Synthesis, biological activity and molecular modelling studies of tricyclic alkylimidazo-, pyrimido- and diazepinopurinediones

Syntheses and biological activities of imidazo-, pyrimido- and diazepino[2,1-f]purinediones containing N-alkyl substituents (with straight, branched or unsaturated chains) are described. Tricyclic derivatives were synthesized by the cyclization of 8-bromo-substituted 7-(2-bromoethyl)-, 7-(3-chloropropyl)- or 7-(4-bromobutyl)-theophylline with primary amines under various conditions. Compound 22 with an ethenyl substituent was synthesized by dehydrohalogenation of 9-(2-bromoethyl)-1,3-dimethyltetrahydropyrimido[2,1-f]purinedione. The obtained derivatives (5–35) were initially evaluated for their affinity at rat A₁ and A_2A adenosine receptors (AR), showing moderate affinity for both adenosine receptor subtypes. The best ligands were diazepinopurinedione 28 (K_i = 0.28 μM) with fivefold A_2A selectivity and the non-selective A₁/A_2A AR ligand pyrimidopurinedione 35 (K_i A₁ = 0.28 μM and K_i A_2A = 0.30 μM). The compounds were also evaluated for their affinity at human A₁, A_2A, A_2B and A₃ ARs. All of the obtained compounds were docked to the A_2A AR X-ray structure in complex with the xanthine-based, potent adenosine receptor antagonist—XAC. The likely interactions of imidazo-, pyrimido- and diazepino[2,1-f]purinediones with the residues forming the A_2A binding pocket were discussed. Furthermore, the new compounds were tested in vivo as anticonvulsants in maximal electroshock, subcutaneous pentylenetetrazole (ScMet) and TOX tests in mice (i.p.). Pyrimidopurinediones showed anticonvulsant activity mainly in the ScMet test. The best derivative was compound 11, showing 100 % protection at a dose of 100 mg/kg without symptoms of neurotoxicity. Compounds 6, 7, 8 and 14 with short substituents showed neurotoxicity and caused death. In rat tests (p.o.), 9 was characterized by a high protection index (>13.3). AR affinity did not apparently correlate with the antiepileptic potency of the compounds.

Electronic supplementary material

The online version of this article (doi:10.1007/s11302-013-9358-3) contains supplementary material, which is available to authorized users.     

Quantitative structure–activity relationship study of new potent and selective antagonists at the 5-HT1A and adrenergic α1d receptors: Derivatives of spiroethyl phenyl(substituted)piperazine

The antagonistic activities of derivatives of spiroethyl phenyl(substituted)piperazine at the 5-HT_1A and adrenergic α_1d receptors is quantitatively analyzed employing physicochemical and structural parameters. The derived correlation equation revealed that a substituent, other than 2-CH₃ in the phenyl ring, having higher molar refraction, MR, and a substituent producing higher positive field effect at the 3-position are beneficial in increasing the binding affinity at the 5-HT_1A receptor. In addition, a less hydrophobic substituent at the 4-position is also helpful in augmenting the binding affinity. The 5-R substituents which have higher MR values, however, elicit a detrimental effect. Two disubstituted compounds which are not present in the original data-set and have higher theoretical binding affinities are designed from the correlation equation. These compounds consisting of 2-OCH(CH₃)₂, 3-Cl and 2-C₃H₇, 3-Cl in the phenyl ring, have theoretical pK_i values 10.57 and 10.12 respectively. For the adrenergic α_1d receptor, a less bulky group at the 3-position with 5-Cl (or simply a 3-Cl) is advantageous in increasing the binding affinity. Likewise, a substituent exhibiting a less negative resonance effect at the 4-position and the substituent with low polarizability and showing more a negative resonance effect at the 5-position are suitable for enhancement of the binding affinity. The analysis provides the grounds for rationalizing substituent selection in designing better potency antagonists in the series.     

