Rational design of an HClO-specific triggered self-immolative fluorescent turn-on sensor and its bioimaging applications.

The development of a rapid and intuitive method for the detection of a specific small molecule biomarker is important for understanding the pathogenesis of relevant diseases. Described here is the design and evaluation of an HClO-specific triggered self-immolative fluorescent sensor (RESClO) based on the structure of an N-protected Resorufin dye. Due to the interrupted π-conjugated structure of the Resorufin dye, the free sensor showed very weak absorption and fluorescence. It can quickly complete the response to HClO (within 10 s) with high selectivity and sensitivity (LOD = 16.8 nM) in aqueous solution. The sensor can be made into test strips to quickly detect HClO in the environment by obvious changes in color and fluorescence. It was successfully used for bioimaging of exogenous and endogenous HClO in cells and zebra fish. More importantly, it can also be used for visual imaging of mouse arthritis models. Thus, sensor RESClO can provide a simple and promising visual analytical tool for the detection of HClO in the environment and the early diagnosis of HClO-mediated related diseases.

Discovery of Umibecestat (CNP520): A Potent, Selective, and Efficacious ß-Secretase (BACE1) Inhibitor for the Prevention of Alzheimer's Disease.

After identification of lead compound 6, 5-amino-1,4-oxazine BACE1 inhibitors were optimized in order to improve potency, brain penetration, and metabolic stability. Insertion of a methyl and a trifluoromethyl group at the 6-position of the 5-amino-1,4-oxazine led to 8 (NB-360), an inhibitor with a pKa of 7.1, a very low P-glycoprotein efflux ratio, and excellent pharmacological profile, enabling high central nervous system penetration and exposure. Fur color changes observed with NB-360 in efficacy studies in preclinical animal models triggered further optimization of the series. Herein, we describe the steps leading to the discovery of 3-chloro-5-trifluoromethyl-pyridine-2-carboxylic acid [6-((3R,6R)-5-amino-3,6-dimethyl-6-trifluoromethyl-3,6-dihydro-2H-[1,4]oxazin-3-yl)-5-fluoro-pyridin-2-yl]amide 15 (CNP520, umibecestat), an inhibitor with superior BACE1/BACE2 selectivity and pharmacokinetics. CNP520 reduced significantly Aß levels in mice and rats in acute and chronic treatment regimens without any side effects and thus qualified for Alzheimer's disease prevention studies in the clinic.

Discovery, Optimization, and Structure-Activity Relationship Study of Novel and Potent RSK4 Inhibitors as Promising Agents for the Treatment of Esophageal Squamous Cell Carcinoma.

Ribosomal S6 protein kinase 4 (RSK4) was identified to be a promising target for the treatment of esophageal squamous cell carcinoma (ESCC) in our previous research, whose current treatments are primarily chemotherapy and radiotherapy due to the lack of targeted therapy. However, few potent and specific RSK4 inhibitors are reported. In this study, a series of 1,4-dihydro-2H-pyrimido[4,5-d][1,3]oxazin-2-ones derivatives were designed and synthesized as novel and potent RSK4 inhibitors. Compound 14f was identified with potent RSK4 inhibitory activity both in vitro and in vivo. 14f significantly inhibited the proliferation and invasion of ESCC cells in vitro with IC50 values of 0.57 and 0.98 µM, respectively. It dose dependently inhibited the phosphorylation of RSK4 downstream substrates while exerting little effect on the substrates of RSK1-3 in ESCC cells. The markedly suppressed tumor growth and no observed toxicity to main organs in the ESCC xenograft mouse model suggested 14f to be a promising RSK4-targeting agent for ESCC treatment.

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.

Phenoxazine nucleoside derivatives with a multiple activity against RNA and DNA viruses.

Emerging and re-emerging viruses periodically cause outbreaks and epidemics all over the world, eventually leading to global events such as the current pandemic of the novel SARS-CoV-2 coronavirus infection COVID-19. Therefore, an urgent need for novel antivirals is crystal clear. Here we present the synthesis and evaluation of an antiviral activity of phenoxazine-based nucleoside analogs divided into three groups: (1) 8-alkoxy-substituted, (2) acyclic, and (3) carbocyclic. The antiviral activity was assessed against a structurally and phylogenetically diverse panel of RNA and DNA viruses from 25 species. Four compounds (11a-c, 12c) inhibited 4 DNA/RNA viruses with EC50 ≤ 20 µM. Toxicity of the compounds for the cell lines used for virus cultivation was negligible in most cases. In addition, previously reported and newly synthesized phenoxazine derivatives were evaluated against SARS-CoV-2, and some of them showed promising inhibition of reproduction with EC50 values in low micromolar range, although accompanied by commensurate cytotoxicity.

