Synthesis of Benzofuran,Benzothiophene, and Benzothiazole‐Based Thioamides and their Evaluation as KATP Channel Openers

Several series of benzofurans, benzothiophenes, and benzothiazoles, all featuring the thioamide group, were synthesized and tested as novel K_ATP channel openers in artificial cell systems: CHO cells transfected with SUR1/Kir6.2, and HEK 293 cells transfected with SUR2B/Kir6.1; these served as model systems for insulin‐secreting pancreatic β cells and smooth muscle cells, respectively. All compounds were investigated with respect to their binding affinity for the SUR2B‐type K_ATP channels using [³H]P1075 as radioligand. Selected compounds were also tested as agonists in intact cells using DiBAC₄(3) and DyeB (R7260) as membrane potential dyes. Remarkable affinity for SUR2B/Kir6.1 channels in the single‐digit micromolar range was observed. In addition, benzothiazole‐derived thioamides with sterically demanding, lipophilic substituents showed >100‐fold selectivity in favor of SUR2B/Kir6.1. A one‐carbon spacer between the heterocyclic skeleton and the thioamide moiety was observed to be crucial for affinity and selectivity. Two of the most potent and selective compounds were studied by crystal structure analyses.     

Rational Design,Synthesis and Biological Evaluation of Modular Fluorogenic Substrates with High Affinity and Selectivity for PTP1B

Protein‐tyrosine phosphatase 1B (PTP1B) is a key regulatory enzyme in several signal transduction pathways, and its upregulation has been associated with type‐2 diabetes, obesity and cancer. Selective determination of the functional significance of PTP1B remains a major challenge because the activity of this crucial enzyme is currently evaluated through the use of fluorescent probes that lack selectivity and are limited to biochemical assays. Here we describe the rational design, synthesis and biological evaluation of new modular PTP1B fluorogenic substrates. The self‐immolative 4‐hydroxybenzyl alcohol has been used as a key component for the design of phosphotyrosine mimics linked to a latent chromophore, which is released through an enzyme‐initiated domino reaction. Preliminary biological investigations showed that, by optimising the stereoelectronic properties and the binding interactions at the enzyme active site, it is possible to achieve substrates with high affinity and promising selectivity. Due to their modular nature, the synthesised fluorogenic probes represent versatile tools; customisation of the different subunits could widen the scope of these probes to a broader range of in vitro assays. Finally, these studies elucidate the critical role played by Asp181 in the PTP1B‐catalysed dephosphorylation mechanism: disruption of the native conformation of this key amino acid residue on the WDP loop yields fluorogenic inhibitors, rather than substrates. For this reason, our studies also represent a step forward for the development of improved PTP1B noncovalent inhibitors.     

A One‐Pot “Triple‐C” Multicyclization Methodology for the Synthesis of Highly Constrained Isomerically Pure Tetracyclic Peptides

A broadly applicable one‐pot methodology for the facile transformation of linear peptides into tetracyclic peptides through a chemoenzymatic peptide synthesis/chemical ligation of peptides onto scaffolds/copper(I)‐catalyzed reaction (CEPS/CLIPS/CuAAC; “triple‐C”) locking methodology is reported. Linear peptides with varying lengths (≥14 amino acids), comprising two cysteines and two azidohomoalanines (Aha), were efficiently cyclized head‐to‐tail by using the peptiligase variant omniligase‐1 (CEPS). Subsequent ligation–cyclization with tetravalent (T4_1/2) scaffolds containing two bromomethyl groups (CLIPS) and two alkyne functionalities (CuAAC) yielded isomerically pure tetracyclic peptides. Sixteen different functional tetracycles, derived from bicyclic inhibitors against urokinase plasminogen activator (uPA) and coagulation factor XIIa (FXIIa), were successfully synthesized and their bioactivities evaluated. Two of these (FF‐T4_1/2) exhibited increased inhibitory activity against FXIIa, compared with a bicyclic control peptide. The corresponding hetero‐bifunctional variants (UF/FU‐T4_1/2), with a single copy of each inhibitory sequence, exhibited micromolar activities against both uPA and FXIIa; thus illustrating the potential of the “bifunctional tetracyclic peptide” inhibitor concept.     

Design and Synthesis of Trypanosoma brucei Active 1‐Alkyloxy and 1‐Benzyloxyadamantano 2‐Guanylhydrazones

Treating African trypanosomiasis : The synthesis and biological evaluation of novel 1‐alkyloxy and 1‐benzyloxyadamantano 2‐guanylhydrazones, their 1‐dioxa congeners and two 1‐benzyladamantano 2‐guanylhydrazones is reported. Preliminary structure–activity relationship data were elucidated and lead compounds suitable for further optimization were discovered.

