We found that amiodarone has potent antifungal activity against a broad range of fungi, potentially defining a new class of antimycotics. Investigations into its molecular mechanisms showed amiodarone mobilized intracellular Ca2+, which is thought to be an important antifungal characteristic of its fungicidal activity. Amiodarone is a synthetic drug based on the benzofuran ring system, which is contained in numerous compounds that are both synthetic and isolated from natural sources with antifungal activity. To define the structural components responsible for antifungal activity, we synthesized a series of benzofuran derivatives and tested them for the inhibition of growth of two pathogenic fungi, Cryptococcus neoformans and Aspergillus fumigatus, to find new compounds with antifungal activity. We found several derivatives that inhibited fungal growth, two of which had significant antifungal activity. We were surprised to find that calcium fluxes in cells treated with these derivatives did not correlate directly with their antifungal effects; however, the derivatives did augment the amiodarone-elicited calcium flux into the cytoplasm. We conclude that antifungal activity of these new compounds includes changes in cytoplasmic calcium concentration. Analyses of these benzofuran derivatives suggest that certain structural features are important for antifungal activity. Antifungal activity drastically increased on converting methyl 7-acetyl-6-hydroxy-3-methyl-2-benzofurancarboxylate (2b) into its dibromo derivative, methyl 7-acetyl-5-bromo-6-hydroxy-3-bromomethyl-2-benzofurancarboxylate (4).
The incidence of invasive fungal infections (IFIs) has risen dramatically because of an increase in the number of people with AIDS and those undergoing bone marrow and solid organ transplantations, high-dose chemotherapy, steroid treatment, and invasive medical procedures (Pfaller and Diekema, 2010; Pagano et al., 2011; Brissaud et al., 2012), and the increased use of immune suppression drugs has increased susceptibility to IFIs (Agrawal et al., 2011; Person et al., 2011). The familiar pathogens Candida, Aspergillus, and Cryptococcus remain significant clinical problems. Fungal resistance to some antifungals has increased in recent years (Pfaller and Diekema, 2010). Hence, there is a need to develop alternative antimycotics, in particular those with novel mechanisms.
Patients with cancer are particularly vulnerable to IFIs, which represent a major cause of morbidity and mortality (Sipsas and Kontoyiannis, 2012). Aspergillus is one of the main causes of IFIs in the intensive-care unit, causing 25% morbidity and 70% mortality (Andes et al., 2012). Invasive aspergillosis primarily affects immunocompromised individuals (Walsh and Stevens, 2011), but is now recognized as an emerging infection in patients in the intensive-care unit (Webb and Vikram, 2010; Stevens and Melikian, 2011).
Cryptococcosis caused by Cryptococcus neoformans occurs worldwide and is one of the common complications in patients with HIV/AIDS, organ transplant recipients, and patients with hematologic malignancies (Person et al., 2011). Cryptococcus gattii causes disease in healthy, immunocompetent people as well as immune-compromised patients (Chaturvedi et al., 2005) and has infected hundreds of patients in recent outbreaks (Datta et al., 2009; Chaturvedi and Chaturvedi, 2011). Cryptococcus is thought to cause perhaps a million cases of cryptococcosis and 600,000 deaths per year worldwide (Park et al., 2009). Despite current antifungal therapy, mortality remains significant, between 10 and 25%, in patients with AIDS (Li and Mody, 2010).
Amiodarone was found to have potent antifungal activity against a broad range of fungi, including Cryptococcus, Aspergillus, and Candida, potentially defining a new class of antimycotics (Courchesne, 2002). Amiodarone activates Ca2+ flux from both extracellular and intracellular stores (Courchesne and Ozturk, 2003).This Ca2+ flux is a major cause of its antifungal activity (Gupta et al., 2003; Muend and Rao, 2008; Zhang and Rao, 2008). Amiodarone also induces stress responses that are modulated by components of the cell wall (Courchesne et al., 2009), which is considered to be a prime target for new antifungals.
