Subcellular multiplex imaging is fundamentally limited by spectral crowding, restricted photostability, and the modality-specific performance of conventional fluorophores, which constrain the simultaneous visualization of multiple organelles in living cells. Here, we report a modular class of organelle-targeting, coumarin-based fluorescent probes that overcome these limitations through a large (up to 150 nm) Stokes shift, high brightness, good photostability, and efficient twophoton excitation. Central to this platform is a synthetically accessible, sulfonamide-functionalized coumarin core that enables convenient late-stage attachment of diverse organelle-targeting motifs. This modular strategy allows selective labeling of mitochondria, endoplasmic reticulum, lysosomes, plasma membrane, and whole cells at nanomolar concentrations with high signal-to-background ratios. The large Stokes shift minimizes spectral crosstalk and enables straightforward multiplex imaging in combination with green-and red-emitting fluorophores using simplified excitation schemes. These probes perform robustly across multiple imaging modalities, including confocal microscopy, two-photon excitation microscopy, fluorescence lifetime imaging microscopy, and flow cytometry. These new dyes provide a versatile toolkit for dynamic subcellular studies in basic research and disease modelling.

Mitochondrial KV1.3 channels (mitoKV1.3) have emerged as promising targets for cancer therapy due to their role in regulating apoptosis, independent of upstream signalling pathways and Bcl-2 family protein levels. Here, we present a new non-psoralene Kv1.3 mitochondria-targeted conjugates. These conjugates, particularly cis-8 and cis-9, exhibit nanomolar affinity and high selectivity for KV1.3 while effectively inducing apoptosis in tumor cells. Unlike their parent KV1.3 inhibitors, which lack cytotoxicity, the mitoKV1.3 conjugates induce rapid mitochondrial depolarization, and caspase-3/7 activation, culminating in dose-dependent tumor cell death in both 2D and 3D models. Mechanistically, cis-8 and cis-9 disrupt mitochondrial membrane potential and selectively target cancer cells, sparing normal cells at lower concentrations. Notably, KV1.3 knockout models confirmed the dependence of cytotoxicity on mitoKV1.3 inhibition. The conjugates demonstrated robust antitumor activity in murine pancreatic intraepithelial neoplasia (PanIN)-derived organoids, with preferential action over normal pancreatic organoids, highlighting their tumor selectivity. Importantly, safety assessments showed no significant DNA damage or chromosomal aberrations at non-cytotoxic doses. This study introduces a new structural class of mitochondria-targeted KV1.3 inhibitors with enhanced solubility compared to psoralen-based analogues. The unique mechanism of action, characterized by rapid depolarization and moderate ROS dependence, underscores their potential as selective anticancer agents. These findings warrant further investigation into in vivo efficacy and potential synergy with existing therapies.

The glutarimide fragment is a key pharmacophore in cereblon-binding immunomodulatory drugs and proteolysis-targeting chimeras, yet exploration of non-glutarimide scaffolds remains limited. Here, we report the design, synthesis, and evaluation of fluorinated aminopiperidones as three-dimensional, non-glutarimide thalidomide analogs. By replacing a single glutarimide carbonyl with a stereogenic C*-CF3 unit, we accessed a previously underexplored chemical space encompassing all regio- and diastereomeric variants of 3- and 5-phthalimido-6-trifluoromethyl-2-piperidones. Stereocontrolled synthetic routes enabled reliable control over relative configurations, and two-dimensional NMR provided unambiguous structural assignment. Experimental profiling of CF3-piperidones relative to glutarimides revealed improved aqueous solubility despite increased logD and enhanced membrane interaction characteristics, without substantial changes in plasma protein binding, consistent with sp3 enrichment and escape-from-flatland principles. To assess whether cereblon recognition is retained after carbonyl-to-C*-CF3 replacement, the compounds were evaluated by microscale thermophoresis. No measurable cereblon binding was detected for the fluorinated aminopiperidones. Docking studies, performed to rationalize this lack of affinity, suggested disruption of key cereblon-ligand interactions and unfavorable binding poses following carbonyl-to-C*-CF3 substitution. Several CF3-piperidones, particularly tetrafluorophthalimide derivatives, exhibited potent anti-angiogenic activity in vitro and ex vivo, indicating a cereblon-independent mechanism.

