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Clinical development of catechol-based orthosteric agonists of the dopamine D1 receptor has thus far been unsuccessful due to multiple challenges. To address these issues, we identified LY3154207 ( 3 ) as a novel, potent, and subtype selective human D1 positive allosteric modulator (PAM) with minimal allosteric agonist activity. Conformational studies showed LY3154207 adopts an unusual boat conformation, and a binding pose with the human D1 receptor was proposed based on this observation. In contrast to orthosteric agonists, LY3154207 showed a distinct pharmacological profile without a bell-shaped dose-response relationship or tachyphylaxis in preclinical models. Identification of a crystalline form of free LY3154207 from the discovery lots was not successful. Instead, a novel cocrystal form with superior solubility was discovered and determined to be suitable for development. This cocrystal form was advanced to clinical development as a potential first-in-class D1 PAM and is now in phase 2 studies for Lewy body dementia.

Allosteric potentiators amplify the sensitivity of physiologic control circuits, a mode of action that could provide therapeutic advantages. This hypothesis was tested with the dopamine D1 receptor potentiator DETQ [2-(2,6-dichlorophenyl)-1-((1S,3R)-3-(hydroxymethyl)-5-(2-hydroxypropan-2-yl)-1-methyl-3,4-dihydroisoquinolin-2(1H)-yl)ethan-1-one]. In human embryonic kidney 293 (HEK293) cells expressing the human D1 receptor, DETQ induced a 21-fold leftward shift in the cAMP response to dopamine, with a K b of 26 nM. The maximum response to DETQ alone was ∼12% of the maximum response to dopamine, suggesting weak allosteric agonist activity. DETQ was ∼30-fold less potent at rat and mouse D1 receptors and was inactive at the human D5 receptor. To enable studies in rodents, an hD1 knock-in mouse was generated. DETQ (3-20 mg/kg orally) caused a robust (∼10-fold) increase in locomotor activity (LMA) in habituated hD1 mice but was inactive in wild-type mice. The LMA response to DETQ was blocked by the D1 antagonist SCH39166 and was dependent on endogenous dopamine. LMA reached a plateau at higher doses (30-240 mg/kg) even though free brain levels of DETQ continued to increase over the entire dose range. In contrast, the D1 agonists SKF 82958, A-77636, and dihydrexidine showed bell-shaped dose-response curves with a profound reduction in LMA at higher doses; video-tracking confirmed that the reduction in LMA caused by SKF 82958 was due to competing stereotyped behaviors. When dosed daily for 4 days, DETQ continued to elicit an increase in LMA, whereas the D1 agonist A-77636 showed complete tachyphylaxis by day 2. These results confirm that allosteric potentiators may have advantages compared with direct-acting agonists., (Copyright © 2016 The Author(s).)

Background: In the adult population, a high rate of discrepancies exists between provider-performed and pharmacist-performed medication histories. Limited data exist regarding pharmacist-performed medication histories in hospitalized pediatric patients. Objective: Identify the incidence and severity of discrepancies in medication histories performed by practitioners compared with pharmacists in the pediatric population. Methods: After institutional review board approval, a retrospective analysis of pediatric patients admitted to inpatient pediatric units in a tertiary hospital was performed. The primary endpoint of the study was the percentage of provider-performed medication histories with any discrepancies compared with the pharmacist-performed medication history. Secondary endpoints included the number and type of discrepancies and the discrepancy's potential risk of patient harm. Results: A total of 101 subjects were included. Nineteen patients (18.8%) had at least one medication discrepancy. Missing medications accounted for the majority of the discrepancies. Advance practice providers performed a small number of the initial medication histories (5%) and had at least one discrepancy for each history performed. The percentages of Grades 1, 2, and 3 discrepancies were 57.2%, 17.1%, and 25.7%, respectively. Medications with the most frequent discrepancies included anticonvulsants, antihistamines, and histamine receptor antagonists. Limitations include the retrospective nature of the study and lower than expected discrepancy rate. Conclusion: In this study, 18.8% of pediatric patients had a discrepancy between medication histories. Missing medications accounted for the largest amount of discrepancies. A large percentage of discrepancies had the potential to cause patient harm., Competing Interests: Declaration of Conflicting Interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (© The Author(s) 2015.)

