Article

Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX pubs.acs.org/jnp

Nonadride and Spirocyclic Anhydride Derivatives from the Plant

Endophytic Fungus Talaromyces purpurogenus
Jing-Yi Zhao, Xiao-Jing Wang, Zhen Liu, Fan-Xing Meng, Sen-Feng Sun, Fei Ye, and Yun-Bao Liu*
State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of
Medical Sciences and Peking Union Medical College, Beijing 100050, People’s Republic of China
*
S Supporting Information
Downloaded via UNIV OF CALIFORNIA SAN FRANCISCO on November 12, 2019 at 02:47:10 (UTC).

ABSTRACT: Six new nonadride derivatives (1−6) and three

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new spirocyclic anhydride derivatives (7−9) were isolated from

the endophytic fungus Talaromyces purpurogenus obtained from
fresh leaves of the toxic medicinal plant Tylophora ovata. The
structures of these compounds were determined by spectro-
scopic analyses including 1D and 2D NMR, HRESIMS, and
ECD techniques. Maleic anhydride derivatives 1−9 were
evaluated for their in vitro anti-inflammatory activities.
Compound 1 showed significant inhibitory activity against
NO production in LPS-induced RAW264.7 cells with an IC50 value of 1.9 μM. Compounds 2 and 6 showed moderate
inhibitory activities toward XOD and PTP1b, respectively, at 10 μM with inhibition rates of 67% and 76%.

E ndophytic fungi, which establish remarkable mutualistic

associations with their host plants, have attracted
considerable attention owing to their ecological and biotechno-
logical potential.1 Their complex relationship with host plants
enables endophytic fungi to produce various biologically active
secondary metabolites to increase their adaptability to both
biotic and abiotic stresses.2,3 Accumulating evidence has shown
that these bioactive secondary metabolites can serve as an
obtained. The in vitro anti-inflammatory activities of these
compounds were evaluated, and compound 1 significantly
inhibited nitric oxide production in lipopolysaccharide-stimu-
lated RAW264.7 mouse macrophages with an IC50 value of 1.9
μM. Additionally, we evaluated their antidiabetic activities in
vitro based on the inhibition of alpha-glucosidase, protein
tyrosine phosphotase 1b (PTP1b), and xanthine oxidase
(XOD).

important source of lead compounds such as pestalone,
amestolkolides, and cytochalasins for the development of anti-
inflammatory, antimicrobial, and anticancer agents.4−6
Nonadrides, a metabolite exclusively found in fungi, have a
nonadride core structure, namely, a C9 ring, bearing one or two
maleic anhydride moieties.7 Some nonadrides are strong
inhibitors of ras farnesyl transferase and squalene synthase
(phomoidrides),8 some show herbicidal activity (cornexistin),9
and other nonadrides, such as rubratoxins and scytalidin, have
shown prominent cytotoxic10 and antifungal activities,11
respectively. Due to their unique structural framework and
powerful biological activities, the biosynthesis12−14 and
chemical synthesis15,16 of nonadrides have attracted substantial
attention.
Recently, we isolated an endophytic fungus, Talaromyces
purpurogenus, from the leaves of the toxic medicinal plant
Tylophora ovate (Lindl.) Hook. ex Steud. Preliminary evaluation
of the extract of T. purpurogenus by LC-MS and LC-UV analysis
revealed that the main secondary metabolites were nonadrides.
Furthermore, previous investigations of this endophytic fungus
led to the isolation of rubratoxins A and B,17,18 both nonadrides,
inhibitors of protein phosphatase 2A and suppressors of cancer
metastasis in vitro.19 As part of our ongoing search for new
biologically active nonadrides from the endophytic fungus
Talaromyces sp., six new nonadride derivatives (1−6) and three
new spirocyclic anhydride derivatives (7−9) (Figure 1) were
© XXXX American Chemical Society and
American Society of Pharmacognosy
■
RESULTS AND DISCUSSION
Compound 1 was isolated as a white powder, and its molecular
formula was determined to be C26H32O12 based on HRESIMS
together with 1H and 13C NMR analyses (Table 1), indicating
that 1 has 11 degrees of unsaturation. Analyses of the 1H NMR,
13
C NMR, and HSQC spectra suggested the presence of five
carbonyl carbons (δC 167.6, 167.2, 166.7, 164.4, and 164.3), six
sp2 carbons (δC 148.2, 147.2, 144.8, 144.0, 140.8, and 121.2), six
methynes (δC 79.0, 76.6, 70.7, 65.1, 48.7, and 37.1), eight
methylenes (δC 36.7, 32.5, 31.5, 29.7, 27.0, 26.7, 25.8, and 23.3),
and one methyl carbon (δC 14.3). The carbonyl and sp2 carbons
accounted for 8 degrees of unsaturation, and the remaining 3
degrees of unsaturation revealed that 1 was tricyclic. The 1H−1H
COSY and HSQC correlations led to the identification of two
spin systems, I (C2−C8, C7(C11′)−C8) and II (C1′−C8′−
C11), as shown in Figure 2. The HMBC correlation among H-2
(δH 5.97)/H-3 (δH 7.03)/H-5 (δH 4.73) and a carbonyl carbon,
C-1 (δC 164.4), suggested that C-1 and C-5 were connected
through an oxygen atom, forming an unsaturated δ-lactone unit
(ring A). In addition, the HMBC correlations between H-8 (δH
2.91, 2.53) and C-9 (δC 144.8)/C-10 (δC 144.0); H-7 (δH
Received: March 6, 2019
A DOI: 10.1021/acs.jnatprod.9b00210
J. Nat. Prod. XXXX, XXX, XXX−XXX
Journal of Natural Products
Figure 1. Structures of compounds 1−9 from Talaromyces purpurogenus.
1.83)/H-11 (δH 5.45) and C-9; H-8′ (δH 3.25)/H-11′ (δH 2.97,
2.69) and C-9′ (δC 140.8)/C-10′ (δC 148.2); and H-7′ (δH
4.26) and C-9′ constructed the nine-membered carbocycle (ring
B).
After the establishment of rings A and B, the third ring (ring C,
a maleic anhydride ring) was elucidated from the IR, MS, and
NMR data. The IR absorptions at 1854 cm−1 (s), 1825 cm−1 (s),
1776 cm−1 (vs), 1706 cm−1 (vs), and 1258 cm−1 (vs) revealed
the presence of a maleic anhydride functional group in 1 (Figure
S5, Supporting Information). The HMBC correlations between
H-8 and C-13 (δC 166.7), H-11 and C-12 (δC 164.3), H-8′ and
C-13′ (δC 167.6), and H-11′ and C-12′ (δC 167.2) showed that
the four carbonyl groups were located at C-9, C-10, C-9′, and C-
10′, respectively. Compared to the downfield carbonyl carbons
at C-12′ (δC 167.2) and C-13′ (δC 167.6), the two upfield
carbonyl carbons at C-12 (δC 164.3) and C-13 (δC 166.7)
indicated that a maleic anhydride moiety (ring C) was formed
through a C12−O−C13 bond. Therefore, the remaining two
carbons, C-12′ and C-13′, were attributed to acid groups.
Compound 1 contains six stereocenters at C-5, C-6, C-7, C-
11, C-7′, and C-8′. The relative configuration of 1 was
determined by the NOESY correlations and the vicinal
1
H−1H coupling constants. The large coupling constant of H-
11′a/H-7 (3JH‑11′a,H‑7 = 9.8 Hz) and the NOESY correlation of
H-11′a/H-11 indicated that H-7 and H-11 were on opposite
faces of the nine-membered carbocycle. Moreover, the anti-
orientation of H-11/H-8′ was deduced from the large 3JH‑11,H‑8′
value of 10.2 Hz (Figure 2). The homonuclear coupling
constants, along with the NOESY correlations, confirmed only
one of the six possible staggered conformations of the flexible
C5−C6 fragment (A1, A2, A3 and B1, B2, B3), as shown in Figure
3. The vicinal coupling constant of H-5/H-6 (3JH‑5,H‑6 = 4.6 Hz)
was assigned by removing all of the couplings to H-4 in the
homonuclear spin-decoupling experiment (Figure S12, Support-
ing Information). The small coupling constant of H-5/H-6
indicated that these two protons have a gauche relationship,
which eliminates A1 and B1. A2 and B2 were excluded by the
NOESY correlation of H-5/H-7. The NOESY correlations of H-
B DOI: 10.1021/acs.jnatprod.9b00210
Article
4 (both a and b)/H-6 eliminated A3. The remaining rotamer, B3,
is consistent with the coupling constant of H-5/H-6 and the
NOESY correlations of H-5/H-4, H-5/H-6, H-5/H-7, and H-6/
H-4. Similarly, the relative configuration of the flexible C6−C7
fragment was identified by analyzing the coupling constants and
NOESY correlations. The large coupling constant of H-6/H-7
(3JH‑6,H‑7 = 10.2 Hz) and the NOESY correlations of H-5/H-8a,
H-5/H-8b, H-6/H-8b, H-6/11′b, H-7/H-8a, and H-7/H-11′b
clearly confirmed that the relative configuration of C6−C7 was
D1. Likewise, the relative configuration of C7′−C8′ (E1) was
determined based on the small coupling constant of 0 Hz for H-
7′/H-8′ and the NOESY correlations of H-11/H-6′ and H-11/
H-7′. Thus, the relative configuration of 1 was shown to be
identical to that previously reported for rubratoxin B.14 In
addition, the electron circular dichroism (ECD) spectra of 1 and
rubratoxin B were acquired, and the results showed similar
Cotton effects (Figure 5). Thus, the absolute configuration of 1
was determined to be identical to that of rubratoxin B, as shown
in Figure 1. Compound 1 was elucidated to be the
monohydrated diacid form of rubratoxin B and was named
rubratoxin acid A.
Compound 2 was isolated as a white powder, and its
molecular formula, C26H38O11, was supported by HRESIMS and
NMR data (Table 2). The 1H and 13C NMR and HSQC data of
2 indicated that the skeleton of 2 was similar to that of 1.
However, the absence of the signals of C-1 (δC 164.4), C-2 (δC
121.2), and C-3 (δC 147.2) and the presence of an additional
methyl signal (δH 0.92, 3H, t, 3J = 7.2 Hz; δC 14.3) suggested that
2 possessed an alkyl side chain at C1−C5 instead of the lactone
unit present in 1 (Figure 4). The 1H−1H COSY correlations of
H-1 (δH 0.92)/H-2 (δH 1.37), H-4 (δH 1.50, 1.42)/H-5 (δH
3.68), and H-6 (δH 3.40)/H-7 (δH 1.71) together with the
HMBC correlations between H-5/H-1 and C-3 (δC 28.2) and
between H-7 and C-5 (δC 72.1) further supported the above
assignments. The relative configuration of 2 was determined to
be the same as that of 1 based on NOESY, coupling constant,
and conformational analyses. The large coupling constant of H-
11′a/H-7 (3JH‑11′a,H‑7 = 10.2 Hz) and the NOESY correlation of
J. Nat. Prod. XXXX, XXX, XXX−XXX
Journal of Natural Products Article

