Phenolic glucosides and chromane analogs from the insect fungus Conoideocrella krungchingensis BCC53666

Article abstract of DOI:10.1016/j.tet.2019.05.007

Six new compounds, named conoideoglucosides A ? C and conoideochromanes A ? C, together with eight known compounds, including eutypinic acid, 2,2-dimethyl-2H-1-chromene-6-carboxylic acid, (?)-luteoskyrin, (?)-4a-oxyluteoskyrin, chrysophanol, islandicin, c

Full text of DOI:10.1016/j.tet.2019.05.007

Accepted Manuscript
Phenolic glucosides and chromane analogs from the insect fungus Conoideocrella
krungchingensis BCC53666
Karoon Sadorn, Siriporn Saepua, Nattawut Boonyuen, Somjit Komwijit, Pranee
Rachtawee, Pattama Pittayakhajonwut
PII:
S0040-4020(19)30514-9
https://doi.org/10.1016/j.tet.2019.05.007
TET 30328
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 9 February 2019
Revised Date: 1 May 2019
Accepted Date: 2 May 2019
Please cite this article as: Sadorn K, Saepua S, Boonyuen N, Komwijit S, Rachtawee P,
Pittayakhajonwut P, Phenolic glucosides and chromane analogs from the insect fungus Conoideocrella
krungchingensis BCC53666, Tetrahedron (2019), doi: https://doi.org/10.1016/j.tet.2019.05.007.
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
Graphical Abstract
Leave this area blank for abstract info.
Phenolic glucosides and chromane analogs from
the insect fungus Conoideocrella krungchingensis BCC53666
Karoon Sadorna,b,*, Siriporn Saepuac, Nattawut Boonyuenc, Somjit
Pittayakhajonwutc
Komwijitc, Pranee Rachtaweec, Pattama
Six new compounds, named conoideoglucosides A − C and conoideochromanes A − C, were isolated
from the insect fungus Conoideocrella krungchingensis BCC53666.

ACCEPTED MANUSCRIPT
Phenolic glucosides and chromane analogs from the insect fungus
Conoideocrella krungchingensis BCC53666
Karoon Sadorna,b,*, Siriporn Saepuac, Nattawut Boonyuenc, Somjit
Rachtaweec, Pattama Pittayakhajonwutc
Komwijitc, Pranee
aIntegrated Applied Chemistry Research Unit, Faculty of Science, King Mongkut’s Institute of Technology
Ladkrabang, Chalongkrung Road, Ladkrabang, Bangkok 10520, Thailand
bDepartment of Chemistry, Faculty of Science, King Mongkut’s Institute of Technology Ladkrabang, Chalongkrung
Road, Ladkrabang, Bangkok 10520, Thailand
cNational Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology
Development Agency (NSTDA), Thailand Science Park, Phaholyothin Road, Klong Luang, Pathumthani 12120,
Thailand
*Corresponding author.
Tel.: +66-2-329-8400, ext. 290. Fax: +66-2-329-8428. E-mail: karoon.sa@kmitl.ac.th.
1

ACCEPTED MANUSCRIPT
Abstract
Six new compounds, named conoideoglucosides A − C and conoideochromanes A − C,
together with eight known compounds, including eutypinic acid, 2,2-dimethyl-2H-1-chromene-
6-carboxylic acid, (−)-luteoskyrin, (−)-4a-oxyluteoskyrin, chrysophanol, islandicin, catenarin,
and (22E)-5α,8α-epidioxyergosta-6,22-dien-3β-ol were isolated from the insect fungus
Conoideocrella krungchingensis BCC53666. (−)-Luteoskyrin exhibited a broad range of
antimicrobial activity such as antimalarial (IC50 0.51 µg/mL), antitubercular (MIC 6.25 µg/mL),
antibacterial (both Gram positive; MIC 0.39 – 1.56 µg/mL and Gram negative; MIC 3.13 – 12.50
µg/mL), and antifungal (against various plant pathogens; MIC 3.13 – 50.00 µg/mL) activities,
while (−)-4a-oxyluteoskyrin and catenarin showed weaker antibacterial activity. Moreover,
eutypinic acid, (−)-luteoskyrin, (−)-4a-oxyluteoskyrin, and catenarin showed cytotoxicity against
NCI-H187 cells with IC50 in a range of 0.16 – 17.99 µg/mL, while eutypinic acid and catenarin
had no cytotoxicity against non-cancerous (Vero) cells at maximum tested concentration (50
µg/mL). The complete NMR spectral data and biological activity of the known (−)-4a-
oxyluteoskyrin was also reported for the first time.
Keywords. Conoideocrella; Conoideoglucosides; Conoideochromanes; (−)-4a-Oxyluteoskyrin;
Biological activity
2

ACCEPTED MANUSCRIPT
1. Introduction
The genus Conoideocrella is a member in the Clavicipitaceae family and has received
considerable attention on the secondary metabolites due to the chemical diversity with a broad
array of biological activities.1-7 By far, only two species were identified, Conoideocrella
luteorostrata and C. tenuis, which were formerly described as Torrubiella luteorostrata and T.
tenuis, respectively.8 Many compounds from Conoideocrella spp. were described along with
their biological activities such as paecilodepsipeptide A and a naphthopyrone glycoside from T.
luteorostrata BCC9617,2 torrubiellutins A − C from T. luteorostrata BCC12904,3 isocoumarin
glucosides from T. tenuis BCC12732,4 conoideocrellides A − D, hopane-type triterpenes and
bioxanthracenes from C. tenuis BCC18627,5 oxanthracenes and bioxanthracene analogs from C.
luteorostrata Zimm. BCC31648,6 and conoideocrellones A and B, conoideocin A,
bioxanthracenes, and isocoumarin derivatives from C. tenuis BCC44534.7 Interestingly, these
compounds showed a broad range of biological activities such as antimalarial against
Plasmodium falciparum (K1, multidrug-resistant strain),5-7 antitubercular against Mycobacterium
tuberculosis H37Ra,5 antiviral against herpes simplex virus type-1 (HSV-1),5 and antibacterial
against Bacillus cereus7 activities as well as cytotoxicity against cancerous (KB, MCF-7, and
NCI-H187) and non-cancerous (Vero) cells.3,5-7
Based on the results from the chemical and biological screenings, the crude extracts from
broth and mycelia of the fungus Conoideocrella krungchingensis BCC53666 contained several
secondary metabolites detected by HPLC analyses and displayed antimalarial activity against P.
falciparum (K1, multidrug-resistant strain) (IC50 3.36 – >10 µg/mL), anti-plant pathogenic
fungal activity against Alternaria brassicicola (MIC 12.50 – 25.00 µg/mL) and antibacterial
activity against B. cereus (MIC 25.00 – >50 µg/mL) as well as cytotoxicity against cancerous
3

