Pseudopterosins P-V, new compounds from the gorgonian octocoral Pseudopterogorgia elisabethae from Providencia island, Colombian Caribbean

Article abstract of DOI:10.1016/j.tet.2004.09.017

Seven new diterpene glycosides, pseudopterosins P (1), Q (2), R (3), S (4), T (5), U (6) and V (7) along with two known compounds PsG and PsK have been isolated from the methanol/dichloromethane extract of the gorgonian octocoral Pseudopterogorgia elisabethae collected off Providencia Island, Colombian Caribbean. The structures of the new metabolites, including their relative and absolute configuration, were established by MS and NMR spectroscopic studies as well as their conversion to known compounds. Seven new diterpene glycosides, pseudopterosins P (1), Q (2), R (3), S (4), T (5), U (6) and V (7) have been isolated from the gorgonian octocoral Pseudopterogorgia elisabethae collected off Providencia Island, Colombian Caribbean.

Full text of DOI:10.1016/j.tet.2004.09.017

Tetrahedron 60 (2004) 10627–10635
Pseudopterosins P–V, new compounds from the gorgonian
octocoral Pseudopterogorgia elisabethae from Providencia island,
Colombian Caribbean
a,*
a
Carmenza Duque, M o´ nica Puyana, Ginna Narv a´ ez, Oscar Osorno, Noriyuki Hara
a
a
b
b
and Yoshinori Fujimoto
a
Departamento de Qu ´ı mica, Universidad Nacional de Colombia, AA 14490 Bogot a´ , Colombia
Department of Chemistry and Materials Science, Tokyo Institute of Technology, Tokyo 152-8551, Japan
b
Received 9 May 2004; revised 31 August 2004; accepted 9 September 2004
Available online 29 September 2004
Abstract—Seven new diterpene glycosides, pseudopterosins P (1), Q (2), R (3), S (4), T (5), U (6) and V (7) along with two known
compounds PsG and PsK have been isolated from the methanol/dichloromethane extract of the gorgonian octocoral Pseudopterogorgia
elisabethae collected off Providencia Island, Colombian Caribbean. The structures of the new metabolites, including their relative and
absolute configuration, were established by MS and NMR spectroscopic studies as well as their conversion to known compounds.
q 2004 Elsevier Ltd. All rights reserved.
1. Introduction
compounds makes these animals an attractive study target
in other areas of the Caribbean.
Pseudopterosins are an interesting group of diterpene
1
glycosides first discovered by Fenical and collaborators
about 15 years ago from specimens of the gorgonian coral
As part of our continuous search for biologically active
compounds from Colombian marine invertebrates, we
8
,9
Pseudopterogorgia elisabethae. So far, 15 pseudopterosins
PsA–PsO) isolated from specimens collected in the
have recently examined Pseudopterogorgia elisabethae
´
specimens from San Andres and Providencia islands from
(
1
2
3
the Colombian Caribbean, finding distinct chemotypes
regarding to the pseudopterosin content, in each island.
Bahamas, Bermuda, and the Florida Keys have been
reported. The structurally related secopseudopterosins A–D
have also been identified in Pseudopterogorgia kallos
1
0
Samples from Providencia island afforded seven new
pseudopterosins, together with the known pseudopterosins
G and K. In this paper, we report the isolation and structure
elucidation of the seven new pseudopterosins.
4
collected near the Marquesas Keys in Florida. These
pseudopterosins and secopseudopterosins exhibit more
potent antiinflammatory and analgesic activities than the
5
,6
common market drug indomethacin. It is suggested that
the mechanism of action of the pseudopterosins may involve
membrane stabilization, different from inhibition of eico-
sanoid release from inflammatory cells mediated by
2
. Results and discussion
6
traditional non-steroidal drugs. Due to their excellent
antiinflammatory and analgesic activity, partially purified
extracts containing pseudopterosins are currently incorpor-
Pseudopterogorgia elisabethae specimens were collected at
Providencia island, Colombian Caribbean, and air-dried.
Animal tissue was extracted with a MeOH/CH Cl (1:1)
mixture and the extract was separated on silica gel CC and
reversed phase HPLC to yield seven new compounds which
we named pseudopterosins P–V (1–7), along with the
2
2
7
ated into skin care preparations. The availability of
pseudopterosins however, is limited by the actual supply
of organic extracts of Pseudopterogorgia elisabethae which
currently only comes from the Bahamas islands. The
complex and expensive chemical synthesis of these
2
2
known compounds pseudopterosins G and K.
Pseudopterosin-P (1) showed a molecular ion at m/z 446 and
intense fragment ions at m/z 300 and 244. The former
fragment ion corresponds to the pseudopterosin aglycone,
suggesting the loss of a deoxyhexose (146 mass unit) and
Keywords: Pseudopterosins; Gorgonian; Pseudopterogorgia elisabethae.
*
Corresponding author. Tel.: C571 3165000x14472; fax: C571 3165220;
e-mail: cduqueb@unal.edu.co
0040–4020/$ - see front matter q 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2004.09.017

1
Table 1. H NMR (500 MHz, CDCl
3
) data for compounds 1–7
H No.