MePPEP ((3R,5R)-5-(3-methoxy-phenyl)-3-((R)-1-phenyl-ethylamino)-1-(4-trifluoromethyl-phenyl)-pyrrolidin-2-one) is an inverse agonist shown to be an effective PET ligand for labeling cannabinoid CB₁ receptors in vivo. [¹¹C]MePPEP and structurally related analogs have been reported to specifically and reversibly label cannabinoid CB₁ receptors in rat and non-human primate brains, and [¹¹C]MePPEP has been used in human subjects as a PET tracer. We have generated [³H]MePPEP, an ortholog of [¹¹C]MePPEP, to characterize the molecular pharmacology of the cannabinoid CB₁ receptor across preclinical and clinical species. [³H]MePPEP demonstrates saturable, reversible, and single-site high affinity binding to cannabinoid CB₁ receptors. In cerebellar membranes purified from brains of rat, non-human primate and human, and cells ectopically expressing recombinant human cannabinoid CB₁ receptor, [³H]MePPEP binds cannabinoid CB₁ receptors with similar affinity with K(d) values of 0.09 nM, 0.19 nM, 0.14 nM and 0.16 nM, respectively. Both agonist and antagonist cannabinoid ligands compete [³H]MePPEP with predicted rank order potency. No specific binding is present in autoradiographic sections from cannabinoid CB₁ receptor knockout mouse brains, demonstrating that [³H]MePPEP selectively binds cannabinoid CB₁ receptors in native mouse tissue. Furthermore, [³H]MePPEP binding to anatomical sites in mouse and rat brain is comparable to the anatomical profiles of [¹¹C]MePPEP in non-human primate and human brain in vivo, as well as the binding profiles of other previously described cannabinoid CB₁ receptor agonist and antagonist radioligands. Therefore, [³H]MePPEP is a promising tool for translation of preclinical cannabinoid CB₁ receptor pharmacology to clinical PET ligand and cannabinoid CB₁ receptor inverse agonist therapeutic development., (Copyright © 2010 Elsevier B.V. All rights reserved.)

We have reported that [methyl- (11)C] (3 R,5 R)-5-(3-methoxyphenyl)-3-[(R)-1-phenylethylamino]-1-(4-trifluoromethylphenyl)pyrrolidin-2-one ([(11)C] 8, [(11)C]MePPEP) binds with high selectivity to cannabinoid type-1 (CB 1) receptors in monkey brain in vivo. We now describe the synthesis of 8 and four analogues, namely, the 4-fluorophenyl (16, FMePPEP), 3-fluoromethoxy (20, FMPEP), 3-fluoromethoxy- d 2 (21, FMPEP- d 2), and 3-fluoroethoxy analogues (22, FEPEP), and report their activity in an ex vivo model designed to identify compounds suitable for use as positron emission tomography (PET) ligands. These ligands exhibited high, selective potency at CB 1 receptors in vitro (K b < 1 nM). Each ligand (30 microg/kg, iv) was injected into rats under baseline and pretreatment conditions (3, rimonabant, 10 mg/kg, iv) and quantified at later times in frontal cortex ex vivo with liquid chromatography-mass spectrometry (LC-MS) detection. Maximal ligand uptakes were high (22.6-48.0 ng/g). Under pretreatment, maximal brain uptakes were greatly reduced (6.5-17.3 ng/g). Since each ligand readily entered brain and bound with high selectivity to CB 1 receptors, we then established and here describe methods for producing [(11)C] 8, [(11)C] 16, and [(18)F] 20- 22 in adequate activities for evaluation as candidate PET radioligands in vivo.

