Trypanocidal labdane diterpenoids from the seeds of Aframomum aulacocarpos (Zingiberaceae)

Article abstract of DOI:10.1016/j.tet.2007.05.120

Two novel labdane type diterpenoids, 8β(17)-epoxy-14,15,16-trihydroxylabd-12(E)-ene (aulacocarpin C) and 15,16-epoxy-14ξ,16ξ-dimethoxylabda-8(17),12-(E)-diene (aulacocarpin D) together with the known aulacocarpin A and B; 14,15-epoxy-8(17),12(E)-labdadien-16-al, coronarin E, and 15,16-epoxy-12β-hydroxy-labda-8(17)-13(16),14-triene were isolated from the seeds of Aframomum aulacocarpos. To the best of our knowledge, the last compound was isolated from a natural source for the first time. Acid hydrolysis of aulacocarpin D led to another new labdane type diterpenoid, 15,16-epoxy-12β-methoxylabda-8(17)-13(16),14-triene. The structures of all compounds were established on the basis of their spectroscopic data. These new compounds exhibit moderate trypanocidal activity.

Full text of DOI:10.1016/j.tet.2007.05.120

Tetrahedron 63 (2007) 8993–8998
Trypanocidal labdane diterpenoids from the seeds of
Aframomum aulacocarpos (Zingiberaceae)
a,b
Sylvain Val ꢀe re T. Sob, Pierre Tane, Bonaventure T. Ngadjui,
Joseph D. Connolly and Dawei Ma
a,*
b,*
c
d
a
Department of Chemistry, University of Dschang, Box 67, Dschang, Cameroon
Department of Organic Chemistry, University of Yaounde I, Box 812, Yaounde, Cameroon
b
c
Chemistry Department, University of Glasgow, G12 8QQ Scotland, UK
d
State Key Laboratory of Bioorganic and Natural Products Chemistry, Shanghai Institute of Organic Chemistry,
Chinese Academy of Sciences, 354 Fenglin Lu, Shanghai 200032, China
Received 24 January 2007; revised 12 May 2007; accepted 31 May 2007
Available online 6 June 2007
Abstract—Two novel labdane type diterpenoids, 8b(17)-epoxy-14,15,16-trihydroxylabd-12(E)-ene (aulacocarpin C) and 15,16-epoxy-
4x,16x-dimethoxylabda-8(17),12-(E)-diene (aulacocarpin D) together with the known aulacocarpin A and B; 14,15-epoxy-8(17),12(E)-
1
labdadien-16-al, coronarin E, and 15,16-epoxy-12b-hydroxy-labda-8(17)-13(16),14-triene were isolated from the seeds of Aframomum
aulacocarpos. To the best of our knowledge, the last compound was isolated from a natural source for the first time. Acid hydrolysis of
aulacocarpin D led to another new labdane type diterpenoid, 15,16-epoxy-12b-methoxylabda-8(17)-13(16),14-triene. The structures of all
compounds were established on the basis of their spectroscopic data. These new compounds exhibit moderate trypanocidal activity.
Ó 2007 Published by Elsevier Ltd.
1. Introduction
2. Results and discussion
The genus Aframomum K. Schum belongs to the econom-
ically and medicinally important family Zingiberaceae.
Over 20 species of this genus are found in Cameroon
where they are widely used for medicinal, ethno dietary,
The dried powdered seeds of A. aulacocarpos were macer-
ated with acetone and the solvent was removed under re-
duced pressure. The crude extract was separated on silica
gel column chromatography to give several fractions, which
were each further purified by open column chromatography,
gel permeation chromatography through Sephadex LH-20,
chromatotron, preparative TLC or re-crystallization, to
afford compounds 1 and 2 together with the previously
1
and spiritual purposes. The seeds of Aframomum aulaco-
carpos (Pellegr. Ex. J. Koechlin) are widely used as a food
spice. A previous study reported the isolation of three
2
diterpenes, aulacocarpinolide, aulacocarpin A, and aulaco-
carpin B from these seeds. The two later compounds
showed antibacterial activity. In our search for larger
4
described aulacocarpin A and B, 14,15-epoxy-8(17),12(E)-
3
5
6–8
labdadien-16-al, coronarin E,
hydroxylabda-8(17)-13(16),14-triene,
lated for the first time from a natural source.
and 15,16-epoxy-12b-
9
–11
quantities of these compounds for bioactivity tests, we
have isolated seven labdane diterpenoids from the seeds
of this plant. This paper describes the isolation and struc-
tural elucidation of three novel compounds, aulacocarpin
C (1) and D (2), and 15,16-epoxy-12b-methoxylabda-
which was iso-
12
11
16
OMe
16
2
0
13 OR
1
2
1
O
8
(17)-13(16),14-triene.
