Lipidyl pseudopteranes A-F: Isolation, biomimetic synthesis, and PTP1B inhibitory activity of a new class of pseudopteranoids from the Gorgonian Pseudopterogorgia acerosa

Article abstract of DOI:10.1021/np800544b

Novel lipidyl pseudopteranoids, lipidyl pseudopteranes A-F (1-6), have been isolated from the soft coral Pseudopterogorgia acerosa collected from the Bahamas. Structure elucidation of the six new compounds was based on 1D and 2D NMR data and mass spectrometry, and a biomimetic synthesis of 1 from pseudopterolide (7) was used to help establish its absolute configuration. These structures represent the first report of a pseudopterane diterpene with a fatty acid moiety. Lipidyl pseudopteranes A and D exhibited modest yet selective inhibitory activity against protein tyrosine phosphatase 1B, a promising drug target.

Full text of DOI:10.1021/np800544b

Copyright 2008 by the American Chemical Society and the American Society of Pharmacognosy
Volume 71, Number 12
December 2008
Full Papers
Lipidyl Pseudopteranes A-F: Isolation, Biomimetic Synthesis, and PTP1B Inhibitory Activity of
a New Class of Pseudopteranoids from the Gorgonian Pseudopterogorgia acerosa
Abhijeet S. Kate,§,‡ Isabelle Aubry,† Michel L. Tremblay,† and Russell G. Kerr*,§
Department of Chemistry and Department of Biomedical Sciences, Atlantic Veterinary College, UniVersity of Prince Edward Island,
Charlottetown, Prince Edward Island, Canada C1A 4P3, Department of Chemistry and Biochemistry, Florida Atlantic UniVersity,
Boca Raton, Florida 33431, and McGill Cancer Centre and Department of Biochemistry, McGill UniVersity, Montreal, Quebec, Canada H3G 1Y6
ReceiVed August 28, 2008
Novel lipidyl pseudopteranoids, lipidyl pseudopteranes A-F (1-6), have been isolated from the soft coral
Pseudopterogorgia acerosa collected from the Bahamas. Structure elucidation of the six new compounds was based on
1D and 2D NMR data and mass spectrometry, and a biomimetic synthesis of 1 from pseudopterolide (7) was used to
help establish its absolute configuration. These structures represent the first report of a pseudopterane diterpene with a
fatty acid moiety. Lipidyl pseudopteranes A and D exhibited modest yet selective inhibitory activity against protein
tyrosine phosphatase 1B, a promising drug target.
The marine gorgonian coral Pseudopterogorgia acerosa is known
for its diversity of diterpenes. Pseudopterolide (7) was isolated by
Fenical and co-workers1 as the first pseudopterane diterpene from
P. acerosa collected in the Florida Keys. In the 1990s, more than
22 new pseudopteranoid and cembranoid diterpenes were reported
from specimens of P. acerosa collected from Puerto Rico and
Tobago.2-4 We have investigated the diterpene chemistry of
samples of P. acerosa collected from The Bahamas and herein
report the isolation of a new family of pseudopteranes, which were
identified from a UHPLCMS analysis of a nonpolar fraction of the
crude extract. The six most abundant members of this group are
pseudopteranoids that we have named lipidyl pseudopteranes A-F
(1-6). Characterization of the compounds was performed by 1D
and 2D NMR and MS analysis.
possess a palmitic acid moiety, in keeping with the observation
that this is the major fatty acid in P. acerosa.5
These compounds were evaluated for their selective inhibitory
activity against protein tyrosine phosphatase 1B (PTP1B). PTP1B
is well established as a critical modulator of metabolic signaling
in mammals, and recent reviews have thoroughly described the PTP
family and PTP1B in particular.6-11 PTP1B null mice display
enhanced sensitivity to insulin.12 This correlates with enhanced
tyrosine phosphorylation of the insulin receptor in muscle and liver.
PTP1B is also involved in leptin signaling.13-16 Ablation of PTP1B
confers resistance to obesity induced by a high-fat diet,12 and weight
gain in leptin-deficient ob/ob mice lacking PTP1B is significantly
decreased as compared to control littermates.13 Moreover, recent
findings also suggest that inhibiting PTP1B may have important
applications in the treatment of breast cancers.17 The well-
established protective effect of PTP1B genetic ablation on the
development of diabetes, obesity, and cancer prompted substantial
commercial interest in PTP1B as a drug target.18,19 Indeed, an
antisense oligonucleotide therapy targeting PTP1B is currently in
phase II clinical trials.20 In the development of small-molecule
PTP1B inhibitors, achieving sufficient target specificity has been
an important challenge. Members of the PTP family share a well-
conserved catalytic domain that can be broadly targeted by
nonspecific inhibitors. However, the phenotypes of mouse models
lacking a variety of individual PTPs indicate that inhibition of many
A biomimetic synthesis of lipidyl pseudopterane A was com-
pleted by the reaction of palmitic acid with pseudopterolide in the
presence of a catalytic amount of triethyl amine, suggesting that
pseudopterolide is a likely precursor in the biosynthesis of this
lipidyl pseudopterane. Four of the lipidyl pseudopteranes (1, 4-6)
* To whom correspondence should be addressed. Tel: (902) 566-0565.
