Anxiolytic effects of benzalphthalides

Article abstract of DOI:10.1016/j.bmcl.2005.06.031

Anxiolytic effects induced by benzalphthalides on mice have been evaluated. The evaluation was based on the spent time and the number of entries of animals into the open arms in the elevated plus maze test. Single administration of benzalphthalides 1 and 11 induced significant increments in both parameters, thus revealing their potentiality as new leads for the development of non-benzodiazepinic and non-nitrogenated antianxiety agents.

Full text of DOI:10.1016/j.bmcl.2005.06.031

Bioorganic & Medicinal Chemistry Letters 15 (2005) 3483–3486
Anxiolytic effects of benzalphthalides
Alejandro Zamilpa,a,b Maribel Herrera-Ruiz,b Esther del Olmo,a, Jose L. Lopez-Perez,
a
*
´
´
´
Jaime Tortoriellob,* and Arturo San Felicianoa
a
´
Departamento de Quımica Farmaceutica, Facultad de Farmacia, 37007 Salamanca, Spain
Centro de Investigacion Biomedica del Sur, IMSS, 62790 Xochitepec, Morelos, Mexico
´
b
´
´
Received 18 April 2005; revised 30 May 2005; accepted 8 June 2005
Abstract—Anxiolytic effects induced by benzalphthalides on mice have been evaluated. The evaluation was based on the spent time
and the number of entries of animals into the open arms in the elevated plus maze test. Single administration of benzalphthalides 1
and 11 induced significant increments in both parameters, thus revealing their potentiality as new leads for the development of non-
benzodiazepinic and non-nitrogenated antianxiety agents.
ꢀ 2005 Elsevier Ltd. All rights reserved.
Anxiety is a rather common disorder affecting people in
both developed and developing countries.1 Pathological
manifestations of anxiety are prevalent, chronic, and
disabling and can include generalized anxiety, obses-
sive–compulsive disorders, panic, post-traumatic stress
disorders, and social and specificphobias.2 Pharmaco-
logical treatment of anxiety is principally based on 1,4-
benzodiazepines (BDZs, diazepam and related drugs)
or 5-HT1A receptor agonists and selective 5-HT reup-
take inhibitors (SSRIs). These approaches have draw-
backs because BDZs have a number of unwanted
sideeffects, including tolerance, sedation, cognitive
impairments, and alcohol interaction and generally,
the onset of action of 5-HT receptor ligands is slow.3
Non-nitrogenated natural compounds such as flavo-
noids and some related synthetic chromenoids have
attracted much attention and have been tested with po-
sitive results as anxiolytics 4,5 and their interactions with
BDZ (GABA) receptors have also been demonstrated.6
ucts7 displaying a wide range of biological activities,
such as antitussive (noscapine),8 anti-HIV (fuscinarin),9
antineoplastic cytotoxicity (vermistatin),10 or antibacte-
rial (cytosporone E).11 Moreover, various naturally
occurring or synthetic 3-alkylidenephthalides12 showed
muscle relaxant and antiasthmatic properties that have
been protected by patents,13 and 3-butylphthalide re-
duced brain damage in mice, showed anticonvulsant ef-
fects, increased the duration of anesthesia, and exhibited
cerebral antiischemic properties.8 The natural benzalph-
thalide thunberginol F showed inhibitory activity on
histamine release from rat peritoneal mast cells.14 In
previous reports, we showed that some benzalphthalides
displayed low levels of in vitro leishmanicidal15 and try-
panocidal activities.16 More recently, we have evaluated
their anti-HIV, vasorelaxant, and antihistaminic activi-
ties and the results will be published elsewhere. In this
article, we present the results of the evaluation of 12 rep-
resentative benzalphthalides as anxiolytics.
The structural analogy between the chromenoids and
the phthalide (isobenzofuranone) nuclei (Fig. 1) suggest-
ed to us the possibility for the latter of serving as sup-
porting skeleton for anxiolytic compounds. This fact
seemed to have not been tested before. Moreover, the
phthalide moiety is also present in various natural prod-
1. Chemistry
The simple and general procedure (Scheme 1) used to
obtain these benzalphthalides was previously applied
and reported by our group.15,17 Phthalic anhydride
derivatives were condensed with differently substituted
phenylacetic acids, leading to the corresponding ben-
zalphthalides 1–9. Oxidation of the sulfane derivative 4
Keywords: Benzalphthalides; Anxiolytic effect; SAR; Apigenin;
Diazepam.
with KIO4 or H2O2/AcOH19 provided sulfoxide 10
18
*
and sulfone 11, respectively. Transformation of the acid
7 into the amide 12 was carried out through the interme-
Corresponding authors. Tel.: +34 923 294528; fax: +34 923
294515; e-mail: olmo@usal.es
0960-894X/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.06.031

