Phytochemistry xxx (2017) 1e9

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Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem

g-Butenolide and furanone derivatives from the soil-derived fungus

Aspergillus sclerotiorum PSU-RSPG178
Patima Phainuphong a, Vatcharin Rukachaisirikul a, *, Kwanruthai Tadpetch a,
Yaowapa Sukpondma a, Saowanit Saithong a, Souwalak Phongpaichit b, Sita Preedanon c,
Jariya Sakayaroj c
a
Department of Chemistry and Center of Excellence for Innovation in Chemistry, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla 90112,
Thailand
b
Natural Products Research Center of Excellence and Department of Microbiology, Faculty of Science, Prince of Songkla University, Hat Yai, Songkhla 90112,
Thailand
c
National Center for Genetic Engineering and Biotechnology (BIOTEC), Thailand Science Park, 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: Chromatographic separation of the broth extract of the soil-derived fungus Aspergillus sclerotiorum PSU-
Received 10 September 2016 RSPG178 resulted in isolation of four g-butenolide-furanone dimers, aspersclerotiorones A-D, a furanone
Received in revised form derivative, aspersclerotiorone E, and two g-butenolide derivatives, aspersclerotiorones F and G, together
26 January 2017
with six known compounds, penicillic acid, dihydropenicillic acid, 5,6-dihydro-6-hydroxypenicillic acid,
Accepted 6 February 2017
Available online xxx
6-methoxy-5,6-dihydropenicillic acid, coculnol and (4R,5R)-4,5-dihydroxy-3-methoxy-5-methylcycl-
ohex-2-en-1-one. Their structures were determined by spectroscopic evidence. For aspersclerotiorones
A and B, the structures were conrmed by single-crystal X-ray diffraction crystallography. Penicillic acid
Keywords:
Aspergillus sclerotiorum
displayed weak antibacterial activity against Staphylococcus aureus and Escherichia coli with equal MIC
g-butenolides values of 128 mg/mL, and it was noncytotoxic towards African green monkey kidney broblast cells.
Furanone derivatives 2017 Elsevier Ltd. All rights reserved.
Antibacterial
Antifungal
Cytotoxic

1. Introduction
Fungi in the genus Aspergillus, which are efcient producers of
various bioactive metabolites, have drawn attention from many
researchers to investigate their metabolites. In recent years, many
new secondary metabolites were isolated from the genus Asper-
gillus. Some exhibited interesting biological activities such as
antitobacco mosaic virus activity (Zhou et al., 2014), anti-
mycobacterium tuberculosis protein tyrosine phosphatase activity
(Liu et al., 2013), antiviral activity against inuenza H1N1 and H3N2
(He et al., 2013) and cytotoxic activity against human gastric
adenocarcinoma (SCG-7901) and human lung adenocarcinoma
(SPC-A-1) cell lines (Wang et al., 2012). In this study, a focus was
placed on the investigation of secondary metabolites from the
fungus Aspergillus sclerotiorum PSU-RSPG178, isolated from a soil
sample collected from the Plant Genetic Conservation Project under
* Corresponding author.
E-mail address: vatcharin.r@psu.ac.th (V. Rukachaisirikul).
the Royal Initiation of Her Royal Highness Princess Maha Chakri
Sirindhorn at Ratchaprapa Dam in Suratthani Province, Thailand.
Recently, three new and four known lovastatin analogues were
isolated from the extracts of the soil-derived fungus A. sclerotiorum
PSU-RSPG178, which showed activity against HMG-CoA reductase
(Phainuphong et al., 2016). As described herein, further chemical
investigation of the ethyl acetate extract of the broth led to isolation
of four new g-butenolide-furanone dimers, aspersclerotiorones A-
D (1e4), one new furanone derivative, aspersclerotiorone E (5), two
new g-butenolide derivatives, aspersclerotiorones F and G (6 and 7)
and six known compounds, penicillic acid (8) (Kimura et al., 1996),
dihydropenicillic acid (9) (Kimura et al., 1996), 5,6-dihydro-6-
hydroxypenicillic acid (10) (Qi et al., 2015), 6-methoxy-5,6-
dihydropenicillic acid (11) (He et al., 2004), coculnol (12) (Nonaka
et al., 2015) and (4R,5R)-4,5-dihydroxy-3-methoxy-5-methylcycl-
ohex-2-en-1-one (13) (Shiono et al., 2005), respectively. In addi-
tion, the absolute congurations of 10 and 11 were reported for
the rst time. Some of the isolated compounds were evaluated
for antimicrobial (Staphylococcus aureus, methicillin-resistance

http://dx.doi.org/10.1016/j.phytochem.2017.02.008
0031-9422/ 2017 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Phainuphong, P., et al., g-Butenolide and furanone derivatives from the soil-derived fungus Aspergillus
sclerotiorum PSU-RSPG178, Phytochemistry (2017), http://dx.doi.org/10.1016/j.phytochem.2017.02.008
2 P. Phainuphong et al. / Phytochemistry xxx (2017) 1e9

O H3CO
12 13 O 3 OH
9
OCH3 2
4 R
8
O O 6
4 5 7 1
1
O 10 O
2 O O 6
3 11
O O
OCH3 8: R = 5
14 1 2 7

O O
8
7 6 7 9: R =
11
10
HO 3 OH 6
9 5
12
13 O O OH 2 OH
O 4
4 5
1 O H3CO O
1
O OCH3 10: R =
14 8
H3CO
3
2 O
4 O 7 OCH3
3
11: R =
12
12 11

O 7 11 H3CO OH HO 7
6 5 HO 6a
HO 4
6
HO
5a 9 10a 4 O 4
5
6
9a O
3 10 14 1
8
H3CO O O
H3CO O O 3 1
9
2
H3CO H3CO O
13 3 O 8
2
13
5 6 12 13

Fig. 1. Structures of compounds 1e13 isolated from Aspergillus sclerotiorum PSU-RSPG178.

S. aureus, Escherichia coli and Microsporum gypseum), anti- specic rotations, as well as their CD data, with those of known or
mycobacterial (Mycobacterium tuberculosis, H37Ra strain), antima- structurally related compounds. The S conguration at C-4 of 10 (Qi
larial (Plasmodium falciparum, K1 strain) and cytotoxic (KB-oral et al., 2015) was established using the CD spectral data which
cavity, MCF-7 breast cancer cells and Vero-African green monkey showed a positive Cotton effect at 220 nm (D 4.28), the same
kidney broblast cells) activities. sign as that of (6Z,9Z,12Z)-(S)-4-methoxyoctadeca-2,6,9,12-tetraen-
4-olide at 205 nm, D 0.63, c 1  104 M, MeOH (Mansoor
2. Results and discussion et al., 2004). It is worth noting that the bathochromic shift of the
p-p* transition absorption maximum in CD spectrum of 10 might
All isolated compounds (1e13) (Fig. 1) were puried using be due to the effect of the methoxy substituent at the b-position of
chromatographic techniques and their structures were elucidated the a,b-unsaturated furanone moiety (Gawronski et al., 1997). To
by using various spectroscopic techniques. The structures of com- establish the absolute conguration at C-5 of 10, it was converted to
pounds 1 and 2 were conrmed by single-crystal X-ray diffraction its acetonide derivative (10a). The relative conguration of 10a was
crystallography (Fig. 2), with relative congurations assigned ac- determined by a coupling constant value as well as NOEDIFF data
cording to the NOEDIFF data. The absolute conguration of the (Fig. 3). A large coupling constant of 11.7 Hz between Ha-6 (dH 3.85)
isolated compounds was established by comparison of their and H-5 (dH 2.30) was consistent with an axial orientation of H-5.

