Synthesis and nootropic activity of some 2,3-Dihydro-1H-isoindol-1-one derivatives structurally related with piracetam

Article abstract of DOI:10.1002/cbdv.200900269

Three 2,3-dihydro-1H-isoindol-1-ones structurally related with piracetam (=2-oxopyrrolidine-1-acetamide) 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 30mg/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.

Full text of DOI:10.1002/cbdv.200900269

2718
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 Huertaa), Marisol Alfaroa), and Andre´s Navarreteb)
a
´
) Facultad de Estudios Superiores Zaragoza, Unidad Multidisciplinaria de Investigacion Experimental,
´
´
Universidad Nacional Autonoma de Mexico, Batalla 5 de Mayo esquina Fuerte de Loreto,
´
´
´
Ejercito de Oriente, Iztapalapa 09230, Mexico D. F., Mexico
(phone: þ(55)56230794; fax: þ(55)57736330; e-mail: adelfo@puma2.zaragoza.unam.mx)
b
´
´
) Facultad de Quꢀmica, Departamento de Farmacia. Universidad Nacional Autonoma de Mexico,
´
´
Mexico D. F., 04510, Mexico
Three 2,3-dihydro-1H-isoindol-1-ones structurally related with piracetam (¼2-oxopyrrolidine-1-
acetamide) 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
Alzheimerꢁs 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 Alzheimerꢁs 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 (¼2-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, acetylcho-
line 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
ꢂ 2010 Verlag Helvetica Chimica Acta AG, Zꢃrich

CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
2719
level allows sound structure–activity correlations only with in vivo behavioral assays,
leading to frustrating consequences in drug design. However, most of the compounds
containing a 2-oxopyrrolidine structure do present cognition-enhancing activity,
suggesting that this feature plays a critical role [5][7].
On the other hand, the development of N-substituted isoindolinone (¼2,3-
dihydroisoindolone) derivatives has attracted considerable attention of chemists,
because it has been found that these compounds exhibit diverse biological activities.
Some isoindolinones have been described as anxiolytics and reverse transcriptase
inhibitors [8][9], antipsychotics [10], tranquilizing agents and sedatives [11], anti-
microbials [12], antitumor agents [13], and 5-HT2C antagonists [14]. Therefore, taking
in count that isoindolinone has the 2-oxopyrrolidine moiety in its structure, and that,
among the large number of structurally related molecules to piracetam, there are no
nootropic isoindolones, in the present work we synthesized three isoindolinones
structurally related with piracetam and tested their ability to revert the scopolamine-
induced amnesia in mice. In addition, a neuropharmacological profile was assessed for
these compounds.
Results and Discussion. – Chemistry. The synthesis of isoindolinone derivatives
(RS)-2, (R,R)-3, and (R,S)-3 is outlined in Scheme 1. 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. Upon reacting (RS)-1 with NH3 , (RS)-2 was formed, whereas its
reaction with (R)-a-phenylethylamine yielded (R,R)-3 and (R,S)-3. These three
amides were obtained with yields higher than 80% (see Scheme 1).
Scheme 1
a) HCl (g), AcOH, Zn, reflux, 5 h; 88.7%. b) NH3 (g), MeOH, room temperature, overnight; 96.8%.
c) (R)-a-Phenylethylamine, 1608, 8 h; 80.0%.

2720
CHEMISTRY & BIODIVERSITY – Vol. 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 (96.8%), whereas, for the
reaction with (R)-a-phenylethylamine, it was necessary to heat the mixture at 1608 to
give the diastereoisomeric compounds (R,R)-3 and (R,S)-3, obtained in acceptable
yields of 80.0 and 66.7% respectively. Compound (RS)-2 has been described in a patent
[11].
To establish the absolute configuration of the aliphatic stereogenic C-atoms of
(R,R)-3 and (R,S)-3, 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]; the second step consisted of the reaction of (S)-6 with SOCl2 and
then with (R)-a-phenylethylamine to give (R,S)-7; the final step was the reduction of
(R,S)-7 according to the modified Brewster procedure [16], employing Zn in AcOH in
the presence of HCl to give (R,S)-3 as the sole product. The configuration of the
compounds (R,R)-3 was established by comparison of its NMR data with those of the
diastereoisomer (R,S)-3 synthesized according the reaction outlined in Scheme 2. The
main differences observed in the 1H-NMR spectra between (R,R)-3 and (R,S)-3 were
the presence of an AB system at d(H) 4.47 (d, J ¼ 17.2, 1 H) and d(H) 4.60 (d, J ¼ 17.2,
1 H) for the two related diastereotopic H-atoms (ꢀCH2N) in (R,R)-3 compared to a
single signal at d(H) 4.35 (s, 2 H) for (R,S)-3.
