PAF-antagonistic bicyclo[3.2.1]octanoid neolignans from leaves of Ocotea macrophylla Kunth. (Lauraceae)

Article abstract of DOI:10.1016/j.phytochem.2009.07.010

Di-nor-benzofuran neolignan aldehydes, Δ⁷-3,4-methylenedioxy-3′-methoxy-8′,9′-dinor-4′,7-epoxy-8,3′-neolignan-7′-aldehyde (ocophyllal A) 1, Δ⁷-3,4,5,3′-tetramethoxy-8′,9′-dinor-4′,7-epoxy-8,3′-neolignan-7′-aldehyde (ocophyllal B) 2,

Full text of DOI:10.1016/j.phytochem.2009.07.010

Phytochemistry 70 (2009) 1309–1314
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
PAF-antagonistic bicyclo[3.2.1]octanoid neolignans from leaves
of Ocotea macrophylla Kunth. (Lauraceae)
a,
b
Ericsson D. Coy-Barrera a, , Luis E. Cuca-Suárez , Michael Sefkow
*
*
a Universidad Nacional de Colombia, Facultad de Ciencias, Departamento de Química, Laboratorio de Investigación en Productos Naturales Vegetales,
Ciudad Universitaria, Cra 30 45-03, AA 14490, Bogotá DC, Colombia
b Universität Potsdam, Institut für Chemie, Karl-Liebknecht-Straße 24–25, 14476 Golm, Germany
a r t i c l e i n f o
a b s t r a c t
Di-nor-benzofuran neolignan aldehydes, D7-3,4-methylenedioxy-30-methoxy-80,90-dinor-40,7-epoxy-8,30-
neolignan-70-aldehyde (ocophyllal A) 1, D7-3,4,5,30-tetramethoxy-80,90-dinor-40,7-epoxy-8,30-neolignan-
70-aldehyde (ocophyllal B) 2, and macrophyllin-type bicyclo[3.2.1]octanoid neolignans (7R, 8R, 30S,
40S, 50R)-D80-40-hydroxy-50-methoxy-3,4-methylenedioxy-20,30,40,50-tetrahydro-20-oxo-7.30,8.50-neolignan
(ocophyllol A) 3, (7R, 8R, 30S, 40S, 50R)-D80-40-hydroxy-3,4,50-trimethoxy-20,30,40,50-tetrahydro-20-oxo-
7.30,8.50-neolignan (ocophyllol B) 4, (7R, 8R, 30S, 40S, 50R)-D80-40-hydroxy-3,4,5,50-tetramethoxy-20,30,40,50-
tetrahydro-20-oxo-7.30,8.50-neolignan (ocophyllol C) 5, as well as 20-epi-guianin 6 and (+)-licarin B 7, were
isolated and characterized from leaves of Ocotea macrophylla (Lauraceae). The structures and configuration
of these compounds were determined by extensive spectroscopic analyses. Inhibition of platelet activating
factor (PAF)-induced aggregation of rabbit platelets were tested with neolignans 1–7. Although compound
6 was the most potent PAF-antagonist, compounds 3–5 showed some activity.
Article history:
Received 5 February 2009
Received in revised form 21 May 2009
Available online 10 August 2009
Keywords:
Ocotea macrophylla
Lauraceae
dinor-Benzofuran neolignan aldehydes
Bicyclo[3.2.1]octanoid neolignans
Platelet activating factor (PAF)
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
neolignans 3–5 (named ocophyllols A–C), the known compounds
20-epi-guianin 6 and (+)-licarin B 7 were also isolated, whose struc-
Ocotea macrophylla Kunth. (Lauraceae) is an endemic plant to
South America. In Colombia, it is mostly found in central and south
regions, known under the names ‘‘canelo” or ‘‘aguacatillo”. Owing to
its fine resistant hardwood, it is very frequently used by the native
people for timber. So far, only one chemical investigation on O.
macrophylla has been reported, which described isolation and iden-
tification of four aporphine alkaloids from extracts of its trunk-
wood (Franca et al., 1975). However, the Lauraceae plants are
well-recognized as a source of bicyclo[3.2.1]octanoid and dihydro-
benzofuran neolignans (Whiting, 1985, 2001), with both of these
being important classes of plant natural compounds having exhib-
ited excellent PAF (platelet activating factor)-antagonistic activity
(Han, 1995). Moreover, PAF has been suggested to be involved in
pathogenesis of many inflammatory disorders and it has been pro-
posed that PAF-antagonists may be of clinical benefit (Koltei et al.,
1991a,b). As part of our research on the chemistry of neolignans
from Lauraceae species, herein described is a phytochemical explo-
ration of leaves of O. macrophylla and which afforded two new din-
or-benzofuran neolignan aldehydes 1–2 (named ocophyllals A–B)
and three new macrophyllin-type bicyclo[3.2.1]octanoid
tures were established on the basis of the spectroscopic means and
by spectroscopic comparison with data in the literature. In addi-
tion, compounds 1–7 were tested for PAF-antagonistic activity.
O
6'
9
O
9
8
7
R1
R3
6'
2
6
8
1'
2'
5'
4'
4'
OH
2
7'
R1
R2
7'
H
7
2'
R2
3'
O
O
6
O
R3
3
4
5
R1,R2 = OCH2O ; R3 = H
R
R
1
2
R1,R2 = OCH2O ; R3 = H
1 = R2 = R3 = OCH3
1 = R2 = OCH3; R3 = H
1 = R2 = R3 = OCH3
R
O
6'
1'
2'
O
O
OH
O
O
O
O
O
* Corresponding authors. Tel.: +57 (1) 3165000x14453/76; fax: +57 (1) 3165220.
E-mail addresses: edcoyb@unal.edu.co (E.D. Coy-Barrera), lecucas@unal.edu.co
(L.E. Cuca-Suárez).
