Synthesis of Some New Hydroxypyranone Derivatives and Evaluation of Their Anticonvulsant Activities

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1 FABAD J. Pharm. Sci., 31, 23-29, 2006 RESEARCH ARTICLE Synthesis of Some New Hydroxypyranone Derivatives and Evaluation of Their Anticonvulsant Activities Mutlu Dilsiz AYTEM R*, Ünsal ÇALIfi* Synthesis of Some New Hydroxypyranone Derivatives and Evaluation of Their Anticonvulsant Activities Summary In this study, Mannich bases of hydroxypyranone derivatives were synthesized by starting from kojic acid. Their anticonvulsant activities were evaluated in in vivo studies. Mannich bases were prepared by the reaction of cyclic amine derivatives with allomaltol and formaline. The chemical structure of synthesized compounds 3a-e was proven by IR, 1H-NMR, mass spectra and elementary analysis data. Anticonvulsant activities of the compounds were examined by maximal electroshock (MES) and subcutaneous Metrazol (scmet) tests. Neurotoxicity was determined by rotorod toxicity test. All these tests were performed in mice according to procedures of the Antiepileptic Drug Development (ADD) program of the National Institutes of Health (NIH). According to the activity studies, compound 3a, which is carrying 4-[(2,3dimethylphenyl)piperazin-1-yl]methyl at 2 position on the pyranone ring, showed activity against scmet at 0 dose at 4 h. Only compound 3b was determined to be protective against MES at 0 dose at 5 h in this series. None of the synthesized compounds was observed to have neurotoxicity. Key Words: Hydroxypyranone derivatives, kojic acid, allomaltol, Mannich reaction, anticonvulsant activity. Received : Revised : Accepted : INTRODUCTION About 0.5 to 1% of the world s population is affected by epilepsy, which is one of the most common neurological disorders and characterized by recurrent seizure attacks. The drugs that are widely used for treating epileptic seizures have failed to adequately control seizures and have unfavorable adverse effects such as ataxia, hepatotoxicity, gingival hyperplasia and megaloblastic anemia. The restrictive treatment of epileptic seizures in patients have led to researches * to discover new agents with more effective anticonvulsant activity and which toxicity1,2. The derivatives of hydroxypyranone exhibit a variety of biological activities, such as herbicidal 3, antimicrobial 4-8, pesticide and insecticide 9,10, antitumor11,12 and tyrosinase inhibitory effect13,14. 5-Hydroxy-2-(hydroxymethyl)-4H-pyran-4-one (kojic acid) and 3-hydroxy-2-methyl-4H-pyran-4-one (mal- Hacettepe University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, Ankara Turkey Corresponding author mutlud@hacettepe.edu.tr 23

2 Aytemir, Çal fl tol) have catechol groups, which possess metal chelating ability. They form stable complexes with several metal ions 15,16. Tris (maltol)aluminium(iii) and -gallium(iii) complexes have lipid solubility; therefore, they can cross the blood-brain barrier with considerable facility 17. Maltol, kojic acid and allomaltol (5-hydroxy-2-methyl- 4H-pyran-4-one) have been reported to show the same anticonvulsant effect 18,19. 2-Butyl-3-hydroxy- 4H-pyran-4-ones was the most potent protective of the pentylenetetrazole-induced convulsion among the 2-alkyl series. The increase in inhibitory effect of these compounds with increasing carbon number of the alkyl group might be due to enhancement of lipid solubility 18. 