Martin J. Schnermann

A Concise Synthesis of (−)-Aplyviolene Facilitated by a Strategic Tertiary Radical Conjugate Addition

Aplyviolene (1) and macfarlandin E (2, Figure 1A) are representative of the more complex members of the rearranged spongian diterpene class of natural products.[1,2] These diterpenes are structurally defined by attached cis-perhydroazulene and 6-acetoxy-2,7-dioxabicyclo[3.2.1]octan-3-one fragments. The substantial challenge in assembling these structures centers on the construction of the sensitive bicyclic lactone subunit and the formation of the C8–C14 σ-bond joining the two ring systems, a challenge augmented by the quaternary nature of C8.[3] We previously reported preparation of the lactone subunits of 1 and 2 by the synthesis of truncated congeners 3 and 4 (Figure 1A),[4] as well as the first total synthesis of aplyviolene (outlined in Figure 1B).[5] In this latter effort, the key C8–C14 σ-bond was formed by Michael addition of tertiary enolate 5 to enone 6.[6] Subsequent elaboration of product 7 provided intermediate 8, which was converted to (−)-aplyviolene along the lines of our earlier synthesis of 3. Undesirable aspects of this first-generation synthesis are the lengthy preparation of the cis-perhydroazulene unit and the need to remove the extraneous ketone carbonyl group from the product of the fragment-coupling step.

A Near-IR Uncaging Strategy Based on Cyanine Photochemistry

The development of photocaging groups activated by near-IR light would enable new approaches for basic research and allow for spatial and temporal control of drug delivery. Here we report a near-IR light-initiated uncaging reaction sequence based on readily synthesized C4′-dialkylamine-substituted heptamethine cyanines. Phenol-containing small molecules are uncaged through sequential release of the C4′-amine and intramolecular cyclization. The release sequence is initiated by a previously unexploited photochemical reaction of the cyanine fluorophore scaffold. The uncaging process is compatible with biological milieu and is initiated with low intensity 690 nm light. We show that cell viability can be inhibited through light-dependent release of the estrogen receptor antagonist, 4-hydroxycyclofen. In addition, through uncaging of the same compound, gene expression is controlled with near-IR light in a ligand-dependent CreERT/LoxP-reporter cell line derived from transgenic mice. These studies provide a chemical foundation that we expect will enable specific delivery of small molecules using cytocompatible, tissue penetrant near-IR light.

Near-IR Light-Mediated Cleavage of Antibody–Drug Conjugates Using Cyanine Photocages

Despite significant progress in the clinical application of antibody drug conjugates (ADCs), novel cleavage strategies that provide improved selectivity are still needed. Herein is reported the first approach that uses near-IR light to cleave a small molecule from a biomacromolecule, and its application to the problem of ADC linkage. The preparation of cyanine antibody conjugates, drug cleavage mediated by 690 nm light, and initial in vitro and in vivo evaluation is described. These studies provide the critical chemical underpinning from which to develop this near-IR light cleavable linker strategy.

Golgi-modifying properties of macfarlandin E and the synthesis and evaluation of its 2,7-dioxabicyclo(3.2.1)octan-3-one core

Golgi-modifying properties of the spongian diterpene macfarlandin E (MacE) and a synthetic analog, t-Bu-MacE, containing its 2,7-dioxabicyclo[3.2.1]octan-3-one moiety are reported. Natural product screening efforts identified MacE as inducing a novel morphological change in Golgi structure defined by ribbon fragmentation with maintenance of the resulting Golgi fragments in the pericentriolar region. t-Bu-MacE, which possesses the substituted 2,7-dioxabicyclo[3.2.1]octan-3-one but contains a tert-butyl group in place of the hydroazulene subunit of MacE, was prepared by chemical synthesis. Examination of the Golgi-modifying properties of MacE, t-Bu-MacE, and several related structures revealed that the entire oxygen-rich bridged-bicyclic fragment is required for induction of this unique Golgi organization phenotype. Further characterization of MacE-induced Golgi modification showed that protein secretion is inhibited, with no effect on the actin or microtubule cytoskeleton being observed. The conversion of t-Bu-MacE and a structurally related des-acetoxy congener to substituted pyrroles in the presence of primary amines in protic solvent at ambient temperatures suggests that covalent modification might be involved in the Golgi-altering activity of MacE.

Enantioselective Total Synthesis of Aplyviolene

The enantioselective total synthesis of the rearranged spongian diterpene aplyviolene has been completed in 14 steps from the known hydroazulenone 8. The key junction of the hydrocarbon and oxygenated fragments to form the critical C8 quaternary carbon stereocenter and set the stage for elaborating the delicate bicyclic lactone functionality was accomplished in high yield and exquisite stereoselectivity by Michael addition of an enantioenriched hydroazulenone enolate to an enantiopure α-bromocyclopentenone.

Electrophile-integrating Smiles rearrangement provides previously inaccessible C4'-O-alkyl heptamethine cyanine fluorophores.

New synthetic methods to rapidly access useful fluorophores are needed to advance modern molecular imaging techniques. A new variant of the classical Smiles rearrangement is reported that enables the efficient synthesis of previously inaccessible C4′-O-alkyl heptamethine cyanines. The key reaction involves N- to O- transposition with selective electrophile incorporation on nitrogen. A representative fluorophore exhibits excellent resistance to thiol nucleophiles, undergoes productive bioconjugation, and can be used in near-IR fluorescence imaging applications.

