New Sterol Derivatives from the Marine Sponge Xestospongia sp.

Article abstract of DOI:10.1002/hlca.201600021

Chemical examination of a marine sponge Xestospongia sp. resulted in the isolation of 20 sterol derivatives (1 – 20), including eight new sterols namely aragusterols J – L (1 – 3), (5α,7α,12β,22E)-7,12,18-trihydroxystigmast-22-en-3-one (4), (5α,7α,12β,24R)- and (5α,7α,12β,24S)-7,12,20-trihydroxystigmastan-3-one (5/6), and (5α,7α,12β,22E,24R)- and (5α,7α,12β,22E,24S)-7,12,20-trihydroxyergost-22-en-3-one (7/8). The structures of new compounds were determined through extensive spectroscopic analyses and chemical conversion. The sterol diversity was mainly characterized by the presence of a cyclopropane unit at side chain, while compound 4 with 18-hydroxymethyl group was found in stigmasterol family for the first time. Cytotoxic test revealed the inhibitory effects of compounds 1, 4, and 17 against human leukemia cell line K562 with IC₅₀values of 18.3, 24.1, and 34.3 μm, respectively.

Full text of DOI:10.1002/hlca.201600021

Article Type: Full Paper
New Sterol Derivatives from the Marine Sponge Xestospongia sp.
By Zhongbin Chenga), Dong Liua), Nicole J. de Voogdb), Peter Prokschc), Wenhan Lina,*)
a) State Key Laboratory of Natural and Biomimetic Drugs, Institute of Ocean Research,
Peking University, Beijing, 100191, People’s Republic of China
b) Department Marine Zoology, Darwinweg 2 - 2333 CR Leiden, Netherland)
c) Institute für Pharmazeutische Biologie und Biotechnologie, Heinrich-Heine- Universität
Düsseldorf, 40225 Düsseldorf, Germany
*Corresponding author: Tel/Fax: +86-10-82806188, E-mail: whlin@bjmu.edu.cn
Abstract
Chemical examination of a marine sponge Xestospongia sp. resulted in the isolation of 20
sterol derivatives (1-20), including eight new sterols namely aragusterols J-L (1−3),
(5α,7α,12β,22E)-7,12,18-trihydroxystigmast-22-en-3-one (4), (5α,7α,12β,24R)- and
(5α,7α,12β,24S)-7,12,20-trihydroxystigmastan-3-one (5/6), and (5α,7α,12β,22E,24R)- and
(5α,7α,12β,22E,24S)-7,12,20-trihydroxyergost-22-en-3-one (7/8). The structures of new
compounds were determined through extensive spectroscopic analyses and chemical
conversion. The sterol diversity was mainly characterized by the presence of a cyclopropane
unit at side chain, while compound 4 with 18-hydroxymethyl group was found in stigmasterol
family for the first time. Cytotoxic test revealed the inhibitory effects of compounds 1, 4, and
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17 against human leukemia cell line K562 with IC50 values of 18.3, 24.1, and 34.3 μM,
respectively.
Keywords: Sterols, Structure elucidation, Cytotoxic activity, Xestospongia sp.
Introduction.-Marine sponges have been proved to be the most diverse array of
unconventional steroids derived from all organisms [1, 2]. The steroids from sponges are
sometimes highly functionalized and displayed unconventional polycyclic core or unusual
side chains [3], while some of them have no terrestrial counterpart. The highly functionalized
steroids have attracted considerable attention due to the potential biological activities [4]. The
sterols possessing a cyclopropane ring at side chain are a group of unusual natural products
mainly distributed in marine sponges, such as aragusterols A-I, xestokerols A-C, 21-O-
octadecanoyl-xestokerol A, 7-oxopetrosterol, 7β- and 7α-hydroxypetrosterol, and petrosterol
from Xestospongia [5-10] and Ianthella sponges [11]. The potent biological activities such as
aragusterols with strong inhibition against the proliferation of tumor KB cells in vitro and
L1210 leukemia in vivo attracted the attention of chemists for synthesis of aragusterols A-D
[12]. The sponge genus Xestospongia (order Haplosclerida, family Petrosiidae) widely
distributed in the Pacific Ocean, Indian Ocean, and Caribbean Sea. Chemical examination of
sponges revealed that the same species of a sponge collected from different location produced
distinct metabolites. With the aim for the discovery of bioactive metabolites from marine
invertebrates, a marine sponge Xestospongia sp. collected from the South China Sea was
undertaken for chemical examination. The 1H NMR spectrum of the AcOEt extract displayed
the resonances ranging from 0 to 2 ppm typical for cyclopropane-bearing steroids.
Chromatographic separation of the AcOEt extract resulted in the isolation of 20 sterol
derivatives (Fig. 1).
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Results and Discussion.-The frozen sponge of Xestospongia sp. was extracted with EtOH
to afford a crude extract, which was desalted by dissolving in MeOH. The residue was
dispersed in H2O and successively partitioned with AcOEt and BuOH. The AcOEt extract
was subjected to extensive column chromatography to afford compounds 1−20.
