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Development of Palladium-Labile Prodrugs for Bioorthogonally-Activated Chemotherapy

Periodic Reporting for period 1 - ChemoBOOM (Development of Palladium-Labile Prodrugs for Bioorthogonally-Activated Chemotherapy)

Periodo di rendicontazione: 2016-02-01 al 2018-01-31

The social and economic relevance of cancer in Europe is continuously growing. Despite relevant advances in early diagnosis and treatment, the highest therapeutic challenges yet resides on treating cancer patients diagnosed with late-stage cancer due to the dose-limiting adverse effects. In this context, based on the biocompatibility and catalytic activity of Pd-functionalized devices in vivo, the ChemoBOOM project was focused on the development of biochemically-stable prodrugs (i.e. bioorthogonal prodrugs) that are specifically activated by bioorthogonal heterogeneous catalysis in order to reduce systemic side effects associated of chemotherapy.

The research program was divided into two specific research objectives:

Objective 1: Development of novel O-Propargylated Pd-labile prodrugs of cytotoxic agents. In recent years several transition metal-mediated bioorthogonal deprotection reactions have been reported, however they can be grouped in 2 classes: the deprotection of carbamate-masked primary amino groups and the N-depropargylation of endocyclic nitrogen atoms with lactam-lactim tautomerism. As the cytotoxicity of many therapeutic agents is modulated by other chemical functionalities, the 1st specific objective of the project was to extend the scope of functional groups amenable to activation by Pd chemistry in order to increase the diversity of bioorthogonally-activated prodrugs. This is, for example, the case of hydroxamic acid-based drugs such as HDAC inhibitor vorinostat whose pharmacological activity is endowed by the metal-chelating capacity of the hydroxamic acid group. During the course of this project, we have developed a novel strategy that enabled -for the first time- to devise a completely inactive precursor of vorinostat that is rapidly uncaged by biocompatible Pd-resins in cell culture models of glioma and lung cancer. To expand the scope of the locally-controlled bioorthogonal organometallic chemistry in biomedicine, we successfully extended this strategy beyond Pd and demonstrated the bioorthogonal activation of this prodrug by a heterogenous Au catalysts.

Objective 2. Development of precursors of combination therapy activated by Pd chemistry. One of the most promising approaches to address cancer heterogeneity is the use of combination therapy, which is based on the simultaneous use of drugs with different mode of actions and synergistic effect. Accordingly, the 2nd research objective of the project was to create an unprecedented class of bioorthogonal prodrug that consists in 2 chemotherapeutic drugs connected by a Pd-labile bioorthogonal promoiety that upon Pd-mediated cleavage release the active forms of each drug. Based on the uncaging chemistry validated in objective 1, we developed a novel anticancer codrug that combined 2 approved drugs into a single molecular entity and we have proved its in vitro activation via Pd-functionalized resins.
To address the stated objectives, we prepared a research program composed of 2 WP. For the implementation of the WP1, I prepared several bioorthogonal prodrugs of the HDAC inhibitor vorinostat. One of these derivatives proved to be highly bioorthogonal and was rapidly uncaged in the presence of Pd-functionalised resins both in vitro and cancer cell culture. The deprotection proceeds via a tandem mechanism triggered by the Pd-catalysed depropargylation of a phenolic ether group and followed by 1,6-elimination of a 4-hydroxybenzyl group directly attached to the OH of the drug’s hydroxamic acid group; a previously unreported process that takes place at physiological pH. These results derived in a scientific publication: Rubio-Ruiz, B. et al. J Med Chem. 2016, 59, 9974-9980.

Connected with the 1st research objective, over the course of the project we investigated new avenues for the activation of our prodrugs: (i) firstly, we explored Pd-nanoparticles in order to perform bioorthogonal organometallic (BOOM) catalysis inside the cancer cells. Although these nanodevices showed a high capacity to activate Pd-labile pro-dyes in PBS, their catalytic activity in PBS supplemented with FBS was significantly lower indicating that these nanoparticles are not suitable to perform intracellular BOOM catalysis. Since these nanomaterials displayed an exceptional biocompatibility and high photothermal conversion capacity, they were used to tag cancer cells and induce photothermal ablation upon short exposure to NIR light. This research was published in ACS Applied Materials and Interfaces (Rubio-Ruiz, B. et al. ACS Appl Mater Interfaces 2018, 10, 3341-3348); (ii) secondly, aiming to expand the scope of locally-controlled BOOM chemistry in biomedicine, we explored metallic gold to catalyse the activation of our prodrugs. To this end, a solid supported gold catalysts was prepared by in situ generation of Au-nanoparticles (Au-NP) within a PEG-grafted low-crosslinked polystyrene matrix. Embedding these Au-NP in a solid support, we protected the metal nanostructures from large thiol-rich biomolecules, while allowing the free entry of alkyne-functionalized small molecules to undergo gold-mediated chemistries in biological systems. The new devices were fully biocompatible and able to catalyse the activation of our previously synthesised vorinostat prodrugs in cancer cells. These results gave rise a scientific publication (Pérez-López, A.M.; Rubio-Ruiz, B. et al. Angewandte Chemie International Edition 2017, 56, 12548-12552). The validation of this Pd/Au-activatable prodrug generated valuable intellectual property (IP) that was protected (Rubio-Ruiz, B. et al. International patent application PCT/GB2017/051379).

Finally, for the implementation of the WP2, we designed and synthesised a novel Pd-labile codrug. Upon the presence of our biocompatible Pd-devices, the propargyl moiety is cleaved causing the self-fragmentation of the linking moiety and releasing of 2 different anticancer drugs in vitro. The validation of this novel codrug in cancer cell culture is still in progress.
During this fellowship, I have validated several prodrug strategies for the treatment of locally-advanced cancers. Particularly, we have prepared inactive precursors of clinically used vorinostat and demonstrated their specific and efficient activation in the presence of either Pd or Au heterogeneous catalysts. From an academic perspective, the novel approaches investigated have given rise to date into 3 world-class research publications that will serve as the basis to inspire others technologies and discoveries, thus contributing to increase the European excellence. Additionally, the validation of this first-in-class therapeutic strategy to treat cancer have resulted in the generation of valuable IP that has been protected. Several venture capital firms have already showed interest and we are under negotiations with them to progress this technology into complex animal models and human clinical trials. The advance of this novel therapeutic modality and its commercialization could have a significant impact both socially and economically in the EU considering the devastating effects that cancer cause in patients and the significant use of the European Health Systems resources for its treatment. This is well integrated into EU strategy, since this technology could not only strengthen the competitiveness and innovative capacity of European health-related institutions but also improve the health of European citizens.
Bioorthogonal activation of an inactive precursor of vorinostat