Phebalosin and its Structural Modifications are Active against the Pathogenic Fungal Causing Paracoccidioidomycosis

1Departamento de Química, Universidade Federal de Santa Catarina, CEP 88040-970, Florianópolis, SC, Brazil 2Departamento de Microbiologia, Universidade Federal de Minas Gerais, CEP 31270-901, Belo Horizonte, MG, Brazil 3Campus Centro Oeste Dona Lindu, Universidade Federal de São João Del Rei, CEP 35501-296, Divinópolis, MG, Brazil 4Campus Itaqui, Universidade Federal do Pampa (UNIPAMPA), CEP 97650-000, Itaqui, RS, Brazil


Introduction
Coumarins are a vast 1,2-benzopyrone group of natural compounds essentially found in green plants. The substitutions into their basic skeleton 1,2-benzopyrone can occur at any of the six available sites providing them extremely variable structures [1]. Although no longer used as food flavouring, coumarin is present in certain tobaccos and alcoholic beverages and is used in various soup, detergent and cosmetic preparations [2].
Hydroxy derivatives of 4-methyl coumarin are important group of coumarin derivatives showing medicinal as well as other biotechnological applications. For example, 5,7-dihydroxy 4-methyl coumarin and 7,8-dihydroxy 4-methyl coumarins are useful precursors to synthesize respective diacetoxy and hydroxyl-amino derivatives of 4-methyl coumarin, which are known to be good antioxidants having excellent radical scavenging properties [3].
Among the various coumarin derivatives, 7-substituted coumarins are important groups showing various kinds of bioactivities and also other applications. For example, 7-hydroxy 4-methyl coumarin is used in fluorometric determination of enzymatic activity, as a starting material for the preparation of insecticides and furano coumarins [4]. Due to their inherent photochemical characteristics, reasonable stability, good solubility and relative ease of synthesis, coumarin derivatives have been extensively investigated for electronic and photonic applications [5].
In this present work we screened the coumarin phebalosin and seven phebalosin -derivatives against four clinical isolates of the pathogenic dimorphic fungus Paracoccidioides brasiliensis, the causing agent of Paracoccidioidomycosis (PMC). PCM is the most prevalent systemic endemic mycosis in South America with most reported cases in Brazil [6]. In the absence of drug therapy the disease is usually fatal. The treatment of PCM is usually long, with many patients receiving therapy for one to two years or even more. It appears that the number of drugs active against PCM is scarce [7]. Although azoles and other drugs can arrest the progression of PCM, the fibrosis sequelae persist, probably constituting a source of fungi that could lead to a relapse in the disease following termination of treatment [8,9]. The strong toxicity of amphotericin B makes the effective management of this severe disease difficult [10]. For this reason the discovery of new drugs for the treatment of PMC is very important.

Materials and Methods
Chemistry 1 H and 13

Epoxy ring opening of phebalosin with sodium n-butoxy (6)
The reaction was carried out using the same procedure as in (4) using t-butoxy a nuccleofilic reagent, to yield 38% of the product like a white solid.

Obtaining cyclic acetal of phebalosin with acetone (7)
The acetalization reaction of phebalosin (1) was performed in dry acetone (100 mg) by dissolution of the 100 mg of phebalosin with addition 1 ml of chloridric acid (HCl) and 5 g of silica gel (catalyst). The mixture was subjected to magnetic agitation for 72 h and temperature control (50ºC). After this period, the reaction mixture was extracted with (CH 2 Cl 2 ), dried with anhydrous sodium sulfate (Na 2 SO 4 ), filtered and the organic solvent was concentrated. The crude product was purified by chromatography on a flash column (230-400 mesh) with hexane/ethyl acetate 40:60 to yield 70% as white solid.

Inoculum preparation
The strains were maintained by weekly passage in solid Yeast-precipitation of a white amorphous solid. The solid was separated from the supernatant solution followed by successive washing with hexane, yielding 2 g of epoxy coumarin, phebalosin (1).

