Sebestenoids A-D, BACE1 inhibitors from Cordia sebestena

Article abstract of DOI:10.1016/j.phytochem.2010.09.008

Bioassay-guided fractionation of an extract prepared from the fruits of Cordia sebestena led to the isolation of sebestenoids A-D (1-4). Their structures were elucidated on the basis of extensive NMR experiments and mass spectroscopic measurements. Compounds 1-4 exhibited moderate inhibition of the aspartic protease BACE1.

Full text of DOI:10.1016/j.phytochem.2010.09.008

Phytochemistry 71 (2010) 2168–2173
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Sebestenoids A–D, BACE1 inhibitors from Cordia sebestena
Jingqiu Dai a, Analia Sorribas a, Wesley Y. Yoshida a, Philip G. Williams a,b,
⇑
a Department of Chemistry, University of Hawaii at Manoa, Honolulu, HI 96822, USA
b The Cancer Research Center of Hawaii, 651 Ilalo Street, Honolulu, HI 96813, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 17 June 2010
Received in revised form 3 September 2010
Accepted 20 September 2010
Available online 15 October 2010
Bioassay-guided fractionation of an extract prepared from the fruits of Cordia sebestena led to the
isolation of sebestenoids A–D (1–4). Their structures were elucidated on the basis of extensive NMR
experiments and mass spectroscopic measurements. Compounds 1–4 exhibited moderate inhibition of
the aspartic protease BACE1.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Cordia sebestena
Boraginaceae
Phenylpropaniods
BACE1
1. Introduction
fruit of C. sebestena showed activity against the aspartic protease
BACE1, which is central to Alzheimer’s disease etiology. Bioassay-
Cordia sebestena (L.) (Boraginaceae family) is commonly known
as the Geiger tree. Hawaiians refer to the plant as Kou Haole though,
which roughly translates to ‘‘foreign plant” (Abbott, 1992). Recent
archaeological evidence indicates that the plant is actually indige-
nous to the islands (Burney et al., 2001). Regardless of its origin,
the plant has a long history of use in Hawaiian culture. The plant’s
large dark green leaves have often been used to dye kappa, or wood
cloth, that was used for both clothing and bedding. C. sebestena’s
dark orange flowers are typically used to make Leis. The plant is
best known in the Hawaiian Island for its wood, which due to its
lightweight, durable and easily workable nature, is used for many
traditional items ranging from canoes to food vessels. The plant
can grow up to 25 feet tall in tropical and sub-tropical areas where
it is widely distributed due to its extensive use in landscaping. De-
spite its prevalence, C. sebestena’s phytochemical or pharmacologi-
cal properties have not been reported. Whether this species was
used in traditional Hawaiian medicine is not clear, as there are va-
gue references stating ‘‘many parts were used medicinally” (Krauss,
1993) although no specific diseases or preparations are mentioned
and the plant is not included in more comprehensive texts on the
subject (Chun and Hawaii, 1994; Chun and Kapunihana, 1998;
Gutmanis, 1976; Kaaiakamanu and Chun, 2003; Kaitin, 2010).
As part of a program to examine Hawaiian plants for bioactive
components, we discovered that the EtOAc extract of the white
guided fractionation of this extract has now led to the isolation
four new active phenylpropanoids, given the trivial names sebest-
eniods A–D (1–4) that are described below. These new metabolites
were accompanied by five known compounds, including: caffeic
acid (5), netpetoidin A–B (6,7) (Arihara et al., 1975; Grayer et al.,
2003), rosmarinic acid (8) and its methyl ester (9). In this paper,
we describe the isolation and structure elucidation of these new
compounds by spectroscopic methods along with an assessment
of their bioactivity against BACE1.
