Perfluorooxosulfate Salts as SOF4-Gas-Free Precursors to Multidimensional SuFEx Electrophiles.

Sulfur(VI) Fluoride Exchange (SuFEx) chemistry stands as a well-established method for swiftly constructing complex molecules in a modular fashion. An especially promising segment of this toolbox is reserved for multidimensional SuFEx hubs: three or more substituents pluggable into a singular SVI centre to make 'beyond-linear' clicked constructions. Sulfurimidoyl difluorides (RNSOF2) stand out as the prime example of this, however their preparation from the scarcely available thionyl tetrafluoride (SOF4) limits this chemistry to only a few laboratories with access to this gas. In this work, we identify silver pentafluorooxosulfate (AgOSF5) as a viable SuFEx hub with reactivity equal to SOF4. The AgF2-mediated oxidation of SOCl2 gives rise to the hexacoordinate AgOSF5 adduct, which in contact with primary amines produces the sulfurimidoyl fluorides in high yields. In addition, we have found this workflow to be fully extendable to the trifluoromethyl homologue, AgOSF4CF3, and we propose the use of AgOSF4X salts as a general route to azasulfur SuFEx electrophiles from commercial starting materials.

An Extrusion Strategy for On-Demand SF5Cl Gas Generation from a Commercial Disulfide.

Herein we report a novel methodology for the ex situ generation of SF5Cl by employing 4,4'-dipyridyl disulfide as a safe commercial reagent, obviating the need for lecture bottles. The method is applicable to certain SF5Cl-involving transformations by using a two-chamber reactor. Moreover, easily applying SF5Cl in different solvents is rendered feasible, while avoiding the use of glovebox techniques. This report also suggests 1H-19F HOESY as a simple and fast stereochemistry indication for chloropentafluorosulfanylated olefins.

Polyketide Synthase-Mediated O-Methyloxime Formation in the Biosynthesis of the Oximidine Anticancer Agents.

Bacterial trans-acyltransferase polyketide synthases (trans-AT PKSs) are modular megaenzymes that employ unusual catalytic domains to assemble diverse bioactive natural products. One such PKS is responsible for the biosynthesis of the oximidine anticancer agents, oxime-substituted benzolactone enamides that inhibit vacuolar H+ -ATPases. Here, we describe the identification of the oximidine gene cluster in Pseudomonas baetica and the characterization of four novel oximidine variants, including a structurally simpler intermediate that retains potent anticancer activity. Using a combination of in vivo, in vitro and computational approaches, we experimentally elucidate the oximidine biosynthetic pathway and reveal an unprecedented mechanism for O-methyloxime formation. We show that this process involves a specialized monooxygenase and methyltransferase domain and provide insight into their activity, mechanism and specificity. Our findings expand the catalytic capabilities of trans-AT PKSs and identify potential strategies for the production of novel oximidine analogues.

De-Risking S-F Bond Formation: A Gas Cylinder-Free Strategy to Access S(IV) and S(VI) Fluorinated Compounds.

The sulfur-fluorine partnership occupies a privileged position in fluorine chemistry given the functional versatility that it imparts to organic structures. Despite this, available methodologies to forge S-F bonds are limited compared to C-F bond formation. Here, we describe a synthetic protocol that selectively enables the oxidative halogenation of aliphatic, aromatic, and heteroaromatic thiols to their corresponding SF4 Cl, SO2 F and SF3 derivatives. Selective oxidation of thiols to either S(IV)-F or S(VI)-F compounds is achieved by employing bench-stable calcium hypochlorite as chlorine surrogate (CLOgen), in the presence of KF as fluoride source. Density functional theory (DFT) calculations provided insight into the mechanistic aspects of the transformation and rationalized the observed isomeric preference towards the SF4 Cl derivatives. Ultimately, this glovebox-free method selectively dispatches three classes of compounds upon reaction condition fine-tuning. Furthermore, first-in-class transformations are reported, including the preparation of aliphatic SF4 Cl intermediates, their transformation into aliphatic sulfur pentafluoride analogs, and post-functionalizations that allow accessing highly complex SF4 -bridged scaffolds.

