Synthesis and evaluation of 4-substituted piperidines and piperazines as balanced affinity μ opioid receptor (MOR) agonist/δ opioid receptor (DOR) antagonist ligands

In this letter, we describe a series of 4-substituted piperidine and piperazine compounds based on tetrahydroquinoline 1, a compound that shows balanced, low nanomolar binding affinity for the mu opioid receptor (MOR) and the delta opioid receptor (DOR). We have shown that by changing the length and flexibility profile of the side chain in this position, binding affinity is improved at both receptors by a significant degree. Furthermore, several of the compounds described herein display good efficacy at MOR, while simultaneously displaying DOR antagonism. The MOR agonist/DOR antagonist has shown promise in the reduction of negative side effects displayed by selective MOR agonists, namely the development of dependence and tolerance.     

Design and synthesis of iodocarborane-containing ligands with high affinity and selectivity toward ERβ

The selectivity and the binding affinity of previously reported carborane-containing ligands 2 and 3 toward ERβ remains to be optimized. To improve their biological profiles, a series of iodinated carboranyl phenol derivatives (4–6) were designed and synthesized as prospective ERβ-selective ligands with high affinity. Several iodinated carboranyl phenols showed high relative binding affinity (RBA) values for both ERs, and especially for ERβ, due to suitable hydrophobic interactions of the iodine atoms with the hydrophobic amino acid residues of the ERβ ligand-binding domains. Among these derivatives, 9,10-diiodo-m-carborane 5f exhibited a more than 100% increase of the RBA values toward ERβ, a 14-fold increased selectivity for ERβ over ERα, and ER-agonistic activity in MCF-7 cell proliferation assays.     

Morpholine-based RGD-cyclopentapeptides as αvβ3/αvβ5 integrin ligands: Role of configuration towards receptor binding affinity

Two c[RGDfX] cyclopeptides, having either l- or d-morpholine-3-COOH (Mor) as the X amino acid were developed as ligands for α_vβ₃/α_vβ₅ integrins. Biological assays showed only d-Mor-containing cyclopentapeptide capable to bind α_vβ₃ integrin with a low nanomolar affinity according to a two-site model, thus revealing a connection between the configuration of Mor and the preferred binding to α_vβ₃ integrin. Conformational analysis showed different structural preferences for the two peptides induced by the two enantiomeric cyclic amino acids, suggesting a role of the stereochemistry of Mor on the overall peptide conformation and on the presentation of the pharmacophoric Arg and Asp side chains.     

Tetrahydroprotoberberine alkaloids with dopamine and σ receptor affinity

Two series of analogues of the tetrahydroprotoberberine (THPB) alkaloid (±)-stepholidine that (a) contain various alkoxy substituents at the C10 position and, (b) were de-rigidified with respect to (±)-stepholidine, were synthesized and evaluated for affinity at dopamine and σ receptors in order to evaluate effects on D3 and σ2 receptor affinity and selectivity. Small n-alkoxy groups are best tolerated by D3 and σ2 receptors. Among all compounds tested, C10 methoxy and ethoxy analogues (10 and 11 respectively) displayed the highest affinity for σ2 receptors as well as σ2 versus σ1 selectivity and also showed the highest D3 receptor affinity. De-rigidification of stepholidine resulted in decreased affinity at all receptors evaluated; thus the tetracyclic THPB framework is advantageous for affinity at dopamine and σ receptors. Docking of the C10 analogues at the D3 receptor, suggest that an ionic interaction between the protonated nitrogen atom and Asp110, a H-bond interaction between the C2 phenol and Ser192, a H-bond interaction between the C10 phenol and Cys181 as well as hydrophobic interactions of the aryl rings to Phe106 and Phe345, are critical for high affinity of the compounds.     

Opioid and chemokine receptor heterodimers: arranged marriages or dangerous liaisons?

Adiponectin is one of the most effective adipokines in the context of correcting obesity-induced insulin resistance. However, adiponectin-deficient animal models show a relatively modest phenotype unless metabolically challenged. This suggests that potent compensatory mechanisms are in place. In this issue of the Biochemical Journal, Wong et al. characterize new members of the CTRPs [C1q-TNFalpha (tumour necrosis factor alpha)-related proteins]. They establish that some CTRPs are produced primarily in the stromal vascular fraction of adipose tissue, and that expression of CRTP1, in particular (like adiponectin), is induced by PPARgamma (peroxisome-proliferator-activated receptor gamma) agonists. Moreover, injection of recombinant CTRP1 displays glucose-lowering effects. These observations suggest that CTRP1 may have partially overlapping functions and, along with other paralogues, may effectively compensate for the chronic loss of adiponectin function.     

