Design of 1-substituted 2-arylmethyl-4,5-methylenedioxybenzene derivatives as antiseizure agents

Article abstract of DOI:10.1016/j.bmc.2004.04.015

A series of 1-substituted 2-[(4-aryl)-methyl]-4,5-methylenedioxybenzene derivatives (13-25), structurally related to model compound 5 (2-[(4-aminophenyl)-(4-methylsemicarbazono)-methyl]-4,5- methylenedioxyphenylacetic acid methyl ester), were synthesized and tested as anticonvulsant agents in DBA/2 mice against sound-induced seizures. The new compounds possess anticonvulsant properties lower than those of prototype 5 but, in some instances, comparable to that of GYKI 52466, a well-known noncompetitive AMPA receptor antagonist.

Full text of DOI:10.1016/j.bmc.2004.04.015

Bioorganic & Medicinal Chemistry 12 (2004) 3703–3709
Design of 1-substituted 2-arylmethyl-4,5-methylenedioxybenzene
derivatives as antiseizure agents
a
b
b
ꢀ a
Nicola Micale, Giovambattista De Sarro, Guido Ferreri, Maria Zappala,
Silvana Grasso,a Giulia Puiac and Carlo De Michelid,*
aDipartimento Farmaco-Chimico, Universitꢀa di Messina, Viale Annunziata 98168, Messina, Italy
bDipartimento di Medicina Sperimentale e Clinica, Universitꢀa di Catanzaro, Via T. Campanella 88100, Catanzaro, Italy
cDipartimento di Scienze Farmaceutiche,Universitꢀa di Modena, Via dei Campi 183, 41100 Modena, Italy
dIstituto di Chimica Farmaceutica, Universitꢀa di Milano, Viale Abruzzi 42, 20121 Milan, Italy
Received 12 December 2003; accepted 7 April 2004
Available online 18 May 2004
Abstract—A series of 1-substituted 2-[(4-aryl)-methyl]-4,5-methylenedioxybenzene derivatives (13–25), structurally related to model
compound 5 (2-[(4-aminophenyl)-(4-methylsemicarbazono)-methyl]-4,5-methylenedioxyphenylacetic acid methyl ester), were syn-
thesized and tested as anticonvulsant agents in DBA/2 mice against sound-induced seizures. The new compounds possess anti-
convulsant properties lower than those of prototype 5 but, in some instances, comparable to that of GYKI 52466, a well-known
noncompetitive AMPA receptor antagonist.
Ó 2004 Elsevier Ltd. All rights reserved.
1. Introduction
52466 (1) and GYKI 53655 (2) (Fig. 1), acting as non-
competitive antagonists of the AMPA receptors,2 dis-
played potent anticonvulsant properties3;4 and behaved as
neuroprotective agents in focal and global ischemia.5
Epilepsies are common neurological disorders, affecting
approximately 2.5 million people solely in the United
States. Epileptic seizures often cause transient impairment
of consciousness, leaving the individual at risk of bodily
harm. Therapy is symptomatic in that available drugs
inhibit seizures, but they neither affect prophylaxis nor
cure the cause. Compliance with medication is a major
problem, due to the need of a long-term therapy, which is
quite often associated with unwanted side effects.1
As part of a program aimed at identifying potent and
selective AMPA receptor antagonists, we have previously
reported6;7 an investigation on the anticonvulsant activ-
ity of a series of 1-aryl-3,5-dihydro-7,8-methylenedioxy-
4H-2,3-benzodiazepin-4-ones, for example, 3, and their
3-N-alkylcarbamoyl derivatives, for example, 4 (Fig. 1),
which are structural analogues of 1 and 2, respectively.
Drugs effective against the most common forms of epi-
leptic seizures, appear to work by limiting the sustained
repetitive firing of a neuron by one of the following
mechanisms: (i) enhancement of GABA-mediated syn-
aptic inhibition, (ii) stabilization of the inactivated state of
voltage-operated Naþ channels, (iii) inactivation of volt-
age-activated Ca2þ channels. Considerable interest has
lately been focused on antagonists of the ionotropic glu-
tamate receptors (NMDA, AMPA, and KA) having sig-
nificant anticonvulsant activity thus providing the basis
for an extensive research in this area. As a matter of fact, a
number of 2,3-benzodiazepine derivatives, that is GYKI
Compounds 3–4 proved to be roughly 2–3-fold more
potent than GYKI 52466 and were provided with a
CH3
O
O
OMe
O
O
O
O
N
R
NR
O
O
O
N
N
H
N
NHMe
N
H2N
H2N
3, R = H
1, GYKI 52466 3,4-∆
H2N
4, R = CONHMe
2, GYKI 53655 3,4-dihydro,
5
R = CONHMe
* Corresponding author. Tel.: +39-02-50317538; fax: +39-02-503175-
74; e-mail: carlo.demicheli@unimi.it
Figure 1.
