Thomas G. LaCour

Chemistry of Trisdecacyclic Pyrazine Antineoplastics: The Cephalostatins and Ritterazines

The search for natural products of medicinal significance led the Pettit group to isolate the cephalostatins1 (from the hemichordate worm Cephalodiscus gilchristi,2 e.g. cephalostatin 1 (1), and the Fusetani team to the ritterazines3 (from the tunicate Ritterella tokioka, e.g. ritterazine B (2)), respectively. The cephalostatins and ritterazines, are a family of 45 trisdecacyclic bissteroidal pyrazines that display striking cytotoxicity against human tumors (~1 nM in the 2-day NCI 60 cell panel;4 and in some cases ~10 fM 6-day in the Purdue mini panel5), thereby ranking them among the most potent anticancer agents tested by the NCI. Computer matching at the NCI using the COMPARE program have revealed several additional compounds exhibiting similar profiles to the Cephalostatin/Ritterazine family. These compounds include OSW-16 (3) a monosteroidal saponin glycoside from the garden perennial Ornithogalum saundersiae (GI50 of 0.8 nM in the NCI 60 cancer cell line), and solamargine7 (4) (from Solanum species) as additional possible candidates for cancer therapy. OSW-1 (3) shows low toxicity to normal human pulmonary cells but encouraging activity against malignant solid tumor cells. Solamargine (4), is an active ingredient of creme Curaderm®, claimed to be 100% effective against melanomas in preliminary clinical trials without significant side effects or recurrence of cancer 10 years after treatment (Figure 1).8 Figure 1 Steroidal anticancer agents. Following Pettits seminal report on cephalostatin 1 (1) in 1988,1 several articles9 have reviewed the structure elucidation, biological activities, and syntheses of cephalostatins. This account will focus on the advances in the syntheses of cephalostatins and ritterazines over the past 15 years (up to ~July 2008) emphasizing the different strategies adopted, key transformations, and methods for achieving the late construction of the dissymmetric bissteroidal pyrazine framework. Classical steroid numbering (carbons 1-27) and ring designations (A-F) are used throughout the text, supplemented by a “prime” designator for the second steroidal hemisphere (e.g. C21′ = 21′Me of the South hemisphere of cephalostatin 1 (1). Steroidal subunit nomenclature follows published practice, e.g. “North 1” indicates the North10 unit of cephalostatin 1 (1), abbreviated to “1N” especially in analog names or tables (Figure 2). Known stereochemistry is always shown. The somewhat controversial use of solid circles and short dashes to indicate β (up, as drawn) and α (down) hydrogens, respectively, will be retained in the absence of a superior alternative. Figure 2 Steroid and bissteroid nomenclature and numbering. 2. Isolation and Biological Activity 2.1. Cephalostatin Family In 1972, Pettit and coworkers first collected a sample of the marine tubeworm Cephalodiscus gilchristi. Two years later, methanol and water extracts proved active invivo in the National Cancer Institutes PS system (murine lymphocytic leukemia) with a significant lifespan increase in mice.1a In 1988, they were “pleased to report that 15 years of relentless research” had culminated in the structure elucidation of the cephalostatins. Currently, 19 cephalostatins have been reported (Figure 3). All cephalostatins possess two highly oxygenated steroidal spiroketal units linked by a central pyrazine ring. Cephalostatin 1 (1) is among the most powerful anticancer agents ever tested, displaying subnanomolar to picomolar cytotoxicity against much of the National Cancer Institutes (NCI) 60-cell line panel,3 with femtomolar activity against the P388 cell line and in the Purdue Cell Culture Laboratory (PCCL) human tumor panel.4 Four cephalostatins 3, 4, 8, and 9 were as potent vs. P388 (10-4-10-6 nM) but 4-30 fold weaker in the NCI human tumor panel, while three more cephalostatins 10, 11, and 17 displayed 3-10 nM GI50s in both tests. Cephalostatin 16 displayed a mean GI50 (1 nM, NCI) similar to cephalostatin 1 but a 104-106 weaker ED50 (P388). Cephalostatin 7 was assumed to have activity comparable to cephalostatin 1 based on the fact that it was championed along with cephalostatin 1 for clinical trials and was reported to display a comparable ~femtomolar ED50 (P388) as well as “remarkable potency…against a number of cell lines; the mean graphs of cephalostatin 1 (1) and cephalostatin 7 (5) were remarkably similar, if not indistinguishable” in the NCI panel. The cephalostatins