Cryogenic contrast-enhanced microCT enables nondestructive 3D quantitative histopathology of soft biological tissues.

Biological tissues comprise a spatially complex structure, composition and organization at the microscale, named the microstructure. Given the close structure-function relationships in tissues, structural characterization is essential to fully understand the functioning of healthy and pathological tissues, as well as the impact of possible treatments. Here, we present a nondestructive imaging approach to perform quantitative 3D histo(patho)logy of biological tissues, termed Cryogenic Contrast-Enhanced MicroCT (cryo-CECT). By combining sample staining, using an X-ray contrast-enhancing staining agent, with freezing the sample at the optimal freezing rate, cryo-CECT enables 3D visualization and structural analysis of individual tissue constituents, such as muscle and collagen fibers. We applied cryo-CECT on murine hearts subjected to pressure overload following transverse aortic constriction surgery. Cryo-CECT allowed to analyze, in an unprecedented manner, the orientation and diameter of the individual muscle fibers in the entire heart, as well as the 3D localization of fibrotic regions within the myocardial layers. We foresee further applications of cryo-CECT in the optimization of tissue/food preservation and donor banking, showing that cryo-CECT also has clinical and industrial potential.

From the North Sea to Drug Repurposing, the Antiseizure Activity of Halimide and Plinabulin.

PharmaSea performed large-scale in vivo screening of marine natural product (MNP) extracts, using zebrafish embryos and larvae, to identify compounds with the potential to treat epilepsy. In this study, we report the discovery of two new antiseizure compounds, the 2,5-diketopiperazine halimide and its semi-synthetic analogue, plinabulin. Interestingly, these are both known microtubule destabilizing agents, and plinabulin could have the potential for drug repurposing, as it is already in clinical trials for the prevention of chemotherapy-induced neutropenia and treatment of non-small cell lung cancer. Both halimide and plinabulin were found to have antiseizure activity in the larval zebrafish pentylenetetrazole (PTZ) seizure model via automated locomotor analysis and non-invasive local field potential recordings. The efficacy of plinabulin was further characterized in animal models of drug-resistant seizures, i.e., the larval zebrafish ethyl ketopentenoate (EKP) seizure model and the mouse 6 Hz psychomotor seizure model. Plinabulin was observed to be highly effective against EKP-induced seizures, on the behavioral and electrophysiological level, and showed activity in the mouse model. These data suggest that plinabulin could be of interest for the treatment of drug-resistant seizures. Finally, the investigation of two functional analogues, colchicine and indibulin, which were observed to be inactive against EKP-induced seizures, suggests that microtubule depolymerization does not underpin plinabulin's antiseizure action.

Solvent-free N-Boc deprotection by ex situ generation of hydrogen chloride gas.

An efficient, scalable and sustainable method for the quantitative deprotection of the tert-butyl carbamate (N-Boc) protecting group is described, using down to near-stoichiometric amounts of hydrogen chloride gas in solvent-free conditions. We demonstrate the ex situ generation of hydrogen chloride gas from sodium chloride and sulfuric acid in a two-chamber reactor, introducing a straightforward method for controlled and stoichiometric release of HCl gas. The solvent-free conditions allow deprotection of a wide variety of N-Boc derivatives to obtain the hydrochloride salts in quantitative yields. The procedure obviates the need for any work-up or purification steps providing an uncomplicated green alternative to standard methods. Due to the solvent-free, anhydrous conditions, this method shows high tolerance towards acid sensitive functional groups and furnishes expanded functional group orthogonality.

Developments in the Discovery and Design of Protein Kinase D Inhibitors.

Protein kinase D (PKD) is a serine/threonine kinase family belonging to the Ca2+/calmodulin-dependent protein kinase group. Since its discovery two decades ago, many efforts have been put in elucidating PKD's structure, cellular role and functioning. The PKD family consists of three highly homologous isoforms: PKD1, PKD2 and PKD3. Accumulating cell-signaling research has evidenced that dysregulated PKD plays a crucial role in the pathogenesis of cardiac hypertrophy and several cancer types. These findings led to a broad interest in the design of small-molecule protein kinase D inhibitors. In this review, we present an extensive overview on the past and recent advances in the discovery and development of PKD inhibitors. The focus extends from broad-spectrum kinase inhibitors used in PKD signaling experiments to intentionally developed, bioactive PKD inhibitors. Finally, attention is paid to PKD inhibitors that have been identified as an off-target through large kinome screening panels.

