Microbial Transformation of Broussochalcones A and B by Aspergillus niger

Article abstract of DOI:10.1021/acs.jnatprod.0c01102

Broussochalcones A (BCA, 1) and B (BCB, 2) are major bioactive constituents isolated from Broussonetia papyrifera, a polyphenol-rich plant belonging to the family Moraceae. Due to their low yields from natural sources, BCA (1) and BCB (2) were prepared sy

Full text of DOI:10.1021/acs.jnatprod.0c01102

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Article
Microbial Transformation of Broussochalcones A and B by
Aspergillus niger
Yina Xiao, Fubo Han, Myeong Ji Kim, Kwang Youl Lee, and Ik-Soo Lee*
Cite This: J. Nat. Prod. 2021, 84, 601−607
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ABSTRACT: Broussochalcones A (BCA, 1) and B (BCB, 2) are major bioactive constituents isolated from Broussonetia papyrifera,
a polyphenol-rich plant belonging to the family Moraceae. Due to their low yields from natural sources, BCA (1) and BCB (2) were
prepared synthetically by employing Claisen−Schmidt condensation, and these were used as substrates for microbial transformation
to obtain novel derivatives. Microbial transformation of BCA (1) and BCB (2) with the endophytic fungus Aspergillus niger KCCM
60332 yielded 10 previously undescribed chalcones (1a−1e and 2a−2e). Their structures were established based on the
spectroscopic methods. The cytotoxicity of BCA (1), BCB (2), and their metabolites (1a−1e and 2a−2e) was determined by
human cancer cell lines A375P, A549, HT-29, MCF-7, and HepG2, with 1e shown to be most cytotoxic.
icrobial transformation has been developed as a
Mpromising approach to generate new natural product-
like structures.1,2 Moreover, microorganisms can also be an
alternative to the employment of small animals in the
identification of drug metabolites.3,4 The oxidation and
reduction capabilities of microorganisms, especially fungi, are
used to enhance the molecular diversity of bioactive
molecules.5−7 For example, microbial transformation of
ginsenoside Rg3 by Myrothecium verrucaria produced ginseno-
side Rh2, which showed more potent cytotoxicity than Rg3.8
Artemisinin, a sesquiterpene lactone with high therapeutic value
in treating malaria, was oxidized to afford 9β-hydroxyartemisi-
nin, 3β-hydroxyartemisinin, and 1α-hydroxydeoxyartemisinin.9
When cultured with Fusarium tricinctum AM16, xanthohumol, a
bioactive prenylflavonoid isolated from hops, was converted into
α,β-dihydroxanthohumol with improved antiproliferative activ-
ity against human cancer cell lines.10
use of plants and herbs for the treatment of different medical
disorders, including cancer, inflammation, ulcers, and diabe-
tes.14 Broussochalcones A (BCA, 1) and B (BCB, 2, also known
as bavachalcone) are major bioactive chalcones identified in
both the cortex of Broussonetia papyrifera L. and the seeds of
Psoralea corylifolia.15−17 Recently, it has been reported that BCA
(1) exhibits cytotoxic effects against human hepatoma HepG2
cells with activation of apoptosis-related proteins.18 BCA (1)
was identified as an antagonist of the Wnt/β-catenin pathway,
which contributes to the development of colon and liver
cancer.19 An analogue, BCB (2), demonstrated cytotoxicity by
inducing autophagy and apoptosis in HepG2 cells.20
Aspergillus niger is widely used in microbial transformation of
natural products and has shown remarkable ability to carry out
diverse chemical modifications including hydroxylation, hydro-
genation, and cyclization.21−23 To discover additional chalcone
Chalcones, sharing the chemical scaffold of 1,3-diphenyl-2-
propen-1-ones, are an abundant subclass of natural flavonoids
distributed in vegetables, fruits, teas, and other plants.11 The
chalcone family has attracted much attention from the synthetic
and biosynthetic perspectives due to its extensive range of
pharmacological activities.12,13 Therapeutic applications of
chalcones can be traced back thousands of years through the
Special Issue: Special Issue in Honor of A. Douglas
Kinghorn
Received: October 13, 2020
Published: February 2, 2021
© 2021 American Chemical Society and
American Society of Pharmacognosy
https://dx.doi.org/10.1021/acs.jnatprod.0c01102
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Table 1. 1H (400 MHz) NMR Spectroscopic Data for Compounds 1a−1e
a
a
a
b
a
C no.
