Synthesis and Evaluation of Anticonvulsant and Antimicrobial Activities of 3-Hydroxy-6- methyl-2-substituted 4H-Pyran-4-one Derivatives

Arch. Pharm. Pharm. Med. Chem. 2004, 337, 281 288 3-Hydroxy-6-methyl-2-substituted 4H-Pyran-4-one Derivatives 281 Mutlu Dilsiz Aytemir a, Ünsal Çalış a, Meral Özalp b a Hacettepe University, Faculty of Pharmacy, Pharmaceutical Chemistry Department, Ankara, Turkey b Hacettepe University, Faculty of Pharmacy, Pharmaceutical Microbiology Department, Ankara, Turkey Synthesis and Evaluation of Anticonvulsant and Antimicrobial Activities of 3-Hydroxy-6- methyl-2-substituted 4H-Pyran-4-one Derivatives In this study, thirteen 3-hydroxy-6-methyl-2-substituted 4H-pyran-4-one derivatives were synthesized for the evaluation of their potential anticonvulsant activity. Mannich bases were prepared by the reaction of substituted piperazine deriva- tives with allomaltol and formaline. The structures of the synthesized compounds were confirmed by IR, 1 H-NMR and elemental analysis. Their anticonvulsant activities were determined in vivo by maximal electroshock (MES), subcutaneous Metrazol (scmet), and rotorod toxicity tests for neurological deficits. The antimicrobial activities of the synthesized compounds were investigated in vitro against some bacteria and fungi using the microdilution broth method. According to the activity studies, 3-hydroxy-6-methyl-2-[4-(2-trifluoromethyl-phenyl)-piperazin-1-ylmethyl]-4H-pyran-4-one (3i) was the compound determined to be most active in the scmet test for all doses at four hours and for the 300 mg/kg dose at half an hour. 2-[4-(4-Chloro-phenyl)-piperazin-1-ylmethyl]-3-hydroxy-6- methyl-4h-pyran-4-one (3f) was found to be protective against MES whereas 2-chlorophenyl derivative (3e) was not. Looking at the antifungal activity results, compounds 3b, 3h, and 3i were determined to have activity against all fungi. Keywords: Allomaltol; 3-Hydroxy-6-methyl-2-substituted 4H-pyran-4-one derivatives; Mannich bases; Anticonvulsant; Antifungal activity Received: December 13, 2002; Accepted July 14, 2003 [FP754] DOI 10.1002/ardp.200200754 Introduction The treatment of epilepsy is still very problematic because of uncontrolled seizures and medication toxicity [1]. Antiepileptics, which are used in the symptomatic treatment of epilepsy, should be administered for a long time, even throughout one s life, as they have to be used chronically. Although, there are a number of antiepileptic drugs available on the market, the development of new compounds is still popular in the anticonvulsant therapy as the presently available drugs possess the risk of tolerance development and side effects. Moreover, it is still not possible to get some types of epilepsy under control [1 4]. Anticonvulsant agents are found in several different chemical classes: hydantoins, barbiturates, oxazolidinedions, succinimides, acylureides, glutarimides, benzodiazepines, secondary or tertiary alcohols, dibenzazepine derivatives, valproic acid and derivatives, γ-aminobutyric acid (GABA) analogs, and miscellaneous agents [5]. GABA Correspondence: Mutlu D. Aytemir, Hacettepe University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 06100 Sıhhiye-Ankara, Turkey. Phone: +90 312 305-1872, Fax: +90 312 311-4777; e-mail: mutlud@hacettepe.edu.tr has been suggested to be a major inhibitory neurotransmitter in the central nervous system [6]. As GABA itself does not cross the blood-brain barrier, there is considerable interest in the development of systemically active GABA-mimetic agents [7]. Kojic amine (2-aminomethyl-5-hydroxy-4H-pyran-4-one) has been found to possess certain properties of a GABA agonist (Figure 1) [8, 9]. The early neuropharmacological profile of kojic amine resulted in its classification as a GA- BA A receptor agonist [7, 10]. Structure-activity relationship studies show that most of the anticonvulsant drugs contain a phenyl ring or equivalent hydrocarbon substitute, and a carbonyl or another electronegative group adjacent to the phenyl ring. Here, the lipophilic aryl portion has the ability to facilitate the penetration of the blood-brain barrier [5, 6]. In the literature, maltol (2-methyl-3-hydroxy-4H-pyran- 4-one), ethylmaltol (2-ethyl-3-hydroxy-4H-pyran-4- one), kojic acid (2-hydroxymethyl-5-hydroxy-4H-pyran-4-one), and 2-alkyl-3-hydroxy-4H-pyran-4-one have been reported as new groups that show an anticonvulsant activity [11, 12]. Maltol was shown to have a central depressing activity in mice, and ethylmaltol was found to be a potent anticonvulsant against convulsions induced by pentetrazole and strychnine [11].

