Antifungal Activity of Xanthones: Evaluation of their Effect on Ergosterol Biosynthesis by High-performance Liquid Chromatography

Article abstract of DOI:10.1111/j.1747-0285.2010.01072.x

The increasing resistance of pathogenic fungi to antifungal compounds and the reduced number of available drugs led to the search for therapeutic alternatives among natural products, including xanthones. The antifungal activity of 27 simple oxygenated xanthones was evaluated by determination of their minimal inhibitory concentration on clinical and type strains of Candida, Cryptococcus, Aspergillus and dermatophytes, and their preponderance on the dermatophytic filamentous fungi was observed. Furthermore, a simple and efficient HPLC method with UV detection to study the effect of the active xanthones on the biosynthesis of ergosterol was developed and validated. Using this methodology, the identification and quantification of fungal sterols in whole cells of Candida albicans, Cryptococcus neoformans, Aspergillus fumigatus, and Trichophyton mentagrophytes were accomplished. In summary, 1,2-dihydroxyxanthone was found to be the most active compound against all strains tested, showing its effect on sterol biosynthesis by reducing the amount of ergosterol detected.

Full text of DOI:10.1111/j.1747-0285.2010.01072.x

ª 2011 John Wiley & Sons A/S
doi: 10.1111/j.1747-0285.2010.01072.x
Chem Biol Drug Des 2011; 77: 212–222
Research Article
Antifungal Activity of Xanthones: Evaluation of
their Effect on Ergosterol Biosynthesis by
High-performance Liquid Chromatography
Eugenia Pinto1,2*, Carlos Afonso1,3*,
broad-spectrum antibiotics (1). The most frequently encountered
infections are caused by the yeast Candida albicans and also by
species of the filamentous fungus Aspergillus (2). On the other
hand, dermatophytes (the genera Epidermophyton, Trichophyton, and
Microsporum), which invade and multiply in keratinized tissues (3)
and cause infection, have a worldwide distribution with geographic
differences in the incidence and prevalence (4).
´
Serafim Duarte1,2, Luı´s Vale-Silva1,2
,
Elisangela Costa1,3, Em´ılia Sousa1,3 and
Madalena Pinto1,3
1Research Center of Medicinal Chemistry - University of Porto
(CEQUIMED-UP), R. Anꢀbal Cunha, 164, 4050-047, Porto, Portugal
2Department of Microbiology, Faculty of Pharmacy, University of
Porto, R. Anꢀbal Cunha, 164, 4050-047, Porto, Portugal
3Department of Organic Chemistry, Faculty of Pharmacy, University
of Porto, R. Anꢀbal Cunha, 164, 4050-047, Porto, Portugal
*Corresponding authors: Eugꢁnia Pinto and Carlos Afonso,
epinto@ff.up.pt; cafonso@ff.up.pt
The majority of clinically used antifungal drugs suffer from various
drawbacks in terms of toxicity, efficacy, and cost, as well as the
emergence of resistant strains caused by their frequent use (5).
Although this was counteracted by new advances in molecular
genetics which have afforded the promise of revealing new antifun-
gal targets together with new agents to inhibit those targets spe-
cifically (6), there is still a great demand for novel and effective
antifungal agents. One of the most promising groups of antifungal
agents is the xanthones (9H-xanthen-9-ones), which are heterocyclic
compounds based on the dibenzo-c-pyrone scaffold (Figure 1). Now-
adays, xanthone derivatives are of relevance because of the several
biologic activities reported (7). Concerning antifungal activity, inves-
tigations have been undertaken in the group of both synthetic (8,9)
and naturally occurring xanthone derivatives. Consequently, in an
attempt to search for naturally occurring antifungal compounds, a
number of xanthones with clinical value against fungal infections,
from higher plants (8,10–26) and microorganisms (27–34) including
marine species (35,36), have been identified. These xanthones are
mainly divided into four subclasses: simple oxygenated
(8,15,17,25,26,35,36), prenylated (8,10–14,16–22,24,26), polycyclic
(27–29), and dehydroxanthones (15,31–34) such as ergochromes and
hemisecalonic acids. Some of these compounds have revealed sig-
nificant antifungal properties, e.g., Sch 54445 that exhibits highly
potent activities against various yeasts and dermatophytes (27).
Nonetheless, little information is available on structure–antifungal
The increasing resistance of pathogenic fungi to
antifungal compounds and the reduced number of
available drugs led to the search for therapeutic
alternatives among natural products, including
xanthones. The antifungal activity of 27 simple
oxygenated xanthones was evaluated by determi-
nation of their minimal inhibitory concentration
on clinical and type strains of Candida, Cryptococ-
cus, Aspergillus and dermatophytes, and their pre-
ponderance on the dermatophytic filamentous
fungi was observed. Furthermore, a simple and
efficient HPLC method with UV detection to study
the effect of the active xanthones on the biosyn-
thesis of ergosterol was developed and validated.
Using this methodology, the identification and
quantification of fungal sterols in whole cells of
Candida albicans, Cryptococcus neoformans,
Aspergillus fumigatus, and Trichophyton ment-
agrophytes were accomplished. In summary,
1,2-dihydroxyxanthone was found to be the most
active compound against all strains tested, show-
ing its effect on sterol biosynthesis by reducing
the amount of ergosterol detected.
Key words: antifungal, Aspergillus, Candida, Cryptococcus, derma-
tophytes, ergosterol, xanthone
Received 4 September 2009, revised 6 July 2010 and accepted for
publication 10 December 2010
It is well recognized that nowadays invasive mycoses have become
important causes of morbidity and mortality in immunocompromised
patients, such as those infected with HIV and those receiving can-
cer chemotherapy, immunosuppressive therapy, or treatment with
Figure 1: Xanthone core and numbering.
212

Antifungal Activity of Xanthones
activity relationship of oxygenated xanthones and ultimately on their
mechanism of action.
(dd, J = 8.2, 7.6, H-C(7)), 6.83 (dd, J = 8.3, 0.6, H-C(2)). 13C-NMR
((D6)DMSO): 181.8 (C(9)), 161.0 (C(1)), 155.8 (C(4a)), 155.7 (C(10a)),
137.6 (C(3)), 136.5 (C(6)), 125.5 (C(8)), 124.7 (C(7)), 119.9 (C(8a)),
118.1 (C(5)), 110.2 (C(2)), 108.4 (C(9a)), 107.3 (C(4)).
Several bioactive xanthones, different in nature and pattern of
substitution in one of the aromatic rings, have already been
reported by our group for their antitumor (37–40), immunomodula-
tory (38,41–43), modulatory activity of protein kinase C (44–46),
antimalarial (47), hepatoprotective (48), and monoaminoxidase
inhibitory effects (49,50). Following up our investigation into bioac-
tive xanthones, we have evaluated 27 hydroxylated ⁄ methoxylated
xanthone derivatives for their antifungal activity. Thus, the aims of
this work are to evaluate the influence of the nature and position
of the substituents on structure–activity relationships of antifungal
xanthones and to investigate the effect of the most active com-
pounds on sterol biosynthesis. The insight into their mechanisms
of action can not only provide important information in an attempt
to improve the antifungal activity of these compounds, but
also allow their combination with other antifungal agents in
therapeutics.
