Mapping the catechol binding site in dopamine D₁ receptors: synthesis and evaluation of two parallel series of bicyclic dopamine analogues

A novel class of isochroman dopamine analogues, originally reported by Abbott Laboratories, have >100‐fold selectivity for D₁‐like over D₂‐like receptors. We synthesized a parallel series of chroman compounds and showed that repositioning the oxygen atom in the heterocyclic ring decreases potency and confers D₂‐like receptor selectivity to these compounds. In silico modeling supports the hypothesis that the altered pharmacology for the chroman series is due to potential intramolecular hydrogen bonding between the oxygen in the chroman ring and the meta‐hydroxy group of the catechol moiety. This interaction realigns the catechol hydroxy groups and disrupts key interactions between these ligands and critical serine residues in TM5 of the D₁‐like receptors. This hypothesis was tested by the synthesis and pharmacological evaluation of a parallel series of carbocyclic compounds. Our results suggest that if the potential for intramolecular hydrogen bonding is removed, D₁‐like receptor potency and selectivity are restored.     

Synthesis of Bitopic Ligands as Potent Dopamine D2 Receptor Agonists

In this study, we designed and synthesized twelve bitopic ligands as dopamine D₂ receptor (D₂R) agonists. The forskolin-induced cAMP accumulation assay revealed that all the finial compounds are able to activate D₂R. Furthermore, bitopic ligand N-((trans)-4-(((2,3-dihydro-1H-inden-2-yl)(propyl)amino)methyl)cyclo-hexyl)-1H-pyrrolo[2,3-b]pyridine-2-carboxamide ( 11 b ) showed 21-fold higher potency than lead compound propyl aminoindane ( 2 ) and 17-fold higher subtype selectivity for D₂R over D₄R, indicating that the optimal length of spacer affects the D₂R functionality. Molecular modeling study exhibited that 11 b formed an electrostatic interaction and two H-bonds with amino acid Asp114, which contributes significantly to the D₂R functional activity. Taken together, we discovered a bitopic ligand 11 b as potent D₂R agonist, which may be used as a tool compound for further study.     

Synthesis, 3D‐QSAR,and Structural Modeling of Benzolactam Derivatives with Binding Affinity for the D2 and D3 Receptors

A series of 37 benzolactam derivatives were synthesized, and their respective affinities for the dopamine D₂ and D₃ receptors evaluated. The relationships between structures and binding affinities were investigated using both ligand‐based (3D‐QSAR) and receptor‐based methods. The results revealed the importance of diverse structural features in explaining the differences in the observed affinities, such as the location of the benzolactam carbonyl oxygen, or the overall length of the compounds. The optimal values for such ligand properties are slightly different for the D₂ and D₃ receptors, even though the binding sites present a very high degree of homology. We explain these differences by the presence of a hydrogen bond network in the D₂ receptor which is absent in the D₃ receptor and limits the dimensions of the binding pocket, causing residues in helix 7 to become less accessible. The implications of these results for the design of more potent and selective benzolactam derivatives are presented and discussed.     

Pramipexole Derivatives as Potent and Selective Dopamine D3 Receptor Agonists with Improved Human Microsomal Stability

Herein we report the synthesis and evaluation of a series of new pramipexole derivatives as highly potent and selective agonists of the dopamine‐3 (D₃) receptor. A number of these new compounds bind to the D₃ receptor with sub‐nanomolar affinity and show excellent selectivity (>10 000) for the D₃ receptor over the D₁ and D₂ receptors. For example, compound 23 (N‐(cis‐3‐(2‐(((S)‐2‐amino‐4,5,6,7‐tetrahydrobenzo[d]thiazol‐6‐yl)(propyl)amino)ethyl)‐3‐hydroxycyclobutyl)‐3‐(5‐methyl‐1,2,4‐oxadiazol‐3‐yl)benzamide) binds to the D₃ receptor with a K_i value of 0.53 nM and shows a selectivity of >20 000 over the D₂ and D₁ receptors in the binding assays using a rat brain preparation. It has excellent stability in human liver microsomes. Moreover, in vitro functional assays showed it to be a full agonist for the human D₃ receptor.     

