). 13C NMR (126 MHz, DMSO): δ 163.57, 153.71,
147.86, 142.72, 134.22, 132.04, 131.37, 126.13 (q, J = 3.9 Hz), 125.73, 124.52 (q, J = 271.3 Hz),
123.90 (q, J = 32.1 Hz), 119.77, 117.18, 42.70, 38.01, 32.29. MSmonoizotopic (calc/meas): 478.0 / 478.1.
Anal. Calcd. for C18H15BrClF3N2O3 (479.68): C, 45.07; H, 3.15; N, 5.84; Found: C, 45.00; H, 3.11; N,
5.63.
4-Bromo-2-[(3,4-dichlorophenyl)carbamoyl]phenyl (2-chloroethyl)carbamate (4h)
White solid; yield: 22%; mp: decomposes above 236.9 °C. IR (ATR): 3319 (NH), 3256 (NH), 1721 (O-
1
CONH-), 1655 (CONH-I), 1208 (C-O). H NMR (500 MHz, DMSO): δ 10.62 (1H, bs, NH amide),
8.11 (1H, t, J = 5.7 Hz, NH carbamate), 8.06 (1H, d, J = 2.0 Hz, H2´), 7.81 (1H, d, J = 1.9 Hz, H3),
7.72 (1H, dd, J = 1.9 Hz, J = 8.5 Hz, H5),7.66-7.54 (2H, m, H6, H6´), 7.22 (1H, d, J = 8.7 Hz, H5´),
13
3.55 (2H, t, J = 6.1 Hz, CH2-Cl), 3.31 (2H, q, J = 5.9 Hz, N-CH2). C NMR (126 MHz, DMSO): δ
163.29, 153.74, 147.65, 139.18, 134.26, 131.86, 131.32, 131.07, 130.81, 130.75, 125.69, 121.15,
119.97, 117.32, 43.15, 42.76. MSmonoizotopic (calc/meas): 463.9 / 464.1. Anal. Calcd. for
C16H12BrCl3N2O3 (466.54): C, 41.19; H, 2.59; N, 6.00; Found: C, 39.80; H, 2.27; N, 5.89.
4-Bromo-2-[(3,4-dichlorophenyl)carbamoyl]phenyl (3-chloropropyl)carbamate (4i)
White solid; yield: 58%; mp: 237.0-238.3 °C. IR (ATR): 3332 (NH), 3261 (NH), 1715 (O-CONH-),
1655 (CONH-I), 1216 (C-O). 1H NMR (300 MHz, DMSO): δ 10.63 (1H, bs, NH amide), 8.05 (1H, d, J
= 1.8 Hz, H2´), 7.92 (1H, t, J = 5.8 Hz, NH carbamate), 7.80 (1H, d, J = 2.4 Hz, H3), 7.72 (1H, dd, J =
2.5 Hz, J = 8.6 Hz, H5), 7.62-7.58 (2H, m, H6, H6´), 7.21 (1H, d, J = 8.6 Hz, H5´), 3.57 (2H, t, J = 6.5
Hz, CH2-Cl), 3.10 (2H, q, J = 6.3 Hz, N-CH2), 1.80 (2H, p, J = 6.6 Hz, C-CH2-C). 13C NMR (75 MHz,
DMSO): δ 163.44, 153.70, 147.87, 139.22, 134.31, 131.88, 131.34, 131.13, 130.80, 125.77, 125.38,
121.06, 119.90, 117.22, 42.69, 38.00, 32.33. MSmonoizotopic (calc/meas): 477.9 / 478.1. Anal. Calcd. for
C17H14BrCl3N2O3 (480.57): C, 42.49; H, 2.94; N, 5.83; Found: C, 42.35; H, 2.82; N, 5.69.
2-[(4-Benzoylphenyl)carbamoyl]-4-chlorophenyl (2-chloroethyl)carbamate (4j)
White solid; yield: 38%; mp: 180.0-217.0 °C. IR (ATR): 3336 (NH), 3272 (NH), 1712 (O-CONH-),
1662 (CONH-I), 1211 (C-O). 1H NMR (300 MHz, DMSO): δ 10.74 (1H, bs, NH amide), 8.13 (1H, t, J
= 5.8 Hz, NH carbamate), 7.91-7.84 (3H, m, H3, H3´, H5´), 7.79-7.51 (8H, m, H5, H2´, H6´, H2´´,
H3´´, H4´´, H5´´, H6´´), 7.29 (1H, d, J = 8.6 Hz, H6), 3.54 (2H, t, J = 6.1 Hz, CH2-Cl), 3.31 (2H, q, J =
5.9 Hz, N-CH2). 13C NMR (75 MHz, DMSO): δ 194.80, 163.55, 153.88, 147.21, 143.27, 137.66,
132.53, 132.02, 131.74, 131.26, 131.17, 129.61, 129.32, 128.69, 125.38, 119.21, 43.14, 42.77.