6
7
0031-9422/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2009.07.010

1310
E.D. Coy-Barrera et al. / Phytochemistry 70 (2009) 1309–1314
2. Results and discussion
dioxyphenyl group [dH 7.05 (1H, d, J = 8.1 Hz), 7.24 (1H, dd,
J = 8.2, 1.8 Hz), 7.29 (1H, d, J = 1.8 Hz)], and a tetrasubstitued aro-
matic ring [dH 7.35 (1H, d, J = 1.6 Hz), 7.62 (1H, d, J = 1.6 Hz]. The
13C NMR spectrum of 1 (Table 1) indicated the presence of a car-
bonyl group (dC 191.8), a 3,4-dioxyphenyl group (dC 108.5, 110.4,
120.3, 124.1, 145.5, 146.5), a tetrasubstitued aromatic ring (dC
108.8, 115.5, 132.2, 134.2, 148.5, 150.5), an aromatic O-methyl
group (dC 55.8), a methyl group (dC 9.1), as well as two olefin car-
bons (dC 109.6 and 152.2). This spectroscopic information was used
to establish its core structure as a dinor-benzofuran neolignan
aldehyde (Anh et al., 1996; Benevides et al., 1999), which was con-
firmed by the HMBC correlation data (Fig. 1a): H3-9 [dH 2.51 (3H,
s)] with C-7 and C-8 [dC 152.2 (C-7); dC 109.6 (C-8)], and the qua-
ternary aromatic carbon at dC 134.2 (C-50); H-2 and H-6 [dH 7.29
(1H, d, J = 1.8 Hz); dH 7.24 (1H, dd, J = 8.2, 1.8 Hz)] were correlated
with C-7 (dC 152.2). H-20 and H-60 [dH 7.35 (1H, d, J = 1.6 Hz); dH
7.62 (1H, d, J = 1.6 Hz)] were correlated with C-70 (dC 191.8) and
C-40 (dC 150.5). Thereby, the structure of 1 was determined to be
D7-3,4-methylenedioxy-30-methoxy-80,90-dinor-40,7-epoxy-8,30-
neolignan-70-aldehyde (ocophyllal A).
Compound 2 has the molecular formula C20H20O6 based on
HRMS-ESI data ([M+H]+ m/z 357.1324, calcd. for C20H21O6,
357.1338). The IR spectrum showed absorption bands at 1683
and 2751 cmꢀ1 due to a formyl group. The 1H NMR spectrum of
2 (Table 1) exhibited a similar signal pattern to that of 1, except
for the presence of a symmetrical 3,4,5-trioxyphenyl group [dH
7.02 (2H, s)] and additional three methoxy groups [dH 3.92 (3H,
s); 3.96 (6H, s,)] as a replacement for the 3,4-dioxyphenyl group
and the methylenedioxy group, respectively. The 13C NMR data of
2 (Table 1) were also similar to those of 1 containing three meth-
oxy carbons [dC 58.2, 56.1(x2)] instead of the methylenedioxy
group, which indicated that compounds 1 and 2 have closely re-
lated structures differentiated by the substitution pattern of the
aryl group. Key HMBC correlations for compound 2 were also
similar to those of 1. Therefore, 2 was defined to be D7-3,4,5,30-tet-
ramethoxy-80,90-dinor-40,7-epoxy-8,30-neolignan-70-aldehyde (oco-
phyllal B).
Repeated chromatography of the EtOH extract from the leaves
of O. macrophylla using either Sephadex LH-20 or silica gel yielded
five new neolignans (1–5), along with two known neolignans (6–
7). Whereas the dihydrobenzofuran neolignans are a class of natu-
ral products prevalent in the Lauraceae as well as in the Ocotea
genus (Lordello and Yoshida, 1997; Felicio et al., 1986; Khan
et al., 1987), dinor-benzofuran neolignan aldehydes (like com-
pounds 1–2) are less-common. Indeed, only two reports have
described isolation and identification of the dinor-benzofuran
neolignan aldehydes 2-(4-hydroxyphenyl)-3-methylbenzofuran-
5-carbaldehyde and perseal D, obtained from the bark of Caryo-
daphnosis tonkinensis (Lauraceae), an endemic plant to Vietnam
(Anh et al., 1996), and the leaves of Persea obovatofolia (Lauraceae),
an endemic plant to Taiwan (Tsai et al., 1998), respectively. How-
ever, closely related structures has been reported such as benzofu-
ran neolignans (or eupomatenoids) (Anh et al., 1997; Himmelreich
et al., 1996; Picker et al., 1973) and dinor-dihydrobenzofuran neo-
lignan aldehydes (Benevides et al., 1999; Nascimento and Lopes,
1999; Chauret et al., 1996; Picker et al., 1973). On the other hand,
compounds 3–5 contain a bicyclo[3.2.1]octanoid neolignan skele-
ton, which can be further subdivided into guianin and macrophyl-
lin classes (Whiting, 1985, 2001; Coy-Barrera and Cuca-Suárez,
2008). Guianin-type neolignans are the most well-known bicyclo-
octanoids, and only few species from the Piperaceae, Magnoliaceae
and Lauraceae contain macrophyllin-type neolignans. The known
metabolites, 20-epi-guianin 6 (De Carvalho et al., 1988) and (+)-lic-
arin B 7 (Aiba and Gottlieb, 1975), were identified by comparison
of their spectroscopic data (1H, 13C NMR, and HRMS) with litera-
ture values. The structural features of the new compounds 1–5
are then described on the basis of spectroscopic analyses.