Structural Elucidation and Synthesis of Eudistidine A: An Unusual Polycyclic Marine Alkaloid that Blocks Interaction of the Protein Binding Domains of p300 and HIF-1α

Low oxygen environments are a hallmark of solid tumors, and transcription of many hypoxia-responsive genes needed for survival under these conditions is regulated by the transcription factor HIF-1 (hypoxia-inducible factor 1). Activation of HIF-1 requires binding of its α-subunit (HIF-1α) to the transcriptional coactivator protein p300. Inhibition of the p300/HIF-1α interaction can suppress HIF-1 activity. A screen for inhibitors of the protein binding domains of p300 (CH1) and HIF-1α (C-TAD) identified an extract of the marine ascidian Eudistoma sp. as active. Novel heterocyclic alkaloids eudistidines A (1) and B (2) were isolated from the extract, and their structures assigned by spectroscopic analyses. They contain an unprecedented tetracyclic core composed of two pyrimidine rings fused with an imidazole ring. Eudistidine A (1) was synthesized in a concise four-step sequence featuring a condensation/cyclization reaction cascade between 4-(2-aminophenyl)pyrimidin-2-amine (3) and 4-methoxy-phenylglyoxal (4), while eudistidine B (2) was synthesized in a similar fashion with glyoxylic acid (5) in place of 4. Naturally occurring eudistidine A (1) effectively inhibited CH1/C-TAD binding with an IC50 of 75 μM, and synthetic 1 had similar activity. The eudistidine A (1) scaffold, which can be synthesized in a concise, scalable manner, may provide potential therapeutic lead compounds or molecular probes to study p300/HIF-1α interactions and the role these proteins play in tumor response to low oxygen conditions. The unique structural scaffolds and functional group arrays often found in natural products make these secondary metabolites a rich source of new compounds that can disrupt critical protein-protein binding events.

An Intrinsically Disordered Peptide Facilitates Non-Endosomal Cell Entry.

Many cell-penetrating peptides (CPPs) fold at cell surfaces, adopting α- or β-structure that enable their intracellular transport. However, the same structural folds that facilitate cellular entry can also elicit potent membrane-lytic activity, limiting their use in delivery applications. Further, a distinct CPP can enter cells through many mechanisms, often leading to endosomal entrapment. Herein, we describe an intrinsically disordered peptide (CLIP6) that exclusively employs non-endosomal mechanisms to cross cellular membranes, while being remarkably biocompatible and serum-stable. We show that a single anionic glutamate residue is responsible for maintaining the disordered bioactive state of the peptide, defines its mechanism of cellular entry, and is central to its biocompatibility. CLIP6 can deliver membrane-impermeable cargo directly to the cytoplasm of cells, suggesting its broad utility for delivery of drug candidates limited by poor cell permeability and endosomal degradation.

Divergent Synthesis and Chemical Reactivity of Bicyclic Lactone Fragments of Complex Rearranged Spongian Diterpenes

The synthesis and direct comparison of the chemical reactivity of the two highly oxidized bicyclic lactone fragments found in rearranged spongian diterpenes (8-substituted 6-acetoxy-2,7-dioxabicyclo[3.2.1]octan-3-one and 6-substituted 7-acetoxy-2,8-dioxabicyclo[3.3.0]octan-3-one) are reported. Details of the first synthesis of the 6-acetoxy-2,7-dioxabicyclo[3.2.1]octan-3-one ring system, including an examination of several possibilities for the key bridging cyclization reaction, are described. In addition, the first synthesis of 7-acetoxy-2,8-dioxabicyclo[3.3.0]octanones containing quaternary carbon substituents at C6 is disclosed. Aspects of the chemical reactivity and Golgi-modifying properties of these bicyclic lactone analogs of rearranged spongian diterpenes are also reported. Under both acidic and basic conditions, 8-substituted 2,7-dioxabicyclo[3.2.1]octanones are converted to 6-substituted-2,8-dioxabicyclo[3.3.0]octanones. Moreover, these dioxabicyclic lactones react with primary amines and lysine side chains of lysozyme to form substituted pyrroles, a conjugation that could be responsible for the unique biological properties of these compounds. These studies demonstrate that acetoxylation adjacent to the lactone carbonyl group, in either the bridged or fused series, is required to produce fragmented Golgi membranes in the pericentriolar region that is characteristic of macfarlandin E.

Forming Tertiary Organolithiums and Organocuprates from Nitrile Precursors and their Bimolecular Reactions with Carbon Electrophiles to Form Quaternary Carbon Stereocenters

The stereoselective formation of quaternary carbons is one of the most demanding challenges in organic synthesis.1 An especially direct way to construct such stereocenters would be to combine a prochiral tertiary organometallic and a carbon-centered electrophile (Figure 1A). However, this strategy is not mentioned in the numerous reviews of stereoselective synthesis of chiral quaternary carbons,1 and to our knowledge has never been employed in target-directed organic synthesis. This omission undoubtedly derives from the challenge in generating tertiary organometallic intermediates, particularly those containing three alkyl substituents.[2–4] We were recently drawn to explore this undeveloped approach for forming quaternary carbon stereocenters in the context of fashioning the demanding C8–C14 bond and the C8 quaternary stereocenter of rearranged spongian diterpenes such as aplyviolene (4) and dendrillolide A (5) by the reaction of tertiary organocuprate 1 and cyclopentenone 2. This coupling was anticipated to take place from the convex face of nucleophile 1 and from the face of 2 opposite the branched side chain (Figure 1B).[5]