Dimero de Aglicona

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Synthesis of a highly potent antitumor saponin OSW-1 and its analogues

Jacek W. Morzycki* & Agnieszka WojtkielewiczInstitute of Chemistry, University of Bialystok, Pilsudskiego 11/4, 15-443, Bialystok, Poland; *Author for

correspondence (Tel: +48-85-7457585; Fax: +48-85-7457581; E-mail: [email protected])

Key words: antitumor activity, cholestane glycosides, OSW-1, saponins, steroids

Abstract

Twelve years ago a group of cholestane glycosides was isolated from the bulbs of Ornithogalum saundersiae,a species of the lily family without any medicinal folklore background. Similar glycosides were recently

isolated from Galtonia candicans. The major component of the mixture of saponins, OSW-1, exhibited sub-

nanomolar antineoplastic activity. While OSW-1 is exceptionally cytotoxic against various tumor cells, it

shows little toxicity with normal human pulmonary cells. In this review article the synthetic efforts towards

OSW-1 and related cholestane glycosides, as well as the preliminary results of the structureactivity rela-

tionship study are presented.

Introduction

The control of cancer, the second leading cause of

death worldwide, may benefit from the potential

that resides in natural products. Some of them,

originating from plants, have been implicated in

cancer treatment without recognizable side effects

(Reddy et al., 2003). One of such very promising

natural products is the saponin OSW-1, isolated

by Japanese scientists in 1992 from the bulbs of

Ornithogalum saundersiae (Kubo et al., 1992). It is

a perennial garden plant of the lily family endemic

to the Drakensberg Mountains in Africa. The

common name of the plant, Ivory Coast Lily, is

geographically incorrect because it is endemic to

the east coast of Africa; the plant is also known asthe giant chincherinchee. O. saundersiae is the

giant member of the Ornithogalum genus, which

belongs to the sub-family Schilloideae in Liliaceae,

comprised of about 150 species (Bryan, 1989). The

plant flowers normally in open fields all year round

in Kenya and it is one of the top ten cut flowers

exported to Europe in this country (Kariuki et al.,

1999). However, there are some problems in

commercial production which include variability

and instability in yield and quality of cut flowers;

the plant is also commercially grown for cut

flowers in Holland, Israel and Tasmania. The

multi flowered clusters are striking because each

white petaled flower has a dark green center. The

flower stems are tall and strong, up to 1.15 m

height which makes it a versatile flower for use in

floral art. Several cardenolide glycosides have been

previously found in some species of Ornithogalum

(Buchvarov et al., 1984; Ghannamy et al., 1987).

A phytochemical screening of the bulbs of

Ornithogalum saundersiae has proven the lack of

cardenolide glycosides and the presence of mono-

and bisdesmosidic cholestane glycosides. The gly-

cosides have shown considerable inhibitory activ-

ity on cyclic AMP phosphodiesterase (Kubo et al.,1992). A few years later it appeared that a

methanolic extract of Ornithogalum saundersiae, a

species without any medicinal folklore back-

ground, showed exceptional cytostatic activity

against various malignant tumor cells (Mimaki

et al., 1997). A group of cholestane glycosides was

isolated from the extract including saponin OSW-1

as the main component of t he mixtur e

(Figure 1).

Phytochemistry Reviews (2005) 4: 259277 Springer 2005

DOI 10.1007/s11101-005-1233-6


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In vitro assays have shown that OSW-1 is

extremely toxic against a broad spectrum of tumor

cells, such as leukemia HL-60, mouse mastrocar-

cinoma, human pulmonary adenocarcinoma, hu-

man pulmonary large cell carcinoma, and human

pulmonary squamous cell carcinoma including

adriamycin-resistant P388 leukemia and campto-

thecin-resistant P388. The IC50s are between 0.1

and 0.7 nM, which means a cytotoxicity about

10100 times more potent than that of the clini-

cally applied anticancer agents, such as mitomycin

C, adriamycin, cisplatin, camptothecin, and taxol

(Table 1). While OSW-1 is exceptionally cytotoxicagainst various malignant tumor cells, it shows

