A series of naphthalenyloxy-arylpropylamines have been prepared and are demonstrated to be inhibitors of both serotonin and norepinephrine reuptake. One member of this series, duloxetine (Cymbalta™) has proven to be effective in clinical trials for the treatment of depression.

Inhibitors of neuronal norepinephrine (NE) uptake are useful for the treatment of a variety of diseases including depression and urinary incontinence. In the present study, we synthesized and evaluated a novel analog of the potent and selective NE uptake inhibitor, nisoxetine. Thionisoxetine more potently inhibited the uptake of [3H]-NE into hypothalamic synaptosomes and [3H]-nisoxetine binding to the NE transporter than (R)-nisoxetine. The (R) enantiomer of this compound was significantly more potent than the (S) enantiomer, having a Ki of 0.20 nM in [3H]-nisoxetine binding. The (R) enantiomer was approximately 70-fold more potent in inhibiting [3H]-NE uptake when compared to [3H]-5HT uptake. In rats, (R)-thionisoxetine prevented hypothalamic NE depletion by 6-hydroxydopamine with an ED50 of 0.21 mg/kg. Depletion of NE in peripheral nerves was accomplished by the administration of metaraminol to rats. In this paradigm, (R)-thionisoxetine prevented the depletion of heart NE with an ED50 of 3.4 mg/kg and urethral NE with an ED50 of 1.2 mg/kg. Thus, (R)-thionisoxetine is a potent and selective inhibitor of NE uptake in both central and peripheral tissues.

Halogenated analogs of the potent norepinephrine (NE) uptake inhibitor, tomoxetine, were synthesized and their affinities for the serotonin (5HT) and NE uptake sites evaluated. One of the most potent was the 2-iodo substituted analog (289306) that inhibited [3H]tomoxetine binding to rat cerebral cortex with a Ki of 0.37 nM. The compound also inhibited the uptake of [3H]NE into rat hypothalamic synaptosomes with a Ki of 3.5 nM. This analog was significantly less potent at the 5HT uptake site, as exhibited by a Ki of 25 nM in the inhibition of [3H]paroxetine binding and a Ki of 121 nM in [3H]5HT uptake. The resolved (R) enantiomer (303926) was 10 times more potent as a [3H]NE uptake inhibitor and 29 times more potent as an inhibitor of [3H]tomoxetine binding than the (S) enantiomer (303884). Administration of 289306 to rats prior to an i.c.v. injection of 6-hydroxydopamine prevented the depletion of hypothalamic NE and Epi with ED50 values of 0.28 and 0.47 mg/kg, respectively. Thus, 289306 was a potent inhibitor of NE uptake in vitro and in vivo. In addition, these compounds provide structures for potential ligands for the study of NE uptake sites by autoradiography, PET or SPECT imaging.

Recent studies have demonstrated that functionalized alpha,alpha-diamino acids (1) display excellent activity when evaluated in the maximal electroshock seizure (MES) test in mice. The synthesis and pharmacological evaluation of 14 select analogues within this series of compounds are detailed. Included in this survey were 10 N-acyl derivatives in which the basic C(alpha) N-group in 1 was replaced by a neutral N-substituent and four dipeptides where the amino acid fusion point was the alpha-carbon site. N-Acylation of 1 led to decreased anticonvulsant activity. The importance of these findings in relation to the requirements of the C(alpha) substituent for anticonvulsant activity in 1 are briefly discussed.

Earlier studies showed that (R,S)-alpha-acetamido-N-benzylacetamides (2) containing a five- and six-membered aromatic or heteroaromatic group appended at the C(alpha) site displayed outstanding activity in the maximal electroshock-induced seizure (MES) test in mice. An expanded set of C(alpha)-heteroaromatic analogues of 2 have been prepared and evaluated. The observed findings extended the structure-activity relationships previously discerned for this novel class of anti-convulsants and have validated previous trends. The alpha-furan-2-yl (4), alpha-oxazol-2-yl (18), and alpha-thiazol-2-yl (19) alpha-acetamido-N-benzylacetamides afforded excellent protection against MES-induced seizures in mice. The ED50 and PI values for these adducts rivaled those reported for phenytoin. The outstanding properties provided by 4 led to an in-depth examination of the effect of structural modification at key sites within this compound on biological activity. The pharmacological data in this series indicated that stringent steric and electronic requirements existed for maximal activity and revealed the outstanding activity of (R)-(-)-alpha-acetamido-N-(4-fluorobenzyl)-alpha-(furan-2-yl)acetamide [(R)-30].