Amiodarone is based on the benzo[b]furan ring system (Fig. 1), which is found in other synthetic and natural compounds that show antimicrobial activity. Egonol (Fig. 1) and its synthetic derivatives are active against Staphylococcus aureus, Bacillus subtilis, Candida albicans, and Escherichia coli (Emirdağ-Öztürk et al., 2011). It is noteworthy that the benzofurans reported by Masubuchi et al. (2001, 2003) are O-alkylamino derivatives of 2-benzofurancarboxylic acids and amides and have antifungal activity. Furthermore, it was shown that esters and amides of substituted 2-benzofurancarboxylic acids, for example, the compound ethyl 3-methyl-4-[3-(pyridin-3-ylmethylamino)propoxy]-1-benzofuran-2- carboxylate (RO-09-4609) (Fig. 1), may act as inhibitors of fungal N-myristoyltransferase, thereby giving them antifungal activity (Masubuchi et al., 2001, 2003; Ebiike et al., 2002; Georgopapadakou, 2002; Kawasaki et al., 2003). Benzofurans substituted at C-5 with OH have more recently been shown to have antifungal activity (Ryu et al., 2010). Finally, research by Kossakowski et al. (2010) presents data on halogenated derivatives of 3-benzofurancarboxylic acids. Methyl esters of 4-bromo-6-(dibromoacetyl)-5-hydroxy-2-methyl-1-benzofuran-3-carboxylic acid (brominated ester I; Fig. 1) and 4-chloro-6-(dichloroacetyl)-5-hydroxy-2-methyl-1-benzofuran-3- carboxylic acid exhibited antifungal activity against C. albicans and Candida parapsilosis.
These heterocyclic compounds show a variety of pharmacological properties, and change of their structure offers a high degree of diversity that has proven useful in the search for new therapeutic agents, including many with antimicrobial activity (De Luca et al., 2009; Jiang et al., 2011; Kamal et al., 2011; Panchabhai et al., 2011).
To further our understanding of the molecular mechanism for amiodarone's antifungal activity, we designed the syntheses of a series of substituted benzofurans with the goal of identifying structural components that account for its antifungal activity. Such compounds will help define the effective structures of amiodarone and potentially lead to new antifungal compounds better suited to clinical use than amiodarone. We found several new derivatives that inhibited fungal growth, two of which displayed significant antifungal activity. Calcium fluxes in cells treated with these derivatives did not correlate directly with their antifungal effects; nonetheless, these compounds potentiated the fungicidal activity or calcium mobilization caused by amiodarone. Analyses of these benzofuran derivatives suggest that certain structural features are important for antifungal activity.
Materials and Methods
Synthesis of Benzofuran Derivatives.
Melting points were determined with ElectroThermal 9001 Digital Melting Point apparatus (ElectroThermal, Essex, UK) and were uncorrected. Microanalysis was carried out at the Department of Analytical Chemistry, Warsaw Technical University by using the Vario EL III (Elementar GmbH, Hanau, Germany). High-resolution mass spectra were recorded on a Quattro LCT (time of flight). 1H NMR, 13C NMR, heteronuclear single quantum correlation, and heteronuclear multiple-bond correlation spectroscopy spectra in solution were recorded at 25°C with a Varian NMRS-300 spectrometer (Varian, Inc., Palo Alto, CA. Chemical shifts δ (ppm) were referenced to tetramethylsilane. IR spectra were recorded on a FT IR Spectrum 2000 PerkinElmer instrument (PerkinElmer Life and Analytical Sciences, Waltham, MA). Thin layer chromatography was carried out by using Kieselgel 60 F254 sheets (Merck, Darmstadt, Germany), and spots were visualized by UV at 254 and 365 nm.
Methyl 4,5-Dibromo-7-Hydroxy-6-Methoxy-2-Benzofurancarboxylate (2e).
To the solution of methyl 5-bromo-7-hydroxy-6-methoxy-2-benzofurancarboxylate (2d; Fig. 2) (0.04 mol) in glacial acetic acid (20 ml) bromine (0.675 g, 0.00425 mol) dissolved in acetic acid (5 ml) was added dropwise with stirring. The reaction mixture was stirred at ambient temperature for 2 h, and then the precipitated oil was separated and purified by column chromatography on silica gel 230 to 400 mesh. Eluent: CHCl3. Yield 51%, solidified oil. IR (KBr) cm−1: 3466 (νOH), 3050 (νC-Harom), 2952 (νC-Hasym), 2845 (νC-Hsym), 1703 (νC = O), 1583, 1507, 1442 (νC = C), 1442, 1435 (δ OH), 1311 (νC-O-Casym), 1279 (νC-O-Casym), 1116 (νC-O-Csym), 977, 886, 850, 829, 759 (γC-H); 1H NMR (200 MHz, CDCl3) δ = 4.05 (m, 6H, H-8, H-9), 7.50 (s, 1H, H-3). Analyzed: C11H8Br2O5 (379.99) C H Br.
