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 forcorrespondence (Tel: +48-85-7457585; Fax: +48-85-7457581; E-mail: morzycki@uwb.edu.pl)

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 recentlyisolated 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, itshows little toxicity with normal human pulmonary cells. In this review article the synthetic efforts towardsOSW-1 and related cholestane glycosides, as well as the preliminary results of the structure–activity rela-tionship study are presented.

Introduction

The control of cancer, the second leading cause ofdeath worldwide, may benefit from the potentialthat resides in natural products. Some of them,originating from plants, have been implicated incancer treatment without recognizable side effects(Reddy et al., 2003). One of such very promisingnatural products is the saponin OSW-1, isolatedby Japanese scientists in 1992 from the bulbs ofOrnithogalum saundersiae (Kubo et al., 1992). It isa perennial garden plant of the lily family endemicto the Drakensberg Mountains in Africa. Thecommon name of the plant, Ivory Coast Lily, isgeographically incorrect because it is endemic tothe east coast of Africa; the plant is also known asthe giant chincherinchee. O. saundersiae is thegiant member of the Ornithogalum genus, whichbelongs to the sub-family Schilloideae in Liliaceae,comprised of about 150 species (Bryan, 1989). Theplant flowers normally in open fields all year roundin Kenya and it is one of the top ten cut flowersexported to Europe in this country (Kariuki et al.,1999). However, there are some problems incommercial production which include variability

and instability in yield and quality of cut flowers;the plant is also commercially grown for cutflowers in Holland, Israel and Tasmania. Themulti flowered clusters are striking because eachwhite petaled flower has a dark green center. Theflower stems are tall and strong, up to 1.15 mheight which makes it a versatile flower for use infloral art. Several cardenolide glycosides have beenpreviously found in some species of Ornithogalum(Buchvarov et al., 1984; Ghannamy et al., 1987).A phytochemical screening of the bulbs ofOrnithogalum saundersiae has proven the lack ofcardenolide 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 amethanolic extract of Ornithogalum saundersiae, aspecies without any medicinal folklore back-ground, showed exceptional cytostatic activityagainst various malignant tumor cells (Mimakiet al., 1997). A group of cholestane glycosides wasisolated from the extract including saponin OSW-1as the main component of the mixture(Figure 1).

Phytochemistry Reviews (2005) 4: 259–277 � Springer 2005DOI 10.1007/s11101-005-1233-6

In vitro assays have shown that OSW-1 isextremely toxic against a broad spectrum of tumorcells, such as leukemia HL-60, mouse mastrocar-cinoma, human pulmonary adenocarcinoma, hu-man pulmonary large cell carcinoma, and humanpulmonary squamous cell carcinoma includingadriamycin-resistant P388 leukemia and campto-thecin-resistant P388. The IC50’s are between 0.1and 0.7 nM, which means a cytotoxicity about

10–100 times more potent than that of the clini-cally applied anticancer agents, such as mitomycinC, adriamycin, cisplatin, camptothecin, and taxol(Table 1). While OSW-1 is exceptionally cytotoxicagainst various malignant tumor cells, it showslittle toxicity (IC50 1500 nM) to normal humanpulmonary cells. The cytotoxicity profile of OSW-1is strikingly similar to that of cephalostatins, agroup of dimeric steroid–pyrazines from marineorganisms (Gryszkiewicz-Wojtkielewicz et al., 2003)with Pearson correlation coefficients between 0.60and 0.83. Structurally, the aglycone of OSW-1 isreminiscent of half of the cephalostatins. Fuchstherefore hypothesized that these compoundsmight have the same mechanism of action (Guoand Fuchs, 1998). The comparison of pGI50 valuesfor saponin OSW-1, cephalostatin 1 and clinicallyapplied anticancer agents is presented in Table 2.

Further analysis of the bulbs of O. saundersiaeresulted 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 (Kurodaet al., 1999).

During examination of plants taxonomicallyrelated to O. saundersiae (liliaceae family) it wasfound that a methanolic extract of the bulbs of

g g y

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 growthinhibitory 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 SouthAfrica. The long flower spike of G. candicans has10–20 flower bells distributed along the flowerspike, and looks like a large elongated Hyacinth.G. candicans has a very pleasant fragrance. Use of acytotoxicity-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-cholestanediglycoside – 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 potentcytotoxicity of the crude extract, suggesting the

g y g y

Table 2. Comparison of cytostatic activity of saponin OSW-1and 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 furthersearch aimed at finding cytotoxic components fromG. candicans bulb extract resulted in the isolationof cholestane glycosides (Kuroda et al., 2001),closely related to saponins found in O. saundersiae(3-O-glucosylated derivatives).

