ABSTRACT
Due to the emergence of multi-drug resistant strains of yeasts belonging to the Candida genus, there is an urgent need to discover antifungal agents directed at alternative molecular targets. The aim of the current study was to evaluate the capacity of synthetic compounds to inhibit the Candida glabrata enzyme denominated 3-hydroxy-methyl-glutaryl-CoA reductase (CgHMGR), and thus affect ergosterol synthesis and yeast viability. One series of synthetic antifungal compounds were analogues to fibrates, a second series had substituted 1,2-dihydroquinolines and the third series included substituted pyrroles. α-asarone-related compounds 1c and 5b with a pyrrolic core were selected as the best antifungal candidates. Both inhibited the growth of fluconazole-resistant C. glabrata 43 and fluconazole-susceptible C. glabrata CBS 138. A yeast growth rescue experiment based on the addition of exogenous ergosterol showed that the compounds act by inhibiting the mevalonate synthesis pathway. A greater recovery of yeast growth occurred for the C. glabrata 43 strain and after the 1c (versus 5b) treatment. Given that the compounds decreased the ergosterol concentration in the yeast strains, they probably target the ergosterol synthesis. According to the docking analysis, the inhibitory effect of the 1c and 5b could possibly be mediated by their interaction with the amino acid residues of the catalytic site of CgHMGR. Since 1c displayed higher binding energy than α-asarone and 5b, it is a good candidate for further research, which should include structural modifications to increase its specificity and potency as well as in vivo studies on its effectiveness at a therapeutic dose.
HIGHLIGHTS
Fibrate-based and pyrrole-containing compounds were tested as C. glabrata inhibitors.
The best inhibitor from fibrate was 1c and from pyrroles was 5b.
These agents inhibited C. glabrata growth better than the reference antifungals.
They also inhibited ergosterol synthesis by the two C. glabrata strains tested. Experimental
INTRODUCTION
The emergence of multi-drug resistant strains of Candida yeasts in recent years has made infections by these pathogens a more serious problem [1]. Although Candida albicans (C. albicans), C. glabrata, C. tropicalis, C. parapsilosis and C. krusei are species isolated from healthy individuals, they can behave as invasive opportunistic pathogens under host conditions of a compromised immune system.
Among the particularly important Candida species with multi-drug resistance are C. auris, the species of the C. haemulonii complex, and C. glabrata. The former two cause in-hospital outbreaks and polymicrobial infections associated with SARS-Cov-2 [2,3]. C. glabrata is intrinsically resistant to azoles, and the recent pan-echinocandin-resistant strains of this species are also associated with the COVID-19 pandemic [4]. C. glabrata has been proposed as a model for the study of statins as antifungal agents [5].
Three main mechanisms of antifungal action have been found to date for antifungal agents: an alteration of the fungal membrane by the binding of polyenes to ergosterol, of the synthesis of ergosterol by the activity of azoles, allylamines and thiocarbamates, and of the generation of the cell wall by echinocandins [6]. A possible alternative target is 3-hydroxy-methyl-glutaryl-CoA (HMGR), an enzyme that catalyzes the synthesis of mevalonate, one of the critical steps in the ergosterol biosynthesis pathway [7-10]. The purpose of developing new antifungals with alternative molecular targets is to provide a wide range of compounds to respond to the multi-drug resistance of Candida spp. and other fungi.
The aim of the present study was to evaluate the capacity of new synthetic compounds to inhibit the C. glabrata HMGR enzyme (CgHMGR) and therefore affect ergosterol synthesis and yeast viability. Two series of compounds were derived from fibrate-based acyl- and alkyl-phenoxyacetic methyl esters, and 1,2-dihydroquinolines [11] and a third series from substituted pyrroles [12,13]. The best compound in each series was subjected to in vitro experiments to assess yeast growth, the level of ergosterol, and yeast growth rescue with the addition of exogenous ergosterol. The experimental data was complemented with docking simulations.
RESULTS
Selection of the best CgHMGR inhibitors
An evaluation was made of the possible antifungal activity of the thirteen compounds of series 1 and 2 and the seven compounds of series 3. The controls were the DMSO solvent and two compounds (α-asarone and fluconazole, at different concentrations) that reduce the synthesis of ergosterol in C. glabrata (Supplementary Figure 1) (Figure 1). The best inhibition of the growth of C. glabrata in solid YPD medium was exhibited by derivative 1c (of series 1 and 2), and the substituted pyrrole derivate 5b (of series 3) (Supplementary Figure 1) (Figure 1).