Fax: (902) 566-7445. E-mail: rkerr@upei.ca.
§ University of Prince Edward Island.
‡ Florida Atlantic University.
† McGill University.
10.1021/np800544b CCC: $40.75
2008 American Chemical Society and American Society of Pharmacognosy
Published on Web 12/05/2008

1978 Journal of Natural Products, 2008, Vol. 71, No. 12
Table 1. NMR Data for Lipidyl Pseudopterane A (1) in CDCl3
Kate et al.
position
13C (mult.)a
1H mult. (J Hz)
1H-1H COSY
HMBC (1H-13C)
C-1
1H-1H NOESY
1
2
43.4 (CH)
32.2 (CH2)
3.33 dd (13.1, 3.7)
3.61 dd (15.7, 13.1)
2.88 dd, (15.7, 3.7)
H2a, H2b
H2b, 1
H2a, 1
H2b
H2b, 12, 14b
H2a, 1
C-12, C-3
3
4
5
6
7
8
9
10
160.5 (C)
116.5 (C)
110.4 (CH)
150.7 (C)
48.6 (CH)
80.1 (CH)
148.2 (CH)
132.0 (C)
75.5 (CH)
75.4 (CH)
6.38 s
C-4, C-6, C-3
7
3.82 d (4.3)b
5.41 dd (4.1, 0.99)b
6.89 br s
8
7, 9
8
8, 19, 5, 9
7, 9, 19
C-10, C-20
C-11, C-8, C-10, C-20
8, 7, 11b, 12
11
12
5.41 d (2.6)b
3.17 br s
12
C-1, C-12, C-10, C-20
12b, 9b
11, 2a
11,12(OH)
12
12(OH)
13
2.43 nre
144.4 (C)
14
117.2 (CH2)
5.30 s
5.17 s
2.07 s
14b, 15
14a
14a
C-15, C-1
C-15, C-1
C-1, C-14, C-13
14b, 15
14a, 2a, 1
1, 14a
15
16
17
18
24.2 (CH3)
164.0 (C)
141.2 (C)
115.6 (CH2)
5.05 s
4.83 s
1.96 s
19
C-19, C-7
C-19, C-7
C-7, C-18, C-17
18b, 19
18a
7, 18a, 8
19
20
21.8 (CH3)
170.6 (C)
51.8 (CH3)
174.0 (C)
34.0 (CH2)
18a
21
3.82 s
C-16
FA 1′c
2′
2.43 m
2.36 m
1.63 m
1.26 m
1.26 m
3′
C-1′, FAC
C-1′, FAC
FAC
2’b, 3′
2’a, FAC
2’a, FAC
3′
24.8(CH2)
32.1 (CH2)
22.9 (CH2)
14.3 (CH3)
2’a
14′d
15′d
16′
0.89 t (6.7)
15′
FAC
FAC
a
13C multiplicities were based on the DEPTQ and HMQC experiments. b CD3OD was used as a solvent for NMR experiments. c Signals for the fatty
acid chain other than mentioned in Table 1: 1H NMR δ 1.26 (H-4′-H-13′); 13C NMR δ 29.1-29.7 (C-4′-13′). d Signals were assigned on the basis of
the HMBC spectrum. Fatty acid chain signals were represented by FAC. e Not resolved.
of these enzymes is not desirable. Thus, numerous groups have
attempted to modify known, general PTP inhibitors to attain
specificity for PTP1B, but no such compounds have succeeded in
late-stage clinical trials. Moreover, the highly charged phosphoty-
rosine substrates of the PTPs have important consequences, and
most of the small-molecule inhibitors identified to date are
themselves highly hydrophilic. Hence, few have been identified with
good membrane diffusible properties. In this report, we have
identified lipidyl pseudopteranes A and D as a new class of PTP
inhibitors that shows modest but selective activity against PTP1B.
We suggest that their lipidyl moiety could aid in their ability to
diffuse into mammalian cells and inhibit the intracellular PTPs such
as PTP1B.
110.4(CH), 150.7(C), 164.0(C), and 51.8 (CH3) suggested the
presence of an R,R′-disubstituted ꢀ-carboxymethoxyfuran moiety.
This was confirmed by HMBC cross-peaks of H-5 with C-4, C-6,
and C-3 and H-21 with C-16 (Table 1). The proton signal at 6.89
showed cross-peaks with 13C at δ 80.1 (CH), 132.0 (C), and 170.6
(C) in an HMBC experiment. These HMBC cross-peaks along with
a comparison of the 1H and 13C signals with those of gorgi-
acerodiol21 (8) revealed the presence of an R,γ-disubstituted R,ꢀ-
unsaturated γ-lactone. The presence of two isopropenyl groups in
1 was evident from the H and 13C NMR data. The H signals at
δH 5.30 (br s, 1H), 5.17 (br s, 1H), and 2.07 (s, 3H) and the 13C
signals at δC 117.2 (CH2), 144.4 (C), and 24.2 (CH3) were assigned
to one isopropenyl group, and the 1H signals at δH 5.05 (br s, 1H),
4.83 (br s, 1H), and 1.96 (s, 3H) and the 13C signals at δC 115.6
(CH2) and 141.2 (C), 21.8 (CH3) were assigned to a second such
group. This analysis, together with 1H-1H COSY correlations
(Table 1), clearly demonstrates the presence of a pseudopterane
skeleton in 1.
