Carbonic Anhydrase Activators

M. Kuzu et al. / Hacettepe J. Biol. & Chem., 2016, 44 (4), 409–417 Novel Carbonic Anhydrase Activators in the Rats Exposed to H2O2 H2O2 Uygulanan Sıçanlarda Yeni Karbonik Anhidraz Aktivatörleri Research Article Müslüm Kuzu1*, Ramazan Demirdağ2, Ahmet Özkaya3, Veysel Çomaklı2, Zafer Şahin4, Ugur Dardağan3, Kader Uzun3, Murat Koca3 Faculty of Pharmacy, University of Ağrı İbrahim Çeçen, Ağrı, Turkey. Department of Nutrition and Dietetics, University of Ağrı İbrahim Çeçen, Ağrı, Turkey. 3 Department of Chemistry, University of Adıyaman, Adıyaman, Turkey. 4 Faculty of Health Sciences, Necmettin Erbakan University, Konya, Turkey. 1 2 ABSTR AC T C arbonic anhydrase has an important role in attentional gating of memory storage, signal process and longterm synaptic transformation beside its pH regulation, HCO3- reabsorption and CO2 expiration tasks. Within the study, in vivo effects of (Benzofurane-2-yl)(3-phenyl-3-methylcyclobutyl)ketoxime (A), (Benzofurane-2-yl) (3-methyl-3-mesitylcyclobutyl)ketone thiosemicarbazone (T), 1,3-bis(2-chlorbenzoyl)imidazoline-2-thione (B) which are synthetic products and H2O2 (H) on the carbonic anhydrase activity in the liver, erythrocyte, heart and kidney tissues of male Wistar rat were analyzed. For this purpose, control group, separate groups for each of the synthetic products, H2O2 group and H2O2 combination group with synthetic products were created. As a result of the obtained evidence, a decrease in carbonic anhydrase activity was observed in examined tissues exposed to H2O2, but A compound has activation effect on enzyme activity in liver, T compound has it in erythrocyte and heart tissues, B compound has it in liver and erythrocytes. Key Words Activation, Carbonic Anhydrase, H2O2, In vivo, thiosemicarbazone. ÖZET K arbonik anhidrazın pH düzenlenmesi, HCO3- geri emilimi, CO2’in solunumla atılması gibi görevlerinin yanısısıra, hafıza, sinyal prosesleri, uzun dönem sinaptik transformasyon olaylarında önemli rolleri vardır. Yapılan çalışmada, sentetik ürünler olan (Benzofuran-2-il)(3-fenil-3-metilsiklobutil) ketoksim (A), (Benzofuran2-il)(3-metil-3-mezitilsiklobutil)keton tiyosemikarbazon (T), 1,3-bis(2-klorobenzoil)imidazolin-2-tiyon (B) ve H2O2’in karbonik anhidraz aktivitesi üzerine erkek Wistar karaciğer, eritrosit, kalp ve böbrek dokularında etkileri incelendi. Bu amaçla, kontrol grubu, her bir sentetik ürün için bir grup, H2O2 grubu ve sentetik ürünler ile H2O2 kombinasyon grupları oluşturuldu. Elde edilen verilerden H2O2 uygulan grupta bütün dokularda karbonik anhidraz aktivitesinde azalma olduğu görüldü. Bununla birlikte A maddesi karaciğerde aktivasyona neden olurken T maddesi eritrosit ve kalp dokularında, B maddesi ise karaciğer ve eritrositlerde aynı etkiyi gösterdi. Anahtar Kelimeler Aktivasyon, Karbonik Anhidraz, H2O2, In vivo, tiyosemikarbazon. Article History: Received: Jun 9, 2016; Revised: Nov 23, 2016; Accepted: Nov 23, 2016; Available Online: Dec 31, 2016. DOI: 10.15671/HJBC.2016.121 Correspondence to: M. Kuzu, Faculty of Pharmacy, University of Ağrı İbrahim Çeçen, Ağrı, Turkey. Tel: +904722156026 Fax: +904722156026 E-Mail: mkuzu@agri.edu.tr 410 M. Kuzu et al. / Hacettepe J. Biol. & Chem., 2016, 44 (4), 409–417 INTRODUCTION C arbonic anhydrase (carbonate hydrolyase E.C.4.2.1.1) is a metalloenzyme which is present commonly in every organisms and consists Zn2+ ion in its active region. Carbonic anhydrase (CA) explored in cattle’s erythrocytes for the first time is an important enzyme that catalyzes reversibly the reactions of hydration of CO2 and dehydration of HCO3-. CA CO2 + H2O ↔ HCO3- + H+ Carbonic anhydrase