Discovery, oral pharmacokinetics and in vivo efficacy of a highly selective 5-HT4 receptor agonist: clinical compound TD-2749

Further application of our multivalent approach to drug discovery directed to 5-HT(4) receptor agonists is described. Optimization of the linker and secondary binding amine in the indazole-tropane primary binding group series, for binding affinity and functional potency at the 5-HT(4) receptor, selectivity over the 5-HT(3) receptor, oral pharmacokinetics, and in vivo efficacy in models of GI motility, resulted in the identification of clinical compound TD-2749.     

The effect of the selective 5-HT(3) receptor agonist on ferret gut motility

The effect of the selective 5-hydroxytryptamine (5-HT)(3) receptor agonist YM-31636 (2-(1H-imidazol-4-ylmethyl)-8H-indeno[1,2-d]thiazole monofumarate) on gut motility of fed ferrets was investigated. YM-31636 (0.1 mg/kg p.o.) induced a giant migrating contraction (GMC)-like, high-amplitude, ungrouped colonic contraction although it did not change the basal colonic motility pattern. This GMC-like contraction was always accompanied by defecation. Both GMC-like contraction and defecation were inhibited with the selective 5-HT(3) receptor antagonist ramosetron. YM-31636 affected gastric, duodenal and ileal motility pattern only slightly. These results suggest that 5-HT(3) receptor agonists such as YM-31636 are useful in treating constipation since they facilitate GMC-like contractions and defecation without undesired changes in gut motility pattern.     

SAR development of a selective 5-HT1D antagonist/serotonin reuptake inhibitor lead using rapid parallel synthesis

Incorporation of an SRI (serotonin reuptake inhibitor) pharmacophore into a selective 5-HT(1D) agonist has led to the discovery of a molecule having both 5-HT(1D) antagonist and SRI activity. RPS methodology was used to develop the SAR and identify potential approaches to reduce unwanted adrenergic alpha 1 and dopamine D(2) cross-reactivities.     

5-HT1B serotonin receptors and antidepressant effects of selective serotonin reuptake inhibitors ]

We used knockout mice and receptor antagonist strategies to investigate the contribution of the serotonin (5-hydroxytryptamine, 5-HT) 5-HT1B receptor subtype in mediating the effects of selective serotonin reuptake inhibitors (SSRIs). Using in vivo intracerebral microdialysis in awake mice, we show that a single systemic administration of paroxetine (1 or 5 mg/kg, i.p.) increased extracellular serotonin levels [5-HT]ext in the ventral hippocampus and frontal cortex of wild-type and mutant mice. However, in the ventral hippocampus, paroxetine at the two doses studied induced a larger increase in [5-HT]ext in knockout than in wild-type mice. In the frontal cortex, the effect of paroxetine was larger in mutants than in wild-type mice at the 1 mg/kg dose but not at 5 mg/kg. In addition, either the absence of the 5-HT1B receptor or its blockade with the mixed 5-HT1B/1D receptor antagonist, GR 127935, potentiates the effect of a single administration of paroxetine on [5-HT]ext more in the ventral hippocampus than in the frontal cortex. Furthermore, we demonstrate that SSRIs decrease immobility in the forced swimming test; this effect is absent in 5-HT1B knockout mice and blocked by GR 127935 in wild-type suggesting therefore that activation of 5-HT1B receptors mediate the antidepressant-like effects of SSRIs. Taken together these data demonstrate that 5-HT1B autoreceptors appear to limit the effects of SSRI on dialysate 5-HT levels particularly in the hippocampus while presynaptic 5-HT1B heteroreceptors are likely to be required for the antidepressant activity of SSRIs.     

Homozygote mice deficient in serotonin 5-HT1B receptor and antidepressant effect of selective serotonin reuptake inhibitors

We use the knockout mice strategy to investigate the contribution of the 5-HT1B receptor in mediating the effects of selective serotonin reuptake inhibitors (SSRI). Using microdialysis in awake 129/Sv mice, we show that the absence of the 5-HT1B receptor in mutant mice (KO 1B -/-) potentiated the effect of paroxetine on extracellular 5-HT levels in the ventral hippocampus, but not in the frontal cortex compared to wild-type mice (WT). Furthermore, using the forced swimming test, we demonstrate that SSRIs decreased immobility of WT mice, and this effect is absent in KO 1B -/- mice showing therefore that activation of 5-HT1B receptors mediate the antidepressant-like effects of SSRIs. Taken together these findings suggest that 5-HT1B autoreceptors limit the effects of SSRI particularly in the hippocampus while postsynaptic 5-HT1B receptors are required for the antidepressant activity of SSRIs.     

