Total synthesis of a furostan saponin, timosaponin BII

Article abstract of DOI:10.1039/b905091d

The natural timosaponin BII, (25S)-26-O-β-D-glucopyranosyl-22-hydroxy- 5β-furostane-3β,26-diol-3-O-β-D-glucopyranosyl-(1→2) -β-D-galactopyranoside, isolated from the rhizomes of Anemarrhena asphodeloides Bunge (Liliaceae), has been efficiently synthesized

Full text of DOI:10.1039/b905091d

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www.rsc.org/obc | Organic & Biomolecular Chemistry
Total synthesis of a furostan saponin, timosaponin BII†
Shuihong Cheng,a,b Yuguo Du,*a,b Baiping Mac and Dawei Tanc
Received 12th March 2009, Accepted 15th May 2009
First published as an Advance Article on the web 15th June 2009
DOI: 10.1039/b905091d
The natural timosaponin BII, (25S)-26-O-b-D-glucopyranosyl-22-hydroxy-5b-furostane-3b,26-diol-3-
O-b-D-glucopyranosyl-(1→2)-b-D-galactopyranoside, isolated from the rhizomes of Anemarrhena
asphodeloides Bunge (Liliaceae), has been efficiently synthesized in ten steps and 18% overall yield. The
strategy of using a partially protected glycosyl donor was applied to facilitate target synthesis. The
cytotoxic activities of structurally related compounds were evaluated against HL-60 human
promyelocytic leukaemia cells.
D-glucopyranosyl-(1→2)-b-D-galactopyranoside based on 1H
Introduction
NMR and mass spectra.6 Timosaponin BII has been reported
as having useful pharmaceutical properties, including activities
against dementia and stroke, and abilities to lower blood sugar
levels, inhibit platelet aggregation and clear free radicals.7 The
relatively small amount of the compound from natural resources
limits the potential commercial development of the compound,
and also the screening potentials for other biological activities.
Here we report a practical route towards the total synthesis of
natural timosaponin BII starting from readily available aglycon
sarsasapogenin, which was extracted and purified via known
processes.8
It has been recognized that natural products are an excellent source
of chemical structures with a broad range of promising phar-
maceutical properties, including antitumor activity.1 Saponins,
a group of secondary metabolites present in a wide variety of
plants, have opened up a new field of investigation of potential
antitumor compounds, for example, OWS-1 and its analogues.2
However, little has been known regarding the action mechanisms
of saponins due to the poor accessibility to homogeneous saponins
of various structures in appreciable amounts.3 This situation has
encouraged the chemical synthesis of saponins,4 and enhances the
collaboration among chemists and pharmacologists. Collaborat-
ing with others on a project involving bioactive saponin screening,
we intend to find a suitable route towards the total synthesis of the
natural product timosaponin BII (Fig 1).
Results and discussion
Timosaponin BII contains a b-D-glucopyranose substitute at the
26-OH and a b-D-Glc-(1→2)-b-D-Gal disaccharide unit at the
3-OH of furostan aglycon. Initially, we intended to attach a
sugar unit on the 3-OH of sarsasapogenin 3 as the first step.
It was reported that coupling a C-2-branched oligosaccharide
to 3-OH of saponin aglycon would produce an inseparable
mixture of C-1 stereo-isomers.9 We have recently developed a
new methodology to overcome this problem, i.e., using partially
protected thioglycoside as a glycosyl donor to facilitate the
continuous C-2¢ glycosylation.10 Thus, condensation of isopropyl
3,6-di-O-benzoyl-1-thio-b-D-galactopyranoside10 (2, Scheme 1)
and 3 in dry CH2Cl2 in the presence of trimethylsilyl triflu-
oromethanesulfonate (TMSOTf) and N-iodosuccinimide (NIS)
at -42 ◦C obtained the desired saponin derivative 4 in an
Timosaponin BII, also called prototimosaponin AIII, was
isolated from the rhizomes of Anemarrhena asphodeloides Bunge
(Liliaceae) for the first time by Toshio Kawasaki and co-workers.5
Its chemical structure (Fig. 1) was elucidated as (25S)-26-O-
b-D-glucopyranosyl-22-hydroxy-5b-furostane-3b,26-diol-3-O-b-
aState Key Lab of Environmental Chemistry and Ecotoxicology, Research
Center for Eco-Environmental Sciences, Chinese Academy of Sciences,
Beijing, 100085, China
bCollege of Chemistry and Chemical Engineering, Graduate University of
Chinese Academy of Sciences, Beijing, 100049, China
cBeijing Institute of Radiation Medicine, Beijing, 100850, China
† Electronic supplementary information (ESI) available: 1H and 13C NMR
spectra for compounds 1, 4, 7, 10, 13, 14, 15, 17, 18, 20. See DOI:
10.1039/b905091d
Fig. 1 Chemical structure of timosaponin BII.
