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

Abstract

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 type – trimecaine [4,5,6], i.e., for compounds with -CONH- groups in their molecules.

In order to bring together the structural features of the abovementioned compounds and our previous results with 2-alkyl-4-pyridinecaboxylic acids anilides [7], some anilides of 2-alkylthio-4-pyridinecarboxylic (1b-4f) and 2-chloro-6-alkylthio-4-pyridinecarboxylic acids (5a-6c) were synthesised.

Results and Discussion

The synthesis of the anilides is shown in Scheme 1 and Scheme 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 Table 2 and Table 4, and IR and ¹H-NMR spectroscopic data in Table 3 and Table 5.

The biological activity of anilides of 2-alkylthio-4-pyridinecarboxylic acids (1b-4f) and 2-chloro-6- alkylthio-4-pyridinecarboxylic acids (5a-6c) with regards to inhibition of oxygen evolution rate in spinach chloroplasts was investigated. The inhibitory activity of the compounds has been expressed by IC₅₀ values (

Table 1. IC₅₀ values for inhibition of oxygen evolution rate in spinach chloroplasts and calculated logP values of the compounds tested.

Compd.	IC50·106 (mol.dm-3)	calculated logP	Compd.	IC50·106 (mol.dm-3)	calculated logP
1b	7.3	4.55 ± 0.44	4b	6.0	5.13 ± 0.46
2b	8.0	4.90 ± 0.45	4f	35.2	7.01 ± 0.55
2f	13.9	6.79 ± 0.54	5a	32.5	4.03 ± 0.44
3a	12.7	4.24 ± 0.42	5b	14.2	5.41 ± 0.46
3b	4.8	5.62 ± 0.44	5c	16.2	4.64 ± 0.45
3d	11.3	6.66 ± 0.58	6a	19.7	4.57 ± 0.44
3f	69.1	7.50 ± 0.54	6b	18.3	5.94 ± 0.46
4a	10.3	3.75 ± 0.43	6c	14.2	5.17 ± 0.45

). The IC₅₀ values, i.e. molar concentrations of the compounds causing 50 % activity decrease with respect to the untreated control, varied in the range of 6.0 - 69.1 μmol.dm^-3 for 1b-4f and 14.2 - 32.5 μmol.dm^-3, respectively, for 5a-6c.

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 which are situated at 161th position in D₁ and D₂ 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/IC₅₀) 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 D₁ and D₂ proteins [11].

Acknowledgements

This study was supported by the Ministry of Education, Youth and Sports of the Czech Republic (Research Plan No. 111600001), by the Grant Agency of Charles University (Grant No. 26/1998- BCH), and by the Scientific Grant Agency VEGA of the Slovak Republic (No. 1/7262/20).

Experimental

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. ¹H-NMR spectra were determined for solutions in CDCl₃ 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 ¹H). 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-4-cyanopyridines and 2-phenylmethylthio-4-cyanopyridine.

2-Chloro-4-cyanopyridine (10 mmol) and the appropriate thiol (10 mmol) were dissolved in 10 mL of anhydrous N,N-dimethylformamide. To this solution sodium methoxide (10 mmol) in 5 mL methanol was added dropwise with stirring under a nitrogen atmosphere at 20 °C. The stirring was continued until TLC (6:1 hexane-ethyl acetate) indicated completion of the reaction. The reaction mixture was concentrated in vacuo and after evaporation of the solvents, the 2-alkylthio-4- cyanopyridines or 2-phenylmethylthio-4-cyanopyridine were distilled off from the oily product. The boiling points corresponded with the previously described [8].

Synthesis of 2-chloro-6-alkylthio-4-carbamoylpyridines

2,6-Dichloro-4-carbamoylpyridine [13] (10 mmol) and the appropriate thiol (10 mmol) were dissolved in anhydrous N,N-dimethylformamide (10 mL). To the stirred solution sodium methoxide (10 mmol) in methanol (5 mL) was added dropwise. The reaction mixture was stirred at room temperature until TLC (2:1 hexane-ethyl acetate) indicated the reaction was complete. The mixture was then poured into cold water. The crude product was filtered off, purified by column chromatography (2:1 hexane-ethyl acetate), and recrystallised from aqueous ethanol. The boiling points and spectral data agreed with those previously described [9].

