Different Binding Modes of Small and Large Binders of GAT1

Well‐known inhibitors of the γ‐aminobutyric acid (GABA) transporter GAT1 share a common scaffold of a small cyclic amino acid linked by an alkyl chain to a moiety with two aromatic rings. Tiagabine, the only FDA‐approved GAT1 inhibitor, is a typical example. Some small amino acids such as (R)‐nipecotic acid are medium‐to‐strong binders of GAT1, but similar compounds, such as proline, are very weak binders. When substituted with 4,4‐diphenylbut‐3‐en‐1‐yl (DPB) or 4,4‐bis(3‐methylthiophen‐2‐yl)but‐3‐en‐1‐yl (BTB) groups, the resulting compounds have similar pK_i and pIC₅₀ values, even though the pure amino acids have very different values. To investigate if small amino acids and their substituted counterparts share a similar binding mode, we synthesized butyl‐, DPB‐, and BTB‐substituted derivatives of small amino acids. Supported by the results of docking studies, we propose different binding modes not only for unsubstituted und substituted, but also for strong‐ and weak‐binding amino acids. These data lead to the conclusion that following a fragment‐based approach, not pure but N‐butyl‐substituted amino acids should be used as starting points, giving a better estimate of the activity when a BTB or DPB substituent is added.     

Design,Synthesis, in vitro MAO‐B Inhibitory Evaluation,and Computational Studies of Some 6‐Nitrobenzothiazole‐Derived Semicarbazones

Monoamine oxidase B (MAO‐B) is an important drug target for the treatment of neurological disorders. A series of 6‐nitrobenzothiazole‐derived semicarbazones were designed, synthesized, and evaluated as inhibitors of the rat brain MAO‐B isoenzyme. Most of the compounds were found to be potent inhibitors of MAO‐B, with IC₅₀ values in the nanomolar to micromolar range. Molecular docking studies were performed with AutoDock 4.2 to deduce the affinity and binding mode of these inhibitors toward the MAO‐B active site. The free energies of binding (ΔG) and inhibition constants (K_i) of the docked compounds were calculated by the Lamarckian genetic algorithm (LGA) of AutoDock 4.2. Good correlations between the calculated and experimental results were obtained. 1‐[(4‐Chlorophenyl)(phenyl)methylene]‐4‐(6‐nitrobenzothiazol‐2‐yl)semicarbazide emerged as the lead MAO‐B inhibitor, with top ranking in both the experimental MAO‐B assay (IC₅₀: 0.004±0.001 μM ) and in computational docking studies (K_i: 1.08 μM ). Binding mode analysis of potent inhibitors suggests that these compounds are well accommodated by the MAO‐B active site through stable hydrophobic and hydrogen bonding interactions. Interestingly, the 6‐nitrobenzothiazole moiety is stabilized in the substrate cavity with the aryl or diaryl residues extending up into the entrance cavity of the active site. According to our results, docking experiments could be an interesting approach for predicting the activity and binding interactions of this class of semicarbazones against MAO‐B. Thus, a binding site model consisting of three essential pharmacophoric features is proposed, and this can be used for the design of future MAO‐B inhibitors.     

Functionalization of high‐density polyethylene in the molten state by glycidyl methacrylate grafting

:This study concerns the melt‐free radical grafting of glycidyl methacrylate (GMA) onto high‐density polyethylene (HDPE). We studied the effect of two initiators (tert‐butyl cumyl peroxide and di‐tert‐butyl peroxide) onto HDPE. Crosslinking of polymer was observed in the presence of 0.3 wt % tert‐butyl cumyl peroxide but not with 0.3 wt % di‐tert‐butyl peroxide. The grafting was carried out in a Brabender batch mixer at 190 °C. The grafting yield of GMA onto HDPE (determined by infrared spectrometry) is weak (<1 wt % for an initial concentration in monomer of 6 wt %). Moreover, it was noted that the degree of grafting did not vary with the concentration and the nature of peroxide used. To increase the grafting yield of GMA, we added to the HDPE/peroxide/GMA system an electron‐donating monomer, such as styrene. Adding this comonomer multiplied the rate of grafted GMA 3‐ or 4‐fold, resulting in a ratio [styrene]_i/[GMA]_i = 1 mol/mol with [GMA]_i = 6 wt %. So, the copolymerization is favored compared with the homopolymerization. This kind of copolymer presenting reactive functions is very attractive in the field of compatibilizing immiscible polymers. © 2001 John Wiley & Sons, Inc. J Appl Polym Sci 81: 581–590, 2001     

