Determination of a potent non-competitive AMPA receptor antagonist in rat brain.

N-acetyl-1-(p-chlorophenyl)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline derivative (PS3Ac) has been determined in brain tissues by high performance liquid chromatography (HPLC) coupled with a diode array detection. In a previous paper we presented a validation method for detecting PS3Ac and its metabolites in plasma samples after intraperitoneal administration to Wistar rats. In the present paper, we report the results of the determination of PS3Ac and its N-deacetyl (PS3) and O-demethyl (PS3OH) metabolites, in the brain after extraction based on a polymeric matrix with a high hydrophilic-lipophilic balance, using Oasis cartridges. The chromatographic separation was performed in an octadecylsilica stationary phase at 25 degrees C using a mixture of 10 mM potassium dihydrogen orthophosphate (pH 2.24) and acetonitrile in ratio of 30:70 (v/v) as mobile phase, with a flow rate of 0.8 ml/min. The method exhibited a large linear range from 0.05 to 2 microg/ml for all studied compounds (n=6). In the within-day assay (n=4), the accuracy ranged from 87.5% determined with 0.05 microg/ml of PS3 to 110.1% determined with 0.2 microg/ml of PS3OH. In the between-day assay the coefficient of variation ranged from 2.4 determined with 0.05 microg/ml of PS3 to 9.7 determined with 0.2 microg/ml of PS3OH. The extraction efficiency ranged from 77.8% for PS3OH at 0.2 microg/ml to 94.3 for PS3Ac at 0.5 microg/ml. The limit of detection for all the tetrahydroisoquinoline derivatives ranged around 50 ng/ml. The method proved to be highly sensitive and specific to determinate PS3Ac and its metabolites and has been successfully applied to value their concentrations in brain matrix over the time.

SPE-HPLC determination of new tetrahydroisoquinoline derivatives in rat plasma.

Recently a novel class of non-competitive AMPA receptor (AMPAR) antagonists, such as, N-acetyl-1-(p-chlorophenyl)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline (PS3Ac) have been developed using molecular modeling studies. In this study we present a validated method for detecting PS3Ac in biological matrices by high performance liquid chromatography with ultraviolet detection. In this study PS3Ac was administered to Wistar rats. After intraperitoneal administration, the plasma concentrations of PS3Ac and its potential metabolic products, i.e., PS3OH, PS3 and PS3OHAc were determined. Serum samples (0.5 ml) were purified by solid-phase extraction of analytes using Oasis cartridges. The chromatographic separation was performed on a LiChrosorb RP-1 at 30 degrees C. The eluent was made of potassium dihydrogen phosphate/acetonitrile in ratio of 50:50 (v/v); the flow rate was 1 ml/min. The detection was performed at 220 nm. The method exhibited a large linear range from 0.05 to 5 microg/ml for all studied compounds. The intra-assay accuracy ranged from 92% determined at 0.1 microg/ml of PS3OH, to 108% determined at 0.05 microg/ml of PS3OHAc. The average coefficient of variation of inter-assay was 6.27%. The average recovery from plasma was 78.5%. The limits of quantification for all the tetrahydroisoquinoline derivatives was 20 ng. The method proved to be highly sensitive and specific for the determination of the studied compounds in rat plasma and has been successfully applied to the evaluation of the pharmacokinetic profile of the inoculated compound.

4,5-Dihydro-7,8-dimethoxy-1-phenyl-3H-2,3-benzodiazepin-4-one.

The title compound, C(17)H(16)N(2)O(3), is an antagonist for AMPA/kainate receptors. The molecule has its seven-membered oxadiazole ring in a boat conformation. Asymmetry of the two methoxy bond angles is evident, with (Me)O-C-C angles of 115.45 (12) and 124.78 (13) degrees, and 114.67 (12) and 125.31 (12) degrees. A centrosymmetric dimer involving the HN-CO moieties, with an N...O distance of 2.876 (2) A, graph set R(2)(2)(8), is further linked into chains through methoxy Csp(3)-H...N hydrogen bonds, with a C...N distance of 3.418 (2) A.

Synthesis and anticonvulsant properties of 2,3,3a,4-tetrahydro-1H-pyrrolo[1,2-a]benzimidazol-1-one derivatives.

