Antibacterial and herbicidal activity of ring-substituted 2-hydroxynaphthalene-1-carboxanilides

Article abstract of DOI:10.3390/molecules18089397

In this study, a series of twenty-two ring-substituted 2- hydroxynaphthalene-1-carboxanilides were prepared and characterized. Primary in vitro screening of the synthesized compounds was performed against Staphylococcus aureus, three methicillin-resistant

Full text of DOI:10.3390/molecules18089397

Molecules 2013, 18, 9397-9419; doi:10.3390/molecules18089397
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Antibacterial and Herbicidal Activity of Ring-Substituted
2-Hydroxynaphthalene-1-carboxanilides
Tomas Gonec 1, Jiri Kos 1, Iveta Zadrazilova 1,2,3, Matus Pesko 4, Rodney Govender 5,
Stanislava Keltosova 6, Barbara Chambel 1, Diogo Pereira 1, Peter Kollar 6, Ales Imramovsky 7,
Jim O’Mahony 5, Aidan Coffey 5, Alois Cizek 2,3, Katarina Kralova 8 and Josef Jampilek 1,*
1
Department of Chemical Drugs, Faculty of Pharmacy, University of Veterinary and
Pharmaceutical Sciences, Palackeho 1/3, 612 42 Brno, Czech Republic
2
Department of Infectious Diseases and Microbiology, Faculty of Veterinary Medicine,
University of Veterinary and Pharmaceutical Sciences, Palackeho 1/3, 612 42 Brno, Czech
Republic
3
CEITEC VFU, University of Veterinary and Pharmaceutical Sciences, Palackeho 1/3, 612 42 Brno,
Czech Republic
4
Department of Environmental Ecology, Faculty of Natural Sciences, Comenius University,
Mlynska dolina Ch-2, 842 15 Bratislava, Slovakia
5
Department of Biological Sciences, Cork Institute of Technology, Bishopstown, Cork, Ireland
6
Department of Human Pharmacology and Toxicology, Faculty of Pharmacy,
University of Veterinary and Pharmaceutical Sciences, Palackeho 1/3, 612 42 Brno, Czech
Republic
7
Institute of Organic Chemistry and Technology, Faculty of Chemical Technology,
University of Pardubice, Studentska 573, 532 10 Pardubice, Czech Republic
8
Institute of Chemistry, Faculty of Natural Sciences, Comenius University, Mlynska dolina Ch-2,
842 15 Bratislava, Slovakia
* Author to whom correspondence should be addressed; E-Mail: josef.jampilek@gmail.com;
Tel.: +420-54-156-2926.
Received: 22 July 2013; in revised form: 1 August 2013 / Accepted: 2 August 2013 /
Published: 6 August 2013
Abstract: In this study, a series of twenty-two ring-substituted 2-hydroxynaphthalene-
1-carboxanilides were prepared and characterized. Primary in vitro screening of the
synthesized compounds was performed against Staphylococcus aureus, three
methicillin-resistant S. aureus strains, Mycobacterium marinum, M. kasasii, M. smegmatis.
and M. avium paratuberculosis. The compounds were also tested for their activity related

Molecules 2013, 18
to inhibition of photosynthetic electron transport (PET) in spinach (Spinacia oleracea L.)
9398
chloroplasts. 2-Hydroxy-N-phenylnaphthalene-1-carboxanilide and 2-hydroxy-N-(3-
trifluoromethylphenyl)naphthalene-1-carboxamide (IC50 = 29 µmol/L) were the most
active PET inhibitors. Some of tested compounds showed the antibacterial and
antimycobacterial activity against the tested strains comparable or higher than the standards
ampicillin or isoniazid. Thus, for example, 2-hydroxy-N-(3-nitrophenyl)naphthalene-1-
carboxamide showed MIC = 26.0 µmol/L against methicillin-resistant S. aureus and
MIC = 51.9 µmol/L against M. marinum, or 2-hydroxy-N-phenylnaphthalene-1-carboxamide
demonstrated MIC = 15.2 µmol/L against M. kansasii. The structure-activity relationships
for all compounds are discussed.
Keywords: hydroxynaphthalenecarboxanilides; lipophilicity; photosynthetic electron
transport inhibition; spinach chloroplasts; in vitro antibacterial activity; in vitro
antimycobacterial activity; in vitro cytotoxicity; structure-activity relationships
1. Introduction
Since the 1980s morbidity due to bacterial infections has been rising again and today it has become
an increasing worldwide threat. Mortality due to respiratory infections, AIDS and tuberculosis (TB)
represents about 85% of all world mortality. The resistance of common pathogens to first-choice drugs
increased up to 100% during the last decade, and resistance of some strains to second- or third-choice
drugs is a great problem [1–3]. This is caused by general immunosuppression, a significant increase in
the number of HIV-positive patients and development of resistance to commonly used drugs. The
success of treatment is also decreased by development of cross-resistance or multidrug-resistant
(MDR) strains. In general, suppressed immunity often leads to more lethal complications. For
example, tuberculosis has re-emerged as a significant cause of global mortality. In 2011, there were an
estimated 8.7 million new cases of TB (13% co-infected with HIV) and 1.4 million people died from
TB, including almost one million deaths among HIV-negative individuals and 430,000 among people
who were HIV-positive. The number of cases of MDR-TB notified in the 27 high MDR-TB burden
countries is increasing and reached almost 60,000 worldwide in 2011. This is only one in five (19%) of
the notified TB patients estimated to have MDR-TB [1,2]. Also, other non-tuberculous mycobacteria
(NTM) are now recognized as significant human pathogens and cause difficult-to-treat or incurable
diseases ending in deaths especially of immunocompromised patients [1,2,4–6]. The increasing
number of infections and resistance of pathogens to drugs underline the importance of searching for
new antimicrobial chemotherapeutics. The current situation even necessitates the re-engineering and
repositioning of some old drug families to achieve effective control [4,6].
Salicylanilides (N-substituted hydroxybenzamides) represent compounds with a wide range of
pharmacological activities, including anti-inflammatory [7], anthelmintic [8] and herbicidal [9–11]
properties. Of these, at present antibacterial [9–14] and antimycobacterial [9–11,14,15] effects are
becoming more and more important. The exact mechanisms of action are still under investigation, but
these compounds are known to act as inhibitors of protein kinase epidermal growth factor receptor

Molecules 2013, 18
9399
(EGFR PTK) [16]. Salicylanilides are generally designed to compete with ATP for binding in catalytic
domain of tyrosine kinase [17,18] The latest studies specified them also as selective inhibitors of
interleukin-12p40 production that plays a specific role in initiation, expansion, and control of cellular
response to tuberculosis [19,20]. It was found that salicylanilides also inhibit bacterial enzymes, e.g.,
transglycosylases [21], D-alanine-D-alanine ligases [22] or sortase A [23].
The presence of an amide (-NHCO-) group is characteristic not only of various antibacterial
drugs [7,24], but also of a number of herbicides acting as photosynthesis inhibitors [9–11,25–27], e.g.,
by reversibly binding to photosystem II [28,29]. As drugs and pesticides are designed to target
particular biological systems, herbicides can also have molecular sites of action in mammals/non-plant
organisms. Therefore many pharmaceutical companies have pesticide divisions and compounds generated
by either division of the company are evaluated for both pesticide and pharmaceutical uses. In the past,
some leading pesticides, (e.g., fluconazole), have become pharmaceuticals and vice versa [30–32].
Moreover, good correlation between microbiological activities and herbicidal or antialgal effects was
found [10,11,27,33–37].
Investigation of synthesis and biological activity of ring-substituted 2-hydroxynaphthalene-1-
carboxanilides is a follow-up contribution to understanding the structure-activity relationship within a
series of modified salicylanilides. The design of these carboxanilides is based on the principle of ring
analogy with salicylanilides. Thus, primary in vitro antibacterial and antimycobacterial screening of
N-substituted 2-hydroxynaphthalene-1-carboxanilides as positional isomers of the N-substituted
3-hydroxynaphthalene-2-carboxanilides described by Kos et al. [11] was performed. A series of twenty-
two compounds was also tested for their inhibitory effects on photosynthetic electron transport in
spinach chloroplasts (Spinacia oleracea L.). The structure-activity relationships between the chemical
structure, physical properties and in vitro biological activities of all the evaluated compounds
are discussed.
2. Results and Discussion
2.1. Chemistry
All the studied compounds were prepared according to Scheme 1. There are many methods for the
preparation of ortho-hydroxycarboxanilides. The discussed compounds were synthesized by using
modified microwave-assisted synthesis [11], thus synthesis of the target compounds was carried out in
only one step with excellent yields. The condensation of 2-hydroxy-1-naphthoic acid with ring-substituted
anilines using phosphorus trichloride in chlorobenzene under microwawe conditions yielded a series of
twenty-two N-substituted 2-hydroxynaphthalene-1-carboxanilides 1–8c.
Scheme 1. Synthesis of ring-substituted 2-hydroxynaphthalene-1-carboxanilides 1–8c.
O
NH
2
R
COOH
OH
a
N
+
H
R
OH
R = H (1), OCH (2a-c), CH (3a-c), F (4a-c), Cl (5a-c), Br (6a-c), CF (7a-c), NO (8a-c)
3
3
3
2
Reagents and conditions: (a) PCl3, chlorobenzene, MW.

Molecules 2013, 18
9400
Within structure-activity relationship investigations various molecular descriptors (e.g., polar
surface area, distributive parameters, electronic parameters, molar volume, number of rotatable bonds,
and number of hydrogen bond donors or hydrogen bond acceptors, etc.), topological indexes or
parameters describing physico-chemical properties (lipophilicity, solubility, ionizability, etc.) are used.
Thus they represent numerical values that characterize properties of molecules that influence
biological activity (receptor binding). In our previous studies biological activity was strongly
influenced especially by lipophilicity and electronic parameters [9–11,14,25–27,38–41], expressed
either as Hammett’s σ parameters or Taft polar substituent constants σ*.
Lipophilicity as an important drug property has been studied and applied for decades, because drugs
mostly cross biological membranes through passive transport, which strongly depends on their
lipophilicity. Lipophilicity of the studied compounds 1–8c and the associated relevant standards
(Figure 1), was determined by RP-HPLC under isocratic conditions as capacity factor logarithm (log k)
values. The results are shown in Table 1. For individual substituents in the anilide part of the discussed
compounds also electronic Hammett’s σ parameters were predicted using the ACD/Percepta software;
they ranged from −0.28/−0.27 (2a, R = 2-OCH3 / 2c, R = 4-OCH3) to 0.71–0.78 (9a–c, R = 2-, 3-, 4-NO2).
Figure 1. Chemical structures of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU),
ampicillin and isoniazid.
NH
O
2
H
N
CH
N
3
H
S
N
CH
CH
3
H
Cl
Cl
N
CH
N
3
N
NH
3
2
O
O
O
COOH
DCMU
ampicillin
isoniazid
2-Hydroxy-N-(4-methylphenyl)naphthalene-1-carboxamide (3c) showed the lowest lipophilicity
while 2-hydroxy-N-phenylnaphthalene-1-carboxamide (1) had the highest, according to log k data. The
whole series demonstrated significantly lower experimental lipophilicity of anilide substituted
derivatives compared with the ring-substituted 3-hydroxynaphthalene-2-carboxanilides [11]. On the
contrary, 2-hydroxy-N-phenylnaphthalene-1-carboxanilide (1) showed in comparison with 3-hydroxy-
N-phenylnaphthalene-1-carboxanilide (log k = 0.6310) significantly higher experimentally determined
lipophilicity (log k = 1.3016). Within the halogenated series the lipophilicity determined by log k
values increases as follows: meta < para < ortho. Nevertheless the influence of R substituents on
lipophilicity is as follows: CH3 < F < OCH3 < NO2 < Cl < Br < CF3 < H, in general.
2.2. Inhibition of Photosynthetic Electron Transport (PET) in Spinach Chloroplasts
The activity of the evaluated 2-hydroxynaphthalene-1-carboxanilides related to inhibition of
photosynthetic electron transport (PET) in spinach (Spinacia oleracea L.) chloroplasts was medium or
relative low with respect to the standard, see Table 1. IC50 value for compound 2c (R = 4-OCH3) was
not possible to determine due to its interaction with 2,6-dichlorophenol-indophenol (DCPIP). The most
active PET inhibitors (IC50 = 29 µmol/L) were found to be compounds 1 (R = H) and 7c (R = 4-CF3).
The PET-inhibiting activity was expressed by negative logarithm of IC50 value (compound
concentration in mol/L causing 50% inhibition of PET). Correlations between log(1/IC50 [mol/L]) and

