Antibacterial and herbicidal activity of ring-substituted 3-hydroxynaphthalene-2-carboxanilides

Article abstract of DOI:10.3390/molecules18077977

In this study, a series of twenty-two ring-substituted 3-hydroxy- Nphenylnaphthalene- 2-carboxanilides were prepared and characterized. The compounds were tested for their activity related to inhibition of photosynthetic electron transport (PET) in spinach (Spinacia oleracea L.) chloroplasts. Primary in vitro screening of the synthesized compounds was also performed against four Staphylococcus strains and against two mycobacterial species. 3-Hydroxy-N-(2-methoxyphenyl)naphthalene-2-carboxamide showed high biological activity (MIC = 55.0 μmol/L) against S. aureus as well as methicillinresistant strains. N-(2-Fluorophenyl)-3-hydroxynaphthalene-2- carboxamide showed higher activity (MIC = 28.4 μmol/L) against M. marinum than the standard isoniazid and 3-hydroxy-N-(4-nitrophenyl)naphthalene-2- carboxamide expressed higher activity (MIC = 13.0 μmol/L) against M. kansasii than the standard isoniazid. Cytotoxicity assay of effective antimicrobial compounds was performed using the human monocytic leukemia THP-1 cell line. The PET-inhibiting activity expressed by IC₅₀ value of the most active compound 3-hydroxy-N-(3-nitrophenyl)naphthalene-2-carboxamide was 16.9 μmol/L. The structure-activity relationships of all compounds are discussed.

Full text of DOI:10.3390/molecules18077977

Molecules 2013, 18, 7977-7997; doi:10.3390/molecules18077977
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Antibacterial and Herbicidal Activity of Ring-Substituted
3-Hydroxynaphthalene-2-carboxanilides †
Jiri Kos 1, Iveta Zadrazilova 1,2,3, Matus Pesko 4, Stanislava Keltosova 5, Jan Tengler 1,
Tomas Gonec 1, Pavel Bobal 1, Tereza Kauerova 5, Michal Oravec 6, Peter Kollar 5,
Alois Cizek 2,3, Katarina Kralova 4 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
Institute of Chemistry, Faculty of Natural Sciences, Comenius University, Mlynska dolina Ch-2,
842 15 Bratislava, Slovakia
5
Department of Human Pharmacology and Toxicology, Faculty of Pharmacy, University of
Veterinary and Pharmaceutical Sciences, Palackeho 1/3, 612 42 Brno, Czech Republic
6
Global Change Research Centre AS CR, Belidla 986/4a, 603 00 Brno, Czech Republic
†
Preliminary results were presented at The Sixteenth Electronic Conference on Synthetic Organic
Chemistry (ECSOC-16, http://www.sciforum.net/presentation/1009), 1–30 November 2012
(paper B1009).
* Author to whom correspondence should be addressed; E-Mail: josef.jampilek@gmail.com;
Tel.: +420-54-156-2926.
Received: 23 May 2013; in revised form: 17 June 2013 / Accepted: 4 July 2013 /
Published: 8 July 2013
Abstract: In this study, a series of twenty-two ring-substituted 3-hydroxy-N-
phenylnaphthalene-2-carboxanilides were prepared and characterized. The compounds were
tested for their activity related to inhibition of photosynthetic electron transport (PET) in
spinach (Spinacia oleracea L.) chloroplasts. Primary in vitro screening of the synthesized
compounds was also performed against four Staphylococcus strains and against two
mycobacterial species. 3-Hydroxy-N-(2-methoxyphenyl)naphthalene-2-carboxamide showed

Molecules 2013, 18
high biological activity (MIC = 55.0 µmol/L) against S. aureus as well as methicillin-
7978
resistant strains. N-(2-Fluorophenyl)-3-hydroxynaphthalene-2-carboxamide showed higher
activity (MIC = 28.4 µmol/L) against M. marinum than the standard isoniazid and
3-hydroxy-N-(4-nitrophenyl)naphthalene-2-carboxamide expressed higher activity
(MIC = 13.0 µmol/L) against M. kansasii than the standard isoniazid. Cytotoxicity assay of
effective antimicrobial compounds was performed using the human monocytic leukemia
THP-1 cell line. The PET-inhibiting activity expressed by IC50 value of the most active
compound 3-hydroxy-N-(3-nitrophenyl)naphthalene-2-carboxamide was 16.9 μmol/L. The
structure-activity relationships of all compounds are discussed.
Keywords: hydroxynaphthalene-2-carboxanilides; lipophilicity; photosynthetic electron
transport inhibition; spinach chloroplasts; in vitro antibacterial activity; in vitro
antimycobacterial activity; in vitro cytotoxicity; structure-activity relationships
1. Introduction
The increasing number of bacterial, mycobacterial and associated fungal infections underlines the
importance of searching for new antimicrobial chemotherapeutics [1]. Tuberculosis and other
mycobacterial diseases are common, and in many cases lethal, infectious illnesses caused by various
strains of pathogenic mycobacteria. The genus Mycobacterium consists of a closely related group of
fast and slow-growing species. Mycobacterium 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 to work in high containment biosafety facilities. To lower risks
and make manipulation in the laboratory easier, surrogate model pathogens for M. tuberculosis can be
used in laboratory studies. M. marinum is very closely related to the M. tuberculosis; it is the cause of
TB-like infections in poikilothermic organisms, especially frogs and fish. M. marinum is a good model
for studying because of the lower risk for laboratory workers, genetic relatedness and similar
pathology to human TB [2,3]. However, because of M. tuberculosis, the pathogenic role of
nontuberculous mycobacteria (NTM) in humans was overshadowed for a long time. M. kansasii, the
most virulent of the NTM, causes nontuberculous mycobacterial lung infections which are very
common nowadays and can be indistinguishable from tuberculosis [4]. That is the reason why M.
marinum and M. kansasii are often chosen as model species for screening of prospective
antimycobacterial drugs to control mycobacterial diseases.
The treatment of tuberculosis and nontuberculous diseases is mediated by administration of various
antimicrobial chemotherapeutics, however massive using of these drugs is considered to be the main
reason for increased antibiotic resistance among bacteria [5,6]. The antibiotic resistance of important
Gram-positive pathogen, Staphylococcus aureus, has become one of the most challenging and
persistent worldwide health problems. Methicillin-resistant S. aureus (MRSA) was first described in
1961 and since then has become one of the most common clinically relevant bacterial pathogens
isolated almost all over the World. Even though originally limited to hospitals, nowadays MRSA is an
increasing cause of infections in the community. Recent studies have shown that, despite antibacterial

Molecules 2013, 18
7979
therapy, MRSA infections are still associated with serious clinical consequences (treatment failure,
higher morbidity and mortality, prolonged hospitalization, etc.). Because of the changing features of
MRSA, it is one of the most difficult bacteria for clinicians to treat. The emergence of resistance to
currently available drugs, their toxicity and general lack of oral agents justify an urgent need for new
anti-MRSA agents [7,8].
Both pharmaceuticals and pesticides are designed to target particular biological functions, and in
some cases these functions overlap in their molecular target sites, or they target similar processes or
molecules. Taking into consideration that herbicides may also have molecular sites of action in
mammals, until recently most pharmaceutical companies had pesticide divisions, sometimes with a
different name. All compounds generated by either division of the company were evaluated for both
pesticide and pharmaceutical uses. In the past, some leading pesticides have become pharmaceuticals
and vice versa [9–11]. Moreover, good correlation between microbiological activities and herbicidal
effects was found [12–15].
The presence of an amide (-NHCO-) group [16–20] is characteristic of a number of herbicides
acting as photosynthesis inhibitors. Over 50% of commercially available herbicides act by reversibly
binding to photosystem II (PS II), a membrane-protein complex in the thylakoid membranes, which
catalyses the oxidation of water and the reduction of plastoquinone [21], and thereby inhibit
photosynthesis [22–24].
Promising results of biological screening of some salicylanilides (their antimicrobial,
antimycobacterial, antifungal, molluscicidal and herbicidal action) [17–19,25–33] inspired us to
prepare and evaluate ring-substituted 3-hydroxynaphthalene-2-carboxanilides. The design of these 3-
hydroxy-N-phenylnaphthalene-2-carboxanilides is based on ring analogy with salicylanilides (2-
hydroxy-N-phenylbenzamide). Thus in the context of the previously-published results [16–20,25–33],
primary in vitro screening of the synthesized compounds was also performed against four
Staphylococcus strains, three of which were methicillin-resistant Staphylococcus aureus strains, and
against two mycobacterial species, such as Mycobacterium marinum and M. kansasii.
2. Results and Discussion
2.1. Chemistry
All the studied compounds were prepared according to Scheme 1. Modified microwave-assisted
synthesis [29] facilitated the process of obtaining 3-hydroxynaphthalene-2-carboxanilides, thus
synthesis of the target compounds was carried out only by one step. At first the carboxyl group was
activated with phosphorus trichloride. The final amide was immediately formed by aminolysis of the
acyl chloride by ring-substituted aniline in dry chlorobenzene. All the compounds were recrystallized
from ethanol; their HPLC purity exceeded 97%. By microwave-assisted synthesis reaction times were
shortened from hours [34–37] to minutes, and yields of most compounds (except 2-NO2 derivative 8a)
were more than 65%, for details see Section 3.2.