282 Aytemir et al. Arch. Pharm. Pharm. Med. Chem. 2004, 337, 281 288 Table 1. Yields and melting points of the synthesized compounds. Figure 1. Structure of GABA, kojic amine and synthesized compounds. Compound R M.p. ( C) Yield ( %) number 2-Butyl and 2-isobutyl were the most potent depressant of pentetrazole-induced convulsions among the 2- alkyl derivatives. It is generally accepted that the lipid solubility of a drug is an important factor in connection with its transfer into the central spinal fluid and brain. The increase of the inhibitory effect of 2-alkyl-3- hydroxy-4h-pyran-4-ones on the pentetrazole-induced convulsion with an increasing number of carbons in the alkyl group might be due to the enhancement of lipid solubility [11, 12]. In addition, kojic acid and derivatives have shown to possess various pharmacological activities such as herbicidal [13], antimicrobial [14 16], pesticidal and insecticidal [14, 17], antitumoral [18]. They can also serve as cosmetic skin whitening agents [19]. In the scope of the present study, we aim to synthesize thirteen new Mannich bases of hydroxypyrone derivatives in order to develop new anticonvulsant and antifungal compounds (Table 1). Mannich bases were designed structurally related to kojic amines. Different lipophylic phenyl derivatives were substituted at the 4 position of the piperazine ring enabling the penetration of the blood-brain barrier. The anticonvulsant activities of the synthesized compounds were examined in test animals in vivo by maximal electroshock (MES) and subcutaneous Metrazol (scmet) tests. Seizure assays and neurotoxicity were determined by rotorod toxicity test according to the phase I tests of the Antiepileptic Drug Development (ADD) program, which were developed by the National Institute of Neurological and Communicative Disorders and Stroke [3, 20]. This program was used for the evaluation in various previous studies [21 24]. In addition, the antifungal properties of synthesized compounds were investigated. Results and discussion Chemistry In 1924, Yabuta [25] synthesized allomaltol (5- hydroxy-2-methyl-4h-pyran-4-one, 2) and his method was improved by Ellis et al. [26]. Allomaltol was synthesized from commercially available kojic acid in a two-step reaction according to the literature [25, 26]. 3a 176 77 95 3b 180 dec 55 3c 174 75 71 3d 159 60 76 3e 144 45 58 3f 190 dec 64 3g 155 56 75 3h 173 74 61 3i 145 46 78 3j 189 90 68 3k 163 64 45 3l 184 85 65 3m 152 53 45

Arch. Pharm. Pharm. Med. Chem. 2004, 337, 281 288 3-Hydroxy-6-methyl-2-substituted 4H-Pyran-4-one Derivatives 283 The methylene group protons of 3a m appeared as a singlet at 3.50-3.80 ppm. 13 C-NMR spectra of 3c and 3l have characteristic peaks for 6-methyl, carbonyl of 4H-pyran-4-one ring at 25.2 and 179.2 ppm, respectively. The characteristic peak for -CH 2 - was observed at 59.7-60.1 ppm. Scheme 1. Synthesis of the compounds 3a-3m. Chlorination of the 2-hydroxymethyl moiety of kojic acid using clean thionyl chloride at room temperature, afforded 2-chloromethyl-5-hydroxy-4H-pyran-4-one (chlorokojic acid) 1, with the ring hydroxyl being uneffected. Subsequently, the chloro derivative 1 was reduced with zinc dust in conc. hydrochloric acid to afford 2 (58% overall yield in two steps) [26]. Because of its phenol-like properties, kojic acid readily undergoes aminomethylation at room temperature in the Mannich reaction ortho to the enolic hydroxyl group [27, 28]. Woods has reported di-mannich derivatives obtained in an acidic medium from kojic acid, formaldehyde and aromatic amine. Using aliphatic or some heterocyclic secondary amines, only position 6 of the kojic acid was substituted by a Mannich group [29]. Some Mannich bases of chlorokojic acid were prepared by O Brien and co-workers [27]. In this study, thirteen 3-hydroxy-6-methyl-2-substituted 4H-pyran-4-one derivatives were synthesized as Mannich bases, which were prepared by the reaction of appropriately substituted piperazine derivatives with 2 and formaldehyde (Scheme 1). Substitution took place preferably