New 7,8-ethylenedioxy-2,3-benzodiazepines as noncompetitive AMPA receptor antagonists

Article abstract of DOI:10.1016/j.bmcl.2005.09.029

A series of 1-aryl-3,5-dihydro-7,8-ethylenedioxy-4H-2,3-benzodiazepin-4- ones 2a-f, were synthesized and screened as anticonvulsant agents in DBA/2 mice against sound-induced seizures. The new compounds display anticonvulsant properties although the ED

Full text of DOI:10.1016/j.bmcl.2005.09.029

Bioorganic & Medicinal Chemistry Letters 16 (2006) 167–170
New 7,8-ethylenedioxy-2,3-benzodiazepines as noncompetitive
AMPA receptor antagonists
a
Maria Zappala, Alessia Pellicano, Nicola Micale, Frank S. Menniti, Guido Ferreri,
a
a,
b
c
*
`
`
Giovambattista De Sarro,c Silvana Grassoa and Carlo De Michelid
a
`
Dipartimento Farmaco-Chimico, Universita di Messina, Viale Annunziata 98168, Messina, Italy
bPfizer Global Research and Development, Groton, CT, USA
c
`
Dipartimento di Medicina Sperimentale e Clinica, Universita di Catanzaro, Via T. Campanella 88100, Catanzaro, Italy
d
`
Istituto di Chimica Farmaceutica, Universita di Milano, Viale Abruzzi 42, 20121 Milano, Italy
Received 13 June 2005; revised 9 September 2005; accepted 12 September 2005
Available online 10 October 2005
Abstract—A series of 1-aryl-3,5-dihydro-7,8-ethylenedioxy-4H-2,3-benzodiazepin-4-ones 2a–f, were synthesized and screened as
anticonvulsant agents in DBA/2 mice against sound-induced seizures. The new compounds display anticonvulsant properties
although the ED50 values are higher than those of prototypes 1-aryl-3,5-dihydro-7,8-methylenedioxy-4H-2,3-benzodiazepin-4-ones
(1) and GYKI 52466, well-known noncompetitive AMPA receptor antagonists. Functional tests were performed to evaluate the
antagonistic activity at the AMPA and kainate receptors.
Ó 2005 Elsevier Ltd. All rights reserved.
There is increasing evidence of the potential therapeutic
utility of AMPA receptor antagonists in the treatment
of several neurodegenerative disorders, including stroke
and epilepsy. The discovery of GYKI 52466 as the pro-
totype of noncompetitive AMPA antagonists endowed
with anticonvulsant and neuroprotective properties, in-
duced wide-ranging research activities focused on 2,3-
benzodiazepines.1
O
CH3
CH3
O
O
O
O
R
O
O
O
NR
N
N
N
N
N
H2N
1a, R=H,
H2N
GYKI 52466
H2N
GYKI 53655, R=NHCH3
GYKI 53405, R=CH3
1b, R=CONHCH3
1c, R=CONHC2H5
Highly active analogs of GYKI 52466 (Fig. 1) have been
found among its 3,4-dihydro-3-N-methylcarbamoyl
(GYKI 53655) and 3,4-dihydro-3-acetyl (GYKI 53405)
derivatives.2 In particular, the 4-R enantiomer of GYKI
53405 was chosen as drug candidate and is now in clin-
ical investigation as LY 300164 (talampanel).3
Figure 1.
In this context we noticed that substitution of the imi-
nohydrazone portion of the diazepine nucleus of GYKI
52466 by the iminohydrazide moiety as well as the pres-
ence of the substituent appended at the N-3-position
positively affect the anticonvulsant activity. Further-
more, we demonstrated that these derivatives compete
with GYKI 52466 for the same allosteric binding site
of the AMPA receptors.5
We have previously investigated4 a new series of 1-aryl-
3,5-dihydro-7,8-methylenedioxy-4H-2,3-benzodiazepin-
4-ones, e.g., 1a, and their 3-N-alkylcarbamoyl derivatives,
e.g., 1b–1c, which have been shown to possess remarkable
anticonvulsant properties and are endowed with a higher
potency compared to GYKI 52466.
As an extension of our studies on the structure–activity
relationships of this set of derivatives, we designed and
screened for anticonvulsant activity new 3,5-dihydro-
7,8-ethylenedioxy-4H-2,3-benzodiazepin-4-ones 2a–c,
in order to check how the substitution of the dioxole
Keywords: 3,5-Dihydro-7,8-ethylenedioxy-4H-2,3-benzodiazepin-4-
ones; Anticonvulsant activity; AMPA receptor antagonists.
