A Structure-Activity Investigation of the Fungal Metabolite (-)-TAN-2483B: Inhibition of Bruton's Tyrosine Kinase.

The natural product (-)-TAN-2483B is a fungal secondary metabolite which displays promising anti-cancer and immunomodulatory activity. Our previous syntheses of (-)-TAN-2483B and sidechain analogues uncovered inhibitory activity against Bruton's tyrosine kinase (Btk), an established drug target for various leukaemia and immunological diseases. A structure-based computational study using ensemble docking and molecular dynamics was performed to determine plausible binding modes for (-)-TAN-2483B and analogues in the Btk binding site. These hypotheses guided the design of new analogues which were synthesised and their inhibitory activities determined, providing insights into the structural determinants of the furopyranone scaffold that confer both activity and selectivity for Btk. These findings offer new perspectives for generating optimised (-)-TAN-2483B-based kinase inhibitors for the treatment of leukaemia and immunological diseases.

Behavioral metabolomics: how behavioral data can guide metabolomics research on neuropsychiatric disorders.

INTRODUCTION: Metabolomics produces vast quantities of data but determining which metabolites are the most relevant to the disease or disorder of interest can be challenging. OBJECTIVES: This study sought to demonstrate how behavioral models of psychiatric disorders can be combined with metabolomics research to overcome this limitation. METHODS: We designed a preclinical, untargeted metabolomics procedure, that focuses on the determination of central metabolites relevant to substance use disorders that are (a) associated with changes in behavior produced by acute drug exposure and (b) impacted by repeated drug exposure. Untargeted metabolomics analysis was carried out on liquid chromatography-mass spectrometry data obtained from 336 microdialysis samples. Samples were collected from the medial striatum of male Sprague-Dawley (N = 21) rats whilst behavioral data were simultaneously collected as part of a (±)-3,4-methylenedioxymethamphetamine (MDMA)-induced behavioral sensitization experiment. Analysis was conducted by orthogonal partial least squares, where the Y variable was the behavioral data, and the X variables were the relative concentrations of the 737 detected features. RESULTS: MDMA and its derivatives, serotonin, and several dopamine/norepinephrine metabolites were the greatest predictors of acute MDMA-produced behavior. Subsequent univariate analyses showed that repeated MDMA exposure produced significant changes in MDMA metabolism, which may contribute to the increased abuse liability of the drug as a function of repeated exposure. CONCLUSION: These findings highlight how the inclusion of behavioral data can guide metabolomics data analysis and increase the relevance of the results to the phenotype of interest.

Unraveling the binding mode of a methamphetamine aptamer: A spectroscopic and calorimetric study.

Nucleic-acid aptamers are bio-molecular recognition agents that bind to their targets with high specificity and affinity and hold promise in a range of biosensor and therapeutic applications. In the case of small-molecule targets, their small size and limited number of functional groups constitute challenges for their detection by aptamer-based biosensors because bio-recognition events may both be weak and produce poorly transduced signals. The binding affinity is principally used to characterize aptamer-ligand interactions; however, a structural understanding of bio-recognition is arguably more valuable in order to design a strong response in biosensor applications. Using a combination of nuclear magnetic resonance, circular dichroism, and isothermal titration calorimetry, we propose a binding model for a new methamphetamine aptamer and determine the main interactions driving complex formation. These measurements reveal only modest structural changes to the aptamer upon binding and are consistent with a conformational-selection binding model. The aptamer-methamphetamine complex formation was observed to be entropically driven, apparently involving hydrophobic and electrostatic interactions. Taken together, our results exemplify a means of elucidating small molecule-aptamer binding interactions, which may be decisive in the development of aptasensors and therapeutics and may contribute to a deeper understanding of interactions driving aptamer selection.

Mechanistic Studies on the Base-Promoted Ring Opening of Glycal-Derived gem-Dibromocyclopropanes.