Synthesis and Pharmacological Evaluation of a series of the Agomelatine Analogues as Melatonin MT1/MT2 Agonist and 5‐HT2C Antagonist

Agomelatine is a naphthalenic analogue of melatonin that is in clinical use for the treatment of major depressive disorders. Interestingly, while agomelatine exhibits potent affinity for melatonin receptors, it binds with only moderate affinity to the serotonin 5‐HT_2C receptor. Optimization of agomelatine toward this target could further potentiate its clinical efficacy. To explore this hypothesis and to access derivatives in which a key point of agomelatine metabolism is blocked, a series of naphthalenic derivatives was designed and synthesized as novel analogues of agomelatine. Most of the prepared compounds exhibited good binding affinity at the melatonin MT₁ and MT₂ receptor subtypes. Two compounds, an acetamide and an acrylamide derivative, exhibited good binding affinities at both the human melatonin (MT) receptors and the serotonin 5‐HT_2C receptor subtype, with pK_i values of 7.96 and 7.95 against MT₁, 7.86 and 8.68 against MT2, and 6.64 and 6.44 against 5‐HT_2C, respectively.     

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.     

Design, synthesis, and pharmacological characterization of novel spirocyclic quinuclidinyl-Δ2-isoxazoline derivatives as potent and selective agonists of α7 nicotinic acetylcholine receptors

A set of racemic spirocyclic quinuclidinyl‐Δ²‐isoxazoline derivatives was synthesized using a 1,3‐dipolar cycloaddition‐based approach. Target compounds were assayed for binding affinity toward rat neuronal homomeric (α7) and heteromeric (α4β2) nicotinic acetylcholine receptors. Δ²‐Isoxazolines 3 a (3‐Br), 6 a (3‐OMe), 5 a (3‐Ph), 8 a (3‐OnPr), and 4 a (3‐Me) were the ligands with the highest affinity for the α7 subtype (K_i values equal to 13.5, 14.2, 25.0, 71.6, and 96.2 nM , respectively), and showed excellent α7 versus α4β2 subtype selectivity. These compounds, tested in electrophysiological experiments against human α7 and α4β2 receptors stably expressed in cell lines, behaved as partial α7 agonists with varying levels of potency. The two enantiomers of (±)‐3‐methoxy‐1‐oxa‐2,7‐diaza‐7,10‐ethanospiro[4.5]dec‐2‐ene sesquifumarate 6 a were prepared using (+)‐dibenzoyl‐L ‐ or (?)‐dibenzoyl‐D ‐tartaric acid as resolving agents. Enantiomer (R)‐(?)‐ 6 a was found to be the eutomer, with K_i values of 4.6 and 48.7 nM against rat and human α7 receptors, respectively.     

E‐64c‐Hydrazide: A Lead Structure for the Development of Irreversible Cathepsin C Inhibitors

Cathepsin C is a papain‐like cysteine protease with dipeptidyl aminopeptidase activity that is thought to activate various granule‐associated serine proteases. Its exopeptidase activity is structurally explained by the so‐called exclusion domain, which blocks the active‐site cleft beyond the S2 site and, with its Asp 1 residue, provides an anchoring point for the N terminus of peptide and protein substrates. Here, the hydrazide of (2S,3S)‐trans‐epoxysuccinyl‐L ‐leucylamido‐3‐methylbutane (E‐64c) (k₂/K_i=140±5 M ^?1 s^?1) is demonstrated to be a lead structure for the development of irreversible cathepsin C inhibitors. The distal amino group of the hydrazide moiety addresses the acidic Asp 1 residue at the entrance of the S2 pocket by hydrogen bonding while also occupying the flat hydrophobic S1′–S2′ area with its leucine‐isoamylamide moiety. Furthermore, structure–activity relationship studies revealed that functionalization of this distal amino group with alkyl residues can be used to occupy the conserved hydrophobic S2 pocket. In particular, the n‐butyl derivative was identified as the most potent inhibitor of the series (k₂/K_i=56 000±1700 M ^?1 s^?1).     