*
Corresponding author. Tel.: +39 090 6766466; fax: +39 090
355613; e-mail: nmicale@pharma.unime.it
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.09.029

`
M. Zappala et al. / Bioorg. Med. Chem. Lett. 16 (2006) 167–170
168
nucleus by the dioxane homologue affects the anticon-
vulsant activity.
in DMF to afford methyl 3,4-ethylenedioxyphenylace-
tate 4. Ketoesters (5a, 5d, 5e) were prepared by acylation
of derivative 4 with the appropriate 4-nitrobenzoic acid
in the presence of an excess of phosphorous pentoxide.
The subsequent treatment of 5a and 5e with hydrazine
afforded 1-aryl-3,5-dihydro-7,8-ethylenedioxy-4H-2,3-
benzodiazepin-4-ones 6a and 6e in satisfactory yields.
Conversely, ketoester 5d in the same reaction conditions
afforded directly 1-(4-amino-2-chlorophenyl)-2,3-benzo-
diazepine 2d through a cyclization and reduction of the
nitro group. Compounds 6a and 6e were then converted
into the corresponding aminoderivatives 2a and 2e by
reduction with ammonium formate in the presence of
Ni/Raney. Compound 2a was reacted with N-chlorosuc-
cinimide (NCS) to give 2f. 1-(4-Nitrophenyl)-2,3-benzo-
diazepine (6a) was also treated with an excess of an
methyl or ethyl isocyanate in the presence of triethyl-
amine to give the corresponding 3-N-methyl or ethylcar-
bamoyl derivatives which afforded compounds 2b–c by
catalytic hydrogenation with 5% Pd/C. Analytical and
spectral data of all the synthesized compounds are in full
agreement with the proposed structures.8
Moreover, we synthesized derivatives 2d–2f since it has
been reported6 that, within a new set of 2,3- benzodiaze-
pines, the introduction of an additional substituent at
the 1-phenyl moiety, e.g., 3-methyl-, markedly length-
ened the time course of AMPA receptor blocking action.
The involvement of AMPA and KA receptors has been
assessed by means of functional tests.
O
NR
O
a, R = R' = H
b, R = CONHCH3, R' = H
N
O
c, R = CONHC2H5, R' = H
d, R = H, R' = 2-Cl
e, R = H, R' = 3-CH3
R'
f, R = H, R' = 3-Cl
H2N
2a-f
The synthesis of 1-(4-aminophenyl)-3,5-dihydro-7,8-
ethylenedioxy-4H-2,3-benzodiazepin-4-ones (2a, 2d–2f)
and their 3-N-alkylcarbamoyl derivatives (2b–c) was
accomplished according to the reaction sequence report-
ed in Scheme 1.
The anticonvulsant activity of derivatives 2a–f against
audiogenic seizures was evaluated 30 min after intraperi-
toneal administration into DBA/2 mice, a strain geneti-
cally susceptible to sound-induced seizures. This test has
been considered the best animal model for generalized
epilepsy and for screening new anticonvulsant drugs.9
The results are compared with those previously reported
for the set of derivatives 1 and reference compound
Methyl 3,4-dihydroxyphenylacetate (3), obtained
according to a reported procedure,7 was reacted with
dibromoethane in the presence of potassium carbonate
4
GYKI 52466 (Table 1).
O
O
O
RO
RO
O
O
COOCH3
COOCH3
O
c
b
NH
N
R'
R
3, R = H
Cl
a
4, RR = CH2CH2
NO2
5a, R=R'=H
H2N
2d
5d, R=H, R'=Cl
5e, R=CH3, R'=H
c
O
O
O
O
O
O
O
f
d, e
NH
NH
NCONHR
N
N
N
O
O
R
R
H2N
O2N
H2N
2b, R=CH3
6a, R=H
2a, R=H
6e, R=CH3
2e, R=CH3
2c, R=C2H5
g
2f, R=Cl
Scheme 1. Reagents and conditions: (a) (CH2Br)2, K2CO3, acetone, D, 42 h, 64% (4); (b) ArCOOH, P2O5, (CH2Cl)2, rt, 16 h, 54% (5a), 60% (5d),
41% (5e); (c) NH2NH2ÆH2O, n-butanol, D, 20 h, 28% (2d), 64% (6a), 47% (6e); (d) CH3NCO or C2H5NCO, CH2Cl2, Et3N, rt, 24 h; (e) H2/5% Pd-C,
CHCl3, rt, 3 h; for the two-step sequence: 18% (2b), 20% (2c); (f) Ni-Raney, HCOONH4, EtOH, D, 2 h, 92% (2a), 100% (2e); (g) NCS, DMF, rt, 20 h,
33% (2f).

