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Structure, Bioactivity, and Chemical Synthesis of OSW‑1 and Other Steroidal Glycosides in the Genus Ornithogalum Yuping Tang,* Nianguang Li, Jin-ao Duan,* and Weiwei Tao Jiangsu Key Laboratory for High Technology of TCM Formulae Research, Nanjing University of Chinese Medicine, Nanjing 210046, China 1. INTRODUCTION The genus Ornithogalum L. belongs to the subclass Monocotyledonae and was originally classified in the family Liliaceae,1 but was later transferred to Hyacinthaceae.2,3 About 200 species are known from Africa, Europe, and Asia.4 Ornithogalum are closely related to Albuca and Lachenalia, the latter of which have been developed as flowering pot plants.5 Only a few species of Ornithogalum are used in the floriculture trade.6 Ornithogalum umbellatum, Ornithogalum pyramidale, and Ornithogalum nutans are used in gardens. Ornithogalum arabicum, indigenous to the Middle East, is used as a cut flower. Ornithogalum saundersiae, a species from the High Veldt region of South Africa and Swaziland, used as a cut flower, produces exceptionally long stems. Ornighogalum thyrsoides, the first fresh cut flower to be traded internationally from South Africa, is the most widely grown cut flower type. 7 Approximately 40 million stems of cut Ornithogalum are sold through the Dutch Auction system annually, with sales occurring over the entire year.7 Ornithogalum for flower trade are generally easily cultivated, requiring full sun and welldrained soil. The majority of the flowers within the genus are white or yellow, with a green/brown stripe on the midrib of the petals. The species used as fresh cut flowers, and more recently as pot plants, have saturated colored petals with no midrib stripe and range in color from white to yellow, orange, and orange-red.8 Although Ornithogalum caudatum Ait. is known in Chinese folk medicine to exhibit anticancer, antimicrobial, and antiinflammatory activities and has been used for the treatment of hepatitis, parotitis, and some tumor types in northern China,9 most Ornithogalum plants have no folkloric medicinal background, and some are even known to be poisonous.10,11 Several cardenolide glycosides were found in some species of Ornithogalum 20−30 years ago.10,11 In 1992, a phytochemical screening of the bulbs of O. saundersiae (Figure 1) by Sashida’s group resulted in the isolation of three acylated cholestane glycosides, including OSW-1, and the glycosides showed considerable inhibition activity against cyclic AMP phosphodiesterase.12 Five years later it was found that the cholestane glycosides, especially OSW-1, showed exceptional cytostatic activity against various malignant tumor cells.13 The important discoveries led to vigorous research on isolation and bioactivity evaluation of the steroidal glycosides and the synthesis of OSW1 and its analogues. Morzycki and Wojtkielewicz summarized the synthesis of OSW-1 and its analogues with 50 references in 2005,14 and in

CONTENTS 1. Introduction 2. Structural Classification 2.1. Cardenolide Glycosides 2.2. Common Cholestane Glycosides 2.3. Rearranged Cholestane Glycosides 2.4. Spirostane Glycosides 2.5. Stigmastane Glycosides 3. Phytochemical Studies 4. Analytical Methods 5. Biological Activities 5.1. Antitumor Activity 5.2. cAMP Phosphodiesterase Inhibition 5.3. Other Activities 6. Chemical Synthesis 6.1. Synthesis of the Aglycon of OSW-1 6.2. Total Synthesis of OSW-1 6.3. Synthesis of OSW-1 Saponin Analogues 6.3.1. Synthesis of OSW-1 Saponin Analogues with Steroidal Nucleus Modification 6.3.2. Synthesis of OSW-1 Saponin Analogues with Side Chain Modification 6.3.3. Synthesis of OSW-1 Saponin Analogues with Disaccharide Modification 6.3.4. Synthesis of OSW-1 Saponin Analogues with Other Structural Modification 7. SAR of OSW-1 8. Conclusions and Outlook Author Information Corresponding Author Notes Biographies Acknowledgments References

A B B E F F G G G H H M O O O P W W W Z Z AE AF AF AF AF AF AG AG

Received: February 20, 2012

© XXXX American Chemical Society

A

dx.doi.org/10.1021/cr300072s | Chem. Rev. XXXX, XXX, XXX−XXX

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aglycon, can be classified into four groups on the basis of the aglycon framework: cardenolide glycosides (A, Figure 2), cholestane glycosides (B, Figures 3 and 4), spirostane glycosides (C, Figure 5), and stigmastane glycosides (D, Figure 6). Many aglycons of cholestane glycosides from Ornithogalum plants contain a 16,23-epoxy hexatomic ring, and some also form 24(23→22) abeo-cholestane by C-rearrangement, so cholestane glycosides can be further divided into common cholestane glycosides (B1, Figure 3) and rearranged cholestane glycosides (B2, Figure 4). 2.1. Cardenolide Glycosides

In 1959, convallotoxin (1) and convalloside (2) were isolated from the bulbs of O. umbellatem, and their structures were elucidated by comparison of their physiochemical properties and TLC data with those of authentic compounds.18 The two compounds were the first steroidal glycosides isolated and identified from Ornithogalum species. Rhodexin A (3) was isolated from the seedpods of Ornithogalum magnum by Komissarenko, and its structure was elucidated by acid hydrolysis and oxidation of the aglycon with chromic oxide in glacial acetic acid, comparison of molecular rotation with those of some known compounds, and elementary analysis.19 Rhodexoside (4) was isolated in crystal form and identified from the bulbs of O. umbellatum.20 A further investigation of the seedpods of O. magnum by Komissarenko resulted in the isolation of two new cardenolide glycosides, ornithogaloside (5) and ornithogalin (6),21,22 whose structures were assigned as 3β-(β- D -arabinopyranosyloxy)-11α,14β-dihydroxy-5β-card20(22)-enolide and 3β-(β-D-glucopyranosyloxy)-14β-hydroxycarda-4,20(22)-dienolide by acid hydrolysis, UV data, elementary analysis, and comparison of other physiochemical properties with those of 1. Rhodexin B (7) was also isolated and identified from O. magnum.23 Ornithogalum nanum was investigated by column and thin-layer chromatography methods, and periplogenin (8), bipindogenin (9), strophanthidol (10), nigrescigenin (11), and peripalloside (12) were found in bulbs of the plant.11 The structures of 1−12 are given in Figure 2. From the leaves and bulbs of Ornithogalum boucheanum, eight new cardenolide glycosides were isolated by a combination of column and droplet countercurrent chromatography (DCCC). Their structure elucidation was performed mainly by means of 1H NMR, 13C NMR, electrospray ionization mass spectrometry (EI-MS), and laser desorption mass spectrometry (LD-MS) studies as well as by acid and/or enzymatic hydrolysis of the glycosides followed by identification of the aglycons and sugar moieties (TLC, GC). Their structures were identified as sarmentogenin 3-O-[4′-O-(3″-O-βD-apiofuranosyl)-β-D-xylopyranosyl]-β-D-allomethylopyranoside (13), sarmentogenin 3-O-[4′-O-(4″-O-α-L-rhamnopyranosyl)β-D-xylopyranosyl]-β-D-allomethylopyranoside (14), 15β,16αdihydroxyuzarigenin 3-O-[4′-O-(4″-O-α-L-rhamnopyranosyl)β-D-xylopyranosyl]-β-D-digitoxopyranoside (15), sarmentogenin 3-O-[4′-O-(3″-O-β-D-apiofuranosyl)-β-D-xylopyranosyl]-βD-digitoxopyranoside (16), syriogenin 3-O-[4′-O-(4″-O-α-Lrhamnopyranosyl)-β-D-xylopyranosyl]-β-D-digitoxopyranoside (17), sarmentogenin 3-O-(4′-O-β-D-apiofuranosyl)-α-L-rhamnopyranoside (18), uzarigenin 3-O-[4′-O-(4″-O-α-L-rhamnopyranosyl)-β-D-xylopyranosyl]-β-D-digitoxopyranoside (19), and digitoxigenin 3-O-(4′-O-β-D-xylopyranosyl)-β-D-allomethylopyranoside (20).10,24 This was the first report of the occurrence of 15β,16α-dihydroxyuzarigenin. The presence of

Figure 1. Flowers (a) and bulbs (b) of O. saundersiae. The photographs were provided by Yoshihiro Mimaki.

the review contributed by Lee, Thomas, and Fuchs in 2009, the chemistry of OSW-1 was also touched upon.15 Y. Mimaki summarized the structure and biological activities of plant glycosides, including cholestane glycosides from O. saundersiae, O. thyrsoides, and Galtonia candicans, and their cytotoxic and antitumor activities with 24 references in 2006.16 He and his colleagues also gave an analysis and prospects for research on OSW-1 in ref 17. This review focuses on the structure, bioactivity, and chemical synthesis of steroidal glycosides from the genus Ornithogalum between 1959 and 2011 and provides a comprehensive overview that could serve as a reference for interested researchers.

2. STRUCTURAL CLASSIFICATION The steroidal glycosides from Ornithogalum plants, possessing a steroid with 4−6 carbocyclic or O-heterocyclic rings as the B


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Figure 2. Cardenolide glycosides from the genus Ornithogalum.

In Kopp’s group, investigations on the bulbs and leaves of O. umbellatum afforded strophalloside (21), convallatoxol (22), lokundjoside (23), tholloside (24), and seven new cardenolide glycosides. The structures of the new compounds were identified mainly by spectroscopic (FAB-MS (FAB = fast atom bombardment) and NMR techniques) and chemical methods as strophanthidin 3-O-[3′-O-acetyl-4′-O-(4″-O-α-L-

three aglycons, including syriogenin, uzarigenin, and digitoxigenin, in the genus Ornithogalum was described for the first time. For cardenolides, not only was the combination of three different monosaccharides in one glycoside unusual, but particularly the occurrence of apiose was not known for cardiac glycosides at that time. The structures of 13−20 are given in Figure 2. C


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Figure 3. Common cholestane glycosides from the genus Ornithogalum.

droxy-15-oxouzarigenin 3-O-[4′-O-(4″-O-β-D-apiofuranosyl)-βD-xylopyranosyl]-β-D-digitoxopyranoside (36), 8β-hydroxy-15oxouzarigenin 3-O-[3′-O-acetyl-4′-O-(4″-O-β-D-apiofuranosyl)β-D-xylopyranosyl]-β-D-digitoxopyranoside (37), 8β-hydroxy15-oxouzarigenin 3-O-[4′-O-(4″-O-α-L-rhamnopyranosyl)-β-Dxylopyranosyl]-β-D-digitoxopyranoside (38), 3β,11β-dihydroxy12-oxo-18-nor-5α-card-13-enolide 3-O-[3′-O-acetyl-4′-O-(4″O-α-L-rhamnopyranosyl)-β-D-xylopyranosyl]-β-D-digitoxopyranoside (39), 3β,11β-dihydroxy-12-oxo-18-nor-5α-card-13-enolide 3-O-[4′-O-(4″-O-α-L-rhamnopyranosyl)-β-D-xylopyranosyl]-β-D-digitoxopyranoside (40), strophanthidin 3-O-[4′-O(3″-O-β-D-apiofuranosyl)-β-D-xylopyranosyl]-β-D-allomethylopyranoside (41), 16β-hydroxysarmentogenin 3-O-(4′-O-β-Dapiofuranosyl)-α-L-rhamnopyranoside (42), oleandrigenin 3-O[4′-O-(3″-O-β-D-apiofuranosyl)-β-D-xylopyranosyl]-β-D-digitoxopyranoside (43), oleandrigenin 3-O-[4′-O-(3″-O-β-D-apiofuranosyl)-β-D-xylopyranosyl]-2′-desoxy-β-D-allopyranoside (44), and 12β-hydroxyoleandrigenin 3-O-[4′-O-(4″-O-β-D-apiofuranosyl)-β-D-xylopyranosyl]-β-D-digitoxopyranoside (45).26 Glycosides of 7β,15β,16α-trihydroxyuzarigenin, 8β,16α-dihydroxy15-oxo-uzarigenin, 3β,11β-dihydroxy-12-oxo-18-nor-5α-card13-enolide, 16β-hydroxysarmentogenin, 12-oxo-5α-adynerigenin, 8β-hydroxy-15-oxouzarigenin, and 12β-hydroxyoleandrige-

rhamnopyranosyl)-β-D-glucopyranosyl]-β-D-digitoxopyranoside (25), sarmentogenin 3-O-(4′-O-β-D-glucopyranosyl)-β-D-quinovopyranoside (26), sarmentogenin 3-O-β-D-quinovopyranoside (27), sarmentogenin 3-O-β-D-allomethylopyranoside (28), sarmentogenin 3-O-β-D-allopyranoside (29), bipindogenin 3-Oβ-D-ribopyranoside (30), and strophanthidin 3-O-β-D-allopyranoside (31).25 β-D-Ribose and 3-acetyldigitoxose were found as sugar moieties in the genus Ornithogalum for the first time. The structures of 21−31 are given in Figure 2. From the leaves and bulbs of O. nutans, 14 cardenolide glycosides were isolated by column chromatography, DCCC, and TLC. The structure elucidation was performed by means of 1 H NMR, 13C NMR, H−H COSY, C−H COSY, and FAB-MS studies and identification of the sugar moieties by GC after acid hydrolysis. Their structures were 7β,15β,16α-trihydroxyuzarigenin 3-O-[4′-O-(4″-O-α-L-rhamnopyranosyl)-β-D-xylopyranosyl]-β-D-digitoxopyranoside (32), 12-oxo-5α-adynerigenin 3-O[3′-O-acetyl-4′-O-(4″-O-α-L-rhamnopyranosyl)-β-D-xylopyranosyl]-β-D-digitoxopyranoside (33), 8β,16α-dihydroxy-15-oxouzarigenin 3-O-[4′-O-(4″-O-α-L-rhamnopyranosyl)-β-D-xylopyranosyl]-β-D-digitoxopyranoside (34), 8β,16α-dihydroxy-15oxouzarigenin 3-O-[3′-O-acetyl-4′-O-(4″-O-α-L-rhamnopyranosyl)-β-D-xylopyranosyl]-β-D-digitoxopyranoside (35), 8β-hyD


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nin were obtained for the first time. The presence of oleandrigenin glycosides in the genus Ornithogalum was not known until that time. O. nutans showed a cardenolide pattern different from that of the second European species of O. boucheanum. The structures of 32−45 are given in Figure 2. A further investigation of the bulbs of O. nutans resulted in the isolation of 11 additional new cardenolide glycosides. They were structurally elucidated as strophanthidin 3-O-α-L-quinovopyranoside (46), strophanthidin 3-O-(4′-O-β-D-xylopyranosyl)-β-D-allopyranoside (47), strophanthidin 3-O-(4′-O-β-Dglucopyranosyl)-β-D-digitoxopyranoside (48), sarmentosigenin 3-O-α-L-quinovopyranoside (49), sarmentogenin 3-O-(4′-O-αL-rhamnopyranosyl)-α-L-rhamnopyranoside (50), sarmentogenin 3-O-[4′-O-(4″-O-α-L-rhamnopyranosyl)-β-D-xylopyranosyl]-β-D-digitoxopyranoside (51), bipindogenin 3-O-β-D-glucopyranoside (52), 2α-hydroxybipindogenin 3-O-2′-desoxy-β-Dallopyranoside (53), 3β,11β-dihydroxy-12-oxo-18-nor-5αcarda-13,20(22)-dienolide 3-O-[4′-O-(4″-O-α-L-rhamnopyranosyl)-β-D-xylopyranosyl]-β-D-digitoxopyranoside (54), 7α-hydroxy-12-oxo-8β,14β-epoxyuzarigenin 3-O-[4′-O-(4″-O-α-Lrhamnopyranosyl)-β-D-xylopyranosyl]-β-D-digitoxopyranoside (55), and syriogenin 3-O-[3′-O-acetyl-4′-O-(4″-O-α-L-rhamnopyranosyl)-β-D-xylopyranosyl]-β-D-digitoxopyranoside (56).27 The glycoside of 3β,11β-dihydroxy-12-oxo-18-nor-5α-carda13,20(22)-dienolide was found for the first time. The structures of 46−56 are given in Figure 2.

