Bioactive alkenylphenols from Piper obliquum

Article abstract of DOI:10.1016/j.bmc.2008.01.018

Various parts of Piper obliquum Ruiz & Pavon yielded the new alkenylphenols obliquol A (1) and obliquol B (2), the new 4-chromanone 3 together with the known compounds 4 and 5. A synthesis of obliquol B (2) was developed in order to confirm its structure and to provide sufficient amounts for biological testing. Compounds 1 and 2 have antibacterial activity comparable to that of ampicillin, 2 in addition possesses potent anti-NF-κB activity by targeting early events in the TNFα-induced NF-κB inflammatory pathway, which may explain the effects reported for the traditional use of the plant.

Full text of DOI:10.1016/j.bmc.2008.01.018

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 4120–4126
Bioactive alkenylphenols from Piper obliquum
Carola Valdivia,a,b Nieves Marquez,c John Eriksson,d Antonio Vilaseca,b
Eduardo Munozc and Olov Sternera,*
˜
aDivision of Organic Chemistry, Lund University, Box 124, Getingevagen 60, SE-221 00 Lund, Sweden
bCentro de Tecnologı´a Agroindustrial, Universidad Mayor de San Simo´n, calle Jordan final este,
casilla 1 Sucursal UMSS, Cochabamba, Bolivia
cDepartamento de Biologı´a Celular, Fisiologı´a e Inmunologı´a, Universidad de Co´rdoba, Facultad de Medicina,
Avda, de Menendez Pidal s/n, 14004 Co´rdoba, Spain
dDepartment of Applied Microbiology, Lund University, Box 124, SE-221 00 Lund, Sweden
Received 11 October 2007; revised 4 January 2008; accepted 11 January 2008
Available online 16 January 2008
´
Abstract—Various parts of Piper obliquum Ruız & Pavon yielded the new alkenylphenols obliquol A (1) and obliquol B (2), the new
4-chromanone 3 together with the known compounds 4 and 5. A synthesis of obliquol B (2) was developed in order to confirm its
structure and to provide sufficient amounts for biological testing. Compounds 1 and 2 have antibacterial activity comparable to that
of ampicillin, 2 in addition possesses potent anti-NF-jB activity by targeting early events in the TNFa-induced NF-jB inflamma-
tory pathway, which may explain the effects reported for the traditional use of the plant.
ꢀ 2008 Elsevier Ltd. All rights reserved.
1. Introduction
inducible transcription factor made up of homo- and
heterodimers that interact with a family of inhibitory
IjB proteins, of which IjBa is the best charac-
terized. In most cell types, these proteins sequester
NF-jB in the cytoplasm by masking its nuclear local-
ization sequence. Stimulation of cells with a variety of
physiological or pathogenic stimuli leads to phosphor-
ylation, ubiquitination, and the subsequent degrada-
tion of IjBa proteins.4 The degradation of IjB
results in the translocation of NF-jB from the cyto-
plasm to the nucleus, and IjBa kinases (IKKs)-depen-
dent phosphorylation of IjBa is a key step involved in
the activation of NF-jB complexes.3 NF-jB is impli-
cated in the regulation of several cytokines, chemo-
kines, adhesion molecules, acute phase proteins, and
inducible effector enzymes that participate in inflam-
matory diseases such as rheumatoid arthritis,
asthma, and inflammatory bowel disease.2,5,6 In addi-
tion, the metabolites were assayed for antibacterial
activity.
