Novel anellated pyrazoloquinolin-3-ones: synthesis and in vitro BZR activity Maria Grazia Ferlin, a, * Gianfranco Chiarelotto, a Stefano DallÕAcqua, a Elisabetta Maciocco, b,c Maria Paola Mascia, b,c Maria Giuseppina Pisu b,c and Giovanni Biggio b,c a Department of Pharmaceutical Sciences, Faculty of Pharmacy, University of Padova, Via Marzolo 5, 35131 Padova, Italy b Centro di Eccellenza per la Neurobiologia della Dipendenza, University of Cagliari, Italy c CNR, Istituto di Neuroscienze, Sezione di Cagliari, Italy Received 19 October 2004; revised 10 February 2005; accepted 18 February 2005 Available online 31 March 2005 Abstract—A series of pyrazolo[4,3-c]pyrrolo[3,2-f]quinolin-3-one derivatives 6, 7a–c, 8a,b, 9a,b and 10–12 were synthesized as modi- fied pyrazoloquinolinone analogs (PQs) and evaluated for their ability to inhibit radioligand to central and peripheral benzodi- azepine receptors (BZRs) and their effect on GABA A a 1 b 2 c 2L receptors expressed in Xenopus laevis oocytes. Multistep synthesis starting from 5-nitroindole, via the Gould–Jacobs reaction to the quinoline nucleus, yielded key intermediates 9-chloro-3H-pyr- rolo[3,2-f]quinoline-8-carboxylates. The reaction of the latter with methyl-hydrazine and various phenyl-hydrazines furnished the final compounds. In order to confirm the expected tetracyclic 2-substituted-2H-pyrazolopyrroloquinolin-3-one structure, IR spec- trophotometric, mono- 1 H and 13 C and bi-dimensional spectrometric and HRMS analyses were carried out: all compounds were found to be 2-substituted 3-keto tautomers; compound 6 only differed because it turned out to be 1-methyl-2H-pyrazolo[4,3-c]pyr- rolo[3,2-f]quinolin-3-olo. The results of this work are consistent with those previously reported for PQs: 7–9 show high potency in displacing specific [ 3 H]flunitrazepam from its receptor site; no compound was active in inhibiting the binding of [ 3 H]PK 11195. They all act as antagonists at central BZR. Ó 2005 Elsevier Ltd. All rights reserved. 1. Introduction Since 1982, 1 2-aryl-pyrazoloquinolin-3-ones (PQs) have been known for their high affinity for central benzodi- azepine receptors (BZRs) and later papers reported spe- cific SARs and QSARs, indicating the structural requirements (lipophilic, electronic and steric) which cause changes in activity from state of an inverse agonist to an antagonist or agonist. 2,3 These compounds belong to a large number of BZ-binding site ligands, both clas- sical benzodiazepine and non-benzodiazepine types, that is, polyheterocyclic structures synthesized and studied for their ability to interact with BZRs and all aimed at discovering more selective drugs. Extensive SARs and QSARs on the classes of compounds were useful in iden- tifying pharmacophore models of the BZ-binding site, important tools for rational drug design, in the lack of sufficient structural insights into various receptor subtypes. 4 The proposed agonist/antagonist pharmacophore model, based on 2-aryl-pyrazoloquinolinones, involves the interaction sites between the receptor and ligand neces- sary for activity, 5 whereas SAR and QSAR findings indicate the importance of various substitutions on the pyrazolo[4,3-c]quinolin-3-one skeleton which determine ligand affinities for various BZR subtypes. 3,5–8 As part of our screening research activity on pyrroloquinoline compounds, 9 we were interested in the synthesis of pyr- azolopyrroloquinolin-3-ones and, in the light of specific literature data, we designed some derivatives conform- ing to the guidelines dictated by the postulated phar- macophore model. With the aim of obtaining ligands with high affinity for BZRs, we chose the angular pyrazolo[4,3-c]pyrrolo[3,2-f]quinolin-3-one tetracycle, because it may be considered as pyrazolo[4,3-c]quino- lin-3-one modified by a fused pyrrole ring at 8 and 9 0968-0896/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.bmc.2005.02.042 Keywords: Pyrazolopyrroloquinolinones; GABA A a 1 b 2 c 2L receptor antagonists; BZR pharmacophore/receptor model. * Corresponding author. Tel.: +39 049 8275718; fax: +39 049 8275366; e-mail: mariagrazia.ferlin@unipd.it Bioorganic & Medicinal Chemistry 13 (2005) 3531–3541