2. Results and discussion
2.1. Structure elucidation of the new compounds
Repeated orthogonal chromatographic separations of the crude
extract led to isolation of the optically inactive compound 1 as an
amorphous light yellow powder. Mass spectrometry analysis of
this sample provided a [M+H]+ pseudo-molecular ion at m/z
507.1288 that defined a molecular formula of C27H22O10. Solutions
of this powder displayed a complex UV chromophore, which in-
cluded allowed absorbances at 334 and 343 nm, that implied the
presence of a highly conjugated system. This conclusion was sup-
ported by prominent vibrations attributable to carbonyls
(1702 cmꢀ1) and aromatic rings (1603 and 1504 cmꢀ1) in the IR
spectrum, in addition to those for hydroxyl groups (3403 cmꢀ1).
Considering the source phylum, the 17 degrees of unsaturation
and the number of oxygens required by the molecular formula, 1
was likely a polyphenol.
⇑
Corresponding author at: Department of Chemistry, University of Hawaii at
Manoa, Honolulu, Hawaii 96822, USA. Tel.: +1 808 956 5720; fax: +1 808 956 5908.
E-mail address: philipwi@hawaii.edu (P.G. Williams).
0031-9422/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2010.09.008

J. Dai et al. / Phytochemistry 71 (2010) 2168–2173
2169
O
9'
OH
3'
OH
3"
7'
1'
OMe
8"
O
O
HO
7
1
HO
O
O
9'
1
3
O
O
1'
1"
OMe
3'
OH
8"
8
3
O
OH
OH
1"
3"
1
2
OH
HO
OH
OH
OH
OH
HO
OH
3''
HO
7
OH
OH
O
OH
OH
O
1''
1
OMe
O
O
O
O
8'"
7" 9"
3'
5
6
7
Δ7,8 = E
HO
Δ7,8
OH
= Z
1'''
O
3'''
OH
1'
HO
O
O
OH
OH
7
3
Δ7,8 = Z R1 = OH R2 = H
1
O
3
HO
4 Δ7,8 = E R1 = H R2 = OH
O
O
OR
R1
R2
8 R = H
9
R = CH3
Analyses of the 1H (Table 1) and 2D NMR spectroscopic data
(Table S1) established three fragments A–C (Fig. 1) which
confirmed the compound was a polyphenol derivative. Each frag-
ment shared a similar catechol core as indicated by the three
ABX systems observed in the 1H NMR spectrum, for example, frag-
ment A was comprised of dH-5 6.73 (d, J = 8.2 Hz), dH-6 6.89 (dd,
J = 8.2, 2.1 Hz), dH-2 7.28 (d, J = 2.1 Hz). On the basis of HMBC corre-
lations observed in the 2D NMR spectroscopic data, two of the frag-
ments, A and B, had disubstituted alkenes appended to aromatic
cores, while C contained a trisubstituted olefin. The two carbonyls
observed in the 13C NMR spectrum were substituents on these al-
kenes on the basis of HMBC correlations from H-70 (B) or H-700 (C)
An atom inventory indicated the remaining oxygens were in the
form of five hydroxy groups and either one or two ether linkages
depending on whether C-90 was an ester or a carboxylic acid. This
latter possibility was excluded on the basis of the HMBC correla-
tion observed from the vinyl proton H-8 (A) to the carbonyl carbon
O
OH
6"
O
3
O
9"
OMe
1"
O
OH
4"
1
O
5'
O
9'
5
8
7"
7'
O
A
B
C
0
00
to carbons at dC-9 165.4 and dC-9 165.6, respectively. This latter
carbonyl was clearly a methyl ester on the basis of a HMBC corre-
lation from a downfield methyl singlet at dH 3.72 (C).
Fig. 1. Partial fragments of 1 (A–C) along with selected HMBC (H ? C) and ROESY
(H M H) correlations.
Table 1
1H NMR spectroscopic data for compounds 1–4 (500 MHz, d ppm, J in Hz).
