Designing Antitrypanosomal and Antileishmanial BODIPY Derivatives: A Computational and In Vitro Assessment.

Leishmaniasis and Human African trypanosomiasis pose significant public health threats in resource-limited regions, accentuated by the drawbacks of the current antiprotozoal treatments and the lack of approved vaccines. Considering the demand for novel therapeutic drugs, a series of BODIPY derivatives with several functionalizations at the meso, 2 and/or 6 positions of the core were synthesized and characterized. The in vitro activity against Trypanosoma brucei and Leishmania major parasites was carried out alongside a human healthy cell line (MRC-5) to establish selectivity indices (SIs). Notably, the meso-substituted BODIPY, with 1-dimethylaminonaphthalene (1b) and anthracene moiety (1c), were the most active against L. major, displaying IC50 = 4.84 and 5.41 µM, with a 16 and 18-fold selectivity over MRC-5 cells, respectively. In contrast, the mono-formylated analogues 2b and 2c exhibited the highest toxicity (IC50 = 2.84 and 6.17 µM, respectively) and selectivity (SI = 24 and 11, respectively) against T. brucei. Further insights on the activity of these compounds were gathered from molecular docking studies. The results suggest that these BODIPYs act as competitive inhibitors targeting the NADPH/NADP+ linkage site of the pteridine reductase (PR) enzyme. Additionally, these findings unveil a range of quasi-degenerate binding complexes formed between the PRs and the investigated BODIPY derivatives. These results suggest a potential correlation between the anti-parasitic activity and the presence of multiple configurations that block the same site of the enzyme.

Construction and evaluation of near-infrared fluorescent probes for imaging lipid droplet and lysosomal viscosity.

Microenvironmental viscosity is a crucial parameter for biological systems, and its abnormal fluctuations are closely associated with various functional disorders and diseases. However, it is still important and urgent to develop improved near-infrared fluorescent probes for micro-viscosity with dual-organelle targeting properties, low background noise, and high sensitivity. Herein, two BODIPY-based small-molecule fluorescent probes were designed and synthesized, which were explored for their viscosity- and polarity-responsive properties, and were further applied to imaging sub-cellular viscosity in living cells. Interestingly, BSZ-Ph and BSZ-R displayed near-infrared fluorescence (more than 650 nm) and were sensitive to environmental viscosity and polarity due to the introduction of a benzothiazole at the 2-position and electron-rich aniline groups at the 5-position of the BODIPY core, respectively. The fluorescence intensity increased exponentially with the viscosity changes. Furthermore, the probe BSZ-Ph could successfully target lipid droplets and image cellular viscosity changes by treating lipopolysaccharides (LPS) and nystatin. Comparatively, the probe BSZ-R could successfully target the dual organelles of lipid droplets and lysosomes and image cellular viscosity changes by treating LPS and monensin. Therefore, in this work, we reported two new BODIPY-based near-infrared fluorescent probes, BSZ-Ph and BSZ-R, for cellular viscosity imaging, which could target lipid droplets and the dual organelles of lysosomes and lipid droplets, respectively. The study could provide a reference for the future development of fluorescent probes for viscosity in lipid droplets and lysosomes.

Fluorine-18 labeling PEGylated 6-boronotryptophan for PET scanning of mice for assessing the pharmacokinetics for boron neutron capture therapy of brain tumors.

Two tryptophan compound classes 5- and 6-borono PEGylated boronotryptophan derivatives have been prepared for assessing their aqueous solubility as formulation of injections for boron neutron capture therapy (BNCT). The PEGylation has improved their aqueous solubility thereby increasing their test concentration in 1 mM without suffering from toxicity. In-vitro uptake assay of PEGylated 5- and 6-boronotryptophan showed that the B-10 concentration can reach 15-50 ppm in U87 cell whereas the uptake in LN229 cell varies. Shorter PEG compound 6-boronotryptophanPEG200[18F] was obtained in 1.7 % radiochemical yield and the PET-derived radioradioactivity percentage in 18 % was taken up by U87 tumor at the limb of xenograft mouse. As high as tumor to normal uptake ratio in 170 (T/N) was obtained while an inferior radioactivity uptake of 3 % and T/N of 8 was observed in LN229 xenografted mouse.

