A novel structural framework for α(1A/D)-adrenoceptor selective antagonists identified using subtype selective pharmacophores

In this study four and five-feature pharmacophores for selective antagonists at each of the three α(1)-adrenoceptor (AR) subtypes were used to identify novel α(1)-AR subtype selective compounds in the National Cancer Institute and Tripos LeadQuest databases. 12 compounds were selected, based on diversity of structure, predicted high affinity and selectivity at the α(1D)- subtype compared to α(1A)- and α(1B)-ARs. 9 out of 12 of the tested compounds displayed affinity at the α(1A) and α(1D) -AR subtypes and 6 displayed affinity at all three α(1)-AR subtypes, no α(1B)-AR selective compounds were identified. 8 of the 9 compounds with α(1)-AR affinity were antagonists and one compound displayed partial agonist characteristics. This virtual screening has successfully identified an α(1A/D)-AR selective antagonist, with low μM affinity with a novel structural scaffold of a an isoquinoline fused three-ring system and good lead-like qualities ideal for further drug development.     

Synthesis and biological affinity of new imidazo- and indol-arylpiperazine derivatives: further validation of a pharmacophore model for alpha(1)-adrenoceptor antagonists

In the continuing search for selective alpha(1)-adrenoceptor (AR) antagonists, new alkoxyarylpiperazinylalkylpyridazinone derivatives were designed and synthesized. The new compounds were tested for their affinity toward alpha(1)-AR, alpha(2)-AR and 5-HT(1A) receptors. The ability of these compounds to inhibit the serotonin transporters (SERT) was also determined. The pharmacological data confirm that increasing the size of the ortho alkoxy substituent on the phenyl ring of the arylpiperazine moiety afforded compounds with enhanced affinity toward the alpha(1)-AR. The isopropoxy group, the largest group evaluated, led the best alpha(1)-AR affinity profile. In contrast, the compounds which have an amide group within of the o-alkoxy-phenylpiperazine fragment showed low affinity toward the receptors studied. Similar results were obtained when the amide group was present in the linker of the junction between the two major constituents of the molecule.     

Pharmacological characterization of [3H]-JTH-601, a novel alpha1-adrenoceptor antagonist: binding to recombinant human alpha1-adrenoceptors and human prostates

Several alpha1-adrenoceptor (AR) selective antagonists are now widely used to improve lower urinary tract symptoms in benign prostatic hyperplasia patients. However, these drugs often result in orthostatic hypotension, because of their poor uroselectivity; the blockade of alpha1-AR not only in prostate but also in vasculature. Here we have investigated uroselectivity of JTH-601, a newly developed antagonist, in radioligand binding experiment using recombinant human alpha1-AR subtypes and human prostate. In saturation experiments, [3H]-JTH-601 showed subtype selectivity: high affinity to alpha1a-AR (pKd; 9.88+/-0.09), lower affinity to alpha1b-AR (pKd; 8.96+/-0.17) and no specific binding at concentrations up to 3000 pM to alpha1d-AR. In competition experiments, JTH-601 and its metabolic compound (JTH-601-G1) also showed alpha1a-AR selectivity, exhibiting approximately 5 times higher affinity for alpha1a-AR than for alpha1b-AR, 10 to 20 times higher affinity than for alpha1d-AR, respectively. [3H]-JTH-601 also bound to human prostate membranes in monophasic manner with high affinity constant (pKd; 9.89+/-0.12, Bmax=123.6+/-16 fmol/mg protein). JTH-601 is a unique alpha1-AR antagonist that shows high affinity and selectivity for human recombinant alpha1a- and human prostate. This new compound is useful for understanding alpha1-AR pharmacology and may have a therapeutic value.     

