Synergic stimulation of serotonin 5-HT1A receptor and α2-adrenoceptors for neuropathic pain relief: Preclinical effects of 2-substituted imidazoline derivatives.

Neuropathic pain affects millions of people causing disability and impairing quality of life. Commonly used analgesics are generally characterized by limited therapeutic outcomes. The serotonin 5-HT1A receptor and the α2 adrenergic receptors are involved in central nociceptive mechanisms with a pivotal role in the inhibitory descending pain pathway. Since their stimulation may modulate the nervous signaling altered by neuropathies, the purpose of the present research is the study of the combined activation of 5-HT1A and α2 receptors by rationally designed imidazoline ligands ((S)-(-)-1 and 2-5) in a rat model of neuropathic pain (chronic constriction injury - CCI). On day 14 after nerve damage, the acute administration per os (p.o.) of low doses of (S)-(-)-1 (0.1-1mg/kg) was able to significantly increase the pain threshold to mechanical noxious stimuli for more than 1h. (S)-(-)-1 efficacy was confirmed by the decrease of spontaneous pain evaluated as hind limb weight bearing alterations. The clinically-used compound gabapentin (100mg/kg p.o.) induced a pain relieving effect similar to (S)-(-)-1 administered at 100 fold lower dose. In the same model, the selected analogues, compounds 2-5 (1mg/kg p.o.) were effective 30min after administration. In particular, 5 fully reverted the CCI-induced hypersensitivity. The pain relieving activity of 5 was significantly prevented by the selective 5-HT1A receptor antagonist WAY 100635 (1mg/kg intraperitoneally, i.p.) and, at a lesser extent, by the α2 antagonist yohimbine (3mg/kg i.p.). A novel pharmacodynamic approach to the treatment of neuropathic pain is presented.

Combined Interactions with I1-, I2-Imidazoline Binding Sites and α2-Adrenoceptors To Manage Opioid Addiction.

Tolerance and dependence associated with chronic opioid exposure result from molecular, cellular, and neural network adaptations. Such adaptations concern opioid and nonopioid systems, including α2-adrenoceptors (α2-ARs) and I1- and I2-imidazoline binding sites (IBS). Agmatine, one of the hypothesized endogenous ligands of IBS, targeting several systems including α2-ARs and IBS, proved to be able to regulate opioid-induced analgesia and to attenuate the development of tolerance and dependence. Interested in the complex pharmacological profile of agmatine and considering the nature of its targets, we evaluated two series of imidazolines, rationally designed to simultaneously interact with I1-/I2-IBS or I1-/I2-IBS/α2-ARs. The compounds showing the highest affinities for I1-/I2-IBS or I1-/I2-IBS/α2-ARs have been selected for their in vivo evaluation on opiate withdrawal syndrome. Interestingly, 9, displaying I1-/I2-IBS/α2-ARs interaction profile, appears more effective in reducing expression and acquisition of morphine dependence and, therefore, might be considered a promising tool in managing opioid addiction.

The Versatile 2-Substituted Imidazoline Nucleus as a Structural Motif of Ligands Directed to the Serotonin 5-HT1A Receptor.

The involvement of the serotonin 5-HT1A receptor (5-HT1A -R) in the antidepressant effect of allyphenyline and its analogues indicates that ligands bearing the 2-substituted imidazoline nucleus as a structural motif interact with 5-HT1A -R. Therefore, we examined the 5-HT1A -R profile of several imidazoline molecules endowed with a common scaffold consisting of an aromatic moiety linked to the 2-position of an imidazoline nucleus by a biatomic bridge. Our aim was to discover other ligands targeting 5-HT1A -R and to identify the structural features favoring 5-HT1A -R interaction. Structure-activity relationships, supported by modeling studies, suggested that some structural cliché such as a polar function and a methyl group in the bridge, as well as proper steric hindrance in the aromatic area of the above scaffold, favored 5-HT1A -R recognition and activation. We also highlighted the potent antidepressant-like effect (mouse forced swimming test) of (S)-(+)-19 [(S)-(+)-naphtyline] at very low dose (0.01 mg kg-1 ). This effect was clearly mediated by 5-HT1A , as it was significantly reduced by pretreatment with the 5-HT1A antagonist WAY100635.

