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CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)

Synthesis and Nootropic Activity of Some 2,3-Dihydro-1H-isoindol-1-one Derivatives Structurally Related with Piracetam
by Adelfo Reyes* a ), Leticia Huerta a ), Marisol Alfaro a ), and Andre ´ s Navarrete b )
a

´ n Experimental, ) Facultad de Estudios Superiores Zaragoza, Unidad Multidisciplinaria de Investigacio ´ noma de Me ´ xico, Batalla 5 de Mayo esquina Fuerte de Loreto, Universidad Nacional Auto ´ rcito de Oriente, Iztapalapa 09230, Me ´ xico D. F., Me ´ xico Eje (phone: þ (55) 56230794; fax: þ (55) 57736330; e-mail: adelfo@puma2.zaragoza.unam.mx) b ´ noma de Me ´ xico, ) Facultad de Química, Departamento de Farmacia. Universidad Nacional Auto ´ xico D. F., 04510, Me ´ xico Me

Three 2,3-dihydro-1H-isoindol-1-ones structurally related with piracetam ( ¼ 2-oxopyrrolidine-1acetamide) have been synthesized and tested for their nootropic effects in the passive avoidance test in mice. Compounds ( RS )-2, ( R,R )-3, and ( R,S )-3 were obtained in good yields in only two steps starting from methyl dl-phthaloylalanine. Compound ( RS )-2 exhibited nootropic activity at lower doses than piracetam, used as reference drug, but it showed lower efficacy. Whereas diastereoisomers ( R,R )-3 and ( R,S )-3 were as potent as piracetam to revert amnesia induced by scopolamine, ( R,S )-3 showed lower efficacy than ( R,R )-3. Only ( R,R )-3 showed myorelaxant effect at doses of 10 and 30 mg/kg; other compounds did not exhibit any anticonvulsant, sedative, myorelaxant, or impaired motor-coordination effect in mice. These synthesized 2,3-dihydro-1H-isoindol-1-one derivatives constitute a new kind of nootropic compounds.

Introduction. – Cognitive dysfunction is one of the clinical manifestations that often appears in age-related pathologies, head injury, and neurodegenerative diseases such as Alzheimers disease. Cognition enhancers, often referred as nootropics, are frequently used to treat such cognitive alterations. In some European countries, piracetam has been used for patients with a moderate degree of Alzheimers disease to increase the cognitive functions [1] with excellent tolerance and security [2]. Although the mode of action of piracetam in the nervous system is not well-defined, its efficacy has been documented in a wide range of clinical indications like cognitive disorders and dementia, vertigo, and dyslexia, as well as cortical myoclonus [3] [4]. The properties of piracetam ( ¼ 2-oxopyrrolidine-1-acetamide) were disclosed in 1967, and this stimulated the design and synthesis of a large number of structurally related molecules that were found to be endowed with a similar pharmacological profile [5]. Much like the cognition enhancers of the first generation, piracetam-like nootropics revert amnesia induced by scopolamine and other amnesing drugs, electroconvulsive shock, and hypoxia with an unknown mechanism [6]. In general, they show no affinity for the most important central receptors ( Ka > 10 mm ), but are able to modulate the action of most central neurotransmitters, in particular, acetylcholine and glutamate [7]. Several biochemical and behavioral findings have been presented for piracetam-like nootropics, but, so far, a common molecular mechanism of action has not been indicated [7]. The lack of a common mechanism at a molecular 
2010 Verlag Helvetica Chimica Acta AG, Zürich

7 (2010) 2719 level allows sound structure – activity correlations only with in vivo behavioral assays. MeOH. ( R. antitumor agents [13]. reflux. in the present work we synthesized three isoindolinones structurally related with piracetam and tested their ability to revert the scopolamineinduced amnesia in mice.R )-3 and ( R. 1608. AcOH. among the large number of structurally related molecules to piracetam. most of the compounds containing a 2-oxopyrrolidine structure do present cognition-enhancing activity. Upon reacting ( RS )-1 with NH3 .7%.0%. because it has been found that these compounds exhibit diverse biological activities. Zn. 8 h. 5 h. overnight. antipsychotics [10]. However.3dihydroisoindolone) derivatives has attracted considerable attention of chemists. the development of N-substituted isoindolinone ( ¼ 2. ( RS )-2 was formed. a neuropharmacological profile was assessed for these compounds. antimicrobials [12]. and that. whereas its reaction with ( R )-a-phenylethylamine yielded ( R. tranquilizing agents and sedatives [11].R )-3.S )-3. Therefore. leading to frustrating consequences in drug design. Results and Discussion. room temperature. c ) ( R )-a-Phenylethylamine. 88. suggesting that this feature plays a critical role [5] [7]. – Chemistry. These three amides were obtained with yields higher than 80% (see Scheme 1). Some isoindolinones have been described as anxiolytics and reverse transcriptase inhibitors [8] [9]. there are no nootropic isoindolones. In addition. The N-substituted isoindolinone ( RS )-1 was obtained by the reduction of a racemic mixture of 5 with Zn in AcOH in the presence of HCl. and 5-HT2C antagonists [14]. . The synthesis of isoindolinone derivatives ( RS )-2.S )-3 is outlined in Scheme 1.CHEMISTRY & BIODIVERSITY – Vol. 96. b ) NH3 (g).8%. and ( R. 80. Scheme 1 a ) HCl (g). On the other hand. taking in count that isoindolinone has the 2-oxopyrrolidine moiety in its structure.

