Facile synthesis of trisaccharide moiety corresponding to antitumor activity in triterpenoid saponins isolated from Pullsatilla roots

Article abstract of DOI:10.1248/cpb.55.1734

A trisaccharide found in triterpenoid saponins isolated from Pullsatilla roots appears as an important promoiety for the enhancement of anticancer activity of their aglycones. Thus a facile synthetic method for a trisaccharide moiety, allyl-2,3,4-tri-O-benzoyl-α-L-rhamnopyranosyl-(1→2)-[2,3,4,6- tetra-O-benzoyl-β-D-glucopyranosyl-(1→4)]-3-O-benzoyl-β-L- arabinopyranoside (3), has been firstly developed through the regio- and stereoselective glycosylations from arabinose in total 16% yield via route 2 (eight steps). In this synthetic procedure, the protection of anomeric -OH of L-arabinose with equatorially oriented allyl group unlike with the axial 4-methoxybenzyl protecting group well promoted glycosyl bond formation between α-L-rhamnopyranosyl trichloroacetimidate and 2-OH of arabinose. As expected, the synthesized trisaccharide moiety 3 has no cytotoxicity (ED ₅₀: >100 μM) against three human cancer cell lines (A-549, SK-OV-3, and SK-MEL-2), respectively.

Full text of DOI:10.1248/cpb.55.1734

1734
Notes
Chem. Pharm. Bull. 55(12) 1734—1739 (2007)
Vol. 55, No. 12
Facile Synthesis of Trisaccharide Moiety Corresponding to Antitumor
Activity in Triterpenoid Saponins Isolated from Pullsatilla Roots
Seong-Cheol BANG, Hyun-Hee SEO, Hwi-Yeol YUN, and Sang-Hun JUNG*
College of Pharmacy, Chungnam National University; Daejon 305–764, Korea.
Received February 7, 2007; accepted August 29, 2007
A trisaccharide found in triterpenoid saponins isolated from Pullsatilla roots appears as an important pro-
moiety for the enhancement of anticancer activity of their aglycones. Thus a facile synthetic method for
a trisaccharide moiety, allyl-2,3,4-tri-O-benzoyl-a-L-rhamnopyranosyl-(1→2)-[2,3,4,6-tetra-O-benzoyl-b-D-glu-
copyranosyl-(1→4)]-3-O-benzoyl-b-L-arabinopyranoside (3), has been firstly developed through the regio- and
stereoselective glycosylations from arabinose in total 16% yield via route 2 (eight steps). In this synthetic proce-
dure, the protection of anomeric –OH of L-arabinose with equatorially oriented allyl group unlike with the axial
4-methoxybenzyl protecting group well promoted glycosyl bond formation between a-L-rhamnopyranosyl
trichloroacetimidate and 2-OH of arabinose. As expected, the synthesized trisaccharide moiety 3 has no cytotoxi-
city (ED50: ꢀ100 mM) against three human cancer cell lines (A-549, SK-OV-3, and SK-MEL-2), respectively.
Key words trisaccharide; Pullsatilla roots; regio- and stereoselective glycosylation
Saponins, the glycosides of steroids or triterpenes, are ana N. root and the structure–activity relationships of anti-
widely distributed in plants and animals.1) They exist in rela- tumoral saponins was published.6,7) Among them, Pullsatilla
tively high quantities in many significant food and beverage saponin D (1, inhibition ratio (IR): 66.9% against LLC lung
plants, including oats, peanuts, soybeans, lentils, mung carcinoma xengraft model at 6.0 mg/kg/d i.p., ED50 3.04—
beans, garlic, onions, spinach, asparagus, jujube, quillaja and 13.17 mM) and oleanolic acid 3-O-a-L-rhamnopyranosyl-
tea. Saponins also are generally found as active constituents (1→2)-[b-D-glucopyranosyl-(1→4)]-a-L-arabinopyranoside
in many well known oriental herbal medicines such as gin- (2, ED50 1.57—8.36 mM) exhibited highly potent anticancer
seng, notoginseng, licorice, horse chestnut, red clover, sene- activity. Pulsatilla saponin D shows five times more active in
gae, and primula.2) Many previous studies reveal that anticancer test than its aglycone (ED50; ꢀ50 mM).8,9) This
saponins show various physiological and pharmacological indicated that the presence of a sugar moiety, O-a-L-
activities, such as anti-cancer, anti-inflammatory, cardiovas- rhamnopyranosyl-(1→2)-[b-D-glucopyranosyl-(1→4)]-a-L-
cular, and cytotoxic activities.3,4) The oligosaccharides inte- arabinopyranoside, at C-3 position enhances the activity. It
grated into the saponins have a very important role in their has been postulated that the linked trisaccharide improves the
bioactivity and thus the interest in this sugar unit is rapidly bioavailability in tumor tissue in vivo. Therefore, this moiety
increasing. For example, when the ether-linked tetrasaccha- could be utilized as a promoiety for the increment of the ac-
ride was removed from julibrosides, its cytotoxicity dramat- tivity of other antitumoral compounds and their solubility in
ically decreased. We recently reported Pulsatilla saponin water as shown in Fig. 1. Indeed, glucose has been integrated
D, hederagenin 3-O-a-L-rhamnopyranosyl-(1→2)-[b-D-glu- into anticancer agents as a conjugate for the improvement of
copyranosyl-(1→4)]-a-L-arabinopyranoside, as an anticancer their activity since cancer cells have increased rate of glucose
component.5) In addition, a total 17 triterpenoid saponins in- metabolism compared with healthy cells and over-expression
cluding six new saponins were isolated from Pullsatilla kore- of GLUT-1.10,11) This approach has been also applied to pep-
Fig. 1. Structures of Pullsatilla Saponin 1 and 2, Target Trisaccharide Template 3, and a Proposing Example (3-1) Conjugated with Drug
∗ To whom correspondence should be addressed. e-mail: jungshh@cnu.ac.kr
© 2007 Pharmaceutical Society of Japan

December 2007
1735
tide drugs12) and cytotoxic agents13,14) for improvement of de- sized readily form L-arabinose in four steps with 40—50%.19)
livery into the central nervous system (CNS). Although there Reaction of 4-methoxybenzyl a-L-arabinopyranoside (4)
are some studies on natural oligosaccharide (i.e., Digi- with 2,2-dimethoxypropane in N,N-dimethylformamide
toxin),15) these attempts have been mainly restricted to conju- (DMF) containing p-toluenesulfonic acid (p-TsOH) at room
gation with a monosaccharide.16) For the concrete investiga- temperature for 3 h, allylation of 2-OH with allyl bromide
tion of pharmacological function as a promoiety of a-L- and NaH at 0 °C for 1 h, and then removal of the acetonide in
rhamnopyranosyl-(1→2)-[b-D-glucopyranosyl-(1→4)]-a-L- 70% aq. acetic acid (AcOH) gave compound 5 in overall
arabinopyranosyl moiety of 1, the simple and effective syn- 71% yield (three steps). The 4-methoxybenzyl 2-O-allyl-a-L-
thetic route of this sugar moiety is urgently needed. Accord- arabinopyranoside (5) was reacted with dibutyltin oxide
ingly, a synthetic method of trisaccharide moiety (3) was de- (Bu2SnO) in MeOH to give the corresponding 3,4-O-stan-
signed and the detail procedure is described in this paper.
