Halogenated 1-Hydroxynaphthalene-2-Carboxanilides Affecting Photosynthetic Electron Transport in Photosystem II †

Series of seventeen new multihalogenated 1-hydroxynaphthalene-2-carboxanilides was prepared and characterized. All the compounds were tested for their activity related to the inhibition of photosynthetic electron transport (PET) in spinach (Spinacia oleracea L.) chloroplasts. 1-Hydroxy-N-phenylnaphthalene-2-carboxamides substituted in the anilide part by 3,5-dichloro-, 4-bromo-3-chloro-, 2,5-dibromo- and 3,4,5-trichloro atoms were the most potent PET inhibitors (IC50 = 5.2, 6.7, 7.6 and 8.0 µM, respectively). The inhibitory activity of these compounds depends on the position and the type of halogen substituents, i.e., on lipophilicity and electronic properties of individual substituents of the anilide part of the molecule. 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. The structure-activity relationships are discussed.


Introduction
In spite of the fact that various classes of herbicides are known and currently can be classified according to about 20 different modes of action [1], over 50% of marketed herbicides act by reversible binding to photosystem II (PS II) [2]. This effect results in the interruption of the photosynthetic electron transport (PET) [3][4][5]. PS II uses light energy to drive two chemical reactions: the oxidation of water and the reduction of plastoquinone. The following redox components of PS II participate in the transfer of electrons from H 2 O to the plastoquinone pool: water oxidizing manganese cluster (Mn) 4 and redox-active tyrosine (Tyr z ) situated on the donor side of PS II, reaction centre chlorophyll (P680) and components situated on the acceptor side of PS II, namely pheophytin and two plastoquinone molecules, Q A and Q B . While the plastoquinone molecule Q A acting as a one-electron acceptor is permanently bound to PS II, the plastoquinone molecule Q B acting as a two-electron acceptor is loosely bound at the Q B -site, and reduced plastoquinone unbinds from the reaction centre and diffuses in the hydrophobic core of the membrane, whereby Q B -binding site will be occupied by an oxidized plastoquinone molecule [6]. Herbicides belonging to photosystem (PS) II inhibitors inhibit photosynthetic electron transport (PET) by binding to the Q B -binding niche on the D 1 protein of the PS II complex in chloroplast thylakoid membranes, which results in the inhibition of photosynthetic electron transport (PET) from Q A to Q B , blockage of CO 2 fixation and inhibition of ATP production. Many QSAR studies of PS II inhibitors with diverse chemical structures have emphasized the hydrophobic nature of the binding domain, with lipophilicity being the dominant determinant of Hill inhibition activity [7], e.g., the review paper of Lambreva et al. [8] presents a compendious overview of the recent improvements in the understanding of the structure and function of PS II donor side with a focus on the interactions of the plastoquinone cofactors with the surrounding environment and operational features. PET inhibitors in the functional dissection of the photosynthetic electron transport system were summarized in the review paper of Trebst [9], and a comprehensive overview of synthetic photosynthetic inhibitors and those based on natural products was published by Teixeira et al. [10].
In the context of the above-mentioned facts, new di-and trichlorinated and brominated 1-hydroxynaphthalene-2-carboxanilides were prepared and characterized and tested for their activity related to the PET inhibition in spinach (Spinacia oleracea L.) chloroplasts. The results were completed by seven recently published unsubstituted and monochlorinated and brominated derivatives [11], and the structure-activity relationships (SAR) of all the mentioned compounds are discussed.

