Protein phosphatase inhibitors isolated from Spongia irregularis collected in Papua New Guinea

Article abstract of DOI:10.1021/np0702887

Irregularasulfate (1), a new nitrogen-containing sesterterpenoid, and the known sesterterpenoids hipposulfate C (2), halisulfate-7 (3), and igernellin (4), have been isolated from the marine sponge Spongia irregularis collected in Papua New Guinea. The st

Full text of DOI:10.1021/np0702887

1812
J. Nat. Prod. 2007, 70, 1812–1815
Protein Phosphatase Inhibitors Isolated from Spongia irregularis Collected in Papua New
Guinea
Gavin Carr,† Mikolaj Raszek,§ Rob Van Soest, Teatulohi Matainaho,‡ Micheal Shopik,§ Charles F. B. Holmes,§ and
Raymond J. Andersen*,†
Departments of Chemistry and Earth & Ocean Sciences, UniVersity of British Columbia, VancouVer, B. C., Canada V6T 1Z1, Canadian
Institutes of Health Research, Group in Protein Structure and Function, Department of Biochemistry, Faculty of Medicine, UniVersity of
Alberta, Edmonton, Alta, Canada T6G 2H7, Institute for Systematics and Ecology, UniVersity of Amsterdam, 1090 GT Amsterdam,
The Netherlands, and Discipline of Pharmacology, UniVersity of Papua New Guinea, N.C.D., Papua New Guinea
ReceiVed June 15, 2007
Irregularasulfate (1), a new nitrogen-containing sesterterpenoid, and the known sesterterpenoids hipposulfate C (2),
halisulfate-7 (3), and igernellin (4), have been isolated from the marine sponge Spongia irregularis collected in Papua
New Guinea. The structure of 1 was elucidated via analysis of its spectroscopic data. Sesterterpenoids 1, 2, and 3 are
moderate inhibitors of the catalytic subunits of the mammalian Ser/Thr protein phosphatases calcineurin, PP-1, and
PP-2A. The phosphate analogue of 3 and the thiophosphate analogue of 2 have been prepared from the corresponding
natural products and evaluated for their ability to inhibit the phosphatase activity of calcineurin.
Calcineurin, a serine/threonine protein phosphatase, is the indirect
cellular target of the two important immunosuppressive natural
product drugs cyclosporine and FK506 (tacrolimus).1 When recep-
tors on the surface of T cells encounter antigens from foreign tissue
associated with organ transplants, a calcium-dependent signal trans-
duction cascade is activated. As part of this process, calcium levels
in the cell increase due to stimulated release of calcium from
intracellular stores in the endoplasmic reticulum and an influx of
extracellular calcium through channels in the plasma membrane.
The increased intracellular calcium binds to calmodulin, and the
calcium/calmodulin complex in turn binds to calcineurin and
activates its phosphatase activity against the principal substrate,
multiply phosphorylated “nuclear factor of activated T cell” (NFAT)
protein in the cytoplasm.2 Once dephosphorylated, NFAT is trans-
located to the nucleus, where it enhances binding of transcription
factors to genes encoding for cytokines such as interleukin 2 (IL-
2). The upregulation of production of IL-2 and other cytokines is
an important component of T cell activation and the immune
response. Cyclosporin and FK506 inhibit calcineurin activity and,
therefore, the immune response to foreign tissue in an indirect
fashion. Cyclosporin binds to its direct molecular target cyclophilin,
and the cyclosporine/cyclophilin complex then binds to calcineurin
and inhibits its phosphatase activity. In a similar manner, FK506
binds to the 12 KDa FK506 binding protein (FKBP-12), and again
this complex binds to calcineurin and inhibits its enzymatic activity.