Molecules 2013, 18
9401
the lipophilicity of compounds expressed as log k or electronic properties of individual anilide
substituents expressed as Hammett’s σ parameters were performed, see Figure 2.
Table 1. Structure of the discussed ring-substituted 2-hydroxynaphthalene-1-
carboxanilides 1–8c, experimentally determined values of lipophilicity log k, predicted
electronic Hammett’s σ parameters, and IC50 values related to PET inhibition in spinach
chloroplasts in comparison with 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) standard,
in vitro antibacterial activity (MIC) of compounds in comparison with ampicillin (APC)
standard; in vitro antimycobacterial activity (MIC) of compounds in comparison with
isoniazid (INH) standard, and in vitro cytotoxicity assay (LD50) of choice compounds.
O
R
N
H
OH
Comp.
[µmol/L]
MIC
R
log k
σa
PET
IC50
LD50
MRSA
63718
>972
>873
436
MRS
A 630
243
MRSA
SA
MM
MK
MS
MAP
3202
122
>873
218
>873
462
231
462
455
228
>910
>860
107
107
>748
>748
94.1
193
94
1
2a
H
2-OCH3
3-OCH3
4-OCH3
2-CH3
3-CH3
4-CH3
2-F
1.3016
0.5121
0.6582
0.6342
0.7962
0.4593
0.3751
0.5664
0.5025
0.5568
0.8984
0.7904
0.7908
0.9509
0.8595
0.8790
0.8353
0.9411
0.9994
0.8501
0.9187
0.6260
0.8801
0.4337
0.0141
0
28.9
477
681
ND
586
372
874
243
213
313
49.6
79.7
59.2
52.2
61.1
102
153
45.6
29.0
121
86.4
37.5
1.9
>972
>873
436
60.7
>873
218
15.2
>873
109
218
115
115
115
114
114
>910
107
53.7
53.7
93.5
46.7
93.5
96.6
93.5
23.3
51.9
104
415
–
486
>873
218
436
462
231
>923
228
>910
>910
107
215
>860
>748
>748
187
386
>748
>748
208
208
208
–
950
852
426
852
451
451
451
203
>889
>889
202
840
420
731
731
175
377
731
731
195
405
811
–
>20
–
−0.28
0.12
−0.27
−0.17
−0.07
−0.17
0.06
0.34
0.06
0.22
0.37
0.23
0.22
0.39
0.23
0.51
0.43
0.51
0.77
0.71
0.78
0.6
>873
873
2b
–
2c
>873
462
>873
462
>873
462
109
–
3a
231
–
3b
231
462
231
231
–
3c
231
462
462
115
–
4a
228
455
455
228
–
4b
3-F
228
>910
>910
860
>910
>910
>860
>860
>860
>748
>748
47.0
97.1
374
>910
>910
107
–
4c
4-F
>910
215
–
5a
2-Cl
>20
–
5b
3-Cl
>860
215
>860
>860
>748
>748
187
>860
>860
>748
>748
187
5c
4-Cl
–
6a
2-Br
>748
>748
47.0
97.1
>748
>748
26.0
208
–
6b
3-Br
–
6c
4-Br
8.0
>20
>20
3.3
>20
>20
2.5
–
7a
2-CF3
3-CF3
4-CF3
2-NO2
3-NO2
4-NO2
–
193
96.6
>748
>748
104
7b
187
7c
94
94
47
8a
415
104
52
8b
26.0
830
208
208
104
–
51.9
51.9
–
8c
>830
–
415
DCMU
APC
INH
–
–
–
–
–
5.7
>45.8
–
>45.8
–
>45.8
–
–
–
–
–
–
–
–
–
–
467
29.2
117
>1823
–
a
calculated using ACD/Percepta ver. 2012 (Advanced Chemistry Development, Inc., Toronto, ON, Canada, 2012);
SA = Staphylococcus aureus ATCC 29213, MRSA = clinical isolates of methicillin-resistant Staphylococcus aureus
63718, SA 630 and SA 3202 (National Institute of Public Health, Prague, Czech Republic); MM = M. marinum CAMP
5644, MK = M. kansasii DSM 44162, MM = M. smegmatis ATCC 700084 and clinical isolate MAP = M. avium
paratuberculosis CIT03; ND = not determined due to its interaction with 2,6-dichlorophenol-indophenol (DCPIP).

Molecules 2013, 18
Figure 2. Relationships between PET inhibition log (1/IC50) [mol/L]) in spinach
9402
chloroplasts and lipophilicity expressed as log k (2A) or N-substituent electronic Hammett’s
σ parameters (2B) of selected studied compounds (eliminated compounds are marked by
crosses, nitro derivatives 8a–c are marked by triangles).
A
4.7
4.5
4.3
4.1
3.9
3.7
3.5
3.3
3.1
2.9
0.2
0.4
0.6
0.8
log k
1.0
1.2
1.4
B
4.7
4.5
4.3
4.1
3.9
3.7
3.5
3.3
3.1
2.9
-0.4
-0.2
0.0
0.2
0.4
0.6
0.8
1.0
σ
The biological activity is affected by lipophilicity (see Table 1 and Figure 2A). In general, when the
inactive anilide 2b (3-OCH3) and 3a (R = 2-CH3) as well as the compound 8c (R = 4-NO2) were
eliminated (marked by crosses), a linear dependence of log(1/IC50 [mol/L]) on log k for 19 ortho-,
meta- and para-substituted derivatives could be observed. The correlation between log(1/IC50 [mol/L])
and log k could be expressed by the following equation:
log(1/IC50) = 2.613(±0.203) + 1.658(±0.251)log k
(1)
r = 0.848, s = 0.244, F = 43.5, n = 19
On the other hand, PET inhibition is also influenced by the electronic properties of individual
substituents, see Figure 2B. After elimination of unsubstituted compound 1 (IC50 = 28.9 µmol/L) and
compound 2b (R = 3-OCH3, IC50 = 681 µmol/L) for compounds with σ varying in the range from
−0.28 to 0.51 a linear correlation between log(1/IC50 [mol/L]) on σ could be expressed by equation (2).
log(1/IC50) = 3.584 (± 0.085) + 1.493 (± 0.280)σ
(2)
r = 0.818, s = 0.272, F = 28.3, n = 16
The linear correlation between log(1/IC50 [mol/L]) and log k for 16 compounds for which
Equation (2) was evaluated is as follows:
log(1/IC50) = 2.449(±0.245) + 1.918(±0.323)log k
(3)
r = 0.846, s = 0.252, F = 35.3, n = 16

Molecules 2013, 18
9403
For compounds with σ > 0.51, i.e., 8a (R = 2-NO2), 8b (R = 3-NO2) and 8c (R = 4-NO2) PET
inhibiting activity decreased with increasing σ parameter. Bilinear dependence of PET inhibiting
activity on electronic properties of R substituent was found for example with 2-benzylsulphanyl-
benzimidazoles [42].
The use of both the abovementioned descriptors (log k and σ) in a multilinear correlation improved
the statistical analysis results:
log(1/IC50) = 2.809(±0.234) + 1.223(±0.356)log k + 0.826(±0.286)σ
(4)
r = 0.909, s = 0.204, F = 31.0, n = 16
The results of the statistical analyses above indicate that the biological activity of the tested
compounds is significantly influenced by their lipophilicity and the σ parameter of the R substituent.
As the studied compounds were found to inhibit the Hill reaction, their site of action is situated in
PS II. For more precise determination of their site of action diphenylcarbazide (DPC), an artificial
electron donor acting in the intermediate Z•/D• on the donor side of PS II [43] can be used. If addition
of DPC to chloroplasts results in practically complete restoration of photosynthetic electron transport
which was inhibited by one PET inhibitor, the inhibitory site of action of this inhibitor is situated
exclusively on the donor side of PS II, in the section between oxygen evolving complex and Z•/D•
intermediate. On the other hand, if the site of action of certain PS II inhibitor would be situated on the
acceptor side of PS II, in the section between PS II reaction centre (P680) and secondary quinone
acceptor QB, the supply of electrons by DPC acting in Z•/D• intermediate would not be able to secure
complete PET restoration. The results of DPC experiment confirmed that the site of action of the
studied compounds is situated only on the donor side of PS II. Similar site of action in the
photosynthetic electron transport chain was found for substituted salicylanilides [38], N-substituted
2-aminobenzothiazoles [27], N-phenylcarbamates [39], and 4-chloro-2-(chlorophenylcarbamoyl)phenyl
alkylcarbamates [40].
The effects of studied compounds on the photosynthetic centres of chloroplasts were investigated
by studying chlorophyll a fluorescence. Figure 3A shows the effect of compound 1 on the chlorophyll
a fluorescence emission band at 686 nm. The decreased intensity of this emission band belonging to
the chlorophyll-protein complexes mainly in photosystem (PS) II [44] suggested PS II as the site of
action of the studied inhibitor.
Binding of PET inhibitors to aromatic amino acids (AAA) occurring in photosynthetic proteins of
spinach chloroplasts situated in PS II contributes to PET inhibition. The interaction of AAA with PET
inhibitors can be studied by the quenching of AAA fluorescence at 334 nm Figure 3B presents
fluorescence emission spectra of aromatic amino acids of untreated spinach chloroplasts and of
chloroplasts treated with increasing concentrations of compound 1 (see Figure 3B). Increasing
concentration of 1 resulted in strong decline of AAA fluorescence and similar effects exhibited also
further studied compounds. Decrease of Chla and AAA fluorescence in spinach chloroplasts was
observed previously in the presence of substituted N-benzylpyrazine-2-carboxamides [41] and
N-substituted 2-aminobenzothiazoles [27].

