Synthesis, cytotoxicity, and haemolytic activity of chacotrioside lupane-type neosaponins and their germanicane-type rearrangement products Charles Gauthier, Jean Legault, Marianne Piochon, Serge Lavoie, Samuel Tremblay, André Pichette * Laboratoire LASEVE, Chaire de Recherche sur les Agents Anticancéreux d’Origine Naturelle, Université du Québec à Chicoutimi, 555 boul. de l’Université, Chicoutimi (Québec), Canada G7H 2B1 article info Article history: Received 15 December 2008 Revised 15 February 2009 Accepted 19 February 2009 Available online 23 February 2009 Keywords: Saponin Chacotrioside Lupane Germanicane Cytotoxicity Haemolytic activity abstract The concise synthesis, via a stepwise glycosylation approach, of lupeol, betulin and betulinic acid O-gly- cosides bearing a chacotriosyl moiety at the C-3 position is described. All neosaponins as well as their rearrangement products of the germanicane-type were evaluated in vitro for their anticancer and haemo- lytic activities. Although betulinic acid and betulin 3b-O-chacotriosides were neither cytotoxic nor hae- molytic, their rearrangement products allobetulin and 28-oxoallobetulin 3b-O-chacotriosides (9 and 10) exhibited a cytotoxicity profile up to fourfold superior to betulinic acid against human breast (MCF7) and prostate (PC-3) adenocarcinomas cell lines (IC 50 = 10–18 lM). One important result was that only chac- otriosides featuring non-polar functions at the C-28 position (6, 9 and 10) exerted a haemolytic activity against red blood cells. Ó 2009 Elsevier Ltd. All rights reserved. The naturally occurring lupane-type triterpenoids lupeol (1), betulin (2) and betulinic acid (3) have been thoroughly investi- gated during the past years for their promising medicinal proper- ties, 1–5 and particularly their chemopreventive 6 and antitumor 7,8 activities. Consequently, several studies were mainly focused on the preparation of anticancer derivatives of triterpenoids 2 and 3 modified at C-3 and/or C-28 positions. 1,8–11 To increase water sol- ubility of these non-polar cholesterol-like triterpenoids as well as to study the structure–activity relationships, a broad library of mono- and bidesmosidic lupane-type saponins were recently syn- thesized in our laboratory. 12–15 These molecules have been evalu- ated for their cytotoxicity against human cancer cells growth and the main observation was that the addition of rhamnose moieties at both C-3 and C-28 positions of triterpenoids 2 and 3 gives anti- cancer agents many fold stronger than betulinic acid (3). 15 Further- more, it was demonstrated that contrary to the majority of naturally occurring saponins, 16,17 most of the lupane-type glyco- sides do not exhibit any haemolytic activity (HD 50 > 100 lM) against red blood cells, 18 which is of a great interest regarding their clinical utilisation as intravenously delivered anticancer agents. Saponins bearing a 2,4-branched trisaccharide containing rhamnose moieties such as chacotriosides are very attractive for their anticancer activity. Indeed, dioscin, namely diosgenyl a-L-rhamnopyranosyl-(1?2)-[a-L-rhamnopyranosyl-(1?4)]-b- D-glucopyranoside, a well-known saponin isolated from several plant species used in traditional oriental medicine, induces apopto- sis within cancerous cells 19,20 and exhibits promising antitumor activities. 21–23 Moreover, the apoptosis-inducing activity of sola- margine, a solasodine steroidal alkaloid bearing the same chacotri- osyl moiety, is correlated with the presence of rhamnose moieties. 24,25 Therefore, we thought that it would be of interest to prepare lupane-type saponins incorporating this particular 2,4-branched trisaccharide. Hence, as shown in Figure 1, we report here the synthesis of lupeol, betulin and betulinic acid monodes- mosidic saponins (6–8) bearing a chacotriosyl moiety at the C-3 position as well as their unexpected germanicane-type rearrange- ment products (9 and 10). The in vitro cytotoxic and haemolytic activities of all synthesized neosaponins are also reported. In this work, a stepwise glycosylation strategy was chosen rather than a convergent one, in order to obtain exclusively a 1,2-trans-glycosidic linkage. 26 Thus, as depicted in Scheme 1, the synthesis began by coupling the lupane-type acceptors lupeol (1), 12 28-tert-butyldiphenylsilyl betulin (11) 14 or allyl betulinate (12) 13 with the donor 2,3,4,6-tetra-O-benzoyl-a-D-glucopyranosyl trichloroacetimidate (13, 1.5 equiv) 12 under the promotion of the Lewis acid trimethylsilyl trifluoromethanesulfonate (TMSOTf, 0.1 equiv). After the glycosylation, removal of the benzoyl groups under standard deprotection conditions (0.5 N NaOH, MeOH/THF/ H 2 O 1:2:1, room temperature) afforded target b-D-glucosides 14 12 (90%), 15 (72%) and 16 (80%) in good to excellent yields after two steps. Then, the regioselective pivaloylation 27 at both C-6 0 and 0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.bmcl.2009.02.076 * Corresponding author. Tel.: +1 418 545 5011; fax: +1 418 545 5012. E-mail address: andre_pichette@uqac.ca (A. Pichette). Bioorganic & Medicinal Chemistry Letters 19 (2009) 2310–2314 Contents lists available at ScienceDirect Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl