Erinacerins c-l, isoindolin-1-ones with α-glucosidase inhibitory activity from cultures of the medicinal mushroom hericium erinaceus

Article abstract of DOI:10.1021/np5004388

The well-known edible and medicinal mushroom Hericium erinaceus produces various bioactive secondary metabolites. Ten new isoindolin-1-ones, named erinacerins C-L (1-10), together with (E)-5-(3,7-dimethylocta-2,6-dien-1-yl)-4-hydroxy-6-methoxy-2-phenethylisoindolin-1-one (11) were isolated from the solid culture of H. erinaceus. The structures of new metabolites were established by spectroscopic methods. The absolute configurations of 3, 4, 9, and 10 were assigned by comparing their specific rotations with those of related phthalimidines (13-20). Compounds 5 and 6, 7 and 8, and 9 and 10 are double-bond positional isomers. In a α-glucosidase inhibition assay, compounds 2-11 showed inhibitory activity with IC₅₀ values ranging from 5.3 to 145.1 μM. Preliminary structure-activity analysis indicated that the terpenoid side chain and the phenolic hydroxy groups contributed greatly to the α-glucosidase inhibitory activity of 1-11. In a cytotoxicity assay, compound 11 also presented weak cytotoxicity against two cell lines, A549 and HeLa, with IC₅₀ values of 49.0 and 40.5 μM.

Full text of DOI:10.1021/np5004388

Article
pubs.acs.org/jnp
Erinacerins C−L, Isoindolin-1-ones with α‑Glucosidase Inhibitory
Activity from Cultures of the Medicinal Mushroom Hericium erinaceus
Kai Wang,†,‡ Li Bao,† Qiuyue Qi,†,‡ Feng Zhao,§ Ke Ma,† Yunfei Pei,† and Hongwei Liu*,†
†State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, No. 1 Beichenxi Road, Chaoyang
District, Beijing 100101, People’s Republic of China
‡University of Chinese Academy of Sciences, Beijing, 100049, People’s Republic of China
§School of Pharmacy, Yantai University, No. 32 Qingquan Road, Laishan District, Yantai, 264005, People’s Republic of China
S
* Supporting Information
ABSTRACT: The well-known edible and medicinal mushroom Hericium
erinaceus produces various bioactive secondary metabolites. Ten new
isoindolin-1-ones, named erinacerins C−L (1−10), together with (E)-5-
(3,7-dimethylocta-2,6-dien-1-yl)-4-hydroxy-6-methoxy-2-phenethylisoindo-
lin-1-one (11) were isolated from the solid culture of H. erinaceus. The
structures of new metabolites were established by spectroscopic methods.
The absolute configurations of 3, 4, 9, and 10 were assigned by comparing
their specific rotations with those of related phthalimidines (13−20).
Compounds 5 and 6, 7 and 8, and 9 and 10 are double-bond positional
isomers. In a α-glucosidase inhibition assay, compounds 2−11 showed
inhibitory activity with IC50 values ranging from 5.3 to 145.1 μM.
Preliminary structure−activity analysis indicated that the terpenoid side
chain and the phenolic hydroxy groups contributed greatly to the α-
glucosidase inhibitory activity of 1−11. In a cytotoxicity assay, compound
11 also presented weak cytotoxicity against two cell lines, A549 and HeLa, with IC50 values of 49.0 and 40.5 μM.
Hericium erinaceus, also known as lion’s mane mushroom, is an
important edible and medicinal mushroom. The fruiting bodies
and mycelia of this mushroom have been used as an herbal
medicine for the treatment of gastricism and hyperglycemia in
China.1 Aromatic compounds and diterpenoids with various
interesting bioactivities have been isolated from H. erinaceus.
Examples include hericenones C−E2−4 and erinacines A−
K,5−10 possessing stimulatory activity for the biosynthesis of
nerve growth factor, isohericenone and isohericerin with
cytotoxicity,11 (E)-5-(3,7-dimethylocta-2,6-dien-1-yl)-4-hy-
droxy-6-methoxy-2-phenethylisoindolin-1-one and (E)-5-(3,7-
dimethylocta-2,6-dien-1-yl)-4-hydroxy-6-methoxyisoindolin-1-
one with α-glucosidase inhibitory activity,12 and methyl 4-
hydroxy-3-(3-methylbutanoyl)benzoate, 2-chloro-1,3-dime-
thoxy-5-methylbenzene, methyl 4-chloro-3,5-dimethoxylben-
zoate, and 4-chloro-3,5-dimethoxylbenzaldehyde, exhibiting a
protective effect against endoplasmic reticulum stress-depend-
ent cell death.13
the solid culture of mushrooms, such as Flammulina velutipes,18
Pleurotus eryngii,19 Pleurotus cornucopiae,20 and Cyathus
gansuensis.21
During our expeditions to explore the fungal resources of the
Tibet plateau region, a strain of H. erinaceus was isolated from
its fruiting body collected in 2012. This fungus was fermented
on rice and extracted with ethyl acetate to afford the organic
solvent extract. High-performance liquid chromatography
(HPLC) and thin-layer chromatography (TLC) analysis of
this extract indicated the presence of many secondary
metabolites. In order to obtain new bioactive secondary
metabolites from this mushroom, a detailed chemical
investigation was conducted, which led to the isolations of 10
new isoindolin-1-ones (1−10) together with the known
compound (E)-5-(3,7-dimethylocta-2,6-dien-1-yl)-4-hydroxy-
6-methoxy-2-phenethylisoindolin-1-one (11). In this work, we
describe the isolation, structural elucidation, α-glucosidase
inhibitory activity, and cytotoxicity of 1−11.