1a
1b
1c
1d
1e
α
3.18, t (7.5)
2.85, t (7.5)
7.60, d (15.1)
7.70, d (15.1)
3.17, t (7.5)
2.85, t (7.5)
7.73, d (15.5)
7.66, d (15.5)
3.09, t (7.5)
2.83, t (7.5)
β
CO
1
2
6.63, d (1.8)
7.21, s
6.67, d (1.7)
7.33, s
6.66, d (1.8)
3
4
5
6
6.67, d (8.1)
6.56, dd (8.1, 1.8)
6.83, d (8.2)
7.13, d (8.2)
6.65, d (8.0)
6.54, d (8.0, 1.8)
6.83, d (7.8)
7.22, d (7.8)
6.63, d (8.1)
6.53, dd (8.1, 1.8)
1′
2′
3′
4′
5′
6′
1″
13.27 (OH)
6.22, s
6.26, s
6.31, s
6.20, s
6.25, s
7.63, s
7.87, s
7.53, s
8.10, s
7.45, s
3.19, d (7.3)
2.97, dd (14.1, 1.6)
2.40, dd (14.1, 10.3)
3.55, dd (10.3, 1.7)
3.06, dd (14.0, 1.2)
2.45, dd (14.0, 10.6)
3.60, dd (10.6, 1.2)
2.95, dd (16.0, 5.1)
2.67, dd (16.0, 7.5)
3.75, dd (7.0, 5.1)
2.97, dd (16.2, 4.7)
2.65, dd (16.2, 7.4)
3.69, dd (7.4, 4.7)
2″
3″
4″
5″
5.28, t (7.3)
1.23, s
1.23, s
1.27, s
1.27, s
1.33, s
1.28, s
1.31, s
1.23, s
1.74, s
1.69, s
a
b
Data were measured in CD3OD. Data were measured in DMSO-d6.
Table 2. 1H (400 MHz) NMR Spectroscopic Data for Compounds 2a−2e
a
a
a
a
b
C no.
2a
2b
2c
2d
2e
α
3.20, t (7.5)
2.82, t (7.5)
7.76, d (15.2)
7.71, d (15.2)
3.22, t (7.6)
2.82, t (7.6)
3.25, t (7.6)
2.82, t (7.6)
7.79, d (15.4)
7.66, d (15.4)
β
CO
1
2
3
7.07, d (8.4)
6.68, d (8.4)
7.73, d (8.5)
6.87, d (8.5)
7.07, d (8.4)
6.68, d (8.4)
7.07, d (8.3)
6.68, d (8.3)
7.64, d (8.6)
6.86, d (8.6)
4
5
6
6.68, d (8.4)
7.07, d (8.4)
6.87, d (8.5)
7.73, d (8.5)
6.68, d (8.4)
7.07, d (8.4)
6.68, d (8.3)
7.07, d (8.3)
6.86, d (8.6)
7.64, d (8.6)
1′
2′
3′
4′
5′
6′
1″
12.49 (OH)
6.28, s
13.47 (OH)
6.32, s
12.98 (OH)
6.27, s
12.27 (OH)
6.20, s
6.25, s
7.69, s
7.98, s
2.94, d (13.7)
2.35, dd (13.7, 10.4)
3.44 (overlapped)
7.76, s
3.07, d (8.6)
7.70, s
7.86, s
2.89, dd (14.1, 1.3)
2.25, dd (14.1, 10.6)
3.35 (overlapped)
2.88, dd (16.3, 5.0)
2.57, dd (16.3, 7.3)
3.65, dd (7.3, 5.0)
3.06, dd (16.2, 5.1)
2.76, dd (16.2, 7.3)
3.79, dd (7.3, 5.1)
2″
3″
4″
5″
4.66, d (8.6)
1.11, s
1.09, s
1.14, s
1.13, s
1.13, s
1.10, s
1.28, s
1.20, s
1.36, s
1.30, s
a
b
Data were measured in DMSO-d6. Data were measured in CD3OD.
analogues for the development of novel therapeutics, the
microbial transformation of BCA (1) and BCB (2) by Aspergillus
niger KCCM 60332 was investigated, yielding 10 previously
undescribed chalcone derivatives (1a−1e and 2a−2e). Their
structures were established based on spectroscopic data, and
their cytotoxicity was evaluated.
chromatography and HPLC yielded 10 novel chalcone
derivatives (1a−1e and 2a−2e) from the extracts. Based on
the metabolites obtained from the microbial transformation of 1
and 2 by A. niger, four main reactions were observed:
regioselective epoxidation, hydrogenation, hydroxylation, and
cyclization.
Compound 1a was obtained as a yellow amorphous solid,
presenting a [M + Na]+ ion peak at m/z 399.1418 in the
HRESIMS spectrum, consistent with a molecular formula of
C20H24O7. The UV spectrum of 1a displayed characteristic
absorption maxima at 214, 279, and 323 nm. Major bands in the
RESULTS AND DISCUSSION
■
Structure Elucidation. Microbial transformations of BCA
(1) and BCB (2) were carried out using the endophytic fungus
A. niger KCCM 60332. Subsequently, a combination of column
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Table 3. 13C (100 MHz) NMR Spectroscopic Data for Compounds 1a−1e and 2a−2e
a
a
a
b
a
b
b
b
b
a
C no.