Mitochondria-targeting moieties (MTMs) are molecular fragments designed to deliver covalently tethered functional cargo to mitochondria, providing a modular strategy for chemical biology tools, imaging agents, and mitochondria-targeted therapies. Phosphonium- or nitrogen cation-based MTMs are not inert vectors and exhibit intrinsic bioactivity on mitochondrial and cellular levels to various extents. Here, we systematically evaluated a panel of N+-based cations to determine how structural features influence subcellular distribution and inherent bioactivity. Live-cell imaging of fluorescent dye conjugates revealed that 3,5-diphenylpyridinium (DPPy+) exhibits cellular uptake and mitochondrial targeting comparable to the benchmark triphenylphosphonium (TPP+), whereas conjugates with unsubstituted pyridinium preferentially accumulate in lysosomes. Profiling of inert cargo derivatives showed that DPPy+ has lower intrinsic activity on mitochondrial membrane potential and oxidative phosphorylation, as well as on cellular respiration and viability than TPP+. The combination of efficient mitochondrial delivery and low intrinsic bioactivity translated to bioactive cargo: a Kv1.3 inhibitor conjugate with DPPy+ induced apoptosis in cancer cell lines and demonstrated improved cancer selectivity relative to the TPP+ conjugate in pancreatic organoid models. These results position lipophilic pyridinium cations as effective TPP+ surrogates with enhanced biocompatibility for mitochondria-targeted therapeutic and diagnostic agents, while revealing the structure-dependent competing lysosomal accumulation of permanent nitrogen cations.

The title compound, C13H15N3O3S·0.25H2O, crystallizes in the triclinic space group P1 and features four organic molecules in the asymmetric unit alongside one water molecule. The extended structure exhibits both hydrogen bonds (O—H...O, N—H...O and N—H...N) and chalcogen (C—S...O) contacts, leading to a complex three-dimensional network.

Mitochondria-targeting technology, in the form of (lipophilic cation)-(small molecule) conjugates, was first discussed more than 50 years ago. Since then, the triphenylphosphonium (TPP) cation has become synonymous with the concept of a mitochondria-targeting moiety (MTM). The discovery of its ability to accumulate in mitochondria occurred alongside research on mitochondrial functions and the mechanisms underlying cation import. The recognition of intrinsic biological effects of TPP, apart from delivery of functional cargo, came much later, prompting the development of novel MTMs beyond the archetypal TPP. In this Perspective, we present the current understanding of the biological mechanisms of action of mitochondria-targeting conjugates, describe the methods used for their validation, and overview the recently developed novel mitochondria-targeting moieties. Building upon the recent advances, we propose a rational approach for the development of novel MTMs to be incorporated into the future MTM-linker-cargo therapeutics (MITACs, MITochondria-TArgeting Conjugates).

Noyori-Ikariya-type ruthenium(II)-catalysts for asymmetric transfer hydrogenation (ATH) have been known for 25 years and have proved as a well-behaved and user-friendly platform for the synthesis of chiral secondary alcohols. A progress has been made in the past five years in understanding the asymmetric reduction of complex ketones, where up to four stereocenters can be controlled in a single chemical transformation. Intriguing multi-chiral molecular architectures are therefore available in few well understood and robust synthetic steps from commercially available building blocks and possess handles for additional functionalization. The aim of this Review is to showcase the availability of three-dimensional scaffolds and homochiral lead-like compounds via ATH and inspire their direct use in drug discovery endeavors. Basic mechanistic insights are provided to demystify the stereo-chemical outcomes, as well as examples of diastereoselective transformations of enantiopure alcohols to give a feeling of how these rigid non-planar molecules can be further elaborated.

Asymmetric transfer hydrogenation (ATH) has been recognized as a highly valuable strategy that allows access to enantioenriched substances and has been widely applied in the industrial production of drug molecules. However, despite the great success in ATH of ketones, highly efficient, regio- and stereoselective ATH on enones remains underdeveloped. Moreover, optically pure acyloins and 1,2-diols are both extremely useful building blocks in organic synthesis, medicinal chemistry, and materials science, but concise asymmetric approaches allowing access to different types of acyloins and 1,2-diols have scarcely been discovered. We report in this paper the first highly efficient ATH of readily accessible beta,gamma-unsaturated alpha-diketones. The protocol affords four types of enantioenriched acyloins and four types of optically pure 1,2-diols in highly regio- and stereoselective fashion. The synthetic value of this work has been showcased by the divergent synthesis of four related natural products. Moreover, systematic mechanistic studies and density functional theory (DFT) calculations have illustrated the origin of the reactivity divergence, revealed the different roles of aromatic and aliphatic substituents in the substrates, and provided a range of unique mechanistic rationales that have not been disclosed in ATH-related studies.

