DEPARTEMENT OF PHARMACY
Ph.D. School
Science of Drug and Bioactive Substances
PRECLINICAL CHARACTERIZATION AND LIPOSOMAL FORMULATIONS OF PYRAZOLO[3,4-d]PYRIMIDINE SRC
INHIBITORS FOR CANCER TREATMENT
Supervisors
Prof. Elena DREASSI
Prof. Tiziano TUCCINARDI
Author
Arianna MANCINI
October, 2019
Pyrazolo[3,4-d]pyrimidines represent a promising class of compounds capable of inhibiting several oncogenic tyrosine kinases, in particular c-Src, which had become an attractive target for the development of new therapeutic agents against cancer.
The members of this family of compounds were found to induce apoptosis and to reduce proliferation in different cell lines in which c-Src is overexpressed or overactivated, both from solid and hematologic tumors.
The first part of this thesis essentially focuses on the preclinical characterization of compound Si306, a pyrazolo[3,4-d]pyrimidine derivative, identified as a very promising agent for the treatment of glioblastoma multiforme (GBM) and neu- roblastoma (NB), two types of cancer arising from nervous system. Si306 and its prodrug pro-Si306 have been demonstrated to inhibit P-glycoprotein, a cellular membrane efflux pump, which is reported to have an important role in multidrug resistance (MDR) for several cancers. Encouraging pharmacokinetic properties and tolerability in vivo were observed for Si306. These 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 central nervous system tumors, such as GBM.
In the second section, the development of a stealth liposomal formulation for Si306 for the passive targeting toward NB is described. Si306-loaded liposomes have been prepared with high drug encapsulation efficacy and characterized dimensionally and morphologically by Dynamic Light Scattering (DLS) and Cryo-TEM. Finally, liposomes have been proved to be a long circulating drug delivery system in vivo, capable of enhancing Si306 accumulation in the tumor area thanks to the enhanced permeability and retention effect (EPR).
quence D-Ala-Phe-Lys, which binds a specific reactive site of plasmin, are described.
This tripeptide was used to decorate the surface of liposomes encapsulating three selected pyrazolo[3,4-d]pyrimidines, including Si113, a dual c-Src/Sgk1 inhibitor with high anticancer activity against hepatocellular carcinoma (HCC). In vitro cell uptake and cytotoxicity data in HCC cell line showed that the presence of the tripeptide on the liposomal membrane surface has improved the cell-penetrating ability of liposomes and has increased the activity of two of the three tested compounds.
List of Tables ix
List of Figures x
Acronyms xii
I Preclinical Characterization of Compound Si306, a Pyrazolo[3,4-d]Pyrimidine Src Inhibitor for the
Treatment of Glioblastoma 1
1 Introduction 3
1.1 Protein Kinases . . . . 3
1.1.1 c-Src . . . . 4
1.1.2 Src Signaling and Tumor Biology . . . . 6
1.1.3 BCR-Abl . . . . 7
1.2 Small-molecule Tyrosine Kinase Inhibitors . . . . 9
1.2.1 Src Inhibitors . . . . 10
1.2.2 Pyrazolo[3,4-d]pyrimidines . . . . 13
1.2.3 Src Inhibitors for Nervous System Cancers: NB and GMB . 16 1.3 Multidrug Resistance in GBM . . . . 18
1.3.1 P-glycoprotein . . . . 19
1.3.2 CYP450 System . . . . 20
1.3.3 Correlation between TKIs and ABC Transporters: Substrates or Potential Inhibitors . . . . 21
1.4.1 Cellular Assays . . . . 23
1.4.2 PK behavior . . . . 24
1.4.3 GBM Xenograft Mice Models . . . . 24
2 Aim of the Project 26 3 Results 28 3.1 Inhibitory Effect of Si306 and Pro-Si306 towards P-gp . . . . 28
3.2 Caco-2 Permeability Assay . . . . 28
3.3 Si306 and pro-Si306 Interaction with CYP3A4 in Human Liver Microsomes . . . . 30
3.4 Protein-Binding Profile of Si306 and proSi306 to HSA and AGP . . 30
3.5 Pharmacokinetics and Biodistribution of Si306 After i.v. and per os Administration . . . . 32
3.6 Acute Toxicity Study in Mice . . . . 35
4 Discussion and Conclusions 36 5 Materials and Methods 40 5.1 Drugs . . . . 40
5.2 Binding Fluorimetric Assay . . . . 40
5.3 CYP450 Inhibition Study . . . . 41
5.4 Animals for PK and BD Studies . . . . 41
5.5 In vivo Administration of Si306 . . . . 42
5.5.1 Sample preparation . . . . 42
5.5.2 Recovery and Matrix Effect . . . . 43
5.5.3 UV/HPLC-MS Instrumentation and Analysis Conditions . . 43
II Stealth Liposome Formulation of Compound Si306 for Passive Targeting of Neuroblastoma 44 6 Introduction 45 6.1 Liposome Formulations in Cancer Therapy . . . . 45
6.1.2 Ligand-Targeted Stealth Liposomes . . . . 48
6.1.3 Approved and Emerging Liposome Formulations for Cancer Treatment . . . . 48
6.1.4 Therapeutic Opportunities of Liposomes for NB Treatment . 49 6.2 Encapsulation of Hydrophobic compounds . . . . 50
6.2.1 Methods of Preparation . . . . 51
6.2.2 Pocket-Forming Phospholipids . . . . 52
6.3 Compound Si306 against NB: State of Art . . . . 53
7 Aim of the Project 55 8 Results 57 8.1 Preparation and Characterization of Si306-loaded Liposomes . . . . 57
8.2 In vitro Cytotoxicity Studies . . . . 58
8.3 Stability Studies and In Vitro Release . . . . 59
8.4 Administration in Healthy Mice of Si306-LP for PK Studies . . . . 60
8.5 Administration in Orthotopic NB-Xenografted Immunodeficient Mice of Si306-LP for PK Studies . . . . 63
9 Discussion and Conclusions 65 10 Materials and Methods 68 10.1 Preparation of Si306-LP . . . . 68
10.2 Characterization of Si306-LP . . . . 68
10.3 Stability Studies . . . . 69
10.4 In vitro Release . . . . 69
10.5 In vivo Administration of Si306-LP for PK Studies . . . . 70