0968-0896/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.04.015

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N. Micale et al. / Bioorg. Med. Chem. 12 (2004) 3703–3709
better protective index. Furthermore, analogously to 1
and 2, they displayed anticonvulsant activity mainly by
blocking the allosteric AMPA-receptor binding site.8 In
view of these results, we subsequently designed and
tested9 a series of alkyl esters of 4,5-methylenedioxy-
phenylacetic acid bearing an N-alkyl-semicarbazono
moiety at position 2, which may be envisaged as ‘open
models’ of reference compound 4. Within this series of
derivatives, 2-[(4-aminophenyl)-(4-methylsemicarbaz-
ono)-methyl]-4,5-methylenedioxyphenylacetic acid me-
thyl ester 5 proved to be the most active compound. It
displayed an anticonvulsant activity slightly higher than
that of its parent 4 and 5-fold higher than that shown by
reference compound 1. It is worth pointing out that,
despite a close structural similarity with 4, derivative 5
possesses significant differences in the pharmacological
profile. Compound 5 does not bind or only marginally
interacts with either the GYKI 52466- or the AMPA-
receptor binding sites. Furthermore, no interaction with
either the glycine-binding site of the NMDA receptors or
the inhibitory GABAA-receptor complex was noticed.8
R
R
O
O
O
O
a, b
R'
O
N
H2N
NO2
22 R = CH2COOMe, R' = NHAc
6-7
23 R = Me, R' = NHAc
24 R = Me, R' = CONHNH2
Scheme 2. (a) AcNHNH2 or NH2NHCOOMe, MeOH/HCl cat.,
reflux, 48 h; (b) Raney-Ni, ammonium formate, EtOH, reflux, 2 h.
O
NHMe
O
O
O
NHMe
O
O
O
O
H
N
a, b
N
N
NHMe
N
H2N
O2N
25
26
Scheme 3. (a) MeNH2, THF, reflux, 1 h; (b) Raney-Ni, ammonium
formate, EtOH, reflux, 2 h.
Since the semicarbazono moiety is an important struc-
tural feature of a number of anticonvulsant agents,10 we
designed the series of derivatives 13–25 as analogues of
the lead compound 5. The objective of this study was to
investigate whether the ester group and the 4-amino-
phenyl moiety were involved in determining the anti-
convulsant potency of the compounds. Therefore,
the methyl ester functionality of 5 was converted into the
bioisosteric amide, that is 25, or removed, that is 21. The
importance of the 4-aminophenyl group was evaluated by
replacing it with a 4-halophenyl moiety, that is 18–20.
Furthermore, in order to test the influence of the meth-
ylsemicarbazono group on the anticonvulsant activity, we
converted such a moiety into its bioisosteric thio-ana-
logue, that is 13–17, inverted it, that is 24, and trans-
formed it into the acetylhydrazono moiety, that is 22–23.
were prepared via a Friedel–Crafts acylation according to
a previously reported methodology.11 Derivatives 11–15
were prepared in good yields by reacting 6–10 with
4-methyl-3-thiosemicarbazide. The 4-nitrophenyl deriva-
tives 11–12 were subsequently transformed into the
4-aminophenyl analogues 16–17 through a reduction
process carried out with Raney-Ni/ammonium formate.
On the other hand, thiosemicarbazono derivatives 13–16
were easily converted into the corresponding semi-
carbazono derivatives 18–21 by treatment with hydrogen
peroxide and sodium methoxide (Scheme 1).
Compound 6 was also treated with acetylhydrazine or
methyl hydrazinocarboxylate to give compounds 23 and
24, respectively,whereas derivative 7 was reacted with
acetylhydrazine to yield derivative 22 (Scheme 2).
2. Chemistry
The synthesis of target compounds 13–25 is outlined in
Schemes 1–3. Compounds 6–10, used as starting material,
Finally, with a procedure similar to that employed for
compound 5,9 derivative 25 was prepared by reacting
R
R
O
O
O
O
S
R
O
O
O
H
N
a
b
H
N
O
NHMe
N
NHMe
N
R'
R'
R'
R
R’
R
R’
R
R’
6
7
8
9
10
—
—
11
12
13
14
15
18
Me
NO2
NO2
Br
Cl
F
Me
NO2
NO2
Br
Cl
F
CH2COOMe
CH2COOMe
CH2COOMe
Me
Br
Cl
F
19
20
21
CH2COOMe
CH2COOMe
CH2COOMe
CH2COOMe
CH2COOMe
CH2COOMe
CH2COOMe
CH2COOMe
Me
c
NH2
→16
→17
NH2
NH2
CH2COOMe
Scheme 1. (a) MeNHCSNHNH2, MeOH, HCl cat., reflux, 48 h; (b) H2O2, MeONa, MeOH, rt, 200; (c) Raney-Ni, ammonium formate, EtOH, reflux,
2 h.

N. Micale et al. / Bioorg. Med. Chem. 12 (2004) 3703–3709
3705
3,5-dihydro-3-N-methylcarbamoyl-7,8-methylenedioxy-
1-(4-nitrophenyl)-4H-2,3-benzodiazepin-4-one (26) with
methylamine and subsequently reducing its nitro group
(Scheme 3).