Methyl 7-Acetyl-5-Bromo-6-Hydroxy-3-Bromomethyl-2-Benzofurancarboxylate (4).
Methyl 7-acetyl-6-hydroxy-3-methyl-2-benzofurancarboxylate (2b) (Fig. 2; 1.98 g, 0.008 mol) was dissolved in glacial acetic acid (10 ml) at ambient temperature. Br2 (1.05 ml, 3.2 g, 0.020 mol) dissolved in acetic acid (10 ml) was added dropwise with stirring for 1 h. The reaction mixture was stirred at ambient temperature for 6 h, then water (50 ml) and saturated Na2S2O3 solution was added portion wise to remove any excess Br2. Then the reaction mixture was extracted with CHCl3 (3 × 60 ml), and the organic layer was dried (MgSO4 anh.). The solvent was evaporated, and the residue was crystallized (CH3COOH). Yield: 40%, mp 167 to 168°C. IR (KBr) cm−1: 3418 (νOH), 3064 (νC-Harom), 2954, 2925 (νC-Hasym), 2851 (νC-Hsym), 1715 (νC = O), 1628, 1603 (νC = C), 1471, 1437 (δC-Hasym), 1385, 1342 (δOH), 1227, 1216 (νC-O-Casym), 1052 (νC-O-Csym), 930, 849, 823, 776 (γC-H); 1H NMR (300 MHz, CDCl3) δ = 2.98 (s, 3H, H-11), 4.02 (s, 3H, H-12), 4.94 (s, 2H, H-8), 8.17 (s, 1H, H-4), 13.98 (s, 1H, OH). 13C NMR (125 MHz, CDCl3) δ ppm: 19.61 (C8), 31.83 (C11), 52.84 (C12), 107.35 (C7), 109.71 (C5), 120.07 (C3a), 125.30 (C3), 131.47 (C4), 140.88 (C2), 153.06 (C7a), 159.51 (C9), 161.21 (C6), 202.49 (C10). Analyzed: C13H10Br2O5 (406.03) C H Br. Time-of-flight mass spectrometry ES+: [M+Na]+ calculated for C13H10NaBr2O5: 426.8803, found 426.8783.
Strains, Media, and Reagents.
The C. neoformans strain used was JEC21 (MATα). The Saccharomyces cerevisiae strain used was FY70 (MATa ade1 trp1 leu2 his3 ura3). The human leukemia cell line, K-562, was from the American Type Culture Collection (Manassas, VA).
Cells were grown in 0.17% (w/v) Difco (Detroit, MI) yeast nitrogen base without amino acids and ammonium sulfate, 0.4% (w/v) ammonium sulfate, and 2% (w/v) glucose. Agar media contained 2% (w/v) Bacto-agar. Adenine (12 mg/l final concentration), uridine (40 mg/l), leucine (30 mg/l), histidine (20 mg/l), and tryptophan (20 mg/l) were added to supplement auxotrophies as needed.
For in vitro studies, amiodarone (MP Biomedicals, Solon, OH) was added from a 10 mM stock in dimethyl sulfoxide (DMSO). DMSO was added to the no-drug cultures as a control. No effect on cell proliferation was observed by the addition of DMSO at the concentrations used (data not shown).
Yeast Growth Rates and Viability.
For quantification of C. neoformans proliferation, cells were grown in 5 ml of liquid medium in Klett test tubes with vigorous shaking in a water bath. Measurement of cell density was done in a Klett-Summerson colorimeter (Klett, New York, NY). Cells were grown overnight in the medium to be tested. Dilutions of the overnight cultures were made into a series of test tubes containing fresh medium and various concentrations of drug or DMSO. The cell densities were monitored at time 0 (typically approximately 1–2 × 106 cells/ml) and over time (typically 8–10 points for each growth curve) up to approximately 10 h. The increase in cell density was plotted versus time, and the resulting curves were used to determine the generation time for each culture (Courchesne, 2002). Each experiment was repeated four to five times, and the mean values for generation times are with their S.E.