It is well known that the different culture media can induce
or inhibit different secondary metabolite biosynthetic gene
clusters in the fermentation process of microorganisms.14,15 In
the past 10 years, mushrooms cultured in liquid have been
studied for their pharmacological effects and bioactive
components.16,17 Solid-state fermentation (SSF) refers to the
fermentation process using moist solid substrates in the absence
or near absence of free-flowing water. Recently, we have
separated a number of bioactive secondary metabolites from
RESULTS AND DISCUSSION
■
The ethyl acetate extract of the mushroom H. erinaceus
fermented on rice was separated by silica gel and reversed-
phase C18 column chromatography followed by semipreparative
HPLC to give secondary metabolites 1−11 (Figure 1). The
Received: May 29, 2014
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
DOI: 10.1021/np5004388
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Journal of Natural Products
Article
Figure 1. Structures of compounds 1−20.
structure of the known compound (E)-5-(3,7-dimethylocta-2,6-
dien-1-yl)-4-hydroxy-6-methoxy-2-phenethylisoindolin-1-one
(11) was determined by comparing the experimental NMR
data obtained with literature values.12
150.4, and 156.2), and two carbonyl groups (δC 168.9 and
170.4). The 1H and 13C spectral data of 1 were similar to those
of the known compound 12,12 except for the loss of one
olefinic methyl group and the presence of an additional
carbonyl moiety. All the above data indicate the presence of an
isoindolinone skeleton in 1. The HMBC correlations from H2-3
to C-1, C-3a, C-4, and C-7a, from H-7 to C-1, C-3a, C-5, C-6,
and C-7a, from H2-1′ to C-2′, C-3′, C-4, C-5, and C-6, from
H2-5′ to C-3′, C-4′, C-6′, and C-7′, from H3-9′ to C-2′, C-3′,
and C-4′, and from H3-10′ to C-6′, C-7′, and C-8′ supported
the structure of 1 as shown in Figure 1. The chemical shifts for
C-4 (δC 150.4), C-6 (δC 156.2), and C-8′ (δC 170.4), as well as
the molecular formula of 1, confirmed the substitution of a
hydroxy group at C-4 and C-6 and the presence of a carboxylic
acid group at C-8′. NOE correlations observed between H-2′
Erinacerin C (1) was obtained as a colorless powder. It has a
molecular formula of C18H2NO5, as determined by the
molecular ion peak at m/z 332.1496 [M + H]+ obtained by
1
HRTOFMS. The H, 13C, and HSQC NMR data of 1 showed
resonances due to two olefinic methyl groups (δH 1.71 3H, s;
1.75, 3H, s; δC 12.2 and 16.0), four methylenes (δH 2.01, 2H, t,
J = 7.4 Hz; 2.20, 2H, m; 3.29, 2H, d, J = 6.8 Hz; 4.13, 2H, s; δC
37.9, 26.8, 22.4, 43.0), one aromatic proton (δH 6.62, 1H, s; δC
100.3), two trisubstituted olefins [δH 5.22, 1H, t, J = 6.8 Hz;
6.60, 1H, t, J = 7.5 Hz; δC 123.0 (CH), 133.2, 141.2 (CH),
127.8], five tertiary aromatic carbons (δC 118.5, 121.0, 131.3,
B
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1
Figure 2. Key H−1H COSY, HMBC, and NOESY correlations of 1−10.
and H2-4′ and between H2-5′ and H3-10′ confirmed the E
configuration for two double bonds. On the basis of above
analysis, the structure of 1 was determined as (2E,6E)-8-(4,6-
dihydroxy-1-oxoisoindolin-5-yl)-2,6-dimethylocta-2,6-dienoic
acid.
and H3-4″ (δH 0.99, 3H, d, J = 6.8 Hz) together with the
HMBC correlations from H2-1″ to C-1 (δC 168.8), C-3 (δC
45.3), C-2″ (δC 28.2), C-3″ (δC 19.5), C-4″ (δC 19.0), and C-5″
(δC 172.0), from H-2″ to C-4″ and C-5″, and from H3-3″(4″)
to C-1″ (δC 59.7) and C-2″ confirmed the connection of a 3-
methylbutyric acid moiety at the nitrogen atom (Figure 2).
Compound 2 was isolated as a yellow powder. The molecular
formula of 2 was determined to be C20H25NO6 by the
molecular ion peak at m/z 376.1757 [M + H]+ in its
1
Assignments of the H and 13C signals in 3 were achieved by
detailed analysis of 1H−1H COSY, HSQC, and HMBC spectra
(Figure 2). In the ROESY spectrum of 3, NOE correlations of
H2-1′ with H3-9′, H-2′ with H2-4′, and H2-5′ with H3-10′ were
observed, which determined the E configuration for two double
bonds. On the basis of the above analysis, the structure of 3 was
determined as (2E,6E)-8-(2-(1-carboxy-2-methylpropyl)-4,6-
dihydroxy-1-oxoisoindolin-5-yl)-2,6-dimethylocta-2,6-dienoic
acid. It was named erinacerin E.
Erinacerin F (4) was obtained as a yellow oil. Its molecular
formula of C24H31NO7 was assigned by the molecular ion peak
for [M + H]+ at m/z 446.2170 in its HRTOFMS. Comparison
of the NMR data between 3 and 4 suggested that they had the
same substructure of (2E,6E)-8-(4,6-dihydroxy-1-oxoisoindo-
lin-5-yl)-2,6-dimethylocta-2,6-dienoic acid, with the main
difference at the nitrogen-linked moiety. Specifically, com-
pound 4 exhibited the presence of an additional methylene (δH
1.28, 2H, m; δC 34.2). The HMBC correlations from H-1″ (δH
4.53, 1H, d, J = 9.7 Hz) to C-1 (δC 168.8), C-3 (δC 45.2), C-2″
(δC 24.9), C-3″ (δC 34.2), C-5″ (δC 16.0), and C-6″ (δC 172.1),
from H-2″ (δH 2.24, 2H, m) to C-1″ (δC 58.2), C-3″, C-4″ (δC
10.7), C-5″, and C-6″, from H2-3″ (δH 1.28, 2H, m) to C-1″, C-
2″, C-4″, and C-5″, from H3-4″ (δH 0.84, 3H, s) to C-2″ and C-
3″, and from H3-5″ (δH 0.96, 3H, d, J = 6.7 Hz) to C-1″, C-2″,
1
HRTOFMS. The H and 13C NMR spectra of 2 were quite
similar to those of 1, except for the presence of two extra
methylenes [δH 3.50 (2H, t, J = 5.3 Hz, H2-1″); 3.57 (2H, t, J =
5.3 Hz, H2-2″); δC 48.8 and 59.4] in 2. The coupling constant
between H2-1″ and H2-2″, as well as the HMBC correlations
from H2-1″ to C-1, C-3, and C-2″ and from H2-2″ to C-1″,
established the 2-hydroxyethyl moiety was linked to the
nitrogen atom. NOE correlations of H2-1′ with H3-9′, H-2′
with H2-4′, and H2-5′ with H3-10′ assigned the E configuration
for two double bonds. Thus, the structure of 2 was determined
as (2E,6E)-8-(4,6-dihydroxy-2-(2-hydroxyethyl)-1-oxoisoindo-
lin-5-yl)-2,6-dimethylocta-2,6-dienoic acid. It was designated
as erinacerin D.