LY248686 is an inhibitor of serotonin (5-hydroxytryptamine; 5-HT) and norepinephrine (NE) uptake in synaptosomal preparations of hypothalamus and cerebral cortex, and 5-HT uptake in human blood platelets, with inhibitor constants near nanomolar concentrations. Upon administration to rats 1 hour before sacrifice, LY248686 caused dose-dependent and parallel decreases of 5-HT and NE uptake in hypothalamus homogenates ex vivo. LY248686 is a positive enantiomer and was slightly more potent than its negative isomer, LY248685, as an inhibitor of 5-HT uptake. Both isomers were only weak inhibitors of dopamine (DA) uptake in striatal synaptosomes. The inhibitory effects on 5-HT and NE uptake after a single administration of LY248686 followed similar time courses and simultaneously persisted for as long as 6 hours. LY248686 in vivo could effectively antagonize the p-chloroamphetamine-induced decreases of 5-HT uptake and levels of 5-HT and 5-hydroxyindoleacetic acid in cerebral cortex, and block the accumulation of 14C-NE in rat hearts. In food deprived rats, LY248686 suppressed food intake synergistically with 5-hydroxytryptophan, a precursor amino acid of 5-HT. Because of its lack of affinity for receptors of 5-HT, NE, DA, acetylcholine, histamine and naloxone, and its ability to inhibit 5-HT and NE uptake simultaneously, LY248686 has a favorable pharmacological profile as a potential antidepressant drug.

We have developed a high specific activity radioiodinated ligand for the biochemical evaluation and autoradiographic localization of 5HT3 receptors in the brain. [125I]-(S)-iodozacopride was synthesized by radioiodination of deschloro-(S)-zacopride using chloramine-T, and the product was purified by HPLC. The equilibrium kinetics and pharmacology of the binding of this radioligand were studied in homogenates of rat cerebral cortex, while the distribution of binding was examined by quantitative autoradiography. [125I]-(S)-iodozacopride bound to a single, saturable, specific binding site (Kd = 192 +/- 9 pM, Bmax = 1.2 +/- 0.2 fmol/mg protein). The binding had the pharmacological properties of a 5HT3 receptor, being potently inhibited by a variety of 5HT3 agonists and antagonists including (S)-zacopride (Ki = 0.032 nM), Quipazine (Ki = 0.45 nM), LY278584 (Ki = 0.5 nM), (1-m-chlorophenyl)-biguanide (Ki = 0.6 nM) and ICS 205-930 (Ki = 1.0 nM). Autoradiographic studies were undertaken by incubating sections with 400 pM [125I]-(S)-iodozacopride and exposing them to film for 3-7 days to obtain suitable autoradiograms. Specific binding of [125I]-(S)-iodozacopride was found at various amounts in a variety of brain regions. The highest levels of binding were found in the brainstem, principally the nucleus of the solitary tract with somewhat lower levels in the area postrema, substantia gelatinosa of the trigeminal nucleus and dorsal motor nucleus of the vagus. In the rat forebrain, moderate levels of specific binding were found in the glomerular layer of the olfactory bulb, anterior olfactory nucleus and various subnuclei of the amygdala. Lower levels of binding were seen in the superficial laminae of the parietal cerebral cortex and diffusely distributed throughout the hippocampal formation. In conclusion, [125I]-(S)-iodozacopride binds to a receptor site with the pharmacological properties and distribution that is consistent with the 5HT3 receptor. [125I]-(S)-iodozacopride represents a significant improvement in autoradiographic studies of the 5HT3 receptor by reducing the required exposure time for producing autoradiograms from the 3-6 months required for [3H]-labeled ligands to 3-7 days.