1a
1b
1c
1d
1e
2a
2b
2c
2d
2e
α
41.0, CH2
31.3, CH2
205.7, C
134.1, C
116.4, CH
146.2, C
118.6, CH
145.8, CH
193.5, C
128.5, C
115.9, CH
146.9, C
149.9, C
116.6, CH
123.5, CH
114.5, C
166.0, C
103.6, CH
164.8, C
120.3, C
134.4, CH
33.3, CH2
79.3, CH
73.9, C
41.1, CH2
31.3, CH2
205.9, C
134.0, C
116.5, CH
146.2, C
117.3, CH
145.8, CH
191.7, C
126.2, C
115.8, CH
149.3, C
41.1, CH2
31.8, CH2
205.6, C
134.0, C
116.4, CH
146.2, C
39.7, CH2
29.1, CH2
203.8, C
117.6, CH
144.0, CH
191.4, C
39.5, CH2
29.5, CH2
203.9, C
39.7, CH2
29.3, CH2
204.1, C
118.4, CH
145.9, CH
193.7, C
β
CO
1
131.2, C
125.8, C
131.1, C
131.1, C
127.8, C
2
3
4
5
129.3, CH
115.2, CH
155.6, C
115.2, CH
129.3, CH
112.1, C
162.5, C
102.1, CH
163.2, C
131.6, CH
115.9, CH
160.2, C
115.9, CH
131.6, CH
112.7, C
164.1, C
102.3, CH
163.5, C
129.3, CH
115.5, CH
155.5, C
115.5, CH
129.3, CH
112.8, C
166.6, C
96.9, CH
164.6, C
129.3, CH
115.1, CH
155.5, C
115.1, CH
129.3, CH
113.7, C
161.7, C
103.2, CH
159.8, C
131.9, CH
116.9, CH
161.6, C
116.9, CH
131.9, CH
115.9, C
165.0, C
103.7, CH
161.6, C
144.5, C
144.6, C
145.0, C
144.6, C
116.6, CH
120.7, CH
113.8, C
164.9, C
103.5, CH
164.9, C
116.3, CH
120.7, CH
115.2, C
164.0, C
104.9, CH
161.5, C
115.8, CH
122.7, CH
114.1, C
163.2, C
103.3, CH
160.0, C
116.5, CH
120.6, CH
113.6, C
164.7, C
103.1, CH
164.3, C
6
1′
2′
3′
4′
5′
6′
1″
2″
3″
4″
5″
120.4, C
113.2, C
112.4, C
121.9, C
119.6, C
119.7, C
120.0, C
112.4, C
113.3, C
134.7, CH
33.2, CH2
79.4, CH
73.9, C
25.6, CH3
25.1, CH3
133.8, CH
31.2, CH2
70.1, CH
79.6, C
26.0, CH3
21.6, CH3
132.7, CH
30.1, CH2
67.7, CH
78.8, C
25.7, CH3
21.2, CH3
132.4, CH
28.5, CH2
123.5, CH
133.6, C
25.9, CH3
17.8, CH3
133.4, CH
31.2, CH2
77.0, CH
71.8, C
26.3, CH3
24.9, CH3
133.3, CH
31.5, CH2
77.0, CH
71.8, C
26.2, CH3
25.0, CH3
126.9, CH
28.4, CH2
91.2, CH
70.1, C
25.9, CH3
24.9, CH3
132.9, CH
30.0, CH2
67.7, CH
78.7, C
25.6, CH3
21.1, CH3
133.4, CH
31.3, CH2
70.2, CH
79.7, C
24.6, CH3
20.2, CH3
24.2, CH3
23.8, CH3
a
b
Data were measured in CD3OD. Data were measured in DMSO-d6.
IR spectrum of 1a included a broad band for a hydroxy group at
3332 cm−1 and a sharp strong carbonyl stretch at 1704 cm−1.
The 1H NMR data of 1a exhibited five aromatic signals (6.2−7.7
ppm), one methine multiplet, three methylene multiplets, and
two methyl singlets (Table 1). The 13C and HSQC
spectroscopic data of 1a displayed resonances for 20 carbons,
including one carbonyl, eight nonprotonated, six methine, three
methylene, and two methyl carbons (Table 3).
2″ and C-3″. The absolute configuration of the 2″,3″-diols in 1a
was determined by analysis of the ECD spectrum obtained using
Snatzke’s method.25−28 Based on the empirical rule, the induced
CD curve of the Mo complex of 1a showed a positive Cotton
effect at around 300 nm, indicating that the O−C−C−O
dihedral angle in the favored conformation was positive for 1a
(Figure 2). Therefore, the absolute configuration at C-2″ was
assigned S. Taken together, the structure of 1a was elucidated as
(2″S)-3,4,2′,4′-tetrahydroxy-5′-(2,3-dihydroxy-3-methylbutyl)-
α,β-dihydrochalcone, or (2″S)-α,β-dihydrobroussachalcone C.
Compound 1b was isolated as a yellow amorphous solid. The
HRESIMS spectrum of 1b exhibited a sodium adduct ion peak
[M + Na]+ at m/z 397.1263, corresponding to the molecular
formula C20H22O7. The UV spectrum displayed characteristic
absorptions of a chalcone moiety at 210, 263, and 385 nm. NMR
data of 1b were similar to those of 1a. Differences were observed
for the proton and carbon signals at C-α,β of 1b (Tables 1 and
3). These were replaced by two typical trans-olefinic protons at
δH 7.70 (d, J = 15.1 Hz) and 7.60 (d, J = 15.1 Hz) correlated to
two respective downfield carbon signals at δH 145.8 and 118.6 in
the HSQC spectrum. The absolute configuration at C-2″ was
assigned R from the ECD measurement on the Mo complex of
1b (Figure S17). Based on these observations, the structure of
1b was deduced as (2″R)-3,4,2′,4′-tetrahydroxy-5′-(2,3-dihy-
droxy-3-methylbutyl)chalcone, which was assigned the trivial
name (2″R)-broussochalcone C.
