Detection of subcellular nitric oxide in mitochondria using a pyrylium probe: assays in cell cultures and peripheral blood.

Fluorescent probes for the detection of intracellular nitric oxide (NO) are abundant, but those targeted to the mitochondria are scarce. Among those molecules targeting mitochondrial NO (mNO), the majority use a triphenylphosphonium (TPP) cation as a vector to reach such organelles. Here we describe a simple molecule (mtNOpy) based on the pyrylium structure, made in a few synthetic steps, capable of detecting selectively NO (aerated medium) over other reactive species. The calculated detection limit for mtNOpy is 88 nM. The main novelty of this probe is that it has a simple molecular architecture and can act both as a fluorogenic and as a mitochondriotropic agent, without using TPP. mtNOpy has been tested in two different scenarios: (a) in a controlled environment of cell line cultures (human colon carcinoma HT-29 cells and mouse macrophage RAW 264.7 cells), using confocal laser scanning microscopy, and (b) on a much more complex sample of peripheral blood, using flow cytometry. In the first context, mtNOpy has been found to be responsive (turn-on fluorescence) to exogenous and endogenous NO stimuli (via SNAP donor and LPS stimulation, respectively). In the second area, mtNOpy has been able to discriminate between NO-generating phagocytes (neutrophils and monocytes) from other leukocytes (NK, B and T cells).

De novo Assembly of the Benzenoid Ring as a Core Strategy for Synthesis of the Isoindolinone Natural Products Isohericerin, Erinacerin A, and Sterenin A.

Here we describe the use of the hexadehydro-Diels-Alder (HDDA) reaction for the de novo construction of the isoindolinone scaffold and its application to the synthesis of the title natural products. The key isoindolinone-forming HDDA reaction involved an unprecedented substrate motif in which an amide carbonyl group was conjugated to the 4π 1,3-diyne component. In addition, a dimethylsilyl (-SiMe2H) substituent was exploited to trigger a Fleming-Tamao-Kumada oxidation for the installation of an essential phenolic hydroxyl group.

Synthesis of the Proposed Structures of Parvistemoamide and Their Transformations to Stemoamide Derivatives.

The proposed structures of parvistemoamide have been achieved by macrolactamization, but none of the characterization data of synthetic samples matched with those of the natural sample. The transformation of the highly strained 10-membered lactam ring in parvistemoamide into the pyrrolo[1,2-a]-azepine nucleus in stemoamide is accomplished for the first time by either transannular cyclization or Pilli's transformation. This research may promote the total synthesis of other more complex stemoamide-type or medium-sized-ring-containing Stemona alkaloids.

[11C]CHDI-626, a PET Tracer Candidate for Imaging Mutant Huntingtin Aggregates with Reduced Binding to AD Pathological Proteins.

The expanded polyglutamine-containing mutant huntingtin (mHTT) protein is implicated in neuronal degeneration of medium spiny neurons in Huntington's disease (HD) for which multiple therapeutic approaches are currently being evaluated to eliminate or reduce mHTT. Development of effective and orthogonal biomarkers will ensure accurate assessment of the safety and efficacy of pharmacologic interventions. We have identified and optimized a class of ligands that bind to oligomerized/aggregated mHTT, which is a hallmark in the HD postmortem brain. These ligands are potentially useful imaging biomarkers for HD therapeutic development in both preclinical and clinical settings. We describe here the optimization of the benzo[4,5]imidazo[1,2-a]pyrimidine series that show selective binding to mHTT aggregates over Aß- and/or tau-aggregates associated with Alzheimer's disease pathology. Compound [11C]-2 was selected as a clinical candidate based on its high free fraction in the brain, specific binding in the HD mouse model, and rapid brain uptake/washout in nonhuman primate positron emission tomography imaging studies.

Total Synthesis, Discovery and Biological Evaluation of Daldinin A Derivatives for Improving Hyperglycemia-Induced Endothelial Cell Death.

Daldinins are a novel type of naturally occurring tricyclic heterocycles isolated from Daldinia concentrica. In this study, four daldinin A derivatives with different alkyl side chains were synthesized using the same synthetic protocol. Bioactivity tests first indicated that the daldinin A derivatives showed significant protection for endothelial cells against damage caused by high glucose. The derivative compound with three carbon atoms on the alkyl side exhibited the best effect.

