Cytotoxic Nitrogenated Azaphilones from the Deep-Sea-Derived Fungus Chaetomium globosum MP4-S01-7

Article abstract of DOI:10.1021/acs.jnatprod.9b01165

Eight new nitrogenated azaphilones (1-8) and two known compounds (chaetoviridin A and chaetoviridin E, 9, 10) were isolated from the culture of the deep-sea-derived fungus Chaetomium globosum MP4-S01-7. The absolute configurations of new compounds were elucidated by HSQC-HECADE NMR data, J-based configuration analysis, and modified Mosher's method and finally verified by comparison of recorded and computed NMR chemical shifts from quantum chemical calculations coupled with a statistical procedure (DP4+). All of the compounds were evaluated for their in vitro cytotoxicities against the gastric cancer cell lines MGC803 and AGS, and most of them showed significant inhibition on cancer cell viability at 10 μM. Among them, compounds 1, 2, and 5 exerted the most potent cytotoxic activities, with IC₅₀ values less than 1 μM. Further studies showed that compound 2 inhibited cell cycle progression, and both compounds 1 and 2 induced apoptosis of gastric cancer cells in a concentration-dependent manner.

Full text of DOI:10.1021/acs.jnatprod.9b01165

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Article
Cytotoxic Nitrogenated Azaphilones from the Deep-Sea-Derived
Fungus Chaetomium globosum MP4-S01‑7
Weiyi Wang,*,∥ Jing Yang,∥ Yan-Yan Liao, Gang Cheng, Jing Chen, Xiang-Dong Cheng,*
Jiang-Jiang Qin,* and Zongze Shao
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ABSTRACT: Eight new nitrogenated azaphilones (1−8) and two
known compounds (chaetoviridin A and chaetoviridin E, 9, 10)
were isolated from the culture of the deep-sea-derived fungus
Chaetomium globosum MP4-S01-7. The absolute configurations of
new compounds were elucidated by HSQC-HECADE NMR data,
J-based configuration analysis, and modified Mosher’s method and
finally verified by comparison of recorded and computed NMR
chemical shifts from quantum chemical calculations coupled with a
statistical procedure (DP4+). All of the compounds were evaluated
for their in vitro cytotoxicities against the gastric cancer cell lines
MGC803 and AGS, and most of them showed significant
inhibition on cancer cell viability at 10 μM. Among them,
compounds 1, 2, and 5 exerted the most potent cytotoxic activities, with IC50 values less than 1 μM. Further studies showed that
compound 2 inhibited cell cycle progression, and both compounds 1 and 2 induced apoptosis of gastric cancer cells in a
concentration-dependent manner.
zaphilones are fungal secondary metabolites having an
oxabicyclic core that bears an oxygenated nonprotonated
derived fungus. The compounds were further evaluated for
their cytotoxicity in HO8910 and MGC803 cell lines, and N-
glutarylchaetoviridin C exhibited the most significant cytotox-
icity with IC50 values less than 10 μM.12 Li et al. also identified
nine new azaphilone alkaloids, termed penazaphilones A−I.13
Qin et al. isolated three azaphilone alkaloids, named
chaetomugilides A−C. Chaetomugilide C significantly blocked
HepG2 cell growth, and the IC50 value was determined to be
1.7 μM.8
Our recent investigation on a marine-derived fungus,
Chaetomium sp. NA-S01-R1, resulted in the isolation of four
new azaphilones, including three azaphilone alkaloids
(chaetoviridides A−C) with antibacterial and cytotoxic
activities.14 The present study was performed to identify new
cytotoxic compounds derived from the culture of a deep-sea
fungus, Chaetomium globosum MP4-S01-7, resulting in the
characterization of eight structurally related nitrogenated
azaphilones, 1−8, along with two known azaphilones,
chaetoviridin A (9) and chaetoviridin E (10). The structures
of the new compounds were determined by analysis of
A
carbon in position 7.1,2 They are often produced by
ascomycetes and have broad-spectrum bioactivities, including
cytotoxicity, anti-inflammatory, antimicrobial, and other
activities.3,4 In view of their promising pharmacological
properties and diverse structures, azaphilones have attracted
the attention of scientists for chemical or biological synthesis.
Chaetoviridins are a class of azaphilones produced by fungi of
the genera Chaetomium, possessing an angular lactone moiety
that bears a syn or anti aldol group, an attached chlorine atom
at C-5, and a pentenyl group attached to C-3.1,5,6
Chaetoviridin A was first elucidated by Natori in 1990, who
assigned the syn aldol side chain as (4′S, 5′R).5 The elucidation
of this configuration also helped the structural assignment of
other epimers.7−9 However, Xavier Franck revised the absolute
configuration of the positions of 4′ and 5′ in chaetoviridin A as
(4′R, 5′R) by total synthesis of (4′R, 5′R)-chaetoviridin A and
its related epimers.1
Naturally nitrogenated azaphilones are usually classified into
two types, Monascus pigments and sclerotioramine colorants.10
Nitrogenated Monascus pigments often contain an N-
containing isoquinoline skeleton bearing a γ-lactone ring, a
1-propenyl chain, and an n-hexanoyl or n-octanoyl side chain.
Nitrogenated sclerotioramine colorants commonly have an N-
containing isoquinoline moiety that harbors a branched heptyl
group.11 In addition, Li et al. isolated N-glutarylchaetoviridins
A−C from Chaetomium globosum HDN151398, a marine-
Received: December 5, 2019
© XXXX American Chemical Society and
American Society of Pharmacognosy
https://dx.doi.org/10.1021/acs.jnatprod.9b01165
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Chart 1
conventional NMR and HRMS data, J-based configuration
analysis (JBCA),15 modified Mosher’s method,16 and computa-
tional methods coupled with a statistical package (DP4+).17
We evaluated their cytotoxicities in two gastric cancer cell
lines, and compounds 1, 2, and 5 showed the most potent
cytotoxic activities. We further determined their effects on cell
cycle distribution and cell apoptosis.
respectively (Table 1 and Figure 1). The absolute config-
uration of C-7 was established as S based on the observation of
a negative Cotton effect at 379 nm in the ECD spectrum
(Figure S96) as reported by Steyn and Vleggaar.18 Oxidation
of compound 1 with chromium trioxide yielded the product of
2-methylbutyric acid with a positive specific rotation5 (Figure
S95), which unambiguously assigned C-11 as S. The relative
configuration of C-4′ and C-5′ was established on the basis of
3
JBCA. Small J values for H-4′/C-6′ and H-5′/C-7′ indicated
RESULTS AND DISCUSSION
■
2
their synclinal dispositions, and the medium JH4′‑C5′ value
Compound 1, obtained as a dark red solid, has a molecular
formula of C33H42ClNO5 measured by HRESIMS, suggesting
13 degrees of unsaturation. The chlorine atom was confirmed
by an isotopic peak for [M + H]+:[M + H + 2]+ with a ratio of
3:1. The 13C, DEPT, and HSQC spectra of 1 exhibited two
keto carbons (δC 181.5 and 201.5), one ester carbonyl carbon
(δC 168.9), two tetrasubstituted double bonds (δC 145.0 and
111.5; δC 122.7 and 169.2), four trisubstituted double bonds
(δC 141.2 and 99.8; δC 147.5 and 111.2; δC 116.4 and 144.5;
δC 123.2 and 132.4), and one disubstituted double bond (δC
119.4 and 149.0) (Table 1). These NMR spectroscopic data
showed that this compound was structurally similar to the co-
isolated compound chaetoviridin A (9). They shared the same
tricyclic core moiety bearing two side chains, a methyl-
branched pentenyl and an aldol group. The major differences
were the chemical shifts of C-1, from δC 151.6 in 9 to δC 141.2
in 1, and of C-3 from δC 157.0 in 9 to δC 147.5 in 1, and 10
extra resonances in 1 assigned as a 3,7-dimethyl-2,6-octadienyl
unit. In view of the changes in δC for C-1 and C-3 as well as its
molecular weight, a nitrogen atom at position 2 was
established, bearing a 3,7-dimethyl-2,6-octadienyl group,
deduced from COSY correlations of H2-1″/H-2″ and H2-4″/
H2-5″/H-6″ and HMBC correlations of H2-1″/C-1, H2-1″/C-
3, H2-1″/C-3″, H-2″/C-10″, H-2″/C-4″, H2-5″/C-3″, H3-8″/
C-6″, and H3-9″/C-6″ (Figure 1).