Positionb
1a
2a
3a
4a
2
4
7.28, d (2.1)
6.69, d (1.5)
6.83, d (1.5)
7.27, d (2.0)
6.67, d (1.7)
6.81, d (1.7)
5
6
7
6.73, d (8.2)
6.89, dd (8.2, 2.1)
5.63, d (7.2)
7.22, d (7.2)
7.27, d (2.0)
6.76, d (8.2)
7.04, dd (8.2, 2.0)
7.75, d (15.8)
6.55, d (15.8)
7.24, d (2.0)
6.73, d (8.4)
7.08, dd (8.4, 2.0)
7.33, s
6.75, d (8.2)
6.92, dd (8.2, 2.0)
5.61, d (7.3)
6.70, br s
6.68, br s
6.35, d (12.8)
7.76, d (12.8)
6.36, d (12.8)
7.78, d (12.8)
7.23, d (2.0)
6.74, d (8.4)
7.00, dd (8.4, 2.0)
7.69, d (15.8)
6.42, d (15.8)
7.26, d (2.0)
6.72, d (8.4)
7.07, dd (8.4, 2.0)
7.33, s
8
7.21, d (7.3)
20
50
6.87, d (8.3)
7.30, d (8.3)
7.80, d (15.9)
6.44, d (15.9)
6.75, d (2.0)
6.74, d (8.2)
6.63, dd (8.2, 2.0)
5.81, d (4.4)
4.42, d (4.4)
6.56, d (2.0)
6.84, d (8.4)
7.22, d (8.4)
7.70, d (15.9)
6.29, d (15.9)
6.73, d (2.1)
6.75, d (8.2)
6.62, dd (8.2, 2.1)
5.80, d (4.6)
4.38, d (4.6)
6.59, d (2.0)
60
70
80
200
500
600
700
800
2000
5000
6000
7000
6.56, d (8.1)
6.62, d (8.1)
6.36, dd (8.1, 2.0)
2.84, dd (14.2, 9.2)
2.96, dd (14.2, 4.5)
5.16, dd (9.2, 4.5)
3.62, s
6.40, dd (8.1, 2.0)
2.91, dd (14.2, 9.2)
3.02, dd (14.2, 4.0)
5.19, dd (9.2, 4.0)
3.67, s
8000
OCH3
3.72, s
3.72, s
a
Data measured in MeOH-d4.
Compounds numbered as in Murata et al. (2009).
b

2170
J. Dai et al. / Phytochemistry 71 (2010) 2168–2173
data, this established the molecular formula of 3 as C36H30O14 indi-
HO
3"
OH
4"
cating 3 was larger than 1 or 2. A comparison of the spectroscopic
data with the other compounds isolated from this extract identi-
fied the presence of fragment A (C-1 to C-8), as observed in 1,
and the alpha-hydroxy ester subunit (C-1000 to C-9000) found in the
known rosmarinic acid derivative 9. This latter unit was also later
confirmed by hydrolysis (vide infra). Analysis of the 2D NMR data
established a catechol system, which had a sp3-hybridized carbon
in the benzylic position rather than the typical sp2-hybridized car-
bon. This unit was expanded into a two carbon unit on the basis of
a COSY correlation between this downfield proton and the H-800
methine. Beginning with these two resonances, analysis of HMBC
correlations established that this two carbon unit was part of a
larger dihydrobenzofuran ring system. Specifically, correlations
observed between H-700/C-30, H-50 to C-30, H-800/C-20, and H-60 to
C-20 were crucial for establishing the basic scaffold, while the addi-
tional correlations (Fig. 2) allowed for the final structure of this
compound to be deduced. An isomer 4 that differed both in the
geometry of the C-7/C-8 enol ether and the substitution pattern
on the aromatic ring was also identified from the same fraction
and identified in a similar fashion.
"
"
5
2
O
1"
6
"
O
O
7"
8"
9'"
8'"
9" O
O
HO
7'"
OH
O
OH
O
HO
HO
(Z)
Fig. 2. Selected 1H–1H COSY (bold solid bars) and 1H–13C HMBC (H ? C) correla-
tions of 3.