nylidene derivative, which was subsequently treated with
benzyl bromide (BnBr) in the presence of tetrabutylammo-
nium (Bu4NBr) in toluene to afford 4-methoxybenzyl 2-O-
Results and Discussion
Although numerous efforts have been devoted to the syn- allyl-3-O-benzyl-a-L-arabinopyranoside 6 in 68% yield. At
thesis of diverse carbohydrate structures which is occurred in first, coupling reaction of 6 with thioglucoside donor 720)
nature, it is still complicated in their synthesis. This can be using N-iodosuccinimide (NIS) and triflic acid (TfOH) as an
attributed mainly to difficulties in synthesizing oligosaccha- iodonium ion was attempted.21) But the desired disaccharide
rides, unlike proteins and nucleic acids, because (i) the mole- 9 was not produced. Therefore the other coupling condition
cules are typically branched rather than linear, (ii) the mono- using trichloroacetimidate donor 818,22,23) and trimethylsilyl
saccharide units can be connected by a- or b-linkages, and trifluoromethanesulfonate (TMSOTf) as catalyst at ꢁ20 °C
(iii) oligosaccharide synthesis requires multiple selective in dry CH2Cl2 was applied. The completed reaction mixture
protection and deprotection steps. Although considerable was purified by silica gel chromatography (3 : 1 cHx–EtOAc)
progress has been made in the field over the past few to give the b-(1→4) linked disaccharide 9 (Glc J1,2 values
decades, there is still no general route for synthesis of 7.6 Hz) as major product (83%) contaminated by a small
oligosaccharide and glycosylation chemistry is often not pre- amount of impurities (about 8%) in NMR spectra. For that
dictable.17) However the method for the synthesis of oligosac- reason, an additional purification step using preparative
charides devised by Schmidt and his coworkers are fre- HPLC elution with 80% acetonitrile (flow rate: 1 ml/min,
quently successful for forming mainly or exclusively a- or b- tRꢂ17.53 min) was performed to provide pure compound 9
linkage product.18)
(isolated yield: 71%). The a-anomer of 9 was not detected.
Based on this strategy, we efficiently performed the syn- This compound 9 was subsequently treated with palladium
thesis of protected trisaccharide moiety of 1. Initially we se- chloride (PdCl2)24) in CH2Cl2–MeOH to afford 2-O-deally-
lected 4-methoxybenzyl a-L-arabinopyranoside (4) as an ef- lated disaccharide 10 in 42% yield. However, as shown in
fective starting point because the synthetic route shown in Chart 1, the final glycosylation reaction of 10 and tri-
Chart 1 could be stereoselectively formed 1→4 linkage and chloroacetimidate 1125) catalyzed by TMSOTf was not ac-
the 4-methoxybenzyl (MPM) group at anomeric position of 4 complished under various conditions. It may be attributed to
can be removed under mild condition. The starting com- the instability of 4-methoxybenzyl group at anomeric posi-
pound 4-methoxybenzyl a-L-arabinopyranoside (4) synthe- tion of the acceptor under acidic conditions. In recovering
Reagents and conditions: (a) 2,2-dimethoxypropropane, p-TsOH, DMF, rt, 6 h; (b) allyl bromide, NaH, DMF, 0 °C, 1 h; (c) 70% aq. AcOH, 70 °C, 1 h, 71% for three steps; (d)
Bu2SnO, MeOH, reflux, 3 h; then BnBr, Bu4NBr, 60 °C, 16 h, 81%; (e) NIS, TfOH, 4Å MS, CH2Cl2, ꢁ20 °C, 1 h, 0%; (f) TMSOTf, 4Å MS, CH2Cl2, ꢁ20 °C, 2 h, 83% for 9 and
0% for 12; (g) PdCl2, MeOH, rt, 48 h, 42%.
Chart 1. Attempted Synthesis towards Trisaccharide Template (Route 1)

1736
Vol. 55, No. 12
Reagents and conditions: (a) 2,2-dimethoxypropropane, p-TsOH, DMF, rt, 6 h, 100%; (b) TMSOTf, 4Å MS, ꢁ20 °C, 2 h, 63%; (c) p-TsOH, CH2Cl2/MeOH 1 : 2, rt, 2 h, 99%; (d)
CH3C(OEt)3, p-TsOH, toluene, rt, 1 h; (e) 80% aq. HOAc, rt, 1 h, 71% for two steps; (f) BzCl, Pyr, 0 °C—rt, 12 h; (g) AcCl, CH2Cl2/MeOH 1 : 2, 0 °C—rt, 12 h, 68% for two steps;
(h) TMSOTf, 4Å MS, CH2Cl2, ꢁ20 °C, 2 h, 62%.