completed a set of values that matched with the pair of
interconvertible conformers in Figure 2A. The above data
allowed us to define the relative 4′R*, 5′R* configuration, the
same as is present in 9. Then, compound 1 was treated with
1
(R)- and (S)-MTPA-Cl separately, and analysis of the H
NMR spectra of the corresponding ester revealed the absolute
configuration of C-5′ was R (Figure 2B)16 and C-4′ was
proposed as R accordingly. Quantum chemical NMR shift
calculations and DP4+ probability analysis confirmed the 4′R*,
5′R* relative configuration (Figures 2C and S4).17 Therefore,
the absolute configuration for 1, named N-(3,7-dimethyl-2,6-
octadienyl)-2-aza-2-deoxychaetoviridin A, can be defined as
4′R, 5′R, 7S, 11S.
Compound 2 was obtained as a dark red powder with a
molecular formula identical to that of 1. An in-depth analysis of
its NMR data disclosed the same planar structure of 2 to 1, and
the variations of chemical shifts from C-4′ to C-7′ indicated an
aldol side chain in 2 with a different relative configuration
(Table 1). The modified Mosher’s method was also performed
to determine the 5′R configuration (Figure 2B), and C-4′ can
be assigned as S accordingly. This proposal was corroborated
by the small value of 3JH4′‑H5′, which indicated a gauche
disposition of H-4′ and H-5′ (Table 1). Further, the small
3
2
values of JH4′‑C6′ and JH4′‑C5′ revealed the syn and anti
orientations for H-4′/C-6′ and H-4′/5′-OH, respectively15
(Figure 2A). In addition, the observed NOE correlation
between H-4′/H3-6′ and no NOE correlation between H-5′/
The configurations of the double bonds C-9−C-10 and C-
3
2″−C-3″ were both determined as E by JH9−H10 (15.6) and
the NOE correlations of H2-1″/H3-10″ and H-2″/H2-4″,
B
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Table 1. NMR Data for 1−4 in Chloroform-d
b
b
a
a
1
2
3
4
no.
δC, type
δH (J in Hz)
δC, type
δH (J in Hz)
δC, type
δH (J in Hz)
δC, type
δH (J in Hz)
1
141.2, CH
8.78, s
141.4, CH
8.79, s
141.1, CH
8.77, s
141.3, CH
8.79, s
2
3
4
4a
5
147.5, C
111.2, CH
145.0, C
147.6, C
111.2, CH
145.1, C
147.5, C
111.2, CH
145.0, C
99.8, C
147.6, C
111.3, CH
145.1, C
99.8, C
6.88, s
6.89, s
6.87, s
6.89, s
99.8, C
99.8, C
6
7
181.5, C
88.9, C
181.3, C
89.1, C
181.5, C
88.9, C
181.4, C
89.1, C
8
8a
9
169.2, C
111.5, C
171.0, C
111.4, C
169.1, C
111.5, C
119.4, CH
149.0, CH
39.4, CH
29.1, CH2
11.8, CH3
19.4, CH3
27.6, CH3
168.9, C
122.7, C
201.6, C
50.7, CH
70.7, CH
21.3, CH3
13.7, CH3
52.3, CH2
116.6, CH
141.3, C
18.4, CH3
25.9, CH3
171.0, C
111.4, C
119.4, CH
149.2, CH
39.4, CH
29.1, CH2
11.8, CH3
19.4, CH3
27.5, CH3
169.0, C
121.3, C
201.8, C
48.2, CH
67.6, CH
19.3, CH3
10.0, CH3
52.3, CH2
116.5, CH
141.6, C
18.4, CH3
25.9, CH3
119.4, CH
149.0, CH
39.3, CH
29.1, CH2
11.8, CH3
19.4, CH3
27.6, CH3
168.9, C
6.22, d (15.6)
6.40, dd (15.6, 7.8)
2.29, m
119.4, CH
149.2, CH
39.4, CH
29.1, CH2
11.8, CH3
19.4, CH3
27.4, CH3
169.0, C
6.24, d (15.3)
6.41, dd (15.3, 7.8)
2.30, m
6.22, d (15.3)
6.40, dd (15.3, 7.8)
2.30, m
6.23, d (15.4)
6.40, dd (15.4, 7.7)
2.30, m
10
11
12
13
14
15
1′
2′
3′
4′
5′
6′
7′
1″
2″
3″
4″
5″
6″
7″
8″
9″
10″
1.47, m
1.48, m
1.47, m
1.48, m
0.94, t (7.5)
1.12, d (6.7)
1.71, s
0.94, t (7.4)
1.12, d (6.8)
1.72, s
0.94, t (7.3)
1.11, d (6.0)
1.71, s
0.94, t (7.3)
1.12, d (6.6)
1.72, s
122.7, C
201.5, C
121.3, C
201.6, C
50.7, CH
70.7, CH
21.3, CH3
13.7, CH3
52.5, CH2
116.4, CH
144.5, C
39.5, CH2
26.1, CH2
123.2, CH
132.4, C
3.71, qd (6.8, 6.6)
3.86, qd (6.5. 6.6)
1.13, d (6.5)
1.17, d (6.8)
4.50, m
48.2, CH
67.6, CH
19.3, CH3
10.1, CH3
52.5, CH2
116.3, CH
144.7, C
39.5, CH2
26.1, CH2
123.1, CH
132.4, C
3.79, qd (7.2, 2.8)
4.27, qd (6.4, 2.8)
1.22, d (6.4)
1.02, d (7.2)
4.52, m
3.70, qd (6.7, 6.5)
3.86, qd (6.4, 6.5)
1.13, d (6.4)
1.18, d (6.7)
4.49, d (6.4)
3.79, qd (7.2, 2.7)
4.28, qd (6.3, 2.7)
1.22, d (6.3)
1.02, d (7.2)
4.50, d (6.7)
5.29, t (6.8)
5.30, t (6.8)
5.29, t (6.4)
5.31, t (6.7)
2.16, m
2.16, m
5.07, m
2.16, m
2.16, m
5.06, m
1.85, s
1.88, s
1.86, s
1.88, s
25.7, CH3
17.8, CH3
16.9, CH3
1.68, s
1.60, s
1.84, s
25.7, CH3
17.8, CH3
16.9, CH3
1.67, s
1.60, s
1.84, s
a
b
1H NMR 600 MHz; 13C NMR 150 MHz. 1H NMR 850 MHz; 13C NMR 213 MHz.
