Antibacterial and cytotoxic phenolic metabolites from the fruits of amorpha fruticosa

Article abstract of DOI:10.1021/acs.jnatprod.6b00809

Fourteen new natural products, namely, 2-[(Z)- styryl]-5-geranylresorcin-1-carboxylic acid (1), amorfrutin D (2), 4-O-demethylamorfrutin D (3), 8-geranyl-3,5,7- trihydroxyflavanone (4), 8-geranyl-5,7,3′-trihydroxy-4′-methoxyisoflavone (5), 6-geranyl-5,7,3

Full text of DOI:10.1021/acs.jnatprod.6b00809

Article
pubs.acs.org/jnp
Antibacterial and Cytotoxic Phenolic Metabolites from the Fruits of
Amorpha fruticosa
†
,‡
†
†,§
⊥
⊥
†
Rini Muharini, Adriana Díaz, Weaam Ebrahim, Attila Man
́
di, Tibor Kurtan
́
, Nidja Rehberg,
†
∥
¶
○
,†
Rainer Kalscheuer, Rudolf Hartmann, Raha S. Orfali, Wenhan Lin,
Zhen Liu,*
,
†
and Peter Proksch*
†
̈
̈ ̈
Institute of Pharmaceutical Biology and Biotechnology, Heinrich-Heine-Universitat Dusseldorf, 40225 Dusseldorf, Germany
‡
Department of Chemistry Education, Faculty of Education, Tanjungpura University, 78124 Pontianak, Indonesia
§
Department of Pharmacognosy, Faculty of Pharmacy, Mansoura University, Mansoura 35516, Egypt
⊥
∥
Department of Organic Chemistry, University of Debrecen, Debrecen 4032, Hungary
Institute of Complex Systems: Strukturbiochemie, Forschungszentrum Juelich, 52428 Juelich, Germany
¶
Department of Pharmacognosy, Faculty of Pharmacy, King Saud University, Riyadh, Saudi Arabia
○
State Key Laboratory of Natural and Biomimetic Drugs, Peking University, Beijing 100191, People’s Republic of China
S Supporting Information
*
ABSTRACT: Fourteen new natural products, namely, 2-[(Z)-
styryl]-5-geranylresorcin-1-carboxylic acid (1), amorfrutin D
(
2), 4-O-demethylamorfrutin D (3), 8-geranyl-3,5,7-
trihydroxyflavanone (4), 8-geranyl-5,7,3′-trihydroxy-4′-me-
thoxyisoflavone (5), 6-geranyl-5,7,3′-trihydroxy-4′-methoxy-
isoflavone (6), 8-geranyl-7,3′-dihydroxy-4′-methoxyisoflavone
(
7), 3-O-demethyldalbinol (8), 6a,12a-dehydro-3-O-demethyl-
amorphigenin (9), (6aR,12aR,5′R)-amorphigenin (10), amor-
phispironones B and C (11 and 12), resokaempferol 3-O-β-D-
glucopyranosyl-(1→2)-β-D-glucopyranoside-7-O-α-L-rhamno-
pyranoside (13), and daidzein 7-O-β-D-glucopyranosyl-(1→2)-
β-D-glucopyranoside (14), together with 40 known compounds, were isolated from the fruits of Amorpha fruticosa. The
structures of the new compounds were elucidated by 1D and 2D NMR spectroscopic analysis as well as from the mass
spectrometry data. ECD calculations were performed to determine the absolute configurations of 11 and 15. Compounds 1, 4−6,
and 16−23 showed potent to moderate antibacterial activities against several Gram-positive bacteria with MIC values ranging
from 3.1 to 100 μM. In addition, compounds 11 and 24−33 were significantly cytotoxic against the L5178Y mouse lymphoma
cell line and exhibited IC values from 0.2 to 10.2 μM.
5
0
Amorpha fruticosa L. (Fabaceae) is a deciduous shrub that is
cytotoxic activities. Detailed investigation of the secondary
metabolites contained in these bioactive fractions afforded 54
phenolic compounds including 14 new natural products, which
included three new bibenzyl compounds, 2-[(Z)-styryl]-5-
geranylresorcin-1-carboxylic acid (1), amorfrutin D (2), and 4-
O-demethylamorfrutin D (3), a new geranylated flavonoid, 8-
geranyl-3,5,7-trihydroxyflavanone (4), three new geranylated
isoflavonoids, 8-geranyl-5,7,3′-trihydroxy-4′-methoxyisoflavone
(5), 6-geranyl-5,7,3′-trihydroxy-4′-methoxyisoflavone (6), and
native to North America and has been introduced to Europe
1
,2
and West Asia as an ornamental plant. Since the beginning of
the 20th century, this plant has attracted phytochemists. The
essential oil from its dry fruits was shown to consist of
monoterpenes and sesquiterpenes such as α-pinene and δ-
3
cadinene as major constituents. The crude extract of dry fruits
4
of A. fruticosa was demonstrated to possess antibacterial,
5
2
insecticidal, and cytotoxic activities. Amorfrutins A and B, two
bibenzyl metabolites, were reported to exhibit promising
antimicrobial, antidiabetic, and anti-inflammatory activities.
Other phenolic compounds classified as flavonoids and
rotenoids were also discovered in previous studies on
8
-geranyl-7,3′-dihydroxy-4′-methoxyisoflavone (7), five new
4
6
7,8
rotenoids, 3-O-demethyldalbinol (8), 6a,12a-dehydro-3-O-
demethylamorphigenin (9), (6aR,12aR,5′R)-amorphigenin
(
10), and amorphispironones B and C (11 and 12), a new
9
−12
flavonoid glycoside, resokaempferol 3-O-β-D-glucopyranosyl-
1→2)-β-D-glucopyranoside-7-O-α-L-rhamnopyranoside (13),
A. fruticosa.
Some of the latter compounds showed
1
1
13,14
15,16
(
antibacterial, antiviral,
and cytotoxic
activities.
In this study, screening of fractions derived from a
dichloromethane (CH Cl )−methanol (MeOH) extract of
Received: September 4, 2016
2
2
dry fruits of A. fruticosa revealed promising antibacterial or
©
XXXX American Chemical Society and
American Society of Pharmacognosy
A
DOI: 10.1021/acs.jnatprod.6b00809
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
and a new isoflavonoid glycoside, daidzein 7-O-β-D-glucopyr-
anosyl-(1→2)-β-D-glucopyranoside (14).
6.20 (s, H-3), and characteristic signals of a geranyl side chain.
The above data were almost identical to those of the known 2-
17
The structures of the new compounds were elucidated by
extensive NMR spectroscopic methods and mass spectrometric
analysis. The absolute configurations of 8, 10, 11, and 15 were
determined employing ECD calculation or by comparison with
literature data. The sugars were characterized following acid
hydrolysis of the respective glycosides, derivatization, and
HPLC analysis with comparison to known standards. In this
paper, we describe the isolation, structure elucidation, and
antibacterial and cytotoxic activities of the isolated compounds.
[(E)-styryl]-5-geranylresorcin-1-carboxylic acid (16). How-
3
ever, the smaller J value of 1 (12.0 Hz) compared to that of
1′,2′
1
6 (16.0 Hz) indicated a Z double bond at C-1′/C-2′ in 1,
which was further supported by the coupling constants in 19
3
3
(
J1′,2′ = 12.2 Hz, 1′Z) and 20 ( J = 16.3 Hz, 1′E). Except for
1′,2′
the configuration of the double bond, 1 was found to share the
same gross structure as 16, as confirmed by 2D NMR analysis.
Thus, 1 was elucidated as 2-[(Z)-styryl]-5-geranylresorcin-1-
carboxylic acid. Since compound 1 was already detected in the
crude extract of fruits of A. fruticosa, it is assumed to be a true
natural product and not to arise from the E isomer of this
compound by light-induced isomerization during the isolation
procedure.
+
The HRESIMS of 2 exhibited a [M + H] peak at m/z
4
25.2319, corresponding to the molecular formula C H O .
26 32 5
Its UV spectrum gave maximal absorbances at 222, 269, and
4
3
09 nm, which are similar to those of amorfrutin B (17). The
NMR data of 2 (Table 1) were very similar to those of 17
except for the detection of a terminal olefinic methylene (δC
1
10.6, δ 4.89 and 4.80, CH -8″) and an oxygenated methine
H
2
(
δ 75.6, δ 4.03, CH-6″) in 2. The HMBC correlations from
C
H
Me-9″ (δ 1.70) to C-6, C-7″ (δ 147.0), and C-8″ and the
H
C
COSY correlations between H-6″/H -5″ (δ 1.65) and H -5″/
2
H
2
H -4″ (δ 2.03) indicated a terminal olefinic double bond
2
H
located at C-7″/C-8″ and a hydroxy group attached at C-6″ in
2
. Thus, 2 was identified as a new amorfrutin derivative as
shown, for which the trivial name amorfrutin D is proposed. By
comparison of its optical rotation value (+31) with those of two
related known compounds, (R)- and (S)-2′-(6-hydroxy-3,7-
dimethyl-2,7-octadien-1-yl)-3′-methoxy-5′,6-dihydroxy-2-aryl-
18
benzofuran (−6.6 and +6.6, respectively), the absolute
configuration of 2 is suggested to be S.
