Antimycobacterial N-alkoxyphenylhydroxynaphthalenecarboxamides affecting photosystem II

Article abstract of DOI:10.1016/j.bmcl.2017.03.050

N-(Alkoxyphenyl)-2-hydroxynaphthalene-1-carboxamides (series A) and N-(alkoxyphenyl)-1-hydroxynaphthalene-2-carboxamides (series B) affecting photosystem (PS) II inhibited photosynthetic electron transport (PET) in spinach chloroplasts. Their inhibitory activity depended on the compound lipophilicity as well as on the position of the alkoxy substituent. The most potent PET inhibitors were 2-hydroxy-N-phenylnaphthalene-1-carboxamide and N-[3-(but-2-yloxy)phenyl]-2-hydroxynaphthalene-1-carboxamide within series A (IC₅₀?=?28.9 and 42.5?μM, respectively) and 1-hydroxy-N-(3-propoxyphenyl)naphthalene-2-carboxamide and 1-hydroxy-N-(3-ethoxyphenyl)-naphthalene-2-carboxamide (IC₅₀?=?2.0 and 3.1?μM, respectively) within series B. The inhibitory activity of C′₍₃₎ or C′₍₄₎ alkoxy substituted compounds of series B was considerably higher than that of C′₍₂₎ ones within series A. The PET-inhibiting activities of both series were compared with the PET inhibition of isomeric N-alkoxyphenyl-3-hydroxynaphthalene-2-carboxamides (series C) reported recently. Interactions of the studied compounds with chlorophyll a and aromatic amino acids present in pigment–protein complexes mainly in PS II were documented by fluorescence spectroscopy. The section between P₆₈₀ and plastoquinone Q_B in the PET chain occurring on the acceptor side of PS?II can be suggested as the site of action of the compounds.

Full text of DOI:10.1016/j.bmcl.2017.03.050

Bioorganic & Medicinal Chemistry Letters 27 (2017) 1881–1885
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Antimycobacterial N-alkoxyphenylhydroxynaphthalenecarboxamides
affecting photosystem II
b
c
d,
Tomas Gonec a, , Katarina Kralova , Matus Pesko , Josef Jampilek
⇑
⇑
a Department of Chemical Drugs, Faculty of Pharmacy, University of Veterinary and Pharmaceutical Sciences, Palackeho 1, 612 42 Brno, Czech Republic
b Institute of Chemistry, Faculty of Natural Sciences, Comenius University, Ilkovicova 6, 842 15 Bratislava, Slovakia
c Department of Environmental Ecology, Faculty of Natural Sciences, Comenius University, Ilkovicova 6, 842 15 Bratislava, Slovakia
d Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Comenius University, Odbojarov 10, 832 32 Bratislava, Slovakia
a r t i c l e i n f o
a b s t r a c t
Article history:
N-(Alkoxyphenyl)-2-hydroxynaphthalene-1-carboxamides (series A) and N-(alkoxyphenyl)-1-hydroxy-
naphthalene-2-carboxamides (series B) affecting photosystem (PS) II inhibited photosynthetic electron
transport (PET) in spinach chloroplasts. Their inhibitory activity depended on the compound lipophilicity
as well as on the position of the alkoxy substituent. The most potent PET inhibitors were 2-hydroxy-N-
phenylnaphthalene-1-carboxamide and N-[3-(but-2-yloxy)phenyl]-2-hydroxynaphthalene-1-carboxam-
ide within series A (IC50 = 28.9 and 42.5 mM, respectively) and 1-hydroxy-N-(3-propoxyphenyl)naph-
thalene-2-carboxamide and 1-hydroxy-N-(3-ethoxyphenyl)-naphthalene-2-carboxamide (IC50 = 2.0 and
3.1 mM, respectively) within series B. The inhibitory activity of C0(3) or C0(4) alkoxy substituted compounds
of series B was considerably higher than that of C0(2) ones within series A. The PET-inhibiting activities of
both series were compared with the PET inhibition of isomeric N-alkoxyphenyl-3-hydroxynaphthalene-
2-carboxamides (series C) reported recently. Interactions of the studied compounds with chlorophyll a
and aromatic amino acids present in pigment–protein complexes mainly in PS II were documented by
fluorescence spectroscopy. The section between P680 and plastoquinone QB in the PET chain occurring
on the acceptor side of PS II can be suggested as the site of action of the compounds.
