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Article

A Potent Fluorescent Derivative of 8-Hydroxyquinoline Suggests Cell Wall Damage as a Possible Cellular Action of the 5-Triazole 8-Hydroxyquinoline Class

by
Caroline de Bem Gentz
1,2,3,*,
Marcela Silva Lopes
2,
Priscilla Maciel Quatrin
1,
Mariana Pies Gionbelli
2,3,
Maycon Antonio de Cesare
2,3,4,
Ana Paula Perin
4,
William Lopes
5,
Alexandre Meneghello Fuentefria
1,2,
Marilene Henning Vainstein
4 and
Saulo Fernandes de Andrade
1,2,3,*
1
Agricultural and Environmental Microbiology Graduate Program, Universidade Federal do Rio Grande do Sul, Porto Alegre 90035-003, Brazil
2
Pharmaceutical Sciences Graduate Program, Universidade Federal do Rio Grande do Sul, Porto Alegre 90160-093, Brazil
3
Pharmaceutical Synthesis Group (PHARSG), Universidade Federal do Rio Grande do Sul, Porto Alegre 90160-093, Brazil
4
Department of Molecular Biology and Biotechnology, Universidade Federal do Rio Grande do Sul, Porto Alegre 91501-970, Brazil
5
Physics of Living Systems, Department of Physics, Massachusetts Institute of Technology, Cambridge, MA 02139, USA
*
Authors to whom correspondence should be addressed.
Appl. Microbiol. 2025, 5(2), 38; https://doi.org/10.3390/applmicrobiol5020038
Submission received: 11 February 2025 / Revised: 1 April 2025 / Accepted: 7 April 2025 / Published: 10 April 2025
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Abstract

:
Fungal infections are a major but often neglected global health challenge, affecting both human health and agricultural productivity. Current treatments are limited by few drug classes and increasing multidrug resistance, exacerbated by the widespread use of antifungal agents in clinical and agricultural settings. This study investigates the antifungal potential of a novel 8-hydroxyquinoline derivative with a triazole core at the 5-position, synthesized to improve both efficacy and mechanistic understanding as a fluorescent chemical probe. Biological assays demonstrated significant antifungal activity of compound 10 against a range of pathogens, which was active against all Candida species, dermatophytes, and Fusarium solani with MIC values ranging from 0.5 to 4 µg/mL. Confocal fluorescence microscopy of treated fungal cells was conducted and showed a high accumulation of compound 10 at the cell edge. To further investigate the mode of action, results from a sorbitol protection assay suggested a possible cell wall action, and scanning electron microscopy (SEM) revealed cell wall disruption, such as cell shrinkage and surface roughness, in treated fungal cells. These findings highlight the 8-hydroxyquinoline-triazole scaffold as a promising antifungal agent with cell wall damage properties, providing a basis for future therapeutic development against human and plant fungal pathogens.

1. Introduction

Fungal infections are widely recognized as a major global health concern. Among other factors, the epidemiology of these infections is affected by geographic location, cultural habits, climate characteristics, and economic status of the country [1,2,3,4]. In addition to human infections, fungi can colonize plants and soil causing low-quality crops and mycotoxin contamination. Both infections bring about serious economic impacts such as medical costs or food waste [5,6,7,8,9,10]. Treatment of fungal infections has become increasingly challenging due to the limited classes of antifungal drugs available, the rise of multidrug-resistant strains, and the widespread prophylactic use of these drugs in clinical settings. The uncontrolled use of agricultural fungicides also contributes to the resistance problem [6,11,12,13,14,15,16,17].
In recent years, drug discovery has been focused on privileged structures, such as the quinoline core [18]. The quinoline nucleus has already shown great pharmacological potential depending on its substitution pattern, and some quinoline derivatives have been reported as antiviral, antioxidant, antibacterial, or antifungal compounds [18,19,20,21,22,23,24,25,26,27,28,29,30]. Among the quinoline derivatives, the 8-hydroxyquinoline class could be highlighted due to its potent antimicrobial activity, which seems to be related to the nature of the substituents at the 5- or 7-position [19,24,25,26,31]. Previous publications from our research group were focused on exploring the 5-position modifications to evaluate the antifungal activity of novel derivatives against important medical strains, including Candida species and dermatophyte species. Joaquim et al. [26] synthesized a novel series of 8-hydroxyquinoline-5-sulfonamide derivatives and evaluated their antifungal potential. The derivatives showed high potency and encouraged us to further explore the 5-position modifications. In a second work, Silva et al. [32] synthesized 8-hydroxyquinoline bearing a substituted triazole ring at the 5-position. The triazole ring was chosen for its versatility, stability, and a broad spectrum of biological activities, including antifungal activity [32,33]. Silva’s work resulted in 5-triazole-8-hydroxy-quinoline derivatives with a MIC range of 1–16 μg/mL against Candida and dermatophyte species but showed no activity against Fusarium species.
Despite the potent antifungal activity demonstrated by the 8-hydroxyquinoline derivatives, few studies have explored the mechanism of action of this class, which is an essential step for the further development of these compounds. Preliminary results from our research group suggest a potential effect on the fungal cell wall. In a pioneering study, Pippi et al. [34] used scanning electron microscopy (SEM) and sorbitol assays to show cell wall damage caused by clioquinol, a well-known commercial 8-hydroxyquinoline derivative with potent antifungal properties. Subsequent studies by Pippi et al. and Silva et al. on 8-hydroxyquinoline-5-sulfonamide and 5-triazole-8-hydroxyquinoline derivatives reported similar results [32,35]. Taken together, these results highlight the 8-hydroxyquinoline scaffold as a promising basis for the development of new antifungal agents.
To elucidate the cellular action of this class, the main goal of this study was to design and synthesize a potent and fluorescent chemical probe of an 8-hydroxyquinoline compound containing a triazole core at the 5-position. The derivative was designed to enable cell studies using confocal fluorescence microscopy while also serving as a tool for the identification of novel antifungals as a hit molecule. Given the critical role of antifungals in both human and plant health, experiments were conducted with strains relevant to both fields, broadening the impact of this work.