For quantification of Aspergillus fumigatus proliferation, liquid cultures were inoculated with 105 conidia plus either 30 μl of DMSO only, 15 μM amiodarone (in DMSO), 25 μM 4 (in DMSO), or amiodarone plus 4. Aspergillus cultures were then grown in minimal medium (5–20 ml) at 30°C with vigorous aeration for 4 days. Cultures were collected by filtration, and the net dry weight of Aspergillus cells was measured. The ratio of the weight of cultures treated with drug versus the weight of control cultures receiving only DMSO was calculated and determined as the percentage of growth of drug-treated cultures.
Human Cell Line Growth Rates.
K-562 cells were grown in Iscove's modified Dulbecco's medium in culture flasks kept at 37°C. Cells were treated with methyl 5-bromo-7-(O-ethyl-2′-diethylamino)-6-methoxy-2-benzofurancarboxylate (3d), compound 4, amiodarone, or the carrier (DMSO) as a control. The growth of the cells was followed over a 3-day period. Control cells increased in density approximately 5-fold during this time (Fig. 3A), whereas cells treated with 10 μM amiodarone increased slightly less than 3-fold (Fig. 3B). Strikingly, cells treated with 10 μM 3d did not grow (Fig. 3C) and appeared dead by trypan blue staining (data not shown). K-562 cells were killed by exposure to either 30 μM amiodarone or 30 μM 3d.
Cytoplasmic calcium concentration ([Ca2+]cyt) was followed by using aequorin as described previously (Courchesne and Ozturk, 2003). In brief, cells expressing apoaequorin in the cytoplasm cells were exposed to 5.9 μM coelenterazine for 1 h to allow the cofactor to diffuse into cells and bind the apoaequorin to generate the holoprotein aequorin. Cells (2.5 × 106) were aliquoted to a microtiter plate for luminescence measurement in a luminometer (Berthold Technologies, Bad Wildbad, Germany) using Winglow monitoring and analysis software (Berthold Technologies). The luminescence of aliquoted cells before any treatment was measured as a control, then at 30 s wells were injected with amiodarone in a volume of buffer equal to that of the cells, resulting in 50 μM final amiodarone concentration.
Compounds 7-acetyl-6-methoxy-3-methyl-2-benzofurancarboxylic acid (1a), methyl 7-acetyl-6-methoxy-3-methyl-2-benzofurancarboxylate (2a), 2b, methyl 7-acetyl-6-(O-ethyl-2′-diethylamino)-3-methyl-2-benzofurancarboxylate (3b), methyl 6-hydroxy-7-(p-methoxycinnamoyl)-3-methyl-2- benzofurancarboxylate (2c), methyl 6-(O-ethyl-2′-diethylamino)-7-(p-methoxycinnamoyl)-3-methyl-2-benzofurancarboxylate (3c), 2d, 3d, methyl 7-acetyl-6-(O-ethyl-2′-diethylamino)-5-methoxy-3-methyl-2-benzofurancarboxylate (3f), and methyl 6-acetyl-5-(O-ethyl-2′-diethylamino)-2-methyl-3- benzofurancarboxylate (3g) were prepared according to previously reported procedures (Kossakowski et al., 2005). The O-ethyl-N,N-diethylamino dervatives 3b, 3c, 3d, 3f, and 3g were converted to their hydrochlorides to improve their solubility in polar solvents.
The analytical data were in agreement with those reported previously (Kossakowski et al., 2005).
The novel 2e was synthesized by bromination of the ester 2d with Br2 in acetic acid.