1
1
Results and Discussion
P. acerosa, collected at Sweetings Cay, Bahamas, was sun-dried
and extracted with ethyl acetate and dichloromethane. The crude
extract was subjected to silica gel flash column chromatography
with a step gradient of solvents, hexanes to ethyl acetate, resulting
in 11 fractions. The 50% hexane fraction was further purified by
semipreparative HPLC using phenyl hexyl as a stationary phase,
which led to the isolation of lipidyl pseudopteranes A-C (1-3).
Lipidyl pseudopteranes D-F (4-6) were isolated from the 60%
hexane fraction using C18 flash chromatography followed by
semipreparative HPLC.
The mass spectrometry analysis indicated that the molecular
weight of 1 was 238 mass units greater than gorgiacerodiol (8).
The negative mode ionization of 1 followed by MS/MS fragmenta-
tion of the [M - H]- ion indicated that the peaks at m/z 387 and
255 corresponded to C21H21O6 and C16H31O2, respectively, indicat-
1
ing the presence of a palmityl group. The H NMR signals at δ
0.89 (t), 1.26 (br s) 1.63 (m), and 2.43 (m) confirmed the presence
of a fatty acid unit. To confirm the identity of this fatty acid moiety,
base hydrolysis of the fraction enriched with lipidyl pseudopterane
A was carried out followed by methyl esterification. This led to
the formation of fatty acid methyl esters that were analyzed by
GC-MS. The MS of the major peak was identical to that of methyl
palmitate in the NIST library.
Lipidyl pseudopterane A (1) was obtained as a colorless oil, and
HRMS analysis (observed [M + Na]+ ) 649.3715, calculated [M
+ Na]+ ) 649.3716) revealed the molecular formula C37H54O8 with
11 degrees of unsaturation. IR absorptions at 3529, 3087, 1737,
and 1720 cm-1 suggested the presence of a hydroxyl group, an
alkene, an unsaturated γ-lactone, and a conjugated ester, which are
3
1
characteristic of the pseudopteranoids. The H signals at δ 6.38
The location of the palmityl group on the pseudopterane ring
was assigned on the basis of the chemical shift of H-11 (5.41 ppm)
(1H, s) and 3.82 (3H, s) with the 13C signals at δ 160.5(C), 116.5(C),

Pseudopteranoids from Pseudopterogorgia acerosa
Journal of Natural Products, 2008, Vol. 71, No. 12 1979
and the comparison of the 1H spectrum of 1 and of gorgiacerodiol
(8).21 All 1H NMR signals in 1 and 8 were within (0.12 ppm with
the exception of H-11, which was at 4.65 ppm in gorgiacerodiol
and at 5.41 ppm (∆0.75) in 1. Somewhat surprisingly, the three-
bond HMBC correlation was not observed between H-11 (5.41
ppm) and C-1′ (174.0 ppm).
Table 2. NMR Data for Lipidyl Pseudopteranes B and C (2, 3)
in CDCl3
2
3
position 13C (mult.)a 1H mult (J Hz) 13C (mult.)a 1H mult (J Hz)
1
2
43.1 (CH)
31.9 (CH2) 3.61 dd (15.7, 13.1) 31.9 (CH2) 3.62 dd (15.7, 13.1)
2.89 dd (15.7, 3.7) 2.88 dd (15.7, 3.5)
3.33 dd (13.4, 3.1) 43.1 (CH)
3.33 dd (12.7, 3.5)
The relative configuration of 1 was established on the basis of
2D NOESY cross-peaks as well as 1H-1H coupling constants. H-5
showed a cross-peak with H-7, which in turn showed a cross-peak
with H-8 in the NOESY spectrum, suggesting a cis orientation of
these hydrogens. This was supported by the observed coupling
constant (4.3 Hz) between H-7 and H-8. The 1H-1H coupling
constant (2.6 Hz) between H-11 and H-12 suggested a dihedral
angle close to 53° with consideration of the NOESY correlation.22
The absence of a NOESY correlation between H-12 and H-1
suggested a trans orientation. These relative configurations and the
absolute configuration of 1 were established on the basis of a
biomimetic synthesis of 1 from pseudopterolide (7). Pseudopterolide