Scheme 2
a) SOCl2, toluene, room temperature, 20 h and then (R)-a-phenylethylamine, room temperature, 3 h;
78.4%. b) HCl (g), AcOH, Zn, 908, 1 h; 92.2%.
Pharmacology. The ability of the synthesized compounds to revert scopolamine-
induced amnesia is shown in Fig. 1. The y-axis expresses D avoidance-latency values
(difference between initial and 24-h latency, expressed in s). In this test, the control
group, treated only with saline solution, showed a D avoidance latency of 49.9ꢁ11.8 s
(Fig. 1). This value represents the normal behavior of mice in this test. Scopolamine
induced amnesia in this test; the mice, treated with 1 mg/kg scopolamine, showed a D
avoidance latency value of 14.3ꢁ5.2 s (Fig. 1). Compound (RS)-2 from 0.3 to 10 mg/kg
reverted scopolamine-induced amnesia [2][17]. At doses higher than 10 mg/kg, (RS)-2
showed toxicity signs in mice, such as ataxia and tremors; therefore, we did not
continue tests with doses higher than 10 mg/kg for nootropic activity. Apparently, the
absolute configuration of the stereogenic center is important for the activity, since
(R,R)-3 was more active as nootropic than (R,S)-3 to inhibit the scopolamine-induced
amnesia in the avoidance test in mice [2][17]. At a dose of 100 mg/kg, (R,R)-3 led to
the same level of D avoidance latency (185ꢁ40.3 s) as piracetam (197.2ꢁ57.1 s) used as
positive nootropic drug, whereas (R,S)-3 showed only one third of D avoidance latency

CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
2721
Fig. 1. D Avoidance latency values [s] for test compounds and piracetam at different doses after
intraperitoneal administration in mice. Bars represent the meanꢁSEM, n¼10. *: P<0.05, vs. respective
control; Dunnꢁs multiple comparison test after Kruskal–Wallis test.
(64.4ꢁ28.3 s) of piracetam at the same dose (Fig. 1), however, it was sufficient to
revert the scopolamine-induced amnesia. Although only three compounds were tested,
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, the introduction of the N-(1-phenylethyl) fragment, 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. The difference between these
compounds can be also explained by the fact that, in the in vivo studies, the biological
activity is the consequence of both the pharmacokinetic and pharmacodynamic
properties, which may be differently affected by structural modifications. It is necessary
to perform additional experiments to find out if differences in the effect are due to their
pharmacokinetic or pharmacodynamic properties.
In addition to nootropic activity, a neuropharmacological profile was performed for
the studied compounds and piracetam; neither the synthesized compounds nor
piracetam showed anticonvulsant (pentylenetetrazole (PTZ)-induced seizures), sed-
ative (exploratory cylinder), or impaired motor-coordination (rotarod test) effect in
mice (data not showed). Only compound (R,R)-3 showed a myorelaxant effect at a
dose of 10 and 30 mg/kg but not in a dose-dependent manner (Fig. 2). 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, sedative, or stimulant
effects [18][19].
In summary, in this work the racemic amide (RS)-2, and diastereoisomeric amides
(R,R)-3 and (R,S)-3 were synthesized by a rapid and low-cost procedure. Compound
(RS)-2 exhibited nootropic activity at low doses but not at the same intensity level of

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

CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
2723
nootropic activity than piracetam, highlighting the importance of the stereogenic
center. The anticonvulsive, sedative, motor-coordination, or myorelaxant effects were
absent or not related with the doses for these compounds. These compounds constitute
a new kind of a nootropic drug.
´
´
This study was supported by two grants from Direccion General de Asuntos del Personal Academico
(DGAPA), projects: IN 223509 and IN 205008. The authors are grateful to R. I. del Villar, V. M.
