ETD

• C-nucleosides
• kinetoplastid parasites
• neglected tropical diseases
• nucleoside analogues

Neglected tropical diseases (NTDs) remain a major global health burden, particularly in low-income regions where limited healthcare infrastructure, inadequate surveillance, and socioeconomic vulnerability facilitate their persistence. Among them, kinetoplastid infections such as Human African trypanosomiasis (HAT), Chagas disease and leishmaniasis, are responsible for extensive morbidity and mortality. These illnesses are caused by protozoa of the Trypanosomatidae family, which share unique biological features such as kinetoplast DNA, polycistronic transcription, glycosome-based glycolysis and sophisticated immune evasion mechanisms. Current treatments for these diseases suffer from severe drawbacks including systemic toxicity, high cost, long or parenteral administration regimens and the emergence of drug resistance. As a result, the discovery of new safe and effective chemotherapeutic agents remains a pressing need.
Kinetoplastids are purine auxotrophs, relying entirely on salvage pathways for purine acquisition from their hosts. This biological dependency has encouraged drug-discovery efforts centered on nucleoside analogues, a structural class proven clinically valuable in antiviral and anticancer therapy. Despite promising enzyme inhibition data, many target-based approaches have failed to translate into in-cell efficacy due to metabolic redundancy. Consequently, phenotypic screening of nucleoside analogues has emerged as a more productive strategy, particularly within the 7-deazapurine family, where multiple analogues have demonstrated potent antiparasitic activity against Trypanosoma and Leishmania species.
Building upon this body of work, the present thesis explores the synthesis and biological evaluation of a new class of C-nucleoside analogues based on the pyrrolo[2,1-f][1,2,4]triazine scaffold. C-nucleosides possess enhanced metabolic stability relative to N-nucleosides due to their C–C glycosidic bond, and their scaffold allows the incorporation of diverse pharmacophores. The central objective of this work was to design a synthetic route allowing late-stage diversification at the 6-position of the heterocycle, enabling efficient construction of a small library of analogues suitable for phenotypic screening against kinetoplastid parasites.
A key strategic element was the installation of the 3,5-bis(trifluoromethyl)phenoxy (BTFP) group in the 6-position. This substituent is electronically activated and remarkably stable across a range of harsh conditions, while still undergoing smooth nucleophilic aromatic substitution (SNAr). Its presence makes it an ideal leaving group for diversification in the final synthetic steps.
The synthetic route began from commercially available pyrrolo[2,1-f][1,2,4]triazin-4(3H)-one, which was first chlorinated to afford the 6-chloro derivative. Nucleophilic substitution with the BTFP phenol yielded the key 6-BTFP intermediate, subsequently brominated at C9 using DBDMH under strictly controlled low-temperature conditions to ensure site-selectivity. The introduction of the ribose moiety required careful optimization: after metalation of the heterocycle, ribonolactone was added to form a hemiketal mixture, which was then stereoselectively reduced with hydrosilane to afford the desired β-C-nucleoside. Final hydrogenolytic removal of benzyl protecting groups yielded a single versatile intermediate, from which a library of analogues was generated.
The late-stage diversification step involved the efficient substitution of the BTFP group with a wide range of nucleophiles, enabling the construction of a 36-member library of C-nucleoside analogues. This strategy allowed the introduction of structurally diverse substituents including primary and secondary amines, aliphatic and aromatic alcohols, and thiols resulting in a broad spectrum of chemical properties across the series. The resulting derivatives span significant variations in polarity, steric demand, hydrophobicity and electronic character, providing a robust platform for exploring preliminary structure–activity relationships (SAR). The size and diversity of this library, achieved through a single convergent late-stage transformation, underscore the synthetic efficiency of the approach and set the basis for subsequent biological evaluation.
The biological evaluation of the synthesized library revealed several promising hits across chemotypes, with compound 25 (n-propyloxy) emerging as a clear lead candidate. Compound 25 demonstrated double-digit nanomolar potency against intracellular parasites and exceptional activity versus extracellular forms, while showing no detectable host-cell cytotoxicity in the assays performed. These features make compound 25 an excellent candidate for immediate follow-up studies, including in-depth DMPK profiling and preliminary in vivo evaluation. In addition to compound 25, a cluster of low- to mid-micromolar hits across the amino, oxy and thio series strengthens the SAR and validates the late-stage diversification strategy as a productive platform for hit discovery.
In conclusion, this thesis presents the first exploration of 6-modified pyrrolo[2,1-f][1,2,4]triazine ribosides as antikinetoplastid agents. It demonstrates that rational scaffold modification, combined with a divergent synthetic design, can generate structurally diverse libraries suitable for phenotypic screening. Although still at the hit-identification stage, the work offers foundational SAR insights and establishes a synthetic framework with clear potential. This study thus contributes constructively to ongoing efforts in nucleoside-based antiparasitic drug discovery and supports the future development of optimized C-nucleoside analogues targeting kinetoplastid parasites.