The HMGR inhibitors affect the viability of Candida glabrata
The phenotype of the strains was verified, being C. glabrata CBS 138 and C. glabrata 43, susceptibility and resistant to fluconazole, respectively. Once this was established, an evaluation was made of the in vitro antifungal activity of 1c, 5b, α-asarone (from which 1c is structurally related), and atorvastatin (an HMGR inhibitor and from which 5b is structurally related). Both test compounds (1c and 5b) and reference compounds (α-asarone and atorvastatin) were able to diminish the viability of the two strains of C. glabrata. 1c at 75 µg/mL provided growth inhibition similar to atorvastatin and α-asarone at the same concentration, reducing yeast growth by up to 90% for the two strains. It was necessary to apply 300 µg/mL of 5b to afford a similar percentage of inhibition (Figure 1) (Figure 2). As the concentration of the compound increased, the growth of the yeast strains decreased (Tables 1 and 2), indicating a dose-response effect. Compound 1c presented lower IC50 and IC70-90 values than its control (α-asarone), 5b and atorvastatin (Table 3).
For Candida glabrata treated with inhibitors, growth recovered after adding ergosterol
A yeast growth rescue experiment was carried out to verify that the inhibition of the HMGR enzyme affects the levels of ergosterol, the final product of the biosynthesis pathway (Figure 2). The compounds were applied at the sublethal concentrations estimated in the previous experiment (MIC70-90). When exogenous ergosterol was subsequently added to the culture medium, yeast growth did indeed occur, in contrast to the lack of growth caused by the inhibitor. In some cases, such as with compound 1c applied to C. glabrata 43, the recovery of yeast growth reached an even higher level than the control (the yeast cultured in the absence of an inhibitor). Thus, this finding confirmed that the compound derived from α-asarone altered the pathway for the production of ergosterol in C. glabrata, and more specifically that it targeted the synthesis of the HMGR enzyme.
The test compounds (CgHMGR inhibitors) affect ergosterol biosynthesis in Candida glabrata
To explore the possible association between the loss of viability of C. glabrata and the inhibition of the production of ergosterol, the level of ergosterol in the yeasts was measured after 18 h of treatment with 1c, 5b, simvastatin or α-asarone (the latter two as reference compounds; data not shown). The corresponding absorption spectra (Figure 3) contained the characteristic four peaks of sterols. The test compounds caused a reduction in the level of sterols in both the fluconazole-susceptibility and -resistant strains of C. glabrata.
The absorption peak corresponding to 281.5 nm was used to quantify the concentration of ergosterol, allowing for the calculation of the percentage of inhibition of its synthesis (Table 4). In general, residual ergosterol levels were higher in the C. glabrata 43 versus C. glabrata CBS 138 strain. In both strains, a greater decrease in ergosterol was caused by 1c than 5b. Simvastatin and α-asarone served as positive controls for the inhibition of CgHMGR, since previous studies demonstrated their capability of inhibiting the recombinant HMGR of C. glabrata [8]. It is observed that the higher the concentration of the inhibitor, the greater the percentage of inhibition of ergosterol synthesis (Table 4).
The level of ergosterol was calculated based on the absorbance obtained at 281.5 nm, expressing it as a percentage of the wet weight of the cells, as described by Arthington-Skaggs et al. [15]. C. glabrata was grown in YPD medium treated with different concentrations (50, 150, 300 and 600 μM) of the inhibitors: simvastatin, α-asarone, 1c and 5b. For the controls, the yeast was grown in YPD medium without any treatment or with DMSO only. The data represent the average of the three independent assays for each treatment. The previous results allowed for the calculation of the IC50, the concentration of the inhibitor that causes 50% inhibition of ergosterol synthesis in C. glabrata (Supplementary Table 1). 1c had lower IC50 values than 5b for both the C. glabrata CBS 138 (125 and 230 µM, respectively) and C. glabrata 43 (260 and >600 µM, respectively) strains.
