Some Anilides of 2-Alkylthio- and 2-Chloro-6-Alkylthio-4- Pyridinecarboxylic Acids: Synthesis and Photosynthesis- Inhibiting Activity

Many compounds containing a -CONH- group display photosynthesis inhibiting activity. Based on this structural feature, a group of anilides of 2-alkylthio-(1b-4f) or 2-chloro-6-alkylthio-4-pyridinecarboxylic acids (5a-6c) was synthesised. The prepared compounds were tested for their inhibition of the oxygen evolution rate (OER) in spinach chloroplasts. A quasi-parabolic dependence between photosynthesis-inhibiting activity and the lipophilicity of the compounds was determined for 1b-4f as well as for 5a-6c. The inhibitory activity of compounds 1b-4f was higher than that of 5a-6c for comparable lipophilicity values.


Introduction
Many herbicides acting as photosynthesis inhibitors possess in their molecules an >N-C(=X)group (X=O or N, not S) and a hydrophobic residue in close vicinity to this group.Shipman concluded that the hydrophilic part of a herbicide binds electrostatically to the terminus of an α-helix at a highly charged amino acid, whereas the hydrophobic part of the inhibitors extends into the hydrophobic part of the membrane [1].Recently, pronounced photosynthesis-inhibiting activity has been found for alkoxy substituted phenylcarbamates [2,3] as well as for the local anesthetic of the anilide typetrimecaine [4,5,6], i.e., for compounds with -CONH-groups in their molecules.

Results and Discussion
The synthesis of the anilides is shown in Schemes 1 and 2. The 2-alkylthio-4-cyanopyridines were synthesised as described previously [8].Subsequent treatment with ethanolic sodium hydroxide solution afforded the corresponding acids 1-4.2-chloro-6-alkylthio-4-pyridinecarboxylic acids 5 and 6 were obtained by a similar procedure from 2-chloro-6-alkylthio-4-carbamoylpyridines [9].The anilides were prepared from the acids by reaction of the corresponding acyl chlorides with substituted anilines and aminophenols (Scheme 3).The melting points, yields, and elemental analyses for compounds are given in Tables 2 and 4, and IR and 1 H-NMR spectroscopic data in Tables 3 and 5.
A quasi-parabolic dependence of photosynthesis-inhibiting activity upon the lipophilicity (log P) of the compounds was determined for 1b-4f as well as for 5a-6c (Table 1).The comparison of the biological activity of compounds 1b-4f and 5a-6c having the same lipophilicity showed that the introduction of a halogen substituent in the 6 position led to a partial decrease of the biological activity.The previous study with anilides of 2-alkyl-4-pyridinecarboxylic acids showed that the site of their inhibitory action is the intermediate Z + /D + corresponding to the tyrosine radicals Tyr Z and Tyr D iii, iv which are situated at 161th position in D 1 and D 2 proteins located on the donor side of photosystem (PS) 2 [10].The same site of action in the photosynthetic apparatus of spinach chloroplasts can also be expected for the studied compounds 1b-4f and 5a-6c.
From the quasi-parabolic course of the dependence log (1/IC 50 ) vs. log P it can be assumed that the most active inhibitors are compounds with sufficiently high lipophilicity for securing their passage through the lipidic parts of the biological membranes, but enabling also their sufficiently high concentration in the aqueous phase.This is necessary for their interaction with the intermediates Z + /D + situated at the lumenal side of photosynthetic membranes in D 1 and D 2 proteins [11].

General
Column chromatography was performed on silica gel (Silpearl, Kavalier Votice).Melting points were determined on a Kofler block, and are uncorrected.IR spectra were recorded on a Nicolet Impact 400 spectrometer in chloroform. 1 H-NMR spectra were determined for solutions in CDCl 3 or DMSO (substituted 4-pyridinecarboxylic acids) with a BS 587 (Tesla, Brno) 80 MHz apparatus or a Varian Mercury -Vx BB 300 spectrometer operating at 300 MHz.Chemical shifts were recorded as δ values in parts per million (ppm), and were indirectly referenced to tetramethylsilane via the solvent signal (7.26 for 1 H).Multiplicities are given together with the coupling constants (in Hz).Elemental analyses were performed on a EA 1110 CHNS-O CE INSTRUMENTS elemental analyser.Lipophilicity of the compounds was computed using a program ACD/LogP version 1.0 (Advanced Chemistry Development Inc., Toronto).

Synthesis of 2-alkylthio, 2-phenylmethylthio and 2-chloro-6-alkylthio-4-pyridinecarboxylic acids 1-6
The 2-or 2,6-substituted 4-cyano or carbamoylpyridine (10 mmol) in 10 mL of ethanol was mixed with 25% aqueous sodium hydroxide (30 mmol) and refluxed until the evolution of the ammonia ceased.The reaction mixture was then diluted with twice its volume of water and acidified with 10% hydrochloric acid to pH 4-5.The crude product was collected, washed with water, and recrystallised from aqueous ethanol.TLC for checking of the purity of final products was performed using hexaneethyl acetate-acetic acid (50:45:5) as the mobile phase.The yields, melting points, and elemental analyses are given in Table 2, IR spectral data and 1 H-NMR chemical shifts in Table 3.

b,e,f; 5a-c; 6a-c).
A mixture of the 2-or 2,6 substituted-4-pyridinecarboxylic acid (10 mmol) and thionyl chloride (15 mmol) in 10 mL of dry benzene was refluxed for about 1 h.The excess of thionyl chloride was removed by repeated evaporation of dry benzene solutions in vacuo.The resulting crude acyl chloride dissolved in 10 mL of dry acetone was added dropwise to a stirred solution of substituted aniline or aminophenol (10 mmol) in 10 mL of dry pyridine keeping the temperature at 10 °C.After addition of the aniline or aminophenol was complete, stirring at 10 °C was continued for another 30 min.The low temperature was essential in the case of aminophenols in order to avoid the partial esterification of acyl chloride.The reaction mixture was poured into 40 mL of cold water.Crude anilide was collected and recrystallised from aqueous ethanol.TLC was performed using hexane-ethyl acetate (50:50) as the mobile phase.The yields, melting points and elemental analyses of the anilides are given in Table 4, IR spectral data and 1 H-NMR chemical shifts in Table 5.
Chlorophyll content in the samples was 30 mg.dm -3 and the samples were irradiated (~ 100 W.m -2 ) from 10cm distance with a halogen lamp (250 W) using a water filter to prevent warming of the samples (suspension temperature 22 o C).The compounds were dissolved in dimethyl sulfoxide (DMSO) because of their limited water solubility.The applied DMSO concentration (up to 5 %) did not affect OER.

Table 1 .
IC 50 values for inhibition of oxygen evolution rate in spinach chloroplasts and calculated logP values of the compounds tested..

Table 4 .
Analytical data of the prepared anilides.

Table 5 .
IR and 1 H-NMR spectroscopic data of the prepared anilides.