spectroscopic analysis and chemical correlations.31 Determination of the absolute configuration at the C-22 hydroxyl position of 64 was achieved by the application of the advanced Mosher’s method to the aglycon of 64 and that of 65−68 by correlating them to 64. The configuration at the C-11 hydroxyl group of 64−68 was assigned as 11β by the conventional comparison of the 1H NMR data with those of cholesterol. However, seven years later, detailed spectroscopic analysis of the aglycon led to a revision of the configuration at the C-11 hydroxyl group of 64 and 67 as 11α.32 On the basis of the spectral data comparison between 64 and 67 and 65, 66, and 68, along with their aglycons, especially the spectral data at C11, it is believable that the configuration at the C-11 hydroxyl group of 65, 66, and 68 is the same as that of 64 and 67; that is, the configuration at the C-11 hydroxyl group of 65, 66, and 68 should also be revised to 11α. Therefore, their structures were finally assigned as (22S)-cholest-5-ene-3β,11α,16β,22-tetrol 16O-α- L -rhamnopyranoside (64), (22S)-cholest-5-ene3β,11α,16β,22-tetrol 16-O-(3-O-acetyl-α-L-rhamnopyranoside) (65), (22S)-cholest-5-ene-3β,11α,16β,22-tetrol 16-O-(2-O-acetyl-α-L-rhamnopyranoside) (66), (22S)-cholesta-5,24-diene3β,11α,16β,22-tetrol 16-O-α-L-rhamnopyranoside (67), and (22S)-cholesta-5,24-diene-3β,11α,16β,22-tetrol 16-O-(3-O-acetyl-α-L-rhamnopyranoside) (68). The structures of 61−68 are given in Figure 3. Bioassay-guided fractionation of the MeOH extract of O. saundersiae bulbs led to the isolation of a new cholestane bisdesmoside with potent cytotoxic activities toward leukemia HL-60 and MOLT-4 cells. The structure was deduced mainly from spectroscopic information as (23E)-cholesta-5,23-diene1β,3β,16β,25-tetrol 1-O-β-D-glucopyranoside 16-O-(2-O-(3,4,5trimethoxybenzoyl)-α-L-arabinopyranoside) (69).33 Further investigation of the n-BuOH extract as the cytostatic-active fraction resulted in the isolation of three new cholestane glycosides by silica gel ODS column chromatography and reversed-phase high-performance liquid chromatography (HPLC). Their structures were characterized as 3β,16β,17αtrihydroxycholest-5-en-22-one 16-O-[3′-O-(2(E)-cinnamoyl-βD -xylopyranosyl)](2-O-acetyl-α- L -arabinopyranoside) (70), 3β,16β,17α-trihydroxycholest-5-en-22-one 3-O-β-D-glucopyran o s i d e 1 6 - O - [ 3 ′ - O - ( 2- O -( p - m e t h o x y b e n z o y l ) - β - D xylopyranosyl)](2-O-acetyl-α-L-arabinopyranoside) (71), and 3β,16β,17α-trihydroxycholest-5-en-22-one 3-O-β-D-glucopyranoside 16-O-[3′-O-(2(E)-cinnamoyl-β-D-xylopyranosyl)](2-Oacetyl-α-L-arabinopyranoside) (72).13 Four new cholestane glycosides were isolated by further analysis of the bulbs of O. saundersiae, and on the basis of spectroscopic analysis, including 2D NMR techniques, and the results of hydrolysis, their structures were elucidated as (22S)-cholest-5-ene3β,11α,16β,22-tetrol 16-O-(2,3-di-O-acetyl-α-L-rhamnopyranoside) (73), (22S)-cholest-5-ene-3β,11α,16β,22-tetrol 16-O-[2O-acetyl-3-O-(3,4,5-trimethoxybenzoyl)-α-L-rhamnopyranoside] (74), (22S)-cholest-5-ene-3β,11α,16β,22-tetrol 16-O-[2O-acetyl-3-O-(p-methoxybenzoyl)-α- L -rhamnopyranoside] (75), and (22S)-cholesta-5,24-diene-3β,11α,16β,22-tetrol 16O-(2,3-di-O-α-L-rhamnopyranoside) (76).32 Further phytochemical analysis of the bulbs of O. saundersiae yielded two new cytotoxic cholestane triglycosides. On the basis of their spectroscopic analysis, including 2D NMR, and the results of hydrolytic cleavage, their structures were determined as 3β,16β,17α-trihydroxycholest-5-en-22-one 16-O-[3′-O-(4″-Oβ-D-glucopyranosyl)((3,4-dimethoxybenzoyl)-β-Dxylopyranosyl)](2-O-acetyl-α-L-arabinopyranoside) (77) and

2.2. Common Cholestane Glycosides

Four new cholestane bisdesmosides were isolated from the fresh bulbs of O. thyrsoides. Their structures were determined by spectroscopic analysis and chemical evidence to be (22S)5α-cholestane-1β,3β,16β,22-tetrol 1-O-α-L-rhamnopyranoside 16-O-β- D -glucopyranoside (57), (22S)-5α-cholestane1β,3β,16β,22-tetrol 1-O-α-L-rhamnopyranoside 16-O-(6-O-acetyl-β- D -glucopyranoside) (58), (22S)-5α-cholest-24-ene1β,3β,16β,22-tetrol 1-O-α-L-rhamnopyranoside 16-O-β-D-glucopyranoside (59), and (22S)-5α-cholest-24-ene1β,3β,16β,22-tetrol 1-O-α-L-rhamnopyranoside 16-O-(6-O-acetyl-β-D-glucopyranoside) (60).28 An advanced Mosher’s method was applied to determine the C-22 absolute configuration. The structures of 57−60 are given in Figure 3. In Sashida’s group, a phytochemical examination of the bulbs of O. saundersiae led to the isolation of three new acylated cholestane glycosides. Their structures were elucidated on the basis of spectroscopic data and chemical evidence and by comparing the data and evidence with those of known compounds as 3β,16β,17α-trihydroxycholest-5-en-22-one 16O-(3′-O-β-D-xylopyranosyl)(2-O-acetyl-α-L-arabinopyranoside) (61), 3β,16β,17α-trihydroxycholest-5-en-22-one 16-O-[3′-O(2-O-(4-methoxybenzoyl)-β-D-xylopyranosyl)](2-O-acetyl-α-Larabinopyranoside) (62), and 3β,16β,17α-trihydroxycholest-5en-22-one 16-O-[3′-O-(2-O-(3,4-dimethoxybenzoyl)-β- D xylopyranosyl)](2-O-acetyl-α-L-arabinopyranoside) (63).12 Compound 62 (called OSW-1) showed very potent cytostatic activity against various malignant tumor cells, and its structure was later confirmed by chemical synthesis.29,30 Further analysis of the 1-butanol-soluble phase of the methanol extract of O. saundersiae bulbs led to the isolation of five new polyhydroxylated cholestane glycosides. Their structures were determined to be (22S)-cholest-5-ene-3β,11β,16β,22-tetrol 16-O-α-L-rhamnopyranoside (64) and its acetyl derivatives 65 and 66, and (22S)-cholesta-5,24-diene-3β,11β,16β,22-tetrol 16-O-α-L-rhamnopyranoside (67) and its acetyl derivative 68 using E


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(94), (22S)-cholest-5-ene-1β,3β,16β,22-tetrol 1-O-α-L-rhamnopyranoside 16-O-α-L-rhamnopyranoside (95), (22S)-5α-cholest-24-ene-1β,3β,16β,22-tetrol 1-O-α-L-rhamnopyranoside 16O-α-L-rhamnopyranoside (96), and (22S)-cholest-5,24-diene1β,3β,16β,22-tetrol 1-O-α-L-rhamnopyranoside 16-O-α-L-rhamnopyranoside (97).36 The structures of 79−97 are given in Figure 3.

3β,16β,17α-trihydroxycholest-5-en-22-one 16-O-[3′-O-(4″-Oβ- D -glucopyranosyl)((3,4,5-trimethoxybenzoyl)-β- D xylopyranosyl)](2-O-acetyl-α-L-arabinopyranoside) (78).34 The structures of 69−78 are given in Figure 3. A cytotoxicity-guided fraction−action procedure of the MeOH extract of O. thyrsoides led to the isolation of 12 cholestane glycosides. Their structures were determined as 3β,16β,17α-trihydroxycholest-5-en-22-one 3-O-β-D-glucopyranoside 16-O-[3′-O-(2-O-(3,4,5-trimethoxybenzoyl)-β- D xylopyranosyl)](2-O-acetyl-α- L -arabinopyranoside) (79), 3β,16β,17α-trihydroxycholest-5-en-22-one 3-O-(6′-O-β-D-glucopyranosyl)-β- D -glucopyranoside 16-O-[3′-O-β- D xylopyranosyl](2-O-acetyl-α- L -arabinopyranoside) (80), 3β,16β,17α-trihydroxycholest-5-en-22-one 3-O-(6′-O-β-D-glucopyranosyl)-β-D-glucopyranoside 16-O-[3′-O-(2-O-(3,4-dimethoxybenzoyl)-β-D-xylopyranosyl)](2-O-acetyl-α-L-arabinopyranoside) (81), 3β,16β,17α-trihydroxycholest-5-en-22-one 3-O(6′-O-β-D-glucopyranosyl)-β-D-glucopyranoside 16-O-[3′-O-(2O-(3,4,5-trimethoxybenzoyl)-β-D-xylopyranosyl)](2-O-acetylα-L-arabinopyranoside) (82), 3β,16β,17α-trihydroxycholest-5en-22-one 3-O-[6′-O-(4″-O-β-D-glucopyranosyl)-β-D-glucopyranosyl]-β-D-glucopyranoside 16-O-[3′-O-β-D-xylopyranosyl](2O-acetyl-α-L-arabinopyranoside) (83), 3β,16β,17α-trihydroxycholest-5-en-22-one 3-O-[6′-O-(4″-O-β-D-glucopyranosyl)-β-Dglucopyranosyl]-β-D-glucopyranoside 16-O-[3′-O-(2-O-(3,4-dimethoxybenzoyl)-β-D-xylopyranosyl)](2-O-acetyl-α-L-arabinopyranoside) (84), 3β,16β,17α-trihydroxycholest-5-en-22-one 3O-[6′-O-(4″-O-β-D-glucopyranosyl)-β-D-glucopyranosyl]-β-Dglucopyranoside 16-O-[3′-O-(2-O-(3,4,5-trimethoxybenzoyl)β-D-xylopyranosyl)](2-O-acetyl-α-L-arabinopyranoside) (85), 3β,16β,17α-trihydroxycholest-5-en-22-one 3-O-[6′-O-(4″-O-βD-glucopyranosyl)-β-D -glucopyranosyl]-β-D -glucopyranoside 16-O-[3′-O-(2-O-(4-hydroxy-3-methoxybenzoyl)-β- D xylopyranosyl)](2-O-acetyl-α- L -arabinopyranoside) (86), 3β,16β,17α-trihydroxycholest-5-en-22-one 3-O-[6′-O-(4″-O-βD-glucopyranosyl)-β-D -glucopyranosyl]-β-D -glucopyranoside 16-O-[3′-O-(3-O-(3,4-dimethoxybenzoyl)-β-D-xylopyranosyl)](2-O-acetyl-α-L-arabinopyranoside) (87), 3β,16β,17α-trihydroxycholest-5-en-22-one 3-O-β-D-glucopyranoside 16-O-α-Larabinopyranoside (88), 3β,16β,17α-trihydroxycholest-5-en-22one 3-O-β-D-glucopyranoside 16-O-(3′-O-β-D-xylopyranosyl)(2-O-acetyl-α-L-arabinopyranoside) (89), and 3β,16β,17αtrihydroxycholest-5-en-22-one 3-O-β-D-glucopyranoside 16-O[3′-O-(2-O-(3,4-dimethoxybenzoyl)-β-D-xylopyranosyl)](2-Oacetyl-α-L-arabinopyranoside) (90).35 79−87 were new cholestane glycosides, and all the new compounds were revealed to be 3β,16β,17α-trihydroxycholest-5-en-22-one by analysis of their spectral data and were different from each other with regard to the structures of the sugar moieties and the acyl groups attached at the C-16 sugar residue. Further phytochemical analysis of the bulb extract with particular attention to the steroidal glycoside constituents led to the isolation of seven cholestane glycosides. On the basis of the spectroscopic data, including 2D NMR, and the results of hydrolytic cleavage, their structures were determined as (22S)-5α-cholestane1β,3β,16β,22-tetrol 1-O-α-L-rhamnopyranoside 16-O-(4″-O-βD-apiofuranosyl)(6-O-acetyl-β-D-glucopyranoside) (91), (22S)5α-cholestane-1β,3β,16β,22-tetrol 1-O-β-D-glucopyranoside 16O-β- D -glucopyranoside (92), (22S)-5α-cholestane1β,3β,16β,22-tetrol 1-O-(2-O-(3,4,5-trimethoxybenzoyl)-β-Dglucopyranoside) 16-O-β-D-glucopyranoside (93), (22S)-5αcholest-24-ene-1β,3β,16β,22-tetrol 1-O-(2-O-(3,4,5-trimethoxybenzoyl)-β- D -glucopyranoside) 16-O-β- D -glucopyranoside

2.3. Rearranged Cholestane Glycosides

The continuous phytochemical investigation of O. saundersiae bulbs resulted in the isolation of nine novel 24(23→22)-abeocholestane glycosides, identified as saundersiosides A−I (98− 106, respectively), together with a new 16,23-epoxy-5βcholestane glycoside, identified as saundersioside J (107).37−44 Their structures were determined on the basis of spectroscopic analysis and the results of hydrolysis. 98 and 99 were rearranged cholestane glycosides with a six-membered hemiacetal ring between C-16 and C-13 and a five-membered acetal ring between C-18 and C-20.37,41,42 The structure of 106 was different from those of 98−105 with a δ-lactone ring system formed from the rearranged side chain and a sugar moiety at C-1 of the aglycon.43 By molecular mechanics and molecular dynamics calculation studies, the E-ring part of 107 existed in a boat form, and the conformation of the sixmembered hemiacetal ring of 100−105 was also found to be almost in a boat form, while the six-membered hemiacetal ring of 97 and 98 existed in a half-chair form.38,39,41−43 The difference was considered to be caused by the five-membered acetal ring formed between C-18 and C-20, which left the sixmembered hemiacetal ring for the half-chair form. The structures of 98−107 are given in Figure 4. 2.4. Spirostane Glycosides

To investigate the chemical constituents of the whole plant of O. caudatum, two spirostane glycosides, caudaside A (108) as a new compound and hecogenin 3-O-[4′-O-(2″-O-β-D-glucopyranosyl-3″-O-β-D-xylopyranosyl)-β-D-glucopyranosyl]-β-D-galactopyranoside (109), were isolated by various chromatographic techniques, and their structures were determined by EI-MS, IR, 1 H NMR, 13C NMR, DEPT, 1H−1H COSY, 1H−13C COSY, HMBC, and NOSEY spectral analyses.45 Phytochemical analysis was carried out on the fresh bulbs of O. thyrsoides with particular attention to the steroidal glycoside constituents, resulting in the isolation of four new spirostanol saponins, (25R)-3β-hydroxy-5α-spirostan-1β-yl 1-O-β-D-glucopyranoside (110), (25R)-3β-hydroxyspirost-5-en-1β-yl 1-O-(2′-O-α-Lrhamnopyranosyl-3′-O-β-D-xylopyranosyl)-β-D-arabinopyranoside (111), (25R,25R)-3β,24-dihydroxyspirost-5-en-1β-yl 1-O(2′-O-α-L-rhamnopyranosyl)-β-D-arabinopyranoside (112), and (25R,25R)-3β,24-dihydroxyspirost-5-en-1β-yl 1-O-(2′-O-α-Lrhamnopyranosyl-3′-O-β-D-xylopyranosyl)-β-D-arabinopyranoside (113), along with a known spirostanol saponin, (25R)-3βhydroxyspirost-5-en-1β-yl 1-O-(2′-O-α-L-rhamnopyranosyl)-β36 D-arabinopyranoside (114). The structures of the four new compounds were determined on the basis of their spectroscopic data, including 2D NMR spectroscopy, and the results of hydrolytic cleavage. By analyzing the steroidal glycoside content of fresh bulbs of O. thyrsoides, four new polyoxygenated steroidal glycosides were isolated and identified as ornithosaponins A−D (115−118) on the basis of extensive spectroscopic analysis, including 2D NMR, and the results of acidic or alkaline hydrolysis.46 The aglycon structure of 115−118 was not previously reported. It was also notable that 116−118 were found to contain 6-deoxy-β-D-gulopyranose (gulomethylose) as F


Chemical Reviews

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phenylsitosterol (124) by spectroscopic data and chemical evidence.48 The structures of 120−124 are given in Figure 6.