MSmonoizotopic (calc/meas): 456.1/456.2. Anal. Calcd. for C23H18Cl2N2O4 (457.06): C, 60.41; H, 3.97; N,
6.13; Found: C, 60.25; H, 3.79; N, 5.84.
2-[(4-Benzoylphenyl)carbamoyl]-4-chlorophenyl (3-chloropropyl)carbamate (4k)
White solid; yield: 64%; mp: 220.0-222.6 °C. IR (ATR): 3336 (NH), 3289 (NH), 1708 (O-CONH-),
1
1655 (CONH-I), 1222 (C-O). H NMR (300 MHz, DMSO): δ 10.74 (1H, bs, NH amide), 7.96-7.84
(4H, m, NH carbamate, H3, H3´, H5´), 7.80-7.51 (8H, m, H5, H2´, H6´, H2´´, H3´´, H4´´, H5´´, H6´´),
7.29 (1H, d, J = 8.7 Hz, H6), 3.57 (2H, t, J = 6.5 Hz, CH2-Cl), 3.31 (2H, q, J = 6.3 Hz, N-CH2), 1.80
(2H, p, J = 6.6 Hz, C-CH2-C). 13C NMR (75 MHz, DMSO): δ 195.05, 163.93, 154.07, 147.67, 143.56,
137.92, 132.78, 132.26, 132.00, 131.53, 131.45, 129.87, 129.47, 128.94, 128.86, 125.69, 119.42, 43.06,
13

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38.28, 32.59. MSmonoizotopic (calc/meas): 470.1 / 470.2. Anal. Calcd. for C24H20Cl2N2O4 (471.33): C,
61.16; H, 4.28; N, 5.94; Found: C, 60.97; H, 3.95; N, 5.77.
2-[(4-Benzoylphenyl)carbamoyl]-4-chlorophenyl propylcarbamate (4l)
White solid; yield: 23%; mp: 215.9-217.2 °C. IR (ATR): 3339 (NH), 3276 (NH), 1713(O-CONH-),
1662 (CONH-I), 1218 (C-O). 1H NMR (300 MHz, DMSO): δ 10.72 (1H, bs, NH), 7.94-7.51 (12H, m,
NH carbamate, H3, H5, H2´, H3´, H5´, H6´, H2´´, H3´´, H4´´, H5´´, H6´´), 7.27 (1H, d, J = 8.7 Hz,
H6), 2.93 (2H, q, J = 6.6 Hz, N-CH2), 1.36 (2H, h, J = 7.2 Hz, C-CH2-C), 0.77 (3H, t, J = 7.3 Hz, CH3).
13C NMR (75 MHz, DMSO): δ 194.78, 163.62, 153.73, 147.42, 143.32, 137.66, 132.52, 131.97,
131.91, 131.23, 131.14, 129.60, 129.11, 128.69, 128.54, 125.46, 119.16, 42.42, 22.52, 11.26.
MSmonoizotopic (calc/meas): 436.1 / 436.3. Anal. Calcd. for C24H21ClN2O4 (436.12): C, 65.98; H, 4.84; N,
6.41; Found: C, 65.74; H, 4.62; N, 6.21.
2-[(4-Benzoylphenyl)carbamoyl]-4-bromophenyl (2-chloroethyl)carbamate (4m)
White solid; yield: 35%; mp: 225.7-227.0 °C. IR (ATR): 3335 (NH), 3270 (NH), 1712 (O-CONH-),
1662 (CONH-I), 1221 (C-O). 1H NMR (300 MHz, DMSO): δ 10.74 (1H, bs, NH amide), 8.13 (1H, t, J
= 5.7 Hz, NH carbamate), 7.92-7.51 (11H, m, H3, H5, H2´, H3´, H5´, H6´, H2´´, H3´´, H4´´, H5´´,
H6´´), 7.23 (1H, d, J = 8.7 Hz, H6), 3.54 (2H, t, J = 6.1 Hz, CH2-Cl), 3.31 (2H, q, J = 5.9 Hz, N-CH2).
13C NMR (75 MHz, DMSO): δ 194.79, 163.45, 153.80, 147.68, 143.27, 137.66, 132.52, 132.10,
132.01, 131.41, 131.23, 131.16, 129.61, 128.69, 125.70, 119.22, 117.33, 43.14, 42.78. MSmonoizotopic
(calc/meas): 500.0 / 500.1. Anal. Calcd. for C23H18BrClN2O4 (501.76): C, 55.06; H, 3.62; N, 5.58;
Found: C, 54.86; H, 3.54; N, 5.46.
