New constituents from the roots of erythrina X bidwillII

Article abstract of DOI:10.3987/COM-07-11065

Three new compounds, erythbidins C-E (1-3), together with five known compounds 4-8 were isolated from the roots of Erythrina x bidwillii. Their structures were established on the basis of spectroscopic evidence. Among the isolated compounds, erythbidin E (3) showed a potent antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA).

Full text of DOI:10.3987/COM-07-11065

HETEROCYCLES, Vol. 71, No. 8, 2007
1779
HETEROCYCLES, Vol. 71, No. 8, 2007, pp. 1779 – 1785. © The Japan Institute of Heterocyclic Chemistry
Received, 26th March, 2007, Accepted, 11th May, 2007, Published online, 15th May, 2007. COM-07-11065
NEW CONSTITUENTS FROM THE ROOTS OF ERYTHRINA X
BIDWILLII
Hitoshi Tanaka,*,a Hisanori Hattori,a Masaru Sato,b Ryozo Yamaguchi,b
Toshio Fukai,c Toshihiro Tanaka,d and Eiji Sakaid
aFaculty of Pharmacy, Meijo University, Yagoto, Tempaku-ku, Nagoya 468-8503,
b
Japan, Department of Oral Pathology, Asahi University School of Dentistry,
1851-Hozumi, Mizuho, Gifu 501-0296, Japan, cSchool of Pharmaceutical Sciences,
Toho University, Miyama, Funabashi, Chiba 274-8510, Japan, dGifu
Pharmaceutical University, Mitahora-higashi, Gifu 502-8585, Japan
E-mail address: hitoshi@ccmfs.meijo-u.ac.jp
Abstract － Three new compounds, erythbidins C-E (1–3), together with five
known compounds 4–8 were isolated from the roots of Erythrina x bidwillii. Their
structures were established on the basis of spectroscopic evidence. Among the
isolated compounds, erythbidin E (3) showed a potent antibacterial activity against
methicillin-resistant Staphylococcus aureus (MRSA).
INTRODUCTION
The genus Erythrina (Leguminosae) is distributed in the tropical and subtropical regions of the world, and
has been used as a folklore medicinal treatment of microbial infections. Erythrina x bidwillii has been grown
as an ornamental plant, a shrub with scarlet flowers, in the southern area of Japan. There have been many
secondary metabolites extracted from the roots, wood and flowers of E. x bidwillii,1-3 among which the
non-alkaloidal component, erycristagallin, showed a potent antibacterial activity against Streptococcus
mutanns.1 In continuation of our screening of antibacterial compounds against MRSA from Erythrina plants,
we describe the isolation and structural elucidation of a new cinnamylphenol, erythbidin C (1), a new
pterocarpan, erythbidin D (2), and a new 2-arylbenzofuran, erythbidin E (3), along with the known
2-arylbenzofuran (4) and four known isoflavonoids (5-8) from the roots of this plant, and report their
antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA).
The five known compounds were identified as 2-(2,4-dihydroxyphenyl)-6-hydroxybenzofuran (4),4,5
erythbidin A (5),2 hydroxycristacarpone (6),6 3,9-dihydroxypterocarp-6a-ene (7)7 and 8-prenyldaidzein (8)8
by comparing their spectroscopic data with those of authentic samples or reported values.