C-90 (B), which linked these two units. Discriminating between the
various possible combinations of ether linkages to C required de-
tailed analyses of the ROESY spectrum. This spectrum displayed a
cross-peak between H-50 of fragment (B) and H-700 (C), which
would only be observed if these two fragments were connected
at the C-40 oxygen of fragment B. Structure 1 was thus established
as depicted after assignment of the olefin geometries on the basis
D7,8 cis, JH7,H8 = 7.2 Hz, D7 ,8 trans,
JH7 ,H8 = 15.8) and ROESY analyses.
Sebestenoid B (2) was obtained as an amorphous light yellow
powder, which had a molecular formula of C27H22O10 consistent
with an isomer of 1. The NMR spectroscopic data of 2 were quite
similar to those of 1, which allowed facile identification of frag-
ments B and C originally found in 1. Differences were noted for res-
onances of fragment A that were attributed to changes in the
substitution pattern of the aromatic ring and the configuration of
the disubstituted alkene. Specifically, the ortho coupling (8.2 Hz)
observed in 1 was missing in 2, replaced instead with a smaller
meta coupling (1.5 Hz) and corresponding upfield shifts of the
three aromatic protons in that moiety (dH-2 6.69, d, J = 1.5 Hz;
dH-4 6.83, d, J = 1.5 Hz; dH-6 6.70, br s) indicative of a 1,3,5-substitu-
tion pattern on the ring. Likewise, the vicinal coupling between the
alkene protons was now consistent with a trans rather than cis sys-
tem (1 JH-7/H-8 = 7.2 Hz; 2 JH-7/H-8 = 12.8 Hz). With these principal
differences established, this 2-(3,5-dihydroxyphenyl)ethenyl frag-
ment was connected to B via the same HMBC correlation observed
for 1 thus defining the planar structure of 2.
The stereochemistry of compound 3 was assigned using several
techniques. The E and Z-configurations of the C-70/C-80 and C-7/C-8
olefins were elucidated on the basis of their characteristic 3JH,H val-
3
ues (3JH-7 ,H-8 = 15.9 Hz, 3JH-7,H-8 = 7.3 Hz) as was the trans relation-
0
0
of coupling constants
(
0
0
3
3
0
0
00
ship of the substituents on the dihydrobenzofuran ring (3 JH-7 ,H-
3
800
= 4.4 Hz, Lit: JH,H = 4.9 Hz) (Wada et al., 1992). To deduce the
absolute configuration a mixture of 3 and 4 was hydrolyzed with
a solution of sodium hydroxide under a N2 atmosphere (Fig. 3).
After acid-exchange and reversed-phase chromatography 10 and
prolithospermic acid (11) were isolated. A 8000R configuration was
established for 10, on the basis of comparison of the optical rota-
tion with a standard prepared from hydrolysis of rosmarinic acid
8 (Fig. 3). The 700S, 800S configuration of 11 was deduced in a similar
manner using optical rotation data previously reported (Wada
et al., 1992; Murata et al., 2009). While epimerization of the cis-
dihydrobenzofuran derivative under basic conditions has been
noted (Wada et al., 1992), under our conditions the corresponding
trans derivative is stable. The nearly identical magnitude of the vic-
inal proton–proton coupling values at the epimerizable stereogenic
center (C-800) before and after hydrolysis for this moiety support
3
3
00
00
00
00
this conclusion (3 JH-7 ,H-8 = 4.4 Hz, 11 JH-7 ,H-8 =5.1 Hz; if cis
3JH,H = 9.2 Hz (Wada et al., 1992)).
