Glioblastoma (GBM) is an extremely aggressive brain tumor and the most common form of astrocytoma, accounting for more than half of all malignant brain tumors. It is particularly difficult to eradicate. The relative survival probability over a period of five years is no more than 7%, and the median estimated survival is approximately 15 to 18 months. Only a limited number of chemotherapeutic drugs have been approved for its treatment, due to the substantial challenges associated with targeting this disease. Over the years, many strategies have been explored with the aim of improving GBM treatment outcomes and increasing patient survival. This Ph.D. thesis focuses on the synthesis and biological evaluation of novel anti-GBM chemotherapeutic agents, pursuing two promising therapeutic approaches: 1. Development of new dopamine D4 receptor (D4R) antagonists. D4R antagonism has been proved to disrupt the autophagy-lysosomal pathway of GBM stem cells, leading to apoptosis. Additionally, the Cancer Genome Atlas data on GBM gene expression has demonstrated that adult patients with high D4R levels have a reduced survival rate in comparison to those exhibiting low expression. The research team with whom I developed my Ph.D. thesis has recently demonstrated that the known M1 muscarinic bitopic agonist 77-LH-28-1 also behaves as a potent and selective D4R antagonist. This compound was taken as a starting point for a structure-activity relationship (SAR) study, which resulted in the identification of its promising 4-benzylpiperidine (10), phenylpiperazine (13) and p-chlorophenylpiperazine (33) derivatives. In my thesis work, these molecules were used as lead compounds for additional SAR studies involving a series of piperadine derivatives, in which the distances between the pharmacophoric features of 77-LH-28-1 and 10 have been modified (compounds 43-54), and a series of piperazine derivatives, in which the aromatic area of 13 has been varied and the p-chloro substituent of 33 has been replaced by different electron withdrawing groups (compounds 55-66). All the synthesised molecules have been evaluated for their affinity at D2-like receptor subtypes. The most potent and effective piperidines proved to be the lower homologue of lead 10 (compound 46) and its regioisomer with the N atom in position 4 instead of position 1 of the piperidine ring (compound 50). Concerning piperazines, the most promising results were obtained by replacing the N4-phenyl terminal of 13 with a naphthyl group: the α-naphthyl derivative 59 showed D4R affinity higher than lead 13, while the β-naphthyl compound 60 demonstrated to be about 10-fold more selective for D4R with respect to 13. From functional studies performed on the most potent and selective compounds, all derivatives behaved as D4R antagonists in both Gi/Go activation and β-arrestin2 recruitment assays. Interestingly, all of them induced a dose- dependent cell viability decrease and cell cycle alteration in U87 MG, T98G, and U251 MG human GBM cell lines, accompanied by induction of cell death and cell cycle arrest, promotion of reactive oxygen species (ROS) production, induction of mitochondrial dysfunction, and inhibition of colony formation. 2. Development of new copper-based antitumor agents. Recent studies have demonstrated that copper (Cu) based antitumor drugs can trigger cuproptosis, a recently identified form of Cu- dependent non-apoptotic cell death, in cancer cells. Over the years, an increasing body of evidence has supported the role of Cu in the development of effective anti-GBM therapies, highlighting its potential against these highly aggressive tumors. The research team with whom I developed my Ph.D. thesis has recently demonstrated that Cu complexes supported by ligands bearing biologically active molecules conjugated to pyrazolyl coordinating groups can affect proliferation, growth and death of GBM cell lines. In this context, in my thesis work the new ligand bis(1H-pyrazol-1-yl)acetyl-3,5- dimethyladamantane-1-amide (LMem) was synthesized by conjugating the drug memantine with the bifunctional species bis(pyrazol-1-yl)acetic acid (LH). LMem was then employed as a supporting ligand for Cu(II) and Cu(I) complexes 97-99 and 100-103, respectively. In the synthesis of the Cu(I) complexes 100-103, the lipophilic triphenylphosphine (PPh3) and hydrophilic 1,3,5-triaza-7- phosphaadamantane (PTA) were selected as co-ligands, in order to stabilize Cu in +1 oxidation state and to confer different solubility properties to the corresponding metal complexes. X-ray diffraction (XRD) studies were carried out to describe the molecular structure and the intermolecular contacts of the LMem ligand. Among all the compounds tested, the lipophilic Cu(I) complexes 100 and 101, bearing one or two PPh3 co-ligands, respectively, exhibited potent antiproliferative and cytotoxic effects in U87 MG, T98G, and U251 MG human GBM cell lines. These effects were associated with increased ROS production and mitochondrial dysfunction, as evidenced by mitochondrial depolarization and altered intracellular distribution. Furthermore, the cytotoxic activity of these compounds was shown to be Cu-dependent, as it was effectively inhibited by the Cu chelator tetrathiomolybdate (TTM), confirming the essential role of Cu in their mechanism of action. Overall, the results presented in this Ph.D. thesis highlight the potential of both Cu-based complexes and D4R antagonists as innovative therapeutic strategies against GBM. The developed compounds demonstrated significant antiproliferative activity and well-defined mechanisms of action in relevant GBM cellular models, supporting the validity of the proposed approaches. Importantly, the identified compounds represent promising leads that may serve as valuable starting points for further chemical optimization and preclinical development, potentially contributing to the advancement of more effective treatments for this highly aggressive tumor.
