Tetrahedron 72 (2016) 1415e1421

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Tetrahedron
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Acremonidins FeH and acremoxanthones FeG, antimicrobial

substances from the insect fungus Verticillium sp. BCC33181
Chakapong Intaraudom, Nantiya Bunbamrung, Aibrohim Dramae, Nattawut Boonyuen,
Somjit Komwijit, Pranee Rachtawee, Pattama Pittayakhajonwut *
National Center for Genetic Engineering and Biotechnology (BIOTEC), Thailand Science Park, Paholyothin Road, Klong Luang, Pathumthani 12120,
Thailand

a r t i c l e i n f o a b s t r a c t

Article history: Six new compounds, including three acremonidins FeH (1, 3, and 5), two acremoxanthones FeG (7 and
Received 17 December 2015 8), and acremonoxide (10), along with eight known compounds (2, 4, 6, 9, 11e14) were isolated from
Received in revised form 11 January 2016 Verticillium sp. BCC33181. Their structures were determined based on extensive 1D and 2D NMR spectral
Accepted 25 January 2016
analyses, mass spectrometric data and by comparison with the known compounds reported previously.
Available online 28 January 2016
The relative configurations were identified based upon NOESY spectroscopic information as well as
comparison of CD spectrum with the known analogs. Both acremonidins and acremoxanthones exhibited
Keywords:
anti-Bacillus cereus activity (MIC 0.39e25 mg/mL), while acremonidins F (1), A (2), G (3), and C (4) also
Anthraquinone-xanthone heterodimers
Acremonidin
displayed anti-Enterococcus faecium activity (MIC 1.56e25 mg/mL). In addition, acremoxanthones F (7)
Acremoxanthone and G (8) showed antimalarial activity against Plasmodium falciparum K1 strain, multidrugeresistant
Verticillium strain, with respective IC50 values of 2.38 and 1.71 mg/mL, while others were inactive.
Cytotoxic Ó 2016 Elsevier Ltd. All rights reserved.
Antimicrobial

1. Introduction In our continuing search for bioactive compounds from micro-

Verticillium species have so far been isolated from soil and insect
(Homopteraeleaf hopper), producing several diverse secondary
metabolites including bioxanthracenes e ES-242s1,2 from a soil
fungus Verticillium sp. SPC-15898, sulfur-containing diketopiper-
azines3 and an ascochlorin glycoside e vertihemipterin A4 from the
insect fungi Verticillium hemipterigenum BCC1449 and BCC2370,
and verticipyrone from a soil fungus Verticillium sp. FKI-1083.5
These compounds displayed various biological activities. Bio-
xanthracene derivatives (ES-242s) inhibited [3H]thienyl cyclo-
hexylpiperidine binding to the N-methyl-D-aspartate receptor.1 A
sulfur-containing diketopiperazine, 1-demethylhyaodendrin tetra-
sulfide, showed antimalarial activity against Plasmodium falciparum
K1 strain with an IC50 value of 2.50 mg/mL.3 Verticipyrone inhibited
NADH-fumarate reductase from Ascaris suum (roundworm) with an
IC50 value of 0.88 nM and also showed nematocidal and insecticidal
activities against Caenorhabditis elegans and Artemia salina with
MICs of 20 mg/mL and 2 mg/mL, respectively.5 Vertihemipterin A
showed cytotoxic activity against KB, BC-1, NCI-H187, and Vero
cells.4
* Corresponding author. Tel.: þ66 2 564 6700x3559; fax: þ66 2 564 6632; e-mail
address: pattamap@biotec.or.th (P. Pittayakhajonwut).
http://dx.doi.org/10.1016/j.tet.2016.01.043
0040-4020/Ó 2016 Elsevier Ltd. All rights reserved.
organisms by employing chemical and biological screening scaf-
folds, the crude extracts from both broth and cells of the insect
fungus Verticillium sp. BCC33181 displayed good HPLC profiles and
exhibited antimalarial (IC50 4.35e>10 mg/mL) and antibacterial
activities against Bacillus cereus (IC50 0.78e1.56 mg/mL), with weak
cytotoxicity against both cancer (IC50 26.79e33.17 mg/mL) and non-
cancerous (IC50 17.81e31.43 mg/mL) cells. Therefore, the fungus was
selected for chemical investigation, which led to the isolation of five
new anthraquinone e xanthone heterodimers (1, 3, 5, 7, and 8),
a new xanthone derivative (10), and eight known compounds. We
herein describe the isolation and structure elucidation of new
compounds. Furthermore, these isolated compounds were evalu-
ated for antimalarial (against P. falciparum K1 strain), and anti-
bacterial activities (against both Gram-positive and Gram-negative
bacteria), and for cytotoxic activity against cancerous and non-
cancerous cells.
2. Results and discussions
Verticillium sp. BCC33181 was cultured on a potato dextrose
broth for 28 days in static conditions. The broth was extracted with
EtOAc and the cells were macerated in organic solvents (MeOH and
CH2Cl2) prior to evaporation, and extracted consecutively with
hexane and EtOAc. The EtOAc crude extracts from both broth and
1416 C. Intaraudom et al. / Tetrahedron 72 (2016) 1415e1421