Table 1. 1H and 13C NMR Data of Compounds 1, 2, and 6

1a 2a 6b
position δC c
δH (J in Hz)
d
δC c
δH (J in Hz)
d
δC e
δHf (J in Hz)
1 164.4 14.3 0.92, t (7.2) 63.2 4.01, d (4.0)
2 121.2 5.97, dd (9.6, 2.4) 23.4 1.37, m 130.6 5.64, m
3 147.2 7.03, ddd (9.6, 6.6, 2.4) 28.2 1.38, m 132.0 5.64, m
4 26.7 2.53, m 33.9 1.50, m 32.3 2.09, m
2.38, ddd (18.6, 6.6, 4.2) 1.42, m
5 79.0 4.73, dt (12.6, 4.2) 72.1 3.68, m 28.7 1.59, m
5-OH 3.00
6 76.6 3.68, m 79.2 3.40, brt (4.8) 34.2 2.32, m
6-OH 3.63 3.32
7 37.1 1.83, m 37.7 1.71, m 148.1 7.14, dt (16.0, 7.5)
8 25.8 2.91, d (13.0) 25.6 2.86, d (13.2) 118.6 6.46, dt (16.0, 1.5)
2.53, overlap 2.50, t (12.6)
9 144.8 145.3 139.3
10 144.0 143.8 137.1
11 65.1 5.45, d (10.2) 65.1 5.43, d (10.2) 28.0 3.11, dd (14.0, 9.5)
2.96, dd (14.0, 6.0)
11-OH 3.98 4.04
12 164.3 164.4 167.1
13 166.7 166.6 165.2
1′ 14.3 0.89, t (7.0) 14.3 0.89, t (7.0) 14.3 0.86, t (7.0)
2′ 23.3 1.31, overlap 23.3 1.29, overlap 23.3 1.27, overlap
3′ 32.5 1.26, overlap 32.5 1.27, overlap 32.5 1.26, overlap
4′ 29.7 1.31, overlap 29.7 1.30, overlap 29.9 1.28, overlap
1.27, overlap 1.27, overlap
5′ 27.0 1.44, m 27.0 1.43, overlap 30.1 1.31, overlap
1.37, m
6′ 36.7 1.40, m 36.7 1.40, overlap 28.1 1.33, overlap
1.26, overlap 1.26, overlap
7′ 70.7 4.26, m 70.7 4.26, m 33.8 1.85, m
7′-OH 3.21 3.22
8′ 48.7 3.25, d (10.2) 48.7 3.24, d (10.2) 36.5 3.33, m
9′ 140.8 140.6 144.4
10′ 148.2 148.6 144.5
11′ 31.5 2.97, dd (14.4, 9.8) 32.3 2.96, dd (14.4, 10.2) 9.7 2.04, s
2.69, d (14.4) 2.65, d (14.4)
12′ 167.2 167.5 166.6
13′ 167.6 167.6 166.1
a
Measured in CD3CN. bMeasured in CD3OCD3. cData measured at 200 MHz. dData measured at 800 MHz. eData measured at 125 MHz. fData
measured at 500 MHz

Figure 2. Key 1H−1H COSY, HMBC, and NOESY correlations of 1.