Molecules 2013, 18
Figure 3. Emission fluorescence spectra of chlorophyll a in suspension of spinach
9404
chloroplasts without and with compound 1 (c = 0, 0.059, 0.117, 0.234 and 0.468 mmol/L;
curves from top to bottom) (A), and emission fluorescence spectra of aromatic amino acids
in suspension of spinach chloroplasts without and with 1 (c = 0, 0.012, 0.029, 0.059,
0.117 mmol/L) (B).
100
1500
A
B
80
60
40
20
0
1000
500
0
660
680
700
300
325
350
375
λ nm
λ nm
2.3. In Vitro Antibacterial Susceptibility Testing
The discussed compounds were tested for their in vitro antibacterial activity against Staphylococcus
aureus ATCC 29213 (SA) and three methicillin-resistant S. aureus (MRSA 63718, SA 630, SA 3202),
see Table 1. S. aureus and especially MRSA strains were selected, because they caused life-threatening
nosocomial infections for decades and have recently become a significant threat for community
acquired infections and livestock associated infections with high levels of morbidity and mortality.
Methicillin resistance is connected with not only clinically inadequate susceptibility to all β-lactam
antibiotics but usually to other antimicrobial drugs as well [45,46]. Therefore there is an urgent need to
develop new, potent, and fast acting anti-tuberculosis and anti-MRSA drugs.
Although salicylanilides seem to be promising antibacterial agent candidates [13], most of the
compounds showed only moderate activity, except for 2-hydroxy-N-(2-nitrophenyl)naphthalene-1-
carboxamide (8a), 2-hydroxy-N-(3-nitrophenyl)naphthalene-1-carboxamide (8b), 2-hydroxy-N-(4-
bromophenyl)naphthalene-1-carboxamide (6c) and 2-hydroxy-N-(4-trifluoromethylphenyl)-naphthalene-
1-carboxamide (7c), whose activity, especially against MRSA, was comparable with or higher than
that of the standard, see Table 1. Nevertheless due to the moderate activity of the rest of the
compounds, no thorough structure-activity relationships could be established. In general, it can be
stated that antibacterial activity is positively influenced by higher lipophilicity of compounds with
simultaneous substitution of the anilide ring by electron-withdrawing moieties.
In our previous study of 3-hydroxynaphthalene-2-carboxanilides [11] only 3-hydroxy-N-(2-
methoxyphenyl)naphthalene-2-carboxamide showed biological activity (MIC = 55.0 µmol/L) against
S. aureus, as well as all the MRSA strains. The lipophilicity of this compound is log k = 0.69, and
2-methoxy moiety is a significant electron donor (σ = −0.28). Inactivity of the rest of those derivatives
may be caused by their relatively low solubility in aqueous media and different properties of
compounds affecting their biological activity. Similarly, as differences in lipo/hydrophilic properties of

Molecules 2013, 18
9405
3-hydroxynaphthalene-2-carboxanilides vs. 2-hydroxynaphthalene-1-carboxanilides were mentioned
above, different substituents in the anilide ring were found as being favourable in other series of
hydroxyarylcarboxanilides [9,10,14,25].
2.4. In Vitro Antimycobacterial Evaluation
The evaluation of the in vitro antimycobacterial activity of the compounds was performed against
Mycobacterium marinum CAMP 5644 (MM), M. kansasii DSM 44162 (MK), M. smegmatis ATCC
700084 (MS) and a clinical isolate M. avium paratuberculosis CIT03 (MAP), see Table 1. The genus
Mycobacterium consists of a closely related group of fast and slow-growing species. M. tuberculosis
causes one of the most serious human infections, (tuberculosis). Difficulties should be considered
while studying M. tuberculosis—especially a slow growth rate and the requirement of working in high
containment biosafety facilities. To lower risk and make manipulation in the laboratory easier,
surrogate model pathogens for M. tuberculosis can be used in laboratory studies. M. smegmatis is an
ideal representative of a fast-growing nonpathogenic microorganism particularly useful in studying
basic cellular processes of special relevance to pathogenic mycobacteria. M. marinum is very closely
related to the M. tuberculosis and it is the cause of TB-like infections in poikilothermic organisms,
especially frogs and fish. M. marinum is a good model for studying especially because of the lower
risk for laboratory workers, genetic relatedness and similar pathology to human TB [47–49]. However,
because of M. tuberculosis, the pathogenic role of NTM in humans was overshadowed for a long time.
M. kansasii, the most virulent of the NTM, causes non-tuberculous mycobacterial lung infections which
are very common nowadays and can be indistinguishable from tuberculosis [50]. M. avium
paratuberculosis is suspected to be a causative agent in gastrointestinal diseases, is resistant to
standard antimycobacterial therapy, but is susceptible to some standard antibiotics. However, the
resistance to these antibiotics develop fast [51].
As shown in Table 1, isoniazid was active only against M. kansasii, whereas the target compounds
showed comparable with or higher effectiveness than that of the isoniazid. Although compounds
showed a wide range of activity that did not reach a level equivalent to “classical antituberculotics”,
some general structure-activity relationships could be established.
Compounds 8b (R = 3-NO2) and 8c (R = 4-NO2) demonstrated about 9-fold higher activity (MIC
ca. 52 µmol/L) against M. marinum than isoniazid, unsubstituted compound 1 and 7c (R = 4-CF3)
showed higher effect against M. kansasii than isoniazid as well as 5a (R = 2-Cl) against M. smegmatis.
Almost all the compounds expressed higher activity against M. avium paratuberculosis than isoniazid,
nevertheless 6c (R = 4-Br) was the most active. Based on these facts and data presented in Table 1 it
can be concluded that activity decreased with change of anilide substituent position as follows:
para > meta > ortho. Further parameters influencing activity can involve lipophilicity and electronic
properties of the individual substituents. In general it can be stated that the effects against all four
mycobacterial strains increased with higher lipophilicity and also electron-withdrawing properties of
individual substituents are more advantageous than electron donor substituents.
Generally it can be stated that from the tested compounds para- and meta-substituted derivatives or
non-substituted compound 1 expressed higher antimycobacterial activity than ortho-substituted ones.
This fact is probably caused by a steric effect of a spatially-close moiety. The proximity of the

Molecules 2013, 18
9406
ortho-substituent to the carboxamide group on the aniline ring led to the twist of the aniline ring plain
towards the carboxamide group, i.e., to the whole naphthalene core, while meta- and especially
para-substitution lead to planar structure that, when the substituent is more lipophilic, easier permeates
through various types of membranes [52]. Thus, the lipophilicity of meta- and para-substituted
derivatives was found as the factor conducive to the activity of such structures. As compounds with
electron-withdrawing substituents expressed antimycobacterial activities, it can be stated that these
components affecting electronic density (charge) at the carbonyl moiety influence the potential of
binding of the carboxamide group to possible binding sites in a mycobacterial cell [52]. Janin suggests
establishing the hypothesis that carboxamides can interfere with the mycobacterial proton pump
F0F1H+ATPase or they can inhibit biosynthesis of amino acid [24]. Kratky et al. recently reported the
ability of salicylanilide derivatives to inhibit two essential mycobacterial enzymes; methionine
aminopeptidase (performs an important function in different growth phases) and isocitrate lyase
(essential for the metabolism of fatty acids). Both are necessary for the maintenance of the latent
tuberculosis infection [16].
Previously presented 3-hydroxynaphthalene-2-carboxanilides [11] with different substituents
showed antimycobacterial activity. As preferred substitutions with activity against M. marinum and M.
kansasii 2-fluoro, 2-methoxy and 3-methyl moieties were found. In general it can be stated that in
both series, derivatives with lipophilicity log k > 0.62 can show antimycobacterial effect. Within
3-hydroxynaphthalene-2-carboxanilides rather electron-neutral or slightly electron-withdrawing
substituents are preferable [11], while in the series of 2-hydroxynaphthalene-1-carboxanilides
substituents with significant electron-withdrawing effect are favoured.
2.5. In Vitro Cytotoxicity Assay
The in vitro primary screening of cytotoxicity of the selected compounds was performed using the
human monocytic leukemia THP-1 cell line. The cytotoxicity was evaluated as the LD50 value
(LD50–lethal dose to 50% of the cell population), see Table 1. Compounds were not soluble above
concentrations 20 μmol/L, the highest concentration prepared in DMSO for the toxicity assay was
20.0 μmol/L. Treatment with 20 μmol/L of 1, 5a, 7a, 7b, 8a and 8b did not lead to significant lethal effects
on THP-1 cells. The strongest effect on cell viability was detected in compounds 6c (LD50 = 8.0 µmol/L),
7c (LD50 = 3.3 µmol/L), and 8c (LD50 = 2.5 µmol/L); however, their toxicity is still not comparable
with those of camptothecin (0.16 ± 0.07 µmol/L) assessed in this line formerly. It is evident that
cytotoxicity within the series of carboxanilides is related only to para-position of substituents and
increases with electron-withdrawing properties of substituents, see Table 1. Based on these observations it
is important to conclude that unsubstituted 1 (R = H), 8a (R = 2-NO2) and 8b (R = 3-NO2) or 5a (R =
2-Cl) can be considered as non-toxic agents for subsequent design of novel antimicrobial agents.
3. Experimental
3.1. General
All reagents were purchased from Aldrich (Sigma-Aldrich, St. Louis, MO, USA) and Merck
(Darmstadt, Germany). Reactions were carried out in StartSYNTH microwave labstation (Milestone,

Molecules 2013, 18
9407
Italy). The melting points were determined on Kofler hot-plate apparatus (HMK Franz Kustner,
Germany) and are uncorrected. Infrared (IR) spectra were recorded on a Smart MIRacle™ ATR ZnSe
for Nicolet™ Impact 400 FT-IR spectrometer (Thermo Electron Corporation, West Palm Beach, FL,
USA). The spectra were obtained by accumulation of 256 scans with 2 cm−1 resolution in the region of
1
4000–600 cm−1. All H and 13C-NMR spectra were recorded on a Bruker Avance III 400 MHz
1
13
FT-NMR spectrometer (400 MHz for H and 100 MHz for C, Bruker Comp., Karlsruhe, Germany).
Chemical shifts are reported in ppm (δ) using signal of the solvent (DMSO-d6) as the reference
(2.500, resp. 39.50). Mass spectra were measured using a LTQ Orbitrap Hybrid Mass Spectrometer
(Thermo Electron Corporation) with direct injection into an APCI source (400 °C) in the positive mode.
3.2. Synthesis
General Procedure for Synthesis of Carboxamide Derivatives 1–8c
2-Hydroxynaphthalene-1-carboxylic acid (5.3 mmol) and the corresponding substituted aniline
(5.3 mmol) were suspended in dry chlorobenzene (30 mL). Phosphorous trichloride (2.65 mmol) was
added dropwise, and the reacting mixture was heated in the microwave reactor at maximal allowed
power 500 W and 130 °C, using infrared flask-surface control of temperature, for 15 min. The solvent
was evaporated under reduced pressure, the solid residue washed with 2M HCl, and the crude product
was recrystallized from aqueous ethanol. Studied compounds 1–8c are presented in Table 1.
2-Hydroxy-N-phenylnaphthalene-1-carboxamide (1). Yield 80%; Mp 172–173 °C (171–172 °C [53],
182 °C [54]); IR (cm−1): 3180, 3050, 1637, 1627, 1597, 1536, 1513, 1497, 1442, 1431, 1328, 1306,
1234, 1203, 963, 813, 740, 693; 1H-NMR (DMSO-d6), δ: 11.11 (s, 1H), 8.20 (d, 1H, J = 8.5 Hz), 7.97
(br. s, 1H), 7.86 (t, 2H, J = 8.2 Hz), 7.62 (d, 2H, J = 7.8 Hz), 7.48 (ddd, 1H, J = 8.0 Hz, J = 7.0 Hz,
J = 1.3 Hz), 7.44–7.36 (m, 3H), 7.25–7.18 (m, 2H); 13C-NMR (DMSO-d6), δ: 168.46, 159.81, 136.90,
134.51, 130.46, 129.73, 129.32, 128.89, 128.63, 125.30, 123.72, 122.29, 120.74, 119.36, 109.91; HR-MS:
for C17H13NO2 [M+H]+ calculated 264.1019 m/z, found 264.1023 m/z.
2-Hydroxy-N-(2-methoxyphenyl)naphthalene-1-carboxamide (2a). Yield 76%; Mp 169–170 °C; IR
(cm−1): 3283, 1645, 1600, 1528, 1513,1478, 1459, 1431, 1337, 1281, 1245, 1113, 1022, 966, 894, 818,
743, 735; 1H-NMR (DMSO-d6), δ: 10.42 (br. s, 1H), 9.34 (s, 1H), 8.28 (dd, 1H, J = 7.8 Hz, J = 1.0 Hz),
8.00 (d, 1H, J = 8.5 Hz), 7.87 (d, 1H, J = 9.0 Hz), 7.84 (d, 1H, J = 8.3 Hz), 7.48 (ddd, 1H, J = 8.0 Hz,
J = 7.0 Hz, J = 1.3 Hz), 7.33 (ddd, 1H, J = 8.0 Hz, J = 7.0 Hz, J = 1.3 Hz), 7.25 (d, 1H, J = 8.8 Hz),
7.16–7.07 (m, 2H), 7.01 (dt, 1H, J = 7.5 Hz, J = 1.0 Hz), 3.82 (s, 3H); 13C-NMR (DMSO-d6), δ:
165.31, 152.19, 149.45, 131.76, 130.90, 128.02, 127.68, 127.64, 127.00, 124.47, 123.95, 123.08,
121.37, 120.45, 118.30, 117.13, 111.25, 55.82; HR-MS: for C18H15NO3 [M+H]+ calculated 294.1125
m/z, found 294.1133 m/z.
2-Hydroxy-N-(3-methoxyphenyl)naphthalene-1-carboxamide (2b). Yield 77%; Mp 147–149 °C; IR
(cm−1): 3115, 1642, 1607, 1538, 1512, 1447, 1329, 1240, 1202, 1157, 1029, 815, 776, 755, 738, 684;
1H-NMR (DMSO-d6), δ: 10.37 (s, 1H), 10.12 (br. s, 1H), 7.87–7.84 (m, 2H), 7.68 (d, 1H, J = 8.3 Hz),
7.55 (s, 1H), 7.46 (ddd, 1H, J = 8.0 Hz, J = 7.0 Hz, J = 1.3 Hz), 7.36–7.31 (m, 2H), 7.26–7.23 (m, 2H),
6.68 (d, 1H, J = 7.8 Hz), 3.76 (s, 3H); 13C-NMR (DMSO-d6), δ: 165.79, 159.54, 151.61, 140.84,