Molecules 2013, 18
Scheme 1. Synthesis of ring-substituted 3-hydroxynaphthalene-2-carboxanilides 1–8c.
7980
NH2
O
R
COOH
OH
a
N
+
R
H
OH
1-8c
R = H, OCH3, CH3, F, Cl, Br, CF3, NO2
Reagents and conditions: (a) PCl3, chlorobenzene, MW.
2.2. Lipophilicities
Lipophilicity is a property that has a major effect on solubility, absorption, distribution and
biotransformation as well as pharmacological activity, because drugs cross biological membranes
through passive transport, which strongly depends on their lipophilicity. Lipophilicity has been studied
and applied as an important drug property for decades. Lipophilicity of the studied compounds was
determined by RP-HPLC as capacity factor logarithm (log k) and calculated as log P using
ACD/Percepta software. The results of ring-substituted 3-hydroxynaphthalene-2-carboxanilides 1–8c are
shown in Table 1 and illustrated in Figure 1. The highest experimental lipophilicity was
found for 3-hydroxy-N-(4-trifluoromethylphenyl)naphthalene-2-carboxamide (7c), while 3-hydroxy-
N-phenylnaphthalene-2-carboxamide (1) showed the lowest log k value. It is important to note that
lipophilicity also has a great impact on target and off-target interactions, as mentioned below.
The results obtained with the discussed compounds show that the experimentally-determined
lipophilicities (log k) of the meta- and para-substituted compounds are in accordance with the
calculated log P values as shown in Figure 1B, while ortho-substituted derivatives showed poor match,
see Figure 1A. The most significant deviations within the dependence illustrated in Figure 1B can be
observed for 4-CF3 (7c) and 4-NO2 (8c). The influence of R substituents on lipophilicity is as follows:
H < OCH3 < CH3 < F < NO2 < Cl < Br ≤ CF3. Within the individual series the lipophilicity determined
by log k values increases for halogens and methyl substituents as follows: ortho < para < meta; for
methoxy substituent as follows: para < ortho < meta, and for CF3 and NO2 as follows: ortho < meta <
para. Generally, it could be concluded that the prediction power of the used experimental log k or
calculated log P values, especially for meta- and para-substituted derivatives, may be a good tool for
searching potential drugs. It can be assumed that log k values specify lipophilicity within individual
series of the studied compounds.
2.3. Inhibition of Photosynthetic Electron Transport (PET) in Spinach Chloroplasts
The activity of the evaluated naphthanilide derivatives related to inhibition of photosynthetic
electron transport (PET) in spinach (Spinacia oleracea L.) chloroplasts was moderate or low relative
to the standard, see Table 1. Generally compounds showed poor aqueous solubility. Only seven
compounds from twenty-two tested compounds could be evaluated. PET inhibition by 1, 2c–5c, 6b, 6c
and 7b, 7c could not be determined due to precipitation of the compounds during the experiments.
With respect to these small but specifically substituted groups of compounds some structure-activity
relationships (SAR) can be proposed. Compound 8b (R = 3-NO2) expressed the highest PET-inhibiting

Molecules 2013, 18
7981
activity
(IC50 = 16.9 µmol/L), while compound 8c (R = 4-NO2) expressed the lowest PET-inhibiting activity
(IC50 = 187.5 µmol/L).
Table 1. Structure of ring-substituted 3-hydroxynaphthalene-2-carboxanilides 1–8c,
experimentally determined values of lipophilicity log k, calculated values of log P and
electronic Hammett’s σ parameters, IC50 [μmol/L] values related to PET inhibition in
spinach chloroplasts in comparison with 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU)
standard, in vitro anti-Staphylococcus activities [MIC (μmol/L)] in comparison with
standards ampicillin (APC), in vitro antimycobacterial activity [MIC (μmol/L)] of
compounds 1–8c compared to isoniazid (INH) standard and in vitro cytotoxicity assay
(LD50) of selected compounds.
O
R
N
H
OH
[μmol/L]
MIC
MRSA MRSA MRSA
LD50
log P a
σ a
Comp.
R
log k
PET
IC50
SA
MM
MK
63718
>972
55.0
630
>972
55.0
>873
>873
>923
>923
462
3202
>972
55.0
>873
>873
462
462
231
228
228
228
215
215
>860
>748
187
187
97
b
H
2-OCH3
3-OCH3
4-OCH3
2-CH3
3-CH3
4-CH3
2-F
0.6310
0.6916
0.6971
0.5951
0.6936
0.8831
0.8753
0.7303
0.8296
0.7317
0.9509
1.0796
1.0687
0.9715
1.1536
1.1459
0.8762
1.2053
1.2835
0.8710
0.8143
0.9175
–
4.52
4.61
4.56
4.37
4.85
4.85
4.85
4.56
4.69
4.70
5.02
5.25
5.24
5.06
5.39
5.28
5.42
5.49
5.33
4.51
4.64
4.65
–
0
>972
55.0
>873
>873
>923
>923
>923
>910
>910
>910
>860
>860
>860
>748
>748
>748
>773
>748
>748
>830
>830
>830
–
122
873
972
218
54.6
873
115
57.7
923
56.9
114
114
860
860
430
748
748
374
193
748
187
830
415
13.0
–
–
1
2a
−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
59.5
>30
53.4
b
>873
>873
>923
>923
>923
>910
>910
>910
>860
>860
>860
>748
>748
>748
>773
>748
>748
>830
>830
>830
–
>873
>873
462
>30
2b
–
2c
b
b
b
b
b
b
b
b
b
–
3a
923
>30
3b
>923
28.4
114
–
3c
455
>30
4a
3-F
455
–
4b
4-F
455
910
–
4c
2-Cl
>860
430
860
–
5a
3-Cl
>860
>860
>748
>748
>748
193
–
5b
4-Cl
>860
>748
>748
>748
>773
374
–
5c
2-Br
43.2
b
–
6a
3-Br
–
6b
b
4-Br
–
6c
2-CF3
3-CF3
4-CF3
2-NO2
3-NO2
4-NO2
–
105.2
b
28.6 ± 0.5
7a
187
187
415
>830
>830
–
>748
>748
830
–
7b
b
374
–
7c
106.6
16.9
187.5
1.9
415
–
8a
>830
>830
–
>830
>830
–
2.5 ± 0.9
8b
<0.37
8c
–
–
–
DCMU
APC
–
–
–
5.7
>45.8
–
>45.8
–
>45.8
–
–
–
–
467
29.2
INH
a
b
calculated using sw. ACD/Percepta ver. 2012; precipitation during experiment; SA = Staphylococcus aureus ATCC
29213; MRSA = clinical isolates of methicillin-resistant S. aureus 63718, SA 630 and SA 3202 (National Institute of
Public Health, Prague, Czech Republic); MM = Mycobacterium marinum CAMP 5644, MK = M. kansasii DSM 44162.

Molecules 2013, 18
Figure 1. Comparison of experimentally found log k values with calculated log P of
7982
ortho-substituted (A) and meta- and para-substituted derivatives (B).
B
A
1.4
1.2
1.0
0.8
0.6
0.4
1.2
1.0
0.8
0.6
0.4
R = 0.9530
R = 0.5271
4.2
4.4
4.6
4.8
5.0
5.2
5.4
5.6
4.2
4.4
4.6
4.8
5.0
5.2
5.4
5.6
log P
log P
The PET-inhibiting activity was expressed by negative logarithm of IC50 value (compound
concentration in mol/L causing 50% inhibition of PET). Despite the relatively low inhibitory activity
of the studied compounds, correlations between log(1/IC50 [mol/L]) and 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. Based on the obtained results it is not possible
to decide, whether some of ortho-, meta- or para-positions are preferred from the point of view of
PET-inhibiting activity.
Figure 2. Relationships between PET inhibition log (1/IC50) [mol/L] in spinach
chloroplasts and lipophilicity expressed as log k (A) or N-substituent electronic Hammett's σ
parameters (B) of selected studied compounds.
A
B
4.8
4.6
4.4
4.2
4.0
3.8
3.6
3.4
4.8
4.6
4.4
4.2
4.0
3.8
3.6
3.4
8b
8b
6a
2b
2a
6a
2a
2b
7a
7a
8a
8a
8c
8c
0.6
0.7
0.8
0.9
1.0
-0.4
-0.2
0.0
0.2
0.4
0.6
0.8
1.0
log k
σ
The biological activity is affected by lipophilicity (see Table 1 and Figure 2A). In general, when the
inactive anilide, i.e., 6a, was eliminated; the dependence of log (1/IC50 [mol/L]) on log k is bilinear
with the optimum log k ca. 0.81 (R = 3-NO2, compound 8b). The corresponding correlation
coefficients were r = 0.9993 and r = −0.9673. On the other hand, PET inhibition also increases with
electron-withdrawing substituent to σ = 0.71 (R = 3-NO2, compound 8b), where the optimum can be
found, and then decreases with a subsequent increase of electron-withdrawing properties of
substituents (σ = 0.78, R = 4-NO2, compound 8c and σ = 0.77, R = 2-NO2, compound 8a). Thus, when
the activity of compound 7a is not considered, the dependence of PET-inhibiting activity on the
electronic σ properties of substituents (Figure 2B) shows a similar bilinear trend as in case of log k