1
2
3
4
5
6
7
1
2
3.66 q-like (8.5)
1.93 dd (10.5, 7.8)
1.21 m
1.22 m
2.05 m
0.92 qd (12.7, 3.2)
3.66 q-like (8.6)
1.93 m
1.22 m
1.22 m
2.06 m
0.93 qd (12.7, 3.2)
3.67 q-like (8.5)
1.93 m
ca. 1.22 m
ca. 1.22 m
ca. 2.05 m
0.93 qd (12.7, 3.2)
3.66 q-like (8.7)
1.94 m
ca. 1.21 m
ca. 1.23 m
ca. 2.05 m
0.93 qd (12.7, 3.0)
3.62 q-like (8.7)
1.90 dd (10.2, 6.3)
ca. 1.22 m
ca. 1.23 m
ca. 2.02 m
0.88 qd (12.7, 3.2)
3.67 m
1.23 m
2.25 m
1.22 m
2.05 m
0.92 qd (12.7, 3.2)
3.67 q (8.7)
1.22 m
1.93 m
1.20 m
2.05 m
0.93 qd (12.7, 3.3)
2.05 m
1.33 m
3
4
5
2.02 m
1.31 m
6
1.31 m
2.13 m
3.19 sextet-like (7.0)
4.98 br d (9.3)
1.67 s
1.32 m
2.14 m
3.20 sextet-like (7.2)
4.97 d (9.1)
1.67 s
1.32 m
2.14 m
3.20 sextet-like (7.0)
4.99 br d (9.2)
1.67 s
1.32 m
2.15 m
3.19 sextet-like (7.2)
4.93 d (9.2)
1.67 s
1.30 m
2.08 m
3.12 sextet-like (7.2)
4.93 br d (9.3)
1.64 s
2.13 m
3.19 m
4.96 d (9.1)
1.66 s
1.72 s
1.02 d (5.9)
1.27 d (1.8)
2.06 s
5.18 d (3.6)
4.10 dd (9.9, 3.6)
4.32 dd (9.9, 3.4)
5.24 m
2.15 m
7
3.20 sextet-like (7.1)
4.98 br d (11.5)
1.67 d (1.0)
1.72 d (1.5)
1.02 d (7.5)
1.26 d (8.5)
2.09 s
5.22 d (3.6)
4.33 dd (10.0, 3.8)
5.31 dd (10.0, 3.8)
4.27 m
1
1
1
1
1
2
1
2
3
4
5
4
6
7
8
9
1.71 s
1.71 s
1.72 s
1.72 s
1.68 s
1.02 d (6.0)
1.25 d (6.8)
2.07 s
5.11 d (3.7)
4.03 dd (9.5, 3.7)
4.13 br d (9.5)
3.89 br
1.02 d (5.9)
1.27 d (6.8)
2.05 s
5.14 d (3.8)
4.04 dd (10.1, 3.8)
4.35 dd (10.1, 3.3)
5.21 br d (3.3)
4.55 q (6.6)
1.02 d (6.0)
1.26 d (6.8)
2.08 s
1.03 d (5.9)
1.25 d (6.8)
2.10 s
5.12 d (3.7)
5.24 dd (10.4, 3.7)
4.26 dd (10.4, 3.2)
3.97 br d (3.2)
4.56 q (6.6)
1.00 d (5.6)
1.18 d (6.6)
1.97 s
5.10 d (2.5)
4.17 br d (7.3)
4.12 br d (7.3)
4.08 br s
0
0
5.18 d (4.0)
0
0
0
0
a
4.30 ddd (10.4, 4.0, 3.0 )
5.29 dd (10.4, 3.0)
4.06 br s
4.52 q (6.7)
4.58 q (6.6)
4.25 d (12.6)
.78 d (12.6)
4.36 br d (13.0)
3.89 dd (13.0, 1.9)
4.42 dd (16.0, 1.5)
3.87 dd (16.0, 3.0)
3
0
6
Ac
1.33 d (6.7)
1.17 d (6.6)
2.19 s
1.33 d (6.6)
2.21 s
1.39 d (6.6)
2.23 s
2.16 s
2.22 s
a
0
The coupling is due to hydroxyl group present at C-2 .

C. Duque et al. / Tetrahedron 60 (2004) 10627–10635
10629
1
3
m/z 244 the aglycone after loosing an isopropylidene unit.
Its molecular formula, C H O , was determined by
C data for the sugar moiety of 1 were in good agreement
with those of a fucose-substituted pseudopterosin, pseudop-
2
6 38 6
2
terosin-E which is a C-1 epimer of 1. Thus, the sugar
HREIMS. The UV spectrum showed maximum absorptions
at 213, 232, 276, and 285 nm due to the presence of a
substituted benzene ring. The H and C NMR data of 1
moiety was determined as a-fucopyranoside. That the sugar
was linked to C-10 rather than C-9 was initially deduced
1
13
1
3
(
family.
Tables 1 and 2) suggested that 1 belongs to pseudopterosin
from comparison of the C data of 1 with those of
compound 2.
1
–4
1
The H NMR spectrum of 1 showed signals of
two singlet methyls at d 1.67 (H -16) and 1.71 (H -17),
3
3
a singlet methyl substituted on an aromatic ring at d 2.07
(
We then investigated the absolute configuration of the
aglycone and fucose moieties of 1, since 1 was available in
good quantity and not acetylated. It should be noted that the
occurrence of an antipode of pseudopterosin aglycone was
reported in pseudopterosins K and L (1S,3R,4S,7R-agly-
H -20), two doublet methyls at d 1.02 (H -18) and 1.25
3 3
(
aglycone part. The C NMR spectrum of 1 showed 26
H -19) and an olefinic proton at d 4.98 (H-14) for the
3
1
3
signals, among which 20 resonances including six aromatic
carbons at d 126.8, 127.8, 129.2, 136.6, 143.2, and 145.2
2
cone, compared to an usual 1R,3S,4R,7S-aglycone). For
1
were assigned to the aglycone. Further analysis of
H
this purpose, 1 was benzylated and the resulting 9-benzyl
ether derivative was hydrolyzed under acidic conditions to
give the aglycone 9-benzyl ether (Scheme 1). Fortunately,
signals, assisted with H–H COSY spectra, allowed assign-
ing all proton signals. In particular, the chemical shifts and
coupling patterns of H-1 (d 3.66, q-like, JZ8.5 Hz), H-7 (d
1
1
Lazerwith et al. reported the synthesis of the same ether of
the aglycone with known 1S,3S,4R,7S absolute configur-
ation together with the NMR and optical data. The NMR
data of the aglycone 9-benzyl ether derived from 1 were in
3
.19, sextet-like, JZ7.0 Hz) and H-14 (d 4.98, br d, JZ
* * * *
.3 Hz) suggested 1S ,3S ,4R ,7S configuration for the
9
aglycone, since the data were close to the values reported for
the aglycone derivative with the known configuration.
