Preparation and Photosynthesis-Inhibiting Activity of N-( n-Alkoxy ) phenylamides of 2-Hydroxynaphthalene-1-carboxylic acid

In this study a series of twelve n-alkoxy-substituted 2-hydroxynaphthalene-1carboxanilides was prepared and characterized. The discussed compounds were prepared by microwave-assisted synthesis. The compounds were tested for their activity related to inhibition of photosynthetic electron transport (PET) in spinach (Spinacia oleracea L.) chloroplasts. The highest PET inhibition was observed for meta-substituted compounds, whereas 2-hydroxy-N-(3-propoxyphenyl)naphthalene-1-carboxamide showed the highest PET inhibition within the whole series. In spite of medium or moderate PET-inhibiting activity it was found that the compounds inhibit PET in photosystem II. The activity of all positional isomers is strongly influenced by lipophilicity and the length/bulkiness of the alkoxy chain.

Lipophilicity of all compounds 1a-4c was calculated as log P using ACD/Percepta ver.2012 (Advanced Chemistry Development, Inc., Toronto, ON, Canada).The results are shown in Table 1.Lipophilicity values of the compounds expressed as log P values ranged from 4.30 (compounds 1c, R = 4-OCH 3 ) to 5.53/5.54(compounds 4a/b, R = 2-and 4-OC 4 H 9 ).Logically lipophilicity increases with lengthening of the alkoxy chain.For individual substituents in the aniline part of the discussed compounds also electronic Hammett's σ parameters were predicted using the same software; they ranged from -0.29 to -0.27 for ortho-and parasubstituted compounds and from 0.10 to 0.14 for meta-substituted compounds.Experience has shown that a parameter representing the volume of the substituents on each compound relative to other members of the same series may often be correlated with biological measurement [23][24][25], therefore molar volume MV [cm -3 ] was also calculated.The predicted molecular descriptors of the studied compounds are shown in Table 1.With respect to these small but specifically substituted groups of compounds some structureactivity relationships (SAR) can be proposed.Correlations between log(1/IC 50 [mol/L]) and lipophilicity (expressed as log P), electronic properties of the individual alkoxy substituents in compounds 1a-4c (expressed as electronic Hammett's σ parameters) and the length (bulkiness) of the alkoxy tail (expressed as molar volume MV [cm -3 ]) of individual alkoxy chains were performed, see Figure 1.Based on the obtained results it is evident that meta substitution of the aniline ring is preferred.Figure 1A (the dependences between PET inhibition and lipophilicity) and Figure 1C (the dependences between PET inhibition and molar volume) illustrate the general quasi-parabolic trend within all three positional isomers.The propoxy substituent in the ortho and meta positions of aniline (compounds 3a, 3b) seems to be the most favourable.Among para-substituted compounds 2c, 3c, 4c butyl derivative 4c is the most effective PET inhibitor (see Figures 1A and 1C, blue line).The dependence of PET inhibition on electronic σ properties of the anilide substituents plays a secondary role, see Figure 1B.It can be stated that weak electron-withdrawing properties of a substituent in the meta position are more preferable than electron-donor properties of alkoxy moieties in the ortho and para positions.

C
Application of artificial electron donors allows specifying the section in the photosynthetic electron transport chain in which PET is stopped by an inhibitor [26].One of such suitable artificial electron donors is 2,5-diphenylcarbazide (DPC) which supplies electrons in the site of Z  /D  intermediate on the donor side of PS II [20].Consequently, in the presence of DPC the PET which was inhibited in the section between the oxygen evolving complex and the Z  /D  intermediate can be restored.On the other hand, PET restoration by DPC does not occur if the site of PET inhibition is situated on the acceptor side of PS II, between P680 and secondary quinone acceptor Q B .However, if PET inhibitors directly interact with the herbicide-binding niche they can be displaced from their binding site similarly as it was demonstrated for atrazine [27] or metribuzin [28], which is also connected with complete restoration of the photochemical activity of chloroplasts.
As application of DPC to chloroplasts, the activity of which was inhibited (up to 15% of the control), caused practically complete PET restoration, it can be concluded that the site of studied n-alkoxy-substituted 2-hydroxynaphthalene-1-carboxanilides is situated mainly on the donor side of PS II.However, for univocal confirmation whether the PET inhibition by the studied compounds occur also on the acceptor side of PS II further investigation would be necessary.The site of action situated on the donor side of PS II was found also for 2-alkylthio-6-R-benzothiazoles (R = 6-formamido-, 6-acetamido-, and 6-benzoylamino-) [29], anilides of 2-alkylpyridine-4-carboxylic acids acting in the intermediates Z  /D  [30] and 2-alkylsulphanyl-4-pyridinecarbothioamides acting in the D  intermediate [31].Studied compounds affected the chlorophyll a (Chla) fluorescence in spinach chloroplasts.As shown in Figure 2, the intensity of the Chla emission band at 686 nm belonging to the pigment-protein complexes in photosystem II decreased in the presence of compound 2b [26].This finding indicates a perturbation of the Chla-protein complexes in the thylakoid membrane caused by the tested compound, which contributes to PET inhibition.A similar Chla fluorescence decrease in spinach chloroplasts was observed previously with several PET inhibitors, e.g., ring-substituted 3-hydroxynaphthalene-2-carboxanilides [6], 2-hydroxynaphthalene-1-carboxanilides [7] and 1-hydroxynaphthalene-2-carboxanilides [8] or ringsubstituted 4-arylamino-7-chloroquinolinium chlorides [32].