little toxicity (IC50 1500 nM) to normal human

pulmonary cells. The cytotoxicity profile of OSW-1

is strikingly similar to that of cephalostatins, a

group of dimeric steroidpyrazines from marine

organisms (Gryszkiewicz-Wojtkielewicz et al., 2003)

with Pearson correlation coefficients between 0.60

and 0.83. Structurally, the aglycone of OSW-1 is

reminiscent of half of the cephalostatins. Fuchs

therefore hypothesized that these compounds

might have the same mechanism of action (Guo

and Fuchs, 1998). The comparison of pGI50 valuesfor saponin OSW-1, cephalostatin 1 and clinically

applied anticancer agents is presented in Table 2.

Further analysis of the bulbs of O. saundersiae

resulted in the isolation of a group of cholest-5-en-

3b,11a,16b,22-tetraol 16-O-rhamnosides, which ap-

peared to be less cytotoxic than OSW-1 (Kuroda

et al., 1999).

During examination of plants taxonomically

related to O. saundersiae (liliaceae family) it was

found that a methanolic extract of the bulbs of

Figure 1. OSW-1 and related saponins.

Table 1. Cytostatic activities of OSW-1 and clinically applied anticancer agents on various malignant tumor cells

(Mimaki et al. 1997).

Malignant cells IC50 (mg/ml)

OSW-1 Mitomycin C Adriamycin Cisplatin Camptothecin Taxol

CCD-19Lu 1.5 2 2 10 2 2

P388 0.00013 0.01 0.003 0.05 0.005 0.01

P388/ADM 0.00077

P388/CPT 0.00010

FM3A 0.00016

A-549 0.00068

Lu-65 0.00020

Lu-99 0.00020 0.01 0.002 0.001 0.001 0.002

RERF-LC-AI 0.00026

CCRF-CEM 0.00016 0.02 0.01 0.005 0.005 0.001

CCD-19Lu (human normal pulmonary cell)P388 (mouse leukemia)

P388/ADM (adriamycin-resistant P388)

P388/CPT (camptothecin-resistant P388)

FM3A (mouse mastrocarcinoma)A-549 (human pulmonary adenocarcinoma)

Lu-65 (human pulmonary large cell carcinoma)

Lu-99 (human pulmonary large cell carcinoma)

RERF-LC-AI (human pulmonary squamous cell carcinoma)CCRF-CEM (human leukemia)

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Galtonia candicans showed potent cell growth

inhibitory activity against HL-60 human prom-

yelocytic leukemia cells. The common name of

G. candicans is summer hyacinth and it is a peren-

nial plant native to the Cape Province of South

Africa. The long flower spike of G. candicans has

1020 flower bells distributed along the flowerspike, and looks like a large elongated Hyacinth.

G. candicans has a very pleasant fragrance. Use of a

cytotoxicity-guided fractionation procedure, com-

bined with a 3-(4,5-dimethylthiazol-2-yl)-2,5-

diphenyl-2H-tetrazolium bromide (MTT) reduc-

tion assay method for cytotoxicity evaluation

(Sargent and Taylor, 1989), led to the isolation

(Figure 2) of a polyoxygenated 5b-cholestane

diglycoside galtonioside A (Kuroda et al., 2000)

and a hexacyclic rearranged cholestane diglyco-

side candicanoside A (Mimaki et al., 2000).

However, the yield and cytotoxic potency of theseproducts were not enough to explain the potent

cytotoxicity of the crude extract, suggesting the

Table 2. Comparison of cytostatic activity of saponin OSW-1

and clinically applied chemotherapeutics (Guo and Fuchs,1998; Weinstein et al., 1992).

Compound pGI50 (mean value for

60 cancer cells)

Cyclophosphamid 3.7

5-Fluorouracyl 4.7

Cisplatin 5.7

Adriamycin 6.9

Taxol 7.9

Cephalostatin 1 8.3

OSW-1 9.1

Figure 2. Other cholestane glycosides from O. saundersiae and G. candicans.

