N-Hydroxypyridone alkaloids, chromone derivatives, and tetrahydroxanthones from the scale-insect pathogenic fungus Orbiocrella sp. BCC 33248

Article abstract of DOI:10.1016/j.tet.2014.10.029

Six new compounds, an N-hydroxypyridone glucoside, orbiocrellin A (1), its aglycone orbiocrellin B (2), chromone glucosides 3 and 4, a dihydrochromone 5a/5b, and a chromone 6, were isolated from the scale-insect pathogenic fungus Orbiocrella sp. BCC 33248. Orbiocrellin A (1) exhibited antimalarial activity against Plasmodium falciparum K1 (IC₅₀ 3.1 μg/mL) while it was non-cytotoxic. In contrast, orbiocrellin B (2) showed both antimalarial (IC₅₀ 2.1 μg/mL) and cytotoxic (NCI-H187 cells, IC₅₀ 0.70 μg/mL) activities.

Full text of DOI:10.1016/j.tet.2014.10.029

Accepted Manuscript
N-Hydroxypyridone alkaloids, chromone derivatives, and tetrahydroxanthones from
the scale-insect pathogenic fungus Orbiocrella sp. BCC 33248
Masahiko Isaka, Rachada Haritakun, Sumalee Supothina, Wilunda Choowong,
Suchada Mongkolsamrit
PII:
S0040-4020(14)01456-2
10.1016/j.tet.2014.10.029
TET 26099
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 19 August 2014
Revised Date: 30 September 2014
Accepted Date: 13 October 2014
Please cite this article as: Isaka M, Haritakun R, Supothina S, Choowong W, Mongkolsamrit S, N-
Hydroxypyridone alkaloids, chromone derivatives, and tetrahydroxanthones from the scale-insect
pathogenic fungus Orbiocrella sp. BCC 33248, Tetrahedron (2014), doi: 10.1016/j.tet.2014.10.029.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT
N-Hydroxypyridone
alkaloids,
chromone
derivatives,
and
tetrahydroxanthones from the scale-insect pathogenic fungus
Orbiocrella sp. BCC 33248
Masahiko Isaka*, Rachada Haritakun, Sumalee Supothina, Wilunda Choowong,
Suchada Mongkolsamrit
National Center for Genetic Engineering and Biotechnology (BIOTEC), 113 Thailand
Science Park, Phaholyothin Road, Klong Luang, Pathumthani 12120, Thailand
* Corresponding author. Tel: +66-25646700x3554; fax: +66-25646707.
E-mail address: isaka@biotec.or.th (M. Isaka).
1

ACCEPTED MANUSCRIPT
ABSTRACT
Six new compounds, an N-hydroxypyridone glucoside, orbiocrellin A (1), its aglycone
orbiocrellin B (2), chromone glucosides 3 and 4, a dihydrochromone 5a/5b, and a
chromone 6, were isolated from the scale-insect pathogenic fungus Orbiocrella sp. BCC
33248. Orbiocrellin A (1) exhibited antimalarial activity against Plasmodium falciparum
K1 (IC50 3.1 µg/mL) while it was non-cytotoxic. In contrast, orbiocrellin B (2) showed
both antimalarial (IC50 2.1 µg/mL) and cytotoxic (NCI-H187 cells, IC50 0.70 µg/mL)
activities.
Keywords: Orbiocrella; Invertebrate pathogenic fungi; Antimalarial activity;
Cytotoxicity
2

ACCEPTED MANUSCRIPT
1. Introduction
Invertebrate pathogenic fungi have recently been proved to be sources of
structurally diverse bioactive compounds.1 Although a number of compounds with
unique skeletons and significant biological activities have been isolated from several
large genera such as Cordyceps, Beauveria, Aschersonia, Isaria, and Hirsutella,2 there
are still many genera/species of entomogenous fungi that remain chemically poorly
explored or unexplored. Orbiocrella is a very small genus of specific scale-insect
pathogen in the family Clavicipitaceae, recently described as a part of taxonomic
reclassification of Torrubiella.3 There is only one currently recorded species,
Orbiocrella petchii, which was formerly named as Torrubiella petchii. There has been
still no report of the secondary metabolites from Orbiocrella, in contrast to the fact that
a variety of bioactive compounds have been isolated from Conoideocrella (also moved
from Torrubiella),4-8 another genus of specific scale-insect pathogen of the family
Clavicipitaceae. As part of our research program on novel bioactive compounds from
invertebrate pathogenic fungi, primarily aimed for utilization of the fungal sources in
Thailand, we selected a strain (BCC 28517) of Orbiocrella sp. for large scale
fermentation and chemical studies. A culture broth extract (screening sample) of this
strain exhibited cytotoxicity to small-cell lung cancer cells (NCI-H187) with an IC50
value of 2.9 µg/mL. We report here the isolation, structure elucidation, and biological
activities of five new compounds, an N-hydroxypyridone glucoside, orbiocrellin A (1),
its aglycone orbiocrellin B (2), chromone glucosides 3 and 4, a dihydrochromone 5a/5b,
and a chromone 6 (Fig. 1). Eugenitol (7)9 and isoeugenitol (8),10 aglycones of 3 and 4,
respectively, were also isolated from the same extract.
3