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M. Zappala et al. / Bioorg. Med. Chem. Lett. 16 (2006) 167–170
169
Table 1. Anticonvulsant activity of compounds 1, 2 and GYKI 52466 against audiogenic seizures in DBA/2 mice and TD50 values on locomotion
assessed by rotarod testa
Compounds
ED50 (lmol/kg)
TD50 (lmol/kg locomotor deficit)
PI,b TD50/ED50
Clonic phase
Tonic phase
1a
1b
15.4 (10.1–23.5)
12.4 (6.44–23.8)
35.0 (18.5–66.3)
61.3 (41.2–91.1)
45.5 (36.1–57.3)
48.1 (32.6–70.9)
>100
10.9 (4.60–24.6)
8.70 (4.61–16.4)
23.8 (13.4–42.1)
44.8 (36.2–58.8)
32.1 (22.0–46.8)
41.7 (28.3–61.4)
>100
99.1 (72.4–135)
48.6 (31.4–54.6)
134 (70.7–255)
102 (80.6–130)
96.4 (62.7–148)
95.3 (64.5–140)
ND
4.5
3.9
3.8
1.7
2.1
2.0
ND
2.4
1.4
2.1
1c
2a
2b
2c
2d
2e
48.2 (32.2–72.3)
70.8 (42.7–117)
35.8 (24.4–52.4)
24.6 (12.1–50.2)
27.2 (15.1–49.0)
25.3 (16.0–40.0)
115 (82.1–163)
99.1 (68.4–143)
76.1 (47.5–122)
2f
GYKI 52466
nd, not detectable.
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 Wilcoxon.12 Ninety-five percent 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).
As shown in Table 1, the new compounds possess anti-
convulsant properties lower than those of prototypes 1
as well as of lead compound GYKI 52466. In particular,
a comparison among the activity of 2a–c with that of
the corresponding derivatives 1a–1c suggests that the
replacement of the methylenedioxy moiety with the
ethylenedioxy homologue is detrimental to the anti-
convulsant activity.
chlorine or a methyl group at C-30 of the phenyl substi-
tuent (2e and 2f) does substantially affect the antagonis-
tic potency of these derivatives, as confirmed by binding
assay.10
For compound 2a, another functional test was carried
out in rat HEK293 cells expressing GluR5, a kainate
receptor subtype, stimulated by domoic acid; SYM
2081 was used as the control.11 Compound 2a did
not inhibit responses in the GluR5 cell line
(IC50 > 10 lM).
The introduction of a methyl group in the 30-position
slightly enhanced the anticonvulsant activity (ED50
48.2 lmol/kg for 2e vs ED50 61.3 lmol/kg for 2a); this
result parallels that previously noticed in the GYKI
52466 series.6 The presence of a chlorine atom (2d, 2f)
in the 4-aminophenyl group negatively influences the
activity.
To sum up, the in vivo results on DBA/2 mice reported
in this study suggest that the replacement of the dioxole
fragment with the dioxane moiety led to a reduction in
the anticonvulsant activity even if the in vitro data from
functional [Ca2+]i measurements clearly indicate for
compounds 2 a selective antagonistic activity at the
AMPA receptor higher than that displayed by 1 and
GYKI 52466.
Compounds 2a–b were tested for their ability to inhibit
the kainate-induced increase of the [Ca2+]i in a primary
culture of rat cerebellar granule cells (CGC) which ex-
press AMPA receptors; GYKI 52466 was used as the
10
control (Table 2).
Acknowledgments
Noteworthy, compounds 2a and 2b showed a concentra-
tion-dependent inhibition of the calcium influx in CGC
test and were more potent than the corresponding deriv-
atives 1a and 1b (IC50 2.9 lM for 2a and 5.4 lM for 2b
vs IC50 12 lM for 1a and 11 lM for 1b) and GYKI
52466 (IC50 22 lM). Furthermore, the insertion of a
This work was financially supported by the Ministero
`
dellꢀIstruzione, dellꢀUniversita
e
della Ricerca
(MIUR—COFIN2003), Rome, Italy. We thank John
T. Lazzaro, Pfizer Inc., for excellent technical assistance.
Table 2. Binding and functional assays for compounds 1,2 and GYKI
52466
References and notes
Compounds [3H]CP-526,427 IC50 (lM) KA-[Ca2+]i IC50 (lM)
1. (a) Bra¨uner-Osborne, H.; Egebjerg, J.; Nielsen, E. Ø.;
Madsen, U.; Krogsgaard-Larsen, P. J. Med. Chem. 2000,
1a
1e
32
12
12
11
`
43, 2609; (b) Zappala, M.; Grasso, S.; Micale, N.;
Polimeni, S.; De Micheli, C. Mini Rev. Med. Chem.