RO 17
20
13
9
15
11
1
0
1
15
17
7
RO
14
3
O
9
5
10
OMe
3
7
19
18
5
19
18
1
R=H
1a R=Ac
2
Keywords: Aframomum aulacocarpos; Zingiberaceae; Labdane diter-
penoids; Aulacocarpin; Trypanocidal.
Compound 1 was obtained as brown needles from EtOAc,
ꢀ
*
Corresponding authors. Tel.: +237 760 58 57; fax: +237 345 12 02
B.T.N.); tel.: +237 761 95 46; fax: +237 345 12 02 (P.T.); e-mail
addresses: ptane@yahoo.com; ngadjuibt@yahoo.fr
mp 160–161 C. Its EIMS showed a pseudo-molecular ion
[MꢁH O] at m/z 320 consistent with the molecular formula
(
+
2
0
040–4020/$ - see front matter Ó 2007 Published by Elsevier Ltd.
doi:10.1016/j.tet.2007.05.120

8994
S. V. T. Sob et al. / Tetrahedron 63 (2007) 8993–8998
1
C H O , which accounted for four degrees of unsaturation.
2
Absorption of hydroxyl groups (n 3474 cm ) and double
max
was deduced by comparison of the H NMR spectroscopic
shifts of the epoxide proton H-17 with those reported for
0 34 4
ꢁ1
ꢁ
1
4,14
bond (n
1640 cm ) was observed in the IR spectrum.
aulacocarpin A and B. Compound 1 was thus established
to be 8b(17)-epoxy-14,15,16-trihydroxylabd-12(E)-ene and
was trivially named as aulacocarpin C.
max
1
The H NMR spectrum of 1 (Table 1) displayed signals of
three tertiary methyl groups at d 0.95, 0.90 and, 0.85 and
a deshielded olefinic proton at d 5.46 (dd, J¼7.8, 4.7 Hz)
4
,12
typical of labdanes type skeleton.
This spectrum also
Acetylation of (1) (in acetic anhydride–pyridine (1:1) at
room temperature, 24 h) and work-up with 10% HCl solu-
tion led to the isolation of the triacetate (1a) as the major
product and compounds 3 and 4, being artefacts resulting
from the opening of the oxonium ion formed by protonation
of the epoxide at C-8 (17) to give the tertiary carbocation and
subsequent elimination of b-protons in the acidic work-up
medium.
showed an oxymethine at d 4.60 (dd, J¼7.4, 4.6 Hz) and sig-
nals for protons geminated to two primary alcohols at d 4.15
(
7
d, J¼12.4 Hz), 4.05 (d, J¼12.4 Hz) and 3.60 (dd, J¼11.3,
.4 Hz), 3.52 (dd, J¼11.3, 4.6 Hz). Chemical shifts at
d 2.70 (d, J¼3.8 Hz, H-17b) and 2.30 (d, J¼3.8 Hz, H-
1
3
1
7a) suggested an 8(17)-epoxide in 1. The C NMR spec-
trum (Table 2) confirmed the presence of these functions
with signals of three oxymethylenes at d 66.5 (C-15), 64.3
(
C-16), and 50.7 (C-17), one trisubstituted double bond at
d 139.2 (C-13) and 133.7 (C-12). The rest of the data (Table
) being comparable to those reported in the literature for
The triacetate (1a) was obtained as syrup. The CIMS of 1a
showed an [M+NH ] peak at m/z 482 indicating an increase
+
4
2
of 126 units attributed to the three acetate groups picked up
by 1 to form 1a. The H NMR spectral data (Table 1) of 1a
1
3
1
1
1
similar compounds. The H– H COSY and HMQC spec-
tral analysis revealed spin systems of –CH–CH –CH–,
–
isolated –CH –. HMBC correlations (Fig. 1) enabled us to
complete the structural elucidation of 1. Pertinent correla-
tions were observed between H-19 and C-18, C-5; H-20
and C-1, C-9; H-9 and C-1; H-11 and C-13 as well as
between H-12 and C-16, C-14.
