Cordycepamides A?E and cordyglycoside A, new alkaloidal and glycoside metabolites from the entomopathogenic fungus Cordyceps sp.

Article abstract of DOI:10.1016/j.fitote.2020.104525

Five new alkaloidal metabolites cordycepamides A?E (1?5), and one glycoside metabolite cordyglycoside A (6), together with six known compounds (7?12) were isolated from the entomopathogenic fungus Cordyceps sp. (LB1.18060004) from unidentified insect collected in Baoshan City, Yunnan Province, People's Republic of China. The structures were characterized by NMR and HRESIMS spectroscopic analyses. Cordycepamides A and B (1 and 2) were mixtures of two isomers in 5:4 ratio by integration of ¹H NMR spectra. In additional, the structure of cordycepamide A (1) was further confirmed by X-ray crystallography as a pair of enantiomers. Absolute configurations of sugar moiety of cordyglycoside A (6) was confirmed by the acid hydrolysis and subsequent HPLC analysis. The isolated metabolites were evaluated for antimicrobial, cytotoxicity, and the DPPH scavenging assay, only 4 showed modest antioxidant effects in the DPPH scavenging assay (IC₅₀ = 51.42 ± 3.08 μM).

Full text of DOI:10.1016/j.fitote.2020.104525

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Cordycepamides A−E and cordyglycoside A, new alkaloidal and
glycoside metabolites from the entomopathogenic fungus
Cordyceps sp.
Wenwen Fan, Erwei Li, Jinwei Ren, Wenzhao Wang, Xingzhong
Liu, Yongjie Zhang
PII:
S0367-326X(20)30107-6
DOI:
https://doi.org/10.1016/j.fitote.2020.104525
FITOTE 104525
Reference:
To appear in:
Fitoterapia
Received date:
Revised date:
Accepted date:
31 December 2019
17 February 2020
19 February 2020
Please cite this article as: W. Fan, E. Li, J. Ren, et al., Cordycepamides A−E and
cordyglycoside A, new alkaloidal and glycoside metabolites from the entomopathogenic
fungus Cordyceps sp., Fitoterapia (2020), https://doi.org/10.1016/j.fitote.2020.104525
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Cordycepamides A−E and cordyglycoside A, new alkaloidal and
glycoside metabolites from the entomopathogenic fungus Cordyceps
sp.
Wenwen Fana,b,c,1, Erwei Lic,1, Jinwei Renc, Wenzhao Wangc, Xingzhong Liuc,d,*
Yongjie Zhanga,*
,
a School of Life Science, Shanxi University, Taiyuan 030006, China.
b Institute of Biotechnology, Shanxi University, Taiyuan 030006, China.
c State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of
Sciences, Beijing 100101, China.
d
Department of Microbiology, College of Life Sciences, Nankai University, Tianjin
300071, China.
Corresponding authors.
E-mail addresses: liuxz@im.ac.cn (X. Liu), zhangyj2008@sxu.edu.cn (Y. Zhang).
1 Wenwen Fan and Erwei Li contributed equally.

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ABSTRACT
Five new alkaloidal metabolites cordycepamides A−E (1−5), and one glycoside
metabolite cordyglycoside A (6), together with six known compounds (7−12) were
isolated from the entomopathogenic fungus Cordyceps sp. (LB1.18060004) from
unidentified insect collected in Baoshan City, Yunnan Province, People's Republic of
China. The structures were characterized by NMR and HRESIMS spectroscopic
analyses. Cordycepamides A and B (1 and 2) were mixtures of two isomers in 5:4
ratio by integration of 1H NMR spectra. In additional, the structure of cordycepamide
A (1) was further confirmed by X-ray crystallography as a pair of enantiomers.
Absolute configurations of sugar moiety of cordyglycoside A (6) was confirmed by
the acid hydrolysis and subsequent HPLC analysis. The isolated metabolites were
evaluated for antimicrobial, cytotoxicity, and the DPPH scavenging assay, only 4
showed modest antioxidant effects in the DPPH scavenging assay (IC50 = 51.42±3.08
μM).
Keywords: Cordyceps sp.; entomopathogenic fungus; alkaloidal metabolite;
enantiomer

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1. Introduction
Entomopathogenic fungi such as species from the genera Cordyceps have been
frequently used as foods and medicines for the treatment of various diseases. The
diversity and complexity of the living environment and metabolic type of
entomogenous fungi lead to them possessing the capability to produce structurally
diverse and biologically active secondary metabolites [1]. One of the well-known
metabolites was ISP-I, isolated from entomogenous Isaria sinclairii, while
investigating structure-activity relationship of its derivatives, fingolimod (FTY720,
Gilenya) was the first US Food and Drug Administration–approved oral therapy for
relapsing forms of multiple sclerosis [2]. Notable other examples include beauvericins
isolated from Cordyceps cicadae with cytotoxic activities [3], cordyformamide
isolated from Cordyceps brunnearubra with antimalarial activity [4], cordypyridones
A−D isolated from Cordyceps nippocica with antimalarial activity [5],
dimethylguanosine isolated from Cordyceps militaris with antioxidant and HIV-1
protease inhibitory activity [6], cicadapeptins I and II isolated from Cordyceps
heteropoda with antibacterial and antifungal activity [7]. Therefore, entomogenous
fungi are considered to be important potential sources of natural medicines [8].
During an ongoing search for new bioactive natural products from entomogenous
and related fungi [9-11], a strain of entomopathogenic fungus Cordyceps sp. from
unidentified insect collected in Baoshan City, Yunnan Province, People's Republic of
China in 2018, was subjected to chemical investigation. Fractionation of an EtOAc
extract from a solid-substrate fermentation culture afforded five alkaloidal metabolites

