Synthesis and pharmacology of 3-hydroxy-delta2-isoxazoline-cyclopentane analogues of glutamic acid.

The synthesis and pharmacology of two potential glutamic acid receptor ligands are described. Preparation of the bicyclic 3-hydroxy-delta2-isoxazoline-cyclopentane derivatives (+/-)-7 and (+/-)-8 was accomplished via 1,3-dipolar cycloaddition of bromonitrile oxide to suitably protected 1-amino-cyclopent-3-enecarboxylic acids. Their structure was established using a combination of 1H NMR spectroscopy and molecular mechanics calculations carried out on the intermediate cycloadducts (+/-)-11 and (+/-)-12. Amino acid derivatives (+/-)-7 and (+/-)-8 were assayed at ionotropic and metabotropic glutamic acid receptor subtypes and their activity compared with that of trans-ACPD and cis-ACPD. The results show that the replacement of the omega-carboxylic group of the model compounds with the 3-hydroxy-delta2-isoxazoline moiety abolishes or reduces drastically the activity at the metabotropic glutamate receptors. Conversely, on passing from cis-ACPD to derivative (+/-)-8, the agonist activity at NMDA receptors is almost unaffected.

Synthesis and anticonvulsant activity of novel and potent 1-aryl-7,8-methylenedioxy-1,2,3,5-tetrahydro-4H-2,3-benzodiazepin-4-ones.

The synthesis and anticonvulsant activity of 1-aryl-7,8-methylenedioxy-1,2,3,5-tetrahydro-4H-2,3-benzodiazepin-4-(thi)ones (4a-d) and their 3-N-alkylcarbamoyl derivatives (4e-h) are reported. The new compounds possess marked anticonvulsant properties, comparable to those of the dehydro analogues 3 and higher than that of GYKI 52466 (1). Noteworthy, compound 4c shows a longer-lasting anticonvulsant activity. Electrophysiological experiments show that derivative 4c is less effective than 1 and 3c to reduce the KA-evoked currents in cerebellar granule neurons.

Synthesis and structure-activity relationships of 2,3-benzodiazepines as AMPA receptor antagonists.

There is increasing evidence of the potential therapeutic utility of glutamate receptor antagonists in the treatment of several neurodegenerative disorders, including stroke and epilepsy. In the last few years noncompetitive AMPA receptor antagonists have received considerable attention due to their therapeutic potentiality. The discovery of GYKI 52466, the prototype of noncompetitive AMPA receptor antagonists endowed with anticonvulsant and neuroprotective properties, induced growing interest on 2,3-benzodiazepine derivatives. This review covers the chemistry and pharmacology of this important class of AMPA receptor antagonists.

Pharmacological profile of enantiomerically pure chiral muscarinic agonists: desoxymuscarines.

The enantiomers desoxymuscarine 6 were tested in vitro on guinea pig tissues, and their muscarinic potency was evaluated at M2 (heart force and rate) and M3 (ileum and bladder) receptor subtypes together with the enantiomers of the parent compound muscarine 1. The eutomers (+)-1 and (+)-6 and distomers (-)-1 and (-)-6 were also assayed in vivo on pithed rat. Affinity, relative efficacy and enantio-selectivity were also determined for the compounds under study at M2 (heart force and rate) and M3 (ileum and bladder), in order to investigate muscarinic receptor heterogeneity. The results of this study have been discussed in comparison with the data previously reported for the structurally related fluoromuscarine (+)-4 and difluoromuscarines (+)-5 and (-)-5.

Synthesis and anticonvulsant activity of novel and potent 6,7-methylenedioxyphthalazin-1(2H)-ones.

In this paper, we describe the synthesis of a series of novel substituted 4-aryl-6,7-methylenedioxyphthalazin-1(2H)-ones. The anticonvulsant activity of these compounds against audiogenic seizures was evaluated in DBA/2 mice after intraperitoneal (ip) injection. Most of these derivatives are more active than 1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine (1, GYKI 52466), a well-known noncompetitive AMPA receptor antagonist. As deduced by the rotarod test, all the compounds exhibit a toxicity lower than that of 1. Within the series of derivatives submitted to investigation, 4-(4-aminophenyl)-2-butylcarbamoyl-6,7-methylenedioxyphthalazin -1(2H)-one (21) proved to be the most active compound and is 11-fold more potent than 1 (i.e., ED50 3.25 micromol/kg for 21 versus ED50 35.8 micromol/kg for 1). When compared to 1, compound 21 as well as its analogue 4-(4-aminophenyl)-6,7-methylenedioxyphthalazin-1(2H)-one (16) show a longer lasting anticonvulsant activity. Compound 21 also effectively suppresses seizures induced in Swiss mice by maximal electroshock (MES) and pentylenetetrazole (PTZ). Furthermore, it antagonizes in vivo seizures induced by 2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionic acid (AMPA), 2-amino-3-(3-hydroxy-5-tert-butyl-isoxazol-4-yl)propionic acid (ATPA), and kainate (KA), and its anticonvulsant activity is reversed by pretreatment with aniracetam. Using the patch-clamp technique, the capability of derivatives 16 and 21 to antagonize KA-evoked currents in primary cultures of granule neurons was tested. They behaved as antagonists, but they proved to be less effective than 1 and 1-(4-aminophenyl)-3,4-dihydro-4-methyl-3-N-methylcarbamoyl-7,8-met hylenedioxy-5H-2,3-benzodiazepine (2, GYKI 53655) to reduce the KA-evoked currents.