3. PHYTOCHEMICAL STUDIES Phytochemical studies on Ornithogalum species are summarized in Table 1, in which the new steroidal glycosides are given as bold compound numbers. It should be noted that the literature was covered from 1959 to 2011, and we apologize for any unintentional omissions. Table 1 shows that most species of Ornithogalum plants were not used for phytochemical investigation, and only 11 species were studied and found to contain steroidal glycosides. Most steroidal glycosides existed in the bulbs of these plants. O. boucheanum, O. nutans, O. umbellatum, O. magnum, and O. nanum yielded many cardenolide glycosides, especially the first three, which indicated that the biogenesis relationships of the five plants might be very close in the Ornithogalum genus from the perspective of chemotaxonomy. In the bulbs of O. saundersiae, there were plenty of cholestane glycosides, including OSW-1 and its analogues. O. thyrsoides also contained many cholestane glycosides along with spirostane glycosides, and both of them contained no cardenolide glycosides, which suggested the biogenesis relationship of O. thyrsoides might be close to that of O. saundersiae in the Ornithogalum genus. Interestingly, in Mimaki and Sashida’s group phytochemical investigation of G. candicans bulbs also resulted in the isolation of five OSW-1 analogues, 72, 88−90, and 97, and two 24(23→22)-abeocholestane glycosides, 101 and 125, which have a monoglucosyl or diglucosyl unit at the hydroxyl group at C-3, without any exceptions.51 Candicanoside A (126) was also obtained from the fresh bulbs of G. candicans, which was unique in this family of saponins with a fused-ring scaffold resulting from acetal formation between the aldehyde group at C-23 and the hydroxyl groups at C-16 and C-18,52 and all 1H and 13C NMR data for 126 were assigned on the basis of the 2D NMR spectroscopic analysis. Furthermore, the structure of 126 was later confirmed via chemical synthesis53,54 by Yu’s research group. Of course, there were many other types of constituents reported in Ornithogalum plants, such as flavone and its glycosides,55−57 sapocarotenoids,58 monoterpenes,59 triterpenes,9 and polysaccharides.60 Figure 4. Rearranged cholestane glycosides from the genus Ornithogalum.

4. ANALYTICAL METHODS HPLC was used as a tool for the isolation of the naturally occurring cholestane glycosides,12,13 and MS was an important technique for the identification of compounds involving synthetic analogues. The coupling of HPLC with MS was a powerful tool for identifying steroidal glycosides. Three cholestane glycosides, including OSW-1 (62) with antitumor activity and two new analogues with modified steroidal side chains, thienyl-OSW-1 (127) and silylated thienyl-OSW-1 (128) (Figure 7), were analyzed by using optimized, reversedphase HPLC with electrospray ionization and atmospheric pressure chemical ionization quadrupole MS.61 The results showed that optimized HPLC/MS analysis could yield useful information for the isolation, identification, and structural elucidation of OSW-1 and its analogues. In Kasai’s study,62 matrix-assisted laser desorption/ionization (MALDI) quadrupole ion trap (QIT) time-of-flight tandem mass spectrometry (TOF-MSn) was also used to identify OSW-1, each molecularly related ion was identified, and subsequent collision-induced dissociation experiments in which a molecularly related ion was

a sugar component, which is rarely encountered in plant glycosides. Methanol extraction of the bulbs of Ornithogalum tenuifolium afforded a novel crystalline steroidal sapogenin, (25R)-5β-spirostane-1β,3α-diol (119).47 Its structure and stereochemistry were unambiguously assigned using X-ray diffraction and multidimensional 1H and 13C NMR data. The structures of 108−119 are given in Figure 5. 2.5. Stigmastane Glycosides

To investigate the chemical constituents of the whole plant of O. caudatum, β-sitosterol (120) was isolated and identified by Liu et al.45 Afterward, daucosterol (121), stigmasterol (122), and stigmasterol 3-O-β-D-glucopyranoside (123) were also isolated from the bulbs of O. caudatum in our laboratory.9 Phytochemical examination of the bulbs of O. umbellatum led to the isolation of a new steroidal compound. Its structure was determined to be 3-O-[2′-methoxy-4′-(2-pentenal)]G


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Figure 5. Spirostane glycosides from the genus Ornithogalum.

more potent activity in comparison with the clinically applied anticancer agents, such as etoposide, adriamycin (ADM), and methotrexate (MTX) used as positive controls. Further detailed examination of OSW-1 (62) as a main constituent in O. saundersiae bulbs showed that it exhibited exceptionally potent cytostatic activities on various malignant tumor cells such as mouse mastrocarcinoma, human pulmonary adenocarcinoma, human pulmonary large cell carcinoma, and human pulmonary squamous cell carcinoma, including ADM-resistant P388 leukemia and camptothecin (CPT)-resistant P388. The activities were around 10−100 times more potent than those of the clinical anticancer agents, such as mitomycin C (MMC), ADM, cisplatin (CDDP), CPT, and paclitaxel (TAX) (Table 2). It should be emphasized that OSW-1 showed little toxicity to normal human pulmonary cells. The deacyl derivatives of OSW-1 (129) were significantly less potent, indicating that a structural requirement for the potent activity was the acyl groups attached at the diglycoside moiety. Later on, many cholestane glycosides from Ornithogalum plants were isolated and assayed to have antitumor activities, but none of them were more potent than OSW-1. Though the higher concentration of OSW-1 showed a weak influence on the endothelium function, OSW-1 at an antitumor dose had no influence on either endothelium or smooth muscle function.63 OSW-1 was also found to be cytotoxic in the U.S. National Cancer Institute 60-cell in vitro screen,64 with a mean GI50 value of 0.00078 μM and a mean TGI of 0.018 μM (Table 3),16 and it was remarkably effective on mouse P388 with an increased life span of 59% by using one administration of 0.01 mg/kg. Striking similarities between the correlation coefficients of 0.60−0.83 were found at all three levels of response (GI50, TGI, and LC50) between OSW-1 and cephalostatins; the latter were isolated from the Indian Ocean hemichordate Cephalodiscus gilchristi and also exhibited remarkable cytotoxic activities in relation to a broad spectrum of malignant tumor cells.65−68 Structurally, OSW-1 could be considered to be half a cephalostatin molecule, with oxidation of the cholestane skeleton at C-16 and C-17, and as bis-steroidal analogues, cephalostatins could be produced from a dihydroaglycon of OSW-1.69

Figure 6. Stigmastane glycosides from the genus Ornithogalum.

selected as a precursor ion produced the characteristic product ions that were essential for structural elucidation (Figure 8).

5. BIOLOGICAL ACTIVITIES The steroidal glycosides from Ornithogalum plants have attracted considerable attention as antitumor agents and cAMP phosphodiesterase inhibitors. Many natural Ornithogalum steroidal glycosides from different species, as well as hemisynthetic derivatives, were tested in laboratory assays. A few showed very potent activity against tumor cell lines, and some could inhibit cAMP, but it is still unknown whether these activities can be retained in vivo. The present review is a systematic summary of the biological activities of steroidal glycosides from the genus Ornithogalum. 5.1. Antitumor Activity

Studies on the antitumor activity of steroidal glycosides from Ornithogalum plants began in the late 1990s, when five cholestane glycosides (62, 63, and 70−72) isolated from the MeOH extract of O. saundersiae bulbs were reported to strongly suppress the growth of leukemia HL-60 cells with IC50 values ranging between 0.0001 and 0.0003 μM.13 They showed much H


Chemical Reviews

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Table 1. Steroidal Glycosides Isolated from Ornithogalum Species structure type

steroidal glycoside O. boucheanum, Leaves and Bulbs sarmentogenin 3-O-[4′-O-(3″-O-β-D-apiofuranosyl)-β-D-xylopyranosyl]-β-D-allomethylopyranoside (13) sarmentogenin 3-O-[4′-O-(4″-O-α-L-rhamnopyranosyl)-β-D-xylopyranosyl]-β-D-allomethylopyranoside (14) 15β,16α-dihydroxyuzarigenin 3-O-[4′-O-(4″-O-α-L-rhamnopyranosyl)-β-D-xylopyranosyl]-β-D-digitoxopyranoside (15) sarmentogenin 3-O-[4′-O-(3″-O-β-D-apiofuranosyl)-β-D-xylopyranosyl]-β-D-digitoxopyranoside (16) syriogenin 3-O-[4′-O-(4″-O-α-L-rhamnopyranosyl)-β-D-xylopyranosyl]-β-D-digitoxopyranoside (17) sarmentogenin 3-O-(4′-O-β-D-apiofuranosyl)-α-L-rhamnopyranoside (18) uzarigenin 3-O-[4′-O-4″-O-α-L-rhamnopyranosyl)-β-D-xylopyranosyl]-β-D-digitoxopyranoside (19) digitoxigenin 3-O-(4′-O-β-D-xylopyranosyl)-β-D-allomethylopyranoside (20) O. caudatum, Whole Herb caudaside A (108) hecogenin 3-O-[4′-O-(2″-O-β-D-glucopyranosyl-3″-O-β-D-xylopyranosyl)-β-D-glucopyranosyl]-β-D-galactopyranoside β-sitosterol O. caudatum, Bulbs daucosterol stigmasterol stigmasterol 3-O-β-D-glucopyranoside O. magnum, Seedpods rohdexin A ornithogaloside (3) ornithogalin (4) rohdexin B rhodexoside O. nanum, Bulbs periplogenin bipindogenin strophanthidol nigrescigenin peripalloside convalloside O. nutans, Leaves and Bulbs 15β,16α-dihydroxyuzarigenin 3-O-[4′-O-(4″-O-α-L-rhamnopyranosyl)-β-D-xylopyranosyl]-β-D-digitoxopyranoside 7β,15β,16α-trihydroxyuzarigenin 3-O-[4′-O-(4″-O-α-L-rhamnopyranosyl)-β-D-xylopyranosyl]-β-D-digitoxopyranoside (32) uzarigenin 3-O-[4′-O-(4″-O-α-L-rhamnopyranosyl)-β-D-xylopyranosyl]-β-D-digitoxopyranoside 12-oxo-5α-adynerigenin 3-O-[3′-O-acetyl-4′-O-(4″-O-α-L-rhamnopyranosyl)-β-D-xylopyranosyl]-β-D-digitoxopyranoside (33) 8β,16α-dihydroxy-15-oxouzarigenin 3-O-[4′-O-(4″-O-α-L-rhamnopyranosyl)-β-D-xylopyranosyl]-β-D-digitoxopyranoside (34) 8β,16α-dihydroxy-15-oxouzarigenin 3-O-[3′-O-acetyl-4′-O-(4″-O-α-L-rhamnopyranosyl)-β-D-xylopyranosyl]-β-D-digitoxopyranoside (35) 8β-hydroxy-15-oxouzarigenin 3-O-[4′-O-(4″-O-β-D-apiofuranosyl)-β-D-xylopyranosyl]-β-D-digitoxopyranoside (36) 8β-hydroxy-15-oxouzarigenin 3-O-[3′-O-acetyl-4′-O-(4″-O-β-D-apiofuranosyl)-β-D-xylopyranosyl]-β-D-digitoxopyranoside (37) 8β-hydroxy-15-oxouzarigenin 3-O-[4′-O-(4″-O-α-L-rhamnopyranosyl)-β-D-xylopyranosyl]-β-D-digitoxopyranoside (38) 3β,11β-dihydroxy-12-oxo-18-nor-5α-card-13-enolide 3-O-[3′-O-acetyl-4′-O-(4″-O-α-L-rhamnopyranosyl)-β-D-xylopyranosyl]-β-D-digitoxopyranoside (39) 3β,11β-dihydroxy-12-oxo-18-nor-5α-card-13-enolide 3-O-[4′-O-(4″-O-α-L-rhamnopyranosyl)-β-D-xylopyranosyl]-β-D-digitoxopyranoside (40) strophanthidin 3-O-[4′-O-(3″-O-β-D-apiofuranosyl)-β-D-xylopyranosyl]-β-D-allomethylopyranoside (41) sarmentogenin 3-O-(4′-O-β-D-apiofuranosyl)-α-L-rhamnopyranoside 16β-hydroxysarmentogenin 3-O-(4′-O-β-D-apiofuranosyl)-α-L-rhamnopyranoside (42) oleandrigenin 3-O-[4′-O-(3″-O-β-D-apiofuranosyl)-β-D-xylopyranosyl]-β-D-digitoxopyranoside (43) oleandrigenin 3-O-[4′-O-(3″-O-β-D-apiofuranosyl)-β-D-xylopyranosyl]-β-D-2-deallopyranoside (44) 12β-hydroxyoleandrigenin 3-O-[4′-O-(3″-O-β-D-apiofuranosyl)-β-D-xylopyranosyl]-β-D-2-deallopyranoside (45) convallatoxin strophanthidin 3-O-α-L-quinovopyranoside (46) convalloside strophanthidin 3-O-(4′-O-β-D-xylopyranosyl)-β-D-allopyranoside (47) strophanthidin 3-O-(4′-O-β-D-glucopyranosyl)-β-D-digitoxopyranoside (48) tholloside sarmentosigenin 3-O-α-L-quinovopyranoside (49) rodexin A rodexoside sarmentogenin 3-O-(4′-O-β-D-apiofuranosyl)-α-L-rhamnopyranoside sarmentogenin 3-O-(4′-O-α-L-rhamnopyranosyl)-α-L-rhamnopyranoside (50) sarmentogenin 3-O-[4′-O-(4″-O-α-L-rhamnopyranosyl)-β-D-xylopyranosyl]-β-D-digitoxopyranoside (51) lokundjoside bipindogenin 3-O-β-D-glucopyranoside (52) I

ref

A A A A A A A A

10 10 10 10 10 10 10 10

C C D

45 45 45

D D D

9 9 9

A A A A A

19 21 22 23 23

A A A A A A

11 11 11 11 11 11

A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A

26 26 26 26 26 26 26 26 26 26 26 26 26 26 26 26 26 27 27 27 27 27 27 27 27 27 27 27 27 27 27


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Table 1. continued structure type

steroidal glycoside

ref

O. nutans, Leaves and Bulbs 2α-hydroxybipindogenin 3-O-β-D-2-deallopyranoside (53) 3β,11β-dihydroxy-12-oxo-18-nor-5α-carda-13,20(22)-dienolide 3-O-[4′-O-(4″-O-α-L-rhamnopyranosyl)-β-D-xylopyranosyl]-β-D-digitoxopyranoside (54) 7α-hydroxy-12-oxo-8β,14β-epoxy-uzarigenin 3-O-[4′-O-(4″-O-α-L-rhamnopyranosyl)-β-D-xylopyranosyl]-β-D-digitoxopyranoside (55) syriogenin 3-O-[3′-O-acetyl-4′-O-(4″-O-α-L-rhamnopyranosyl)-β-D-xylopyranosyl]-β-D-digitoxopyranoside (56) O. saundersiae, Bulbs 3β,16β,17α-trihydroxycholest-5-en-22-one 16-O-(3′-O-β-D-xylopyranosyl)(2-O-acetyl-α-L-arabinopyranoside) (61) 3β,16β,17α-trihydroxycholest-5-en-22-one 16-O-[3′-O-(2-O-(4-methoxybenzoyl)-β-D-xylopyranosyl)](2-O-acetyl-α-L-arabinopyranoside) (62) 3β,16β,17α-trihydroxycholest-5-en-22-one 16-O-[3′-O-(2-O-(3,4-dimethoxybenzoyl)-β-D-xylopyranosyl)](2-O-acetyl-α-L-arabinopyranoside) (63) (22S)-cholest-5-ene-3β,11α,16β,22-tetrol 16-O-α-L-rhamnopyranoside (64) (22S)-cholest-5-ene-3β,11α,16β,22-tetrol 16-O-(3-O-acetyl-α-L-rhamnopyranoside) (65) (22S)-cholest-5-ene-3β,11α,16β,22-tetrol 16-O-(2-O-acetyl-α-L-rhamnopyranoside) (66) (22S)-cholesta-5,24-diene-3β,11α,16β,22-tetrol 16-O-α-L-rhamnopyranoside (67) (22S)-cholesta-5,24-diene-3β,11α,16β,22-tetrol 16-O-(3-O-acetyl-α-L-rhamnopyranoside) (68) (23E)-cholesta-5,23-diene-1β,3β,16β,25-tetrol 1-O-β-D-glucopyranoside 16-O-(2-O-(3,4,5-trimethoxybenzoyl)-α-L-arabinopyranoside) (69) 3β,16β,17α-trihydroxycholest-5-en-22-one 16-O-[3′-O-(2(E)-cinnamoyl-β-D-xylopyranosyl)](2-O-acetyl-α-L-arabinopyranoside) (70) 3β,16β,17α-trihydroxycholest-5-en-22-one 3-O-β-D-glucopyranoside 16-O-[3′-O-(2-O-(4-methoxybenzoyl)-β-D-xylopyranosyl)](2-O-acetyl-α-Larabinopyranoside) (71) 3β,16β,17α-trihydroxycholest-5-en-22-one 3-O-β-D-glucopyranoside 16-O-[3′-O-(2(E)-cinnamoyl-β-D-xylopyranosyl)](2-O-acetyl-α-L-arabinopyranoside) (72) (22S)-cholest-5-ene-3β,11α,16β,22-tetrol 16-O-(2,3-di-O-acetyl-α-L-rhamnopyranoside) (73) (22S)-cholest-5-ene-3β,11α,16β,22-tetrol 16-O-[2-O-acetyl-3-O-(3,4,5-trimethoxybenzoyl)-α-L-rhamnopyranoside] (74) (22S)-cholest-5-ene-3β,11α,16β,22-tetrol 16-O-[2-O-acetyl-3-O-(p-methoxybenzoyl)-α-L-rhamnopyranoside] (75) (22S)-dholesta-5,24-diene-3β,11α,16β,22-tetrol 16-O-(2,3-di-O-α-L-rhamnopyranoside) (76) 3β,16β,17α-trihydroxycholest-5-en-22-one 16-O-[3′-O-(4″-O-β-D-glucopyranosyl)((3,4-dimethoxybenzoyl)-β-D-xylopyranosyl)](2-O-acetyl-α-Larabinopyranoside) (77) 3β,16β,17α-trihydroxycholest-5-en-22-one 16-O-[3′-O-(4″-O-β-D-glucopyranosyl)((3,4,5-trimethoxybenzoyl)-β-D-xylopyranosyl)](2-O-acetyl-α-Larabinopyranoside) (78) saundersioside A (98) saundersioside B (99) saundersioside C (100) saundersioside D (101) saundersioside E (102)