Even though cyclosporine and FK506 are important drugs that have
revolutionized organ transplantation, they are not without side
effects.3,4 These include nephrotoxicity (kidney), hypertension,
neurotoxicity, and diabetogenesis. Despite the obvious clinical
importance of calcineurin inhibitors, there are few known potent
and selective active-site inhibitors of this phosphatase.5,6 This can
be contrasted with the existence of okadaic acid,7 motuporin,8
spirastrellolide A,9 and several other highly potent natural product
inhibitors of the serine/threonine protein phosphatases PP-1 and
PP-2A.
substrate inhibitor-1, was used to screen a library of marine
invertebrate and microbial extracts, resulting in the discovery of
several hits, including the MeOH extract from the marine sponge
Spongia irregularis. Bioassay-guided fractionation of the S. ir-
regularis extract led to the isolation of the new sesterterpenoid
alkaloid irregularasulfate (1) and the known metabolites hipposulfate
C (2)11 and halisulfate-7 (3),12 which all showed moderate cal-
cineurin inhibition, along with igernellin (4),13 which was inactive.
The details of the isolation and structure elucidation of irregulara-
sulfate (1), some SAR-driven chemical modifications of 3 and 4,
and the phosphatase inhibition activities of 1, 2, 3, and their
transformation products are presented below.
Specimens of S. irregularis (670 g wet wt) were collected by
hand using scuba near Keviang, Papua New Guinea, frozen on site,
and transported frozen to Vancouver. Thawed sponge tissue was
exhaustively extracted with MeOH, which was filtered and con-
centrated in Vacuo to give the crude extract. This extract was
partitioned between H2O and CH2Cl2, and the bioactive CH2Cl2-
soluble portion was first fractionated on Si gel flash chromatography
(step gradient elution: hexanes to EtOAc to MeOH). The active
fraction, eluting with 10% MeOH in EtOAc, was subjected to
reversed-phase flash chromatography (step gradient elution: H2O
to MeOH) to give a calcineurin inhibitory fraction (7:3 MeOH/
H2O) that was subjected to reversed-phase HPLC (7:3 MeOH/H2O),
yielding irregularasulfate (1, 1.6 mg) and the known compounds
hipposulfate C (2, 20 mg)11 and halisulfate-7 (3, 63 mg).12 Igernellin
(4)13 was isolated from an inactive fraction by Si gel flash chro-
matography. The known compounds 2, 3, and 4 were identified by
comparison of their spectroscopic data with the literature values.
Recently, an assay suitable for detecting direct inhibitors of
calcineurin has been developed in one of our laboratories.10 This
assay, based on the singly phosphorylated physiological calcineurin
* To whom correspondence should be addressed. Tel: 604 822 4511.
Fax: 604 822 6091. E-mail: randersn@interchange.ubc.ca.
† Chemistry and EOS, University of British Columbia.
§ Biochemistry, University of Alberta.
University of Amsterdam.
‡ University of Papua New Guinea.
10.1021/np0702887 CCC: $37.00
2007 American Chemical Society and American Society of Pharmacognosy
Published on Web 11/03/2007

Notes
Journal of Natural Products, 2007, Vol. 70, No. 11 1813
Table 1. 1H and 13C NMR Data for Irregularasulfate (1) and Halisulfate-7 (3) Recorded in CDCl3 at 600 MHz
irregularasulfate (1)
halisulfate 7 (3)
mult.
position
δC
mult.