was treated with palmitic acid in the presence of a catalytic quantity
of triethyl amine to afford a trace amount of lipidyl pseudopterane
A (0.03% yield). Most of the product mixture was unreacted
pseudopterolide. The production of 1 was confirmed by LC-MS
analysis and comparison with an authentic sample of lipidyl
pseudopterane A. The product and authentic standard of 1 had
identical retention times and MS fragmentation patterns. The
assignments of stereocenters depicted in structure 1 (1R, 7R, 8R,
11S, 12R) are therefore based on the absolute configuration of
pseudopterolide.1 Further confirmation of the absolute configuration
at C-8 was achieved using CD data. Previous studies with the R,ꢀ-
unsaturated γ-lactone moiety have shown that the sign of the Cotton
effect due to the n-π* and π-π* transitions can be correlated
directly with the absolute configuration of the stereogenic center
at the γ-carbon.23 The CD data for compound 1 (measured in
chloroform at concentration 3.6 × 10-3 M: ∆ε (270 nm, n-π*) )
+3.10, ∆ε (231 nm, π-π*) ) +11.28) revealed that the config-
uration at C-8 was R.21
HRMS analysis (observed [M + Na]+ ) 675.3854, calculated
[M + Na]+ ) 675.3867) of 2 indicated a molecular formula of
C39H56O8, which suggested a molecular weight of 26 mass units
greater than 1 with an additional degree of unsaturation. The APCI-
MS/MS fragmentation pattern of ions less than m/z 371 was exactly
the same as that of 1, while the peaks greater than m/z 371 in the
MS/MS spectrum were all (26) greater than those of 1. This
suggested the presence of the same pseudopteranoid structure as
in 1 but with a different lipid side chain. 1H and 13C NMR spectra
of 2 (Table 2) showed the same chemical shifts of the pseudopterane
core of 1, which was confirmed by 2D NMR experiments. The 1H
NMR of 2 showed signals at δ 0.89 (t), 1.26 (m), 1.61 (m), 2.00
(4H, m), 2.38 (1H, m), 2.43 (1H, m), and 5.35 (2H, m), which,
together with the mass spectrometry analysis, revealed the presence
of a C-18 chain with one double bond. A base hydrolysis of 2 and
subsequent GC-MS comparison identified the fatty acid as oleic
acid. On the basis of the chemical shift of H-11 in 2 and comparison
with 1, the oleoyl group was assigned to C-11 in 2.
3
4
5
6
7
8
9
10
11
12
160.2 (C)
116.3 (C)
110.2 (CH) 6.38 s
150.4 (C)
48.3 (CH)
79.9 (CH)
147.9 (CH) 6.89 br s
131.7 (C)
160.2 (C)
116.3 (C)
110.2 (CH) 6.38 s
150.4 (C)
48.3 (CH)
79.9 (CH)
148.0 (CH) 6.89 br s
132.4 (C)
3.82 nrd
5.41 nr
3.82 nr
5.41 nr
75.2 (CH)
75.2 (CH)
5.40 nr
3.16 br d (7.9)
2.43 nr
75.2 (CH)
75.2 (CH)
5.40 nr
3.14 dd (10.5, 2.3)
2.43 nr
13
14
144.2 (C)
116.9 (CH2) 5.30 s
5.17 s
24.0 (CH3) 2.07 s
163.8 (C)
141.0 (C)
115.4 (CH2) 5.06 s
4.83 s
21.6 (CH3) 1.96 s
170.4 (C)
144.2 (C)
116.9 (CH2) 5.31 s
5.16 s
24.0 (CH3) 2.07 s
163.8 (C)
141.0 (C)
115.4 (CH2) 5.06 s
4.83 s
21.6 (CH3) 1.96 s
170.4 (C)
51.6 (CH3) 3.82 s
173.8 (C)
33.8 (CH2) 2.43 m
2.39 m
15
16
17
18
19
20
21
51.5 (CH3) 3.82 s
FA 1′b 173.8(C)
2′
33.7 (CH2) 2.43 m
2.38 m
24.6 (CH2) 1.61 m
27.2 (CH2) 2.00 m
129.9 (CH) 5.35 nr
129.8 (CH) 5.35 nr
27.2 (CH2) 2.00 m
31.9 (CH2) 1.26 m
22.7 (CH2) 1.26 m
14.1 (CH3) 0.89 t (6.8)
3′
8′
9′
10′
11′
16′c
17′c
18′
24.6 (CH2) 1.62 m
31.9 (CH2) 1.26 m
22.7 (CH2) 1.26 m
14.1 (CH3) 0.89 t (6.9)
a Multiplicities were obtained from DEPTQ experiment. b Signals for
the fatty acid chain other than mentioned in Table 2: For compound 2
1H NMR δ 1.26 (H-4′-H-7′, H-12′-H-15′); 13C NMR δ 29.1-29.8
(C-4′-C-7′, C-12′-C-15′); for compound
3 δ 1.26
1H NMR
(H-4′-H-15′); 13C NMR δ 29.1-29.7 (C-4′-C-15′). c Signals were
assigned on the basis of the HMBC spectrum. d Not resolved.