Inotropic effects of prokinetic agents with 5-HT(4) receptor agonist actions on human isolated myocardial trabeculae

AimsBesides acting as gastrointestinal prokinetic agents, 5-hydroxytryptamine₄ (5-HT₄) receptor agonists can induce positive inotropism in human isolated atrium, but not in ventricles. We pharmacologically evaluated the gastroprokinetic 5-HT₄ receptor agonists tegaserod, prucalopride, R199715, cisapride, the cisapride metabolite norcisapride, and the 5-HT₃ receptor agonist MKC773 on human isolated myocardial trabeculae, and compared their effects with those induced by 5-HT and 5-methoxytryptamine (5-MeOT).Main methodsAtrial and ventricular trabeculae were paced and changes in contractile force were studied in the absence or presence of the 5-HT₄ receptor antagonist GR113808. Partial agonism was assessed using 5-HT₄ receptor agonists as antagonists against 5-HT. To test the contribution of L-type calcium channels, the inotropic responses to 5-HT and 5-MeOT were studied in the absence or presence of verapamil.Key findingsLike 5-HT and 5-MeOT, cisapride and tegaserod, but not prucalopride, R19971 and MKC­733, induced concentration-dependent positive inotropic responses on atrial trabeculae, which were abolished by GR113808. The L-type calcium channel blocker verapamil attenuated inotropic responses to 5-HT and 5-MeOT. None of the agonists affected the contraction of left ventricular trabeculae. Concentration response curves to 5-HT were shifted to the right in the presence of prucalopride, cisapride, tegaserod and R199715, but not MKC-773.SignificanceWe conclude that (i) inotropic responses to 5-HT and 5-MeOT seem to depend on L-type calcium channels, (ii) tegaserod and cisapride behave as partial 5-HT₄ receptor agonists, while prucalopride, norcisapride and MKC-733 cause no significant effects on human atrial trabeculae, (iii) R199715 seems to behave as a 5-HT₄ receptor antagonist.     

Desensitization of the peristaltic reflex induced by mucosal stimulation with the selective 5-HT4 agonist tegaserod

The intestinal peristaltic reflex induced by mucosal stimulation is mediated by mucosal release of serotonin (5-HT), which acts on 5-HT(4) receptors located on CGRP-containing afferent nerve terminals. Exposure of the colonic mucosa to the 5-HT(4) receptor agonist tegaserod in the range of 1 nM to 10 muM elicits a peristaltic reflex and stimulates colonic propulsion. The present study was designed to identify the 5-HT(4) receptor subtype mediating the reflex and determine whether functionally effective concentrations of tegaserod desensitize the reflex induced by mucosal stimulation. Exposure of rat colonic mucosa to tegaserod in the range of 5 nM to 5 muM for 5 or 10 min caused rapid time- and concentration-dependent desensitization of the peristaltic reflex induced by mucosal stroking, consistent with the operation of a rapidly desensitizing 5-HT(4b) receptor subtype. Desensitization was accompanied by a decrease in CGRP release. The rate of recovery of peristaltic response depended on the desensitizing concentration of tegaserod: ascending contraction and descending relaxation recovered within 15 min after 5-50 nM tegaserod, 30 min after 0.5 muM, and 60 min after 5 muM. Neither CGRP release nor the peristaltic reflex induced by muscle stretch was affected by 5-HT(4) receptor desensitization, providing further evidence that 5-HT does not mediate the reflex induced by muscle stretch. These results suggest in cases of increased 5-HT availability or prolonged exposure, such as colitis, that it is likely the peristaltic reflex will be blunted.     

Bridgehead-methyl analog of SC-53116 as a 5-HT4 agonist

Pyrrolizidine benzamide (+/-)-2, the bridgehead-methyl analog of SC-53116, was prepared and evaluated for 5-HT(4) agonism activity in the rat tunica muscularis (TMM) mucosae assay. Compound (+/-)-2 has an EC(50) of 449 nM in the TMM assay, as compared to 23 nM for SC-53116, and 66 nM for the racemate of SC-53116.     