Compound 1 was assigned the molecular formula C18H14O5 as
determined by HRMS-ESI ([M+H]+ m/z 311.0901, calcd. for
C18H15O5, 311.0919), and whose IR spectrum showed absorption
bands at 1688 and 2745 cmꢀ1 due to a formyl group. The 1H
NMR spectrum (Table 1) suggested the presence of an aromatic
O-methyl group [dH 3.93 (3H, s)], a methylenedioxy group [dH
5.98 (2H, s)], a proton as part of a formyl group [dH 10.03 (1H,
s)], a methyl group attached to an olefin [dH 2.51 (3H, s)], a 3,4-
Compound 3 was assigned the molecular formula C20H22O5 by
HRMS-ESI analysis ([M+H]+ m/z 343.1552, calcd. for C20H23O5,
343.1545). The IR spectrum showed absorption bands at 3455
and 1681 cmꢀ1 due to a hydroxyl group and an
a,b-unsaturated ke-
tone functionalities, respectively. The 1H NMR spectrum (Table 2)
evidenced the presence of a methoxy group attached to an ali-
phatic carbon [dH 3.43 (3H, s)], a methylenedioxy group [dH 5.90
(2H, s)], an allyl group [dH 5.15–5.10 (2H, m), 5.87–5.80 (1H, ddt,
J = 17.0, 9.0, 6.7 Hz), 3.12–3.05 (2H, mdd, J = 16.8, 6.7 Hz)], a methyl
group [dH 0.93 (3H, d, J = 6.8 Hz)], a 3,4-dioxyphenyl group [dH 7.08
(1H, d, J = 1.6 Hz), 6.75 (1H, dd, J = 7.5, 1.6 Hz), 6.68 (1H, d,
J = 7.5 Hz)], an olefinic proton as part of an enone system [dH
6.55 (1H, d, J = 1.8 Hz)], a benzylic methine proton [dH 2.90 (1H,
d, J = 7.0 Hz)], a carbinol proton [dH 4.55 (1H, dd, J = 4.7, 1.8 Hz)],
and two methine protons [dH 2.98 (1H, d, J = 4.7 Hz); dH 2.35–
2.42 (1H, m)]. The 13C NMR spectrum of 3 (Table 2) exhibited the
presence of a carbonyl group (dC 194.6), a 3,4-dioxyphenyl group
(dC 109.3, 118.5, 121.3, 135.2, 146.3, 148.2), a carbinol carbon (dC
81.2), a carbinyl carbon (dC 89.9), a methylenedioxy group (dC
101.2), and a methoxy group attached to an aliphatic carbon (dC
53.8). This spectroscopic information was used for establishing
the core structure as a bicyclo[3.2.1]octane derivative (Han et al.,
1992; Zhang et al., 1995; Kuroyanagi et al., 2000; De Carvalho
et al., 1988), which was confirmed by the HMBC data (Fig. 1b):
H-7 [dH 2.90 (1H, d, J = 7.0 Hz)] was correlated with C-40 (dC
81.2). H2-70 [dH 3.12–3.05 (2H, mdd, 16.8, 6.7 Hz)] and H-60 [dH
6.55 (1H, d, 1.8 Hz)] were correlated with C-20 (dC 194.6), which
confirmed the connection of two C6C3 moieties. The 1H NMR chem-
ical shift of the C-8 methyl group [dH 0.93 (3H, d, J = 6.8 Hz)] indi-
Table 1
1H and 13C NMR chemical shiftsa (d) of compounds 1–2 (400 MHz 1H, 100 MHz 13C,
CDCl3).
No.
1
2
R
R
dC
dC
dH
(
, m, J Hz)
dH
(
, m, J Hz)
1
2
3
4
5
6
7
8
124.1
108.5
145.5
146.5
110.4
120.3
152.2
109.6
9.1
–
135.3
106.3
159.6
137.5
159.6
106.3
152.8
109.1
9.1
–
7.29 (1H, d, 1.8)
7.02 (2H, s)b
–
–
–
–
7.05 (1H, d, 8.1)
7.24 (1H, dd, 8.2, 1.8)
–
b
–
–
–
–
9
2.51 (3H, s)
2.52 (3H, s)
10
132.2
108.8
148.5
150.5
134.2
115.5
191.8
55.8
–
132.3
109.2
147.2
151.2
134.3
114.9
191.2
55.6
–
20
7.35 (1H, d, 1.6)
–
–
–
7.62 (1H, d, 1.6)
10.03 (1H, s)
4.06 (3H, s)
5.98 (2H, s)
–
7.38 (1H, d, 1.3)
–
–
–
7.69 (1H, d, 1.3)
10.06 (1H, s)
4.09 (3H, s)
–
3.92 (3H, s)
3.96 (6H, s)
30
40
50
60
70 (CHO)
OMe-30
OCH2O
OMe-4
OMe-3, OMe-5
101.4
–
–
–
58.2
56.1
–
a
Assignments made by 1H–1H COSY and HMQC experiments.
This signal includes protons H-2 and H-6.
b

E.D. Coy-Barrera et al. / Phytochemistry 70 (2009) 1309–1314
1311
O
O
H
O
OH
H
H
H
O
H
H3
H
H2
O
4'
H
OH
2'
O
7
9
H
O
O
O
H
O
H
O
6'
H
8
O
O
3
H3
3
1
H
C HMBC
b)
H
C HMBC
a)
NOESY
H
H
c)
Fig. 1. Key (a) HMBC correlations of compound 1. (b) HMBC correlations of compound 3. (c) NOESY correlations of compound 3.
Table 2
1H and 13C NMR chemical shiftsa (d) of compounds 3–5 (400 MHz 1H, 100 MHz 13C, CDCl3).
No.