2-[(4-Benzoylphenyl)carbamoyl]-4-bromophenyl (3-chloropropyl)carbamate (4n)
White solid; yield: 54%; mp: 225.0-226.9 °C. IR (ATR): 3339 (NH), 3294 (NH), 1708 (O-CONH-),
1
1655 (CONH-I), 1220 (C-O). H NMR (300 MHz, DMSO): δ 10.74 (1H, bs, NH amide), 7.96-7.51
(12H, m, NH carbamate,H3, H5, H2´, H3´, H5´, H6´, H2´´, H3´´, H4´´, H5´´, H6´´), 7.22 (1H, d, J =
8.6 Hz, H6), 3.57 (2H, t, J = 6.5 Hz, CH2-Cl), 3.10 (2H, q, J = 6.3 Hz, N-CH2), 1.80 (2H, p, J = 6.6 Hz,
C-CH2-C). 13C NMR (75 MHz, DMSO): δ 194.79, 163.57, 153.74, 147.89, 143.30, 137.66, 134.23,
132.52, 132.11, 131.99, 131.41, 131.19, 129.61, 128.68, 125.77, 119.16, 117.21, 42.80, 38.03, 32.32.
MSmonoizotopic (calc/meas): 514.0 / 514.2. Anal. Calcd. for C24H20BrClN2O4 (515.78): C, 55.89; H, 3.91;
N, 5.43; Found: C, 55.64; H, 3.84; N, 5.36.
4.2. Determination of in vitro antimycobacterial activity
In vitro antimycobacterial activity of the compounds was determined on fast growing
Mycobacterium abscessus strain (ATCC 19977), on Mycobacterium tuberculosis H37Rv (ATCC
27294) and Mycobacterium tuberculosis A8 MDR strain (ATCC 35822, resistant to isoniazid, INH,
and rifampicin, RIF) by serial dilution in Sula semisynthetic medium, which was prepared in-house
(pH 6.5) [35,36]. Compounds were added to the medium as DMSO solutions at 10 various doses (range
of final concentration was between 0.05 and 100 ꢀg/mL). Minimal inhibitory concentrations (MIC)
were determined after incubation at 37 °C for 28 days in the case of M. tuberculosis H37Rv and A8
MDR, and for 7 days in the case of M. abscessus. MIC was the lowest concentration of a compound at
which the visible inhibition of the growth of M. abscessus, M. tuberculosis H37Rv and A8 MDR strain
occurred. In order to confirm the growth inhibition, colony forming unit (CFU) was determined by
subculturing from the Sula medium onto drug-free Löwenstein-Jensen solid medium. Samples were
incubated for further 28 days in the case of M. tuberculosis H37Rv and A8 MDR, and for 7 days in the
case of M. abscessus (CFU means the number of the colonies that developed from the viable bacteria)
(Table 3). Experiments were repeated at least two times. Isoniazid (INH), gentamicin (GEN) and
ciprofloxacin (CIPX) were involved as reference antimycobacterial drugs.
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A selected set of compounds were tested for their in vitro activity against multidrug-resistant M.
tuberculosis 9449/2006 strain and against M. tuberculosis Praha 131 (XDR-TB) with different
resistance patterns: both strains are resistant to INH, RIF, rifabutine, and streptomycin; an additional
resistance was present in the case of Praha 131 strain: ethambutol, ofloxacin, gentamicin and amikacin
(i.e., XDR-TB strain). The micromethod for the determination of MICs was used. Antimycobacterial
activities were determined in the Sula semisynthetic medium (SEVAC, Prague, Czech Republic). The
tested compounds were added to the medium as solutions in DMSO. The following concentrations
were used: 32, 16, 8, 4, 2, 1, 0.5, 0.25, 0.125, 0.064 and 0.03 ꢀM. The MICs were determined after
incubation at 37 °C for 14 and 21 days. MIC (in ꢀM) was the lowest concentration at which the
complete inhibition of mycobacterial growth occurred (Table 4).
4.3. Determination of in vitro cytotoxic activity on MonoMac6 cells
MonoMac6 cell cultures (DSMZ no.: ACC 124, Deutsche Sammlung von Mikroorganismen and
Zellkulturen GmbH, Braunschweig, Germany) were maintained in RPMI-1640 medium containing
10% FCS, 2 mM L-glutamine, and 160 ꢀg/mL gentamycin at 37 °C in 5% CO2 atmosphere. The cells
were plated on 96-well plates one day before the treatment (105 cells/1 ml medium/well). After 24 h
incubation at 37 °C, cells were treated for 16 h (overnight) with the compounds dissolved in serum free
RPMI-1640 medium (cDMSO = 1.0 v/v %) at 5 x10-2 to 102 µM concentration range. Control cells were
treated with serum free medium only or with DMSO containing serum free medium (c = 1.0 v/v %) at
37 °C. After the treatment and incubation, cells were washed twice with serum-free medium
(centrifugation: 1000 rpm, 5 min) and the cell viability was determined with (4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide (MTT)-assay.