H3-7′ demonstrated that H-4′ and C-6′ were close in space
while H-5′ and C-7′ were distant with an antiperiplanar
orientation (Figure 2A). The DP4+ analysis of compound 2
illuminated that the Conf2 showed a probability of 100% given
that both 1H and 13C NMR chemical shift values were
considered (Figures 2C and S6). Thus, compound 2 was
elucidated as 4′-epi-N-(3,7-dimethyl-2,6-octadienyl)-2-aza-2-
deoxychaetoviridin A.
Compound 3 was isolated as a dark red solid, and its
molecular formula was deduced as C28H34ClNO5 by
HRESIMS. The generally similar NMR data of 3 relative to
1 suggested that they shared the same stereogenic centers
while differing in the side chain attached to N-2. The loss of
five carbon resonances relative to 1, the trisubstituted double
bond (δC 116.6 and 141.3) (Table 1), COSY correlations of
H2-1″/H-2″, and HMBC correlations of H2-1″/C-1, H2-1″/C-
3, H2-1″/C-3″, H-2″/C-4″ and H-2″/C-5″ established that a
3-methyl-2-butenyl group was attached to N-2 (Figure 1).
Further 2D NMR analyses confirmed the structure as N-(3-
methyl-2-butenyl)-2-aza-2-deoxychaetoviridin A.
same stereogenic centers as 2, except for a 3-methyl-2-butenyl
unit attached to N-2 instead of a 3,7-dimethyl-2,6-octadienyl
group (Table 1 and Figure 1). Therefore, 4 was assigned as 4′-
epi-N-(3-methyl-2-butenyl)-2-aza-2-deoxychaetoviridin A.
Compounds 5 and 6 had the molecular formulas of
C33H40ClNO4 and C28H32ClNO4, respectively. Both com-
pounds shared similar NMR data, except for a 3,7-dimethyl-
2,6-octadienyl group in 5 and a 3-methyl-2-butenyl group in 6,
respectively. Comparing the spectroscopic data of 5 with 1, 5
possessed two extra olefinic carbons, C-4′ (δC 137.5) and C-5′
(δC 146.2), and lost two methine groups (Table 2). This
observation together with loss of a 18 Da mass revealed that 5
was a dehydrated derivative of 1 with a 2-methylbut-2-enoyl
group attached to the γ-lactone, which was authenticated by
HMBC correlations of H-5′ with C-3′/C-4′, H-6′ with C-4′/
C-5′, and H-7′ with C-4′/C-5′ (Figure 1). By careful
comparison with the spectroscopic data of chaetoviridin E
(10), combined with an NOE correlation between H3-7′/H-1
and no NOE correlation between H-5′/H-1 (Figure 1), the E
configuration was assigned to the double bond C4′−C5′.
Consequently, 5 was determined as N-(3,7-dimethyl-2,6-
octadienyl)-2-aza-2-deoxychaetoviridin E and 6 was estab-
Compound 4 was isolated as a dark red solid with a
molecular formula of C28H34ClNO5 deduced by HRESIMS.
An in-depth analysis of NMR data revealed that it shared the
C
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Figure 1. Selected key NOESY, COSY, and HMBC correlations of compounds 1, 2, 5, 6, and 8.
and Figure 1), and named 4′,5′-dinor-5′-deoxy-N-(3,7-
dimethyl-2,6-octadienyl)-2-aza-2-deoxychaetoviridin A.
Although the NMR spectra of 7 and 8 were generally similar,
they differed mainly in the moiety attached to N-2. Detailed
HMBC and COSY correlations confirmed a 3-methyl-2-
butenyl group attached to N-2 in 8 instead of a 3,7-
dimethyl-2,6-octadienyl group in 7 (Figure 1 and Table 1).
The structure of 8 was established and termed 4′,5′-dinor-5′-
deoxy-N-(3-methyl-2-butenyl)-2-aza-2-deoxychaetoviridin A.
The structures of chaetoviridin A (9) and chaetoviridin E
(10) were determined by comparison of their NMR data to
those reported in the literature as well as the single-crystal X-
ray diffraction study for chaetoviridin A (9) (Figure 3, S89−
S92 and Table S24).5,19
Compounds 1−10 were evaluated for their effects on the
viability of gastric cancer MGC803 and AGS cell lines. The
majority of the compounds exhibited significant cytotoxicities
against both cell lines, and their IC50 values were determined
and are summarized in Table 3. Importantly, compounds 1, 2,
and 5 exhibited the most potent cytotoxic activities with IC50
values less than 1 μM. In addition, compound 2 exhibited
similar inhibitory effects on both MGC803 and AGC cells,
whereas compounds 1 and 5 showed more significant
cytotoxicities against AGS cells.
We evaluated the effects of compound 2 on the cell cycle
distribution of MGC803 cells. Compound 2 arrested MGC803
cells at the G1 phase in a concentration-dependent way
(Figure 4). Compared to the control group, compound 2 at 1
μM increased accumulation of the cells in the G1 phase from
45% to 58% (p < 0.01).
Figure 2. Structural elucidation of compounds 1 and 2 at C-4′ and C-
5′. (A) Conformers around the C4′−C5′ bond compatible with the
JBCA. (B) MTPA ester analysis (values are ΔδH = δS − δR) of
compounds 1 and 2 in chloroform-d. (C) DP4+ analysis results for
compounds 1 and 2.
lished as N-(3-methyl-2-butenyl)-2-aza-2-deoxychaetoviridin
E.
We further found that compounds 1 and 2 concentration-
dependently induced apoptosis in both MGC803 and AGS cell
lines (Figure 5). After treatment with 1 and 5 μM compound 1
for 48 h, 11% (p < 0.05) and 95% (p < 0.01) of MGC803 cells
and 47% (p < 0.01) and 90% (p < 0.01) of AGS cells
underwent apoptosis, respectively, which were significantly
higher than that of control cells. Similar results have also been
Compounds 7 and 8 were purified as dark red, amorphous
powders, and their molecular formulas were determined to be
C31H38ClNO4 and C26H30ClNO4, respectively. Comparing the
NMR data of 7 with 1, 7 was characterized as an analogue of 1
with the aldol side chain attached to C-3′ replaced by an ethyl
unit, confirmed by the HMBC correlations of H2-4′/C-3′ and
H3-5′/C-3′ and COSY correlation of H2-4′/H3-5′ (Table 2
D
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Table 2. NMR Data for 5−8 in Chloroform-d
b
a
b
a
5
6
7
8
no.