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. Analytical data of the prepared 2-alkylthio- and 2-chloro-6-alkylthio-4-pyridinecarboxylic acids.

Compd.	Formula M. w.	R X	M. p. °C Yield %	Calculated / Found
				% C	% H	% N	%S	%Cl
1	C9H11NO2S	C3H7	141-143	54.80	5.62	7.10	16.25	-
	197.3		80	54.55	5.79	6.95	16.08
2	C10H13NO2S	iC4H9	137-139	56.85	6.20	6.63	15.17	-
	211.3		78	56.61	6.41	6.46	14.93
3	C11H15NO2S	C5H11	135-137	58.64	6.71	6.22	14.23	-
	225.3		82	58.48	6.89	6.05	14.02
4	C13H11NO2S	CH2C6H6	196-197 a)	-	-	-	-	-
	245.3		76
5	C9H10ClNO2S	C3H7	119-120	46.66	4.35	6.05	13.84	15.30
	231.7	Cl	77	46.45	4.21	6.19	13.65	15.55
6	C10H12ClNO2S	C4H9	93-95	48.88	4.92	5.70	13.05	14.43
	245.7	Cl	75	48.67	4.85	5.82	12.87	14.65

a) ref. [12] M. p. 195-196°C

, IR spectral data and ¹H-NMR chemical shifts in

Table 3. IR and ¹H-NMR spectroscopic data of the 2-alkylthio and 2-chloro-6-alkylthio-4-pyridinecarboxylic acids (DMSO).

Compd.	IR (cm-1)	δ 1H-NMR (ppm)
1	2975, 2935, 2890 (CH-aliph.)	(CDCl3): 1.06 t, J=7.3 Hz, 3H, CH3; 1.76 m, 2H, CH2; 3.18 t,
	2480 (COOH)	J= 7.3 Hz, 2H, SCH2; 7.56 m, 1H, H-5; 7.78 m, 1H, H-3; 8.59
	1725 (CO)	m, 1H, H-6 a)
2	2960, 2930, 2870 (CH-aliph.)	(CDCl3): 1.05 d, J=6.6 Hz, 6H, 2xCH3; 1.96 m, 1H, CH<; 3.11
	2470 (COOH)	d, J= 6.9 Hz, 2H, SCH2; 7,53 d, J=4.95 Hz, 1H, H-5; 7.78 m,
	1725 (CO)	1H, H-3; 8.59 d, J=4.95 Hz, 1H, H-6 a)
3	2970, 2925, 2860 (CH-aliph.)	(CDCl3 ): 0.91 t, J=7 Hz, 3H, CH3; 1.3 - 1.5 m, 4H, 2xCH2; 1.73
	2450 (COOH)	m, 2H, CH2; 3.19 t, J=7.3 Hz, 2H, SCH2; 7.56 d, J=4.95 Hz, 1H,
	1730 (CO)	H-5; 7.79 m, 1H, H-3; 8.62 d, J=4.95 Hz, 1H, H-6 a)
5	2970, 2934, 2874 (CH-aliph.)	(DMSO-d6): 1.05 t, J=7.2 Hz, 3H, CH3; 1.47-2.10 m, 2H, CH2;
	2658 (COOH)	3.18 t, J=7.1 Hz, 2H, SCH2; 7.54 d, J=0.7 Hz, 1H, H-5; 7.67 d,
	1705 (CO)	J=0.7 Hz, 1H, H-3; 11.65 s, 1H, COOH
6	2962, 2933, 2873 (CH-aliph.)	(DMSO-d6): 0.96 t, J=6.2 Hz, 3H, CH3; 1.21-1.98 m, 4H, CH2;
	2541 (COOH)	3.20 t, J=7.1 Hz, 2H, SCH2; 7.53 d, J=1.1 Hz, 1H, H-5; 7.66 d,
	1707 (CO)	J=1.1 Hz, 1H, H-3; 11.62 s, 1H, COOH

a) A COOH signal was not observed in the ¹H-NMR spectrum.