δH (J in Hz)
δC
δH (J in Hz)
1
2
3
4
5
6
7
8
117.0
23.5
31.5
31.5
43.7
30.4
31.5
44.9
42.9
146.4
28.7
25.0
38.7
31.0
24.9
26.6
140.6
134.5
50.9
27.9
28.2
16.8
23.5
70.9
CH
CH2
CH2
C
CH
CH2
CH2
CH
C
5.28 br, s
1.98, m; 2.02, m
1.06, m; 1.36, m
117.1
23.4
31.5
31.5
43.8
31.0
31.5
44.9
42.8
146.3
28.6
24.6
38.7
30.4
26.9
25.1
125.2
111.3
142.8
27.9
28.2
16.7
23.4
72.3
CH
CH2
CH2
C
CH
CH2
CH2
CH
C
5.29 br, s
1.97, m; 2.02, m
1.05, m; 1.34, m
1.51, m
1.50, m
1.06, m; 1.78, m
1.43, m; 1.50, m
1.23, m
1.06, m; 1.77, m
1.43, m; 1.50, m
1.23, m
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
C
C
CH2
CH2
CH
CH2
CH2
CH2
C
CH
CH2
CH3
CH3
CH3
CH3
CH2
1.56, m
1.05, m
1.53, m
1.25, m
1.49, m
2.23, m
CH2
CH2
CH
CH2
CH2
CH2
C
CH
CH
CH3
CH3
CH3
CH3
CH2
1.55, m
1.05, m
1.51, m
1.24, m
1.50, m
2.36, t (7.5)
6.62, s
3.80, s
0.83, s
0.84, s
0.81, d (7.0)
0.95, s
3.87, t (8.6)
3.97, dd (9.3, 3.7)
6.23 br, s
7.32 br, s
0.83, s
0.84, s
0.82, d (7.2)
0.95, s
3.86, t (8.3)
3.95, dd (9.1, 4.7)
7.20, s
25
26
27
28
29
30
172.0
40.9
37.6
26.0
22.8
22.8
C
139.1
CH
CH2
CH2
CH
CH3
CH3
3.43, t (7.5)
1.41, m
1.54, m
0.92, d (6.6)
0.92, d (6.6)
Irregularasulfate (1) gave a [M - H]- ion at m/z 536.3407 in
the negative ion HRESIMS appropriate for a molecular formula of
C30H51NO5S (calcd for C30H50NO5S, 536.3410), requiring six sites
of unsaturation. It was possible to identify 30 carbon resonances
(5 × C, 6 × CH, 13 × CH2, 6 × CH3) and 50 protons attached to
carbon from the 1D 13C NMR, DEPT, and HSQC data (Table 1)
in agreement with the HRESIMS measurement. Analysis of the
COSY, HSQC, and HMBC data obtained for 1 and comparison
with the same data set obtained for halisulfate-7 (3) showed that
assigned to the methylene carbon terminus (C-26) of the isopentyl
fragment, showed HMBC correlations to the lactam carbonyl (δ
172.0; C-25) and γ-methylene carbon (δ 50.9; C-19) resonances,
demonstrating that the isopentyl moiety was attached to the lactam
nitrogen. The near identity of the carbon and proton chemical shifts
assigned to the decalin ring systems in 1 and 3 showed that the
relative configurations in 1 at C-5, C-8, and C-9 were identical to
those in 3, as shown.12b,14 The relative configuration at C-13 has
not been assigned in either 1 or 3.
Irregularasulfate (1) inhibited calcineurin in Vitro with an IC50
of 59 µM, while hipposulfate C (2) and halisulfate 7 (3) showed
calcineurin inhibition with IC50’s of 66 and 69 µM, respectively.
To test for selectivity, hipposulfate C (2) and halisulfate-7 (3) were
also tested against pure preparations of the catalytic subunits of
protein phosphatases PP-1 and PP-2A.5 They both showed similar
potency against PP-1 (IC50’s: 2 71 µM; 3 64 µM) as they did against
calcineurin, but were less active against PP-2A (IC50’s: 2 130 µM;
3 140 µM). It seemed possible that the sulfated C-24 hydroxymethyl
fragment in 1, 2, and 3 mimicked a phosphorylated serine residue
in the natural substrates for calcineurin, PP-1, and PP-2A. In order
to explore this further, phosphate and thiophosphate analogues of
the major S. irregularis metabolites hipposulfate C (2) and hal-
isulfate-7 (3) were prepared in an attempt to increase their potency
as calcineurin inhibitors.