The cannabinoid CB(1) receptor is one of the most abundant G protein-coupled receptors in the brain and is a promising target of therapeutic drug development. Success of drug development for neuropsychiatric indications is significantly enhanced with the ability to directly measure spatial and temporal binding of compounds to receptors in central compartments. We assessed the utility of a new positron emission tomography (PET) radioligand to image CB(1) receptors in monkey brain. [(11)C]MePPEP ((3R,5R)-5-(3-methoxy-phenyl)-3-((R)-1-phenyl-ethylamino)-1-(4-trifluoromethyl-phenyl)-pyrrolidin-2-one) has high CB(1) affinity (K(b)=0.574+/-0.207 nM) but also moderately high lipophilicity (measured LogD(7.4)=4.8). After intravenous injection of [(11)C]MePPEP, brain activity reached high levels of almost 600% standardized uptake value (SUV) within 10-20 min. The regional uptake was consistent with the distribution of CB(1) receptors, with high radioactivity in striatum and cerebellum and low in thalamus and pons. Injection of pharmacological doses of CB(1)-selective agents confirmed that the tracer doses of [(11)C]MePPEP reversibly labeled CB(1) receptors. Preblockade or displacement with two CB(1) selective agents (ISPB; (4-(3-cyclopentyl-indole-1-sulfonyl)-N-(tetrahydro-pyran-4-ylmethyl)-benzamide) and rimonabant) showed that the majority (>89%) of brain uptake in regions with high receptor densities was specific and reversibly bound to CB(1) receptors in the high binding regions. [(11)C]MePPEP was rapidly removed from arterial plasma. Regional brain uptake could be quantified as distribution volume relative to the concentration of parent radiotracer in plasma. The P-glycoprotein (P-gp) inhibitor DCPQ ((R)-4-[(1a,6,10b)-1,1-dichloro-1,1a,6,10b-tetrahydrodibenzo[a,e]cyclopropa[c]cyclohepten-6-yl]-[(5-quinolinyloxy)methyl]-1-piperazineethanol) did not significantly increase brain uptake of [(11)C]MePPEP, suggesting it is not a substrate for this efflux transporter at the blood-brain barrier. [(11)C]MePPEP is a radioligand with high brain uptake, high specific signal to CB(1) receptors, and adequately fast washout from brain that allows quantification with (11)C (half-life=20 min). These promising results in monkey justify studying this radioligand in human subjects.

Analogues of pindolol, 1-(1H-indol-4-yloxy)-3-isopropylamino-propan-2-ol, were synthesized and evaluated as 5-HT(1A) receptor antagonists. The structural features required for optimal binding to the 5-HT1A receptor are as follows: S-2-propanol linker, 4-indoloxy substituent, and a large lipophilic cyclic amine substituent.

LY334370 is a high affinity, selective agonist at the 5-HT(1F) receptor. On this basis, the tritiated compound was examined for its utility in autoradiography to localize the 5-HT(1F) receptor in rat and guinea pig brain regions. Specific 5-HT(1F) receptor binding in rat brain was found in layers 4-5 of all cortical regions examined, as well as olfactory bulb and tubercle, nucleus accumbens, caudate putamen, parafascicular nucleus of the thalamus, medial mammillary nucleus, the CA3 region of the hippocampus, subiculum, and several amygdaloid nuclei. In guinea pig brain, the [(3)H]LY334370 binding sites were found at highest density in claustrum, but also in a layer of the cortex, caudate putamen, nucleus accumbens, thalamus, and medial mammillary nucleus. Some species differences in the distribution of the 5-HT(1F) receptor were noted. Side by side comparison of rat brain autoradiography with [(3)H]LY334370 and [(3)H]sumatriptan showed labeling in the same brain regions. Preliminary binding studies in rhesus monkey and human brain sections showed [(3)H]LY334370 binding in cortical layers 4-5, subiculum (in the monkey), and the granule cell layer of the cerebellum. These findings suggest a discrete localization of the 5-HT(1F) receptor in the rat, guinea pig, monkey and human brain, and confirms the utility of [(3)H]LY334370 as a potential tool to explore further the localization and possible functions of the 5-HT(1F) receptor.

