Full text of DOI:10.1016/S0040-4039(00)01015-7

Tetrahedron Letters 41 (2000) 6035±6040
New antiproliferative epoxysecosterols from
Pseudopterogorgia americana
Samina Naz,a Russell G. Kerra,* and Ramaswamy Narayananb
aDepartment of Chemistry and Biochemistry, Center for Molecular Biology and Biotechnology,
Florida Atlantic University, 777 Glades Road, Boca Raton, FL 33431-0991, USA
bDepartment of Biological Sciences, Center for Molecular Biology and Biotechnology, Florida Atlantic University,
777 Glades Road, Boca Raton, FL 33431-0991, USA
Received 4 May 2000; revised 5 June 2000; accepted 6 June 2000
Abstract
Two new 9(11)-secogorgosterols containing oxirane rings at the C-5/C-6 position (1 and 2) were isolated
from Pseudopterogorgia americana along with a known secogorgosterol (3). The structures were elucidated
using detailed spectroscopic studies and the epoxysecosteroids were found to exhibit activity against pros-
tate (LnCap) and lung cancer (Calu-3) cell lines. # 2000 Elsevier Science Ltd. All rights reserved.
Keywords: NMR; mass spectrometery; steroids; anti-tumor compounds and anti-in¯ammatory compounds.
Pseudopterogorgia americana, an abundant marine gorgonian on reefs in Florida and the
Caribbean, produces 9(11)-secosterols and polyoxygenated sterols, which exhibit inhibitory
activity against protein kinase C, potent anti-proliferative and promising anti-in¯ammatory
activity.1 3 Our recent detailed chemical studies on P. americana have resulted in the isolation of
two new 9(11)-secogorgosterols containing an epoxide ring at the C-5/C-6 position, 1b,3b-di-
hydroxy-5a,6a-epoxy-9-oxo-9,11-secogorgostan-11-ol (1), 3b-hydroxy-5a,6a-epoxy-9-oxo-9,11-
secogorgostan-11-ol (2) and the known 3b-hydroxy-9-oxo-9,11-secogorgostan-11-ol (3). Their
structures were established on the basis of spectral studies.
Methanol extraction of the freeze-dried gorgonian (88.9 g) yielded a crude extract (26.8 g)
which was fractionated by solvent partitioning between water and CHCl3:MeOH (2:1). The
organic fraction was subjected to silica gel column chromatography and reversed-phase HPLC to
a€ord two new secosterols with an unusual oxygenation pattern 1 (4.2 mg) and 2 (3.6 mg) along
with a known metabolite, secogorgosterol 3 (5.1 mg).6
1b,3b-Dihydroxy-5a,6a-epoxy-9-oxo-9,11-secogorgostan-11-ol (1), C30H50O5, was isolated as a
colorless gum. The UV spectrum showed a terminal absorption, indicating the lack of a
* Corresponding author. Tel: 561 297-3356; fax: 561 297-2759; e-mail: rkerr@fau.edu
0040-4039/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(00)01015-7

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conjugated p system. The IR spectrum displayed intense absorption bands at 3317 (O±H), 1710
(CˆO), and 1103 (C±O±C) cm^1. The FABMS of 1 showed a molecular ion peak at m/z 490.3653
which is consistent with the elemental formula C30H50O5 (calcd 490.3658) and indicated the
presence of six degrees of unsaturation, ascribed to one carbonyl group, one epoxide ring, and the
four secogorgosterol rings. A signal at m/z 472 (C30H48O4) was ascribed to M+^H2O.
1
The presence of a secogorgosterol derivative was evident from the H, 13C, and COSY data
1
(Table 1). The H NMR spectrum (500 MHz, CDCl3) of 1 featured signals as doublets of
doublets at ꢀ ^0.14 and 0.45 (J=9.0, 4.5 Hz), integrating for one hydrogen each, which are
characteristic signals of the C-30 methylene protons of a cyclopropyl ring, while a two-hydrogen
resonance at ꢀ 0.27 was ascribed to the C-22 and C-24 methine protons. These characteristic
signals suggested the presence of a cyclopropane system substituted at C-22 and C-23 of the side
chain of a secogorgosteroidal skeleton.4 Three singlets, integrating for three hydrogens each,
resonated at ꢀ 0.67, 0.85 and 1.24 were ascribed to hydrogens of the C-18, C-29 and C-19 methyl
groups,4,5 while four three-hydrogen doublets at ꢀ 0.82 (J=6.5 Hz), 0.89 (J=7.0 Hz), 0.91 (J=6.5
Hz) and 1.01 (J=6.5 Hz) were assigned to the protons of C-26, C-28, C-27 and C-21 methyl
groups, respectively.4,5 Another one-proton signal appeared as a broad doublet of triplets at ꢀ
2.81 was diagnostic of the C-8 methine proton,4,5 while another down®eld signal at ꢀ 3.22 (d,
J=4.5 Hz) was assigned to the C-6 methine proton. A four-hydrogen multiplet at ꢀ 3.65±3.76 was
assigned to the C-1 methine, C-3 methine, and C-11 methylene protons. Their down®eld chemical
shift values suggested the presence of a geminal oxygen functionality at each above mentioned
carbon atom. The chemical shift values of the C-11 methylene protons (ꢀ 3.72±3.76) are
characteristic of 9(11)-secosterols with a hydroxyl functionality at C-11.4 6

