(7-Chloroquinolin-4-yl)arylhydrazones: Candida albicans enzymatic repression and cytotoxicity evaluation, Part 2

Abstract

Objective: This work describes the anti-enzymatic activity of (7-chloroquinolin-4-yl)arylhydrazones against Candida albicans and examines their cytotoxicity.

Material and methods: Ten C. albicans strains [nine isolates and one azole-resistant standard strain (ATCC 62342)] were used to assess the anti-enzymatic activity. Fifteen compounds at sub-antifungal concentrations ranging from 12.5 to 100 µg/ml were assessed after a 30-min exposure. The strains were seeded onto petri dishes with selective agar media for aspartyl proteases (Saps) and phospholipases (PLs). Enzymatic inhibition was measured by the reduction of the precipitation zone (Pz) against untreated strains (positive control). A colorimetric MTT assay was used with 3T3/NIH mouse fibroblasts to evaluate cytotoxicity. Cells were exposed to 15 compounds in concentrations from 6.25 to 100 µg/ml for 24 and 48 h.

Results: Four hydrazones showed enzymatic repression values over 40% to Pl and three over 20% to Saps. The cell viability was over 50% at hydrazone concentrations of 25–100 µg/ml.

Conclusion: These results revealed that select (7-chloroquinolin-4-yl)arylhydrazones may be potential antifungal agents for the control of C. albicans infections.

• 7-Chloroquinoline
• Candida albicans
• hydrazones
• phospholipases
• secreted aspartyl proteases
• virulence factors

Introduction

The increase of Candida albicans strains that are resistant to antifungal azoles is one of the major reasons for the investigation of new antimicrobial agents1. Candida spp. pathogenicity has been related to an imbalance between the host and the opportunist fungus2. In addition, it has been associated with the expression of virulence factors by the yeast, including the production of factors related to adhesion, adherence and biofilm formation as well as exoenzymes.

Recent studies support investigating drugs with specific targets, suggesting a change of focus to the development of new antifungal agents capable of inhibiting virulence factors. Moreover, industry has stimulated the development of new methods to identify and inhibit these virulence factors with lower doses of antimicrobials, aiming to avoid an increase in antifungal resistance3–5.

The prevalence of exoenzyme production by C. albicans and the effect of several antimicrobials on the enzymatic secretion of these yeasts have been investigated4^,6^,7. The most frequent enzymes produced by C. albicans are Secreted Aspartyl Proteases (Saps) and Phospholipases (PLs)5. However, no difference in the production of PLs or Saps was reported between healthy patient isolates and isolates from patients with denture stomatitis4 or between isolates from diabetic patients with candidiasis and non-diabetic patient isolates6^,8.

Saps and PLs have many functions in the virulence of C. albicans, such as the degradation of immunoglobulin and proteins of the extracellular matrix, inhibition of neutrophil phagocytosis, epithelial/mucosal invasion and tissue degradation5^,9.

Hydrazones and hydrazides are important in medicinal chemistry because of their versatile biological properties10^,11. These molecules are easily prepared and have diverse pharmacological potentials, which lead to the synthesis of various novel hydrazone compounds. Numerous studies have centered on hydrazones for the development of better biological molecules with low toxicity profiles12^,13.

The biological potential of hydrazone derivatives has been widely described in the literature; considering the variety of biological activities described, the antimicrobial potential seems to be very significant12–14. Biological activity has been described against many strains. However, the strains that hydrazones are most frequently reported to have biological activities against are Staphylococcus aureus, Escherichia coli, Candida albicans, Bacillus subtilis and Pseudomonas aeruginosa. In addition to the antimicrobial activity, hydrazones have also been reported to have anticancer, anti-inflammatory, antiprotozoal, antiplatelet and cardio-protective properties14. Many effective compounds, such as iproniazid and isocarboxazid, are synthesized by the reduction of hydrazide-hydrazones and are used in the treatment of tuberculosis (TB) and display an anti-depressant effect12.

The most significant reactivity of hydrazones is the nucleophilicity of the carbon atom. It appears that hard nucleophiles preferentially attack nitrogen, while soft nucleophiles preferentially attack carbon. The functional substituents retain their established reactivity pattern, although they generally become more electrophilic14.

Recently, our research group reported on the in vitro antifungal activity of (7-chloroquinolin-4-yl)hydrazones against strains of Candida spp. and Rhodotorula spp.7 The aim of this study was to evaluate the activity of such hydrazones on the production of PLs and Saps by various strains of C. albicans and their cytotoxicity in 3T3/NIH mouse fibroblasts cultures.