     

Evaluation of (4‐Arylpiperidin‐1‐yl)cyclopentanecarboxamides As High‐Affinity and Long‐Residence‐Time Antagonists for the CCR2 Receptor

Animal models suggest that the chemokine ligand 2/CC‐chemokine receptor 2 (CCL2/CCR2) axis plays an important role in the development of inflammatory diseases. However, CCR2 antagonists have failed in clinical trials because of a lack of efficacy. We previously described a new approach for the design of CCR2 antagonists by the use of structure–kinetics relationships (SKRs). Herein we report new findings on the structure–affinity relationships (SARs) and SKRs of the reference compound MK‐0483, its diastereomers, and its structural analogues as CCR2 antagonists. The SARs of the 4‐arylpiperidine group suggest that lipophilic hydrogen‐bond‐accepting substituents at the 3‐position are favorable. However, the SKRs suggest that a lipophilic group with a certain size is desired [e.g., 3‐Br: K_i=2.8 nM , residence time (t_res)=243 min; 3‐iPr: K_i=3.6 nM , t_res=266 min]. Alternatively, additional substituents and further optimization of the molecule, while keeping a carboxylic acid at the 3‐position, can also prolong t_res; this was most prominently observed in MK‐0483 (K_i=1.2 nM , t_res=724 min) and a close analogue (K_i=7.8 nM ) with a short residence time.     

Synthesis, σ Receptor Affinity,and Pharmacological Evaluation of 5‐Phenylsulfanyl‐ and 5‐Benzyl‐Substituted Tetrahydro‐2‐benzazepines

In accordance with a novel strategy for generating the 2‐benzazepine scaffold by connecting C6–C1 and C3–N building blocks, a set of 5‐phenylsulfanyl‐ and 5‐benzyl‐substituted tetrahydro‐2‐benzazepines was synthesized and pharmacologically evaluated. Key steps of the synthesis were the Heck reaction, the Stetter reaction, a reductive cyclization, and the introduction of diverse N substituents at the end of the synthesis. High σ₁ affinity was achieved for 2‐benzazepines with linear or branched alk(en)yl residues containing at least an n‐butyl substructure. The butyl‐ and 4‐fluorobenzyl‐substituted derivatives, (±)‐5‐benzyl‐2‐butyl‐2,3,4,5‐tetrahydro‐1H‐2‐benzazepine ( 19 b ) and (±)‐5‐benzyl‐2‐(4‐fluorobenzyl)‐2,3,4,5‐tetrahydro‐1H‐2‐benzazepine ( 19 m ), show high selectivity over more than 50 other relevant targets, including the σ₂ subtype and various binding sites of the N‐methyl‐D ‐aspartate (NMDA) receptor. In the Irwin screen, 19 b and 19 m showed clean profiles without inducing considerable side effects. Compounds 19 b and 19 m did not reveal significant analgesic and cognition‐enhancing activity. Compound 19 m did not have any antidepressant‐like effects in mice.     

A Novel Competitive Class of α‐Glucosidase Inhibitors: (E)‐1‐Phenyl‐3‐(4‐Styrylphenyl)Urea Derivatives

Competitive glycosidase inhibitors are generally sugar mimics that are costly and tedious to obtain because they require challenging and elongated chemical synthesis, which must be stereo‐ and regiocontrolled. Here, we show that readily accessible achiral (E)‐1‐phenyl‐3‐(4‐strylphenyl)ureas are potent competitive α‐glucosidase inhibitors. A systematic synthesis study shows that the 1‐phenyl moiety on the urea is critical for ensuring competitive inhibition, and substituents on both terminal phenyl groups contribute to inhibition potency. The most potent inhibitor, compound 12 (IC₅₀=8.4 μM , K_i=3.2 μM ), manifested a simple slow‐binding inhibition profile for α‐glucosidase with the kinetic parameters k₃=0.005256 μM ^?1 min^?1, k₄=0.003024 min^?1, and ${K{{{\rm app}\hfill \atop {\rm i}\hfill}}}$ =0.5753 μM .     

Synthesis and Structure–Activity Relationships of 1‐Benzyl‐4,5,6‐trimethoxyindoles as a Novel Class of Potent Antimitotic Agents

Combretastatin A‐4 derivatives : A series of combretastatin A‐4‐derived 1‐benzyl‐4,5,6‐trimethoxyindoles was designed and prepared as a novel class of potent antimitotic agents acting through the colchicine binding site on the microtubule.

