Synthesis and in vitro evaluation of tetrahydroisoquinolinyl benzamides as ligands for sigma receptors

5-Bromo-N-[4-(6,7-dimethoxy-3,4-dihydro-1H-isoquinolin-2-yl)-butyl)]-2,3-dimethoxy-benzamide (1) is one of the most potent and selective sigma(2) receptor ligands reported to date. Previous structure-activity relationship studies of such tetrahydroisoquinolinyl benzamides have focused on the linker that connects the ring systems and the effects of benzamide ring substituents. The present study explores the effects of fusing methylene-, ethylene-, and propylenedioxy rings onto the tetrahydroisoquinoline in place of the two methoxy groups. These modifications decreased sigma(2) affinity by 8- to 12-fold, with no major differences noted with ring size. By contrast, the methylenedioxy analog showed a 10-fold greater sigma(1) affinity than 1, and progressively lower sigma(1) affinities were then noted with increasing ring size. We also opened the tetrahydroisoquinoline ring of 1 to study the effects of greater conformational fluidity on sigma receptor binding. The sigma(2) affinity of the open-ring compound decreased by 1700-fold, while sigma(1) affinity was not changed. Thus, a constrained tetrahydroisoquinoline ring system is key to the exceptional sigma(2) receptor binding affinity and selectivity of this active series.     

Syntheses of novel high affinity ligands for opioid receptors

A series of novel high affinity opioid receptor ligands have been made whereby the phenolic-OH group of nalbuphine, naltrexone methiodide, 6-desoxonaltrexone, hydromorphone and naltrindole was replaced by a carboxamido group and the furan ring was opened to the corresponding 4-OH derivatives. These furan ring ‘open’ derivatives display very high affinity for μ and κ receptors and much less affinity for δ. The observation that these target compounds have much higher receptor affinity than the corresponding ring ‘closed’ carboxamides significantly strengthens our underlying pharmacophore hypothesis concerning the bioactive conformation of the carboxamide group.     

Synthesis and evaluation of galactofuranosyl N,N-dialkyl sulfenamides and sulfonamides as antimycobacterial agents

The recent emergence of clinically oppressive superbugs, some with resistance to nearly all frontline drug therapies, has challenged our ability to combat such infectious organisms as Mycobacterium tuberculosis, the causative agent of tuberculosis (TB). Our medicinal chemistry program targeting this pathogen has identified several potent galactofuranose-based in vitro inhibitors of mycobacterial growth. The most potent compound, the Galf N,N-didecyl sulfenamide 8d, displayed anti-mycobacterial activity (MIC) of 1 microg/mL in a cell based assay against a representative strain of Mycobacterium smegmatis.     

Design of high affinity cyclic pentapeptide ligands for kappa-opioid receptors.

Using results from our previously reported cyclic opioid peptide series and reliable models for mu-, delta-, and kappa-opioid receptors (MOR, DOR, and KOR, respectively) and their complexes with peptide ligands, we have designed and synthesized a series of cyclic pentapeptides of structure Tyr-C[D-Cys-Phe-Phe-X]-NH2, cyclized via disulfide, methylene, or ethylene dithioethers, and where X = D- or L-Cys; or D- or L-penicillamine (Pen; beta,beta-dimethylcysteine). Determination of binding affinities to MOR, DOR, and KOR revealed that members of this series with X = D- or L-Cys display KOR affinities in the low nanomolar range, demonstrating that a 'DPDPE-like' tetrapeptide scaffold is suitable not only for DOR and MOR ligands, but also for KOR ligands. The cyclic pentapeptides reported here are not, however, selective for KOR, rather they display significant selectivity and high affinity for MOR. Indeed, peptide 8, Tyr-C[D-Cys-Phe-Phe-Cys]-NH2-cyclized via a methylene dithioether, shows picomolar binding affinity for MOR ( = 16 pm) with more than 100-fold selectivity for MOR vs. DOR or KOR, and may be of interest as a high affinity, high selectivity MOR ligand. Nonetheless, the high affinity KOR peptides in this series represent excellent leads for the development of structurally related, selective KOR ligands designed to exploit structurally specific features of KOR, MOR, and DOR.     