2. Materials and Methods

2.1. Chemistry

Reactants were obtained from commercial suppliers and used without further purification. Column chromatography was performed on silica gel Fluka (Sigma-Aldrich, St. Louis, MO, USA) 0.035–0.070 mm. Solvents were distilled, when necessary, before use. The 1H and 13C NMR spectra were obtained on a Bruker 400 nuclear magnetic resonance spectrometer (Billerica, MA, USA). Proton and carbon shifts (δ) are given concerning TMS (tetramethylsilane). The compound’s purity was determined using high-performance liquid chromatography (HPLC) with a Shimazdu equipped with a UV DAD detection (Kyoto, Japan) and Phenomenex C8 100A column (250 mm × 4.6 mm, 5 μm). Detection was performed at 254 nm wavelength. The characterization data of intermediates and final compound are available in the Supplementary Information in Figure S1 (1H NMR of 5-nitro-8-hydroxyquinoline), Figure S2 (zoom of the 1H NMR of 5-nitro-8-hydroxyquinoline spectrum), Figure S3 (13C NMR of 5-nitro-8-hydroxyquinoline), Figure S4 (HPLC trace of 5-nitro-8-hydroxyquinoline), Figure S5 (IR spectrum of 5-nitro-8-hydroxyquinoline), Figure S6 (HRMS spectrum of 5-nitro-8-hydroxyquinoline), Figure S7 (1H NMR of 5-azide-8-hydroxyquinoline spectrum), Figure S8 (zoom of the 1H NMR of 5-azide-8-hydroxyquinoline), Figure S9 (13C NMR of 5-azide-8-hydroxyquinoline spectrum), Figure S10 (HPLC trace of 5-azide-8-hydroxyquinoline), Figure S11 (IR spectrum of 5-azide-8-hydroxyquinoline), Figure S12 (HRMS spectrum of 5-azide-8-hydroxyquinoline), Figure S13 (1H NMR of 5-(4-(4-chlorophenyl)-1H-1,2,3-triazol-1-yl)quinoline-8-ol spectrum), Figure S14 (zoom of the 1H NMR of 5-(4-(4-chlorophenyl)-1H-1,2,3-triazol-1-yl)quinoline-8-ol spectrum), Figure S15 (13C NMR of 5-(4-(4-chlorophenyl)-1H-1,2,3-triazol-1-yl)quinoline-8-ol spectrum), Figure S16 (HPLC trace of 5-(4-(4-chlorophenyl)-1H-1,2,3-triazol-1-yl)quinoline-8-ol), Figure S17 (IR spectrum of 5-(4-(4-chlorophenyl)-1H-1,2,3-triazol-1-yl)quinoline-8-ol) and Figure S18 (HRMS spectrum of 5-(4-(4-chlorophenyl)-1H-1,2,3-triazol-1-yl)quinoline-8-ol).

2.1.1. Synthesis of 5-Nitro-8-Hydroxyquinoline (2)

It was obtained as previously described by Mazumder et al. [36]. Bright yellow crystal; 53% yield; mp: 179–181 °C; 1H NMR (400 MHz, CDCl3) δ (ppm): 9.34 (dd, 1H, J = 1.4 Hz, 8.8 Hz), 8.89 (dd, 1H, J = 1.4 Hz, 4.4 Hz), 8.60 (d, 1H, J = 8.6 Hz), 7.74 (dd, 1H, J = 4.4 Hz, 8.8 Hz), 7.20 (d, 1H, J = 8.6 Hz). 13C NMR (100 MHz, CDCl3) δ (ppm): 157.68, 148.76, 137.04, 136.10, 133.74, 129.06, 125.21, 122.53, 108.12. IR (ῡ/cm−1): 3113, 1571, 1510, 1471, 1418, 1401. HRMS (m/s): [MH+] calc for C9H7N2O3, 191.0451; found, 191.0453.

2.1.2. Synthesis of 5-Azido-8-Hydroxyquinoline (4)

To a stirred solution of 5-nitro-8-hydroxyquinoline (3.45 g, 18.40 mmol) in isopropanol (175 mL), palladium-on-carbon 10% (440 mg) and hydrazine solution (16% w/v, 13.10 mL, 64.40 mmol) were added. The mixture was heated to 82 °C and refluxed for 3 h. Then, the mixture was hot filtered and washed with hot isopropanol, and the filtrate was concentrated at reduced pressure to give 5-amino-8-hydroxyquinoline (2.85 g) as a black solid used in the next step without further purification, with 74% yield. The 5-amino-8-hydroxyquinoline (2.85 g, 18.73 mmol) was dissolved in concentrated hydrochloric acid (1.66 mL) and water (20.7 mL), cooled to −3 °C in an ice bath, and stirred for 10 min. Then, a solution of sodium nitrite (2.07 g, 29.97 mmol) in cold water (20.7 mL) was added dropwise to the mixture. After 20 min, a solution of sodium azide (2.44 g, 37.46 mmol) in water (89.10 mL) was added dropwise to the mixture and stirred at 0 °C for 1.5 h. The reaction was kept at room temperature for 24 h in the dark while stirring. After 24 h, ethyl acetate (100 mL) was added to the resulting mixture, filtered, and extracted. The organic layer was washed out with water, dried over with Na2SO4, filtered, and concentrated under reduced pressure to give 5-azido-8-hydroxyquinoline (2.45 g) as a brown solid used in the next step without further purification; 74% yield; mp: 130–134 °C; 1H NMR (400 MHz, DMSO-d6) δ (ppm): 9.92 (bs, 1H), 8.91 (dd, 1H, J = 1.6 Hz, 4.2 Hz), 8.34 (dd, 1H, J = 1.6 Hz, 8.4 Hz), 7.60 (dd, 1H, J = 4.2 Hz, 8.4 Hz), 7.37 (d, 1H, J = 8.4), 7.13 (d, 1H, J = 8.4 Hz). 13C NMR (100 MHz, DMSO-d6) δ (ppm): 151.74, 150.02, 139.51, 131.74, 126.20, 122.92, 122.47, 117.10, 112.27. IR (ῡ/cm−1): 3309, 2144, 2113, 1591, 1578, 1511, 1473, 1411. HRMS (m/s): [MH+] calc for C9H7N4O, 187.0614; found, 187.0613.