´
Arredondo, M. Gutierrez, and G. Duarte-Lisci, USAI-Facultad de Quꢀmica for recording the NMR, IR,
and mass spectra.
Experimental Part
General. Solvents were distilled from the appropriate drying agents prior to use. All reactions were
monitored by TLC (sheets coated with silica gel 60 F254 (Merck); spots were visualized with UV light).
Products were purified by open column chromatography (CC) on silica gel 60 (SiO2, 230–400 mesh
ASTM; Merck). M.p.: Electrothermal Digital IA9100 melting-point apparatus; uncorrected. Optical
rotations: Perkin-Elmer 241 polarimeter. IR Spectra: Perkin-Elmer model 599 spectrophotometer, in
KBr; in cmꢀ1. 1H- and 13C-NMR spectra: Varian VXR-300S spectrometer, in CDCl3; chemical shifts (d)
in ppm, with TMS as internal reference; coupling constants (J) in Hz. MS: Hewlett-Packard model 5890
spectrometer.
Methyl N-Phthaloyl-dl-alaninate (¼(ꢁ)-Methyl 2-(1,3-Dihydro-1,3-dioxo-2H-isoindol-2-yl)propa-
noate; dl-5). This racemic ester was prepared according to the procedure that we reported in [20]. M.p.
65–678 ([20]: m.p. 66–678).
Methyl (RS)-2-(1,3-Dihydro-1-oxo-2H-isoindol-2-yl)propanoate ((RS)-1). In a modification of the
procedure reported by Brewster et al. [16], HCl was bubbled at r.t. into a stirred mixture of dl-5 (13.1 g,
56.13 mmol) and Zn dust (24.0 g, 0.367 mol) in glacial AcOH (175 ml). The mixture was heated at reflux
for 5 h, filtered, and evaporated under reduced pressure to give crude product (11.8 g), which was
purified by open CC (AcOEt/hexane 1:1) to yield (RS)-1 (10.9 g, 88.7%). Colorless oil. Rf (AcOEt/
1
hexane 1:1) 0.37. IR (KBr): 2952, 1745, 1693, 1681. H-NMR (400 MHz, CDCl3): 1.59 (d, J¼5.7, 3 H);
3.73 (s, 3 H); 4.43 (d, J ¼ 12.3, 1 H); 4.59 (d, J¼12.3, 1 H); 5.22 (q, J¼5.7, 1 H); 7.40–7.50 (m, 2 H); 7.50–
7.60 (m, 1 H); 7.80–7.90 (m, 1 H). 13C-NMR (100 MHz, CDCl3): 15.8; 46.7; 49.0; 52.3; 122.8; 123.9; 128.0;
131.6; 132.0; 141.6; 168.6; 172.2. EI-MS: 219 (Mþ ).
(RS)-2-(1,3-Dihydro-1-oxo-2H-isoindol-2-yl)propanamide ((RS)-2). A soln. of (RS)-1 (3.0 g,
13.7 mmol) in MeOH (60 ml) saturated with NH3 was stirred at r.t. overnight. The solvent was removed
in vacuo, and the remaining residue (3.0 g) was crystallized from MeOH to afford (RS)-2 (2.7 g, 96.8%).
White solid. M.p. 188–1898 ([11]: m.p. 184–1878). Rf (AcOEt/hexane/CHCl3 15 :6 :4) 0.32. IR (KBr):
3320, 3168, 1693, 1658. 1H-NMR (300 MHz, CDCl3): 1.54 (d, J ¼ 7.2, 3 H); 4.49 (s, 2 H); 5.04 (q, J¼7.2,
1 H); 5.40 (br. s, 1 H); 6.40 (br. s, 1 H); 7.45–7.60 (m, 3 H); 7.83–7.87 (m, 1 H). 13C-NMR (75.5 MHz,
CDCl3): 14.4; 47.0; 49.9; 123.0; 123.8; 128.2; 131.8; 131.9; 141.5; 169.1; 172.7. EI-MS: 204 (Mþ ).