There are few reports describing the isolation of natural prod-
ucts possessing 2-(3,5-dihydroxyphenyl)ethenyl ethers (Nakanishi
et al., 1990), making this a relatively distinct aspect of 2 and 4. In
Compound 3 provided a pseudo-molecular ion peak at m/z
709.1532 [M+Na]+. In conjunction with the 13C NMR spectroscopic
HO
OH
3''
O
1''
OMe
HO
OH
O
3''
O
'"
8
O
7" 9"
3'
10% NaOH
2h, N2
HO
3
OH
OH
1'''
O
'"
8
1''
HO
3'''
1'
O
7" 9"
OH
OH
OH
1'''
3'
O
3'''
OH
HO
7
O
3
Δ7,8 = Z
Δ7,8 = E
1
O
4
HO
1'
11
10
O
HO
OH
]
22 +13
D
Exp: [α]D22 +137
Lit: [α]D22 +142
α
Exp: [
Std: [α]D22 +13
Fig. 3. Degradation of 3 and 4 to assign absolute configuration.

J. Dai et al. / Phytochemistry 71 (2010) 2168–2173
2171
HO
(A)
OH
O
O
COOCH3
COOH
OH
HO
O
O
O
R1
O
O
COOH
O
O
OH
HO
HO
HO
O
OH
12
(B)
(C)
O
S-Enz
O
O
S-Enz
Ph
R2
Ph
O
R
Ph
H
OH
Ph
13
14
15
16
R
O
Oxidative
Decarboxylation
R2
R1
O
R2
R1
R3
R4
R3
R4
R3
OH
O
O
OH
O
O
OH
R4
O
17
18
3 R2= OH R3 = OH R4 = H
4
R2= OH R3 = H R4 = OH
Fig. 4. Proposed biosynthesis of A) dihydrobenzofuran core 12 B) enol ether in 6 and 7 (Grayer et al., 2003) (C) enol ether in 3 and 4 via oxidative decarboxylation.
our hands, the 3,4-dihydroxyphenyl derivatives 1 and 3 were sta-
ble, while the corresponding 3,5-dihydroxyphenyl analogs 2 and
4 decomposed rapidly after isolation in pure form. Discoloration
began almost immediately upon isolation, resulting in the forma-
tion of a complex mixture of products as determined by LC–MS
with both higher and lower molecular weight products present.
Mixtures of 3 and 4 were stable enough for prolonged storage in
the freezer. Presumably, hydrogen bonding which is only possible
in the 3,4-dihydroxyl isomer and a strongly electron-donating
group para to the side-chain in 1 and 3 both stabilize the enol
ether, relative to 2 and 4. Consistent with this latter proposal, are
the observations that electronegative substituents significantly in-
crease the rate of vinyl ester hydrolysis (Euranto, 1977). Circum-
stantial evidence suggests a similar phenomenon may occur with
the appropriately substituted caffeic acid derivatives as well. While
the 2-(3,4-dihydroxyphenyl)ethenyl derivative has been reported
frequently (Grayer et al., 2003), there is only one report of the cor-
responding (3,5-dihydroxyphenyl)ethenyl analog, and both are re-
ported only detectable when extracted with organic solvent
(Grayer et al., 2003) as they do not survive the herbarium sample
preparation of freeze-drying.
is esterified (15) and the thiol eliminated (16). An alternatively
proposal is shown in Fig. 4C, which involves simple esterification
of the corresponding a-hydroxyacid (17), found in 3 and 4 already,
followed by oxidative decarboxylation (Boland and Mertes, 1985;
Neumann and Boland, 1990; Dewick, 2002). Stereospecific loss of
the pro-R or pro-S methylene proton during this process would re-
sult in either E- or Z-olefin.