Triazoloquinazolinediones as novel high affinity ligands for the benzodiazepine site of GABA(A) receptors

Based on a pharmacophore model of the benzodiazepine-binding site of GABA_A receptors, a series of 2-aryl-2,6-dihydro[1,2,4]triazolo[4,3-c]quinazoline-3,5-diones (structure type I) were designed, synthesized, and identified as high-affinity ligands of the binding site. For several compounds, K_i values of around 0.20 nM were determined. They show a structural resemblance with the previously described 2-phenyl-2H-pyrazolo[4,3-c]quinolin-3(5H)-ones (II) and 2-phenyl-[1,2,4]triazolo[1,5-a]quinoxalin-4(5H)-one (III). The 9-bromo substituted compounds 8a-d were prepared in an 8-step synthesis in an overall yield of approximately 40%, and a library of 9-substituted analogues was prepared by cross-coupling reactions. Compound 8e, 21, 22, and 24 were tested on recombinant rat ??₁??₃??₂, ??₂??₃??₂, ??₃??₃??₂, and ??₅??₃??₂ subtypes, and displayed selectivity for the ??₁??₃??₂ isoform.     

Chlorophenylpiperazine analogues as high affinity dopamine transporter ligands

Selective σ₂ ligands continue to be an active target for medications to attenuate the effects of psychostimulants. In the course of our studies to determine the optimal substituents in the σ₂-selective phenyl piperazines analogues with reduced activity at other neurotransmitter systems, we discovered that 1-(3-chlorophenyl)-4-phenethylpiperazine actually had preferentially increased affinity for dopamine transporters (DAT), yielding a highly selective DAT ligand.     

Synthesis and structure-activity relationships of N-(3-phenylpropyl)-N'-benzylpiperazines: Potent ligands for sigma1 and sigma2 receptors

Ten N-(3-phenylpropyl)-N'-benzylpiperazines having different substituents on the benzyl moiety were synthesized and evaluated for sigma(1) and sigma(2) receptor binding. The sigma(1) affinities were 0.37-2.80nM, sigma(2) affinities were 1.03-34.3nM, and selectivities, as sigma(2)/sigma(1) affinity ratios, ranged from 1.4 to 52. Three compounds tested in a phenytoin shift binding assay profiled as probable sigma(1) antagonists. Quantitative structure-activity relationships depended on pi(x), MR or E(s) and Hammett sigma values. The hydrophobicity term is negative for sigma(1) binding but positive for sigma(2) binding, indicating a major difference between the pharmacophores.     

Identification of 9-fluoro substituted (-)-cytisine derivatives as ligands with high affinity for nicotinic receptors

(-)-9-Fluorocytisine, (-)-9-methylcytisine and (-)-9-trifluoromethylcytisine were synthesized from the natural product (-)-cytisine. 9-Methyl and 9-trifluoromethyl cytisines display a remarkable affinity at the α(4)β(2) nicotinic receptor subtype (0.2 nM) with a high selectivity versus the α(7) nAChR subtype. Comparison of the affinity values suggests that the size of the substituent at the 9 position of (-)-cytisine seems more important than electronic factors for efficient binding and selectivity at α(4)β(2) nAChRs.     

N6-functionalized congeners of adenosine with high potency at A2-adenosine receptors: potential ligands for affinity chromatography

Adenosine analogues substituted at N6 with spacer arms designed for attachment to soluble macromolecules or to solid supports for affinity chromatography are agonists at the A2-adenosine receptor that mediates coronary vasodilation in the dog. The most active analogues had spacer arms terminating in -NH2, -NHCH3 or in a biotin residue. Comparisons of coronary vasoactivity with affinity for brain A1 adenosine receptors identified one biotin-containing analogue as relatively selective for coronary A2 receptors. The complex of this analogue with avidin retained coronary vasoactivity.     

New piperidine-based derivatives as sigma receptor ligands. Synthesis and pharmacological evaluation

The sigma receptor (σR) family has been considered mysterious for a long time. In fact, the σ2R subtype has been cloned only recently, revealing its identity as TMEM97, a NPC1-binding protein involved in cholesterol biosynthesis and implicated in the pathogenesis of cancer and neurologic disorders. With the aim of developing new chemical entities gifted with σR affinity, herein we report the design and synthesis of new piperidine-based alkylacetamide derivatives with mixed affinity towards both σ1 and σ2R subtypes.     

