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Stephacidin B and avrainvillamide are complex indole alkaloids that have been separately identified in culture media from various strains of Aspergillus. Both compounds have been shown to inhibit the growth of cultured human cancer cells, with IC50 values in the ~50-100 nM range. Avrainvillamide is apparently the first natural product known to contain the 3-alkylidene-3H-indole 1-oxide function (shown in red below). In early studies we prepared this function in a model system and showed that it is capable of reversible, covalent bond formation with oxygen- and sulfur-based nucleophiles. In addition, we have recently completed an efficient and convergent synthesis of the reported structure of avrainvillamide that proceeds in 17 steps and 4.2% overall yield (scheme below). We have observed that synthetic "avrainvillamide" readily dimerizes to form stephacidin B and have clear evidence that the reverse transformation occurs as well. It is therefore possible that the observed biological activity of stephacidin B may be attributable to the formation of avrainvillamde in vivo. This in turn may relate to the ability of the latter substance to react with hydroxyl and thiol nucleophiles in vivo.
The tetracyclines are broad-spectrum antimicrobial agents that are are composed of four linearly-fused six membered rings with a high density of polar functionality. Extensive use of the tetracyclines in human and veterinary medicine over the past 50 years has led to significant bacterial resistance. Prior research has shown great opportunity for antibiotic development by modification of the C and D rings of the tetracycline structure (1). Most tetracyclines of clinical importance are characterized as "6-deoxy" tetracyclines, the C6 hydroxy group of tetracycline being a site of chemical lability.
A convergent and enantioselective total synthesis of the tetracyclines has been achieved by late-stage assembly of the C-Ring. Application of the synthetic route to (–)-doxycycline (2), one of the most clinically significant tetracyclines, and (–)-6-deoxytetracycline (3), provided the antibiotics in 18 steps (8.3%) and 14 steps (7.0%), respectively, from benzoic acid.
By choice of its late-stage bond constructions, as well as by its brevity, the route is amenable to the rapid preparation of new tetracycline derivatives that are inaccessible by semi-synthetic methods, such as the pyridine derivative 4 and the pentacyclic derivative 5. The latter compound (5) is active against Gram-positive bacterial strains that are resistant to tetracycline, methicillin, and vancomycin.
Kedarcidin is one of the most complex and reactive of the natural enediyne antitumor agents isolated to date. These natural products are among the most potent antitumor agents known. A chromoprotein antibiotic, kedarcidin is manufactured by the producing organism as a 1:1 complex of the biologically active chromophore component (kedarcidin chromophore) and a binding protein, believed to stabilize the chromophore in vivo. Kedarcidin chromophore is structurally related to another enediyne antitumor agent that we have studied, neocarzinostatin chromophore (see below), but differs importantly in the site of epoxidation within the highly unsaturated bicyclic core. As a consequence, kedarcidin contains a conjugated (Z)-enediyne group within a nine-membered ring - a group that undergoes spontaneous biradical-forming cycloaromatization at 37 °C. This extraordinary reactivity profile, coupled with the inherent structural complexity of the unsaturated core, the ansa bridge, the macrolactone, and carbohydrate residues, define one of the most challenging synthetic targets undertaken in our laboratory to date. Our goal is to develop an efficient, enantioselective laboratory route to this highly reactive target. We plan to implement a new strategy for the synthesis of the reactive core by a one-step transannular cyclization of a 12-membered ring precursor. The strategy has been shown to work in an advanced model study, leading to a fully functional core structure. Our efforts now are focused on defining the optimal sequence for assembling the 12-membered ring precursor and macrolactone components, as well as the carbohydrate and naphthoic acid ester appendages.