2
,11
The sugar portion showed signals of an anomeric proton,
0
0
H-1 (d 5.11), a doublet methyl (d 1.33) assignable to H -6
3
and four oxymethine protons. Decoupling experiments and
correlations in the H–H COSY spectrum established the
0
0
0
values were 3.7 and
assignments of H-2 (d 4.03), H-3 (d 4.13), H-4 (d 3.89),
0
0
.5 Hz, respectively, and H-4 was observed as a broad
and H-5 (d 4.52). J
9
0
0
and J
0
H-2 ,H-3
H-2 ,H-3
0
singlet. These data evidenced axial–axial relationships for
0
0
0
0
suggesting a-fucose as a sugar structure. Furthermore, the
H-2 and H-3 and equatorial orientation of H-1 and H-4 ,
Scheme 1. Conversion of pseudopterosin-P (1) to the C-9 benzyl ether
derivative.
13
3
Table 2. C NMR (500 MHz, CDCl ) spectral data of compounds 1–7
Carbon No.
1
2
3
4
5
6
7
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
1
2
3
4
5
6
37.3
40.2
33.9
44.6
27.8
31.7
28.4
126.8
145.2
143.2
127.8
136.6
129.2
131.3
128.1
25.4
17.5
20.1
23.3
13.5
103.0
70.4
69.6
72.2
67.3
37.3
40.2
33.9
44.7
27.8
31.8
28.4
126.9
145.2
143.2
127.8
136.7
129.3
131.4
128.2
25.4
17.5
20.1
23.3
13.6
103.0
69.5
69.1
73.6
66.3
37.3
40.2
33.9
44.6
27.8
31.8
28.5
127.0
145.0
143.1
127.6
136.6
129.2
131.4
128.2
25.4
17.5
20.1
23.2
13.5
102.8
70.4
74.3
67.7
67.1
37.3
40.1
33.9
44.7
27.8
31.9
28.6
126.7
144.5
143.0
127.3
136.6
129.8
131.2
128.4
25.4
17.5
20.0
23.1
13.7
101.3
72.3
68.8
71.4
67.4
37.3
40.2
33.9
44.6
27.8
31.7
28.4
126.8
145.0
143.1
127.9
136.6
129.3
131.3
128.1
25.4
37.3
40.2
33.9
44.7
27.8
31.8
28.5
126.9
145.2
143.1
127.8
136.8
129.2
131.3
128.2
25.4
17.5
20.1
23.2
13.9
103.1
70.0
68.2
71.5
61.9
37.3
40.2
33.9
44.7
27.8
31.8
28.5
127.1
145.0
142.9
127.6
136.7
129.2
131.4
128.2
25.5
17.5
20.1
23.2
13.8
103.1
68.1
73.3
67.5
63.7
0
1
2
3
4
5
6
7
8
9
17.5
20.0
23.3
13.8
0
0
103.8
69.5
69.5
69.4
64.0
0
0
0
0
0
a
a
a
16.2
16.3
20.8
16.1
20.8
171.7
16.1
21.0
170.9
Ac
21.1
171.3
21.1
171.6
1
71.9
1
Assignments for compound 2 and 6 were based on H– H COSY, DEPT, HMQC and HMBC spectra, while the other compounds were assigned from their
1
1
analogy and based on H– H COSY and DEPT spectra.
1
a
Assignments may be reversed.

10630
C. Duque et al. / Tetrahedron 60 (2004) 10627–10635
1
1
0
0
0
excellent agreement with those reported. This unequi-
vocally determined the relative stereochemistry at the chiral
centers of 1. Further, the sign and magnitude of the a value
for the ether were similar to the reported values, thus
establishing the absolute configuration of the aglycone as
H-4 , although the J value between H-4 and H-5 was nearly
0
zero. Further, an NOE correlation was observed from H-3
0
0
to H-5 , indicating an axial orientation of H-5 . Thus, the
sugar moiety was unambiguously established as 4-O-acetyl-
a-L-fucopyranoside (L configuration of the sugar was
assigned by converting 2 to peracetylated derivative (vide
infra)). The C NMR signals for the sugar, assigned by
HMQC spectrum, were in good agreement with those of
pseudopterosin J which has an 4-O-acetyl-a-fucopyranoside
moiety. Finally, the sugar was linked to the C-10 position
on the basis of an HMBC correlation from the anomeric
proton to C-10 (d 143.2). The structure of compound 2 was
C
1S,3S,4R,7S. Conversion of 1 to the known benzyl ether also
proved the position of glycosylation.
1
3
Fucose was identified by TLC in the aqueous phase of the
above acidic hydrolysis. Chirality of the fucose sample was
2
1
2
analyzed by the method of Hara et al. GLC analysis of the
trimethylsilyl ether of the corresponding methyl 2-(poly-
hydroxyalkyl)-thiazolidine-4(R)-carboxylate derivative
revealed that the fucose is in L-form. The results agree
with earlier findings in which fucose-bearing pseudopter-
osins utilize the L-form of fucose. The structure of
compound 1 was, therefore, established to be as shown in
Figure 1, which depicts the correct absolute configuration.
established to be as shown in Figure 1, which depicts the
1
3
correct absolute configuration. The C signals were
completely assigned with the aid of the HMBC spectrum
(Fig. 2). Pseudopterosin-Q is a regioisomer of pseudopter-
osin-J which is glycosylated at C-9. Originally assigned
1R,3S,4R,7R stereochemistry of pseudopterosin-J was
2
,3,11
2
1
1
revised to 1S,3S,4R,7S by synthetic work.
Figure 1. New pseudopterosins isolated from Pseudopterogorgia elisa-
bethae from Providencia island.
Figure 2. HMBC correlations from H to C for pseudopterosin-Q (2).