In comparison with the NMR data of 1,24 the typical trans-
olefinic signals of a chalcone together with the characteristic
olefinic methine signal of a prenyl group were absent in the 1H
NMR spectrum of 1a. Two coupled upfield triplets at δH 3.18 (t,
J = 7.5 Hz) and 2.85 (t, J = 7.5 Hz) were observed,
corresponding to two carbon signals at the upfield region of
δC 41.0 and 31.3 in the HSQC spectrum. Together with the
HMBC correlation between the upfield protons at δH 2.85 and
the carbonyl signal at δC 205.7 (Figure 1), the location of the
Compound 1c was isolated as a yellow amorphous solid with a
molecular formula of C20H22O6 on the basis of its HRESIMS
molecular ion [M + Na]+ at m/z 381.1316, which lacks one H2O
moiety compared with that of 1a. The UV spectrum of 1c was
only slightly shifted from that of 1a, with absorption maxima at
211, 281, and 324 nm. The 1H and 13C NMR data of compound
1c closely resembled those of 1a (Tables 1 and 3). The only
difference was in the presence of two individual methyl singlets
at δH 1.33 (3H, s) and 1.28 (3H, s) instead of the combined
methyl singlet at δH 1.23 (6H, s). Additionally, the HMBC
spectrum showed the two individual methyl singlets at δH 1.33
and 1.28 correlated together with the oxygenated methine
Figure 1. Key HMBC correlations of 1a (H→C).
hydrogenation was determined to be at C-α,β. Meanwhile, the
two methyl proton signals of the prenyl group were shifted
upfield to δH 1.23 (6H, s), and an additional oxymethine proton
signal at δH 3.55 (dd, J = 10.3, 1.7 Hz) was observed, suggesting
the generation of two hydroxy groups from the double bond of
prenyl group. HMBC correlations from the oxymethine proton
signal δH 3.55 to 120.4 (C-5′) supported dihydroxylation at C-
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Article
Figure 2. (a) ICD spectrum of the Mo complex of 1a. (b) Determination of the absolute configuration at C-2″ of 1a by Snatzke’s method.
carbon signal at δC 70.1 and the oxygenated carbon signal at δC
79.6. Thus, the prenyl substituent of 1c was found to be a 2,3-
epoxy-3-methylbutyl moiety instead of a 2,3-dihydroxy-3-
methylbutyl group, as in 1a. The attachment of the 2,3-epoxy-
3-methylbutyl moiety was confirmed to be located at C-5′ via
HMBC analysis. On the basis of the literature,29−31 the
configuration of several known epoxides of the prenyl group
with positive or negative specific rotation was proposed to be R
or S, respectively. In the case of compound 1c, the specific
rotation was identified to be [α]2D0 +13.9, suggesting the
configuration at C-2″ was R. Hence, the structure of 1c was
deduced as (2″R)-3,4,2′,4′-tetrahydroxy-5′-(2,3-epoxy-3-meth-
ylbutyl)-α,β-dihydrochalcone, or (2″R)-α,β-dihydrobroussa-
chalcone D.
Compound 1d was obtained as a yellow amorphous solid, and
its molecular formula was assigned as C20H20O6 based on the [M
+ Na]+ ion peak at m/z 379.1157 in the HRESIMS spectrum.
Compound 1d had the same 3,4,2′,4′-tetrahydroxychalcone
skeleton as 1b according to the 1H NMR signals at δH 8.10 (s),
7.73 (d, J = 15.5 Hz), 7.66 (d, J = 15.5 Hz), 7.33 (s), 7.22 (d, J =
7.8 Hz), 6.83 (d, J = 7.8 Hz), and 6.22 (s) (Table 1).
Additionally, the 1H NMR spectrum indicated the presence of a
2,3-epoxy-3-methylbutyl group by the observations of a doublet
of doublets signal at δH 3.69 (1H, J = 7.4, 4.7 Hz, H-2″), two
doublet signals at δH 2.97 (1H, J = 16.2, 4.7 Hz, H-1″a) and 2.65
(dd, J = 16.2 Hz, 7.4, H-1″b), and two singlet signals at δH 1.31
(3H, H-4″) and 1.23 (3H, H-5″). This moiety was attached to
C-5′ according to the HMBC correlation from H-2″ (δH 3.69)
to C-5′ (δC 112.4). The absolute configuration of C-2″ was
established as S by comparison of its specific rotation with that of
1c and the data reported in the literature.29−31 Therefore,
compound 1d was identified as (2″S)-3,4,2′,4′-tetrahydroxy-5′-
(2,3-epoxy-3-methybutyl)chalcone and named (2″S)-brousso-
chalcone D.
to C-5′ (δC 121.9) indicated a connectivity between the 3,3-
dimethylallyl group and C-5′. Compound 1e was therefore
identified as α,β-dihydrobroussochalcone A.
Compound 2a was obtained as a pale yellow amorphous solid.