Molecular Recognition of Two 2,4‐syn‐Functionalized (S)‐Glutamate Analogues by the Kainate Receptor GluK3 Ligand Binding Domain

The kainate receptors are the least studied subfamily of ionotropic glutamate receptors. These receptors are thought to have a neuromodulatory role and have been associated with a variety of disorders in the central nervous system. This makes kainate receptors interesting potential drug targets. Today, structures of the ligand binding domain (LBD) of the kainate receptor GluK3 are only known in complex with the endogenous agonist glutamate, the natural product kainate, and two synthetic agonists. Herein we report structures of GluK3 LBD in complex with two 2,4‐syn‐functionalized (S)‐glutamate analogues to investigate their structural potential as chemical scaffolds. Similar binding affinities at GluK3 were determined for the 2‐(methylcarbamoyl)ethyl analogue (K_i=4.0 μM ) and the 2‐(methoxycarbonyl)ethyl analogue (K_i=1.7 μM ), in agreement with the similar positioning of the compounds within the binding pocket. As the binding affinity is similar to that of glutamate, this type of Cγ substituent could be used as a scaffold for introduction of even larger substituents reaching into unexplored binding site regions to achieve subtype selectivity.     

Alternating copolymerization and terpolymerization of vinyl‐substituted phenolic antioxidants with propene and carbon monoxide by a palladium(II)‐based catalyst: polyketones containing intramolecular stabilizers

The copolymerization and terpolymerization reactions of the vinyl‐substituted phenolic stabilizers, 6‐tert‐butyl‐2‐(1,1‐dimethylhept‐6‐enyl)‐4‐methylphenol, o‐allylphenol, 4‐methylstyrene‐2,6‐di‐tert‐butylphenol and 2,6‐di‐tert‐butyl‐4‐allylphenol, with propene and carbon monoxide, by using the solvent‐stabilized palladium(II ) phosphine complex [Pd(dppp)(NCCH₃)₂](BF₄)₂ (dppp, 1,3‐bis(diphenylphosphino)propane) as a catalyst precursor and methanol as a co‐catalyst, is described. The influence of functional α‐olefins/CO units, distributed statistically along the propene/carbon monoxide (P/CO) copolymer backbone, on the molecular weight, glass transition temperature (T_g), elastic behavior and stability of the high‐molecular‐weight P/CO copolymer has been investigated. Loss of both elasticity and transparency were observed upon incorporating o‐allylphenol as a termonomer. The terpolymers, which contain phenolic stabilizers, were shown to be more stable when compared to the stabilizer‐free polyketones. In contrast to the propene/carbon monoxide copolymer, no degradation was observed for the 2,6‐di‐tert‐butyl‐4‐allylphenol/P/CO terpolymer; instead, the molar masses increased. Copyright © 2004 Society of Chemical Industry     

Acetylcholine Promotes Binding of α‐Conotoxin MII at α3β2 Nicotinic Acetylcholine Receptors