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Employing a similar procedure to that developed by Bovicelli
et al.,12 the 3-O-tert-butyldimethylsilyl ether of sarsasapogenin (3)
was oxidized with Oxone to afford an interconvertible mixture of
compounds 11 and 12 in 81% total yield. Treatment of the mixture
(11, 12) with glycosyl donor 5 in the presence of a catalytic amount
of TMSOTf in CH2Cl2 provided 26-O-glycosylated product 13 in
a yield of 80%. TLC detection clearly indicated the transformation
between 11 to 12 and 12 to 13. Desilylation13 of 13 with BF3·Et2O
in CH2Cl2 gave 14, which was subjected to glycosylation with 2
as described in the preparation of 4, affording latent acceptor
15 in an acceptable yield (62%). A second glycosylation between
15 and 6 under similar conditions to those employed in the
synthesis of 7, followed by acetylation with Ac2O in pyridine,
gained the key diketone intermediate 17 (75% for two steps). It has
been well documented that selective reduction of the 16-ketone
of the cholestan-16,22-dione with NaBH4 in i-PrOH provided
the corresponding furostan through a concurrent intramolecular
hemiketal formation.14 Applying the same chemistry, dione 17 was
successfully converted into hemiketal 18 in a moderate yield of
68%. Removal of all acyl protecting groups from 18 using NaOMe
in MeOH/CH2Cl2 afforded a furostan saponin mixture 19 after
column separation using MeOH/EtOAc as eluent. Refluxing of 19
in acetone/H2O (3/7, v/v) furnished target molecule 1 as the sole
product, which showed identical physical data to our authentic
sample.8
The cytotoxic activities of (25S)-26-O-b-D-glucopyranosyl-
22-hydroxy-5b-furostane-3b,26-diol-3-O-b-D-glucopyranosyl-
(1→2)-b-D-galactopyranoside (1), sarsasapogenin (3), sarsasa-
pogenyl b-D-glucopyranosyl-(1→2)-b-D-galactopyranoside (20,
obtained from deacylation of compounds
7 and/or 8
(Scheme 1), and diosgenyl a-L-rhamnopyranosyl-(1→2)-b-D-
galactopyranoside (21)15 were evaluated against HL-60 human
promyelocytic leukaemia cells (Table 1 and Fig. 2).16 The saponins
1 and 20 from this project exhibited weak cytotoxic activity with
IC50 values at 15.5 and 12.7 mg/mL, respectively. Aglycon 3
was not cytotoxic under our experimental conditions, while the
positive control CDDP (cis-diaminodichloroplatinum) presented
an IC50 value at 0.7 mg/mL. Compared to compound 20, structural
analogue 21 showed moderate activity under the same testing
conditions, suggesting that the a-L-rhamnopyranosyl residue
might play an important role in triggering cytotoxicity to the
tumor cells.17 In a parallel bioactivity screening, we also found
that compound 1, at a concentration of 10-5 M, could exert a
direct relaxation effect on vascular smooth muscle in a significant
dose-dependent way. More details will be published in due course.
Scheme 1 Attempted synthetic strategy: (a) NIS, TMSOTf, CH2Cl2,
-42 ◦C, 67%; (b) TMSOTf, CH2Cl2, -42 ◦C, no reaction using 5, while 65%
for 7 from 6; (c) Ac2O, Pyr, 93%; (d) oxone, NaHCO3, 1.0 mM Na2EDTA,
CH2Cl2, acetone.
acceptable yield (67%). A small amount of a-isomer might
also be generated, but the presence of inseparable contami-
nants did not permit us to rule out its formation. Taking
advantage of the reactivity between the 2-OH and 4-OH of
the galactose residue in 4, we decided to try a regioselective
coupling reaction using glycosyl trichloroacetimidate as a donor.
It was found, surprisingly, that employing 2,3,4,6-tetra-O-benzoyl-
a-D-glucopyranosyl trichloroacetimidate (5) gave no expected
product. Replacement of the donor with the sterically more favor-
able 2,3,4,6-tetra-O-acetyl-a-D-glucopyranosyl trichloroacetimi-
date (6), the desired sarsasapogenyl 2,3,4,6-tetra-O-acetyl-b-D-
glucopyranosyl-(1→2)-3,6-di-O-benzoyl- b-D-galactopyranoside
(7) was unambiguously obtained in 65% yield, as its acetylated
derivative 8 showed H-4 of galactosyl residue at 5.58 ppm in
Table 1 Cytotoxicity of compounds 1, 3, 20, 21, and CDDP against
HL-60 cellsa
Entry
Compound
IC50 (mg/mL)
1
2
5
6
7
1
15.5
> 30
12.7
7.1
3
20
21
CDDP
1
the H NMR spectrum. However, attempts to introduce 16-OH
0.7
to compounds 7 or 8 using published Oxone chemistry11 were
frustrating, and no major component was observed under reaction
conditions. We thus turned our attention to a more formal strategy
(Scheme 2).
a The cells were continuously treated with each sample for 72 h, and the
cell growth was evaluated using an MTT reduction assay. Data are mean
values of three experiments performed in triplicate.
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Scheme 2 Reagents and conditions: (a) TBSCl, imidazole, DMF, 97%; (b) oxone, NaHCO3, 1.0 mM Na2EDTA, CH2Cl2, acetone, 81%; (c) TMSOTf,
CH2Cl2, 0 ◦C, 80%; (d) Et2O·BF3, CH2Cl2, 95%; (e) 2, NIS, TMSOTf, CH2Cl2, -20 ◦C, 62%; (f) 6, TMSOTf, CH2Cl2, -42 ◦C, 30 min; (g) pyridine, Ac2O,
75% in two steps; (h) NaBH4, i-PrOH, CH2Cl2, 8 h, 68%; (i) NaOMe, CH2Cl2–MeOH (2:1, v/v); (j) acetone–H2O (3:7, v/v), reflux, 95%.