cells exhibited antimalarial (IC50 4.35e>10 mg/mL) and anti-B. ce-
reus (MIC 0.781e1.56 mg/mL) activities with weak cytotoxicity
against MCF-7, KB, NCI-H187, and Vero cells (IC50 17.8e>50 mg/
mL). Therefore, these crude extracts were subjected to chromato-
graphic purification, yielding three new acremonidins F (1), G (3), H
(5), two new acremoxanthones F (7), G (8), and one new xanthone
derivative 10, along with eight known compounds, including
acremonidins A (2), C (4), acremoxanthones A (6), B (9), mon-
iliphenone (11), acremonidin E (12), penicillanone (13), and 7-
hydroxy-2-(2-hydroxypropyl)-5-methylchromone (14).
Compound 1 was obtained as a brown solid. The 1H and 13C NMR
spectral data (Table 1) were similar to those of acremonidin A (2),6
except that the oxymethine at H-6 resonated upfield at dH 4.48
(Dd1.51 ppm) and one methyl signal was absent from the 1H NMR
spectrum. HRESIMS data revealed the mass ion peak at m/z
573.1397 [MþH]þ, suggesting the loss of an acetyl group in com-
pound 1, compared to that of 2. In the COSY spectrum, H-6 was
coupled to an additional hydroxyl proton at dH 5.57 and the HMBC
spectrum showed correlations from H-6 to C-8, C-12, and C-14. The
spectroscopic evidence indicated that an acetyl group in acre-
monidin A was replaced with a hydroxyl group at C-6. The rest of
the structure was confirmed by 2D spectral analyses, including
COSY, HMQC, and HMBC. Moreover, the NOESY spectrum showed
cross-peak correlations from H-6 to Ha-150 (dH 2.68) and Hb-150 (dH
2.31) with a stronger intensity to Ha-150 ; and from H-6 to H-40 ,
indicating an identical relative configuration to compound 2. Thus,
the chemical structure of compound 1 was assigned as shown in
Fig. 1 and given a trivial name of acremonidin F.
Likewise, 1H and 13C NMR spectral data of compound 3 were
similar to those of aremonidin C (4), except that in the 1H NMR
spectrum the oxymethine at dH 4.81 (H-6) resonated at a higher
field than that of 4 (dH 6.29),6 and one methyl signal was absent in
compound 3. Moreover, in the 13C NMR spectrum, two carbons
belonging to the acetyl group in 3 were absent. HRESIMS data also
confirmed the loss of an acetyl group, revealing a mass ion peak at
m/z 587.1192 [MH]. In the COSY spectrum, the H-6 proton was
coupled to an additional hydroxyl proton at dH 6.24, and in the
HMBC spectrum there were correlations from H-6 to C-4, C-5, C-7,

Table 1
1
H and 13C NMR spectroscopic assignments for acremonidins FeH (1, 3, and 5) and acremoxanthones F (7) and G (8)

Position Acremonidin F (1)a Acremonidin G (3)a Acremonidin H (5)b Acremoxanthone F (7)b Acremonidin G (8)c

