ISMN- CNR Palermo a Istituto per lo Studio dei Materiali Nanostrutturati (ISMN), Consiglio Nazionale delle Ricerche (CNR), Via U. La Malfa 153, Palermo, Italy. b Dipartimento di Scienze e Tecnologie Biologiche Chimiche e Farmaceutiche (STEBICEF), Sezione di Chimica Farmaceutica e Biologica, Università di Palermo, via Archirafi , Palermo, Italy. References [1] A.M. Almerico, F. Mingoia, P. Diana, P. Barraia, A. Lauria, A. Montalbano, G. Cirrincione, G. Dattolo, J. Med. Chem., 2005, 48, [2] A. Lauria, M. Tutone, A.M. Almerico, Eur. J. Med. Chem., 2011, 46, [3] F. Mingoia, C. Di Sano, F. Di Blasi, M. Fazzari, A. Martorana, A.M. Almerico, A. Lauria, Eur. J. Med Chem., 2013, 64, [4] A.M. El Massry, A. M. Asal, S.N. Khattab, N. S. Haiba, H. A. Hawney, M. Helmy, V. Langer, A. Amer, Bioorg. Med.Chem., 2012, 20, [5] K. Sztanke, et al, Eur. J. Med. Chem.,2008, 43, ; [6] G. Ciciani,et al., Bioorg.Med. Chem., 2008, 16, The pyrazolo[1,2-a]benzo[1,2,3,4]tetrazin-3-one derivatives 1 have been previously designed by us as new alkylating agent because of their peculiar chemical behavior in soft acid environment towards charged or neutral nucleophiles. The tricycle is related to azolo-fused tetrazines such as Temozolomide, currently employed in cancer diseases. A preliminary NCI (USA) screening performed against more than 50 types of human tumor cell lines showed antiproliferative activity reaching in some cases sub-micromolar values [1]. Recently, through the in silico VLAK protocol [2], a second series of new derivatives 1 have been designed. Four derivatives have been selected, synthesized and evaluated on peculiar cellular events including proliferation, apoptosis induction and cell cycle [3]. Several of the new synthesized derivatives screened against apoptosis induction (HeLa) showed promising anticancer potential reaching in some cases activity in the micromolar range and arresting the cell in the S phase. The pyrazolo[1,2-a]benzo[1,2,3,4]tetrazin-3-one derivatives 1 have been previously designed by us as new alkylating agent because of their peculiar chemical behavior in soft acid environment towards charged or neutral nucleophiles. The tricycle is related to azolo-fused tetrazines such as Temozolomide, currently employed in cancer diseases. A preliminary NCI (USA) screening performed against more than 50 types of human tumor cell lines showed antiproliferative activity reaching in some cases sub-micromolar values [1]. Recently, through the in silico VLAK protocol [2], a second series of new derivatives 1 have been designed. Four derivatives have been selected, synthesized and evaluated on peculiar cellular events including proliferation, apoptosis induction and cell cycle [3]. Several of the new synthesized derivatives screened against apoptosis induction (HeLa) showed promising anticancer potential reaching in some cases activity in the micromolar range and arresting the cell in the S phase. Here, with the aim of tuning the biological profile, in terms of activity and selectivity, we planned to switch our interest to a deaza analog of the tricyclic pyrazolo[1,2- a]benzo[1,2,4]triazin-3-one 2, either to verify the electronic effect resulting from a deaza isosterism and to exploit the advantages of introducing a new point of chemical diversity (in R 3 ), useful for combinatorial developments. Variously fused pyrazolo[x,y-z]triazines endowed with antiproliferative activity as novel potent inhibitors of kinase CK2 [4] and of CYP1A1, including enzymes involved in the metabolism of chemical carcinogens, are reported [5]. Moreover, hydrazide derivatives have exhibited remarkable inhibitory activity against SiHa and LS180 human tumor cell lines, with no toxicity towards the HSF control cell line [6]. Thus, in the present work, we designed and synthesized a new series of 4,8,9- Substituted-PYRAZOLO[1,2-a]BENZO[1,2,4]TRIAZIN-3-ONE derivatives of type 2 containing selected functional groups in order to evaluate their anticancer potential on human tumor cell line. Here, with the aim of tuning the biological profile, in terms of activity and selectivity, we planned to switch our interest to a deaza analog of the tricyclic pyrazolo[1,2- a]benzo[1,2,4]triazin-3-one 2, either to verify the electronic effect resulting from a deaza isosterism and to exploit the advantages of introducing a new point of chemical diversity (in R 3 ), useful for combinatorial developments. Variously fused pyrazolo[x,y-z]triazines endowed with antiproliferative activity as novel potent inhibitors of kinase CK2 [4] and of CYP1A1, including enzymes involved in the metabolism of chemical carcinogens, are reported [5]. Moreover, hydrazide derivatives have exhibited remarkable inhibitory activity against SiHa and LS180 human tumor cell lines, with no toxicity towards the HSF control cell line [6]. Thus, in the present work, we designed and synthesized a new series of 4,8,9- Substituted-PYRAZOLO[1,2-a]BENZO[1,2,4]TRIAZIN-3-ONE derivatives of type 2 containing selected functional groups in order to evaluate their anticancer potential on human tumor cell line. The amino key intermediate 8 has been prepared by us according to a previously optimized procedure [1]. The easy condensation with selected commercial aldehydes afforded the di-hydro- 1,2,4-triazine derivatives (40-80% yields) which after treatment with DDQ afforded compounds 2. The method, other than the high overall yields provided, allows the easy introduction of selected moieties on crucial positions (R, 1 R 2 and R 3 ), simply choosing from the large amount of commercial availability of orto-nitro amines and aldehydes. Such a type of synthetic approach can be suitably exploited in combinatorial chemistry for the synthesis of small or virtual library of compounds to be employed in computer aided drug design for specific biological target. REMARKS & PERSPECTIVES The preliminary NCI one dose screening evidenced promising antiproliferative activity (IC 50 ≈ 10  M) against UO31 (renal cancer) for the 3- piperonyl-2-yl derivative. Same activity against Leukemia cell (CCRF-CEM) and prostate cancer PC-3 was observed for the 3-pyridil-2-yl derivative. Taking into account all the chemical diversity points in the scaffold, along with the commercial availability of the reactants, the extension of the library of compounds coupled with further in silico optimization studies could bring to improved results. REMARKS & PERSPECTIVES The preliminary NCI one dose screening evidenced promising antiproliferative activity (IC 50 ≈ 10  M) against UO31 (renal cancer) for the 3- piperonyl-2-yl derivative. Same activity against Leukemia cell (CCRF-CEM) and prostate cancer PC-3 was observed for the 3-pyridil-2-yl derivative. Taking into account all the chemical diversity points in the scaffold, along with the commercial availability of the reactants, the extension of the library of compounds coupled with further in silico optimization studies could bring to improved results. Compounds selected: a) R 1 = R 2 = Me; b) R 1 = R 2 = Cl; c) R 1 = H, R 2 = CN; d) R 1 = H, R 2 = CF3; And others most active of ref. [1] e) R 1 = H, R 2 = Me f) R 1 = H, R 2 = Cl g) R 1 = Cl, R 2 = H Apoptosis HeLa Cell Cycle (arrest S phase HeLa) Cell proliferation From in silico VLAK protocol MTT based assays SRD based assays NCI Control