A Novel Class of Dopamine D4 Receptor Ligands Bearing an Imidazoline Nucleus.

Over the years, the 2-substituted imidazoline nucleus has been demonstrated to be a bioversatile structural motif. In this study, novel imidazoline derivatives bearing a 3- and/or 4-hydroxy- or methoxy-substituted phenyl ring, linked by an ethylene bridge to position 2 of an N-benzyl- or N-phenethyl-substituted imidazoline nucleus, were prepared and studied against D2 -like receptor subtypes. Binding studies highlighted that a set of N-phenethylimidazoline compounds are selective for D4 over D2 and D3 receptors. In functional assays, the 3-methoxy-substituted derivative, endowed with the highest D4 affinity value, and its 3-hydroxy analogue behaved as partial agonists with low intrinsic efficacy and as competitive D4 antagonists when tested in the presence of the D2 -like receptor agonist quinpirole. Molecular docking analysis, performed using a homology model of the human D4 receptor developed using the X-ray crystal structure of the antagonist-bound human D3 receptor as a template, was in accordance with the binding results and provided useful information for the design of novel imidazoline D4 receptor ligands based on this new scaffold.

Dual Alpha2C/5HT1A Receptor Agonist Allyphenyline Induces Gastroprotection and Inhibits Fundic and Colonic Contractility.

BACKGROUND: Allyphenyline, a novel α2-adrenoceptor (AR) ligand, has been shown to selectively activate α2C-adrenoceptors (AR) and 5HT1A receptors, but also to behave as a neutral antagonist of α2A-ARs. We exploited this unique pharmacological profile to analyze the role of α2C-ARs and 5HT1A receptors in the regulation of gastric mucosal integrity and gastrointestinal motility. METHODS: Gastric injury was induced by acidified ethanol in Wistar rats. Mucosal catalase and superoxide dismutase levels were measured by assay kits. The effect of allyphenyline on electrical field stimulation (EFS)-induced fundic and colonic contractions was determined in C57BL/6 mice. RESULTS: Intracerebroventricularly injected allyphenyline (3 and 15 nmol/rat) dose dependently inhibited the development of mucosal damage, which was antagonized by ARC 239 (α2B/C-AR and 5HT1A receptor antagonist), (S)-WAY 100135 (selective 5HT1A receptor antagonist), and JP-1302 (selective α2C-AR antagonist). This protection was accompanied by significant elevation of mucosal catalase and superoxide dismutase levels. Allyphenyline (10(-9)-10(-5) M) also inhibited EFS-induced fundic contractions, which was antagonized by ARC 239 and (S)-WAY 100135, but not by JP-1302. Similar inhibition was observed in the colon; however, in this case only ARC 239 reduced this effect, while neither selective inhibition of α2C-ARs and 5HT1A receptors nor genetic deletion of α2A- and α2B-ARs influenced it. CONCLUSIONS: Activation of both central α2C-ARs and 5HT1A receptors contributes to the gastroprotective action of allyphenyline in rats. Its inhibitory effect on fundic contractions is mediated by 5HT1A receptors, but neither α2-ARs nor 5HT1A receptors take part in its inhibitory effect on colonic contractility in mice.

Biological profile and bioavailability of imidazoline compounds on morphine tolerance modulation.

Tolerance to opioid administration represents a serious medical alert in different chronic conditions. This study compares the effects of the imidazoline compounds 1, 2, and 3 on morphine tolerance in an animal model of inflammatory pain in the rat. 1, 2, and 3 have been selected in that, although bearing a common scaffold, preferentially bind to α2-adrenoceptors, imidazoline I2 receptors, or both systems, respectively. Such compounds have been tested in vivo by measuring the paw withdrawal threshold to mechanical pressure after complete Freund's adjuvant injection. To determine the ligand levels in rat plasma, an HPLC-mass spectrometry method has been developed. All the compounds significantly reduced the induction of morphine tolerance, showing different potency and duration of action. Indeed, the selective imidazoline I2 receptor interaction (2) restored the analgesic response by maintaining the same time-dependent profile observed after a single morphine administration. Differently, the selective α2C-adrenoceptor activation (1) or the combination between α2C-adrenoceptor activation and imidazoline I2 receptor engagement (3) promoted a change in the temporal profile of morphine analgesia by maintaining a mild but long lasting analgesic effect. Interestingly, the kinetics of compounds in rat plasma supported the pharmacodynamic data. Therefore, this study highlights that both peculiar biological profile and bioavailability of such ligands complement each other to modulate the reduction of morphine tolerance. Based on these observations, 1-3 can be considered useful leads in the design of new drugs able to turn off the undesired tolerance induced by opioids.