Received 9 February 2017
Revised 16 March 2017
Accepted 20 March 2017
Available online 21 March 2017
Keywords:
Hydroxynaphthalenecarboxamides
Hill reaction
Photosystem II
Structure–activity relationships
Ó 2017 Elsevier Ltd. All rights reserved.
Substituted salicylanilides are known to exhibit many biological
activities, including antibacterial, antitubercular, antimycotic and
antihelmintic,1,2 and were found also to inhibit photosynthetic
electron transport (PET)3,4 and reduce chlorophyll content in green
alga, Chlorella vulgaris.5 Also cyclic analogues of salicylanilides –
II.14,15 Due to the formation of hydrogen bonds between the CONH
group of amides and the target proteins in photosynthetic centres
of thylakoid membranes, changes in protein conformation may
occur, which results in PET inhibition. Amides were found to be
PS II inhibitors, where they cause displacement of plastoquinone
(QB) from its binding pocket in one of the protein subunits of PS
II, the D1 protein.16 Based on the fact that for many years, virtually
all pharmaceutical companies had an agrochemical division in
order to maximize benefits of expensive chemical synthesis efforts
by searching for many types of useful biological activities and due
to previous findings that leads for pharmaceuticals and pesticides
often overlap, in some cases even leading to similar compounds
used for human health and weed management purposes,17 the bio-
logical activity of these novel N-(alkoxyphenyl)-hydroxynaph-
thalenecarboxamides as PS II herbicides was studied as well.
N-(Alkoxyphenyl)-2-hydroxynaphthalene-1-carboxamides and
ring-substituted
1-hydroxynaphthalene-2-carboxamides,
2-
hydroxynaphthalene-1-carboxamides,6–8 2-hydroxynaphthalene-
3-carboxamides9,10 and 6-hydroxynaphthalene-2-carboxamides11
were found to exhibit potent antibacterial and antimycobacte-
rial6–11 as well as herbicidal6,7,9,10,12 activity due to the presence
of an NHCO group and a hydroxy group in the naphthalene moiety
of these molecules that can interact with cell proteins forming
hydrogen bonds. It could be mentioned that vicinal hydroxynaph-
thalenecarboxamides can be considered as cyclic analogues of sal-
icylanilides, and the properties of the amide moiety can be easily
modified by various substitutions.13 The amide moiety also occurs
in many herbicides acting as PET inhibitors in photosystem (PS)
N-(alkoxyphenyl)-1-hydroxynaphthalene-2-carboxamides
were
prepared according to Scheme 1 and published recently.8 Based
on the promising antimycobacterial activity of these compounds,
we focused our attention on the study of their effects on PET
through PS II, from H2O to plastoquinone QB, by monitoring
⇑
Corresponding authors.
E-mail addresses: t.gonec@seznam.cz (T. Gonec), josef.jampilek@gmail.com
(J. Jampilek).
http://dx.doi.org/10.1016/j.bmcl.2017.03.050
0960-894X/Ó 2017 Elsevier Ltd. All rights reserved.