H-11′a/H-11 verified that H-7 and H-11 had an anti-
orientation, while the anti-orientation of H-11/H-8′ was
deduced from the large 3JH‑11,H‑8′ value of 10.2 Hz. The relative
configurations of C5−C6, C6−C7, and C7′-C8′ were confirmed
as B3, D1, and E1 by analysis of the coupling constants of H-5/H-
6 (3JH5,H6 = 5.4 Hz), H-6/H-7 (3JH6,H7 = 8.2 Hz), and H-7′/H-8′
(3JH7′,H8′ = 0 Hz) (Figure S23, Supporting Information) along
with the NOESY correlations of H-5/H-4, H-5/H-6, H-5/H-7,
H-5/H-8a, H-5/H-8b, H-6/H-4, H-6/H-8b, H-6/11′b, H-7/H-
8a, H-7/H-11′b, H-11/H-6′, and H-11/H-7′, as described for 1.
C DOI: 10.1021/acs.jnatprod.9b00210
The same relative configuration and similar ECD spectra of 2
and 1 (Figure 5) revealed that these two compounds have the
same absolute configuration. Thus, the structure of 2 was
confirmed, and it was named rubratoxin acid B.
Compounds 3 and 4 were isolated as white powders with the
formulas C27H28O8 and C29H40O9, respectively, as determined
from their HRESIMS and NMR data (Table 2). The 1D NMR
data in combination with the HSQC spectrum indicated the
presence of four carbonyl carbons, six olefinic carbons, two
methines, 14 methylenes including one oxygenated methylene,
J. Nat. Prod. XXXX, XXX, XXX−XXX
Journal of Natural Products
Figure 3. Assignment of the relative configuration of the C5−C6, C6−C7, and C7′−C8′ fragments of 1.
Figure 4. Key HMBC and 1H−1H COSY correlations of 2, 3, 4, and 6.
Figure 5. Experimental ECD spectra of rubratoxin B and 1−5.
and one methyl group in 3. The 1H−1H COSY correlations of
H-1 (δH 3.97)/H-2 (δH 5.62), H-3 (δH 5.67)/H-4 (δH 2.10),
and H-6 (δH 1.55, 1.51)/H-7 (δH 1.58) together with the
HMBC correlations between H-3/H-7 and C-5 (δC 27.3)
revealed that the only carbon−carbon double bond was located
at C-2 (δC 131.3) and C-3 (δC 131.8). Extensive analysis showed
that the structure of 3 was similar to that of prerubratoin A1.14
However, one fewer degree of unsaturation and the presence of
an additional methylene group in 3 relative to that of
prerubratoin A1 suggested that one of the maleic anhydride
moieties of 3 was hydrolyzed into a diacid, and the alkyl side
chain in its structure consisted of eight carbons. The 1H NMR
data of 4 were similar to those of 3, except for a singlet at δH 2.00
(3H), which showed HMBC correlations with the signals at δC
171.4 and δC 65.6 (C-1), revealing that the hydroxy group at C-1
was acetylated in 4 (Figure 4).
D DOI: 10.1021/acs.jnatprod.9b00210
Article
To establish the absolute configuration of 3, the ECD spectra
of four simplified isomers (3a, 3b, 3a′, and 3b′) of 3 were
determined at the Cam-B3LYP/6-31+G(d,p) level of theory in
acetonitrile, and these calculated spectra were compared with
the experimental spectrum of 3. The experimental ECD
spectrum of 3 showed an excellent fit with the calculated ECD
curve of 3a′ (Figure 6b), especially at 310 nm, which revealed
that the absolute configuration of 3 was 7S, 9′R. The absence of a
NOESY correlation between H-7 and H-9′ also supports the
anti-orientation of those two protons. In addition, the ECD
spectrum of compound 4 was similar to that of compound 3,
revealing the absolute configuration of 4 to be 7S, 9′R (Figure
5). The double bonds in 3 and 4 were each assigned an E
configuration based on the NOESY correlation of H-1/H-3.
Thus, the structures of compounds 3 and 4 were determined,
and they were named rubratoxin acids C and D, respectively.
J. Nat. Prod. XXXX, XXX, XXX−XXX
Journal of Natural Products Article

Table 2. 1H (800 MHz) and 13C (200 MHz) NMR Data of Compounds 3−5 (in CD3CN)
3 4 5
position δC δH (J in Hz) δC δH (J in Hz) δC δH (J in Hz)
1 63.2 3.97, d (4.0) 65.6 4.46, d (6.4) 13.8 0.91, t (6.4)
1a 171.4
1a-CH3 21.1 2.00, s
2 131.3 5.62, m 125.6 5.61, m 22.8 1.29−1.33, overlap
3 131.8 5.67, m 136.3 5.82, m 31.9 1.26−1.33, overlap
4 32.5 2.10, m 32.5 2.12, m 29.2 1.34, overlap
5 27.3 1.59, overlap 27.0 1.58, overlap 27.8 1.31−1.38, overlap
6 27.3 1.55, m 27.0 1.55, m 27.1 1.54, m
1.51, m 1.50, m 1.49, overlap
7 37.5 1.58, m 37.4 1.82, brs 37.6 1.82, brs
8 30.4 2.61, d (13.6) 30.4 2.61, d (13.6) 29.5 2.62, d (12.8)
2.40, dd (13.6, 9.6) 2.40, dd (13.6, 9.6) 2.39, dd (12.8, 9.6)
9 145.3 145.0 145.1
10 144.9 144.9 144.5
11 28.3 2.98, t (12.6) 28.3 2.98, t (12.6) 27.8 2.97, t (12.6)
2.83, brs 2.83, brs 2.83, brs
12 166.8 166.8 166.4
13 167.0 167.2 166.7
1′ 14.3 0.88, t (7.2) 14.3 1.88, t (7.2) 13.8 0.88, t (7.2)
2′ 23.3 1.29, overlap 23.3 1.29, overlap 22.8 1.29−1.33, overlap
3′ 32.3 1.27, overlap 32.5 1.27, overlap 31.9 1.26−1.33, overlap
4′ 29.7 1.26, overlap 29.7 1.26, overlap 29.2 1.34, overlap
5′ 30.0 1.29, overlap 30.0 1.29, overlap 27.8 1.31−1.38, overlap
6′ 23.3 1.29, overlap 23.3 1.29, overlap 23.3 1.29, overlap
7′ 27.7 1.38, m 27.7 1.38, m 27.2 1.38, overlap
1.34, m 1.34, m 1.31, overlap
8′ 27.7 1.68, m 27.7 1.68, m 27.2 1.68, m
9′ 35.6 3.22, brs 35.6 3.22, brs 35.1 3.22, brs
10′ 144.9 145.1 144.5
11′ 144.9 144.9 144.5
12′ 31.0 2.58, d (13.6) 30.7 2.57, d (13.6) 30.4 2.58, d (13.6)
2.53, brs 2.53, brs 2.52, brs
13′ 167.5 167.5 167.0
14′ 167.4 167.5 167.0

Figure 6. (a) Structures of four isomers, 3a, 3b, 3a′, and 3b′; (b) experimental ECD curve of 3 and calculated ECD spectra of 3a, 3b, 3a′ and 3b′.