Structural modification
Epoxy Ring Hydrolysis of the phebalosin (2): For ring epoxy hydrolysis of phebalosin (1), 100 mg was dissolved in dichloromethane (CH 2 Cl 2 ), 10 mlof distilled water, 1 mlof chloridric acid (HCl) and 5 g of silica gel (catalyst). The mixture was subjected to magnetic agitation for 72 h and temperature control (50ºC). After this period, the reaction mixture was extracted with CH 2 Cl 2 , dried with anhydrous sodium sulfate (Na 2 SO 4 ), filtered and the organic solvent was concentrated. The crude product was purified by chromatography on a flash column (230-400 mesh) with hexane/ethyl acetate 10:90 to yield 19% as a white solid.

Epoxy ring opening of phebalosin with sodium methoxy (4)
For this reaction, 20 mg of metallic sodium was added to 15 ml of dry methanol. In this solution, 100 mg phebalosin was added and the reaction was terminated after 15 minutes. The reaction medium was acidified with methanol-chloridric acid to pH 2-3. The reaction mixture was extracted with (CH 2 Cl 2 ), dried with anhydrous sodium sulfate (Na 2 SO 4 ), filtered and the organic solvent was concentrated in a vacuum desiccator. The crude product was purified by chromatography on a flash column (230-400 mesh) with hexane/ethyl acetate 50:50 to yield 71% as white solid.

Epoxy ring opening of phebalosin with sodium isopropoxy (5)
For this reaction, the same method of (4) was used with the appropriate nucleophile isoproxy to yield 44% of the product as white solid. Peptone-Dextrose medium, at 37ºC and were used after 7-10 days of fungus growth. Yeast cells in the exponential phase were collected aseptically with a platinum loop and re-suspended in a tube containing 5 ml of sterile saline. If large aggregates existed, they were allowed to settle for several minutes, and the supernatants were collected. The suspensions were then diluted in synthetic RPMI medium (Sigma, St. Louis, MO, USA) with L-glutamine buffered to pH 7.0 with 0.165 morpholine propanesulfonic acid (MOPS, Sigma), and prepared according to the CLSI document M 27-A 2 [11] to obtain a final inoculum size suitable for the strains [12]. After homogenization by vortexing, transmittance was measured at 520 nm and adjusted to 69 to 70% [13].

Susceptibility test
The compounds were dissolved in dimethylsulfoxide (DMSO). Serial dilutions were then performed, using RPMI as a diluent, maintaining a constant volume of 1 ml per tube. The extracts were tested at eight different concentrations that ranged from 500-1.9 µg/ ml Volumes of 100 µl of each dilution were distributed in sterile flatbottom 96-well microplates (Difco Laboratories, Detroit, MI, USA).
Susceptibility was determined by the broth microdilution method. Broth microdilution testing was performed in accordance with the guidelines in the CLSI M27-A 2 document [11] and Nakai et al. [12] RPMI medium was used without compounds or solvents as a control for growth and sterility. Solvent DMSO at the same volumes used in the assay was used as control for toxicity. Amphotericin B (Sigma, St Louis, USA) (25 to 0.03 µg/ml and (25 to 0.03 µg/ml in DMSO was included as positive antifungal control with the stock solutions prepared in DMSO and water, respectively. After inoculation of fungal strains the plates were incubated at 37°C for 14 days. The tests were performed in triplicate in at last two independent experiments. The endpoints were determined visually by comparison with the drug-free growth control well. MICs were expressed in µg/ml and defined as the lowest compound concentration for which the well was optically clear. All assays were performed in triplicate and repeated at least once.

Theoretical Chemistry methodology and chemometrics
Structural and electronic properties were obtained from classical mechanics [14] calculations (AMBER force field, at 298 K, in a vacuum) implemented in HYPERCHEM 7.5 molecular modelling program [15] and HF/6-31G** electronic structure calculations (ab initio method of Quantum Chemistry [16], 298 K, in a vacuum), implemented in GAUSSIAN program [17]. The obtained data were compared and correlated with antifungal activity by using chemometrics analysis [18]: Hierarchical Clustering Analysis (HCA), Partial Least Squares (PLS) and Principal Component Analysis (PCA) methods by using MINITAB 15 ® program [19].