Synthesis,modelling and biological characterization of 3-substituted-1H-indoles as ligands of GluN2B-containing N-methyl-d-aspartate receptors

A three-step synthetic pathway has been employed to synthesize a small library of 2-(4-arylpiperidin-1-yl)-1-(1H-indol-3-yl)ethanone and 2-(4-arylpiperidin-1-yl)-1-(1H-indol-3-yl)ethane-1,2-dione derivatives that have been screened in [³H]ifenprodil competition binding assay. Some compounds exhibited significant binding affinity at nanomolar concentration, the most active being ligand 35 (IC₅₀ = 5.5 nM). Docking experiments suggested the main interactions between 35 and GluN2B-containing NMDA receptors. Notably, the compound 35 reduced NMDA-mediated excitatory post-synaptic currents recorded in mouse hippocampal slices indicating antagonistic effects (50 nM). Moreover, the compound 35 has shown antioxidant effects in a preliminary screening, thus suggesting that it might be considered prototype for future drug development of novel ‘dual target’ neuroprotective agents.     

Synthesis and evaluation of phenylcarbamate derivatives as ligands for nicotinic acetylcholine receptors

Phenylcarbamate derivatives were synthesized and evaluated in radioligand binding assays for different nicotinic acetylcholine receptor (nAChR) subtypes. Carbamate derivatives bearing a pyrrolidine or piperidine moiety 8-20 exhibited much lower affinity for alpha7* nAChR than the analogues in the quinuclidine series 21-25, although the same structural elements are present. Furthermore, in contrast to the quinuclidine analogues 21-25, all (S)-pyrrolidine derivatives 8-12 and the piperidine analogues 15 and 16 exhibited higher affinities for alpha4beta2* nAChR.     

The analgesic tropane analogue (+/-)-SM 21 has a high affinity for sigma2 receptors

The analgesic properties of the tropane analogue (+/-)-SM 21 have been attributed to the antagonism of presynaptic m2 receptors resulting in a potentiation of acetylcholine release. However, drugs targeting a number of other neurotransmitter receptors have been shown to enhance acetylcholine release. In the current study, in vitro studies were conducted in order to determine the affinity of (+/-)-SM 21 for serotonin 5-HT3, 5-HT4, and sigma receptors. Our results indicate that (+/-)-SM 21, and its structural congeners, have a relatively high affinity for sigma2 receptors relative to their reported affinity for muscarinic receptors. The higher affinity for sigma2 versus sigma1 receptors indicates that (+/-)-SM 21 may be a suitable lead compound for developing sigma2-selective ligands.     

Isolation and partial characterization of high affinity laminin receptors in neural cells

Neural cells in culture (NG-108, PC12, chick dorsal root ganglion, chick spinal cord, and rat astrocytes) bind laminin with an apparent Kd of congruent to 10(-9) M. Laminin affinity chromatography of chick brain membranes washed with 150 mM NaCl and eluted with 0.2 M glycine buffer, pH 3.5, yields a single protein with an apparent molecular mass of 67 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Isoelectric focusing and peptide mapping indicate that the 67-kDa protein is distinct from bovine serum albumin (68 kDa) but indistinguishable from high affinity laminin receptors isolated from skeletal muscle. After electroblotting onto nitrocellulose paper and probing with 125I-laminin, this putative laminin receptor binds laminin specifically (100 ng/ml). A second protein (congruent to 120-140 kDa) is also detected with 125I-laminin (100 ng/ml) in the laminin affinity-purified membrane proteins. Both 67- and congruent to 120-140-kDa proteins can be laminin affinity-purified from cultures enriched for neurons (greater than 90%) following metabolic labeling with [35S]methionine. Our data suggest that neural cells (dorsal root ganglion, central nervous system neurons, astrocytes, and several neural cell lines) have high affinity binding sites for laminin and that two membrane proteins, 67- and congruent to 120-140-kDa, are responsible at least in part for this binding.     

Synthesis and evaluation of N(1)-alkylindole-3-ylalkylammonium compounds as nicotinic acetylcholine receptor ligands

In this study thirty-three novel indole derivatives were designed and synthesized based on the structure of deformylflustrabromine B (1), a metabolite isolated from the marine bryozoan Flustra foliacea L. The syntheses were carried out using standard methodologies and in good yields. The molecules were tested for their affinities for the α4β2¹, α3β4¹, α7¹ and (α1)₂β1γδ nicotinic acetylcholine receptor (nAChR) subtypes. Binding assays showed that, among these ligands, compound 7c exhibited the highest affinity with K_i = 136.1, 93.9 and 862.4 nM for the α4β2¹, α3β4¹, and α7¹ nAChRs subtypes, respectively. These results indicated that the indole core might be a useful scaffold for the development of new potent and selective nAChR ligands.     

