CHEMISTRY & BIODIVERSITY Vol.

12 (2015) 133

Acremoxanthone E, a Novel Member of Heterodimeric Polyketides with a

Bicyclo[3.2.2]nonene Ring, Produced by Acremonium camptosporum W.
Gams (Clavicipitaceae) Endophytic Fungus
by Claudio Melendez-Gonzalez a ), M. Jordi Muria-Gonzalez a ), Ana Luisa Anaya b ), Blanca E.
Hernandez-Bautista b ), Simon Hernandez-Ortega c ), Mara C. Gonzalez d ), Anthony E. Glenn e ),
Richard T. Hanlin f ), and Martha L. Macas-Rubalcava* a )
a
) Instituto de Qumica, Departamento de Productos Naturales, Ciudad Universitaria, Coyoacan, D.F.,
04510, Mexico (phone: 52-55-56 22 44 32; fax: 52-55-56 16 22 17; e-mail: mlmacias@unam.mx;
mamaciasr@gmail.com)
b
) Instituto de Ecologa, Departamento de Ecologa Funcional, Universidad Nacional Autonoma de
Mexico (UNAM), Ciudad Universitaria, Coyoacan, D.F., 04510, Mexico
c
) Laboratorio de Rayos X, Universidad Nacional Autonoma de Mexico (UNAM), Ciudad
Universitaria, Coyoacan, D.F., 04510, Mexico
d
) Instituto de Biologa, Departamento de Botanica, Laboratorio de Micromicetos, Universidad
Nacional Autonoma de Mexico (UNAM), Ciudad Universitaria, Coyoacan, D.F., 04510, Mexico
e
) USDA, ARS, Toxicology & Mycotoxin Research Unit, Russell Research Center, Athens,
Georgia 30604, USA
f
) University of Georgia, Museum of Natural History Annex, 4435 Atlanta Highway, Bogart,
Georgia 30622, USA

Bioactivity-directed fractionation of the organic mycelium extract of the endophytic fungus

Acremonium camptosporum W. Gams (Clavicipitaceae), isolated from the leaves of Bursera simaruba
(Burseraceae), led to the isolation of six major heterodimeric polyketides, including one not previously
characterized acremoxanthone derivative. In addition, the already known acremoxanthone C, acre-
monidins A and B, and acremoxanthones A and B were obtained. The structure of the new compound
was established by extensive NMR studies, including DEPT, COSY, NOESY, HSQC, and HMBC
methods. The trivial name proposed for this compound is acremoxanthone E. In addition, the structure of
acremoxanthone C was unequivocally established for the first time, through X-ray crystal-structure
analysis. The anti-oomycete activities of the pure compounds were tested against four economically
important phytopathogenic oomycetes. Inhibitory concentration for 50% diameter growth reduction,
IC50 , values for the four phytopathogens ranged from 6 to 38 mm. Also, in parallel, the cytotoxic activities
against six cancer cell lines were evaluated showing IC50 values similar to those of cisplatin. To the best of
our knowledge, this is the first report on three different groups of heterodimeric polyketides, linked by a
bicyclo[3.2.2]nonene, such as xanthoquinodins, acremonidins, and acremoxanthones, which are isolated
from an endophytic fungus. In addition, a common biosynthetic origin could be proposed.

Introduction. In recent years, research on natural products from fungi has

revealed that secondary metabolite production is not a random phenomenon, but
rather that there is close correlation with the ecological niche of the producer fungus
[1] [2]. In addition, it is widely accepted that fungal secondary metabolites (SMs) play a
key role as defensive or offensive strategies for survival in their niche [3], such as
phytotoxins produced by several fungal plant pathogens or insecticide compounds
produced by entomopathogenic fungi. In this manner, the application of fungal ecology
in the search for new bioactive natural products is extremely attractive [4].

 2015 Verlag Helvetica Chimica Acta AG, Zrich

134 CHEMISTRY & BIODIVERSITY Vol. 12 (2015)

Fungal diversity is five times greater than vegetal diversity and fifty times greater
than bacterial diversity. Fungi are the second major producers of SMs, after
actinomycetes [4], and, given that only 5% of fungal diversity has been described
[5], fungi represent a promising alternative source of useful compounds in medicine
and agriculture [6]. In particular, endophytic fungi, these microscopic inhabitants
hidden inside vegetal tissues, have recently been recognized as a new tool in the
production of bioactive SMs [7].
As part of our search for bioactive compounds deriving from Mexican biodiversity,
we previously reported the discovery of novel fungal genus and species associated with
plants from the El Eden Ecological Reserve in the state of Quintana Roo, Mexico.
These endophytic fungi also produce some novel SMs [8] [9]. In the present study, we
describe the isolation, structure elucidation, and biological activity of major com-
pounds from the mycelium organic extract of the endophytic fungus Acremonium
camptosporum isolated from the leaves of Bursera simaruba (Burseraceae): six
bioactive heterodimeric polyketides; one novel acremoxanthone analog (1), and five
known compounds, 2 6. In addition, the structure of acremoxanthone C (2) was
determined unequivocally by X-ray crystallography. These compounds exhibit anti-
oomycete and cytotoxic activities. On the other hand, we discuss a possible biosynthetic
link among xanthoquinodins, acremonidins, and acremoxanthones.

Results and Discussion. 1. GenBank Numbers to DNA Sequence Submissions. The

morphological and molecular analyses indicated that culture B101 is a member of the
Clavicipitaceae family (Ascomycota) belonging to Acremonium camptosporum W.
Gams. The sequences of ITS rDNA/28S rDNA regions were deposited with GenBank
under accessions Nos. JQ811555/JQ811556.
2. Anti-Oomycete and Antifungal Activities of the Endophyte Mycelium Extract.
Acremonium camptosporum was cultivated using fermentation in Potato dextrose
liquid (PD) medium under static conditions. Antimicrobial activity of the mycelium
extract was preliminarily evaluated against the following four economically important
 CHEMISTRY & BIODIVERSITY Vol. 12 (2015) 135