ACCEPTED MANUSCRIPT
(MCF-7 and NCI-H187) and non-cancerous (Vero) cells (IC50 3.34 – >50 µg/mL). The chemical
investigation of constituents of extracts from cultures of the fungus BCC53666 were thus
conducted and led to the isolation of six new compounds, including conoideoglucosides A − C (1
− 3) and conoideochromanes A − C (5 − 7), along with eight known compounds such as
eutypinic acid (4), 2,2-dimethyl-2H-1-chromene-6-carboxylic acid (8), (−)-luteoskyrin (9), (−)-
4a-oxyluteoskyrin (10), chrysophanol (11), islandicin (12), catenarin (13), and (22E)-5α,8α-
epidioxyergosta-6,22-dien-3β-ol (14). Moreover, the isolated compounds were tested for
antimicrobial activity, e.g., antimalarial against P. falciparum (K1, multidrug-resistant strain),
antitubercular against M. tuberculosis H37Ra, antifungal against Candida albicans, antibacterial
against Gram-positive (B. cereus, Enterococcus faecium, Staphylococcus aureus) and Gram-
negative (Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae, Pseudomonas
aeruginosa) bacteria, and anti-plant pathogenic fungal against A. brassicicola, Colletotrichum
acutatum, Curvularia lunata, Magnaporthe grisea activities, and for cytotoxicity against
cancerous (MCF-7 and NCI-H187) and non-cancerous (Vero) cells.
2. Results and discussion
The fungus Conoideocrella krungchingensis BCC53666 was collected from an armored
scale insect (Hemiptera: Diaspididae) at Khao Luang National Park, Nakhon Si Thammarat
province, Thailand. It was cultivated in potato dextrose broth (PDB) under shaking condition at
200 rpm, 25 °C for 5 days. After separation of mycelia from broth by simple filtration, the broth
was extracted with EtOAc, while the mycelia were macerated with organic solvents (MeOH and
CH2Cl2), followed by evaporation and extraction with n-hexane and EtOAc, respectively. All
4

ACCEPTED MANUSCRIPT
crude extracts were then chromatographed to provide fourteen compounds, including six new
and eight known compounds.
Compound 1 was obtained as a yellow oil and gave the molecular formula C19H22O8,
deduced from the sodium-adduct mass ion peak at m/z 401.1208 [M+Na]+ in the HRESIMS
spectrum. Compound 1 displayed a broad hydroxyl absorption at
strong absorption at νmax 1703 cm-1, suggesting a conjugated carboxylic acid carbonyl in the IR
c 166.3 in the 13C NMR spectrum.
ν
3000 – 3600 cm-1 and a
spectrum, which also corresponded to a carbon resonating at
δ
1
13
The H and C NMR spectra (Table 1) of compound 1 were similar to those of the known co-
metabolite, eutypinic acid (4),9 except the presence of seven additional signals of a sugar unit,
including one methoxy (at
δH 3.46), one oxymethylene (at δH 3.50 / 3.60), and five oxymethines
(at H 3.08, 3.33, 3.40 − 3.47, and 5.15). The connectivity, provided by COSY and HMBC
δ
spectral information (Fig. 2), showed correlations from H-1′ to H-2′; from H-2′ to H-3′; from H-
3′ to H-4′; from H-4′ to H-5′; and from H-5′ to H2-6′ in the COSY spectrum and in the HMBC
spectrum from H-2′ to C-1′ and C-3′; from H-3′ to C-2′; from H-4′ to C-3′, C-5′, and 4′-OCH3;
and from 4′-OCH3 to C-4′. Moreover, the HMBC spectrum showed a correlation from an
anomeric proton resonating at
the replacement of a phenolic hydroxyl group at C-4 in compound 4 with the sugar unit.
Furthermore, after an addition of D2O, the large vicinal coupling constants of J1′2′ = 7.7 Hz, J2′3′
δH 5.15 (H-1′) to the aromatic carbon at δC 160.2 (C-4), suggesting
=
9.0 Hz, and J3′4′ and J4′5′ = 9.3 Hz were observed and in the NOESY spectrum the cross-peak
correlations from H-1′ to H-3′ and H-5′ and from H-2′ to H-4′ were seen, confirming the axial
position of all extra oxymethine protons (H-1′ – H-5′). Thus, the sugar unit was 4-O-methyl-β-
glucopyranose and compound 1 could be depicted as shown in Fig. 1. Its trivial name is
conoideoglucoside A.
5

ACCEPTED MANUSCRIPT
Fig. 1. Chemical structures of compounds 1 – 14.
Fig. 2. COSY, key HMBC and selected NOESY correlations of compounds 1.
6

ACCEPTED MANUSCRIPT
Table 1
1H (500 MHz) and 13C (125 MHz) NMR assignments for compounds 1 − 3 in DMSO-d6.
Compound 1
δC, type
Compound 2
δC, type
Compound 3
δC, type
Position
δH, mult (J in Hz)
δH, mult (J in Hz)
δH, mult (J in Hz)
1
125.0a, qC
133.9, CH
112.0, qC
160.2, qC
114.0, CH
131.9, CH
166.3, qC
84.1, qC
135.7, qC
130.7, CH
111.8, qC
156.0, qC
114.6, CH
127.9, CH
61.9, CH2
85.2, qC
122.6, qC
−
−
−
2
3
7.88 d (2.1)
7.30 d (1.6)
−
122.8, CH 7.34 s
135.9, qC
146.7, qC
151.3, qC
−
−
−
−
4
−
−
5
7.22 d (8.8)
7.09 d (8.7)
7.23 dd (8.7, 1.6)
4.40 d (5.2)
6
7
7.86 dd (8.8, 2.1)
111.6, CH 7.38 s
167.0, qC
28.3, CH2
−
−
8
3.35 − 3.45 m
−
−
9
95.0, qC
94.1, qC
122.6, CH 5.28 t (7.0)
−
−
10
11
12
1′
2′
3′
4′
5′
6′
126.4, qC
122.6, CH2
23.0, CH3
98.9, CH
73.4, CH
75.6, CH
78.7, CH
76.5, CH
60.0, CH2
126.5, qC
121.7, CH2
22.9, CH3
99.4, CH
73.5, CH
75.5, CH
78.9, CH
76.5, CH
60.2, CH2
131.7, qC
17.7, CH3
25.5, CH3
−
−
−
5.40 s / 5.42 s
1.96 s
5.15 d (7.7)
3.33 dd (9.0, 7.7)b
5.36 s / 5.38 s
1.95 s
5.02 d (7.7)
3.33 dd (8.6, 7.7)b
1.68 s
1.70 s
102.5, CH 4.91 d (7.7)
74.5, CH
76.2, CH
79.1, CH
75.8, CH
60.4, CH2
3.26 dd (9.0, 7.7)b
3.40 − 3.47 m
3.08 dd (9.3, 9.3)
3.38 − 3.41 m
3.05 dd (9.3, 9.3)
3.35 − 3.40 m
3.00 dd (9.2, 9.2)
3.08 ddd (11.0, 4.2, 1.5)
3.45 dd (11.0, 4.2)b
3.52 dd (11.0, 1.5)b
3.81 s
3.40 − 3.47 m
3.50 dd (11.7, 5.1)b
3.60 d (11.7)b
−
3.42 − 3.45 m
3.49 dd (11.2, 5.4)b
3.59 d (11.2)b
−
5.16 t (5.2)
5.28 br t (4.0)
3.45 s
5-OCH3
7-OH
56.1, CH3
59.5, CH3
−
−
5.31 br s
3.46 s
5.20 br s
3.44 s
2′-OH
59.6, CH3
59.3, CH3
4′-OCH3
6′-OH
a Observed in the HMBC spectrum.
4.70 t (5.1)
4.68 t (5.4)
4.47 t (4.2)
b Coupling constants (J) appeared in the presence of D2O.
Compound 2 was obtained as a yellow oil. Its molecular formula was determined to be
C19H24O7, showing the sodium-adduct mass ion peak at m/z 387.1413 [M+Na]+ in the HRESIMS
1
spectrum. The H and 13C NMR spectral data (Table 1) of compound 2 showed similarity to
those of compound 1, except for the upfield shift of two aromatic protons at H-2 (∆
δ −0.58) and
H-6 (∆
δ
−0.63) in the 1H NMR spectrum and the presence of an additional oxymethylene signal
1
in both H and 13C NMR spectra resonating at δH 4.40 (H2-7) and δC 61.9 (C-7), respectively.
Moreover, in the 13C NMR spectrum, the carbon belonging to carboxylic acid observed in
7