Molecules 2013, 18
7983
(Figure 2A), with correlations coefficients r = 0.9313 (for the σ range from −0.28 to 0.71) and
r = −0.9956 for σ > 0.71).
Using artificial electron donors with the known site of action can help in the determination of
inhibitory site of action of the studied compound in photosynthetic apparatus. For example, the
artificial electron donor 1,5-diphenylcarbazide (DPC) acts in Z•/D• intermediate [38] and by supply of
electrons it can restore PET in chloroplasts which was inhibited on the donor side of PS II in the section
from the oxygen evolving complex to the Z•/D• intermediate. However, if PET is inhibited at the
acceptor side of PS II, PET will not be restored. Application of the artificial electron donor 1,5-
diphenylcarbazide acting in Z•/D• intermediate [38] to chloroplasts the activity of which was inhibited
by the studied compounds (75% inhibition related to the control) practically completely restored PET
(up to 97.8% of the control), indicating that PET on the acceptor side of PS II between the core of PS
II (P680) and the secondary plastoquinone molecules/acceptor QB was not inhibited by the tested
compounds. Consequently, it can be assumed that the site of action of the studied compounds is
situated on the donor side of PS II in the section between the primary electron donor of PS II (H2O)
and Z•/D• intermediate.
The bilinear course of the dependence of log (1/IC50) on σ indicates that for the PET-inhibiting
activity not only sufficient lipophilicity (enabling easier penetration of the compounds into the lipids
of photosynthetic membranes) but also sufficient electronegativity of the R substituent (enabling
interactions with proteins located near oxygen evolving complex present at the luminal side of
thylakoid membrane) is necessary. 3-Hydroxy-N-(3-nitrophenyl)naphthalene-2-carboxamide (8b) was
the most active compound from the series, and this result can indicate that PET inhibition can be
associated with additional interaction of the nitro moiety with photosynthetic proteins, nevertheless the
role of the nitro group in the meta-position might originate also from its electron withdrawing
properties that influence electron distribution in the aromatic ring. A strong dependence of PET-
inhibiting activity on σ was also found for 2-benzylsulphanylbenzimidazoles [39]. The site of action
situated on the donor side of PS II was found also for 2-alkylthio-6-R-benzothiazoles (R = 6-
formamido-, 6-acetamido-, and 6-benzoylamino-) [40], anilides of 2-alkylpyridine-4-carboxylic acids
acting in intermediates Z•/D• [41], 5-bromo-N-phenylbenzamides [42] and 2-alkylsulphanyl-4-
pyridinecarbothioamides acting in the D• intermediate [43].
The effects of the studied compounds on the photosynthetic apparatus of spinach chloroplasts were
investigated by studying chlorophyll a (Chla) fluorescence. The decreased intensity of the emission
band at 686 nm belonging to the pigment-protein complexes in photosystem II [44] (Figure 3A)
suggested PS II as the site of action of the studied inhibitors. Lower solubility of compound 2b did not
allow to record fluorescence emission spectra of chloroplasts treated with compound concentration
higher than 0.780 mmol/L. The extent of perturbation of chlorophyll a-protein complexes in the
thylakoid membrane reflected as decreased fluorescence (Figure 3B) correlated with PET inhibiting
activity of compounds 2b and 7a (IC50 = 53.4 and 105.2 μmol/L, respectively). A similar decrease of
Chla fluorescence in plant chloroplasts was observed after treatment with HgCl2 [45], non-ionic
surfactant Triton X 100 [46] as well as substituted benzanilides [47] and salicyanilides [48].

Molecules 2013, 18
Figure 3. Fluorescence emission spectra of chlorophyll a in untreated spinach chloroplasts
7984
in presence of compound 7a: 0, 0.195, 0.390, 0.780, 1.170 and 1.560 mmol/L (curves from
top to bottom; λex = 436 nm) (A) and dependence of fluorescence intensity of chlorophyll a
on concentration of compounds 2b (triangles) and 7a (circles) (B).
100
90
80
70
60
50
40
100
80
60
40
20
0
A
B
7a
2b
660
680
λ [nm]
700
720
0.00 0.25 0.50 0.75 1.00 1.25 1.50
c [mmol/L]
2.4. In Vitro Antibacterial Susceptibility Testing
Although salicylanilides seem to be promising candidates of antibacterial agents [31,32], all the
compounds showed only moderate activity, except for 3-hydroxy-N-(2-methoxyphenyl)naphthalene-
2-carboxamide (2a). As MIC = 55 µmol/L of compound 2a was the same for all four strains, it can be
speculated about specific effectivity against Staphylococcus sp. From Table 1 it is obvious that
compound 2a exhibited activity comparable with the standards. 3-Hydroxy-N-(2-trifluoromethyl-
phenyl)naphthalene-2-carboxamide (7a) seems to be another noteworthy compound. Nevertheless due
to moderate activity of the rest of the compounds, no thorough SAR could be established.
2.5. In Vitro Antimycobacterial Evaluation
Although all the compounds were evaluated for their in vitro antimycobacterial activity against both
mycobacterial strains, most of compounds showed only moderate or no activity, see Table 1.
Nevertheless N-(2-fluorophenyl)-3-hydroxynaphthalene-2-carboxamide (4a) expressed the activity
comparable with or higher than the used standard isoniazid against both strains, and 3-hydroxy-N-
(4-nitrophenyl)naphthalene-2-carboxamide (8c) showed relatively high activity against M. kansasii.
According to the results, it can be generally concluded that activity against M. marinum can be
observed especially for ortho-substituted anilides. When the least active anilides 3b and 4c are
eliminated, the dependences of activity on log k and on electronic σ properties are bilinear with the
optima log k ca. 0.73 and σ = 0.06 (R = 2-F, compound 4a). After the optima the antimycobacterial
activity against M. marinum decreases with an increase of lipophilicity and/or electron-withdrawing
properties of the R substituent.
Within activity against M. kansasii, it can be generally concluded that for anilides substituted in
meta- or para-position by methyl and halogens (except the fluoro moiety) the activity slightly
increases with increasing lipophilicity and electron-withdrawing properties. For ortho-substituted
anilides similar bilinear relationships can be found, as discussed above, with the optima log k ca. 0.73
and σ = 0.06 (R = 2-F, compound 4a).

Molecules 2013, 18
7985
2.6. In Vitro Cytotoxicity Assay
The most effective compounds 2a, 2b, 3b, 4a, 7a, 8b and 8c were tested for their in vitro
cytotoxicity LD50 (µmol/L) using human monocytic leukemia THP-1 cells. These cells were used,
because they represent human cell line [49]. In the past several works were published, where the
toxicity of tested compounds (including antibacterial agents [20,50]) was assessed on THP-1 cells. The
highest dose of compounds 2a,b (R = 2-, 3-OCH3), 3b (R = 3-CH3) and 4a (R = 2-F) in the medium
(30 μmol/L, it was not possible to solve higher amount) did not lead to significant lethal
effect on THP-1 cells, i.e., LD50 > 30 µmol/L. Compound 7a (2-CF3) demonstrated low toxicity
(LD50 = 28.6 ± 0.5 µmol/L) against the human monocytic leukemia THP-1 cell line. Compounds
containing 3-NO2 (8b) and 4-NO2 (8c) exerted fairly high toxicity LD50 = 2.5 ± 0.9 µmol/L for 8b and
LD50 < 0.37 µmol/L for 8c (for example, LD50 of oxaliplatin and camptothecin assessed in this line
formerly were much lower: 1.7 ± 0.6 µmol/L and 0.20 ± 0.07 µmol/L respectively), which correlates
with the positions of these substituents: toxicity increased with the shift of NO2 moiety from ortho to
para position (8b is less toxic than 8c). Based on these observations it can be concluded that the
discussed amides 2a, 2b, 3b and 4a can be considered as promising agents for subsequent design of
novel antibacterial and antimycobacterial agents, respectively.
3. Experimental
3.1. General
All reagents were purchased from Aldrich (Sigma-Aldrich, St. Louis, MO, USA). TLC experiments
were performed on alumina-backed silica gel 40 F254 plates (Merck, Darmstadt, Germany). The plates
were illuminated under UV (254 nm) and evaluated in iodine vapour. The melting points were
determined on Kofler hot-plate apparatus HMK (Franz Kustner Nacht KG, Dresden, Germany) and are
uncorrected. Infrared (IR) spectra were recorded on a Smart MIRacle™ ATR ZnSe for Nicolet™
Impact 410 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).
Chemicals shifts are reported in ppm (δ) using internal Si(CH3)4 as the reference with diffuse, easily
exchangeable signals being omitted. 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. The purity of the compounds was checked by the HPLC method using the same
conditions as described in Section 3.3. The detection wavelength of 210 nm was chosen. The peaks in
the chromatogram of the solvent (blank) were deducted from the peaks in the chromatogram of the
sample solution. A purity of individual compounds was determined from area peaks in the
chromatogram of the sample solution.