As shown in Table 1, the new compounds possess anti-
convulsant properties lower than those of prototype 5 but,
in some instances, comparable to those of GYKI 52466.
In particular, with respect to compound 5, the activity is
significantly reduced by replacing the semicarbazono
moiety with the corresponding thiosemicarbazono one
(17) or the acetylhydrazono group (22). Noteworthy, at
variance with derivative 5, the anticonvulsant activity of
compound 17 is enhanced if the audiogenic seizure test is
carried out 15 min rather than 30 min after ip adminis-
tration (see footnotes in Table 1), indicating that such a
bioisosteric modification produces a short-time activity.
A conceivable explanation could be that compound 17 is
more easily diffused across the blood–brain barrier than
its analogue 5.
The structure and stereochemistry of new derivatives
11–25, always obtained as as single isomer, rely on
NMR data. The stereochemistry around the C@N bond
of derivative 5 was previously investigated using a
1
combination of modeling studies and H NMR spec-
troscopy.9 Molecular modeling studies showed that the
rotation around the single bond connecting the semi-
carbazono moiety to C-2 is hindered, making derivative
5 a chiral molecule in the NMR time scale.
As a matter of fact, the geminal hydrogens of the two
methylene groups of derivative 5, that is the one in the a-
position with respect to the ester function and the other
of the dioxole ring, are diastereotopic and resonate as
AB systems or multiplets. The stereochemistry around
the C@N bond was assigned as Z. These spectroscopic
features were always found in derivatives 11–25.
Therefore, by analogy with derivative 5, we assigned the
Z configuration to all new derivatives.
The replacement of the 4-aminophenyl group with a
4-halophenyl moiety (18–20) also negatively influences
activity. However, contrarily to the trend observed in
compound 17, thioderivatives 13–15 are more active
than their carbonyl analogues 18–20.
Removal of the ester functionality (21) or its transfor-
mation into an amide (25) destroys or reduces the anti-
convulsant activity (ED50 >100 lmol/kg for 21 and
31.8 lmol/kg for 25 vs 7.87 lmol/kg for 5) except for
acetylhydrazono derivative 23, which is slightly more
potent than its ester counterpart 22. It is worth pointing
out that the anticonvulsant activity of the compounds
has been evaluated on a in vivo assay and, consequently,
the observed variations in potency could simply be due to
differences in the pharmacokinetic parameters, such as
metabolism or diffusion across blood–brain barrier.
3. Results and discussion
The anticonvulsant activity of derivatives 13–25 against
audiogenic seizures was evaluated 30 min after intra-
peritoneal administration to DBA/2 mice, a strain
genetically susceptible to sound-induced seizures. This
test has been considered an excellent animal model for
generalized epilepsy and for screening new anticonvul-
sant drugs.12 The results are compared with those pre-
viously reported for 5 and reference compound 1 (Table
1).9
The anticonvulsant activity of the tested compounds was
effective at doses, which did not cause sedation and
Table 1. Anticonvulsant activity of compounds 1, 5, and 13–25 against audiogenic seizures in DBA/2 mice and TD50 values on locomotion assessed
by rotarod testa
PI,b TD50/ED50
Compds
ED50 (lmol/kg)
Tonic phase
TD50 (lmol/kg)
locomotor deficit
Clonic phase
1
35.8 (24.4–52.4)
7.87 (4.68–13.2)
37.8 (17.6–81.3)
24.7 (11.0–55.3)
36.5 (11.8–112)
42.0 (30.2–58.3)
39.4 (26.5–58.8)
43.5 (22.1–85.5)
73.0 (28.6–186)
53.6 (24.2–117)
>100
25.3 (16.0–40.0)
4.62 (2.47-8.61)
26.2 (16.3–42.2)
19.6 (10.6–36.0)
20.5 (11.4–37.1)
29.1 (16.0–53.1)
24.7 (14.1–43.3)
36.0 (19.9–64.9)
44.7 (21.2–94.5)
37.8 (17.6–81.3)
>100
76.1 (47.5–122)
28.3 (19.0–42.3)
121 (82.6–163)
69.2 (49.4–96.9)
113 (82–156)
2.1
3.6
3.2
2.8
3.1
2.8
2.7
2.7
2.3
2.5
ND
2.5
2.6
ND
1.8
5c
13
14
15
16
17d
18
19
20
21
22
23
24
25
110 (113–153)
113 (82.5–156)
117 (85.6–151)
168 (135–208)
134 (110–163
197 (162–239)
138 (111–171)
119 (84.6–167)
204 (164–254)
57.3 (34.2–96.1)
55.1 (21.9–138)
45.7 (18.9–110)
>100
17.1 (7.02–42.4)
16.9 (6.94–41.5)
>100
31.8 (16.8–60.2)
27.2 (10.5–69.9)
a All compounds were given ip (at doses spanning the range 3.3–200 lmol/kg) 30 min before auditory stimulation. All data were calculated according
to the method of Litchfield and Wilcoxon19 95% confidence limits are given in parentheses. At least 32 animals were used to calculate each ED50 and
TD50 value.
b PI, protective index, represents the ratio between TD50 and ED50 (from the clonic phase of the audiogenic seizures). ND, not detectable.
c ED50 for clonus 24.1 (13.6–42.6) lmol/kg at 15 min after ip administration.9
d ED50 for clonus 24.7 (15.3–48.2) lmol/kg at 15 min after ip administration.

