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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.