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cordycepamides A−E (1−5) and one glycoside metabolite cordyglycosides A (6),
together with six known compounds, cordytakaoamide B (7) [12], beauvericin (8)
[13], beauvericin A (9) [14], beauvericin E (10) [15], 1,4-dimethoxy-2, 4',
4"-trihydroxy-p-terphenyl (11) [16] and cyclo(L-phenylalaninyl-L-leucinyl) (12) [17].
Details of the isolation, structure elucidation, and biological activities of these
compounds are reported herein.
2. Experimental
2.1 General experimental procedures
Optical rotations were measured on an Anton Paar MCP200 polarimeter (Graz,
1
Austria). H and 13C NMR data were acquired with Bruker Avance-500 spectrometers
(Rheinstetten, Germany). The HSQC and HMBC experiments were optimized for
125.0 and 8.0 Hz, respectively. HRESIMS data were recorded on an Agilent
Accurate-Mass-Q-TOF LC/MS 6520 instrument (Santa Clara, CA, USA) in positive
ion mode. HPLC data were obtained with a Waters 2695 instrument (Milford, MA,
USA). Preparative HPLC was performed on an Agilent 1200 HPLC system using a
C18 column (Reprosil-Pur Basic C18 column; 5 μm; 10×250 mm; detector: UV) with
a flow rate of 2.0 mL/min. The absorbance of contents in the 96-well clear plate was
detected by a SpectraMax Paradigm microplate reader (Sunnyvale, CA, USA).
2.2 Fungal material
The strain of Cordyceps sp. (LB1.18060004) was isolated from unidentified
insect collected in Baoshan City, Yunnan Province, People's Republic of China, in
July 2018. The isolated strain was identified by sequence analysis (Genbank

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Accession Number MN606111) of the rDNA internal transcribed spacer (ITS) region.
The strain was firstly cultivated on culture dish of potato-dextrose agar at 25 °C for 10
days. Then the agar was cut into grain size to inoculate in two conical flasks (500 mL)
that each containing 100 mL autoclave sterilized potato-dextrose broth. The flasks
containing inoculated potato-dextrose broth were cultivated at 25 °C on a rotary
shaker at 200 rpm for 6 days. Forty 500 mL Fernbach flasks, each containing 80 g of
rice and 120 ml of distilled water, were autoclaved at 120 °C for 30 min, in which the
fermentation proceeded. After cooling to room temperature, each of the flasks was
inoculated with 10 ml of the spore inoculum and cultured at 25 °C for 40 days.
2.3 Extraction and isolation
The ferment culture was extracted with EtOAc (three times, each 6 L) and
vacuum-dried to afford the crude extract (15 g). The extract was fractionated by silica
gel
vacuum
liquid
chromatography
(VLC)
in
petroleum
ether-dichloromethane-MeOH gradient elution to afford eighteen primary fractions
(Frs.1−18). The fraction Fr.10 (2.3 g) was loaded on Sephadex LH-20 column
chromatography eluted with MeOH to afford 43 subfractions Frs.10.1−10.43. Fr.10.14
was purified by RP HPLC (Reprosil-Pur Basic C18 column; 5 μm; 10×250 mm; 74%
MeOH in H2O with 0.1% HCOOH for 50 min; 2.0 mL/min) to afford 1 (9.3 mg, tR
32.5 min). The subfraction Fr.10.15 was purified by RP HPLC (Reprosil-Pur Basic
C18 column; 5 μm; 10×250 mm; 76% MeOH in H2O with 0.1% HCOOH for 50 min;
2.0 mL/min) to afford 2 (3.9 mg, tR 34.1 min). The subfraction Fr.10.16 was purified
by RP HPLC (Reprosil-Pur Basic C18 column; 5 μm; 10×250 mm; 83% MeOH in

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H2O with 0.1% HCOOH for 60 min; 2.0 mL/min) to afford 8 (14.3 mg, tR 35.3 min)
and 9 (4.5 mg, tR 44.5 min). The subfraction Fr.10.18 was purified by RP HPLC
(Reprosil-Pur Basic C18 column; 5 μm; 10×250 mm; 83% MeOH in H2O with 0.1%
HCOOH for 60 min; 2.0 mL/min) to afford 10 (3.0 mg, tR 31.2 min). The subfractions
Frs.10.25−10.43 were combined to give 1.3 g was loaded on a reverse silica gel
column chromatography (ODS) eluted with 50%-100% MeOH to afford seven tertiary
fractions (Frs.10.25.1−10.25.7). Fr.10.25.4 was purified by RP HPLC (Reprosil-Pur
Basic C18 column; 5 μm; 10×250 mm; 45%MeOH in H2O with 0.1% HCOOH for 50
min; 2.0 mL/min) to afford 5 (5.8 mg, tR 31.1 min). Fr.10.25.5 after elution was
purified by RP HPLC (Reprosil-Pur Basic C18 column; 5 μm; 10×250 mm;
52%MeOH in H2O with 0.1% HCOOH for 60 min; 2.0 mL/min) to afford 11 (3.8 mg,
tR 42.4 min). Fr.10.25.7 was purified by RP HPLC (Reprosil-Pur Basic C18 column; 5
μm; 10×250 mm; 52%MeOH in H2O with 0.1% HCOOH for 60 min; 2.0 mL/min) to
afford 3 (2.3 mg, tR 52.2 min). The fractions Frs.11−13 were combined to give 5.4 g
was loaded on Sephadex LH-20 column chromatography eluted with MeOH to afford
26 subfraction Frs.11.1−11.26. The subfraction Fr.11.8 was purified by RP HPLC
(Reprosil-Pur Basic C18 column; 5 μm; 10×250 mm; 47% MeOH in H2O with 0.1%
HCOOH for 60 min; 2.0 mL/min) to afford 12 (2.5 mg, tR 48.8 min). The subfraction
Fr.11.13 was purified by RP HPLC (Reprosil-Pur Basic C18 column; 5 μm; 10×250
mm; 49% MeOH in H2O with 0.1% HCOOH for 70 min; 2.0 mL/min) to afford 7 (4.3
mg, tR 64.3 min). The subfraction Fr. 11.14 was purified by RP HPLC (Reprosil-Pur
Basic C18 column; 5 μm; 10×250 mm; 56% MeOH in H2O with 0.1% HCOOH for