The UV spectrum of 3 was similar to that of 2. Its molecular
formula was determined as C H O by analysis of its
2
5
30
5
HRESIMS data, lacking a CH unit compared to 2. On the
2
basis of the disappearance of the methoxy signal in 3 and the
similarity of the remaining NMR data between 2 and 3 (Table
1
), compound 3 was determined to be the 4-O-demethyl
derivative of 2, which was confirmed by detailed analysis of the
2
D NMR spectra of 3.
Compound 4 was obtained as a yellow, amorphous solid. Its
molecular formula was determined to be C H O on the basis
25
28
5
of the HRESIMS data. The NMR data of 4 (Tables 2 and 3)
19
resembled those of 6-geranyl-3,5,7-trihydroxyflavanone. How-
ever, the HMBC correlations from H-1″ (δ 3.41) to C-7 (δ
H
C
1
62.6), C-8 (δC 113.6), and C-9 (δC 154.9) indicated the
geranyl moiety to be attached at the C-8 position in 4. Thus, 4
RESULTS AND DISCUSSION
Chromatographic fractionation of the CH Cl −MeOH (1:1)
extract of air-dried fruits of A. fruticosa using vacuum-liquid
chromatography (VLC) gave 25 fractions. All fractions were
tested for their antibacterial activity. Further fractionation and
purification of the bioactive fractions yielded 54 phenolic
natural products, of which 14 are new.
was characterized as 8-geranyl-3,5,7-trihydroxyflavanone. The
■
3
small
J
2,3 value (4.6 Hz) indicated the cis configuration
2
2
19
between H-2 and H-3. The absolute configuration of 4 was
suggested to be the same as that of 6-geranyl-3,5,7-
19
trihydroxyflavanone based on their similar optical rotation.
Compound 5 gave the molecular formula C H O as
26
28
6
established by HRESIMS, containing an additional oxygen
Compound 1 was obtained as an amorphous solid. Its
atom compared to olibergin B (8-geranyl-5,7-dihydroxy-4′-
20
molecular formula was established as C H O by HRESIMS
methoxyisoflavone). The NMR data of 5 (Tables 2 and 3)
were also closely related to those of olibergin B. The singlets at
δH 12.86, 7.91, and 6.33 were assigned as the chelated hydroxy
group OH-5 and as H-2 and H-6, respectively. A series of
2
5
28
4
1
data, indicating 12 degrees of unsaturation. The H NMR
spectrum of 1 (Table 1) showed a chelated hydroxy signal at δH
1.93 (s, OH-6), signals of a monosubstituted benzene ring
1
around δ 7.13−7.17 (5H, H-4′−H-8′), two olefinic protons at
proton resonances at δ 3.51 (d, H-1″), 5.25 (d, H-2″), 2.07
H
H
δ 6.86 (d, H-1′) and 6.58 (d, H-2′), an aromatic proton at δ
(m, H -4″), 2.11 (m, H -5″), 5.05 (t, H-6″), 1.67 (s, Me-8″),
H
H
2
2
B
DOI: 10.1021/acs.jnatprod.6b00809
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Journal of Natural Products
Article
1
13
Table 1. H and C NMR Data for Compounds 1−3
a
b
a
1
2
3
d
d
d
position
δC, type
δH (J in Hz)
δC, type
δH (J in Hz)
δC, type
δH (J in Hz)
1
2
3
4
5
6
7
102.4, C
103.2, C
145.4, C
103.1, C
145.8, C
c
n.d.
110.5, CH
160.7, C
112.9, C
163.4, C
6.20, s
105.9, CH
161.7, C
6.20, s
110.5, CH
159.8, C
112.5, C
161.8, C
6.24, s
114.7, C
162.5, C
c
c
n.d.
174.2, C
n.d.
1
2
3
4
5
6
1
2
3
4
5
6
7
8
′
′
′
130.7, CH
129.3, CH
6.86, d (12.0)
6.58, d (12.0)
39.0, CH2
37.8, CH2
141.7, C
3.25, m
2.92, m
38.8, CH2
37.6, CH2
142.2, C
3.18, m
2.88, m
c
n.d.
′, 8′
′, 7′
′
128.0, CH
129.1, CH
126.9, CH
21.8, CH2
120.8, CH
139.3, C
7.17, m
128.5, CH
128.3, CH
125.7, CH
21.7, CH2
122.7, CH
134.5, C
7.20, m
128.4, CH
128.3, CH
126.0, CH
22.1, CH2
122.1, CH
138.0, C
7.20, m
7.13, m
7.29, m
7.29, m
7.15, m
7.20, m
7.20, m
″
″
″
″
″
″
″
″
3.45, d (7.2)
5.27, t (7.2)
3.36, d (7.0)
5.25, t (7.0)
3.43, d (7.0)
5.32, t (7.0)
39.5, CH2
26.1, CH2
123.5, CH
132.0, C
2.06, m
35.6, CH2
32.6, CH2
75.6, CH
147.0, C
2.03, m
35.8, CH2
32.7, CH2
76.1, CH
147.4, C
2.11, m
2.08, m
1.65, m
1.69, m
5.04, t (6.3)
4.03, t (6.2)
4.06, t (6.1)
25.5, CH3
1.64, s
110.6, CH2
4.89, s
111.2, CH2
4.91, s
4.82, s
1.71, s
1.84, s
4
.80, s
9″
17.5, CH3
16.0, CH3
1.58, s
1.79, s
17.3, CH3
15.8, CH3
55.2, CH3
1.70, s
1.80, s
3.78, s
17.7, CH3
16.6, CH3
1
0″
OMe-4
OH-6
11.93, s
11.67, s
11.97, s
a
Recorded at 300 MHz for H and 75 MHz for 13C in CDCl . Recorded at 600 MHz for H and 150 MHz for C in CDCl . n.d. = not detected.
1
b
1
13
c
3
3
d
Data extracted from HSQC and HMBC spectra.
1
.59 (s, Me-9″), and 1.83 (s, Me-10″) due to the presence of a
for the replacement of the chelated hydroxy signal by an
geranyl moiety were also observed, with the latter being linked
additional aromatic proton (δ 8.10, d, H-5), which exhibited a
H
to C-8 based on the HMBC correlations from H-1″ to C-7 (δ
COSY correlation with H-6 (δH 6.91, d) and HMBC
correlations to C-4 (δ 175.9), C-7 (δ 158.8), and C-9 (δ
C
C
1
60.9), C-8 (δ 104.9), and C-9 (δ 154.9). The E-configurated
C C
C
C
double bond at C-2″/C-3″ was deduced from the cross-peaks
155.0). Taking these data into consideration, 7 was identified as
the new 8-geranyl-7,3′-dihydroxy-4′-methoxyisoflavone.
Compound 8 was obtained as a yellow, amorphous solid. On
the basis of the HRESIMS data, its molecular formula was
determined to be C H O , missing one CH unit compared
between H-2″/H -4″ and H-1″/Me-10″ in the ROESY
2
spectrum. However, instead of ortho-coupled A B -type protons
2
2
in olibergin B, an ABX aromatic spin system of the B-ring at δH
.09 (d, J = 2.0 Hz, H-2′), 6.93 (d, J = 8.3 Hz, H-5′), and 7.07
7
22
20
8
2
2
1
(
dd, J = 8.3, 2.0 Hz, H-6′) was observed in 5. The HMBC
to that of dalbinol (31). Its UV spectrum showed typical
21
correlations from H-2′, H-6′, and the methoxy group (δ 3.93,
absorption maxima of a rotenoid at 212, 242, and 296 nm.
H
s) to C-4′ (δ 146.8) and from H-5′ to C-3′ (δ 145.7), as well
The NMR data of 8 (Tables 4 and 5) were similar to those of
C
C
as the NOE relationship between H-5′ and the methoxy group,
indicated a hydroxy group and a methoxy group to be attached
at C-3′ and C-4′, respectively. Thus, 5 was elucidated as 8-
geranyl-5,7,3′-trihydroxy-4′-methoxyisoflavone.
31 except for the presence of only one methoxy group (δ 56.9
C
and δ 3.64, respectively) in 8 rather than two methoxy groups
H
in 31. The proton at δ 6.63, showing an HMBC correlation to
H
C-12a (δ 68.6), was assigned to H-1, while the proton at δH
C
Compound 6 showed the same molecular formula as 5. Both
6.39, showing an HMBC correlation to C-1a (δ 109.6), was
C
1
13
compounds also had almost identical H and C NMR data
Tables 2 and 3). Interpretation of the 2D NMR spectra of 6
assigned to H-4. In the ROESY spectrum of 8, the methoxy
protons exhibited a NOE correlation with H-1, indicating the
methoxy group to be attached at C-2 and consequently a
hydroxy group to be attached at C-3, in consideration of its
molecular formula. Thus, 8 was elucidated as 3-O-demethyl-
dalbinol. The relative configuration of 8 was suggested to be the
same as that of 31 on the basis of their similar coupling
constants and NOE correlations. Furthermore, on the basis of
the very similar ECD spectra of 8 and 31, the absolute
configuration of 8 was determined to be identical to that of 31.