1
2-Hydroxyxanthone (3) 74%yield. H-NMR ((D6)DMSO) d: 10.00 (s,
OH-C(2)), 8.19 (dd, J = 8.0, 1.7, H-C(8)), 7.85 (ddd, J = 8.2, 7.6, 1.7,
H-C(6)), 7.63 (dd, J = 8.2, 0.9, H-C(5)), 7.56 (d, J = 9.0, H-C(4)), 7.48
(d, J = 3.0, H-C(1)), 7.45 (ddd, J = 8.0, 7.6, 0.9, H-C(7)), 7.32 (dd,
J = 9.0, 3.0, H-C(3)). 13C-NMR ((D6)DMSO): 175.9 (C(9)), 155.6
(C(10a)), 153.9 (C(2)), 149.2 (C(4a)), 135.2 (C(6)), 125.9 (C(8)), 124.6
(C(3)), 124.0 (C(7)), 121.7 (C(9a)), 120.4 (C(8a)), 119.5 (C(4)), 118.2
(C(5)), 108.5 (C(1)).
1
3-Hydroxyxanthone (4) 92%yield. H-NMR ((D6)DMSO) d: 11.00 (s,
OH-C(3)), 8.15 (dd, J = 7.9, 1.7, H-C(8)), 8.04 (d, J = 8.6, H-C(1)),
7.82 (ddd, J = 8.2, 7.6, 1.7, H-C(6)), 7.61 (d, J = 8.2, H-C(5)), 7.44
(dd, J = 7.9, 7.6, H-C(7)), 6.91 (dd, J = 8.6, 2.2, H-C(2)), 6.88 (d,
J = 2.2, H-C(4)). 13C-NMR ((D6)DMSO): 174.8 (C(9)), 164.0 (C(3)),
157.6 (C(4a)), 155.6 (C(10a)), 134.9 (C(6)), 128.0 (C(1)), 125.9 (C(8)),
124.2 (C(7)), 121.2 (C(8a)), 117.9 (C(5)), 114.2 (C(2)), 114.0 (C(9a)),
102.1 (C(4)).
On the one hand, it is well established that ergosterol is the pre-
dominant sterol in fungal cell membrane and is responsible for
maintaining cell integrity and function as well as for the normal
growth. Thus, most of the current frontline antifungal agents act
either on ergosterol biosynthesis (azoles, allylamines) or on ergos-
terol itself (amphotericin B). GC-MS is the most current method
used for the quantification of sterols. Although this method provides
greater sensitivity, it requires sample derivatization. Consequently, it
is very complex and takes longer time to execute. We hereby report
the development of a simpler and quicker method to separate and
calculate the sterol content in fungal whole cell assays using a
normal-phase HPLC without prior tetramethylsilane (TMS) derivatiza-
tion (51).
1
4-Hydroxyxanthone (5) 59%yield. H-NMR ((D6)DMSO) d: 10.51 (s,
OH-C(4)), 8.19 (dd, J = 7.8, 1.6, H-C(8)), 7.88 (ddd, J = 8.0, 7.6, 1.6,
H-C(6)), 7.73 (dd, J = 8.0, 0.8, H-C(5)), 7.61 (dd, J = 7.8, 1.8, H-C(1)),
7.48 (ddd, J = 7.8, 7.6, 0.8, H-C(7)), 7.34 (dd, J = 7.8, 1.8, H-C(3)),
7.26 (t, J = 7.8, H-C(2)). 13C-NMR ((D6)DMSO): 176.2 (C(9)), 155.4
(C(10a)), 146.7 (C(4)), 145.2 (C(4a)), 135.4 (C(6)), 126.0 (C(8)), 124.3
(C(7)), 124.1 (C(2)), 122.2 (C(9a)), 120.9 (C(8a)), 120.2 (C(3)), 118.3
(C(5)), 115.2 (C(1)).
1-Methoxyxanthone (6) 86%yield. 1H-NMR ((D6)DMSO) d: 8.09
(dd, J = 7.7, 1.6, H-C(8)), 7.80 (ddd, J = 7.8, 7.6, 1.6, H-C(6)), 7.74
(dd, J = 8.4, 8.3, H-C(3)), 7.57 (dd, J = 7.8, 0.7, H-C(5)), 7.42 (ddd,
J = 7.7, 7.6, 0.7, H-C(7)), 7.00 (d, J = 8.3, H-C(2)), 3.91 (s, MeO-
C(1)). 13C-NMR ((D6)DMSO): 174.7 (C(9)), 160.2 (MeO-C(1)), 157.4
(C(4a)), 154.4 (C(10a)), 135.7 (C(3)), 134.8 (C(6)), 125.9 (C(8)), 124.2
(C(7)), 122.4 (C(8a)), 117.5 (C(5)), 111.6 (C(9a)), 109.6 (C(4)), 106.4
(C(2)).
Experimental Section
Chemistry
Melting points were obtained in a Kçfler microscope and are uncor-
1
rected. IR spectra were recorded on a Perkin Elmer 257 in KBr. H
and 13C NMR spectra were taken in DMSO-d6 at room temperature,
on Bruker DRX 300 instrument. Chemical shifts are expressed in d
(ppm) values relative to TMS. HR-MS results were obtained in
CACTI services, Vigo, Spain.
2-Methoxyxanthone (7) 66%yield. 1H-NMR ((D6)DMSO) d: 8.18
(dd, J = 7.7, 1.7, H-C(8)), 7.86 (ddd, J = 8.2, 7.4, 1.7, H-C(6)), 7.64
(dd, J = 8.2, 1.9, H-C(5)), 7.63 (d, J = 9.1, H-C(4)), 7.54 (d, J = 3.2,
H-C(1)), 7.47 (dd, J = 9.1, 3.2, H-C(3)), 7.46 (ddd, J = 7.7, 7.4, 1.0,
H-C(7)), 3.87 (s, MeO-C(2)). 13C-NMR ((D6)DMSO): 175.8 (C(9)), 155.7
(C(2)), 155.5 (C(10a)), 150.3 (C(4a)), 135.4 (C(6)), 126.0 (C(8)), 124.7
(C(3)), 124.2 (C(7)), 121.5 (C(9a)), 120.5 (C(8a)), 119.8 (C(4)), 118.2
(C(5)), 105.7 (C(1)), 55.7 (MeO-C(2)).
Xanthone (1) and 2,2¢,4,4¢-tetrahydroxybenzophenone (28) were pur-
chased from Sigma Chemical Co., St. Louis, USA. The natural prod-
ucts 2-hydroxy-1-methoxyxanthone (24), 1,7-dihydroxyxanthone (25),
2-hydroxy-1,8-dimethoxyxanthone (26), and 1,2,8-trimethoxyxanthone
(27) were kindly provided by Prof. Anake Kijjoa, ICBAS-Instituto de
CiÞncias Biomꢀdicas de Abel Salazar, Portugal, and were isolated
from Calophyllum teysmannii var. inophylloide (41,52). The following
xanthone derivatives were synthesized according to previously
described procedures (38,49,53).