dH, mult (J in Hz) dC dH, mult (J in Hz) dC dH, mult (J in Hz) dC dH, mult (J in Hz) dC dH, mult (J in Hz) dC
1 d 187.7, qC d 204.4, qC d 202.0, qC d 204.2, qC d 202.7, qC
2 4.66 (dd, 5.2, 2.6) 38.0, CH 4.58 (d, 6.2) 44.0, CH 4.59 (d, 6.6) 44.3, CH 4.65 (d, 6.4) 44.2, CH 4.88 (d, 6.4) 43.6, CH
3 6.40e6.45 (m) 130.6, CH 6.46 (dd, 9.1, 6.2) 134.3, CH 6.49 (dd, 9.1, 6.6)d 135.3, CH 6.48 (dd, 9.1, 6.4) 133.8, CH 6.63 (dd, 9.1, 6.4) 134.5, CH
4 6.40e6.45 (m) 135.1, CH 5.78 (d, 9.1) 130.3, CH 5.76 (d, 9.1) 128.9, CH 5.98 (d, 9.1) 130.7, CH 5.98 (d, 9.1) 129.5, CH
5 d 42.9, qC d 48.1, qC d 47.3, qC d 48.2, qC d 47.0, qC
6 4.48 (d, 5.4) 72.0, CH 4.81 (d, 7.5) 68.3, CH 6.29 (s) 70.8, CH 4.86 (d, 6.8) 68.3, CH 6.56 (s) 70.4, CH
7 d 143.2, qC d 145.9, qC d 140.2, qC d 145.9, qC d 140.2, qC
8 6.81 (s) 122.7, CH 7.10 (s) 119.8, CH 6.63 (s) 118.6, CH 7.12 (s) 119.9, CH 6.75 (s) 119.3, CH
9 147.4, qC d 149.2, qC d 149.8, qC d 149.3, qC d 150.1, qC
10 6.74 (s) 117.8, CH 6.74 (s) 116.7, CH 6.82 (s) 117.8, CH 6.75 (s) 117.6, CH 6.82 (s) 117.7, CH
11 d 161.0, qC d 162.9, qC d 163.2, qC d 162.9, qC d 163.8, qC
12 d 112.1, qC d 110.7, qC d 110.5, qC d 110.6, qC d 110.3, qC
13 d 185.1, qC d 197.0, qC d 196.2, qC d 196.8, qC d 196.0, qC
14 d 106.6, qC d 82.4, qC d 82.0, qC d 82.5, qC 82.1, qC
15 2.34 (s) 22.0, CH3 2.37 (s) 22.4, CH3 2.36 (s) 22.3, CH3 2.38 (s) 22.5, CH3 2.41e (s) 21.7, CH3
10 d 159.9, qC d 159.5, qC d 157.3, qC d 156.8, qC d 156.9, qC
20 d 114.3, qC d 109.3, qC d 115.6, qC d 119.2, qC d 119.3, qC
30 d 146.7, qC d 146.6, qC d 143.0, qC d 148.3, qC d 147.7, qC
40 5.75 (s) 110.0, CH 6.02 (s) 108.9, CH 6.01 (s) 108.4, CH 6.94 (s) 109.4, CH 6.85 (s) 109.9, CH
50 d 158.8, qC d 159.0, qC d 158.3, qC d 154.3, qC d 154.8, qC
60 d 109.3, qC d 116.0, qC d 113.0, qC d 106.2, qC d 105.7, qC
70 d 200.2, qC d 200.1, qC d 196.2, qC d 180.9, qC d 186.1, qC
80 d 131.7, qC d 131.6, qC d 131.6, qC d 117.7, qC d 108.1, qC
90 d 146.1, qC d 146.1, qC d 144.4, qC d 149.5, qC d 153.2, qC
100 6.79 (d, 8.9) 122.9, CH 6.94 (d, 8.9) 122.9, CH 6.64 (d, 9.0) 119.9, CH 7.62 (d, 9.13) 120.5, CH 6.65 (d, 8.8) 109.8, CH
110 6.91 (d, 8.9) 118.1, CH 6.82 (d, 8.9) 118.2, CH 6.54 (d 9.0) 116.1, CH 7.49 (d, 9.13) 126.1, CH 7.33 (d, 8.8) 124.9, CH
120 d 151.8, qC d 151.8, qC d 153.9, qC d 151.3, qC d 137.5, qC
130 d 112.7, qC d 112.8, qC d 119.7, qC d 116.8, qC d 144.0, qC
140 d 168.3, qC d 168.3, qC d 171.4, qC d 167.0, qC d d
150 2.31 (b, d, 18.1) 35.0, CH2 3.09 (a, d, 19.0) 32.6, CH2 2.84 (b, d, 18.4) 31.9, CH2 3.35 (b, d, 19.3) 33.1, CH2 3.29 (b, d, 18.8) 32.5, CH2
2.68 (a, d, 18.1) 3.28 (b, d, 19.0) 3.08 (a, d, 18.4) 3.61 (a, d, 19.3 3.52 (a, d, 18.8)
6-OH 5.57 (d, 5.42) d 6.24 (d, 7.5) d d 10.34 (d, 6.80) d d d
6-OCOCH3 d d d d d 171.4, qC d d d 170.9, qC
6-OCOCH3 d d d d 2.35 (s) 21.3, CH3 d d 2.40e (s) 20.3, CH3
11-OH 11.40 (s) d 11.45 (s) d d d 11.41 (s) d 11.54 (s) d
13-OH d d d d d d d d df d
14-OH d d 7.20 (s) d d d 7.29 (s) d 6.40 (br s) d
10 -OH 13.38 (s) d 13.37 (s) d d d d d 10.93 (s) d
50 -OH 10.05 (s) d 10.17 (s) d d d d d d d
90 -OH 9.15 (s) d 9.21 (s) d d d d d d d
120 -OH 9.60 (s) d 9.62 (s) d d d d d 8.12f (s) d
140 -OCH3 3.56 (s) 52.4, CH3 3.57 (s) 56.3, CH3 d d 3.86 (s) 52.7, CH3 d d
a
Recorded in DMSO-d6, 400 MHz.
b
Recorded in DMSO-d6, 500 MHz.
c
Recorded in acetone-d6, 400 MHz.
d
Worked out by 1H decoupling experiment.
e
Exchangeable.
f
Exchangeable.
 C. Intaraudom et al. / Tetrahedron 72 (2016) 1415e1421 1417

Fig. 1. Chemical structures of compounds 1e14 isolated from the insect fungus Verticillium sp. BCC33181.