Compound 5 was isolated as a white powder with a molecular (Table 2). The 1H and 13C NMR data of 5 were similar to those
formula of C27H40O7 according to its HRESIMS and NMR data of compound 3. The lack of signals for C-1 (δC 63.2), C-2 (δC
E DOI: 10.1021/acs.jnatprod.9b00210
J. Nat. Prod. XXXX, XXX, XXX−XXX
Journal of Natural Products Article

131.3), and C-3 (δC 131.8) and the presence of a methyl signal Table 3. 1H and 13C NMR Data of Compounds 7−9 (in
(δH 0.91, t, 3J = 6.4 Hz) indicated that the alkyl chain in 5 was CD3CN)
hydrogenated at C-1, C-2, and C-3. The 2D NMR (1H−1H
7 8 9
COSY, HSQC, and HMBC) experiments further verified these
assignments. The ECD spectrum of 5 was consistent with that of δH (J in
b
δHd
(J in δHb (J in
position δC a Hz) δC c Hz) δCa Hz)
3 (Figure 5), indicating that their absolute configurations were
1 180.6 177.3 180.5
identical. Therefore, compound 5 was characterized and named
3 96.9 5.75, d (6.4) 98.6 5.93, d (6.5) 96.8 5.73, d
rubratoxin acid E. (6.0)
Compound 6 had the molecular formula C26H34O7 on the 4 39.5 2.86, dd 54.3 3.72, d (6.5) 39.6 2.85, m
basis of its HRESIMS and NMR data (Table 1), which suggest (13.6, 6.4)
that two rings are present in 6. Analysis of the 1H−1H COSY and 1.76, d 1.75, d
HSQC data of 6 indicated the presence of two isolated spin (13.6) (13.2)
4a 170.6
systems, I (C1−C8) and II (C1′−C8′−C11), as shown in
Figure 4. The UV absorption spectrum showed λmax at 252 nm, 4a-
OCH3
53.2 3.61, s

