# Exploring the antifungal potential of novel carbazate derivatives as promising drug candidates against emerging superbug, Candida auris

Tazeen Fatima, Zeeshan Fatima, Muriel Billamboz, Saif Hameed

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Tazeen Fatima, Zeeshan Fatima, Muriel Billamboz, Saif Hameed. Exploring the antifungal potential of novel carbazate derivatives as promising drug candidates against emerging superbug, Candida auris. Bioorganic Chemistry, 2024, 153, pp.107782. 10.1016/j.bioorg.2024.107782. hal-04920525

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# Exploring the antifungal potential of novel carbazate derivatives as promising drug candidates against emerging superbug, *Candida auris*

Tazeen Fatima<sup>a</sup>, Zeeshan Fatima<sup>a</sup>, Muriel Billamboz<sup>b,*</sup>, Saif Hameed<sup>a,*</sup>

## ARTICLE INFO

**Keywords:**

Candida

Efflux pump

Ergosterol

Oxidative stress

Biofilm

C. elegans

## ABSTRACT

*Candida auris* (*C. auris*) has caused notable outbreaks across the globe in last decade and emerged as a life-threatening human pathogenic fungus. Despite significant advances in antifungal research, the drug resistance mechanisms in *C. auris* still remain elusive. Under such pressing circumstances, research on identification of new antifungal compounds is of immense interest. Thus, our studies aimed at identifying novel drug candidates and elucidate their biological targets in *C. auris*. After screening of several series of synthetic and hemisynthetic compounds from JUNIA chemical library, compounds C4 (butyl 2-(4-chlorophenyl)hydrazine-1-carboxylate) and C13 (phenyl 2-(4-chlorophenyl) hydrazine-1-carboxylate), belonging to the carbazate series, were identified to display considerable antifungal activities against *C. auris* as well as its fluconazole resistant isolates. Elucidation of biological targets revealed that C4 and C13 lead to changes in polysaccharide composition of the cell wall and disrupt vacuole homeostasis. Mechanistic insights further unravelled inhibited efflux pump activities of ATP binding cassette transporters and depleted ergosterol content. Additionally, C4 and C13 cause mitochondrial dysfunction and confer oxidative stress. Furthermore, both C4 and C13 impair biofilm formation in *C. auris*. The *in vivo* efficacy of C4 and C13 were demonstrated in *Caenorhabditis elegans* model after *C. auris* infection showing reduced mortality of the nematodes. Together, promising antifungal properties were observed for C4 and C13 against *C. auris* that warrant further investigations. To summarise, collected data pave the way for the design and development of future first-in-class antifungal drugs.

## 1. Introduction

There has been considerable rise in the global burden of fungal disease due to marked increase in immunocompromised patients particularly those undergoing chemotherapy or organ transplantation. Outbreaks of invasive fungal infections can result from susceptibility to infectious environmental sources or possibly contaminated medical items. Among the invasive fungal infections caused by opportunistic pathogenic fungi, Candidiasis cause by genus *Candida* is the most threatening across the globe. Among them, *Candida albicans* is the most common species of *Candida* [13] along with *non-albicans* species such as *Candida glabrata*, *Candida parapsilosis*, *Candida tropicalis* and *Candida krusei* [68]. However, *Candida auris* (*C. auris*), which was first isolated in 2009 from the external auditory canal of a patient at hospital in Japan [67], is emerging as new superbug gaining attention world-wide [10]. Due to its inherent multi drug-resistance, capability to survive on surface and misidentification as Candida haemulonii, *C. auris* poses new threats

to public health [16]. Therefore, the World Health Organization (WHO) has recently listed *C. auris* as a fungal pathogen of great importance in its list of important pathogens for leading research, development, and public health [83]. *C. auris* is common in some long-term care and intensive care facilities where it can spread among patients, causing infection and systemic illness [75]. Hence, more effective antifungal strategies are needed to fight against this emerging and drug-resistant fungal infections [24,22].

In our continuous attempt to discover new antifungal agents to fight against *C. auris*, a panel of synthetic compounds from the JUNIA-HEI laboratory has been evaluated. Among all tested chemical families, carbazate derivatives exhibited the highest potential as antimicrobial agents. Carbazate derivatives are obtained from the reaction of hydrazines (R²-NH-NH₂) with chloroformates (R¹OCOCl) as described in Fig. 1. Their scaffold is based on a R²-NH-NH-CO-O-R¹ skeleton, where R¹ and R² could be alkyl, aryl or heterocyclic moieties (Fig. 1). The carbazate can drive redox reactions, generally mediated by metals (Kim

<footnote><sup>a</sup> Amity Institute of Biotechnology, Amity University Haryana, Gurugram Manesar 122413, India</footnote>

<footnote><sup>b</sup> ICL, JUNIA, Université Catholique de Lille, LITL, F-59000 Lille, France</footnote>

<footnote>* Corresponding authors.</footnote>

<footnote>E-mail addresses: muriel.billamboz@junia.com (M. Billamboz), shameed@ggn.amity.edu (S. Hameed).</footnote>
et al, 2018), chelate metallic cations [53] or be modified to scavenge proteins [88]. Carbazate derivatives are also high radical scavenging agents. It is notable that the selectivity and affinity towards the biological target can be tuned by modulation of R¹ and R² moieties. The first applications of carbazate derivatives was reported for plant protection, such as seed disinfectants and compounds able to control cereal rusts [37]. In 2020, a new acaricidal composition containing bifenazate was filed [23,28](Fig. 1). Bifenazate is a hydrazine carbazate acaricide first commercialized in 1999 by Crompton Corporation. It is nowadays used worldwide for the control of all growth stages of spider mites (Tetra-nychus spp., Panonychus spp. and Oligonychus spp.) on a wide variety of crops including fruits and ornamentals. Its mode of action was described through inhibition of the complex III at the Qo site [29]. Other carbazate derivatives, such as cadralazine, have been developed as therapeutic agents in the treatment of hypertension [48](Fig. 1).

Overall, carbazate derivatives have been widely used as chemical precursors but rarely applied for biological applications. To the best of our knowledge, no report on their potential to fight against Candida species and particularly C. auris has been done. As many catalytic processes in fungal survival and replication is based on redox metallic exchanges, carbazate derivatives were investigated as potential antifungal agents against C. auris.

