Synthesis of 5-substituted 7,9-dihydro-8 H -[1,3]dioxolo[4,5-h][2,3]benzodiazepin-8-ones as anticonvulsant agents

5-(4-Aminobenzyl)-7,9-dihydro-8 H -[1,3]dioxolo[4,5-h][2,3]benzodiazepin-8-one ( 3 ) and 7,9- dihydro-5-[2-(pyridin-2-yl)-vinyl]-8 H -[1,3]dioxolo[4,5-h][2,3]benzodiazepin-8-one (4) were synthesized and screened as anticonvulsant agents in DBA/2 mice against sound-induced seizures. The new compounds are provided with anticonvulsant properties even if ED 50 values are lower than those of prototype 5-(4-aminophenyl)-7,9-dihydro-8 H -[1,3]dioxolo[4,5-h][2,3]benzodiazepin-8-one ( 2) and of GYKI 52466 ( 1 ), a well-known noncompetitive AMPA receptor antagonist. Binding assays and functional tests were performed to evaluate the affinity for AMPA and kainate receptors.


Introduction
Ionotropic glutamate receptors (iGluRs) are the major excitatory neurotransmitter receptor proteins in the mammalian brain. 1,2As a class of membrane proteins, iGluRs are composed of subunits that span the membrane to form a small pore or channel, which is gated by glutamate, a neurotransmitter.When glutamate is released from a presynaptic neuron and binds to a postsynaptic glutamate receptor, the receptor rapidly changes its conformation and transiently forms an open ion channel, thus resulting in a change of the postsynaptic membrane potential.A postsynaptic potential of sufficient strength triggers an action potential, which will in turn propagate the initial nerve impulse.The major function of iGluRs is to mediate fast synaptic neurotransmission underlying the basic activities of the brain, such as memory and learning.Overactivation of the receptors, on the other hand, has been implicated in a number of neurological diseases such as post-ischemia cell death, epilepsy, Huntington's chorea, and amyotrophic lateral sclerosis. 1To date, three major subtypes of iGluRs have been identified: NMDA (N-methyl-D-aspartate), kainate, and AMPA (2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionic acid) receptors, and classified on the basis of amino acid sequences, pharmacological profiles, and biological functions. 1,2-(4-Aminophenyl)-8-methyl-9H- [1,3]dioxolo [4,5-h][2,3]benzodiazepine (1, GYKI 52466, Figure 1), a 2,3-benzodiazepine derivative, is the prototype of selective noncompetitive AMPA receptor antagonists acting via an allosteric site on the receptor complex.3 It possesses remarkable anticonvulsant properties 4,5 and behaves as a neuroprotective agent in focal and global ischemia.6 Based on the anticonvulsant properties of the previously described 7,8 5-(4-aminophenyl)-7,9dihydro-8H- [1,3]dioxolo [4,5-h][2,3]benzodiazepin-8-one (2), where the iminohydrazone portion of 1 was replaced by the iminohydrazide moiety, we designed 5-(4-aminobenzyl)-7,9-dihydro-8H- [1,3] 1) in order to explore the influence of the substituent appended at position 1 on the anticonvulsant activity.In particular we inserted in such a position the 4-aminobenzyl group, in order to interrupt the electronic connection between the aryl group and the unsaturated heterocyclic ring, and the 2-(pyridin-2-yl)-vinyl moiety which seems to play a role of utmost importance in a strictly-related class of new noncompetitive AMPA receptor antagonists.9 Herein, we report the synthesis and the anticonvulsant properties of derivatives 3 and 4 along with the results of binding assays and functional tests performed at both AMPA and kainate receptors.Physical and spectral data ( 1 H NMR) of the synthesized compounds are in agreement with the proposed structures.In compound 4, the stereochemistry around the C=C bond was assigned as E, on the basis of the coupling constant value (J = 16.2Hz) between the two protons of the vinyl moiety.

Pharmacology
The anticonvulsant activity of derivatives 3 and 4 against audiogenic seizures was evaluated 30 min after intraperitoneal administration to DBA/2 mice, a strain genetically susceptible to soundinduced seizures.This test has been considered an excellent animal model for generalized epilepsy and for screening new anticonvulsant drugs. 10The results are compared with those previously reported for derivative 2 and reference compound 1 (Table 1). 7,8s shown in Table 1, compound 4 possesses anticonvulsant properties lower than those of prototype 2 as well of lead compound 1 whereas derivative 3, even if unable to prevent the clonic phase of the audiogenic seizures, significantly reduces the tonic phase of the seizures; its ED 50 value is comparable to that of GYKI 52466.For this reason, compound 3 was also examined for its ability to displace [ 3 H]CP-526,427 from the corresponding binding site of the AMPA receptor complex (Table 2).The inhibition of [ 3 H]CP-526,427 specific binding (3 nM) to rat forebrain membranes was evaluated as previously described. 11,12In this assay, the quinazolinone CP-465,022 was used as a positive control; its IC 50 values spanned the range 20-40 nM.Compound 3 turned out to be totally inactive (IC 50 >100 µM), differently to that observed 13 for compound 2 which showed IC 50 = 32 µM, a value similar to that reported for GYKI 52466 (IC 50 = 12.6 µM).
Furthermore, compound 3 was tested for its ability to inhibit the kainate-induced increase of the [Ca 2+ ]i in a primary culture of rat cerebellar granule cells (CGC) which express AMPA receptors (Table 2); GYKI 52466 was used as the control.The same 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.
The results of the CGC test confirmed the data of the binding experiments, namely compound 3 at 100 µM produced solely a 20% inhibition of calcium influx, whereas derivative 2 turned out to be slightly more potent than 1 (IC 50 13 µM for 2 vs 22 µM for 1) (Table 2).In the iGluR5/HEK293 cells test, compound 3, assayed up to 10 µM, showed neither agonistic nor antagonistic activity.To sum up, the results reported in this study suggest that the 4-aminophenyl group directly linked to the unsaturated heterocycle is a fragmental requirement necessary to confer anticonvulsant activity to compounds structurally related to derivative 2. The insertion of a methylene between the aryl group and the unsaturated heterocyclic ring to interrupt their electronic connection or an extension of their conjugation through the insertion of a vinyl moiety, in both cases, bring about a drastic reduction in the anticonvulsant activity.

Experimental Section
General Procedures.Melting points were determined on a Kofler hot stage apparatus and are uncorrected.Elemental analyses were carried out on a C. Erba Model 1106 Elemental Analyzer for C, H and N) and the results are within ±0.4% of the theoretical values.Merck silica gel 60 F 254 plates were used as analytical TLC; column chromatography was performed on Merck silica gel 60 (70-230 mesh).IR spectra were obtained on a Perkin Elmer Spectrum BX FT-IR as nujol mulls. 1 H-NMR spectra were recorded in CDCl 3 by means of a Varian Gemini 300 spectrometer.Chemical shifts are expressed in δ (ppm) relative to TMS as internal standard and coupling constants (J) in Hz.

Table 1 .
Anticonvulsant activity of compounds 1-4 against audiogenic seizures in DBA/2 mice and TD 50 values on locomotion assessed by rotarod test a a All compounds were given ip (at doses spanning the range 3.3-200 µmol/kg) 30 min before auditory stimulation.All data were calculated according to the method of Litchfield and Wilcoxon; 17 95% confidence limits are given in brackets.At least 32 animals were used to calculate each ED 50 and TD 50 value.