A molecular formula of C23H29NO7 was assigned for 3 on the
1
basis of its HRTOFMS and NMR data analysis. The H and
13C NMR NMR and IR data for 3 showed that it was a
structural analogue of 1, sharing the same substructure of
(2E,6E)-8-(4,6-dihydroxy-1-oxoisoindolin-5-yl)-2,6-dimethyloc-
ta-2,6-dienoic acid. Compound 3 differs from 1 in the nitrogen-
1
linked moiety. The H−1H COSY correlations between H-1″
(δH 4.45, d, J = 8.1 Hz) and H2-2″ (δH 2.22, 2H, m), between
H-2″ and H3-3″ (δH 0.82, 3H, d, J = 6.8 Hz), and between H-2″
C
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Scheme 1. Hypothetical Biogenetic Pathway of 1−11.
and C-3″ indicated the linkage of a 1-carboxy-2-methylbutyl
moiety at the nitrogen atom. NOE correlations of H2-1′ with
H3-9′, H-2′ with H2-4′, and H2-5′ with H3-10′ confirmed the E
configuration for two double bonds. Therefore, the structure of
4 was concluded as (2E,6E)-8-(2-(1-carboxy-2-methylbutyl)-
4,6-dihydroxy-1-oxoisoindolin-5-yl)-2,6-dimethylocta-2,6-di-
enoic acid.
dihydroxy-2-(2-hydroxyethyl)-1-oxoisoindolin-5-yl)-4-methyl-
hex-3-enoate (Figure 2). Compounds 7 and 8 are a pair of
double-bond positional isomers. They were designated as
erinacerins I and J, respectively.
Erinacerins K (9) and L (10) were isolated as yellow oils.
They possessed the same molecular formula of C26H29NO7, as
determined by the HRTOFMS and NMR data analysis. In the
1H and 13C NMR spectra of 9, signals corresponding to the
substructure of (E)-methyl 6-(4,6-dihydroxy-1-oxoisoindolin-5-
yl)-4-methylhex-4-enoate moiety, a monosubstituted aromatic
ring [δH 7.23 (4H, overlapped); 7.16 (1H, m); δC 126.6, 128.4
(two carbons), 128.6 (two carbons), 137.1], a methoxyl group
(δH 3.66, 3H, s; δC 52.3), a methine group (δH 5.15, 1H, m; δC
54.7), a methylene [δH 3.14, 1H, dd, J = 11.0, 14.5 Hz, 3.37
(overlapped); δC 34.7], and an ester ketone (δC 170.9) were
The same molecular formula of C16H19NO5 was determined
for erinacerin G (5) and erinacerin H (6) by HRTOFMS data
analysis. The 1H and 13C NMR spectra of 5 and 6 indicated the
presence of a 5-substituted-4,6-dihydroxyisoindolin-1-one moi-
ety in their structures. A comparison of NMR data of 5 and 6
with those obtained for 1 revealed the presence of an extra
methoxyl group (5: δH 3.52, 3H, s; δC 51.6; 6: δH 3.59, 3H, s;
δC 51.9) and the loss of a trisubstituted double bond and a
methyl group, which indicated the structural variation in the
side chain located at C-5 in the benzene ring for these two
compounds. The important HMBC correlations of H2-1′ with
C-4, C-5, C-6, C-2′, and C-3′; H-2′ with C-5, C-1′, C-3′, C-4′,
and C-7′; H2-4′ with C-2′, C-3′, C-5′, C-6′, and C-7′; H2-5′
with C-3′, C-4′, and C-6′; and H3-8′ with C-6′ (Figure 2),
together with the NOE correlations of H-2′ with H2-4′ and H2-
1′ with H3-7′ (Figure 2), assigned the structure of 5 as (E)-
methyl 6-(4,6-dihydroxy-1-oxoisoindolin-5-yl)-4-methylhex-4-
enoate. Similarly, a detailed interpretation of the HMBC and
ROESY spectra of 6 determined its structure as (E)-methyl 6-
(4,6-dihydroxy-1-oxoisoindolin-5-yl)-4-methylhex-3-enoate
(Figure 2). Compounds 5 and 6 are a pair of double-bond
positional isomers.
1
present. Furthermore, the H−1H COSY and HMBC data
analysis supported the presence of a methyl 3-phenyl-
propanoate moiety in 9. The HMBC correlations from H-1″
to C-1 and C-3 confirmed the connectivity of the methyl 3-
phenylpropanoate moiety with the nitrogen atom in 9 (Figure
2). NOE correlations of H-2′ with H2-4′ and of H-1′ with H3-7′
assigned the E configuration for the double bond. Thus, the
structure of 9 was established as described in Figure 1. In a
similar way, compound 10 was determined as shown by analysis
of its 2D NMR data (Figure 2).
To determine the absolute configuration in compounds 3, 4,
9, and 10, phthalimidines 13−20 were synthesized by the
reaction of o-phthalaldehyde with the α-amino acid (L-valine, D-
valine, L-phenylalanine, D-phenylalanine, D-isoleucine, L-iso-
leucine, D-alloisoleucine, and L-alloleucine).22 The absolute
configurations at C-1″ in compounds 3, 9, and 10 were
determined by comparsion of their specific optical rotations
with those of the synthetic phthalimidines 13−16. Compounds
3, 9, and 10 showed negative specific optical rotations,
indicating the S configuration at C-1″ in compounds 3, 9,
and 10. The chemical shifts of the amino acid moiety in 4 were
consistent with those of 17 and 18. The stereochemistry in 4
was established as 1″S and 2″S by comparing its specific optical
rotation (−20.1) with that of 17 (−40.3) and 18 (+58.09).
The possible biogenetic pathway of 1−11 is proposed as
shown in Scheme 1. The key presumed intermediate orsellicic
acid (21) is biosynthesized from the acetyl malonyl pathway.
