Antimicrobial, antimalarial, and cytotoxic substances from the insect pathogenic fungus Beauveria asiatica BCC 16812

Article abstract of DOI:10.1016/j.phytol.2021.03.003

Six new compounds 1–6 and two new naturally occurring compounds 7 and 8, together with nine known compounds 9–14, beauverioride I, dipicolinic acid, and 6-(methoxycarbonyl) picolinic acid were isolated from a wasp-pathogenic fungus Beauveria asiatica, strain BCC 16812. Their structures were determined by extensive spectroscopic analyses. The absolute configurations of compound 6 were established by the application of modified Mosher's method and ECD calculation. Compounds 9–11 exhibited antimalarial (IC₅₀ 0.47–1.79 u g/mL), antimicrobial (MIC 3.13–50.0 u g/mL), and cytotoxic (IC₅₀ 0.26–1.23 u g/mL) activities, while compounds 1, 2, 8, and 14 showed only cytotoxicity against Vero cell lines, with IC₅₀ values range 12.96–47.03 u g/mL.

Full text of DOI:10.1016/j.phytol.2021.03.003

Phytochemistry Letters 43 (2021) 8–15
Contents lists available at ScienceDirect
Phytochemistry Letters
journal homepage: www.elsevier.com/locate/phytol
Antimicrobial, antimalarial, and cytotoxic substances from the insect
pathogenic fungus Beauveria asiatica BCC 16812
Jittra Kornsakulkarna, Thapanee Pruksatrakula, Panida Surawatanawongb,
Chattawat Thangsrikeattigunb, Somjit Komwijita, Nattawut Boonyuena,
,
Chawanee Thongpanchanga *
a National Center for Genetic Engineering and Biotechnology (BIOTEC), 113 Thailand Science Park, Phaholyothin Road, Klong Luang, Pathumthani, 12120, Thailand
b Department of Chemistry and Center of Excellence for Innovation in Chemistry, Faculty of Science, Mahidol University, Bangkok, 10400, Thailand
A R T I C L E I N F O
A B S T R A C T
Keywords:
Six new compounds 1–6 and two new naturally occurring compounds 7 and 8, together with nine known
compounds 9–14, beauverioride I, dipicolinic acid, and 6-(methoxycarbonyl) picolinic acid were isolated from a
wasp-pathogenic fungus Beauveria asiatica, strain BCC 16812. Their structures were determined by extensive
spectroscopic analyses. The absolute configurations of compound 6 were established by the application of
modified Mosher’s method and ECD calculation. Compounds 9–11 exhibited antimalarial (IC50 0.47–1.79 u g/
mL), antimicrobial (MIC 3.13–50.0 u g/mL), and cytotoxic (IC50 0.26–1.23 u g/mL) activities, while compounds
1, 2, 8, and 14 showed only cytotoxicity against Vero cell lines, with IC50 values range 12.96–47.03 u g/mL.
2-Pyridone alkaloids
Alloxazines
Insect-pathogenic fungi
Beauveria asiatica
Bioactive secondary metabolites
1. Introduction
presented herein.
Entomopathogenic fungi are widespread in the environment and act
as a pathogen on various substrates, including the plant pests. Many
species of these fungi are widely utilized as biological agents for agri-
cultural and forest pest control, such as Matarhizium anisopliae, Beauveria
bassiana, Beauveria brongniartii, Paecilomyces fumosoroseus, Lecanicilium
longisporum, and Lecanicillium muscarium (Charnley and Collins, 2007).
These fungal strains produce various secondary metabolites, for exam-
ples, oxalic acid, bassianin, pyridovericin, tenellin, beauvericins, beau-
2. Results and discussion
Compound 1 with the molecular formula C27H33NO10, determined
by HRESIMS, was obtained as an amorphous yellow solid. The IR
spectrum displayed the major absorption bands at 3351, 1649 and 1613
cmꢀ 1. The 13C NMR spectrum showed 27 carbons, of which 21 carbons
were assigned for the aglycone unit and the rest were assigned for the
sugar moiety. In the aglycone part, the 1H,13C and HSQC spectroscopic
data indicated the presence of two D2O exchangeable protons (δH 9.58
and δH 17.54 in DMSO), three methyls, one methylene, one methine,
eight olefinic protons, six quaternary olefinic carbons, one carbonyl
group at δC 175.4 and one conjugated carbonyl group at δC 195.2.
Extensive 2D analysis of the NMR spectroscopic data revealed that the
aglycone unit was closely related to that of the known co-metabolite
tenellin (9) (McInnes et al., 1974) except for the presence of the sugar
moiety instead of the hydroxyl group at position 1. The COSY data and
the HMBC correlations from H3-16 to C-9/C-10/C-11 and from H-8/H-9
to C-7 (Fig. 2) constructed the side chain structure C-8 ‒ C-16, which
connected to the carbonyl group (C-7). All E-geometry of the diene was
evident from the large coupling constant between H-8 and H-9 (J =15.4
Hz) and the NOESY correlations from H-8 to H3-16 and H-9 to H-11. The
´
veriolides, and oosporein (Molnar et al., 2010; Roberts, 1981; Wang and
Xu, 2012). However, to the best of our knowledge, there has been no
report to date on the study of bioactive compounds from Beauveria asi-
atica. As part of our ongoing research on the discovery of chemically
diverse and bioactive compounds from insect pathogenic fungi, we came
across the crude extracts of the fungus B. asiatica, strain BCC 16812,
showing a metabolic profile suggestive of novel compounds. The
chemical constituents from the crude extract of the fungus B. asiatica,
strain BCC 16812 were, therefore, investigated. The study led to the
isolation of six new compounds, including alkaloids 1–3 and beauver-
inones A–C (4–6), and two new naturally occurring compounds 7 and 8,
Fig. 1, together with nine known compounds. Details of isolation,
structure elucidation, and biological activities of these compounds are
* Corresponding author.
E-mail address: chawanee@biotec.or.th (C. Thongpanchang).
https://doi.org/10.1016/j.phytol.2021.03.003
Received 21 October 2020; Received in revised form 25 February 2021; Accepted 2 March 2021
Available online 8 March 2021
1874-3900/© 2021 Phytochemical Society of Europe. Published by Elsevier Ltd. All rights reserved.

J. Kornsakulkarn et al.
Phytochemistry Letters 43 (2021) 8–15
appearance of a typical para-disubstituted phenyl signals (AAʹBBʹ sys-
tem) in the 1H NMR spectrum and the HMBC correlations from H-2ʹ/H-6ʹ
to C-4ʹ/C-5, H-3ʹ/H-5ʹ to C-1ʹ and 4ʹ-OH to C-4ʹ/C-5ʹ deduced a
p-hydroxyphenyl group which linked to sp2 quaternary carbon (C-5).
