www.elsevier.nl/locate/farmac Il Farmaco 54 (1999) 375–389 Benzodiazepine receptor ligands III. Synthesis and biological evaluation of 2- and/or 3-substituted pyrazolo[5,1-c ][1,2,4]benzotriazine 5-oxides G. Guerrini a , A. Costanzo a, *, F. Bruni a , G. Ciciani a , S. Selleri a , P. Gratteri a , B. Costa b , C. Martini b , A. Lucacchini b a Dipartimento di Scienze Farmaceutiche, Uniersita ` degli Studi di Firenze, Via Gino Capponi 9, 50121 Florence, Italy b Istituto Policattedra Discipline Biologiche, Uniersita ` degli Studi di Pisa, Via Bonanno 6, 56100 Pisa, Italy Received 2 September 1998; accepted 30 March 1999 Abstract A new series of 2- and/or 3-substituted pyrazolo[5,1-c ][1,2,4]benzotriazine 5-oxides and their 8-chloro derivatives were synthesized, and their benzodiazepine receptor (BZR) affinities were evaluated in vitro in comparison to lead compound 3-ethoxycarbonyl-8-chloropyrazolo[5,1-c ][1,2,4]benzotriazine 5-oxide (29) [1,2]. None of the new compounds showed significant affinity for BZR. On the basis of a pharmacophore/receptor model suggested for lead compound 29, some hypotheses to explain the inactivity of new derivatives are discussed. © 1999 Elsevier Science S.A. All rights reserved. Keywords: 2- and/or 3-substituted pyrazolo[5,1-c ][1,2,4]benzotriazine 5-oxide; Benzodiazepine receptor; Receptor binding 1. Introduction In our studies on tricyclic heterocycles containing a pyrazole moiety with potential CNS activity, we fo- cused on central benzodiazepine receptor (BZR) ligands [1,2]. In addition to the classic 1,4-benzodiazepines (1,4-BZs), a large number of ligands belonging to dif- ferent chemical families have been shown to bind with high affinity to the GABA A –BZR. BZ ligands act as allosteric modulators of the GABA A /chloride channel supramolecular complex and have been classified as agonists, inverse agonists and antagonists if they en- hance, reduce or have no influence on the GABA A - gated chloride current. Agonists exhibit anxiolytic, anticonvulsant, muscle relaxant and sedative/hypnotic activity, the inverse ago- nists cause the opposite behavioral effects, such as anxiogenesis and proconvulsant action, the antagonists inhibit the action of both agonists and inverse agonists. Recently, we reported the synthesis and evaluation of the BZR affinity of a series of pyrazolo[5,1-c ][1,2,4]- benzotriazines and corresponding 5-oxides 3-, 7- and 8-substituted with a variety of groups different for lipophilic and electronic features and amounts of steric hindrance [2]. From binding data we found that the best ligand was the 3-ethoxycarbonyl-8-chloropyra- zolo[5,1-c ][1,2,4]benzotriazine 5-oxide (K i 35 nM), even if compounds bearing at the 3-position bromine and at the 8-position also a chlorine or a small lipophilic group such as ethoxy- or methyl group showed good affinity. The pharmacological profile of these new lig- ands, evaluated by GABA-ratio (GR), was from ago- nists for 3-ethoxycarbonyl derivatives (GR 1.61 – 2.67) to partial agonists for 3-bromine derivatives (GR 1.14 – 1.31) [2]. As part of a wider program of SAR studies, in order to explain the BZR binding of the new pyrazolo[5,1-c ]- [1,2,4]benzotriazine ligands, it is very interesting to focus the role of the substituent at 2- and/or 3-position on receptor affinity and on intrinsic activity in the presence of a favorable substituent at the 8-position. The importance of the rotable aromatic rings in BZ-ligands to recognize and activate BZR and the rela- * Corresponding author. Tel.: +39-55-2757296; fax: +39-55- 240776. E-mail address: costanzo@farmfi.scifarm.unifi.it (A. Costanzo) 0014-827X/99/$ - see front matter © 1999 Elsevier Science S.A. All rights reserved. PII:S0014-827X(99)00044-0