6‑Bromo-8-(4‑[³H]methoxybenzamido)-4-oxo‑4<i>H</i>‑chromene-2-carboxylic Acid: A Powerful Tool for Studying Orphan G Protein-Coupled Receptor GPR35

The potent and selective GPR35 agonist 6-bromo-8-(4-methoxybenzamido)-4-oxo-4<i>H</i>-chromene-2-carboxylic acid (<b>12</b>) was obtained in tritium-labeled form, designated [³H]­PSB-13253, with a specific activity of 36 Ci (1.33 TBq)/mmol. Radiolabeling was achieved by methylation of ethyl 6-bromo-8-(4-((<i>tert</i>-butyldimethylsilyl)­oxy)­benzamido)-4-oxo-4<i>H</i>-chromene-2-carboxylate (<b>19</b>) with [³H]­methyl tosylate followed by ester hydrolysis. The radioligand was characterized by kinetic, saturation, and competition assays at membrane preparations of Chinese hamster ovary cells recombinantly expressing the human GPR35. [³H]<b>12</b> labeled the receptor with high affinity (<i>K</i>_D = 5.27 nM). Binding was saturable (<i>B</i>_max = 12.6 pmol/mg of protein) and reversible. Affinities of selected standard ligands and a library of amidochromen-4-one-2-carboxylates were determined. Binding data mostly correlated with potencies determined in β-arrestin assays. On the basis of the test results, several new fluorine-substituted 6-bromo-8-benzamidochromen-4-one-2-carboxylic acids were obtained, which represent the most potent GPR35 agonists known to date. 6-Bromo-8-(2,6-difluoro-4-methoxybenzamido)-4-oxo-4<i>H</i>-chromene-2-carboxylic acid (<b>83</b>; <i>K</i>_i = 0.589 nM, EC₅₀ = 5.54 nM) showed the highest affinity with a <i>K</i>_i value in the subnanomolar range

General Synthesis of Unsymmetrical 3,3′-(Aza)diindolylmethane Derivatives

Diindolylmethane (DIM) and its derivatives have recently been in the focus of interest due to their significant biological activities, specifically in cancer prevention and therapy. Molecular targets of DIM have been identified, e.g., the immunostimulatory G protein-coupled receptor GPR84. However, most of the reported and investigated DIM derivatives are symmetrical because general methods for obtaining unsymmetrical DIMs have been lacking. To optimize the interaction of DIM derivatives with their protein targets, unsymmetrical substitution is required. In the present study we developed a new, mild and efficient access to unsymmetrically substituted 3,3′-DIMs by reaction of (3-indolylmethyl)­trimethylammonium iodides with a wide range of substituted indole derivatives. 7-Azaindole also led to the 3,3′-connected DIM analogue, while 4- and 5-azaindoles reacted at the <i>N</i>1-nitrogen atom as confirmed by X-ray crystallography. The reactions were performed in water without the requirement of a catalyst or other additives. Wide substrate scope, operational simplicity, environmentally benign workup, and high yields are further advantages of the new method. The synthetic protocol proved to be suitable for upscaling to yield gram amounts for pharmacological studies. This procedure will allow the preparation of a broad range of novel, unsymmetrical DIM derivatives to exploit their potential as novel drugs

Indazole- and Indole-5-carboxamides: Selective and Reversible Monoamine Oxidase B Inhibitors with Subnanomolar Potency

Indazole- and indole-carboxamides were discovered as highly potent, selective, competitive, and reversible inhibitors of monoamine oxidase B (MAO-B). The compounds are easily accessible by standard synthetic procedures with high overall yields. The most potent derivatives were <i>N</i>-(3,4-dichlorophenyl)-1-methyl-1<i>H</i>-indazole-5-carboxamide (<b>38a</b>, PSB-1491, IC₅₀ human MAO-B 0.386 nM, >25000-fold selective versus MAO-A) and <i>N</i>-(3,4-dichlorophenyl)-1<i>H</i>-indole-5-carboxamide (<b>53</b>, PSB-1410, IC₅₀ human MAO-B 0.227 nM, >5700-fold selective versus MAO-A). Replacement of the carboxamide linker with a methanimine spacer leading to (<i>E</i>)-<i>N</i>-(3,4-dichlorophenyl)-1-(1<i>H</i>-indazol-5-yl)­methanimine (<b>58</b>) represents a further novel class of highly potent and selective MAO-B inhibitors (IC₅₀ human MAO-B 0.612 nM, >16000-fold selective versus MAO-A). In <i>N</i>-(3,4-difluorophenyl-1<i>H</i>-indazole-5-carboxamide (<b>30</b>, PSB-1434, IC₅₀ human MAO-B 1.59 nM, selectivity versus MAO-A >6000-fold), high potency and selectivity are optimally combined with superior physicochemical properties. Computational docking studies provided insights into the inhibitors’ interaction with the enzyme binding site and a rationale for their high potency despite their small molecular size