     

Synthesis and Biological Characterization of (3R,4R)‐4‐(2‐(Benzhydryloxy)ethyl)‐1‐((R)‐2‐hydroxy‐2‐phenylethyl)‐piperidin‐3‐ol and Its Stereoisomers for Activity toward Monoamine Transporters

A novel series of optically active molecules based on a 4‐(2‐(benzhydryloxy)ethyl)‐1‐((R)‐2‐hydroxy‐2‐phenylethyl)‐piperidin‐3‐ol template were developed. Depending on stereochemistry, the compounds exhibit various degrees of affinity for three dopamine, serotonin, and norepinephrine transporters. These molecules have the potential for treating several neurological disorders such as drug abuse, depression, and attention deficit hyperactivity disorder.

     

Heme Oxygenase Inhibition by 1‐Aryl‐2‐(1H‐imidazol‐1‐yl/1H‐1,2,4‐triazol‐1‐yl)ethanones and Their Derivatives

Previous studies by our research group have been concerned with the design of selective inhibitors of heme oxygenases (HO‐1 and HO‐2). The majority of these were based on a four‐carbon linkage of an azole, usually an imidazole, and an aromatic moiety. In the present study, we designed and synthesized a series of inhibition candidates containing a shorter linkage between these groups, specifically, a series of 1‐aryl‐2‐(1H‐imidazol‐1‐yl/1H‐1,2,4‐triazol‐1‐yl)ethanones and their derivatives. As regards HO‐1 inhibition, the aromatic moieties yielding best results were found to be halogen‐substituted residues such as 3‐bromophenyl, 4‐bromophenyl, and 3,4‐dichlorophenyl, or hydrocarbon residues such as 2‐naphthyl, 4‐biphenyl, 4‐benzylphenyl, and 4‐(2‐phenethyl)phenyl. Among the imidazole‐ketones, five ( 36 – 39 , and 44 ) were found to be very potent (IC₅₀<5 μM ) toward both isozymes. Relative to the imidazole‐ketones, the series of corresponding triazole‐ketones showed four compounds ( 54 , 55 , 61 , and 62 ) having a selectivity index >50 in favor of HO‐1. In the case of the azole‐dioxolanes, two of them ( 80 and 85 ), each possessing a 2‐naphthyl moiety, were found to be particularly potent and selective HO‐1 inhibitors. Three non‐carbonyl analogues ( 87 , 89 , and 91 ) of 1‐(4‐chlorophenyl)‐2‐(1H‐imidazol‐1‐yl)ethanone were found to be good inhibitors of HO‐1. For the first time in our studies, two azole‐based inhibitors ( 37 and 39 ) were found to exhibit a modest selectivity index in favor of HO‐2. The present study has revealed additional candidates based on inhibition of heme oxygenases for potentially useful pharmacological and therapeutic applications.     

Synthesis and in Vitro Characterisation of Ifenprodil‐Based Fluorescein Conjugates as GluN1/GluN2B N‐Methyl‐D‐aspartate Receptor Antagonists

GluN2B‐containing NMDA receptors are involved in many important physiological functions and play a pivotal role in mediating pain as well as in several neurodegenerative disorders. We aimed to develop fluorescent probes to target the GluN2B subunit selectively in order to allow better understanding of the relationships between receptor localisation and physiological importance. Ifenprodil, known as the GluNR2B antagonist of reference, was chosen as the template for the elaboration of probes. We had previously reported a fluorescein conjugate that was shown (by confocal microscopy imaging of DS‐red‐labelled cortical neurons) to bind specifically to GluN2B. To elaborate this probe, we explored the influence of both the nature and the attachment point of the spacer between the fluorophore and the parent compound, ifenprodil. We performed chemical modifications of ifenprodil at the benzylic position and on the phenol ring by introducing secondary amine or amide functions and evaluated alkyl chains from two to 20 bonds either including or not including secondary amide functions as spacers. The previously developed probe was found to display the greatest activity in the inhibition of NMDA‐induced Ca²⁺ influx by calcium imaging experiments on HEK293 cells transfected with the cDNA encoding for GluN1‐1A and GluN2B. Further investigations revealed that this probe had a neuroprotective effect equivalent to that of ifenprodil in a standard test for neurotoxicity. Despite effects of lesser amplitude with these probes relative to ifenprodil, we demonstrated that they displaced [³H]ifenprodil in mouse brain slices in a similar manner.     

(2,2‐Diphenyl‐[1,3]oxathiolan‐5‐ylmethyl)‐(3‐phenyl‐propyl)‐amine: a Potent and Selective 5‐HT1A Receptor Agonist

A selective 5‐HT _1A receptor agonist : A new series of ligands acting at 5‐HT_1A serotonin receptor were identified. Among them (2,2‐diphenyl‐[1,3]oxathiolan‐5‐yl‐methyl)‐(3‐phenyl‐propyl)amine (shown) possesses outstanding activity (pK_i=8.72, pD₂=7.67, E_max=85) and selectivity (5‐HT_1A/α_1D>150), and represents a new 5‐HT_1A agonist chemotype.