Chart 2. Completion of the Trisaccharide Template (Route 2)
process of starting material, a by-product lost 4-methoxyben- ride in CH2Cl2–MeOH (1 : 2) to afford allyl-2,3,4-tri-O-ben-
zyl group from 10 was obtained in 11% yield. Therefore, we zoyl-a-L-rhamnopyranosyl-(1→2)-3-O-benzoyl-b-L-ara-
needed the revised synthetic route with starting material of L- binopyranoside (20) in 68% yield in two steps. In the follow-
arabinose having more stable and solid protecting group than ing step, coupling of appropriately protected compound 20
4-methoxybenzyl group at anomeric position. Therefore, the with 2,3,4,6-tetra-O-benzoyl-a-D-glucopyranosyl trichloro-
more stable and the less crowded allyl group directed equato- acetimidate 8 was successfully underwent to give the desired
rially in place of 4-methoxybenzyl group at anomeric posi- trisaccharide 3 (62%) containing a trace amount of impurities
tion was selected as shown in Chart 2. The starting material (ꢅ3%) in NMR spectra. The impurities were readily elimi-
compound 13 having an allyl group at 1-OH was readily pre- nated by recrystallization from 3 : 1 petroleum ether–MeOH
pared in 85% yield under the Paul’s condition.19) Treatment (isolated yield of 3: 53%).
of allyl b-L-arabinopyranoside (13) with 2,2-dime-
Although compound 3 is the protected derivative of
toxypropane in dry DMF containing p-TsOH at room tem- sugar unit of Pulsatilla saponin D (hederagenin 3-O-a-L-
perature for 6 h gave the 3,4-isopropylidene derivative (14), rhamnopyranosyl-(1→2)-[b-D-glucopyranosyl-(1→4)]-a-L-
which was used without purification in the next step. Lewis arabinopyranoside), its cytotoxicity was measured against
acid-catalyzed assembly of 14 with the donor 2,3,4-tri-O- three human cancer cell lines (A-549, SK-OV-3, and SK-
benzoyl-a-L-rhamnopyranosyl tirchloroacetimidate (11) by MEL-2) for the possible prediction of cytotoxic effect of this
TMSOTf at a low temperature gave disaccharide 15 (63%) sugar motif. As expected, this compound did not show any
exclusively. The success of the reaction was evident from the activity (ED50: ꢀ100 mM). This result might indicate that the
down field shift in the 1H-NMR signal of H-2ꢃ in L-arabinose trisaccharide moiety of Pulsatilla saponin D would only have
(dH 3.53 ppm in 13 to dH 5.88 ppm in 15) and the chemical the role of carrier moiety of prodrug. Therefore this trisac-
shift 5.34 (d, Jꢂ2.0 Hz, 1H) of H-1ꢄ in rhamnopyranosyl charide 3 could be used as a nontoxic promoiety26) for the en-
moiety. The following deisopropylidenation with p-TsOH in hancement of activity of anticancer drugs.
CH2Cl2–MeOH (1 : 2) afforded allyl-2,3,4-tri-O-benzoyl-a-L-
In conclusion, a highly efficient and concise synthesis of
rhamnopyranosyl-(1→2)-b-L-arabinopyranoside (16) in 99% trisaccharide moiety 3 was developed through the regio- and
yield. For the synthesis of allyl-2,3,4-tri-O-benzoyl-a-L- steroselective glycosylations from arabinose in total 16%
rhamnopyranosyl-(1→2)-4-O-acetyl-b-L-arabinopyranoside yield via route 2 in eight steps. To prepare the full protected
(17) monoacetylated on the axial hydroxyl group of arabi- and practical trisaccharide moiety allyl-2,3,4-tri-O-benzoyl-
nose C-4, compound 16 was treated triethyl orthoacetate a-L-rhamnopyranosyl-(1→2)-[2,3,4,6-tetra-O-benzoyl-b-D-
(CH3C(OEt)3) and p-TsOH as acid catalyst in toluene and glucopyranosyl-(1→4)]-3-O-benzoyl-b-L-arabinopyranoside
subsequently cleaved the intermediate cyclic ortho ester (3) closely related with biological activity in Pulsatilla
under acidic condition (80% aq. HOAc). As a result, com- saponins, the protection of anomeric –OH of L-arabinose
pound 18 was attained in the good yield (71%) in two steps. with equatorially oriented allyl group unlike with the axial 4-
The structure of 18 was unambiguously confirmed by methoxybenzyl protecting group well promoted glycosyl
400 MHz 1H-NMR spectroscopy with a singlet peak of acetyl bond formation between a-L-rhamnopyranosyl trichloroace-
at d 2.17. Benzoylation of 18 with benzoyl chloride in pyri- timidate and 2-OH of arabinose. The (1→4) coupling reac-
dine, and then selective 4-O-deacetylation with acetyl chlo- tion with b-D-glucopyranose in route 2 gave nearly pure

December 2007
1737
was performed to give full-purified 9 (405 mg, isolated yield: 71.4%). But
the a-anomer of 9 was not detected: mp 63—65 °C; [a]D25 0.03° (cꢂ1,
CHCl3); IR (cmꢁ1, KBr) 2925 (sp3–H stretch), 1730 (conj. CꢂO stretch),
1600 (CꢂC stretch), 1520 and 1450 (aromatic CꢂC stretch), 1260 and 1100
product 3 and possible benzoyl group transfer products or
ortho esters have not been detected. Currently, studies on in-
tegrating the synthesized trisaccharide template 3 which has
a possibility as a targeting soluble carrier of diverse drugs
1
(C–O stretch); H-NMR (400 MHz, CDCl3) d 8.02—7.22 (m, 27H, Ar-H),
with linkable anticancer agents are underway, and the result 6.82 (d, Jꢂ8.4 Hz, 2H, Ar-H), 5.90 (t, Jꢂ9.2 Hz, 1H, H-3ꢄ), 5.63 (m, 2H, H-
4ꢄ and –CH2CHꢂCH2), 5.56 (t, Jꢂ9.1 Hz, 1H, H-2ꢄ), 5.13 (d, Jꢂ7.6 Hz,
will be reported in due course. On the other hand, this result
provides a basis of the total synthesis of Pulsatilla saponin D
possessing a potent anticancer activity.