Fig. 2. ORTEP drawings of compounds 1 and 2.

Please cite this article in press as: Phainuphong, P., et al., g-Butenolide and furanone derivatives from the soil-derived fungus Aspergillus
sclerotiorum PSU-RSPG178, Phytochemistry (2017), http://dx.doi.org/10.1016/j.phytochem.2017.02.008
 P. Phainuphong et al. / Phytochemistry xxx (2017) 1e9 3

Fig. 3. Key NOEDIFF data of the acetonide derivative (10a) of compound 10.
This assignment was conrmed by irradiation of H-5 which
enhanced the signal intensity of Hb-6, but not that of Ha-6, together
with irradiation of an equatorial methyl group (H3-7, dH 0.70) which
affected the signal intensity of Hab-6 (dH 3.85 and 3.74). Conse-
quently, the absolute conguration at C-5 was determined to be S.
The absolute congurations at C-4 and C-5 of 11 were assigned to
be identical to those of 10 on the basis of the positive Cotton effect
at 222 nm (D 19.01) in CD spectrum as well as the positive
specic rotation of 11, a24
D 20 (c 0.20, CHCl3), which was almost
identical to that of 10, a24
D 23 (c 0.20, CHCl3).
Aspersclerotiorone A (1) was obtained as colorless crystals with
the molecular formula C14H16O5 deduced from HRESIMS peak at m/
z 265.1076 [MH]. The UV spectrum showed an absorption band
at 269 nm, indicating the presence of a conjugated lactone chro-
mophore (Nonaka et al., 2015). The IR spectrum displayed ab-
sorption bands at 1749 cm1 for an a,b-unsaturated lactone
carbonyl functional group and 1698 cm1 for an a,b-unsaturated
carbonyl ketone functional group (He et al., 2004). The 1H NMR
spectroscopic data (Table 1) contained signals for two olenic
protons (dH 7.93 and 5.27, each s, 1H), one methoxy group (dH 3.92,
s, 3H), one set of nonequivalent methylene protons (dH 3.08 and
2.34 each d, J 15.0 Hz, 1H) and three methyl groups (dH 2.01, 1.71
and 1.35 each s, 3H). The 13C NMR spectrum (Table 1) consisted of
two carbonyl carbons (dC 206.9 and 167.7 for ketone and a,b-un-
saturated lactone carbonyl carbons, respectively), ve quaternary
carbons (dC 170.9, 141.4, 119.9, 113.4 and 88.4), two methine carbons
(dC 172.0 and 90.6), one methoxy carbon (dC 59.2), one methylene
carbon (dC 39.9) and three methyl carbons (dC 21.2, 17.3 and 5.4). A
g-butenolide ring with a methoxy group and an exocyclic tetra-
substituted double bond at C-3 (dC 170.9) and C-4 (dC 141.4) of the
g-butenolide moeity, respectively, was constructed on the basis of
HMBC correlations of the olenic proton (H-2, dH 5.27) with C-1 (dC
167.7), C-3 and C-4, the methoxy protons (H3-14, dH 3.92) with C-3
(Fig. 4), as well as the chemical shifts of C-1 and C-4. In additions,
the same correlations from the methyl protons (H3-11, dH 2.01) to C-
4, C-5 (dC 119.9) and C-6 (dC 39.9) attached a methyl group and a
methylene unit at C-5 of the exocyclic double bond. According to
HMBC correlations from the olenic proton (H-10, dH 7.93) to C-7
(dC 88.4), C-8 (dC 206.9) and C-9 (dC 113.4), from the methyl protons
(H3-13, dH 1.71) to C-8, C-9 and C-10 (dC 172.0) and from the methyl
protons (H3-12, dH 1.35) to C-7 and C-8, together with the chemical
shifts of C-7 and C-10, a furanone ring with two methyl groups at C-
7 and C-9 was constructed. HMBC correlations of Hab-6 (dH 3.08 and
2.34) with C-7, C-8 and C-12 (dC 21.2) linked C-6 of the g-butenolide
with C-7 of the furanone unit. In the NOEDIFF experiment (Fig. 5),
irradiation of H3-14 enhanced signal intensity of H-2 and H3-11,
indicating a Z-conguration of the exocyclic double bond. The
assigned structure of 1 was conrmed by X-ray data (Fig. 2). The
absolute conguration at C-7 was assigned to be R according to a
negative Cotton effect (D 2.48, at 267 nm), similar to that of
spheciospongone A (D 1.02, at 265 nm) (Liu et al., 2009). This
conclusion was consistent with the observed specic rotation of 1,
a26
D 42 (c 0.5, CHCl3), which was similar to that of (5R)-2-
methoxy-3,5-dimethyl-5-pentyl-4-(5H)-furanone, aD 50.6
(c 0.7, CHCl3) (Kapferer et al., 2005). Therefore, 1 was assigned as
a new dimeric g-butenolide-furanone derivative.
Aspersclerotiorone B (2) was obtained as colorless crystals with
the identical molecular formula to 1. Their IR, UV, 1H NMR and 13C
NMR data (Table 1) were similar. The difference was observed in the
NOEDIFF experiment (Fig. 5). Irradiation of H3-14 enhanced only
the signal intensity of H-2, but not that of H3-11. These results
established an E-conguration of the exocyclic double bond.
Moreover, the structure of 2 was conrmed by X-ray data (Fig. 2).
Finally, the absolute conguration at C-7 was proposed to be R,

Table 1
1
H and 13C NMR spectroscopic data of compounds 1, 2 and 7 in CDCl3.