Aragusterol J (1) has a molecular formula of C29H46O4, as established by the HR-ESI-MS
(m/z 459.3472 [M + H]+) and NMR data. The IR absorptions at 3358 and 1716 cm-1
1
suggested the presence of hydroxyl and carbonyl functionalities. The H-NMR spectrum
exhibited the resonances including four Me groups (δ(H) 0.81, s, H3-C(18); 1.23, s, H3-C(19);
0.95, d, J = 6.7 Hz, H3-C(28); 1.03, d, J = 6.0 Hz, H3-C(29)), three oxymethines (δ(H) 3.79,
dt, H-C(6); 3.52, dd, J = 11.1, 4.5 Hz, H-C(12); 4.36, dd, J = 8.0, 4.4 Hz, H-C(22)), an
exomethylene (δ(H) 4.96, 5.13, s), while the shielded protons at δ(H) 0.15 (m), 0.23 (m), and
0.57 (m) were featured by the presence of cyclopropane ring. The 13C-NMR and DEPT
spectra displayed a total of 29 carbon resonances, involving a ketone and two olefinic
carbons for a double bond. Analyses of 1D and 2D NMR (COSY, HMQC and HMBC) data
established a sterol-based gross structure, which was closely related to aragusterol E (11) [7],
a coexisted sterol in the same fraction. The difference was attributed to a hydroxymethine C(6)
(δ(C) 70.6) of 1 to replace a methylene group of 11, as evident from the COSY correlations of
H-C(6) (δ(H) 3.79, dt, J = 2.0, 4.0 Hz) with H-C(5) and H2-C(7). The relative configuration
of 1 was assigned by the NOE interactions. The NOE correlations from H-C(8) to H3-C(18)
and H3-C(19) and between H-C(9) and H-C(5) clarified the trans fusion of the tetraocyclic
nucleus, the same as that of 11. Thus, the NOE relationships between H3-C(19)/Hβ-C(4) and
Hα-C(4)/H-C(6) in association with the JH-5/H-6 value (4.0 Hz) assigned an equatorial
orientation of H-C(6), indicating OH-C(6) to be β-oriented. Additional NOE correlations
from H-C(12) to H-C(9), H-C(14), and H-C(17) reflected OH-C(12) to be β-oriented, while
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the side chain at C(17) was β-oriented. This was also supported by the observation of the
NOE relationship between H3-C(18) and H2-C(21) (Fig. 2). The complete agreement of the
NMR data for the side chain including the NOE interaction and J values led to the assignment
of the same configurations for the side chain of 1 and 11.
The molecular formula of aragusterol K (2) was determined to be C31H50O2 on the basis of
the HR-ESI-MS (m/z 471.3830 [M + H]+) and NMR data. The NMR data of 2 (Tables 1 and 2)
featured a sterol-type analogue, while analyses of 1D and 2D NMR data resulted in the gross
structure of 2 to be closely related to 5α,6α-epoxy-petrosterol (12) [13]. The distinction was
found by the presence of an acetyl group (δ(H) 2.0, s, δ(C) 21.3, 170.2) in 2. The HMBC
correlation of H-C(3) (δ(H) 4.95) with the acetyl carbonyl carbon deduced the location of
acetoxy group at C(3). The similar NMR data and NOE interactions of 2 and 12 reflected the
same configurations of both compounds. Conversion of 2 to 12 by deactylation with alkaline
hydrolysis (Fig. 3) further supported both 2 and 12 sharing the same configurations.
The 1D and 2D NMR data in association with the HR-ESI-MS data provided the gross
structure of aragusterol L (3) to be the same as 5α,6α-epoxy-petrosterol (12) [13]. The
distinction was found by the NMR resonances of the epoxy group, of which the shielded C(5)
(δ(C) 62.9) and deshielded C(6) (δ(C) 63.7) of 3 were observed to replace δ(C) 65.2 (C(5))
and 59.2 (C(6)) of 2 and 12. These findings suggested that the configuration of the epoxy
group in 3 differed from that of 12. The NOE interaction from Hα-C(4) to H-C(3) and H-C(6)
deduced the β-orientation of the epoxide ring [14,15]. The similar NOE interactions and
NMR data regarding the tetracyclic nucleus and side chain indicated the same configurations
of remaining stereogenic centers in both 3 and 12. In addition, the structure of petrosterol (16)
was assigned by the single-crystal X-ray diffraction using Cu Kα radiation in this work (Fig.
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4). Epoxidation of petrosterol (16) by 3-chloroperbenzoic acid oxidation yielded two products
with a ratio of 5:1, which were identical to 3 and 12 by the comparison of their NMR, MS
and specific rotation. This finding further confirmed the configurational assignment of 3.