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N. Micale et al. / Bioorg. Med. Chem. 12 (2004) 3703–3709
Table 2. Reduction of KA-evoked currents induced by 1, 5, 16–17, and
21–24a
4.1. (Z)-2-[(4-Methylthiosemicarbazono)-(4-nitrophenyl)-
methyl]-4,5-methylenedioxytoluene (11)
Compds
% Reductionb
Compds
% Reductionb
4-Methyl-3-thiosemicarbazide (1.4 mmol) and HCl 10 N
(0.2 mL) were added to a stirred solution of 6 (0.7 mmol)
in MeOH (40 mL). The reaction mixture was refluxed for
48 h, cooled to room temperature to afford a yellow solid
that was recrystallized from methanol. Mp 111–114 °C;
Rf ¼ 0:36 (diethyl ether/petroleum ether, 6:4); yield 75%.
1H NMR 1.99 (s, 3H, CH3-1), 3.31 (d, 3H, J ¼ 4:9 Hz
NHCH3), 6.07 (m, 2H, OCH2O), 6.52 (s, 1H, H-3), 6.86
(s, 1H, H-6), 7.66 (m, 1H, NHCH3), 7.68 (d, 2H, J ¼ 8:8
Hz, H-20,60), 8.20 (d, 2H, J ¼ 4:9 Hz, H-30,50), 8.59 (bs,
1H, NH). MS (ESI+) 373 (Mþ+1). Anal. Calcd for
C17H16N4O4 S: C, 54.83; H, 4.33; N, 15.04. Found C,
55.06; H, 4.28; N, 14.88.
1
47 4 (n ¼ 5)
60 1 (n ¼ 5)
47 5 (n ¼ 5)
30 5 (n ¼ 44)
21
22
23
24
25 3 (n ¼ 7)
55 4 (n ¼ 16)
28 4 (n ¼ 3)
32 5 (n ¼ 8)
5
16
17
a All drugs were tested at 100 lM.
b Each value is the mean SE. The number of cells is given in paren-
theses.
ataxia. It is noteworthy that several derivatives pos-
sess a protective index (PI) higher than that of 1 (Table 1).
Recent binding studies8 indicate that the lead compound
5 does not interact with neither the GYKI 52466- nor
the AMPA-receptor binding site whereas electrophysio-
logical experiments demonstrated that compound 5 was
able to reduce KA-evoked currents.9 Consequently, the
ability of derivatives 16, 17, and 21–24 to affect KA-
evoked currents was assessed in cerebellar granule neu-
rons grown in primary cultures by using the patch clamp
technique. As shown in Table 2, KA-evoked currents are
marginally affected by the application of compounds 17,
21, 23, and 24 (100 lM) and reduced by the application
of compounds 16 and22. However, the high concentra-
tion (100 lM) needed to detect, for compounds 16 and
22, a sizeable reduction in the KA-evoked currents does
not allow to relate their anticonvulsant activity to the
involvement of KA receptors.
4.2. (Z)-2-[(4-Methylthiosemicarbazono)-(4-nitrophenyl)-
methyl]-4,5-methylenedioxyphenylacetic acid methyl ester
(12)
Compound 12 was prepared from 7 with a similar pro-
cedure. Mp 126–129 °C; Rf ¼ 0:58 (cyclohexane/EtOAc,
5:5); yield 85%. 1H NMR 3.26 (m, 2H, CH2), 3.31 (d, 3H,
J ¼ 4:7 Hz NHCH3), 3.55 (s, 3H, OCH3), 6.12 (m, 2H,
OCH2O), 6.57 (s, 1H, H-3), 6.96 (s, 1H, H-6), 7.68 (d,
2H, J ¼ 8:5 Hz, H-20,60), 7.69 (m, 1H, NHCH3) 8.20 (d,
2H, J ¼ 8:5 Hz, H-30,50), 8.63 (bs, 1H, NH). MS (ESI+)
431 (Mþ +1). Anal. Calcd for C19H18N4O6S: C, 53.02; H,
4.22; N, 13.02. Found: C, 52.87; H, 4.31; N, 13.11.
Further investigations on the mechanism of action of
these compounds have been undertaken and the results
will be presented in due course.
4.3. (Z)-2-[(4-Bromophenyl)-(4-methylthiosemicarbazono)-
methyl]-4,5-methylenedioxyphenylacetic acid methyl
ester (13)
To sum up, the results of the present investigation reveal
that the methyl ester at position 1 as well as the semi-
carbazono moiety and the 4-aminophenyl group at
position 2 seem to play a crucial role in determining the
anticonvulsant activity of the lead compound 5.