Different response of ANP secretion to adrenoceptor stimulation in renal hypertensive rat atria

Sympathetic nervous system and atrial natriuretic peptide (ANP) system play fundamental roles in the regulation of cardiovascular functions. Overactivity of sympathetic nervous system can lead into cardiovascular diseases such as heart failure and hypertension. The present study aimed to define which adrenergic receptors (ARs) affect atrial contractility and ANP release and to determine their modification in renal hypertensive rat atria. An alpha(1)-AR agonist, cirazoline increased ANP release with positive inotropism. These alpha(1)-AR agonist-mediated responses were attenuated by the alpha(1A)-AR antagonist, but not by the alpha(1B)- or alpha(1D)-AR antagonist. An alpha(2)-AR agonist, guanabenz and clonidine increased ANP release with negative inotropism and decreased cAMP level. The order of potency for the increased ANP release was cirazoline>phenylephrine=guanabenz>clonidine. In contrast, a beta-AR agonist, isoproterenol decreased ANP release with positive inotropism and these responses were blocked by the beta(1)-AR antagonist but not by the beta(2)-AR antagonist. The increased cAMP level by isoproterenol was suppressed by pretreatment with both beta(1)- and beta(2)-AR antagonists. In renal hypertensive rat atria, the effects of isoproterenol on atrial contractility, ANP release, and cAMP level were attenuated whereas the effect of cirazoline on ANP release was unaltered. Atrial beta(1)-AR mRNA level but not alpha(1A)-AR mRNA level was decreased in renal hypertensive rats. These findings suggest that alpha(1A)- and beta(1)-AR oppositely regulate atrial ANP release and that atrial beta(1)-AR expression/function is impaired in renal hypertensive rats.     

Novel aromatic residues in transmembrane domains IV and V involved in agonist binding at alpha(1a)-adrenergic receptors

We examined the role that aromatic residues located in the transmembrane helices of the alpha(1a)-adrenergic receptor play in promoting antagonist binding. Since alpha(1)-antagonists display low affinity binding at beta(2)-adrenergic receptors, two phenylalanine residues, Phe-163 and Phe-187, of the alpha(1a)-AR were mutated to the corresponding beta(2)-residue. Neither F163Q nor F187A mutations of the alpha(1a) had any effect on the affinity of the alpha(1)-antagonists. However, the affinity of the endogenous agonist epinephrine was reduced 12.5- and 8-fold by the F163Q and F187A mutations, respectively. An additive loss in affinity (150-fold) for epinephrine was observed at an alpha(1a) containing both mutations. The loss of agonist affinity scenario could be reversed by a gain of affinity with mutation of the corresponding residues in the beta(2) to the phenylalanine residues in the alpha(1a). We propose that both Phe-163 and Phe-187 are involved in independent aromatic interactions with the catechol ring of agonists. The potency but not the efficacy of epinephrine in stimulating phosphatidylinositol hydrolysis was reduced 35-fold at the F163Q/F187A alpha(1a) relative to the wild type receptor. Therefore, Phe-163 and Phe-187 represent novel binding contacts in the agonist binding pocket of the alpha(1a)-AR, but are not involved directly in receptor activation.     

Synthesis of new piperazine-pyridazinone derivatives and their binding affinity toward alpha1-, alpha2-adrenergic and 5-HT1A serotoninergic receptors

We report the design and synthesis of a new class of piperazine-pyridazinone analogues. The arylpiperazine moiety, the length of the spacer, and the terminal molecular fragment were varied to evaluate their influence in determining the affinity of the new compounds toward the alpha1-adrenergic receptor (alpha1-AR), alpha2-adrenergic receptor (alpha2-AR), and the 5-HT1A serotoninergic receptor (5-HT1AR). Biological data showed that most of the compounds have an alpha1-AR affinity in the nanomolar or subnanomolar range, while affinity toward the other two receptors was lower in most cases. However, several of the tested compounds also showed very good (in the nanomolar range) or moderate affinity toward the 5-HT1AR subtype.     