Taking the biosynthesis of compound 1 as an example,
compound 21 could be oxidized into 3,5-dihydroxyphthalic
acid (22), which subsequently reacts with geranyl pyrophos-
phate (GerPP) and ammonia to give (E)-5-(3,7-dimethylocta-
2,6-dien-1-yl)-4,6-dihydroxyisoindoline-1,3-dione (23). Com-
Compounds 7 and 8 were assigned the same molecular
formula of C18H23NO6 on the basis of their HRTOFMS and
1
NMR data analysis. The H and 13C NMR spectra of 7 were
very similar to those of compound 5, with the exception of the
presence of two additional methylenes (δH 3.69, 2H, t, J = 5.4
Hz, H2-1″; 3.79, 2H, t, J = 5.4 Hz, H2-2″; δC 46.3 and 61.3).
1
The H−1H COSY between H2-1″ and H2-2″ in combination
with the HMBC correlations from H2-1″ to C-1, C-3, and C-2″
and from H2-2″ to C-1″ confirmed the linkage of the 2-
hydroxyethyl moiety at the nitrogen atom. The NOE
correlations of H-2′ with H2-4′ and of H-1′ with H3-7′
determined the E configuration for the double bond between
C-2′ and C-3′. Accordingly, the structure of 7 was elucidated as
(E)-methyl 6-(4,6-dihydroxy-2-(2-hydroxyethyl)-1-oxoisoindo-
1
lin-5-yl)-4-methylhex-4-enoate. Compound 8 has similar H
and 13C NMR spectra to those of compound 6, except for two
extra methylenes. The interpretation of HMBC and ROESY
spectra of 8 assigned its structure as (E)-methyl 6-(4,6-
D
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1
Table 1. H and 13C NMR Data of Compounds 1−4
1 (DMSO-d6)
δH (m, J in Hz)
2 (DMSO-d6)
δH (m, J in Hz)
3 (DMSO-d6)
δH (m, J in Hz)
4 (DMSO-d6)
δH (m, J in Hz)
no.
δC
δC
δC
δC
1
2
3
168.9
167.8
168.3
168.3
8.27 s
4.13 s
43.0
131.3
150.4
121.0
156.2
100.3
118.5
22.4
44.7
131.3
150.1
119.0
156.1
100.3
118.5
22.4
4.32 s
45.3
130.0
150.3
119.0
156.4
100.5
119.1
22.5
4.42 d (17.0); 4.24 d (17.0)
45.2
130.0
150.2
119.0
156.4
100.5
119.1
22.4
4.45 d (17.0); 4.22 d (17.0)
3a
4
5
6
7
6.62 s
6.63 s
6.65 s
6.66 s
7a
1′
2′
3′
4′
3.29 d (6.8)
5.22 t (6.8)
3.29 d (6.9)
5.21 t (6.9)
3.30 d (7.1)
5.21 t (7.1)
3.30 d (7.1)
5.22 t (7.1)
123.0
133.2
37.9
123.0
133.2
37.9
122.8
133.3
37.9
122.8
133.3
37.9
2.01 t (7.4)
2.20 m
2.01 t (7.3)
2.20 m
2.00 t (7.4)
2.19 m
2.01 t (7.4)
2.20 m
5′
26.8
26.9
26.9
26.9
6′
7′
8′
9′
10′
1″
2″
3″
4″
5″
6″
4-OH
6-OH
141.2
127.8
170.4
16.0
6.60 t (7.5)
141.2
127.8
169.0
16.0
6.60 t (7.4)
141.3
127.7
168.8
16.0
6.60 t (7.3)
141.3
127.7
168.8
15.7
6.61 t (7.3)
1.75 s
1.71 s
1.75 s
1.74 s
1.75 s
12.2
12.2
1.71 s
12.2
1.70 s
12.2
1.71 s
48.8
3.50 t (5.3)
3.57 t (5.3)
59.7
4.45 d (8.1)
2.22 m
58.2
4.53 d (9.7)
2.24 m
59.4
28.2
24.9
19.5
0.82 d (6.7)
0.99 d (6.7)
34.2
1.28 m
19.0
10.7
0.84 t (7.0)
0.96 d (6.7)
172.0
16.0
172.1
9.21 s
9.48 s
9.26 s
9.50 s
9.31 s
9.57 s
9.31 s
9.58 s
pound 23 is transformed into isoindolin derivative 24 by
reduction. Finally, compound 1 was synthesized from 24 by
oxidation. Compounds 2−11 may be biosynthesized from 21
by the same route as that proposed for 1.
Isoindolin-1-ones incorporating a terpenoid moiety are a
group of fungal metabolites isolated from the fungus of H.
erinaceus12 and Stachybotrys species.23−26 They have been
reported to possess various interesting bioactivities, such as
inhibition of thromboxane A2-induced vasoconstriction,23
inhibitory activity against α-glucosidase,12 inhibition of tyrosine
kinase,24 inhibition of HIV-1 protease,25 and antichloester-
olemic, anti-inflammatory, and cytotoxic activities.26
activities than 1, suggesting that the length and stereochemistry
of the terpenoid side chain on the benzene ring also influence
the activity.
To evaluate the anticancer activity for all isolates, we tested
the cytotoxicity of compounds 1−11 and 13−17 toward two
cancer cell lines, human lung adenocarcinoma cell line A549
and cervical cancer cell line HeLa. As seen in Table 4, the
known compound 11 presented much stronger activity against
the growth of A549 (IC50 = 49.0 μM) and HeLa (IC50 = 40.5
μM) than the other isolates. Compounds 8, 9, 13, and 14
showed weak cytotoxic activity against A549 with IC50 values of
96.1, 89.7, 89.7, and 41.5 μM, respectively.
In summary, 10 new isoindolin-1-ones (1−10) with α-
glucosidase inhibitory activity were obtained from the solid
culture of the medicinal mushroom H. erinaceus, which
supported the medicinal value of this mushroom and expanded
the chemistry of isoindolin-1-ones. The known compound 11
that was previously isolated from the fruiting bodies of H.
erinaceus was found to possess strong α-glucosidase inhibitory
activity and weak cytotoxicity. On the basis of the current work
and our previous publications,18−21 the solid-state fermentation
technique is demonstrated as a useful method to stimulate the
biosynthesis of secondary metabolites in edible and medicinal
mushrooms.