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Fig. 2 Chemical structures of compounds 20 and 21.
In conclusion, the finding of a new synthetic route to timo-
saponin BII opens up a possibility to develop novel physiologically
active analogues and derivatives of sarsasapogenin. We have
proved here that the target compound can be obtained in ten steps
and in around 18% overall yield. The use of a partially protected
glycosyl donor significantly simplified the tedious carbohydrate
chemistry process. The current results should be valuable for
related molecule design, synthesis, and bioactivity screening.18
15.9, 15.5, 13.8, 13.7. Anal. Calcd for C47H62O10: C, 71.73; H, 7.94;
Found: C, 71.50; H, 8.15%.
Synthesis of compound 7. To a mixture of compound 6
(376 mg, 0.76 mmol) and 4 (500 mg, 0.64 mmol) in anhydrous
CH2Cl2 (15 mL) was added Me3SiOTf (14 mL, 0.08 mmol) under
N2 atmosphere at -42 ◦C. The mixture was stirred under these
conditions for 30 min, after which TLC (petroleum ether–EtOAc,
1:1) indicated that all starting materials were consumed. The
reaction mixture was neutralized with TEA and concentrated.
The syrup was purified by column chromatography (petroleum
ether–EtOAc, 4:1) to give 7 as a white foamy solid (464 mg, 65%):
[a]2D5 -19 (c 2, CHCl3); 1H NMR (400 MHz, CDCl3): d 8.03 (d, 2H,
J 7.6 Hz, Bz), 7.97 (d, 2H, J 7.6 Hz, Bz), 7.60–7.37 (m, 6H, Bz),
5.13 (dd, 1H, J 3.6, 9.9 Hz, H-3Gal), 4.92 (t, 1H, J 10.4 Hz, H-3Glc),
4.87 (t, 1H, J 9.2 Hz, H-2Glc), 4.83 (d, 1H, J 8.4 H, H-4Glc), 4.74
(d, 1H, J= 8.0 Hz, H-1Glc), 4.55 (dd, 1H, J 6.4, 11.2 Hz, H-6a),
4.49 (d, 1H, J 7.6 Hz, H-1Gal), 4.45 (dd, 1H, J 6.7, 11.3 Hz, H-6b),
4.39–4.36 (m, 1H, H-16Sar), 4.27 (dd, 1H, J 5.2, 12.0 Hz, H-6a¢),
4.15–3.87 (m, 6H, H-6b¢, H-5, H-2Gal, H-4Gal, H-3Sar, H-26aSar),
3.66–3.64 (m, 1H, H-5¢), 3.27 (d, 1H, J 11.0 Hz, H-26bSar), 2.04,
1.99, 1.93, 1.86 (4 s, 4 ¥ 3 CH3CO), 1.04 (d, 3H, J 7.2 Hz, 21-
CH3), 0.96 (d, 3H, J 7.2 Hz, 27-CH3), 0.94 (s, 3H, 19-CH3), 0.72
(s, 3H, 18-CH3). 13C NMR (100 MHz, CDCl3): d 170.6, 170.2,
169.5, 168.9, 165.9, 165.2, 133.2, 132.8, 129.2, 129.1, 129.0, 128.8,
128.2, 127.9, 109.2, 100.4, 99.5, 80.5, 72.4, 71.4, 71.3, 70.7, 68.2,
66.7, 64.6, 62.1, 62.0, 61.6, 59.8, 55.9, 41.6, 40.1, 39.8, 39.6, 35.7,
34.8, 34.4, 31.2, 29.5, 26.5, 26.0, 25.7, 25.4, 25.2, 23.4, 22.1, 20.5,
20.3, 20.0, 19.6, 15.9, 15.5, 13.8, 13.6. Anal. Calcd for C61H80O19:
C, 65.57; H, 7.22; Found: C, 65.28; H, 7.25%.
General methods
◦
Optical rotations were determined at 25 C with a Perkin-Elmer
1
Model 241-Mc automatic polarimeter. H NMR and13C NMR
spectra were recorded with a Bruker ARX 400 spectrometer for
solutions in CDCl3 or C5D5N. Chemical shifts are given in ppm
downfield from internal Me4Si. Mass spectra were measured using
a MALDI TOF-MS with a-cyano-4-hydroxycinnamic acid (CCA)
as matrix. Thin-layer chromatography (TLC) was performed on
silica gel HF254 with detection by charring with 30% (v/v) H2SO4 in
MeOH or in some cases by UV detector. Column chromatography
was conducted by elution of a column of silica gel (100–200 mesh)
with EtOAc–petroleum ether (60–90 ◦C) as the eluent. Solutions
were concentrated at <60 ◦C under reduced pressure.
Synthesis of compound 4. To a mixture of compound 2
(750 mg, 1.68 mmol) and 3 (583 mg, 1.40 mmol) in anhydrous
CH2Cl2 (20 mL) was added NIS (564 mg, 2.52 mmol) and
◦
Me3SiOTf (30 mL, 0.17 mmol) under N2 atmosphere at -42 C.