and C-150 ; and from 6-OH to C-6 and C-7. The spectroscopic evi-
dence indicated that the acetyloxy group at C-6 was replaced with
a hydroxyl group. The NOESY spectrum showed cross-peak corre-
lations from H-40 and H-6 to H2-150 , suggesting that they were syn-
facial. Obviously, the different correlation intensity in the NOESY
spectrum to Ha-150 (dH 3.09) and Hb-150 (dH 3.28) was observed,
indicating a pseudoequatorial position of H-6.7 Its CD spectrum was
almost identical to that of the known acremonidin C (4) (Fig. 2).
Therefore, the relative configuration at C-14 was determined to be
the same as that of acremonidin C, reported previously. Compound
3 was then named for acremonidin G.
Compound 5 was obtained as a brown solid. The 1H and 13C NMR
spectra were also similar to that of acremonidin C (4), apart from
the absence of a methoxy signal in 5. Moreover, two methine
protons at H-100 (dH 6.64) and H-110 (dH 6.54) resonated at a higher
field than those of 4 (dH 6.93 and 6.81 for H-100 and H-110 ) and in
the 13C NMR spectrum, the carbonyl carbon at C-140 (dC 171.4) also
appeared at a lower field than that of 4 (dC 167.7).6 The FT-IR
spectrum showed a broad absorption at n 2500e3600 cm1 and
a strong carbonyl absorption for the carboxylic acid carbonyl at nmax
1734 cm1. These spectroscopic evidences indicated the re-
placement of a carbomethoxy group at C-130 with a carboxylic acid.
The remaining structure was confirmed by extensive 2D NMR
spectral analyses including HMQC, COSY, and HMBC. HRESIMS data
confirmed the molecular formula C32H24O13, establishing the mass
ion peak at m/z 639.1110 [MþNa]þ. Moreover, NOESY spectral data
1418 C. Intaraudom et al. / Tetrahedron 72 (2016) 1415e1421
Fig. 2. CD spectra of acremonidins C (3), G (4), and H (5).
showed a cross-peak correlation from H-6 to H2-150 . The CD spec-
trum (Fig. 2) revealed the same pattern to those of acremonidins C
(4) and G (3), therefore compound 5 possessed the relative con-
figuration as shown in Fig. 1 and was given the name as acre-
monidin H.
Compound 7 was obtained as a yellow powder and its UV
spectrum suggested a chemical structure related to acre-
moxanthone B (9),8 displaying UV maxima at 220, 237, 268, 352,
and 394 nm. The 1H NMR spectrum was also similar to that of
compound 9, except for an upfield shift of the oxymethine proton
(H-6) at dH 4.86 (resonating at dH 6.33 in 9),8 and the absence of
a methyl signal. Also, an additional hydroxyl proton at dH 6.34 was
coupled to H-6 in the COSY spectrum and showed HMBC correla-
tion to C-6. The evidence indicated that the acetyloxy group at C-6
of compound 9 was replaced with a hydroxyl group. Moreover, the
two carbons belonging to an acetyl unit were absent in the 13C NMR
spectrum (Table 1). HRESIMS data confirmed the molecular for-
mula to be C31H22O11 by giving the mass ion peak at m/z 593.1059
[MþNa]þ. The NOESY spectrum showed the cross-peak correlations
from H-6 to H-8, and H2-150 ; and from H-40 to H2-150 . The intensity
of the cross-peak correlation from H-6 to Ha-150 (dH 3.61) was
stronger than that to Hb-150 (dH 3.35). Although the cross-peak
correlation between a hydroxyl proton at C-14 and H-6 was not
observed in NOESY spectrum, the NOESY information8 and CD
spectrum (Fig. 3) of compound 7 were consistent with the data
proposed for the relative configuration at C-14 and C-6 of acre-
moxanthone B. Therefore, compound 7 was named acre-
moxanthone F with the chemical structure as shown in Fig. 1.
Fig. 3. CD spectra of acremoxanthones F (7), G (8), and B (9).
Compound (8) was obtained as a yellow powder. HRESIMS data
suggested that it was an isomer of acremoxanthone F (7). The 1H
NMR spectrum was also similar to that of acremoxanthone B (9),8
except that one of the doublet methine signals resonated upfield
at dH 6.65 (Table 1). This methine proton was attributed to the
carbon at dC 109.8 (C-100 ) in the HMQC spectrum and coupled to the
proton at dH 7.33 with a coupling constant of 8.8 Hz, suggesting an
ortho-coupling. In addition, in the 13C NMR spectrum, the acetyl
group signals were absent and the carbon signals at C-100 , C-120 ,
and C-80 of the xanthone moiety were shifted upfield (Table 1),
compared with compound 7. The HMBC spectrum showed corre-
lations from H-100 to C-120 , C-80 , C-90 ; from H-110 (dH 7.33) to C-130 ,
C-90 , and C-120 . Moreover, C-130 and C-70 resonated downfield at dC
144.0 and 186.1, respectively, in the 13C NMR spectrum, suggesting
that the methyl ester at C-130 was replaced with a hydroxyl group.
The NOESY spectrum showed cross-peak correlations from H-6 to
H-8 and H2-150 (with a stronger intensity to Ha-150 ); and from H-40
to H2-150 , the same correlations observed for acremoxanthone F (7).
The CD spectrum (Fig. 3) was similar to that of compounds 7 and 9,
indicating the same relative configuration given for compounds 7
and 9. Thus, compound 8 had the chemical structure as shown in
Fig. 1, and was given the trivial name of acremoxanthone G. In
addition, reaction of compound 7 with Ac2O in the presence of
BF3$Et2O at 0  C yielded acremoxanthone G (8).
Compound 10 was obtained as a yellow powder. HRESIMS data
deduced the molecular formula of compound 10 to be C16H14O8,
establishing the mass ion peak at m/z 357.0580 [MþNa]þ. The 1H
NMR spectrum showed signals of two singlet methyls at dH 2.30 (s)
and 3.55 (s), one singlet sp3 oxymethine at dH 5.28, and four
methines at dH 6.03 (dd, J¼10.6, 2.5 Hz), 6.37 (s), 6.40 (s), and 6.70
(dd, J¼10.6, 2.0 Hz). Two doublet methine protons at dH 6.03 and
6.70 were coupled with the coupling constant of 10.6 Hz, indicating
cis-olefinic protons. Moreover, three signals at dH 5.62, 7.04, and
11.42 disappeared after addition of D2O, indicating the hydroxyl
protons. The 13C NMR spectrum gave 16 carbons, which were dif-
ferentiated by DEPT-135, consisting of two methyl, one sp3 oxy-
methine, four methine, two sp3 quaternary and seven sp2
quaternary signals. Two sp2 quaternary carbons resonating at dC
158.6 and 163.1 must be attached to hydroxyl groups. The IR
spectrum gave the absorption peaks at nmax 1728 (for ester car-
bonyl) and 1703 (for ketone) cm1. The HMBC spectrum displayed
the two ring systems by showing correlations from 3-CH3 to C-2, C-
3, and C-4; from H-2 to C-1, C-4, C-6, and 3-CH3; from H-4 to C-2, C-
5, C-6, and 3-CH3; from 1-OH (dH 11.42) to C-1, C-2, and C-6 for ring
A and correlations from H-10 to C-8, C-9, and C-12; from H-11 to C-
9, C-12, and C-13; and from H-12 to C-10, C-11, C-13, and C-14 for
ring B. In addition, the two rings were linked by a carbonyl carbon
at dC 192.0 (C-7) as a result of a weak correlation from H-4 to C-7
observed in the HMBC spectrum. The methoxy signal at dH 3.55 also
showed the HMBC correlation to the carbonyl carbon at dC 168.4 (C-
14). Together with the mass spectral data, it was deduced that the
molecule possesses an epoxide moiety. Reactions with R/S e
MTPACl indicated the presence of the epoxide at C-8 and C-13 due
to the downfield shift of H-12 (Ddy1.5 ppm). The result (Fig. 4) also
confirmed the absolute configuration at C-12 as S. Furthermore, in
the NOESY spectrum, a cross-peak correlation between H-12 and
14-OCH3 was not observed, thus compound 10 possessed the ab-
solute configuration as 8S, 12R, 13S (Fig. 1). Compound 10 was
named acremonoxide and could be a precursor of the xanthone
unit in anthraquinoneexanthone heterodimers.
Fig. 4. DdSeR of R and S e MTPA derivatives of acremonoxide (10).
 C. Intaraudom et al. / Tetrahedron 72 (2016) 1415e1421 1419