1).4%.S )-3. Compound ( RS )-2 has been described in a patent [11].R )-3 was established by comparison of its NMR data with those of the diastereoisomer ( R.8%). 1 H) for the two related diastereotopic H-atoms ( À CH2N) in ( R. the final step was the reduction of ( R. 3 h. toluene. showed a D avoidance latency value of 14.S )-3 to inhibit the scopolamine-induced amnesia in the avoidance test in mice [2] [17]. The ability of the synthesized compounds to revert scopolamineinduced amnesia is shown in Fig. treated with 1 mg/kg scopolamine. whereas ( R.S )-3.R )-3 and ( R. The main differences observed in the 1H-NMR spectra between ( R. room temperature.35 (s.9 Æ 11. showed a D avoidance latency of 49. for the reaction with ( R )-a-phenylethylamine. the absolute configuration of the stereogenic center is important for the activity.3 Æ 5. 20 h and then ( R )-a-phenylethylamine. such as ataxia and tremors. This value represents the normal behavior of mice in this test.S )-3 synthesized according the reaction outlined in Scheme 2. 2 H) for ( R. 1 H) and d(H) 4.R )-3 and ( R. treated only with saline solution. expressed in s). Compound ( RS )-2 from 0.S )-7 according to the modified Brewster procedure [16]. In this test. employing Zn in AcOH in the presence of HCl to give ( R.7% respectively. the mice. ( R. Pharmacology. To establish the absolute configuration of the aliphatic stereogenic C-atoms of ( R. the control group. J ¼ 17. b ) HCl (g).2720 CHEMISTRY & BIODIVERSITY – Vol.S )-3. we did not continue tests with doses higher than 10 mg/kg for nootropic activity.R )-3 compared to a single signal at d(H) 4. the latter was prepared by an independent method in three steps: the first one consisted of the reaction of l -alanine with phthalic anhydride to afford ( S )-6 ( Scheme 2 ) [15].R )-3 and ( R.2 Æ 57. Zn. Apparently.S )-7. Scopolamine induced amnesia in this test.2%. At doses higher than 10 mg/kg.R )-3 led to the same level of D avoidance latency (185 Æ 40. Scheme 2 a ) SOCl2 . since ( R.S )-3 as the sole product.2 s ( Fig. 908. The configuration of the compounds ( R.2.S )-3 were the presence of an AB system at d(H) 4. AcOH.2. room temperature.60 (d. 7 (2010) The ammonolysis of ( RS )-1 at room temperature using MeOH saturated with NH3 led to the racemic amide 2 in almost quantitative yield (96. 1 h. ( RS )-2 showed toxicity signs in mice. obtained in acceptable yields of 80.0 and 66. 92. 1.8 s ( Fig. 78.S )-3 showed only one third of D avoidance latency . whereas. J ¼ 17.R )-3 was more active as nootropic than ( R. 1).47 (d.1 s) used as positive nootropic drug. it was necessary to heat the mixture at 1608 to give the diastereoisomeric compounds ( R. At a dose of 100 mg/kg.3 to 10 mg/kg reverted scopolamine-induced amnesia [2] [17].3 s) as piracetam (197. the second step consisted of the reaction of ( S )-6 with SOCl2 and then with ( R )-a-phenylethylamine to give ( R. The y-axis expresses D avoidance-latency values (difference between initial and 24-h latency. therefore.

