Benzodiazepine receptor ligands. Synthesis and pharmacological evaluation of 6-aryl-3,8-disubstituted-1,2,4-triazolo[3,4-a]phthalazines

A series of 6-aryl-3,8-disubstituted-1,2,4-triazolo[3,4-a]phthalazines was synthesised starting from suitable o-benzoylbenzoic acids. The compounds were tested in vitro for inhibition of the specific binding of 3H-Diazepam to benzodiazepine receptors in preparations of membranes from rat brain synaptosomes. None of the compounds was notably active in this test.     

Benzodiazepine receptor ligands. Synthesis and in vitro binding activity of some 6-substituted-3-aryl-1,2,4-triazolo[3,4-a]phthalazines

Some 3-aryl-6-hydroxymethyl-1,2,4-triazolo[3,4-a]phthalazines (VI a,b) and 3-aryl-6-(2-hydroxyethyl)-1,2,4-triazolo[3,4-a]phthalazines (XVIII) have been synthesized. From (VI b) some 3-aryl-6-(alkylamino)methyl- and 3-aryl-6-(alkoxy)methyl-1,2,4-triazolo[3,4-a]phthalazines have been prepared and evaluated in vitro for their ability to inhibit selective 3H-diazepam (3H-DZ) binding to benzodiazepine-receptors (BZRs) from homogenates of synaptosomes from rat brains. Several compounds have been shown to displace 3H-DZ from BZRs with Ki (nM) values ranging from 2.2 to 88, comparable to those of the reference drugs including Diazepam.     

Benzodiazepine receptor ligands. Synthesis and preliminary pharmacological evaluation of some 3-aryl-6-thioalkyl-, 3-aryl-6-alkylsulphinyl-, 3-aryl-6-alkylsulphonyl-, and 3-aryl-6-alkoxy-1,2,4-triazolo[3,4-a]phthalazines

A series of 3-aryl-6-alkoxy- and some 3-aryl-6-thioalkyl-, 3-aryl-6-alkylsulphinyl-, and 3-aryl-6-alkylsulphonyl-1,2,4-triazolo[3,4-a]phthalazines were synthesised and tested for inhibition of the in vitro binding of 3H-Diazepam to benzodiazepine receptors in membranes isolated from rat brain synaptosomes. 6-Alkoxy-3-(4'-methoxy)phenyl-1,2,4-triazolo[3,4-a]phthalazines were more active than or as active as diazepam in the binding assay (Ki nM) but unlike diazepam their binding to the benzodiazepine receptors was not enhanced by 4-aminobutyric acid. These compounds did not antagonize pentylenetetrazole induced convulsions and were inactive in modifying the conditioned behaviour of rats. Compound (II a) counteracted the muscle relaxant effects of diazepam (traction test). These results suggest that (II a) may be a benzodiazepine receptor antagonist.     

3-phenyl-6-(2-pyridyl)methyloxy-1,2,4-triazolo[3,4-a]phthalazines and analogues: high-affinity gamma-aminobutyric acid-A benzodiazepine receptor ligands with alpha 2, alpha 3, and alpha 5-subtype binding selectivity over alpha 1

Studies with our screening lead 5 and the literature compound 6 led to the identification of 6-benzyloxy-3-(4-methoxy)phenyl-1,2,4-triazolo[3,4-a]phthalazine 8 as a ligand with binding selectivity for the gamma-aminobutyric acid-A (GABA-A) alpha 3- and alpha 5-containing receptor subtypes over the GABA-A alpha 1 subtype (K(i): alpha 2 = 850 nM, alpha 3 = 170 nM, alpha 5 = 72 nM, alpha 1 = 1400 nM). Early optimization studies identified the close analogue 10 (K(i): alpha 2 = 16 nM, alpha 3 = 41 nM, alpha 5 = 38 nM, alpha 1 = 280 nM) as a suitable lead for further study. High-affinity ligands were identified by replacing the 6-benzyloxy group of compound 10 with 2-pyridylmethoxy (compound 29), but binding selectivity was not enhanced (K(i): alpha 2 = 1.7 nM, alpha 3 = 0.71 nM, alpha 5 = 0.33 nM, alpha 1 = 2.7 nM). Furthermore, on evaluation in xenopus oocytes,(22) 29 was discovered to be a weak to moderate inverse agonist at all four receptor subtypes (alpha 1, -7%; alpha 2, -5%; alpha 3, -16%; alpha 5, -5%). Replacement of the 3-phenyl group of 29 with alternatives led to reduced affinity, and smaller 3-substituents led to reduced efficacy. Methyl substitution of the benzo-fused ring of 29 at the 7-, 8-, and 10-positions resulted in increased efficacy although selectivity was abolished. Increased efficacy and retention of selectivity for alpha 3 over alpha 1 was achieved with the 7,8,9,10-tetrahydro-(7,10-ethano)-phthalazine 62. Compound 62 is currently one of the most binding selective GABA-A alpha 3-benzodiazepine-site partial agonists known, and although its selectivity is limited, its good pharmacokinetic profile in the rat (33% oral bioavailability after a 3 mg/kg dose, reaching a peak plasma concentration of 179 ng/mL; half-life of 1 h) made it a useful pharmacological tool to explore the effect of a GABA-A alpha 2/alpha 3 agonist in vivo.     