Chemistry
The discussed anilides were synthesized using microwave-assisted synthesis as described in Gonec et al. [11]. All the studied compounds were prepared according to Scheme 1. The condensation of 1-hydroxy-2-naphthoic acid and ring-substituted aniline using phosphorus trichloride in chlorobenzene gave a series of twenty-four 1-hydroxynaphthalene-2-carboxanilides 1-24.
Physicochemical descriptors-lipophilicity of the compounds expressed as log P and electronic σ parameters of individual anilides-were calculated for all the investigated compounds by means of the ACD/Percepta ver. 2012 program, see Table 1. Log P values ranged from 4.52 (unsubstituted anilide 1) to 6.34 (2,4,. In general, it can be stated that the lipophilicity is rather high; nevertheless, the recommended log P value for agrochemicals is ≤5 [35]. Beside unsubstituted compound 1, monohalogenated anilides 2-7 expressed the lowest lipophilicity, while trisubstituted derivatives 14-16 and 20 possessed the highest log P values, and in general brominated compounds showed higher lipophilicity than chlorinated. For individual N-aryl parts of the anilides also electronic properties expressed as electronic σ constants of the whole substituted phenyl ring were predicted; they ranged from 0.60 (compound 1) to 1.56 (2,4,5-Cl substituted derivative 16). Scheme 1. Synthesis of ring-substituted 1-hydroxynaphthalene-2-carboxanilides 1-24. Reagents and conditions: (a) PCl3, chlorobenzene, microwave synthesis.
The dependences of the PET-inhibiting activity on the lipophilicity of the discussed mono-, diand tri-N-aryl substituted anilides are shown in Figure 1A. Similar trends for mono-and disubstituted compounds can be observed-the PET-inhibiting activity increases with increasing lipophilicity, while an opposite trend can be found for trisubstituted derivatives. Thus, it seems that for PET-inhibiting activity, a lipophilicity optimum is in the range of log P from 6.01 (compound 13) to 6.28 (compound 16), see Figure 1A. It is important to note that a PET inhibition depends also on the electronic effects of individual substituents within series of different PET inhibitors [11,12,[21][22][23]. The dependences of the PET-inhibiting activity on the electron σ parameters of the whole N-aryl part of individual anilides are illustrated in Figure 1B. Practically the same trends can be found for mono-, di-and trisubstituted derivatives; the PET inhibition sharply decreases with the increasing electron-withdrawing effect of individual anilides (i.e., with high amide bond electron-deficiency) as follows: compound 3 (R = 3-Cl, σ = 0.85, IC 50  The dependences of the PET-inhibiting activity on the lipophilicity of the discussed mono-, diand tri-N-aryl substituted anilides are shown in Figure 1A. Similar trends for mono-and disubstituted compounds can be observed-the PET-inhibiting activity increases with increasing lipophilicity, while an opposite trend can be found for trisubstituted derivatives. Thus, it seems that for PET-inhibiting activity, a lipophilicity optimum is in the range of log P from 6.01 (compound 13) to 6.28 (compound 16), see Figure 1A. It is important to note that a PET inhibition depends also on the electronic effects of individual substituents within series of different PET inhibitors [11,12,[21][22][23]. The dependences of the PET-inhibiting activity on the electron σ parameters of the whole N-aryl part of individual anilides are illustrated in Figure 1B [36][37][38]. This intermediate ensures electron transport from the OEC to the core of PS II (P680) [39]. Consequently, an addition of DPC can completely restore the activity of chloroplasts, which was inhibited by PET inhibitors acting on the donor side of PS II. However, the addition of DPC to chloroplasts, the activity of which was inhibited by tested compounds approximately up to 80% (i.e., corresponding to 20% activity of the control), caused gradual PET restoration with increasing DPC concentration, and complete activity restoration was obtained only with 12-fold higher DPC concentration compared to the concentration of the applied PET inhibitor. Based on this finding, it could be suggested that the site of inhibitory action of the halogenated  [36][37][38]. This intermediate ensures