Immediately after recognition of the highcytotoxic activity of saponin OSW-1, numerousgroups of chemists around the world undertookthe synthesis of this compound. Saponin OSW-1can be logically disconnected into two parts: thecholestane aglycone and the disaccharide moiety.The first synthesis of the OSW-1 aglycone wasaccomplished 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 aglyconewith the sugar part. New approaches for the syn-thesis of this natural product were presented byJin’s (Yu and Jin, 2001a, 2002), Morzycki’s(Morzycki and Wojtkielewicz, 2002) and Tian’s(Xu et al., 2003) groups.

In the section below various syntheses ofsaponin OSW-1 and its analogues will be pre-sented. Then the structure–biological acitivityrelationships will be discussed.

Synthesis of saponin OSW-1 and its analogues

Fuch’s synthesis of the aglycone of saponin OSW-1(Guo and Fuchs, 1998)

Construction of the 22-oxygenated side chain wasperformed by several steps starting from thecommercially available material – androst-5-en-3b-ol-17-one (1). Wittig olefination was followedby acetylation and a stereoselective ene reaction ofcompound 2 (Figure 3). The obtained 22-alcohol 3was oxidized with a Jones reagent to the ketone 4

and then the carbonyl group was protected as aketal. The key step in the synthesis of the aglyconeof saponin OSW-1 was the introduction of thetrans diol function in ring D (16b,17a-diol). Fuchsinitially attempted opening of the 16a,17a-epox-ide. However, all attempts to cleave the oxiranering in the presence of the side chain failed.Altering the sequence of reactions did not solve theproblem. Due to these difficulties, the D16olefinwas subjected to dihydroxylation with a stoichi-ometric amount of OsO4. In the next step the cisdiol 5 was oxidized with the Swern reagent to the16-ketone. The stereoselective reduction of theketone with NaBH4/CeCl3 at low temperatureafforded a 16b,17a-diol 6 – the aglycone of sapo-nin OSW-1 in the protected form.

g y

Figure 3. Fuch’s synthesis of the OSW-1 aglycone.

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Yu and Hui strategy – the first synthesis of saponinOSW-1 (Deng et al., 1999)

The desired steroid aglycone was also synthesizedfrom androst-5-en-3b-ol-17-one (1) by Yu andHui, who used a strategy similar to that employedby Fuchs (Figure 4). The major difference con-sisted in the construction of the side chain, wherethe ene reaction was performed with paraformal-dehyde (in Fuch’s method with 4-methylvaleral-dehyde). The obtained primary alcohol 7 was thenoxidized to the aldehyde 8, which in turn reactedwith a Grignard reagent. The secondary alcoholobtained in this way was further transformed intothe OSW-1 aglycone using Fuch’s method.

The sugar moiety of OSW-1 consists of acyl-ated L-arabinose and D-xylose. For synthesis of thedisaccharide, the choice of protecting groups wasvery important. The xylose donor for couplingwith arabinose was prepared in the following way(Figure 5). Upon the protection of the anomericgroup as a benzyl ether the 2-OH was regioselec-tively masked as a 4-methoxybenzoate and the restof the hydroxyl groups (3-OH and 4-OH) weretransformed into TES-ethers. In the next step, the1-OH group was deprotected (50 atm. H2; Pd/C)and then converted into a trichloracetimidate 11, aconvenient 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-hydroxylgroups, acetylation of the 2-OH and the final re-moval of the acetonide protection. Although theacceptor prepared in this manner contained twohydroxyl groups, selective glycosylation of the diol13 at the equatorial 3-OH group with the xylosyldonor 11 catalyzed by BF3*Et2O (Schmidt andMichel, 1980) afforded the desired disaccharide.The remaining hydroxyl group in this compoundwas then protected as a TES-ether and the benzylprotecting group was removed. In the last step thetrichloroacetimidate 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 protectedsaponin OSW-1. All protecting groups (TBS, TES,ethylene glycol ketal) were removed in one stepwith Pd(MeCN)2Cl2 as catalyst (Lipshutz et al.,1985). Saponin OSW-1 was synthesized in 27 stepsin 6% overall yield.