Four-membered carbocycles are fundamental substructures in bioactive molecules and approved drugs and serve as irreplaceable building blocks in organic synthesis. However, developing efficient protocols furnishing diversified four-membered ring compounds in a highly regio-, diastereo-, and enantioselective fashion remains challenging but very desirable. Here, we report the unprecedented asymmetric transfer hydrogenation of cyclobutenediones. The reaction can selectively afford three types of four-membered products in high yields with high stereoselectivities, and the highly functionalized products enable a series of further transformations to form more diversified four-membered compounds. Asymmetric synthesis of di-, tri-, and tetrasubstituted bioactive molecules has also been achieved. Systematic mechanistic studies and theoretical calculations have revealed the origin of the regioselectivity, the key hydrogenation transition state models, and the sequence of the double and triple hydrogenation processes. The work provides a new choice for the catalytic asymmetric synthesis of cyclobutanes and related structures and demonstrates the robustness of asymmetric transfer hydrogenation in the accurate selectivity control of highly functionalized substrates.

This study presents the discovery of a new series of

The ATP binding site located on the subunit B of DNA gyrase is an attractive target for the development of new antibacterial agents. In recent decades, several small-molecule inhibitor classes have been discovered but none has so far reached the market. We present here the discovery of a promising new series of N-phenylpyrrolamides with low nanomolar IC50 values against DNA gyrase, and submicromolar IC50 values against topoisomerase IV from Escherichia coli and Staphylococcus aureus. The most potent compound in the series has an IC50 value of 13 nM against E. coli gyrase. Minimum inhibitory concentrations (MICs) against Gram-positive bacteria are in the low micromolar range. The oxadiazolone derivative 11a, with an IC50 value of 85 nM against E. coli DNA gyrase displays the most potent antibacterial activity, with MIC values of 1.56 μM against Enterococcus faecalis, and 3.13 μM against wild type S. aureus, methicillin-resistant S. aureus (MRSA) and vancomycin-resistant Enterococcus (VRE). The activity against wild type E. coli in the presence of efflux pump inhibitor phenylalanine-arginine β-naphthylamide (PAβN) is 4.6 μM.

Developing a general, highly efficient, and enantioselective catalytic method for the synthesis of chiral alcohols is still a formidable challenge. We report in this article the asymmetric transfer hydrogenation (ATH) of N-methyliminodiacetyl (MIDA) acylboronates as a general substrate-independent entry to enantioenriched secondary alcohols. ATH of acyl-MIDA-boronates with (het)aryl, alkyl, alkynyl, alkenyl, and carbonyl substituents delivers a variety of enantioenriched α-boryl alcohols. The latter are used in a range of stereospecific transformations based on the boron moiety, enabling the synthesis of carbinols with two closely related α-substituents, which cannot be obtained with high enantioselectivities using direct asymmetric hydrogenation methods, such as the (R)-cloperastine intermediate. Computational studies illustrate that the BMIDA group is a privileged enantioselectivity-directing group in Noyori-Ikariya ATH compared to the conventionally used aryl and alkynyl groups due to the favorable CH-O attractive electrostatic interaction between the η6-arene-CH of the catalyst and the σ-bonded oxygen atoms in BMIDA. The work expands the domain of conventional ATH and shows its huge potential in addressing challenges in symmetric synthesis.

N-(Benzothiazole-2-yl)pyrrolamide DNA gyrase inhibitors with benzyl or phenethyl substituents attached to position 3 of the benzothiazole ring or to the carboxamide nitrogen atom were prepared and studied for their inhibition of Escherichia coli DNA gyrase by supercoiling assay. Compared to inhibitors bearing the substituents at position 4 of the benzothiazole ring, the inhibition was attenuated by moving the substituent to position 3 and further to the carboxamide nitrogen atom. A co-crystal structure of (Z)-3-benzyl-2-((4,5-dibromo-1H-pyrrole-2-carbonyl)imino)-2,3-dihydrobenzo[d]-thiazole-6-carboxylic acid (I) in complex with E. coli GyrB24 (ATPase subdomain) was solved, revealing the binding mode of this type of inhibitor to the ATP-binding pocket of the E. coli GyrB subunit. The key binding interactions were identified and their contribution to binding was rationalised by quantum theory of atoms in molecules (QTAIM) analysis. Our study shows that the benzyl or phenethyl substituents bound to the benzothiazole core interact with the lipophilic floor of the active site, which consists mainly of residues Gly101, Gly102, Lys103 and Ser108. Compounds with substituents at position 3 of the benzothiazole core were up to two orders of magnitude more effective than compounds with substituents at the carboxamide nitrogen. In addition, the 6-oxalylamino compounds were more potent inhibitors of E. coli DNA gyrase than the corresponding 6-acetamido analogues.