Neocarzinostatin is the prototypical chromoprotein antitumor antibiotic, and the only member of the series whose structure has been solved (x-ray crystal structure with Doug Rees' laboratory, Caltech). Our group has for some time been involved in mechanism of action studies of the antitumor agent neocarzinostatin and recently completed a synthesis of the highly unstable chromophore component. We are now involved in efforts to adapt our synthesis for the incorporation of a radiolabel into the synthetic material to conduct mechanism of action studies in vivo. Thus far, we have been successful in preparing fully synthetic neocarzinostatin chromophore with a tritium label on the N-methylfucosamine residue and have reconstituted the chromophore with apoprotein. Incubations with various cancer and bacterial cell lines have been conducted and products isolated. To provide a complete picture of the course of reaction of this agent in vivo it is necessary to conduct a parallel series of experiments in which a radiolabel is incorporated within the carbocyclic core of the chromophore. This presents an enormous synthetic challenge, but one which is attainable on the basis of current experiments. With this material we hope to be able to elucidate the detailed course of reaction of neocarzinostatin in various cell lines and also to determine how the producing organism protects itself from self poisoning. These are questions uniquely addressable by efforts in complex synthesis.
The saframycins are a series of natural bisquinone antibiotics with potent antitumor activity. They are believed to undergo bioreductive activation leading to the covalent modification of DNA in vivo. We recently completed an exceedingly concise, enantioselective synthesis of saframycin A using a new synthetic strategy involving the directed coupling of C- and N-protected a-amino aldehyde components. The eight-step synthetic route that we developed is highly efficient (~15% overall yield, >200 mg enantiomerically pure saframycin prepared to date). In addition, all steps in the sequence proceed readily at or below 35 °C. These features make the route ideally suited for adaptation to the solid phase. Efforts to accomplish this are underway. One goal of this project is to prepare large numbers structural analogs by solid-phase synthesis, an exciting prospect given the potency of known members of the saframycin family in anticancer screens.
(a) Na2SO4, CH2Cl2, 23 °C, >90%; LiBr, DME, 35 °C, 65-72%. (b) CH2O-H2O, NaBH(OAc)3, CH3CN, 23 °C, 94%. (c) HOAc, TBAF, THF, 23 °C; DBU, CH2Cl2, 23 °C, 92%. (d) Na2SO4, CH2Cl2, 23 °C, 66%. (e) ZnCl2, TMSCN, CF3CH2OH-THF, 23 °C, 86%. (f) DBU, CH2Cl2, 23 °C, 88%. (g) ClCOCOCH3, PhNEt2, CH2Cl2, 0 °C, 89%. (h) PhIO, CH3CN-H2O, 0 °C, 66%.
Cyanocycline A, Quinocarcin, and Related Antitumor Agents
The anticancer activity of this class of natural products is believed to derive from the ability of these agents to alkylate double-stranded DNA after reductive activation in vivo, similar to the proposed mechanism of action of saframycin A. We believe that each member of the series is potentially synthesized in the laboratory by the stepwise condensation of optically active a-amino aldehyde precursors. There are many potential routes to each agent using such a strategy; the optimal sequences will likely vary for each target. The development of enantioselective routes to both cyanocycline A and quinocarcin are currently in progress. The goal is to develop exceedingly short routes that are modifiable so as to provide a large number of biologically active analogs, as well as any particular agent in quantity.
Terpestacin, L-696,474, and Fluorinated Inhibitors of HIV Protease
Several ongoing projects target complex molecules active against the human immunodeficiency virus (HIV). Among these targets are the syncytium-forming inhibitor terpestacin, the HIV-protease inhibitor L-696,474, and a series of complex fluorinated inhibitors of HIV-protease. Challenging features of these problems include, in the former series, the development of efficient pathways for macro(carbo)cycle synthesis and, in the latter case, the asymmetric construction of fluorinated carbon centers, a largely unsolved problem in synthesis. We have made great progress in both areas with the construction of macrocyclic precursors to both terpestacin and L-696,474 and with the development of highly practical methods for the asymmetric synthesis of monoorganofluorine compounds. Highly complex, nanomolar inhibitors of HIV protease have been synthesized in gram quantities using this methodology. Efforts to obtain structural information for bound inhibitors are also under way.
Gene Transcription Analysis of S. cerevisiae Exposed to Neocarzinostatin Protein-Chromophore Complex Reveals Evidence of DNA Damage, a Potential Mechanism of Resistance, and Consequences of Prolonged Exposure. Scott E. Schaus, Duccio Cavalieri and Andrew G. Myers, PNAS 2001, 20, 11075 download data
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