3-Methoxyxanthone (8) 45%yield. 1H-NMR ((D6)DMSO) d: 8.17
(dd, J = 7.7, 1.7, H-C(8)), 8.10 (d, J = 8.9, H-C(1)), 7.85 (ddd,
J = 8.0, 7.6, 1.7, H-C(6)), 7.63 (d, J = 8.0, H-C(5)), 7.47 (dd, J = 7.7,
7.6, H-C(7)), 7.16 (d, J = 2.4, H-C(4)), 7.05 (dd, J = 8.9, 2.4, H-C(2)),
3.93 (s, MeO-C(3)). 13C-NMR ((D6)DMSO, 75.47 MHz): 174.9 (C(9)),
165.0 (C(3)), 157.6 (C(4a)), 155.6 (C(10a)), 135.1 (C(6)), 127.6 (C(1)),
125.9 (C(8)), 124.4 (C(7)), 121.2 (C(8a)), 117.9 (C(5)), 114.9 (C(9a)),
113.7 (C(2)), 100.6 (C(4)), 56.2 (MeO-C(3)).
1
1-Hydroxyxanthone (2) 48%yield. H-NMR ((D6)DMSO) d: 12.56 (s,
OH-C(1)), 8.18 (dd, J = 8.2, 1.7, H-C(8)), 7.92 (ddd, J = 8.1, 7.6, 1.7,
H-C(6)), 7.74 (dd, J = 8.3, 8.2, H-C(3)), 7.67 (d, J = 8.1, H-C(5)), 7.51
Chem Biol Drug Des 2011; 77: 212–222
213

Pinto et al.
4-Methoxyxanthone (9) 14%yield. 1H-NMR ((D6)DMSO) d: 8.20
(dd, J = 8.0, 1.6, H-C(8)), 7.88 (dt, J = 8.1, 1.6, H-C(6)), 7.73 (dd,
J = 7.9, 1.4, H-C(1)), 7.72 (d, J = 8.1, H-C(5)), 7.52 (dd, J = 7.9, 1.4,
H-C(3)), 7.49 (dd, J = 8.0, 7.8, H-C(7)), 7.40 (t, J = 7.9, H-C(2)), 3.99
(s, MeO-C(4)). 13C-NMR ((D6)DMSO): 176.0 (C(9)), 155.4 (C(10a)),
148.4 (C(4)), 145.8 (C(4a)), 135.5 (C(6)), 125.9 (C(8)), 124.5 (C(7)),
124.0 (C(2)), 121.0 (C(8a)), 121.9 (C(9a)), 118.4 (C(5)), 116.4 (C(3)),
116.4 (C(1)), 56.2 (MeO-(C(4)).
(C(7)), 121.7 (C(1)), 120.8 (C(8a)), 118.2 (C(5)), 115.9 (C(9a)), 109.7
(C(2)), 60.9 (MeO-C(4)), 56.1 (MeO-C(3)).
3-Hydroxy-4-methoxyxanthone
(16)
51%yield.
1H-NMR
((D6)DMSO) d: 10.79 (s, OH-C(3)), 8.16 (dd, J = 7.7, 1.7, H-C(8)), 7.84
(ddd, J. 8.1, 7.6, 1.7, H-C(6)), 7.80 (d, J.8.9, H-C(1)), 7.69 (dd, J.8.1,
1.0, H-C(5)), 7.46 (ddd, J.7.7, 7.6, 1.0, H-C(7)), 7.00 (d, J.8.9, H-C(2)),
3.92 (s, MeO-C(4)). 13C-NMR ((D6)DMSO): 175.0 (C(9)), 156.3 (C(3)),
155.5 (C(10a)), 150.7 (C(4a)), 135.0 (C(6)), 134.6 (C(4)), 125.9 (C(8)),
124.3 (C(7)), 121.6 (C(1)), 120.9 (C(8a)), 118.2 (C(5)), 114.8 (C(9a)),
114.1 (C(2)), 60.9 (MeO-C(4)).
1,2-Dihydroxyxanthone (10) 48%yield. 1H-NMR ((D6)DMSO) d:
12.45 (OH-C(1)), 9.42 (OH-C(2)), 8.16 (dd, J = 7.9, 1.6, H-C(8)), 7.81
(ddd, J = 8.4, 7.0, 1.6, H-C(6)), 7.60 (dd, J = 8.4, 0.8, H-C(5)) 7.46
(ddd, J = 7.9, 7.0, 0.8 H-C(7)), 7.32 (d, J = 9.0, H-C(4)), 6.96 (d,
J = 9.0, H-C(3)), 13C-NMR ((D6)DMSO): 182.3 (C(9)), 155.9 (C(10a)),
148.3 (C(4a)), 147.6 (C(1)), 140.2 (C(2)), 136.4 (C(6)), 125.4 (C(8)),
124.5 (C(3)), 124.2 (C(7)), 119.3 (C(8a)), 118.0 (C(5)), 108.8 (C(9a)),
106.2 (C(4)).
4-Hydroxy-3-methoxyxanthone
(17)
39%yield.
1H-NMR
((D6)DMSO) d: 9.66 (s, OH-C(4)), 8.16 (dd, J = 7.8, 1.7, H-C(8)), 7.84
(ddd, J = 8.3, 7.6, 1.7, H-C(6)), 7.68 (d, J = 9.0, H-C(1)), 7.65 (dd,
J = 8.3, 1.0, H-C(5)), 7.44 (ddd, J = 7.8, 7.6, 1.0, H-C(7)), 7.19 (d,
J = 9.0, H-C(2)), 3.96 (s, MeO-C(3)). 13C-NMR ((D6)DMSO): 175.6
(C(9)), 155.7 (C(10a)), 152.5 (C(3)), 145.5 (C(4a)), 135.1 (C(6)), 134.1
(C(4)), 126.0 (C(8)), 124.1 (C(7)), 120.8 (C(8a)), 118.1 (C(5)), 116.3
(C(1)), 115.9 (C(9a)), 109.0 (C(2)), 56.4 (MeO-C(3)).
2,3-Dihydroxyxanthone (11) 90%yield. 1H-NMR ((D6)DMSO) d:
8.12 (dd, J = 7.5, 1.6, H-C(8)), 7.78 (ddd, J = 8.4, 6.8, 1.7, H-C(6)),
7.58 (d, J = 8.4 Hz, H-C(5)), 7.43 (s, H-C(1)), 7.42 (ddd, J = 7.5,
6.8, 1.9 Hz, H-C(7)), 6.92 (s, H-C(4)), 13C-NMR ((D6)DMSO): 175.0
(C(9)), 155.7 (C(10a)), 154.2 (C(4a)), 151.3 (C(3)), 144.1 (C(2)), 134.7
(C(6)), 125.9 (C(8)), 124.1 (C(7)), 120.9 (C(8)), 118.1 (C(5)), 113.7
(C(9a)), 108.9 (C(1)), 103.0 (C(4)).
3,5-Dihydroxyxanthone (18) 55%yield. 1H-NMR ((D6)DMSO) d:
8.02 (d, J = 9.4, H-C(1)), 7.56 (dd, J = 7.5, 1.8, H-C(8)), 7.27 (dd,
J = 7.8, 1.8, H-C(6)), 7.21 (t, J = 7.7, H-C(7)), 6.89 (dd, J = 7.4, 2.1,
H-C(2)), 6.88 (s, H-C(4)). 13C-NMR ((D6)DMSO): 175.0 (C(9)), 163.9
(C(3)), 157.3 (C(4a)), 146.3 (C(5)), 145.8 (C(10a)), 127.9 (C(1)), 123.8
(C(7)), 122.0 (C(8a)), 116.1 (C(6)), 115.2 (C(8)), 114.2 (C(2)), 113.8
(C(9a)), 102.1 (C(4)).