Molecules 2013, 18
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3.2. Synthesis
3.2.1. General Procedure for Synthesis of Carboxamide Derivatives 1–8c
3-Hydroxynaphtalene-2-carboxylic acid (1.0 g, 5.3 mmol) was suspended in dry chlorobenzene
(30 mL) at ambient temperature and phosphorus trichloride (0.23 mL, 2.7 mmol, 0.5 eq.), and the
corresponding substituted aniline (5.3 mmol, 1 eq.) was added dropwise. The reaction mixture was
transferred to the microwave reactor, where the synthesis was performed (1st phase: 10 min, 100 °C,
100 W; 2nd phase: 15 min, 120 °C, 500 W; 3rd phase: 20 min, 130 °C, 500 W). Then the mixture was
cooled to 60 °C, and then the solvent was removed to dryness under reduced pressure. The residue was
washed with hydrochloride acid and water. The crude product was recrystallized from EtOH. Studied
compounds 1–8c are presented in Table 1.
3-Hydroxy-N-phenylnaphthalene-2-carboxamide (1). Yield 76%; Mp. 245–246 °C (Mp. 242–243 °C
[34]); HPLC purity 99.41%; IR (Zn/Se ATR, cm−1): 3291w, 1620m, 1556m,1494w, 1448w, 1396w,
1
1344m, 1250w, 1209m, 1173m, 1064w, 950w, 915w, 870m, 842m, 771w, 739s, 712m, 687m; H-
NMR (DMSO-d6), δ: 11.36 (s, 1H), 10.60 (s, 1H), 8.53 (s, 1H), 7.93 (d J = 8.1 Hz, 1H), 7.78 (m, 3H),
7.51 (ddd, J = 8.1 Hz, J = 6.8 Hz, J = 1.3 Hz, 1H), 7.44–7.33 (m, 4H), 7.15 (t, J = 7.26 Hz, 1H); 13C-
NMR (DMSO-d6), δ: 165.67, 153.79, 138.37, 135.76, 130.42, 128.72, 128.66, 128.08, 126.83, 125.72,
124.03, 123.69, 121.55, 120.55, 110.57; HR-MS: for C17H14NO2[M+H]+ calculated 264.1019 m/z,
found 264.1023 m/z.
3-Hydroxy-N-(2-methoxyphenyl)naphthalene-2-carboxamide (2a). Yield 80%; Mp. 165–166 °C (Mp.
164–166 °C [34]); HPLC purity 99.28%; IR (Zn/Se ATR, cm−1): 3191w, 1624m, 1593s, 1549m,
1487w, 1435m, 1393w, 1393w, 1342m, 1284m, 1248w, 1227m, 1173m, 1115m, 1067m, 1031w,
1
922w, 864m, 837m, 801w, 771m, 735s, 685m; H-NMR (DMSO-d6), δ: 11.78 (s, 1H), 11.08 (s, 1H),
8.72 (s, 1H), 8.51 (d, J = 7.7 Hz, 1H), 7.98 (d, J = 8.1 Hz, 1H), 7.77 (d, J = 8.1 Hz, 1H), 7.51 (ddd,
J = 8.1 Hz, J = 6.8 Hz, J = 0.9 Hz, 1H), 7.40–7.32 (m, 2H), 7.11–6.92 (m, 3H), 3.92 (s, 3H);
13C-NMR (DMSO-d6), δ: 162.27, 152.61, 148.64, 135.82, 132.50, 128.94, 128.22, 127.91, 127.19,
125.58, 123.94, 123.79, 121.29, 120.62, 120.19, 111.01, 110.71, 56.05; HR-MS: for C18H16NO3
[M+H]+ calculated 294.1124 m/z, found 294.1130 m/z.
3-Hydroxy-N-(3-methoxyphenyl)naphthalene-2-carboxamide (2b). Yield 75%; Mp. 194–195 °C;
HPLC purity 99.05%; IR (Zn/Se ATR, cm−1): 3316w, 3047w, 1642m, 1622s, 1593s, 1556s, 1456m,
1398w, 1365w, 1346w, 1266m, 1225m, 1157m, 1134w, 1067m, 1046s, 962w, 913m, 873s, 799w,
1
770m, 752m, 682m; H-NMR (DMSO-d6), δ: 11.30 (s, 1H), 10.56 (s, 1H), 8.51 (s, 1H), 7.93 (d,
J = 8.1 Hz, 1H), 7.77 (d, J = 8.1 Hz, 1H), 7.55–7.47 (m, 2H), 7.40–7.24 (m, 4H), 6.76–6.70 (m, 1H),
3.78 (s, 3H); 13C-NMR (DMSO-d6), δ: 165.64, 159.52, 153.67, 139.58, 135.73, 130.42, 129.50,
128.64, 128.06, 126.83, 125.72, 123.69, 121.75, 112.72, 110.54, 109.50, 106.28, 55.03; HR-MS: for
C18H16NO3 [M+H]+ calculated 294.1124 m/z, found 294.1131 m/z.
3-Hydroxy-N-(4-methoxyphenyl)naphthalene-2-carboxamide (2c). Yield 66%; Mp. 235–236 °C;
HPLC purity 99.53%; IR (Zn/Se ATR, cm−1): 3283w, 3013w, 1636m, 1618s, 1564m, 1510m, 1393w,
1357m, 1303w, 1247m, 1170m, 1146w, 1116w, 1070m, 1030m, 950m, 878m, 856m, 830s, 795w,
1
775w, 737m; H-NMR (DMSO-d6), δ: 11.47 (s, 1H), 10.50 (s, 1H), 8.56 (s, 1H), 7.92 (d, J = 7.7 Hz,

