New pyrazolo[1,2-a]benzo[1,2,5]triazepine-3,6(7H)dione derivatives: The influence of the side chains in the tuning of antiproliferative activity Francesco Mingoia, a Riccardo Delisi, b Carla Gentile, b Annamaria Martorana, b Anna Maria Almerico, b Antonino Lauria b a Istituto per lo Studio dei Materiali Nanostrutturati, uos Palermo (ISMN-CNR), Via Ugo La Malfa, 153 – Palermo, ITALY a Istituto per lo Studio dei Materiali Nanostrutturati, uos Palermo (ISMN-CNR), Via Ugo La Malfa, 153 – Palermo, ITALY b Dipartimento di Scienze e Tecnologie Biologiche Chimiche e Farmaceutiche (STEBICEF), Università di Palermo, Via Archirafi 32, Palermo,Italy Recently, a new series of pyrazolo[1,2-a]benzo-[1,2,3,4]tetrazin-3-one derivatives 1 (PBTs) has been synthesized and evaluated for their anticancer potential. Several of the new synthesized derivatives screened against a panel of more than 50 types of human tumor cell lines (NCI), showed promising antiproliferative activity reaching in some cases sub-micromolar values [1]. By optimization studies, new designed derivatives, effective on apoptosis induction, cell cycle arrest and antiproliferative activity, were proposed as new promising anticancer candidates [2]. Furthermore, by ring expansion, the new Pyrazolo[1,2- a]benzo[1,2,5]triazepine-3,6(7H)dione (PBTZP) ring system 2 was obtained. The preliminary NCI one dose screening (10  M) evidenced for the 9,10-dimethyl derivative a moderate antiproliferative activity with GI%= 23 against SNB75 (CNS cancer); and 13% against CCRF-CEM cells (leukemia). Analogously, the 10-Chloro derivative displayed only a GI% of 18 against CCRF-CEM cells (leukemia) and a GI% =15 against NCI-H322M (NSCLC) lung tumor cell. Curiously, contrarily to the observation made for the PBTs series, in this class of ring expanded derivatives, the chlorinated compounds resulted less active than the dimethyl one. It is to note that, to date, only two derivatives have been screened. Other series of seven derivatives PBTZP are currently under screening at NCI. References: References: [1] A.M. Almerico et al, J. Med. Chem., 2005, 48, [2] F. Mingoia et al, Eur. J. Med. Chem., 2013, 64, [3] A. Lauria et al., Eur. J. Med. Chem., 2013, 62, i) DMF, p-TSA, °C, 1-3h. The anti-proliferative activity of some 6-(3-R2-propoxy)-1-methyl-9-R1-3H,5H- pyrazolo[1,2-a]benzo[1,2,5]triazepin-3-one derivatives and the related precursors 6-(3-chloropropoxy)-1-methyl-9-R-3H,5H-pyrazolo[1,2- a]benzo[1,2,5]triazepin-3-one, were tested against HeLa tumor cell line for 48 h, using MTT based cell viability assay. The GI 50 values of the tested compounds are shown below.. Although many of the synthesized derivatives resulted inactive at 50 μM, few tested compounds showed concentration-dependent antiproliferative activity. The 3 c-1 derivative was the most active with a GI50 value of 27.3 μM. Interestingly, none of the screened derivatives, tested in the concentration range 50-1 μM affected the cell membrane integrity as preliminary determined by the Trypan Blu exclusion method. Although the preliminary results evidenced moderate antiproliferative activity against HeLa cells, we are waiting for the results of NCI one dose screening of the parent derivatives PBTZPs to set further optimization studies. Therefore, in the present study, we designed and synthesized a set of new derivatives of type 3 containing selected biologically inspired side chains in order to modulate the biological profile, as previously stated [3]. Cpds3 a-2’3 d-4’3 a-4’3 b-3’3 a-5’3 f-2’3 d-3’3 b-1’3 a-1’3 b-4’ GI 50 (  M) >50 Cpds3 d-1’3 c-3’3 c-4’36??3 d-5’3 d-2’3 a-3’3 b-5’3 b-2’ GI 50 (  M) > / / / /- 1.8