The molecular formula of 9 was determined to be C H O
(
revealed that it shares the same 5,7,3′-trihydroxy-4′-methox-
yisoflavone substructure with 5. However, H-1″ (δ 3.48, d) of
H
the geranyl side chain in 6 exhibited HMBC correlations to C-5
(
δ 159.6), C-6 (δ 109.6), and C-7 (δ 161.4), indicating that
C C C
the geranyl side chain is attached at the C-6 position. Thus, 6
was assigned as 6-geranyl-5,7,3′-trihydroxy-4′-methoxyisofla-
vone, an isomer of 5.
The molecular formula of 7 was deduced as C H O by the
2
6
28
5
+
pseudomolecular ion peak at m/z 421.2010 [M + H] in the
2
2
18
7
1
HRESIMS, missing one oxygen atom compared to 5. The H
by its HRESIMS data, missing a H O unit compared to that of
2
NMR data of 7 (Table 2) were comparable to those of 5 except
8. The oxygenated methylene CH -6 in 9 showed two doublet
2
C
DOI: 10.1021/acs.jnatprod.6b00809
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
Table 2. 13C NMR Data for Compounds 4−7
a
,
c
b
b
,
c
7 ,
ac
position
4
5
6
2
3
4
5
6
7
8
9
84.1, CH
73.5, CH
189.0, C
152.7, CH
123.3, C
152.6, CH
123.2, C
180.6, C
159.6, C
109.6,C
152.2, CH
124.2, C
181.1, C
175.9, C
162.0, C
160.8, C
125.5, CH
114.5,CH
158.8, C
103.2, CH
162.6, C
99.8, CH
160.9, C
161.4, C
94.0, CH
155.8, C
105.6, C
123.7, C
114.9, CH
145.7, C
146.5, C
110.4, CH
121.3, CH
21.2, CH2
120.8, CH
140.0, C
39.5, CH2
26.1, CH2
123.4, CH
132.0, C
25.6, CH3
17.6, CH3
16.1, CH3
55.8, CH3
113.6, C
104.9, C
113.9, C
154.9, C
154.9, C
155.0, C
1
1
2
3
4
5
6
1
2
3
4
5
6
7
8
9
1
4
0
104.9, C
106.3, C
118.4, C
′
′
′
′
′
′
″
″
″
″
″
″
″
″
″
137.6, C
123.9, C
125.0, C
126.6, CH
128.4, CH
128.2, CH
128.4, CH
126.6, CH
21.3, CH2
120.0, CH
140.2, C
115.1, CH
145.7, C
114.8, CH
145.4, C
146.8, C
146.4, C
110.7, CH
121.0, CH
21.6, CH2
120.9, CH
139.3, C
110.4, CH
120.8, CH
22.0, CH2
120.0, CH
139.6, C
39.4, CH2
26.1, CH2
123.4, CH
132.3, C
39.7, CH2
26.3, CH2
123.6, CH
132.1, C
39.5, CH2
26.0, CH2
123.5, CH
132.0, C
25.8, CH3
17.6, CH3
16.0, CH3
25.7, CH3
17.7, CH3
16.2, CH3
25.5, CH3
17.5, CH3
16.2, CH3
55.7, CH3
0″
′-OMe
56.0, CH3
a
b
Recorded at 175 MHz in CDCl . Recorded at 150 MHz in CDCl .
3
3
c
Data extracted from HSQC and HMBC spectra.
proton signals at δ 4.99 and 4.97 in contrast to two dd proton
H
signals in 8, suggesting C-6a to be a quaternary carbon. In
addition, in the HMBC spectrum of 9, H -6 exhibited
2
correlations to C-4a (δ 146.6), C-6a (δ 156.0), and C-12a
C
C
(
δC 111.7), indicating a new double bond at C-6a/C-12a in 9.
Analysis of the 2D NMR spectra revealed that 9 shares the
1
Table 3. H NMR Data for Compounds 4−7
a
b
b
a
position
4
5
6
7
2
3
5
6
8
5.56, d (4.6)
5.19, d (4.6)
7.91, s
6.33, s
7.84, s
6.39, s
7.97, s
8.10, d (8.7)
6.91, d (8.7)
5.90, s
2
3
4
5
6
1
2
4
5
6
8
9
1
′
′
′
′
7.40, m
7.38, m
7.09, d (2.0)
7.08, d (2.1)
7.12, d (2.2)
7.38, m
7.38, m
6.93, d (8.3)
7.07, dd (8.3, 2.0)
3.51, d (7.1)
5.25, d (7.1)
2.07, m
6.92, d (8.3)
7.05, dd (8.3, 2.1)
3.48, d (7.1)
5.28, d (7.1)
2.09, m
6.92, d (7.8)
′
7.40, m
7.12, dd (7.8, 2.2)
3.64, d (6.9)
5.29, t (6.9)
2.09, m
″
″
″
″
″
″
″
0″
3.41, d (7.2)
5.21, t (7.2)
2.06, m
2.10, m
2.11, m
2.12, m
2.12, m
5.03, t (6.6)
1.67, s
5.05, t (6.8)
1.67, s
5.05, t (6.6)
1.68, s
5.05, t (6.7)
1.67, s
1.58, s
1.59, s
1.60, s
1.59, s
1.80, s
1.83, s
1.83, s
1.86, s
OH-5
12.02, s
12.86, s
13.28, s
OMe-4′
3.93, s
3.93, s
3.92, s
a
b
Recorded at 700 MHz in CDCl . Recorded at 600 MHz in CDCl .
3
3
D
DOI: 10.1021/acs.jnatprod.6b00809
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Journal of Natural Products
Article
Table 4. 13C NMR Data for Compounds 8−12
a
9 ,
bd
10 ,
bd
c
12 ,
ad
position
1
8
11
111.8, CH
109.6, C
143.1, C
149.3, C
104.7, CH
149.9, C
64.5,CH2
77.1, CH
158.4, C
114.0, C
168.0, C
105.4, CH
130.2, CH
113.6, C
191.6, C
68.6, C
109.1, CH
110.2, C
141.6, C
145.7, C
102.6, CH
146.8, C
64.2, CH2
156.0, C
152.1, C
112.7, C
164.2, C
108.2, CH
127.3, CH
112.7, C
174.2, C
111.7, C
31.3, CH2
85.0, CH
146.2, C
112.6, CH2
62.5, CH2
55.9, CH3
110.2, CH
104.5, C
113.1, CH
86.7, C
75.3, CH
94.6, C
1
2
3
4
4
6
6
7
8
9
1
1
1
1
1
4
5
6
7
8
a
143.7, C
152.4, C
186.5, C
53.0, CH2
198.5, C
75.6, CH2
81.7, CH
156.3, C
109.3, C
160.8, C
112.2, CH
127.9, CH
113.7, C
185.9, C
56.2, CH
115.3, CH
129.5, CH
78.1, C
192.1, C
164.1, C
110.2, CH
190.6, C
74.3, CH2
82.2, CH
157.7, C
110.2, C
159.9, C
111.0, CH
127.6, CH
116.8, C
189.5, C
48.6, CH
116.2, CH
129.5, CH
78.0, C
149.3, C
100.8, CH
147.2, C
a
66.1, CH2
72.3, CH
157.8, C
a
a
112.7, C
166.7, C
0
1
1a
2
2a
′
′
′
′
′
104.8, CH
129.9, CH
113.4, C
188.7, C
44.5, CH
31.6, CH2
85.4, CH
146.4, C
32.3, CH2
86.2, CH
149.1, C
110.5, CH2
62.3, CH2
56.9, CH3
112.5, CH2
62.8, CH2
56.2, CH3
55.7, CH3
28.3, CH3
28.1, CH3
55.3, CH3
28.4, CH3
28.1, CH3
OMe-2
OMe-3
57.1, CH3
a
b
c
d
Recorded at 150 MHz in CD COCD . Recorded at 175 MHz in CDCl . Recorded at 75 MHz in CDCl . Data extracted from HSQC and
3
3
3
3
HMBC spectra.