2.1.3. Synthesis of 1-Chloro-4-Ethynylbenzene (8)

It was obtained as previously described by Zhao et al. [37], and all the NMR data are in conformity with the previous literature [38,39,40,41].

2.1.4. Synthesis of 5-(4-(4-Chlorophenyl)-1H-1,2,3-Triazol-1-yl)Uinoline-8-ol (10)

First, the 5-azido-8-hydroxyquinoline (0.105 g, 0.81 mmol) was solubilized in acetic anhydride (114.20 μL, 1.21 mmol), and pyridine (39.15 μL, 0.486 mmol) was added to the solution at room temperature. After 1 h, EtOAc was added (10 mL) to the mixture, and acetic acid (1M) and sodium acetate were used to adjust the pH to 7. The organic layer was washed with water (3 × 10 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure to give 5-azidoquinolin-8-yl acetate as a brown solid, which was used in the next step without purification. Next, to a solution of 5-azidoquinolin-8-yl acetate (5) (1.0 eq.) in t-BuOH at room temperature, in order, 1-chloro-4-ethynylbenzene (8) (1.5 eq.), CuSO4.5H2O (0.4 eq.), and an aqueous solution of ascorbic acid (0.6 eq.) and NaHCO3 (0.6 eq.) were added. After 24 h, EtOAc was added, and the organic layer was washed out with water dried over Na2SO4, filtered, and concentrated under reduced pressure. The crude was purified by silica gel column chromatography (cyclohexane/ethyl acetate). Due to the instable nature of the acetylated derivative, it was not possible to characterize it by NMR techniques. After the purification, the derivative was hydrolyzed to give the hydroxyl group at 8-position. To a stirred solution of 5-(4-(4-chlorophenyl)-1H-1,2,3-triazol-1-yl)uinoline-8-yl acetate (9) (1.0 eq.) in absolute ethanol at room temperature, KOH (2.0 eq.) was added. After 1 h, the mixture was concentrated under pressure, water was added, and acetic acid (1 M) was used to adjust to pH 7. EtOAc was added, and the organic layer was washed out with water, dried over Na2SO4, filtered, and concentrated under reduced pressure to give the final compound as a white solid; 65% yield (two steps); mp: 140–143 °C; 1H NMR (400 MHz, CDCl3) δ (ppm): 8.89 (dd, 1H, J = 1.4 Hz, 4.4 Hz), 8.15 (dd, 1H, J = 1.4 Hz, 8.6 Hz), 8.09 (s, 1H), 7.88 (d, 2H, J = 8.6 Hz), 7.62 (d, 1H, J = 8.2 Hz), 7.55 (dd, 1H, J = 4.4 Hz, 8.8 Hz), 7.45 (d, 2H, J = 8.8 Hz), 7.28 (d, 1H, J = 8.2 Hz). 13C NMR (100 MHz, CDCl3) δ (ppm): 153.9, 148.9, 146.8, 137.8, 134.3, 131.9, 129.2, 128.6, 127.1, 125.0, 124.4, 124.2, 123.5, 122.1, 108.8. IR (ῡ/cm−1): 3084, 1621, 1580, 1520, 1479, 1438, 1420, 1408. HRMS (m/s): [MH+] calc for C17H12ClN4O, 323.0694; found, 323.0693.

2.2. Fungal Strains

All microorganisms selected for this study are deposited in the library of the Applied Mycology Research Group laboratory at UFRGS. The sets of strains include Candida albicans: CA10; C. glabrata: CGM28; C. parapsilosis: CP10; C. tropicalis: CT04; Trycophyton mentagrophytes: TMEATCC; T. rubrum: TRU51; Microsporum canis: MCA01; Nannizia gypsea: MGYATCC; Fusarium solani: ATCC36031 and F. oxysporum: HCF46. The ATCC strains were acquired from the American Type Culture Collection (USA). The other strains were isolated from patients at HCPA (Hospital de Clínicas de Porto Alegre). All strains were chosen based on their relevance to this study.

Susceptibility Tests

The protocols of the Clinical Laboratory Standards Institute (CLSI) were applied to assess the susceptibility of the 8-hydroxyquinoline derivative against fungal species. The protocol M27-A3 was applied to yeast strains and the protocol M38-A2 to filamentous fungi [41,42]. All micro dilutions were performed in 96-well polystyrene microplates at inoculum concentration of 5 × 103 CFU/mL for Candida species and 6 × 103 CFU/mL for dermatophytes and Fusarium species.
The compound was solubilized in dimethyl sulfoxide and diluted in RPMI. The maximum DMSO concentration in the assay was limited to 2%. The experiments were conducted in triplicate and incubated for 48 h (30 °C for Fusarium spp. and 35 °C for yeasts) and 120 h (30 °C for dermatophytes). The minimum inhibitory concentration (MIC) was defined as the lowest concentration of compound 10 and other antifungal agents in which microorganisms did not grow. The concentration range evaluated for the novel derivative was 64 μg/mL–0.015 μg/mL. The positive controls were evaluated at 32 μg/mL–0.062 μg/mL (fluconazole), 64 μg/mL–0.125 μg/mL (voriconazole), and 8 μg/mL–0.015 μg/mL (ciclopirox olamine).