Like fluoxetine, the N-demethylated metabolite norfluoxetine exists in R- and S-enantiomeric forms. S-Norfluoxetine inhibited serotonin (5-HT) uptake and [3H]paroxetine binding to 5-HT uptake sites with a pKi of 7.86 and 8.88 or 14 and 1.3 nM, respectively, whereas R-norfluoxetine was 22 and 20 times, respectively, less potent. R- and S-Norfluoxetine were less potent than the corresponding enantiomers of fluoxetine as inhibitors of norepinephrine uptake and [3H]tomoxetine binding to norepinephrine uptake sites. Ex vivo studies showed that S-norfluoxetine inhibited 5-HT uptake with an ED50 of 3 mg/kg intraperitoneally, 4.7 mg/kg subcutaneously, and 9 mg/kg orally (7.3, 11.4 and 21.9 mumol/kg, respectively), while the ED50 for R-norfluoxetine exceeded 20 mg/kg intraperitoneally (48.6 mumol/kg). Inhibition of 5-HT uptake in cerebral cortex ex vivo and decrease in 5-HIAA levels in hypothalamus persisted for 24 hours after administration of S-norfluoxetine as demonstrated with the administration of fluoxetine. Thus, S-norfluoxetine is the active N-demethylated metabolite responsible for the persistently potent and selective inhibition of 5-HT uptake in vivo.

Zatosetron is a potent, orally active 5‐HT3 receptor antagonist with a long duration of activity in laboratory animals and humans. Several metabolites have been detected in plasma and urine of humans and experimental animals receiving zatosetron. The present study was designed to explore the pharmacological activity of the detected metabolites, 3‐hydroxyzatosetron, 3‐ketozatosetron, and N‐desmethylzatosetron, relative to the parent molecule. These three metabolites had relatively high affinity at 5‐HT3 receptors based on in vitro radioligand binding and inhibited serotonin‐induced bradycardia in urethane‐anesthetized rats after intravenous administration. However, these metabolites had lower affinity and were less potent than zatosetron. Of these metabolites, 3‐hydroxyzatosetron (ED50 = 4.0 μg/kg iv) was approximately 5‐fold less potent than zatosetron (ED50 = 0.8 μg/kg iv) in vivo and had approximately 10‐fold lower affinity at 5‐HT3 receptors in vitro relative to zatosetron. N‐desmethylzatosetron and 3‐ketozatosetron were approximately 15‐fold less potent than zatosetron in vivo as 5‐HT3 receptor antagonists. With regard to duration of activity in vivo, after intravenous administration, 3‐hydroxyzatosetron and 3‐ketozatosetron blocked 5‐HT3 receptors longer than zatosetron, whereas N‐desmethylzatosetron showed a duration of pharmacological activity similar to zatosetron. A fourth metabolite, zatosetron‐N‐oxide can exist in two isomeric forms, with stereoselective N‐oxidation of zatosetron resulting in formation of only one isomer in humans, zatosetron‐β‐N‐oxide. Zatosetron‐β‐N‐oxide had approximately 100‐fold lower 5‐HT3 receptor affinity relative to zatosetron and was approximately 150‐fold less active as an antagonist at 5‐HT3 receptors in vivo (ED50 = 115 μg/kg iv). Thus, although pharmacological activity was observed with all four metabolites, they were all less active in vivo than zatosetron. Therefore, these metabolites would contribute significantly to the activity of zatosetron only if plasma (and tissue) levels greatly exceeded those of zatosetron. © 1993 Wiley‐Liss, Inc.