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presence of other, more potent principles. A further

search aimed at finding cytotoxic components from

G. candicans bulb extract resulted in the isolation

of cholestane glycosides (Kuroda et al., 2001),closely related to saponins found in O. saundersiae

(3-O-glucosylated derivatives).

Immediately after recognition of the high

cytotoxic activity of saponin OSW-1, numerous

groups of chemists around the world undertook

the synthesis of this compound. Saponin OSW-1

can be logically disconnected into two parts: the

cholestane aglycone and the disaccharide moiety.

The first synthesis of the OSW-1 aglycone was

accomplished by Fuchs in 1998 (Guo and Fuchs,

1998), a year after the discovery of the extraordi-

nary cytotoxicity of saponin OSW-1 (Mimakiet al., 1997). A few months later Yu et al. pub-

lished the first total synthesis of saponin OSW-1

(Deng et al., 1999) by coupling of the aglycone

with the sugar part. New approaches for the syn-

thesis of this natural product were presented by

Jins (Yu and Jin, 2001a, 2002), Morzyckis

(Morzycki and Wojtkielewicz, 2002) and Tians

(Xu et al., 2003) groups.

In the section below various syntheses of

saponin OSW-1 and its analogues will be pre-

sented. Then the structurebiological acitivity

relationships will be discussed.

Synthesis of saponin OSW-1 and its analogues

Fuchs synthesis of the aglycone of saponin OSW-1

(Guo and Fuchs, 1998)

Construction of the 22-oxygenated side chain was

performed by several steps starting from the

commercially available material androst-5-en-

3b-ol-17-one (1). Wittig olefination was followed

by acetylation and a stereoselective ene reaction of

compound 2 (Figure 3). The obtained 22-alcohol 3

was oxidized with a Jones reagent to the ketone 4

and then the carbonyl group was protected as a

ketal. The key step in the synthesis of the aglycone

of saponin OSW-1 was the introduction of the

trans diol function in ring D (16b,17a-diol). Fuchsinitially attempted opening of the 16a,17a-epox-

ide. However, all attempts to cleave the oxirane

ring in the presence of the side chain failed.

Altering the sequence of reactions did not solve the

problem. Due to these difficulties, the D16olefin

was subjected to dihydroxylation with a stoichi-

ometric amount of OsO4. In the next step the cis

diol 5 was oxidized with the Swern reagent to the

16-ketone. The stereoselective reduction of the

ketone with NaBH4/CeCl3 at low temperature

afforded a 16b,17a-diol 6 the aglycone of sapo-

nin OSW-1 in the protected form.

Figure 3. Fuchs synthesis of the OSW-1 aglycone.

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Yu and Hui strategy the first synthesis of saponin

OSW-1 (Deng et al., 1999)

The desired steroid aglycone was also synthesizedfrom androst-5-en-3b-ol-17-one (1) by Yu and

Hui, who used a strategy similar to that employed

by Fuchs (Figure 4). The major difference con-

sisted in the construction of the side chain, where

the ene reaction was performed with paraformal-

dehyde (in Fuchs method with 4-methylvaleral-

dehyde). The obtained primary alcohol 7 was then

oxidized to the aldehyde 8, which in turn reacted

with a Grignard reagent. The secondary alcohol

obtained in this way was further transformed into

the OSW-1 aglycone using Fuchs method.

The sugar moiety of OSW-1 consists of acyl-ated L-arabinose and D-xylose. For synthesis of the

disaccharide, the choice of protecting groups was

very important. The xylose donor for coupling

with arabinose was prepared in the following way

(Figure 5). Upon the protection of the anomeric

group as a benzyl ether the 2-OH was regioselec-

tively masked as a 4-methoxybenzoate and the rest

of the hydroxyl groups (3-OH and 4-OH) were

transformed into TES-ethers. In the next step, the

1-OH group was deprotected (50 atm. H2; Pd/C)

and then converted into a trichloracetimidate 11, a

convenient glycosyl donor. The arabinosyl accep-

tor 13 was synthesized from L-arabinose in foursteps: benzylation of the anomeric hydroxyl group,

isopropylidenation of the 3- and 4-hydroxyl

groups, acetylation of the 2-OH and the final re-

moval of the acetonide protection. Although the

acceptor prepared in this manner contained twohydroxyl groups, selective glycosylation of the diol

13 at the equatorial 3-OH group with the xylosyl

donor 11 catalyzed by BF3*Et2O (Schmidt and

Michel, 1980) afforded the desired disaccharide.