3
4
5
R
R
R
dC
dC
dC
dH
(
, m, J Hz)
dH
(
, m, J Hz)
dH
(
, m, J Hz)
1
2
3
4
5
6
7
8
135.2
118.5
146.3
148.2
109.3
121.3
51.7
46.7
13.2
139.5
194.6
53.5
81.2
89.9
148.2
32.4
134.3
118.0
53.8
–
–
135.8
119.2
146.7
148.3
111.2
120.4
52.2
46.8
13.5
140.2
194.4
53.7
81.4
89.3
148.6
32.5
134.7
117.7
53.8
56.1
56.1
–
–
134.1
105.8
152.7
136.7
152.7
105.8
52.4
46.5
13.1
139.8
194.7
53.4
–
7.08 (1H, d, 1.6)
7.05 (1H, d, 1.7)
6.58 (2H, d, 1.6)b
–
–
–
–
–
–
6.68 (1H, d, 7.5)
6.75 (1H, dd, 7.5, 1.6)
2.90 (1H, d, 7.0)
2.35–2.42 (1H, m)
6.70 (1H, d, 7.4)
6.75 (1H, dd, 7.4, 1.7)
2.91 (1H, d, 7.1)
2.35–2.43 (1H, m)
–
b
2.93 (1H, d, 7.0)
2.30–2.38 (1H, m)
0.95 (3H, d, 6.7)
–
9
0.93 (3H, d, 6.8)
0.90 (3H, d, 6.8)
10
–
–
–
–
20
–
30
2.98 (1H, d, 4.7)
4.55 (1H, dd, 4.7, 1.8)
3.00 (1H, d, 5.2)
4.58 (1H, dd, 5.2, 1.7)
2.97 (1H, d, 5.2)
4.59 (1H, dd, 5.2, 1.8)
–
40
80.9
89.5
50
–
–
60
6.55 (1H, d, 1.8)
3.05–3.12 (2H, mdd, 16.8, 6.7)
5.80–5.87 (1H, ddt, 17.0, 9.0, 6.7)
5.10–5.15 (2H, m)
6.58 (1H, d, 1.7)
3.02–3.08 (2H, mdd, 16.9, 6.8)
5.78–5.85 (1H, ddt, 16.8, 9.4, 6.8)
5.10–5.14 (2H, m)
3.44 (3H, s)
3.80 (3H, s)
3.81 (3H, s)
149.3
33.1
133.9
118.8
53.7
56.2
60.3
56.2
–
6.60 (1H, d, 1.8)
70
3.03–3.08 (2H, mdd, 17.0, 6.6)
5.82–5.88 (1H, ddt, 17.1, 9.4, 6.7)
5.08–5.13 (2H, m)
3.40 (3H, s)
3.83 (6H, s)
3.85 (3H, s)
80
90
OMe-50
OMe-3
OMe-4
OMe-5
OCH2O
3.43 (3H, s)
–
–
–
–
–
–
–
3.83 (6H, s)
101.2
5.90 (2H, s)
–
–
a
Assignments made by 1H–1H COSY and HMQC experiments.
This signal includes protons H-2 and H-6.
b
cated a trans-relationship between C-7 aryl group and C-8 methyl
group (Felicio et al., 1986). Compound 3 has similar 1H and 13C
NMR data to those of kadsurenin B, a bicyclo[3.2.1]octanoid neolig-
nan isolated from the Chinese plant Piper kadsura (Han et al., 1992),
whose molecular formula is identical to that of 3. However, the hy-
droxyl group at C-40 of 3 was defined to be oriented toward enone
system at bicyclooctane moiety, on the basis of two NMR evi-
0.9 mg of this compound was derivatized with (R)- and (S)-MTPA
-methoxy- -(trifuoromethyl)phenylacetyl], following the re-
[a
a
ported methodology (Seco et al., 2001; Ohtani et al., 1991). The 1H
NMR
D
dRS values for H-7 and H-30 were ꢀ0.07 and ꢀ0.10 ppm,
respectively, while for H-8, H3-9, H-60 and OCH3-50 were +0.03,
+0.08,+0.07,+0.04 ppm, respectively. This observation indicated that
the absolute configuration was (S) at the C-40 chiral center and sup-
ported the above-mentioned orientation of C-40 hydroxyl group.
Comparison of the circular dichroism (CD) data of 3 (negative Cotton
effects at 330 and 262 nm) to that of the related kadsurenin C, an-
other bicyclo[3.2.1]octanoid neolignan isolated from Piper kadsura
(Han et al., 1992), whose absolute configuration was previously
establishedbysingle-crystalX-rayanalysis(Jiang etal., 2003), there-
13
dences: 1) the C NMR chemical shift of C-40 at dC 81.2 (Alegrio
et al., 1981), and 2) the multiplicity of H-40 [dH 4.55 (1H, dd,
J = 4.7, 1.8 Hz)], which is attributable to the expected vicinal cou-
pling with H-30 [dH 2.98 (1H, d, J = 4.7 Hz)] on one side, and the
occurrence of a W-coupling with H-60 [dH 6.55 (1H, d, J = 1.8 Hz)]
on the other (De Carvalho et al., 1988). In the case of kadsurenin
B no coupling is observed in these systems (H-40 proton signal is
a singlet), since the dihedral angle for H-40–H-30 system in kad-
surenin B is approximately 90°, whose C-40 hydroxyl group is ori-
ented toward aryl group (Han et al., 1992). These facts can be
explained solely in terms of the opposite location of H-40 in 3
and kadsurenin B. The key NOESY correlation between H-8 [dH
2.35–2.42 (1H, m)] and H-40 confirmed this relative configuration
of the C-40 hydroxyl group. As well, the key NOESY correlation be-
tween H3-9 and H-60 indicated an endo configuration of the C-8
methyl group, which was supported by its 13C NMR chemical shift
(dC 13.2), according to the information reported by Guilhon et al.,
1992. In order to establish the absolute configuration for 3,
bydefiningtheabsoluteconfiguration attheother chiralcentersof 3.
0
Thereby, structure 3 was determined to be (7R, 8R, 30S, 40S, 50R)-D8
-
40-hydroxy-50-methoxy-3,4-methylenedioxy-20,30,40,50-tetrahydro-
20-oxo-7.30,8.50-neolignan (ocophyllol A).