ACCEPTED MANUSCRIPT
Fig. 1. Structures of compounds 1–4, 5a/5b, and 6–8 isolated from Orbiocrella sp.
BCC 33248.
2. Results and discussion
Orbiocrellin A (1) was isolated as a yellow solid, and its molecular formula was
determined to be C25H33NO10 on the basis of the HR-ESI-MS. Interpretation of 2D
NMR spectroscopic data suggested the presence of a pyranose unit (Table 1). The
proton at δH 4.97 (d, J = 7.8 Hz, H-1″), which was attached to the downfield carbon at
δC 100.9 (C-1″), was assigned to the anomeric position. The connectivity from C-1″ to
C-6″ was revealed by analyses of COSY and HMQC spectra. Intense HMBC correlation
from methoxy protons (δH 3.57, 3H, s) to C-4″ and the correlation from H-4″ to the
methoxy carbon (δC 59.7) indicated that 4″-OH was methylated. Vicinal coupling
constants of J1″,2″ = 7.8, J2″,3″ = 9.1, J3″,4″ = 9.3, and J4″,5″ = 9.3 Hz indicated axial
orientations of these protons, which was further confirmed by the NOESY correlations
4

ACCEPTED MANUSCRIPT
from H-1″ to H-3″ and H-5″. Consequently, the sugar unit was identified as
1
4″-O-methyl-β-glucopyranose. The rest of the H and 13C NMR resonances were
assigned to those of the aglycone, a C18 alkaloid. The 13C NMR, DEPT135, and HMQC
spectroscopic data indicated that the aglycone contained five sp2 quaternary carbons,
five sp2 methines, two oxygenated methines (δC 80.8 and 75.4), a methine, two
methylenes, and two methyl groups. The presence of a para-oxygenated phenyl group
was revealed by two pairs of ortho-coupled (J = 8.7 Hz) symmetrical aromatic methines
(H-2′/H-6′ and H-3′/H-5′) and a downfield sp2 quaternary carbon (δC 157.3, C-4′). An
intense HMBC correlation from the sugar anomeric proton (H-1″) to C-4′ indicated the
location of the sugar unit, which was further confirmed by the NOESY correlations
from H-1″ to H-3′/H-5′. A terminal tetrahydropyran ring bearing two methyl groups was
demonstrated by COSY correlations. A key HMBC correlation from H-7 (δH 4.71) to
C-11 (δC 75.4) indicated the ether linkage to form a tetrahydropyran ring. The remaining
five sp2 carbons, together with one nitrogen and three oxygen atoms, should constitute
the central unit of the aglycone. Analysis of the HMBC correlations revealed
1,4-dihydroxy-2-pyridone substituted at C-3 and C-5 (Fig. 2). Key HMBC correlations
4
were those from H-6 to C-2, C-3 (weak, J), C-4, C-5, and C-1, from H-7 to C-2, C-3,
and C-4, and from H-1′/H-6′ to C-5. The C-3–C-1 bond was further supported by the
NOESY correlations from H-6 to H-1′/H-6′.
The relative configuration of the tetrahydropyran unit was assigned to be 7S*,
8S*, and 11S* on the basis of the vicinal coupling constant values and NOESY
correlations (Fig. 3). The axial orientations (antiperiplanar relation) of H-7 and H-8 was
evident from the large J-value (10.1 Hz) and a weaker intensity of the NOESY
cross-peak of these protons relative to that for H-7 and H3-13. NOESY correlations
5

ACCEPTED MANUSCRIPT
from H-7 to Hax-9 and H-11, and the correlation from Hax-9 to H-11 demonstrated the
coplanar relation and that the tetrahydropyran ring adopts a chair conformation, wherein
the pyridone ring and two methyl groups are equatorial. The
D-configuration of the
4-O-methylglucopyranose was determined by acid hydrolysis of 1.11 The anomeric
mixture of sugar fragment obtained from the hydrolysate aqueous layer showed positive
sign of specific rotation, [α]26 +71 (c 0.30, MeOH), which was consistent with the
D
literature data for 4-O-methylglucopyranose, [α]20 D +80 (c 1.3, MeOH).12
Fig. 2. Selected HMBC correlations for 1.
Fig. 3. Key NOESY correlations for the tetrahydropyran unit of 1.
6