The HRMS (observed [M + Na]+ ) 633.3746, calculated [M
+ Na]+ ) 633.3762) of lipidyl pseudopterane D (4; molecular
formula C37H54O7) suggested one oxygen atom less than 1. The
comparison of the H and 13C NMR spectra of 1 and 4 (Table 3)
1
indicated the hydroxyl group at C-12 in lipidyl pseudopterane A
(1) was absent in 4 (Figure 1). The 13C chemical shift assigned to
C-12 at δ 39.0 (CH2) with HMQC cross-peaks with protons at δ
2.24 (nr) and 1.2 (d, J ) 15.5 Hz) supported the structure. COSY
and HMBC correlations confirmed the overall structure of lipidyl
pseudopterane D as 4. The relative configuration of 4 was
established on the basis of comparison with 1-3; however the
relative configuration of C-11 could not be unambiguously
established.
Lipidyl pseudopterane E (5) was isolated as a colorless oil and
found to have a molecular formula of C37H52O7, indicating 12
degrees of unsaturation (observed [M + Na]+ ) 631.3586,
calculated [M + Na]+ ) 631.3605). The MS/MS fragmentation of
5 showed m/z 353.13 as a daughter ion (base peak) with the
associated neutral loss of 256, corresponding to palmitic acid. Full
MS (APCI mode) with source fragmentation comparison of 5 and
4 suggested that both possess the same fatty acid chain with one
extra double bond in the pseudopterane skeleton of 5. HMBC
correlations between C-1′ at δ 173.4 (C) and H-15 at δ 4.74 (2H,
s) confirmed the position of the palmityl group at C-15 (Figure 1).
The location of the double bond in the pseudopterane ring was
The HRMS analysis (observed [M + Na]+ ) 677.3978,
calculated [M + Na]+ ) 677.4024) of compound 3 (molecular
formula C39H58O8) revealed that the molecular weight of 3 was
two mass units greater than 2 with one less degree of unsaturation,
which suggested the hydrogenation of one double bond. The MS/
MS pattern suggested the difference of two mass units was
contributed by hydrogenation of the double bond in the fatty acid
1
chain of 2. Comparison of the H and 13C chemical shifts and 2D
NMR data of 1 and 3 confirmed the structure (Table 2) of lipidyl
pseudopterane C as the stearyl analogue of 1. The diterpene moiety
is common in lipidyl pseudopteranes A-C, and hence it was
assumed that the absolute and relative configuration of 2 and 3 is
the same as in 1.
assigned on the basis of H and 13C chemical shifts (Table 3) as
well as COSY correlations. On the basis of NMR data, 5 can be
1

1980 Journal of Natural Products, 2008, Vol. 71, No. 12
Kate et al.
Table 3. NMR Data for Lipidyl Pseudopteranes D and E (4, 5)
in CDCl3
4
5
position 13C (mult.)a 1H mult (J Hz) 13C (mult.)a 1H mult (J Hz)
1
2
39.0 (CH)
32.4 (CH2) 3.33 dd (14.4, 12.8) 29.7 (CH2) 3.57 dd (15.4, 12.8)
2.83 dd (14.5, 3.3) 2.90 (15.3, 3.7)
3.23 m
39.7 (CH)
4.12 m
3
4
161.3 (C)
115.7 (C)
161.2 (C)
115.3 (C)
5
6
7
8
110.1 (CH) 6.37 s
150.3 (C)
48.5 (CH)
79.7 (CH)
111.5 (CH) 6.41 s
150.3 (C)
3.81 nrd
5.38 dd (4.1, 1.3)
49.7 (CH)
79.8 (CH)
3.83 d (4.8)
5.39 d (4.7)
9
146.6 (CH) 6.78 br s
134.8 (C)
145.5 (CH) 6.58 dd (1.4, 1.3)
131.8 (C)
119.5 (CH) 5.96 dd (11.6, 0.9)
137.1 (CH) 5.47 (11.3, 10.4)
10
11
12
69.9 (CH)
39.0 (CH2) 2.24 nr
1.20 d (15.5)
5.49 br s
13
14
148.5 (C)
111.6 (CH) 5.04 s
4.84 s
145.9 (C)
111.3 (CH2) 5.20 s
5.19 s
15
16
17
18
19.9 (CH3) 1.84 s
164.0 (C)
141.1 (C)
115.3 (CH) 5.06 s
4.87 s
65.5 (CH2) 4.74 s
163.9 (C)
140.4 (C)
115.2 (CH2) 5.05 s
4.86 s
19
20
21
21.6 (CH3) 1.97 s
171.0 (C)
21.6 (CH3) 1.99 s
171.5 (C)
51.4 (CH3) 3.80 s
173.4 (C)
51.4 (CH3) 3.81 s
FA 1′b 173.8 (C)
2′
33.9 (CH2) 2.37 m
34.3 (CH2) 2.41 m
2.32 m
3′
24.5 (CH2) 1.62 m
25.0 (CH2) 1.66 m
31.9 (CH2) 1.26 m
22.7 (CH2) 1.26 m
14.1 (CH3) 0.89 t (6.7)
14′c
15′c
16′
31.9
22.7
14.1
1.26 m
1.26 m
0.89 t (6.4)
a Multiplicities were obtained from DEPTQ and HMQC experiments.
b Signals for the fatty acid chain other than mentioned in Table 2: for
compounds 4 and 5 1H NMR δ 1.26 (H-4′-H-13′); 13C NMR δ
29.1-29.8 (C-4′-C-13′). c Signals were assigned on the basis of the
HMBC spectrum. d Not resolved.