Benzodiazepine receptor ligands. Synthesis and preliminary pharmacological evaluation of 2-aminoalkyl-8-chloro- and 2-aryl-1,2,4-triazolo[3,4-a]phthalazine-3(2H)-ones

Some 2-aminoalkyl-8-chloro- and 2-aryl-1,2,4-triazolo[3,4-a]-phthalazine-3-(2H)-ones were synthesized and preliminarily tested in vitro and in vivo as potential benzodiazepine-receptor (BZRs) ligands. 2-Aryl-1,2,4-triazolo[3,4-a]-phthalazine-3(2H)-ones displaced in vitro 3H-diazepam (3H-DZ) from rat brain specific binding sites with Ki (nM) comparable to DZ and chlordiazepoxide used as reference compounds. The specific binding of the triazolones of this study was not enhanced in vitro by 4-aminobutyric acid (GABA) and in vivo they did not show any activity in counteracting the pentylenetetrazole (PTZ) induced convulsions (mice). One of these compounds (IV a) antagonized the effects of DZ in the bicuculline (BIC) induced convulsions test (mice) and the DZ induced muscle relaxant effects in the horizontal wire test.     

Discovery of functionally selective 7,8,9,10-tetrahydro-7,10-ethano-1,2,4-triazolo[3,4-a]phthalazines as GABA A receptor agonists at the alpha3 subunit

We have previously identified the 7,8,9,10-tetrahydro-7,10-ethano-1,2,4-triazolo[3,4-a]phthalazine (1) as a potent partial agonist for the alpha(3) receptor subtype with 5-fold selectivity in binding affinity over alpha(1). This paper describes a detailed investigation of the substituents on this core structure at both the 3- and 6-positions. Despite evaluating a wide range of groups, the maximum selectivity that could be achieved in terms of affinity for the alpha(3) subtype over the alpha(1) subtype was 12-fold (for 57). Although most analogues showed no selectivity in terms of efficacy, some did show partial agonism at alpha(1) and antagonism at alpha(3) (e.g., 25 and 75). However, two analogues tested (93 and 96), both with triazole substituents in the 6-position, showed significantly higher efficacy for the alpha(3) subtype over the alpha(1) subtype. This was the first indication that selectivity in efficacy in the required direction could be achieved in this series.     

Benzodiazepine receptor ligands. Synthesis and preliminary pharmacological evaluation of some 3,7-disubstituted-4H-[1,2,4]triazino[3,4-a]phthalazines and 3,7-disubstituted-4H-[1,2,4]triazino[3,4-a]phthalazine-4-ones

Some disubstituted triazinophthalazines and traizinophthalazinones were synthesized and tested in vitro for inhibition of the 3H-diazepam (3H-DZ) specific binding to benzodiazepine receptors (BZRs) in membranes from synaptosomes of rat brain and in vivo for their effects on the conditioned behaviour (rat). These compounds showed Ki values (nM) in the in vitro test ranging from 220 to more than 3000 and some of them had approximate ED50S of 30 mg/kg, i.p. (rat) in the conditioned avoidance behaviour test (CR2).     