The remaining hydroxyl group in this compound

was then protected as a TES-ether and the benzyl

protecting group was removed. In the last step the

trichloroacetimidate 14 was prepared as the reac-

tive disaccharide donor for glycosylation reac-

tions.

The glycosylation (Figure 6) of the aglycone 6

under TMSOTf catalysis (Schmidt and Toepfer,1991) proceeded smoothly affording the protected

saponin OSW-1. All protecting groups (TBS, TES,

ethylene glycol ketal) were removed in one step

with Pd(MeCN)2Cl2 as catalyst (Lipshutz et al.,

1985). Saponin OSW-1 was synthesized in 27 steps

in 6% overall yield.

Yu and Jin strategy for the synthesis of saponin

OSW-1 (Yu and Jin, 2001a, 2002)

A completely different approach for the synthesis

of OSW-1 was proposed by American chemists in

2001 (Figure 7). The strategy was based on the1,4-addition of an a-alkoxy vinyl cuprate to the

Figure 4. Yu et al. synthesis of the OSW-1 aglycone.

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steroid enone. The starting material was androst-

5-en-3b-ol-17-one (1), as in the previous cases,

which was subjected to Wittig olefination

followed by allylic oxidation at C-16 with SeO2/

t-BuOOH (Snider and Shi, 1999), without pro-

tection of the double bond in ring B. Swern

Figure 6. Glycosylation reaction.

Figure 5. Synthesis of the OSW-1 disaccharide.

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oxidation of the obtained 16b-alcohol 15 afforded

the enone 16 in nearly quantitative yield. TMSCl-

activated (Corey and Boaz, 1985; Alexakis et al.,1986) 1,4-addition of a suitable a-alkoxy vinyl

cuprate to this compound gave a silyl ether 17,

which was converted to the enol acetate 18

without isolation. This conversion allowed for the

chemoselective transformation of the 22-enol ether

18 to the cyclic ketal 19. In the next step the 17a-

hydroxyl group was introduced by Davis oxidation

(Davis and Sheppard, 1989) of the enolate, obtained

from enol acetate 19 by treatment with t-BuOK

(Duhamel et al., 1993; Yu and Jin, 2001b). Stereo-

selective reduction of the 17a-hydroxy-16-ketone 20

with LAH at )78 C afforded the desired 16b,17a-

diol 6 protected aglycone of saponin OSW-1. This

synthesis is illustrated in Figure 7.

Yu and Jin also elaborated a new method for the

synthesis of the disaccharide (Figure 8). The xylosyl

donor 25 was prepared from 1,2,3,4-tetra-O-acetyl-

D-xylopyranose, which wasinitially converted into a

thio-orthoester 22 via substitution with bromide at

the anomeric position followed by reaction of 21

with EtSH in presence of 2,6-lutidine and MeNO2(Nicolaou et al., 1997). 4-Methoxybenzylation

followed by an intramolecular ring opening of

thio-orthoester 23 gave the thioglycoside 24. This

compound was acylated with 4-methoxybenzoylchloride and finally the desired active donor 25

(Nicolaou et al., 1998) was prepared for coupling

with the arabinosyl unit. The other sugar unit was

prepared from 1,2,3,4-tetra-O-acetyl-L-arabinopi-

ranose in five steps. The starting material was

transformed into the thioglycoside 26 with PhSH

in presence of SnCl4 followed by the protection of

the remaining hydroxyl groups: the 3-OH and 4-

OH as an acetonide, and the 2-OH as an acetate

(compound 27). Deprotection of the acetonide

group afforded a 3,4-diol. Although it is known

that in many sugars, the equatorial 3-OH group is

more reactive than the axial 4-OH group, in this

case regioselective triethylsilylation of the 4-OH

group was successfully performed (compound 28).