The mixture was stirred under these conditions for 50 min,
then neutralized with TEA, and concentrated. Purification of the
resulting residue on column chromatography (petroleum ether–
EtOAc, 3:1) gave 4 as a white foamy solid (737 mg, 67%): [a]D25
Synthesis of compound 20. Compound 6 (100 mg, 0.089 mmol)
was dissolved in anhydrous CH2Cl2–MeOH (18 mL, 2:1, v/v),
and then 1.0 M NaOMe in MeOH (0.1 mL) was added slowly
at room temperature. After stirring at room temperature for
3.5 h, TLC (n-BuOH : EtOH : H2O = 4:2 : 0.5) indicated that
all starting material was consumed. The reaction mixture was
neutralized with Dowex 50 ion-exchange resin (H+), then filtered
and concentrated to dryness. Purification of the residue on Bio-gel
P2 column using double distilled water as eluent afforded, after
freeze drying, compound 7 as an amorphous solid (64 mg, 95%):
[a]2D5 -18 (c 1, H2O); selected1H NMR (400 MHz, C5D5N): d 5.48
(d, 1H, J 7.6 Hz, H-1Glc), 5.04 (d, 1H, J 8.0 Hz, H-1Gal), 1.17 (d,
3H, J 6.8 Hz, 21-CH3), 1.09 (d, 3H, J 6.8 Hz, 27-CH3), 0.98 (s,
3H, 19-CH3), 0.83 (s, 3H, 18-CH3). 13C NMR (100 MHz, C5D5N):
1
-21 (c 1, CHCl3); H NMR (400 MHz, CDCl3): d 8.08 (d, 2H,
J 7.6 Hz, Bz), 8.00 (d, 2H, J 8.0 Hz, Bz), 7.58–7.53 (m, 2H, Bz),
7.44–7.40 (m, 4H, Bz), 5.19 (dd, 1H, J 3.2, 10.0 Hz, H-3), 4.62 (dd,
1H, J 6.6, 11.4 Hz, H-6a), 4.56 (dd, 1H, J 6.4, 11.3 Hz, H-6b),
4.47 (d, 1H, 7.7 Hz, H-1), 4.44–4.37 (m, 1H, H-16Sar), 4.21 (d, 1H,
J 2.3 Hz, H-4), 4.06–3.94 (m, 4H, H-2, H-5, H-3Sar, H-26aSar), 3.30
(d, 1H, J 11.0 Hz, H-26bSar), 1.07 (d, 3H, J 7.1 Hz, 21-CH3), 0.98
(d, 3H, J 6.7 Hz, 27-CH3), 0.94 (s, 3H, 19-CH3), 0.75 (s, 3H, 18-
CH3). 13C NMR (100 MHz, CDCl3): d 165.8, 165.5, 132.7, 129.4,
129.2, 129.1, 127.9, 109.2, 101.4, 80.5, 74.9, 74.8, 71.8, 69.0, 66.8,
64.6, 62.3, 61.6, 59.9, 55.9, 41.6, 40.9, 40.1, 39.8, 39.6, 36.6, 34.7,
34.5, 31.2, 29.9, 29.7 26.5, 26.1, 25.4, 25.2, 23.3, 22.1, 20.5, 20.3,
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d 110.1, 106.6, 103.0, 82.3, 81.8, 78.8, 77.4, 77.0, 75.9, 75.6, 72.1,
70.3, 65.6, 63.4, 63.2, 62.6, 56.9, 50.1, 42.9, 41.9, 41.3, 40.8, 40.7,
37.4, 36.0, 35.7, 32.6, 31.4, 28.0, 27.5, 27.2, 26.9, 26.7, 24.5, 21.6,
17.8, 17.4, 17.1, 16.7, 15.4. MALDITOF-MS: Calcd for C39H64O13:
740.4 [M]+; Found 763.4 [M + Na]+.
0.72 (s, 3H, 18-CH3), 0.01 (s, 6H, 2CH3). 13C NMR (100 MHz,
CDCl3): d 218.4, 213.6, 166.1, 165.8, 165.2, 165.1, 133.3, 133.1,
133.1, 133.0, 129.7, 129.7, 129.5, 129.3, 128.8, 128.7, 128.3, 128.3,
128.5, 101.4, 75.3, 72.9, 72.0, 71.9, 69.8, 67.2, 66.4, 63.2, 51.2, 43.2,
42.0, 39.8, 39.5, 39.1, 37.2, 36.3, 35.1, 34.7, 34.3, 32.6, 29.6, 28.5,
26.7, 26.6, 26.5, 25.9, 25.8, 25.8, 25.6, 23.8, 22.6, 20.5, 18.0, 16.8,
15.3, 13.1, -4.86, -4.89. Anal. Calcd for C67H84O13Si: C, 71.50; H,
7.52; Found: C, 71.19; H, 7.40%.
Synthesis of compound 10. Compound 3 (13.0 g, 31.2 mmol)
was dissolved in dry DMF (100 mL), and TBSCl (6.0 g, 39.8 mmol)
◦
was added to the solution. The mixture was stirred at 80 C for
8 h, after which TLC (petroleum ether–EtOAc, 30:1) indicated
that all starting materials were consumed. The reaction mixture
was diluted with petroleum ether (300 mL), washed with water
(450 mL ¥ 2) and brine (300 mL). The organic phase was dried
over anhydrous Na2SO4 and concentrated under vacuum to give
a white solid 10 (16.1 g, 97%) that was directly subjected to the
next reaction. Part of the crude product (50 mg) was purified
with column chromatography (petroleum ether–EtOAc, 30:1) for
Synthesis of compound 14. To a solution of compound 13
(3.6 g, 3.2 mmol) in dry CH2Cl2 (40 mL) was added BF3·Et2O
(1.0 mL, 7.3 mmol), and the mixture was stirred at room
temperature for 4 h. The mixture was diluted with CH2Cl2 (60 mL),
washed with saturated aqueous NaHCO3 (100 mL) and then
brine (80 mL). The organic layer was dried and concentrated.