Compound 4 has a molecular formula of C29H44O10 as determined by the HR-ESI-MS (m/z
1
13
461.3629 [M + H]+) and NMR data. The H- and C-NMR spectra featured a stigmasterol
nucleus, based on the resonances for an ethyl group, an ispropane unit, a methyl doublet, and
two olefinic protons at δ(H) 5.52 (dd, J = 8.2, 15.4 Hz, H-22) and 5.14 (dd, J = 9.0, 15.4 Hz,
H-23) for E-geometry of the double bond in the side chain, in association with their COSY
and HMBC interactions. A ketone group resided at C(3) (δ(C) 214.4) was evident from the
HMBC interactions from C(3) to H2-C(1), H2-C(2), H2-C(4) and H-C(5). In addition, the
chemical shifts of C(7) (δ(C) 67.6)/H-C(7) (δ(H) 3.79) and C(12) (δ(C) 82.1)/H-C(12) (δ(H)
3.45) indicated C(7) and C(12) to be hydroxylated. The absence of methyl group C(18) and
the presence of hydroxymethylene (δ(C) 61.7; δ(H) 3.74, 3.97), in addition to the HMBC
correlations from the hydroxymethylene protons to C(12), C(13), C(14) and C(17) clarified
C(18) to be hydroxylated. The trans fusions of the backbone were determined by the NOE
interactions as mentioned for 1. The small J values of H-C(7) in association with the NOE
interaction between H-C(7) and H-C(8) assigned H-C(7) to be in equatorial orientation (β-
orientation), whereas the J values of H-C(12) (dd, J = 4,4, 11.2 Hz) reflected axial-orientation
(α-orientation). The NOE interactions between H2-C(18)/H-C(20) and H-C(12)/H-C(17)
assigned α-orientation of H-C(17). The comparable NMR data of 4 and 22E,24R-stigmast-
22-ene-3,7-dione [16] regarding to the side chain conducted to assign the same configuration
of C(20) and C(24) of both compounds. Thus, the structure of 4 was assigned as
(5α,7α,12β,22E)-7,12,18-trihydroxystigmast-22-en-3-one.
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Compounds 5 and 6 were obtained as a pair of inseparable analogues, whose molecular
formula (C29H50O4) was determined by the HR-ESI-MS data. The NMR data of 5 and 6 were
characteristic of sitosterol-type analogues, based on the typical NMR resonances of two
methyl doublets, a methyl triplet, and a methyl singlet in the side chain. Detailed analyses of
2D NMR data determined the partial structure of tetracyclic nucleus to be closely related to
that of 4, with the exception of C(18) being a methyl group instead of hydroxymethyl group.
This finding was supported by the presence a ketone at C(3) (δ(C) 214.4), hydroxy groups at
C(7) (δ(C) 67.7) and C(12) (δ(C) 78.7). An additional hydroxy group at C(20) (δ(C)
75.7/75.8) was defined by the HMBC correlations from H3-C(21) (δ(H) 1.21, s) to C(17) (δ(C)
66.0), C(20), and C-22 (δ(C) 35.6/35.7). The duplicated NMR data from C(1) to C(19) (Table
2) indicated both 5 and 6 sharing the same partial structure of tetracyclic nucleus. However,
the doubling NMR resonances at side chain of 5 and 6 were observed. The NOE interactions
from H-C(17) to H-C(12) and H3-C(21) were indicative of the same configuration at C(20) of
5 and 6. In addition, the NOE correlations between H-C(12)/H3-C(21) and H3-C(18)/OH-
C(20) in association with the absence of NOE correlation between H-C(12)/OH-C(20)
indicated the side chain maintaining a dominant conformer, while OH-C(20) was spatially
approximated to H3-C(18). Thus, both 5 and 6 were supposed to be a pair of C(24) epimers.
Pairwise comparison of the 13C-NMR signals for side chain of 5/6 with those of sitosterol and
its C(24) epimer clionasterol [17] assigned both 5 and 6 to be C(24) epimers. Thus, the
structures of 5 and 6 were assigned as (5α,7α,12β,24R)- and (5α,7α,12β,24S)-7,12,20-
trihydroxystigmastan-3-one.
Compounds 7 and 8 were a pair of inseparable sterols with a ratio of 2:1 as detected by the
NMR spectra, while the molecular formula of C28H46O4 as determined by the HR-ESI-MS
(m/z 447.3474 [M + H]+) data. Their NMR data (Tables 1 and 2) featured the signals of
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ergosterol-type analogues. Comparison of the NMR data between 7/8 and 5/6 revealed that
they possessed the same tetracyclic nucleus. The distinction was attributed to the resonances
of side chain where doubling NMR resonances were observed. The position of a hydroxyl
group at C(20) (δ(C) 75.1) was assigned by the HMBC interactions from H3-C(21) (δ(H) 1.21,
s) to C(17), C(20), and C(22), while the COSY and additional HMBC correlations established
the side chain to be related to that of ergosterol with a 20-hydroxy group. The coupling
constant JH-22/H-23 = 15.4 Hz was in agreement with 22E geometry. The NOE interaction
between H-C(17) and H3-C(21) reflected the same configuration of C(20) in 7 and 8.
