The development of covalent inhibitors represents an important strategy in the search for new
therapeutic agents for various diseases, with achieving adequate selectivity and optimal
reactivity remaining key challenges. In recent years, considerable attention has been devoted to
carbamoyl fluorides, whose chemical properties makes them promising covalent warheads for
targeting various proteins, including caspases. Caspases are cysteine proteases that play a central
role in the regulation of apoptosis and neuroinflammation. One of the best-studied members is
caspase-1, which triggers inflammatory responses by activating the pro-inflammatory cytokines
IL-1β and IL-18 and cleaving gasdermin D, thereby inducing pyroptotic cell death. Caspase-1
inhibitors hence hold considerable potential for the development of novel therapeutic agents for
neurodegenerative diseases.
As part of this master’s thesis, two model probes, carbamoyl fluorides 14 and 15., were
synthesized. The key synthetic step was a copper(I)-catalysed azide–alkyne cycloaddition
(CuAAC), a so-called click reaction that afforded a 1,4-disubstituted 1,2,3-triazole. In addition
to the synthesised probes, we investigated the reactivity of three smaller carbamoyl fluorides 1–
3, in the presence of various nucleophiles. The results showed that the reactivity of carbamoyl
fluorides depends on their structural scaffold. Compound 1, an isoindoline derivative, was the
least reactive, followed by compound 2, a benzylpiperidine derivative. Compound 2 underwent
rapid degradation in the presence of thiol nucleophiles, particularly dithiothreitol, and was also
more reactive in the presence of other amino acids. Indoline derivative 3, was the most reactive,
as the resonance effect of the aromatic ring increases the susceptibility of the carbamoyl fluoride
group to nucleophilic attack. In the presence of thiol-containing amino acids, it decomposed
completely within only a few hours. All three compounds were highly stable in ethanol. Model
probe 15 was less reactive than probe 14. They differ only in their linker: 14 contains an aliphatic
chain, whereas 15 contains tetraethylene glycol linker. In addition to thiol nucleophiles, 15
reacted rapidly with L-lysine, indicating that under the conditions employed it can also react
with amino groups and that its reactivity is not restricted to thiols. These findings contribute to
a better understanding of how the structure of carbamoyl fluorides affects their reactivity and
provide a basis for designing new covalent probes with appropriate stability and reactivity.
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