Towards combining backbone and sugar constraint in 3'-3' bis-phosphonate tethered 2'-4' bridged LNA oligonucleotide trimers.

Therapeutic oligonucleotides are chemically modified to enhance their drug-like properties - including binding affinity for target RNA. Many nucleic acid analogs that enhance RNA binding affinity constrain the furanose sugar in an RNA-like sugar pucker. The improvements in binding affinity result primarily from increased off-rates with minimal effects on on-rates for hybridization. To identify alternate chemical modification strategies that can modulate on- and off-rates for oligonucleotide hybridization, we hypothesized that extending conformational restraint across multiple nucleotides could modulate hybridization kinetics by restricting rotational freedom of the sugar-phosphate backbone. As part of that effort, we recently reported that using hydrocarbon tethers to bridge adjacent phosphodiester linkages as phosphonate tethered bridges can pre-organize nucleic acids in conformations conducive for Watson-Crick base-pairing and modulate hybridization kinetics. In this report, we describe the synthesis of locked nucleic acid (LNA) trimers linked through alkylphosphonate tethers which restrict conformation of the furanose sugar in addition to restricting conformational mobility of the sugar-phosphate backbone across three nucleotide units.

Revisiting the dipeptidyl carboxypeptidase inhibitor captopril as a source of pan anti-trypanosomatid agents.

The protozoan parasites Trypanosoma brucei, Trypanosoma cruzi and Leishmania spp. are responsible for continued propagation of neglected tropical diseases such as African sleeping sickness, Chagas disease and leishmaniasis respectively. Following a report that captopril targets Leishmania donovani dipeptidyl carboxypeptidase, a series of simple proline amides and captopril analogues were synthesized and found to exhibit 1-2 µM in vitro inhibition and selectivity against Trypanosoma brucei, Trypanosoma cruzi and Leishmania spp. The results were corroborated with computational docking studies. Arguably, the synthetic proline amides represent the structurally simplest examples of in vitro pan antiprotozoal compounds.

My 50-Plus Years of Academic Research Collaborations with Industry. A Retrospective.

A retrospective is presented highlighting the synthesis of selected "first-in-kind" natural products, their synthetic analogues, structure elucidations, and rationally designed bioactive synthetic compounds that were accomplished because of collaborations with past and present pharmaceutical and agrochemical companies. Medicinal chemistry projects involving structure-based design exploiting cocrystal structures of small molecules with biologically relevant enzymes, receptors, and bacterial ribosomes with synthetic small molecules leading to marketed products, clinical candidates, and novel drug prototypes were realized in collaboration. Personal reflections, historical insights, behind the scenes stories from various long-term projects are shared in this retrospective article.

Pseudodiproline (Pro-Cyp) Oligomers Fold into Helical Polyproline Type secondary structures.

The synthesis and conformational properties of oligo-proline mimetics composed of dimeric and tetrameric Pro-Cyp constructs linked by a hydroxymethylene unit are reported. Oligomers were studied both in the solid state and in solution, unveiling right-handed helical conformation depending on the configuration of the vicinally substituted trans-cyclopentane carboxylic acid unit (Cyp). Unlike polyproline oligomers, the alternating synthetic Pro-Cyp counterparts are not stabilized by n-π* interactions but rely instead on the steric demands of the extended backbone conformation within the hydroxymethylene-linked Pro-Cyp repeating units.

Synthesis of Aza-Bridged Perhydroazulene Chimeras of Tropanes and Hederacine A.

We report the synthesis of two novel azaperhydroazulene tropane-hederacine chimeras A and B, which contain an 8-azabicyclo[3.2.1]octane ring and a 7-azabicyclo[4.1.1]octane ring, respectively. The synthesis of both chimeras was achieved by epoxide ring opening and was governed by the stereochemistry of the hydroxy-epoxide unit. Finally, a density functional theory study was conducted to explain the regioselectivity of the cyclization and the importance of the stereochemistry of the hydroxyl group.