Compound 4 was assigned the molecular formula C21H26O5 as
determined by HRMS-ESI analysis ([M+H]+ m/z 359.1839, calcd.
for C21H27O5, 359.1858). The IR data of 4 was similar to that of 3,
as well as the 1H and 13C NMR spectroscopic data (Table 2) except
for the presence of two aromatic O-methyl groups [dH 3.80 (6H, s);
dc 56.1 (ꢁ2)] instead of the methylenedioxy group. Hence, the 1H
and 13C NMR spectroscopic data of the bicyclo[3.2.1]octane moiety
of 4 were identical to those of 3, this being in agreement with the

1312
E.D. Coy-Barrera et al. / Phytochemistry 70 (2009) 1309–1314
reported data for closely related molecules (Zhang et al., 1995; Han
et al., 1992). HMBC and NOESY correlations of 4 were also similar
to those of 3, indicating the origin of two C3C6 units as in a C-7 aryl/
C-8 methyl trans-relationship, and the endo configuration of the C-
8 methyl group. Neolignan 4 was a 40-epimer of kadsurenin C, be-
cause the NMR differences between them are very similar to that
of between 3 and kadsurenin B. The absolute configuration of 4
was identical to that of 3, since the CD spectrum of 4 showed
negative Cotton effects at 259 and 335 nm. From these data, the
structure of 4 was determined to be (7R, 8R, 30S, 40S, 50R)-D80-40-hy-
droxy-3,4,50-trimethoxy-20,30,40,50-tetrahydro-20-oxo-7.30,8.50-neo-
lignan (ocophyllol B).
Compound 5 was assigned the molecular formula C22H28O6
based on HRMS-ESI ([M+H]+ m/z 389.1945, calcd. for C22H29O6,
389.1964). The spectroscopic comparison (IR, 1H NMR and 13C
NMR) (Table 2) also indicated that 5 differs from 3 and 4 solely
by the substitution pattern on the aryl group: the 3,4-dioxyphenyl
group of 3–4 was replaced by a symmetrical 3,4,5-trioxyphenyl
group of 5, which was substituted by three methoxy groups. HMBC
and NOESY correlations for compound 5 were also similar to those
of 3–4, confirming the origin of two C3C6 units and the endo config-
uration of the C-8 methyl group. The absolute configuration of 5
was identical to that of 3–4, since the CD spectrum of 5 showed
negative Cotton effects at 265 and 332 nm. Therefore structure 5
was determined to be (7R, 8R, 30S, 40S, 50R)-D80-40-hydroxy-
3,4,5,50-tetramethoxy-20,30,40,50-tetrahydro-20-oxo-7.30,8.50-neolig-
nan (ocophyllol C).
3–5 about 30.0%, 31.0% and 40.4% of its potency (Table 3). The re-
lated neolignan kadsurenin C has an IC50 5.1 M (Ma et al., 1993),
l
equivalent to 31.4% of 20-compound 60s potency. To the best of our
knowledge, there is no previous report on the anti-PAF activity by
the known compound 20-epi-guianin 6. Hence, compounds 3–6
would be good candidates toward PAF inhibition.
4. Experimental
4.1. General experimental procedures
Melting points were determined on a Fisher–Johns melting
point apparatus without correction. Optical rotations were mea-
sured on a JASCO DIP-1000 digital polarimeter in CHCl3 at 25 °C.
CD spectra were obtained using a JASCO spectropolarimeter, model
J-715. IR spectra were recorded on a Thermo Nicolet 6700 spectro-
photometer. 1H and 13C NMR spectra were acquired on a Bruker
Avance 400 spectrometer, using TMS as internal shift reference.
High resolution mass spectra (HRMS) were determined on a
qTOF-micro mass spectrometer, Waters Inc., with an ESI source
in the positive ion mode. Thin layer chromatography (TLC) was
performed with commercial silica gel 60 F254 on aluminum sheets
(Merck). Column chromatography (CC) was carried out using silica
gel 60 (70–230 mesh ASTM, Merck).
4.2. Plant material
PAF-antagonistic action was tested with neolignans 1–7 using
the inhibition of PAF-induced aggregation on rabbit platelets
method (Koch, 2005). Whereas ocophyllal A–B 1–2 and (+)-licarin
B 7 showed weakest anti-PAF activity, 20-epi-guianin 6 was found
Whole plants of O. macrophylla Kunth. were collected in June
2006 at side of Nocaima–Vergara road at Nocaima county [coordi-
nates: 05°704800N 74°2905300W], Department of Cundinamarca,
Colombia, and identified by biologist A. Jara Muñoz. A voucher
specimen (number COL517191) was placed at Herbario Nacional
Colombiano, Universidad Nacional de Colombia.
to be the most potent antagonist (IC50 1.6 lM) (see Table 3), but
its activity was no better to that of the positive control ginkgolide
B, a recognized PAF-antagonist from extracts of Ginkgo biloba
(Steinke et al., 1993).