respective control. sedative. or stimulant effects [18] [19]. vs. (64. and diastereoisomeric amides ( R. Only compound ( R. it was sufficient to revert the scopolamine-induced amnesia.05. 1.3 s) of piracetam at the same dose ( Fig. in this work the racemic amide ( RS )-2. 7 (2010) 2721 Fig. 1). The difference between these compounds can be also explained by the fact that. Bars represent the mean Æ SEM.CHEMISTRY & BIODIVERSITY – Vol. and the fusion of the benzo ring to the pyrrolidinone did not affect the nootropic effect in much as the absolute configuration of the stereogenic center. a neuropharmacological profile was performed for the studied compounds and piracetam. sedative (exploratory cylinder). In addition to nootropic activity. These results are in agreement with those previously reported for nootropic drugs that have low levels of neurotoxicity and do not induce any behavioral impairment. Although only three compounds were tested. in the in vivo studies. 2 ). they provide a clear indication of the structure – activity relationship with respect to the structural modification of piracetam: the presence of a Me group in the lateral chain of the isoindolinone. *: P < 0.S )-3 were synthesized by a rapid and low-cost procedure. Dunns multiple comparison test after Kruskal – Wallis test. neither the synthesized compounds nor piracetam showed anticonvulsant (pentylenetetrazole (PTZ)-induced seizures).R )-3 and ( R.4 Æ 28. In summary. which may be differently affected by structural modifications. or impaired motor-coordination (rotarod test) effect in mice (data not showed).R )-3 showed a myorelaxant effect at a dose of 10 and 30 mg/kg but not in a dose-dependent manner ( Fig. the introduction of the N-(1-phenylethyl) fragment. Compound ( RS )-2 exhibited nootropic activity at low doses but not at the same intensity level of . however. the biological activity is the consequence of both the pharmacokinetic and pharmacodynamic properties. n ¼ 10. It is necessary to perform additional experiments to find out if differences in the effect are due to their pharmacokinetic or pharmacodynamic properties. D Avoidance latency values [s] for test compounds and piracetam at different doses after intraperitoneal administration in mice.

piracetam in the passive avoidance test in mice.R )-3 showed nootropic activity with the same potency as piracetam.2722 CHEMISTRY & BIODIVERSITY – Vol. and ( R. i. On the other hand.p. Diazepam (2 mg/kg.R )-3 (a and c ) and piracetam ( b and d ) . ( R. 7 (2010) Fig. Each point represents the mean Æ SEM of six animals. respectively. 2.S )-3 showed lower .) was used as positive myorelaxant and impaired coordination control drug. Myorelaxant effect in the traction test and effect on motor coordination in rotarod test for ( R.

50 ( m.. 3168. 66 – 678 ).6.6. MeOH).8%). highlighting the importance of the stereogenic center. MS: Hewlett-Packard model 5890 spectrometer. . 22. J ¼ 5.57 (dt.3-Dihydro-1-oxo-2H-isoindol-2-yl)-N-[ (1R )-1-phenylethyl]propanamide (( R. 1693.90 ( m.5. 1686.p.8. IR (KBr): 2952. A soln. 126. 1 H).50 – 7. J ¼ 7.45 – 7.9 g.0. 49. 131. J ¼ 7. 1 H). Optical rotations: Perkin-Elmer 241 polarimeter.S )-3 (1. 1 H). 230 – 400 mesh ASTM. 3. uncorrected.2.68 (c ¼ 0. Diastereoisomer ( R. and G.6.3.1. Methyl N-Phthaloyl-dl-alaninate ( ¼ ( Æ )-Methyl 2-(1. of ( RS )-1 (3.32.9.7. IR.p.47 (t. 1H-NMR (400 MHz. J ¼ 7. 3 H).5. into a stirred mixture of dl -5 (13.3-Dihydro-1-oxo-2H-isoindol-2-yl)propanamide (( RS )-2 ). The authors are grateful to R. 1681. or myorelaxant effects were absent or not related with the doses for these compounds.9. 6.1. 7.0. 13C-NMR (75. 