(2R)- and (2S)-2-(1,3-Dihydro-1-oxo-2H-isoindol-2-yl)-N-[(1R)-1-phenylethyl]propanamide
((R,R)-3 and (R,S)-3, resp.). A stirred mixture of (RS)-1 (3.2 g, 14.6 mmol) and (R)-a-phenylethyl-
amine (2.1 ml, 16.5 mmol) was heated to 1608 for 8 h. After cooling to r.t., the resulting mixture was
dissolved in a minimum of hot AcOEt and then kept for 12 h at 08. The resulting precipitate was collected
by filtration to give (R,R)-3 (1.8 g, 80.0%). White solid. The diastereoisomer (R,S)-3 was obtained by
evaporation of the mother liquor, followed by purification by open CC (AcOEt/hexane/CH2Cl2 1:1:1) to
afford pure (R,S)-3 (1.5 g, 66.7%).
Diastereoisomer (R,R)-3. M.p. 174–1768. Rf (AcOEt/hexane/CH2Cl2 1:1:1) 0.32. [a]D ¼ þ101.68
(c ¼ 0.53, MeOH). IR (KBr): 3322, 1686, 1658. 1H-NMR (300 MHz, CDCl3): 1.38 (d, J¼7.2, 3 H); 1.52
(d, J¼7.2, 3 H); 4.47 (d, J¼17.2, 1 H); 4.60 (d, J¼17.2, 1 H); 5.023 (q, J¼7.2, 1 H); 5.024 (q, J¼7.2, 1 H);
6.92 (br. d, J¼7.2, 1 H); 7.22–7.36 (m, 5 H); 7.47 (t, J¼7.5, 2 H); 7.57 (dt, J¼7.5, 1.6, 1 H); 7.83 (dd, J¼7.5,
1.6, 1 H). 13C-NMR (75.5 MHz, CDCl3): 14.8; 22.0; 47.1; 49.0; 50.5; 122.9; 123.7; 126.0; 127.3; 128.1;
128.6; 131.7; 132.0; 141.7; 143.1; 169.0; 169.8. EI-MS: 308 (Mþ ).

2724
CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
Diastereoisomer (R,S)-3: M.p. 95–968. Rf (AcOEt/hexane/CH2Cl2 1:1:1) 0.24. [a]D ¼ þ64.73 (c ¼
0.52, MeOH). IR (KBr): 3303, 2927, 1664. 1H-NMR (300 MHz, CDCl3): 1.46 (d, J¼7.2, 3 H); 1.49 (d, J¼
7.2, 3 H); 4.35 (s, 2 H); 5.05 (q, J ¼ 7.2, 1 H); 5.12 (q, J¼7.2, 1 H); 7.02–7.08 (m, 3 H); 7.12–7.18 (m, 2 H);
7.24–7.38 (m, 2 H); 7.47 (dt, J¼7.2, 1.2, 1 H); 7.56 (br. d, J¼7.2, 1 H); 7.65 (dd, J¼7.2, 1.2, 1 H). 13C-NMR
(100 MHz, CDCl3): 14.3; 22.3; 46.9; 49.1; 50.3; 122.9; 123.7; 125.6; 127.0; 128.1; 128.4; 131.7; 131.9; 141.5;
143.4; 169.2; 169.6. EI-MS: 308 (Mþ ).
(2S)-2-(1,3-Dihydro-1,3-dioxo-2H-isoindol-2-yl)propanoic Acid ((S)-6). This acid was prepared
according to a modification of the procedure described by Zeng et al. [15]. Briefly, a stirred mixture of
phthalic anhydride (4.1 g, 27.7 mmol) and l-alanine (2.22 g, 25 mmol) was heated at 135–1408 at
atmospheric pressure for 30 min. The resulting product was crystallized from EtOH/H2O to give (S)-6
(4.5 g, 82.2%). White solid. M.p. 145–1468. [a]D ¼ ꢀ21.82 (c¼0.82, EtOH) ([15]: m.p. 149–1518; [a]D ¼
ꢀ23.0 (c¼0.8, EtOH)).
(2S)-2-(1,3-Dihydro-1,3-dioxo-2H-isoindol-2-yl)-N-[(1R)-1-phenylethyl]propanamide ((R,S)-7).