1H, H-1ꢄ), 5.02 (d, Jꢂ18.4 Hz, 1H, –CH2CHꢂCH2), 4.96 (d, Jꢂ10.0 Hz,
1H, –CH2CHꢂCH2), 4.75 (d, Jꢂ11.6 Hz, 1H, –CH2MPM), 4.61 (dd,
Jꢂ12.4 Hz, Jꢃꢂ3.6 Hz, 1H, H-5ꢄ), 4.52—4.45 (m, 4H, H-6ꢄ (2H),
–CH2MPM (1H), and –CH2Ph (1H)), 4.24 (d, Jꢂ6.0 Hz, 1H, H-1ꢃ), 4.08—
4.04 (m, 2H, –CH2Ph and H-5aꢃ), 3.94 (dd, Jꢂ12.8 Hz, Jꢃꢂ5.6 Hz, 1H,
–CH2CHꢂCH2), 3.90 (br s, 1H, H-4ꢃ), 3.78 (s, 3H, –OCH3), 3.57 (dd,
Jꢂ12.8 Hz, Jꢃꢂ5.6 Hz, 1H, –CH2CHꢂCH2), 3.40—3.34 (m, 3H, H-5bꢃ, H-
3ꢃ, and H-2ꢃ); ESI-MS: m/zꢂ945 [MꢇNa]ꢇ; HR-FAB-MS: m/zꢂ944.9480
[MꢇNa]ꢇ (Calcd for C54H49O14Na, 944.9482).
Experimental
All reagents and solvents were dried prior to use according to standard
methods (Perrin, D. D.; Amarego, W. L.; Perrin, D. R., Purification of labora-
tory chemicals; Pergamon: London, 1996). Commercial reagents were used
without further purification unless otherwise stated. Reactions were followed
by thin-layer chromatography (TLC) on Merck glass silica gel (Kieselgel 60
F254) plates that were visualized under a UV lamp. Chromatographic separa-
tions were performed on silica gel columns by flash (Kieselgel 40, 0.040—
0.063 mm; Merck) or gravity column (Kieselgel 60, 0.063—0.200 mm;
Merck). HPLC was performed using a Shimadzu liquid chromatograph
model Class-vp version 6.12, equipped with a SPD-10A UV–vis detector
(Shimadzu). The preparative HPLC separation of compound 9 was per-
formed using a Spherisorb® S5 ODS2 column (250 mmꢆ10 mm, RP-C18,
5 mm, Waters, Milford, MA, U.S.A.), and all solvents for HPLC were filtered
through a 0.45 mm membrane filter (Waters). Melting points were measured
on an Electrothermal melting point apparatus. IR spectra were obtained on
KBr disks using a JASCO Report 100 spectrophotometer. NMR spectra
were recorded on Bruker MSL-300 or -500 instrument operating at
400 MHz, the chemical shift (d) is reported in parts per million downfield
from tetramethylsilane (TMS) and from solvent references. Electron impact
(EI) mass spectra were obtained on a HP-5988A mass spectrometer, and
HR-FAB-MS were recorded on a JMS-HX110/110A spectrometer.
4-Methoxybenzyl 2,3,4,6-Tetra-O-benzoyl-b-D-glucopyranosyl-(1→4)-
3-O-benzyl-a-L-arabinopyranoside (10) Palladium chloride (9 mg,
0.05 mmol) was added to a solution of compound 9 (159 mg, 0.16 mmol) in
dry MeOH (10 ml), and the reaction mixture was stirred for 48 h at room
temperature. After starting material was completely disappeared, the mixture
was filtered through Celite, evaporated, and then the residue was purified by
column chromatography using the 2 : 1 cHx–EtOAc as eluent to give color-
less solid 10 (63 mg, 41.9%): mp 59—61 °C; [a]D25 1.0° (cꢂ1, CHCl3); IR
(cmꢁ1, KBr) 3525 (O–H stretch), 2920 (sp3–H stretch), 1730 (conj. CꢂO
stretch), 1600 and 1450 (aromatic CꢂC stretch), 1280 and 1100 (C–O
1
stretch); H-NMR (400 MHz, CDCl3): d 8.02—7.23 (m, 27H, Ar-H), 6.84
(d, Jꢂ8.4 Hz, 2H, Ar-H), 5.88 (t, Jꢂ9.6 Hz, 1H, H-3ꢄ), 5.65 (t, Jꢂ9.6 Hz,
1H, H-4ꢄ), 5.56 (dd, Jꢂ9.6 Hz, Jꢃꢂ8.0 Hz, 1H, H-2ꢄ), 5.07 (d, Jꢂ7.6 Hz,
1H, H-1ꢄ), 4.78 (d, Jꢂ11.2 Hz, 1H, –CH2MPM), 4.63 (dd, Jꢂ12.0 Hz,
Jꢃꢂ3.2 Hz, 1H, H-5ꢄ), 4.51—4.45 (m, 4H, H-6ꢄ (2H), –CH2MPM (1H), and
–CH2Ph (1H)), 4.14 (d, Jꢂ7.2 Hz, 1H, H-1ꢃ), 4.05—4.03 (m, 2H, –CH2Ph
and H-5aꢃ), 3.91 (br s, 1H, H-4ꢃ), 3.79 (s, 3H, –OCH3), 3.48 (t, Jꢂ6.8 Hz,
1H, H-3ꢃ), 3.37 (d, Jꢂ12.4 Hz, 1H, H-2ꢃ), 3.31 (dd, Jꢂ9.4 Hz, Jꢃꢂ3.2 Hz,
1H, H-5bꢃ); ESI-MS: m/zꢂ962 [MꢇNa]ꢇ; HR-FAB-MS: m/zꢂ961.9561
[MꢇNa]ꢇ (Calcd for C54H50O15Na, 961.9556).