Development of [³H]2-Carboxy-4,6-dichloro‑1<i>H</i>‑indole-3-propionic Acid ([³H]PSB-12150): A Useful Tool for Studying GPR17

The recently described synthetic GPR17 agonist 2-carboxy-4,6-dichloro-1<i>H</i>-indole-3-propionic acid (<b>1</b>) was prepared in tritium-labeled form by catalytic hydrogenation of the corresponding propenoic acid derivative <b>8</b> with tritium gas. The radioligand [³H]­PSB-12150 (<b>9</b>) was obtained with a specific activity of 17 Ci/mmol (629 GBq/mmol). It showed specific and saturable binding to a single binding site in membrane preparations from Chinese hamster ovary cells recombinantly expressing the human GPR17. A competition assay procedure was established, which allows the determination of ligand binding affinities

Virtual Screening Identifies Novel Sulfonamide Inhibitors of <i>ecto</i>-5′-Nucleotidase

We aimed to identify inhibitors of <i>ecto</i>-5′-nucleotidase (<i>ecto</i>-5′-NT, CD73), a membrane-bound metallophosphoesterase that is implicated in the control of purinergic receptor signaling and a number of associated therapeutically relevant effects. Currently, only very few compounds, including ADP, its more stable analogue α,β-methylene-ADP, ATP, and anthraquinone derivatives are known to inhibit this enzyme. In the search for inhibitors with more drug-like properties, we applied a model structure-based virtual screening approach augmented by chemical similarity searching. On the basis of this analysis, 51 candidate compounds were finally selected for experimental evaluation. A total of 13 of these molecules were confirmed to have competitive inhibitory activity. The most potent inhibitor, 6-chloro-2-oxo-<i>N</i>-(4-sulfamoylphenyl)-2<i>H</i>-chromene-3-carboxylic acid amide (<b>17</b>), showed an IC₅₀ value of 1.90 μM. In contrast to the nucleotide- and anthraquinone-derived antagonists, the newly identified competitive inhibitors are uncharged at physiological pH values, possess a drug-like structure, and are structurally distinct from known active compounds

Antagonists for the Orphan G‑Protein-Coupled Receptor GPR55 Based on a Coumarin Scaffold

The orphan G-protein-coupled receptor GPR55, which is activated by 1-lysophosphatidylinositol and interacts with cannabinoid (CB) receptor ligands, has been proposed as a new potential drug target for the treatment of diabetes, Parkinson’s disease, neuropathic pain, and cancer. We applied β-arrestin assays to identify 3-substituted coumarins as a novel class of antagonists and performed an extensive structure–activity relationship study for GPR55. Selectivity versus the related receptors CB₁, CB₂, and GPR18 was assessed. Among the 7-unsubstituted coumarins selective, competitive GPR55 antagonists were identified, such as 3-(2-hydroxybenzyl)-5-isopropyl-8-methyl-2<i>H</i>-chromen-2-one (<b>12</b>, PSB-SB-489, IC₅₀ = 1.77 μM, p<i>A</i>₂ = 0.547 μM). Derivatives with long alkyl chains in position 7 were potent, possibly allosteric GPR55 antagonists which showed ancillary CB receptor affinity. 7-(1,1-Dimethyloctyl)-5-hydroxy-3-(2-hydroxybenzyl)-2<i>H</i>-chromen-2-one (<b>69</b>, PSB-SB-487, IC₅₀ = 0.113 μM, <i>K</i>_B = 0.561 μM) and 7-(1,1-dimethylheptyl)-5-hydroxy-3-(2-hydroxybenzyl)-2<i>H</i>-chromen-2-one (<b>67</b>, PSB-SB-1203, IC₅₀ = 0.261 μM) were the most potent GPR55 antagonists of the present series

Identification of a Potent and Selective Cannabinoid CB₁ Receptor Antagonist from <i>Auxarthron reticulatum</i>