Structure–affinity relationships and pharmacological characterization of new alkyl-resorcinol cannabinoid receptor ligands: Identification of a dual cannabinoid receptor/TRPA1 channel agonist

In our ongoing program aimed at deeply investigating the endocannabinoid system (ES), a set of new alkyl-resorcinol derivatives was prepared focusing on the nature and the importance of the carboxamide functionality. Binding studies on CB₁ and CB₂ receptors, monoacylglycerol lipase (MAGL) and fatty acid amide hydrolase (FAAH) showed that some of the newly developed compounds behaved as very potent cannabinoid receptor ligands (K_i in the nanomolar range) while, however, none of them was able to inhibit MAGL and/or FAAH.Derivative 11 was a potent CB₁ and CB₂ ligand, with K_i values similar to WIN 55,212, exhibiting a CB₁ and CB₂ agonist profile in vitro. In the formalin test of peripheral acute and inflammatory pain in mice, this compound showed a weak and delayed antinociceptive effect against the second phase of the nocifensive response, exhibiting, interestingly, a quite potent transient receptor potential ankyrin type-1 (TRPA1) channel agonist activity. Moreover, derivative 14, characterized by lower affinity but higher CB₂ selectivity than 11, proved to behave as a weak CB₂ competitive inverse agonist.     

Functionalized cyclic RGD peptidomimetics: conjugable ligands for αvβ3 receptor imaging

The α(v)β(3) integrin is an adhesion molecule involved in physiological and pathological angiogenesis as well as in tumor invasion and metastasis, and therefore, there is a strong interest in developing novel agents interacting with this molecule. We report the synthesis and characterization of fluorescent α(v)β(3) integrin probes and their use to visualize integrin α(v)β(3) expression on human normal and cancer cells. The fluorescent probes we describe here may be of use for noninvasive imaging of α(V)β(3) integrin expression also in vivo.     

Regio and stereospecific synthesis of 11β-substituted 19-norsteroids: Influence of 11β-substitution on progesterone receptor affinity — (1)

The convenient synthesis of a series of lie-substituted 17-ethynyl-17β-hydroxy-4, 9-estradien-3-ones and their corresponding estradiols is reported. Relative affinities for the progestin and estrogen receptors showed very specific interactions between the progesterone receptor and the unsaturated substituents in the 11β-position of the steroid.     

Computer modeling of δ opioid receptor and interaction with ligands

Summary Human σ opioid receptor (σOR), a G-protein-coupled receptor, has been modeled using the helix axes as revealed by the crystallographic structure of bacteriorhodopsin and ligand binding profiles of single-point mutants of σOR. The model revealed feasibility of existence of a second disulfide bridge between the transmembrane helices (TMHs) 6 and 7, Cys²⁷³-Cys³⁰³. A common binding site has been suggested for high-affinity selective agonists DPDPE, DPLPE, DTLET, BW373U86 and antagonist Naltrindole. Docking calculations have shown that the amino group of the ligands forms a hydrogen bond with the imidazole ring of His³⁰¹ (TMH7) rather than with Asp¹²⁸ (TMH3) and is not a cation counterpart of this highly conserved aspartyl residue. All the findings and the model shed light on the putative structure and functioning of opioid receptors and can be used for designing further mutagenesis experiments.     

Synthesis of spirocyclic σ1 receptor ligands as potential PET radiotracers,structure–affinity relationships and in vitro metabolic stability

Several 3H-spiro[[2]benzofuran-1,4′-piperidines] bearing a p-fluorobenzyl residue at the N-atom and various substituents in position 3 of the benzofuran system were synthesized. The crucial reaction steps are the addition of a lithiated benzaldehyde derivative to the p-fluorobenzylpiperidone 5 and the BF₃·OEt₂ catalyzed substitution of the methoxy group of 2a by various nucleophiles. Structure–affinity relationship studies revealed that compounds with two protons (2d), a methoxy group (2a), and a cyano group (2e) in position 3 possess subnanomolar σ₁ affinity (K_i = 0.18 nM, 0.79 nM, 0.86 nM) and high selectivity against the σ₂ subtype. The metabolites of 2a, 2d, and 2e, which were formed upon incubation with rat liver microsomes, were identified. Additionally, the rate of metabolic degradation of 2a, 2d, and 2e was determined and compared with the degradation rate of the non-fluorinated spirocyclic compound 1. For the synthesis of the potential PET tracers [¹⁸F]2a and [¹⁸F]2e two different radiosynthetic approaches were followed.     