The downfield-shifted of the OH group at δH 17.54 (in DMSO) as well as
the chemical shifts of the C-3, C-4, and C-7 strongly suggested the enol
form of a β-diketone system. The requirement of the N atom from the
molecular formula in combination with the chemical shifts of the het-
eroaromatic sp2 carbon (C-2 and C-6) and the correlations from H-6 to
C-2/C-4/C-1ʹ in the HMBC spectrum proposed the structure of 4-hydrox-
y-2-pyridone ring. The rest of the proton resonances, which exhibited
cross peaks in the COSY spectrum, were attributed to a sugar unit. The
axial orientation of protons 1ʹʹ‒5ʹʹ of the sugar moiety were deduced on
the basis of large vicinal coupling constants (J1ʹʹ,2ʹʹ =8.1 Hz, J2ʹʹ,3ʹʹ =7.5
Hz, J3ʹʹ,4ʹʹ =9.8 Hz, and J4ʹʹ,5ʹʹ = 9.8). These results indicated that the
sugar unit was a β-glucopyranose. The attachment of the sugar moiety at
position 1 of the tenellin was suggested by the NOESY correlation from
Fig. 2. Selected COSY, HMBC, and NOESY correlations of compound 1.
spectrum [(MeCN) Δε (nm) ꢀ 0.24 (345), 0 (285), ꢀ 0.13 (251), 0.06
the anomeric proton (δ 5.10) to H-6 (δ 8.10). Comparison of the
specific rotations of the Haqueous layer of itHs hydrolysate ([
α
]
+48, c
D
(239)] of the aglycone unit, obtained by acid hydrolysis of compound 2,
24
were in good agreement with those of tenellin (McInnes et al., 1974).
20
0.2, MeOH) with that of D-glucopyranose ([
α]
+52, c 10.0, H2O)
(Shiono et al., 2009) established the D configuratioDn of β-glucopyranose.
The 1H NMR spectroscopic data and the optical rotation of the aglycone
unit, obtained by acid hydrolysis of compound 1, were identical to those
of tenellin (McInnes et al., 1974). The CD spectroscopic data of the
Compound
2
was,
therefore,
identified
as
tenellin-N-(4-O-methyl)-β-D-glucopyranoside.
Compound 3 was obtained as a yellow powder. The NMR spectro-
scopic data revealed one methyl, one methylene, two singlet aromatic
protons, three D2O exchangeable protons, and two carbonyl carbons.
The alloxazine skeleton was established on the basis of the molecular
formula of C12H10N4O3, determined by HRESIMS, together with their
chemical shifts and the HMBC correlations from H-6 to C-8/C-9a and H-
9 to C-5a/C-7. The correlations between H2-11 and 11-OH in the COSY
spectrum and the correlations from H-6 to C-11, H-9 to C-12, H2-11 to C-
6/C-7/C-8, H3-12 to C-7/C-8/C-9 in the HMBC spectrum indicated the
positions of the hydroxymethylene group and the methyl group at C-7
and C-8, respectively. The NMR spectroscopic data of 3 corresponded
well to those of the known metabolite 1-methyl-11-hydroxylumichrome
(Andrioli et al., 2017) except for the missing of the methyl group at
position 1 in 3. The UV and IR spectrum of these two compounds were
also almost identical. Therefore, compound 3 was identified as
11-hydroxylumichrome.
aglycone unit [(MeCN) Δε (nm) ꢀ 0.25 (344), 0 (287), ꢀ 0.11 (251), 0.04
(240)] and compound 9 [tenellin, (MeCN) Δ
ε (nm) ꢀ 0.75 (340),
0 (286), ꢀ 0.19 (251), 0.26 (239)] were also similar. Therefore, com-
pound 1 was identified as tenellin-N-β-D-glucopyranoside.
The UV and IR spectra of compound 2 were almost identical to those
of 1. The 1H and 13C NMR spectra were also similar except for the
presence of one more methoxy signal in compound 2. The molecular
formula of 2 was established as C28H35NO10 by HRESIMS, 14 amu
higher than that of 1, which indicated that 2 has an additional methyl
group. The correlation from these methoxy protons (δH 3.53) to C-4ʹ in
the HMBC spectrum led to the deduction of 4-O-methyl-β-glucopyranose
for the sugar unit in 2. The D configuration of 4-O-methyl-β-glucopyr-
anose was deduced by comparing the specific rotations of the aqueous
23
20
layer of its hydrolysate ([
α]
+21.0, c 0.2, MeOH) with that of 4-O-
D
The molecular formula of beauverinone A (4) was determined as
methyl-D-glucopyranose ([α] D +80, c 1.30, MeOH) (Smith, 1951). The
C
17H20N2O5 by HRESIMS in combination with 13C NMR spectroscopic
1H NMR spectroscopic data and the optical rotaion as well as the CD
Fig. 1. Chemical structures of compounds 1–15.
9

J. Kornsakulkarn et al.
Phytochemistry Letters 43 (2021) 8–15
data. The 1H and 13C NMR spectra exhibited the signals for two methyls,
one methoxy, one methylene, one oxymethylene, five aromatic/olefinic
protons, two D2O exchangeable protons, and three carbonyl carbons.
The typical pattern for the AAʹBBʹ system of the four aromatic protons in
the 1H NMR spectrum and the HMBC correlations from H-9 /H-13 to C-
11, H-10/H-12 to C-8 revealed the para-disubstituted phenyl ring. The
presence of an oxygenated isoprene moiety attached at C-11 of the
phenyl ring was indicated by the corresponding chemical shifts, the
cross peak between H2-14 and H-15 in the COSY spectrum, and the
correlations from H2-14 to C-11/C-16, H-15 to C-17/C-18, and H3-17/
H3-18 to C-15/C-16 in the HMBC spectrum. The three carbonyl groups
and the two N atoms were assigned to a piperazinetrione unit, in which
displayed the correlations from 4-NH to C-3/C-5 in the HMBC spectrum.
The HMBC correlations from the methoxy protons to C-6, H2-7 to C-5/C-
6/C-8/C-9/C-13, and H-9/H-13 to C-7 established the position of
methoxy group at C-6 and the CH2-7 linkage between the piperazine and
the phenyl ring at C-6 and C-8, respectively. The gross structure of
beauverinone A (4) was similar to that of 6-[4-(3-methyl-but-2-eny-
loxy)-benzyl]-6-methylsulfanyl-piperazine-2,3,5-trione (Wang et al.,
2015) except for the presence of OMe group at C-6 in 4 instead of SMe
group. The 6S configuration of beauverinone A was suggested by
Fig. 3. Selected COSY and HMBC correlations of compound 6.
25
comparing the optical rotation ([
α]
D ꢀ 21) with that of the known S-1,
4-dimethyl-6-[4-(3-methyl-but-2-enylox-
y)-benzyl]-6-methylsulfanyl-piperazine-2,3,5-trione
(Wang et al., 2015).
in the COSY spectrum and the correlations from H-3ʹ to C-1ʹ/C-5ʹ, H-4ʹ to
C-2ʹ/C-6ʹ, H-5ʹ to C-1ʹ/C-3ʹ, H-6ʹ to C-2ʹ/C-4ʹ, H-4 to C-1ʹ, and 4-NH to C-
2ʹ/C-4ʹ in the HMBC spectrum. The side chain at C-2ʹ of the phenyl ring
was deduced by the cross peaks between H2-8ʹ, H-9ʹ and 9ʹ-OH in the
COSY spectrum and the HMBC correlations from H-3ʹ to C-7ʹ, H2-8ʹ to C-
7ʹ/C-10ʹ, H-9ʹ to C-10ʹ, and 10ʹ-OCH3 to C-10ʹ. Application of the
modified Mosher’s method (Dale and Mosher, 1973; Hoye et al., 2007)
to beauverinone C (6) by conversion into bis (S)- and (R)- MTPA esters
6a and 6b (Fig. 4) led to the deduction of 3S and 9ʹS configurations. The
absolute configurations at C-4 and C-9 were addressed by ECD calcu-
lations. As shown in Fig. 5, the calculated ECD spectrum of (3S,4R,9R,
9ʹS)-6 matched with the experimental curve, suggested the 4R,9R con-
figurations. The co-occurrence with the synthetically known cepha-
losporolide A (7) (Ackland et al., 1984) was also supported the
assignment of 3S and 9R configurations.
