Lichen Xanthones as Models for New Antifungal Agents

Due to the emergence of multidrug-resistant pathogenic microorganisms, the search for new antimicrobial compounds plays an important role in current medicinal chemistry research. Inspired by lichen antimicrobial xanthones, a series of novel chlorinated xanthones was prepared using five chlorination methods (Methods A–E) to obtain different patterns of substitution in the xanthone scaffold. All the synthesized compounds were evaluated for their antimicrobial activity. Among them, 3-chloro-4,6-dimethoxy-1-methyl-9H-xanthen-9-one 15 showed promising antibacterial activity against E. faecalis (ATCC 29212 and 29213) and S. aureus ATCC 29213. 2,7-Dichloro-3,4,6-trimethoxy-1-methyl-9H-xanthen-9-one 18 revealed a potent fungistatic and fungicidal activity against dermatophytes clinical strains (T. rubrum, M. canis, and E. floccosum (MIC = 4–8 µg/mL)). Moreover, when evaluated for its synergistic effect for T. rubrum, compound 18 exhibited synergy with fluconazole (ΣFIC = 0.289). These results disclosed new hit xanthones for both antibacterial and antifungal activity.


Introduction
Bacterial and fungal infections constitute a serious challenge due to the increasing number of multidrug resistant organisms that consequently can lead to treatment failure. The discovery of new antimicrobial drugs which can overcome problems of resistance to current anti-infective drug therapies is urgent and requires efforts in industry and scientific research communities [1]. The rapidly evolving recognition that a significant number of natural products used as anti-infective drugs/leads are actually produced by microbes [2] has led medicinal chemists to rediscover this traditional source of antimicrobial agents. Xanthones are a well-known class of secondary metabolites found in a restricted assembly of higher plants, fungi, and lichens [3]. Over the preceding decade, more than one hundred of xanthones of lichen sources were identified [4], but only a limited number have been found attractive for their antibacterial and antifungal activities, such is the case of thiophanic and thiophaninic acids with potent fungicidal effects [6], and of cladoxanthone A with antibacterial effects towards Staphylococcus minimoides [7] (Figure 1). Other interesting chlorinated xanthones have been recently isolated from marine organisms. For example, penicillixanthone isolated from a marinederived fungus Aspergillus terreus from the gorgonian coral Echinogorgia aurantiaca exhibited potent antifouling activity against larvae of the barnacle Balanus amphitrite [8]. Two other chlorinated metabolites, 4-chloro-1-hydroxy-3-methoxy-6-methyl-8-methoxycarbonyl-xanthen-9-one and chloroisosulochrin dehydrate were isolated from the extract of the endophytic fungus Penicillium citrinum HL-5126 from the mangrove Bruguiera sexangula; however, these revealed low antibacterial activity with MIC values of 50 µM [9]. The total synthesis of these metabolites can be quite complex, involving several steps to achieve the intricate substitution pattern. Although a number of natural chlorinated lichen xanthones, including thiophanic acid [10][11][12] have already their total synthesis described [5,[13][14][15][16], these natural products can serve also as models in order to explore structure-activity relationship and improve their biological activities.
Inspired by the molecules of chlorinated natural xanthones, in the present paper we focused on the design and synthesis of new chlorinated derivatives with different patterns of substitution for their antimicrobial activity evaluation against seven Gram-positive and Gram-negative bacteria strains and five yeast and filamentous fungi strains.