3,4-Dihydroxyxanthone (12) 78%yield. 1H-NMR ((D6)DMSO) d:
8.14 (dd, J = 8.1, 1.6, H-C(8)), 7.81 (ddd, J = 8.6, 6.9, 1.7, H-C(6)),
7.62 (dd, J = 8.6, 0.9, H-C(5)), 7.56 (d, J = 8.6, H-C(1)), 7.42 (ddd,
J = 8.1, 6.9, 0.9, H-C(7)), 6.93 (d, J = 8.6, H-C(2)), 13C-NMR
((D6)DMSO): 175.3 (C(9)), 155.5 (C(10a)), 151.6 (C(3)), 146.4 (C(4a)),
134.8 (C(6)), 132.7 (C(4)), 125.9 (C(8)), 124.0 (C(7)), 120.8 (C(8a)),
118.0 (C(5)), 116.6 (C(1)), 114.7 (C(9a)), 113.2 (C(2)).
3,5-Dimethoxyxanthone (19) 35%yield. 1H-NMR ((D6)DMSO) d:
8.09 (d, J = 8.9, H-C(1)), 7.70 (dd, J = 7.9, 1.7, H-C(8)), 7.49 (dd,
J = 7.9, 1.7, H-C(6)), 7.38 (t, J = 7.9, H-C(7)), 7.20 (d, J = 2.4, H-
C(4)), 7.05 (dd, J = 8.8, 2.4, H-C(2)), 3.97 (s, MeO-C(5)), 3.94 (s,
MeO-C(3)). 13C-NMR ((D6)DMSO): 175.0 (C(9)), 165.0 (C(3)), 157.4
(C(4a)), 148.3 (C(5)), 145.8 (C(10a)), 127.5 (C(1)), 124.0 (C(7)), 122.0
(C(8a)), 116.4 (C(8)), 116.1 (C(6)), 114.8 (C(2)), 114.1 (C(9a)), 100.6
(C(4)), 56.2 (MeO-C(3), MeO-C(5)).
1,2-Dimethoxyxanthone (13) 74%yield. 1H-NMR ((D6)DMSO) d:
8.12 (dd, J = 7.9 and 1.7, H-C(8)), 7.81 (ddd, J = 7.6, 7.4, 1.7, H-
C(6)), 7.63 (d, J = 9.3, H-C(4)), 7.56 (d, J = 7.4, H-C(5)), 7.44 (dd,
J = 7.9, 7.6, 1.1, H-C(7)), 7.40 (d, J = 9.3, H-C(3)), 3.86 (s, MeO-
C(1)), 3.80 (s, MeO-C(2)), 13C-NMR ((D6)DMSO): 175.2 (C(9)), 154.7
(C(10a)), 150.3 (C(2)), 148.9 (C(1)), 147.5 (C(4a)), 135.0 (C(6)), 126.0
(C(8)), 123.9 (C(7)), 121.4 (C(8a)), 120.7 (C(5)), 117.6 (C(3)), 116.4
(C(9a)), 113.2 (C(4)), 61.0 (MeO-C(1)), 56.6 (MeO-C(2)).
3-Hydroxy-5-methoxyxanthone
(20)
42%yield.
1H-NMR
((D6)DMSO) d: 8.03 (d, J = 8.6, H-C(1)), 7.70 (dd, J = 7.9, 1.2, H-
C(8)), 7.46 (dd, J = 7.9, 1.2, H-C(6)), 7.35 (t, J = 7.9, H-C(7)), 6.91
(dd, J = 8.6, 2.0, H-C(2)), 6.88 (d, J = 2.0, H-C(4)), 3.96 (s, MeO-
C(5)). 13C-NMR ((D6)DMSO): 174.8 (C(9)), 164.1 (C(3)), 157.4 (C(4a)),
148.2 (C(5)), 145.7 (C(10a)), 128.0 (C(1)), 123.8 (C(7)), 122.0 (C(8a)),
116.4 (C(8)), 115.9 (C(6)), 114.4 (C(2)), 113.9 (C(9a)), 102.24 (C(4)),
55.2 (MeO-C(5)).
2,3-Dimethoxyxanthone (14) 61%yield. 1H-NMR ((D6)DMSO) d:
8.16 (dd, J = 7.9, 1.5, H-C(8)), 7.82 (ddd, J = 8.4, 7.4, 1.5, H-C(6)),
7.60 (d, J = 8.4, H-C(5)), 7.49 (s, H-C(1)), 7.44 (ddd, J = 7.9, 7.4,
0.9 H-C(7)), 7.20 (s, H-C(4)), 3.94 (s, MeO-C(3)), 3.87 (3H, s, MeO-
C(2)), 13C-NMR ((D6)DMSO): 174.8 (C(9)), 155.7 (C(10a)), 155.6
(C(4a)), 152.0 (C(3)), 146.7 (C(2)), 134.8 (C(6)), 125.9 (C(8)), 124.3
(C(7)), 120.9 (C(8)), 118.0 (C(5)), 114.0 (C(9a)), 104.7 (C(1)), 100.5
(C(4)), 56.6 (MeO-C(3)), 55.9 (MeO-C(2)).
1,3-Dimethoxyxanthone (21) 30%yield. 1H-NMR ((D6)DMSO) d:
8.06 (dd, J = 7.7, 1.7, H-C(8)), 7.75 (ddd, J = 7.9, 7.0, 1.8, H-C(6)),
7.51 (dd, J = 7.9, 1.8, C(5)), 7.39 (ddd, J = 7.9, 7.0, 1.8, H-C(7)),
6.69 (d, J = 2.2, C(4)), 6.50 (d, J = 2.2, H-C(2)), 3.90 (s, MeO-C(1)),
3.86 (s, MeO-C(3)). 13C-NMR ((D6)DMSO, 200 MHz): 180.0 (C.O),
164.8 (C(3)), 161.5 (C(1)), 159.1 (C(4a)), 154.3 (C(10a)), 134.3 (C(6)),
125.9 (C(8)), 124.1 (C(7)), 122.4 (C(8a)), 117.2 (C(5)), 107.9 (C(9a)),
95.4 (C(2)), 93.2 (C(4)), 56.2 (MeO-C(1)), 56.0 (MeO-C(3)).