Discovery of a highly efficient nitroaryl group for detection of nitroreductase and imaging of hypoxic tumor cells.

Hypoxia is a pathological hallmark of solid tumors. Detection of hypoxia is therefore of great interest for tumor diagnosis and treatment. As a well-established biomarker of hypoxia, nitroreductase (NTR) has been widely exploited in the development of hypoxia-responsive fluorescent probes on the basis of its enzymatic activity to reduce nitroaryl groups. However, studies on the relationship between the nitroaryl structure and the probe performance for optimal probe design are still rare. Here we report a comparative investigation of nitroaryl groups and identification of the optimal nitroaryl structure for developing new fluorescent probes with extremely high efficiency in the detection of NTR and the imaging of hypoxic tumor cells. Specifically, we synthesized a series of resorufin-based fluorescent probes containing different nitroaryl groups, compared their fluorescence responses to NTR, and identified 2-nitro-N-methyl-imidazolyl as the optimal nitroaryl group that is much more efficient than the most widely used 4-nitrophenyl for NTR detection. The structure-performance relationship was then studied by theoretical molecular docking, revealing the unique features of 2-nitro-N-methyl-imidazolyl in binding and reaction with NTR. We further incorporated the 2-nitro-N-methyl-imidazolyl group into a near-infrared (NIR) hemicyanine fluorophore and developed a NIR fluorescent probe NFP-7 for the detection of NTR and hypoxic tumor cells. NFP-7 exhibits a strong fluorescence increase toward NTR in vitro with an ultrafast (within 40 seconds to fluorescence maximum) and ultrasensitive (0.2 ng mL-1 detection limit) response. NFP-7 has also been demonstrated for imaging the degree of hypoxia in live tumor cells and, more importantly, in a murine tumor model. Our study provides important insights into hypoxia probe development and new tools for hypoxia imaging.

In-depth characterization of ubiquitin turnover in mammalian cells by fluorescence tracking.

Despite almost 40 years having passed from the initial discovery of ubiquitin (Ub), fundamental questions related to its intracellular metabolism are still enigmatic. Here we utilized fluorescent tracking for monitoring ubiquitin turnover in mammalian cells, resulting in obtaining qualitatively new data. In the present study we report (1) short Ub half-life estimated as 4 h; (2) for a median of six Ub molecules per substrate as a dynamic equilibrium between Ub ligases and deubiquitinated enzymes (DUBs); (3) loss on average of one Ub molecule per four acts of engagement of polyubiquitinated substrate by the proteasome; (4) direct correlation between incorporation of Ub into the distinct type of chains and Ub half-life; and (5) critical influence of the single lysine residue K27 on the stability of the whole Ub molecule. Concluding, our data provide a comprehensive understanding of ubiquitin-proteasome system dynamics on the previously unreachable state of the art.

1,3-Oxazine-2-one derived dual-targeted molecules against replicating and non-replicating forms of Mycobacterium tuberculosis.

The high mortality rate and increasing prevalence of resistant Mtb are the major concerns for the Tuberculosis (TB) treatment in this century. To curtail the prevalence of resistant Mtb, we have prepared 1,3-oxazine-2-one based dual targeted molecules. Compound 67 and 68 were found to be equally active against replicating and non-replicatiing form of Mtb (MICMABA 3.48 and 2.97 µg/ml; MICLORA 2.94 and 2.15 µg/ml respectively). They had found to suppress the biosynthesis of alfa, methoxy and keto-mycolate completely, as well as inhibit enzymatic activity of MenG (IC50 = 9.11 and 6.25 µg/ml respectively for H37Ra; IC50 = 11.76 and 10.88 µg/ml respectively for M smegmatis).

Discovery of Novel Antibiotics as Covalent Inhibitors of Fatty Acid Synthesis.