25
([α]
ꢀ 25)
D
Beauverinone B (5) was obtained as a light brown solid, possessing
the molecular formula C24H28O11 as determined from HRESIMS. The
major absorption bands for hydroxyl (3327 cmꢀ 1), carbonyl (1712
cmꢀ 1), and conjugated carbonyl (1633 cmꢀ 1) were observed in the IR
spectrum. The 1H NMR and the HSQC spectroscopic data indicated the
presence of one methyl, two methoxys, three methylenes, six methines,
three singlet aromatic protons, and four D2O exchangeable protons. The
downfield hydroxyl proton at δH 12.69 suggested the intramolecular H-
bonding with a carbonyl, which corresponded to low absorption fre-
quency of the conjugated carbonyl group at 1633 cmꢀ 1 in the IR spec-
trum. The HMBC correlations from H-4 to C-3/C-4a/C-5/C-10a, H-5 to
C-4/C-6/C-9a, H-6 to C-7/C-8/C-9a, H-8 to C-6/C-7/C-9/C-9a, 7-OCH3
to C-7, and 10-OH to C-9a/C-10/C-10a established the aglycone core
structure of compound 5, which closely resembled that of semi-
vioxanthin (Yada et al., 2001; Zeeck et al., 1979) except for the sub-
stituent at C-3. The cross peaks between H2-11, H-3, and H2-4 in the
COSY spectrum and the correlations from H2-11 to C-3/C-4/C-12 and
H3-13 to C-11/C-12 deduced the 2-oxypropyl side chain at C-3 of
beauverinone B (5) instead of the methyl group in semivioxanthin. The
sugar unit was established to be the same as that in compound 2 based
on the NMR spectroscopic analysis as previously described for com-
pound 2. Attachment of the sugar unit at C-9 was confirmed by the
correlation from the anomeric proton H-1ʹ to C-9 in the HMBC spectrum.
The configuration of the sugar unit, 4-O-methyl-β-glucopyranose, was
The spectroscopic data of compound 7, including UV, IR, 1H, and 13
C
NMR were resembled those of the semi-synthetic cephalosporolide A
(Ackland et al., 1984). These two compounds were possess the same
deduced as D by comparing the specific rotations of the aqueous layer of
25
its hydrolysate ([
α]
+57, c 0.14, MeOH) with that of 4-O-methyl--
D
25
D-glucopyranose ([
α
]
+80, c 1.30, MeOH) (Smith, 1951). The CD
D
spectrum of beauverinone B (5) [Δ
ε
ꢀ 38.4 (266), 0 (238), ꢀ 32.79
(220)] was in good agreement with that of semivioxanthin isolated from
Penicillium citro-viride [Δε ꢀ 8.18 (270), +1.67 (248), ꢀ 7.79 (222)]
(Zeeck et al., 1979), therefore, the absolute configuration at C-3 of
beauverinone B was suggested as S.
Beauverinone C (6) was obtained as a yellow, amorphous solid. Its
the molecular formula was determined by HRESIMS as C21H27NO8. The
1H and 13C NMR spectra exhibited the presence of one methyl, one
methoxy, five methylenes, four methines, four aromatic protons, three
D2O exchangeable protons, and four carbonyl groups. The COSY cor-
relations and the HMBC correlations from H2-2 to C-1, H2-7/H2-8 to C-6,
and 9-CH3 to C-8/C-9 established the structural feature of 10-membered
macrolide part (Fig. 3). The attachment of the phenyl ring to the NH was
proposed by the correlations between the aromatic protons (H-3ʹ‒H-6ʹ)
Fig. 4. Δδ-Values (δS–δR) of bis (S)- and bis (R)-MTPA esters 6a and 6b.
10

J. Kornsakulkarn et al.
Phytochemistry Letters 43 (2021) 8–15
H3-3ʹ to C-2ʹ, H2-1ʹʹ to C-5/C-3ʹʹ, H2-2ʹʹ to C-5, H-3ʹʹ to C-1ʹʹ, and H3-4ʹʹ to
C-2ʹʹ/C-3ʹʹ confirmed the structural feature of 8 as shown in Fig. 1. The
25
poor specific rotation of compound 8 ([
α]
ꢀ 1.8) suggested that it
D
might be a scalemix mixture. Although methyl 2-(5-(3-hydroxybutyl)
furan-2-yl)acetate has been previously been synthesized (Katsuta et al.,
2015), it is now isolated and reported for the first time from natural
source.
The nine known compounds were identified as tenellin (9) (McInnes
et al., 1974; Wat et al., 1977), pretenellin B (10) (Andrioli et al., 2017;
Halo et al., 2008), 15-hydroxytenellin (11) (Halo et al., 2008), pyr-
idovericin (12) (Andrioli et al., 2017), pyridovericin-N-O-glucopyrano-
side (13) (Andrioli et al., 2017), lumichrome (14) (Andrioli et al., 2017),
beauverioride I (Namatame et al., 1999), dipicolinic acid, and
6-(methoxycarbonyl)picolinic acid (Schmuck and Machon, 2005),
respectively, by analysis of their spectroscopic data which were identical
to those reported in the literature data.
Fig. 5. Comparison of the calculated ECD spectra for (3S,4R,9R,9ʹS)-6,
(3S,4R,9S,9ʹS)-6, (3S,4S,9R,9ʹS)-6, and (3S,4S,9S,9ʹS)-6 with the experimental
CD spectrum for 6.
Compounds 1, 2, 4‒11, 13, and 14 were subjected to assays for
antimalarial (Plasmodium falciparum K1) and antimicrobial (Staph-
ylococus aureus, Acinetobacter baumanii, Colletotrichum acutatum, Curvu-
laria lunata, and Alternaria brassicicola) activities and cytotoxicity
against noncancerous Vero cells. Compounds 9‒11 exhibited weak to
moderate antimalarial (IC50 0.47–1.79 u g/mL), antimicrobial (MIC
3.13–50.0 u g/mL), and cytotoxic (IC50 0.26–1.23 u g/mL) activities
(Table 1). For antimicrobial activity, compound 9 was active against
almost all tested strains except A. baumanii, compound 10 was active
only against S. aureus and C. acutatum while compound 11 was not
active against A. brassicicola. Compounds 1, 2, 8, and 14 showed only
cytotoxic activity against Vero cell lines, with IC50 values of
12.96–49.03 u g/mL.
molecular formula C10H16O4 as deduced by HRESIMS. Analysis of the
2D NMR spectroscopic data supported the structural feature of com-
pound 7 to be the same as that of cephalosporolide A and also corre-
sponded to the 10-membered macrolide part of beauverinone C (6). The
25
good agreement of the optical rotation of 7 ([
α]
D ꢀ 54, c 0.33, CHCl3)
2
◦
with that of cephalosporolide A ([α] D ꢀ 17.8, c 0.5 in CHCl3) (Ackland
et al., 1984) suggested the same absolute configurations at C-3 and C-9
(3S,9R) of these two compounds. Cephalosporolide A has been obtained
from the reduction of thiobiscephalosporolide A, produced by Cepha-
losporium aphidicola (Ackland et al., 1984), however, it was not previ-
ously been isolated from the nature.