Purification by column chromatography (petroleum ether–EtOAc,
1:1) gave 14 as a white foamy solid (3.07 g, 95%): [a]2D5 -56 (c 2,
CHCl3); 1H NMR (400 MHz, CDCl3): d 8.02–7.27 (m, 20H,
4PhCO), 5.89 (t, 1H, J 9.7 Hz, H-3Glc), 5.67 (t, 1H, J 9.7 Hz,
H-4Glc), 5.53 (dd, 1H, J 7.8, 9.7 Hz, H-2Glc), 4.84 (d, 1H, J 7.8 Hz,
H-1Glc), 4.62 (dd, 1H, J 3.3, 12.0 Hz, H-6aGlc), 4.50 (dd, 1H, J 5.1,
12.0 Hz, H-6bGlc), 4.17–4.12 (m, 2H, H-5Glc, H-3Sar), 3.88 (dd, 1H,
J 5.2, 9.4 Hz, H-26aSar), 3.29 (dd, 1H, J 7.4, 9.4 Hz, H-26bSar),
0.98 (s, 3H, 19-CH3), 0.94 (d, 3H, J 6.6 Hz, 21-CH3), 0.81 (d, 3H,
J 6.6 Hz, 27-CH3), 0.73 (s, 3H, 18-CH3). 13C NMR (100 MHz,
CDCl3): d 218.1, 213.4, 166.0, 165.7, 165.1, 165.0, 133.3, 133.1,
133.0, 133.0, 129.7, 129.6, 129.6, 129.6, 129.5, 129.3, 128.8, 128.8,
128.3, 128.2, 128.2, 101.5, 75.28, 72.9, 72.0, 71.9, 69.9, 66.7, 66.4,
63.2, 60.3, 51.1, 43.2, 42.0, 39.6, 39.5, 39.0, 38.9, 37.1, 36.2, 35.1,
34.6, 33.4, 32.6, 29.5, 29.5, 29.4, 27.7, 26.7, 26.3, 26.3, 23.7, 22.6,
20.5, 19.1, 16.8, 15.2, 14.1, 13.1. Anal. Calcd for C61H70O13: C,
72.45; H, 6.98; Found: C, 72.21; H, 7.09%.
1
analysis: [a]2D5 -47 (c 2, CHCl3); H NMR (400 MHz, CDCl3): d
4.37–4.42 (m, 1H, H-16), 4.01 (br s, 1H, H-3), 3.95 (dd, 1H, J 2.6,
10.9 Hz, H-26a), 3.29 (d, 1H, J 11.8 Hz, H-26b), 1.07 (d, 3H,
J 7.1 Hz, 21-CH3), 0.98 (d, 3H, J 6.7 Hz, 27-CH3), 0.94 (s, 3H,
19-CH3), 0.87 (s, 9H, t-Bu), 0.75 (s, 3H, 18-CH3), 0.00 (s, 6H, 2
CH3).13C NMR (100 MHz, CDCl3): d 109.6, 81.0, 67.3, 65.0, 62.1,
56.5, 42.1, 40.6, 40.0, 36.4, 35.3, 35.1, 27.0, 26.8, 26.7, 25.8, 25.7,
23.9, 20.9, 18.0, 16.4, 16.0, 14.3. Anal. Calcd for C33H58O3Si: C,
74.66; H, 11.01; Found: C, 75.02; H, 10.91%.
Synthesis of compounds 11 and 12. To a mixture of compound
10 (16.1 g, 30 mmol) and NaHCO3 (49 g, 0.58 mmol) in CH2Cl2–
acetone–1.0 mM aqueous Na2EDTA (450 mL, 1:1:1) was added
dropwise a solution of Oxone (2KHSO5·KHSO4·K2SO4) (105 g,
0.169 mol) in a minimum amount of 1.0 mM aqueous Na2EDTA.
The reaction mixture was stirred at room temperature for 16 h,
after which TLC (petroleum ether–EtOAc, 10:1) indicated that
all starting materials were consumed. Removal of acetone and
CH2Cl2 under diminished pressure was followed by dilution of the
residue with CH2Cl2 and washing with water; the organic phase
was dried over anhydrous Na2SO4 and concentrated to dryness.
The crude product was purified with column chromatography
(petroleum ether–EtOAc, 20:1) to afford a mixture of 11 and 12 as
white solid (13.5 g, 81%). MALDITOF-MS: Calcd for C33H58O4Si:
546.4 [M]+; Found 549.2 [M + Na]+.
