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

Article abstract of DOI:10.1016/j.bmc.2013.08.030

In this study, a series of 22 ring-substituted 1-hydroxynaphthalene-2- carboxanilides were prepared and characterized. Primary in vitro screening of the synthesized compounds was performed against Mycobacterium marinum, Mycobacterium kansasii and Mycobacterium smegmatis. The compounds were also tested for their activity related to inhibition of photosynthetic electron transport (PET) in spinach (Spinacia oleracea L.) chloroplasts. Most of tested compounds showed the antimycobacterial activity against the three strains comparable or higher than the standard isoniazid. N-(3-Fluorophenyl)-1- hydroxynaphthalene-2-carboxamide showed the highest biological activity (MIC = 28.4 μmol/L) against M. marinum, N-(4-fluorophenyl)-1-hydroxynaphthalene-2- carboxamide showed the highest biological activity (MIC = 14.2 μmol/L) against M. kansasii, and N-(4-bromophenyl)-1-hydroxynaphthalene-2-carboxamide expressed the highest biological activity (MIC = 46.7 μmol/L) against M. smegmatis. This compound and 1-hydroxy-N-(3-methylphenyl)naphthalene-2- carboxamide were the most active compounds against all three tested strains. The PET inhibition expressed by IC₅₀ value of the most active compound 1-hydroxy-N-(3-trifluoromethylphenyl)naphthalene-2-carboxamide was 5.3 μmol/L. The most effective compounds demonstrated insignificant toxicity against the human monocytic leukemia THP-1 cell line. For all compounds, structure-activity relationships are discussed.

Full text of DOI:10.1016/j.bmc.2013.08.030

Bioorganic & Medicinal Chemistry 21 (2013) 6531–6541
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Antimycobacterial and herbicidal activity of ring-substituted
1-hydroxynaphthalene-2-carboxanilides
Tomas Gonec a, Jiri Kos a, Iveta Zadrazilova a,b, Matus Pesko c, Stanislava Keltosova d, Jan Tengler a,
Pavel Bobal a, Peter Kollar d, Alois Cizek b, Katarina Kralova e, Josef Jampilek a,
⇑
a Department of Chemical Drugs, Faculty of Pharmacy, University of Veterinary and Pharmaceutical Sciences, Palackeho 1/3, 612 42 Brno, Czech Republic
b Department of Infectious Diseases and Microbiology, Faculty of Veterinary Medicine, University of Veterinary and Pharmaceutical Sciences,
Palackeho 1/3, 612 42 Brno, Czech Republic
c Department of Environmental Ecology, Faculty of Natural Sciences, Comenius University, Mlynska dolina Ch-2, 842 15 Bratislava, Slovakia
d Department of Human Pharmacology and Toxicology, Faculty of Pharmacy, University of Veterinary and Pharmaceutical Sciences, Palackeho 1/3, 612 42 Brno, Czech Republic
e Institute of Chemistry, Faculty of Natural Sciences, Comenius University, Mlynska dolina Ch-2, 842 15 Bratislava, Slovakia
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 7 July 2013
Revised 10 August 2013
Accepted 16 August 2013
Available online 12 September 2013
In this study, a series of 22 ring-substituted 1-hydroxynaphthalene-2-carboxanilides were prepared and
characterized. Primary in vitro screening of the synthesized compounds was performed against Mycobac-
terium marinum, Mycobacterium kansasii and Mycobacterium smegmatis. The compounds were also tested
for their activity related to inhibition of photosynthetic electron transport (PET) in spinach (Spinacia oler-
acea L.) chloroplasts. Most of tested compounds showed the antimycobacterial activity against the three
strains comparable or higher than the standard isoniazid. N-(3-Fluorophenyl)-1-hydroxynaphthalene-2-
carboxamide showed the highest biological activity (MIC = 28.4 lmol/L) against M. marinum, N-(4-fluo-
rophenyl)-1-hydroxynaphthalene-2-carboxamide showed the highest biological activity (MIC = 14.2 l-
mol/L) against M. kansasii, and N-(4-bromophenyl)-1-hydroxynaphthalene-2-carboxamide expressed
Keywords:
Hydroxynaphthalene-2-carboxanilides
Lipophilicity
Photosynthetic electron transport inhibition
Spinach chloroplasts
the highest biological activity (MIC = 46.7 lmol/L) against M. smegmatis. This compound and 1-
hydroxy-N-(3-methylphenyl)naphthalene-2-carboxamide were the most active compounds against all
three tested strains. The PET inhibition expressed by IC50 value of the most active compound 1-
hydroxy-N-(3-trifluoromethylphenyl)naphthalene-2-carboxamide was 5.3 lmol/L. The most effective
In vitro antimycobacterial activity
In vitro cytotoxicity
Structure–activity relationships
compounds demonstrated insignificant toxicity against the human monocytic leukemia THP-1 cell line.
For all compounds, structure–activity relationships are discussed.
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
skeletal infections. The number of infections that can be associated
with specific species as well as the number of new species as etio-
Tuberculosis (TB) is more prevalent in the world today than at
any other time. Mycobacterium tuberculosis, the pathogen responsi-
ble for TB, uses diverse strategies to survive in a variety of host le-
sions and to evade immune surveillance.1 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 mul-
ti-drug-resistant strains of M. tuberculosis (MDR-TB) notified in 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 noti-
fied TB patients estimated to have MDR-TB.2 Nontuberculous
mycobacteria (NTM) are important environmental pathogens that
can cause a broad spectrum of diseases, such as pulmonary disease,
lymphadenitis, skin and soft tissue disease, gastrointestinal and
logical agents has exploded the last few years due to the increase of
the number of immunocompromised patients.3 The emergence of
MDR-TB makes the discovery of new molecular scaffolds a priority,
and the current situation even necessitates the re-engineering and
repositioning of some old drug families to achieve effective control.
Also more effective drugs are needed for the treatment of NTM.1,3
Salicylanilides (N-substituted hydroxybenzamides) are com-
pounds with a wide range of pharmacological activities, whereas
antibacterial/antimycobacterial4–10 and antifungal6,7 effects be-
come more and more important. They also showed herbicidal6–9
activity. Their mechanism of action is still under investigation;
they also serve as inhibitors of protein kinase epidermal growth
factor receptor (EGFR PTK).11 They are generally designed to com-
pete with ATP for binding in catalytic domain of tyrosine kinase.12
The latest studies specified them also as selective inhibitors of pro-
duction of interleukin-12p40 that plays a specific role in initiation,
expansion and control of cellular response to tuberculosis.13
⇑
Corresponding author. Tel.: +420 541 562 926.
E-mail address: josef.jampilek@gmail.com (J. Jampilek).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.08.030

6532
T. Gonec et al. / Bioorg. Med. Chem. 21 (2013) 6531–6541
The presence of an amide group is characteristic not only in var-
target compounds was carried out only in one step with satisfac-
tory yields. All the studied compounds were prepared according
to Scheme 1. The condensation of commercially available 1-hydro-
xy-2-naphthoic acid and ring-substituted aniline using phosphorus
trichloride in chlorobenzene under microwawe conditions yielded
a series of 22 N-substituted 1-hydroxynaphthalene-2-carboxam-
ides 1–8c.
ious antimycobacterial drugs14 but also in a number of herbicides
acting as photosynthesis inhibitors.6–9,15–18 Over 50% of commer-
cially available herbicides act by reversible binding to photosystem
II (PS II), a membrane–protein complex in the thylakoid mem-
branes, which catalyses the oxidation of water and the reduction
of plastoquinone,19 and thereby inhibit photosynthesis.20–22
Both pharmaceuticals and pesticides are designed to target par-
ticular biological functions, and in some cases these functions
overlap in their molecular target sites or they target similar pro-
cesses or molecules. However, the consequences of inhibition of
the overlapping target site can be completely different for plants
and animals. Therefore a compound that has lethal action on plants
may be beneficial for mammals.23 Taking into consideration that
herbicides may also have molecular sites of action in mammals,
until recently most pharmaceutical companies had pesticide divi-
sions, sometimes with a different name. All compounds generated
by either division of the company were evaluated for both pesti-
cide and pharmaceutical uses. In the past, some leading pesticides
have become pharmaceuticals and vice versa. However, little infor-
mation of this type was published and must usually be deduced
from patent literature. One of the exceptions is fluconazole, a fun-
gicide product discovered by the pharmaceutical sector that is now
used both as a pharmaceutical and also patented as a chemical
with applications in crop production.23–25 Moreover, good correla-
tion between antimicrobial activities and herbicidal effects was
found [6–9,26 and refs. therein].
2.2. Lipophilicity
Lipophilicity is a property that has a major effect on absorption,
distribution, metabolism, excretion and toxicity properties as well
as pharmacological activity, because drugs cross biological mem-
branes through passive transport, which strongly depends on their
lipophilicity. Lipophilicity has been studied and applied as an
important drug property for decades.
Lipophilicity of studied compounds 1–8c was predicted as logP
using ACD/Percepta software and determined by RP-HPLC as
capacity factor logarithm (logk). The procedure was performed un-
der isocratic conditions with methanol as an organic modifier in
the mobile phase using an end-capped nonpolar C18 stationary
RP column. The results are shown in Table 1 and illustrated in
Figure 2.
The results obtained with all the ring-substituted 1-hydroxy-
naphthalene-2-carboxanilides show that the experimentally-
determined lipophilicity (logk) of the discussed compounds poorly
correlates with the calcd values of compounds 1–8c as shown in
Figure 2, contrary to ring-substituted 3-hydroxynaphthalene-2-
carboxanilides investigated by Kos et al.8 or chlorosalicylanilide
derivatives.7 Figure 2A illustrates the match of logk to logP of
the ortho-substituted derivatives, while Figure 2B illustrates the
match of logk to logP of the meta- and para-substituted com-
pounds. Similar poor correlation between experimentally and pre-
dicted lipophilicity values was described by Gonec et al.9 and
Promising results of antimicrobial and herbicidal screening of
some salicylanilides-like compounds,5–10 styrylhydroxyquinolines
and hydroxyquinolinecarboxamides16,27,28 inspired us to prepare
and evaluate ring-substituted 1-hydroxynaphthalene-2-carboxani-
lides. The design of these N-substituted 1-hydroxynaphthalene-2-
carboxanilides is based on the principle of bioisosterism and ring
analogy with salicylanilides and quinoline-like compounds, see
Figure 1. Thus in the context of the previously-published re-
sults,5–9,16,17,27,28 primary in vitro screening of the synthesized
compounds was performed against Mycobacterium marinum, Myco-
bacterium kansasii and Mycobacterium smegmatis. A series of 22
compounds was also tested for their inhibitory effect on photosyn-
thetic electron transport in spinach chloroplasts (Spinacia oleracea
L.). As many low-molecular-weight drugs cross biological mem-
branes through passive transport, which strongly depends on their
lipophilicity, the experimental logk values were determined by
means of the HPLC method. The structure–activity relationships
between the chemical structure, physical properties and in vitro
biological activities of all the evaluated compounds are discussed.