2.2. Biological activity evaluation
Sebestenoids A–D (1–4) were tested for their ability to inhibit
b-secretase. In the Amyloid Cascade Hypothesis (Hardy, 2006) of
Alzheimer’s disease progression, cleavage of amyloid precursor
protein (APP) by BACE1 begins a cascade leading to formation of
soluble polypeptide oligomers that trigger neurodegeneration
(Shankar et al., 2008). Compounds 3 and 4 were the most potent
with IC50 values of 20 and 22
the smaller compounds 1 and 2 were less active at 32 and
116 M, respectively.
lM, respectively. In comparison,
l
Phenylpropanoids have been reported to display a wide range of
activities, raising questions regarding the specificity of 3 and 4. Re-
cently, Shoichet et al. have demonstrated that many compounds
that frequently hit in enzyme assays are promiscuous inhibitors
that aggregate with enzymes in a non-stoichiometric fashion (Feng
and Shoichet, 2006), which disrupts protein folding (Coan et al.,
2009). To determine if the observed effects were due to aggrega-
tion, our BACE1 assay was repeated with addition of detergent to
disrupt these non-specific interactions. Unfortunately, our original
assay is unable to tolerate the suggested conditions (0.01% or 0.1%
Triton). Presumably, this additive is also disrupting the crucial pro-
tein aggregation step in our complementation assay (Eglen, 2002).
The activities of 3 and 4 were therefore investigated using a surro-
gate system. The compounds were assayed against the serine pro-
The structures of 3 and 4 raise an interesting question regarding
the biosynthesis (Fig. 4) of enol ethers. The substituted dihydro-
benzofuran core likely arises through radical coupling of the sin-
gle-electron oxidation products of
5 and 9 followed by
nucleophilic attack of the C-3 hydroxyl group in 5 on C-7 of 9 to
generate 12 (Dewick, 2002) (Fig. 4A). The carbons of the 2-(3,5-
dihydroxyphenyl)ethenyl ether found in 4 presumably originate
via an acetate-derived pathway, while the 2-(3,4-dihydroxy-
phenyl)ethenyl ether found in 3 arise via a more traditional shiki-
mic acid biosynthesis. On causal inspection it appears these
moieties then couple with the corresponding aldehyde acting a
nucleophile through its enol form, a rather unlikely proposal. To
avoid this issue, Brown and coworkers (Grayer et al., 2003) origi-
nally hypothesized an addition, esterification, and elimination se-
tease chymotrypsin in
a standard chemiluminescent assay
(Gunasekera et al., 2010) with and without the addition of deter-
gent. The IC50 values of both compounds were strongly affected
quence involving
a transient biogenetic equivalent of the
aldehyde 13 in the biosynthesis of 6 and 7 (Fig. 4B). This interme-
diate (13) reacts with a thiol to generate 14 after which the alcohol
by this additive. For 3, the IC50 value was 33
but in the presence of 0.01% Triton (v/v) the potency dropped to
lM without detergent,

2172
J. Dai et al. / Phytochemistry 71 (2010) 2168–2173
134
l
M. The effect with compound 4 was similar, as this com-
M, which
M in the presence of detergent. This behavior is
and IR spectra were measured as a thin film on a CaF2 disk. NMR
pound inhibited chymotrypsin with an IC50 value of 27
increased to 65
similar to that observed for Congo Red in this same assay, which
is a known aggregator, suggesting that these compounds also
interacting with both enzymes in a similar non-specific fashion.
l
spectra were acquired on a 500 MHz spectrometer operating at
500 (1H) or 125 (13C) MHz using the residual solvent signals as
an internal reference (CD3OD dH 3.30 ppm, dC 49.0 ppm). High-res-
olution mass spectroscopic data were obtained on a LC–MSTOF
with ES ionization in the positive mode. Gradient separations were
performed on a system consisting of solvent delivery modules, a
photodiode diode array detector, an evaporating light scattering
detector, and a system controller. TLC analyses was performed on
Si60F254 plates and visualized under UV or by heating after spraying
with a 1% anisaldehyde solution in acetic acid/H2SO4 (50:1). Ros-
marinic acid (8) was purchased from Sigma–Aldrich.
l
3. Conclusion
From a Hawaiian sample of C. sebestena, four new metabolites
were isolated and their structures defined. Compounds 2 and 4
are relatively rare examples of 2-(3,5-dihydroxyphenyl)ethenyl
ethers. In our hands, these units were unstable and degraded to
complex mixtures rapidly. This finding may partially explain why
there are so few reports of compounds containing this moiety. Bio-
logical evaluation against serine and aspartic proteases showed
that these compounds inhibited both enzymes. While this activity
was dose-dependent, it was also strongly influence by the addition
of detergents suggesting a non-specific inhibition. The operational
simplicity of this counter screen suggests it should be incorporated
early in any screening campaign involving plant natural products.