III Plasmin-binding Tripeptide-decorated Liposomes Loading Pyrazolo[3,4-d]pyrimidines for Targeting Hepa-
tocellular Carcinoma 71
11 Introduction 73
11.1.1 Sgk1 Signaling and Tumor Biology . . . . 73
11.1.2 Compound Si113 Against HCC: State of Art . . . . 74
11.2 Peptide-Targeted Liposomes . . . . 75
11.2.1 Plasmin-Binding Tripeptide D-Ala-Phe-Lys . . . . 76
11.2.2 Role of Plasmin in Cancer Progression . . . . 76
12 Aim of the Project 79 13 Results 82 13.1 Synthesis of Tripeptide-Linker (TRP) . . . . 82
13.2 LC-MS/MS characterization of TRP . . . . 82
13.3 Preparation and characterization of LPs . . . . 84
13.4 Preparation and characterization of TRP-LPs . . . . 84
13.5 Cytotoxicity studies . . . . 85
13.6 Confocal microscopy study for the in vitro uptake of TRP-liposomes 87 14 Discussion and Conclusions 89 15 Materials and Methods 92 15.1 Materials . . . . 92
15.2 MS/MS characterization of TRP-linker . . . . 92
15.3 Preparation of liposomes encapsulationg pyrazolo[3,4-d]pyrimidines . . . . 93
15.4 Encapsulation efficiency and drug loading capacity . . . . 94
15.5 Particle size and ζ-potential measurements . . . . 94
List of Publications 95
Bibliography & Sitography 98
1.1 Si163, Si223 and S13: Inhibitory activity toward c-Src . . . . 14
1.2 Si214 and Si192: Inhibitory activity toward c-Src; ADME properties 15 1.3 Si306 and pro-Si306: Inhibitory activity toward c-Src; ADME prop- erties . . . . 16
1.4 Cytotoxic effect of Si306 and pro-Si306 . . . . 23
3.1 Papp coefficients and efflux ratio for compound Si306 . . . . 29
3.2 Plasmatic proteins binding parameters for Si306 and pro-Si306 . . . 31
3.3 Matrix effect % and Recovery % for the analysis of Si306 . . . . 32
3.4 Plasma PK parameters of Si306-Tween80 . . . . 34
8.1 Properties of Si306-LP . . . . 58
8.2 Cytotoxicity of Si306 . . . . 59
8.3 Stability of Si306-LP over a period of 14 days . . . . 59
8.4 Plasma PK parameters of Si306 . . . . 61
8.5 AUC0→48 for plasma, spleen, liver, kidneys and lungs for Si306 . . . 63
8.6 AUC0→48 for plasma and tumor for Si306 . . . . 64
12.1 Compounds Si113, S29, Si163: inhibitory activity toward Src and Sgk1; ADME properties. . . . 80
13.1 Properties of pyrazolo[3,4-d]pyrimidines-loaded LPs . . . . 84
13.2 Properties of pyrazolo[3,4-d]pyrimidines-loaded TRP-LPs . . . . 85
13.3 IC50 values calculated 48h after HepG2 cells treatment with Si113, S29 and Si163 . . . . 86
1.1 Structural domains of Src protein . . . . 5 1.2 Contribution of Src-mediated pathways to tumor progression . . . . 7 1.3 BCR-Abl . . . . 8 1.4 Structures of some Src/multikinase inhibitors . . . . 11 1.5 Structures of PP1 and PP2, the first selective Src inhibitors . . . . 12 1.6 Structure of some pyrazolo[3,4-d]pyrimidines: Si163 (a), Si223 (b),
S13 (c) . . . . 14 1.7 Structures of some pyrazolo[3,4-d]pyrimidines: Si214 (a), Si192 (b) 15 1.8 Structures of some pyrazolo[3,4-d]pyrimidines: Si306 (a), pro-Si306 16 1.9 Biological Activity of Si306 . . . . 23 1.10 Plasma concentration-time curves of pro-Si306 and Si306 . . . . 24 1.11 Si306 efficacy in mice models of GBM . . . . 25 3.1 Inhibition of CYP3A4 isoform activity by Si306 and pro-Si306 . . . 30 3.2 Plasma protein binding for Si306 and pro-Si306 . . . . 31 3.3 PK and tissue distribution of Si306 after i.v. and per os administration 33 3.4 Microscopic analysis of tissues after Si306 administration in mice . . 35 6.1 Structure of liposomes . . . . 46 6.2 Thin layer evaporation technique passeges . . . . 51 6.3 Various types of phospholipids and their packing structure; repre-
sentation of cavities formed by pocket-forming lipids in a liposomal bilayer. . . . 53 6.4 Biodistribution of compound Si306 after administration of free drug
and liposomal drug . . . . 54
8.2 Plasma concentration-time curves of Si306 . . . . 60 8.3 Biodistribution of Si306 for the dosage of 5 and 25 mg/kg over 48 h 62 8.4 Tumor and plasma concentration-time curves of Si306 in orthotopic
NB mice model . . . . 64 11.1 Chemical structure of compound Si113 . . . . 75 11.2 Predicted complex of plasmin with D-Ile-Phe-Lys substrates . . . . 77 11.3 Graphical representation of PM activation system . . . . 78 12.1 Structures of pyrazolo[3,4-d]pyrimidines: S29 (a) and Si163 (b) . . 80 13.1 Structure of TRP . . . . 83 13.2 Schematic representation of TRP and the sites that undergo MS/MS
fragmentation . . . . 83 13.3 RWPE-1 cell line viability 48h after the treatment with S29 (a),
Si163 (b), Si113 (c) . . . . 86 13.4 Fluorescence microscopy images of HepG2 cells following LPs and
TLPs administration . . . . 87 13.5 Relative fluorescence intensity per cell area histograms for each
formulation. . . . 88
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(carboxyfluorescein) ammonium salt (PE-CF); 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine semisynthetic (DPPC);
1,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-[maleimide(polyethylene gly- col)-2000] ammonium salt (DSPE-PEG2000-Mal); 1-palmitoyl-2-oleoyl-glycero-3- phosphocholine (POPC); -enolase (ENO-1); β6-hydroxytestosterone (β6-OH-TST);
ADME (absorption, distribution, metabolism, elimination); amiloride-sensitive sodium channel (ENaC); alpha-1-acid glycoprotein (AGP); ATP-binding cassette (ABC); area under the curve(AUC); Biopharmaceutics Classification System (BCS);
blood brain barrier (BBB); breast cancer resistance protein (BCRP, ABCG2);