ACCEPTED MANUSCRIPT
Table 1
13C (100 MHz) and 1H (400 MHz) NMR data for 1 and 2 in acetone-d6
Position
Orbiocrellin A (1)
H, mult. (J in Hz) HMBC
Orbiocrellin B (2)
δ
C, mult.
δ
δ
C, mult.
δ
H, mult. (J in Hz)
2
3
157.0,a C
109.3, C
160.6,a C
112.2, C
157.1,b C
109.5, C
4
160.8,b C
112.9, C
5
6
130.8, CH 7.83, s
2, 4, 5, 1′
130.8, CH
80.9, CH
35.7, CH
32.5, CH2
33.7, CH2
75.5, CH
21.3, CH3
17.2, CH3
124.9, C
7.78, s
7
80.8, CH
35.5, CH
4.71, d (10.1)
1.82, m
2, 3, 4, 8, 9, 11
4.71, d (10.1)
1.80, m
8
9
32.4, CH2 1.87, m; 1.41, m
33.5, CH2 1.76, m; 1.47, m
7, 8, 10, 11
8
1.87, m; 1.41, m
1.76, m; 1.48, m
3.68, m
10
11
12
13
1′
75.4, CH
3.68, m
21.1, CH3 1.24, d (6.1)
17.1, CH3 0.76, d (6.2)
127.4, C
10, 11
7, 8, 9
1.24, d (6.1)
0.77, d (6.3)
2′,6′
3′,5′
4′
130.3, CH 7.43, d (8.7)
116.3, CH 7.08, d (8.7)
157.3, C
5, 2′, 4′, 6′
1′, 3′, 4′, 5′
130.6, CH
115.1, CH
157.0, C
7.34, d (8.4)
6.87, d (8.4)
1″
2″
3″
4″
5″
6″
100.9, CH 4.97, d (7.8)
4′, 2″, 5″
74.1, CH
77.1, CH
79.3, CH
76.2, CH
3.47, m
1″, 3″
3.64, t (9.1)
3.23, t (9.3)
3.49, m
2″, 4″
2″, 5″, 6″, 4″-OCH3
6″
4″
61.2, CH2 3.84, dd (12.0, 1.9)
3.69, dd (12.0, 4.8)
4″-OCH3
59.7, CH3 3.57, s
4″
a,b The assignment of carbons can be interchanged.
7

ACCEPTED MANUSCRIPT
Orbiocrellin B (2), the aglycone of 1, was also isolated in relatively lower
quantity. The 2D NMR spectroscopic data were consistent with those of the aglycone
unit in 1. 4-Hydroxy-2-pyridone alkaloids are a family of fungal secondary metabolites
that are rich in diversity and biological activity.13 Biosynthetically, orbiocrellin B (2) is
a tetraketide fused to
possessing the same
L
-tyrosine.14 A number of 4-hydroxy-2-pyridone alkaloids
L
-tyrosine-derived unit as 2, such as tenellins, bassianins,
militarinones, farinosones, and torrubiellones, have been isolated from invertebrate
pathogenic fungi of the genera Beauveria, Cordyceps, Isaria, Torrubiella, all are in the
family Cordycipitaceae. These compounds possess a larger polyketide 3-acyl chain,
while compounds 1 and 2 from Orbiocrella (family Clavicipitaceae) have a C-7 reduced
structure forming a tetrahydropyran ring. To the best of our knowledge, orbiocrellide A
(1) is the first fungal 4-hydroxy-2-pyridone alkaloid possessing a sugar unit.
The molecular formula of a new chromone glycoside 3 was determined to be
C18H22O9 by HR-ESI-MS. Interpretation of the NMR spectroscopic data revealed that
the sugar unit was identical to that of 1, 4-O-methyl-β-glucopyranose (Table 2). The
aglycone was identified as 5,7-dihydroxy-2,6-dimethylchromone (eugenitol) based on
the HMBC correlations (Fig. 4). An intense HMBC correlation from the glucose
anomeric proton (H-1′) to C-7 (δC 161.1) and a NOESY correlation from H-1′ to H-8
indicated the location of the sugar linkage.
The molecular formula of another chromone glycoside 4 was the same as 3
(HR-ESI-MS). Analysis of 2D NMR spectroscopic data revealed the same sugar moiety
as 3, 4′-O-methyl-β-glucopyranose, while the aglycone was identified to be
5,7-dihydroxy-2,8-dimethylchromone (isoeugenitol). An HMBC correlation from the
H-1′ to C-7 (δC 160.9) and an NOESY correlation from H-1′ to H-6 indicated the
8

ACCEPTED MANUSCRIPT
glucosidation at C-7. The D-configuration of the 4′-O-methyl-β-glucopyranose in 4 was
confirmed by acid hydrolysis. The sugar fragment obtained from the hydrolysate
aqueous layer showed positive sign of specific rotation, [α]27D +93 (c 0.48, MeOH).
Fig. 4. Selected HMBC correlations for 3 amd 4.
9