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 20. In our previous studies 21, we have reported the anticonvulsant and antifungal activities of novel Mannich base derivatives possessing piperazine groups. This study describes the synthesis of Mannich base derivatives possessing piperidine, piperazine and morpholine rings and investigates their anticonvulsant activity. Anticonvulsant activities of the synthesized compounds were examined by maximal electroshock (MES) and subcutaneous Metrazol (scmet) tests. Seizure assays and neurotoxicity were determined according to the phase I tests of the Antiepileptic Drug Development (ADD) program, which were developed by the National Institute of Neurological Disorders and Stroke (NINDS) and National Institutes of Health (NIH) MATERIALS and METHODS Chemistry 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 (Beaconsfield, UK) as KBr disc (γ, cm -1 ). 1 H-NMR spectra were obtained on a Bruker AC 80 MHz spectrophotometer and Bruker GMBH DPX-400 MHz High Performance Digital FT NMR spectrophotometer (Karlsruhe, Germany) using TMS as an internal standard (chemical shift in δ, ppm). Mass spectra were recorded on Scientific Instrument Service HPP7-M direct insertion probe spectrometer using Agilent 5973 network mass selective electron impact detector and Agilent 6890 network GC system. The elemental analyses were performed with a Leco CHNS-932 (St. Joseph, MI, USA) at The Scientific & Technological Research Council of Turkey-Ankara Testing and Analyses Laboratory (TÜB TAK-ATAL). The purity of the compounds was assessed by TLC on Kieselgel 60 F254 (Merck, Darmstadt, Germany). Seven compounds were synthesized according to the procedures as shown below. 2-(Chloromethyl)-5-hydroxy-4H-pyran-4-one 27 (1) Kojic acid (25 g, mol) was dissolved in distilled thionyl chloride (100 ml) and stirred for 1 h. The product formed, a yellow crystalline solid, was collected by filtration and washed with petroleum ether and then recrystallized from water to give colorless needles (23.5 g, 74%), mp C (lit. value C). IR (KBr disc) 32 (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, 60 MHz) δ 4.55 (2H; s; 2-CH 2 Cl), 6.45 (1H; s; H 3 ), 8.00 (1H; s; H 6 ), (1H; broad; 5-OH). 5-Hydroxy-2-methyl-4H-pyran-4-one 27 (2) Chlorokojic acid (1) (20 g, mol) was added to 100 ml of distilled water and heated to 50ºC with stirring. Zinc dust (16.29 g, 0.25 mol) was added followed by the dropwise addition of concentrated hydrochloric acid (37 ml) over 1 h with vigorous stirring maintaining the temperature between 70-80ºC. The reaction mixture was left stirring for 2-3 h 24

3 FABAD J. Pharm. Sci., 31, 23-29, 2006 at 70 C. The excess zinc was removed by hot filtration and the filtrate extracted with dichloromethane (3 x 150 ml). The combined organic extracts were dried over anhydrous sodium sulphate, filtered and concentrated in vacuum to yield the crude product. Recrystallization from isopropanol afforded allomaltol (2) as colorless plates (9.5 g, 60%): mp ºC (lit. value C). IR (KBr disc) (O-H st), 1640 (C=O st), 1587, 1384 (C=C st), 1223, 1150 cm -1 (C-O st). 1 H-NMR (DMSO-d 6, 60 MHz) δ 2.25 (3H; s; 2-CH 3 ), 6.10 (1H; s; H 3 ), (broad, 1H, 5-OH), 7.80 (1H; s; H 6 ). General preparation of Mannich bases of allomaltol derivatives (3a-e) Mannich bases were prepared by the reaction of substituted cyclic amine derivatives (0.01 mol) and allomaltol (0.01 mol) in methanol (15 ml) with 37% formaline (1 ml). The reaction mixture was stirred vigorously for 15 to 25 min. The resulting precipitate was collected by filtration and washed with cold methanol. The crude product recrystallized from appropriate solvents. 2-{[4-(2,3-Dimethylphenyl)piperazin-1-yl]methyl}- 3-hydroxy-6-methyl-4H-pyran-4-one (3a) 1-(2,3-xylyl)piperazine, allomaltol and formaline. Recrystallization from methanol gave a white crystalline solid. IR (KBr disc) 1659 (C=O st), 1587 (C=C st) and 1219 cm -1 (C-O st). 