were comparable with that of 1 except the increases ob-
served in the chemicals shifts of the protons at positions
14, 15, and 16 due to the greater deshielding effect of the
acetoxy group; and three tertiary methyl signals at d 2.0,
2.0, and 1.9, attributed to the methyl groups of the acetyl
functions. According to the data above, compound 1a
was found to be 8b(17)-epoxy-14,15,16-triacetoxylabd-
2
CH –CH –CH –, –CH–CH –CH –, –CH–CH –, and two
2 2 2 2 2 2
2
1
2(E)-ene.
The stereochemistry of 1 was deduced from its NOESY
spectrum. Spatial connectivities were observed between H-
19 and H-20, H-5 and H-18, H-9. Further connectivity
between H-16a and H-12 indicated the double bond being
in the E-configuration. The b-orientation of the 8(17)-epoxide
The two other triacetates, 3 and 4 were also obtained as
syrups. The CIMS spectra of these showed the same
+
[M+NH ] peak at m/z 482 as 1a. However, some differences
4
1
13
were observed in their H NMR (see Table 1) and C NMR
1
Table 1. H NMR data for compounds 1–5 and 1a (J in Hz)
a
1
b
2
b
1a
b
3
b
4
c
5
Proton
1
2
3
5
6
7
9
1.0 (m), 1.85 (m)
1.55 (m)
1.05 (dd, 4.0, 12.6), 1.77 (m) 0.90 (m), 1.70 (m)
1.52 (m), 1.57 (dt, 3.3, 7.0) 1.40 (m), 1.55 (ov)
1.70 (m)
1.40 (m)
1.25 (m), 1.45 (ov) 1.21 (dd, 4.1, 13.3), 1.42 (m) 1.15 (m), 1.35 (br t) 1.10 (m), 1.30 (m)
1.80 (m)
1.50 (m)
1.40 (m)
1.6 (m)
2.1 (m)
5.70 (m)
1.95 (m)
1.77 (m)
1.56 (m)
1.18 (m)/1.58 (m)
1.20 (dd, 2.9, 12.8)
1.75 (m)
1.93 (m), 2.42 (m)
2.08 (m)
1.55 (m), 1.97 (m)
1.65 (ov)
1.50 (m), 1.75 (m)
1.99 (ov), 1.89 (m) 2.03 (m), 2.41 (m)
1.1 (m) 1.80 (m)
1.75 (m), 2.00 (ov) 2.32 (m), 2.48 (m)
1.12 (dd, 2.6, 12.7)
1.36 (ov) (m), 1.74 (m)
1.45 (br s)
1.60 (ov)
1.10 (m)
1.50 (m), 1.80 (m)
2.1 (m), 2.20 (m)
—
1.30 (ov), 1.85 (m)
1.45 (br s)
1.80 (m)
1
1
2.75 (dd, 12.0, 4.3), 2.20 (m),
.10 (dd, 12.0, 3.3) 2.35 (m)
3
1
1
1
2
4
5
5.46 (dd, 7.8, 4.7)
4.60 (dd, 7.4, 4.6)
5.92 (dd, 7.2, 6.0)
4.45 (d, 3.8)
5.55 (dd, 7.4, 5.0)
5.65 (dd, 8.3, 3.9)
4.1 (ov)
5.60 (dd, 4.3, 3.3)
5.85 (dd, 7.8, 3.8)
4.15 (m)
5.85 (m)
5.80 (m)
3.8 (br d),
4.0 (br d)
4.12 (br d, 10.1)
6.39 (br s)
7.40 (s)
3.52 (dd, 11.3, 4.6), 3.95 (dd, 10.3, 3.8),
.60 (dd, 11.3, 7.4) 4.07 (d, 10.3)
4.05 (d, 12.4), 4.15 5.26 (s)
d, 12.4)
2.30 (d, 3.8), 2.70
d, 3.8)
3
1
6
7
4.35 (d, 12.2),
4.55 (d, 12.2)
2.20 (d, 3.8),
2.58 (d, 3.8)
0.80 (s)
0.70 (s)
0.85 (s)
—
4.30 (d, 12.0), 4.55 4.40 (d, 12.0), 7.35 (s)
(d, 12.0) 4.60 (d, 12.0)
3.82 (d, 11.7), 3.95 4.2 (ov)
(d, 11.7)
0.85 (s)
0.7 (s)
0.90 (s)
—
(
1
4.63 (d, 1.2), 4.87 (d, 1.2)
4.44 (s), 4.84 (s)
(
1
1
2
1
1
1
1
1
1
8
9
0
0.90 (s)
0.85 (s)
0.95 (s)
—
—
—
—
—
—
0.91 (s)
0.84 (s)
0.67 (s)
—
3.34 (s)
3.39 (s)
—
0.83 (s)
0.78 (s)
0.89 (s)
—
0.89 (s)
0.81 (s)
0.66 (s)
3.17 (s)
—
—
—
—
—
2-OMe
4-OMe
6-OMe
4-COCH
5-COCH
6-COCH
—
—
2.01 (s)
2.00 (s)
1.99 (s)
—
—
2.05 (s)
2.00 (s)
1.95 (s)
—
—
2.10 (s)
2.00 (s)
1.90 (s)
d
d
d
d
d
d
d
3
3
3
d
—
—
d
(
ov)—Overlap; (m)—multiplet.
