Schiff bases of sulphonamides as a new class of antifungal agent against multidrug‐resistant Candida auris

Abstract Invasive Candida infections in hospitalized and immunocompromised or critically ill patients have become an important cause of morbidity and mortality. There are increasing reports of multidrug resistance in several Candida species that cause Candidemia, including C. glabrata and C. auris, with limited numbers of antifungal agents available to treat patients with invasive Candida infections. Therefore, there is an urgent need to discover new antifungal agents that work against multidrug‐resistant Candida species, particularly C. auris, which has been identified as an emerging global pathogen. In this article, we report a new class of antifungal agents, the Schiff bases of sulphonamides, that show activity against all Candida species tested, with an MIC range of 4–32 µg/ml. Compound 2b showed activity against C. glabrata and a panel of fluconazole‐resistant C. auris strains, with MICs of 4–16 µg/ml. The drug‐like nature of these Schiff bases offers opportunities to optimize these compounds with medicinal chemistry techniques to obtain more potent analogs that can be progressed toward pre‐clinical evaluation.

Candida auris is an emerging fungal pathogen that was firstly identified in Asia in 2009. According to data from the CDC, the case count was increased by 328% in 2018, and until August 2020, more than 1000 cases were reported (Bhattacharya et al., 2020). The major concerns for C. auris are its multidrug resistance, high mortality rate, difficulty in fast identification, and high risks of healthcare outbreaks (Nett, 2019). In 2020, several cases of hospital candidemia outbreaks related to COVID-19 in Intensive Care Units and severe fungal co-infections have been reported across the world including UK (White et al., 2020), India (Chowdhary et al., 2020), and China (Song et al., 2020). Treatment of candidiasis, including infections caused by C. auris, relies on very few classes of antifungal drugs. The vast majority of C. auris isolates sent to CDC possessed resistance to fluconazole and up to one third are resistant to amphotericin B (Borman et al., 2016). Therefore, there is an urgent need to identify new antifungal agents containing new chemical classes to treat drug-resistant Candida infections.
Sulphonamides are a versatile class of drug-like chemical scaffolds that have shown a wide range of therapeutic activities including antimicrobial (Seydel, 1968), antitumor (Bouissane et al., 2006), antiviral (Gawin et al., 2008), and anti-inflammatory (Weber et al., 2004) effects. More recently sulphonamide derivatives like phosphodiesterase-5 inhibitor sildenafil (Kim et al., 2001) are being used in the treatment of erectile dysfunction. In addition, a wide variety of sulphonamide derivatives have been evaluated as experimental agents against ulcerative colitis (Wilson et al., 2004), rheumatoid arthritis (Levin et al., 2002), obesity (Hu et al., 2001), anticancer agents (Ma et al., 2012), and in Alzheimer's disease (Roush et al., 1998). One of the widely employed techniques to explore the therapeutic utility of this chemical class is the conversion of sulphonamides into Schiff bases by condensing them with different aldehyde derivatives. The Schiff bases have been reported as anti-infective agents including antimalarial (Rathelot et al., 1995), antimicrobial (Shi et al., 2007), antifungal (Guo et al., 2007), antiviral (Wang et al., 1990), anticonvulsant (Verma et al., 2004), and antiplasmodial (Adams et al., 2016) agents.
In this study, we explored the antifungal potential of three marketed sulfa drugs and their Schiff bases to identify new antifungal agents containing a sulphonamide chemical scaffold. While parent sulfa drugs were found to be inactive against all pathogenic Candida species tested, their Schiff bases showed promising antifungal activities against a panel of pathogenic Candida strains including an extended panel of multidrug-resistant C. auris.