Halisulfate-7 (3) was first desulfated by treatment with refluxing
HCl in MeOH to give the alcohol 5 (Scheme 1). Reaction of alcohol
5 with dibenzyl phosphite, CBrCl3, and diisopropylethylamine gave
the dibenzyl phosphate derivative of 5.15 Removal of the benzyl
protecting groups by reaction with bromotrimethylsilane gave hali-
phosphate-7 (6). Igernellin (4) was reacted with bis(2-cyanoethyl)-
N,N-diisopropylphosphoramidite and tetrazole overnight at room
temperature followed by addition of sulfur to give the bis(2-cyanoet-
hyl)-protected thiophosphate 7.16 Removal of the protecting groups
by treatment of 7 with KOH in MeOH gave the thiophosphate 8.
As anticipated, the desulfated analogue 5 of halisulfate-7 (3),
like igernellin (4), was completely inactive as a calcineurin inhibitor.
1
the two molecules were closely related. In particular, the H and
13C NMR resonances assigned to the C-1 to C-16 fragment
including the attached C-20, C-21, C-22, C-23 methyl, and C-24
sulfated methyl branches in 3 (Table 1) and their COSY, HSQC,
and HMBC correlations were nearly identical to the data for 1,
indicating that 1 and 3 contained identical C-1 to C-16 diterpenoid
fragments.
1
The H-16 resonance at δ 2.23 in the H NMR spectrum of 1
showed HMBC correlations to carbon resonances at δ 134.5 (C-
18), 140.6 (C-17), and 172.0 (C-25), suggesting the attachment of
C-16 to the R-carbon (C-17) of an R,ꢀ-unsaturated ester or amide.
In agreement with this assignment, the olefinic resonance at δ 6.62
(H-18), which was correlated in the HSQC spectrum to δ 134.5
(C-18), showed HMBC correlations to carbon resonances at δ 172.0
(C-25), 140.6 (C-17), and 26.6 (C-16). The H-18 resonance (δ 6.62)
was correlated in the COSY spectrum to a methylene resonance at
δ 3.80 (H-19), which was in turn correlated to a carbon resonance
at δ 50.9 (C-19) in the HSQC spectrum. The chemical shift of C-19
indicated that it was attached to nitrogen, and HMBC correlations
from the H-19 resonance (δ 3.80) to the carbonyl resonance at δ
172.0 (C-25) showed that the nitrogen atom and carbonyl carbon
were joined to form an R,ꢀ-unsaturated γ-lactam, accounting for
the last site of unsaturation required by the molecular formula
of 1.
COSY and HMBC correlations routinely identified an isopentyl
fragment (C-26-C-30) that accounted for the remaining atoms in
1 (Table 1). A proton resonance at δ 3.43 (t, J ) 7.5 Hz; H-26),

1814 Journal of Natural Products, 2007, Vol. 70, No. 11
Notes
Scheme 1
equipped with a Waters 2487 dual λ absorbance detector and a CSC-
Inertsil 150A/ODS2 column was used for HPLC.
The phosphate analogue 6 (IC50 230 µM) was significantly less
active than the sulfated natural product 3 (IC50 66 µM) against
calcineurin but was more active against PP-1c (IC50 36 µM). One
possible explanation for the lower potency of the phosphate
analogue 6 against calcineurin was that it was acting as a substrate
and was being converted to the inactive alcohol 5 during the assay.
This prompted the preparation of the thiophosphate 8, which should
not be as good a substrate for calcineurin. The thiophosphate 8
had an IC50 of 70 µM against calcineurin, which was comparable
to the IC50 for the corresponding sulfate 2.