and the IR spectrum presented bands at νmax 1822 (s), 1766 (vs), 5 54.9 59.3 54.9
and 1277 (s) cm−1, which together with the four carbonyl signals 6 58.9 59.6 58.9
at δC 167.1 (C-12), 165.2 (C-13), 166.6 (C-12′), and 166.1 (C- 6a 172.7 172.8 173.0
13′) in the 13C NMR spectrum, suggested the presence of two 6a- 52.9 3.59, s 53.2 3.63, s 52.9 3.59, s
maleic anhydride moieties. The HMBC correlations between H- OCH3
8 and C-9 (δC 139.3)/C-10 (δC 137.1)/C-13, H-11 and C-9/C- 7 38.3 2.57, dd 38.5 2.63, m 38.4 2.52, m
(12.8, 9.6)
10/C-12, and H-7 and C-9 indicated that one of the maleic
2.17, overlap 2.24, m 2.10, m
anhydride moieties was located at C-9 and C-10. The other
8 39.3 2.77, m 38.9 2.86, m 40.0 2.56, m
maleic anhydride moiety was determined to be at C-9′ and C-
9 40.3 2.43, dd 33.8 2.15, dd 40.3 2.38, d
10′ based on the HMBC correlations between H-8′ and C-9′ (14.4, 4.0) (14.5, 2.5) (14.4)
(δC 144.4)/C-10′ (δC 144.5)/C-13′ and between H-7′ and C- 2.12, overlap 1.94, overlap 2.04, m
9′. The HMBC correlations between the methyl signal (δH 2.04, 2′ 172.9 173.3 172.8
s) and C-9′, C-10′, and C-12′ suggested that the methyl group 3′ 134.0 134.2 134.1
was connected to C-10′. Δ2,3 and Δ7,8 were confirmed to be Z 4′ 154.6 7.47, d (1.6) 154.5 7.44, d (1.0) 154.5 7.48, brs
and E by the small coupling constant of H-2/H-3 (3JH‑2, H‑3 < 9.6 5′ 82.3 5.06, m 82.5 5.00, m 82.3 5.04, m
Hz) and the NOESY correlation of H-6/H-8, respectively. The 6′ 33.4 1.83, m 33.3 1.82, m 33.4 1.80, m
absolute configuration of 6 was determined by means of the 1.63, m 1.65, m 1.63, m
ECD exciton chirality method.20 The ECD spectrum of 6 7′ 25.2 1.37, m 25.3 1.41, m 25.2 1.36, m
showed a negative signal resulting from the exciton coupling 8′ 29.7 1.35, m 29.7 136, m 29.7 1.32, m
between two similar chromophores of the maleic anhydride 9′ 32.3 1.28, m 32.3 1.31, m 32.3 1.27, m
moieties at 343 nm (Δε = −0.14, π → π*) and 308 nm (Δε = 10′ 23.2 1.31, m 23.2 1.32, m 23.2 1.30, m
+0.43, π → π*), which revealed that the absolute configuration 11′ 14.3 0.89, t (7.0) 14.3 0.89, t (6.5) 14.4 0.89,
overlap
of 6 was 8′S (Figure S56, Supporting Information). Thus, 2″ 164.8 164.8 14.3 0.89,
compound 6 was named talarodride. overlap
Compound 7 was isolated as a white powder, and its 3″ 120.9 5.89, dd 120.9 5.90, dd 23.4 1.30, m
molecular formula, C26H34O12 (10 degrees of unsaturation), was (9.6, 2.4) (10.0, 2.5)
derived from its HRESIMS and NMR data (Table 3). Its 13C 4″ 147.7 6.99, ddd 147.8 6.99 ddd 28.9 1.36, m
(9.6, 6.2, (10.0, 6.5,
NMR spectrum showed four carbonyl groups at δC 180.6, 172.9, 2.4) 2.0)
172.7, and 164.8 and four sp2 carbons at δC 154.6, 147.7, 134.0, 5″ 26.9 2.65, m 27.0 2.65, m 34.7 1.44, m
and 120.9, which accounted for 6 of the 10 degrees of 2.26, m 2.27, m
unsaturation, implying a tetracyclic molecular structure. Three 6″ 79.0 4.45, ddd 78.6 4.45, ddd 72.0 3.38, m
structural fragments, C3−C4, C4′−C11′, C3″−C7″−C8, and (12.6, 3.6, (12.5, 4.0,
1.8) 1.5)
C7−C9 (Figure 7), were revealed by analyzing the 1H−1H
7″ 75.3 3.61, d (9.0) 75.1 3.59, d (8.5) 76.8 3.34, m
COSY and HSQC spectra. The C3−C4 fragment and the 7″-OH 3.18
HMBC correlation between H-3 (δH 5.75)/H-4 (δH 2.86, 1.76) a
and C-1 (δC 180.6)/C-5 (δC 54.9) revealed that C-1 and C-3 Data measured at 200 MHz. bData measured at 800 MHz. cData
measured at 125 MHz. dData measured at 500 MHz.
were connected through an oxygen atom to form a five-
membered ring (ring A) and that a hemiacetal was located at C-3
(δC 96.9). The C4′−C5′ fragment and the HMBC correlation spirocyclic system with a hemiacetal unit. Through detailed
between H-5′ (δH 5.06)/H-4′ (δH 7.47) and C-2′ (δC 172.9)/C- analysis of its NMR data, compound 7 was determined to have
3′ (δC 134.0) indicated that an unsaturated γ-lactone moiety the same carbon skeleton as that of penicillactone A.21 This
(ring C) was present in the molecule. The presence of an conclusion was further confirmed by the HMBC correlations
unsaturated δ-lactone unit (ring D) was confirmed by the C3″− between H-4 and C-6/C-9, H-7 and C-3′/C-5/C-6, and H-9
C6″ fragment and the HMBC correlations between H-3″ (δH and C-4/C-5/C-6. However, an additional methyl signal at δH
5.89)/H-4″ (δH 6.99)/H-6″ (δH 4.45) and C-2″ (δC 164.8). The 3.59 (3H, s), which showed an HMBC correlation to the signal
fragment C7−C9 and HMBC correlation between H-7 (δH 2.57, at δC 172.7, revealed that the carboxyl at C-6a was formylated in
2.17)/H-9 (δH 2.43, 2.12) and C-5/C-6 (δC 58.9) indicated that 7.
there should be another ring (ring B) in 7. These data showed The relative configuration of 7 was determined via the
that 7 may be an analogue of penicillactone, which has a unique coupling constants, NOESY correlations, and conformational
F DOI: 10.1021/acs.jnatprod.9b00210
J. Nat. Prod. XXXX, XXX, XXX−XXX
Journal of Natural Products
Figure 7. Key 1H−1H COSY, HMBC, and NOESY correlations of 7 and 8.
Figure 8. Assignment of the relative configuration of C6″−C7″ and C7″−C8 of 7.
analysis. Homonuclear spin-decoupling experiments rapidly
identified the vicinal coupling constants of H-3/H-4 (3JH‑3,H‑4a =
6.4 Hz, 3JH‑3,H‑4b = 0 Hz) (Figure S74, Supporting Information).
Using ChemBio 3D software to minimize the molecular energy
of 7, we were able to elucidated the dihedral angles (φH-3, H-4a
= 11° and φH-3, H-4b = 100°), which were consistent with the
vicinal 1H−1H coupling constants of H-3 and H-4 and the
NOESY correlation of H-3/H-4a. The NOESY correlation
between H-8 and H-4′ indicated that ring C and H-8 were on the
same face of ring B. In addition, the NOESY correlations of H-
9b/H-4a and H-9b/H-8 revealed that rings A and B are fused, as
shown in Figure 8. The small vicinal coupling constant of H-6″/
H-7″ (3JH‑6″,H‑7″ = 0 Hz) and the NOESY correlations of H-8/H-
6″, H-6″/H-7″, H-6″/H-5″b, and H-5″ (a and b)/H-7″
confirmed that the relative configuration of C6″−C7″ was B3.
The relative configuration of C7″−C8 was identified as D1 based
on the large coupling constant of H-7″/H-8 (3JH‑7″,H‑8 = 9.0 Hz)
(Figure S74, Supporting Information) and the NOESY
correlations of H-7 (a and b)/H-6″, H-8/H-7b, H-8/H-9b, H-
7″/H-7a, and H-7″/H-9a (Figure 8).
Considering the similarities between the ECD spectra of
compound 7 (Figure S65, Supporting Information) and
penicillactone A,20 the absolute configuration of 7 was
determined to be 3R, 5S, 6S, 8S, 5′S, 6″R, 7″R. Thus, compound
7 was characterized and named 6a-methylpenicillactone A.
G DOI: 10.1021/acs.jnatprod.9b00210
Article
Compound 8 was obtained as a white powder and showed a
deprotonated ion at m/z 563.2131 [M − H]−, corresponding to
a formula of C28H36O12. The 1H and 13C NMR data (Table 3) of
8 closely resembled those of 7, which indicated that compounds
7 and 8 have the same carbon skeleton. The HMBC correlation
between H-4 (δH 3.72)/H-3 (δH 5.93)/δH 3.61 (3H) and δC
170.6 (C-4a) revealed that the carbonyl located at C-4 was
formylated in 8.
The relative configuration of 8 was confirmed by the NOESY
correlations, coupling constants, and conformational analysis.
The NOESY correlation between H-8 and H-4′ indicated that
ring C and H-8 were in the same orientation relative to ring B.
The NOESY correlations of H-9b/H-4 and H-9b/H-8 indicated
that rings A and B are fused as shown in Figure 8. H-9a and H-3