Chemistry
The introduction of one or more chlorine atoms into the xanthone scaffold can be achieved either by junction of chlorinated building blocks of by aromatic chlorination of the xanthone core. Generally, the chlorination of aromatic compounds is achieved with molecular Cl2. Although the use of chlorine gas has some drawbacks regarding toxicity and hazardousness, ecofriendly procedures involving the in situ generation of Cl2 based on the use of NaCl/p-TsOH/NCS in aqueous media have already been described [17]. On the other hand, in the last years, a number of green procedures based on the generation of the electrophilic reagent Cl + by an ecofriendly oxidation of chlorine ions was also reported in the literature. These include the use of benign oxidants like dimethyldioxirane (DMD), potassium peroxymono-sulfate (Oxones ® ) or NaCl, aqueous H2O2 and acetic acid [18]. Other methods use harsh conditions like neat thionyl chloride [19] or sulfuryl chloride in tetrahydrofuran [11].
Inspired by the molecules of chlorinated natural xanthones, in the present paper we focused on the design and synthesis of new chlorinated derivatives with different patterns of substitution for their antimicrobial activity evaluation against seven Gram-positive and Gram-negative bacteria strains and five yeast and filamentous fungi strains.

Chemistry
The introduction of one or more chlorine atoms into the xanthone scaffold can be achieved either by junction of chlorinated building blocks of by aromatic chlorination of the xanthone core. Generally, the chlorination of aromatic compounds is achieved with molecular Cl 2 . Although the use of chlorine gas has some drawbacks regarding toxicity and hazardousness, ecofriendly procedures involving the in situ generation of Cl 2 based on the use of NaCl/p-TsOH/NCS in aqueous media have already been described [17]. On the other hand, in the last years, a number of green procedures based on the generation of the electrophilic reagent Cl + by an ecofriendly oxidation of chlorine ions was also reported in the literature. These include the use of benign oxidants like dimethyldioxirane (DMD), potassium peroxymono-sulfate (Oxones ® ) or NaCl, aqueous H 2 O 2 and acetic acid [18]. Other methods use harsh conditions like neat thionyl chloride [19] or sulfuryl chloride in tetrahydrofuran [11].

Microbiology
In order to evaluate the antimicrobial activity of compounds 6-20 against Gram-positive and Gram-negative bacteria, an initial activity screening was performed by disk diffusion method for different reference strains and environmental multidrug-resistant strains. The results are presented in Table 3. Any of the tested compounds revealed antibacterial activity against Gram-negative bacteria. Regarding Gram-positive bacteria, compound 15 was effective with an inhibition halo of 10 mm for E. faecalis ATCC 29212 and 9.5 mm for S. aureus ATCC 29213. However, 15 was not effective when tested with either methicillin-resistant S. aureus (MRSA) or vancomycin-resistant enterococci (VRE). Despite these encouraging results, it was not possible to determine a MIC for any compound in any of the strains in the range of the tested concentrations. This might be related to the fact that some compounds are poorly soluble in the culture media used for the determination of MIC, and the amount of available compound in the solution was probably lower than anticipated. Especially for hydrophobic compounds, such is the case of chlorinated xanthones, the diffusion through the agar media tends to be slower [22][23][24]. Regarding the screening for potential synergies with the multidrugresistant bacterial strains and the tested compounds in combination with clinically relevant antibiotics, none of the compounds revealed a synergistic association with antibiotics (data not shown).
The results for the antifungal activity of the tested compounds against yeast and filamentous fungi are presented in Table 4. None of the compounds tested showed activity against C. albicans nor A. fumigatus strains. Nevertheless, compounds 8 and 9 revealed variable inhibitory effect on dermatophytes with MIC and MFC ranging from 128 to 256 (≥128) µg/mL, depending of the compounds and the species used for testing.