1 H-NMR δ (CDCl 3, 400 MHz) 2.22 (3H; s; -CH 3 ), 2.27 (3H; s; -CH 3 ), 2.33 (3H; s; 6-CH 3 ), (4H; m; piperazine), 2.95 (4H; t; J= 4.66, piperazine), 3.75 (2H; s; -CH 2 -), 6.26 (1H; s; H 5 ), 6.92 (2H; d; J= 6.87, phenyl), 7.08 ppm (1H; t; J= 7.72, phenyl). EI m/e 328 (M + ), 329 (M + +1), 189, 160, 148 (base peak), 132, 117, 105, 91, 77, 56, 43. Anal. Cal. for C 19 H 24 N 2 O 3 M.W.: 328 Cal. C: H: 7.37 N: 8.53 Found C: H: 8.03 N: {[4-(3-Chlorophenyl)piperazin-1-yl]methyl}-3- hydroxy-6-methyl-4h-pyran-4-one (3b) 1-(3-chlorophenyl)piperazine, allomaltol and formaline. Recrystallization from chloroform gave a white crystalline solid. IR (KBr disc) 16 (C=O st), 1488 (C=C st) and 1224 cm -1 (C-O st). 1 H-NMR δ (DMSOd 6, 400 MHz) 2.27 (3H; s; 6-CH 3 ), 2.57 (4H; t; J= 4.60, piperazine), 3.16 (4H; t; J= 4.56, piperazine), 3.55 (2H; s; -CH2-), 6.24 (1H; s; H 5 ), 6.77 (1H; d; J= 7.68, phenyl), 6.88 (1H; d; J= 8.34, phenyl), 6.92 (1H; s; phenyl), 7.20 ppm (1H; t; J= 8.10, phenyl). EI m/e 334 (M + ), 335 (M + +1), 264, 195, 179, 166, 154 (base peak), 140, 111, 85, 69, 56, 43. Cal. for C 17 H 19 ClN 2 O 3 M.W.: Cal. C: H: 5.72 N: 8.36 Found C: H: 5.24 N: Hydroxy-6-methyl-2-[(4-pyrimidin-2-ylpiperazin- 1-yl)methyl]-4H-pyran-4-one (3c) N-(2-pyrimidinyl)piperazine, allomaltol and formaline. Recrystallization from chloroform/petroleum ether (40-60 C) gave a white powder. IR (KBr disc) 1621 (C=O st), 1587 (C=C st) and 1218 cm -1 (C-O st). 1 H-NMR δ (CDCl3, 80 MHz) 2. (3H; s; 6-CH 3 ), 2.60 (4H; t; piperazine), 3.60 (2H; s; -CH 2 -), 3.80 (4H; t; piperazine), 6.20 (1H; s; H 5 ), 6.40 (1H; t; pyrimidine), 8.25 ppm (2H; d; pyrimidine). EI m/e 2 (M + ), 3 (M + +1), 264, 177, 163 (base peak), 140, 122, 108, 96, 56, 43. Anal. Cal. for C 15 H 18 N 4 O 3 M.W.: 2 Cal. C: H: 6.00 N: Found C: H: 5.87 N: (1,4'-Bipiperidin-1'-ylmethyl)-3-hydroxy-6-methyl- 4H-pyran-4-one (3d) 4-piperidinopiperidine, allomaltol and formaline. Recrystallization from chloroform/petroleum ether (40-60ºC) gave an orange powder. IR (KBr disc) 1622 (C=O st), 1459 (C=C st) and 1198 cm -1 (C-O st). 1 H-NMR δ (DMSO-d 6, 400 MHz) (11H; m; piperidine), 2.17 (3H; s; 6-CH 3 ), (8H; m; piperidine), 3.37 (2H; s; -CH 2 -), 6.13 ppm (1H; s; H 5 ). GC(MS) m/e 253, 235, 210 (base peak), 193, 158, 139, 111, 99, 86, 57. Anal. Cal. for C 17 H 26 N 2 O 3 M.W.: 6 Cal. C: H: 8.55 N: 9.14 Found C: H: 7.99 N:

4 Aytemir, Çal fl 3-Hydroxy-6-methyl-2-(morpholin-4-ylmethyl)-4Hpyran-4-one (3e) morpholino, allomaltol and formaline. Recrystallization from methanol gave a white crystalline solid. IR (KBr disc) 1621 (C=O st), 1461 (C=C st) and 1200 cm -1 (C-O st). 1 H-NMR δ (CDCl 3, 80 MHz) 2.40 (3H; s; 6-CH 3 ), (4H; t; morpholine), (6H; m; morpholine and -CH 2 -), 6.40 ppm (1H; s; H 5 ). GC(MS) m/e 151, 140 (base peak), 111, 86, 69, 56. Anal. Cal. for C 11 H 15 NO 4 M.W.: Cal. C: H: 6.71 N: 6.21 Found C: H: 6.31 N: Anticonvulsant Activity The compounds were tested for their anticonvulsant activity against MES- and scmet-induced seizures, and rotorod toxicity test was performed for neurological toxicity according to the phase I tests of the ADD program 24. 