Design of new analogues of glutamic acid with a conformationally restricted structure.

Regioisomeric 3-carboxyisoxazolinyl prolines (CIP-A and CIP-B) and 3-hydroxyisoxazolinyl prolines [(+/-)-8 and (+/-)-9] were synthesized and assayed for glutamate receptor activity. CIP-A [(+/-)-6] showed a convulsant activity evaluated in vivo on DBA/2 mice, higher than AMPA and similar to kainic acid. The eutomer of CIP-A [CIP-AS, (-)-6], obtained from (S)-3,4-didehydroproline, evidenced common stereochemical requirements with AMPA and kainic acid.

Synthesis and pharmacological characterization of new chiral derivatives of muscarine and allo-muscarine.

Novel derivatives of natural muscarine and allo-muscarine, i.e. the benzyl ethers (-)-10 and (-)-12 and the benzoate (-)-13, were synthesized in very high enantiomeric excess. Target compounds were tested in vitro on guinea pig tissues, and their muscarinic potency was evaluated at M2 (heart force and rate) and M3 (ileum and bladder) receptor subtypes. The derivatives under study were also assayed in vivo on pithed rat. In addition, muscarinic receptor heterogeneity was investigated by determining the affinity and the relative efficacy of compounds (-)-10, (-)-12 and (-)-13 at M2 (heart force and rate) and M3 (ileum and bladder) receptor subtypes.

New analogues of oxotremorine and oxotremorine-M: estimation of their in vitro affinity and efficacy at muscarinic receptor subtypes.

Two subsets of tertiary amines (1a-6a) and methiodides (1b-6b) with a structural resemblance to oxotremorine and oxotremorine-M were tested at rabbit vas deferens (M1), guinea pig left atrium (M2), guinea pig ileum and urinary bladder (M3) muscarinic receptor subtypes. The pharmacological profile of the derivatives under study has been discussed by evaluating their potency, affinity and efficacy as well as the regional differences in muscarinic receptor occupancy.

Synthesis and anticonvulsant activity of novel and potent 2,3-benzodiazepine AMPA/kainate receptor antagonists.

We have previously shown that 1-aryl-3,5-dihydro-7, 8-methylenedioxy-4H-2,3-benzodiazepin-4-ones (3) possess marked anticonvulsant properties and antagonize seizures induced by 2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionic acid (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 noncompetitive AMPA/kainate receptor antagonist. We now report the synthesis of 3-(N-alkylcarbamoyl)-1-aryl-3,5-dihydro-7, 8-methylenedioxy-4H-2,3-benzodiazepin-4-ones (4a-h) and 1-aryl-3, 5-dihydro-7,8-methylenedioxy-4H-2,3-benzodiazepine-4-thiones (5a-c). The activity of all compounds, intraperitoneally (ip) injected, was evaluated against audiogenic seizures in DBA/2 mice and against seizures induced by maximal electroshock (MES) and pentylenetetrazole (PTZ) in Swiss mice. Some of the new compounds 4 and 5 showed remarkable anticonvulsant activity, and their toxicity, as evidenced by the rotarod test, is lower than that of 1. The time course of anticonvulsant activity of derivatives 4b and 5b,c was studied and compared to that of 1 and 3b,c. Compounds 4a,b and 5a-c antagonize seizures induced by AMPA and kainate (KA) and their anticonvulsant activity is reversed by pretreatment with aniracetam. Using the patch-clamp technique, the capability of derivatives 3c, 4b, and 5c to antagonize KA-evoked currents in primary cultures of granule neurons was tested and compared with that of the parent compounds 1 and 1-(4-aminophenyl)-3, 4-dihydro-4-methyl-3-methylcarbamoyl-7,8-methylenedioxy-5H-2, 3-benzodiazepine (2, GYKI 53655).