Compound 4 was obtained by the multistep synthesis. 8-Acetyl-7-hydroxy-4-methylcoumarin was reacted with bromine in acetic acid to give 8-acetyl-3-bromo-7-hydroxy-4-methylcoumarin. Compound was converted to 7-acetyl-6-hydroxy-3-methyl-2-benzofurancarboxylic acid (1b) by reaction with sodium hydroxide. The acid 1b was esterifed with methanol to give its methyl ester 2b (Kossakowski et al., 2005). Bromination of the compound 2b with bromine in acetic acid at ambient temperature afforded the novel benzofuran derivative 4 (Scheme 1). The structures are presented in Fig. 2.
In Vitro Antifungal Activity of Benzofuran Derivatives.
We screened the benzofuran derivatives 1a, 2a to 2e, 3b, 3c, 3d, 3f, 3g, and 4 for antifungal activity. We tested whether these derivatives could inhibit the growth of the opportunistic pathogen C. neoformans and found effects ranging from nearly none to severe growth inhibition. Table 1 shows the generation times of C. neoformans strain JEC21 treated with these derivatives growing in minimal media in the presence of 20 μM benzofuran derivatives or just the carrier (DMSO) as a control. Cells treated with derivatives 1 and 3f had generation times like the control cells treated with DMSO, whereas cells treated with derivatives 2a, 2c, 2d, 2e, 3c, and 3g had generation times only approximately 10 to 20% slower than controls. Strikingly, cells treated with 20 μM 3d and 4 arrested growth immediately upon addition. The viability of the growth-arrested cells was tested by the live/dead lumofungin assay, and the derivative-treated cells were found to be metabolically dead (data not shown). Because of the potent antifungal activity exhibited by 3d and 4 we examined the effects these two derivatives in greater detail (Table 2).
JEC21 cells treated with 10 μM 3d did not grow during the 8- to 10-h period of the experiment, whereas cells treated with 5 and 2 μM 3d had generation times 600 and 12% slower than controls, respectively (Table 2). Cells treated with 10 and 5 μM 4 had generation times 200 and 15% slower than controls, respectively.
Effects of 3d and 4 on Mammalian Cell Growth In Vitro.
Because it is desirable to identify benzofuran derivatives that retain antifungal activity but have reduced toxicity toward mammalian cells we tested the effects of 3d and 4 on the growth of the human leukemia cell line K-562.
K-562 cells were treated with 3d, 4, amiodarone, or the carrier (DMSO) as a control. The growth of the cells was followed over 3 days. Control cells treated with two amounts of DMSO increased in density approximately 5-fold during this time (Fig. 3A), whereas cells treated with 10 μM amiodarone increased slightly less than 3-fold (Fig. 3B). Strikingly, cells treated with 10 μM 3d did not grow (Fig. 3C) and appeared dead by trypan blue staining (data not shown). K-562 cells were killed by exposure to either 30 μM amiodarone or 30 μM 3d.
Compound 4 was less toxic to the K-562. Cells treated with both 10 and 30 μM 4 increased more than 4-fold during the period of the experiment, similar to the growth of the control (Fig. 3D). When 4 was added at 50 μM, cell growth was slowed slightly. Compound 4-treated cells had a 54.2 h (S.E. of 10.4) generation time compared with 34.0 (2.5) for control cells, a 60% increase in the time required per-cell generation.
Thus, amiodarone is moderately toxic to K-562 cells, whereas 3d showed significantly more toxicity than amiodarone. In contrast, 4 showed no effects on cell growth at concentrations (10–30 μM) that are completely inhibitory to yeast growth and showed only mild growth inhibition at a higher concentration.
3d and 4 Affect Yeast Cytoplasmic Ca2+.
We have shown that amiodarone causes a major rise in [Ca2+]cyt in S. cerevisiae cells (Courchesne and Ozturk, 2003). We tested for similar effects on [Ca2+]cyt in S. cerevisiae by 3d and 4 (Fig. 4). S. cerevisiae cells [FY70 (pEVP11/AEQ)] expressing apoaequorin were assayed for their response to amiodarone, 3d, and 4. Cells were treated with 50 μM amiodarone, 3d, or 4 at time 0, and the increases in relative light units (RLUs)/s were monitored for 3.5 min. The increase in RLUs was proportional to the increase in [Ca2+]cyt. Amiodarone elicited an immediate and very large increase in RLUs, whereas 3d elicited a much smaller increase, approximately 5-fold smaller than amiodarone (Fig. 4, A and B, respectively). In contrast, 4 elicited no increase in the RLUs (Fig. 4C), producing a response like that of the control cells treated only with carrier (Fig. 4D). An inability to stimulate Ca2+ flux also occurred with each of the other derivatives shown in Table 1 (data not shown). Thus, the various benzofuran derivatives displayed distinct abilities to elicit increases in [Ca2+]cyt that did not correspond with their relative growth inhibitory effects.