Selective Pharmacophore Models of Dopamine D1 and D2 Full Agonists Based on Extended Pharmacophore Features

This study is focused on the identification of structural features that determine the selectivity of dopamine receptor agonists toward D₁ and D₂ receptors. Selective pharmacophore models were developed for both receptors. The models were built by using projected pharmacophoric features that represent the main agonist interaction sites in the receptor (the Ser residues in TM5 and the Asp in TM3), a directional aromatic feature in the ligand, a feature with large positional tolerance representing the positively charged nitrogen in the ligand, and sets of excluded volumes reflecting the shapes of the receptors. The sets of D₁ and D₂ ligands used for modeling were carefully selected from published sources and consist of structurally diverse, conformationally rigid full agonists as active ligands together with structurally related inactives. The robustness of the models in discriminating actives from inactives was tested against four ensembles of conformations generated by using different established methods and different force fields. The reasons for the selectivity can be attributed to both geometrical differences in the arrangement of the features, e.g., different tilt angels of the π system, as well as shape differences covered by the different sets of excluded volumes. This work provides useful information for the design of new D₁ and D₂ agonists and also for comparative homology modeling of D₁ and D₂ receptors. The approach is general and could therefore be applied to other ligand–protein interactions for which no experimental protein structure is available.     

Structure–Activity Relationships of Quinoxaline‐Based 5‐HT3A and 5‐HT3AB Receptor‐Selective Ligands

Until recently, discriminating between homomeric 5‐HT₃A and heteromeric 5‐HT₃AB receptors was only possible with ligands that bind in the receptor pore. This study describes the first series of ligands that can discriminate between these receptor types at the level of the orthosteric binding site. During a recent fragment screen, 2‐chloro‐3‐(4‐methylpiperazin‐1‐yl)quinoxaline (VUF10166) was identified as a ligand that displays an 83‐fold difference in [³H]granisetron binding affinity between 5‐HT₃A and 5‐HT₃AB receptors. Fragment hit exploration, initiated from VUF10166 and 3‐(4‐methylpiperazin‐1‐yl)quinoxalin‐2‐ol, resulted in a series of compounds with higher affinity at either 5‐HT₃A or 5‐HT₃AB receptors. These ligands reveal that a single atom is sufficient to change the selectivity profile of a compound. At the extremes of the new compounds were 2‐amino‐3‐(4‐methylpiperazin‐1‐yl)quinoxaline, which showed 11‐fold selectivity for the 5‐HT₃A receptor, and 2‐(4‐methylpiperazin‐1‐yl)quinoxaline, which showed an 8.3‐fold selectivity for the 5‐HT₃AB receptor. These compounds represent novel molecular tools for studying 5‐HT₃ receptor subtypes and could help elucidate their physiological roles.     

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.     

Targeting Serotonin 2A and Adrenergic α1 Receptors for Ocular Antihypertensive Agents: Discovery of 3,4‐Dihydropyrazino[1,2‐b]indazol‐1(2H)‐one Derivatives

Glaucoma affects millions of people worldwide and causes optic nerve damage and blindness. The elevation of the intraocular pressure (IOP) is the main risk factor associated with this pathology, and decreasing IOP is the key therapeutic target of current pharmacological treatments. As potential ocular hypotensive agents, we studied compounds that act on two receptors (serotonin 2A and adrenergic α₁) linked to the regulation of aqueous humour dynamics. Herein we describe the design, synthesis, and pharmacological profiling of a series of novel bicyclic and tricyclic N2‐alkyl‐indazole‐amide derivatives. This study identified a 3,4‐dihydropyrazino[1,2‐b]indazol‐1(2H)‐one derivative with potent serotonin 2A receptor antagonism, >100‐fold selectivity over other serotonin subtype receptors, and high affinity for the α₁ receptor. Moreover, upon local administration, this compound showed superior ocular hypotensive action in vivo relative to the clinically used reference compound timolol.     