Microbiology
In order to evaluate the antimicrobial activity of compounds 6-20 against Gram-positive and Gram-negative bacteria, an initial activity screening was performed by disk diffusion method for different reference strains and environmental multidrug-resistant strains. The results are presented in Table 3. Any of the tested compounds revealed antibacterial activity against Gram-negative bacteria. Regarding Gram-positive bacteria, compound 15 was effective with an inhibition halo of 10 mm for E. faecalis ATCC 29212 and 9.5 mm for S. aureus ATCC 29213. However, 15 was not effective when tested with either methicillin-resistant S. aureus (MRSA) or vancomycin-resistant enterococci (VRE). Despite these encouraging results, it was not possible to determine a MIC for any compound in any of the strains in the range of the tested concentrations. This might be related to the fact that some compounds are poorly soluble in the culture media used for the determination of MIC, and the amount of available compound in the solution was probably lower than anticipated. Especially for hydrophobic compounds, such is the case of chlorinated xanthones, the diffusion through the agar media tends to be slower [22][23][24]. Regarding the screening for potential synergies with the multidrug-resistant bacterial strains and the tested compounds in combination with clinically relevant antibiotics, none of the compounds revealed a synergistic association with antibiotics (data not shown).
The results for the antifungal activity of the tested compounds against yeast and filamentous fungi are presented in Table 4. None of the compounds tested showed activity against C. albicans nor A. fumigatus strains. Nevertheless, compounds 8 and 9 revealed variable inhibitory effect on dermatophytes with MIC and MFC ranging from 128 to 256 (≥128) µg/mL, depending of the compounds and the species used for testing.    Concerning structure-activity relationship (SAR) analysis, the obtained results were consistent with data previously reported for some natural products (Figure 3). For antibacterial activity, the chlorine atom at C-3 seems to have some influence since 3-chloro-4,6-dimethoxy-1-methyl-9Hxanthen-9-one 15 showed promising antibacterial activity against E. faecalis (ATCC 29212) and S. aureus ATCC 29213.
Regarding antifungal activity, SAR suggests that the presence of a chlorine atom at C-2, C-3, C-5, or C-7 plays an important role towards this activity. Interestingly, the fact that only 2,7-dichloro-3,4,6-trimethoxy-1-methyl-9H-xanthen-9-one 18, with chlorine atoms at both C-2 and C-7, exhibited potent antifungal activity suggests that their joint presence may be required for this effect, similarly to the natural product thiophanic acid.   Concerning structure-activity relationship (SAR) analysis, the obtained results were consistent with data previously reported for some natural products (Figure 3). For antibacterial activity, the chlorine atom at C-3 seems to have some influence since 3-chloro-4,6-dimethoxy-1-methyl-9Hxanthen-9-one 15 showed promising antibacterial activity against E. faecalis (ATCC 29212) and S. aureus ATCC 29213.
Regarding antifungal activity, SAR suggests that the presence of a chlorine atom at C-2, C-3, C-5, or C-7 plays an important role towards this activity. Interestingly, the fact that only 2,7-dichloro-3,4,6-trimethoxy-1-methyl-9H-xanthen-9-one 18, with chlorine atoms at both C-2 and C-7, exhibited potent antifungal activity suggests that their joint presence may be required for this effect, similarly to the natural product thiophanic acid.

General
All reagents and solvents were purchased from TCI (Tokyo Chemical Industry Co. Ltd., Chuoku, Tokyo, Japan), Acros Organics (Geel, Belgium), Sigma-Aldrich (Sigma-Aldrich Co. Ltd., Gillinghan, UK), or Alfa Aesar (Thermo Fisher GmbH, Kandel, Germany) and had no further purification process. Solvents were evaporated using rotary evaporator under reduced pressure, Buchi Waterchath B-480. Microwave (MW) reactions were performed using an Ethos MicroSYNTH 1600 Microwave Labstation from Milestone (Thermo Unicam, Portugal). The internal reaction temperature was controlled by a fiber optic probe sensor. All reactions were monitored by TLC