phytopathogens: two fungoid oomycetes, Phytophthora parasitica and Phytophthora

capsici, and two true fungi, Fusarium oxysporum and Pestalotiopsis sp. The mycelium
extract significantly inhibits the growth of the two oomycetes, reducing, in general,
radial growth by 50% to 100 mg/ml; however, neither of the two true fungi were
significantly inhibited. These results showed strong bioactivity against oomycetes (data
not shown); thus, the selective anti-oomycete activity of the mycelium was subse-
quently explored against other phytopathogenic oomycetes using the commercial anti-
oomycete metalaxyl as positive control. Four Pythium species, five Phytophthora
species, and two true fungi, Fusarium oxysporum and Pestalotiopsis sp. were selected
for bioassays. The mycelium extract was more active against the nine oomycetes than
against the true fungi. Pythium aphanidermatum, Phytophthora capsici, and Phytoph-
thora cinnamomi were the most inhibited species, followed by Phytophthora parasitica,
Pythium ultimum, and Phytophthora cactorum (Table 1).
3. Isolation of Bioactive Compounds. Bioassay-guided fractionation of the mycelial
extract from endophytic fungus Acremonium camptosporum led to the isolation of six
heterodimeric polyketides. These compounds included one new acremoxanthone
derivative, which was characterized by spectroscopic and spectrometric methods. The
trivial name acremoxanthone E was proposed for the new oxanthrone-hydroxanthone
heterodimer 1. In addition, the already known acremoxanthone C (2), acremonidins A
and B (3 and 4, resp.), and acremoxanthones A and B (5 and 6, resp.) were obtained.
4. Structure Elucidation of Compounds. Acremoxanthone E (1) was obtained as
yellow amorphous solid, and the molecular formula of 1 was determined as C31H24O11
by ESI-MS (m/z 627.2 ([M MeOH Na] ; calc. 627.1472), 595.2 ([M Na] ; calc.
595.1210), implying an unsaturation index of 20. The NMR spectra were recorded in
(D6 )acetone and later in CDCl3 , because the chemical shift of the tautomeric C-atom
C(6) was unusual (upfield shift; Tables 2 and 3). According to Meyers rule, the
tautomeric equilibrium is shifted in the direction of the keto form with increasing

Table 1. Radial Growth-Inhibitory Activity ( IC50 [mg/ml]) of the Mycelial Extracts from A. camptospo-
rum against Phytopathogenic Microorganisms

Microorganism IC50 [mg/ml] a )

Mycelium extract Metalaxyl
b
Pythium ultimum ) 166.1 296.9
Pythium debaryanum b ) 371.0 531.0
Pythium polytylum b ) 363.2 513.7
Pythium aphanidermatum c ) 36.7 0.05
Phytophthora cactorum b ) 182.2 502.6
Phytophthora cinnamomi b ) 54.3 8.1
Phytophthora palmivora b ) 263.6 494.6
Phytophthora capsici b ) 52.4 0.28
Phytophthora parasitica d ) 117.9 0.07
Fusarium oxysporum d ) 420.0 339.9
Pestalotiopsis sp. b ) 438.6 105.0
a
) The effective concentration for 50% diameter growth reduction. b ) Results after 3 d of incubation.
c
) Results after 1 d of incubation. d ) Results after 4 d of incubation.
136 CHEMISTRY & BIODIVERSITY Vol. 12 (2015)

Table 2. 1H-NMR (500 MHz) Data for Acremoxanthone E (1). d(H ) in ppm, J in Hz.

H-atom 1 in CDCl3 1 in ( D6 )acetone HMBC ( H ! C ) a )

HC(3) 5.39 (dt, J 2.3, 3.0, 5.6) 5.29 (pt, J 2.5) C(2), C(4), C(5), C(15)
HC(4) 6.62 (dd, J 2.3, 10.3) 6.50 (dd, J 1.7, 10.2) C(2), C(6)
HC(5) 6.17 (dd, J 10.3, 3.0) 6.13 (dd, J 10.2, 2.8) C(2), C(3), C(7)
HC(11) 6.15 (s) 6.12 (s) C(9), C(10), C(12), C(13), C(15)
Me(16) 3.63 (s) 3.69 (s) C(15)
HC(1) 4.65 (d, J 4.5) 4.55 (s) C(2), C(3), C(7), C(9), C(14),
C(15)
HC(3) 6.87 (s) 6.79 b ) C(6), C(7), C(16)
HC(5) 6.74 (s) 6.79 b ) C(3), C(7), C(16)
HC(11) 4.98 (dd, J 6.5, 1.3) 4.80 (pt, J 3.72) C(9), C(10), C(12), C(12),
C(13), C(13), C(14)
HC(12) 6.40 (dd, J 8.4, 6.5) 6.46 a ) C(10), C(11), C(14)
HC(13) 6.48 (dd, J 8.4, 1.3) 6.46 a ) C(1), C(9), C(11), C(13), C(14)
CH2(15) 2.77 (d, J 18.2, Ha ), 2.73 (d, J 17.8, Ha ), C(1), C(9), C(11), C(13), C(12),
2.56 (d, J 18.2, Hb ) 2.62 (d, J 17.8, Hb ) C(14)
Me(16) 2.39 (s) 2.39 (s) C(3), C(4), C(5), C(7)
c
HOC(3) 5.97 (d, J 5.6) )
HOC(6) 14.25 (br. s) 14.17(s) C(6), C(5)
HOC(10) 11.06 (s) 11.09 (s) C(10), C(11)
c
HOC(1) 4.58 (d, J 5.0) )
HOC(6) 11.61 (s) 11.39 (s) C(6)
HOC(8) 14.43 (br. s)
a
) Determined in CDCl3 and ( D6 )acetone. b ) Multiplicities not shown, when the chemical shifts overlap.
c
) Not observed under experimental conditions.

13
Table 3. C-NMR (125 MHz) Data for Acremoxanthone E (1). d(C ) in ppm.

Position 1 in ( D6 )acetone 1 in CDCl3 Position 1 in ( D6 )acetone 1 in CDCl3

2 86.2 84.6 2 135.8 140.7
3 75.0 74.6 3 123.0 121.9
4 147.3 143.6 4 148.5 147.9
5 123.5 123.6 5 118.4 119.1
6 163.7 170.9 6 162.7 162.0
7 100.3 99.1 7 112.9 111.4
8 185.2 185.1 8 185.3 185.1
9 105.9 105.1 9 107.5 105.4
10 160.4 159.8 10 186.7 185.9
11 114.3 114.2 11 38.4 38.2
12 150.0 147.5 12 131.2 131.7
13 117.0 115.2 13 135.8 133.3
14 155.6 154.1 14 43.7 42.7
15 169.6 168.6 15 36.0 35.4
16 52.7 53.1 16 22.0 22.1
1 73.4 73.4
 CHEMISTRY & BIODIVERSITY Vol. 12 (2015) 137