Piper species are known as ‘thuda’ by the Tacana
community in Bolivia. The Tacana recognize different
classes of thuda by the smell, color, shape, and place
of collection. Several thudas have been used for many
medicinal purposes, for example as antiseptics, to
treat skin diseases, as painkillers, and against fever,1
although it is difficult to determine exactly which spe-
cies that were used traditionally due to the number of
Piper species that grow in the area. Here we wish to
report a chemical and biological investigation of Piper
obliquum, from which two new alkenylphenols and a
new 4-chromanone derivative were obtained together
with known metabolites. Motivated by the traditional
use of thuda, the isolated metabolites were assayed for
inhibition of the NF-jB pathway. The transcription
factor NF-jB plays a key role for the inducible
expression of genes mediating proinflammatory ef-
fects, and is thus an important target for the develop-
ment of anti-inflammatory drugs.2,3 NF-jB is an
2. Chemistry
Keywords: Piper obliquum; Alkenylphenols; Obliquol A and B; Struc-
ture determination; Synthesis; Antibacterial; Anti-inflammatory.
An ethanol extract of the inflorescences of P. obliquum
yielded the two new alkenylphenols obliquol A (1), and
obliquol B (2) (see Fig. 1 for structures, names pro-
*
Corresponding author. Tel.: +46 462228213; fax: +46 462228209;
e-mail: Olov.Sterner@organic.lu.se
0968-0896/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.01.018

C. Valdivia et al. / Bioorg. Med. Chem. 16 (2008) 4120–4126
4121
1'
O
O
14'
4'
4
HO
HOOC
3
R
1 R = H
2 R = OH
:
1H-1H COSY
: HMBC
3'
5'
1'
7'
9'
8
O
Figure 2. Pertinent COSY and HMBC correlations observed with
chromanone 3.
8'
2
4'
6'
2'
6
HOOC
4
O
3
cient information to position it, as an hydroxyl group,
on C-3. For the 4-chromanone 3, the elemental com-
position was by HRESIMS experiments suggested to
be C22H28O4. COSY and HMBC correlations estab-
lished the presence of a disubstituted phenolic moiety,
substituted with an acyl group in position 2 and a car-
boxylic functionality in position 4. In addition, a C15
isoprenoid chain ending with the acyl carbonyl could
be deduced. C-2 is oxygenated, as evidenced by its
chemical shift and the fact that 2-CH3 only give
HMBC correlations to C-2, C-3, and C-10. As the
unsaturation index (9) of 3 required an additional
ring, this has to be between C-2 and C-8a. Pertinent
COSY and HMBC correlations for 3 are shown in
Figure 2.
OH
4
COOH
H3CO
OCH3
OCH3
OH
H3CO
OCH3O
5
Obliquol B (2) was isolated in quite small amounts and
in order to facilitate the biological testing we decided
to synthesize it. Natural alkenylphenols have been syn-
thesized previously,19,20 but we were interested in mak-
ing use of the efficient metathesis reaction. Veratrole
(6) underwent a Friedel–Crafts acylation with 4-pente-
noyl chloride to provide the coupled product 7
(Scheme 1), and a Clemensen reduction afford 4-pent-
4-enyl-1,2-dimethoxybenzene (8). A cross-metathesis
reaction between the two terminal alkenes 8 and 1-
undecen gave 10 in good yields. The subsequent
demethylation of the catechol ring gave obliquol B
(2) as the only product, in all ways identical to the nat-
ural product.
Figure 1. Secondary metabolites isolated from the inflorescences (1
and 2) and the leaves (3, 4, and 5) of Piper obliquum.
posed by us), while an ethanol extract of the leaves
gave the new 4-chromanone (3) together with the
known 3-farnesyl-4-hydroxybenzoic acid7–12 (4) and
20-hydroxy-3,4,40,60-tetramethoxychalcone13–15 (5). De-
spite the fact that the genus Piper has been intensively
studied, alkenylphenols have rarely been isolated. In
fact, only the occurrence of a C16 alkenylphenol in Pi-
per hispidum16 and the C8 and C10 alkenylphenols gib-
bilimbols A–D from Piper gibbilimbum17 have been
reported.