Yu and Jin strategy for the synthesis of saponinOSW-1 (Yu and Jin, 2001a, 2002)

A completely different approach for the synthesisof OSW-1 was proposed by American chemists in2001 (Figure 7). The strategy was based on the1,4-addition of an a-alkoxy vinyl cuprate to the

g y g y y

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

263

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

g y y g y y

Figure 6. Glycosylation reaction.

Figure 5. Synthesis of the OSW-1 disaccharide.

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oxidation of the obtained 16b-alcohol 15 affordedthe enone 16 in nearly quantitative yield. TMSCl-activated (Corey and Boaz, 1985; Alexakis et al.,1986) 1,4-addition of a suitable a-alkoxy vinylcuprate to this compound gave a silyl ether 17,which was converted to the enol acetate 18

without isolation. This conversion allowed for thechemoselective transformation of the 22-enol ether18 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, obtainedfrom 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. Thissynthesis is illustrated in Figure 7.

Yu and Jin also elaborated a newmethod for thesynthesis of the disaccharide (Figure 8). The xylosyldonor 25 was prepared from 1,2,3,4-tetra-O-acetyl-D-xylopyranose,whichwas initially converted into athio-orthoester 22 via substitution with bromide atthe 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 ofthio-orthoester 23 gave the thioglycoside 24. Thiscompound was acylated with 4-methoxybenzoylchloride and finally the desired active donor 25

(Nicolaou et al., 1998) was prepared for couplingwith the arabinosyl unit. The other sugar unit wasprepared from 1,2,3,4-tetra-O-acetyl-L-arabinopi-ranose in five steps. The starting material wastransformed into the thioglycoside 26 with PhSHin presence of SnCl4 followed by the protection ofthe remaining hydroxyl groups: the 3-OH and 4-OH as an acetonide, and the 2-OH as an acetate(compound 27). Deprotection of the acetonidegroup afforded a 3,4-diol. Although it is knownthat in many sugars, the equatorial 3-OH group ismore reactive than the axial 4-OH group, in thiscase regioselective triethylsilylation of the 4-OHgroup was successfully performed (compound 28).The coupling reaction and glycosylation of theOSW-1 aglycone 6 were carried out under condi-tions analogous to those used by Chinese chemists(Deng et al., 1999). To summerize, the Americanchemists synthesized saponin OSW-1 in 10 linearsteps 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 theChinese one is eliminating the use of expensiveand toxic OsO4.

Morzycki’s strategy to saponin OSW-1 (Morzyckiand Wojtkielewicz, 2002)

Still another strategy was elaborated by Polishchemists. They developed the cleavage of the

16a,17a-epoxide ring to the desired trans diol inring D (Morzycki et al., 2000) (Figure 9). Thestarting material, as in the previous cases, wasandrost-5-en-3b-ol-17-one (1). The B-ring doublebond 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 inthe presence of NaOEt. The resulting unsaturated

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

Figure 9. Morzycki’s 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 shiftto the ring D to afford the desired (20S)-product30 (Piatak and Wicha, 1978; Wicha and Bal, 1978;Ibuka et al., 1988a, b). The D-ring double bondwas subjected to epoxidation with MCPBA. Theobtained 16a,17a-epoxide 31 underwent ringopening with LiOH/H2O2 via an intramolecularmechanism (Morzycki et al., 2001b). The primaryreaction product, 16b,17a-dihydroxy-22-carbox-ylic acid, spontaneously cyclized to the 17a-hy-droxy lactone 32, the key-precursor in furthersynthesis of saponin OSW-1. The lactone 32 waseasily transformed into the OSW-1 aglycone in its

hemiketal form 33 by reaction with isoamyllithium(Morzycki and Gryszkiewicz, 2001a; Morzyckiand 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 compoundafforded the protected saponin OSW-1 35 and the22-O-glycoside of the hemiketal 34 (Figure 10).

Two other routes from the lactone 32 to thesaponin OSW-1 were also explored. In one of them32 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

g 6 y y g p py

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.

267

free aldehyde group 37, the 22-O-glycoside 38 andortho-acetate 39. To convert the 16b-O-glycoside37 into saponin OSW-1, its reaction with isoam-ylmagnesium bromide was carried out followed byoxidation with PDC. The protected saponin OSW-135 was obtained and the protecting groups wereremoved (simultaneous desilylation of the sugarmoiety and cycloreversion of the steroid) withp-TsOH in dioxane/water at 70 �C in nearlyquantitatively yield. The drawback of thisapproach was side reaction, retroaldol fragmen-tation, occurring when 37 was treated with theGrignard reagent. The saponin OSW-1 was alsoobtained by glycosylation of the 22-O-benzyl ether43 obtained in three steps from lactone 32

(Figure 12). Glycosylation was followed by etherhydrogenolysis, PDC oxidation the 22-alcohol tothe ketone 35 and final removal of the protectinggroups.