7.7.6 mmol) and ( R )-a-phenylethylamine (2. 14.54 (d. M. 13. 1 H).2. projects: IN 223509 and IN 205008. resp. 123.87 ( m. Rf (AcOEt/hexane/CH2Cl2 1 : 1 : 1) 0. 132. 1 H). 184 – 1878 ). coupling constants ( J ) in Hz. d.36 ( m. 13C-NMR (100 MHz. 7. with TMS as internal reference. J ¼ 7. 122.59 (d.2.and 13C-NMR spectra: Varian VXR-300S spectrometer. 1658. 127. J ¼ 7. 174 – 1768. in CDCl3 . 1H-NMR (300 MHz. 168.t. in KBr.). 3 H).367 mol) in glacial AcOH (175 ml). 188 – 1898 ([11]: m.p. chemical shifts (d ) in ppm. dl -5 ). CDCl3 ): 14. 46. CDCl3 ): 14.47 (d. motor-coordination.3-Dihydro-1-oxo-2H-isoindol-2-yl)propanoate (( RS )-1). 1693. M. M.49 (s. 50.1 ml.92 (br. EI-MS: 219 ( M þ ). 56. sedative.1 g. White solid. spots were visualized with UV light).2. 169.R )-3 and ( R. 141. HCl was bubbled at r. IR (KBr): 3320.2 g. followed by purification by open CC (AcOEt/hexane/CH2Cl2 1 : 1 : 1) to afford pure ( R. The resulting precipitate was collected by filtration to give ( R. M. 47.7%).5 MHz. 5. Experimental Part General.13 mmol) and Zn dust (24.5 MHz. which was purified by open CC (AcOEt/hexane 1 : 1) to yield ( RS )-1 (10. USAI-Facultad de Química for recording the NMR. 2 H).8 g). The mixture was heated at reflux for 5 h. IR Spectra: Perkin-Elmer model 599 spectrophotometer.22 (q. Rf (AcOEt/ hexane 1 : 1) 0. and evaporated under reduced pressure to give crude product (11.60 (d. 1. 169. J ¼ 7.and (2S )-2-(1. Products were purified by open column chromatography (CC) on silica gel 60 (SiO2 . 3 H). 1 H).7. J ¼ 7.0%). CDCl3 ): 1.R )-3 (1. J ¼ 7.8.3. All reactions were monitored by TLC (sheets coated with silica gel 60 F254 ( Merck ). 1.5.0. 131. 96. 1 H).0.2. 1 H). Solvents were distilled from the appropriate drying agents prior to use. overnight.9. 5. The diastereoisomer ( R. 0. 65 – 678 ([20]: m. 128. EI-MS: 308 ( M þ ).32.0. 5 H).2. M. 7. 1 H). Rf (AcOEt/hexane/CHCl3 15 : 6 : 4) 0. 7.73 (s. M. 47. 1658. 123. Merck ).5 mmol) was heated to 1608 for 8 h. 5.2. 1 H).5 g. Gutie and mass spectra. 1 H). 123. 2 H). 169. 128. CDCl3 ): 15. 128.4.0.R )-3.43 (d. 88. Colorless oil. (2R ).CHEMISTRY & BIODIVERSITY – Vol.7 g. 4. 7.0. 1 H).6. 4. 132.59 (d.83 – 7.40 – 7.p. 122. 49.p.37. 141.t.53. ( RS )-2-(1. This racemic ester was prepared according to the procedure that we reported in [20].8. 16.: Electrothermal Digital IA9100 melting-point apparatus.3-Dihydro-1. In a modification of the procedure reported by Brewster et al.1. CDCl3 ): 1.83 (dd. J ¼ 17. J ¼ 5. filtered.6. 80.52 (d.38 (d.7. in cm À 1. 5. 1.0 g.2. 13C-NMR (75. The solvent was removed in vacuo. 131.0 g. 52. 1 H).S )-3. 1H-NMR (300 MHz. These compounds constitute a new kind of a nootropic drug.9. 4.7.60 ( m.80 – 7. and the remaining residue (3. 3 H).S )-3 was obtained by evaporation of the mother liquor. 123.2. J ¼ 12. 128.0. 7.8. 1 H). J ¼ 7. 172. 3 H). Duarte-Lisci.7.04 (q.3. 1 H). 5. 131. J ¼ 7. EI-MS: 204 ( M þ ).023 (q.8. White solid. The anticonvulsive. 66.6.2. 6.1. 1 H). s. A stirred mixture of ( RS )-1 (3. 7 (2010) 2723 nootropic activity than piracetam. J ¼ 12. 1745. 7. ´n General de Asuntos del Personal Acade ´ mico This study was supported by two grants from Direccio (DGAPA) . 172.40 (br. 143. After cooling to r.024 (q. 7. 4. 49. IR (KBr): 3322.22 – 7.0. V. 2 H).3. 4.7%). J ¼ 17. Methyl ( RS )-2-(1. [16].8 g.p.0 g) was crystallized from MeOH to afford ( RS )-2 (2. I. CDCl3 ): 1. [a ]D ¼ þ 101. s.2.t. 1H.7 mmol) in MeOH (60 ml) saturated with NH3 was stirred at r.5.40 (br.0.7. 141. the resulting mixture was dissolved in a minimum of hot AcOEt and then kept for 12 h at 08. del Villar. Arredondo. ´ rrez.60 ( m.5.8.3-dioxo-2H-isoindol-2-yl)propanoate . 3 H).

