Discovery of novel selective norepinephrine inhibitors: 1-(2-morpholin-2-ylethyl)-3-aryl-1,3-dihydro-2,1,3-benzothiadiazole 2,2-dioxides (WYE-114152).

Sequential modification of the previously identified 4-[3-aryl-2,2-dioxido-2,1,3-benzothiadiazol-1(3H)-yl]-1-(methylamino)butan-2-ols led to the identification of a new series of 1-(2-morpholin-2-ylethyl)-3-aryl-1,3-dihydro-2,1,3-benzothiadiazole 2,2-dioxides that are potent and selective inhibitors of the norepinephrine transporter over both the serotonin and dopamine transporters. One representative compound 10b (WYE-114152) had low nanomolar hNET potency (IC(50) = 15 nM) and good selectivity for hNET over hSERT (>430-fold) and hDAT (>548-fold). 10b was additionally bioavailable following oral dosing and demonstrated efficacy in rat models of acute, inflammatory, and neuropathic pain.

Discovery of novel selective norepinephrine reuptake inhibitors: 4-[3-aryl-2,2-dioxido-2,1,3-benzothiadiazol-1(3H)-yl]-1-(methylamino)butan-2-ols (WYE-103231).

Structural modification of a virtual screening hit led to the identification of a new series of 4-[3-aryl-2,2-dioxido-2,1,3-benzothiadiazol-1(3H)-yl]-1-(methylamino)butan-2-ols which are potent and selective inhibitors of the norepinephrine transporter over both the serotonin and dopamine transporters. One representative compound S-17b (WYE-103231) had low nanomolar hNET potency (IC(50) = 1.2 nM) and excellent selectivity for hNET over hSERT (>1600-fold) and hDAT (>600-fold). S-17b additionally had a good pharmacokinetic profile and demonstrated oral efficacy in rat models of ovariectomized-induced thermoregulatory dysfunction and morphine dependent flush as well as the hot plate and spinal nerve ligation (SNL) models of acute and neuropathic pain.

Heterocyclic cycloalkanol ethylamines as norepinephrine reuptake inhibitors.

A series of heterocyclic cycloalkanol ethylamines have been prepared to expand our norepinephrine reuptake inhibitor (NRI) program. Synthesis of a variety of heterocycles identified (+)-S-21, a potent NRI efficacious in an animal model for thermoregulatory dysfunction.

1-(Indolin-1-yl)-1-phenyl-3-propan-2-olamines as potent and selective norepinephrine reuptake inhibitors.

Efforts to identify new selective and potent norepinephrine reuptake inhibitors (NRIs) for multiple indications by structural modification of the previous 3-(arylamino)-3-phenylpropan-2-olamine scaffold led to the discovery of a novel series of 1-(indolin-1-yl)-1-phenyl-3-propan-2-olamines (9). Investigation of the structure-activity relationships revealed that small alkyl substitution at the C3 position of the indoline ring enhanced selectivity for the norepinephrine transporter (NET) over the serotonin transporter (SERT). Several compounds bearing a 3,3-dimethyl group on the indoline ring, 9k, 9o,p, and 9s,t, exhibited potent inhibition of NET (IC(50) = 2.7-6.5 nM) and excellent selectivity over both serotonin and dopamine transporters. The best example from this series, 9p, a potent and highly selective NRI, displayed oral efficacy in a telemetric rat model of ovariectomized-induced thermoregulatory dysfunction, a mouse p-phenylquinone (PPQ) model of acute visceral pain, and a rat spinal nerve ligation (SNL) model of neuropathic pain.

Structure-activity relationships of norepinephrine reuptake inhibitors with benzothiadiazine dioxide or dihydrosulfostyril cores.

Two related series of selective norepinephrine reuptake inhibitors were synthesized based on 3,4-dihydro-1H-2,1,3-benzothiadiazine 2,2-dioxide or 3,4-dihydrosulfostyril cores, and screened for monoamine reuptake inhibition. Structure-activity relationships were determined for the series' in vitro potency and selectivity versus serotonin or dopamine transporter inhibition, and analogs based on both cores were identified as potent and selective NRIs. The 3,4-dihydrosulfostyril series was further tested for microsome stability, and compound 16j, which was optimized for both potency and stability, showed efficacy in an in vivo model of thermoregulatory dysfunction.

Discovery of WAY-260022, a Potent and Selective Inhibitor of the Norepinephrine Transporter.