Molecules 2013, 18
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1H), 7.76 (d, J = 8.6 Hz, 1H), 7.71–7.64 (m, 2H), 7.51 (ddd, J = 8.1 Hz, J = 6.8 Hz, J = 1.3 Hz, 1H),
7.40–7.33 (m, 2H), 7.01–6.93 (m, 2H), 3.77 (s, 3H); 13C-NMR (DMSO-d6), δ: 165.65, 155.91, 154.18,
135.78, 131.25, 130.12, 128.62, 128.06, 126.75, 125.70, 123.64, 122.31, 120.87, 113.87, 110.59,
55.16; HR-MS: for C18H16NO3 [M+H]+ calculated 294.1124 m/z, found 294.1130 m/z.
3-Hydroxy-N-(2-methylphenyl)naphthalene-2-carboxamide (3a). Yield 74%; Mp. 195–196 °C (Mp.
194–196 °C [34]); HPLC purity 98.84%; IR (Zn/Se ATR, cm−1): 3325w, 3115w, 1622s, 1586m,
1548m, 1456m, 1385w, 1355w, 1356w, 1248w, 1173m, 1065m, 954w, 915w, 873m, 844w, 803w, 773w,
742s, 679m; 1H-NMR (DMSO-d6), δ: 11.80 (s, 1H), 10.53 (s, 1H), 8.69 (s, 1H), 7.96 (d, J = 7.7 Hz, 2H),
7.78 (d, J = 8.1 Hz, 1H), 7.52 (ddd, J = 8.1 Hz, J = 6.8 Hz, J = 1.3 Hz, 1H), 7.41–7.36 (m, 2H), 7.33–
7.22 (m, 2H), 7.16–7.08 (m, 1H), 2.34 (s, 3H); 13C-NMR (DMSO-d6), δ: 164,58, 153.57, 136.36,
135.87, 131.45, 130.29, 129.99, 128.82, 128.22, 126.99, 126.24, 125.67, 124.86, 123.76, 123.36,
120.60, 110.72, 17.73; HR-MS: for C18H16NO2 [M+H]+ calculated 278.1176 m/z, found 278.1182 m/z.
3-Hydroxy-N-(3-methylphenyl)naphthalene-2-carboxamide (3b). Yield 73%; Mp. 207–208 °C; HPLC
purity 98.73%; IR (Zn/Se ATR, cm−1): 3297w, 3048w, 1607s, 1557 m, 1489m, 1451w, 1397w, 1357w,
1
1344w, 1259m, 1210m, 1173w, 1076w, 951m, 923m, 859s, 836m, 783s, 744s, 689s; H-NMR
(DMSO-d6), δ: 11.38 (s, 1H), 10.53 (s, 1H), 8.54 (s, 1H), 7.93 (d, J = 8.6 Hz, 1H), 7.77 (d, J = 8.1 Hz,
1H), 7.60–7.47 (m, 3H), 7.40–7.32 (m, 2H), 7.27 (t, J = 7.7 Hz, 1H), 6.97 (d, J = 7.3 Hz, 1H), 2.34
(s, 3H); 13C-NMR (DMSO-d6), δ: 165.62, 153.84, 138.26, 137.96, 135.77, 130.42, 128.66, 128.56,
128.08, 126.82, 125.71, 124.74, 123.69, 121.40, 121.06, 117.73, 110.59; 21.11; HR-MS: for
C18H16NO2 [M+H]+ calculated 278.1176 m/z, found 278.1181 m/z.
3-Hydroxy-N-(4-methylphenyl)naphthalene-2-carboxamide (3c). Yield 68%; Mp. 220–221 °C (Mp.
221 °C [35]); HPLC purity 99.56%; IR (Zn/Se ATR, cm−1): 3290w, 3008w, 1619s, 1556m, 1516w,
1450w, 1357m, 1252m, 1208m, 1175m, 1121w, 1070m, 951m, 913m, 869s, 832m, 810s, 761w, 741s,
716s; 1H-NMR (DMSO-d6), δ: 11.46 (s, 1H), 10.57 (s, 1H), 8.55 (s, 1H), 7.93 (d, J = 8.1 Hz, 1H), 7.77
(d, J = 8.1 Hz, 1H), 7.67 (d, J = 8.6 Hz, 2H), 7.51 (ddd, J = 8.1 Hz, J = 6.8 Hz, J = 1.3 Hz, 1H),
13
7.40–7.32 (m, 2H), 7.19 (d, J = 8.6 Hz, 2H), 2.29 (s, 3H); C-NMR (DMSO-d6), δ: 165.64, 154.01,
135.78, 133.12, 130.32, 129.10, 128.64, 128.06, 126.80, 125.70, 123.66, 121.16, 120.62, 110.60,
20.42; HR-MS: for C18H16NO2 [M+H]+ calculated 278.1176 m/z, found 278.1182 m/z.
N-(2-Fluorophenyl)-3-hydroxynaphthalene-2-carboxamide (4a). Yield 65%; Mp. 222–223 °C (Mp.
226–228 °C [36]); HPLC purity 97.21%; IR (Zn/Se ATR, cm−1): 2981w, 1625s, 1605s, 1556m,
1488w, 1458m, 1345m, 1262m, 1219m, 1206w, 1191w, 1102w, 1065m, 1033m, 951w, 916e, 867m,
1
838w, 811m, 774w, 740s, 693w; H-NMR (DMSO-d6), δ: 11.86 (s, 1H), 10.95 (s, 1H), 8.69 (s, 1H),
8.36 (dt, J = 7.7 Hz, J = 2.2 Hz, 1H), 7.98 (d, J = 8.1 Hz, 1H), 7.78 (d, J = 7.7 Hz, 1H), 7.52 (ddd,
J = 8.1 Hz, J = 6.8 Hz, J = 1.3 Hz, 1H), 7.41–7.33 (m, 2H), 7.30–7.25 (m, 1H), 7.24–7.16 (m, 2H);
13C-NMR (DMSO-d6), δ: 163.90, 153.08, (d, J = 243.8 Hz), 152.88, 135.97, 132.17, 128.93, 128.03,
127.08, 126.27 (d, J = 10.7 Hz), 125.66, 124.98 (d, J = 6.9 Hz), 124.62 (d, J = 3.4 Hz), 123.88, 122.87
(d, J = 0.8 Hz), 120.46, 115.18 (d, J = 19.1 Hz), 110.81; HR-MS: for C17H13FNO2 [M+H]+ calculated
282.0925 m/z, found 282.0931 m/z.
N-(3-Fluorophenyl)-3-hydroxynaphthalene-2-carboxamide (4b). Yield 67%; Mp. 248–249 °C; HPLC
purity 97.78%; IR (Zn/Se ATR, cm−1): 3305w, 3033w, 1622s, 1557w, 1516w, 1489w, 1449w, 1401w,

Molecules 2013, 18
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1361w, 1334w, 1253m, 1212s, 1178m, 952m, 915m, 872m, 840s 771m, 742s, 715m, 685m; 1H-NMR
(DMSO-d6), δ: 11.19(s, 1H), 10.73 (s, 1H), 8.45 (s, 1H), 7.93 (d, J = 7.7 Hz, 1H), 7.82 (t, J = 2.1 Hz,
13
1H), 7.79–7.75 (m, 1H), 7.51 (t, J = 7.1 Hz, 2H), 7.44–7.32 (m, 3H), 7.02–6.92 (m, 1H); C-NMR
(DMSO-d6), δ: 165.70, 162.09 (d, J = 241.5 Hz), 153.38, 140.24 (d, J = 11.1 Hz), 135.73, 130.54,
130.48, 130.30 (d, J = 9.5 Hz), 128.63, 128.08, 126.84, 125.73, 123.71, 122.09, 116.06 (d, J = 2.7 Hz),
110.32 (d, J = 18.7 Hz), 107.10 (d, J = 25.9 Hz); HR-MS: for C17H13FNO2 [M+H]+ calculated
282.0925 m/z, found 282.0932 m/z.
N-(4-Fluorophenyl)-3-hydroxynaphthalene-2-carboxamide (4c). Yield 69%; Mp. 264–265 °C (Mp.
264.5–265.5 °C [36]); HPLC purity 98.03%; IR (Zn/Se ATR, cm−1): 3289w, 2994w, 1615s, 1569m,
1505m, 1445w, 1409w, 1357m, 1250w, 1206m, 1171w, 1100m, 1068m, 1011w, 952w, 915m, 871m,
1
829s, 799m, 766m, 739m, 706m; H-NMR (DMSO-d6), δ: 11.31 (s, 1H), 10.64 (s, 1H), 8.49 (s, 1H),
7.93 (d, J = 8.12 Hz, 1H), 7.83–7.75 (m, 3H), 7.51 (ddd, J = 8.1 Hz, J = 6.8 Hz, J = 1.3 Hz, 1H),
7.40–7.32 (m, 2H), 7.23 (t, J = 9.0 Hz, 2H); 13C-NMR (DMSO-d6), δ: 165.71, 158.50 (d, J = 240.7 Hz),
153.79, 135.76, 134.72 (d, J = 2.7 Hz), 130.29, 128.62, 128.06, 126.79, 125.72, 123.68, 122.44 (d, J = 8.0
Hz), 121.47, 115.28 (d, J = 22.1 Hz), 110.55; HR-MS: for C17H13FNO2 [M+H]+ calculated 282.0925
m/z, found 282.0930 m/z.
N-(2-Chlorophenyl)-3-hydroxynaphthalene-2-carboxamide (5a). Yield 50%; Mp. 226–227 °C (Mp.
225 °C [35]); HPLC purity 98.70%; IR (Zn/Se ATR, cm−1): 3164w, 1625m, 1591s, 1546s, 1439m,
1345m, 1293w, 1243w, 1192m, 1171m, 1034m, 840m, 740s, 668m;1H-NMR (DMSO-d6), δ: 11.91 (s,
1H), 11.13 (s, 1H), 8.73 (s, 1H), 8.51 (dd, J = 8.1 Hz, J = 1.3 Hz, 1H), 7.99 (d, J = 7.69 Hz, 1H), 7.78
(d, J = 8.12 Hz, 1H), 7.59–7.52 (m, 2H), 7.49–7.45 (m, 1H), 7.41–7.33 (m, 2H), 7.22–7.14 (m, 1H)
13C-NMR (DMSO-d6), δ: 163.49, 152.65, 135.99, 135.29, 132.62, 129.29, 128.99, 128.42, 127.77,
127.13, 125.62, 125.12, 123.88, 123.35, 122.69, 120.55, 110.78; HR-MS: for C17H13ClNO2 [M+H]+
calculated 298.0629 m/z, found 298.0637 m/z.
N-(3-Chlorophenyl)-3-hydroxynaphthalene-2-carboxamide (5b). Yield 69%; Mp. 257–258 °C (Mp.
258–261 °C [37]); HPLC purity 98.82%; IR (Zn/Se ATR, cm−1): 3299w, 3054w, 1623s, 1545m,
1476w, 1426m 1364m, 1278w, 1248m, 1214m, 1174w, 1065w, 913w, 866m, 776m, 744m, 708m,
1
680m; H-NMR (DMSO-d6), δ: 11.16 (s, 1H); 10.68 (s, 1H), 8.46 (s, 1H), 7.99 (t, J = 1.9 Hz, 1H),
7.93 (d, J = 8.1 Hz, 1H), 7.76 (d, J = 7.7 Hz, 1H), 7.67 (d, J = 8.1 Hz, 1H), 7.51 (ddd, J = 8.1 Hz,
J = 6.8 Hz, J = 1.3 Hz, 1H), 7.47–7.34 (m, 3H), 7.2 (d, J = 8.1 Hz, 1H); 13C-NMR (DMSO-d6),
δ: 165.77, 153.42, 139.95, 135.73, 133.06, 130.45, 130.35, 128.61, 128.09, 126.81, 125.73, 123.71,
123.60, 122.03, 119.80, 118.73, 110.52; HR-MS: for C17H13ClNO2 [M+H]+ calculated 298.0629 m/z,
found 298.0637 m/z.
N-(4-Chlorophenyl)-3-hydroxynaphthalene-2-carboxamide (5c). Yield 71%; Mp. 263–264 °C (Mp.
260 °C [34]); HPLC purity 99.35%; IR (Zn/Se ATR, cm−1): 3283w, 3052w, 1612s, 1549m, 1488m,
1400m, 1359w, 1334w, 1252w, 1209m, 1170m, 1116w, 1068w, 1012m, 749s, 711m, 680m;1H-NMR
(DMSO-d6), δ: 11.24 (s, 1H), 10.68 (s, 1H), 8.47 (s, 1H), 7.93 (d, J = 7.7 Hz, 1H), 7.8 (t, J = 9.6 Hz,
2H), 7.54–7.46 (m, 2H), 7.42–7.32 (m, 4H); 13C-NMR (DMSO-d6), δ: 165.69, 153.56, 137.42, 135.74,
130.45, 130.40, 128.61, 128.08, 127.61, 126.81, 125.73, 123.70, 121.97, 121.83, 110.53; HR-MS: for
C17H13ClNO2 [M+H]+ calculated 298.0629 m/z, found 298.0636 m/z.