Musiol et al.16 where also a phenolic moiety was in the
a-position
of the aromatic ring and a carboxamide group in the b-position.
Different properties of 1-OH and 3-OH moieties resulting in differ-
ent behaviour of the whole molecules within lipophilicity mea-
surement are related to the different position of the b-ring of
naphthalene towards phenolic moieties. Different electron densi-
ties in position 1 or 3 are connected with this fact, resulting in dif-
ferent acidity and reactivity of individual 1-OH and 3-OH
moieties,29–32 therefore the
a-position of the hydroxyl moiety in
the naphthalene scaffold is responsible for stronger intramolecular
interactions than in the case of ring-substituted 3-hydroxynaph-
thalene-2-carboxanilides, and these interactions were not involved
in prediction by the used software.
2. Results and discussion
2.1. Chemistry
When compared calcd logP with experimentally determined
logk (see Table 1 and Fig. 2), the most significant deviations can
be found for nitro and methoxy moieties. Nevertheless it is
important to note that the software was not able to distinguish
lipophilicity values of methyl derivatives 3a–c. 1-Hydroxy-N-(2-
nitrophenyl)naphthalene-2-carboxamide (8a) showed the lowest
lipophilicity, while 1-hydroxy-N-(2-methoxyphenyl)naphthalene-
There are many methods for the preparation of o-hydroxy-car-
boxanilides. The discussed compounds were synthesized by using
modified microwave-assisted synthesis,8,9 thus synthesis of the
1
H
H
N
R
N
H C
N
3
2
R
R
OH
N
O
X
OH
O
H
N
I
III
R
OH
O
O
1
2
H
R
R
1-8c
Y
N
N
H
R
3
OH
OH
O
R
II
IV
Figure 1. Design of compounds 1–8c on the basis of structural analogy/bioisosterism to hydroxyquinolinecarboxamides (I)16 styrylhydroxyquinolines (II)27,28 and
salicylanilides (III and IV).5–7

T. Gonec et al. / Bioorg. Med. Chem. 21 (2013) 6531–6541
6533
of special relevance to pathogenic mycobacteria. M. marinum is
very closely related to the M. tuberculosis and 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.33–35 However, because of M. tuberculosis,
the pathogenic role of nontuberculous mycobacteria (NTM) in hu-
mans was overshadowed for a long time. M. kansasii, the most vir-
ulent of the NTM, causes nontuberculous mycobacterial lung
infections which are very common nowadays and can be indistin-
guishable from tuberculosis.36 That is the reason why M. smegma-
tis, M. marinum and M. kansasii were chosen as model species for
screening of prospective antimycobacterial drugs to control myco-
bacterial diseases.
Most of tested compounds showed the antimycobacterial activ-
ity against the three strains comparable or higher than the stan-
dard isoniazid. Most compounds were active against M. marinum,
but the highest effectivity of the compounds was against M. kans-
asii. The lowest activity of compounds was observed against M.
smegmatis. 1-Hydroxy-N-(3-methylphenyl)naphthalene-2-carbox-
amide (3b) and N-(4-bromophenyl)-1-hydroxynaphthalene-2-car-
boxamide (6c) showed the highest activity against all three tested
strains.
OH
NH
2
OH
O
R
COOH
a
N
H
+
R
R = H (1), OCH (2a-c), CH (3a-c), F (4a-c), Cl (5a-c), Br (6a-c),
3
3
CF (7a-c), NO (8a-c)
3
2
Scheme 1. Synthesis of ring-substituted 1-hydroxynaphthalene-2-carboxanilides
1–8c: (a) PCl3, chlorobenzene, MW.
2-carboxamide (2a) demonstrated the highest lipophilicity accord-
ing to logk data. The influence of R substituents on experimentally
determined
lipophilicity
is
as
follows:
NO2 < F < CF3 -
ꢀ Cl < Br < H < CH3 < OCH3. Within the individual series the lipo-
philicity determined as logk values increases for halogens,
methyl, CF3 and NO2 substituents as follows: ortho < para < meta;
and for methoxy substituent as follows: para < meta < ortho. Simi-
lar unexpected lipophilicity contributions of individual substituent
were described also in refs.9,16 nevertheless these results are prob-
ably caused by the effect of spatially-close phenolic moiety that af-
fects charge distribution within the discussed molecules29–32 and
thus interactions leading to a change in supposed lipo/hydrophilic
properties. Ab initio/DFT calculations of charges in the individual
structures could provide more detailed information, nevertheless
such investigation was not the aim of this paper.
Compounds 4b (R = 3-F), 3a (R = 2-CH3) and 3b (R = 3-CH3)
demonstrated about 16-fold higher activity (MIC ca.
28–29 lmol/L) against M. marinum than isoniazid. From the
data presented in Table 1 it can be concluded that the activity
decreased with a change of the position of the anilide substitu-
ent as follows: meta > ortho > para. Further parameters influenc-
ing activity can involve lipophilicity and electronic properties of
individual substituents. Despite high variance of MIC values, it
seems that the effect against M. marinum increased with higher
lipophilicity but decreased with increasing electron-withdrawing
2.3. Biological activities
The discussed compounds were evaluated for their antimyco-
bacterial activities and inhibition of photosynthetic electron trans-
port (PET) in spinach (Spinacia oleracea L.) chloroplasts, and for the
compounds with the highest effects in vitro cytotoxicity was deter-
mined. The compounds showed a wide range of biological activi-
ties, and some interesting structure–activity relationships were
observed. All the results are summarized in Table 1.
properties (expressed as Hammett’s
substituents, see Table 1.
r parameters) of individual
Compounds 4c (R = 4-F) and 1 (R = H) showed the highest activ-
ity against M. kansasii. The effect of substitution of the position in
the anilide part of the molecule on activity against M. kansasii de-
creased as follows: para > meta > ortho, contrary to the above men-
tioned discussion concerning M. marinum. Lipophilicity seems to
be only a secondary parameter influencing of the activity, never-
theless anti-M. kansasii effect was demonstrated by compounds
with lipophilicity logk ranging from 1.05 to 1.16. A bilinear depen-
2.3.1. In vitro antimycobacterial evaluation
The evaluation of the in vitro antimycobacterial activity of the
compounds was performed against M. marinum CAMP 5644
(MM), M. kansasii DSM 44162 (MK) and M. smegmatis ATCC
700084 (MS), see Table 2. Genus Mycobacterium consists of a clo-
sely related group of fast and slow-growing species. M. tuberculosis
causes one of the most serious human infections, tuberculosis. Dif-
ficulties should be considered while studying M. tuberculosis—
especially a slow growth rate and the requirement of working 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. smegmatis
is an ideal representative of a fast-growing nonpathogenic micro-
organism particularly useful in studying basic cellular processes
dence between activity and electronic Hammett’s
was found after elimination of the least active ortho-derivative
5a (R = 2-Cl), see Figure 3. The optimum of is ca. 0.06. A further
increase of resulted in a linear decrease of activity.
For compounds with
r parameters
r
r
r
varying in the range from ꢁ0.27 to 0.06
the linear correlation between log(1/IC50 [mol/L]) and
r
could be
expressed by the following equation:
A
B
1.6
1.6
3b
2a
1.4
1.4
2b
3c
6b
7c
5b
5c
1
1.2
1.0
0.8
0.6
0.4
1.2
1.0
0.8
0.6
0.4
4b
1
3a
2c
6c
7b
6a
4c
4a
5a
8b
7a
8c
8a
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
logP
logP
Figure 2. Match of experimentally found logk values with calculated logP data of ortho-substituted (A) and meta- and para-substituted derivatives (B).

6534
T. Gonec et al. / Bioorg. Med. Chem. 21 (2013) 6531–6541
logð1=MICÞ ¼ 4:727ðꢂ0:085Þ þ 3:579ðꢂ0:736Þ
r
compounds can interfere with the mycobacterial proton pump F0-
ð1Þ
F1H+ATPase or inhibit biosynthesis of amino acid.14 Kratky et al. re-
cently published an article about the ability of salicylanilide
derivatives to inhibit two essential mycobacterial enzymes: methi-
onine 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.10
r ¼ 0:942; s ¼ 0:150; F ¼ 23:6; n ¼ 5
while for compounds with
tion was found:
r > 0.06 the following correlation equa-
logð1=MICÞ ¼ 4:811ðꢂ0:048Þ ꢁ 0:631ðꢂ0:123Þ
r ¼ 0:902; s ¼ 0:081; F ¼ 26:3; n ¼ 8
r
ð2Þ
Although all the compounds were evaluated for their in vitro
antimycobacterial activity against M. smegmatis ATCC 700084,
most compounds did not show any activity. Only 6c (R = 4-Br)
and 3b (R = 3-CH3) demonstrated about 2.5-fold higher activity
than isoniazid against M. smegmatis. Due to a small number of
effective compounds, a distinct dependence of the activity on the
studied physico-chemical characteristics could not be determined,
nevertheless based on the data from Table 1 the preferred position
of anilide substituents can be determined as follows
para > meta > ortho.
Generally it can be stated that from the tested compounds para-
and meta-substituted derivatives or nonsubstituted compound 1
expressed higher antimycobacterial activity than orto-substituted
ones. This fact is probably caused by a steric effect of a spatially-
close moiety. The proximity of the ortho-substituent to the carbox-
amide group on the aniline ring led to the twist of the aniline ring
plain towards the carboxamide group, that is, to the whole naph-
thalene core. The described process resulted in the planarity viola-
2.3.2. Inhibition of photosynthetic electron transport (PET) in
spinach chloroplasts
The activity of the evaluated 1-hydroxynaphthalene-2-carbox-
anilide derivatives related to inhibition of photosynthetic electron
transport (PET) in spinach (Spinacia oleracea L.) chloroplasts was
medium or relatively high related to the standard, see Table 1. It
was not possible to determine IC50 values for compound 8a
(R = 2-NO2) due to its interaction with 2,6-dichlorophenol-
indophenol (DCPIP). Compound 2a (R = 2-OCH3) expressed the
lowest PET-inhibiting activity (IC50 = 199.6
droxy-N-(3-trifluoromethylphenyl)naphthalene-2-carboxamide
(7b) the highest (IC50 = 5.3 mol/L).
lmol/L), while 1-hy-
l
The PET-inhibiting activity was expressed by negative loga-
rithm of IC50 value (compound concentration in mol/L causing
50% inhibition of PET). Dependences of log(1/IC50) on the lipophil-
icity of compounds expressed as logP (Fig. 4A) as well as logk
(Fig. 4B) and electronic properties of individual anilide substituents
expressed as Hammett’s
r parameters (Fig. 4C) are presented in
tion of the molecule, implying subsequent conjugation of p-bonds
Figure 4. Based on the obtained results it can be stated that deriv-
atives with ortho-substituents showed the lowest PET activity,
while meta-substituted compounds expressed the highest PET-
inhibiting activity.