3-Aryl-[1,2,4]triazino[4,3-a]benzimidazol-4(10H)-ones: tricyclic heteroaromatic derivatives as a new class of benzodiazepine receptor ligands

A series of 3-substituted [1,2,4]triazino[4,3-c]benzimidazoles V were prepared and tested at the central benzodiazepine receptor (BzR). These compounds were designed as rigid analogues of the previously described N-benzylindolylglyoxylylamide derivatives IV. The title compounds V showed an affinity which depended directly on the presence of the N(10)-H group and an aromatic ring at position 3. Some of them elicited a 2- or 3-fold higher affinity with respect to that of the indolylglyoxylylamide derivatives IV (R = H). The GABA ratio and [(35)S]-tert-butylcyclophosphorothionate binding data revealed an efficacy profile of partial inverse agonists/antagonists for compounds 1c,e,f,j,k, and of a partial agonist for 2c. This last compound proved to be effective in antagonizing pentylenetetrazole-induced seizures in mice. Attempts were made to interpret the structure-affinity relationships of compounds V in the light of possible tautomeric equilibria involving the ligands.     

Synthesis and biological evaluation of 3-heterocyclyl-7,8,9,10-tetrahydro-(7,10-ethano)-1,2,4-triazolo[3,4-a]phthalazines and analogues as subtype-selective inverse agonists for the GABA(A)alpha5 benzodiazepine binding site

The identification of a novel series of 7,8,9,10-tetrahydro-(7,10-ethano)-1,2,4-triazolo[3,4-a]phthalazines as GABA(A)alpha5 inverse agonists, which have both binding and functional (efficacy) selectivity for the benzodiazepine binding site of alpha5- over alpha1-, alpha2-, and alpha3-containing GABA(A) receptor subtypes, is described. Binding selectivity was determined to a large part by the degree of planarity of the fused ring system whereas functional selectivity was dependent on the nature of the heterocycle at the 3-position of the triazolopyridazine ring. 3-Furan and 5-methylisoxazole were shown to be optimal for GABA(A)alpha5 functional selectvity. 3-(5-Methylisoxazol-3-yl)-6-(2-pyridyl)methyloxy-1,2,4-triazolo[3,4-a]phthalazine (43) was identified as a full inverse agonist at the GABA(A)alpha5 subtype with functional selectivity over the other GABA(A) receptor subtypes and good oral bioavailability.     

Synthesis of 6-(Ethoxymethylenimino)-1,2,4-triazolo[3′,4′:5,1]-[1,2,4]triazolo[4,3-a]quinazolin-7(6H)-one

As a progressive continuation of our study in the condensed quinazolinone series^{1, 2)}, we recently described the synthesis and antihypertensive activity of some 4-amino[1,2,4]triazolo[4,3-a]quinazolin-5(4H)-one derivatives ( 1a-c , R=H, OH, SH). Now, we wish to report the conversion of one of these derivatives, namely 4-amino-1-mercapto-[1,2,4]-triazolo[4,3-a]quinazolin-5(4H)-one (1c) into a more complicate condensed quinazolinone system.     

Synthesis and antifungal activity of 2-aryl-1,2,4-triazolo[1,5-a]pyridine derivatives

A series of novel antifungal triazole derivatives 2-aryl-1,2,4-triazolo[1,5-a]pyridine 9a-m were synthesized and tested in vitro for their growth inhibitory activities against C. albicans and T. rubrum. The MIC values indicate that the activities of three compounds were superior or comparable to fluconazole against both tested fungi, worthy of further investigation of its antifungal activities.     

Synthesis and antiviral activity evaluation of some new 6-substituted 3-(1-adamantyl)-1,2,4-triazolo[3,4-b][1,3,4]thiadiazoles

A number of novel 3,6-disubstituted 1,2,4-triazolo[3,4-b][1,3,4]thiadiazole derivatives, containing the adamantyl moiety, were synthesized and examined in various viral test systems. No antiviral effects were noted with any of the compounds at subtoxic concentrations in cell culture.     

Synthesis and benzodiazepine receptor affinity of pyrazolo[1,5-a]pyrimidine derivatives. 3. New 6-(3-thienyl) series as alpha 1 selective ligands

New 3-aryl-6-(3-thienyl)pyrazolo[1,5-a]pyrimidin-7-ones (2a-j) are synthesized and evaluated in vitro on Bz/GABA(A) receptors and on recombinant benzodiazepine receptors (alpha x beta 2/3 gamma 2; x = 1-3, 5) expressed in HEK293 cells. SAR studies on the new compounds are conducted and molecular modeling is accomplished to better investigate requirements leading to subtype selectivity. Some of the synthesized compounds are tested in vivo to explore their pharmacological effect as a consequence of their high alpha 1 beta 2 gamma 2 subtype selectivity observed in vitro.     