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50 min; 2.0 mL/min) to afford 4 (5.9 mg, tR 38 min).The subfraction Fr.11.26 was
purified by RP HPLC (Reprosil-Pur Basic C18 column; 5 μm; 10×250 mm; 69%
MeOH in H2O with 0.1% HCOOH for 50 min; 2.0 mL/min) to afford 6 (4.0 mg, tR
49.5 min).
2.3.1 Cordycepamide A (1)
White powder; [α]25 +13.57 (c 0.28, MeOH); UV (MeOH) λmax (log ε): 224 (2.84)
D
nm; IR (neat) νmax 3307, 2954, 2926, 2855, 1666, 1622, 1539, 1478, 1350, 1279, 1248,
1201, 1066, 1032, 976, 726 cm–1; 1H and 13C NMR data (500 MHz, CDCl3) see Table 1;
HMBC correlations (500 MHz, CDCl3): H-1 → C-9; H-2 → C-3, 4; H2-3 → C-1, 2, 4,
9; H-5 → C-4, 9; H-6 → C-5; H-7 → C-6, 8, 9; H-11 → C-10, 13; H-12 → C-10, 13, 14;
H2-13 → C-11, 12; H2-14 → C-15; H2-15 → C-13; H2-16 → C-18; H2-17 → C-18;
H2-18 → C-19; H3-19 → C-18; HRESIMS: m/z 356.1841 [M+Na]+ (calcd for
C19H27NO4Na 356.1838).
2.3.1.1 X-ray Crystallographic Analysis of 1
Upon crystallization from MeOH–H2O (3:1) using the vapor diffusion method,
colorless crystals were obtained for 1. A crystal (0.32 × 0.02 × 0.31 mm) was
separated from the sample and mounted on a glass fiber, and data were collected using
the Agilent CrysAlisPro software (Version 1.171.40.39) with
graphite-monochromated Cu Kα radiation, λ = 1.54184 Ǻ at 107.0(10) K. Crystal data:
C19H27NO4, M = 333.42, space group orthorhombic, P-1; unit cell dimensions a =
9.0892(2) Ǻ, b = 13.7368(3) Ǻ, c = 15.4522(3) Ǻ, V = 1812.42(7) Ǻ3, Z = 4, Dcalcd
1.222 mg/m3, µ = 0.687 mm–1, F(000) = 720.0. The structure was solved by direct
=

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methods using SHELXL-97 [18] and refined by using full-matrix least-squares
difference Fourier techniques. All non-hydrogen atoms were refined with anisotropic
displacement parameters, and all hydrogen atoms were placed in idealized positions
and refined as riding atoms with the relative isotropic parameters. Absorption
corrections were performed using spherical harmonics, implemented in SCALE3
ABSPACK scaling algorithm [19]. The 6318 measurements yielded 5750 independent
reflections after equivalent data were averaged, and Lorentz and polarization
corrections were applied. The final refinement gave R1 = 0.0484 and wR2 = 0.1425 [I >
2σ(I)]. Crystallographic data for 1 has been deposited with the Cambridge
Crystallographic Data Centre as supplementary publications Nos. CCDC 1973842.
These data can be obtained free of charge from the Cambridge Crystallographic Data
Centre via www.ccdc.cam.ac.uk/data_request/cif.
2.3.2. Cordycepamide B (2)
White powder; [α]25 -4.87 (c 0.39, MeOH); UV (MeOH) λmax (log ε): 219 (2.87)
D
nm; IR (neat) νmax 3307, 2927, 2855, 1666, 1629, 1545, 1457, 1221, 1074, 980, 751
cm–1; 1H and 13C NMR data (500 MHz, CDCl3) see Table 1; HMBC correlations (500
MHz, CDCl3): H-1 → C-3, 9; H-2 → C-1, 3, 4; H2-3 → C-1, 2, 4, 5, 9; H-5 → C-3, 7,
9; H-6 → C-7; H-7 → C-5, 9; H-8 → C-4, 7, 9; H-11 → C-10, 13; H-12 → C-10, 13;
H2-13 → C-11, 12, 14; H2-14 → C-12, 16; H2-15 → C-13; H2-16 → C-17; H2-17 →
+
C-18; H2-18 → C-19; H3-19 → C-18; HRESIMS: m/z 318.2066 [M+H] (calcd for
C19H28NO3 318.2069).
2.3.3. Cordycepamide C (3)

