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Discovery and structure elucidation

Evolution of the proposed structure of kedarcidin chromophore

Kedarcidin was first discovered in 1992, when bioassays conducted at Bristol-Myers Squibb indicated the presence of a DNA-damaging chromoprotein in the fermentation broth of a novel actinomycete strain. The involvement of a non-peptidic chromophore was deduced by UV spectroscopy, and reverse-phase chromatogrpahy effected the dissociation of this noncovalently bound chromophore from its apoprotein host. This isolate decomposed readily in ambient conditions, and was shown to posess exquisite cytotoxicity (IC₅₀ 0.4 ng/mL, HCT-116 human colorectal carcinoma cell line).

Subsequent NMR, mass spectrometry, chemical degradation, and derivatization experiments enabled the isolation team to correctly identify all of the key structural features of kedarcidin chromophore, including the enediyne bicyclic core, the ansa-bridging chloropyridyl ring, mycarose and kedarosamine sugars, and naphthoamide appendage. Unfortunately, owing to the unique challenges posed by the unprecedented structure, the initial report had several errors. The bicyclic core proved particularly difficult to deconvolute, as interpretation NOE correlations led the researchers to misassign the relative stereochemistry of the core stereotetrad. Moreover, as global absolute chemistry was assigned on the basis of NOE correlations between the L-mycarose sugar and the aglycone, the errors of the stereotetrad propagated to the other two stereocenters of the aglycone. Connectivity of the naphthoamide group to the ansa bridge was also misjudged in the initial report.

These errors were later corrected by the independent synthetic efforts of researchers at Tohoku University and Harvard University. In 1997, en route to the originally reported structure, researchers under the direction of Prof. Hirama discovered that the spectroscopic data of the proposed chloroazatyrosyl (S)-α-amino acid derivative were not consistent with those of the degradation product characterized by Leet et al. Instead, an (R)-β-amino acid derivative was proposed and validated by the Hirama group. This revision led Hirama et al. to invert the other aglycone stereocenters as well, affording a revised structure of kedarcidin chromophore that differed only in the relative stereochemistry of the mycarose-bearing carbon, C10. Finally, in 2007, Prof. Myers and co-workers synthesized the structure proposed by Hirama et al.; the corresponding NMR spectroscopic data were distinct from that of the natural product, leading the Myers group to revise the stereochemistry of the mycarose-bearing carbon to 10-(S).

Mechanism of Action

The general mechanism by which kedarcidin chromophore damages DNA is known; however, the details of this process—particularly the necessity of nucleophilic activiation—have been disputed.

Equilibrium of kedarcidin chromophore core and Bergman-cycloaromatized biradical.

As with other enediyne secondary metabolites, kedarcidin chromophore exhibits biological effects through radical-induced cleavage of duplex DNA. With considerable sequence selectivity, kedarcidin chromophore binds and cleaves DNA preferentially at TCCTn-mer sites, producing single-strand breaks. Puzzlingly, while the structure of kedarcidin chromophore is most closely related to that of neocarzinostatin chromophore, the former shares sequence-specificity with the structurally distinct calicheamicin enediyne antitumor antibiotic. The naphthoic acid substructure has been implicated in DNA binding, as divalent cations such as Ca²⁺ and Mg²⁺ chelatively bind this group and in so doing, decrease kedarcidin chromophore–induced DNA cleavage. Moreover, competition experiments with netropsin, a known binder of the DNA minor groove, indicate that kedarcidin likely binds the minor groove as well.

Borohydride-induced tandem epoxide opening–Bergman cycloaromatization of kedarcidin chromophore. Analogous nucleophilic "bioactiviation" was initially implicated in the mechanism of action as well.

The unifying mechanism of bioactivity in all enediyne antibiotics is the Bergman cyclization, wherein the enediyne portion undergoes spontaneous cycloaromatization to generate a para-benzyne biradical activated toward homolytic abstraction of hydrogen from suitable donors, including the deoxyribose sugars of DNA. In vivo nucelophilic addition of thiolates to C12 and consequent opening of the core epoxide has been hypothesized to trigger Bergman cyclization in kedarcidin chromophore—a process that is thought to diminish the ring strain incurred by formation of the cycloaromatized product, and thus activiate kedarcidin chromophore toward DNA scission. This proposal is encountered extensively in review literature, in no small part due to the finding that C12-sodium borohydride reduction of kedarcidin chromophore induces rapid cycloaromatization; this product is vastly more stable under ambient conditions and thus facilitated the structural characterization carried out by Leet et al.

Ring strain associated with the C1-C12 double bond in kedarcidin chromophore core.

