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Our previous studies with shishijimicin A resulted in the total synthesis of this scarce marine natural product and a number of its simpler analogues endowed with picomolar potencies against certain cancer cell lines. Herein, we describe the design, synthesis, and biological evaluation of four linker-drugs, anticipating the construction of antibody-drug conjugates (ADCs) as the ultimate goal of this research program. Using a common payload, the assembly of these linker-drugs utilized different linkers and attachment points, providing opportunities to probe the optimal molecular design of the intended ADCs as targeted cancer therapies. In the course of ADC generation and in vitro evaluation, we identified two linker-drugs with a promising in vitro plasma stability profile and excellent targeted cytotoxicity and specificity. Conjugation of shishijimicin A enediyne payloads through their phenolic moiety represents a novel approach to enediyne ADC creation, while the pharmacological profiles of at least two of the generated ADCs compare well with the profiles of the corresponding clinically approved ADC Kadcyla.

Although shishijimicin A and its extreme potencies against an array of cancer cell lines have been known for more than a decade, its assumed DNA-cleaving mechanism has not been substantiated as yet. Herein we report studies that reveal binding and scission of double-stranded DNA by shishijimicin A. The results of these studies support the proposed hypothesis that DNA strand scissions are caused by 1,4-benzenoid diradicals formed by Bergman cycloaromatization of the enediyne core of shishijimicin A upon activation by thiols. In addition, double-stranded supercoiled DNA-cleavage experiments with shishijimicin A in competition with known minor groove binders, UV spectroscopic studies, and electrophoretic analysis were utilized to clarify the binding mode of the molecule to DNA. These investigations indicate that shishijimicin A binds to the minor groove of double-stranded DNA and that its β-carboline moiety plays a role in the binding through intercalation. In addition, due to the fact that naked linker regions of DNA in the interphase and metaphase of eukaryotic cells are unprotected by histone proteins during entire cell cycles and because these unprotected regions of DNA are vulnerable to attack by DNA binders, it was concluded that the observed double-strand DNA cleavage and very low sequence selectivity by shishijimicin A may account for its extraordinary cytotoxicity.

Shishijimicin A is a scarce marine natural product with highly potent cytotoxicities, making it a potential payload or a lead compound for designed antibody-drug conjugates. Herein, we describe an improved total synthesis of shishijimicin A and the design, synthesis, and biological evaluation of a series of analogues. Equipped with appropriate functionalities for linker attachment, a number of these analogues exhibited extremely potent cytotoxicities for the intended purposes. The synthetic strategies and tactics developed and employed in these studies included improved preparation of previously known and new sulfenylating reagents such as PhthNSSMe and related compounds.

The total synthesis of the rare but extremely potent antitumor agent shishijimicin A has been achieved via a convergent strategy involving carboline disaccharide 3 and hydroxy enediyne thioacetate 4.