cell-penetrating peptide (CPP); cell-targeting peptide (CTP); chemokine receptors (CXCR1 and CXCR2); chronic myeloid leukemia (CML); central nervous system (CNS); clearance (CL); Cryo Transmission Electron Microscopy (Cryo-TEM); Csk (C-terminal Src kinase); cytochrome P450 (CYP450); D-Ala-Phe-Lys-linker (TRP);
Dynamic Light Scattering (DLS); encapsulation efficacy (EE); enhanced permeabil- ity and retention (EPR) effect; epidermal growth factor (EGF); epidermal growth factor receptor (EGFR); epithelial-to-mesenchimal transition (EMT); extracellular matrix (ECM); ephrin type-A receptor 2 (EphA2); fibroblast growth factor (FGF);
fibroblast growth factor receptor (FGFR); focal adhesion kinase (Fak); folate re- ceptors (FRs); Food and Drug Administration (FDA); gastrointestinal tract (GI);
glioblastoma multiforme (GBM); half-life (t1/2); hormone receptor positive (HR+);
human epidermal growth factor receptor-2 (HER2); human serum albumin (HSA);
IL2 (interleukin2); immunoliposomes (IL); internal standard (IS); intravenous in- jection (i.v.); ketoconazole (KTZ); large unilamellar vesicles (LUV); lymphoblastic leukemias (ALLs); maleimide (Mal); matrix metallo-proteases (MMPs); maximum concentration observed (Cmax); mean residence time (MRT); metabolic stability
cytic system (MPS); Mouse Double Minutes 2 (MDM2); multidrug resistance (MDR); multi-drug resistance protein 1 (MRP1, ABCC1); multilamellar vesicles (MLV); N-(carbonyl methoxypolyethylenglycol 2000)-1,2-dipalmitoyl-snglycero-3-
phosphoethanolamine sodium salt (MPEG-2000-DPPE Na); neuroblastoma (NB);
non-small-cell lung cancer (NSCLC); non-receptor tyrosine kinases (nRTKs); oral administration (p. os); paclitaxel (PTX); PAMPA (Parallel Artificial Membrane Permeability Assay); Papp (apparent permeability); P-glycoprotein (P-gp, MDR1, ABCB1), pharmacokinetics (PK); Philadelphia chromosome (Ph); Philadelphia positive (Ph+); phosphate buffer (PBS); platelet-derived growth factor (PDGF);
plasmin (PM); plasminogen (PG); plasminogen activators (PAs); plasminogen receptors (PG-Rs); platelet-derived growth factor receptor (PDGFR); polydisper- sity index (PDI); polyethylene glycols (PEGs); pyrazolo[3,4-d]pyrimidine-loaded bare liposomes (LP); pyrazolo[3,4-d]pyrimidine-loaded tripeptide-conjugated li- posomes (TLP); radiotherapy (RT); RANBP1 (Ran-specific binding protein 1);
reticuloendothelial system (RES); SH (Src homology); Si306 formulated as mixture of Tween80/ benzyl alcohol/ citric acid solution (Si306-Tween80); Si306-loaded liposomes (Si306-LP); small unilamellar vesicles (SUV); Src family kinases (SFKs);
structure–activity relationships (SAR); subcutaneous (s.c.); temozolomide (TMZ);
time of maximum concentration observed (Tmax); tissue-type plasminogen activa- tor (tPA); transactivator of transcription (TAT); transarterial chemoembolization (TACE); transferrin (Tf); transferrin receptors (TfRs); transforming growth factor-b (TGF-b); transmembrane receptor tyrosine kinases (RTKs); tyrosine 416 (Tyr416);
tyrosine kinases (TK); tyrosine kinase inhibitors (TKIs); tyrosine kinase-like (TKL);
urinary-type plasminogen activator (uPA); vascular endothelial growth factor re- ceptor (VEGFR); volume of distribution (V).
Preclinical Characterization of Compound Si306, a Pyrazolo[3,4-d]Pyrimidine Src Inhibitor for the
Treatment of Glioblastoma
Fallacara, A.L.; Zamperini, C.; Podolski-Renić, A.; Dinić, J.; Stanković, T.;
Stepanović, M.; Mancini, A.; Rango, E.; Iovenitti, G.; Molinari, A.; Bugli, F.;
Sanguinetti, M.; Torelli, M.; Martini, M.; Maccari, L.; Valoti, M.; Dreassi, E.;
Botta, M.; Pesić, M.; Schenone, S. 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. Cancers (Basel) 2019, 11 (6), 848. https//- doi.org/10.3390/cancers11060848.
Introduction
1.1 Protein Kinases
To date, 518 protein kinase genes have been identified (478 typical and 40 atypical), comprising one of the largest gene families in human (about 1.7 % of the genome)1. This protein superfamily catalyzes protein phosphorylation:
MgATP1− + protein-OH → protein-OPO32− + MgADP + H+
which represents the most widespread class of post-translational modification used in signal transduction. Based upon the nature of the phosphorylated -OH group, these enzymes are classified as tyrosine kinases (TK), serine/threonine kinases and a small group of dual specificity kinases, and further divided on the basis of structure and function into 9 groups: TK, tyrosine kinase-like (TKL), STE, CSNK1, AGC, CAMK, CMGC, RGC and Other. Most of these proteins have been grouped into families and subfamilies.
Since protein phosphorylation controls nearly every process of cellular biology, deregulation and abnormalities in protein kinase activity can lead to a wide variety of diseases, including cancer, inflammatory disorders, diabetes, neurodegeneration and central nervous system (CNS) diseases. Protein kinases involvement in cell motility, cell cycle progression and apoptosis makes them an important class of targets for the development of new small molecules inhibitors as therapeutics for the treatment of several diseases and conditions.
TKs are one of the most studied and important groups of protein kinases and are divided into two subclasses:
• Transmembrane receptor tyrosine kinases (RTKs), such as the insulin receptor, the epidermal growth factor (EGF), platelet-derived growth factor (PDGF), fibroblast growth factor (FGF). They consist of an extracellular portion that binds ligands, a transmembrane helix and a cytoplasmic portion with TK catalytic activity for transmembrane signaling downstream.