B2 B2

saundersioside saundersioside saundersioside saundersioside saundersioside

F (103) G (104) H (105) I (106) J (107)

A A A A

27 27 27 27

B1 B1 B1 B1 B1 B1 B1 B1 B1 B1 B1

12 12, 12, 31, 31 31 31, 31 33 13 13

B1

13

B1 B1 B1 B1 B1

32 32 32 32 34

B1

34

B2 B2 B2

B2 B2 B2 B2 B2

37, 42 41, 42 39, 41, 43 39, 43 39, 40, 43 43 43 43 43 38, 42

A

49

C

47

B1 B1 B1 B1 B1

28, 36 28, 36 28 28 35

B1

35

B1

35

B1

35

B1

35

B1

35

B1

35

B1

35

13 13 32

32

O. schelkovnikovii, Seeds and Bulbs rohdexin A O. tenuifolium, Bulbs (25R)-5β-spirostane-1β,3α-diol (119) O. thyrsoides, Bulbs (22S)-5α-cholestane-1β,3β,16β,22-tetrol 1-O-α-L-rhamnopyranoside 16-O-β-D-glucopyranoside (57) (22S)-5α-cholestane-1β,3β,16β,22-tetrol 1-O-α-L-rhamnopyranoside 16-O-(6-O-acetyl-β-D-glucopyranoside) (58) (22S)-5α-cholest-24-ene-1β,3β,16β,22-tetrol 1-O-α-L-rhamnopyranoside 16-O-β-D-glucopyranoside (59) (22S)-5α-cholest-24-ene-1β,3β,16β,22-tetrol 1-O-α-L-rhamnopyranoside 16-O-(6-O-acetyl-β-D-glucopyranoside) (60) 3β,16β,17α-trihydroxycholest-5-en-22-one 3-O-β-D-glucopyranoside 16-O-[3′-O-(2-O-(3,4,5-trimethoxybenzoyl)-β-D-xylopyranosyl)](2-O-acetyl-α-Larabinopyranoside) (79) 3β,16β,17α-trihydroxycholest-5-en-22-one 3-O-(6′-O-β-D-glucopyranosyl)-β-D-glucopyranoside 16-O-[3′-O-β-D-xylopyranosyl](2-O-acetyl-α-Larabinopyranoside) (80) 3β,16β,17α-trihydroxycholest-5-en-22-one 3-O-(6′-O-β-D-glucopyranosyl)-β-D-glucopyranoside 16-O-[3′-O-(2-O-(3,4-dimethoxybenzoyl)-β-Dxylopyranosyl)](2-O-acetyl-α-L-arabinopyranoside) (81) 3β,16β,17α-trihydroxycholest-5-en-22-one 3-O-(6′-O-β-D-glucopyranosyl)-β-D-glucopyranoside 16-O-[3′-O-(2-O-(3,4,5-trimethoxybenzoyl)-β-Dxylopyranosyl)](2-O-acetyl-α-L-arabinopyranoside) (82) 3β,16β,17α-trihydroxycholest-5-en-22-one 3-O-[6′-O-(4″-O-β-D-glucopyranosyl)-β-D-glucopyranosyl]-β-D-glucopyranoside 16-O-[3′-O-β-Dxylopyranosyl](2-O-acetyl-α-L-arabinopyranoside) (83) 3β,16β,17α-trihydroxycholest-5-en-22-one 3-O-[6′-O-(4″-O-β-D-glucopyranosyl)-β-D-glucopyranosyl]-β-D-glucopyranoside 16-O-[3′-O-(2-O-(3,4dimethoxybenzoyl)-β-D-xylopyranosyl)](2-O-acetyl-α-L-arabinopyranoside) (84) 3β,16β,17α-trihydroxycholest-5-en-22-one 3-O-[6′-O-(4″-O-β-D-glucopyranosyl)-β-D-glucopyranosyl]-β-D-glucopyranoside 16-O-[3′-O-(2-O-(3,4,5trimethoxybenzoyl)-β-D-xylopyranosyl)](2-O-acetyl-α-L-arabinopyranoside) (85) 3β,16β,17α-trihydroxycholest-5-en-22-one3-O-[6′-O-(4″-O-β-D-glucopyranosyl)-β-D-glucopyranosyl]-β-D-glucopyranoside 16-O-[3′-O-(2-O-(4-hydroxy3-methoxybenzoyl)-β-D-xylopyranosyl)](2-O-acetyl-α-L-arabinopyranoside) (86)

J


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Table 1. continued structure type

steroidal glycoside O. thyrsoides, Bulbs 3β,16β,17α-trihydroxycholest-5-en-22-one 3-O-[6′-O-(4″-O-β-D-glucopyranosyl)-β-D-glucopyranosyl]-β-D-glucopyranoside 16-O-[3′-O-(3-O-(3,4dimethoxybenzoyl)-β-D-xylopyranosyl)](2-O-acetyl-α-L-arabinopyranoside) (87) 3β,16β,17α-trihydroxycholest-5-en-22-one 3-O-β-D-glucopyranoside 16-O-α-L-arabinopyranoside 3β,16β,17α-trihydroxycholest-5-en-22-one 3-O-β-D-glucopyranoside 16-O-(3′-O-β-D-xylopyranosyl)(2-O-acetyl-α-L-arabinopyranoside) 3β,16β,17α-trihydroxycholest-5-en-22-one 3-O-β-D-glucopyranoside 16-O-[3′-O-(2-O-(3,4-dimethoxybenzoyl)-β-D-xylopyranosyl)](2-O-acetyl-α-Larabinopyranoside) (22S)-5α-cholestane-1β,3β,16β,22-tetrol 1-O-α-L-rhamnopyranoside 16-O-(4″-O-β-D-apiofuranosyl)(6-O-acetyl-β-D-glucopyranoside) (91) (22S)-5α-cholestane-1β,3β,16β,22-tetrol 1-O-β-D-glucopyranoside 16-O-β-D-glucopyranoside (92) (22S)-5α-cholestane-1β,3β,16β,22-tetrol 1-O-(2-O-(3,4,5-trimethoxybenzoyl)-β-D-glucopyranoside) 16-O-β-D-glucopyranoside (93) (22S)-5α-cholest-24-ene-1β,3β,16β,22-tetrol 1-O-(2-O-(3,4,5-trimethoxybenzoyl)-β-D-glucopyranoside) 16-O-β-D-glucopyranoside (94) (22S)-cholest-5-ene-1β,3β,16β,22-tetrol 1-O-α-L-rhamnopyranoside 16-O-α-L-rhamnopyranoside (95) (22S)-5α-cholest-24-ene-1β,3β,16β,22-tetrol 1-O-α-L-rhamnopyranoside 16-O-α-L-rhamnopyranoside (96) (22S)-cholest-5,24-diene-1β,3β,16β,22-tetrol 1-O-α-L-rhamnopyranoside 16-O-α-L-rhamnopyranoside (97) (25R)-3β-hydroxy-5α-spirostan-1β-yl 1-O-β-D-glucopyranoside (110) (25R)-3β-hydroxyspirost-5-en-1β-yl 1-O-(2′-O-α-L-rhamnopyranosyl-3′-O-β-D-xylopyranosyl)-β-D-arabinopyranoside (111) (25R,25R)-3β,24-dihydroxyspirost-5-en-1β-yl 1-O-(2′-O-α-L-rhamnopyranosyl)-β-D-arabinopyranoside (112) (25R,25R)-3β,24-dihydroxyspirost-5-en-1β-yl 1-O-(2′-O-α-L-rhamnopyranosyl-3′-O-β-D-xylopyranosyl)-β-D-arabinopyranoside (113) (25R)-3β-hydroxyspirost-5-en-1β-yl 1-O-(2′-O-α-L-rhamnopyranosyl)-β-D-arabinopyranoside ornithosaponin A (115) ornithosaponin B (116) ornithosaponin C (117) ornithosaponin D (118) O. umbellatum, Bulbs and Leaves convallotoxin

ref

B1

35

B1 B1 B1

35 35 35

B1 B1 B1 B1 B1 B1 B1 C C C C C C C C C

36 36 36 36 36 36 36 36 36 36 36 36 46 46 46 46

A

18, 25, 50 18, 25, 50 18, 25, 50 20, 25 25 25 25 25 25 25 25 25 25 25 25 25 48

convalloside

A

rohdexin A

A

rhodexoside strophalloside convallatoxol lokundjoside tholloside peripalloside strophanthidin 3-O-[3′-O-acetyl-4′-O-(4″-O-α-L-rhamnopyranosyl)-β-D-glucopyranosyl]-β-D-digitoxopyranoside (25) sarmentogenin 3-O-(4′-O-β-D-glucopyranosyl)-β-D-quinovopyranoside (26) sarmentogenin 3-O-β-D-quinovopyranoside (27) sarmentogenin 3-O-β-D-allomethylopyranoside (28) sarmentogenin 3-O-β-D-allopyranoside (29) bipindogenin 3-O-β-D-ribopyranoside (30) strophanthidin 3-O-β-D-allopyranoside (31) 3-O-[2′-methoxy-4′-(2-pentenal)]phenylsitosterol (124)

A A A A A A A A A A A A A D

of leukemia cells with mitochondrial DNA defects and respiration deficiency that adapted the ability to survive in culture without mitochondrial respiration were also resistant to OSW-1.70 In vitro analysis revealed that OSW-1 effectively killed primary leukemia cells from chronic lymphocytic leukemia patients with disease refractory to fludarabine. The promising anticancer activity of OSW-1 and its unique mechanism of action make this compound worthy of further investigation for its potential to overcome drug resistance. Zhou et al. reported that OSW-1 induced apoptosis of mammalian cells in a concentration- and time-dependent manner.71 The drug-induced apoptosis was mediated through the mitochondrial pathway, involving the cleavage of Bcl-2. This drug-induced Bcl-2 cleavage in Chinese hamster ovary (CHO) cells could be suppressed by either dominant-negative caspase-8 or a caspase-8 inhibitor, suggesting that the Bcl-2 cleavage was dependent on caspase-8. In contrast, the Bcl-2

Figure 7. Chemical structures of thienyl-OSW-1 and silylated thienylOSW-1.

Electron microscopy and biochemical analyses revealed that OSW-1 damaged the mitochondrial membrane and cristae in both human leukemia and pancreatic cancer cells, leading to the loss of transmembrane potential, an increase of cytosolic calcium, and activation of calcium-dependent apoptosis. Clones K


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Figure 8. Fragment pathway of OSW-1 and its analogues in MALDI-QIT-TOF-MSn.

Table 2. Cytostatic Activity of 62 and Clinically Applied Anticancer Agents on Various Malignant Tumor Cells IC50 (μg/mL) malignant cell

62

mitomycin C

adriamycin

cisplatin

camptothecin

paclitaxel

CCD-19Lua P388b P388/ADMc P388/CPTd FM3Ae A-549f Lu-65g Lu-99h RERF-LC-AIi CCRF-CEMj

1.5 0.00013 0.00077 0.00010 0.00016 0.00068 0.00020 0.00020 0.00026 0.00016

2.0 0.010

2.0 0.003

10.0 0.050

2.0 0.005

2.0 0.010

0.010

0.002

0.001

0.001

0.002

0.020

0.010

0.005

0.005

0.001

a

CCD-19Lu = human normal pulmonary cell. bP388 = mouse leukemia. cP388/ADM = adriamycin-resistant mouse leukemia. dP388/CPT = camptothecin-resistant mouse leukemia. eFM3A = mouse mastrocarcinoma. fA-549 = human pulmonary adenocarcinoma. gLu-65 = human pulmonary large-cell carcinoma. hLu-99 = human pulmonary large-cell carcinoma. iRERF-LC-AI = human pulmonary squamous cell carcinoma. j CCRF-CEM = human leukemia.

caspase-8 activity in the processes of the OSW-1-induced apoptosis was further examined by using caspase-8-deficient Jurkat T cells. It was found that the caspase-8-deficient cells were resistant to OSW-1-induced Bcl-2 cleavage or apoptosis.

cleavage was independent of caspase-3 activity. The inhibition of caspase-8 activity also resulted in the reduction of apoptotic cells, indicating that Bcl-2 cleavage induced by caspase-8 promotes the progression of apoptosis. The involvement of the L


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Table 3. GI50, TGI, and LC50 Values of OSW-1 against the 60 Cell Lines panel/cell line CCRF-CEM HL-60 K-562 MOLT-4 RPMI-8226 SR A549/ATCC EKVX HOP-62 HOP-92 NCI-H23 NCI-H322M NCI-H460 NCI-H522 COLO 205 HCC-2998 HCT-116 HCT-15 HT29 KM12 SW-620 SF-268 SF-295 SF-539 SNB-19 SNB-75 U251 LOX IMVI MALME-3M M14 SK-MEL-2 SK-MEL-28

log GI50a(M)

log TGIb(M)

Leukemia −10.00 −8.15 −10.46 −8.51 −9.66 −7.85 −10.01 −8.57 −10.42 −9.66 −9.92 −7.38 Non-Small-Cell Lung Cancer −9.34 −7.00 −9.74 −7.19 −9.80 −8.36 −8.96 −7.01 −9.44 −7.18 −7.57 −7.00 −9.80 −8.80 −9.44 −8.52 Colon Cancer −9.00 −8.23 −7.52 −7.00 −9.42 −7.00 −7.96 −7.19 −9.44 −7.48 −9.39 −7.92 −9.89 −9.26 CNS Cancer −8.62 −7.00 −10.32 −8.64 −10.00 −8.35 −7.89 −7.00 −10.38 −7.85 −10.59 −7.44 Melanoma −9.60 −8.07 −9.20 −8.41 −9.57 −9.02 −9.46 −8.02 −9.74 −8.96

log LC50c(M)

panel/cell line

log GI50a(M)

log TGIb(M)

−7.00 −7.37 −7.42 −7.00 −7.00 −7.00

MCF7 MCF7/ADR-RES MDA-MB-231/ATCC HS 578T MDA-MB-435 MDA-N BT-549 T-47D

Melanoma −9.12 −9.03 −9.51 Ovarian Cancer −8.82 −8.02 −8.44 −7.30 −7.82 −8.40 Renal Cancer −10.21 −9.62 −8.01 −8.77 −10.15 −7.00 −7.11 −9.21 Prostate Cancer −9.68 −8.43 Breast Cancer −9.09 −7.62 −7.72 −10.27 −9.24 −9.11 −8.74 −8.29

−7.15 −7.00 −8.48 −7.00 −8.31

MG-MID Delta Range

−9.11 1.48 3.59

−7.00 −7.00 −7.00 −7.82 −7.00 −7.00

SK-MEL-5 UACC-257 UACC-62 IGROV1 OVCAR-3 OVCAR-4 OVCAR-5 OVCAR-6 SK-OV-3

−7.00 −7.00 −7.02 −7.00 −7.00 −7.00 −7.00 −7.17

786-0 A498 ACHN CAKI-1 RXF-393 SN12C TK-10 UO-31

−7.34 −7.00 −7.00 −7.00 −7.00 −7.38 −7.48

PC-3 DU-145

log LC50c(M)