Position 1 2 7
a b a b
dC, dH, mult. dC, dH, mult. dC,c dH,d mult.
type (J, Hz) type (J, Hz) type (J, Hz)

1 167.7, C 167.8, C 170.1, C

2 90.6, CH 5.27, s 90.6, CH 5.28, s 119.0, CH 5.88, brs
3 170.9, C 170.6, C 165.1, C
4 141.4, C 141.2, C 104.3, C
4-OH 4.10, brs
5 119.9, C 120.2, C 157.1, C
6 39.9, CH2 a: 3.08, d (15.0) 37.9, CH2 a: 3.33, d (14.1) 84.3, CH2 a: 4.49, d (3.5)
b: 2.34, d (15.0) b: 2.39, d (14.1) b: 4.28, d (3.5)
7 88.4, C 88.4, C 12.7, CH3 2.04, d (1.5)
8 206.9, C 207.0, C 55.8, CH3 3.64, s
9 113.4, C 113.6, C
10 172.0, CH 7.93, s 172.1, CH 7.92, s
11 17.3, CH3 2.01, s 19.1, CH3 1.94, s
12 21.2, CH3 1.35, s 20.9, CH3 1.30, s
13 5.4, CH3 1.71, s 5.3, CH3 1.70, s
14 59.2, CH3 3.92, s 59.2, CH3 3.95, s
a
Recorded at 125 MHz.
b
Recorded at 500 MHz.
c
Recorded at 75 MHz.
d
Recorded at 300 MHz.

Please cite this article in press as: Phainuphong, P., et al., g-Butenolide and furanone derivatives from the soil-derived fungus Aspergillus
sclerotiorum PSU-RSPG178, Phytochemistry (2017), http://dx.doi.org/10.1016/j.phytochem.2017.02.008
4 P. Phainuphong et al. / Phytochemistry xxx (2017) 1e9

Fig. 4. Key HMBC correlations of compounds 1, 2 and 7.

identical to that of 1, according to its CD spectrum which showed a
negative Cotton effect at 264 nm (D 2.37), the same sign as that
of 1. This assignment was consistent with the specic rotation of 2,
a26
D 43 (c 0.5, CHCl3), which was almost identical to that of 1.
Thus, 2 was assigned as a geometrical isomer of 1.
Aspersclerotiorone C (3) was obtained as a colorless solid with
the molecular formula C14H18O6 determined by the HRESIMS peak
at m/z 283.1176 [MH]. The IR spectrum showed absorption bands
at 3418 and 1746 cm1, suggesting the presence of hydroxy and a,b-
unsaturated lactone groups. The UV spectrum exhibited an ab-
sorption band at 224 nm, corresponding to an a,b-unsaturated
lactone chromophore (Liu et al., 2005). The 1H NMR spectroscopic
data (Table 2) contained signals for one olenic proton (dH 5.19, s,
1H), two methine protons [dH 4.17 (dd, J 5.4 and 4.5 Hz) and 2.14
(m), each 1H], one methoxy group (dH 3.94, s, 3H), one hydroxy
group (dH 2.95, brs, 1H), one set of nonequivalent methylene pro-
tons [dH 2.78 (d, J 13.2 Hz, 1H) and 1.42 (ddd, J 13.2, 5.4 and
1.5 Hz, 1H)] and three methyl groups [dH 1.49 (s, 3H), 1.02 (d,
J 6.9 Hz, 3H) and 0.98 (s, 3H)]. The 13C NMR spectrum (Table 2)
contained one a,b-unsaturated lactone carbonyl carbon (dC 168.4),
ve quaternary carbons (dC 175.9, 112.2, 108.1, 92.3 and 53.2), three
methine carbons (dC 92.3, 78.0 and 47.5), one methoxy carbon (dC
59.9), one methylene carbon (dC 34.6) and three methyl carbons (dC
16.3, 11.1 and 7.8). The g-butenolide moiety with a methoxy group
at C-3 (dC 175.9) was constructed by HMBC correlations (Table 2) of
the olenic proton (H-2, dH 5.19) with C-1 (dC 168.4), C-3 and C-4 (dC
108.1) and that of the methoxy protons (H3-14, dH 3.94) with C-3 as
well as the chemical shifts of C-1 and C-4. A 1,2,5-trimethyl-7-
oxabicyclo[2.2.1]heptanyl unit was established by the following
1
H-1H COSY correlations: H3-12 (dH 1.02)/H-8 (dH 2.14), H-8/H-7 (dH
4.17) and H-7/Hb-6 (dH 1.42) together with HMBC correlations from
H3-11 (dH 0.98) to C-5 (dC 53.2), C-6 (dC 34.6) and C-10 (dC 92.3),
from H3-12 to C-7 (dC 78.0), C-8 (dC 47.5) and C-9 (dC 112.2) and from
H3-13 (dH 1.49) to C-5, C-9 and C-10 (dC 92.3). According to the
chemical shifts of C-7 and C-10, as well as an HMBC correlation of
H-7 with C-10, an ether linkage between C-7 and C-10 was formed.
This 7-oxabicyclo[2.2.1]heptanyl unit was connected to C-4 of the
g-butenolide moiety due to HMBC correlations of Hab-6 and H3-11
with C-4. Moreover, the second ether linkage between C-4 and C-9
was established on the basis of the molecular formula and the
chemical shift of C-4. These results along with the chemical shift of
C-9 indicated that the hydroxy group resonating at dH 2.95 was
attached at C-9. In the NOEDIFF experiment (Fig. 5), irradiation of
H3-13 enhanced signal intensity of H3-11, indicating their

Fig. 5. Key NOEDIFF data of compounds 1e6.

Please cite this article in press as: Phainuphong, P., et al., g-Butenolide and furanone derivatives from the soil-derived fungus Aspergillus
sclerotiorum PSU-RSPG178, Phytochemistry (2017), http://dx.doi.org/10.1016/j.phytochem.2017.02.008
 P. Phainuphong et al. / Phytochemistry xxx (2017) 1e9 5

Table 2
1
H, 13C NMR and HMBC spectroscopic data of compounds 3 and 4 in CDCl3.

Position 3 4

dC,a dH,b mult. HMBC dC,a dH,b mult. HMBC

type (J, Hz) type (J, Hz)

1 168.4, C 169.3, C
2 92.3, CH 5.19, s 1, 3, 4 90.8, CH 5.12, s 1, 3, 4
3 175.9, C 178.6, C
4 108.1, C 106.7, C
4-OH 6.09, brs
5 53.2, C 47.6, C
6 34.6, CH2 a: 2.78, d (13.2) 4, 5, 8, 10, 11 33.9, CH2 a: 3.00, dd (14.1, 6.0) 4, 5, 7
b: 1.42, ddd (13.2, 5.4, 1.5) 4, 5, 7, 8, 11 b: 1.67, d (14.1) 4, 7, 8, 11
7 78.0, CH 4.17, dd (5.4, 4.5) 5, 9, 10 77.2, CH 4.71, t (6.0) 5, 6, 8, 9, 10
8 47.5, CH 2.14, m 6, 7, 9, 12 48.0, CH 2.66, m 6, 7, 9, 12
9 112.2, C 212.4, C
9-OH 2.95, brs
10 92.3, C 92.4, C
11 16.3, CH3 0.98, s 4, 5, 6, 10 17.5, CH3 0.88, s 4, 5, 6, 10
12 7.8, CH3 1.02, d (6.9) 7, 8, 9 10.0, CH3 1.10, d (7.2) 7, 8, 9
13 11.1, CH3 1.49, s 5, 9, 10 13.3, CH3 1.65, s 5, 9, 10
14 59.9, CH3 3.94, s 3 59.8, CH3 3.90, s 3
a
Recorded at 75 MHz.
b
Recorded at 300 MHz.