We also investigated whether pretreatment of yeast cells with benzofuran derivatives could affect the calcium response elicited by subsequent amiodarone treatment (Fig. 5). S. cerevisiae cells were prepared for aequorin assays as described above. As a control, cells were pretreated only with carrier (DMSO) at zero time, and then amiodarone was added at 30 s. This resulted in the same increase in RLUs elicited by amiodarone addition with no pretreatment (Fig. 5A). Pretreatment with 2a did not change the maximum level of RLUs but shifted the response approximately 10 s sooner (Fig. 5B). Pretreatment with 3f and 3g increased the maximum RLUs approximately 40% versus the control (Fig. 5, C and D, respectively), and 3d increased the maximum RLUs by approximately 55% and shifted the response by approximately 10 s sooner (Fig. 5E). In contrast, pretreatment with 4 increased the maximum RLUs approximately 100% and shifted the response approximately 20 s earlier (Fig. 5F), demonstrating a significant stimulating effect by 4 on the rise in [Ca2+]cyt caused by amiodarone.
4 Stimulates Amiodarone's Antifungal Activity.
We then checked whether 4 also stimulated amiodarone's antifungal activity against the pathogenic fungi A. fumigatus. Growth of Aspergillus was measured by inoculating 105 spores into liquid minimal growth medium containing only amiodarone, only 4, or amiodarone plus 4. The control culture was treated only with the carrier DMSO. After 4 days of growth, the cultures were removed from the liquid medium and dried, and total culture was weighed to measure the amount of growth. The ratios of the weight of drug-treated versus control cultures were calculated. The mean ratios and S.E. from three independent experiments were determined.
Amiodarone (15 μM) treatment had no deleterious effect on the growth of Aspergillus, possibly even increasing growth slightly to 113% (S.E. = 18) of control. Cultures treated with 25 μM 4 produced 77% (S.E. = 14) of the weight of control cultures, revealing a slight growth inhibition. In striking contrast, cultures treated with a combination of 25 μM 4 plus 15 μM amiodarone produced only 3.7% (S.E. = 1.3) of the weight of the control culture, showing a marked growth inhibition, which was more than 20-fold lower than either drug by itself. These results show that 4 acts in concert with amiodarone to have a major inhibitory effect on the growth of Aspergillus.
Amiodarone is a potent antiarrythmic drug, which is used to treat all forms of supraventricular and ventricular tachycardia. Although widely used, it is known to have significant adverse side effects on various organs, such as the liver, neuromuscular system, thyroid, cornea, skin, and lungs (for a review see Connolly, 1999). Although several organ systems can be affected, pulmonary complications are perhaps the most deleterious and occur in approximately 10% of patients, usually after 2 or more months of treatment (Kennedy et al., 1987), and clinically significant lung damage seems to require a very high total dose of amiodarone (as much as 200 g; Dusman et al., 1990). Nonetheless, acute presentations can occur within weeks of initiating treatment (Ashrafian and Davey, 2001; Lardinois et al., 2002), and the problem of adverse effects is a limiting factor for long-term utilization of amiodarone. Benzofuran-based compounds related to amiodarone with improved safety profiles are being developed to replace amiodarone. For example, in 2009 the Food and Drug Administration approved dronedarone, a benzofuran-based compound closely related to amiodarone, for clinical use as an antiarrhythmic drug. In an analogous approach, the goal of our research was to identify novel benzofuran derivatives that retained amiodarone's antifungal activity but with reduced toxicity.