1882
T. Gonec et al. / Bioorganic & Medicinal Chemistry Letters 27 (2017) 1881–1885
O
R
series A
N
COOH
H
OH
R = H
(1/8/15)
O
2a-c 9a-c 16a-c
/ / )
OCH3
(
OH
COOH
NH2
R
OC2H5
(3a-c /10a-c /17a-c )
(4a-c /11a-c /18a-c )
N
H
OH
a
OC3H7
series B
+
5a-c 12a-c 19a-c
)
OC4H9
(
/
/
R
OH
OCH(CH3)2
(6a-c /13a-c /20a-c )
OCH(CH3)C2H5 (7a-c /14a -c/21a-c )
O
COOH
R
N
series C
H
OH
OH
Scheme 1. Synthesis of N-(alkoxyphenyl)-2-hydroxynaphthalene-1-carboxamides 1–7c (series A), N-(alkoxyphenyl)-1-hydroxynaphthalene-2-carboxamides 8–14c (series
B) and N-alkoxyphenyl-3-hydroxynaphthalene-2-carboxanilides 15–21c (series C). Reagents and conditions: (a) PCl3, chlorobenzene, microwave irradiation.8,10
photoreduction of 2,6-dichlorophenolindophenol (DCPIP), an artifi-
cial electron acceptor, in spinach chloroplasts according to Kralova
et al.18 As the site of DCPIP action is the plastoquinone pool on the
acceptor side of PS II,19 this method is suitable for PET monitoring
through PS II. DMSO solutions of the studied compounds were
added to chloroplasts due to the limited solubility of the com-
pounds in water, and the applied DMSO concentration (up to 4
vol%) did not affect the photochemical activity of spinach (Spinacia
oleracea L.) chloroplasts. The activity of the studied compounds
was compared with diuron (3-(3,4-dichlorophenyl)-1,1-dimethy-
lurea; DCMU), a commercial PET inhibitor, and relationships
between the structure and the PET inhibiting activity of N-
alkoxyphenylhydroxynaphthalenecarboxamides are discussed.
N-ortho/meta/para-substituted 2-hydroxynaphthalene-1- (ser-
ies A) and 1-hydroxynaphthalene-2-carboxamides (series B), see
Scheme 1, inhibited PET in spinach chloroplasts, which was
reflected in the inhibition of DCPIP photoreduction. The PET
inhibiting activity of the tested compounds was expressed by
IC50 values, i.e. compound concentration in mol/L causing 50% inhi-
bition of PET (Table 1). The IC50 values estimated for compounds of
series A were relatively low and varied from 42.5 mM (R = 3-OCH
(CH3)CH2CH3) to 681 mM (R = 3-OCH3). The IC50 values estimated
for compounds of series B ranged from 2.0 mM (R = 3-OC3H7) to
781.9 mM (R = 2-OC2H5), and the inhibitory activity of 3-propyloxy
derivative was comparable with that of standard DCMU. The PET-
inhibiting activity of the tested compounds strongly depended on
the lipophilicity and the position of substitution of R substituents
on the phenyl ring.
The dependences of the PET-inhibiting activity on the
lipophilicity of compounds for 2-, 3- and 4-substituted derivatives
of both series completed with PET-inhibiting activities of recently
published isomeric N-(alkoxyphenyl)-3-hydroxynaphthalene-2-
carboxamides10 (series C, see Scheme 1) are shown in Fig. 1. It
could be noted that the aqueous solubility of the C0(4) substituted
compounds of series C was limited, and therefore IC50 values could
not be determined due to precipitation of compounds during the
experiment; however, some C0(3) substituted compounds of this
series were found to be effective PET inhibitors (17b, R = 3-
OC2H5, IC50 = 4.5
lM; 18b, R = 3-OC3H7, IC50 = 4.8
lM; 20b, R = 3-
OCH(CH3)2, IC50 = 6.9
lM). While the dependence of the PET-
inhibiting activity of the C0(2) substituted derivatives of 3-hydroxy-
naphthalene-2-carboxamides on the lipophilicity of the com-
pounds linearly decreased (Fig. 1A, circles), for C0(3) substituted
compounds, this dependence was quasi-parabolic. However, the
PET inhibiting activity of compounds with log P ranging from
4.88 to 5.54 was relatively high with IC50 values in the range from
4.5
lM (17b, R = 3-OC2H5) to 8.3 lM (21b, R = 3-OCH(CH3)CH2-
CH3)10 (Fig. 1B, circles).