also catalyzes cyanate’s carbamic acid or urea’s cyanamide, aldehyde’s geminal diole hydration reactions besides hydration reaction of CO2. This enzyme also catalyzes carboxylic, sulfonic, and phosphoric acid ester hydrolysis [1]. CA enzyme is characterized as an enzyme that regulates pH in various tissues along with erythrocytes. It has a role in various metabolic issue notably acid-base balance. It takes charge in the critic physiological process related to CO2/bicarbonate respiration and transport between tissues/organs and lung, in pH and CO2 hemostasis, electrolyte secretion, biosynthetic reactions (gluconeogenesis, lipogenesis and urea synthesis), ostosis, calcification, tumorigenesis and in many other physiological and pathological process [2,3]. Benzofuranes which are present in many natural products attract the attention of chemistry because of their biological activities. It is known that there are many natural products consisting of benzofurane rings. These are particularly the products isolated from the species like Machilus glaucescens, Ophryosporus charua, Ophryosporus lorentzii, Krameria ramosissima and Zanthoxylum ailanthoidol [4]. Benzofurane-derived products are important molecules in terms of pharmaceutics. It is known that benzofurane-derivatives constituting a considerable part of heterocyclic compound have important biological features. Many studies have still be conducted to produce more active structures of these molecules by diversifying them in medical word [5,6]. Benzofurane-derived products are stated to be important biologically and their features like antimicrobial agent, enzyme inhibitor and activator, agonist and antagonist receptor, anti-inflammatory agent, anticancer, antiviral, antitubercular, antioxidant, diagnostic imaging in Alzheimer’s disease, complement system inhibitor, anti-ulcerogenic, ischemic cell death inhibitor and dopamine uptake inhibitor have been expressed [7]. Imidazole ring is present in many compounds in the nature and in the structure of various drugs. Imidazole is present in histidine –an amino acid, adenine and guanine bases and uric acid. This compound can be seen in the structure of many enzymes and have a role in the active center of these enzymes [8]. Imidazole-derived products have been reported to have antioxidant activities besides their anti-parasite, antiviral, antibacterial, antihypertensive, analgesic effects [9]. Thiosemicarbazones present their biological activities by inhibiting ribonucleotide reductase enzyme which is necessary for synthesizing DNA precursors. The inhibition is achieved by that the non-heme subunit of the enzyme is inhibited/inactivated by thiosemicarbazones. Because of these features, it is reported that thiosemicarbazones show antitumor activity and also intifungal features [10]. In another study, it is determined that thiosemicarbazone-derivatives have metal chelation features and antimicrobial effect [11]. Hydrogen peroxide (H2O2) is among the potential reactive oxygen species sources. Reactive oxygen species damage enzymes, cell membrane, DNA, proteins and lipids from the macro molecules. H2O2 causes oxidative stress in metabolism by transforming into hydroxyl radical (.OH). As a result of oxidative stress, many diseases such as cardiovascular, diabetes and cancer occur [12,13]. Within this study, CA activity in the erythrocyte, liver, heart and kidney tissues of the rat groups that were treated with H2O2, synthetic products {(Benzofurane-2-il)(3-phenyl-3-methylcyclobutyl) ketoxim e, (B enzofuran e -2-il) (3-m ethyl-3- M. Kuzu et al. / Hacettepe J. Biol. & Chem., 2016, 44 (4), 409–417 Figure 