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HETEROCYCLES, Vol. 71, No. 8, 2007
3
6
3'
6'
HO
OH
4'
5'
2'
1'
4
5
2
1
2a
1"
2"
1a
1'a
4"
1
3"
HO
5"
4
5'
MeO
O
OR2
4a
4'
6
6'
1'
3
2
B
H
7
7
6a
11a
R1O
O
8
9
3'
6b
8
9
11b
6
2
3
1
2'
H
A
4
C
OMe
O
5
OR2
10a
OH
10
3 R1 = Me, R2 = H
3a R1 = Me, R2 = Ac
4 R1 = R2 = H
2
RESULTS AND DISCUSSION
Silica gel chromatography of the CH2Cl2-soluble portion of the acetone extract of the roots of E. x bidwillii
gave three new compounds 1–3, together with five known compounds 5–8.
Erythbidin C (1) was assigned the molecular formula of C20H24O3 ([M]+ m/z 312.1734) based on the
HREIMS spectrum. This compound was found to have an E-cinnamylphenol skeleton from the characteristic
UV spectrum (λmax 203, 250, 285 and 293) and a typical ABX2 system associated with a E-CH=CH–CH2-
1
1
structure in the H NMR spectrum (δ 3.45, 6.36 and 6.46).9,10 The H NMR spectrum showed two singlet
aromatic protons (δ 6.37 and 6.82) and a 3-hydroxy-3-methylbutyl moiety (δ 1.27, 1.76 and 2.63), along with
an unsubstituted phenyl group (δ 7.19, 7.28 and 7.34). The placement of the 3-hydroxy-3-methylbutyl group
at the C-5 position was determined from the HMBC spectrum, which revealed correlations between H-6/C-1",
H-1"/C-4, H-1"/C-5, H-1"/C-6 and H-2"/C-5. The attachment of a 2,4-dihydroxy-5-(3-hydroxy-3-
methylbutyl)benzene ring to the C-1a position was established from both the NOESY data (NOE interaction:
H-6/H-1a) and the HMBC spectrum (correlations: H-6/C-1a, H-1a/C-1, H-1a/C-2 and H-1a/C-6). The
assignment of the unsubstituted phenyl moiety at the C-1'a position was obtained from both the NOESY data
(NOE interactions: H-2'/H-2a and H-6'/H-1'a) and the HMBC spectrum (correlations: H-2'(6')/C-1'a,
H-3'(5')/C-1', H-1'a/C-1', H-1'a/C-2'(6') and H-2a/C-1'). Thus, erythbidin C was characterized as E-1-[2,4-
dihydroxy-5-(3- hydroxy-3-methylbutyl)benzyl]-2-phenylethylene (1).
This is the first report of the naturally-occurring cinnamylphenol derivative that possesses a 3-hydroxy-3-
methylbutyl group.
Erythbidin D (2), C17H16O5 ([M]+ m/z 300.0998), was revealed to have a pterocarpan structure on the basis of

HETEROCYCLES, Vol. 71, No. 8, 2007
1781
13
Table 1. C NMR spectral data for 1－4
its UV spectrum data and a set of four aliphatic
a
b
b
b
proton signals (δ 3.41, 3.54, 4.21 and 5.55) in the
position
1
2
3
4
1
1H NMR spectrum.11 The H NMR spectrum
1
117.3
153.2
104.0
153.4
161.9
92.7
2
153.4
103.9
121.4
153.0
104.0
121.4
displayed metha-coupled aromatic protons (δ 6.07
and 6.22) and three aromatic protons in an AMX
system (δ 6.29, 6.36 and 7.13), as well as two
methoxyl groups (δ 3.78 and 3.87). The locations
of the AMX-type aromatic protons at the C-7, C-8
and C-10 positions were based on both the COSY
spectrum (correlation: H-7/H-8) and the NOESY
data (NOE interaction: H-6a/H-7). The further
assignment of the three aromatic protons was
obtained by the HMBC technique (correlations:
3
162.7
94.2
4
4a
158.4
5
121.2
131.4
112.2
158.6
112.6
156.0
6
67.1
39.5
6a
6b
119.1
125.7
108.0
159.5
98.5
7
96.2
155.3
124.1
98.2
155.5
123.5
8
9
10
H-7/C-9,
H-7/C-10a,
H-8/C-6b, H-8/C-9,
10a
161.9
75.9
H-8/C-10, H-10/C-6b, H-10/C-8, H-10/C-9 and
H-10/C-10a). The locations of the two methoxyl
groups at the C-1 and C-3 positions were achieved
from both the NOESY data (NOE interactions:
OMe-1/H-2 and OMe-1/H-11a for the methoxyl
group at C-1; OMe-3/H-2 and OMe-3/H-4 for the
methoxyl group at C-3) and the HMBC spectrum
11a
11b
103.0
1'
137.3
126.2
128.5
127.2
128.5
126.2
24.0
110.9
156.3
103.8
159.3
108.3
128.0
111.1
156.3
104.0
159.2
108.3
128.0
2'
3'
4'
5'
6'
1"
(correlations:
OMe-1/C-1
for
OMe-C(1);
2"
43.4
OMe-3/C-3 for OMe-C(3)). The absolute
stereochemistry at C-6a and C-11a was assigned
as 6a R: 11a R from the negative optical rotation
value.12 Thus, erythbidin D was characterized as
(6aR,11aR)-9-hydroxy-1,3-dimethoxypterocarpan
(2)
Erythbidin E (3), C15H12O4 ([M]+ m/z 256.0726),
was found to be a 2-arylbenzofuran derivative
based on the UV spectral data and the
characteristic olefinic proton signal (δ 7.17) in the
1H NMR spectrum.13,14 The acetylation of 3 with
acetic anhydride and pyridine produced a
3"
71.9
4"
29.5
5"
29.5
6"
1a
33.4
128.8
130.9
2a
1'a
1-OMe
3-OMe
6-OMe
a: CDCl3.
56.0
55.6
55.9
b: acetone-d6.