In the presence of a nucleophilic base, ring-fused gem-dibromocyclopropanes derived from d-glycals undergo ring opening to give 2-deoxy-2-(E-bromomethylene)glycosides. Such cleavage of an exocyclic cyclopropane bond contrasts with the more usual silver-promoted ring-expansion reactions in which endocyclic bond cleavage occurs. Experimental and theoretical studies are reported which provide insights into the reaction mechanism and the origin of its kinetic selectivity for E-configured bromoalkene products. Density functional theory computations (M06-2X) predict that the reaction commences with alkoxide-induced HBr elimination from the dibromocyclopropane to form a bromocyclopropene. Ring opening then gives a configurationally stable zwitterionic (oxocarbenium cation/vinyl carbanion) intermediate, which undergoes nucleophilic addition and protonation to give the bromoalkene. There are two competing sources of the proton in the final step: One is the alcohol (co)solvent, and the other is the molecule of alcohol produced during the initial deprotonation step. The roles of the formed alcohol molecule and the bulk (co)solvent are demonstrated by isotope-labeling studies performed with deuterated solvents. The acid-promoted isomerization of the E-bromoalkene product into the corresponding Z-bromoalkene is also described. The mechanistic knowledge gained in this investigation sheds light on the unusual chemistry of this system and facilitates its future application in new settings.

Kinase-Inhibitory Nucleoside Derivatives from the Pacific Bryozoan Nelliella nelliiformis.

Marine organisms are a valuable source of bioactive natural products, yet bryozoan invertebrates have been relatively understudied. Herein, we report nelliellosides A and B, new secondary metabolites of the Pacific bryozoan Nelliella nelliiformis, found using NMR-guided isolation. Their structures, including absolute configurations, were elucidated using spectroscopic and chromatographic techniques. Total synthesis of the natural products and four analogues was also achieved, in addition to an assessment of their biological activity, especially kinase inhibition.

Synthesis of a simplified triazole analogue of pateamine A.

Pateamine A is a naturally occurring metabolite extracted from the marine sponge Mycale hentscheli. It exhibits potent cytotoxicity towards cancer cell lines and has been shown to target protein translation initiation via inhibition of the function of eukaryotic initiation factor 4A proteins. We have synthesised a simplified analogue of pateamine A, consisting of the skeletal core of the natural product but with the thiazole heterocycle replaced by a triazole. The convergent design of the synthesis features a base-induced opening of a δ-valerolactone to access the Z,E-dienoate moiety, Julia-Kocienski olefination and copper-catalysed azide-alkyne cycloaddition. Bioactivity testing of the simplified pateamine A analogue (3) indicated a significant reduction in cytotoxicity, compared to natural pateamine A. We propose that this reduced activity is due mainly to the substitution of the thiazole for the triazole heterocycle. This supports the hypothesis that the thiazole of pateamine A is important for binding to its biological target.

Synthesis of mycothiol conjugate analogues and evaluation of their antimycobacterial activity.

Drug-resistant Mycobacterium tuberculosis is a growing health problem. As proof of principle that the bacterial-specific metabolite mycothiol could be used as a delivery agent for antimycobacterial agents, simplified analogues of mycothiol were synthesised containing an S-trichloroethenyl substituted cysteine residue. It was envisaged that uptake of the mycothiol analogue would be followed by release of the known cytotoxin S-trichloroethenyl cysteine by the action of mycothiol S-conjugate amidase or its paralog, mycothiol deacetylase MshB. Promising activity was displayed against model Mycobacteria, although further development will be required to improve selectivity.

Preparation of conjugated dienoates with Bestmann ylide: Towards the synthesis of zampanolide and dactylolide using a facile linchpin approach.

Bestmann ylide [(triphenylphosphoranylidene)ketene] acts as a chemical linchpin that links nucleophilic entities, such as alcohols or amines, with carbonyl moieties to produce unsaturated esters and amides, respectively. In this work, the formation of α,ß,γ,δ-unsaturated esters (dienoates) is achieved through the coupling of Bestmann ylide, an alcohol and an α,ß-unsaturated aldehyde. Primary and secondary alcohols, including allylic alcohols, are suitable substrates; the newly formed alkene has an E-geometry. Strategically, this represents a highly efficient route to unsaturated polyketide derivatives. A linchpin approach to the synthesis of a major fragment of the natural products zampanolide and dactylolide is investigated using Bestmann ylide to link the C16-C20 alcohol with the C3-C8 aldehyde fragment.

Synthesis and solution aggregation studies of a suite of mixed neutral and zwitterionic chromophores for second-order nonlinear optics.