proximity. Irradiation of Ha-6 enhanced signal intensity of H3-12
but did not affect the signal intensity of H3-11. These results sug-
gested that H3-12 was trans to H3-11. In addition, a positive Cotton
effect at 218 nm (D 13.43) in the CD spectrum, the same sign as
that in 10 and 11, indicated S-conguration at C-4. Therefore, the
absolute congurations at C-5 and C-7 were assigned to be both S
whereas those at C-8, C-9 and C-10 were assigned to be all R. Thus, 3
was assigned as a new spirofuranone-g-butenolide derivative.
Aspersclerotiorone D (4) was obtained as a colorless gum with
the molecular formula C14H18O6 determined by the HRESIMS peak
at m/z 283.1176 [MH]. Its UV spectrum was almost identical to
those of 3, indicating that they possessed the same chromophore.
The IR spectrum was similar to that of 3 except for one additional
absorption band at 1715 cm1 for a ketone carbonyl functional
group. In addition, their 1H and 13C NMR spectroscopic data
(Table 2) were similar except for the replacement of an oxy-
quaternary carbon at dC 112.2 (C-9) in the 13C NMR spectrum of 3
with a ketone carbonyl carbon (dC 212.4) in 4. These data suggested
that the hemi-ketal moiety in 3 was converted to a ketone func-
tional group. This conclusion was conrmed by HMBC correlations
(Table 2) from H3-12 (dH 1.10) to C-9 (dC 212.4) and from H3-13 (dH
1.65) to C-5 (dC 47.6), C-9 and C-10 (dC 92.4). Compound 4 had the
same relative conguration as 3 according to their NOEDIFF data
(Fig. 5). It showed a positive Cotton effect at 218 nm (D 6.12) in
CD spectrum, similar to that of 10, indicating the identical S-
conguration at C-4. Accordingly, the remaining absolute congu-
rations in 4 were proposed to be 5S, 7S, 8R and 10R, identical to
those of 3. Therefore, aspersclerotiorone D had the structure 4.
Aspersclerotiorone E (5) was obtained as a colorless gum with
the molecular formula C14H16O5 determined by the HRESIMS peak
at m/z 287.0899 [MNa]. Its UV spectrum exhibited absorption
bands at 204, 219 and 287 nm, indicating the presence of a benzene
ring chromophore. The IR spectrum showed absorption bands at
3455 and 1735 cm1 for hydroxy and ketone carbonyl functional
groups, respectively. The 1H NMR spectroscopic data (Table 3)
contained signals for one aromatic proton of a pentasubstituted
benzene (dH 6.28, s, 1H), one oxymethine proton (dH 5.87, d,
J 5.4 Hz, 1H), one hydroxy group (dH 5.39, brs, 1H), one methoxy
group (dH 3.84, s, 3H), one methine proton (dH 2.76, m, 1H) and
three methyl groups [dH 2.31 (s, 3H), 1.89 (s, 3H) and 1.14 (d,
J 7.5 Hz, 3H)]. The 13C NMR spectrum (Table 3) indicated the
presence of fourteen carbon resonances, including one ketone
carbonyl carbon (dC 206.5), six quaternary carbons (dC 147.5, 144.9,
139.0, 121.0, 113.8 and 80.6), three methine carbons (dC 98.6, 97.1
and 48.0), one methoxy carbon (dC 56.0) and three methyl carbons
(dC 18.9, 13.1 and 11.1). The aromatic proton of the pentasubstituted
benzene (dH 6.28) was assigned as H-9 and displayed HMBC cross-
peaks with C-5a (dC 113.8), C-7 (dC 139.0), C-8 (dC 147.5) and C-9a (dC
144.9). A methoxy group was attached at C-8 according to an HMBC
correlation from the methoxy protons (H3-13, dH 3.84) to C-8. Signal
enhancement of H3-13 after irradiation of H-9 in the NOEDIFF
experiment (Fig. 5) supported the above assignment. The substit-
uent at C-7 was a hydroxy group according to HMBC correlations
from the hydroxy proton (7-OH, dH 5.39) to C-6 (dC 121.0), C-7 and
C-8. The methyl group at dH 2.31 (H3-12) was attached to C-6 due to
its HMBC correlations with C-5a, C-6 and C-7. A furanone moiety
with two methyl groups at C-3 (dC 48.0) and C-5 (dC 80.6) was
established according to the 1H-1H COSY correlations of H-2 (dH
5.78)/H-3 (dH 2.76) and H-3/H3-10 (dH 1.14), and the HMBC corre-
lations (Table 3) from: H3-10 to C-2 (dC 98.6), C-3 and C-4 (dC 206.5);
H3-11 (dH 1.89) with C-4 and C-5 and H-2 to C-5, as well as the
chemical shifts of C-2 and C-5. A linkage between C-5 of the fur-
anone ring and C-5a of the pentasubstituted benzene was estab-
lished due to the HMBC of H3-11 with C-5a. This formation was
conrmed by signal enhancement of H3-12 after irradiation of H3-
11 in the NOEDIFF experiment (Fig. 5). An ether linkage between C-
2 and C-9a was constructed on the basis of an HMBC correlation of
H-2 with C-9a, as well as from the chemical shifts of C-2 and C-9a.
The relative conguration of 5 was determined by NOEDIFF data.
Irradiation of the H-2 enhanced signal intensity of H-3 and H3-10,
whereas irradiation of H3-10 enhanced the signal intensity of H-2,
H-3 and H-9. These results were consistent with an endo-position
of H3-10. The absolute congurations of both C-3 and C-5
were assigned to be R according to a negative Cotton effect at
295 nm (D 9.15), the same sign to that of (R,R)-trans-2,6-
dimethylcyclohexanedione monoethylene acetal (D 215, at
295 nm) (Fleischhauer et al., 2000). It is worth noting that the
absolute conguration at C-5 was identical to that of C-7 in the
cometabolites 1 and 2. Consequently, the remaining absolute
conguration at C-2 was then assigned to be R. Thus, 5 was assigned
as a new fused benzene-furanone derivative.
Aspersclerotiorone F (6) was obtained as a colorless gum with
the molecular formula C16H18O6, as deduced from the HRESIMS
peak at m/z 329.0999 [MNa]. Its UV and IR spectra were similar

Please cite this article in press as: Phainuphong, P., et al., g-Butenolide and furanone derivatives from the soil-derived fungus Aspergillus
sclerotiorum PSU-RSPG178, Phytochemistry (2017), http://dx.doi.org/10.1016/j.phytochem.2017.02.008
6 P. Phainuphong et al. / Phytochemistry xxx (2017) 1e9

Table 3
1
H, 13C NMR and HMBC spectroscopic data of compounds 5 and 6 in CDCl3.