The PET-inhibiting activity expressed by log(1/IC50) of 2-substi-
tuted derivatives of series A increased linearly ca. up to log P = 5.47
Table 1
Discussed N-(alkoxyphenyl)-2-hydroxynaphthalene-1-carboxamides 1–7c, N-(alkoxyphenyl)-1-hydroxynaphthalene-2-carboxamides 8–14c and N-alkoxyphenyl-3-hydroxy-
naphthalene-2-carboxanilides 15–21c,10 calculated values of lipophilicity (logP) and IC50 values related to PET inhibition in isolated spinach chloroplasts in comparison with 3-
(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) standard (IC50 = 1.9 mM, logP = 2.75). LogP was predicted using ACD/Percepta ver. 2012 (Advanced Chemistry Development, Inc.,
Toronto, ON, Canada). ND = not determined due to precipitation during experiment.
R
Comp.
LogP
IC50
[lM]
Comp.
LogP
IC50
[
lM]
Comp.
LogP
IC50 [lM]
H
1
4.49
4.54
4.51
4.30
4.88
4.83
4.76
5.22
5.14
5.21
5.53
5.49
5.54
5.15
5.06
5.04
5.47
5.40
5.40
28.97
4777
6817
ND7
279.1
117.3
492.7
152.0
77.3
170.7
237.6
94.5
151.2
181.1
65.6
8
9a
9b
9c
4.52
4.61
4.56
4.37
4.92
4.88
4.67
5.26
5.21
5.27
5.60
5.54
5.60
5.18
5.13
5.11
5.52
5.47
5.46
31.36
15
4.52
4.61
4.56
4.37
4.92
4.88
4.67
5.26
5.21
5.27
5.60
5.54
5.60
5.18
5.13
5.11
5.52
5.47
5.46
ND10
2-OCH3
3-OCH3
4-OCH3
2-OC2H5
3-OC2H5
4-OC2H5
2-OC3H7
3-OC3H7
4-OC3H7
2-OC4H9
3-OC4H9
4-OC4H9
2-OCH(CH3)2
3-OCH(CH3)2
4-OCH(CH3)2
2-OCH(CH3)CH2CH3
3-OCH(CH3)CH2CH3
4-OCH(CH3)CH2CH3
2a
2b
2c
3a
3b
3c
4a
4b
4c
5a
5b
5c
6a
6b
6c
7a
7b
7c
199.66
23.56
79.56
781.9
3.1
16a
16b
16c
17a
17b
17c
18a
18b
18c
19a
19b
19c
20a
20b
20c
21a
21b
21c
59.510
53.410
ND10
10a
10b
10c
11a
11b
11c
12a
12b
12c
13a
13b
13c
14a
14b
14c
76.110
4.510
35.7
463.8
2.0
ND
675.6
20.9
490.5
151.0
15.5
38.6
161.7
16.6
ND10
128.510
4.810
ND10
182.010
7.810
ND10
138.210
6.910
452.4
47.8
42.5
ND10
134.610
8.310
86.3
52.4
ND10

T. Gonec et al. / Bioorganic & Medicinal Chemistry Letters 27 (2017) 1881–1885
1883
Fig. 1. Dependence of PET-inhibiting activity on lipophilicity of tested compounds: N-(alkoxyphenyl)-1-hydroxynaphthalene-2-carboxamides (series A, squares), N-
(alkoxyphenyl)-2-hydroxynaphthalene-1-carboxamides (series B, triangles) and N-(alkoxyphenyl)-3-hydroxynaphthalene-2-carboxamides10 (series C, circles) substituted in
ortho- (A), meta- (B) and para- (C) position.