at position 2 and not at position 5 because of the electronegative hydroxyl group at position 3 on the pyranone ring. The mono-substituted Mannich bases 3a m readily formed in methanol even at room temperature. The structures of the synthesized compounds 3a m were confirmed by IR, 1 H-NMR, 13 C-NMR, 13 C-DEPT, mass spectra, and elementary analysis. Yields and the melting point properties of the synthesized compounds are presented in Table 1. In the IR spectra of 3a m the O-H stretching bands were observed at 3400-3300 cm 1. All compounds showed stretching bands associated with C=O, C=C and C-O stretching at 1674-1620, 1584-1439 and 1300-1200 cm 1, respectively. With 1 H-NMR spectra, characteristic singlet peaks of the 4H-pyran-4-one (H 5 ) ring proton were found in the region 6.11-6.50 ppm in accordance with the literature. 6-Methyl-4H-pyran-4-one derivatives showed the methyl group protons as a singlet at 2.20-2.50 ppm. Anticonvulsant activity Mannich bases carrying phenyl piperazine derivatives are more lipophilic, therefore, these compounds may possibly be easily transfered into the central spinal fluid and brain. 3b j were prepared to examine the effects of mono substitution of an electron-donating or an electron-withdrawing group at the ortho, meta and para position of the phenyl group of 3a. Mannich bases with electronegative groups such as fluoro, chloro and trifluoromethyl adjacent to the phenyl ring, were planned in order to increase the anticonvulsant activity, according to the literature [5]. These activities of 3a m were initially evaluated against MES- and scmet-induced seizures using male Swiss albino mice (20 ± 2 g). The results are shown in Table 2. The activity studies revealed that 3-hydroxy-6-methyl-2-[4- (2-trifluoromethyl-phenyl)-piperazin-1-ylmethyl]-4Hpyran-4-one (3i) was the most active compound in the scmet test at all doses at four hours and for the 300 mg/kg dose at half an hour. 2-[4-(4-Chlorophenyl)-piperazin-1-ylmethyl]-3-hydroxy-6-methyl-4Hpyran-4-one (3f) and 2-(4-benzo[1,3]-dioxol-5-ylmethyl-piperazin-1-ylmethyl)-3-hydroxy-6-methyl-4Hpyran-4-one (3l) were protective against scmet with a 300 mg/kg dose at four hours. 3f was found to be protective against MES at all doses at half an hour, whereas 2-chlorophenyl derivative (3e) was not. Also, 2-[4-(4-acetyl-phenyl)-piperazin-1-ylmethyl]-3- hydroxy-6-methyl-4h-pyran-4-one (3b) showed activity against MES at 100 and 300 mg/kg doses at half an hour. Both, 3a and 3b, showed selective activity against MES except with the 30 mg/kg dose. 3a, 3f, 3i, 3j, 3m were active against MES at 300 mg/kg dose at four hours. When the phenyl group (3a) was changed to pyridine-2-yl (3m), the activity against MES decreased. Piperazine derivatives bearing a methoxy group in position 2 and 3 of the phenyl ring (3c, 3d) were inactive. 3k and 3l have a methylene bridge between two rings. Although 3k was completely inactive, 3l, which has a 1,3-benzodioxole ring, showed activity against scmet at the high dose. None of 3c, 3d, 3e, 3g, 3h, and 3k showed anticonvulsant activity. Neurotoxicity was observed for the synthesized compounds 3a, 3i and 3k, which were administered to mice at the 300 mg/kg dose level.

284 Aytemir et al. Arch. Pharm. Pharm. Med. Chem. 2004, 337, 281 288 Table 2. Phase I anticonvulsant screening of the compounds. MES ScMet Toxicity Compound 0.5 h 4 h 0.5 h 4 h 0.5 h 4 h number mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg 30 100 300 30 100 300 30 100 300 30 100 300 30 100 300 30 100 300 1 0/1 0/1 1/1 0/1 0/1 1/1 0/1 0/1 0/1 0/1 0/1 0/1 0/4 0/4 0/4 0/2 0/2 0/2 2 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 1/1 0/1 0/1 0/1 0/4 0/4 0/4 0/2 0/2 0/2 3a 0/1 0/1 0/1 0/1 1/1 1/1 0/1 0/1 0/1 0/1 0/1 0/1 0/4 0/4 4/4 0/2 0/2 0/2 3b 0/1 1/1 1/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/4 0/4 0/4 0/2 0/2 0/2 3c 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/4 0/4 0/4 0/2 0/2 0/2 3d 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/4 0/4 0/4 0/2 0/2 0/2 3e 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/4 