5-Bromo- and 3,5-dibromo-2-hydroxy-N-phenylbenzamides - Inhibitors of photosynthesis

Article abstract of DOI:10.2478/s11696-013-0416-7

5-Bromo-(Br-PBA) and 3,5-dibromo-2-hydroxy-N-phenylbenzamides (Br ₂-PBA) inhibited photosynthetic electron transport (PET) and their inhibitory efficiency depended on the compound lipophilicity as well as on the electronic properties of the R substituent in the N-phenyl moiety. Br-PBA showed higher PET inhibiting activity than Br₂-PBA with the same R substituent. The most effective inhibitors in the tested series were the derivatives with R = 3-F (Br-PBA; IC₅₀ = 4.3 μmol dm^-3) and R = 3-Cl (Br₂-PBA; IC₅₀ = 8.6 μmol dm ^-3). Bilinear dependence of the PET inhibiting activity on the lipophilicity of the compounds as well as on the Hammett constant, σ, of the R substituent was observed for both investigated series. Using EPR spectroscopy it was found that the site of action of the tested compounds in the photosynthetic apparatus is situated on the donor side of PS 2, in D ^· or in the Z^·/D^· intermediates. Interaction of the studied compounds with chlorophyll a and aromatic amino acids present in the pigment-protein complexes mainly in photosystem 2 was documented by fluorescence spectroscopy.

Full text of DOI:10.2478/s11696-013-0416-7

Chemical Papers
DOI: 10.2478/s11696-013-0416-7
ORIGINAL PAPER
5-Bromo- and 3,5-dibromo-2-hydroxy- -phenylbenzamides –
inhibitors of photosynthesis
a
b
c
aKatarína Kráľová*, František Šeršeň, Matúš Peško, Karel Waisser,
dLenka Kubicová
aInstitute of Chemistry, bDepartment of Ecosozology and Physiotactics, Faculty of Natural Sciences, Comenius University,
SK-842 15 Bratislava, Slovakia
cDepartment of Inorganic and Organic Chemistry, Faculty of Pharmacy, Charles University,
CZ-501 65 Hradec Králové, Czech Republic
dDepartment of Molecular Systems Biology, Faculty of Life Sciences, University of Vienna, A-1090 Vienna, Austria
Received 21 January 2013; Revised 2 April 2013; Accepted 5 April 2013
Dedicated to the memory of Professor Ľudovít Krasnec (1913–1990)
5-Bromo- (Br-PBA) and 3,5-dibromo-2-hydroxy-N-phenylbenzamides (Br2-PBA) inhibited pho-
tosynthetic electron transport (PET) and their inhibitory efficiency depended on the compound
lipophilicity as well as on the electronic properties of the R substituent in the N-phenyl moiety.
Br-PBA showed higher PET inhibiting activity than Br2-PBA with the same R substituent. The
most effective inhibitors in the tested series were the derivatives with R = 3-F (Br-PBA; IC50
=
4.3 µmol dm−3) and R = 3-Cl (Br2-PBA; IC50 = 8.6 µmol dm−3). Bilinear dependence of the PET
inhibiting activity on the lipophilicity of the compounds as well as on the Hammett constant, σ, of
the R substituent was observed for both investigated series. Using EPR spectroscopy it was found
that the site of action of the tested compounds in the photosynthetic apparatus is situated on the
.
.
.
donor side of PS 2, in D or in the Z /D intermediates. Interaction of the studied compounds
with chlorophyll a and aromatic amino acids present in the pigment–protein complexes mainly in
photosystem 2 was documented by fluorescence spectroscopy.
c
ꢀ 2013 Institute of Chemistry, Slovak Academy of Sciences
Keywords: salicylanilides, EPR spectroscopy, fluorescence spectroscopy, photosynthetic electron
transport, site of action
2-Hydroxybenzoic acid (2-HBA, salicylic acid) and
related compounds exhibit a wide spectrum of bi-
ological activities. 2-HBA acts as an endogenous
plant growth regulator with significant impact on
the growth and development of plants (Hayat et al.,
2010); plants treated with this bioactive compound
are characterised by higher tolerance to abiotic stress
such as chilling (Janda et al., 1999; Promyou et al.,
2012), ultraviolet-B radiation (Zhang & Li, 2012),
salt stress (Nazar et al., 2011) or cadmium stress
(Popova et al., 2009), and by increased induced an-
tioxidant capacity in leaves of herbicide-treated plants
(Ananieva et al., 2004). On the other hand, appli-
cation of higher 2-HBA concentrations resulted in
swelling of the thylakoid grana in various degrees, co-
agulation of the stroma, increase in the chloroplast
volume (Uzunova & Popova, 2000), reduced chloro-
phyll (Chl) content and inhibition of the stomata
function, photosynthetic gas-exchange and the activ-
ity of ribulose-1,5-biphosphate carboxylase/oxygenase
(Pancheva et al., 1996; Pancheva & Popova, 1998).