In conclusion, ten alkaloids (1–4 and 9–14) together with the other
seven compounds were isolated from a wasp-pathogenic fungus
B. asiatica, strain BCC 16812. Among all isolated compounds, com-
pounds 9-11 exhibited antimalarial, antimicrobial, and cytotoxic ac-
tivities, while compounds 1, 2, 8, and 14 showed only cytotoxic activity.
The HRESIMS data of compound 8 indicated a C11H16O4 molecular
formula, requiring four degree of unsaturations. The 13C NMR and the
HSQC spectroscopic data revealed only four olefinic carbons and one
carbonyl carbon. Therefore, the need for one more degree of unsatura-
tion led to the monocyclic structural feature of 8. The 1H NMR spectrum
resembled that of the semi-synthetic and the synthetic methyl 2-(5-(3-
3. Experimental
´
hydroxybutyl)furan-2-yl)acetate (Katsuta et al., 2015; Oller-Lopez et al.,
2005). Their chemical shifts as well as the COSY correlations from H-3 to
H-4, H2-1ʹʹ to H2-2ʹʹ, H2-2ʹʹ to H-3ʹʹ, H-3ʹʹ to H3-4ʹʹ and the HMBC cor-
relations from H-3 to C-2/C-5, H-4 to C-2/C-5, H2-1ʹ to C-2/C-3/C-2ʹ,
3.1. General procedures
Melting points were measured using a Metler MP90 melting point
Table 1
Biological activities of compounds 1, 2, 4–11, 13, and 14.
compounds
antimalarial P. falciparum, K1 IC50
,
μ
g/
antimicrobial, MIC,
μ
g/mL
cytotoxicity Vero cells IC50
mL
, μg/
mL
S. aureus
A. baumannii
C. acutatum
C. lunata
A. brassicicola
compound 1
compound 2
compound 4
compound 5
compound 6
compound 7
compound 8
compound 9
compound 10
compound 11
compound 13
compound 14
dihydroartemisinina
chloroquine
>10
>10
>10
>10
>10
>10
>10
0.51
0.47
1.79
>10
>10
0.0001
0.184
>50
>50
>50
>50
>50
>50
>50
6.25
6.25
25.0
>50
>50
–
>50
>50
>50
>50
>50
>50
>50
>50
>50
25.0
>50
>50
–
>50
>50
>50
>50
>50
>50
>50
3.13
50
>50
>50
>50
>50
>50
>50
>50
3.13
>50
12.5
>50
>50
–
>50
>50
>50
>50
>50
>50
>50
50.0
>50
>50
>50
>50
–
49.0
16.9
>50
>50
>50
>50
33.0
0.26
0.55
1.23
>50
12.9
–
12.5
>50
>50
–
–
–
–
–
–
–
diphosphatea
rifamycinb
–
–
–
–
–
0.08
3.13
–
–
–
–
vancomycinb
erythromycinb
amphotericin Bb
ellipticinec
1.00
–
–
–
–
–
–
–
–
12.50
–
–
–
–
–
–
1.56
3.13
1.56
–
–
–
–
1.81
a
Positive control for the antimalarial assay.
Positive control for the antimicrobial assay.
Positive control for the cytotoxicity assay.
b
c
11

J. Kornsakulkarn et al.
Phytochemistry Letters 43 (2021) 8–15
apparatus and are uncorrected. Optical rotation measurements were
conducted by using a JASCO P-1030 digital polarimeter. UV and FT-IR
spectra were recorded on an Analytic Jena Specord 50 P spectropho-
tometer and a Bruker Alpha spectrometer. The CD spectra were recorded
on a JASCO J-810 spectropolarimeter. NMR spectra were recorded on
Bruker Advance III 400 and Bruker Advance 500 spectrometers. ESITOF
MS data were obtained on a Bruker micrOTOF mass spectrometer.
Column chromatography was performed on silica gel 60 (70–230 Mesh
ASTM, Merck). HPLC were performed using Dionex-Ultimate 3000 se-
ries equipped with a binary pump, an autosampler, and a diode array
detector.
concentrated filtrate (7.24 g) was fractionated using Sephadex LH 20
(7.0 ☓ 35 cm), eluted with 100 % MeOH, to give five fractions (B1–B5).
Fraction B-2 (6.21 g) was separated by combination of CC on Sephadex
LH-20 (MeOH), silica gel (MeOH/CH2Cl2) and preparative HPLC MeCN/
H2O) to give compounds 6 (28.3 mg), 7 (31.3 mg), 8 (26.5 mg), dipi-
colinic acid (20.7 mg), and 6-(methoxycarbonyl) picolinic acid (172.8
mg). Compound 3 (0.9 mg) was obtained from fraction B4 (35.1 mg)
after purification by preparative HPLC (step gradient elution with 8 ꢀ 38
% MeCN/H2O, flow rate 12 mL/min, 35 min).
3.3.1. Compound 1
23
Yellow amorphous solid; [
α]
D –58.0 (c 0.30, MeOH); UV (MeOH)
(nm)
λmax (log
ε
) 201 (4.67), 249 (4.36), 343 (4.23) nm; CD (MeCN) Δ
ε
3.2. Fungal material
ꢀ 0.30 (350), 0.18 (292), ꢀ 0.56 (250); IR (ATR) νmax 3351, 2961, 2925,
1649, 1613, 1513, 1459, 1366, 1270, 1229, 1177 cmꢀ 1; 1H NMR (500
MHz, Acetone-d6) δ 17.54 (1H, s, 4-OH, in DMSO- d6), 8.71 (1H, s, 4ʹ-
OH), 8.10 (1H, s, H-6), 8.01 (1H, d, J =15.4 Hz, H-8), 7.63 (1H, d, J
=15.4 Hz, H-9), 7.36 (2H, d, J =8.5 Hz, H-2ʹ, H-6ʹ), 6.88 (2H, d, J =8.5
Hz, H-3ʹ, H-5ʹ), 5.97 (1H, d, J =9.8 Hz, H-11), 5.20 (1H, br s, 3ʹʹ-OH),
5.10 (1H, d, J =8.1 Hz, H-1ʹʹ), 4.50 (1H, br s, 4ʹʹ-OH), 4.40 (1H, d, J =3.7
Hz, 2ʹʹ-OH), 3.87–3.91 (1H, m, Ha-6ʹʹ), 3.70–3.74 (1H, m, Hb-6ʹʹ), 3.53
(1H, t, J =9.7 Hz, H-3ʹʹ), 3.48–3.51 (1H, m, H-5ʹʹ), 3.41 (1H, dd, J = 7.5,
9.7 Hz, H-4ʹʹ), 3.38 (1H, dd, J = 7.5, 8.1 Hz, H-2ʹʹ), 2.54–2.63 (1H, m, H-
12), 1.93 (3H, s, H-16), 1.43–1.50 (1H, m, Ha-13), 1.33–1.37 (1H, m,
Hb-13), 1.03 (3H, d, J =6.6 Hz, H-15), 0.87 (3H, t, J =7.51 Hz, H-14);
13C-NMR (125 MHz, Acetone-d6) δ 195.2 (C-7), 177.1 (C-2), 159.7 (C-4),
158.2 (C-4ʹ), 152.3 (C-11), 151.7 (C-7), 143.0 (C-6), 134.0 (C-10), 131.5
(C-2ʹ, C-6ʹ), 123.9 (C-1ʹ), 123.8 (C-8), 115.9 (C-3ʹ, C-5ʹ), 114.2 (C-5),
108.9 (C-1ʹʹ), 107.3 (C-3), 78.6 (C-5ʹʹ), 77.4 (C-3ʹʹ), 73.0 (C-2ʹʹ), 70.6 (C-
4ʹʹ), 62.5 (C-6ʹʹ), 35.9 (C-12), 30.3 (C-13), 20.3 (C-15), 12.8 (C-16), 12.2
(C-14); HRMS (ESITOF) m/z 554.1997 [M + Na]+ (calcd. for
C27H33NO10Na, 554.1995).