1. Structure of organic synthesis matters. Figure 2. Synthesis of (Benzofurane-2-il)(3-metil-3- mesitylcyclobutyl)ketone thiosemicarbazone. mesitylcyclobutyl) ketone thiosemicarbazone and 1,3-bis(2clorbenzoyl)imidazoline-2tion)} (Figure 1) and the combination of these compounds. (Benzofurane-2-il)(3-metil-3-mesitylcyclobutyl) ketone thiosemicarbazone which is another compound used in the study was synthesized with the method summarised in Figure 2. MATERIALS AND METHODS Chemical Protein assay reagents and 4-nitrophenylacetate were obtained from Sigma-Aldrich Co. The other chemicals were of analytical grade and obtained from Merck. The Synthesis of the Compounds The synthetic products used in this study were synthesized before with 1,3-bis(2-clorbenzoyl) imidazoline-2-tion [14] and (Benzofurane-2-il) (3-phenyl-3-mesitylcyclobutyl)ketoxime[4]. All the chemicals were reagent grade as received from commercial sources (Sigma–Aldrich and Fluka) unless otherwise stated. 1-mesityl-1methyl-3-(2-chloro-1-oxoethyl) cyclobutane 1 and (benzofuran-2-yl) (3- mesityl 3- methylcyclobutyl) methanone 2 were prepared according to the literature [4,15,16]. Melting points (uncorrected) were determined with a Gallenkamp apparatus. The IR spectra were measured with Mattson 1000 FT-IR spectrophotometer (potassium bromide disks). 411 412 M. Kuzu et al. / Hacettepe J. Biol. & Chem., 2016, 44 (4), 409–417 The 1H-NMR spectra were recorded on a VarianGemini 200 MHz spectrometer and were reported in ppm (d) relative to tetramethylsilane (TMS) as the internal standard and 13C NMR (50.34 MHz) were referenced to deuterochloroform(CDCl3). Synthesis of (Benzofuran-2-yl)(3-mesityl-3methylcyclobutyl) Ketone Thiosemicarbazone A mixture of (2) (0.3324 g, 1.0 mmol), thiosemicarbazide (0.091 g, 1.0 mmol) and p-toluene sulphonic acid (0.010 g, as catalyst) in absolute ethanol (150 mL) was refluxed. The progress of reaction was monitored by FT-IR by following the disappearance of 1687 cm-1 ketone (carbonyl) peak. After the solvent was removed under reduced pressure, the residue was treated with water and the final crude product (3) thus obtained by recrystallizing from ethanol [4,15,16]. Spectral data: (benzofuran-2-yl)(3-mesityl-3phenylcyclobutyl) ketone thiosemicarbazone (3). Yield: 0.39 g (97%); m.p.: 256-258°C ; IR (KBr cm-1,): 3432-3155 (four sharp peaks, thiosemicarbazide), 1660 (C=N), 1251 (C-O, on furan ring), 1048 (C=S). 1H-NMR(200 MHz; CDCl3): 1.63 (s, 3H, CH3), 2.24(s,9H, CH3), 2.59-2.80 (m, 4H, -CH2-), 3.523.75(p, 1H, >CH-), 6.29-6.32 (broad s,2H, -NH2), 6.78(s.2H, mesityl), 7.11-7.70 (m, 5H, benofurane), 10.93 (s, 1H, -NH-); 13C-NMR (50.34 MHz; CDCl3): 181.45 (C=S), 156.63, 150.83, 151.87, 145.66(2C), 141.77(2C), 137.03, 136.99, 132.97, 129.20, 128.37, 126.24, 124.14, 114.07, 112.75, 44.00, 42.68(3C), 35.72, 26.90, 23.48, 22.38. Preparation of the Hemolysate and Lysate Fresh rat blood samples were collected in tubes containing EDTA, then centrifuged (15 min, 2.500xg) and plasma and buffy coat (leucocytes) were removed. The packed red cells were washed three times with physiological serum, homolyzed with 5 volume of ice-cold water and then centrifuged (10.000xg, for 30 min) to remove the ghosts and intact cells. Tissue samples were lysed with liquid nitrogen, then taken into 2-3 ml per gram tamp (50 mM TrisSO4 pH: 7.4). Then, they were centrifuged (30 min, 10.000xg), collected for supernatant experiments, and the remaining cell trashes were removed. Laboratory Animals In the study, male Wistar albino rats were used. Laboratory animals were obtained from Fırat University Experimental Research Center (FUERC) and within this