Systematic Investigation of Tether Length and Phosphorus Configuration in Backbone Constrained Macrocyclic Nucleic Acids to Modulate Binding Kinetics for RNA.

We recently described a chemical strategy to pre-organize a trinucleotide subunit in a conformation suitable for Watson-Crick base pairing for modulating the binding kinetics of single-stranded oligonucleotides (ONs) using bis-phosphonate esters bridging hydrocarbon tethers to provide 11- and 15-membered macrocyclic analogues. In this manuscript, we describe the synthesis of all eight P-stereoisomers of macrocyclic 12-, 13-, 14-, and 16-membered hydrocarbon-bridged nucleotide trimers, their incorporation into ONs, and biophysical characterization of the modified ONs. The size of the macrocyclic tether and configuration at phosphorus had profound effects on hybridization kinetics. ONs containing 12- and 13-membered rings exhibited faster on-rates (up to 5-fold) and off-rates (up to 161-fold). In contrast, ONs using the larger ring size macrocycles generally exhibited smaller changes in binding kinetics relative to unmodified DNA. Interestingly, several of the analogues retained significant binding affinity for RNA based on their dissociation constants, despite being modestly destabilizing in the thermal denaturation experiments, highlighting the potential utility of measuring dissociation constants versus duplex thermal stability when evaluating novel nucleic acid analogues. Overall, our results provide additional insights into the ability of backbone-constrained macrocyclic nucleic acid analogues to modulate hybridization kinetics of modified ONs with RNA.

Simultaneous inhibition of endocytic recycling and lysosomal fusion sensitizes cells and tissues to oligonucleotide therapeutics.

Inefficient endosomal escape remains the primary barrier to the broad application of oligonucleotide therapeutics. Liver uptake after systemic administration is sufficiently robust that a therapeutic effect can be achieved but targeting extrahepatic tissues remains challenging. Prior attempts to improve oligonucleotide activity using small molecules that increase the leakiness of endosomes have failed due to unacceptable toxicity. Here, we show that the well-tolerated and orally bioavailable synthetic sphingolipid analog, SH-BC-893, increases the activity of antisense oligonucleotides (ASOs) and small interfering RNAs (siRNAs) up to 200-fold in vitro without permeabilizing endosomes. SH-BC-893 treatment trapped endocytosed oligonucleotides within extra-lysosomal compartments thought to be more permeable due to frequent membrane fission and fusion events. Simultaneous disruption of ARF6-dependent endocytic recycling and PIKfyve-dependent lysosomal fusion was necessary and sufficient for SH-BC-893 to increase non-lysosomal oligonucleotide levels and enhance their activity. In mice, oral administration of SH-BC-893 increased ASO potency in the liver by 15-fold without toxicity. More importantly, SH-BC-893 enabled target RNA knockdown in the CNS and lungs of mice treated subcutaneously with cholesterol-functionalized duplexed oligonucleotides or unmodified ASOs, respectively. Together, these results establish the feasibility of using a small molecule that disrupts endolysosomal trafficking to improve the activity of oligonucleotides in extrahepatic tissues.

Polygonapholine: A Total Synthesis Questions the Identity for the Purported Structure of the Natural Product.

Polygonapholine was isolated in 1997 from the Polygonatum alte-lobatum rhizome. Based on spectroscopic data, it was assigned a structure comprising an unusual cis-2,6-disubstituted bis-aryl morpholine ring to which is attached a (Z)-4-hydroxycinnamate as an amide and an (E)-4-hydroxycinnamate as an ester. Being a meso compound, polygonapholine should not exhibit an optical rotation as reported. A total synthesis of the purported morpholine alkaloid presented herein reveals discrepancies between the synthetic and the natural product casting doubt on the originally proposed structure.

Targeting non-alcoholic fatty liver disease: Design, X-ray co-crystal structure and synthesis of 'first-in-kind' inhibitors of serine/threonine kinase25.