82.3 mA. EtOH) ([15]: m. All experiments were performed on adult male ICR (Institute for Cancer Research) mice ´ xico. EtOH)).6.1. SOCl2 (1.49 (d. 1 H). After 10 s. and stirred for further 3 h.0 (c ¼ 0. Procedures weighing 25 – 34 g purchased from Centro UNAM-Harlan ( Harlan Me involving animals and their care were conducted in conformity with the Mexican Official Norm for Animal Care and Handing (NOM-062-ZOO-1999) currently adopted in our laboratory.5 ml) was added dropwise to a suspension of ( S )-6 (2. 2 H).t.08 ( m.9%).2%). 7. was then treated with ( R )-a-phenylethylamine (1. CDCl3 ): 15. Animals.05 (q.5% Tween 80 in saline or saline soln.9.S )-7 (0. 1661.73 (c ¼ 0.2. 95 – 968. 3 H). 127. the animals had free access to water and food.2. M. 5 H).5 Â 16 cm). (22 Æ 28 ). 2 H). 22. MeOH). J ¼ 7.24. 1664. 7 (2010) Diastereoisomer ( R. 27.3-dioxo-2H-isoindol-2-yl)propanoic Acid (( S )-6 ).6 ml) at r. The solvent was removed in vacuo. 169.8. 160 – 1618. Control animals received the same volume of vehicle (0. The time required for each mouse to enter the dark compartment was recorded (time of initial latency in s). phase was washed with H2O (2 Â 20 ml).46 (d.70 – 7. dried (Na2SO4 ). The org.6.25 g. and the remaining residue was dissolved in dry THF (30 ml). 149 – 1518 .9. 7. 25 mmol) was heated at 135 – 1408 at atmospheric pressure for 30 min.4 ml. [a ]D ¼ þ 54. with a 12 h light-dark cycle at r. J ¼ 7.0. M.3-Dihydro-1. 49. 128.2. 21.3 cm of diameter separated by 1. 1. Rf (AcOEt/hexane/CH2Cl2 1 : 1 : 1) 0.2 cm) [21]. 145 – 1468. 2 H).3. 131.R )-3 (1.2.24. Passive Avoidance Test. 4.7 g).73 (c ¼ 0. . the animals were returned to their home cage and injected with scopolamine (1 mg/kg) to induce amnesia.12 (q. 1.24 – 7. 1718.0 to 100 mg/kg). 49. 2927. J ¼ 7. 3 H). The passive avoidance apparatus ( Ugo Basile 7550) consists of two compartments: a white and illuminated compartment (of 18 Â 9.1. 123. d.p.52. 6. only).S )-7 (2.0 to 300 mg/kg).2. 128.3. The solvent was removed in vacuo.8. 1.0 to 100 mg/kg). 1.2.0 mg/kg) were prepared in saline soln. 2 mg/kg) were suspended in 0. The same procedure was repeated 24 h after (latency at . 1 H).2. 2. ( R.p.A. 7.V. 143. J ¼ 7.2.5. EI-MS: 308 ( M þ ). [a ]D ¼ À 21.38 ( m.49 (d. HCl was bubbled at r. and the remaining residue was partitioned with AcOEt (20 ml) and 1m HCl (20 ml). 126. IR (KBr): 3303.1 ml/10 g body weight. and an electrical shock (0. As soon as the mouse entered the dark compartment with its four paws. 1 H).0 mmol) and pyridine (0.2.3-Dihydro-1-oxo-2H-isoindol-2-yl)-N-[(1R )-1-phenylethyl]propanamide (( R.2.81 – 7. and scopolamine ( Fluka Co..82 (c ¼ 0. The drugs were freshly prepared each time and intraperitoneally injected in a volume of 0.7. 