4.3. Extraction and isolation
3. Conclusion
Dried, ground leaves of O. macrophylla (212 g) were macerated
with EtOH (300 mL per day for a week) at 18 °C. The combined eth-
anolic extracts were evaporated by drying (36.3 g) under vacuum,
following which 30 g residing was fractionated by column chroma-
tography (CC) on LH-20 (n-hexane:CHCl3:MeOH 3:1:1) collecting
62 fractions (100 mL each one). These were then combined into
nine fractions 1–9 according to CC similarities in TLC profile. Frac-
tion 2 (389 mg) was subjected silica gel using EtOAc–cyclohexane
(26:74, v/v) and fraction 2.4 (75.8 mg) was further purified by silica
gel CC using MeCN–EtOAc–n-hexane (3:20:77, v/v) as eluent to
afford 3 (18.1 mg). Fraction 3 (225.9 mg) was subjected to silica
gel CC (eluted with acetone–toluene, 12:88, v/v) and then silica
gel CC (MeOH–CH2Cl2–n-hexane, 1:9:90, v/v) to give 4 (12.6 mg)
and 6 (28.5 mg). Fraction 6 (1.25 g) was applied to a silica gel col-
umn (eluted with EtOAc–cyclohexane 28:72, v/v) and following sil-
ica gel CC (EtOAc–petroleum ether (5:5, v/v) as eluent) (MeOH–
CH2Cl2–toluene 5:20:75, v/v) to give 5 (12.3 mg). Fraction 7
(1.74 g) was subjected to silica gel CC obtaining eight fractions
7.1–7.8. Fraction 7.3 (175.6 mg) was applied to a silica gel column
(Me2CO–Et2O–n-hexane 7:13:80, v/v) and following silica gel CC
(MeCN–CHCl3–cyclohexane 2:5:93, v/v) to give 1 (35.8 mg) and 2
(8.5 mg). Fraction 7.7 (73.2 mg) was purified by silica gel CC
(Me2CO–CHCl3–n-hexane 12:12:76 v/v) to afford 7.
Ocotea plants are a good source of neolignans related to hydro-
benzofuran and bicyclo[3.2.1]octanoid classes, which have
PAF-antagonistic activities. In this work, 5 new neolignans 1–5
are reported, two of which were identified as dinor-benzofuran
neolignan aldehydes (ocophyllals A–B 1–2), having a less-common
core. Moreover, these were found to be non-PAF-antagonists. The
other three isolated new neolignans (ocophyllols A–C 3–5) were
related to the macrophyllin-type bicyclo[3.2.1]octane class, whose
common structural feature involves the configuration at C-40.
These were found to be 40-epimers of neolignans isolated from
the Chinese plant Piper kadsura. Ocophyllols A–C 3–5 exhibited
good anti-PAF activity, however, the known neolignan 20-epi-guia-
nin 6 was the most potent PAF-antagonist, having ocophyllols A–C
Table 3
PAF-antagonistic activity of compounds 1–7.
Compound
IC50
l
Ma
Potency (%)b
1
2
3
4
5
6
7
1102 10.2
847.3 8.8
3.1 0.3
3.0 0.5
2.3 0.4
1.6 0.3
564.8 12.3
0.93 0.2
0.10
0.12
30.0
31.0
40.4
58.1
0.16
100
4.3.1. Ocophyllal A [D7-3,4-methylenedioxy-30-methoxy-80,90-dinor-
Ginkgolide Bc
40,7-epoxy-8,30-neolignan-70-aldehyde] (1)
a
The data were expressed as means 95% confidence intervals of four rabbits.
Potency of inhibition was calculated on the IC50 (lM). The effect of gingkolide B
film
Orangish oil; IR
m
cmꢀ1: 2955, 2745, 1688, 1456, 1160,
max
b
977; for 1H and 13C NMR (CDCl3) spectroscopic data, see Table 1.
was arbitrarily set at 100%.
c
HRMS-ESI m/z 311.0901 [M+H]+ (calcd. for C18H15O5, 311.0919).
Ginkgolide B (BN52021) as positive control.

E.D. Coy-Barrera et al. / Phytochemistry 70 (2009) 1309–1314
1313
4.3.2. Ocophyllal B [D7-3,4,5,30-tetramethoxy-80,90-dinor-40,7-epoxy-
4.5. Inhibition of (PAF)-induced aggregation of rabbit platelets assay
8,30-neolignan-70-aldehyde] (2)
film
Orangish oil; IR
m
cmꢀ1: 2968, 2751, 1683, 1477, 1133,
Anti-PAF activity was carried out according to the method re-
ported by Koch (2005). Briefly, convenient platelet rich plasma
(PRP) suspensions from rabbit’s blood were stirred at 800 rpm
and maintained at 37 °C. Samples of PRP were preincubated for
5 min at 37 °C with tested compounds in dimethyl sulfoxide
(DMSO). Aggregation was induced by addition of diluted PAF
max
1050, 920, 654; for 1H and 13C NMR (CDCl3) spectroscopic data,
see Table 1. HRMS-ESI m/z 357.1324 [M+H]+ (calcd. for C20H21O6,
357.1338).
4.3.3. Ocophyllol A [(7R, 8R, 30S, 40S, 50R)-D80-40-hydroxy-50-methoxy-
3,4-methylenedioxy-20,30,40,50-tetrahydro-20-oxo-7.30,8.50-neolignan]
(10 lL). The final PAF concentration was 1 ng/mL for PRP. In order
to eliminate effects of solvent on aggregation, the final concentra-
tion of DMSO was fixed at 0.5%, and did not affect the aggregation
measured. Inhibition of platelet aggregation versus solvent control
was calculated in percent. Half-maximal inhibition concentrations
(IC50) were determined by non-linear regression analysis using the
software GraphPad prism 5.00 (GraphPad software, San Diego, CA,
USA).
(3)
Colourless oil; ½a D25
= ꢀ27.4 (c 0.41, CHCl3); CD (c 0.03, MeOH)
film
ꢂ
[h]262 ꢀ18144, [h]330 ꢀ10425; IR
m
cmꢀ1: 3455, 2988, 1681,
max
1470, 1121, 1050; for 1H and 13C NMR (CDCl3) spectroscopic data,
see Table 2. HRMS-ESI m/z 343.1552 [M+H]+ (calcd. for C20H23O5,
343.1545).
