Carbon monoxide (CO) is an endogenous signaling molecule and has therapeutic values. However, the application of CO in the development of therapeutic options is hampered by the lack of pharmaceutically acceptable delivery methods. Inhalation of CO is not an ideal option for wide-spread clinical applications. Existing CO releasing molecules (CORMs) are mostly metal complexes, which have toxicity concerns to overcome. Some metal free CORMs have been developed. However, they all require light as a trigger to release CO, which limits their applications in vivo. Herein, we describe a metal-free CO prodrug approach using an intramolecular inverse electron-demanded Diels-Alder reaction. Such prodrugs can release CO spontaneously under physiological conditions with tunable release rates with the concomitant formation of a fluorescent reporter after CO releases. This intramolecular “click and release” strategy represents a milestone in the development of CO based therapeutics.

Résumé confidentiel / Confidential abstract

This thesis deals with the synthesis of prodrugs based on analogs of nucleoside phosphonates derived from 6-fluoro-3-hydroxypyrazine-2-carboxamide (T-705) and 3- hydroxypyrazine-2-carboxamide (T-1105). T-705 and T-1105 act as inhibitors of an influenza RNA polymerase. Both compounds mimic naturally occurring nucleobases, so their fluorinated nucleoside phosphonates could also be biologically active. Derivatives of 2-deoxy-2-fluoro-D-ribose (2-FdR) were prepared in this work. Because of complications during the syntthesis of 3-deoxy-3-fluoro-D-ribose (3-FdR) derivatives, 5- deoxy-5-fluoro-D-xylose (5-FdX) derivatives were prepared instead. Deoxyfluorination was done after incorporation of suitable protecting groups followed by selective deprotection and phosphonate binding. Furthermore nucleosides were synthetised using silyl-Hilbert-Johnson method and their bis-POM derivattives were also prepared. Key words: favipiravir (T-705), T-1105, prodrugs, phosphonates, fluorinated nucleosides

6-Diazo-5-oxo-L-norleucine (DON, 1) is a non-standard amino acid with proven antitumor activity found in soil bacteria of the genus Streptomyces. However, due to the considerable systemic toxicity manifested mainly in the gastrointestinal tract, DON alone is not a suitable clinical candidate for the treatment of cancer. One of the ways to solve the problem of its toxicity is the reversible structural modification of this molecule by protecting both its amino group and carboxyl functional group, by preparing the so-called prodrug of DON. The prepared prodrug may suitably alter the distribution of DON in the body and at the same time increase its permeability to brain tissue. Due to this structural modification, its side effects can be eliminated and a substance for the treatment of brain tumors, such as glioblastoma multiforme (GBM), can potentially be formed. In my dissertation, five strategies for the specific delivery of DON to the brain using different types of its prodrugs are discussed. The new prodrugs are designed to be either capable of spontaneous penetration across the blood-brain barrier or of being a substrate for one of its influx transporters. At the same time, these prodrugs should be stable in other metabolically active organs and blood plasma in order to sufficiently reduce the...