The potency and selectivity of a series of 1-{(1S)-2-[amino]-1-[3-(trifluoromethoxy)phenyl]ethyl}cyclohexanol analogues are described. These compounds were prepared to improve in vitro metabolic stability and achieve brain penetration. Compound 13 (WAY-260022, NRI-022) was found to be a potent inhibitor of norepinephrine reuptake and demonstrated excellent selectivity over the serotonin and dopamine transporters. Additionally, 13 exhibited oral efficacy in a rat model of thermoregulatory dysfunction.

Dual acting norepinephrine reuptake inhibitors and 5-HT(2A) receptor antagonists: Identification, synthesis and activity of novel 4-aminoethyl-3-(phenylsulfonyl)-1H-indoles.

The discovery of a series of 4-aminoethyl-3-(phenylsulfonyl)-1H-indoles, dual acting norepinephrine reuptake inhibitors (NRIs) and 5-HT(2A) receptor antagonists, is described. The synthesis and structure-activity relationship (SAR) of this novel series of compounds is also presented.

Structure-activity relationships of the 1-amino-3-(1H-indol-1-yl)-3-phenylpropan-2-ol series of monoamine reuptake inhibitors.

The SAR of a series of 1-amino-3-(1H-indol-1-yl)-3-phenylpropan-2-ols as monoamine reuptake inhibitors, with a goal to improve both potency toward inhibiting the norepinephrine transporter and selectivity over the serotonin transporter, is reported. The effect of specific substitution on both the 3-phenyl group and the indole moiety were explored. This study led to the discovery of compound 20 which inhibited the norepinephrine transporter with an IC50 value of 4 nM while exhibiting 86-fold selectivity over the serotonin transporter.

Discovery of a new series of monoamine reuptake inhibitors, the 1-amino-3-(1H-indol-1-yl)-3-phenylpropan-2-ols.

A novel series of monoamine reuptake inhibitors, the 1-amino-3-(1H-indol-1-yl)-3-phenylpropan-2-ols, have been discovered by combining virtual and focused screening efforts with design techniques. Synthesis of the two diastereomeric isomers of the molecule followed by chiral resolution of each enantiomer revealed the (2R,3S)-isomer to be a potent norepinephrine reuptake inhibitor (IC(50)=28 nM) with excellent selectivity over the dopamine transporter and 13-fold selectivity over the serotonin transporter.

Estradiol increases catecholamine levels in the hypothalamus of ovariectomized rats during the dark-phase.

Estrogens modulate critical homeostatic functions of the hypothalamus such as temperature regulation, sexual behavior and sleep with the most pronounced effects in rats occurring during the dark-phase. The neurochemical signals underlying estrogenic regulation of these hypothalamic functions have not been clearly identified, possibly due to the fact that previous studies have not explored the effects of estrogen treatments on neuronal signaling during the dark-phase. In the present study, ovariectomized rats received estradiol benzoate (5 microg/rat for 7 days, s.c.) and norepinephrine and dopamine levels were measured in the preoptic area of the hypothalamus across the light/dark cycle using in vivo microdialysis. Estradiol benzoate treatment increased extracellular norepinephrine and dopamine levels relative to vehicle treatment during the dark-phase. Increases in norepinephrine and dopamine were first detected by 30 min and 5.5h after lights-off, respectively. Subsequent increases in norepinephrine and dopamine were also noted throughout the 9.5-h collection period. The effect of estradiol benzoate on catecholamine release did not correlate with increases in either tyrosine hydroxylase (TH) protein expression or activity levels in the anterior hypothalamus, although a marked decrease in TH activity correlated with a rise in extracellular norepinephrine at the beginning of the dark-phase. We conclude that subchronic estradiol benzoate treatment increases extracellular catecholamine levels in the preoptic area of the hypothalamus during the dark-phase without a concomitant increase in neurotransmitter biosynthesis. The estradiol benzoate-induced increases in norepinephrine and dopamine levels in the preoptic area during the dark-phase may play an important role in modulating critical hypothalamic functions.

3-(Arylamino)-3-phenylpropan-2-olamines as a new series of dual norepinephrine and serotonin reuptake inhibitors.

A series of 3-(arylamino)-3-phenylpropan-2-olamines was prepared and screened for their ability to inhibit monoamine reuptake. A number of analogues displayed significant dual norepinephrine and serotonin reuptake inhibition. Compounds in this class exhibited minimal affinity for the dopamine transporter.

Simultaneous telemetric monitoring of tail-skin and core body temperature in a rat model of thermoregulatory dysfunction.