ACCEPTED MANUSCRIPT
Table 2
13C (125 MHz) and 1H (500 MHz) NMR data for 3 and 4 in DMSO-d6
Position
3
4
δ
C, mult.
δ
H, mult. (J in Hz)
HMBC
2, 4a, 9
δ
C, mult.
δ
H, mult. (J in Hz)
HMBC
2
168.6, C
168.7, C
3
108.7, CH 6.25, s
182.5, C
108.4, CH 6.24, s
182.9, C
2, 4, 4a, 9
4
4a
105.1, C
105.2, C
5
158.4, C
159.5, C
6
109.1, C
98.3, CH
160.9, C
104.6, C
155.0, C
6.57, s
4a, 5, 7, 8
7
161.1, C
8
93.3, CH
155.9, C
6.75, s
4, 4a, 6, 7, 8a
8a
9
20.5, CH3 2.38, s
2, 3
20.5, CH3 2.41, s
2, 3
10
7.9, CH3
2.03, s
5, 6, 7
4a, 5, 6
7
8.1, CH3
2.16, s
7, 8, 8a
4a, 5, 6
7
5-OH
1′
13.03, s
12.78, s
100.2, CH 5.05, d (7.8)
100.3, CH 5.02, d (7.8)
2′
73.8, CH
76.5, CH
79.4, CH
76.1, CH
3.30, m
1′, 3′
2′, 4′
5′, 4′-OCH3
4′
73.9, CH
76.5, CH
79.4, CH
76.1, CH
3.30, m
1′, 3′
3′
3.43, m
3.44, m
2′
4′
3.05, t (9.1)
3.49, m
3.05, t (9.2)
3.47, m
5′, 4′-OCH3
5′
6′
60.7, CH2 3.63, m; 3.50, m
5.50, d (5.3)
60.7, CH2 3.63, m; 3.50, m
5.47, d (5.4)
2′-OH
3′-OH
4′-OCH3
6′-OH
1′, 2′, 3′
2′, 3′, 4′
4′
1′, 2′, 3′
2′, 3′, 4′
4′
5.31, d (5.5)
5.29, d (5.5)
60.1, CH3 3.46, s
4.74, dd (6.0, 5.1)
60.1, CH3 3.46, s
4.73, dd (5.8, 4.7)
5′, 6′
5′, 6′
10

ACCEPTED MANUSCRIPT
A pair of dihydrochromone isomers, 5a/5b, was obtained as a 3:2 mixture by
silica gel column chromatography. Clear peak separation could be achieved by
preparative HPLC; however, concentration of the fractions corresponding to each peak
gave the same mixture of 5a and 5b. These results strongly suggested their
interconversion during the concentration of the aqueous solution. Thus, structure
elucidation was achieved using a mixture. Both isomers had a hemiacetal functionality
(
δC 100.9 and 100.8, C-2) whose location was indicated by the HMBC correlations from
the methyl protons (H3-9) and diastereotopic methylene protons (H2-3) to the hemiacetal
quaternary carbon. The structure of 5a was assigned by the HMBC correlations from the
aromatic methine proton H-8 (δH 5.98) to C-4a, C-6, C-7, and C-8a, and the correlations
from the benzylic methyl protons H3-10 (δH 1.95) to C-5, C-6, and C-7. On the other
hand, 5b showed key HMBC correlations from H-6 (δH 5.99) to C-4a, C-5, C-7, and
C-8, and the correlations from H3-10 to C-7, C-8, and C-8a. The interconversion of
5a/5b indicated that the hemiacetal formations are reversible. Hydrated chromones
5a/5b should be the biosynthetic precursors for the chromones 7 and 8 and their
glucoside derivatives 3 and 4. The interconversion of 3/4 was not observed during the
isolation procedures. Even under the acid hydrolysis conditions for 4, the chromone
isomerisation from the aglycone 8 to 7 was not observed, which indicated the absence
of the chromone hydration/dehydration equilibrium.
The molecular formula of compound 6 was determined as C11H8O5 based on its
1
HR-ESI-MS and NMR data. The H and 13C NMR spectroscopic data suggested a
chromone skeleton similar to 7 and 8. A significant difference was the presence of an
aldehyde (δC 191.1; δH 10.35, s), replacing the benzylic methyl group in 7 and 8. Since
the HMBC and NOESY spectroscopic data for 6 were not informative enough to assign
11

ACCEPTED MANUSCRIPT
the location of the formyl group of 6, this compound was converted into its
5,7-O-dimethyl derivative 9 (Fig. 5). The HMBC correlation from the formyl proton
to C-8a (δC 160.8) and the NOESY correlations from H-6 (δH 6.72) to both two
methoxy groups (δH 4.09 and 4.02) indicated the connection of the formyl group to C-8.
Therefore, compound 6 was identified as 5,7-dihydroxy-8-formyl-2-methychromone.
O
H
H3CO
H
O
O
CH3
H
HMBC
NOESY
H3CO
9
Fig. 5. Key HMBC and NOESY correlations for 9.
The new compounds, except for 6 (sample shortage), were tested for
cytotoxicity to cancer cell-lines (NCI-H187, MCF-7, and KB) and nonmalignant Vero
cells, and antimalarial activity against Plasmodium falciparum K1 (Table 3).
Orbiocrellide A (1) was non-cytotoxic up to a concentration of 50 µg/mL, while it
exhibited antimalarial activity (IC50 3.1 µg/mL). By contrast, orbiocrellin B (2) showed
both cytotoxicity and antimalarial activity. Compounds 3, 4, and 5a/5b, were inactive in
these assays.
12