The crude extracts of H. erinaceus have been reported to
possess antihyperglycemic effects.27 In this report, we utilized
an in vitro α-glucosidase inhibition assay to evaluate the
antihyperglycemic effect of compounds 1−11. The known
compound 11 showed the strongest inhibitory activity with an
IC50 of 5.3 μM, which was consistent with the early report.12
Compound 1 showed weak inhibition against α-glucosidase
with an IC50 value larger than 200 μM. New isoindolin-1-ones
2−10 exhibited α-glucosidase inhibitory activity with IC50
values of 24.2, 12.5, 39.6, 145.1, 10.3, 97.8, 18.6, 15.5, and
19.8 μM, respectively (Table 4). On the basis of the above
activity data, a preliminary structure−activity relationship was
deduced. The substitution on the nitrogen atom can increase
the inhibitory activity, as indicated by comparing the inhibitory
activity of 1 with that of 2−4. Compounds 3, 4, 9, and 10
showed stronger inhibitory activity than compounds 13−17,
which indicated the significance of the terpenoid side chain and
the phenolic hydroxy groups for the α-glucosidase inhibitory
activity. Compounds 5 and 6 showed stronger inhibitory
EXPERIMENTAL SECTION
■
General Experimental Procedures. Solvents used for extraction
and chromatographic separation were analytical grade. TLC was
carried out on silica gel HSGF254, and the spots were visualized by
spraying with 10% H2SO4 and heating. Silica gel (Qingdao Haiyang
Chemical Co., Ltd., People’s Republic of China) and Sephadex LH-20
E
DOI: 10.1021/np5004388
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
(Amersham Biosciences) were used for column chromatography.
HPLC separation was performed on an Agilent 1200 HPLC system
using an ODS column (C18, 250 × 9.4 mm, YMC Pak, 5 μm; detector:
UV) with a flow rate of 2.0 mL/min. UV and IR spectral data were
acquired using a ThermoGenesys-10S UV−vis and Nicolet IS5FT-IR
spectrophotometer, respectively. Specific rotations were recorded on a
PerkinElmer 241 polarimeter. NMR spectral data were obtained with a
Bruker Avance-500 spectrometer (DMSO-d6, δH 2.50/δC 40.0;
methanol-d4, δH 3.30/δC 49.9). The HSQC and HMBC experiments
were optimized for 145.0 and 8.0 Hz, respectively. HR-TOF-MS data
were measured using an Agilent Accurate-Mass-Q-TOF LC/MS 6520
instrument.
Fungal Material. The strain used in this work was isolated from
the fruiting body of H. erinaceus and identified by one of the authors
(Y.P.). The fungus was identified on the basis of the DNA sequences
of the ITS1-5.8S-ITS2 regions of their rRNA gene. The ITS gene
sequence obtained in this study (accession number: KJ627741)
showed 99% homology with that of the fungus H. erinaceus in
GenBank with the accession number GU566758. H. erinaceus was
cultured on slants of potato dextrose agar at 25 °C for 10 days. Agar
plugs were inoculated in 500 mL Erlenmeyer flasks containing 120 mL
of media (0.4% glucose, 1% malt extract, and 0.4% yeast extract; the
final pH of the media was adjusted to 6.5 before sterilization) and
incubated at 25 °C on a rotary shaker at 170 rpm for 1 week. The
scale-up fermentation was carried out in 30 500 mL Fernbach culture
flasks each containing 80 g of rice and 120 mL of distilled water. Each
flask was inoculated with 5.0 mL of the culture medium and incubated
at 25 °C for 40 days.
1464, 1349, 1204, 1142, 1061, 833, 800, 735, 612 cm−1; for 1H and 13
C
1
NMR data measured in DMSO-d6 see Table 1; for H and 13C NMR
data measured in CD3OD see Table S1 in the Supporting Information;
positive HRTOFMS m/z [M + H]+376.1757 (calcd for C20H26NO6,
376.1755).
Erinacerin E (3): yellow oil; [α]2D5 −19.5 (c 1.0, methanol); UV
(methanol) λmax nm (log ε) 213 (4.63), 262 (4.29), 305 (2.12); IR
(neat) νmax 3222, 2965, 2925, 1673, 1462, 1393, 1358, 1203, 1024
cm−1; for 1H and 13C NMR data see Table 1; positive HRTOFMS m/
z [M + H]+ 432.2021 (calcd for C23H30NO7, 432.2017).
Erinacerin F (4): yellow oil; [α]2D5 −20.1 (c 0.6, methanol);
UV(methanol) λmax nm (log ε) 213 (4.63), 262 (4.29), 305 (2.12); IR
(neat) νmax 3210, 2938, 2920, 1675, 1463, 1350, 1222, 1020, 853, 750,
620 cm−1; for 1H and 13C NMR data see Table 1; positive HRTOFMS
m/z [M + H]+ 446.2170 (calcd for C24H34NO7, 446.2173).
Erinacerin G (5): colorless oil; UV (methanol) λmax nm (log ε) 213
(4.63), 262 (4.29), 305 (2.12); IR (neat) νmax 3273, 2948, 2842, 1680,
1
1470, 1328, 1240, 1020, 856, 789, 610 cm−1; for H and 13C NMR
data see Table 2; positive HRTOFMS m/z [M + H]+ 306.1338 (calcd
for C16H20NO5, 306.1336).
1
Table 2. H and 13C NMR Data of Compounds 5−7
5 (DMSO-d6)
6 (DMSO-d6)
7 (CD3OD)
δH (m, J in
Hz)
δH (m, J in
Hz)
δH (m, J in
position
δC
δC
δC
Hz)
1
170.9
170.9
171.4
3
43.4 4.14 s
131.8
43.4 4.13 s
131.8
50.6 4.43 s
132.0
Extraction and Isolation. The fermented rice substrate was
extracted repeatedly with EtOAc (3 × 4L), and the organic solvent was
evaporated to dryness under vacuum to afford the crude extract (19.6
g). The residue (18.0 g) was subjected to silica gel column
chromatography (CC) using hexane−ethyl acetate in a gradient
elution (v/v, 100:0, 100:2, 100:5, 100:8, 100:10, 100:15, 100:20,
100:40, 100:50), followed by dichloromethane−methanol elution (v/
v, 100:1, 100:2, 100:3, 100:5, 100:10, 100:15, 100:20, 0:100) to give
20 fractions (HE-1−HE-20).