3. Results and discussion
High resolution MS experiments suggested that the
elemental composition of obliquol A (1) is C20H32O,
and this was confirmed by 1D NMR data. HMBC
correlations between 10-H2 and C-1, C-2, and C-6
established a para substituted phenol. The position
of the double bond in the chain was determined by
the COSY correlations between the benzylic methylene
(10-H2) at d 2.54 with 20-H2 at d 1.63 and further on to
30-H2 which, in turn, correlate to the olefinic hydro-
gens (40-H/50-H), confirming that the double bond is
between C-40 and C-50. The E stereochemistry was as-
signed by comparison of the chemical shifts of C-30
and C-60 at d 31.9 and d 32.7, respectively, for a Z
configuration chemical shifts between d 26 and 27
would be expected.18 The NMR data of obliquol B
(2) are very similar to those of 1, although it contains
one additional oxygen according to the MS data. An
analysis of the HMBC spectrum of 2 provided suffi-
The isolated compounds were tested in a NF-jB-depen-
dent luciferase gene reporter assay, where the amount
of the luciferase gene product reflects the extent of
NF-jB transcriptional activation. The HIV-1 promoter
is highly responsive to TNFa-induced NF-jB pathway
and by using the 5.1 stably transfected cell line it could
be shown that obliquol B (2), in a concentration-depen-
dent manner, inhibited TNFa-induced HIV-1-LTR
transactivation (Fig. 3).
Interestingly, obliquol A (1) did not show a significant
anti-NF-jB activity, demonstrating that the 1,2-dihy-
droquinone moiety is critical for the biological activity
of obliquol B in the NF-jB pathway. Compounds 3, 4,
and 5 were inactive in this biological assay. The IC50
inhibitory activity for obliquol B (2) was calculated

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C. Valdivia et al. / Bioorg. Med. Chem. 16 (2008) 4120–4126
O
a
b
H3CO
H3CO
H3CO
7
8
OCH3
6
OCH3
OCH3
c
9
d
2
H3CO
10
OCH3
Scheme 1. Reagents and conditions: (a) 1-pentenoyl-chloride, AlCl3, CH2Cl2, 0 ꢁC (74%); (b) Zn, HgCl2, 50% aq HCl (85%); (c)
CH2@CH(CH2)8CH3 (9), Grubbs catalyst, 2nd generation, CH2Cl2, reflux (74%); (d) TMSI, 20% mol 2,6-di-tert-butyl-4-pyridine, CDCl3 (87%)
(the reaction was carried out in a NMR tube with a sealed cap).
80
70
60
50
40
30
20
10
0
_
_
µM
10
25
10
25
10
25
10
25
10
25
1
2
3
4
5
TNFα
Figure 3. Effects of Piper obliquum compounds on NF-jB activation. The 5.1 cells were pre-treated with increasing concentrations of the isolated
compounds for 15 min and then treated with TNFa for 6 h, after which the luciferase activities were measured in the cell lysates and expressed as a
fold induction.
to be close to 5 lM. We also determined that 2 does
not affect the luciferase activity induced by doxycy-
cline in the HeLa-Tet-On-Luc cells, ruling out the pos-
sibility that the NF-jB inhibitory activity of 2 is
mediated by either non-specific inhibition of the lucif-
erase activity or cytotoxicity (Fig. 4A). It has been
shown that natural compounds isolated from medici-
nal plants with anti-inflammatory activities, such as
Salix alba and Tanacetum parthenium, have potent
anti-NF-jB activity by acting a different levels of the
NF-jB pathway.6 Therefore the biochemical pathways
affected by obliquol B (2) was investigated by immu-
noanalysis of proteins involved in the NF-jB path-
way. In 5.1 cells we detected that TNFa induced a
rapid phosphorylation and degradation of the cyto-
plasmic IjBa protein, and this was prevented by pre-
incubation of the cells with obliquol B (10 lM) prior
to TNFa stimulation (Fig. 4B).
This result indicates that 2 targets the IKK complex,
and we are currently investigating this possibility.