ACCEPTED MANUSCRIPT
Table 3
Biological activities of compounds 1–4 and 5a/5b
Compound
Cytotoxicity (IC50, µg/mL)a
NCI-H187 MCF-7 KB
>50
Anti-malariab
(IC50, µg/mL)
Vero
1
>50
0.70
>50
7.3
>50
0.92
>50
3.1
2.1
2
>50
>50
>50
>50
3
>50
>50
>50
>50
>50
>50
>10
>10
>10
4
>50
>50
5a/5b
a
The IC50 values of a standard compound, doxorubicin hydrochloride, against NCI-H187, MCF-7,
and KB cells were 0.18, 9.3, and 0.96 µg/mL, respectively. Ellipticine was used as a standard
compound for the cytotoxicity assay against Vero cells (IC50 0.73 µg/mL).
b
Antimalarial activity against Plasmodium falciparum K1. The standard antimalarial drug,
dihydroartemisinin, showed an IC50 value of 0.75 ng/mL.
3. Conclusion
The present study demonstrates that the genus Orbiocrella is a unique source
of novel bioactive compounds. The biological activities of the N-hydroxypyridones 1
and 2 are noteworthy. Orbiocrellin A (1) selectively inhibited the proliferation of
malarial parasite, while orbiocrellin B (2) deserves further biological evaluation as an
anticancer agent.
4. Experimental Section
4.1. General procedures
Melting points were measured with an Electrothermal IA9100 digital melting
point apparatus. Optical rotations were measured with a JASCO P-1030 digital
polarimeter. UV spectra were recorded on a GBC Cintra 404 spectrophotometer. IR
spectra were taken on a Bruker ALPHA spectrometer. NMR spectra were recorded on
Bruker AV500D and DRX400 spectrometers. ESITOF mass spectra were measured with
13

ACCEPTED MANUSCRIPT
a Bruker micrOTOF mass spectrometer.
4.2. Fungal material
The fungus used in this study was isolated from a scale insect (Hemiptera)
collected in Khlong Lan National Park, Kamphaeng Phet Province, Thailand, on
September 26, 2008 by one of the authors (S.M.). This fungus was deposited in the
BIOTEC Culture Collection (BCC) as BCC 33248. On the basis of the ITS rDNA
(GenBank accession number, KJ138267) and LSU rRNA (GenBank accession number,
KJ138268) gene sequence data and the results of the BLAST search, this strain was
assigned to the genus Orbiocrella within the family Clavicipitaceae.
4.3. Fermentation and isolation
The fungus BCC 33248 was maintained on Potato Dextrose Agar (PDA) at
25°C. The agar was cut into small plugs and inoculated in 4 × 250 mL Erlenmeyer
flasks containing 25 mL of DifcoTM Potato Dextrose Broth (PDB; composition, potato
starch 4 g/L, dextrose 20 g/L). After incubation at 25°C for 5 days on a rotary shaker
(200 rpm), each primary culture was transferred into a 1000 mL Erlenmeyer flask
containing 250 mL of PDB, and incubated at 25°C for 5 days on a rotary shaker (200
rpm). These secondary cultures were pooled and each 25 mL portion was transferred
into 40 × 1000 mL Erlenmeyer flasks, each containing 250 mL of M102 medium
(composition, sucrose 30 g/L, malt extract 20 g/L, Bacto-peptone 2.0 g/L, yeast extract
1.0 g/L, KCl 0.5 g/L, MgSO4·7H2O 0.5 g/L, KH2PO4 0.5 g/L), and final fermentation
was carried out at 25°C for 33 days under static conditions. The cultures were filtered to
separate mycelia and filtrate. The filtrate was extracted with ethyl acetate (3 × 10 L),
14

ACCEPTED MANUSCRIPT
and concentrated under reduced pressure to leave a brown gum (5.8 g). This extract was
triturated in MeOH at room temperature and filtered. The residual solid was
recrystallized from MeOH-H2O to give 4 (92 mg). The filtrate from trituration (MeOH
solution) was subjected to column chromatography on Sephadex LH-20 (3.8 × 60 cm,
MeOH) to obtain ten pooled fractions. Fractions 3 (756 mg) and 4 (929 mg) were
combined and fractionated again by Sephadex LH-20 (MeOH), and the fractions were
purified by preparative HPLC (Dionex SunFire C18 OBD, 19 × 150 mm, 5 µm; step
gradient elution with 20-100% MeCN/H2O, 0–30 min, then MeCN 100%; flow rate 10
ml/min) to furnish 1 (133 mg, retention time 19 min). Fractions 5 (1.20 g) and 6 (599
mg) were combined and further fractionated and purified by Sephadex LH-20 (MeOH)
and preparative HPLC (MeCN/H2O) to give 2 (7.2 mg). Fraction 7 (501 mg) from the
Sephadex LH-20 column chromatography was recrystallized from MeOH/CH2Cl2 to
furnish 3 (31 mg). Fraction 8 (792 mg) was chromatographed again on Sephadex LH-20
(MeOH) and the subfractions were further separated by preparative HPLC
(MeCN/H2O) to furnish 5a/5b (17 mg), 6 (4.9 mg), 7 (2.2 mg), and 8 (4.4 mg).
4.3.1. Orbiocrellin A (1)
Yellow solid; mp 134–135 °C; [α]28D –131 (c 0.12, MeOH); UV (MeOH) λmax
(log ε) 214 (3.99), 247 (4.10), 310 sh (3.37) nm; IR (ATR) νmax 3455, 1739, 1641, 1511,
1367, 1230, 1217, 1061 cm-1; 1H NMR (400 MHz, acetone-d6) and 13C NMR (100 MHz,
acetone-d6) data, see Table 1; HRMS (ESI-TOF) m/z 508.2172 [M + H]+ (calcd for
C25H34NO10, 508.2177).
4.3.2. Orbiocrellin B (2)
15