45 ꢀL MTT solution (2 mg/mL) was added to each well (final concentration 367 ꢀg/mL) and during
3.5 h incubation purple crystals were formed by mitochondrial dehydrogenase enzyme present in the
living cells. After incubation cells were centrifuged for 5 min at 2000 rpm and the supernatant was
removed. Crystals were dissolved in DMSO and the optical density (OD) of the samples was
determined at λ= 540 and 620 nm using an ELISA Reader (Labsystems MS reader, Helsinki, Finland).
OD620 was subtracted from OD540. The percent of cytotoxicity was calculated using the following
equation: cytotoxicity (%) = 100 × (1-ODtreated/ODcontrol), where ODtreated and ODcontrol correspond to the
optical densities of treated and control cells, respectively. Cytotoxicity (%) was plotted as a function of
concentration, fitted to a sigmoidal curve and the 50% inhibitory concentration (IC50) value was
determined from these curves. Each experiment was repeated at least 2 times and the average IC50
values are presented in Table 6.
In each case two independent experiments were carried out with 4–8 parallel measurements. The
50% inhibitory concentration (IC50) values were determined from the dose-response curves. The curves
were defined using MicrocalTM Origin1 (version 9.0) software.
Acknowledgements
This work was financially supported by IGA NT 13346 (2012); Hungarian Research Fund, OTKA
104275; travel grants of Campus Hungary Short Term Study Program and Pázmány-Eötvös Foundation
for Natural Sciences and Informatics; and co-financed by the European Social Fund and the state
budget of the Czech Republic Project No. CZ.1.07/2.3.00/20.0235, the title of the project: TEAB. The
authors thank Katalin Uray, PhD (MTA-ELTE Research Group of Peptide Chemistry) for helpful
comments and critical reading of the manuscript.
15

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Conflict of Interest
The authors declare no conflict of interest.
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Schemes and Tables
Scheme 1. Preparation of 5-chloropyrazine-2-carboxylic acid 1 (DMF: N,N-dimethylformamide).
Scheme 2. Synthesis of substituted salicylanilides 2 (Ph-Cl: chlorobenzene).
Scheme 3. Esterification of salicylanilides 2 by 5-chloropyrazine-2-carboxylic acid 1 (DMF: N,N-
dimethylformamide; DCC: N,N’-dicyclohexylcarbodiimide).
Scheme 4. Synthesis of salicylanilide carbamates 4 (TEA: triethylamine; MeCN: acetonitrile).
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Table 1. The previously published most potent substituted salicylanilides, salicylanilide esters and
salicylanilide carbamates, and the structure of PZA. Assignation of the R groups was chosen according
to the cited references.
R1
R2
R3
R4
R5
Method / In vitro efficacy
Ref.
[6]
4-Cl
4-Cl
4-Cl
5-Cl
5-Cl
5-Br
5-Br
4-Cl
4-Br
5-Cl
5-Cl
5-Cl
4-Cl
5-Cl
5-Cl
5-Cl
5-Cl
4-Cl
4-Cl
5-Cl
5-Br
4-Br
4-Cl
4-Cl
4-Cl
-
-
-
4-CF3
-
-
-
[6]
3,4-diCl
4-CF3
-
-
-
[6]
-
-
-
[6]
in vitro antimycobacterial assay (Sula) /
growth inhibition,
M. tuberculosis,
M. kansasii,
M. avium
3,4-diCl
4-CF3
-
-
-
[6]
-
-
-
[6]
3,4-diCl
4-CF3
-
-
-
[6]
2-pyrazinyl (pyrazinoate)
-
-
[10]
[10]
[11]
[32]
[32]
[10]