The use of plant extracts in the healing of inflamma-
tory diseases is millenia old and continues to this
day. Alkenylphenols such as gibbilimbols show potent
antibacterial properties toward Staphylococcus epider-
mis and B. cereus, and also display cytotoxic proper-
ties toward KB nasopharyngal carcinoma cells.17
Compounds 1, 2, 3, and 5 were evaluated for antibac-
terial activity (the antibacterial properties of com-
pound 4 have been reported previously),21 and the
minimum inhibitory concentration (MIC) with Esche-
richia coli and S. epidermis was determined by the
broth dilution method22 (see Table 3). It is obvious
that both 1 and 2 possess potent antibacterial activity,
comparable to that of ampicillin which was used as
positive control. Compound 5 is less potent while 3
is only weakly active.

C. Valdivia et al. / Bioorg. Med. Chem. 16 (2008) 4120–4126
4123
60
50
5.1
HeLa TET-ON-LUC
40
30
20
10
0
-
-
25
1
5
10
15
Obliquol B (µM)
TNF / DOX
α
-
-
-
-
-
+
5
+
+
Obliquol B
TNF (min)
α
5
15
30
15
30
κ
α
P-I B
κ
I B
α
α
-Tubulin
Figure 4. (A) Determination of the IC50 inhibitory anti-NF-jB activity. Both the 5.1 and the HeLa-Tet-On-Luc were pre-incubated increasing
concentrations of obliquol B (2) for 15 min and then stimulated with TNFa (2 ng/mL) or Doxycyclin (DOX; 1 lg/mL) for 6 h, after which the
luciferase activities (R.L.U.; relative light units) were measured in the cell lysates. (B) Effects of obliquol B (2) on the NF-jB activation pathway 5.1
cells were incubated with the compounds for 15 min and then treated with TNFa for the indicated times and the then tested for IjBa
phosphorylation and degradation, and a-tubulin expression by Western blots.
4. Conclusion
5.2. Plant material
´
A large number of compounds isolated from plants are
currently known as NF-jB modulators,6 and our re-
sults have set the biochemical principles that explain
the anti-inflammatory activity of P. obliquum, a plant
used in traditional medicine. In addition, obliquol B
(2) should be expected to have also an antioxidative
activity, although this was not tested. However, it
should be noted that 2 is a catechol and that similar
compounds have been shown to possess allergenic
properties.
Piper obliquum Ruız & Pavon, was collected in the
region of Valle del Sacta in Cochabamba, Bolivia in
November 2003 a voucher specimen (RB-1111) was
authenticated by Rosario Barco, botanist at Martin
Cardenas National Herbarium, where it is deposited.
5.3. Extraction and isolation
The dried inflorescences of P. obliquum (200 g) were ex-
tracted with ethanol (95%) at room temperature for 3
days, and after the extract was concentrated under re-
duced pressure, 5 g of crude extract was obtained. This
extract was subjected to silica gel column chromatogra-
phy, eluting with a petroleum ether/EtOAc gradient
(0%, 25%, 50%, and 75% EtOAc). Four fractions were
obtained, fractions 3 and 4 were further purified by flash
column chromatography using petroleum ether/EtOAc
(3:2), to afford obliquol A (1) (300 mg) and obliquol B
(2), which was difficult to isolate as a pure compound
so further purifications were done by preparative thin
layer chromatography using petroleum ether/EtOAc
(3:2). Running the plate two times afforded 6 mg of obli-
quol B (2). The dried leaves of P. obliquum (300 g) were
extracted with ethanol at room temperature for 3 days.
After evaporation of the solvent using a rotavaporator
8 g of crude extract was obtained. The ethanolic extract
was fractionated by flash column chromatography using
petroleum ether/EtOAc gradient (0%, 25%, 50%, 75%,
and 100% EtOAc) to afford 5 fractions. From fractions
4 and 5, 20 mg of the known 20-hydroxy-3,4,40,6-tetra-
methoxychalcone (5) were isolated as orange needles.