Aptamers as affinity ligands for downstream processing

Aptamers are single‐stranded synthetic oligonucleotides that are able to capture their target molecule with high affinity and specificity. Therefore, they can be thought of as nucleic acid‐based alternative to antibodies, which have several advantages over their amino acid‐based counterparts. Consequently, aptamers can be used in different applications based on molecular recognition including affinity separations. This review will summarize the state‐of‐the‐art in aptamer‐based affinity separations; will discuss the current limitations and will highlight possible future prospects. The first part will point out the advantages of aptamers in downstream processing. Here, the properties of aptamers will be discussed along with their implications on downstream processing from a user's point of view. In the second part, a brief summary of the literature is given with focus on aptamer‐based separation of proteins. Finally, some drawbacks of aptamers will be illustrated and possibilities to overcome these limitations will be suggested. New technologies in the fields of aptamer selection and synthesis are expected to further promote the use of aptamers as affinity ligands in downstream processing.     

Synthesis of N-phenyl-N-(3-(piperidin-1-yl)propyl)benzofuran-2-carboxamides as new selective ligands for sigma receptors

Novel benzofuran-2-carboxamide ligands, which are selective for sigma receptors, have been synthesized via a microwave-assisted Perkin rearrangement reaction and a modified Finkelstein halogen-exchange used to facilitate N-alkylation. The ligands synthesized are the 3-methyl-N-phenyl-N-(3-(piperidin-1-yl)propyl)benzofuran-2-carboxamides (KSCM-1, KSCM-5 and KSCM-11). The benzofuran-2-carboxamide structure was N-arylated and N-alkylated to include both N-phenyl and N-(3-(piperidin-1-yl)propyl substituents, respectively. These new carboxamides exhibit high affinity at the sigma-1 receptor with K_i values ranging from 7.8 to 34 nM. Ligand KSCM-1 with two methoxy substituents at C-5 and C-6 of the benzofuran ring, and K_i = 27.5 nM at sigma-1 was found to be more selective for sigma-1 over sigma-2.     

Specific, high affinity relaxin-like factor receptors.

The relaxin-like factor (RLF) is a circulating hormone that binds to specific membrane-bound uterine receptors in the mouse. Mono-iodinated RLF tracers were produced and characterized specifically to study the properties of the RLF receptor. The tracers bound to the RLF receptor in uterine crude membrane preparations with high affinity (73 nM for (125)I-Tyr(A9) RLF and 90 nM for (125)I-Tyr(A26) RLF) as determined by Scatchard analysis. The specificity of binding was confirmed by chemical cross-linking experiments. Binding of (125)I-Tyr(A9) RLF to the putative receptor was inhibited in the presence of a 640-fold excess of unlabeled human RLF but not by the same excess of human relaxin. SDS-gel electrophoresis of the RLF-receptor complex revealed a molecular mass of >200 kDa, which remained unchanged upon reduction. The size and the lack of subunit structure of the receptor is similar to the features reported for the relaxin receptor. In this regard both, the RLF and the relaxin receptor are different from the insulin- and the insulin-like growth factor-type 1 receptors. This observation supports the relaxin-likeness of this new factor not only toward potential target tissues but also as regards receptor features.     

Synthesis and pharmacological evaluation of Tic-hydantoin derivatives as selective sigma1 ligands. Part 1

Herein is described a new class of selective sigma1 ligands consisting of tetrahydroisoquinoline-hydantoin (Tic-hydantoin) derivatives. Compound 3a has high affinity (IC50 = 16 nM) for the sigma1 receptor and is selective in a large panel of therapeutic targets. This first study presents structural changes around the Tic-hydantoin core, leading to a Tic-hydantoin analogue with a higher sigma1 affinity (IC50 approximately 1 nM).     

Synthesis,binding affinity and SAR of new benzolactam derivatives as dopamine D3 receptor ligands

A series of new benzolactam derivatives was synthesized and the derivatives were evaluated for their affinities at the dopamine D₁, D₂, and D₃ receptors. Some of these compounds showed high D₂ and/or D₃ affinity and selectivity over the D₁ receptor. The SAR study of these compounds revealed structural characteristics that decisively influenced their D₂ and D₃ affinities. Structural models of the complexes between some of the most representative compounds of this series and the D₂ and D₃ receptors were obtained with the aim of rationalizing the observed experimental results. Moreover, selected compounds showed moderate binding affinity on 5-HT_2A which could contribute to reducing the occurrence of extrapyramidal side effects as potential antipsychotics.