Antagonism/Agonism modulation to build novel antihypertensives selectively triggering i1-imidazoline receptor activation.

Pharmacological studies have suggested that I1-imidazoline receptors are involved in the regulation of cardiovascular function and that selective I1-agonists, devoid of the side effects associated with the common hypotensive α2-adrenoreceptor agonists, might be considered as a second generation of centrally acting antihypertensives. Therefore, in the present study, inspired by the antihypertensive behavior of our selective I1-agonist 4, we designed, prepared, and studied the novel analogues 5-9. A selective I1-profile, associated with significant hemodinamic effects, was displayed by 5, 8, and 9. Interestingly, the highest potency and longest lasting activity displayed by 8 (carbomethyline) suggested that van der Waals interactions, promoted by the ortho methyl decoration of its aromatic moiety, are particularly advantageous. In addition, in analogy to what was noted for (S)-(+)-4, the observation that only (S)-(+)-8 displayed significant hemodynamic effects unequivocally confirmed the stereospecific nature of the I1 proteins.

Investigation of allyphenyline efficacy in the treatment of alcohol withdrawal symptoms.

We have recently demonstrated that allyphenyline, behaving as α2C-adrenoceptor/serotonin 5-HT1A receptor agonist and α2A-adrenoceptor antagonist, in mice enhanced morphine analgesia, attenuated morphine withdrawal symptoms, showed significant antidepressant-like activity and was devoid of sedative side effects. Opioid and alcohol withdrawal shares several common neurobiological and molecular mechanisms. Therefore, in this study we expanded our analysis of the pharmacological properties of allyphenyline by investigating its ability to prevent the expression of somatic withdrawal signs, anxiety-like behavior and hyperlocomotion associated with chronic ethanol intoxication. Rats were subjected to induction of ethanol dependence via repeated daily intragastric ethanol (20%) administration for 4 consecutive days. Twelve hours after the last alcohol administration, somatic alcohol withdrawal signs were scored. Results revealed a significant expression of physical withdrawal signs that were not affected by intraperitoneal (i.p.) administration of allyphenyline at the doses of 0.05, 0.275 and 0.5 mg/kg. In contrast, allyphenyline (0.05 and 0.275 mg/kg i.p.) significantly reduced hyperanxiety-like behavior observed 6 days after alcohol intoxication as measured using the defensive burying test. Allyphenyline also reduced open field hyperlocomotor activity associated with alcohol withdrawal. Notably, the anxiolytic effect of the compound, as well as the already reported antidepressant action, was observed at very low doses, suggesting the involvement of its α2C-adrenoceptor/serotonin 5-HT1A receptor agonism. Therefore, the present investigation suggests that allyphenyline might represent an interesting pharmacological tool to investigate the potential of compounds exhibiting α2C-adrenoceptor/serotonin 5-HT1A receptor agonism and α2A-adrenoceptor antagonism in the treatment of hyperanxiety and hyperlocomotion occurring during alcohol withdrawal in dependent subjects.

Exploring multitarget interactions to reduce opiate withdrawal syndrome and psychiatric comorbidity.