BODIPY(aryl)iodonium salts in the efficient synthesis of diversely functionalized BODIPYs and selective detection of serum albumin.

BODIPY(aryl)iodonium salts were readily accessible from the high-yielding reaction of BODIPY with iodoarenes or hydroxyl(tosyloxy)iodoarenes in the presence of m-CPBA. The prepared BODIPY(aryl)iodonium salts bearing substituents of varied electronic nature were utilized for the direct syntheses of thiocyanate, azide, amine and acrylate functionalized BODIPYs and ß,ß'-bis-BODIPYs. The regioselective syntheses of α-piperidinyl and ß-piperidinyl substituted BODIPYs were achieved through the reaction of BODIPY(aryl)iodonium salts with piperidine in the absence and presence of copper(I). Expeditious and high yielding (79-82%) synthesis of ß,ß'-bis-BODIPYs was also developed through the palladium-catalyzed reductive coupling of the easily accessible BODIPY(aryl)iodonium salts. Some of the indole-appended BODIPYs and bis-BODIPYs displayed strong absorption in the visible region (∼610 nm). The BODIPY(aryl)iodonium salts also showed significant binding with serum albumin and were observed to be selective serum protein sensors with estimated limits of detection as low as 7 µg mL-1 in some cases.

Discovery of an Amino Acid-Modified Near-Infrared Aza-BODIPY Photosensitizer as an Immune Initiator for Potent Photodynamic Therapy in Melanoma.

The discovery of novel photosensitizers with potent phototoxicity and desirable water solubility is an urgent task for photodynamic therapy. Herein, a series of amino acid-modified aza-BODIPY photosensitizers were synthesized and evaluated. These new PSs exhibited enhanced aqueous solubility, increased 1O2 generation efficiency, and an improved photo-dark toxicity ratio. Aspartic acid-modified PS of 1a, which possessed intense NIR absorption and high 1O2 quantum yield, demonstrated the most potent efficacy toward the investigated tumor cell lines without using an emulsifier. Subcellular localization, cell-based ROS production, and cell death pathway of 1a were studied. In vivo fluorescence imaging and ex vivo organ distribution assays manifested that 1a possessed reasonable distribution and clearance. In vivo PDT studies indicated that 1a revealed advantages over Ce6 and our previously optimized PS of BDP-4. It not only afforded an excellent PDT effect with a low drug dose under only single-time photoirradiation but also induced an antitumor immunological response.

Development of Chiral Ligands for the Transition-Metal-Catalyzed Enantioselective Silylation and Borylation of C-H Bonds.

Enantioselective reactions that install functional groups at the positions of unactivated C-H bonds can be envisioned to produce intermediates for the synthesis of the active ingredients in pharmaceuticals and agrochemicals directly from simple feedstocks. Among these C-H bond functionalization reactions, those that form carbon-silicon (C-Si) and carbon-boron (C-B) bonds have been pursued because the products of these reactions can be converted to those containing a wide range of functional groups and because compounds containing silicon and boron possess unique properties that can be valuable for medicinal and materials chemistry. Although the silylation and borylation of C-H bonds have undergone extensive development during the past two decades, enantioselective versions of these reactions were not known until a few years ago. In this Minireview, we present the rapid development of enantioselective silylation and borylation of C-H bonds, with an emphasis on the design and development of the types of chiral ligands needed to achieve these reactions and an intention to inspire an expansion of these types of transformations.

Recent Advances in Hydrogen Peroxide Responsive Organoborons for Biological and Biomedical Applications.