The fungus <i>Auxarthron reticulatum</i> derived from the marine sponge <i>Ircinia variabilis</i> produced the diketopiperazine alkaloid amauromine (<b>1</b>) and the quinolinone methyl-penicinoline (<b>2</b>). Compound <b>2</b> is identical to the reported methyl-marinamide, whose structure is herewith revised. In radioligand binding studies at human cannabinoid CB₁ and CB₂ receptors recombinantly expressed in Chinese hamster ovary (CHO) cells, amauromine (<b>1</b>) was found to exhibit high affinity and selectivity for the CB₁ receptor (<i>K</i>_i = 178 nM). The compound was shown to be a neutral CB₁ antagonist with a <i>K</i>_b value of 66.6 nM determined in cAMP assays. Compound <b>2</b> exhibited only weak or no effects at CB receptors. To the best of our knowledge, compound <b>1</b> is the first fungal natural product that shows affinity for cannabinoid CB₁ receptors. Because of its high antagonistic potency and selectivity, it may have potential for use as a drug and/or serve as a lead structure for drug development

Dual Targeting of Adenosine A_2A Receptors and Monoamine Oxidase B by 4<i>H</i>‑3,1-Benzothiazin-4-ones

Blockade of A_2A adenosine receptors (A_2AARs) and inhibition of monoamine oxidase B (MAO-B) in the brain are considered attractive strategies for the treatment of neurodegenerative diseases such as Parkinson’s disease (PD). In the present study, benzothiazinones, e.g., 2-(3-chlorophenoxy)-<i>N</i>-(4-oxo-4<i>H</i>-3,1-benzothiazin-2-yl)­acetamide (<b>13</b>), were identified as a novel class of potent MAO-B inhibitors (IC₅₀ human MAO-B: 1.63 nM). Benzothiazinones with large substituents in the 2-position, e.g., methoxycinnamoylamino, phenylbutyrylamino, or chlorobenzylpiperazinylbenzamido residues (<b>14</b>, <b>17</b>, <b>27</b>, and <b>28</b>), showed high affinity and selectivity for A_2AARs (<i>K</i>_i human A_2AAR: 39.5–69.5 nM). By optimizing benzothiazinones for both targets, the first potent, dual-acting A_2AAR/MAO-B inhibitors with a nonxanthine structure were developed. The best derivative was <i>N</i>-(4-oxo-4<i>H</i>-3,1-benzothiazin-2-yl)-4-phenylbutanamide (<b>17</b>, <i>K</i>_i human A_2A, 39.5 nM; IC₅₀ human MAO-B, 34.9 nM; selective versus other AR subtypes and MAO-A), which inhibited A_2AAR-induced cAMP accumulation and showed competitive, reversible MAO-B inhibition. The new compounds may be useful tools for validating the A_2AAR/MAO-B dual target approach in PD

N‑Substituted Phenoxazine and Acridone Derivatives: Structure–Activity Relationships of Potent P2X4 Receptor Antagonists

P2X4 receptor antagonists have potential as drugs for the treatment of neuropathic pain and neurodegenerative diseases. In the present study the discovery of phenoxazine derivatives as potent P2X4 antagonists is described. N-Substituted phenoxazine and related acridone and benzoxazine derivatives were synthesized and optimized with regard to their potency to inhibit ATP-induced calcium influx in 1321N1 astrocytoma cells stably transfected with the human P2X4 receptor. In addition, species selectivity (rat, mouse, human) and receptor subtype selectivity (versus P2X1,2,3,7) were investigated. The most potent P2X4 antagonist of the present series was <i>N</i>-(benzyloxycarbonyl)­phenoxazine (<b>26</b>, PSB-12054) with an IC₅₀ of 0.189 μM and good selectivity versus the other human P2X receptor subtypes. <i>N</i>-(<i>p</i>-Methylphenylsulfonyl)­phenoxazine (<b>21</b>, PSB-12062) was identified as a selective P2X4 antagonist that was equally potent in all three species (IC₅₀: 0.928–1.76 μM). The compounds showed an allosteric mechanism of action. The present study represents the first structure–activity relationship analysis of P2X4 antagonists

Molecular Recognition of Agonists and Antagonists by the Nucleotide-Activated G Protein-Coupled P2Y₂ Receptor

A homology model of the nucleotide-activated P2Y₂R was created based on the X-ray structures of the P2Y₁ receptor. Docking studies were performed, and receptor mutants were created to probe the identified binding interactions. Mutation of residues predicted to interact with the ribose (Arg110) and the phosphates of the nucleotide agonists (Arg265, Arg292) or that contribute indirectly to binding (Tyr288) abolished activity. The Y114F, R194A, and F261A mutations led to inactivity of diadenosine tetraphosphate and to a reduced response of UTP. Significant reduction in agonist potency was observed for all other receptor mutants (Phe111, His184, Ser193, Phe261, Tyr268, Tyr269) predicted to be involved in agonist recognition. An ionic lock between Asp185 and Arg292 that is probably involved in receptor activation interacts with the phosphate groups. The antagonist AR-C118925 and anthraquinones likely bind to the orthosteric site. The updated homology models will be useful for virtual screening and drug design