ACCEPTED MANUSCRIPT
compound 1 was absent in compound 2. The HMBC spectrum showed correlations from H2-7 to
C-1, C-2, and C-6, indicating the replacement of the carboxylic group with a hydroxymethyl
group. In addition, after an addition of D2O, the large vicinal coupling constants of J1′2′ (7.7 Hz),
J2′3′ (8.6 Hz), and J3′4′ and J4′5′ (9.3 Hz) together with the NOESY correlations from H-1′ to H-3′
and H-5′ and from H-2′ to H-4′ suggested the axial position of all oxymethine protons (H-1′ to
H-5′) in the sugar unit. Therefore, compound 2 was a reduced form of compound 1 (as shown in
Fig. 1) and is named conoideoglucoside B.
Compound 3 was obtained as a yellow oil. The IR spectrum indicated the presence of a
carboxylic group (ν ν
3000 – 3600 cm-1 and max 1703 cm-1). The 1H and 13C NMR spectra (Table
1) of compound 3 were in good agreement with those of trollioside,10 except an extra methoxy
1
signal at δH 3.44 (4′-OCH3) in the H NMR spectrum, which attributed to the carbon at δC 59.5
by the HSQC correlation. In the HMBC spectrum, the additional methoxy protons at δH 3.44 (4′-
OCH3) correlated to an oxymethine carbon at δC 79.1 (C-4′), suggesting that the methoxy group
was attached to C-4′ of the sugar unit. Thus, compound 3 also had 4-O-methyl-β-glucopyranose
moiety. The HRESIMS spectrum confirmed the molecular formula C20H28O9 by showing the
sodium-adduct mass ion peak at m/z 435.1621 [M+Na]+. Thus, compound 3 had the chemical
structure as shown in Fig. 1 and is given the trivial name conoideoglucoside C.
Moreover, compound 3 was subjected to acid hydrolysis to give the aglycone unit and
glucosidic part (detail given in Experimental section 4.4). The sugar residue of compound 3
ꢀ ꢁ20
showed the positive optical rotation value ( α +61.4), which was consistent with the reported
ꢂ
data of 4-O-methyl-D-glucopyranose ( α +80).11 The absolute configuration of its sugar unit
ꢀ ꢁ25
ꢂ
could thus be assigned as the D configuration. In addition, the similar spectral data of the sugar
ꢀ ꢁ24
unit (Table 1) and the negative optical rotations of compounds 1 ( α
ꢀ ꢁ25
−24.0) and 2 ( α
ꢂ
ꢂ
8

ACCEPTED MANUSCRIPT
ꢀ ꢁ24
−16.9) to those of the co-metabolite 3 ( α −8.9) suggested the same configuration of the sugar
ꢂ
moiety as that of compound 3.
Compound 5 was obtained as a yellow oil. HRESIMS data showing the sodium-adduct
mass ion peak at m/z 247.0940 [M+Na]+ revealed the molecular formula of C12H16O4, indicating
1
five degrees of unsaturation. The H and 13C NMR spectra (Table 2) of compound 5, which
incorporated with HSQC correlations, disclosed signals of two methyls, one methylene, one
oxymethylene, one oxymethine, two sp2 methines, and three hydroxyl protons. Moreover, the 13C
NMR spectrum, differentiated by the DEPT-135 spectrum, gave additional signals of one sp3
quaternary and four quaternary carbons. The COSY correlations from H-3 to H2-4 and 3-OH
together with the HMBC correlations from H2-4 to C-2, C-3, C-4a, C-5, and C-8a; from H-5 to
C-4, C-7, and C-8a; from H-7 to C-5, C-8, and C-8a; from H3-9 to C-2, C-3, and C-10; and from
H3-10 to C-2, C-3, and C-9 gave 3-hydroxy-2,2-dimethylchromane ring similar to that of
acremine R (Fig. 3).12 In addition, the cross-peaks correlations between H2-11 and 11-OH in the
COSY spectrum and the HMBC correlations from H-5 and H-7 to C-11; from H2-11 to C-5, C-6,
and C-7; and from 8-OH to C-7 and C-8a suggested that the hydroxymethyl and hydroxyl groups
should be placed at C-6 and C-8, respectively. Moreover, compound 5 showed the positive
ꢀ ꢁ24
optical rotation value ( α +52.1) similar to those of 3-hydroxy-2,2-dimethylchromane analogs
ꢂ
bearing S configuration,13 which was opposite to that of acremine R having R configuration
−8.0).12 In order to confirm the absolute configuration at C-3, compound 5 was treated
ꢀ ꢁꢃꢄ
( α
ꢂ
with the Mosher’s reagents, (R)- and (S)-MTPACl (α-methoxy-α-trifluoromethylphenylacetyl
chloride). The reaction resulted in diastereomeric mixtures of corresponding MTPA esters,
indicating an enantiomeric mixture of compound 5. Thus, it could then be summarized that
compound 5 contained a non-equivalent mixture of enantiomers by having a larger amount of
9

ACCEPTED MANUSCRIPT
(S)-stereoisomer. Unfortunately, the attempt to separate the enantiomers by using the chiral
HPLC analysis was unsuccessful. At present, the chemical structure of compound 5 was
therefore depicted as shown in Fig. 1 and conoideochromane A is named for compound 5.
Table 2
1H and 13C NMR assignments for compounds 5 − 7 in DMSO-d6.
Compound 5a,c
Compound 6b
δC, type
Compound 7a,d
δC, type
Position
δC, type
δH, mult (J in Hz)
δH, mult (J in Hz)
δH, mult (J in Hz)
2
3
4
76.7, qC
68.4, CH
31.2, CH2
76.8, qC
68.2, CH
31.1, CH2
77.4, qC
67.8, CH
31.0, CH2
−
−
−
3.54 − 3.62 m
2.53 dd (16.7, 8.2)
3.58 − 3.63 m
2.54 dd (16.6, 8.1)
3.60 − 3.65 m
2.61 dd (16.8, 7.7)
2.82 dd (16.7, 5.4)
2.83 dd (16.6, 5.4)
2.92 dd (16.8, 5.1)
4a
5
6
120.4, qC
117.8, CH
133.5, qC
111.9, CH
145.4, qC
139.7, qC
20.0, CH3
25.7, CH3
62.9, CH2
−
120.6, qC
119.2, CH
129.2, qC
112.8, CH
145.5, qC
140.4, qC
20.1, CH3
25.7, CH3
73.7, CH2
−
122.3, qC
124.2, CH
134.2, qC
117.0, CH
142.0, qC
142.7, qC
20.3, CH3
25.4, CH3
62.2, CH2
150.0, qC
100.5, CH2
−
−
−
6.42 d (1.6)
6.43 d (1.6)
6.87 d (1.5)
−
−
−
7
8
6.55 d (1.6)
6.53 d (1.6)
6.76 d (1.5)
−
−
−
8a
9
10
11
12
13
−
−
−
1.12 s
1.28 s
4.27 s
−
1.13 s
1.28 s
4.18 s
−
1.10 s
1.22 s
4.35 s
−
4.57 d (2.3)
−
−
−
−
5.45 d (2.3)
14
162.4, qC
−
−
−
−
−
−
−
−
−
−
3-OH
5.09 s
8.54 s
4.90 s
5.09 s
8.60 s
−
−
−
8-OH
−
−
−
11-OH
11-OCH3
14-OCH3
5.08 br s
−
−
−
57.1, CH3
3.21 s
−
−
−
−
52.3, CH3
3.77 s
−
−
a 400 MHz for 1H and 100 MHz for 13C.
b 500 MHz for 1H and 125 MHz for 13C.
c Approx. 92% purity examined by HPLC analysis.
d Approx. 89% purity examined by HPLC analysis.
10