Molecules 2013, 18
9408
131.40, 130.13, 129.47, 127.97, 127.38, 126.97, 123.41, 123.00, 118.62, 118.35, 111.60, 108.71,
105.07, 54.99; HR-MS: for C18H15NO3 [M+H]+ calculated 294.1125 m/z, found 294.1132 m/z.
2-Hydroxy-N-(4-methoxyphenyl)naphthalene-1-carboxamide (2c). Yield 74%; Mp 168-170°C; IR
1
(cm−1): 3262, 1625, 1538, 1506, 1462, 1413, 1247, 1232, 1204, 1178, 1027, 828, 792, 712; H-NMR
(DMSO-d6), δ: 10.24 (s, 1H), 10.08 (br. s, 1H), 7.84 (d, 2H, J = 8.8 Hz), 7.75–7.72 (m, 2H), 7.69 (d,
2H, J = 8.5 Hz), 7.46 (ddd, 1H, J = 8.0 Hz, J = 7.0, J = 1.3 Hz), 7.32 (ddd, 1H, J = 8.0 Hz, J = 7.0 Hz,
J = 1.3 Hz), 7.25 (d, 1H, J = 8.8 Hz), 6.94 (d, 1H, J = 8.8 Hz), 3.75 (s, 3H); 13C-NMR (DMSO-d6), δ:
165.17, 155.24, 151.56, 132.93, 131.50, 130.00, 127.95, 127.41, 126.90, 123.52, 122.96, 120.72, 118.76,
118.38, 113.81, 55.21; HR-MS: for C18H15NO3 [M+H]+ calculated 294.1125 m/z, found 294.1133 m/z.
2-Hydroxy-N-(2-methylphenyl)naphthalene-1-carboxamide (3a). Yield 69%; Mp 127–130 °C; IR
(cm−1): 3176, 1615, 1599, 1574, 1519, 1456, 1433, 1315, 1276, 1252, 1212, 1148, 1040, 969, 900,
818, 752, 743; 1H-NMR (DMSO-d6), δ: 10.16 (br. s, 1H), 9.80 (s, 1H), 7.88–7.84 (m, 3H), 7.60 (d, 1H,
J = 7.8 Hz), 7.50 (ddd, 1H, J = 8.0 Hz, J = 7.0 Hz, J = 1.3 Hz), 7.34 (ddd, 1H, J = 8.0 Hz, J = 7.0 Hz,
J = 1.3 Hz), 7.28–7.23 (m, 3H), 7.15 (t, 1H, J = 7.3 Hz) 2.35 (s, 3H); 13C-NMR (DMSO-d6), δ: 165.64,
151.79, 136.45, 132.54, 131.67, 131.31, 131.30, 130.09, 127.97, 127.49, 126.89, 125.88, 125.86,
125.41, 123.64, 122.96, 118.36, 18.08; HR-MS: for C18H15NO2 [M+H]+ calculated 278.1176 m/z, found
278.1183 m/z.
2-Hydroxy-N-(3-methylphenyl)naphthalene-1-carboxamide (3b). Yield 74%; Mp 145–148 °C; IR (cm−1):
3343, 3206, 3029, 1628, 1614, 1601,1575, 1527, 1435, 1274, 1229, 1211, 1146, 1042, 967, 819, 773,
1
745, 687; H-NMR (DMSO-d6), δ: 10.32 (s, 1H), 10.10 (br. s, 1H), 7.87–7.84 (m, 2H), 7.71 (s, 1H),
7.69 (d, 1H, J = 8.5 Hz), 7.57 (d, 1H, J = 8.0 Hz), 7.46 (ddd, 1H, J = 8.0 Hz, J = 7.0 Hz, J = 1.3 Hz),
7.33 (ddd, 1H, J = 8.0 Hz, J = 7.0 Hz, J = 1.3 Hz), 7.26 (d, 1H, J = 8.8 Hz), 8.23 (t, 1H, J = 7.5 Hz),
6.92 (d, 1H, J = 7.5 Hz), 2.32 (s, 3H); 13C-NMR (DMSO-d6), δ: 165.69, 151.59, 139.60, 137.84,
131.45, 130.06, 128.52, 127.97, 127.40, 126.94, 123.98, 123.44, 122.99, 119.81, 118.72, 118.38,
116.53, 21.32; HR-MS: for C18H15NO2 [M+H]+ calculated 278.1176 m/z, found 278.1182 m/z.
2-Hydroxy-N-(4-methylphenyl)naphthalene-1-carboxamide (3c). Yield 77%; Mp 143–145 °C; IR (cm−1):
1
3291, 1637, 1599, 1537, 1513, 1435, 1323, 1299, 1252, 1210, 1044, 963, 892, 806, 739; H-NMR
(DMSO-d6), δ: 10.30 (s, 1H), 10.09 (br. s, 1H), 7.86-7.83 (m, 2H), 7.72–7.68 (m, 3H), 7.46 (ddd, 1H,
J = 8.0 Hz, J = 7.0 Hz, J = 1.3 Hz), 7.32 (ddd, 1H, J = 8.0 Hz, J = 7.0 Hz, J = 1.3 Hz), 7.25 (d, 1H,
13
J = 9.0 Hz), 7.16 (d, 1H, J = 8.3 Hz), 2.29 (s, 3H); C-NMR (DMSO-d6), δ: 165.49, 151.58, 137.23,
132.15, 131.48, 130.04, 129.06, 127.97, 127.41, 126.93, 126.92, 123.47, 122.98, 119.27, 118.38,
20.54; HR-MS: for C18H15NO2 [M+H]+ calculated 278.1176 m/z, found 278.1180 m/z.
N-(2-fluorophenyl)-2-hydroxynaphthalene-1-carboxamide (4a). Yield 64%; Mp 150–152 °C; IR
(cm−1): 3256, 1622, 1605, 1580, 1524, 1502, 1460, 1403, 1327, 1266, 1220, 1201, 1148, 1105, 970,
861, 819, 795, 763, 753, 704; 1H-NMR (DMSO-d6), δ: 10.22 (br. s, 1H), 10.17 (s, 1H), 7.99–7.95 (m,
2H), 7.88-7.83 (m, 3H), 7.49 (ddd, 1H, J = 8.0 Hz, J = 7.0 Hz, J = 1.3 Hz), 7.34 (ddd, 1H, J = 8.0 Hz,
J = 7.0 Hz, J = 1.3 Hz), 7.33–7.27 (m, 1H), 7.26–7.22 (m, 3H); 13C-NMR (DMSO-d6), δ: 156.92,
154.48 (d, J = 246.5 Hz), 151.95, 131.60, 130.41, 127.99, 127.47, 126.96, 126.13 (d, J = 12.5 Hz),
125.88 (d, J = 8.1 Hz), 125.35 (d, J = 1.5 Hz), 124.32 (d, J = 3.7 Hz), 123.56, 122.99, 118.33, 117.72,
115.68 (d, J = 19.1 Hz); HR-MS: for C17H12NO2F [M+H]+ calculated 282.0925 m/z, found 282.0931 m/z.