4-Methoxybenzyl 2-O-Allyl-a-L-arabinopyranoside (5) Compound 5
was prepared from 4-methoxybenzyl a-L-arabinopyranoside (4, 1.5 g,
5.55 mmol) by following the same procedure described in ref. 8. The crude
product was purified by gradient column chromatography (cyclohexnae
(cHx)/ethyl acetate (EtOAc), 3 : 1→1 : 1) to yield pure 5 (1.22 g, 3.94 mmol,
71%) as white solid, and the physical, chemical, and spectral NMR data
were exactly accorded with the above reference.
Allyl-2,3,4-tri-O-benzoyl-a-L-rhamnopyranosyl-(1→2)-3,4-O-iso-
propylidene-b-L-arabinopyranoside (15) A suspension of 13 (300 mg,
1.58 mmol),12) 2,2-dimethoxypropane (388 ml, 3.16 mmol), and p-TsOH
(30 mg, 0.16 mmol) in dry DMF (20 ml) was stirred for 6 h at room tempera-
ture. The reaction mixture was stopped by the addition of Et3N (100 ml) and
then the mixture was diluted with diethyl ether (Et2O, 100 ml) and washed
with H2O (100 ml). The aqueous layer was extracted with Et2O (100 mlꢆ3).
The organic layers was combined, washed with satd. NaHCO3 (400 ml) and
satd. NaCl (400 ml), and dried with Na2SO4. Solvents were evaporated and
the residue (allyl-(1→2)-3,4-O-isopropylidene-b-L-arabinopyranoside, 14)
was used without further purification in the following step.
4-Methoxybenzyl 2-O-Allyl-3-O-benzyl-a-L-arabinopyranoside (6)
A solution of 5 (125 mg, 0.4 mmol) and Bu2SnO (120 mg, 0.48 mmol) in dry
MeOH (10 ml) was refluxed for 3 h until the solution became clear. Solvents
were evaporated under reduced pressure with toluene. The residue was dis-
solved in dry toluene, benzyl bromide (120 ml, 1.0 mmol) and Bu4NBr
(156 mg, 0.48 mmol) were added and the mixture was stirred at 60 °C for
16 h. Solvents were evaporated and residue was separated by column chro-
matography using cHx–EtOAc (3 : 1) to afford compound 6 (130 mg, 81.2%)
as colorless syrup: [a]D25 ꢁ0.3° (cꢂ1, CHCl3); IR (cmꢁ1, CH2Cl2) 3530
(O–H stretch), 2930 (sp3–H stretch), 1715 (conj. CꢂO stretch), 1600 (CꢂC
stretch), 1530 and 1440 (aromatic CꢂC stretch), 1260 and 1100 (C–O
A solution of this isopropylidene 14 (278 mg, 1.21 mmol), trichloroace-
timidate 11 (977 mg, 1.57 mmol),18) and 4 Å molecular sieves (500 mg) in
dry CH2Cl2 (20 ml) was treated with TMSOTf (0.1 eq) in the same manner
as that described for compound 9. After stirring for 2 h at this temperature,
the mixture was neutralized with triethylamine (Et3N), filtered with Celite,
and then concentrated in vacuo. The residue was purified by a Si gel column
chromatography using the 5.5 : 1 cHx–EtOAc as eluent to give colorless
syrup 15 (434 mg, 63.0%): [a]D25 1.4° (cꢂ1, CHCl3); IR (cmꢁ1, CH2Cl2)
2920 (sp3–H stretch), 1730 (conj. CꢂO stretch), 1600 (CꢂC stretch), 1580
1
stretch); H-NMR (400 MHz, CDCl3) d 7.35—7.24 (m, 7H, Ar-H), 6.85 (d,
Jꢂ8.8 Hz, 2H, Ar-H), 5.88 (m, 1H, –CH2CHꢂCH2), 5.23 (d, Jꢂ17.2 Hz,
1H, –CH2CHꢂCH2), 5.14 (d, Jꢂ10.4 Hz, 1H, –CH2CHꢂCH2), 4.81 (d,
Jꢂ11.6 Hz, 1H, –CH2MPM), 4.74 (d, Jꢂ12.0 Hz, 1H, –CH2Ph), 4.66 (d,
Jꢂ11.6 Hz, 1H, –CH2Ph), 4.54 (d, Jꢂ11.6 Hz, 1H, –CH2MPM), 4.34 (d,
Jꢂ6.4 Hz, 1H, H-1ꢃ), 4.31 (dd, Jꢂ12.8 Hz, Jꢃꢂ5.6 Hz, 1H, –CH2CHꢂCH2),
4.15 (dd, Jꢂ12.8 Hz, Jꢃꢂ5.6 Hz, 1H, –CH2CHꢂCH2), 3.97 (dd, Jꢂ10.4 Hz,
Jꢃꢂ3.2 Hz, 1H, H-5aꢃ), 3.89 (br s, 1H, H-4ꢃ), 3.78 (s, 3H, –OCH3), 3.56 (t,
Jꢂ7.6 Hz, 1H, H-3ꢃ), 3.46 (dd, Jꢂ8.4 Hz, Jꢃꢂ3.6 Hz, 1H, H-5bꢃ), 3.38 (d.
Jꢂ12.4 Hz, 1H, H-2ꢃ); ESI-MS: m/zꢂ366 [MꢇNa]ꢇ; HR-FAB-MS:
m/zꢂ366.3831 [MꢇNa]ꢇ (Calcd for C20H23O5Na, 366.3834).