Derivative 13 was prepared from 8 with a similar pro-
cedure. Mp 99–102 °C; Rf ¼ 0:39 (cyclohexane/EtOAc,
1
7:3); yield 78%. H NMR 3.27 (m, 2H, CH2), 3.28 (d,
3H, J ¼ 4:9 Hz, NHCH3), 3.53 (s, 3H, OCH3), 6.09 (m,
2H, OCH2O), 6.56 (s, 1H, H-3), 6.93 (s, 1H, H-6), 7.37
(d, 2H, J ¼ 8:5 Hz, H-30,50), 7.47 (d, 2H, J ¼ 8:5 Hz,
H-20,60), 7.52 (m, 1H, NHCH3), 8.51 (bs, 1H, NH).
MS (ESI+) 464 and 466 (Mþ+1). Anal. Calcd for
C19H18BrN3O4S: C, 49.15; H, 3.91; N, 9.05. Found: C,
48.93; H, 3.96; N, 9.19.
4. Experimental
Melting points were determined on a Kofler hot stage
apparatus and are uncorrected. Elemental analyses were
carried out on a Carlo Erba 1106 elemental analyzer for
C, H, and N, and the results are within 0.4% of the
theoretical values. Merck silica gel 60 F254 plates were
used for analytical TLC; column chromatography was
performed on Merck silica gel 60 (70–230 mesh). 1H
NMR spectra were recorded in CDCl3 by means of a
Varian Gemini-300 spectrometer. Chemical shifts are
expressed in d (ppm) relative to TMS as internal stan-
dard, and coupling constants (J) are in Hz. All
exchangeable protons were confirmed by addition of
D2O. Mass spectra of the compounds were recorded
under positive electrospray ionization (ESI+) with a
ThermoQuest LCQ mass spectrometer. The synthesis,
physical, and analytical properties of compound 22 have
been previously described.13
4.4. (Z)-2-[(4-Chlorophenyl)-(4-methylthiosemicarbazono)-
methyl]-4,5-methylenedioxyphenylacetic acid methyl
ester (14)
Derivative 14 was prepared from 9 with a similar
procedure. Mp 93–96 °C; Rf ¼ 0:42 (cyclohexane/
1
EtOAc, 7:3); yield 80%. H NMR 3.26 (m, 2H, CH2
and d, 3H, J ¼ 4:7 Hz, NHCH3), 3.54 (s, 3H, OCH3),
6.08 (m, 2H, OCH2O), 6.55 (s, 1H, H-3), 6.93 (s, 1H,
H-6), 7.30 (d, 2H, J ¼ 8:5 Hz, H-30,50), 7.45 (d, 2H,
J ¼ 8:5 Hz, H-20,60), 7.69 (m, 1H, NHCH3), 8.51 (bs,
1H, NH). MS (ESI+) 420 (Mþ+1). Anal. Calcd for
C19H18ClN3O4 S: C, 54.35; H, 4.32; N, 10.01. Found:
C, 54.51; H, 4.21; N, 10.09.

N. Micale et al. / Bioorg. Med. Chem. 12 (2004) 3703–3709
3707
4.5. (Z)-2-[(4-Fluorophenyl)-(4-methylthiosemicarbazono)-
methyl]- 4,5-methylenedioxyphenylacetic acid methyl
ester (15)
toring). The solvent was evaporated in vacuo and the
residue, dissolved in EtOAc, was washed with H2O to
remove MeONa. The organic layer was dried (Na2SO4)
and purified by silica gel column chromatography using
EtOAc as eluent. Mp 113–116 °C; Rf ¼ 0:54 (EtOAc);
Compound 15 was prepared from 10 with a similar
procedure. Mp 79–82 °C; Rf ¼ 0:41 (cyclohexane/
EtOAc, 7:3); yield 80%. 1H NMR 3.27 (m, 2H, CH2 and
d, 3H, J ¼ 4:4 Hz, NHCH3), 3.54 (s, 3H, OCH3), 6.08
(m, 2H, OCH2O), 6.56 (s, 1H, H-3), 6.93 (s, 1H, H-6),
7.03 (dd, 2H, JH–H and JH–F ¼ 8:5 Hz, H-30,50), 7.51 (dd,
2H, JH–H ¼ 8:5 Hz and JH–F ¼ 5:2 Hz, H-20,60), 7.65 (m,
1H, NHCH3), 8.49 (bs, 1H, NH). MS (ESI+) 404
(Mþ+1). Anal. Calcd for C19H18FN3O4S: C, 56.57; H,
4.50; N, 10.42. Found: C, 56.66; H, 4.58; N, 10.25.
