Study of Microwave Irradiation Synthesis, Solvatochromism And Photosynthetic Activity of the 2-(4H-4-Oxo-Benzopyran-3-Yl) Benzothiazolium Salts

M. Lácová^a, D. Loos^b, M. Klestinec^a, A. Gáplovsky^b, K. Králová^b**, F. Sersen^b, J. Chovancová^a

^aDepartment of Organic chemistry, Faculty of Science, Communes University, SK-842 15 Bratislava, Slovakia, ^bInstitute of Chemistry, Faculty of Science, Comenius University, SK-84215 Bratislava, Slovakia, Tel. +421 7 60296338, Fax +421 7 65429064, E-mail: lacova@fns.uniba.sk

Introduction
The synthesis of different types 4-oxo-4H-1-benzopyrans (chromones) with electron - withdrawing substituents at C-3 has attracted a great deal of attention over the past several years [1 - 4]. These chromone derivatives are highly versatile molecules because their reactivity towards nucleophiles provides useful route to preparation of a variety of heterocyclic systems [1].
This work was aimed to prepare chromone systems bonding benzothiazolium or benzoxazolium groups at position 3 of chromone ring.
On the basis of our experimental work it was found that the prepared condensation products IV are very sensitive to water. We oriented our study to theoretical calculation of heats of formation and charge densities of the products. In the second part of this work we investigated condensation of starting compounds by focused microwave irradiation and the kinetics properties and photosynthetic activity of products.
Benzothiazolium salts and several natural and synthetic chromone derivatives were found to exhibit a wide spectrum of biological activities including antimicrobial and photosynthesis-inhibiting effectiveness as well [5 - 8]. It was found that these heterocyclic inhibitors inhibit photosynthetic electron transport in photosynthesizing organisms what was reflected in the inhibition of oxygen evolution rate.
The aim of this study was to investigate the inhibition of photosynthetic electron transport in the algal suspensions of Chlorella vulgaris and spinach chloroplasts by the studied 3-R₂-2-[2-6-R¹-chromon-3-yl)ethenyl]benzothiazolium halides (IV), i.e. by compounds containing in their molecules two heterocyclic skeletons and to determine the site of their action in the photosynthetic apparatus.

Experimental

Melting points of synthesized compounds were determined on a Kofler block and are uncorrected (Table 1.). Data of elemental analysis are in Table 1 and 2. ¹H NMR spectral were measured in DMSO - d₆ or CF₃COOH - d solution, on a TESLA BS 487 (80 MHz) instrument (Table 3.). UV VIS spectral measurements were realized on HP 8452 A - spectrophotometer and on fluorescence spectra on spectrofluorimeter Hitachi F - 2000.
All microwave assisted reactions were carried out in a Lavis - 1000 multi Quant microwave oven. The apparaturs has been adapted for laboratory application with magnetic stirring and an external reflux condenser.
Chemical Part
3-Benzyl-2-methylbenzothiazolium bromide III
A stirred mixture of 2-methylbenzothiazole (0.5 g, 3.35 mmol) and benzylbromide (0.573 g, 3.35 mmol) in anhydrous nitromethane (2 ml) or acetonitrile (2 ml) was irradiated for 20 minutes at 270 W in microwave oven. Pale - green precipitate was diluted by acetone, filtered off and dried. Yields were about 55 % (acetonitrile) 70 % (nitromethane), m.p. 241 - 243 °C.
Synthesis of 3-benzyl-2-(6-R¹-chromon-3-yl)ethenyl benzothiazolium bromides IV
Method A (by irradiation). A stirred mixture of 3-benzyl-2-methyl benzothiazolium bromide (1 mmol) and appropriate 3-formylchromone derivative (1 mmol) in 2 ml dry nitromethane was irradiated at 270 W for 2 - 8 minutes (Table 1). Solid products were filtered off and washed with warm acetone.
Method B (one - pot - reaction, by irradiation). A stirred mixture of benzylbromide (1 mmol), 2-methylbenzothiazole (1 mmol) and appropriate 3-formylchromone derivative (1 mmol) in dry nitromethane (2 ml) was irradiated at 270 W for 2 - 8 minutes. The isolation of products was the same as for method A.
Method C (the classic conditions). The same mixture as in method A was refluxed for 4 - 6 hrs. The crystals were filtered off and recrystallized from acetonitrile. Yields about 50 - 60 %.
Benzoxazole derivatives IVk - IVo can be prepared by the same method B.
Hydrolysis of compounds IVa
The same mixture as above in method A was irradiated at 270 W for 10 minutes. Then water was added (2 mmol) and irradiation was prolonged for 20 minutes. After cooling the yellow crystals were filtered off and washed with warm acetone. Yield of compound V was 60 %, m.p. 220 - 223 °C.

