Development of Chemical Probes for Functional Analysis of Anticancer Saponin OSW-1.

Chemical probe-based approaches have proven powerful in recent years in the target identification studies of natural products. OSW-1 is a saponin class of natural products with highly potent and selective cytotoxicity against various cancer cell lines. Understanding its mechanism of action is important for the development of anticancer drugs with potentially novel target pathways. This account reviews recent progress in the development of OSW-1 derived probes for exploring the mechanism of its action. The key to the probe development is a judicious choice of functionalization sites and a selective functionalization strategy. The types of probes include fluorescent probes for cellular imaging analysis and affinity probes for target identification analysis.

Chemical synthesis of 7α-hydroxycholest-4-en-3-one, a biomarker for irritable bowel syndrome and bile acid malabsorption.

7α-Hydroxy-cholest-4-en-3-one is a biomarker for bile acid loss, irritable bowel syndrome, and other diseases associated with defective bile acid biosynthesis. Furthermore, 7α-hydroxy-cholest-4-en-3-one is the physiological substrate for cytochrome P450 8B1 (P450 8B1 or CYP8B1), the oxysterol 12α-hydroxylase enzyme implicated in obesity and cardiovascular health. We report the chemical synthesis of this physiologically important oxysterol beginning with cholesterol. The key feature of this synthesis involves a regioselective C3-allylic oxidation of a 3-desoxy-Δ4-7α-formate steroid precursor to form 7α-formyloxy-cholest-4-en-3-one, which was saponified to yield 7α-hydroxy-cholest-4-en-3-one.

Chemical synthesis of marine saponins.

Covering: 1989-2017 Saponins are characteristic metabolites of starfish and sea cucumbers, and occasionally are also found in sponges, soft coral, and small fish. These steroid or triterpenoid glycosides often show remarkable biological and pharmacological activities, such as antifungal, antifouling, shark repellent, antitumor and anti-inflammatory activities. Over one thousand marine saponins have been characterized; the majority of them can be categorized into three major structural types, i.e., asterosaponins, polyhydroxysteroid glycosides, and holostane glycosides. Thus far, only 12 marine saponins have been synthesized; those representing the major types were successfully synthesized recently. The syntheses involve preparation of the aglycones from the terrestrial steroid or triterpene materials, installation of the carbohydrate units, and manipulation of the protecting groups. Herein, we provide a comprehensive review on these syntheses.

Synthesis of a fluorescent photoaffinity probe of OSW-1 by site-selective acylation of an inactive congener and biological evaluation.

A novel fluorescent photoaffinity probe of OSW-1 was prepared in two steps from a naturally occurring inactive congener by a sequential site-selective acylation strategy using Me2SnCl2. It displayed highly potent anticancer activity and a similar intracellular localization property to that of a fluorescently-tagged OSW-1, thereby demonstrating its potential utility in live cell studies.

Synthesis of OSW-1 derivatives by site-selective acylation and their biological evaluation.

A strategy to site-selectively monoacylate an antitumor saponin OSW-1 was developed using an organotin reagent to rapidly access its derivatives that are useful as chemical probes. 4″-O-Acylated OSW-1 derivatives bearing a fluorophore, an alkyne tag, or biotin were prepared in good yields and were shown to maintain highly cytotoxic activity.

Synthesis of a smoothened cholesterol: 18,19-di-nor-cholesterol.

Herein, we report the first synthesis of a demethylated form of cholesterol (18,19-di-nor-cholesterol), in which the C18 and C19 methyl groups of the ß-face were eliminated. Recent molecular simulations modeling 18,19-di-nor-cholesterol have suggested that cholesterol's opposing rough ß-face and smooth α-face play necessary roles in cholesterol's membrane condensing abilities and, additionally, that specific facial preferences are displayed as cholesterol interacts with different neighboring lipids and transmembrane proteins. Inspired by these poorly characterized biochemical interactions, an extensive 18-step synthesis was completed as part of a collaborative effort, wherein synthesizing a "smoothened" cholesterol analogue would provide a direct way to experimentally measure the significance of the ß-face methyl groups. Starting from known perhydrochrysenone A, the synthesis of 18,19-di-nor-cholesterol was accomplished with an excellent overall yield of 3.5%. The use of the highly stereoselective Dieckmann condensation and the employment of Evans' chiral auxiliary were both key to ensuring the success of this synthesis.