Results
Whole P. paniculata plants were extracted with hexane to afford the coumarin phebalosin as a white amorphous solid, m.p. 124°C (Lit. 121-123°C); Rf=0.39 (eluent 60:40 hexane/ethyl acetate) and was identified by spectroscopic analysis and comparison with literature data [20]. A study of the reactivity of phebalosin (1) was planned, preparing a series of derivatives involving the opening epoxide ring by nucleophilic substitution reactions using different reagents, acetylation and formation of the cyclic acetal as shown in Figure 1.
The phebalosin (1) was treated with water and silica gel under The reaction with sodium n-buthoxy gives the 1'-hydroxy-2'-nbuthoxy derivative 6 (38%). The compound was presented in the form of yellow solid, with MP 97-99°C, and Rf=0.52 (eluent hexane/ethyl acetate 50:50). The spectrum in the IR region in KBr pellets showed the appearance of hydroxyl absorption at υ 3435 cm -1 , showing the opening of the epoxide ring, keeping other absorption characteristics of coumarin. The 1 H NMR spectrum of the reaction product kept the absorption characteristics of the coumarin nucleus. Halfway prenyl, the displacement of hydrogen atoms H-1' and H-2' of 3.92 and 3.99 ppm (epoxide) to 5.14 ppm (1H, d, J = 8.8 Hz) and 4.92 ppm (1H, d, J = 8.8 Hz) was observed respectively. We also observed the appearance of the signals 0.82 ppm (3H, t), 1.31 ppm (2H, m), 1.53 ppm (2H, m) and 3.33 ppm (2H, t) of the n-butoxy group inserted.
The treatment of phebalosin with acetone and silica gel under agitation lead to formation of the respective cyclic derivative (20%) as a colorless crystal, with MP 111-112°C, with Rf=0.58 (eluent hexane/ ethyl acetate 50:50). The spectrum in the IR region in KBr pellets showed the appearance of intense hydroxyl absorption at υ 2980 cm -1 , showing the opening of the epoxide ring, while it maintained the other absorption characteristics of coumarin. The NMR spectrum of the reaction product kept the absorption characteristics of the coumarin nucleus. Halfway prenyl, a pair of doublet absorbing at δ H1' 5.57 and δ H2' 5.02 ppm (1H each, d, J = 9.60 Hz) was displaced in relation to epoxy moiety. The singlets that absorb at 1.70 ppm and 1.74 ppm, with integration for three hydrogen atoms each, were assigned to the two methyl H-8' and H-7' of the acetal group.
Under acetylation with acetic acid and acetic anhydride under agitation, the phebalosin was partially converted to a diacetyl derivative 8 (65%) which presented itself in the form of solid white, with MP 120-121°C, with Rf=0.40 (eluent hexane/ethyl acetate 50:50). The spectrum in the IR region in KBr pellets showed no absorption of hydroxyl and retained all other characteristic absorptions of coumarin. The 1 H NMR spectrum of the reaction product kept the absorption characteristics of the coumarin nucleus. Halfway prenyl, we observed the displacement of hydrogen atoms H-1' and H-2' of 3.92 and 3.99 ppm (epoxide) to 6.08 ppm (1H, d, J = 12.0 Hz) and 5.99 ppm (1H, d, J = 12.0) respectively, due to the formation of acetyl derivative. The acetylation is confirmed by the presence of signals 1.87 ppm (3H, s) and 1.92 ppm (3H, s) relative to the acetyl groups.

Antifungal activity
In this work, the phebalosin was active against all isolates of P. brasiliensis tested ( Table 1). The MIC values ranged from 31.2 to 500 µg/ml In relation to the phebalosin-derivative compounds, an increase of antifungal activity was observed for compounds 3, 5 and 6 against isolate Pb03 of P. brasiliensis. However, Pb18 did not present significant susceptibility against the compounds 7, 8 and 4. Isolate Pb03 was more susceptive to drugs tested but not for trimethoprim-sulfamethoxazole (300 µg/ml) in the tested conditions.