were shown to have the same relative orientation by the NOESY
correlation between H-9a and H-3. The NOESY correlations
between 4a-OCH3 and H-5′ revealed that H-4 and H-3 were on
the same face of ring A, while H-5′ and ring A were on the same
face of ring C (Figure 7). The relative configurations of C6″−
C7″ and C7″−C8 were confirmed as B3 and D1 by analysis of
their coupling constants (3JH,H) (Figure S86, Supporting
Information) and NOESY correlations as described for 7. The
similarities of the ECD spectra of compounds 7 and 8 (Figure
S77, Supporting Information) revealed that they shared the
same absolute configuration and that the configuration at C-4
J. Nat. Prod. XXXX, XXX, XXX−XXX
Journal of Natural Products
Figure 9. Proposed dimerization pathway.
was S. Hence, the structure of compound 8 was established, and
it was named penicillactone D.
The HRESIMS data of compound 9 showed a molecular
formula of C26H40O9. Extensive analysis of the 1H and 13C NMR
data (Table 3) of 9 showed that they were similar to those of 7.
The presence of an additional methyl group (δH 0.89, 3H) and
the absence of signals C-2″ (δC 164.8), C-3″ (δC 120.9), and C-
4″ (δC 147.7) for 9 compared with 7 suggested that the lactone
(ring D in 7) was hydrolyzed and the double bond at C-3″ and
C-4″ along with the carboxyl at C-2″ was hydrogenated to alkyl
groups in 9. The 1H−1H COSY correlations of H-2″ (δH 0.89)/
H-3″ (δH 1.30), H-5″ (δH 1.44)/H-6″ (δH 3.38), and H-7″ (δH
3.34)/H-8 (δH 2.56) together with the HMBC correlations
between H-2″/H-6″ and C-4″ (δC 28.9) and between H-8 and
C-6″ (δC 72.0) further supported the above assignments. The
relative configuration was confirmed to be the same as that of 7
by NOSEY analysis. According to Snatzke’s rule, the negative
signal at 312 nm observed in the circular dichroism spectrum of
9 in DMSO in the presence of dimolybdenum tetraacetate
indicated that the configurations of C-6″ and C-7″ were both R
(Figure S89, Supporting Information). Thus, the configuration
of 9 was confirmed, and it was named penicillactone E.
In previously reported studies, nonadrides were found to be
formed by the dimerization of two maleic anhydride
monomers.13 Herein, the discovery of compound 6 further
revealed that the formation of the nine-membered ring may be
catalyzed by two ketosteroid isomerase (KI)-like proteins in two
steps: two maleic anhydride monomers are first linked and then
cyclized (Figure 9). Nonadrides with two maleic anhydride
moieties exist in equilibrium with their monohydrated diacid
forms in aqueous solvents,12 and more interestingly, the
dicarboxylic acid in rubratoxin A was the active form for its
PP2A-specific inhibition.19 According to the literature, nona-
drides are usually isolated as follows: the broth is extracted with
an organic solvent and/or the sample is purified by HPLC at low
pH.14,22 However, in our research, the compounds were all
isolated under neutral pH conditions (both extraction and
purification), and under these conditions, the ring-opened form
was the stable form. All these compounds were tested for in vitro
anti-inflammatory activities, and compound 1 showed signifi-
cant inhibitory activity against nitric oxide production in
liposaccharide (LPS)-induced RAW264.7 cells, with an IC50
value of 1.9 μM, while the inhibition rate of compound 1 on cell
proliferation was −14.5%. They were also evaluated for in vitro
antidiabetic activities based on the inhibition of alpha-
H DOI: 10.1021/acs.jnatprod.9b00210
Article
glucosidase, PTP1b, and XOD. Compounds 2 and 6 showed
moderate inhibitory activities toward XOD and PTP1b,
respectively, with inhibition rates of 67% and 76% at 10 μM.
■ EXPERIMENTAL SECTION
General Experimental Procedures. Optical rotations were
measured with a Rudolph Autopol V automatic polarimeter at 20 °C
(Rudolph Research Analytical, Hackettstown, NJ, USA). UV spectra
were recorded using a V-670 UV−visible/NIR spectrophotometer
(JASCO, Tokyo, Japan). ECD spectra were obtained on a JASCO J-815
CD spectropolarimeter (JASCO, Tokyo, Japan). IR analysis was
performed using a Nicolet 5700 FT-IR spectrometer (FT-IR
microscope transmission, Thermo Electron Corporation, Madison,
WI, USA). NMR spectra were recorded on a Bruker AVIIIHD-600
spectrometer (Bruker Corp. Karlsruhe, Germany), a Bruker AVIIIHD-
800 spectrometer (Bruker Corp. Karlsruhe, Germany), and an Agilent
Technologies DD2-500 spectrometer (Agilent Technologies, Ltd.,
Santa Clara, CA, USA). The HRESIMS experiments were carried out
on an Agilent Technologies 6250 Accurate-Mass Q-TOF LC/MS
spectrometer (Agilent Technologies, Ltd.) and a Thermo Scientific Q-
Exactive Fouce LC/MS spectrometer (Thermo Fisher Scientific, USA).
HPLC separations were performed using a Shimadzu LC-6AD
instrument with an SPD-20 detector (Shimadzu, Tokyo, Japan) and a
YMC-Pack ODS-A column (250 × 10 mm, 5 μm, YMC Co., Ltd.,
Kyoto, Japan). ODS (45−70 μm, Merck, Darmstadt, Germany) and
silica gel (200−300 mesh, Qingdao Marine Chemical Inc. China) were
used for column chromatography. Thin-layer chromatography (TLC)
separations were conducted on glass precoated with silica gel GF254
(Qingdao Marine Chemical Inc. China).
Isolation and Identification of Endophytic Fungi. Fresh leaves
of Tylophora ovate were collected from Guangxi Province, People’s
Republic of China, in May 2015. Plant samples were washed in tap
water for 15 min and air-dried. Plant fragments were sequentially
immersed in 75% EtOH for 10 min, 1% HgCl2 solution for 10 min, and
sterile distilled H2O for 1 min (five times). Then, the surface-sterilized
fragments were cut into small pieces (ca. 1 cm2) using a sterile blade and
placed on potato dextrose agar (PDA) plates for incubation at 28 °C.
The hyphal tips of the endophytic fungus growing out from the plant
tissue were cut using a sterile pipet and transferred onto a new PDA
plate. After incubation at 28 °C for 2−4 d, the culture purity was
determined based on colony morphology. The endophytic fungus was
identified by internal transcribed spacer (ITS) sequencing and
morphology (GenBank accession no. KM507168) by Beijing Sunbio
Tech Co., Ltd. The fungus was deposited in Liu’s lab at the Institute of
Materia Medica, Chinese Academy of Medical Sciences and Peking
Union Medical College.
Fermentation, Extraction, and Isolation. The strain was
cultured in modified Martin broth in a conical flask (500 mL) at 24
°C on a shaker at 110 rpm for 3 d to obtain the seed culture. The large-
scale fermentation involved the incubation of 5 mL of seed culture on
J. Nat. Prod. XXXX, XXX, XXX−XXX
Journal of Natural Products
solid rice medium (100 mL of water, 100 g of rice) at 24 °C for 30 d in
conical flasks (1 L). The fermented material was extracted with ethyl
acetate (EtOAc) three times at room temperature to yield a red extract
(130 g). The extract was suspended in water and partitioned
successively into petroleum ether and EtOAc. The EtOAc fraction
(77.9 g) was subjected to silica gel column chromatography and eluted
by a stepwise CH2Cl2−CH3OH gradient (200:1−0:1) to afford
fractions A and B. Fraction A (11.82 g) was separated stepwise on an
ODS column using 10% to 100% CH3OH in water to give compound 3
(10 mg) and another three subfractions (A1−A3). Fraction A1 was