The steady increase in the prevalence of multidrug-resistant Staphylococcus aureus has made the search for novel antibiotics to combat this clinically important pathogen an urgent matter. In an effort to discover antibacterials with new chemical structures and mechanisms, we performed a growth inhibition screen of a synthetic library against S. aureus and discovered a promising scaffold with a 1,3,5-oxadiazin-2-one core. These compounds are potent against both methicillin-sensitive and methicillin-resistant S. aureus strains. Isolation of compound-resistant strains followed by whole genome sequencing revealed its cellular target as FabH, a key enzyme in bacterial fatty acid synthesis. Detailed mechanism of action studies suggested the compounds inhibit FabH activity by covalently modifying its active site cysteine residue with high selectivity. A crystal structure of FabH protein modified by a selected compound Oxa1 further confirmed covalency and suggested a possible mechanism for reaction. Moreover, the structural snapshot provided an explanation for compound selectivity. On the basis of the structure, we designed and synthesized Oxa1 derivatives and evaluated their antibacterial activity. The structure-activity relationship supports the hypothesis that noncovalent recognition between compounds and FabH is critical for the activity of these covalent inhibitors. We believe further optimization of the current scaffold could lead to an antibacterial with potential to treat drug-resistant bacteria in the clinic.

Scaffolding-Induced Property Modulation of Chemical Space.

Physicochemical property switching of chemical space is of great importance for optimization of compounds, for example, for biological activity. Cyclization is a key method to control 3D and other properties. A two-step approach, which involves a multicomponent reaction followed by cyclization, is reported to achieve the transition from basic moieties to charge neutral cyclic derivatives. A series of multisubstituted oxazolidinones, oxazinanones, and oxazepanones as well as their thio and sulfur derivatives are synthesized from readily available building blocks with mild conditions and high yields. Like a few other methods, MCR and cyclization allow for the collective transformation of a large chemical space into a related one with different properties.

Phenoxazinone synthase-like catalytic activity of novel mono- and tetranuclear copper(ii) complexes with 2-benzylaminoethanol.

Three novel coordination compounds, [Cu(ca)2(Hbae)2] (1), [Cu(va)2(Hbae)2] (2) and [Cu4(va)4(bae)4]·H2O (3), have been prepared by self-assembly reactions of copper(ii) chloride (1 and 2) or tetrafluoroborate (3) and CH3OH (1 and 3) or CH3CN (2) solution of 2-benzylaminoethanol (Hbae) and cinnamic (Hca, 1) or valeric (Hva, 2 and 3) acid. Crystallographic analysis revealed that both 1 and 2 have mononuclear crystal structures, wherein the complex molecules are H-bonded forming extended supramolecular chains. The tetranuclear structure of 3 is based on the {Cu4(µ3-O)4} core, wherein the metal atoms are bound together by µ3 oxygen bridges from 2-benzylaminoethanol forming an overall cubane-like configuration. The strong hydrogen bonding in 1-3 leads to the joining of the neighbouring molecules into 1D chains. Concentration-dependent ESI-MS studies disclosed the equilibria between di-, tri- and tetranuclear species in solutions of 1-3. All three compounds act as catalysts for the aerobic oxidation of o-aminophenol to the phenoxazinone chromophore (phenoxazinone synthase-like activity), with the maximum reaction rates of 4.0 × 10-7, 2.5 × 10-7 and 2.1 × 10-7 M s-1 for 1, 2 and 3, respectively, supported by the quantitative yield of the product after 24 h. The dependence of the reaction rates on catalyst concentrations is evidence of reaction orders higher than one relative to the catalyst. Kinetic and ESI-MS data allowed us to assume that the tetranuclear species, originating from 1, 2 and 3 in solution, possess considerably higher activity than the species of lower nuclearity. Mechanistic and isotopic 18O-labelling experiments suggested that o-aminophenol coordinates to CuII species with the formation of reactive intermediates, while the oxygen from 18O2 is not incorporated into the phenoxazinone chromophore.

Benzo[a]phenoxazinium chlorides: Synthesis, antifungal activity, in silico studies and evaluation as fluorescent probes.