A number of novel 1H-pyrrolo[1,2-a]benzimidazol-1-one derivatives were prepared and their anticonvulsant properties evaluated. The new synthesized compounds proved to possess anticonvulsant effects depending on the nature of substituents at C-6, C-2, and C-3a positions of the polycyclic system. In particular, the 6-chloro-3a-(p-tolyl)-2,3,3a,4-tetrahydro-1H-pyrrolo[1,2-a]benzimidazol-1-one derivative (22) displayed potency fivefold higher than unsubstituted compound (13).

Synthesis and evaluation of pharmacological and pharmacokinetic properties of 11H-[1,2,4]triazolo[4,5-c][2,3]benzodiazepin-3(2H)-ones.

A series of 2,3-benzodiazepine derivatives has been previously described as noncompetitive AMPA-type glutamate receptor antagonists potentially useful for treatment of epilepsy. To further explore the structure-activity relationships of AMPA antagonists, a series of 11H-[1,2,4]triazolo[4,5-c][2,3]benzodiazepin-3(2H)-ones (6) was synthesized starting from the corresponding bicyclic 1-aryl-3, 5-dihydro-7,8-dimethoxy-4H-2,3-benzodiazepin-4-ones (2, CFM). The new compounds were found to possess anticonvulsant effects against seizures induced both by means of auditory stimulation in DBA/2 mice and by pentylenetetrazole or maximal electroshock in Swiss mice. In addition, they antagonize the AMPA-induced seizures, and their anticonvulsant activity is reversed by pretreatment with aniracetam, thus suggesting the involvement of AMPA receptors. The pharmacological studies revealed that the 11H-[1,2,4]triazolo[4, 5-c][2,3]benzodiazepin-3(2H)-ones (6) herein reported show anticonvulsant activity comparable to that of their bicyclic precursors. Furthermore, an HPLC study put in evidence that these tricyclic derivatives 6 were converted in vivo into the corresponding 2, the agents likely to be mainly responsible for the anticonvulsant properties observed.

Anticonvulsant activity and plasma level of 2,3-benzodiazepin-4-ones (CFMs) in genetically epilepsy-prone rats.

Anticonvulsant properties of some 2,3-benzodiazepine derivatives acting as alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA) antagonists have been examined in vivo in the genetically epilepsy-prone rats using an audiogenic seizures assay. 2,3-Benzodiazepin-4-ones (CFMs) are nonselective AMPA antagonists that have been found to be potent anticonvulsant compound is in acute models of epilepsy. Because very little is known about their actions in a chronic model of epilepsy, and no correlations exist between anticonvulsant potency and plasma levels of these derivatives, we planned to investigate such a relationship. Maximal anticonvulsant protection occurred 15-60 min after the IP administration of GYKI 52466, 30-90 min after CFM-2, and 45-120 min after CFM-3. In addition, maximal anticonvulsant effect was observed 60-120 min after the IP administration of CFM-4 and at 90 min after CFM-5. The therapeutic index revealed that GYKI 52466 was slightly more toxic than CFM-2 and CFM-3. The time course of plasma levels of rats treated showed that peak plasma concentration was observed 45 min after IP administration of CFM-2 and CFM-3 and 75 min after CFM-4 and CFM-5. Following IP administration of CFM-3 two curves were detected, one is referred to the injected compound, and the other to its demethylated metabolite, which corresponds to CFM-2. Also. for the nitroderivative CFM-4 two curves were detected: one of an injected compound and the second due to its reduced metabolite (CFM-2). Finally, three different metabolites were detected in rat plasma after IP administration of CFM-5. The present study demonstrated that CFMs showed a significant protection against auditory stimulation during the period of peak plasma concentrations, suggesting a marked inhibition of those brain structures involved in the initiation and/or spreading of the audiogenic seizures.

Determination of 2,3-benzodiazepine derivatives in rat plasma by high-performance liquid chromatography.

A simple high-performance liquid chromatographic method with ultraviolet detection at 240 nm for determination of a novel AMPA/kainate antagonist 1-(4'-aminophenyl)-3,5-dihydro-7,8-dimethoxy-2,3-benzodiazepine (2,3-BZ 6), and its derivatives in rat plasma is described. The procedure involves a fast extraction of the drugs from the plasma spiked with an internal standard. The samples are applied to a pre-packed glass column and drugs are eluted using ethyl acetate. A linear response was observed over the examined concentration range. The lower limit of detection of 2,3-BZ 6 was 5.5 ng/ml. The assay has been used to determine the time course of plasma levels of the 2,3-benzodiazepine derivatives in Sprague-Dawley rats.