1
and 1460 (aromatic CꢂC stretch), 1260 and 1120 (C–O stretch); H-NMR
(400 MHz, CDCl3) d 8.12—7.22 (m, 15H, Ar-H), 6.07—5.97 (m, 1H,
–CH2CHꢂCH2), 5.89 (dd, Jꢂ10 Hz, Jꢃꢂ3.6 Hz, 1H, H-2ꢄ), 5.81—5.79 (m,
1H, H-4ꢄ), 5.64 (t, Jꢂ9.6 Hz, 1H, H-3ꢄ), 5.44 (dd, Jꢂ17.4 Hz, Jꢃꢂ1.6 Hz,
1H, –CH2CHꢂCH2), 5.34 (d, Jꢂ2.0 Hz, 1H, H-1ꢄ), 5.29 (dd, Jꢂ10.4 Hz,
Jꢃꢂ1.2 Hz, 1H, –CH2CHꢂCH2), 4.98 (d, Jꢂ3.6 Hz, 1H, H-1ꢃ), 4.44 (dd,
Jꢂ7.6 Hz, Jꢃꢂ5.6 Hz, 1H, H-5ꢄ), 4.31—4.24 (m, 3H, –CH2CHꢂCH2, H-4ꢃ,
and H-2ꢃ), 4.05 (dd, Jꢂ13.2 Hz, Jꢃꢂ6.4 Hz, 1H, –CH2CHꢂCH2), 4.00 (s,
2H, H-5ꢃ), 3.87 (dd, Jꢂ7.8 Hz, Jꢃꢂ3.6 Hz, 1H, H-3ꢃ), 1.55 (s, 3H,
–O(CH3)2CO–), 1.37 (s, 3H, –O(CH3)2CO–), 1.33 (d, Jꢂ6.0 Hz, 1H, H-6ꢄ);
ESI-MS: m/zꢂ712 [MꢇNa]ꢇ; HR-FAB-MS: m/zꢂ711.7066 [MꢇNa]ꢇ
(Calcd for C38H40O12Na, 711.7068).
Allyl-2,3,4-tri-O-benzoyl-a-L-rhamnopyranosyl-(1→2)-b-L-ara-
binopyranoside (16) p-TsOH (77 mg, 0.41 mmol) was add to a solution of
15 (400 mg, 0.58 mmol) in CH2Cl2–MeOH (1 : 2, 30 ml) and the mixture was
stirred at room temperature for 6 h when the deprotection had completed on
TLC (cHx–EtOAcꢂ3 : 1), and then Et3N (0.1 ml) was added and the mixture
was concentrated and purified by a Si gel column chromatography using the
4-Methoxybenzyl 2,3,4,6-Tetra-O-benzoyl-b-D-glucopyranosyl-(1→4)-
2-O-allyl-3-O-benzyl-a-L-arabinopyranoside (9) A suspension of com-
pound
6
(240 mg, 0.60 mmol), trichloroacetimidate 813,17) (533 mg,
0.72 mmol), and 4 Å molecular sieves (500 mg) in dry CH2Cl2 (20 ml) was
stirred for 1 h at room temperature under N2 atmosphere. The mixture was
cooled to ꢁ20 °C, then was added TMSOTf (0.1 eq, 13 ml) as catalyst via sy-
ringe. After stirring for 2 h at same temperature, the mixture was neutralized
with triethylamine (Et3N), filtered with Celite, and then concentrated in
vacuo. The residue was purified on a Si gel column chromatography using
the 3 : 1 cHx–EtOAc as eluent to give colorless solid 9 (483 mg, 82.7%) con-
taminated with a little impurity. An additional purification step using prepar-
ative HPLC elution with 80% MeCN (flow rate: 1 ml/min, tRꢂ17.53 min)

1738
Vol. 55, No. 12
3 : 1 cHx–EtOAc as eluent to give 16 (373 mg, 99.0%) as a white amorphous
solid: mp 130—133 °C; [a]D25 0.9° (cꢂ1, CHCl3); IR (cmꢁ1, KBr) 3400
chromatography using the gradient solvent system (cHx–EtOAc, 3 : 1→1 : 1)
as eluent to give the desired compound 3 (99 mg, 62.0%) and is finally re-
(O–H stretch), 2930 (sp3–H stretch), 1720 (conj. CꢂO stretch), 1600 (CꢂC crystallized as a colorless amorphous solid (86 mg, 52.9%) from petroleum
stretch), 1580 and 1460 (aromatic CꢂC stretch), 1270 and 1120 (C–O
stretch); 1H-NMR (400 MHz, CDCl3) d 8.05—7.23 (m, 15H, Ar-H), 6.06— KBr) 2920 (sp3–H stretch), 1730 (conj. CꢂO stretch), 1600 (CꢂC stretch),
5.96 (m, 1H, –CH2CHꢂCH2), 5.85 (dd, Jꢂ10 Hz, Jꢃꢂ3.6 Hz, 1H, H-2ꢄ),
1450 (aromatic CꢂC stretch), 1260 and 1110 (C–O stretch); 1H-NMR
5.80—5.79 (m, 1H, H-4ꢄ), 5.6 (t, Jꢂ10.0 Hz, 1H, H-3ꢄ), 5.40 (dd, (400 MHz, CDCl3) d 8.09—6.94 (m, 40H, Ar-H), 6.02—5.95 (m, 1H,
Jꢂ17.2 Hz, Jꢃꢂ1.6 Hz, 1H, –CH2CHꢂCH2), 5.25—5.23 (m, 2H, H-1ꢄ and –CH2CHꢂCH2), 5.83—5.76 (m, 2H, H-3ꢈ and H-2ꢄ), 5.69—5.58 (m, 4H,
–CH2CHꢂCH2), 5.10 (d, Jꢂ3.6 Hz, 1H, H-1ꢃ), 4.43—4.36 (m, 1H, H-5ꢄ), H-4ꢈ, H-4ꢄ, H-3ꢄ, and H-2ꢈ), 5.37 (d, Jꢂ17.2 Hz, –CH2CHꢂCH2), 5.24 (s,
4.29 (dd, Jꢂ13.0 Hz, Jꢃꢂ5.0 Hz, 1H, –CH2CHꢂCH2), 4.21 (d, Jꢂ9.2 Hz, 1H, H-1ꢄ), 5.21 (d, Jꢂ10.8 Hz, 1H, –CH2CHꢂCH2), 5.02 (d, Jꢂ3.6 Hz, 1H,
ether–MeOH (3 : 1): mp 94—97 °C; [a]D25 0.9° (cꢂ1, CHCl3); IR (cmꢁ1
,
1H, H-4ꢃ), 4.03—3.95 (m, 3H, H-2ꢃ, –CH2CHꢂCH2 and H-3ꢃ), 3.88 (d, H-1ꢃ), 4.88 (s, 1H, H-1ꢈ), 4.75 (dd, Jꢂ14.0 Hz, Jꢃꢂ5.2 Hz, 1H, H-4ꢈ), 4.55
Jꢂ12.4 Hz, 1H, H-5aꢃ), 3.76 (dd, Jꢂ12.4 Hz, Jꢃꢂ1.6 Hz, 1H, H-5bꢃ), 1.32 (d, Jꢂ9.2 Hz, 2H, H-6ꢈ), 4.42—4.36 (m, 2H, H-5ꢈ and H-5ꢄ), 4.25 (dd,
(d, Jꢂ6.4 Hz, 1H, H-6ꢄ); ESI-MS: m/zꢂ972 [MꢇNa]ꢇ; HR-FAB-MS: Jꢂ13.2 Hz, Jꢃꢂ5.2 Hz, 1H, –CH2CHꢂCH2), 4.08 (s, 1H, H-2ꢃ), 3.98 (dd,
m/zꢂ671.6423 [MꢇNa]ꢇ (Calcd for C35H36O12Na, 671.6429).