Synthesis
Alkyloxy anilines (except commercially available o-, m-and p-anisidine) were prepared from corresponding aminophenols and alkyl bromides using the method described by De Marco et al. [33].Freshly distilled crude products were used directly in the next step.
General procedure for synthesis of carboxamide derivatives (1a-4c): 2-Hydroxynaphthalene-1-carboxylic acid (5.3 mmol) and the corresponding substituted aniline (5.3 mmol) were suspended in dry chlorobenzene (30 mL).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 2M HCl, and the crude product was recrystallized from aqueous ethanol.Studied compounds 1a-4c are presented in Table 1.

Study of photosynthetic electron transport (PET) inhibition in spinach chloroplasts
Chloroplasts were prepared from spinach (Spinacia oleracea L.) according to Masarovicova and Kralova [34].The inhibition of photosynthetic electron transport (PET) in spinach chloroplasts was determined spectrophotometrically (Genesys 6, Thermo Scientific), using an artificial electron acceptor 2,6-dichlorophenol-indophenol (DCPIP) according to Kralova et al. [35], and the rate of photosynthetic electron transport was monitored as a photoreduction of DCPIP.The measurements were carried out in phosphate buffer (0.02 mol/L, pH 7.2) containing sucrose (0.4 mol/L), MgCl 2 (0.005 mol/L) and NaCl (0.015 mol/L).The chlorophyll content was 30 mg/L in these experiments, and the samples were irradiated (~100 W/m 2 with 10 cm distance) with a halogen lamp (250 W) using a 4 cm water filter to prevent warming of the samples (suspension temperature 4 °C).The studied compounds were dissolved in DMSO due to their limited water solubility.The applied DMSO concentration (up to 4%) did not affect the photochemical activity in spinach chloroplasts.The inhibitory efficiency of the studied compounds was expressed by IC 50 values, i.e., by molar concentration of the compounds causing 50% decrease in the oxygen evolution rate relative to the untreated control.The comparable IC 50 value for a selective herbicide 3-(3,4-dichlorophenyl)-1,1-dimethylurea, DCMU (Diuron ® ) was about 1.9 μmol/L.The results are summarized in Table 1.

Study of fluorescence of chlorophyll a in spinach chloroplasts
The fluorescence emission spectra of chlorophyll a (Chla) in spinach chloroplasts were recorded on fluorescence spectrophotometer F-2000 (Hitachi, Tokyo, Japan) using excitation wavelength λ ex = 436 nm for monitoring fluorescence of Chla, excitation slit 20 nm and emission slit 10 nm.The samples were kept in the dark for 2 min before measuring.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 the chloroplast suspension in DMSO solution.The DMSO concentration in all samples was the same as in the control (10% (v/v)).The chlorophyll concentration in the chloroplast suspension was 10 mg/L.

Table 1 .
Structure 2.Fluorescence emission spectra of chlorophyll a in untreated spinach chloroplasts in presence of compound 2b: 0, 102, 204, 408 and 612 μmol/L (curves from top to bottom;  ex = 436 nm; chlorophyll concentration 10 mg/L).All reagents were purchased from Aldrich (Sigma-Aldrich, St. Louis, MO, USA) and Merck (Merck, Darmstadt, Germany).TLC experiments were performed on alumina-backed silica gel 40 F254 plates (Merck, Darmstadt, Germany).The plates were illuminated under UV (254 nm) and evaluated in iodine vapour.The melting points were determined on 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 FT-IR spectrometer (Thermo Electron Corporation, West Palm Beach, FL, USA).The spectra were by accumulation of 256 scans with 2 cm −1 resolution in the region of 4000-600 cm −1 .All 1 H-and 13 C-NMR spectra were recorded on an Agilent 300 MHz VNMR spectrometer (299.96MHz for 1 H and 75.43 MHz for  [nm]EXPERIMENTAL General13C, Agilent Technologies, Santa Clara, CA, USA) in DMSO-d 6 .Chemical shifts () are reported in ppm.Mass spectra were measured using a LTQ Orbitrap Hybrid Mass Spectrometer (Thermo Electron Corporation) with direct injection into an APCI source (400 °C) in the positive mode.