General
All reagents and solvents were purchased from TCI (Tokyo Chemical Industry Co. Ltd., Chuo-ku, Tokyo, Japan), Acros Organics (Geel, Belgium), Sigma-Aldrich (Sigma-Aldrich Co. Ltd., Gillinghan, UK), or Alfa Aesar (Thermo Fisher GmbH, Kandel, Germany) and had no further purification process. Solvents were evaporated using rotary evaporator under reduced pressure, Buchi Waterchath B-480. Microwave (MW) reactions were performed using an Ethos MicroSYNTH 1600 Microwave Labstation from Milestone (Thermo Unicam, Portugal). The internal reaction temperature was controlled by a fiber optic probe sensor. All reactions were monitored by TLC carried out on precoated plates with 0.2 mm thickness using Merck silica gel 60 (GF254) with appropriate mobile phases and detection at 254 and/or 365 nm. Purification of the synthesized compounds was performed by chromatography flash column using Merck silica gel 60 (0.040-0.063 mm). Melting points (m.p.) were measured in a Köfler microscope (Wagner and Munz, Munich, Germany) and are uncorrected. 1 H-and 13 C-NMR spectra were taken in CDCl 3 (Deutero GmbH, Kastellaun, Germany) at room temperature on Bruker Avance 300 instrument (300.13 or 500.13 MHz for 1 H and 75.47 or 125.77 MHz for 13 C, Bruker Biosciences Corporation, Billerica, MA, USA). Chemical shifts are expressed in δ (ppm) values relative to tetramethylsilane (TMS) as an internal reference. Coupling constants are reported in hertz (Hz). 13 C-NMR assignments were made by 2D HSQC and HMBC experiments. HRMS mass spectra were measured on a Bruker FTMS APEX III mass spectrometer (Bruker Corporation, Billerica, MA, USA) recorded as ESI (Electrospray) mode in Centro de Apoio Cientifico e Tecnolóxico á Investigation (CACTI, University of Vigo, Pontevedra, Spain).

Antimicrobial Susceptibility Testing
Antibacterial Activity An initial screening of the antibacterial activity of the compounds was performed by the disk diffusion method as previously described [27,28]. Briefly, sterile 6 mm blank paper disks (Oxoid, Basingstoke, UK) impregnated with 15 µg of each compound were placed on MH agar plates inoculated with the bacteria. A blank disk with DMSO was used as a negative control. MH inoculated plates were incubated for 18-20 h at 37 • C. At the end of incubation, the inhibition halos where measured. The minimum inhibitory concentration (MIC) was determined for each compound, in accordance with the recommendations of the Clinical and Laboratory Standards Institute (CLSI) [29]. For each compound, a stock solution of 10 mg/mL was prepared in dimethylsulfoxide (DMSO, Alfa Aesar, Kandel, Germany). For compounds 6, 11, 13, 15-17, 19, and 20, which were less soluble in DMSO than the other compounds, a stock solution of 2 mg/mL was prepared. In the case of compounds 10, 12, and 18 the stock solution prepared was 1 mg/mL. Two-fold serial dilutions of the compounds were prepared in Mueller-Hinton broth 2 (MHB2, Sigma-Aldrich, St. Louis, MO, USA) within the concentration range of 0.062 to 64 µg/mL. The highest concentration tested was chosen in order to maintain DMSO in-test concentration below 1% (v/v), as recommended by the CLSI [29]. At this concentration DMSO did not affected bacterial growth. Cefotaxime (CTX) ranging between 0.031 and 16 µg/mL was used as a quality control for E. coli reference strain ATCC 25922. Sterility and growth controls were included in each assay. Purity check and colony counts of the inoculum suspensions were also evaluated in order to ensure that the final inoculum density closely approximates the intended number (5 × 10 5 CFU/mL). The MIC was determined as the lowest concentration at which no visible growth was observed. The minimum bactericidal concentration (MBC) was assessed by spreading 10 µL of culture collected from wells showing no visible growth on MH agar plates. The MBC was determined as the lowest concentration at which no colonies grew after 16-18 h incubation at 37 • C. These assays were performed in duplicate.