Hydrogen peroxide is the most stable reactive oxygen species generated endogenously, participating in numerous physiological processes and abnormal pathological conditions. Mounting evidence suggests that a higher level of H2 O2 exists in various disease conditions. Thus, H2 O2 functions as an ideal target for site-specific bioimaging and therapeutic targeting. The unique reactivity of organoborons with H2 O2 provides a method for developing chemoselective molecules for biological and biomedical applications. This review highlights the design and application of boron-derived molecules for H2 O2 detection, and the utility of boron moieties toward masking reactive compounds leading to the development of metal prochelators and prodrugs for selectively delivering an active species at the target sites with elevated H2 O2 levels. Additionally, the emergence of H2 O2 -responsive theranostic agents consisting of both therapeutic and diagnostic moieties in one integrated system are discussed. The purpose of this review is to provide a better understanding of the role of boron-derived molecules toward biological and pharmacological applications.

Synthesis and Preliminary Biological Assessment of Carborane-Loaded Theranostic Nanoparticles to Target Prostate-Specific Membrane Antigen.

Boron neutron capture therapy (BNCT) is an encouraging therapeutic modality for cancer treatment. Prostate-specific membrane antigen (PSMA) is a cell membrane protein that is abundantly overexpressed in prostate cancer and can be targeted with radioligand therapies to stimulate clinical responses in patients. In principle, a spatially targeted neutron beam together with specifically targeted PSMA ligands could enable prostate cancer-targeted BNCT. Thus, we developed and tested PSMA-targeted poly(lactide-co-glycolide)-block-poly(ethylene glycol) (PLGA-b-PEG) nanoparticles (NPs) loaded with carborane and tethered to the radiometal chelator deferoxamine B (DFB) for simultaneous positron emission tomography (PET) imaging and selective delivery of boron to prostate cancer. Monomeric PLGA-b-PEGs were covalently functionalized with either DFB or the PSMA ligand ACUPA. Different nanoparticle formulations were generated by nanoemulsification of the corresponding unmodified and DFB- or ACUPA-modified monomers in varying percent fractions. The nanoparticles were efficiently labeled with 89Zr and were subjected to in vitro and in vivo evaluation. The optimized DFB(25)ACUPA(75) NPs exhibited strong in vitro binding to PSMA in direct binding and competition radioligand binding assays in PSMA(+) PC3-Pip cells. [89Zr]DFB(25) NPs and [89Zr]DFB(25)ACUPA(75) NPs were injected to mice with bilateral PSMA(-) PC3-Flu and PSMA(+) PC3-Pip dual xenografts. The NPs demonstrated twofold superior accumulation in PC3-Pip tumors to that of PC3-Flu tumors with a tumor/blood ratio of 25; however, no substantial effect of the ACUPA ligands was detected. Moreover, fast release of carborane from the NPs was observed, resulting in a low boron delivery to tumors in vivo. In summary, these data demonstrate the synthesis, characterization, and initial biological assessment of PSMA-targeted, carborane-loaded PLGA-b-PEG nanoparticles and establish the foundation for future efforts to enable their best use in vivo.

Homocystamide Conjugates of Human Serum Albumin as a Platform to Prepare Bimodal Multidrug Delivery Systems for Boron Neutron Capture Therapy.

Boron neutron capture therapy is a unique form of adjuvant cancer therapy for various malignancies including malignant gliomas. The conjugation of boron compounds and human serum albumin (HSA)-a carrier protein with a long plasma half-life-is expected to extend systemic circulation of the boron compounds and increase their accumulation in human glioma cells. We report on the synthesis of fluorophore-labeled homocystamide conjugates of human serum albumin and their use in thiol-'click' chemistry to prepare novel multimodal boronated albumin-based theranostic agents, which could be accumulated in tumor cells. The novelty of this work involves the development of the synthesis methodology of albumin conjugates for the imaging-guided boron neutron capture therapy combination. Herein, we suggest using thenoyltrifluoroacetone as a part of an anticancer theranostic construct: approximately 5.4 molecules of thenoyltrifluoroacetone were bound to each albumin. Along with its beneficial properties as a chemotherapeutic agent, thenoyltrifluoroacetone is a promising magnetic resonance imaging agent. The conjugation of bimodal HSA with undecahydro-closo-dodecaborate only slightly reduced human glioma cell line viability in the absence of irradiation (~30 µM of boronated albumin) but allowed for neutron capture and decreased tumor cell survival under epithermal neutron flux. The simultaneous presence of undecahydro-closo-dodecaborate and labeled amino acid residues (fluorophore dye and fluorine atoms) in the obtained HSA conjugate makes it a promising candidate for the combination imaging-guided boron neutron capture therapy.