The coupling reaction and glycosylation of the

OSW-1 aglycone 6 were carried out under condi-

tions analogous to those used by Chinese chemists

(Deng et al., 1999). To summerize, the American

chemists synthesized saponin OSW-1 in 10 linear

steps from commercially available androst-5-en-

3b-ol-17-one (1) in 28% overall yield. The

Figure 7. Synthesis of the OSW-1 aglycone by Yu and Jin.

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important advantage of this method over the

Chinese one is eliminating the use of expensive

and toxic OsO4.

Morzyckis strategy to saponin OSW-1 (Morzycki

and Wojtkielewicz, 2002)

Still another strategy was elaborated by Polish

chemists. They developed the cleavage of the

16a,17a-epoxide ring to the desired trans diol in

ring D (Morzycki et al., 2000) (Figure 9). The

starting material, as in the previous cases, was

androst-5-en-3b-ol-17-one (1). The B-ring double

bond and 3b-OH group were protected simulta-

neously as a 3a,5a-cyclo-6b-methoxy derivativeand this compound was subjected to a Wittig

Horner reaction with triethylphosphonoacetate in

the presence of NaOEt. The resulting unsaturated

Figure 8. An alternative approach to the OSW-1 disaccharide synthesis.

Figure 9. Morzyckis synthesis of the OSW-1 aglycone hemiketal 33.

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ester was methylated with MeI (LDA, )78 C).

The reaction proceeded with a double bond shift

to the ring D to afford the desired (20S)-product

30 (Piatak and Wicha, 1978; Wicha and Bal, 1978;Ibuka et al., 1988a, b). The D-ring double bond

was subjected to epoxidation with MCPBA. The

obtained 16a,17a-epoxide 31 underwent ring

opening with LiOH/H2O2 via an intramolecular

mechanism (Morzycki et al., 2001b). The primary

reaction product, 16b,17a-dihydroxy-22-carbox-

ylic acid, spontaneously cyclized to the 17a-hy-

droxy lactone 32, the key-precursor in further

synthesis of saponin OSW-1. The lactone 32 was

easily transformed into the OSW-1 aglycone in its

hemiketal form 33 by reaction with isoamyllithium

(Morzycki and Gryszkiewicz, 2001a; Morzycki

and Wojtkielewicz, 2002).

The aglycone 33 was subjected to glycosylationwith the disaccharide trichloroacetimidate 14 un-

der standard conditions (Schmidt and Toepfer,

1991). The direct glycosylation of this compound

afforded the protected saponin OSW-1 35 and the

22-O-glycoside of the hemiketal 34 (Figure 10).

Two other routes from the lactone 32 to the

saponin OSW-1 were also explored. In one of them

32 was reduced with DIBAL-H to the lactol 36,

which was then glycosylated (Figure 11) with 14.

The reaction afforded the 16b-O-glycoside with a

Figure 10. Synthesis of OSW-1 by direct glycosylation of the OSW-1 aglycone hemiketal (33).

Figure 11. Synthesis of OSW-1 by glycosylation of lactol 36.

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free aldehyde group 37, the 22-O-glycoside 38 and

ortho-acetate 39. To convert the 16b-O-glycoside

37 into saponin OSW-1, its reaction with isoam-

ylmagnesium bromide was carried out followed byoxidation with PDC. The protected saponin OSW-1

35 was obtained and the protecting groups were

removed (simultaneous desilylation of the sugar

moiety and cycloreversion of the steroid) with

p-TsOH in dioxane/water at 70 C in nearly

quantitatively yield. The drawback of this

approach was side reaction, retroaldol fragmen-

tation, occurring when 37 was treated with the

Grignard reagent. The saponin OSW-1 was also

obtained by glycosylation of the 22-O-benzyl ether

43 obtained in three steps from lactone 32

(Figure 12). Glycosylation was followed by etherhydrogenolysis, PDC oxidation the 22-alcohol to

the ketone 35 and final removal of the protecting

groups.