4.3.4. Ocophyllol B [(7R, 8R, 30S, 40S, 50R)-D80-40-hydroxy-3,4,50-
tri-methoxy-20,30,40,50-tetrahydro-20-oxo-7.30,8.50-neolignan] (4)
Acknowledgments
Colourless oil; ½a D25
ꢂ
= ꢀ25.3 (c 0.37, CHCl3); CD (c 0.02, MeOH)
We thank the Chemistry Department and the División de
Investigación – Sede Bogotá (DIB) (Call 2009, Project Code No.
8003383) at Universidad Nacional de Colombia-Bogotá for financ-
ing this work, the NMR laboratory at Universidad Nacional de
Colombia-Bogotá for the NMR spectroscopy, and the Institute für
Chemie at Universität Potsdam for analytical support. E.D. Coy-Bar-
rera acknowledges the DAAD for a research stay at Universität
Potsdam.
film
[h]259 ꢀ17163, [h]335 ꢀ11234; IR
m
cmꢀ1: 3449, 2978, 1680,
max
1456, 1093; 1H and 13C NMR (CDCl3) spectroscopic data, see Table
2. HRMS-ESI m/z 359.1839 [M+H]+ (calcd. for C21H27O5, 359.1858).
4.3.5. Ocophyllol C [(7R, 8R, 30S, 40S, 50R)-D80-40-hydroxy-3,4,5,50-
tet-ramethoxy-20,30,40,50-tetrahydro-20-oxo-7.30,8.50-neolignan] (5)
Needles; mp 188–190 °C; ½a D25
ꢂ
= ꢀ29.8 (c 0.63, CHCl3); CD (c
KBr
0.04, MeOH) [h]265 ꢀ20567, [h]332 ꢀ10745; IR
m
cmꢀ1
:
max
3455, 2983, 1675, 1473, 1199, 1093, 878; for 1H and 13C NMR
(CDCl3) spectroscopic data, see Table 2. HRMS-ESI m/z 389.1945
[M+H]+ (calcd. for C22H29O6, 389.1964).
References
Aiba, C.J., Gottlieb, O.R., 1975. Synthesis of ( )-licarin B. Phytochemistry 14, 253–255.
Alegrio, L.V., Braz-Filho, R., Gottlieb, O.R., Maia, J.G.S., 1981. Lignans and neolignans
from Licaria armeniaca. Phytochemistry 20, 1963–1965.
Anh, N.H., Ripperger, H., Sung, T.V., Adam, G., 1996. Neolignans and a sesquiterpene
from Caryodaphnosis tonkinensis. Phytochemistry 42, 1167–1169.
Anh, N.H., Ripperger, H., Porzel, A., Sung, T.V., Adam, G., 1997. Neolignans from
Caryodaphnosis baviensis. Phytochemistry 46, 569–571.
Benevides, P.J.C., Sartorelli, P., Kato, M.J., 1999. Phenylpropanoids and neolignans
from Piper regnellii. Phytochemistry 52, 339–343.
Chauret, D.C., Bernard, C.B., Arnason, J.T., Durst, T., Krishnamurty, H.G., Sanchez-
Vindas, P., Moreno, N., San Roman, L., Poveda, L., 1996. Insecticidal neolignans
from Piper decurrens. J. Nat. Prod. 59, 152–155.
Coy-Barrera, E.D., Cuca-Suárez, L.E., 2008. Chemical constituents from Pleurothyrium
cinereum (van der Werff) (Lauraceae) from Colombia. Biochem. Syst. Ecol. 36,
674–677.
4.4. Preparation of (R)- and (S)-MTPA esters of 3 (3a and 3b)
(S)- and (R)-MTPA esters of 3 were individually prepared by
reacting 3 (0.9 mg) in pyridine (30
lL) with (R)- and (S)-MTPA
chlorides (1.5 L), respectively, at room temperature overnight.
l
The solvents were removed by evaporation to afford each desired
ester as a colourless oil in good yield, with these individually dis-
solved in CDCl3 and subjected to NMR spectroscopic analyses.
Assignments were made according to the reported method
(Seco et al., 2001).
De Carvalho, M.G., Yoshida, M., Gottlieb, O.R., Gottlieb, H.E., 1988. Bicyclooctanoid,
carinatone and megaphone type neolignans from Ocotea porosa.
Phytochemistry 27, 2319–2323.
Felicio, J.D., Motidome, M., Yoshida, M., Gottlieb, O.R., 1986. Further neolignans
from Ocotea aciphylla. Phytochemistry 25, 1707–1710.
Franca, N.C., Giesbrecht, A.M., Gottlieb, O.R., Magalhaes, A.F., Magalhaes, E.G., Maia,
J.G.S., 1975. Benzylisoquinolines from Ocotea species. Phytochemistry 14, 1671–
1672.
Guilhon, G.M.S.P., Conserva, L.M., Maia, J.G.S., Yoshida, M., Gottlieb, O.R., 1992.
Bicyclo[3.2.1]octanoid neolignans from Licaria brasiliensis. Phytochemistry 31,
2847–2850.
Han, G., 1995. PAF receptor antagonistic principles from chinese traditional
drugs. Progr. Nat. Sci. 5, 299–306.
Han, G.Q., Dai, P., Xu, L., Wang, S.C., Zheng, Q.T., 1992. Bicyclooctanoid neolignans
from Piper kadsura. Chin. Chem. Lett. 3, 521–524.
Himmelreich, U., Ripperger, H., Adam, G., Anh, N.H., Sung, T.V., 1996.
Eupomatenoids from Caryodaphnosis tonkinensis and their complete NMR
assignment. Magn. Reson. Chem. 33, 280–282.