Moreover, the uncoupler-mediated stimulation of the
*Corresponding author, e-mail: kralova@fns.uniba.sk

ii
K. Kráľová et al./Chemical Papers
Table 1. Spectral data of studied compounds
Compound
Spectral data
FTIR ν˜/cm−1: 3319 (NH), 2988 (C O· · ·HO), 1618 (CONH), 1552, 1445 (C Carom), 1324 (C—O), 1220 (OH)
—
—
—
I
—
1H NMR (DMSO-d6), δ: 11.88 (s, 1H), 10.41 (s, 1H) 8.09 (d, J = 2.6 Hz, 1H), 7.70 (m, 2H), 7.38 (m, 2H), 7.15 (m,
1H), 6.96 (d, J = 8.8 Hz, 1H)
IR ν˜/cm−1: 3322 (NH), 2996 (C O···HO), 2842 (CH3), 1621 (CONH), 1548, 1415 (C Carom), 1324 (C—O), 1227
—
—
—
II
—
(OH)
1H NMR (DMSO-d6), δ: 11.97 (bs, 1H), 10.40 (s, 1H), 8.10 (d, J = 2.6 Hz, 1H), 7.57 (m, 3H), 7.18 (m, 2H), 6.97
(d, J = 8.8 Hz, 1H), 2.29 (s, 3H)
—
—
—
III
IV
FTIR, ν˜/cm−1: 3317 (NH), 3114 (C O· · ·HO), 1628 (CONH), 1550, 1487 (C Carom), 1318 (C—O), 1219 (OH)
—
1H NMR (DMSO-d6), δ: 11.96 (s, 1H), 10.47 (s, 1H), 8.12 (d, J = 2.6 Hz, 1H), 7.77 (m, 2H), 7.60 (m, 1H), 7.49 (m,
2H), 7.03 (d, J = 8.8 Hz, 1H)
FTIR, ν˜/cm−1: 3306 (NH), 3072 (C O···HO), 2835 (OCH3), 1622 (CONH), 1566, 1417 (C Carom), 1333 (C—O),
—
—
—
—
1220 (OH)
1H NMR (DMSO-d6), δ: 12.05 (bs, 1H), 10.33 (s, 1H), 8.12 (d, J = 2.6 Hz, 1H), 7.59 (m, 3H), 6.94 (m, 3H), 3.75
(s, 3H)
FTIR, ν˜/cm−1: 3317 (NH), 3105 (C O· · ·HO), 1626 (CONH), 1544, 1489 (C Carom), 1319 (C—O), 1219 (OH)
—
—
—
V
—
1H NMR (DMSO-d6), δ: 11.72 (s, 1H), 10.47 (s, 1H), 8.02 (d, J = 2.6 Hz, 1H), 7.75 (m, 2H), 7.58 (m, 1H), 7.43 (m,
2H), 6.96 (d, J = 8.8 Hz, 1H)
FTIR, ν˜/cm−1: 3311 (NH), 3065 (C O· · ·HO), 1622 (CONH), 1533, 1489 (C Carom), 1366 (C—O), 1224 (OH)
—
—
—
VI
—
1H NMR (DMSO-d6), δ: 11.56 (s, 1H), 10.58 (s, 1H), 8.11 (d, J = 2.3 Hz, 1H), 7,98 (d, J = 2.4 Hz, 1H), 7.67 (m,
2H), 7.59 (m, 1H), 6.98 (d, J = 8.8 Hz, 1H)
—
—
—
—
—
VII
VIII
FTIR, ν˜/cm−1: 3301 (NH), 3065 (C O· · ·HO), 1626 (CONH), 1551, 1487 (C Carom), 1315 (C—O), 1221 (OH)
—
FTIR, ν˜/cm−1: 3295 (NH), 3088 (C O···HO/NO2 ···HO), 1633 (CONH), 1559, 1477 (C Carom), 1522 (C—NO2),
—
—
1343 (C—O), 1220 (OH)
FTIR, ν˜/cm−1: 3338 (NH), 2989 (C O· · ·HO), 1632 (CONH), 1569, 1507 (C Carom), 1331 (C—O), 1226 (OH)
—
—
—
IX
—