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HETEROCYCLES, Vol. 71, No. 8, 2007
1
diacetate (3a), indicating the presence of two hydroxyl groups. The H NMR spectrum revealed three
aromatic protons in an AMX-system (δ 6.82, 7.10 and 7.43) and methoxyl group (δ 3.84) on an A-ring, along
with other AMX-type aromatic protons (δ 6.51, 6.57 and 7.73) on a B-ring. The location of the methoxyl
group at the C-6 position was determined from both the NOESY data (NOE interactions: OMe-6/H-5 and
OMe-6/H-7) and the HMBC experiment (correlation: OMe-6/C-6). The assignment of the AMX-type
aromatic protons at the C-3', C-5' and C-6' positions on the B-ring was obtained from the HMBC spectrum
which indicated correlations between H-3'/C-1', H-3'/C-2', H-3'/C-4', H-3'/C-5', H-5'/C-1', H-5'/C-3',
H-5'/C-4', H-6'/C-2' and H-6'/C-4'. The attachment of the B-ring to the arylbenzofuran moiety at the C-2
position was established by identification of the cross-peak between the aromatic proton at H-6' (δ 7.73) and
an sp2 quaternary carbon at C-2 (δ 153.4) in the HMBC spectrum. Thus, erythbidin E was characterized as
2-(2,4-dihydroxyphenyl)-6-methoxybenzofuran (3).
To the best of our knowledge, erythbidin E (3) is the first naturally-occurring product, which had been
previously prepared by the hydrogenolysis of 2-benzyloxy-4-methoxybenzyl-4-benzyloxy-2-
hydroxyphenylketone.15 The 1H and 13C NMR spectra of 3 are reported here for the first time.
Compound 4, 2-(2,4-dihydroxyphenyl)-6-hydroxybenzofuran, has been isolated from the heartwood of
Lespedeza cyrtobotrya and its acetylated compound was identified with that in the literature.4 However, in
the reported 13C NMR spectral data of 4 several erroneous assignments have been found.5 The revised
assignments of the 13C NMR spectrum are demonstrated in this paper.
The assignment of all the 1H and 13C NMR signals of 1–4 was accomplished by analyses of the 1H-1H COSY,
NOESY, HSQC and HMBC spectra.
Minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) against
MRSA of the isolated compounds 1–8, vancomycin and methicillin, are summarized in Table 2. Among the
isolated compounds, erythbidin E (3) showed the highest antibacterial potency against the MRSA strains,
followed by erythbidin A (5). These compounds also exhibited MBC values comparable to or lower than
those of vancomycin, indicating their potent ability to kill MRSA cells.
EXPERIMENTAL
General Experimental Procedures. The Optical rotation was measured using a JASCO DIP-370 digital
polarimeter. The IR spectra were recorded by a JASCO IR-810 spectrophotometer, and the UV spectra were
obtained in MeOH using a Beckman DU-530 spectrophotometer. The MS spectra were obtained using a
1
13
JEOL JMS-SX102A spectrometer. The H and C NMR spectra were measured by a JEOL ALPHA-600
MHz spectrometer. Column chromatography was performed using Merck silica gel (230–400 mesh). The
MICs and MBCs of the isolated compounds against 13 MRSA strains were determined as previously
reported.16