Pseudopterosin Q (2) showed a molecular ion at m/z 488 and
intense fragment ions at m/z 300 and 244 in EI-MS. The
former fragment ion corresponds to the pseudopterosin
aglycone, suggesting a loss (188 mass unit) of a mono-
acetylated deoxyhexose. The ion at m/z 244 could be due to
a loss of an isopropylidene unit from the aglycone. The
molecular formula of 2 was established as C H O by
Pseudopterosin-R (3) showed a molecular ion at m/z 488 and
intense fragment ions at m/z 300 and 244 in EI-MS. The
molecular formula, C H O , was deduced from HREIMS.
2
8
40
13
7
1
Analysis of the H and C NMR data (Tables 1 and 2)
indicated that the aglycone structure of 3 is identical to that
of 2. It became also clear from C NMR data that the C-10
13
2
8 40 7
HREIMS. The general characteristics of the UV and NMR
data (Tables 1 and 2) suggested that 2 belongs to the
phenol is glycosidated. Compound 3 showed an anomeric
0
proton at d 5.18 (d) which was coupled to H-2 (d 4.30)
0
1
13
pseudopterosin family too. The H and C NMR spectra of
resembled those of 1, but with an additional signal for an
with JZ3.0 Hz. The latter was coupled to H-3 (d 5.29) with
2
0
JZ10.4 Hz, which was then coupled to H-4 (d 4.06) with
0
methyl at d 1.33. J
acetyl group. It is obvious from the similarity of the NMR
data for the aglycone part of 1 and 2 that compound 2 has the
aglycone with the same stereochemistry as in compound 1.
JZ3.0 Hz. H-5 (d 4.58, q, 6.6 Hz) was coupled to a doublet
0
0
value was nearly zero. The acetyl
H-4 ,H-5
methyl signal resonated at d 2.21. These data established
that the sugar moiety at C-10 of 3 is 3-O-acetyl-a-
fucopyranoside. Chirality of 3-O-acetyl-fucose was deter-
mined to be L, since 3 and 1 furnished the same peracetate
(vide infra). Hence, the structure of 3 was established to be
as shown in Figure 1. Pseudopterosin-R is a regioisomer of
1
The H NMR spectrum of 2 exhibited signals of an
anomeric proton (d 5.14), a doublet methyl assignable to
0
H -6 (d 1.17) and an acetyl methyl (d 2.19) along with
3
H
four oxymethine proton signals for the sugar moiety.
0
2
The anomeric proton was coupled to H-2 (d 4.04) with
0
pseudopterosin-I which is glycosylated at C-9 (initially
reported 1R,3S,4R,7R configuration was revised to
JZ3.8 Hz, which in turn coupled to H-3 (d 4.35) with
0
0
11
1S,3S,4R,7S ).
JZ10.1 Hz. H-3 further coupled to H-4 (d 5.21) with
0
JZ3.3 Hz. The downfield shift of H-4 evidenced that the
acetoxy group was attached to C-4 of the sugar. The large
Pseudopterosin-S (4) showed a molecular ion at m/z 488 in
EI-MS and its molecular formula was determined to be
C H O by HREIMS. The NMR data revealed that
0
0
coupling constant between H-2 and H-3 indicated axial–
0
0
axial relationships for these protons, while H-1 and H-4
must bear equatorial orientation. These data established an
2
8 40 7
compound 4 is an isomer of 2 and 3 in terms of the position
of the acetyl group. Signals of an anomeric proton, a
doublet methyl and acetyl methyl were observed at d 5.12
0
a-glycosidic linkage. H-5 (d 4.55, q, JZ6.6 Hz), coupled to
H -6, was sharpened by a decoupling experiment irradiating
3

C. Duque et al. / Tetrahedron 60 (2004) 10627–10635
10631
0
0
0
(
d, JZ3.7 Hz), 1.39 and 2.23, in that order. The H–H COSY
H-4 (d 5.24, m)–H-5 a (d 4.36, br d, JZ13.0 Hz)/H-5 b
(d 3.89, dd, JZ13.0, 1.9 Hz). These data clearly indicated
that sugar is 4-O-acetyl-b-D-arabinopyranoside. D configur-
ation of the sugar was assigned by converting 6 to
peracetylated derivative (vide infra). HMBC correlations
(Fig. 3) corroborated the sugar structure and C-10
substitution of the sugar. Hence, the structure of compound
6 was established to be as shown in Figure 1.
spectrum allowed us to connect oxymethine protons to form
the network: H-1 –H-2 (d 5.24, dd, JZ10.4, 3.7 Hz)–H-3
(
(
is therefore clear that the sugar moiety at C-10 of 4 is 2-O-
acetyl-a-L-fucopyranoside (L configuration of the sugar was
assigned by converting 3 to peracetylated derivative (vide
infra)). Hence, compound 4 was elucidated to be as shown
in Figure 1. Pseudopterosin-S is a regioisomer of pseudop-
0
0
0
0
0
d 4.26, dd, JZ10.4, 3.2 Hz)–H-4 (d 3.97, JZ3.2 Hz). H-5
d 4.56, q, JZ6.6 Hz) was coupled to the methyl doublet. It
Pseudopterosin-V (7) had the same molecular formula
(C H O ) as for 6, and the mass spectral pattern was
essentially identical to that of 6. These data, together with
2
terosin-H which is glycosylated at C-9 (initially assigned
1
27 38 7
1
R,3S,4R,7R configuration was revised to 1S,3S,4R,7S ).
1
1
H NMR data, indicated that 7 is an isomer in terms of the
position of acetyl group. The H–H COSY spectrum of 7
Pseudopterosin-T (5) showed a molecular ion peak at m/z
32 and intense fragment ions at m/z 300 and 244. The loss
0
0
showed coupling networks: H-1 (d 5.22)–H-2 (d 4.33)–H-
0
4
0 0 0
3
(d 5.31)–H-4 (d 4.27)–H-5 a (d 4.42)/H-5 b (d 3.87).
of 132 mass units suggested the presence of a pentose. Its
molecular formula, C H O , was determined by
HREIMS. Comparison of the NMR data of 5 with those
of 1 indicated that 5 has the same aglycone as in 1, but the
structure of the sugar moiety substituted at C-10 is different
These data clearly indicated that the sugar structure is 3-O-
acetyl-b-D-arabinopyranoside. D configuration of the sugar
was assigned by converting 6 to peracetylated derivative
(vide infra). Hence, the structure of 7 was established to be
as depicted in Figure 1.