167. (2S )-2-(1. 7.12 – 7.t. J ¼ 7. 13C-NMR (75. 7. (2S )-2-(1. 3 H). and in compliance with international rules on care and use of laboratory animals.86 ( m.5 MHz. 131.7.03 to 10 mg/kg).65 (dd.3 mmol) in toluene (40 ml) at r. 142. [a ]D ¼ þ 64.4. The mixture was heated at 908 for 1 h. into a stirred mixture of ( R. [a ]D ¼ þ 64.02 – 7. 46. J ¼ 7. 131. 1.75 ( m. 128. The training session started by placing one mouse in the illuminated compartment. J ¼ 7.t.5. CDCl3 ): 1. 5.598 g). 13C-NMR (100 MHz. pentylenetetrazole (PTZ. Drugs and Dosage. J ¼ 7. 11.3.6. 2 H). de C. 127. The floor of the dark compartment is a grid with steel bars (0. 168.568 g.13 (q. the sliding door was automatically opened. S. M. During the experiment. 1 H). and diazepam ( Roche S.93 (q.18 ( m.p. J ¼ 7. Sigma Co.2% yield). 1H-NMR (300 MHz. 49.35 (s. 125. The resulting product was crystallized from EtOH/H2O to give ( S )-6 (4. 1 H). Zn dust (0. 169. . Briefly.85 g. CDCl3 ): 14.3-dioxo-2H-isoindol-2-yl)-N-[ (1R )-1-phenylethyl]propanamide (( R.2. 82. 7.22 – 7. [a ]D ¼ À 23. . 1 H). and evaporated under reduced pressure to give a crude product (0. White solid. 10.4.S )-3 (1. the door was automatically closed.22 g. 78. This acid was prepared according to a modification of the procedure described by Zeng et al. White solid.p. Rf (AcOEt/hexane/CH2Cl2 1 : 1 : 1) 0.0 mmol). J ¼ 7.9.56 (br. CHCl3 ). 5. 1 H).1 g. leaving free access to the dark compartment.52. J ¼ 7. 50. Crystallization from MeOH afforded ( R. 7. [15].36 ( m.2724 CHEMISTRY & BIODIVERSITY – Vol.7 mmol) and l -alanine (2.2. CDCl3 ): 1. 92.p. J ¼ 7. 80 mg/kg). EI-MS: 322 ( M þ ). 4. The resulting soln. (0.1.7.5 Â 16 cm) separated by a guillotine door from the second dark compartment (18 Â 9.S )-3 : M.2. filtered. 1 H). 95 – 968. ). and the mixture was stirred for 20 h.A. 7.69 (d. 141.2.38 (br.1. 2 H).5 g.S )-3 ).6 g. with 2 s of duration) was delivered.S )-3 (0.2. 5.5.52. 123. (2S )-2-(1. The animals that needed more than 100 s to enter the dark compartment were eliminated from the test [22]. . 3 H). Compounds ( RS )-2 (0.5% Tween 80 in saline soln.9. 3 H).2. 1.3-Dihydro-1. 134. and glacial AcOH (10 ml).S )-7 ) .2. Piracetam ( Sigma Co.0 mmol). 122. 13.t. IR (KBr): 3350.47 (dt.4%). 1H-NMR (400 MHz. ( R.6.4.3. which was purified by open CC (AcOEt/hexane/CH2Cl2 1 : 1 : 1) to give ( R. 7.33 (c ¼ 0. Immediately afterwards. and evaporated to give the crude product (3. d.644 g. MeOH). a stirred mixture of phthalic anhydride (4.