1,3-Dialkyl-8-N-substituted benzyloxycarbonylamino-9-deazaxanthines as potent adenosine receptor ligands: Design,synthesis, structure–affinity and structure–selectivity relationships

A number of 1,3-dialkyl-9-deazaxanthines (9-dAXs), bearing a variety of N-substituted benzyloxycarbonylamino substituents at position 8, were prepared and evaluated for their binding affinity to the recombinant human adenosine receptors (hARs), chiefly to the hA_2B and hA_2A AR subtypes. Several ligands endowed with excellent binding affinity to the hA_2B receptors, but low selectivity versus hA_2A and hA₁ were identified. Among these, 1,3-dimethyl-N-3′-thienyl carbamate 15 resulted as the most potent ligand at hA_2B (K_i = 0.8 nM), with a low selectivity versus hA_2A (hA_2A/hA_2B = 12.6) and hA₁ (hA₁/hA_2B = 12.5) and a higher selectivity versus hA₃ (hA₃/hA_2B = 454). When tested in functional assays in vitro, compound 15 exhibited high antagonist activities and efficacies versus both the A_2A and A_2B receptor subtypes, with pA₂ values close to the corresponding pK_is. A comparative analysis of structure–affinity and structure–selectivity relationships of the similar analogues 8-N-substituted benzyloxycarbonylamino- and 8-N-substituted phenoxyacetamido-9-dAXs suggested that their binding modes at the hA_2B and hA_2A ARs may strongly differ. Computational studies help to clarify this striking difference arising from a simple, albeit crucial, structural change, from CH₂OCON to OCH₂CON, in the para-position of the 8-phenyl ring.     

Opioid Receptor Trafficking and Signaling: What Happens After Opioid Receptor Activation?

Prolonged opioid treatment leads to a comprehensive cellular adaptation mediated by opioid receptors, a basis to understand the development of opioid tolerance and dependence. However, the molecular mechanisms underlying opioid-induced cellular adaptation remain obscure. Recent advances in opioid receptor trafficking and signaling in cells have extensively increased our insight into the network of intracellular signal integration. This review focuses on those important intracellular biochemical processes that play critical roles in the development of opioid tolerance and dependence after opioid receptor activation, and tries to explain what happens after opioid receptor activation, and how the cellular adaptation develops from cell membrane to nucleus. Decades of research have delineated a network on opioid receptor trafficking and signaling, but the challenge remains to explain opioid tolerance and dependence from a single cellular signal network.     

Nuclear receptor ligands and cofactor recruitment: is there a coactivator "on deck"?

Farnesoid X receptor (FXR) is a bile acid sensor that regulates lipid homeostasis. New structural data suggest that, unlike other nuclear receptors, FXR contains a second coactivator binding site and binds bile acids with the steroid backbone flipped head to tail, both of which have important functional ramifications.     

Agouti-related protein: more than a melanocortin-4 receptor antagonist?

It is well established that agouti-related protein (AGRP) can act as a competitive antagonist to proopiomelanocortin (POMC)-derived peptides at the melanocortin-4 receptor (MC4R), and that this homeostatic mechanism is important as a means of coordinating appetite with perceived metabolic requirement. However, there are clearly additional facets to the physiological role of AGRP, given that it is active in MC4R knockout mice and it has strikingly long-lasting effects on food intake, compared with MC4R agonists. In this review we focus on: (i) evidence that AGRP is more sensitive to perturbations in energy balance than POMC and is therefore the primary basis of melanocortinergic regulation. (ii) Evidence that the bioactive peptide AGRP83-132, acts by alternate mechanism(s) to elicit its long-term effects on food intake. (iii) Evidence that AGRP is post-translationally cleaved to generate AGRP83-132 and one or more N terminal peptides, which may have an important physiological role(s) that are independent of the melanocortin system. A clear understanding of how proAGRP processing is regulated, and the role of resultant peptides, may define additional therapeutic targets in the treatment of obesity.     

