Small‐Molecule Inhibitors Targeting Sterol 14α‐Demethylase (CYP51): Synthesis, Molecular Modelling and Evaluation Against Candida albicans

Abstract Fungal infections are a global issue affecting over 150 million people worldwide annually, with 750 000 of these caused by invasive Candida infections. Azole drugs are the frontline treatment against fungal infections; however, resistance to current azole antifungals in C. albicans poses a threat to public health. Two series of novel azole derivatives, short and extended derivatives, have been designed, synthesised and investigated for CYP51 inhibitory activity, binding affinity and minimum inhibitory concentration (MIC) against C. albicans strains. The short derivatives were more potent against the C. albicans strains (e. g., MIC 2‐(4‐chlorophenyl)‐N‐(2,4‐dichlorobenzyl)‐3‐(1H‐imidazol‐1‐yl)propanamide (5 f) <0.03 μg/mL, N‐(4‐((4‐chlorophenyl)sulfonamido)benzyl)‐2‐phenyl‐3‐(1H‐1,2,4‐triazol‐1‐yl)propanamide (12 c), 1 μg/mL, fluconazole 0.125 μg/mL) but both displayed comparable enzyme binding and inhibition (5 f K d 62±17 nM, IC50 0.46 μM; 12 c K d 43±18 nM, IC50 0.33 μM, fluconazole K d 41±13 nM, IC50 0.31 μM, posaconazole K d 43±11 nM, IC50 0.2 μM). The short series had poor selectivity for CaCYP51 over the human homologue, whereas the selectivity of the extended series, for example, compound 12 c, was higher (21.5‐fold) than posaconazole (4.7‐fold) based on K d values, although posaconazole was more selective (615‐fold) than 12 c (461‐fold) based on IC50 values. Based on inhibitory activity and selectivity profile, the extended series are the better of the two series for further development.


Supporting Information
S2 Figure S1. Spectral characterization of CaCYP51 and Δ60HsCYP51 S3-S4 Figure S2. CaCYP51 type II azole binding difference spectra S5 Figure S3. CaCYP51 azole saturation curves S6 Figure S4. Δ60HsCYP51 azole inhibition profiles S7 Figure S5. A schematic of detailed ligand atom interactions of representative (S)-enantiomers of short derivative 5f and extended derivative 12c with the protein residues of wild-type CaCYP51 active site. Interactions that occur more than 30.0% of the simulation time in the selected trajectory (0 through 100 ns) are shown.
S8-S19 Procedures and characterisation of synthesised compounds S20-S27 HPLC traces of final 5 and 12 series compounds

(S)--5f
(S)--12c Figure S5. A schematic of detailed ligand atom interactions of representative (S)-enantiomers of short derivative 5f and extended derivative 12c with the protein residues of wild-type CaCYP51 active site.
Interactions that occur more than 30.0% of the simulation time in the selected trajectory (0 through 100 ns) are shown. .

Procedures and characterisation of synthesised compounds:
General procedure for the preparation of amides (3).
To a suspension of acid (1) (5 mmol) in dry CPME (15 mL) and activated 4Å molecular sieves (~ 1 mg/mmol) was added benzylamine (2) (5.5 mmol) followed by B(OMe)3 (1.14 mL, 10 mmol). The resulting mixture was heated at 100 o C overnight. Upon completion, the reaction mixture was cooled to room temperature and diluted with acetone (10 mL) and H2O (1 mL). Amberlite IRA743 (0.5 g), Amberlyst 15 (0.5 g) and Amberlyst A26 (OH) (0.5 g) resins were added and the resulting suspension was stirred for 2 h. After disappearance of any remaining starting materials (monitored by TLC), anhydrous MgSO4 was added to the mixture. The reaction mixture was filtered, and the residue washed with acetone (2 x 10 mL), and the combined filtrates concentrated under reduced pressure to give the product, which was washed with Et2O/petroleum ether to remove any remaining CPME.