The biological activity is affected by lipophilicity (see Table 1
and Fig. 4A and B). Ortho-substituted compounds showed quasi-
parabolic dependence with the optimum of lipophilicity logP ca.
5.02 and logk ca. 0.88 (R = 2-Cl, compound 5a). In general, when
inactive anilide 7c (R = 4-CF3) was eliminated (marked by cross)
the dependence of log(1/IC50 [mol/L]) on logP of meta- and para-
substituted compounds was linear, and the correlation could be
expressed by the following equation:
of the phenyl ring over the NH fragment up to the carbonyl
group.37 The result was the different electronic density (charge)
at the carbonyl moiety, which can influence the potential of bind-
ing of the carboxamide group to possible binding sites in a myco-
bacterial cell. In the case of meta- and para-derivatives, the
described secondary steric effect did not manifest. para-Substi-
tuted derivatives should have practically a linear/planar structure
as was, for example, described for a similar type of molecule,
where the X-ray analysis indicated a planar structure.27 Thus, it
seems that the mode of action of this compound type should not
be very different from other planar antimycobacterial agents. The
lipophilicity of meta- and para-substituted derivatives was found
as the factor conducive to the activity of such structures. More
lipophilic structures displayed relatively higher efficiency, and
simultaneously planar structures easier permeate through various
types of membranes.
logð1=IC50Þ ¼ 1:061ðꢂ0:483Þ þ 0:758ðꢂ0:099Þ log P
r ¼ 0:912; s ¼ 0:130; F ¼ 59:0; n ¼ 14
ð3Þ
On the other hand, the corresponding dependence of PET-
inhibiting activity on logk after excluding (marked by cross)
inactive anilides 2c (R = 4-OCH3) and 7c (R = 4-CF3) showed qua-
si-parabolic course (Fig. 4B) with optimum of lipophilicity logk
about 1.13 (7b, R = 3-CF3). However, it could be noted that high
variance of log(1/IC50) values within a relatively narrow range of
logk (from 1.0533 to 1.2849) was observed.
An excellent review by Janin discusses a similar type of carbox-
amides and suggests establishing the hypothesis that all these
5.0
4.8
4.6
4.4
4.2
4.0
3.8
3.6
3.4
3.2
3.0
2.8
The dependence of PET-inhibiting activity on the electronic
properties of individual anilide substituents expressed as
Hammett’s
r parameters, see Figure 4C, showed quasi-parabolic
course for ortho-substituted derivatives with the optimum
r
= 0.22 (R = 2-Cl, 5a).
On the other hand, the dependence of log(1/IC50) on
r showed
to be bilinear for meta- and para-substituted derivatives
(Fig. 4C): it increased with electron-withdrawing substituent
to
A further increase of
For compounds with
the linear correlation between log(1/IC50 [mol/L]) and
expressed by the following equation:
r
= 0.43 (R = 3-CF3, 7b), where the optimum can be found.
r
resulted in a linear decrease of activity.
r
varying in the range from ꢁ0.27 to 0.43
-0.4
-0.2
0.0
0.2
0.4
0.6
para
0.8
1.0
σ
r
could be
H
ortho
meta
Figure 3. Relationships between in vitro antimycobacterial activity against M.
kansasii DSM 44162 log(1/MIC [mol/L]) and N-substituent electronic Hammett’s
parameters of selected studied compounds. (Eliminated compound 5a is marked by
logð1=IC50Þ ¼ 4:608ðꢂ0:048Þ þ 1:280ðꢂ0:183Þ
r ¼ 0:911; s ¼ 0:141; F ¼ 48:7; n ¼ 12
r
r
ð4Þ
cross).

T. Gonec et al. / Bioorg. Med. Chem. 21 (2013) 6531–6541
6535
B 5.4
A
5.4
5.2
5.0
4.8
4.6
4.4
4.2
4.0
3.8
3.6
3.4
3.2
5.2
5.0
4.8
4.6
4.4
4.2
4.0
3.8
3.6
3.4
3.2
4.0
4.5
5.0
5.5
6.0
0.4
0.6
0.8
H
1.0
log k
1.2
1.4
1.6
log P
H
ortho meta para
ortho
meta
para
5.4
C
5.2
5.0
4.8
4.6
4.4
4.2
4.0
3.8
3.6
3.4
-0.4
-0.2
0.0
H
0.2
σ meta
0.4
0.6
0.8
ortho
para
Figure 4. Relationships between PET inhibition log(1/IC50) [mol/L] in spinach chloroplasts and lipophilicity expressed as logP (A) or logk (B) as well as N-substituent
electronic Hammett’s parameters (C) of selected studied compounds. (Eliminated compounds 2c and 7c are marked by cross).
r
The linear correlation between log(1/IC50 [mol/L]) and logP for
12 compounds for which Eq. (4) was found is as follows:
served for 5-bromo- and 3,5-dibromo-2-hydroxy-N-phenylbenza-
mides.40 On the other hand, for PET inhibiting activity of 3- and
4-halogeno substituted benzanilides and thiobenzanilides the total
lipophilicity of the inhibitor was decisive; however the introduc-
tion of the Hammett constants into the correlation equation par-
tially improved the results of statistical analysis.41
logð1=IC50Þ ¼ 0:903ðꢂ0:529Þ þ 0:787ðꢂ0:107Þ log P
r ¼ 0:919; s ¼ 0:134; F ¼ 54:1; n ¼ 12
ð5Þ
The use of both above mentioned descriptors (logP and
r) im-
Because the studied compounds inhibited Hill reaction, they
can be considered to be PS II herbicides. Upon the addition of
diphenylcarbazide (DPC, an artificial electron donor with the
known site of action in the intermediate ZÅ/DÅ on the donor side
of PS II42) to chloroplasts, the activity of which was inhibited by
the studied compounds (up to 10% of the control), practically com-
plete PET restoration (up to 95% of the control) occurred. This indi-
cates that the site of inhibitory action of ring-substituted
1-hydroxynaphthalene-2-carboxanilide derivatives in photosyn-
thetic apparatus is situated only on the donor side of PS II and
the studied compounds did not damage the section between the
photosynthetic centre of PS II (P680) and the secondary quinone
acceptor QB on the acceptor side of PS I.
proved the results of statistical analysis:
logð1=IC50Þ ¼ 2:400ðꢂ0:397Þþ0:463ðꢂ0:083ÞlogP þ0:717ðꢂ0:136Þ
r ¼ 0:981;s ¼ 0:070;F ¼ 113:1;n ¼ 12
r
ð6Þ
Good results of the above presented statistical analyses indi-
cate a significant effect of lipophilicity and the parameter of
the substituent on the biological activity of the tested
compounds.
The bilinear course of the dependence of log(1/IC50) on
r
R
r
indicates that for the PET-inhibiting activity not only sufficient
lipophilicity (enabling easier penetration of compounds into the
lipids of photosynthetic membranes) but also sufficient electro-
negativity of the
R substituent affecting interactions with
The effects of studied compounds on the photosynthetic centers
of chloroplasts were investigated by studying chlorophyll a fluo-
rescence. Figure 5A shows how the chlorophyll a fluorescence
emission band at 686 nm is influenced in the presence of 1-hydro-
xy-N-(2-trifluoromethylphenyl)naphthalene-2-carboxamide (7a).
The decreased intensity of this emission band belonging to the
chlorophyll–protein complexes mainly in photosystem II43 sug-
gested PS II as the site of action of the studied inhibitor. As shown
in Figure 5B, the intensity of the chlorophyll a fluorescence emis-
sion band at 686 nm in the suspension of spinach chloroplasts de-
creased with the increasing concentration of the studied
compounds, and the rate of fluorescence decline correlated with
the effectiveness of PET inhibition, while stronger decline was ob-
photosynthetic proteins is necessary. 1-Hydroxy-N-(3-trifluoro-
methylphenyl)naphthalene-2-carboxamide (7b) was the most
active compound from the series, and this result can indicate that
PET inhibition can be associated with additional interaction of the
trifluoromethyl moiety with photosynthetic proteins.
Lower PET-inhibiting activity of ortho-substituted derivatives
with respect to meta and para substituted ones was observed for
several esters of 2-, 3- and 4-substituted alkoxyphenyl-carbamic
acids.26,38 The lower inhibitory activity of 2-alkoxy substituted
derivatives with respect to their 3- and 4-substituted analogues
can be explained by secondary steric effect, which is induced due
to mutual interactions between the alkoxy substituent and the car-
bamate group (for details see39). The bilinear dependence of the
PET inhibiting activity on the lipophilicity of the compounds as
tained with compound 4b (IC50 = 15.1
pound 1 (IC50 = 31.3 mol/L).
lmol/L) than with com-
l
well as on the Hammett constant, r, of the R substituent was ob-

6536
T. Gonec et al. / Bioorg. Med. Chem. 21 (2013) 6531–6541
2.3.3. In vitro cytotoxicity assay
cytotoxicity against THP-1 cells. It can be concluded that the above
discussed anilides can be considered as promising agents for sub-
sequent design of novel antimycobacterial agents.
The preliminary in vitro 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 2. Com-
4. Experimental
4.1. Chemistry
pounds were not soluble at concentrations above 20
highest concentration prepared in DMSO for the toxicity assay
was 20.0 mol/L. Treatment with 20 mol/L of 1, 3a, 3b, 5b, 5c,
lmol/L; the
l
l
6b, 6c and 7a did not lead to significant lethal effect on THP-1 cells.