The molecular formula of C20H24O6 was assigned on the basis of
its HRESIMS data [M + Na]+ at m/z 383.1472, which suggested
nine indices of hydrogen deficiency. The strong IR bands at
3423 and 1639 cm−1 suggested the presence of hydroxy and
carbonyl absorptions. The UV spectrum showed absorption
maxima at 215, 277, and 324 nm. Analysis of 1H NMR data for
2a revealed four aromatic proton resonances at δH 7.07 and 6.68
(2H each, J = 8.4 Hz) characterized by an AA′XX′-type aromatic
signal for ring B and two other aromatic singlets at δH 7.69 and
6.28 for ring A. Moreover, two sets of triplets at δH 3.20 and 2.82
(3H each, J = 7.5 Hz) corresponded to the α,β-saturated protons
of a chalcone. And characteristic signals at δH 3.35 (1H, m, H-
2″), 2.89 (1H, dd, J = 14.1, 1.3 Hz, H-1″a), 2.25 (1H, dd, J =
14.1, 10.6 Hz, H-1″b), 1.11 (3H, s, H-4″), and 1.09 (3H, s, H-
5″) were assigned a 2,3-dihydroxy-3-methylbutyl group. These
data, along with analysis of the 13C and HSQC NMR data,
corresponded to a chalcone derivative with a 2,3-dihydroxy-3-
methylbutyl group. An HMBC correlation of H-1″ with C-5′
supported the 2,3-dihydroxy-3-methylbutyl group fused to C-5′.
The absolute configuration of 2a was determined as 2″S by a
positive Cotton effect at 300 nm in its Mo complex CD spectrum
(Figure S38), which was consistent with that for 1a. Thus, the
structure of 2a was elucidated as (2″S)-4,2′,4′-trihydroxy-5′-
(2,3-dihydroxy-3-methylbutyl)-α,β-dihydrochalcone, or (2″S)-
α,β-dihydrobroussochalcone E.
Compound 2b was obtained as a yellow amorphous solid. Its
molecular formula was determined to be C20H22O6 according to
its sodium adduct ion [M + Na]+ at m/z 381.1313 observed in
the HRESIMS spectrum, which suggested 10 indices of
hydrogen deficiency. The NMR data indicated that 2b was
structurally similar to the parent compound 2, except that the
prenyl group in 2 was replaced with the 2,3-dihydroxy-3-
methylbutyl group in 2b (Tables 2 and 3). This was confirmed
by the HMBC correlations from H-1″ to C-5′ and from H-2″ to
C-4″/5″. The absolute configuration was assigned 2″R from the
ECD spectrum of the Mo complex of 2b by the negative Cotton
effect at 300 nm (Figure S44). The structure of 2b was thus
elucidated as (2″R)-4,2′,4′-trihydroxy-5′-(2,3-dihydroxy-
methylbutyl)chalcone, and the trivial name was given as
(2″R)-broussochalcone E.
Compound 1e was obtained as a yellow amorphous solid. Its
HRESIMS spectrum exhibited a molecular ion peak at m/z
365.1364, corresponding to a molecular formula of C20H22O5.
The UV spectrum showed absorption maxima at 210, 280, and
1
325 nm. The H NMR data of 1e revealed two coupled
methylene signals at δH 3.09 and 2.83 (J = 7.5 Hz), characteristic
of the α,β-dihydrochalcone skeleton (Table 1). Meanwhile, a
3,3-dimethylallyl group was indicated due to a set of proton
signals resonating as two methyl singlets 1.74 (s) and 1.69 (s)
and a triplet at δH 5.28 (t, J = 7.3 Hz) with a coupled doublet at
δH 3.19 (d, J = 7.3 Hz). HMBC correlation from H-1″ (δH 3.19)
604
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Chart 1
Compound 2c appeared as a pale yellow amorphous solid
with a molecular formula of C20H22O5 established from the [M +
Na]+ ion peak at m/z 365.1365 in its HRESIMS data. The NMR
data (Tables 2 and 3) of 2c were comparable to those of 2a with
a skeleton of 4,2′,4′-trihydroxy-α,β-dihydrochalcone, except for
the substituent at C-5′. Different from those of 2a, four proton
signals at δH 4.66 (1H, J = 8.6 Hz, oxymethine), 3.07 (2H, d, J =
8.6 Hz, methylene), and 1.13 and 1.10 (each 3H, s, gem-
dimethyl) indicated that compound 2c was based on a 4,2′,4′-
trihydroxy-α,β-dihydrochalcone with a 1-hydroxy-1-methyle-
thyldihydrofuran group. The HMBC data showed a correlation
between the methylene proton signal at δH 3.07 and the aromatic
methine at C-5′, suggesting direct attachment of the
dihydrofuran group to C-5′ of the 4,2′,4′-trihydroxy-α,β-
dihydrochalcone moiety. The configuration at C-2″ was
considered to be R by comparison of its optical rotation
([α]D20 −28.1) with those of (−)-R-coryaurone A ([α]D25
−44.9),32 (−)-R-albanin T ([α]D25 −17.0),33 (+)-S-methyl
wutaiensate ([α]2D4 +17.4) and (+)-S-methyl 7-hydroxyanoden-
droate ([α]2D4 +52.4).34 Thus, the structure of 2c was determined
to be a new prenyl-derived dihydrofuran-containing chalcone
and named (2″R)-4,2′-dihydroxy-4′,5′-[2-(1-hydroxy-1-meth-
ylethyl)-2,3-dihydrofuran]-α,β-dihydrochalcone.
as (2″R)-4,2′,4′-trihydroxy-5′-(2,3-epoxy-3-methylbutyl)-α,β-
dihydrochalcone, or (2″R)-α,β-dihydrobroussochalcone F.