a
Spectra recorded in CD
3
OD, 400 MHz.
Spectra recorded in CDCl , 400 MHz.
Spectra recorded in CDCl , 500 MHz.
The values are interchangeable in the same column.
b
c
d
3
3

S. V. T. Sob et al. / Tetrahedron 63 (2007) 8993–8998
Table 2. C NMR data for compounds 1–5 and 1a
8995
1
3
a
1
b
2
b
1a
b
3
b
4
c
5
Carbon
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
1
1
1
1
1
1
40.7
21.6
43.6
34.9
54.9
20.1
37.5
59.7
56.6
41.3
21.2
133.7
139.2
72.3
66.5
64.3
50.7
34.5
22.6
15.7
—
39.5
19.7
42.5
33.9
55.3
24.6
38.4
148.6
55.3
39.9
39.6
19.0
42.3
33.9
53.7
20.5
36.3
57.7
55.4
40.2
20.6
140.1
129.1
70.5
64.5
65.4
49.4
33.9
22.1
15.0
—
36.9
19.3
41.9
33.7
52.1
19.3
30.3
132.6
143.0
39.5
25.8
139.9
128.0
69.9
64.7
65.6
63.6
33.6
22.0
20.2
—
35.6
17.8
41.1
35.6
51.6
17.8
39.0
19.3
42.1
33.6
55.3
24.4
38.3
149.3
52.7
39.2
126.1
136.7
48.7
35.6
24.1
139.4
126.7
69.2
64.9
64.3
63.1
32.1
20.9
13.0
—
0
1
2
3
4
5
6
7
24.5
31.9
74.1
134.7
138.5
78.2
127.1
108.6
143.3
139.4
106.2
33.3
21.7
14.5
56.4
70.7
104.8
108.1
34.0
22.1
14.7
8
9
0
2-OMe
4-OMe
6-OMe
4-COCH
5-COCH
6-COCH
56.9
55.7
—
—
—
—
—
—
—
—
—
d
d
d
d
d
d
d
d
d
d
d
d
3
3
3
—
—
—
170.9 , 21.4
170.9 , 21.2
171.0 , 21.5
171.0 , 21.3
170.7 , 21.2
169.7 , 20.0
169.6 , 19.9
169.5 , 19.8
d
d
d
d
170.3 , 21.2
d
d
a
b
c
d
Spectra recorded in CD
Spectra recorded in CDCl
Spectra recorded in CDCl
3
OD, 100 MHz.
3
, 100 MHz.
, 125 MHz.
3
The values are interchangeable in the same column.
(see Table 2). This revealed the presence of a second double
bond, a primary alcohol as well as the three acetoxy func-
tions and the absence of the epoxide. The H NMR spectrum
of 4 indicated the presence of a second olefinic proton, which
was absent in that of 3. Further analyses of their HMBC
spectra led to the unambiguous positioning of the double
bond in the two compounds.