Synthesis of Catechol Derived Rosamine Dyes and Their Reactivity toward Biogenic Amines.

Functional organic dyes play a key role in many fields, namely in biotechnology and medical diagnosis. Herein, we report two novel 2,3- and 3,4-dihydroxyphenyl substituted rosamines (3 and 4, respectively) that were successfully synthesized through a microwave-assisted protocol. The best reaction yields were obtained for rosamine 4, which also showed the most interesting photophysical properties, specially toward biogenic amines (BAs). Several amines including n- and t-butylamine, cadaverine, and putrescine cause spectral changes of 4, in UV-Vis and fluorescence spectra, which are indicative of their potential application as an effective tool to detect amines in acetonitrile solutions. In the gas phase, the probe response is more expressive for spermine and putrescine. Additionally, we found that methanolic solutions of rosamine 4 and n-butylamine undergo a pink to yellow color change over time, which has been attributed to the formation of a new compound. The latter was isolated and identified as 5 (9-aminopyronin), whose solutions exhibit a remarkable increase in fluorescence intensity together with a shift toward more energetic wavelengths. Other 9-aminopyronins 6a, 6b, 7a, and 7b were obtained from methanolic solutions of 4 with putrescine and cadaverine, demonstrating the potential of this new xanthene entity to react with primary amines.

Aptamer-PROTAC Conjugates (APCs) for Tumor-Specific Targeting in Breast Cancer.

Development of proteolysis targeting chimeras (PROTACs) is emerging as a promising strategy for targeted protein degradation. However, the drug development using the heterobifunctional PROTAC molecules is generally limited by poor membrane permeability, low in vivo efficacy and indiscriminate distribution. Herein an aptamer-PROTAC conjugation approach was developed as a novel strategy to improve the tumor-specific targeting ability and in vivo antitumor potency of conventional PROTACs. As proof of concept, the first aptamer-PROTAC conjugate (APC) was designed by conjugating a BET-targeting PROTAC to the nucleic acid aptamer AS1411 (AS) via a cleavable linker. Compared with the unmodified BET PROTAC, the designed molecule (APR) showed improved tumor targeting ability in a MCF-7 xenograft model, leading to enhanced in vivo BET degradation and antitumor potency and decreased toxicity. Thus, the APC strategy may pave the way for the design of tumor-specific targeting PROTACs and have broad applications in the development of PROTAC-based drugs.

Palladium-Catalyzed Annulations of Strained Cyclic Allenes.

We report Pd-catalyzed annulations of in situ generated strained cyclic allenes. This methodology employs aryl halides and cyclic allene precursors as the reaction partners in order to generate fused heterocyclic products. The annulation proceeds via the formation of two new bonds and an sp3 center. Moreover, both diastereo- and enantioselective variants of this methodology are validated, with the latter ultimately enabling the rapid enantioselective synthesis of a complex hexacyclic product. Studies leveraging transition metal catalysis to intercept cyclic allenes represent a departure from the more common, historical modes of cyclic allene trapping that rely on nucleophiles or cycloaddition partners. As such, this study is expected to fuel the development of reactions that strategically merge transition metal catalysis and transient strained intermediate chemistry for the synthesis of complex scaffolds.

Preparation of the Key Dolutegravir Intermediate via MgBr2-Promoted Cyclization.

A novel approach for synthesizing the key dolutegravir intermediate is described via MgBr2-promoted intramolecular cyclization. Condensation of commercially available methyl oxalyl chloride and ethyl 3-(N,N-dimethylamino)acrylate afforded the vinylogous amide in an excellent yield. Subsequent substitution by aminoacetaldehyde dimethyl acetal and methyl bromoacetate gave rise to the expected precursor for cyclization, which was promoted by MgBr2 to highly selectively convert into pyridinone diester. The key dolutegravir intermediate was finally prepared by the selective hydrolysis of the corresponding diester via LiOH.

Acceleration of 1,3-Dipolar Cycloadditions by Integration of Strain and Electronic Tuning.