HETEROCYCLES, Vol. 71, No. 8, 2007
1783
Table 2. Minimum inhibitory and minimum bactericidal concentrations (MICs and MBCs) of
compounds 1–8 against 13 MRSA strains
MICs (µg/mL)
MBCs (µg/mL)
a
a
b
b
Compounds
MIC range
> 50
MBC range
MIC
> 50
50
MIC
> 50
50
MBC
MBC
90
50
90
50
c
c
c
1
–
–
–
2
25–50
50–> 50
12.5–25
25–> 50
12.5–25
> 50
12.5
50
> 50
25
3
3.13–6.25
6.25–25
6.25–12.5
> 50
6.25
12.5
12.5
> 50
25
6.25
25
4
50
5
12.5
> 50
25
12.5
12.5
c
c
c
6
–
–
–
25－50
7
12.5–25
> 50
50
50
c
c
c
8
> 50
1.56
> 50
> 50
3.13
> 50
–
–
–
Vancomycin
Methicillin
0.78–1.56
12.5–> 50
3.13–25
> 50
6.25
> 50
25
> 50
a: Minimum concentration needed to inhibit growth of 50 and 90% of strains tested.
b: Minimum concentration needed to inhibit bacterial recovery of 50 and 90% of strains tested.
c: Not tested.
Plant material. The roots were collected in February 2005 from an E. x bidwillii planted in the Ogasawara
Islands, Tokyo, Japan. A voucher specimen (No.050201) was deposited at the Department of Natural
Product Chemistry, Faculty of Pharmacy, Meijo University.
Extraction and isolation. The fine powdered roots (2.34 kg) were macerated with acetone (48 L) at 23 ℃
for 48 h (2 times) and the solvent was removed to give a residue that was divided into the n-hexane-, CH2Cl2-,
and EtOAc-soluble fractions. The CH2Cl2-soluble fraction (24.44 g) was first applied to a silica gel column
eluted with CHCl3-acetone (20 : 1 → 10 : 1 → 5 : 1 → 1 : 1) and acetone (each volume; 1 L, Column A) to
afford 9 fractions. Fraction A4 (656 mg) was purified by repeated silica gel chromatography using
n-hexane-acetone (3 : 1) to give 2 (15 mg). Fractions A5 and A6 (6.47 g) were subjected to silica gel column
chromatography using CHCl3-acetone (20 : 1 → 5 : 1) (each volume; 30 mL, Column B) to give 40 fractions.
Fractions B20-40 (3.76 g) were separated by silica gel column chromatography using benzene-EtOAc (5 : 1)
and n-hexane-acetone (3 : 1) to give 5 (200 mg) and 7 (3.7 mg). Fraction A7 (2.56 g) was purified by silica
gel column chromatography using CHCl3-acetone (10 : 1.5) and n-hexane-acetone (3 : 1 → 2 : 1) to give 3
(22 mg). Fraction A9 (5.82 g) was analyzed by silica gel chromatography using n-hexane-acetone (2 : 1 →
1 : 1) (each volume; 30 mL, Column C) to give 30 fractions. Fractions C10-14 (173 mg) were separated by
silica gel column chromatography using benzene-acetone (5 : 1 → 1 : 1) to give 1 (6.8 mg), 6 (14 mg) and 8