Synthesis and enantiopharmacology of new AMPA-kainate receptor agonists.

Regioisomeric 3-carboxyisoxazolinyl prolines [CIP-A (+/-)-6 and CIP-B (+/-)-7] and 3-hydroxyisoxazolinyl prolines [(+/-)-8 and (+/-)-9] were synthesized and assayed for glutamate receptor activity. The tests were carried out in vitro by means of receptor binding techniques, second messenger assays, and the rat cortical wedge preparation. CIP-A showed a good affinity for both 2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionic acid (AMPA) and kainic acid (KAIN) receptors. These results were confirmed in the cortical slice model where CIP-A displayed an EC(50) value very close to that of AMPA. The convulsant properties of all the compounds were evaluated in vivo on DBA/2 mice after icv injection. CIP-A showed a convulsant activity, measured as tonus and clonus seizures, 18-65 times higher than that produced by AMPA. It was also quite active after ip administration, since it induced seizures in mice at doses as low as 3.2 nmol/mouse. On the basis of the above-reported results we prepared and tested the enantiomers of CIP-A and CIP-B, obtained by reacting (S)-3,4-didehydroproline and (R)-3,4-didehydroproline, respectively, with ethoxycarbonylformonitrile oxide. In all the tests the S-form, CIP-AS [(-)-6], emerged as the eutomer evidencing common stereochemical requirements with the reference compounds AMPA and KAIN. Through modeling studies, carried out on CIP-A, AMPA, and KAIN, active conformations for CIP-AS and AMPA at AMPA receptors as well as for CIP-AS and KAIN at KAIN receptors are suggested.

Synthesis and functional characterization of novel derivatives related to oxotremorine and oxotremorine-M.

Two subseries of nonquaternized (5a-10a) and quaternized derivatives (5b-10b) related to oxotremorine and oxotremorine-M were synthesized and tested. The agonist potency at the muscarinic receptor subtypes of the new compounds was estimated in three classical in vitro functional assays: M1 rabbit vas deferens, M2 guinea pig left atrium and M3 guinea pig ileum. In addition, the occurrence of central muscarinic effects was evaluated as tremorigenic activity after intraperitoneal administration in mice. In in vitro tests a nonselective muscarinic activity was exhibited by all the derivatives with potencies values that, in some instances, surpassed those of the reference compounds (i.e. 8b). Functional selectivity was evidenced only for the oxotremorine-like derivative 9a, which behaved as a mixed M3-agonist/M1-antagonist (pD2 = 5.85; pA2 = 4.76, respectively). In in vivo tests non-quaternary compounds were able to evoke central muscarinic effects, with a potency order parallel to that observed in vitro.

Synthesis of new delta 2-isoxazoline derivatives and their pharmacological characterization as beta-adrenergic receptor antagonists.

A series of delta 2-isoxazoline derivatives structurally related to Broxaterol 1 and Falintolol 3 has been prepared and evaluated for their binding affinity to beta 1- and beta 2-adrenergic receptors. Among the tested compounds only the 3-isopropenyl anti derivative 4d is as active as the reference compounds. An electron-releasing group, probably operating through a pi-pi interaction, in the 3-position of the isoxazoline nucleus greatly enhances the affinity of the compounds. Conversely, the closest analogs of Broxaterol (3-bromo delta 2-isoxazolines 4a and 5a) are at least one order of magnitude less active than the model compound 1. Throughout the series of derivatives the anti stereoisomers are invariably more active than their syn counterparts.

Chemoenzymatic synthesis of chiral biologically active heterocycles.

The chemoenzymatic approach to the preparation of some chiral biologically active heterocycles is discussed. Synthetic strategies took advantage of enantioselective bioconversion processes carried out on suitable reaction intermediates. Reductions of carbonyl compounds catalyzed by different alcohol dehydogenases (TBADH from Thermoanaerobium brockii, 20 beta-HSDH from Streptomyces hydrogenans, beta-HSDH from Pseudomonas testosteroni) allowed the preparation with high enantiomeric purity of the eutomer of broxaterol (a selective beta 2-adrenergic agonist) and six out of the eight muscarine stereoisomers. On the other hand, hydrolyses, catalyzed by lipase PS (from Pseudomonas cepacia), of racemic butyrates were the key step in the synthesis of both the enantiomers of two muscarinic antagonists. Finally, the preparation of acetyl cycloserine antipodes was attained by means of a highly enantioselective hydrolysis catalyzed by lipase from Chromobacterium viscosum.

Synthesis and pharmacological characterization of enantiomerically pure muscarinic agonists: difluoromuscarines.