Jꢂ9.2 Hz, Jꢃꢂ3.6 Hz, 1H, H-3ꢃ), 3.92 (dd, Jꢂ12.8 Hz, Jꢃꢂ6.0 Hz, 1H,
–CH2CHꢂCH2), 3.78 (d, Jꢂ12.4 Hz, 1H, H-5aꢃ), 3.68 (d, Jꢂ12.4 Hz, 1H,
H-5bꢃ), 1.32 (d, Jꢂ6.4 Hz, 1H, H-6ꢄ); ESI-MS: m/zꢂ1354 [MꢇNa]ꢇ; HR-
FAB-MS: m/zꢂ1354.3129 [MꢇNa]ꢇ (Calcd for C76H66O22Na, 1354.3138).
Cell Culture Assay The cytotoxicity assay was carried out according to
Allyl-2,3,4-tri-O-benzoyl-a-L-rhamnopyranosyl-(1→2)-4-O-acetyl-b-
L-arabinopyranoside (18)
A solution of 16 (346 mg, 0.53 mmol),
CH3C(OEt)3 (483 ml, 2.65 mmol) and p-TsOH (50 mg, 0.265 mmol) in dry
toluene (10 ml) was stirred at room temperature for 2 h. The reaction mix-
ture was neutralized by the addition of Et3N (300 ml) at the same tempera- the SRB assay described previously.27) Three human cancer cell lines, A-549
ture. The solvent was removed in vacuo, the residue was dissolved in (Lung cancer), SK-OV-3 (Ovarian cancer) and SK-MEL-2 (Skin cancer)
CH2Cl2 (100 ml), washed with water (100 mlꢆ3). After drying over Na2SO4, were examined, and doxorubicin was used as the positive control (ED50:
the extract was concentrated to give cyclic ortho ester intermediate 17 as a
colorless syrup. The crude was then dissolved in 80% aq. HOAc (10 ml) and
0.02, 0.1, 0.04 mM, respectively). Growth inhibition of 50% (ED50) of 3 cal-
culated using the method described elsewhere.28) Briefly, the cells were di-
stirred at room temperature for 1.5 h. Coevaporation with toluene yielded a vided into 96-well plates and preincubated on the plates for 24 h. The com-
colorless syrup, which was then subjected to Si gel column chromatography pounds were added to the wells and incubated for 48 h. After incubation, the
using the 2 : 1 cHx–EtOAc as eluent to give 18 (259 mg, 70.8%) as a white culture medium in each well was removed, and the cells were fixed with cold
foam: mp 69—71 °C; [a]D25 1.0° (cꢂ1, CHCl3); IR (cmꢁ1, KBr) 3450 (O–H
10% trichloroacetic acid. Subsequently, a 0.4% SRB solution in 1% acetic
stretch), 2950 (sp3–H stretch), 1730 (conj. CꢂO stretch), 1600 (CꢂC acid was added to each well. The optical density was measured in a mi-
stretch), 1580 and 1460 (aromatic CꢂC stretch), 1260 and 1120 (C–O
stretch); 1H-NMR (400 MHz, CDCl3) d 8.08—7.24 (m, 15H, Ar-H), 6.06—
crotiter plate reader at 540 nm.
5.97 (m, 1H, –CH2CHꢂCH2), 5.85 (dd, Jꢂ10.2 Hz, Jꢃꢂ3.4 Hz, 1H, H-2ꢄ), References
5.77 (m, 1H, H-4ꢄ), 5.67 (t, Jꢂ10.0 Hz, 1H, H-3ꢄ), 5.42 (dd, Jꢂ17.2 Hz,
Jꢃꢂ1.6 Hz, 1H, –CH2CHꢂCH2), 5.28 (d, Jꢂ1.6 Hz, H-1ꢄ), 5.27 (dd,
Jꢂ9.2 Hz, Jꢃꢂ1.2 Hz, 1H, –CH2CHꢂCH2), 5.11 (d, Jꢂ4.0 Hz, 1H, H-1ꢃ),
4.37 (dd, Jꢂ9.6 Hz, Jꢃꢂ6.4 Hz, 1H, H-5ꢄ), 4.32 (dd, Jꢂ10.0 Hz, Jꢃꢂ4.0 Hz,
1H, –CH2CHꢂCH2), 4.27 (d, Jꢂ4.8 Hz, 1H, H-4ꢃ), 4.01—3.97 (m, 3H, H-
2ꢃ, –CH2CHꢂCH2 and H-3ꢃ), 3.92 (d, Jꢂ12.0 Hz, 1H, H-5aꢃ), 3.74 (dd,
Jꢂ13.2 Hz, Jꢃꢂ1.6 Hz, 1H, H-5bꢃ), 1.34 (d, Jꢂ6.0 Hz, 1H, H-6ꢄ); ESI-MS:
m/zꢂ714 [MꢇNa]ꢇ; HR-FAB-MS: m/zꢂ713.6799 [MꢇNa]ꢇ (Calcd for
C37H38O13Na, 713.6796).