Compound 2e, a yellow amorphous powder, was assigned the
molecular formula C20H20O5 on the basis of its HRESIMS data.
Its UV spectrum showed characteristic absorptions at 211 and
375 nm for a chalcone core. The 1H NMR data of 2e (Table 2)
also demonstrated the typical signals for the chalcone skeleton at
δH 7.86 (1H, s), 7.79 (1H, d, J = 15.4 Hz), 7.66 (1H, d, J = 15.4
Hz), 7.64 (2H, d, J = 8.6 Hz), 6.86 (2H, d, J = 8.6 Hz), and 6.25
(1H, s), similar to those of 2. Remaining 1H NMR resonances
were attributed to a 2,3-epoxy-3-methylbutyl group. This
substituent was assigned to be at C-5′ by the HMBC correlation
between H-1″ and C-5′. Compound 2e showed a specific
rotation of [α]2D0 −15.4, similar to that of 1d ([α]2D4 −14.5),
which indicated an S configuration. Hence, 2e was identified as
(2″S)-4,2′,4′-trihydroxy-5′-(2,3-epoxy-3-methylbutyl)-
chalcone, and the trivial name was assigned (2″S)-broussochal-
cone F.
Cytotoxic Activity. The in vitro cytotoxic activities of 1a−
1e and 2a−2e were determined with human cancer cell lines
A375P, A549, HT-29, MCF-7, and HepG2 by the MTT method
(see Table S1). 1e showed the most potent cytotoxicity against
A375P, A549, HT-29, and MCF-7 cells with IC50 values ranging
from 3.8 to 6.2 μM. Against HepG2 cells, 1e was less active than
parent compound 1. The remaining compounds were weakly
active.
Compound 2d was isolated as a pale yellow amorphous solid
and was found to have identical mass and molecular formula of
1
C20H22O5 to that of 2c. Comparison of H and 13C NMR
spectroscopic data (Tables 2 and 3) of 2c and 2d indicated that
the 1-hydroxy-1-methylethyldihydrofuran group in 2c was
replaced by a 2,3-epoxy-3-methylbutyl group in 2d. This was
further confirmed by HSQC and HMBC data. The HMBC
correlation of H-1″ with C-5′ proved that the 2,3-epoxy-3-
methylbutyl group was fused to C-5′. The absolute config-
uration of C-2″ was established as R by comparison of its specific
rotation with that of 1c. Therefore, compound 2d was identified
EXPERIMENTAL SECTION
■
General Experimental Procedures. Optical rotations were
recorded using a 343 Plus polarimeter (PerkinElmer, Waltham, MA,
USA). UV spectra were recorded on a V-530 spectrophotometer
(JASCO, Tokyo, Japan). CD spectra were obtained on a J-815 CD
spectrophotometer (JASCO, Tokyo, Japan). IR spectra were recorded
on a FT/IR 300-E spectrophotometer (JASCO, Tokyo, Japan). NMR
spectra were measured on an Avance III 400 spectrometer (Bruker,
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̈
Fallanden, Switzerland) with TMS as the internal standard. HRESIMS
211 (1.8), 281 (0.7), 324 (0.4) nm; IR νmax 3750, 3018, 1698, 1133, 689
cm−1; 1H NMR (CD3OD, 400 MHz) see Table 1; 13C NMR (CD3OD,
100 MHz) see Table 3; HRESIMS m/z 381.1316 [M + Na]+ (calcd for
C20H22O6Na, 381.1314).
were determined on a Waters Synapt G2 QTOF (Waters, Manchester,
UK). Chromatography was performed on a Waters 1525 Binary HPLC
pump equipped with a 996 photodiode array (PDA) detector using Isco
Allsphere ODS-2 (10 μm, 10 × 250 mm) and Nova-Pak C18 (4 μm, 3.9
× 150 mm) columns. 2′,4′-Dihydroxyacetophenone, 3,4-dihydrox-
ybenzaldehyde, and 4-hydroxybenzaldehyde were purchased from
Tokyo Chemical Industry Co., Ltd. Demethylzeylasteral (DZ) used as
control in the bioassays was purchased from Sigma-Aldrich, Inc.
General Procedure for the Synthesis of Broussochalcones A
(1) and B (2). Synthesis of BCA (1) and BCB (2) was conducted for
microbial transformation studies due to their low yields from natural
sources. Briefly, BCA (1) and BCB (2) were synthesized through
Claisen−Schmidt condensations using 2′,4′-dihydroxyacetophenone
with 3,4-dihydroxybenzaldehyde or 4-hydroxybenzaldehyde as starting
materials.35−37 The detailed procedure and spectroscopic character-
ization data of the intermediates and final products 1 and 2 are given in
the Supporting Information.
(2″S)-Broussochalcone D (1d): yellow amorphous solid; [α]D20
−14.5 (c 0.06, MeOH); UV (c 0.1, MeOH) λmax (log ε) 209 (1.5),
1
389 (0.4) nm; IR νmax 3744, 3198, 1743, 1649, 1422, 853 cm−1; H
NMR (DMSO-d6, 400 MHz) see Table 1; 13C NMR (DMSO-d6, 100
MHz) see Table 3; HRESIMS m/z 379.1157 [M + Na]+ (calcd for
C20H20O6Na, 379.1158).