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 electrodes were used for the evaluation of anticonvulsant activity against MES test. All synthesized compounds were suspended in % aqueous PEG 400 and administered to the mice intraperitoneally in a volume of 0.01 ml/g body weight. Twelve Swiss albino male mice (20 ± 2 g) were used for each compound (mice were obtained from the Hacettepe University Animal Farm according to the NINDS-ADD program 24 and used according to Hacettepe University s `Laboratory Animals Ethics Committee decision ( date 2002/24-3 number). Control animals received % aqueous PEG 400. Pentetrazol (metrazol) was administer ed subcutaneously (s.c.) on 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 Phase I test was made by Hacettepe University s Technical Department). Maximal Electroshock Seizure (MES) test Maximal electroshock seizures were elicited with a 60-cycle alternating current of 50 ma intensity (5-7 times more than that required to elicit minimal electroshock seizures) delivered for 0.2 sec via corneal electrodes. A drop of 0.9% saline was instilled into 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 was defined as protection. Subcutaneous Pentetrazol (Metrazol) (ScMet) test 85 of pentetrazol (produces seizures in more than 95% of mice) was administered as a 0.5% solution s.c. into the posterior midline. The animal was observed for min to decide whether the failure of the threshold seizure (a single episode of clonic spasms of at least 5 sec duration) could be defined as protection. Neurotoxicity The rotorod test was used to evaluate neurotoxicity. The animal was placed on a 1 inch diameter knurled wooden 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. RESULTS and DISCUSSION Chemistry Allomaltol, which was used as a starting material, was synthesized from commercially available kojic acid in accordance with previous studies 27,28. Chlorokojic acid (1) was prepared with quite a significant yield (75%) by chlorination of kojic acid using neat thionyl chloride at room temperature. Reduction of chlorokojic acid with zinc dust in concentrated hydrochloric acid resulted in the production of allomaltol (2) 27,28. In the hydroxypyran form, only one enolic hydroxyl group is present and the position ortho to this hydroxyl group is readily subject to aminomethylation in the Mannich reaction at room temperature 29,. In this study, the synthesis of 2-substituted-3-hydroxy- 26

5 FABAD J. Pharm. Sci., 31, 23-29, methyl-4H-pyran-4-one derivatives (3a-e) was prepared by the reaction of appropriate cyclic amine derivatives with allomaltol and formaldehyde at room temperature as shown in Scheme 1. Yields and the melting point properties of the synthesized compounds are presented in Table 1. Table 1. Yields and melting points of the synthesized compounds (3a-e) The structures of the synthesized compounds were confirmed by IR, 1 H-NMR, mass spectra and elementary analysis. All the spectral data of the compounds were in accordance with the assigned structures. In the IR spectra, the synthesized compounds have O-H stretching bands at cm 1. All compounds were associated with C=O, C=C and C-O stretching bands at , and cm 1, respectively. With 1 H-NMR spectra, allomaltol derivatives showed methyl group protons as a singlet at ppm. The methylene group protons of 3a-e appeared as a singlet at ppm. H 5 protons of the 4H-pyran-4-one ring were found as a singlet peak in the region ppm in accordance with literature 21. EI mass spectra s of 3a, 3b and 3c showed the molecular ion (M + ) and (M + +1) peaks of the compounds. Anticonvulsant Activity Compound Number 3a 3b 3c 3d 3e R Melting points ( C) Scheme 1. Schematic representation of the synthesized compounds. Yield (%) The anticonvulsant activity results of the synthesized compounds are shown in Table 2. According to the activity studies, chlorokojic acid (1) has anticonvulsant activity against MES seizures at 0 dose at 5 and 4 h. Allomaltol (2) showed activity against scmet at 0 at 4 h. Compound 3a, which is carrying 4-(2,3-dimethylphenyl)piperazinylmethyl at second position on the 4H-pyran-4-one ring, showed activity against scmet at 0 dose at 4 h. Compound 3b, carrying 4-(3-chlorophenyl)piperazine