We describe the synthesis of a series of 3-t-butyl 5-aminopyrazole p-substituted arylamides as inhibitors of serine-threonine25 (STK25), an enzyme implicated in the progression of non-alcoholic fatty liver disease (NAFLD). Appending a p-N-pyrrolidinosulphonamide group to the arylamide group led to a 'first-in kind' inhibitor with IC50 = 228 nM. A co-crystal structure with STK 25 revealed productive interactions which were also reproduced using molecular docking. A new series of triazolo dihydro oxazine carboxamides of 3-t-butyl 5-aminopyrazole was not active against STK25.

2-Oxa-5-azabicyclo[2.2.1]heptane as a Platform for Functional Diversity: Synthesis of Backbone-Constrained γ-Amino Acid Analogues.

We communicate a versatile synthetic approach to C-3 disubstituted 2-oxa-5-azabicyclo[2.2.1]heptanes as carbon-atom bridged morpholines, starting with 4R-hydroxy-l-proline as a chiron. Attaching an acetic acid moiety on the C-3 carbon of the 2-oxa-5-azabicyclo[2.2.1]heptane core reveals the framework of an embedded γ-amino butyric acid (GABA). Variations in the nature of the substituent on the tertiary C-3 atom with different alkyls or aryls led to backbone-constrained analogues of the U.S. Food and Drug Administration-approved drugs baclofen and pregabalin.

Targeting NOX2 via p47/phox-p22/phox Inhibition with Novel Triproline Mimetics.

On the basis of the knowledge that the proline-rich hot spot PPPRPP region of P(151)PSNPPPRPP(160), an oligopeptide derived from the cytosolic portion of p22phox (p22), binds to the single functional bis-SH3 domain of the regulatory protein p47phox (p47), we designed a mimetic of the tripeptide PPP based on NMR and X-ray crystallographic data for the p22(151-161) peptide PPSNPPPRPPA with a peptide construct. Incorporation of the synthetic pseudo-triproline mimetic Pro-Pro-Cyp in a molecule derived from molecular modeling studies led to only a 7-fold diminution in activity in a surface plasmon resonance assay relative to the same molecule containing the natural Pro-Pro-Pro tripeptide. The alternative sequence corresponding to a Pro-Cyp-Pro insertion was inactive. This is a first example of the use of a triproline mimetic to interfere with the formation of the p47-p22 complex, which is critical for the activation of NOX, leading to the production of reactive oxygen species as superoxide anions.

Surprising Chemistry of 6-Azidotetrazolo[5,1-a]phthalazine: What a Purported Natural Product Reveals about the Polymorphism of Explosives.

6-Azidotetrazolo[5,1-a]phthalazine (ATPH) is a nitrogen-rich compound of surprisingly broad interest. It is purported to be a natural product, yet it is closely related to substances developed as explosives and is highly polymorphic despite having a nearly planar structure with little flexibility. Seven solid forms of ATPH have been characterized by single-crystal X-ray diffraction. The structures show diverse patterns of molecular organization, including both stacked sheets and herringbone packing. In all cases, N···N and C-H···N interactions play key roles in ensuring molecular cohesion. The high polymorphism of ATPH appears to arise in part from the ability of virtually every atom of nitrogen and hydrogen in the molecule to take part in close N···N and C-H···N contacts. As a result, adjacent molecules can adopt many different relative orientations that are energetically similar, thereby generating a polymorphic landscape with an unusually high density of potential structures. This landscape has been explored in detail by the computational prediction of crystal structures. Studying ATPH has provided insights into the field of energetic materials, where access to multiple polymorphs can be used to improve performance and clarify how it depends on molecular packing. In addition, our work with ATPH shows how valuable insights into molecular crystallization, often gleaned from statistical analyses of structural databases, can also come from in-depth empirical and theoretical studies of single compounds that show distinctive behavior.

Backbone Hydrocarbon-Constrained Nucleic Acids Modulate Hybridization Kinetics for RNA.