4.2. Plant material
The fruits of Cordia sebestena were collected in Honolulu, Ha-
waii, in August 2009 adjacent to the University of Hawaii at Manoa
campus. A voucher specimen (No. 0901) is maintained in the
Department of Chemistry at the University of Hawaii at Manoa.
4.3. Extraction and isolation
The fruits (2.5 kg) were extracted with EtOAc (3 ꢁ 2L, 12 h each
time) at room temperature. The combined EtOAc extract (10 g) was
separated over silica gel (300–400 mesh) with a step-gradient of
increasing amounts of EtOAc in hexane (1:10, 1:7, 1:5, 1:3, 1:1,
1:0). The gummy fraction (Fraction 15) containing 1 and 2 eluted
with 1:1 hexane:EtOAc, while compounds 3 and 4 eluted with
EtOAc (Fraction 17). Fraction 15 (200 mg) was separated by RP-
HPLC [Luna C8, 250 ꢁ 10 mm, a linear gradient from 5% to 100%
CH3CN in H2O over 40 min, flow rate 3 mL/min, PDA and ELSD
detection] to afford sebestenoid A (1) (tR 26.0 min, 2.0 mg) and
sebestenoid B (2) (tR 25.0 min, 1.0 mg). Sebestenoid C (3) (tR
24.0 min, 2.0 mg) and sebestenoid D (4) (tR 23.5 min, 1.0 mg) were
isolated from Fraction 17 (150 mg) by RP-HPLC [Luna C8,
250 ꢁ 10 mm, a linear gradient from 5% to 100% MeOH in H2O over
40 min, flow rate 3 mL/min, PDA and ELSD detection].
4. Experimental
4.1. General experimental procedures
Optical rotations were measured on a polarimeter at the sodium
line (589 nm). UV spectra were obtained on a spectrophotometer
Table 2
13C NMR spectroscopic data for compounds 1–4 (125 MHz, d ppm).
Positionb
1a
2a
3a
4a
1
2
3
4
5
6
7
127.7
117.3
146.1
148.5
116.1
122.7
113.3
132.8
130.8
116.8
145.5
148.4
115.5
122.3
148.0
115.8
165.4
125.4
118.2
146.1
149.2
116.4
125.2
129.8
138.3
165.6
127.3
116.6
146.3
113.6
146.5
119.6
116.8
135.4
130.8
116.8
146.6
148.8
115.5
122.3
147.6
115.7
165.9
125.4
118.1
148.0
149.2
116.4
125.2
129.8
138.2
165.5
127.8
117.4
146.0
145.7
116.5
122.8
113.2
132.9
124.5
126.5
149.1
145.9
118.6
122.2
144.9
115.7
165.5
133.5
113.3
146.1
146.8
116.3
118.3
88.1
127.5
116.5
146.2
113.6
146.3
119.6
116.8
135.5
124.4
126.3
149.2
145.7
118.6
122.3
144.5
115.7
165.9
133.5
113.3
146.5
146.6
116.4
118.3
88.2
4.4. Characterization
8
4.4.1. Sebestenoid A (1)
10
Amorphous light yellow powder; UV (MeOH) kmax (log e) 243
20
30
(4.1), 252 (4.1), 301 (4.1), 334 (4.2), 343 (4.2) nm; IR (CaF2) mmax
3403, 1702, 1603, 1504, 1260, 1123 cmꢀ1. For 1H (500 MHz,
MeOH-d4) and 13C (125 MHz, MeOH-d4) NMR spectroscopic data,
see Tables 1 and 2; HRESI-TOFMS obsd m/z: 507.1288 [M+H]+,
calcd for C27H23O10+, 507.1291.