5. Experimental
5.1. General experimental procedure
The NMR spectra were recorded in a Bruker DRX300
spectrometer at 300 MHz (1H) and at 75 MHz (13C)
and
a
Bruker DRX400 spectrometer (at 400/
100 MHz). The chemical shifts (d) are reported in parts
per million relative to chloroform (7.27 and 77.0 ppm
for CDCl3). HRESIMS spectra (H3PO4 for calibration
and as internal standard) were recorded with a Micro-
mass Q-tof Micro spectrometer and FABMS spectra
were recorded with a JEOL SX 102 spectrometer. Or-
ganic extracts were dried over MgSO4. All chromatog-
˚
raphy was performed on 60 A 35–70 lm Matrex silica
gel (Grace Amicon). Preparative TLC were performed
on 1 mm plates (20 · 20 cm) (Aldrich). TLC analyses
were made on Silica Gel 60 F254 (Merck) plates and
visualized withp-anisaldehyde and heating.

4124
C. Valdivia et al. / Bioorg. Med. Chem. 16 (2008) 4120–4126
Fractions 4 and 5 were re-purified by flash column chro-
matography using petroleum ether/EtOAc (2:3) from
which a mixture of two compounds was obtained. Fur-
ther purification by preparative thin layer chromatogra-
phy using CH2Cl2/MeOH (9:1) as eluent afforded 50 mg
of a new 4-chromanone derivative (3) and 100 mg of the
known 3-farnesyl-p-hydroxybenzoic acid (4).
5.7. Western blots
5.1 cells (1 · 106 cells/mL) were stimulated with TNFa
(2 ng/mL) in the presence or absence of the compounds
for the indicated period of time. Cells were then washed
with PBS and proteins extracted in 50 ll of lysis buffer
(20 mM Hepes, pH 8.0, 10 mM KCl, 0.15 mM EGTA,
0.15 mM EDTA, 0.5 mM Na3VO4, 5 mM NaFl, 1 mM
DTT, leupeptin 1 lg/mL, pepstatin 0.5 lg/mL, aprotinin
0.5 lg/mL, and 1 mM PMSF) containing 0.5% NP-40.
Protein concentration was determined by the Bradford
assay (Bio-Rad, Richmond, CA, USA) and 30 lg of
proteins were boiled in Laemmli buffer and electropho-
resed in 10% SDS/polyacrylamide gels. Separated pro-
teins were transferred to nitrocellulose membranes (0.5
A at 100 V; 4 ꢁC) for 1 h. Blots were blocked in TBS
solution containing 0.1% Tween 20 and 5% non-fat
dry milk overnight at 4 ꢁC, and immunodetection of spe-
cific proteins was carried out with primary antibodies
using an ECL system (Amersham, UK).
5.4. Antibacterial activities
The antibacterial properties of compounds 1, 2, 3, and 5
were evaluated against E. coli and S. epidermis (isolates
characterized and stored at the division of Applied
Microbiology, Lund University). E. coli were grown in
LB-broth medium, S. epidermis were grown in a buffered
peptone water medium at 37 ꢁC. The minimal inhibitory
concentration of the compounds previously dissolved in
DMSO (dimethyl sulfoxide) was estimated by the broth
dilution method, using an inoculum of 106–107 cells/mL.
The antibacterial properties were determined by measur-
ing the optical density at 620 nm (OD620) read after 24 h
incubation at 37 ꢁC. All the experiments were performed
in duplicate.
5.8. Obliquol A (1)
Yellowish oil; see Table 1 for H and 13C NMR data;
1
5.5. Cell lines and reagents
HRESIMS m/z M+H+ observed 289.2518, Calcd for
C20H33O 289.2531.