Molecules 2013, 18
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N-(2-Bromophenyl)-3-hydroxynaphthalene-2-carboxamide (6a). Yield 61%; Mp. 215–216 °C; HPLC
purity 97.75%; IR (Zn/Se ATR, cm−1): 3158w, 1623s, 1582s, 1537s, 1446w, 1434m, 1388w, 1343m,
1312w, 1291w, 1193w, 1071w, 1047w, 1024m, 916m, 873m, 845m, 750s, 698m; 1H-NMR (DMSO-d6), δ:
11.88 (s, 1H), 11.00 (s, 1H), 8.72 (s, 1H), 8.42 (dd, J = 8.1 Hz, J = 1.3 Hz, 1H), 7.98 (d, J = 8.1 Hz
1H), 7.80–7.70 (m, 2H), 7.57–7.50 (m, 2H), 7.45–7.34 (m, 2H), 7.13 (dt J = 7.7 Hz, J = 1.7 Hz, 1H);
13C-NMR (DMSO-d6), δ: 163.67, 152.77, 136.53, 136.02, 132.59, 132.54, 128.99, 128.45, 128.28,
127.10, 125.82, 125.64, 123.88, 123.50, 120.47, 114.35, 110.76; HR-MS: for C17H13BrNO2 [M+H]+
calculated 342.0124 m/z, found 342.0133 m/z.
N-(3-Bromophenyl)-3-hydroxynaphthalene-2-carboxamide (6b). Yield 73%; Mp. 251–252 °C; HPLC
purity 98.67%; IR (Zn/Se ATR, cm−1): 3295w, 3075w, 1622m, 1583m, 1553w, 1479m, 1403w, 1239w,
1209m, 1095w, 1073w, 992w, 953w, 923w, 867m 852s, 837m, 780s, 745s, 667m; 1H-NMR (DMSO-d6),
δ: 11.16 (s, 1H), 10.00 (s, 1H), 8.46 (s, 1H), 8.13 (s, 1H), 7.92 (d, J = 7.69 Hz, 1H), 7.78-7.68 (m, 2H),
7.51 (ddd, J = 8.1 Hz, J = 6.8 Hz, J = 1.3 Hz, 1H), 7.40–7.33 (m, 4H); 13C-NMR (DMSO-d6), δ:
165.79, 153.44, 140.08, 135.74, 130.66, 130.43, 128.62, 128.09, 126.80, 126.51, 125.74, 123.72,
122.66, 122.00, 121.48, 119.14, 110.52; HR-MS: for C17H13BrNO2 [M+H]+ calculated 342.0124m/z,
found 342.0130 m/z.
N-(4-Bromophenyl)-3-hydroxynaphthalene-2-carboxamide (6c). Yield 69%; Mp. 253–254 °C (Mp.
248–249 °C [35]); HPLC purity 99.02%; IR (Zn/Se ATR, cm−1): 3288w, 3052w, 1605s, 1544m, 1486s,
1447w, 1394m, 1360w, 1251w, 1209m, 1169w, 1075m, 1010m, 955w, 915w, 871m, 828s, 810s, 785m,
1
749s, 712m; H-NMR (DMSO-d6), δ: 11.22 (s, 1H), 10.67 (s, 1H), 8.46 (s, 1H) 7.92 (d, J = 7.7 Hz,
13
1H), 7.76 (d, J = 8.6 Hz, 3H), 7.59–7.46 (m, 3H), 7.39–7.32 (m, 2H); C-NMR (DMSO-d6), δ: 165.66,
153.51, 137.83, 135.72, 131.52, 130.39, 128.60, 128.06, 126.80, 125.70, 123.69, 122.30, 121.87, 115.63,
110.52; HR-MS: for C17H13BrNO2 [M+H]+ calculated 342.0124m/z, found 342.0132 m/z.
3-Hydroxy-N-(2-trifluoromethylphenyl)naphthalene-2-carboxamide (7a). Yield 49%; Mp. 209–210 °C;
HPLC purity 99.66%; IR (Zn/Se ATR, cm−1): 3274w, 1660w, 1626w, 1597w, 1515m, 1466s, 1352w,
1278s, 1217w, 1198w, 1146m, 1071w, 956m, 874m, 833m, 788m, 748w, 722m; 1H-NMR (DMSO-d6),
δ: 11.67 (s, 1H), 10.43 (s, 1H), 8.72 (s, 1H), 7.99 (d, J = 8.1 Hz, 1H), 7.86 (d, J = 7.8 Hz, 1H),
7.83–7.77 (m, 2H), 7.70–7.66 (m, 1H), 7.62–7.57 (m, 1H), 7.53 (ddd, J = 7.0 Hz, J = 5.5 Hz,
J = 1.1 Hz, 1H), 7.40 (s, 1H), 7.36 (ddd, J = 8.4 Hz, J = 7.0 Hz, J = 1.1 Hz); 13C-NMR (DMSO-d6), δ:
167.12, 153.35, 135.78, 135.47 (q, J = 3.9 Hz), 133.15, 131.16, 130.42, 128.63, 128.12, 126.86, 126.45
(q, J = 4.6 Hz), 126.16 (q, J = 29.6 Hz), 125.75, 123.75, 123.70, (q, J = 276.2 Hz), 122.11, 120.19,
110.55; HR-MS: for C18H13NO2F3 [M+H]+ calculated 332.0893 m/z, found 332.0898 m/z.
3-Hydroxy-N-(3-trifluoromethylphenyl)naphthalene-2-carboxamide (7b). Yield 64%; Mp. 239–240 °C;
HPLC purity 98.81%; IR (Zn/Se ATR, cm−1): 3296w, 3108w, 1626s, 1575m, 1494m, 1428w, 1399w,
1361w, 1327s, 1207m, 1178s, 1148m, 1118s, 1100m, 1076m, 925w, 893m, 867s, 840m, 803s, 771m,
1
750s, 696s, 680m; H-NMR (DMSO-d6), δ: 11.16 (s, 1H), 10.83 (s, 1H), 8.45 (s, 1H), 8.29 (s, 1H),
7.96 (t, J = 8.1 Hz, 2H), 7.97 (d, J = 8.1 Hz, 1H), 7.62 (t, J = 7.9 Hz, 1H), 7.55–7.47 (m, 2H),
13
7.40–7.32 (m, 2H); C-NMR (DMSO-d6), δ: 166.06, 153.46, 139.32, 135.79, 130.45, 129.93, 129.51
(q, J = 31.3 Hz), 128.63, 128.13, 126.84, 125.76, 124.11 (q, J = 277.7 Hz), 123.94, 123.76, 122.09,