2.3. Cellular Action Studies

2.3.1. Sorbitol Protection Assays

A sorbitol protection assay was conducted to clarify the cellular action of the synthesized molecules on fungi cell walls. The MIC value of 10 was determined by microdilution in broth (CLSI M27-A3 for yeasts and CLSI M38-A2 for filamentous fungi) in the absence and presence of 0.8 M sorbitol (Sigma-Aldrich, St. Louis, MO, USA). The experiments were carried out in 96-well polystyrene microplates in duplicate. Anidulafungin (Pfizer®) was chosen for the positive control due to its action on the cellular wall. MICs were determined in two separate readings: after 2 and 7 days for the yeasts (35 °C) and after 5 and 7 days for the dermatophytes (30 °C) [43].

2.3.2. Ergosterol Effect Assay

An ergosterol effect assay was conducted to try to elucidate the effects of 10 on the cell membrane. Both 10 and the positive control MIC values were determined via broth microdilution. The micro dilutions were conducted twice—with and without exogenous ergosterol (Sigma-Aldrich, St. Louis, MO, USA). Ergosterol was added at varying concentrations: 50 μg/mL, 100 μg/mL, 150 μg/mL, and 200 μg/mL. Commercial-grade ergosterol was dissolved in DMF (Sigma-Aldrich, St. Louis, MO, USA) and diluted in RPMI 1640 culture medium to a final concentration of 0.1%. Duplicate 96-well polystyrene microplates were used for the experiment. Amphotericin B (União Química, São Paulo, Brazil) was used as the positive control due to its affinity for ergosterol and its action on the cell membrane. The MIC values were determined after 2 days at 35 °C for the yeasts and 5 days at 30 °C for the dermatophytes [41].

2.3.3. Confocal Fluorescence Microscopy of Fungal Strains Treated with Compound 10

A qualitative assay was carried out using confocal fluorescence microscopy to explore the action of derivative 10 on fungal cells. Monocultures of Candida albicans, Fusarium solani, and Trichophyton rubrum at 106 CFU/mL were treated with sub-inhibitory concentrations of 10. The fungal inoculum was prepared under the same standards for the evaluation of the susceptibility profile. The microscopy reading was performed up to 30 min after the administration of compound 10 in the inoculum.

2.3.4. Confocal Fluorescence Microscopy with Calcofluor White (CFW)

To gain deeper insight into the cellular effects of compound 10, a qualitative assay using Calcofluor White (CFW), a chitin-specific stain, was performed to assess potential changes in fungal cell wall chitin density following treatment. Candida albicans ATCC 24433 was selected for this assay. First, the MIC value of 10 was determined by broth microdilution (CLSI M27-A3), and the fungal culture was incubated for 24 h at 35 °C. Cells were then treated with 0.12 and 0.06 µg/mL of 10 (MIC/2 and MIC/4, respectively) to facilitate microscopic evaluation. Following treatment, cells were pelleted, stained with CFW, washed, and resuspended in PBS. DMSO 2% was used as a control.

2.3.5. Scanning Electron Microscopy

The effect of derivative 10 on the morphology of Candida albicans CA10 and Trichophyton rubrum TRU51 strains was evaluated by scanning electron microscopy (SEM). Inoculums of C. albicans and T. rubrum were incubated for 48 h at 35 °C and 96 h at 30 °C in sub-inhibitory concentration of derivatives. After the incubation period, treated and control cells were washed three times with PBS, followed by centrifugation. C. albicans cells were fixed in 1 mL of modified Karnovsky’s fixative composed of 2.5% glutaraldehyde and 0.1 M sodium cacodylate buffer for 1 h. T. rubrum cells were fixed in 1mL of the same solution supplemented with 4% paraformaldehyde for 1.5 h. After fixation, cells were washed three times in 1 mL of 0.1 M sodium cacodylate solution buffered at pH 7.2 containing 0.2 M sucrose and 2 mM MgCl2 and adhered in 18 mm round coverslips previously functionalized with poly-L-lysine. The adhered cells were dehydrated in solutions of increasing concentrations of absolute ethyl alcohol for analysis EMSURE® (30, 50, 70, 90, 95, and 100% for 10 min and 100% again for 20 min) and then subjected to critical point drying (EM CPD 300, Leica, Wetzlar, Germany). Samples were mounted on metallic stubs and sputter-coated with a 15–20 nm gold layer (Q150R Plus, Quorum Tech, Sacramento, CA, USA) and visualized in a scanning electron microscope (Carl Zeiss EVO® MA10 Carl, Oberkochen, Germany) operating at 10 kV.