solvent polarity [10]. Thus, additional NMR experiments in CDCl3 revealed that the
degree of tautomerism of C(6) is solvent-dependent. However, the structure
elucidation was accomplished by NMR data of compound 1 in (D6 )acetone, because
a lesser overlapping of peaks and more clear multiplicities were achieved: also two
additional OH H-atom signals were detected. The 13C-NMR spectrum of 1 showed the
following 31 resonances corresponding to three of CO groups (d(C) 169.6, 185.3, and
186.7), 13 aromatic quaternary C-atoms; two O-bearing CH groups (d(C) 73.4 and
75.0), two aliphatic quaternary C-atoms (d(C) 43.7 and 86.2), one MeO group (d(C)
52.7), one Me group (d(C) 22.0), one CH2 group (d(C) 36.0), three aromatic CH
groups (d(C) 114.3, 117.0, and 123.0), one aliphatic CH group (d(C) 38.4), and four
CH groups (d(C) 123.5, 131.2, 135.8, and 147.3), by DEPT spectra. The OH signals
were assigned according to 1H-NMR data and HMBC experiments. Analysis of the 1H-
and 13C-NMR, COSY, HSQC, and HMBC data revealed the presence of an oxanthrone
moiety (ABC rings) and hydroxanthone moiety (DEF rings) as a heterodimer
(Tables 2 and 3, and Fig. 1).
The linkage between oxanthrone (ABC rings) and hydroxanthone (DEF rings)
moieties was determined as a bicyclo[3.2.2]nonene based on HMBCs HC(11)/C(15)
and nonequivalent HaC(15) and HbC(15)/C(9), C(12), C(13), and C(14) (Fig. 1).
A NOESY correlation between HC(13) of the ethylene bridge was observed
exclusively with the diastereotopic HbC(15), while HC(1) and HC(11) displayed
correlations with both diastereotopic CH2 H-atoms, HaC(15) and HbC(15),
indicating that HC(1) and C(15) should be of syn-facial orientation, and that
HC(1) occupies a pseudoequatorial position. The C(8) and C(8) signals were absent
in the 13C-NMR spectrum; however, their chemical shifts were estimated by the clear
correlations observed in the HMBC spectrum.
The proposed structure for 1 is related to acremoxanthone C [11] [12] and the family
of xanthoquinodin antibiotics [12 15]. In the NOESY spectrum of 1, there are clear
correlations HC(3)/HC(11) and HOC(10)/HC(11), suggesting a para arrange-
ment according to Tabata et al. [13] [14]. This compound was named acremoxanthone E
to maintain consistency with the acremoxanthones previously reported [11] [16],
although this compound is structurally closely related to xanthoquinodins due the
presence of a hydroxanthone instead of a xanthone moiety.
Acremoxanthone C (2) was the major compound obtained as fine yellow needles.
The molecular formula of this compound was determined as C33H26O12 by ESI-MS (m/z
615.2 ([M H] ; calc. 615.1503), implying an unsaturation index of 21. The NMR
spectra of 2 were similar to those of 1, except for the presence of the signals associated

Fig. 1. Key HMBCs of Acremoxanthone E (1)

138 CHEMISTRY & BIODIVERSITY Vol. 12 (2015)

with CO(17) and Me(18) in the 13C-NMR spectrum, and the simple signal associated
with Me(18) in the 1H-NMR spectrum. The difference of 42 mass units indicated that
the AcO group at C(1) in compound 2 was replaced with a OH group in 1. The NMR
data of compound 2 matched those of the acremoxanthone C isolated from an extract
of an unidentified fungus of the order Hypocreales (MSX 17022) that was initially
reported by Ayers et al. [11] and subsequently reisolated from the fungus Purpur-
eocillium lilacinum [12]. Moreover, for the first time, we unambiguously determined
the structure of compound 2 by X-ray crystal-structure analysis. Single yellow crystals
were obtained by slow evaporation from CH2Cl2 /hexane. Compound 2 crystallized in a
monoclinic system (P21). An ORTEP representation is displayed in Fig. 2 [17]. The
asymmetric unit is constituted of two crystallographically independent molecules of
acremoxanthone. The structure of 2 is a bicyclo[3.2.2] compound formed by an
oxanthrone (ABC rings) and a hydroxanthone (DEF rings) moiety. The relationship
between ABC and DEF ring systems is nearly perpendicular, forming a dihedral angle
of 90.5(1)8. The ethylene bridge bisects these systems with dihedral angles of 29.1(4)8
with the oxanthrone system and of 64.7(2)8 with the hydroxanthone system. The
geometry adopted by the acremoxanthone confirms that the AcO group at C(1) is b-
oriented and syn with the ethylene system, the MeOCO group at C(2) and the OH
group at C(3) are in a-orientation, and anti with the ethylene system in accordance with
the NMR analyses. The relative configuration suggested by Ayers et al. [11] mismatch,
since he proposed a b-orientation for MeOCO at C(2) and a-orientation for OH at
C(3); thus, the expected coupling constant 3J(2,3) should be near to 6 Hz, such as in
nidulalin A [18] and phomalevone A [19]. However, the experimental value was
2.1 Hz. Madariaga-Mazon et al. [12] addressed this issue, comparing experimental
results with theoretical calculations about optical rotations (OR) and coupling
constants (C,H and H,H) for the sixteen possible stereoisomers of acremoxanthone
C. The authors reported that xanthoneanthraquinone heterodimers with a b-oriented
C(11)C(14) ethylene bridge exhibited positive OR, and stereoisomers MeOCO
group at C(2) and OH group at C(3) at the same face have 3J(2,3) values of ca. 2.5 Hz.
Therefore, the relative configuration of acremoxanthone C (2) was established as rel-

Fig. 2. ORTEP Representation of Acremoxanthone C (2)

 CHEMISTRY & BIODIVERSITY Vol. 12 (2015) 139

(2R,3R,1S,11S,14R). In agreement with the positive experimental value of OR for

compounds 1 ( 536) and 2 ( 519), the 3J(2,3) values (2.3 and 2.5, resp.), and X-ray
analysis, the same relative configuration is assumed for 1 and 2.
The spectral properties of the known acremonidins A and B (3 and 4, resp.), and
acremoxanthones A and B (5 and 6, resp.), including IR, MS, and 1H- and 13C-NMR
data, were identical to those previously described in the literature [16] [20].
5. Monitoring Polyketides Production and Biosynthetic Considerations. The high-
performance liquid chromatography (HPLC) profile of liquid cultures showed that, at
day 30, 2 was the major compound, while at day 60, 3 was most abundant, and 2 was
barely detectable, suggesting the decomposition or degradation process of 2 to 3 (data
not shown). This process of transformation from hydroxanthone to benzophenone was
documented previously [11] [12] as a result of an aromatization reaction that is favored
under alkaline conditions from acremoxanthone C to acremonidin A. Additionally,
other related hydroxanthones, such as nidulalins A and B [18] and phomalevones A
and B [19], exhibit the same rearrangement. In our opinion, transformation in liquid
cultures from 2 to 3 could be associated with the lability of 2 favored in aqueous
medium and temperature; as a consequence, the acremonidins will be artifacts; in order
to establish the latter, solid substrate cultivations were analyzed three times by HPLC
(on days 20, 30, and 40), with low water availability and fresh and extracts concentrated
(rotary evaporator). Identical chromatographic profiles were obtained with both fresh
and exhaustive extracts. The results (Fig. 3, left) indicated that acremoxanthone C (2)
was the most abundant compound at day 20, and that 2 was barely detectable at day 30,
in contrast to the acremonidin A (3), of which the signal was the most intense (Fig. 3,
left), while at day 40, acremoxanthone A (5) exhibited the most intense signal,
indicating that 2 had been transformed to benzophenone 3, and subsequently
dehydrated and converted to 5, as depicted in Fig. 3 (right). According to these
results, if acremoxanthone A (5) were an artifact, then the dehydratation of 2 5 would
occur in the culture conditions or in the extraction process, and 5 should be present in
ortho/para arrangement at a 1 : 1 ratio; however, only the ortho acremoxanthone A was
isolated. Indeed, cyclization from benzophenones to xanthones is not a trivial reaction;
it requires a strong dehydrating agent, such as P2O5 or polyphosphoric acid. In our
hands, the attempts to obtain 5 from 3 employing these dehydrating agents according to
the procedure of Gil et al. [21] failed. Therefore, benzophenoneoxanthrone hetero-
dimers (acremonidins) must be natural products and intermediates in the biosynthesis
of xanthoneoxanthrone heterodimers (such as acremoxanthones). Hence, acremoni-
dins and acremoxanthones should be formed from the xanthoquinodin precursors
(hydroxanthoneanthraquinone heterodimers) by post-polyketide synthetase modifi-
cations.
6. Bioactivities of Compounds 1 6. The bioactivities of heterodimeric polyketides,
1 6, isolated from the endophytic fungus A. camptosporum, were evaluated by the
radial growth of the following four phytopathogenic oomycetes: Pythium aphanider-
matum, Phytophthora cinnamomi, P. capsici, and P. parasitica. The oomycetes were
most strongly inhibited by the A. camptosporum mycelium extract and by metalaxyl.
The inhibitory concentration for 50% diameter-growth reduction (IC50) values
indicate that compounds 1 6 reduced the diameter growth of all target species in a
concentration-dependent manner. The most active polyketides were the compounds
140 CHEMISTRY & BIODIVERSITY Vol. 12 (2015)