Also compounds 4b and 4c demonstrated low toxicity (LD50
All reagents were purchased from Aldrich and Merck. Reactions
were carried out in StartSYNTH microwave labstation (Milestone,
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 Scientific,
USA). The spectra were obtained by accumulation of 256 scans
with 2 cmꢁ1 resolution in the region of 4000–600 cmꢁ1. All 1H
and 13C NMR spectra were recorded on a Bruker Avance III
400 MHz FT-NMR spectrometer (400 MHz for 1H and 100 MHz
for 13C, Bruker Comp., Karlsruhe, Germany). Chemical shifts are re-
ported in ppm (d) using signal of the solvent (DMSO-d6) as the ref-
erence (2.500, resp. 39.50). Mass spectra were measured using a
LTQ Orbitrap Hybrid Mass Spectrometer (Thermo Electron Corpo-
ration, USA) with direct injection into an APCI source (400 °C) in
the positive mode.
>10
were found for compounds with trifluoromethyl moiety located
in meta 7b (LD50 = 1.0 0.11 mol/L) and para 7c (LD50 = 0.6 0.25 -
mol/L) position, which indicates fairly high toxicity (e.g., LD50 of
oxaliplatin and of camptothecin assessed in this line formerly
showed similar values: 1.7 0.64 and 0.16 0.07 mol/L, respec-
lmol/L). Conversely, a significant changes in viability of cells
l
l
l
tively). Based on these observations it can be concluded that all
tested substances, except 7b and 7c, can be considered as nontoxic
agents for subsequent design of novel antimycobacterial agents.
3. Conclusions
A series of 22 ring-substituted 1-hydroxynaphthalene-2-car-
boxanilides were prepared by means of microwave synthesis and
characterized. All the compounds were tested for their ability to in-
hibit photosynthetic electron transport (PET) in spinach chloro-
plasts (Spinacia oleracea L.) and for their in vitro
antimycobacterial activity against M. marinum, M. kasasii and M.
smegmatis. The ortho-substituted compounds showed lower PET
inhibiting activity than the meta- and para-substituted ones, and
1-hydroxy-N-(3-trifluoromethylphenyl)naphthalene-2-carboxam-
ide (7b) was found to be the most efficient PET inhibitor
4.1.1. General procedure for synthesis of N-(substituted
phenyl)-1-hydroxynaphthalene-2-carboxamides 1–8c
1-Hydroxynaphthalene-2-carboxylic acid (5.3 mmol) and the
corresponding substituted aniline (5.3 mmol) were suspended in
30 mL of dry chlorobenzene. 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 res-
idue washed with 2 M HCl, and the crude product was recrystal-
lized from aqueous ethanol. Studied compounds 1–8c are
presented in Table 1.
(IC50 = 5.3
carboxamide (4b) showed the highest biological activity
(MIC = 28.4 mol/L) against M. marinum, N-(4-fluorophenyl)-1-
hydroxynaphthalene-2-carboxamide (4c) showed the highest bio-
logical activity (MIC = 14.2 mol/L) against M. kansasii, and N-(4-
bromophenyl)-1-hydroxynaphthalene-2-carboxamide (6c) ex-
pressed the highest biological activity (MIC = 46.7 mol/L) against
lmol/L). N-(3-Fluorophenyl)-1-hydroxynaphthalene-2-
l
l
l
4.1.2. 1-Hydroxy-N-phenylnaphthalene-2-carboxamide (1)
Yield 78%; mp 142–145 °C (155–156 °C44,45); IR (cmꢁ1): 3422,
3049, 1624, 1597, 1532, 1499, 1465, 1442, 1385, 1298, 1243,
1208, 1134, 1088, 871, 789, 754, 683; 1H NMR (DMSO-d6), d:
7.20 (t, 1H, J = 7.3 Hz), 7.42 (t, 2H, J = 7.9 Hz), 7.47 (d, 1H,
J = 8.8 Hz), 7.58 (ddd, 1H, J = 8.3, 7.0, 1.3 Hz), 7.67 (ddd, 1H,
J = 8.3, 7.0, 1.3 Hz), 7.75 (d, 2H, J = 7.5 Hz), 7.91 (d, 1H, J = 8.3 Hz),
8.14 (d, 1H, J = 8.8 Hz), 8.32 (d, 1H, J = 8.3 Hz), 10.49 (s, 1H),
14.07 (s, 1H); 13C NMR (DMSO-d6), d: 107.51, 117.82, 122.20,
M. smegmatis. This compound and 1-hydroxy-N-(3-methylhe-
nyl)naphthalene-2-carboxamide (3b) were the most active com-
pounds against all three tested strains. Meta- or para-position of
substituents on anilide is essential for biological effect. Further-
more, it can be stated that the dependence of activity on the lipo-
philicity was parabolic or bilinear, and the dependence of activity
on electron properties of individual substituents was bilinear.
The most effective compounds were tested for their in vitro
80
100
A
B
70
90
80
70
60
50
60
50
40
30
20
10
0
620 640 660 680 700 720 740
0.0
0.2
0.4
0.6
0.8
1.0
1.2
λ [nm]
c [mmol dm-3]
Figure 5. Emission fluorescence spectra of chlorophyll a in suspension of spinach chloroplasts without and with 7a (c = 0, 0.1, 0.2, 0.4, 0.8 and 1.2 mmol/L; curves from top to
bottom) (A) and dependence of intensity of chlorophyll a fluorescence emission band at 686 nm in suspension of spinach chloroplasts on concentration of studied compounds
1 (square) and 4b (triangle) (B) (kex = 436 nm).

T. Gonec et al. / Bioorg. Med. Chem. 21 (2013) 6531–6541
6537
123.07, 123.09, 124.68, 124.79, 125.94, 127.50, 128.68, 129.15,
136.00, 137.59, 160.02, 169.51; HR-MS: for C17H13NO2 [M+H]+
calcd 264.1019 m/z, found 264.1022 m/z.
129.17, 129.47, 136.00, 138, 81, 159.42, 159.98, 169.50; HR-MS:
for C18H15NO3 [M+H]+ calcd 294.1125 m/z, found 294.1132 m/z.
4.1.5. 1-Hydroxy-N-(4-methoxyphenyl)naphthalene-2-
carboxamide (2c)
4.1.3. 1-Hydroxy-N-(2-methoxyphenyl)naphthalene-2-
carboxamide (2a)
Yield 78%; mp 126–129 °C (129–130 °C44); IR (cmꢁ1): 3413,
2995, 2948, 2832, 1622, 1597, 1575, 1535, 1505, 1464, 1406,
1384, 1299, 1232, 1209, 1181, 1032, 877, 829, 806, 788, 762,
714; 1H NMR (DMSO-d6), d: 3.77 (s, 3H), 6.98 (d, 2H, J = 9.3 Hz),
7.45 (d, 1H, J = 8.8 Hz), 7.57 (ddd, 1H, J = 8.3, 7.0, 1.3 Hz), 7.63 (d,
2H, J = 9.0 Hz), 7.66 (ddd, 1H, J = 8.3, 7.0, 1.3 Hz), 7.91 (d, 1H,
J = 8.5 Hz), 8.11 (d, 1H, J = 8.8 Hz), 8.30 (d, 1H, J = 8.3 Hz), 10.41
(s, 1H), 14.22 (s, 1H); 13C NMR (DMSO-d6), d: 55.22, 107.40,
113.82, 117.72, 122.95, 123.05, 123.89, 124.69, 125.88, 127.48,
129.04, 130.37, 135.91, 156.42, 159.95, 169.19; HR-MS: for
Yield 73%; mp 159–161 °C (161–162 °C44); IR (cmꢁ1): 3427,
2837, 1622, 1605, 1593, 1538, 1490, 1457, 1390, 1352, 1317,
1289, 1252, 1245, 1209, 1115, 1045, 1026, 804, 786, 758, 740,
718; 1H NMR (DMSO-d6), d: 3.85 (s, 3H), 7.01 (td, 1H, J = 7.6,
1.4 Hz), 7.14 (dd, 1H, J = 8.3, 1.3 Hz), 7.27 (td, 1H, J = 7.5, 1.8 Hz),
7.47 (d, 1H, J = 8.8 Hz), 7.58 (ddd, 1H, J = 8.3, 7.0, 1.3 Hz), 7.67
(ddd, 1H, J = 8.3, 7.0, 1.3 Hz), 7.92 (d, 1H, J = 8.5 Hz), 8.10 (d, 1H,
J = 9.0 Hz), 8.32 (d, 1H, J = 8.3 Hz), 10.27 (s, 1H), 13.91 (s, 1H); 13C
NMR (DMSO-d6), d: 55.72, 108.32, 111.67, 118.22, 120.29, 123.09,
123.41, 124.80, 125.54, 125.91, 126.11, 126.97, 127.58, 128.96,
135.98, 152.66, 159.03, 168.77; HR-MS: for C18H15NO3 [M+H]+
calcd 294.1125 m/z, found 294.1132 m/z.
C
18H15NO3 [M+H]+ calcd 294.1125 m/z, found 294.1131 m/z.
4.1.6. 1-Hydroxy-N-(2-methylphenyl)naphthalene-2-
carboxamide (3a)
4.1.4. 1-Hydroxy-N-(3-methoxyphenyl)naphthalene-2-
carboxamide (2b)
Yield 70%; mp 107–109 °C (89–90 °C44); IR (cmꢁ1): 3338,
3056, 3021, 1640, 1622, 1580, 1524, 1501, 1470, 1413, 1347,
1286, 1212, 952, 919, 815, 761, 745, 720; 1H NMR (DMSO-d6),
d: 2.27 (s, 3H), 7.23–7.30 (m, 2H), 7.32–7.30 (m, 2H), 7.47 (d,
1H, J = 8.8 Hz), 7.58 (ddd, 1H, J = 8.2, 6.8, 1.3 Hz), 7.67 (ddd,
1H, J = 8.2, 6.8, 1.3 Hz), 7.92 (d, 1H, J = 8.0 Hz), 8.11 (d, 1H,
J = 8.8 Hz), 8.29–8.32 (m, 1H), 10.40 (s, 1H), 14.26 (s, 1H); 13C
NMR (DMSO-d6), d: 17.79, 107.16, 117.19, 122.98, 123.05,
124.71, 125.92, 126.24, 126.89, 127.39, 127.53, 129.06, 130.44,
134.49, 135.18, 135.98, 159.99, 169.62; HR-MS: for C18H15NO2
[M+H]+ calcd 278.1176 m/z, found 278.1182 m/z.