In summary, the new sesterterpenoid alkaloid irregularasulfate
(1) and the known analogues hipposulfate C (2) and halisulfate-7
(3) have been isolated as the phosphatase inhibitory components
of the crude MeOH extract from S. irregularis collected in Papua
New Guinea. Chemical transformation of the natural product 3 to
the alcohol 5 and the lack of activity observed for igernellin (4)
showed that the sulfate functionality in the natural products is
essential for phosphatase inhibition. The phosphate analogue 6 of
halisulfate-7 was less active than the natural sulfate 3, while the
thiophosphate analogue 8 was comparable in activity to the natural
product 2 against calcineurin. The phosphate and thiophosphate
analogues of 2 and 3, which might more closely resemble the natural
substrates of calcineurin, were no better inhibitors than the natural
sulfates. However, the phosphate analogue 6 of halisulfate-7 (3)
(6: IC50 36 µM) was slightly more active against PP-1c than the
natural product (3: IC50 64 µM), suggesting that it might be a
substrate for calcineurin but not PP-1c. This is the first report of
sulfated natural products as active site directed protein phosphatase
inhibitors. Observed alterations in the selectivity of the chemically
altered compounds toward either calcineurin, PP-1, and/or PP-2A
may facilitate rational design of selective inhibitors of these
phosphatases based on marine natural products as a starting
template.
Isolation of Sesterterpenoids. Specimens of S. irregularis (Le-
denfeld) (670 g) were collected by hand using scuba near Keviang,
Papua New Guinea (2°45.1′ S, 150°43′ E). A voucher sample has been
deposited at the University of Amsterdam (ZMAPOR18522). The
sponge was frozen on site and transported frozen to Vancouver. Thawed
specimens were extracted exhaustively with MeOH, and the combined
MeOH extracts were concentrated in Vacuo to give the crude extract.
The crude extract was partitioned between H2O and CH2Cl2, and the
CH2Cl2-soluble materials were subjected to Si gel chromatography (step
gradient: hexanes to EtOAc to MeOH). The active fraction, eluting
with 10% MeOH in EtOAc, was subjected to reversed-phase separation
on a 10 g Waters C18 Sep-pak (step gradient: H2O to MeOH). The
fraction eluting with 70% MeOH/H2O was subjected to isocratic
reversed-phase HPLC (eluent: 70% MeOH/H2O) to give irregulara-
sulfate (1: 1.6 mg), hipposulfate C (2: 20 mg), and halisulfate-7 (3: 63
mg). Igernellin (4) was isolated from an inactive fraction from the first
Si gel column by further isocratic flash Si gel chromatography (eluent:
10% EtOAc/hexanes).
1
Irregularasulfate (1): clear glass; [R]D +39.8 (c 0.4 MeOH); H
NMR and 13C NMR, see Table 1; HRESIMS(-) m/z 536.3407 (calcd
for C30H5oNO5S, 536.3410).
Synthetic Transformations. Alcohol 5. Halisulfate-7 (3) was
dissolved in 1 mL of MeOH, and 0.2 mL of 1 N HCl was added. The
solution was stirred and heated to reflux for 2 h. The reaction mixture
was cooled, and water (5 mL) was added before extraction with CH2Cl2
(3 × 5 mL). The combined CH2Cl2 layers were dried over MgSO4,
filtered, concentrated in Vacuo, and subjected to Si gel chromatography
1
(eluent: 10% EtOAc/hexanes) to give alcohol 5: H NMR (400 MHz,
CDCl3) δ 7.32 (br. s, H-19), 7.18 (s, H-25), 6.24 (br s, H-18), 5.29 (br
s, H-1), 3.50 (m, H2-24), 2.39 (t, J ) 5.0 Hz, H2-16), 1.98–2.01 (m,
H2-2), 1.77 (m, H-6a), 1.45–1.60 (m, H-5, H2-7, H2-11, H-13, H2-15),
1.20–1.40 (m, H-3b, H-8, H2-14), 1.00–1.10 (m, H-3a, H-6b, H2-12),
0.96 (s, H3-23), 0.83 (s, H3-20, H3-21), 0.81 (d, J ) 6.9 Hz, H3-22);
13C NMR (100 MHz, CDCl3) δ 146.4 (C, C-10), 142.9 (CH, C-19),
139.0 (CH, C-25), 125.3 (C, C-17), 117.1 (CH, C-1), 111.1 (CH, C-18),
66.1 (CH2, C-24), 44.9 (CH, C-8), 43.8 (CH, C-5), 42.8 (C, C-9), 41.5
(CH, C-13), 31.5 (CH2, C-3), 31.5 (C, C-4), 31.5 (CH2, C-7), 31.0 (CH2,
C-6), 30.4 (CH2, C-14), 28.3 (CH2, C-11), 28.2 (CH3, C-21), 27.9 (CH3,
C-20), 27.6 (CH2, C-15), 25.3 (CH2, C-16), 24.7 (CH2, C-12), 23.4
(CH2, C-2), 23.4 (CH3, C-23), 16.7 (CH3, C-22); HRESIMS(+) m/z
395.2937 (calcd for C25H40O2Na 395.2926).