Synthesis of three OSW-1 analogs with maltose side chains bearing different protection groups.

In order to simplify the synthesis of OSW-1's disaccharide side chain and explore the structure-activity relationship of OSW-1, three 16α-O-maltose OSW-1 analogs carrying three maltose side chains bearing different protections were designed and synthesized.

An efficient synthesis of 7α,12α-dihydroxy-4-cholesten-3-one and its biological precursor 7α-hydroxy-4-cholesten-3-one: Key intermediates in bile acid biosynthesis.

This paper describes a method for the chemical synthesis of 7α,12α-dihydroxy-4-cholesten-3-one (1a) and its biological precursor, 7α-hydroxy-4-cholesten-3-one (1b), both of which are key intermediates in the major pathway of bile acid biosynthesis from cholesterol. The principal reactions involved were (1) building of the cholesterol (iso-octane) side chain by 3-carbon elongation of the cholane (iso-pentane) one, (2) oxidation sequence to transform the 3α-hydroxy group of the steroidal A/B-ring to the desired 4-en-3-one system, and (3) appropriate protection strategy for hydroxy groups in the positions at C-7 and C-12 in the steroid nucleus. The absolute structure of 1a and 1b were confirmed by NMR and X-ray crystallography. The targeted compounds 1a and 1b, prepared in 11 steps from 2a and 2b respectively, should be useful for biochemical studies of bile acid biosynthesis or clinical studies of bile acid metabolism, as plasma levels of 1b (also termed C4) have been shown to correlate highly with the rate of bile acid biosynthesis in man.

Synthesis of 5(6)-dihydro-OSW-1 analogs bearing three kinds of disaccharides linking at 15-hydroxy and their antitumor activities.

In order to study the SAR of 5(6)-dihydro-OSW-1, eight 15(α)ß-O-glycosyl analogs (26-33) carrying three kinds of disaccharides including [ß-d-Xylp-(1-3)-α-l-Arap], [ß-d-Xylp-(1-4)-α-l-Arap] and [α-l-Rhap-(1-2)-(α)ß-d-Glcp] were designed and synthesized. Their in vitro antitumor activities were tested by the standard MTT assay which disclosed that compound 33 (IC(50)=0.28-0.52 µM) showed potential antitumor activities.

Synthesis and biological activity of 22-deoxo-23-oxa analogues of saponin OSW-1.

Analogues of the potent cytotoxic saponin OSW-1 were prepared from the readily available steroidal 16ß,17α,22-triol. The new 22-deoxo-23-oxa analogues of OSW-1 were screened against eight cancer cell lines and normal human fibroblasts. The analogues proved to be slightly less active than OSW-1 but also less toxic to normal cells. They induce concentration- and time-dependent apoptosis of mammalian cancer cells with caspase-3 activation.

Ergosta-4,6,8(14),22-tetraen-3-one induces G2/M cell cycle arrest and apoptosis in human hepatocellular carcinoma HepG2 cells.