• Non-receptor tyrosine kinases (nRTKs) are localized in cytoplasm and function in signal transduction to the nucleus. Some of them possess some amino terminal modifications, such as myristoylation or palmitoylation, which allow them to be anchored to cell membrane and be activated. In addition to TK domain, nRTKs possess domains that mediate protein-protein, protein-lipid, and protein-DNA interactions. Examples of nRTKs are Abl, Csk, Fak, Jak, Hck, Ack and Src families.
1.1.1 c-Src
c-Src is a nRTK, largely involved in intracellular signaling transduction pathways, performing essential roles in the regulation of a wide variety of cellular processes, such as apoptosis, cell cycle progression, differentiation, proliferation, motility, adhesion and transcription. Src dysregulation occurs in several diseases, including cancers and inflammatory disorders. Src is expressed ubiquitously, but brain cells, osteoclasts, and platelets express 5–200-fold higher levels of this protein than others2.
Src has been the subject of intense investigation for decades because of its proto- oncogenic activity. The interest in this kinase occurred since the discovery of Rous sarcoma virus, a chicken tumor virus discovered in 1911 by Peyton Rous. The viral protein v-Src is encoded by the avian cancer-causing oncogene of Rous sarcoma virus, and c-Src, the cellular homologue in humans, chickens, and other animals, is encoded by a physiological gene, the first proto-oncogene discovered3.
Src family kinases (SFKs) include 11 members with highly conserved domain organization: Src, Fyn, Yes, Fgr (group I); Blk, Hck, Lck, Lyn (group II); Frk, Srm and Brk (group III). Their structure consists of several functional domains,
as illustrated in Figure 1.1. Src contains a myristoylated N-terminal segment attached to SH4 (Src homology 4), which is responsible for Src cell membrane association, SH2 (Src homology 2) and SH3 (Src homology 3) domains which mediate Src-substrate or intramolecular interactions, a TK domain (or SH1, Src homology 1), and a C-terminal negative regulatory domain. SH1, the catalytic domain, is composed of two lobes (N-terminal and C-terminal), separated by a catalytic cleft, which can be occupied by ATP and where there are substrate-binding sites for PO32− transfer. The cleft forms an activation loop that contains Tyrosine 416 (Tyr416), which is the positive regulatory site, responsible for promoting the kinase activity.
Figure 1.1: Structural domains of Src protein
Phosphorylation at the C-terminal end Tyr527, the negative regulatory site, by Csk (C-terminal Src kinase)4 leads to conformational changes inducing the catalytic cleft to be inaccessible to external ligands and Tyr416 to be masked, attaining an inactive (or closed) conformation. Src exists in equilibrium between phosphorylated or free Tyr527 forms, with the bound state greatly favored (under basal condition, about 90-95% of Src is phosphorylated at Tyr527)2.
The activation of Src is finely regulated by a variety of mechanisms and mediated by many upstream kinases and phosphatases. Activation is promoted by dephosphory- lation of Tyr527 by various protein tyrosine phosphatases3, allowing Src structure to
unfold into a configuration that permits the autophosphorylation of Tyr416 residue and the access of substrates to the kinase domain. Src can also be activated by the displacement of the SH3 and SH2 domains mediated by intermolecular interactions with activated TK growth factor receptors, e.g., EGF receptor (EGFR), human epidermal growth factor receptor-2 (HER2), PDGF receptor (PDGFR), vascular endothelial growth factor receptor (VEGFR), and FGF receptor (FGFR)5, integrin cell adhesion receptors6, steroid hormone receptors, G protein-coupled receptors, focal adhesion kinase (Fak)7 and cytoskeleton components.
1.1.2 Src Signaling and Tumor Biology
Tumor growth is typically associated with increased proliferation, reduced apoptosis, inhibition of autophagy, and increased angiogenesis. Also, invasion of primary tumors and metastasis involves altered cell adhesion processes, increased motility and high grade of intravasation/extravasation of cells. Src participates in numerous signaling pathways8, generating a huge flow of information (Figure 1.2).
In normal cells, Src is mostly in inactive state, being only transiently activated during the cellular events in which it is involved. By contrast, Src protein over- expression and hyperactivation are associated to malignant potential, having a role in progression, maintenance and survival in several tumor types: solid cancers as colon9, breast10, lungs11, liver12, prostate13 and pancreatic14 cancers; nervous system cancers, such as glioblastoma multiforme (GBM)15,16 and neuroblastoma (NB)17 and hematologic tumors such as chronic myeloid leukemia (CML)18 and lymphomas19. Src plays an important role in promoting tumor cell proliferation, mediating signaling from growth factor receptors. Src activation leads to down- stream signaling through the RAS/mitogen-activated protein kinase (MAPK) and PI3 pathways. In addition, Src has been shown to stimulate proliferation by inducing cyclin expression, by JAK/STAT pathway madiation20. There is also evidence that Src activates antiapoptotic and pro-survival signal transduction pathways, including those mediated by PI3 and Akt signaling21. Src can mediate disruption of cell-cell adhesion and increase of turnover of cell- extracellular matrix (ECM) contacts, thereby promoting tumor cell migration, invasion and metastasis.
Src-mediated decrease in cellular adhesiveness result from multiple mechanisms, including redistribution of focal adhesion component proteins22, altered regulation
of integrin signal and decreased deposition of ECM proteins23.
Src activity can affect extravasation through its downstream role in VEGF, chemokine receptors (CXCR1 and CXCR2)24 and urokinase receptors25 signaling pathways. Disruption of vascular endothelial cell-cell junctions following VEGF- activated Src signaling, increases vascular permeability, permitting tumor cell extravasation. Crosstalk between Src and Fak also promotes tumor invasion.
In addition, Src-dependent expression of membrane bound and secreted matrix metallo-proteases (MMPs), has a well established role in tumor invasion26. Because Src can induce VEGF expression, it has a potential role in the angiogenic process:
it has been proved that in human solid tumor cell lines, Src inhibition reduces VEGF expression, reducing proangiogenetic activity27.
Figure 1.2: Contribution of Src-mediated pathways to tumor progression
1.1.3 BCR-Abl
The Abl is a nRTK with a predominant role in the pathogenesis of CML. It shares significant sequence homology with Src and bears remarkable structural resem- blances with most SFKs (Figure 1.3a). c-Abl is localized at several subcellular sites, including the nucleus, cytoplasm, mitochondria, the endoplasmic reticulum and the cell cortex, where it interacts with a large variety of cellular proteins, includ- ing signaling adaptors, kinases, phosphatases, cell-cycle regulators, transcription factors and cytoskeletal proteins. Abl contributes to the leukemogenic formation of
the fusion protein BCR-Abl, with deregulated constitutive kinase activity. BCR-Abl fusion gene resulted from a reciprocal translocation between chromosomes 9 and 22, generating a shortened chromosome 22 containing the chimeric gene BCR-Abl (Figure 1.3b). This is commonly known as the Philadelphia chromosome (Ph)
and found to be in over 90% of CML patients and also detectable in a subset (25-30%) of patients with acute lymphoblastic leukemias (ALLs)28.