´
2001, 1, 243; (c) Solyom, S.; Tarnawa, I. Curr. Pharm.
Design 2002, 8, 913.
2a
2b
2.9
5.4
2c
2d
9.3
>20
1.8
2. Szabados, T.; Gigler, G.; Gacsalyi, I.; Gyertan, I.; Levay,
G. Brain Res. Bull. 2001, 55, 387.
3. (a) Chappell, A. S.; Sander, J. W.; Brodie, M. J.;
Chadwick, D.; Liedo, A.; Zhang, D.; Bjerke, J.; Kiesler,
G. M.; Arroyo, S. Neurology 2002, 58, 1680; (b) Lagan, Y.
2e
2f
GYKI52466
2.0
12.6
22

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M. Zappala et al. / Bioorg. Med. Chem. Lett. 16 (2006) 167–170
170
JAA XX ¼ 8:5 Hz, H-20 and H-60), 8.70 (br s, 1H, NH).
Anal. Calcd for C20H20N4O4: C, 63.15; H, 5.30; N, 14.73.
Found: C, 63.28; H, 5.39; N, 14.80.
0
0
M.; Lucas, R.; Jewell, H.; Toublane, N.; Schaefer, H.;
Sander, J. W.; Patsalos, P. N. Epilepsia 2003, 44, 46.
4. (a) De Sarro, A.; De Sarro, G.; Gitto, R.; Grasso, S.;
`
Micale, N.; Quartarone, S.; Zappala, M. Bioorg. Med.
Chem. Lett. 1998, 8, 971; (b) Grasso, S.; De Sarro, G.; De
`
Sarro, A.; Micale, N.; Zappala, M.; Puja, G.; Baraldi, M.;
De Micheli, C. J. Med. Chem. 1999, 42, 4414.
Compound 2d: mp 256–258 °C; Rf = 0.40 (CHCl3/EtOAc
50/50); 1H NMR (300 MHz, CDCl3): 3.53 (s, 2H, CH2-5),
3.91 (s, 2H, NH2), 4.22–4.29 (m, 4H, OCH2CH2O), 6.55
(s, H, H-9), 6.63 (dd, 1H, J0 = 8.0 Hz, Jm = 2.0 Hz, H-50),
6.66 (d, 1H, Jm = 2.0 Hz, H-30), 6.83 (s, 1H, H-6), 7.35 (d,
1H,J0 = 8.0 Hz, H-60), 8.64 (br s, 1H, NH). Anal. Calcd
for C17H14ClN3O3: C, 59.40; H, 4.10; N, 12.22. Found:
C, 59.56; H, 4.21; N, 12.09.
`
5. Grasso, S.; Micale, N.; Zappala, M.; Galli, A.; Costagli,
C.; Menniti, F. S.; De Micheli, C. Bioorg. Med. Chem.
Lett. 2003, 13, 443.
6. (a) Szenasi, G.; Harsing, L. G. Drug Discov. Today 2004,
´
´
1, 69; (b) Solyom, S.; Abraham, G.; Hamori, T.; Berzs-
enyi, P.; Andrasi, F.; Kurucz, I.; U.S. Patent 152693, 2004;
´
´
Compound 2e: mp 203–205 °C; Rf = 0.48 (EtOAc/cyclo-
hexane 90/10); H NMR (300 MHz, CDCl3): 2.19 (s, 3H,
1
´
Chem. Abstr. 2004, 141, 174197.
CH3), 3.43 (s, 2H, CH2-5), 3.85 (br s, 2H, NH2), 4.23–
4.32 (m, 4H, OCH2CH2O), 6.66 (d, 1H, J0 = 8.2 Hz, H-
50), 6.80 (s, 1H, H-9), 6.85 (s, 1H, H-6), 7.24 (dd, 1H,
J0 = 8.2 Hz, Jm = 2.2 Hz, H-60), 7.37 (d, 1H, Jm = 2.2 Hz,
H-20), 8.20 (br s, 1H, NH). Anal. Calcd for C18H17N3O3:
C, 66.86; H, 5.30; N, 13.00. Found: C, 66.71; H, 5.18; N,
13.19.