Structure--activity relationships among novel phenoxybenzamine-related beta-chloroethylamines

A series of beta-chloroethylamines 5--18, structurally related to the irreversible alpha(1)-adrenoceptor antagonist phenoxybenzamine [PB, N-benzyl-N-(2-chloroethyl)-N-(1-methyl-2-phenoxyethyl)amine hydrochloride, 1] and the competitive antagonist WB4101 [N-(2,3-dihydro-1,4-benzodioxin-2-ylmethyl)-N-[2-(2,6-dimethoxyphenoxy)ethyl]amine hydrochloride, 2], were synthesized and evaluated for their activity at alpha-adrenoceptors of the epididymal and the prostatic portion of young CD rat vas deferens. All compounds displayed irreversible antagonist activity. Most of them showed similar antagonism at both alpha(1)- and alpha(2)-adrenoceptors, whereas compounds 13 and 18, lacking substituents on both the phenoxy group and the oxyamino carbon chain, displayed a moderate alpha(1)-adrenoceptor selectivity (10--35 times), which was comparable to that of PB. Compounds 14 and 15, belonging to the benzyl series and bearing, respectively, a 2-ethoxyphenoxy and a 2-i-propoxyphenoxy moiety, were the most potent alpha(1)-adrenoceptor antagonists with an affinity value similar to that of PB (pIC(50) values of 7.17 and 7.06 versus 7.27). Interestingly, several compounds were able to distinguish two alpha(1)-adrenoceptor subtypes in the epididymal tissue, as revealed by the discontinuity of their inhibition curves. A mean ratio of 24:76 for these alpha(1)-adrenoceptors was determined from compounds 8--10, 12, and 15--17. Furthermore, compounds 9, 10, 12, 16a, and 16b showed higher affinity towards the minor population of receptors, whereas compounds 8, 15, and 17 preferentially inhibited the major population of alpha(1)-adrenoceptors. In addition, selected pharmacological experiments demonstrated the complementary antagonism of the two series of compounds and their different, preferential affinity for one of the two alpha(1)-adrenoceptor subtypes. In conclusion, we found beta-chloroethylamines that demonstrate a multiplicity of alpha(1)-adrenoceptors in the epididymal portion of young CD rat vas deferens and, as a consequence, they are possible useful tools for alpha(1)-adrenoceptor characterization.     

QSAR study for a novel series of ortho monosubstituted phenoxy analogues of alpha1-adrenoceptor antagonist WB4101

A number of (S)- and (R)-2-[(2-phenoxyethyl)aminomethyl]-1,4-benzodioxanes unsubstituted or ortho monosubstituted at the phenoxy moiety were synthesized and tested in binding assays on the alpha(1a)-AR, alpha(1b)-AR, alpha(1d)-AR and the 5-HT(1A) receptor. The affinity values of the new compounds 1-16 were compared with those of the enantiomers of the 2,6-dimethoxyphenoxy analogue, the well-known alpha(1) antagonist WB4101, finding that the unsubstituted derivative (S)-1 and the o-methyl, the o-t-butyl, the o-fluoro and the o-methoxy derivatives, (S)-2, (S)-4, (S)-8 and (S)-16, respectively, display a significantly specific 5-HT(1A) affinity, very close, with the exception of (S)-4, to the almost nanomolar one of (S)-WB4101. Otherwise, sensible affinity decreases were recorded for the three alpha(1)-AR subtypes. A classical quantitative structure-activity relationship (Hansch) analysis was successfully applied to compounds (S)-1 to (S)-16 and (S)-WB4101 to rationalize such binding data.     

Sex-dependent thermogenesis,differences in mitochondrial morphology and function,and adrenergic response in brown adipose tissue

Gender-related differences in brown adipose tissue (BAT) thermogenesis of 110-day-old rats were studied by determining the morphological and functional features of BAT. The adrenergic control was assessed by studying the levels of beta(3)- and alpha(2A)-adrenergic receptors (AR) and by determining the lipolytic response to norepinephrine (beta(1)-, beta(2)-, beta(3)-, and alpha(2)-AR agonist), isoprenaline (beta(1)-, beta(2)-, and beta(3)-AR agonist), and CGP12177A (selective partial beta(3)-AR agonist but beta(1)- and beta(2)-AR antagonist) together with post-receptor agents, forskolin and dibutyryl cyclic AMP. The female rats that had greater oxygen consumption showed higher UCP1 content, a higher multilocular arrangement, and both longer cristae and higher cristae dense mitochondria in BAT indicating heightened thermogenic capacity and activity; this picture is accompanied by a more sensitive beta(3)-AR to norepinephrine signal (EC(50) 10-fold lower for CGP12177A) and a lower expression of alpha(2A)-AR than male rats. Taken together, our results support the idea that the BAT hormonal environment could be involved in the control of different elements of lipolytic and thermogenic adrenergic pathways. Gender dimorphism is both at receptor (changing alpha(2A)-AR density and beta(3)-AR affinity) and post-receptor (modulating the links involved in the adrenergic signal transduction) levels. These changes in adrenergic control could be responsible, at least in part, both for the important mitochondrial recruitment differences and functional and morphological features of BAT in female rats under usual rodent housing temperatures.     