[10]
[32]
[14]
[10]
[10]
[14]
[11]
[14]
[14]
[14]
[14]
4-CF3
2-pyrazinyl (pyrazinoate)
-
-
4-Cl
-
-
-
CH3
CH3
3,4-diCl
3,4-diCl
C2H5
H
H
-
C6H13
3,4-diCl 2-pyrazinyl (pyrazinoate)
3,4-diCl 2-pyrazinyl (pyrazinoate)
-
in vitro antimycobacterial assay (Sula) /
growth inhibition,
MDR and XDR-TB
-
-
3,4-diCl
4-CF3
-
-
C5H11
H
-
-
-
-
-
3,4-diCl 2-pyrazinyl (pyrazinoate)
-
mycobacterial isocitrate lyase inhibition
assay (ICL1) /
enzyme inhibition
4-CF3
3,4-diCl
5-F
2-pyrazinyl (pyrazinoate)
phenyl (benzoate)
-
-
-
phenyl phenyl
4-CF3
4-CF3
4-CF3
4-CF3
-
-
-
-
-
-
-
-
-
mycobacterial methionine
aminopeptidase inhibition assay /
enzyme inhibition
2-pyrazinyl (pyrazinoate)
2-pyrazinyl (pyrazinoate)
phenyl (benzoate)
20

ACCEPTED MANUSCRIPT
Table 2. Physicochemical properties of 5-chloropyrazine-2-carboxylic acid 1 and salicylanilide
derivatives 2, 3, 4
code
R1
R2
R3
-
Mwmo calc/measa
158.0 / 158.0
315.0 / 315.1
315.0 / 315.0
359.0 / 359.1
358.9 / 359.0
LogPb
0.24
3.93
4.12
4.20
4.40
4.22
4.49
4.45
4.65
4.72
4.92
solubility (µM)c
<500
<100
<100
<100
<100
<50
-
-
1
2a
2b
2c
2d
2e
2f
-
Cl
Cl
Br
Br
Cl
Br
Cl
Cl
Br
Br
Cl
Cl
Cl
Cl
Cl
Br
Br
Br
Br
Cl
Cl
Cl
Br
Br
4-CF3
-
3,4-diCl
4-CF3
-
-
3,4-diCl
4-CO-Ph
4-CO-Ph
4-CF3
-
351.1 / 351.3
395.0 / 395.2
455.0 / 455.2
454.9 / 455.0
499.0 / 499.1
499.0 / 499.0
420.0 / 420.2
434.0 / 434.2
420.0 / 420.1
434.0 / 434.1
400.0 / 400.2
464.0 / 464.1
478.0 / 478.1
463.9 / 464.1
477.9 / 478.1
456.1/456.2
-
<50
-
<100
<100
<100
<100
<50
3a
3b
3c
3d
4a
4b
4c
4d
4e
4f
-
3,4-diCl
4-CF3
-
-
3,4-diCl
4-CF3
Cl
CH2Cl
Cl
4.52
4.63
4.72
4.82
4.83
4.79
4.90
4.99
5.09
4.81
4.92
4.93
5.08
5.19
<50
4-CF3
<50
3,4-diCl
3,4-diCl
3,4-diCl
4-CF3
<50
CH2Cl
CH3
Cl
<50
<50
<50
4-CF3
CH2Cl
Cl
4g
4h
4i
<50
3,4-diCl
3,4-diCl
4-CO-Ph
4-CO-Ph
4-CO-Ph
4-CO-Ph
4-CO-Ph
<50
CH2Cl
Cl
<50
4j
<50
CH2Cl
CH3
Cl
470.1 / 470.2
436.1 / 436.3
500.0 / 500.1
514.0 / 514.2
4k
4l
<50
<50
4m
4n
<50
CH2Cl
a Calculated and measured monoisotopic molecular mass (acquired by Bruker Esquire 3000+ ESI-MS)
The log P values were calculated using the program CS ChemOffice Ultra ver. 11.0 (CambridgeSoft, Cambridge, MA,
b
USA).
c
Determined solubility using in vitro conditions (RPMI and Sula media (cDMSO= 1 v/v %)). The undissolved compound, if
any, was filtered using the PVDF filter. To 120 ꢀL of the filtrate, 80 ꢀL of acetonitrile was added and mixed thoroughly.
The concentration of the compounds was determined spectrophotometrically.
21

ACCEPTED MANUSCRIPT
Table 3. Antimycobacterial activity of 5-chloropyrazine-2-carboxylic acid 1 and salicylanilide
derivatives 2, 3, 4
M. abscessus
M. tuberculosis H37Rv
M. tuberculosis A8 MDR
Code
R1
R2
R3
MICa
MICa
MIC
MICa
MIC
MIC
CFUb
CFUb
CFUb
γ (µg/mL)
γ (µg/mL)
γ (µg/mL)
(µM)
(µM)
(µM)
no
inhibition
no
inhibition
no
inhibition
controlc
-
+++d
-
+++d
+++d
-
-
-
INHe,f
1
-
-
40
>100
1
291.8
>632.9 +++d
26
0.16
>100
0.5
0.5
0.5
0.5
5
1.17
>632.9 +++d
0
1
>100
0.5
0.5
1
7.3
12
-
-
-
-
-
-
-
-
-
-
-
-
-
-
>632.9 +++d
Cl
Cl
Br
Br
Cl
4-CF3
3,4-diCl
4-CF3
3,4-diCl
4-CO-Ph
3.2
3.2
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1.6
1.6
1.4
1.4
14.2
12.7
1.1
1.1
1.0
2.0
0.5
0.5
1.2
2.3
0.2
0.4
1.0
0.2