The four homochiral 4-deoxy-4,4-difluoromuscarine stereoisomers (difluoromuscarines) were prepared in very high enantiomeric excess. A convenient sequence based on the use of natural as well as "unnatural" ethyl lactate allowed the synthesis of target compounds, whose absolute configuration is dictated by that of the starting synthon. Quaternary ammonium salts (+)-5, (-)-5, (-)-6, and (+)-6 were tested in vitro on guinea pig tissues, and their muscarinic potency was evaluated at M2 (heart) and M3 (ileum and bladder) muscarinic receptor subtypes. The eutomer (+)-5 and distomer (-)-5 were also tested in vivo on pithed rat, and their muscarinic activity at the M1 receptor subtype was compared with those of racemic muscarine [(+/-)-1] and (2S,4R,5S)-4-deoxy-4-fluoromuscarine [(+)-4]. Further pharmacological parameters such as affinity, relative efficacy, and enantioselectivity have been determined for compounds (+)-5 and (-)-5 at M2 (heart force and rate) and M3 (ileum and bladder) receptors in order to investigate muscarinic receptor heterogeneity. The four homochiral difluoromuscarines behave as muscarinic agonists in all the tests with a potency trend which is different from that previously observed with the 4-deoxy-4-fluoromuscarines and (+/-)-1, thus indicating the intervention of the second fluorine atom on the receptor-ligand interaction. Moreover, the second fluorine atom produces significant differences in the affinity and relative efficacy values of compounds (+)-5 and (-)-5 at M2 and M3 subtypes, which could be attributed to a heterogeneity between the muscarinic receptors mediating heart rate and heart force and those involved in the contraction of ileum and bladder.

Synthesis and pharmacological investigation of new chiral muscarinic antagonists.

The two pairs of enantiomers of isoxazolidin-3-ones 3 and 4 were synthesized by means of Lipase PS-catalyzed hydrolyses of suitable racemic butyrates. The same butyrates were also employed as key intermediates in the preparation of racemic 3 and 4. The antimuscarinic potency of the new compounds was assayed in two in vitro functional tests. The eutomers (-)-3 and (-)-4 share the same stereochemistry (5R) of the most potent enantiomer of "azamuscarone" 2, a structurally related muscarinic agonist. Such a spatial arrangement around the chiral center of 2-4, coupled with the low values of eudismic ratio, represents an anomaly among the chiral muscarinic ligands. This anomaly was accounted for by the absence of a chiral center at C-2, a position whose configuration is crucial in determining the high enantioselectivity of muscarinic agonists and antagonists.

Synthesis of a metabolite of metoclopramide and its detection in human urine.

The synthesis of 2-(4-amino-5-chloro-2-methoxybenzamido)acetic acid 2, a metabolite of metoclopramide 1, has been accomplished through the coupling of 4-amino-5-chloro-2-methoxybenzoic acid 4 with glycine benzyl ester followed by a catalytic hydrogenation. Such a metabolite could not be detected directly in the human urines but only after its transformation into the corresponding methyl ester 6. Compound 6 was prepared both by condensing acid 4 with glycine methyl ester and by reacting acid 2 with diazomethane. HPLC analyses of biological samples were therefore performed after treatment with diazomethane. In five healthy volunteers, the percentage of 6 spanned the range 0.6-1.2%.

Synthesis and muscarinic activity of some muscarone analogs.

The synthesis of some muscarone analogs has been accomplished and their muscarinic activity has been assayed in three in vitro functional tests. The results of such an investigation put in evidence the active role played by the methylene moiety, placed exocyclic to the pentaatomic ring, on the interaction of the ligands with the different muscarinic receptor subtypes. Worth noting is the ileum-atria selectivity displayed by methylene derivative 7.

Synthesis and muscarinic activity of the chiral forms of methylenemuscarones.

The synthesis of the two pairs of enantiomers of methylenemuscarones 3 and 4 has been accomplished by using (R)- and (S)-lactic esters as starting materials. Due to the existence of different muscarinic receptor subtypes, the compounds were examined for their ability to bind membranes from cerebral cortex (M1), heart (M2), and salivary glands (M3) and were assayed in "in vitro" functional tests as well. The results of such an investigation put in evidence that, in both binding and functional tests, (-)-3 (2S,5S) and (-)-4 (2R,5S) were the eutomers and shared the stereochemistry of the eutomer of muscarone and allomuscarone respectively. It is worth noting that the distomer of 3 and 4 behaves as a partial agonist in the cardiac tissue and as a full agonist in the other preparations. This peculiarity of the chiral forms of 3 and 4 could account for the remarkable selectivity for the ileum observed in the corresponding racemates.