Four new benzo[a]phenoxazinium chlorides with combinations of chloride, ethyl ester and methyl as terminals of the amino substituents were synthesized. These compounds were characterized and their optical properties were studied in absolute dry ethanol and water. Their antiproliferative activity was tested against Saccharomyces cerevisiae in a broth microdilution assay, along with an array of 36 other benzo[a]phenoxazinium chlorides. Minimum Inhibitory Concentration (MIC) values between 1.56 and >200 µM were observed. Fluorescence microscopy studies, used to assess the intracellular distribution of the dyes, showed that these benzo[a]phenoxazinium chlorides function as efficient and site specific probes for the detection of the vacuole membrane. The added advantage of some of the compounds, that displayed the lower MIC values, was the simultaneous staining of both the vacuole membrane and the perinuclear membrane of endoplasmic reticulum (ER). Molecular docking studies were performed on the human membrane protein oxidosqualene cyclase (OSC), using the crystal structure available on PDB (code 1W6K). The results showed that these most active compounds accommodated better in the active sites of ER enzyme OSC suggesting this enzyme as a potential target. As a whole, the results demonstrate that the benzo[a]phenoxazinium chlorides are interesting alternatives to the available commercial dyes. Changes in the substituents of these compounds can tailor both their staining specificity and antimicrobial activity.

A One-pot Synthesis of Novel Derivatives of Oxadiazine-4-thione, and its Antibacterial Activity, and Molecular Modeling Studies.

BACKGROUND: The synthesis of a novel series of oxadiazine-4-thione biological molecules was executed through the incorporation of the ortho-, meta-, and para-benzoyl isocyanates to the tetrabromophthalimide nucleus. OBJECTIVES: A one-pot multicomponent methodology in a solvent-free microwave irradiation environment was employed to afford this series of oxadiazine-4-thione, deriving a comparison with the conventional method. Subsequently, the yielded derivatives were subjected to further biological assessment. MATERIALS AND METHODS: The acquired results denoted that the one-pot procedure, which delivered products in a 2-4 min. interval, was more efficient in evaluation against the classical method, which consumed a 1-2:30 hr. interval. RESULTS: The application of the antibacterial analyses was subjected to all the compounds, resulting in molecules 6a and 6c demonstrating the highest activity regarding Aspergillus Favus; molecules 5b and 5c exhibiting an equivalent level of activity towards E-coli and Fusarium Moniliform; and molecules 4b, 4c, 5b, and 5c presenting an identical level of activity to the aforementioned derivatives involving Staphylococcus. Concluison: Molecular modeling studies by the MOE, the preceding antibacterial behavior was conducted to advocate the newly prepared compounds. Moreover, the spectroscopic approaches were exploited to verify and establish the structures and mechanisms of the synthesized derivatives' reactions.

First synthesis of merged hybrids phosphorylated azirino[2,1-b]benzo[e][1,3]oxazine derivatives as anticancer agents.

This work describes a straightforward diastereoselective synthetic access to azirino[2,1-b]benzo[e][1,3]oxazines containing phosphorus substituents such as phosphonate or phosphine oxide, by means of nucleophilic addition of functionalized phenols to the C-N double bond of 2H-azirine derivatives. In addition, the cytotoxic effect on cell lines derived from human lung adenocarcinoma (A549) and human embryonic kidney (HEK293) was also screened. Some azirino[2,1-b]benzo[e][1,3]oxazines 4 and 6 exhibited very good activity against the A549 cell line in vitro. Furthermore, selectivity towards cancer cell (A549) over (HEK293), and non-malignant cells (MCR-5) has been detected.

Optimisation of novel 4, 8-disubstituted dihydropyrimido[5,4-b][1,4]oxazine derivatives as potent GPR 119 agonists.

GPR119 is a promising target for discovery of anti-type 2 diabetes mellitus agents. We described the optimisation of a novel series of pyrimido[5,4-b][1,4]oxazine derivatives as GPR119 agonists. Most designed compounds exhibited good agonistic activities. Among them, compound 10 and 15 demonstrated the potent EC50 values (13 and 12 nM, respectively) and strong inherent activities. Moreover, significant hypoglycaemic effect of compound 15 was observed by reducing the blood glucose AUC0-2h at the dose of 30 mg/kg, which is stronger than Vildagliptin (23.4% reduction vs. 17.9% reduction).

A simple hypochlorous acid signaling probe based on resorufin carbonodithioate and its biological application.