The 1,3-dipolar cycloaddition between azides and alkynes provides new means to probe and control biological processes. A major challenge is to achieve high reaction rates with stable reagents. The optimization of alkynyl reagents has relied on two strategies: increasing strain and tuning electronics. We report on the integration of these strategies. A computational analysis suggested that a CH → N aryl substitution in dibenzocyclooctyne (DIBO) could be beneficial. In transition states, the nitrogen of 2-azabenzo-benzocyclooctyne (ABC) engages in an n→π* interaction with the C=O of α-azidoacetamides and forms a hydrogen bond with the N-H of α-diazoacetamides. These dipole-specific interactions act cooperatively with electronic activation of the strained π-bond to increase reactivity. We found that ABC does indeed react more quickly with α-azidoacetamides and α-diazoacetamides than its constitutional isomer, dibenzoazacyclooctyne (DIBAC). ABC and DIBAC have comparable chemical stability in a biomimetic solution. Both ABC and DIBO are accessible in three steps by the alkylidene carbene-mediated ring expansion of commercial cycloheptanones. Our findings enhance the accessibility and utility of 1,3-dipolar cycloadditions and encourage further innovation.

New Approaches for the Synthesis of Heterocyclic Compounds Derived from Cyclohexan-1,3-dione with Anti-proliferative Activities.

In the present work a series of heterocyclization reactions were adopted using cyclohexan-1,3-dione through its reaction with either furan-2-carbaldehyde or thiophene-2-carbaldehyde to give the corresponding ylidene derivatives 3a,b. The latter compounds underwent heterocyclization reactions to give thiophene and pyran derivatives 5a-d and 6a-d, respectively. Moreover, compounds 3a,b reacted with elemental sulfur and phenyl isothiocyanate to give the fused thiazole derivatives 8a,b. In addition, the reaction with either of hydrazine hydrate or phenylhydrazine has given the 4-hydrazono-4,5,6,7-tetrahydro-2H-indazole derivatives 10a-d, respectively. Similarly, the reaction of either 3a or 3b with hydroxylamine hydrochloride gave the 6,7-dihydrobenzo[c]isoxazol-4(5H)-one oxime derivatives 12a and 12b, respectively. Other fused heterocyclic compounds were produced and their structures were elucidated. Evaluation of the synthesized compounds against selected cancer cell lines was performed. The most active compounds were further evaluated against tyrosine kinases and Pim-1 kinase inhibitions.

Synthesis of Simple Strigolactone Mimics.

Strigolactones (SLs) are natural compounds occurring in plants which have a numerous functions in plant development; therefore, they are plant hormones. Unfortunately, their natural abundance is very low and because of their structure complexity it is difficult to prepare them in big quantities; alternatives with simpler structures and similar biological activity was developed. SLs mimics are compounds with simple synthesis. Methods for preparation of basic SLs mimics are described here.

Synthesis of Analogs of Strigolactones and Evaluation of Their Stability in Solution.

Strigolactones (SLs) are new plant hormones that play an important role in the control development of plants. They are germination stimulants for seed of parasitic weeds, are the branching factor of arbuscular mycorrhizal fungi and inhibitors for bud outgrowth and shoot branching. Natural SLs contain an annulated system of three rings (ABC scaffold) connected to a furanone (the D-ring) by an enol ether unit. The natural distribution of strigolactones is low, and their synthesis is long and difficult. Therefore, SL analogs are designed to have the same bioactiphore as natural SLs and an appreciable bioactivity. For the design a model is used based on the natural bioactiphore. Typical SL analogs are GR24, Nijmegen-1, and EM1 (derived from ethyl 2-phenylacetate). The synthesis of these SL analogs is reported together with their stability in aqueous solution.

Evaluation of the Effect of Strigolactones and Synthetic Analogs on Fungi.

Strigolactones (SLs) are components of root exudates as a consequence of active release from the roots into the soil. Notably, they have been described as stimulants of seed germination in parasitic plants and of the presymbiotic growth in arbuscular mycorrhizal (AM) fungi, which are a crucial component of the plant root beneficial microbiota. SLs have therefore the potential to influence other microbes that proliferate in the soil around the roots and may interact with plants. A direct effect of SL analogs on the in vitro growth of a number of saprotrophic or plant pathogenic fungi was indeed reported.Here we describe a standardized method to evaluate the effect of SLs or their synthetic analogs on AM and filamentous fungi. For AM fungi, we propose a spore germination assay since it is more straightforward than the hyphal branching assay and it does not require deep expertise and skills. For filamentous fungi that can grow in axenic cultures, we describe the assay based on SLs embedded in the solid medium or dissolved in liquid cultures where the fungus is inoculated to evaluate the effect on growth, hyphal branching or conidia germination. These assays are of help to test the activity of natural SLs as well as of newly designed SL analogs for basic and applied research.

