Src is a non-receptor tyrosine kinase (TK) whose involvement in cancer, including glioblastoma (GBM), has been extensively demonstrated. In this context, we started from our in-house library of pyrazolo[3,4-d]pyrimidines that are active as Src and/or Bcr-Abl TK inhibitors and performed a lead optimization study to discover a new generation derivative that is suitable for Src kinase targeting. We synthesized a library of 19 compounds, 2a-s. Among these, compound 2a (SI388) was identified as the most potent Src inhibitor. Based on the cell-free results, we investigated the effect of SI388 in 2D and 3D GBM cellular models. Interestingly, SI388 significantly inhibits Src kinase, and therefore affects cell viability, tumorigenicity and enhances cancer cell sensitivity to ionizing radiation.

The therapeutic use of tyrosine kinase inhibitors (TKIs) represents one of the successful strategies for the treatment of glioblastoma (GBM). Pyrazolo[3,4-d]pyrimidines have already been reported as promising small molecules active as c-Src/Abl dual inhibitors. Herein, we present a series of pyrazolo[3,4-d]pyrimidine derivatives, selected from our in-house library, to identify a promising candidate active against GBM. The inhibitory activity against c-Src and Abl was investigated, and the antiproliferative profile against four GBM cell lines was studied. For the most active compounds endowed with antiproliferative efficacy in the low-micromolar range, the effects toward nontumoral, healthy cell lines (fibroblasts FIBRO 2-93 and keratinocytes HaCaT) was investigated. Lastly, the in silico and in vitro ADME properties of all compounds were also assessed. Among the tested compounds, the promising inhibitory activity against c-Src and Abl (Ki 3.14 µM and 0.44 µM, respectively), the irreversible, apoptotic-mediated death toward U-87, LN18, LN229, and DBTRG GBM cell lines (IC50 6.8 µM, 10.8 µM, 6.9 µM, and 8.5 µM, respectively), the significant reduction in GBM cell migration, the safe profile toward FIBRO 2-93 and HaCaT healthy cell lines (CC50 91.7 µM and 126.5 µM, respectively), the high metabolic stability, and the excellent passive permeability across gastrointestinal and blood-brain barriers led us to select compound 5 for further in vivo assays.

The Bcr-Abl tyrosine kinase (TK) is the molecular hallmark of chronic myeloid leukemia (CML). Src is another TK kinase whose involvement in CML was widely demonstrated. Small molecules active as dual Src/Bcr-Abl inhibitors emerged as effective targeted therapies for CML and a few compounds are currently in clinical use. In this study, we applied a target-oriented approach to identify a family of pyrazolo[3,4-d]pyrimidines as dual Src/Bcr-Abl inhibitors as anti-leukemia agents. Considering the high homology between Src and Bcr-Abl, in-house Src inhibitors 8a-l and new analogue compounds 9a-n were screened as dual Src/Bcr-Abl inhibitors. The antiproliferative activity on K562 CML cells and the ADME profile were determined for the most promising compounds. Molecular modeling studies elucidated the binding mode of the inhibitors into the Bcr-Abl (wt) catalytic pocket. Compounds 8j and 8k showed nanomolar activities in enzymatic and cellular assays, together with favorable ADME properties, emerging as promising candidates for CML therapy. Finally, derivatives 9j and 9k, emerging as valuable inhibitors of the most aggressive Bcr-Abl mutation, T315I, constitute a good starting point in the search for compounds able to treat drug-resistant forms of CML. Overall, this study allowed us to identify more potent compounds than those previously reported by the group, marking a step forward in searching for new antileukemic agents.

Pyrazolo[3,4-d]pyrimidines are potent protein kinase inhibitors with promising antitumor activity but suboptimal aqueous solubility, consequently worth being further optimized. Herein, we present the one-pot two-step procedure for the synthesis of a set of pyrazolo[3,4-d]pyrimidine prodrugs (1a-8a and 9a-e) with higher aqueous solubility and enhanced pharmacokinetic and therapeutic properties. ADME studies demonstrated for the most promising prodrugs a better aqueous solubility, a favorable hydrolysis in human and murine serum, and an increased ability to cross cell membranes with respect to the parental drugs, explaining their better 24 h in vitro cytotoxicity against human glioblastoma U87 cell line. Finally, the 4-4a couple of drug/prodrug was also evaluated in vivo, revealing a profitable pharmacokinetic profile of the prodrug associated with a good efficacy. The application of the prodrug approach demonstrated to be a successful strategy for improving aqueous solubility of the parental drugs, determining a positive impact also in their biological efficacy.

Some novel 6-substituted pyrazolo[3,4-d]pyrimidines 4, 5, 6a-d, 7a-c, 8 and pyrazolo[4,3-e][1,2,4]triazolo[4,3-a]pyrimidines 9a-c, 10a-c, 11, 12a,b, 13a-c and 14 were synthesized and characterized by spectral and elemental analyses. They were screened for their biological activity in vitro against Abl and Src kinases. Compounds 7a and 7b revealed the highest activity against both wild and mutant Abl kinases as well as the Src kinase and the leukemia K-562 cell line. They can be considered as new hits for further structural optimization to obtain better activity.

Neuroblastoma (NB) represents the most common extracranial paediatric solid tumor for which no specific FDA-approved treatment is currently available. The tyrosine kinase c-Src has been reported to play an important role in the differentiation, cell-adhesion and survival of NB cells. Starting from dual Src/Abl inhibitors previously found active in NB cell lines (1-3), small modification of the original structures almost abolished the Abl activity with a contemporary improvement of affinity and specificity for c-Src. Among the synthesized compounds, the most potent c-Src inhibitor (10a) showed a very interesting antiproliferative activity in SH-SY5Y cells with an IC(50) of 80 nM and a favourable ADME profile. A 3D SAR analysis was also attempted and may guide the design of more potent c-Src inhibitors as potential agents for NB treatment.

Abstract A family of dual Src/Abl inhibitors characterized by a substituted pyrazolo[3,4-d]pyrimidine scaffold was previously reported by us and proved to be active against several tumor cell lines. Among these compounds, a promising antileukemia lead (1) has been recently identified, but, unfortunately, it suffers from substandard pharmaceutical properties. Accordingly, an approach for the optimization of the lead 1 is described in the present work. A series of more soluble pyrazolo[3,4-d]pyrimidine derivatives were rationally designed and proved to maintain the dual Src/Abl activity of the lead. Selected compounds showed an interesting activity profile against three different leukemic cells also in hypoxic conditions, which are usually characterized by imatinib-resistance. Finally, in vitro ADME properties (PAMPA permeation, water solubility, microsomal stability) for the most promising inhibitors were also evaluated, thus allowing the identification of a few optimized analogues of lead 1 as promising antileukemia agents.