40
50
60
70
80
90
100
200
300
400
500
600
700
800
900
1000
2000
3000
4000
5000
6000
7000
8000
9000
OCH3
4.4.2. Sebestenoid B (2)
Light yellow powder; UV (MeOH) kmax (log
(3.9) nm; IR (CaF2) mmax 3385, 1709, 1598, 1501, 1441, 1259,
1122 cmꢀ1 For 1H (500 MHz, MeOH-d4) and 13C (125 MHz,
e) 333 (4.0), 292
.
MeOH-d4) NMR spectroscopic data, see Tables 1 and 2; HRESI-TOF-
MS obsd m/z: 507.1290 [M+H]+, calcd for C27H23O10+, 507.1291.
57.4
57.6
172.3
128.5
117.2
146.6
145.2
116.5
121.8
37.4
172.3
128.5
117.3
146.8
145.3
116.5
121.8
37.5
4.4.3. Sebestenoid C (3)
Light yellow powder; ½a D22
ꢂ
+ 32 (c 0.2, MeOH); UV (MeOH) kmax
(log e) 335 (4.3), 290 (4.2), 257 (4.3) nm; IR (CaF2) mmax 3401, 1728,
1606, 1504, 1440, 1280, 1155 cmꢀ1. For 1H (500 MHz, MeOH-d4)
and 13C (125 MHz, MeOH-d4) NMR spectroscopic data, see Tables
1 and 2; HRESI-TOFMS obsd m/z: 709.1532 [M+Na]+, calcd for
36H30O14Na+, 709.1533.
75.7
171.2
52.8
75.7
171.2
52.9
C
52.8
52.8
4.4.4. Sebestenoid D (4)
Light yellow powder; ½a D22
ꢂ
+ 77 (c 0.2, MeOH); UV (MeOH) kmax
(log e) 223 (4.7), 253 (4.4), 292 (4.4), 337 (4.4) nm; IR (CaF2) mmax
a
Data measured in MeOH-d4.
Compounds numbered as in Murata et al. (2009).
b

J. Dai et al. / Phytochemistry 71 (2010) 2168–2173
2173
3398, 1730, 1608, 1508, 1260, 1176 cmꢀ1. For 1H (500 MHz,
MeOH-d4) and 13C (125 MHz, MeOH-d4) NMR spectroscopic data,
see Tables 1 and 2; HRESI-TOFMS obsd m/z: 709.1526 [M+Na]+,
calcd for C36H30O14Na+, 709.1533.
National Institute of Aging (R20072671). Funds for the upgrades
of the NMR instrumentation were provided by the CRIF program
of the National Science Foundation (CH E9974921) and the Elsa
Pardee Foundation. The purchase of the Agilent LC–MS was funded
by grant W911NF-04-1-0344 from the Department of Defense. We
thank T. Hemscheidt for helpful discussions regarding the biosyn-
thesis and B. Rubio for the chymotrypsin data.
4.4.5. Alkaline hydrolysis of rosmarinic acid (8)
Rosmarinic acid (100 mg) was dissolved in 10% aqueous NaOH
(5 mL) and the mixture stirred for 1 h at room temperature under
N2. The reaction mixture was passed through a DOWEX 50W-X2
column (3 ꢁ 6 cm) and eluted with H2O (200 mL). The eluate was
concentrated under reduced pressure to give a brown residue
(50 mg). The latter was then purified by RP-HPLC [Luna C8,
250 ꢁ 10 mm, a linear gradient from 5% to 10% CH3CN in H2O
(0.1% HCOOH) over 40 min, flow rate 3 mL/min, PDA and ELSD
detection] to give (2R)-3-(3,4-dihydroxyphenyl)-2-hydroxypropa-
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.phytochem.2010.09.008.
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