The binding affinity of therapeutic oligonucleotides (ONs) for their cognate RNA is determined by the rates of association (ka) and dissociation (kd). Single-stranded ONs are highly flexible and can adopt multiple conformations in solution, some of which may not be conducive for hybridization. We investigated if restricting rotation around the sugar-phosphate backbone, by tethering two adjacent backbone phosphonate esters using hydrocarbon bridges, can modulate hybridization kinetics of the modified ONs for complementary RNA. Given the large number of possible analogues with different tether lengths and configurations at the phosphorus atoms, we employed molecular dynamic simulations to optimize the size of the hydrocarbon bridge to guide the synthetic efforts. The backbone-constrained nucleotide trimers with stereodefined configurations at the contiguous backbone phosphorus atoms were assembled using a ring-closing metathesis reaction, then incorporated into oligonucleotides by an in situ synthesis of the phosphoramidites followed by coupling to solid supports. Evaluation of the modified oligonucleotides revealed that 15-membered macrocyclic-constrained analogues displayed similar or slightly improved on-rates but significantly increased off-rates compared to unmodified DNA ONs, resulting in reduced duplex stability. In contrast, LNA ONs with conformationally preorganized furanose rings showed similar on-rates to DNA ONs but very slow off-rates, resulting in net improvement in duplex stability. Furthermore, the experimental data generally supported the molecular dynamics simulation results, suggesting that this strategy can be used as a predictive tool for designing the next generation of constrained backbone ON analogues with improved hybridization properties.

Computational and Further Experimental Explorations of the Competing Cascades Following Claisen Rearrangements of Aryl Propargyl Ethers: Substituent Effects on Reactivity and Regioselectivity.

We report a computational investigation of two reaction cascades occurring following the Claisen rearrangements of aryl propargyl ethers to the alternate ortho positions in unsymmetrical reactants. Our computations explain how substituents influence reactivity and regioselectivity. Rearrangement to the substituted ortho carbon leads to a tricyclo[3.2.1.0]octane core, while rearrangement to an unsubstituted ortho carbon leads to a benzopyran. Density functional theory with ωB97X-D indicates that these reactions involve rate-determining Claisen rearrangements followed by subsequent reaction cascades of the Claisen rearrangement products depending on the presence or absence of a substituent at the ortho carbon.

Design of Pseudodiproline Dimers as Mimetics of Pro-Pro Units: Stereocontrolled Synthesis, Configurational Relevance, and Structural Properties.

Stereocontrolled methods are described for the synthesis of hitherto unreported pseudodiproline dimers in which a cyclopentane carboxylic acid is linked to a pyrrolidine residue by a stereochemically defined hydroxymethylene tether. These proline-cyclopentane (Pro-Cyp) dimers have interesting structural characteristics as seen in their X-ray crystal structures as well as their nuclear magnetic resonance (NMR) spectra in CDCl3. They can be considered to be novel Pro-Pro mimetics, which can be used to replace natural diproline sequences with potential applications in medicinal chemistry. They also represent a new concept in the peptidomimetic design of chimeric proline-based amino acids as carbocyclic hydroxyethylene isosteres of inhibitor molecules, in which the stereodefined bridging hydroxyl group can simulate a tetrahedral intermediate in an enzyme complex.

Drug-like sphingolipid SH-BC-893 opposes ceramide-induced mitochondrial fission and corrects diet-induced obesity.

Ceramide-induced mitochondrial fission drives high-fat diet (HFD)-induced obesity. However, molecules targeting mitochondrial dynamics have shown limited benefits in murine obesity models. Here, we reveal that these compounds are either unable to block ceramide-induced mitochondrial fission or require extended incubation periods to be effective. In contrast, targeting endolysosomal trafficking events important for mitochondrial fission rapidly and robustly prevented ceramide-induced disruptions in mitochondrial form and function. By simultaneously inhibiting ARF6- and PIKfyve-dependent trafficking events, the synthetic sphingolipid SH-BC-893 blocked palmitate- and ceramide-induced mitochondrial fission, preserved mitochondrial function, and prevented ER stress in vitro. Similar benefits were observed in the tissues of HFD-fed mice. Within 4 h of oral administration, SH-BC-893 normalized mitochondrial morphology in the livers and brains of HFD-fed mice, improved mitochondrial function in white adipose tissue, and corrected aberrant plasma leptin and adiponectin levels. As an interventional agent, SH-BC-893 restored normal body weight, glucose disposal, and hepatic lipid levels in mice consuming a HFD. In sum, the sphingolipid analog SH-BC-893 robustly and acutely blocks ceramide-induced mitochondrial dysfunction, correcting diet-induced obesity and its metabolic sequelae.