The fungus used in this study was found on a wasp (Hymenoptera) at
Sip Et Chan waterfall, Khao Sok National Park, Surat Thani Province,
Thailand. The living culture was deposited in the BIOTEC Culture
Collection (BCC) as BCC 16812 on January 5, 2005. This fungus was
isolated and morphologically identified as Beauveria sp. by Dr. Nigel
Hywel-Jones, BIOTEC.
The ITS region was amplified using primers: ITS1 and ITS4 primers
(White et al., 1990). DNA sequences similarities of this locus was used to
search for similar sequences in the NCBI (GenBank) using BLASTN
2.2.18 (Altschul et al., 1997). Sequence of ITS data is matched
(99.26–99.44 % homology) with accession numbers of B. asiatica
(MN401634, MG345071, MN401644 and MN401641). The ITS
sequence of B. asiatica isolate BCC16812 (Cordycipitaceae, Hypocreales,
Hypocreomycetidae, Sordariomycetes, Pezizomycotina, Ascomycota)
has been deposited in GenBank with accession number MW139305.
3.3. Fermentation, extraction, and isolation
B. asiatica BCC 16812 was developed on potato dextrose agar at 25 ◦C
for 14 days, and the agar was cut into pieces (1 × 1 cm) and inoculated
into 8 × 250 mL Erlenmeyer flasks containing 25 mL of yeast extract
broth (YES: yeast extract 20.0 g/L, sucrose 150 g/L). After incubation at
25 ◦C for 5 days on a rotary shaker (200 rpm), each primary culture was
transferred into 1 L Erlenmeyer flask containing 250 mL of the same
medium and incubated under the same conditions for 5 days. Each 25
mL portion of the secondary culture was transferred into 80 × 1 L
Erlenmeyer flasks containing 250 ml of YES medium and was statically
incubated at 25 ◦C for 35 days.
3.3.2. Compound 2
23
Yellow amorphous solid; [
α]
D –54.0 (c 0.23, MeOH); UV (MeOH)
(nm)
λmax (log
ε) 201 (4.36), 249 (4.10), 346 (3.93) nm; CD (MeCN) Δ
ε
ꢀ 0.23 (349), 0.23 (292), ꢀ 0.69 (249); IR (ATR) νmax 3377, 2961, 2925,
1650, 1613, 1514, 1461, 1365, 1271, 1226, 1177 cmꢀ 1; 1H NMR (400
MHz, Acetone-d6) δ 17.53 (1H, s, 4-OH, in DMSO- d6), 8.56 (1H, s, 4ʹ-
OH), 8.07 (1H, s, H-6), 8.01 (1H, d, J =15.4 Hz, H-8), 7.63 (1H, d, J
=15.4 Hz, H-9), 7.36 (2H, d, J =8.4 Hz, H-2ʹ, H-6ʹ), 6.89 (2H, d, J =8.5
Hz, H-3ʹ, H-5ʹ), 5.97 (1H, d, J =9.8 Hz, H-11), 5.15 (1H, br s, 3ʹʹ-OH),
5.08 (1H, d, J =8.3 Hz, H-1ʹʹ), 4.48 (1H, d, J =3.9 Hz, 2ʹʹ-OH), 3.92 (1H,
t, J =6.1 Hz, 6ʹʹ-OH), 3.83 (1H, dd, J = 4.9, 10.9 Hz, Ha-6ʹʹ), 3.72 (1H,
dd, J = 6.0, 11.8 Hz, Hb-6ʹʹ), 3.65 (1H, t, J =10.2 Hz, H-3ʹʹ), 3.53 (3H, s,
4ʹʹ-OCH3), 3.46 (1H, ddd, J = 2.0, 4.9, 9.6 Hz, H-5ʹʹ), 3.38 (1H, t, J =8.6
Hz, H-2ʹʹ), 3.17 (1H, t, J =9.3 Hz, H-4ʹʹ), 2.55–2.63 (1H, m, H-12), 1.93
(3H, s, H-16), 1.42–1.51 (1H, m, Ha-13), 1.34–1.40 (1H, m, Hb-13), 1.03
(3H, d, J =6.6 Hz, H-15), 0.88 (3H, t, J =7.3 Hz, H-14); 13C-NMR (100
MHz, Acetone-d6) δ 195.2 (C-7), 177.2 (C-2), 159.7 (C-4), 158.2 (C-4ʹ),
152.3 (C-11), 151.6 (C-9), 143.0 (C-6), 134.0 (C-10), 131.5 (C-2ʹ, C-6ʹ),
124.1 (C-1ʹ), 123.9 (C-8), 116.0 (C-3ʹ, C-5ʹ), 114.2 (C-5), 108.6 (C-1ʹʹ),
107.4 (C-3), 79.5 (C-4ʹʹ), 77.8 (C-5ʹʹ), 77.5 (C-3ʹʹ), 73.2 (C-2ʹʹ), 62.1 (C-
6ʹʹ), 60.6 (4ʹʹ-OCH3), 35.0 (C-12), 30.8 (C-13), 20.3 (C-15), 12.8 (C-16),
12.2 (C-14); HRMS (ESITOF) m/z 568.2183 [M + Na]+ (calcd. for
C28H35NO10Na, 568.2153).