It has since been suggested that spontaneous cycloaromatization of kedarcidin chromophore is competitive with nucleophilic bioactivation, if not the predominant mechanism in vivo. While MM2 calculations show that the C1-C12 double bond in the bicyclic core imparts a considerable amount of ring strain (ca. 14 kcal·mol^–1) to the [6,5,5] tricycle formed upon Bergman cyclization–reduction, Hirama et al. note that the [9,5] enediyne core is susceptible to cycloaromatization–redution in the absence of both thiol "activating agents" and (non-solvent) hydrogen donors. The kedarcidin chromophore aglycone similarly undergoes reductive cycloaromatization at comparable rates, irrespective of the presence of β-mercaptoethanol, a common thiol reductant. In a model system, it was found that the [9,5] core of kedarcidin chromophore exists in equilibrium with the corresponding [6,5,5] cycloaromatized biradical. The rate of pseudo-first-order decay of this model enediyne is highly dependent on the solvent hydrogen-donor ability, indicating that the hydrogen abstraction step following biradical formation is kinetically significant in the cycloaromatization of the enediyne, as opposed to acyclic systems, where formation of the biradical itself is known to be the rate-limiting step. It is noteworthy that of the solvents examined, tetrahydrofuran—structurally homologous with deoxyribose—led to comparatively fast decomposition of the [9,5] enediyne scaffold (t_½ = 68 min); Zein et al. independently remark that deoxyribose 4'-hydrogen abstraction is most likely operative in kedarcidin chromophore bioactivity.

Synthesis of the core

Transannular cyclization in the synthesis of the bicyclic core of kedarcidin chromophore.

Myers and co-workers have pioneered the application of transannular anionic cyclization reactions in the synthesis of the fused [9,5] bicyclic core of kedarcidin chromophore. In the first incarnation, hydride delivery to a cyclic tetrayne was guided by aluminum coordination to a proximal alkoxide, thus generating the desired enediyne core in one step via two successive 5-exo-dig–type cyclizations. Later-generation syntheses of the core "intercept" this cascade cyclization, relying on lithium-halogen exchange on a cyclic vinyl bromide to generate the vinyl anion precursor to the bicyclic product.

Synthesis of epi-kedarcidin chromophore

In 2007, Myers and co-workers at Harvard University reported the synthesis of C10-epi-kedarcidin chromophore, corresponding to the 1997 revised structure advanced by Hirama et al. Critical to the success of this endeavor was systematic application of retrosynthetic analysis, which served to iteratively diminish the complexity of the final target and thus pave a clear path forward. Several of the major challenges of C10-epi-kedarcidin chromophore, as well as the strategies used in addressing these difficulties are discussed below.

Retrosynthetic approach to 10-epi-kedarcidin chromophore employed by Ren, Hogan, Anderson, and Myers (2007).

Inherent instability of the enediyne core

The instability toward Bergman cyclization–reduction decomposition pathways poses a major threat to any proposed synthesis of enediynes. Myers and co-works addressed this liability by late-stage dehydrative installation of the olefin. Without this unsaturation linking the two alkynyl bridges, synthetic intermediates are not disposed toward Bergman-type decomposition, and risk of decomposition is mitigated. In this case, dehydration of a propargylic alcohol was induced by treatment with Martin sulfurane.

Epoxide stereochemistry

In targeting 10-epi-kedarcidin chromophore, Myers et al. sought to install the epoxide functionality syn to the adjacent C10 hydroxyl group. This was accomplished by vanadium-catalyzed epoxidation directed by the C10 hydroxyl group; had the natural C10-(S)-epimer been desired, it is conceivable that protection of the C10 hydroxyl would lead to the desired α-face epoxidation product by steric occlusion of the β face of the olefin.

Construction of the bicyclic core

The bicyclic core of C10-epi-kedarcidin chromophore was prepared by the sequential application of three carbon-carbon bond forming reactions. First, a Sonogashira coupling was carried out between a bromovinyl electrophile and alkynyl nucleophile; ring closure to give a cyclic triyne was then accomplished by Glaser coupling of two terminal alkynes. Lastly, as discussed above, the [5,9] bicyclic core was established by in situ generation of a vinyllithium species that underwent transannular 5-exo-dig cyclization.

Ansa-bridging macrolactone

The ansa-bridgging macrolactone was constructed following the first Sonogashira coupling, using Shiina's lactonization conditions. This protocol, similar to Yamaguchi esterification, employs 2-methyl-6-nitrobenzoic anhydride, 4-dimethylaminopyridine, and triethylamine as a base to promote intramolecular esterification.