| RE SULTS AND D ISCUSS I ON
The Schiff base derivatives of the sulfa drugs were synthesized by condensation of commercially available 4-amino-N-(5-methylisox azol-3-yl)benzenesulfonamide (sulphamethoxazole), 4-amino-N-( 4,6-dimethylpyrimidin-2-yl)benzenesulfonamide (sulfamethazine), and 4-amino-N-(6-methoxypyridazin-3-yl)benzenesulfonamide (Sulfamethoxypyridazine) with appropriate substituted aromatic aldehydes ( Figure 1) (Hamad, Abbas Khan, et al., 2020;, and the NMR data were compared with the literature, where applicable. The synthesized compounds ( Figure 2)  base analog of sulfamethoxypyridazine, 2c, containing the same aromatic aldehyde was synthesized to assess the importance of ring C (i.e., hydroxy and chloro-substituted phenyl ring, Figure 1b) in conferring antifungal activity to the sulfa drugs. The Schiff base 2c was also found to be active against the Candida strains with MICs ranging from 16 to 128 µg/ml. However, it was less active against all strains tested compared to 2b, but more active than fluconazole against the C. auris strain TDG1912. This suggests the heteroaryl ring A also plays a role in antifungal activity in addition to ring C of these Schiff bases ( Figure 1b).
To assess the effect of the electronegative halogen atom on the antifungal activity of the Schiff bases, compound 2d was synthesized in which the chlorine was substituted with more electronegative fluorine. Both compounds 2c and 2d were Schiff bases of sulfamethoxypyridazine with the fluorine substitution in ring C in place of chlorine which allowed direct comparison of the activity F I G U R E 2 Structures of the Schiff bases evaluated for antifungal activities against the Candida panel of these two compounds. The Schiff base 2d was slightly less active compared to 2c against all Candida strains (MIC range 16 to >128 µg/ml), and it was found to be inactive against the C. krusei NCPF3876 strain. This suggests that the introduction of the more electronegative fluorine in ring C has a negative impact on antifungal activity for this chemical scaffold.
To assess the importance of halogen substitution in ring C of the Schiff bases, compound 2e was synthesized in which the halogen atom in position-4 was replaced with another hydroxy substituent. Interestingly, the compound was found to be inactive against all Candida strains tested (Table 1) suggesting the presence of the halogen atom in the ring is essential for antifungal activity. The Schiff base 2f was synthesized to determine the effect of multiple halogen substitutions on activity. In this compound, the hydroxy group in position-2 was replaced with a chlorine substituent allowing a direct comparison between 2d and 2f. Surprisingly, compound 2f was also found to be completely inactive against all After observing promising activity against the multi-species Candida panel, we decided to focus more specifically on the ability of these Schiff bases to kill multidrug-resistant C. auris strains.
The Schiff bases 2b-2e were tested against an extended panel of fluconazole-resistant C. auris strains. All Schiff bases were found to be active against the extended C. auris panel, with MICs ranging from 8 to 128 µg/ml ( Table 2). The activity pattern of the compounds somewhat mirrored their activities against the multi-species Candida panel with 2b emerging as the most active compound with MICs in the range of 8-32 µg/ml. 2d was least active with an MIC range of 32-128 µg/ml. Interestingly, the Schiff base 2h with the halogen (bromine) substitution at the C5-position was found to be more active compared to 2g, and its activity against the C. auris panel was comparable to that observed for compound 2b. Overall, the activity of these Schiff bases against the C. auris panel is encouraging and provides a new chemical scaffold to develop more potent antifungal agents against a pathogen of global concern. This is the first report  (Figure 3 and Figures S10-S14) while the parent sulfa drugs did not interact with the Erg11. This suggests inhibition of Erg11 as a potential mechanism of action of these Schiff bases. However, more studies are required to determine the molecular mechanism of action of these Schiff bases against C. auris, which might initiate future drug discovery efforts using the Schiff bases of sulphonamides as a new antifungal chemical scaffold.

| CON CLUS ION
A new sulphonamide-based chemical scaffold has been identified with broad-spectrum antifungal activity against major Candida species, including multidrug-resistant C. auris strains. The Schiff bases are non-toxic against healthy human cell lines at the concentrations tested, which offers excellent opportunities to develop more potent analogs of this chemical class as antifungal agents. It was possible to establish a limited structure-activity relationship that shows the importance of both halogen and hydroxy substituents on antifungal activity. Molecular modeling suggests Erg11 inhibition as a potential mechanism of action, but further work is necessary to determine the target and mechanism of this chemical class.

| General Experimental
All the solvents and reagents were commercially available from

| Determination of minimum inhibitory concentration
The MIC was determined according to modified EUCAST guidelines for azoles, echinocandins, and flucytosine in which we culture the test organisms overnights in liquid rather than on agar for MIC testing and adjust to the correct cell concentration using absorbance. Briefly, strains were grown overnight in RPMI-1640-MOPS containing 2% glucose and back-diluted to a concentration of 0.5−5 × 10 5 mCFU/ml.

| Molecular modeling
The 3D structures of wild type and F126L, F132Y, and K143R mutant ERG11 were obtained from homology modeling using the retrieved amino acid sequence from gene sequence with the code of A0A2H4QC40 (Template PDB id code: 5v5z, Seq. identity: 71.46%). Three forms of the mutations were generated using the PyMol program with an appropriate rotamer of mutated amino acid, which does not lead to any steric clash with the neighboring residues. All the structures were minimized and equilibrated using the AMBER program. Then molecular docking was performed by GOLD software by applying ChemScore as the scoring function.
The cavity definition for the target protein was performed based on the known binding site of the corresponding crystal structures applied in this study.

ACK N OWLED G M ENTS
We thank HEC Pakistan for supporting AH's doctoral placement at King's College London. We acknowledge funding from PHE Pipeline (project 109502) and grant-in-aid projects (Project 109505 and 111742) for this work. We also thank Ginny Moore of Public Health England for providing C. auris strains isolated from UK hospitals.

CO N FLI C T O F I NTE R E S T
None declared.

E TH I C S S TATEM ENT
None required.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data generated or analyzed during this study are included in this published article and its supplementary material.