Effect of Linker Length and Composition on Heterobivalent Ligand‐Mediated Receptor Cross‐Talk between the A1 Adenosine and β2 Adrenergic Receptors

Heterobivalent ligands that possess pharmacophores designed to interact with both the A₁ adenosine receptor (A₁AR) and the β₂ adrenergic receptor (β₂AR) were prepared. More specifically, these ligands contain an adenosine moiety that is linked via its N⁶‐position to the amino group of the saligenin‐substituted ethanolamine moiety present in the well‐known β₂AR agonist, salbutamol. The affinities of these ligands were determined at both receptors and found to vary with linker length and composition. With all compounds, affinity and functional potencies were found to have selectivity for the A₁AR over the β₂AR. In all cases, cAMP accumulation (a β₂AR‐mediated response) was mainly observed when the A₁AR was blocked or its function decreased by pertussis toxin or chronic agonist treatment. This suggests that heterobivalent compounds for receptors that mediate opposite responses might be useful for elucidating the mechanisms of receptor cross‐talk and how this interaction, in terms of responsiveness, may change under pathophysiological conditions.     

Development of Benzophenone‐Alkyne Bifunctional Sigma Receptor Ligands

Sigma (σ) receptors are unique non‐opioid binding sites that are associated with a broad range of disease states. Sigma‐2 receptors provide a promising target for diagnostic imaging and pharmacological interventions to curb tumor progression. Most recently, the progesterone receptor (PGRMC1, 25 kDa) has been shown to have σ2 receptor‐like binding properties, thus highlighting the need to understand the biological function of an 18 kDa protein that exhibits σ2‐like photoaffinity labeling (denoted here as σ2‐18k) but the amino acid sequence of which is not known. In order to provide new tools for the study of the σ2‐18k protein, we have developed bifunctional σ receptor ligands each bearing a benzophenone photo‐crosslinking moiety and an alkyne group to which an azide‐containing biotin affinity tag can be covalently attached through click chemistry after photo‐crosslinking. Although several compounds showed favorable σ2 binding properties, the highest affinity (2 nM ) and the greatest potency in blocking photolabeling of σ2‐18k by a radioactive photoaffinity ligand was shown by compound 22 . These benzophenone‐alkyne σ receptor ligands might therefore be amenable for studying the σ2‐18k protein through chemical biology approaches. To the best of our knowledge, these compounds represent the first reported benzophenone‐containing clickable σ receptor ligands, which might potentially have broad applications based on the “plugging in” of various tags.     

Carbohydrate–PNA and Aptamer–PNA Conjugates for the Spatial Screening of Lectins and Lectin Assemblies

Nucleic acid architectures offer intriguing opportunities for the interrogation of structural properties of protein receptors. In this study, we performed a DNA‐programmed spatial screening to characterize two functionally distinct receptor systems: 1) structurally well‐defined Ricinus communis agglutinin (RCA₁₂₀), and 2) rather ill‐defined assemblies of L‐selectin on nanoparticles and leukocytes. A robust synthesis route that allowed the attachment both of carbohydrate ligands—such as N‐acetyllactosamine (LacNAc), sialyl‐Lewis‐X (sLe^X), and mannose—and of a DNA aptamer to PNAs was developed. A systematically assembled series of different PNA–DNA complexes served as multivalent scaffolds to control the spatial alignments of appended lectin ligands. The spatial screening of the binding sites of RCA₁₂₀ was in agreement with the crystal structure analysis. The study revealed that two appropriately presented LacNAc ligands suffice to provide unprecedented RCA₁₂₀ affinity (K_D=4 μM ). In addition, a potential secondary binding site was identified. Less dramatic binding enhancements were obtained when the more flexible L‐selectin assemblies were probed. This study involved the bivalent display both of the weak‐affinity sLe^X ligand and of a high‐affinity DNA aptamer. Bivalent presentation led to rather modest (sixfold or less) enhancements of binding when the self‐assemblies were targeted against L‐selectin on gold nanoparticles. Spatial screening of L‐selectin on the surfaces of leukocytes showed higher affinity enhancements (25‐fold). This and the distance–activity relationships indicated that leukocytes permit dense clustering of L‐selectin.     