Additional NOE correlations between H-C(12)/H3-C(21) and H3-C(18)/OH-C(20) and the
absence of NOE correlation between H-C(12)/OH-C(20) with the same as those observed in
the NOESY spectrum of 5/6, indicated OH-C(20) was resided in the same orientation as that
of 5/6. The doubling of side chain signals at C(22) (δ(C) 134.9/135.1), C(23) (δ(C)
132.6/132.3), C(24) (δ(C) 34.5/34.6), C(25) (δ(C) 20.2/20.3), C(25) (δ(C) 34.5/34.6), C(26)
(δ(C) 20.4/20.7), and C(28) (δ(C) 17.6/17.9), was indicative of C(24) epimers of both 7 and 8.
Thus, the structures of 7 and 8 were assigned as (5α,7α,12β,22E,24R)- and
(5α,7α,12β,22E,24S)-7,12,20-trihydroxyergost-22-en-3-one.
In addition, 3α-aragusterol I (9) [12] and petrosteryl acetate (10) [18], as synthetic
intermediates, were isolated from nature for the first time. Chemical transformation of 14 to 9
(Fig. 6) and 16 to 10 (Fig. 7), clarified the stereogenic centers of 9 and 10. Ten additional
known analogues were identical to aragusterol E (11) [7], 5α,6α-epoxy-petrosterol (12) [11],
aragusterol I (13) [7], aragusterol B (14) [8], xestokerol B (15) [10], petrosterol (16) [19],
aragusterol A (17) [9], 7-oxopetrosterol (18) [20], 7β-hydroxypetrosterol (19) [7], and 7α-
hydroxypetrosterol (20) [9], on the basis of the comparison of their NMR, MS, and specific
rotation data with those published in literature.
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The X-ray crystal data clarified petrosterol (16) bearing trans-fusion of rings A/B, B/C,
and C/D, while the absolute configurations of the stereogenic centers were determined to be
3S, 8S, 9S, 10R, 13R, 14S, 17R, 20R, 24R, 25R, and 27R. These assignments were in
agreement with known natural sterols which exclusively presented trans-fusion of nucleus
rings. The coexistence of the cycloprepane-bearing sterols with petrosterol in the sponge
conducted the biogenetic assumption of the same configurations of sterol backbone among
the isolated analogues.
All steroids (1−20) were evaluated for their inhibitory activity against the human leukemia
cell line K562 at an initial concentration of 10 μM by the MTT method. Adriamycin was used
as the positive control. The bioassay results showed that nine compounds showed inhibitions
more than 20% at a single dose of 10 μM (Table 3), while compounds 1, 4, 14, and 17
showed moderate/weak activity with IC50 values of 18.3, 24.2, 34.3, and 95.6 μM,
respectively.
Conclusion.-Present work provided a group of new steroids which enriched the number of
sterol family derived from marine sponges. Steroids obtained from Xestospongia sponges
exclusively possessed the analogues with a cyclopropane unit, suggesting the 26,27-
cyclopropane-bearing steroids to be one of the chemotaxonomic marks of Xestospongia
species. The weak cytotoxic activity of the isolates implied that the steroids from the sponge
may play ecological role other than toxic effect.
ACKNOWLEDGMENT
This work was supported by the grants from 973 program (2015CB755906), NSFC-
Shangdong Join Fund for Marine Science (U1406402), and NSFC (41376127).
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Experimental Part
General
TLC: HF254 silica gel (SiO2, Qingdao Marine Chemistry Co. Ltd.) Column
chromatography (CC): silica gel (SiO2, 200 − 300 mesh; Qingdao Marine Chemistry Co.
Ltd.), Sephadex LH-20 (18 − 110 μm; Pharmacia). HPLC: semi-prep. Prevail C18 column (5
μm), Alltech 426 pump, UV detector (Pharmacia). Optical rotations: Autopol III automatic
polarimeter (Rudolph Research Co., Ltd.). IR Spectra: Thermo Nicolet Nexus 470 FT-IR
spectrometer; ṽ in cm–1. 1H- and 13C-NMR spectra: Bruker Avance-400FT NMR spectrometer;
δ in ppm rel. to Me4Si as internal standard, J in Hz. HR-ESI-MS: Bruker APEX IV 70 eV FT-
MS spectrometer and on a Thermo DFS spectrometer using a matrix of 3-nitrobenzyl alcohol;
in m/z. EI-MS (70 eV): Finnigan MAT 95 mass spectrometer; in m/z.
Animal Material
The sponge Xestospongia sp. was collected at a depth of 10 m water in Yongxin Island,
Hainan Province of China, in June 2012, and the fresh sample was frozen immediately. The
specimen was identified by Dr. N. J. de V. (Department Marine Zoology, The Netherland). A
voucher specimen (XSA-16) was deposited with the State Key Laboratory of Natural and
Biomimetic Drugs, Peking University, P. R. China.