Analyzing the Effect of Strigolactones on the Motility Behavior of Rhizobia.

In the Rhizobium-legume symbiosis, strigolactones (SLs) promote root nodule formation; however, the exact mechanism underlying this positive effect remains unknown. The recent finding that an SL receptor legume mutant shows a wild-type nodulation phenotype suggests that SLs influence the symbiosis by acting on the bacterial partner. In agreement with this, the application of the synthetic SL analog GR24 on the alfalfa symbiont Sinorhizobium (Ensifer) meliloti has been shown to stimulate swarming, a specialized bacterial surface motility, which could influence infection of legumes by Rhizobia. Surface motility assays for many bacteria, and particularly for Rhizobia, are challenging. The establishment of protocols to study bacterial surface motility is key to decipher the role of SLs as rhizosphere cues for rhizobacteria. In this chapter, we describe a set of protocols implemented to study the different types of motility exhibited by S. meliloti.

Antibody-Mediated Delivery of Chimeric BRD4 Degraders. Part 1: Exploration of Antibody Linker, Payload Loading, and Payload Molecular Properties.

The biological and medicinal impacts of proteolysis-targeting chimeras (PROTACs) and related chimeric molecules that effect intracellular degradation of target proteins via ubiquitin ligase-mediated ubiquitination continue to grow. However, these chimeric entities are relatively large compounds that often possess molecular characteristics, which may compromise oral bioavailability, solubility, and/or in vivo pharmacokinetic properties. We therefore explored the conjugation of such molecules to monoclonal antibodies using technologies originally developed for cytotoxic payloads so as to provide alternate delivery options for these novel agents. In this report, we describe the first phase of our systematic development of antibody-drug conjugates (ADCs) derived from bromodomain-containing protein 4 (BRD4)-targeting chimeric degrader entities. We demonstrate the antigen-dependent delivery of the degrader payloads to PC3-S1 prostate cancer cells along with related impacts on MYC transcription and intracellular BRD4 levels. These experiments culminate with the identification of one degrader conjugate, which exhibits antigen-dependent antiproliferation effects in LNCaP prostate cancer cells.

Antibody-Mediated Delivery of Chimeric BRD4 Degraders. Part 2: Improvement of In Vitro Antiproliferation Activity and In Vivo Antitumor Efficacy.

Heterobifunctional compounds that direct the ubiquitination of intracellular proteins in a targeted manner via co-opted ubiquitin ligases have enormous potential to transform the field of medicinal chemistry. These chimeric molecules, often termed proteolysis-targeting chimeras (PROTACs) in the chemical literature, enable the controlled degradation of specific proteins via their direction to the cellular proteasome. In this report, we describe the second phase of our research focused on exploring antibody-drug conjugates (ADCs), which incorporate BRD4-targeting chimeric degrader entities. We employ a new BRD4-binding fragment in the construction of the chimeric ADC payloads that is significantly more potent than the corresponding entity utilized in our initial studies. The resulting BRD4-degrader antibody conjugates exhibit potent and antigen-dependent BRD4 degradation and antiproliferation activities in cell-based experiments. Multiple ADCs bearing chimeric BRD4-degrader payloads also exhibit strong, antigen-dependent antitumor efficacy in mouse xenograft assessments that employ several different tumor models.

Receptor-Ligand Kinetics Influence the Mechanism of Action of Covalently Linked TLR Ligands.

We report a mechanistic study comparing the immune activation of conjugated Toll-like receptor (TLR) agonists and their unlinked mixtures. Herein, we synthesized a set of six linked dual agonists with different ligands, molecular structures, receptor locations, and biophysical characteristics. With these dimers, we ran a series of in vitro cell-based assays, comparing initial and overall NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) activation, cytokine expression profiles, as well as time-resolved TNF-α (Tumor Necrosis Factor alpha) expression. We show that initial activation kinetics, ligand specificity, and the dose of the agonist influence the activity of these linked TLR systems. These results can help improve vaccine design by showing how linked TLR agonists can improve their potency with the appropriate selection of key criteria.