ACCEPTED MANUSCRIPT
Fig. 3. COSY and key HMBC correlations of compounds 5.
1
13
Compound 6 was obtained as a yellow oil. The H and C NMR spectra (Table 2) were
almost identical to those of compound 5, apart from the presence of an additional signal of a
1
methoxy group (11-OCH3) resonating at δH 3.21 and δC 57.1 in the H and 13C NMR spectra,
respectively. The extra methoxy protons (11-OCH3) showed HMBC correlation to an
oxymethylene carbon at δC 73.7 (C-11), bearing the methylene protons at δH 4.18 (H2-11) in the
HSQC spectrum, which was in turn correlated in HMBC spectrum to C-5, C-6, C-7, and 11-
OCH3, corroborating the position of the methoxy group at C-11. The remaining structure was
reassured by an extensive analysis of 2D NMR spectroscopic information (HSQC, HMBC, and
COSY). HRESIMS data confirmed the structure by revealing the sodium-adduct mass ion peak
at m/z 261.1099 [M+Na]+, corresponding to the molecular formula of C13H18O4. The reaction
with Mosher’s reagents also resulted in diastereomeric mixtures of corresponding MTPA esters,
suggesting an enantiomeric form of compound 6. The positive optical rotation of compound 6
ꢀ ꢁ25
( α +5.1) suggested a non-racemic mixture of compound 6, which was slightly rich quantity in
ꢂ
(S)-stereoisomer. The chemical structure of compound 6 was thus illustrated as shown in Fig. 1
and the name conoideochromane B is given.
1
Compound 7 was obtained as a yellow oil. The H and 13C NMR spectra (Table 2) of
1
compound 7 contained those signals present in compound 5 and three extra signals in the H
NMR spectrum including one methoxy (at δH 3.77, s) and two olefinic protons [at δH 4.57 (d,
11

ACCEPTED MANUSCRIPT
2.3) and 5.45 (d, 2.3)], which attributed in the HSQC spectrum to the carbons resonating at δC
52.3 and 100.5, respectively. In the HMBC spectrum, the extra methoxy at δH 3.77 (14-OCH3)
correlated to C-14, while two additional olefinic protons at δH 4.57 and 5.45 (H2-13) correlated
to C-12 (δC 150.0) and C-14 (δC 162.4). The spectroscopic information suggested the presence of
an additional methyl acrylate unit in compound 7. The carbon resonating at δC 150.0 (C-12)
indicating an attachment to an oxygen together with the evidence from NOESY spectrum
showing correlations from H3-9 (δH 1.10) to Hb-13 (δH 4.57) suggested that the methyl acrylate
moiety was linked to an oxygen at C-3. The remaining chemical structure was affirmed by
extensive 2D NMR spectroscopic analyses (HSQC, HMBC, and COSY). The molecular formula
of C16H20O6 was approved by HRESIMS spectral data, establishing the sodium-adduct mass ion
peak at m/z 331.1151 [M+Na]+. In addition, compound 7 should be derived from the same
biosynthesis as the co-metabolite 5, thus it was likely to obtain as an enantiomeric mixture,
although it was fail to separate by chiral HPLC analysis. Compound 7 displayed the positive
ꢀ ꢁꢃꢅ
ꢀ ꢁꢃꢄ
ꢂ
optical rotation value ( α +35.4), which was the same sign as that of compound 5 ( α
ꢂ
+52.1), suggesting the enantiomeric mixture with a larger amount of (S)-stereoisomer. The
chemical structure of compound 7 was therefore depicted as shown in Fig. 1 and
conoideochromane C is given as its informal name.
Compound 10 was obtained as a yellowish brown solid with the molecular formula of
C30H22O13, deduced from the negative mass ion peak at m/z 589.0987 [M−H]− in the HRESIMS
1
spectrum. The molecular formula suggested twenty degrees of unsaturation. The H NMR
spectrum (Table 3) showed signals of two methyls, four sp3 methines, two sp3 oxymethines, and
13
two sp2 methines. In the C NMR spectrum, there were additional signals of fifteen quaternary
and five carbonyl carbons, which was differentiated by the DEPT-135 spectrum. The 2D NMR
12

ACCEPTED MANUSCRIPT
spectral information, including COSY, HSQC, HMBC spectra led to a bisanthraquinone
heterodimer, whose two subunit were linking at C-1−C-1′, C-3−C-9′a, and C-9a−C-3′.
Compound 10 (as illustrated in Fig. 1), known as (−)-4a-oxyluteoskyrin, was previously isolated
only from pigments of the fungus Penicillium islandicum Sopp NRRL1036 as a minor
constituent, reported only 1H NMR data by Takeda and co-workers in 1973.14 Compound 10 was
also given from the reaction of (−)-luteoskyrin (9) with pertrifluoroacetic acid, reported by
Takeda et al.14 Moreover, the CD spectrum of compound 10 showed a similar pattern to that
previously reported for compound 9 (Fig. 4),15 demonstrating the same absolute stereochemistry
of a central cage structure. However, it cannot be concluded for the absolute configuration at C-
4a, which probably causing a strong positive peak at λ 356 nm in the CD spectrum. Herein we
also describe the complete 1H and 13C NMR spectral data in the Table 3.
Fig. 4. CD spectra of compounds 9 and 10.
13

ACCEPTED MANUSCRIPT
Table 3
1H (400 MHz) and 13C (100 MHz) NMR assignments for compound 10 in acetone-d6.
Compound 10a
δC, type
Position
δH, mult (J in Hz)
1
45.0, CH
3.99 dd (3.7, 1.7)
2
71.1, CH
5.02 dd (4.9, 3.7)
3
66.8, CH
3.22 br d (4.9)
4
198.4, qC
−
4a
5
75.5, qC
−
157.1 / 157.2, qC
128.6(5) / 128.7(3), CH
140.7 / 140.8, qC
155.0 / 155.3, qC
113.4, qC
−
6
7
7.25 s
−
8
−
8a
9
−
201.3 / 201.4, qC
65.4, qC
−
9a
10
10a
11
1′
−
193.7 / 193.8, qC
112.3, qC
−
−
16.4, CH3
50.9, CH
2.34 s
3.62 dd (3.5, 1.8)
4.62 dd (4.1, 3.5)
3.16 br d (4.1)
71.5, CH
2′
58.0, CH
3′
177.5, qC
4′
−
108.3, qC
4′a
5′
−
156.6(7) / 156.7(4), qC
129.9, CH
−
7.30 s
6′
140.4 / 140.5, qC
156.9 / 157.0, qC
112.0, qC
7′
−
8′
−
8′a
9′
−
201.0(7) / 201.1(3), qC
55.2, qC
−
9′a
10′
10′a
11′
5-OH
8-OH
−
187.3, qC
−
113.0, qC
−
16.4, CH3
2.34 s
11.27 s
11.56 s
4.77 br s
14.15 s
11.89 br s
12.42 s
−
−
−
−
−
−
2′-OH
4′-OH
5′-OH
8′-OH
a The 1H NMR spectral information of compound 10 was previously reported in DMSO-d6.14 In our structural determination, the
1D and 2D NMR spectral data of this compound 10 were recorded in both DMSO-d6 and acetone-d6. It was found that acetone-d6
14