The synthesis and biological evaluation of 2-amino-4,5,6,7,8,9-hexahydrocycloocta[b]thiophenes as allosteric modulators of the A1 adenosine receptor

A series of 2-amino-4,5,6,7,8,9-hexahydrocycloocta[b]thiophenes were prepared and evaluated as potential allosteric modulators of the A₁ adenosine receptor (AR). The structure-activity relationships of the 3-position were explored along with varying the size of the cycloalkyl ring. 2-Aminothiophenes with amide and hydrazide groups in the 3-position were completely inactive in an A₁-AR-mediated ERK1/2 phosphorylation assay, yet most of the 3-benzoyl substituted compounds exhibited allosteric effects on responses mediated by the orthosteric agonist, R-PIA. Despite finding an increase in both agonistic and allosteric activities by going from a cyclopentyl ring to a cyclohexyl ring in the 3-benzoyl series, decreases were observed when further increasing the ring size. Varying the substituents on the phenyl ring of the 3-benzoyl group also affected the activity of these compounds.     

A new D₂ dopamine receptor agonist allosterically modulates A(2A) adenosine receptor signalling by interacting with the A(2A)/D₂ receptor heteromer

The structural and functional interaction between D₂ dopamine receptor (DR) and A_2A adenosine receptor (AR) has suggested these two receptors as a pharmacological target in pathologies associated with dopamine dysfunction, such as Parkinson's disease. In transfected cell lines it has been demonstrated the activation of D₂DR induces a significant negative regulation of A_2AAR-mediated responses, whereas few data are at now available about the regulation of A_2AAR by D₂DR agonists at receptor recognition site. In this work we confirmed that in A_2AAR/D₂DR co-transfected cells, these receptors exist as homo- and hetero-dimers. The classical D₂DR agonists were able to negatively modulate both A_2AAR affinity and functionality. These effects occurred even if any significant changes in A_2AAR/D₂DR energy transfer interaction could be detected in BRET experiments.Since the development of new molecules able to target A_2A/D₂ dimers may represent an attractive tool for innovative pharmacological therapy, we also identified a new small molecule, 3-(3,4-dimethylphenyl)-1-(2-piperidin-1-yl)ethyl)piperidine (compound 1), full agonist of D₂DR and modulator of A_2A-D₂ receptor dimer. This compound was able to negatively modulate A_2AAR binding properties and functional responsiveness in a manner comparable to classical D₂R agonists. In contrast to classical agonists, compound 1 led to conformational changes in the quaternary structure in D₂DR homomers and heteromers and induced A_2AAR/D₂DR co-internalization. These results suggest that compound 1 exerts a high control of the function of heteromers and could represent a starting point for the development of new drugs targeting A_2AAR/D₂ DR heteromers.     

Design,synthesis and evaluation of 2-aryl benzoxazoles as promising hit for the A2A receptor

The development of adenosine A_2A receptor antagonists has received much interest in recent years for the treatment of neurodegenerative diseases. Based on docking studies, a new series of 2-arylbenzoxazoles has been identified as potential A_2AR antagonists. Structure-affinity relationship was investigated in position 2, 5 and 6 of the benzoxazole heterocycle leading to compounds with a micromolar affinity towards the A_2A receptor. Compound F1, with an affinity of 1?μm, presented good absorption, distribution, metabolism and excretion properties with an excellent aqueous solubility (184?μm) without being cytotoxic at 100?μm. This compound, along with low-molecular weight compound D1 (K_i?=?10?μm), can be easily modulated and thus considered as relevant starting points for further hit-to-lead optimisation.     