1H NMR (DMSO-d6), δ: 11.82 (bs, 1H), 10.44 (s, 1H), 8.06 (d, J = 2.57 Hz, 1H), 7.72 (dd, J = 5.00 Hz, J = 9.10 Hz,
2H), 7.58 (dd, J = 2.57 Hz, J = 8.78 Hz, 1H), 7.22 (t, J = 8.80 Hz, 2H), 6.96 (d, J = 9.54 Hz, 1H)
FTIR, ν˜/cm−1: 3312 (NH), 3087 (C O· · ·HO), 1629 (CONH), 1548, 1490 (C Carom), 1338 (C—O), 1220 (OH)
—
—
—
X
XI
—
1H NMR (DMSO-d6), δ: 13.02 (bs, 1H), 10.66 (s, 1H), 8.31 (d, J = 2.4 Hz, 1H), 8.03 (d, J = 2.3 Hz, 1H), 7.66 (m,
2H), 7.20 (m, 1H)
XII
XIII
XIV
XV
1H NMR (DMSO-d6), δ: 13.16 (bs, 1H), 10.60 (s, 1H), 8.32 (d, J = 2.3 Hz, 1H), 8.03 (d, J = 2.3 Hz, 1H) 7.55 (m,
2H), 7.21 (d, J = 8.3 Hz, 2H), 2.30 (s, 3H)
1H NMR (DMSO-d6), δ: 12.72 (bs, 1H), 10.74 (s, 1H), 8.26 (d, J = 2.3 Hz, 1H), 8.04 (d, J = 2.3 Hz, 1H), 7.67 (m,
2H), 7.59 (m, 2H)
1H NMR (DMSO-d6), δ: 13.28 (bs, 1H), 10.58 (s, 1H), 8.32 (d, J = 2.3 Hz, 1H), 8.02 (d, J = 2.3 Hz, 1H), 7.58 (m,
2H), 6.97 (m, 2H), 3.77 (s, 3H)
1H NMR (DMSO-d6), δ: 12.52 (bs, 1H), 10.74 (s, 1H), 8.26 (d, J = 2.3 Hz, 1H), 8.03 (d, J = 2.3 Hz, 1H), 7.68 (m,
2H), 7.53 (m, 2H)
XVI
XVII
XVIII
XIX
1H NMR (DMSO-d6), δ: 12.40 (s, 1H), 10.58 (s, 1H), 8.20 (d, J = 2.4 Hz, 1H), 8.04 (m, 1H), 7.68 (m, 2H), 7.58 (m,
1H)
1H NMR (DMSO-d6), δ: 12.53 (s, 1H), 10.76 (s, 1H), 8.24 (d, J = 2.3 Hz, 1H), 8.03 (d, J = 2.3 Hz, 1H), 7.86 (t, J
= 2.0 Hz, 1H), 7.64 (m, 1H), 7.47 (t, J = 8.1 Hz, 1H), 7.26 (m, 1H)
1H NMR (DMSO-d6), δ: 12.38 (bs, 1H), 11.02 (s, 1H), 8.68 (t, J = 2.0 Hz, 1H), 8.25 (d, J = 2.3 Hz, 1H), 8.13 (m,
1H), 8.05 (m, 2H), 7.71 (t, J = 8.2 Hz, 1H)
FTIR, ν˜/cm−1: 3398 (NH), 3072 (C O· · ·HO), 1651 (CONH), 1544, 1507 (C Carom), 1331 (C—O), 1209 (OH)
—
—
—
—
1H NMR (DMSO-d6), δ: 12.60 (bs, 1H), 10.79 (s, 1H), 8.23 (d, J = 2.38 Hz, 1H), 8.03 (d, J = 2.38 Hz, 1H), 7.59–7.68
(m, 1H), 7.38–7.54 (m, 2H), 6.98–7.09 (m, 1H)
FTIR, ν˜/cm−1: 3401 (NH), 3066 (C O· · ·HO), 1639 (CONH), 1537, 1487 (C Carom), 1322 (C—O), 1223 (OH)
—
—
—
XX
—
1H NMR (DMSO-d6), δ: 12.93 (bs, 1H), 10.79 (s, 1H), 8.27 (d, J = 2.38 Hz, 1H), 8.02 (d, J = 2.38 Hz, 1H), 7.69
(dd, J = 5.14 Hz, J = 9.17 Hz, 2H), 7.25 (t, J = 8.80 Hz, 2H)
electron flow was considerably reduced in thylakoids
isolated from the leaves of seedlings grown using high
concentrations of 2-HBA (Sahu et al., 2002).