α‐Conotoxin MII (α‐CTxMII) is a 16‐residue peptide with the sequence GCCSNPVCHLEHSNLC, containing Cys2–Cys8 and Cys3–Cys16 disulfide bonds. This peptide, isolated from the venom of the marine cone snail Conus magus, is a potent and selective antagonist of neuronal nicotinic acetylcholine receptors (nAChRs). To evaluate the impact of channel–ligand interactions on ligand‐binding affinity, homology models of the heteropentameric α₃β₂‐nAChR were constructed. The models were created in MODELLER with the aid of experimentally characterized structures of the Torpedo marmorata‐nAChR (Tm‐nAChR, PDB ID: 2BG9) and the Aplysia californica‐acetylcholine binding protein (Ac‐AChBP, PDB ID: 2BR8) as templates for the α₃‐ and β₂‐subunit isoforms derived from rat neuronal nAChR primary amino acid sequences. Molecular docking calculations were performed with AutoDock to evaluate interactions of the heteropentameric nAChR homology models with the ligands acetylcholine (ACh) and α‐CTxMII. The nAChR homology models described here bind ACh with binding energies commensurate with those of previously reported systems, and identify critical interactions that facilitate both ACh and α‐CTxMII ligand binding. The docking calculations revealed an increased binding affinity of the α₃β₂‐nAChR for α‐CTxMII with ACh bound to the receptor, and this was confirmed through two‐electrode voltage clamp experiments on oocytes from Xenopus laevis. These findings provide insights into the inhibition and mechanism of electrostatically driven antagonist properties of the α‐CTxMIIs on nAChRs.     

Prospective Validation of a Comprehensive In silico hERG Model and its Applications to Commercial Compound and Drug Databases

Ligand‐based in silico hERG models were generated for 2 644 compounds using linear discriminant analysis (LDA) and support vector machines (SVM). As a result, the dataset used for the model generation is the largest publicly available (see Supporting Information). Extended connectivity fingerprints (ECFPs) and functional class fingerprints (FCFPs) were used to describe chemical space. All models showed area under curve (AUC) values ranging from 0.89 to 0.94 in a fivefold cross‐validation, indicating high model consistency. Models correctly predicted 80 % of an additional, external test set; Y‐scrambling was also performed to rule out chance correlation. Additionally models based on patch clamp data and radioligand binding data were generated separately to analyze their predictive ability when compared to combined models. To experimentally validate the models, 50 of the predicted hERG blockers from the Chembridge database and ten of the predicted non‐hERG blockers from an in‐house compound library were selected for biological evaluation. Out of those 50 predicted hERG blockers, tested at a concentration of 10 μM , 18 compounds showed more than 50 % displacement of [³H]astemizole binding to cell membranes expressing the hERG channel. K_i values of four of the selected binders were determined to be in the micromolar and high nanomolar range (K_i (VH 01 )=2.0 μM , K_i (VH 06 )=0.15 μM , K_i (VH 19 )=1.1 μM and K_i (VH 47 )=18 μM ). Of these four compounds, VH 01 and VH 47 showed also a second, even higher affinity binding site with K_i values of 7.4 nM and 36 nM , respectively. In the case of non‐hERG blockers, all ten compounds tested were found to be inactive, showing less than 50 % displacement of [³H]astemizole binding at 10 μM . These experimentally validated models were then used to virtually screen commercial compound databases to evaluate whether they contain hERG blockers. 109 784 (23 %) of Chembridge, 133 175 (38 %) of Chemdiv, 111 737 (31 %) of Asinex and 11 116 (18 %) of the Maybridge database were predicted to be hERG blockers by at least two of the models, a prediction which could, for example, be used as a pre‐filtering tool for compounds with potential hERG liabilities.     

Potent,Metabolically Stable 2‐Alkyl‐8‐(2H‐1,2,3‐triazol‐2‐yl)‐9H‐adenines as Adenosine A2A Receptor Ligands

Inhibition of adenosine A_2A receptors has been shown to elicit a therapeutic response in preclinical animal models of Parkinson’s disease (PD). We previously identified the triazolo‐9H‐purine, ST1535, as a potent A_2AR antagonist. Studies revealed that ST1535 is extensively hydroxylated at the ω‐1 position of the butyl side chain. Here, we describe the synthesis and evaluation of derivatives in which the ω‐1 position has been substituted (F, Me, OH) in order to block metabolism. The stability of the compounds was evaluated in human liver microsomes (HLM), and the affinity for A_2AR was determined. Two compounds, (2‐(3,3‐dimethylbutyl)‐9‐methyl‐8‐(2H‐1,2,3‐triazol‐2‐yl)‐9H‐purin‐6‐amine ( 3 b ) and 4‐(6‐amino‐9‐methyl‐8‐(2H‐1,2,3‐triazol‐2‐yl)‐9H‐purin‐2‐yl)‐2‐methylbutan‐2‐ol ( 3 c ), exhibited good affinity against A_2AR (K_i=0.4 nM and 2 nM , respectively) and high in vitro metabolic stability (89.5 % and 95.3 % recovery, respectively, after incubation with HLM for two hours).     