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Table 1
1H and 13C NMR chemical shift assignments of 1 and 2
ꢀ
1
The COSY-45 spectrum was used for the complete H NMR chemical shift assignments and
identi®ed four isolated spin systems in 1. The ®rst spin system consisted of the C-1 to C-4 carbon
fragment. H-1 (ꢀ 3.65) exhibited COSY-45ꢀ interactions with the C-2 methylene protons (ꢀ 1.50
and 2.12). The latter further exhibited vicinal couplings with the C-3 methine proton (ꢀ 3.67)
which showed H±1H spin correlations with the C-4 methylene protons (ꢀ 1.45 and 1.93). The
1
second spin system is comprised of C-11 and C-12. The COSY-45ꢀ spectrum featured cross-peaks
of the C-11 methylene protons (ꢀ 3.72 and 3.76) with the C-12 methylene protons (ꢀ 1.40 and
1.79). Geminal couplings of the C-11 and C-12 methylene protons were also observed. The third,
and largest fragment traced from C-6 to C-30 and started with the C-6 methine proton (ꢀ 3.22)

6038
which showed vicinal coupling with the C-7 methylene protons (ꢀ 1.80 and 2.40) which in turn
exhibited cross-peaks with the C-8 methine proton (ꢀ 2.81). The latter further showed cross-peaks
with the C-14 methine proton (ꢀ 2.60) which exhibited vicinal couplings with the C-15 methylene
protons (ꢀ 1.25 and 1.40). The C-15 methylene protons showed COSY-45ꢀ interactions with the
C-16 methylene protons (ꢀ 1.65 and 1.99), which in turn exhibited vicinal couplings with the C-17
methine proton (ꢀ 1.23). The C-17 methine proton further showed cross-peaks with the C-20
methine proton (ꢀ 0.99). Cross-peaks of the C-20 methine proton with the C-21 methyl protons (ꢀ
1.01) and C-22 methine proton (ꢀ 0.27) were also observed in the COSY-45ꢀ spectrum. The latter
exhibited vicinal couplings with the C-30 methylene protons (ꢀ -0.14 and 0.45). Geminal
couplings between the C-7, C-15, C-16 and C-30 methylene protons were also observed in the
COSY-45ꢀ spectrum. A fourth spin system composed of the C-24 to C-28 fragment and begins
with the C-24 methine proton (ꢀ 0.27) which showed cross-peaks with the C-25 methine proton (ꢀ
1.50) and C-28 methyl protons (ꢀ 0.89). The former further exhibited vicinal couplings with the
1
C-26 (ꢀ 0.82) and C-27 (ꢀ 0.91) methyl protons. H NMR chemical shift assignments of 1 are
summarized in Table 1.
The 13C NMR spectrum (125 MHz, CDCl3) of 1 showed distinct resonances of all 30 carbons.
An attached proton test (APT) experiment indicated the presence of ten CH, eight CH2, seven
CH3 and ®ve quaternary carbon atoms in 1. Interpretation of 13C NMR data further suggested
that the compound is a secosterol as chemical shift values of the majority of the carbon atoms
were found to be nearly identical to those of previously reported secogorgosterols of the series,4 6
greatly facilitating the 13C NMR chemical shift assignments of 1. A resonance at ꢀ 69.9 was
assigned to C-1 while a signal at ꢀ 69.4 was due to C-3. Other down®eld signals at ꢀ 61.0 and 60.4
were diagnostic signals for the epoxy-bearing C-5 and C-6. A resonance at ꢀ 59.1 is characteristic
for the C-11 hydroxy-bearing carbon in secosterols.2,4 6 Complete 13C NMR chemical shift
assignments of 1 are shown in Table 1.
The stereochemistry at various chiral centers was established with the aid of NOESY, coupling
constants, optical rotations and by comparing the 13C NMR chemical shift values with known
20
D
secosterol (3) of the series. Compound 1 showed an ‰ꢁŠ value of +21. The positive sign of optical
rotation suggested that the C-3 methine proton has an a-orientation, while the C-8 methine
proton, C-18 and C-19 methyl groups are b-oriented as in other secosterols of this series. The 13C
NMR chemical shift values of C-3, C-8, C-10, C-13, C-14, C-17, C-20, C-22, C-23 and C-24 were
identical to those reported for secosterol 3 which further suggested that these centers have the
same orientation as in 3.4 6 NOE cross-peaks between H-8 (ꢀ 2.81), H3-18 (ꢀ 0.67) and H3-19 (ꢀ
1.24) were also observed in the NOESY spectrum which indicated that they are cis to each other
and thus all oriented b. With the stereochemistry at these chiral centers established, the NOESY
spectrum was helpful for the assignment of stereochemistry at other chiral centers. The C-1
methine proton (ꢀ 3.65) showed a cross-peak with the C-3 methine proton (ꢀ 3.67) which
suggested the a-stereochemistry for the C-1 methine proton and thus the b-stereochemistry for
the C-1 hydroxyl. The C-6 methine proton (ꢀ 3.22) exhibited an NOE with the C-8 methine
proton (ꢀ 2.81) and the C-19 methyl protons (ꢀ 1.24), indicating that the C-5/C-6 epoxy
functionality has the a-orientation. Tori and co-workers7 have shown that for 9b,11b-epoxy
steroids, J11a,12a and J11a,12b are ꢁ1.5 Hz while for the 9a,11a isomer, J11b,12a ꢁ0, J11b,12b ꢁ4.5
Hz. Hence the single 4.5 Hz coupling observed for the H-6 resonance in 1 further con®rmed the a
stereochemistry for the oxirane ring. Probable conformation of 1 and its important NOE
interactions are shown in Fig. 1. Based on these spectral data, structure 1 was established for this
new natural product.