3,4-Dimethoxyxanthone (15) 61%yield. 1H-NMR ((D6)DMSO) d:
8.16 (dd, J = 7.9, 1.7, H-C(8)), 7.94 (d, J = 9.0, H-C(1)), 7.85 (ddd,
J = 8.1, 7.5, 1.7, H-C(6)), 7.69 (d, J = 8.1, H-C(5)), 7.47 (dd, J = 7.9,
7.5, H-C(7)), 7.26 (d, J = 9.0, H-C(2)), 3.97 (s, MeO-C(3)), 3.92 (s,
Me-C(4)). 13C-NMR ((D6)DMSO): 175.3 (C(9)), 157.5 (C(3)), 155.6
(C(10a)), 149.9 (C(4a)), 135.9 (C(4)), 135.2 (C(6)), 125.9 (C(8)), 124.4
2,7-Dimethoxyxanthone (22) 40%yield. 1H-NMR ((D6)DMSO) d:
7.45 (dd, J = 9.0, 3.0, H-C(3) and H-C(6)), 7.33 (d, J = 9.1, H-4 and
214
Chem Biol Drug Des 2011; 77: 212–222

Antifungal Activity of Xanthones
H-5), 7.80 (d, J = 9.0, H-C(1) and H-C(8)), 3.87 (s, MeO-C(2), MeOH-
C(7)). 13C-NMR ((D6)DMSO): 175.5 (C(9)), 155.6 (C(2), C(7)), 150.3
(C(4a) C(10a)), 124.7 (C(3), C(6)), 120.8 (C(8a), C(9a)), 119.8 (C(1),
C(4)), 55.7 (MeO-C(2), MeOH-C(7)).
A. fumigatus), and 7 days (dermatophytes). MICs were determined
as the lowest concentrations capable of substantially inhibiting fun-
gal growth in comparison with controls. All results are from three
independent and concordant experiments, performed in duplicate. In
addition, the antifungal compound fluconazole was used as the
standard antifungal drug. Twofold serial dilutions ranging from 3.9
to 250 lg mL)1 were used. Quality control determinations of the
fluconazole MICs were ensured by testing C. parapsilosis ATCC
90018. The results obtained were within the recommended limits
(data not shown).
Synthesis of 3,6-dihydroxyxanthone (23)
The heating of 2,2¢,4,4¢-tetrahydroxybenzophenone (28, 500 mg;
2 mmol) at 180 ꢀC, in a furnace, overnight, afforded the pure prod-
uct.
23. Brown solid. 85% yield. Mp > 330/C; IR (KBr): 3383; 3135;
1
1610, 1579, 1454; 1251; 1170 cm)1; H-NMR ((D6)DMSO) d: 10.88
Sterol extraction
(s, OH-C(3) and OH-C(6)), 8.00 (d, J = 8.7, H-C(1) and H-C(8)), 6.88
(dd, J = 8.7, 2.2, H-C(2) and H-C(7)), 6.84 (d, J = 2.2, H-C(4) and H-
C(5)). 13C NMR ((D6)DMSO): 174.0 (C(9)), 163.4 (C(3), C(6)), 157.5
(C(4a), C(10a)), 127.8 (C(1), C(8)), 114.0 (C(8a), C(9a)), 113.7 (C(2),
Cell suspensions of the different strains of fungi were prepared on
distilled water from recent cultures on SDA with chloramphenicol,
at a density of 106 CFU mL)1. Fifty microliters of these cell suspen-
sions was diluted in RPMI-1640 medium, yielding a final inoculum
size of 103 CFU mL)1. Several twofold dilutions of xanthones 10
and 13 and fluconazole were prepared and added to the cell sus-
pensions. Cultures were incubated with shaking at 35/C (for C. albi-
cans, C. neoformans, and A. fumigatus) or 25 ꢀC (for
dermatophytes) during 48 h (C. albicans), 72 h (C. neoformans and
A. fumigatus), and 7 days (dermatophytes). A quantification of
ergosterol amount was performed after incubation with xanthones
10 and 13 or fluconazole and without as control, at different con-
centrations.
+
C(7)), 102.1 (C(4), C(5)). HRMS-FAB+ m ⁄ z calcd for C13H8O4 :
229.0501, found: 229.0504.
Microorganisms
The antifungal activity of the xanthones was evaluated against Can-
dida, Cryptococcus, Aspergillus, and dermatophyte strains: clinical
isolates (C. glabrata D10R, from recurrent cases of oral candidosis;
Cryptococcus neoformans PH1, from cerebrospinal fluid; Microspo-
rum canis FF1, M. gypseum FF3, Trichophyton mentagrophytes FF7,
T. rubrum FF5, and Epidermophyton floccosum FF9, all isolated from
nails and skin) and ATCC (American Type Culture Collection) type
strains (C. albicans ATCC 10231 and Aspergillus fumigatus ATCC
46645). Strains were stored in Sabouraud dextrose broth with glyc-
erol, at )70 ꢀC. To ensure optimal growth, they were subcultured
twice in Sabouraud dextrose agar (SDA) with chloramphenicol (Bio-
Mꢀrieux) prior to testing and the cultures were incubated during
24 h (for Candida spp.), 48 h (for C. neoformans and A. fumigatus),
and 7 days for dermatophytes.
The sterol extraction was adapted from Arthington-Skaggs et al.
(56). Briefly, fungal cells were harvested by centrifugation (C. albi-
cans and C. neoformans) at 980· g for 5 min or by filtration (A. fu-
migatus and T. mentagrophytes), and the pellets were washed
twice with sterile distilled water. The wet pellet weight was
adjusted to 0.100 g, and 3 mL of 25% alcoholic potassium hydrox-
ide solution was added, followed by a vigorous agitation in a vortex
for 1 min. Cell suspensions were incubated in a water bath at
85 ꢀC during 60 min. Following incubation, tubes were removed
and left to cool at room temperature. Sterols were then extracted
by addition of 1 mL of sterile distilled water and 3 mL of n-heptane
(Romil Chemicals, Leics., England) to each tube, followed by a vigor-
ous vortex agitation for 3 min. The organic phase (n-heptane) was
then transferred to a clean glass tube, and the n-heptane was
evaporated to dryness, under a nitrogen stream. The extracted ster-
ols were redissolved in 1 mL of dichloromethane (Merck) prior to
HPLC-UV analysis.
Susceptibility testing
Broth microdilution methods based on the CLSI (formerly NCCLS)
reference documents M27A-2 (54) and M38-A (55) for yeasts and
filamentous fungi, respectively, with minor modifications, were used
to determine minimum inhibitory concentrations (MIC).
Briefly, cell or spore suspensions were prepared from recent cul-
tures on SDA with chloramphenicol of the different strains of fungi
and diluted to final inoculum sizes of 103 CFU (colony forming
units) mL)1 in RPMI-1640 broth (Sigma), with glutamine, without
bicarbonate and with phenol red as the pH indicator, buffered to
pH 7.0 with MOPS (Sigma). Serial twofold dilutions of each xan-
thone (1–27) in DMSO (Sigma) were prepared over the range 3.9–
250 lg mL)1. Testing was performed in 96-well round-bottom mic-
rotitration plates. Two growth controls, using test medium alone
and with 2.0% (v ⁄ v) DMSO, and a sterility control (drug-free med-
ium only and medium with the xanthones) were included in all
assays. The plates were incubated at 35 ꢀC (for C. albicans,
C. glabrata, C. neoformans, and A. fumigatus) or 25 ꢀC (for derma-
tophytes) during 48 h (Candida spp.), 72 h (C. neoformans and
Sterol analysis
Ergosterol was analyzed by HPLC with ultraviolet (UV) detection
based on the method proposed by Peacock and Goosey (57). The
chromatography was performed with a system consisting of two
high-pressure pumps (Jasco 880-PU intelligent HPLC pump I), a
manual injector (rheodyne 7125), and a spectrophotometer detector
(Jasco 875-UV intelligent UV ⁄ Vis). The column was stainless steel
(250 · 4.6 mm), packed with Hypersil silica 3 lm (Hichrom). A
guard column (10 · 4.6 mm) filled with Hypersil silica 5 lm was
used. The mobile phase was a solution of methanol (Merck) in di-
chloromethane 0.025% (v ⁄ v), and the flow rate was 1.0 mL min)1
with an injection volume of 100 lL and a race time of about
Chem Biol Drug Des 2011; 77: 212–222
215

Pinto et al.