The first selective agonist for the neuropeptide YY5 receptor increases food intake in rats

The first Y(5) receptor-selective analog of neuropeptide Y (NPY), [Ala(31),Aib(32)]NPY, has been developed and biologically characterized. Using competition binding assays on cell lines that express different Y receptors, we determined the affinity of this analog to be 6 nm at the human Y(5) receptor, >500 nm at the Y(1) and Y(2) receptors, and >1000 nm at the Y(4) receptor. Activity studies performed in vitro using a cAMP enzyme immunoassay, and in vivo using food intake studies in rats, showed that the peptide acted as an agonist. Further peptides obtained by the combination of the Ala(31)-Aib(32) motif with chimeric peptides containing segments of NPY and pancreatic polypeptide displayed the same selectivity and even higher affinity (up to 0.2 nm) for the Y(5) receptor. In vivo administration of the new Y(5) receptor-selective agonists significantly stimulated feeding in rats. The NMR solution structures of NPY and [Ala(31),Aib(32)]NPY showed a different conformation in the C-terminal region, where the alpha-helix of NPY was substituted by a more flexible, 3(10)-helical turn structure.     

Comparison of 5-HT4 and 5-HT7 receptor expression and function in the circular muscle of the human colon

Serotonin receptors are potential targets for treating functional bowel disorders. This study investigated the functional roles and expression of the 5-HT4 and the 5-HT7 receptor, which coexist in human colon circular smooth muscle. 5-HT3 receptor expression was also investigated. Part of the relaxant response to 5-HT was due to activation of 5-HT4 receptors as the apparent pKB value of the selective 5-HT4 antagonist, GR 113808, was 9.36. 5-HT4 mRNA levels were low in five tissues and undetectable in four others, but all responded to 5-HT with an EC50 value of 102.54+/-19.32 nM. The contribution of 5-HT7 receptors to the response was not readily demonstrated using the selective 5-HT7 antagonist, SB-269970, as its apparent pKB value of 7.19 (5-HT4 block with 1 microM GR 113808) was lower than the value obtained using the 5-HT7 guinea pig ileum assay (8.62). Nevertheless, the 5-HT7 receptor was expressed more consistently than the 5-HT4, but at similar levels. The 5-HT(3Ashort) and 5-HT(3B) subunits were co-expressed at similar levels, but the 5-HT(3Along) subunit was detected in only five of the nine samples tested. The findings show that 5-HT4-induced relaxation occurs at low to undetectable levels of tissue mRNA, as measured by qPCR. Although 5-HT7 receptor mRNA is detected at low, but consistent levels, the functional activity of this receptor is not readily identified given the currently available drugs.     

SAR of psilocybin analogs: discovery of a selective 5-HT 2C agonist

An SAR study of psilocybin and psilocin derivatives reveals that 1-methylpsilocin is a selective agonist at the h5-HT(2C) receptor. The corresponding phosphate derivative, 1-methylpsilocybin, shows efficacy in an animal model for obsessive-compulsive disorder, as does 4-fluoro-N,N-dimethyltryptamine. These results suggest a new area for development of novel 5-HT(2C) agonists with applications for drug discovery.     

Discovery of TD-8954, a clinical stage 5-HT4 receptor agonist with gastrointestinal prokinetic properties

The discovery of a series of 5-HT₄ receptor agonists based on a novel 2-alkylbenzimidazole aromatic core is described. Optimization of the 2-substituent of the benzimidazole ring led to a series of agonists with subnanomolar binding affinity and moderate-to-high intrinsic activity relative to that of 5-HT. Consistent with our previously described multivalent design approach to this target, subsequent optimization of the linker and secondary binding group regions of the series afforded compound 18 (TD-8954), a potent and selective 5-HT₄ receptor agonist in vitro with demonstrated prokinetic activity in multiple species.     

DAU 6285: a novel antagonist at the putative 5-HT4 receptor.

The antagonistic properties of DAU 6285, an azabicycloalkyl benzimidazolone derivative, at putative 5-hydroxytryptamine4 (5-HT4) receptors were investigated in in vitro preparations of guinea-pig ileum and human atrium, in comparison to ICS 205-930. DAU 6285 behaved as a competitive antagonist in all the preparations examined. Its affinity (pA2) ranged between 6.50 and 7.12 in the test models considered. The affinity of ICS 205-930 was 2-3 fold lower. At variance with ICS 205-930, DAU 6285 displayed a weak affinity for 5-HT3 receptors (pKi = 6.1, rat cortex; pA2 less than 5, guinea-pig ileum). In the guinea-pig ileum, DAU 6285 (10 microM) did not exert antimuscarinic, antihistaminic, antinicotinic or myolytic activity. Moreover, it did not bind to other 5-HT receptor subtypes, or to adrenergic, dopaminergic, benzodiazepine, nicotine, GABA receptors. DAU 6285 may represent a suitable tool for studies in the field of 5-HT4 receptors.     