Molecular modelling and chemometrics
The coumarin phebalosin compound and its seven phebalosin derivatives had the optimization of their geometries realized through Classical Mechanics (AMBER force field, vacuum, 298 K) aimed at obtaining the structure with a minimum of energy. Then used the PM3 level of theory [15] (semiempirical quantum mechanics) at 298 K, vacuum, in order to determine the physico-chemical properties of interest (descriptors). All protocol for obtaining descriptors was held in the programmes HyperChem Professional 7 5 and GAUSSIAN 09. In this work, the following descriptors were determined through the protocol described in the previous section ( Table 2): enthalpy of formation (ΔH f o ), variation of energy between the frontier orbitals (the highest occupied energy -HOMO and lowest unoccupied energy -LUMO), ΔE HOMO-LUMO, lipophilicity parameter (log P), dipole moment (µ), polarizability, molecular volume, solvent accessible area (SA) and system energy (Hartree Fock energy, at 6-31G** level of theory). The values of activity were converted into log(1/a) from each antifungal analysis, where a = experimental activity.
Initially it was applied a hierarchical method agglomerative (HCA) with all submissions, each forming its own grouping and by similarity, the final grouping of the clusters (also called final partition) which must, as expected, identify groups whose comments or variables have characteristics in common was determined. PCA and PLS techniques were performed in order to separate the compound as function of their antifungal activities.
Despite a few numbers of compounds and a low range of activity values observed, a chemometric analysis was performed: in a general sense, it seems that polarizability is the most important descriptor for antifungal activity against Pb03, Pb339, Pb18 and Pb01, respectively. In this case, the antifungal activity should be increased by increasing the values of the cited properties.

Discussion
Currently, there are few works on the search for new drugs against P. brasiliensis. San-Blas et al. [21] was one of the prime works with a natural product against this fungus. Recently, Thomaz et al. [22]    showed that alone from garlic therapy was effective in Balb/c mice infected intra tracheally with the virulent isolate P. brasiliensis Pb18.
Pelegrini et al. [23] have tested peptides from Passiflora edulis against this fungus, but they did not find any effective activity. Marques et al. [24] studied a peptide vaccine with P10, derived from gp43 (major diagnostic antigen), as an adjuvant to chemotherapy, which reduced treatment time, and prevented relapsing disease. In another work, Martins et al. [25], studied the antifungal activity of curcumin, against 23 fungal strains and observed that P. brasiliensis were the most susceptive to curcumin. In present work, the phebalosin was active against all isolates of P. brasiliensis tested. Due to the great relationship between the coumarins and the biological activities mentioned above, a series of modifications were made to the majority (phebalosin) of Polygala paniculata for the evaluation of a possible improvement in antifungal activity displayed by these compounds. The compound of reaction of phebalosin with ethanol (3) presented strong activity against isolate Pb03 of P. brasiliensis with a MIC values of the 1.9 µg/ml (6.5 µmol/L), sixteen times more active than phebalosine. The isopropoxy (5) and n-butoxy (6) derivatives were respectively four times and twice more active than phebalosine against Pb03 (Table 1). These experimental results suggest important hydrophobic and steric interaction simultaneously with alkyl groups in the moiety isoprenyl of the phebalosin. This result indicates an activity intermediate to the two drugs used in the treatment of PCM, and the most used in Brazil by the National Health Care System (SUS) is trimethoprim-sulfamethoxazole [25]. Other authors also tested the antifungal activity of amphotericin B and trimethoprim-sulfamethoxazole against several isolates of    Paracoccidioides, with MIC values close to those found in present work, using RPMI medium, range 0.25-2 µg/ml and 300-75 µg/ml for amphotericin B and trimethoprim-sulfamethoxazole, respectively [25].
The chemometric results, derived from quantum chemistry calculations, revealed the same tendency observed from experimental data: structural properties are responsible for the antifungal activity. Polarizability, surface area and volume showed a good correlation with activity. In spite of all the observations, it's important to remark that the variation of energy between the frontier orbitals (ΔE HOMO-LUMO ) is an important property related to activity, either.
Despite the chemometric analysis is not completely conclusive, PCA technique was able to separate the compounds as function of antifungal activity. In order to increase the antifungal activity, it can be suggested that structural modifications of phebalosin should have the following properties: greater values for polarizability, volume and the variation of energy between the frontier orbitals (ΔE HOMO-LUMO ). It pointed to steric influence (volume), lipophilicity character (polaizability and volume) and compound stability during the interaction (electronic influence, ΔE HOMO-LUMO ) for the substituint alkyl groups.
In conclusion, phebalosin showed promised antifungal activity against isolates of P. brasiliensis tested. The structural modifications of phebalosin with different nucleophiles yielded seven compounds, four being new compounds (8,4,5,6). Overall, these data open new possibilities for the potential use of phebalosin and its structural modifications as possible antifungal drugs against P. brasiliensis. However, new studies are necessary to characterize the mechanism of action of these compounds and produce more conclusive chemometric data.