1
Table 5. H NMR Data for Compounds 8−12
a
b
b
c
a
position
8
9
10
11
12
1
4
6.63, s
6.39, s
8.45, s
6.59, s
6.78, s
6.45, s
5.49, s
4.25, d (17.7)
.63, d (17.7)
4.75, s
6.21, s
3
6
4.55, dd (12.2, 2.5)
.45, dd (12.2, 1.1)
4.99, d (14.8)
4.97, d (14.8)
4.61, dd (12.1, 3.1)
4.18, dd (12.1, 1.0)
4.94, ddd (4.1, 3.1, 1.0)
6.52, d (8.5)
4.67, d (10.6)
4.30, dd (10.6, 2.5)
5.26, dd (4.5, 2.5)
6.52, d (8.7)
4.39, d (10.8)
3.82, dd (10.8, 3.2)
5.31, dd (6.4, 3.2)
6.43, d (8.6)
4
6
1
1
1
4
a
4.68, dd (2.5, 1.1)
6.54, d (8.5)
0
1
2a
′
6.94, d (8.6)
8.14, d (8.6)
7.77, d (8.5)
7.85, d (8.5)
7.66, d (8.7)
7.57, d (8.6)
3.86, d (4.1)
3.34, d (4.5)
4.31, d (6.4)
3.31, dd (15.7, 9.9)
3.60, dd (15.6, 9.9)
3.34, dd (15.6, 8.3)
5.58, dd (9.9, 8.3)
5.33, s
3.37, dd (15.7, 9.9)
3.09, dd (15.7, 8.3)
5.46, dd (9.9, 8.3)
5.25, s
6.66, d (10.1)
6.63, d (10.1)
3.04, dd (15.7, 8.3)
5
′
′
5.45, dd (9.9, 8.3)
5.23, s
5.66, d (10.1)
1.48, s
5.72, d (10.1)
1.44, s
7
5
.17, s
4.21, d (14.6)
.17, d (14.6)
3.64, s
5.32, s
5.23, s
8
′
4.33, d (13.6)
4.30, d (13.6)
3.96, s
4.25, d (13.6)
4.21, d (13.6)
3.77, s
1.45, s
3.27, s
1.42, s
3.90, s
4
OMe-2
OMe-3
3.81, s
a
b
c
Recorded at 600 MHz in CD COCD . Recorded at 700 MHz in CDCl . Recorded at 300 MHz in CDCl .
3
3
3
3
same substructure as 8 except for this C-6a/C-12a double bond.
Considering the structural relationship between 9 and
amorphigenin, 9 was named 6a,12a-dehydro-3-O-demethyla-
morphigenin. Its absolute configuration at C-5′ is suggested to
be identical to that of 8, 31, and amorphigenin due to their
close biosynthetic relationships.
exhibited almost a mirror image electronic circular dichrosim
(ECD) spectrum compared to that of (6aS,12aS,5′R)-
1
5
22
amorphigenin. Considering that the CD curves are mainly
governed by the chirality of C-6a and C-12a, it is assumed that
(6aS,12aS,5′R)-amorphigenin and compound 10 differ by their
opposite absolute configurations at these positions. Thus, 10
was determined to be (6aR,12aR,5′R)-amorphigenin.
Compound 10 shared the same gross structure as
1
5
(
6aS,12aS,5′R)-amorphigenin, as confirmed by analysis of
Compound 11 was obtained as a yellow, amorphous solid. Its
HRESIMS spectrum revealed a pseudomolecular ion peak at
the HRESIMS and the 2D NMR data. Both compounds also
+
were assigned the same cis configuration for H-6a and H-12a on
m/z 397.1281 [M + H] , corresponding to the molecular
3
the basis of the small value of J
(4.1 Hz). However, 10
formula C H O . The NMR data of 11 (Tables 4 and 5)
6
a,12a
22 20
7
E
DOI: 10.1021/acs.jnatprod.6b00809
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
9
resembled those of amorphispironone except for the
disappearance of a double bond and a methoxy group and
neglected. The ECD study of 11 supports the idea that there is
no superior single method for ECD calculations and the
experimental ECD spectra could be reproduced best by
different functionals, basis sets, and solvent models as also
indicated previously in the literature.
The molecular formula of amorphispironone C (12) was
the presence of additional methylene (δ 53.0, δH 4.25 and
.63, CH -4) and carbonyl groups (δ 186.5, C-3) instead in
C
3
1
1
2
C
25,28
1. The HMBC correlations from H-12a (δ 3.34) to C-1 (δC
H
13.1) and C-4a (δC 198.5) of 11 indicated an aromatic
methine and a carbonyl group at C-1 and C-4a, respectively.
The presence of the C-1/C-2 double bond and a methoxy
group attached at the C-2 position were confirmed by the
HMBC correlation from the protons of the methoxy group (δH
established as C H O , containing an additional oxygen atom
compared to 11. Comparison of their NMR data (Tables 4 and
22
20
8
5) indicated that the methylene group (CH -4) in 11 was
2
replaced by an oxygenated methine (δ 75.3, δ 4.75, CH-1) in
C
H
3
.27) to C-2 (δ 152.4) as well as by the NOE relationship
12. Analysis of the 2D NMR spectra of 12 disclosed that it
shares the same amorphispironone substructure with 11 except
for the substituent in ring A. In the HMBC spectrum of 12, H-
12a (δH 4.31) exhibited correlations to C-1 and C-4a (δC
190.6), suggesting a hydroxy and a carbonyl group to be located
at the C-1 and C-4a positions, respectively. The HMBC
C
between the protons of the methoxy group and H-1 (δ 5.49).
Furthermore, the HMBC correlations from H-1 to C-3 and
from H -4 to C-1 and C-3 indicated that an additional
H
2
methylene and a carbonyl group are present at the C-4 and C-3
positions, respectively. Detailed analysis of the 2D NMR
spectra revealed that 11 shares the same substructure with
amorphispironone regarding the rings B−E. Thus, 11 was
identified as a new amorphispironone derivative and was given
the name amorphispironone B. On the basis of the similar
coupling constants and NOE relationships, compound 11 was
expected to share the same relative configuration with
amorphispironone. In order to determine the absolute
configuration of 11, a Merck molecular force field (MMFF)
conformational search, density functional theory (DFT)
optimizations, and ECD calculations were performed on the
correlations from the singlet olefinic proton at δ 6.21 (H-4) to
H
C-1a (δ 94.6) and C-3 (δ 164.1) and from protons of the
C
C
methoxy group at δH 3.90 to C-3 as well as the NOE
relationship between H-4 and protons of the methoxy group
indicated a double bond at C-3/C-4 with a methoxy group
linked to C-3. In addition, both H-1 and H-4 showed HMBC
correlations to C-2 (δ 192.1), revealing the presence of an
C
additinal carbonyl group at the C-2 position. The absolute
configuration at C-1a, C-6a, and C-12a of 12 was proposed to
be the same as 11 considering their similar coupling constants
and NOE correlations as well as their close biogenetic
relationship. Thus, the NOE cross-peak between H-1 and H-
23
arbitrarily chosen (1aR,6aS,12aR) enantiomer (Figure 1).
1
2a of 12 suggested the 1R configuration, and the structure of
amorphispironone C (12) was elucidated as shown. The ECD
spectra of 11, 12, 32, and amorphispironone showed similar
patterns, with two intense negative Cotton effects (CEs) above
2
70 nm and a positive CE around 240 nm, which suggested
that they have identical absolute configurations at C-6a, C-12a,
and C-1a.
Compound 13 was obtained as a yellow, amorphous solid,
and its UV spectrum suggested it to be a kaempferol derivative
2
9
with maximal absorbances at 266 and 348 nm. The
HRESIMS exhibited a pseudomolecular ion peak at m/z
+
7
41.2237 [M + H] , which established the molecular formula,
C H O . Further analysis of ESIMS demonstrated fragment
3
3
40 19
+
peaks at m/z 579 [M + H − 162] , 417 [M + H − 162 −
+
+
1
62] , and 271 [M + H − 162 − 162 − 146] , suggesting the
presence of two hexose residues and one deoxyhexose residue
Figure 1. Experimental ECD spectrum of 11 in MeCN compared with
the Boltzmann-weighted B3LYP/TZVP ECD spectrum of (1aR,6a-
S,12aR)-11 computed for the B3LYP/6-31G(d) in vacuo conformers.
Bars represent the rotational strength of the lowest energy conformer.
1
in the molecule. The H NMR spectrum (Table 6) showed
signals for a 1,4-disubstituted benzene ring at δ 8.10 (d, J = 8.8
H
Hz, H-2′ and 6′) and 6.93 (d, J = 8.8 Hz, H-3′ and 5′), a 1,2,4-
trisubstituted benzene ring at δ 8.10 (d, J = 8.8 Hz, H-5), 7.16
H
(
dd, J = 8.8, 2.1 Hz, H-6), and 7.33 (d, J = 2.1 Hz, H-8), and
Reoptimization of the initial 12 MMFF conformers yielded five
and one low-energy (≥2%) conformer at B3LYP/6-31G(d) in
vacuo and the B97D/TZVP (PCM/MeCN) level, respec-
three anomeric protons at δ 5.64 (d, J = 1.8 Hz, H-1⁗), 5.47
H
(d, J = 7.6 Hz, H-1″), and 4.76 (d, J = 7.8 Hz, H-1‴), indicating
the presence of resokaempferol as aglycone and three sugar
residues. Combining the TOCSY and HSQC spectra, three
sugar units were recognized and their chemical shifts and
coupling constants were in good agreement with two units of
glucopyranose and one unit of rhamnopyranose, which was
confirmed by HPLC analysis of the acid hydrolysis products of
13 in comparison to known standards. The J values of the
anomeric protons of the glucopyranose (7.6 or 7.8 Hz) and
rhamnopyranose (1.8 Hz) were in accordance with those of β-
D-glucopyranose and α-L-rhamnopyranose, respectively. The
2
4,25
tively.