We report details of the synthesis of a series of bi- and trichromophores. These compounds contain mixtures of chromophores that have zwitterionic (ZWI) and neutral ground state (NGS) components covalently attached to each other. The neutral ground state moieties are based on dyes with aniline donors--such as Disperse Red 1--whereas the zwitterionic components are derived from chromophores with pro-aromatic donors such as 1,4-dihydropyridinylidene. By combining both ZWI and NGS components, we aim to develop novel compounds for nonlinear optics in which there is an enhancement of the overall hyperpolarizability coupled with a decrease in the net dipole moment. Thus, this approach should eliminate the electrostatic effects that result when only one type of chromophore is used, and so reduce the likelihood of undesirable aggregation occurring. This, in turn, should enable us to realize organic materials with large macroscopic optical nonlinearities. An analysis of the UV-vis results suggests that there is a strong dependence on solvent polarity that determines whether the embedded constituents should be treated as discrete elements; in low polarity solvents, there appear to be strong intramolecular interactions occurring, particularly when a 1,4-quinolinylidene-based donor is used in the ZWI component.

13C NMR analysis of 3,6-dihydro-2H-pyrans: assignment of remote stereochemistry using axial shielding effects.

The rational analysis of (13)C NMR axial shielding effects has enabled the assignment of remote relative stereochemistry in 3,6-oxygen-substituted 3,6-dihydro-2H-pyrans. Comparison of the (13)C NMR shifts of equivalent centers in cis- and trans-substituted 3,6-dihydro-2H-pyrans allows the relative configuration at the C3 and C6 positions to be defined in diastereoisomeric mixtures. Density functional calculations were used to validate this method and assess the conformational bias present in the ring system. Ultimately, the coupling of computational chemistry with this (13)C NMR-based method provided a reliable and convenient method for stereochemical assignment of a single diastereomer. This approach provides a facile and complementary alternative to the practices previously employed for determining the relative configuration in 3,6-dihydro-2H-pyrans.

Preparation, analysis and toxicity characterisation of the redox metabolites of the azo food dye tartrazine.

Tartrazine (E102, FD&C Yellow 5) is a vibrant yellow azo dye added to many processed foods. The safety of this ubiquitous chemical has not been fully elucidated, and it has been linked to allergic reactions and ADHD in some individuals. In our study, bacterial species isolated from human stool decolourised tartrazine and, upon exposure to air, a purple compound formed. Tartrazine is known to undergo reduction in the gut to sulfanilic acid and 4-amino-3-carboxy-5-hydroxy-1-(4-sulfophenyl)pyrazole (SCAP). These metabolites and their derivatives are relevant to the toxicology of tartrazine. The toxicity of sulfanilic acid has been studied before, but the oxidative instability of SCAP has previously prevented full characterisation. We have verified the chemical identity of SCAP and confirmed that the purple-coloured oxidation derivative is 4-(3-carboxy-5-hydroxy-1-(4-sulfophenyl)-1H-pyrazol-4-yl)imino-5-oxo-1-(4-sulfophenyl)-4,5-dihydro-1H-pyrazole-3-carboxylic acid (purpurazoic acid, PPA), as proposed by Westöö in 1965. A yellow derivative of SCAP is proposed to be the hydrolysed oxidation product, 4,5-dioxo-1-(4-sulfophenyl)-4,5-dihydro-1H-pyrazole-3-carboxylic acid. SCAP and PPA are moderately toxic to human cells (IC50 89 and 78 µM against HEK-293, respectively), but had no apparent effect on Escherichia coli and Bacillus subtilis bacteria. These results prompt further analyses of the toxicology of tartrazine and its derivatives.

Comparative B cell and antibody responses induced by adenoviral vectored and mRNA vaccines against COVID-19.

Both vector and mRNA vaccines were an important part of the response to the COVID-19 pandemic and may be required in future outbreaks and pandemics. However, adenoviral vectored (AdV) vaccines may be less immunogenic than mRNA vaccines against SARS-CoV-2. We assessed anti-spike and anti-vector immunity among infection-naïve Health Care Workers (HCW) following two doses of AdV (AZD1222) versus mRNA (BNT162b2) vaccine. 183 AdV and 274 mRNA vaccinees enrolled between April and October 2021. Median ages were 42 and 39 years, respectively. Blood was collected at least once, 10-48 days after vaccine dose 2. Surrogate virus neutralization test (sVNT) and spike binding antibody titres were a median of 4.2 and 2.2 times lower, respectively, for AdV compared to mRNA vaccinees (p<0.001). Median percentages of memory B cells that recognized fluorescent-tagged spike and RBD were 2.9 and 8.3 times lower, respectively for AdV compared to mRNA vaccinees. Titres of IgG reactive with human Adenovirus type 5 hexon protein rose a median of 2.2-fold after AdV vaccination but were not correlated with anti-spike antibody titres. Together the results show that mRNA induced substantially more sVNT antibody than AdV vaccine due to greater B cell expansion and targeting of the RBD. Pre-existing AdV vector cross-reactive antibodies were boosted following AdV vaccination but had no detectable effect on immunogenicity.