Of these three routes to OSW-1, the one based

on direct glycosylation of the aglycone in hemik-

etal form (Figure 10) is the shortest (9 steps) and

the most efficient (5% overall yield). This method

provided a very convenient tool for the synthesis

of analogues of saponin OSW-1. The other

advantage is that it eliminates the use of osmium

tetroxide.

Tians strategy for the aglycone of saponin OSW-1

(Xu et al., 2003)

Tians group obtained the aglycone of saponinOSW-1 by degradation of diosgenin 45, an easily

available and cheap substrate (Figure 13). To open

ring F, diosgenin was treated with PhSH in the

presence of BF3*Et2O. The product, 26-thioacetal

46 underwent reductive desulfurization catalyzed

by Raney nickel. For further modification of the

ring E, the double bond and the 3a-hydroxyl

group were protected as an i-steroid ether. The

oxidation of the obtained compound 47 with

dimethyldioxirane gave the diketone 48, the

opened E-ring product (Murray, 1989; Bovicelli

et al., 1994). The 16,22-diketone 48 was regiose-lectively converted into a 16-thioketal in reaction

with ethanedithiol in the presence of BF3*Et2O,

which was then treated with Ac2O to afford an

enol thioether 49. Desulfurization of this com-

pound yielded the 16-en-22-one 4, the known

intermediate in the earlier presented synthetic

strategies. This compound was further converted

into the protected OSW-1 aglycone 6 according to

Fuchs method (Guo and Fuchs, 1998). The agly-

cone was obtained in 13 linear steps in 9.5% yield.

Figure 12. Synthesis of OSW-1 via 16b,17a,22n-triol 22-benzyl ether 43.

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Chemical synthesis and biological properties of

saponin OSW-1 analogues

The successful crusade for the synthesis of the

natural product saponin OSW-1, opened the way

for chemical synthesis of a large number of ana-

logues for biological studies. All of the research

groups that were involved in the synthesis of

OSW-1 continued their studies towards synthesis

of OSW-1 analogues.

The Fuchs group accomplished the synthesis of

the first OSW-1 analogues in 1999 (Guo et al.,1999). With regard to the high Pearson correlation

coefficient of cephalostatins and saponin OSW-1,

it seemed likely that these two types of compounds

shared a similar mode of action. Fuchs postulated

that the oxocarbenium ion, which could be gen-

erated from both natural products as an interme-

diate was responsible for cytotoxicity. For this

reason Fuchs synthesized a hybrid of these two

compounds dihydroornithostatin O11N 50 (the

pyrazine dimer from the North unit of cephalostin

1 and dihydroaglycone of OSW-1) (Figure 14).

Ornithozine (the pyrazine dimer from the Northunit of ritterazine G and dihydroaglycone of

OSW-1) was also obtained. The activity of these

synthetic compounds was significantly lower than

that of cephalostatin and OSW-1. Fuchs et al. also

studied the biological properties of various agly-

cones. They noted that 3-O-acylated compounds

generally showed lower cytotoxicity than their

counterparts with a free hydroxyl group. They also

proved that the configuration of the 16-OH is very

important for biological activity.

An important contribution to this field was

made by Yu and co-workers. From the early

studies, it was known that removal of the acyl

groups on the disaccharide moiety diminished the

cytotoxicity 1000 times (Mimaki et al., 1997). This

result implied that the disaccharide part was

essential to the antitumor activity of OSW-1.

Therefore the Chinese researchers synthesized

glycosides bearing the OSW-1 disaccharide from

steroid aglycones with a simplified structure or

simple molecules such as nonyl 51 or benzyl 52alcohols (Ma et al., 2000). The study proved that

the glycosides of simple alcohols and of 3a-ste-

roidal alcohols showed only marginal cytotoxicity.

The compounds with the disaccharide attached at

C-16 or at a position close to the D ring (ring C or

side chain) showed stronger activity than those of

the saponin bearing a sugar at C-3 (Ma et al.,

2001a). However, they lost their activity at lower

(10)6 M) concentrations. The 1 fi 4 linked ana-

logues (61, 62) also showed low activity (Ma et al.,

Figure 13. Tians synthesis of the OSW-1 aglycone.