3. Results

3.1. Chemistry

The synthesis route was based on the Ramirez–Corey–Fuchs reaction for the preparation of the appropriate terminal alkyne for the new 1,2,3-triazole derivative (Scheme 1). The first step was to synthesize 1,1-dibromoolefin 7 from 4-chlorobenzaldehyde. The 1,1-dibromoolefin were then converted to the terminal alkyne. Several bases such as n-BuLi, carbonates, or triethylamine could be used to eliminate the bromine hydrogen, which provided the desired terminal alkyne [37,38,39,40].
After the synthesis and elucidation of the desired terminal alkyne, the synthesis of 1,2,3-triazole derivative 10 was carried out. Initially, 5-nitro-8-hydroxyquinoline was prepared as previously described by Mazumder et al. [36]. Briefly, commercially available 8-hydroxyquinoline was dissolved in HCl (37%) and treated with NaNO2 to give 5-nitroso-8-hydroxyquinoline, which was oxidized with HNO3 to give the nitro intermediate. Palladium on carbon 10% catalyzed the reduction of 5-nitro-8-hydroxyquinoline with hydrazine solution (16%) to give 5-amino-8-hydroxyquinoline 3. Then, 5-azido-8-hydroxyquinoline was formed by diazotization of 3 with NaNO2 and concentrated HCl, followed by treatment with sodium azide. The triazole derivative was prepared by click chemistry, as previously described by Silva [32]. The cycloaddition of 5-azido-8-hydroxyquinoline and alkyne could not be carried out using the 8-hydroxyquinoline intermediate. Previous studies have shown that 8-hydroxyquinolines are bidentate chelators, preferentially binding to copper (II) and zinc (II) via the phenolic oxygen and pyridine nitrogen. Based on these literature reports, it was decided to introduce an acetyl group at OH-8 prior to cycloaddition to prevent copper chelation and a low-yielding reaction. The 5-azido-8-hydroxyquinoline was then treated with acetic anhydride and pyridine to give 5-azidoquinolin-8-yl acetate.
After acetylation of the hydroxyl group, a copper (I) catalyzed azide-alkyne cycloaddition reaction was conducted, giving the formation of 1,4–disubstituted 1,2,3–triazole by coupling a terminal alkyne and organic azide. Ascorbic acid and NaHCO3 in a water medium gave sodium ascorbate, which was used as a reducing agent to generate copper (I) in situ from copper (II) sulfate (CuSO4.5H2O). A nitrogen atmosphere was not required despite the instability of the oxidation state of copper (I) in the presence of oxygen. After deprotection, the derivative was confirmed by 1H and 13C NMR spectra (Supplementary Materials). The hydrogen signal of the 1,4-disubstituted triazole group was detected as a singlet, confirming the synthesis of compound 10.

3.2. Biological Assays

3.2.1. Fungal Susceptibility Test

Compound 10 was evaluated against a variety of fungal strains using available commercial drugs as positive controls: fluconazole, voriconazole, and ciclopirox olamine (Table 1).
The MIC values indicate the great potential of this compound as it showed activity against clinical yeast and dermatophyte strains.
Evaluation against Candida species showed low MIC values against all strains tested. In particular, compound 10 exhibited promising activity against C. glabrata and C. krusei—important pathogens with intrinsic resistance to fluconazole. Given the increasing prevalence of these fungi in immunocompromised patients, it is important to find alternatives to fluconazole-based treatments.
In addition, compound 10 showed potent activity against dermatophyte species, with MIC values ranging from 0.5 to 16 µg/mL. Furthermore, clinical infections caused by Fusarium species pose a significant therapeutic challenge. Remarkably, compound 10 showed activity against Fusarium species, successfully inhibiting the growth of F. solani at half the concentration required by the positive control, voriconazole.

3.2.2. Cellular Action Assays

Sorbitol Protection Assay

If the MIC value of a compound increases in the presence of sorbitol in the culture medium, this suggests that the compound may be acting on the fungal cell wall. As shown in Table 2, there was a slight increase in MIC values in the presence of sorbitol against C. albicans. On the other hand, the dermatophyte tests showed an increase in MIC values only after four days of treatment.

Exogenous Ergosterol Effect Assay

Compound 10 showed no change in MIC values with the addition of exogenous ergosterol against the three selected strains (Table 3).
It is expected that compounds acting on the fungal cell membrane may show higher MIC values in the presence of higher concentrations of ergosterol.

Confocal Fluorescence Microscopy

Confocal fluorescence microscopy of three different fungal species treated with compound 10 showed that the compound fluorescence appears to be concentrated at the edge of the fungal cells (Figure 1). A control image (Figure S19) of the fungal cell alone (without compound 10) showed very little fluorescence, confirming that the fluorescence was due to the presence of compound 10. These results are consistent with the sorbitol protection assay.
Interestingly, the same behavior can be observed in both yeast and filamentous fungi, corroborating with the previous studies of 8-hydroxyquinoline-5-substituted class.

Confocal Fluorescence Microscopy with Calcofluor White (CFW)

The Calcofluor White (CFW) staining assay was performed to evaluate the effect of compound 10 on the chitin in the Candida albicans cell wall (Figure 2). In the control group, CFW staining clearly highlighted the cell edges, marking the chitin-rich regions of the cell wall. Similarly, in cells treated with compound 10, the fluorescence was also concentrated along the cell edges, showing no noticeable difference in chitin density compared to the control.
These results indicate that treatment with compound 10 does not appear to affect the density of chitin in the fungal cell wall despite the morphological changes, suggesting that its antifungal activity may not involve direct interference with chitin synthesis or deposition.

Scanning Electron Microscopy

Several morphological changes were observed when C. albicans and T. rubrum strains were exposed to sub-inhibitory concentrations of compound 10 by analyzing the SEM images (Figure 3).
In C. albicans-treated cells, it was possible to observe the presence of cell clusters and changes in cell morphology, such as shrinkage and loss of smooth oval shape. In T. rubrum, treated hyphae showed roughness, grooves, cell wall damage, and changes in hypha width.