−8.04 −8.52 −8.89

−7.44 −8.12 −8.21

−7.00 −7.00 −7.38 −7.00 −7.00 −7.17

−7.00 −7.00 −7.00 −7.00 −7.00 −7.00

−7.00 −8.40 −7.00 −7.17 −7.74 −7.00 −7.00 −8.54

−7.00 −7.04 −7.00 −7.00 −7.14 −7.00 −7.00 −7.77

−8.07 −7.00

−7.00 −7.00

−7.00 −7.00 −7.00 −9.13 −8.11 −7.48 −7.00 −7.00

−7.00 −7.00 −7.00 −7.00 −7.57 −7.12 −7.00 −7.00

−7.74 1.92 2.66

−7.18 1.31 1.48

a

GI50 is the concentration that yields 50% growth. bTGI (total growth inhibition) is the concentration at which no growth is observed. cLC50 is the concentration at which only 50% of the cells are viable.

transduction, lipid transport and metabolism, vesicular traffic, and nonvesicular sterol transport. Although OSBP was the first protein discovered to bind endogenenous oxysterols,74,75 its function and that of its ORP paralogs remain largely unclear.73 OSBP and ORP4L are receptors of ORPphilins, and by uncovering the cellular targets of the ORPphilins, it was revealed that OSBP and ORP4L are involved in cancer cell survival, which provides another link between cancer cell proliferation and lipid processing.76

Furthermore, the small subunit of caspase-8 was found to interact with Bcl-2 by yeast two-hybrid and coimmunoprecipitation assays. Overexpression of the caspase-8 small subunit reduced the cleavage of Bcl-2 and inhibited the apoptosis induced by OSW-1. Taken together, these results demonstrated that OSW-1 was capable of inducing apoptosis in mammalian cells, in which the caspase-8-dependent cleavage of Bcl-2 played an important role. In the study of Sakurai et al.,72 the 3D structures of OSW-1 (62) and its closely related congeners 61, 70, 129, and 130 (Figure 9) were investigated by NMR studies and an X-ray crystallographic analysis (Figures 10 and 11). The disaccharide moiety was found as a structural scaffold for the formation of a hydrophobic cluster by the biologically required functionalities. Burgett et al. discovered that the molecular targets of OSW-1 were woxysterol binding protein (OSBP) and its closest paralogue, OSBP-related protein 4L (ORP4L)proteins not known to be involved in cancer cell survival.73 OSBP and the ORPs constitute an evolutionarily conserved protein superfamily, members of which have been implicated in signal

5.2. cAMP Phosphodiesterase Inhibition

The cyclic AMP phosphodiesterase inhibition test provided a useful means for the screening of biologically active compounds present in natural sources. The inhibitory activity of many cholestane glycosides from Ornithogalum plants on cAMP phosphodiesterase was assayed. Compound 60 showed inhibitory activity (IC50 = 0.153 μM), while 57−59 were inactive;28 compounds 62 and 63 showed considerable inhibitory activity (62, IC50 = 0.055 μM; 63, IC50 = 0.005 μM), while 61 was inactive,12 which suggested that the benzoyl derivative attached to the sugar moiety seemed to enhance the M


Chemical Reviews

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Figure 11. Molecular structure of the OSW-1 congener 70 in stick drawing (a) and in CPK representation (b) shown in two perspectives. The dotted line in (a) shows the triangular shape for 70. Hydrophobic side chains are colored in magenta in (b). The experimental conditions are given in ref 72.

Figure 9. Chemical structures of OSW-1 and its congeners.

the acetyl group linked to the C-3 hydroxyl position of the rhamnose might contribute to the inhibitory activity. The inhibitory activity of 98 on cAMP phosphodiesterase was tested from a sample concentration of 0.20 mg/mL at an interval of 0.02 mg/mL.37 The activity increased when the

activity. Compounds 65 and 68 showed potent inhibitory activity (65, IC50 = 0.00099 μM; 68, IC50 = 0.109 μM), while compounds 64, 66, and 67 were inactive,31 which indicated that

Figure 10. (a) Crystal packing with the unit cell viewed along the a axis. The OSW-1 congener 70 is shown in stick representation, and water molecules are shown as cyan balls. (b) Hydrophobic dimer. (c) Water-medicated sugar−sugar interaction shown in two perspectives. The experimental conditions are given in ref 72. N


Chemical Reviews

Review

Scheme 1. Synthesis of the 16β,17α-Dihydroxycholest-22-one Aglycon (138)

Scheme 2. Reactions of 16a,17a-Oxidosteroid with Lithium Hydroperoxide

sample concentration was raised from 0.02 to 0.08 mg/mL. However, it decreased from 0.10 mg/mL, with the minimum activity (16.4%) at 0.16 mg/mL, and in turn increased from 0.18 mg/mL. This phenomenon should encourage further detailed studies of inhibitory effects of the cholestanes from Ornithogalum plants on phosphodiesterase.

OSW-129 by coupling of the aglycon with the sugar part. New approaches for the synthesis of this natural product were presented by Jin’s79,80 and Morzycki’s30 groups. In addition, many other research groups have made some important achievement in the synthesis of the sugar part81 and nucleus82−86 of OSW-1.

5.3. Other Activities

6.1. Synthesis of the Aglycon of OSW-1

The study of Mimaki’s group showed that OSW-1 inhibited ovarian E2 secretion and induced an irregular oestrous cycle, which was recovered within a few days depending on the time of administration.77 The decrease in E2 secretion induced by OSW-1 probably contributed to the decreased thoracic aorta relaxation. Furthermore, the inhibitory actions of OSW-1 on the gene expression of steroidal enzyme and on the growth of ovarian follicles might at least in part mediate a blockage of E2 secretion, independent of the central hypothalamus−pituitary system.

Notably, Guo and Fuchs achieved the first synthesis of the protected 16β,17α-dihydroxycholest-22-one aglycon 138 in nine steps from 5-androsten-3β-ol-17-one (131) in 55% overall yield in 1998 (Scheme 1).78 They assembled the side chain onto 5-androsten-3β-ol-17-one via a sequence of Wittig olefination and ene reactions and then introduced the 16β,17α-diol by dihydroxylation (of the 16,17-ene with 1 equiv of OsO4) followed by inversion of the resulting 16α-OH. After Wittig olefination of ketone 131, the C-22-oxygenated side chain was introduced by diastereoselective ene reaction.87 The C-22-oxygenated center in 133 was next protected because the C-22 alcohol would interfere during later transformations, and C-22 ketal 134 was subjected to the osmylation reaction and was found to give 16,17-diols 135 in good yield (∼80%). Fortunately, Swem oxidation (oxalyl chloride/DMSO)88 of cisdiols 135 afforded the desired α-hydroxy ketones 136 in excellent yield (90%). The C-3 acetate 136 was hydrolyzed upon treatment with basic methanol, the resulting alcohols were protected as tert-butyldiphenylsilyl (TBDPS) ethers 137 in excellent yield,89 and ketal 137 then underwent reduction with CeCl3/NaBH4 at low temperature to exclusively afford the

6. CHEMICAL SYNTHESIS 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 aglycon and the disaccharide moiety. The first synthesis of the OSW-1 aglycon was accomplished by the Fuchs group in 1998,78 three years after the discovery of the extraordinary cytotoxicity of saponin OSW-1.13 A few months later Yu’s group published the first total synthesis of saponin O


Chemical Reviews

Review

Scheme 3. Synthesis of Aglycon 157 from the Intact Sapogenin Skeleton of Diosgenin (145)

thioacetal 146.94,95 The 26-thioacetal 146 underwent reductive desulfurization catalyzed by W-2 Raney nickel96 to realize the needed conversion at C-26. For further modification of the E ring, the Δ5(6)-double bond and C-3 hydroxyl group in the A/B ring of 147 were protected via a classical carbonation rearrangement.97 The oxidation of the A/B-ring-protected compound 148 by dimethyldioxirane, which was generated in situ from Oxone and acetone in a buffer of 0.4 μM aqueous ethylenediaminetetraacetic acid disodium salt (EDTADS),98,99 directly resulted in the formation of the expected E-ring-opened compound 149 (32%). After deprotection of the double bond, compound 150 was obtained, which had the same side chain and A/B ring as OSW-1. They also found that 150 could be converted to 151 in one pot with BF3·Et2O catalyzing the acetylization, thioketalization, and thioketal-opening−acetylization (TOA reaction). Desulfurization of 151 with W-2 Raney nickel at room temperature produced compound 152, which was a known intermediate in the synthetic strategies of the Fuchs group78 and Yu’s group.29 They performed further transformations according to the literature,29,78 that is, ethylene glycol ketal protection of the 22-ketone 152, dihydroxylation of the 16,17-alkene function in 153 with OsO4, Swern oxidation of the 16α-OH group in 154, conversion of 3-OAc in 154 to 3O(TBS) (TBS = tert-butyldimethylsilyl) in 156, and stereoselective reduction of the 16-keto group in 156 to the 16β-OH group in 157. Therefore, they completed the synthesis of the protected aglycon of saponin OSW-1 (compound 157).

trans-diol 138 in excellent yield (95%). The stereochemistry of 138 was confirmed by single-crystal X-ray diffraction. In principle, base-catalyzed hydrolysis of the epoxide was expected to afford the 16β,17α-diol, and this would be the best way to synthesize this system. However, it was reported that the oxirane ring of 16α,17α-epoxides was resistant to alkaline cleavage, while acid-catalyzed cleavage resulted in “total decomposition of the starting steroid”.78 In spite of these unpromising results, Morzycki’s group decided to examine the reactions of some 16α,17α-oxidosteroids in detail in 2000 (Scheme 2).90,91 141 was conveniently prepared from the corresponding 3β-hydroxy-5,16-dienes 13992,93 by epoxidation with m-CPBA preceded by selective protection of the B-ring double bond as 3α,5α-cyclo-6β-methoxy derivatives. The reaction with LiOH/H2O2 led to the desired epoxide cleavage product in the case of compound 141. Reaction of epoxy ester 141 with this reagent slowly but steadily afforded dihydroxy acid 142, and monitoring of the reaction by TLC proved that cleavage of epoxide was preceded by ester hydrolysis. Dihydroxy acid 142 was isolated by column chromatography, but it appeared to be stable only in solution. Evaporation of the chromatographic fractions resulted in the spontaneous lactonization to 143. Hydroxy lactone 143 afforded the triol 144 upon LAH reduction in almost quantitative yield. In 2003, Tian’s group82 synthesized the aglycon of OSW-1 and its analogues with related side chains from the intact sapogenin skeleton of diosgenin (145) (Scheme 3). The opening of the E/F ring was the first key step for utilizing the intact skeleton of sapogenins to synthesize the aglycon of OSW-1. 145, an abundant and cheap plant-derived steroidal sapogenin, readily reacted with thiols or dithiols in the presence of a Lewis acid such as BF3·Et2O to afford directly the 26-

6.2. Total Synthesis of OSW-1

In 1999, Yu’s group29 reported the first total synthesis of OSW1 from cholestane aglycon 157 and disaccharide moiety 181 (Scheme 4). In their synthetic method, judicious choice of protecting groups is of great importance for the syntheses of P


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Scheme 4. Total Synthesis of OSW-1 by Yu’s Group

of alcohol 160 with Dess−Martin periodinane102 provided aldehyde 161 smoothly and in good yield (86%), and Grignard addition of aldehyde 161 with (3-methylbutyl)magnesium bromide afforded the expected adduct in high yield (96%), based on its 1H NMR spectrum, containing only one isomer (162). Finally, the installation of the C-17 side chain was completed by oxidation of alcohol 162 with PDC, providing C22 keto 163 in 83% yield. The C-22 carbonyl of 163 was masked as an ethylene glycol ketal under very mild conditions (catalytic TsOH, HC(OEt)3, and room temperature), giving

polyhydroxyl glycoconjugates; therefore, protecting groups (silyl groups for hydroxyls and ethylene glycol ketal for keto) were selected to allow complete removal under neutral or nearneutral conditions. Cholestane aglycon 157 was synthesized starting from the industrial material dehydroisoandrosterone (158). Wittig olefination of ketone 158100 followed by reaction with (TBDPS)Cl provided 159, which underwent an ene reaction with paraformaldehyde in the presence of catalytic BF3·OEt2 to generate the desired homoallylic alcohol 160 stereoselectively and in satisfactory yield (75%).87,101 Oxidation Q


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Scheme 5. Total Synthesis of OSW-1 by Jin’s Group

164 slowly (4 days) and in high yield (96%). Then the 3TBDPS ether of 164 was converted to the 3-TBS ether in two steps, giving 165. Finally, diene 165 was subjected to OsO4 (1.2 equiv),103−106 affording the corresponding 16α,17α-diol 166 in

moderate yield (41%). Treatment of 166 with DMSO/ ClCOCOCl107 conveniently afforded the desired C-16 keto 156 in 78% yield, which was then reduced under NaBH4/CeCl3 to provide the required 16β-OH aglycon 157 exclusively.78 R


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Scheme 6. Total Synthesis of OSW-1 by Morzycki’s Group

one,79,80 in which epiandrosterone was still used as the starting material (Scheme 5). The key steps in the total synthesis included a highly regio- and stereoselective selenium dioxidemediated allylic oxidation of 184 and a highly stereoselective 1,4-addition of (R)-alkoxyvinyl cuprates 187 to steroid 17(20)en-16-one 186 to introduce the steroid side chain. 62 was disconnected into the dissacharide 206 and the steroid aglycon 157. 157 was envisaged to be prepared by the 1,4-addition of the α-alkoxyvinyl cuprate 187 to 186, which was from commercially available 5-androsten-3β-ol-17-one (158). Compound 184 was prepared from 158 according to the literature procedure (Scheme 5).115 Swern oxidation of 185 afforded enone 186 in nearly quantitative yield. 115 (TMS)Clactivated116,117 1,4-addition of α-alkoxyvinyl cuprate 187 to enone 186 went smoothly to give silyl enol ether intermediate 188, which was converted to enol acetate 189 in a single operation without the isolation of 188. The conversion of silyl enol ether 188 to enol acetate 189 enabled them to achieve chemoselective transformation of the enol ether to cyclic acetal 190. Generation of the enolate from 190 by potassium ethoxide or potassium tert-butoxide118 followed by in situ oxidation by Davis reagent119 stereoselectively gave α-hydroxy ketone 156 in 76% yield. Stereoselective reduction of compound 156 by LiAlH4 at −78 °C provided the requisite trans-16β,17α-diol 157 in 97% yield.120 Thus, the protected aglycon of 62 was synthesized with eight operations in 48.4% overall yield. Synthesis of the disaccharide 206 was outlined in Scheme 5. Thioglycoside 192 was prepared from tetraacetyl-L-arabinose (191). Regioselective protection of the cis-diol 192 followed by protection of the C-2 hydroxy group gave 193 in 90% yield. Deprotection of the acetonide afforded diol 194. When 194 was treated with (TES)OTf and lutidine at low temperature, the desired product 195 was obtained in 90% yield. The thio ortho ester 198 was prepared from tetraacetyl-D-xylose (196).121 Protecting group manipulations followed by zinc chloride-promoted intramolecular ring opening of the thio ortho ester 200 gave thioglycoside 201 in excellent yield. After deacetylation, a p-methoxybenzoyl group was introduced, and 203 was converted to 204 in 84% yield.122 Glycosylation of 195 with 204 afforded the β-disaccharide 205, which was converted to 206. Coupling of 206 with the steroid aglycon 157 under

Thus, they achieved the preparation of 157 in 12 steps and 10% yield overall from 158. The xylosyl donor 171 was readily prepared from D-xylose in five steps as shown in Scheme 4. Therein, the 2-O-MBz (4-methoxybenzoyl) group was regioselectively introduced by treatment of triol 167 under MBzCl/Py, affording 168 in 65% yield.108 Afterward the diol of 168 was masked as the bis-TES ether to afford 169 in excellent yield (93%). The anomeric benzyl group of 169 was found to be rather resistant to hydrogenolysis; therefore, stronger conditions (10% Pd−C, 40 atm of H2, 40 °C, 24 h) were employed to generate the hemiacetal 170 (76% yield, 20% of 169 recovered), which was then converted 109 to the corresponding trichloroacetimidate 171. Meanwhile, the arabinosyl acceptor 175 was readily prepared from L-arabinose in four steps and in 67% yield, i.e., benzylation of 1-OH, isopropylidenation of 3,4-OH, acetylation of 2-OH, and final removal of isopropylidene. Glycosylation of diol 175 with 171 under BF3·Et2O110 catalysis gave the desired 1→3-linked disaccharide 176 as the major product, which could not be separated from its 1→4-linked isomer 177 through silica gel column chromatography on a large scale. The mixture of 176 and 177, without separation, was further subjected to (TES)OTf,111,112 affording disaccharides 178 (70%) and 179 (21%), which were readily separable on a silica gel column (Scheme 4). Under forcing conditions (10% Pd−C, 50 atm of H2, 50 °C, 3 days), the corresponding hemiacetal 180 was generated in 50% yield and 36% of 178 was recovered. Then treatment of 180 with CCl3CN/DBU109 provided the expected disaccharide donor 181 (65%), and glycosylation of the 16β,17α-diol 157 with disaccharide imidate 181 proceeded smoothly under the promotion of trimethylsilyl triflate [(TMS)OTf]113 to provide the fully protected OSW-1 182 in satisfactory yield (69%). Finally, all of the protecting groups (one TBS group, three TES groups, and one ethylene glycol ketal group ) were removed smoothly and cleanly in a single step by employing Pd(MeCN)2Cl2 as a catalyst114 to furnish the final target OSW-1 (62) in satisfactory yield (79%) (Scheme 4). In 2001 Jin’s group presented a somewhat different approach to the synthesis of OSW-1 employing a stereoselective 1,4addition of α-alkoxyvinyl cuprate to a steroid Δ17(20)-en-16S


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Scheme 7. Total Synthesis of OSW-1 by Yu’s Group

standard conditions123 gave compound 207 in 71% yield. Removal of all of the protecting groups by sequential treatment of compound 207 with 2,3-dichloro-5,6-dicyanobenzoquinone (DDQ) and bis(acetonitrile)dichloropalladium(II) in one operation afforded 62 in 81% yield. In 2002, Morzycki’s group30 reported the results of glycosylation of the protected OSW-1 aglycon 209 (Scheme 6). This method proved that direct glycosylation of the aglycon in its hemiketal form could be achieved, affording the protected OSW-1 in a moderate yield. LiAlH4 reduction of 209 afforded the triol 210 as a C-22 diastereoisomeric mixture with one epimer prevailing. Triol 210 was regioselectively protected at O-22 as the benzyl ether 211. Glycosylation proceeded smoothly under the promotion of (TMS)OTf, yielding 213. Hydrogenolysis of the benzyl ether was followed by oxidation of the 22-OH group to the ketone 214 with pyridinium dichromate. Finally, all protective groups (3α,5α-cyclo-6βmethoxy protection of the aglycon and TES groups on the sugar moiety) were simultaneously removed using a catalytic amount of p-TsOH in dioxane/water at 75 °C, affording OSW1 in a high yield.