Docking suggests the interaction of the test compounds with HMGR of Candida glabrata
Docking simulations displayed the hypothetical interaction of the compounds with CgHMGR. The related values for 1c and 5b are shown in Table 5. 1c has the highest binding energy in silico, which correlates with the in vitro results (Table 1). Atorvastatin had the lowest binding energy (Table 5). The interaction of compounds 1c and 5b with the amino acid residues in the catalytic site is depicted in Figure 4. 1c exhibited hydrogen bonds with a length of 2.58-2.99 Å between the hydroxyl groups at C-5 and C-8 and Glu93 and Asn192, respectively, as well as an electrostatic interaction of the O11 methoxy group with Met191. For 5b, there were hydrogen bonds 2.19 and 19.7 Å in length between the hydroxyl group at C-5 and Met191, and between the carboxyl group at C-7 and Asp303, respectively. The interaction between atorvastatin and the HMGR catalytic site revealed that van der Waals interactions are predominant, although two hydrogen bonds are detected (19.7 and 22.7 Å) between the carboxyl group at C-17 and Gly341. Additionally, Asp303 interacted by hydrogen bonds with the carboxyl group at C-17 and the hydroxyl group at C-15 (Figure 4). The calculated binding energies of 1c and 5b (−5.99 and −5.71 kcal/mol, respectively) were better than those found for α-asarone and atorvastatin (4.53 and −2.13 kcal/mol, respectively) (Table 5).
DISCUSSION
The problem of drug-resistant strains will always exist due to the process of natural evolution and selection of yeasts and bacteria [25]. Therefore, the probability of applying an effective treatment to patients would be increased by having a broad battery of antifungal agents from which to choose as well as distinct molecular targets among such drugs.
The HMGR enzyme (particularly CgHMGR) has for some time been proposed as a possible target, leading to the study of some cholesterol-lowering drugs (e.g., simvastatin and atorvastatin) as inhibitors of the growth of pathogenic yeasts [10,26]. According to in vitro evolutionary experiments, treatment of C. glabrata with some statins may allow for the selection of mutants. However, gene sequencing has not detected any changes in the catalytic domain of CgHMGR, indicating no effect on HMGR activity. C. glabrata is a useful model for examining resistance to statins and the precise molecular mechanisms of resistance to compounds that inhibit the CgHMGR enzyme [5].
In the current effort, three series of compounds were evaluated as inhibitors of C. glabrata. Two of the best derivatives were selected to determine their effect on yeast growth and ergosterol synthesis. Complementary studies were carried out with yeast growth rescue assays and docking simulations.
The compounds presently investigated were originally designed as lipid-lowering [11] and anti-inflammatory agents [12]. Their chemical structure could plausibly enable them to inhibit the activity of the CgHMGR enzyme. In fact, substituted pyrroles have been considered as antifungals [13,23] and their fungicidal activity is reported. However, the possible molecular target has not been previously explored in an in-depth manner.
Compounds such as statins (e.g., simvastatin and atorvastatin) and fibrates that inhibit HMGR have been administered to lower cholesterol levels in humans [27]. Additionally, they have been assessed as growth inhibitors of Candida spp., Aspergillus spp. and Ustilago maydis [7-10,14,26]. Based on its hypercholesterolemic activity, α-asarone underwent initial studies [27,28] that resulted in a finding of high toxicity. Thus, new derivative compounds have been designed and synthesized, and these have produced good activity against different fungi, such as C. glabrata and Ustilago maydis [9,14].
When the test compounds were examined in vitro, the growth inhibition of both strains of C. glabrata was better for 1c than 5b and α-asarone. On the other hand, 5b did not induce a greater growth inhibition than its reference compound, atorvastatin. The latter statin, bearing a substituted pyrrolic ring, has already been proposed as an antifungal agent to inhibit the growth of Candida spp. [26]. Although the antifungal activity of 1c has already been studied [11], this is the first evaluation, to our knowledge, of its effect on an opportunistic pathogenic yeast. Furthermore, the current investigation constitutes the first in-depth exploration of the mechanism of action and molecular target of the inhibitors.
According to the yeast growth rescue experiment, the test compounds likely inhibited the pathway for sterol biosynthesis [9,26]. The addition of ergosterol to C. glabrata CBS 138 resulted in a recovery of growth at a level below that of the control (without treatment with an inhibitor), while its addition to C. glabrata 43 led to growth that overcame the control level. This behavior can be explained by what is observed in the fluconazole-resistant C. glabrata strains, in which the consumption and metabolism of sterols might be affected by mutations in the ERG11 gene. Moreover, the exposure of susceptible C. glabrata strains to fluconazole (an inhibitor of ergosterol synthesis) causes a coordinated action between the consumption and production of ergosterol. Hence, the present test compounds probably inhibit the pathway for sterol biosynthesis, as fluconazole does [30,31].
Since 1c and 5b inhibited ergosterol synthesis, they may reduce the activity of the CgHMGR enzyme [26]. A better inhibition of the production of ergosterol was found for 1c in both strains of C. glabrata compared to its control (α-asarone) and 5b. Of these compounds, 1c had the lowest IC50. Previous publications have documented the capability of simvastatin, α-asarone, and derivatives of the latter to inhibit recombinant CgHMGR [8,9].