A novel hypochlorous-acid-selective signaling probe based on the carbonodithioate derivative of resorufin (RT-1) was developed. Probe RT-1 showed prominent colorimetric and turn-on type fluorescence signaling behavior exclusively toward hypochlorous acid, induced by oxidative hydrolysis, to regenerate resorufin dye. Hypochlorous acid signaling was not affected by the presence of common metal ions and anions as background, except for the redox active bromide and iodide anions. The detection limit of RT-1 for hypochlorous acid was found to be 2.18 × 10-9 M (0.11 ppb), and the signaling was completed within 3 min. It was also confirmed that hypochlorous acid signaling by the carbonodithioate-based probe RT-1 was superior to that of the closely related carbonothioate derivative RT-2. This was rationalized by density functional theory calculations, which demonstrated that the C[double bond, length as m-dash]S sulfur atom of the former is more negatively charged than that of the latter. Finally, as a biological application of the probe, visualization of hypochlorous acid in RAW 264.7 murine macrophages and HeLa cells was successfully conducted to detect the cellular response to hypochlorous acid.

Rational Design of Nile Red Analogs for Sensing in Membranes.

Development of next-generation fluorescent probes is a key element in the quest for a greater understanding of complex biological environments (e.g., membranes) by bioimaging. Such fluorescence-based techniques rely on specialized small molecules that possess excellent fluorescent properties but also do not perturb the native biological environment in which they reside. Herein we present a theoretical/computational strategy for the design of novel optical probes for sensing in membranes based on the parent chromophore Nile Red. Using a combination of time-dependent density functional theory (TD-DFT) and molecular dynamics (MD), we have studied the optical properties and accommodation in a model membrane of Nile Red and eight analogs. Special attention has been given to the design of probes with improved solvatochromism and two-photon absorption (2PA) without altering the membrane properties. Of the eight studied analogs, two probes were found to possess attractive probe features and are hence suggested to be taken forward to chemical synthesis and experimental exploration.

Simple Fluorescence Turn-On Chemosensor for Selective Detection of Ba2+ Ion and Its Live Cell Imaging.

A phenoxazine-based fluorescence chemosensor 4PB [(4-(tert-butyl)-N-(4-((4-((5-oxo-5H-benzo[a]phenoxazin-6-yl)amino)phenyl)sulfonyl)phenyl)benzamide)] was designed and synthesized by a simple synthetic methods. The 4PB fluorescence chemosensor selectively detects Ba2+ in the existence of other alkaline metal ions. In addition, 4PB showed high selectivity and sensitivity for Ba2+ detection. The detection limit of 4PB was 0.282 µM and the binding constant was 1.0 × 106 M-1 in CH3CN/H2O (97.5:2.5 v/v, HEPES = 1.25 mM, pH 7.3) medium. This chemosensor functioned through the intramolecular charge transfer (ICT) mechanism, which was further confirmed by DFT studies. Live cell imaging in MCF-7 cells confirmed the cell permeability of 4PB and its capability for specific detection of Ba2+ in living cells.

Design of a Nile red-based NIR fluorescent probe for the detection of hydrogen peroxide in living cells.

In this article, a novel fluorescent probe (NRBE) for detecting H2O2 was developed using benzyl boronic ester as the H2O2-recognized group and Nile red as the matrix. The probe has several advantages, such as good selectivity, high sensitivity (LOD = 75 nM), good water solubility and emission in the near-infrared region (ex/em:585/670 nm). With the NRBE probe, the endogenous H2O2 in human hepatocellular carcinoma cells BEL-7402, was detected, and the H2O2 generated during the ischemia-reperfusion of the cells was imaged. These results show that NRBE can be applied for real-time detection of H2O2 in biological systems.

Synthesis of anti-1,3 Amino Alcohol Motifs via Pd(II)/SOX Catalysis with the Capacity for Stereodivergence.

We report the development of a Pd(II)/(±)-MeO-SOX/2,5-dimethylbenzoquinone system that enables unprecedented access to anti-1,3 amino alcohol motifs in good yields (33 substrates, avg. 66% isolated yield, >20:1 dr) and high selectivities (avg. 10:1 dr). Switching ligands to (±)-CF3-SOX with the use of a less bulky quinone oxidant, the kinetic syn-1,3 amino alcohol motif can be accessed in comparable yields and selectivities. Advantages of the stereodivergent nature of this reaction are seen in the synthesis of anti- and syn-1,3 amino alcohol vitamin D3 analogue intermediates in half the steps and higher overall yield relative to previous routes. Additionally, all eight possible stereoisomers of a chiral diamino alcohol core are generated from two amino acids. Mechanistic studies reveal that the anti-isomer is furnished through concurrent Pd(II)(SOX) catalyzed C-H amination and Pd(0)(SOX) catalyzed isomerization cycles.