ACCEPTED MANUSCRIPT
provided better resolution NMR spectra. Therefore, the NMR characterization of compound 10 was performed in acetone-d6,
which should be the preferred solvent for future studies.
The chemical structures of other known compounds (4, 8, 9, 11 – 14) were established by
comparison of the 1H and 13C NMR spectroscopic information with those formerly reported data
for eutypinic acid (4),9 2,2-dimethyl-2H-1-chromene-6-carboxylic acid (8),16 (−)-luteoskyrin
(9),14,17 chrysophanol (11),18 islandicin (12),19 catenarin (13),20 and (22E)-5
-ol (14)21,22 along with their physical properties.
α,8α-
epidioxyergosta-6,22-dien-3
β
The isolated compounds were assessed for antimicrobial activity, including antimalarial
against P. falciparum (K1, multidrug-resistant strain), antitubercular against M. tuberculosis
H37Ra, antifungal against C. albicans, A. brassicicola, C. acutatum, C. lunata, and M. grisea,
antibacterial activities against Gram-positive (B. cereus, E. faecium, S. aureus) and Gram-
negative (A. baumannii, E. coli, K. pneumoniae, P. aeruginosa) bacteria, and for cytotoxicity
against cancerous (MCF-7 and NCI-H187) and non-cancerous (Vero) cells. Since compounds 1,
3, and 8 were obtained in poor amounts, their biological activity was then not evaluated.
Compound 14 was not subjected for biological evaluation due to its biological activity has
previously been done by our group.23
Only compounds 9 and 12 exhibited antimalarial activity against P. falciparum with IC50
values of 0.51 and 8.81 µg/mL, respectively (Table 4). (−)-Luteoskyrin (9) possessed a broad
range of antimicrobial activity (MIC 0.39 – 50.00 µg/mL), while (−)-4a-oxyluteoskyrin (10) had
anti-M. tuberculosis H37Ra (MIC 50.00 µg/mL) and antibacterial (against B. cereus, E. faecium
and S. aureus) activities (MIC12.50 – 25.00 µg/mL) (Table 4 and 5). Both compounds were
inactive to E. coli and K. pneumoniae at the maximum tested concentration of 50 µg/mL, but in
combination with agent that increases permeability of outer bacterial membrane such as
15

ACCEPTED MANUSCRIPT
phenylalanine-arginine β-naphthylamide (PAβN), these compounds became active against E. coli
with respective MIC values of 12.50 and 25.00 µg/mL. The results (Table 5) suggested that anti-
Gram negative bacterial activity of compound 9 against K. pneumoniae and A. baumannii (MIC
of 3.13 and 6.25 µg/mL) was improved in the presence of PA
displayed anti-Gram positive bacterial activity against B. cereus, E. faecium, and S. aureus with
MIC values in a range of 6.25 − 25.00 µg/mL. In combination with PA N, compound 13 was
βN. Furthermore, catenarin (13)
β
also active to Gram-negative bacterial such as E. coli and K. pneumoniae with MIC values of
12.50 and 50.00 µg/mL, respectively. Moreover, compound 13 exhibited anti-plant pathogen
activity against M. grisea at the maximum tested concentration (MIC of 50 µg/mL) (Table 4).
For cytotoxicity (Table 6), compounds 4, 9, 10, and 13 were toxic to NCI-H187 cells with IC50
values ranging from 0.16 to 17.99 µg/mL. Only compounds 10 and 13 had weak cytotoxicity
against MCF-7 cells with respective IC50 values of 12.49 and 34.49 µg/mL. Compounds 9, 10,
and 12 showed cytotoxicity against non-cancerous (Vero) cells with IC50 values of 0.48, 17.38,
and 17.03 µg/mL, respectively. All tested compounds were inactive for anti-C. albicans and anti-
P. aeruginosa at the maximum tested concentration (50 µg/mL).
16

ACCEPTED MANUSCRIPT
Table 4
Antimalarial, antitubercular, and anti-plant pathogen activities of compounds 2, 4 − 7, and 9 −
13.
Compounds
Anti-P. falciparuma
(IC50, µg/mL)
Anti-M. tuberculosis
Anti-plant pathogen activityb (MIC, µg/mL)
H37Rab
(MIC, µg/mL)
>50
A. brassicicola
C. acutatum
M. grisea
>10
>10
>10
>10
>10
0.51
>10
>50
>50
>50
>50
>50
3.13
>50
>50
>50
>50
−
>50
>50
>50
>50
>50
50.00
>50
>50
>50
>50
−
>50
>50
>50
>50
>50
12.50
>50
>50
>50
50.00
−
2
4
5
6
7
9
10
11
>50
>50
>50
>50
6.25
50.00
>50
>50
>50
>10
8.81
>10
12
13
Dihydroartemisinin
Chloroquine
Rifampicin
Ofloxacin
Streptomycin
Isoniazid
Ethambutol
Amphotericin B
4.35 × 10-4
0.10
−
−
−
−
−
0.01 − 0.05
0.39 − 0.78
0.63
0.05
0.94
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
1.56
0.78 − 3.13
0.78 − 1.56
a Maximum tested concentration was done at 10 µg/mL.
b Maximum tested concentration was done at 50 µg/mL.
Table 5
Antibacterial activity of compounds 2, 4 − 7, and 9 − 13.
Compounds
Antibacterial activity (MIC, µg/mL)a
B. cereus
E. faecium
S. aureus
A. baumannii
A. baumannii
E. coli
K. pneumoniae
+PA
>50
>50
>50
>50
>50
6.25
>50
>50
>50
>50
0.10
β
Nb
+PA
>50
>50
>50
>50
>50
β
Nb
+PA
>50
>50
>50
>50
>50
3.13
>50
>50
>50
β
Nb
>50
>50
>50
>50
>50
1.56
25.00
>50
>50
25.00
0.31 − 0.63
4.00
−
−
>50
>50
>50
>50
>50
>50
>50
>50
>50
1.56
12.50
>50
>50
6.25
>50
>50
>50
>50
>50
12.50
>50
>50
>50
>50
2
4
5
6
7
9
10
11
>50
0.39
25.00
>50
>50
12.50
3.13
12.50
25.00
>50
>50
12.50
0.02 − 0.05
−
−
12
13
50.00
0.39
−
Rifampicin
Vancomycin
Tetracycline HCl
Erythromycin
0.08 − 0.16 1.56 − 3.13
1.00
−
−
−
−
−
−
−
0.10 − 0.20
−
−
25.00
12.50
1.56 − 3.13
0.78 − 1.56
a Maximum tested concentration was done at 50 µg/mL.
b PA
βN = Phenylalanine-arginine β-naphthylamide.
17

ACCEPTED MANUSCRIPT
Table 6
Cytotoxicity of all tested compounds.
Compounds Cytotoxicitya (IC50, µg/mL)
MCF-7
>50
>50
>50
>50
>50
>50
12.49
>50
>50
NCI-H187 Vero
>50
17.99
>50
>50
>50
0.16
2.27
>50
>50
8.21
>50
>50
>50
>50
>50
0.48
17.38
>50
17.03
>50
−
2
4
5
6
7
9
10
11
12
13
34.49
Doxorubicin
Tamoxifen
Ellipticine
7.01 − 9.24 0.08 − 0.12
6.52 − 7.36
−
−
2.23 − 3.43 0.98 − 1.94
−
a Maximum tested concentration was done at 50 µg/mL.
3. Conclusion
Six new compounds, which included conoideoglucosides A − C (1 − 3) and
conoideochromanes A − C (5 − 7), were isolated from the insect fungus Conoideocrella
krungchingensis BCC53666 along with eight known compounds [eutypinic acid (4), 2,2-
dimethyl-2H-1-chromene-6-carboxylic acid (8), (−)-luteoskyrin (9), (−)-4a-oxyluteoskyrin (10),
chrysophanol (11), islandicin (12), catenarin (13), and (22E)-5
α,8α-epidioxyergosta-6,22-dien-
3
β-ol (14)]. Compound 9 had a wide range of antimicrobial activity against P. falciparum, M.
tuberculosis, B. cereus, E. faecium, S. aureus, A. baumannii, E. coli, K. pneumoniae, A.
brassicicola, C. acutatum, and M. grisea (Table 4 and 5). Compounds 10 and 13 showed
antibacterial activity against Gram-positive bacteria with MIC values of 6.25 – 25.00 µg/mL and
had anti-Gram negative bacterial activity such as E. coli in the presence of PAβN agent with
MIC values of 25.00 and 12.50 µg/mL, respectively. Compound 12 was active against P.
falciparum with an IC50 value of 8.81 µg/mL. In addition, compound 13 exhibited anti-K.
pneumoniae activity in the presence of PAβN and anti-M. grisea activity at the maximum tested
concentration (50 µg/mL). Compounds 4, 9, 10, and 13 displayed cytotoxicity against NCI-H187
18