The 1H and 13C NMR spectral data of compounds 2, 4, 6, 9, and
11e14 closely agreed with the published data for acremonidin A,6
acremonidin C,6 acremoxanthones A and B,8 moniliphenone,9
acremonidin E,10 penicillanone,11 and 7-hydroxy-2-(2-
hydroxypropyl)-5-methylchromone,12,13 respectively. The isolated
acremonidins (1e5), acremoxanthones (7 and 8) and acremonoxide
(10) were tested for biological activity against Candida albicans,
Gram-positive (B. cereus and Enterococcus faecium) and Gram-
negative (Escherichia coli, Acinetobacter baumannii, and Klebsiella
pneumoniae) bacteria, and for cytotoxicity against cancerous (KB,
MC-F, NCI-H187) and non-cancerous (Vero) cells. Acremoxanthones
A (6) and B (9) were previously isolated from Acremonium sp.
BCC31806 and their biological activities for anti-B. cereus and anti-C.
albicans have already been reported.8 Compound 6 showed anti-C.
albicans with IC50 value of 1.7 mg/mL and compound 9 showed anti-
B. cereus with MIC value of 6.25 mg/mL. All acremonidins (1e5) ex-
cept acremonidin H (5) gave positive results with antibacterial ac-
tivity against Gram-positive bacteria, exhibiting anti-E. faecium
activity with MIC in a range of 1.56e25 mg/mL and anti-B. cereus with
MIC in a range of 0.39e6.25 mg/mL (Table 2). All tested compounds
were inactive for C. albicans and Gram-negative bacteria at the
maximum tested concentrations (50 mg/mL). Compound 4 displayed
antibacterial activity against both B. cereus and E. faecium with low
cytotoxicity, while compound 5 showed only anti-B. cereus activity
without cytotoxicity at the maximum tested concentration (Table 2).
Compound 10 exhibited cytotoxic activity against KB, MCF-7,
NCIeH187 and Vero cells (IC50 5.50e19.77 mg/mL). Only acre-
moxanthones F (7) and G (8) showed antimalarial activity against P.
falciparum K1 strain at IC50 values of 2.38 and 1.71 mg/mL.
isolated from Acremonium sp. BCC31806 and showed moderate
antibacterial activity against S. aureus (MIC 12.5 and 6.25 mg/mL)
and B. cereus (MIC>100 and 6.25 mg/mL). Acremoxanthone A also
displayed significant anti-C. albicans activity with IC50 value of
1.7 mg/mL.8 Acremoxanthones A (6), B (9), C and E together with
acremonidins A (2) and B were also reported from the endophytic
fungus A. camptosporum W. Gams.7 Acremoxanthones C and E and
acremonidins A (2) and B exhibited a broad range of anti-oomycete
activity against four phytopathogenic oomycetes including Pythium
aphanidermatum, Phytophthora cinnamomi, Phytophthora capsici,
and Phytophthora parasitica, while acremoxanthones A and B were
inactive.7
Anthraquinone e xanthone heterodimers, such as acre-
monidins, acremoxanthones, xanthoquinodins etc., were derived
from the same biosynthesis.15 In addition, compounds 10e14 could
be precursors of acremonidins and acremoxanthones.
Compounds 11 (moniliphenone) and 13 (penicillanone) were
originally isolated from Monilinia fructicola and Penicillium cit-
rinum PSU-RSPG95.9,11 Compound 13 was inactive against both
cancerous and non-cancerous cells at maximum tested concen-
tration (50 mg/mL).11 Acremonidin E was previously isolated from
the lichen Graphis tetralocularis BCC12103 and showed weak
cytotoxic tests against KB, MCF-7 and NCI-H187 (IC50
12.78e27.12 mg/mL) and was active for anti-TB with MIC value of
50 mg/mL).10 Compound 14 [7-hydroxy-2-(2-hydroxypropyl)-5-
methylchromone] was formerly isolated from the mangrove en-
dophytic fungus Pestalotiopsis sp.13 and from rhubarb Rhei Rhi-
zoma (Rheum officinale).12 It was inactive against the murine
cancer cell line L5178Y.13