BCR-Abl, the final product of this genetic rearrangement, is responsible for the phenotypic abnormalities of chronic phase CML. It activates signal transduction pathways such as RAS/MAPK, PI3 kinase, JAK-STAT and the Src pathways, me- diating altered cellular adhesion, activation of mitogenic signaling, and inhibition of apoptosis, leading to the transformation of hematopoietic stem cells. BCR- ABL-mediated signaling is also associated with defective DNA repair, resulting in additional chromosomal alterations and mutations, which may partly explain the aggressive nature of advanced CML29.
(a)
(b)
Figure 1.3: Structural domains of BCR-Abl (a) and schematic representation of the formation of BCR-Abl gene, the Philadelphia chromosome (b).
1.2 Small-molecule Tyrosine Kinase Inhibitors
Kinases have been established as promising drug targets for the treatment of various types of human diseases because of their pivotal roles in signal transductions and regulation of a wide range of cellular activities.
The concept of kinase inhibition started during the 1950s, when pioneering re- searches about kinase characterization and signaling cascade identification were carried out. The strategy of kinase inhibition was initiated during the late 1980s, when inhibitors against EGFR were described30. Since then, a large number of tyrosine kinase inhibitors (TKIs) based on diverse molecular scaffolds and pharma- cological profiles have been reported, accompanied by a rapidly increasing number of publications on this topic (about 500,000 in 2019).
The approval of imatinib (Gleevec®, Novartis, Figure 1.4a), a multikinase in- hibitor, by the US Food and Drug Administration (FDA) in 2001 for the treatment of patients with CML was not only a breakthrough in targeted cancer therapy, but also paved the way for future research in the field. FDA has approved 48 small molecules TKIs as of March 2019, nearly all of which are orally effective and about 231 different TKIs are in clinical trials worldwide (a complete and regularly updated list can be found at PKIDB Database31). Of the 48 approved drugs, the majority (25) inhibit RTKs, 10 inhibit nRTKs, 13 are directed at protein-serine/threonine protein kinases and at least 18 of these drugs are multikinase inhibitors. Data indicate that 43 TKIs are directed toward malignancies (37 against solid tumors in- cluding lymphomas and 8 against non-solid tumors). To date, most of the approved TKIs are ATP competitive inhibitors, which include:
• Type I inhibitors, which bind within and around the adenine site of the ATP-binding pocket of active protein kinase;
• Type II inhibitors, which bind with both the ATP pocket and an adjacent allosteric pocket, in the inactive forms of kinase;
• Lenvatinib showed both type I and type II binding features32 and is grouped as a type V inhibitor;
• Type III antagonists, which bind to an allosteric site that does not overlap the adenine-binding pocket (trametinib, binimetinib, cobimetinib);
• No type IV inhibitors, which bind into a pocket remote from the ATP-binding pocket33, has been approved yet;
• Afatinib and ibrutinib represent a group of inhibitors that incorporate chem- ically active Michael Acceptor electrophiles that react with a cysteine nu- cleophile adjacent to the hinge region of the ATP-binding pocket to form a covalent adduct. Thus, they are named “covalent inhibitors”34.
The structures of selected pharmacophores that make up the currently FDA- approved small molecule TKIs consist of a small number of nitrogen-containing compounds such as quinazolines, quinolines, isoquinolines, pyrimidines, and indoles.
TKIs represent a new and improved step toward targeted therapy and toward a new generation of anticancer drugs that has the potential for avoiding some of the drawbacks associated with cytotoxic chemotherapy. Nevertheless, at the present time, TKIs serve more as second- or third-line therapies rather than as primary therapy, being useful in combination with traditional cytotoxic chemotherapy. For the TKIs to have a primary role in therapy, there has to be a clear hypothesis for their use, relevant preclinical data, and a demonstrated use in well characterized groups of patients.
1.2.1 Src Inhibitors
In the last three decades, extensive work on the development of Src inhibitors has been performed. Src is a participant in many pathways promoting cell division and survival and its inhibition may play an important auxiliary role in various cancer treatments. Four orally effective Src/multikinase inhibitors are FDA-approved for the treatment of various malignancies: dasatinib, vandenatinib, ponatinib and bosutinib (Figure 1.4b, c, d, f ), all approved for the treatment of Philadelphia positive (Ph+) CML and ALLs.
Several Src inhibitors are currently in phase II/III clinical trials for solid tumor patients. As an example, Saracatinib (AZD0530; AstraZeneca, Wilmington; Figure 1.4e), an orally active, highly selective dual Src-Abl inhibitor, has shown promising results in preclinical and clinical studies for the treatment of non small cell lung cancer (NSCLC), prostate cancer, head and neck cancer and breast cancer35.
(a)Imatinib (b) Dasatinib
(c) Vandenatinib (d) Ponatinib
(e) Saracatinib (f) Bosutinib
Figure 1.4: Structures of some Src/multikinase inhibitors
PP1 and PP2
Inhibitors of SFK activity were first utilized two decades ago. However, the earliest inhibitors suffered from toxicity and poor selectivity. The pyrazolopyrimidines PP1 and PP2 (Figure 1.5) were the first small molecule inhibitors that could truly be classified as selective for SFK and have remained important to study SFK inhibition in tissue cultures36,37.
(a) PP1 (b) PP2
Figure 1.5: Structures of PP1 and PP2, the first selective Src inhibitors
Dasatinib
Dasatinib (BMS-354825; Bristol-Myers Squibb, New York; Figure 1.4b) is an orally bioavailable ATP-site competitive 2-(aminopyrimididinyl)thiazole-5-carboxamide, FDA-approved Src/Abl inhibitor for use in patients with CML or Ph+ ALL. It is the most well studied Src/Abl inhibitor, displaying a 325-fold greater potency for Bcr-Abl inhibition than imatinib. It is fairly unselective, inhibiting an array of additional downstream tyrosine kinases including Lck, Fyn, Yes, c-Kit, ephrin type-A receptor 2 (EphA2) and PDGFR.