Synthesis of compound 15. To a mixture of compound 2 (1.38 g,
3.08 mmol) and 14 (2.6 g, 2.6 mmol) in anhydrous CH2Cl2
(50 mL) was added NIS (1.04 g, 4.6 mmol) and Me3SiOTf (55 mL,
0.30 mmol) under N2 atmosphere at -20 ◦C. The mixture was
stirred under these conditions for 60 min, then neutralized with
TEA, and concentrated. Purification of the resulting residue on
column chromatography (petroleum ether–EtOAc, 2:1) gave 15
1
as a foamy solid (2.23 g, 62%): [a]2D5 -20 (c 2, CHCl3); H NMR
(400 MHz, CDCl3): d 8.11–7.28 (m, 30H, 6PhCO), 5.90 (t, 1H,
J 9.6 Hz, H-3Glc), 5.67 (t, 1H, J 9.6 Hz, H-4Glc), 5.53 (dd, 1H,
J 7.8, 9.6 Hz, H-2Glc), 5.18 (dd, 1H, J 3.2, 10.0 Hz, H-3Gal), 4.84 (d,
Synthesis of compound 13. To a mixture of compounds 11,
12 (2.25 g, 4.2 mmol), and 5 (3.56 g, 4.8 mmol) in anhydrous
CH2Cl2 (40 mL) was added Me3SiOTf (86 mL, 0.47 mmol) under
N2 atmosphere at 0 ◦C. The mixture was stirred under these
conditions for 40 min, after which TLC (petroleum ether–EtOAc,
4:1) indicated that all starting materials were consumed. The
reaction mixture was neutralized with triethylamine (TEA), then
concentrated. Column chromatography (petroleum ether–EtOAc,
6:1) of the residue gave 13 as a foamy solid (3.78 g, 80%): [a]D25
1H, J 7.8 Hz, H-1Glc), 4.64–4.48 (m, 4H, H-6aGlc, H-6aGal, H-6bGlc
,
H-6bGal), 4.48 (d, 1H, J 7.8 Hz, H-1Gal), 4.23 (br d, 1H, J 3.2 Hz,
H-4Gal), 4.17–4.15 (m, 1H, H-5Gal), 4.10 (br s, 1H, H-3Sar), 4.05 (dd,
1H, J 7.8, 10.0 Hz, H-2Gal), 3.96 (t, 1H, J 6.6 Hz, H-5Glc), 3.88 (dd,
1H, J 5.2, 9.4 Hz, H-26aSar), 3.30 (dd, 1H, J 7.4, 9.3 Hz, H-26bSar),
0.95 (s, 3H, 19-CH3), 0.94 (d, 3H, J 7.3 Hz, 21-CH3), 0.82 (d, 3H,
J 6.6 Hz, 27-CH3), 0.73 (s, 3H, 18-CH3). 13C NMR (100 MHz,
CDCl3): d 218.0, 213.3, 171.0, 166.2, 166.0, 165.9, 165.7, 165.1,
164.9, 133.2, 133.1, 133.1, 133.0, 132.9, 129.7, 129.6, 129.6, 129.5,
129.5, 129.2, 128.7, 128.7, 128.2, 128.1, 101.8, 101.4, 75.4, 75.2,
75.0, 72.9, 72.2, 71.9, 71.8, 69.8, 69.3, 67.1, 66.3, 63.1, 62.8, 60.2,
51.0, 43.1, 41.9, 39.7, 39.4, 38.9, 37.0, 36.7, 34.8, 34.5, 32.5, 30.1,
29.8, 26.6, 26.3, 26.2, 26.1, 23.6, 20.8, 20.4, 16.7, 15.2, 14.0, 13.0.
Anal. Calcd for C81H88O20: C, 70.42; H, 6.42; Found: C, 70.06; H,
6.64%.
1
-26 (c 2, CHCl3); H NMR (400 MHz, CDCl3): d 8.02–7.27 (m,
20H, 4PhCO), 5.90 (t, 1H, J 9.7 Hz, H-3Glc), 5.67 (t, 1H, J 9.7 Hz,
H-4Glc), 5.53 (dd, 1H, J 7.8, 9.7 Hz, H-2Glc), 4.86 (d, 1H, J 7.8 Hz,
H-1Glc), 4.63 (dd, 1H, J 3.3, 12.1 Hz, H-6aGlc), 4.51 (dd, 1H, J 5.3,
12.1 Hz, H-6bGlc), 4.19–4.14 (m, 1H, H-5Glc), 4.05 (br s, 1H, H-
3Sar), 3.87 (dd, 1H, J 5.3, 9.4 Hz, H-26aSar), 3.31 (dd, 1H, J 7.3,
9.4 Hz, H-26bSar), 0.95 (s, 3H, 19-CH3), 0.89 (d, 3H, J 6.6 Hz, 21-
CH3), 0.88 (s, 9H, t-Bu of TBS), 0.79 (d, 3H, J 6.6 Hz, 27-CH3),
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Synthesis of compound 17. To a mixture of compound 6
(685 mg, 1.39 mmol) and 15 (1.6 g, 1.2 mmol) in anhydrous
CH2Cl2 (15 mL) was added Me3SiOTf (30 mL, 0.17 mmol) under
N2 atmosphere at -42 ◦C. The mixture was stirred under these
conditions for 30 min, after which TLC (petroleum ether–EtOAc,
1:1) indicated that all starting materials were consumed. The
reaction mixture was neutralized with TEA and concentrated to
dryness. The resulting mixture was kept in pyridine (15 mL) and
Ac2O (5 mL) at room temperature for about 3 h, then the mixture