## 2. Materials and methods

### 2.1. Materials

All Media chemicals YPD (Yeast Extract Peptone Dextrose), yeast nitrogen base w/o amino acid and ammonium sulphate (YNB w/o amino acid and ammonium sulphate), nutrient broth, agar, 2-deoxy glucose (2-DOG), 2,4 dinitrophenol (2,4 DNP), n-heptane, rhodamine 6G (R6G), propidium iodide (PI), Brain Heart Infusion (BHI), were purchased from Himedia (Mumbai, India). Sodium chloride (NaCl), mannitol, potassium chloride (KCl), di-sodium hydrogen orthophosphate (Na₂HPO₄), di-potassium hydrogen orthophosphate (K₂HPO₄), potassium di-hydrogen orthophosphate (KH₂PO₄), sodium hydroxide (NaOH), potassium hydroxide (KOH), D-glucose, dimethyl sulphoxide (DMSO), sodium dodecyl sulphate (SDS), ammonium sulphate (NH₄)₂SO₄, ammonium chloride (NH₄Cl), Triton-X were obtained from Fischer Scientific. Tetrazolium salt 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl-tetrazolium bromide (MTT), Trypan blue, were obtained from SRL, Mumbai. Calcofluor white (CFW), Congo red (CR), Fluconazole (FLC), Caspofungin (CAS), Amphotericin B (AMP B), Rhodamine 123, DCFDA (2,7-dichlorofluorescein diacetate), DAPI (4',6-diamidino-2-phenylindole) and Aniline Blue (AB) were obtained from Sigma Chemical Co. (St. Louis, MO, USA). FM4-64 dye was procured from Invitrogen, Thermo Fischer Scientific, USA. Quinacrine dihydrochloride was procured from Merk, Germany.

<figure><img src="image_4.png" /><figcaption>**Fig. 1.** Synthesis and selection of compounds as antifungal agents against C. auris.</figcaption></figure>
## 2.2. Chemistry and synthesis of carbazate derivatives

Reagents and solvents were purchased from common commercial suppliers (TCI, Sigma Aldrich). The carbazate derivatives were synthesized by adaptation of reported protocols ([4,38]). The chloroformate derivatives (1.0 equiv) were added dropwise to a mixture of the corresponding hydrazine (1.0 equiv) and pyridine (2.0 equiv) in dry CH₂Cl₂ at 0–5 °C. The working concentration ranged from 1 to 5 mmol/L in hydrazine, depending on its solubility. If hydrazine salts is used, a preliminary step to free the hydrazine with sodium methylate has been realized. The addition is highly exothermic. When addition is completed, the mixture is stirred one hour at 0–5 °C then two hours at room temperature. The completion of the reaction is checked by TLC with a CH₂Cl₂/CH₃OH 95/05 solvent. The mixture is then concentrated under vacuum and the residue is diluted with ethylacetate. The reaction medium is then washed with water and brine. The organic layer is dried over sodium sulphate and concentrated under vacuum. The carbazate derivatives are finally purified by flash chromatography using a gradient of Heptane/AcOEt. The structures of all obtained compounds were determined by examining their nuclear magnetic resonance (¹H NMR and ¹³C NMR) spectra recorded by a Brücker 400 spectrometer (internal standard tetramethylsilane) and their melting points. All data are gathered in *Supporting Information*. It is notable than some compounds are obtained as mixture of conformers (namely, C3 and C6-9) as evidenced by the ¹H NMR record of the same samples before and after gentle heating for C6 (see SI). Some compounds have already been reported [18,36,69,79].

## 2.3. Candida culture conditions

The Candida strains were cultured in YPD (yeast extract peptone dextrose) broth with the composition of yeast extract 1 % (w/v), dextrose 2 % (w/v), and peptone 2 % (w/v) with addition of 2 % (w/v) agar for plates. Yeast extract 1 % (w/v), glycerol 2 % (v/v), peptone 2 % (w/v) and 2 % (w/v) agar were used to prepare YPG plates for nonfermentable source of carbon of. All Candida strains were stored in 30 % (v/v) glycerol stocks at –80 °C. The cells were revived freshly at 30 °C on YPD broth and agar plate before each experiment to make sure the restoration of the strains. C. auris CBS10913T (Clade II) [3] strain was used in all the experiments and if any other strains were used it is mentioned in the respective sections.

## 2.4. Broth dilution assay

We followed the guidelines mentioned in method M27-A3 from the Clinical and Laboratory Standards Institute (CLSI) formerly NCCLS [30,34,51,52,63] to determine MIC by broth microdilution method with slight modifications (see SI).

## 2.5. Spot assay

This method was used by drop dilution assay as described previously [30]. The YPD agar plates were prepared adding the compounds C4 and C13 at concentrations specified in the figure legend along with control plates (without compounds). For spot assay, 5 µl Candida cell culture (0.1 OD₆₀₀) used and spotted on the agar plate in the presence and absence of compounds in fivefold serial dilution. The plates were incubated at 30 °C and differences in growth were recorded after 48 h.

## 2.6. Static/cidal determination assay

C. auris cells (OD₆₀₀ = 0.1) were inoculated in the presence of C4 and C13 at MIC and incubated overnight at 30 °C. On second day, about 100-µl cultures were drawn and re-inoculated into the fresh YPD media without C4 and C13 and further incubated overnight at 30 °C. The following day, OD₆₀₀ was measured with a spectrophotometer (VSI

Electronics Pvt Ltd/model no. VSI-501) to assess the static-cidal activity of C4 and C13 [30].

## 2.7. Drug synergism

This method used to determine the synergistic nature of compounds C4 and C13 with the known antifungal drugs [54]. The serial twofold dilutions and stocks were prepared as per the recommendations of NCCLS immediately before testing (NCCLS, 2017). 100 µl YPD media was distributed into every row and columns of the 96 well microdilution plate leaving the first column and last two columns. The compounds and media making up a total volume of 200 µl were added in the first column. The last two columns consist of negative control (containing media only) and positive control (containing Candida cells). The first compound of the combination was serially diluted along the ordinate, while the second antifungal drug was diluted along the abscissa. Each well was inoculated with 100 µl of Candida cells (0.1 OD₆₀₀) and incubated for 48 h at 30 °C. The checkerboard contains each combination of two drugs, with wells that contain the highest concentration of each drug at opposite corners. According to the NCCLS guidelines for broth microdilution, the MIC defined as the lowest concentration of antibiotic that completely inhibited the growth of the organism as detected with the naked eye (see SI).

