Total synthesis of complex 2,5-diketopiperazine alkaloids.

The 2,5-diketopiperazine (DKP) motif is present in many biologically relevant, complex natural products. The cyclodipeptide substructure offers structural rigidity and stability to proteolysis that makes these compounds promising candidates for medical applications. Due to their fascinating molecular architecture, synthetic organic chemists have focused significant effort on the total synthesis of these compounds. This review covers many such efforts on the total synthesis of DKP containing complex alkaloid natural products.

Molecular Dynamics Simulations Guide Chimeragenesis and Engineered Control of Chemoselectivity in Diketopiperazine Dimerases.

In the biosynthesis of the tryptophan-linked dimeric diketopiperazines (DKPs), cytochromes P450 selectively couple DKP monomers to generate a variety of intricate and isomeric frameworks. To determine the molecular basis for selectivity of these biocatalysts we obtained a high-resolution crystal structure of selective Csp2 -N bond forming dimerase, AspB. Overlay of the AspB structure onto C-C and C-N bond forming homolog NzeB revealed no significant structural variance to explain their divergent chemoselectivities. Molecular dynamics (MD) simulations identified a region of NzeB with increased conformational flexibility relative to AspB, and interchange of this region along with a single active site mutation led to a variant that catalyzes exclusive C-N bond formation. MD simulations also suggest that intermolecular C-C or C-N bond formation results from a change in mechanism, supported experimentally through use of a substrate mimic.

Total Synthesis of (-)-Voacinol and (-)-Voacandimine C.

We describe the first total synthesis of complex aspidosperma alkaloids (-)-voacinol and (-)-voacandimine C via a late-stage C7-methylenation strategy inspired by a biogenetic hypothesis. We envisioned rapid access to these natural alkaloids from a common, symmetrical precursor assembled by methylenation of a D-ring-oxidized variant of the structurally related natural product (-)-deoxoapodine. Chemoselective N9-oxidation of a pentacyclic deoxoapodine precursor enabled the synthesis of the corresponding hexacyclic C8-aminonitrile. Stereocontrolled methylenation of a C8-enamine derivative of deoxoapodine, accessed by ionization of the C8-aminonitrile, afforded a symmetrical dodecacyclic bisaminonitrile as a versatile precursor to these bisindole alkaloids. The final-stage, biosynthesis-inspired, controlled reductive opening of the oxolane substructures of this dodecacyclic intermediate provided a unified approach to (-)-voacinol and (-)-voacandimine C, while direct reduction of the same intermediate afforded the structurally related (-)-methylenebisdeoxoapodine.

Total Synthesis and Stereochemical Assignment of (-)-Psychotridine.

We report the first enantioselective total synthesis and stereochemical assignment of (-)-psychotridine. The application of our diazene-directed assembly of enantiomerically enriched cyclotryptamines afforded a highly convergent synthesis of the pentameric alkaloid, allowing its detailed structural assignment. Highlights of the synthesis include the introduction of four quaternary stereocenters with complete stereochemical control in a single step via the photoextrusion of three molecules of dinitrogen from an advanced intermediate and metal-catalyzed C-H amination reactions in challenging settings.

Total synthesis of himastatin.

The natural product himastatin has an unusual homodimeric structure that presents a substantial synthetic challenge. We report the concise total synthesis of himastatin from readily accessible precursors, incorporating a final-stage dimerization strategy that was inspired by a detailed consideration of the compound's biogenesis. Combining this approach with a modular synthesis enabled expedient access to more than a dozen designed derivatives of himastatin, including synthetic probes that provide insight into its antibiotic activity.

Directed Palladium-Catalyzed Acetoxylation of Indolines. Total Synthesis of N-Benzoylcylindrocarine.

We describe a palladium-catalyzed C7-acetoxylation of indolines with a range of amide directing groups. While a variety of substituents are tolerated on the indoline-core and the N1-acyl group, the acetoxylation is most sensitive to the C2- and C6-indoline substituents. The practicality of this indoline C7-acetoxylation is demonstrated using a cinnamamide substrate on a mmol scale. Several N1-acyl groups, including those present in natural alkaloids, guide C7-acetoxylation of indoline substrates over a competitive C5-oxidation. The application of this chemistry allowed for the first synthesis of N-benzoylcylindrocarine by late-stage C17-acetoxylation of N-benzoylfendleridine.

Total Synthesis of (-)-Kopsifoline A and (+)-Kopsifoline E.