Biological Part

Inhibition of oxygen evolution rate in Chlorella vulgaris
The oxygen evolution rate (OER) in algal suspensions (Chlorella vulgaris) was measured at 24^oC by a Clark type electrode (SOPS 31 atp, Chemoprojekt, Prague) in a chamber constructed according to Barto_ et al. [9]^.Prior to the OER measurements the suspensions were accommodated in the dark (4 h). The samples were then illuminated with a 250 W halogen lamp through a water filter (5 cm). Irradiance was 90 W m^-2. The composition of the algal cultivation medium was the same as described in [10], the chlorophyll content of the suspension was 20 mg dm^-3 and its pH was 7.2. The values of the logarithms of partition coefficients (log P) were calculated using Crippen method [11].
Determination of the site of action of IV in the photosynthetic apparatus of spinach chloroplasts
The chloroplasts applied for the study of IV effects upon photosynthetic centers were prepared according to the procedure described in [13]. The site of action of the studied IV concerning inhibition of photosynthetic electron transport in spinach chloroplasts was investigated by fluorescence and EPR spectroscopies.
Fluorescence study
The effects of the studied compounds on photosynthetic centres of chloroplasts were investigated by studying of fluorescence of chlorophyll a (excitation wavelength [lambda]_ex= 436 nm). The measurements were carried out at room temperature, using excitation and emission slits of 10 nm. Chlorophyll (Chl) content in the samples was 10 mg Chl dm^-3. Thechloroplast suspensions used for the study of Chl_a fluorescence were accommodated in the dark 10 min. prior to the measurements.
In the suspensions of spinach chloroplasts the interaction of IV with photosynthetic centres was manifested by the quenching of the chlorophyll a fluorescence at 685 nm with increasing IV concentration. For example, in the presence 2, 10 and 20 µmol dm^-3 of compound IV₈ the fluorescence intensity at 685 nm reached only 75.4, 56.0 and 44.8 % of the value obtained with the untreated suspension of spinach chloroplasts. The fluorescence emission band at 685 nm belongs to Chl_a which is present in the pigment-protein complexes, situated mainly in photosystem 2 [13].

Scheme 1

Table 1. Reaction time, yields and melting points of 3-benzyl-2-[2-(6-R¹, 7-R², 8-R³-chromone-3-yl) ethenyl] benzothiazolium bromide (IVa - IVj) and 3-benzyl-2-[2-(6-R¹, 7-R², 8-R³-chromone-3-yl) ethenyl] benzothiazolium bromide (IVk - IVo)

compounds	IVa	IVb	IVc	IVd	IVe
reaction time [min.]	5	4.5	2	2 - 3.5	1.5 - 2
yields [%]	45.9	28.2	56	78 - 81	81
melting point [C]	215-217	171-173	242-245	253-256	294-297
compounds	IVf	IVg	IVh	IVi	IVj
reaction time [min.]	5b	3.5a	7b	2b	8b
yields [%]	45b	79.5a	52b	53b	61b
melting point [C]	231-233	255-258	239-242	174-177	229-232
compounds	IVk	IVl	IVm	IVn	IVo
reaction time [min]	10	3	15	12	10
yields [%]	62	51	38	41	42
melting point [C]	221-223	175-177	275-278	277-279	203-205

Table 2. Characterisation of the prepared compounds IVa - IVg.