A correlation has been detected in C. albicans strains between their sensitivity to azoles and their total ergosterol concentration [15]. Therefore, it was important to demonstrate that the test compounds were capable of inhibiting the synthesis of ergosterol in both strains of C. glabrata (the fluconazole-susceptibility and -resistant strains).
The experimental results from the assays on yeast growth inhibition and the inhibition of ergosterol synthesis were complemented by docking simulations based on molecular coupling between the test compounds and CgHMGR. The binding energy values calculated for 1c and 5b were congruent with the in vitro findings for these two compounds. 1c exhibited the lowest binding energies and the best in vitro inhibition of yeast growth. Better binding to the active site of CgHMGR was displayed by 1c and 5b than α-asarone and its derivates, based on the calculated binding energies of the present study for the former two and reports in the literature for the latter [9]. This supports the in vitro results, in which 1c and 5b showed the greatest inhibition of yeast growth and of ergosterol synthesis.
The high binding energy determined from the docking of 1c and 5b into the active site of CgHMGR may stem from the addition of the ester and hydroxyl groups to the molecule, elements that do not exist in the structure of α-asarone. The hydroxyl group of 1c might play a crucial role in the proper binding mode of the compounds with HMGR [28,29]. Perhaps the chemical structure also confers a strong binding mode, considering the generation of hydrogen bonds with a short distance between atoms. On the other hand, the unsuitable binding mode of atorvastatin with CgHMGR possibly owes itself to steric interference of the chemical structure with a proper approach to the catalytic site of HMGR, as well as to the longer distance of the hydrogen bonds observed in the atorvastatin-CgHMGR complex (19.7 and 22.9 Å), which would confer weaker binding. Actually, atorvastatin has exhibited weak binding energy (−2.89 kcal/mol) with the catalytic site of human HMGR [32], substantiating the results obtained in this work with atorvastatin and CgHMGR. Interestingly, α-asarone, simvastatin and the substrate HMG-CoA presented almost identical high binding energy for the catalytic site of human HMGR [29]. Hence, the structural differences between human HMGR and CgHMGR may influence the binding mode.
Molecular modeling of proteins is a useful analytical technique that in the future should allow for the characterization of mutants in the CgHmgr gene, a phenotype resistant to antifungal inhibitors of the HMGR enzyme. Such resistance could be explained by changes in the protein related to its tertiary structure or by the capacity of inhibitors to bind with the amino acids of the catalytic site, among other possibilities. Indeed, molecular modeling analysis and mutations in the ERG11 gene, encoding for the enzyme 14-alpha-lanosterol demethylase (CYP51), have already been carried out with distinct C. albicans strains. Thus, a molecular explanation can be provided for the resistance or sensitivity of these strains to different azoles [33].
CONCLUSIONS
Three series of plausible inhibitors of the CgHMGR enzyme were designed, synthesized and tested for the inhibition of yeast growth. The two best candidates, 1c (structurally related to fibrates) and 5b (structurally related to atorvastatin), were chosen for further experiments. When comparing the results of these two compounds, treatment with the former led to a greater inhibition of yeast growth and ergosterol synthesis. The fact that the target of 1c is the pathway for the synthesis of ergosterol was demonstrated by the decrease it caused in the level of ergosterol as well as the posterior rescue of yeast viability by the addition of exogenous ergosterol. According to the docking analysis, the present test compounds displayed a better binding mode with CgHMGR than α-asarone and atorvastatin, supporting the experimental results.
There are many advantages to the rational design of antifungal compounds that are derived from known drugs (statins, fibrates, etc.), have a defined chemical structure, and are directed at a specific target. Their pharmacokinetics and pharmacodynamics can be inferred, suggesting potential redesign strategies to make them more specific, more potent and less toxic. Based on the molecular modeling analysis, a plausible interaction of the inhibitory compound with the target protein is visualized and analyzed, thus providing insights into the possible mechanisms of resistance of a yeast to an antifungal agent. Such resistance might be explained on the basis of changes in the tertiary structure of the protein or in the binding mode of inhibitors with their target. The fibrate-related compound, 1c, herein proved to be a good candidate for further research on its antifungal activity. Modifications of the compound should be considered to achieve greater specificity and potency. The derivatives could then be examined with in vivo animal models at a therapeutic dose. Other important areas to be explored are its toxicity and the inhibition of the recombinant CgHMGR enzyme.