electron transport from the OEC to the core of PS II (P680) [39]. Consequently, an addition of DPC can completely restore the activity of chloroplasts, which was inhibited by PET inhibitors acting on the donor side of PS II. However, the addition of DPC to chloroplasts, the activity of which was inhibited by tested compounds approximately up to 80% (i.e., corresponding to 20% activity of the control), caused gradual PET restoration with increasing DPC concentration, and complete activity restoration was obtained only with 12-fold higher DPC concentration compared to the concentration of the applied PET inhibitor. Based on this finding, it could be suggested that the site of inhibitory action of the halogenated 1-hydroxynaphthalene-2-carboxanilides is situated on the acceptor side of PS II, at the section between P680 (primary donor of PS II) and Q B . The complete restoration of PET using DPC concentration exceeding that of the applied inhibitor by more than one order of magnitude indicates that, due to direct interaction of DPC with the herbicide binding niche, these PET inhibitors were displaced from their binding site, as was similarly shown for atrazine [40] or metribuzin [41]. The plastoquinone Q B at the acceptor side of PS II was found to be the site of inhibitory action of N-benzylpyrazine-2-carboxamides [42] and N-alkoxyphenyl-3-hydroxy-naphthalene-2-carboxamides [16] as well as ring-substituted salicylanilides, carbamoylphenyl-carbamates [29] and 8-hydroxyquinoline-2-carboxamides [23].
In addition, the interaction of PET inhibitors with aromatic amino acids (AAA), mainly tryptophan and tyrosine, occurring in photosynthetic proteins situated mainly in PS II can be investigated using the fluorescence method. In the presence of the tested halogenated 1-hydroxynaphthalene-2-carboxanilides, quenching of AAA fluorescence at 334 nm was observed, which showed an increase with increasing concentration of PET inhibitors, which is presented in Figure 2B for the model compound 23. It could be assumed that the decrease in fluorescence is caused by a change in the environment of AAAs upon interaction with the tested compounds. The quenching of the AAA fluorescence in the presence of various PET inhibitors such as ring-substituted 8-hydroxyquinoline-2-carboxamides [23], 5-bromo-and 3,5-dibromo-2-hydroxy-N-phenylbenzamides [45], N-substituted 5-amino-6-methylpyrazine-2,3-dicarbonitriles [47] and 2-substituted 6-fluoro-benzothiazoles [48] was observed previously. 1-hydroxynaphthalene-2-carboxanilides is situated on the acceptor side of PS II, at the section between P680 (primary donor of PS II) and QB. The complete restoration of PET using DPC concentration exceeding that of the applied inhibitor by more than one order of magnitude indicates that, due to direct interaction of DPC with the herbicide binding niche, these PET inhibitors were displaced from their binding site, as was similarly shown for atrazine [40] or metribuzin [41]. The plastoquinone QB at the acceptor side of PS II was found to be the site of inhibitory action of N-benzylpyrazine-2-carboxamides [42] and N-alkoxyphenyl-3-hydroxy-naphthalene-2-carboxamides [16] as well as ring-substituted salicylanilides, carbamoylphenyl-carbamates [29] and 8-hydroxyquinoline-2-carboxamides [23].
In addition, the interaction of PET inhibitors with aromatic amino acids (AAA), mainly tryptophan and tyrosine, occurring in photosynthetic proteins situated mainly in PS II can be investigated using the fluorescence method. In the presence of the tested halogenated 1-hydroxynaphthalene-2-carboxanilides, quenching of AAA fluorescence at 334 nm was observed, which showed an increase with increasing concentration of PET inhibitors, which is presented in Figure 2B for the model compound 23. It could be assumed that the decrease in fluorescence is caused by a change in the environment of AAAs upon interaction with the tested compounds. The quenching of the AAA fluorescence in the presence of various PET inhibitors such as ring-substituted 8-hydroxyquinoline-2-carboxamides [23], 5-bromo-and 3,5-dibromo-2-hydroxy-N-phenylbenzamides [45], N-substituted 5-amino-6-methylpyrazine-2,3-dicarbonitriles [47] and 2-substituted 6-fluoro-benzothiazoles [48] was observed previously.