unit the experimental applications were done. The rats were fed in cages which were peculiarly designed and their daily cleanings were done regularly in a special environment with ventilation system. The rats were given tap water with nursing bottles with stainless steel balls. They were fed with special steel dishes by giving pellet feed. Before starting experimental studies, preparatory study was done. The environment in which the animals were kept stabilized to 2225oC. The rats were observed for 12 hours under the light and for 12 hours in the dark. A total of 56 Wistar albino male rats whose average weight is between 200-250 g were used in the study. The rats were divided into 8 groups. These groups and the matter concentrations given to these groups are indicated below: 1. Control group (K) 2. H2O2 group (H) 3. Benzofurane-2-il)(3-phenyl-3- methylcyclobutyl) ketoxime (A) 4. (Benzofurane-2-il(3-methyl-3-methylcyclobutyl) ketone thyosemicarbazone (T) 5. 1,3-bis(2-clorbenzoyl)imidazoline-2-tion (B) 6. A + H group 7. T + H group 8. B + H group The synthetic products were dissolved in DMSO. Control group was given DMSO. H2O2 in 12 mg/kg distillate waters, A in 12 mg/kg, B in 12 mg/kg and C in 12 mg/kg DMSO were dispersed and were administrated to the rats via intraperitoneally. After the 30-day period, the rats were decapitated and the organs (heart, liver, etc. and tissues and plasma) were kept in –50oC after being removed surgically until the analysis. Protein Determination Quantitative protein determination was spectrophotometrically measured at 595 nm M. Kuzu et al. / Hacettepe J. Biol. & Chem., 2016, 44 (4), 409–417 according to Bradford’s method [17], with bovine serum albumin being used as a standard. Esterase Activity Assay The esterase activity was assayed by following the change in absorbance of 4-nitrophenylacetate to 4-nitrophenylate ion at 348 nm over a period of 3 min at 25ºC using a spectrophotometer (Shimadzu UV-VIS Spectrophotometer, UV-1800) according to the method described by Verpoorte et al. [18]. The enzymatic reaction, in a total volume of 3.0 mL, contained 1.4 mL 0.05 M Tris-SO4 buffer (pH 7.4), 1.0 mL 3 mM 4-nitrophenylacetate, 0.5 mL H2O and 0.1 mL enzyme solution. A reference measurement was obtained by preparing the same cuvette without enzyme solution. 413 oximes, it was found that such organic matters have a higher interaction capacity with Fe2+ in liver, kidney and brain homogenates of rats. It was explained that the reason of this is the reactions and bonding of such compounds with the oxygen, sulfur and nitrogen atoms in the electron-donor center of such compounds [19]. Besides, it was reported that benzofurane-derived compounds have anticancer, antimicrobial [20], antitumor [21,22], anti-HIV [23] and antioxidant [24] activities. Also, it was determined that imidazolederived compounds have antihypertensive, antiviral, antibacterial and antioxidant activities [9]. Thiosemicarbazone-derived products were reported to have antiviral, anticancer, antitumor, antibacterial, anti-inflammatory and antiamibic effects [25]. RESULTS AND DISCUSSION CA activity of H group was decreased in all tissues compared to C group (p<0.05, p<0.01, p<0.001). The enzyme activity of A and B groups was increased in liver tissue compared to C group (p<0.01, p<0.001). With the addition of A and B compounds, enzyme activities of HA and HB groups were increased compared to H group (p<0.05, p<0.001). In erythrocytes, enzyme activities of B and T groups were increased compared to C group (p<0.001). In erythrocytes, enzyme activities of all combination groups were observed to increase