Molecules 2013, 18
7990
120.21 (q, J = 3.8 Hz), 116.47 (q, J = 3.8 Hz), 110.54; HR-MS: for C18H13NO2F3 [M+H]+ calculated
332.0893 m/z, found 332.0900 m/z.
3-Hydroxy-N-(4-trifluoromethylphenyl)naphthalene-2-carboxamide (7c). Yield 58%; Mp. 281–282 °C;
HPLC purity 99.46%; IR (Zn/Se ATR, cm−1): 3292w, 3021w, 1623s, 1548m, 1450m, 1410m, 1324m,
1255w, 1212m, 1175m, 1112s, 1065s, 1016m, 960w, 916w, 873m, 841s, 822m, 791w, 752s, 707m;
1H-NMR (DMSO-d6), δ: 11.17 (s, 1H), 10.85 (s, 1H), 8.45 (s, 1H), 8.03–7.91 (m, 3H), 7.79–7.72 (m,
3H), 7.51 (ddd, J = 8.1 Hz, J = 6.8 Hz, J = 1.3 Hz, 1H), 7.40–7.32 (m, 2H); 13C-NMR (DMSO-d6), δ:
165.87, 153.28, 142.15 (q, J = 1.5 Hz), 135.76, 130.61, 128.64, 128.12, 126.86, 125.98 (q, J = 3.8 Hz),
125.74, 124.30 (q, J = 271.2Hz), 123.88 (q, J = 32.0 Hz), 123.74, 122.28, 120.18, 110.53;
HR-MS: for C18H13NO2F3 [M+H]+ calculated 332.0893 m/z, found 332.0899 m/z.
3-Hydroxy-N-(2-nitrophenyl)naphthalene-2-carboxamide (8a). Yield 29%; Mp. 174–175 °C; HPLC
purity 97.17%; IR (Zn/Se ATR, cm−1): 3240w, 1627m,1581m, 1557w, 1494m, 1450w, 1434w, 1393w,
1
1341m, 1270m, 1203w, 1147m, 870w, 840m, 771w, 736s, 691w; H-NMR (DMSO-d6), δ: 12.05 (s,
1H), 11.79 (s, 1H), 8.73 (s, 1H), 8.60 (dd, J = 7.2 Hz, J = 1.2 Hz, 1H), 8.16 (dd, J = 8.2 Hz, J = 1.5 Hz,
1H), 7.97 (t, J = 8.0 Hz, 2H), 7.82–7.78 (m, 1H), 7.51 (ddd, J = 8.1 Hz, J = 6.8 Hz, J = 1.3 Hz, 1H),
7.39–7.34 (m, 3H); 13C-NMR (DMSO-d6), δ: 164.21, 155.98, 152.85, 137.18, 136.17, 132.97, 132.43,
129.16, 128.94, 127.04, 126.62, 125.89, 125.63, 123.75, 120.74, 115.27, 110,75; HR-MS: for
C17H13N2O4 [M+H]+ calculated 309.0870 m/z, found 309.0875 m/z.
3-Hydroxy-N-(3-nitrophenyl)naphthalene-2-carboxamide (8b). Yield 58%; Mp. 250–251 °C; (Mp.
242–244 °C [34]); HPLC purity 97.62%; IR (Zn/Se ATR, cm−1): 3394w, 1658m, 1586m, 1525m,
1463m, 1345s, 1297s, 1230m, 1209m, 1145m, 1033w, 911w, 876m, 808m, 747m, 738s, 668m;
1H-NMR (DMSO-d6), δ: 11.12 (s, 1H), 10.93 (s, 1H), 8.83 (t, J = 2.14 Hz, 1H), 8.45 (s, 1H), 8.11 (dd,
J = 8.12 Hz, J = 1.28 Hz, 1H), 8.01–7.91 (m, 2H), 7.77 (d, J = 8.1 Hz, 1H), 7.66 (t, J = 8.1 Hz, 1H),
7.51 (ddd, J = 8.1 Hz, J = 6.8 Hz, J = 1.3 Hz, 1H), 7.40–7.32 (m, 2H); 13C-NMR (DMSO-d6), δ:
166.08, 153.30, 147.95, 139.72, 135.76, 130.48, 130.08, 128.61, 128.12, 126.80, 126.24, 125.76,
123.76, 122.22, 118.32, 114.35, 110.50; HR-MS: for C17H13N2O4 [M+H]+ calculated 309.0870 m/z,
found 309.0876 m/z.
3-Hydroxy-N-(4-nitrophenyl)naphthalene-2-carboxamide (8c). Yield 72%; Mp. 265–266 °C; HPLC
purity 99.03%; IR (Zn/Se ATR, cm−1): 3285w, 2981w, 1618s, 1599m, 1568w, 1513s, 1449w, 1407w,
1
1337s, 1260w, 1210m, 1171m, 1147w, 1112m, 1033m, 916w, 851s, 772w, 747s, 704w; H-NMR
(DMSO-d6), δ: 11.10 (s, 1H), 11.03 (s, 1H), 8.41 (s, 1H), 8.28 (d, J = 9.4 Hz, 2H), 8.03 (d, J = 9.0 Hz,
2H), 7.93 (d, J = 8.1Hz, 1H), 7.77 (d, J = 8.6 Hz, 1H), 7.51 (ddd, J = 8.1 Hz, J = 6.8 Hz, J = 1.3 Hz,
1H), 7.39–7.32 (m, 2H); 13C-NMR (DMSO-d6), δ: 165.89, 152.98, 144.80, 142.58, 135.76, 130.73,
128.63, 128.15, 126.85, 125.75, 124.82, 123.76, 122.68, 119.79, 110.49; HR-MS: for C17H13N2O4
[M+H]+ calculated 309.0870 m/z, found 309.0875 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

Molecules 2013, 18
7991
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)
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 [51]. The inhibition of photosynthetic electron transport (PET) in spinach chloroplasts was
determined spectrophotometrically (Genesys 6, Thermo Scientific), using an artificial electron
acceptor 2,6-dichlorophenol-indophenol (DCIPP) according to Kralova et al. [52], 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 Fluorescence in Spinach Chloroplasts
The fluorescence emission spectra of chlorophyll a (Chla) in spinach chloroplasts were recorded on
fluorescence spectrophotometer F-2000 (Hitachi, Tokyo, Japan) using excitation wavelength λex = 436 nm
for monitoring fluorescence of Chla, 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,

Molecules 2013, 18
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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
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 broth dilution micro-method modified according to NCCLS guidelines
[53,54] 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 and M. kansasii DSM 44162. The broth dilution micro-method
in Middlebrook 7H9 medium (Difco, Lawrence, KS, USA) supplemented with ADC Enrichment
(BBL, USA) was used to determine the minimum inhibitory concentration (MIC) as previously
described [55]. The tested compounds were dissolved as described above in chapter 3.6. Isoniazid
(Sigma-Aldrich) was used as reference antibacterial drug. 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). The final inoculum was made by 1:1,000 dilution
of the suspension with sterile water. Drug-free controls, sterility controls and controls consisted of
medium and DMSO alone were included. The determination of results was performed visually after 7
days of static incubation in the darkness at 37 °C in an aerobic atmosphere for M. kansasii and after 21
days of static incubation in the darkness at 28 °C in an aerobic atmosphere for M. marinum. 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.
3.8. In Vitro Cytotoxicity Assay
Human monocytic leukemia THP-1 cells were used for in vitro toxicity assay. Cells were obtained
from the European Collection of Cell Cultures (ECACC, Salisbury, UK) and routinely cultured in
RPMI medium supplemented with 10% fetal bovine serum, 2% L-glutamine, 1% penicillin and
streptomycin at 37 °C with 5% CO2. Cytotoxicity was determined using a WST-1 assay kit (Roche
Diagnostics, Mannheim, Germany) according to the manufacturer’s instructions. The tested
compounds were dissolved in DMSO and added in five increasing concentrations (0.37, 1.1, 3.3, 10,
and 30 μmol/L) to the cell suspension in the culture medium. Subsequently, the cells were incubated
for 24 h at 37 °C with 5% CO2. WST-1 assays conducted in triplicates were performed as previously
described [49]. The median lethal dose values, LD50, were deduced through the production of a dose-