β-Peptides with improved affinity for hDM2 and hDMX

We previously described a series of 3₁₄-helical β-peptides that bind the hDM2 protein and inhibit its interaction with a p53-derived peptide in vitro. Here we present a detailed characterization of the interaction of these peptides with hDM2 and report two new β-peptides in which non-natural side chains have been substituted into the hDM2-recognition epitope. These peptides feature both improved affinity and inhibitory potency in fluorescence polarization and ELISA assays. Additionally, one of the new β-peptides also binds the hDM2-related protein, hDMX, which has been identified as another key therapeutic target for activation of the p53 pathway in tumors.     

Synthesis and structure–activity relationships of N-substituted spiropiperidines as nociceptin receptor ligands: Part 2

A series of N-8 substituted analogs based upon the spiropiperidine core of the original lead compound 1 was synthesized. This lead has been elaborated to compounds to give compounds 2 and 3 (R = H) that exhibited high NOP binding affinity as well as selectivity against other known opioid receptors. These two series have been further functionalized at the amido nitrogen. The synthesis and structure–activity relationship (SAR) of these and related compounds are discussed.     

A progesterone receptor affinity chromatography reagent: 17α-Hexynyl nortestosterone sepharose

Several affinity chromatography reagents have been proposed for purification of progesterone receptor (PgR), and significant results have been achieved with some of these. None, however, have approached the results achieved in affinity chromatography of estrogen receptor. We have therefore synthesized a number of new 19-nortestosterone derivatives capable of chemically stable linkage with Sepharose beads, and have identified one with very high PgR affinity for further study. We first synthesized the epoxides of 17α-allyl nortestosterone, by analogy with the estradiol derivatization of Greene and Jensen. The relative affinity of these epoxides for PgR from T47D human breast cancer cells, however, was only around 5% that of R5020, and affinity beads prepared from them bound very little PgR. We then reacted appropriately protected 17α-ethynyl-nortestosterone with a series of diiodo alkanes, and found that 17α-(6'-iodohex-1'-ynyl)nortestosterone had an affinity of 22% relative to R5020, equal to the affinity of progesterone itself. Reaction with Thiopropyl-Sepharose 6B yielded hexynyl-nortestosterone-Sepharose beads with a ligand density of about 7 micromoles/ml beads. One-hundred μl of these beads adsorbed 71% of the PgR present in 1 ml ofcytosol from T47D cells. This adsorption was inhibited by 10 μM progesterone but not Cortisol, indicating the specificity of the binding. Comparisions with NADAC and Sterogel, other affinity beads used for PgR purification, show that the former takes up much less receptor, while the latter takes up and releases similar amounts of receptor but more extraneous protein, and is less stable. We therefore believe that hexynyl-nortestosterone-Sepharose, having a high density of a high affinity ligand, and having chemically and biochemically stable covalent bonds, should be a good reagent for affinity purification of PgR.     

Relationships between the structure of 6-allyl-6,8-diazabicyclo[3.2.2]nonane derivatives and their σ receptor affinity and cytotoxic activity

A series of bridged piperazine derivatives was prepared and the affinity toward σ₁ and σ₂ receptors by means of radioligand binding assays as well as the inhibition of the growth of six human tumor cell lines was investigated. All possible stereoisomers of the 2-hydroxy, 2-methoxy, 2,2-dimethoxy, 2-oxo, and 2-unsubstituted 6,8-diazabicyclo[3.2.2]nonanes were prepared in a chiral pool synthesis starting with (S)- and (R)-glutamate. A Dieckmann analogous cyclization was the key step in the synthesis of the bicyclic framework. The configuration in position 2 was established by a diastereoselective LiBH₄ reduction and subsequent Mitsunobu inversion. Structure–affinity relationships demonstrate that substituents in position 2 decrease σ₁ receptor affinity which might be due to unfavorable interactions with the σ₁ receptor protein. Without a substituent in position 2 high σ₁ affinity was obtained (23a ((+)-(1S,5S)-6-allyl-8-(4-methoxybenzyl)-6,8-diazabicyclo[3.2.2]nonane): K_i = 11 nM). Experiments with six human tumor cell lines showed a weak but selective growth inhibition of the human small cell lung cancer cell line A-427 by the methyl ethers ent-16b (IC₅₀ = 18.9 μM), 21a (IC₅₀ = 16.4 μM), ent-21a (IC₅₀ = 20.4 μM), and 21b (IC₅₀ = 27.1 μM) and the unsubstituted compounds 23a and 23b (42% inhibition at 20 μM).