     

Rational Design,Synthesis, and Biological Evaluation of Lactam‐Bridged Gramicidin A Analogues: Discovery of a Low‐Hemolytic Antibacterial Peptide

A linear peptide, gramicidin A (GA), folds into a β^6.3‐helix, functions as an ion channel in the cell membrane, and exerts antibacterial activity. Herein we describe the rational design, synthesis, and biological evaluation of lactam‐bridged GA analogues. The GA analogue with a 27‐membered macrolactam was found to adopt a stable β^6.3‐helical conformation and exhibits higher ion‐exchange activity than GA. Furthermore, this GA analogue retains the potent antibiotic activity of GA, but its hemolytic activity and toxicity toward mammalian cells are significantly lower than those of GA. This study thus dissociates the antibacterial and hemolytic/cytotoxic activities of GA, and charts a rational path forward for the development of new ion‐channel‐based antibiotics.     

Defining the Potential of Aglycone Modifications for Affinity/Selectivity Enhancement against Medically Relevant Lectins: Synthesis,Activity Screening,and HSQC‐Based NMR Analysis

The emerging significance of lectins for pathophysiological processes provides incentive for the design of potent inhibitors. To this end, systematic assessment of contributions to affinity and selectivity by distinct types of synthetic tailoring of glycosides is a salient step, here taken for the aglyconic modifications of two disaccharide core structures. Firstly we report the synthesis of seven N‐linked‐lactosides and of eight O‐linked N‐acetyllactosamines, each substituted with a 1,2,3‐triazole unit, prepared by copper‐catalyzed azide–alkyne cycloaddition (CuAAC). The totally regioselective β‐D ‐(1→4) galactosylation of a 6‐O‐TBDPSi‐protected N‐acetylglucosamine acceptor provided efficient access to the N‐acetyllactosamine precursor. The resulting compounds were then systematically tested for lectin reactivity in two binding assays of increasing biorelevance (inhibition of lectin binding to a surface‐presented glycoprotein and to cell surfaces). As well as a plant toxin, we also screened the relative inhibitory potential with adhesion/growth‐regulatory galectins (total of eight proteins). This type of modification yielded up to 2.5‐fold enhancement for prototype proteins, with further increases for galectins‐3 and ‐4. Moreover, the availability of ¹⁵N‐labeled proteins and full assignments enabled ¹H,¹⁵N HSQC‐based measurements for hu‐ man galectins‐1, ‐3, and ‐7 against p‐nitrophenyl lactopyranoside, a frequently tested standard inhibitor containing an aromatic aglycone. The measurements confirmed the highest affinity against galectin‐3 and detected chemical shift differences in its hydrophobic core upon ligand binding, besides common alterations around the canonical contact site for the lactoside residue. What can be accomplished in terms of affinity/selectivity by this type of core extension having been determined, the applied combined strategy should be instrumental for proceeding with defining structure–activity correlations at other bioinspired sites in glycans and beyond the tested lectin types.     

siRNA Modified with 2′‐Deoxy‐2′‐C‐methylpyrimidine Nucleosides

(2′S)‐2′‐Deoxy‐2′‐C‐methyluridine and (2′R)‐2′‐deoxy‐2′‐C‐methyluridine were incorporated in the 3′‐overhang region of the sense and antisense strands and in positions 2 and 5 of the seed region of siRNA duplexes directed against Renilla luciferase, whereas (2′S)‐2′‐deoxy‐2′‐C‐methylcytidine was incorporated in the 6‐position of the seed region of the same constructions. A dual luciferase reporter assay in transfected HeLa cells was used as a model system to measure the IC₅₀ values of 24 different modified duplexes. The best results were obtained by the substitution of one thymidine unit in the antisense 3′‐overhang region by (2′S)‐ or (2′R)‐2′‐deoxy‐2′‐C‐methyluridine, reducing IC₅₀ to half of the value observed for the natural control. The selectivity of the modified siRNA was measured, it being found that modifications in positions 5 and 6 of the seed region had a positive effect on the ON/OFF activity.     

α‐Bromoacrylamido N‐Substituted Isatin Derivatives as Potent Inducers of Apoptosis in Human Myeloid Leukemia Cells

A novel series of α‐bromoacryloyl N‐substituted isatin analogues were found to inhibit the growth and viability of human myeloid leukemia HL‐60 and U‐937 cells as well as human lymphoid leukemia MOLT‐3 cells. Cell death induced by these molecules was preceded by a rapid release of cytochrome c from mitochondria into the cytosol and subsequent caspase activation involving caspase‐3, to cleave poly(ADP‐ribose) polymerase (PARP). These findings suggest that these compounds present antiproliferative activity which may be mediated by apoptosis caused by cytochrome c release and caspase activation in human leukemia cells.