New 2-pyrrolamidobenzothiazole-based inhibitors of mycobacterial DNA gyrase were discovered. Among these, compounds 49 and 51, show excellent antibacterial activity against Mycobacterium tuberculosis and Mycobacterium abscessus with a notable preference for mycobacteria. Both compounds can penetrate infected macrophages and reduce intracellular M. tuberculosis load. Compound 51 is a potent inhibitor of DNA gyrase (M. tuberculosis DNA gyrase IC50 = 4.1 nM, Escherichia coli DNA gyrase IC50 of <10 nM), selective for bacterial topoisomerases. It displays low MIC90 values (M. tuberculosis: 0.63 M; M. abscessus: 2.5 mu M), showing specificity for mycobacteria, and no apparent toxicity. Compound 49 not only displays potent antimycobacterial activity (MIC90 values of 2.5 mu M for M. tuberculosis and 0.63 mu M for M. abscessus) and selectivity for mycobacteria but also exhibits favorable solubility (kinetic solubility = 55 mu M) and plasma protein binding (with a fraction unbound of 2.9 % for human and 4.7 % for mouse). These findings underscore the potential of fine-tuning molecular properties to develop DNA gyrase B inhibitors that specifically target the mycobacterial chemical space, mitigating the risk of resistance development in non-target pathogens and minimizing harm to the microbiome.

Background: The novel bacterial topoisomerase inhibitors (NBTIs) developed in our laboratory show potent on-target enzyme inhibition but suffer from low activity against Gram-negative bacteria. Methods: With the aim of improving the antibacterial activity of our compounds against Gram-negative bacteria, we tested them in combination with different efflux pump inhibitors (EPIs), a strategy that showed promise in several other classes of antimicrobials. We also investigated the combined effect of NBTIs with ATP-competitive inhibitors of bacterial type II topoisomerases (ACIs), as well as the antibiofilm properties of our compounds and the combination with EPIs against early and mature Acietobacter baumannii biofilm. Results: Our results demonstrate that combinations of NBTIs with EPI Phenylalanine-arginyl-β-naphthylamide significantly reduce the corresponding NBTIs’ minimal inhibitory concentration values and show potentiation of A. baumannii biofilm inhibition as compared to NBTIs alone. Although combinations of NBITs and ACIs did not show synergistic effects, the FIC index value calculations revealed additive effects for all the combinations of a selected NBTI in combination with three ACIs in all the assayed Gram-negative bacteria from the ESKAPE group. Conclusions: These results show for the first time that combinations of NBTIs with either EPIs or a different class of the topoisomerase inhibitors may be a beneficial strategy to combat difficult-to-treat bacterial infections.

In this work, we describe an improved series of N-phenylpyrrolamide inhibitors that exhibit potent activity against DNA gyrase and are highly effective against high-priority gram-positive bacteria. The most potent compounds show low nanomolar IC50 values against Escherichia coli DNA gyrase, and in addition, compound 7c also inhibits E. coli topoisomerase IV in the nanomolar concentration range, making it a promising candidate for the development of potent dual inhibitors for these enzymes. All tested compounds show high selectivity towards the human isoform DNA topoisomerase IIα. Compounds 6a, 6d, 6e and 6f show MIC values between 0.031 and 0.0625 μg/mL against vancomycin-intermediate S. aureus (VISA) and Enterococcus faecalis strains. Compound 6g shows an inhibitory effect against the methicillin-resistant S. aureus strain (MRSA) with a MIC of 0.0625 μg/mL and against the E. faecalis strain with a MIC of 0.125 μg/mL. In a time-kill assay, compound 6d showed a dose-dependent bactericidal effect on the MRSA strain and achieved bactericidal activity at 8 × MIC after 8 hours. The duration of the post-antibiotic effect (PAE) on the MRSA strain for compound 6d was 2 hours, which corresponds to the PAE duration for ciprofloxacin. The compounds were not cytotoxic at effective concentrations, as determined in an MTS assay on the MCF-7 breast cancer cell line.