was concentrated with the help of toluene. The syrup was purified
by column chromatography (petroleum ether–EtOAc, 3:1) to give
17 as a white foamy solid (1.58 g, 75%): [a]2D5 -23 (c 2, CHCl3);
1H NMR (400 MHz, CDCl3): d 8.01–7.28 (m, 30H, 6PhCO), 5.90
(t, 1H, J 9.6 Hz, H-3Glc-I), 5.67 (t, 1H, J 9.6 Hz, H-4Glc-I), 5.58
(dd, 1H, J 3.4, 4.1 Hz, H-4Gal), 5.53 (dd, 1H, J 7.6, 9.6 Hz, H-
2Glc-I), 5.30 (dd, 1H, J 3.4, 9.9 Hz, H-3Gal), 4.99 (t, 1H, J 9.2 Hz,
H-3Glc-II), 4.92 (t, 1H, J 9.2 Hz, H-4Glc-II), 4.85 (dd, 1H, J 7.8,
9.2 Hz, H-2Glc-II), 4.84 (d, 1H, J 7.8 Hz, H-1Glc-II), 4.70 (d, 1H,
J 7.6 Hz, H-1Glc-I), 4.61 (dd, 1H, J 8.8, 12.0 Hz, H-6a), 4.56
(d, 1H, J 7.7 Hz, H-1Gal), 4.53–4.48 (m, 2H, H-6a, H-6b), 4.36
(dd, 1H, J 7.2, 12.0 Hz, H-6a), 4.29 (dd, 1H, J 6.8, 11.1 Hz,
1.98, 1.90, 1.75 (5 s, 5 ¥ 3CH3CO), 0.99 (s, 3H, 19-CH3), 0.94 (d,
3H, J 7.3 Hz, 21-CH3), 0.80 (d, 3H, J 6.6 Hz, 27-CH3), 0.73 (s,
3H, 18-CH3).13C NMR (100 MHz, CDCl3): d 174.1, 170.9, 170.5,
169.9, 169.7, 169.2, 169.2, 165.9, 165.8, 165.6, 165.2, 165.0, 164.9,
163.9, 133.0, 133.0, 132.9, 129.6, 129.5, 129.5, 129.4, 129.3, 129.2,
129.2, 128.7, 128.5, 128.2, 128.2, 128.1, 110.1, 101.5, 100.8, 99.6,
81.1, 75.4, 75.4, 75.2, 73.8, 72.8, 72.8, 72.7, 72.0, 71.9, 71.8, 71.5,
71.0, 70.5, 69.8, 69.7, 68.6, 67.5, 63.0, 62.7, 62.3, 61.7, 60.2, 56.2,
41.45, 40.8, 39.9, 39.6, 36.0, 35.1, 34.8, 33.3, 31.5, 29.8, 29.7, 27.0,
26.3, 26.3, 26.0, 23.7, 20.8, 20.8, 20.8, 20.6, 20.5, 20.3, 20.0, 16.6,
16.5, 16.2, 15.3, 14.0, 13.5. Anal. Calcd for C97H110O30: C, 66.35;
H, 6.31; Found: C, 66.09; H, 6.40%.
Synthesis of compound 1. Compound 18 (202 mg, 0.114 mmol)
was dissolved in anhydrous CH2Cl2–MeOH (36 mL, 1:2, v/v), and
then 1.0 M NaOMe in MeOH (0.2 mL) was added slowly at 0 ◦C.
After stirring at room temperature for 5 h, TLC (n-BuOH : EtOH :
H2O = 4:2 : 1) indicated that all starting material was consumed.
The reaction mixture was neutralized with Dowex 50 ion-exchange
resin (H+), then filtered and concentrated to dryness. MS spectrum
analyses suggested the resulting syrup is a mixture of 1 and its
22-methyl ether derivative. The above mixture in acetone/H2O
(40 mL, 3:7, v/v) was stirred under reflux conditions for 12 h, then
concentrated to dryness under diminished pressure. Purification
of the residue on Bio-gel P2 column using double distilled water
as eluent afforded, after freeze drying, target compound 1 as an
H-6b), 4.19–4.12 (m, 1H, H-5), 4.10–4.04 (m, 4H, H-6b, H-3Sar
,
H-5, H-2Gal), 3.90 (dd, 1H, J 5.2, 9.4 Hz, H-26aSar), 3.72–3.69 (m,
1H, H-5), 3.30 (dd, 1H, J 7.4, 9.3 Hz, H-26bSar), 2.14, 2.08, 1.98,
1.89, 1.75 (5 s, 5 ¥ 3 CH3CO), 0.99 (s, 3H, 19-CH3), 0.94 (d, 3H,
J 7.3 Hz, 21-CH3), 0.82 (d, 3H, J 6.6 Hz, 27-CH3), 0.73 (s, 3H, 18-
CH3). 13C NMR (100 MHz, CDCl3): d 218.0, 213.3, 170.5, 169.9,
169.7, 169.3, 169.2, 166.0, 165.9, 165.7, 165.3, 165.1, 165.0, 133.6,
133.3, 133.2, 133.1, 133.0, 133.0, 129.7, 129.6, 129.6, 129.5, 129.5,
129.5, 129.5, 129.4, 129.4, 129.3, 129.3, 129.1, 128.8, 128.6, 128.3,
128.3, 128.3, 128.2, 128.2, 101.4, 100.9, 99.5, 75.4, 75.2, 75.1, 73.9,
72.9, 72.8, 72.0, 71.9, 71.6, 71.1, 70.6, 69.9, 69.5, 68.7, 67.5, 66.4,
63.2, 62.5, 61.8, 60.2, 51.1, 43.2, 42.0, 41.3, 39.8, 39.5, 39.0, 37.1,
35.9, 34.9, 34.9, 34.6, 32.6, 29.7, 29.6, 26.7, 26.3, 26.2, 26.1, 25.6,
23.7, 22.5, 20.9, 20.7, 20.6, 20.5, 20.4, 20.1, 16.8, 15.2, 14.1, 13.0.