compared to H group (p<0.001). It was observed that the enzyme activity of T group was increased in heart tissue compared to C group (p<0.05). It was determined that the enzyme activities of the heart tissue combination groups were increased compared to H group (p<0.05). It was determined that there is no statistically difference between the CA activity of C group and A, B and T groups (p>0.05). It was determined that the CA activity of kidney tissue HB group was increased compared to H group (p<0.001), however it was relatively decreased compared to HA and HT groups (p>0.05, Table 1). Discussion The synthetic products used in this study are thiosemicarbazone, benzofurane and imidazolederived compounds and these structures have oxygen, sulfur and nitrogen within themselves. In a study conducted with thiosemicarbazone Memory acquisition and strengthening recollection are an important pharmacological objective in the treatment of cognitive diseases. CA has an important role in attentional gating of memory storage, signal process and long-term synaptic transformation beside its pH regulation, HCO3- reabsorption and CO2 expiration tasks. CA function disorders lead to cognitive disorders and this is related to Alzheimer, mental retardation and aging [26]. CA inhibitors were analyzed in detail in many studies and it was determined that these have been used to prevent some diseases and clinical treatments. Yet, the studies on CA activators are quiet limited in the literature [27]. However, it was reported that the three series of the derivatives obtained through the reaction of amino-5(2aminoethyl)- and 2-amino-5(3-aminopropyl)-1,3,4thiadiazolün 2,4,6-trisubstituted pyrylium salts activates CA II enzyme [28]. It is also known that many amine and amino acids such as noradrenaline, adrenaline, histamine, hystidine, imidazoles, phenylalanine and 5-Ht are CA activators. It was also reported that CA activators may have and important use in the treatment genetic CA deficiency and mental disorders [26]. The central administration of MCD (mast-celldegranulating peptide) which is a neurotoxin from bee venom that releases histamine was reported to induces a quasi-permanent hippocampal waves and generates arousal at low concentrations. A 414 M. Kuzu et al. / Hacettepe J. Biol. & Chem., 2016, 44 (4), 409–417 Table 1. CA enzyme activities in tissues. Groups Liver Erythrocyte Heart Kidney C 0.649035±0.03 0.119346±0.006 0.039445±0.002 0.654126±0.02 A 0.866513±0.02bt 0.128330±0.005t 0.035886±0.001 0.678422±0.02t B 1.421233±0.04ct 0.150378±0.008ct 0.037168±0.004 0.706442±0.03t T 0.616211±0.05 0.153321±0.005ct 0.045016±0.002at 0.737921±0.04t H 0.504052±0.01a 0.099572±0.003a 0.033351±0.001b 0.374625±0.07c HA 0.669332±0.03y 0.145819±0.003bt 0.040145±0.001y 0.312100±0.01c HB 1.214423±0.02ct 0.130479±0.004t 0.047586±0.006ct 0.695647±0.03t HT 0.566532±0.03 0.130109±0.006t 0.038508±0.001y 0.248609±0.03c Statistical significancy compared to the C group: a:P<0.05, b:P<0.01, c:P<0.001 Statistical significancy compared to the H group: y:P<0.05, z:P<0.01, t:P<0.001 MCD-like endogenous was determined in extract of rat brain [29]. It was determined that the concentration of nerve endings in hippocampus containing histamine was decreased in the brain tissues of Alzheimer patients [30]. The importance of GABAergic synaptic transformation in the control of signal process in hippocampal network was determined with conducted studies showing that strengthening of these transformations can lead to enhance memory and learning [31]. Besides, in the study conducted by Sun and Alkon, it was reported that the strengthening of these transformation