In 2005, Yu’s group124 described an aldol approach to the stereoselective construction of the 16α,17α-dihydroxycholest22-one structure from 16α-hydroxy-5-androsten-17-ones and propionates (Scheme 7). Elaboration of the aldol adducts toward OSW-1, involving installation of the isoamyl ketone side chain, inversion of the 16-hydroxyl configuration, and selective protection of the C-22 oxy function, has been explored and accomplished. In particular, the present route was found convenient for the synthesis of OSW saponin analogues with a C-22 ester side chain. The required 16α-hydroxy-5-androsten17-one derivative 217, with its 3-OH being protected with a robust TBS ether, was readily prepared from the industrial acetate 131 following modification of a literature procedure.125 Then reaction of the 3-O-TBS 17-ketone 217 under similar conditions in the absence of HMPA afforded the desired 218 in a satisfactory 75% yield. Treatment of diol 218 with TPAP (tetrapropylammonium perruthenate) and NMO (N-methylmorpholine N-oxide) in the presence of 4 Å molecular sieves gave C-16 ketone 219 in 93% yield.126 Reduction of ketone 219 with NaBH4/CeCl3 led stereoselectively to 16β,17α-diol 220. However, only the intramolecular lactonization product 221 could be isolated (92%) when this reaction was quenched T


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Scheme 8. Total Synthesis of OSW-1 by Tsubuki’s Group

was subjected to Swern oxidation133 to furnish diketone 233 in 85% yield (two steps). Reduction of 233 with NaBH4 in MeOH−CH2Cl2 (1:1, v/v) at −15 °C occurred chemoselectively to afford the desired trans-diol 234 in 82% yield. Glycosylation of steroid aglycon 234 with disaccharide imidate 181 in the presence of (TMS)OTf provided β-glycoside 235 in 72% yield. All the protecting groups, one MOM group and three TES groups, were removed by treatment of 235 with (TMS)Br134 to give OSW-1 thiophene analogue 236 in 73% yield. Finally, the thiophene ring in 236 was reductively desulfurized with W-2 Raney Ni135,136 under an atmosphere of hydrogen, furnishing OSW-1 in 79% yield. In 2008, Guo’s group137 developed a new and practical method to synthesize OSW-1 from (+)-dehydroisoandrosterone, L-arabinose, and D-xylose on a gram scale (Scheme 9). OSW-1 was synthesized from (+)-dehydroisoandrosterone (158) in 10 linear steps and an overall yield of 6.4%, with new and efficient procedures developed to prepare the aglycon as well as the monosaccharide and disaccharide building blocks. This synthesis is highlighted by the reliability of all involved transformations and the simple workup procedures. Using commercially available 158 as the starting material, they first added propanenitrile to 158 at −78 °C by an aldol condensation reaction catalyzed by LDA to obtain an epimeric mixture, 237, in excellent yield upon recrystallization. Further elongation of the side chain by refluxing 237 with a Grignard reagent in benzene proceeded smoothly to give 238. Selective protection of 3-OH in 238 by TBS via reaction with (TBS)Cl and imidazole in DMF and then protection of the C-22 carbonyl group by ethylene glycol formed ketal 165. Compound 165 was transformed into 157 in three steps as described in the literature.29,78 After 240 was obtained from Larabinose as reported,138 it was regioselectively p-methoxybenzylated with the assistance of a dibutyltin complex to afford 241, which was directly subjected to acetolysis to give the

at room temperature with addition of methanol and water. This required them to quench the reaction at low temperature (−40 to −78 °C) to obtain the desired diol 220 (76%). Both lactone 221 and ester 220 could be converted into hemiketal 222 quantitatively upon addition of isoamyllithium. Reduction of 222 with LiAlH4 in Et2O afforded triol 223 in a good 87% yield. Treatment of triol 223 with 3 equiv of (AZMB)Cl [2(azidomethyl)benzoyl chloride] in the presence of 4-(N,Ndimethylamino)pyridine (DMAP) in THF afforded the 22-OAZMB product 224 mainly in 48% yield.127,128 Glycosylation of 16β,17α-diol 224 with the disaccharide trichloroacetimidate 181 by use of (TMS)OTf as a promoter in the presence of 4 Å molecular sieves at −20 °C afforded the desired glycoside 225 in 61% yield.29 Then the 22-O-AZMB group was selectively removed with PBu3, providing 22-ol 226 in a good 82% yield. Subsequent oxidation of the resulting 22-OH with PDC afforded ketone 227 in 97% yield. Finally, the silyl groups on 227 were removed with Pd(MeCN)2Cl2 in acetone−water, furnishing OSW-1 in 82% yield. In 2008, Tsubuki’s group129 embarked on the synthesis of OSW-1 employing the Wittig rearrangement of thiophene-yl methyl ether (Scheme 8). The key feature of his synthesis is based on stereoselective conversion of 22-hydroxy-22-(4′methylthienyl) steroid 230 into OSW-1 thiophene analogue 236 by introduction of a trans-diol functionality at the C-16 and -17 positions and glycosylation of aglycon with disaccharide. Requisite thiophene-yl methyl ether 229 was prepared by etherification of the known allylic alcohol130,131 228 with (4methyl-2-thiophene-yl)methyl bromide132 in the presence of 18-crown-6 in 92% yield. Treatment of 229 with t-BuLi (5 equiv) in THF at −78 °C gave [2,3]-rearranged product 230 (22α- and 22β-alcohols in a ratio of 78:22) in 59% yield. Oxidation of 230 with Dess−Martin periodinane102 in CH2Cl2 afforded ketone 231 quantitatively. Dihydroxylation of 231 with OsO4 in the presence of pyridine gave cis-diol 232, which U


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Scheme 9. Total Synthesis of OSW-1 by Guo’s Group

Figure 12. Chemical structures of OSW-1 saponin analogues with steroidal nucleus modification.

the glycosylation of 242 with 244 as a glycosyl donor went well to give 245 in a good yield and stereoselectivity, and the reaction was also proved to be very reliable. Finally, 245 was converted to glycosyl donor 247 as an α- and β-anomeric mixture upon selective removal of the anomeric acetyl group and then trichloroacetimidation of the resultant hemiacetal 246.

desired glycosyl acceptor 242. D-Xylose was transformed into 243 through a series of reactions without isolation of the reaction intermediates; then 243 was converted to Schmidt donor 244 following oxidative deprotection of the anomeric center with N-bromosuccinimide (NBS) and H2O and then trichloroacetimidation of the resultant hemiacetal. Fortunately, V


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commercially available estrone and might provide some insights into the effect of modification of the aglycon moiety for the bioactivity. In 2005, they described the preparation of the estrane analogue 251 and its cytostatic activity against several human malignant tumor cells. They found that the estrane analogue 251 had anticancer effects on all assayed cancer cell lines (Table 5). 6.3.2. Synthesis of OSW-1 Saponin Analogues with Side Chain Modification. In 2004, Yu’s group reported a novel and efficient approach to the construction of 16β,17αdihydroxycholest-22-one structures 252−254 (Figure 12).142 23-Oxa-OSW-1 (252) strongly suppressed the growth of the three types of malignant tumor cells tested. The concentration of 252 required for 50% inhibition (IC50) was 0.031−3.1 μM (Table 4), which indicated that this compound was as potent as OSW-1. The short congener 253 was slightly less potent than 252. Interestingly, the longer congener 254 was considerably more potent than OSW-1 (as much as 25 times more potent against the human liver carcinoma cell line 7404). To examine the importance of the side chain, especially of the 22-one function, in 2004, Yu’s group synthesized OSW-1 analogues 255−258 (Figure 12)143 with modified side chains on the steroidal skeleton and tested their antitumor activities against AGS (stomach cancer cells), 7404 (liver carcinoma cells), and MCF-7 (breast cancer cells). Surprisingly, analogue 255,144 with the 22-one (of OSW-1) being saturated into a CH2, was slightly more potent than OSW-1 against the three tested cancer cell lines (Table 4), while the 22-OH analogue 256 was less potent by 30-fold (for 7407 and MCF-7) than 255. The full length of the cholestane side chain was also not essential to the antitumor activity; congener 257, with the two terminal methyl groups (of OSW-1) being removed, was slightly more potent than OSW-1. However, the shorter congener 258, with the terminal isobutyl group (of OSW-1) being removed, was significantly less potent than OSW-1. In 2004, Morzycki and Wojtkielewicz’s group synthesized a series of side chain analogues (257, 259−262) (Figure 12)145 and tested them for cytotoxicity against two breast cancer cell lines (MCF-7 and MDA-MB-231) and the endometrial cancer Ishikawa cell line. They found that the analogues with a linear side chain (257, 261, and 262) showed much lower cytotoxicity than the saponin OSW-1 (Table 6). In 2005, Yu’s group reported the in vitro activities of the OSW-1 side chain modified analogues (252−254, and 263− 272) (Figure 13)124 against the proliferation of several human cancer cell lines, including HeLa, Jurkat T cells, and the human MCF-7 breast cancer cell line. They found that the 23-oxa analogue of OSW-1 (252), which has a C-22 isobutyl ester in place of the isoamyl ketone in OSW-1, showed strong activity against the growth of tumor cells (Table 7). The isosteres of OSW-1 with its 23-CH2 group replaced by a S or NH group (compounds 268 and 269) showed similar antiproliferative

The glycosylation of 157 by 247 was achieved with (TMS)OTf as a promoter to produce the protected OSW-1 248. Global deprotection of 248 was accomplished in two separate steps. First, the TBS and ketal groups at the O-3 and C-22 positions of the aglycon, respectively, were removed under mild acidic conditions. Next the PMB groups on the disaccharide moiety of 249 were removed by DDQ oxidation to afford the synthetic target OSW-1. 6.3. Synthesis of OSW-1 Saponin Analogues

While OSW-1 is exceptionally cytotoxic against various tumor cells, it showed little toxicity to normal human cells. Much attention has been paid to the synthesis of OSW-1 analogues because of their extraordinary potent activity. A number of OSW-1 saponin analogues with steroidal nuclei, side chains, disaccharides, and other structural modifications have been obtained by means of chemical synthesis for SAR (structure− activity relationship) studies. 6.3.1. Synthesis of OSW-1 Saponin Analogues with Steroidal Nucleus Modification. In 2004, Yu’s group anticipated that saturation of the 5,6-double bond,139 a position distant from the disaccharide residue,140 would not significantly alter the cytotoxicity of OSW-1, so they synthesized 5,6dihydro-OSW-l (250) (Figure 12) and tested its antitumor activity. They found that 250 with a simplified and easily prepared structure was slightly more potent than OSW-1 against the three tested cancer cell lines (Table 4) [including AGS (stomach cancer cells), IC50 = 0.71 μM, 7404 (liver carcinoma cells), IC50 = 0.025 μM, and MCF-7 (breast cancer cells), IC50 = 0.029 μM]. Table 4. Cytotoxic Activities of OSW-1, 250, and 252−258 against Tumor Cells IC50 (μM) compd

AGSa

7404b

MCF-7c

OSW-1 cisplatin 250 252 253 254 255 256 257 258

1.42 24.1 0.71 3.1 11.82 0.4 1.38 7.26 1.92 6.98

0.1 8.37 0.025 0.031 0.3 0.004 0.063 1.86 0.032 2.9

0.27 18.7 0.029 0.31 1.48 0.052 0.06 1.79 0.02 6.61

a

AGS = human stomach cancer cell line. b7404 = human liver carcinoma cell line. cMCF-7 = human breast cancer cell line.

To search for new anticancer drugs based on OSW-1, in 2005, Nemoto’s group speculated that the estrane analogue 251 (Figure 12)141 of OSW-1 might be readily accessible from

Table 5. Cytotoxic Activities of the OSW-1 Aglycon and 251 against Tumor Cells IC50 (μM) compd OSW-1 aglycon cisplatin 251

NCI-H460a >10 0.660 0.439

T-470b

MDA-MB-231b

A498c

PC-3d

DLD-1e

NT 7.85 1.01

NT 2.11 0.43

>10 3.62 1.4

>10 2.21 1.18

f

NT 11.5 0.709

a

NCI-H460 = human lung cancer. bT-470 and MDA-MB-231 = human breast cancer. cA498 = human renal cancer. dPC-3 = human prostate cancer. DLD-1 = human colon cancer. fNT = not tested.

e

W


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Table 6. Growth Inhibition Ratio (%) for Cancer Cell Necrosis compd (10−3 μM) OSW-1 257 261 262

MCF-7a 100 5 15 10

MDA-MB-231b 100 7 12 14

Table 7. Cytotoxic Activities of OSW-1 Analogues against Tumor Cells

Ishikawa cells, 24 h

IC50 (μM)

100 10 17 12

a MCF-7 = human breast cancer cell line. bMDA-MB-231 = human breast cancer.

potencies toward HeLa and Jurkat T cells. However, 268 and 269 suffered from an over 20-fold decrease in potency in MCF7 cells. A variety of the C-22 ester changes, that is, from ethyl (in 253), allyl (in 264), heptyl (in 265), and dodecyl (in 266) to the 9-decen-1-yl benzoate (in 271), did not dramatically affect their antitumor activities, except for 253 and 266, toward MCF-7 cells with a significant decrease in activity. In 2007, Wojtkielewicz and Morzycki’s group synthesized a series of novel analogues of the compound (257, 273−275) (Figure 14)146 with modified side chains and evaluated their toxicity toward a battery of cancer cell lines. The cancer cell lines exhibited distinct sensitivity to OSW-1 and its analogues, with CEM, K562, and A549 cell lines being the most sensitive and G361 human melanoma cells being the least sensitive (Table 8). In contrast, no compounds tested showed cytotoxicity to the normal mouse NIH 3T3 fibroblasts. Among the tested analogues, the most active appeared to be compound 273, which showed antitumor potency similar to that of OSW-1. Against the ARN 8 cell line, it was even 10 times more active than natural saponin. However, shortening of the alkyl side chain (257) led to a slight loss of activity. The results demonstrated that small variations in the structure, for example, in the size of the cholestane side chain, did not affect antitumor activity significantly. Contrary to other published assertions,143 the presence of a carbonyl group at C-22 also seemed to be a pharmacophore requirement. Compound 274

a

compd

Hela

OSW-1 252 253 254 263 264 265 266 267 268 269 270 271 272

0.012 0.23 0.24 0.0033 0.034 0.065 0.002 >10 10 0.0013 0.0084 0.012 0.04 0.07

Jurkat Tb

MCF-7c

0.0022 0.00068 0.0053 0.0073 0.042 0.0027 0.0014 >10 0.073 0.0009 0.0053 0.035 0.003 0.066

0.094 0.9 >10 0.14 0.13 0.1 2.6 >10 1.4 5.2 1.8 16.5 0.19 2.8

a Hela = human epithelial cervical cancer. bJurkat T = human T cell lymphoblast-like cell. cMCF-7 = human breast cancer cell line.

showed much lower activity in anticancer tests than OSW-1. Similarly, the OSW-1 ether analogue 275 appeared to be much less active than the very potent ester analogue (23-oxa analogue of OSW-1) described recently.142 In 2011, Wojtkielewicz and Morzycki’s group147 synthesized three series of ester analogues with saturated (276−281), unsaturated (282−284), and aromatic (285, 286) (Figure 14) side chains and screened these analogues against various tumor cells (Table 9). They found that the alkenyl side chains of corresponding lengths (283, 284) had similar effects on the analogues’ anticancer activities. It seemed that small variations in the structure and length of the alkyl side chain did not affect the compounds’ antiproliferative activities significantly.146 The cytotoxicity (IC50) values of 285 and 286, bearing a benzyl substituent, varied between 0.057 and 0.070 μM and were

Figure 13. Chemical structures of OSW-1 saponin analogues with side chain modification. X


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Figure 14. Chemical structures of OSW-1 saponin analogues with side chain and disaccharide modification.