## 2.8. Cell viability assay

This method used to determine the viability of Candida cells in the presence of C4 and C13. It is performed by using trypan blue dye for the exclusion assay [72,70]. Trypan blue works on the principle that in live cells, the membrane remains intact hence cannot take up the dye as they allow the exclusion of the dye whereas dead cells can take up the dye as they cannot exclude the dye from the cell. Candida cell suspension inoculated with C4 and C13 at MIC along with untreated cells as a control and incubated overnight at 30 °C. The next day, cells are washed with PBS and resuspended. The cells and dye were prepared in a 1:1 ratio by mixing 10 µl of Candida cell (all test samples) with 10 µl trypan blue (0.4 %) solution and incubated for 15 min at 30 °C with shaking. The dead cell will appear blue and live cells will appear colourless. The number of viable cells and dead cells were determined by examining under light microscope using haemocytometer and the percentage of viable cells and dead cells was calculated by using the following formula.

$$ \%Cell \ Viability = [Total \ Viable \ cells \ (Unstained) / Total \ cells \ (Viable + Dead)] \times 100. $$

## 2.9. Cell wall staining (Chitin, β-1,3-glucan and mannan)

To determine the cell wall components, 2.5 × 10⁶ exponential cells of control, C4 and C13 treated cells were achieved from an overnight culture. After harvesting the cells were fixed with 4 % paraformaldehyde for 30 min in PBS. Fixed cells after washing were stained with 50 µg/ml ConA-fluorescein conjugate for mannans, 100 µg/ml of AB for β-1,3-glucan, 5 µg/ml of CFW for chitin, and incubated at room temperature for 30 min. Fluorescence-stained cells were seen by a fluorescence microscope (Nikon, Japan) at 100X magnification. Images were taken using NIS element software [80].

## 2.10. Cell sedimentation

Cell sedimentation was measured spectrophotometrically as described previously [27,7]. Briefly, 0.1 OD₆₀₀ culture of control, C4 and C13 treated C. auris cells were inoculated and allowed to grow till the OD₆₀₀ reaches 1.0. The OD₆₀₀ from each culture were measured at regular intervals of 10 min till 60 min. Sedimentation rates were calculated by the differences in growth from zero time point to 60 min per unit time interval as shown below:
$$ Sedimentation rate = \frac{\text{Initial OD}(0\text{min}) - \text{Final OD}(60\text{min})}{60} $$

## 2.11. PI uptake

PI (Propidium Iodide) is a fluorescent nucleic acid binding dye that is generally impermeable to cell membrane and primarily used to differentiate between healthy and damaged cells [70]. Candida cells of 0.1 OD₆₀₀ were inoculated with C4 and C13 at 30 °C and allowed to grow for 3 h with gentle shaking. After incubation, cells were washed and harvested by centrifugation (5000 g for 3 min) stained with PI (1 µg/ml) for 15 min and observed under fluorescence microscope (Nikon, Japan).

## 2.12. Quantitation of ergosterol

The alcoholic KOH method was used to extract sterols and the percentage of ergosterol was calculated as previously described [8]. Briefly, 0.1 OD₆₀₀ C. auris cells were inoculated in 50 ml of YPD in the presence of C4 (64.2 µM) and C13 (59.3 µM). Ergosterol content (see SI) was calculated as a percentage of the wet weight of the cells with the following equations:

% Ergosterol + % 24(28)-DHE = [(A281.5/290). F] / pellet weight; % 24(28)-DHE = [(A230/518). F] / pellet weight and % Ergosterol = [% ergosterol + % 24(28) DHE]-% 24(28) DHE, where F is the factor for dilution in petroleum ether and 290 and 518 are the E values (in percent per centimetre) determined for crystalline ergosterol and 24(28)-DHE, respectively.

## 2.13. Vacuolar morphology and acidification

The control, C4 and C13 treated C. auris cells were cultured overnight in YPD. The cells were harvested and washed with 1x PBS and resuspended in fresh YPD for 4 h. Then 40 µM FM4-64 and 10 µl of the Quinacrine solution (10 mg/ml; Molecular Probes) were added for morphology and acidification respectively and the cells were further incubated for 15 min at 30 C, then washed and resuspended in fresh YPD, incubated for 60 min at 30 C, and images were obtained using a fluorescent microscope at 100x magnification [44,41].

## 2.14. R6G extracellular efflux assay

The efflux pump activities were estimated by Rhodamine 6G (R6G) efflux as described previously [30]. About 1x10⁶ yeast cells were inoculated to YPD medium and allowed to grow for 5 h in the presence of C4 (64.2 µM) and C13 (59.3 µM). Cells were harvested with phosphate-buffered saline (PBS) (without glucose), and resuspended as a 2 % cell suspension, corresponding to 10⁸ cells (wt/vol) (see SI). Glucose-free negative controls were also included in all the experiments. For competition assays, C4 (64.2 µM) and C13 (59.3 µM) was added to the deenergized cells 30 min before the addition of R6G at various concentrations (10 µM, 20 µM, 30 µM, 40 µM) and allowed to equilibrate to follow glucose for energy-dependent efflux as above.

## 2.15. R6G intracellular accumulation

Accumulation assays were performed according to previously described protocol with minor modifications [86,32]. The protocol is like R6G extracellular efflux assay (see SI) except that final pellet instead of supernatant was collected for fluorescence microscopy analysis at 100x magnification (Nikon, Japan).

## 2.16. Biofilm formation

### 2.16.1. Biofilm metabolic activity

For estimating biofilm metabolic activity [30], 50 µl (stock solution containing 5 mg/ml, diluted 1:5 in prewarmed 0.15 M PBS prior to

addition) of tetrazolium salt, 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) was added in the presence and absence of C4 and C13 in each well and incubated for 5 h at 37 °C. MTT formazan product was solubilized in DMSO (200 µl) and OD was measured at 450 nm. The metabolic activity of biofilm was represented as absorbances.

### 2.16.2. Biofilm biomass quantification

Biofilm biomass was measured as described previously [30]. The total biomass of each biofilm (see SI) was calculated by subtracting the weight of the silicon square after biofilm formation from the preweighed silicon square.

## 2.17. Adherence to epithelial cells

Adherence assays were performed as described previously [6]. Epithelial cells were voluntarily donated by one of the co-author via soft scraping of the cheek mucous membrane with sterile cotton swabs and adherence assays were performed (see SI).

## 2.18. Lipid peroxidation

Oxidation of lipids was determined by quantitative measurement of thiobarbituric acid reactive substances (TBARS) as described previously (Zoric et al, 2017) in presence of C4 and C13 for 4 h (see SI).

## 2.19. DCFDA and DAPI staining

Experimental C. auris cultures were grown in presence of C4 and C13 at 30 °C for 3 h with shaking. Cells were harvested and washed three times with phosphate buffered saline (PBS) stained with DCFDA and DAPI for 20 min. Subsequently, the unbound dye was removed by washing with the same buffer. Specimens were then examined under fluorescence microscope (Nikon, Japan) at 100x [64].