20 min. Detection was performed at 245 nm. Methanol, dichlorome-
thane, and n-heptane were HPLC grade. Ergosterol (E) and lanos-
terol (L) (Sigma) were analytical grade.
the previously described procedures (60,61) because no purification
is needed to furnish compound 23 in quantitative yields.
Antifungal activity
Validation procedure
To investigate the antifungal activity of simple oxygenated xanthon-
es, 8 monosubstituted, 16 disubstituted, and 2 trisubstituted xant-
hones along with the xanthone (1) were evaluated for their growth
inhibitory effect against three yeasts (C. albicans, C. glabata,
C. neoformans), a non-dermatophyte filamentous fungi (A. fumiga-
tus), and five dermatophyte filamentous fungi (M. gypseum,
M. canis, E. floccosum, T. mentagrophytes, and T. rubrum). The
results (oxygenated xanthones 1–27) are presented in Table 1.
The method was validated by ICH described parameters (58). Linear-
ity was evaluated in triplicates of at least five calibration standard
solutions of ergosterol. The regression line was calculated as
y = a + bx, by plotting analyte concentration (x), namely ergosterol
(E) (expressed as mg ⁄ 100 g wet fungal cells), versus average peak
area (y) (expressed as mAU). The calibration curves were obtained
using the linear least squares regression procedure. The RSD (rela-
tive standard deviation) values for the response factors of three
experiments were calculated. For intraday precision, three concen-
trations of the extracted sterols were analyzed within 24 h and
injected at least five times. For intermediate precision, samples
from three concentrations of the extracted sterols were analyzed on
five consecutive days assayed in quintuplicate. Precision was
expressed as RSD. The limits of detection (LOD) and quantification
(LOQ) were evaluated by analyzing ten blank samples. The specific-
ity of the analytical method in this study was determined by the
analysis of two other sterols involved in the ergosterol pathway:
lanosterol (early sterol pathway intermediate) and 24 (28) dehydr-
oergosterol (late sterol pathway intermediate) which absorb in the
same k values. Under the applied conditions, no interference from
these mycosterols was observed at the retention time for ergos-
terol. Recovery studies were performed using the minimum, maxi-
mum, and an intermediary concentration; accuracy was determined
by spiking three known concentrations of ergosterol (9.6, 96,
192 lg mL)1) obtained from the test samples.
Xanthone (1) did not inhibit the growth of the examined strains
even when tested at concentrations higher than 250 lg mL)1. How-
ever, introduction of oxygenated substituents on the xanthone
nucleus has led to the appearance of an inhibitory effect
(MIC<250 lg mL)1). Xanthones 3–6,10–12,16,19,23,25–27 were
found to inhibit the growth of the fungal strains tested (Table 1).
Although the majority of these xanthones did not show a very
strong activity, some of them (3–5,10,12) exhibited striking inhibi-
tory effects with MIC values <10 lg mL)1, while compounds 1–
2,7–9,13–15,17,18,20–22,24 were found to be inactive against
all the tested organisms (MIC>250 lg mL)1).
From the results shown in Table 1, it was found that except for
1,7-dihydroxyxanthone (25) that was selective to E. floccosum
(MIC = 15.6 lg mL)1) and for 3-hydroxy-4-methoxyxanthone (16),
all the other active xanthones (3–6,10–12,19,23,26–27) inhibited
the growth of the five dermatophyte filamentous fungi, with MIC
values ranging from 7.8 to 250 lg mL)1. They were, in general,
more active against E. floccosum (7.8–125 lg mL)1) and less active
against T. rubrum (15.2–250 lg mL)1). 4-Hydroxy-(5) and 1,2-di-
hydroxyxanthone (10) also inhibited C. neoformans (MIC =
Results
Chemistry
Among the investigated xanthones (1–27), four are from higher
plants of the Clusiacea family: 2-hydroxy-1-methoxyxanthone (24)
(41), 1,7-dihydroxyxanthone (euxanthone, 25), 2-hydroxy-1,8-dimeth-
oxyxanthone (26) (52), and 1,2,8-trimethoxyxanthone (27) from Calo-
phyllum teysmannii var. inophylloide (52). The majority of the
oxygenated xanthones (2–22) were obtained by classical methods
via benzophenone or a biphenyl ether intermediates (59), and their
syntheses are described elsewhere (38,49,53). In this study, a one
pot synthesis for 3,6-dihydroxyxanthone (23) was performed through
a dehydrative cyclization of the commercially available 2,2¢,4,4¢-tet-
rahydroxybenzophenone (28, Figure 2). This method is preferred to
31.3 lg mL)1) and A. fumigatus (MIC = 62.5 and 31.3 lg mL)1
,
respectively); however, only compound 10 was active against
Candida species (MIC = 31.3 lg mL)1).
Sterol content
Sterols were extracted from cultures of four fungal strains (C. albi-
cans, C. neoformans, A. fumigatus, and T. mentagrophytes) and ana-
lyzed by normal-phase HPLC. Separations of sterols were optimized
for the described experimental conditions used in the validation
procedure. The assay was successfully validated with respect to
Figure 2: Synthesis of 3,6-dihydroxyxanthone (23) from 2,2¢,4,4¢-tetrahydroxybenzophenone (28).
216
Chem Biol Drug Des 2011; 77: 212–222

Antifungal Activity of Xanthones
a
Table 1: Antifungal activity (MIC , lg mL)1) of 27 xanthones and fluconazole against Candida, Cryptococcus, Aspergillus, and dermato-
phyte strainsb
Xanthone
R8
O
O
R1
R4
R7
R6
R2
R3
R5
No
C. albicans C. glabrata C. neoformans A. fumigatus M. gypseum M. canis E. floccosum T. mentagrophytes T. rubrum
1
2
3
4
5
6
7
8
9
Xanthone
1-hydroxy
2-hydroxy
3-hydroxy
4-hydroxy
1-methoxy
2-methoxy
3-methoxy
4-methoxy
>250
>250
>250
>250
>250
>250
>250
>250
>250
31.3
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
31.3
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
64
>250
>250
>250
>250
31.3
>250
>250
>250
>250
31.3
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
32
>250
>250
>250
>250
62.5
>250
>250
>250
>250
31.3
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>128
>250
>250
15.6
15.6
15.6
31.3
>250
>250
>250
7.8
>250
>250
15.6
250
15.6
31.3
>250
>250
>250
15.6
31.3
15.6
>250
>250
>250
>250
>250
>250
125
>250
>250
>250
250d
>250
>250
125
125
8
>250
>250
7.8
>250
>250
7.8
>250
>250
15.6
250
7.8
7.8
7.8
15.6
31.3
>250
>250
>250
7.8
31.3
62.5
>250
>250
>250
31.3
31.3
31.3
>250
>250
>250
>250
>250
>250
125
>250
>250
>250
250d
>250
>250
125
125
0.5
31.3
>250
>250
>250
15.6
15.6
7.8
10 1,2-dihydroxy
11 2,3-dihydroxy
12 3,4-dihydroxy
13 1,2-dimethoxy
14 2,3-dimethoxy
15 3,4-dimethoxy
16 3-hydroxy -4-methoxy
17 4-hydroxy -3-methoxy
18 3,5-dihydroxy
31.3
15.6
>250
>250
>250
125
>250
>250
125
>250
>250
>250
125d
>250
>250
125
125
4
31.3
31.3
>250
>250
>250
125
>250
>250
125
>250
>250
>250
250d
>250
>250
125
62.5–125
2
250
>250
>250
31.3
>250
>250
62.5
>250
>250
>250
250d
>250
15.6
125
62.5
2
19 3,5-dimethoxy
20 3-hydroxy-5-methoxy
21 1,3-dimethoxy
22 2,7-dimethoxy
23 3,6-dihydroxy
24 2-hydroxy-1-methoxyc
25 1,7-dihydroxyc
26 2-hydroxy-1,8-dimethoxyc >250
27 1,2,8-trimethoxyc
>250
64
fluconazole
aResults are expressed as MIC (minimal inhibitory concentration) in lg mL)1 and show means of three independent observations made in duplicate.