While solvent model calculations usually result in
similar or a larger number of low-energy conformers than the
2
5−27
gas-phase ones,
11 represented an interesting counter-
example where all minor conformers had negligible contribu-
tions in the solvent model calculation. Although solvent model
ECD spectra computed for the single B97D/TZVP (PCM/
MeCN) conformer also allowed the assignment of the absolute
configuration as (1aR,6aS,12aR), the in vacuo ECD calculations
computed for the B3LYP/6-31G(d) in vacuo conformers gave
a far better overall agreement, indicating that the minor
conformers were better estimated and so they cannot be
HMBC correlations from the H-1″ anomeric proton to C-3 (δ
C
136.6) and from H-1‴ to C-2″ (δ 82.3) confirmed that a β-D-
C
F
DOI: 10.1021/acs.jnatprod.6b00809
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Journal of Natural Products
Article
Table 6. H and 13C NMR Data for Compounds 13 and 14
1
a
b
1
3
14
δH (J in Hz)
8.21, s
c
c
position
δC, type
δH (J in Hz)
δC, type
2
3
4
5
6
7
8
9
158.9, C
136.6, C
175.8, C
154.8, C
126.0, C
177.8, C
127.6, CH
116.4, CH
162.0, C
8.10, d (8.8)
128.0, CH
116.7, CH
163.0,C
8.15, d (8.9)
7.16, dd (8.8, 2.1)
7.24, dd (8.9, 2.3)
104.1, CH
157.8, C
7.33, d (2.1)
104.7, CH
158.8, C
7.27, d (2.3)
1
1
2
3
4
1
2
3
4
5
6
0
119.3, C
119.9, C
′
122.7, C
123.7, C
′, 6′
′, 5′
′
″
″
″
″
″
″
132.2, CH
116.0, CH
161.2, CH
100.7, CH
82.3, CH
77.7, CH
71.0, CH
78.0, CH
62.1, CH2
8.10, d (8.8)
6.93, d (8.8)
131.2, CH
116.0, CH
158.5, CH
100.1, CH
83.3, CH
77.3, CH
70.7, CH
78.0, CH
61.9, CH
7.39, d (8.6)
6.85, d (8.6)
5.47, d (7.6)
3.76, dd (9.0, 7.6)
3.60, dd (9.0, 8.7)
3.36, m
5.31, d (7.4)
3.77, dd (9.0, 7.4)
3.72, dd (9.0, 8.8)
3.47, dd (9.8, 8.8)
3.17, m
3.56, ddd (9.8, 5.8, 2.3)
3.93, dd (12.2, 2.3)
3.72, dd (12.2, 5.8)
4.69, d (7.9)
3.66, m
3
.48, m
1‴
2‴
3‴
4‴
5‴
6‴
104.5, CH
75.3, CH
77.6, CH
71.0, CH
77.9, CH
62.5, CH2
4.76, d (7.8)
3.35, m
105.3, CH
75.8, CH
77.5, CH
70.9, CH
77.7, CH
61.7, CH2
3.24, dd (9.0, 7.9)
3.41, t (9.0)
3.39, dd (9.1, 7.6)
3.36, m
3.35, dd (9.6, 9.0)
3.27, ddd (9.6, 4.5, 2.4)
3.61, dd (11.8, 4.5)
3.52, dd (11.8, 2.4)
3.29, m
3.78, dd (11.9, 2.7)
3
.68, dd (11.9, 5.0)
1
2
3
4
5
6
⁗
⁗
⁗
⁗
⁗
⁗
99.7, CH
71.4, CH
71.9, CH
73.3, CH
71.0, CH
17.8, CH3
5.64, d (1.8)
4.06, dd (3.3, 1.8)
3.86, dd (9.3, 3.3)
3.50, t (9.3)
3.60, dq (9.3, 6.2)
1.25, d (6.2)
a
1
13
b
1
13
c
Recorded at 700 MHz for H and 175 MHz for C in CD OD. Recorded at 600 MHz for H and 150 MHz for C in CD OD. Data extracted
3
3
from HSQC and HMBC spectra.
glucopyranosyl-(1→2)-β-D-glucopyranosidic fragment was
linked to the 3-hydroxy group of resokaempferol. In addition,
the anomeric proton H-1⁗ showed an HMBC correlation to C-
HPLC analysis of the acid hydrolysis products of 14. The
coupling constants of the anomeric protons of the glucoses
were in accordance with that of the β-D anomer. In the HMBC
spectrum, the two anomeric protons H-1″ and H-1‴ showed
correlations to C-7 (δ 163.0) and C-2″ (δ 83.3), respectively,
indicating that the inner β-D-glucopyranosyl moiety was
attached to C-7 of the daidzein aglycone and that the second
β-D-glucopyranosyl residue and the inner one were 1,2-linked.
Taking these data into consideration, 14 was elucidated as
daidzein 7-O-β-D-glucopyranosyl-(1→2)-β-D-glucopyranoside.
7
(δ 162.0), indicating the α-L-rhamnopyranose to be linked
C
to the 7-hydroxy group of resokaempferol. Thus, the structure
of 13 was identified as resokaempferol 3-O-β-D-glucopyranosyl-
C
C
(1→2)-β-D-glucopyranoside-7-O-α-L-rhamnopyranoside.
The UV spectrum of compound 14 exhibited almost
30
identical absorbances to the isoflavone daidzein. Its molecular
formula was determined as C H O based on the HRESIMS
2
7
30 14
data. The positive and negative ESIMS data displayed fragment
The NMR data and optical rotation value of 15 were in
+
−
31,32
peaks at m/z 255 [M + H − 324] and 253 [M − H − 324] ,
agreement with those of (+)-puerol A,
for which the
1
respectively, corresponding to the loss of two hexoses. The H
absolute configuration has not been reported yet. For the
configurational assignment of 15, the same solution TDDFT-
ECD calculation protocol (Figure 2) was carried out on (S)-15
as on 11. The TDDFT-ECD method has been proved to be an
efficient and generally applied method for the elucidation of
absolute configuration of natural products with varying
NMR spectrum showed signals characteristic for daidzein
including an ortho-coupled A B aromatic spin system at δ
2
2
H
7
.39 (d, J = 8.6 Hz, H-2′ and 6′) and 6.85 (d, J = 8.6 Hz, H-3′
and 5′), an ABX aromatic spin system at δ 8.15 (d, J = 8.9 Hz,
H-5), 7.24 (dd, J = 8.9, 2.3 Hz, H-6), and 7.27 (d, J = 2.3 Hz,
H
21
H-8), and an olefinic proton at δ 8.21 (s, H-2). In addition,
conformational flexibility and complexity. Reoptimization of
the initial 48 MMFF conformers resulted in 11 and 15 low-
energy (≥2%) conformers at B3LYP/6-31G(d) in vacuo and
H
two anomeric protons at δ 5.31 (d, J = 7.4 Hz, H-1″) and 4.69
H
(
d, J = 7.9 Hz, H-1‴) as well as 12 additional protons between
H
24,25
δ 3 and 4 were observed due to the presence of two hexose
the B97D/TZVP
PCM/MeCN levels, respectively. Both
units, which were identified further as two glucose moieties by
gas-phase and solvent model calculations reproduced well the
G
DOI: 10.1021/acs.jnatprod.6b00809
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Journal of Natural Products
Article
15
30
glucopyranosyldalpanol (33), daidzein, 5,7-dihydroxy-4′-
4
4
45
47
methoxyisoflavone, 5-hydroxy-7,4′-dimethoxyisoflavone,
4
6
afrormorsin,
7-hydroxy-2′,4′,5′-trimethoxyisoflavone,
48 39
7
,2′,4′,5′-tetramethoxyisoflavone, munduserone, cis-12a-
39 49
hydroxymunduserone, 6-deoxyclitoriacetal, cis-12a-hydrox-
3
8
50
40
yrot-2′-enonic acid, sumatrolic acid, tephrosin, 11-
40
15
51
hydroxytephrosin, 12a-hydroxydalpanol, rotenolone, 9 11-
52
15
hydroxyrotenone, amorphigenin, amorphispironone, 11-
53
licoagroside F, kaempferol 3-O-α-L-rhamnopyranosyl-(1→2)-
β-D-glucopyranoside-7-O-α-L-rhamnopyranoside (13a), and
′-O-β-D-glucopyranosyl-12a-hydroxydalpanol (33a) by com-
parison with the literature data.
29
2
6
All isolated compounds were evaluated for their antimicrobial
activity against a set of human pathogenic Gram-positive
bacteria. Among them, the geranylated bibenzyl derivatives 16,
1
7, 20, and 21 and the likewise geranylated chalcone 23
Figure 2. Experimental ECD spectrum of 15 in MeCN compared with
the Boltzmann-weighted BH&HLYP/TZVP PCM/MeCN ECD
spectrum of (S)-15 computed for the B97D/TZVP PCM/MeCN
conformers. Bars represent the rotational strength of the lowest energy
conformer.
showed significant antibacterial activities with MIC values
ranging from 3.1 to 25 μM, while compounds 1, 4−6, 18, 19,
and 22 displayed moderate activities with MIC values ranging
from 25 to 100 μM (Table 7). Compounds 17 and 20−23 were
previously reported for their antibacterial activity against S.