	R¹	R²	R³	Formula	calc. / found
				M_r[g.mol^-1]	%C	%H	%N	%S	%Br	%Cl
IVa	H	H	H	C₂₅H₁₈BrNO₂S 476.3927	63.03 63.21	3.81 3.86	2.94 2.82	6.73 6.35	16.77 17.06
IVb	CH₃	H	H	C₂₆H₂₀BrNO₂S 490.4195	63.68 63.89	4.11 4.32	2.86 2.73	6.54 6.38	16.30 16.58
IVc	Cl	H	H	C₂₅H₁₇BrClNO₂S 510.8378	58.78 58.25	3.35 3.42	2.74 2.61	6. 28 6.06	15.64 15.21	6.94 6.58
IVd	Br	H	H	C₂₅H₁₇Br₂NO₂S 555.2888	54.08 53.84	3.09 3.12	2.52 2.47	5.77 5.68	28.78 29.12
IVe	NO₂	H	H	C₂₅H₁₇BrN₂O₄S 521.3903	57.59 57.11	3.28 3.35	5.37 5.14	6.15 5.85	15.33 14.82
IVf	H	OH	H	C₂₅H₁₈BrNO₃S 492.3921	60.98 60.68	3.68 3.62	2.84 3.02	6.51 6.32	16.23 16.09
IVg	CH₃	CH₃	H	C₂₇H₂₂BrNO₂S 504.4463	64.29 64.03	4.40 4.49	2.78 2.54	6.36 6.48	15.84 16.12

Table 2. Continued. Characterisation of the prepared compounds IVh - IVj and V.

	R¹	R²	R³	Formula	calc. / found
				M_r[g.mol^-1]	%C	%H	%N	%S	%Br	%Cl
IVh	H	OH	OH	C₂₅H₁₈BrNO₄S 508.3915	59.06 58.64	3.57 3.73	2.76 2.65	6.31 6.01	15.72 15.23
IVi	Cl	H	CH₃	C₂₆H₁₉BrClNO₂S 524.8646	59.50 59.28	3.65 3.57	2.67 2.76	6.11 5.89	15.22 15.84	6.75 6.25
IVj	OH H H			C₂₉H₂₀BrNO₂S 526.4525	66.16 65.39	3.83 3.64	2.66 2.53	6.09 5.83	15.18 15.32
V	H H H			C₂₅H₂₀BrNO₃ S	60.67	4.04	2.83	6.47	16.18
				532.4519	60.87	3.85	2.54	6.70	16.50

Table 2. Continued. Characterisation of the prepared compounds IVk - IVo.

	R¹	R²	R³	Formula	calc. / found
				M_r [g.mol-1]	%C	%H	%N	%Br	%Cl
IVk	H	H	H	C₂₅H₁₈BrNO₃460.3261	65.23 64.34	3.94 3.82	3.04 3.04	17.36 17.56
IVl	CH₃	H	H	C₂₆H₂₀BrNO₃ 526.2411	59.34 58.67	3.83 3.73	2.66 2.61	15.18 14.68
IVm	Cl	H	H	C₂₅H₁₇BrClNO₃494.7712	60.69 60.64	3.46 3.82	2.83 2.81	16.15 16.19	7.16 7.05
IVn	Br	H	H	C₂₅H₁₇Br₂NO₃539.2222	55.69 54.92	3.18 3.31	2.60 2.73	29.64 28.92
IVo	H	OH	H	C₂₅H₁₈BrNO₄476.3255	63.04 62.84	3.81 3.69	2.94 2.87	16.77 17.06

Tab. 3. ¹H NMR spectra [delta] (ppm) (CF₃COOH - d, 80 MHz)