Table 8. Cytotoxic Activities of OSW-1 Analogues against Tumor Cells TCS50 (10−3 μM) a

compd

CEM

OSW-1 257 273 274 275 289 290 318 daunorubicin

0.3 0.5 0.2 1300 50000 >50000 >50000 >50000 >50000 NT

a

CEM = T-lymphoblastic leukemia cell line. bMCF7 = breast carcinoma cell line. cK 562 = chronic myelogenous leukemia cell line. dARN 8 = melanoma cell line. eG 361 = malignant melanoma cell line. fHeLa = epitheloid carcinoma cell line. gHOS = osteosarcoma cell line. hA 549 = lung carcinoma cell line. iNIH 3T3 = mouse fibroblast. jNT = not tested.

Table 9. Cytotoxic Activities of OSW-1 Analogues against Tumor Cells IC50 (μM) compd

CEMa

MCF7b

G361c

Helad

HOSe

OSW-1 276 277 278 279 280 281 282 283 284 285 286

0.3 60 10 7.2 6.3 16 58 340 21 6.1 70 57

2.4 500 400 26 21 48 195 840 54 29 700 410

1000 390 1280 15 8 30 300 890 44 6.7 1660 170

3.4 170 30 47 42 67 435 1940 61 103 240 340

8200

2800 1900 2700 >50000 >50000 6500 1940 5400

A549f

T98g

HCT116h

27 160 20 590 930 720 550 550 840 600 170 570

70

8400

11 7 28 210 940 43 7

4900 2800 2800 >50000 >50000 11000 2600

55

5000

BJi 0.2 500 600 7 5 83 80 230 20 3 100 39

a

CEM = T-lymphoblastic leukemia cell line. bMCF7 = breast carcinoma cell line. cG361 = malignant melanoma cell line. dHeLa = epitheloid carcinoma cell line. eHOS = osteosarcoma cell line. fA549 = lung carcinoma cell line. gT98 = human glioblastoma cell line. hHCT116 = human colon carcinoma cells. iBJ = human fibroblasts.

Y


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Figure 15. Chemical structures of 12 OSW-1 saponin analogues synthesized by Yu’s group.

6.3.4. Synthesis of OSW-1 Saponin Analogues with Other Structural Modification. Candicanoside A (126),52 which was isolated from the fresh bulbs of G. candicans and totally synthesized by Yu’s group recently,53,54 was unique in this family of saponins with a fused-ring scaffold resulting from acetal formation between the aldehyde group at C-23 and the hydroxyl groups at C-16 and C-18. In addition, it is the only congener to show remarkable cytostatic activity (IC50 = 0.032 μM against the HL-60 cells) without benzoate substitution on the saccharide moiety.37,41−44,51,52 In 2000, Yu’s group synthesized 12 glycosides (291−302) (Figure 15)148 bearing the disaccharide of OSW-1, namely, 2O-(4-methoxybenzoyl)-β-D-xylopyranosyl-(1→3)-2-O-acetyl-αL-arabinopyranoside or its 1→4-linked analogue. They found that the diosgenyl glycoside 293 showed the strongest activities (Table 10), with 51.9% inhibition against P388 and 61.3% inhibition against A-549 at 0.01 μM. However, it lost its activity at 0.001 μM. This activity was much less potent than that of OSW-1, which had been reported to have an IC50 around 10−4 μM.12 This result demonstrated that the aglycon was also essential to the antitumor activities of glycosides. Herein, the glycosides of cholesterol (291 and 296) and simple alcohols (nonol, benzyl, and phenylthiol (300, 301, and 302, respectively)) were just inactive at 0.01 μM.

about 100-fold lower than that of OSW-1. The compounds were also tested for cytotoxicity to normal BJ human fibroblasts and proved to be (except compounds 278, 279, 282, 283, and 284) substantially (3−360-fold) less toxic toward them than toward malignant cell lines. OSW-1 was more cytotoxic toward normal human fibroblasts than its derivatives, and the ester analogue 277 exhibited the highest selectivity toward cancer cells versus normal cells. 6.3.3. Synthesis of OSW-1 Saponin Analogues with Disaccharide Modification. In 2004, Morzycki and Morzycki’s group synthesized 16β-O-L-arabinosyl (287) and 16β-O145 D-xylosyl (288) (Figure 14). They found that these two analogues of OSW-1 were not biologically active against breast and endometrial cancer cell lines compared to the natural product, and the data were not shown. In 2007, Wojtkielewicz and Morzycki’s group synthesized a series of novel analogues (289−290) (Figure 14)146 with different sugar moieties and evaluated their toxicity toward a battery of cancer cell lines (Table 8). The monosaccharide analogues of OSW-1 (289, 290) appeared to be lethal to 50% of the tumor cells at concentrations of 0.2−7.7 μM; that is, they were about 1000 times less active than OSW-1. These findings clearly indicated that the disaccharide moiety was essential for the antitumor activities of OSW-1. Z


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general, the present compounds showed stronger activities than those of the saponins bearing the disaccharide of OSW-1 at the C-3 position on the A ring,148 with up to 100% growth inhibition at 0.01 μM. However, they also lost their activity at 0.001 μM. These results further demonstrated that the aglycon was also essential to the antitumor activities of OSW-1. In 2001, Yu’s group synthesized compounds151 with structures more close to that of OSW-1 and tested their antitumor activities. The in vitro antitumor activities of OSW-1 and its analogues 312−316 and the dimer 317 (Figure 17) against P388 (mouse leukemia) and A-549 (human lung adenocarcinoma) were evaluated by the standard MTT assay (Table 10).42 Its 16-epimer 315 showed only marginal activities at 0.1 μM. This result demonstrated that the C16 configuration was essential to the antitumor activities of OSW-1. It was therefore reasonable that compounds 312−314, having both an opposite configuration at C-16 and a modified C-17 side chain, were much less potent, which showed little or no inhibition against P388 and A-549 at 1.0 μM concentration. It was reported that a 3-O-glucopyranosyl derivative of OSW-1 was as active as OSW-1 against HL-60 cells.13 However, the 3-Odisaccharide derivative of OSW-1 (316) showed a high growth inhibition rate only at 1.0 μM, about 1000 times less active than OSW-1. Interestingly, the 3-O-teraphthaloyl-linked dimer 317 showed 65.6% and 52.8% growth inhibition rates against P388 and A-549 cells, respectively, at 0.01 μM. Thus far, it has been clearly demonstrated that the acyl groups on the disaccharide moiety and the C-16 configuration were essential to the antitumor activities of OSW-1. The importance of the C17 side chain to the antitumor activities of OSW-1 has also been implied. In 2007, Wojtkielewicz and Morzycki’s group synthesized a structural isomer of saponin 318 (Figure 18)146 and tested its antitumor activity by using the panel of cancer and normal cells. The cytotoxicity (TCS50) values of 318 (Table 8) varied between 0.28 and 14.4 μM and were about 1000 times lower than that of OSW-1, which demonstrated that the position of the disaccharide moiety is also important for the antitumor activity. In an attempt to identify structurally simpler analogues of OSW-1 with full antitumor activity, in 2007, Yu’s group turned their attention to the disaccharide moiety, so they synthesized the monosaccharide derivative 319 and its congeners 320−326 (Figure 18).152 The in vitro activities of the synthetic monosaccharide analogues 319−326 of OSW saponins against

Table 10. Cytotoxic Activities of OSW-1 Analogues against Tumor Cells P388a

A549b

compd

10−1 μM

10−2 μM 10−3 μM 10−1 μM 10−2 μM 10−3 μM

OSW-1 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317

100 39.4 100.0 100.0 68.7 51.0 38.9 80.4 60.5 85.4 85.6 44.9 54.1 99.6 100.0 98.1 98.1 100.0 100.0 100.0 83.8 100.0

100 1.8 20.9 51.9 13.9 10.9 6.2 6.8 16.3 36.1 6.2 6.5 6.2 41.3 39.6 0 98.1 30.2 100.0 96.9 100.0 100.0

100 1.9 11.9 16.6 9.7 4.7 4.8 7.7 11.6 6.5 4.3 5.4 2.4 18.8 5.7 3.8 7.5 3.8 0 15.9 3.7 0

99.3 43.6 98.6 98.6 97.9 98.7 15.1 98.7 96.2 98.2 97.9 8.2 25.5 98.2 87.8 79.6 89.8 89.8 98.2 98.5 98.7 98.1

99.1 0.0 8.6 61.3 0.0 0.0 17.2 0.0 2.4 20.7 0.2 0.0 2.7 19.3 10.2 0 71.4 24.5 57.0 39.7 88.7 0

20.4

15.5

17.9

6.2

0.8

66.7 90.7 99.7

35.4 52.0 98.7

15.5

53.8 80.9 89.7

29.5 6.7 74.1

65.6

98.2 0.0 0.0 1.6 0.0 9.9 0.0 0.0 10.9 0.0 0.0 0.0 7.3 0 0 0 0 4.1 0 0 0 0

0.7 52.8

a

P388 = mouse leukemia. bA-549 = human pulmonary adenocarcinoma.

In 2001, Yu’s group thought149 that the steroidal glycosides with the disaccharide of OSW-1 attached at a position at or close to the D ring (on the C ring or on the C17 side chain) would probably show strong cytotoxic activities against tumor cells, so they reported the synthesis of such glycosides 303− 311 (Figure 16) and their antitumor activities. The in vitro antitumor activities of the resulting glycosides 303−311 against mouse leukemia P388 and human lung adenocarcinoma A-549 cell lines were tested by using the MTT assay (Table 10).150 In

Figure 16. Chemical structures of nine OSW-1 saponin analogues synthesized by Yu’s group. AA


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Figure 17. Chemical structures of six OSW-1 saponin analogues synthesized by Yu’s group.

Figure 18. Chemical structures of OSW-1 saponin analogues.

the sugar moiety in the OSW saponins was essential for their potent antitumor activity. In 2007, Eustache’s group synthesized and evaluated a short series of simplified OSW-1 analogues153 which were modified on the disaccharide moiety (Figure 18). In compound 327, the role of the 4-methoxybenzoyl groups (pMBz) was examined. In compounds 328 and 329 the role of the substituent on C-4 was examined. In compound 330, the key acetate and pMBz groups were present, but the xylose moiety was replaced by a simple, semirigid spacer. The antitumor activity of the four new OSW-1

the proliferation of several human cancer cell lines, including RKO (colon carcinoma), Jurkat (human T cell leukemia), and HeLa (human cervical cancer) cell lines, with the 22-ester analogue of OSW-1 (254) as a positive control124,142 were determined by following the incorporation of [3H]thymidine (Table 11). One analogue, 2-O-acetyl-α-L-arabinopyranoside (320), showed antiproliferative activity against the Jurkat cells (IC50 = 0.078 μM) comparable to that of the disaccharide derivative 254 (IC50 = 0.015 μM). These results suggested that AB


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showed much weaker activity against the Jurkat cells when compared to the steroid counterpart (IC50 = 0.0015 μM). However, they still possessed considerable activity, with IC50 at the micromolar level. For HeLa cells, the hydrindane glycosides 331−333) showed activity that was only slightly weaker than that of the parent saponin derivative (IC50 = 0.24 μM). These results suggested that an intact steroid ring was not absolutely required for the biological activity of the OSW-1 family of saponin analogues. To determine the molecular target of OSW-1 in cells, a biotinylated OSW-1 (334) (Figure 18) was designed by Jin’s group in 2009.156 The biotinylated OSW-1 offered the feature of a reversible solid support reagent when combined with monomeric avidin−Sepharose or an avidin-coated solid surface.157 To test whether biotinylated OSW-1 334 had any biological activity, they used the MTT assay to evaluate its antiproliferative effect on several human cancer cell lines (Table 14). They found that the biotinylated OSW-1 exhibited potent

Table 11. Cytotoxic Activities of OSW-1 Analogues against Tumor Cells IC50 (μM) cell line

254

319

320

321

322−326

RKOa Jurkatc HeLad

0.0007 0.015 0.071

NDb ND ND

ND 0.078 1.2

1.7 ND 1.1

ND ND ND

a RKO = colon carcinoma cell line. bND = not determined. cJurkat = human T cell leukemia cell line. dHeLa = human cervical cancer cell line.

analogues was first evaluated using non-small-cell lung cancer cells (NCI-H460) and breast cancer cells (MDAMB-231) (Table 12). The OSW-1 estrane analogue 251 and cisplatin Table 12. Cytotoxic Activities of OSW-1 Analogues against Tumor Cells IC50 (μM)

Table 14. Cytotoxic Activities of 334 against Tumor Cells

cell line

251

327

328

329

330

NCI-H460a MDA-MB-231b

0.26 0.68

9.98 6.94

13.7 10.9

2.59 1.71

5.75 18.2

a

cell line a

HL-60 Panc1b AsPC1b

b

NCI-H460 = human lung cancer. MDA-MB-231 = human breast cancer.

332

333

252

8.9 14.9

14.5 2.6

21.1 0.8

0.0013 0.24

A375 WM35c

IC50 (10−3 μM) 0.2 0.2

inhibitory activity against human leukemia cells (HL-60), pancreatic cancer cells (Panc1 and AsPC1), and melanoma cells (A375 and WM35). The IC50 values ranged from 0.0001 to 0.0011 μM, indicating that this compound was highly potent in inhibiting cancer cell growth, like the parent natural product OSW-1. The biological testing suggested that the attachment of biotin to the C-3 position of the aglycon did not significantly affect the interaction of OSW-1 with its putative target in the cells. For further SAR research of OSW saponins and simplifying the synthesis procedure, in 2010, Lei’s group designed and synthesized 3β,16β,26-trihydroxycholest-5-en-22-one (17deoxy-26-hydroxyl-OSW-1 aglycon)158 from diosgenin, which was then attached with OSW-1 disaccharide and its 1→4-linked analogue at 16β-OH, affording three OSW-1 analogues, 335− 337 (Figure 19). The in vitro antitumor activities of the synthetic glycosides against HCT-8, BEL-7402, BGC-823, A2780, and A-549 were evaluated by the standard MTT assay using paclitaxel as a positive control (Table 15). It was shown that compound 335, 17-deoxy-26-hydroxyl-OSW-1, exhibited potent cytotoxic activity on various cancer cell lines, especially BGC-832 (IC50 = 0.0013 μM, which is comparable to that of OSW-1). This result indicated the 17-OH could be truncated without great loss of activity. On the other hand, compound 336, the 1→4-linked disaccharide isomer of 335, was inactive at a concentration of 10−5 μM, as was compound 337. The results implied that the 1→3 glycosidic linkage in disaccharide is critical to the cytotoxicities, and the 17-OH might not be of importance for antitumor activity. To examine the importance of the link position of the sugar chain, fulfill the SAR of OSW-1, and aim at finding the simplest portion that maintains most of the biological activities, in 2011, Lei’s group designed and synthesized two 7-OH steroid aglycons:159 (25R)-3β,7β,26-trihydroxycholest-5-ene and (25R)-3β,7β,16β,26-tetrahydroxycholest-5-ene, plus one 16-

IC50 (μM) 331

c

HL-60 = human leukemia cells. bPanc1 and AsPC1 = pancreatic cancer cells. cA375 and WM35 = melanoma cells.

