021 | Novel para-phenylenediamine-based derivatives as receptor tyrosine kinase-like orphan receptor 1 inhibitors: an in vitro preliminary characterization

Maria Rosaria Miranda¹, Gerardina Smaldone¹, Francesca Di Matteo¹, Valeria Napolitano¹, Michela Aliberti¹, Simona Musella¹, Veronica Di Sarno¹, Gianluigi Lauro¹, Giuseppe Bifulco¹, Giacomo Pepe¹, Giovanna Aquino¹, Mario Felice Tecce¹, Isabel Maria Gomez-Monterrey², Pietro Campiglia¹, Carmine Ostacolo¹, Alessia Bertamino¹, Tania Ciaglia¹, Vincenzo Vestuto¹ | ¹Department of Pharmacy, University of Salerno, Italy; ²Department of Pharmacy, University of Naples Federico II, Naples, Italy.

ROR1 kinase is an underexplored yet promising target for the development of novel anticancer agents, as it is highly expressed in several cancer cell lines while showing limited expression in non-tumor cells. This selective expression, together with the limited number of effective ROR1 inhibitors currently available, motivated the design and development of a research program aimed at identifying new chemical entities capable of inhibiting ROR1 and interfering with its protumoral activity. Step-by-step in silico studies guided the design and synthesis of para-phenylenediamine-based compounds. The synthesized derivatives were first evaluated for their ability to directly bind the ROR1 kinase domain using surface plasmon resonance (SPR) assays, allowing the selection of the most promising candidates for cellular studies. Antiproliferative activity was then assessed in SH-SY5Y neuroblastoma and JeKo-1 mantle cell lymphoma cell lines, revealing a markedly stronger effect in JeKo-1 cells. Based on this higher sensitivity, further biological investigations were performed in JeKo-1 cells, demonstrating a significant induction of apoptosis upon treatment. Cellular studies confirmed direct ROR1 engagement and a consequent reduction in its phosphorylation, leading to modulation of downstream survival signaling pathways and activation of pro-apoptotic mechanisms. Among the synthesized compounds, derivative 17 emerged as the most promising candidate, also displaying a favorable in vitro pharmacokinetic stability profile. Overall, these findings identify compound 17 as a novel chemotype targeting ROR1, providing a solid basis for further structure–activity relationship optimization and hit-to-lead development.