Biological Evaluation and X‐ray Co‐crystal Structures of Cyclohexylpyrrolidine Ligands for Trypanothione Reductase,an Enzyme from the Redox Metabolism of Trypanosoma

The tropical diseases human African trypanosomiasis, Chagas disease, and the various forms of leishmaniasis are caused by parasites of the family of trypanosomatids. These protozoa possess a unique redox metabolism based on trypanothione and trypanothione reductase (TR), making TR a promising drug target. We report the optimization of properties and potency of cyclohexylpyrrolidine inhibitors of TR by structure‐based design. The best inhibitors were freely soluble and showed competitive inhibition constants (K_i) against Trypanosoma (T.) brucei TR and T. cruzi TR and in vitro activities (half‐maximal inhibitory concentration, IC₅₀) against these parasites in the low micromolar range, with high selectivity against human glutathione reductase. X‐ray co‐crystal structures confirmed the binding of the ligands to the hydrophobic wall of the “mepacrine binding site” with the new, solubility‐providing vectors oriented toward the surface of the large active site.     

Synthesis and properties of ethylene‐substituted styrene copolymers

The copolymerization of ethylene and substituted styrenes [RSt's; p‐methylstyrene (MSt), p‐tert‐butylstyrene (BSt), 2‐vinylnaphthalene (VN), and p‐(tert‐butyldimethylsilyloxy)styrene (BMSiOSt)] were investigated with dimethylsilylene(tetramethylcyclopentadienyl)(N‐tert‐butyl)titanium dichloride to yield the corresponding ethylene–RSt copolymers. The substituent on the styrene (St) monomers did not affect the monomer reactivity ratio. The effect of the substituent structure of RSt on the thermal and mechanical properties was studied with differential scanning calorimetry, dynamic mechanical thermal spectroscopy, and elongation testing. The glass‐transition temperature (T_g) of the copolymers increased with increasing RSt content, and the order of T_g was as follows: BSt > VN > MSt = St. A copolymer with p‐hydroxystyrene (HOSt) was successively synthesized by means of deprotection of the copolymer with BMSiOSt. The copolymer showed a much higher T_g than the other copolymers because of the hydrogen connection of its OH groups. The mechanical properties of the copolymer in the glass state, at a lower temperature than T_g, were almost independent of the nature of the RSt. The substituent of the St monomers affected the pattern of the stress–strain curve in the elongation testing in the amorphous state. An improvement in the shape memory effect was observed in poly(ethylene‐co‐BSt). © 2008 Wiley Periodicals, Inc. J Appl Polym Sci, 2008     

Preparation and characterization of poly(silyl ester)s containing 2,2‐bis(p‐dimethylsiloxy‐phenyl)propane units in the polymer backbones

The two poly(silyl ester)s containing 2,2‐bis(p‐dimethylsiloxy‐phenyl)propane units in the polymer backbones have been prepared via polycondensation reaction of di‐tert‐butyl adipate and di‐tert‐butyl fumarate with 2,2‐bis(p‐chloro dimethylsiloxy‐phenyl)propane to give tert‐butyl chloride as the condensate. The polymerizations were performed under nitrogen at 110°C for 24 h without addition of solvents and catalysts to obtain the poly(silyl ester)s with weight average molecular weights typically ranging from 5000 to 10,000 g/mol. Characterization of the poly(silyl ester)s included ¹H NMR and ¹³C NMR spectroscopies, infrared spectroscopy, ultraviolet spectroscopy, differential scanning calorimetry, thermogravimetric analysis (TGA), gel permeation chromatography, and Ubbelohde viscometer. The glass transition temperatures (T_g) of the obtained polymers were above zero because of the introducing 2,2‐bis(p‐dimethylsiloxy‐phenyl)propane units in the polymer backbones. The TGA/DTG results showed that the obtained poly(silyl ester)s were stable up to 180°C and the residual weight percent at 800°C were 18 and 9%, respectively. © 2006 Wiley Periodicals, Inc. J Appl Polym Sci 101: 1937–1942, 2006     