1
yield 83%. H NMR 2.96 (d, 3H, J ¼ 4:9 Hz, NHCH3),
3.27 (s, 2H, CH2), 3.51 (s, 3H, OCH3), 6.07 (m, 2H,
OCH2O), 6.19 (m, 1H, NHCH3), 6.54 (s, 1H, H-3), 6.92
(s, 1H, H-6), 7.36 (d, 2H, J ¼ 8:8 Hz, H-30,50), 7.46 (d,
2H, J ¼ 8:8 Hz, H-20,60), 7.52 (bs, 1H, NH). MS (ESI+)
448 and 450 (Mþ+1). Anal. Calcd for C19H18BrN3O5: C,
50.91; H, 4.05; N, 9.37. Found: C, 50.78; H, 4.11; N, 9.21.
4.9. (Z)-2-[(4-Chlorophenyl)-(4-methylsemicarbazono)-
methyl]-4,5-methylenedioxyphenylacetic acid methyl
ester (19)
4.6. (Z)-2-[(4-Aminophenyl)-(4-methylthiosemicarbazono)-
methyl]-4,5-methylenedioxytoluene (16)
Compound 19 was prepared from 14 with a similar
procedure. Mp 109–111 °C; Rf ¼ 0:59 (EtOAc); yield
Raney-Ni (40 mg) and an excess of ammonium formate
(1.00 mmol) were added to a slurry of 11 (0.50 mmol) in
ethanol (40 mL). The reaction mixture was stirred under
reflux for 2 h, then filtered off on a Celite pad and the
solvent was removed under reduced pressure. The resi-
due, dissolved in CHCl3, was washed with saturated
NaCl to remove ammonium formate. The organic layer
was evaporated under reduced pressure and the residue
was purified by silica gel column chromatography using
the same eluent employed for TLC. Mp 117–120 °C;
Rf ¼ 0:44 (cyclohexane/EtOAc, 5:5); yield 80%. 1H
NMR 2.00 (s, 3H, CH3-1), 3.27 (d, 3H, J ¼ 4:7 Hz
NHCH3), 3.90 (bs, 2H, NH2) 6.02 (m, 2H, OCH2O),
6.52 (s, 1H, H-3), 6.61 (d, 2H, J ¼ 8:8 Hz, H-30,50), 6.81
(s, 1H, H-6), 7.32 (d, 2H, J ¼ 8:8 Hz, H-20,60), 7.63 (m,
1H, NHCH3) 8.37 (bs, 1H, NH). MS (ESI+) 343 (Mþ
+1). Anal. Calcd for C17H18N4O2S: C, 59.63; H, 5.30; N,
16.36. Found: C, 59.49; H, 5.38; N, 16.49.
1
82%. H NMR 2.95 (d, 3H, J ¼ 4:9 Hz, NHCH3), 3.27
(m, 2H, CH2), 3.51 (s, 3H, OCH3), 6.07 (m, 2H,
OCH2O), 6.21 (m, 1H, NHCH3), 6.55 (s, 1H, H-3), 6.92
(s, 1H, H-6), 7.29 (d, 2H, J ¼ 8:8 Hz, H-30,50), 7.43 (d,
2H, J ¼ 8:8 Hz, H-20,60), 7.54 (bs, 1H, NH). MS (ESI+)
404 (Mþ+1). Anal. Calcd for C19H18ClN3O5: C, 56.51;
H, 4.49; N, 10.41. Found: C, 56.34; H, 4.59; N, 10.55.
4.10. (Z)-2-[(4-Fluorophenyl)-(4-methylsemicarbazono)-
methyl]-4,5-methylenedioxyphenylacetic acid methyl ester
(20)
Derivative 20 was prepared from 15 with a similar
procedure. Mp 190–193 °C; Rf ¼ 0:59 (EtOAc); yield
1
85%. H NMR 2.95 (d, 3H, J ¼ 4:9 Hz, NHCH3), 3.28
(m, 2H, CH2), 3.51 (s, 3H, OCH3), 6.06 (m, 2H,
OCH2O), 6.19 (m, 1H, NHCH3), 6.55 (s, 1H, H-3), 6.92
(s, 1H, H-6), 7.02 (dd, 2H, JH–H ¼ 8:8 Hz and JH–F ¼ 8:5
Hz, H-30,50), 7.48 (dd, 2H, JH–H ¼ 8:8 Hz and JH–F ¼ 5:5
Hz, H-20,60 and bs, 1H, NH). MS (ESI+) 388 (Mþ+1).
Anal. Calcd for C19H18FN3O5 : C, 58.91; H, 4.68; N,
10.85. Found: C, 58.77; H, 4.75; N, 10.73.
4.7. (Z)-2-[(4-Aminophenyl)-(4-methylthiosemicarbazono)-
methyl]-4,5-methylenedioxyphenylacetic acid methyl
ester (17)
Compound 17 was prepared from 12 with a similar
procedure. Mp 110–112 °C; Rf ¼ 0:65 (EtOAc/cyclo-
hexane/i-PrOH, 6:3:1); yield 74%. 1H NMR 3.26 (d, 3H,
J ¼ 4:9 Hz, NHCH3), 3.29 (m, 2H, CH2), 3.56 (s, 3H,
OCH3), 3.98 (bs, 2H, NH2), 6.07 (m, 2H, OCH2O), 6.56
(s, 1H, H-3), 6.59 (d, 2H, J ¼ 8:5 Hz, H-30,50), 6.90 (s,
1H, H-6), 7.30 (d, 2H, J ¼ 8:5 Hz, H-20,60), 7.63 (m, 1H,
NHCH3) 8.40 (bs, 1H, NH). MS (ESI+) 401 (Mþ +1).