Table 13. Cytotoxic Activities of OSW-1 Analogues against Tumor Cells

Jurkata HeLab

0.1 1.1 0.7

cell line

a

were used as positive controls. In the primary assay, the analogue 327 lacking the pMBz group showed only weak activity. Surprisingly, an even lower level of activity was observed for 328, despite the presence of the key Ac and pMBz groups. This strongly suggested that the 4-OH group played a crucial role for the biological activity (perhaps as a H-bond donor or acceptor). Introduction of the polar fluorine atom at C-4 partially restored activity. Indeed 329 was the only analogue with activity approaching that of the parent molecule 251 in the MDA-MB-231 assay (see Table 12). Finally, compound 330, which featured the key Ac and pMBz groups as well as the arabinose moiety found in OSW-1, showed only weak activity in the NCIH460 assay and was almost inactive on MDA-MB-231 cells. To study whether removal of the steroidal A/B ring of the 23-oxa-OSW-1 would cause a complete loss of the antitumor activities, in 2007, Yu’s group reported the synthesis of such compounds (e.g., 331−333) (Figure 18)154 and the determination of their inhibitory activities against the growth of HeLa and Jurkat cells. The in vitro activities of the synthetic A/Bring-truncated OSW saponin analogues 331−333 against the growth of two human cancer cell lines, Jurkat (human T cell leukemia) and HeLa (human cervical cancer), with 23-oxaOSW-1 as a positive control, were determined by following the incorporation of [3H]thymidine (Table 13).155 As shown in Table 13, the simplified hydrindane glycosides 331−333

cell line

IC50 (10−3 μM)

a

Jurkat T = human T cell lymphoblast-like cell. bHela = human epithelial cervical cancer. AC


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Figure 19. Chemical structures of 17 OSW-1 saponin analogues synthesized by Lei’s group.


HCT-8a

BEL-7402b

BGC-823c

A2780d

A-549e

paclitaxel 335 336 337 338 339 340 341 342 343

5.1 × 10−5 2.3 × 10−5 >10−2 >10−2 7.0 × 10−5 >10−2 7.5 × 10−5 >10−2 >10−2 >10−2

6.0 × 10−6 3.9 × 10−5 >10−2 >10−5 2.3 × 10−5 >10−2 5.0 × 10−4 >10−2 >10−2 >10−2

10−2 >10−2 2.0 × 10−6 >10−2 7.8 × 10−5 >10−2 >10−2 >10−2

10−2 >10−2 6.0 × 10−6 >10−2 1.9 × 10−3 >10−2 >10−2 >10−2

1.6 × 10−5 4.6 × 10−5 >10−2 >10−2 9.3 × 10−6 >10−2 8.7 × 10−5 >10−2 >10−2 >10−2

a

HCT-8 = colon carcinoma cell line. bBEL-7402 = liver cancer cell line. cBGC-823 = stomach carcinoma cell line. dA2780 = ovarian cancer cell line. A-549 = lung carcinoma cell line.

e


344

345

346

347

348

349

350

351

dioscin

HCT-8a BEL-7402b Ketr3c A2780d MCF-7e A549f BGC-823g

>10 >10 >10 >10 >10 >10 >10

>10 >10 >10 >10 >10 >10 >10

>10 >10 >10 >10 >10 >10 >10

>10 >10 >10 >10 >10 >10 >10

>10 >10 >10 >10 >10 >10 >10

>10 >10 >10 >10 >10 >10 >10

>10 >10 >10 >10 >10 >10 >10

1 > IC50 > 0.1 0.522 1 > IC50 > 0.1 0.545 0.499 0.843 0.283

0.351 0.930 0.447 0.581 0.879 0.454 0.268

a HCT-8 = colon carcinoma cell line. bBEL-7402 = liver cancer cell line. cKetr3 = renal cancer cell line. dA2780 = ovarian cancer cell line. eMCF-7 = breast cancer cell line. fA549 = lung cancer cell line. gBGC-823 = stomach cancer cell line.

AD


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Figure 20. SAR of OSW-1 in antitumor activity.

Analogues with modified sugar residue showed much lower activities. (2) Especially, removal of the acetyl (Ac) and the 4methoxybenzoyl (MBz) groups on the disaccharide moiety diminished the cytotoxicity by 3 orders of magnitude.13,34,35,51 However, synthetic glycosides bearing the acyl disaccharide but disparate steroid aglycons of OSW-1 did not show any cytotoxicity.138,148 (3) The 1→3 glycosidic linkage in the disaccharide is critical to the cytotoxicity.158 A change of the linkage of L-arabinopyranose and D-xylopyranose derivatives (to 1→4) resulted in a dramatic decrease of activity. (4) 4′-OH clearly appears to be crucial, and the integrity of the acylated xylopyranoside moiety is equally important.153 The presence of an additional glucopyranosyl residue on the 4″-OH,34 or truncation on the xylopyranosyl residue, greatly diminishes the activity.152,153 (5) An α-glycoside bond between a 16βhydroxysteroid and acylated sugar moieties is a pharmacophore requirement.145 Inversion of the C-16 configuration, where the disaccharide is attached, was also not allowed to retain the significant cytotoxic activity of OSW-1.151 (6) The steroidal C17 side chain can tolerate certain modifications without significant loss in antitumor potency.124,142,143 These results suggested that the 17-OH might not be of importance for antitumor activity.158 (7) The steroidal A/B ring can also be modified without a significant loss in antitumor potency. An intact steroid ring is not absolutely required for the biological activity of the OSW-1 family of saponin analogues.154 (8) An analogue with an aromatic A ring is as potent as cisplatin, although it is much less potent than the parent OSW-1.141,153 (9) 5,6-Dihydro-OSW-1139 derivatives are as active as the parent compound. (10) Substitution with a glucose on the 3OH, a site remote to the 16-O-disaccharide, apparently did not affect the cytotoxic activity.13,34,35,51 Although a number of synthetic and pharmacologic studies of OSW-1 and its analogues have been reported, the SAR still needs further investigation. For example, the necessity of a C22 carbonyl group is still controversial (Figure 20). Wojtkielewicz and Morzycki’s group146 commented that the presence of a carbonyl group at C-22 was a pharmacophore requirement, but Yu’s group143 found that the 22-deoxy-OSW1 was slightly more potent than OSW-1.124,142 In addition, candicanoside A (126) was the only congener to show remarkable cytostatic activity (IC50 = 0.032 μM) against the HL-60 cells without benzoate substitution on the saccharide moiety.51,52 More interestingly, candicanoside A displays differential cytotoxicities against various tumor cell lines, and its antitumor profile does not correlate with that of OSW-1 and its analogues.52

OH aglycon, (25R)-3β,16β,26-trihydroxycholest-5-ene. By attaching these three aglycons to OSW-1 disaccharide (2-O(4-methoxybenzoyl)-β-D-xylopyranosyl-(1→3)-2-O-acetyl-α-Larabinopyranosyl) and its 1→4-linked analogue, six cholestane saponins, 338−343 (Figure 19), as mimics of OSW-1 were afforded. All the compounds were assayed for in vitro antitumor activities against a panel of five human cancer cell lines, including HCT-8, BEL-7402, BGC-823, A2780, and A-549 using paclitaxel as a positive control (Table 15). From the results, it was clear that compound 338, with OSW-1 disaccharide linked at the 16-OH of sapogenin, showed potent cytotoxicity with IC50 from 0.002 to 0.07 μM. For the compounds with OSW-1 disaccharide linked at 7-OH, compound 339, a 16-deoxy analogue, was inactive (IC50 >10 μM), but its 16-OH derivative 340 exhibited significant activities with a minimum IC50 of 75 nM. The results implied that the 22-one function was not necessary for cytotoxicity. The disaccharide moiety could be linked to a position other than 16OH, but the oxygen-bearing substituent at C-16 might be required for the antitumor activity. To study the SAR of 5(6)-dihydro-OSW-1, eight 15(α)β-Oglycosyl analogues (344−351) (Figure 19) carrying three kinds of disaccharides, including β-D-Xylp-(1−3)-α-L-Arap, β-D-Xylp(1−4)-α-L-Arap, and α-L-Rhap-(1−2)-(α)β-D-Glcp were designed and synthesized by Lei’s group in 2011.160 The in vitro antitumor activities of compounds 344−351 against HCT-8, BEL-7402, Ketr3, A2780, MCF-7, A549, and BGC-823 were evaluated by the standard MTT assay using dioscin as a positive control (Table 16). Unexpectedly, compounds 344−347 bearing the disaccharide α-L-Rhap-(1−2)-(α)β-D-Glcp linked at 15-OH showed no inhibition against the seven tumor cells. The consequences proved that the introduction of α-L-Rhap(1−2)-(α)β-D-Glcp did not improve the cytotoxicities of the whole molecule. The IC50 values of compounds 348 and 350 carrying the disaccharide β-D-Xylp-(1−4)-α-L-Arap were beyond 10 μM, which indicated the alteration of glycosylation in arabinose can diminish the activities, whereas compound 351 (IC50 = 0.28−0.52 μM) bearing the disaccharide β-D-Xylp-(1− 3)-α-L-Arap displayed potential activities against the seven cancer lines. Nevertheless, its 15-epimer 349 was not biologically active. This result revealed that the β configuration of 15-OH is essential to maintain the antitumor activities of these kinds of saponins.

7. SAR OF OSW-1 Comparing the SAR of OSW-1 and its natural congeners showed (Figure 20) the following: (1) The 16-O-disaccharide part is crucial to the antitumor activities of the molecule. AE


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8. CONCLUSIONS AND OUTLOOK This first review concerning the steroidal glycosides in Ornithogalum plants covering the literature from 1959 to November 2011 has summarized the results on their structure, bioactivity, and chemical synthesis and will hopefully attract more attention of the biological and chemical communities to these interesting compounds. Steroidal saponins, widespread secondary metabolites with a variety of bioactivities, are believed to be principal constituents in many medicinal plants and are described as being responsible for most of the observed biological effects. Several cardenolide glycosides have been found in some species of Ornithogalum. However, no biological activity investigation on them has been reported. Some cardenolide glycosides from other plants showed potent cytotoxic activity,161−167 which suggested that cardenolide glycosides from Ornithogalum plants might possess similar bioactivity. The discovery of OSW-1, a valuable cholestane glycoside from the bulbs of O. saundersiae, attracted great attention to the cytotoxicity of this kind of compound. Pharmacological studies showed that OSW-1 exhibited extremely potent cytotoxicity against various malignant tumor cells in vitro that was superior to that of the prominent cancer therapeutic agents presently in use, such as paclitaxel. Apart from excellent activity, OSW-1 also has high selectivity for tumor cells. Although a number of synthetic and pharmacologic studies in vitro of OSW-1 and its analogues have been reported, its pharmacology in vivo and pharmacokinetics have rarely been studied, the precise and unambiguous mechanism needs further exploration, and the SAR needs more investigation. In addition, an industrialized sample preparation method also should be considered to form an applicable medicine for treating tumor patients.

engineer, and mainly conducted the research and development of new drug from TCM. Two years later, he came back to China Pharmaceutical University and obtained his Ph.D. (major medicinal chemistry, supervisor Professor Fengchang Lou) in 2000, and his doctoral dissertation ‘Isolation and structure identification of the chemical constituents from Ginkgo biloba leaves and Sophora japonica pericarps’ was awarded the excellent doctoral dissertation by College of Traditional Chinese Medicine, China Pharmaceutical University. As a postdoctoral researcher in Professor Biao Yu’s group, he worked for two years on the study of bioactive constituents from Ornithogalum caudatum and Valerinana jatamansi at the State Key Laboratory of Bioorganic and Natural Products Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences. He visited the Cancer Research Institute, Arizona State University, as a postdoctoral and faculty research fellow in Professor George R. Pettit’s group for four years, devoting his time to studying the isolation and structure of the antitumor constituents from some marine organisms and microorganisms there. In 2006 he came back to China, joined the Nanjing University of Chinese Medicine, and was given an accelerated promotion to a full professor one year later. His research interests mainly focus on the isolation, structure, analysis, bioactivity evaluation, and structure−activity relationship of the chemical constituents in medicinal plants, with over 200 publications including review articles. He obtained the Program for New Century Excellent Talents by the Ministry of Education of China in 2009, and in the same year his group was awarded the Excellent Scientific and Technological Innovation Team of Jiangsu Higher Education of China.

AUTHOR INFORMATION Corresponding Author

*Phone/fax: 86-25-8581-1916. E-mail: yupingtang@njutcm. edu.cn (Y.T.); [email protected] (J.-a.D.). Notes

The authors declare no competing financial interest. Biographies

Nianguang Li (born 1977 in Laiwu, Shandong Province), Associate Professor of the Department of Medicinal Chemistry, Nanjing University of Chinese Medicine, received his B.S. degree (major chemical engineering) from Shandong Polytechnic University in 2002 and obtained his Ph.D. degree (major medicinal chemistry, supervisor Professor Qidong You) from China Pharmaceutical University in 2007. He became a senior lecturer at the Nanjing University of Chinese Medicine in 2007 and was promoted to an associate professor three years later. His research interests include the total synthesis and structure−activity relationship of complex natural products with interesting biological activities and development of new synthetic methodologies. He has published over 20 academic papers and is the holder of one national patent of invention.

Yuping Tang (born 1970 in Bazhong, Sichuan Province), Professor and Executive Director of the Jiangsu Key Laboratory for High Technology of TCM Formulae Research, Nanjing University of Chinese Medicine, obtained his bachelor’s degree (major medicobotany) from China Pharmaceutical University in 1993. Afterward, he joined Jiangsu Tianqing Pharmaceutical Co. Ltd. as an assistant AF


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bioactive constituents in blood activation and stasis elimination. Now he is studying for his doctorate (supervisor Professor Jin-ao Duan) with combined training at China Pharmaceutical University and the Nanjing University of Chinese Medicine.

ACKNOWLEDGMENTS This research work was financially supported by the National Key Technology R&D Program (Grant 2008BAI51B01), National Natural Science Foundation of China (Grants 81001382 and 81274058), and 2009’ Program for New Century Excellent Talents by the Ministry of Education (Grant NCET-09-0163). This research was also financially supported by a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (ysxk-2010). We also thank Professor Yoshihiro Mimaki (Tokyo University of Pharmacy and Life Sciences, Japan) for providing the photographs of Ornithogalum saundersiae flowers and bulbs and helpful discussions on OSW-1 and other steroidal glycosides in Ornithogalum plants. We also thank Professor Biao Yu (Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, China) for help and support in our preparation of the paper. We thank Dr. Li Zhang (Department of Analysis of Chinese Medicine, Nanjing University of Chinese Medicine, China) for helping with material collection.

Jin-ao Duan (born 1956 in Zhongwei, Ningxia Province), Professor and Vice-president of the Nanjing University of Chinese Medicine, obtained his bachelor’s degree (major pharmacy) from Shenyang Pharmaceutical University in 1980. He worked for 10 years at the Ningxia Institute for Drug Control. He received his master’s degree (1993, pharmacognosy, Professor Ronghan Zhou) and Ph.D. (1996, phytochemistry, Professor Shouxun Zhao and Professor Ronghan Zhou) from China Pharmaceutical University. Afterward, he assumed the position of Deputy Division Chief of Scientific and Technical Department, China Pharmaceutical University. He visited the Department of Chemistry, Hong Kong University of Science & Technology, as a visiting researcher in Professor Chun-Tao Che’s group in 1997−1998. He became Research Professor in Natural Medicine in 1999. He became Director for the Jiangsu Province Academy of Traditional Chinese Medicine in 1999. Nearly six years later, he moved to the Nanjing University of Chinese Medicine as the Vice-president and the Academy Leader of the Science of Chinese Material Medica. His research interests mainly focus on natural resources and their chemistry in Chinese medicinal materials and their bioactive constituents and the compatibility mechanism of traditional Chinese medicine (TCM) formulas under the direction of TCM theory. He has published over 200 academic papers and 4 academic books. He is also the holder of eight national patents of invention and five clinic approval documents of new drugs of TCM. He was the Academic Specialist of Jiangsu Province with State Department special allowance, and as the chief scientist he obtained the National Basic Research Program of China (973 Program) in 2010. He also won the National Scientific and Technological Progress Award at the end of 2011.

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Weiwei Tao (born 1984 in Lianyungang, Jiangsu Province) received his bachelor’s and master’s degrees from the College of Pharmacy, Nanjing University of Chinese Medicine, in 2007 and 2010. His master’s thesis was on the resource chemistry of Pollen Typhae and its AG


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