The presence of benzophenone in sunscreens and cosmetics containing the organic UV filter octocrylene: A laboratory study.

BACKGROUND: The reason why patients photosensitized to the drug ketoprofen (KP) may develop severe photoallergic skin reactions to octocrylene (OCT), an organic ultraviolet filter in sunscreens and cosmetics, remains largely unknown. OCT can be synthesized by using unsubstituted benzophenone (BP), a possible human carcinogen. OBJECTIVES: To verify if, and to what extent, BP residues are present in OCT-containing consumer products. METHODS: The raw material of OCT and 39 skincare products, of which 28 contain OCT, were chemically analysed for the presence of BP by means of liquid chromatography. RESULTS: In the OCT raw material and in all 28 OCT-containing products the presence of BP could be demonstrated, mostly in concentrations above 10 ppm (0.001%), whereas a majority of OCT-free products (8/11, 73%) did not contain BP. Moreover, BP concentrations significantly increased, in a time- and temperature-dependent manner, likely due to the additional degradation of OCT. CONCLUSIONS: Photoallergic contact dermatitis from OCT in patients photosensitized to KP might rely on residual BP impurities. Toxicological and ecological studies that evaluate the safety of OCT might also need to consider the concomitant presence of BP.

Design, synthesis and biological evaluation of pyrazolo[3,4-d]pyrimidine-based protein kinase D inhibitors.

The multiple roles of protein kinase D (PKD) in various cancer hallmarks have been repeatedly reported. Therefore, the search for novel PKD inhibitors and their evaluation as antitumor agents has gained considerable attention. In this work, novel pyrazolo[3,4-d]pyrimidine based pan-PKD inhibitors with structural variety at position 1 were synthesized and evaluated for biological activity. Starting from 3-IN-PP1, a known PKD inhibitor with IC50 values in the range of 94-108 nM, compound 17m was identified with an improved biochemical inhibitory activity against PKD (IC50 = 17-35 nM). Subsequent cellular assays demonstrated that 3-IN-PP1 and 17m inhibited PKD-dependent cortactin phosphorylation. Furthermore, 3-IN-PP1 displayed potent anti-proliferative activity against PANC-1 cells. Finally, a screening against different cancer cell lines demonstrated that 3-IN-PP1 is a potent and versatile antitumoral agent.

Acylated sulfonamide adenosines as potent inhibitors of the adenylate-forming enzyme superfamily.

The superfamily of adenylate-forming enzymes all share a common chemistry. They activate a carboxylate group, on a specific substrate, by catalyzing the formation of a high energy mixed phosphoanhydride-linked nucleoside intermediate. Members of this diverse enzymatic family play key roles in a variety of metabolic pathways and therefore many have been regarded as drug targets. A generic approach to inhibit such enzymes is the use of non-hydrolysable sulfur-based bioisosteres of the adenylate intermediate. Here we compare the activity of compounds containing a sulfamoyl and sulfonamide linker respectively. An improved synthetic strategy was developed to generate inhibitors containing the latter that target isoleucyl- (IleRS) and seryl-tRNA synthetase (SerRS), two structurally distinct representatives of Class I and II aminoacyl-tRNA synthetases (aaRSs). These enzymes attach their respective amino acid to its cognate tRNA and are indispensable for protein translation. Evaluation of the ability of the two similar isosteres to inhibit serRS revealed a remarkable difference, with an almost complete loss of activity for seryl-sulfonamide 15 (SerSoHA) compared to its sulfamoyl analogue (SerSA), while inhibition of IleRS was unaffected. To explain these observations, we have determined a 2.1 Šcrystal structure of Klebsiella pneumoniae SerRS in complex with SerSA. Using this structure as a template, modelling of 15 in the active site predicts an unfavourable eclipsed conformation. We extended the same modelling strategy to representative members of the whole adenylate-forming enzyme superfamily, and were able to disclose a new classification system for adenylating enzymes, based on their protein fold. The results suggest that, other than for the structural and functional orthologues of the Class II aaRSs, the O to C substitution within the sulfur-sugar link should generally preserve the inhibitory potency.