Molecules 2013, 18
7993
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.
4. Conclusions
A series of twenty-two ring-substituted 3-hydroxynaphthalene-2-carboxanilides were prepared and
characterized. The prepared compounds were tested for their ability to inhibit photosynthetic electron
transport (PET) in spinach chloroplasts (Spinacia oleracea L.) and for their antibacterial and
antimycobacterial activity against four Staphylococcus strains, Mycobacterium marinum and
M. kansasii. 3-Hydroxy-N-(3-nitrophenyl)naphthalene-2-carboxamide (8b) showed relatively high
PET inhibition. 3-Hydroxy-N-(2-methoxyphenyl)naphthalene-2-carboxamide (2a) showed the high
biological activity against S. aureus as well as methicillin-resistant strains. N-(2-Fluorophenyl)-3-
hydroxynaphthalene-2-carboxamide (4a) and N-(3-fluorophenyl)-3-hydroxynaphthalene-2-carboxamide
(4b) expressed high activity against M. marinum. 3-Hydroxy-N-(4-nitrophenyl)naphthalene-2-
carboxamide (8c) showed high activity against M. kansasii. All the above-mentioned compounds
exhibited activity comparable with or higher than the standards ampicillin or isoniazid. Lipophilicity
and electronic properties of anilide substituents influenced the biological activities of compounds in all
biological assays. It can be stated that the dependences of activities on lipophilicity and on electronic
properties show bilinear trends. The most effective compounds 2a, 4a, 7a, 8b and 8c were tested for
their in vitro cytotoxicity against THP-1 cells. It can be concluded that the discussed anilides 2a and
4a can be considered as promising agents for subsequent design of novel antibacterial and
antimycobacterial 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 CzechGlobe (Centre for Global Climate Change Impacts Studies),
Reg. No. CZ.1.05/1.1.00/02.0073, and 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.
Conflict of Interest
The authors declare no conflict of interest.
References
1. Baquero, F.; Coque, T.M.; de la Cruz, F. Ecology and evolution as targets: the need for novel
eco-evo drugs and strategies to fight antibiotic resistance. Antimicrob. Agents Chemother. 2011,
55, 3649–3660.
2. 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.

Molecules 2013, 18
7994
3. 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.
4. 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.
5. Laughon, E.L. New tuberculosis drugs in development. Curr. Top. Med. Chem. 2007, 7, 463–473.
6. Levy, S.B. Antibiotic resistance-the problem intensifies. Adv. Drug. Delivery Rev. 2005, 57,
1446–1450.
7. 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.
8. 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.
9. Shaner, D.L. Herbicide safety relative to common targets in plants and mammals. Pest. Manag. Sci.
2004, 60, 17–24.
10. Delaney, J.; Clarke, E.; Hughes, D.; Rice, M. Modern agrochemical research: A missed
opportunity for drug discovery? Drug Discov. Today 2006, 11, 839–845.
11. Duke, S.O. Herbicide and pharmaceutical relationships. Weed Sci. 2010, 58, 334–339.
12. Kralova, K.; Sersen, F.; Cizmarik, J. Inhibitory effect of piperidinoethylesters of
alkoxyphenylcarbamic acids on photosynthesis. Gen. Physiol. Biophys. 1992, 11, 261–267.
13. 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.
14. 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.
15. Kralova, K.; Bujdakova, H.; Cizmarik, J. Antifungal and antialgal activity of piperidinopropyl
esters of alkoxy substituted phenylcarbamic acids. Pharmazie 1995, 50, 440–441.
16. 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.
17. Imramovsky, A.; Vinsova, J.; Monreal-Ferriz, J.; Buchta, V.; Jampilek, J. Salicylanilide esters of
N-protected amino acids as novel antimicrobial agents. Bioorg. Med. Chem. Lett. 2009, 19,
348–351.
18. 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.

Molecules 2013, 18
7995
19. 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.
20. 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.
21. Draber, W.; Tietjen, K.; Kluth, J.F.; Trebst, A. Herbicides in photosynthesis research.
Angew. Chem. 1991, 3, 1621–1633.
22. Tischer, W.; Strotmann, H. Relationship between inhibitor binding by chloroplasts and inhibition
of photosynthetic electron-transport. Biochim. Biophys. Acta 1977, 460, 113–125.
23. 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.
24. 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.
25. Koreyoshi, S.; Masato, M.; Tetsuji, I. (Yoshitomi Pharmaceutical Industries) Hydroxynaphthoic
acid anilides, microbicides and slimicides. Japan. Kokai JP 52 076 430, 27 June 1977.
26. 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.
27. Kauppi, A.M.; Nordfelth, R.; Uvell, H.; Wolf-Watz, H.; Elofsson, M. Targeting bacterial
virulence: Inhibitors of type III secretion in Yersinia. Chem. Biol. 2003, 10, 241–249.
28. Vinsova, J.; Imramovsky, A.; Buchta, V.; Ceckova, M.; Dolezal, M.; Staud, F.; Jampilek, J.;
Kaustova, J. Salicylanilide acetates: Synthesis and antibacterial evaluation. Molecules 2007, 12,
1–12.
29. 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.
30. 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.
31. Kratky, M.; Vinsova, J. Salicylanilide ester prodrugs as potential antimicrobial agents – a review.
Curr. Pharm. Des. 2011, 17, 3494–3505.
32. Kratky, M.; Vinsova, J. Buchta, V. In vitro antibacterial and antifungal activity of salicylanilide
benzoates. Sci. World J. 2012, 2012, 290628.
33. 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.
34. Hartrodt, V.W.; Woohsmann, H. Ein Beitrag zur Darstellun von Alkalisalzen saurer
Schwefelsäureester verschiedener Naphthol AS-Verbindungen. J. Prakt. Chem. 1965, 27, 99–107.

Molecules 2013, 18
7996
35. Tripathy, H.; Pradhan, D.G.; Dash, B.C.; Mahapatra, G.N. Synthesis of some new halogenated
N-thiazolyl substituted hydroxy acid amides and their use as possible fungicides. Agric. Biol. Chem.
1973, 6, 1375–1383.
36. Hitch, F.E.; Dahlen, A.M.; Friedrich, M.E. (E.I. du Pont de Nemours & Company). Fluorinated
arylamides. US 1982661, 4 December 1934.
37. 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.
38. 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.
39. 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.
40. Kralova, K.; Sersen, F.; Sidoova, E. Effect of 2-alkylthio-6-aminobenzothiazoles and their 6-N-
substituted derivatives on photosynthesis inhibition in spinach chloroplasts. Gen. Phys. Biophys.
1993, 12, 421–427.
41. Kralova, K.; Sersen, F.; Miletin, M.; Hartl, J. Inhibition of photosynthetic electron transport by
some anilides of 2-alkylpyridine-4-carboxylic acids in spinach chloroplasts. Chem. Pap. 1998, 52,
52–55.
42. Kralova, K.; Sersen, F.; Pesko, M.; Waisser, K.; Kubicova, L. 5-Bromo- and 3,5-dibromo-2-
hydroxy-N-phenylbenzamides—Inhibitors of photosynthesis. Chem. Pap. 2013, 67, in press
43. Kralova, K.; Sersen, F.; Klimesova, V.; Waisser, K. 2-Alkylsulphanyl-4-pyridine-
carbothioamides—Inhibitors of oxygen evolution in freshwater alga Chlorella vulgaris.
Chem. Pap. 2011, 65, 909–912.
44. Atal, N.; Saradhi, P.P.; Mohanty, P. Inhibition of the chloroplast photochemical reactions by
treatment of wheat seedlings with low concentrations of cadmium: Analysis of electron transport
activities and changes in fluorescence yields. Plant Cell Physiol. 1995, 32, 943–951.
45. Sersen, F.; Kralova, K.; Bumbalova A. Action of mercury on the photosynthetic apparatus of
spinach chloroplasts. Photosynthetica 1998, 35, 551–559.
46. Apostolova, E.L.; Ivanov, A.G. Influence of Triton X-100on the structure and functions of pea
thylakoid membranes. J. Plant Physiol. 1995, 145, 239–244.
47. Kralova, K.; Sersen, F.; Kubicova, L.; Waisser, K. Inhibitory effects of substituted benzanilides
on photosynthetic electron transport in spinach chloroplasts. Chem. Pap. 1999, 53, 328–331.
48. 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.
49. Kollar, P.; Zavalova, V.; Barta, T.; Smejkal, K.; Hampl, A. Geranylated flavanone tomentodiplacone
B inhibits proliferation of human monocytic leukaemia (THP-1) cells. Br. J. Pharmacol. 2011,
162, 1534–1541.
50. 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.

Molecules 2013, 18
7997
51. 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.
52. Kralova, K.; Sersen, F.; Sidoova, E. Photosynthesis inhibition produced by 2-alkylthio-6-R-
benzothiazoles. Chem. Pap. 1992, 46, 348–350.
53. National Committee for Clinical Laboratory Standards. Methods for Dilution Antimicrobial
Susceptibility Tests for Bacteria that Grow Aerobically, 5th Edition; Approved Standard M7-A5.
NCCLS: Wayne, PA, USA, 2000.
54. National Committee for Clinical Laboratory Standards. Performance Standards for Antimicrobial
Susceptibility Testing; 12th Informational Supplement M100-S12. NCCLS: Wayne, PA, USA, 2002.
55. 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.
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