Materials and methods

Chemistry

Fifteen hydrazones (1a–1o) with the same basic structure were synthesized from the reaction of 7-chloro-4-hydrazinoquinoline and arenealdehydes refluxed in toluene for 3 hours, according to our previous method7. The compounds differ from one another by the type (electron-withdrawing or electron-donating) and the position (ortho, meta or para) of the substituent attached to the benzene ring. The general structure of the hydrazones is shown in Figure 1.

Figure 1. General structure of the tested 7-chloro-4-quinolinylarylhydrazones.

Strain culture conditions

Tests were carried out with 10 different strains of C. albicans, including one azole-resistant strain (ATCC 62342). Candida isolates were obtained from the mouth of patients with denture stomatitis at multiple affected sites and identified as Candida albicans. The strains are part of a fungi bank at the Oral Microbiology Lab at the Federal University of Pelotas, which includes over 100 isolates. For these assays, strains were maintained at −80 °C, defrosted at room temperature and suspended in Sabouraud Dextrose Broth (Acumedia Manufacturers, Inc., Lansing, MI) for overnight incubation. Next, a 20-µl aliquot was seeded in Sabouraud Dextrose Agar with 0.01% chloramphenicol and incubated for 24 h at 37 °C.

Anti-enzymatic assay

For the preparation of inocula, the strains were individually suspended in 5 ml of 1% sterile phosphate-buffered saline (PBS), with turbidity and visual refraction corresponding to 0.5 on the McFarland scale (1–5 × 10⁶ microorganisms/ml).

The chemical compounds (Table 1) were initially diluted in dimethyl sulfoxide (DMSO) at a concentration of 1 mg/ml and then serially diluted by half until 10 different concentrations were obtained. Yeasts were then exposed to two different concentrations below the minimal inhibitory concentration, as previously described7. For the exposure, 20 µl aliquots of each concentration were added to 1980 µl of PBS. Immediately, 0.5 ml of the strain suspension was added to the PBS + anti-fungal agent and incubated for 30 min at 37 °C. DMSO exposure was used as a control.

Table 1. Comparison between Pz and inhibition ratio results in percentage for PL production.

Comp.	R	Concentration 1 (µg/ml)	Concentration 2 (µg/ml)	Control Pz (mean ± SD)	Concentration 1 Pz (mean ± SD)*	Concentration 2 Pz (mean ± SD)	Inhibition Ratio 1 (%)	Inhibition Ratio 2 (%)
1a	2-F	25	12.5	^a0.55 ± 0.04	^b0.88 ± 0.06A	^c0.87 ± 0.11A	74.25 ± 12.54	71.13 ± 22.07
1b	3-F	100	50	^a0.64 ± 0.11	^a0.71 ± 0.08B	^a0.72 ± 0.08B	14.14 ± 29.33	15.76 ± 31.66
1c	4-F	100	50	^a0.64 ± 0.11	^a0.69 ± 0.05B	^a0.64 ± 0.05B	6.35 ± 28.83	(−)5.08 ± 27.35†
1d	2-Cl	100	50	^a0.64 ± 0.27	^b0.77 ± 0.2B	^b0.68 ± 0.24B	23.16 ± 28.12	7.35 ± 14.26†
1e	2-OH	50	25	^a0.62 ± 0.11	^b0.78 ± 0.08C	^a0.7 ± 0.07B	41.77 ± 20.35	21.13 ± 11.97†
1f	3-OH	100	50	^a0.46 ± 0.07	^a0.47 ± 0.07D	^a0.44 ± 0.06C	0.26 ± 3.50	(−)3.82 ± 7.71
1g	4-OH	50	25	^a0.47 ± 0.04	^b0.69 ± 0.16B	^c0.55 ± 0.06D	41.73 ± 27.68	14.22 ± 15.16†
1h	2-OCH3	100	50	^a0.43 ± 0.06	^a0.44 ± 0.07D	^a0.43 ± 0.07C	0.97 ± 9.99	(−)0.87 ± 10.64
1i	3-OCH3	100	50	^a0.56 ± 0.13	^b0.73 ± 0.17B	^b0.65 ± 0.14B	44.63 ± 29.47	23.44 ± 16.90†
1j	4-OCH3	50	25	^a0.54 ± 0.08	^b0.73 ± 0.17B	^b0.62 ± 0.08B	43.59 ± 30.51	16.92 ± 6.48†
1k	2-CN	100	50	^a0.48 ± 0.08	^a0.46 ± 0.08D	^a0.45 ± 0.04C	(−)3.42 ± 8.33	(−)7.21 ± 10.86†
1l	3-CN	50	25	^a0.50 ± 0.06	^b0.71 ± 0.08B	^b0.62 ± 0.08B	42.42 ± 13.84	23.86 ± 8.28†
1m	4-CN	50	25	^a0.43 ± 0.06	^a0.46 ± 0.07D	^a0.45 ± 0.09C	3.14 ± 17.53	3.47 ± 10.25
1n	3-NO2	100	50	^a0.47 ± 0.03	^b0.57 ± 0.07E	^c0.54 ± 0.03D	17.99 ± 16.93	13.62 ± 4.80
1o	4-CH3	25	12.5	^a0.66 ± 0.11	^b0.75 ± 0.07F	^a0.7 ± 0.08B	25.67 ± 16.53	10.02 ± 8.30†