δC, type
δH (J in Hz)
δC, type
δH (J in Hz)
δC, type
δH (J in Hz)
δC, type
δH (J in Hz)
1
137.5, CH
7.96, s
137.4, CH
7.95, s
141.2, CH
8.83, s
141.1, CH
8.83, s
2
3
4
4a
5
147.7, C
110.5, CH
144.8, C
99.8, C
147.7, C
110.6, CH
144.8, C
99.8, C
147.6, C
111.0, CH
145.1, C
99.7, C
147.5, C
111.0, CH
145.1, C
99.7, C
6.84, s
6.84, s
6.88, s
6.87, s
6
7
181.6, C
88.8, C
181.6, C
88.8, C
181.5, C
88.7, C
181.5, C
88.7, C
8
8a
9
164.3, C
112.2, C
119.5, CH
148.9, CH
39.3, CH
29.1, CH2
11.8, CH3
19.4, CH3
26.4, CH3
168.3, C
127.7, C
191.2, C
137.5, C
164.2, C
112.2, C
119.5, CH
148.9, CH
39.3, CH
29.1, CH2
11.8, CH3
19.4, CH3
26.4, CH3
168.3, C
124.7, C
191.2, C
137.5, C
168.7, C
111.5, C
119.5, CH
149.0, CH
39.4, CH
29.2, CH2
11.8, CH3
19.4, CH3
27.3, CH3
168.8, C
168.8, C
111.5, C
119.5, CH
149.0, CH
39.4, CH
29.2, CH2
11.8, CH3
19.4, CH3
27.3, CH3
168.7, C
122.4, C
197.7, C
35.7, CH2
6.20 (15.3)
6.37, dd (15.3, 7.9)
2.28, m
6.20, d (15.6)
6.37, dd (15.6, 7.8)
2.28, m
6.22, d (15.6)
6.39, dd (15.3, 7.8)
2.29, m
6.23, d (15.4)
6.39, dd (15.3, 7.8)
2.30, m
10
11
12
13
14
15
1′
2′
3′
4′
1.47, m
1.46, m
1.47, m
1.47, m
0.93, t (7.4)
1.10, d (6.8)
1.70, s
0.93, t (7.3)
1.11, d (6.6)
1.70, s
0.94, t (7.5)
1.12, d (6.8)
1.70, s
0.94, t (7.4)
1.12, d (6.6)
1.70, s
122.4, C
197.7, C
35.7, CH2
2.78, qd (7.1, 19.1);
3.26, qd (7.1, 19.1)
1.07, t (7.1)
2.78, qd (7.1, 19.0);
3.26, qd (7.1, 19.0)
1.07, t (7.1)
5′
6′
7′
146.2, CH
15.4, CH3
10.9, CH3
52.2, CH2
116.4, CH
144.5, C
39.5, CH2
26.1, CH2
123.1, CH
132.4, C
6.55, q (6.9)
1.87, d (7.0)
1.80, s
4.43, m
5.21, t (6.8)
146.3, CH
15.4, CH3
10.8, CH3
51.9, CH2
116.5, CH
141.5, C
6.55, q (7.0)
1.88, d (7.0)
1.84, s
4.4, m
5.22, t (6.5)
7.4, CH3
7.4, CH3
1″
2″
3″
4″
5″
6″
7″
8″
9″
10″
52.5, CH2
116.5, CH
144.3, C
39.5, CH2
26.1, CH2
123.2, CH
132.4, C
25.7, CH3
17.7, CH3
16.9, CH3
4.52, m
5.30, t (6.8)
52.4, CH2
116.7, CH
141.2, C
18.4, CH3
25.8, CH3
4.5, d (6.8)
5.31, t (6.4)
2.15, m
2.15, m
5.06, m
18.3, CH3
25.8, CH3
1.82, s
1.87, s
2.17, m
2.16, m
5.07, m
1.86, s
1.88, s
25.7, CH3
17.7, CH3
16.9, CH3
1.67, s
1.60, s
1.83, s
1.67, s
1.60, s
1.85, s
a
b
1H NMR 600 MHz; 13C NMR 150 MHz. 1H NMR 850 MHz; 13C NMR 213 MHz.
Table 3. IC50 Values of Compounds 1−10 in Human Gastric
Cancer Cells
IC50 (μM)
compound
MGC803
AGS
1
2
3
0.78
0.46
2.7
0.12
0.62
6.5
4
3.0
2.9
5
6
0.72
6.8
0.12
2.0
7
2.2
1.2
8
9
10
5.8
>10
>10
>10
53
>10
>10
3.8
Figure 3. X-ray structure of compound 9.
a
paclitaxel (nM)
a
Positive control.
observed for compound 2 in both MGC803 and AGS cell
lines.
In the present study, we identified four N-(3,7-dimethyl-2,6-
octadienyl)azaphilone alkaloids, four N-(3-methyl-2-butenyl)-
azaphilone alkaloids, and two known compounds produced by
the deep-sea-derived fungus C. globosum MP4-S01-7. All of the
compounds were examined for cytotoxicities in gastric cancer
cells. Compounds 1, 2, and 5 showed the most effective
antigastric cancer activities with IC50 values less than 1 μM. We
also found that compound 2 arrested gastric cancer cells in the
E
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Figure 4. Compound 2 alters the cell cycle distribution of MGC803 cells (#p < 0.01).
MeOH and filtered through Whatman filter paper three times. The
organic extract was reduced in vacuo and then partitioned between
H2O and EtOAc. The EtOAc partition was reduced in vacuo to give
an extract (50 g). It was separated by silica gel CC (4.5 × 20 cm;
200−300 mesh) using petroleum ether−EtOAc (9.5:0.5, 9.0:1.0,
8.5:1.5, 8.0:2.0, v:v) to yield four fractions (Frs1−4). Fr1 was found
to mainly consist of aliphatic compounds and not further processed.
Frs2−4 were combined and applied to an MCI gel column (4.5 × 45
cm, 75−150 μM) and eluted using a MeOH−H2O gradient system
(from 1:1 to 1:0, v/v) to yield four subfractions (SFs1−4), which
were pooled based on TLC analysis. SF1 was further resolved by flash
silica gel chromatography (90% petroleum ether in acetone),
preparative HPLC (75% MeCN in H2O, 8 mL/min), and Sephadex
LH-20 (1.0 × 120 cm; MeOH) to yield 3 (8.0 mg) and compound 2
(9.8 mg). SF2 was further subjected to Sephadex LH-20 (2.0 × 150
cm; MeOH) to obtain two subfractions (SF2a and SF2b). SF2a was
chromatographed by preparative HPLC (88% MeCN in H2O, 8 mL/
min) to yield compound 1 (35 mg, tR 19.1 min). SF2b was applied to
preparative HPLC (70% MeCN in H2O, 8 mL/min) to give 4 (3.0
mg, tR 10.4 min) and 10 (3.1 mg, tR 17.2 min). SF3 was
chromatographed with a gradient elution of MeOH−H2O (1:1 to
1:0) on RP-C18 CC to give two subfractions (SF3a and SF3b). SF3a
was purified by Sephadex LH-20 (1.0 × 120 cm; MeOH) to obtain 9
(25.0 mg), and SF3b was further separated by Sephadex LH-20 (1.0 ×
120 cm; MeOH) and preparative HPLC (88% MeCN in H2O, 8 mL/
min) to obtain compounds 5 (5.2 mg, tR 22.6 min) and 7 (4.5 mg, tR
25.2 min). SF4 was separated by preparative HPLC (70% MeOH in
H2O) and Sephadex LH-20 (1.0 × 120 cm; MeOH) to obtain
compounds 6 (3.0 mg) and 8 (7.2 mg).
G1 phase and compounds 1 and 2 induced gastric cancer cell
apoptosis in a concentration-dependent manner. A primary
analysis of the structure−activity relationships revealed that the
3,7-dimethyl-2,6-octadienyl group attached to N-2 contributed
to the potent cytotoxic activities against gastric cancer
MGC803 and AGS cell lines.
EXPERIMENTAL SECTION
■
General Experimental Procedures. The optical rotations were
determined by an Anton Paar MCP 500 polarimeter. The UV spectra
were obtained on a UV-1800 spectrophotometer (Shimadzu). ECD
spectra were recorded on a J-715 spectropolarimeter (JASCO
Corporation). IR spectra were recorded on a Bruker Tensor 27
spectrometer using KBr pellets. NMR data were measured on a
Bruker Avance 600 and/or 850 MHz spectrometer. HRESIMS were
determined by a Xevo G2 Q-TOF mass spectrometer (Waters).