Fig. 3. Left: HPLC-UV Profile of compounds from solid cultures of A. camptosporum at 265 nm. a) 20 d,
b) 30 d, and c) 40 d. Right: Possible biosynthetic link among xanthoquinodins, acremonidins, and
acremoxanthones.

formed by a hydroxanthone subunit (i.e., 1 and 2), while the compounds formed by a
xanthone subunit (i.e., 5 and 6) did not show any significant antifungal activity in the
test with the highest concentration (50 mm). Moreover, polyketides with a benzophe-
none subunit (i.e., 3 and 4) in general were slightly less active than their analogs 1 and 2.
Polyketides 1 4 were less active than metalaxyl, except for Phytophthora cinnamomi,
which was most strongly inhibited by 1 and 2 (Table 4).
On the other hand, preliminary evaluation of mycelium extract from endophytic
fungus A. camptosporum exhibited growth inhibitions in the range of 70 100% of the
six cancer cell lines assayed at 50 mg/ml. Consequently, the cytotoxicities of pure
compounds 1 6 were also evaluated, showing similar cytotoxic activities for the three
classes of polyketides, ranging from 3 to 16 mm, and exhibiting a similar potential to that
of cisplatin and a moderate one with respect to camptothecin (Table 5). Similar
behaviors had been observed previously in the cytotoxicities of xanthoquinodins [15],
acremonidins, and acremoxanthones [16], and in xanthoquinodins-like compounds
[22], indicating that stereochemical changes (epimers or enantiomers), structural
 CHEMISTRY & BIODIVERSITY Vol. 12 (2015) 141

Table 4. Anti-Oomycete Activity of Compounds 1 6 on the Diameter Growth ( IC50 [mm]) of Selected
Phytopathogenic Oomycetes

Microorganism IC50 [mm]

1 2 3 4 5 6 Mt a )
P. aphanidermatum b ) 5.9 11.8 9.7 17.4 > 50 > 50 0.2
P. cinnamomi c ) 16.8 24.3 49.8 33.9 > 50 > 50 29.1
P. capsici d ) 37.1 29.3 > 50 27.2 > 50 > 50 1.0
P. parasitica c ) 33.7 30.8 31.2 23.8 > 50 > 50 0.3
a
) Metalaxyl. b ) Results after 1 d of incubation. c ) Results after 4 d of incubation. d ) Results after 3 d of
incubation.

Table 5. Cytotoxicities for Compounds 1 6 against Human Tumor Cell Lines ( IC50 [mm])

Compound Human tumor cell lines a ) b )

U251 PC-3 K562 HCT-15 MCF-7 SKLU-1
c
Extract ) 99.91 87.9 70.35 93.41 100 95.54
1 5.32  0.4 8.62  0.4 ND 10.27  0.5 7.39  0.7 1.16  0.1
2 6.37  0.5 6.51  0.5 6.55  0.2 5.98  0.3 5.16  0.1 5.88  0.6
3 7.61  0.1 8.64  0.2 ND 8.83  0.8 7.79  0.2 7.56  0.6
4 6.37  0.5 16.1  0.6 ND 3.3  0.3 15.8  1.3 10.90  0.9
5 3.33  0.2 3.1  0.2 5.72  0.1 5.76  0.1 5.9  0.5 4.53  0.2
6 6.16  0.3 4.51  0.3 ND 5.76  0.1 5.05  0.1 8.13  0.8
cisPt d ) 9.09  0.8 15.94  1.2 15.20  1.4 13.83  0.7 13.03  1.3 7.13  0.2
CPTe ) 0.024  0.005 0.12  0.010 0.59  0.020 0.13  0.005 0.16  0.001 0.15  0.009
a
) U251, Central nervous system cancer; PC-3, human prostatic adenocarcinoma; K562, human chronic
myelogenous leukemia; HCT-15, human colorectal adenocarcinoma; MCF-7, human mammary
adenocarcinoma; and SKLU, human lung adenocarcinoma. b ) ND, Not determined. c ) Cytotoxicity
data of organic extract from mycelium at 50 mg/ml, expressed in %. d ) cisPt, Cisplatin. e ) CPT,
Camptothecin.

changes such as ortho/para arrangements, and rearrangement of the hydroxanthone

subunit to chromanone-g-lactone (i.e., xanthoquinodins A3 and B3) or benzophenone
(acremonidins), and also tailtail or tailhead fusion between both subunits are
irrelevant for cytotoxic activity. Indeed, it is outstanding that this class of heterodimers
has positive optical rotations and, therefore, has a retained configuration of the
ethylene bridge between both subunits in all compounds isolated to date, suggesting
that the pharmacophore is the bicyclo[3.2.2]nonene and the residue of anthraquinone
(ABC rings). This is highly significant in the design of synthetic routes for optimizing
the biological activity of this class of compounds.