ACCEPTED MANUSCRIPT
Yellow solid; mp 137–138 °C; [α]28 –89 (c 0.12, MeOH); UV (MeOH) λmax
D
(log
ε
) 214 (3.90), 254 (3.92), 317 sh (3.34) nm; IR (ATR) νmax 1740, 1366, 1229, 1217
1
13
cm-1; H NMR (400 MHz, acetone-d6) and C NMR (100 MHz, acetone-d6) data, see
Table 1; HRMS (ESI-TOF) m/z 332.1499 [M + H]+ (calcd for C18H22NO5, 332.1492).
4.3.3. 7-O-(4-O-Methyl-β-glucopyranosyl)eugenitol (3)
Colorless solid; mp 167–168 °C; [α]27D –81 (c 0.11, MeOH); UV (MeOH) λmax
ε) 213 (3.98), 231 (4.05), 254 (4.05), 288 (3.75), 324 sh (3.42) nm; IR (ATR) νmax
(log
1
13
3375, 1662, 1626, 1341, 1119, 1093 cm-1; H NMR (500 MHz, DMSO-d6) and C
NMR (125 MHz, DMSO-d6) data, see Table 2; HRMS (ESI-TOF) m/z 383.1335 [M +
H]+ (calcd for C18H23O9, 383.1337).
4.3.4. 7-O-(4-O-Methyl-
β-glucopyranosyl)isoeugenitol (4)
Pale yellow solid; mp 237–238 °C; [α]27 –81 (c 0.11, MeOH); UV (MeOH)
D
λmax (log
ε
) 227 sh (3.81), 253 (3.94), 293 sh (3.32), 326 (3.32) nm; IR (ATR) νmax 3307,
1
1667, 1621, 1595, 1424, 1315, 1276, 1109, 1083, 818 cm-1; H NMR (500 MHz,
13
DMSO-d6) and C NMR (125 MHz, DMSO-d6) data, see Table 2; HRMS (ESI-TOF)
m/z 383.1344 [M + H]+ (calcd for C18H23O9, 383.1337).
4.3.5. Compound 5a/5b
Yellow solid; UV (MeCN/H2O) λmax 210, 259, 273 sh nm; IR (ATR) νmax 3444,
1
1738, 1638, 1366, 1229, 1217, 1161, 1112 cm-1; H NMR (400 MHz, acetone-d6)
δ 5a,
12.36 (1H, s, 5-OH), 5.98 (1H, s, H-8), 2.99 (1H, d, J = 16.8 Hz, Ha-3), 2.71 (1H, d, J =
16.8 Hz, Hb-3), 1.95 (3H, s, H-10), 1.66 (3H, s, H-9), 5b, 12.04 (1H, s, 5-OH), 5.99 (1H,
16

ACCEPTED MANUSCRIPT
s, H-6), 2.99 (1H, d, J = 16.8 Hz, Ha-3), 2.71 (1H, d, J = 16.8 Hz, Hb-3), 1.95 (3H, s,
13
H-10), 1.72 (3H, s, H-9); C NMR (100 MHz, acetone-d6)
δ 5a, 195.9 (C, C-4), 164.0
(C, C-7), 161.1 (C, C-5), 158.5 (C, C-8a), 103.5 (C, C-6), 101.9 (C, C-4a), 100.9 (C,
C-2), 95.1 (CH, C-8), 27.5 (CH3, C-9), 6.3 (CH3, C-10), 5b, 196.3 (C, C-4), 164.3 (C,
C-7), 161.4 (C, C-5), 157.9 (C, 8a), 103.7 (C, C-8), 102.1 (C, C-4a), 100.8 (C, C-2),
95.1 (CH, C-6), 27.6 (CH3, C-9), 7.1 (CH3, C-10); HRMS (ESI-TOF) m/z 247.0578 [M
+ Na]+ (calcd for C11H12O5Na, 247.0577).
4.3.6. Compound 6
Pale yellow solid; UV (MeOH) λmax (log
ε
) 240 (3.83), 265 (3.86), 300 (3.82)
1
nm; IR (ATR) νmax 3399, 1666, 1615, 1588, 1433, 1360, 1286, 1209 cm-1; H NMR
(400 MHz, acetone-d6)
δ
10.35 (1H, s, H-10), 6.34 (1H, s, H-3), 6.21 (1H, s, H-6), 2.53
191.1 (CH, C-10), 182.1 (C, C-4),
13
(3H, s, H-9); C NMR (100 MHz, acetone-d6)
δ
169.2 (C) and 168.6 (C) (C-5 and C-7), 168.4 (C, C-2), 160.3 (C, C-8a), 109.8 (CH,
C-3), 104.3 (C, C-4a), 98.9 (CH, C-6), 19.4 (CH3, C-9); HRMS (ESI-TOF) m/z
243.0277 [M + Na]+ (calcd for C11H8O5Na, 243.0264).
4.4. Hydrolysis of 1.
Compound 1 (20.5 mg) was hydrolyzed in 5% aqueous hydrochloric acid (4
mL) at 90 °C for 15 h. The mixture was washed with EtOAc (2 × 3 mL), and the
aqueous layer was concentrated under reduced pressure and purified by column
chromatography on Sephadex LH-20 (MeOH) to furnish an anomeric mixture of
4-O-methyl-D
-glucopyranose (5.9 mg, 1H NMR in D2O–acetone-d6); [α]26 D +71 (c 0.30,
MeOH). The EtOAc layer was concentrated in vacuo to give a yellow gum (12.7 mg),
17