Yield 81%; mp 135–139 °C; IR (cmꢁ1): 3430, 1622, 1592, 1575,
1538, 1503, 1447, 1409, 1289, 1273, 1245, 1198, 1168, 1155,
1050, 937, 869, 852, 812, 793, 755, 747, 682; 1H NMR (DMSO-
d6), d: 3.79 (s, 3H), 6.78 (ddd, 1H, J = 8.2, 2.6, 1.0 Hz), 7.31 (t, 1H,
J = 8.0 Hz), 7.38 (ddd, 1H, J = 8.0, 1.9, 1.1 Hz), 7.41 (t, 1H,
J = 2.0 Hz), 7.47 (d, 1H, J = 8.5 Hz), 7.58 (ddd, 1H, J = 8.2, 6.8,
1.3 Hz), 7.67 (ddd, 1H, J = 8.2, 6.8, 1.3 Hz), 7.91 (d, 1H, J = 8.0 Hz),
8.13 (d, 1H, J = 9.0 Hz), 8.31 (dt, 1H, J = 8.3, 0.5 Hz), 10.43 (s, 1H),
14.00 (s, 1H); 13C NMR (DMSO-d6), d: 55.12, 107.56, 107.75,
110.26, 114.21, 117.82, 123.06, 123.09, 124.66, 125.95, 127.51,
Table 1
Structure of discussed ring-substituted 1-hydroxynaphthalene-2-carboxanilides 1–8c, experimentally determined values of lipophilicity logk, calculated values of logP, electronic
Hammett’s 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
r
antimycobacterial activity (MIC) of compounds in comparison with isoniazid (INH) standard; and in vitro cytotoxicity assay (LD50) of chosen compounds. ND = not determined
due to interaction with 2,6-dichlorophenol-indophenol (DCPIP)
Compd
R
logk
logPa
ra
(lmol/L)
PET IC50
MIC
MK
LD50
MM
MS
1
H
1.2302
1.4716
1.2849
1.1882
1.1176
1.4254
1.4008
0.8296
1.0763
1.0533
0.8764
1.1615
1.1314
0.9234
1.1784
1.1224
0.6899
1.1270
1.1158
0.5014
0.6609
0.5298
—
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
31.3
199.6
23.5
79.5
62.8
20.0
28.7
57.0
15.6
20.3
29.4
7.9
10.8
61.9
8.2
9.6
126.1
5.3
60.7
>873
54.5
>873
28.8
28.8
115
56.8
28.4
114
15.2
>873
>873
>873
115
28.8
57.7
>910
28.4
14.2
860
26.8
26.8
>748
>748
23.3
>773
>748
23.3
>830
>830
51.9
—
243
>20
—
—
2a
2b
2c
3a
3b
3c
4a
4b
4c
5a
5b
5c
6a
6b
6c
7a
7b
7c
8a
8b
8c
DCMU
INH
2-OCH3
3-OCH3
4-OCH3
2-CH3
3-CH3
4-CH3
2-F
3-F
4-F
2-Cl
3-Cl
4-Cl
2-Br
3-Br
4-Br
2-CF3
3-CF3
4-CF3
2-NO2
3-NO2
4-NO2
—
ꢁ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
—
>873
>873
>873
231
57.7
923
228
910
>910
860
>860
215
>748
>748
46.7
>773
>748
>748
>830
>830
>830
—
—
>20
>20
—
—
>10
>10
—
>20
>20
—
>20
>20
>20
1.0 0.11
0.6 0.25
—
107
107
215
>748
>748
46.7
96.6
46.7
187
>830
>830
104
36.5
ND
19.9
24.0
1.9
—
—
—
—
—
467
—
—
—
—
—
29.2
117
a
Calculated using ACD/Percepta ver. 2012 (Advanced Chemistry Development, Inc., Toronto, ON, Canada, 2012); MM = M. marinum CAMP 5644, MK = M. kansasii DSM
44162, MS = M. smegmatis ATCC 700084.

6538
T. Gonec et al. / Bioorg. Med. Chem. 21 (2013) 6531–6541
4.1.7. 1-Hydroxy-N-(3-methylphenyl)naphthalene-2-
carboxamide (3b)
(m,2H), 7.46 (d, 1H, J = 8.8 Hz), 7.58 (ddd, 1H, J = 8.3, 7.0, 1.3 Hz),
7.67 (ddd, 1H, J = 8.3, 7.0, 1.3 Hz), 7.73–7.79 (m, 2H), 7.91 (d, 1H,
J = 8.0 Hz), 8.11 (d, 1H, J = 9.0 Hz), 8.31 (d, 1H, J = 8.3 Hz), 10.53
(s, 1H), 14.01 (s, 1H); 13C NMR (DMSO-d6), d: 107.33, 115.33 (d,
J = 22.0 Hz), 117.84, 123.03 (d, J = 11.0 Hz), 124.15, 124.23,
124.65, 125.95, 127.50, 129.15, 133.88 (d, J = 2.2 Hz), 136.00,
158.94 (d, J = 241.4 Hz), 159.92, 169.43; HR-MS: for C17H12NO2F
[M+H]+ calcd 282.0925 m/z, found 282.0929 m/z.
Yield 70%; mp 107–110 °C (118–119 °C44); IR (cmꢁ1): 3441,
1624, 1589, 1542, 1503, 1470, 1412, 1390, 1292, 1244, 1206,
933, 875, 857, 780, 758, 746, 688; 1H NMR (DMSO-d6), d:
2.34 (s, 3H), 7.01 (d, 1H, J = 7.5 Hz), 7.29 (t, 1H, J = 7.9 Hz),
7.46 (d, 1H, J = 8.8 Hz), 7.52–7.60 (m, 3H), 7.67 (ddd, 1H,
J = 8.2, 7.0, 1.3 Hz), 7.91 (d, 1H, J = 8.3 Hz), 8.14 (d, 1H,
J = 9.0 Hz), 8.31 (d, 1H, J = 8.5 Hz), 10.41 (s, 1H), 14.10 (s, 1H);
13C NMR (DMSO-d6), d: 21.13, 107.51, 117.79, 119.32, 122.69,
123.05, 123.08, 124.68, 125.46, 125.90, 127.48, 128.50, 129.11,
135.98, 137.49, 137.90, 160.00, 169.45; HR-MS: for C18H15NO2
[M+H]+ calcd 278.1176 m/z, found 278.1183 m/z.
4.1.12. N-(2-Chlorophenyl)-1-hydroxynaphthalene-2-
carboxamide (5a)
Yield 81%; mp 162–164 °C (140 °C45); IR (cmꢁ1): 3433, 1633,
1589, 1581, 1532, 1502, 1472, 1438, 1414, 1390, 1327, 1297,
1269, 1243, 1207, 1150, 1031, 940, 840, 801, 789, 763, 751, 722,
686; 1H NMR (DMSO-d6), d: 7.34–7.39 (m,1H), 7.44 (td, 1H,
J = 7.7, 1.5 Hz), 7.49 (d, 1H, J = 8.8 Hz), 7.59 (ddd, 1H, J = 8.3, 7.0,
1.3 Hz), 7.61–7.63 (m, 1H), 7.66–7.70 (m, 2H), 7.93 (d, 1H,
J = 8.3 Hz), 8.11 (d, 1H, J = 9.0 Hz), 8.32 (d, 1H, J = 8.3 Hz), 10.70
(s, 1H), 14.01 (s, 1H); 13C NMR (DMSO-d6), d: 107.56, 118.28,
123.12, 123.20, 124.70, 126.02, 127.59, 127.72, 128.19, 128.85,
129.18, 129.68, 129.95, 134.12, 136.09, 159.50, 169.34; HR-MS:
for C17H12NO2Cl [M+H]+ calcd 298.0629 m/z, found 298.0635 m/z.
4.1.8. 1-Hydroxy-N-(4-methylphenyl)naphthalene-2-
carboxamide (3c)
Yield 68%; mp 148–150 °C (155–156 °C44); IR (cmꢁ1): 3441,
1627, 1598, 1577, 1532, 1504, 1469, 1407, 1391, 1304, 1246,
1206, 1087, 864, 813, 800, 781, 755, 723, 716; 1H NMR
(DMSO-d6), d: 2.31 (s, 3H), 7.21 (d, 2H, J = 8.3 Hz), 7.46 (d,
1H, J = 8.8 Hz), 7.58 (ddd, 1H, J = 8.3, 7.0, 1.3 Hz), 7.62 (d, 2H,
J = 8.5 Hz), 7.66 (ddd, 1H, J = 8.3, 7.0, 1.3 Hz), 7.91 (d, 1H,
J = 8.0 Hz), 8.13 (d, 1H, J = 8.8 Hz), 8.31 (d, 1H, J = 8.3 Hz),
10.42 (s, 1H), 14.15 (s, 1H); 13C NMR (DMSO-d6), d: 20.52,
107.47, 117.74, 122.17, 123.01, 123.06, 124.68, 125.89, 127.47,
129.07, 129.08, 133.91, 134.99, 135.95, 159.99, 169.34; HR-
4.1.13. N-(3-Chlorophenyl)-1-hydroxynaphthalene-2-
carboxamide (5b)
C
18H15NO2 [M+H]+ calcd 278.1176 m/z, found
Yield 88%; mp 165–168 °C (198 °C45); IR (cmꢁ1): 3433, 1626,
1591, 1577, 1532, 1465, 1424, 1410, 1387, 1326, 1278, 881, 801,
788, 775, 756, 723, 681; 1H NMR (DMSO-d6), d: 7.25 (ddd, 1H,
J = 8.0, 2.0, 1.0 Hz), 7.44 (t, 1H, J = 8.2 Hz), 7.48 (d, 1H, J = 8.8 Hz),
7.58 (ddd, 1H, J = 8.1, 6.8, 1.4 Hz), 7.67 (ddd, 1H, J = 8.1, 6.8,
1.4 Hz), 7.72 (ddd, 1H, J = 8.2, 2.0, 0.9 Hz), 7.90–7.93 (m, 2H),
8.10 (d, 1H, J = 9.0 Hz), 8.31 (d, 1H, J = 8.3 Hz), 10.57 (s, 1H),
13.77 (s, 1H); 13C NMR (DMSO-d6), d: 107.47, 117.96, 120.26,
121.40, 123.03, 123.12, 124.37, 124.62, 126.02, 127.52, 129.28,
130.36, 132.94, 136.07, 139.22, 159.97, 169.62; HR-MS: for C17H12-
NO2Cl [M+H]+ calcd 298.0629 m/z, found 298.0634 m/z.
MS: for
278.1182 m/z.