Temperature dysfunction, clinically described as hot flashes/flushes and night sweats, commonly occur in women transitioning through menopause. Research in this field has yet to fully elucidate the biological underpinnings explaining this dysfunction. The need to develop animal models that can be used to study hormone-dependent temperature regulation is essential to advancing this scientific area. Development of telemetric transmitters for monitoring tail-skin (TST) and core body (CBT) temperatures for animal research has increased the accuracy of data by reducing extraneous factors associated with previous methods. However, until recently, TST and CBT could not be simultaneously measured telemetrically within the same animal. In this report, new dual temperature monitoring transmitters were validated by simultaneously evaluating them with the single measurement transmitters using the ovariectomized (OVX) rat thermoregulatory dysfunction model. A major advantage of measuring TST and CBT in the same animal is the ability to relate temporal changes on these two temperature parameters. Comparative experimentation was performed by single administration of clonidine (alpha(2) adrenergic agonist), MDL-100907 (5-HT(2a) antagonist), or a 7-day treatment of 17alpha-ethinyl estradiol (EE). Clonidine caused decreases in TST and CBT, MDL-100907 caused increases in TST while decreasing CBT, and EE caused decreases in TST with minor CBT decreases only at the higher dose. Data from either probe type showed similar results on temperature parameters regardless of transmitter used. These findings support the use of the new dual temperature transmitters and should enhance the quality and interpretation of data being generated in thermoregulation studies.

Characterization of ligands for thyroid receptor subtypes and their interactions with co-regulators.

Thyroid hormone receptors (TRs) are nuclear receptors that are activated by thyroid hormone ligands and co-regulator proteins. Two receptor subtypes, TRalpha and TRbeta, have been suggested to play a role in numerous physiological functions. However, specificity of receptor subtype function and co-regulator interaction is unclear due to the lack of TR subtype-specific ligands. Five TR ligands were evaluated for their selectivity and interaction with the TR subtypes. A multiplex assay was used to identify co-regulator peptide interaction, and biochemical assays were used to characterize ligand-receptor specificity. In the biochemical assay, rank order ligand potencies were similar in the presence of co-activator peptides, SRC1-2 and SRC3-2, and the co-repressor peptide, NCoR1-2, with T3 and Triac potencies greater in the presence of the co-repressor. The potency of Tetrac was similar regardless of the co-regulator used while T4 and rT3 demonstrated selectivity for TRalpha subtype. The rank order among TR ligands at either receptor subtype in the biochemical assay correlated with the multiplex assay. These assays can be used to identify new ligands that can provide further insight into TR biology.

Synthesis and activity of 1-(3-amino-1-phenylpropyl)indolin-2-ones: a new class of selective norepinephrine reuptake inhibitors.

Norepinephrine and serotonin play an important role in a wide variety of biological processes and are implicated in a number of neurological disorders. A novel class of 1-(3-amino-1-phenylpropyl)indolin-2-ones was designed and synthesized that displays potent norepinephrine reuptake inhibition while maintaining high selectivity (>100-fold) against the human serotonin and dopamine transporters.

ICI 182,780 penetrates brain and hypothalamic tissue and has functional effects in the brain after systemic dosing.

Previous reports suggest the antiestrogen ICI 182,780 (ICI) does not cross the blood-brain barrier (BBB). However, this hypothesis has never been directly tested. In the present study, we tested whether ICI crosses the BBB, penetrates into brain and hypothalamic tissues, and affects known neuroendocrine functions in ovariectomized rats. Using HPLC with mass spectrometry, ICI (1.0 mg/kg.d, 3 d) was detected in plasma and brain and hypothalamic tissues for up to 24 h with maximum concentrations of 43.1 ng/ml, and 31.6 and 38.8 ng/g, respectively. To evaluate antiestrogenic effects of ICI in the brain after systemic dosing, we tested its ability to block the effect of 17 alpha-ethinyl estradiol (EE) (0.3 mg/kg, 8 d) on tail-skin temperature abatement in the morphine-dependent model of hot flush and on body weight change. In the morphine-dependent model, EE abated 64% of the naloxone-induced tail-skin temperature increase. ICI pretreatment (1.0, 3.0 mg/kg.d) dose dependently inhibited this effect. ICI (3.0 mg/kg.d) alone showed estrogenic-like actions, abating 30% the naloxone-induced flush. In body weight studies, EE-treated rats weighed 58.5 g less than vehicle-treated rats after 8 d dosing. This effect was partially blocked by ICI (3.0 mg/kg.d) pretreatment. Similar to EE treatment, rats receiving 1.0 or 3.0 mg/kg.d ICI alone showed little weight gain compared with vehicle-treated controls. Thus, ICI crosses the BBB, penetrates into brain and hypothalamic tissues, and has both antiestrogenic and estrogenic-like actions on neuroendocrine-related functions.

Effect of 17alpha-ethinyl estradiol on active phase rapid eye movement sleep microarchitecture.