Concluding Remarks. Endophytic fungus Acremonium camptosporum from

tropical plant Bursera simaruba turned out to be a promising microorganism producer
of heterodimeric polyketides. It is noteworthy that, in the solid media culture, we
detected the presence of some unidentified metabolites, possibly polyketides on the
basis of their UV/VIS HPLC profile. A. camptosporum is a source of bioactive
142 CHEMISTRY & BIODIVERSITY Vol. 12 (2015)

compounds, and, in liquid culture, this fungus produces 2 in a high yield (35.5 mg/l).
Heterodimeric polyketides possess promising potential in modern agriculture for the
development of strategies to control plant pathogenic oomycetes such as Pythium
aphanidermatum, Phytophthora cinnamomi, Phytophthora capsici, and Phytophthora
parasitica.
Phytophthora spp. and Pythium spp. are among the worlds most destructive plant
pathogens, which cause multibillion-dollar losses in crops worldwide; these also cause
significant environmental damage in natural ecosystems [23] [24]. Zoospores are the
most important propagules for the epidemic development of Phytophthora spp. and
Pythium spp. diseases in crop production [25] [26]. Further studies of heterodimeric
polyketides at several points in the reproductive cycle of pathogenic plant oomycetes,
including inhibition of sporangia germination, inhibition of sporangia formation,
release and germination of zoospores, and inhibition of zoospore movement, may allow
us to establish the mechanism of action of isolated bioactive compounds.
On the other hand, this class of heterodimeric polyketides has been evaluated in
several organisms, such as pathogenic bacteria and fungi, and in protozoan parasites
such as Plasmodium falciparum and Plasmodium spp. and in Coccidia [13] [16] [20]
[27]. Additionally, the activities of some of these compounds have been evaluated
against human cancer cell lines [11] [15] [16] [22], but, to the best of our knowledge,
their anti-oomycete activities against some plant pathogens have not been explored,
before.
This is the first report on three different groups of heterodimeric polyketides linked
by a bicyclo[3.2.2]nonene to form xanthoquinodins, acremonidins, and acremoxan-
thones, which are produced by one unique fungus. Accordingly, this is the first occasion
on which it is possible to assume a common biosynthetic origin of these heterodimers.

This work was supported by grant CONACyT 81017 and 179194 and DGAPA-UNAM (IN217603).
We wish to thank Dra. Bertha Tlapal Bolanos and Dra. Olga Gomez from Instituto de Fitosanidad,
Colegio de Postgraduados, Montecillo, Estado de Mexico, for the donation of plant-pathogenic
microorganism used in the bioassays. We are also grateful to Hector Ros, Beatriz Quiroz, Isabel Chavez,
Elizabeth Huerta, Ma. de los Angeles Pena, Luis Velasco-Ibarra, Javier Perez Flores, Roco Patino,
Erendira Garca, and Ma. Teresa Ramrez Apan. Instituto de Qumica, UNAM, for recording NMR, IR,
UV, and mass spectra and cytotoxicity determinations. Claudio Melendez and Jordi M. Muria
acknowledge a fellowship from CONACyT to carry out graduate studies.

Experimental Part
General. Open column chromatography (CC): silica gel 60 (SiO2 , 70 230 mesh; Merck). Anal.
TLC: pre-coated silica-gel 60 F254 plates (Merck); visualization with UV (254 and 360 nm) and by
spraying with 10% H2SO4 , followed by heating at 808. HPLC: Waters (Alliance 2695) system equipped
with a photodiode array detector (model 2996). Anal. HPLC: reversed-phase (RP) column (Atlantis C18,
5 mm  150 mm  i.d. 4.6 mm), at r.t.; flow rate, 1 ml/min. Semi-prep. HPLC: C18 column (Clipeus, 5 mm 
150 mm  i.d. 10 mm); at r.t., isocratic elution condition or gradient system; flow rate, 5 ml/min; samples
filtered through a 0.4 mm Nylon filter before HPLC analysis; control of the equipment, data acquisition,
and processing and management of chromatographic information performed by the Empower 2.0
software program. M.p.: Fisher-Johns apparatus; uncorrected. UV Spectra: HPLC photodiodes array
detector at 190 500 nm in H2O/MeCN 1 : 1. IR Spectra: KBr disks; Perkin-Elmer 599-B spectropho-
tometer. Optical rotations: JASCO DIP 360 polarimeter. 1H- and 13C-NMR, COSY, NOESY, HMBC,
 CHEMISTRY & BIODIVERSITY Vol. 12 (2015) 143

and HMQC: Bruker DMX500, in CDCl3 , (D6 )acetone, or CD3OD either at 500 (1H) or 125 (13C) MHz;
tetramethylsilane (TMS) as internal standard. ESI-MS: Bruker Daltonics Esquire 6000 system (ion trap
mass analyzer) in positive and negative mode. X-Ray analysis of compound 2: Bruker SMART APEX
diffractometer equipped with graphite-monochromated and Mo radiation.
Isolation, Description, and Preservation of Fungi. A Mexican new fungal species culture was
obtained from uninjured and symptomless leaves of Bursera simaruba (Burseraceae; chaka or palo
mulato) collected from the semideciduous dry tropical forest of the Ecological Reserve El Eden in the
state of Quintana Roo, Mexico. The leaves were processed as described in [28]. Among the fungi
recovered was an interesting new fungal specimen denominated B101. To identify this novel Mexican
fungus, a molecular analysis was performed. The genomic DNA was extracted from the endophytic
fungus culture, and nuclear ribosomal DNA internal transcribed spacer (ITS) regions (ITS1-5.8S rDNA-
ITS2) from culture B101 were amplified and sequenced using primers ITS5 and ITS4 and analyzed as
described in [29]. Sequencing was performed by the United States Department of Agriculture-
Agricultural Research Service South Atlantic Area Sequencing Facility (Athens, GA, USA). ClustalW2
(www.ebi.ac.uk/Tools/clustalw2/) was used to generate a DNA sequence alignment between strain B101
and other GenBank accessions. Distance-based analysis of ITS sequence alignment was performed using
MEGA 4.1. Morphological and molecular analyses suggested that culture B101 is a member of the
Clavicipitaceae family (Ascomycota). Comparison of the B101 sequences with accessions in GenBank
indicates that the fungus is Acremonium camptosporum W. Gams. The Mexican endophytic fungal
culture was deposited with the Coleccion de Hongos of the Herbario Nacional de Mexico (MEXU
26354) and the sequences of ITS rDNA/28S rDNA regions in GenBank under the accessions
Nos. JQ811555/JQ811556. In addition, a specimen of Bursera simaruba (L.) Sarg. (Burseraceae), the
plant associated with the endophyte fungus MEXU 26354, was deposited in the University of California,
Riverside Herbarium (G. P. Schultz & R. Palestina # 1092/UCR 110695).
Fungal Fermentations. Acremonium camptosporum was grown in liquid and solid media. Liquid
culture was carried out to isolate the different secondary metabolites produced. The fungus was cultured
on five glass carboys (20 l), each containing 6.5 l of potato dextrose broth (PDB). Each carboy was
individually inoculated with five 10-mm2 agar plugs taken from the borders of 15-day-old mycelium
growing on a PDA plate maintained at 288. Carboys were incubated for 30 d. Additional liquid
fermentations were performed to analyze the secondary metabolite production (SMP) by this fungus in
eight Fernbach flasks (2.8 l) containing 1 l of PDB culture medium. Each flask was inoculated with three
5-mm2 agar plugs taken from a stock culture of the fungus growing on PDA; four flasks were incubated
for 20 d, and the remaining flasks for 40 d. Carboy and flask cultures were incubated under static
conditions at r.t. (25 288) and a 12-h/12-h light/dark photoperiod under fluorescent light Octron 4100 K,
Ecologic, 32 W).
The solid cultures were conducted simultaneously in Petri dishes to perform SMP analyses. Four
15-cm Petri dishes for each incubation period containing PDA medium were inoculated with one 5 mm2
plug of the growing periphery of 5-d mycelium of fungi growing in PDA and placed in the center of a
Petri dish (mycelium side down). Petri dishes were incubated at r.t. for 20, 30, and 40 d in a 12-h/12-h
light/dark photoperiod under fluorescent light.
Extraction and Isolation of SM from A. camptosporum. At the end of the fermentation process, the
mycelium of each glass carboy was separated from the culture medium by simple filtration. The mycelium
was macerated three times with 10 l of CH2Cl2 , and then three times with 10 l of AcOEt. The combined
org. phases were filtered over anh. Na2SO4 and concentrated in vacuo to yield 50.68 g of yellowish oil.
The mycelial extract was subjected to CC (SiO2 ; gradient of hexane/CH2Cl2/MeOH of increasing
polarity). In total, 180 fractions (200 ml each) were collected and pooled on the basis of their TLC
profiles to yield 14 major fractions (I XIV). Bioactivity of the antifungal bioassay revealed four active
pools: Fr. XI (5.059 g); Fr. XII (4.896 g); Fr. XIII (258 mg), and Fr. XIV (198 mg).
Fr. XII (4.896 g), eluted with CH2Cl2/MeOH 7 : 3, spontaneously precipitated as a yellow amorphous
solid and was purified by successive recrystallization from CH2Cl2/MeOH 1 : 1 to achieve yellowish
needle crystals (2, 155 mg). A 300-mg sample from the residual mother liquor of Fr. XII, constituted of a
complex mixture of several compounds, was analyzed by RP-HPLC (MeCN/H2O 25 : 75 to 50 : 50 (0
5 min), 50 : 50 to 65 : 35 (5 30 min), 50 : 50 at 100% MeCN (35 min), and maintaining this for up to
144 CHEMISTRY & BIODIVERSITY Vol. 12 (2015)