ACCEPTED MANUSCRIPT
cells with IC50 values ranging from 0.16 to 17.99 µg/mL, while only compounds 10 and 13
exhibited cytotoxicity against MCF-7 cells with IC50 values of 12.49 and 34.49 µg/mL,
respectively. Compounds 9, 10, and 12 had cytotoxicity against Vero cells with respective IC50
values of 0.48, 17.38, 17.03 µg/mL.
4. Experimental
4.1. General experimental procedures
A melting point was detected using a melting point MP90 apparatus from Mettler Toledo.
Optical rotations were performed on a JASCO P-1030 digital polarimeter. UV spectra were taken
in MeOH on a Spekol 1200 from Analytik Jena UV–Vis spectrophotometer. FTIR spectra were
detected on a Bruker ALPHA spectrometer. CD spectra were done in MeOH on a JASCO J-810
spectropolarimeter. NMR spectra were carried out on either Bruker Avance-III 400 (400 MHz
1
13
1
13
for H and 100 MHz for C) or Bruker Avance 500 (500 MHz for H and 125 MHz for C)
NMR spectrometers. HRESIMS data were recorded on a Bruker MicrOTOF mass spectrometer.
Semi-preparative HPLC were performed by a reverse phase column (SunFire C18 OBD, particle
size 5 µm, diam. 19 mm × 150 mm) at the flow rate of 8 mL/min, while preparative HPLC were
taken by a reverse phase column (SunFire C18 OBD, particle size 10 µm, diam. 19 mm × 250
mm) at the flow rate of 15 mL/min. Chiral HPLC analysis was performed using a chiral CD-Ph
column (Shiseido chiral CD-Ph, particle size 5 µm, diam. 4.6 mm × 250 mm) at the flow rate of
1 mL/min. All HPLC were carried out on a Dionex–Ultimate 3000 series equipped with a binary
pump, an autosampler, and a diode array detector. Purity of isolated compounds was detected
using analytical reversed phase HPLC (Merck LiChroCART C18, particle size 3 µm, diam. 2
19

ACCEPTED MANUSCRIPT
mm × 55 mm), eluted with a linear gradient system (0 – 100% MeCN in water containing 0.05%
formic acid) at the flow rate 0.5 mL/min over 15 min.
4.2. Fungal material
The fungus Conoideocrella krungchingensis was isolated from an insect in the Hemiptera
order, collected at Khao Luang National Park, Nakhon Si Thammarat province, Thailand and
deposited at BIOTEC Culture Collection (BCC), the National Center for Genetic Engineering
and Biotechnology (BIOTEC), Thailand with a registration number BCC53666. The fungus was
previously reported as new species from Thailand.24 The fungus was identified by based on
morphology and molecular phylogenetic relationships of the partial nuclear large subunit rRNA
(LSU, accession number KJ435,070) and the elongation factor (EF-1α, accession number
KJ435,099) genes.
4.3. Fermentation, extraction, and isolation
The fungus Conoideocrella krungchingensis BCC53666 was maintained on potato
dextrose agar (PDA) plates at 25 °C, which was then cut into small pieces (1 cm × 1 cm each)
and transferred into 8 × 250 mL Erlenmeyer flasks, which each contained 25 mL of potato
dextrose broth (PDB, containing potato starch 4.0 g/L, dextrose 20.0 g/L in distilled water). After
incubation at 25 °C for 5 days on a rotary shaker at 200 rpm, each flask was equally transferred
into 4 × 1 L Erlenmeyer flasks, containing 250 mL of PDB medium. The production culture (20
L) was cultivated at 25 °C for 5 days under shake condition (200 rpm). The broth and mycelia
were separated by simple filtration. The culture broth was extracted three times with an equal
volume of EtOAc. The combined organic layer was dried over Na2SO4 and then concentrated
under reduced pressure to provide a brown gum (3.3 g). The mycelia were macerated in MeOH
20

ACCEPTED MANUSCRIPT
(1 L) for 3 days and consecutively in CH2Cl2 (1 L) for 3 days. The combined organic solvent was
then evaporated to dryness in vacuo. Water (500 mL) was added and the mixture was extracted
three times with an equal volume of n-hexane, followed by three times with an equal volume of
EtOAc. Later, n-hexane and EtOAc extracts were separately dried over Na2SO4 and concentrated
under reduced pressure to yield crude extracts as brown gum from n-hexane (6.5 g) and from
EtOAc (0.6 g), respectively.
The crude broth extract (3.3 g) was fractionated by a Sephadex LH-20 column (4.2 cm ×
36.5 cm), eluted with 100% MeOH to give 9 fractions. The first and seventh − ninth fractions
were not further purified due to no compound of interest. The second fraction (0.9 g) was then
subjected to a preparative HPLC using a linear gradient elution of 10 − 100% aqueous MeCN
over 40 min to provide twenty subfractions (2.1 − 2.20), from which compounds 5 (7.8 mg), 6
(12.9 mg), and 7 (7.6 mg) were obtained in subfractions 2.2, 2.6, and 2.8, respectively.
Subfraction 2.5 (14.7 mg) was further purified by a semi-preparative HPLC using a linear
gradient elution of MeCN:H2O + 0.05% formic acid (10:90 to 30:70) over 50 min to give
compound 2 (4.2 mg). Compound 3 (3.2 mg) was obtained from subfraction 2.7 (10.4 mg) after
separation by a preparative HPLC using a linear gradient elution of 15 − 50% aqueous MeCN
over 40 min. The third fraction (0.5 g) was subjected to a preparative HPLC using a linear
gradient elution of 10 − 100% aqueous MeCN over 40 min to yield twenty-two subfractions (3.1
− 3.22), from which compound 5 (4.4 mg) was obtained in subfraction 3.6. Separation of
subfraction 3.9 (11.4 mg) by a preparative HPLC using a linear gradient elution of MeCN:H2O +
0.05% formic acid (15:85 to 35:65) over 50 min afforded compounds 6 (3.7 mg) and 1 (1.3 mg),
respectively, while purification of subfraction 3.17 (14.3 mg) by a preparative HPLC using a
linear gradient elution of MeCN:H2O + 0.05% formic acid (40:60 to 50:50) over 50 min
21