Anal. Calcd for C19H20N4O4S: C, 56.99; H, 5.03; N,
13.99. Found: C, 57.10; H, 4.84; N, 13.82.
4.11. (Z)-2-[(4-Aminophenyl)-(4-methylsemicarbazono)-
methyl]-4,5-methylenedioxytoluene (21)
Compound 21 was prepared from 16 with a similar
procedure. Mp 112–115 °C; Rf ¼ 0:51 (EtOAc); yield
83%. 1H NMR 1.99 (s, 3H, CH3-1), 2.94 (d, 3H, J ¼ 4:9
Hz, NHCH3), 4.00 (bs, 2H, NH2) 6.00 (m, 2H, OCH2O),
6.27 (m, 1H, NHCH3), 6.50 (s, 1H, H-3), 6.61 (d, 2H,
J ¼ 8:2 Hz, H-30,50), 6.78 (s, 1H, H-6), 7.31 (d, 2H,
J ¼ 8:2 Hz, H-20,60), 7.47 (bs, 1H, NH). MS (ESI+) 327
(Mþ +1). Anal. Calcd for C17H18N4O3:C, 62.57; H, 5.56;
N, 17.17. Found: C, 62.81; H, 5.46; N, 17.21.
4.8. (Z)-2-[(4-Bromophenyl)-(4-methylsemicarbazono)-
methyl]-4,5-methylenedioxyphenylacetic acid methyl
ester (18)
4.12. (Z)-2-[(4-Acetylhydrazono)-(4-aminophenyl)-
methyl]-4,5-methylenedioxytoluene (23)
An excess of 30% H2O2 (10 mL) and MeONa (100 mg)
were added to a solution of 13 (0.4 mmol) in MeOH
(30 mL); the mixture was stirred at room temperature
until disappearance of the starting material (TLC moni-
Acetylhydrazine (104 mg, 1.4 mmol) and 10 N HCl
(0.2 mL) were added to a solution of 6 (200 mg,

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N. Micale et al. / Bioorg. Med. Chem. 12 (2004) 3703–3709
0.7 mmol) in MeOH (25 mL). The reaction mixture was
refluxed for 48 h. After cooling, the solvent was removed
under reduced pressure and the residue was subjected to
chromatography eluting with EtOAc/cyclohexane, 1:1 to
afford the acetylhydrazono derivative, which was suc-
cessively reduced, with the procedure reported for
compound 16, to give compound 23. Mp 103–106 °C;
mice of either sex were exposed to auditory stimulation
30 min following administration of vehicle or each dose
of drugs studied. The compounds were given ip (0.1 mL/
10 g of body weight of the mouse) as a freshly prepared
solution in 50% DMSO and 50% sterile saline (0.9%
NaCl). Individual mice were placed under a hemispheric
perspex dome (diameter 58 cm) and 60 s were allowed
for habituation and assessment of locomotor activity.
Auditory stimulation (12–16 kHz, 109 dB) was applied
for 60 s or until tonic extension occurred and induced a
sequential seizure response in control DBA/2 mice,
consisting of an early wild running phase, followed by
generalized myoclonus and tonic flexion and extension,
sometimes followed by respiratory arrest. The control
and drug-treated mice were scored for latency to and
incidence of the different phases of the seizures.15
1
Rf ¼ 0:67 (EtOAc); yield 46%. H NMR 2.00 (s, 3H,
CH3-1), 2.41(s, 3H, COCH3), 3.87 (bs, 2H, NH2), 6.01
(m, 2H, OCH2O), 6.51 (s, 1H, H-3), 6.62 (d, 2H, J ¼ 8:8
Hz, H-30,50), 6.80 (s, 1H, H-6), 7.36 (d, 2H, J ¼ 8:8 Hz,
H-20,60), 8.17 (bs, 1H, NH). MS (ESI+) 312 (Mþ +1).
Anal. Calcd for C17H17N3O3: C, 65.58; H, 5.50; N,
13.50. Found: C, 65.36; H, 5.38; N, 13.74.
4.13. (Z)-2-[(4-Aminophenyl)-(4-semicarbazono)-methyl]-
4,5-methylenedioxytoluene (24)
4.16. Electrophysiological recordings
Methylhydrazino-carboxylate (1.4 mmol) and 10 N HCl
(0.2 mL) were added to a stirred solution of 6 (0.7 mmol)
in MeOH (40 mL) The reaction mixture was refluxed for
48 h. After removal of the solvent under reduced pres-
sure, the oily residue was treated with diethyl ether to
afford a solid, which by successive reduction with the
procedure reported for 16, gave compound 24. Mp 111–
114 °C; Rf ¼ 0:80; yield 68% (EtOAc); 1H NMR 2.01 (s,
3H, CH3), 3.83 (m, 4H, NH2-40 and NHNH2), 6.02 (m,
2H, OCH2O), 6.53 (s, 1H, H-3), 6.60 (d, 2H, J ¼ 8:2 Hz,
H-30,50), 6.81 (s, 1H, H-6), 7.38 (d, 2H, J ¼ 8:2 Hz,
H-20,60), 7.58 (bs, 1H, NH). MS (ESI+) 313 (Mþ +1).
