Virtual Screening of a Chemically Diverse "Superscaffold" Library Enables Ligand Discovery for a Key GPCR Target.

The advent of ultra-large libraries of drug-like compounds has significantly broadened the possibilities in structure-based virtual screening, accelerating the discovery and optimization of high-quality lead chemotypes for diverse clinical targets. Compared to traditional high-throughput screening, which is constrained to libraries of approximately one million compounds, the ultra-large virtual screening approach offers substantial advantages in both cost and time efficiency. By expanding the chemical space with compounds synthesized from easily accessible and reproducible reactions and utilizing a large, diverse set of building blocks, we can enhance both the diversity and quality of the discovered lead chemotypes. In this study, we explore new chemical spaces using reactions of sulfur(VI) fluorides to create a combinatorial library consisting of several hundred million compounds. We screened this virtual library for cannabinoid type II receptor (CB2) antagonists using the high-resolution structure in conjunction with a rationally designed antagonist, AM10257. The top-predicted compounds were then synthesized and tested in vitro for CB2 binding and functional antagonism, achieving an experimentally validated hit rate of 55%. Our findings demonstrate the effectiveness of reliable reactions, such as sulfur fluoride exchange, in diversifying ultra-large chemical spaces and facilitate the discovery of new lead compounds for important biological targets.

Discovery of a novel androgen receptor antagonist, MEL-6, with stereoselective activity and optimization of its metabolic stability.

A new chemical scaffold with antagonistic activity towards the androgen receptor (AR) was identified. The parent compound, (3-Methoxy-N-[1-methyl-2-(4-phenyl-1-piperazinyl)-2-(2-thienyl)ethyl]benzamide) referred to as MEL-6, binds in the ligand binding pocket of AR and induces an antagonistic conformation of the ligand binding domain, even in presence of the antagonist-to-agonist switch mutations W741C, T877A and F876L-T877A. MEL-6 has antiproliferative effects on several AR positive prostate cancer cell lines. We further identified AR as the specific target of MEL-6 since it demonstrates little effect on other steroid receptors. In LNCaP cells it also inhibits the androgen-regulated transcriptome. These findings identify MEL-6 as a promising candidate for treatment of patients with prostate tumors that have become resistant to current clinically used AR antagonists. Analytical studies on the chemical composition of MEL-6 identified the presence of four isomers (two enantiomeric pairs), among which one isomer is responsible for the antiandrogenic activity. We therefore developed a synthetic route towards the selective preparation of the active enantiomeric pair. Various MEL-6-like analogues had improved metabolic stability while maintaining antiandrogenic activity. Metabolite identification of MEL-6 derivatives pinpointed N-dealkylation of the piperazine as the main mode for inactivation by liver enzymes. For further structural optimization, MEL-6 derivatives were purchased or synthesized having alterations on the N-phenyl group of the piperazine, the benzoyl group and additionally substituting the thiophen-2-yl ring of MEL-6 to a phenyl ring. This optimization process resulted in compound 12b with sustained AR inhibition and a 4-fold increased half-life due to the 1-(5-chloro-2-methylphenyl)-piperazine substitution, thienyl-to-phenyl substitution and chloro in para-position of the benzoyl group.

Advances in the Synthesis of Fused 1,2,3-Triazoles via a MCR-Intramolecular Azide-Alkyne Cycloaddition Approach.

The present review narrates several reports which deal with the synthesis of fused 1,2,3-triazole containing scaffolds following a sequential multicomponent reaction (MCR)-intramolecular azide-alkyne cycloaddition (IAAC) approach. The reviewed reactions were cleverly designed so as to incorporate azide and alkyne functionalities in the MCR product which was then subjected to IAAC. The review is divided into two sections based on the number of components in the multicomponent reaction. We have aimed at a critical discussion and also have highlighted either advantages or disadvantages of each methodology.

Synthesis of homochiral sulfanyl- and sulfoxide-substituted naphthyltriazoles and study of the conformational stability.

