Synthesis of 2-phenyl-5,6,7,8-tetrahydroquinoxaline derivatives and screening for P2X1-purinoceptor antagonist activity in isolated preparations of rat vas deferens, for translation into a male contraceptive†

Sympathetically mediated contractions of smooth muscle cells in the vasa deferentia are mediated by neuronally released adenosine 5′-triphosphate (ATP) and noradrenaline, which stimulate P2X1-purinoceptors and α1A-adrenoceptors, respectively. This process is crucial for sperm transport, as demonstrated in knockout mouse studies where simultaneous genetic deletion of P2X1-purinoceptors and α1A-adrenoceptors resulted in male infertility. We hypothesize that dual pharmacological antagonism of these two receptors could inhibit sperm transport sufficiently to provide a novel nonhormonal method of male contraception. To generate a suitable P2X1-purinoceptor antagonist, substituents were introduced on the phenyl moiety of 2-phenyl-5,6,7,8-tetrahydroquinoxaline to create a series of analogues that were tested for P2X1-purinoceptor antagonism in isolated preparations of rat vas deferens. Novel compounds were initially screened for their ability to attenuate contractile responses to electrical field stimulation (EFS: 60 V, 0.5 ms, 0.2 Hz). The addition of polar substituents to the meta, but not ortho, position markedly increased the inhibition of contractions, as did the addition of both polar and aliphatic substituents to the para position. Di-substituted compounds were also synthesized and tested, resulting in a compound 31 (2-hydroxy, 4-fluoro), which exhibited the greatest potency, with an IC50 of 14 μM (95% confidence limits: 12–16 μM). Additionally, compound 31 noncompetitively antagonized contractions mediated by exogenously administered αß-methylene ATP (10 nM–30 μM) but had no inhibitory effect on contractions mediated by exogenously administered noradrenaline (30 nM–100 μM) or acetylcholine (30 nM–100 μM). These results have contributed to a structure–activity relationship profile for the P2X1-purinoceptor that will inform future designs of more potent antagonists.

Castration Induces Down-Regulation of A-Type K+ Channel in Rat Vas Deferens Smooth Muscle

A-type K+ channels contribute to regulating the propagation and frequency of action potentials in smooth muscle cells (SMCs). The present study (i) identified the molecular components of A-type K+ channels in rat vas deferens SMs (VDSMs) and (ii) showed the long-term, genomic effects of testosterone on their expression in VDSMs. Transcripts of the A-type K+ channel α subunit, Kv4.3L and its regulatory β subunits, KChIP3, NCS1, and DPP6-S were predominantly expressed in rat VDSMs over the other related subtypes (Kv4.2, KChIP1, KChIP2, KChIP4, and DPP10). A-type K+ current (IA) density in VDSM cells (VDSMCs) was decreased by castration without changes in IA kinetics, and decreased IA density was compensated for by an oral treatment with 17α-methyltestosterone (MET). Correspondingly, in the VDSMs of castrated rats, Kv4.3L and KChIP3 were down-regulated at both the transcript and protein expression levels. Changes in Kv4.3L and KChIP3 expression levels were compensated for by the treatment with MET. These results suggest that testosterone level changes in testosterone disorders and growth processes control the functional expression of A-type K+ channels in VDSMCs.

Evaluation of Antidepressant Effects on Recovery of Electrical Field Stimulation-Induced Contractions that have been Suppressed by Clonidine in Isolated Rat Vas Deferens

Background: A report examining whether clinically available antidepressants increase urethral smooth muscle contraction via antagonistic effects on the α2-adrenoceptor (α2-AR) is lacking. Objectives: The present study was performed to evaluate the potential of clinically available antidepressants to reverse α2-AR-mediated contractile inhibition in rat vas deferens, in order to predict whether they can induce voiding impairment. Method: The effects of 18 antidepressants of different classes on electrical field stimulation (EFS)-induced contractions suppressed by 10–8 mol/L clonidine (a selective α2-AR agonist) in isolated rat vas deferens were investigated and related to their respective clinical blood concentrations. Results: The EFS-induced contractions suppressed by clonidine were recovered by amitriptyline (a tricyclic antidepressant), mirtazapine (a noradrenergic and specific serotonergic antidepressant), and trazodone (a serotonin 5-HT2A receptor antagonist) at concentrations close to the clinical blood levels. EFS-induced contractions were also recovered by trimipramine, clomipramine (tricyclic antidepressants), mianserin (a tetracyclic antidepressant), sertraline (a selective serotonin reuptake inhibitor [SSRI]), and sulpiride (a dopamine D2-receptor antagonist), albeit at concentrations that substantially exceeded their clinically-achievable blood levels. EFS-induced contractions were not significantly affected by imipramine, nortriptyline, amoxapine (tricyclic antidepressants), maprotiline (a tetracyclic antidepressant), fluvoxamine, paroxetine, escitalopram (SSRIs), milnacipran, duloxetine (serotonin and noradrenaline reuptake inhibitors), and aripiprazole (a dopamine partial agonist). Conclusions: These findings suggest that amitriptyline, mirtazapine, and trazodone induce voiding impairment caused by increased urethral resistance by enhancing sympathetic nerve activities attributed to α2-AR antagonism.