## 2.20. C. Elegans studies

C. elegans nematodes studies were performed according to the standard Kaplan-Meier method [12,30]. About, 50 young adult hermaphrodite worms at L4 stage were transferred from a lawn of E. coli OP50 to BHI medium in the presence of C4 (64.2 µM) and C13 (59.3 µM). The infected C. elegans were incubated at 25 °C for 7 days and scored as live or dead daily and images were taken on the 7th day of infection. Images were taken from a stereo microscope (Olympus, India) equipped with a Coslab camera at 20x magnification. For visualization of intestinal persistence, C. auris lawns were prepared and worms were placed overnight, Fluorescence of CFW-stained C. auris was observed in the intestine of C. elegans through a fluorescence microscope (Nikon, Japan) at 100x. Magnification using NIS element software (see SI).

## 2.21. Haemolytic activity assay

The haemolytic activity of C4 and C13 was performed as described elsewhere [9] with modifications. The release of haemoglobin (see SI) was monitored by measuring the absorbance (A sample) of the supernatant at 540 nm. For negative and positive controls, RBCs in PBS (A blank) and in 1 % (final concentration v/v) Triton X-100 were used, respectively. The percentage of haemolysis was calculated according to the following equation:

$$ Haemolysis(\%) = \left[ \frac{(A_{\text{sample}} - A_{\text{blank}})}{(A_{\text{Triton}} - A_{\text{blank}})} \right] * 100 $$

## 2.22. Statistical analysis

All experiments were performed in triplicates (n = 3). Student's t test was applied to analyze the differences between control and treated groups. The results reported as mean ± standard deviation (SD) were
considered to be statistically significant only when *P*<0.05.

### 3. Results

#### 3.1. Synthesis and screening of panel of carbazate derivatives for considerable antifungal activity

From our preliminary screening, addition of a para-chlorophenyl moiety allow to reach a good antifungal activity. Therefore para-chlorophenylhydrazine was selected as the main R² provider. In addition, the chloroformate moiety has been investigated to identify the most powerful R¹ group. A diversity of aliphatic so as aromatic chloroformate derivatives were coupled with para-chlorophenylhydrazine to conduct to compounds C1-C13. Carbazate derivatives C14-C16, where para-chlorophenylhydrazine has been replaced by phenylhydrazine have been synthetized to confirm the role of the chlorine. The biological activity of this series of carbazate derivatives was then evaluated against C. auris. As showed in Fig. 1, three compounds exhibited a MIC value lower or equal to 62.5 µg/mL (namely C4, C12 and C13). Whereas C4 came from aliphatic chloroformates, C12 and C13 are aromatic ones. To decipher which carbazate drug candidates will enter further evaluation, a series of in silico modelling of their ADME properties was performed using the admetSAR free tool. This software furnished a complete study and predicted about 50 ADMET endpoints with published QSAR models. From these predicted data, compounds C4, C12 and C13 showed low predictable toxicity. However, from a synthetic view, C13 was considered as a best model. Consequently, C4 and C13 were selected for further evaluation. It must be noticed, from this in silico data, that aliphatic derivatives were a little more skilled than aromatic ones.

#### 3.2. Selected carbazate derivatives display fungicidal activity and drug synergism against C. Auris

The efficacy of our selected carbazate derivatives (C4 and C13) was tested against C. auris and its two fluconazole resistant clinical isolates (C34 and C33) by microbroth dilution assay. We observed the MIC₉₀ of

C4 against C. auris was 128.7 µM (Fig. 2a) while for the fluconazole resistant isolates was 257.5 µM (Fig. 2a). In similar fashion we explored the anticandidal activity of other selected carbazate derivative against C. auris and found that MIC₉₀ of C13 was 237.9 µM (Fig. 2a) while for both the fluconazole resistant isolates (C34 and C33) was 475.8 µM (Fig. 2a). The broth microdilution assay was validated by spot assay depicting no growth at the respective MIC₉₀ of C4 and C13 (Fig. 2b). We further explored the static or cidal nature of C4 and C13 by a spectrophotometric based assay and found that growth was inhibited at MIC₉₀ of C4 and C13 on day 1 as expected but after reinoculation growth was not reverted on day 2 at 257.5 µM depicting fungicidal nature of C4 (Fig. 2c). Contrary C13 exhibited a fungistatic nature as the growth was reverted even till 3806.6 µM on day 2 (Fig. 2c). In addition, the checkerboard microdilution assay depicted that C4 was synergistic with all three classes of known antifungal drugs i.e. Fluconazole (FLC), Amphotericin B (AMP B) and Caspofungin (CAS) with fractional inhibitory concentration index (FICI) less than 0.5 whereas C13 showed synergism only with CAS (Fig. 2d). Furthermore, we investigated the impact of C4 and C13 on cell viability in C. auris at their respective MIC's of C4 and C13 and the outcome stipulated that C4 and C13 significantly decreased the cellular viability to 18 % and 23 % respectively in contrast to control and negligible viability at a dose of 257.5 µM (C4 MFC) where all dead cells were observed (Fig. 2e).

#### 3.3. C4 and C13 changes polysaccharide composition of the cell wall and disrupts vacuole homeostasis

Next, the effect of C4 and C13 on the three constituents of the cell wall (chitin, β-1,3-glucan and mannan) were investigated. The chitin, β-1,3-glucan and mannan were stained with CFW, AB and ConA-fluorescein conjugate respectively. We noticed enhanced fluorescence in chitin (Fig. 3a) and β-1,3-glucan (Fig. 3b) stained cells with their respective dyes in the presence of C4 and C13, contrary to control cells, while mannan staining by ConA-fluorescein conjugate was diminished (Fig. 3c) in the presence of C4 and C13. The phenotypic susceptibility spot assay also revealed sensitivity in the presence of C4 and C13 along