Synthesis of Zwitter-Ionic Conjugate of Nido-Carborane with Cholesterol.

9-HC≡CCH2Me2N-nido-7,8-C2B9H11, a previously described carboranyl terminal alkyne, was used for the copper(I)-catalyzed azide-alkyne cycloaddition with azido-3ß-cholesterol to form a novel zwitter-ionic conjugate of nido-carborane with cholesterol, bearing a 1,2,3-triazol fragment. The conjugate of nido-carborane with cholesterol, containing a charge-compensated group in the linker, can be used as a precursor for the preparation of liposomes for BNCT (Boron Neutron Capture Therapy). The solid-state molecular structure of a nido-carborane derivative with the 9-Me2N(CH2)2Me2N-nido-7,8-C2B9H11 terminal dimethylamino group was determined by single-crystal X-ray diffraction.

Cytotoxic and apoptotic effects of 1,2-diborolanes with strong donor substitutes on human cancer cells.

In recent years, boron compounds have become more common as chemotherapy agents against certain types of cancers. Along with the development of boron-based therapeutic agents have come investigations into the various cancers and biochemical and molecular mechanisms affected by boron compounds and the relationships between boron compounds and chemical protection against cancer. In this preliminary study, the effects of new 1,2-N-substituted-1,2-diborolane derivatives on types of breast and liver cancers were examined for the first time. Four were found to significantly affect the cell viabilities and mitochondrial membrane potential changes in MCF-7, HepG2 and Hep3B cancer cells. Each was prepared in n-hexane at various concentrations (5, 10, 25, 50, 75 and 100 µg/mL). Human peripheral blood lymphocytes were used as control cells. Compounds 1, 2, 3a, and 3b 1,2-diborolane derivatives selectively killed cancer cells, but compound 1 was cytotoxic in a concentration-dependent manner on HepG2 and Hep3B and only at concentrations of at least 75 µg/mL on MCF-7 cells. Compound 3a exhibited cytotoxic effect on lymphocytes at 75 and 100 µgmL-1 concentrations, but compounds 1, 2 and 3b, 3c and 3d have not possessed significant cytotoxic effect on lymphocytes. Compounds 3c and 3d have not possessed significant cytotoxic effects. Mitochondrial membrane potential assay results supported these findings. Our results reveal that 1,2-diborolane derivates have high cytotoxic and apoptotic activities on human hepatocarcinoma cells and are therefore potential candidates in the development of new drugs against liver cancer.

Facile synthesis of near-infrared bodipy by donor engineering for in vivo tumor targeted dual-modal imaging.

Bodipy is one of the most popular dyes for bioimaging, however, a complicated synthetic protocol is needed to create and isolate ideal near-infrared (NIR) emissive Bodipy derivatives for optical bioimaging. It is noticed that the donor species impact the wavelength when the π-conjugation system of green light emissive Bodipy is elongated via a one-step reaction. Herein, several Bodipy dyes bearing different common donors are synthesized. Their optical properties confirm that both absorption and emission peaks of the synthesized Bodipy could be tuned to NIR wavelength by using stronger donors via a facile reaction. The synthesized monocarboxyl Bodipy could conjugate with aminated PEG to yield an amphiphilic polymer, which further self-assembles into a NIR nanoparticle (NP). The NIR NP exhibits preferential tumor accumulation via the enhanced permeation and retention (EPR) effect, making it useful for tumor diagnosis by both fluorescence imaging and photoacoustic tomography.

Synthesis and biological evaluation of cationic TopFluor cholesterol analogues.