3a
4
150.8
151.0
151.6
5
118.8
119.6
121.3
6
156.6
156.7
158.3
7
100.7 6.63 s
121.4
100.7 6.63 s
121.4
101.8 6.74 s
120.8
7a
1′
22.9 3.28 d
(7.1)
22.5 2.69 t
(7.1)
23.6 3.40 d
(7.1)
Fraction 14 (1.45 g), eluted with dichloromethane−methanol (v/v,
100:2), was further separated on Sephadex LH-20 CC eluted with 50%
methanol in water to give 20 subfractions (HE-14-1−HE-14-20).
Fraction HE-14-12 (89 mg) was further separated by Sephadex LH-20
CC eluted with 50% methanol in water to afford compounds 3 (7 mg)
and 4 (5.4 mg). Compound 11 (12.1 mg, tR 40 min) was obtained
from fraction HE-14-16 (45.2 mg) by RP-HPLC using 65% methanol
in water. Fraction HE-19 (3.45g), eluted with dichloromethane−
methanol (v/v, 100:5), was first separated by ODS CC using a
gradient of methanol−water (20−100%) to afford 30 subfractions
(HE-19-1−HE-19-30). Compounds 1 (82 mg, tR 35.3 min) and 2 (5.3
mg, tR 34.2 min) were obtained from fraction HE-19-15 (213.5 mg) by
RP-HPLC using 30% acetonitrile in water. Fraction HE-19-12 (51.2
mg) was subjected to Sephadex LH-20 CC eluted with 50% methanol
in water to afford four subfractions (HE-19-12-1−HE-19-12-4).
Compounds 5 (3.6 mg, tR 36.3 min) and 6 (1.2 mg, tR 35.5 min)
were obtained from subfraction HE-19-12-4 (12.5 mg) by RP-HPLC
using 22% acetonitrile in water. Compounds 7 (5.1 mg, tR 26.5 min)
and 8 (2.8 mg, tR 25.7 min) were purified from HE-19-12-3 (13.1 mg)
by RP-HPLC using 22% acetonitrile in water. Subfraction HE-19-16
(21.5 mg) was separated by RP-HPLC using 40% acetonitrile in water
to afford compounds 9 (5.2 mg, tR 40.6 min) and 10 (1.8 mg, tR 42.1
min). The physical properties and spectroscopic data of the new
compounds are as follows.
2′
123.6 5.20 t
(7.1)
38.6 2.10 t
(7.1)
124.6 5.28 t
(7.1)
3′
4′
132.9
139.2
134.2
34.7 2.17 t
(7.6)
116.0 5.26 t
(7.0)
36.0 2.26 t
(7.5)
5′
32.8 2.35 t
(7.6)
33.4 3.06 d
(7.0)
33.9 2.39 t
(7.5)
6′
7′
8′
1″
173.5
172.5
175.1
16.3 1.73 s
51.6 3.52 s
16.8 1.67 s
51.9 3.59 s
16.1 1.80 s
51.9 3.56 s
46.3 3.69 t
(5.4)
2″
61.3 3.79 t
(5.4)
4-OH
6-OH
NH
9.25 s
9.51 s
8.28 s
9.18 s
9.47 s
8.27 s
Erinacerin H (6): colorless oil; UV (methanol) λmax nm (log ε) 213
(4.63), 262 (4.29), 305 (2.12); IR (neat) νmax 3270, 2948, 2832, 1667,
1470, 1340, 1246, 1018, 1007, 880, 790, 625 cm−1; for H and 13C
1
NMR data see Table 2; positive HRTOFMS m/z [M + H]+ 306.1338
(calcd for C16H20NO5, 306.1336).
Erinacerin C (1): colorless powder; UV (methanol) λmax nm (log
ε) 213 (4.63), 262 (4.29), 305 (2.12); IR (neat) νmax 3272, 2924,
2855, 1685, 1604, 1460, 1381, 1271, 1164, 1060, 825, 738, 600 cm−1;
for 1H and 13C NMR data measured in DMSO-d6 see Table 1; for 1H
and 13C NMR data measured in CD3OD see Table S1 in the
Supporting Information; positive HRTOFMS m/z [M + H]+ 332.1498
(calcd for C18H22NO5, 332.1496).
Erinacerin I (7): colorless powder; UV (methanol) λmax nm (log ε)
213 (4.63), 262 (4.29), 305 (2.12); IR (neat) νmax 3270, 2940, 2854,
1711, 1658, 1605, 1465, 1348, 1304, 1205, 1164, 1061, 860, 788, 620
cm−1; for 1H and 13C NMR data see Table 2; positive HRTOFMS m/
z [M + H]+ 350.1593 (calcd for C18H24NO6, 350.1598).
Erinacerin J (8): yellow oil; UV (methanol) λmax nm (log ε) 213
(4.63), 262 (4.29), 305 (2.12); IR (neat) νmax 3267, 2952, 2860, 1659,
1459, 1364, 1300, 1240, 870, 756 cm−1; for 1H and 13C NMR data see
Erinacerin D (2): yellow powder; UV (methanol) λmax nm (log ε)
213 (4.63), 262 (4.29), 305 (2.12); IR (neat) νmax 3306, 2928, 1673,
F
DOI: 10.1021/np5004388
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
1
Table 3. H and 13C NMR Data of Compounds 8−10
8 (DMSO-d6)
9 (DMSO-d6)
δH (m, J in Hz)
10 (DMSO-d6)
δH (m, J in Hz)
no.
δC
δH (m, J in Hz)
δC
δC
1
3
167.8
48.8
168.1
45.5
168.6
46.0
4.33 s
4.14 d (16.6); 4.23 d (16.6)
4.16 d (16.6); 4.24 d (16.6)
3a
131.4
150.2
119.0
156.3
100.3
119.1
22.1
130.0
150.2
119.0
156.4
100.5
119.2
22.4
130.5
150.8
120.4
156.9
100.9
119.4
22.5
4
5
6
7
6.64 s
6.58 s
6.60 s
7a
1′
2′
3′
4′
2.69 t (7 0.0)
2.10 t (7.0)
3.25 d (8.1)
2.67 t (7,0)
2.08 t (7.0)
b
38.2
122.9
132.7
32.3
5.15
38.5
139.0
115.5
33.0
139.1
116.0
33.4
5.26 t (7.0)
3.05 d (7.0)
2.33 t (7.6)
2.15 t (7.6)
5.22 t (6.5)
3.05 d (6.5)
5′
34.3
6′
7′
8′
1″
172.1
16.3
173.0
15.9
172.5
16.8
1.66 s
1.71 s
3.49 s
1.66 s
a
51.5
3.58
51.1
51.9
3.58 s
b
44.7
3.50 t (5.5)
54.7
5.15
55.2
5.16 dd (6.0, 11.0)
a
c
c
2″
59.5
3.58
34.7
3.14 dd (11.2, 14.5); 3.37
35.2
3.16 dd (11.0, 14.5) 3.37
3″
137.1
128.6
128.4
126.6
170.9
52.3
137.5
128.9
129.0
127.1
171.4
52.8
d
e
4″, 8″
5″, 7″
6″
7.23
7.24
d
e
7.23
7.24
7.16 m
7.17 m
9″
10″
4-OH
6-OH
3.66 s
9.28 s
9.57 s
3.66 s
9.26 s
9.58 s
9.23 s
9.49 s
a,b,d,e
c
Signals overlapped with each other. Signals overlapped with residual H2O signal.