Position 5 6

dC,a dH,b mult. HMBC dC,a dH,b mult. HMBC

type (J, Hz) type (J, Hz)

1 169.7, C
2 98.6, CH 5.87, d (5.4) 3, 4, 5, 9a 90.5, CH 5.19, s 1, 3, 4
3 48.0, CH 2.76, m 2, 10, 4 177.2, C
4 206.5, C 103.7, C
5 80.6, C 30.5, CH 2.39, m 4, 6, 6a, 11
5a 113.8, C
6 121.0, C 27.0, CH2 a: 2.71, dd (16.0, 5.5) 4, 5, 6a, 7, 10a, 11
b: 2.50, dd (16.0, 13.0) 4, 5, 6a, 7, 10, 10a, 11
6a 112.6, C
7 139.0, C 121.4, C
7-OH 5.39, brs 6, 7, 8
8 147.5, C 138.7, C
8-OH 5.36, brs 7, 8, 9
9 97.1, CH 6.28, s 5, 5a, 7, 8, 9a 145.30, C
9a 144.9, C
10 11.1, CH3 1.14, d (7.5) 2, 3, 4 97.6, CH 6.32, s 6, 6a, 8, 9, 10a
10a 144.2, C
11 18.9, CH3 1.89, s 4, 5, 5a 14.8, CH3 1.01, d (6.5) 4, 5, 6, 6a
12 13.1, CH3 2.31, s 5a, 6, 7 11.0, CH3 2.13, s 6a, 7, 8
13 56.0, CH3 3.84, s 8 59.8, CH3 3.98, s 3
14 56.0, CH3 3.80, s 9
a
Recorded at 75 MHz.
b
Recorded at 300 MHz.

to those of 5. Apart from 1H NMR signals of the furanone ring of 3
and the pentasubstituted aromatic ring of 5, compound 6 consisted
of signals for a eCH2CH(CH3)e unit [dH 2.71 (dd, J 16.0 and 5.5 Hz,
1H), 2.50 (dd, J 16.0 and 13.0 Hz, 1H), 2.39 (m, 1H) and 1.01 (d,
J 6.5 Hz, 3H)]. This unit was conrmed by 1H-1H COSY correla-
tions of H3-11 (dH 1.01)/H-5 (dH 2.39) and H-5/Hab-6 (dH 2.71 and
2.50), as well as by the HMBC (Table 3) from H3-11 to C-5 (dC 30.5)
and C-6 (dC 27.0). The HMBC of Hab-6 with C-4 (dC 103.7), C-6a (dC
112.6), C-7 (dC 121.4) and C-10a (dC 144.2) linked C-6 and C-5 with
C-6a of the pentasubstituted benzene and C-4 of the furanone ring,
respectively. An ether linkage between C-4 and C-10a was estab-
lished on the basis of their chemical shifts and the molecular for-
mula of 6. The relative conguration was determined by coupling
constants, as well as from NOEDIFF data (Fig. 5). A large coupling
constant of 13.0 Hz between Hb-6 (dH 2.50) and H-5 was consistent
with an axial orientation of H-5. This assignment was conrmed by
irradiation of the equatorial H3-11 (dH 1.01) which affected the
signal intensity of H-5, Hab-6 and H3-13 (dH 3.98). Its CD spectrum
showed a positive Cotton effect at 240 nm (D 3.89), which was
similar to the quantum chemical TDDFT calculated ECD spectrum of
(2S,3S)-isomer of aspergispiroketal (Chen et al., 2013). From this
result, the absolute conguration at C-4 was proposed to be S,
identical to that of the cometabolites 3, 4 and 8e11. Consequently,
the absolute conguration at C-5 was then assigned to be S.
Aspersclerotiorone G (7) was obtained as a colorless gum with
the molecular formula C8H10O4 determined by the HRESIMS peak at
m/z 193.0477 [MNa]. Its IR spectrum showed absorption bands
at 3370 and 1747 cm1, suggesting the presence of hydroxy and a,b-
unsaturated lactone groups (He et al., 2004). The UV spectrum
exhibited an absorption band at 212 nm for a conjugated carbonyl
chromophore of an a,b-unsaturated lactone (Xu et al., 2011). The 1H
and 13C NMR data (Table 1) suggested that 4 and 7 possessed the
similar g-butenolide moiety. The obvious differences were the
replacement of the methoxy signal at C-3 in 4 with a methyl signal
(dH 2.04, d, J 1.5 Hz, 3H) in 7, and resonances of the 1,2,5-
trimethyl-7-oxabicyclo[2.2.1]-heptanonyl unit in 4 with signals
for e(CH3O)C]CH2 unit [dH 4.49 (d, J 3.5 Hz, 1H), 4.28 (d,
J 3.5 Hz, 1H) and 3.64 (s, 3H)] in 7. This unit was established by the
HMBC of H3-8 (dH 3.63) with C-5 (dC 157.1) and C-6 (dC 84.3). The
HMBC of the gem-olenic protons (Hab-6, dH 4.49 and 4.28) with C-
4 (dC 104.3) supported the attachment of the e(CH3O)C]CH2 unit
at C-4. According to the chemical shift of C-4, the hydroxy group
resonating at dH 4.10 was then attached at this carbon. Signal
enhancement of H3-7 (dH 2.04) upon irradiation of H-2 (dH 5.88) in
the NOEDIFF experiment and HMBC correlations of H3-7 to C-2 (dC
119.0), C-3 (dC 165.1) and C-4 conrmed the attachment of the
methyl group at C-3. The similar CD data of 7 (D 1.37, at
213 nm) to that of (2S)-2,5-diydro-2-hydroxy-3,4-dimethyl-5-oxo-
2-furantridecanoic acid methyl ester (D 0.3, at 213 nm) (Lee
et al., 2007) suggested that 7 should possess S conguration at C-4.
Compounds 1e2, 4 and 8e12 were tested for antimycobacterial
(M. tuberculosis, H37Ra strain), antimalarial (P. falciparum, K1 strain)
and cytotoxic (KB, MCF-7 and Vero cell lines) activities, while 8e12
with sufcient amount were also tested for antimicrobial activity
against S. aureus (ATCC25923), methicillin-resistance S. aureus,
E. coli (ATCC25) and M. gypseum (Table 4). Compounds 1e2, 4, 9,
and 12 were inactive in all tested assays. Only compound 8 dis-
played mild antimalarial activity with an IC50 value of 16.8 mM, mild
antimycobacterial activity with an MIC value of 25.0 mg/mL, and
weak antimicrobial activity against S. aureus, methicillin-resistance
S. aureus, E. coli and M. gypseum with the MIC values of 128, 200, 128
and 200 mg/mL, respectively. For cytotoxic activity, compound 10
was completely inactive against both KB and MCF-7 cell lines
whereas compound 8 showed no activity against only KB cell lines.
Although compounds 8 and 11 gave IC50 values in the range of
88.6e234.6 mM, they were considered to be non-cytotoxic towards
the tested cancer cell lines, compared to the standard drugs. In
addition, compounds 8, 10 and 11 displayed non-cytotoxic activity
to Vero cells according to their IC50 values which were higher than
10 mM.
3. Conclusion
This work reported the isolation of 13 secondary metabolites
from the broth extract of the soil-derived fungus A. sclerotiorum
PSU-RSPG178, including four new g-butenolide-furanone dimers