Ketone Synthesis by a Nickel-Catalyzed Dehydrogenative Cross-Coupling of Primary Alcohols.

An intermolecular coupling of primary alcohols and organotriflates has been developed to provide ketones by the action of a Ni(0) catalyst. This oxidative transformation is proposed to occur by the union of three distinct catalytic cycles. Two competitive oxidation processes generate aldehyde in situ via hydrogen transfer oxidation or (pseudo)dehalogenation pathways. As aldehyde forms, a Ni-catalyzed carbonyl-Heck process enables formation of the key carbon-carbon bond. The utility of this rare alcohol to ketone transformation is demonstrated through the synthesis of diverse complex and bioactive molecules.

Identification of fukinolic acid from Cimicifuga heracleifolia and its derivatives as novel antiviral compounds against enterovirus A71 infection.

Human enterovirus 71 (EV-A71) infections cause a wide array of diseases ranging from diarrhoea and rashes to hand-foot-and-mouth disease and, in rare cases, severe neurological disorders. No specific antiviral drug therapy is currently available. Extracts from 75 Chinese medicinal plants selected for antiviral activity based on the Chinese pharmacopeia and advice from traditional Chinese medicine clinicians were tested for activity against EV-A71. The aqueous extract of the rhizome of Cimicifuga heracleifolia (Sheng Ma) and Arnebia euchroma (Zi Cao) showed potent antiviral activity. The active fractions were isolated by bioassay-guided purification, and identified by a combination of high-resolution mass spectrometry and nuclear magnetic resonance. Fukinolic acid and cimicifugic acid A and J, were identified as active anti-EV-A71 compounds for C. heracleifolia, whereas for A. euchroma, two caffeic acid derivatives were tentatively deduced. Commercially available fukinolic acid analogues such as L-chicoric acid and D-chicoric also showed in vitro micromolar activity against EV-A71 lab-strain and clinical isolates.

Methylated flavonoids as anti-seizure agents: Naringenin 4',7-dimethyl ether attenuates epileptic seizures in zebrafish and mouse models.

Epilepsy is a neurological disease that affects more than 70 million people worldwide and is characterized by the presence of spontaneous unprovoked recurrent seizures. Existing anti-seizure drugs (ASDs) have side effects and fail to control seizures in 30% of patients due to drug resistance. Hence, safer and more efficacious drugs are sorely needed. Flavonoids are polyphenolic structures naturally present in most plants and consumed daily with no adverse effects reported. These structures have shown activity in several seizure and epilepsy animal models through allosteric modulation of GABAA receptors, but also via potent anti-inflammatory action in the brain. As such, dietary flavonoids offer an interesting source for ASD and anti-epileptogenic drug (AED) discovery, but their pharmaceutical potential is often hampered by metabolic instability and low oral bioavailability. It has been argued that their drug-likeness can be improved via methylation of the free hydroxyl groups, thereby dramatically enhancing metabolic stability and membrane transport, facilitating absorption and highly increasing bioavailability. Since no scientific data is available regarding the use of methylated flavonoids in the fight against epilepsy, we studied naringenin (NRG), kaempferol (KFL), and three methylated derivatives, i.e., naringenin 7-O-methyl ether (NRG-M), naringenin 4',7-dimethyl ether (NRG-DM), and kaempferide (4'-O-methyl kaempferol) (KFD) in the zebrafish pentylenetetrazole (PTZ) seizure model. We demonstrate that the methylated flavanones NRG-DM and NRG-M are highly effective against PTZ-induced seizures in larval zebrafish, whereas NRG and the flavonols KFL and KFD possess only a limited activity. Moreover, we show that NRG-DM is active in two standard acute mouse seizure models, i.e., the timed i.v. PTZ seizure model and the 6-Hz psychomotor seizure model. Based on these results, NRG-DM is proposed as a lead compound that is worth further investigation for the treatment of generalized seizures and drug-resistant focal seizures. Our data therefore highlights the potential of methylated flavonoids in the search for new and improved ASDs.