In vivo regulation of dopamine and noradrenaline release by alpha2A-adrenoceptors in the mouse nucleus accumbens

The present study investigated the role of alpha2A-adrenoceptor (alpha2A-AR) subtype in the regulation of noradrenaline (NA) and dopamine (DA) release in the nucleus accumbens (NAc). The effect of locally infused and systemically injected alpha2-AR agonist, dexmedetomidine (DMT), and alpha2-AR antagonist, atipamezole, on NA and DA release was investigated in alpha2A-AR knockout and control mice by using in vivo microdialysis. In addition, we compared the drug effects on DA and NA release in the NAc to their effect on locomotor activity. Baseline NA and DA concentrations did not differ between genotypes. Local infusion of DMT decreased, in a concentration-dependent manner, NA, but not DA, levels in the control mice. However, systemic injection of DMT decreased both NA and DA levels in the control mice. In both cases DMT had no effects on transmitter release in alpha2A-AR knockout mice. Our results suggest that alpha2-ARs regulate the release of NA, but not DA, at the terminal level in the NAc. However, alpha2-ARs regulate DA release in the NAc indirectly by their effect on DA neurones in the ventral tegmental area via an unknown mechanism. In both cases the regulation is mediated by alpha2A-adrenoceptor subtype. Also the modulation of locomotor activity by alpha2-AR agonist and antagonist seems to be mediated via alpha2A-adrenoceptors.     

Binding site analysis of full-length alpha1a adrenergic receptor using homology modeling and molecular docking

The recent availability of crystal structure of bovine rhodopsin offers new opportunities in order to approach the construction of G protein coupled receptors. This study focuses the attention on the modeling of full-length alpha(1a) adrenergic receptor (alpha(1a)-AR) due to its biological role and significant implications in pharmacological treatment of benign prostate hyperplasia. This work could be considered made up by two main steps: (a) the construction of full structure of alpha(1a)-AR, through homology modeling methods; (b) the automated docking of an endogenous agonist, norepinephrine, and of an antagonist, WB-4101, using BioDock program. The obtained results highlight the key residues involved in binding sites of both agonists and antagonists, confirming the mutagenesis data and giving new suggestions for the rational design of selective ligands.     

Constitutive precoupling to G(i) and increased agonist potency in the alpha(2B)-adrenoceptor

The human alpha(2B)-adrenoceptor (alpha(2B)-AR) was mutated by substituting the D(3.49) aspartate in position 109 with an alanine (alpha(2B)-D109A) in the conserved DRY sequence at the cytoplasmic face of TM3. We studied the effects of the mutation on agonist binding and on receptor activation in CHO cells, including possible inverse agonism monitored by measuring intracellular Ca(2+) concentrations ([Ca(2+)](i)). The mutated receptor had increased binding affinity for agonists, especially dexmedetomidine (3.8-fold). The increased affinity was abolished by pretreatment of the cells with pertussis toxin. The mutation produced constitutive receptor activity evidenced as increased basal [Ca(2+)](i) and increased potency and efficacy of agonists to elicit Ca(2+) responses. The imidazoline derivative RX821002 functioned as an inverse agonist only through the alpha(2B)-D109A, reducing [Ca(2+)](i). The results thus indicate that this mutation causes constitutive receptor-G(i)-protein precoupling, and that the D(3.49) aspartate residue of the DRY motif is involved in controlling coupled and uncoupled conformations of alpha(2B)-AR.     