We report the first total synthesis of (-)-kopsifoline A and (+)-kopsifoline E. Our synthetic strategy features a biogenetically inspired regioselective C17-functionalization of a versatile intermediate containing the pentacyclic core of aspidosperma alkaloids. The vinylogous urethane substructure of this intermediate affords (-)-kopsifoline D via C3-C21 bond formation under the Mitsunobu reaction conditions, while it enables selective C17-functionalization en route to (-)-kopsifoline A and (+)-kopsifoline E.

Chemoselective α-Sulfidation of Amides Using Sulfoxide Reagents.

The direct α-sulfidation of tertiary amides using sulfoxide reagents under electrophilic amide activation conditions is described. Employing convenient and readily available reagents, selective functionalization takes place to generate isolable sulfonium ions en route to α-sulfide amides. Mechanistic studies identified activated sulfoxides as promoters of the desired transformation and enabled the extension of the methodology from benzylic to aliphatic amide substrates.

Structure and Function of NzeB, a Versatile C-C and C-N Bond-Forming Diketopiperazine Dimerase.

The dimeric diketopiperazine (DKPs) alkaloids are a diverse family of natural products (NPs) whose unique structural architectures and biological activities have inspired the development of new synthetic methodologies to access these molecules. However, catalyst-controlled methods that enable the selective formation of constitutional and stereoisomeric dimers from a single monomer are lacking. To resolve this long-standing synthetic challenge, we sought to characterize the biosynthetic enzymes that assemble these NPs for application in biocatalytic syntheses. Genome mining enabled identification of the cytochrome P450, NzeB (Streptomyces sp. NRRL F-5053), which catalyzes both intermolecular carbon-carbon (C-C) and carbon-nitrogen (C-N) bond formation. To identify the molecular basis for the flexible site-selectivity, stereoselectivity, and chemoselectivity of NzeB, we obtained high-resolution crystal structures (1.5 Å) of the protein in complex with native and non-native substrates. This, to our knowledge, represents the first crystal structure of an oxidase catalyzing direct, intermolecular C-H amination. Site-directed mutagenesis was utilized to assess the role individual active-site residues play in guiding selective DKP dimerization. Finally, computational approaches were employed to evaluate plausible mechanisms regarding NzeB function and its ability to catalyze both C-C and C-N bond formation. These results provide a structural and computational rationale for the catalytic versatility of NzeB, as well as new insights into variables that control selectivity of CYP450 diketopiperazine dimerases.

n→π* Interactions Modulate the Disulfide Reduction Potential of Epidithiodiketopiperazines.

Epithiodiketopiperazines (ETPs) are a structurally complex class of fungal natural products with potent anticancer activity. In ETPs, the diketopiperazine ring is spanned by a disulfide bond that is constrained in a high-energy eclipsed conformation. We employed computational, synthetic, and spectroscopic methods to investigate the physicochemical attributes of this atypical disulfide bond. We find that the disulfide bond is stabilized by two n→π* interactions, each with large energies (3-5 kcal/mol). The n→π* interactions in ETPs make disulfide reduction much more difficult, endowing stability in physiological environments in a manner that could impact their biological activity. These data reveal a previously unappreciated means to stabilize a disulfide bond and highlight the utility of the n→π* interaction in molecular design.

Synthesis of Potent Cytotoxic Epidithiodiketopiperazines Designed for Derivatization.

We describe our design, synthesis, and chemical study of a set of functional epidithiodiketopiperazines (ETPs) and evaluation of their activity against five human cancer cell lines. Our structure-activity relationship-guided substitution of ETP alkaloids offers versatile derivatization while maintaining potent anticancer activity, offering exciting opportunity for their use as there are no examples of complex and potently anticancer (nM) ETPs being directly used as conjugatable probes or warheads. Our synthetic solutions to strategically designed ETPs with functional linkers required advances in stereoselective late-stage oxidation and thiolation chemistry in complex settings, including the application of novel reagents for dihydroxylation and cis-sulfidation of diketopiperazines. We demonstrate that complex ETPs equipped with a strategically substituted azide functional group are readily derivatized to the corresponding ETP-triazoles without compromising anticancer activity. Our chemical stability studies of ETPs along with cytotoxic evaluation of our designed ETPs against A549, DU 145, HeLa, HCT 116, and MCF7 human cancer cell lines provide insights into the impact of structural features on potency and chemical stability, informing future utility of ETPs in chemical and biological studies.