Anal. Calcd for C16H16N4O3: C, 61.53; H, 5.16; N,
17.94. Found: C, 61.69; H, 5.01; N, 17.81.
Recordings were performed on single cerebellar granule
neurons after 7 days in culture16 using the voltage-clamp
technique in the whole-cell configuration.17 Electrodes
were pulled from borosilicate glass on a vertical puller
and had a resistance of 5–7 MX when filled with KCl
internal solution. Currents were amplified with an
Axopatch 1D amplifier, filtered at 5 kHz, and digitized
at 10 kHz by using pClamp software. Intracellular
solution contained (mM): KCl 140, MgCl2 3, ethyl-
ene glycol-bis-(b-aminoethylether)N,N,N0,N0-tetraacetic
acid (EGTA) 5, N-(2-hydroxyethyl)piperazine-N0-(2-
ethanesulfonic acid) (HEPES) 5, ATP-Na 2, pH 7.3 with
KOH. The cells were continuously perfused with the
external solution (mM): NaCl 145, KCl 5, CaCl2 1,
HEPES 5, glucose 5, sucrose 20, pH 7.4 with NaOH. All
drugs were dissolved in DMSO and diluted at the final
concentration (<1%) in extracellular solution. KA was
also dissolved in the extracellular solution. All drugs
were applied directly by gravity through a Y-tube per-
fusion system18 at the same concentration (100 lM).
After a wash-out of test compounds from the cell cul-
ture, the responses to KA returned to the control level.
4.14. (Z)-2-{2-[(4-Aminophenyl)-(4-methylsemicarbaz-
ono)-methyl]-4,5-methylenedioxyphenyl}-N-methyl-
acetamide (25)
An excess methylamine (1.5 mmol) was added to a stirred
solution of 269 (0.5 mmol) in dry THF (30 mL); the
reaction mixture was refluxed for 1 h. After removal of
the solvent under reduced pressure, the crude was puri-
fied by silica gel column chromatography using EtOAc/
MeOH 90:10 as eluent to give the intermediate nitro
derivative. The successive reduction was accomplished
with the procedure reported for 16. The oily residue,
dissolved in 2 mL of acetone, by treatment with light
petroleum gave 25 as pale yellow solid. Mp 166–168 °C;
Rf ¼ 0:28 (EtOAc/MeOH); yield 75%. 1H NMR 2.57 (d,
3H, J ¼ 4:9 Hz, NHCH3-1), 2.92 (d, 3H, J ¼ 4:9 Hz, NH
CH3-2), 3.13, and 3.20 (dd, 2H, J ¼ 15:4 Hz, CH2CO),
3.88 (bs, 2H, NH2), 5.37 (m, 1H, NHCH3-1), 6.04 (m, 2H,
OCH2O), 6.22 (m, 1H, NHCH3-2), 6.57 (s, 1H, H-3), 6.61
(d, 2H, J ¼ 8:5 Hz, H-30,50), 7.00 (s, 1H, H-6), 7.28 (d,
2H, J ¼ 8:5 Hz, H-20,60), 7.63 (bs, 1H, NH). MS (ESI+)
384 (Mþ +1). Anal. Calcd for C19H21N5O4: C, 59.52; H,
5.52; N, 18.27. Found: C, 59.32; H, 5.68; N, 18.42.
4.17. Statistical analysis
The ED50 values of each phase of the audiogenic seizure
were determined for each dose of compound adminis-
tered, and dose–response curves were fitted using a
computer program by the method of Litchfield and
Wilcoxon.19 The median toxic dose (TD50) values were
estimated using the method of Litchfield and Wilco-
xon.19 Electrophysiological data were analyzed using the
software Clampex (Axon Instrument). Results are
expressed as mean SE.
Acknowledgements
4.15. Audiogenic seizures test in DBA/2 mice14
This work was financially supported by the Ministero
ꢀ
dell’Istruzione, dell’Universita e della Ricerca (MIUR––
COFIN 2003)––Rome, Italy.
DBA/2 mice (8–12 g, 22–25 days old) were purchased
from Charles River (Calco, Como, Italy). Groups of 10

N. Micale et al. / Bioorg. Med. Chem. 12 (2004) 3703–3709
3709
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G.; Ferreri, G.; De Sarro, A.; Toma, L.; De Micheli, C.
J. Med. Chem. 2002, 45, 4433.
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