GluN2B‐Selective N‐Methyl‐d‐aspartate (NMDA) Receptor Antagonists Derived from 3‐Benzazepines: Synthesis and Pharmacological Evaluation of Benzo[7]annulen‐7‐amines

Given their high neuroprotective potential, ligands that block GluN2B‐containing N‐methyl‐D ‐aspartate (NMDA) receptors by interacting with the ifenprodil binding site located on the GluN2B subunit are of great interest for the treatment of various neuronal disorders. In this study, a novel class of GluN2B‐selective NMDA receptor antagonists with the benzo[7]annulene scaffold was prepared and pharmacologically evaluated. The key intermediate, N‐(2‐methoxy‐5‐oxo‐6,7,8,9‐tetrahydro‐5H‐benzo[7]annulen‐7‐yl)acetamide ( 11 ), was obtained by cyclization of 3‐acetamido‐5‐(3‐methoxyphenyl)pentanoic acid ( 10 b ). The final reaction steps comprise hydrolysis of the amide, reduction of the ketone, and reductive alkylation, leading to cis‐ and trans‐configured 7‐(ω‐phenylalkylamino)benzo[7]annulen‐5‐ols. High GluN2B affinity was observed with cis‐configured γ‐amino alcohols substituted with a 3‐phenylpropyl moiety at the amino group. Removal of the benzylic hydroxy moiety led to the most potent GluN2B antagonists of this series: 2‐methoxy‐N‐(3‐phenylpropyl)‐6,7,8,9‐tetrahydro‐5H‐benzo[7]annulen‐7‐amine ( 20 a , K_i=10 nM ) and 2‐methoxy‐N‐methyl‐N‐(3‐phenylpropyl)‐6,7,8,9‐tetrahydro‐5H‐benzo[7]annulen‐7‐amine ( 23 a , K_i=7.9 nM ). The selectivity over related receptors (phencyclidine binding site of the NMDA receptor, σ₁ and σ₂ receptors) was recorded. In a functional assay measuring the cytoprotective activity of the benzo[7]annulenamines, all tested compounds showed potent NMDA receptor antagonistic activity. Cytotoxicity induced via GluN2A subunit‐containing NMDA receptors was not inhibited by the new ligands.     

Improving the Binding Affinity of in‐Vitro‐Evolved Cyclic Peptides by Inserting Atoms into the Macrocycle Backbone

Cyclic peptides binding to targets of interest can be generated efficiently with powerful in vitro display techniques, such as phage display or mRNA display. The cyclic peptide libraries screened with these methods are generated by altering in a combinatorial fashion the amino acid sequence of the peptides, the number of amino acids in the macrocycle rings, and the cyclization chemistry. A structural element that cannot easily be varied in the cyclic peptides is the backbone, which is built from amino acids, each of which contributes three atoms to the macrocyclic ring structure. Here, we proposed to improve the affinity of a phage‐selected bicyclic peptide inhibitor of coagulation factor XII (FXII) by screening variants with one or two carbon atoms inserted into different positions of the backbone, and thus tapping into a structural space that was not sampled by phage display. Two mutants showed 4.7‐ and 2.5‐fold improved K_i values. The better one blocked FXII with a K_i of 1.5±0.1 nm and inhibited activation of the intrinsic coagulation pathway (EC_2x 1.7 μm) . The strategy of ring size variation by one or several atoms should be generally applicable for the affinity maturation of in‐vitro‐evolved cyclic peptides.     