3-Arylpiperazinylethyl-1H-pyrrolo[2,3-d]pyrimidine-2,4(3H,7H)-dione derivatives as novel, high-affinity and selective alpha(1)-adrenoceptor ligands

The discovery of a new series of selective and high-affinity alpha(1)-adrenoceptor (alpha(1)-AR) ligands, characterized by a 1H-pyrrolo[2,3-d]-pyrimidine-2,4(3H,7H)-dione system, is described in this paper. Some synthesized compounds, including 20, 22, and 30, displayed affinity in the nanomolar range for alpha(1)-ARs and substantial selectivity with respect to 5-HT(1A) and dopaminergic D(1) and D(2) receptors. Functional assays, performed on selected derivatives, showed antagonistic properties.     

Pharmacophore-based design, synthesis, biological evaluation, and 3D-QSAR studies of aryl-piperazines as alpha(1)-adrenoceptor antagonists

Phenyl-piperazines were designed and synthesized based on pharmacophore for uro-selective alpha(1)-adrenoceptor antagonists and 3D chemical database searching. Within this series, three compounds, 2, 3, and 13, showed similar or better alpha(1)-AR antagonistic activity compared with prazosin. The 3D-QSAR study of these compounds may provide useful information for the development of novel aryl-piperazines as uro-selective alpha(1)-adrenoceptor antagonists, which can be used for the treatment of BPH with fewer side effects.     

α2—肾上腺素体介导大鼠主动脉收缩的特性

为阐明功能性α2－肾上腺素受体（α2－AR）的特性及其与α1－AR的相互关系,以离体大鼠主动脉为模型,进行收缩功能实验,发现在大鼠主动脉中,α1－AR和α2－AR产可介导其收缩效应并以α1－AR折作用为主,α1－AR可增强α2－AR介导的收缩效应,而α2－AR则对α1－AR介导的收缩效应无影响,在不可逆阻断α1－AR而保留α2－AR的条件下,α2－AR介导的缩血管效应消失,仅在阈值浓度的KCl存在下才能显示,且收缩幅度较对照组显著降低,结果表明,在离体大鼠主体动脉中存在功能性α2－AR,α2－AR的缩血管作用依赖于α1－AR的激动,其最大收缩反应远远小于α1－AR。     

New 4-(4-methyl-phenyl)phthalazin-1(2H)-one derivatives and their effects on alpha1-receptors

Continuing our research aimed at obtaining new compounds with high affinity and selectivity toward alpha(1)-AR, a new series of arylpiperazine derivatives was designed, synthesized, and biologically tested. The new compounds 1-17 are characterized by a phenylphthalazin-1(2H)-one fragment connected through an alkyl chain to an arylpiperazine residue. The pharmacological profile of these compounds was evaluated for their affinity and selectivity toward alpha(1)-AR, alpha(2)-AR and toward 5HT(1A) serotoninergic receptor. A discussion on the structure-activity relationship (SAR) of these compounds is also reported.     

Pre- and postjunctional alpha(2)-adrenergic receptors in fetal and adult ovine cerebral arteries

In ovine cerebral arteries, adrenergic-mediated vasoconstrictor responses differ significantly with developmental age. We tested the hypothesis that, in part, these differences are a consequence of altered alpha(2)-adrenergic receptor (alpha(2)-AR) density and/or affinity. In fetal (approximately 140 days) and adult sheep, we measured alpha(2)-AR density and affinity with the antagonist [(3)H]idazoxan in main branch cerebral arteries and other vessels. We also quantified contractile responses in middle cerebral artery (MCA) to norepinephrine (NE) or phenylephrine in the presence of the alpha(2)-AR antagonists yohimbine and idazoxan and contractile responses to the alpha(2)-AR agonists clonidine and UK-14304. In fetal and adult cerebral artery homogenates, alpha(2)-AR density was 201 +/- 18 and 52 +/- 6 fmol/mg protein, respectively (P < 0.01); however, antagonist affinity values did not differ. In fetal, but not adult, MCA, 10(-7) M yohimbine significantly decreased the pD(2) for NE-induced tension in the presence of 3 x 10(-5) M cocaine, 10(-5) M deoxycorticosterone, and 10(-6) M tetrodotoxin. In fetal, but not adult, MCA, UK-14304 induced a significant decrease in pD(2) for the phenylephrine dose-response relation. In addition, stimulation-evoked fractional NE release was significantly greater in fetal than in adult cerebral arteries. In the presence of 10(-6) M idazoxan to block alpha(2)-AR-mediated inhibition of prejunctional NE release, the fractional NE release was significantly increased in both age groups. We conclude that in fetal and adult ovine cerebral arteries, alpha(2)-AR appear to be chiefly prejunctional. Nonetheless, the fetal cerebral arteries appear to have a significant component of postjunctional alpha(2)-AR.     