<figure><img src="image_5.png" /><figcaption>**Fig. 2.** Antifungal activity of C4 and C13 (a) Broth microdilution assay with Candida auris (CBS10913T) and Flucanazole resistant isolates (C34 and C33) in the presence of C4 and C13 to determine the MIC₉₀. Data were quantitatively displayed with color (see color bar), where each shade of color represents relative optical densities of the cell. The minimum drug concentration that inhibits growth by 90 % relative to the drug-free growth control is indicated as MIC₉₀ for each strain. b) Spot assay of CBS10913T, C34 and C33 in the absence (control) and presence of C4 (128.7 µM and 257.5 µM) and C13 (237.9 µM and 475.8 µM). c) Static cidal assay of C4 and C13. Bar graph depicting the revival of untreated candidal cells (Control) and non-revival of C4 and C13 treated (MIC₉₀) cells in normal YPD media confirming the fungicidal nature of C4 and fungistatic nature of C13. d) FICI by checkerboard assay. Drug synergism activity of C4 and C13 to the FLC (azole), CAS (echinocandin) and AMP B (polyene). e) Cell viability assay of C4 and C13. Bar graph depicting the cell viability at MIC and MFC of C4 and C13.</figcaption></figure>
<figure><img src="image_6.png" /><figcaption>**Fig. 3.** Effect of C4 and C13 on cell wall. (a) chitin (CFW staining) scale bar = 10 µm, (b) β1,3-glucan (AB staining) scale bar = 10 µm and (c) mannan (ConA staining) scale bar = 20 µm. d) Sensitivity to cell wall perturbing agents in the presence of C4 and C13. Spot assay of control, C4 and C13 in presence of CR (10 µg/ml) and CFW (10 µg/ml). e) Effect of C4 and C13 on cell sedimentation of C. auris. Left panel shows the time kinetics of cell sedimentation in C4 and C13 Treated cells. Right panel shows the significantly enhanced sedimentation rate of C4 and C13 treated cells measuring the difference in absorbance from 0 till 30 min per unit time interval.</figcaption></figure>

with CFW and CR (Fig. 3d). Furthermore, a spectrophotometric based assay was performed to determine the sedimentation rates of C. auris cells in the presence of C4 and C13. We found that the rate of sedimentation was enhanced in the presence of C4 with moderate effect on C13 (Fig. 3e).

Further, the vacuole function was examined by observing the morphology and acidification. The vacuole morphology was assessed by FM4-64 lipophilic dye that is effectively endocytosed in the vacuole film. It was observed that control cell showed regular ring staining design depicting ordinary morphology contrary to the C4 and C13 treated cells showing diffusely distributed staining pattern of deformed morphology (Fig. 4a). For vacuolar acidification, quinacrine dye was used and being

a weak base remains inside the vacuole and fluoresces when protonated under acidic conditions. Therefore, in control cells as expected it showed high fluorescence which confirmed the acidic environment of the vacuole is intact whereas in C4 and C13 less fluorescence was observed due to elevated vacuolar pH (Fig. 4b).

### 3.4. C4 and C13 confers ergosterol depletion and inhibits R6G efflux of ABC transporters

To study the cell membrane more closely, we checked the sensitivity of C4 and C13 in presence of SDS being a membrane perturbing agent. The phenotypic susceptibility assay revealed the sensitivity towards SDS

<figure><img src="image_7.png" /><figcaption>**Fig. 4.** Effect of C4 and C13 on Vacuolar homeostasis. (a) Vacuolar morphology of control, C4 (64.2 µM) and C13 (59.3 µM) treated C. auris observed by FM4-64 staining. Scale bar = 20 µm. (b) Vacuolar acidification of control, C4 (64.2 µM) and C13 (59.3 µM) C. auris observed using quinacrine staining. Scale bar = 20 µm.</figcaption></figure>
in the presence of C4 and C13 (Fig. 5a). Subsequently, we checked the PI uptake and observed enhanced fluorescence depicting the non-intact membrane (Fig. 5b). Furthermore, we checked the ergosterol level as it is one of the major components of cell membrane. We found that ergosterol level was depleted by 42 % and 88 % in the presence of C4 and C13 respectively (Fig. 5c). Further, we checked the functionality of efflux pumps located in the cell membrane and observed that in the presence of C4 and C13 R6G efflux is inhibited (Fig. 6a). Additionally, to confirm the above results, accumulation of R6G was assessed in the presence of C4 and C13 where enhanced red fluorescence was seen depicting more accumulation contrary to control cells (Fig. 6b). Next, we did phenotypic susceptibility assay in the presence of R6G and Nile Red and observed that growth is inhibited in the presence of C4 and C13 and R6G but not Nile red (Fig. 6c). Kinetic studies for the mode of inhibition via Line Weaver Burk (LB) plot revealed that C4 competitively inhibits R6G efflux of ABC transporters as the apparent Km increased while Vmax remained unchanged (Fig. 6d). On the other hand, C13 non-competitively inhibits R6G efflux of ABC transporter since Vmax was changed while Km remains unchanged (Fig. 6d).

### 3.5. C4 and C13 causes mitochondrial dysfunction

To investigate the function of mitochondria, we performed the phenotypic susceptibility assay in the presence of C4 and C13 with non-fermentative carbon source and noticed that growth of C. auris cells were inhibited (Fig. 7a). In support of this hypothesis mitochondrial potential was examined by incorporating Rhodamine 123 in C. auris cells in the presence of C4 and C13. Rhodamine 123 acts as a probe of mitochondrial membrane potential used for estimation of mitochondrial potential of cells. The enhanced red fluorescence was observed in the presence of

C4 and C13 unlike untreated (control) cells where no fluorescence was observed reflecting mitochondrial dysfunction (Fig. 7b).

### 3.6. C4 and C13 cause oxidative stress leading to lipid peroxidation

Subsequently we investigated the generation of oxidative stress in C. auris. Firstly, we did phenotypic susceptibility assay in the presence of C4 and C13 at their MIC$_{90}$ and found as expectedly the cells showed no growth. Interestingly, after the addition of an antioxidant Ascorbic acid (AA) the growth of Candida cells reverted (Fig. 8a) that was also confirmed by DCFDA staining (Fig. 8b). Next, we examined lipid peroxidation and DNA damage, an attributes of reactive oxygen species (ROS) generation and discovered that C4 and C13 treated cells resulted in elevated TBARS levels that could be reverted in the presence of AA (Fig. 8c) and enhanced DAPI staining (Fig. 8d).