0/4 0/4 0/2 0/2 0/2 3f 1/1 1/1 1/1 0/1 0/1 1/1 0/1 0/1 0/1 0/1 0/1 1/1 0/4 0/4 0/4 0/2 0/2 0/2 3g 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/4 0/4 0/4 0/2 0/2 0/2 3h 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/4 0/4 0/4 0/2 0/2 0/2 3i 0/1 0/1 0/1 0/1 0/1 1/1 0/1 0/1 1/1 1/1 1/1 1/1 0/4 0/4 4/4 0/2 0/2 0/2 3j 0/1 0/1 0/1 0/1 0/1 1/1 0/1 0/1 0/1 0/1 0/1 0/1 0/4 0/4 0/4 0/2 0/2 0/2 3k 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/4 0/4 3/4 0/2 0/2 0/2 3l 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 1/1 0/4 0/4 0/4 0/2 0/2 0/2 3m 0/1 0/1 0/1 0/1 0/1 1/1 0/1 0/1 0/1 0/1 0/1 0/1 0/4 0/4 0/4 0/2 0/2 0/2 MES maximal electroshock seizure test; scmet subcutaneous pentylenetetrazole (metrazol) seizure test; toxicity rotorod test. 0/1 no activity at dose level, 1/1 noticeable activity at dose level, 3/4 neurotoxic at dose level for three of all tested animals, 4/4 neurotoxic at dose level for all tested animals, 0/4 no neurotoxicity at dose level at 1 / 2 hour, 0/2 no neurotoxicity at dose level at four hour. Table 3. Antifungal activity of the synthesized compounds (MIC in µg/ml). Compound Fungi (MIC-µg/mL) number C. albicans C. krusei C. parapsilosis ATCC 90028 ATCC 6258 ATCC 22019 1 128 32 32 2 128 128 128 3a 64 64 128 3b 64 64 64 3c 128 128 128 3d 128 128 128 3e 128 64 64 3f 64 128 128 3g 64 64 64 3h 64 64 64 3i 128 128 128 3j 64 64 128 3k 128 128 128 3l 128 128 128 3m 128 128 128 Fluconazole 0.25 64 8

Arch. Pharm. Pharm. Med. Chem. 2004, 337, 281 288 3-Hydroxy-6-methyl-2-substituted 4H-Pyran-4-one Derivatives 285 Antifungal activity The in vitro antifungal activities of the synthesized compounds were determined as MIC values by using the microdilution broth method. Fluconazole was used as the reference compound. All Mannich bases showed similar antifungal activity (MIC: 64-128 µg/ml) against C. albicans. Among this Mannich bases series, especially 3a, 3b, 3e, 3g, 3h, and 3j (MIC: 64 µg/ml) were determined to have a higher activity against C. krusei. 3b, 3g and 3h, which carry acetyl and fluoro phenyl moieties, showed the same activity (MIC: 64 µg/ml) against all fungi (Table 3). Conclusion In the Mannich base series, anticonvulsant activity has increased. Among the synthesized compounds, especially 3i, carrying 4-(3-trifluoromethylphenyl)piperazin-1-ylmethyl group at position 2 on the pyranone ring, was found to have significantly high anticonvulsant activity against the scmet seizures, while 3f which carry a 4-chlorophenyl moiety was found to be protective against MES at all doses of half an hour. Although 3i showed neurotoxicity at the high dose, 3f did not. Therefore, 3f is the most promising compound among these Mannich bases. 3c and 3d, possessing methoxy phenyl groups and 3k, benzyl group, haven t shown any anticonvulsant and antifungal activities. Concerning structure-activity relationship, while chloro and fluoro at ortho position of the phenyl ring (3e, 3g) have decreased anticonvulsant activity, para-chloro and meta-trifluoromethyl substituents (3f, 3i) have increased the activity. Generally, the newly synthesized compounds seem to be really promising compounds for their anticonvulsant activity. In the light of those findings we will undertake further synthetic studies on the new compounds in the future. Acknowledgments This study was supported by a grant from TUBITAK (Project no: TBAG 2021). Experimental All chemicals used in this study were supplied by Merck (Darmstadt, Germany) and Aldrich Chemical Co. (Steinheim, Germany). Melting points were determined with a Thomas Hoover capillary melting point apparatus (Philadelphia, PA, USA) and were uncorrected. IR spectra were recorded on a Perkin Elmer FT-IR Spectrometer 1720 X (Perkin Elmer, Beaconsfield, UK) as KBr disc (γ cm -1 ). 1 H-NMR spectra were obtained in CDCl 3 on a Bruker AC 80 MHz spectrophotometer and Bruker GMBH DPX-400 MHz High Performance Digital FT NMR spectrophotometer (Bruker, Karlsruhe, Germany) using TMS as an internal standard (chemical shift in δ/ppm). 