The preparation of a series of novel homochiral atropisomeric sulfanyl- and sulfoxide-substituted naphthyltriazoles is described. The triazolization methodology used presents a new way towards novel and highly stable 1,2,3-triazole-based atropisomers, and introduces a new and complementary synthetic pathway towards 4-sulfanyl substituted 1,2,3-triazoles. Starting from sulfanyl-substituted naphthyl ketones, enantiopure amines, and 4-nitrophenyl azide, a collection of 16 sulfanyl-substituted naphthyltriazoles were obtained via the triazolization reaction in which the homochiral diastereomers are readily isolated. Subsequent monooxidation results in the preparation of several sulfoxide-substituted naphthyltriazoles. The absolute configuration of a set of diastereomeric sulfanyl- and sulfoxide-appended naphthyltriazoles was deduced via X-ray crystallography. Furthermore, the conformational stability of the atropisomers was determined experimentally, and further confirmed and analyzed with the aid of computational DFT calculations.

Betulonic Acid Derivatives Interfering with Human Coronavirus 229E Replication via the nsp15 Endoribonuclease.

To develop antiviral therapeutics against human coronavirus (HCoV) infections, suitable coronavirus drug targets and corresponding lead molecules must be urgently identified. Here, we describe the discovery of a class of HCoV inhibitors acting on nsp15, a hexameric protein component of the viral replication-transcription complexes, endowed with immune evasion-associated endoribonuclease activity. Structure-activity relationship exploration of these 1,2,3-triazolo-fused betulonic acid derivatives yielded lead molecule 5h as a strong inhibitor (antiviral EC50: 0.6 µM) of HCoV-229E replication. An nsp15 endoribonuclease active site mutant virus was markedly less sensitive to 5h, and selected resistance to the compound mapped to mutations in the N-terminal part of HCoV-229E nsp15, at an interface between two nsp15 monomers. The biological findings were substantiated by the nsp15 binding mode for 5h, predicted by docking. Hence, besides delivering a distinct class of inhibitors, our study revealed a druggable pocket in the nsp15 hexamer with relevance for anti-coronavirus drug development.

DNA-SIP and repeated isolation corroborate Variovorax as a key organism in maintaining the genetic memory for linuron biodegradation in an agricultural soil.

The frequent exposure of agricultural soils to pesticides can lead to microbial adaptation, including the development of dedicated microbial populations that utilize the pesticide compound as a carbon and energy source. Soil from an agricultural field in Halen (Belgium) with a history of linuron exposure has been studied for its linuron-degrading bacterial populations at two time points over the past decade and Variovorax was appointed as a key linuron degrader. Like most studies on pesticide degradation, these studies relied on isolates that were retrieved through bias-prone enrichment procedures and therefore might not represent the in situ active pesticide-degrading populations. In this study, we revisited the Halen field and applied, in addition to enrichment-based isolation, DNA stable isotope probing (DNA-SIP), to identify in situ linuron-degrading bacteria in linuron-exposed soil microcosms. Linuron dissipation was unambiguously linked to Variovorax and its linuron catabolic genes and might involve the synergistic cooperation between two species. Additionally, two novel linuron-mineralizing Variovorax isolates were obtained with high 16S rRNA gene sequence similarity to strains isolated from the same field a decade earlier. The results confirm Variovorax as a prime in situ degrader of linuron in the studied agricultural field soil and corroborate the genus as key for maintaining the genetic memory of linuron degradation functionality in that field.

Culture-Independent Analysis of Linuron-Mineralizing Microbiota and Functions in on-Farm Biopurification Systems via DNA-Stable Isotope Probing: Comparison with Enrichment Culture.