Molecules 2013, 18
9409
N-(3-fluorophenyl)-2-hydroxynaphthalene-1-carboxamide (4b). Yield 68%; Mp 187–190 °C; IR
(cm−1): 3371, 3284, 1640, 1611, 1586, 1514, 1445, 1430, 1279, 1238, 1212, 1145, 1039, 973, 956,
1
807, 766, 676; H-NMR (DMSO-d6), δ: 10.62 (s, 1H), 10.18 (br. s, 1H), 7.89–7.81 (m, 3H), 7.67 (d,
1H, J = 8.5 Hz), 7.53–7.51 (m, 1H), 7.47 (ddd, 1H, J = 8.0 Hz, J = 7.0 Hz, J = 1.3 Hz), 7.39 (ddd, 1H,
J = 8.0 Hz, J = 7.0 Hz, J = 1.3 Hz), 7.38–7.31 (m, 1H), 7.26 (d, 1H, J = 9.0 Hz), 6.93 (ddd, 1H, J = 8.7 Hz,
J = 2.8 Hz, J = 0.7 Hz); 13C-NMR (DMSO-d6), δ: 166.10, 162.20 (d, J = 241.4 Hz), 151.73, 141.33 (d,
J = 11.0 Hz), 131.32, 130.37, 130.37 (d, J = 9.5 Hz), 128.02, 127.37, 127.10, 123.31, 123.09, 118.33,
118.23, 115.03 (d, J = 2.9 Hz), 109.75 (d, J = 21.3 Hz), 105.99 (d, J = 26.4 Hz); HR-MS: for
C17H12NO2F [M+H]+ calculated 282.0925 m/z, found 282.0933 m/z.
N-(4-fluorophenyl)-2-hydroxynaphthalene-1-carboxamide (4c). Yield 72%; Mp 210–212 °C; IR
(cm−1): 3266, 1638, 1615, 1547, 1507, 1434, 1404, 1211, 1147, 1098, 1040, 961, 827, 810, 739;
1H-NMR (DMSO-d6), δ: 10.46 (s, 1H), 10.13 (br. s, 1H), 7.87-7.82 (m, 4H), 7.68 (d, 1H, J = 8.3 Hz),
7.46 (ddd, 1H, J = 8.0 Hz, J = 7.0 Hz, J = 1.3 Hz), 7.33 (ddd, 1H, J = 8.0 Hz, J = 7.0 Hz, J = 1.3 Hz),
7.26 (d, 1H, J = 9.0 Hz), 7.23–7.18 (m, 2H); 13C-NMR (DMSO-d6), δ: 165.60, 158.04 (d, J = 239.2 Hz),
151.65, 136.08 (d, J = 2.9 Hz), 131.41, 130.20, 127.99, 127.40, 127.02, 123.41, 123.04, 120.94 (d,
J = 8.1 Hz), 118.46, 118.36, 115.27 (d, J = 22.0 Hz); HR-MS: for C17H12NO2F [M+H]+ calculated
282.0925 m/z, found 282.0930 m/z.
N-(2-chlorophenyl)-2-hydroxynaphthalene-1-carboxamide (5a). Yield 62%; Mp 138–139 °C; IR
(cm−1): 3271, 1625, 1614, 1580, 1518, 1431, 1402, 1321, 1283, 1229, 1196, 1156, 1032, 821, 793,
1
762, 689; H-NMR (DMSO-d6), δ: 10.38 (br. s, 1H), 9.99 (s, 1H), 8.00 (d, 1H, J = 8.3 Hz), 7.97 (d,
1H, J = 7.8 Hz), 7.88 (d, 1H, J = 9.0 Hz), 7.85 (d, 1H, J = 8.5 Hz), 7.55 (d, 1H, J = 8.0 Hz), 7.50 (ddd,
1H, J = 8.0 Hz, J = 7.0 Hz, J = 1.3 Hz), 7.42 (t, 1H, J = 7.5 Hz), 7.34 (ddd, 1H, J = 8.0 Hz,
J = 7.0 Hz, J = 1.3 Hz), 7.28–7.24 (m, 2H); 13C-NMR (DMSO-d6), δ: 165.81, 152.29, 135.15, 131.70,
130.80, 129.56, 128.03, 127.57, 127.50, 127.02, 126.46, 126.41, 126.40, 123.81, 123.07, 118.30,
117.09; HR-MS: for C17H12NO2Cl [M+H]+ calculated 298.0629 m/z, found 298.0636 m/z.
N-(3-chlorophenyl)-2-hydroxynaphthalene-1-carboxamide (5b). Yield 64%; Mp 191–194 °C; IR
(cm−1): 3358, 3297, 1640, 1589, 1538, 1514, 1474, 1422, 1353, 1312, 1265, 1212, 1145, 1038, 963,
1
911, 808, 770, 746, 676; H-NMR (DMSO-d6), δ: 7.61 (s, 1H), 10.19 (br. s, 1H), 8.06 (t, 1H,
J = 1.9 Hz), 7.89–7.85 (m, 2H), 7.69–7.64 (m, 2H), 7.47 (ddd, 1H, J = 8.0 Hz, J = 7.0 Hz, J = 1.3 Hz),
7.38 (t, 1H, J = 8.2 Hz), 7.33 (ddd, 1H, J = 8.0 Hz, J = 7.0 Hz, J = 1.3 Hz), 7.26 (d, 1H, J = 9.0 Hz),
13
7.16 (ddd, 1H, J = 8.0 Hz, J = 2.0 Hz, J = 1.0 Hz); C-NMR (DMSO-d6), δ: 166.13, 151.75, 141.05,
133.11, 131.32, 130.46, 130.41, 128.03, 127.38, 127.13, 123.31, 123.11, 123.03, 118.70, 118.34,
118.18, 117.66; HR-MS: for C17H12NO2Cl [M+H]+ calculated 298.0629 m/z, found 298.0636 m/z.
N-(4-chlorophenyl)-2-hydroxynaphthalene-1-carboxamide (5c). Yield 61%; Mp 193–195 °C (193 °C
[55]); IR (cm−1): 3293, 1639, 1597, 1538, 1514, 1492, 1435, 1397, 1321, 1300, 1269, 1250, 1211,
1
1092, 810; H-NMR (DMSO-d6), δ: 10.55 (s, 1H), 10.17 (br. s, 1H), 7.88–7.84 (m, 4H), 7.67 (d, 1H,
J = 8.5 Hz), 7.46 (ddd, 1H, J = 8.0 Hz, J = 7.0 Hz, J = 1.3 Hz), 7.42 (d, 2H, J = 9.0 Hz), 7.33 (ddd, 1H,
J = 8.0 Hz, J = 7.0 Hz, J = 1.3 Hz), 7.26 (d, 1H, J = 8.8 Hz); 13C-NMR (DMSO-d6), δ: 165.88, 151.70,
138.60, 131.36, 130.31, 128.64, 128.02, 127.39, 127.08, 126.83, 123.35, 123.07, 120.79, 118.36,
118.33; HR-MS: for C17H12NO2Cl [M+H]+ calculated 298.0629 m/z, found 298.0635 m/z.

Molecules 2013, 18
9410
N-(2-bromophenyl)-2-hydroxynaphthalene-1-carboxamide (6a). Yield 74%; Mp 158–160 °C; IR
(cm−1): 3267, 1625, 1615, 1575, 1516, 1460, 1425, 1401, 1319, 1281, 1221, 1195, 1155, 1141, 1022,
1
821, 762; H-NMR (DMSO-d6), δ: 10.40 (br. s, 1H), 9.90 (s, 1H), 8.04 (d, 1H, J = 8.3 Hz), 7.93 (d,
1H, J = 7.8 Hz), 7.88 (d, 1H, J = 9.0 Hz), 7.85 (d, 1H, J = 8.3 Hz), 7.72 (d, 1H, J = 7.8 Hz), 7.52–7.45
(m, 2H), 7.34 (ddd, 1H, J = 8.0 Hz, J = 7.0 Hz, J = 1.3 Hz), 7.26 (d, 1H, J = 8.8 Hz), 7.19 (d, 1H,
J = 7.4 Hz); 13C-NMR (DMSO-d6), δ: 165.81, 152.33, 136.54, 132.75, 131.68, 130.83, 128.12, 128.02,
127.58, 127.01, 126.99, 126.84, 123.91, 123.08, 118.29, 117.82, 117.04; HR-MS: for C17H12NO2Br
[M+H]+ calculated 342.0124 m/z, found 342.0133 m/z.
N-(3-bromophenyl)-2-hydroxynaphthalene-1-carboxamide (6b). Yield 72%; Mp 195–198 °C; IR
(cm−1): 3354, 3302, 1640, 1586, 1532, 15313, 1471, 1436, 1418, 1353, 1317, 1271, 1264, 1252, 1212,
1
963, 901, 857, 808, 766, 745, 674; H-NMR (DMSO-d6), δ: 10.59 (s, 1H), 10.19 (br. s, 1H), 8.20 (s,
1H), 7.88–7.84 (m, 2H), 7.69–7.66 (m, 2H), 7.47 (ddd, 1H, J = 8.0 Hz, J = 7.0 Hz, J = 1.3 Hz),
7.35–7.24 (m, 4H); 13C-NMR (DMSO-d6), δ: 166.10, 151.73, 141.18, 131.30, 130.76, 130.39, 128.02,
127.36, 127.12, 125.92, 123.30, 123.09, 121.61, 121.54, 118.33, 118.15, 118.03; HR-MS: for
C17H12NO2Br [M+H]+ calculated 342.0124 m/z, found 342.0132 m/z.
N-(4-bromophenyl)-2-hydroxynaphthalene-1-carboxamide (6c). Yield 68%; Mp 188–190 °C; IR
(cm−1): 3285, 1639, 1585, 1530, 1514, 1487, 1435, 1393, 1302, 1271, 1249, 1210, 1073, 1010, 963,
1
812, 739; H-NMR (DMSO-d6), δ: 10.54 (s, 1H), 10.17 (br. s, 1H), 7.88–7.84 (m, 2H), 7.79 (d, 2H,
J = 8.5 Hz), 7.66 (d, 1H, J = 8.3 Hz), 7.54 (d, 2H, J = 8.5 Hz), 7.46 (ddd, 1H, J = 8.0 Hz, J = 7.0 Hz,
13
J = 1.3 Hz), 7.33 (ddd, 1H, J = 8.0 Hz, J = 7.0 Hz, J = 1.3 Hz), 7.25 (d, 1H, J = 8.5 Hz); C-NMR
(DMSO-d6), δ: 165.92, 151.70, 139.00, 131.55, 131.34, 130.32, 128.02, 127.38, 127.08, 123.34,
123.07, 121.18, 118.35, 118.31, 114.86; HR-MS: for C17H12NO2Br [M+H]+ calculated 342.0124 m/z,
found 342.0133 m/z.
2-Hydroxy-N-(2-trifluoromethylphenyl)naphthalene-1-carboxamide (7a). Yield 92%; Mp 124–125 °C;
IR (cm−1): 3235, 1626, 1615, 1579, 1515, 1459, 1312, 1280, 1247, 1198, 1162, 1107, 1061, 1035, 825,
756; 1H-NMR (DMSO-d6), δ: 10.32 (br. s), 10.05 (s, 1H), 7.93–7.76 (m, 6H), 7.52–7.48 (m, 2H), 7.34
13
(ddd, 1H, J = 8.0 Hz, J = 7.0 Hz, J = 1.3 Hz), 7.26 (d, 1H, J = 9.0 Hz); C-NMR (DMSO-d6), δ:
166.86, 152.29, 135.83 (q, J = 2.2 Hz), 133.10, 131.59, 130.64, 129.91, 128.02, 127.51, 126.94,
126.62, 126.37 (q, J = 5.1 Hz), 124.70 (q, J = 29.3 Hz), 123.69 (q, J = 272.9 Hz), 123.59, 123.03,
118.29, 117.25; HR-MS: for C18H12NO2F3 [M+H]+ calculated 332.0893 m/z, found 332.0901 m/z.
2-Hydroxy-N-(3-trifluoromethylphenyl)naphthalene-1-carboxamide (7b). Yield 72%; Mp 153–156 °C;
IR (cm−1): 3296, 3060, 1660, 1651, 1622, 1583, 1548, 1541, 1450, 1435, 1332, 1240, 1205, 1157,
1116, 816, 792, 753, 739, 693; 1H-NMR (DMSO-d6), δ: 10.77 (s, 1H), 10.22 (br. s, 1H), 8.38 (s, 1H),
7.97 (d, 1H, J = 8.3 Hz), 7.90–7.85 (m, 2H), 7.70 (d, 1H, J = 8.5 Hz), 7.62–7.58 (m, 1H), 7.49–7.45
(m, 2H), 7.34 (ddd, 1H, J = 8.0, J = 7.0, J = 1.3 Hz), 7.27 (d, 1H, J = 8.8 Hz); 13C-NMR (DMSO-d6),
δ: 166.35, 151.82, 140.36, 131.31, 130.49, 130.00, 129.52 (q, J = 31.6 Hz), 128.03, 127.39, 127.16,
124.22 (q, J = 272.9 Hz), 123.32, 123.12, 122.78, 119.67 (q, J = 3.7 Hz), 118.35, 118.06, 115.26
(q, J = 3.7 Hz); HR-MS: for C18H12NO2F3 [M+H]+ calculated 332.0893 m/z, found 332.0900 m/z.