0.4
8.7
8.5
9.2
8.0
8.7
0
2
2
2
3
2
3
10
0
1
1
1
2
0
0
0
8
2
2
2
3
3
2
2
1.6
1.6
0
8
2a
2b
2c
2d
2e
2f
1
5
13.9
7.0
2.8
0
2.5
10
10
2.5
0.5
2.5
0.5
0.5
0.1
0.5
0.5
0.5
2
1
2.8
0
28.5
25.3
5.5
5
14.2
12.7
1.1
50
1
Br 4-CO-Ph
5
5
Cl
Cl
Br
Br
Cl
Cl
Cl
Cl
Cl
Br
Br
Br
Br
Cl
Cl
Cl
4-CF3
3,4-diCl
4-CF3
0.5
0.5
0.5
1
0.5
2
1
3a
3b
3c
3d
4a
4b
4c
4d
4e
4f
1.1
4.4
0
5.0
1
2.0
22
11
0
3,4-diCl
4-CF3
1.0
1
2.0
Cl
CH2Cl
Cl
1.2
0.2
0.2
0.5
1
0.5
0.5
1
1.2
4-CF3
0.2
1.2
0
3,4-diCl
3,4-diCl
3,4-diCl
4-CF3
1.2
2.4
0
CH2Cl
CH3
Cl
1.2
0.5
0.5
0.5
0.5
1
1.2
1
1.3
0.1
0.2
0.5
0.1
0.2
4
1.3
2
4.3
1.1
9
4-CF3
CH2Cl
Cl
0.1
0.1
1
0.2
1.0
0
4g
4h
4i
3,4-diCl
3,4-diCl
4-CO-Ph
4-CO-Ph
4-CO-Ph
0.2
2.2
0
CH2Cl
Cl
2.1
0.5
5
1.0
2
20
20
10
20
20
43.6
42.5
22.9
40.0
38.9
10.9
21.3
11.5
20.0
1.9
21
0
4j
CH2Cl
CH3
Cl
4
10
5
4k
4l
4
6
Br 4-CO-Ph
Br 4-CO-Ph
4
10
1
0
4m
4n
CH2Cl
4
1
a MIC (minimal inhibitory concentration) was determined on the M. tuberculosis H37Rv, MDR-TB strain A8 (4 weeks) and
M. abscessus strain (1 week) in Sula semisynthetic medium, pH 6.5.
b To confirm the growth inhibition, CFU (colony forming unit) was determined on Löwenstein-Jensen solid media. In the
case of M. tuberculosis H37Rv and MDR-TB strain A8 4 weeks and in the case of M. abscessus strain 1 week further
incubation was applied.
22

ACCEPTED MANUSCRIPT
c As positive control, bacteria were inoculated in the absence of drugs.
d +++: confluent colonies.
e isoniazid
f The MIC values against M. abscessus of the control drugs CIPX and GEN were 40 ꢀg/mL (291.8 ꢀM), 1 ꢀg/mL (3.0 ꢀM)
and 5 ꢀg/mL (10.5 ꢀM) respectively.
Table 4. Activity of selected salicylanilide derivatives 3 and 4 against MDR- and XDR-TB strains
M. tuberculosis 9449/2006a
14 d 21 d
MICc MICc
γ (µg/mL) (µM) γ (µg/mL) (µM) γ (µg/mL) (µM) γ (µg/mL) (µM)
M. tuberculosis Praha 131b
14 d 21 d
MICc MICc
code R1
R2
R3
MIC
MIC
MIC
MIC
-
-
-
Cl
4-CF3
0.5
0.5
0.5
0.2
0.4
0.4
0.5
0.5
0.5
0.5
1
1
0.9
0.9
1.0
0.4
0.9
0.4
0.9
1.0
0.9
1.0
2
2
2
1
2
1
2
2
2
2
0.5
0.5
1.0
0.4
0.4
0.4
0.5
0.5
0.5
0.5
1
1
2
1
1
1
1
1
1
1
0.5
0.9
1.0
0.8
0.9
0.4
0.9
1.0
0.9
0.5
1
2
2
2
2
1
2
2
2
1
3a
3b
3c
4a
4b
4e
4f
Cl 3,4-diCl
Br
Cl
Cl
4-CF3
4-CF3
4-CF3
1
Cl
CH2Cl
CH3
Cl
0.5
1
Cl 3,4-diCl
1
Br
Br
4-CF3
4-CF3
1
CH2Cl
Cl
1
4g
4h
4i
Br 3,4-diCl
1
Br 3,4-diCl CH2Cl
a MDR-TB strain 9449/2006 resistant to INH, RIF, rifabutine and streptomycin.
XDR-TB strain Praha 131 resistant to INH, RIF, rifabutine, streptomycin, ethambutol, ofloxacin, gentamicin and
amikacin.
c MIC was determined after 14 days and 21 days of incubation in Sula semisynthetic media, pH 6.5.
1
b
Table 5. Comparison of the in vitro MDR- and XDR-TB inhibition of the selected salicylanilide 5-
chloropyrazinoates 3a-c with their previously described salicylanilide pyrazinoate analogs
M. tuberculosis 9449/2006 M. tuberculosis Praha 131
compound
code
R1
R2
21 d MIC (µM)
21 d MIC (µM)
SAL 5-chloropyrazinoate
SAL pyrazinoate
Cl
Cl
Cl
Cl
Br
Br
4-CF3
4-CF3
2
0.5*
2
1
1*
3a
-*
SAL 5-chloropyrazinoate
SAL pyrazinoate
3,4-diCl
3,4-diCl
4-CF3
2
3b
-*
0.25*
2
0.125*
2
SAL 5-chloropyrazinoate
SAL pyrazinoate
3c
-*
4-CF3
0.5*
0.5*
* Compounds and their MIC values were cited from [10].