4.4.1. (R)-MTPA ester of 3 (3a)
1H NMR (CDCl3, 400 MHz) d 7.06 (1H, d, J = 1.6 Hz, H-2), 6.76
(1H, dd, J = 7.5, 1.6 Hz, H-6), 6.67 (1H, d, J = 7.5 Hz, H-5), 6.59 (1H,
d, J = 1.8 Hz, H-60), 5.93 (2H, s, OCH2O), 5.82–5.87 (1H, ddt,
J = 17.1, 8.9, 6.7 Hz, H-80), 5.08–5.13 (2H, m, H-90), 5.40 (1H, dd,
J = 4.7, 1.8 Hz, H-40), 3.59 (3H, br s, OMe-700), 3.45 (3H, s, OMe-50),
3.05–3.12 (2H, mdd, J = 16.8, 6.7 Hz, H-70), 2.95 (1H, d, J = 4.7 Hz,
H-30), 2.85 (1H, d, J = 7.0 Hz, H-7), 2.36–2.43 (1H, m, H-8), 0.96
(3H, d, J = 6.8 Hz, H-9). HRMS-ESI m/z 559.1953 [M+H]+ (calcd. for
C30H30F3O7, 559.1944).
Jiang, R.W., Mak, T.C.W., Fung, K.P., 2003. Molecular structures of two bicyclo-
(3.2.1)-octanoid neolignans from Piper kadsura. J. Mol. Struct. 654, 177–182.
Khan, M.R., Gray, A.I., Waterman, P.G., 1987. Neolignans from stem bark of Ocotea
veraguensis. Phytochemistry 4, 1155–1158.
Koch, E., 2005. Inhibition of platelet activating factor (PAF)-induced aggregation of
human thrombocytes by ginkgolides: considerations on possible bleeding
complications after oral intake of Ginkgo biloba extracts. Phytomedicine 12, 10–
16.
Koltei, M., Hosford, D., Guinot, P., Esanu, A., Braquet, P., 1991a. PAF. A review of its
effects, antagonists and possible future clinical implications (Part I). Drugs 42,
9–29.
Koltei, M., Hosford, D., Guinot, P., Esanu, A., Braquet, P., 1991b. PAF. A review of its
effects, antagonists and possible future clinical implications (Part II). Drugs 42,
174–204.
4.4.2. (S)-MTPA ester of 3 (3b)
1H NMR (CDCl3, 400 MHz) d 7.07 (1H, d, J = 1.6 Hz, H-2), 6.75
(1H, dd, J = 7.5, 1.6 Hz, H-6), 6.67 (1H, d, J = 7.5 Hz, H-5), 6.52 (1H,
d, J = 1.8 Hz, H-60), 5.91 (2H, s, OCH2O), 5.78–5.86 (1H, ddt,
J = 17.0, 9.1, 6.8 Hz, H-80), 5.09–5.14 (2H, m, H-90), 5.37 (1H, dd,
J = 4.7 Hz, 1.8, H-40), 3.57 (3H, br s, OMe-700), 3.41 (3H, s, OMe-50),
3.05–3.12 (2H, mdd, J = 16.8, 6.7 Hz, H-70), 3.02 (1H, d, J = 4.7 Hz,
H-30), 2.95 (1H, d, J = 7.0 Hz, H-7), 2.33–2.40 (1H, m, H-8), 0.88
(3H, d, J = 6.8 Hz, H-9). HRMS-ESI m/z 559.1956 [M+H]+ (calcd. for
C30H30F3O7, 559.1944).

1314
E.D. Coy-Barrera et al. / Phytochemistry 70 (2009) 1309–1314
Kuroyanagi, M., Yoshida, K., Yamamoto, A., Miwa, M., 2000. Bicyclo[3.2.1]octane and
6-oxabicyclo[3.2.2]nonane type neolignans from Magnolia denudata. Chem.
Pharm. Bull. 48, 832–837.
Seco, J.M., Quiñoá, E., Riguera, R., 2001. A practical guide for the assignment of the
absolute configuration of alcohols, amines and carboxylic acids by NMR.
Tetrahedron: Asymm. 12, 2915–2925.
Lordello, A.L.L., Yoshida, M., 1997. Neolignans from leaves of Ocotea catharinensis.
Phytochemistry 46, 741–744.
Steinke, B., Müller, B., Wagner, H., 1993. Biologische standardisierungsmethode für
ginkgo-extrakte. Planta Med. 59, 155–160.
Ma, Y., Han, G.Q., Liu, Z.J., 1993. Studies on PAF antagonistic bicyclo[3.2.1]octanoid
neolignans from Piper kadsura. Acta Pharmaceutica Sinica 28, 207–211.
Nascimento, I.R., Lopes, L.M.X., 1999. 2,3-Dihydrobenzofuran neolignans from
Aristolochia pubescens. Phytochemistry 52, 345–350.
Ohtani, I., Kusumi, T., Kashman, Y., Kakisawa, H., 1991. High-field FT NMR
application of Mosher’s method. The absolute configurations of marine
terpenoids. J. Am. Chem. Soc. 113, 4092–4096.
Picker, K., Ritchie, E., Taylor, W.C., 1973. Constituents of Eupomatia species 3. New
eupomatenoid lignans from the leaves and wood of Eupomatia laurina. Aust. J.
Chem. 26, 1111–1119.
Tsai, I.L., Hsieh, C.F., Duh, C.Y., 1998. Additional cytotoxic neolignans from
Persea obovatifolia. Phytochemistry 48, 1371–1375.
Whiting, D.A., 1985. Lignans and neolignans. Nat. Prod. Rep. 2, 191–211.
Whiting, D.A., 2001. Natural phenolic compounds 1900–2000: a bird’s eye view of a
century’s chemistry. Nat. Prod. Rep. 18, 583–606.
Zhang, S.X., Chen, K., Liu, X.J., Zhang, D.C., Tao-Wiedmann, T.W., Leu, S.L.,
McPhail, A.T., Lee, K.H., 1995. The isolation and structural elucidation of
three new neolignans, puberulins A, B, and C, as platelet activating
factor receptor antagonists from Piper puberulum. J. Nat. Prod. 58, 540–
547.