Role of Disulfides and Sulfhydryl Groups in Agonist and Antagonist Binding in Serotonin1A Receptors from Bovine Hippocampus

SUMMARY 1. The serotonin_1A (5-HT_1A) receptors are members of a superfamily of seven-transmembrane-domain receptors that couple to G-proteins. They appear to be involved in various behavioral and cognitive functions. Mutagenesis and modeling studies point out that the ligand-binding sites in serotonin receptors are located in the transmembrane domain. However, these binding sites are not very well characterized. Since disulfide bonds and sulfhydryl groups have been shown to play vital roles in the assembly, organization, and function of various G-protein-coupled receptors, we report here the effect of disulfide and sulfhydryl group modifications on the agonist and antagonist binding activity of 5-HT_1A receptors from bovine hippocampus.2. DTT or NEM treatment caused a concentration-dependent reduction in specific binding of the agonist and antagonist in 5-HT_1A receptors from bovine hippocampal native and solubilized membranes. This is supported by a concomitant reduction in binding affinity.3. Pretreatment of the receptor with unlabeled ligands prior to chemical modifications indicate that the majority of disulfides or sulfhydryl groups that undergo modification giving rise to inhibition in binding activity could be at the vicinity of the ligand-binding sites.4. In addition, ligand-binding studies in presence of GTP--S, a nonhydrolyzable analogue of GTP, indicate that sulfhydryl groups (and disulfide bonds to a lesser extent) are vital for efficient coupling between the 5-HT_1A receptor and the G-protein.5. Our results point out that disulfide bonds and sulfhydryl groups could play an important role in ligand binding in 5-HT_1A receptors.     

Activation of 5-HT1A receptors inhibits carbachol-stimulated inositol 1,4,5-trisphosphate mass accumulation in the rodent hippocampus

We have previously demonstrated that 5-HT_1A receptor agonists partially prevent the stimulation by carbachol of [³H]-phosphoinositide hydrolysis in immature rat hippocampal slices. This negative modulation has been investigated further by measuring, using a radioreceptor assay, the mass accumulation of IP₃. In hippocampal slices from developing rats and in hippocampal neurons, carbachol enhanced the accumulation of IP₃ and this response was partially inhibited by 8-OH-DPAT with a potency compatible with the affinity of this agonist for 5-HT_1A receptors. The inhibition of the carbachol response by 8-OH-DPAT was non-competitive in nature and 8-OH-DPAT did not affect the inhibitory potency of pirenzepine. The inhibitory effect of 8-OH-DPAT was maintained after washing the slices preincubated with this compound but was not observed on the carbachol-stimulated PIP₂ hydrolysis in hippocampal membranes, suggesting that this compound induces long lasting changes of nuscarinic receptors and/or their effector mechanism by an indirect action.     

Design, synthesis, and evaluation of fused heterocyclic analogs of SCH 58261 as adenosine A2A receptor antagonists

SCH 58261 is a reported adenosine A_2A receptor antagonist which is active in rat in vivo models of Parkinson’s Disease upon ip administration. However, it has poor selectivity versus the A₁ receptor and does not demonstrate oral activity. Quinoline analogs have improved upon the selectivity and pharmacokinetics of SCH 58261, but were difficult to handle due to poor aqueous solubility. We report the design and synthesis of fused heterocyclic analogs of SCH 58261 with aqueous solubility as well as improved A_2A receptor binding selectivity and pharmacokinetic properties. In particular, the tetrahydronaphthyridine 4s has excellent A_2A receptor in vitro binding affinity and selectivity, is active orally in a rat in vivo model of Parkinson’s Disease, and has aqueous solubility of 100 μM at physiological pH.     

Limonene, a natural cyclic terpene, is an agonistic ligand for adenosine A(2A) receptors

Limonene is a major aromatic compound in essential oils extracted from citrus rind. The application of limonene, especially in aromatherapy, has expanded significantly, but its potential effects on cellular metabolism have been elusive. We found that limonene directly binds to the adenosine A_2A receptor, which may induce sedative effects. Results from an in vitro radioligand binding assay showed that limonene exhibits selective affinity to A_2A receptors. In addition, limonene increased cytosolic cAMP concentration and induced activation of protein kinase A and phosphorylation of cAMP-response element-binding protein in Chinese hamster ovary cells transfected with the human adenosine A_2A receptor gene. Limonene also increased cytosolic calcium concentration, which can be achieved by the activation of adenosine A_2A receptors. These findings suggest that limonene can act as a ligand and an agonist for adenosine A_2A receptors.     