Extraction and Isolation
The frozen animal of (700 g, wet weight) was extracted with EtOH to give a crude extract,
which was desalted by dissolving in MeOH to obtain a residue (12.5 g). The residue was
dispersed in H2O and successively partitioned with AcOEt and BuOH. The concentrated
AcOEt solution (2.5 g) was subjected to a RP-18 gel column chromatography eluting
with MeOH/H2O (1:4) to afford five fractions (Fr. F1 – F5). Fr. F5 (1.1 g) was
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separated on a silica gel column with petroleum ether PE/CH2Cl2 (4:1) as eluent to
obtain 10 (5.0 mg), 2 (4.4 mg), and 16 (286 mg). Fr. F4 (150 mg) was subjected to a
silica gel column eluting with CH2Cl2/MeOH (3:1, 4 ml/min) to afford 20 (16 mg), 3
(1.5 mg), 11 (2 mg), and 14 (10 mg). Fr. F3 (90 mg) was followed by the same
separation protocol as for Fr. F1 to be separated by a silica gel column eluting with
PE/AcOEt (4:1, 2 ml/min) to obtain 18 (1.5 mg), 19 (2 mg), 9 (1.8 mg), 13 (1.5 mg), 12
(8.0 mg), and 17 (3 mg). Fr. F2 (76 mg) was separated on semi-preparative HPLC (YMC
column) using MeOH/H2O (4:1, 2 ml/min) as the mobile phase to yield 5/6 (4 mg, tR 35.5
min), 15 (6 mg, tR 35.8 min), 4 (1.5 mg, tR 25.5 min), 1 (1.5 mg, tR 22.4 min), and 7/8 (2
mg, tR 27.2 min).
Aragusterol J (= (5S,6R,8R,9S,10R,12R,13S,14S,17R)-6,12-Dihydroxy-17-
{(3R,5R)-3-hydroxy-5-[(1R,2R)-2-methylcyclopropyl]hex-1-en-2-yl}-10,13-
20
dimethylhexadecahydro-3H-cyclopenta[a]phenanthren-3-one; 1). White powder. [α]D
=
−12.0 (c = 0.05, CH2Cl2). IR (KBr): 3358, 2924, 2856, 1716, 1459, 1375, 1247. 1H- and 13C-
+
NMR data: see Tables 1 and 2, resp. HR-ESI-MS: 441.3368 ([M − H2O + H]+, C29H45O3 ;
+
calc. 441.3369). HR-ESI-MS: 459.3472 ([M + H]+, C29H47O4 ; calc. 459.3474). HR-ESI-MS:
+
481.3297 ([M + Na]+, C29H46NaO4 ; calc. 481.3294).
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Aragusterol K (= (3S,4aR,5aS,6aS,6bS,9R,9aR,11aS,11bR)-9a,11b-Dimethyl-9-
{(2R,5R)-5-[(1R,2R)-2-methylcyclopropyl]hexan-2-
yl}hexadecahydrocyclopenta[1,2]phenanthro[8a,9-b]oxiren-3-yl Acetate; 2). White
powder. [α]D20 = −51.0 (c = 0.87, CH2Cl2). IR (KBr): 2944, 2868, 1734, 1451, 1371, 1247,
1034. 1H- and 13C-NMR: see Tables 1 and 2, resp. HR-ESI-MS: 471.3830 ([M + H]+,
+
C31H51O3 ; calc. 471.3838).
Aragusterol L (= (3S,4aS,5aR,6aS,6bS,9R,9aR,11aS,11bR)-9a,11b-Dimethyl-9-
{(2R,5R)-5-[(1R,2R)-2-methylcyclopropyl]hexan-2-
20
yl}hexadecahydrocyclopenta[1,2]phenanthro[8a,9-b]oxiren-3-ol; 3). White powder. [α]D
= −4.8 (c = 0.46, CH2Cl2). IR (KBr): 3398, 2926, 2863, 1456, 1370, 1248, 1022. 1H- and 13C-
NMR: see Tables 1 and 2. HR-ESI-MS: 411.3624 ([M − H2O + H]+, C29H47O+; calc.
+
411.3627). HR-ESI-MS: 429.3739 ([M + H]+, C29H49O2 ; calc. 429.3727).
(5α,7α,12β,22E)-7,12,18-Trihydroxystigmast-22-en-3-one (4). White powder.
[α]D20 = +24.7 (c = 0.48, MeOH). IR (KBr): 3234, 2944, 2922, 2854, 1712, 1447, 1370, 1270.
1H- and 13C-NMR: see Tables 1 and 2, resp. HR-ESI-MS: 443.3518 ([M − H2O + H]+,
+
+
C29H47O3 ; calc. 443.3525). HR-ESI-MS: 461.3629 ([M + H]+, C29H49O4 ; calc. 461.3631).
(5α,7α,12β,24R)- and (5α,7α,12β,24S)-7,12,20-Trihydroxystigmastan-3-one (5/6).
White powder. [α]D20 = +7.9 (c = 0.28, MeOH). IR (KBr): 3306, 2924, 2856, 1737, 1717,
1450, 1369, 1221, 1021. 1H- and 13C-NMR: see Tables 1 and 2, resp. HR-ESI-MS: 507.3687
−
([M + HCOO]−, C30H51O6 ; calc. 507.3686).