Column chromatography (CC) was carried out on silica gel (100−
200, 200−300 mesh, Yantai) and Sephadex LH 20 (GE Healthcare
Bio-Sciences AB), RP-C18 (ODA-A, 50 μM, YMC), and MCI gel
(CHP20/P120, 75−150 μM, Mitsubishi Chemical Corporation).
Preparative HPLC was carried out with a P3000 pump (CXTH) and
a UV3000 ultraviolet−visible detector (CXTH) with a preparative
RP-C18 column (5 μM, 20 mm × 250 mm, YMC). X-ray
crystallography analysis was carried out on a Bruker APEX-II CCD
diffractometer.
Fungal Strain. The strain Chaetomium globosum MP4-S01-7 was
obtained from a water sample collected at a depth of 4300 m
(19°57′32.9205″ N, 161°51′47.3181″ E) in the West Pacific Ocean in
2017. The ITS sequence has been deposited at GenBank (accession
number MN099445). The fungus was deposited at the China Center
for Type Culture Collection (CCTCC) under the accession number
CCTCC M 2019509.
Fermentation, Extraction, and Isolation. The fermentation
was performed on a rice solid medium statically (each flask contained
70 g of rice, 2 g of D-glucose, 1 g of maltose, 1 g of mannitol, 0.3 g of
peptone, 0.1 g of corn flour, 0.1 g of sodium glutamate, and 100 mL of
seawater collected from Baicheng Beach near the author’s institution,
adjusting its pH to 6.5) in 40 × 1 L Erlenmeyer flasks for 25 days at
room temperature (rt). The fermented broth was extracted with
N-(3,7-Dimethyl-2,6-octadienyl)-2-aza-2-deoxychaetoviridin A
(1): dark red, amorphous powder (CHCl3); [α]2D5 +592 (c 0.024,
CHCl3); UV (MeOH) λmax (log ε) 294 (4.21), 379 (3.72) nm; ECD
(c 0.33 mg/mL, MeOH) λmax (Δε) 379 (−23.4), 308.5 (29.1), 269
(−4.6), 230.5 (−22.9) nm; IR (KBr) νmax 3462, 2965, 2928, 2875,
1755, 1678, 1596, 1485, 1460, 1375, 1251, 1191, 1134, 985, 931, 880,
694 cm−1; 1H and 13C NMR data, Table 1; HRESIMS m/z 568.2835
[M + H]+ (calcd for C33H4335ClNO5, 568.2830), m/z 570.2821 [M +
H]+ (calcd for for C33H4337ClNO5, 570.2818).
4′-epi-N-(3,7-Dimethyl-2,6-octadienyl)-2-aza-2-deoxychaetoviri-
din A (2): dark red, amorphous powder (CHCl3); [α]2D5 +344 (c
0.038, CHCl3); UV (MeOH) λmax (log ε) 293 (4.25), 379 (3.73) nm;
F
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#
Figure 5. Compounds 1 and 2 induced apoptosis in MGC803 and AGS cells (*p < 0.05; p < 0.01).
ECD (c 0.33 mg/mL, MeOH) λmax (Δε) 382.5 (−29.3), 309.5 (34.9),
270.5 (−5.8), 235 (−22.0) nm; IR (KBr) νmax 3417, 2966, 2926,
1756, 1680, 1597, 1485, 1462, 1374, 1252, 1191, 1134, 1111, 1014,
985, 932, 880, 696 cm−1; 1H and 13C NMR data, Table 1; HRESIMS
m/z 568.2851 [M + H]+ (calcd for C33H4335ClNO5, 568.2830), m/z
570.2836 [M + H]+ (calcd for for C33H4337ClNO5, 570.2818).
N-(3-Methyl-2-butenyl)-2-aza-2-deoxychaetoviridin A (3): dark
red, amorphous powder (CHCl3); [α]2D5 +26 (c 0.02, CHCl3); UV
(MeOH) λmax (log ε) 298 (4.22), 382 (3.85) nm; ECD (c 0.50 mg/
mL, MeOH) λmax (Δε) 380 (−21.7), 308.5 (25.7), 268.5 (−4.1), 230
(−22.2) nm; IR (KBr) νmax 3455, 2965, 2928, 2875, 1755, 1684,
1596, 1485, 1460, 1376, 1252, 1192, 1135, 1108, 1015, 985, 932, 880
cm−1; 1H and 13C NMR data, Table 1; HRESIMS m/z 500.2199 [M
+ H]+ (calcd for C28H3535ClNO5, 500.2204), m/z 502.2183 [M + H]+
(calcd for for C28H3537ClNO5, 502.2188).
4′-epi-N-(3-Methyl-2-butenyl)-2-aza-2-deoxychaetoviridin A (4):
dark red, amorphous powder (CHCl3); [α]2D5 +37 (c 0.01, CHCl3);
UV (MeOH) λmax (log ε) 293 (4.35), 380 (3.87) nm; ECD (c 0.50
mg/mL, MeOH) λmax (Δε) 384 (−32.2), 309.5 (37.1), 269 (−8.0),
234.5 (−27.1) nm; IR (KBr) νmax 3418, 2965, 2927, 2875, 1754,
G
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1595, 1526, 1484, 1376, 1256, 1192, 1136, 1018, 979, 933, 880, 800,
695 cm−1; 1H and 13C NMR data, Table 1; HRESIMS m/z 500.2197
[M + H]+ (calcd for C28H3535ClNO5, 500.2204), m/z 502.2182 [M +
H]+ (calcd for for C28H3537ClNO5, 502.2188).
5.33 (1H, qd, J = 6.3, 8.9 Hz, H-5′), 5.28 (1H, t, J = 6.7 Hz, H-2″),
5.07 (1H, m, H-6″), 4.47 (2H, m, H-1″), 3.90 (1H, m, H-4′), 3.31
(3H, s, OMe of MTPA), 2.30 (1H, m, H-11), 2.17 (2H, m, H-5″),
2.16 (2H, m, H-4″), 1.84 (3H, s, H-10″), 1.70 (3H, s, H-15), 1.67
(3H, s, H-8″), 1.60 (3H, s, H-9″), 1.48 (2H, m, H-12), 1.34 (3H, d, J
= 6.3 Hz, H-6′), 1.19 (3H, d, J = 6.9 Hz, H-7′), 1.13 (3H, d, J = 6.7
Hz, H-14), 0.94 (3H, t, J = 7.4 Hz, H-13); HRESIMS m/z 784.3236
[M + H]+ (calcd for C43H5035ClF3NO7, 784.3228), m/z 786.3229 [M
+ H]+ (calcd for C43H5037ClF3NO7, 786.3225).