SOCl2 (1.5 ml) was added dropwise to a suspension of (S)-6 (2.25 g, 10.3 mmol) in toluene (40 ml) at
r.t., and the mixture was stirred for 20 h. The solvent was removed in vacuo, and the remaining residue
was dissolved in dry THF (30 ml). The resulting soln. was then treated with (R)-a-phenylethylamine
(1.4 ml, 11.0 mmol) and pyridine (0.6 ml) at r.t. and stirred for further 3 h. The solvent was removed in
vacuo, and the remaining residue was partitioned with AcOEt (20 ml) and 1m HCl (20 ml). The org.
phase was washed with H2O (2ꢂ20 ml), dried (Na2SO4), and evaporated to give the crude product
(3.7 g). Crystallization from MeOH afforded (R,S)-7 (2.6 g, 78.4%). White solid. M.p. 160–1618. [a]D ¼
þ54.33 (c ¼ 0.52, CHCl3). IR (KBr): 3350, 1718, 1661. 1H-NMR (400 MHz, CDCl3): 1.49 (d, J¼7.2, 3 H);
1.69 (d, J¼7.2, 3 H); 4.93 (q, J¼7.2, 1 H); 5.13 (q, J¼7.2, 1 H); 6.38 (br. d, J¼7.2, 1 H); 7.22–7.36 (m,
5 H); 7.70–7.75 (m, 2 H); 7.81–7.86 (m, 2 H). 13C-NMR (75.5 MHz, CDCl3): 15.4; 21.5; 49.1; 49.6; 123.5;
126.1; 127.3; 128.6; 131.8; 134.2; 142.7; 167.9; 168.2. EI-MS: 322 (Mþ ).
(2S)-2-(1,3-Dihydro-1-oxo-2H-isoindol-2-yl)-N-[(1R)-1-phenylethyl]propanamide ((R,S)-3). HCl
was bubbled at r.t. into a stirred mixture of (R,S)-7 (0.644 g, 2.0 mmol), Zn dust (0.85 g, 13.0 mmol),
and glacial AcOH (10 ml). The mixture was heated at 908 for 1 h, filtered, and evaporated under reduced
pressure to give a crude product (0.598 g), which was purified by open CC (AcOEt/hexane/CH2Cl2
1:1:1) to give (R,S)-3 (0.568 g, 92.2% yield). M.p. 95–968. Rf (AcOEt/hexane/CH2Cl2 1:1:1) 0.24.
[a]D ¼ þ64.73 (c¼0.52, MeOH).
Animals. All experiments were performed on adult male ICR (Institute for Cancer Research) mice
´
weighing 25–34 g purchased from Centro UNAM-Harlan (Harlan Mexico, S.A. de C.V.). Procedures
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, and in
compliance with international rules on care and use of laboratory animals. During the experiment, the
animals had free access to water and food, with a 12 h light-dark cycle at r.t. (22ꢁ28).
Drugs and Dosage. Compounds (RS)-2 (0.03 to 10 mg/kg), (R,R)-3 (1.0 to 300 mg/kg), (R,S)-3 (1.0
to 100 mg/kg), and diazepam (Roche S.A.; 2 mg/kg) were suspended in 0.5% Tween 80 in saline soln.
Piracetam (Sigma Co.; 1.0 to 100 mg/kg), pentylenetetrazole (PTZ; Sigma Co.; 80 mg/kg), and
scopolamine (Fluka Co.; 1.0 mg/kg) were prepared in saline soln. (0.9%). The drugs were freshly
prepared each time and intraperitoneally injected in a volume of 0.1 ml/10 g body weight. Control
animals received the same volume of vehicle (0.5% Tween 80 in saline or saline soln. only).
Passive Avoidance Test. The passive avoidance apparatus (Ugo Basile 7550) consists of two com-
partments: a white and illuminated compartment (of 18ꢂ9.5ꢂ16 cm) separated by a guillotine door
from the second dark compartment (18ꢂ9.5ꢂ16 cm). The floor of the dark compartment is a grid with
steel bars (0.3 cm of diameter separated by 1.2 cm) [21]. The training session started by placing one
mouse in the illuminated compartment. After 10 s, the sliding door was automatically opened, leaving
free access to the dark compartment. As soon as the mouse entered the dark compartment with its four
paws, the door was automatically closed, and an electrical shock (0.3 mA, with 2 s of duration) was
delivered. The time required for each mouse to enter the dark compartment was recorded (time of initial
latency in s). Immediately afterwards, the animals were returned to their home cage and injected with
scopolamine (1 mg/kg) to induce amnesia. The animals that needed more than 100 s to enter the dark
compartment were eliminated from the test [22]. The same procedure was repeated 24 h after (latency at

CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
2725
24 h), but without the electrical shock and without scopolamine administration. The test session is
finished when the animal enters the dark compartment, or remains in the illuminated compartment for
more than 420 s. The test compounds or vehicle were intraperitoneally administered 20 min before
training session. A control group treated exclusively with saline and without scopolamine injection was
included to evaluate the normal behavior of the mice in this test.