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(5α,7α,12β,22E,24R)- and (5α,7α,12β,22E,24S)-7,12,20-Trihydroxyergost-22-en-
3-one (7/8). White powder. [α]D20 = +32.0 (c = 0.15, MeOH). IR (KBr): 3278, 2924, 2869,
1712, 1448, 1369, 1226, 1019. 1H- and 13C-NMR: see Tables 1 and 2, resp. HR-ESI-MS:
+
429.3355 ([M − HO]+, C28H45O3 ; calc. 429.3363). HR-ESI-MS: 447.3474 ([M + H]+,
+
C28H47O4 , calc. 447.3469).
3α-Aragusterol I (9). White powder. [α]D20 = +46.2 (c = 0.39, CH2Cl2).
Petrosteryl Acetate (10). White powder. [α]D20 = −37.5 (c = 0.22, CH2Cl2).
Chemical Transformation of
2 to 12. To a stirred solution of 2 (1 mg) in 1 ml MeOH,
0.5 mg NaOH was added. The mixture was stirred at r.t. for 0.5 h and then evaporated.
The residue was subjected to Sephadex LH-20 using EtOH as eluent to afford a
1
product, which was identified to 12 by the comparison of the H-NMR data, Rf, and
[α]
25 values with those reported in literature.
D
Acetylation of 16 to 10. Acetic anhydride (200 μl) was added to a stirred solution of
compound 16 (1 mg) in freshly distilled pyridine (0.5 ml). The reaction was stirred at r.t. for
12 h and quenched by adding 0.1 ml of H2O. After removal of solvent under vacuum, the
residue was purified on a flash silica gel column eluting with CH2Cl2 to afford 10.
Epoxidation of 16
To a stirred solution of 16 (10 mg, 0.02 mmol) in CH2Cl2 (1 ml) at 0°, 3-chloroperbenzoic
acid (10 mg) was added. The mixture was stirred at r.t. for 2 h and then evaporated. The
mixture was subject to a silica gel CC (CH2Cl2/MeOH, 100:1, 2 ml/min) to afford 3 (1 mg)
and 12 (5 mg).
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Chemical Transformation of 14
To a stirred solution of 12 (3 mg) in MeOH (0.5 ml), NaBH4 (2 mg) was added and was
stirred for 15 min at r.t. The mixture was stirred sequentially at r.t. for 20 min and then
purified on Sephadex LH-20 using EtOH as eluent to afford two products (8:1). The mixture
was further purified using a slica gel CC (CH2Cl2/MeOH, 80:1) to obtain 9 (1.5 mg) and 13
(0.4 mg).
Cytotoxicity Assays
The cytotoxicity against human leukemia cell line K562 cell lines was evaluated using the
MTT (= 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide) method [21].
Adriamycin was used as a positive control.
X-Ray Crystal Data of 16
Colorless crystals of 16 were obtained from PE/AcOEt (1:1) using the vapor diffusion
method. A colourless crystal (0.65 × 0.65 × 0.08) was used for X-ray diffraction on an Agilent
Gemini E single crystal X-ray diffractometer with graphite monochromated CuKα radiation at
103.5 K. Crystal Data: C29H48O, M = 412.67, monoclinic, a = 11.6546(3) Å, b = 5.99382(15)
Å, c = 18.3691(6) Å, β = 96.828(3)°, U = 1274.08(6) Å3, T = 103.5, space group P21 (no. 4),
Z = 2, μ (CuKα ) = 0.462, 8054 reflections measured, 4782 unique (Rint = 0.0270) which were
used in all calculations. The final wR(F2) was 0.1157 (all data). Flack parameters = −0.2 (3).
The Crystallographic data for 16 have been deposited in Cambridge Crystallographic Data
Center [deposition number: CCDC 1417552]. Copy of the data can be obtained free of charge
from the CCDC via www.ccdc.cam.ac.uk.
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Received January 23, 2016
Accepted Febuary 26, 2016
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Table 1. 1H-NMR Data (400 MHz, in CDCl3) of 1 − 8. δ in ppm, J in Hz.