α,β-Dihydrobroussochalcone A (1e): yellow amorphous solid; UV
(c 0.1, MeOH) λmax (log ε) 210 (1.3), 280 (0.3), 325 (0.2) nm; IR νmax
3587, 2920, 1736, 1357, 757 cm−1; 1H NMR (CD3OD, 400 MHz) see
Table 1; 13C NMR (CD3OD, 100 MHz) see Table 3; HRESIMS m/z
365.1364 [M + Na]+ (calcd for C20H22O5Na, 365.1365).
(2″S)-α,β-Dihydrobroussochalcone E (2a). pale yellow amorphous
solid; [α]2D0 −12.6 (c 0.02, MeOH); UV (c 0.1, MeOH) λmax (log ε) 215
(1.9), 277 (1.1), 324 (0.6) nm; IR νmax 3621, 3423, 1639, 1396, 874,
Fungal Strain and Culture Media. The microorganism Aspergillus
niger KCCM 60332 was obtained from Korean Culture Center of
Microorganisms (KCCM, Seoul, Korea). All the ingredients for
microbial media, including peptone, malt extract, and dextrose, were
purchased form Becton, Dickinson and Co. (Sparks, MD, USA). A.
niger KCCM 60332 was cultured on malt medium (malt extract 20 g/L,
dextrose 20 g/L, peptone 1g/L).
1
766 cm−1; H NMR (DMSO-d6, 400 MHz) see Table 2; 13C NMR
(DMSO-d6, 100 MHz) see Table 3; HRESIMS m/z 383.1472 [M +
Na]+ (calcd for C20H24O6Na, 383.1471).
(2″R)-Broussochalcone E (2b): yellow amorphous solid; [α]2D0 +17.3
(c 0.06, MeOH); UV (c 0.1, MeOH) λmax (log ε) 211 (1.4), 374 (0.4)
nm; IR νmax 3420, 3212, 3136, 1704, 1631, 956, 712 cm−1; 1H NMR
(DMSO-d6, 400 MHz) see Table 2; 13C NMR (DMSO-d6, 100 MHz)
see Table 3; HRESIMS m/z 381.1313 [M + Na]+ (calcd for
C20H22O6Na, 381.1314).
Microbial Transformation Procedures. Microbe A. niger KCCM
60332 was cultured according to the standard two-stage procedure in
150 mL of malt medium held in 500 mL Erlenmeyer flasks.38 The
cultures were maintained at 25 °C with gentle agitation at 200 rpm in a
temperature-controlled shaking incubator. A 1 mL amount of A. niger
KCCM 60332 inoculum derived from 24-h-old stage I culture was used
to initiate the stage II cultures. After a further 24 h incubation, the
ethanol solution (20 mg/mL, 150 μL) of each substrate 1 or 2 was
added to each flask. Culture controls consisting of medium only and
substrate controls consisting of sterile medium and the substrate
without microorganisms were incubated under the same conditions.
Extraction and Isolation. Cultures containing 1 or 2 were
incubated for 2 days and extracted with equal volumes of EtOAc two
times; the organic layers were concentrated under reduced pressure.
The EtOAc extract of 1 incubated with A. niger was subjected to
reversed-phased HPLC with a gradient solvent system from 55%
MeOH to 70% MeOH to yield 1a (5.0 mg, tR = 18.2 min), 1b (6.0 mg,
tR = 26.0 min), and 1d (6.5 mg, tR = 45.2 min), together with fractions
A1 (tR = 35.3 min) and A2 (tR = 54.2 min). Fraction A1 was further
separated using HPLC eluted with an isocratic solvent system of 52%
MeOH to yield 1c (4.2 mg, tR = 49.6 min). Fraction A2 was further
separated using HPLC with a gradient solvent system from 55% MeOH
to 74% MeOH to yield 1e (4.0 mg, tR = 29.4 min). The EtOAc extract of
2 incubated with A. niger was subjected to reversed-phased HPLC with
a gradient solvent system from 58% MeOH to 65% MeOH to afford 2b
(4.0 mg, tR = 25.0 min), 2e (3.0 mg, tR = 40.8 min), and fractions B1 (tR
= 19.3 min) and B2 (tR = 32.2 min). Fraction B1 was further separated
using HPLC eluted with an isocratic solvent system of 60% MeOH to
yield 2a (3.0 mg, tR= 29.8 min). Fraction B2 was further separated using
HPLC with a gradient solvent system from 58% MeOH to 62% MeOH
to yield 2c (1.8 mg, tR = 36.6 min) and 2d (2.6 mg, tR = 38.5 min).
(2″S)-α,β-Dihydrobroussochalcone C (1a): yellow amorphous
solid; [α]2D0 −13.3 (c 0.05, MeOH); UV (c 0.1, MeOH) λmax (log ε)
214 (1.8), 279 (1.0), 323 (0.6) nm; IR νmax 3619, 3332, 1704, 1626,
1010, 731 cm−1; 1H NMR (CD3OD, 400 MHz) see Table 1; 13C NMR
(CD3OD, 100 MHz) see Table 3; HRESIMS m/z 399.1418 [M + Na]+
(calcd for C20H24O7Na, 399.1420).