MATERIALS AND METHODS
Strains and culture media
C. glabrata CBS 138 and C. glabrata 43 are susceptibility and resistant to fluconazole, respectively [9]. They were employed to examine the antifungal effect and ergosterol inhibition produced by the current test compounds. C. glabrata CBS 138 was donated by Dr. Bernard Dujon of the Pasteur Institute, Paris. C. glabrata, C. albicans and C. krusei strains were stored at −70 °C in 50% (v/v) anhydrous glycerol (Sigma-Aldrich). They were recovered in yeast extract-peptone-dextrose (YPD) medium (1% yeast extract, 2% casein peptone, and 2% dextrose anhydrous powder; J.T. Baker) at 37 °C under orbital shaking at 120 rpm, to be used as inoculum in the assays. The RPMI-1640 medium (Sigma-Aldrich) was prepared in accordance with the standard procedures of the Clinical and Laboratory Standards Institute (CLSI). For growth rescue assays, stock solutions of the yeasts were elaborated at a final concentration of 2.5% (v/v) in a mixture of Tween 80 and ethanol (1:1) (Sigma-Aldrich).
Evaluation of the growth inhibition of C. albicans and C. glabrata
To identify the compounds with the greatest potential antifungal activity, all the compounds in the three series were examined, together with three reference compounds (fluconazole, α-asarone and simvastatin), for their effect on the growth of two strains of C. albicans and two strains of C. glabrata. Yeast cells were cultured in slightly stirred YPD medium at 37 °C for 24 h, and later adjusted to a density of 0.5 (As600) to obtain a new inoculum. A stock solution, prepared with dimethyl sulfoxide (DMSO) and 10 mM of each of the inhibitors, was added (50 μL) in a Petri dish to afford a final inhibitor concentration of 50, 300 or 600 μM. Subsequently, YPD medium (25 mL) was added, and the mixture was slightly stirred until a homogenous solid was formed. The solidified media were inoculated with 20 μL of each of the Candida strains, previously adjusted, in a section of the Petri dish and incubated at 37 °C for 24 h [14]. Based on this procedure, two inhibitors were selected for further experiments, 1c from the fibrate derivates and 5b from the substituted pyrroles.
In vitro activity of the synthetic compounds against Candida spp
The effect of 1c and 5b on the growth of C. glabrata CBS 138 and C. glabrata 43 was evaluated by using the CLSI M27-A3 microdilution method. Briefly, stock solutions of antifungal compounds were prepared, from which the experimental concentrations were obtained in RPMI-1640 medium (Sigma-Aldrich). Fluconazole, simvastatin, atorvastatin and α-asarone served as reference compounds for examining susceptibility. C. albicans ATCC 10231 and C. krusei ATCC 14423 were the controls for sensitivity and resistance, respectively. The synthetic compounds were dissolved in DMSO at the time they were placed on the microplates, followed by incubation for 24 h at 37 °C. The volume of the solvent was less than 10% of the total volume to avoid problems of inhibition by the solvent. Growth was quantified by optical density in a Thermo Scientific™ Multiskan™ FC microplate spectrophotometer at 620 nm. The values of yeast growth are expressed as the average of three independent assays.
Candida glabrata growth rescue
To verify that inhibitors affect yeast viability by inhibiting ergosterol synthesis, a growth rescue experiment was conducted. Growth was first stopped by subjecting yeasts to the sublethal concentration (IC70-90) of one of the inhibitors, determined by the CLSI M27-A3 protocol (see section 2.3), and then ergosterol was added. Briefly, to each well of 96-well microplates were added 100 µL of one of the antifungal solutions (2x) prepared in RPMI-1640 medium (Sigma-Aldrich), followed by 80 µL of a yeast suspension adjusted to 1-5 × 106 UFC/mL and diluted 1:1000 with RPMI-1640 medium (Sigma-Aldrich). A stock solution of ergosterol was prepared by dissolving 11 µg/mL in Tween 80/ethanol, and 20 µL of this solution was added to each well. The controls were yeasts cultured without any inhibitor (growth control) and those with an inhibitor but without sterol (growth rescue control).
Statistical analysis
Data are expressed as the mean of three replicates ± standard deviation (SD). Differences between groups were examined with two-way analysis of variance (ANOVA), with the Bonferroni correction, and a 95% confidence interval. Statistical analyses were performed and graphs constructed with GraphPad Prism 5.0. Statistical significance was considered at P<0.001.