To exploit the potential antifungal activity of amiodarone and synthesize new derivatives better suited to clinical use, it is essential to identify the structures that function in this capacity. Amiodarone is a complex synthetic drug based on a benzofuran ring system, which is an important pharmacophore contained in numerous compounds that can be isolated from natural sources as well as in synthetic products. Many of these heterocyclic compounds are simpler in structure than amiodarone and have proven to be useful in the search for new therapeutic agents, including many with antifungal activity (Masubuchi et al., 2003; Kossakowski et al., 2010; Ryu et al., 2010). Comparison of amiodarone and these other compounds strongly supports the hypothesis that biological activity and therapeutic application of amiodarone relies in particular on the pattern of substitution on the aromatic ring.
To investigate the role of the benzofuran ring system, in particular substitutions on the aromatic ring, we synthesized and screened a series of benzofuran derivatives for antifungal activity. Although several derivatives did not significantly inhibit yeast cell growth, cryptococcal cells treated with derivatives 2a, 2b, 2c, 2d, 2e, 3c, and 3g were growth-inhibited and had generation times ranging from 10 to 20% slower than control cells treated with DMSO. These are modest effects on fungal growth but demonstrate that different derivatives can have antifungal effects. The substitutions can now be evaluated for the synthesis of future derivatives.
It is noteworthy that we found two derivatives with significant antifungal activity. Derivatives 3d and 4 had substantial fungicidal activity against C. neoformans. 3d showed inhibition of cryptococcal growth at 2 μM, and growth was completely inhibited at 10 μM, whereas 4 required 5 μM to cause significant inhibition and 20 μM prevented cryptococcal growth. Thus, 3d possesses the more potent antifungal activity that is effective in approximately the same concentrations as is amiodarone. Although 4 has slightly less active antifungal activity on its own, it was found to have a major stimulating activity in combination with amiodarone. It is noteworthy that 4 showed low toxicity to a human leukemia cell line, suggesting a potential for therapeutic use. Thus, 4 has the desired characteristics of a second-generation amiodarone derivative. We first saw stimulating activity when 4 was tested for its effects on the ability of amiodarone to mobilize Ca2+ in yeast cells. Yeast cells treated with both drugs increased their [Ca2+]cyt to approximately twice the level, and more rapidly, as cells treated with amiodarone alone. 3d also showed such stimulation but to a significantly lesser extent than 4.
We explored the molecular characteristics of 3d and 4, testing their abilities to elicit calcium fluxes in yeast cells, as amiodarone does. The ability to mobilize Ca2+ is thought to be an important antifungal characteristic of amiodarone (Courchesne and Ozturk, 2003; Gupta et al., 2003). We were surprised to find that calcium fluxes in cells treated with these derivatives were significantly different from those for amiodarone and did not correlate with their antifungal effects. Although the antifungal activity of 3d was similar to amiodarone and 3d's toxicity to human cells even greater, its ability to elicit a calcium flux in yeast was approximately 4-fold lower than amiodarone. Although 4 demonstrated significant antifungal activity, it was ineffective, by itself, in promoting calcium flux in yeast, yet stimulated calcium flux by amiodarone. These results provide a basic understanding of the mechanism of action for the compounds studied here; they act similarly but not identically to amiodarone. We speculate their antifungal activity may include activities in addition to dramatic changes in cytoplasmic calcium concentration.
In addition to its in vitro antifungal activity, amiodarone has shown antiprotozoal activity, by itself and in combination with certain antifungal drugs, in vitro (Benaim et al., 2006; Serrano-Martín et al., 2009a; de Macedo-Silva et al., 2011) and in vivo in murine models (Benaim et al., 2006; Serrano-Martín et al., 2009b; Bobbala et al., 2010). It is noteworthy that there have been clinical case reports showing amiodarone efficacy in humans against Chagas' disease (Paniz-Mondolfi et al., 2009) and cutaneous leishmaniasis (Paniz-Mondolfi et al., 2008). Given the lack of good alternatives, those authors speculated that amiodarone might be considered for clinical treatment of these parasites. In this regard, we have shown that compound 4 dramatically stimulates amiodarone's antifungal activity to inhibit Aspergillus. Thus, we speculate that compound 4 would also stimulate amiodarone's antiparasitic activity as well. It should be noted that the recently approved amiodarone replacement drug, dronedarone, has also been shown to have antiparasitic activity in vitro (Benaim et al., 2012), raising the possibility that it may be effective in vivo against these parasites and be a less toxic alternative to amiodarone. This hope may be premature even though dronedarone was Food and Drug Administration-approved after reports of an improved safety profile (Hohnloser et al., 2009), because more recent studies cast doubt on its safety (Said et al., 2012). Should additional studies show dronedarone to truly be safer than amiodarone, particularly during short-term use against parasitic agents, it could, in theory, be tested for antifungal activity by itself and in combination with antifungal drugs, in particular compound 4.