2
5
36
6
1
from 1. The H NMR spectrum of 5 exhibited an anomeric
proton at d 5.10 in addition to the five signals assignable to
It should be mentioned that Jacobs et al. reported in their
patent the isolation of pseudopterosins M, N and O, which
are listed in SciFinder as 2-O-acetyl-xyloside, 3-O-acetyl-
xyloside and 4-O-acetyl-xyloside at C-10 of 1R ,3S ,
4R ,7S -aglycone. They recently published details of these
compounds, reporting that pseudopterosins M, N and O are
acetyl-arabinoside derivatives (the structures shown in the
paper are not acetyl-arabinoside but acetyl-xyloside deriva-
tives). Our H and C NMR data for compounds 6 and 7
are not consistent with the values reported for pseudopter-
osins M and O.
the sugar moiety. The H–H COSY spectrum correlated these
0
13
0
signals: H-1 (d, JZ2.5 Hz)–H-2 (d 4.17, br d, JZ7.3 Hz)–
0
0
0
H-3 (d 4.12, br d, JZ7.3 Hz)–H-4 (d 4.08, br s)–H-5 a
(
data clearly indicated that H-2 and H-3 have an axial
*
*
0
d 4.25, d, JZ12.5 Hz)/H-5 b (d 3.78, d, JZ12.5 Hz). These
*
*
0
0
0
0
orientation while H-1 and H-4 must take an equatorial
position and that the anomeric configuration is b. The data
further indicated that sugar is b-arabinopyranoside. In order
to establish the chirality of arabinose, 5 was subjected to
acidic hydrolysis. Arabinose was identified by TLC in the
hydrolysate. Analysis of the sample in the same manner as
described for fucose revealed that the arabinose is in D-form.
Hence, the structure of 5 was established to be as shown in
Figure 1.
3
1
13
14
Compounds 1–4 yielded a common peracetylated derivative
upon acetylation, which secured the stereochemical (absol-
ute configuration) identity for the aglycone and sugar
moieties (L-fucose derivatives) among the four pseudopter-
Pseudopterosin-U (6) showed a molecular ion at m/z 474
and intense fragment ions at m/z 300 and 244. The loss of
1
osins. The H NMR spectrum of the tetra-acetate showed a
unique pattern due to the presence of two conformers as
illustrated in Figure 4. When recorded at 20 8C, the
spectrum showed two sets (ca. 45:55 ratio) of signals in
some peaks which might correspond to two conformers. For
example, the signal for H-7 was clearly observed at d 2.70
and d 3.18 as broad peaks. When measured at 60 8C, the
two H-7 signals turned to one very broad signal, and one of
the acetate signals was similarly very broad, suggesting that
the two conformers were not rapidly interchanged at this
temperature. Compounds 5–7 furnished the other common
peracetylated derivative, confirming the absolute stereo-
chemistry of the aglycone and sugar moieties (D-arabinose
174 mass units could be due to a mono-acetylated pentose.
The molecular formula of 6 was determined to be C H O
7 38 7
2
by HREIMS. Comparison of the NMR data of 6 and 5
indicated that 6 is a mono-acetyl derivative in the sugar
moiety of 5.
1
5
1
The H signals for the sugar moiety of 6 were correlated by
H–H COSY spectrum: H-1 (5.18, d, JZ3.6 Hz)–H-2 (d
0
0
0
4
.10, dd, JZ9.9, 3.6 Hz)–H-3 (d 4.32, dd, JZ9.9, 3.4 Hz)–
1
derivatives). When the H NMR spectrum of the acetate was
recorded at 20 8C, the signal for H-7 appeared very broad
around at d 2.8–3.1, also the H -19 and one of the acetates
3
H were not clear due to their broad and complex signals. At
3
60 8C, the spectrum became clearer except for the some
broadness of H-7 (Fig. 4). The 9-benzyl ether of
pseudopterosin-P (see Scheme 1) did not show such
broadening of the H NMR signals. It is, therefore,
1
1
conceivable that these H NMR behaviors are due to
restricted rotation of the glycosidic bond caused by
acetylation of the C-10 hydroxyl as well as the hydroxyl
groups at the sugar moiety. Furthermore, higher energy
Figure 3. HMBC correlations from H to C for pseudopterosin-U (6).

10632
C. Duque et al. / Tetrahedron 60 (2004) 10627–10635
1
Figure 4. H NMR spectra of the peracetylated derivatives of compounds 1–4 and of compounds 5–7, at different temperature.
barrier for the rotation of the fucose derivative, compared to
that of the arabinose derivative, allowed us to detect two
distinct conformers at 20 8C.
molecules, and this rule seems to be applicable, irrespective
of C-1 stereochemistry of the aglycone.
In addition to the new pseudopterosins reported in this
paper, new seco-type pseudopterosins were isolated as
minor constituents. Further isolation and structure analysis
of these compounds are in progress in our laboratory.