Synthesis and pharmacological evaluation of novel 1- and 8-substituted-3-furfuryl xanthines as adenosine receptor antagonists

The synthesis of an important set of 3-furfurylxanthine derivatives is described. Binding affinities were determined for rat A₁ and human A_2A, A_2B and A₃ receptors. Several of the 3-furfuryl-7-methylxanthine derivatives showed moderate-to-high affinity at human A_2B receptors, the most active compound (10d) having a K_i of 7.4 nM for hA_2B receptors, with selectivities over rA₁ and hA_2A receptors up to 14-fold and 11-fold, respectively. Affinities for hA₃ receptors were very low for all members of the set.     

Modulation of Antagonist Binding to Serotonin1A Receptors from Bovine Hippocampus by Metal Ions

1. The serotonin_1A(5-HT_1A) receptors are members of a superfamily of seven transmembrane domain receptors that couple to G-proteins. They appear to be involved in various behavioral and cognitive functions. Although specific 5-HT_1Aagonists have been discovered more than a decade back, the development of selective 5-HT_1Aantagonists has been achieved only recently.2. We have examined the modulation of the specific antagonist [³H]p-MPPF binding to 5-HT_1Areceptors from bovine hippocampal membranes by monovalent and divalent metal ions. Our results show that the antagonist binding to 5-HT_1Areceptors is inhibited by both monovalent and divalent cations in a concentration-dependent manner. This is accompanied by a concomitant reduction in binding affinity.3. Our results also show that the specific antagonist p-MPPF binds to all available receptors in the bovine hippocampal membrane irrespective of their state of G-protein coupling and other serotonergic ligands such as 5-HT and OH-DPAT effectively compete with the specific antagonist [³H]p-MPPF.4. These results are relevant to ongoing analyses of the overall modulation of ligand binding in G-protein-coupled seven transmembrane domain receptors.     

Quantitative structure-activity relationship study on affinity profile of a series of 1,8-naphthyridine antagonists toward bovine adenosine receptors

The affinity profiles for the bovine adenosine receptors, A(1) and A(2A), of a series of 1,8-naphthyridine derivatives were quantitatively analyzed using physicochemical and structural parameters of the substituents, present at varying positions of the molecules. The derived significant correlation, for bovine A(1) receptor, suggested that a R(1) substituent having a higher van der Waals volume, a R(2) substituent being a hydrogen-bond donor and a R(3) substituent able to transmit a higher field effect are helpful in augmenting the pK(i) of a compound. Similarly the study, pertaining to bovine A(2A) receptor, revealed that a less bulky substituent at R(2) and a strong electron-withdrawing substituent at R(3) are desirable in improving the binding affinity of a compound while substituents at R(1) remain insignificant to any interaction.     

The Serotonin 1A A Receptor: A Representative Member of the Serotonin Receptor Family

1. Serotonin is an intrinsically fluorescent biogenic amine that acts as a neurotransmitter and is found in a wide variety of sites in the central and peripheral nervous system. Serotonergic signaling appears to play a key role in the generation and modulation of various cognitive and behavioral functions.2. Serotonin exerts its diverse actions by binding to distinct cell surface receptors which have been classified into many groups. The serotonin_1A (5-HT_1A) receptor is the most extensively studied of the serotonin receptors and belongs to the large family of seven transmembrane domain G-protein coupled receptors.3. The tissue and sub-cellular distribution, structural characteristics, signaling of the serotonin_1A receptor and its interaction with G-proteins are discussed.4. The pharmacology of serotonin_1A receptors is reviewed in terms of binding of agonists and antagonists and sensitivity of their binding to guanine nucleotides.5. Membrane biology of 5-HT_1A receptors is presented using the bovine hippocampal serotonin_1A receptor as a model system. The ligand binding activity and G-protein coupling of the receptor is modulated by membrane cholesterol thereby indicating the requirement of cholesterol in maintaining the receptor organization and function. This, along with the reported detergent resistance characteristics of the receptor, raises important questions on the role of membrane lipids and domains in the function of this receptor.     