Position
1
2
3
4
5/6
7/8
1
2
1.93 – 1.87 (m),
1.34 – 1.30 (m)
2.41 – 2.37 (m),
2.34 – 2.30 (m)
1.69 – 1.65 (m),
1.42 – 1.39 (m)
1.97 – 1.95 (m),
1.26 – 1.23 (m)
2.06 – 2.02 (m),
1.54 – 1.50 (m)
2.04 – 2.01 (m),
1.44 – 1.40 (m)
2.04 – 2.00 (m),
1.41 – 2.38 (m)
1.69 – 1.63 (m),
1.42 – 1.38 (m)
1.81 – 1.78 (m),
1.45 – 1.42 (m)
2.61 (dt, J = 12.0,4.0),
2.26 – 2.22 (m)
2.50 (dt, J = 12.0,4.0),
2.25 – 2.20 (m)
2.49 (dt, J = 12.0,4.0),
2.25 – 2.21 (m)
3
4
4.95 – 4.90 (m)
3.75 – 3.70 (m)
2.80 (t, J = 12),
2.16 (t, J = 12.0),
1.32 (dd, J = 12.0, 4.0)
2.19 (t, J = 12.0),
1.43 (dd, J = 12.0, 4.0)
2.36 (t, J = 12.0),
1.95 (dd, J = 12.0, 4.0)
2.02 – 1.98 (m),
1.57 – 1.53 (m),
1.46 (m)
2.36 (t, J = 12.0),
1.95 (dd, J = 12.0, 2.0)
2.04 – 2.00 (m)
2.35 (t, J = 12.0),
1.95 (dd, J = 12.0, 2.0)
2.04 – 2.00 (m)
2.12 (dd, J = 12.0, 2.0)
1.57 – 1.50 (m)
5
6
3.79 (td, J = 2.0, 4.0)
2.89 (d, J = 4.4)
3.06 (d, J = 2.3)
1.59 – 1.55 (m),
1.48 – 1.45 (m)
1.57 – 1.54 (m),
1.47 – 1.44 (m)
7
1.87 – 1.82 (m),
1.18 – 1.12 (m)
1.81 – 1.75 (m)
0.92 – 0.85 (m)
1.72 – 1.68 (m),
1.46 – 1.40 (m)
3.52 (dd, J = 11.1, 4.5)
1.91 – 1.87 (m),
1.49 – 1.45 (m)
1.35 – 1.31 (m)
1.30 – 1.25 (m)
1.37 – 1.32 (m),
1.27 – 1.23 (m)
1.95 – 1.92 (m),
1.12 – 1.08 (m)
0.98 – 0.95 (m)
1.56 – 1.52 (m),
2.08 – 2.05 (m),
1.40 – 1.35 (m)
1.48 – 1.44 (m)
0.61 – 0.58 (m)
1.38 – 1.34 (m),
1.26 – 1.24 (m)
1.97 – 1.94 (m),
1.06 – 1.03 (m)
0.87 – 0.83 (m)
1.58 – 1.54 (m),
3.79 (td, J = 3.0, 4.0)
3.82 – 3.80 (m)
3.81 – 3.79 (m)
8
1.40 – 1.37 (m)
1.42 – 1.39 (m)
1. 42 – 1.38 (m)
1.42 – 1.38 (m)
9
1.45 – 1.41 (m)
1.43 – 1.40 (m)
11
1.85 – 1.82 (m),
1.70 – 1.66 (m)
1.72 – 1.68 (m),
1.36 – 1.33 (m)
1.73 – 1.69 (m),
1.33 – 1.30 (m)
12
3.45 (dd, J = 11.2, 4.4)
3.33 (dd, J = 11.2, 4.4)
3.31 (dd, J = 11.2, 4.4)
14
15
1.21 – 1.17 (m)
1.72 – 1.67 (m),
1.40 – 1.36 (m)
1.78 – 1.75 (m),
1.43 – 1.40 (m)
1.86 – 1.82 (m),
1.42 – 1.39 (m)
1.85 – 1.81 (m),
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1.41 – 1.38 (m)
1.91 – 1.85 (m),
1.65 – 1.60 (m)
2.37 – 2.30 (m)
0.81 (s)
0.98 – 0.94 (m)
1.83 – 1.80 (m),
1.22 – 1.18 (m)
1.07 – 1.04 (m)
0.61 (s)
1.07 – 1.03 (m)
1.83 – 1.80 (m),
1.26 – 1.22 (m)
1.07 – 1.03 (m)
0.64 (s)
1.10 – 1.08 (m)
1.63 – 1.60 (m),
1.47 – 1.43 (m)
1.62 – 1.58 (m)
3.97 (d, J = 11.3),
3.74 (d, J = 11.3)
1.09 (s)
1.20 – 1.17 (m)
1.78 – 1.74 (m),
1.55 – 1.51 (m)
1.69 – 1.65 (m)
0.85 (s)
1.25 – 1.22 (m)
1.78 – 1.75 (m),
1.62 – 1.59 (m)
1.71 – 1.67 (m)
0.73 (s)
16
17
18
19
20
21
22
1.23 (s)
1.07 (s)
1.00 (s)
1.07 (s)
1.05 (s)
1.32 – 1.29 (m)
0 .89 (s)
1.34 – 1.30 (m)
0.90 (d, J = 6.5)
1.46 – 1.42 (m),
1.00 – 0.95 (m)
1.29 – 1.25 (m),
1.26 – 1.23 (m)
0.60 – 0.56 (m)
0.13 – 0.10 (m)
0.08 – 0.04 (m),
0.14 – 0.11 (m)
0.45 – 0.42 (m)
0.88 (d, J = 6.5)
2.89 – 2.84 (m)
1.19 (d, J = 6.8)
5.52 (dd, J = 15.4, 8.2)
4.96 (s), 5.13 (s)
1.13 (s)