Antifungal Activity
The antifungal activity of all tested compounds was evaluated against C. albicans, A. fumigatus, and T. rubrum. For compounds showing some activity in the dermatophyte T. rubrum the activity was enlarged to other genus of dermatophytes (M. canis and E. floccosum). The MIC of each compound was determined by the broth microdilution method according to CLSI guidelines (reference documents M27-A3 for yeasts [30] and M38-A2 for filamentous fungi [31]). Briefly, cell or spore suspensions were prepared in RPMI-1640 broth medium supplemented with MOPS (Sigma-Aldrich, St. Louis, MO, USA) from fresh cultures of the different strains of fungi. In the case of filamentous fungi, the inoculum was adjusted to 0.4-5 × 10 4 CFU/mL for A. fumigatus ATCC 46645 and to 1-3 × 10 3 CFU/mL for the dermatophytes. The inoculum of C. albicans was adjusted to 0.5-2.5 × 10 3 CFU/mL. Two-fold serial dilutions of the compounds were prepared in RPMI-1640 broth medium supplemented with MOPS within the concentration range of 1 to 128 µg/mL, with maximum DMSO concentration not exceeding 2.5% (v/v). Sterility and growth controls were also included in each assay. The plates were incubated for 48 h at 35 • C (C. albicans and A. fumigatus) or for 5 days at 25 • C (T. rubrum, M. canis and E. floccosum). MICs were recorded as the lowest concentrations resulting in 100% growth inhibition in comparison to the compound-free controls. Voriconazole MIC for reference strain Candida krusei ATCC 6258 was used as quality control [30,31]. The results obtained were within the recommended limits. The minimum fungicidal concentration (MFC) was determined by spreading 20 µL of culture collected from wells showing no visible growth on SDA plates. The MFC was determined as the lowest concentration showing 100% growth inhibition after 48 h at 35 • C (for C. albicans and A. fumigatus) or 5 days incubation at 25 • C (T. rubrum, M. canis and E. floccosum). All the experiments were repeated independently at least two times.

Antibiotic Synergy
In order to evaluate the combined effect of the compounds and clinically relevant antimicrobial drugs, a screening was conducted using the disk diffusion method, as previously described [27,28]. A set of antibiotic disks (Oxoid, Basingstoke, UK), to which the isolates were resistant, was selected: cefotaxime (CTX, 30 µg) for E. coli SA/2, oxacillin (OX, 1 µg) for S. aureus 66/1, and vancomycin (VA, 30 µg) for E. faecalis B3/101. Antibiotic disks alone (controls) and antibiotic disks impregnated with 15 µg of each compound were placed on MH agar plates seeded with the respective bacteria. Sterile 6 mm blank paper impregnated with 15 µg of each compound alone were also tested. A blank disk with DMSO was used as a negative control. MH inoculated plates were incubated for 18 to 20 h at 37 • C. Potential synergism was recorded when the halo of an antibiotic disk impregnated with a compound was greater than the halo of the antibiotic or compound-impregnated blank disk alone.

Antifungal Synergy
In order to evaluate the combined effect of the compounds and clinically relevant antifungal drugs, checkerboard assay was conducted, as previously described [32]. Fluconazole was used in a range between 0.062 and 4 µg/mL and compounds were tested in a range between their MIC and progressive two-fold dilutions. Potential synergism was recorded when the inhibitions of the combined compounds with antifungals is greater than the compounds or the antifungals alone.

Conclusions
A series of xanthones (6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20) was synthesized and evaluated for its antibacterial and antifungal activity. Some of the methodologies used, such as NaCl, p-TsOH, and NCS, were more selective and produced higher yields while others, like the use of thionyl chloride gave a higher diversity of compounds but with lower yields. The presence of the chlorine isotopic pattern in the HRMS spectra was crucial to identify the presence of one or two chlorine atoms and bidimensional NMR to disclose the position of those atoms and the substitution pattern. Although some of the compounds exhibited great potential as antibacterial or antifungal agents, the low solubility displayed by some derivatives limited further screenings. Nevertheless, compounds 15 and 18 can be used in the future as models in order to improve drug-like properties.