Some time ago, salicylanilides were found to be
very effective uncoupling agents of oxidative phospho-
rylation (Williamson & Metcalf, 1967; Renger, 1975)
and acceleration of the deactivation reactions of the
water splitting enzyme system Y by 5-chloro-3-tert-
butyl-2ꢀ-chloro-4ꢀ-nitrosalicylanilide was also observed
(Renger, 1975). In general, salicylanilides are known
to exhibit antibacterial, antituberculotic, antimycotic,
antihelmintic (Kubicová & Waisser, 1992), and photo-
synthetic electron transport inhibiting activities (Ku-
bicová et al., 2000a; Otevřel et al., 2010). Reduced
chlorophyll content in green alga, Chlorella vulgaris,
due to the treatment with substituted salicylanilides

K. Kráľová et al./Chemical Papers
iii
Table 2. Photosynthesis inhibiting activity (IC50), lipophilicity (logP), and Hammett constants (σ) of R substituent of compounds
I–XX
Compound
R
X
σa
logP
IC50/(µmol dm−3
)
I
II
III
IV
V
H
4-CH3
4-Br
4-OCH3
4-Cl
3,4-Cl2
3-Cl
3-NO2
4-F
3-F
H
4-CH3
4-Br
4-OCH3
4-Cl
H
H
H
H
H
H
H
H
H
0
4.38
4.65
5.01
4.35
4.86
5.62
5.11
4.44
4.53
4.59
5.13
5.46
5.78
5.11
5.66
6.41
5.78
5.29
5.25
5.40
28.0
51.4
5.6
92.8
5.0
12.2
5.3
19.2
21.9
4.3
60.9
89.9
14.1
199.6
24.6
10.7
8.6
10.7
56.8
17.7
–0.17
0.23
–0.27
0.23
0.60
0.37
0.71
0.06
0.34
0
–0.17
0.23
–0.27
0.23
0.60
0.37
0.71
0.06
0.34
VI
VII
VIII
IX
X
XI
XII
XIII
XIV
XV
XVI
XVII
XVIII
XIX
XX
H
Br
Br
Br
Br
Br
Br
Br
Br
Br
Br
3,4-Cl2
3-Cl
3-NO2
4-F
3-F
a) Values of σ were taken from Norrington et al. (1975).
was observed as well (Kubicová et al., 2000b).
NMR spectral data of compounds VII, VIII, and X
(Waisser et al., 2003). Unpublished spectral data of
the tested compounds are presented in Table 1.
1H NMR spectra (300 MHz) were obtained on a
VARIAN VNMRS 300 spectrometer in DMSO-d6 with
tetramethylsilane as an internal standard. FTIR spec-
tra (in solid phase) were recorded on a Nicolet 6700
spectrometer (Nicolet, USA) using the ATR tech-
nique.
The goal of the present work was to investi-
gate the inhibitory effect of ten 5-bromo-2-hydroxy-
N-phenylbenzamides (Br-PBA) as well as ten 3,5-
dibromo-2-hydroxy-N-phenylbenzamides (Br2-PBA)
on the photosynthetic electron transport in spinach
chloroplasts using the Hill reaction, EPR spectroscopy,
and fluorescence spectroscopy. The N-phenyl moiety
of the compounds was modified by groups (R) with
electron-accepting and electron-donating properties.
Inhibition of the photosynthetic electron trans-
port (PET) in spinach (Spinacia oleracea L.) chloro-
plasts by the studied compounds was determined
spectrophotometrically (Genesys 6, Thermo Scien-
tific, USA) using an artificial electron acceptor, 2,6-
dichlorophenol-indophenol (DCPIP) (Kráľová et al.,
1992, 2012). The rate of PET was monitored as a pho-
toreduction of DCPIP. The chlorophyll content was
Experimental
All reagents used in this study were of analyti-
cal grade and were purchased from Centralchem (Slo-
vakia). Tested compounds I–XX (see Table 2) were
prepared by heating the mixture of substituted aniline
and 5-bromo- or 3,5-dibromosubstituted salicylic acid
under reflux in the presence of phosphorus trichloride
for 3 h. Products obtained after cooling of the reaction
mixture were recrystallised from an ethanol/water so-
lution (Waisser et al., 2001).