ring, was protective against MES at 0 dose at 5 h. None of compounds 3c, 3d and 3e showed anticonvulsant activity. In our previous study 21, derivatives of Mannich bases possessing piperazine ring were found to be protective against MES and scmet at different doses. In this study, when the piperazine ring (3a and 3b) was changed with piperidine (3d) and morpholine (3e) rings, Mannich bases were inactive. Neurotoxicity was not observed with any of the synthesized compounds that wer e administered to mice in the dose range of -0. Acknowledgement This research was supported by a grant from The Scientific & Technological Research Council of Turkey (TÜB TAK) (Project no: TBAG 2021). 27

6 Aytemir, Çal fl Table 2. Phase I anticonvulsant screening of the synthesized compounds Compound Number 1 1/2 h MES 4 h /2 h 100 ScMet 0 4 h Toxicity 1/2 h 4 h a 3b 3c 3d 3e MES: Maximal electroshock seizure test. scmet: Subcutaneous pentetrazole (metrazol) seizure test. Toxicity: Rotorod test. : No activity at dose level. : Noticeable activity at dose level. REFERENCES 1. Malawska B. New anticonvulsant agents, Curr. Top. Med. Chem., 5(1), 69-85, Shindikar AV, Khan F, Viswanathan CL. Design, synthesis and in vivo anticonvulsant screening in mice of novel phenylacetamides, Eur. J. Med. Chem., 41, , Veverka M, Kralovicova E. Synthesis of some biologically active derivatives of 2-hydroxymethyl-5-hydroxy-4H-pyran-4-one, Collect. Czech. Chem. Commun., 55, , Kayahara H, Shibata N, Tadasa K, Maeda H, Kotani T, Ichimoto I. Amino acid and peptide derivatives of kojic acid and their antifungal properties, Agr. Biol. Chem., 54(9), , Kotani T, Ichimoto I, Tatsumi C, Fujita T. Structure-activity study of bacteriostatic kojic acid analogs, Agr. Biol. Chem., 39(6), , Aytemir MD, Hider RC, Erol DD, Özalp M, Ekizo lu M. Synthesis of new antimicrobial agents; amide derivatives of pyranones and pyridinones, Turk J. Chem., 27(4), , Aytemir MD, Erol DD, Hider RC, Özalp M. Synthesis and evaluation of antimicrobial activity of new 3-hydroxy-6-methyl-4-oxo-4H-pyran-2- carboxamide derivatives, Turk J. Chem., 27(6), , Wan HM, Chen CC, Giridhar R, Chang TS, Wu WT. Repeated-batch production of kojic acid in a cell-retention fermenter using Aspergillus oryae M3B9, J. Ind. Microbiol. Biotechnol., 32(6), , Veverka M. Synthesis of some biologically active derivatives of 2-hydroxymethyl-5-hydroxy-4Hpyran-4-one. II. Synthesis and biological properties of S-substituted 2-thiomethyl-5-O-acyl derivatives, Chem. Papers, 46(3), , Uher M, Konecny V, Rajniakova O. Synthesis of 5-hydroxy-2-hydroxymethyl-4H-pyran-4-one derivatives with pesticide activity, Chem. Papers, 48(4), , Yamato M, Yasumoto Y, Sakai J, Luduena RF, Baneerjee A, Tashiro T. Synthesis and antitumor activity of tropolone derivatives. 6. Structureactivity relationships of antitumor-active tropolone and 8-hydroxyquinoline derivatives, J. Med. Chem.,, , Tamura T, Mitsumori K, Totsuka Y, Wakabayashi K, Kido R, Kasai H, Nasu M, Hirose M. Absence of in vivo genotoxic potential and tumor initiation activity of kojic acid in the rat thyroid, Toxicology, 222(3), , Cabanes J, Chazarra S, Garcia-Carmona F. Kojic acid, a cosmetic skin whitening agent, is a slowbinding inhibitor of catecholase activity of tyrosinase, J. Pharm. Pharmacol., 46, , Kim H, Choi J, Cho JK, Kim SY, Lee YS. Solidphase synthesis of kojic acid-tripeptides and their tyrosinase inhibitory activity, storage stability, and toxicity, Bioorg. Med. Chem. Letters, 14, , Moggia F, Brisset H, Fages F, Chaix C, Mandrand B, Dias M, Levillain E. Design, synthesis and redox properties of two ferrocene-containing iron 28

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