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Improving the therapeutic efficacy and quality of life of patients by reducing the side effects caused by non-specificity of cytotoxic drugs has been a challenge in cancer treatment. A hypothesis was developed where integrin binding induced conformational change in a drug conjugated to hairpin peptide with an integrin binding ligand can lead to preferential accumulation of drug and reduced collateral damage by decreased premature prodrug activation. A model drug, MTX and a tripeptide ligand, RGD, known to specifically bind tumor overexpressing α v β 3 integrin receptors, were selected to test the hypothesis. A twelve amino acid sequence that has been previously shown to preferentially adopt an anti-parallel beta hairpin conformation in aqueous environment was flanked with MTX and RGD on N and C termini respectively by solid phase peptide synthesis to form a labile link between Arg-Glu specifically cleaved by SGPE, a Streptomyces griseus derived endopeptidase. Adenoviral vector was developed using AdEasy system for β 3 cDNA transfection to overexpress integrin α v β 3 receptor. MTX-α-RGD and MTX-β-hairpin-RGD were characterized using MALDI-TOF (MTX-α-RGD, 782.6(M+H + ); MTX-β-hairpin-RGD, 2272.1(M+H + )). Cell adhesion assay using HUVEC and A549 cells that overexpress α v β 3 showed that RGD conjugated prodrugs recognize and preferentially bind to integrin α v β 3 in RGD dependent manner. In rabbit plasma, MTX-β-hairpin-RGD was found to be 3 times more stable than MTX-α-RGD. In the absence of α v β 3 binding, SGPE mediated hydrolysis rate of MTX-β-hairpin-RGD was 0.7±0.1 ng/hr, that was significantly (P<0.025) lower than that of MTX-α-RGD (1.0±0.1ng/hr), a prodrug without hairpin structure. In presence of α v β 3 over-expressing cells, significant increase (P<0.025) in hydrolysis rate of MTX-β-hairpin-RGD to 1.0±0.1 ng/hr was observed, not significantly (P=0.6) different from that of MTX-α-RGD (1.1±0.1ng/hr). In addition, there was 400% increase in the fluorescence when FRET based quenching was abolished by the binding induced unfolding. These experiments along with docking studies using molecular modeling support the binding induced unfolding. Results from this investigation suggest that drugs conjugated to peptide ligands such as RGD may reduce the dose needed to achieve therapeutic concentrations by preferential recognition and binding to overexpressed integrin markers. Secondly, reduction of premature activation of prodrugs and thus reduced collateral damage may be achieved by making the the drug release to occurs preferentially upon binding to cells expressing specific integrin markers.

The full text will be available at the end of the embargo period: 28th March 2027

Introduction Cytochromes P450 (CYPs) are the major family of enzymes responsible for detoxification and metabolism of a wide range of both endogenous and xenobiotics chemicals in living organisms. The use of CYPs to activate prodrugs to cytotoxins selectively in tumours has been explored including AQ4N, Phortress and Aminoflavone. CYP1A1, CYP1B1, CYP2W1, and CYP4F11 have been identified as expressed in tumour tissue and surrounding stroma at high frequency compared to most normal tissues. Aim is to investigate the differential metabolism of novel chloromethylindoline by high frequency expressed CYPs in tumours. This differential may be exploited to elicit a selective chemotherapeutic effect by metabolising inert small molecules to potent cytotoxins within the tumour environment. Materials and Methods Sensitive and specific LC/MS/MS techniques have been developed to investigate the metabolism of chloromethylindolines. Recombinant enzymes and transfected cell lines were used to investigate the metabolic profiles with a focus on production of the cytotoxic derivatives of chloromethylindolines. Results Detailed metabolic studies show that (1-(Chloromethyl)-1,2-dihydropyrrolo [3,2-e]indol-3(6H)-yl)(5-methoxy-1H-indol-2-yl) methanone (ICT2700) and other chloromethylindolines are converted by CYP1A1 mediated hydroxylation at the C-5 position leading to highly potent metabolites. In vitro cytotoxicity studies showed differentials of up to 1000-fold was achieved between CYP1A1 activated compared to the non-metabolised parent molecules. The reactivity of metabolites of ICT2700 was also explored using glutathione as a nucleophile. The metabolites were identified by a combination of LC/MS and LC MS/MS techniques. Investigations using mouse and human liver microsomes show that a large number of metabolites are created though none were shown to be associated with a potential anticancer effect. Studies focused on CYP2W1 show that this isoform metabolised ICT2706 to a cytotoxic species and a pharmacokinetic study showed a good distribution of ICT2706 into mouse tissues including tumour. However metabolism of ICT2726 by CYP2W1 resulted only in a non-toxic metabolite profile and may have potential as a biomarker for functional CYP2W1 in tissues. Preliminary studies show that palmitic acid hydroxylation is a useful marker of functional CYP4F11. Summary and conclusion The in vitro results show that the chloromethylindolines are a novel class of agent with potential as prodrugs that following specific hydroxylation by CYP1A1 and CYP2W1 are converted to ultra-potent cytotoxins. Other metabolites are also evident which are not cytotoxic. Studies in vivo show that selected chloromethylindolines possess a good pharmacokinetic profile and show potential as prodrug anticancer agents that require activation by CYP1A1 or CYP2W1. The methods, results, progress and suggestions for future work are presented in this thesis.

The full text will be available at the end of the embargo period: 3rd April 2025 / The author's name as given on this thesis is Francis MPRAH BARNIEH. His publications use the name format Francis M. Barnieh.

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