bIsolate sources: C. glabrata D10R, from recurrent cases of oral candidosis; Cryptococcus neoformans PH1, from cerebrospinal fluid; Microsporum canis FF1,
M. gypseum FF3, Trichophyton mentagrophytes FF7, T. rubrum FF5, and Epidermophyton floccosum FF9, all isolated from nails and skin and ATCC (American Type
Culture Collection) type strains (C. albicans ATCC 10231 and Aspergillus fumigatus ATCC 46645).
cIsolated from natural sources.
dShowed a slight inhibitory effect at this concentration against these strains; MICs were not determined.
specificity, linearity (y = )54.3 + 16.0x; R = 0.998; relative standard
deviation RSD £ 3.5%), range, precision (RSD £ 7.5%), and limits
of detection (LOD = 0.71 lM) and quantification (LOQ = 2.15 lM),
according to ICH guidelines (58). Recovery data were within the
range of 61.2–75.9%.
obtained for controls, compound 10 and fluconazole. For compound
13, a relationship between xanthone concentration and amount of
ergosterol was not observed (data not shown). For 1,2-dihydroxyx-
anthone (10), the relationship between concentration and amount
of ergosterol is visible for all the fungi studied.
Ergosterol (E, tR 17.8–18.2 min) and lanosterol (L, tR 10.5–
10.7 min) were detected at their maxima absorption (245 nm), and
compounds were identified by coinjection with real standards. Both
compounds were well separated, without interferences, in all sam-
ples. This method was applied to the analysis of the ergosterol
content from fungal cells treated with different concentrations of
1,2-dihydroxyxanthone (10), which is active against all the tested
fungi, and its dimethoxylated analog 1,2-dimethoxyxanthone (13),
which is inactive against all tested fungi and fluconazole, a well-
known inhibitor of ergosterol biosynthesis. A model of chromato-
grams is presented in Figure 3, and Table 2 displays the results
The effect of 1,2-dihydroxyxanthone (10) on ergosterol level varies
with its concentrations and the tested organisms. In C. albicans
and C. neoformans, the lowest concentration (3.9 lg mL)1) of 1,2-
dihydroxyxanthone (10) does not appear to show a difference or, if
anything, a slightly higher level of ergosterol, while at higher con-
centrations (7.8 and 15.6 lg mL)1), ergosterol levels appear to be
lower than those of control. On the contrary, ergosterol levels in
A. fumigatus were higher than those of the control for all concen-
trations (3.9, 7.8, and 15.6 lg mL)1) of 1,2-dihydroxyxanthone (10).
On the other hand, T. mentagrophytes was found to be more sensi-
tive to 1,2-dihydroxyxanthone (10), which was able to inhibit ergos-
Chem Biol Drug Des 2011; 77: 212–222
217

Pinto et al.
Figure 3: HPLC chromatograms obtained from extracts of cells of T. mentagrophytes A) untreated or B) treated with 3.9 lg mL)1, C)
1.9 lg mL)1, and D) 0.98 lg mL)1 of 1,2-dihydroxyxanthone (10). L = lanosterol, E = ergosterol. Conditions: silica: methanol in dichlorome-
thane 0.025% (v ⁄ v), 1.0 mL min)1
.
terol synthesis at a concentration of 3.9 lg mL)1 (Table 2). At
lower concentrations (1.95 and 0.98 lg mL)1), the levels of ergos-
terol were found to be higher than those of the control.
to investigate a series of simple oxygenated xanthones for their
potential antifungal properties.
All dermatophytes investigated were found to be sensitive to fluco-
nazole (which is in accordance with CLSI guidelines), and results
revealed that, among the 27 xanthones tested, the number of
xanthones capable of inhibiting their growth is much higher than
that for the non-dermatophytes group (Table 1). Among the investi-
gated xanthones, 1,2-dihydroxyxanthone (10) showed a broad spec-
trum of activity, being active against all the nine fungal strains
tested.
For fluconazole, the relationship between concentration and amount
of ergosterol is visible for all the fungi under study. All concentra-
tions (0.5, 1.0, 2.0 lg mL)1) of fluconazole gave lower ergosterol
levels than those of the controls in C. albicans, while this happened
only at the concentrations of 1.0 and 2.0 lg mL)1 in C. neoformans
(Table 2). Only at a concentration as high as 128 lg mL)1 of fluco-
nazole, the amount of ergosterol was lower than that in the control
for A. fumigatus. T. mentagrophytes was most sensitive to fluconaz-
ole as well, showing lower ergosterol contents than in the control
when incubated with fluconazole at the concentrations of 0.125 and
Interestingly, with the exception of compounds 5 and 10, some
selectivity toward dermatophyte filamentous fungi was observed,
with compounds 3,4,6,11,12,16,19,23,25–27 showing no activ-
ity against Candida species, C. neoformans, or A. fumigatus even at
the maximum concentrations tested (250 lg mL)1). In contrast,
compounds 5 and 10 were active in the same range of concentra-
tions as fluconazole to C. neoformans and were found to inhibit the
growth of A. fumigatus (MIC = 62.5 and 31.3 lg mL)1, respec-
tively), which displays the lowest susceptibility to fluconazole
(MIC > 128 lg mL)1). It is also interesting to point out that xan-
thone (1), 2-hydroxy-1-methoxyxanthone (24), and 1,7-dihydroxy-
xanthone (25) did not show any growth inhibitory activity against
C. albicans, C. glabrata, or C. neoformans (Table 1), these results
being in agreement with those reported previously for these com-
pounds (8,10,12,20). Although compound 24 has been previously
reported as responsible for the antifungal activity of the extract of
Kielmeyera coriacea against Cladosporium cucumerinum (12), it
showed no antifungal activity against all the nine fungal strains
0.25 lg mL)1
.