Synthesis and anticonvulsant activity of new 2,3-benzodiazepines as AMPA receptor antagonists.

Novel 1-aryl-3,5-dihydro-7,8-methylenedioxy-4H-2,3-benzodiazepine-4-ones (12a-j) were prepared and their anticonvulsant effects were evaluated by using various models of experimental epilepsy. The seizures were evoked both by means of auditory stimulation in DBA/2 mice and by pentylenetetrazole or maximal electroshock in Swiss mice. Some of these compounds possess marked anticonvulsant properties in all tests employed. Compounds 12 antagonise seizures induced by AMPA in analogy to the structurally-related 1-(4'-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3- benzodiazepine (1) (GYKI 52466), a well-known non-competitive AMPA-receptor antagonist. On the other hand, these novel 2,3-benzodiazepines exhibit anticonvulsant properties that are not affected by flumazenil, but are reversed by aniracetam. In addition, when compared to model compound 1, compounds 12 show a longer-lasting anticonvulsant activity and a lower toxicity. A structure-activity relationship study carried out on compounds 12 as well as analogous 7,8-dimethoxy derivatives 2 offers an approach for designing more potent agents.

Relationship between anticonvulsant activity and plasma level of some 2,3-benzodiazepines in genetically epilepsy-prone rats.

The anticonvulsant effects of some novel 2,3-benzodiazepines acting as alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid/kainate (AMPA/KA) antagonists were evaluated in genetically epilepsy prone rats. The ED50 values against clonic and tonic seizures (in micromol/kg) revealed that the rank order of anticonvulsant activity was: GYKI 52466 > 2,3BZ-2 > 2,3 MBZ-2 > NBQX. Maximal anticonvulsant protection was observed 15-45 min after the i.p. administration of NBQX and GYKI 52466, 30-90 min after the i.p. administration of 2,3BZ-2, and 45-120 min after the i.p. administration of 2,3MBZ-2. The time course of plasma levels of rats treated with GYKI 52466 showed that peak plasma concentration was observed 15 min after i.p. administration, 2,3BZ-2 revealed that peak plasma concentration was achieved 45 min after i.p. administration, whereas following 2,3MBZ-2 administered i.p., two curves were detected; one is referred to the parent compound and the other to its demethylate metabolite that corresponds to 2,3BZ-2. The therapeutic index (ratio of TD50 values for impaired rotarod performance and ED50 values for anticonvulsant activity) revealed that NBQX and GYKI 52466 were slighly more toxic than 2,3BZ-2 and 2,3MBZ-2. The present data suggest that 2,3-benzodiazepines acting at AMPA/kainate receptors play an important role in the generation and/or propagation of the audiogenic seizures in genetically epilepsy-prone rats.

3,5-Dihydro-4H-2,3-benzodiazepine-4-thiones: a new class of AMPA receptor antagonists.

Synthesis and evaluation of anticonvulsant activity of a series of 2,3-benzodiazepin-4-ones (2) chemically related to 1-(4'-aminophenyl)-4-methyl-7,8-(methylenedioxy)-5H-2,3-benzodiazepine (1, GYKI 52466) have been reported in our recent publications. Compounds 2 manifested marked anticonvulsant properties acting as 2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionic acid (AMPA) receptor antagonists. In an attempt to better define the structure-activity relationships (SAR) and to obtain more potent and selective anticonvulsant agents, 1-aryl-3,5-dihydro-4H-2, 3-benzodiazepine-4-thiones 3 were synthesized from the corresponding isosteres 2. The evaluation is reported of their anticonvulsant effects, both in the audiogenic seizures test with DBA/2 mice and against the maximal electroshock- and pentylenetetrazole-induced seizures in Swiss mice. New derivatives 3 showed higher potency, less toxicity and longer-lasting anticonvulsant action than those of the parent compounds 2 in all tests employed. Analogous to derivatives 2, new compounds 3 do not affect the benzodiazepine receptor (BZR) while they do antagonize AMPA-induced seizures; their anticonvulsant activity is reversed by pretreatment with aniracetam but not with flumazenil, thus suggesting a clear involvement of AMPA receptors. Electrophysiological data indicate a noncompetitive blocking mechanism at the AMPA receptor sites for 3i, the most active of the series and over 5-fold more potent than 1.