[(3)H]LY334370 was developed as a radioligand to study the characteristics of this compound's interaction with the 5-HT(1F) receptor. Monovalent or divalent cations did not enhance the binding of [(3)H]LY334370 to the cloned human 5-HT(1F) receptor. In the presence of MgCl(2), the time to reach equilibrium was approximately 2 h, while in its absence equilibrium was reached in less than 1 h. [(3)H]LY334370 had high affinity for the cloned human 5-HT(1F) receptor (K(d)=0.446 nM) and the 5-HT(1F) receptor in rat brain (K(d)=0.388 nM). The expression density of 5-HT(1F) receptors, as determined by binding to homogenates of cortical regions from rat, was low (B(max)=79.1 fmol/mg protein). There was a statistically significant correlation between the apparent pK(i) for inhibition of [(3)H]LY334370 binding and the pEC(50) for stimulation of [(35)S]GTPgammaS binding to homogenates of cells expressing the cloned human 5-HT(1F) receptor. In addition, there was a statistically significant correlation between the apparent pK(i) for inhibition of [(3)H]LY334370 binding to the cloned human 5-HT(1F) receptor and the pID(50) for inhibition of trigeminal nerve stimulated dural plasma protein extravasation in the guinea pig. The conclusion from these studies is that [(3)H]LY334370 is a high affinity radioligand which can be used for the study of the 5-HT(1F) receptor in rat brain or in cells transformed with the human 5-HT(1F) receptor.

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.

The conversion of testosterone (T) to dihydrotestosterone (DHT) has been demonstrated to be catalysed by at least two isoforms of human steroid 5 alpha-reductase, designated types I and II. Type II 5 alpha-reductase expression predominates in human accessory sex tissues, localized to the fibromuscular stromal compartment. The type I isoform predominates in skin, prostatic epithelia and, to a lesser extent, in prostatic fibromuscular stroma. The significance of the type I isoform to prostatic cellular growth and function remains undefined. In cultured DU145 cells, we evaluated the metabolism of [14C]-T and demonstrated the time-dependent formation of [14C]-DHT. Oxidative metabolism (conversion of [14C]-T to [14C]-androstenedione) and the formation of conjugated androgen metabolites occurred at a relatively low rate in the DU145 cells. Using human type I 5 alpha-reductase cDNA, Northern blot analysis of DU145 cell mRNA revealed high levels of type I isoform expression. Analogous probing of the DU145 cells with a human 5 alpha-reductase II cDNA failed to reveal expression of the type II isoform. The expression of functional type I activity has been confirmed pharmacologically using isoform-selective 5 alpha-reductase inhibitors. Reductive metabolism of [3H]-T in the DU145 cells was inhibited in a concentration-dependent manner by LY306089, a potent non-steroidal type I-selective inhibitor (IC50 = 10.0 nM). SKF105657, a steroidal type II-specific inhibitor was distinctly less active at inhibiting [3H]-DHT formation. LY306089 was a non-competitive inhibitor of type I 5 alpha-reductase in DU145 cellular homogenates with an apparent Ki value of 4.0 nM. These studies have identified and pharmacologically defined type I 5 alpha-reductase activity in an androgen-insensitive prostatic cancer cell line and provide the basis for additional investigations into the significance of type I 5 alpha-reductase to human prostatic pathophysiology.

Early structure-activity studies on racemic tryptophan ester and amide NK-1 antagonists 5-7 led to the discovery that the potency of the series could be markedly increased by moving the carbonyl function in these molecules to an off-chain position as in the 3-aryl-1,2-diacetamidopropane 9. Further medicinal chemistry incorporating this change resulted in the discovery of a novel series of highly potent aryl amino acid derived NK-1 antagonists of the R stereoisomeric series (IC50's = 100 pM to > 5 microM). Compounds in this series were shown to be competitive antagonists using an in vitro NK-1 smooth muscle assay, and this data correlated well with observed human NK-1 binding affinities. Two of these agents, (R)-25 and (R)-32, blocked intrathecal NK-1 agonist-driven [Ac-[Arg6,Sar9,Met(O2)11]- substance P 6-11 (Ac-Sar9)] nociceptive behavior in mice. Both compounds potently blocked the neurogenic dural inflammation following trigeminal ganglion stimulation in the guinea pig after intravenous administration. Further, upon oral administration in this model, (R)-32 was observed to be very potent (ID50 = 91 ng/kg) and have a long duration of action (> 8 h at 1 micrograms/kg). Compound (R)-32, designated LY303870, is currently under clinical development as an NK-1 antagonist with a long duration of action.