4. Discussion

Susceptibility tests were conducted against eleven fungal species capable of infecting humans or plants. Overall, compound 10 showed remarkable potency, being active against 10 of the 11 strains at low concentrations. In addition, compound 10 showed significant antifungal activity against Fusarium solani, with a MIC value lower than voriconazole. Based on its potency and fluorescence emission, compound 10 proved to be promising for further studies.
Previous studies by our research group have suggested that 8-hydroxyquinoline derivatives may damage the fungal cell wall. When compound 10 was tested in sorbitol protection and ergosterol assays, the results indicated that it likely affects the cell wall rather than the plasma membrane, as expected. These findings are consistent with those reported in the literature [25,26,32,35]. The planar structure and π-conjugated system of these aromatic compounds offer a promising approach to elucidate their cellular action through a qualitative assay, using the inherent fluorescence of the derivatives as chemical probes. To advance the study of the 8-hydroxyquinoline class, confocal fluorescence microscopy was proposed as it allows visualization of compound localization within fungal cells due to fluorescence accumulation. Confocal microscopy images of treated cells revealed a fluorescence accumulation along hyphae and at cell edges, while in untreated cells, only weak or no fluorescence was observed, showing that the intense fluorescence in treated cells is due to compound 10. The concentration of this derivative at the cell surface corroborates with the previously proposed effect of compound 10 on the fungal cell wall, consistent with sorbitol assay results and previous studies on non-fluorescent 8-hydroxyquinolines from our group. The Calcofluor White assay showed no significant difference in chitin density between treated and untreated Candida albicans cells, as indicated by similar staining patterns along the cell edges—this finding may suggest that the antifungal activity may be based on another component of the fungal cell wall.
To further investigate the morphological changes in fungal cells after treatment with compound 10, scanning electron microscopy (SEM) was used. SEM images showed remarkable structural changes in C. albicans and T. rubrum compared to untreated controls. In C. albicans, extensive cellular damage was observed in the blastoconidia, including significant changes in size and shape, multiple non-polar bud scars, and loss of the typical oval shape, indicating potential problems in the division process and signs of endopolyploidy. Altered cell shapes, suggesting changes in cell permeability and cell wall damage, were also noted. SEM images of T. rubrum showed severe hyphal damage compared to untreated cells, with irregularities, roughness, and visible cracks and pits on the hyphal surface. The rate of hyphae was also significantly reduced, which correlated with the low MIC value of compound 10 against T. rubrum.
Another important point to emphasize is that the three major classes of antifungals currently available (polyenes, echinocandins, and azoles) have significant limitations. Amphotericin (the major polyene agent) and echinocandins are natural or derivatives of natural products with limited physicochemical properties, mainly due to their higher molar mass (compared to most synthetic drugs) and the higher presence of hydrogen bond donor groups—such as hydroxyl. They are also expensive drugs limited to only hospital use. Echinocandins have a narrow spectrum, while amphotericin can cause severe side effects due to off-target binding of host membranes, resulting from its ability to form membrane pores [44]. On the other hand, the major class to control fungal infection, the azole derivatives, is associated with the development of fungal resistance due to the extensive use of this class in clinical and agricultural fields [45]. Compound 10 is a low-molecular-weight synthetic compound with relevant MIC values against 10 of the 11 strains tested, suggesting broad-spectrum activity. Its promising efficacy highlights its potential as an alternative to currently available drugs. In addition, cellular studies indicated an effect on the cell wall, which is a microorganism-specific structure, further increasing interest in this derivative.
Other results reported in the literature support the findings of this study. 8-hydroxyquinoline-5-sulfonamides have previously been evaluated against fungi responsible for dermatomycoses in the presence and absence of sorbitol [35]. Similar to the results found in this study, 8HQ derivatives containing sulfonamide substituents at the 5-position were less active in the presence of sorbitol, indicating a cellular effect on the fungal wall, which was also supported by the SEM assay. Although sulfonamides and triazoles are different functional groups, triazoles can be considered bioisosteres of sulfonamides, and similar results are, therefore, a good indication of validation of the results. In another relevant study, Silva et al. showed that 8HQ derivatives containing other triazole substituents at the 5-position gave similar results in the sorbitol and SEM assays [32].
However, the results reported in this article are groundbreaking because, to the best of our knowledge, this is the first time that compound 10 has been reported in the literature, and, in general, the derivative was the most potent antifungal among the other 5-triazole-8-hydroxyquinolines already described. In addition, the accumulation of an 8HQ at the cell edge was described for the first time by confocal fluorescence microscopy, confirming previous results. It was also shown for the first time that the effect of 8HQ is not related to chitin synthesis. All these results contribute to a better understanding of the antifungal action of 5-triazole-8-hydroxyquinoline and allow us to advance the state of the art.

5. Conclusions

Overall, compound 10 was successfully synthesized and characterized, and it showed significant activity against several fungal strains. The new compound has great potential as a hit molecule, especially due to its low MIC values against T. rubrum, C. albicans, and F. solani. In addition, we successfully designed and synthesized a fluorescent chemical probe, which allowed a better understanding of the cellular action of the 8-hydroxyquinoline core. The novel derivative 10 showed strong potential, suggesting an action on the fungal cell edge due to fluorescence accumulation observed in confocal microscopy and corroborated by structural cell wall damage evident in SEM images.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/applmicrobiol5020038/s1, Figure S1: 1H NMR of 5-nitro-8-hydroxyquinoline; Figure S2: Zoom of the 1H NMR of 5-nitro-8-hydroxyquinoline spectrum; Figure S3: 13C NMR of 5-nitro-8-hydroxyquinoline; Figure S4: HPLC trace of 5-nitro-8-hydroxyquinoline; Figure S5: IR spectrum of 5-nitro-8-hydroxyquinoline; Figure S6: HRMS spectrum of 5-nitro-8-hydroxyquinoline; Figure S7: 1H NMR of 5-azide-8-hydroxyquinoline spectrum; Figure S8: Zoom of the 1H NMR of 5-azide-8-hydroxyquinoline; Figure S9: 13C NMR of 5-azide-8-hydroxyquinoline spectrum; Figure S10: HPLC trace of 5-azide-8-hydroxyquinoline; Figure S11: IR spectrum of 5-azide-8-hydroxyquinoline; Figure S12: HRMS spectrum of 5-azide-8-hydroxyquinoline; Figure S13: 1H NMR of 5-(4-(4-chlorophenyl)-1H-1,2,3-triazol-1-yl)quinoline-8-ol spectrum; Figure S14: Zoom of the 1H NMR of 5-(4-(4-chlorophenyl)-1H-1,2,3-triazol-1-yl)quinoline-8-ol spectrum; Figure S15: 13C NMR of 5-(4-(4-chlorophenyl)-1H-1,2,3-triazol-1-yl)quinoline-8-ol spectrum; Figure S16: HPLC trace of 5-(4-(4-chlorophenyl)-1H-1,2,3-triazol-1-yl)quinoline-8-ol; Figure S17: IR spectrum of 5-(4-(4-chlorophenyl)-1H-1,2,3-triazol-1-yl)quinoline-8-ol; Figure S18: HRMS spectrum of 5-(4-(4-chlorophenyl)-1H-1,2,3-triazol-1-yl)quinoline-8-ol; Figure S19: Fluorescence microscopy of Candida albicans, Fusarium solani and Trichophyton rubrum cells untreated in bright field (left) and UV filter (right).