The 5.1 cell line is a Jurkat derived clone stably transfec-
ted with a plasmid containing the luciferase gene driven
by the HIV-1-LTR promoter and was maintained in
exponential growth in RPMI 1640 (Invitrogen Life
Technologies) supplemented with 10% heat inactivated
FCS, 2 mM L-glutamine, 1 mM Hepes and antibiotics.23
The HeLa-Tet-On-Luc cell line was previously de-
scribed.24 Briefly, this cell line contains two plasmids:
the pTET-ON codifies constitutively for rtTA protein
that in response to doxycycline, gets active, and binds
to the pTRE2hyg-Luc starting the synthesis of luciferase
gene. The anti-IjBa mAb 10B was a gift from R.T. Hay
(St. Andrews, Scotland), the mAb anti-tubulin was pur-
chased from Sigma Co. (St. Louis, MO, USA), and anti-
phospho-IjBa was from New England Biolabs (Hitchin,
UK).
5.9. Obliquol B (2)
Yellowish oil; see Table 1 for H and 13C NMR data;
1
HRESIMS m/z M+Na+ observed 327.2281, Calcd for
C20H32NaO2 327.2300.
5.10. 2-Methyl-2,2[(30E)-40,80-dimethylnona-30,70-dienyl]-
2,4-dihydro-2H-1-benzopyran-6-carboxylic acid-4-one (3)
22
Colorless oil; ½aꢀ +8.5ꢁ (c 4, chloroform); see Table 2
D
1
for H and 13C NMR data; HRESIMS m/z M+H+ ob-
served 357.2076, Calcd for C22H29O4 357.2066.
5.11. 4-(1-Oxo-4-pentenyl)veratrole (7)
Veratrole (1 g, 7.2 mmol) was dissolved in dry CH2Cl2
(15 mL). The mixture was cooled to 0 ꢁC and AlCl3 (2 g,
13 mmol) were added in portions over 10 min with vigor-
ous stirring. After 5 min, 4-pentenoyl chloride (859 mg,
7.2 mmol) in CH2Cl2 (10 mL) was added dropwise via
addition funnel. The resulting brown solution was stirred
at 0 ꢁC for 3 h, then poured onto conc. hydrochloric acid
(20 mL) and ice (20 g). The organic layer was separated,
the aqueous layer was extracted with ether (2· 15 mL)
and the combined organic phases were dried (MgSO4)
and concentrated under reduced pressure. The residue
was fractionated by chromatography on silica gel (petro-
leum ether/EtOAc, 4:1) to give 1.2 g (74%) of 7 as a color-
less oil. 1H NMR (300 MHz, CDCl3) d 2.34 (2H, d,
J = 7.7 Hz, 30-H), 2.89 (2H, dd, J1 = 7.6 Hz,
J2 = 2.5 Hz, 20-H), 3.79 (6H, s, OCH3), 4.87 (1H, d,
J = 10.2 Hz, 50-H), 4.96 (1H, d, J = 17.1, 50-H), 5.76
(1H, m, 40-H ), 6.74 (1H, dd, J = 8.3 Hz, J = 2.5 Hz, 5-
H), 7.4 (1H, d, J = 2.3 Hz, 3-H), 7.45 (1H, d, J = 8.3 Hz,
5.6. Luciferase assays
To determine NF-jB-dependent transcription of the
HIV-1-LTR-luc, 5.1 cells were preincubated for 30 min
with the compounds tested as indicated, followed by
stimulation with TNFa (2 ng/mL) for 6 h. Then, the
cells were lysed in 25 mM Tris–phosphate, pH 7.8,
8 mM MgCl2, 1 mM DTT, 1% Triton X-100, and 7%
glycerol. Luciferase activity was measured using an
Autolumat LB 9510 (EG&G Berthold, USA) following
the instructions of the luciferase assay kit (Promega,
Madison, WI, USA) and protein concentration was
measured by the Bradford method. The background ob-
tained with the lysis buffer was subtracted from each
experimental value, the relative light units (RLU)/mg
of protein was calculated and the specific transactivation
expressed as the percentage of transcriptional activity
compared to TNF-a alone (fold induction) or by show-
ing the absolute RLU numbers. All the experiments
were repeated at least four times.