.

General method for the synthesis of anilides of 2-alkylthio, 2-phenylmethylthio and 2-chloro-6- alkylthio-4-pyridinecarboxylic acids (1b,d; 2b,f; 3a,b,d-f; 4a,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. Analytical data of the prepared anilides.

Compd.	Formula M. w.	R Y, Z	X1 X2	M. p. °C Yield %	Calculated / Found
					% C	% H	% N	% S	% Cl(Br,F)
1b	C15H15ClN2O2S	SC3H7	5´-Cl	161-163	55.81	4.68	8.68	9.93	10.98
	322.8	2´-OH, H	H	57	55.96	4.52	8.49	10.06	10.76
1d	C15H14Br2N2O2S	SC3H7	3´-Br	152-153	40.38	3.16	6.28	7.19	35.82
	446.2	4´-OH, H	5´-Br	65	40.41	3.23	6.22	7.12	35.71
2b	C16H17ClN2O2S	S-iC4H9	5´-Cl	162-164	57.05	5.09	8.32	9.52	10.53
	336.8	2´-OH, H	H	58	57.11	5.07	8.27	9.56	10.45
2f	C18H16F6N2OS	S-iC4H9	3´-CF3	164-165	51.18	3.82	6.63	7.59	26.99
	422.4	H, H	5´-CF3	54	51.31	3.72	6.48	7.75	26.78
3a	C17H20N2O2S	SC5H11	H	123-125	64.53	6.37	8.85	10.13	-
	316.4	2´-OH, H	H	60	64.48	6.45	8.71	10.28
3b	C17H19ClN2O2S	SC5H11	5´-Cl	153-155	58.19	5.46	7.98	9.14	10.10
	350.9	2´-OH, H	H	58	58.23	5.39	7.88	9.19	10.01
3d	C17H18Br2N2O2S	SC5H11	3´-Br	120-122	43.06	3.83	5.91	6.76	33.70
	474.2	4´-OH, H	5´-Br	67	43.15	3.81	5.77	6.67	33.50
3e	C17H19BrN2OS	SC5H11	4´-Br	94-95	53.83	5.05	7.39	8.45	21.07
	379.3	H, H	H	52	53.97	4.93	7.23	8.31	20.88
3f	C19H18F6N2OS	SC5H11	3´-CF3	122-124	52.29	4.16	6.42	7.35	26.12
	436.4	H, H	5´-CF3	56	52.16	4.21	6.36	7.21	25.95
4a	C19H16N2O2S	SCH2C6H5	H	149-150	67.84	4.79	8.33	9.53	-
	336.4	2´-OH, H	H	60	67.57	4.97	8.09	9.75
4b	C19H15ClN2O2S	SCH2C6H5	5´-Cl	194-196	61.54	4.08	7.55	8.66	9.56
	370.9	2´-OH, H	H	78	61.65	3.86	7.41	8.72	9.38
4e	C19H15BrN2OS	SCH2C6H5	4´-Br	108-109	57.15	3.79	7.02	8.03	20.01
	339.3	H, H	H	55	57.31	3.71	6.87	8.14	19.85
4f	C21H14F6N2OS	SCH2C6H5	3´-CF3	141-143	55.26	3.09	6.14	7.02	24.98
	456.4	H, H	5´-CF3	61	55.38	3.01	6.03	7.18	24.75
5a	C15H15ClN2O2S	SC3H7	H	138-139	55.81	4.68	8.68	9.93	10.98
	322.81	2´-OH, Cl	H	44	55.68	4.51	8.79	9.72	11.21
5b	C15H14Cl2N2O2S	SC3H7	5´-Cl	152-153	50.43	3.95	7.84	8.97	19.85
	357.25	2´-OH, Cl	H	53	50.33	3.91	7.72	8.85	20.07
5c	C15H14Cl2N2O2S	SC3H7	3´-Cl	144-146	50.43	3.95	7.84	8.97	19.85
	357.25	4´-OH, Cl	H	34	50.29	3.86	7.95	8.81	20.03
6a	C16H17ClN2O2S	SC4H9	H	123-125	57.05	5.09	8.32	9.52	10.53
	336.84	2´-OH, Cl	H	56	56.91	5.02	8.48	9.39	10.79
6b	C16H16Cl2N2O2S	SC4H9	5´-Cl	158-160	51.76	4.34	7.55	8.63	19.10
	371.28	2´-OH, Cl	H	59	51.58	4.31	7.68	8.41	19.31
6c	C16H16Cl2N2O2S	SC4H9	3´-Cl	115-117	51.76	4.34	7.55	8.63	19.10
	371.28	4´-OH, Cl	H	53	51.63	4.23	7.71	8.38	19.33