Modulation of myocardial alpha 1- but not beta-adrenoceptors after 90-day tail-suspension

We have previously demonstrated that prolonged simulated microgravity (tail-suspension) leads to cardiac alterations with increased resting heart rate, myocardial degradation changes and attenuated myocardial contractility. The present study investigated the potential role of adrenoceptor mechanisms underlying them. Changes of myocardial alpha 1-adrenoceptor (alpha 1-AR) and beta 1-adrenoceptor (beta-AR) in 90-day tail-suspended rats was investigated by the method of radioligand binding assay and application of Scatchard's method. The results showed significantly decreased quantity of specific binding of 125I-BE[2-beta-(4-hydroxy-3-[125I]indophenyl)-ethylaminomethyltetralone] to alpha 1-AR present in membrane derived from ventricular myocardium of the suspended animals, despite the affinity of the alpha 1-AR to 125I-Be was unchanged. But neither the quantity nor the affinity of beta-AR binding to 125I-Pindolol was significantly altered. In addition, the spontaneously beating rate of isolated right atria from tail-suspended animals showed little change in sensitivity and reactivity to the stimulations of graded phenylephrine (alpha-agonist, measured in the presence of beta-antagonist propranolol) and isoproterenol (beta-agonist), compared with the control rats. There were also no obvious differences of the effects of the isoproterenol on the contractility of isolated left ventricular papillary muscles between the two groups. Since myocardial alpha 1-AR mediated-effects include production of cardiac hypertrophy and enhancement of myocardial glucose uptake and glycolysis, the down-regulation of the alpha 1-AR may be a contributor to the cardiac cellular accumulation and the myocardial degradation changes as found in our tail-suspended rats. The data from this study also suggest that the myocardial beta-adrenoceptors are not affected by the prolonged tail-suspension.     

Presynaptic alpha-adrenoceptors in median preoptic nucleus modulate inhibitory neurotransmission from subfornical organ and organum vasculosum lamina terminalis

The median preoptic nucleus (MnPO) in the lamina terminalis receives a prominent catecholaminergic innervation from the dorsomedial and ventrolateral medulla. The present investigation used whole cell patch-clamp recordings in rat brain slice preparations to evaluate the hypothesis that presynaptic adrenoceptors could modulate GABAergic inputs to MnPO neurons. Bath applications of norepinephrine (NE; 20-50 microM) induced a prolonged and reversible suppression of inhibitory postsynaptic currents (IPSCs) and reduced paired-pulse depression evoked by stimulation in the subfornical organ and organum vasculosum lamina terminalis. These events were not correlated with any observed changes in membrane conductance arising from NE activity at postsynaptic alpha(1)- or alpha(2)-adrenoceptors. Consistent with a role for presynaptic alpha(2)-adrenoceptors, responses were selectively mimicked by an alpha(2)-adrenoceptor agonist (UK-14304) and blockable with an alpha(2)-adrenoceptor antagonist (idazoxan). Although the alpha(1)-adrenoceptor agonist cirazoline and the alpha(1)-adrenoceptor antagonist prazosin were without effect on these evoked IPSCs, NE was noted to increase (via alpha(1)-adrenoceptors) or decrease (via alpha(2)-adrenoceptors) the frequency of spontaneous and tetrodotoxin-resistant miniature IPSCs. Collectively, these observations imply that both presynaptic and postsynaptic alpha(1)- and alpha(2)-adrenoceptors in MnPO are capable of selective modulation of rapid GABA(A) receptor-mediated inhibitory synaptic transmission along the lamina terminalis and therefore likely to exert a prominent influence in regulating cell excitability within the MnPO.