Ni-Catalyzed Reductive and Merged Photocatalytic Cross-Coupling Reactions toward sp3/sp2-Functionalized Isoquinolones: Creating Diversity at C-6 and C-7 to Address Bioactive Analogues.

Naturally occurring isoquinolones have gained considerable attention over the years for their bioactive properties. While the late-stage introduction of various functionalities at certain positions, namely, C-3, C-4, and C-8, has been widely documented, the straightforward introduction of challenging sp3 carbon-linked acyclic aminoalkyl or aza- and oxacyclic appendages at C-6 and C-7 remains largely underexplored. Interest in 6-substituted azacyclic analogues has recently garnered attention in connection with derivatives exhibiting anticancer activity. Reported here is the first application of the versatile and recently emerging field of Ni-catalyzed reductive cross-coupling reactions to the synthesis of 6- and 7- hetero(cyclo)alkyl-substituted isoquinolones. In a second and complementary approach, a new set of C-6- and C-7-substituted positional isomers of hetero(cyclo)alkyl appendages were obtained from the merging of photocatalytic and Ni-catalyzed coupling reactions. In both cases, 6- and 7-bromo isoquinolones served as dual-purpose reacting partners with readily available tosylates and carboxylic acids, respectively.

Cyanide-Free Synthesis of Air Stable N-Substituted Li and K Cyanamide Salts from Tetrazoles. Applications toward the Synthesis of Primary and Secondary Cyanamides as Precursors to Amidines.

A practical two-step synthesis of N,N'-disubstituted cyanamides consists in the low-temperature metalation of N-substituted 5H-tetrazoles that undergo spontaneous cycloreversion at 0 °C releasing dinitrogen, and forming N-metalated cyanamides that can be reacted in situ with a variety of electrophiles. Remarkably, the N-substituted Li and K cyanamides are air stable white solids at room temperature. Addition of lithium organometallics to the N,N'-disubstituted cyanamides provides a new method for accessing N,N'-disubstituted amidines.

Synthetic Sphingolipids with 1,2-Pyridazine Appendages Improve Antiproliferative Activity in Human Cancer Cell Lines.

A synthetic sphingolipid related to a ring-constrained hydroxymethyl pyrrolidine analog of FTY720 that was known to starve cancer cells to death was chemically modified to include a series of alkoxy-tethered 3,6-substituted 1,2-pyridazines. These derivatives exhibited excellent antiproliferative activity against eight human cancer cell lines from four different cancer types. A 2.5- to 9-fold reduction in IC50 in these cell lines was observed relative to the lead compound, which lacked the appended heterocycle.

Total Synthesis and Stereochemical Confirmation of (-)-Olivil, (+)-Cycloolivil, (-)-Alashinols F and G, (+)-Cephafortin A, and Their Congeners: Filling in Biosynthetic Gaps.

For the first time, we describe the stereocontrolled total syntheses of olivil, cephafortin A, 4-des-O-methyl-4-O-rhamnosyl cephafortin A, and alashinol F from a common precursor using a combination of chemoenzymatic and biomimetic methods for the systematic introduction of functional groups on three vicinal stereogenic carbon atoms. We revised the previously assigned stereochemistry of (+)-cephafortin A, which was reported as the enantiomer. Natural and unnatural congeners provide insights into the biogenetic interrelations of members of this family.