30 mg dm−3
.
EPR spectra of chloroplast suspensions were recor-
ded by an ERS 230 apparatus (ZWG AdW, Berlin,
Germany) operating in the X-band at 24◦C (Kráľová
et al., 2012).
Some characteristics of the studied compounds
were published previously, namely the melting points
of I–X (Waisser et al., 2001), melting points and IR
spectral data of XI–XVIII (Waisser et al., 1998), and
Fluorescence emission spectra of chlorophyll a
(Chla) and aromatic amino acids in spinach chloro-
plasts were recorded on a fluorescence spectropho-
tometer F-2000 (Hitachi, Tokyo, Japan) applying a

iv
K. Kráľová et al./Chemical Papers
published method (Servusová et al., 2012).
The studied compounds were added to chloroplasts
as dimethyl sulfoxide (DMSO) solutions due to their
limited solubility in water. The applied DMSO con-
centration (up to 5 vol. % in the experiment with
DCPIP or 10 vol. % in the EPR and fluorescence ex-
periments) did not affect the photochemical activity
in spinach chloroplasts and it had no observable effect
on the EPR or fluorescence spectra of chloroplasts.
The logarithm of the partition coefficient for octan-
1-ol/water (logP) was calculated using the program
ACD/LogP DB version 12.01 (Advanced Chemistry
Development Inc., Toronto, Canada).
Results and discussion
Substituted Br-PBA and Br2-PBA inhibited PET
in spinach chloroplasts and the most effective in-
hibitors in the individual tested series were Br-PBA
derivative X with R = 3-F (IC50 = 4.3 µmol dm−3
)
and Br2-PBA derivative XVII with R = 3-Cl (IC50
= 8.6 µmol dm−3), while derivatives with R = 4-
OCH3 (compounds IV and XIV, respectively) showed
the lowest inhibitory activity in both series (Table 2).
Fig. 1 presents the dependence of the PET in-
hibiting activity of the tested compounds on their
lipophilicity expressed as logP (Fig. 1a) and its depen-
dence on the Hammett constant, σ, of R substituents
in the N-phenyl moiety (Fig. 1b).
In general, Br-PBA showed higher PET inhibiting
activity than Br2-PBA with the same R substituent.
This can be connected with the decreased solubility of
Br2-PBA due to the increased compound lipophilicity
caused by a second Br substituent in the acid moi-
ety. Bilinear dependence of the inhibitory activity on
the lipophilicity of the compounds (Fig. 1a) as well
as on the substituent constant σ of the R substituent
(Fig. 1b) was observed in both investigated series. The
most active inhibitor among Br-PBA was X (R = 3-F;
IC50 = 4.3 µmol dm−3) with logP = 4.59 and σ = 0.32,
while the most active inhibitor of the Br2-PBA series
was XVII (R = 3-Cl; IC50 = 8.6 µmol dm−3) with logP
= 5.78 and σ = 0.37. For Br-PBA with logP > 4.59, a
further increase of logP or σ resulted in linear activity
decrease (Fig. 1a, black circles). On the other hand,
differences in the PET inhibiting activity for Br2-PBA
with logP > 5.29 were relatively low (Fig. 1a, white
circles). As shown in Fig. 1b, for Br-PBA compounds
with σ > 0.37, the PET inhibiting activity strongly
decreased with the increasing σ value (Fig. 1b, black
circles) while practically no differences in the PET in-
hibiting activity of Br2-PBA compounds with σ ≥ 0.37
were observed (Fig. 1b, white circles). When excluding
compounds VI (R = 3,4-Cl2; σ = 60) and VIII (R = 3-
NO2; σ = 0.71) from the study, the linear dependence
of the PET inhibiting activity on the σ parameter of
the R substituent for the other eight compounds of the
Br-PBA series can be expressed by Eq. (1). Similarly,
Fig. 1. Dependence of the PET inhibiting activity of the tested
compounds on the compound lipophilicity (logP) (a)
and the electronic properties (σ) of the R substituent
(b); 5-bromo-2-hydroxy-N-phenylbenzamides ( ), 3,5-
•
dibromo-2-hydroxy-N-phenylbenzamides ( ).
