Microsoft Word - Gasparova NB.doc Nova Biotechnologica VII-I (2007) 115 EFFECT OF 2-[3-(TRIFLUOROMETHYL)PHENYL]- 4H-FURO[3,2-b]PYRROLE-5-CARBOXHYDRAZIDES ON PHOTOSYNTHETIC PROCESSES KATARÍNA KRÁĽOVÁ1, RENATA GAŠPAROVÁ2, MARTIN MONCMAN2 1Institute of Chemistry, Faculty of Natural Sciences, Comenius University, Mlynská dolina CH-2, SK-842 15 Bratislava, Slovak Republic (katarina.kralova@fns.uniba.sk) 2Department of Chemistry, Faculty of Natural Sciences, University of SS. Cyril and Methodius, J. Herdu 2, SK-917 01 Trnava, Slovak Republic (gasparor@ucm.sk) Abstract: A new series of carboxhydrazides 6-8 was synthesized under microwave irradiation by reaction of carboxhydrazide 1 with heterocyclic aldehydes 2-4 in the presence of p-toluenesulfonic acid in ethanol. N- Benzoylcarboxhydrazide 9 was prepared by reaction of 1 with benzoylchlorid 5 in THF at room temperature. The effects of 6-9 on inhibition of photosynthetic electron transport in spinach chloroplasts and chlorophyll content in the antialgal suspensions of Chlorella vulgaris were investigated. Keywords: 2-[3-(trifluoromethyl)phenyl]furo[3,2-b]pyrrole-5-carboxhydrazide, Chlorella vulgaris, spinach chloroplasts, photosynthetic electron transport 1. Introduction Carboxhydrazides and their derivatives represent an interesting class of compounds which exhibits antimicrobial (EL-SHAAER et al., 1998), antifungal (DUTTA et al., 1986), analgesic and anti-inflammatory (SANTOS et al., 1997) activities. The presence of trifluoromethyl group in the molecule leads often to the biologically active compounds. Many heterocycles bearing the trifluoromethyl group possess antiprotozoal (NAVARRETE-VAZQUEZ et al., 2006), antimalarial (MADRID et al., 2005) or antibacterial (WOLFART et al., 2004) activities. Substituted furans are structural units in natural products and pharmaceuticals (KRUTOŠÍKOVÁ, 1996) and have been widely used as synthetic intermediates (LIPSHUTZ, 1986). 5-Substituted furan-2-carboxaldehydes and some of their derivatives show antibacterial (SHINDHAR et al., 1980) or antiviral (SHIGETAKA et al., 1970) activities The present study is a follow-up paper to our previous research dealing with the synthesis and reactions of furo[3,2-b]pyrrole system and the study of its biological activity (GAŠPAROVÁ et al., 2005; MONCMAN, 2006). 2. Materials and methods 2.1 Chemistry Scheme 1 shows the synthetic pathway to hydrazides 6-9. 116 Kráľová, K. et al. O N HN O NH2 A O H R O N HN O N A O N O H CO2Me R1 O N H HN O N O N CO2Me R 8a-8c R =CH3, CH2-O-CH3, CH2-Ph 1 4 F3C F3C F3C 6a - 6f A =O, R =4-Cl, 3-CF3, 4-NO2, 4-CH3, 4-I, 4-Br 7 A = S, R = H 2 A =O, R = Aryl 3 A = S, R = H Scheme 1 H H 6 R COCl O N HN O NH F3C H O 5 9 THF pyridin 2.1.1 General procedure for synthesis of 6-8 The mixture carboxhydrazide 1 (0.3 g, 1 mmol), 5-arylfuran-2-carboxaldehyde 2 (or thiophen-2-carboxaldehyde 3, methyl 2-formylfuro[3,2-b]pyrrole-5-carboxylates 4) (0.21 g, 1 mmol) and catalytic amount of p-toluenesulfonic acid in ethanol (5 cm3) was irradiated in microwave oven at 90W for 0.5-2 min. After cooling, the solid product was filtered off, dried and crystallized from ethanol to give 51-87 % yields of products. 2.1.2 Synthesis of 9 The solution of carboxhydrazide 1 (0.5 g, 1.6 mmol) in THF (5 cm3) and catalytic amount of pyridin was cooled in the ice bath to 0 °C. Then benzoylchlorid 5 (0.18g, 1.3 mmol) was added and the reaction mixture was stirred at room temperature for 5 h. The solid product was filtered off, dried and crystallized from ethanol to give 60 % of product. 