1. ISMN- CNR Palermo a Istituto per lo Studio dei Materiali Nanostrutturati, CNR, Via U. La Malfa 153, 90146 Palermo, Italy. mingoia@pa.ismn.cnr.itmingoia@pa.ismn.cnr.it b Dipartimento di Scienze e Tecnologie Molecolari e Biomolecolari (STEMBIO), Sezione di Chimica Farmaceutica e Biologica, Università di Palermo, via Archirafi 32. 90123, 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, 2859-2866. [2] 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, 2624-2637. [3] Z. Nie, C. Peretta, P. Erikson, S. Margosiak, gei Lu, A. Averill, R. Almassy, S. Chu, Bioorg. Med. Chem. Lett., 2008,18,619-623. [4] S.T. Davis, B.G. Benson, H.N. Bramson, D.E. Chapman, S.H. Dickerson, K.M. Dold, D.J. Eberwein, M. Edelstein, S.V. Frye, R.T. Gampe Jr, R.J. Griffin, P.A. Harris, A.M. Hassell, W.D. Holmes, R.N. Hunter, V.B. Knick, K. Lackey, B. Lovejoy, M.J. Luzzio, D Murray, P Parker, WJ Rocque, L Shewchuk, JM Veal, DH Walker, LF Kuyper. Science 2001 291 (5501): 134–7 [5] Dolzhenko, A. V.; Chui, W. K. J. Heterocycl. Chem. 2006, 43, 95–100. [6] Kompis, I. M.; Islam, K.; Then, R. L. Chem. Rev. 2005, 105, 593–620. [7] Anton V. Dolzhenko, Anna V. Dolzhenko, Wai-Keung Chui, Tetrahedron 2007, 63, 12888–12895. [8] Li R, Sirawaraporn R, Chitnumsub P, et al., J. Mol. Biol. 2000, 295, 307–23. [9] Benkovic SJ, Fierke CA, Naylor AM, Science 1988, 239 (4844): 1105–10. [10] Verdel, B. M.; Souverein, P. C.; Egberts, A. C. G.; Leufkens, H. G. M. Ann. Pharm. 2006, 40, 1040. 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 related to azolo-fused tetrazines such as Mitozolomde and Temozolomide, currently used in cancer therapy. The 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]. Like tetrazines, variously fused pyrazolo-triazines derivatives are also of interest for their antiproliferative activity. In particular they behave as novel potent inhibitors of CYP1A1, a cytochrome P450 family of enzymes involved in the metabolism of chemical carcinogens [2] and inhibit CdK2 involved in the transition from G1 to S phase [3]. Drugs that inhibit Cdk2 and arrest the cell cycle represent a strategy for prevention of chemotherapy induced alopecia [4]. Recently, dihydro-1,3,5-triazino[1,2-a]benzimidazoles have also been found to possess antifolate activity [5]. Antifolate drugs have been developed as anticancer, antibacterial, antifungal, and antiparasitic agents [6]. However, antifolate activity of the compounds with benzo-pyralzol-3-one scaffold has not been yet investigated. 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 related to azolo-fused tetrazines such as Mitozolomde and Temozolomide, currently used in cancer therapy. The 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]. Like tetrazines, variously fused pyrazolo-triazines derivatives are also of interest for their antiproliferative activity. In particular they behave as novel potent inhibitors of CYP1A1, a cytochrome P450 family of enzymes involved in the metabolism of chemical carcinogens [2] and inhibit CdK2 involved in the transition from G1 to S phase [3]. Drugs that inhibit Cdk2 and arrest the cell cycle represent a strategy for prevention of chemotherapy induced alopecia [4]. Recently, dihydro-1,3,5-triazino[1,2-a]benzimidazoles have also been found to possess antifolate activity [5]. Antifolate drugs have been developed as anticancer, antibacterial, antifungal, and antiparasitic agents [6]. However, antifolate activity of the compounds with benzo-pyralzol-3-one scaffold has not been yet investigated. Here, with the aim to modulate the biological profile, we planned to switch our interest to the deaza tricycle 1,2-dihydropyrazolo[1,2- a]benzo[1,2,4]triazine-3-one 2 exploiting the advantage of introducing new moieties in R 2 useful for carrying out SAR studies. In the present work, we designed and synthesized a new series of 1,2-DIHYDROPYRAZOLO[1,2-A]BENZO[1,2,4]TRIAZINE-3-ONE derivatives of type 2 containing selected functional groups in order to evaluate their anticancer potential on human tumor cell line. Although in some cases derivatives 2 are metabolically unstable, because of their nature of cyclic aminals, there are relatively common structural elements with some commercial drugs, including Aquamox and Thiabutazide, two