Table 2
Antimicrobial activity and cytotoxicity of the isolated compounds

Compound Anti-P. falciparum Antibacterial activity (MIC, mg/mL) Cytotoxicity (IC50, mg/mL)
(IC50, mg/mL)
B. cereus E. faecium MCF-7 KB NCI-H187 Vero
1 >10 3.13 12.50 36.69 15.08 6.52 16.60
2 >10 0.39 1.56 >50 15.90 4.18 5.98
3 >10 6.25 25.0 >50 44.21 17.65 18.48
4 >10 1.56 6.25 >50 37.83 >50 47.89
5 >10 6.25 >50 >50 >50 >50 >50
7 2.38 12.50 >50 1.83 18.78 15.84 14.83
8 1.71 25.0 >50 >50 15.14 18.53 15.65
10 >10 >50 >50 19.05 19.77 5.50 5.50
Dihydroartemisinin 7.82104 nt nt nt nt nt nt
Mefloquine 0.011 nt nt nt nt nt nt
Rifampicin nt nt 12.50 nt nt nt nt
Tetracycline HCl nt nt 0.098 nt nt nt nt
Ellipticine nt nt nt nt 2.78 3.15 1.34
Doxorubicin nt nt nt 8.97 0.69 0.04 nt
Tamoxifen nt nt nt 6.84 nt nt nt
Vancomycin nt 1.0e2.0 nt nt nt nt nt

nt¼not being tested.