SAL: corresponding salicylanilide
23

ACCEPTED MANUSCRIPT
Table 6. In vitro cytotoxic activity of salicylanilide derivatives 2, 3, 4 on MonoMac6 cells
SIa for
M.
tuberculosis tuberculosis tuberculosis tuberculosis
IC50
R1
R2
R3
MonoMac6
M.
M.
M.
Code
M.
abscessus
(µM)b
H37Rv
A8 MDR
9449/2006c Praha 131c
-
-
-
-
-
-
-
-
-
-
2a
2b
2c
2d
2e
2f
Cl
Cl
Br
Br
Cl
Br
Cl
Cl
Br
Br
Cl
Cl
Cl
Cl
Cl
Br
Br
Br
Br
Cl
Cl
Cl
Br
Br
4-CF3
3,4-diCl
4-CF3
5.3±0.8
3.6±1.4
7.6±2.2
3.5±2.4
48±3.7
1.7
1.1
0.5
0.5
1.7
0.9
0.2
9.2
0.2
5.7
6.3
44.0
3.8
2.1
5.4
2.2
28.5
34.5
4.5
0.9
0.4
0.8
0.6
0.3
3.3
2.3
3.3
2.3
2.7
1.3
3.4
1.7
1.0
2.3
0.6
2.9
6.3
7.3
1.9
2.1
5.4
8.5
5.7
3.1
9.5
3.5
0.8
1.6
1.2
6.6
ndd
nd
nd
nd
5.4
nd
nd
3,4-diCl
4-CO-Ph
4-CO-Ph
4-CF3
2.5
nd
nd
3.4
nd
nd
21.8±4.3
1.1±1.6
10.1±0.5
1.1±0.5
5.7±4.9
7.6±5.6
8.8±6.0
4.6±0.2
2.5±2.3
7.0±6.3
9.3±1.7
5.7±0.1
6.9±4.5
9.5±6.1
37.8±4.3
16.1±6.6
18.7±4.5
24.9±4.5
12.6±0.1
1.7
nd
nd
3a
3b
3c
3d
4a
4b
4c
4d
4e
4f
1.0
0.6
5.1
0.6
nd
1.1
5.1
0.6
nd
3,4-diCl
4-CF3
9.2
1.1
3,4-diCl
4-CF3
2.9
Cl
CH2Cl
Cl
15.2
17.6
3.8
7.6
4.4
nd
3.8
4.4
nd
4-CF3
3,4-diCl
3,4-diCl CH2Cl
1.1
nd
nd
3,4-diCl
4-CF3
CH3
Cl
35.0
23.3
5.7
7.0
4.7
2.9
3.5
4.8
nd
7.0
4.7
2.9
3.5
9.5
nd
4g
4h
4i
4-CF3
CH2Cl
Cl
3,4-diCl
34.5
23.8
4.3
3,4-diCl CH2Cl
4-CO-Ph Cl
4-CO-Ph CH2Cl
4j
4k
4l
1.9
nd
nd
4-CO-Ph
4-CO-Ph
CH3
Cl
2.0
nd
nd
4m
4n
3.1
nd
nd
4-CO-Ph CH2Cl
a selectivity index, SI = IC50 (ꢀM)/MIC (ꢀM)
b 5-chloropyrazine-2-carboxylic acid 1 had no cytostatic activity at the measured concentration range, up to 250 ꢀM
c SI for M. tuberculosis 9449/2006 and Praha 131 calculated to the 21 day data
d nd: not determined
1.4
nd
nd
24

ACCEPTED MANUSCRIPT
Highlights
•
•
•
•
•
Salicylanilide 5-chloropyrazinoates, carbamates were synthesised and characterised.
In vitro activity against fast-growing M. abscessus from 0.2 µM.
MICs against M. tuberculosis H37Rv with MICs from 0.2 µM.
MICs against multidrug-resistant (MDR) M. tuberculosis A8 from 1 µM.
In vitro activity against MDR and extremely-resistant M. tuberculosis from 1 µM.

Supplementary Information
ACCEPTED MANUSCRIPT
Stability study of salicylanilide 5-chloropyrazinoate 3c and salicylanilide carbamate 4g
Experimental Methods and Conditions
The stability of the compounds 3c and 4g (Figure 1) was monitored using analytical RP-HPLC.