◦
for compounds of the series Br2-PBA, when excluding
compounds XVI (R = 3,4-Cl2; σ = 0.60) and XVII
(R = 3-NO2; σ = 0.71), the above mentioned linear
dependence can be expressed by Eq. (2).
log(1/IC50) = 4.646( 0.043) + 2.167( 0.181)σ (1)
r = 0.980, s = 0.113, F = 143.4, n = 8
log(1/IC50) = 4.262( 0.045) + 1.884( 0.186)σ (2)
r = 0.972, s = 0.117, F = 102.7, n = 8
Good results of the above presented statistical
analyses indicate a significant effect of the σ param-
eter of the R substituent on the biological activity of
the tested compounds. However, the dependence of
the PET inhibiting activity on the calculated values

K. Kráľová et al./Chemical Papers
v
compound VII (b) or compound XVII (c), which were
recorded in the dark (solid lines) and in the light
(dotted lines). The signal recorded in the dark (sig-
.
nal IIslow) is attributed to oxidised tyrosine (D inter-
.
mediate or TyrD) located on the D2 protein on the
donor side of PS 2 (Svensson et al., 1991). A compari-
son with the control chloroplasts (Fig. 2a) showed that
treatment with VII resulted in reduced intensity of the
EPR signal IIslow (g = 2.0046; ∆Bpp = 2 mT) (Fig. 2,
solid line) indicating an interaction of this compound
.
with TyrD or its protein surroundings. On the other
hand, in the presence of compound XVII (Fig. 2c), not
only the intensity of signal IIslow (solid line) but also
that of signal IIvery fast (g = 2.0046; ∆Bpp = 2 mT;
signal recorded in the light, dotted line) were reduced.
Signal IIvery fast, which approximately represents the
difference between the signals recorded in the light
and in the dark with a small contribution from sig-
nal I, belongs to the intermediate Z (TyrZ) which is
an electron donor for the core of PS 2 (P 680) and is
located on the D1 protein on the donor side of PS 2
(Svensson et al., 1991).
Based on the EPR experiments with the eight
tested compounds showing high PET inhibiting ac-
.
tivity, interaction with the D intermediate was con-
firmed for the following Br-PBA compounds: III (R =
4-Br), V (R = 4-Cl), VII (R = 3-Cl) as well as for XIII
(R = 4-Br), XVI (R = 3,4-Cl2) and XIX (R = 4-F)
from the Br2-PBA series. However, only XVII (3-Cl)
and XX (3-F) of the Br2-PBA series showed inter-
Fig. 2. EPR spectra of untreated spinach chloroplasts (a) and
chloroplasts treated with 0.05 mmol dm−3 of VII (b)
or XVII (c); solid lines correspond to chloroplasts kept
in the dark, dotted lines to the illuminated chloro-
plasts; modulation amplitude: 0.5 mT; Chl content:
3.0 g dm−3. All spectra were recorded at the same am-
plification.
.
.
action with both intermediates (Z and D ). It seems
that for the interaction with both above mentioned in-
termediates, 3,5-dibromo substitution in the salicylic
part of the compound and a halogen substituent at
position 3 in the N-phenyl ring is favourable. Inter-
of logP was characterised by substantially higher vari-
ance (see Fig. 1a).
.
.
Similarly to the presented results, PET inhibit-
ing activity of the substituted salicylanilides (Ku-
bicová et al., 2000a) also depends on the com-
pound lipophilicity (logP) and the electronic prop-
erties of the R substituent, σ. On the other hand,
the PET inhibiting activity of 2-alkylsulfanyl sub-
stituted 4-pyridinecarbothioamides (with a biologi-
cally active CSNH2 group) (Kráľová et al., 2011)
or of 2-alkylsulfanyl substituted anilides of pyridine-
4-carboxylic acids with substituents in the anilide
moiety (Kráľová et al., 2001) clearly depends on
the compound lipophilicity, probably also due to the
membrane-active 2-alkylsulfanyl substituents.