was done for the first time by using CA activators [26]. The critic role of CA activation which is the observed effect of CA activators was determined directly with the effect of acetazolamide -a CA inhibitor- in stopping the synaptic transformation. It was shown that HCO3- flow can be eliminated in hippocampal pyramidal neurons [32]. In CA1 pyramidal cells, CA activation was reported to be essential in intracellular applications of benzolamide due to blocking GABAergic synaptic transformation [33]. The evidence as a result of the conducted studies supports that A matter statistically activates carbonic anhydrase enzyme in liver in a considerable extent (p<0.05), B matter substantially activates it in liver and erythrocytes (p<0.001), and T matter show quiet more activation effect in erythrocytes (P<0.001) compared to heart tissue (P<0.05) (Figure 3). Moreover, it was determined that H2O2 statistically inhibits CA enzyme in liver and erythrocytes in a considerable extent compared to control group (P<0.05), and this inhibition effect was observed more in kidney and heart tissues (P<0.01, P<0.001, respectively). As a result of the evidence obtained through the study, it is thought that the synthetic products used in the study can be used as CA activators and found a base for the further therapeuticintended CA activator synthesis studies. Reactive oxygen species like H2O2 are produced in normal cell metabolism process and they get important roles in signal paths. However, H2O2 shows toxicological effects and induces the damage on the cell components as it causes new radicals to occur. Beyond this, it was determined that exogen H2O2 disrupt the balance between formation of reactive oxygen species and transformation to the pro-oxidative state [34]. Hydrogen peroxide forms hydroxide radical by reacting with metal ions like ferrous, copper as an oxidizing species. When H2O2 reacts with ferro (Fe2+) which is present in the heme group of proteins radical reactions start. H2O2 forms the most harmful radical, hydroxyl radical, by reacting with superoxide radical. This reaction is called as Haber-Weiss reaction. If this reaction is with catalyzer, the reaction is faster. Ferri (Fe3+) is degraded to ferro (Fe2+) by superoxide. With the present ferro (Fe2+), hydroxyl radical is produced from H2O2 by Fenton reaction [35]. M. Kuzu et al. / Hacettepe J. Biol. & Chem., 2016, 44 (4), 409–417 Figure 3. % Activity in different tissues of the group compared to the control group (The control group activity was taken as 100%). Figure 4. % Activity in different tissues of the group compared to the H group (The H group activity was taken as 100%). Within the study, in vivo effect of H2O2 on CA was analyzed and for this purpose, H2O2 was administrated to healthy adult male Wistar albino rats. It was determined that administration of H2O2 inhibits CA in all tissues. H2O2 statistically inhibits CA enzyme in liver and erythrocytes in a considerable extent compared to control group (P<0.05), and this inhibition effect was observed more in kidney and heart tissues (P<0.01, P<0.001, respectively). However, it was observed that when the rats on which H2O2 administration was done were administered with A matter, CA activity was increased in a considerable extent in liver (P<0.05), erythrocyte (P<0.001) and heart (P<0.05) tissues compared to H group. It was determined that B matter increased CA activity (P<0.001) in all tissues compared to H group while T matter increased the activity only in erythrocyte (P<0.001) and heart (P<0.05) tissues (Figure 4). ACKNOWLEDGEMENTS This study was funded by Adıyaman University Scientific Research Projects Coordination Unit, Project No FEFYL/2013-0005. Authors wish to extend gratitude to Ağrı İbrahim Çeçen University Directorship of Central Research and Application Laboratories for providing laboratories and equipment. 