4
,54
55
aureus ATCC13709
and ATCC25923, two standard
main features of the experimental ECD spectrum, with
BH&HLYP/TZVP (PCM/MeCN) ECD calculation of the
B97D/TZVP (PCM/MeCN) conformers giving the best
agreement (Figure 2). In the low-energy conformers, the two
benzene rings adopted a folded orientation, in which the OH-
laboratory strains of S. aureus. However, their antibacterial
activity against the methycilin-resistant strain S. aureus
ATCC700699, the vancomycin-resistant strains E. faecalis
ATCC51299 and E. faecium ATCC700221 as well as against
two standard laboratory strains, E. faecalis ATCC29212 and
E. faecium ATCC35667, is reported for the first time in this
study. Compounds 17 (MIC value of 6.3 μM against
vancomycin-resistant strain E. faecium ATCC700221), 20
(MIC values of 3.1 and 6.3 μM against methicillin-resistant
S. aureus strain ATCC700699 and vancomycin-resistant
E. faecium strain ATCC700221, respectively), and 23 (MIC
values of 3.1 against both methicillin-resistant S. aureus strain
ATCC700699 and vancomycin-resistant E. faecium strain
ATCC700221) were similar in regard to their antibiotic activity
to the positive control moxifloxacin. However, none of the
isolated compounds exhibited activity against Mycobacterium
tuberculosis or against the Gram-negative bacterium Acineto-
bacter baumannii ATCCBAA1605 (MIC > 50 μM).
2″ was oriented above the plane of the other benzene ring
assuming a bonding interaction. The low-energy conformers
showed similar ECD spectra, and there was a good overall
agreement between the experimental and Boltzmann-weighted
computed ECD spectra at all the applied levels; thus the
absolute configuration of (+)-15 could be assigned unambig-
uously as S.
The remaining known compounds were identified as 2-[(E)-
17
styryl]-5-geranylresorcin-1-carboxylic acid (16), amorfrutin B
4
1
(
17), 2-carboxy-3,5-dihydroxy-4-geranylbibenzyl (18), 2-
33 34
geranyl-5-[(Z)-styryl]resorcin (19), amorphastilbol (20),
3
5
2
(
-geranyl-5-(2-phenylethyl)resorcin (21), amorfrutin A
4 36 37
22), xanthoangelol (23), sermundone (24), rot-2′-enonic
38 39 40
acid (25), deguelin (26), α-toxicarol (27), 6a,12a-
41
42
43
dehydrodeguelin (28), dalpanol (29), rotenone (30),
dalbinol (31), hydroxyamorphispironone (32), 6′-O-β-D-
All antibacterially active compounds (Table 7) were further
2
1
1
tested for their cytotoxicity against eukaryotic cells using the
Table 7. Antimicrobial (MIC, μM) and Cytotoxic Activities (IC , μM)
5
0
S. aureus
E. faecalis
E. faecium
compound
ATCC 25923
ATCC 700699
ATCC 29212
ATCC 51299
ATCC 35667
ATCC 700221
MRC-5
THP-1
1
4
5
6
1
1
1
1
2
2
2
2
2
50
100
100
100
100
100
100
100
12.5
12.5
100
100
3.1
50
50
100
100
100
50
50
100
25
100
100
25
100
100
50
100
100
50
50
25
25
12.5
25
50
50
6
7
8
9
0
12.5
12.5
100
100
6.3
25
25
25
100
100
100
100
50
100
100
100
100
25
6.3
6.3
6.3
6.3
100
100
12.5
>100
25
100
100
6.3
100
100
100
100
6.3
6.3
100
12.5
a
0a
>100
25
>100
6.3
>100
12.5
25
>100
12.5
25
100
100
50
1
2
3
50
6.3
100
12.5
0.9
50
50
25
3.1
6.3
12.5
1.9
3.1
25
25
b
moxifloxacin
<0.9
3.8
0.9
7.8
a
b
2
0a is a dimethoxy product of 20. Positive control.
H
DOI: 10.1021/acs.jnatprod.6b00809
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
5
8
12
MRC-5 human lung fibroblast cell line and the THP-1 human
monocytic cell line as models. The IC50 values of 16 and 17
were 100 μM against MRC-5 and THP-1 cells for both
compounds, indicating a high degree of selectivity for the
antibacterial versus cytotoxic activity, which makes these two
compounds interesting candidates for further studies on the
molecular targets and models of action underlying the antibiotic
activity. For compounds 20 and 23, the IC values against the
231 and MCF-7 human breast cancer cell lines, the HCT-
12
116 human colon cancer cell line, and the HEL299 human
lung normal cell line.
The most potent cytotoxic activity was observed in this study
for compounds 24−27 and 29−33, which exhibited IC values
2
5
0
ranging from 0.2 to 1.7 μM, respectively. These compounds
were more potent than the positive control kahalalide F. 12a-H-
substituted rotenoids seem to be more active than correspond-
ing 12a-OH-substituted derivatives by comparison of 24 vs 6-
deoxyclitoriacetal, 25 vs cis-12a-hydroxyrot-2′-enonic acid, 26
vs tephrosin, 27 vs 11-hydroxytephrosin, 29 vs 12a-hydrox-
ydalpanol, 30 vs rotenolone, and 33 vs 33a. The presence of a
double bond at C-6a/C-12a (28 vs 26) decreased activity, while
the loss of a methoxy group (8 vs 31) led to total loss of
cytotoxicity. Spirorotenoid compounds 32 and amorphispir-
onone were less active compared to their corresponding
biosynthetic precursors 27 and 26. In addition, the rotenoid
glycoside 33 was less cytotoxic compared to its aglycone 29.
In summary, bioactivity-guided isolation of fruits of
A. fruticosa gave 54 phenolic metabolites including 11 bibenzyl
compounds, two chalcone derivatives, three flavonoids, 10
isoflavonoids, and 28 rotenoids, of which 14 were new
compounds. Several geranylated bibenzyl compounds, espe-
cially 16, 17, 20, 21, and 23, exhibited significant antibacterial
activity. Rotenoids and their derivatives, being present as
dominant substances in the crude extract of fruits from
A. fruticosa, displayed significant cytotoxicity against the
L5178Y mouse lymphoma cell line. This study revealed the
presence of numerous additional new and known phenolic
metabolites in fruits of A. fruticosa compared to previous
reports and provided several potential antibacterial and
cytotoxic substances.
5
0
two eukaryotic cell lines varied from 25 to 50 μM.
A preliminary structure−activity relationship for the
antibacterial activity of the geranylated bibenzyl derivatives is
proposed. The chain length of the isoprenoid derived side chain
seems to be important for antibacterial activity against E. faecalis
and E. faecium strains since compound 22, which differs from
1
7 only by an isoprene versus a geranyl side chain, was largely
inactive, whereas 17 showed strong antibiotic activity. The
presence of a double bond at the C-1/C-2 position of the
analyzed bibenzyl derivatives is not essential, as indicated by the
strong antibiotic activity of the hydrogenated compounds 17
and 21. Compounds with an E-configurated double bond at the
C-1/C-2 position such as 16 and 20 were more active than the
corresponding Z isomers, as exemplified by comparison of 1
and 19. Addition of an oxo function to the central chain linking
the aromatic rings of bibenzyl derivatives as present in
compound 23 showed no significant effect on the antibiotic
activity in comparison to 20. The presence of a carboxylic
group at C-1 of one of the aromatic rings is not required for
activity, as shown by comparison of 16 versus 20, which
4
corroborated earlier results by Mitscher et al. Methylation of
phenolic OH groups like for compound 17 increased its
antibiotic activity when compared to the demethylated
derivative 18.
In addition to studying the antibiotic activity, all compounds
isolated in this study were subjected to an MTT assay using the
L5178Y mouse lymphoma cell line as a model for evaluating
EXPERIMENTAL SECTION
■
56,57
cytotoxicity (Table 8).