It represents an important option for the treatment of patients with newly diagnosed chronic-phase CML and for imatinib-resistant or -intolerant patients with chronic- or advanced-phase CML or Ph+ ALL. In addition, clinical data demonstrated the activity of dasatinib against several solid tumors: castration resistant prostate cancer38, hormone receptor positive (HR+) breast cancer39, NSCLC 40. Many phase I and phase II trials studying dasatinib in combination with other agents are also in progress for other cancers, including melanoma, colorectal cancer, and GBM.
Bosutinib
Bosutinib (SKI-606; Wyeth/Pfizer, New York; Figure 1.4f ) is a 4-anilino-3- quinoline-carconitrile BCR-Abl, Src, Lyn, Hck, c-Kit, and PDGFR inhibitor that is approved for the treatment of Ph+ CML and ALL. It displays 200-fold more potent inhibition of Bcr/Abl than imatinib. This drug is currently in clinical trials for the
treatment of metastatic solid tumors in combination with pemetrexed (recruiting)41. A phase II trial for the treatment of GBM has recently been completed42.
1.2.2 Pyrazolo[3,4-d]pyrimidines
Pyrazolo[3,4-d]pyrimidines represent a promising class of compounds capable of inhibiting several oncogenic TKs, which became an attractive target for the devel- opment of new therapeutic agents against cancer.
In the last years, the research group of Professor Maurizio Botta has developed a huge library of pyrazolo[3,4-d]pyrimidines compounds, which have been found to act as ATP-competitive c-Src inhibitors, thanks to their isosterism with adenine.
The first small group of pyrazolo[3,4-d]pyrimidines synthesized by this research group has been published in 2004: the compounds showed inhibitory activity towards Src phosphorylation and towards cell proliferation for A-431 (epidermoid carcinoma cell line)43. Since then, once understood the potential of the pyrazolo[3,4- d]pyrimidine core, a large library of compound has been synthesized and several
members of this family were found to induce apoptosis and to reduce proliferation in different cell lines in which c-Src is overexpressed or overactivated, both from solid tumors (osteosarcoma44, prostate45, NB46−48, GBM49−51, rhabdomyosarcoma52, mesothelioma53, medulloblastoma54, medullary thyroid carcinoma55) and hemato- logical tumors (leukemia56 and Burkitt lymphomas57).
Many members of the family showed interesting dual Abl/Src inhibition proper- ties and strong antiproliferative activity towards human leukemia cell lines58. In addition, some compounds have been found to inhibit the Bcr-Abl T315I mutant (Table 1.1): in particular, Si163 (Figure 1.6a) and Si223 (Figure 1.6b) has been tested in mice inoculated with 32D-T315I cells, showing a more than 50% reduction in tumor volumes56. On the other hand, S13 (Figure 1.6c) seemed to be one of the best candidates for development as selective c-Src inhibitor59. Interestingly, S13 showed a synergic activity in combination treatment with paclitaxel (PTX) against hormone-insensible prostate cancer both in cell and in subcutaneous (s.c.) PC3 xenograft model mice model59.
Despite their remarkable activity, these compounds suffer from a low water solu- bility and unfavorable ADME (absorption, distribution, metabolism, elimination) properties which preclude oral administration and further development.
(a) (b) (c)
Figure 1.6: Structure of some pyrazolo[3,4-d]pyrimidines: Si163 (a), Si223 (b), S13 (c)
Table 1.1: Si163, Si223 and S13: Inhibitory activity toward c-Src, Abl and Abl T315I expressed as Ki (inhibition constant)
Compound c-Src (µM) Abl (µM) Abl T315I (µM)
Si163 0.045 0.055 0.036
Si223 0.064 0.610 0.090
S13 3.0 / /
Hence, a series of more soluble pyrazolo[3,4-d]pyrimidine derivatives have been designed and synthesized through the introduction of polar groups. More recently, the development of these molecules led to the synthesis of a small library with increased activity against NB and GBM. Starting from the hit compound Si214 (Figure 1.7a)58, which showed remarkable activity, but low water solubility, our studies led to the identification of Si192 (Figure 1.7b), which showed a beneficial profile in term of ADME properties with potent inhibitory activity against isolated c- Src46(Table 1.2). Further optimization based on structure–activity relationships (SAR) and computational studies has identified compound Si306 (Figure 1.8a) as the best candidate for development against NB46 and GBM50, thanks to its favorable ADME and inhibition properties46.
(a) (b)
Figure 1.7: Structures of some pyrazolo[3,4-d]pyrimidines: Si214 (a), Si192 (b)
Table 1.2: Si214 and Si192: Inhibitory activity toward c-Src; ADME properties
Compound c-Src (Ki) Solubility (µg/mL)
PAMPA Papp (10−6 cm/sec)
Met. Stab.
Human (%)
Si214 0.09 0.12 0.2 99
Si192 0.21 1.7 10.0 95
PAMPA: Parallel Artificial Membrane Permeability Assay; Papp: apparent permeability;
Met. Stab. Human (%): Metabolic stability in presence of human microsomes expressed as percentage of unmodified drug.
The compound exhibited the ability to inhibit c-Src in cell-free assays at submicro- molar concentration and demonstrated a good aqueous solubility profile and high metabolic stability determined in vitro (Table 1.3).
In the last years, a prodrug strategy was applied with the aim of overcoming the lack of aqueous solubility that characterizes the pyrazolo[3,4-d]pyrimidine class of compounds51. The secondary amine in the C4 position was selected to accomodate a promoiety, as a consequence of being the moiety that most of the previously synthesized pyrazolo[3,4-d]pyrimidines shared. The NH group on C4 position represents an essential feature for favorable interactions within the ATP-binding site of TKs, as demonstrated by SAR and computational studies. Indeed, prodrugs are unable to inhibit the enzymatic activity as they are, but only after hydrolysis and release of the parental drug. Taken the water solubility issue into account, a N-methylpiperazino moiety, protonated at physiological pH (pKa 9.27 ± 0.1) and characterized by high water solubility (molar solubility 9.98 mol L–1, pH
7), was chosen. All the synthesized prodrugs demonstrated to have higher water solubility when compared with parental drugs with consequent increase of their bioavailability and biological activity. Among them, pro-Si306 (Figure 1.8b), the prodrug of Si306, has been selected as the most promising for its good aqueous solubility, favorable hydrolysis in human serum, and an increased ability to cross cell membranes (Table 1.3).