40 min). Compounds were detected at wavelengths of 272 and 365 nm. Six peaks were collected, and,
after processing in the usual manner, these yielded 46 mg of 4 (tR 10.60 min), 29 mg of 1 (16.25 min),
85 mg of 3 (19.45 min), 24 mg of 6 (27.83 min), 80 mg of 2 (26.64 min), and 23 mg of 5 (tR , 20.27 min).
Antifungal and Anti-Oomycete Assays. The inhibitory effect of the mycelium extract from
endophytic fungus A. camptosporum was tested on the radial growth of the following phytopathogenic
microorganisms of economic importance: nine oomycetes, including Pythium ultimum, Pythium
debaryanum, Pythium aphanidermatum, Phytophthora cactorum, Phytophthora cinnamomi, Phytoph-
thora palmivora, Phytophthora capsici, and Phytophthora parasitica, and two true fungi, including
Fusarium oxysporum and Pestalotiopsis sp. The extract was evaluated at a wide range of concentrations
(2 500 mg/ml) by dilution in agar for IC50 calculations. In each treatment, sterile PDA (408) was added
prior to agar solidification in 5-cm Petri dishes. Fractions were evaluated at 250 mg/ml against P. capsici, P.
parasitica, and F. oxysporum as test microorganisms. Four microorganisms were the most sensitive to
mycelium extract; therefore, these were assayed to evaluate the pure compounds employing different
concentrations (1 50 mm) with respect to each microorganism. Extract, fractions, and compounds were
dissolved in MeOH or acetone, not exceeding 0.5%. The commercial fungicide Ridomil Gold 4E,
Metalaxyl-m (Mefenoxam) was utilized as positive control, and PDA plates with 0.5% MeOH or acetone,
and without org. solvents were used as negative control. Bioassays were performed as in [8] [9]. Results
were analyzed by ANOVA and Tukey statistical tests using GraphPad Prism ver.5.01 computer software.
The IC50 values for org. extracts were calculated based on the average diameter growth of the mycelium
with the same statistical software. Data are represented as mean  standard deviation (SD). A P value of
0.05 or less was employed to indicate significance.
Cytotoxicity Assay. Cytotoxicity determinations of pure compounds against U251 (central nervous
system cancer), PC-3 (human prostatic adenocarcinoma), K562 (human chronic myelogenous
leukemia), HCT-15 (human colorectal adenocarcinoma), MCF-7 (human mammary adenocarcinoma),
and SKLU (human lung adenocarcinoma) human solid tumor cell lines were performed in a 7-d 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay at the Institute of Chemistry,
Biological Activity Screening laboratory, in accordance with the protocol described by the United States
National Cancer Research, with cisplatin (cisPt) and camptothecin (CPT) as positive controls.
HPLC Analysis Monitoring of Secondary Metabolites Production. a) Solid Cultures. PDA Plate
cultures of A. camptosporum of different growth times (20, 30, and 40 d) were processed simultaneously.
The mycelia were mechanically fragmented, subsequently macerated for 4 h with 25 ml of CH2Cl2 , and
analyzed in fresh as follows: 5 ml of each crude extract was evaporated at r.t. and resuspended in 0.5 ml of
MeCN to inject it. Subsequently, in order to explore the likely MS lability due to rotavapor temp., the
mycelia were exhaustively extracted (for 2 d) with the same solvent, and then the extracts were prepared.
From each extract, 1 mg was dissolved in 0.5 ml of MeCN. After filtering, these were analyzed by RP-
HPLC with the same gradient system, starting with 25 : 75 50 : 50 (MeCN/H2O) for 0 5 min, 50 : 50
65 : 35 for 5 30 min, 50 : 50 to 100% MeCN for 30 35 min, and constant until 40 min.
b) Liquid Cultures. Liquid flask cultures (30 and 60 d) were processed in the usual manner. The org.
extracts from mycelium were obtained after successive maceration and extracted with CH2Cl2 . Ten-mg
samples were taken, dissolved in 1 ml of MeCN, and were subsequently analyzed by HPLC (isocratic
solvent system H2O/MeCN 30 : 70). Chromatograms were overlapped and color bars added with Gimp
2.8.8 computer software, no modification of scale in x or y axis was made. The peaks identity was assessed
comparing the retention times and UV profiles of pure compounds injected under the same
chromatographic conditions.
Acremoxanthone E ( Methyl rel-(1R,8aR,9S,16S,17aR)-1,5,8,9,15,16-Hexahydro-1,4,6,9,13,14-hexa-
hydroxy-11-methyl-5,15-dioxo-17aH-8a,16-ethenonaphtho[2,3:5,6]cyclohepta[1,2-c]xanthene-17a-car-
boxylate; 1). Pale-yellow powder. M.p. 226 2278. [a]D 536 (c 0.08, acetone). UV (H2O/MeCN
1 : 1): 369, 276, 213. IR (KBr): 3430, 1740, 1640, 1575. 1H- and 13C-NMR: see Tables 2 and 3, resp. ESI-MS
(pos.): 627.2 ([M MeOH Na] ), 611.2 ([M K] ), 595.2 ([M Na] ).
Acremoxanthone C ( Methyl rel-(1R,8aR,9S,16S,17aR)-9-(Acetyloxy)-1,5,8,9,15,16-hexahydro-
1,4,6,13,14-pentahydroxy-11-methyl-5,15-dioxo-17aH-8a,16-ethenonaphtho[2,3:5,6]cyclohepta[1,2-
c]xanthene-17a-carboxylate; 2). Yellow crystalline needles. M.p. 228 2308. [a]D 519 (c 0.05,
CHCl3 ). UV (H2O/MeCN 1 : 1): 370, 275, 215. IR (KBr): 3435, 1740, 1637, 1573. 1H-NMR (CDCl3 ,
 CHEMISTRY & BIODIVERSITY Vol. 12 (2015) 145