Superior immunogenicity of mRNA over adenoviral vectored COVID-19 vaccines reflects B cell dynamics independent of anti-vector immunity: Implications for future pandemic vaccines.

Both vector and mRNA vaccines were an important part of the response to the COVID-19 pandemic and may be required in future outbreaks and pandemics. The aim of this study was to validate whether immunogenicity differs for adenoviral vectored (AdV) versus mRNA vaccines against SARS-CoV-2, and to investigate how anti-vector immunity and B cell dynamics modulate immunogenicity. We enrolled SARS-CoV-2 infection-naïve health care workers who had received two doses of either AdV AZD1222 (n = 184) or mRNA BNT162b2 vaccine (n = 274) between April and October 2021. Blood was collected at least once, 10-48 days after vaccine dose 2 for antibody and B cell analyses. Median ages were 42 and 39 years, for AdV and mRNA vaccinees, respectively. Surrogate virus neutralization test (sVNT) and spike binding antibody titres were a median of 4.2 and 2.2 times lower, respectively, for AdV compared to mRNA vaccinees (p < 0.001). Median percentages of memory B cells that recognized fluorescent-tagged spike and RBD were 2.9 and 8.3 times lower, respectively for AdV compared to mRNA vaccinees. Titres of IgG reactive with human adenovirus type 5 hexon protein rose a median of 2.2-fold after AdV vaccination but were not correlated with anti-spike antibody titres. Together the results show that mRNA induced substantially more sVNT antibody than AdV vaccine, which reflected greater B cell expansion and targeting of the RBD rather than an attenuating effect of anti-vector antibodies. ClinicalTrials.gov Identifier: NCT05110911.

Isolation and characterization of degradation products of moxidectin using LC, LTQ FT-MS, H/D exchange and NMR.

This study aimed to evaluate the degradation profile and pathways, and identify unknown impurities of moxidectin under stress conditions. During the experiments, moxidectin samples were stressed using acid, alkali, heat and oxidation, and chromatographic profiles were compared with known impurities given in European Pharmacopeia (EP) monograph. Moxidectin has shown good stability under heat, while reaction with alkali produced 2-epi and ∆2,3 isomers (impurities D and E in EP) by characteristic reactions of the oxahydrindene (hexahydrobenzofuran) portion of the macrocyclic lactone. Two new, previously unreported, unknown degradation products, i.e. impurity 1 and impurity 2, detected after acid hydrolysis of moxidectin (impurity 2 was also observed to a lesser extent after oxidation), were isolated from sample matrices and identified using liquid chromatography, NMR, high-resolution FT-ICR MS, and hydrogen/deuterium exchange studies. FTMS analysis showed accurate mass of molecular ion peaks for moxidectin at m/z 640.38412, impurity 1 at m/z 656.37952 and impurity 2 at m/z 611.35684, giving rise to daughter ions traceable up to the seventh levels of MS(n) experiments and supporting the proposed structures. Both unknown impurities along with moxidectin were fully characterized by (1)H, (13)C, 1D HMBC and 2D (NOESY, COSY and HSQC) NMR experiments. The interpretation of experimental data positively identified impurity 1 as 3,4-epoxy-moxidectin and impurity 2 as 23-keto-nemadectin. The identification of new impurities and correlation of their chromatographic profiles with the EP method is very useful to establish the stability profile of moxidectin and its preparations, as well as add value to the forthcoming moxidectin finished product European Pharmacopeia monographs.

An overview on chemical derivatization and stability aspects of selected avermectin derivatives.