Compound 2 was obtained as white needles in methanol, mp
114–115 C. Its EIMS showed a pseudo-molecular ion peak
ꢀ
1
+
at m/z 316 [MꢁMeOH] compatible with the molecular for-
mula C H O , which accounted for one more degree of un-
2
2 36 3
saturation compared to 1, indicating one additional ring or
1
13
double bond. The IR, H, and C NMR spectra of 2 (Tables
1 and 2) showed some similarities with those of 1, suggesting
that they were analogs. In the H NMR data of 2 (Table 1),
1
olefinic proton signals were observed at d 5.92 (dd, J¼7.2,
OAc
6.0 Hz, H-12), and at d 4.87 (d, J¼1.2 Hz, H-17a) and 4.63
15,16
OAc
(
d, J¼1.2 Hz, H-17b) characteristic of the exomethylene
AcO
OH
AcO
OH
moiety at C-8 found in labdane diterpenoids.
This spec-
OAc
OAc
trum also showed two methoxyl groups at d 3.39 (s, 16-OMe),
d 3.34 (s, 14-OMe), a deshielded ketalic proton at d 5.26 (s,
H-14); an oxymethylene [d 4.07 (d, J¼10.3 Hz, H-15b),
3
4
3
.95 (dd, J¼10.3, 3.8 Hz, H-15a)], and one oxymethine at
1
3
d 4.45 (d, J¼3.8 Hz, H-16). The C NMR spectrum (Table
HMBC correlations were observed between H-9 (d 1.95)
and the methyl carbon at d 13.0 (C-20) for 4 but no correla-
tion from this methyl group to H-9 proton was observed for
compound 3. Their structures were thus established to be 17-
hydroxy-14,15,16-triacetoxylabda-8(E),12(Z)-diene (3) and
2
) indicated the presence of one ketal carbon atom at
d 104.8 (C-16), one oxymethine carbon at d 78.2 (C-14),
one disubstituted double bond [d 108.1 (t, C-17) and 148.6
(s, C-8)], and one trisubstituted double bond [d 134.7 (d, C-
1
2) and 138.5 (s, C-13)]. The rest of the data were in agree-
1
7-hydroxy-14,15,16-triacetoxylabda-7(E),12(Z)-diene (4).
13
ment with those published for labdane skeletons. The two
methoxyl groups were positioned at C-14 and C-16 on the ba-
sis of correlations observed on the HMBC spectrum (Fig. 1).
Further correlations were observed between 2H-11 and C-9,
H-12, C-13 and C-14. The H– H COSY clearly demon-
strated the connectivities H-9/2H-11/H-12. From the above
evidence, the trisubstituted double bond was located at C-
OMe
O
1
1
OH
HO
OMe
HO
O
12/C-13. The E-configuration of the hitherto mentioned dou-
ble bond was deduced from the connectivities observed
between H-12 and H-16. A judicious analyses of the HMBC
(Fig. 1), H– H COSY, DEPT 135, NOESY (Fig. 2), and
1
1
Figure 1. HMBC correlations for compounds 1 and 2.

8996
S. V. T. Sob et al. / Tetrahedron 63 (2007) 8993–8998
HMQC data led to the unambiguous assignment of struc-
ture 2, 15,16-epoxy-14x,16x-dimethoxylabda-8(17),12-(E)-
diene. Compound 2 was trivially named as aulacocarpin D.
determined on a Reitcher Nr-229 micro-melting point appa-
ratus and were uncorrected. Mass spectra (70 eV) were
recorded in the EI and CI modes on a Jeol JMS 700 apparatus
and no fragments below m/z 40 were registered. The UV
spectra were obtained with a Shimadzu 3101 PC
instrument and the IR spectra determined with a JASCO
Acid hydrolysis of (2) and work-up with solid NaHCO led
3
to the isolation of a new labdane diterpenoid (5) in 2% yield
and the known compounds (6): 15,16-epoxy-12a-hydr-
1
FTIR 410 apparatus. H NMR (400.6 and 500 MHz) and
C NMR (100.13 and 125 MHz) spectra with DEPT pro-
9
–11
13
oxylabda-8(17)-13(16),14-triene
previously isolated (7): 15,16-epoxy-12b-hydroxylabda-
8
(21% yield) and the
gram were recorded in CDCl (with its signal at d 7.25 and
3
77.0 ppm as standard reference) or in CD OD (with its sig-
3
9
–11
(17)-13(16),14-triene (44% yield). The furanolabdane
5) was obtained as colorless oil. Its CIMS showed an
M+NH ] peak at m/z 334 indicating an increase of 14 units
4
(
[
nals at d 3.21 and 49.4 ppm as standard reference), unless
otherwise stated, on Br €u kers DPX 400 and 500 apparatus.