Phosphate 6. To a flask containing alcohol 5 (19.0 mg) dissolved
in MeCN (5 mL), under N2, at -10 °C, were added CBrCl3 (0.1 mL)
and DIEA (0.1 mL), followed by dibenzyl phosphite (0.3 mL,
dropwise). The reaction was stirred at -10 °C for 1.5 h, at which point
0.5 M NaH2PO4 (5 mL) was added. The reaction mixture was then
extracted with CH2Cl2 (3 × 5 mL), and the combined CH2Cl2 layers
were dried over MgSO4, filtered, concentrated, and subjected to Si gel
chromatography (eluent: 5% EtOAc/hexanes) to give dibenzyl-protected
Experimental Section
General Experimental Procedures. Optical rotations were recorded
with a JASCO P-1010 polarimeter equipped with a halogen lamp (589
nm) and a 10 mm microcell. NMR spectra were recorded on Bruker
Avance 400 and Bruker Avance 600 (equipped with a cryoprobe)
spectrometers at 400 and 600 MHz, respectively, in CDCl3. Chemical
shifts are given in δ (ppm) with the residual CDCl3 solvent peak
referenced to δH 7.24 and δC 77.23 as the internal reference. ESIMS
spectra were obtained with Kratos MS-50, Micromass LCT, and Bruker
Esquire-LC mass spectrometers. Si gel (Silicycle, 230–400 mesh) and
Sephadex LH20 were used for column chromatography; precoated Si
gel plates (Merck, Kieselgel 60 F254, 0.25 mm and Whatman, MKC18F
60 A) were used for TLC analysis. A Waters 1500 Series pump system

Notes
Journal of Natural Products, 2007, Vol. 70, No. 11 1815
haliphosphate-7 (14.1 mg, 44%). To a flask containing dibenzyl-
protected haliphosphate-7 (2.6 mg) under N2 was added CH2Cl2 (1 mL),
and the solution was cooled to 0 °C. Bromotrimethylsilane (0.05 mL)
was added, and the reaction was stirred at 0 °C for 45 min before the
solvent and reagent were removed in Vacuo to give crude haliphos-
phate-7 (6). The crude phosphate 6 was dissolved in MeOH and purified
via Sephadex LH-20 chromatography (eluent: MeOH) to give pure 6
(1.4 mg, 75%): 1H NMR (600 MHz, CDCl3) δ 7.30 (br s, H-19), 7.18
(s, H-25), 6.22 (br s, H-18), 5.29 (br s, H-1), 3.91 (m, H2-24), 2.36 (m,
H2-16), 1.96–2.02 (m, H2-2), 1.76 (m, H-6a), 1.45–1.55 (m, H-5, H2-
7, H2-11, H-13, H2-15), 1.20–1.40 (m, H-3b, H-8, H2-14), 1.00–1.10
(m, H-3a, H-6b, H2-12), 0.94 (s, H3-23), 0.81 (s, H3-20, H3-21), 0.80
(d, J ) 6.9 Hz, H3-22); 13C NMR (150 MHz, CDCl3) δ 146.2 (C, C-10),
142.9 (CH, C-19), 139.0 (CH, C-25), 125.2 (C, C-17), 117.1 (CH, C-1),
111.2 (CH, C-18), 70.9 (CH2, C-24), 44.9 (CH, C-8), 43.8 (CH, C-5),
42.8 (C, C-9), 39.5 (CH, C-13), 31.5 (CH2, C-3), 31.5 (C, C-4), 31.5
(CH2, C-7), 30.8 (CH2, C-6), 30.4 (CH2, C-14), 28.2 (CH2, C-11), 28.1
(CH3, C-21), 27.8 (CH3, C-20), 27.2 (CH2, C-15), 25.1 (CH2, C-16),
24.3 (CH2, C-12), 23.4 (CH2, C-2), 23.4 (CH3, C-23), 16.7 (CH3, C-22);
HRESIMS(-) m/z 451.2615 (calcd for C25H40O5P 451.2613).