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.

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.

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.

The 4-aminobenzamides have provided several anticonvulsants that have been extensively investigated. Ameltolide, 4-amino-N-(2,6-dimethylphenyl)benzamide (compound 2,LY201116), is the most potent analogue studied to date. This drug is inactivated in vivo by metabolic N-acetylation and addition of a hydroxy moiety to one of the methyl substituents, resulting in compound 7,N-[4-[[[2-(hydroxymethyl)-6- methylphenyl] amino] carbonyl] phenyl] acetamide. This metabolite was prepared in five steps from a readily available starting material. Compound 7 and its nonacetylated analogue 6 were compared to ameltolide as anticonvulsants. After oral administration to mice, the MES ED50 values of ameltolide, 6, and 7 were 1.4, 10.9, and greater than 100 mg/kg, respectively, demonstrating that hydroxylation and acetylation dramatically decrease the anticonvulsant potency of ameltolide. This rank order of MES anticonvulsant potency was also seen after iv administration to mice, suggesting that these data reflect intrinsic pharmacological activities. After oral administration of 2.0 mg/kg of ameltolide to mice, parent drug, N-acetyl metabolite 3, and the hydroxy metabolite 7 were detected in plasma; the Cmax values were 572, 387, and 73 ng/mL, respectively. Compound 7 was the primary metabolite excreted in urine. These data indicate that 7 is a major metabolite of ameltolide, but does not contribute significantly to the pharmacological effects seen after administration of ameltolide to mice.

We have radiolabeled a structural analogue of indolidan, a potent phosphodiesterase-inhibitor cardiotonic, to permit biochemical studies regarding the interaction of this class of drugs with their pharmacological receptor. [3H]-LY186126 (1,3-dihydro-3,3-dimethyl-1-[3H3]methyl-5-(1,4,5,6-tetrahydro-4-me thyl-6- oxo-3-pyridazinyl)-2H-indol-2-one; [3H]-3) was selected as a synthetic target because of its potency as a cardiotonic and the ability to readily incorporate three tritia via the indolone N-CH3 substituent. Alkylation of a desmethyl precursor with tritium-labeled iodomethane resulted in [3H]-3 with a radiochemical purity of 98% and a specific activity of 79.2 Ci/mmol. This radioligand binds with high affinity to myocardial membrane vesicles. The binding was saturable, and Kd and Bmax values of 4.1 nM and 383 fmol/mg protein were obtained. A series of indolidan congeners displaced [3H]-3 bound to myocardial vesicles, and Ki values for inhibition of binding were highly correlated with canine inotropic ED50 values, suggesting the specific binding of [3H]-3 to cardiac vesicles is pharmacologically relevant.

Recently several noncatecholamine, nonglycoside cardiotonic drugs have been discovered that possess both inotropic and vasodilator activities in experimental animals and man. Prototypical compounds include amrinone, sulmazole, and fenoximone. We investigated the structural requirements necessary for optimal inotropic activity in a series of molecules containing a heterocyclic ring fused to 2-phenylimidazole and discovered that 2-phenylimidazo[4,5-c]pyridines were generally 5-10-fold more potent than analogous 2-phenylimidazo[4,5-b]pyridines (e.g., sulmazole) or 8-phenylpurines. Furthermore, all imidazo[4,5-c]pyridine analogues we tested were orally active; in contrast, only one of the imidazo[4,5-b]pyridine derivatives, sulmazole, was significantly active. One of several highly active compounds in the [4,5-c] series was 50 (LY175326, 2-[2-methoxy-4-(methylsulfinyl)phenyl]-1H-imidazo[4,5-c]pyridine hydrochloride). The structure-activity relationship of this series is presented and compared to that of the imidazo[4,5-b]pyridine and purine series.