Molecules 2013, 18
9411
2-Hydroxy-N-(4-trifluoromethylphenyl)naphthalene-1-carboxamide (7c). Yield 70%; Mp 216–218 °C;
IR (cm−1): 3267, 3053, 1660, 1631, 1598, 1583, 1513, 1409, 1322, 1198, 1116, 1108, 1067, 1017, 836,
813, 800, 752; 1H-NMR (DMSO-d6), δ: 10.79 (s, 1H), 10.22 (s, 1H), 8.03 (d, 2H, J = 8.5 Hz), 7.90–7.85
(m, 2H), 7.74 (d, 2H, J = 8.5 Hz), 7.68 (d, 1H, J = 8.3 Hz), 7.47 (ddd, 1H, J = 8.0 Hz, J = 7.0 Hz,
13
J = 1.3 Hz), 7.34 (ddd, 1H, J = 8.0 Hz, J = 7.0 Hz, J = 1.3 Hz), 7.27 (d, 2H, J = 9.0 Hz); C-NMR
(DMSO-d6), δ: 166.40, 151.82, 143.15, 131.30, 130.49, 128.05, 127.39, 127.16, 126.09 (q, J = 3.67 Hz),
124.51 (q, J = 263.4 Hz), 123.36 (q, J = 31.5 Hz), 123.35, 123.12, 119.16, 118.35, 118.09; HR-MS: for
C18H12NO2F3 [M+H]+ calculated 332.0893 m/z, found 332.0900 m/z.
2-Hydroxy-N-(2-nitrophenyl)naphthalene-1-carboxamide (8a). Yield 73%; Mp 139–141 °C; IR (cm−1):
1
3329, 1651, 1634, 1578, 1496, 1462, 1428, 1341, 1269, 1195, 1143, 823, 790, 735; H-NMR
(DMSO-d6), δ: 10.35 (s, 1H), 10.12 (s, 1H), 8.03 (d, 1H, J = 9.3 Hz), 7.89–7.80 (m, 4H), 7.69 (d, 1H,
J = 8.5 Hz), 7.53–7.50 (m, 1H), 7.46 (ddd, 1H, J = 8.0 Hz, J = 7.0 Hz, J = 1.3 Hz), 7.32 (ddd, 1H,
J = 8.0 Hz, J = 7.0 Hz, J = 1.3 Hz), 7.25 (d, 1H, J = 9.0 Hz); 13C-NMR (DMSO-d6), δ: 166.80, 152.30,
143.06, 133.12, 131.56, 130.60, 129.70, 128.00, 127.36, 127.11, 125.73, 124.85, 124.56, 123.56,
123.04, 118.30, 117.36; HR-MS: for C17H12N2O4 [M+H]+ calculated 309.0870 m/z, found 309.0877 m/z.
2-Hydroxy-N-(3-nitrophenyl)naphthalene-1-carboxamide (8b). Yield 60%; Mp 255–257 °C; IR (cm−1):
3403, 2837, 2539, 1629, 1601, 1571, 1455, 1429, 1304, 1248, 1206, 1167, 1149, 1089, 911, 794, 760,
721; 1H-NMR (DMSO-d6), δ: 10.88 (s, 1H), 10.41 (s, 1H), 8.03 (dd, 1H, J = 8.3, J = 1.5 Hz), 7.95 (d,
1H, J = 8.5 Hz), 7.92–7.85 (m, 3H), 7.77 (ddd, 1H, J = 8.3 Hz, J = 7.0 Hz, J = 1.3 Hz), 7.51 (ddd, 1H,
J = 8.0 Hz, J = 7.0 Hz, J = 1.3 Hz), 7.41 (ddd, 1H, J = 8.3 Hz, J = 7.0 Hz, J = 1.3 Hz), 7.36 (ddd, 1H,
J = 8.0 Hz, J = 7.0 Hz, J = 1.3 Hz), 7.26 (d, 1H, J = 8.8 Hz); 13C-NMR (DMSO-d6), δ: 165.79, 152.49,
142.11, 134.13, 131.72, 131.54, 131.22, 128.07, 127.51, 127.17, 125.10, 125.01, 124.92, 123.54,
123.23, 118.25, 116.45; HR-MS: for C17H12N2O4 [M+H]+ calculated 309.0870 m/z, found 309.0874 m/z.
2-Hydroxy-N-(4-nitrophenyl)naphthalene-1-carboxamide (8c). Yield 81%; Mp 195–197 °C; IR (cm−1):
3345, 1648, 1614, 1552, 1506, 1435, 1329, 1300, 1257, 1212, 1176, 1111, 960, 895, 847, 808, 748,
1
738; H-NMR (DMSO-d6), δ: 11.05 (s, 1H), 10.31 (br. s, 1H), 8.29 (d, 2H, J = 8.3 Hz), 8.05 (d, 2H,
J = 8.3 Hz), 7.92–7.86 (m, 2H), 7.67 (d, 1H, J = 8.3 Hz), 7.48 (ddd, 1H, J = 8.0 Hz, J = 7.0 Hz,
13
J = 1.3 Hz), 7.34 (ddd, 1H, J = 8.0 Hz, J = 7.0 Hz, J = 1.3 Hz), 7.27 (d, 1H, J = 8.3 Hz); C-NMR
(DMSO-d6), δ: 166.73, 152.00, 145.72, 142.31, 131.23, 130.77, 128.13, 127.39, 127.31, 125.09,
123.21, 123.16, 119.02, 119.00, 118.33; HR-MS: for C17H12N2O4 [M+H]+ calculated 309.0870 m/z,
found 309.0876 m/z.
3.3. Lipophilicity Determination by HPLC (Capacity Factor k/Calculated log k)
A HPLC system Agilent 1200 equipped with DAD detector (Agilent, Santa Clara, CA, USA) was
used. A chromatographic column Symmetry® C18 5 μm, 4.6 × 250 mm, Part No. WAT054275, (Waters
Corp., Milford, MA, USA) was used. The HPLC separation process was monitored and evaluated by
EZChrom Elite software ver. 3.3.2 (Agilent). Isocratic elution by a mixture of MeOH p.a. (60%) and
H2O-HPLC Mili-Q grade (40%) as a mobile phase was used. The total flow of the column was
1.0 mL/min, injection 20 μL, column temperature 40 °C and sample temperature 10 °C. The detection
wavelength 210 nm was chosen. The KI methanolic solution was used for the dead time (tD)

Molecules 2013, 18
9412
determination. Retention times (tR) were measured in minutes. The capacity factors k were calculated
according to formula k = (tR − tD)/tD, where tR is the retention time of the solute, whereas tD denotes the
dead time obtained using an unretained analyte. Log k, calculated from the capacity factor k, is used as
the lipophilicity index converted to log P scale. The log k values of the individual compounds are
shown in Table 1.
3.4. Study of Inhibition of Photosynthetic Electron Transport (PET) in Spinach Chloroplasts
Chloroplasts were prepared from spinach (Spinacia oleracea L.) according to Masarovicova and
Kralova [56]. The inhibition of photosynthetic electron transport (PET) in spinach chloroplasts was
determined spectrophotometrically (Genesys 6, Thermo Electron Scientific Instruments, Madison, WI,
USA), using an artificial electron acceptor 2,6-dichlorophenol-indophenol (DCPIP) according to
Kralova et al. [57], and the rate of photosynthetic electron transport was monitored as a photoreduction
of DCPIP. The measurements were carried out in phosphate buffer (0.02 mol/L, pH 7.2) containing
sucrose (0.4 mol/L), MgCl2 (0.005 mol/L) and NaCl (0.015 mol/L). The chlorophyll content was
30 mg/L in these experiments and the samples were irradiated (~100 W/m2 with 10 cm distance) with a
halogen lamp (250 W) using a 4 cm water filter to prevent warming of the samples (suspension
temperature 22 °C). The studied compounds were dissolved in DMSO due to their limited water
solubility. The applied DMSO concentration (up to 4%) did not affect the photochemical activity in
spinach chloroplasts. The inhibitory efficiency of the studied compounds was expressed by IC50
values, i.e., by molar concentration of the compounds causing 50% decrease in the oxygen evolution
rate relative to the untreated control. The comparable IC50 value for a selective herbicide 3-(3,4-
dichlorophenyl)-1,1-dimethylurea, DCMU (Diurone®) was about 1.9 μmol/L. The results are
summarized in Table 1.
3.5. Study of Chlorophyll a and Aromatic Amino Acids Fluorescence in Spinach Chloroplasts
The fluorescence emission spectra of chlorophyll a (Chla) and aromatic amino acids (AAA) in
spinach chloroplasts were recorded on fluorescence spectrophotometer F-2000 (Hitachi, Tokyo, Japan)
using excitation wavelength λex = 436 nm for monitoring fluorescence of Chla and λex = 275 nm for
monitoring AAA fluorescence, excitation slit 20 nm and emission slit 10 nm. The samples were kept in
the dark for 2 min before measuring. The phosphate buffer used for dilution of the chloroplast
suspension was the same as described above. Due to low aqueous solubility the compounds were
added to chloroplast suspension in DMSO solution. The DMSO concentration in all samples was the
same as in the control (10%). The chlorophyll concentration in chloroplast suspension was 10 mg/L.
3.6. In Vitro Antibacterial Susceptibility Testing
The synthesized compounds were evaluated for in vitro antibacterial activity against representatives
of multidrug-resistant bacteria, clinical isolates of methicillin-resistant Staphylococcus aureus
(MRSA) 63718, SA 630 and SA 3202 that were obtained from the National Institute of Public Health,
Prague, Czech Republic. Staphylococcus aureus ATCC 29213 was used as a reference and quality
control strain. Ampicillin (Sigma-Aldrich) was used as the standard. Prior to testing, each strain was

Molecules 2013, 18
9413
passaged onto nutrient agar (Oxoid, Hampshire, UK) with 5% of bovine blood, and bacterial inocula
were prepared by suspending a small portion of bacterial colony in sterile phosphate buffered saline
(pH 7.2–7.3). The cell density was adjusted to 0.5 McFarland units using a densitometer
(Densi-La-Meter, LIAP, Riga, Latvia). The final inoculum was made by 1:20 dilution of the
suspension with the Mueller-Hinton broth (MH broth). The compounds were dissolved in DMSO
(Sigma), and the final concentration of DMSO in the MH broth (Oxoid) did not exceed 2.5% of the
total solution composition. The final concentrations of the evaluated compounds ranging from 256
μg/mL to 0.008 μg/mL. The broth dilution micro-method modified according to NCCLS guidelines
[58,59] in MH broth was used to determine the minimum inhibitory concentration (MIC). Drug-free
controls, sterility controls and controls consisted of MH broth and DMSO alone were included. The
determination of results was performed visually after 24 h of static incubation in the darkness at 37 °C
in an aerobic atmosphere. The MICs were defined as the lowest concentration of the compound at
which no visible bacterial growth was observed. The results are summarized in Table 1.
3.7. In Vitro Antimycobacterial Evaluation
The evaluation of in vitro antimycobacterial activity of the compounds was performed against
Mycobacterium marinum CAMP 5644, M. kansasii DSM 44162 and M. smegmatis ATCC 700084.
The broth dilution micro-method in Middlebrook 7H9 medium (Difco, Lawrence, KS, USA)
supplemented with BD BBL™ Middlebrook ADC Enrichment (Becton, Dickinson & Comp., Franklin
Lakes, NJ, USA) was used to determine the minimum inhibitory concentration (MIC) as previously
described [60]. The compounds were dissolved in DMSO (Sigma-Aldrich), and the final concentration
of DMSO did not exceed 2.5% of the total solution composition. The final concentrations of the
evaluated compounds ranging from 256 μg/mL to 0.125 μg/mL were obtained by twofold serial
dilution of the stock solution in microtiter plate with sterile medium. Bacterial inocula were prepared
by transferring colonies from culture to sterile water. The cell density was adjusted to 0.5 McFarland
units using a densitometer (Densi-La-Meter, LIAP, Riga, Latvia). The final inoculum was made by
1:1000 dilution of the suspension with sterile water. Drug-free controls, sterility controls and controls
consisted of medium and DMSO alone were included. Results were recorded visually after 3 days of
static incubation in the darkness at 37 °C aerobically for M. smegmatis, after 7 days of static
incubation in the darkness at 37 °C aerobically for M. kansasii and after 21 days of static incubation in
the darkness at 28 °C aerobically for M. marinum.
A well characterised clinical isolate of Mycobacterium avium subsp. paratuberculosis (CIT03) was
grown in Middlebrook broth (MB), supplemented with Oleic-Albumin-Dextrose-Catalase supplement
(OADC, Becton, Dickinson & Comp., Franklin Lakes, NJ, USA) and mycobactin J (2 µg/mL).
Identification of this isolate was performed using biochemical and molecular protocols. At log phase
growth, a culture sample (10 mL) was centrifuged at 15,000 rpm/20 min using a bench top centrifuge
(Model CR 4-12, Jouan Inc., Winchester, VA, USA). Following removal of the supernatant, the pellet
was washed in fresh Middlebrook 7H9GC broth and re-suspended in fresh supplemented MB (10 mL).
The turbidity was adjusted to match McFarland standard No. 1 (3 × 108 cfu) with MB broth. A further
1:20 dilution of the culture was then performed in MB broth. The antimicrobial susceptibility of the
mycobacterial species was investigated in a 96-well plate format. In these experiments, sterile