(7a, R = 2-OCH(CH3)CH2CH3, IC50 = 47.8
lipophilicity increase to 5.53 led to a strong decrease of activity
(5a, R = 2-OC4H9, IC50 = 237.6 M) (Fig. 1A, squares). On the other
l
M), and
a
further
significantly affect the PET-inhibiting activity. Besides, as men-
tioned above, significant differences in PET-inhibiting activity were
observed depending on the position of the R substituent on the
phenyl ring, and the C0(3) substituted derivatives were the most
active, while C0(2) substituted ones were the least active. The lower
PET-inhibiting activity of 2-substituted derivatives compared to 3-
and 4-substituted ones was previously estimated also for dimethy-
laminoethylesters,20 piperidinoethylesters21 and morpholi-
noethylesters of alkoxyphenylcarbamic acids.22 The lower
inhibitory activity of 2-alkoxy-substituted derivatives with respect
to their 3- and 4-alkoxy-substituted analogues can be explained by
a secondary steric effect that is induced due to mutual interactions
between the alkoxy substituent and the carbamate group.23 The
close vicinity of the alkoxy substituent to the carbamate bond in
2-substituted derivatives may provoke a secondary steric effect,
distortion of the benzene ring plane against that of the carbamate
group resulting in perturbation of molecule planarity. It is reflected
l
hand, the activity of C0(2) substituted compounds of series C
decreased linearly from logP 4.88 to 5.52,10 and of C0(2) substituted
compounds of series B, 2-OC2H5 (10a), 2-OC3H7 (11a) and 2-OC4H9
(12a) derivatives partially precipitated during the experiment due
to low aqueous solubility, which was then reflected in significantly
higher values of estimated IC50 (Fig. 1A, empty triangles) compared
to those of 2-OCH3 (9a), 2-OCH(CH3)2 (13a) and 2-OCH(CH3)CH2-
CH3 (14a) substituted derivatives (IC50 = 59.5, 138 and 134 mM,
respectively) (Fig. 1A, triangles). The dependences of log(1/IC50
)
on logP for 3-substitued derivatives of all three discussed series
were quasi-parabolic, compounds of series A being the less active.
In general, the activity of C0(3) substituted compounds of series B up
to logP ca. 5.2 (except 13b, R = 3-OCH(CH3)2 showing lower aque-
ous solubility; empty triangle) was moderately higher (Fig. 1B, tri-
angles) than that of 3-substituted compounds of series C (Fig. 1B,
circles). Among C0(4) substituted derivatives, the most active com-
pounds were those of series B showing quasi-parabolic depen-
dence of PET-inhibiting activity on compound lipophilicity
(Fig. 1C, triangles), while the PET inhibiting activity of C0(4) substi-
tuted compounds of series A increased linearly up to logP 5.40, and
with a further lipophilicity increase, a loss of the activity was
observed (Fig. 1C, squares). It should be noted that the IC50 value
for compound 11a (R = 2-OC3H7) could not be determined due to
its low aqueous solubility.
With respect to considerable differences in activities of struc-
turally similar compounds of the three above discussed series
showing comparable lipophilicity, in which the OH moiety on the
naphthalene moiety is vicinal to the CONH group, it could be
assumed that the position of the substitution of the CONH group
on the naphthalene moiety, which determines the spatial orienta-
tion of naphthalene in the molecule and so, the access of the mole-
cule to the site of action in the photosynthetic apparatus, will
also in conjugation of p-bonds of the benzene ring through NH to
the CO group in the molecule, thus changing electron density
(charge) on the carbonyl group.24
In order to reach the site of action in the thylakoid membrane,
compounds must penetrate through a series of lipid bilayers sepa-
rated by aqueous layers, and thus, too high lipophilicity of the
compound results in an activity decrease. This so-called ‘‘cut-off”
effect, i.e. decreased activity for more lipophilic compounds within
homologous series, occurring frequently with alkyl-substituted
compounds20 and surfactants25,26 and also observed for the
