The design of covalent drugs has gained significant attention, shifting from targeting enzymatic catalytic sites to non-catalytic residues. Reversible covalent inhibitors are particularly promising due to their reduced toxicity compared to irreversible counterparts. Fluorinated ketones have shown promise as reversible covalent inhibitors, primarily via hemiketal formation at catalytic sites. However, their use in targeting non-catalytic residues, such as cysteine, remains rare. Cysteine, with its high nucleophilicity and relatively low abundance, is an ideal target for specific and long-lasting covalent inhibitors. This research explores the potential of fluorinated geminal diketones (FDKs) as multifaceted reversible covalent warheads. The addition of a second carbonyl group to the α-fluorinated keto moiety creates structures with increased complexity and unique properties. While difluorostatone (fluorinated β-keto amide) has been used as a warhead, it only utilizes one carbonyl. FDKs, with their two independent carbonyl electrophiles, offer increased flexibility. This study aims to evaluate the CF(CO)2 function as a multifaceted warhead by examining the complex equilibria compositions of FDK model compounds in octanol-water phases, measuring species-specific lipophilicities, and investigating their reactivity with nucleophilic amino acids like cysteine.

Existing literature extensively covers the design of covalent inhibitors, particularly reversible ones using fluorinated ketones. These studies primarily focus on targeting catalytic sites of enzymes through hemiketal formation. Examples include the inhibition of acetylcholinesterase, phospholipases, and dengue NS2B-NS3pro serine protease using trifluoromethyl ketones. Difluorinated carbonyl functions have also been incorporated into inhibitors for various proteases. However, the literature lacks comprehensive studies on the lipophilicity of these compounds, especially those existing in multiple equilibrium forms. Recently, a direct measurement method for species-specific log P values of α-di- and trifluoromethyl phenyl ketones was developed, opening new possibilities for studying such systems. This study builds upon this advancement, exploring the unique properties of FDKs as a novel class of reversible covalent inhibitors.

The study employed a multi-pronged approach combining synthesis, NMR spectroscopy, and computational methods. Six FDKs (1–6) were synthesized, with variations in fluorination patterns to systematically study their properties. A direct and simultaneous determination of species-specific log P values was achieved using a 19F-NMR-based method. This method involves measuring the equilibrium distribution of each species between octanol and water, and utilizing a reference compound to determine individual log P values, correcting for systematic errors. NMR techniques, including 19F-NMR, 13C-NMR, and 19F-DOSY-NMR, were used for species identification and signal assignment, distinguishing between ketones, enols, and hydrated forms. The 19F-DOSY-NMR was also used to probe species-solvent interactions. DFT calculations were performed at the m062x/6-311++g(d,p) level to study the conformations, polarity, and charge distributions of FDKs and their hydrated forms. Additionally, solid-state 19F-MAS-NMR measurements were performed using artificial membranes to investigate water-membrane partitioning. Finally, model reactions were conducted with protected cysteine and serine to study the regio- and chemoselectivity of the FDK warhead.

The study revealed that FDKs exist in complex equilibria systems, with some compounds having up to nine species in equilibrium in octanol-water mixtures. The 19F-NMR-based method successfully determined species-specific log P values for most components, demonstrating significant lipophilicity variation across different species within a single FDK system. In some cases, this resulted in a lipophilic-to-hydrophilic shift, showcasing the potential for adaptation to different environments. 19F-DOSY-NMR spectroscopy helped assign signals and understand species-solvent interactions, particularly H-bonding. The DFT studies showed the influence of fluorination and hydration on the polarity of FDKs and the formation of intramolecular hydrogen bonds in some hydrated forms, which can affect lipophilicity. Solid-state 19F-MAS-NMR experiments demonstrated the adaptation of FDK 1 and its hydrated forms to the different environments of liposomes and membranes. Model reactions showed high chemoselectivity of FDK 2 towards thiols, with reaction occurring at both carbonyls to form hemi-thioketal regioisomers when reacting with protected cysteine. This result highlights the warhead's potential flexibility in binding to noncatalytic cysteine residues in proteins. The use of computational methods provided better predictions than traditional log P calculation methods.

The findings address the research question by demonstrating the potential of FDKs as multifaceted warheads for reversible covalent drugs. The ability of FDKs to exist in multiple forms with varying lipophilicities allows them to adapt to different environments, from aqueous solutions to hydrophobic pockets within proteins. This adaptability is crucial for drug efficacy and pharmacokinetic properties. The observed regioselectivity and chemoselectivity towards cysteine residues are highly significant for designing specific and long-lasting covalent inhibitors targeting non-catalytic sites. The use of multiple analytical techniques and computational modelling provided a comprehensive understanding of the complex equilibria and the factors governing lipophilicity. This integrated approach allows for better prediction and manipulation of FDK properties.

This study introduces the CF2(CO)2 group as a novel multifaceted reversible covalent warhead. The ability of FDKs to adapt to various environments via equilibrium shifts between different species with varying lipophilicities makes them highly promising for drug development. Future studies should investigate the application of FDKs in targeting specific proteins, assessing in vivo efficacy and toxicity. Further exploration into the structure-activity relationships of FDKs, including additional modifications to optimize potency and selectivity, is warranted.

The study primarily used model compounds and in vitro experiments. The in vivo performance and potential toxicity of FDKs still require further investigation. The focus on cysteine as the primary nucleophilic target may limit the applicability to other residues. The DFT calculations were conducted in the gas phase and might not fully capture the complexities of solvation effects. The solid-state NMR experiments provided preliminary results on membrane partitioning, requiring further investigation using other methods such as fluorescence spectroscopy.