Novel pyrazolo[3,4-d]pyrimidines as dual Src/Bcr-Abl kinase inhibitors: Synthesis and biological evaluation for chronic myeloid leukemia treatment.

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.

Si113-prodrugs selectively activated by plasmin against hepatocellular and ovarian carcinoma.

Si113, a pyrazolo[3,4-d]pyrimidine derivative, gained more attention as an anticancer agent due to its potent anticancer activity on both in vitro and in vivo hepatocellular carcinomas (HCC) and ovarian carcinoma models. But the drawback is the low water solubility which prevents its further development. In this context, we successfully overcame this limitation by synthesizing two novel prodrugs introducing the amino acid sequence D-Ala-Leu-Lys (TP). Moreover, TP sequence has a high affinity with plasmin, a protease recognized as overexpressed in many solid cancers, including HCC and ovarian carcinoma. The prodrugs were synthesized and fully characterized in terms of in vitro ADME properties, plasma stability and plasmin-induced release of the parent drug. The inhibitory activity against Sgk1 was evaluated and in vitro growth inhibition was evaluated on ovarian carcinoma and HCC cell lines in the presence and absence of human plasmin. In vivo pharmacokinetic properties and preliminary tissue distribution confirmed a better profile highlighting the importance of the prodrug approach. Finally, the prodrug antitumor efficacy was evaluated in an HCC xenografted murine model, where a significant reduction (around 90%) in tumor growth was observed. Treatment with ProSi113-TP in combination with paclitaxel in a paclitaxel-resistant ovarian carcinoma xenografted murine model, resulted in an impressive reduction of tumor volume greater than 95%. Our results revealed a promising activity of Si113 prodrugs and pave the way for their further development against resistant cancer.

AuNP Pyrazolo[3,4-d]pyrimidine Nanosystem in Combination with Radiotherapy against Glioblastoma.

Gold-nanoparticle (AuNP)-conjugated drugs represent a promising and innovative antitumor therapeutic approach. In our study, we describe the design, the synthesis, the preparation, and the characterization of AuNPs conjugated with the pyrazolo[3,4-d]pyrimidine derivative SI306, a c-Src inhibitor. AuNPs-SI306 showed a good loading efficacy (65%), optimal stability in polar media and in human plasma, and a suitable morphological profile: a ζ-potential of -43.9 mV, a nanoparticle diameter of 48.6 nm, and a 0.441 PDI value. The antitumoral activity of AuNPs-SI306 was evaluated in vitro in the glioblastoma model, by the low-density growth assay, and also in combination with radiotherapy (RT). Results demonstrated that AuNPs had a basal radiosensitization ability and that AuNPs-SI306, when used in combination with RT, were more effective in inhibiting tumor cell growth with respect to AuNPs and free SI306.

A New Strategy for Glioblastoma Treatment: In Vitro and In Vivo Preclinical Characterization of Si306, a Pyrazolo[3,4-d]Pyrimidine Dual Src/P-Glycoprotein Inhibitor.

Overexpression of P-glycoprotein (P-gp) and other ATP-binding cassette (ABC) transporters in multidrug resistant (MDR) cancer cells is responsible for the reduction of intracellular drug accumulation, thus decreasing the efficacy of chemotherapeutics. P-gp is also found at endothelial cells' membrane of the blood-brain barrier, where it limits drug delivery to central nervous system (CNS) tumors. We have previously developed a set of pyrazolo[3,4-d]pyrimidines and their prodrugs as novel Src tyrosine kinase inhibitors (TKIs), showing a significant activity against CNS tumors in in vivo. Here we investigated the interaction of the most promising pair of drug/prodrug with P-gp at the cellular level. The tested compounds were found to increase the intracellular accumulation of Rho 123, and to enhance the efficacy of paclitaxel in P-gp overexpressing cells. Encouraging pharmacokinetics properties and tolerability in vivo were also observed. Our findings revealed a novel role of pyrazolo[3,4-d]pyrimidines which may be useful for developing a new effective therapy in MDR cancer treatment, particularly against glioblastoma.

Recent Studies on Ponatinib in Cancers Other Than Chronic Myeloid Leukemia.