Please cite this article in press as: Phainuphong, P., et al., g-Butenolide and furanone derivatives from the soil-derived fungus Aspergillus
sclerotiorum PSU-RSPG178, Phytochemistry (2017), http://dx.doi.org/10.1016/j.phytochem.2017.02.008
 P. Phainuphong et al. / Phytochemistry xxx (2017) 1e9 7

Table 4
Antimicrobial, antimycobacterial, antimalarial, and cytotoxic activities for compounds 8, 10 and 11.

Compound Antimicrobial (MIC, mg/mL) Antimycobacterial (MIC, mg/mL) Antimalarial (IC50, mM) Cytotoxic (IC50, mM)

S. aureus methicillin-resistant E. coli M. gypseum M. tuberculosis H37Ra P. falciparum KB MCF-7 Vero

S. aureus strain

8 128 200 128 200 25.00 16.8 IN 234.6 16.5

10 IN IN IN IN IN IN IN IN 259.5
11 IN IN IN IN IN IN 88.6 160.5 75.6
vancomycin 1.00 0.50
gentamicin 0.50
miconazol 2.00
rifampicin 0.0063
streptomycin 0.6250
isoniazid 0.0469
ooxacin 0.391
ethambutol 0.469
dihydroarte-misinine 0.0023
meoquine 0.0269
ellipticine 8.24 4.06
doxorubicin 2.19 12.42
tamoxifen 18.81

IN inactive.

(1-4), one new furanone derivative (5), and two new g-butenolide
derivatives (6 and 7) together with six known compounds, which
are ve g-butenolide derivatives (8e12) and one cyclohexanone
derivative (13). Compound 8, a major component produced by this
fungus, has been reported to be derived from orsellinic acid (Seto
et al., 1974) which is obtained from the condensation of an
acetyl-CoA and three malonyl-CoA units (Ding et al., 2010). The
remaining compounds might have the similar biosynthesis as
compound 8. Accordingly, their biosynthetic pathway is of interest.
4. Experimental
4.1. General experimental procedures
Melting points were determined on an Electrothermal 9100
melting point apparatus and reported without correction. Optical
rotations were recorded on a JASCO P-1020 polarimeter. The ul-
traviolet (UV) absorption spectra were measured in MeOH on a
Perkin-Elmer Lambda 45 spectrophotometer. The infrared (IR)
spectra were recorded neat using a Perkin-Elmer 783 FTS165 FT-IR
spectrometer. Mass spectra were obtained using a MAT 95 XL mass
spectrometer (Thermo Finnigan), Bruker MicrOTOF mass spec-
trometer or a liquid chromatograph-mass spectrometer (2090, LCT,
Waters, Micromass). The 1H and 13C NMR spectra were recorded on
a 300 or 500 MHz Bruker FTNMR Ultra Shield spectrometer.
Chemical shifts are expressed in d (parts per million, ppm) referring
to the tetramethylsilane peak. Thin-layer chromatography (TLC)
and preparative TLC (PTLC) were performed on silica gel 60 GF254
(Merck). Column chromatography (CC) was carried out on Sepha-
dex LH-20 with MeOH, silica gel (Merck) type 60 (230e400 mesh
ASTM) or type 100 (70e230 mesh ASTM), or on reversed phase C18
silica gel, respectively.
4.2. Fungal material
The soil fungus PSU-RSPG178 was isolated from a soil sample
from Suratthani province, Thailand and deposited as BCC56851 at
BIOTEC Culture Collection, National Center for Genetic Engineering
and Biotechnology (BIOTEC), Thailand. PSU-RSPG178 was identied
by morphological characteristics and the analysis of an internal
transcribed spacer (ITS1-5.8S-ITS2) rDNA using universal fungal
primers (White et al., 1990). Its colony was yellow, occose and
rapid growing. The microscopic features showed hyaline and
septate hyphae, unbranched conidiophores with a vesicle. Phialides
were borne directly on metulae. Phylogenetic analysis using
maximum parsimony method indicated that the fungus RSPG178
(Genbank accession number KC478521) was placed in a subclade
with several strains of Aspergillus sclerotiorum with A. sclerotiorum
AY373866 the most similar taxon (99% nucleotide identity).
Therefore, the fungus RSPG178 could be identied as
A. sclerotiorum.
4.3. Fermentation, extraction and isolation
A. sclerotiorum PSU-RSPG178 was grown on potato dextrose agar
at 25  C for 5 days. Five pieces (0.5  0.5 cm2) of mycelial agar plugs
were inoculated into 500 mL Erlenmeyer asks containing potato
dextrose broth (300 mL) at room temperature for 3 weeks. The ask
culture (16 L) was ltered to separate into the ltrate and wet
mycelia. The ltrate was extracted twice with EtOAc (2  300 mL).
The organic layer was dried (anhyd. Na2SO4) and evaporated to
dryness under reduced pressure to afford a dark brown gum
(9.43 g). The broth extract was then separated by Sephadex LH-20
CC using MeOH to give three fractions (A-C). Fraction B (8.26 g) was
subjected to silica gel CC using a gradient of EtOAc:petroleum ether
(3:17 / 1:0) to afford nine fractions (B1-B9). Fraction B4 (4.48 g)
was further puried by silica gel CC using a gradient of aceto-
ne:CHCl3 (0:1 / 1:0) to give eight fractions (B4A-B4H). Fraction
B4C (188.5 mg) was subjected to silica gel CC using a gradient of
acetone:CHCl3 (1:4 / 1:0) to give six fractions (B4C1-B4C6).
Fraction B4C3 (114.6 mg) was applied to a silica gel column, using
the same solvent system as fraction B4C to afford seven fractions
(B4C3A-B4C3G). Fraction B4C3B (16.2 mg) was further puried by
PTLC using CHCl3 as a mobile phase (5 runs) to provide 1 (4.7 mg)
and 2 (6.3 mg). Fraction B4C3C (38.3 mg) was puried by silica gel
CC using CHCl3 as an eluent, with subsequent PTLC using EtOAc:-
petroleum ether (3:7) as a mobile phase (4 runs), followed by PTLC
using CH2Cl2:MeOH:EtOAc (8:11:1) as a mobile phase (3 runs) to
afford 11 (7.6 mg). Fraction B4D (228.3 mg) was applied to a silica
gel column using a gradient of EtOAc:petroleum ether (1:4 / 1:0)
to give six fractions (B4D1-B4D6). Fraction B4D2 (8.3 mg) was
puried by PTLC using acetone:petroleum ether (3:7) as a mobile
phase (2 runs), followed by PTLC using CHCl3 as a mobile phase (5
runs) and PTLC using EtOAc/CH2Cl2 (1:9) as a mobile phase (3 runs),
to give 7 (0.6 mg). Compound 3 (2.9 mg) was obtained from fraction
B4D4 (55.7 mg) after purication by silica gel CC using a gradient of