Figure 1. Structures of lipidyl pseudopteranes A-F.
considered to be the palmitate ester of 15-hydroxypseudopteradi-
ene.24 The relative configuration in 5 was assigned on the basis of
the comparison of 13C chemical shifts of 5 and pseudopteradiene.24
The MS/MS fragmentation of [M + H]+ ion of compound 6,
molecular formula C37H54O9 (observed [M + Na]+ ) 665.3651,
calculated [M + Na]+ ) 665.3660), gave a base peak at m/z 387.17,
representing a loss of 256 mass units, corresponding to palmitic
acid. The fragmentation pattern of 6 was substantially different from
that of 1, suggesting a structural difference in the pseudopterane
ring. A comparison of the 13C spectra of 4 and 6 revealed that most
of the chemical shifts were very similar other than the signals at δ
161.3 (C), 115.7 (C), 110.1 (CH), and 150.3 (C) of 4 were shifted
to 102.0 (C), 60.5 (C), 54.7 (CH), and 96.2 (C) in 6. The molecular
formula revealed 11 degrees of unsaturation with the addition of
1
Figure 2. H-13C HMBC correlations of 6.
of P. acerosa. The MS analysis, GC-MS and LC-MS, along with
1H NMR of some of these minor compounds indicated the presence
of pseudopteranoids with the same diterpene moiety as 1 but with
different fatty acid side chains including C16:2, C17:0, C18:2, and
C18:3. Unfortunately, there was insufficient sample to allow full
characterization of these compounds.
1
two oxygen atoms relative to 4. 13C and H chemical shifts of 6
with HMBC (Figure 2) and COSY correlations (Table 4) suggested
the structure shown in Figure 1.
Lipidyl pseudopteranes A-F (1-6) as well as pseudopterolide
(7) and gorgiceradiol (8) were subjected to a primary screen (at a
concentration of 100 µg/mL) for inhibition of a panel of protein
tyrosine phosphatases. Compounds 1 and 4 were the most active,
and interestingly, these diterpenes were active against PTP1B and
inactive against other members of the PTP family (including LAR,
SHP-1, and MKPX). The most active compound was lipidyl
pseudopterane A, with an IC50 of 71 µg/mL. It is important to note
that genetic as well as pharmacological studies demonstrated that
inhibiting PTP1B at various levels has led to a corresponding
beneficial effect in animal models of diabetes, obesity, and cancer.
Therefore, the therapeutic window of inhibition for PTP1B is
relatively large. The intrinsic qualities of selectivity and diffusibility
of the lipidyl pseudopteranes even within relatively modest inhibi-
tory activity may still be of great value for therapeutic intervention.
1
The comparison of the assigned H and 13C chemical shifts of
the diepoxyfuran ring of 6 with that of the previously reported
diepoxygorgiacerone19,25 confirmed the presence of the diepoxy-
furan moiety in lipidyl pseudopterane F. The relative configuration
of 6 was assigned on the basis of 1H and 13C NMR chemical shifts
(Table 4) as well as 2D NOESY correlations. The two epoxide
functionalities are shown as being on the R-face of 6, which was
suggested by 1H-1H NOESY cross-peaks between H-5 (4.23 ppm)
and one of the C-18 exomethylene protons (5.34 ppm). The 13C
NMR signals ascribed to C-3, C-4, C-5, and C-6 in 6 were similar
to those reported for diepoxygorgiacerone, whose epoxy groups
were demonstrated to be R-oriented by X-ray crystallographic
analysis.25
Several other less abundant lipidyl pseudopteranoids were
observed in the relatively nonpolar fractions of the organic extract

Pseudopteranoids from Pseudopterogorgia acerosa
Journal of Natural Products, 2008, Vol. 71, No. 12 1981
and 13C NMR (CDCl3) refer to Table 2; Mass (LRMS) obsd [M +
H]+ 655.18 m/z, MS2 fragments (CID ) 35) 637.38, 623.25 (BP),
567.33, 371.14, 353.15, 321.15, 247.15; HRESIMS calcd [M + Na]+
677.4024, obsd 677.3978.