Our findings suggest that certain structural features are important for antifungal activity. As indicated earlier, compounds 2a and 2b have modest effects on fungal growth. Changing the position of the acetyl substituent (at C7 in 2a and 2b), and methylation the C-6 phenolic group as well as the modification of the C-7 acetyl group in the compound 2b leading to 2c, does not result in any significant effect toward the antifungal activity.
In view of the influence of bromine atoms in the aromatic ring on the antifungal activity we have shown that introducing additional bromine does not work. Both benzofurans derivatives, monobrominated at C-5 compound 2d and C-4 and C-5 o-dibrominated compound 2e, are modestly active.
The introduction of an ethyl-N,N-diethylamino group to the moderately active compound 2d resulted in improving its antifungal activity, as was proved for 3d. However, similar modification carried out in 2c did not yield the active compound.
Double bromination (in the aromatic ring and C-3 methyl group) of the moderately active compound 2b afforded the active compound 4. Compounds 3d and 4 have similar substitution pattern on benzofuran moiety (drawn in bold in Scheme 1) that may be responsible for antifungal activity. However, the lack of alkyl substituent on C-3 in 3d may be associated with high toxicity. Likewise, 4-methylcoumarin is 3.5 to 8.5 times less toxic than coumarin (Feuer, 1974; Fernyhough et al., 1994) but 4-methylumbelliferone (7-hydroxy-4-methylcoumarin) is approved as food additive and a drug. We hypothesize that the key moiety responsible for compound 4's antifungal activity is the CH2Br substituent at C-3. It increases the lipophilicity and gives compound 4 the opportunity to act as an alkylating agent. Future studies will investigate the role of this key moiety.
Participated in research design: Hejchman and Courchesne.
Conducted experiments: Ostrowska, Kossakowski, and Courchesne.
Performed data analysis: Hejchman, Maciejewska, and Courchesne.
Wrote or contributed to the writing of the manuscript: Hejchman, Maciejewska, and Courchesne.
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
- invasive fungal infection
- cytoplasmic calcium concentration
- dimethyl sulfoxide
- relative light unit
- 7-acetyl-6-methoxy-3-methyl-2-benzofurancarboxylic acid
- methyl 7-acetyl-6-methoxy-3-methyl-2-benzofurancarboxylate
- methyl 7-acetyl-6-hydroxy-3-methyl-2-benzofurancarboxylate
- methyl 6-hydroxy-7-(p-methoxycinnamoyl)-3-methyl-2- benzofurancarboxylate
- methyl 5-bromo-7-hydroxy-6-methoxy-2-benzofurancarboxylate
- methyl 4,5-dibromo-7-hydroxy-6-methoxy-2-benzofurancarboxylate
- methyl 7-acetyl-6-(O-ethyl-2′-diethylamino)-3-methyl-2-benzofurancarboxylate
- methyl 6-(O-ethyl-2′-diethylamino)-7-(p-methoxycinnamoyl)- 3-methyl-2-benzofurancarboxylate
- methyl 5-bromo-7-(O-ethyl-2′-diethylamino)-6-methoxy-2-benzofurancarboxylate
- methyl 7-acetyl-6-(O-ethyl-2′-diethylamino)-5-methoxy-3-methyl-2-benzofurancarboxylate
- methyl 6-acetyl-5-(O-ethyl-2′-diethylamino)-2-methyl-3- benzofurancarboxylate
- methyl 7-acetyl-5-bromo-6-hydroxy-3-bromomethyl-2-benzofurancarboxylate
- ethyl 3-methyl-4-[3-(pyridin-3-ylmethylamino)propoxy]-1-benzofuran-2-carboxylate.
- Received May 29, 2012.
- Accepted August 13, 2012.
- Copyright © 2012 by The American Society for Pharmacology and Experimental Therapeutics