Molecules 2013, 18
9414
deionised water (300 µL) was added to all outer-perimeter wells of the plates to minimize evaporation
of the medium in the test wells during incubation. Each evaluated compound (100 µL) was incubated
with each of the mycobacterial species (100 µL). Dilutions of each compound were prepared in
duplicate. For all synthesized compounds, final concentrations ranged from 1,000 µg/mL to 8 µg/mL.
All compounds were prepared in DMSO and subsequent dilutions were made in supplemented MB.
The plates were sealed with parafilm and incubated at 37 °C, for 11 days in the case of M. avium
paratuberculosis. Following incubation, a 10% addition of alamarBlue (AbD Serotec, Kidlington, UK)
was mixed into each well and readings at 570 nm and 600 nm were taken, initially for background
subtraction and subsequently after 24 h re-incubation. The background subtraction is necessary for
strongly coloured compounds, where the colour may interfere with the interpretation of any colour
change. For non-interfering compounds, a blue colour in the well was interpreted as an absence of
growth and a pink colour was scored as growth. The minimum inhibitory concentrations (MICs) were
initially defined as the lowest concentration which prevented a visual colour change from blue to pink.
The MICs were defined as the lowest concentration of the compound at which no visible bacterial
growth was observed. The MIC value is routinely and widely used in bacterial assays and is a standard
detection limit according to the Clinical and Laboratory Standards Institute (CLSI). Isoniazid
(Sigma-Aldrich) was used as reference antimycobacterial drug. The results are summarized in Table 1.
3.8. In Vitro Cytotoxicity Assay
Human monocytic leukemia THP-1 cells were obtained from the European Collection of Cell
Cultures (ECACC, Salisbury, UK; Methods of characterization: DNA Fingerprinting (Multilocus
probes) and isoenzyme analysis). These cells were routinely cultured in RPMI 1640 (Lonza, Verviers,
Belgium) medium supplemented with 10% fetal bovine serum (FBS, Sigma-Aldrich), 2% L-glutamine,
1% penicillin and streptomycin (Lonza, Verviers, Belgium) at 37 °C with 5% CO2. Cells were
passaged at approximately 1 week intervals. Cells were routinely tested for the absence of mycoplasma
(Hoechst 33258 staining method). The tested compounds were dissolved in DMSO (Sigma-Aldrich)
and added in five increasing concentrations to the cell suspension in the culture medium. The
maximum concentration of DMSO in the assays never exceeded 0.1%. Subsequently, the cells were
incubated for 24 h at 37 °C with 5% CO2 to various compound concentrations ranging from 0.37 to
20 μmol/L in RPMI 1640 medium. Cell toxicity was determined using a Cytotoxicity Detection
KitPLUS Lactate dehydrogenase (LDH) assay kit (Roche Diagnostics, Mannheim, Germany) according
to the manufacturer’s instructions, as described previously [11,26,27]. For LDH assays, cells were
seeded into 96-well plates (5 × 104 cells·well−1 in 100 μL culture medium) in triplicate in serum-free
RPMI 1640 medium, and measurements at 492 nm wavelength (Synergy 2 Multi-Mode Microplate
Reader, BioTek, Winooski, VT, USA) were taken 24 h after the treatment with tested compounds. The
median lethal dose values, LD50, were deduced through the production of a dose-response curve. All
data were evaluated using GraphPad Prism 5.00 software (GraphPad Software, San Diego, CA, USA).
The results are summarized in Table 1.

Molecules 2013, 18
9415
4. Conclusions
A series of twenty-two 2-hydroxy-N-phenylnaphthalene-1-carboxanilides were prepared by means
of microwave synthesis and subsequently characterized. All the compounds were tested for their
ability to inhibit photosynthetic electron transport (PET) in spinach chloroplasts (Spinacia oleracea L.),
and for their in vitro antimicrobial activity against S. aureus, three methicillin-resistant S. aureus
strains, M. marinum, M. kasasii, M. smegmatis. and a clinical isolate of M. avium paratuberculosis.
The ortho-substituted compounds showed lower biological activity than the meta- and para-
substituted ones, therefore it can be concluded that, meta- or para-position of substituents on anilide is
essential for biological effect. Furthermore, it can be stated that the dependence of activity on the
lipophilicity was parabolic or bilinear and mostly the dependence of activity on electron parameters of
individual substituents increased with electron-withdrawing properties that also influenced in vitro
cytotoxicity against THP-1 cells. In conclusion it can be stated that unsubstituted 1, 8a (R = 2-NO2) and
8b (R = 3-NO2) can be considered as non-toxic lead compounds for the subsequent design of novel
antimicrobial agents.
Acknowledgements
This study was supported by the IGA VFU Brno 96/2012/FaF and 65/2012/FVL, the Slovak Grant
Agency VEGA, Grant No. 1/0612/11, by the Project APVV-0061-11, by Sanofi-Aventis Pharma
Slovakia, by the project CEITEC (Central European Institute of Technology), Reg. No.
CZ.1.05/1.1.00/02.0068 from the European Regional Development Fund and by the Irish Department
of Agriculture Fisheries and Food (FIRM): Refs 08RDCIT601 & 08RDCIT617.
Conflict of Interest
The authors declare no conflict of interest.
References
1. World Health Organization. WHO Global Strategy for Containment of Antimicrobial Resistance
2001; WHO Press: Geneva, Switzerland, 2001.
2. World Health Organization. Global Tuberculosis Report 2012; WHO Press: Geneva, Switzerland,
2012.
3. World Health Organization. World Health Statistics 2013; WHO Press: Geneva, Switzerland,
2013.
4. Wagner, D.; Young, L.S. Nontuberculous mycobacterial infections: A clinical review. Infection
2004, 32, 257–270.
5. Martinez, S.; McAdams, H.P.; Batchu, C.S. The many faces of pulmonary nontuberculous
mycobacterial infection. Am. J. Roentgenol. 2007, 189, 177–186.
6. Koul, A.; Arnoult, E.; Lounis, N.; Guillemont, J.; Andries, K. The challenge of new drug
discovery for tuberculosis. Nature 2011, 469, 483–490.
7. Roth, H.J.; Fenner, H. Arzneistoffe, 3rd ed.; Deutscher Apotheker Verlag: Stuttgart, Germany, 2000.

Molecules 2013, 18
9416
8. Sjogren, E.B.; Rider, M.A.; Nelson, P.H.; Bingham, S.; Poulton, A.L.; Emanuel, M.A.;
Komuniecki, R. Synthesis and biological activity of a series of diaryl-substituted alpha-cyano-
beta-hydroxypropenamides, a new class of anthelmintic agents. J. Med. Chem. 1991, 34, 3295–3301.
9. Otevrel, J.; Mandelova, Z.; Pesko, M.; Guo, J.; Kralova, K.; Sersen, F.; Vejsova, M.; Kalinowski, D.;
Kovacevic, Z.; Coffey, A.; et al. Investigating the spectrum of biological activity of
ring-substituted salicylanilides and carbamoylphenylcarbamates. Molecules 2010, 15, 8122–8142.
10. Imramovsky, A.; Pesko, M.; Kralova, K.; Vejsova, M.; Stolarikova, J.; Vinsova, J.; Jampilek, J.
Investigating spectrum of biological activity of 4- and 5-chloro-2-hydroxy-N-[2-(arylamino)-1-
alkyl-2-oxoethyl]benzamides. Molecules 2011, 16, 2414–2430.
11. Kos, J.; Zadrazilova, I.; Pesko, M.; Keltosova, S.; Tengler, J.; Gonec, T.; Bobal, P.; Kauerova, T.;
Oravec, M.; Kollar, P.; Cizek, A.; Kralova, K.; Jampilek, J. Antibacterial and herbicidal activity
of ring-substituted 3-hydroxynaphthalene-2-carboxanilides. Molecules 2013, 18, 7977–7997.
12. Macielag, M.J.; Demers, J.P.; Fraga-Spano, S.A.; Hlasta, J.D.; Johnson, G.S.; Kanojia, M.R.;
Russell, K.R.; Sui, Z.; Weidner-Wells, A.M.; Werblood, H.; et al. Substituted salicylanilides as
inhibitors of two-component regulatory systems in bacteria. J. Med. Chem. 1998, 41, 2939–2945.
13. Kratky, M.; Vinsova, J. Buchta, V. In vitro antibacterial and antifungal activity of salicylanilide
benzoates. Sci. World J. 2012, 2012, 290628.
14. Imramovsky, A.; Vinsova, J.; Monreal-Ferriz, J.; Dolezal, R.; Jampilek, J.; Kaustova, J.; Kunc, F.
New antituberculotics originated from salicylanilides with promising in vitro activity against
atypical mycobacterial strains. Bioorg. Med. Chem. 2009, 17, 3572–3579.
15. Kratky, M.; Vinsova, J.; Novotna, E.; Mandikova, J.; Wsol, V.; Trejtnar, F.; Ulmann, V.;
Stolarikova, J.; Fernandes, S.; Bhat, S.; et al. Salicylanilide derivatives block Mycobacterium
tuberculosis through inhibition of isocitrate lyase and methionine aminopeptidase. Tuberculosis
2012, 92, 434–439.
16. Kamat, S.; Buolamwini, J.K. Targeting EGFR and HER-2 receptor tyrosine kinases for cancer
drug discovery and development. Med. Res. Rev. 2006, 26, 569–594.
17. Liechty, C.H.; Sequin, U.; Bold, G.; Furet, P.; Meyer, T.; Traxler, P. Salicylanilides as inhibitors
of the protein tyrosine kinase epidermal growth factor receptor. Eur. J. Med. Chem. 2004, 39, 11–26.
18. Guo, L.; Wang, Q.L.; Jiang, Q.Q.; Jiang, J.J.; Jiang, Y.B. Anion-triggered substituent-dependent
conformational switching of salicylanilides. New hints for understanding the inhibitory
mechanism of salicylanilides. J. Org. Chem. 2007, 72, 9947–9953.
19. Brown, M.E.; Fitzner, J.N.; Stevens, T.; Chin, W.; Wright, C.D.; Boyce, J.P. Salicylanilides:
Selective inhibitors of interleukin-12p40 production. Bioorg. Med. Chem. 2008, 16, 8760–8764.
20. Boyce, J.P.; Brown, M.E.; Chin, W.; Fitzner, J.N.; Paxton, R.J.; Shen, M.; Stevens, T.; Wolfson,
F.; Wright, C.D. Identification of 14–3-3ζ by chemical affinity with salicylanilide inhibitors of
interleukin-12p40 production. Bioconjugate Chem. 2008, 19, 1775–1784.
21. Cheng, T.J.R.; Wu, Y.T.; Yang, S.T.; Lo, K.H.; Chen, S.K.; Chen, Y.H.; Huang, W.I.; Yuan,
C.H.; Guo, C.W.; Huang, L.Y.; et al. High-throughput identification of antibacterials against
methicillin-resistant Staphylococcus aureus (MRSA) and the transglycosylase. Bioorg. Med.
Chem. 2010, 18, 8512–8529.