Cholesterol is not only a major component of the cell membrane, but also plays an important role in a wide range of biological processes and pathologies. It is therefore crucial to develop appropriate tools for visualizing intracellular cholesterol transport. Here, we describe new cationic analogues of BODIPY-Cholesterol (TopFluor-Cholesterol, TF-Chol), which combine a positive charge on the sterol side chain and a BODIPY group connected via a C-4 linker. In contrast to TF-Chol, the new analogues TF-1 and TF-3 possessing acetyl groups on the A ring (C-3 position on steroid) internalized much faster and displayed slightly different levels of intracellular localization. Their applicability for cholesterol monitoring was indicated by the fact that they strongly label compartments with accumulated cholesterol in cells carrying a mutation of the Niemann-Pick disease-associated cholesterol transporter, NPC1.

Nucleophilic radiosynthesis of boron neutron capture therapy-oriented PET probe [18F]FBPA using aryldiboron precursors.

A reliable copper-mediated nucleophilic radiosynthesis of the PET imaging probe [18F]FBPA was developed using novel aryldiboron precursors. The carrier-free [18F]FBPA with radiochemical purity >99% was prepared routinely via the two-step synthesis with an automatic module and can be used for clinical PET imaging of tumours.

A bright, red-emitting water-soluble BODIPY fluorophore as an alternative to the commercial Mito Tracker Red for high-resolution mitochondrial imaging.

With the emergence and rapid development of super-resolution fluorescence microscopy, monitoring of mitochondrial morphological changes has aroused great interest for exploring the role of mitochondria in the process of cell metabolism. However, in the absence of water-soluble, photostable and low-toxicity fluorescent dyes, ultra-high-resolution mitochondrial imaging is still challenging. Herein, we designed two fluorescent BODIPY dyes, namely Mito-BDP 630 and Mito-BDP 760, for mitochondrial imaging. The results proved that Mito-BDP 760 underwent aggregation-caused quenching (ACQ) in the aqueous matrix owing to its hydrophobicity and was inaccessible to the cells, which restricted its applications in mitochondrial imaging. In stark contrast, water-soluble Mito-BDP 630 readily penetrated cellular and mitochondrial membranes for mitochondrial imaging with high dye densities under wash-free conditions as driven by membrane potential. As a comparison, Mito Tracker Red presented high photobleaching (the fluorescence intensity dropped by nearly 50%) and high phototoxicity after irradiation by a laser for 30 min. However, Mito-BDP 630 possessed excellent biocompatibility, photostability and chemical stability. Furthermore, clear and bright mitochondria distribution in living HeLa cells after incubation with Mito-BDP 630 could be observed by CLSM. Convincingly, the morphology and cristae of mitochondria could be visualized using an ultra-high-resolution microscope. In short, Mito-BDP 630 provided a powerful and convenient tool for monitoring mitochondrial morphologies in living cells. Given the facile synthesis, photobleaching resistance and low phototoxicity of Mito-BDP 630, it is an alternative to the commercial Mito Tracker Red.

Development of an Easily Bioconjugatable Water-Soluble Single-Photon Emission-Computed Tomography/Optical Imaging Bimodal Imaging Probe Based on the aza-BODIPY Fluorophore.

A water-soluble fluorescent aza-BODIPY platform (Wazaby) was prepared and functionalized by a polyazamacrocycle agent and a bioconjugable arm. The resulting fluorescent derivative was characterized and bioconjugated onto a trastuzumab monoclonal antibody as a vector. After bioconjugation, the imaging agent appeared to be stable in serum (>72 h at 37 °C) and specifically labeled HER-2-positive breast tumors slices. The bioconjugate was radiolabeled with [111In] indium and studied in vivo. The developed monomolecular multimodal imaging probe (MOMIP) is water-soluble and chemically and photochemically stable, emits in the near infrared (NIR) region (734 nm in aqueous media), and displays a good quantum yield of fluorescence (around 15%). Single-photon emission-computed tomography and fluorescence imaging have been performed in nude mice bearing HER2-overexpressing HCC1954 human breast cancer xenografts and have evidenced the good tumor targeting of the [111In] In bimodal agent. Finally, the proof of concept of using it as a new tool for fluorescence-guided surgery has been shown.