*Different lowercase letters indicate significant difference in rows (p < 0.05) and different capital letters indicate significant difference in columns.

†Indicates the cases with significant difference between Inhibition Ratio for the tested concentration 1 and Inhibition Ratio for the tested concentration 2.

After incubation, the solutions were centrifuged at 3000 rpm for 10 min, the supernatant was removed and the pellet was washed in 2 ml of sterile PBS. This process was repeated twice to remove the antifungal agent. The pellet was then suspended in 2 ml of sterile PBS.

For the enzyme production measurement, 20 µl of the final suspension was plated in specific agar medium for PLs (Sabouraud Dextrose Agar 65 g, sodium chloride 57.3 g, calcium chloride 0.55 g and 40 ml of sterile egg yolk) and Saps (Agar–agar 20 g, glucose 20 g, yeast nitrogen base [Difco, Difco Laboratories Detroit, MI. ref. 233520, without amino acids and ammonium sulfate] 1.45 g and bovine serum albumin 2 g).

Results were measured at 48 h for Saps and 96 h for PLs to analyse the enzyme production. Pz was calculated as previously described by Barros et al.5 Colony diameter (cd) and the diameter of the colony plus the precipitation diameter around the colony (cd + p) were measured and Pz was calculated using Equation (1). (1)

For further analysis, the results of the control Pz_c and the test Pz_t were compared to calculate the enzymatic inhibition percentage (Ei%) using Equation (2). (2)

Cell culture

The NIH/3T3 cell strain was obtained from the Rio de Janeiro Cell Bank (PABCAM, Federal University of Rio de Janeiro, RJ, Brazil). The cells were cultured in Dulbecco's modified Eagle's medium (DMEM), supplemented with 10% fetal bovine serum (FBS) purchased from Vitrocell Embriolife (Campinas, Brazil) and Gibco (Grand Island, NY), respectively. Cells were grown at 37 °C in an atmosphere of 95% humidified air and 5% CO₂, as described previously15. The experiments were performed with cells in the logarithmic phase of growth.

Cytotoxicity assay

Briefly, cells were seeded at a density of 2 × 10⁴ cells per well in a volume of 100 µl in 96-well plates and grown at 37 °C in a humidified atmosphere of 5% CO₂/95% air for 24 h. Cells were incubated with 11 different hydrazone compounds for 24 h at the following concentrations: 6.25, 12.5, 25 and 50 µg/ml. These compounds were previously dissolved in DMSO and added to DMEM supplemented with 10% FBS to the desired concentrations. The final DMSO concentration in the culture medium never exceeded 0.5%, and a control group exposed to an equivalent concentration of DMSO was evaluated. After incubation, 20 µl of MTT (5 mg MTT/ml solution) was added to each well. The plates were incubated for an additional 3 h, and the medium was discarded. About 200 µl of DMSO was added to each well, and the formazan was solubilized on a shaker for 5 min at 100 × g. The viability of the NIH/3T3 cells was determined by measuring the reduction of soluble MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] to water-insoluble formazan16. The absorbance of each well was read on a microplate reader (Thermo Plate TP-Reader, Thermo Fisher Scientific, Waltham, MA) at a wavelength of 492 nm15–17.

The rate of cell growth inhibition was determined using Equation (3):

(3)

Statistical analysis

Data were typed twice and descriptive and statistical analyses were carried out using the Stata 12.0 software package (StataCorp LP; College Station, TX). To compare all groups, one-way anova was performed followed by Tukey's test. A p value of <0.05 was considered statistically significant.