Discussion
Plants present a unique pool of compounds in the search for new
antifungal lead structures because of the variety and chemical com-
plexity of their constituents. Previous studies have reported some
xanthone derivatives as remarkable antifungal agents. The antifun-
gal profile of the described xanthones suggests that, in the majority
of cases, hydroxyl groups are important for activity (8,13,62,63).
However, because of biosynthesis limitations of these natural prod-
ucts, the pattern of oxygenation is most frequently restricted to
positions 1,3,5,6 for simple oxygenated and prenylated xanthones
and to 1,4,8 for polycyclic and dehydroxanthones. These facts, allied
with our experience in this class of compounds, have prompted us
218
Chem Biol Drug Des 2011; 77: 212–222

Antifungal Activity of Xanthones
Table 2: Ergosterol content (mg ⁄ 100 g wet fungal cells) from fungal cells treated with different concentrations of 1,2-dihydroxyxanthone
(10) and fluconazole
Compound
Ergosterol
Fungi strains
Compound
concentration (lg mL)1
)
concentration € SDa
Candida albicans
Control
10
0.0
15.6
7.8
3.9
2.0
25.80 € 7.31
15.94 € 4.61
20.34 € 2.83
26.60 € 2.17
5.88 € 3.04
Fluconazole
1.0
7.32 € 2.56
0.5
0.0
15.6
7.8
3.9
2.0
1.0
0.5
0.0
15.6
7.8
3.9
128
32
8
15.86 € 2.32
23.22 € 0.24
20.00 € 5.58
22.96 € 3.79
29.58 € 8.30
15.24 € 1.77
21.48 € 2.55
27.39 € 1.01
39.30 € 7.54
39.97 € 7.61
43.41 € 10.85
54.42 € 4.23
29.02 € 6.13
68.96 € 7.80
70.59 € 6.83
43.44 € 8.16
16.84 € 0.61
43.79 € 7.25
56.55 € 15.28
30.23 € 1.25
37.24 € 3.85
79.07 € 4.52
Cryptococcus neoformans
Aspergillus fumigatus
Control
10
Fluconazole
Control
10
Fluconazole
Trichophyton mentagrophytes
Control
10
0.0
3.9
1.95
0.98
0.25
0.125
0.063
Fluconazole
aResults are presented as mg ⁄ 100 g wet fungal cells and show means of three independent analyses € SD.
investigated here (Table 1). Also, 2-hydroxyxanthone (3) was previ-
ously described to inhibit A. fumigatus with a MIC value of
31 lg mL)1 (64). Various factors may contribute to this discrepancy,
namely different pH conditions (65).
(19) and 1,2,8-trimethoxyxanthone (27) showed only a mild inhibi-
tory effect against dermatophytes. Oddly enough, the only active
methoxyxanthone was 1-methoxyxanthone (6), possibly because of
the close proximity of the methoxyl group with the carbonyl group.
In this study, antifungal activity profiles of simple oxygenated xant-
hones and the correlation with their structures also suggest that
free hydroxyl groups in one ring of the xanthone nucleus are impor-
tant for optimal activity, as observed for monohydroxyxanthones 3–
5 and for dihydroxyxanthones 10–12. The quinone substructure
has been frequently characterized in compounds having strong anti-
candidal activity. A keto-enol tautomerism-based formation of a
reactive quinone methide intermediate was previously described to
explain the antifungal activity of flavonoids (65). In a similar way,
Interestingly, Gopalakrishnan et al. (8) have suggested, from the cor-
relation of the antifungal activity profiles and the structures of xan-
thone (1) and euxanthone (25), that the presence of hydroxyl
groups in rings A and B was important for the antifungal activity.
Furthermore, the nature of the substituents seems to influence the
growth inhibitory effect of xanthones (Table 1). Thus, most mono-
(7–9) and dimethoxylated (13–15,21,22) xanthones were found to
be inactive against the test fungi, while 3,5-dimethoxyxanthone
A
B
Figure 4: Qualitative structure–activity relationship for antifungal activity most favorable molecular substitutions for (A) prenylated (7,13)
and (B) simple oxygenated xanthones.
Chem Biol Drug Des 2011; 77: 212–222
219

Pinto et al.
the ability of hydroxyxathones to form quinone substructures (66,67)
may explain the fact that the highest effect was observed for com-
pounds 3–5 and 10–12. These findings support the data from the
recently reported antifungal activity of prenylated xanthones iso-
lated from Cratoxylum cochinchinense, in which only compounds
with a catechol moiety exhibited strong activity (11). The absence
of the effect observed for 1-hydroxyxanthone (2) can be justified by
the formation of hydrogen bonding between the carbonyl and hydro-
xyl groups at C-1. In contrast, hydroxylation in both rings (A and B)
of the xanthone nucleus does not favor the antifungal activity, as
can be observed for compounds 18, 23, and 25. Additionally, it
can be affirmed that monomethylation of the catechol moiety (in
position 2 for 24 with respect to 10 and in position 3 for 17 with
respect to 12) could be the reason for the loss of the antifungal
activity. However, this effect was less pronounced for position 4
(from the observation of 12 and 16). Opinions diverge concerning
the effect of hydroxyl groups on the antifungal activity of xanthon-
es. While some authors have stated that only hydroxyl groups in
the xanthone rings could increase the antifungal activity (13), fur-
ther suggesting that hydroxyl groups in the side chains did not
seem to affect the activity, others (63) reported as likely that anti-
fungal xanthones from plants required three or four hydroxyl groups,
in which one or two of them must be at C-5 (C-4) and ⁄ or C-6 (C-3),
and a hydrophobic group must be on one of the aromatic rings as
illustrated in Figure 4A. Herein, some relationships previously estab-
lished for anticandidal flavonoids (65) and prenylated xanthones can
be extrapolated to xanthones (Figure 4B): a hydroxyl group in posi-
tion 3 and ⁄ or 4 seems to be favorable and a keto group must be
present in position 9. Additionally, a catechol group is important for
activity, and substitution of hydroxyl by methoxyl groups is associ-
ated with the decrease in the activity.
influence their antifungal inhibitory effect. In fact, inhibitors of fun-
gal iron acquisition may provide attractive antifungal therapies
(69,70), and this hypothesis deserves to be explored in the future.
Conclusion
In conclusion, the investigation into the antifungal activity of 27
simple oxygenated xanthones has led us to obtain some interesting
structure–activity relationships for this class of compounds. The
simple, rapid, and efficient HPLC-UV method for the identification
and quantification of fungal sterols in whole cells of Candida, Cryp-
tococcus, Aspergillus, and dermatophyte strains has been developed
and validated, and this method can be used as a tool to evaluate
the antifungal activity of the compounds that inhibit the ergosterol
biosynthesis. 1,2-Dihydroxyxanthone (10) was identified as a valu-
able scaffold for further development of more complex and diverse
xanthone derivatives for broad-spectrum antifungal activity.
Acknowledgments
This work was supported by Fundażo para a CiÞncia e a Tecnolo-
gia (FCT) grant I&D, n/ 4040 ⁄ 07, FEDER and POCI. Elisangela Costa
and Luꢁs Vale-Silva acknowledge FCT for their respective PhD
(SFRH ⁄ BD ⁄ 30615 ⁄ 2006) and Postdoctoral (SFRH ⁄ BPD ⁄ 29112 ⁄ 2006)
grants.
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