As expected from the results of the
General Experimental Procedures. A JASCO P-2000 polar-
imeter was used for measuring optical rotations. A UV/vis
spectrophotometer (PerkinElmer Instruments) was utilized to obtain
UV absorbance spectra. ECD spectroscopy was conducted using a
JASCO J-810 CD spectropolarimeter. NMR experiments were carried
out on a Bruker 300, Bruker Avance III 600, or Bruker Biospin 700
spectrometers, with chemical shifts given in δ (ppm) values referring
to the solvent peaks at δ 7.26 and δ 77.0 for CDCl , δ 3.31 and δ
Table 8. Cytotoxicity against the Mouse Lymphoma Cell
Line L5178Y
compound
IC50 (μM)
1
2
2
2
2
2
2
3
3
3
3
1
4
5
6
7
8
9
0
1
2
3
7.6
0.2
0.6
0.2
0.2
10.2
0.7
0.3
0.2
1.3
1.7
4.3
H
C
3
H
C
4
9.0 for CD OD, and δH 2.05 and δ 29.8 for CD COCD . ESIMS
3 C 3 3
were acquired from an LC-MS HP1100 Agilent Finnigan LCQ Deca
XP Thermoquestman spectrometer, and HRESIMS were recorded on
a UHR-QTOF maXis mass 4G spectrometer. HPLC analysis was
carried out using a Dionex P580 system connected to a photodiode
array detector (UVD340s), and the separation column (125 × 4 mm)
was prefilled with Eurospher 10 C18 (Knauer, Germany). Semi-
preparative RP-HPLC purification was achieved by an HPLC
Lachrom-Merck Hitachi system (pump L7100 and UV detector
L7400) with a Eurospher 100p10 C18 (300 × 8 mm) column. Open
column chromatography was applied for fractionation using Merck
MN silica gel 60 M (0.04−0.063 mm), Sephadex LH-20 (Sigma-
Aldrich), Diaon HP-20 (Merck), or LiChroprep Diol (40−63 μm,
Merck) as stationary phase. Thin layer chromatography (TLC) was
a
kahalalide F
a
Positive control.
used for monitoring and accomplished on precoated silica gel 60 F254
plates (Macherey-Nagel GmBH, Germany) with detection at 254 and
cytotoxicity counter-screen carried out for the bibenzyl
derivatives (1−3, 16−22), none of these latter metabolites
showed activity against the mouse lymphoma cell, thus
corroborating the results obtained with the MRC-5 and
THP-1 cell lines. From all compounds analyzed only the
rotenoid derivatives were active against the L5178Y cell line,
which is in agreement with previous reports on the cytotoxicity
of rotenoids against human cell lines such as the MDB-MB-
3
65 nm. Distillated solvents were used during isolation, and spectral
grade solvents were employed for spectroscopic measurements.
Plant Material. Air-dried fruits of A. fruticosa used in this study
were purchased from Friedrich Nature Discovery Company,
Euskirchen, Germany. A voucher specimen (PB1601) was deposited
at the Institute of Pharmaceutical Biology and Biotechnology,
Heinrich-Heine University Duesseldorf, Germany.
I
DOI: 10.1021/acs.jnatprod.6b00809
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
Extraction and Isolation. The dry fruits of A. fruticosa (500 g)
were powdered and extracted three times with CH Cl −MeOH (1:1)
isolated form subfraction 18-8-10 (31.9 mg); 29 (1.1 mg) and 21 (0.4
mg) were obtained from subfraction 18-8-12 (17 mg).
Fraction 24 (6 g) was fractionated by a Diaion HP-20 column using
2
2
at room temperature for 24 h each. The solution was dried in vacuo to
yield 72.5 g of a pale yellow crude extract, 55 g of which was subjected
initially to silica gel VLC, eluting with n-hexane, ethyl acetate (EtOAc),
CH Cl , and MeOH in order of polarity to afford 25 fractions. All
a gradient of MeOH−H O (0:100, 20:80, 40:60, 80:20, 100:0) to give
2
12 subfractions (24-1 to 24-12). Subfraction 24-7 (425.6 mg) was
subjected to a Sephadex LH-20 column with MeOH to give nine
subfractions (24-7-1 to 24-7-9). Subfraction 24-7-4 (27.0 mg) was
2
2
fractions were tested for their antituberculosis and antibacterial
activity. A growth inhibition against Gram-positive bacteria was
exhibited by fractions 11, 12, and 14−19, whereas none of the analyzed
fractions showed growth inhibition toward M. tuberculosis or against
Gram-negative A. baumannii ATCCBAA1605.
purified by semipreparative HPLC using MeOH−H O (gradient
2
30:70 to 100:0) to yield 32 (11.6 mg). Subfraction 24-11 (23.3 mg)
was purified by semipreparative HPLC using MeOH−H O (gradient
2
40:60 to 100:0) to give 33 (1.3 mg).
Fraction 11 (1.8 g) was fractionated using a Sephadex LH-20
column with MeOH as eluent to give nine subfractions (11-1 to 11-9).
Subfractions 11-3 (96 mg) and 11-8 (74 mg) were further subjected to
a Sephadex LH-20 column using acetone as mobile phase followed by
2-[(Z)-Styryl]-5-geranylresorcin-1-carboxylic acid (1): yellow,
amorphous solid; UV (MeOH) λ (log ε) 224 (2.89), 301 (2.46)
max
1
13
nm; H and C NMR, see Table 1; HRESIMS m/z 393.2059 [M +
H] (calcd for C H O , 393.2060).
Amorfrutin D (2): white, amorphous solid; [α]
+
25
29
4
22
purification using semipreparative HPLC (gradient MeOH−H 0,
+31 (c 0.14,
D
2
5
0:50 to 100:0) to yield 19 (2.0 mg) and 3 (2.8 mg), respectively.
Fraction 12 (1.3 g) was subjected to passage over a Sephadex LH-20
acetone); UV (MeOH) λ (log ε) 222 (3.34), 269 (2.94), 309 (1.96)
max
1
13
nm; H and C NMR, see Table 1; HRESIMS m/z 425.2319 [M +
+
column using MeOH as mobile phase to afford eight subfractions (12-
to 12-8). Subfraction 12-2 (21.8 mg) was purified by semipreparative
H] (calcd for C H O , 425.2323).
26
33
5
2
2
D
1
4-O-Demethylamorfrutin D (3): white, amorphous solid; [α]
HPLC (gradient MeOH−H O, 50:50 to 100:0) to give 2 (2.7 mg).
+13 (c 0.13, acetone); UV (MeOH) λ (log ε) 230 (3.34), 266
max
2
1
13
Fraction 13 (370 mg) was subjected to purification over a
(2.94), 304 (1.96) nm; H and C NMR, see Table 1; HRESIMS m/z
+
LiChroprep Diol column eluting with CH Cl2 to afford four
411.2164 [M + H] (calcd for C H O , 411.2166).
2
25 31
5
subfractions (13-1 to 13-4). Subfraction 13-1 (194.0 mg) was
8-Geranyl-3,5,7-trihydroxyflavanone (4): yellow, amorphous solid;
[α]21 −24 (c 0.15, CHCl ); UV (MeOH) λ (log ε) 219 (4.53),
fractionated over a Sephadex LH-20 column using CH Cl −MeOH
2
2
D
3
max
1
13
(
1:1) as eluent, followed by purification with semipreparative HPLC to
263 (4.05), 293 (3.73) nm; H and C NMR, see Tables 2 and 3;
HRESIMS m/z 409.2011 [M + H] (calcd for C H O , 409.2010).
+
yield 24 (3.0 mg). Following similar procedures, 10 (3.0 mg) was
obtained from subfraction 13-2, while 5 (21.0 mg), 9 (0.5 mg), 14 (2.0
mg), and 18 (1.0 mg) were isolated from subfraction 13-3 (41 mg).
Fraction 14 (330 mg) was chromatographed over a LiChroprep
Diol column using CH Cl to yield nine subfractions (14-1 to 14-9).
25
29
5
8-Geranyl-5,7,3′-trihydroxy-4′-methoxyisoflavone (5): yellow,
amorphous solid; UV (MeOH) λ (log ε) 213 (3.69), 268 (3.53)
max
1
13
nm; H and C NMR, see Tables 2 and 3; HRESIMS m/z 437.1961
[M + H] (calcd for C H O , 437.1959).
+
2
2
26 29
6
Subfraction 14-2 (59 mg) was purified by semipreparative HPLC to
yield 25 (0.5 mg), 26 (2.0 mg), and 27 (0.8 mg). Further purification
of subfraction 14-7 (24.0 mg) with semipreparative HPLC gave 5
6-Geranyl-5,7,3′-trihydroxy-4′-methoxyisoflavone (6): yellow,
amorphous solid; UV (MeOH) λ (log ε) 206 (3.67), 267 (3.58)
max
1
13
nm; H and C NMR, see Tables 2 and 3; HRESIMS m/z 437.1966
+
(
14.0 mg) and 15 (2.0 mg).
[M + H] (calcd for C H O , 437.1959).
26
29
6
Fraction 15 (600 mg) was subjected to a Sephadex LH-20 column
8-Geranyl-7,3′-dihydroxy-4′-methoxyisoflavone (7): yellow, amor-
1
eluting with CH Cl −MeOH (1:1) to give nine subfractions (15-1 to
phous solid; UV (MeOH) λ (log ε) 251 (3.73), 292 (3.35) nm; H
2
2
max
13
1
5-9). Subfraction 15-4 (95.6 mg) was further separated by a silica gel
and C NMR, see Tables 2 and 3; HRESIMS m/z 421.2010 [M +
+
column with CH Cl −MeOH (99:1) as mobile phase, followed by
H] (calcd for C H O , 421.2010).
2
2
26 29
5
2
0
purification using semipreparative HPLC (gradient MeOH−H O,
3-O-Demethyldalbinol (8): yellow, amorphous solid; [α] −105
2
D
5
(
(
0:50 to 100:0) to afford 23 (1.3 mg). Separation of subfraction 15-6
(c 0.11, MeOH); UV (MeOH) λmax (log ε) 212 (2.86), 242 (2.74),
−4
142.5 mg) by a Sephadex LH-20 column using CH Cl −MeOH
296 (2.65) nm; ECD {MeCN, λ [nm] (Δε), c = 1.82 × 10 M} 324
2
2
1:1), followed by a silica gel column using n-hexane−EtOAc (6:4),
(−4.48), 277 (−2.62), 243 (5.36), 221sh (−8.27), 205 (−15.70),
1
13
gave 12 (2.5 mg) and 6 (4.8 mg).
negative below 198 nm; H and C NMR, see Tables 4 and 5;
+
Fraction 17 (800 mg) was separated by a Sephadex LH-20 column
using acetone as eluent to afford 16 subfractions (17-1 to 17-16),
among which subfraction 17-13 was pure compound 17 (1.0 mg).