members of the benzo(thia)diazine class of cyclic aminals used for the treatment of high blood pressure [10]. In our opinion, a good balance between the appropriate selection of R 1 and R 2 moities could bring more stable derivatives. Here, with the aim to modulate the biological profile, we planned to switch our interest to the deaza tricycle 1,2-dihydropyrazolo[1,2- a]benzo[1,2,4]triazine-3-one 2 exploiting the advantage of introducing new moieties in R 2 useful for carrying out SAR studies. In the present work, we designed and synthesized a new series of 1,2-DIHYDROPYRAZOLO[1,2-A]BENZO[1,2,4]TRIAZINE-3-ONE derivatives of type 2 containing selected functional groups in order to evaluate their anticancer potential on human tumor cell line. Although in some cases derivatives 2 are metabolically unstable, because of their nature of cyclic aminals, there are relatively common structural elements with some commercial drugs, including Aquamox and Thiabutazide, two members of the benzo(thia)diazine class of cyclic aminals used for the treatment of high blood pressure [10]. In our opinion, a good balance between the appropriate selection of R 1 and R 2 moities could bring more stable derivatives. 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 the di-hydro-1,2,4-triazine derivatives (40-80% yields). The method, other than the high overall yields, allows the easy introduction of selected moieties on crucial positions (R 1 and R 2 ), simply choosing from the large amount of commercial availability of orto-nitro amines and aldehydes. Additionally, such a type of approach can be suitably exploited in combinatorial chemistry for the synthesis of a small or virtual library of compounds to be employed in computer aided drug design for specific biological target. In this first step, we are optimizing the yields, we are testing the compound stability, in a second step we will be engaged to set up a stereoselective approach. 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 the di-hydro-1,2,4-triazine derivatives (40-80% yields). The method, other than the high overall yields, allows the easy introduction of selected moieties on crucial positions (R 1 and R 2 ), simply choosing from the large amount of commercial availability of orto-nitro amines and aldehydes. Additionally, such a type of approach can be suitably exploited in combinatorial chemistry for the synthesis of a small or virtual library of compounds to be employed in computer aided drug design for specific biological target. In this first step, we are optimizing the yields, we are testing the compound stability, in a second step we will be engaged to set up a stereoselective approach. There are some common structural features, which are unique to the triazine antifolates: -(1) one of the carbon atoms of triazine ring should be in sp 3 hybridization (gem-dimethyl substitution is usually preferred); -(2) the other two carbon atoms ought to be connected with nitrogen atoms of amino group or fused ring; -(3) lypophilic aromatic moiety is required at the distal part of molecule [7]. There are some common structural features, which are unique to the triazine antifolates: -(1) one of the carbon atoms of triazine ring should be in sp 3 hybridization (gem-dimethyl substitution is usually preferred); -(2) the other two carbon atoms ought to be connected with nitrogen atoms of amino group or fused ring; -(3) lypophilic aromatic moiety is required at the distal part of molecule [7]. DHFR is responsible for the levels of tetrahydrofolate in a cell, and their inhibition can limit the growth and proliferation of cells that are characteristic of cancer. Since folate is needed by rapidly dividing cells to make thymine, this effect may be used to therapeutic advantage. Thus, human DHFR can be targeted in the treatment of cancer. A competitive inhibitor of human DHFR, is methotrexate, used as anticancer drug [8]. Other drugs, acting only to bacterial DHFR and used as antimicrobial agents include trimethoprim and pyrimethamine [9]. Synthesis Remarks Antiproliferative screening of dervatives 2 are currently in progress. Further target specifically testing will be made on human DHFR Human DHFR with bound dihydrofolate and NADPH