The coupling constants (J) are given in hertz. NMR data
acquisition and processing were performed with the aid of
+
as compared to 6 and 7. The obtained 5 also exhibited spec-
tral properties very similar to those reported for 6 and 7
except for the chemical shifts at d 3.17 in H NMR and d 56.4
9
–11
1
1
1
the XWIN NMR software package. H– H COSY, HMBC,
and HMQC experiments were recorded with gradient
enhancements using sine shaped gradient pulses. NOESY
experiments were carried out using a Br €u ker AM 360 instru-
ments. For the MPLC, the chromatotron ser. no. 36B con-
nected to a FMI pump QD (flow rate 10 ml/mn) was used
with the plates (e 2 mm) prepared with silica gel PF254 con-
1
3
in C NMR attributed to a methoxy group. The positioning
of this methoxy group at C-12 was in agreement with the de-
crease observed in the chemical shift of the H-12 proton due
to the shielding effect of the methoxy group; and the increase
in that of C-12. Its b-orientation was deduced by comparison
1
of the H NMR shifts of the methylene protons H-17 with
those reported for compounds 6 and 7. All these data,
together with the analyses of its H– H COSY, HMBC,
9
–11
taining CaSO . Column chromatographies were run with
4
1
1
Merck silica gel 60 and Sephadex LH-20, while TLC was
carried out on silica gel 60 GF254 pre-coated plates with de-
tection accomplished by spraying with 50% H SO followed
and HMQC spectra led to the structure of 5: 15,16-epoxy-
1
2b-methoxy-labda-8(17)-13(16),14-triene.
2
4
ꢀ
by heating at 100 C, or by visualizing with an UV lamp at
254 and 366 nm.
OMe 16
16
R1 R2
O
O
1
2
1
2
13
15
13
15
20
11
20 11
1
4
14
1
4
14
17
17
3.2. Plant material
2
3
9
6
2
3
9
1
0
8
10
8
7
7
5
5
The fruits of Aframomum aulacocarpos were collected at
Bamenda, North-West province, Cameroon in March 2000.
Authentication was done by Mr Paul Mezili, a retired
botanist of the Cameroon National Herbarium, Yaound ꢁe .
Voucher specimen (BUD 0513) was deposited at the Herbar-
ium of the Botany Department of the University of Dschang,
Cameroon.
6
19
18
19
18
5
6: R = OH, R = H
1 2
7
: R = H, R = OH
1 2
4
–6
5,6
Aulacocarpin A and B, coronarin E, 14,15-epoxy-
(17),12-(E)-labdadien-16-al, and 15,16-epoxy-12b-hydr-
1
4
8
oxylabda-8(17)-13(16),14-triene
agreement with those reported in the literature.
9
–11
spectral data were in
3
.3. Extraction and isolation
Compounds 1 and 2 exhibited inhibitory activity against four
strains of bloodstream trypomastigotes Trypanosoma brucei
rhodesiense with IC50 values in the range 15–29 mg/ml. The
tests were carried out by Professor Cyrus Bacchi, Pace
University, Haskins Laboratory, New York, NY 10038 (see
Section 3).
The seeds (500 g) obtained from the fresh fruits of A. aula-
cocarpos were air-dried, ground into a fine powder, and mac-
erated three times with acetone at room temperature. The
solvent was removed under reduced pressure to afford
1
03 g of crude extract. This extract was subjected to open
column chromatography on silica gel and eluted with
a step gradient of hexane–EtOAc to yield five major frac-
tions (A–E): A (5 g) eluted with n-hexane, B (1.60 g) eluted
with n-hexane–EtOAc (9:1), C (2.5 g) eluted with n-hexane–
EtOAc (7:3), D (23.2 g) eluted with n-hexane–EtOAc (1:1),
and E (4.2 g) eluted with EtOAc. A portion of fraction A
H
H
H
OMe
O
H
H
HA
OH
OH
HB
H
H
OMe
(800 mg) was subjected to gel permeation chromatography
through Sephadex LH-20 [(n-hexane–EtOAc (99:1)] to
O
OH
H
H
give three sub-fractions (A –A ). A was further chromato-
1
3
1
1
2
graphed on silica gel using gradients of n-hexane–acetone to
yield compounds 2 (75 mg) and 15,16-epoxy-12b-hydroxy-
labda-8(17)-13(16),14-triene (6 mg). Compound 1 (137 mg)
crystallized out of fraction E. Fraction B (1.60 g) was passed
through a chromatotron, using an isocratic system of
Figure 2. Important NOESY correlations for compound 1 and 2.