(2″R)-4,2′-Dihydroxy-4′,5′-[2-(1-hydroxy-1-methylethyl)-2,3-
dihydrofuran]-α,β-dihydrochalcone (2c): pale yellow amorphous
solid; [α]2D0 −28.1 (c 0.03, MeOH); UV (c 0.1, MeOH) λmax (log ε)
213 (1.6), 280 (0.6), 327 (0.5) nm; IR νmax 3677, 3451, 3026, 1707,
877, 703 cm−1; 1H NMR (DMSO-d6, 400 MHz) see Table 2; 13C NMR
(DMSO-d6, 100 MHz) see Table 3; HRESIMS m/z 365.1365 [M +
Na]+ (calcd for C20H22O5Na, 365.1365).
(2″R)-α,β-Dihydrobroussochalcone F (2d): pale yellow amorphous
solid; [α]2D0 +11.5 (c 0.06, MeOH); UV (c 0.1, MeOH) λmax (log ε) 215
(1.7), 280 (1.1), 324 (0.6) nm; IR νmax 3589, 2943, 2605, 1641, 1048
cm−1; 1H NMR (DMSO-d6, 400 MHz) see Table 2; 13C NMR
(DMSO-d6, 100 MHz) see Table 3; HRESIMS m/z 365.1367 [M +
Na]+ (calcd for C20H22O5Na, 365.1365).
(2″S)-Broussochalcone F (2e): yellow amorphous solid; [α]2D0 −15.4
(c 0.03, MeOH); UV (c 0.1, MeOH) λmax (log ε) 211(1.5), 375 (0.9)
nm; IR νmax 3556, 3195, 2945, 2604, 1642, 1515, 697 cm−1; 1H NMR
(CD3OD, 400 MHz) see Table 2; 13C NMR (CD3OD, 100 MHz) see
Table 3; HRESIMS m/z 363.1208 [M + Na]+ (calcd for C20H20O5Na,
363.1208).
Cytotoxicity Assay. Cytotoxic activities of 1, 2, 1a−1e, and 2a−2e
were evaluated against A375P (human melanoma), A549 (human lung
carcinoma), HT-29 (human colorectal adenocarcinoma), MCF-7
(human breast adenocarcinoma), and HepG2 (human hepatocellular
carcinoma) cell lines through measuring the reduction of the
tetrazolium salt MTT (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetra-
zolium bromide) by metabolically active cells following standard
procedures.39 Demethylzeylasteral was used as a positive control for the
cytotoxicity assay. The IC50 values of all tested compounds and positive
control are shown in Table S1.
ASSOCIATED CONTENT
■
sı
* Supporting Information
(2″R)-Broussochalcone C (1b): yellow amorphous solid; [α]2D0 +17.6
(c 0.05, MeOH); UV (c 0.1, MeOH) λmax (log ε) 210 (1.7), 263 (0.5),
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jnatprod.0c01102.
385 (0.9) nm; IR νmax 3612, 2996, 1846, 1687, 1417, 715 cm−1; H
1
NMR (CD3OD, 400 MHz) see Table 1; 13C NMR (CD3OD, 100
MHz) see Table 3; HRESIMS m/z 397.1263 [M + Na]+ (calcd for
C20H22O7Na, 397.1263).
Synthetic procedures of broussochalcones A and B;
spectroscopic data of 1a−1e and 2a−2e; cytotoxic
activity of compounds 1, 2, 1a−1e, and 2a−2e (Table
S1) (PDF)
(2″R)-α,β-Dihydrobroussochalcone D (1c): yellow amorphous
solid; [α]2D0 +13.9 (c 0.04, MeOH); UV (c 0.1, MeOH) λmax (log ε)
606
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AUTHOR INFORMATION
Corresponding Author
Ik-Soo Lee − College of Pharmacy, Chonnam National
University, Gwangju 61186, Republic of Korea; orcid.org/
0000-0003-4903-7106; Email: islee@chonnam.ac.kr
■
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Authors
Yina Xiao − College of Pharmacy, Chonnam National
University, Gwangju 61186, Republic of Korea
Fubo Han − College of Pharmacy, Chonnam National
University, Gwangju 61186, Republic of Korea
Myeong Ji Kim − College of Pharmacy, Chonnam National
University, Gwangju 61186, Republic of Korea
Kwang Youl Lee − College of Pharmacy, Chonnam National
University, Gwangju 61186, Republic of Korea
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Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jnatprod.0c01102
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Notes
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The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
■
The authors are grateful for the NMR experimental support of
the Center for Research Facilities, Chonnam National
University, and for the HRESIMS analyses of Korea Basic
Science Institute (KBSI). The authors also would like to thank
Gwangju Institute of Science and Technology (GIST) for their
help in the operation of the CD spectrophotometer and data
processing. This study was supported by the Basic Science
Research Program through the National Research Foundation
of Korea (NRF), funded by the Ministry of Education (NRF-
2019R1I1A3A01043084 and NRF-2019R1I1A1A01059410).
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DEDICATION
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Nat. Prod. 2008, 71, 1146−1151.
Dedicated to Dr. A. Douglas Kinghorn, The Ohio State
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