Estrogen treatment decreases active phase rapid eye movement (REM) sleep in ovariectomized rats. Here we explored further the effect of 17alpha-ethinyl estradiol (17alpha-EE) on active phase REM sleep in ovariectomized rats by analyzing spectral properties and the number and length of REM sleep bouts. The greatest suppression of REM sleep occurred on day 4 of 17alpha-EE treatment, was due to decreases in bout length, and was accompanied by decreased EEG theta power. These results further elucidate 17alpha-EE's effects on REM sleep and provide greater understanding of the mechanisms by which estrogens alter sleep-wakefulness patterns.

Structure-activity relationships of the cycloalkanol ethylamine scaffold: discovery of selective norepinephrine reuptake inhibitors.

Further exploration of the cycloalkanol ethylamine scaffold, of which venlafaxine ( 1) is a member, was undertaken to develop novel and selective norepinephrine reuptake inhibitors (NRIs) for evaluation in a variety of predictive animal models. These efforts led to the discovery of a piperazine-containing analogue, 17g (WY-46824), that exhibited potent norepinephrine reuptake inhibition, excellent selectivity over the serotonin transporter, but no selectivity over the dopamine transporter. Synthesis and testing of a series of cyclohexanol ethylpiperazines identified ( S)-(-)- 17i (WAY-256805), a potent norepinephrine reuptake inhibitor (IC 50 = 82 nM, K i = 50 nM) that exhibited excellent selectivity over both the serotonin and dopamine transporters and was efficacious in animal models of depression, pain, and thermoregulatory dysfunction.

Subchronic 17alpha-ethinyl estradiol differentially affects subtypes of sleep and wakefulness in ovariectomized rats.

In ovariectomized (OVX) Sprague-Dawley rats, estradiol benzoate (EB) has been reported to decrease rapid eye movement (REM) and non-REM (NREM) sleep during the dark phase for up to 3 days. It is unknown, however, if estrogenic effects on sleep extend beyond 3 days or if other estrogens could induce the same changes. Furthermore, it is unclear whether the increased wakefulness in the dark phase was due to changes in active or quiet wakefulness. Therefore, we examined the effects of daily injections of 17alpha-ethinyl estradiol (EE) for 6 days on sleep and wakefulness in the OVX rat. After 3 days of baseline recording using a telemetric system, rats were administered sesame oil (sc) for 3 days followed by injection with EE (20 mug/rat/day, sc) for 6 days. After treatment, sleep was recorded during hormone withdrawal for an additional 5 days. A few sporadic but statistically significant increases in light phase sleep occurred during the last 3 days of EE treatment. Starting on day 2 of the study, EE caused statistically significant decreases in dark phase REM sleep that were maintained throughout the treatment period and persisted until the 3rd day of hormone withdrawal. During the dark phase, statistically significant decreases in NREM sleep and increases in active wakefulness started on the second day of treatment and abated by the end of treatment. This study demonstrated that EE had similar effects on sleep-wakefulness to EB and demonstrates the utility of telemetric polysomnographic recording of the female OVX rat as a model for understanding the estrogen-induced changes on sleep-wakefulness.

Desvenlafaxine succinate identifies novel antagonist binding determinants in the human norepinephrine transporter.

Desvenlafaxine succinate (DVS) is a recently introduced antagonist of the human norepinephrine and serotonin transporters (hNET and hSERT, respectively), currently in clinical development for use in the treatment of major depressive disorder and vasomotor symptoms associated with menopause. Initial evaluation of the pharmacological properties of DVS (J Pharmacol Exp Ther 318:657-665, 2006) revealed significantly reduced potency for the hNET expressed in membranes compared with whole cells when competing for [(3)H]nisoxetine (NIS) binding. Using hNET in transfected human embryonic kidney-293 cells, this difference in potency for DVS at sites labeled by [(3)H]NIS was found to distinguish DVS, the DVS analog rac-(1-[1-(3-chloro-phenyl)-2-(4-methylpiperazin-1-yl)-ethyl]cyclohexanol (WY-46824), methylphenidate, and the cocaine analog 3beta-(4-iodophenyl)tropane-2beta-carboxylic acid methyl ester (RTI-55) from other hNET antagonists, such as NIS, mazindol, tricyclic antidepressants, and cocaine. These differences seem not to arise from preparation-specific perturbations of ligand intrinsic affinity or antagonist-specific surface trafficking but rather from protein conformational alterations that perturb the relationships between distinct hNET binding sites. In an initial search for molecular features that differentially define antagonist binding determinants, we document that Val148 in hNET transmembrane domain 3 selectively disrupts NIS binding but not that of DVS.