Cumulative NMR data for pseudopterosins including the
present study provided a simple clue to differentiate C-10
glycosylation from C-9 glycosylation. The chemical shift
1
3
for C-11 in C NMR spectra showed the most diagnostic
difference: C-11 appears at d 121–122 for C-9 glycosylated
compounds, whereas it resonates at d 126–127 for C-10
glycosylated pseudopterosins. Fortunately, there are no
Our preliminary results in the neutrophil degranulation
inhibition assay have revealed interesting activity of the
new pseudopterosins reported here, but varying effects in
neutrophil activation or inhibition depending on
1
3
disturbing C signals in this area in pseudopterosin

C. Duque et al. / Tetrahedron 60 (2004) 10627–10635
10633
concentration tested. Therefore, determination of antiin-
flammatory activity will be matter of further research and
results will be published elsewhere.
were present, as shown by HPLC-MS in APCI mode, in
fraction 6 which eluted with EtOAc/CH Cl (6:4) and in
fraction 8 which eluted with EtOAc. Fraction 6 was
subjected first to preparative HPLC using a Nucleosil 120
2
2
1
0 C-18 column with a solvent gradient of acetonitrile–
water to yield pure compounds 2 (6 mg), 3 (2 mg), 4
1.5 mg), 6 (4.5 mg) and 7 (1.5 mg). On the other hand,
3
. Experimental
(
3
.1. General
fraction 8 was subjected to preparative HPLC, as mentioned
above, and then for final purification to HPLC on a
Optical rotations were measured on a JASCO DIP-360
polarimeter. UV spectra were recorded on a Shimadzu
Shimadzu Shim-Pack CLC-ODS with MeO–H O as solvent
2
system to yield compounds 1 (5 mg) and 5 (6 mg) and
pseudopterosins G (4 mg), and K (7 mg). Structures of the
known three compounds were determined by comparison of
1
13
UV-1600PC spectrophotometer. H NMR and C NMR
one- and two-dimensional) spectra were recorded on a
Bruker DRX500 (500 MHz for H and 125 MHz for C)
(
1
13
2,4
the spectral data with reported values.
1
spectrometer in CDCl solution. H chemical shifts are
3
25
expressed with tetramethylsilane as an internal standard
(
3.3.1. Pseudopterosin P (1). White powder; [a] K298
D
1
3
dZ0.00), while C chemical shifts are referenced to the
(c, 0.52, MeOH); UV lmax (MeOH) 213 (3 24,300), 232
(shoulder), 276 (shoulder), 285 (3 1400) nm; EI-MS m/z
(relative intensity): 446 (M , 2), 300 (4.7), 285 (8), 244
solvent signal (dZ77.0). EI-MS and HREIMS were
obtained on a JEOL JMS-700 spectrometer. HPLC-MS on
APCI mode was carried out on a Shimadzu QP-8000a
spectrometer with a Thermo Hypersil-Keystone RP-18
C
C
(100), 229 (78); HREIMS m/z: 446.2669 (M ), C26H O
38 6
1
13
requires 446.2668; H NMR: see Table 1; C NMR: see
Table 2.
(
100!2 mm i.d., 3 mm) column. Preparative HPLC was
conducted with a Merck-Hitachi instrument with a UV/VIS
L-4250 detector (detected at 230 nm) using a Nucleosil 120
2
5
3.3.2. Pseudopterosin Q (2). White powder; [a]D K538
(c, 0.56, MeOH); UV lmax (MeOH) 214 (3 23,700), 232
(shoulder), 276 (shoulder), 285 (3 1670) nm; EI-MS m/z
1
0 C-18 (300!8 mm i.d., 10 mm) column with a 30 min
gradient of acetonitrile–water (70–100%) to 100% aceto-
nitrile as mobile phase at a flow rate of 1 ml/min. Final
HPLC purification was performed with a Shimadzu LC-6A
apparatus equipped with a UV detector (detected at 230 nm)
under a Shimadzu Shim-Pack CLC-ODS (15!6 mm i.d.,
C
(relative intensity): 488 (M , 8), 300 (71), 285 (9), 244 (100),
229 (38); HREIMS 488.2762 (M ), C H O requires
C
2
8 40 7
1 13
488.2774; H NMR: see Table 1; C NMR: see Table 2.
25
5
mm) using MeOH/water (9:1) as mobile phase at a flow
3.3.3. Pseudopterosin R (3). White powder; [a] K348
D
rate of 1 ml/min. The following adsorbents were used for
purification: column chromatography, Merck Kieselgel 60;
preparative TLC, Merck Kieselgel 60 F254. TLC plates were
visualized by dipping with 5% phosphomolybdic acid in
EtOH followed by heating.
(c, 0.28, MeOH); UV lmax (MeOH) 208 (3 25,900), 234
(shoulder), 276 (shoulder), 285 (3 1150) nm; EI-MS m/z
(relative intensity): 488 (M , 2), 300 (40), 285 (10), 244
C
C
(100), 229 (56); HREIMS m/z: 488.2779 (M ), C H O
8 40 7
2
1
13
requires 488.2774; H NMR: see Table 1; C NMR: see
Table 2.
3
.2. Animal material
2
D
5
3
.3.4. Pseudopterosin S (4). White powder; [a] K488
Fragments of several Pseudopterogorgia elisabethae colo-
nies were collected by SCUBA at a range depth of 20–30 m
at various sites around the island of Providencia. Sample
collection never implied removing whole colonies, only a
terminal fragment of each individual colony was cut off the
main gorgonian axis with sharp scissors. Gorgonian
fragments were air-dried and stored in the freezer. Samples
were kept frozen until the moment of extraction. Animals
were identified as Pseudopterogorgia elisabethae by M.
Puyana, and voucher specimens were deposited at the
invertebrate collection of Museo de Historia Natural Marina
Colombiano (MHNMC) at Instituto de Investigaciones
Marinas de Punta de Bet ´ı n (INVEMAR), coded as INV
CNI 1612, INV CNI 1613 and INV CNI 1614.
(c, 0.18, MeOH); UV lmax (MeOH) 208 (3 20,500), 232
(shoulder), 276 (shoulder), 284 (3 1200) nm; EI-MS m/z
(relative intensity): 488 (M , 1), 300 (30), 285 (7), 244
C
C
(100), 229 (48); HREIMS m/z: 488.2773 (M ), C H O
8 40 7
2
1
13
requires 488.2774; H NMR: see Table 1; C NMR: see
Table 2.
2
5
3.3.5. Pseudopterosin T (5). White powder; [a]D K388
(c, 0.89, MeOH); UV lmax (MeOH) 212 (3 15,100), 232
(shoulder), 276 (shoulder), 285 (3 1770) nm; EI-MS m/z
C
(relative intensity): 432 (M , 22), 300 (94), 285 (32), 244
C
(100), 229 (88); HREIMS m/z: 432.2544 (M ), C H O
5 36 6
2
1
13
requires 432.2512; H NMR: see Table 1; C NMR: see
Table 2.