Synthesis of triterpenoid triazine derivatives from allobetulone and betulonic acid with biological activities.

The synthetic transformation and modification of natural products with the aim to improve the biological properties is an area of current interest. The triterpenoids betulin and betulinic acid are very abundant in nature and now are commercially available. In our study, starting from betulin and betulinic acid, we obtained allobetulone and betulonic acid in a few synthetic steps. The ketone function at the A-ring was used as the starting point for the synthesis of a series of 1,2,4-triazine-fused triterpenoids. The alkylation and Liebeskind-Srogl coupling were used for further substitution of 1,2,4-triazines, and the intramolecular hetero Diels-Alder reaction leads to interesting fused thienopyridine derivatives. All new compounds were tested for their cytostatic activities against murine leukemia L1210, human cervix carcinoma HeLa and human lymphoblast CEM tumor cells. The results show that some triterpenoid triazine betulonic acid derivatives have a promising cytostatic activity in vitro and could be used as potential leads for the development of new type of anti-cancer agents. Several compounds were also endowed with anti-HCMV activity in the low micromolar range.

Alpha-helical destabilization of the Bcl-2-BH4-domain peptide abolishes its ability to inhibit the IP3 receptor.

The anti-apoptotic Bcl-2 protein is the founding member and namesake of the Bcl-2-protein family. It has recently been demonstrated that Bcl-2, apart from its anti-apoptotic role at mitochondrial membranes, can also directly interact with the inositol 1,4,5-trisphosphate receptor (IP3R), the primary Ca(2+)-release channel in the endoplasmic reticulum (ER). Bcl-2 can thereby reduce pro-apoptotic IP3R-mediated Ca(2+) release from the ER. Moreover, the Bcl-2 homology domain 4 (Bcl-2-BH4) has been identified as essential and sufficient for this IP3R-mediated anti-apoptotic activity. In the present study, we investigated whether the reported inhibitory effect of a Bcl-2-BH4 peptide on the IP 3R1 was related to the distinctive α-helical conformation of the BH4 domain peptide. We therefore designed a peptide with two glycine "hinges" replacing residues I14 and V15, of the wild-type Bcl-2-BH4 domain (Bcl-2-BH4-IV/GG). By comparing the structural and functional properties of the Bcl-2-BH4-IV/GG peptide with its native counterpart, we found that the variant contained reduced α-helicity, neither bound nor inhibited the IP 3R1 channel, and in turn lost its anti-apoptotic effect. Similar results were obtained with other substitutions in Bcl-2-BH4 that destabilized the α-helix with concomitant loss of IP3R inhibition. These results provide new insights for the further development of Bcl-2-BH4-derived peptides as specific inhibitors of the IP3R with significant pharmacological implications.

Tanshinone IIA exhibits anticonvulsant activity in zebrafish and mouse seizure models.

Danshen or Chinese red sage (Salvia miltiorrhiza, Bunge) is used by traditional Chinese medicine (TCM) practitioners to treat neurological, cardiovascular, and cerebrovascular disorders and is included in some TCM formulations to control epileptic seizures. In this study, acetonic crude extracts of danshen inhibited pentylenetetrazol (PTZ)-induced seizure activity in zebrafish larvae. Subsequent zebrafish bioassay-guided fractionation of the extract resulted in the isolation of four major tanshinones, which suppressed PTZ-induced activity to varying degrees. One of the active tanshinones, tanshinone IIA, also reduced c-fos expression in the brains of PTZ-exposed zebrafish larvae. In rodent seizure models, tanshinone IIA showed anticonvulsive activity in the mouse 6-Hz psychomotor seizure test in a biphasic manner and modified seizure thresholds in a complex manner for the mouse i.v. PTZ seizure assay. Interestingly, tanshinone IIA is used as a prescription drug in China to address cerebral ischemia in patients. Here, we provide the first in vivo evidence demonstrating that tanshinone IIA has anticonvulsant properties as well.