Of these three routes to OSW-1, the one basedon direct glycosylation of the aglycone in hemik-etal form (Figure 10) is the shortest (9 steps) andthe most efficient (5% overall yield). This methodprovided a very convenient tool for the synthesisof analogues of saponin OSW-1. The otheradvantage is that it eliminates the use of osmiumtetroxide.

Tian’s strategy for the aglycone of saponin OSW-1(Xu et al., 2003)

Tian’s group obtained the aglycone of saponinOSW-1 by degradation of diosgenin 45, an easilyavailable and cheap substrate (Figure 13). To openring F, diosgenin was treated with PhSH in thepresence of BF3*Et2O. The product, 26-thioacetal46 underwent reductive desulfurization catalyzedby Raney nickel. For further modification of thering E, the double bond and the 3a-hydroxylgroup were protected as an i-steroid ether. Theoxidation of the obtained compound 47 withdimethyldioxirane gave the diketone 48, theopened E-ring product (Murray, 1989; Bovicelliet al., 1994). The 16,22-diketone 48 was regiose-lectively converted into a 16-thioketal in reactionwith ethanedithiol in the presence of BF3*Et2O,which was then treated with Ac2O to afford anenol thioether 49. Desulfurization of this com-pound yielded the 16-en-22-one 4, the knownintermediate in the earlier presented syntheticstrategies. This compound was further convertedinto the protected OSW-1 aglycone 6 according toFuchs’ 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 thenatural product – saponin OSW-1, opened the wayfor chemical synthesis of a large number of ana-logues for biological studies. All of the researchgroups that were involved in the synthesis ofOSW-1 continued their studies towards synthesisof OSW-1 analogues.

The Fuchs group accomplished the synthesis ofthe first OSW-1 analogues in 1999 (Guo et al.,1999). With regard to the high Pearson correlationcoefficient of cephalostatins and saponin OSW-1,it seemed likely that these two types of compoundsshared a similar mode of action. Fuchs postulatedthat the oxocarbenium ion, which could be gen-erated from both natural products as an interme-diate was responsible for cytotoxicity. For thisreason Fuchs synthesized a hybrid of these twocompounds – dihydroornithostatin O11N 50 (thepyrazine dimer from the North unit of cephalostin1 and dihydroaglycone of OSW-1) (Figure 14).Ornithozine (the pyrazine dimer from the Northunit of ritterazine G and dihydroaglycone ofOSW-1) was also obtained. The activity of thesesynthetic compounds was significantly lower thanthat of cephalostatin and OSW-1. Fuchs et al. alsostudied the biological properties of various agly-cones. They noted that 3-O-acylated compoundsgenerally showed lower cytotoxicity than theircounterparts with a free hydroxyl group. They alsoproved that the configuration of the 16-OH is veryimportant for biological activity.

An important contribution to this field wasmade by Yu and co-workers. From the earlystudies, it was known that removal of the acylgroups on the disaccharide moiety diminished thecytotoxicity 1000 times (Mimaki et al., 1997). Thisresult implied that the disaccharide part wasessential to the antitumor activity of OSW-1.Therefore the Chinese researchers synthesizedglycosides bearing the OSW-1 disaccharide fromsteroid aglycones with a simplified structure orsimple molecules such as nonyl 51 or benzyl 52alcohols (Ma et al., 2000). The study proved thatthe glycosides of simple alcohols and of 3a-ste-roidal alcohols showed only marginal cytotoxicity.The compounds with the disaccharide attached atC-16 or at a position close to the D ring (ring C orside chain) showed stronger activity than those ofthe 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. Tian’s 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 anarabinosyl trichloroacetimidate as the glycosyldonor in the presence of TMSOTf. The couplingof the aglycone and the 1 fi 4 linked disaccharidein the presence of NIS/AgOTf gave the corre-sponding glycoside in a moderate yield. All of theanalogues obtained by Chinese scientists are pre-sented in Table 3.

Among the synthesized analogues, the mostactive 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 at0.01 lM against P388 and A549 cell, respectively).The synthesis of the dimer is presented inFigure 15.