2
5
3
.3. Extraction and separation of pseudopterosins
3.3.6. Pseudopterosin U (6). White powder; [a]
K908
D
(c, 0.88, MeOH); UV lmax (MeOH) 211 (3 21,900), 232
(shoulder), 276 (shoulder), 285 (3 2020) nm; EI-MS m/z
Dried gorgonian tissue (10 g) were repeatedly extracted
with a CH Cl /MeOH (1:1) mixture. The resultant extract
C
(relative intensity): 474 (M , 13), 300 (91), 285 (11), 244
2
2
C
was filtered and concentrated by rotary evaporation
obtaining a dark green oily extract (4 g). This crude extract
was subjected to silica gel column chromatography using
CH Cl and EtOAc mixtures of increasing polarity, and
(100), 229 (32); HREIMS m/z: 474.2626 (M ), C27H O
38 7
1
13
requires 474.2618; H NMR: see Table 1; C NMR: see
Table 2.
2
2
25
finally pure EtOAc, to yield eight fractions. Pseudopterosins
3.3.7. Pseudopterosin V (7). White powder; [a] K638
D

10634
C. Duque et al. / Tetrahedron 60 (2004) 10627–10635
(
(
(
(
c, 0.31, MeOH); UV lmax (MeOH) 207 (3 21,100), 232
shoulder), 274 (shoulder), 285 (3 2130) nm; EI-MS m/z
mixture was diluted with chloroform and water. The water
layer was washed with chloroform once more and then
lyophilized. TLC analysis of the residue indicated the
presence of arabinose. Chirality of the sugar was examined
as described for fucose. Retention times of the D- and
L-arabinose derivatives were 11.5 and 10.4 min in the same
GC conditions as described above. The derivative of
compound 5 was detected at 11.5 min.
C
relative intensity): 474 (M , 15), 300 (91), 285 (8), 244
C
92), 229 (13); HREIMS m/z: 474.2582 (M ), C H O
7 38 7
2
1
13
requires 474.2618; H NMR: see Table 1; C NMR: see
Table 2.
3.4. Conversion of Pseudopterosin P (1) to the C-9 benzyl
ether derivative
3
.5.1. Conversion of compounds 1–4 and compounds 5–7
Potassium carbonate (8.8 mg) was added to a solution of
compound 1 (8 mg, combined amount from two extraction
processes) in dry acetone (0.5 ml). The mixture was stirred
at room temperature under nitrogen for 10 min. Benzyl
bromide (5.0 ml) was added to this suspension and the
mixture was heated at reflux for 7 h. Solvent was removed
by flushing nitrogen and the residue was partitioned
to the peracetylated derivatives. Each compound
(1–2 mg) was treated with pyridine (40 ml) and acetic
anhydride (20 ml) at room temperature overnight. Addition
of methanol followed by evaporation of the solvent gave an
oily residue, which was partitioned with ether and water.
The ether layer was washed with 2 N-HCl, satd NaHCO3
and brine, dried over MgSO , evaporated and analyzed by
4
1
between CHCl and water. The water layer was washed
3
H NMR. The peracetylated derivatives of compounds 1–4
showed identical behavior on TLC (hexane/EtOAc, 4:1). H
1
with CHCl once more, and the combined CHCl layer was
3
3
dried over Na SO and concentrated to dryness. The crude
4
benzyl ether (8 mg) was dissolved in MeOH (0.5 ml) and
NMR (recorded at 60 8C) d: 1.03 (d, JZ7.0 Hz, H -18), 1.16
2
3
0
(d, JZ6.5 Hz, H -19 and H -6 ), 1.67 (d, JZ1.0 Hz, H -16),
3
3
3
3
5
N-HCl (0.5 ml) and the solution was allowed to react at
0 8C under nitrogen for 3 h. The solution was cooled to
1.70 (d, JZ1.1 Hz, H -17), 1.90 (broad, Ac), 1.99 (s, Ac),
3
2.146 (s, Ac), 2.151 (s, Ac), 2.29 (s, H -20), 2.96 (very
broad, H-7), 3.68 (q-like, JZ8.5 Hz, H-1), 4.50 (q-like,
3
room temperature, diluted with water, and extracted with
CHCl (3!10 ml). The CHCl layer was washed with 5%
aq NaHCO and water, dried over Na SO , and concen-
3
trated to give a crude aglycone benzyl ether, which was
chromatographed on silica gel with hexane/ether (10:1) to
0
JZ6.6 Hz, H-5 ), 4.93 (br d, JZ8.9 Hz, H-14), 5.20 (dd,
3
3
0
0
2
4
JZ10.9, 3.4 Hz, H-2 ), 5.35 (d, JZ3.3 Hz, H-4 ), 5.54 (d,
0
0
m/z: 615.3156 (MH ), C H O requires 615.3169.
JZ3.4 Hz, H-1 ), 5.62 (br d, JZ10.9 Hz, H-3 ). HRFABMS
C
3
4 47 10
furnish pure aglycone benzyl ether (3 mg). [a] ZC83
D
1
1
c, 0.24, CHCl ) [lit.C103 ]; H NMR d: 0.97, 1.03
1
(
(
(
(
(
7
2
1
Compounds 5–7 were similarly converted to the peracetyl-
ated derivatives. Identity of them was confirmed by TLC
3
d, 3H), 1.25 (m, 2H), 1.34 (d, 3H), 1.36 (m, 1H), 1.67
s, 3H), 1.74 (s, 3H), 2.01 (m, 1H), 2.06 (s, 3H), 2.10
m, 3H), 3.24 (sextet, JZ7.3 Hz, 1H), 3.74 (m, 1H), 4.73
d, JZ11.2 Hz, 1H), 4.97 (d, JZ11.2 Hz, 2H), 5.51 (s, 1H),
1
1
(hexane/EtOAc 4:1) and H NMR. H NMR (recorded at
60 8C) d: 1.03 (d, JZ5.9 Hz, H -18), 1.15 (d, JZ6.8 Hz,
3
H -19), 1.67 (d, JZ1.0 Hz, H -16), 1.70 (d, JZ1.1 Hz,
3
3
1
3
.42 (m, 5H); C NMR d: 12.1, 17.7, 20.1, 23.8, 25.6, 27.8,
9.3, 31.6, 36.4, 37.1, 40.2, 43.8, 75.3, 121.0, 127.9, 128.3,
28.5, 128.7, 131.0, 131.2, 131.8, 135.1, 137.3, 142.3, 145.3.