4.1.9. N-(2-Fluorophenyl)-1-hydroxynaphthalene-2-
carboxamide (4a)
Yield 77%; mp 114–117 °C; IR (cmꢁ1): 3456, 1631, 1616, 1593,
1538, 1483, 1455, 1390, 1334, 1307, 1251, 1243, 1181, 1149,
1099, 825, 788, 750, 718; 1H NMR (DMSO-d6), d: 7.26–7.30
(m,1H), 7.35–7.38 (m, 2H), 7.48 (d, 1H, J = 8.8 Hz), 7.57–7.62 (m,
2H), 7.68 (ddd, 1H, J = 8.3, 7.0, 1.3 Hz), 7.92 (d, 1H, J = 8.3 Hz),
8.10 (d, 1H, J = 8.8 Hz), 8.32 (d, 1H, J = 8.3 Hz), 10.60 (s, 1H),
13.91 (s, 1H); 13C NMR (DMSO-d6), d: 107.20, 115.99 (d,
J = 19.8 Hz), 118.15, 123.11, 123.10 (d, J = 2.2 Hz), 124.40 (d,
J = 12.5 Hz), 124.52 (d, J = 3.7 Hz), 124.63, 126.00, 127.56, 128.04
(d, J = 4.0 Hz), 128.08, 129.20, 126.08, 156.39 (d, J = 247.2 Hz),
4.1.14. N-(4-Chlorophenyl)-1-hydroxynaphthalene-2-
carboxamide (5c)
Yield 75%; mp 165–170 °C (175 °C,45 179 °C46); IR (cmꢁ1): 3440,
1626, 1595, 1575, 1528, 1490, 1469, 1391, 1300, 1244, 1208, 1086,
820, 794, 782, 757, 723; 1H NMR (DMSO-d6), d: 7.47 (d, 3H,
J = 8.8 Hz), 7.58 (ddd, 1H, J = 8.3, 7.0, 1.3 Hz), 7.67 (ddd, 1H,
J = 8.3, 7.0, 1.3 Hz), 7.79 (d, 2H, J = 8.8 Hz), 7.91 (d, 1H, J = 8.0 Hz),
8.11 (d, 1H, J = 8.8 Hz), 8.31 (d, 1H, J = 8.3 Hz), 10.56 (s, 1H),
13.89 (s, 1H); 13C NMR (DMSO-d6), d: 107.43, 117.88, 123.01,
123.09, 123.55, 124.63, 125.97, 127.49, 128.49, 128.58, 129.21,
136.02, 136.63, 159.97, 169.50; HR-MS: for C17H12NO2Cl [M+H]+
calcd 298.0629 m/z, found 298.0632 m/z.
159.77, 169.54; HR-MS: for
C
17H12NO2F [M+H]+ calcd
282.0925 m/z, found 282.0929 m/z.
4.1.10. N-(3-Fluorophenyl)-1-hydroxynaphthalene-2-
carboxamide (4b)
Yield 69%; mp 169–172 °C; IR (cmꢁ1): 3431, 1644, 1627, 1606,
1578, 1538, 1465, 1446, 1413, 1386, 1327, 1283, 1244, 1147,
968, 871, 800, 787, 776, 751, 682; 1H NMR (DMSO-d6), d: 7.03
(ddd, 1H, J = 8.5, 2.5, 0.8 Hz), 7.42–7.47 (m, 1H), 7.48 (d, 1H,
J = 8.8 Hz), 7.58 (ddd, 1H, J = 8.2, 7.0, 1.3 Hz), 7.58–7.61 (m, 1H),
7.67 (ddd, 1H, J = 8.2, 7.0, 1.3 Hz), 7.71 (dt, 1H, J = 11.5, 2.5 Hz),
7.91 (d, 1H, J = 8.0 Hz), 8.10 (d, 1H, J = 8.8 Hz), 8.32 (d, 1H,
J = 8.5 Hz), 10.59 (s, 1H), 13.78 (s, 1H); 13C NMR (DMSO-d6), d:
107.51, 108.68 (d, J = 25.7 Hz), 111.16 (d, J = 21.3 Hz), 117.57 (d,
J = 2.9 Hz), 117.92, 123.03, 123.10, 124.62, 125.99, 127.50,
129.25, 130.27 (d, J = 9.5 Hz), 136.05, 139.47 (d, J = 11.0 Hz),
159.94, 161.95 (d, J = 241.3 Hz), 169.61; HR-MS: for C17H12NO2F
[M+H]+ calcd 282.0925 m/z, found 282.0929 m/z.
4.1.15. N-(2-Bromophenyl)-1-hydroxynaphthalene-2-
carboxamide (6a)
Yield 76%; mp 175–178 °C; IR (cmꢁ1): 3418, 1634, 1593, 1575,
1532, 1502, 1434, 1411, 1390, 1328, 1298, 1243, 1208, 1150,
1019, 839, 799, 789, 763, 750, 721; 1H NMR (DMSO-d6), d: 7.30
(td, 1H, J = 7.7, 1.6 Hz), 7.48 (td, 1H, J = 7.8, 1.3 Hz), 7.49 (d, 1H,
J = 8.8 Hz), 7.59 (ddd, 1H, J = 8.3, 7.0, 1.3 Hz), 7.64 (dd, 1H, J = 7.8,
1.5 Hz), 7.68 (ddd, 1H, J = 8.3, 7.0, 1.3 Hz), 7.77 (dd, 1H, J = 8.0,
1.5 Hz), 7.92 (d, 1H, J = 8.0 Hz), 8.11 (d, 1H, J = 8.8 Hz), 8.32 (d,
1H, J = 8.3 Hz), 10.66 (s, 1H), 13.90 (s, 1H); 13C NMR (DMSO-d6),
d: 107.41, 118.20, 121.03, 123.09, 123.10, 124.68, 126.00, 127.58,
128.32, 128.64, 129.17, 129.31, 132.80, 135.58, 136.07, 159.65,
169.42; HR-MS: for C17H12NO2Br [M+H]+ calcd 342.0124 m/z,
found 342.0127 m/z.
4.1.11. N-(4-Fluorophenyl)-1-hydroxynaphthalene-2-
carboxamide (4c)
Yield 61%; mp 166–169 °C; IR (cmꢁ1): 3430, 1626, 1612, 1594,
1538, 1504, 1467, 1408, 1386, 1306, 1238, 1205, 1144, 1103,
1083, 855, 832, 800, 786, 752; 1H NMR (DMSO-d6), d: 7.22–7.29

T. Gonec et al. / Bioorg. Med. Chem. 21 (2013) 6531–6541
6539
4.1.16. N-(3-Bromophenyl)-1-hydroxynaphthalene-2-
carboxamide (6b)
107.54, 118.00, 121.72, 123.12, 123.13, 124.31 (q, J = 271.4 Hz),
124.54 (q, J = 31.5 Hz), 124.60, 125.91 (q, J = 3.7 Hz), 126.04,
127.53, 129.34, 136.12, 141.49 (q, J = 1.5 Hz), 160.02, 169.83; HR-
MS: for
332.0899 m/z.
Yield 66%; mp 166–170 °C; IR (cmꢁ1): 3433, 1625, 1589, 1575,
1526, 1464, 1409, 1387, 1322, 1276, 998, 882, 852, 803, 788,
773, 759, 722, 680; 1H NMR (DMSO-d6), d: 7.37–7.39 (m, 2H),
7.47 (d, 1H, J = 8.8 Hz), 7.58 (ddd, 1H, J = 8.3, 7.0, 1.3 Hz), 7.67
(ddd, 1H, J = 8.3, 7.0, 1.3 Hz), 7.77 (ddd, 1H, J = 5.5, 3.6, 2.0 Hz),
7.91 (d, 1H, J = 8.0 Hz), 8.06 (t, 1H, J = 1.3 Hz), 8.09 (d, 1H,
J = 9.0 Hz), 8.31 (d, 1H, J = 8.3 Hz), 10.55 (s, 1H), 13.77 (s, 1H); 13C
NMR (DMSO-d6), d: 107.45, 117.95, 120.64, 121.34, 123.01,
123.11, 124.22, 124.61, 126.00, 127.25, 127.51, 129.27, 130.64,
136.06, 139.35, 159.97, 169.58; HR-MS: for C17H12NO2Br [M+H]+
calcd 342.0124 m/z, found 342.0128 m/z.
C
18H12NO2F3 [M+H]+ calcd 332.0893 m/z, found
4.1.21. 1-Hydroxy-N-(2-nitrophenyl)naphthalene-2-
carboxamide (8a)
Yield 74%; mp 198–200 °C (190 °C47); IR (cmꢁ1): 3405, 1630,
1602, 1540, 1396, 1330, 1318, 1290, 1270, 1248, 841, 809, 790,
762, 757, 678; 1H NMR (DMSO-d6), d: 7.50-7.55 (m, 2H), 7.61
(ddd, 1H, J = 8.3, 7.0, 1.3 Hz), 7.70 (ddd, 1H, J = 8.3, 7.0, 1.3 Hz),
7.80–7.87 (m, 2H), 7.94 (d, 1H, J = 8.0 Hz), 8.04 (d, 2H, J = 9.3 Hz),
8.09 (d, 1H, J = 8.3 Hz), 8.32 (d, 1H, J = 8.5 Hz), 11.16 (s, 1H),
13.40 (s, 1H); 13C NMR (DMSO-d6), d: 107.63, 118.07, 123.17,
124.56, 124.62, 124.85, 125.73, 125.93, 126.09, 127.57, 129.70,
131.00, 133.12, 136.18, 143.06, 160.06, 169.85; HR-MS: for
4.1.17. N-(4-Bromophenyl)-1-hydroxynaphthalene-2-
carboxamide (6c)
Yield 81%; mp 167–171 °C (188 °C45); IR (cmꢁ1): 3443, 1630,
1595, 1574, 1527, 1505, 1488, 1467, 1391, 1300, 1244, 1073,
818, 792, 782, 757, 722; 1H NMR (DMSO-d6), d: 7.47 (d, 1H,
J = 8.8 Hz), 7.58 (ddd, 1H, J = 8.3, 7.0, 1.3 Hz), 7.59 (d, 2H,
J = 8.8 Hz), 7.67 (ddd, 1H, J = 8.3, 7.0, 1.3 Hz), 7.74 (d, 2H,
J = 8.8 Hz), 7.91 (d, 1H, J = 8.3 Hz), 8.10 (d, 1H, J = 8.8 Hz), 8.31 (d,
1H, J = 8.3 Hz), 10.55 (s, 1H), 13.88 (s, 1H); 13C NMR (DMSO-d6),
d: 107.45, 116.65, 117.89, 123.01, 123.09, 123.87, 124.62, 125.97,
127.49, 129.22, 131.51, 136.02, 137.08, 159.97, 169.49; HR-MS:
for C17H12NO2Br [M+H]+ calcd 342.0124 m/z, found 342.0127 m/z.