Figure 14. Dihydroornithostatin O11N.

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2000). The 1 fi 4 linked disaccharide was pre-

pared from a D-xylopyranose thioglycoside and an

arabinosyl trichloroacetimidate as the glycosyl

donor in the presence of TMSOTf. The couplingof the aglycone and the 1 fi 4 linked disaccharide

in the presence of NIS/AgOTf gave the corre-

sponding glycoside in a moderate yield. All of the

analogues obtained by Chinese scientists are pre-

sented in Table 3.

Among the synthesized analogues, the most

active one, (Ma et al., 2001b) appeared to be a 3-

O-terephtalic acid dimer of OSW-1 (compound 77)

(65.6% and 52.8% growth inhibition rate at

0.01 lM against P388 and A549 cell, respectively).

The synthesis of the dimer is presented in

Figure 15.Generally, the Chinese chemists employed the

same glycosylation and deprotection conditions in

the synthesis of analogues as used in their synthesis

of saponin OSW-1 (Deng et al., 1999). Yu and Hui

have demonstrated that the structure of the agly-

cone is essential for the antitumor activity of

saponin OSW-1. The importance of the configu-

ration at C-16 for biological activity was also

proved.

Further studies, led almost simultaneously by

Yus (Deng et al., 2004) and Morzyckis

(Morzycki et al., 2004) groups were aimed at

determining the influence of the side chain struc-ture on cytotoxicity. These OSW-1 analogues are

presented in Table 4.

For the synthesis of the side chain analogues,

Morzycki used one of the methods elaborated

(Morzycki and Wojtkielewicz, 2002) for the syn-

thesis of saponin OSW-1, i.e. direct glycosylation

of the aglycone hemiketal form. The Chinese

chemists also used their own previously described

method (Deng et al., 1999). These methods were

then suitably modified to synthesize analogues

without a carbonyl group at C-22 (Figures 16, 17).

The biological tests performed by the Chinesescientists demonstrated that the side chain of

OSW-1 tolerates certain modifications (even loss

of the C-22 carbonyl group) without affecting the

significant antitumor potency of saponin OSW-1.

The studies by Morzyckis group did not confirm

this conclusion. The OSW-1 analogue 83 was ob-

tained by both groups. However, the results of

cytotoxicity tests against breast cancer line MCF7

were contradictory. The studies were repeated in

frame of the ChinesePolish cooperation proving

the chemical identity of both samples of com-

pound 83. The cytotoxicity tests were performed in

an independent laboratory (Profesor Jun Liu,

Johns Hopkins University, USA). Compound 83did not show high cytotoxicity against MCF7

cancer line but appeared to be highly toxic against

Jurcat cells.

A structural isomer of saponin OSW-1 with the

disaccharide attached to C-22 and a carbonyl

group at C-16 94 was also synthesized based on

stoichiometric glycosylation of the 16,17,22-triol

42 (Morzycki et al., 2004) (Figure 18).

The Polish chemists also obtained two ana-

logues (95 and 96) differing in the sugar moiety in

which the OSW-1 aglycone was coupled with an

acylated monosaccharide: 2-acetylarabinopyranoseor 2-(4-methoxybenzoyl)xylopyranose, respec-

tively. Unfortunately, they appeared to be inactive

against cancer cells.

A synthesis of 23-oxa analogues (22-carboxylic

acid esters) was recently published by Yu (Shi

et al., 2004). One of them, dodecyl ester 87, has

shown an even higher activity than OSW-1

(Table 4).

The strategy used for synthesis of these esters is

illustrated in Figure 19.

The synthesis is based on an aldol condensa-

tion. The starting androst-5-en-3b-ol-17-one (1)

was converted to the 16b-hydroxy-17-ketone 102with the known method (Numazawa et al., 1982).