Table 4. NMR Data for Lipidyl Pseudopterane F (6) in CDCl3
position 13C (mult.)a 1H mult (J Hz) 1H-1H COSY 1H-13C HMBC
1
2
38.0 (CH)
36.7 (CH2) 2.33 nr
1.75 nr
2.50 nrd
2a, 2b, 12a
2b, 1
2a, 1
Lipidyl pseudopterane D (4): colorless oil (4.34 mg), [R]D +120
(c 0.45, CHCl3); UV (acetonitrile) λmax (ε), 216 nm (10 616), 248 nm
(4623); IR (neat), 3510, 3082, 2930, 2859, 1765, 1721, 1649, 1615,
3
3
102.0 (C)
4
60.5 (C)
1
1579, 1439; H NMR and 13C NMR (CDCl3) refer to Table 3; Mass
5
6
54.7 (CH)
96.2 (C)
4.23 s
4, 3
(LRMS) obsd [M + H]+ 610.87 and its fragment 355.01 m/z, MS2
fragments of 355.01 (CID ) 35) 340.98, 323.09 (BP), 295.12, 246.00,
191.05; HRESIMS calcd [M + Na]+ 633.3762, obsd 633.3746.
Lipidyl pseudopterane E (5): colorless oil (4.00 mg), [R]D +110
(c 0.39, CHCl3); UV (acetonitrile) λmax (ε), 244 nm (7196); IR (neat),
3453, 3093, 2929, 2858, 1765, 1720, 1655, 1619, 1579, 1439; 1H NMR
and 13C NMR (CDCl3) refer to Table 3; Mass (LRMS) obsd [M +
H]+ 609.18 m/z, MS2 fragments (CID ) 35) 576.95, 353.13 (BP),
321.05, 275.13, 247.1, HRESIMS calcd [M + Na]+ 631.3605, obsd
631.3586.
7
8
9
10
11
12
48.2 (CH)
80.2 (CH)
150.3 (CH) 7.20 dd (1.53, 1.09) 8
132.8 (C)
69.2 (CH)
41.4 (CH2) 2.50 nr
1.70 nr
3.28 br s
5.32 dd (<1)
8(w)
9, 7(w)
19, 5, 6, 18, 17
6, 7
8, 20
5.64 d (2.6)
12a, 12b
12b, 11, 1
12a, 11
13
13
14
148.7 (C)
111.0 (CH2) 4.79 s
4.70 s
18.9 (CH3) 1.67 s
164.5 (C)
141.0 (C)
115.7 (CH2) 5.34 s
5.04 s
24.5(CH3)
170.4(C)
14b, 15
14a, 15
14a, 14b
15, 1
15, 1
1, 14, 13
Lipidyl pseudopterane F (6): colorless oil (3.99 mg), [R]D +60 (c
15
16
17
18
0.46, CHCl3); UV (acetonitrile) λmax (ε), 216 nm (11783); IR (neat),
1
3509, 3082, 2929, 2858, 1765, 1720, 1651, 1439; H NMR and 13C
NMR (CDCl3) refer to Table 4; Mass (LRMS) obsd [M + H]+ 643.08
m/z, MS2 fragments (CID ) 35) 625.26, 611.31, 405.06, 387.03(BP),
337.09, 259.15; HRESIMS calcd [M + Na]+ 665.3660, obsd 665.3651.
Base Hydrolysis of Fraction Enriched with 1. Three milliliters of
0.5 M methanolic NaOH was added to the fraction enriched with 1
(100 µg), and the mixture was heated over a steam bath for 3 min. A
solution of 5 mL of BF3-methanol (14% solution) was then added to
the mixture, which was boiled for another 3 min. The sample mixture
was cooled and transferred into a separatory funnel containing 25 mL
of hexane. A NaCl solution was added, and fatty acid methyl esters
were extracted in hexane and analyzed by GC-MS. EIMS fragmentation
patterns were compared with the NIST library database. The same
protocol was used to identify the fatty acid components of 2-6.
Synthesis of Lipidyl Pseudopterane A (1) from Pseudopterolide.
A solution of palmitic acid (420 µg, 1.6 µmol) in dichloromethane (250
µL) with a catalytic amount of triethyl amine was added to
pseudopterolide (590 µg, 1.6 µmol) in 250 µL of dichloromethane. The
reaction mixture was stirred at ambient temperature, and after 26 h,
methanol (50 µL) was added and the solvent evaporated under a stream
of nitrogen. The product mixture was reconstituted with 0.5 mL of
acetonitrile, and 20 µL was analyzed by LCMS. The production of 1
was confirmed by comparison of retention time, full MS, and MS2
spectra of [M - 18]+ (609 m/z) with an authentic standard. The
percentage yield (0.03%) was calculated by plotting a calibration curve
18b, 19
18a, 19
18a, 18b
19, 7
19, 7
7, 17, 18
19
20
21
1.85 s
53.2 (CH3) 3.87 s
16
FA 1′b 173.8 (C)
2′
33.8 (CH2) 2.33 nr
3′
3′
1′
1′
2.33 nr
24.9 (CH2) 1.60 nr
31.9 (CH2) 1.26 br m
22.9 (CH2) 1.26 br m
14.1 (CH3) 0.89 t (6.7)
3′
2’a, 2’b, FAC
14′c
15′c
16′
FAC
14′, 15′
a Multiplicities were obtained from DEPTQ experiment. b Signals for
the fatty acid chain other than mentioned in Table 3: 1H NMR δ 1.26
(H-4′-H-13′); 13C NMR δ 29.1-29.7 (C-4′-C-13′). c Signals were
assigned on the basis of the HMBC spectrum. Fatty acid chain
represented as FAC. d Not resolved.