PTZ-Induced Seizures. The mice were injected with PTZ (80 mg/kg) and immediately placed in
individual plastic boxes, and observed for at least 30 min to record the occurrence of the first episode of
clonic or tonic seizures and mortality. Mice living longer than 30 min following PTZ administration were
considered to be protected [23]. The drugs were administered in different doses 30 min before PTZ-
induced seizures.
Exploratory Cylinder Test [24][25]. The apparatus consisted of a glass cylinder (30 cm in height,
11 cm in diameter, with a wall of 3 mm). The cylinder was placed on filter paper in a room with constant
lighting and isolated from external noise [23][26]. An individual naꢄve mouse was put on the filter paper-
covered floor of the glass cylinder, and the number of rearings performed over a 5-min period was
recorded. The inner side of the apparatus and floor were cleaned with alcoholic soln. (10% v/v in H2O),
and filter paper was changed between each animal test session [23][24]. The drugs were administered
30 min before testing in different doses. During observation, the experimenter stood next to the
apparatus always in the same place. The observations were made without prior knowledge of the
experimental conditions applied to the animal. Reduced exploratory rearing shown by naꢄve mice after
placement in an unfamiliar environment reveals a sedative effect [23][24][26][27].
Rotarod Test. Mice remaining for at least 2 min on the rod (Rotarod Treadmills for mice, constant
speed model 7600, Ugo Basile; 4-cm diameter, 16 rpm) were selected and allocated to a group of six
animals each. Immediately after administration of the test compound, reference drug or vehicle mice
were placed on the rod for a maximum of 120 s, and the length of time each mouse remained there was
recorded (ꢅtime on rodꢁ). The time on the rod was registered each 10 min for the first 2 h and each 30 min
for the following 2 h, for a total recording time of 4 h. The effect on motor coordination was considered
when animals remained on the rod less than 120 s [28].
Traction Performance. The traction test was conducted with a stainless bar (diameter 1.5 mm, length
35 cm) set horizontally at an elevation of 40 cm. The mice were hung with their forelimbs on the
horizontal bar. A 30-s performance time was considered normal. Only animals that fulfill this criterion
were included in the experiment. The performance time was registered each 10 min for the first 2 h and
each 30 min for the following 2 h, for a total recording time of 4 h [23].
Statistical analysis. The results are presented as meanꢁSEM. Data were analyzed using one-way
ANOVA, followed by Dunnettꢁs t-test for neuropharmacological tests. For nootropic activity evaluation,
the statistically significant differences between the treatments were tested by Kruskal–Wallis test (non-
parametric one-way analysis of variance), followed by Dunnꢁs multiple comparison test. A value of P<
0.05 was considered significant.
REFERENCES
[1] M. I. Turan, E. Eralp, I. Ahmed, A. Kunitz, K. Itil, Psychopharmacol. Bull. 1998, 34, 391.
[2] M. Pinza, C. Farina, A. Cerri, U. Pfeiffer, M. T. Riccaboni, S. Banfi, R. Biagetti, O. Pozzi, M.
Magnani, L. Dorigotti, J. Med. Chem. 1993, 36, 4214.
[3] D. Ince Gunal, K. Agan, N. Afsar, D. Borucu, O. Us, J. Clin. Pharm. Ther. 2008, 33, 175.
[4] C. R. Wilsher, Int. J. Psychophysiol. 1986, 4, 29.
[5] F. Gualtieri, D. Manetti, M. N. Romanelli, C. Ghelardini, Curr. Pharm. Des. 2002, 8, 125.
[6] D. Manetti, C. Ghelardini, A. Bartolini, C. Bellucci, S. Dei, N. Galeotti, F. Gualtieri, M. N.
Romanelli, S. Scapecchi, E. Teodori, J. Med. Chem. 2000, 43, 1969.