Acremonidins AeE were previously isolated from Acremonium 3. Conclusion

sp. LL-Cyan416.6 Acremonidin A showed moderate Gram-positive
bacterial activity against both methicillin-resistant Staphylococci
and vancomycin-resistant Enterococci, with MIC in a range of
8e16 mg/mL. Acremonidins A (2) and C (4) together with acre-
moxanthones C and D were also isolated from an unidentified
fungus in the order Hypocreales MSX17022.14 These compounds
showed moderate cytotoxic activity (IC50 13.6e20.4 mM) and were
inactive in the NF-kB and mitochondria transmembrane potential
inhibition tests. Acremoxanthones A (6) and B (9) were originally
Five new xanthone e anthraquinone heterodimers (1, 3, 5, 7, and
8) and its xanthone monomer (10) were isolated from the insect
fungus Verticillium sp. BCC33181. All isolated acremonidins, except
acremonidin H (5), exhibited antibacterial activity against Gram-
positive bacteria (B. cereus and E. faecium) with MIC of
0.39e6.25 mg/mL and 1.56e25.0 mg/mL, respectively, while acre-
moxanthones F (7), G (8), and compound 5 showed antibacterial
activity against B. cereus with MIC of 6.25e25.0 mg/mL with low
1420 C. Intaraudom et al. / Tetrahedron 72 (2016) 1415e1421
cytotoxicity against both cancerous and non-cancerous cells. In
addition, compounds 7 and 8 displayed antimalarial activity against
multidrugeresistant P. falciparum K1 strain with IC50 values of 2.38
and 1.71 mg/mL, respectively.
4. Experimental section
4.1. General experimental procedures
Mps were recorded on a melting point MP90 apparatus from
Mettler Toledo. UV spectra were obtained using a Spekol 1200,
Analytik Jena AG, in MeOH. IR spectra were taken on a Bruker AL-
PHA FT-IR spectrometer. Optical rotations were recorded on
a JASCO P-1030 polarimeter in either CHCl3 or MeOH. CD spectral
data were collected on a JASCO J-810 spectropolarimeter in MeOH.
1D and 2D NMR spectra, including 1H, 13C, DEPT-135, COSY, NOESY,
DEPT-135, HMQC and HMBC experiments, were recorded on either
Bruker Avance 500 NMR spectrometer (at 500 MHz for 1H and
125 MHz for 13C) or Bruker Avance III 400 NMR spectrometer (at
400 MHz for 1H and 100 MHz for 13C), using either acetone-d6 or
DMSO-d6 as internal standards. HR-ESI-MS data were determined
on a Bruker MicrOTOF mass spectrometer. Semi-preparative and
preparative HPLC were performed using Dionex, Ultimate 3000
series model, which was equipped with a binary pump, an auto-
sampler and a diode array detector. Semipreparative HPLC and
Preparative HPLC were carried out using a Waters Sunfire C18 OBD
column (diam. 19150 mm2, particle size 5 mm) at a flow rate of
9 mL/min, and a Waters Sunfire C18 OBD column (diam.
19250 mm2, particle size 10 mm) at a flow rate of 15 mL/min,
respectively, unless otherwise mentioned.
4.2. Fungal material
The fungus was collected from an insect in the family Hyme-
noptera at Doi Suthep-Pui National Park, Chiang Mai province,
Thailand. It was identified based on the analysis of the internal
transcribed spacer (ITS1-5.8S-ITS2) rDNA by using universal fungal
primers. The ITS sequence (GenBank accession number KU292006)
had affinity within fungal taxa Class Sordariomycetes and showed
97e98% similarity with four Verticillium spp. of GenBank accession
numbers GU062214, GQ379907, AF108467, and EF513023. There-
fore, the fungus was identified as Verticillium sp. in the fungal
taxonomic placement of Plectosphaerellaceae, Incertae sedis,
Hypocreomycetidae, Sordariomycetes, Pezizomycotina, Ascomy-
cota. The fungus was then deposited at BIOTEC Culture Collection
(BCC) with the registration code BCC33181.
4.3. Fermentation, extraction, and isolation
The fungus was grown on PDA (potato dextrose agar) and then
cut into pieces. Each piece (11 cm2) was transferred into
20,250 mL Erlenmeyer flasks, which each contained 100 mL PDB
(potato dextrose broth). The seed fungus was grown for four days
on a rotary shaker at 200 rpm. Each flask was then transferred
into four 1L Erlenmeyer flasks, which each contained 250 mL
PDB. The production scale (20 L) was cultivated for 28 days in
static condition at 25  C. The broth and cell were then separated
by simple filtration. The broth was then extracted three times
with equal volume of EtOAc. The EtOAc was dried over Na2SO4
and evaporated to dryness to yield a brown gum (1.74 g). The cell
was macerated with MeOH for three days, then with CH2Cl2 for
three days. The organic solvents were then combined and con-
centrated by rotary evaporator. Water (100 mL) was added and
the mixture was then extracted with hexane twice and followed
by extraction with EtOAc three times. The hexane extraction was
discarded and EtOAc was evaporated to dryness to give a brown
gum (4.50 g).
The crude from the broth was passed through a Sephadex LH20
column (4.5 cm40 cm), using 100% MeOH as an eluent. Six frac-
tions were obtained. The first fraction (64.0 mg) was purified by
semipreparative HPLC using a linear gradient of 5e35 % acetonitrile
in water over 20 min to afford compounds 14 (5.9 mg) and 10
(12.5 mg). The second fraction (0.3 g) was subjected to preparative
HPLC using a linear gradient of 5e65% acetonitrile in water over
20 min to afford compounds 13 (4.5 mg) and 11 (6.2 mg), re-
spectively. The third fraction (0.2 g) was further purified by pre-
parative HPLC using a linear gradient of 10e55% acetonitrile in water
over 20 min to furnish compounds 12 (5.9 mg) and 2 (11.0 mg), re-
spectively. The fourth fraction (0.2 g) was not purified due to con-
taining the known compounds 4 and 2 from analytical HPLC, which
were obtained from other fractions. The fifth fraction (0.2 g) was
further purified by preparative HPLC using a linear gradient of