BACKGROUND: Mushrooms have been used in Asia as traditional foods and medicines for a long time. Ergosta-4,6,8(14),22-tetraen-3-one (ergone) is one of the well-known bioactive steroids, which exists widely in various medicinal fungi such as Polyporus umbellatus, Russula cyanoxantha, and Cordyceps sinensis. Ergone has been demonstrated to possess cytotoxic activity. However, the molecular mechanisms by which ergone exerts its cytotoxic activity are currently unknown. METHODS: In the present study, ergone possessed a remarkable anti-proliferative activity toward human hepatocellular carcinoma HepG2 cells. We assayed the cell cycle by flow cytometry using PI staining; investigated the exposure of phosphatidylserine at the outer layer of the cytoplasmic membrane by the FITC-annexin V/PI staining; observed the nuclear fragmentation by Hoechst 33258 staining and studied the protein expression of Bax, Bcl-2, p-53, procaspase-3, -8, -9, PARP and cleaved PARP by Western blotting analysis. RESULTS: Cells treated with ergone showed typical markers of apoptosis: G2/M cell cycle arrest, chromatin condensation, nuclear fragmentation, and phosphatidylserine exposure. Furthermore, PARP-cleavage; activation of caspase-3, -8, -9; up-regulation of Bax and down-regulation of Bcl-2 were observed in HepG2 cells treated with ergone, which show that both the intrinsic and extrinsic apoptotic pathways are involved in ergone-induced apoptosis in HepG2 cells. Ergosta-4,6,8(14),22-tetraen-3-one induces G2/M cell cycle arrest and apoptosis in HepG2 cells in a caspase-dependent manner. GENERAL SIGNIFICANCE: In this study, we reported for the first time that ergone-induced apoptosis through activating the caspase. These results would be useful for the further utilization of many medicinal fungi in cancer treatment.

Synthesis of cholestane glycosides bearing OSW-1 disaccharide or its 1-->4-linked analogue and their antitumor activities.

For further structure-activity relationship (SAR) research of OSW saponins, a cholestane glycoside, namely 3beta, 16beta, 26-trihydroxycholest-5-en-22-one 16-O-(2-O-4-methoxybenzoyl-beta-D-xylopyranosyl)-(1-->3)-2-O-acetyl-alpha-L-arabinopyranoside (1) together with two 1-->4-linked disaccharide analogues (2 and 3) were synthesized. Their cytotoxic activities were evaluated by the standard MTT assay. Compound 1 showed potent cytotoxicity against five types of human tumor cells, with IC50 ranging between 1.3 and 73 nM.

Unusual oxidative transformations of a steroidal 16alpha,17alpha,22-triol.

A number of unexpected reactions were observed during attempts to invert configuration at C16 in 16alpha,17alpha,22-triol 3a. The PDC oxidation of 3a produced the D-seco-aldehyde 4a. Analogous compound 4b was obtained by Swern oxidation of the 16alpha,17alpha-dihydroxy-22-O-TES-ether 3b in addition to the desired 16-ketone 7. The unprotected triol 3a yielded pentacyclic products 5 and 6 under similar conditions. The Mitsunobu reaction of the triol 3a afforded 16-ketone 8 with inverted configuration of the side chain. During heating of a solution of 3a in THF with NaH at reflux autoxidation to the 16-ketone cyclic hemiketal 5, identical to one of the Swern oxidation products, took place.

Anti-HBV agents. Part 3: preliminary structure-activity relationships of tetra-acylalisol A derivatives as potent hepatitis B virus inhibitors.

Thirty-two tetra-acylated derivatives of alisol A were synthesized and evaluated for their anti-hepatitis B virus (HBV) activities and cytotoxicities in vitro. Among the series of alisol A derivatives examined, five analogues were active against HBV surface antigen (HBsAg) and HBV e antigen (HBeAg) secretion in HepG 2.2.15 cells. These results also provide interesting structure-activity relationships of tetra-acylalisol A derivatives. Compounds tetra-acetyl alisol A (A1), tetra-methoxyacetyl alisol A (A23), and tetra-ethoxyacetyl alisol A (A24) exhibited high activities against secretion of HBsAg with IC(50) values of 0.0048, 0.0044, and 0.014 mM, respectively, HBeAg with IC(50) values of 0.011, 0.012, and 0.018 mM, respectively, and remarkable selective index values SI(HBsAg)>333, SI(HBeAg)>145; SI(HBsAg)=209, SI(HBeAg)=77; and SI(HBsAg)>200, SI(HBeAg)>156, respectively. Additional studies in rats showed that compound A1 has favorable pharmacokinetic prosperities for further development purpose, with elimination half-time (t(1/2)) of 1.63 h and oral bioavailability (F) of 40.9%.