Opioid addiction is often characterized as a chronic relapsing condition due to the severe somatic and behavioral signs, associated with depressive disorders, triggered by opiate withdrawal. Since prolonged abstinence remains a major challenge, our interest has been addressed to such objective. Exploring multitarget interactions, the present investigation suggests that 3 or its (S)-enantiomer and 4, endowed with effective α2C-AR agonism/α2A-AR antagonism/5-HT1A-R agonism, or 7 and 9-11 producing efficacious α2C-AR agonism/α2A-AR antagonism/I2-IBS interaction might represent novel multifunctional tools potentially useful for reducing withdrawal syndrome and associated depression. Such agents, lacking in sedative side effects due to their α2A-AR antagonism, might afford an improvement over current therapies with clonidine-like drugs.

Structure-activity relationships in 1,4-benzodioxan-related compounds. 11. (1) reversed enantioselectivity of 1,4-dioxane derivatives in α1-adrenergic and 5-HT1A receptor binding sites recognition.

5-HT(1A) receptor and α(1)-adrenoreceptor (α(1)-AR) binding sites recognized by the 1,4-dioxanes 2-4 display reversed stereochemical requirements. (S)-2 proved to be a potent 5-HT(1A) receptor agonist highly selective over α(1)-AR subtypes. Chirality influenced the anticancer activity of 3 and 4 in human prostate cancer cells (PC-3): (R)-4, eutomer at the α(1d)-AR subtype, was the most potent. The decreased effect of 4 and (R)-4 in α(1d)-AR silenced PC-3 cells confirmed that their anticancer activity was α(1d)-AR-dependent.

Characterization of the hypothermic effects of imidazoline I2 receptor agonists in rats.

BACKGROUND AND PURPOSE Imidazoline I(2) receptors have been implicated in several CNS disorders. Although several I(2) receptor agonists have been described, no simple and sensitive in vivo bioassay is available for studying I(2) receptor ligands. This study examined I(2) receptor agonist-induced hypothermia as a functional in vivo assay of I(2) receptor agonism. EXPERIMENTAL APPROACH Different groups of rats were used to examine the effects of I(2) receptor agonists on the rectal temperature and locomotion. The pharmacological mechanisms were investigated by combining I(2) receptor ligands and different antagonists. KEY RESULTS All the selective I(2) receptor agonists examined (2-BFI, diphenyzoline, phenyzoline, CR4056, tracizoline, BU224 and S22687, 3.2-56 mg·kg(-1) , i.p.) dose-dependently and markedly decreased the rectal temperature (hypothermia) in rats, with varied duration of action. Pharmacological mechanism of the observed hypothermia was studied by combining the I(2) receptor agonists (2-BFI, BU224, tracizoline and diphenyzoline) with imidazoline I(2 ) receptor/ α(2) adrenoceptor antagonist idazoxan, selective I(1) receptor antagonist efaroxan, α(2) adrenoceptor antagonist/5-HT(1A) receptor agonist yohimbine. Idazoxan but not yohimbine or efaroxan attenuated the hypothermic effects of 2-BFI, BU224, tracizoline and diphenyzoline, supporting the I(2) receptor mechanism. In contrast, both idazoxan and yohimbine attenuated hypothermia induced by the α(2) adrenoceptor agonist clonidine. Among all the I(2) receptor agonists studied, only S22687 markedly increased the locomotor activity in rats. CONCLUSIONS AND IMPLICATIONS Imidazoline I(2) receptor agonists can produce hypothermic effects, which are primarily mediated by I(2) receptors. These data suggest that I(2) receptor agonist-induced hypothermia is a simple and sensitive in vivo assay for studying I(2) receptor ligands.

Favourable involvement of α2A-adrenoreceptor antagonism in the I2-imidazoline binding sites-mediated morphine analgesia enhancement.

Aim of the present study was to obtain novel α(2)-adrenoreceptor (α(2)-AR) antagonists, possibly endowed with subtype-selectivity. Therefore, inspired by the non subtype-selective α(2)-AR antagonist idazoxan, we designed 1,4-dioxane derivatives bearing an aromatic area in position 5 or 6 and the imidazoline nucleus in position 2. Among the novel molecules 1-6, compound 2, with a trans stereochemical relationship between 5-phenyl and 2-imidazoline groups, was able to antagonize the sole α(2A)-subtype. Moreover, 2 showed an affinity at I(2)-imidazoline binding sites (I(2)-IBS) comparable to that at α(2A)-AR. In in vivo studies 2 strongly increased morphine analgesia. This interesting behaviour appeared to be induced by the favourable involvement of α(2A)-AR antagonism in the I(2)-IBS-mediated morphine analgesia enhancement.