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Yellow oil; [α]25 -39.56 (c 0.23, MeOH); UV (MeOH) λmax (log ε) 215 (2.69),
D
274 (0.41) nm; IR (neat) νmax 3296, 2934, 1737, 1667, 1622, 1539, 1456, 1365, 1242,
1034, 755 cm–1; 1H and 13C NMR data (500 MHz, acetone-d6) see Table 1; HMBC
correlations (500 MHz, acetone-d6): H-1 → C-2, 3; H-2 → C-1, 3; H2-3 → C-1, 2, 4, 5,
9; H-6 → C-4, 8; H-7 → C-5; H-8 → C-6, 9; H-9 → C-3, 5, 7; H-11 → C-10, 13; H-12
→ C-10, 13; H2-13 → C-11, 12, 14, 15; H2-14 → C-12, 13, 15; H2-16 → C-14, 15, 17;
+
H2-17 → C-15, 1'; H3-2' → C-1'; NH → C-10; HRESIMS: m/z 350.1971 [M+H]
(calcd for C19H28NO5 350.1967).
2.3.4. Cordycepamide D (4)
Yellow oil; [α]25 -27.28 (c 0.59, MeOH); UV (MeOH) λmax (log ε): 212 (2.36),
D
296 (0.38) nm; IR (neat) νmax 3306, 2931, 2856, 1716, 1667, 1621, 1543, 1506, 1455,
1366, 1251, 1204, 1149, 1045, 978 cm–1; 1H and 13C NMR data (500 MHz, DMSO-d6)
see Table 1; HMBC correlations (500 MHz, DMSO-d6): H-1 → C-2, 3; H2-3 → C-1, 2,
4, 5, 9; H-6 → C-4, 7, 8; H-7 → C-5, 9; H-9 → C-3; H-11 → C-10, 13; H-12 → C-10,
13, 14; H2-13 → C-11, 12, 14, 15; H2-14 → C-12, 13, 16; H2-17 → C-16, 19; H2-18 →
C-17, 19; H2-19 → C-17, 18, 1'; H3-2' → C-1'; OH-1 → C-1, 2; OH-5 → C-4, 5, 6;
+
OH-8 → C-7, 8, 9; NH → C-2, 10; HRESIMS: m/z 416.2046[M+Na] (calcd for
C21H31NO6Na 416.2049).
2.3.5. Cordycepamide E (5)
White powder; [α]25 +173.01 (c 0.56, MeOH); UV (MeOH) λmax (log ε): 229
D
(3.61), 276 (0.91) nm; IR (neat) νmax 3368, 2969, 2933, 1746, 1648, 1614, 1516, 1446,
1368, 1340, 1259, 1231, 1205, 1172, 1128, 1036, 826, 599, 576 cm–1; 1H and 13C NMR

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data (500 MHz, CDCl3) see Table 2; HMBC correlations (500 MHz, CDCl3): H-3 →
C-2, 5, 10, 11, 17; H-6 → C-2, 5, 7, 8, 9; H-7 → C-5, 8, 9; H3-8 → C-6, 7, 9; H3-9 →
C-6, 7, 8; H2-10 → C-2, 3, 11, 16; H-12 → C-10, 13, 14; H-13 → C-11, 14; H3-17 →
C-3, 5; HRESIMS: m/z 278.1391 [M+H]+ (calcd for C15H20NO4 278.1392).
2.3.6. Cordyglycoside A (6)
Yellow oil; [α]25 -7.50 (c 0.2, MeOH); UV (MeOH) λmax (log ε): 229 (1.24) nm;
D
IR (neat) νmax 3391, 2929, 2857, 1714, 1456, 1374, 1198, 1109, 1080 cm–1; 1H and 13C
NMR data (500 MHz, DMSO-d6) see Table 3; HMBC correlations (500 MHz,
DMSO-d6): H2-2 → C-1, 3; H2-6 → C-7, 8; H-7 → C-6, 9; H-8 → C-6, 10; H-9 → C-7,
8, 11; H-10 → C-8, 11; H-11 → C-9, 12, 1'; H2-12 → C-14; H3-18 → C-17; H-1' →
C-11; H-2' → C-1', 3', H-3' → C-2', 4'; H-4' → C-5', 4'-OCH3; H-5' → C-4'; H2-6a' →
C-4', 5'; 4'-OCH3 → C-4'; HRESIMS m/z 495.2937 [M+Na]+ (calcd for C25H44O8Na
495.2934).
2.3.6.1 Acid hydrolysis and determination of the absolute configuration of glucose
Compound 6 (2.0 mg) was added with hydrochloric acid (3 M, 3 mL), and
reacted in a water bath at 90 °C for 2 h. After the reaction was completed, an equal
volume of ethyl acetate was added for extraction twice, and the aqueous layer was
evaporated to dryness to obtain a sugar residue. The sugar residue was added with
L-cysteine methyl ester hydrochloride (2.5 mg) and pyridine (1 mL), and then heated
to 60 °C for 1 h. σ-Tolylisothiocyanate (10 μL) was added to the mixture, and heating
was continued for an additional 1 h. The reaction mixture was directly analyzed using
HPLC (10% to 100% MeCN gradient with 0.1% formic acid over 40 min) [20].

