In Vivo Labeling and Detection of Circulating Tumor Cells in Mice Using OTL38.

PURPOSE: We recently developed an optical instrument to non-invasively detect fluorescently labeled circulating tumor cells (CTCs) in mice called 'Diffuse in vivo Flow Cytometry' (DiFC). OTL38 is a folate receptor (FR) targeted near-infrared (NIR) contrast agent that is FDA approved for use in fluorescence guided surgery of ovarian and lung cancer. In this work, we investigated the use OTL38 for in vivo labeling and detection of FR + CTCs with DiFC. PROCEDURES: We tested OTL38 labeling of FR + cancer cell lines (IGROV-1 and L1210A) as well as FR- MM.1S cells in suspensions of Human Peripheral Blood Mononuclear cells (PBMCs) in vitro. We also tested OTL38 labeling and NIR-DIFC detection of FR + L1210A cells in blood circulation in nude mice in vivo. RESULTS: 62% of IGROV-1 and 83% of L1210A were labeled above non-specific background levels in suspensions of PBMCs in vitro compared to only 2% of FR- MM.1S cells. L1210A cells could be labeled with OTL38 directly in circulation in vivo and externally detected using NIR-DiFC in mice with low false positive detection rates. CONCLUSIONS: This work shows the feasibility of labeling CTCs in vivo with OTL38 and detection with DiFC. Although further refinement of the DiFC instrument and signal processing algorithms and testing with other animal models is needed, this work may eventually pave the way for human use of DiFC.

Fragtory: Pharmacophore-Focused Design, Synthesis, and Evaluation of an sp3-Enriched Fragment Library.

Fragment-based drug discovery has played an important role in medicinal chemistry and pharmaceutical research. Despite numerous demonstrated successes, the limited diversity and overrepresentation of planar, sp2-rich structures in commercial libraries often hamper the full potential of this approach. Hence, the thorough design of screening libraries inevitably determines the probability for meaningful hits and subsequent structural elaboration. Against this background, we present the generation of an exclusive fragment library based on iterative entry nomination by a specifically designed computational workflow: "Fragtory". Following a pharmacophore diversity-driven approach, we used Fragtory in an interdisciplinary academic setting to guide both tailored synthesis efforts and the implementation of in-house compounds to build a curated 288-member library of sp3-enriched fragments. Subsequent NMR screens against a model protein and hit validation by protein crystallography led to the identification of structurally novel ligands that were further characterized by isothermal titration calorimetry, demonstrating the applicability of our experimental approach.

Water-soluble chromenylium dyes for shortwave infrared imaging in mice.

In vivo imaging using shortwave infrared light (SWIR, 1000-2000 nm) benefits from deeper penetration and higher resolution compared to using visible and near-infrared wavelengths. However, the development of biocompatible SWIR contrast agents remains challenging. Despite recent advancements, small molecule SWIR fluorophores are often hindered by their significant hydrophobicity. We report a platform for generating a panel of soluble and functional dyes for SWIR imaging by late-stage functionalization of a versatile fluorophore intermediate, affording water-soluble dyes with bright SWIR fluorescence in serum. Specifically, a tetra-sulfonate derivative enables clear video-rate imaging of vasculature with only 0.05 nmol dye, and a tetra-ammonium dye shows strong cellular retention for tracking of tumor growth. Additionally, incorporation of phosphonate functionality enables imaging of bone in awake mice. This modular design provides insights for facile derivatization of existing SWIR fluorophores to introduce both solubility and bioactivity towards in vivo bioimaging.

sp3 -Rich Glycyrrhetinic Acid Analogues Using Late-Stage Functionalization as Potential Breast Tumor Regressing Agents.

Late-stage functionalization (LSF) aids drug discovery efforts by introducing functional groups onto C-H bonds on pre-existing skeletons. We adopted the LSF strategy to synthesize analogues of the abundantly available triterpenoid, glycyrrhetinic acid (GA), by introducing aryl groups in the A-ring, expanding the A-ring and selectively activating one methyl group of the gem-dimethyl groups. Intriguingly, two compounds were found to preferentially accumulate in the mitochondrial compartment of MDA-MB-231 breast cancer cells, to cause depolarization of mitochondrial membrane potential and to induce antiproliferative and anti-invasive effects through enhanced mitochondrial superoxide production with parallel depletion of GSH levels. Furthermore, intraperitoneal administration of these two compounds, in comparison with GA, greatly regressed breast tumor growth and metastasis in a SCID mouse model bearing labeled MDA-MB-231 cells.

Benzyne Insertion onto ß-Keto Esters of Polycyclic Natural Products: Synthesis of Benzo Octacyclo Scaffolds.

Benzyne insertion to build the privileged scaffold of a [6.8.6]-tricyclic framework on polycyclic and sp3-rich natural products has been developed. The formation of the [6.8.6]-tricyclic ring system is solvent dependent.

Total Synthesis of Desmethyl Jahanyne and Its Lipo-Tetrapeptide Conjugates Derived from Parent Skeleton as BCL-2-Mediated Apoptosis-Inducing Agents.

The total synthesis of highly potent and scarcely available marine natural product (-)-jahanyne was attempted resulting in a solution-phase synthesis of pruned versions with comparable activity. A simple and facile synthetic route was employed for the preparation of pruned congeners and would be scalable. The lipophilic tail of the natural product was synthesized from R-(+)-citronellol, utilizing easily available chemicals. All the synthesized compounds were screened for apoptotic activity against a panel of cell lines. These compounds depicted marked binding to B cell lymphoma 2 till 50 °C in cellular thermal shift analysis.

Synthetic Strategy toward the Pentacyclic Core of Melodinus Alkaloids.

The three-component Povarov reaction is efficiently utilized for construction of the pentacyclic framework of complex Melodinus alkaloids, which is amenable to expansion to other complex natural products. The key steps were Povarov reaction, one-pot reductive cyclization, and ring-closing metathesis (RCM) reaction.

Carbonyl-assisted reverse regioselective cascade annulation of 2-acetylenic ketones triggered by Ru-catalyzed C-H activation.

The first reverse regioselective intermolecular annulation of aryl substituted 2-acetylenic ketones with O-substituted N-hydroxybenzamides or acrylamides followed by tandem cyclization via ruthenium-catalyzed C-H activation, is reported. Excellent reverse selectivity of alkyne insertion was induced by the weak coordination between the carbonyl group and ruthenium complex. This highly efficient and practical reaction has a broad range of substrate scope with excellent functional-group tolerance. The tandem reaction provides a wide range of polycyclic products that have an indozilidine structural motif, and are found to potentially be synthetically and pharmaceutically valuable.

Synthesis of highly strained bicyclic[3.n.1]alkenes by a metal-catalyzed Conia-ene reaction.

A high yielding metal-catalysed Conia-ene reaction of 2-acetylenic ketones for the synthesis of bicyclo[3.n.1]alkenes has been developed. This simple and efficient 6-endo-dig-cyclization protocol enables the synthesis of a wide variety of bicyclic systems, present in many natural products.