7. Cabedo, N.; Andreu, I.; Ramirez de Arellano, M. C.;
Chagraoui, A.; Serrano, A.; Bermejo, A.; Protais, P.;
Cortes, D. J. Med. Chem. 2001, 44, 1794.
8. Compound 2a: mp 222–224 °C; Rf = 0.50 (EtOAc/cyclo-
hexane 90/10); H NMR (300 MHz, CDCl3): 3.43 (s, 2H,
1
CH2-5), 3.92 (br s, 2H, NH2), 4.24–4.31 (m, 4H,
OCH2CH2O), 6.68 (d, 2H, JAA XX ¼ 8:5 Hz, H-30 and H-
0
0
Compound 2f: mp 256–258 °C; Rf = 0.60 (EtOAc/cyclo-
hexane, 70/30); H NMR (300 MHz, CDCl3): 3.43 (s, 2H,
1
50), 6.80 (s, 1H, H-9), 6.86 (s, 1H, H-6), 7.42 (d, 2H,
JAA XX ¼ 8:5 Hz, H-20 and H-60), 8.24 (br s, 1H, NH).
Anal. Calcd for C17H15N3O3: C, 66.01; H, 4.89; N, 13.58.
Found: C, 65.88; H, 4.96; N, 13.74.
CH2-5), 4.25–4.30 (m, 6H, OCH2CH2O and NH2), 6.76
(d, 1H, J0 = 8.2 Hz, H-50), 6.78 (s, 1H, H-9), 6.86 (s, 1H,
H-6), 7.33 (dd, 1H, J0 = 8.2 Hz, Jm = 2.0 Hz, H-60), 7.55
(d, 1H, Jm = 2.0 Hz, H-20), 8.26 (br s, 1H, NH). Anal.
Calcd for C17H14ClN3O3: C, 59.40; H, 4.10; N, 12.22.
Found: C, 59.54; H, 4.22; N, 12.05.
0
0
Compound 2b: mp 193–195 °C; Rf = 0.34 (CHCl3/EtOAc
50/50) 1H NMR (300 MHz, CDCl3): 2.90 (d, 3H,
J = 4.7 Hz, CH3), 3.50 (s, 2H, CH2), 4.19–4.31 (m, 6H,
0
0
OCH2CH2O and NH2), 6.68 (d, 2H, JAA XX ¼ 8:8 Hz, H-
9. (a) Chapman, A. G.; Croucher, M. J.; Meldrum, B. S.
Arzneim. Forsch. 1984, 34, 1261; (b) Engstrom, F. L.;
Woodbury, D. M. Epilepsia 1988, 29, 389.
10. Menniti, F. S.; Chenard, M. B.; Collins, M. F.; Ducat, M.
F.; Elliot, M. L.; Ewing, F. E.; Huang, J. I.; Kelly, K. A.;
Lazzaro, J. T.; Pagnozzi, M. J.; Weeks, J. L.; Welch, W.
M.; White, W. F. Mol. Pharmacol. 2000, 58, 1310.
11. Parks, T. N.; Artman, L. D.; Alasti, N.; Nemeth, E. F.
Brain Res. 1991, 552, 13.
20 and H-60), 6.82 (s, 1H, H-9), 6.89 (s, 1H, H-6), 7.58 (d,
2H, JAA XX ¼ 8:8 Hz, H-30 and H-50), 8.63 (br s, 1H, NH).
Anal. Calcd for C19H18N4O4: C, 62.29; H, 4.95; N, 15.29.
Found: C, 62.43; H, 4.80; N, 15.17.
0
0
Compound 2c: mp 185–187 °C; Rf = 0.37 (CHCl3/EtOAc
50/50) 1H NMR (300 MHz, CDCl3): 1.19 (t, 3H,
J = 7.1 Hz, CH3), 3.35 (m, 2H, CH2), 3.50 (m, 2H,
CH2), 3.96 (br s, 2H, NH2), 4.24–4.31 (m, 4H,
OCH2CH2O), 6.67 (d, 2H, JAA XX ¼ 8:5 Hz, H-30 and
12. Litchfield, J.; Wilcoxon, F. J. Pharmacol. Exp. Ther. 1949,
96, 99.
0
0
H-50), 6.82 (s, 1H, H-9), 6.89 (s, 1H, H-6), 7.57 (d, 2H,