Might the observed α(2A)-adrenoreceptor agonism or antagonism of allyphenyline analogues be ascribed to different molecular conformations?

We recently reported that the α(2)-adrenoreceptor (AR) ligand allyphenyline (9) significantly enhanced morphine analgesia (due to its α(2C)-AR agonism), was devoid of sedative side effects (due to its α(2A)-AR antagonism), prevented and reversed morphine tolerance and dependence. To highlight the molecular characteristics compatible with this behaviour and to obtain novel agents potentially useful in chronic pain and opioid addiction management, the allyl group of 9 was replaced by substituents of moderate steric bulk (MR) and positive or negative lipophilic (π) and electronic (σ) contributions in all the possible combinations. Effective novel α(2C)-agonists/α(2A)-antagonists (2, 3, 10, 12, and 17) were obtained. This study also demonstrated that contradictory combinations of the physicochemical parameters were similarly able to induce the α(2A)-activation. Since we had previously observed that the absolute configuration affected only the potency, but not the functional profile of the ligands, we hypothesized that the α(2A)-activation was governed by a ligand preferred conformation. From a structural overlay investigation it emerged that an extended conformation appeared to be associated with dual α(2C)-agonism/α(2A)-antagonism, whereas a folded conformation associated with α(2C)-/α(2A)-agonism.

1,4-dioxane, a suitable scaffold for the development of novel M3 muscarinic receptor antagonists.

In this study the modulation of the pharmacological profile from agonist to antagonist was successfully obtained by replacing the methyl group in position 6 of the 1,4-dioxane scaffold of the potent M(2)/M(3) muscarinic agonist 1 with bulkier groups. In particular, the 6,6-diphenyl substitution provided the potent M(3) preferring antagonist (±)-17, which in in vivo study proved to be effective in reducing the volume-induced contractions of rat urinary bladder and was devoid of cardiovascular effects.

Low Doses of Allyphenyline and Cyclomethyline, Effective against Morphine Dependence, Elicit an Antidepressant-like Effect.

This study demonstrated that cyclomethyline (2) and the corresponding enantiomers (R)-(-)-2 and (S)-(+)-2, displaying α2C-adrenoreceptor (AR) agonism/α2A-AR antagonism, similarly to allyphenyline (1) and its enantiomers, significantly decreased the naloxone-precipitated withdrawal symptoms in mice at very low doses. It also highlighted that such positive effects on morphine dependence can even be improved by additional serotoninergic 5-HT1A receptor (5-HT1A-R) activation. Indeed, 1 or the single (S)-(+)-1, 2, or both its enantiomers, all behaving as α2C-AR agonists/α2A-AR antagonists/5-HT1A-R agonists, alone and at the same low dose, improved morphine withdrawal syndrome and exerted a potent antidepressant-like effect. Therefore, considering the elevated comorbidity between opiate abuse and depressed mood and the benefit of these multifunctional compounds to both disorders, it is possible that they prove more efficacious and less toxic than a cocktail of drugs in managing opioid addiction.

α2C-adrenoceptor modulators: a patent review.

INTRODUCTION: α2-Adrenoceptors (α2-ARs) are membrane proteins belonging to the superfamily of GPCRs. Detailed studies have shown three different subtypes, namely α2A, α2B and α2C. Although numerous α2-AR ligands exist, only a small set of compounds have shown even a degree of selectivity among the three α2-AR subtypes. Moreover, these compounds suffer from binding to receptor sites outside the α2-AR subfamily. Efforts made to understand the biological significance of each α2-AR subtype have greatly been assisted by genetically engineered mice. The main results obtained suggest that α2C-AR stimulation may represent a therapeutic strategy to get an analgesic response with reduced sedative effects and undesirable changes in blood pressure due to α2A-AR activation. AREAS COVERED: This review summarizes the patent literature about the development of α2C-AR modulators from 2000 to early 2010 and their therapeutic effects evoked by the interaction with this receptor subtype. EXPERT OPINION: Over 90 patents have been deposited in the last 10 years regarding different methods of α2C-AR modulation (use of agonists or antagonists, nucleic acids and polypeptides) for diagnosis, prognosis and treatment of disorders involving this receptor. Nevertheless, despite the numerous published patents, ligands highly selective for the α2C-AR subtype, which continues to be enigmatic, are lacking.