General Information
All reagents were purchased from Merck (Sigma-Aldrich, St. Louis, MO, USA) and Alfa (Alfa-Aesar, Ward Hill, MA, USA). Reactions were performed using a CEM Discover SP microwave reactor (CEM, Matthews, NC, USA). The melting points were determined on a Kofler hot-plate apparatus HMK (Franz Kustner Nacht KG, Dresden, Germany) and are uncorrected. Infrared (IR) spectra were recorded on a Smart MIRacle™ ATR ZnSe for Nicolet™ Impact 410 Fourier-transform IR spectrometer (Thermo Scientific, West Palm Beach, FL, USA). The spectra were obtained by the accumulation of 256 scans with 2 cm −1 resolution in the region of 4000-650 cm −1 . All 1 H-and 13 C-NMR spectra were recorded on an Agilent VNMRS 600 MHz system (600 MHz for 1 H and 150 MHz for 13 C, Agilent Technologies, Santa Clara, CA, USA) in dimethyl sulfoxide-d 6 (DMSO-d 6 ). 1 H and 13

Synthesis
General Procedure for Synthesis of N-(substituted phenyl)-1-hydroxynaphthalene-2-carboxamides 1-24 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 residue washed with 2 M HCl, and the crude product was recrystallized from aqueous ethanol. All the studied compounds are presented in Table 1.

Study of Fluorescence of Chlorophyll a and Aromatic Amino Acids in Spinach Chloroplasts
The fluorescence emission spectra of chlorophyll a (Chla) and aromatic amino acids in spinach chloroplasts were recorded on fluorescence spectrophotometer F-2000 (Hitachi, Tokyo, Japan) using excitation wavelength λ ex = 436 nm for monitoring the fluorescence of Chla and λ ex = 275 nm for monitoring the fluorescence of aromatic amino acids, excitation slit 20 nm and emission slit 10 nm. The samples were kept in the dark for 2 min prior to the measurement. The phosphate buffer used for dilution of the chloroplast suspension was the same as described above. Due to low aqueous solubility, the compounds were added to a chloroplast suspension in DMSO solution. The DMSO concentration in all samples was the same as in the control (10%). The chlorophyll concentration in chloroplast suspension was 10 mg/L.

Conclusions
Series of seventeen new di-and trichlorinated and brominated 1-hydroxynaphthalene-2-carboxanilides was prepared and characterized. These compounds were completed by seven recently published unsubstituted and monochlorinated and brominated derivatives, and all the compounds were tested for their in vitro activity related to the inhibition of photosynthetic electron transport (PET) in spinach (Spinacia oleracea L.) chloroplasts. N-(3,5-dichlorophenyl)- (13), N-(4-bromo-3-chlorophenyl)- (24), N-(2,5-dibromophenyl)-(18) and 1-hydroxy-N-(3,4,5-trichlorophenyl)naphthalene-2-carboxamide (16) were the most potent PET inhibitors with IC 50 = 5.2, 6.7, 7.6 and 8.0 µM, respectively. In general, it can be stated that the chlorinated derivatives demonstrated higher potency than the bromine derivatives. The inhibitory activity of these compounds depends on the position and the type of halogen substituents, e.g., all 2,4-disubstituted compounds 9, 17, 21, 22 and 2,3-disubstituted compound 8 showed limited aqueous solubility resulting in moderate PET-inhibiting activity compared with that of 2,5-, 2,6-, 3,4-or 3,5-disubstituted derivatives or other investigated compounds. It can be stated that the PET inhibition increases with increasing lipophilicity for monoand disubstituted analogues, while an opposite trend is observed for trisubstituted derivatives. Thus, it seems that for PET-inhibiting activity, a lipophilicity optimum is in the range of log P from 6.01 to 6.28. On the other hand, the PET inhibition decreases with increasing electron-withdrawing properties of individual anilides, and this trend is similar for all the series. 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 P 680 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.