Total Synthesis and Anti-Cancer Activity of All Known Communesin Alkaloids and Related Derivatives.

A unified enantioselective total synthesis and anticancer evaluation of all known epoxide-containing communesin alkaloids and related derivatives is described. Our synthesis is predicated on the convergent and modular diazene-directed assembly of two complex fragments to secure the critical C3a-C3a' linkage followed by a guided biomimetic aminal reorganization to deliver the heptacyclic core of these alkaloids. Concise enantioselective syntheses of the fragments were devised, with highlights including the application of a rationally designed sulfinamide chiral auxiliary, an efficient calcium trifluoromethanesulfonate promoted intramolecular amination, and a diastereoselective epoxidation that simultaneously converts the new chiral auxiliary to a versatile amine protective group. The modularity of our convergent approach enabled the rapid synthesis of all epoxide-containing members of the communesin family from a single heterodimeric intermediate, including the first total synthesis of communesins C-E, and G-I, and facilitated our stereochemical revision of (-)-communesin I, the most recently isolated communesin alkaloid. Furthermore, the generality of our biogenetically inspired heterodimer rearrangement was demonstrated in a guided synthesis of a communesin derivative with an unnatural topology. Finally, we report the first comparative analysis of the anticancer activities of all naturally occurring communesin alkaloids A-I and eight complex derivatives against five human cancer cell lines. From these data, we have identified (-)-communesin B as the most potent natural communesin and discovered that derivatives with N8'-sulfonamide substitution exhibit up to a 10-fold increase in potency over the natural alkaloids.

Mechanism of Permanganate-Promoted Dihydroxylation of Complex Diketopiperazines: Critical Roles of Counter-cation and Ion-Pairing.

The mechanism of permanganate-mediated dual C-H oxidation of complex diketopiperazines has been examined with density functional theory computations. The products of these oxidations are enabling intermediates in the synthesis of structurally diverse ETP natural products. We evaluated, for the first time, the impact of ion-pairing and aggregation states of the permanganate ion and counter-cations, such as bis(pyridine)-silver(I) (Ag+) and tetra- n-butylammonium (TBA+), on the C-H oxidation mechanism. The C-H abstraction occurs through an open shell singlet species, as noted previously, followed by O-rebound and a competing OH-rebound pathway. The second C-H oxidation proceeds with a second equivalent of oxidant with lower free energy barriers than the first C-H oxidation due to directing effects and the generation of a more reactive oxidant species after the first C-H oxidation. The success and efficiency of the second C-H oxidation are found to be critically dependent on the presence of an ion-paired oxidant. We used the developed mechanistic knowledge to rationalize an experimentally observed oxidation pattern for C3-indole-substituted diketopiperazine (+)-5 under optimal oxidation conditions: namely, the formation of diol (-)-6 as a single diastereomer and lack of the ketone products. We proposed two factors that may impede the ketone formation: (i) the conformational flexibility of the diketopiperazine ring, and (ii) hindrance of this site, making it less accessible to the ion-paired oxidant species.

Enantioselective Synthesis of (-)-Vallesine: Late-Stage C17-Oxidation via Complex Indole Boronation.

The first enantioselective total synthesis of (-)-vallesine via a strategy that features a late-stage regioselective C17-oxidation followed by a highly stereoselective transannular cyclization is reported. The versatility of this approach is highlighted by the divergent synthesis of the archetypal alkaloid of this family, (+)-aspidospermidine, and an A-ring-oxygenated derivative, (+)-deacetylaspidospermine, the precursor to (-)-vallesine, from a common intermediate.

Concise total synthesis of (+)-asperazine A and (+)-pestalazine B.

The highly convergent total synthesis of dimeric diketopiperazine alkaloids (+)-asperazine A and (+)-pestalazine B is described. A critical aspect of our expedient route was the development of a directed regio- and diastereoselective C3-N1' coupling of complex tetracyclic diketopiperazine components. This late-stage heterodimerization reaction was made possible by design of tetracyclic diketopiperazines that allow C3-carbocation coupling of the electrophilic component to the N1' locus of the nucleophilic fragment. The application of this new coupling reaction to the first total synthesis of (+)-asperazine A led to our revision of the sign and magnitude of the optical rotation for the reported structure.

Quantitative Modeling of Bis(pyridine)silver(I) Permanganate Oxidation of Hydantoin Derivatives: Guidelines for Predicting the Site of Oxidation in Complex Substrates.