The ruthenium complex of the tethered syn-ULTAM ligand, i.e. syn-3-(α-aminobenzyl)-benzo-γ-sultam, has been evaluated for the asymmetric transfer hydrogenation (ATH) of a variety of ketones in formic acid/triethylamine mixture. Its performance was similar to the established Noyori–Ikariya-type catalysts for the reduction of benzo-fused cyclic ketones, but the enantioselectivity for the reduction of acetophenone was only moderate. The syn-ULTAM-based catalyst was particularly efficient and enantioselective for ATH of the sterically demanding α,α-disubstituted ketones. Moreover, we report a divergent ATH of 2-phenyl-1,3-indandione to either cis-2-phenyl-1-indanol, or cis,trans-2-phenyl-1,3-indandiol.

The development of new anticancer agents is one of the most urgent topics in drug discovery. Inhibition of molecular chaperone Hsp90 stands out as an approach that affects various oncogenic proteins in different types of cancer. These proteins rely on Hsp90 to obtain their functional structure, and thus Hsp90 is indirectly involved in the pathophysiology of cancer. However, the most studied ATP-competitive inhibition of Hsp90 at the N-terminal domain has proven to be largely unsuccessful clinically. Therefore, research has shifted towards Hsp90 C-terminal domain (CTD) inhibitors, which are also the focus of this study. Our recent discovery of compound C has provided us with a starting point for exploring the structure-activity relationship and optimising this new class of triazole-based Hsp90 inhibitors. This investigation has ultimately led to a library of 33 analogues of C that have suitable physicochemical properties and several inhibit the growth of different cancer types in the low micromolar range. Inhibition of Hsp90 was confirmed by biophysical and cellular assays and the binding epitopes of selected inhibitors were studied by STD NMR. Furthermore, the most promising Hsp90 CTD inhibitor 5x was shown to induce apoptosis in breast cancer (MCF-7) and Ewing sarcoma (SK-N-MC) cells while inducing cause cell cycle arrest in MCF-7 cells. In MCF-7 cells, it caused a decrease in the levels of ERα and IGF1R, known Hsp90 client proteins. Finally, 5x was tested in zebrafish larvae xenografted with SK-N-MC tumour cells, where it limited tumour growth with no obvious adverse effects on normal zebrafish development.

The crystal structure of the title enantiopure tetralol derivative {systematic name: (1S,2S)-2-[(S)-2,2,2-trifluoro-1-hydroxyethyl]-1,2,3,4-tetrahydronaphthalen-1-ol}, C12H13F3O2, synthesized by asymmetric transfer hydrogenation, was elucidated by low-temperature single-crystal X-ray diffraction. The enantiopure compound crystallizes in the Sohncke space group P212121 with one molecule in the asymmetric unit and features intramolecular as well as intermolecular O—H...O hydrogen bonding. The absolute configuration was established from anomalous dispersion effects.

We present a new series of 2-aminobenzothiazole-based DNA gyrase B inhibitors with promising activity against ESKAPE bacterial pathogens. Based on the binding information extracted from the cocrystal structure of DNA gyrase B inhibitor A, in complex with Escherichia coli GyrB24, we expanded the chemical space of the benzothiazole-based series to the C5 position of the benzothiazole ring. In particular, compound E showed low nanomolar inhibition of DNA gyrase (IC50 < 10 nM) and broad-spectrum antibacterial activity against pathogens belonging to the ESKAPE group, with the minimum inhibitory concentration < 0.03 μg/mL for most Gram-positive strains and 4-16 μg/mL against Gram-negative E. coli, Acinetobacter baumannii, Pseudomonas aeruginosa, and Klebsiella pneumoniae. To understand the binding mode of the synthesized inhibitors, a combination of docking calculations, molecular dynamics (MD) simulations, and MD-derived structure-based pharmacophore modeling was performed. The computational analysis has revealed that the substitution at position C5 can be used to modify the physicochemical properties and antibacterial spectrum and enhance the inhibitory potency of the compounds. Additionally, a discussion of challenges associated with the synthesis of 5-substituted 2-aminobenzothiazoles is presented.