Naturally occurring avermectins (AVMs) and its derivatives are potent endectocide compounds, well-known for their novel mode of action against a broad range of nematode and anthropod animal parasites. In this review, chemical and pharmaceutical aspects of AVM derivatives are described including stability, synthetic and purification processes, impurities and degradation pathways, and subsequent suggestions are made to improve the chemical stability. It has been found out that unique structure of AVM molecules and presence of labile groups facilitated the derivatization of AVM into various compounds showing strong anthelmintic activity. However, the same unique structure is also responsible for labile nature related to sensitive stability profile of molecules. AVMs are found to be unstable in acidic and alkaline conditions. In addition, these compounds are sensitive to strong light, and subsequently presence of photo-isomer in animals treated topically with AVM product is well known. The pharmacoepial recommendations for addition of antioxidant into drug substance, as well as its products, arises from the fact that AVM are very sensitive to oxidation. Formations of solvates, salts, epoxides, reduction of double bonds and developing liquid formulation around pH 6.2, were some chemical approaches used to retard the degradation in AVM. This coherent review will contribute towards the better understanding of the correlation of chemical processes, stability profile and biological activity; therefore, it will help to design the shelf-life stable formulations containing AVMs.

The role of extracellular serotonin and MDMA in the sensitizing effects of MDMA.

MDMA is a non-selective monoamine releasing stimulant with potent serotonergic effects - a pharmacological effect not typically associated with drugs of misuse or efficacious reinforcers. Nonetheless, MDMA is misused by humans and self-administered by laboratory animals. We have previously shown that repeated exposure to MDMA sensitized both the locomotor activating and reinforcing effects of MDMA in rats. Because repeated MDMA exposure often results in decreased markers of serotonin neurotransmission, it is possible that this might underlie the sensitizing effects of MDMA. This was examined in the current study. Male Sprague-Dawley rats were stereotaxically implanted with guide cannula in the medial striatum. They were then pre-treated with saline (n =  11) or MDMA (10 mg/kg, i.p.; n =  10), once daily for five days. Two-days later, all rats received ascending doses of MDMA (0.0, 5.0, 10.0, mg/kg, i.p.) administered at 2 hr intervals, during which locomotor activity was measured and microdialysis samples were collected. Microdialysates were analyzed using liquid chromatography-mass spectrometry and the concentrations of serotonin and MDMA were quantified. Acute MDMA administration produced dose-dependent increases in locomotor activity, which was significantly enhanced by MDMA pre-treatment. Acute MDMA also produced dose-dependent increases in medial-striatal serotonin and MDMA, but this was not impacted by MDMA pre-treatment. These results suggest that the sensitizing effects of MDMA are not due to changes in MDMA-produced synaptic overflow of serotonin in the medial striatum or the absorption/elimination of systemically administered MDMA. More likely candidates are alterations in serotonin receptor mechanisms and/or dopamine neurotransmission following repeated exposure.

Synthesis of the (-)-TAN-2483B ring system via a D-mannose-derived cyclopropane.

The ring system of the fungal metabolite (-)-TAN-2483B has been synthesised, for the first time, from d-mannose, utilising a cyclopropanation/ring expansion sequence.

Alkenylphosphonates: unexpected products from reactions of methyl 2-[(diethoxyphosphoryl)methyl]benzoate under Horner-Wadsworth-Emmons conditions.

Methyl 2-[(diethoxyphosphoryl)methyl]benzoate reacts with several aldehydes to produce an alkenylphosphonate as the major product, together with varying amounts of the expected Horner-Wadsworth-Emmons product, a 1,2-disubstituted E-alkene. Use of a bulky aldehyde or the tert-butyl ester favours the normal HWE product.

Synthesis of oxepines and 2-branched pyranosides from a d-glucal-derived gem-dibromo-1,2-cyclopropanated sugar.

The conversion of cyclopropane-fused carbohydrates into oxepines is an attractive method for accessing diverse members of the septanoside family of carbohydrate mimetics. 2-Bromooxepines are obtained through silver(I)-promoted thermal ring expansion of a d-glucal-derived gem-dihalocyclopropanated sugar. In contrast, cyclopropane ring cleavage under basic conditions leads to 2-C-branched pyranosides, not the 2-bromooxepine structures assigned in an earlier report.

Total Synthesis and Bioactivity Studies of Fungal Metabolite (-)-TAN-2483B.

The first total synthesis of (-)-TAN-2483B, a fungal metabolite possessing a densely functionalized furo[3,4-b]pyran-5-one framework, is achieved in 14 steps from d-mannose. Generation of the 2,6-trans-pyran is by cyclopropane ring expansion followed by α-selective alkynylation. Julia-Kocienski olefination introduces the E-propenyl side chain. Alkyne functionalization and carbonylation stereoselectively establish the bicyclic core of (-)-TAN-2483B. Inhibition of kinases Btk and Bmx, bacterial priority pathogens, and cytokine production in splenocytes indicates promising therapeutic potential.