Ergosterol quantification
Total sterols were extracted with a slightly modified version of the methodology reported by Arthington-Skaggs et al. [15]. Briefly, C. glabrata yeasts were grown in YPD medium by incubation at 37 °C for 24 h under constant agitation at 200 rpm. The cell culture was prepared by adjusting it to a density of 0.3 (As600) in different flasks containing 5 mL of YPD medium, followed by the addition of DMSO solvent as the control (Sigma-Aldrich, USA) or one of the inhibitors (simvastatin, α-asarone, 1c or 5b at 50, 150, 300 or 600 μM). For each treatment, the yeasts were incubated at 37 °C for 18 h under constant shaking at 200 rpm. Cells were harvested by centrifugation and washed with distilled water. After establishing the net weight of the pellet, it was mixed with 3 mL of an alcoholic solution of potassium hydroxide (KOH) (25 g of KOH and 35 mL of distilled water, brought to 100 mL with absolute ethanol) in a vortex for 1 min to extract the sterols [14-16]. The cell suspensions were incubated at 85 °C for 1 h, and then the sterols were extracted with a mixture of 1 mL of sterile distilled water and 3 mL of n-heptane by vigorously mixing in a vortex for 3 min. The n-heptane layer was spectrophotometrically scanned between 230 and 300 nm (BioSpectrometer, Eppendorf). The presence of ergosterol (As281.5 peak) and 24 (28) dihydroxy-ergosterol (24 (28) DHE), a late intermediate (As230 peak), can be appreciated by the characteristic four-peaked spectrum indicating sterol absorption. The technique is also capable of revealing a decrease in the level of ergosterol. The absence of detectable levels is evidenced by a flattening of the curve [14-16].
Docking of the test compounds on CgHMGR
The hypothetical three-dimensional structure of CgHMGR was obtained by homology modeling with MODELLER 9.13 software [17], using the crystallographic structure of human HMGR as the template (PDB entry: 1DQ8). The quality of the resulting model was evaluated by determining the stereochemical restrictions with a Ramachandran plot constructed on Procheck v.3.5.4 [18]. The structure was energetically minimized and equilibrated through molecular dynamic simulations on the NAMD2 program [19], which were performed in 2,000,000 steps for a total run time of 1 ns. The three-dimensional structure of the ligands, obtained with the ChemSketch program (www.acdlabs.com), was subjected to energy optimization and minimization with AVOGADRO software [20]. Docking simulations were conducted on AUTODOCK 4 [21], employing the parameters established by Andrade-Pavón et al. [9]. Docking results were computed based on a total of 100 runs and 1,250,000,000 generations, analyzed in AutodockTools and visualized on LigProt+ software [22].
Synthesis of the compounds tested as potential antifungal agents
The fibrate-based derivates 1a-c, 2a-c and 3a-c, and 1,2-dihydroquinolines 4a-d, constituted the first two series of compounds [11] (Figure 5). The substituted pyrrole derivatives comprised the third series, being 5a-d and 6b-d [12] (Figure 6). The brominated pyrroles 5b, 5c and 6b-d were designed because of its similarity to some pyrrole-based marine alkaloids known to exert both antifungal and antibacterial activity [13, 23-24].
Synthesis of bromopyrroles 5b and 5c
The synthesis of 5a, 5d and 6c-d has been previously reported [11,12]. The preparation of bromopyrroles 5b and 5c was achieved by treatment of compound 5a [12] with N-bromosuccinimide (NBS) as the brominating agent under mild reaction conditions (Scheme 1). Even though l.0 mol equivalent of NBS was employed, a mixture of bromopyrroles 5b and 5c was obtained. Due to the fact that they were easily separated by column chromatography, an excess of NBS (2.5 mol equiv.) was added to the reaction mixture to give 5b and 5c in 32% and 58% yields, respectively.
The synthesis of dibromopyrrole 6b was carried out by a two-step methodology. The first step consisted of a Knoevenagel reaction of 5a with malononitrile under acid conditions [12] to provide 6a in high yield (Scheme 1). Bromination of the latter with NBS (2.0 mol equiv.) in DMF, as the solvent, led to the desired product 6b in good yield (88%) (Scheme 1).
General information
Melting points were determined on a Krüss KSP 1N capillary melting point apparatus. IR spectra (ATR-FT or KBr) were recorded on a Perkin-Elmer 2000 spectrophotometer. 1H and 13C NMR spectra were captured on a Varian Mercury (300 MHz) instrument, with CDCl3 as the solvent and TMS as internal standard. Signal assignments were based on 2D NMR spectra (HMQC and HMBC). High-resolution mass spectra (HRMS) were obtained (in electron impact mode) on a Jeol JSM-GCMateII spectrometer. Analytical thin-layer chromatography was carried out using E. Merck silica gel 60 F254 coated 0.25 plates, visualized by using a long- and short-wavelength UV lamp. Flash column chromatography was conducted over Natland International Co. silica gel (230-400 and 230-400 mesh). All air moisture sensitive reactions were carried out under N2 using oven-dried glassware. CH2Cl2 and DMF (Sigma-Aldrich) were distilled over CaH2 (Sigma-Aldrich) prior to use. All other reagents (Sigma-Aldrich) were employed without further purification.