Author Contributions

C.d.B.G. designed and performed experiments, collected data, analyzed the results, and wrote and reviewed the manuscript. M.S.L. designed and performed experiments, collected data, analyzed the results, and reviewed the manuscript. P.M.Q. performed experiments, collected data, and wrote the manuscript. M.P.G. performed experiments and collected data. M.A.d.C. performed experiments and collected data. A.P.P. performed experiments and collected data. W.L. designed and performed the experiments, collected data, analyzed the results, and reviewed the manuscript. A.M.F. designed the experiments, provided resources, analyzed the results, and reviewed the manuscript. M.H.V. designed the experiments, provided resources, analyzed the results, and reviewed the manuscript. S.F.d.A. designed the experiments, provided resources, analyzed the results, and wrote and reviewed the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the Brazilian agencies Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Chamada CNPq/MCTI/FNDCT No. 18/2021-Faixa A-Grupos Emergentes, grant 406112/2021-5; CNPq, Apoio a projetos de pesquisa científica, tecnológica e de inovação-Bolsas de Doutorado, grant 140293/2021-3) and Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul (FAPERGS-EDITAL 05/2019—PROGRAMA PESQUISADOR GAÚCHO-PQG-19 grant 2551-0001972-6; EDITAL FAPERGS 06/2021—PROGRAMA DE REDES INOVADORAS DE TECNOLOGIAS ESTRATÉGICAS DO RIO GRANDE DO SUL—RITEs-RS, 22/2551-0000396-6) by financial support and research fellowships. This study was financed, in part, by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior-Brasil (CAPES)-Finance Code 001.

Data Availability Statement

Data are contained within the article and Supplementary Materials.

Conflicts of Interest

The authors declare no conflicts of interest.