SYNTHESIS AND BIOLOGICAL EVALUATION OF INNOVATIVE AGENTS POTENTIALLY USEFUL FOR THE TREATMENT OF GLIOBLASTOMA
MATTEUCCI, FEDERICA
2026-04-27
Abstract
Glioblastoma (GBM) is an extremely aggressive brain tumor and the most common form of astrocytoma, accounting for more than half of all malignant brain tumors. It is particularly difficult to eradicate. The relative survival probability over a period of five years is no more than 7%, and the median estimated survival is approximately 15 to 18 months. Only a limited number of chemotherapeutic drugs have been approved for its treatment, due to the substantial challenges associated with targeting this disease. Over the years, many strategies have been explored with the aim of improving GBM treatment outcomes and increasing patient survival. This Ph.D. thesis focuses on the synthesis and biological evaluation of novel anti-GBM chemotherapeutic agents, pursuing two promising therapeutic approaches: 1. Development of new dopamine D4 receptor (D4R) antagonists. D4R antagonism has been proved to disrupt the autophagy-lysosomal pathway of GBM stem cells, leading to apoptosis. Additionally, the Cancer Genome Atlas data on GBM gene expression has demonstrated that adult patients with high D4R levels have a reduced survival rate in comparison to those exhibiting low expression. The research team with whom I developed my Ph.D. thesis has recently demonstrated that the known M1 muscarinic bitopic agonist 77-LH-28-1 also behaves as a potent and selective D4R antagonist. This compound was taken as a starting point for a structure-activity relationship (SAR) study, which resulted in the identification of its promising 4-benzylpiperidine (10), phenylpiperazine (13) and p-chlorophenylpiperazine (33) derivatives. In my thesis work, these molecules were used as lead compounds for additional SAR studies involving a series of piperadine derivatives, in which the distances between the pharmacophoric features of 77-LH-28-1 and 10 have been modified (compounds 43-54), and a series of piperazine derivatives, in which the aromatic area of 13 has been varied and the p-chloro substituent of 33 has been replaced by different electron withdrawing groups (compounds 55-66). All the synthesised molecules have been evaluated for their affinity at D2-like receptor subtypes. The most potent and effective piperidines proved to be the lower homologue of lead 10 (compound 46) and its regioisomer with the N atom in position 4 instead of position 1 of the piperidine ring (compound 50). Concerning piperazines, the most promising results were obtained by replacing the N4-phenyl terminal of 13 with a naphthyl group: the α-naphthyl derivative 59 showed D4R affinity higher than lead 13, while the β-naphthyl compound 60 demonstrated to be about 10-fold more selective for D4R with respect to 13. From functional studies performed on the most potent and selective compounds, all derivatives behaved as D4R antagonists in both Gi/Go activation and β-arrestin2 recruitment assays. Interestingly, all of them induced a dose- dependent cell viability decrease and cell cycle alteration in U87 MG, T98G, and U251 MG human GBM cell lines, accompanied by induction of cell death and cell cycle arrest, promotion of reactive oxygen species (ROS) production, induction of mitochondrial dysfunction, and inhibition of colony formation. 2. Development of new copper-based antitumor agents. Recent studies have demonstrated that copper (Cu) based antitumor drugs can trigger cuproptosis, a recently identified form of Cu- dependent non-apoptotic cell death, in cancer cells. Over the years, an increasing body of evidence has supported the role of Cu in the development of effective anti-GBM therapies, highlighting its potential against these highly aggressive tumors. The research team with whom I developed my Ph.D. thesis has recently demonstrated that Cu complexes supported by ligands bearing biologically active molecules conjugated to pyrazolyl coordinating groups can affect proliferation, growth and death of GBM cell lines. In this context, in my thesis work the new ligand bis(1H-pyrazol-1-yl)acetyl-3,5- dimethyladamantane-1-amide (LMem) was synthesized by conjugating the drug memantine with the bifunctional species bis(pyrazol-1-yl)acetic acid (LH). LMem was then employed as a supporting ligand for Cu(II) and Cu(I) complexes 97-99 and 100-103, respectively. In the synthesis of the Cu(I) complexes 100-103, the lipophilic triphenylphosphine (PPh3) and hydrophilic 1,3,5-triaza-7- phosphaadamantane (PTA) were selected as co-ligands, in order to stabilize Cu in +1 oxidation state and to confer different solubility properties to the corresponding metal complexes. X-ray diffraction (XRD) studies were carried out to describe the molecular structure and the intermolecular contacts of the LMem ligand. Among all the compounds tested, the lipophilic Cu(I) complexes 100 and 101, bearing one or two PPh3 co-ligands, respectively, exhibited potent antiproliferative and cytotoxic effects in U87 MG, T98G, and U251 MG human GBM cell lines. These effects were associated with increased ROS production and mitochondrial dysfunction, as evidenced by mitochondrial depolarization and altered intracellular distribution. Furthermore, the cytotoxic activity of these compounds was shown to be Cu-dependent, as it was effectively inhibited by the Cu chelator tetrathiomolybdate (TTM), confirming the essential role of Cu in their mechanism of action. Overall, the results presented in this Ph.D. thesis highlight the potential of both Cu-based complexes and D4R antagonists as innovative therapeutic strategies against GBM. The developed compounds demonstrated significant antiproliferative activity and well-defined mechanisms of action in relevant GBM cellular models, supporting the validity of the proposed approaches. Importantly, the identified compounds represent promising leads that may serve as valuable starting points for further chemical optimization and preclinical development, potentially contributing to the advancement of more effective treatments for this highly aggressive tumor.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