, IR spectral data and ¹H-NMR chemical shifts in

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

Compd.	IR (cm-1)	δ 1H-NMR (ppm)
1b	2965, 2932, 2873	(DMSO-d6): 1.00 t, J=7, 3H, CH3; 1.66 m, 2H, CH2; 3.18 t, J=7, 2H, SCH2;
	(CH aliph.),	6.93 d, J=8.5, 1H, H-3´; 7.12 dd, J=8.5, J=2.4, 1H, H-4´; 7.53 dd, J=5.1,
	1651 (CO)	J=1.5, 1H, H-5; 7.73 qs, 2H, H-3 and H-6´; 8.60 d, J=5.1, 1H, H-6; 9.82 s,
		1H, OH or NH; 10.08 s, 1H, NH or OH
1d	2975, 2945, 2890	(DMSO-d6): 1.00 t, J=7, 3H, CH3; 1.75 m, 2H, CH2; 3.18 t, J=7, 2H, SCH2;
	(CH aliph.),	7.52 d, J=5.1, 1H, H-5; 7.71 s, 1H, H-3; 8,00 s, 2H, H-2´and H-6´; 8.62 d,
	1650 (CO)	J=5.1, 1H, H-6; 10.45 s, 1H, OH or NH; 10.51 s, 1H,NH or OH
2b	2975, 2940, 2890	(DMSO-d6): 1.01 d, J=7, 6H, 2xCH3; 1.92 m, 1H, -CH<; 3.13 t, J=7.5, 2H,
	(CH aliph.),	SCH2; 6.95 d, J=8.5, 1H, H-3´; 7.13 dd, J=8.5, J=2.5, 1H, H-4´; 7.52 dd, J=5,
	1655 (CO)	J=1, 1H, H-5; 7.73 dd, J=1, J<1, 1H, H-3; 7.74 d, J=2.5, 1H, H-6´; 8.59 dd,
		J=5, J<1, 1H, H-6
2f	2962, 2929, 2869	(CDCl3) 1.05 d, J=6.4, 6H, 2xCH3; 1.94 m, 1H, CH; 3.14 d, J=6.7, 2H,
	(CH aliph.),	SCH2; 7.31 dd, J=5.2, J=1.5, 1H, H-5; 7.55 d, J=1.5, 1H H-3; 7.68 s, 1H, H-
	1662 (CO)	4´; 8.17 s, 3H, H-2´, H-6´ and NH; 8.57 d, J=5.2, 1H, H-6
3a	2958, 2931, 2859	(DMSO-d6): 0.88 dist.t, CH3; 1.38 m, 4H, 2xCH2; 1.66 m, 2H, CH2; 3.19 t,
	(CH aliph.),	2H, CH2; 6.96 m, 3H, arom.; 7.55 m, 2H, H-5 and 1H arom.; 7.74 s, 1H, H-
	1652 (CO)	3; 8.59 d, J=5.1, 1H, H-6; 9.66 s, 1H, OH; 9.77 bs, 1H, NH
3b	2975, 2940, 2870	(DMSO-d6): 0.88 dist. t, 3H, CH3; 1.36 m, 4H, (CH2)2; 1.63 m, 2H, CH2;
	(CH aliph.),	3.20 t, J=7, 2H, SCH2; 7.13 dd, J=8.5, J=2.5, 1H, H-4’; 7.53 dd, J=5, J=1.5,
	1640 (CO)	1H, H-5; 7.72 dd, J=1.5, J=1, 1H, H-3; 7.75 d, J=2.5, 1H, H-6´; 8.61 dd, J=5,
		J=1, 1H, H-6
3d	2980, 2945, 2875	(DMSO-d6): 0.88 dist. t, 3H, CH3; 1.37 m, 4H, (CH2)2; 1.66 m, 2H, CH2;