	(ppm )
III	3.3 (s, 3H, CH₃); 6.13 (s, 2H, CH₂); 7.37 (s, 5H, Ph); 7.76 - 7.88 (m., 2H, H-arom.); 8.49 - 8.61(m., 2H, H-arom.).
IVa	6.178 (s, 2H, CH₂); 7.466 (s, 5H, Ph); 7.871-8.472 (m, 9H, H-5-9, H-11-14); 8.811 (s, 1H, H-2); 9.130 (d, ³J=15.38 Hz, 1H, H-10)
IVb	2.590 (s, 3H, CH₃); 6.120 (s, 2H, CH₂); 7.270-7.508 (m, 5H, Ph); 7.597-8.222 (m, 8H, H-5,7-9,11-14); 8.768 (s, 1H, H-2); 9.026 (d, ³J=15.38 Hz, 1H, H-10)
IVc	6.111 (s, 2H, CH₂); 7.307-7.505 (m, 5H, Ph); 7.761-8.326 (m, 8H, H-5, 7-9, 11-14); 8.744 (s, 1H, H-10); 9.008 (d, ³J=15.38 Hz, 1H, H-10)
IVd	6.145 (s, 2H, CH₂); 7.420 (s, 5H, Ph); 7.688-8.463 (m, 8H, H-5,7-9,11-14); 8.872 (s, 1H,H-2); 8.969 (d,³J=15.38 Hz, 1H, H-10)
IVe	6.172 (s, 2H, CH2); 7.435 (s, 5H, Ph); 7.908-8.344 (m, 6H, H-8,9,11-14); 8.882 (d-d, J¹=15.38 Hz, J²=2.44 Hz, 1H, H-7); 8.930 (s,1H, H-2); 8.989 (d, ³J=15.38 Hz, 1H, H-10); 9.281 (d, J=2.44 Hz, 1H, H-5)
IVf	6.120 (s, 2H, CH₂); 7.221-7.420 (m, 7H ); 7.841- 8.301 (m, 7H ); 8.710 (s,1H, H-2); 9.004 (d, ³J= 15.38 Hz, 1H, H-10)
IVg	2.340 (s, 3H, CH3); 2.532 (s, 3H, CH3); 6.125 (s, 2H, CH2); 7.235 (s, 5H, Ph); 7.659-8.521 (m, 7H, H-5, 8, 9, 11-14); 8.865 (s, 1H, H-2); 9.105 (d, ³J=15.38 Hz, 1H, H-10)
IVh	6.004 (s, 2H, CH₂); 7.209-7.578 (m, 7H); 7.957-8.384 (m, 7H); 8.979 (s, 1H, H-2); 10.251 (s, 1H, OH)
IVi	2.584 (s, 3H, CH₃); 6.141 (s, 2H, CH₂); 7.447 (s, 5H, Ph); 7.871-8.323 (m, 7H,H- 5,7,9, 11-14); 8.817 (s, 1H, H-2); 9.037 (d, ³J=15.62 Hz, 1H, H-10)
IVj	6.148 (2H, s,CH2); 7.444 (5H, s, Ph); 7.650-8.393 (10H, m,); 8.775 (1H, s, H-2); 8.869 (1H, d, J=15 Hz); 9.769 (1H, d, J=9 Hz)
V^a	6.15 (s, 2H); 7.39 (s, 5H, H-Ph); 7.61 (t, 1H, H-5); 7.81 - 7.89 (m, 4H, H-Bt-het.); 8.06 (d, 1H, H-9, ³J=15Hz); 8.702 (d, 1H, H-10, ³J=15Hz); 8.19 (dd, 1H, H-6, ³J=8Hz, ⁴J=1.43Hz); 8.32 (d, 1H, H-8, ³J=8Hz); 8.50 (dd, 1h, H-7, ³J=8Hz, ⁴J=1.44Hz); 9.174 (s, 1H, H-2)

^aDMSO - d₆, 300 MHz (Varian GEMINI 2000)
Table 4. Enthalpy reaction ([Delta]H), for preparing salts IV, compounds V and heats of formation ([Delta]H_f) for compounds V and VI (in Kcal mol^-1)

R¹	[Delta]H(IV)	[Delta]H(V)	[Delta]H_f(V)	[Delta]H_f(VI)
H	-7.107	-194.219	-15.857	-7.714
NO₂	-3.131	-201.382	-12.520	-4.140
Cl	-6.097	-195.945	-14.260	-14.260

Table 5. IC₅₀ values of IV₁ - IV₁₅^a causing 50 % decrease of oxygen evolution rate in the algal suspensions of Chlorella vulgaris.