13
6-H). C NMR (75 MHz, CDCl3) d 28.0 (C-30), 36.8
(C-20), 55.4 (OCH3), 55.6 (OCH3), 109.6 (C-6), 114.8

C. Valdivia et al. / Bioorg. Med. Chem. 16 (2008) 4120–4126
4125
Table 1. 1H (300 MHz) and 13C (75 MHz) NMR spectral data for obliquol A (1) and obliquol B (2) (CDCl3, d 7.27 and 77.0)
Position
1H (d, multiplicity, J [Hz])
13C
1
2
1
2
1
2
—
—
6.73 (d, J = 1.8)
153.3 (s)
129.5 (d)
115.2 (d)
134.9 (s)
115.2 (d)
129.5 (d)
34.4 (t)
31.6 (t)
31.9 (t)
129.8 (d)
131.0 (d)
32.7 (t)
29.4 (t)
29.2 (t)
29.6 (t)
29.7 (t)
29.7 (t)
31.9 (t)
22.7 (t)
14.2 (q)
143.3 (s)
141.3 (s)
120.7 (d)
135.8 (s)
115.1 (d)
115.5 (d)
34.6 (t)
31.4 (t)
32.0 (t)
129.7 (d)
130.9 (d)
32.6 (t)
29.3 (t)
29.2 (t)
29.5 (t)
29.6 (t)
29.6 (t)
31.9 (t)
22.7 (t)
14.1 (q)
7.06 (d, J = 8.1)
6.76 (d, J = 8.1)
—
3
—
—
4
5
6.76 (d, J = 8.1)
7.06 (d, J = 8.1)
2.54 (t, J = 7.5)
1.63 (m)
6.79 (d, J = 8)
6.62 (dd, J1 = 8, J2 = 1.8)
2.50 (t, J = 7.5)
1.64 (m)
6
10
20
30
1.99 (m)
5.41 (m)
2.01 (m)
5.40 (m)
40
50
5.41 (m)
1.99 (m)
5.40 (m)
2.01 (m)
60
70
1.29 (m)
1.29 (m)
1.29 (m)
1.29 (m)
80
90
1.29 (m)
1.29 (m)
1.29 (m)
1.29 (m)
100
110
120
130
140
1.29 (m)
1.29 (m)
1.29 (m)
1.29 (m)
1.29 (m)
0.89 (t, J = 7)
1.29 (m)
0.90 (t, J = 6.7)
Table 2. 1H (400 MHz) and 13C (100 MHz) NMR data for 2-methyl 2-
[(30E)-40,80-dimethylnona-30,70-dienyl]-2,4-dihydro-2H-1-benzopy-
ran-6-carboxylic acid-4-one (3) (CDCl3, d 7.27 and 77.0)
(C-3), 122.2 (C-4), 129.7 (C-40), 137.1 (C-50), 148.6 (C-2),
152.8 (C-1), 197.5 (C-10). HRESIMS m/z M+Na+ ob-
served 243.1002, calculated for C13H16NaO3 243.0997.
