Preparation and Photosynthesis-Inhibiting Activity of 1-[(2-Chlorophenyl)carbamoyl]naphthalen-2-yl Alkylcarbamates

In this study a series of eight 1-[(2-chlorophenyl)carbamoyl]naphthalen-2-yl alkylcarbamates was prepared and characterized. The discussed compounds were prepared by microwave-assisted and conventional synthesis. The compounds were tested for their activity related to inhibition of photosynthetic electron transport (PET) in spinach (Spinacia oleracea L.) chloroplasts. The PET-inhibiting activity of the compounds was moderate; the highest activity within the series of compounds was observed for 1-[(2-chlorophenyl)carbamoyl]naphthalen-2-yl propylcarbamate, and the compounds were found to inhibit PET in photosystem II.

All the predicted molecular descriptors (lipophilicity, hydrophobic distributive parameters, molar volumes and surface) were calculated using the ACD/Percepta ver.2012 program (Advanced Chemistry Development, Toronto, ON, Canada), see Table 1.The lipophilicity of compounds 2-9, expressed as log P values, ranged from 3.58 (compound 2, R = C 2 H 5 ) to 7.22 (compound 9, R = C 8 H 17 ).Logically, lipophilicity increases with lengthening of the alkyl tail.Isopropyl showed lower lipophilicity value than propyl.For individual substituentsalkyl chains of the discussed compoundsalso hydrophobic properties expressed as distributive parameters π were predicted; they ranged from 0.99 to 4.18.Bulkiness (i.e.tail length/branching) of individual substituents expressed as molar volume MV [cm -3 ] and surface activity expressed as surface tension ST [dyne/cm] of the discussed carbamates were determined as other parameters that could influence PET-inhibiting activity.1D.Bilinear dependences of activity on individual descriptors can be observed in all Figures.The PET-inhibiting activity within the series linearly increased with the increase of log P values (as lipophilicity of the whole structure including intramolecular interactions, correlation coefficient r = 0.9541, n = 4, Fig. 1A), distributive parameter (influence of substituent R hydrophobicity, r = 0.9949, n = 4, Fig. 1B), molar volume (influence of substituent R bulkiness, r = 0.9949, n = 4, Fig. 1C) and with increasing surface tension (i.e. with a decrease of surface activity, r = 0.9851, n = 4, Fig. 1D) up to pentyl derivative 6.After this optimum, activity linearly decreased with the subsequent increase of lipophilicity (r = -0.9666,n = 4, Fig. 1A), distributive parameter (r = -0.9919,n = 4, Fig. 1B), molar volume (r = -0.9923,n = 4, Fig. 1C) and surface tension (i.e. with a surface activity decrease, r = -0.9956,n = 4, Fig. 1D).A better correlation coefficient estimated for the dependence of log(1/IC 50 ) on distributive parameter π characterizing the hydrophobicity of R substituent compared to that of log(1/IC 50 ) on log P characterizing the lipophilicity of the whole molecule indicates that the inhibitory activity is dominantly affected by R substituent causing perturbation of thylakoid membranes.On the other hand, in general limited PET-inhibiting activity of the compounds can be caused by possible interactions of amide and carbamate groups (responsible also for interactions with photosynthetic apparatus) with the spatially close NO 2 moiety in the ortho position of anilide ring [16][17][18][19]31].An increase of PET-inhibiting activity with the prolongation of the alkyl tail estimated for compounds 2, 3, 5, 6 is connected with the fact that a longer alkyl chain can be incorporated in the thylakoid membrane to a greater extent and subsequently causes membrane damage.This biological activity is connected with the surface activity of these compounds (they can be considered as non-ionic surfactants) and with the alkyl tail length (molar volume), which is again reflected in lipophilicity.Nevertheless, besides the above-mentioned physicochemical parameters, also the appropriate concentration of the compound at the site of action in the photosynthetic apparatus is important for PET-inhibiting activity.Consequently, a compound having poor water solubility cannot pass through the hydrophilic regions of the thylakoid membrane to reach the site of action, which results in a significant decrease of inhibitory activity.
The solubility of hexyl derivative 7 and especially derivatives 8 and 9 with longer alkyl chains was significantly lower than that of pentyl derivative 6, which resulted in a notable activity decrease.From the aspect of PET-inhibiting activity, the lipophilicity optimum can be found for C 5 , see Fig. 1.With the further elongation of the alkyl chain (hydrophobic part) to octyl, so called 'cut-off' effect, i.e. the loss of biological activity with the increasing lipophilicity of the compounds usually observed for amphiphilic compounds was manifested [5,28,[32][33][34].