	(CH aliph.),	3.19 t, J=7, 2H, SCH2; 7.52 d, J=5.1, 1H, H-5; 7.71 s, 1H, H-3; 8,00 s, 2H,
	1655 (CO)	H-2´and H-6´; 8.61 d, J=5.1, 1H, H-6; 9.84 s, 1H, OH or NH; 10.44 s, 1H,
		NH or OH
3e	2952, 2926, 2852	(CDCl3) 0.91 dist. t, 3H, CH3; 1.38 m, 4H 2xCH2; 1.71 m, 2H, CH2;
	(CH aliph.),	3.19 t, J=7, 2H, SCH2; 7.27 dd, J=5, J=1.5, 1H, H-5; 7.49 qs, 5H, H-3 and
	1658 (CO)	C6H4; 8.01 bs, 1 H, NH; 8.52 d, J= 5, 1H, H-6
3f	2958, 2930, 2858	(CDCl3) 0.91 dist. t, 3H, CH3; 1.38 m, 4H, 2xCH2; 1.71 m, 2H, CH2; 3.22 t,
	(CH aliph.),	J=7, 2H, SCH2; 7.32 dd, J=5.2, J=1.5, 1H, H-5; 7.55 d, J=1.5, 1H, H-3; 7.68
	1669 (CO)	s, 1H, H-4´; 8.17 qs, 3H, H-2´, H-6´and NH; 8.58 d, J=5.2, 1H, H-6
4a	1660 (CO)	(CDCl3) 4.49 s, 2H, SCH2; 6.95 m, 1H, H-4´ or H-5´; 7.03 m, 1H, H-3´ or H-
		6´; 7.16 m, 1H, H-5´ or H-4´; ca.7.25 m overlapped with the signal of
		solvent, H-6´ or H-3´; 7.27-7.45 m, 5H, C6H5; 7.39 overlapping dd, J=5.2
		Hz, J=1.6 Hz, 1H, H-5; 7.58 m, 1H, H-3; 7.75 bs, 1H, NH; 8.14 bs, 1H, OH;
		8.63 d, J=5.2 Hz, 1H, H-6
4b	1655 (CO)	(CDCl3) 4.49 s, 2H, SCH2; 6.95 d, J=8.7 Hz, 1H, H-3´; 7.11 dd, J=8.7 Hz,
		J=2.5, 1H, H-4´; 7.27-7.45 m, 5H, C6H5; 7.37 overlapping dd, J=5.2 Hz,
		J=1.6 Hz, 1H, H-5; 7.55 m, 1H, H-6´; 7.56 m, 1H, H-3; 8.09 bs, 1H, NH;
		8.64 dd, J=5.2 Hz, J=0.8 Hz,1H, H-6
4e	1653 (CO)	(CDCl3) 4.45 s, 2H, SCH2; 7.31 m, 6H, H-5 and C6H5; 7.45 qs, 5H, H-3 and
		C6H4; 7.98 bs, 1 H, NH; 8.53 d, J= 5.2, 1H, H-6
4f	1668 (CO)	(CDCl3) 4.46 s, 2H, SCH2; 7.36 m, 7H, H-3, H-5 and C6H5; 7.67 s, 1H, H-4´;
		8.10 s, 2H, H-2´and H-6´; 8.24 bs, 1 H, NH; 8.57 d, J=5.2, 1H, H-6
5b	2965, 2931, 2872	(DMSO-d6): 0.86-1.33 m, 3H, CH3; 1.50-2,02 m, 2H, CH2; 3.20 t,
	(CH aliph.),	J=7.2 Hz, 2H, CH2; 6.90 d, J=8.5 Hz, 1H, H-3´; 7.00-7.23 m, 1H,
	1655 (CO)	NH; 7.10 dd, J1=2.2 Hz, J2=8.5 Hz, 1H, H-4´; 7.34 d, J=1.2 Hz, 1H,
		H-5; 7.46 d, J=1.2 Hz, 1H, H-3; 7.69 d, J=2.2 Hz, 1H, H-6´; 8.18 s,