Molecules 2013, 18
9417
22. Triola, G.; Wetzel, S.; Ellinger, B.; Koch, M.A.; Hubel, J.; Rauh, D.; Waldmann, H. ATP
competitive inhibitors of D-alanine-D-alanine ligase based on protein kinase inhibitor scaffolds.
Bioorg. Med. Chem. 2009, 17, 1079–1087.
23. Chenna, B.C.; Shinkre, B.A.; King, J.R.; Lucius, A.L.; Narayana, S.V.L.; Velu, S.E. Identification
of novel inhibitors of bacterial surface enzyme Staphylococcus aureus Sortase A. Bioorg. Med.
Chem. Lett. 2008, 18, 380–385.
24. Janin, Y.L. Antituberculosis drugs: Ten years of research. Bioorg. Med. Chem. 2007, 15, 2479–2513.
25. Musiol, R.; Tabak, D.; Niedbala, H.; Podeszwa, B.; Jampilek, J.; Kralova, K.; Dohnal, J.; Finster,
J.; Mencel, A.; Polanski, J. Investigating biological activity spectrum for novel quinoline
analogues 2: Hydroxyquinolinecarboxamides with photosynthesis inhibiting activity. Bioorg.
Med. Chem. 2008, 16, 4490–4499.
26. Gonec, T.; Bobal, P.; Sujan, J.; Pesko, M.; Guo, J.; Kralova, K.; Pavlacka, L.; Vesely, L.;
Kreckova, E.; Kos, J.; et al. Investigating the spectrum of biological activity of substituted
quinoline-2-carboxamides and their isosteres. Molecules 2012, 17, 613–644.
27. Fajkusova, D.; Pesko, M.; Keltosova, S.; Guo, J.; Oktabec, Z.; Vejsova, M.; Kollar, P.; Coffey, A.;
Csollei, J.; Kralova, K.; Jampilek, J. Anti-infective and herbicidal activity of N-substituted
2-aminobenzothiazoles. Bioorg. Med. Chem. 2012, 20, 7059–7068.
28. Trebst, A.; Draber, W. Structure activity correlations of recent herbicides in photosynthetic
reactions. In Advances in Pesticide Science; Greissbuehler, H., Ed.; Pergamon Press: Oxford, UK,
1979; pp. 223–234.
29. Bowyer, J.R.; Camilleri, P.; Vermaas, W.F.J. Photosystem II and its interaction with herbicides.
In Herbicides, Topics in Photosynthesis; Baker, N.R., Percival, M.P., Eds.; Elsevier: Amsterdam,
The Netherlands, 1991; Volume 10, pp. 27–85.
30. Shaner, D.L. Herbicide safety relative to common targets in plants and mammals. Pest. Manag. Sci.
2004, 60, 17–24.
31. Delaney, J.; Clarke, E.; Hughes, D.; Rice, M. Modern agrochemical research: A missed
opportunity for drug discovery? Drug Discov. Today 2006, 11, 839–845.
32. Duke, S.O. Herbicide and pharmaceutical relationships. Weed Sci. 2010, 58, 334–339.
33. Kralova, K.; Sersen, F.; Cizmarik, J. Inhibitory effect of piperidinoethylesters of
alkoxyphenylcarbamic acids on photosynthesis. Gen. Physiol. Biophys. 1992, 11, 261–267.
34. Kralova, K.; Bujdakova, H.; Kuchta, T.; Loos, D. Correlation between biological activity and the
structure of 6-amino-2-R-thiobenzothiazoles. Anti-yeast activity and inhibition of photochemical
activity of chloroplasts. Pharmazie 1994, 49, 460–461.
35. Kralova, K.; Kallova, J.; Loos, D.; Devinsky, F. Correlation between biological activity and the
structure of N,N'-bis(alkyldimethyl)-1,6-hexanediammonium dibromides. Antibacterial activity
and inhibition of photochemical activity of chloroplasts. Pharmazie 1994, 49, 857–858.
36. Bujdákova, H.; Kralova, K.; Sidoova, E. Antifungal and antialgal activity of 3-(2-alkylthio-6-
benzothiazolylaminomethyl)-2-benzoxazolinethiones. Pharmazie 1995, 50, 156–156.
37. Kralova, K.; Bujdakova, H.; Cizmarik, J. Antifungal and antialgal activity of piperidinopropyl
esters of alkoxy substituted phenylcarbamic acids. Pharmazie 1995, 50, 440–441.
38. Kubicova, L.; Kralova, K.; Sersen, F.; Gregor, J.; Waisser, K. Effects of substituted salicylanilides on
the photosynthetic apparatus of spinach chloroplasts. Folia Pharm. Univ. Carol. 2000, 25, 89-96.

Molecules 2013, 18
9418
39. Sersen, F.; Kralova, K.; Macho, V. New findings about the inhibitory action of phenylcarbamates
and phenylthiocarbamates on photosynthetic apparatus. Pestic. Biochem. Physiol. 2000, 68, 113–118.
40. Imramovsky, A.; Pesko, M.; Monreal-Ferriz, J.; Kralova, K.; Vinsova, J.; Jampilek, J.
Photosynthesis-Inhibiting efficiency of 4-chloro-2-(chlorophenylcarbamoyl)phenyl alkylcarbamates.
Bioorg. Med. Chem. Lett. 2011, 21, 4564–4567.
41. Servusova, B.; Eibinova, D.; Dolezal, M.; Kubicek, V.; Paterova, P.; Pesko, M.; Kralova, K.
Substituted N-benzylpyrazine-2-carboxamides: Synthesis and biological evaluation. Molecules
2012, 17, 13183–13198.
42. Kralova, K.; Sersen, F.; Pesko, M.; Klimesova, V.; Waisser, K. Photosynthesis-inhibiting effects
of 2-benzylsulphanylbenzimidazoles in spinach chloroplasts. Chem. Pap. 2012, 66, 795–799.
43. Izawa, S. Acceptors and donors for chloroplast electron transport. In Methods in Enzymology;
Colowick, P., Kaplan, N.O., Eds.; Academic Press: London, UK, 1980; Volume 69, Part C, pp.
413–434.
44. Govindjee, A. Sixty-three years since Kautsky: Chlorophyll a fluorescence. Aust. J. Plant Physiol.
1995, 22, 131–160.
45. Kallen, A.J.; Mu, Y.; Bulens, S.; Reingold, A.; Petit, S.; Gershman, K.; Ray, S.M.; Harrison,
L.H.; Lynfield, R.; Dumyati, G.; et al. Health care-associated invasive MRSA infections, 2005–
2008. JAMA 2010, 304, 641–647.
46. Liu, C.; Bayer, A.; Cosgrove, S.E.; Daum, R.S.; Fridkin, S.K.; Gorwitz, R.J. Kaplan, S.L.;
Karchmer, A.W.; Levine, D.P.; Murray, B.E.; et al. Clinical practice guidelines by the Infectious
Diseases Society of America for the treatment of methicillin-resistant Staphylococcus aureus
infections in adults and children. Clin. Infect. Dis. 2011, 52, 285–292.
47. Acharya, N.; Varshney, U. Biochemical properties of single-stranded DNA-binding protein from
Mycobacterium smegmatis, a fast-growing Mycobacterium and its physical and functional
interaction with uracil DNA glycosylases. J. Mol. Biol. 2002, 318, 1251–1264.
48. Broussard, G.W.; Ennis, D.G. Mycobacterium marinum produces long-term chronic infections in
medaka: A new animal model for studying human tuberculosis. Comp. Biochem. Phys. C 2007,
145, 45–54.
49. Valente, W.J.; Pienaar, E.; Fast, A.; Fluitt, A.; Whitney, S.E.; Fenton, R.J.; Barletta, R.G.;
Chacon, O.; Viljoen, H.J. A kinetic study of in vitro lysis of Mycobacterium smegmatis. Chem.
Eng. Sci. 2009, 64, 1944–1952.
50. Matveychuk, A.; Fuks, L.; Priess, R.; Hahim, I.; Shitrit D. Clinical and radiological features of
Mycobacterium kansasii and other NTM infections. Resp. Med. 2012, 106, 1472–1477.
51. Rath, T.; Roderfeld, M.; Blocher, S.; Rhode, A.; Basler, T.; Akineden, O.; Abdulmawjood, A.;
Halwe, J.M.; Goethe, R.; Bulte, M.; Roeb, E. Presence of intestinal Mycobacterium avium
subspecies paratuberculosis (MAP) DNA is not associated with altered MMP expression in
ulcerative colitis. BMC Gastroenterol. 2011, 11, 34.
52. Malik, I.; Bukovsky, M.; Andriamainty, F.; Galisinova, J. Antimicrobial activity of
meta-alkoxyphenylcarbamates containing substituted N-phenylpiperazine fragment. Braz. J.
Microbiol. 2012, 43, 959–965.
53. Katritzky, A.R.; Singh, S.K.; Cai, C.; Bobrov, S. Direct synthesis of esters and amides from
unprotected hydroxyaromatic and—aliphatic carboxylic acids. J. Org. Chem. 2006, 71, 3364–3374.

Molecules 2013, 18
9419
54. Hahn, G. (Naphtol-Chemie Offenbach). Verfahren zur Herstellung von Arylamiden der
2-Oxynaphthalin-1-carbonsäure. PCT Int. Appl. D 838290, 8 May 1949.
55. Chipalkatti, V.B.; Manivannan, K.; Desai, R.M.; Gopal, M. (Shriram Institute for Industrial
Research). Hydroxy aromatic acid amides. PTC Int. Apl. 69680, 5 August 1962.
56. Masarovicova, E.; Kralova, K. Approaches to measuring plant photosynthesis activity. In
Handbook of Photosynthesis, 2nd ed.; Pessarakli, M., Ed.; Taylor & Francis Group: Boca Raton,
FL, USA, 2005; pp. 617–656.
57. Kralova, K.; Sersen, F.; Sidoova, E. Photosynthesis inhibition produced by 2-alkylthio-6-R-
benzothiazoles. Chem. Pap. 1992, 46, 348–350.
58. National Committee for Clinical Laboratory Standards. Methods for Dilution Antimicrobial
Susceptibility Tests for Bacteria that Grow Aerobically, In Approved Standard—Fifth Edition,
CISI document M7-A5; NCCLS: Wayne, PA, USA, 2000.
59. National Committee for Clinical Laboratory Standards. Performance Standards for Antimicrobial
Susceptibility Testing: 12th Informational Supplement M100-S12; NCCLS: Wayne, PA, USA, 2002.
60. Schwalbe, R.; Steele-Moore, L.; Goodwin, A.C. Antimicrobial Susceptibility Testing Protocols;
CRC Press: Boca Raton, FL, USA, 2007.
Sample Availability: Samples of the compounds are available from the authors.
© 2013 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
(http://creativecommons.org/licenses/by/3.0/).