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sclerotiorum PSU-RSPG178, Phytochemistry (2017), http://dx.doi.org/10.1016/j.phytochem.2017.02.008
8 P. Phainuphong et al. / Phytochemistry xxx (2017) 1e9
acetone:petroleum ether (3:7 / 1:0), followed by PTLC using
acetone:petroleum ether (3:7) as a mobile phase (2 runs). Fraction
B4F (2.30 g) was subjected to silica gel CC using a gradient of ace-
tone:hexane (3:7 / 1:0) to give six fractions (B4F1-B4F6). Fraction
B4F3 (61.2 mg) was puried by silica gel CC using a gradient of
EtOAc:petroleum ether (2:3 / 1:0) to give 9 (36.4 mg). Compound
8 (1.68 g) was obtained from fraction B4F5. Fraction B5 (2.12 g) was
applied to a silica gel column using a gradient of acetone:petroleum
ether (3:7 / 1:0) to give seven fractions (B5A-B5G). Fraction B5D
(131.6 mg) was fractionated by silica gel CC using a gradient of
acetone:petroleum ether (2:7 / 1:0) to give seven fractions (B5D1-
B5D7). Fraction B5D3 (20.9 mg) was puried by silica gel CC using
MeOH:CH2Cl2 (3:97) to give four fractions (B5D3A-B5D3D). Com-
pound 4 (5.5 mg) was obtained from fraction B5D3B. Fraction
B5D3D (11.6 mg) was puried by PTLC using acetone:petroleum
ether (3:7) as a mobile phase (3 runs) to give 12 (5.6 mg). Fraction
B6 (1.32 g) was separated by silica gel CC using a gradient of ace-
tone:petroleum ether (3:7 / 1:0) to afford six fractions (B6A-B6F).
Fraction B6D (714.8 mg) was separated using the same procedure as
fraction B6 to give ve fractions (B6D1-B6D5). Compound 10
(55.7 mg) was obtained from fraction B6D4. Fraction B7 (283.1 g)
was fractionated by silica gel CC using a gradient of MeOH:CH2Cl2
(1:24 / 1:0) to afford six fractions (B7A-B6G). Fraction B7F
(37.2 mg) was separated using the same procedure as fraction B7 to
give ve fractions (B7F1-B7F5). Fraction B7F3 was further puried
by PTLC using acetone:petroleum ether (2:3) as a mobile phase (5
runs) to give 13 (3.5 mg). Fraction C (40.0 mg) was subjected to
silica gel CC using a gradient of MeOH:CH2Cl2 (1:99 / 1:0) to give
six fractions (C1-C6). Fraction C1 (5.1 mg) was further puried by
PTLC using EtOAc:petroleum ether (1:4) as a mobile phase (2 runs)
to give 5 (2.6 mg). Compound 6 (1.5 mg) was obtained from fraction
C2 (2.9 mg) after purication by PTLC using EtOAc:petroleum ether
(2:3) as a mobile phase (2 runs).
4.3.1. Aspersclerotiorone A (1)
Colorless crystals; mp 163e165  C; a26
D 42 (c 0.5, CHCl3); UV
(MeOH) lmax (log ): 269 (3.05) nm; CD (MeOH, c 0.0032) lmax (D)
267 (2.48) nm; IR (neat)nmax 2924, 1749, 1698 cm1; For 1H and
13
C NMR spectroscopic data, see Table 1; HRESIMS m/z: [MH]
265.1076 (calcd for C14H17O5, 265.1076).
4.3.2. Aspersclerotiorone B (2)
Colorless crystals; mp 155e156  C; a26
D 43 (c 0.5, CHCl3); UV
(MeOH) lmax (log ): 271 (3.28) nm; CD (MeOH, c 0.0039) lmax (D)
264 (2.37) nm; IR (neat)nmax 2929, 1751, 1697 cm1; For 1H and 13C
NMR spectroscopic data, see Table 1; HRESIMS m/z: [MH]
265.1076 (calcd for C14H17O5, 265.1076).
4.3.3. Aspersclerotiorone C (3)
Colorless solid; mp 177e178  C; a24
D 56 (c 0.5, CHCl3); UV
(MeOH) lmax (log ): 224 (2.91) nm; CD (MeOH, c 0.0051) lmax (D)
218 (13.43) nm; IR (neat)nmax 3418, 2943, 1746, 1641 cm1; For 1H
and 13C NMR spectroscopic data, see Table 2; HRESIMS m/z:
[MH] 283.1176 (calcd for C14H19O6, 283.1182).
4.3.4. Aspersclerotiorone D (4)
Colorless gum; a24
D 77 (c 0.5, CHCl3); UV (MeOH) lmax (log ):
226 (2.70) nm; CD (MeOH, c 0.0070) lmax (D) 218 (6.12) nm; IR
(neat)nmax 3370, 2942, 1745, 1715, 1638 cm1; For 1H and 13C NMR
spectroscopic data, see Table 2; HRESIMS m/z: [MH] 283.1176
(calcd for C14H19O6, 283.1181).
4.3.5. Aspersclerotiorone E (5)
Colorless gum; a24
D -41 (c 0.5, CHCl3); UV (MeOH) lmax (log ):
204 (3.19), 219 (2.87), 287 (2.23) nm; CD (CH3CN, c 0.0017) lmax (D)
Please cite this article in press as: Phainuphong, P., et al., g-Butenolide and furanone derivatives from the soil-derived fungus Aspergillus
sclerotiorum PSU-RSPG178, Phytochemistry (2017), http://dx.doi.org/10.1016/j.phytochem.2017.02.008
295 (9.15) nm; IR (neat)nmax 3455, 2938, 1735 cm1; For 1H and
13
C NMR spectroscopic data, see Table 3; HRESIMS m/z: [MNa]
287.0899 (calcd for C14H16O5Na, 287.0895).
4.3.6. Aspersclerotiorone F (6)
Colorless gum; a24
D 41 (c 0.5, CHCl3); UV (MeOH) lmax (log ):
203 (3.88), 220 (3.54), 288 (2.90) nm; CD (MeOH, c 0.0021) lmax
(D) 240 (3.89) nm; IR (neat)nmax 3468, 2936, 1755 cm1; For 1H
and 13C NMR spectroscopic data, see Table 3; HRESIMS m/z:
[MNa] 329.0999 (calcd for C16H18O6Na, 329.1001).
4.3.7. Aspersclerotiorone G (7)
Colorless gum; a24
D 14 (c 0.2, CHCl3); UV (MeOH) lmax (log ):
212 (3.59) nm; CD (MeOH, c 0.0035) lmax (D) 213 (1.37) nm; IR
(neat)nmax 3370, 2919, 1747, 1639 cm1; For 1H and 13C NMR
spectroscopic data, see Table 1; HRESIMS m/z: [MNa] 193.0477
(calcd for C8H10O4Na, 193.0477).
4.4. Preparation of the acetonide derivative (10a)
Compound 10 (3.3 mg, 0.017 mmol) was dissolved with acetone
(0.5 mL). To the solution of 10, p-TsOH (1 mg) and 2,2-
