DOI: 10.1002/cmdc.201200465 C3’/C4’-Stereochemical Effects of Digitoxigenin a-l-/a-d-Glycoside in Cancer Cytotoxicity John W. Hinds, [a] Sean B. McKenna, [a] Ehesan U. Sharif, [a] Hua-Yu L. Wang, [a] Novruz G. Akhmedov,* [b] and George A. O’Doherty* [a] Digitoxin, a potent cardiotonic agent that has been used con- tinuously over centuries for the treatment of congestive heart failure, has more recently been recognized for its potential ap- plication in oncology. [1–3] The mechanism of action for digitoxin cardiotonic effect occurs via an increase of Ca 2 + influx as a result of inhibition of the extracellular Na + /K + -ATPase pump. [4] In contrast to the cardiotonic effects, less detail is known for the mechanism of antitumor activity. [5] It has been shown that cell death occurs via apoptosis. Several studies led by Xie [6–8] have suggested that, in cancer cells, a cardiac glyco- side-bound Na + /K + -ATPase complex signals via Src-tyrosine kinase and 1,4,5-triphosphoinositol (IP3), several downstream events that ultimately lead to apoptosis. This signaling path- way is believed to occur through specific isoforms of the Na + /K + -ATPase pump, which are associated with cancer and not heart tissue. These promising findings of potent cytotoxicity against cancer cells inspired many structural–activity relationship (SAR) studies, primarily aimed at understanding the role that the car- bohydrate plays in the cytotoxicity of the cardiac glycosides. These studies include direct comparison of glycosidic link- age, [9, 10] the study of sugar-chain length, [10, 11] the stereochemi- cal survey of digitoxin monosaccharides, [12] and the study of C5’-alkyl steric effect. [13] These carbohydrate-based SAR studies have led to the discovery of new sugar motifs that significantly improved the cytotoxicity across a range of cancer cell lines. [12] To date, we have found that digitoxin a-l-rhamno-monosac- charide 1 to be the most active analogue across the widest range of cell lines, with a-l-amiceto 4 having the next best re- sults. This surprising similarity in anticancer activity of the C2’/ C3’-dideoxy-rhamno-analogue (a-l-amiceto 4) motivated us to explore other deoxygenation patterns that could further en- hance the cytotoxicity. Using our lead digitoxin analogue (a-l-rhamno 1) as a start- ing point, we conducted a systematic SAR study, focusing on the stereochemistry and functionality of the C3’- and C4’-ste- reocenters. Using de novo carbohydrate synthetic methodolo- gy, [14, 15] the C3’- and C4’- hydroxy groups can be inverted or re- moved to study the importance of the stereochemistry and pattern of oxygenation (Figure 1). For example, inversion of the C3’-hydroxy group of rhamno 1 gives altro 2, and removal of the C3’-hydroxy group of altro 2 gives ascarylo 3. Similarly, inversion followed by a removal of the C4’-hydroxy group of ascarylo 3 gives C2’-epi-colito 5 and C3’-C4’-dideoxy-rhamno 6. As a control, we also planned to prepare and test several d- sugar diastereomers, which should have very similar pharma- cokinetic properties but occupy drastically different three-di- mensional space. Herein, we report the successful syntheses of new digitoxin C3’/C4’-sugar analogues and the evaluation of their cytotoxicity against four different cancer cell lines, includ- ing lung cancer (NCI-H460 and A549) and breast cancer (MCF- 7 and MDA-MB-231). Retrosynthetically, synthesis of the desired C3’/C4’-sugar congeners of digitoxin a-l-rhamno 1 can be prepared from a common allylic alcohol intermediate (7) through a series of oxidation and/or reduction sequences to install the required hydroxy or hydride functional group (X = OH or H) (Scheme 1). [16, 17] The stereochemically pure C2’-allylic alcohol could be delivered via a 1,3-allylic transposition of the epoxide ketone (8) using a Wharton rearrangement. [15, 18] Epoxide ketone intermediate 8, in turn, could be derived from a diaste- reoselective epoxidation of the pyran enone, which could be easily furnished by using a palladium-catalyzed glycosylation to couple tert-butyloxycarbonyl (Boc)-protected pyranone 9 to digitoxigenin (DigOH). The pyran sugar building blocks with Figure 1. C3’/C4’-stereochemical study of digitoxin rhamno 1. [a] J. W. Hinds, S. B. McKenna, E. U. Sharif, Dr. H.-Y. L. Wang, Prof. G. A. O’Doherty Department of Chemistry & Chemical Biology, Northeastern University 117 Hurtig Hall, 360 Huntington Ave., Boston, MA 02115 (USA) E-mail : g.odoherty@neu.edu [b] Dr. N. G. Akhmedov Department of Chemistry, West Virginia University 217 Clark Hall, Prospect Street, Morgantown, WV 26506 (USA) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cmdc.201200465. ChemMedChem 2012, 7, 1 – 7  2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 1 These are not the final page numbers! ÞÞ MED