Table 3; positive HRTOFMS m/z [M + H]+ 350.1595 (calcd for
C18H24NO6, 350.1598).
positive HRTOFMS m/z [M + H]+ 468.2017 (calcd for C26H30NO7,
468.2017).
Erinacerin L (10): yellow oil; [α]2D5 −65.3 (c 1.2, methanol); UV
(methanol) λmax nm (log ε) 213 (4.63), 262 (4.29), 305 (2.12); IR
(neat) νmax 3275, 2923, 2863, 1659, 1464, 1325, 1298, 1220, 1154,
Erinacerin K (9): yellow oil; [α]2D5 −65.4 (c 1.2, methanol); UV
(methanol) λmax nm (log ε) 213 (4.63), 262 (4.29), 305 (2.12); IR
(neat) νmax 3270, 2952, 2925, 1737, 1667, 1459, 1355, 1295, 1210,
1
1
880, 728, 628 cm−1; for H and 13C NMR data see Table 3; positive
1153, 875, 740, 615 cm−1; for H and 13C NMR data see Table 3;
HRTOFMS m/z [M + H]+ 468.2019 (calcd for C26H30NO7,
468.2017).
Synthesis of Phthalimidines (13−20).22 L-Valine (2 mmol) was
added into a solution of phathalic dicarboxaldehyde (2 mmol) in 30
mL of acetonitrile. The mixture was heated under reluxing for 16 h;
then the reaction mixture was filtered while hot. The solvent was
allowed to cool to yield the desired phathalimidine 13 as crystalline
solids (0.42 g). Using the same method, compounds 14 (0.40 g), 15
(0.52 g), 16 (0.48 g), 17 (0.46 g), 18 (0.23 g), 19 (0.15 g), and 20
(0.23 g) were synthesized from L-valine, D-valine, L-phenylalanine, D-
phenylalanine, D-isoleucine, L-isoleucine, D-alloisoleucine, and L-
alloleucine, respectively.
Table 4. α-Glucosidase Inhibtory and Cytotoxic Activity of
Compounds 1−11 and 13−17
cytotoxic activity
α-glucosidase inhibition (IC50,
μM)
A549 (IC50,
HeLa (IC50,
compound
μM)
μM)
1
>200
24.2
>100
>100
>100
>100
>100
>100
>100
96.1
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
40.5
2
3
12.8
4
39.6
Compound 13: colorless crystalline; [α]D25 −41.8 (c 1.0,
methanol); IR (neat) νmax 1654,1434, 1320, 1229, 1124, 860, 732,
624 cm−1; 1H NMR (500 MHz, DMSO-d6) 13.01 (1H, s, H-5″), 7.71
(1H, d, J = 7.4 Hz, H-7), 7.63 (1H, d, J = 7.4 Hz, H-4), 7.62 (1H, m,
H-5), 7.50 (1H, m, H-6), 4.63 (1H, d, J = 17.5 Hz, H-3), 4.53 (1H, d, J
= 17.5 Hz, H-3), 4.51 (1H, d, J = 6.3 Hz, H-1″), 2.28 (1H, m, H-2″),
1.01 (3H, d, J = 6.7 Hz, H-3″), 0.83 (3H, d, J = 6.7 Hz, H-4″); 13C
NMR (125 MHz, DMSO-d6) 172.3 (C-5″), 168.4 (C-1), 142.6 (C-
3a), 132.1 (C-5), 131.8 (C-7a), 128.4 (C-6), 124.1 (C-4), 123.5 (C-7),
60.2 (C-1″), 47.7 (C-3), 28.6 (C-2″), 19.9 (C-3″), and 19.6 (C-4″);
positive HRTOFMS m/z [M + H]+ 234.1128 (calcd for C13H15NO3,
234.1125).
5
145.1
10.3
6
7
97.8
8
18.6
9
15.5
89.7
10
19.8
>100
49.0
11
5.3
13
>200
>200
>200
>200
>200
acarbose
382.7
89.7
>100
>100
>100
>100
>100
cisplatin
14.4
14
41.5
15
>100
>100
>100
cisplatin
12.6
16
Compound 14: colorless crystalline; [α]2D5 +40.9 (c1.0, methanol);
the IR and 1H and 13C NMR data of 14 are identical with those of 13;
positive HRTOFMS m/z [M + H]+ 234.1130 (calcd for C13H15NO3,
234.1125).
17
positive
control
G
DOI: 10.1021/np5004388
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
Compound 15: yellow crystalline; [α]2D5 −75.1 (c 1.0, methanol);
IR (neat) νmax 1664, 1460, 1324, 1220, 1124, 864, 632 cm−1; 1H NMR
(500 MHz, DMSO-d6) 13.16 (1H, s, H-9″), 7.62 (1H, d, J = 7.1 Hz,
H-7), 7.58 (1H, m, H-4), 7.56 (1H, m, H-5), 7.45 (1H, t, J = 7.1 Hz,
H-6), 7.23 (4H, m, H-4″, H-5″, H-7″, H-8″), 7.14 (1H, t, J = 7.1 Hz),
5.14 (1H, dd, J = 11.4, 4.7 Hz, H-1″), 4.44 (2H, s, H-3), 3.40 (1H, dd,
J = 14.7, 4.7 Hz, H-2″), 3.21 (1H, m, H-2″); 13C NMR (125 MHz,
DMSO-d6) 172.0 (C-9″), 167.8 (C-1), 137.5 (C-3″), 131.7 (C-5),
131.6 (C-7a), 128.5 (C-4″, C-8″), 128.4 (C-5″, C-7″), 127.9 (C-6),
126.5 (C-6″), 123.5 (C-7), 122.9 (C-4), 54.8 (C-1″), 47.4 (C-3), 34.7
(C-2″); positive HRTOFMS m/z [M + H]+ 282.1122 (calcd for
C17H15NO3, 282.1125).