N-(3,7-Dimethyl-2,6-octadienyl)-2-aza-2-deoxychaetoviridin E
(5): orange, amorphous powder (CHCl3); [α]2D5 +924 (c 0.025,
CHCl3); UV (MeOH) λmax (log ε) 292 (4.27), 385 (3.71) nm; ECD
(c 0.33 mg/mL, MeOH) λmax (Δε) 387.5 (−11.1), 305 (13.9), 269.5
(−1.0), 221 (−9.0) nm; IR (KBr) νmax 2963, 2926, 2874, 1760, 1638,
1597, 1529, 1486, 1461, 1376, 1250, 1189, 1140, 1017, 982, 934, 879,
1
(R)-MTPA ester of 1 (1b): H NMR (chloroform-d, 850 MHz) δH
1
704, 667 cm−1; H and 13C NMR data, Table 2; HRESIMS m/z
8.89 (1H, s, H-1), 7.34 (5H, m, phenyl protons), 6.89 (1H, s, H-4),
6.39 (1H, dd, J = 15.4, 7.8 Hz, H-10), 6.22 (1H, d, J = 15.5 Hz, H-9),
5.34 (1H, qd, J = 6.3, 9.4 Hz, H-5′), 5.29 (1H, t, J = 6.7 Hz, H-2″),
5.08 (1H, m, H-6″), 4.47 (2H, m, H-1″), 3.89 (1H, qd, J = 6.8, 9.3
Hz, H-4′), 3.07 (3H, s, OMe of MTPA), 2.29 (1H, m, H-11), 2.17
(2H, m, H-5″), 2.17 (2H, m, H-4″), 1.84 (3H, s, H-10″), 1.72 (3H, s,
H-15), 1.67 (3H, s, H-8″), 1.61 (3H, s, H-9″), 1.47 (2H, m, H-12),
1.25 (3H, d, J = 6.3 Hz, H-6′), 1.23 (3H, d, J = 6.8 Hz, H-7′), 1.12
(3H, d, J = 6.7 Hz, H-14), 0.93 (3H, t, J = 7.4 Hz, H-13); HRESIMS
m/z 784.3223 [M + H]+ (calcd for C43H5035ClF3NO7, 784.3228), m/
z 786.3216 [M + H]+ (calcd for C43H5037ClF3NO7, 786.3225).
550.2711 [M + H]+ (calcd for C33H4135ClNO4, 550.2724), m/z
552.2695 [M + H]+ (calcd for for C33H4137ClNO4, 552.2712).
N-(3-Methyl-2-butenyl)-2-aza-2-deoxychaetoviridin E (6): or-
ange, amorphous powder (CHCl3); [α]2D5 +14 (c 0.02, CHCl3); UV
(MeOH) λmax (log ε) 298 (4.03), 390 (3.75) nm; ECD (c 0.50 mg/
mL, MeOH) λmax (Δε) 381.5 (−7.7), 304 (9.8), 272.5 (−0.7), 221.5
(−7.7) nm; IR (KBr) νmax 2961, 2926, 2855, 1759, 1639, 1597, 1527,
1485, 1461, 1377, 1253, 1190, 1177, 1139, 1109, 1018, 980, 934, 879,
1
705, 663 cm−1; H and 13C NMR data, Table 2; HRESIMS m/z
482.2102 [M + H]+ (calcd for C28H3335ClNO4, 482.2098), m/z
484.2086 [M + H]+ (calcd for for C28H3337ClNO4, 484.2082).
4′,5′-dinor-5′-Deoxy-N-(3,7-dimethyl-2,6-octadienyl)-2-aza-2-
deoxychaetoviridin A (7): dark red, amorphous powder (CHCl3);
[α]2D5 +947 (c 0.023, CHCl3); UV (MeOH) λmax (log ε): 294 (4.18),
379 (3.69) nm; ECD (c 0.33 mg/mL, MeOH) λmax (Δε) 379.5
(−24.4), 309 (33.0), 269 (−5.0), 229.5 (−21.1) nm; IR (KBr) νmax
2963, 2928, 2875, 1756, 1686, 1602, 1487, 1460, 1377, 1246, 1192,
1124, 1033, 978, 927, 880, 690 cm−1; 1H and 13C NMR data, Table 2;
HRESIMS m/z 524.2597 [M + H]+ (calcd for C31H3935ClNO4,
524.2856), m/z 526.2584 [M + H]+ (calcd for for C31H3937ClNO4,
526.2554).
1
(S)-MTPA ester of 2 (2a): H NMR (chloroform-d, 850 MHz) δH
8.61 (1H, s, H-1), 7.42 (5H, m, phenyl protons), 6.90 (1H, s, H-4),
6.40 (1H, dd, J = 15.5, 7.8 Hz, H-10), 6.21 (1H, d, J = 15.4 Hz, H-9),
5.58 (1H, m, H-5′), 5.27 (1H, t, J = 6.7 Hz, H-2″), 5.06 (1H, m, H-
6″), 4.46 (2H, m, H-1″), 3.93 (1H, qd, J = 7.1, 5.3 Hz, H-4′), 3.53
(3H, s, OMe of MTPA), 2.28 (1H, m, H-11), 2.14 (2H, m, H-5″),
2.14 (2H, m, H-4″), 1.80 (3H, s, H-10″), 1.74 (3H, s, H-15), 1.67
(3H, s, H-8″), 1.60 (3H, s, H-9″), 1.47 (2H, m, H-12), 1.43 (3H, d, J
= 6.4 Hz, H-6′), 1.11 (3H, d, J = 6.7 Hz, H-14), 0.93 (3H, t, J = 7.1
Hz, H-7′), 0.92 (3H, d, J = 7.1 Hz, H-13); HRESIMS m/z 784.3237
[M + H]+ (calcd for C43H5035ClF3NO7, 784.3228), m/z 786.3231 [M
+ H]+ (calcd for C43H5037ClF3NO7, 786.3225).
4′,5′-dinor-5′-Deoxy-N-(3-methyl-2-butenyl)-2-aza-2-deoxy-
chaetoviridin A (8): dark red, amorphous powder (CHCl3); [α]D25
+66 (c 0.02, CHCl3); UV (MeOH) λmax (log ε) 299 (4.18), 385
(3.81) nm; ECD (c 0.50 mg/mL, MeOH) λmax (Δε) 380.5 (−20.5),
308.5 (28.9), 269 (−4.5), 229.5 (−19.4) nm; IR (KBr) νmax 2966,
2930, 2874, 1756, 1683, 1601, 1486, 1458, 1376, 1247, 1194, 1124,
1031, 978, 926, 868, 753, 690 cm−1; 1H and 13C NMR data, Table 2;
HRESIMS m/z 456.1935 [M + H]+ (calcd for C26H3135ClNO4,
456.1942), m/z 458.1913 [M + H]+ (calcd for for C26H3137ClNO4,
458.1924).
X-ray Crystallography for Chaetoviridin A (9). Crystal data for
C23H25ClO6 (M = 432.88 g/mol): monoclinic, space group P21 (no.