This compound was subjected to aldol conden-

sation with propionate enolates. The reaction

predominately led to the desired product with the

20S configuration. The obtained 16a,17a-dihydr-

oxy ester 103 was subjected to inversion of con-

figuration at C-16 by oxidation with TPAP/NMO

to the ketone 104 and reduction with NaBH4/

CeCl3. In order to prevent lactonization, the

reaction was quenched at )40 C. The aglycone

obtained in this way was immediately subjected

to glycosylation. Finally, the protecting groupswere removed in the usual manner affording the

oxa analogue 105.

Analogues of OSW-1 have also been described

with A-nor-B-aromatic aglycone (Matsuya et al.,

2003). No results have been reported from their

biological tests. A novel synthetic approach to the

OSW-1 aglycone from diosgenin was recently at-

tempted (Chaosuancharoen et al., 2004) but the

synthesis failed in the final step (i.e. isomerization

of the 20,22-epoxide into the 22-ketone).

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Table 3. Analogues synthesized by Yu Hui et al.

Reference Structure Reference Structure

Ma et al. (2000) C9H19OR 51 Ma et al. (2000) C6H5CH2OR 52

Ma et al. (2000) C6H5SR 53 Ma et al. (2000)

Ma et al. (2000) Ma et al. (2000)

Ma et al. (2000) Ma et al. (2000)

Ma et al. (2000) Ma et al. (2000)

Ma et al. (2000) Ma et al. (2001a)

Ma et al. (2001a) Ma et al. (2001a)

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Table 3. Continued

Reference Structure Reference Structure




Ma et al. (2001a) Ma et al. (2001b)

Ma et al. (2001b) Ma et al. (2001b)

Ma et al. (2001b) Ma et al. (2001b)

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Summary and outlook

As it has been presented, chemical synthesis

allowed to obtain naturally occurring saponin

OSW-1 and a large number of OSW-1 analogues

specially designed for the structure-activity rela-

tionship (SAR) studies. The preliminary investi-

gation of structural requirements for biological

activity reveals that:

Both aglycone and sugar were proved impor-tant for cytotoxic activity;

In aglycone, the inversion of 16-OH configura-

tion resulted in significantly reduced potency.

Similar lost of activity was observed when the

3-hydroxyl group was acylated. There is con-

troversy about the importance of the side chain

(its length, shape and presence of the 22-car-

bonyl group). The 23-oxa analogues showed

similar or even higher antitumor potency to

that of OSW-1.

Acyl groups in the sugar part are necessary for

strong cytotoxic activity. The replacement of

OSW-1 disaccharide with acylated monosac-

charide led to the lost of activity of the sapo-nin. The position of the sugar moiety is also

important. The compounds with disaccharide

attached at positions other than 16b proved

Table 3. Continued

Reference Structure

Ma et al. (2001b)

Figure 15. Synthesis of dimer of OSW-1 aglycone.

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Table 4. Side chain analogues and structural isomer of OSW-1.

Reference Structure

YuDeng et al. (2004) R = CH2CH2CH2CH(CH3)2 (81)

CH(OH)CH2CH2CH(CH3)2 (82)

C(O)CH2CH2CH3(83)

C(O)CH3(84)

Shi et al. (2004) R=CH2CH(CH3)2 (85)

CH2CH3 (86)

(CH2)11CH3(87)

Morzycki

Morzycki et al. (2004) R=C(O)CH2CH2CH3(83)

C(O)CH2CH2CH2CH3 (88)

C(O)CH2CH2CH2CH2CH3 (89)

C(O)CH(CH3)2 (90)

CH(OH)CH2CH2CH(CH3)2 (91)

CHO (92)

CH3(93)

Morzycki et al. (2004)



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Figure 16. Morzyckis synthesis of an OSW-1 analogue with an isopropyl side chain.

Figure 17. Yus synthesis of an OSW-1 analogue devoid of the carbonyl group in the side chain.

Figure 18. Synthesis of an OSW-1 isomer (94).

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biologically inactive or showed only marginal

activity.

However, the studies on the structurebiologi-

cal activity relationship for saponin OSW-1 has

not been fully completed and further investiga-tions in this field are needed.

Acknowledgements

This work was supported by the State Committee

for Scientific Research. The authors thank Mrs.

J. Maj for assistance in preparation of the manu-

script.

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