The fact that undamaged chloroplasts of higher
plants exhibit stable EPR signals in the region of free
radicals (g ∼ 2.0) (Hoff, 1979), associated with photo-
systems PS 2 (signals IIvery fast and IIslow) and PS 1
(signal I) and which can be affected by PET inhibitors,
enables to determine the site of action of these com-
pounds in the photosynthetic apparatus using EPR
spectroscopy.
action of the studied inhibitors with the Z and D
intermediates results in PET interruption between PS
2 and PS 1, which is reflected in significant increase
of the intensity of signal I for illuminated chloroplasts
(Fig. 2b and 2c, dotted lines; g = 2.0026, ∆Bpp = 0.7
mT). This signal is attributed to the cation-radical
of the chlorophyll a dimer in the core of PS 1 (Hoff,
1979).
.
In our previous studies, the D intermediate
was determined as the site of inhibitory action of
substituted benzanilides (Kráľová et al., 1999), 2-
alkylsulfanyl-4-pyridinecarbothioamides (Kráľová et
al., 2011), and anilides of substituted pyrazine-2-
carboxylic acids (Doležal et al., 2001), while anilides
of 2-alkylpyridine-4-carboxylic acids (Kráľová et al.,
.
.
1998) acted in the Z /D intermediates. However,
for some ring-substituted salicylanilides, the second
quinone acceptor, QB, situated on the acceptor side
of PS 2 was determined by the EPR experiments as
the site of their inhibitory action (Otevřel et al., 2010).
The site of action of the studied compounds on the
acceptor side of PS 2 was excluded by applying an ar-
tificial electron-donor, 1,5-diphenylcarbazide (DPC),
Fig. 2 presents the EPR spectra of untreated
spinach chloroplasts (a) and chloroplasts treated with

vi
K. Kráľová et al./Chemical Papers
orescence but also that of aromatic amino acids
in spinach chloroplasts (Fig. 3). In the presence of
XVIII,the intensity of the Chla emission band at
686 nm belonging to the pigment–protein complexes in
photosystem 2 (Atal et al., 1991) decreased (Fig. 3a)
and a similar decrease was observed for the intensity
of the fluorescence emission band at 334 nm belonging
to aromatic amino acids (Fig. 3b). These findings in-
dicate a perturbation of the Chla–protein complexes
in the thylakoid membrane caused by the tested com-
pound as well as its interaction with aromatic amino
acids occurring in the proteins of spinach chloro-
plasts. Similar effects, which enhanced with the in-
creasing compound concentration, were also observed
for other tested Br-PBA and Br2-PBA compounds
as well as for previously investigated substituted
benzanilides (Kráľová et al., 1999), salicylanilides
(Kubicová et al., 2000a), and N-benzylpyrazine-2-
carboxamides (Servusová et al., 2012).
Conclusions
5-Bromo- (Br-PBA) and 3,5-dibromo-2-hydroxy-
N-phenylbenzamides (Br2-PBA) were found to inhibit
PET in spinach chloroplasts acting on the donor side
.
.
.
of PS 2, in D or Z /D intermediates. For interaction
.
.
with the Z /D intermediates, 3,5-dibromo substitu-
tion in the salicylic part of the compound and the pres-
ence of a halogen substituent at position 3 in the N-
phenyl ring were found to be desirable. The inhibition
efficiency of Br-PBA and Br2-PBA depended both on
the electronic properties of the R substituent, σ, in
the N-phenyl moiety and on the compound lipophilic-
ity (logP). For compounds of both series, bilinear de-
pendences of the inhibitory activity on σ as well as
on logP were observed. The interaction of the tested
compounds with chlorophyll a molecules and aromatic
amino acids present mainly in PS 2 was also con-
firmed.
Fig. 3. Fluorescence emission spectra of Chla in untreated
spinach chloroplasts in the presence of XVIII (c in µmol
dm−3): 0, 23, 46, 91, 183, 346 (curves from top to
bottom; λex = 436 nm) (a) and fluorescence emis-
sion spectra of aromatic amino acids (AAA) in un-
treated spinach chloroplasts in the presence of XVIII
(c in µmol dm−3): 0, 2.3, 4.6, 9.1, 22.8, 45.7 (curves
from top to bottom; λex = 275 nm) (b). Chl content:
Acknowledgements. This study was financially supported by
the VEGA grant of the Ministry of Education of Slovak Re-
public and Slovak Academy of Sciences, no. 1/0612/11 and by
Sanofi Aventis Pharma Slovakia.
10 mg dm−3
.
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