Probing the Peptidylglycine α‐Hydroxylating Monooxygenase Active Site with Novel 4‐Phenyl‐3‐butenoic Acid Based Inhibitors

Specific inhibition of the copper‐containing peptidylglycine α‐hydroxylating monooxygenase (PHM), which catalyzes the post‐translational modification of peptides involved in carcinogenesis and tumor progression, constitutes a new approach for combating cancer. We carried out a structure–activity study of new compounds derived from a well‐known PHM substrate analogue, the olefinic compound 4‐phenyl‐3‐butenoic acid (PBA). We designed, synthesized, and tested various PBA derivatives both in vitro and in silico. We show that it is possible to increase PBA affinity for PHM by appropriate functionalization of its aromatic nucleus. Compound 2 d , for example, bears a meta‐benzyloxy substituent, and exhibits better inhibition features (K_i=3.9 μM , k_inact/K_i=427 M ^?1 s^?1) than the parent PBA (K_i=19 μM , k_inact/K_i=82 M ^?1 s^?1). Docking calculations also suggest two different binding modes for PBA derivatives; these results will aid in the development of further PHM inhibitors with improved features.     

Synthesis and Pharmacological Evaluation of SNC80 Analogues with a Bridged Piperazine Ring

To discover novel δ‐opioid receptor ligands derived from SNC80 ( 1 ), a series of 6,8‐diazabicyclo[3.2.2]nonane derivatives bearing two aromatic moieties was prepared, and the affinity toward δ, μ, and κ receptors, as well as σ receptors, was investigated. After removal of the 4‐methoxybenzyl and 2,4‐dimethoxybenzyl protecting groups, the pharmacophoric N,N‐diethylcarbamoylbenzyl residue was attached to the 6,8‐diazabicyclo[3.2.2]nonane framework to yield the designed δ receptor ligands. In a first series of compounds the benzhydryl moiety of SNC80 was dissected, and one phenyl ring was attached to the bicyclic framework. In a second series of δ ligands the complete benzhydryl moiety was introduced into the bicyclic scaffold. The determined δ receptor affinities show that compounds based on an (R)‐glutamate‐derived bicyclic scaffold possess higher δ receptor affinity than their (S)‐glutamate‐derived counterparts. Furthermore, an intact benzhydryl moiety leads to δ receptor ligands that are more potent than compounds with two separated aromatic moieties. Compound 24 , with the same spatial arrangement of substituents around the benzhydryl stereocenter as SNC80, shows the highest δ receptor affinity of this series: K_i=24 nM . Whereas the highly potent δ ligands reveal good selectivity against μ and κ receptors, the σ₁ and/or σ₂ affinities of some compounds are almost in the same range as their δ receptor affinities, such as compound 25 (σ₂: K_i=83 nM ; δ: K_i=75 nM ). In [³⁵S]GTPγS assays the most potent δ ligands 24 and 25 showed almost the same intrinsic activity as the full agonist SNC80, proving the agonistic activity of 24 and 25 . The enantiomeric 4‐benzylidene derivatives 15 and ent‐ 15 showed selective cytotoxicity toward the 5637 (bladder) and A‐427 (small‐cell lung) human tumor cell lines.     

Selective Covalent Protein Modification by 4‐Halopyridines through Catalysis

We have investigated 4‐halopyridines as selective, tunable, and switchable covalent protein modifiers for use in the development of chemical probes. Nonenzymatic reactivity of 4‐chloropyridine with amino acids and thiols was ranked with respect to common covalent protein‐modifying reagents and found to have reactivity similar to that of acrylamide, but could be switched to a reactivity similar to that of iodoacetamide upon stabilization of the positively charged pyridinium. Diverse, fragment‐sized 4‐halopyridines inactivated human dimethylarginine dimethylaminohydrolase‐1 (DDAH1) through covalent modification of the active site cysteine, acting as quiescent affinity labels that required off‐pathway catalysis through stabilization of the protonated pyridinium by a neighboring aspartate residue. A series of 2‐fluoromethyl‐substituted 4‐chloropyridines demonstrated that the pK_a and k_inact/K_I values could be predictably varied over several orders of magnitude. Covalent labeling of proteins in an Escherichia coli lysate was shown to require folded proteins, indicating that alternative proteins can be targeted, and modification is likely to be catalysisdependent. 4‐Halopyridines, and quiescent affinity labels in general, represent an attractive strategy to develop reagents with switchable electrophilicity as selective covalent protein modifiers.