Membrane Organization of the Serotonin<Subscript><Emphasis Type="Bold">1A</Emphasis></Subscript> Receptor Monitored by a Detergent-Free Approach

1. Insolubility of membrane constituents in nonionic detergents such as Triton X-100 has been a widely used biochemical criterion to indicate their localization in membrane domains. However, concerns on the possibility of membrane perturbation in the presence of detergents have led to the development of detergent-free approaches. 2. We have explored the organization of the serotonin_1A receptor, an important G-protein coupled receptor, from bovine hippocampus and CHO cells using a detergent-free approach in order to address the points of agreement with our previous results using Triton X-100. 3. A significant fraction of the serotonin_1A receptor has been found to be localized in a heavy density fraction obtained using a detergent-free approach to isolate membrane domains. In addition, we have characterized the membrane fractions isolated in terms of their lipid composition and membrane physical properties. 4. The results obtained on the membrane localization of the serotonin_1A receptor from the present experiments using a detergent-free approach correlate well with our earlier findings obtained using a detergent-based method (Kalipatnapu, S., and Chattopadhyay, A., FEBS Lett. 576:455–460, 2004). These results provide important information on the membrane organization of the hippocampal serotonin_1A receptor and are relevant in view of the concerns on the use of detergent in determination of membrane organization of constituent proteins and lipids.     

Biaryl and heteroaryl derivatives of SCH 58261 as potent and selective adenosine A2A receptor antagonists

SCH 58261 is a reported adenosine A_2A receptor antagonist, which is active in rat in vivo models of Parkinson’s Disease upon ip administration. However, it has poor selectivity versus the A₁ receptor and does not demonstrate oral activity. We report the design and synthesis of biaryl and heteroaryl analogs of SCH 58261 which improve the A_2A receptor binding selectivity as well as the pharmacokinetic properties of SCH 58261. In particular, the quinoline 25 has excellent A_2A receptor in vitro binding affinity and selectivity, sustained rat plasma levels upon oral dosing, and is active orally in a rat behavioral assay.     

Neuroprotection by adenosine in the brain: From A<Subscript>1</Subscript> receptor activation to A<Subscript>2A</Subscript> receptor blockade

Adenosine is a neuromodulator that operates via the most abundant inhibitory adenosine A₁ receptors (A₁Rs) and the less abundant, but widespread, facilitatory A_2ARs. It is commonly assumed that A₁Rs play a key role in neuroprotection since they decrease glutamate release and hyperpolarize neurons. In fact, A₁R activation at the onset of neuronal injury attenuates brain damage, whereas its blockade exacerbates damage in adult animals. However, there is a down-regulation of central A₁Rs in chronic noxious situations. In contrast, A_2ARs are up-regulated in noxious brain conditions and their blockade confers robust brain neuroprotection in adult animals. The brain neuroprotective effect of A_2AR antagonists is maintained in chronic noxious brain conditions without observable peripheral effects, thus justifying the interest of A_2AR antagonists as novel protective agents in neurodegenerative diseases such as Parkinsons and Alzheimers disease, ischemic brain damage and epilepsy. The greater interest of A_2AR blockade compared to A₁R activation does not mean that A₁R activation is irrelevant for a neuroprotective strategy. In fact, it is proposed that coupling A_2AR antagonists with strategies aimed at bursting the levels of extracellular adenosine (by inhibiting adenosine kinase) to activate A₁Rs might constitute the more robust brain neuroprotective strategy based on the adenosine neuromodulatory system. This strategy should be useful in adult animals and especially in the elderly (where brain pathologies are prevalent) but is not valid for fetus or newborns where the impact of adenosine receptors on brain damage is different.     