Reactions with 3-amino-1,2,4-triazole: Synthesis of several new 1,2,4-triazolo[5,1-a]pyrimidine derivatives

Several new 1,2,4-triazolo[5,1-a]pyrimidine derivatives were synthesized via the reactions of 3-amino-1,2,4-triazole with active methylene nitriles and thier ylidene derivatives. The structures assigned for the reaction products were based on elemental analyses and spectral data.     

3-Aryl[1,2,4]triazino[4,3-a]benzimidazol-4(10H)-ones: a new class of selective A1 adenosine receptor antagonists.

Radioligand binding assays using bovine cortical membrane preparations and biochemical in vitro studies revealed that various 3-aryl[1,2,4]triazino[4,3-a]benzimidazol-4(10H)-one (ATBI) derivatives, previously reported by us as ligands of the central benzodiazepine receptor (BzR) (Primofiore, G.; et al. J. Med. Chem. 2000, 43, 96-102), behaved as antagonists at the A1 adenosine receptor (A1AR). Alkylation of the nitrogen at position 10 of the triazinobenzimidazole nucleus conferred selectivity for the A1AR vs the BzR. The most potent ligand of the ATBI series (10-methyl-3-phenyl[1,2,4]triazino[4,3-a]benzimidazol-4(10H)-one 12) displayed a Ki value of 63 nM at the A1AR without binding appreciably to the adenosine A2A and A3 nor to the benzodiazepine receptor. Pharmacophore-based modeling studies in which 12 was compared against a set of well-established A1AR antagonists suggested that three hydrogen bonding sites (HB1 acceptor, HB2 and HB3 donors) and three lipophilic pockets (L1, L2, and L3) might be available to antagonists within the A1AR binding cleft. According to the proposed pharmacophore scheme, the lead compound 12 engages interactions with the HB2 site (via the N2 nitrogen) as well as with the L2 and L3 sites (through the pendant and the fused benzene rings). The results of these studies prompted the replacement of the methyl with more lipophilic groups at the 10-position (to fill the putative L1 lipophilic pocket) as a strategy to improve A1AR affinity. Among the new compounds synthesized and tested, the 3,10-diphenyl[1,2,4]triazino[4,3-a]benzimidazol-4(10H)-one (23) was characterized by a Ki value of 18 nM which represents a 3.5-fold gain of A1AR affinity compared with the lead 12. A rhodopsin-based model of the bovine adenosine A1AR was built to highlight the binding mode of 23 and two well-known A1AR antagonists (III and VII) and to guide future lead optimization projects. In our docking simulations, 23 receives a hydrogen bond (via the N1 nitrogen) from the side chain of Asn247 (corresponding to the HB1 and HB2 sites) and fills the L1, L2, and L3 lipophilic pockets with the 10-phenyl, 3-phenyl, and fused benzene rings, respectively.     

10-(Alkylamino)-4H-thieno[3,4-b][1,5]benzodiazepines. A novel class of potential neuroleptic agents.

An investigation of the structural requirements for CNS activity of the title compounds was undertaken. A synthesis of the precursor dihydro-10H-thieno[3,4-b][1,5]benzodiazepin-10-ones was achieved and three routes for their conversion to the title compounds were developed. The compounds were tested for neuroleptic activity by means of the blockade or d-amphetamine lethality in aggregated mice and/or effects on locomotor activity in rats. Antidepressant activity was examined using inhibition of tetrabenazine-induced depression in mice. Most of the compounds were found to be potent neuroleptic agents with several exhibiting additional antidepressant activity.     

6-(Alkylamino)-9-benzyl-9H-purines. A new class of anticonvulsant agents

Several 9-alkyl-6-substituted-purines were synthesized and tested for anticonvulsant activity against maximal electroshock-induced seizures (MES) in rats. Most compounds were prepared in three steps from 5-amino-4,6-dichloropyrimidine or in two steps via alkylation of 6-chloropurine. Potent anticonvulsant activity against MES resided in compounds that contain a benzyl substituent at the 9-position of 6-(methylamino)- or 6-(dimethyl-amino)purine. Among commonly used agents for control of seizures, this type of structure represents a new class of potent anticonvulsant agents.