Semisynthesis and biological evaluation of a focused library of unguinol derivatives as next-generation antibiotics.

In this study, we report the semisynthesis and in vitro biological evaluation of thirty-four derivatives of the fungal depsidone antibiotic, unguinol. Initially, the semisynthetic modifications were focused on the two free hydroxy groups (3-OH and 8-OH), the three free aromatic positions (C-2, C-4 and C-7), the butenyl side chain and the depsidone ester linkage. Fifteen first-generation unguinol analogues were synthesised and screened against a panel of bacteria, fungi and mammalian cells to formulate a basic structure activity relationship (SAR) for the unguinol pharmacophore. Based on the SAR studies, we synthesised a further nineteen second-generation analogues, specifically aimed at improving the antibacterial potency of the pharmacophore. In vitro antibacterial activity testing of these compounds revealed that 3-O-(2-fluorobenzyl)unguinol and 3-O-(2,4-difluorobenzyl)unguinol showed potent activity against both methicillin-susceptible and methicillin-resistant Staphylococcus aureus (MIC 0.25-1 µg mL-1) and are promising candidates for further development in vivo.

Lessons from Natural Product Total Synthesis: Macrocyclization and Postcyclization Strategies.

Macrocyclic natural products are plentiful in the bacteria, archaea, and eukaryote domains of life. For the significant advantages that they provide to the producing organisms, evolution has learned how to implement various types of macrocyclization reactions into the different biosynthetic pathways and how to effect them with remarkable ease. Mankind greatly benefits from nature's pool, not least because naturally occurring macrocycles or derivatives thereof serve as important drugs for the treatment of many serious ailments.In stark contrast, macrocyclization reactions are usually perceived as difficult to accomplish by purely chemical means. While it is true that ring closure necessarily entails an entropic loss and may result in the buildup of (considerable) ring strain that must be compensated for in one way or the other, it is also fair to note tremendous methodological advances during the last decades that greatly alleviated this traditional "macrocycle challenge". It is therefore increasingly possible to explore the advantages provided by large as well as medium-size ring systems in a more systematic manner. This venture also holds the promise of increasing the "chemical space" amenable to drug development to a considerable extent.In consideration of this and other important long-term perspectives, it is appropriate to revisit the current state of the art. To this end, a number of vignettes are presented, each of which summarizes a total synthesis project targeting macrocyclic natural products of greatly different chemotypes using a variety of transformations to reach these goals. Although we were occasionally facing "dead ends", which are also delineated for the sake of a complete picture, these case studies illustrate the notion that the formation of a certain macrocyclic perimeter is (usually) no longer seriously limiting. In addition to substantial progress in the "classical" repertoire (macrolactonization and macrolactamization (pateamine A, spirastrellolide, and belizentrin)), various metal-catalyzed reactions have arguably led to the greatest leaps forward. Among them, palladium-catalyzed C-C bond formation (roseophilin and nominal xestocyclamine A) and, in particular, alkene and alkyne metathesis stand out (iejimalide, spirastrellolide, enigmazole, ingenamine, and sinulariadiolide). In some cases, different methods were pursued in parallel, thus allowing for a critical assessment and comparison.To the extent that the macrocyclic challenge is vanishing, the opportunity arises to focus attention on the postmacrocyclization phase. One may stipulate that a well-designed cyclization precursor does not only ensure efficient ring closure but also fosters and streamlines the steps that come after the event. One way to do so is dual (multiple) use in that the functional groups serving the actual cyclization reaction also find productive applications downstream from it rather than being subject to simple defunctionalization. In this context, better insight into the conformational peculiarities of large rings and the growing confidence in their accessibility in a stereochemically well defined format rejuvenate the implementation of transannular reactions or reaction cascades that can lead to rapid and substantial increases in molecular complexity. The examples summarized herein showcase such possibilities, with special emphasis on tranannular gold catalysis and the emerging ruthenium-catalyzed trans-hydrometalation chemistry for the selective functionalization of alkynes.