above-discussed N-alkoxyphenylhydroxynaphthalenecarboxam-
ides was comprehensively explained by Balgavy & Devinsky.27
For more precise determination of the site of action of the tested
compounds in the PET chain, an artificial electron donor 1,5-
diphenylcarbazide (DPC) acting in Z/D intermediate that is situated
on the donor side of PS II has been used.28,29 This intermediate
ensures electron transport from the oxygen evolving complex to
the core of PS II (P680).30 Therefore, the activity of chloroplasts that

1884
T. Gonec et al. / Bioorganic & Medicinal Chemistry Letters 27 (2017) 1881–1885
was inhibited by compounds acting on the donor side of PS II can
be completely restored by DPC addition. Based on the facts that
DPC produced only gradual restoration of PET in chloroplasts, the
activity of which was inhibited by the tested compounds up to
90% (i.e. reached 10% activity of the control), and the complete
restoration of PET occurred only when the concentration of DPC
exceeded the concentration of the applied inhibitor by more than
one order of magnitude, it could be assumed that the section
between P680 (primary donor of PS II) and plastoquinone QB occur-
ring on the acceptor side of PS II was damaged by N-(alkoxyphe-
11b). The quenching of AAA fluorescence at 334 nm increased with
the increasing concentration of the tested compound. It could be
assumed that the decrease in fluorescence is caused by a change
in the environment of AAAs upon interaction with tested com-
pounds. The quenching of the AAA fluorescence in the presence
of various PET inhibitors such as 5-bromo- and 3,5-dibromo-2-
hydroxy-N-phenylbenzamides,38 ring substituted 8-hydrox-
yquinoline-2-carboxamides,33 2-substituted 6-fluoro-benzothia-
zoles39
and
N-substituted
5-amino-6-methylpyrazine-2,3-
dicarbonitriles40 was observed previously.
nyl)-1-hydroxynaphthalene-2-carboxamides
and
N-
The previous investigation of the antimycobacterial activity of
the above-discussed compounds showed that the compounds of
series B in the range of logP values from ca. 4.8 to 5.5 exhibited
potency against M. tuberculosis, and quasi-parabolic dependences
of activity on logP (with logP optimum ca. 5) for the C0(2) and
C0(4) substituted derivatives were observed, while for C0(3) substi-
tuted derivatives the activity increased up to logP ca. 5.1, and then
only its insignificant increase up to 5.49 was estimated.8 The most
active compound of this series was 2-hydroxy-N-(4-
propoxyphenyl)naphthalene-1-carboxamide (4c, MIC = 12 mM). In
general, the C0(3) substituted derivatives of series A demonstrated
the same course of dependence (activity increase from logP ca.
5.2) as the C0(3) substituted derivatives of series B; however, the
C0(2) and C0(4) substituted derivatives of series A expressed no
effect.8 On the other hand, most compounds of series C did not
show any antimycobacterial activity, except C0(2) and C0(3) position
(alkoxyphenyl)-2-hydroxynaphthalene-1-carboxamides. The com-
plete restoration of PET at high DPC concentrations can be
explained by the replacement of tested PET inhibitors by sym-
diphenylcarbazide from their binding site due to their direct inter-
action with the herbicide-binding niche, similarly as it was demon-
strated for atrazine31 and metribuzin.32 Inhibition of electron
transport in PS II at the QB site, i.e. at the acceptor side of PS II
was observed also for ring-substituted salicylanilides and car-
bamoylphenylcarbamates,3 ring-substituted 8-hydroxyquinoline-
2-carboxamides,33 N-benzylpyrazine-2-carboxamides34 and N-
alkoxyphenyl-3-hydroxynaphthalene-2-carboxamides.10
The effect of the studied compounds on the fluorescence of
chlorophyll a (Chla) and aromatic amino acids (AAAs) in spinach
chloroplasts was investigated as well, applying
a published
method.34 The DMSO concentration in all samples was the same
as in the control (10% (v/v)). The intensity of the Chla emission
band at 686 nm belonging to pigment–protein complexes in PS II
decreased with the increasing concentration of 3-(propoxyphe-