AUTHOR INFORMATION
■
Corresponding Author
*Tel/Fax: +86-10-62566577. E-mail: liuhw@im.ac.cn.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Financial support of the Ministry of Science and Technology of
China (2014CB138304) and the National Natural Science
Foundation (21472233) is acknowledged. Dr. J. Ren and Dr.
W. Wang (State Key Laboratory of Mycology, Institute of
Microbiology, Chinese Academy of Sciences) are appreciated
for their help in measuring the NMR and MS data.
Compound 16: yellow crystalline; [α]2D5 +91.0 (c 1.0, methanol);
the IR and 1H and 13C NMR data of 16 are identical with those of 15;
positive HRTOFMS m/z [M + H]+ 282.1130 (calcd for C17H15NO3,
282.1125).
Compound 17: colorless crystalline; [α]D25 −40.3 (c 1.0,
methanol); IR (neat) νmax 1665,1464, 1324, 1256, 1210, 728, 628
cm−1; 1H NMR (500 MHz, DMSO-d6) 13.01 (1H, s, H-5″), 7.71 (1H,
d, J = 7.4 Hz, H-7), 7.63 (1H, d, J = 7.4 Hz, H-4), 7.62 (1H, m, H-5),
7.50 (1H, m, H-6), 4.65 (1H, d, J = 17.5 Hz, H-3), 4.48 (1H, d, J =
17.5 Hz, H-3), 4.61 (1H, d, J = 9.7 Hz, H-1″), 2.11 (1H, m, H-3″),
1.33 (1H, m, H-2″), 1.09 (1H, m, H-2″), 0.98 (3H, d, J = 6.7 Hz, H-
5″), 0.83 (3H, t, J = 7.4 Hz, H-4″); 13C NMR (125 MHz, DMSO-d6)
172.0 (C-5″), 167.9 (C-1), 142.5 (C-3a), 131.7 (C-5), 131.3 (C-7a),
128.0 (C-6), 123.6 (C-4), 123.0 (C-7), 58.3 (C-1″), 47.2 (C-3), 34.2
(C-3″), 25.0 (C-2″), 15.7 (C-5″), 10.6 (C-4″); positive HRTOFMS
m/z [M + H]+ 248.1280 (calcd for C14H17NO3, 248.1281).
Compound 18: colorless crystalline; [α]D25 +58.09 (c 1.0,
methanol); the IR and 1H and13C NMR data of 18 are identical
with those of 17; positive HRTOFMS m/z [M + H]+ 248.1282 (calcd
for C14H17NO3, 248.1281).
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Compound 19: colorless crystalline; [α]2D5 +17.8 (c 1.0, methanol);
IR (neat) νmax 1665, 1423, 1310, 1256, 1220, 725, 625 cm−1; 1H NMR
(500 MHz, DMSO-d6) 13.01 (1H, s, H-5″), 7.71 (1H, d, J = 7.4 Hz,
H-7), 7.63 (1H, d, J = 7.4 Hz, H-4), 7.62 (1H, m, H-5), 7.50 (1H, m,
H-6), 4.71 (1H, d, J = 9.7 Hz, H-1″), 4.64 (1H, d, J = 17.5 Hz, H-3),
4.51 (1H, d, J = 17.5 Hz, H-3), 2.11 (1H, m, H-3″), 1.33 (1H, m,, H-
2″), 1.09 (1H, m, H-2″), 0.92 (3H, t, J = 7.4 Hz, H-4″), 0.86 (3H, d,
J=6.7 Hz, H-5″); 13C NMR (125 MHz, DMSO-d6) 172.1 (C-5″),
168.1 (C-1), 142.2 (C-3a), 131.7 (C-5), 131.2 (C-7a), 127.9 (C-6),
123.6 (C-4), 123.0 (C-7), 57.8 (C-1″), 47.7 (C-3), 34.6 (C-3″), 25.8
(C-2″), 15.6 (C-4″), 11.1 (C-5″); positive HRTOFMS m/z [M + H]+
248.1285 (calcd for C14H17NO3, 248.1281).
Compound 20: colorless crystalline; [α]D25 −65.89 (c 1.0,
methanol); the IR and 1H and13C NMR data of 20 are identical
with those of 19; positive HRTOFMS m/z [M + H]+ 248.1282 (calcd
for C14H17NO3, 248.1281).
α-Glucosidase Inhibitory Assay. As described in our early
work,28 the bioassay was conducted using a 96-well plate, and the
absorbance was determined at 405 nm using a Spectra Max 190
microplate reader (Molecular Devices Inc.). The control was prepared
by adding phosphate buffer instead of the sample in the same way as
the test. The blank was prepared by adding phosphate buffer instead of
the α-glucosidase. The inhibition rates (%) = [(ODcontrol
−
ODcontrol blank) − (ODtest − ODtest blank)]/(ODcontrol − ODcontrol blank
)
× 100%. Acarbose was utilized as the positive control with an IC50 of
382.7 μM.
Cytotoxicity Assay. The cytotoxicity against A549 and HeLa cell
lines of compounds 1−11 and 13−17 was tested using the MTT
method as previously reported.20
Statistical Analysis. The bioactivity values were expressed as
means of three independent experiments, and each was carried out in
triplicate.
(23) Ogawa, K.; Nakamura, M.; Hayashi, M.; Yaginuma, S.;
Yamamoto, S.; Furihata, K.; Shinya, K.; Seto, H. J. Antibiot. 1995,
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(24) Roggo, B. E.; Petersen, F.; Sills, M.; Roesel, J. L.; Moerker, T.;
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ASSOCIATED CONTENT
■
S
* Supporting Information
NMR spectral data of compounds 1−10. This material is
available free of charge via the Internet at http://pubs.acs.org.
H
DOI: 10.1021/np5004388
J. Nat. Prod. XXXX, XXX, XXX−XXX

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I
DOI: 10.1021/np5004388
J. Nat. Prod. XXXX, XXX, XXX−XXX