Gliomas (GB) account for nearly 50% of brain tumors. Numerous genetic alterations responsible for their development have been identified. The most frequently mentioned are mutations and overexpression of receptor tyrosine kinases, such as EGFR, as well as alterations in the RAS/MAPK signaling pathway and the PI3K/AKT/mTOR pathway, which regulate cell proliferation and apoptosis. Depending on the stage of tumor progression, the treatment of GB is based on surgical resection, optionally combined with chemotherapy and radiotherapy. In recent years, the development of targeted therapies has been observed. However, the use of selective kinase inhibitors has proven to be largely ineffective at the clinical trial stage. The mechanistic target of rapamycin (mTOR) drives several physiological and pathological cellular processes and is frequently deregulated in various types of cancer, including glioblastoma multiforme (GBM). To date, no attempts to target this pathway using kinase inhibitors have improved treatment outcomes for patients with GBM.

Serotonin (5-HT) is considered a growth factor for several types of cancer cells. It has been shown to stimulate the proliferation, migration, and invasion of glioma cells. In contrast to other serotonin receptors, the expression of 5-HT₆R is restricted to the central nervous system. Depending on time and subcellular localization, this receptor may engage different signaling pathways. 5-HT₆R modulates various key developmental targets, such as mTOR. Constitutively active 5-HT₆R contributes to increased mTOR activity in the brain. In the present project, the use of 5-HT₆R ligands is proposed as indirect modulators of the mTOR and CDK-5 kinases. In contrast to existing approaches in glioma pharmacotherapy, these compounds combine the inhibition of signaling pathways associated with glioma growth with a reduction in serotonin levels in the tumor microenvironment.

The aim of the project is to increase the commercialization potential of basic research conducted at the Cracow University of Technology within the NCN Preludium project entitled “New ligands of the 5-HT₆R receptor from the group of sulfonamide derivatives of cyclic arylguanidines in the treatment of glioblastoma multiforme.” As part of the Proof of Concept (PoC) activities, optimization of the synthesis methods for selected molecules is planned, using sonochemically or microwave-assisted synthesis, as well as a detailed evaluation of the anticancer potential of 5-HT₆R ligands in selected glioma cell lines in vitro. The main objective of the project is to confirm the antineoplastic activity of the developed compounds, using, among others, a 3D research model which, according to the literature, is considered to closely resemble the in vivo tumor microenvironment. It is planned to obtain biological material from patients diagnosed with GB through cooperation with the Medical University of Łódź and to perform in vitro tests using the acquired cell lines. The potential of the compounds to inhibit proliferation, migration, and invasion, as well as to promote apoptosis, will be evaluated. The compounds intended for biological studies will partly originate from the previously conducted Preludium project and will partly be modified following in silico analyses. This approach aims to potentially increase their modulatory capacity toward selected signaling pathways and to improve penetration of the blood–brain barrier. The outcome of the project will be compounds with confirmed anticancer activity against GB at the early preclinical research stage. In addition, their synthesis methods will be optimized, and their release profile and impurity profile will be characterized. The project is carried out with advisory support from the commercial entities Farmina sp. z o.o. and AT Analytica Prosta Spółka Akcyjna.

As part of the project, eight molecules with high affinity for serotonin receptors were selected and subsequently subjected to detailed analysis of their anticancer activity in vitro. Three of the investigated compounds originated from the previously completed Preludium project carried out at the Cracow University of Technology. Five new ligands were designed based on earlier results, supported by molecular modeling methods.

One of the outcomes of the PoC project is the development of a new, solvent-free method for the synthesis of these molecules, conducted under sonochemical conditions. Despite the absence of a solvent, liquefaction of the reagents in the reaction environment enables the effective use of ultrasound as a medium for energy transfer to the system. Application of the developed method allowed the products to be obtained with high yields ranging from 64% to 87%, with reaction times of less than 2 hours. In vitro studies were performed using both commercial glioma cell lines and cells isolated from oncological patients, in cooperation with the Medical University of Łódź. The tested compounds exhibited high anticancer activity. Two of them, characterized by the highest selectivity toward cancer cells relative to normal cells (astrocytes), were selected for detailed studies.

The two selected molecules, L184 and JK75, were further analyzed using 2D and 3D cellular models, employing four glioma cell lines, including two derived from tumor material obtained from oncological patients. It was found that the tested compounds exhibit pro-apoptotic and anti-proliferative activity and are capable of reducing the invasiveness of GB cells in vitro, although individual GB cell lines showed different, concentration-dependent sensitivities. The results also revealed the ability of L184 and JK75 to affect the cell cycle, suggesting a partial arrest in the G0/G1 phase. Both compounds are promising candidates for further studies as potential chemotherapeutic agents. Due to their likely distinct mechanism of action compared to currently used therapies, L184 and JK75 may significantly contribute to addressing the problem of drug resistance.