500 MHz): 2.01 (s, Me(18)); 2.38 (s, Me(16)); 2.69 (d, J 18.0, HaC(15)); 2.79 (d, J 18.0, HbC(15));
3.70 (s, Me(16)); 4.82 (dd, J 6.7, 1.0, HC(11)); 5.30 (dt, J 2.5, HC(3)); 5.79 (s, HC(1)); 6.08 (dd,
J 8.3, 1.0 HC(13)); 6.14 (s, HC(11)); 6.43 (dd, J 8.3, 6.7, HC(12)); 6.80 (d, J 1.5, HC(3)); 6.89
(d, J 1.5, HC(5)); 6.13 (dd, J 10.5, 3.0, HC(5)); 6.51 (dd, J 10.5, 3.0, HC(4)); 11.11 (s,
HOC(10)); 11.35 (s, HOC(6)); 14.18 (s, HOC(6)); 14.40 (s, HOC(8)). 13C-NMR (CDCl3 ,
125 MHz) 21.1 (C(18)); 22.1 (C(16)); 35.1 (C(15)); 38.1 (C(11)); 41.5 (C(14)); 53.1 (C(16)); 73.0
(C(1)); 74.5 (C(3)); 84.9 (C(2)); 99.0 (C(7)); 105.4 (C(9)); 105.6 (C(9)); 112.5 (C(7)); 114.2 (C(11));
114.9 (C(13)); 119.4 (C(5)); 123.3 (C(3)); 123.5 (C(5)); 143.8 (C(4)); 147.6 (C(12)); 171.0 (C(6)); 131.8
(C(12)); 132.5 (C(13)); 136.3 (C(2)); 147.8 (C(4)); 154.2 (C(14)); 159.8 (C(10)); 161.5 (C(6)); 168.7
(C(15)); 170.4 (C(17)); 185.2 (C(8)); 186.4 (C(10)); 185.1 (C(8)). ESI-MS (pos.): 637.2 ([M Na] ),
615.2 ([M H] ).
Acremonidin A ( Methyl 2-{[(5aR,6S,13S)-6-(Acetyloxy)-5,6,12,13-tetrahydro-1,3,10,11-tetrahy-
droxy-8-methyl-12-oxo-5a,13-ethenobenzo[4,5]cyclohepta[1,2-b]naphthalen-2-yl]carbonyl}-3,6-dihy-
droxybenzoate; 3). Pale-yellow powder. UV (H2O/MeCN 1 : 1): 358, 288, 278, 219. IR (KBr): 3359, 3025,
2955, 1735, 1680, 1614, 1579, 1464, 1414, 1369, 1217. 1H-NMR (CDCl3 , 500 MHz): 1.99 (s, Me(18)); 2.35
(s, Me(16)); 2.58 (d, J 17.7, HaC(15)); 2.69 (d, J 17.7, HbC(15)); 3.61 (s, Me(16)); 4.86 (d, J 6.7,
HC(11)); 5.77 (s, HC(11)); 5.93 (s, HC(1)); 6.06 (dd, J 8.4, 1.0, HC(13)); 6.44 (dd, J 8.4, 6.7,
HC(12)); 6.78 (s, HC(5)); 6.85 (s, HC(3)); 6.93 (d, J 9.1, HC(3)); 7.05 (d, J 8.8, HC(4)); 10.39
(s, HOC(5)); 11.58 (s, HOC(6)); 14.17 (s, HOC(10)). 13C-NMR (CDCl3 , 125 MHz) 21.1 (C(18));
22.0 (C(16)); 34.6 (C(15)); 37.7 (C(11)); 41.5 (C(14)); 52.4 (C(16)); 73.1 (C(1)); 105.3 (C(9)); 109.3
(C(7)); 109.4 (C(9)); 110.5 (C(11)); 112.7 (C(7)); 115.8 (C(13)); 119.3 (C(5)); 120.4 (C(3)); 123.2
(C(3)); 125.4 (C(4)); 128.1 (C(6)); 132.0 (C(12)); 132.2 (C(13)); 136.4 (C(2)); 145.0 (C(2)); 146.3
(C(12)); 147.6 (C(4)); 155.9 (C(5)); 156.6 (C(10)); 159.3 (C(14)); 161.6 (C(6)); 169.1 (C(15)); 170.5
(C(17)); 185.7 (C(8)); 186.6 (C(10)); 198.3 (C(8)). ESI-MS (neg.): 613.2 ([M  H]  ).
Acremonidin B ( Methyl 3,6-Dihydroxy-2-{[(5aR,6S,13S)-5,6,12,13-tetrahydro-1,3,6,10,11-penta-
hydroxy-8-methyl-12-oxo-5a,13-ethenobenzo[4,5]cyclohepta[1,2-b]naphthalen-2-yl]carbonyl}benzoate;
4). Yellow powder. UV (H2O/MeCN 1 : 1): 357, 286, 281, 210. IR (KBr): 3428, 3236, 3049, 2953, 1723,
1616, 1579, 1461, 1415, 1281, 1209. 1H-NMR ((D4 )MeOH, 500 MHz): 2.31 (s, Me(16)); 2.43 (d, J 17.8,
HbC(15)); 2.57 (d, J 17.8, HaC(15)); 3.54 (s, Me(16)); 4.43 (s, HC(1)); 4.79 (dd, J 1.3, 6.4,
HC(11)); 5.69 (s, HC(11)); 6.38 (dd, J 8.4, 1.3, HC(13)); 6.42 (dd, J 6.4, 8.4, HC(12)); 6.68 (s,
HC(3)); 6.69 (s, HC(5)); 6.78 (d, J 9.0, HC(4)); 6.93 (d, J 9.0, HC(3)). 13C-NMR (CDCl3 ,
125 MHz): 21.8 (C(16)); 35.1 (C(15)); 37.7 (C(11)); 42.8 (C(14)); 52.1 (C(16)); 73.1 (C(1)); 105.1
(C(9)); 109.2 (C(7)); 109.4 (C(9)); 109.9 (C(11)); 111.8 (C(7)); 115 (C(13)); 118.3 (C(3)); 118.5 (C(4));
121.9 (C(5)); 124.5 (C(3)); 130.1 (C(6)); 131.6 (C(12)); 133.2 (C(13)); 141.2 (C(2)); 145.2 (C(5)); 146.3
(C(12)); 147.4 (C(4)); 154.8 (C(2)); 157.7 (C(10)); 159.4 (C(14)); 161.4 (C(6)); 169.6 (C(15)); 185.4
(C(8)); 187.2 (C(10)); 199.4 (C(8)). FAB-MS: 595 (9, [M Na] ), 573 (6, [M H] ). ESI-MS (pos.):
573.2 ([M H] ).
Acremoxanthone A ( Methyl (7aR,8S,15S)-8-(Acetyloxy)-7,14,15,17-tetrahydro-2,12,13,16-tetrahy-
droxy-10-methyl-14,17-dioxo-8H-7a,15-ethenonaphtho[2,3:4,5]cyclohepta[1,2-b]xanthene-1-carboxy-
late; 5). Yellow crystalline needles. UV (H2O/MeCN 1 : 1): 362, 303, 271, 239. IR (KBr): 3425, 1731, 1641,
1612, 1577, 1220, 1015. 1H-NMR (CDCl3 , 500 MHz): 2.01 (s, Me(18)); 2.39 (s, Me(16)); 2.80 (d, J 18.1,
HbC(15)); 2.93 (d, J 17.8, HaC(15)); 3.98 (s, Me(16)); 4.93 (dd, J 1.1, 6.6, HC(11)); 6.00 (s,
HC(1)); 6.11 (dd, J 8.5, 1.1, HC(13)); 6.48 (s, HC(13)); 6.48 (dd, J 6.6, 8.5, HC(12)); 6.80 (s,
HC(5)); 6.91 (s, HC(3)); 7.34 (d, J 9.25, HC(4)); 7.41 (d, J 9.25, HC(3)); 11.57 (s, HOC(8));
12.79 (s, HOC(10)). 13C-NMR (CDCl3 , 125 MHz): 21.1 (C(18)); 22.1 (C(16)); 35.2 (C(15)); 37.6
(C(11)); 41.6 (C(14)); 53.1 (C(16)); 73.0 (C(1)); 105.2 (C(9)); 107.1 (C(9)); 109.8 (C(13)); 112.7
(C(7)); 113.4 (C(6)); 117.4 (C(11)); 118.4 (C(7)); 119.4 (C(5)); 122.3 (C(3)); 123.2 (C(3)); 125.7 (C(4));
131.8 (C(12)); 132.9 (C(13)); 136.4 (C(2)); 146.4 (C(12)); 147.7 (C(4)); 150.7 (C(2)); 152.7 (C(5)); 153.9
(C(14)); 157.7 (C(10)); 161.8 (C(6)); 169.0 (C(15)); 170.5 (C(17)); 180.0 (C(8)); 186.1 (C(10)); 186.8
(C(8)). ESI-MS (pos.): 619.2 ([M Na] ). ESI-MS (neg.): 595.1 ([M  H]  ).
Acremoxanthone B ( Methyl (7aS,8S,13aS,15S)-8-(Acetyloxy)-7,13,13a,14,15,17-hexahydro-
2,12,13a,16,17-pentahydroxy-10-methyl-13,14-dioxo-8H-7a,15-ethenonaphtho[2,3:4,5]cyclohepta[1,2-
b]xanthene-1-carboxylate; 6). Yellow powder. UV (H2O/MeCN 1 : 1): 272, 234, 222. IR (KBr): 3443, 1737,
146 CHEMISTRY & BIODIVERSITY Vol. 12 (2015)