13 C-NMR and 13 C-DEPT spectra were recorded on a Bruker DPX-400 MHz High Performance Digital FT NMR spectrophotometer. Fast atom bombardment (FAB) mass spectra were recorded via VG Analytical ZAB-SE with Matrix m- nitrobenzylalcohol+sodium and Xenon Gas @ 8 KV. Electroimpact mass spectrometry (EIMS) spectra were obtained on a Micromass UK Platform-II. The elemental analyses were performed at Leco CHNS-932 Elemental Analyzer (Philadelphia, PA, USA) by The Scientific and Technical Research Council of Turkey Instrumental Analysis Laboratories (Ankara, Turkey). 2-Chloromethyl-5-hydroxy-4H-pyran-4-one (Chlorokojic acid) (1) was synthesized as described by Ellis et al. [26]. Yield 76%, mp 166 167 C (lit. 166 167 C). IR (KBr disc) 3230 (O-H st), 1657 (C=O st), 1623, 1586, 1455 (C=C st), 1286, 1166 (C-O st), 767 cm 1 (C-Cl st). 1 H-NMR (DMSO-d 6,60MHz)δ 4.55 (2H; s; 2-CH 2 Cl), 6.45 (1H; s; H 3 ), 8.00 (1H; s; H 6 ), 9.0-9.4 (1H; broad; 5-OH). 2-Methyl-5-hydroxy-4H-pyran-4-one (Allomaltol) (2) Chlorokojic acid 1 (30 g, 0.187 mol, 1 equiv.) was added to 100 ml of distilled water and heated to 50 C while stirring. Zinc dust (24.4 g, 0.375 mol, 2 equiv.) was added followed by the dropwise addition of conc. hydrochloric acid (56.1 ml, 3 equiv.) over 1 h with vigorous stirring, maintaining the temperature between 70 80 C. The reaction mixture was stirred for another 3 h at 70 C. The excess zinc was removed by hot filtration and the filtrate extracted with dichloromethane (3 200 ml). The combined organic extracts were dried over anhydrous sodium sulphate, filtered, and concentrated in vacuum to yield the crude product. Recrystallisation from isopropanol afforded allomaltol as colourless plates (14.8 g, 63%): mp 152-153 C (lit. 153 155 C). IR (KBr disc) 3300-3100 (O-H st), 1640 (C=O st), 1587, 1384 (C=C st), 1223, 1150, 1050 cm -1 (C-O st). 1 H-NMR (DMSO-d 6, 60 MHz) 2.25 (3H; s; 2-CH 3 ), 6.10 (1H; s; H 3 ), 6.30-7.15 (broad, 1H, -OH), 7.80 (1H; s; H 6 ). MS (FAB): m/z 127 (M + +H, 100 %). General synthesis of Mannich bases (3a 3m) Mannich bases were prepared by the reaction of substituted piperazine derivatives (0.01 mol) and allomaltol (0.01 mol) in methanol (20 ml) with 37% formaline (1 ml). The mixture was stirred vigorously for 15 to 25 min. The resulting precipitate was collected by filtration and the crude product recrystallized from chloroform/petroleum spirit. 3-Hydroxy-6-methyl-2-(4-phenyl-piperazin-1-ylmethyl)-4Hpyran-4-one (3a) IR (KBr disc) 1628 (C=O st), 1461 (C=C st), 1378 cm 1 (C-O st). 1 H-NMR δ (CDCl 3, 80 MHz) 2.50 (3H; s; 6-CH 3 ), 3.10 (4H; t; piperazine), 3.50 (4H; t; piperazine), 4.00 (2H; s; -CH 2 -), 6.50 (1H; s; H 5 ), 7.20 7.60 ppm (5H; m; phenyl). MS(EI) m/z 300 (M + ), 301 (M + +1), 160, 140, 120 (base peak), 104. Anal. Cal. for C 17 H 20 N 2 O 3.H 2 O M.W.: 300.35 Cal. C: 64.13 H: 6.96 N: 8.79. Found C: 63.90 H: 6.32 N: 8.95. 2-[4-(4-Acetyl-phenyl)-piperazin-1-ylmethyl]-3-hydroxy-6- methyl-4h-pyran-4-one (3b) IR (KBr disc) 1658, 1612 (C=O st), 1461 (C=C st), 1379, 1215 cm 1 (C-O st). 1 H-NMR δ (CDCl 3, 80 MHz) 2.80 (3H; s; 6- CH 3 ), 3.10 (3H; s; -CO-CH 3 ), 3.30 (4H; t; piperazine), 3.90

286 Aytemir et al. Arch. Pharm. Pharm. Med. Chem. 2004, 337, 281 288 (4H; t; piperazine), 4.20 (2H; s; -CH 2 -), 6.70 (1H; s; H 5 ), 7.30 (2H; d; J= 8 Hz, phenyl), 8.40 ppm (2H; d; J= 8 Hz, phenyl). Anal. Cal. for C 19 H 22 N 2 O 4 M.W.: 342.38 Cal. C: 66.65 H: 6.47 N: 8.18. Found C: 67.11 H: 6.47 N: 8.25. 3-Hydroxy-2-[4-(2-methoxy-phenyl)-piperazin-1-ylmethyl]-6- methyl-4h-pyran-4-one (3c) IR (KBr disc) 1658 (C=O st), 1612, 1460 (C=C st), 1379, 1216, 1138 cm 1 (C-O st). 1 H-NMR δ (CDCl 3, 400 MHz) 2.33 (3H; s; 6-CH 3 ), 2.83 (4H; t; piperazine), 3.14 (4H; t; piperazine), 3.74 (2H; s; -CH 2 -), 3.88 (2H; s; -OCH 3 ), 6.23 (1H; s; H 5 ), 6.80 7.10 ppm (4H; m; phenyl). 