(a) (b)
Figure 1.8: Structures of some pyrazolo[3,4-d]pyrimidines: Si306 (a), pro-Si306
Table 1.3: Si306 and pro-Si306: Inhibitory activity toward c-Src; ADME properties
Compoud c-Src (Ki)
Abl (Ki)
Solubility (µg/mL)
PAMPA Papp (10−6 cm/sec)
Met. Stab.
Human (%)
Plasma t1/2 (h)
Si306 0.13 0.12 3.7 5.3 96 /
pro-Si306 >100 >100 8.7 2.1 94 3.48
t1/2: hydrolisis half life calculated as ln 2/Kobs. Prodrug was incubated in human plasma at 37 °C, and the formation of parent drug was monitored by UV/LC-MS.
1.2.3 Src Inhibitors for Nervous System Cancers: NB and GMB
The role of SFK in the brain and neuronal tumors is not as extensively studied as solid tumors deriving from the breast, lung, colon, prostate, or pancreas. However, neuronal tissues do exhibit increased expression of Src and Fyn, and it is likely that
these proteins play a role in the deregulated proliferation and uninhibited growth of tumors arising in the nervous system60. This paragraph will focus on the role of c-Src in the development and progression of two solid tumors arising from nervous system: NB and GBM.
NB is the most common extracranial solid tumor of childhood and it can originate anywhere along the sympathetic chain (most frequently it arises from the adrenal gland). Several studies reported a correlation between c-Src overexpression and the ability of NB to differentiate in vitro and in vivo61. NB cell lines have been shown to express Src at levels much higher than that observed in primary cultures from noncancerous tissues62. The same is also true for many other neuroendocrine tumors, where Src was found to be in correlation with the differentiation state of the tumor63. In addition, overexpression of the Src kinase in advanced NB patients has been associated with poor outcome64.
Inhibition of Src TK activity by dasatinib and bosutinib resulted in decreased proliferation and apoptosis induction in NB cells and demonstrated anti-tumor efficacy in NB mouse models65,66. Therefore, targeting Src tyrosine kinase may represent a feasible solution in NB therapy.
Furthermore, in the past two decades, there has been a tremendous influx of data describing genomic alterations in gliomas and particularly in GBM, which is the most aggressive malignant form of primary brain tumor (WHO grade IV) with an extremely poor prognosis: a 1-year and 5-years survival rate of 35.7% and 4.7%
respectively)67. Since its low incidence rate, it is considered an orphan disease with an annual prevalence of 1-9/100,000 people68. The standard-of-care is the surgical resection followed by adjuvant radiotherapy combined with chemotherapy. Temo- zolomide (TMZ) is and alkylating agent and represents the first line in treatment.
Investigators have found elevated Src activity in various CNS tumor, including GBM, compared with normal brain samples69 (Src activity/phosphorylation is elevated in GBM samples and cell lines, including T98G and U87)16. In preclinical GBM models, Src inhibition reduces cell proliferation and viability, and increases glioma cell apoptosis16. Also, Src inhibition reduces vessel density and VEGF- induced vascular permeability. The role of Src in GBM development was further supported by a study in transgenic mice expressing v-Src. These mice spontaneously developed low-grade tumors that progressed to a morphology resembling human
GBM (14% of mice at 65 weeks)70. Many studies also showed that Src is activated in primary GBM patient samples42.
Taking together all these evidence supporting the role of SFKs in glioma biology, it is hardly surprising that the number of clinical trials for SFK inhibitors for the treatment of GBM had an exponential growth in the recent years.
1.3 Multidrug Resistance in GBM
Multidrug resistance (MDR) is a severe and pervasive clinical problem and actually represents the leading cause of cancer treatment failure. Response failures in brain tumor chemotherapy are frequently observed and result from GBM unique characteristics. The heterogeneous nature of GBM cells, its infiltrative, invasive, and aggressive nature together with the inherent issues of treating any cancer of the CNS due to anatomic complexities, make the treatment of GBM a difficult goal. Chemotherapy failure against GBM has partially been attributed to intrinsic and/or acquired drug resistance and unsuccessful drug delivery across blood brain barrier (BBB).
Drug resistance can generally be categorized as either acquired or intrinsic. Ac- quired drug resistance occurs when a tumor that initially responded to treatment is no longer sensitive to the anticancer agent. Intrinsic drug resistance refers to a tumor that shows insignificant or no response to the therapy at the onset of treatment.
MDR occurs when cancer cells develop simultaneous resistance to structurally and mechanistically unrelated classes of anticancer drugs and involves several complex mechanisms:
• Efflux of drugs out of cells by increasing the activity and the expression of efflux pumps, such as ATP-dependent transporters;
• Reduction of drug influx for agents that are transported by intracellular carriers or enter the cell by means of endocytosis;
• Activation of detoxifying proteins, such as cytochrome P450 (CYP450) mixed- function oxidases;
• Activation of mechanisms that repair drug-induced DNA damage;
• Disruptions in apoptotic signaling pathways (e.g., p53 or ceramide) that allow cells to become resistant to drug-induced cell death;
• Tumor induced hypoxic barriers.
1.3.1 P-glycoprotein
One of the most prominent mechanisms involved in conferring MDR is the over- expression of ATP-binding cassette (ABC) transporters, which have been linked to resistance to multiple chemotherapeutic drugs. ABC transporters regulate the ATP-dependent efflux of toxic endogenous molecules and chemotherapeutics out of the cell. There are 49 known ABC transporters present in humans, which are classified into seven families (A–G) by the Human Genome Organization (HUGO).
Many of these ABC transporters were reported to confer resistance to anticancer drugs in cellular models. Among them, P-glycoprotein (P-gp, MDR1, ABCB1), multi-drug resistance protein 1 (MRP1, ABCC1) and breast cancer resistance protein (BCRP, ABCG2) appear to account for most of the reported MDR in cancer71.