Results

Anti-enzymatic activity

The compounds with significant results had inhibition values over 40% for PLs and 20% for Saps. The most effective compound was hydrazone 1a, which showed a PL inhibition of 71% at the lowest concentration (12.5 µg/ml) and inhibited Saps by 23% at 25 µg/ml. Compound 1o showed little inhibition of PL (25%); however, 1o inhibited 20% of Saps production at 25 µg/ml. Furthermore, four compounds (1e, 1g, 1j and 1l) also showed important enzymatic suppression of PLs, and one compound (1l) suppressed Saps at 50 µg/ml (Tables 1 and 2).

Table 2. Comparison between Pz and inhibition ratio results in percentage for Spas production.

Comp.	R	Concentration 1 (µg/ml)	Concentration 2 (µg/ml)	Control Pz (mean ± SD)	Concentration 1 Pz (mean ± SD)*	Concentration 2 Pz (mean ± SD)	Inhibition Ratio 1 (%)	Inhibition Ratio 2 (%)
1a	2-F	25	12.5	^a0.19 ± 0.02	^b0.38 ± 0.1A	^c0.29 ± 0.06A	23.64 ± 10.44	12.93 ± 5.53†
1b	3-F	100	50	^a0.15 ± 0.02	^a0.16 ± 0.02BC	^a0.16 ± 0.03B	0.26 ± 1.94	0.39 ± 2.89
1c	4-F	100	50	^a0.15 ± 0.02	^a0.15 ± 0.03B	^a0.15 ± 0.03C	(−)0.32 ± 3.21	(−)0.54 ± 4.51
1d	2-Cl	100	50	^a0.12 ± 0.01	^b0.17 ± 0.04C	^c0.14 ± 0.01D	5.48 ± 4.29	2.43 ± 1.67†
1e	2-OH	50	25	^a0.15 ± 0.03	^b0.26 ± 0.08DI	^c0.21 ± 0.05B	12.26 ± 8.65	6.16 ± 5.88†
1f	3-OH	100	50	^a0.13 ± 0.03	^b0.14 ± 0.03E	^a0.12 ± 0.02E	0.80 ± 1.22	(−)1.13 ± 2.63
1g	4-OH	50	25	^a0.13 ± 0.02	^b0.22 ± 0.1F	^c0.19 ± 0.04F	11.05 ± 11.72	6.99 ± 5.69†
1h	2-OCH3	100	50	^a0.11 ± 0.02	^a0.11 ± 0.03G	^a0.11 ± 0.03E	(−)0.52 ± 3.92	(−)0.25 ± 4.02
1i	3-OCH3	100	50	^a0.19 ± 0.05	^b0.24 ± 0.05D	^a0.23 ± 0.09B	23.78 ± 50.56	4.04 ± 8.58†
1j	4-OCH3	50	25	^a0.18 ± 0.04	^b0.26 ± 0.08DI	^c0.22 ± 0.05B	9.03 ± 6.08	4.64 ± 2.17†
1k	2-CN	100	50	^a0.13 ± 0.03	^a0.13 ± 0.02G	^a0.12 ± 0.02E	0.23 ± 1.31	(-)1.22 ± 1.52
1l	3-CN	50	25	^a0.2 ± 0.04	^b0.4 ± 0.26H	^c0.27 ± 0.07G	25.01 ± 32.36	8.1 ± 10.81†
1m	4-CN	50	25	^a0.11 ± 0.02	^a0.12 ± 0.03G	^a0.12 ± 0.02E	1.21 ± 0.93	0.3 ± 3.03
1n	3-NO2	100	50	^a0.12 ± 0.01	^b0.15 ± 0.02B	^a0.13 ± 0.03E	3.61 ± 1.65	1.83 ± 3.05†
1o	4-CH3	25	12.5	^a0.14 ± 0.036	^b0.31 ± 0.15I	^c0.23 ± 0.06B	19.93 ± 16.70	9.78 ± 6.91†

*Different lowercase letters indicate significant difference in rows (p < 0.05) and different capital letters indicate significant difference in columns.

†Indicates the cases with significant difference between Inhibition Ratio for the tested concentration 1 and Inhibition Ratio for the tested concentration 2.

Cytotoxicity

To evaluate whether the hydrazones had cytotoxic effects, the MTT assay was carried out (Figure 2). The cytotoxic activities of compounds 1a, 1g, 1l and 1o were published in our previous work7. In this study, 1e, 1j and 1m were the least cytotoxic. Interestingly, cell proliferative activity was observed for compounds 1e and 1m at the lowest concentration tested (6.25 µg/ml). Compound 1e also increased the cell count at 12.5 µg/ml. The highest cytotoxicity was presented by 1i, as shown by the following inhibitory ratios: 57.3% (6.25 µg/ml), 81.4% (12.5 µg/ml), 91.1% (25 µg/ml) and 97.3% (50 µg/ml).