13 C-NMR δ (CDCl 3 + DMSO-d 6 ) 25.2 (6-CH 3 ), 55.7 and 58.2 (piperazine, -CH 2 -), 59.7 (-CH 2 -), 60.6 (-OCH 3 ), 116.6, 116,9, 123.3 and 126.2 (phenyl, -CH-), 128.0 (4H-pyran-4-one -CH-, C 5 ), 146.5 C 6, 149.0 (phenyl, C-), 151.5 C 3, 157.4 (phenyl, C-O-), 169.9 C 2, 179.2 C 4 ppm. 13 C-DEPT δ (CDCl 3 + DMSO-d 6 ) 25.2 ppm 6-CH 3, 55.7 and 58.2 (piperazine CH 2, -3.7, -3.7), 59.7 (-CH 2 -, -1.8), 60.6 (-OCH 3 ), 116.6, 116.9, 123.3, 126.2, 128.0 ppm -CH. MS (EI) m/z 330 (M + ), 331 (M + +1), 191, 139, 140, 106, 42 (base peak). Anal. Cal. for C 18 H 22 N 2 O 4 M.W.: 330.37 Cal. C: 65.43 H: 6.71 N: 8.47. Found C: 65.34 H: 6.35 N: 8.37. 3-Hydroxy-2-[4-(3-methoxy-phenyl)-piperazin-1-ylmethyl]-6- methyl-4h-pyran-4-one (3d) IR (KBr disc) 1674 (C=O st), 1460 (C=C st), 1378, 1046 cm 1 (C-O st). 1 H-NMR δ (CDCl 3, 80 MHz) 2.30 (3H; s; 6-CH 3 ), 2.70 (4H; t; piperazine), 3.10 (4H; t; piperazine), 3.70 (2H; s; -CH 2 -), 3.80 (3H; s; -OCH 3 ), 6.20 (1H; s; H 5 ), 6.40-7.30 ppm (4H; m; phenyl). Anal. Cal. for C 18 H 22 N 2 O 4 M.W.: 330.37 Cal. C: 65.43 H: 6.71 N: 8.47. Found C: 64.94 H: 6.83 N: 8.23. 2-[4-(2-Chloro-phenyl)-piperazin-1-ylmethyl]-3-hydroxy-6- methyl-4h-pyran-4-one (3e) IR (KBr disc) 1650 (C=O st), 1460 (C=C st), 1378, 1010 cm 1 (C-O st). 1 H-NMR δ (CDCl 3, 80 MHz) 2.30 (3H; s; 6-CH 3 ), 2.70 (4H; t; piperazine), 3.10 (4H; t; piperazine), 3.70 (2H; s; -CH 2 -), 6.20 (1H; s; H 5 ), 7.00 7.40 ppm (4H; m; phenyl). Anal. Cal. for C 17 H 19 ClN 2 O 3 M.W.: 334.79 Cal. C: 60.98 H: 5.72 N: 8.36. Found C: 60.72 H: 5.82 N: 8.21. 2-[4-(4-Chloro-phenyl)-piperazin-1-ylmethyl]-3-hydroxy-6- methyl-4h-pyran-4-one (3f) IR (KBr disc) 1621 (C=O st), 1461 (C=C st), 1378, 1216 cm 1 (C-O st). 1 H-NMR δ (CDCl 3, 80 MHz) 2.30 (3H; s; 6-CH 3 ), 2.70 (4H; t; piperazine), 3.20 (4H; t; piperazine), 3.70 (2H; s; -CH 2 -), 6.20 (1H; s; H 5 ), 6.90 (2H; d; J= 8 Hz, phenyl), 7.20 ppm (2H; d; J= 8 Hz, phenyl). Anal. Cal. for C 17 H 19 ClN 2 O 3 M.W.: 334.79 Cal. C: 60.98 H: 5.72 N: 8.36. Found C: 60.70 H: 6.20 N: 8.12. 2-[4-(2-Fluoro-phenyl)-piperazin-1-ylmethyl]-3-hydroxy-6- methyl-4h-pyran-4-one (3g) IR (KBr disc) 1624 (C=O st), 1511, 1461 (C=C st), 1380, 1207 cm 1 (C-O st). 1 H-NMR δ (CDCl 3, 80 MHz) 2.30 (3H; s; 6- CH 3 ), 2.70 (4H; t; piperazine), 3.10 (4H; t; piperazine), 3.75 (2H; s; -CH 2 -), 6.20 (1H; s; H 5 ), 6.80 7.20 ppm (4H; m; phenyl). Anal. Cal. for C 17 H 19 FN 2 O 3 M.W.: 318.34 Cal. C: 64.13 H: 6.01 N: 8.79. Found C: 63.65 H: 6.46 N: 8.64. 2-[4-(4-Fluoro-phenyl)-piperazin-1-ylmethyl]-3-hydroxy-6- methyl-4h-pyran-4-one (3h) IR (KBr disc) 1612 (C=O st), 1460 (C=C st), 1378, 1013 cm 1 (C-O st). 1 H-NMR δ (CDCl 3, 80 MHz) 2.30 (3H; s; 6-CH 3 ), 2.80 (4H; t; piperazine), 3.20 (4H; t; piperazine), 3.80 (2H; s; -CH 2 -), 6.30 (1H; s; H 5 ), 6.80 7.20 ppm (4H; m; phenyl). Anal. Cal. for C 17 H 19 FN 2 O 3 M.W.: 318.34 Cal. C: 64.13 H: 6.01 N: 8.79. Found C: 63.86 H: 6.06 N: 8.82. 3-Hydroxy-6-methyl-2-[4-(2-trifluoromethyl-phenyl)-piperazin- 1-ylmethyl]-4H-pyran-4-one (3i) IR (KBr disc) 1632 (C=O st), 1458 (C=C st), 1363, 1253 cm 1 (C-O st). 1 H-NMR δ (CDCl 3, 80 MHz) 2.25 (3H; s; 6-CH 3 ), 2.70 (4H; t; piperazine), 3.15 (4H; t; piperazine), 3.70 (2H; s; -CH 2 -), 6.20 (1H; s; H 5 ), 7.00 7.40 ppm (4H; m; phenyl). Anal. Cal. for C 18 H 19 F 3 N 2 O 3 M.W.: 368.35 Cal. C: 58.69 H: 5.20 N: 7.60. Found C: 58.61 H: 5.46 N: 7.60. 3-Hydroxy-6-methyl-2-[4-(4-nitro-phenyl)-piperazin-1-ylmethyl]-4H-pyran-4-one (3j) IR (KBr disc) 1623 (C=O st), 1495, 1460 (C=C st), 1336, 1207 cm 1 (C-O st). 1 H-NMR δ (CDCl 3, 80 MHz) 2.30 (3H; s; 6- CH 3 ), 2.70 (4H; t; piperazine), 3.40 (4H; t; piperazine), 3.80 (2H; s; -CH 2 -), 6.25 (1H; s; H 5 ), 6.80 (2H; d; J= 8 Hz, phenyl), 8.20 ppm (2H; d; J= 8 Hz, phenyl). Anal. Cal. for C 17 H 19 N 3 O 5 M.W.: 345.35 Cal. C: 59.12 H: 5.54 N: 12.16. Found C: 58.69 H: 5.62 N: 11.94. 2-(4-Benzyl-piperazin-1-ylmethyl)-3-hydroxy-6-methyl-4Hpyran-4-one (3k) IR (KBr disc) 1645 (C=O st), 1460 (C=C st), 1376 cm -1 (C-O st). 1 H-NMR δ (CDCl 3, 80 MHz) 2.25 (3H; s; 6-CH 3 ), 2.30-2.80 (8H; m; piperazine), 3.55 (2H; s; -CH 2 -), 3.65 (2H; s; -CH 2 -), 6.25 (1H; s; H 5 ), 7.30 ppm (5H; s; phenyl). Anal. Cal. for C 18 H 22 N 2 O 3 M.W.:314.37Cal.C:68.76H:7.05N:8.91. Found C: 68.66 H: 7.04 N: 8.84. 