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A. Zamilpa et al. / Bioorg. Med. Chem. Lett. 15 (2005) 3483–3486
Figure 1. Structural relationship between anxiolytic natural and synthetic flavonoids and benzalphthalides.
Scheme 1. Benzalphthalides tested as anxiolytics.
diate acid chloride that was prepared by reaction with
SOCl2/benzene, by treatment with ammonia solution.
Phthalides 7, 8, 9, and 12 were obtained and evaluated
as unresolved regioisomeric (approximately 1:1)
mixtures.
It can be observed in table and figures that for com-
pounds containing natural oxygenated substituents as
p-methoxy (1), 3,4-methylenedioxy (2), or 3,4,5-trimeth-
oxy (3) group, the monosubstitution seems to be better
for the activity than those di- or tri-substitutions. On
the other hand, the replacement of the p-methoxy group
by other non-natural substituents as methylsulfanyl (4),
chloro (5), or nitro (6) leads to lowering the values of
anxiolytic parameters, while oxidation of the sulfane
group of 4 to the corresponding sulfoxide 10 and sulfone
11 gradually increases the anxiolytic effects to levels even
higher than those observed for the methoxy derivative 1.
2. Biological assays
The evaluation of anxiolytic effects was performed on
albino ICR mice through the elevated plus maze test.20
In this assay, anxiolytic drugs prolong the time spent
and the number of entries into the open arms of the
maze, whereas anxiogenic compounds produced a fair
reduction of these parameters. The test group was treat-
ed with compounds 1–12 (15 mg/kg, ip); three other
groups were, respectively, treated with the anxiolytic ref-
erence drug (diazepam, DZP, 1 mg/kg, ip), the anxio-
genic reference drug (picrotoxin, 2 mg/kg, ip), and
vehicle (Tween 20, 5%, ip).21 Tests begun 1 h after
administration, and mice evolutions were recorded for
5 min using a video camera. The anxiolytic activity
was evaluated through the number of entries and the
time spent into the open arms of the maze.22
With respect to the influence of substituents on ring A, on
the light of the results observed for compounds 7, 8, 9, and
12 and within the scarce number of compounds tested, it
can be noted that the presence of an electron-withdrawing
group linked at position 5 or 6, such as a free carboxyl
(compound 7), a nitro (compound 8), or an amide (com-
pound 12) group, provided less active derivatives if com-
pared with the corresponding unsubstituted phthalide 1.
The most relevant fact within the results observed is that
the percentage of entries into the open arms for the sul-
fone derivative 11 was significantly higher, in compari-
son with that observed for mice administered with the
vehicle only, thus demonstrating for the first time in this
type of compounds a certain level anxiolytic activity.
3. Results and discussion
The effects of single administration of benzalphthalides
1–12, DZP, PTX, vehicle on the spent time, and the
number of entries into the arms of plus maze are shown
in Table 1. Figures 2A–D are arranged to provide a
graphical comparison related with the influence of sub-
stituents attached to ring B on the activity.
The results observed are in agreement with the close
structural proximity between benzalphthalides and fla-
vonoids. This fact can easily be confirmed by simple
visual inspection of the superimpositions of the lowest
energy calculated structures for the most potent phtha-

A. Zamilpa et al. / Bioorg. Med. Chem. Lett. 15 (2005) 3483–3486
3485
Table 1. Effects of benzalphthalides 1–12 on the relevant anxiolytic parameters in the elevated plus maze test
Compound
Spent time*
into open arms
Number of entries
into open arms
Number of entries
into closed arms
Total number
of entries
% Number of entries
into open arms
1
2
177.3 33.5
150.6 27.9
106.6 66.1
130.6 46.5
159.8 82.5
158.7 27.4
137.5 71.1
107.8 50.1
129.1 45.5
153.8 29.4
187.1 44.2
125.2 43.9
230.4 16.9
122.4 17.4
26.6 17.6
13.0 4.2
11.2 2.8
8.3 3.8
7.9 3.1
8.0 2.8
9.0 3.3
9.8 5.3
9.5 5.2
11.7 7.2
10.8 4.1
10.6 2.8
13.0 5.1
15.8 4.5
9.6 3.0
2.5 2.0
7.1 2.1
9.0 3.0
9.0 4.8
9.9 3.3
8.5 5.7
7.3 3.1
9.0 4.1
11.7 3.4
10.5 2.1
9.0 3.1
5.6 4.5
11.5 2.8
3.7 2.5
8.9 1.7
20.1 5.1
20.2 4.9
17.3 5.3
17.7 5.1
16.5 7.4
16.3 5.1
18.8 6.7
21.2 6.4
22.2 8.5
19.8 5.5
16.4 7.1
24.0 6.4
19.5 5.7
18.5 4.0
11.8 5.6
64.1 10.8*
54.4 7.3
3
44.8 19.8
49.5 14.8
55.8 24.8
57.2 11.3
51.2 20.5
42.8 15.7
51.7 10.1
55.6 10.9
69.9 15.2*
50.2 12.1
82.0 11.4
51.2 7.2
4
5
6
7
8
9
10
11
12
DZP
VEH
PTX
9.4
4
19.0
8
Data represent the mean SEM; n = 8. DZP, diazepam; VEH, Tween 20 (5%); PTX, picrotoxin.
* s/5 min.
Figure 2. Effect produced by benzalphthalides, without (A,B) or with (C,D) substituents on ring A, upon the spent time (A,C) and the percentage of
entries into the open arms (B,D) in elevated plus maze test. DZP, diazepam; VEH, Tween 20 (5%); PTX, picrotoxin. *p < 0.05, **p < 0.001.
Figure 3. Superimposition of calculated structures: (A) apigenin (yellow) with compound 1 (blue); (B) 6-bromo-30-nitroflavone (yellow) with
compound 11 (blue).
lides found in this work and the flavonoids apigenin and
bromonitroflavone mentioned above (Fig. 3).
lide skeleton supporting adequate functionalities could
provide new potential candidates for developing new
anxiolytic drugs. Nevertheless, with the model of 1
and 11 more compounds with electron donating or with-
drawing groups attached to the same or other positions
After this preliminary prospection of anxiolytic activity,
it can be concluded that, as expected, the benzalphtha-