### 3.7. C4 and C13 impairs biofilm formation and buccal epithelial cell adherence

The ability of C4 and C13 to inhibit biofilm formation was determined by crystal violet staining and it was observed that C4 and C13 impaired biofilm formation (Fig. 9a). Next, we performed the MTT assay to check the metabolic activity of cells and noticed that metabolic activity was decreased by 79 % in the case of C4 and 85 % in the case of C13 (Fig. 9a). Additionally, we checked the biofilm biomass and observed that there was considerable reduction of biofilm biomass by 75 % in presence of C4 and 85 % in the presence of C13 (Fig. 9a). Furthermore, we deciphered the impact of C4 and C13 on the adherence of C. auris colonization and found both the compounds, C4 and C13, showed substantial disruption of Candida cell adherence to buccal

<figure><img src="image_8.png" /><figcaption>**Fig. 5.** Effect of C4 and C13 on the membrane integrity. a) Spot assay in the absence and presence of C4 (64.23 µM) and C13 (59.3 µM) with membrane damaging agent SDS (0.02 %). b) Propidium iodide (PI) staining of C. auris cells in the presence and absence of C4 and C13. Bar in the image depicts 20 µm. c) Upper panel showing UV spectrophotometric ergosterol profiles of C. albicans scanned between 220 and 300 nm in the absence and presence of C4 and C13. Lower panel showing relative percentages of ergosterol content absence and presence of C4 and C13. A mean of % ergosterol levels is calculated and normalized by considering the untreated control as 100 ± SD of three independent sets of experiments are depicted on y-axis and * depicts P<0.05.</figcaption></figure>
<figure><img src="image_9.png" /><figcaption>**Fig. 6.** Effect of C4 and C13 on efflux pump activity. a) Extracellular R6G concentrations in control, C4, C13 treated *C. auris* cells. The energy dependent R6G efflux initiated by adding glucose (2 %) and quantified by measuring the absorbance of the supernatant at 527 nm. The values are the means and standard deviations (indicated by error bars) from three independent experiments. b) R6G accumulation depicted by fluorescence images of R6G staining in the presence of C4 and C13. Scale bar = 20 µm c) Phenotypic susceptibility assay of R6G and Nile Red in the presence and absence of C4 and C13. d) Lineweaver-Burk plot in the presence of C4. The x-axis (1/S) represents the various concentrations (µM) of R6G used, and the y-axis (1/V) shows the rate of release of R6G in the presence of C4. e) Lineweaver-Burk plot in the presence of C13. The x-axis (1/S) represents the various concentrations (µM) of R6G used, and the y-axis (1/V) shows the rate of release of R6G in the presence of C13. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>

epithelial in contrast to untreated cells (Fig. 9b).

### 3.8. C4 and C13 prolonged the survival of C. Auris infected C. Elegans

Finally, the *in-vivo* efficacy of C4 and C13 as potent antifungals against *C. auris* was evaluated in a widely accepted nematode model, *C. elegans*. Initially, the host toxicity of both the compounds was tested in *C. elegans* at its sub inhibitory concentrations (64.2 µM for C4 and 59.3 µM for C13) and we noticed that there were no significant growth differences between treated (75 %) and untreated (80 %) worms upto 7 days (Fig. 10a). Later, for determining the antimycotic effect, nematodes were introduced to *C. auris* in the absence and presence of C4 and C13. It was revealed that the survival of *C. elegans* was significantly increased from 25 % to 75 % and 65 % in the presence of C4 and C13 respectively (Fig. 10b). Furthermore, infected *C. elegans* were stained with CFW and intestinal persistence of *C. auris* was clearly visible in the control in contrast to C4 and C13 treated nematodes which showed reduced fluorescence (Fig. 10c). Subsequently we evaluated the haemolytic activity of both our compounds against erythrocytes. Haemolytic assay revealed that both the tested compounds C4 and C13 display negligible haemolytic activity till 4x MIC. We observed only 12.9 % haemolysis at 4x MIC of C4 and 5 % haemolysis at 4x MIC of C13 in contrast to Triton-X as a positive haemolytic agent which displayed 100 % haemolysis (Fig. 10d).

## 4. Discussion

The availability of limited repertoire of antifungal armamentarium against the emerging superbug, *C. auris* and increasing frequency of drug resistance poses an alarming threat to human health and healthcare system globally [10,11,62]. Hence it is imperative to develop novel antifungals that will help to circumvent the above problem. This is

evident by several recent studies dealing with the design of antifungal agents or repositioning of drugs. For instance, anticancer small molecule 3-bromopyruvate exhibited fungicidal activity [42]. 1,2,4-Triazole benzamide derivative was recently reported as a novel dual target fungicide [81]. Thiazole-, hydrazone-, and amide-containing derivatives of laurene exhibited fungicidal activities [85], 4-chlorobenzyl *p*-coumarate exhibited strong fungicidal activity [49]. 2-Chloro-*N*-phenyl-acetamide exhibited antifungal and cidal activity against different strains of *Aspergillus niger* [31]. Synthetic sulfonate derivatives evaluated for potent fungicidal activity against four plant pathogenic fungi (*Botrytis* (B.) cinerea, *Valsa* (V.) mali, *Fusarium* (F.) graminearum, *Sclerotinia* (S.) sclerotiorum) [74]. Hinokitiol, Hydrazine-based, Pyridinone, Triazine and γ-lactam heterocycles have been identified as potent antifungal agents [14,15,50,20]. Following our effort to develop novel and effective antifungal compounds, in the present study we explored a panel of 16 carbazate derivatives from the JUNIA chemical library for their antifungal properties. From this panel, two compounds namely (C4 (butyl 2-(4-chlorophenyl) hydrazine-1-carboxylate) and C13 (phenyl 2-(4-chlorophenyl) hydrazine-1-carboxylate) displayed considerable antifungal activity against *C. auris* and FLC-resistant clinical *C. auris* isolates.

The screening results allow to set some conclusions, useful to design further development. Concerning R¹, two sub-classes have been attempted, e.g. aliphatic or aromatic moieties. When a linear alkyl chain has been introduced, the length of the chain dramatically impacted the activity. Indeed, antifungal activity against *C. auris* reached an optimum by a butyl chain, whereas shorter chain (ethyl) or longer chain (pentyl to dodecanoyl) were less potent. This could be related to the lipophilicity, expressed by their *logP* (Fig. 1). Previous data reported for the development of antifungal drugs against crop pathogens already underlined such tendency [21]. In that case, compounds exhibited *logP* ranging from 2 to 3 were the most interesting, with no impact of the steric
<figure><img src="image_10.png" /><figcaption>**Fig. 7.** Effect of C4 and C13 on mitochondria. a) Spot assays in the absence and presence of C4 and C13 in YPD (fermentable carbon source) as controls and YPG media (non-fermentable carbon source). b) Fluorescent microscopy images at 10x magnification showing Rhodamine 123 (40 mM) staining for monitoring mitochondrial membrane potential. Scale bar = 10 µm.</figcaption></figure>