10e80% acetonitrile in water over 20 min to afford compounds 3
(2.1 mg), 4 (9.2 mg), 9 (27.5 mg) and 6 (9.3 mg), respectively. The
sixth fraction (0.2 g) was subjected to preparative HPLC using a lin-
ear gradient of 15e70% acetonitrile in water over 20 min to yield
compounds 5 (26.5 mg), 7 (7.1 mg) and 8 (3.2 mg), respectively.
The crude from the cell extraction (4.5 g) was passed through
a Sephadex column (4.5 cm90 cm) to give three main fractions.
The first fraction (0.2 g) was further purified by preparative HPLC
using a linear gradient of 10e80% acetonitrile in water over 20 min
to afford compounds 2 (4.2 mg) and 6 (18.0 mg), respectively. The
second fraction (2.8 mg) was subjected to preparative HPLC using
a linear gradient of 20e75% acetonitrile in water over 20 min to
furnish compounds 1 (70.0 mg), 7 (21.2 mg), 4 (0.5 g), 2 (1.56 mg)
and 6 (81.2 mg), respectively. The third fraction (0.1 g) was purified
by preparative HPLC using a linear gradient of 15e60% acetonitrile
in water over 20 min to give compounds 3 (7.9 mg), 7 (7.0 mg) and 8
(1.3 mg), respectively.
4.3.1. Acremonidin F (1). Brown solid, mp 202  C (dec); [a]22 D
þ275.0 (c 0.16, MeOH); UV (MeOH) lmax (log 3 ) 231 (4.32), 277
(4.21), 354 (4.26) nm; FTIR (ATR) nmax 3257 (br), 1669 (w), 1608,
1578, 1463, 1414, 1363, 1286, 1205 cm1; 1H NMR (500 MHz, ace-
tone-d6) and 13C NMR (125 MHz, acetone-d6) data, see Table 1;
HRESIMS m/z 573.1397 [MþH]þ (calcd for C31H25O11, 573.1391).
4.3.2. Acremonidin G (3). Yellow powder; [a]24 D þ95.8 (c 0.08,
MeOH); UV (MeOH) lmax (log 3 ) 228 (4.38), 274 (4.35), 348 (4.18)
nm; FTIR (ATR) nmax 3388 (br), 1622, 1461, 1417, 1208, 1210 cm1; 1H
NMR (400 MHz, acetone-d6) and 13C NMR (100 MHz, acetone-d6)
data, see Table 1; HRESIMS m/z 587.1192 [MH] (calcd for
C31H23O12, 587.1195).
4.3.3. Acremonidin H (5). Brown solid, m.p. 201  C (dec); [a]26 D
þ30.4 (c 0.12, MeOH); UV (MeOH) lmax (log 3 ) 222 (4.27), 270 (4.25),
333 (3.97) nm; FTIR (ATR) nmax 2500e3600 (br), 1734, 1625, 1585,
1470, 1372, 1236 cm1; 1H NMR (500 MHz, DMSO-d6) and 13C NMR
(125 MHz, DMSO-d6) data, see Table 1; HRESIMS m/z 639.1110
[MþNa]þ (calcd for C32H24O13Na, 639.1109).
4.3.4. Acremoxanthone F (7). Yellow powder, mp 212  C (dec); [a]25 D
þ68.3 (c 0.08, MeOH); UV (MeOH) lmax (log 3 ) 237 (3.56), 268
(3.64), 390 (3.13) nm; FTIR (ATR) nmax 3430 (br), 1728, 1637, 1615,
1568, 1503, 1433, 1332, 1296, 1240, 1211, 1178, 1099, 1078, 1014, 856,
740 cm1; 1H NMR (500 MHz, DMSO-d6) and 13C NMR (125 MHz,
 C. Intaraudom et al. / Tetrahedron 72 (2016) 1415e1421
DMSO-d6) data, see Table 1; HRESIMS m/z 593.1059 [MþNa]þ (calcd
for C31H22O11Na, 593.1054).
4.3.5. Acremoxanthone G (8). Yellow powder, mp 204  C (dec);
[a]25
D þ66.3 (c 0.11, MeOH); UV (MeOH) lmax (log 3 ) 226 (4.20), 266
(4.34), 347 (4.00), 420 (3.41) nm; FTIR (ATR) nmax 3447 (br), 1737,
1629, 1587, 1498, 1435, 1368, 1287, 1251, 1210, 1094, 1043 cm1; 1H
NMR (400 MHz, acetone-d6) and 13C NMR (100 MHz, acetone-d6)
data, see Table 1; HRESIMS m/z 571.1233 [MþH]þ (calcd for
C31H23O11, 571.1235).
4.3.6. Acremonoxide (10). Yellow powder, mp 172e173  C; [a]23 D pounds 1, 3, 5, 7, 8, and 10.) associated with this article can be found
66.2 (c 0.16, MeOH); UV (MeOH) lmax (log 3 ) 227 (4.01), 284 (4.00),
354 (3.54), 423 (2.66) nm; FTIR (ATR) nmax 3374 (br), 1728, 1703,
1639, 1567, 1439, 1361, 1267, 1203, 1075, 836, 817 cm1; 1H NMR
(400 MHz, acetone-d6) 2.30 (3H, s, 3-CH3), 3.55 (3H, s, 14-OCH3),
5.28 (1H, s, 12-H), 5.62 (1H, s, OH), 6.03 (1H, dd, J 10.56, 2.51 Hz, 10-
H), 6.37 (1H, s, 2-H), 6.40 (1H, s, 4-H), 6.70 (1H, dd, J 10.56, 2.00 Hz,
11-H), 7.04 (1H, s, OH), 11.42 (1H, s, 1-OH); 13C NMR (100 MHz,
acetone-d6) 21.7 (3-CH3), 52.5 (14-OCH3), 69.2 (12-CH), 70.1 (8-C),
90.5 (13-C), 105.0 (6-C), 108.2 (4-CH), 110.7 (2-CH), 128.5 (10-CH),
144.7 (11-CH), 151.0 (3-C), 158.6 (5-C), 163.1 (1-C), 168.4 (14-C),
187.9 (9-C), 192.0 (7-C); HRESIMS m/z 357.0580 [MþNa]þ (calcd for
C16H14O8Na, 357.0581).
4.4. Biological assays
All compounds, except compounds 6 and 9, were assessed for
antibacterial activity against Gram-positive (B. cereus and E. fae-
cium) and Gram-negative (E. coli, A. baumannii and K. pneumoniae)
bacteria, and for anti-fungal C. albicans, and for cytotoxic activity
against both cancerous (KB, MCF-7, NCI-H187) and non-cancerous
(Vero) cells. Antibacterial activity against E. faecium, E. coli, A.
baumannii, K. pneumoniae was performed based on the standard
protocols published by Clinical and Laboratory Standard In-
stitute.16,17 Rifampicin and erythromycin (or tetracycline HCl) were
used as positive controls. The resazurin microplate assay (REMA)
was employed to evaluate anti-C. albicans, anti-B. cereus and cyto-
toxicity against KB (human epidermoid carcinoma), MCF-7 (human
breast cancer) and NCI-H187 (human small lung cancer) cells.18,19
Amphotericin B and vancomycin were used as positive controls
for anti-C. albicans and anti-B. cereus, respectively. Tamoxifen and
ellipticine were used as positive controls for anti-MCF-7 and anti-
KB activities. Doxorubicin was used as positive control for anti-
MCF-7, anti-KB and anti-NCI-H187 activities. The green fluores-
cent protein microplate assay (GFPMA) was employed to evaluated
cytotoxicity against Vero cell line.20 Ellipticine was used as positive
1421
control. Maximum tested concentration was done at 50 mg/mL for
all tests, except for B. cereus was done at 25 mg/mL.
Acknowledgements
Financial support from National Center for Genetic Engineering
and Biotechnology (BIOTEC) is acknowledged.
Supplementary data
Supplementary data (1D and 2D NMR spectral data of com-
in the online version, at http://dx.doi.org/10.1016/j.tet.2016.01.043.
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