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A. Zamilpa et al. / Bioorg. Med. Chem. Lett. 15 (2005) 3483–3486
14. Matsuda, H.; Shimoda, H.; Yoshikawa, M. Bioorg. Med.
Chem. 1999, 7, 1445.
on ring A or B need to be prepared and evaluated. It is
also interesting to note that the absence of nitrogen in
the structure could prevent some of those secondary ad-
verse effects displayed by the currently most used DZP
and its related drugs.
´
´
´
15. Del Olmo, E.; Garcıa, M.; Lopez-Perez, J. L.; Mun˜oz, V.;
Deharo, E.; San Feliciano, A. Bioorg. Med. Chem. Lett.
2001, 11, 2123.
´
16. Del Olmo, E.; Garcıa, M.; Lopez-Perez, J. L.; Grace, R.;
Vargaz, F.; Gimenez, A.; Deharo, E.; San Feliciano, A.
´
´
´
On the basis of the results of this research, apart from
the progression of studies on the benzalphthalide group,
several other structurally related heterocycles are being
evaluated in vivo for their anxiolytic activity.
Bioorg. Med. Chem. Lett. 2001, 11, 2755.
17. Phthalic anhydride (3.0 g, 24.0 mmol) and phenyl acetic
acid (3.0 g, 22.0 mmol) in addition to sodium acetate
(60 mg, 1.2 mmol) and 5 mL of benzene were placed into a
flask provided of a Dean-Stark system and the mixture
was maintained at 230–240 ꢁC during 6 h. The reaction
was controlled by TLC. After completion of the reaction,
the mixture was cooled to room temperature, poured into
water (100 mL), and extracted with 2· with EtOAc. The
organic layer was washed with aqueous 10% Na2CO3 and
water, dried, filtered and evaporated off. The crude
material crystalised from methanol afforded compound 1
Acknowledgments
Financial support came from Spanish MCYT (Grant:
AGL2000-0039-P4-02). A.Z. thanks the CIS-IMSS
(Mexico) for a Post-doctoral fellowship. Collaborative
work performed under the auspices of ÔPrograma Ibero-
as a white powder (4.3 g, 70%); mp = 79–80 ꢁC. IR kmax
:
´
americano de Ciencia y Tecnologıa para el Desarrollo
2974, 2844, 1786, 1603, 1514, 1260, 1029, 978, 859, 823,
1
and 762 cmꢀ1. H NMR (200 MHz, CDCl3): d 3.84 (3H,
(CYTED), SubPrograma XÕ.
s, OCH3), 6.37 (1H, s, H-8), 6.93 (2H, d, J = 8.8 Hz,
H-11 + H-13), 7.50 (1H, m, H-6), 7.68 (1H, m, H-5), 7.73
(1H, m, H-4), 7.79 (2H, d, J = 8.8 Hz, H-10 + H-14) and
7.91 ppm (1H, d, J = 7.7 Hz, H-7). 13C NMR (50.3 MHz,
CDCl3): d 55.3, 106.9, 114.2, 119.5, 123.0, 125.5, 125.8,
129.2, 131.7, 134.3, 140.7, 143.0, 159.7, and 167.2 ppm.
MS (FAB) m/z 253 (M+H)+. Anal. Calcd for C16H12O3:
C, 76.18; H, 4.79. Found: C, 76.07; H, 4.55.
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