Synthesis of bromopyrroles 5b and 5c
Methyl 2-(4-bromo-2-formyl-1H-pyrrol-1-yl)acetate (5b). Methyl 2-(2,3-dibromo-5-formyl-1H-pyrrol-1-yl)acetate (5c). To a stirring solution of 5a (0.100 g, 0.60 mmol) in anhydrous DMF (5 mL) at 0 °C, a solution of NBS (0.267 g, 1.50 mmol) in anhydrous DMF (2 mL) was added dropwise, and the mixture stirred at 0 ° C for 12 h. A mixture of water/hexane/EtOAc (1:0.5:0.5) (20 mL) was added, the organic layer dried (Na2SO4) and the solvent removed under vacuum. The residue was purified by column chromatography over silica gel (30 g/g crude, hexane/EtOAc, 9:1) leading to 5b (0.062 g, 32%) as a yellow solid and 5c (0.112 g, 58%) as a yellow oil.
Data of 5b: Rf 0.43 (hexane/EtOAc, 7:3); mp 203-205 °C. IR (film): 3121, 2954, 1754, 1666, 1392, 1365, 1219, 1092, 923, 771 cm-1. 1H NMR (300 MHz, CDCl3): δ 3.78 (s, 3H, CO2CH3), 5.03 (s, 2H, CH2), 6.92 (br dd, J = 1.8, 1.2 Hz, 1H, H-5’), 6.98 (d, J = 1.8 Hz, 1H, H-3’), 9.47 (d, J = 0.9 Hz, 1H, CHO). 13C NMR (75.4 MHz, CDCl3): δ 50.1 (CH2), 52.7 (CO2CH3), 97.5 (C-4’), 125.2 (C-3’), 131.2 (C-5’), 131.6 (C-2’), 168.2 (CO2CH3), 179.3 (CHO). HRMS (EI): m/z [M+] calcd for C8H8BrNO3: 244.9688; found: 244.9690.
Data of 5c: Rf 0.69 (hexane/EtOAc, 7:3); IR (film): 2955, 1755, 1668, 1450, 1397, 1363, 1218, 1005, 810, 776 cm-1. 1H NMR (300 MHz, CDCl3): δ 3.78 (s, 3H, CO2CH3), 5.25 (s, 2H, CH2), 7.05 (s, 1H, H-4’), 9.32 (s, 1H, CHO). 13C NMR (75.4 MHz, CDCl3): δ 49.0 (CH2), 52.7 (CO2CH3), 101.1 (C-3’), 118.6 (C-5’), 125.3 (C-4’), 132.4 (C-2’), 167.5 (CO2CH3), 178.2 (CHO). HRMS (EI): m/z [M+] calcd for C8H7Br2NO3: 322.8793; found: 322.8791.
Synthesis of pyrroles 6a and 6b
Methyl 2-(2-(2,2-dicyanovinyl)-1H-pyrrol-1-yl)acetate (6a). In a threaded ACE glass pressure tube with a sealed Teflon screw cap and magnetic stirring bar, a solution of 5a (0.100 g, 0.60 mmol), malononitrile (0.044 g, 0.66 mmol), piperidine (0.026 g, 0.30 mmol) and glacial AcOH (0.029 g, 0.48 mmol) in anhydrous CH2Cl2 (5 mL) was heated at 70 °C for 24 h. The reaction mixture was diluted with CH2Cl2 (50 mL) and washed with water (25 mL) and an aqueous saturated solution of NaHCO3 until neutral. The organic layer was dried (Na2SO4) and the solvent removed under vacuum. The residue was purified by column chromatography over silica gel (20 g/g crude, hexane/EtOAc, 9:1) to afford 6a (0.12 g, 93%) as a yellow solid. Rf 0.51 (hexane/EtOAc, 8:2); mp 203-205 °C. IR (KBr): 3132, 2992, 2220, 1751, 1583, 1476, 1399, 1350, 1328, 1239, 1169, 1132, 1088, 994, 758, 732 cm-1. 1H NMR (300 MHz, CDCl3): δ 3.83 (s, 3H, CO2CH3), 4.80 (s, 2H, CH2CO2Me), 6.49 (ddd, J = 4.5, 2.4, 0.6 Hz, 1H, H-4’), 7.10 (dd, J = 2.4, 1.5 Hz, 1H, H-5’), 7.38 (s, 1H, H-1”), 7.73 (ddd, J = 4.5, 1.5, 0.6 Hz, 1H, H-3’). 13C NMR (75.4 MHz, CDCl3): δ 48.3 (CH2CO2Me), 53.3 (CO2CH3), 72.5 (C-2”), 113.4 (C-4’), 114.0 (CN), 115.3 (CN), 121.1 (C-3’), 127.2 (C-2’), 131.6 (C-5’). 142.7 (C-1”), 167.3 (CO2CH3). HRMS (EI): m/z [M+] calcd for C12H9N3O2: 215.0695; found: 215.0694.