Fruitful adrenergic α(2C)-agonism/α(2A)-antagonism combination to prevent and contrast morphine tolerance and dependence.

The functional in vitro study of the enantiomers of imidazolines 4-7 highlighted the role played by the nature of the ortho phenyl substituent in determining the preferred α(2C)-AR configuration. Indeed, the (S) enantiomers of 4-6 or (R) enantiomer of 7 behave as eutomers and activate this subtype as full agonists; the corresponding distomers are partial agonists. Because in clinical pain management with opioids α(2C)-AR agonists, devoid of the α(2A)-AR-mediated side effects, may represent an improvement over current therapies with clonidine like drugs, 4 and its enantiomers, showing α(2C)-agonism/α(2A)-antagonism, have been studied in vivo. The data suggest that partial α(2C)-activation is compatible with effective enhancement of morphine analgesia and reduction both of morphine tolerance acquisition and morphine dependence acquisition and expression. On the contrary, full α(2C)-activation appears advantageous in reducing morphine tolerance expression. Interestingly, the biological profile displayed by 4 (allyphenyline) and its eutomer (S)-(+)-4 has been found to be very unusual.

Novel imidazoline compounds as partial or full agonists of D2-like dopamine receptors inspired by I2-imidazoline binding sites ligand 2-BFI.

Based on the well known biological versatility of the imidazoline nucleus, we prepared the novel derivatives 3a-k inspired by 2-BFI scaffold to assess imidazoline molecules as D(2)-like dopamine receptor ligands. Conservative chemical modifications of the lead structure, such as the introduction of an hydroxy group in the aromatic ring alone or associated with N-benzyl substitution, provided partial (3f) or nearly full (3e and 3h) agonists, all endowed with D(2)-like potency comparable to that of dopamine.

Structure-activity relationships in 1,4-benzodioxan-related compounds. 10. Novel α1-adrenoreceptor antagonists related to openphendioxan: synthesis, biological evaluation, and α1d computational study.

A series of novel openphendioxan analogues were synthesized and tested at α(1)-adrenoreceptor (AR) subtypes by binding and functional assays. The α(1d)-AR binding profile was also examined by means of 2D, 3D-QSAR together with docking studies. Multiple regression analysis suggested the relevance of adequate number of heteroatoms in the whole molecule and of passive membrane diffusion to enhance α(1d)-AR affinity. Docking simulations against a computational structural model of the biological target further proved this evidence and furnished support for chemiometric analysis, where polar, electrostatic, hydrophobic and shape effects of the ortho substituents in the phenoxy terminal, most likely governing ligand binding, helped the depiction of pharmacophore hypothesis for the examined ligands data set.

Novel highly potent and selective sigma 1 receptor antagonists related to spipethiane.

Conservative chemical modifications of the core structure of the lead spipethiane (1) afforded novel potent sigma(1) ligands. sigma(1) affinity and sigma(1/)sigma(2) selectivity proved to be favored by the introduction of polar functions (oxygen atom or carbonyl group) in position 3 or 4 (4-6) or by the elongation of the distance between the two hydrophobic portions of the molecule with the simultaneous presence of a carbonyl group in position 4 (8 and 9). The observed cytostatic effect against the human breast cancer cell line MCF-7/ADR, highly expressing sigma(1) receptors, and not against MCF-7, as well as the enhancement of morphine analgesia highlighted the sigma(1) antagonist profile of this series of compounds. In particular, due to its high sigma(1) affinity (pK(i) = 10.28) and sigma(1)/sigma(2) selectivity ratio (29510), compound 9 might be a novel valuable tool for sigma receptor characterization and a suitable template for the rational design of potential therapeutically useful sigma(1) antagonists.