415 416 M. Kuzu et al. / Hacettepe J. Biol. & Chem., 2016, 44 (4), 409–417 References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. C.T. Supuran, A. Scozzafava, Carbonic Anhydrase Inhibitors, Curr. Med. Chem., 1 (2001), 61-97. W.R. Chegwidden, Y. Edwards, N. Carter, The Carbonic Anhydrases-New Horizons. Molecular Bases of Inherited Disease (Scriver CR; Beaudet AL, Sly WS, Valle D, eds) 8th Ed., 2165–2204, McGraw-Hill, Inc., NewYork, 2000. C.T. Supuran, A. Scozzafava. Carbonic anhydrase inhibitors –Part 94. 1,3,4-Thiadiazole-2-sulfonamide derivatives as antitumor agents? Eur. J. Med. Chem., 35 (2000), 867-874. M. Koca, S. Servi, C. Kirilmis, M. Ahmedzade, C. Kazaz, B. Özbek, G. Ötük, Synthesis and antimicrobial activity of some novel derivatives of benzofuran: part 1. Synthesis and antimicrobial activity of (benzofuran2-yl)(3-phenyl-3-methylcyclobutyl) ketoxime derivatives, Eur. J. Med. Chem., 40 (2005), 1351–1358. V. Ugale, H. Patel, B. Patel, S. Bari, Benzofuranoisatins: Search for antimicrobial agents, Arabian J. Chem., doi:10.1016/j.arabjc.2012.09.011, (2012). C. Kirilmis, M. Ahmedzade, S. Servi, M. Koca, A. Kizirgil, C. Kazaz, Synthesis and antimicrobial activity of some novel derivatives of benzofuran: Part 2. The synthesis and antimicrobial activity ofsome novel 1-(1-benzofuran-2-yl)-2-mesitylethanone derivatives, Eur. J. Med. Chem., 43 (2008), 300-308. R.J. Nevagi, S.N. Dighe, S.N. Dighe, Biological and medicinal significance of benzofuran, Eur. J. Med. Chem., 97 (2015), 561-581. N. İkizoğlu, Reactions of Imidazole and Oxindole wtih Metal Compounds, Characterization of Structures of Synthesized Complexes. Master’s thesis, Kocaeli University: Kocaeli, (2012). İ. Sarı, The investigation of the effect of component containing benzofurane and benzimidazol on the some parameters at blood of rats (Wistar). Master’s thesis, Fırat University: Elazığ, (2009). A.E. Liberta, D.X. West, Antifungal and antitumor activity of heterocyclic thiosemicarbazones and their metal complexes: current status, Biometals, 5 (1992), 121-126. T. Rosu, A. Gulea, A. Nicolae, R. Georgescu, Complexes of 3dn Metal Ions with Thiosemicarbazones: Synthesis and Antimicrobial Activity. Molecules, 12 (2007), 782796. J.M.C. Gutteridge, Free Radicals in Diseases Processes: A Compilation of Cause and Consequence, Free Radical Res. Com., 19 (1993), 141– 158. P. Storz, Reactive Oxygen Species in Tumor Progression, Frontiers in Bioscience, 10 (2005), 1881– 1896. S. Öztürk Yıldırım, M. Akkurt, M. Genç, M. Şekerci, H.K. Fun, 1,3-bis(2-klorbenzoil)imidazolin-2-tiyon, Anal. Sci., 22 (2006), 171. M.A. Ahmedov, I.M. Ahmedov, A.V. Kisin, R.R Kostikov, I.K. Sardarov, N.M. Babaev, Formation of substituted cyclobutanes in the reaction of 2-(2-methyl-2prophenyl)-3-chloromethyloxirane witharomatic hydrocarbons, Zhr. Org. Chem., 27 (1991), 1254–1262. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. M. Koca, M. Ahmedzade, A. Çukurovali, C. Kazaz, Studies on the Synthesis and Reactivity of Novel Benzofuran-2-yl-[3-Methyl-3-Phenylcyclobutyl] Methanones and their Antimicrobial Activity, Molecules, 10 (2005), 747-754. M.M. Bradford. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding, Anal. Biochem., 72 (1976), 248–251. J.A. Verpoorte, S. Mehta, J.T. Edsall, Esterase activities of human carbonic anhydrases B and C. J. Biol. Chem., 242 (1967), 4221-4229. V.S. Carratu, L. Bresolin, J.B.T.D. Rocha, F.A.A Soares, Butane-2,3-dionethiosemicarbazone: An oxime with antioxidant properties, Chem-Biol. Interact., 177 (2009), 153-160. Y.S. Xie, D. Kumar, V.D.V. Bodduri, P.S. Tarani, B.X. Zhao, J.Y. Miao , K. Jang, D.S. Shin, Microwaveassisted parallel synthesis of benzofuran-2carboxamide derivatives bearing anti-inflammatory, analgesic and antipyretic agents, Tetrahedron Lett., 55 (2014), 2796–2800. P.G. Baraldi, R. Romagnoli, M.G. Pavani, Benzoyl and cinnamoyl nitrogen mustard derivatives of benzoheterocyclic analogues of the tallimustine: Synthesis and antitumour activity, Bioorgan. Med. Chem., 10 (2002), 1611-1618. I. Hayakawa, R. Shioya, T. Agatsuma, Thienopyridine and benzofuran derivatives as potent anti-tumor agents possessing different structure-activity relationships, Bioorg. Med. Chem. Lett., 14 (2004), 3411-3414. S. Parekh, D. Bhavsar, M. Savant, S. Thakrar, A. Bavishi, M. Parmar, H. Vala, A. Radadiya, N. Pandya, J. Serly, J. Molnár, A. Shah, Synthesis of some novel benzofuran-2-yl(4,5-dihyro-3,5-substituted diphenylpyrazol-1-yl) methanones and studies on the antiproliferative effects and reversal of multidrug resistance of human MDR 1-gene transfected Mouse lymphoma cells in vitro, Eur. J. Med. Chem, 46 (2011), 1942-1948. J.M. Grisar, F.N. Bolkenius, M.A. Petty, 2,3-Dihydro1-benzofuran-5-ols as analogs of a-tocopherol that ınhibit in-vitro and ex-vivo lipid autoxidation and protect mice against centralnervous-system trauma, J. Med. Chem., 38 (1995), 453-458. B. Prathima, Y.S. Rao, S.A. Reddy, Y.P. Reddy, A.V. Reddy, Copper(II) and nickel(II) complexes of benzyloxybenzaldehyde-4-phenyl-3thiosemicarbazone: Synthesis, characterization and biological activity, Spectrochim. Acta A, 77 (2010), 248-252. M.K. Sun, D.L. Alkon, Pharmacological Enhancement of Synaptic Efficacy, Spatial Learning, and Memory through Carbonic Anhydrase Activation in Rats, J. Pharmacol. Exp. Ther., 297 (2002), 961-967. C.T. Supuran, Carbonic anhydrases: novel therapeutic applications for inhibitors and activators, Nat. Rev. Drug. Discov., 7 (2008), 1-14. M. Kuzu et al. / Hacettepe J. Biol. & Chem., 2016, 44 (4), 409–417 28. C.T. Supuran, M. Barboiu, C. Luca, E. Pop, M.E. Brewster, A. Dinculescu, Carbonic anhydrase activators. Part 14*. Syntheses of mono and bis pyridinium salt derivatives of 2-amino-5 (2-aminoethyl)- and 2-amino-5(3-aminopropyl)-1,3,4-thiadiazole and their interaction with isozyme II. Eur. J. Med. Chem., 31 (1996), 597-606. 29. E. Cherubini, Y.B. Ari, M. Gho, J.N. Bidard, M. Lazdunski, Long-term potentiation of synaptic transmission in the hippocampus induced by a bee venom peptide, Nature, 328 (1987), 70-73. 30. P. Panula, J. Rinne, K. Kuokkanen, K.S. Eriksson, T. Sallmen, H. Kalimo, M. Relja, Neuronal histamine deficit in Alzheimer’s disease. Neuroscience, 82 (1997), 993-997. 31. M-K. Sun, T.J. Nelson, D.L. Alkon, Functional switching of GABAergic synapses by ryanodine receptor activation, Proc. Natl. Acad. Sci., 97 (2000), 12300–12305. 32. K.J. Staley, B.L. Soldo, W.R. Proctor, Ionic mechanisms of neuronal excitation by inhibitory GABAA receptors, Science, 269 (1995), 977–981. 33. M-K. Sun, T.J. Nelson, H. Xu, D.L. Alkon, Calexcitin transformation of GABAergic synapses from excitation filter to amplifier, Proc Natl Acad Sci., 96 (1999), 7023–7028. 34. A. Quincozes-Santos, L.D. Bobermin, A. Latini, M. Wajner, D.O. Souza, C-A. Gonçalves, C. Gottfried, Resveratrol Protects C6 Astrocyte Cell Line against Hydrogen Peroxide-Induced Oxidative Stress through Heme Oxygenase 1, PloS One, 8 (2013), 1-1. 35. E.M. Gözükara, Biyokimya, 5. Baskı, Nobel Tıp Kitabevleri, Hadımköy-İstanbul (2011). 417