2
Position
1H
13C
d, multiplicity, J (Hz)
d
5.12. 4-(4-Pentenyl)veratrole (8)
2
—
2.85 (d, J = 16.4); 2.72 (1H, d, J = 16.4)
82.4 (s)
3
47.2 (d)
191.4 (s)
119.9 (s)
129.9 (d)
122.0 (s)
137.2 (s)
118.8 (d)
163.7 (s)
39.3 (t)
A solution of 7 (900 mg, 4 mmol) in ethanol (10 mL) was
added to freshly prepared amalgamated zinc (2 g) in con-
cd HCl (10 mL) and water (10 mL), and the mixture was
stirred vigorously under nitrogen for 16 h. After the
extraction of the mixture with ether, the extract was
washed with water, dried (MgSO4), and the solvent was
evaporated to leave a yellow oil of 4-(4-pentenyl)veratrole
(8) (700 mg, 85%). NMR dH (300 MHz, CDCl3): 1.66
(2H, pt, J1 = 8.1, J2 = 7.0 Hz, 20-H), 2.05 (2H, q,
J = 7.2 Hz, 30-H), 2.53 (2H, t, J = 8.1 Hz, 10-H), 3.78
(3H,s, OMe), 3.81 (3H, s, OMe), 4.94 (1H, d,
J = 10.2 Hz, 50-H), 4.99 (1H, d, J = 17.7, 50-H), 5.79
(1H, m, 40-H), 6.67 (2H, br s, 5-H and 3-H), 6.73 (1H, d,
J = 8.4 Hz, 6-H). NMR (75 MHz, CDCl3) d 30.4 (C-20),
32.9 (C-30), 34.5 (C-10), 55.3 (OCH3), 55.4 (OCH3),
110.7 (C-6), 111.3 (C-3), 114.3 (C-4), 119.8 (C-5), 134.6
(C-50), 138.1 (C-40), 146.7 (C-1), 148.3 (C-2). HRESIMS
m/z M+Na+ observed 229.1204, calculated for
C13H18NaO2 229.1204.
4
4a
—
—
5
8.64 (d, J = 2.2)
—
6
7
8.19 (dd, J1 = 8.7, J2 = 2.2)
7.01 (d, J = 8.7)
—
8
8a
10
1.84 (m); 1.73 (m)
2.13 (m)
5.08 (t, J = 6.6)
—
20
22.1 (t)
30
122.8 (d)
136.2 (s)
39.6 (t)
40
50
1.96 (m)
2.04 (m)
5.06 (t, J = 6.6)
—
60
26.6 (t)
70
124.1 (d)
131.4 (s)
25.6 (q)
15.9 (q)
17.6 (q)
24.0 (q)
170.9 (s)
80
90
1.66 (s)
40-CH3
80-CH3
2-CH3
6-COOH
1.57 (br s)
1.57 (br s)
1.45 (s)
—
5.13. 4-(4-Tetradecenyl)veratrole (10)
4-(40-Pentenyl)veratrole (8) (125 mg, 0.61 mmol) and 1-
undecene (260 lL, 1.2 mmol) were simultaneously
added via a syringe to a stirring solution of Grubb’s cat-
alyst (53 mg, 0.06 mmol, 10% mol) in dry CH2Cl2
(15 mL). The flask was fitted with a condenser and re-
fluxed under N2 for 12 h. The reaction mixture was then
reduced in volume and purified directly on a silica gel
column eluting with 9.5:0.5 petroleum ether/EtOAc. 4-
(4-Tetradecenyl)veratrole (10) was obtained as a light
Table 3. MIC’s values of compounds 1, 2, 3, and 5
Compound
MIC (lg/mL)
E. coli
S. epidermis
Ampicillin
5
5
1
2.5
5
1
2
3
5
5
100
10
50
10

4126
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the relative intensity of alkene 13C peaks at 129.6 and
129.1 ppm). NMR dH (300 MHz, CDCl3): 0.87 (3H, t,
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for the OCH3 protons in the NMR spectrum had disap-
peared and a signal at 2.15 ppm indicated the presence
of CH3I. The brown solution was poured into 5 mL
MeOH and the volatile components were removed at re-
duced pressure. The residue was purified by column chro-
matography using petroleum ether/EtOAc (4:1) as eluent,
to afford obliquol B (2) as a yellowish oil (24 mg, 87%),
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Financial support from the Swedish Board for Scientific
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(CVI-304), and the Alfa Programme (EU) is gratefully
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References and notes
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