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2.4 Cell Culture
Humanbreast cancer cells MCF-7, human liver cancer cells HepG-2, human colon
cancer cells HT29, and humankidney normal cells 293t were purchased fromShanghai
Institute of Biological Sciences (SIBS), Chinese Academy of Sciences. The cell line
was added to RPMI-1640 medium (Hyclone) containing 10% fetal bovine serum, and
all cells were cultured in a 5% CO2, 37°C incubator.
2.5 Cytotoxicity assay
The cytotoxicity evaluations of 3−5 and 7 against normal human breast cancer
cells MCF-7, human liver cancer cells HepG-2, human colon cancer cells HT29, and
human kidney normal cells 293t
were
carried
out
using
the
3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.
Doxorubicin was used as positive control drug and deionized H2O with the same
DMSO concentration was used as parallel control. All experiments were repeated
three times [21].
2.6 Antimicrobial assays
This experiment mainly tested the antibacterial activity of compounds 3−7
against three pathogenic bacteria, Staphylococcus aureus, Bacillus subtilis and E. coli.
Each pathogen was cultured on a LA plate at 37°C overnight. A single bacterial
colony was picked and suspended in LB medium to about 1×104 cfu/mL. Vancomycin
and ampicillin were used as positive controls, and DMSO was used as a negative
control. Prepare two-fold serial dilutions of each test compound (20 to 1.25 µg/mL in
DMSO) and add an aliquot (2 µL) of each dilution to a 96-well containing 98 µL LB

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medium In the board. An aliquot (100 µL) of the bacterial suspension was then added
to each well (to make the final compound concentration of 200 to 12.5 µg/mL) and
incubated at 37 °C for 12 h. Finally, the optical density of each well at 600 nm was
measured with an EnVision 2103 Multilabel Plate Reader. MIC values were defined
as the minimum concentration of compound that inhibited visible bacterial growth.
All experiments were repeated three times.
2.7 DPPH Scavenging assay
The mixture containing 50 μLof each compound in DMSO (final concentrations
of 6.25, 12.5, 25, 50, 100 μM) and 50 μL of DPPH (sigma) radicals in ethanolic
solution (final concentration of 100 μM) were placed in a 96-well microplate and
incubate in the dark for 30 minutes, and measurement of absorbance at 517 nm using
a Spectra Max 190 microplate reader (Molecular Devices Inc.). The scavenging
ability was calculated as follows: scavenging ability (%)= [(A517 of control - A517 of
sample)/A517 of control] × 100. Ascorbic acid was used as positive control. All experiments
were repeated three times.
2. Results and discussion
Cordycepamide A (1) was obtained as a mixture of two isomers (1a and 1b) in a
1
ratio of approximately 4:5 by analysis of its H NMR spectrum (Fig. S1). Although
1
the two isomers were well-resolved using a chiral HPLC column (Fig. S2), H NMR
spectra of each collected peak showed the presence of both isomers (Fig. S3),
suggesting the occurrence of a spontaneous equilibration. Therefore, the structure was
elucidated on the equilibrium mixture. Compound 1 has a molecular formula of

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C19H27NO4 (seven degrees of unsaturation) on the basis of its HRESIMS
pseudomolecular ion peak ([M+Na]+ at m/z 356.1841) and NMR data (Table 1). The
1H and 13C NMR data (Table 1) of the major isomer 1b showed the presence of one
carboxylic carbon (δC 167.8), eight aromatic/olefinic carbons with five protonated,
one hemiacetal carbon (δC/H 93.5/5.52), one sp3 methine carbon (δC/H 47.1/4.51),
seven sp3 methylene carbons, one methyl. According to its NMR data and the
requirement of unsaturation degree, 1b required to be bicyclic structure. Interpretation
1
of the H–1H COSY NMR data led to the identification of three isolated proton
spin-systems corresponding to the C-1–C-3, C-5–C-7 and C-11–C-19 fragments.
HMBC correlations from H-5 to C-4 and C-9, from H-7 to C-8 and C-9, from H-1 to
C-9, and from H2-3 to C-4 and C-9 established one chromane moiety with two
hydroxyl groups attached to C-1 and C-8, respectively. HMBC correlations from H-11
and H-12 to C-10, and the large coupling constant (J = 15.2 Hz) observed between
H-11 and H-12 established (11E)-dec-2-enoic acid subunit. Consideration of the
chemical shifts of C-10 (δC 167.8) and C-2 (δC/H 47.1/4.51), and the requirement of
molecular formula, the chromane moiety and (11E)-dec-2-enoic acid subunit was
linked together by amide bond. On the basis of these data, the gross structure of
cordycepamide A was established as 1 (Fig. 1).
The proposed structure for 1 was confirmed by single-crystal X-ray diffraction
analysis (Fig. 3). Compound 1 was found to crystallize as a mixture of enantiomers
(1R, 2S and 1S, 2R). In additional, the presence of internolecular hydrogen bond and
shielding or deshielding effects of benzene ring by observing X-ray diffraction

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explained the phenomena that the 1D NMR spectra of 1a and 1b as a pair of
enantiomers were not overlap.
Cordycepamide B (2) was also obtained as a mixture of two isomers (2a and 2b)
1
in a ratio of approximately 5:4 by analysis of its H NMR spectrum (Fig. S8).
Compound 2 has a molecular formula of C19H27NO3 (seven degrees of unsaturation)
on the basis of its HRESIMS pseudomolecular ion peak ([M+H]+ at m/z 318.2066).
Compared to that of 1, the less 16 mass units suggested the absence of an oxygen
1
atom. The H and 13C NMRdata (Table 1) of 2 revealed the presence of structural
features similar to those found in 1, except that the oxygenated aromatic carbon (δC
145.1) was replaced by a protonated aromatic carbon (δH/C 6.83, 117.0) in the NMR
spectra of 2, and this observation was supported by HMBC correlations from the
newly observed H-8 to C-4, C-7 and C-9. Therefore, the gross structure of
cordycepamide B was proposed as 2.
Cordycepamide C (3) was obtained as a yellow oil. It has a molecular formula of
C19H27NO5 (seven degrees of unsaturation) on the basis of its HRESIMS
pseudomolecular ion peak ([M+H]+ at m/z 350.1971). Its 1H and 13C NMR data (Table
1) revealed the presence of two carboxylic carbons (δC 167.6, 171.1, respectively),
eight aromatic/olefinic carbons with six protonated, one sp3 methine carbon (δC/H
54.3/3.87), seven sp3 methylene carbons with two oxygenated, and one methyl.
Analysis of its NMR data (δC/H 131.9, 157.2, 120.2/6.74, 128.8/7.06, 116.9/6.84,
125.5/7.09, respectively) and the requirement of unsaturation degree, 3 contained
1
an ortho-disubstituted benzene ring moiety. Analysis of the H–1H COSY NMR data

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led to the identification of three isolated proton spin-systems corresponding to the
C-1–C-3, C-6–C-9 and C-11–C-17 fragments. HMBC correlations from H-3 to C-4,
C-5 and C-9, from H-6 to C-4, and from H-9 to C-3 and C-5 established one
3-phenylpropan-1-ol subunit with two hydroxyl groups attached to C-1 and C-5,
respectively. HMBC correlations from H-11 and H-12 to C-10, from H2-17 and H3-2'
to C-1' (δC 171.1) and the large coupling constant (J = 15.3 Hz) observed between
H-11 and H-12 established (11E)-8-hydroxyoct-2-enoic acid subunit with an acetyl
attached to the terminal of the unsaturated fatty acid. Consideration of the chemical
shifts of C-10 (δC 167.6) and C-2 (δC/H 54.3/3.87), and the requirement of molecular
formula, the 3-phenylpropan-1-ol moiety and (11E)-8-hydroxyoct-2-enoic acid
subunit was linked together by amide bond. On the basis of these data, the gross
structure of cordycepamide C was established as 3 (Fig. 1). 3 possessed structural
similarity to those of cordytakaoamide B, an alkaloidal metabolite from
entomopathogenic fungus, Cordyceps takaomontana NBRC 101754 [12]. The
negative optical rotation ([α]D -39.56, MeOH) of 3 was agreement with those of
cordytakaoamide B ([α] D -23.22, MeOH), indicating that the absolute configuration
of C-2 is the same to cordytakaoamide B as 2S.
Cordycepamide D (4) was obtained as a yellow oil. It has a molecular formula of
C21H31NO6 (seven degrees of unsaturation) on the basis of its HRESIMS
pseudomolecular ion peak ([M+Na]+ at m/z 416.2046). Its 1H and 13C NMR data
(Table 1) revealed the presence of two carboxylic carbons (δC 164.8, 170.4,
respectively), eight aromatic/olefinic carbons with five protonated, one sp3 methine

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carbon (δC/H 51.5/3.93), nine sp3 methylene carbons with two oxygenated, one methyl
1
carbon. Its H NMR spectrum showed the presence of an ABX coupling system of
benzene ring (δH 6.57, d, J = 8.5 Hz; 6.40, dd, J = 8.5, 2.9 Hz; 6.50, d, J = 2.9 Hz),
indicating that 4 contained a 1,2,4-trisubstituted benzene ring moiety. Compared
NMR data with 3, 4 possessed structural similarity to 3 except extra an hydroxyl
group attached at C-8, these confirmed by the HMBC correlations from OH-8 to C-7,
C-8 and C-9. In addition, two more sp3 methylenes than 3 was further confirmed by
1
its H–1H COSY and HMBC correlations. The negative optical rotation ([α]D -27.28,
MeOH) of 4 was agreement with those of 3 ([α] -39.56, MeOH) and
D
cordytakaoamide B ([α]D -23.22, MeOH), indicating that the absolute configuration of
C-2 is the same to 3 and cordytakaoamide B as 2S.
Cordycepamide E (5) was obtained as a white powder. It has a molecular formula
of C15H19NO4 (seven degrees of unsaturation) on the basis of its HRESIMS
pseudomolecular ion peak ([M+H]+ at m/z 278.1391). Its 1H and 13C NMR data (Table
2) revealed the presence of two carboxylic carbons (δC 167.7 and 166.7, respectively),
six aromatic carbons with four protonated, one sp3 methylene, three sp3 methines with
one oxygenated and one nitrogenated, three methyls including one N-methyl (δH/C
3.06/33.0). These accounted for all the NMR resonances of 5 except for one
exchangeable proton (OH-14), and required 5 to be a monocyclic compound. One
1
p-substituted benzene ring was confirmed by analysis of its H NMR spectrum (δH
6.77, 2H, br d, J = 8.5 Hz; 6.97, 2H, br d, J = 8.5 Hz). HMBC correlations from H-3
to C-2, C-5, C-10 and C-11, from H-6 to C-2, C-5, C-7, C-8, and C-9, from H-7 to