4), a = 12.1671(8) Å, b = 5.4176(4) Å, c = 16.7266(11) Å, β =
107.932(4)°, V = 1049.00(13) Å3, Z = 2, T = 100.0 K, μ(Cu Kα) =
1.935 mm−1, Dcalc = 1.370 g/cm3, 6402 reflections measured (5.554°
≤ 2θ ≤ 130.064°), 3380 unique (Rint = 0.0692, Rsigma = 0.0864),
which were used in all calculations. The final R1 was 0.0620 (I >
2σ(I)) and wR2 was 0.1701 (all data). The goodness of fit on F2 was
1.094. Flack parameter = 0.076(19).
1
(R)-MTPA ester of 2 (2b): H NMR (chloroform-d, 850 MHz) δH
8.55 (1H, s), 7.45 (5H, m, phenyl protons), 6.90 (1H, s, H-4), 6.40
(1H, dd, J = 15.5, 7.8 Hz, H-10), 6.19 (1H, d, J = 15.4 Hz, H-9), 5.62
(1H, qd, J = 6.3, 4.4 Hz, H-5′), 5.21 (1H, t, J = 6.6 Hz, H-2″), 5.05
(1H, tq, J = 5.3, 1.5 Hz, H-6″), 4.38 (2H, m, H-1″), 3.98 (1H, qd, J =
7.1, 4.4 Hz, H-4′), 3.52 (3H, s, OMe of MTPA), 2.28 (1H, m, H-11),
2.14 (2H, m, H-5″), 2.11 (2H, t, J = 5.0 Hz, H-4″), 1.77 (3H, s, H-
10″), 1.73 (3H, s, H-15), 1.67 (3H, s, H-8″), 1.59 (3H, s, H-9″), 1.47
(2H, m, H-12), 1.37 (3H, d, J = 6.3 Hz, H-6′), 1.11 (3H, d, J = 6.8
Hz, H-14), 1.08 (3H, d, J = 7.1 Hz, H-7′), 0.93 (3H, t, J = 7.4 Hz, H-
13); HRESIMS m/z 784.3237 [M
+
H]+ (calcd for
C43H5035ClF3NO7, 784.3228), m/z 786.3224 [M + H]+ (calcd for
C43H5037ClF3NO7, 786.3225).
Chromium Trioxide Oxidation of Compound 1. Oxidation of
compound 1 (21.0 mg) was carried out using a reagent prepared from
CrO3 (30.0 mg), HOAC (900 μL), H2SO4 (25 μL), and H2O (25
μL). After being stirred for 20 h at rt, the reaction mixture was
extracted with EtOAc three times. The combined organic layer was
washed with H2O, evaporated, and applied to preparative HPLC
(30% MeCN in H2O, 10 mL/min, 205 nm) to afford the compound
(S)-2-methylbutyric acid (1.0 mg, tR 13.9 min) (Figure S113), which
was identified by comparison of the NMR data (Figures S93 and S94)
and specific rotation value (Figure S95) to those reported in the
literature.5
The crystallographic data for 9 have been deposited at the
Cambridge Crystallographic Data Centre with the deposition number
of CCDC 1918313. Copies of the data can be obtained, free of
charge, on application to the Director, Crystallographic Data Center
(CCDC), 12 Union Road, Cambridge CB2 1EZ, UK (fax: + 44-0-
1223-336033 or e-mail: deposit@ccdc.cam.ac.uk).
Quantum Chemical Calculations. The conformational search
was carried out using the conformer rotamer ensemble sampling tool
(crest).20 All theoretical calculations were carried out using the
Gaussian 09 program package.21 More details about the computa-
tional method, the optimized conformation geometries, thermody-
namic parameters, population of all conformations, and the DP4+
probability analysis are provided in the Supporting Information.
Cell Lines and Culture. Human gastric cancer MGC803 and
AGS cell lines were purchased from the Cell Bank of the Chinese
Academy of Science. Both cell lines were cultured in RPMI 1640
media (Hyclone) supplemented with 10% fetal bovine serum and 1%
penicillin/streptomycin from Northend Biotechnology and kept in an
incubator with 5% CO2 at 37 °C.
Preparation of (S)- and (R)-MTPA Ester Derivatives of 1 and
2. The Mosher’s esters were prepared based on the standard
procedure.16 Compound 1 (2.1 mg) was dissolved in 500 μL of
anhydrous CHCl3. Then dry pyridine (1.2 mg) and (R)-MTPA-Cl
(1.9 mg) were added to the solution. The reaction was stirred at rt for
2 h. The target product was purified by preparative HPLC (85%
MeOH in H2O) to get the (S)-MTPA ester 1a (0.8 mg). Identical
conditions were used to prepare the (R)-MTPA ester 1b (0.7 mg)
with (S)-MTPA-Cl. (S)-MTPA and (R)-MTPA esters of 2, 2a (0.8
mg), and 2b (0.6 mg) were prepared based on the above protocol.
1
(S)-MTPA ester of 1 (1a): H NMR (chloroform-d, 850 MHz) δH
8.88 (1H, s, H-1), 7.29 (5H, m, phenyl protons), 6.88 (1H, s, H-4),
6.39 (1H, dd, J = 15.4, 7.9 Hz, H-10), 6.21 (1H, d, J = 15.3 Hz, H-9),
H
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Cell Viability Assay. The Cell Counting Kit 8 (Nuoyang Biotech)
assay was performed to examine the cytotoxicity of compounds 1−10
in gastric cancer cells. Briefly, MGC803 and AGS cells were cultured
in a 96-well plate (3 × 103 cells per well) overnight and then exposed
to compounds 1−10 at different concentrations (0.1−10 μM). After
72 h of exposure, the cells were incubated with CCK8 solution and
examined at 450 nm. The IC50 values were calculated as described
previously.22
Jing Chen − College of Pharmaceutical Sciences, Zhejiang
Chinese Medical University, Hangzhou 310053, People’s
Republic of China
Zongze Shao − Key Laboratory of Marine Biogenetic Resources,
Third Institute of Oceanography, Ministry of Natural Resources,
Xiamen 361005, People’s Republic of China
Complete contact information is available at:
Cell Cycle Distribution Analysis. The effects of compound 2 on
cell cycle distribution were determined as described previously.22
Briefly, MGC803 cells were kept in a six-well plate (3 × 105 cells per
well) overnight and exposed to compound 2 (0, 0.2, 0.5, or 1 μM) for
24 h. Further, MGC803 cells were trypsinized and maintained in
fixing buffer. The fixed cells were further incubated with RNase and
staining reagents (4A Biotech Co., Ltd.) and analyzed by flow
cytometry.
https://pubs.acs.org/10.1021/acs.jnatprod.9b01165
Author Contributions
∥W. Wang and J. Yang contributed equally to this work.
Notes
The authors declare no competing financial interest.
Detection of Apoptosis. The effects of compounds 1 and 2 on
cell apoptosis were determined as described previously.22 Briefly,
MGC803 and AGS cells in six-well plates (3 × 105 cells/well) were
treated with compound 1 or 2 (0, 0.2, 0.5, 1, or 5 μM) for 48 h. Upon
treatment, the cells were collected and washed after trypsinization.
The cells were further suspended in the mixture of the binding buffer
and staining reagents from Apoptosis Detection Kit I (BD
Pharmingen). All apoptotic cells were detected by the flow cytometer.
ACKNOWLEDGMENTS
■
The authors thank the specialists from Academy of Chinese
Medical Sciences, Zhejiang Chinese Medical University, for
their technical support of this work. This research was funded
by the Natural Science Foundation of Fujian Province
(2018J01064), COMRA program (DY135-B2-01 and
DY135-B2-05), the Foundation of Third Institute of Ocean-
ography SOA (2018021 and 2017001), National Natural
Science Foundation of China (81903842, 81573953), Program
of Zhejiang Provincial TCM Sci-tech Plan (2020ZZ005,
2016ZZ012), Zhejiang Chinese Medical University Startup
Funding (111100E014), Zhejiang Provincial Science and
Technology Projects (2019C03049), Medical Science and
Technology Project of Zhejiang Province (WKJ-ZJ-1728), and
Traditional Chinese Medical Science and Technology Major
Project of Zhejiang Province (2018ZY006). The authors also
thank the assistant from High-Field Nuclear Magnetic
Resonance Research Center of Xiamen University for
providing kind help during the experiments.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jnatprod.9b01165.