No	R₂	R₁	X^-	log P	IC₅₀(µmol dm^-3)
IV₁	CH₃	Cl	I	3.62	153
IV₂	C₂H₅	H	I	3.44	219
IV₃	CH₂-C₆H₅	H	Br	4.88	97
IV₄	CH₂-C₆H₅-4-NO₂	H	Cl	4.83	115
IV₅	CH₂-CH=CH₂	Cl	Br	4.36	138
IV₆	CH₂-C[equivalence]CH	H	Br	3.38	229
IV₇	CH₂-C[equivalence]CH	CH₃	Br	3.84	94
IV₈	C₈H₁₇	H	I	5.89	36
IV₉	C₈H₁₇	Cl	I	6.41	123
IV₁₀	CH₂CH₂-C₆H₅	H	Br	5.13	46
IV₁₁	CH₂CH₂-C₆H₅	Br	Br	5.92	76
IV₁₂	CH₂COOC₂H₅	Cl	Cl	3.54	195
IV₁₃	CH₂CH₂CH₂CH₂COOCH₃	Cl	Br	4.02	124
IV₁₄	CH₂CH₂CH₂COOC₂H₅	Cl	Br	3.97	169
IV₁₅	C₂H₅COO-CH-COOC₂H₅	Cl	Br	3.90	119

^aPhysical constants and analytical data are published in ref. 4.
Results and Discussion
We report herein the fast, facile and one - pot - synthesis of 2-[(4H-4-oxobenzopyran-3-yl) ethenyl]benzothiazolium (or bezoxazolium) salts by condensation of three starting compounds, e.g. 2-methylbenzothiazole (or 2-methylbenzoxazole), 3-formylchromones and benzylbromide in apropriate solvents.
We compared the results of classic methods with results using microwave irradiation. It was found, that N-substituted 2-methylbenzothiazolium salts were very convenient components for condensations with 3-formylchromones by both classic and microwaves assisted aldol synthesis. The both used methods were carried out in five solvents (C₂H₅OH, CH₃NO₂, CHCl₃, DMSO, CH₃CN) in order to find favorable reaction conditions for high yields. Using a microwave oven the reaction time was considerable shorter (5-6 hrs for classical and 8-10 minutes for irradiation conditions). The investigation of the formed salt in various solvents initiated the study of solvatochromism.
We found that the products from dry nitromethane afforded the higher yields. The starting compounds as polar molecules absorbed efficiently microwaves and then reacted very rapidly (8 - 15 minutes) without catalyst and yielded IV (70 - 90 %).
3-Formylchromones I can be prepared by convenient Vilsmeier double formylation of appropriate 2-hydroxyacetophenones [17].The preparation of benzothiazolium or benzoxazolium bromides is published using classic procedures [4].
We found also that the formation of bezothiazolium bromides III takes place rapidly under focused microwave irradiation. Creation of compounds IV by one - pot - synthesis is very effective (method B).
The one - pot - synthesis of benzoxazole analogues of compounds IV was the most convenient method, the two - step - process (Method A) gave only (20 %) yields. The method for preparation of compounds IV by classic conditions was generally less effective, yields were about 50 - 60 %.
The optimal structures and quantum chemical parameters (heat of formation and charge densities) of starting compounds I, III and products IV, V and VI were calculated by AM1 method with standard parametrization (keyword PRECISE) [18]. We have found that creation of componds IV and V are exothermic processes (Table 4.).
Compounds V are more stable than compounds VI (8.1 - 8.3 kcal/mol). Subsituents R¹ (H, NO₂, Cl) at position 6 of [gamma]-pyrone ring have a very negligible effect on thermodynamic characteristics of the reactions and nondecisive influence at reaction sites of compounds IV (Fig. 1). The distribution of the molecular electrostatic potential on the van der Waals surface for all compounds IV are very similar (the most positive values of MEP are in blue area). The reactivity of all salts IV are also very similar.

Fig 5. Molecular electrostatic potential on the van der Waals surface for salts IV (The most positive values are in blue area, R¹ = H, NO₂, Cl).
The nucleophilic reactions at C-2 of compounds IV in the presence any traces of water in solvent led to compounds V or VI. These reactions were indicated by coloring of samples from yellow to red or violet.
The UV - VIS spectra were used for investigation of interactions compounds IVa and IVk with solvents (DMF, DMSO, CHCl₃) and especially with water present in solvents (Fig. 2, 3, 4). The absorbance at ¹_max(378 nm) decreases and at ²_max(275 and 502 nm) increases with increasing of water concentration in methanolic solution of IVa and IVk (Fig. 3, 4). We found relative small shifts of wavelenght in solvents with different polarity.