1) Hostettmann K., Marston A., “Chemistry and Pharmacology of Nat-
ural Products; Saponins,” Cambridge University Press, London, 1995,
p. 18.
2) Shaojiang D., Biao Y., Jianming X., Yongzheng H., J. Org. Chem., 64,
7265—7266 (1999).
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Allyl-2,3,4-tri-O-benzoyl-a-L-rhamnopyranosyl-(1→2)-3-O-benzoyl-
b-L-arabinopyranoside (20) To a solution of 18 (240 mg, 0.35 mmol) in
dry pyridine (10 ml), BzCl (122 ml, 1.05 mmol) was added slowly at 0 °C.
After addition complete, the reaction mixture was warmed up to room tem-
perature and stirred for overnight. Water (0.40 ml) was added slowly to
quench the reaction and the solvent was removed in vacuo. The resulting
residue was dissolved in CH2Cl2 (50 ml), washed with water (50 mlꢆ3), and
then dried over Na2SO4 to afford crude compound 19 as a white amorphous
solid, which was then dissolved in dry CH2Cl2–MeOH (1 : 2, 15 ml). To the
solution was added AcCl (300 ml) at 0 °C, and the solution was allowed to
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warm to room temperature and stirred for 12 h when starting material was 10) Brown R. S., Wahl R. I., Cancer, 72, 2979—2983 (1993).
completely disappeared. Et3N (1.20 ml) was added to neutralize the acid. 11) Yamamoto I., Sekine M., Hata T., J. Chem. Soc. Perkin 1, 1980, 306—
The solution was then concentrated and purified with a Si column chro-
310 (1980).
matography using the 1 : 1 cHx–EtOAc as eluent to give 20 (180 mg, 68.3%)
12) Polt R., Porreca F., Szabo L. Z., Bilsky E. J., Davis P., Abbruscato T. J.,
Davis T. P., Horvath R., Yamamura H. I., Hruby V. J., Proc. Natl. Acad.
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Pharmacol., 318, 477—484 (1996).
as a colorless solid: mp 75—78 °C; [a]D25 0.4° (cꢂ1, CHCl3); IR (cmꢁ1
,
KBr) 3450 (O–H stretch), 2920 (sp3–H stretch), 1740 (conj. CꢂO stretch),
1600 (CꢂC stretch), 1520 and 1460 (aromatic CꢂC stretch), 1260 and 1100
1
(C–O stretch); H-NMR (400 MHz, CDCl3) d 8.12—7.23 (m, 20H, Ar-H),
6.06—5.96 (m, 1H, –CH2CHꢂCH2), 5.87—5.82 (m, 2H, H-2ꢄ and H-4ꢄ),
5.64 (t, Jꢂ9.6 Hz, 1H, H-3ꢄ), 5.41 (dd, Jꢂ17.2 Hz, Jꢃꢂ1.6 Hz, 1H,
14) Uriel C., Egron M. J., Santarromana M., Scherman D., Antonakis K.,
Herscovici J., Bioorg. Med. Chem., 4, 2081—2090 (1996).
–CH2CHꢂCH2), 5.25 (s, 1H, H-1ꢄ), 5.24 (dd, Jꢃꢂ10.4 Hz, Jꢃꢂ1.6 Hz, 1H, 15) James K. T., Wang S. P., Mary B. T., Shawn B., Daniel J. R., Dexter L.
–CH2CHꢂCH2), 5.12 (d, Jꢂ3.6 Hz, 1H, H-1ꢃ), 4.44—4.36 (m, 1H, H-5ꢄ),
4.29 (dd, Jꢂ13.2 Hz, Jꢃꢂ5.2 Hz, 1H, –CH2CHꢂCH2), 4.22 (d, Jꢂ7.6 Hz,
M., David S. W., Donald C. L., Proc. Natl. Acad. Sci. U.S.A., 103,
10461—10466 (2006).
1H, H-4ꢃ), 4.11 (s, 1H, H-2ꢃ), 4.03 (dd, Jꢂ9.8 Hz, Jꢃꢂ3.4 Hz, 1H, H-3ꢃ), 16) Maoquan Z., George A. O’Doherty, J. Org. Chem., 72, 2485—2493
3.97 (dd, Jꢂ13.0 Hz, Jꢃꢂ5.8 Hz, 1H, –CH2CHꢂCH2), 3.88 (d, Jꢂ12.8 Hz,
1H, H-5aꢃ), 3.77 (dd, Jꢂ12.4 Hz, Jꢃꢂ1.6 Hz, 1H, H-5bꢃ), 1.33 (d, Jꢂ6.4 Hz,
1H, H-6ꢄ); ESI-MS: m/zꢂ776 [MꢇNa]ꢇ; HR-FAB-MS: m/zꢂ775.7493
[MꢇNa]ꢇ (Calcd for C42H40O13Na, 775.7490).
Allyl-2,3,4-tri-O-benzoyl-a-L-rhamnopyranosyl-(1→2)-[2,3,4,6-tetra-
O-benzoyl-b-D-glucopyranosyl-(1→4)]-3-O-benzoyl-b-L-arabinopyra-
noside (3) A suspension of 20 (90 mg, 0.12 mmol), trichloroacetimidate 8
(107 mg, 0.14 mmol), and 4 Å molecular sieves (300 mg) in dry CH2Cl2
(10 ml) was treated with TMSOTf (2 ml, 0.1 eq) in the same manner as that
described for compound 9. The product was purified by a Si gel column
(2007).
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1739
New York, 2005, pp. 739—740.
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