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Applmicrobiol 05 00038 sch001
Scheme 1. Synthesis of compound 10. Reactants and conditions: (a) NaNO3/HCl, 0 °C, 15 min; (b) HNO3/H2O, 17 °C, 1 h 15min; (c) Pd/C, 16% hydrazine aqueous solution, isopropyl alcohol, 82 °C, 3 h; (d) NaNO3, NaN3, HCl, −3 °C then 0 °C for 1.5 h, rt for 24 h; (e) acetic anhydride, pyridine, rt, 1 h; (f) CBr4, PPh3, 0 °C, 3 h; (g) Cs2CO3, 110 °C, overnight; (h) alkyne 8, CuSO4.5H2O, ascorbic acid, NaHCO3, t-BuOH/H2O, rt, 24 h; (i) KOH, EtOH, rt, 1 h.
Scheme 1. Synthesis of compound 10. Reactants and conditions: (a) NaNO3/HCl, 0 °C, 15 min; (b) HNO3/H2O, 17 °C, 1 h 15min; (c) Pd/C, 16% hydrazine aqueous solution, isopropyl alcohol, 82 °C, 3 h; (d) NaNO3, NaN3, HCl, −3 °C then 0 °C for 1.5 h, rt for 24 h; (e) acetic anhydride, pyridine, rt, 1 h; (f) CBr4, PPh3, 0 °C, 3 h; (g) Cs2CO3, 110 °C, overnight; (h) alkyne 8, CuSO4.5H2O, ascorbic acid, NaHCO3, t-BuOH/H2O, rt, 24 h; (i) KOH, EtOH, rt, 1 h.
Applmicrobiol 05 00038 sch001
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Figure 1. Fluorescence microscopy of Candida albicans, Fusarium solani, and Trichophyton rubrum cells treated with compound 10 in MIC/2 concentrations in bright field (left) and UV filter (right).
Figure 1. Fluorescence microscopy of Candida albicans, Fusarium solani, and Trichophyton rubrum cells treated with compound 10 in MIC/2 concentrations in bright field (left) and UV filter (right).
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Figure 2. Fluorescence microscopy of Candida albicans treated with CFW and compound 10 in MIC/2 and MIC/4 concentrations, respectively, in bright field (left) and UV filter (right).
Figure 2. Fluorescence microscopy of Candida albicans treated with CFW and compound 10 in MIC/2 and MIC/4 concentrations, respectively, in bright field (left) and UV filter (right).
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Figure 3. Scanning electron microscopy of Candida albicans and Trichophyton rubrum treated with a sub-inhibitory concentration of 10 and untreated cells. (A) Untreated cells of C. albicans; (A1) (Bar = 10 μm): large number of regular cells; (A2) (Bar = 2 μm): same field of (A1), showing the oval-shaped cells. (B) C. albicans treated with 10; (B1) (Bar = 10 μm): fewer cells with cluster formation; (B2) (Bar = 2 μm): loss of natural shape and presence of large round cells. (C) Untreated T. rubrum; (C1) (Bar = 10 μm): intense formation of hyphae; (C2) (Bar = 2 μm): regular and intact hyphae. (D) Hyphal cells treated with 10; (D1) (Bar = 10 μm); (D2) (Bar = 2 μm): irregular hyphae, showing rough cell walls with grooves, pitting, and tears.
Figure 3. Scanning electron microscopy of Candida albicans and Trichophyton rubrum treated with a sub-inhibitory concentration of 10 and untreated cells. (A) Untreated cells of C. albicans; (A1) (Bar = 10 μm): large number of regular cells; (A2) (Bar = 2 μm): same field of (A1), showing the oval-shaped cells. (B) C. albicans treated with 10; (B1) (Bar = 10 μm): fewer cells with cluster formation; (B2) (Bar = 2 μm): loss of natural shape and presence of large round cells. (C) Untreated T. rubrum; (C1) (Bar = 10 μm): intense formation of hyphae; (C2) (Bar = 2 μm): regular and intact hyphae. (D) Hyphal cells treated with 10; (D1) (Bar = 10 μm); (D2) (Bar = 2 μm): irregular hyphae, showing rough cell walls with grooves, pitting, and tears.
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Table 1. MIC results of compound 10 and antifungal control against fungal species.
Table 1. MIC results of compound 10 and antifungal control against fungal species.
CA 10CK ATCC 6258CG M28CP 10CT 04TME ATCCMCA 01MGY ATCCTRU 51FS
ATCC
36031		FO
HCF46
10111120.5120.54>64
FCZ16>3240.54------
CICLO-----<0.125>10.251--
VCZ---------84
FCZ—fluconazol (µg/mL): 50% of inhibition; CICLO: ciclopirox; VCZ: voriconazol; CA: Candida albicans; CK: Candida krusei; CG: Candida glabrata; CP: Candida parapsilosis; CT: Candida tropicalis. TME: Trichophyton mentagrophytes; MCA: Microsporum canis; MGY: Nannizzia gypsea; TRU: Trichophyton rubrum; FS: Fusarium solani; FO: Fusarium oxysporum.
Table 2. Minimum inhibitory concentration (µg/mL) for 10 and Micafungin obtained by sorbitol protection assay without and with sorbitol addition.
Table 2. Minimum inhibitory concentration (µg/mL) for 10 and Micafungin obtained by sorbitol protection assay without and with sorbitol addition.
Fungi StrainsReadings10MCF
−/Sorbitol+/Sorbitol−/Sorbitol+/Sorbitol
CAMS5Day 2240.0150.03
Day 7480.03>2
MCA 01Day 4120.1252
Day 7220.1252
TME 40Day 4240.1252
Day 7440.1250.5
MCF: Micafungin; −/Sorbitol: without sorbitol; +/Sorbitol: with sorbitol.
Table 3. Minimum inhibitory concentration (µg/mL) obtained by ergosterol binding assay for 10 and Amphotericin B before and after adding ergosterol.
Table 3. Minimum inhibitory concentration (µg/mL) obtained by ergosterol binding assay for 10 and Amphotericin B before and after adding ergosterol.
CompoundsFungi StrainsMIC1MIC2MIC3MIC4MIC5
10CAMS522222
MCA 0121222
TME 4021222
AFBCAMS52>8>8>8>8
MCA 018>8>8>8>8
TME 40>8>8>8>8>8
CAMS5: Candida albicans; MCA 01: Microsporum canis; TME 40: Trichophyton mentagrophytes. MIC1 corresponds to MIC without the addition of commercial ergosterol. MIC2, MIC3, MIC4, and MIC5 correspond to MIC with the addition of ergosterol at the concentrations of 100 μg/mL, 150 μg/mL, 200 μg/mL, and 250 μg/mL, respectively.
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Gentz, C.d.B.; Lopes, M.S.; Quatrin, P.M.; Gionbelli, M.P.; de Cesare, M.A.; Perin, A.P.; Lopes, W.; Fuentefria, A.M.; Vainstein, M.H.; Andrade, S.F.d. A Potent Fluorescent Derivative of 8-Hydroxyquinoline Suggests Cell Wall Damage as a Possible Cellular Action of the 5-Triazole 8-Hydroxyquinoline Class. Appl. Microbiol. 2025, 5, 38. https://doi.org/10.3390/applmicrobiol5020038

AMA Style

Gentz CdB, Lopes MS, Quatrin PM, Gionbelli MP, de Cesare MA, Perin AP, Lopes W, Fuentefria AM, Vainstein MH, Andrade SFd. A Potent Fluorescent Derivative of 8-Hydroxyquinoline Suggests Cell Wall Damage as a Possible Cellular Action of the 5-Triazole 8-Hydroxyquinoline Class. Applied Microbiology. 2025; 5(2):38. https://doi.org/10.3390/applmicrobiol5020038

Chicago/Turabian Style

Gentz, Caroline de Bem, Marcela Silva Lopes, Priscilla Maciel Quatrin, Mariana Pies Gionbelli, Maycon Antonio de Cesare, Ana Paula Perin, William Lopes, Alexandre Meneghello Fuentefria, Marilene Henning Vainstein, and Saulo Fernandes de Andrade. 2025. "A Potent Fluorescent Derivative of 8-Hydroxyquinoline Suggests Cell Wall Damage as a Possible Cellular Action of the 5-Triazole 8-Hydroxyquinoline Class" Applied Microbiology 5, no. 2: 38. https://doi.org/10.3390/applmicrobiol5020038

APA Style

Gentz, C. d. B., Lopes, M. S., Quatrin, P. M., Gionbelli, M. P., de Cesare, M. A., Perin, A. P., Lopes, W., Fuentefria, A. M., Vainstein, M. H., & Andrade, S. F. d. (2025). A Potent Fluorescent Derivative of 8-Hydroxyquinoline Suggests Cell Wall Damage as a Possible Cellular Action of the 5-Triazole 8-Hydroxyquinoline Class. Applied Microbiology, 5(2), 38. https://doi.org/10.3390/applmicrobiol5020038

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