		1H, -OH
5c	2965, 2931, 2872	(DMSO-d6): 0.90-1.18 m, 3H, CH3; 1.46-2.04 m, 2H, CH2; 3.18 t,
	(CH aliph.),	J=7.1 Hz, CH2; 5.57 s, 1H, NH; 6.98 d, J=8.8 Hz, 1H, H-5´; 7.28 dd,
	1654 (CO)	J1=2.4 Hz, J2=8.8 Hz, 1H, H-6´; 7.29 d, J=1.2 Hz, 1H, H-5; 7.41 d,
		J=1.2 Hz, 1H, H-3; 7.73 d, J=2.4 Hz, 1H, H-2´; 7.83 s, 1H, OH
6a	2958, 2931, 2872	(DMSO-d6): 0.78-1.10 m, 3H, CH3; 1.5-1.95 m, 4H, CH2CH2; 3.18 t,
	(CH aliph.),	J=7.0 Hz, 2H, CH2; 6.70-7.26 m, 3H, H-4´, H-5´, H-6´; 7.32 d, J=1.0
	1646 (CO)	Hz, 1H, H-5; 7.43 d, J=1,2 Hz, 1H, H-3; 7.58 d, J=7.8 Hz, 1H, H-3´;
		7.72 s, 1H, NH; 8.47 s, 1H, OH
6b	2959, 2931, 2872	(DMSO-d6): 0.73-1.32 m, 3H, CH3; 1.50-2.02 m, 4H, CH2CH2; 3.22 t,
	(CH aliph.),	J=7.2 Hz, 2H, CH2; 6.90 d, J=8.3 Hz, 1H, H-3´; 7.00-7.23 m, 1H,
	1656 (CO)	NH; 7.10 dd overlapped by another signal, J1=2.2 Hz, J2=8.5 Hz, 1H,
		H-4´; 7.35 d, J=1.2 Hz, 1H, H-5; 7.45 d, J=1.2 Hz, 1H, H-3; 7.69 d,
		J=2.2 Hz, 1H, H-6´; 8.14 s, 1H, OH
6c	2959, 2930, 2872	(DMSO-d6): 0.96 t, J=6.4 Hz, 3H, CH3; 1.14-1.93 m, 4H, CH2CH2;
	(CH aliph.),	3.19 t, J=7.1 Hz, CH2; 5.62 s, H, NH; 7.27 dd ovelapped by d, J1=2.4
	1649 (CO)	Hz, J2=8.8 Hz, 2x1 H, H-5´, H-6´; 7.29 d, J=1.2 Hz, 1H, H-5; 7.40 d,
		J=1.2 Hz, 1H, H-3; 7.73 d, J=2.4 Hz, 1H, H-2´; 7.91 s, 1H, OH

.

Biological assays

Chloroplasts were prepared from locally purchased spinach according to the procedure described by Walker [14]. The oxygen evolution rate (OER) in spinach chloroplasts was determined spectrophotometrically (Specord UV VIS Zeiss Jena, Germany) by the Hill reaction. The measurements were carried out in phosphate buffer (20 mmol, pH = 7.2) containing sucrose (0.4 mol.dm^-3), MgCl₂ (5 mmol.dm^-3) and NaCl (15 mmol.dm^-3) using 2,6-dichlorophenol-indophenol as electron acceptor.

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 °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.