C
17H12N2O4 [M+H]+ calcd 309.0870 m/z, found 309.0873 m/z.
4.1.22. 1-Hydroxy-N-(3-nitrophenyl)naphthalene-2-
carboxamide (8b)
Yield 74%; mp 252–255 °C; IR (cmꢁ1): 3402, 1628, 1601, 1549,
1528, 1390, 1345, 1321, 1284, 1249, 891, 836, 809, 790, 765,
732, 722, 664; 1H NMR (DMSO-d6), d: 7.48 (d, 1H, J = 8.8 Hz),
7.57 (ddd, 1H, J = 8.3, 7.0, 1.3 Hz), 7.67 (ddd, 1H, J = 8.3, 7.0,
1.3 Hz), 7.68 (t, 1H, J = 8.3 Hz), 7.90 (d, 1H, J = 8.03 Hz), 8.01 (ddd,
1H, J = 8.3, 2.3, 0.8 Hz), 8.10 (d, 1H, J = 9.0 Hz), 8.21 (ddd, 1H,
J = 8.0, 2.3, 0.8 Hz), 8.30 (d, 1H, J = 8.3 Hz), 8.73 (t, 1H, J = 2.1 Hz),
10.80 (s, 1H), 13.62 (s, 1H); 13C NMR (DMSO-d6), d: 107.39,
115.83, 118.05, 118.97, 123.00, 123.15, 124.59, 126.05, 127.52,
127.56, 129.37, 130.06, 136.13, 139.04, 147.80, 160.03, 169.82;
4.1.18. 1-Hydroxy-N-(2-trifluoromethylphenyl)naphthalene-2-
carboxamide (7a)
Yield 66%; mp 116–119 °C; IR (cmꢁ1): 3478, 1643, 1634, 1613,
1588, 1538, 1505, 1450, 1391, 1330, 1280, 1249, 1208, 1153,
1097, 1087, 1031, 800, 790, 760, 720; 1H NMR (DMSO-d6), d:
7.49 (d, 1H, J = 8.8 Hz), 7.57–7.62 (m, 2H), 7.66–7.70 (m, 2H),
7.78–7.82 (m, 1H), 7.86 (d, 1H, J = 7.8 Hz), 7.93 (d, 1H, J = 8.3 Hz),
8.08 (d, 1H, J = 8.8 Hz), 8.31 (d, 1H, J = 8.3 Hz), 10.70 (s, 1H),
13.87 (s, 1H); 13C NMR (DMSO-d6), d: 107.18, 118.20, 123.03,
123.08, 123.54 (q, J = 272.9 Hz), 124.63, 126.00, 126.50 (q,
J = 29.3 Hz), 126.64 (q, J = 4.4 Hz), 127.56, 128.02, 129.19, 131.20,
133.35, 134.64 (q, J = 1.5 Hz), 136.08, 159.71, 170.41; HR-MS: for
HR-MS: for
C
17H12N2O4 [M+H]+ calcd 309.0870 m/z, found
309.0873 m/z.
4.1.23. 1-Hydroxy-N-(4-nitrophenyl)naphthalene-2-
carboxamide (8c)
Yield 67%; mp 245–248 °C (225 °C47); IR (cmꢁ1): 3405, 1638,
1610, 1538, 1503, 1487, 1468, 1408, 1391, 1325, 1291, 1269,
1248, 1207, 1178, 1144, 1109, 1088, 844, 808, 789, 749, 684; 1H
NMR (DMSO-d6), d: 7.48 (d, 1H, J = 9.0 Hz), 7.57 (ddd, 1H, J = 8.3,
7.0, 1.3 Hz), 7.67 (ddd, 1H, J = 8.3, 7.0, 1.3 Hz), 7.90 (d, 1H,
J = 8.0 Hz), 8.05 (d, 2H, J = 9.0 Hz), 8.10 (d, 1H, J = 8.8 Hz), 8.27 (d,
2H, J = 9.0 Hz), 8.30 (d, 1H, J = 8.5 Hz), 10.85 (s, 1H), 13.49 (s,
1H); 13C NMR (DMSO-d6), d: 107.67, 118.09, 121.19, 123.17,
124.56, 124.60, 124.61, 126.07, 127.53, 129.44, 136.18, 143.02,
144.20, 160.03, 169.90; HR-MS: for C17H12N2O4 [M+H]+ calcd
309.0870 m/z, found 309.0871 m/z.
C
18H12NO2F3 [M+H]+ calcd 332.0893 m/z, found 332.0899 m/z.
4.1.19. 1-Hydroxy-N-(3-trifluoromethylphenyl)naphthalene-2-
carboxamide (7b)
Yield 60%; mp 156–158 °C (166 °C46); IR (cmꢁ1): 3412, 1626,
1596, 1553, 1445, 1410, 1337, 1315, 1290, 1267, 1246, 1159,
1148, 1110, 1101, 1067, 891, 867, 806, 800, 785, 752, 710, 694;
1H NMR (DMSO-d6), d: 7.49 (d, 1H, J = 8.8 Hz), 7.54 (d, 1H,
J = 7.8 Hz), 7.59 (ddd, 1H, J = 8.3, 7.0, 1.3 Hz), 7.65 (t, 1H,
J = 8.3 Hz), 7.68 (ddd, 1H, J = 8.3, 7.0, 1.3 Hz), 7.92 (d, 1H,
J = 8.0 Hz), 8.07 (d, 1H, J = 8.3 Hz), 8.12 (d, 1H, J = 8.8 Hz), 8.19 (s,
1H), 8.32 (d, 1H, J = 8.0 Hz), 10.71 (s, 1H), 13.71 (s, 1H); 13C NMR
(DMSO-d6), d: 107.46, 118.00, 118.02 (q, J = 3.7 Hz), 120.95 (q,
J = 3.7 Hz), 123.03, 123.13, 124.10 (q, J = 270.8 Hz), 124.62,
125.43, 126.04, 127.53, 129.31, 129.39 (q, J = 31.6 Hz), 129.94,
136.09, 138.59, 159.98, 169.74; HR-MS: for C18H12NO2F3 [M+H]+
calcd 332.0893 m/z, found 332.0900 m/z.
4.2. Lipophilicity determination using HPLC (capacity factor k/
calculated logk)
A HPLC system Agilent 1200 equipped with DAD detector (Agi-
lent, USA) was used. A chromatographic column SymmetryÒ C18
5
l
m, 4.6 ꢃ 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,
USA). Isocratic elution by a mixture of MeOH pa (60%) and H2O-
HPLC Mili-Q grade (40%) as a mobile phase was used. The total flow
4.1.20. 1-Hydroxy-N-(4-trifluoromethylphenyl)naphthalene-2-
carboxamide (7c)
of the column was 1.0 mL/min, injection 20 lL, column tempera-
Yield 70%; mp 204–205 °C; IR (cmꢁ1): 3320, 1621, 1596, 1572,
1529, 1459, 1409, 1324, 1295, 1252, 1209, 1147, 1108, 1066,
1015, 940, 845, 832, 791, 764; 1H NMR (DMSO-d6), d: 7.48 (d,
1H, J = 8.8 Hz), 7.58 (ddd, 1H, J = 8.3, 7.0, 1.3 Hz), 7.68 (ddd, 1H,
J = 8.3, 7.0, 1.3 Hz), 7.77 (d, 2H, J = 8.8 Hz), 7.92 (d, 1H, J = 8.0 Hz),
8.00 (d, 2H, J = 8.8 Hz), 8.12 (d, 1H, J = 9.0 Hz), 8.32 (d, 1H,
J = 8.0 Hz), 10.76 (s, 1H), 13.83 (s, 1H); 13C NMR (DMSO-d6), d:
ture 40 °C, and sample temperature 10 °C. The detection wave-
length 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. logk, calculated from the capacity factor k, is

6540
T. Gonec et al. / Bioorg. Med. Chem. 21 (2013) 6531–6541
used as the lipophilicity index converted to logP scale. The logk
values of individual compounds are shown in Table 1.
excited at 436 nm using a slit width of 10 nm and were kept in the
dark for 2 min prior to the measurement. Due to low aqueous sol-
ubility the compounds were added to a chloroplast suspension in
DMSO solution. The DMSO concentration in all samples was the
same as in the control (10%).
4.3. In vitro antimycobacterial evaluation
The evaluation of in vitro antimycobacterial activity of the com-
pounds was performed against M. marinum CAMP 5644, M. kansasii
DSM 44162 and M. smegmatis ATCC 700084. The broth dilution mi-
cro-method in Middlebrook 7H9 medium (Difco, USA) supple-
mented with ADC Enrichment (BBL, USA) was used to determine
the minimum inhibitory concentration (MIC) as previously
described.48 The compounds were dissolved in DMSO (Sigma, Ger-
many), and the final concentration of DMSO did not exceed 2.5% of
the total solution composition. The final concentrations of the eval-
4.5. 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 cul-
tured in RPMI 1640 (Lonza, Verviers, Belgium) medium supple-
mented with 10% fetal bovine serum (FBS, Sigma–Aldrich, St.
uated compounds ranging from 256 to 0.125
lg/mL were obtained
Louis, MO, USA), 2% L-glutamine, 1% penicillin and streptomycin
by two-fold serial dilution of the stock solution in microtiter plate
with sterile medium. Isoniazid (Sigma, Germany) was used as ref-
erence 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, 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. The determination of results was per-
formed visually after 3 days of static incubation in the darkness at
37 °C in an aerobic atmosphere for M. smegmatis, 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 minimum
inhibitory concentrations (MICs) were defined as the lowest con-
centration of the compound at which no visible bacterial growth
was observed. The MIC value is routinely and widely used in bac-
terial assays and is a standard detection limit according to the Clin-
ical and Laboratory Standards Institute (CLSI, www.clsi.org). The
results are summarized in Table 1.
(Lonza) 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 dis-
solved in DMSO (Sigma–Aldrich) and added in five increasing con-
centrations 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
using
l
mol/L in RPMI 1640 medium. Cell toxicity was determined
a
Cytotoxicity Detection KitPLUS Lactate dehydrogenase
(LDH) assay kit (Roche Diagnostics, Mannheim, Germany) accord-
ing to the manufacturer’s instructions, as described previously.18
For LDH assays, cells were seeded into 96-well plates (5 ꢃ 104 -
cellsꢄwellꢁ1 in 100
lL culture medium) in triplicate in serum-free
RPMI 1640 medium, and measurements at 492 nm wavelength
(Synergy
2 Multi-Mode Microplate Reader, BioTek, Vermont,
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.