Exformma HPLC system (Exformma Technology, ASIA Co., Limited, Hong Kong, China) was used; the
column was Agilent Zorbax SB-C18 4.6mmx150mm, 100Å. The applied gradient elution was 0 min 0%
B, 5 min 0% B, 15 min 60% B, 25 min 100% B with eluent A (0.1% TFA in water) and eluent B (0.1%
TFA in acetonitrile-water (80:20, V/V)) was used at a flow rate of 1 ml/min at ambient temperature. The
detection was carried out at λ = 220 nm or 280 nm. The peaks were collected and after freeze-drying
characterized by mass spectrometry using ESI-MS. Positive and negative electrospray ionization mass
spectrometric analysis was performed on a Bruker Daltonics Esquire 3000 plus (Germany). The samples
were dissolved in acetonitrile–water (50:50, V/V), containing 0.1% acetic acid.
DMSO stock solution was used in the in vitro studies, therefore the chemical stability of ester 3c and
carbamate 4g was studied in DMSO solution (0.75 mg/ml compound concentration) and in serum-free
RPMI-1640 medium containing 10% DMSO (0.5 mg/ml). In the in vitro experiments serum-free RPMI-
1640 medium containing 1% DMSO was used, but the HPLC studies required higher concentration, and
because of its moderate solubility in medium the percentage of DMSO needed to be increased to 10% for
the stability analysis.
CF3
O
CF3
O
O
Br
CF3
N
Br
O
H
N
H
N
COOH
Br
O
N
H
N
N
O
Cl
N
OH
O
N
H
Cl
Cl
1
2c
4g
3c
Figure 1. The structures of the compounds 1, 2c, 3c and 4g
Results and Discussion
The ESI-MS spectra of the compounds 1, 2c, 3c and 4g are summarized for comparison in Figure 2. The
compounds 3c and 4g are not stable under the ESI-MS circumstances, a part of them decomposed to the
corresponding salicylanilide 2c.
The DMSO stability chromatograms are presented in Figure 3. Both compounds 3c and 4g were
stable in the DMSO stock solution, no decomposition was observed even after 29 h.
The stability of the compound 3c in serum-free RPMI-1640 medium containing 10% DMSO is
presented in Figure 4. The salicylanilide-5-chloropyrazinoate 3c decomposed in serum-free RPMI-1640
medium containing 10% DMSO after 29 hours of incubation. The ESI-MS spectra of the corresponding
peaks are presented in Figure 5. The released parent salicylanilide (2c) was co-eluted with its ester, the
releasing of 5-chloropyrazine-2-carboxylic acid 1 indicated the decomposition. The half-time of 3c was
determined according to the percentage of the released 5-chloropyrazine-2-carboxylic acid (Figure 6), and
it was estimated to be above 3 h.

The stability of the compound 4g in serum-free RPMI-1640 medium containing 10% DMSO is
ACCEPTED MANUSCRIPT
presented in Figure 7. The carbamate 4g started to decompose immediately in serum-free RPMI-1640
medium containing 10% DMSO. No intact 4g was observed after 6 h (Figure 7). The ESI-MS spectra of
the corresponding peaks are presented in Figure 8. The half-time of 4g was determined according to the
percentage of the intact carbamate 4g and the corresponding salicylanilide 2c released (Figure 9). The
half-time of the carbamate was markedly low, less than 1 hour.
Figure 2. ESI-MS spectra of the compounds , and
,
1 2c 3c 4g
(A) Mwmo calc/meas: 158.0/158.0, negative ionization,
1
(B) 2c Mwmo calc/meas: 359.0/359.2, positive ionization,
(C) 3c Mwmo calc/meas: 499.0/499.1, positive ionization, the compound 3c is not stable under the ESI-
MS circumstances, a part of it decomposes to the corresponding salicylanilide 2c
(B) 4g Mwmo calc/meas: 478.0/478.1, positive ionization, the compound 4g is not stable under the ESI-
MS circumstances, a part of it decomposes to the corresponding salicylanilide 2c
Experimental methods and conditions: see above.

ACCEPTED MANUSCRIPT
1,6
1,4
1,2
1,0
0,8
0,6
0,4
0,2
0,0
1,6
B
A
1,4
1,2
1,0
0,8
0,6
0,4
0,2
0,0
0
5
10
15
20
25
30
0
5
10
15
20
25
30
time (min)
time (min)
1,8
1,6
1,4
1,2
1,0
0,8
0,6
0,4
0,2
0,0
1,8
1,6
1,4
1,2
1,0
0,8
0,6
0,4
0,2
0,0
D
C
0
5
10
15
20
25
30
0
5
10
15
20
25
30
time (min)
time (min)
Figure 3. The analytical RP-HPLC chromatograms of the salicylanilide ester 3c and carbamate 4g in
DMSO, (A) 3c in DMSO at 0 min, Rt: 24.0 min, λ = 280 nm, (B) 3c in DMSO after 29 h, Rt: 24.0 min, λ
= 280 nm (C) 4g in DMSO at 0 min, Rt: 22.6 min, λ = 220 nm, (D) 4g in DMSO after 29 h, Rt: 22.6 min,
λ = 220 nm. Experimental methods and conditions: see above.