Insight into the Interactions between Novel Coumarin Derivatives and Human A3 Adenosine Receptors

A study focused on the discovery of new chemical entities based on the 3‐arylcoumarin scaffold was performed with the aim of finding new adenosine receptor (AR) ligands. Thirteen synthesized compounds were evaluated by radioligand binding (A₁, A_2A, and A₃) and adenylyl cyclase activity (A_2B) assays in order to study their affinity for the four human AR (hAR) subtypes. Seven of the studied compounds proved to be selective A₃AR ligands, with 3‐(4′‐methylphenyl)‐8‐(2‐oxopropoxy)coumarin ( 12 ) being the most potent (K_i=634 nM ). None of the compounds showed affinity for the A_2B receptor, while four compounds were found to be nonselective AR ligands for the other three subtypes. Docking simulations were carried out to identify the hypothetical binding mode and to rationalize the interaction of these types of coumarin derivatives with the binding site of the three ARs to which binding was observed. The results allowed us to conclude that the 3‐arylcoumarin scaffold composes a novel and promising class of A₃AR ligands. ADME properties were also calculated, with the results suggesting that these compounds are promising leads for the identification of new drug candidates.     

Synthesis and Pharmacological Evaluation of α4β2 Nicotinic Ligands with a 3‐Fluoropyrrolidine Nucleus

Nicotinic acetylcholine receptors (nAChRs) play an important role in many central nervous system disorders such as Alzheimer’s and Parkinson’s diseases, schizophrenia, and mood disorders. The α₄β₂ subtype has emerged as an important target for the early diagnosis and amelioration of Alzheimer’s disease symptoms. Herein we report a new class of α₄β₂ receptor ligands characterized by a basic pyrrolidine nucleus, the basicity of which was properly decreased through the insertion of a fluorine atom at the 3‐position, and a pyridine ring carrying at the 3‐position substituents known to positively affect affinity and selectivity toward the α₄β₂ subtype. Derivatives 3‐(((2S,4R)‐4‐fluoropyrrolidin‐2‐yl)methoxy)‐5‐(phenylethynyl)pyridine ( 11 ) and 3‐((4‐fluorophenyl)ethynyl)‐5‐(((2S,4R)‐4‐fluoropyrrolidin‐2‐yl)methoxy)pyridine ( 12 ) were found to be the most promising ligands identified in this study, showing good affinity and selectivity for the α₄β₂ subtype and physicochemical properties predictive of a relevant central nervous system penetration.     

Multitarget‐Directed Tricyclic Pyridazinones as G Protein‐Coupled Receptor Ligands and Cholinesterase Inhibitors

By following a multitarget ligand design approach, a library of 47 compounds was prepared, and they were tested as binders of selected G protein‐coupled receptors (GPCRs) and inhibitors of acetyl and/or butyryl cholinesterase. The newly designed ligands feature pyridazinone‐based tricyclic scaffolds connected through alkyl chains of variable length to proper amine moieties (e.g., substituted piperazines or piperidines) for GPCR and cholinesterase (ChE) molecular recognition. The compounds were tested at three different GPCRs, namely serotoninergic 5‐HT_1A, adrenergic α_1A, and dopaminergic D₂ receptors. Our main goal was the discovery of compounds that exhibit, in addition to ChE inhibition, antagonist activity at 5‐HT_1A because of its involvement in neuronal deficits typical of Alzheimer’s and other neurodegenerative diseases. Ligands with nanomolar affinity for the tested GPCRs were discovered, but most of them behaved as dual antagonists of α_1A and 5‐HT_1A receptors. Nevertheless, several compounds displaying this GPCR affinity profile also showed moderate to good inhibition of AChE and BChE, thus deserving further investigations to exploit the therapeutic potential of such unusual biological profiles.     