■
sı
DFT computational data and results of DP4+ analysis of
compounds 1 and 2; detailed 1D and 2D NMR,
HRESIMS, UV, IR, and ECD spectra for compounds 1−
8 (PDF)
Crystallographic data for 9 (CIF)
AUTHOR INFORMATION
Corresponding Authors
■
REFERENCES
Weiyi Wang − Key Laboratory of Marine Biogenetic Resources,
Third Institute of Oceanography, Ministry of Natural Resources,
Xiamen 361005, People’s Republic of China; orcid.org/
0000-0003-3163-9491; Phone: +86-592-2195518;
Email: wywang@tio.org.cn
■
(1) Makrerougras, M.; Coffinier, R.; Oger, S.; Chevalier, A.; Sabot,
C.; Franck, X. Org. Lett. 2017, 19, 4146−4149.
(2) Luo, X.; Lin, X.; Tao, H.; Wang, J.; Li, J.; Yang, B.; Zhou, X.; Liu,
Y. J. Nat. Prod. 2018, 81, 934−941.
(3) Gao, J. M.; Yang, S. X.; Qin, J. C. Chem. Rev. 2013, 113, 4755−
4811.
Xiang-Dong Cheng − Institute of Cancer and Basic Medicine,
Chinese Academy of Sciences; Cancer Hospital of the University
of Chinese Academy of Sciences, Zhejiang Cancer Hospital,
Hangzhou 310022, People’s Republic of China; Phone: +86-
571-87070965; Email: chengxd516@126.com
Jiang-Jiang Qin − College of Pharmaceutical Sciences, Zhejiang
Chinese Medical University, Hangzhou 310053, People’s
Republic of China; Institute of Cancer and Basic Medicine,
Chinese Academy of Sciences; Cancer Hospital of the University
of Chinese Academy of Sciences, Zhejiang Cancer Hospital,
Hangzhou 310022, People’s Republic of China; Phone: +86-
571-86633013; Email: jqin@zcmu.edu.cn
(4) Mapari, S. A.; Meyer, A. S.; Thrane, U. J. Agric. Food Chem.
2006, 54, 7027−7035.
(5) Takahashi, M.; Koyama, K.; Natori, S. Chem. Pharm. Bull. 1990,
38, 625−628.
(6) Chen, G. D.; Li, Y. J.; Gao, H.; Chen, Y.; Li, X. X.; Li, J.; Guo, L.
D.; Cen, Y. Z.; Yao, X. S. Planta Med. 2012, 78, 1683−1689.
(7) Borges, W. S.; Mancilla, G.; Guimaraes, D. O.; Duran-Patron, R.;
Collado, I. G.; Pupo, M. T. J. Nat. Prod. 2011, 74, 1182−1187.
(8) Qin, J. C.; Zhang, Y. M.; Gao, J. M.; Bai, M. S.; Yang, S. X.;
Laatsch, H.; Zhang, A. L. Bioorg. Med. Chem. Lett. 2009, 19, 1572−
1574.
(9) Muroga, Y.; Yamada, T.; Numata, A.; Tanaka, R. Tetrahedron
2009, 65, 7580−7586.
(10) Jung, H.; Kim, C.; Kim, K.; Shin, C. S. J. Agric. Food Chem.
2003, 51, 1302−1306.
Authors
Jing Yang − College of Pharmaceutical Sciences, Zhejiang
Chinese Medical University, Hangzhou 310053, People’s
Republic of China
Yan-Yan Liao − Key Laboratory of Urban Environment and
Health, Institute of Urban Environment, Chinese Academy of
Sciences, Xiamen 361021, People’s Republic of China
Gang Cheng − College of Pharmaceutical Sciences, Zhejiang
Chinese Medical University, Hangzhou 310053, People’s
Republic of China
(11) Wei, M. Y.; Wang, C. F.; Wang, K. L.; Qian, P. Y.; Wang, C. Y.;
Shao, C. L. Mar. Biotechnol. 2017, 19, 372−378.
(12) Sun, C.; Ge, X.; Mudassir, S.; Zhou, L.; Yu, G.; Che, Q.; Zhang,
G.; Peng, J.; Gu, Q.; Zhu, T.; Li, D. Mar. Drugs 2019, 17, 253.
(13) Tang, J. L.; Zhou, Z. Y.; Yang, T.; Yao, C.; Wu, L. W.; Li, G. Y.
J. Agric. Food Chem. 2019, 67, 2175−2182.
(14) Wang, W.; Liao, Y.; Chen, R.; Hou, Y.; Ke, W.; Zhang, B.; Gao,
M.; Shao, Z.; Chen, J.; Li, F. Mar. Drugs 2018, 16, 61.
I
https://dx.doi.org/10.1021/acs.jnatprod.9b01165
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
pubs.acs.org/jnp
Article
(15) Bifulco, G.; Dambruoso, P.; Gomez-Paloma, L.; Riccio, R.
Chem. Rev. 2007, 107, 3744−3779.
(16) Hoye, T. R.; Jeffrey, C. S.; Shao, F. Nat. Protoc. 2007, 2, 2451−
2458.
(17) Grimblat, N.; Zanardi, M. M.; Sarotti, A. M. J. Org. Chem. 2015,
80, 12526−12534.
(18) Steyn, P. S.; Vleggaar, R. J. Chem. Soc., Perkin Trans. 1 1976,
204−206.
(19) Phonkerd, N.; Kanokmedhakul, S.; Kanokmedhakul, K.;
Soytong, K.; Prabpai, S.; Kongsearee, P. Tetrahedron 2008, 64,
9636−9645.
(20) Grimme, S. J. Chem. Theory Comput. 2019, 15, 2847−2862.
(21) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.;
Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Mennucci,
B.; Petersson, G. A.; Nakatsuji, H.; Caricato, M.; Li, X.; Hratchian, H.
P.; Izmaylov, A. F.; Bloino, J.; Zheng, G.; Sonnenberg, J. L.; Hada, M.;
Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.;
Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Vreven, T.;
Montgomery, J. A., Jr.; J, E. P.; Ogliaro, F.; Bearpark, M.; Heyd, J.
J.; Brothers, E.; Kudin, K. N.; Staroverov, V. N.; Keith, T.; Kobayashi,
R.; Normand, J.; Raghavachari, K.; Rendell, A.; Burant, J. C.; Iyengar,
S. S.; Tomasi, J.; Cossi, M.; Rega, N.; Millam, J. M.; Klene, M.; Knox,
J. E.; Cross, J. B.; Bakken, V.; Adamo, C.; Jaramillo, J.; Gomperts, R.;
Stratmann, R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.;
Ochterski, J. W.; Martin, R. L.; Morokuma, K.; Zakrzewski, V. G.;
Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Dapprich, S.; Daniels, A.
D.; Farkas, O.; Foresman, J. B.; Ortiz, J. V.; Cioslowski, J.; Fox, D. J.
Gaussian 09; Gaussian, Inc.: Wallingford, CT, 2010.
(22) Wang, W.; Qin, J. J.; Voruganti, S.; Nijampatnam, B.; Velu, S.
E.; Ruan, K. H.; Hu, M.; Zhou, J. W.; Zhang, R. W. Cancer Res. 2018,
78, 5656−5667.
J
https://dx.doi.org/10.1021/acs.jnatprod.9b01165
J. Nat. Prod. XXXX, XXX, XXX−XXX