Anal. Calcd for C97H108O30: C, 66.43; H, 6.21; Found: C, 66.05; H,
6.27%.
1
amorphous solid (99 mg, 95%): [a]2D5 -6 (c 1, H2O); selected H
NMR (400 MHz, C5D5N): d 5.27 (d, 1H, J 7.5 Hz, H-1Glc-I), 4.91
(d, 1H, J 7.5 Hz, H-1Gal), 4.82 (d, 1H, J 7.6 Hz, H-1Glc-I), 1.15
(d, 3H, J 6.9 Hz, 21-CH3), 1.10 (s, 3H, 27-CH3), 1.02 (d, 3H,
J 6.5 Hz, 19-CH3), 0.98 (s, 3H, 18-CH3). 13C NMR (100 MHz,
C5D5N): d 110.5, 105.8, 104.9, 102.3, 81.5, 81.0, 78.4, 78.2, 78.2,
77.8, 76.7, 76.4, 75.3, 75.2, 75.1, 75.0, 71.5, 71.5, 69.6, 63.8,
62.6, 62.0, 56.2, 41.0, 40.5, 40.2, 40.1, 36.9, 36.7, 35.3, 35.1, 34.2,
32.2, 30.7, 30.7, 28.1, 26.8, 26.6, 26.6, 23.8, 21.0, 17.3, 16.5, 16.3.
MALDITOF-MS: Calcd for C45H76O19: 920.5 [M]+; Found 944.0
[M + Na]+.
Synthesis of compound 18. The mixture of compound 17
(300 mg, 0.17 mmol) and NaBH4 (200 mg, 5 mmol) in
i-propanol/CH2Cl2 (18 mL, 8:1, v/v) was stirred at room tempera-
ture for about 8 h. The reaction mixture was then poured into cold
water, then extracted with CH2Cl2 (100 mL ¥ 2). The combined
organic layer was washed with water (100 mL ¥ 3), dried over
anhydrous Na2SO4, and concentrated to dryness. The resulting
colorless oil was subjected to column chromatography using
petroleum ether/EtOAc (v/v, 1:1) as eluent, to give compound
18 as a white solid (204 mg, 68%): [a]2D5 -5 (c 2, CHCl3); 1H NMR
(400 MHz, CDCl3): d 8.00–7.28 (m, 30H, 6PhCO), 5.88 (t, 1H,
J 9.7 Hz, H-3Glc-I), 5.67 (t, 1H, J 9.7 Hz, H-4Glc-I), 5.58 (dd, 1H,
J 3.4, 4.1 Hz, H-4Gal), 5.53 (dd, 1H, J 7.8, 9.7 Hz, H-2Glc-I), 5.30 (dd,
1H, J 3.4, 9.8 Hz, H-3Gal), 5.00 (t, 1H, J 9.6 Hz, H-3Glc-II), 4.93 (t,
1H, J 9.6 Hz, H-4Glc-II), 4.86 (dd, 1H, J 7.8, 9.6 Hz, H-2Glc-II), 4.81
(d, 1H, J 7.8 Hz, H-1Glc-II), 4.70 (d, 1H, J 7.8 Hz, H-1Glc-I), 4.61 (dd,
1H, J 3.0, 12.1 Hz, H-6a), 4.55 (d, 1H, J 7.5 Hz, H-1Gal), 4.53–4.51
(m, 2H, H-6a, H-6b), 4.40 (dd, 1H, J 7.2, 12.1 Hz, H-6a), 4.30
(dd, 1H, J 6.8, 11.1 Hz, H-6b), 4.14–4.03 (m, 5H, H-5, H-6b, H-
3Sar, H-5, H-2Gal), 3.87 (dd, 1H, J 5.2, 9.4 Hz, H-26aSar), 3.75–3.48
(m, 1H, H-5), 3.25 (dd, 1H, J 7.4, 9.3 Hz, H-26bSar), 2.14, 2.09,
Cell culture assay
HL-60 cells were maintained in the RPMI 1640 medium con-
taining 10% fetal bovin serum supplemented with L-glutamine,
100 units/mL penicillin, and 100 mg/mL streptomycin. The
leukaemia cells were washed and suspended in the above medium
to 1 ¥ 105 cells/mL, and 100 mL of this cell suspension was placed in
each well of a 96-well plate. The cells were incubated in 5% CO2/air
for 24 h at 37 ◦C. After incubation, 3 mL/mL of DMSO–H2O
(1:20, v/v) solution containing the sample was added to give the
final concentration of 0.1–30 mL/mL, while 3 mL/mL of DMSO–
H2O (1:20, v/v) was added into control wells. The cells were further
incubated for 72 h in the presence of each agent, and then cell
growth was evaluated using a modified 3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay, and
the IC50 values were calculated accordingly.16
Acknowledgements
This work was partially supported by NNSF of China (Projects
20621703, 20872172, and 20732001).
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