High-performance liquid chromatographic determination of new 2,3-benzodiazepines.

A simple high-performance liquid chromatographic assay with ultraviolet detection at 254 nm for simultaneous determination of 2,3-benzodiazepine derivatives (2,3-BZ2 and 2,3-BZ2Me) and their metabolites in rat plasma is described. The procedure involves a fast extraction of the drugs from the buffered sample using methanol. The extract is evaporated to dryness at 45 degrees C and the residue is redissolved in methanol (twice). A 20-microl aliquot is injected into the liquid chromatograph and eluted with methanol-water (65:35, v/v) on a C18 reversed-phase column. At a flow-rate of 1.5 ml/min the detection time was 3.1 min for 2,3-BZ2, 5.06 min for 2,3-BZ2Me and 10.9 min for prazepam, used as internal standard for the quantification of the studied compounds. The method has been used to investigate the steady-state concentrations of two 2,3-benzodiazepine derivatives in Sprague-Dawley rat plasma.

7,8-Methylenedioxy-4H-2,3-benzodiazepin-4-ones as novel AMPA receptor antagonists.

The synthesis and anticonvulsant activity of novel 7,8-methylenedioxy-4H-2,3-benzodiazepin-4-ones 3a-e, structurally-related to GYKI 52466 1, a well-known noncompetitive AMPA-receptor antagonist, are reported. The new compounds possess marked anticonvulsant properties and, in analogy to 1, antagonize seizures induced by AMPA. In addition, when compared to the model compound 1, compounds 3 show a longer-lasting anticonvulsant activity and a lower toxicity.

1-Aryl-3,5-dihydro-4H-2,3-benzodiazepin-4-ones: novel AMPA receptor antagonists.

Our previous publication (Eur. J. Pharmacol. 1995, 294, 411-422) reported preliminary chemical and biological studies of some 2,3-benzodiazepines, analogues of 1-(4-aminophenyl)-4-methyl-7,8-(methylenedioxy)-5H-2,3-benzodiazepine (1, GYKI 52466), which have been shown to possess significant anticonvulsant activity. This paper describes the synthesis of new 1-aryl-3,5-dihydro-4H-2,3-benzodiazepin-4-ones and the evaluation of their anticonvulsant effects. The observed findings extend the structure-activity relationships previously suggested for this class of anticonvulsants. The seizures were evoked both by means of auditory stimulation in DBA/2 mice and by pentylenetetrazole or maximal electroshock in Swiss mice. 1-(4'-Aminophenyl)- (38) and 1-(3'-aminophenyl)-3,5-dihydro-7,8-dimethoxy-4H-2,3-benzodiazepin- 4-one (39), the most active compounds of the series, proved to be more potent than 1 in all tests employed. In particular, the ED50 values against tonus evoked by auditory stimulation were 12.6 micromol/kg for derivative 38, 18.3 micromol/kg for 39, and 25.3 micromol/kg for 1. Higher doses were necessary to block tonic extension induced both by maximal electroshock and by pentylenetetrazole. In addition these compounds exhibited anticonvulsant properties that were longer lasting than those of compound 1 and were less toxic. The novel 2,3-benzodiazepines were also investigated for a possible correlation between their anticonvulsant activities against convulsions induced by 2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionic acid (AMPA) and their affinities for benzodiazepine receptors (BZR). The 2,3-benzodiazepines did not affect the binding of [3H]flumazenil to BZR, and conversely, their anticonvulsant effects were not reversed by flumazenil. On the other hand the 2,3-benzodiazepines antagonized seizures induced by AMPA and aniracetam in agreement with an involvement of the AMPA receptor. In addition, both the derivative 38 and the compound 1 markedly reduced the AMPA receptor-mediated membrane currents in guinea-pig olfactory cortical neurons in vitro in a noncompetitive manner. The derivatives 25 and 38-40 failed to displace specific ligands from N-methyl-D-aspartate (NMDA), AMPA/kainate, or metabotropic glutamate receptors.

1,4-Benzodiazepine derivatives as anticonvulsant agents in DBA/2 mice.