Fig. 2. UV VIS spectra of 3-benzyl-2-[2-(chromon-3-yl)ethenyl]benzothiazólium bromid (IVa).
Absorbance in various solvents 1. DMF
¹_max=386 nm, ²_max=490 nm
2. DMSO
¹_max=386 nm, ²_max=498 nm
3. CHCl₃
_max=396 nm

Fig. 3. UV VIS spectra of methanolic solution of 3-benzyl-2-[2-(chromon-3-yl)ethenyl]benzothiazolium bromid (IV_a) with increasing concentration of water.
Water concentration [mol.dm^-3]: 0.044, 0.088, 0.133, 0.177, 0.222, 0.266, 0.311, 0.355, 0.400

Fig. 4. The dependence of concentration changes of compounds IVa and Va on square of water concentration in methanolic solution of samples.
Biological Part
The inhibition of photosynthetic electron transport by the studied compounds was reflected in the inhibition of OER in the algal suspensions of Chlorella vulgaris. The IC₅₀ values, i.e. concentrations of the compounds causing 50 % decrease of OER with respect to the control varied in the range 36 - 229 µmol dm^-3 (Table). The inhibitory effectiveness depended upon the lipophilicity of the studied compounds and showed an increase with the increasing lipophilicity in the range of log P = 3.38 - 5.89. With the further increase of the lipophilicity (log P > 5.89) the inhibitory activity showed a decrease. Quasi-parabolic dependence of biological activity upon the lipophilicity of the compounds has been found also for several inhibitors with heterocyclic skeleton, including benzothiazole and chromone [8 - 12].
The paramagnetic constituents occurring in spinach chloroplasts have been investigated by EPR spectroscopy. The chloroplasts of higher plants exhibit in the region of free radicals (g ~ 2.00) EPR signals, so called signal I and signal II, belonging to the photosynthetic centres (PS) 1 and PS 2 respectively. The signal II consists from two components, namely from the signal II_{very fast} and the signal II_slow, belonging to the intermediates Z⁺/D⁺ (i.e. tyrosine radicals Tyr_Z and Tyr_D which are present in 161 position in D₁ and D₂ proteins) on the donor side of PS 2 [15, 16]. The both constituents of the signal II are manifested in Fig. 1A (EPR spectra of untreated chloroplasts) whereby the full line corresponds to the signal II_slow(g = 2.0046; [Delta]B_pp= 2.0 mT) and the difference between the dashed and the full line corresponds to the signal II_{very fast} (g = 2.0046; [Delta]B_pp= 2.0 mT).
EPR measurements were carried out with an instrument ERS 230 (WG, Akademie der Wissenschaften, Berlin, Germany) operating in X-band at 5 mW of microwave power and 0.5 mT modulation amplitude. EPR spectra of untreated spinach chloroplasts and in the presence of studied compounds (0.05 mol dm^-3) were recorded in the dark and in the light. Chl content in the samples was 4 g dm^-3. The samples were irradiated with a 250 W halogen lamp (~ 60 W m^-2) through a water filter (5 cm). Due to lower aqueous solubility of the studied compounds (in comparison to 3-substituted benzothiazolium salts with R²= H or thioalkyl) these were dissolved in dimethyl sulfoxid.

Fig. 5. EPR spectra of untreated spinach chloroplasts (A) and of chloroplasts treated with 0.05 mol dm^-3of compound IV₈ (B). The full lines correspond to chloroplasts kept in the dark, the dashed lines to the illuminated chloroplasts. The arrows denote g=2.0026.
EPR spectra of spinach chloroplasts without (Fig. 5A) and in the presence of compound IV₈ with R¹ = C₈H₁₇, R² = H and X = I (Fig. 1B) were measured in the dark (full line) and in the light (dashed line). It is evident that in the presence of this compound a decrease of the intensity of EPR signal II_slowcan be observed (Fig. 5B, full line). Thus, it can be concluded that this compound interacts with the intermediate D⁺(Tyr_D) which is present in D₂protein on the donor side of PS 2. On the other hand, the intensity of the signal I with g = 2.0026 and [Delta]B_pp= 0.8 mT (belonging to the dimer of chlorophyll a in the core of PS 1) in IV-treated chloroplasts showed a pronounced increase in the light due to interruption of the electron flow from PS 2 to PS 1 (Fig. 5B, dashed line).
Similar EPR study carried out in the suspension of spinach chloroplasts in the presence of structurally similar compounds - 3-alkylcarbonylmethyl substituted benzothiazolium salts and derivatives of 3-formylchromone (the condensation products of 6-R¹-3-formylchromone with 4-aminosalicylic acid and the adducts of 6-R¹-3-formylchromone with n-alcohols and aminosalicylic acids ) as well - showed that the site of inhibitory action of these inhibitors are the intermediates Z⁺/D⁺[7, 8]. The above benzothiazolium salts caused also the damage of the water-splitting complex, namely of its manganese cluster [7]. Summarizing it can be concluded that the site of action of the studied IV is also located on the donor side of PS 2, however it is limited only on the intermediate D⁺ and IV do not interact with the intermediate Z⁺ and the water-splitting complex (the release of manganese ions was not observed).