LDH Test Principle: The LDH activity is determined by a coupled
enzymatic reaction, whereby the tetrazolium salt INT is reduced to
formazan. An increase in the amount of dead or plasma membrane
damaged cells results in an increase of LDH enzyme activity in the
culture supernatant. This increase in the amount of enzyme activ-
ity in the supernatant directly correlates to the amount of forma-
zan formed during a limited time period. The formazan dye
formed is water soluble and shows a broad absorption maximum
at approximately 500 nm.
4.4. Study of inhibition photosynthetic electron transport (PET)
in spinach chloroplasts
Chloroplasts were prepared from spinach (Spinacia oleracea L.)
according to Masarovicova and Kralova.49 The inhibition of photo-
synthetic electron transport (PET) in spinach chloroplasts was
determined spectrophotometrically (Genesys 6, Thermo Scientific,
USA), using an artificial electron acceptor 2,6-dichlorophenol–
indophenol (DCIPP) according to Kralova et al.50 and the rate of
photosynthetic electron transport was monitored as photoreduc-
tion 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 irradi-
ated (ꢀ100 W/m2 with 10 cm distance) with a halogen lamp
(250 W) using a 4 cm water filter to prevent warming of the sam-
ples (suspension temperature 22 °C). The studied compounds were
dissolved in DMSO due to their limited water solubility. The ap-
plied DMSO concentration (up to 4%) did not affect the photochem-
ical activity in spinach chloroplasts. The inhibitory efficiency of the
studied compounds was expressed by IC50 values, that is, by molar
concentration of the compounds causing 50% decrease in the oxy-
gen evolution rate relative to the untreated control. The compara-
ble IC50 value for a selective herbicide 3-(3,4-dichlorophenyl)-1,1-
Acknowledgments
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-0416-10, and by Sanofi-Aventis Pharma
Slovakia.
References and notes
1. Koul, A.; Arnoult, E.; Lounis, N.; Guillemont, J.; Andries, K. Nature 2011, 469,
483.
2. World Health Organization Global Tuberculosis Report 2012; WHO Press:
Geneva, Switzerland, 2012.
3. Wagner, D.; Young, L. S. Infection 2004, 32, 257.
4. Koreyoshi, S.; Masato, M.; Tetsuji, I. (Yoshitomi Pharmaceutical Industries).
Hydroxynaphthoic acid anilides, microbicides and slimicides. JP Pat. JP 52 076
430, 1977.
dimethylurea, DCMU (DiuroneÒ) was about 1.9
are summarized in Table 1.
lmol/L. The results
5. Imramovsky, A.; Vinsova, J.; Monreal-Ferriz, J.; Dolezal, R.; Jampilek, J.;
Kaustova, J.; Kunc, F. Bioorg. Med. Chem. 2009, 17, 3572.
6. Otevrel, J.; Mandelova, Z.; Pesko, M.; Guo, J.; Kralova, K.; Sersen, F.; Vejsova, M.;
Kalinowski, D.; Kovacevic, Z.; Coffey, A.; Csollei, J.; Richardson, D. R.; Jampilek, J.
Molecules 2010, 15, 8122.
The emission fluorescence spectra were recorded on a fluores-
cence spectrophotometer F-2000 (Hitachi, Tokyo, Japan) at room
temperature (24 °C). The samples of chloroplast suspension
(10 mg chlorophyll/L) with and without the studied inhibitor were

T. Gonec et al. / Bioorg. Med. Chem. 21 (2013) 6531–6541
6541
7. Imramovsky, A.; Pesko, M.; Kralova, K.; Vejsova, M.; Stolarikova, J.; Vinsova, J.;
Jampilek, J. Molecules 2011, 16, 2414.
28. Cieslik, W.; Musiol, R.; Nycz, J.; Jampilek, J.; Vejsova, M.; Wolff, M.; Machura, B.;
Polanski, J. Bioorg. Med. Chem. 2012, 20, 6960.
8. 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. Molecules
2013, 18, 7977.
9. Gonec, T.; Kos, J.; Zadrazilova, I.; Pesko, M.; Govender, R.; Keltosova, S.;
Chambel, B.; Pereira, D.; Kollar, P.; Imramovsky, A.; O’Mahony, J.; Coffey, A.;
Cizek, A.; Kralova, K.; Jampilek, J. Molecules 2013, 18, 9397.
10. Kratky, M.; Vinsova, J.; Novotna, E.; Mandikova, J.; Wsol, V.; Trejtnar, F.;
Ulmann, V.; Stolarikova, J.; Fernandes, S.; Bhat, S.; Liu, J. O. Tuberculosis 2012,
92, 434.
29. Fedorov, I. A.; Zhuravlev, Y. N.; Berveno, V. P. Phys. Chem. Chem. Phys. 2011, 13,
5679.
30. Harris, C. M.; Selinger, B. K. J. Phys. Chem. 1980, 84, 1366.
31. Boyer, R.; Deckey, G.; Marzzacco, C.; Mulvaney, M.; Schwab, C.; Halpern, A. M. J.
Chem. Educ. 1985, 62, 630.
32. De Vleeschouwer, F.; Yang, W.; Beratan, D. N.; Geerlings, P.; De Proft, F. Phys.
Chem. Chem. Phys. 2012, 14, 16002.
33. Acharya, N.; Varshney, U. J. Mol. Biol. 2002, 318, 1251.
34. Broussard, G. W.; Ennis, D. G. Comp. Biochem. Phys. C 2007, 145, 45.
35. Valente, W. J.; Pienaar, E.; Fast, A.; Fluitt, A.; Whitney, S. E.; Fenton, R.
J.; Barletta, R. G.; Chacon, O.; Viljoen, H. J. Chem. Eng. Sci. 2009, 64,
1944.
11. Kamatb, S.; Buolamwini, J. K. Med. Res. Rev. 2006, 26, 569.
12. Liechti, C.; Sequin, U.; Bold, G.; Furet, P.; Meyer, T.; Traxler, P. Eur. J. Med. Chem.
2004, 39, 11.
13. Brown, M. E.; Fitzner, J. N.; Stevens, T.; Chin, W.; Wright, C. D.; Boyce, J. P.
Bioorg. Med. Chem. 2008, 16, 8760.
36. Matveychuk, A.; Fuks, L.; Priess, R.; Hahim, I.; Shitrit, D. Resp. Med. 2012, 106,
1472.
14. Janin, Y. L. Bioorg. Med. Chem. 2007, 15, 2479.
15. Good, N. E. Plant Physiol. 1961, 36, 788.
37. Malik, I.; Bukovsky, M.; Andriamainty, F.; Galisinova, J. Br. J. Microbiol. 2012, 43,
959.
16. Musiol, R.; Tabak, D.; Niedbala, H.; Podeszwa, B.; Jampilek, J.; Kralova, K.;
Dohnal, J.; Finster, J.; Mencel, A.; Polanski, J. Bioorg. Med. Chem. 2008, 16, 4490.
17. Gonec, T.; Bobal, P.; Sujan, J.; Pesko, M.; Guo, J.; Kralova, K.; Pavlacka, L.; Vesely,
L.; Kreckova, E.; Kos, J.; Coffey, A.; Kollar, P.; Imramovsky, A.; Placek, L.;
Jampilek, J. Molecules 2012, 17, 613.
18. Fajkusova, D.; Pesko, M.; Keltosova, S.; Guo, J.; Oktabec, Z.; Vejsova, M.; Kollar,
P.; Coffey, A.; Csollei, J.; Kralova, K.; Jampilek, J. Bioorg. Med. Chem. 2012, 20,
7059.
38. Kralova, K.; Sersen, F.; Cizmarik, J. Chem. Pap. 1992, 46, 266.
39. Cizmarik, J.; Borovansky, A.; Svec, P. Acta Fac. Pharm. Univ. Comenianae 1976,
29, 53.
40. Kralova, K.; Sersen, F.; Pesko, M.; Waisser, K.; Kubicová, L. Chem. Pap. 2013.
http://dx.doi.org/10.2478/s11696-013-0416-7. in press.
41. Kralova, K.; Sersen, F.; Kubicova, L.; Waisser, K. J. Trace Microprobe Technol.
2000, 18, 251.
42. Izawa, S. Acceptors and Donors for Chloroplast Electron Transport In Methods in
Enzymology; Part, C., Colowick, P., Kaplan, N. O., Eds.; Academic Press: New
York, London, UK, 1980; Vol. 69, pp 413–434.
19. Draber, W.; Tietjen, K.; Kluth, J. F.; Trebst, A. Angew. Chem., Int. Ed. Engl. 1991, 3,
1621.
20. Tischer, W.; Strotmann, H. Biochim. Biophys. Acta 1977, 460, 113.
21. 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.
43. Govindjee, A. Aus. J. Plant Physiol. 1995, 22, 131.
44. Jadhav, G. V.; Rao, S. N.; Hirwe, N. W. Chem. Zentralbl. 1938, 109, 65.
45. Tripathy, H.; Pradhan, D. G.; Dash, B. C.; Mahapatra, G. M. Agric. Biol. Chem.
1973, 37, 1375.
22. 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; Vol. 10, pp 27–85.
23. Shaner, D. L. Pest Manage. Sci. 2004, 60, 17.
24. Delaney, J.; Clarke, E.; Hughes, D.; Rice, M. Drug Discovery Today 2006, 11, 839.
25. Duke, S. O. Weed Sci. 2010, 58, 334.
26. Kralova, K.; Sersen, F.; Cizmarik, J. Gen. Physiol. Biophys. 1992, 11, 261.
27. Musiol, R.; Jampilek, J.; Buchta, V.; Silva, L.; Niedbala, H.; Podeszwa, B.; Palka,
A.; Majerz-Maniecka, K.; Oleksyn, B.; Polanski, J. Bioorg. Med. Chem. 2006, 14,
3592.
46. van Allan, J. A. J. Org. Chem. 1954, 19, 56.
47. Dubey, S. K.; Singh, A. K.; Singh, S.; Sharma, S.; Iyer, R. N. J. Med. Chem. 1978, 21,
1178.
48. Schwalbe, R.; Steele-Moore, L.; Goodwin, A. C. Antimicrobial Susceptibility
Testing Protocols; CRC Press: Boca Raton, FL, USA, 2007.
49. Masarovicova, E.; Kralova, K. Approaches to Measuring Plant Photosynthesis
Activity. In Handbook of Photosynthesis; Pessarakli, M., Ed., 2nd ed.; Taylor &
Francis Group: Boca Raton, FL, USA, 2005; pp 617–656.
50. Kralova, K.; Sersen, F.; Sidoova, E. Chem. Pap. 1992, 46, 348.