1. The behavioural and anticonvulsant effects of 10 1,4-benzodiazepine derivatives were studied after intraperitoneal administration in DBA/2 mice, a strain genetically susceptible to sound-induced seizures. 2. The anticonvulsant effects were evaluated on seizures evoked by means of auditory stimulation (109 dB, 12-16 kHz) in animals placed singly under a perspex dome. The rank order of potency for anticonvulsant activity was alprazolam > clonazepam > flunitrazepam > diazepam > pinazepam > desmethyldiazepam > oxazepam > prazepam > halazepam > camazepam. 3. The impairment of locomotor performance following IP administration of the above reported derivatives was also evaluated by means of the rotarod test. 4. Hypothermic activity was observed after the highest doses of the benzodiazepines studied. 5. The potency of various 1,4-benzodiazepines as inhibitors of specific [3H] flumazenil binding to membranes from cerebellum or cortex was evaluated. In general, 1,4-benzodiazepines were active as anticonvulsants at micromolar range and inhibited [3H] flumazenil binding at nanomolar range. 6. The different degree of anticonvulsant activity and impairment of coordinated motor movements cannot be directly related to the benzodiazepine binding affinity or to the lipophilicity of the compounds studied.

GYKI 52466 and related 2,3-benzodiazepines as anticonvulsant agents in DBA/2 mice.

The behavioural and anticonvulsant effects of several 1-aryl-3,5-dihydro-4H-2,3-benzodiazepin-4-ones (2,3-BZs) and of 11b-aryl-7,11-dihydro-3-phenyl[1,2,4]oxadiazolo[5,4-a][2,3]benz odiazepin-6-ones (2,3-OBZs) were studied after intraperitoneal (i.p.) administration in DBA/2 mice, a strain genetically susceptible to sound-induced seizures. The seizures were evoked by means of auditory stimulation (109 dB, 12-16 kHz) in animals placed singly under a hemispheric Perspex dome. The 2,3-benzodiazepines studied after 30 min pretreatment were generally less potent than the related derivative 1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine hydrochloride (GYKI 52466) except 3,5-dihydro-7,8-dimethoxy-1-phenyl-4H-2,3-benzodiazepin-4-one (2,3-BZ-2) and 2,3-BZ-2M (3-methyl derivative of 2,3-BZ-2) which showed comparable activity. Thirty minutes after i.p. administration of 2,3-benzodiazepines, the rank order of potency for anticonvulsant activity against clonus was 2,3-BZ-2 > GYKI 52466 > 2,3-BZ-2M > 2,3-BZ-1 > 2,3-BZ-3, > 2,3-OBZ-1, > 2,3-OBZ-2 2,3-OBZ-3. The intracerebroventricular (i.c.v.) injection of aniracetam on it own (12.5 - 100 nmol/mouse) had no convulsant activity, but it reversed the anticonvulsant effects of some 2,3-benzodiazepines. In particular, the pharmacological actions of GYKI 52466, 2,3-BZ-2 and 2,3-BZ-2M, which proved to be the most potent 2,3-benzodiazepine derivatives as anticonvulsants, were significantly reduced by an i.c.v. pretreatment with aniracetam (50 nmol/mouse). Concomitant treatment with aniracetam (50 nmol/mouse) shifted to the right the dose-response curves and significantly increased the ED50 values for GYKI 52466, 2,3-BZ-2 and 2,3-BZ-2M. After 30 min pretreatment 2,3-BZ-2 showed a similar potency to GYKI 52466 in antagonizing seizures induced by i.c.v. administration of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), thus suggesting a clear involvement of AMPA receptors in the anticonvulsant activity of these compounds. In addition, 2,3-BZ-2 and 2,3-BZ-2M showed anticonvulsant properties longer lasting than GYKI 52466.

Synthesis and antitumor activity evaluation of 1-[(arylidene)amino]adamantanes.

Several 1-[(arylidene)amino]adamantanes were synthesized and evaluated in vitro as antitumor agents against 60 human tumor cell lines. Some of these derivatives showed specificity for human leukemia cell lines and 1-[(2-bromobenzylidene)amino]adamantane (2) (NSC 631076-L), the most active compound, was selected by the Biological Evaluation Committee (BEC), for in vivo testing.

Structure-activity relationships in thienodiazepine and benzodiazepine derivatives.

The structural and electronic characteristics as well as the anticonvulsant properties and benzodiazepine receptor binding of thieno[3,4-b][1,4]diazepine and 1,5-benzodiazepine derivatives are compared and discussed. The data obtained suggest that the electronic rather than the structural properties appear mainly responsible for the variant degree of anticonvulsant activity exhibited by the tested compounds.