Summary

The synthesis of the biologically active novel systems prepared by reaction of 3-formylchromones with 3-benzyl-2-methylbenzothiazolium (or benzoxazolium) bromides IV was studied. Compounds IV showed very interesting photochromic properties (Scheme 1).
The heats of formation, charge densities, and fully optimization of geometry of compounds I, III, IV, V and VI was investigated using quantum chemical AM1 method [18].
The 3-R₂-2-[2-(6-R¹-chromon-3-yl)ethenyl] benzothiazolium halides (IV) inhibited photosynthetic electron transport in spinach chloroplasts and in algal suspensions of Chlorella vulgaris. Using EPR spectroscopy it was confirmed that these compounds, containing two heterocyclic skeletons - benzothiazole and chromone, interact with the intermediate D⁺(corresponding to the tyrosine radical Tyr_D) situated in D₂ protein on the donor side of photosystem 2. The smaller inhibitory effects of the compounds with lower partition coefficients can be connected with the fact that the passage of these compounds through the lipidic parts of thylakoid membranes is limited and consequently, the number of inhibitors reaching the site of action in proteins situated on the inner side of thylakoid membranes is insufficient. On the other hand, due to intense interaction with membrane lipids, the compounds with high log P values remain predominantly incorporated in the lipidic part of the membrane without reaching and damaging the corresponding membrane proteins.

References

1. Haas, G., Stanton, J.L., Sprecher, A., Wenl, P. J. Heterocyclic Chem. 18, 607 (1981)
2. Coutts, S.J., Wallace, T.W. Tetrahedron 50, 11755 (1994)
3. Gasparová, R., Lácová, M. Collect. Czech. Chem. Commun. 60, 1178 (1995)
4. Gasparová, R., Lácová, M., El-Shaaer, H.M., Odlerová, _. IlFarmaco 52, 251 (1997)
5. Sutoris, V., Halgas, J., Sekerka, V., Foltínová, P., Gáplovsky, A. Chem. Zvesti 37, 653 (1983)
6. Lácová, M., Stankovièová, H., Odlerová, Z. Il Farmaco 50, 885 (1995).
7. Králová, K., Serseò, F. Zbornik príspevkov 3, 49. Zjazd chemickych spoloèností, Bratislava, 4. - 7. september 1995, p.335, Uher, M., Hlousková, Z. editors, STU Bratislava (1995)
8. Králová, K., Serseò, F., Lácová, M., Stankovièová, H. Biol.Plant. 38, 397 (1996)
9 Bartos, J., Berková, E., Setlík I. Photosynthetica 9, 395 (1975)
10. Sidóová E., Králová, K., Mitterhauszerová, L. Chem. Pap. 46, 55 (1992)
11. Ertl, P. Chem. Listy 86, 465 (1992)
12. Králová, K., Sersen, F., Sidóová, E. Gen. Physiol. Biophys. (in press)
13. Sersen, F., Balgavy, P., Devínsky, F. Gen. Physiol. Biophys. 9, 395 (1990)
14. Govindjee, Aust. J. Plant Physiol. 22, 131 (1995)
15. Noren, G. H., Barry, B.A. Biochemistry (USA) 31, 3335 (1992)
16. Barry, B.A., Babcock, G.T. Chem. Scr. A 28, 117 (1998)
17. Nohara, A., Ishiguto, T., Sanno, Y. Tetrahedron 13, 1183 (1974)
18. Dewar, M.J.S., Zoebisch, E., Stewart, J.P.P. J. Am. Chem. Soc. 107, 3902 (1985)