We discovered that 6 (N-[4-(1,4,5,6-tetrahydro-6-oxo-3-pyridazinyl)phenyl]acetamide) is a potent positive inotrope in dogs, and we have prepared several lactam analogues of this agent. These included 16 (1,3-dihydro-5-(1,4,5,6-tetrahydro-6-oxo-3-pyridazinyl)-2H-indol-2-one), 32 (the analogous quinolin-2-one), and 37 (the analogous benzazepin-2-one). The inotropic ED50's of these compounds were 24, 3.3, and 5.2 micrograms/kg, respectively, after iv administration to pentobarbital-anesthetized dogs. Compound 20 (LY195115, 1,3-dihydro-3,3-dimethyl-5-(1,4,5,6-tetrahydro-6-oxo-3-pyridazinyl)-2H-i ndol-2- one), the geminal dimethyl analogue of 16, was 3.5-fold more potent than 16 when administered iv (ED50 = 6.8 micrograms/kg). However, the most profound effect of the geminal alkyl substitution was on oral activity. The approximate ED50's of 20 and 16 after oral administration to conscious dogs were 25 and 400 micrograms/kg, respectively. The increase in contractility produced by 25 micrograms/kg of 20 was maximally sustained in excess of 8 h. Thus, 20 is one of the most potent and long-acting oral inotropes described to date.

A recently discovered and structurally distinct class of antiepileptic drugs is the (arylalkyl)imidazoles. Two independently discovered representatives of this class, denzimol (alpha-[4-(2-phenylethyl)phenyl]-1H-imidazole-1-ethanol) and nafimidone (2-(1H-imidazol-1-yl)-1-(2-naphthalenyl)ethanone), are undergoing clinical evaluation. Our structure-activity relationship (SAR) studies revealed that in addition to the naphthalenyl and phenethylphenyl aryl moieties of nafimidone and denzimol, respectively, fluorenyl, benzo[b]thienyl, and benzofuranyl aryl groups provided several highly active (arylalkyl)imidazole anticonvulsants. These structurally diverse aryl moieties, and comparable anticonvulsant activities, lend credence to the hypothesis that the pharmacophore of this class of anticonvulsants is the alkylimidazole portion of the molecule, with the lipophilic aryl portion enabling penetration of the blood-brain barrier. We focused our SAR studies on the (fluorenylalkyl)imidazole series. A representative compound from this series is 1-(9H-fluoren-2-yl)-2-(1H-imidazol-1-yl)ethanone. This agent was twice as potent as nafimidone in inhibiting maximal electroshock seizures in mice (po ED50's = 25 and 56 mg/kg, respectively) and considerably less toxic in the rat (po LD50's = 4550 and 504 mg/kg, respectively). The tertiary alcohol alpha-(9H-fluoren-2-yl)-alpha-methyl-1H-imidazole-1-ethanol was as potent as denzimol in mice (po ED50's = 10 and 12 mg/kg, respectively). This series of imidazole anticonvulsants was highly selective; while many compounds displayed potent antielectroshock activity, little or not activity was observed against pentylenetetrazole-induced clonic seizures or in the horizontal screen test for ataxia. All active compounds that we tested in this series, as well as denzimol and nafimidone, potentiated hexobarbital-induced sleeping time in mice, probably by imidazole-mediated inhibition of cytochrome P-450. The SAR's for the anticonvulsant activity and the sleeping time potentiation were similar. The propensity of these (arylalkyl)imidazole anticonvulsants to interact strongly with cytochrome P-450 and thereby impair the metabolism of other antiepileptic drugs may severely limit their clinical utility as anticonvulsants.