High-Throughput Crystallography Reveals Boron-Containing Inhibitors of a Penicillin-Binding Protein with Di- and Tricovalent Binding Modes.

The effectiveness of ß-lactam antibiotics is increasingly compromised by ß-lactamases. Boron-containing inhibitors are potent serine-ß-lactamase inhibitors, but the interactions of boron-based compounds with the penicillin-binding protein (PBP) ß-lactam targets have not been extensively studied. We used high-throughput X-ray crystallography to explore reactions of a boron-containing fragment set with the Pseudomonas aeruginosa PBP3 (PaPBP3). Multiple crystal structures reveal that boronic acids react with PBPs to give tricovalently linked complexes bonded to Ser294, Ser349, and Lys484 of PaPBP3; benzoxaboroles react with PaPBP3 via reaction with two nucleophilic serines (Ser294 and Ser349) to give dicovalently linked complexes; and vaborbactam reacts to give a monocovalently linked complex. Modifications of the benzoxaborole scaffold resulted in a moderately potent inhibition of PaPBP3, though no antibacterial activity was observed. Overall, the results further evidence the potential for the development of new classes of boron-based antibiotics, which are not compromised by ß-lactamase-driven resistance.

Enantiomerically Enriched α-Borylzinc Reagents by Nickel-Catalyzed Carbozincation of Vinylboronic Esters.

In this paper is described a synthesis of enantiomerically enriched, configurationally stable organozinc reagents by catalytic enantioselective carbozincation of a vinylboronic ester. This process furnishes enantiomerically enriched α-borylzinc intermediates that are shown to undergo stereospecific reactions, producing enantioenriched secondary boronic ester products. The properties of the intermediate α-borylzinc reagent are probed and the synthetic utility of the products is demonstrated by application to the synthesis of (-)-aphanorphine and (-)-enterolactone.

The Versatile Reaction Chemistry of an Alpha-Boryl Diazo Compound.

The first α-boryl diazo compound that is capable of engaging in classic synthetic organic diazo reaction chemistry is described. The diazomethyl-1,2-azaborine 1, which is a BN isostere of phenyldiazomethane, is significantly more stable than phenyldiazomethane; its reaction chemistry ranges from C-H activation, O-H activation, [3+2] cycloaddition, and halogenation, to Ru-catalyzed carbonyl olefination. The demonstrated broad range of reactivity of diazomethyl-1,2-azaborine 1 makes it an exceptionally versatile synthetic building block for the 1,2-azaborine heterocyclic motif.

Probing Variations of Reduction Activity at the Plasma Membrane Using a Targeted Ratiometric FRET Probe.

Plasma membrane (PM) is the turntable of various reactions that regulate essential functionalities of cells. Among these reactions, the thiol disulfide exchange (TDE) reaction plays an important role in cellular processes. We herein designed a selective probe, called membrane reduction probe (MRP), that is able to report TDE activity at the PM. MRP is based on a green emitting BODIPY PM probe connected to rhodamine through a disulfide bond. MRP is fluorogenic as it is turned off in aqueous media due to aggregation-caused quenching, and once inserted in the PM, it displays a bright red signal due to an efficient fluorescence energy resonance transfer (FRET) between the BODIPY donor and the rhodamine acceptor. In the PM model, the MRP can undergo TDE reaction with external reductive agents as well as with thiolated lipids embedded in the bilayer. Upon TDE reaction, the FRET is turned off and a bright green signal appears allowing a ratiometric readout of this reaction. In cells, the MRP quickly labeled the PM and was able to probe variations of TDE activity using ratiometric imaging. With this tool in hand, we were able to monitor variations of TDE activity at the PM under stress conditions, and we showed that cancer cell lines presented a reduced TDE activity at the PM compared to noncancer cells.