H -17), 1.95 (s, Ac), 2.02 (s, Ac), 2.13 (s, Ac), 2.15 (s, Ac),
2.27 (s, H -20), 2.96 (br, H-7), 3.69 (q-like, JZ8.7 Hz,
3
3
H-1), 3.75 (dd, JZ14.2, 2.3 Hz, H-5a), 4.27 (dd, JZ14.2,
1.5 Hz, H-5b), 4.94 (d, JZ9.1 Hz, H-14), 5.28 (dd, JZ10.6,
1
1
These data were in good agreement with reported values.
0
0
0
3
.2 Hz, H-2 ), 5.39 (m, H-4 ), 5.51 (d, JZ3.2 Hz, H-1 ),
0
(MNa ), C H O Na requires 623.2832.
3.5. Analysis of the sugar portion of Pseudopterosins
P (1) and T (5)
5.61 (dd, JZ10.5, 3.5 Hz, H-3 ). HRFABMS m/z: 623.2864
C
3
3 44 10
The water layer of the acidic treatment of the C-9 benzyl
ether of compound 1 was freeze-dried. Fucose was
identified by TLC (CH Cl /MeOH/H O, 8:7:1 and visual-
Acknowledgements
2
2
2
ized with p-anisaldehyde reagent) analysis of the residue.
Chirality of the sugar was determined according to the
This work was financially supported by grants from
Colciencias, Fundaci o´ n para la Promoci o´ n de la Investiga-
ci o´ n y la Tecnolog ´ı a del Banco de la Rep u´ blica and DIB
(
Divisi o´ n de Investigaciones-sede Bogot a´ , de la Universi-
dad Nacional de Colombia).
1
2
protocol of Hara et al. To the dried sugar sample dissolved
in pyridine (0.2 ml) was added L-cysteine methyl ester
hydrochloride (2 mg), and the mixture was allowed to react
at 60 8C for 1.5 h. The solvent was evaporated by flushing
nitrogen and the residue was treated with hexamethyldisi-
lazane–trimethylchlorosilane (HMDS–TMCS) (0.1 ml) at
6
(
0 8C for 1 h. The mixture was partitioned between hexane
0.3 ml) and water (0.3 ml) and an aliquot of the hexane
References and notes
layer was injected to GC [column, DB-1, 0.25 mm!30 m,
oven temp 200 8C]. The derivatives from D-fucose and
L-fucose had retention times of 13.4 and 15.2 min,
respectively, and the sample from compound 1 eluted at
1. Look, S. A.; Fenical, W.; Matsumoto, G. K.; Clardy, J. J. Org.
Chem. 1986, 51, 5140–5145.
2. Roussis, V.; Wu, Z.; Fenical, W.; Strobel, S. A.; Van Duyne,
G. D.; Clardy, J. J. Org. Chem. 1990, 55, 4916–4922.
1
5.2 min.
3
. Ata, A.; Kerr, R. G.; Moya, C. E.; Jacobs, R. S. Tetrahedron
2003, 59, 4215–4222.
4. Look, S. A.; Fenical, W. Tetrahedron 1987, 43, 3363–3370.
Compound 5 (4 mg) was hydrolyzed in MeOH (0.5 ml) and
N-HCl (0.5 ml) at 50 8C under nitrogen for 3 h. The
3

C. Duque et al. / Tetrahedron 60 (2004) 10627–10635
10635
5
6
. Look, S. A.; Fenical, W.; Jacobs, R. S.; Clardy, J. Proc. Natl.
Acad. Sci. U.S.A. 1986, 83, 6238–6240.
12. Hara, S.; Okabe, H.; Mihashi, K. Chem. Pharm. Bull. 1987, 35,
501–506.
. Mayer, A. M. S.; Jacobson, P. B.; Fenical, W.; Jacobs, R. S.;
Glaser, K. B. Life Sci. 1998, 62, 401–407.
13. Jacobs, R. S.; Kerr, R. G. U.S. Pat. Appl. Publ. US
2002091093 A1 20020711, 2002; Chem. Abstr. 137, 98953.
14. Direct comparison of compounds 6 and 7 with pseudopterosins
M and O were not possible, since authentic samples were not
available to us.
7
. Pomponi, S. A. J. Biotechnol. 1999, 70, 5–13.
. Petrichtcheva, N. V.; Duque, C.; Due n˜ as, A.; Zea, S.;
Fujimoto, Y. J. Nat. Prod. 2002, 65, 851–855.
. Mart ´ı nez, A.; Duque, C.; Sato, N.; Fujimoto, Y. Chem. Pharm.
Bull. 1997, 45, 181–184.
8
1
9
15. Look and Fenical (Ref. 4) reported on the H NMR
measurement of seco-pseudopterosin peracetate derivatives
1
1
0. Puyana, M.; Narvaez, G.; Paz, A.; Osorno, O.; Duque, C.
J. Chem. Ecol. 2004, 30, 1183–1201.
1. Lazerwith, S. E.; Johnson, T. W.; Corey, E. J. Org. Lett. 2000,
3
at 60 8C (CDCl ), probably due to the broadening of the
signals at room temperature. Their results may rule out the
possibility that the presently observed two conformers are
associated with the aglycone moiety.
2, 2389–2392.