Ponatinib is a third line drug for the treatment of chronic myeloid leukemia patients, especially those that develop the gatekeeper mutation T315I, which is resistant to the first and the second line drugs imatinib, nilotinib, dasatinib and bosutinib. The compound was first identified as a pan Bcr-Abl and Src kinase inhibitor. Further studies have indicated that it is a multitargeted inhibitor that is active on FGFRs, RET, AKT, ERK1/2, KIT, MEKK2 and other kinases. For this reason, the compound has been evaluated on several cancers in which these kinases play important roles, including thyroid, breast, ovary and lung cancer, neuroblastoma, rhabdoid tumours and in myeloproliferative disorders. Ponatinib is also being tested in clinical trials to evaluate its activity in FLT3-ITD acute myelogenous leukemia, head and neck cancers, certain type of lung cancer, gastrointestinal stromal tumours and other malignancies. In this review we report the most recent preclinical and clinical studies on ponatinib in cancers other than CML, with the aim of giving a complete overview of this interesting compound.

Efficient optimization of pyrazolo[3,4-d]pyrimidines derivatives as c-Src kinase inhibitors in neuroblastoma treatment.

The proto-oncogene c-Src is a non-receptor tyrosine kinase which is involved in the regulation of many cellular processes, such as differentiation, adhesion and survival. c-Src hyperactivation has been detected in many tumors, including neuroblastoma (NB), one of the major causes of death from neoplasia in infancy. We already reported a large family of pyrazolo[3,4-d]pyrimidines active as c-Src inhibitors. Interestingly, some of these derivatives resulted also active on SH-SY5Y NB cell line. Herein, starting from our previous Free Energy Perturbation/Monte Carlo calculations, we report an optimization study which led to the identification of a new series of derivatives endowed with nanomolar Ki values against c-Src, interesting antiproliferative activity on SH-SY5Y cells and a suitable ADME profile.

Optimization of Aminoimidazole Derivatives as Src Family Kinase Inhibitors.

Protein kinases have emerged as crucial targets for cancer therapy over the last decades. Since 2001, 40 and 39 kinase inhibitors have been approved by FDA and EMA, respectively, and the majority are antineoplastic drugs. Morevoer, many candidates are currently in clinical trials. We previously reported a small library of 4-aminoimidazole and 2-aminothiazole derivatives active as Src family kinase (SFK) inhibitors. Starting from these results, we decided to perform an optimization study applying a mix and match strategy to identify a more potent generation of 4-aminoimidazoles. Firstly, a computational study has been performed, then compounds showing the best predicted docking scores were synthesized and screened in a cell-free assay for their SFK inhibitory activity. All the new chemical entities showed IC50s in the nanomolar range, with 2⁻130 fold increased activities compared to the previously reported inhibitors. Finally, the most active compounds have been tested on three cancer cell lines characterized by Src hyperactivation. Compounds 4k and 4l showed an interesting antiproliferative activity on SH-SY5Y neuroblastoma (NB) cell line. In this assay, the compounds resulted more potent than dasatinib, a tyrosine kinase inhibitor approved for the treatment of leukemias and in clinical trials for NB.

Prodrugs of Pyrazolo[3,4-d]pyrimidines: From Library Synthesis to Evaluation as Potential Anticancer Agents in an Orthotopic Glioblastoma Model.

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.

Hematopoietic cell kinase (HCK) is a potential therapeutic target for dysplastic and leukemic cells due to integration of erythropoietin/PI3K pathway and regulation of erythropoiesis: HCK in erythropoietin/PI3K pathway.

New drug development for neoplasm treatment is nowadays based on molecular targets that participate in the disease pathogenesis and tumor phenotype. Herein, we describe a new specific pharmacological hematopoietic cell kinase (HCK) inhibitor (iHCK-37) that was able to reduce PI3K/AKT and MAPK/ERK pathways activation after erythropoietin induction in cells with high HCK expression: iHCK-37 treatment increased leukemic cells death and, very importantly, did not affect normal hematopoietic stem cells. We also present evidence that HCK, one of Src kinase family (SFK) member, regulates early-stage erythroid cell differentiation by acting as an upstream target of a frequently deregulated pathway in hematologic neoplasms, PI3K/AKT and MAPK/ERK. Notably, HCK levels were highly increased in stem cells from patients with some diseases, as Myelodysplastic Syndromes (MDS) and Acute Myeloid Leukemia (AML), that are associated with ineffective erythropoiesis These discoveries support the exploration of the new pharmacological iHCK-37 in future preclinical and clinical studies.