Synthesis of biotinylated OSW-1.

OSW-1 is a highly potent anticancer natural saponin with an unknown mode of action. To determine its cellular target(s) biotinylated OSW-1 was successfully synthesized in nine steps.

Anti-HBV agents. Part 2: synthesis and in vitro anti-hepatitis B virus activities of alisol A derivatives.

Chemical modifications were performed on hydroxyl groups at C-11,23,24,25 positions and C-13(17) double bond of alisol A for structure-activity relationship study. Forty-one derivatives of alisol A were synthesized and assayed for their in vitro anti-hepatitis B virus (HBV) activities and cytotoxicities. Of them, 14 compounds were active against HBV surface antigen (HBsAg) and HBV e antigen (HBeAg) secretion in HepG 2.2.15 cells, and the most promising compound 25 exhibited high activities against secretion of HBsAg (IC(50)=0.028 mM), HBeAg (IC(50)=0.027 mM) and remarkable selective indices (SI(HBsAg) >90, SI(HBeAg) >93).

Anti-HBV agents. Part 1: Synthesis of alisol A derivatives: a new class of hepatitis B virus inhibitors.

A series of alisol A derivatives were synthesized and evaluated for their anti-hepatitis B virus (HBV) activities and cytotoxicities in vitro. The preliminary investigation demonstrates that simple modifications of the parent structure of alisol A can produce a number of potentially important derivatives against HBV. The most active anti-HBV compound 6a showed high activities against the secretion of HBV surface antigen (IC(50)=0.024 mM), HBV e antigen (IC(50)=0.028 mM) and remarkable selective indices (SI(HBsAg)>108, SI(HBeAg)>93), which was selected for further evaluation as a novel HBV inhibitor.

Synthesis and cytotoxicity evaluation of 22,23-oxygenated stigmastane derivatives.

Starting from (22E)-3alpha,5alpha-cyclo-6beta-methoxystigmast-22-ene eighteen derivatives of (22S,23S)-22,23-oxidostigmastane, (22R,23R)-22,23-oxidostigmastane, and (22R,23R)-22,23-dihydroxystigmastane were synthesized and screened for cytotoxicity in human hepatoma Hep G2 cells and human breast carcinoma MCF-7 cells using MTT assay. Four compounds of this series exhibited high cytotoxicity in both cells; three compounds were selectively toxic in MCF-7 cells, one compound was toxic in Hep G2 cells, rather than in MCF-7 cells; four compounds at low concentrations increased MTT test values over the control.

A facile and efficient synthesis of some (6E)-hydroximino-4-en-3-one steroids, steroidal oximes from Cinachyrella spp. sponges.

Using beta-sitosterol as a starting material, (6E)-hydroximino-24-ethylcholest-4-en-3-one (1), a natural steroidal oxime from Cinachyrella alloclada and C. apion, was synthesized in four steps with a high overall yield. First, beta-sitosterol (5a) is transformed into the corresponding 24-ethylcholest-4-en-3,6-dione (6a) via oxidation with pyridinium chlorochromate (PCC). Selective reduction of 6a by NaBH(4) in the presence of CoCl(2) gives 24-ethylcholest- 4-en-3beta-ol-6-one (7a). The reaction of 7a with hydroxylamine hydrochloride offers the oxime 8a and the oxidation of 8a by Jones reagent gives the target steroid 1. (6E)-Hydroximinocholest-4-en-3-one (2) and (6E)-hydroximino-24-ethylcholest-4,22-dien-3-one (4) were synthesized by a similar method. The cytotoxicity of the synthesized compounds against sk-Hep-1 (human liver carcinoma cell line), H-292 (human lung carcinoma cell line), PC-3 (human prostate carcinoma cell line) and Hey-1B (human ovarian carcinoma cell line) cells were investigated. The presence of a cholesterol-type side chain appears to be necessary for the biological activity.