3. Experimental
3
.1. General experimental procedures
n-hexane–EtOAc (97:3) to yield two sub-fractions (B and
1
B ). B was then purified by preparative TLC plates using
2
2
Optical rotations were measured on an AA Series Automatic
ꢀ
n-hexane–acetone (95:5) and extracted with CH Cl solvent
2 2
to yield 14,15-epoxy-8(17),12-(E)-labdadien-16-al (18 mg).
Polarimeter Polaar-2000 at 22 C. Melting points were

S. V. T. Sob et al. / Tetrahedron 63 (2007) 8993–8998
8997
Fraction C was treated on a silica gel column chromato-
graphy using n-hexane–EtOAc (95:5) as eluant to yield
coronarin E (72 mg), while fraction D treated in the same
manner gave aulacocarpin A (3.71 g) [n-hexane–EtOAc
100 MHz) data see Table 2. HRCIMS m/z 465.6051
[M+H] (calcd for C H O , 465.6057); CIMS m/z
[M+NH ] 482.
4
+
2
6 41 7
+
(
7:3)] and aulacocarpin B (5.05 g) [n-hexane–EtOAc (6:4)].
3.5. Acid hydrolysis of 2
Some of the compounds were further purified by re-crystalli-
zation in appropriate solvent systems before analyses.
A solution of 2 (0.328 mmol) in a mixture of dioxane (3 ml)
and 1 M HCl (0.66 ml) was stirred at room temperature as
1
7
3
.3.1. Aulacocarpin C (1): 8b(17)-epoxy-14,15,16-trihy-
droxylabd-12-(E)-ene. Brown needles from EtOAc; mp
reported in the litterature. The reaction was monitored
by TLC and found to be completed after 49 h. The reaction
mixture was then neutralized with solid NaHCO , the filtrate
ꢀ
22
D
ꢀ
1
lmax nm (log 3); no absorption above 210. IR (KBr) nmax
60–161 C; [a] +13.71 (c 2.27, CH Cl ); UV (MeOH)
2
2
3
being evaporated. The residue was chromatographed on
silica gel with n-hexane–acetone (95:5) as eluant to give
the furanolabdane 5 (2 mg; 1.9%) as the minor product.
Compounds 6 (44 mg; 44.5%) and 7 (21 mg; 21.2%) were
obtained using n-hexane–acetone (88:12).
3
474 (OH), 1603 (C]C), 1445, 1204, 1093, 1027, 962,
14 and 545 cm ; for H NMR (CD OD, 400 MHz) data
ꢁ
1
1
9
see Table 1. For C NMR (CD OD, 100 MHz) data see Ta-
ble 2. HRCIMS m/z 339.4939 [M+H] (calcd for C H O ,
2
3
3
3
1
3
3
+
0 35 4
+
39.4941); EIMS (70 eV) m/z (rel int.) [MꢁH O] 320 (1),
2
02 (9), 284 (13), 277 (22), 259 (42), 203 (20), 189 (18), 131
3.5.1. Compound 5: 15,16-epoxy-12b-methoxylabda-
8(17)-13(16),14-triene. Colorless oil; H NMR (CDCl ,
1
(
83), 117 (78), 63 (100), 61 (96).
3
1
3
500 MHz) data see Table 1. C NMR (CDCl , 125 MHz)
data see Table 2. HRCIMS m/z 317.4898 [M+H] (calcd
3
+
3.3.2. Aulacocarpin D (2): 15,16-epoxy-14x,16x-di-
methoxylabda-8(17),12-(E)-diene. White needles from
+
for C H O , 317.4905); CIMS m/z [M+NH ] 334.
4
2
1
33
2
ꢀ
22
ꢀ
MeOH; mp 114–115 C; [a]D +36.8 (c 0.38, CH Cl );
2
2
UV (CH Cl ) l nm (log 3) no absorption above 210. IR
max
3.5.2. Compound 6. Compound 6 was isolated as colorless
oil and identified on the basis of its NMR, MS spectra data,
and comparison with previously reported data.