P-gp is the most prominent and best characterized of the mammalian ABC trans- porters. P-gp is expressed in normal tissues, such as excretory epithelial cells of the gastrointestinal tract (GI), kidneys, liver and as endothelial cells of physiologi- cal barriers such as the BBB72,73, playing a very important physiological role by protecting the body from xenobiotics and other natural product toxins. Altered function or expression of P-gp has been reported to have an important role in MDR for several cancers, such as lung, breast, colon, ovarian, CNS cancers, and melanomas and it also has been shown to influence variability in pharmacokinetics (PK) and the pharmacodynamics (PD) of many drugs that are handled by it,
substantially affecting intestinal absorption and elimination and reducing intra- cellular drug concentrations74. P-gp confers resistance to and transports a wide variety of compounds that are central to most of the cytotoxic chemotherapeutic
regimens such as vinca alkaloids, anthracyclines, epipodophyllotoxins and taxanes75. Furthermore, in recent years, a number of studies have suggested that P-gp is also a transporter that effluxes molecularly targeted drugs such as TKIs.
Insufficient drug delivery to the brain has been recognized as a major cause of failure in CNS development programs. Besides having optimal PK properties, CNS drug candidates must be able to cross the protective barriers of the brain. The BBB and the blood–cerebrospinal fluid barrier are the main functional interfaces that regulate the transfer of solutes between the systemic circulation and the brain. These barriers express a wide variety of efflux transport proteins, the most prominent of those being P-gp and BCRP, that extrude their substrates against a steep concentration gradient, limiting the brain uptake of many lipophilic com- pounds. A large amount of data suggests that active efflux is a relevant obstacle to treating GBM and provides a plausible mechanistic basis for the clinical failure of numerous drugs that are BCRP/P-gp substrates. Thus, drug efflux pumps are key elements for drug delivery and efficacy in target tumor cells, as well as for CNS distribution. Therefore, the modulation of ABC transporters offers a novel strategy to enhance penetration and improve efficacy of drugs into the brain, especially for drug-resistant CNS tumors.
1.3.2 CYP450 System
CYP450 is a hemeprotein that plays a key role in the metabolism of drugs and other xenobiotics, with significant involvement in drug-drug interactions.
Primary sites of drug metabolism are liver, intestinal wall, lungs, kidneys, and plasma. Predominant isoforms of CYP450 involved in drug metabolism are CYP2C9, CYP2D6 and CYP3A4 which metabolize up to 80-90% of all xenobiotics76. In particular, it has been estimated that CYP3A4 metabolizes up to 50% of all reported drugs and a few of them are also metabolized by other isoforms. CYP3A4 is responsible for many drug–drug interactions: drugs with CYP activity may be inhibitors, inducers, or substrates for a specific CYP enzymatic pathway, thus altering the metabolism of concomitantly administered agents. Drugs that inhibit an enzymatic pathway of CYP may cause increased concentrations of other drugs metabolized by the same pathway, resulting in drug toxicity. Likewise, drugs that induce an enzymatic pathway of CYP may reduce concentrations of drugs
metabolized by the same pathway, leading to subtherapeutic drug levels or treatment failure.
CYP450 and P-gp are highly expressed along the GI tract (especially on the brush border membrane of enterocytes, oral mucosa and intestinal lumen77) and are two central factors controlling the oral bioavailability of drugs and subsequently their therapeutic efficacy. CYP450 enzymes and P-gp proteins act synergistically to reduce drug absorption of substrates with repeated cycles of active efflux- metabolism: the efflux of xenobiotics back to intestinal lumen exposes drugs to further metabolism. Not surprisingly, a huge number of publications, clinical reports and in vitro studies indicate that many drugs are substrates for both P-gp and CYP3A4. These overlapping substrate specificities of these proteins result in complex and sometimes perplexing PK profiles of multidrug regimens.
1.3.3 Correlation between TKIs and ABC Transporters:
Substrates or Potential Inhibitors
Based on several published studies, TKIs are both substrates and inhibitors of ABC drug transporters, depending on the concentration of the inhibitor used and its affinity to the transporter78. Several TKIs are proved to be substrates or modulators of P-gp and this may explain the role of these transporters in the development of resistance to specific TKIs, and the effect on the alteration of the PK and toxicity of clinically important TKIs79,80. For example, the Abl/Src inhibitor dasatinib is one of the most known ABC transporters substrates: plasma concentration of dasatinib is significantly altered by P-gp and ABCG2, as well as its brain penetration80. Important PK aspects, e. g., short half-life, sub-optimal CNS penetration, and inadequate delivery to the tumor were hypothesized to be the explanation for a recent failure for this drug in a phase II clinical trial as monotherapy for recurrent GBM81. It is possible that the brain concentration was insufficient for antineoplastic effect and the cause may be a limited accumulation of dasatinib into the brain and brain tumor due to active efflux by P-gp82.
In this context, the drug dosing schedule, systemic concentrations of the TKI, its affinity to ABC drug transporters and expression levels of these transporters in tumor cells can be important factors in clinical resistance to TKIs. These can
eventually determine the potential of ABC transporters to confer resistance to TKIs in patients.
This suggests that an ideal TKI would be a molecule that can inhibit the activity of the target TK but would not be a substrate or modulator for ABC drug transporters.
Therefore, synthetic efforts and preclinical studies in the field should now be focused on developing such molecules. This will not only increase the probability of avoiding development of resistance to TKIs due to overexpression of ABC drug transporters, but would also lead to development of novel TKIs that kill tumor cells at much lower concentrations.
As said before, TKIs can also show inhibitor properties for the function of the transporter. Based on this assumption, another area of translational research that requires more in vivo studies is the use of TKIs as agents that can potentially modify the functional activity of ABC transporters. TKIs can possibly be exploited to improve PK or overcome resistance to chemotherapeutics that are substrates of ABC drug transporters in cells, tissues or tumors that express high levels of these transporters. A combination of TKIs inhibitors of ABC transporters with cytotoxic anticancer drugs that are their substrates, especially P-gp and ABCG2, may offer an advantage for treating the drug-resistant cancers83,84.
1.4 Compound Si306 against GBM: State of Art
As already said in paragraph 1.2.2, Src inhibitors pyrazolo[3,4-d]pyrimidine deriva- tives have shown interesting antitumor activity towards several cancer cells such as osteosarcoma, leukemia, GBM and NB.
In 2017 the couple Si306/pro-Si306 (Figure 1.8a, b), patented by Professor Mau- rizio Botta (WO2016066755), has been selected for an in depth investigation for their use against GBM. Both compounds have been fully characterized for their ADME properties and enzymatic activity (Table 1.3). Since their identification and selection, several iterative cycles of synthesis and biological testing, both in vitro and in vivo, have been performed to confirm the activity of these molecules.