1.21 (s)
4.36 (dd, J = 8.0, 4.4)
1.46 – 1.42 (m),
0.98 – 0.95 (m)
1.33 – 1.30 (m),
1.23 – 1.20 (m)
0.59 – 0.54 (m)
0.13 – 0.10 (m)
0.09 – 0.05 (m),
0.15 – 0.12 (m)
0.44 – 0.41 (m)
0.88 (d, J = 6.7)
1.61 – 1.57 (m),
1.46 – 1.42 (m)
1.57 – 1.53 (m)
5.70 (d, J = 15.8)
23
1.73 – 1.65 (m),
1.42 – 1.38 (m)
0.83 – 0.78 (m)
0.23 – 0.21 (m)
0.15 – 0.13 (m),
0.23 – 0.20 (m)
0.57 – 0.52 (m)
0.95 (d, J = 6.7)
5.14 (dd, J = 15.4, 9.0)
5.56 (dd, J = 15.8, 7.7)
24
25
26
1.62 – 1.58 (m)
1.57 – 1.52 (m)
0.83 (d, J = 6.6)
0.98 – 0.95 (m)
1.70 – 1.65 (m)
0.88 (d, J = 6.6)
2.0 – 1.97 (m)
1.58 – 1.54 (m)
0.89 (d, J = 6.8)
27
28
0.89 (d, J = 6.6)
1.46 – 1.43 (m),
1.25 – 1.21 (m)
0.86 (t, J = 7.4)
0.88 (d, J = 6.6)
1.38 – 1.35 (m),
1.25 – 1.21 (m)
0.89 (t, J = 7.0)
0.89 (d, J = 6.8)
0.97 (d, J = 6.8)
29
1.03 (d, J = 6.0)
0.99 (d, J = 5.9)
2.00 (s)
1.01 (d, J = 6.0)
AcO
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Table 2. 13C-NMR Data (100 MHz, in CDCl3) of 1 − 8. δ in ppm.
Position
1
2
3
4
5/6
39.6
7/8
39.5
1
39.7
38.1
212.2
41.9
48.8
70.6
39.4
29.9
52.9
35.7
30.0
78.5
49.3
54.6
24.2
32.0
47.9
8.3
32.1
27.2
71.4
36.1
65.2
59.2
28.8
29.9
42.5
35.0
20.6
39.4
42.3
56.8
24.1
28.1
55.9
11.9
37.2
31.1
69.4
42.2
62.9
63.7
32.6
29.8
51.4
34.9
22.0
39.9
42.3
56.3
24.2
28.2
56.2
11.8
39.5
38.9
214.4
45.0
40.6
37.9
67.6
39.1
45.8
36.8
33.4
82.1
50.2
50.0
24.4
26.9
58.4
61.7
2
38.9
214.4
45.0
38.9
214.4
45.0
3
4
5
40.6
40.6
6
38.0
37.9
7
67.7
67.7
8
39.3
39.3
9
45.5
45.4
10
11
12
13
14
15
16
17
18
36.8
36.8
29.8
29.9
78.7
78.6
48.9
49.5
50.1
49.9
23.8
23.9/23.7
25.5
26.0
66.0/66.1
10.0
64.3
9.98/10.0
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19
14.8
151.1
113.8
76.9
45.0
34.9
27.3
11.7
12.9
20.1
19.1
15.9
35.9
18.6
33.5
34.0
38.7
27.4
12.8
11.6
19.8
19.1
21.3
170.2
17.0
35.9
18.7
33.4
33.9
38.7
27.4
11.6
12.8
19.8
19.1
10.6
39.2
23.7
138.6
131.4
53.0
33.1
19.4
21.5
26.7
12.8
10.6
10.6
20
75.7/75.8
27.9/27.9
35.6/35.7
25.1/25.2
47.7/47.8
30.6/30.7
19.79/19.83
19.8/20.0
24.3
75.1
21
31.2
22
134.9/135.1
132.6/132.3
44.1/44.8
34.5/34.6
20.2/20.3
20.4/20.7
17.6/17.9
23
24
25
26
27
28
29
12.5/12.7
3-AcO
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Table 3. Inhibitory effects of compounds against human tumor cells K562.
Inhibition ([%], 10 μM)
Compound
IC50 [μM]
34.31
18.32
> 50
50.28
65.24
25.73
41.32
46.43
33.47
38.11
48.73
53.21
1
4
5/6
7/8
9
> 50
> 50
> 50
11
13
14
17
> 50
> 50
24.19
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Captions:
Fig. 1. Structures of sterols isolated from Xestospongia sp.
Fig. 2. Key NOE correlations of 1 – 4.
Fig. 3. Alkaline conversion of 2 to 12.
Fig. 4. X-ray structure of 16.
Fig. 5. Epoxidation of 16 to 3 and 12.
Fig. 6. Chemical conversion of 14 to 9 and 13.
Fig. 7. Acetylation 16 to 10.
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