[7] S. Scapecchi, C. Martelli, C. Ghelardini, L. Guandalini, E. Martini, F. Gualtieri, Farmaco 2003, 58,
715.
[8] I. Takahashi, T. Kawakami, E. Hirano, H. Yokota, H. Kitajima, Synlett 1996, 353.

2726
CHEMISTRY & BIODIVERSITY – Vol. 7 (2010)
[9] A. Mertens, H. Zilch, B. Kçnig, W. Schꢆfer, T. Poll, W. Kampe, H. Seidel, U. Leser, H. Leinert, J.
Med. Chem. 1993, 36, 2526.
[10] M. H. Norman, G. C. Rigdon, F. Navas III, B. R. Cooper, J. Med. Chem. 1994, 37, 2552.
[11] N. A. Jonsson, L. Mikiver, U. V. Selberg, to Aktiebolag Kabi, SE 333734 (Chem. Abst. 1973, 78,
16033).
[12] J. C. Breytenbach, S. van Dyk, I. van den Heever, S. M. Allin, C. C. Hodkinson, C. J. Northfield,
M. I. Page, Bioorg. Med. Chem. Lett. 2000, 10, 1629.
[13] I. R. Hardcastle, S. U. Ahmed, H. Atkins, G. Farnie, B. T. Golding, R. J. Griffin, S. Guyenne, C.
Hutton, P. Kꢆllblad, S. J. Kemp, M. S. Kitching, D. R. Newell, S. Norbedo, J. S. Northen, R. J. Reid,
K. Saravanan, H. M. G. Willems, J. Lunec, J. Med. Chem. 2006, 49, 6209.
[14] D. Hamprecht, F. Micheli, G. Tedesco, A. Checchia, D. Donati, M. Petrone, S. Terreni, M. Wood,
Bioorg. Med. Chem. Lett. 2007, 17, 428.
[15] Q. Zeng, Z. Liu, B. Li, F. Wang, Amino Acids 2004, 27, 183.
[16] J. H. Brewster, A. M. Fusco, L. E. Carosino, B. G. Corman, J. Org. Chem. 1963, 28, 498.
´
[17] E. Everss, C. Arenas, S. Vinader-Caerols, S. Monleon, A. Parra, Behav. Brain Res. 2005, 159, 235.
[18] G. Heise, Trends Pharmacol. Sci. 1987, 8, 65.
[19] C. Farina, S. Gagliardi, C. Ghelardini, M. Martinelli, M. Norcini, C. Parini, P. Petrillo, S. Ronzoni,
Bioorg. Med. Chem. 2008, 16, 3224.
´
´
[20] A. Reyes, I. Regla, M. C. Fragoso, L. A. Vallejo, P. Demare, H. A. Jimenez-Vazquez, Y. Ramꢀrez, E.
Juaristi, J. Tamariz, Tetrahedron 1999, 55, 11187.
´
´
ˇ ´
´
[21] M. M. Savic, D. I. Obradovic, N. D. Ugresic, J. M. Cook, W. Yin, D. R. Bokonjic, Behav. Brain Res.
2005, 158, 293.
[22] I. Misane, S. O. ꢇgren, Neuropsychopharmacology 2003, 28, 253.
´
[23] I. Oliva, M. E. Gonzalez-Trujano, J. Arrieta, R. Enciso-Rodrꢀguez, A. Navarrete, Phytother. Res.
2004, 18, 290.
´
[24] M. Ugalde, V. Reza, M. E. Gonzalez-Trujano, B. Avula, I. A. Khan, A. Navarrete, J. Pharm.
Pharmacol. 2005, 57, 631.
´
[25] M. E. Gonzalez-Trujano, A. L. Martꢀnez, A. Reyes-Ramꢀrez, B. Reyes-Trejo, A. Navarrete, Planta
Med. 2006, 72, 703.
[26] K.-O. Hiller, G. Zetler, Phytother. Res. 1996, 10, 145.
[27] A. Rolland, J. Fleurentin, M.-C. Lanhers, C. Younos, R. Misslin, F. Mortier, J. M. Pelt, Planta Med.
1991, 57, 212.
[28] N. W. Durham, T. S. Miya, J. Am. Pharm. Assoc. 1957, 46, 208.
Received August 31, 2009