ACCEPTED MANUSCRIPT
1
whose H NMR spectrum (CDCl3) indicated that compound 2 was the major
component.
4.5. Hydrolysis of 4.
The same acid hydrolysis procedure as described above for 1 was applied to 4
(20.0 mg). The aqueous layer was concentrated in vacuo to yield
4-O-methyl-D
-glucopyranose (9.5 mg, 1H NMR in D2O–acetone-d6); [α]27 D +93 (c 0.48,
MeOH). The EtOAc layer (11.3 mg) was identified as isoeugenitol (1H NMR in CDCl3).
4.6. Synthesis of 9.
A mixture of compound 6 (1.6 mg), MeI (100 µL), and K2CO3 (30 mg) in
DMSO-d6 (0.5 ml) was stirred at room temperature for 15 hours. The mixture was
diluted with EtOAc and washed with H2O. The organic layer was concentrated in vacuo
to obtain a yellow gum, which was purified by preparative HPLC (MeCN/H2O) to
furnish the 5,7-O-dimethyl derivative 9 (0.8 mg).
4.6.1. Compound 9
Pale yellow solid; 1H NMR (400 MHz, acetone-d6)
δ 10.45 (1H, s, H-10), 6.72
(1H, s, H-6), 5.99 (1H, s, H-3), 4.09 (3H, s) and 4.02 (3H, s) (5-OCH3 and 7-OCH3),
2.32 (3H, s, H-9); 13C NMR (100 MHz, acetone-d6)
δ
185.4 (CH, C-10), 175.6 (C, C-4),
167.1 (C) and 166.5 (C) (C-5 and C-7), 164.1 (C, C-2), 160.8 (C, C-8a), 112.8 (CH,
C-3), 109.3 (C, C-4a), 92.6 (CH, C-6), 57.1 (CH3) and 56.9 (CH3) (5-OCH3 and
7-OCH3),, 19.4 (CH3, C-9); HRMS (ESI-TOF) m/z 271.0578 [M + Na]+ (calcd for
C13H12O5Na, 271.0577).
18

ACCEPTED MANUSCRIPT
4.6. Biological assays
Cytotoxic activities against human cancer cell-lines, NCI-H187 cells
(small-cell lung cancer), MCF-7 cells (breast cancer), and KB cells (oral cavity cancer),
were evaluated using the resazurin microplate assay.15 Cytotoxicity to Vero cells
(African green monkey kidney fibroblasts) was performed using the green fluorescent
protein microplate assay (GFPMA).16 Assay for activity against Plasmodium falciparum
(K1, multi-drug resistant strain) was performed in duplicate using the microculture
radioisotope technique.17
Acknowledgments
Financial support from the National Center for Genetic Engineering and
Biotechnology is gratefully acknowledged.
Supplementary data
NMR spectra of compounds 1–4, 5a/5b, 6, and 9. Supplementary data
associated with this article can be found in the online version, at doi:.
References and notes
1.
2.
Molnár, I.; Gibson, D. M.; Krasnoff, S. B. Nat. Prod. Rep. 2010, 27, 1241–1275.
Isaka, M.; Kittakoop, P.; Kirtikara, K.; Hywel-Jones, N. L.; Thebtaranonth, Y. Acc.
Chem. Res. 2005, 38, 813–823.
3.
Johnson, D.; Sung, G.-H.; Hywel-Jones, N. L.; Luangsa-ard, J. J.; Bischoff, J. F.;
Kepler, R. M.; Supatafora, J. W. Mycol. Res. 2009, 113, 279–289.
19