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C-5 established morpholine-2,5-dione moiety with one 4-methenylphenol and one
isopropyl attached to C-3 and C-6, respectively. The N-methyl (δH/C 3.06/33.0) was
attached to nitrogen atom by HMBC correlations from H3-17 to C-3 and C-5. On the
basis of these data, the gross structure of cordycepamide E was established as 5 (Fig.
1). 5 possessed structural similarity to those of bassiatin, an anticoagulant metabolite
from soil fungus Beauveria bassiana K-717 [22]. Bassiatin and its stereoisomers have
been synthesized [23]. Thus, compared the optical rotation of 5 ([α]D +173.01, MeOH)
to those of reported compounds (natural original bassiatin: +181.05, CHCl3; synthetic
bassiatin: +166, CHCl3, +165.9, CH2Cl2) [22, 23], the absolute configurations of C-3
and C-6 were assigned as 3S, 6R.
Cordyglycoside A (6) was obtained as a yellow powder. It has a molecular
formula of C25H44O8 (four degrees of unsaturation) on the basis of its HRESIMS
pseudomolecular ion peak ([M+Na]+ at m/z 495.2937). Its 1H and 13C NMR data
(Table 3) revealed the presence of one carboxylic carbon (δC 174.8), four protonated
olefinic carbons, one hemiacetal carbon (δC/H 99.4/4.06), five sp3 oxygenated methine
carbons, 12 sp3 methylene carbons with one oxygenated, one methoxyl (δC/H 59.5/3.40)
1
and a methyl. Its H and 13C NMR spectral data showed the presence of a
glucopyranose moiety (δC/H 99.4/4.06, 73.7/2.94, 76.6/3.22, 79.5/2.90, 75.5/2.99,
1
60.7/3.47, 3.59, respectively). Analysis of the H–1H COSY NMR data led to the
identification of two isolated proton spin-systems corresponding to the C-2–C-18 and
C-1'–C-6' fragments. HMBC correlations from H2-2 to C-1, from H2-6 to C-7 and C-8,
from H-11 to C-9, and the large coupling constant (J = 15.3 Hz) observed between

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H-7 and H-8, H-9 and H-10 established (7E, 9E)-octadeca-7, 9-dienoic acid subunit.
HMBC correlations from H-11 to C-1' and from H-1' to C-11 indicated that
glucopyranose moiety attached to C-11. The methoxyl was located at C-4' of the sugar
by the HMBC correlation of OCH3 to C-4'. The coupling constant (J = 7.8 Hz) of the
anomeric proton (δH 4.06) indicated that C-1' of glucopyranose was β-configuration.
According the reported procedure [20], the absolute configuration of glucose was
determined by acid hydrolysis followed by chemical derivatization and comparison
with authentic D- and L-glucose samples using HPLC (Fig. S32), the glucose in 6 was
assigned as D-glucose. Due to few amount of its hydrolysis products, the
configuration of C-11 were not determined by using the modified Mosher’s method.
Thus, the configuration of C-11 in 6 still remains unknown.
The bioactivities of isolated metabolites were evaluated for antimicrobial,
cytotoxicity, and the DPPH scavenging assay. Compounds 3−5 and 7 showed no
cytotoxic activity against the human breast cancer cells MCF-7, human liver cancer
cells HepG-2, human colon cancer cells HT29, and human kidney normal cells 293t at
the concentration of 20 μM. Compounds 3−7 also showed no antibacterial activity
against Bacillus subtilis, E. coli, and Staphylococcus aureus at the concentration of
200 μg/mL. In the DPPH assay, compound 4 showed moderate free radical
scavenging activity with IC50 = 51.42±3.08 μM, using ascorbic acid as a positive
control (IC50 = 30.75±0.10 μM).
4. Conclusion
In summary, chemical investigation of solid-substrate fermentation of

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entomopathogenic fungus, Cordyceps sp. led to the isolation of five new alkaloidal
metabolites, cordycepamides A−E (1−5) and one glycoside metabolite cordyglycoside
A (6). Cordycepamides A and B (1 and 2) are a class of chromane analogs with
(11E)-dec-2-enoic acid moiety linked C-2 through an amide bond. Cordycepamides C
and D (3 and 4) possess similar structural feature to 1 and 2, except the
tetrahydro-2H-pyran ring cleaved and the terminal of the long chain unsaturated fatty
acids acetylated. Biogenetically, compounds 1−4 could be derived from phenylalanine
and dec-2-enoic acid/oct-2-enoic acid [12]. The carboxyl group of phenylalanine has
been reduced to aldehyde and alcohol, then though various degrees of oxygenation,
dehydration, and cyclisation, compounds 1−4 could been product. Cordycepamide E
(5) is a hydroxylated analogue of bassiatin, and cordyglycoside A (6) is a glycosylated
derivate at C-11 of (7E, 9E)-octadeca-7, 9-dienoic acid. In the DPPH assay,
compound 4 showed moderate free radical scavenging activity.
Conflict of interest
The authors declare no conflict of interest.
Acknowledgements
This work was supported by National Special Project for Key Science and
Technology of Food Safety (No. 2017YFC1601300), National Natural Science
Foundation of China (No. 31872162), and the Research Project Supported by Shanxi
Scholarship Council of China (2017-015).
Appendix A. Supplementary data
Supplementary data (1D, 2D NMR spectra of compounds 1–6, the chiral HPLC

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chromatogram of 1 and 2, and the HPLC chromatography of sugar residue of 6) to
this article can be found online at https://.
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Figure 1. Structures of compounds 1–6.

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1
Figure 2. Selected H- 1H COSY and key HMBC correlations of compounds 1−6.

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Figure 3. Thermal ellipsoid representation of 1.

