The Apoptolidin Project
The ability to achieve selectivity in therapeutic function is one of the major and largely unmet challenges of medicine and is a motivation for our interest in the apoptolidin project. In this effort, advances in synthesis are being used to elucidate the molecular basis for the activity of this fascinating new therapeutic lead. In 1997, Seto and coworkers reported the structure of apoptolidin (1, Figure 1), which was isolated from Nocardiopsis sp. in a screening program designed to identify new compounds that selectively induce apoptosis in E1A transformed cells.1 Apoptolidin was subsequently shown to be among the top 0.1% most selective agents tested in the NCI's 60 human cancer cell line panel out of the more than 37,000 compounds analyzed to date.2 The ability of apoptolidin to induce apoptosis potently and with remarkable selectively make it an exciting lead compound for the treatment of cancer. The complexity of the molecule furthermore makes manipulation of its structure a formidable synthetic challenge! Our goals include use of synthetic chemistry to learn more about the chemistry of 1 and its derivatives and to use that synthetic expertise to study the mode of action of these molecules. Furthermore, this knowledge can be applied to the design of superior agents for treatment of human disease.
Figure 1. Structure of apoptolidin (1).
Because of apoptolidin's promising biological activity and structural complexity, it has attracted considerable attention from the synthetic community.3. An alternative approach to establishing the structural basis for apoptolidin's activity and for identifying related clinical leads is modification of apoptolidin itself, available in substantial quantity (109 mg/L) through fermentation. By evaluation of derivatives of 1, a structure-activity relationship (SAR) for apoptolidin can be formulated that will facilitate the design of apoptolidin analogues that share apoptolidin's unique biological activity, but are more amenable to therapeutic use. Analogues of 1 also serve as useful scientific tools to establish the mechanism of action of apoptolidin.
Isoapoptolidin
Our initial attempts to isolate apoptolidin from crude cell extract were complicated by the presence of an isomer, isoapoptolidin (2), which had chromatographic properties very similar to 1.4 The structure of 2 was determined by extensive 1D and 2D NMR experiments to contain a ring-expanded macrolactone, formed via a transesterification at C19/C20 (Figure 2).
Figure 2. Structure of isoapoptolidin (2).
The identification of isoapoptolidin raises the possibility that an interconversion between 1 and 2 could take place under the conditions of biological assay or chemical isolation. Although both 1 and 2 were found to be stable at -20oC in organic solvents for several months, a dilute aqueous solution of apoptolidin at ambient temperature can be observed to convert to isoapoptolidin. Similarly, an aqueous solution of 2 can be observed to convert to 1. Starting from pure 1 or 2 in aqueous solution, the kinetics of this interconversion were measured. Significantly, an equilibrium mixture of 1 and 2 in roughly equal amounts is attained in less than 24 hours, a result which must be taken seriously when interpreting data from cell-based assays.
Apoptolidin was shown by Khosla and coworkers to be an inhibitor of mitochondrial F0F1-ATPase.5 While this did not entirely explain the observed biological activity of 1, F0F1-ATPase inhibition was used as a preliminary assay for testing derivatives of apoptolidin. This assay can be performed in less than 10 minutes, during which time little isomerization of 1 and 2 can be expected. Apoptolidin inhibits ATPase in intact mitochondria isolated from yeast with an IC50 value of 0.7 microM. In contrast, 2 inhibits ATPase with an IC50 of 17 microM. Although this difference is significant, it does not directly address the issue of which compound, 1 or 2, is responsible for the induction of apoptosis observed in sensitive cells.