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HETEROCYCLES, Vol. 71, No. 8, 2007
(4.4 mg). Fractions C18-28 (1.06 g) were separated by silica gel column chromatography using
CHCl3-acetone (10 : 1.5) and benzene-EtOAc (5 : 1) to give 4 (13 mg) and 6 (26 mg).
Erythbidin C (1). Amorphous powder; IR (KBr) νmax cm–1: 3370, 1620; UV λmax nm (log ε): 203 (4.27),
250 (3.79), 285 (3.54), 293 (sh, 3.49); EIMS m/z (rel. int.): 312 ([M]+, 19), 294 (42), 239 (100), 225 (10), 147
(9), 117 (16), 91 (32); HREIMS m/z 312.1734 (M+, Calcd for C20H24O3, 312.1724); 1H NMR (CDCl3): δ 1.27
(6H, s, H-4" and H-5"), 1.76 (2H, t, J = 7.4 Hz, H-2"), 2.63 (2H, t, J = 7.4 Hz, H-1"), 3.45 (2H, d, J = 6.2 Hz,
H-1a), 6.36 (1H, dt, J = 15.9, 6.2 Hz, H-2a), 6.37 (1H, s, H-3), 6.46 (1H, d, J = 15.9 Hz, H-1'a), 6.82 (1H, s,
H-6), 7.19 (1H, t, J = 7.4 Hz, H-4'), 7.28 (2H, t, J =7.4 Hz, H-3' and H-5'), 7.34 (2H, d, J = 7.4 Hz, H-2' and
H-6'); 13C NMR: see Table 1.
Erythbidin D (2). Amorphous powder; [α]D –385° (c 0.1, MeOH); IR (KBr) νmax cm–1: 3420; UV λmax
nm (log ε): 210 (4.96), 239 (sh, 4.22), 285 (3.92); EIMS m/z (rel. int.): 300 ([M]+, 100), 285 (11), 178 (13),
167 (7), 150 (8); HREIMS m/z 300.0998 (M+, Calcd for C17H16O5, 300.0997); 1H NMR (acetone-d6): δ 3.41
(1H, m, H-6a), 3.54 (1H, t-like, J = 11.0 Hz, H-6α), 3.78 (3H, s, OMe-3), 3.87 (3H, s, OMe-1), 4.21 (1H, dd,
J = 11.0, 4.4 Hz, H-6β), 5.55 (1H, d, J = 6.6 Hz, H-11a), 6.07 (1H, d, J = 2.2 Hz, H-4), 6.22 (1H, d, J = 2.2
Hz, H-2), 6.29 (1H, d, J = 2.2 Hz, H-10), 6.36 (1H, dd, J = 8.1, 2.2 Hz, H-8), 7.13 (1H, d, J = 8.1 Hz, H-7),
8.34 (1H, br s, OH); 13C NMR: see Table 1.
Erythbidin E (3). Amorphous powder; IR (KBr) νmax cm–1: 3380, 1620; UV λmax nm (log ε): 211 (4.46),
229 (sh, 4.14), 274 (sh, 4.16), 282 (4.19), 306 (sh, 4.30), 320 (4.55), 334 (4.53); EIMS m/z (rel. int.): 256
([M]+, 100), 241 (83), 149 (5), 128 (9), 101 (10); HREIMS m/z 256.0726 (M+, Calcd for C15H14O4,
256.0735); 1H NMR (acetone-d6): δ 3.84 (3H, s, OMe-6), 6.51 (1H, dd, J = 8.8, 2.2 Hz, H-5'), 6.57 (1H, d, J
= 2.2 Hz, H-3'), 6.82 (1H, dd, J = 8.8, 2.2 Hz, H-5), 7.10 (1H, d, J = 2.2 Hz, H-7), 7.17 (1H, s, H-3), 7.43
(1H, d, J = 8.8 Hz, H-4), 7.73 (1H, d, J = 8.8 Hz, H-6'), 8.58 (1H, br s, OH-4'), 9.14 (1H, br s, OH-2'); 13C
NMR: see Table 1.
Acetylation of 3. A mixture of 3 (8.3 mg), Ac2O (0.5 mL) and pyridine (0.5 mL) was stirred overnight at rt.
The reaction mixture was worked up as usual and the resulting residue was purified by silica gel column
chromatography [benzene-EtOAc (40 : 1)] to yield a diacetate derivative (3a) (10 mg) as an amorphous
powder. IR (KBr) νmax cm–1: 1770, 1620, 1590; UV λmax nm (log ε): 203 (4.41), 237 (sh, 3.94), 281 (sh, 3.94),
315 (4.44), 327 (sh, 4.37); EIMS m/z (rel. int.): 340 ([M]+, 44), 298 (36), 256 (100), 241 (31), 212 (6), 128
1
(3); HREIMS m/z 340.0917 (M+, Calcd for C19H16O6, 340.0946); H NMR (acetone-d6): δ 2.29 (3H, s,
OAc-4'), 2.44 (3H, s, OAc-2'), 3.87 (3H, s, OMe-6), 6.90 (1H, dd, J = 8.8, 2.0 Hz, H-5), 7.12 (1H, d, J = 2.0
Hz, H-3'), 7.17 (1H, d, J = 2.0 Hz, H-7), 7.19 (1H, s, H-3), 7.20 (1H, dd, J = 8.8, 2.0 Hz, H-5'), 7.54 (1H, d, J
= 8.8 Hz, H-4), 8.00 (1H, d, J = 8.8 Hz, H-6').
2-(2,4-Dihydroxyphenyl)-6-hydroxybenzofuran (4). Amorphous powder; IR (KBr) νmax cm–1: 3340,
1630; UV λmax nm (log ε): 209 (4.55), 231 (sh, 4.17), 282 (4.11), 308 (sh, 4.19), 320 (4.39), 335 (4.38);

HETEROCYCLES, Vol. 71, No. 8, 2007
1785
EIMS m/z (rel. int.): 242 ([M]+, 100), 213 (22), 197 (8), 185 (14), 171 (7), 149 (17), 139 (15), 121 (28), 115
(26); HREIMS m/z 242.0574 (M+, Calcd for C14H10O4, 242.0578); 1H NMR (acetone-d6): δ 6.50 (1H, dd, J =
8.5, 2.3 Hz, H-5'), 6.56 (1H, d, J = 2.3 Hz, H-3'), 6.77 (1H, dd, J = 8.5, 2.3 Hz, H-5), 6.97 (1H, br s, H-7),
7.14 (1H, s, H-3), 7.36 (1H, d, J = 8.5 Hz, H-4), 7.72 (1H, d, J = 8.5 Hz, H-6'), 8.41 (1H, br s, OH), 9.10 (1H,
br s, OH); 13C NMR: see Table 1.
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