After filtration of the culture, the cells were macerated in MeOH
(20.0 L) for 3 days, and then in CH2Cl2 (20.0 L) for 3 days. The MeOH
and CH2Cl2 extracts were combined and evaporated under reduced
pressure. Water (3.0 L) was added and the mixture was extracted with
hexanes (3 × 3.0 L), followed by EtOAc (3 × 3.0 L). Trituration of the
EtOAc extract (a brown solid, 14.12 g) in MeOH followed by filtration
provided a brown solid (7.40 g) in which compound 5 (13.0 mg) and
beauveriolide I (1.54 g) were obtained after further purification by silica
gel CC, step gradient elution with 0 ꢀ 10 % MeOH/CH2Cl2. The filtrate
(5.96 g) was fractionated using Sephadex LH 20 (7.0 ☓ 35 cm), eluted
with 100 % MeOH, to provide five fractions (A1–A5). Fraction A3 (1.44
g) was subjected to preparative HPLC using reverse phase column
(SunFire C18 OBD, 10 μm, 19☓250 mm, step gradient elution with 15 ꢀ
80 % MeCN/H2O, flow rate 12 mL/min, 65 min), and the fractions were
further purified by preparative thin layer chromatography, using 10 %
MeOH/CH2Cl2 as an eluent, to furnished compounds 1 (3.5 mg), 2 (7.2
mg), 4 (3.7 mg), 5 (8.4 mg), and 13 (10.0 mg). Purification of fraction
A4 (310.0 mg) by preparative HPLC (step gradient elution with 20 ꢀ 100
% MeCN/H2O, flow rate 12 mL/min, 45 min) afforded compounds 3 (2.4
mg), 9 (18.5 mg), 10 (5.5 mg), 11 (9.7 mg), 12 (1.6 mg), and 14 (4.4
mg).
3.3.3. Compound 3
Yellow solid solid; UV (MeOH) λmax (log ε) 206 (4.01), 245 (3.80),
260 (3.78), 284 (3.57) nm; IR (ATR) νmax 3429, 2924, 1709, 1578, 1455,
1434, 1362, 1289, 1207 cmꢀ 1; 1H NMR (500 MHz, DMSO-d6) δ 11.87
(1H, s, NH-1), 11.69 (1H, s, NH-3), 7.91 (1H, s, H-9), 7.89 (1H, s, H-6),
5.56 (1H, t, J =5.0 Hz, 11-OH), 4.72 (2H, d, J =5.0 Hz, H-11), 2.44 (3H,
s, H-12); 13C-NMR (125 MHz, DMSO-d6) δ 160.6 (C-4), 150.0 (C-2),
148.2 (C-8), 146.5 (C-10a), 141.6 (C-5a), 138.5 (C-9a), 136.8 (C-7),
130.4 (C-4a), 128.9 (C-9), 122.5 (C-6), 60.8 (C-11), 18.1 (C-12); HRMS
The culture broth was extracted with EtOAc (3 × 20 L) and evapo-
rated to dryness, leaving a brown solid (8.33 g). Trituration of the crude
extract in MeOH followed by filtration gave a brown solid (1.08 g). The
12

J. Kornsakulkarn et al.
Phytochemistry Letters 43 (2021) 8–15
(ESITOF) m/z 281.0648 [M + Na]+ (calcd. for C12H10N4O3Na,
2.02–2.11 (1H, m, Ha-8), 1.93–2.00 (1H, m, Hb-8), 1.47–1.55 (1H, m,
Hb-4), 1.23 (3H, d, J =6.3 Hz, 9-CH3); 13C-NMR (100 MHz, Acetone-d6)
δ 210.9 (C-6), 170.4 (C-1), 72.1 (C-9), 64.9 (C-3), 42.4 (C-2), 40.1 (C-7),
38.7 (C-5), 33.6 (C-8), 31.8 (C-4), 19.6 (9-CH3); HRMS (ESITOF) m/z
223.0973 [M + Na]+ (calcd. for C10H16O4Na, 223.0941).
281.0645).
3.3.4. Compound 4
24
Light brown solid; [
α
]
–21.0 (c 0.08, MeOH); UV (MeOH) λmax
(log
ε
) 201 (4.48), 219 (4.20D), 268 (3.48) nm; IR (ATR) νmax 3205, 2924,
2853, 1709, 1613, 1511, 1434, 1368, 1250, 1179 cmꢀ 1; 1H NMR (400
MHz, Acetone-d6) δ 10.89 (1H, br s, NH-1), 8.61 (1H, s, NH-4), 7.10 (2H,
d, J =9.5 Hz, H-9, H-13), 6.82 (2H, d, J =9.5 Hz, H-10, H-12), 5.42 (1H,
t, J =6.6 Hz, H-15), 4.49 (2H, d, J =6.6 Hz, H-14), 3.34 (3H, s, 6-OCH3),
3.33 (1H, d, J =13.3 Hz, Ha-7), 3.09 (1H, d, J =13.3 Hz, Hb-7), 1.75 (3H,
s, H-17), 1.71 (3H, s, H-18); 13C-NMR (100 MHz, Acetone-d6) δ 168.5 (C-
5), 159.4 (C-11), 156.4 (C-3), 154.5 (C-2), 137.7 (C-16), 132.6 (C-9, C-
13), 125.4 (C-8), 120.9 (C-15), 115.5 (C-10, C-12), 90.9 (C-6), 65.3 (C-
14), 52.2 (6-OCH3), 46.0 (C-7), 25.7 (C-17), 18.1 (C-18); HRMS (ESI-
TOF) m/z 355.1246 [M + Na]+ (calcd. for C17H20N2O5Na, 355.1264).
3.3.8. Compound 8
25
Yellow oil; [
α
]
D –1.8 (c 0.21, MeOH); UV (MeOH) λmax (log
ε) 202
(3.78), 228 (3.67), 360 (2.88) nm; IR (ATR) νmax 3486, 2969, 1737,
1644, 1437, 1378, 1345, 1299, 1131, 1096 cmꢀ 1; 1H NMR (400 MHz,
CDCl3) δ 6.09 (1H, d, J = 3.0, Hz, H-3), 5.94 (1H, d, J =3.0 Hz, H-4),
3.78–3.87 (1H, m, H-3ʹʹ), 3.71 (3H, s, H-3ʹ), 3.63 (2H, s, H-1ʹ), 2.60–2.78
(2H, m, H-1ʹʹ), 1.73–1.79 (2H, m, H-2ʹʹ), 1.21 (3H, d, J =6.2 Hz, H-4ʹʹ);
13C-NMR (100 MHz, Acetone-d6) δ 170.1 (C-2ʹ), 155.4 (C-5), 145.8 (C-
2), 108.6 (C-3), 105.8 (C-4), 67.3 (C-3ʹʹ), 52.2 (C-3ʹ), 37.3 (C-2ʹʹ), 33.9
(C-1ʹ), 24.4 (C-1ʹʹ), 23.4 (C-4ʹʹ); HRMS (ESITOF) m/z 235.0947 [M +
Na]+ (calcd. for C11H16O4Na, 235.0941).
