Development of squaramide-based self-immolative spacers for drug delivery

Abstract

This PhD thesis entitled “Squaramide-Based Self-Immolative Spacers for Drug Delivery” describes the development of squaramide spacers able to undergo triggereddisassembly for the release of bioactive compounds. As a starting point, we synthesised a small collection of squaramate esters to study their hydrolytic stability in biologically simulated conditions. These compounds bear different side chains with chemical functionalities that may assist the hydrolysis or cyclization reaction. The kinetic study has permitted to find that the propanediamino and the ethyl sulphur side chains successfully undergo the cyclization reaction releasing ethanol as the leaving group. Based on the results found for squaramate ester derivatives, we designed and synthesized squaramide-based self-immolative models bearing amines as the leaving group. We explored both anilines and aliphatic amines to this purpose. The kinetic study revealed that the rate of disassembly was strongly dependent on the pH and the nucleofugacity of the leaving group. Henceforth the release of anilines was faster than n-butylamine. Additionally, N-methylated models showed faster cyclization rates and better aqueous solubility. Overall, the most suitable squaramide-based self-immolative linker should present the following key features: 1) Propanediamino chain as the nucleophile 2) Aniline as the chemical nature of the leaving group or cargo 3) Nmethylation of the squaryl ring. As proof of concept, we applied the optimised design in the synthesis of a prodrug. We selected the p-nitrobenzyl carbamate residue as the trigger group and phenylenediamine mustard as the active drug. The disassembly process was enzymatically triggered by a nitroreductase using NADH as the reductive agent. We tested the biological activity of the prodrug against glioblastoma cancer cells. The clonogenic assay performed with LN229 cells showed that the treatment with the parent mustard drug had no effect on the cell growth but, when treated with the mustard prodrug, we could observe the significant decrease in the survival factor (down to 24 %). This result was outstanding since the treatment with temozolomide, a common antitumor agent used in the treatment of brain cancer, showed a survival factor of 58 %.