Our understanding of the microorganisms involved in in situ biodegradation of xenobiotics, like pesticides, in natural and engineered environments is poor. On-farm biopurification systems (BPSs) treat farm-produced pesticide-contaminated wastewater to reduce surface water pollution. BPSs are a labor and cost-efficient technology but are still mainly operated as black box systems. We used DNA-stable isotope probing (DNA-SIP) and classical enrichment to be informed about the organisms responsible for in situ degradation of the phenylurea herbicide linuron in a BPS matrix. DNA-SIP identified Ramlibacter, Variovorax, and an unknown Comamonadaceae genus as the dominant linuron assimilators. While linuron-degrading Variovorax strains have been isolated repeatedly, Ramlibacter has never been associated before with linuron degradation. Genes and mobile genetic elements (MGEs) previously linked to linuron catabolism were enriched in the heavy DNA-SIP fractions, suggesting their involvement in in situ linuron assimilation. BPS material free cultivation of linuron degraders from the same BPS matrix resulted in a community dominated by Variovorax, while Ramlibacter was not observed. Our study provides evidence for the role of Variovorax in in situ linuron biodegradation in a BPS, alongside other organisms like Ramlibacter, and further shows that cultivation results in a biased representation of the in situ linuron-assimilating bacterial populations.

Synthesis and post-functionalization of alternate-linked-meta-para-[2 n .1 n ]thiacyclophanes.

In recent decades, considerable research attention has been devoted to new synthetic procedures for thiacyclophanes. Thiacyclophanes are widely used as host molecules for the molecular recognition of organic compounds as well as metals. Herein, we report the selective and high-yielding synthesis of novel alternate-linked-meta-para-thiacyclophanes. These novel thiacyclophanes are selectively synthesized in high-yielding procedures. Furthermore, post-functionalization of the phenolic moieties was successfully performed. The 3D structure of the alternate-linked-meta-para-[22.12]thiacyclophane was further elucidated via X-ray crystallographic analysis.

Regioselective Synthesis of Fluorosulfonyl 1,2,3-Triazoles from Bromovinylsulfonyl Fluoride.

A regioselective metal-free preparation of 4-fluorosulfonyl 1,2,3-triazoles from organic azides and a hitherto underexplored bromovinylsulfonyl fluoride building block is described. This reaction is very general and was extended to the synthesis of various sulfonates, sulfonamides, and sulfonic acid derivatives of triazoles and other azole heterocycles which would otherwise be difficult to access by existing methods.

Sequential Ugi reaction/base-induced ring closing/IAAC protocol toward triazolobenzodiazepine-fused diketopiperazines and hydantoins.

A practical three-step protocol for the assembly of triazolobenzodiazepine-fused diketopiperazines and hydantoins has been developed. The synthesis of these tetracyclic ring systems was initiated by an Ugi reaction, which brought together all necessary functionalities for further transformations. The Ugi adducts were then subjected to a base-induced ring closing and an intramolecular azide-alkyne cycloaddition reaction in succession to obtain highly fused benzodiazepine frameworks.

Straightforward synthesis of enantiomerically pure 1,2,3-triazoles derived from amino esters.

A practical and straightforward approach that enables, for the first time, the synthesis of enantiomerically pure 1,4,5-trisubstituted, 1,5-disubstituted, and fused 1,2,3-triazole derivatives has been developed. The synthesis employs enantiomerically pure amino esters derived from amino acids and commercially available ketones under metal-free conditions.

2-Amino-3-methylcarboxy-5-heptyl-thiophene (TJ191) is a selective anti-cancer small molecule that targets low TßRIII-expressing malignant T-cell leukemia/lymphoma cells.

Current chemotherapy regimens often include non-specific cytostatic/cytotoxic drugs, which do not distinguish between normal and tumor cells, therefore causing considerable systemic toxicity. We previously reported the synthesis and anti-proliferative activity of a novel synthetic 2-aminothiophene-3-carboxylic acid ester derivative TJ191 that selectively targets certain cancer cells without affecting the proliferation of other cancer cells or normal fibroblasts or immune cells (over 600-fold selectivity). In a panel of ten human T-cell leukemia/lymphoma cell lines and peripheral blood mononuclear cells (PBMCs), we now found that transforming growth factor ß type III receptor (TßRIII) expression correlates inversely with TJ191 sensitivity, but not with sensitivity against classical chemotherapeutic drugs, thus serving as a predictive marker for TJ191 sensitivity. Accordingly, CRISPR/Cas9-mediated knock-out of TßRIII partially restored the susceptibility of TJ191-resistant cells to this novel compound. Our findings highlight TJ191 as a potent and selective anti-cancer molecule with pronounced activity against human malignant T-cells expressing low levels of TßRIII.