purified by RP-HPLC to give compounds 1 (10.0 mg, 45% CH3CN, tR
= 31.4 min) and 2 (5.0 mg, 45% CH3CN, tR = 32.6 min). The
purification of fraction A2 by HPLC gave 6.0 mg of compound 7 (43%
CH3CN, tR = 23.3 min). Fraction A3 was separated by HPLC using
43% CH3CN in water to afford compounds 8 (5 mg, tR = 31.3 min) and
9 (5 mg, tR = 35.5 min). Fraction B (3.74 g) was subjected to silica gel
column chromatography using a stepwise petroleum ether−acetone
gradient (1:0−0:1) to afford two subfractions, B1 and B2. Fraction B1
was purified by HPLC to yield 5.4 mg of compound 6 (80% CH3CN, tR
= 37.2 min). Fraction B2 was separated by HPLC using 70% CH3CN in
water to afford compound 4 (6.0 mg, tR = 33.8 min) and compound 5
(6.0 mg, tR = 48.5 min).
Rubratoxin acid A (1): white, amorphous powder; [α]20 D +37.24 (c
0.29, CH3CN); UV (CH3CN) λmax (log ε) 205 (4.46) and 251 (4.01)
nm; CD (CH3CN) 190 (Δε +97.31), 252 (Δε −47.43), and 284 (Δε
−27.72) nm; IR νmax 3525, 2929, 2860, 1776, 1706, 1258, and 1072
cm−1; 1H and 13C NMR, see Table 1; and HRESIMS m/z 559.1774 [M
+ Na]+ (calcd for C26H32O12Na, 559.1786).
Rubratoxin acid B (2): white, amorphous powder; [α]20 D +19.16 (c
0.12, CH3CN); UV (CH3CN) λmax (log ε) 203 (4.17) and 252 (3.83)
nm; CD (CH3CN) 194 (Δε +3.86), 221 (Δε −4.29), 251 (Δε −2.21),
and 283 (Δε +2.58) nm; IR νmax 3399, 2958, 2930, 2860, 1847, 1828,
1775, 1712, 1287, 1254, and 1047 cm−1; 1H and 13C NMR, see Table 1;
and HRESIMS m/z 539.2496 [M − H]− (calcd for C27H39O11,
539.2498).
Rubratoxin acid C (3): white, amorphous powder; [α]20 D −44.29 (c
0.07, CH3CN); UV (CH3CN) λmax (log ε) 198 (4.40) and 250 (4.11)
nm; CD (CH3CN) 200 (Δε +1.59), 219 (Δε −4.25), 247 (Δε +0.89),
and 270 (Δε −2.31) nm; IR νmax 3568, 3384, 2931, 2857, 1846, 1825,
1769, 1725, and 1255 cm−1; 1H and 13C NMR, see Table 2; and
HRESIMS m/z 489.2493 [M − H]− (calcd for C27H37O8, 489.2494).
Rubratoxin acid D (4): white, amorphous powder; [α]20 D −23.33 (c
0.09, CH3CN); UV (CH3CN) λmax (log ε) 197 (4.22) and 250 (3.92)
nm; CD (CH3CN) 199 (Δε +0.72), 218 (Δε −1.84), 246 (Δε +0.95),
and 270 (Δε −2.08) nm; IR νmax 2929, 2857, 1847, 1825, 1769, 1734,
and 1254 cm−1; 1H and 13C NMR, see Table 2; and HRESIMS m/z
531.2582 [M − H]− (calcd for C27H39O9, 531.2599).
Rubratoxin acid E (5): white, amorphous powder; [α]20 D −39.00 (c
0.10, CH3CN); UV (CH3CN) λmax (log ε) 203 (4.25) and 250 (4.02)
nm; CD (CH3CN) 197 (Δε +4.05), 216 (Δε −6.96), 245 (Δε +3.52),
and 270 (Δε −7.80) nm; IR νmax 2928, 2857, 1847, 1788, 1766, and
1252 cm−1; 1H and 13C NMR, see Table 2; and HRESIMS m/z
475.2697 [M − H]− (calcd for C27H39O7, 475.2701).
Talarodride (6): yellowish oil; [α]20D −2.86 (c 0.07, CH3CN); UV
(CH3CN) λmax (log ε) 198 (4.31), 252 (3.83), and 316 (3.92) nm; CD
(CH3CN) 202 (Δε +1.07), 254 (Δε −1.09), 307 (Δε +0.43), and 343
(Δε −0.14) nm; IR νmax 2928, 2857, 1822, 1766, and 1277 cm−1; 1H
and 13C NMR, see Table 1; and HRESIMS m/z 457.2204 [M − H]−
(calcd for C26H33O7, 457.2232).
6A-Methylpenicillactone A (7): white, amorphous powder; [α]20 D
+61.25 (c 0.08, CH3CN); UV (CH3CN) λmax (log ε) 207 (4.26) nm;
CD (CH3CN) 195 (Δε +28.04), 215 (Δε +30.83), and 263 (Δε
+5.99) nm; IR νmax 3436, 2928, 2859, 1748, and 1257 cm−1; 1H and 13C
NMR, see Table 3; and HRESIMS m/z 529.2060 [M + Na]+ (calcd for
C26H34O10, 529.2044).
Penicillactone D (8): white, amorphous powder; [α]20 D +84.28 (c
0.07, CH3CN); UV (CH3CN) λmax (log ε) 206 (4.40) and 250 (4.02)
nm; CD (CH3CN) 217 (Δε +18.93) and 266 (Δε +2.99) nm; IR νmax
3460, 2953, 2828, 1744, and 1257 cm−1; 1H and 13C NMR, see Table 3;
I DOI: 10.1021/acs.jnatprod.9b00210
Article
and HRESIMS m/z 563.2131 [M − H]− (calcd for C28H35O12,
563.2134).
Penicillactone E (9): white, amorphous powder; [α]20 D +38.57 (c
0.07, CH3CN); UV (CH3CN) λmax (log ε) 200 (3.93) and 210 (3.95)
nm; CD (CH3CN) 216 (Δε +15.80) and 247 (Δε −1.57) nm; IR νmax
3452, 2956, 2928, 2859, 1753, and 1250 cm−1; 1H and 13C NMR, see
Table 3; and HRESIMS m/z 495.2598 [M − H]− (calcd for C26H39O9,
495.2599).
ECD Calculation. The ECD curves of 3 were calculated using
Gaussian 09 software. The conformation analysis was performed in DS
(Discovery Studio) 2018 with the MMFF94s force field. The stable
conformers subjected to ECD calculations were optimized by time-
dependent density functional theory at the B3LYP/6-31G(d) level with
the polarized continuum model in acetonitrile. The 80 lowest-energy
conformers were calculated using Cam-B3LYP/6-31+G(d,p) with a
half-bandwidth of no more than 0.35 eV. The final calculated ECD
spectra were obtained according to the Boltzmann-calculated
contribution of each conformer.
NO Inhibition Assay. RAW264.7 mouse macrophages were
cultured in DMEM supplemented with 10% fetal bovine serum, 100
U/mL penicillin, and 100 μg/mL streptomycin. After preincubation for
24 h in a 96-well plate, the cells were treated with the test compounds
and then stimulated with LPS (300 ng/mL) for 24 h. The production of
NO was determined by measuring the concentration of nitrite in the
supernatant using a Griess assay. The absorbance was measured at 550
nm, and dexamethasone was used as the positive control.
α-Glucosidase Inhibition Assay. The activity of α-glucosidase
was determined spectrophotometrically using a 96-well microplate
reader. In brief, 10 μM (final concentration) solutions of the test
compounds were preincubated with 0.1 U/mL α-glucosidase in
phosphate buffer at 37 °C for 5 min, and then the substrate p-
nitrophenyl-α-D-glucopyranoside was added. The reaction was
incubated at 37 °C for 15 min and stopped by the addition of 0.4 M
Na2CO3. A similar assay buffer without α-glucosidase was used as the
compound blank. Additionally, a similar system without the compound
was utilized as a control. The absorbance (A) was determined at 400
nm.
PTP1b Inhibition Assay. Recombinant human PTP1b protein was
overexpressed by hNi-PTP1B-BL21 E. coli and purified by Ni-NTA
agarose. 4-Nitrophenyl phosphate disodium salt (pNPP) was used as
the substrate. Compounds at a final concentration of 10 μM were
preincubated with the PTP1b protein at room temperature for 5 min in
an assay buffer containing 50 mM HEPES, 150 mM NaCl, 2 mM
EDTA, and 5 mM DTT (pH 7.0). Then, 2 mM pNPP was added, and
the mixture was incubated at 30 °C for 10 min, and the reaction was
stopped by the addition of 3 M NaOH. A similar assay buffer without
the PTP1b protein was used as the compound blank. Additionally, a
similar system without the compound was utilized as a control. The
absorbance was determined at 405 nm.
XOD Inhibition Assay. The effect of each compound on XOD-
catalyzed xanthine (XAN) hydrolysis was determined in a 96-well plate.
Compounds at a final concentration of 10 μM were incubated with
XOD at 3 mU/mL (final concentration) in an assay buffer (containing
3.5 mM KH2PO4, 15.2 mM K2HPO4, 0.25 mM EDTA, and 50 μM
XAN, pH 7.4) at 37 °C for 15 min. A similar assay buffer without the
enzyme XOD was used as the compound blank. Additionally, a similar
system without the compound was utilized as a control. The absorbance
was determined at 400 nm.
■
ASSOCIATED CONTENT
*
S Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jnat-
prod.9b00210.
HRESIMS, UV, IR, 1D NMR, 2D NMR, and ECD
spectra for compounds 1−9 (PDF)
J. Nat. Prod. XXXX, XXX, XXX−XXX
Journal of Natural Products