6039
Figure 1. Probable conformation of compound 1 and its important NOE interactions as observed in the NOESY
spectrum
The second compound, 3b-hydroxy-5a,6a-epoxy-9-oxo-9,11-secogorgostane-11-ol (2), was also
isolated as a colorless gum. The UV and IR spectra of compound 2 were identical to those of 1.
The EIMS of 1 showed a molecular ion peak at m/z 474, which is consistent with an elemental
formula C30H50O4 and indicated the presence of six degrees of unsaturation.
The 1H NMR spectrum (500 MHz, CDCl3) of 2 was very similar to that of 1 except the multiplet
at ꢀ 3.66±3.76 integrated for three hydrogens instead of four hydrogens as discussed previously for
compound 1. This indicated that the signal for the C-1 methine proton, geminal to the hydroxyl
group, is absent in this region. The C-1 methylene protons were observed as a multiplet at ꢀ 1.79
and 1.39 as observed in the COSY-45ꢀ spectrum by tracing 1H±1H coupling frame work of the C-
1
3 methine proton (ꢀ 3.66) in order to assign complete H NMR chemical shift assignments of 2
(Table 1). The 13C NMR spectrum of 2 (Table 1) indicates that chemical shift values of all the
carbon atoms are nearly identical to those as previously discussed for compound 1 except for C-1
which resonated at ꢀ 29.9. The high resolution electron impact mass spectrum of 2 showed an M+
at m/z 474.3715, which is in agreement with the molecular formula C30H50O4 (calcd 474.3709).
The mass spectrum also exhibited a similar fragmentation pattern to that of 1 with many ions
1
observed at 16 amu lower than that of 1. This combination of H, 13C NMR and MS data con-
®rmed that compound 2 is the C-1 deoxy derivative of 1. The optical rotation, NOESY and 13C
NMR spectra also favors similar stereochemistry at all chiral centers as established for compound
1. Based on these spectroscopic studies, structure 2 was established for this new secosterol.
Compound 1 exhibited signi®cant activity against prostate cancer (LnCap) and lung cancer cell
lines (Calu-3) in an MTT assay with observed IC50 values of 15.49 mg/ml and 11.0 mg/ml,
respectively. Compound 2 also showed similar bioactivity against the above mentioned cancer cell
lines with observed IC50 values of 18.43 mg/ml and 12.0 mg/ml, respectively. Interestingly, com-
pound 3 exhibited IC50 values of 41.0 mg/ml and 38.12 mg/ml against the LnCap and Calu-3 cell
lines, respectively, suggesting that the epoxide ring is at least partly responsible for the observed
activity. The similarity of the IC50 values of 1 and 2 indicates that the C-1 hydroxyl is not
responsible for the observed activity.
Acknowledgements
We are very grateful to the Camille and Henry Dreyfus Foundation for providing ®nancial
support for this study. We also thank Harbor Branch Oceanographic Institution, Florida for

6040
permitting us to use their polarimeter for measuring optical rotations, and the Midwest Center
for Mass Spectrometry at the University of Nebraska±Lincoln for mass measurements.
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