ACCEPTED MANUSCRIPT
provided compound 8 (3.9 mg). After separation by a preparative HPLC using a linear gradient
elution of MeCN:H2O + 0.05% formic acid (5:95 to 15:85) over 40 min, the fourth fraction (0.3
g) gave respective compounds 4 (17.5 mg) and 11 (6.4 mg), while the fifth fraction (0.2 g)
afforded compounds 11 (6.7 mg) and 12 (7.8 mg), respectively. The sixth fraction (0.2 g) was
applied to a preparative HPLC using a linear gradient elution of 40 − 70% aqueous MeCN over
40 min to provide compounds 10 (14.0 mg), 9 (27.7 mg), and 13 (3.0 mg), respectively.
The n-hexane crude extract from mycelia (6.5 g) was passed through a Sephadex LH-20
column (4.2 cm × 36.5 cm), eluted with 100% MeOH to yield 10 fractions. The first, second, and
eighth − ninth fractions were discarded since there was no compound of interest. The third
fraction (1.6 g) was further fractionated by a Sephadex LH-20 column (4.2 cm × 36.5 cm), eluted
with 100% MeOH to yield 4 subfractions (3.1 − 3.4). Purification of subfraction 3.2 (1.2 g) by a
preparative HPLC using a linear gradient elution of 70 − 100% aqueous MeCN over 40 min
provide compound 14 (62.5 mg). The fourth fraction (0.5 g) was further subjected to a
preparative HPLC using a linear gradient elution of 30 − 70% aqueous MeCN over 40 min to
furnish compounds 9 (2.1 mg), 11 (3.7 mg), and 12 (50.6 mg), respectively. Moreover, using the
same separation method for the fourth fraction, the fifth fraction (0.2 g) gave compounds 10 (2.2
mg), 11 (2.3 mg), and 12 (2.3 mg), respectively, while the sixth fraction (0.1 g) provided
respective compounds 10 (3.4 mg), 9 (12.8 mg), 11 (1.4 mg), and 12 (3.7 mg). The seventh
fraction (0.3 g) was applied to a preparative HPLC using a linear gradient elution of 40 − 70%
aqueous MeCN over 40 min to afford compounds 10 (22.5 mg), 9 (36.9 mg), 13 (6.1 mg), 11
(0.9 mg), and 12 (27.0 mg), respectively.
The EtOAc crude extract from mycelia (0.6 g) was triturated from MeOH to give
compound 12 (131.9 mg). The filtrate was further purified by a preparative HPLC using a linear
22

ACCEPTED MANUSCRIPT
gradient elution of 40 − 100% aqueous MeCN over 40 min to afford compounds 10 (4.7 mg) and
12 (7.2 mg), respectively.
4.3.1 Conoideoglucoside A (1)
ꢀ ꢁ24
−24.0 (c 0.13, MeOH); UV (MeOH) λmax (log ε) 240 (4.09), 262 (4.03),
ꢂ
Yellow oil;
α
424 (3.21) nm; FTIR (ATR): νmax 3385 (br), 2957, 2924, 2853, 2195, 1703, 1641, 1599, 1535,
1 1
13
1456, 1387, 1250, 1107, 1084, 1059, 1040 cm− ; H and C NMR data, see Table 1; HRESIMS
m/z 401.1208 [M+Na]+ (calcd for C19H22NaO8, 401.1207).
4.3.2 Conoideoglucoside B (2)
ꢀ ꢁ25
−16.9 (c 0.19, MeOH); UV (MeOH) λmax (log ε) 228 (3.90), 259 (3.91),
ꢂ
Yellow oil;
α
297 (3.74) 422 (3.39) nm; FTIR (ATR): νmax 3364 (br), 2953, 2922, 2855, 2193, 1659, 1605,
1 1
1497, 1452, 1416, 1377, 1240, 1099, 1078, 1053, 1018 cm− ; H and 13C NMR data, see Table 1;
HRESIMS m/z 387.1413 [M+Na]+ (calcd for C19H24NaO7, 387.1414).
4.3.3 Conoideoglucoside C (3)
ꢀ ꢁ24
−8.9 (c 0.32, MeOH); UV (MeOH) λmax (log ε) 240 (3.54), 260 (3.53),
ꢂ
Yellow oil;
α
290 (3.35) 420 (2.46) nm; FTIR (ATR): νmax 3402 (br), 2956, 2924, 2854, 1703, 1650, 1591,
1 1
13
1458, 1419, 1377, 1299, 1206, 1072, 1024 cm− ; H and C NMR data, see Table 1; HRESIMS
m/z 435.1621 [M+Na]+ (calcd for C20H28NaO9, 435.1626).
4.3.4 Eutypinic acid (4)
1
Reddish brown solid; H and 13C NMR data in acetone-d6, see Figs. S89 and S90;
HRESIMS m/z 201.0559 [M−H]− (calcd for C12H9O3, 201.0557).
4.3.5 Conoideochromane A (
5)
ꢀ ꢁ24
Yellow oil ( 92% purity);
α
+52.1 (c 0.06, MeOH); UV (MeOH) λmax (log ε) 207
ꢂ
(4.08), 238 (3.35), 287 (3.05) nm; FTIR (ATR): νmax 3353 (br), 2928, 2874, 1599, 1497, 1449,
23

ACCEPTED MANUSCRIPT
1 1
1380, 1371, 1319, 1272, 1252, 1235, 1218, 1188, 1135, 1062, 1025 cm− ; H and 13C NMR data,
see Table 2; HRESIMS m/z 247.0940 [M+Na]+ (calcd for C12H16NaO4, 247.0941).
4.3.6 Conoideochromane B (6)
ꢀ ꢁ25
+5.1 (c 0.19, MeOH); UV (MeOH) λmax (log ε) 208 (4.40), 236 (3.68),
ꢂ
Yellow oil;
α
286 (3.28) nm; FTIR (ATR): νmax 3410 (br), 2954, 2924, 2854, 1599, 1497, 1459, 1382, 1370,
1
1316, 1267, 1253, 1231, 1218, 1189, 1137, 1066 cm− ; 1H and 13C NMR data, see Table 2;
HRESIMS m/z 261.1099 [M+Na]+ (calcd for C13H18NaO4, 261.1097).
4.3.7 Conoideochromane C (
7)
ꢀ ꢁ25
Yellow oil ( 89% purity);
α
+35.4 (c 0.07, MeOH); UV (MeOH) λmax (log ε) 203
ꢂ
(4.21), 234 (3.77), 287 (3.39) nm; FTIR (ATR): νmax 3374 (br), 2955, 2925, 2856, 1730, 1632,
1 1
1592, 1488, 1441, 1415, 1371, 1323, 1249, 1203, 1168, 1133, 1068, 1025 cm− ; H and 13C NMR
data, see Table 2; HRESIMS m/z 331.1151 [M+Na]+ (calcd for C16H20NaO6, 331.1152).
4.3.8 2,2-Dimethyl-2H-1-chromene-6-carboxylic acid (8)
Brown solid; 1H and 13C NMR data in acetone-d6, see Figs. S91 and S92; HRESIMS m/z
227.0680 [M+Na]+ (calcd for C12H12NaO3, 227.0679).
4.3.9 (−)-Luteoskyrin (9)
1
Yellow solid;
α
−728.5 (c 0.11, MeOH); H and 13C NMR data in DMSO-d6, see
ꢀ ꢁ24
ꢂ
Figs. S93 and S94; HRESIMS m/z 573.1035 [M−H]− (calcd for C30H21O12, 573.1038).
4.3.10 (
−
)-4a-Oxyluteoskyrin (10
Yellowish brown solid; mp 199.1 °C (dec);
) 215 (+7.1), 234 (−1.2), 255 (−11.5), 292 (+5.5), 356 (+11.4), 429
) 218 (4.13), 238 (4.27), 267 (4.18), 422 (4.20) nm; FTIR
)
ꢀ ꢁ24
α
−28.4 (c 0.92, MeOH); CD (c 5.08 ×
ꢂ
5
10− M, MeOH): λmax (∆
ε
(−9.3) nm; UV (MeOH) λmax (log
ε
24