hindrance of the aliphatic chains. In our study, even if C4 logP is equal to 2.67, which perfectly fits with the reported data, it must be noted than the biological activity is strongly impacted by the steric hindrance. Indeed, compound C10, bearing an isobutyle chain instead of the *n*-butyle moiety, is 4-fold less potent than C4, even if their logP are similar. Moreover, by comparing C2 and C4, it is obvious that increasing the hydrophilicity (replacement of a –CH₂– by an –O– with the same chain length) negatively affects the biological potential. Only three aromatic chloroformates have been introduced, e.g. R¹ = phenyl, *para*-chlorophenyl and *para*-methoxyphenyl. From these preliminary data, introduction of electrodonating such a methoxy one seemed better than electrowithdrawing one. This tendency should be further investigated. Concerning R², *para*-chlorophenyl allowed to reach a high activity towards *C. auris*. Using R² = phenyl with R¹ = *n*-butyl conducted to an 8-fold decrease in activity. From its chemical skeleton, C16 could be considered as the “reverse” C13. Indeed, both compounds possess the same carbazate linker but R¹ and R² groups were inversed. Comparing these two compounds confirm that, from an electronic point of view, R² should be electro withdrawing whereas R¹ should be neutral or electrodonating. The importance of the linker has also been tested with the evaluation of *HydrazineCarboxamide* (HC) and *Urethane* (UR) derivatives. In particular, the UR molecule proved the great importance of the second –NH– in the carbazate linker, as its depletion conducted to a complete loss in antifungal activity (comparison UR with C13). Gathering all results, the following early SAR could be proposed (Fig. 11).

We established that C4 and C13 displayed considerable antimycotic activity against *Candida auris* at 128.7µM and 237.9µM, respectively. Moreover, we observed one well shift for both the carbazate derivatives against the two tested fluconazole resistant isolates (C34 and C33) at 257.5µM and 475.8µM, respectively (Fig. 2a and b). Interestingly, we explored that only C4 displayed fungicidal activity at 257.5 µM while C13 remained fungistatic even till 3806.6 µM (Fig. 2c). This difference

in mode of action could be linked with the flexibility of the butyl chain. Indeed, the carbazate linker is planar. C13 is then a planar molecule, with low steric hindrance but restricted flexibility. In contrary, the R¹ butyl chain exhibited low steric hindrance and high flexibility, which could help to fit with a narrow recognition site in the biological target. This hypothesis is also stressed by the lost in activity observed for C10 (R¹ isobutyl flexible but sterically hindered chain). This difference in structure could also explain all the differences in mode of action observed for each compound.

As combination therapy is a good option to fight against drug resistant fungal pathogens to improve the efficacy of known antifungal drugs ([45,54]), we sought to explore the possibility of synergism with FLC, AMP B and CAS. We found that C4 displayed synergism with all three classes of known antifungal drugs namely FLC, AMPB and CAS while C13 depicted synergism with CAS only (Fig. 2d). Upon challenging of C. auris with tested carbazate derivatives, we could observe significant reduction in cell viability (Fig. 2e). These results indicated that the selected synthetic compounds have potential antifungal properties against C. auris.

Cell wall is an important structure of fungi being the first interface between the host and utilization as antifungal target since it is absent in humans and therefore the components of fungal cell wall are crucial for antifungal therapy [33,39]. Hence, we checked the cell wall composition in the presence of both C4 and C13 and observed significant remodelling (Fig. 3 a, b and c) in the cell composition. Of note, we found that β-1,3-glucan and chitin were enhanced while mannan was diminished which was in concurrence to the antifungal properties of C4 and C13 due the fact that C. auris usually masks the β-1,3-glucan to evade the immune response [55,25] and in the present study we observed enhanced β-1,3-glucan which will lead to unmasking and easier immune recognition. The compositional changes in the cell wall could also be the reason for the observed hypersensitivities in presence of cell wall
<figure><img src="image_11.png" /><figcaption>**Fig. 8.** Effect of C4 and C13 on reactive oxygen species. a) Spot assays showing hypersensitivity of *C. auris* cells in the presence of C4 and C13 at MIC alleviated by AA (20 mM), which is an antioxidant. b) DCFDA staining of *C. auris* cells in the presence of C4 and C13 at MIC alleviated by AA (20 mM). c) TBARS /MDA level as a measure of lipid peroxidation depicted as bar graph in the presence of C4 and C13, AA (antioxidant) and H₂O₂ (oxidant). Mean of O.D₅₀₀ nm ± SD of three independent sets of experiments are depicted on Y-axis and * depicts P<0.05. d) DAPI staining of *C. auris* cells in the presence of C4 and C13.</figcaption></figure>

<figure><img src="image_12.png" /><figcaption>**Fig. 9.** Effect of C4 and C13 on biofilm formation. a) MTT assay depicting the disrupted biofilm formation in C4 and C13 treated cells by left y axis * depicts P<0.05. Depleted biomass measurement of C4 and C13 treated cells by right y axis * depicts P<0.05. Inset in the graph showing crystal violet stained *C. auris* in C4 and C13 treated cells. b) Effect of C4 and C13 on cell adherence using buccal epithelial cells. Bar graph depicts percent cell adherence. Inset in the graph showing Inhibition of buccal epithelial cell adhesion by C4 and C13 treated cells. Arrows indicating the non-attachment of *C. auris* cells with buccal epithelial cells. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)</figcaption></figure>

perturbing agents such as CFW and CR (Fig. 3d) and enhanced cell sedimentation rates (Fig. 3e). Cellular homeostasis is maintained by the functionality of the highly complex organelle fungal vacuole [1]. Moreover, it has been studied that cell wall remodelling could also be attributed to dysfunctional vacuole [17,26]. Therefore, the vacuole morphology and acidification were also examined in the presence of C4 and C13 and found that both the morphology and the acidification of the vacuole was abrogated (Fig. 4a and b).