2
2
(
(
KBr) nmax 3454, 1685 (C]C), 1642 (C]C), 1459, 1387
C–O), 1100, 1038, 979, 929, 675 and 578 cm ; for H
ꢁ
1
1
9–11
1
3
NMR (CDCl , 400 MHz) data see Table 1. For C NMR
3
(
[
(
CDCl , 100 MHz) data see Table 2. HRCIMS m/z 349.5321
3
3.5.3. Compound 7. After purification through chromato-
graphy 7 was isolated as colorless needles and identified by
comparison of its NMR data with literature values as being
15,16-epoxy-12b-hydroxylabda-8(17)-13(16),14-triene.
This is the first report of 7 from natural source.
+
M+H] (calcd for C H O , 349.5325); EIMS (70 eV) m/z
2
2 37 3
+
rel int.) [M] 348 (2), 316 (100), 285 (52), 269 (17), 215 (8),
79 (40), 137 (68), 119 (33), 81 (48), 69 (54), 55 (36).
9
–11
1
3
.4. Acetylation of 1
3.6. Biological tests
The triol (1) (0.074 mmol) was shaken in 2 ml of the acetic
anhydride–pyridine (1:1) mixture and allowed to stand at
room temperature for 24 h. The reaction mixture was then
subjected to a 10% HCl solution treatment followed by
a chloroform extraction. The organic phase was further chro-
matographed on an open silica gel column chromatography
using CH Cl –acetone (95:5) to yield the triacetate 1a
The compounds were tested against trypanosome isolates
grown in bloodforms in an HMI-18 medium containing 20%
fetal bovine serum. Coulter counts were made daily and IC50
values determined after 48 h. The strains used in the tests in-
cluded Lab 110 EATRO, KETRI 243, 269, and 243 As 103.
2
2
(
(
24.6 mg; 71.7%) as the major product and compounds 3
5.8 mg; 16.9%) and 4 (3.8 mg; 11.0%), being artefacts
Compound 1 was weakly active against Lab 110 EATRO
(IC50 15.5 mg/ml), KETRI 243 (IC50 29 mg/ml), and KETRI
243 As 103 (IC50 24 mg/ml).
resulting from the opening of the oxonium ion formed by
protonation of the epoxide at C-8 (17) to give the tertiary
carbocation and subsequent elimination of b-protons in the
acidic work-up.
Compound 2 moderately inhibited the growth of the same
pathogenic microbes with IC50 values of 17.5, 21.0, and
24 mg/ml. No activity was shown against KETRI 269.
3
.4.1. Compound 1a: 8b(17)-epoxy-14,15,16-triacetoxy-
labd-12(E)-ene. Syrup; for H NMR (CDCl , 400 MHz)
1
3
1
3
data see Table 1. For C NMR (CDCl , 100 MHz) data
3
see Table 2. HRCIMS m/z 465.6056 [M+H] (calcd for
C H O , 465.6057); CIMS m/z [M+NH ] 482.
Acknowledgements
+
+
The authors are grateful to the International Science Pro-
gram (ISP) (grant No CAM: 02, Uppsala University, Swe-
den.), the Chinese Academy of Sciences (CAS) and the
Third World Academy of Sciences (TWAS) through the
CAS-TWAS fellowship program for their financial support.
2
6
41
7
4
3.4.2. Compound 3: 17-hydroxy-14,15,16-triacetoxy-
labda-8(E),12(Z)-diene. Syrup; for H NMR (CDCl3,
1
1
3
4
1
00 MHz) data see Table 1. For C NMR (CDCl3,
00 MHz) data see Table 2. HRCIMS m/z 465.6047 [M+H]
+
+
(
calcd for C H O , 465.6057); CIMS m/z [M+NH ] 482.
2
6
41
7
4
References and notes
3.4.3. Compound 4: 17-hydroxy-14,15,16-triacetoxy-
labda-7(E),12(Z)-diene. Syrup; for H NMR (CDCl3,
1
1. Badr ꢁe , F. Flore du Cameroun; Mus ꢁe um National de l’Histoire
Naturelle: Paris, 1972.
1
3
4
00 MHz) data see Table 1. For C NMR (CDCl3,

8998
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