Synthesis and anticancer activity of novel aza-artemisinin derivatives.

Three series of aza-artemisinin derivatives were synthesized for studies of anticancer activity. The first series of compounds were prepared via copper(I)-catalyzed azide alkyne cycloaddition, so called "click reaction", starting from propargyl derivatives of 11-aza-artemisinin and various azides, whereas the second and third series of compounds were prepared by triazolization reaction starting from enolizable ketones and primary amines connected to artemisinin. In vitro studies of the 23 synthesized artemisinin derivatives unveiled that 9 compounds displayed antiproliferative activity in the low micromolar range, with 5d being the most promising compound showing 50% inhibition of Cem and HeLa cell growth at 0.92 and 1.2µM, respectively.

A new four-component reaction involving the Michael addition and the Gewald reaction, leading to diverse biologically active 2-aminothiophenes.

The Gewald three-component reaction yielding highly substituted 2-aminothiophene heterocycles has been known for a long time and holds an extraordinary potential in the pharmaceutical industry. Herein, we describe a four-component reaction initiated by the conjugate addition of different indole derivatives to α,ß-unsaturated carbonyl compounds. This is followed by an in situ Gewald three-component reaction which results in the formation of a compound containing an indole and a 2-aminothiophene moiety separated by a methylene spacer. We also examined the impact of the use of other nucleophilic components such as pyrrole derivatives on this MG-4CR (Michael-Gewald four component reaction). All these synthesized compounds were tested for anti-proliferative activity and three of them showed activity in the nanomolar range.

Pronounced anti-proliferative activity and tumor cell selectivity of 5-alkyl-2-amino-3-methylcarboxylate thiophenes.

5-(2-(4-Methoxyphenyl)ethyl)-2-amino-3-methylcarboxylate thiophene (TR560) is the prototype drug of a recently discovered novel class of tumor-selective compounds that preferentially inhibit the proliferation of specific tumor cell types (e.g. leukemia/lymphoma). Here, we further increased tumor selectivity by simplification of the molecule through replacing the 4-methoxyphenyl moiety by an alkyl chain. Several 2-amino-3-methylcarboxylate thiophene derivatives containing at C-5 an alkyl group consisting of at least 6 (hexyl) to 9 (nonyl) carbon units showed pronounced anti-proliferative activity in the mid-nanomolar range with 500- to 1000-fold tumor cell selectivity. The compounds preferentially inhibited the proliferation of T-lymphoma CEM and Molt/4, prostate PC-3, kidney Caki-1 and hepatoma Huh-7 tumor cells, but were virtually inactive against other tumor cell lines including B-lymphoma Raji and cervix carcinoma HeLa cells. The novel prototype drug 3j (containing a 5-heptyl chain) elicited a cytotoxic, rather than cytostatic activity, already after 4 h of exposure. The unusual tumor selectivity could not be explained by a differential uptake (or efflux) of the drug by sensitive versus resistant tumor cells. Exposure of a fluorescent derivative of 3j revealed pronounced uptake of the drug in the cytoplasm, no visible appearance in the nucleus, and a predominant localization in the endoplasmic reticulum. These observations may be helpful to narrow down the intracellular localization and identification of the molecular target of the 5-substituted thiophene derivatives.

Selective alkaline stripping of metal ions after solvent extraction by base-stable 1,2,3-triazolium ionic liquids.

