Preparation and Herbicidal Activity of Halogenated 8-Hydroxyquinoline-2-carboxanilides

In this study a series of twelve ring-substituted 8-hydroxyquinoline2-carboxanilides was prepared and characterized. The discussed compounds were prepared by using 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 compounds were found to inhibit PET in photosystem II. Significant PET-inhibiting activity was observed for meta-substituted compounds showing IC50 values close to that of photosystem II herbicide DCMU (3-(3,4-dichlorophenyl)-1,1-dimethylurea, IC50 = 1.9 μmol/L). N-(3-Fluorophenyl)-8-hydroxyquinoline-2-carboxamide showed the highest PET inhibition. The activity of metaand para-substituted compounds strongly decreased with lipophilicity increase and electron-withdrawing effect. No influence of any of these parameters on PET-inhibiting activity of ortho-substituted compounds was observed.

Although at present approximately 20 mechanisms of action of herbicides are known [21], over 50% of commercially available herbicides act by reversible binding to photosystem II (PS II), a membrane-protein complex in the thylakoid membranes, which catalyses the oxidation of water and the reduction of plastoquinone [22], and thereby inhibit photosynthesis [23][24][25].Some organic compounds, possessing an amide (-NHCO-) group, e.g., substituted anilides [11,[15][16][17][18]20], or a wide variety of compounds containing the quinoline system [9,10,[12][13][14]19] were found to interact with tyrosine radicals Tyr Z and Tyr D (or their surroundings) which are situated in D 1 and D 2 proteins on the donor side of PS II.Due to this interaction, interruption of the photosynthetic electron transport occurs.In the context of the previously-described azanaphtalenes and their isosteres [7][8][9][10][11][12][13][14][15][16][17][18][19][20], new simple modifications of quinoline that can possess interesting biological activity were investigated.The compounds were tested for their photosynthesis-inhibiting activitythe inhibition of photosynthetic electron transport in spinach chloroplasts (Spinacia oleracea L.).Relationships between the structure and the inhibitory activity related to inhibition of photosynthetic electron transport (PET) in spinach chloroplasts of the new compounds are discussed.

RESULTS AND DISCUSSION
All the studied compounds were prepared according to Scheme 1. Microwave-assisted synthesis [17,18,20] facilitated the process of obtaining ring-substituted 8-hydroxyquinoline-2-carboxanilides, thus synthesis of the target compounds was carried out only by one step.At first the carboxyl group was activated with phosphorus trichloride.The final anilide was immediately formed by aminolysis of the acyl chloride by ring-substituted aniline in dry chlorobenzene.All the compounds were recrystallized from ethanol.Scheme 1. Synthesis of ring-substituted 8-hydroxyquinoline-2-carboxanilides 1a-4c: (a) PCl 3 , chlorobenzene, MW.
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.Compounds showed a wide range of lipophilicities, with log P values from 2.59 (compounds 2a, R = 2-F and 2c, R = 4-F) to 3.44 (compound 4b, R = 3-CF 3 ) within the series of ring-substituted 8-hydroxyquinoline-2-carboxanilides.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 0.06 (compound 1a, R = 2-F) to 0.51 (compounds 4a, R = 2-CF 3 and 4c, R = 4-CF 3 ).) and the lipophilic or electronic properties of the individual anilide substituents in compounds 1a-4c were performed, see Fig. 1 and Fig. 2. Based on the obtained results it is evident that meta substitution of aniline ring is preferred.Figure 1 (dependences between PET inhibition and electronic σ properties of the anilide substituents) illustrates the general trend: PET inhibition strongly decreases within individual meta-and para-series with electron-withdrawing substituent (F > Cl > Br > CF 3 ).On the other hand, the biological activity is also affected by the lipophilicity of studied compounds 1a-4c, see Figure 2, where dependences of PET inhibition on log P are illustrated.In general, the dependences of log (1/IC 50 ) on log P show a similar trend as in case of electronic σ properties.The activity of the discussed compounds decreases with lipophilicity increase.It can be stated that both parameters had considerable influence on PET-inhibiting activity of meta-and para-substituted compounds, while in case of ortho-substituted compounds the influence of both observed parameters on PET inhibition was insignificant.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.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 .Because 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 halogenated 8-hydroxyquinoline-2-carboxanilides is situated 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-) [27], anilides of 2-alkylpyridine-4-carboxylic acids acting in the intermediates Z  /D  [28] and 2-alkylsulphanyl-4-pyridinecarbothioamides acting in the D  intermediate [29].
Interaction of the studied compounds with aromatic amino acids, which are present in the proteins of spinach chloroplasts situated in PS II, was documented by quenching their fluorescence at 340 nm. Figure 3 presents fluorescence emission spectra of aromatic amino acids of untreated spinach chloroplasts and of chloroplasts treated with increasing concentrations of compound 3c.Binding of these compounds to aromatic amino acids occurring in photosynthetic proteins contributes to PET inhibition.Similar fluorescence quenching of aromatic amino acids present in PS II was observed for other tested halogenated 8-hydroxyquinoline-2-carboxanilides as well as previously studied N-benzylpyrazine-2-carboxamides [30] and 5-bromo-and 3,5-dibromo-2-hydroxy-N-phenylbenzamides [31].C spectra with digital resolution 0.3 Hz or better.Chemical shifts () are reported in ppm.When necessary, additional experiments were done: 13 C-APT (Attached Proton Test) for discrimination between CH and quaternary carbons; DQF COSY, HSQC and HMBC for through-bond 1 H-1 H and one-and multiple-bond 1 H- 13 C correlations.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.

Study of photosynthetic electron transport (PET) inhibition in spinach chloroplasts
Chloroplasts were prepared from spinach (Spinacia oleracea L.) according to Masarovicova and Kralova [32].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 (DCIPP) according to Kralova et al. [33], 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 (Diurone ® ) was about 1.9 μmol/L.The results are summarized in Table 1.

Study of fluorescence of aromatic amino acids in spinach chloroplasts
The fluorescence emission spectra of aromatic amino acids in spinach chloroplasts were recorded on fluorescence spectrophotometer F-2000 (Hitachi, Tokyo, Japan) using excitation wavelength  ex = 275 nm for monitoring fluorescence of aromatic amino acids, excitation slit 20 nm and emission slit 10 nm.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 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.