■
Article

AUTHOR INFORMATION (21) Liu, Y. B.; Ding, G. Z.; Li, Y.; Qu, J.; Ma, S. G.; Lv, H. N.; Liu, Y.;
Wang, W. J.; Dai, J. G.; Tang, Y.; Yu, S. S. Org. Lett. 2013, 15, 5206−
Corresponding Author 5209.
*E-mail: liuyunbao@imm.ac.cn. Tel: +86-10-83162679. Fax: (22) Han, C. G.; Hiroyuki, F.; Tomohiko, T.; Ryosuke, F.; Kenichi, K.;
+86-10-63017757. Bong-Keun, C.; Genji, I.; Makoto, O. J. Nat. Prod. 2015, 78, 639−644.
ORCID
Jing-Yi Zhao: 0000-0001-6057-1831
Zhen Liu: 0000-0002-1763-4953
Yun-Bao Liu: 0000-0002-1338-0271
Author Contributions
Jing-Yi Zhao and Xiao-Jing Wang contributed equally.
Notes
The authors declare no competing financial interest.

■ ACKNOWLEDGMENTS
This study was supported by grants from the National Natural
Science Foundation of China (No. 21572276), the Fundamen-
tal Research Funds for the Central Institutes (2018RC350007),
and the Young Elite Scientists Sponsorship Program by CAST
(YESS, No. 2016QNRC001).

■ REFERENCES
(1) Kusari, S.; Spiteller, M. Nat. Prod. Rep. 2011, 28, 1203−1207.
(2) Aly, A. H.; Debbab, A.; Proksch, P. Pharmazie 2013, 68, 499−505.
(3) Jia, M.; Chen, L.; Xin, H. L.; Zheng, C. J.; Rahman, K.; Han, T.;
Qin, L. P. Front. Microbiol. 2016, 27, 906.
(4) Chen, S. H.; Ding, M.; Liu, W. Y.; Huang, X. S.; Liu, Z. M.; Lu, Y.
J.; Liu, H. J.; She, Z. G. Phytochemistry 2018, 146, 8−15.
(5) Deshmukh, S. K.; Verekar, S. A.; Bhave, S. V. Front. Microbiol.
2015, 5, 715.
(6) Kharwar, R. N.; Mishra, A.; Gond, S. K.; Stierle, A.; Stierle, D. Nat.
Prod. Rep. 2011, 28, 1208−1228.
(7) Barton, D. H. R; Sutherland, J. K. J. Chem. Soc. 1965, 1769−1772.
(8) Dabrah, T. T.; Kaneko, T.; Massefski, W. Jr.; Whipple, E. B. J. Am.
Chem. Soc. 1997, 119, 1594−1598.
(9) Nakajima, M.; Itoi, K.; Takamatsu, Y.; Sato, S.; Furukawa, Y.;
Furuya, K.; Honma, T.; Kadotanic, J.; Kozasa, M.; Haneishi, T. J.
Antibiot. 1991, 44, 1065−1072.
(10) Wang, T.; Zhang, Y.; Wang, Y.; Pei, Y. H. Toxicol. In Vitro 2007,
21, 646−650.
(11) Strunz, G. M.; Kakushima, M.; Stillwell, M. A. J. Chem. Soc.,
Perkin Trans. 1 1972, 18, 2280.
(12) Szwalbe, A. J.; Williams, K.; O’Flynn, D. E.; Bailey, A. M.;
Mulholland, N. P.; Vincent, J. L.; Willis, C. L.; Cox, R. J.; Simpson, T. J.
Chem. Commun. 2015, 51, 17088−17091.
(13) Williams, K.; Szwalbe, A. J.; Mulholland, N. P.; Vincent, J. L.;
Bailey, A. M.; Willis, C. L.; Simpson, T. J.; Cox, R. J. Angew. Chem., Int.
Ed. 2016, 55, 6784−6788.
(14) Bai, J.; Yan, D. J.; Zhang, T.; Guo, Y. Z.; Liu, Y. B.; Zou, Y.; Tang,
M. C.; Liu, B. Y.; Wu, Q.; Yu, S. H.; Tang, Y.; Hu, Y. C. Angew. Chem.,
Int. Ed. 2017, 56, 4782−4786.
(15) Clark, J. S.; Marlin, F.; Nay, B.; Wilson, C. Org. Lett. 2003, 5, 89−
92.
(16) Leung, J. C.; Bedermann, A. A.; Njardarson, J. T.; Spiegel, D. A.;
Murphy, G. K.; Hama, N.; Twenter, B. M.; Dong, P.; Shirahata, T.;
McDonald, I. M.; Inoue, M.; Taniguchi, N.; McMahon, T. C.;
Schneider, C. M.; Tao, N.; Stoltz, B. M.; Wood, J. L. Angew. Chem., Int.
Ed. 2018, 57, 1991−1994.
(17) Townsend, R. J.; Moss, M. O.; Peck, H. M. J. Pharm. Pharmacol.
1966, 18, 471−473.
(18) Natori, S.; Sakaki, S.; Kurata, H.; Udagawa, S.; Ichinoe, M.; Saito,
M.; Umeda, M.; Ohtsuba, K. Appl. Microbiol. 1970, 19, 613−617.
(19) Wada, S. I.; Usami, I.; Umezawa, Y.; Inoue, H.; Ohba, S. I.;
Someno, T.; Kawada, M.; Ikeda, D. Cancer Sci. 2010, 101, 743−750.
(20) Harada, N.; Nakanishi, K. J. Am. Chem. Soc. 1969, 91, 3989−
3991.

J DOI: 10.1021/acs.jnatprod.9b00210
J. Nat. Prod. XXXX, XXX, XXX−XXX