complex, unprecedented structure and promise as an anticancer lead compound inspired attention by several groups. Figure 3 Cephalostatin family. Clinical trials of a cephalostatin (or analog) will require several grams of material. Pettits fourth and most prodigious collection afforded only ~0.1 g of cephalostatin 1 (1) from half a ton (450 kg) of this tiny (<5 mm) worm, which hides as colonies in small calcium carbonate sheaths. The harvest involved repeated SCUBA operations at ~25 m depth in waters off East Africa patrolled by the great white shark. The bioassay-guided isolation followed a complex, evolving protocol of extraction (whole worm, several months with aq. MeOH), multiple large scale solvent partitionings, and protracted chromatographic separations. Clearly, chemical synthesis is the only solution to the availability problem. Early speculation on the mode of action of the cephalostatins centered around; i) the likelihood of cell membrane penetration due to the steroidal nature and dimensions (~30 A x 9 A x 5 A) of cephalostatin 1 (1);11 ii) the possibility that the compounds serve as a spatially-defined set of hydrogen-bond donors/acceptors for enzyme binding,12 and iii) the importance of the Δ14 moiety,13 perhaps due to a chemical role of a derived β-epoxide and the C-ring ketone in the South half of cephalostatin 1 or 7 (Scheme 1).14 Scheme 1 Possible C/D ring alkylating sites generated from a cephalostatin. The Purdue group initially speculated that reaction of the C/D homoallylic alcohol array of South 7 generated similar potential alkylating centers. However, the 1997 revelation15 that OSW-1 (3), a monosteroidal glycoside lacking a South unit, displayed a profile and potency similar to cephalostatin 1 against human tumor lines, prompting consideration of an equilibrium between the North spiroketal and its E-ring oxacarbenium ion as a potential alkylating agent (Scheme 2). Scheme 2 The E-ring oxacarbenium ion. The antineoplastic mechanism of the cephalostatins is presently largely unknown. The fingerprint of cephalostatin activity in the NCI 60-tumor panel is quite different from known anticancer agents, likely indicating a new mechanism of action. The cephalostatin pattern was most similar to the topoisomerase II inhibitors, but Pettit relates that cephalostatins 1 (1) and 7 (5) are neither topoisomerase inhibitors nor serve as antimicrotuble agents like taxol.16 Studies using synthetic cephalostatin 7 (5) indicate that this compound is not an inhibitor of protein Kinase C nor does it inhibit the tyrosine phosphatase cdc25. A recent biological study17 revealed that cephalostatin 1 affects cells by disrupting the mitochondrial transmembrane potential. Dirsch et al. in collaboration with Pettit documented18 that cephalostatin 1 triggers the release of Smac/DIABLO, a pro-apoptotic mitochondrial signaling factor which induces receptor-independent apoptosis. Muller and coworkers demonstrated16 that cephalostatin 1 inactivate Bcl-2, an anti-apoptotic protein, by activating JNK (c-Jun N-terminal Kinase). In 2006, Vollmar et al. reported19a that cephalostatin 1 utilizes the endoplasmic reticulum stress pathway rather than the intrinsic mitochondrial pathway. Cephalostatin 1 (1) not only induces classical apoptosis parameters (e.g. cell shrinkage, increased cellular granularity, DNA fragmentation, caspase activation) but also shows very unusual apoptosis signaling events (e. g. selective Smac/DIABLO release, no cytochrome c release from mitochondria, and apoptosome-independent activation of caspase-9).18b This unique apoptotic pathway triggered by cephalostatins implies that they could be used to treat drug-resistant cancers.

On the relationship of OSW-1 to the cephalostatins.

Antineoplastic bis-steroidal (cephalostatin-type) analogues of the saponin OSW-1 were produced from a dihydroaglycone of OSW-1. The key aglycone 6H was obtained from 5alpha-androstan-3beta-ol-17-one in 8 steps (38% yield). The SAR of the aglycones, intermediates, and hybrid analogues provide insights regarding the proposed common role of C22-oxocarbenium ions in the bioactivity of both OSW-1 and cephalostatins.

Concurrent ring opening and halogenation of spiroketals

Abstract Ring opening of various spiroketals with triphenylphosphine dihalides under neutral conditions produced ω-halo-enolethers in good to excellent yield. The method transformed even the very stable spiroketal of hecogenin acetate at temperatures below any previously reported for such isomerative opening.