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Table 1. NMR Data for 1−4.
No
1b
2a
3
4
f
δCa, mult.
93.5, CH
47.1, CH
29.4, CH2
δHb (J in Hz)
5.52, mc
4.51, mc
δCa, mult.
92.2, CH
46.1, CH
28.3, CH2
δHb (J in Hz)
5.47, d(2.0)
4.52, mc
δCd, mult.
δHe (J in Hz)
δC , mult.
δHg (J in Hz)
1
3.61, qd(11.3,4.6)
3.87, tdd(9.9,8.0,6.3,4.3)
2.77, dd(13.9,8.7)
2.91, dd(13.8,4.1)
62.6, CH2
3.33, d
63.0, CH2
54.3, CH
2
3.93, q(7.6,7.2)
2.47, s
51.5, CH
31.4, CH2
3a
3b
4
2.85, dd(16.4,7.8)
3.09, dd(16.4,5.5)
2.88, dd(16.1,8.9)
3.06, dd(16.1,5.7)
33.0, CH2
2.69, dd(13.7,6.5)
119.4,qC
113.7,CH
120.5, CH
119.1,qC
129.4, CH
121.3, CH
131.9, qC
125.8, qC
5
6.80-6.83, mc
6.59-6.63, mc
6.80-6.83, mc
7.07, mc
6.83-6.95, mc
7.14, mc
6.83-6.95, mc
157.2, qC
120.2, CH
128.8, CH
147.8, qC
115.3,CH
113.3,CH
6
6.74, td(7.4,1.3)
6.57, d(8.5)
7
8
122.5, CH
145.1, qC
127.9, CH
117.0,CH
7.06, m
6.84, m
7.09, m
6.40, dd(8.5,2.9)
116.9,CH
125.5, CH
167.6, qC
149.5, qC
117.2,CH
164.8, qC
9
139.2, qC
167.8, qC
151.2, qC
166.8, qC
6.50, d(2.9)
10
11
12
13
14
15
16
17
18
19
1'
121.8, CH
147.5, CH
32.3, CH2
29.2, CH2
31.9, CH2
28.5, CH2
28.2, CH2
22.8, CH2
14.2, CH3
5.80, d(15.2)
6.92, mc
2.16, mc
122.5, CH
146.6, CH
32.0, CH2
28.2, CH2
29.1, CH2
29.0, CH2
28.1, CH2
22.6, CH2
14.0, CH3
5.78, dt (15.1, 1.4)
6.83-6.95, mc
2.18, mc
6.06, d(15.3)
6.81, m
5.89, d(15.4)
6.53, m
125.0, CH
144.7, CH
32.5, CH2
28.8, CH2
26.3, CH2
29.3, CH2
64.7, CH2
124.7, CH
142.1, CH
31.2, CH2
28.1, CH2
27.8, CH2
28.4, CH2
25.3, CH2
2.21, m
2.10, m
1.42, mc
1.43, mc
1.50, p (7.3)
1.39, p (7.4)
1.63, p (6.9)
4.03, t (6.7)
1.38, p (7.0)
1.25, mc
1.27, mc
1.25-1.28, mc
1.25-1.28, mc
1.25-1.28, mc
1.25-1.28, mc
0.87, mc
1.24-1.29, mc
1.24-1.29, mc
1.24-1.29, mc
1.24-1.29, mc
0.87, mc
1.26, mc
1.54, q(6.8)
3.97, t (6.7)
28.5, CH2
63.8, CH2
171.1, qC
20.9, CH3
170.4, qC
20.7, CH3
2'
1.98, s
1.99 s
4.70
OH-1
OH-5
8.63
OH-8
NH
8.53
7.73, d(8.0)
7,52, d(6.3)

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c
a Recorded at 125 MHz in CDCl3. b Recorded at 500 MHz in CDCl3. Signals overlapped. d Recorded at 125 MHz in acetone-d6. e Recorded at 500 MHz in acetone-d6.
f Recorded at 125 MHz in DMSO-d6. g Recorded at 500 MHz in DMSO-d6..

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Table 2. NMR Data for 5.
No
5
δCa, mult.
δHb (J in Hz)
2
3
5
167.7, qC
63.2, CH
166.7, qC
4.39, t (4.1)
6
7
8
81.8, CH
30.4, CH
19.0, CH3
3.15, m
2.32, m
0.87, d(7.0)
9
15.5, CH3
36.6, CH2
0.77, d(6.7)
3.13, m
10a
10b
11
12
13
14
15
16
17
3.25, dd(14.3,3.7)
125.5, qC
131.3, CH
116.5, CH
156.9, qC
116.5,CH
131.1, CH
33.0, CH3
6.97, brd(8.5)
6.77, brd(8.5)
6.77, brd(8.5)
6.97, brd(8.5)
3.06, s
a Recorded at 125 MHz in CDCl3. b Recorded at 500 MHz in CDCl3.

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Table 3. NMR Data for 6.
No
6
δCa, mult.
δHb (J in Hz)
1
2
3
4
5
174.8, qC
33.9, CH2
24.8, CH2
28.9, CH2
28.6, CH2
2.16, t (7.4)
1.47, t (6.9)
1.23-1.30, mc
1.34, mc
6
7
8
32.0, CH2
134.8, CH
129.7, CH
2.04, t (7.4)
5.68, m
6.03, dd(15.2,10.5)
9
132.6, CH
131.1, CH
6.18, dd(15.3,10.4)
5.37, dd(15.3,7.8)
10
11
12a
12b
13
14
15
16
17
18
1’
76.7, CH
35.4, CH2
4.13, q(7.1)
1.52, m
1.40, m
30.9, CH2
28.4, CH2
28.8, CH2
24.6, CH2
22.0, CH2
13.9, CH3
99.4, CH
73.7, CH
76.6, CH
79.5, CH
75.5, CH
60.7, CH2
1.23-1.30, mc
1.23-1.30, mc
1.23-1.30, mc
1.23-1.30, mc
1.23-1.30, mc
0.86, t (6.9)
4.06, d(7.8)
2.94, m
2’
3’
3.22, t (8.9)
2.90, t (9.0)
2.99, m
4’
5’
6a’
6b’
3.59, d(10.8)
3.47, d(5.3)
3.40, s
4'-OCH3 59.5, CH3

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a Recorded at 125 MHz in DMSO-d6. b Recorded at 500 MHz in DMSO-d6. c Signals overlapped.