2-(4-Benzo[1,3]-dioxol-5ylmethyl-piperazin-1-ylmethyl)-3- hydroxy-6-methyl-4h-pyran-4-one (3l) IR (KBr disc) 1624 (C=O st), 1459 (C=C st), 1375, 1199, 1150, 1042 cm -1 (C-O st). 1 H-NMR δ (CDCl 3, 400 MHz) 2.20 (3H; s; 6-CH 3 ), 2.41 (4H; t; piperazine), 2.55 (4H; t; piperazine), 3.34 (2H; s; -CH 2 -), 3.56 (2H; s; -CH 2 -), 5.85 (2H; s; -OCH 2 O-), 6.11 (1H; s; H 5 ), 6.65 (2H; s; 1,3-benzodioxole), 6.75 ppm (1H; s; 1,3-benzodioxole). 13 C-NMR (CDCl 3 + DMSO-d 6 ) 25.20 (6-CH 3 ), 57.8 and 57.9 (piperazine CH 2 ), 60.1 (-CH 2 -), 67.6 (-CH 2 -), 106.0 (-OCH 2 O-, C 2 ), 112.9, 114.5, 116.7 (phenyl, -CH-, C 4,5,7 ), 127.3 (4H-pyran-4-one, -CH-, C 5 ), 137.0 C 6, 148.9 C 6, 151.0 C 3a, 151.7 C 3, 152.7 C 7a, 169.9 C 2, 179.2 C 4 ppm. 13 C-DEPT (CDCl 3 + DMSOd 6 ) 25.2 ppm 6-CH 3, 57.8 and 57.9 (piperazine CH 2, -1.9, -2.4), 60.10 (-CH 2 -, -1.7), 60.6 (-CH 2 -, -1.67), 106.0 (-OCH 2 O-, -0.83), 112.2, 114.5, 116.7, 127.3 ppm -CH. EIMS m/e 358.38 (M + ), 359.71 (M + +1), 219, 140, 139 (base peak). Anal. Cal. for C 19 H 22 N 2 O 5 M.W.: 358.38 Cal. C: 63.67 H: 6.18 N: 7.81. Found C: 63.21 H: 5.74 N: 7.76. 3-Hydroxy-6-methyl-2-(4-pyridin-2-yl-piperazin-1-ylmethyl)- 4H-pyran-4-one (3m) IR (KBr disc) 1630 (C=O st), 1461 (C=C st), 1380, 1204, 1045 cm 1 (C-O st). 1 H-NMR δ (CDCl 3, 80 MHz) 2.30 (3H; s; 6- CH 3 ), 2.60 (4H; t; piperazine), 3.20-4.00 (6H; m; piperazine and -CH 2 -), 6.20 (1H; s; H 5 ), 6.60-7.60 (3H; m; pyridyl), 8.20

Arch. Pharm. Pharm. Med. Chem. 2004, 337, 281 288 3-Hydroxy-6-methyl-2-substituted 4H-Pyran-4-one Derivatives 287 ppm (1H; d; J= 4 Hz, pyridyl). Anal. Cal. for C 16 H 19 N 3 O 3 M.W.: 301.34 Cal. C: 63.77 H: 6.35 N: 13.94. Found C: 63.98 H: 5.83 N: 13.90. Anticonvulsant activity Stimulator (Grass S88, Astro-Med. Inc. Grass Instrument Division, W. Warwick, RI, USA), constant current unit (Grass CCU1A, Grass Medical Instrument, Quincy, MA, USA), and corneal electrode were used for the evaluation of anticonvulsant activity. All synthesized compounds were suspended in 30 % aqueous of PEG 400 and administered intraperitoneally in a volume of 0.01 mg/kg body weight to the mice. Twelve Swiss albino male mice (20 ± 2 g) were used for each compound (mice were obtained from the Hacettepe University Animal Farm and used in our laboratory according to the NINCDS-ADD program [20] and according to the Hacettepe University, Test Animals Ethic Committee 17. 04. 2002 date 2002/ 24-3 number decision). Control animals received 30 % aqueous PEG 400. The compounds were tested for their anticonvulsant activity against MES and scmet induced seizures and the rotorod toxicity test was performed to check for neurological toxicity according to the phase I tests of the ADD (Antiepileptic Drug Development) program [20]. Pentylenetetrazole (Metrazol) was administered s.c. from the back of the neck. The rotorod toxicity test was performed on a 1 inch diameter knurled wooden rod; rotating at 6 rpm (the rotorod used in Screening I test was made by Hacettepe University Technical Department). MES maximal electroshock seizure test Maximal electroshock seizures are elicited with a 60 Hz/50 ma alternating current (5-7 times is required to elicit minimal electroshock seizures) delivered for 0.2 s via corneal electrodes. A drop of 0.9% saline is instilled in the eye prior to application of the electrodes in order to prevent the death of the animal. Abolition of the hind limb tonic extension component of the seizure is defined as protection. ScMet subcutaneous pentylenetetrazole (Metrazol) 85 mg/kg of pentylenetetrazole (produces seizures in more than 95% of mice) is administered as a 0.5% solution subcutaneously in the posterior midline. The animals were observed for 30 min; failure to observe even a threshold seizure (a single episode of clonic spasms of at least 5 s duration) was defined as protection. Neurotoxicity The rotorod test was used to evaluate neurotoxicity. The animalwasplacedona1inchdiameterknurledwooden rod rotating at 6 rpm. Normal mice remain on a rod rotating at this speed indefinitely. Neurologic toxicity was defined as the failure of the animal to remain on the