The distribution of binding sites for the potent inhibitor of norepinephrine (NE) reuptake, [3H]tomoxetine, was examined in rat brain using quantitative autoradiography. Scatchard analysis of [3H]tomoxetine-binding to slide-mounted sections of rat forebrain indicated that the ligand bound to two sites, a high-affinity site with a Kd of 0.29 nM and a lower-affinity site with a Kd of 16 nM. Pharmacological characterization of this high-affinity site was consistent with labelling a NE-uptake site in brain. Autoradiographic localization of the binding sites for [3H]tomoxetine was performed at a ligand concentration of 1 nM representing the distribution of high-affinity sites. The radioligand bound with a distribution of binding sites that was consistent with the known distribution of NE-containing neurons. The highest levels of binding were seen in regions, such as the locus coeruleus, bed nucleus of the stria terminalis, anterior ventral nucleus of the thalamus and the paraventricular nucleus of the hypothalamus. Low levels were seen in regions such as the caudate-putamen, ventral tegmental area and zona reticulata of the substantia nigra, where NE-containing neurons have been reported to be low. Binding to all these sites was inhibited by 1 μM desipramine which produced autoradiograms with a uniform nonspecific binding. These results indicate that low concentrations of [3H]tomoxetine can be used to localize and characterize NE-binding sites. Further study will be necessary to determine the nature of the low-affinity binding site.

Both alpha-methylserotonin and 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) are agonists at 5-hydroxytryptamine2 (5-HT2) receptors. The present study compared these agonists for their binding affinities at the high- and low-affinity states of the 5-HT2 receptor and for their contractile activities in certain smooth muscle preparations. Both agonists contracted the rat aorta and rat jugular vein, tissues possessing 5-HT2 receptors, and contraction was blocked by ketanserin. However, alpha-methylserotonin produced greater maximal response (80-90% maximum response to serotonin) than DOI. In the rat jugular vein, the calculated dissociation constant of DOI (-log Kb = 7.7) corresponded well with its affinity for [3H]ketanserin- (pKi = 7.5) but not [125I]DOI- (pKi = 8.6) radiolabeled sites. This might suggest that binding to the agonist low-affinity state of the 5-HT2 receptor is more relevant to vascular agonist activity. alpha-Methylserotonin was slightly more potent than serotonin in contracting the rat aorta but not the jugular vein, whereas DOI was more potent in the jugular vein than in the aorta. In the jugular vein but not the aorta, the relative potency of these agents corresponded well with their relative affinities for either the [3H]ketanserin- or the [125I]DOI-labeled 5-HT2 receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

Charybdotoxin, a 37 amino acid peptide isolated from scorpion venom, is a potent inhibitor of potassium channel function. [125I]charybdotoxin was originally believed to be a selective ligand for the Ca(2+)-sensitive channel in many tissues, but it appears to bind only to a voltage-sensitive potassium channel in brain. We found high densities of [125I]charybdotoxin binding in lateral olfactory tract, interpeduncular nucleus and a variety of mesencephalic nuclei. Moderate levels were found in the cerebral cortex, medial thalamus, hypothalamus and selected thalamic nuclei. These results indicate that [125I]charybdotoxin identifies a potassium channel or channels with a unique distribution in the brain.

Antagonists of 5HT3 receptors are clinically effective in treating nausea and emesis associated with certain oncolytic drugs, including cisplatin. Moreover, these agents may be useful in pharmacological management of several central nervous system disorders, including anxiety, schizophrenia, dementia, and substance abuse. Our studies on aroyltropanamides led to the discovery that dihydrobenzofuranyl esters and amides are potent 5HT3 receptor antagonists. Simple benzoyl derivatives of tropine and 3 alpha-aminotropane possessed weak 5HT3 receptor antagonist activity, as judged by blockade of bradycardia produced by iv injection of serotonin (5HT) to anesthetized rats. Within this series, use of benzofuran-7-carboxamide as the aroyl moiety led to a substantial increase of 5HT3 receptor affinity. The optimal 5HT3 receptor antagonist identified via extensive SAR studies was endo-5-chloro-2,3-dihydro-2,2-dimethyl-N-(8-methyl-8-azabicyclo[3.2.1]oc t- 3-yl)-7-benzofurancarboxamide (Z)-2-butenedioate (zatosetron maleate). The 7-carbamyl regiochemistry, dimethyl substitution, chloro substituent, and endo stereochemistry were all crucial elements of the SAR. Zatosetron maleate was a potent antagonist of 5HT-induced bradycardia in rats (ED50 = 0.86 micrograms/kg i.v.). Low oral doses of zatosetron (30 micrograms/kg) produced long-lasting antagonism of 5HT3 receptors, as evidenced by blockade of 5HT-induced bradycardia for longer than 6 h in rats. Moreover, this compound did not produce hemodynamic effects after i.v. administration to rats, nor did it block carbamylcholine-induced bradycardia in doses that markedly blocked 5HT3 receptors. Thus, zatosetron is a potent, selective, orally effective 5HT3 receptor antagonist with a long duration of action in rats.