Subfraction 17-4 (112.9 mg) was further purified by a Sephadex LH-20
column with acetone to give 31 (41.0 mg). Subfraction 17-8 (35.4 mg)
HRESIMS m/z 413.1227 [M + H] (calcd for C H O , 413.1231).
22
21
8
6a,12a-Dehydro-3-O-demethylamorphigenin (9): yellow, amor-
phous solid; [α]21 −17 (c 0.19, MeOH); UV (MeOH) λ (log ε)
D
max
232 (3.86), 280 (3.74), 341 (3.65) nm; ECD {MeCN, λ [nm] (Δε), c
−4
= 1.90 × 10 M} 302sh (−0.82), 279 (−1.26), 249 (0.06), 235
1
13
was purified with semipreparative HPLC (gradient MeOH−H O,
(−0.71), 215 (0.28), 197 (−1.60); H and C NMR, see Tables 4 and
2
+
6
0:40 to 100:0) to yield 16 (0.5 mg) and 4 (1.4 mg).
5; HRESIMS m/z 395.1128 [M + H] (calcd for C H O ,
2
2
19
7
Fractions 18 and 19 (3 g) were combined and chromatographed by
395.1125).
silica VLC using n-hexane, EtOAc, CH Cl , and MeOH by increasing
(6aR,12aR,5′R)-Amorphigenin (10): yellow, amorphous solid;
2
2
[α]22 −4 (c 0.11, MeOH); UV (MeOH) λ (log ε) 204 (2.99),
the polarity of eluting solvents, to afford 17 subfractions (18-1 to 18-
7). After purification by semipreparative HPLC with a gradient of
D
max
1
13
1
236 (2.99), 295 (2.59) nm; H and C NMR, see Tables 4 and 5;
HRESIMS m/z 411.1437 [M + H] (calcd for C H O , 411.1438).
Amorphispironone B (11): yellow, amorphous solid; [α] −175
(c 0.49, MeOH); UV (MeOH) λmax (log ε) 256 (4.05), 265 (4.05),
319 (3.66) nm; ECD {MeCN, λ [nm] (Δε), c = 1.89 × 10 M} 369
(−0.74), 344sh (−0.74), 316 (−7.63), 267 (−9.77), 239 (7.33), 203
(9.84); 1H and C NMR, see Tables 4 and 5; HRESIMS m/z
397.1281 [M + H] (calcd for C H O , 397.1282).
Amorphispironone C (12): yellow, amorphous solid; [α] −92 (c
0.37, MeOH); UV (MeOH) λmax (log ε) 257 (3.44), 265 (3.57), 320
+
MeOH−H O (50:50 to 100:0) as eluent system, 7 (0.6 mg) and 8
2
23 23
7
2
1
(
(
17.0 mg) were obtained from subfraction 18-3 (32.8 mg), while 13
0.6 mg) and 1 (0.3 mg) were isolated from subfraction 18-4 (53.8
D
−
4
mg). Subfraction 18-6 (559 mg) was subjected to passage over a
LiChroprep Diol column using n-hexane−acetone (80:20, 60:40,
0:80, 0:100), followed by purification with semipreparative HPLC
13
2
(
+
gradient MeOH−H O, 50:50 to 100:0) to give 20 (0.6 mg).
2
22 21
7
2
1
Subfraction 18-7 (1.3 g) was separated by a silica gel column using a
D
gradient of n-hexane−acetone (6:1 to 0:100), followed by purification
−
4
with semipreparative HPLC using MeOH−H O (40:60 to 100:0) to
(2.84) nm; ECD {MeCN, λ [nm] (Δε), c = 1.82 × 10 M} 359sh
2
afford 11 (3.4 mg). Subfraction 18-8 (251 mg) was fractionated using
(−1.16), 312 (−6.78), 274 (−9.18), 255 (6.52), 235 (−3.30), 203
1
13
a Sephadex LH-20 column with acetone to give 12 subfractions (18-8-
(13.77); H and C NMR, see Tables 4 and 5; HRESIMS m/z
+
1
1
to 18-8-12). Semipreparative HPLC using MeOH−H O (50:50 to
413.1231 [M + H] (calcd for C H O , 413.1231).
2
22 21
8
00:0) was applied for further purification: 22 (0.5 mg) was obtained
Resokaempferol 3-O-β-D-glucopyranosyl-(1→2)-β-D-glucopyra-
noside-7-O-α-L-rhamnopyranoside (13): yellow, amorphous powder;
from subfraction 18-8-6 (36.9 mg); 28 (4.9 mg) and 30 (0.6 mg) were
J
DOI: 10.1021/acs.jnatprod.6b00809
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
[
3
7
α]19 −51 (c 0.10, MeOH); UV (MeOH) λ (log ε) 266 (4.38),
1 13
D
max
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jnat-
prod.6b00809.
■
*
48 (4.11) nm; H and C NMR, see Table 6; HRESIMS m/z
S
+
41.2237 [M + H] (calcd for C H O , 741.2237).
33
41 19
Daidzein 7-O-β-D-glucopyranosyl-(1→2)-β-D-glucopyranoside
2
1
(14): yellow, amorphous solid; [α]
−34 (c 0.56, MeOH); UV
D
1 13
(
MeOH) λmax (log ε) 251 (4.11), 294 (3.65) nm; H and C NMR,
+
see Table 6; HRESIMS m/z 579.1706 [M + H] (calcd for C H O ,
27
31 14
UV, HRESIMS, and NMR spectra of all the new
compounds and ECD calculations for compounds 11
and 15 as well as dose−response curves of antibacterial
active compounds 16, 17, 20, and 23 (PDF)
5
79.1708).
+)-Puerol A (15): white, amorphous solid; [α]21D +32 (c 0.18,
MeOH); UV (MeOH) λmax (log ε) 287 (2.74), 326 (2.84) nm; ECD
(
−
4
{
(
MeCN, λ [nm] (Δε), c = 2.52 × 101 M} 312 (3.75), 283 (6.14), 248
13
−3.27), 230 (1.30), 202 (−4.34); H and C NMR were identical
31,32
+
with those previously published;
ESIMS m/z 299 [M + H] .
AUTHOR INFORMATION
Corresponding Authors
Tel: +49 211 81 15979. Fax: +49 211 81 11923. E-mail:
zhenfeizi0@sina.com. (Z. Liu).
Tel: +49 211 81 14163. Fax: +49 211 81 11923. E-mail:
proksch@uni-duesseldorf.de. (P. Proksch).
ORCID
Wenhan Lin: 0000-0002-4978-4083
Zhen Liu: 0000-0003-3314-7853
Notes
■
*
Acid Hydrolysis and HPLC Analysis of Compounds 13 and
4. Compounds 13 and 14 were separately hydrolyzed with 2 N HCl
0.1 mL) at 80 °C for 4 h. After drying in vacuo, the residue was
1
(
dissolved in pyridine (0.1 mL) containing L-cysteine methyl ester
hydrochloride (0.5 mg) and heated at 60 °C for 1 h. A solution of p-
tolylisothicyanate (0.5 mg) in pyridine (0.1 mL) was added to the
mixture, which was heated at 60 °C for 1 h. After the solvent was
removed, the residue was dissolved in MeOH and analyzed by HPLC
*
(
MeOH−H O, 1 mL/min). The peaks at 24.580 and 25.587 min were
2
identical to the derivatives of pure standard β-D-glucose and α-L-
rhamnose that had been prepared using the same method as described
for compounds 13 and 14.
59
The authors declare no competing financial interest.
Methylation of Compound 20. A mixture of potassium
carbonate (254 mg) and dry methyl iodide (0.12 mL) was added to
an acetone solution (2 mL) of 20 (3.0 mg). After stirring at room
temperature for 10 h, the remaining acetone was evaporated in vacuo.
The residue was then dissolved in 15 mL of ethyl acetate, and 5 mL of
water was added. The organic layer was separated and dried over
anhydrous magnesium sulfate. The dried organic layer was filtered and
concentrated to afford 3.8 mg of a brown, amorphous solid.
ACKNOWLEDGMENTS
■
P.P. thanks BMBF (project BALIPEND) for financial support.
R.M. wishes to thank DIKTI (Directorate General Higher
Education) Indonesia for a scholarship. T.K. and A.M. thank
the European Regional Development Fund (GINOP-2.3.2-15-
016-00008) for financial support and the National Informa-
tion Infrastructure Development Institute (NIIFI 10038) for
CPU time.
2
Computational Section. Mixed torsional/low-frequency mode
conformational searches were carried out by means of the Macro-
model 9.9.223 software using the MMFF with an implicit solvent
60
model for CHCl3. Geometry reoptimizations were carried out at the
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