Generally, the Chinese chemists employed thesame glycosylation and deprotection conditions inthe synthesis of analogues as used in their synthesisof saponin OSW-1 (Deng et al., 1999). Yu and Huihave demonstrated that the structure of the agly-cone is essential for the antitumor activity ofsaponin OSW-1. The importance of the configu-ration at C-16 for biological activity was alsoproved.

Further studies, led almost simultaneously byYu’s (Deng et al., 2004) and Morzycki’s(Morzycki et al., 2004) groups were aimed atdetermining the influence of the side chain struc-ture on cytotoxicity. These OSW-1 analogues arepresented 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 glycosylationof the aglycone hemiketal form. The Chinesechemists also used their own previously describedmethod (Deng et al., 1999). These methods werethen suitably modified to synthesize analogueswithout a carbonyl group at C-22 (Figures 16, 17).

The biological tests performed by the Chinesescientists demonstrated that the side chain ofOSW-1 tolerates certain modifications (even lossof the C-22 carbonyl group) without affecting thesignificant antitumor potency of saponin OSW-1.The studies by Morzycki’s group did not confirmthis conclusion. The OSW-1 analogue 83 was ob-tained by both groups. However, the results ofcytotoxicity tests against breast cancer line MCF7were contradictory. The studies were repeated inframe of the Chinese–Polish cooperation proving

the chemical identity of both samples of com-pound 83. The cytotoxicity tests were performed inan independent laboratory (Profesor Jun Liu,Johns Hopkins University, USA). Compound 83

did not show high cytotoxicity against MCF7cancer line but appeared to be highly toxic againstJurcat cells.

A structural isomer of saponin OSW-1 with thedisaccharide attached to C-22 and a carbonylgroup at C-16 94 was also synthesized based onstoichiometric glycosylation of the 16,17,22-triol42 (Morzycki et al., 2004) (Figure 18).

The Polish chemists also obtained two ana-logues (95 and 96) differing in the sugar moiety inwhich the OSW-1 aglycone was coupled with anacylated monosaccharide: 2-acetylarabinopyranoseor 2-(4¢-methoxybenzoyl)xylopyranose, respec-tively. Unfortunately, they appeared to be inactiveagainst cancer cells.

A synthesis of 23-oxa analogues (22-carboxylicacid esters) was recently published by Yu (Shiet al., 2004). One of them, dodecyl ester 87, hasshown an even higher activity than OSW-1(Table 4).

The strategy used for synthesis of these esters isillustrated 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 102

with the known method (Numazawa et al., 1982).This compound was subjected to aldol conden-sation with propionate enolates. The reactionpredominately led to the desired product with the20S configuration. The obtained 16a,17a-dihydr-oxy ester 103 was subjected to inversion of con-figuration at C-16 by oxidation with TPAP/NMOto the ketone 104 and reduction with NaBH4/CeCl3. In order to prevent lactonization, thereaction was quenched at )40 �C. The aglyconeobtained in this way was immediately subjectedto glycosylation. Finally, the protecting groupswere removed in the usual manner affording theoxa analogue 105.

Analogues of OSW-1 have also been describedwith A-nor-B-aromatic aglycone (Matsuya et al.,2003). No results have been reported from theirbiological tests. A novel synthetic approach to theOSW-1 aglycone from diosgenin was recently at-tempted (Chaosuancharoen et al., 2004) but thesynthesis failed in the final step (i.e. isomerizationof 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. (2001a)

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

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

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 synthesisallowed to obtain naturally occurring saponinOSW-1 and a large number of OSW-1 analoguesspecially designed for the structure-activity rela-tionship (SAR) studies. The preliminary investi-gation of structural requirements for biologicalactivity 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 the3-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 showedsimilar or even higher antitumor potency tothat of OSW-1.

• Acyl groups in the sugar part are necessary forstrong cytotoxic activity. The replacement ofOSW-1 disaccharide with acylated monosac-charide led to the lost of activity of the sapo-nin. The position of the sugar moiety is alsoimportant. The compounds with disaccharideattached 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

Yu

Deng 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)

Morzycki et al. (2004)

Morzycki et al. (2004)

274

Figure 16. Morzycki’s synthesis of an OSW-1 analogue with an isopropyl side chain.

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

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

275

biologically inactive or showed only marginalactivity.

However, the studies on the structure–biologi-cal activity relationship for saponin OSW-1 hasnot been fully completed and further investiga-tions in this field are needed.

Acknowledgements

This work was supported by the State Committeefor Scientific Research. The authors thank Mrs.J. Maj for assistance in preparation of the manu-script.

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