J. The time on the rod was registered each 10 min for the first 2 h and each 30 min for the following 2 h. A. Galeotti. Kunitz. A. Med. Afsar. The mice were injected with PTZ (80 mg/kg) and immediately placed in individual plastic boxes. E. Riccaboni. constant speed model 7600. Int. followed by Dunns multiple comparison test. A 30-s performance time was considered normal. During observation. E. 33. Data were analyzed using one-way ANOVA. the statistically significant differences between the treatments were tested by Kruskal – Wallis test (nonparametric one-way analysis of variance). Traction Performance. R. and filter paper was changed between each animal test session [23] [24]. For nootropic activity evaluation. Ghelardini. 58. The test session is finished when the animal enters the dark compartment. F. J. The inner side of the apparatus and floor were cleaned with alcoholic soln. Takahashi. Pharm. S. T. M. Guandalini. Exploratory Cylinder Test [24] [25]. J. [2] M. R. Teodori. Farina. and the length of time each mouse remained there was recorded (time on rod). Synlett 1996. Itil. M. PTZ-Induced Seizures. followed by Dunnetts t-test for neuropharmacological tests. The drugs were administered in different doses 30 min before PTZinduced seizures. Ahmed. O. Rotarod Test. O. and the number of rearings performed over a 5-min period was recorded. Biagetti. The drugs were administered 30 min before testing in different doses. The traction test was conducted with a stainless bar (diameter 1. the experimenter stood next to the apparatus always in the same place. [7] S. Hirano. Bellucci. Mice remaining for at least 2 min on the rod (Rotarod Treadmills for mice. 16 rpm) were selected and allocated to a group of six animals each.CHEMISTRY & BIODIVERSITY – Vol. Agan. with a wall of 3 mm). Manetti. 36. Martelli. Bartolini. Mice living longer than 30 min following PTZ administration were considered to be protected [23]. Des. L. N. Ghelardini. for a total recording time of 4 h [23]. 4-cm diameter. Farmaco 2003. I. 1986. Eralp. Scapecchi. H. 715. Kawakami. Us. 1993. 1969. Cerri. C. D. . C. N. N. Psychopharmacol. Pfeiffer. 29. A control group treated exclusively with saline and without scopolamine injection was included to evaluate the normal behavior of the mice in this test. 7 (2010) 2725 24 h). H. The mice were hung with their forelimbs on the horizontal bar. Borucu. I. Magnani. Scapecchi. J. S. 43. C. C. A value of P < 0. 4214. Curr. 4. Yokota. Clin. Romanelli. K. Turan. Martini. 11 cm in diameter. N. C. Wilsher. The apparatus consisted of a glass cylinder (30 cm in height. 2002. 2008. 353. Dorigotti. [6] D. Romanelli. Ugo Basile . L. Ther. Manetti. Bull. but without the electrical shock and without scopolamine administration. Gualtieri. Dei. Immediately after administration of the test compound. U. 2000. S. Kitajima. A. Pozzi. for a total recording time of 4 h. C. The results are presented as mean Æ SEM. (10% v/v in H2O). Statistical analysis. E. The cylinder was placed on filter paper in a room with constant lighting and isolated from external noise [23] [26]. and observed for at least 30 min to record the occurrence of the first episode of clonic or tonic seizures and mortality. Chem. or remains in the illuminated compartment for more than 420 s. Only animals that fulfill this criterion were included in the experiment. 125. 391. Ghelardini. M. Chem. REFERENCES [1] M. T. length 35 cm) set horizontally at an elevation of 40 cm. The effect on motor coordination was considered when animals remained on the rod less than 120 s [28]. F. Psychophysiol. The test compounds or vehicle were intraperitoneally administered 20 min before training session. [5] F. An individual naïve mouse was put on the filter papercovered floor of the glass cylinder. Pinza. 34. M. Gualtieri. Banfi. 1998. Gualtieri. [8] I. [3] D. D. reference drug or vehicle mice were placed on the rod for a maximum of 120 s. Reduced exploratory rearing shown by naïve mice after placement in an unfamiliar environment reveals a sedative effect [23] [24] [26] [27].05 was considered significant. Ince Gunal. Pharm. Med. E. The performance time was registered each 10 min for the first 2 h and each 30 min for the following 2 h.5 mm. 8. [4] C. K. The observations were made without prior knowledge of the experimental conditions applied to the animal. 175.

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