Figure 2. Fibroblast cytotoxicity of the tested compounds, Y axis represents the absorbance at 492 nm and X axis the tested concentration in µg/ml. Bars with different letters indicate statistically significant intragroup differences by one-way ANOVA followed by Tukey's test (p < 0.05).

Discussion

Although the number of antifungal drugs has increased, the clinical need for novel antifungals remains important in today’s medical practice. Considering the worldwide increase in fungal infections associated with patients with impaired immunity, a new gamma of antifungal drugs has been developed in the last two decades. Unfortunately, the increased use of antifungal drugs caused an expansion of fungal resistance to most commercially available drugs18.

In humans, Candida spp. are the most common fungal pathogens. Moreover, Candida albicans is known as the main fungus associated with human candidiasis, and it is the most virulent species of Candida18.

Protein and enzyme secretion is considered an essential process in fungal survival, and the characteristics of the secreted proteins define many of the functional capabilities of these microorganisms. The secretion of hydrolytic enzymes by C. albicans, the expression of surface adhesins, morphological mutation between hyphae and yeast, phenotypic switch and biofilm formation are crucial for species perpetuation19.

The methodology used was previously validated by Kadir et al.4 for the inhibition of PLs using chlorhexidine in various concentrations. In this study, the authors observed statistically significant reductions of PLs at concentrations of 0.0012% and 0.002% chlorhexidine against 10 oral isolates of C. albicans. However, their data are presented in Pz results, impairing observations of the amount of enzymatic inhibition and comparisons with percentage values4.

The hydrazones are a class of compounds cited as biologically valuable for pharmaceutical applications due to their anti-inflammatory20, anticancer21, anti-tuberculosis22, anti-HIV23 and anti-Alzheimer's disease (cholinesterase inhibitors) properties24. The literature also shows important antifungal properties of hydrazones25–27. In addition, the quinoline ring is also an important group in medicinal chemistry and has particular utility in antimalarial28, antituberculosis²⁹, antileishmania³⁰ and antimicrobial³¹ agents.

In this study, the minimal concentrations needed to inhibit the enzymatic activity ranged from 6.25 to 100 µg/ml. It is important to highlight that the basic chain structure was maintained and that only the substituent attached to the heterocyclic ring was changed. These results suggest an effect dependent on the association between the general structure and the substituent.

Although the focus in this study was the inhibition of Candida species growth, it is important to note that compounds 1e and 1m were able to stimulate cell growth. This result indicates that these hydrazones could be used to inhibit fungal growth and to stimulate tissue repair.

The most effective heterocyclic ring substitution for the repression of the PL production appeared to be compound 1a (71% inhibition at 12.5 μg/ml). Unfortunately, this compound also showed cytotoxicity at this concentration7. This result is important because compound 1a showed the best MIC value against C. albicans strains (25 μg/ml) in our previous study7. Compound 1a also had the best inhibitory activity against the Sap production. However, hydrazone 1o appears to be the best compound because, at 25 μg/ml, it reduced the production of Saps by 20% and did not display cytotoxicity at 50 μg/ml in 3T3/NIH fibroblast cells7.

These results do not allow us to determinate a structure–activity relationship for the arylhydrazone moiety. Both electron-donating (1g, 1j and 1o) and electron-withdrawing (1a, 1i and 1l) groups showed PL activity. In addition, the position does not affect the activity. The hydrogen bond-donating property of the R–OH groups might be important to compound–enzyme affinity because only hydrazones with R=2–OH (1e) and R=4–OH (1g) have activity.

Exoenzyme production and secretion in Candida and other fungi have many mechanisms. The main mechanism for Sap production is the chain production between the endoplasmic reticulum and the Golgi complex, a system involving vesicle secretion. PL production is most likely a membrane release mechanism19. These different pathways may underlie the discrepancy in the results between PL inhibition and Sap inhibition in our study.

Conclusion

According to our findings, (7-chloroquinolin-4-yl)arylhydrazones can potently inhibit enzymatic processes in C. albicans and have low cytotoxicity at sub-antifungal concentrations. Based on these results, the quinolin-4-yl hydrazones are promising agents for pharmaceutical use, and the methodology used supports a new target of action in antifungal studies.

Declaration of interest

The authors are thankful to UFPel, Fundação de Amparo a Pesquisa do Estado do Rio Grande do Sul (FAPERGS) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq APE Grant No. 462866/2014-9), for the financial support of the research. The authors report no declarations of interest.