The bis(pyridine)silver(I) permanganate promoted hydroxylation of diketopiperazines has served as a pivotal transformation in the synthesis of complex epipolythiodiketopiperazine alkaloids. This late-stage C-H oxidation chemistry is strategically critical to access N-acyl iminium ion intermediates necessary for nucleophilic thiolation of advanced diketopiperazines en route to potent epipolythiodiketopiperazine anticancer compounds. In this study, we develop an informative mathematical model using hydantoin derivatives as a training set of substrates by relating the relative rates of oxidation to various calculated molecular descriptors. The model prioritizes Hammett values and percent buried volume as key contributing factors in the hydantoin series while correctly predicting the experimentally observed oxidation sites in various complex diketopiperazine case studies. Thus, a method is presented by which to use simplified training molecules and resulting correlations to explain and predict reaction behavior for more complex substrates.

Concise Synthesis of (-)-Hodgkinsine, (-)-Calycosidine, (-)-Hodgkinsine B, (-)-Quadrigemine C, and (-)-Psycholeine via Convergent and Directed Modular Assembly of Cyclotryptamines.

The enantioselective total synthesis of (-)-hodgkinsine, (-)-calycosidine, (-)-hodgkinsine B, (-)-quadrigemine C, and (-)-psycholeine through a diazene-directed assembly of cyclotryptamine fragments is described. Our synthetic strategy enables multiple and directed assembly of intact cyclotryptamine subunits for convergent synthesis of highly complex bis- and tris-diazene intermediates. Photoextrusion of dinitrogen from these intermediates enables completely stereoselective formation of all C3a-C3a' and C3a-C7' carbon-carbon bonds and all the associated quaternary stereogenic centers. In a representative example, photoextrusion of three dinitrogen molecules from an advanced intermediate in a single-step led to completely controlled introduction of four quaternary stereogenic centers and guided the assembly of four cyclotryptamine monomers en route to (-)-quadrigemine C. The synthesis of these complex diazenes was made possible through a new methodology for synthesis of aryl-alkyl diazenes using electronically attenuated hydrazine-nucleophiles for a silver-promoted addition to C3a-bromocyclotryptamines. The application of Rh- and Ir-catalyzed C-H amination reactions in complex settings were used to gain rapid access to C3a- and C7-functionalized cyclotryptamine monomers, respectively, used for diazene synthesis. This convergent and modular assembly of intact cyclotryptamines offers the first solution to access these alkaloids through completely stereoselective union of monomers at challenging linkages and the associated quaternary stereocenters as illustrated in our synthesis of five members of the oligocyclotryptamine family of alkaloids.

Enantioselective Total Synthesis of (-)-Deoxoapodine.

The first enantioselective total synthesis of (-)-deoxoapodine is described. Our synthesis of this hexacyclic aspidosperma alkaloid includes an efficient molybdenum-catalyzed enantioselective ring-closing metathesis reaction for the desymmetrization of an advanced intermediate that introduces the C5-quaternary stereocenter. After C21-oxygenation, the pentacyclic core was accessed by electrophilic C19-amide activation and transannular spirocyclization. A biogenetically inspired dehydrative C6-etherification reaction proved highly effective to secure the F-ring and the fourth contiguous stereocenter of (-)-deoxoapodine with complete stereochemical control.

Synthesis and Evaluation of Agelastatin Derivatives as Potent Modulators for Cancer Invasion and Metastasis.

The synthesis of new agelastatin alkaloid derivatives and their anticancer evaluation in the context of the breast cancer microenvironment is described. A variety of N1-alkyl and C5-ether agelastatin derivatives were accessed via application of our strategy for convergent imidazolone synthesis from a common thioester along with appropriately substituted urea and alcohol components. These agelastatin derivatives were evaluated in our three-dimensional coculture assay for the effects of mammary fibroblasts on associated breast cancer cells. We have discovered that agelastatin alkaloids are potent modulators for cancer invasion and metastasis at noncytotoxic doses. Herein, we discuss the increased potency of (-)-agelastatin E as compared to (-)-agelastatin A in this capacity, in addition to identification of new agelastatin derivatives with activity that is statistically equivalent to (-)-agelastatin E. The chemistry described in this report provides a platform for the rapid synthesis of agelastatin derivatives with excellent potency (50-100 nM) as modulators for cancer invasion and metastasis.