Apoptolidin Derivatives
To address potential differences in activity between 1 and 2 in cells, as well as to collect data that will allow the construction of a structure-activity relationship for 1, selective modification of functionality in 1 was next pursued.6 Because of their participation as both hydrogen-bond donors and acceptors, as well as their ease of functionalization, selective modification of the hydroxyl group array in 1 was performed. Five of the hydroxyl groups could be selectively modified by sequential silylation, acetylation, and deprotection to produce compounds 13-17 (Scheme 1A). The C-21 and C-20 alcohols could also be selectively methylated to yield products 18 and 22 respectively (Scheme 1B). Compound 22 was of particular interest due to the participation of the C-20 alcohol in the isomerization of apoptolidin to isoapoptolidin.
Scheme 1. Selective Functionalization of the hydroxyl group array.
Compounds 13 - 20 and 22 have been tested for their ability to inhibit F0F1-ATPase and were found to have activities roughly similar to 1. The only exceptions to this were compounds functionalized at C-20 (17 and 22) or functionalized at C-21 (compound 18).
As an alternative means of determining the structural basis for the activity of 1, a divide and diversify strategy, facilitated by oxidative cleavage of the C-20/C-21 diol functionality, was employed.7 While this cleavage can be performed by treating 1 with NaIO4, significantly higher yields can be obtained when the penta-silylated compound 7 is oxidized with Pb(OAc)4 (Scheme 3). This also avoids the concomitant cleavage of the C-2'/C-3' diol. In this manner, compounds 25 and 29 - 31 can be obtained after further functionalization and deprotection. Interestingly, excision of the C-21 through C-28 portion of 1, along with the attached pyranose residues, does not eliminate its ability to inhibit F0F1-ATPase. Despite this, compounds 29 - 31 lose all activity in cell-based assay. Delta-lactone 25, also retains some residual activity against this enzyme, although with greatly reduced potency as compared to 1.
Scheme 2. Divide-and-diversify strategy.
Additional Members of the Apoptolidin Family
Recently, we detected the existence of compounds similar in molecular weight to apoptolidin from the crude ethyl acetate extract of fermentation of Nocardiopsis sp. but lacking one or two oxygen mass equivalents.8 The molecular formulas of these agents, named apoptolidin B (32) and apoptolidin C (33) due to their structural relationship to the parent compound, were determined to be C58H96O20 and C58H96O19 respectively. The primary structures of these new apoptolidins (Figure 3) were established by two-dimensional NMR experiments (COSY, TOCSY, HMQC, HMBC). Proton and carbon NMR spectra show extensive similarities to apoptolidin with respect to chemical shifts and coupling constants. While the overall spin systems for the macrolactone ring, the hemi-ketal moiety and the three glycoside linkages remain unchanged, differences were found in additional methylene groups within the macrolactone structure. In particular, unlike apoptolidin, apoptolidin B lacks a hydroxyl group at position C-16 and apoptolidin C lacks hydroxyl groups at positions C-16 and C-20 (Figure 3).
Figure 3. Structures of apoptolidins B (32) and C (33).
To determine the biological activities of apoptolidins A, B and C a cell proliferation assay with H292 cancer cells (lung carcinoma) was used. The cells were treated with different concentrations of the compounds (0.5 nM to 10 microM) for 48 hours. The data from these assays indicate that neither the hydroxyl group at C-16 nor at C-20 is essential for activity in this assay. Furthermore, apoptolidin B, which lacks the C-16 hydroxyl group, is more potent than apoptolidin A in our cell-based assay (7 nm vs. 32 nm). This suggests that the C-16 hydroxyl group in apoptolidin A might interfere with target binding. Apoptolidin C is less active than B but similar in potency to A.
Continuing Work
The chemistry and biology of apoptolidin continues to be explored in the Wender lab. Additional derivatives are being synthesized, further studies into apoptolidin's mode of action are underway, and characterization of additional natural products is ongoing.
1 (a) Kim, J. W.; Adachi, H.; Shin-ya, K.; Hayakawa, Y.; Seto, H. J. Antibiotics 1997, 50, 628.
(b) Hayakawa, Y.; Kim, J. W.; Adachi, H.; Shin-ya, K.; Fujita, K.; Seto, H. J. Am. Chem Soc. 1998, 120, 3524.