2.2 Study of inhibition of photosynthetic electron transport in spinach chloroplasts Spinach chloroplasts were prepared according to WALKER (1980). The effect of the compounds 5-7 on the inhibition of photosynthetic electron transport (PET) in Nova Biotechnologica VII-I (2007) 117 spinach chloroplasts was investigated spectrophotometrically in the presence of electron acceptor 2,6-dichlorophenol indophenol (DCPIP) (30 μmol.dm−3). Before measurements, the chloroplasts were resuspended in phosphate buffer (20 mmol.dm−3; pH = 7.2) containing 5 mmol.dm−3 MgCl2 and 15 mmol.dm −3 NaCl. The chlorophyll content in the suspension was adjusted to 30 mg.dm−3. Samples were irradiated at 25 °C with a halogen lamp (250 W) at a distance of 1 dm. A 4 cm water filter was used to prevent overheating of the samples. The PET-inhibitory activity of the compounds studied was expressed in term of IC50 values as their negative logarithms thus, corresponding to molar concentrations of inhibitors causing a 50% decrease of oxygen evolution rate (OER) with respect to the untreated control sample. Due to lower aqueous solubility of the compounds studied, these were dissolved in dimethyl sulfoxide. The effect of DMSO on OER in the suspensions of spinach chloroplasts was in the range of experimental error and could be neglected. 2.3 Study of chlorophyll content in Chlorella vulgaris The algae Chlorella vulgaris were statically cultivated (photoperiod: 16h light / 8h dark; illumination: 5 000 lx; temperature: 23 ±1 °C) in liquid cultivation medium (pH = 7.2) (KRÁĽOVÁ et al., 1998). The effect of the compounds applied at three stepwise increasing concentrations (10, 50 and 100 μmol.dm−3) on the total chlorophyll content of algal suspension was determined after 7 days of cultivation spectrophotometrically (Kontron Uvikon 800) and after extraction into methanol according to WELLBURN, 1994. The chlorophyll content in the suspensions at the beginning of cultivation was 0.1 mg.dm−3. The effect of the compounds studied and applied in the concentration range 1–100 μmol.dm−3 on the content of chlorophyll in the suspensions was expressed as the percentage from the corresponding value obtained for the control (Table 2). 3. Results and discussion 3.1 Study of inhibition of photosynthetic electron transport in spinach chloroplasts HILL and SCARISBRICK (1940) showed that isolated chloroplasts and chloroplast fragments could release O2 in the light if they were given a suitable acceptor for the electrons being removed from H2O. DCPIP (2,6-dichlorophenol indophenol) is often used as an synthetic electron acceptor for this reaction for measuring oxygen evolution rate (OER) in isolated plant chloroplasts (e.g. ŠERŠEŇ et al., 1990). PET-inhibitory activity is exhibited by many compounds possessing X = C-NH group with a sp2 hybridized carbon atom i.e. ureas, triazines or anilides (KRÁĽOVÁ et al., 1999; MILETIN et al., 2001). Due to formation of hydrogen bonds between this group and the target proteins in photosynthetic centers of thylakoid membranes, changes in protein conformation may occur resulting in inhibition of photosynthetic electron transport (DRABER et al., 1991). 118 Kráľová, K. et al. Table 1. Inhibition of photosynthetic electron transport in spinach chloroplasts. compound R log(1 / IC50) [mol.dm-3] IC50 [mmol.dm-3] 6a 4-Cl 3.8975 0.127 6b 3-CF3 4.1513 0.071 6c 4-NO2 4.0244 0.095 6d 4-CH3 2.8936 1.278 6e 4-I 3.7615 0. 173 6f 4-Br 3.0702 0.851 7 - 3.2194 0.604 8a CH3 2.2948 5.180 8b CH2OCH3 2.2857 5.178 8c CH2Ph 3.2857 0.518 9 - 3.6583 2.220 IC50 values of the standards, used for testing of herbicidal as well as antialgal activity (KUBICOVÁ et al., 2003; CONRAD et al., 1993), related to inhibition of photosynthetic electron transport in plant chloroplasts determined for these herbicides that act in the photosystem 2, varied in the range 0.25 – 0.79 μmol.dm-3 for atrazine (2- chloro-4-ethylamino-6-isopropylamino-1,3,5-triazine), 0.40 – 3.98 μmol.dm-3 for simazine (2-chloro-4,6-bis(ethylamino)-1,3,5-triazine) and 0.032-0.200 μmol.dm-3 for diurone (1-(3,4-dichlorophenyl)-3,3-dimethylurea), as described by FEDTKE (1982). In comparison with these values, carboxhydrazides 6 – 9 showed relatively low inhibitory effect on photosynthetic electron transport (PET) in spinach chloroplasts (Table 1). The most effective inhibitors were compounds 6a (R = 4-Cl), 6b (R = CF3), 6c (R = 4-NO2) and 6e (R = 4-I). The influence of the electron acceptor properties of R substituent on PET-inhibitory activity was not significant. 3.2 Study of chlorophyll content in Chlorella vulgaris In the concentration range (1 – 100 μmol.dm-3) the majority of carboxhydrazides 6 – 9 only slightly reduced chlorophyll content in statically cultivated algal suspensions of Chlorella vulgaris (Table 2). The most effective inhibitors were compounds 6c (R = 4-NO2) a 6e (R = 4-I). Table 2. Effect of compounds 6-8 on chlorophyll content in algal suspensions of Chlorella vulgaris Compound R Concentration (μmol.dm-3) Concentration of chlorophyll (mg .dm-3) Average in 10 – 100 μM % of control 0 7.333 control 100 5.561 50 6.424 6a 4-Cl 10 6.342 84.3±4.8 continued on next page Nova Biotechnologica VII-I (2007) 119 Compound R Concentration (μmol.dm-3) Concentration of chlorophyll (mg .dm-3) Average in 10 – 100 μM % of control 100 6.610 50 6.629 6b 3-CF3 10 6.836 91.3±1.7 100 1.877 50 2.609 6c 4-NO2 10 5.539 30.9* 100 6.516 50 6.691 6d 4-CH3 10 6.964 91.7±3.1 100 3.552 50 4.979 6e 4-I 10 5.741 100* 100 5.917 50 5.909 6f 4-Br 10 6.379 82.8±3.7 100 7.217 50 6.682 7 - 10 5.917 90.1±8.9 100 5.714 50 6.570 8a CH3 10 6.946 87.4±7.6 100 4.655 50 6.317 8b CH2OCH3 10 6.222 78.2±4.3 100 6.958 50 6.400 8c CH2Ph 10 6.409 93.2±5.1 100 5.833 50 8.564 9 - 10 6.267 81.7±3.3 * IC50 in μM 4. Conclusions N′-{[5-(R-Phenyl)furan-2-yl]methylene}-2-[3-(trifluoromethyl)phenyl]-4H-furo [3,2-b] pyrrole-5-carboxhydrazides 6a – 6f, N′-[(thiophen-2-yl)methylene]-2-[3- (trifluoromethyl) phenyl] - 4H – furo [3,2-b] pyrrole – 5 - carboxhydrazide 7, N′ - {[5 - (methoxycarbonyl) – 4 - R1 – furo [3,2-b] pyrrol – 2 - yl] methylene} – 2 - [3 - (trifluoromethyl) phenyl]-4H-furo[3,2-b]pyrrole-5-carboxhydrazides 8a – 8c and N'- benzoyl-2-[3-(trifluoromethyl)phenyl]-4H-furo[3,2-b]pyrrole-5-carboxhydrazide 9 showed relative-ly low inhibitory effect on the photosynthetic electron transport of spinach chloroplasts and only slightly reduced chlorophyll content in statically cultivated algal suspensions of Chlorella vulgaris. The most effective inhibitors of the PET were compounds 6a (R = 4-Cl), 6b (R = CF3), 6c (R = 4-NO2) and 6e (R = 4-I). 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