Fluoxetine is a potent and selective inhibitor of the neuronal serotonin-uptake carrier and is a clinically effective antidepressant. Although fluoxetine is used therapeutically as the racemate, there appears to be a small but demonstrable stereospecificity associated with its interactions with the serotonin-uptake carrier. The goals of this study were to determine the absolute configurations of the enantiomers of fluoxetine and to examine whether the actions of fluoxetine in behavioral tests were enantiospecific. (S)-Fluoxetine was synthesized from (S)-(-)-3-chloro-1-phenylpropanol by sequential reaction with sodium iodide, methylamine, sodium hydride, and 4-fluorobenzotrifluoride. (S)-Fluoxetine is dextrorotatory (+1.60) in methanol, but is levorotatory (-10.85) in water. Fluoxetine enantiomers were derivatized with (R)-1-(1-naphthyl)ethyl isocyanate, and the resulting ureas were assayed by 1H NMR or HPLC to determine optical purities of the fluoxetine samples. Both enantiomers antagonized writhing in mice; following sc administration of (R)- and (S)-fluoxetine, ED50 values were 15.3 and 25.7 mg/kg, respectively. Moreover, both enantiomers potentiated a subthreshold analgesic dose (0.25 mg/kg) of morphine, and ED50 values were 3.6 and 5.7 mg/kg, respectively. Following ip administration to mice, the two stereoisomers antagonized p-chloroamphetamine-induced depletion of whole brain serotonin concentrations. ED50 values for (S)- and (R)-fluoxetine were 1.2 and 2.1 mg/kg, respectively. The two enantiomers decreased palatability-induced ingestion following ip administration to rats; (R)- and (S)-fluoxetine reduced saccharin-induced drinking with ED50 values of 6.1 and 4.9 mg/kg, respectively. Thus, in all biochemical and pharmacological studies to date, the eudismic ratio for the fluoxetine enantiomers is near unity.

In the 1,3-dihydro-5-(1,4,5,6-tetrahydro-6-oxo-3-pyridazinyl)-2H-indol-2-one series of cardiotonics, we found that a spirocycloalkyl ring may be annealed to the 3-position of the indolone moiety while retaining inotropic activity. An inverse relationship was found between spirocyloalkyl ring size and inotropic potency. ED50 values of the spirocyclopropane 10, spirocyclobutane 12, and spirocyclopentane 13 were 2.7, 35, and 133 micrograms/kg, respectively, following iv administration to pentobarbital-anesthetized dogs. The most potent compound prepared was 11 (5'-(1,4,5,6-tetrahydro-4-methyl-6-oxo-3-pyridazinyl)spiro[cyclopropane- 1,3'-[3H]indol]-2'(1'H)-one), the 4-methyl analogue of 10. This compound had an iv ED50 of 1.5 microgram/kg. Oral activity was evaluated by administering 50 micrograms/kg of 10 to conscious, chronically instrumented dogs. A 39% increase in LV dP/dt60 was observed, and an inotropic effect was demonstrable in excess of 7 h. Thus, the spirocyclic dihydropyridazinone inotropes are potent, long-acting, orally effective cardiotonics. Compound 11 was a potent inhibitor (IC50 = 13 nM) of cAMP phosphodiesterase derived from canine cardiac sarcoplasmic reticulum (SR-PDE). Importantly, -log IC50 values for inhibition of SR-PDE for this entire series of compounds were highly correlated (r = 0.949, p less than 0.02) with their inotropic -log ED50 values, supporting the hypothesis that inhibition of this enzyme contributes to the mechanism of action of the spirocyclic dihydropyridazinones.