Metal Ion and Guanine Nucleotide Modulations of Agonist Interaction in G-Protein-Coupled Serotonin1A Receptors from Bovine Hippocampus

1. The serotonin type 1A (5-HT_1A) receptors are members of a superfamily of seven transmembrane domain receptors that couple to GTP-binding regulatory proteins (G-proteins). We have studied the modulation of agonist binding to 5-HT_1A receptors from bovine hippocampus by metal ions and guanine nucleotide.2. Bovine hippocampal membranes containing the 5-HT_1A receptor were isolated. These membranes exhibited high-affinity binding sites for the specific agonist [³H]OH-DPAT.3. The agonist binding is inhibited by monovalent cations Na⁺, K⁺, and Li⁺ in a concentration-dependent manner. Divalent cations such as Ca²⁺, Mg²⁺, and Mn²⁺, on the other hand, show more complex behavior and induce enhancement of agonist binding up to a certain concentration. The effect of the metal ions on agonist binding is strongly modulated in the presence of GTP--S, a nonhydrolyzable analogue of GTP, indicating that these receptors are coupled to G-proteins.4. To gain further insight into the mechanisms of agonist binding to bovine hippocampal 5-HT_1A receptors under these conditions, the binding affinities and binding sites have been analyzed by Scatchard analysis of saturation binding data. Our results are relevant to ongoing analyses of the overall regulation of receptor activity for G-protein-coupled seven transmembrane domain receptors.     

The discovery of novel 8-azabicyclo[3.2.1]octan-3-yl)-3-(4-chlorophenyl) propanamides as vasopressin V1A receptor antagonists

The discovery of a novel series of 8-azabicyclo[3.2.1]octan-3-yl)-3-(4-chlorophenyl) propanamide antagonists of the vasopressin V_1A receptor is disclosed. Compounds 47 and 48 were found to be high affinity, selective vasopressin V_1A antagonists.     

Pharmacophore models based studies on the affinity and selectivity toward 5-HT1A with reference to α1-adrenergic receptors among arylpiperazine derivatives of phenytoin

The study is focused on (2-alkoxy)phenylpiperazine derivatives of 1-(2-hydroxy-3-(4-arylpiperazin-1-yl)propyl)-5,5-diphenylimidazolidine-2,4-dione with alkyl or ester substituents at N3 of hydantoin ring, as well as a new designed and synthesized series of compounds with a free N3H group or N3-acetic acid terminal fragment. The compounds were assessed on their affinity for 5-HT_1A and ??₁-adrenoceptors and evaluated in functional bioassays for antagonistic properties. Classical molecular mechanics (MMFFs force field, MCMM, MacroModel) and DFT methods (B3LYP functional, Gaussian 0.3) were used to investigate 3D structure of the compounds. SAR analysis was based on two pharmacophore models, the one described by Barbaro et al. for ??₁-adenoceptor antagonist and the model of Lepailleur et al. for 5-HT_1A receptor ligands. All compounds exhibited significant to moderate affinities for 5-HT_1A receptors in nanomolar range (7-610 nM). The highest activity (7 nM) and selectivity (17.38) for 5-HT_1A was observed for 1-(3-(4-(2-ethoxyphenyl)piperazin-1-yl)-2-hydroxypropyl)-3-methyl-5,5-diphenylimidazolidine-2,4-dione (13a). Among new synthesized compounds 1-(2-hydroxy-3-(4-(2-methoxyphenyl)piperazin-1-yl)propyl)-5,5-diphenylimidazolidine-2,4-dione hydrochloride (20a) displayed the highest affinity (16.6 nM) and selectivity (5.72) for ??₁-AR.