Synthesis and Characterization of New Bivalent Agents as Melatonin- and Histamine H3-Ligands

Melatonin is an endogenous molecule involved in many pathophysiological processes. In addition to the control of circadian rhythms, its antioxidant and neuroprotective properties have been widely described. Thus far, different bivalent compounds composed by a melatonin molecule linked to another neuroprotective agent were synthesized and tested for their ability to block neurodegenerative processes in vitro and in vivo. To identify a novel class of potential neuroprotective compounds, we prepared a series of bivalent ligands, in which a prototypic melatonergic ligand is connected to an imidazole-based H₃ receptor antagonist through a flexible linker. Four imidazolyl-alkyloxy-anilinoethylamide derivatives, characterized by linkers of different length, were synthesized and their binding affinity for human MT₁, MT₂ and H₃ receptor subtypes was evaluated. Among the tested compounds, 14c and 14d, bearing a pentyl and a hexyl linker, respectively, were able to bind to all receptor subtypes at micromolar concentrations and represent the first bivalent melatonergic/histaminergic ligands reported so far. These preliminary results, based on binding affinity evaluation, pave the way for the future development of new dual-acting compounds targeting both melatonin and histamine receptors, which could represent promising therapeutic agents for the treatment of neurodegenerative pathologies.     

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.     

Optimizing the Expression of Human Dopamine Receptors in Escherichia coli

The human dopamine receptors D_2S and D₃ belong to the group of G protein-coupled receptors (GPCRs) and are important drug targets. Structural analyses and development of new receptor subtype specific drugs have been impeded by low expression yields or receptor instability. Fusing the T4 lysozyme into the intracellular loop 3 improves crystallization but complicates conformational studies. To circumvent these problems, we expressed the human D_2S and D₃ receptors in Escherichia coli using different N- and C-terminal fusion proteins and thermostabilizing mutations. We optimized expression times and used radioligand binding assays with whole cells and membrane homogenates to evaluate K_D-values and the number of receptors in the cell membrane. We show that the presence but not the type of a C-terminal fusion protein is important. Bacteria expressing receptors capable of ligand binding can be selected using FACS analysis and a fluorescently labeled ligand. Improved receptor variants can thus be generated using error-prone PCR. Subsequent analysis of clones showed the distribution of mutations over the whole gene. Repeated cycles of PCR and FACS can be applied for selecting highly expressing receptor variants with high affinity ligand binding, which in the future can be used for analytical studies.     

Application of the guanidine-acylguanidine bioisosteric approach to argininamide-type NPY Y₂ receptor antagonists

Strongly basic groups such as guanidine moieties are crucial structural elements, but they compromise the drug‐likeness of numerous biologically active compounds, including ligands of G‐protein‐coupled receptors (GPCRs). As part of a project focused on the search for guanidine bioisosteres, argininamide‐type neuropeptide Y (NPY) Y₂ receptor (Y₂R) antagonists related to BIIE0246 were synthesized. Starting from ornithine derivatives, N^G‐acylated argininamides were obtained by guanidinylation with tailor‐made mono‐Boc‐protected N‐acyl‐S‐methylisothioureas. The compounds were investigated for Y₂R antagonism (calcium assays), Y₂R affinity, and NPY receptor subtype selectivity (flow cytometric binding assays). Most of the N^G‐substituted (S)‐argininamides showed Y₂R antagonistic activities and binding affinities similar to those of the parent compound, whereas N^G‐acylated or ‐carbamoylated analogues with a terminal amine were superior (Y₂R: K_i and K_B values in the low nanomolar range). This demonstrates that the basicity of the compounds, although 4–5 orders of magnitude lower than that of guanidines, is sufficient to form key interactions with acidic amino acids of the Y₂R. The acylguanidines bind with high affinity and selectivity to Y₂R over the Y₁, Y₄, and Y₅ receptors. As derivatization of the amino group is tolerated, these compounds can be considered building blocks for the preparation of versatile fluorescent and radiolabeled pharmacological tools for in vitro studies of the Y₂R. The results support the concept of bioisosteric guanidine–acylguanidine exchange as a broadly applicable approach to retain pharmacological activity despite decreased basicity.