We previously reported the structure-activity relationships (SAR), molecular structure, pharmacology, and molecular pharmacology of indolidan (LY195115), a potent and long-acting dihydropyridazinone cardiotonic. Our 6-phenyldihydropyridazinone SAR studies revealed the critical nature of the substituent at the para position of the phenyl ring. An acetamido substituent provided potent cardiotonic activity and we hypothesized that this may relate to the ability of the acetamide carbonyl to function as a hydrogen-bond acceptor. To further address this question, we prepared 15 (4,5-dihydro-6-[4-(3-pyridinyl)phenyl]-3(2H)-pyridazinone), the 3-pyridyl analogue of imazodan. As is the case with imazodan, this (pyridylphenyl)dihydropyridazinone possesses a nitrogen three atoms removed from the phenyl ring, but the molecular framework through which it is attached to the phenyldihydropyridazinone moiety is altered. After iv administration to pentobarbital-anesthetized dogs, inotropic ED50 values of 15, imazodan, and the parent compound, 4,5-dihydro-6-phenyl-3(2H)-pyridazinone, were 19.4, 50.1, and 6330 micrograms/kg, respectively. Thus, 15 is over 2-fold more potent than imazodan and 326-fold more potent than the parent, unsubstituted compound. These data, as well as data obtained with other congeners, are consistent with the hypothesis that a suitably oriented hydrogen-bond-acceptor site contributes to the high degree of inotropic potency observed with these dihydropyridazinone cardiotonics.

LY227942, (+/-)-N-methyl-3-(1-naphthalenyloxy)-3-(2-thiophene)propanamine ethanedioate, is a new, competitive inhibitor of monoamine uptake in synaptosomal preparations of rat brain. LY227942 inhibits uptake of serotonin (5-hydroxytryptamine, 5HT) and norepinephrine (NE) in cortical synaptosomes and uptake of dopamine (DA) in striatal synaptosomes with inhibitor constants (Ki values) of 8.5, 45 and 300 nM, respectively. Upon administration in vivo, LY227942 lowers 5HT and NE uptake in hypothalamus homogenates to half their respective control activities (ED50) at 0.74 and 1.2 mg/kg s.c., 7 and 12 mg/kg i.p., and 12 and 22 mg/kg p.o., but LY227942 at doses up to 30 mg/kg p.o. does not change DA uptake in striatal homogenates. Lowering of 5HT and NE uptake is demonstrated after 15 min and 6 hr, but has dissipated by 16 hr after oral administration. According to radioligand binding determinations, LY227942 possesses only weak affinity for muscarinic receptors, histamine-1 receptors, adrenergic receptors, dopamine receptors and serotonin receptors. These findings suggest that LY227942 has the pharmacological profile of an antidepressant drug and is useful to study the pharmacological responses of concerted enhancement of serotonergic and noradrenergic neurotransmission.

The cardiotonic 1,3-dihydro-3,3-dimethyl-5-(1,4,5,6-tetrahydro-6-oxo-3- pyridazinyl)-2H-indol-2-one (1, LY195115) is a potent, competitive inhibitor (Ki = 80 nM) of sarcoplasmic reticulum derived phosphodiesterase (SR-PDE). Moreover, the compound is a potent positive inotrope both in vitro and in vivo. To assist further cardiotonic drug-design studies, we have mapped the three-dimensional structure of 1 using X-ray crystallography. From a global viewpoint, this drug was essentially planar, but two small regions of nonplanarity were apparent. These involved the geminal methyl substituents in the indol-2-one moiety and the C5' methylene unit of the dihydropyridazinone ring. Because of our previous studies involving the bipyridine cardiotonics amrinone and milrinone, the conformational relationship between the plane of the phenyl ring and the horizontal symmetry plane defined by N2', C3', and C4' of 1 was of particular interest. The C6-C5-C3'-C4' dihedral angle was -2.7 degrees, whereas the C6-C5-C3'-N2' dihedral angle was 174.6 degrees. Therefore the two rings maintain a high degree of coplanarity. Compound 4, the congener of 1 possessing a completely unsaturated pyridazinone ring was also studied. In terms of inotropic activity, this compound, devoid of any puckering in the pyridazinone moiety, was equipotent with 1. Methyl substitution at the 4-position of the dihydropyridazinone and pyridazinone rings provided disparate results. Compound 2, the 4-methyl analogue of 1, was 2-fold more potent than 1, and the methyl substituent probably caused only minor perturbations in overall molecular topology. However 5, the 4-methyl analogue of the pyridazinone 4, was 4.4-fold less active than 4, perhaps as a result of methyl-induced molecular nonplanarity.