3.3.5. Compound 5
23
Light brown solid; [
α]
–44.6 (c 0.12, DMSO); UV (MeOH) λmax
D
(log
ε
) 201 (3.90), 219 (3.94), 262 (4.45), 308, (3.48), 358 (3.69) nm;
(nm) ꢀ 38.4 (266), 0 (238), ꢀ 32.79 (220); IR (ATR) νmax
3.4. Hydrolysis of compound 1
CD (MeOH) Δ
ε
3327, 2925, 2853, 1712, 1633, 1613, 1580, 1456, 1380, 1208, 1164,
1107, 1092 cmꢀ 1; 1H NMR (500 MHz, DMSO-d6) δ 12.69 (1H, s, 10-OH),
7.04 (1H, s, H-5),6.90 (1H, s, H-6), 6.78 (1H, s, H-8), 5.29 (1H, d, J =5.2
Hz, 3ʹ-OH), 5.07 (1H, br s, 2ʹ-OH), 5.03 (1H, d, J =7.0 Hz, H-1ʹ),
4.98–5.02 (1H, m, H-3), 4.71 (1H, t, J =5.5 Hz, 6ʹ-OH), 3.86 (3H, s, 7-
OCH3), 3.66 (1H, dd, J = 4.5, 11.6 Hz, Ha-6ʹ), 3.52 (1H, dd, J = 5.5, 11.6
Hz, Hb-6ʹ), 3.45 (3H, s, 4ʹ-OCH3), 3.45 (1H, m, H-3ʹ), 3.41–3.44 (2H, m,
H-2ʹ, H-5ʹ), 3.05 (1H, t, J =9.5 Hz, H-4ʹ), 3.02–3.09 (2H, m, H-4),
2.94–3.02 (2H, m, H-11), 2.18 (3H, s, H-13); 13C-NMR (125 MHz,
DMSO-d6) δ 205.2 (C-12), 169.9 (C-1), 162.8 (C-10), 161.1 (C-7), 157.7
(C-9), 140.6 (C-5a), 134.3 (C-4a), 115.2 (C-5), 109.8 (C-9a), 101.8 (C-8),
100.9 (C-1ʹ), 100.8 (C-6), 100.6 (C-10a), 79.1 (C-4ʹ), 76.1 (C-5ʹ), 75.8 (C-
3ʹ), 75.0 (C-3), 73.6 (C-2ʹ), 60.3 (C-6ʹ), 59.6 (4ʹ-OCH3), 55.44 (7-OCH3),
47.3 (C-11), 32.1 (C-4), 30.2 (C-13); HRMS (ESITOF) m/z 515.1527 [M
+ Na]+ (calcd. for C24H28O11Na, 515.1524).
Compound 1 (1.5 mg, 2.82 mmol) was hydrolyzed with 6 M aqueous
HCl (0.2 mL) in MeOH (0.1 mL) at 80 ◦C for 2 h. The reaction mixture
was then diluted with H2O (2.0 mL) and extracted with EtOAc (2.0 mL).
The aqueous layer was concentrated in vacuo to yield D-glucopyranose
24
(0.5 mg, 100 % yield, [
α
]
D +48.0, c 0.2, MeOH). The organic layer was
evaporated to dryness under reduced pressure to obtain the aglycone
24
[0.9 mg, 88 % yield, [
α
]
ꢀ 23.0 (c 0.1, MeOH), CD (MeCN) Δε (nm)
ꢀ 0.25 (344), 0 (287), ꢀ 0.D11 (251), 0.04 (240)], whose 1H NMR spec-
trum as well as optical rotation were in good agreement with those of
tenellin (McInnes et al., 1974).
3.5. Hydrolysis of compound 2
Compound 2 (1.2 mg, 2.2 mmol) was hydrolyzed by the method
described for compound 1. The aqueous layer was concentrated in vacuo
23
3.3.6. Compound 6
to yield 4-O-methyl-D-glucopyranose (0.4 mg, 92 % yield, [
α
]
D +21.0,
25
Yellow amorphous solid; [
α]
D –61.0 (c 0.20, CHCl3); UV (CH3CN)
c 0.2, MeOH). The organic layer was evaporated to dryness under
24
λmax (log
ε
) 191 (4.48), 231 (4.31), 260 (3.88), 379, (3.68) nm; CD
(nm) +27.81 (266), 0 (247), ꢀ 39.32 (236); IR (ATR) νmax
reduced pressure to obtain the aglycone unit [0.7 mg, 81 % yield, [
α]
D
(CH3CN) Δ
ε
ꢀ 18 (c 0.1, MeOH), CD (MeCN) Δ
ε
(nm) ꢀ 0.24 (345), 0 (285), ꢀ 0.13
1
3300, 2928, 1730, 1708, 1633, 1576, 1520, 1435, 1362, 1253, 1222,
1167, 1044, 1025 cmꢀ 1; 1H NMR (400 MHz, Acetone-d6) δ 9.17 (1H, d, J
=8.2 Hz, 4-NH), 7.88 (1H, dd, J = 1.5, 8.1 Hz, H-3ʹ), 7.36 (1H, t, J =8.3
Hz, H-5ʹ), 6.96 (1H, d, J =8.6 Hz, H-6ʹ), 6.60 (1H, t, J =7.6 Hz, H-4ʹ),
5.08 (1H, m, H-9), 4.69 (1H, dd, J = 5.6, 11.3 Hz, H-9ʹ), 4.44–4.49 (1H,
m, H-3), 4.40–4.42 (1H, m, H-4), 4.39–4.42 (1H, m, 9ʹ-OH), 4.23 (1H, d,
J =4.7 Hz, 3-OH), 3.71 (3H, s, 10ʹ-OCH3), 3.46 (1H, dd, J = 6.6, 16.9 Hz,
Ha-8ʹ), 3.40 (1H, dd, J = 4.7, 16.9 Hz, Hb-8ʹ), 2.64–2.72 (2H, m, H-5),
2.63–2.68 (2H, m, H-2), 2.54 (1H, ddd, J = 3.0, 10.3, 13.4 Hz, Ha-7),
2.22–2.26 (1H, m, Hb-7), 2.06–2.11 (1H, m, Ha-8), 1.99–2.03 (1H, m,
Hb-8), 1.23 (3H, d, J =6.3 Hz, 9-CH3); 13C-NMR (100 MHz, Acetone-d6)
δ 209.8 (C-6), 200.1 (C-7ʹ), 174.9 (C-10ʹ), 170.2 (C-1), 151.7 (C-1ʹ),
135.9 (C-5ʹ), 133.0 (C-3ʹ), 118.5 (C-2ʹ), 115.1 (C-4ʹ), 113.2 (C-6ʹ), 72.6
(C-9), 68.2 (C-9ʹ), 67.5 (C-3), 52.8 (C-4), 52.2 (10ʹ-OCH3), 45.4 (C-2),
43.8 (C-8), 40.5 (C-7), 38.8 (C-5), 34.4 (C-8), 19.7 (9-CH3); HRMS
(ESITOF) m/z 444.1631 [M + Na]+ (calcd. for C21H27NO8Na,
444.1629).
(251), 0.06 (239)], whose H NMR spectrum and the optical rotation
were identical to those of tenellin (McInnes et al., 1974).
3.6. Hydrolysis of compound 5
Compound 5 (8.4 mg, 17.1 mmol) was hydrolyzed with 10 % H2SO4
(2.0 mL) in EtOH (2.0 mL) under reflux for 2 h. The reaction mixture was
evaporated to remove EtOH and then diluted with H2O (2.0 mL) and
extracted with EtOAc (2.0 mL). The aqueous layer was concentrated in
25
vacuo to yield 4-O-methyl-D-glycopyranose (1.2 mg, 36 % yield, [α]
+57.0, c 0.14, MeOH). The organic layer was evaporated to drynesDs
under reduced pressure and then purified by prep. HPLC (step gradient
elution with 30 ꢀ 100 % MeCN/H2O, flow rate 10 mL/min, 50 min) to
obtain the aglycone 15 (2.5 mg, 46 % yield).