Novel 1,2,3-triazolium ionic liquids with a high base stability were synthesized for use in solvent extraction of first-row transition elements and rare earths from chloride media. The synthesis of these ionic liquids makes use of a recently reported, metal-free multicomponent reaction that allows full substitution of the 1,2,3-triazolium skeleton. The physical and chemical properties of these ionic liquids are compared with those of a trisubstituted analog. Peralkylation of the 1,2,3-triazolium skeleton leads to ionic liquids with superior properties, such as low viscosity, low solubility in water and higher thermal and base stability. Iodide and thiocyanate ionic liquids with peralkylated cations were applied to the solvent extraction of metal ions, and their stability in alkaline media was exploited in the selective stripping of the metals from the loaded ionic liquid phase by alkaline solutions. EXAFS and Raman spectroscopy were performed to gain insight into the extraction mechanism. The applicability of these extraction systems was demonstrated in separations relevant for the recovery of metals from ores and end-of-life products: Fe(iii)/Cu(ii)/Zn(ii) (copper ores, brass scraps) and Fe(iii)/Nd(iii) (rare earth magnets).

Application of the Triazolization Reaction to Afford Dihydroartemisinin Derivatives with Anti-HIV Activity.

Artemisinin and synthetic derivatives of dihydroartemisinin are known to possess various biological activities. Post-functionalization of dihydroartemisinin with triazole heterocycles has been proven to lead to enhanced therapeutic potential. By using our newly developed triazolization strategy, a library of unexplored fused and 1,5-disubstituted 1,2,3-triazole derivatives of dihydroartemisinin were synthesized in a single step. All these newly synthesized compounds were characterized and evaluated for their anti-HIV (Human Immunodeficiency Virus) potential in MT-4 cells. Interestingly; three of the synthesized triazole derivatives of dihydroartemisinin showed activities with half maximal inhibitory concentration (IC50) values ranging from 1.34 to 2.65 µM.

Acid-Sensitive BODIPY Dyes: Synthesis through Pd-Catalyzed Direct C(sp3 )-H Arylation and Photophysics.

A novel palladium-catalyzed direct C(sp3 )-H arylation of the methyl group at the 8-position of BODIPY by bromoarenes was established. A deprotonative cross-coupling process was supposed to be involved in the reaction. This approach allowed us to attach electron-donating/withdrawing, halogen substituted aryls and a heteroaryl with a yield running from 55 to 99 %. Novel pH sensors, which in the absence of acid showed the occurrence of photoinduced electron transfer, were synthesized by attaching dimethylaniline to the methyl at the C8-position of BODIPY. The reference compounds with dimethylaniline directly attached to the C8-position were also synthesized and besides photoinduced electron transfer also showed a charge-transfer emission. Their photophysical properties were investigated by steady-state fluorescence, time-correlated single-photon counting and femtosecond fluorescence up-conversion. Time-dependent density functional (TD-DFT) electronic-structure calculations on the properties of the excited states corresponding to local excitation of the BODIPY core and to charge transfer were conducted. Upon addition of trifluoroacetic acid in toluene and ethanol, the partial fluorescence intensity recovery was at least an order of magnitude more efficient with the newly synthesized sensors compared to the traditional reference sensors. The improved sensitivity of these novel BODIPY-based pH sensors was attributed to less efficient proton-coupled electron transfer of the protonated species.

A One-Pot Procedure for the Synthesis of "Click-Ready" Triazoles from Ketones.

A practical, straightforward, and highly regioselective Zn(OAc)2-mediated method toward propargyl triazoles has been developed for the first time from commercially available enolizable ketones and propargyl amine. Postfunctionalization of this triazole leads to unique N- and C-linked bis-triazoles in excellent yields.

A single-step acid catalyzed reaction for rapid assembly of NH-1,2,3-triazoles.

NH-1,2,3-Triazole moieties are a part of the design of various biologically active compounds, pharmaceutical agents and functional materials. Unfortunately, the applications of this heterocycle are still underexplored due to the lack of a general synthetic protocol. Here we outline a novel, general and facile metal-free pathway that enables the direct synthesis of these heterocycles by combining readily accessible and abundant precursors such as enolizable ketones and NH4OAc with high levels of regioselectivity via an organocascade process. The developed chemistry has been successfully applied to the synthesis of several structurally diverse products, pharmaceutical agents and supramolecular receptors.