1639, 1433, 1233, 1211. 1H-NMR (CDCl3 , 500 MHz): 2.38 (s, Me(18)); 2.39 (s, Me(16)); 3.05 (d, J 19,
HaC(15)); 3.50 (d, J 18.5, HbC(15)); 3.99 (s, Me(16)); 4.35 (s, HOC(9)); 5.04 (dd, J 0.9, 6.6,
HC(11)); 5.95 (dd, J 9.2, 0.9, HC(13)); 6.50 (s, HC(3)); 6.52 (dd, J 6.6, 9.2, HC(12)); 6.52 (s,
HC(1)); 6.68 (s, HC(13)); 6.78 (s, HC(5)); 7.34 (d, J 9.3, HC(4)); 7.42 (d, J 9.15, HC(3)); 11.48
(s, HOC(8)); 12.80 (s, HOC(10)). 13C-NMR (CDCl3 , 125 MHz): 22.5 (C(16)); 28.3 (C(18)); 32.3
(C(15)); 42.7 (C(11)); 46.1 (C(14)); 53.1 (C(16)); 70.6 (C(1)); 81.4 (C(9)); 107 (C(9)); 109 (C(13));
110.2 (C(7)); 113.6 (C(6)); 118.1 (C(5)); 118.3 (C(3)); 118.6 (C(7)); 118.6 (C(11)); 122.2 (C(3)); 125.0
(C(4)); 129.0 (C(13)); 134.2 (C(12)); 139.3 (C(2)); 145.6 (C(12)); 149.5 (C(4)); 150.6 (C(2)); 152.7
(C(5)); 154.2 (C(14)); 157.2 (C(10)); 164.0 (C(6)); 168.8 (C(15)); 170.7 (C(17)); 180.0 (C(8)); 194.7
(C(8)); 204.0 (C(10)). EI-MS: 612 (1, M ), 524 (2.5), 492 (2), 432 (4.2), 341 (4), 313 (1.2), 222 (6), 189
(3), 149 (6), 135 (10.5), 119 (13.8), 91 (24.1), 69 (11), 57 (13.2), 43 (9.5), 32 (25). ESI-MS (pos.): 667.2
([M MeOH Na] ), 631.1 ([M K] ), 635.1 ([M Na] ).
X-Ray Crystallographic Analysis of Acremoxanthone C (2). Empirical formula C33H26O12 2.5 H2O,
Mr 649.06, 0.42  0.24  0.18 mm, yellow prism, from CH2Cl2 monoclinic, P21 (No. 4), a 8.4138(5), b
27.326(16), c 15.2985(9) , b 98.067(1)8, V 3482.9(4) 3, Z 4, Dcalc. 1.238 Mg/m3, mm
0.098 mm  1, F(000) 1354. Temp., 298(2) K, MoKa 0.71073 , 2.00 25.39 of q collection; Bruker smart
program APEX AXS CCD area detector. The data reduction was carried out with the SAINT program
[30], 38598 reflections were collected, of which 12741 (Rint 0.0471) were independent reflections. The
structure was solved using direct methods and refined using full-matrix least-squares on F 2 on the
programs SHELXS-2013 and SHELXL-2008, resp. [31]. The program XSHELL was used as an interface
to the SHELX programs, and to prepare the figures. Two independent molecules were found in the unit
cell. The final values of S 1.029, R1 0.0696, wR2 0.1729, were based on 9711 reflections observed
with [I > 2s(I)], 924 parameters, the largest diff. peak and hole were 0.522 and  0.327 e   3, resp.
CCDC-940531 contains the supplementary crystallographic data for acremoxanthone C (2). These data
can be obtained free of charge from the Cambridge Crystallographic Data Center via www.ccdc.cam.
ac.un/data_request/cif.

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Received November 27, 2013