isomers 21a,b with R = 2-, 3-OCH(CH3)C2H5 (MIC = 23
lM) and
compound 20b with R = 3-OCH(CH3)2 (MIC = 24 M) that demon-
l
strated the highest activity against M. tuberculosis among N-alkox-
yphenyl-3-hydroxynaphthalene-2-carboxamides.10 Based on the
results of MTT assay assessing cell growth through measurement
of respiration performed with the selected most efficient com-
pounds of the above discussed three series of N-alkoxyphenylhy-
droxynaphthalenecarboxamides,8,10 it may be hypothesized that
the mechanism of action of these compounds could be connected
with an effect on mycobacterial energy metabolism.41 It could be
mentioned that structurally similar salicylanilides are able to
induce the uncoupling of oxidative phosphorylation and pho-
tophosphorylation in mitochondria, chloroplasts and other
energy-transducing membranes1,42,43 and belong to class 1 proto-
nophoric uncouplers causing collapse of the electrical potential of
membranes and showing linear concentration dependent beha-
viour of membrane conductivity.43 For example, 40,5-dichloro-2-
hydroxy-N-(2,4,5-trichlorophenyl)biphenyl-3-carboxamide effec-
tively inhibited photosynthetic phosphorylation coupled to the
nyl)-1-hydroxynaphthalene-2-carboxamide
(compound
4b)
(Fig. 2A), indicating a perturbation of Chla–protein complexes in
the thylakoid membrane35 caused by this compound. A similar
Chla fluorescence decrease in spinach chloroplasts was observed
previously for several PET inhibitors, such as amides of 2-alkylpyr-
idine-4-carboxylic acids,36 substituted benzanilides,37 ring-substi-
tuted 3-hydroxynaphthalene-2-carboxamides,9 ring-substituted
8-hydroxyquinoline-2-carboxamides,33 3,5-dibromo-2-hydroxy-
N-phenylbenzamides38 and N-benzylpyrazine-2-carboxamides.34
The tested compounds also interacted with residues of AAAs,
mainly tryptophan and tyrosine, occurring in photosynthetic pro-
teins situated mainly in PS II. This was documented by quenching
of AAA fluorescence at 334 nm. Fig. 2B presents the fluorescence
emission spectra of AAAs of untreated spinach chloroplasts and
chloroplasts treated with increasing concentrations of 3-(propy-
loxyphenyl)-2-hydroxynaphthalene-1-carboxamide
(compound
Fig. 2. Fluorescence emission spectra of chlorophyll a in suspension of spinach chloroplasts without and with compound 4b (R = 3-OC3H7; c = 0, 102, 204. 408 and 612 mM,
curves from top to bottom; excitation wave length k = 436 nm) (A) and fluorescence emission spectra of aromatic amino acids in suspension of spinach chloroplasts without
and with compound 11b (c = 0, 5.1, 15.3, 20.4 and 25.5 mM, curves from top to bottom; excitation wave length k = 275 nm) (B). Chlorophyll concentration in chloroplast
suspension: 10 mg/L.

T. Gonec et al. / Bioorganic & Medicinal Chemistry Letters 27 (2017) 1881–1885
13. Pattabiraman VR, Bode JW. Nature. 2011;480:471.
1885
reduction of ferricyanide, and complete inhibition occurred already
at 0.4 mM. The maximum rate of ferricyanide reduction was esti-
mated at the same concentration, which indicates that phosphory-
lation is uncoupled from electron flow.44 However, the possibility
that the studied N-alkoxyphenyl-hydroxynaphthalenecarboxam-
ides may act also upon a salicylanilides-like site present in the
mycobacteria1,2 cannot be ruled out.
Summarizing, it can be concluded that similarly to the antimy-
cobacterial activity, the PET inhibiting activity of the three above-
discussed series of N-alkoxyphenylhydroxynaphthalenecarboxam-
ides significantly depends on compound lipophilicity, the position
of the alkoxy substituent on the phenyl ring as well as on the posi-
tion of the OH group on the naphthalene moiety with respect to
the CONH group.
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This study was supported by IGA VFU Brno 320/2015/FAF and
by the Slovak Research and Development Agency (Grant No.
APVV-0516-12). This contribution utilizes also research results of
the CEBV project (ITMS 26240120034).
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