Methyl 2-(2,3-dibromo-5-(2,2-dicyanovinyl)-1H-pyrrol-1-yl)acetate (6b)
To a stirring solution of 6a (0.100 g, 0.47 mmol) in anhydrous DMF (3 mL) at 0 °C, a solution of NBS (0.166 g, 0.93 mmol) in anhydrous DMF (3 mL) was added dropwise, and the mixture stirred at 20 ° C for 16 h. A mixture of water/hexane/EtOAc (0.5:1:1) (30 mL) was added, the organic layer dried (Na2SO4) and the solvent removed under vacuum. The residue was purified by column chromatography over silica gel (20 g/g crude, hexane/EtOAc, 7:3) to give 6b (0.25 g, 88%) as a yellow solid. Rf 0.44 (hexane/EtOAc, 8:2); mp 267-269 °C. IR (KBr): 3004, 2956, 2223, 1741, 1583, 1420, 1391, 1339, 1243, 1167, 1125, 1004, 983, 815, 738, 687 cm-1. 1H NMR (300 MHz, CDCl3): δ 3.85 (s, 3H, CO2CH3), 4.89 (s, 2H, CH2CO2Me), 7.31 (d, J = 0.3 Hz, 1H, H-1”), 7.76 (d, J = 0.3 Hz, 1H, H-4’). 13C NMR (75.4 MHz, CDCl3): δ 47.8 (CH2CO2Me), 53.5 (CO2CH3), 75.1 (C-2”), 104.8 (C-3’), 113.3 (CN), 114.4 (CN), 118.6 (C-5’), 121.5 (C-4’), 128.3 (C-2’), 141.4 (C-1”), 166.1 (CO2CH3). HRMS (EI): m/z [M+] calcd for C11H7Br2N3O2: 370.8905; found: 370.8905.
Reference compounds for the tests of the three series of potential antifungal compounds 1a-c, 2a-c, 3a-c, 4a-d, 5a-d and 6b-d
Depending on the experiment, different inhibitors served as the reference compounds. In the case of sensitivity tests and docking analysis, α-asarone was the control for the fibrate-based derivatives 1a-c, 2a-c, 3a-c (series 1) and 1,2-dihydroquinolines 4a-d (series 2) and atorvastatin for the substituted pyrroles 5a-d and 6b-d (series 3). In the experiment to determine the effect of the compounds on the biosynthesis of ergosterol, simvastatin and α-asarone were employed. It has been reported that these two compounds are capable of inhibiting recombinant Cg-HMGR, thus affecting the production of ergosterol [9].
Funding
This work was supported by CONACyT (grants CB283225, 300520 and A1-S-17131) and the SIP-IPN (grants SIP20200775, SIP20210508, SIP20200227 and SIP20210700).
Declarations of Interest
None.
SUPPLEMENTAL MATERIAL
Acknowledgments
DMA, AGS, BRA, JOA, CBC and CHE appreciate graduate scholarship awarded by CONACyT as well as the scholarship complements furnished by the SIP-IPN (BEIFI). The authors would like to thank Bruce Allan Larsen for proofreading the manuscript. CH-R, GC-C, JT. and LV-T. are fellows of the Estímulos al Desempeño de los Investigadores (EDI)-IPN and Comisión de Operación y Fomento de Actividades Académicas (COFAA)-IPN programs.
ABBREVIATIONS
- ANOVA
- analysis of variance
- CgHMGR
- 3-hydroxy-methyl-glutaryl-CoA reductase in Candida glabrata
- CLSI
- Clinical and Laboratory Standards Institute
- DHE
- dihydroxy-ergosterol
- DMSO
- dimethyl sulfoxide
- HMGR
- 3-hydroxy-methyl-glutaryl-CoA reductase
- KOH
- potassium hydroxide
- NBS
- N-bromosuccinimide
- SD
- standard deviation
- YPD
- yeast extract-peptone-dextrose medium