Norfluoxetine, the N-desmethyl metabolite of fluoxetine, has been reported to resemble fluoxetine in being a potent and selective inhibitor of the serotonin uptake carrier. The enantiomers of norfluoxetine have now been compared as serotonin uptake inhibitors in vivo, based on their antagonism of p-chloroamphetamine-induced depletion of serotonin in brain and their lowering of concentrations of the metabolite of serotonin, 5-hydroxyindoleacetic acid (5-HIAA) in brain. In rats, S-norfluoxetine (ED50 3.8 mg/kg) was more potent than R-norfluoxetine (ED50 > 20 mg/kg) in blocking the depletion of serotonin by p-chloroamphetamine after intraperitoneal administration. The S enantiomer decreased concentrations of 5-HIAA in whole brain after doses of 2.5 20 mg/kg, whereas the R enantiomer did not. The concentrations of both enantiomers in brain increased in proportion to dose and the R enantiomer disappeared from the brain at a slightly slower rate than the S enantiomer. The relative inability of the R enantiomer to block the uptake of serotonin was therefore not a result of smaller concentrations of drug in the brain. In mice, S-norfluoxetine was also more potent than R-norfluoxetine in blocking depletion of serotonin by p-chloroamphetamine (ED50 values 0.82 and 8.3 mg/kg, respectively). Thus, in contrast to the relatively similar potencies of the enantiomers of fluoxetine in blocking the uptake of serotonin, the enantiomers of norfluoxetine have markedly different potencies as inhibitors of the uptake of serotonin.

MPTP-3-ol injected s.c. once daily for 4 days resulted in a dose-dependent depletion of striatal dopamine and cortical norepinephrine one week after the last dose. MPTP-3-ol was approximately one-fourth as potent as MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) in causing these effects. MPTP-3-ol was oxidized by monoamine oxidase in mouse brain in vitro and resulted in MPP+ (1-methyl-4-phenylpyridinium) formation in brain in vivo, both at about one-fourth the rates with MPTP. The in vitro metabolism of MPTP-3-ol was inhibited by deprenyl, a selective inhibitor of monoamine oxidase type B, and deprenyl pretreatment also blocked the depletion of striatal dopamine and cortical norepinephrine in vivo. Pretreatment with EXP 561, an inhibitor of catecholamine uptake, also prevented the dopamine- and norepinephrine-depleting effects of MPTP-3-ol. Thus, substitution of a hydroxy group on the 3-position of MPTP retains its neurotoxic potential toward catecholamine neurons but reduces potency by decreasing the rate of oxidation via monoamine oxidase type B.

Sumatripan and 5-benzyloxytryptamine are ligands with high affinity for 5-HT1D receptors in the caudate nucleus. Both compounds contracted canine saphenous veins, in vitro. Benzyloxytryptamine was less potent as a contractile agonist than sumatriptan which was less potent than serotonin. In high concentrations (>10−5 M) serotonin-induced contraction resulted, in part, from activation of α-adrenoceptors as determined by blockade of contraction with prazosin (10−6 M) and idazoxan (10−6 M). Likewise, benzyloxytryptamine but not sumatripan also activated contractile α-receptors in the canine saphenous vein. Furthermore, benzyloxytryptamine antagonized contraction to sumatriptan in an apparently non-competitive fashion. Thus, benzyloxytryptamine, although possessing some α-receptor agonist activity, like sumatriptan, can interact with serotonin receptors in canine saphenous veins. Although effects of sumatriptan and benzyloxytryptamine quantitatively differed in canine saphenous veins, both agents showed similar affinity and agonist efficacy at 5-HT1D receptors in brain. These studies may reflect potential differences between the 5-HT1D receptor in brain and the 5-HT1-like receptor in canine saphenous veins.