ACCEPTED MANUSCRIPT
4.
5.
Isaka, M.: Palasarn, S.; Kocharin, K.; Hywel-Jones, N. L. J. Antibiot. 2007, 60,
577–581 (2007).
Isaka, M.; Palasarn, S.; Supothina, S.; Komwijit, S.; Luangsa-ard, J. J. J. Nat.
Prod. 2011, 74, 782–789.
6.
7.
Asai, T.; Yamamoto, T.; Oshima, Y. Tetrahedron Lett. 2011, 52, 7042–7045.
Kornsakulkarn, J.; Thongpanchang, C.; Lapanun, S.; Srichomthong, K. J. Nat.
Prod. 2009, 72, 1341–1343.
8.
Isaka, M.; Sappan, M.; Luangsa-ard, J. J.; Hywel-Jones, N. L.; Mongkolsamrit, S.;
Chunhametha, S. Fungal Biol. 2011, 115, 401–405.
9.
Fox, C. H.; Siegfried, H. Phytochemistry 1969, 8, 1301–1304.
10. Schmid, H; Bolleter, A. Helv. Chim. Acta 1949, 32, 1358–1360.
11. Bunyapaiboonsri, T.; Yoiprommarat, S.; Intereya, K.; Kocharin, K. J. Nat. Prod.
2007, 55, 304–307.
12. Smith, F. J. Chem. Soc. 1951, 2646–2652.
13. Jessen, H. J.; Gademann, K. Nat. Prod. Rep. 2010, 27, 1168–1185.
14. Fisch, K. M.; Bakeer, W.; Yakasai, A. A.; Song, Z.; Pedrick, J.; Wasil, Z.; Bailey, A.
M.; Lazarus, C. M.; Simpson, T. J.; Cox, R. J. J. Am. Chem. Soc. 2011, 133,
16635–16641.
15. O’Brien, J.; Wilson, I.; Orton, T.; Pognan, F. Eur. J. Biochem. 2000, 267,
5421–5426.
16. Changsen, C.; Franzblau, S. G.; Palittapongarnpim, P. Antimicrob. Agents
Chemother. 2003, 47, 3682–3687.
17. Desjardins, R. E.; Canfield, C. J.; Haynes, J. D.; Chulay, J. D. Antimicrob. Agents
Chemother. 1979, 16, 710–718.
20

ACCEPTED MANUSCRIPT
Graphical Abstract
N-Hydroxypyridone
alkaloids,
chromone
derivatives,
and
tetrahydroxanthones from the scale-insect pathogenic fungus
Orbiocrella sp. BCC 33248
Masahiko Isaka*, Rachada Haritakun, Sumalee Supothina, Wilunda Choowong,
Suchada Mongkolsamrit
21

ACCEPTED MANUSCRIPT
Supporting Information for:
N-Hydroxypyridone alkaloids, chromone derivatives, and tetrahydroxanthones
from the scale-insect pathogenic fungus Orbiocrella sp. BCC 33248
Masahiko Isaka*, Rachada Haritakun, Sumalee Supothina, Wilunda Choowong, Suchada
Mongkolsamrit
Contents
NMR Spectra
S1
S2
S3
S4
S5
S6
S7
S8
S9
1H NMR spectrum of orbiocrellin A (1) (acetone-d6, 400 MHz)
13C NMR spectrum of orbiocrellin A (1) (acetone-d6, 100 MHz)
DEPT135 spectrum of orbiocrellin A (1) (acetone-d6, 100 MHz)
COSY spectrum of orbiocrellin A (1) (acetone-d6, 400 MHz)
HMQC spectrum of orbiocrellin A (1) (acetone-d6, 400 MHz)
HMBC spectrum of orbiocrellin A (1) (acetone-d6, 400 MHz)
NOESY spectrum of orbiocrellin A (1) (acetone-d6, 400 MHz)
1H NMR spectrum of orbiocrellin B (2) (acetone-d6, 400 MHz)
13C NMR spectrum of orbiocrellin B (2) (acetone-d6, 100 MHz)
S10 1H NMR spectrum of compound 3 (DMSO-d6, 500 MHz)
S11 13C NMR spectrum of compound 3 (DMSO-d6, 125 MHz)
S12 1H NMR spectrum of compound 4 (DMSO-d6, 500 MHz)
S13 13C NMR spectrum of compound 4 (DMSO-d6, 125 MHz)
S14 1H NMR spectrum of compound 5a/5b (acetone-d6, 400 MHz)
S15 13C NMR spectrum of compound 5a/5b (acetone-d6, 100 MHz)
S16 1H NMR spectrum of compound 6 (acetone-d6, 400 MHz)
S17 13C NMR spectrum of compound 6 (acetone-d6, 100 MHz)
S18 1H NMR spectrum of compound 9 (acetone-d6, 400 MHz)
S19 13C NMR spectrum of compound 9 (acetone-d6, 100 MHz)
1

ACCEPTED MANUSCRIPT
1H NMR spectrum of orbiocrellin A (1) (acetone-d6, 400 MHz)
S1
S2
13C NMR spectrum of orbiocrellin A (1) (acetone-d6, 100 MHz)
2

ACCEPTED MANUSCRIPT
S3
DEPT135 spectrum of orbiocrellin A (1) (acetone-d6, 100 MHz)
S4
COSY spectrum of orbiocrellin A (1) (acetone-d6, 400 MHz)
3

ACCEPTED MANUSCRIPT
S5
HMQC spectrum of orbiocrellin A (1) (acetone-d6, 400 MHz)
S6
HMBC spectrum of orbiocrellin A (1) (acetone-d6, 400 MHz)
4

ACCEPTED MANUSCRIPT
5

ACCEPTED MANUSCRIPT
NOESY spectrum of orbiocrellin A (1) (acetone-d6, 400 MHz)
S7
6

ACCEPTED MANUSCRIPT
1H NMR spectrum of orbiocrellin B (2) (acetone-d6, 400 MHz)
S8
S9
13C NMR spectrum of orbiocrellin B (2) (acetone-d6, 100 MHz)
7

ACCEPTED MANUSCRIPT
S10 1H NMR spectrum of compound 3 (DMSO-d6, 500 MHz)
S11 13C NMR spectrum of compound 3 (DMSO-d6, 125 MHz)
8