dimethoxypropane (40 mL) were added (Ding et al., 2013). The re-
action mixture was stirred at room temperature overnight. After
removal of solvent, compound 10a was obtained as sole product.
4.5. X-ray crystallography
The crystal data of 1 and 2 (Fig. 2) were selected for data
collection which was performed on a Bruker Smart Apex CCD
diffractometer equipped with a graphite-monochromatic Mo Ka
radiation (l 0.71073 ) at 293(2) K. Cell renement, the data
reductions and absorption correction were performed by SAINT and
SADABS. The structure was solved using direct methods by SHELXS
(Sheldrick, 2008) and rened with full-matrix least-squares
methods based on F2 with the SHELXL program (Sheldrick, 2015).
Non hydrogen atoms were allowed to vibrate anisotropically in
cycles of renement. All hydrogen atoms were placed in calculated,
ideal positions and rened as riding model approximation on their
respective parent atoms. The WinGX v2014.1 (Farrugia, 2012) and
Mercury (Macrae et al., 2008) programs were used to prepare the
materials and molecular graphic for publication. Crystallographic
data have been deposited with the Cambridge Crystallographic
Data Center (deposit No. CCDC 1457768 for 1 and deposit No. CCDC
1457767 for 2). Copies of the data can be obtained, free of charge, on
application to the Director, CCDC, 12 Union Road, Cambridge CB2
1EZ, U.K. (fax: 44e(0)223e336033 or e-mail: deposit@ccdc.cam.
ac.uk).
4.5.1. Crystal data of compound 1
C14H16O5, M 264.27, T 293(2) K, monoclinic space group P21/
c, a 6.5648(10) , b 8.1751(13) , c 25.439(4) , V 1361.7(4)
3, a g 90 , b 94.105(3) , Z 4, Dcalc 1.289 Mg/m3, crystal
dimensions 0.389  0.194  0.141 mm3, m 0.098 mm1,
F(000) 560, 12878 reections measured, 2368 unique
(Rint 0.1091). The nal renement gave R1 0.0858 and
wR2 0.2056 [I > 2s(I)].
4.5.2. Crystal data of compound 2
C14H16O5, M 264.27, T 293(2) K, Triclinic space group P1,
a 6.5371(7) , b 8.4488(9) , c 12.6395(14) , V 1361.7(4)
3, a 86.903(2) , b 86.116(2)o, g 86.774(2) , Z 2,
Dcalc 1.264 Mg/m3, crystal dimensions 0.345  0.216  0.113 mm3,
m 0.096 mm1, F(000) 280, 7606 reections measured, 2440
unique (Rint 0.0.0241). The nal renement gave R1 0.0416 and
 P. Phainuphong et al. / Phytochemistry xxx (2017) 1e9
wR2 0.1099 [I > 2s(I)].
4.6. Bioassays
Antimicrobial activity was determined as described by the
Clinical and Laboratory Standards Institute (Phongpaichit et al.,
2006). Antibacterial activity was evaluated against S. aureus
ATCC25923 and methicillin-resistant S. aureus SK1 (a clinical
isolate) and E. coli while antifungal activity was determined against
a clinical isolate of M. gypseum SH-MU-4. Vancomycin (MIC 0.50
and 1.00 mg/mL) and gentamicin (MIC 0.50 mg/mL) were used as
positive controls for bacteria whereas miconazole (MIC 2.00 mg/mL)
was used for M. gypseum.
Antimycobacterial activity was determined against
M. tuberculosis H37Ra using green uorescent protein (GFP)-based
uorescent detection (Changsen et al., 2003). The standard drugs
were rifampicin (MIC 0.0063 mg/mL), streptomycin (MIC 0.6250 mg/
mL), isoniazid (MIC 0.0469 mg/mL), ooxacin (MIC 0.391 mg/mL)
and ethambutol (MIC 0.469 mg/mL).
Antimalarial activity was evaluated against the parasite
P. falciparum (K1, multi-drug-resistant strain), using the microcul-
ture radioisotope technique based on the method described
(Desjardins et al., 1979). Dihydroartemisinine and meoquine were
used as standard compounds and exhibited the IC50 values of
0.0023 and 0.0269 mM, respectively.
The cytotoxicity assay against African green monkey kidney
broblast (Vero) cells was performed in triplicate employing the
method described by Hunt and co-workers (Hunt et al., 1999), and
the positive control ellipticine displayed the IC50 value of 4.06 mM.
The activities against KB and MCF-7 cell lines were evaluated using
the resazurin microplate assay (O'Brien et al., 2000). Ellipticine
(IC50 8.24 mM) and doxorubicin (IC50 2.19 mM) were used as stan-
dard drugs for KB cell lines while the standard compounds for MCF-
7 cell lines were doxorubicin (IC50 12.42 mM) and tamoxifen (IC50
18.81 mM).
Acknowledgments
V. Rukachaisirikul thanks the Royal Property Bureau Foundation
and National Science and Tecnology Development Agency for the
NSTDA Chair Professor grant (The Fourth Grant). P. Phainuphong is
grateful to the TRF through the Royal Golden Jubilee Ph.D. program
(Grant number PHD/0031/2555) and Prince of Songkla University
(PSU-Ph.D. scholarship) for a joint funding scholarship. The Center
of Excellence for Innovation in Chemistry (PERCH-CIC) and Prince
of Songkla University Graduate School are acknowledged for partial
support. Finally, the National Center for Genetic Engineering and
Biotechnology (BIOTEC) is acknowledged for antimycobacterial,
antimalarial, anticancer and cytotoxic assays.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.phytochem.2017.02.008.
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