Next, we aimed to study the cell membrane of *C. auris* as many antifungals act on the cell membrane [71,47]. The phenotypic

susceptibility assay with SDS a membrane disrupting agent and observed hypersensitivity with SDS in the presence of C4 and C13 (Fig. 5a) confirmed membrane disruption. Further we checked the fluorescence of cells in the presence of C4 and C13 with Propidium iodide (PI) as this dye is impermeable to cell membrane and binds to nucleic acid only inside the dead cell [73]. The enhanced red fluorescence in the presence of C4 and C13 confirmed non intact membrane (Fig. 5b). The above membrane perturbations encouraged us to estimate ergosterol content as ergosterol biosynthesis is the key feature in cell membranes of fungi which is absent in humans and serves as another potential target for
<figure><img src="image_13.png" /><figcaption>**Fig. 10.** Effect of C4 and C13 on nematode survival. a) Toxicity test of C4 and C13 on *C. elegans* depicted by the Kaplan-Meier curve showing number of *C. elegans* in the presence of C4 and C13 at sub-MIC concentration. Worm survival was determined based on movement. The toxicity of C4 and C13 was studied on nematodes by determining survival rates until 7 days. b) Survival of *C. albicans*-infected *C. elegans*. Kaplan-Meier curve showing % survival of *C. albicans*-infected *C. elegans* in the presence of C4 and C13 until 7 days. c) Intestinal persistence of *C. auris* represented by the CFW-stained *C. auris* visualized in intestine of *C. elegans* treated with C4 and C13 showing lesser fungal burden. Magnification 100x.</figcaption></figure>

<figure><img src="image_14.png" /><figcaption>**Fig. 11.** Early SAR useful for future development.</figcaption></figure>

antifungals [84,57]. We found that C4 and C13 confer ergosterol depletion, but the decrease was more prominent in presence of C13 (Fig. 5c). The observed membrane perturbations led us to study the membrane more intricately. Overexpression of efflux pumps is the major mechanism of multidrug resistance in *Candida* species [58]. It is also reported that approximately 20 transmembrane proteins present in the genome of *C. auris* are responsible for azole resistance [82]. Therefore, we checked functionality of efflux pumps in presence of C4 and C13 and found that the efflux pumps activity was impaired (Fig. 6a and b). Interesting, the inhibition of activity was specific to the ABC superfamily transporters as revealed from phenotypic susceptibility in presence of R6G and NR (Fig. 6c). This is because R6G is the specific substrate for ABC superfamily transporters while NR is the substrate for both ABC as well as MFS superfamily transporters [35,40]. Furthermore, LB plot revealed that the mode of inhibitions for C4 and C13 were competitive and non-competitive in nature respectively as the Vmax remains unaltered in presence of C4 (Fig. 6d) and Km remains unaltered in presence of C13 (Fig. 6e). Additionally, we explored that mitochondrial functioning was disturbed in presence of C4 and C13 as revealed by phenotypic susceptibility in presence of non-fermentable sugar (Fig. 7a)

and rhodamine 123 staining (Fig. 7b). Mitochondrial dysfunction has led to inhibition of efflux pumps as revealed by other studies [77,87]. Hence impaired efflux activities observed herein may be due to dysfunctional mitochondria and could also be attributed to the fact that ABC transporters are preferentially localized in ergosterol rich regions [56] and the present study already established that ergosterol levels are decreased in presence of C4 and C13.

Recent study reveals the interaction of lansoprazole metabolite(s) with mitochondrial respiratory chain results in the enhanced oxidative stress in fungal cells [65]. Similarly, it is reported that mitochondria play an important role in the production of ROS thereby rendering oxidative stress [578,43]. This hypothesis was verified by the phenotypic susceptibility of C4 and C13 and DCFDA staining in presence of antioxi-dant, AA where we observed growth reversion (Fig. 8a and b) hinting towards the fact that antifungal activity of both C4 and C13 involves ROS generation which was neutralised by AA. One of the hallmark features of ROS generation inside the cell are lipid peroxidation and DNA damage [19,64] which was confirmed by enhanced TBARS levels (Fig. 8c) and enhanced DAPI staining (Fig. 8d) as described in methods.

There are numerous findings where it has been reported that compositional changes in cell wall, overexpression of efflux pumps and increased cell density leads to biofilm formation [61,2,46]. Therefore, we also investigated the effects of C4 and C13 in biofilm formation and observed that C4 and C13 effectively inhibits biofilm formation (Fig. 9a). When the yeast cells get adhered to the host cells such as buccal epithelial cells or acrylic denture surfaces forming colonies it leads to the development of clinical manifestation [76]. As the biofilm formation starts with the yeast cell adherence [66] therefore, we checked the buccal epithelial cell adherence and observed that the presence of C4 and C13 decreased the adherence (Fig. 9b). These findings revealed that C4 and C13 as potent inhibitors of virulence traits. The inhibition of biofilm formation may also be attributed to the fact that C4 and C13 causes oxidative stress as revealed in the present study and enhanced oxidative stress also regulates genes involved in biofilm
formation and alternate carbon metabolism [60].

Eventually, the in-vivo efficacy was performed using widely accepted model of *C. elegans* [12,59]. We demonstrated that both C4 and C13 were non-toxic to the host, *C. elegans* (Fig. 10a) and protected the host upon *C. auris* infection (Fig. 10b) that was also confirmed by intestinal persistence depicting less infection in presence of C4 and C13 (Fig. 10c) due to less staining of CFW. Lastly, the selected carbazate derivatives display negligible haemolytic activity towards erythrocyte cells up to 4x MICs (Fig. 10d).

## 5. Conclusions and future perspectives

The evolution of drug resistance against current antifungals has prompted the requirement for safe and effective broad-spectrum antifungal agents with potent pharmaceutical properties and provoked the design and development of new antifungal agents. In conclusion, two carbazate derivatives were selected to elucidate their antifungal potential. This study proved that carbazate derivatives are good drug candidates for the development of antifungals against *C. auris*. In summary, C4 and C13 showed considerable antifungal activity against *C. auris*, including two clinical isolates that were resistant to the most common antifungal drugs. Although at this stage it is difficult to comment as to what are the primary targets for C4 and C13 and what could be the chronology of mode of actions, nevertheless, the biological targets such inhibiting efflux pumps, ergosterol and biofilm formation are of immense pharmacological interest. From a chemical perspective, early SAR have been addressed and could serve as platform for the design of more potent derivatives. An investigation of the chemical diversity in R² is envisioned with the introduction of aliphatic and heteroaromatic hydrazine derivatives. The incoming challenges rely on the optimization of R² moiety to reach a higher biological activity with controlled solubility and stability. Insights on ADME properties of the compounds are also needed to enter the preclinical steps. Globally, these results pave the way for the design and development of future first-in-class antifungal drugs.

## Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

## CRediT authorship contribution statement

**Tazeen Fatima:** Writing – original draft, Visualization, Validation, Methodology, Investigation, Formal analysis, Data curation. **Zeeshan Fatima:** Writing – review & editing, Supervision, Resources, Project administration. **Muriel Billamboz:** Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Resources, Project administration. **Saif Hameed:** Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Resources, Project administration, Investigation, Formal analysis, Conceptualization.

## Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

## Acknowledgment

We are grateful to Arunaloke Chakrabarty for providing *C. auris* strains CBS10913T, C33 and C34 as generous gift. We thank Central Instrumentation Research Facility (CIRF), Amity University Haryana for facilitating fluorescence microscopy.

# Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.bioorg.2024.107782.

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