Acknowledgment. Financial support was provided by the Natural
Sciences and Engineering Research Council of Canada (R.J.A.) and
CIHR (C.H.).
1
Supporting Information Available: H and 13C NMR spectra for
irregularasulfate (1). This material is available free of charge via the
Internet at http://pubs.acs.org.
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Thiophosphate 8. Igernellin (4) (12 mg) was dissolved in CH2Cl2
(3 mL), and to this solution was added bis(2-cyanoethyl)-N,N-
diisopropylphosphoramidite (0.1 mL) and tetrazole (0.7 mL) (1 M) in
MeCN. The reaction mixture was stirred overnight at room temperature.
Sulfur (75 mg) was added, and the reaction was allowed to stir for a
further 4 h before being concentrated in Vacuo and subjected to Si gel
chromatography (eluent: step gradient hexanes to 25% EtOAc/hexanes)
to give 7 (9.5 mg, 57%). Protected thiophosphate 7 (4.9 mg) was
dissolved in 2 mL of 1 N KOH in MeOH, and the reaction was stirred
for 2 h at room temperature. The solution was concentrated in Vacuo
and purified on a Waters 10 g reversed-phase Sep pak (eluent: step
gradient H2O to MeOH). The fraction eluting with 90% MeOH gave
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1
pure thiophosphate 8 (4.6 mg, 100%): H NMR (400 MHz, CDCl3) δ
7.30 (br s, H-19), 7.26 (s, H-25), 6.21 (br s, H-18), 5.23 (br. s, H-1),
5.03 (br s, H-10), 3.74 (br s, H-24a), 3.65 (br s, H-24b), 2.34 (br s,
H2-16), 1.85–1.95 (m, H2-2, H-8, H2-11), 1.62 (s, H3-22), 1.55 (m,
H-13), 1.52 (s, H3-23), 1.20–1.50 (m, H-3a, H-5, H2-7, H2-12, H2-14,
H2-15), 1.07 (br d, J ) 12.9 Hz, H-3b), 0.87 (s, H3-20), 0.82 (s, H3-
21); 13C NMR (100 MHz, CDCl3) δ 142.9 (CH, C-19), 139.3 (CH,
C-25), 136.9 (C, C-6), 136.1 (C, C-9), 125.4 (C, C-17), 124.6 (CH,
C-10), 120.1 (CH, C-1), 111.5 (CH, C-18), 67.9 (CH2, C-24), 49.3 (CH,
C-5), 40.9 (CH2, C-8), 38.6 (CH, C-13), 32.8 (C, C-4), 31.9 (CH2, C-3),
31.0 (CH2, C-7), 30.4 (CH2, C-12), 30.1 (CH2, C-14), 27.7 (CH3, C-20),
27.7 (CH3, C-21), 26.7 (CH2, C-15), 25.4 (CH2, C-11), 25.1 (CH2, C-16),
23.7 (CH3, C-22), 23.3 (CH2, C-2), 16.3 (CH3, C-23); HRESIMS(-) m/z
467.2387 (calcd for C25H40O4PS 467.2385).
NP0702887