ACCEPTED MANUSCRIPT
(ATR): νmax 3417 (br), 2960, 2925, 2869, 1731, 1651, 1622, 1579, 1457, 1435, 1394, 1297,
1
1262, 1202, 1182, 1117 cm− ; 1H and 13C NMR data, see Table 3; HRESIMS m/z 589.0987
[M−H]− (calcd for C30H21O13, 589.0988).
4.3.11 Chrysophanol (11
)
1
Orange brown solid; H and 13C NMR data in acetone-d6, see Figs. S95 and S96;
HRESIMS m/z 253.0509 [M−H]− (calcd for C15H9O4, 253.0506).
4.3.12 Islandicin (12
)
Reddish brown solid; 1H and 13C NMR data in CDCl3, see Figs. S97 and S98; HRESIMS
m/z 271.0603 [M+H]+ (calcd for C15H11O5, 271.0601).
4.3.13 Catenarin (13
)
1
Reddish brown solid; H and 13C NMR data in DMSO-d6, see Figs. S99 and S100;
HRESIMS m/z 285.0410 [M−H]− (calcd for C15H9O6, 285.0405).
4.3.14 (22E)-5
α
,8
α
-Epidioxyergosta-6,22-dien-3
β-ol (14
)
13
−31.9 (c 0.15, CHCl3); 1H and C NMR data in CDCl3, see Figs.
ꢀ ꢁ27
Colourless solid;
α
ꢂ
S101 and S102; HRESIMS m/z 451.3179 [M+Na]+ (calcd for C28H44NaO3, 451.3183).
4.4. Hydrolysis of compound 3
Compound 3 (2.0 mg, 0.005 mmol) was hydrolyzed by 3 M aqueous HCl (0.5 mL) at
90 °C for 15 h. After cooling, the reaction mixture was diluted with water (2.0 mL) and then
extracted with EtOAc (3 × 2 mL). The aqueous layer of hydrolysate was evaporated to dryness
ꢀ ꢁ2ꢆ
under reduced pressure to provide 4-O-methyl-D-glucopyranose (0.70 mg, 72% yield,
α
ꢂ
+61.4, c 0.07, MeOH). The combined organic layer was dried over Na2SO4 and then
25

ACCEPTED MANUSCRIPT
concentrated in vacuo to give 4-hydroxy-3-(3-hydroxy-3-methylbutyl)-5-methoxybenzoic acid
(0.97 mg, 76% yield); 1H NMR (500 MHz, acetone-d6 + D2O)
δ 1.32 (6H, s), 1.81 (2H, t, J = 6.7
Hz), 2.80 (2H, t, J = 6.7 Hz), 3.79 (3H, s), 7.33 (1H, d, J = 1.2 Hz), 7.40 (1H, d, J = 1.2 Hz);
HRESIMS m/z 259.0946 [M−H2O+Na]+ (calcd for C13H16NaO4, 259.0941).
4.5. Biological assays
Antimalarial activity against P. falciparum (K1, multidrug-resistant strain) was treated by
using the microculture radioisotope technique 25
. Dihydroartemisinin and chloroquine were used
as standard references. Antitubercular activity against M. tuberculosis H37Ra (ATCC25177) and
cytotoxicity against non-cancerous Vero cells (African green monkey kidney fibroblasts, ATCC
CCL-81) were carried out by using the green fluorescent protein microplate assay (GFPMA).26
Rifampicin, ofloxacin, streptomycin, isoniazid, and ethambutol were use as standard references
for anti-TB, while ellipticine was used as a standard reference for cytotoxicity against Vero cells.
Antifungal activity against C. albicans (ATCC90028) and cytotoxicity against cancerous cells,
including MCF-7 (human breast cancer, ATCC HTC-22) and NCI-H187 (human small-cell lung
cancer, ATCC CRL-5804) were evaluated based on the resazurin microplate assay (REMA).27
Amphotericin B was used as a standard reference for antifungal test. Doxorubicin and tamoxifen
were used as standard references for anti-MCF-7, while doxorubicin and ellipticine were used as
standard references for activity against NCI-H187 cells. The optical density microplate assay, the
standard protocols published by Clinical and Laboratory Standard Institute,28,29 was applied for
an evaluation of antibacterial activity against Gram-positive bacteria [B. cereus (ATCC11778),
E. faecium (ATCC51559), S. aureus (ATCC29213)] and Gram-negative bacteria [A. baumannii
(ATCC19606), E. coli (ATCC25922), K. pneumoniae (ATCC700603), P. aeruginosa (wild type
26

ACCEPTED MANUSCRIPT
strain PAO1, ATCC15692)]. Rifampicin and vancomycin were used as standard references for
activity against B. cereus and S. aureus. Rifampicin and tetracycline HCl were used as standard
references for anti-E. faecium. Moreover, rifampicin and erythromycin were used as standard
references for assay against A. baumannii, E. coli, and K. pneumoniae, while chloramphenicol
and erythromycin were used as standard references for anti-P. aeruginosa. Anti-plant pathogenic
fungal activity against A. brassicicola (BCC42724), C. acutatum (BCC58146), C. lunata
(BCC15558), and M. grisea (BCC10261) was done by employing the 5(6)-carboxyfluorescein
diacetate (CFDA) fluorometric assay30-32 and amphotericin B was used as a standard reference.
Maximum tested concentration for all assays was performed at 50 µg/mL, except that
antimalarial activity against P. falciparum was done at 10 µg/mL. All tested compounds were
added in triplicate in a well plate.
Acknowledgements
Financial support from the Office of the Higher Education Commission and the Thailand
Research Fund (Grant number MRG6080228 and MRG6280005) and all facilities provided by
BIOTEC are gratefully acknowledged.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://dx.doi.org/xxx.
27

ACCEPTED MANUSCRIPT
References
1.
2.
3.
4.
5.
6.
7.
8.
Isaka, M.; Kittakoop, P.; Kirtikara, K.; Hywel-Jones, N. L.; Thebtaranonth, Y. Acc.
Chem. Res. 2005, 38, 813 – 823.
Isaka, M.; Palasarn, S.; Kocharin, K.; Hywel-Jones, N. L. J. Antibiot. 2007, 60, 577 –
581.
Pittayakhajonwut, P.; Usuwan, A.; Intaraudom, C.; Khoyaiklang, P.; Supothina, S.
Tetrahedron 2009, 65, 6069 – 6073.
Kornsakulkarn, J.; Thongpanchang, C.; Lapanun, S.; Srichomthong, K. J. Nat. Prod.
2009, 72, 1341 – 1343.
Isaka, M.; Palasarn, S.; Supothina, S.; Komwijit, S.; Luangsa-ard, J. J. J. Nat. Prod. 2011,
74, 782 – 789.
Saepua, S.; Kornsakulkarn, J.; Choowong, W.; Supothina, S.; Thongpanchang, C.
Tetrahedron 2015, 71, 2400 – 2408.
Saepua, S.; Kornsakulkarn, J.; Somyong, W.; Laksanacharoen, P.; Isaka, M.;
Thongpanchang, C. Tetrahedron 2018, 74, 859 – 866.
Johnson, D.; Sung, G.-H.; Hywel-Jones, N. L.; Luangsa-Ard, J. J.; Bischoff, J. F.; Kepler,
R. M.; Spatafora, J. W. Mycol. Res. 2009, 113, 279 – 289.
Abraham, W.-R.; Arfmann, H.-A. Phytochemistry 1990, 29, 2641 – 2644.
Wang, R. F.; Yang, X. W.; Ma, C. M.; Liu, H. Y.; Shang, M. Y.; Zhang, Q. Y.; Cai, S.
Q.; Park, J. H. J. Asian. Nat. Prod. Res. 2004, 6, 139 – 144.
Smith, F. J. Chem. Soc. 1951, 2646 – 2652.
9.
10.
11.
12.
Suciati; Fraser, J. A.; Lambert, L. K.; Pierens, G. K.; Bernhardt, P. V.; Garson, M. J. J.
Nat. Prod. 2013, 76, 1432 – 1440.
28