Synthesis and in vitro evaluation of a PDT active BODIPY-NLS conjugate.

Two new photosensitizers based on the BODIPY scaffold have been synthesized, of which one bears an NLS peptide, which is linked to the BODIPY's core using the copper catalysed azide-alkyne click reaction. The phototoxicities of these BODIPY based photosensitizers have been determined, as well as their dark toxicities. Although the conjugation of a single NLS peptide to the BODIPY did not lead to any observable nuclear localization, the photosensitizer did exhibit a superior photoxicity. Cellular co-localization experiments revealed a localization of both dyes in the lysosomes, as well as a partial localization within the ER (for the peptide-bearing BODIPY).

A tripodal ruthenium-gadolinium metallostar as a potential α(v)ß(3) integrin specific bimodal imaging contrast agent.

Gd(III)-containing metallostar contrast agents are gaining increased attention, because their architecture allows for a slower tumbling rate, which, in turn, results in larger relaxivities. So far, these metallostars find possible applications as blood pool contrast agents. In this work, the first example of a tissue-selective metallostar contrast agent is described. This RGD-peptide decorated Ru(II)(Gd(III))(3)metallostar is synthesized as an α(v)ß(3)-integrin specific contrast agent, with possible applications in the detection of atherosclerotic plaques and tumor angiogenesis. The contrast agent showed a relaxivity of 9.65 s(-1) mM(-1), which represents an increase of 170%, compared to a low-molecular-weight analogue, because of a decreased tumbling rate (τ(R) = 470 ps). The presence of the MLCT band (absorption 375-500 nm, emission 525-850 nm) of the central Ru(II)(Ph-Phen)(3)-based complex grants the metallostar attractive luminescent properties. The (3)MLCT emission is characterized by a quantum yield of 4.69% and a lifetime of 804 ns, which makes it an interesting candidate for time-gated luminescence imaging. The potential application as a selective MRI contrast agent for α(v)ß(3)-integrin expressing tissues is shown by an in vitro relaxometric analysis, as well as an in vitroT(1)-weighted MR image.

The C terminus of Bax inhibitor-1 forms a Ca2+-permeable channel pore.

Bax inhibitor-1 (BI-1) is a multitransmembrane domain-spanning endoplasmic reticulum (ER)-located protein that is evolutionarily conserved and protects against apoptosis and ER stress. Furthermore, BI-1 is proposed to modulate ER Ca(2+) homeostasis by acting as a Ca(2+)-leak channel. Based on experimental determination of the BI-1 topology, we propose that its C terminus forms a Ca(2+) pore responsible for its Ca(2+)-leak properties. We utilized a set of C-terminal peptides to screen for Ca(2+) leak activity in unidirectional (45)Ca(2+)-flux experiments and identified an α-helical 20-amino acid peptide causing Ca(2+) leak from the ER. The Ca(2+) leak was independent of endogenous ER Ca(2+)-release channels or other Ca(2+)-leak mechanisms, namely translocons and presenilins. The Ca(2+)-permeating property of the peptide was confirmed in lipid-bilayer experiments. Using mutant peptides, we identified critical residues responsible for the Ca(2+)-leak properties of this BI-1 peptide, including a series of critical negatively charged aspartate residues. Using peptides corresponding to the equivalent BI-1 domain from various organisms, we found that the Ca(2+)-leak properties were conserved among animal, but not plant and yeast orthologs. By mutating one of the critical aspartate residues in the proposed Ca(2+)-channel pore in full-length BI-1, we found that Asp-213 was essential for BI-1-dependent ER Ca(2+) leak. Thus, we elucidated residues critically important for BI-1-mediated Ca(2+) leak and its potential channel pore. Remarkably, one of these residues was not conserved among plant and yeast BI-1 orthologs, indicating that the ER Ca(2+)-leak properties of BI-1 are an added function during evolution.