2 Developmental Therapeutics Program NCI/NIH. http://dtp.nci.nih.gov (accessed April 2003).
3 Total syntheses: (a) Nicolaou, K. C.; Fylakidou, K. C.; Monenschein, H.; Li, Y.; Weyershausen, B.; Mitchell, H. J.; Wei,
H.-X.; Guntupalli, P.; Hepworth, D.; Sugita, K. J. Am. Chem. Soc. 2003, 125, 15433.
(b) Nicolaou, K. C.; Li, Y.; Sugita, K. Monenschein, H.; Guntupalli, P.; Mitchell, H. J.; Fylakidou, K. C.; Vourloumis, D.; Giannakakou, P.; OÕBrate, A.
J. Am. Chem. Soc. 2003, 125, 15443 and references cited therein.
(c) Wehlan, H.; Dauber, M.; Mujica Fernaud, M. T.; Schuppan, J.; Mahrwald, R.; Ziemer, B.; Juarez, Garciz, M. E.; Koert, U.
Angew. Chem., Int. Ed. 2004, 43, 4597.
Apoptolidinone: (a) Schuppan, J.; Wehlan, H.; Keiper, S.; Koert, U. Angew. Chem. Int. Ed. 2001, 40, 2063.
(b) Nicolaou, K. C.; Li, Y.; Weyershausen, B.; Wei, H. Chem. Commun. 2000, 307.
Synthesis studies: (a) Toshima, K.; Arita, T.; Kato, K.; Tanaka, D.; Matsumura, S. Tetrahedron Lett. 2001, 42, 8873.
(b) Schuppan, J.; Ziemer, B.; Koert, U. Tetrahedron Lett. 2000, 41, 621.
(c) Sulikowski, G. A.; Jin, B. H.; Lee, W. M.; Wu, B. Org. Lett. 2000, 2, 1439.
(d) Chng, S.-S.; Xu, J.; Loh, T. P. Tetrahedron Lett. 2003, 44, 4997.
(e) Chen, Y.; Evarts Jr. J. B.;, Torres, E.; Fuchs, P. L.; Org. Lett. 2002, 4, 3571.
(f) Paquette, W. D; Taylor, R. E. Org. Lett. 2004, 6, 103.
(g) Abe, K.; Kato, K.; Arai, T.; Rahim, M. A.; Sultana, I.; Matsumura, S.; Toshima K. Tetrahedron Lett. 2004, 45, 8849.
(h) Jin, B.; Liu, Q.; Sulikowski, G. A. Tetrahedron 2005, 61, 401.
(i) Bouchez, L. C.; Vogel, P. Chem., Eur. J. 2005, 11, 4609.
4 (a) Wender, P. A.; Gulledge, A. V.; Jankowski; O. D.; Seto, H. Org. Lett. 2002, 4, 3819.
(b) Pennington, J. D.; Williams, H. J.; Salomon, A. R.; Sulikowski, G. A. Org. Lett. 2002, 4, 3823.
5 (a) Salomon, A. R.; Voehringer, D. W.; Herzenberg, L. A.; Khosla, C. Proc. Natl. Acad. Sci. U. S. A. 2000, 97, 14766.
(b) Salomon, A. R.; Voehringer, D. W.; Herzenberg, L. A.; Khosla, C. Chem. Biol. 2001, 8, 71.
(c) Salomon, A. R.; Zhang, Y.; Seto, H.; Khosla, C. Org. Lett. 2001, 3, 57.
6 Wender, P. A.; Jankowski, O. D.; Tabet, E. A.; Seto, H. Org. Lett. 2003, 5, 487.
7 Wender, P. A.; Jankowski, O. D.; Tabet, E. A.; Seto, H. Org. Lett. 2003, 5, 2299.
8 Wender, P. A.; Sukopp, M.; Longcore, K. Org. Lett. 2005, 7, 3025.