Further studies in cells and animals will thus be required for
clarifying their true potential as PTP1B inhibitors.
Experimental Section
Collection and Extraction Procedure. The coral specimens were
collected from Sweetings Cay, Bahamas, in May 2005, and a voucher
(#090607-02-009) is maintained at UPEI. Sun-dried coral (367 g) was
blended with ethyl acetate and dichloromethane, and blending continued
with fresh solvents until the color of the solvent became faint yellow
(8 × 1 L) and then it was filtered. Evaporation of solvent in vacuo
resulted in 27.6 g of crude extract. Separation was carried out by silica
(170 g) flash column chromatography using a stepwise gradient (hexane
to ethyl acetate). Lipidyl pseudopteranoids 1-3 were isolated from the
50% hexane extract, while 4-6 were isolated from the 60% hexane
extract. These fractions were then subjected to silica column chroma-
tography using an isocratic solvent system optimized by thin-layer
chromatography (TLC) experiments. The aliquots were collected and
combined on the basis of the TLC of each aliquot, which resulted in
seven different fractions. Compounds 1-3 were isolated by semi-
preparative HPLC (CH3CN-H2O gradient with a phenyl hexyl column)
from fraction number 2 of the 50% hexane extract, and 4-6 were
obtained from fraction numbers 3 and 4 of the 60% hexane extract.
Lipidyl pseudopterane A (1): colorless oil (8.48 mg), [R]D +150
(c 0.27, CHCl3); UV (acetonitrile) λmax (ε), 218 nm (13 743), 254 nm
generated from an authentic standard (range ) 0.1-10 ppm, R2
0.9992).
)
PTP Assays. Assay buffer at pH 7.0 was prepared using HEPES
(50 mM), DTT (3 mM final), and BSA (0.1 mg/mL). Assays were
conducted at 25 °C in 96-well plates (Falcon) in a volume of 100 uL.
Reaction rates were determined using a Varioskan plate reader (Thermo
Electron). Using pNPP as a substrate, absorbance was monitored at
405 nm and measured every 30 s over 10 min, and the reaction rates
were calculated by linear regression. Enzymes used were the catalytic
domains of GST-tagged protein tyrosine phosphatases: GST-PTP1B,
GST-LAR, GST-SHP-1, and GST-MKPX. PP1 was purchased at New
England Biolabs. All inhibitor assays contained 1% DMSO (final). IC50
assays were conducted at substrate concentrations equal to the Km value
for PTP1B. IC50 values were derived by fitting data to a sigmoidal
dose-response (variable slope) curve (Prism software).
Acknowledgment. Funding was provided by NSERC (R.G.K.), the
Canada Research Chair Program (R.G.K.), the University of Prince
Edward Island (R.G.K.), the Canada Foundation for Innovation
(R.G.K.), the Atlantic Innovation Fund (R.G.K.), CIHR (M.L.T.), and
the Jeanne and Jean-Louis Le´vesque Foundation (M.L.T., R.G.K.). We
acknowledge A. Thompson and A. Ali, Dalhousie University, Halifax,
for assistance with measurements of optical rotation and CD. We thank
D. Lund (UPEI) and the Atlantic Region Magnetic Resonance Centre
for NMR support. HRESIMS data were obtained by the Maritime Mass
Spectrometry Laboratories at Dalhousie University, Halifax.
1
(4949); IR (neat) 3529, 3087, 2929, 2858, 1765, 1737 and 1720; H
NMR and 13C NMR (CDCl3) refer to Table 1; Mass (LRMS) obsd [M
+ H]+ 627.17 m/z, MS2 fragments (CID ) 35) 609.3, 595.31 (BP),
371.1, 353.15, 321.16, 247.12; HRESIMS calcd [M + Na]+ 649.3716,
obsd 649.3715.
Lipidyl pseudopterane B (2): colorless oil (2.98 mg), [R]D +120
(c 0.19, CHCl3); UV (acetonitrile) λmax (ε), 214 nm (18 325); 1H NMR
and 13C NMR (CDCl3) refer to Table 2; Mass (LRMS) obsd [M +
H]+ 652.94 m/z, MS2 fragments (CID ) 35) 635.28, 621.41 (BP),
603.41, 442.35, 353.22, 321.19, 247.14; HRESIMS calcd [M + Na]+
675.3867, obsd 675.3854.
Supporting Information Available: Copies of the HRMS, MS, 1H
Lipidyl pseudopterane C (3): colorless oil (2.45 mg), [R]D +130
1
(c 0.19, CHCl3); UV (acetonitrile) λmax (ε), 214 nm (7714); H NMR
NMR (500 and 300 MHz), 13C NMR (125 and 75 MHz), DEPTQ,

1982 Journal of Natural Products, 2008, Vol. 71, No. 12
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