rod for 1 min. Antifungal activity Minimal inhibitory concentrations (MICs) were determined by microdilution broth following the procedures recommended by the National Committee for Clinical Laboratory Standards (NCCLS) [30]. Fluconazole was used as reference compound for the fungi. For testing antifungal activities of the compounds, these reference strains were tested: Candida albicans ATCC 90028, Candida krusei ATCC 6258 and Candida parapsilosis ATCC 22019 [30]. MIC values of compounds are presented in Table 3. Fluconazole was dissolved in sterile distilled water. The stock solutions of the all com- pounds were prepared in dimethylsulfoxide (DMSO). The dilutions in the test medium were prepared at the required concentration of 512-0.5 µg/ml and for fluconazole; 64 0.0625 µg/ml. It was established that the dilution of DMSO lacked antifungal activity against any of the test microorganisms Antifungal activity assay All fungi were cultivated on Sabouraud Dextrose Agar (Merck). The final inoculum was prepared to be 0.5 2.5 10 3 cfu/ml in each well. RPMI-1640 medium (ICN-Flow, Aurora, OH, USA) with L-glutamine, buffered with 3-(N-morpholino) propanesulphonic acid (MOPS) (Buffer-ICN-Flow, Aurora, OH, USA) at ph = 7.4 is used in the test. The microtiter plates were incubated at 35 C and evaluated visually at 24 h. The MIC values were recorded as the lowest concentrations of the substances that had no visible turbidity. References [1] J. B. Martin, Epilepsy Res. 2001, 45, 3 6. [2] M. J. Liebman, A. J. Schneider, Ann. Rep. Med. Chem. 1985, 20, 11 20. [3] H. Schäfer, Handbook of Experimental Pharmacology- Antiepileptic Drugs, Springer-Verlag, Berlin, 1985, pp. 199 210. [4] C. O. Wilson, O. Gisvold, R. F. Doerge, Textbook of Organic, Medicinal and Pharmaceutical Chemistry, 7 th Ed. Lippincott Co., Philadelphia, 1971. [5] A. Kordkovas, Essentials of Medicinal Chemistry, 2 nd Ed., John Wiley Sons, New York, 1988. [6] S. J. Enna, A. Maggi, Life Sci. 1979, 24, 1727 1738. [7] J. G. Atkinson, Y. G. J. Rokach, C. S. Rooney, C. S. McFarlane, A. Rackham, N. N. Share, J. Med. Chem. 1979, 22, 99 106. [8] J. W. Ferkany, T. H. Andree, D. E. Ciarke, S. J. Enna, Neuropharmacology 1981, 20, 1177 1182. [9] G. E. Martin, R. J. Bendesky, Neurosci. Lett 1981, 27(1), 37 40. [10] K. A. Pelley, J. L. Vaught, Neuropharmacology 1987, 26, 301 307. [11] R. Kimura, S. Matsui, S. Ito, T. Aimoto, T. Murata, Chem. Pharm. Bull. 1980, 28, 2570 2579. [12] N. Aoyagi, R. Kimura, T. Murata, Chem. Pharm. Bull. 1974, 22(5), 1008 1013. [13] M. Veverka, E. Kralovicova, Collect. Czech. Chem. Commun. 1990, 55, 833 840. [14] M. Veverka, Chem. Papers 1992, 46(3), 206 210. [15] P. A. Wolf, W. M. Westveer, Arch. Biochem, 1950, 28, 201 206. [16] T. Kotani, I. Ichimoto, C. Tatsumi, T. Fujita, Agr. Biol. Chem. 1975, 39(6), 1311 1317. [17] M. Uher, V. Konecny, O. Rajniakova, Chem. Papers 1994, 48(4), 282 284. [18] M. Yamato, Y. Yasumoto, J. Sakai, R. F. Luduena, A. Baneerjee, T. Tashiro, J. Med. Chem. 1987, 30, 1897 1900.

288 Aytemir et al. Arch. Pharm. Pharm. Med. Chem. 2004, 337, 281 288 [19] J. Cabanes, S. Chazarra, F. Garcia-Carmona, J. Pharm. Pharmacol. 1994, 46, 982 985. [20] R. L. Krall, J. K. Penry, B. G. White, J. H. Kupferberg, E. A. Swinyard, Epilepsia 1978, 9, 409 428. [21] Ü. Çalış, S. Dalkara, M. Ertan, R. Sunal, Arch. Pharm. (Weinheim) 1988, 321, 841 846. [22] Ü. Çalış, S. Dalkara, M. Ertan, Arzneim.-Forsch./Drug Res. 1992, 42(1), 592 594. [23] F. Özkanlı, S. Dalkara, Ü. Çalış, A. Willke, Arzneim.- Forsch../Drug Res. 1994, 44(II), 920 924. [24] Ü. Çalış, M. Köksal, Arzneim.-Forsch./Drug Res. 2001, 51(II), 523 528. [25] T. Yabuta, J. Chem. Soc. 1924, 125, 575-587. [26] B. L. Ellis, A. K. Duhme, R. C. Hider, M. B. Hossain, S. Rizvi, D. van der Helm, J. Med. Chem. 1996, 39, 3659 3670. [27] G. O Brien, J. M. Patterson, J. R. Meadow, J. Org. Chem. 1960, 25,86 89. [28] M. K. Patel, R. Fox, P. D. Taylor, Tetrahedron 1996, 52, 1835 1840. [29] L.L.WoodsJ. Am. Chem. Soc. 1946, 68, 2744 2745. [30] NCCLS Approved Standard M-27-A, 17(9) Villanova, PA, 1997.