3.6.1. Compound 15
24
White solid; [
α]
+27.0 (c 0.06, MeOH); 1H NMR (500 MHz,
D
3.3.7. Compound275
Acetone-d6) δ 13.82 (1H, s, 10-OH), 9.43 (1H, s, 9-OH), 7.07 (1H, s, H-
5),6.79 (1H, d, J =2.3 Hz, H-6), 6.46 (1H, d, J =2.3 Hz, H-8), 5.11–5.15
(1H, m, H-3), 3.90 (3H, s, 7-OCH3), 3.19 (1H, ddd, J = 0.6, 3.3, 16.4 Hz,
Ha-4), 3.15 (1H, dd, J = 7.2, 16.4 Hz, Hb-4), 3.06 (1H, ddd, J = 1.4,
10.9, 16.8 Hz, Ha-11), 3.01 (1H, dd, J = 5.6, 16.8 Hz, Hb-11), 2.22 (3H,
s, H-13); HRMS (ESITOF) m/z 339.0841 [M + Na]+ (calcd. for
Yellow oil; [α] D –54.0 (c 0.33, CHCl3); UV (CH3CN) λmax (log ε) 191
(3.22), 263 (2.61) nm; IR (ATR) νmax 3435, 2958, 2931, 1727, 1708,
1442, 1355, 1253, 1166, 1049 cmꢀ 1; 1H NMR (400 MHz, CDCl3) δ 5.46
(1H, br s 3-OH), 5.01–5.09 (1H, m, H-9), 4.23 (1H, ddd, J = 3.3,
10.3,10.3 Hz, H-3), 2.75 (1H, dd, J = 2.2, 16.2 Hz, Ha-2), 2.58 (1H, dd, J
= 3.0, 5.1 Hz, Ha-5), 2.26–2.43 (5H, m, Hb-2, Ha-4, Hb-5, H-7),
C17H16O6Na, 339.0839).
13

J. Kornsakulkarn et al.
Phytochemistry Letters 43 (2021) 8–15
3.7. Preparation of the Mosher ester derivatives 6a and 6b
experimental ECD spectrum.
Compound 6 (2.1 mg, 0.0050 mmol) was treated with (-)-(R)-
3.9. Biological assays
MTPACl (33 μL) in CH2Cl2 (0.2 mL) and pyridine (0.1 mL) at room
temperature for 16 h. The mixture was evaporated under reduced
pressure. The crude reaction was purified by preparative HPLC (step
gradient elution with MeCN/H2O, 40–100 %, flow rate 10 mL/min, 40
min) to obtain the bis (S)-MTPA ester 6a (2.2 mg, 0.0026 mmol, 52 %
yield). Similarly, bis (R)-MTPA ester 6b was prepared from compound 6
Antimalarial activity against P. falciparum K1 was evaluated using
the microculture radioisotope technique (Desjardins et al., 1979).
Antimicrobial activity against S. aureus, A. baumannii, C. acutatum,
C. lunata, and A. brassicicola were evaluated by using the standard
protocols published by the Clinical and Laboratory Standard Institute
(CLSI, 2006a, 2006b). Cytotoxicity to Vero cells (African green monkey
kidney fibroblast) was performed by using the green fluorescent protein
(GFP) based method (Changsen et al., 2003; Hunt et al., 1999).
(1.9 mg, 0.0045 mmol) and (+)-(S)-MTPACl (33 μL) and was obtained in
1.4 mg (0.0016 mmol, 36 % yield) after purification by preparative
HPLC. The assignment of 6a and 6b were achieved by analysis of NMR
spectroscopic and mass spectrometry data.
Maximum test concentration for the antimalarial test was at 10
and for the other tests was at 50 g/mL.
μg/mL
μ
3.7.1. Bis (S)-MTPA ester 6a
White solid; 1H NMR (500 MHz, CDCl3) δ 9.03 (1H, d, J =8.8 Hz, 4-
NH), 7.60–7.62 (2H, m, ArH of MTPA), 7.58 (1H, d, J =8.4 Hz, H-3ʹ),
7.20–7.37 (8H, m, ArH of MTPA), 7.26 (1H, obsc., H-5ʹ), 6.72 (1H, d, J
=7.7 Hz, H-6ʹ), 6.58 (1H, t, J =7.6 Hz, H-4ʹ), 5.91 (1H, dd, J = 2.9, 8.6
Hz, H-9ʹ), 5.74 (1H, br s, H-3), 5.10–5.15 (1H, m, H-9), 4.70–4.75 (1H,
m, H-4), 3.81 (3H, s, 10ʹ-OCH3), 3.62 (3H, s, OCH3 of MTPA), 3.60 (1H,
dd, J = 8.6, 17.6 Hz, Ha-8ʹ), 3.43 (1H, dd, J = 2.9, 17.6 Hz, Hb-8ʹ), 3.35
(3H, s, OCH3 of MTPA), 2.88–2.93 (1H, m, Ha-2), 2.74 (1H, d, J =16.7
Hz, Hb-2), 2.68 (2H, br s, H-5), 2.33–2.45 (2H, m, H-7), 2.15–2.23 (1H,
m, Ha-8), 2.01–2.07 (1H, m, Hb-8), 1.31 (3H, d, J =6.2 Hz, 9-CH3);
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper
Acknowledgments
This work was supported by National Center for Genetic Engineering
and Biotechnology (BIOTEC), National Science and Technology Devel-
opment Agency (NSTDA), and Thailand Research Fund (Grant No.
P2050093).
HRMS (ESITOF) m/z 876.2421 [M
41H41F6NO12F6+Na, 876.2425).
+
Na]+ (calcd. for:
C
3.7.2. Bis (R)-MTPA ester 6b
White solid; 1H NMR (500 MHz, CDCl3) δ 9.02 (1H, d, J =8.5 Hz, 4-
NH), 7.58–7.60 (2H, m, ArH of MTPA), 7.59 (1H, obsc., H-3ʹ), 7.26–7.47
(8H, m, ArH of MTPA), 7.17–7.26 (2H, m, H-5ʹ, H-6ʹ), 6.59 (1H, t, J =7.5
Hz, H-4ʹ), 5.85 (1H, dd, J = 2.5, 9.5 Hz, H-9ʹ), 5.73 (1H, br s, H-3),
5.10–5.16 (1H, m, H-9), 4.58–4.63 (1H, m, H-4), 3.79 (3H, s, 10ʹ-OCH3),
3.61 (1H, dd, J = 9.5, 17.6 Hz, Ha-8ʹ), 3.54 (3H, s, OCH3 of MTPA), 3.44
(1H, dd, J = 2.5, 17.6 Hz, Hb-8ʹ), 3.37 (3H, s, OCH3 of MTPA), 2.92–2.97
(1H, m, Ha-2), 2.84–2.87 (1H, m, Hb-2), 2.66 (2H, br s, H-5), 2.32–2.42
(2H, m, H-7), 2.13–2.22 (1H, m, Ha-8), 2.01–2.07 (1H, m, Hb-8), 1.31
(3H, d, J =6.3 Hz, 9-CH3); HRMS (ESITOF) m/z 876.2429 [M + Na]+
(calcd. for: C41H41F6NO12F6+Na, 876.2425).
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.phytol.2021.03.003.
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