D
The application of 2,5-diphenylcarbazide (DPC, artificial electron donor) that supplies electrons in the site of Z  /D  intermediate, i.e. tyrosine radicals Tyr Z and Tyr D (or their surroundings) that are situated in D 1 and D 2 proteins on the donor side of PS II [10], to chloroplasts, the activity of which was inhibited by the most active compound 6 (up to 30% of the control), caused practically complete PET restoration already at addition of 3-fold DPC concentration with regard to the applied concentration of compound 6.Therefore it can be concluded that the site of action of the studied 1-[(2-nitrophenyl)carbamoyl]naphthalen-2-yl alkylcarbamates is situated mainly on the donor side of PS II.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-) [35], anilides of 2-alkylpyridine-4-carboxylic acids [36], cationic surfactants [37,38] acting in the intermediates Z  /D  and 2-alkylsulphanyl-4-pyridinecarbothioamides acting in the D  intermediate [39].

General
All reagents were purchased from Aldrich (Sigma-Aldrich, St. Louis, MO, USA) and Alfa (Alfa-Aesar, Ward Hill, Massachusetts, USA).TLC experiments were performed on alumina-backed silica gel 60 F254 plates (Merck, Darmstadt, Germany).The plates were illuminated under UV (254 nm) and evaluated in iodine vapour.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 FT-IR spectrometer (Thermo Scientific, West Palm Beach, FL, USA).The spectra were obtained by 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 a JEOL ECZR 400 MHz NMR spectrometer (400 MHz for 1 H and 100 MHz for 13 C, JEOL, Tokyo, Japan) in DMSO-d 6 . 1 H and 13 C chemical shifts () are reported in ppm.Highresolution mass spectra were measured using a high-performance liquid chromatograph Dionex UltiMate ® 3000 (Thermo Scientific) coupled with a LTQ Orbitrap XL™ Hybrid Ion Trap-Orbitrap Fourier Transform Mass Spectrometer (Thermo Scientific) with injection into HESI II in the positive mode.The lipophilicity (log P) of the final compounds, the surface tension and the molar volume of R substituents were predicted using ACD/Percepta ver.2012 (Advanced Chemistry Development, Inc., Toronto, ON, Canada).
General procedure for synthesis of alkylcarbamates 2-9: N-(2-nitrophenyl)-2hydroxynaphthalene-1-carboxamide (1, 1.0 mmol) and triethylamine (1.1 mmol) were suspended in dry acetonitrile (10 mL).The solution of the appropriate alkyl isocyanate (1.2 mmol) in acetonitrile (5 mL) was added in four portions within 2 h, and the reacting mixture was stirred for 24 h at ambient temperature.The solvent was evaporated under reduced pressure, and the solid residue was washed with methanol and ethyl acetate to give pure product.Studied compounds 2-9 are presented in Table 1. 1

Study of photosynthetic electron transport (PET) inhibition in spinach chloroplasts
Chloroplasts were prepared from spinach (Spinacia oleracea L.) according to Masarovicova and Kralova [40].The inhibition of photosynthetic electron transport (PET) in spinach chloroplasts was determined spectrophotometrically (Genesys 6, Thermo Scientific), using the artificial electron acceptor 2,6-dichlorophenol-indophenol (DCPIP) according to Kralova et al. [41], 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 a 50% decrease in the oxygen evolution rate relative to the untreated control.The comparable IC 50 value for the selective herbicide 3-(3,4-dichlorophenyl)-1,1-dimethylurea, DCMU (Diuron ® ), was about 0.002 mmol/L.The results are summarized in Table 1.