Effects of 5-HT1A receptor agonist and antagonist on anxiety in intact and ovariectomized female rats

The purpose of this work was to study the role of 5-HT1A receptors on the level of anxiety in adult intact and ovariectomized (OVX) female rats. The influence of chronic administration of 5-HT1A receptor agonist 8-OH-DPAT (0.05 mg/kg, s.c.) and 5-HT1A receptor antagonist NAN-190 (0.1 mg/kg, i.p.) given for 14 days alone or in combination with 17beta-estradiol (0.5 microg i.m./rat/day) was studied on behavior in the elevated plus maze. In intact females administration of NAN-190 resulted in significant increase in the number of enterings and the time spent on the open arms in every phase of the estrous cycle, however, 8-OH-DPAT failed to modify these parameters. In OVX females 8-OH-DPAT alone or in combination with 17beta-estradiol significantly increased the number of enterings and time spent on the open arms. On the contrary, NAN-190 alone or in combination with 17beta-estradiol in OVX females failed to evoke behavioral changes in the elevated plus maze. Thus, the 5-HT1A receptor antagonist NAN-190 induced anxiolytic effect in intact female rats, while 5-HT1A receptor agonist 8-OH-DPAT produced an anxiolytic profile on OVX rats. Results of this work specify the involvement of 5-HT1A receptors in behavioral mechanisms of anxiety in OVX female rats.     

New aza(nor)adamantanes are agonists at the newly identified serotonin 5-HT4 receptor and antagonists at the 5-HT3 receptor

New aza(nor)adamantanes , , and are described which exhibit properties of both 5-HT4 agonism and 5-HT3 antagonism. In particular, compound [SC-52491], an azanoradamantane, exhibits an EC₅₀ of 51 nM in a functional model of 5-HT₄ agonism and potent antagonism, Ki = 1.2 nM, at the 5-HT₃ receptor.     

Effects of highly or relatively selective 5-HT1A receptor agonists on lordosis in female rats

To investigate the role of serotonin (5-HT) receptor 1A or 7 in regulating lordosis behavior in female rats, ovariectomized rats were treated with 3 kinds of receptor agonists and lordosis behavior was observed. The injected agents were the selective 5-HT1A receptor agonist, buspirone (BUS), the highly selective 5-HT1A receptor agonist, 8-hydroxy-2-(di-n-propylamino)tetralin ((+/-)8-OH-DPAT), and the 5-HT1A and 5-HT7 receptor agonist, (R)-8-hydroxy-2-(di-n-propylamino)tetralin ((+)8-OH-DPAT). A behavioral test was performed after ovariectomy and subcutaneous implantation of a silicon tube containing estradiol. Female rats in which the lordosis quotient (LQ) was over 70 were intraperitoneally injected with several doses of these agents. As a result, in the BUS group, the dose of 3 mg/kg bw, but not 1 mg/kg was effective for suppressing lordosis. On the other hand, an inhibitory effect was observed from 0.25 mg/kg and 0.5 mg/kg in the (+)8-OH-DPAT and (+/-)8-OH-DPAT groups, respectively. In the time-course experiment, in all drug-treated groups, LQ decreased to lower than 20 after 15 min and low LQ continued for 1 hr at least. Measurement of locomotor activity using an infrared sensor system showed no relation between the decrease in lordosis by these agents and spontaneous locomotion. These results indicate that 5-HT1A is strongly involved in the lordosis-inhibiting circuit of the serotonin neurons.     

Identification of a novel series of selective 5-HT7 receptor antagonists

Novel 5-HT(7) receptor antagonists containing the benzocycloheptanone core were identified from high throughput screening. Molecular modelling and SAR studies have converted these intractable hits into a more potent, selective and tractable series, exemplified by compound (25), SB-691673.     

Effects of novel 5-HT1A receptor antagonists on measures of postsynaptic 5-HT1A receptor activation in vivo

The effects of two putative 5-HT_1A antagonists, 4-(2′-methoxyphenyl)-1-[2′-[N-(2″-pyridinyl)-p-iodobenzamido]ethyl]piperazine (p-MPPI) and 4-(2′-methoxyphenyl)-1-[2′-[N-(2″-pyridinyl)-p-flourobenzamido]ethyl]piperazine (p-MPPF), were examined in vivo in two tests of postsynaptic 5-HT_1A receptor activation, hypothermia and reciprocal forepaw treading, in the rat. Both p-MPPI (10 mg/Kg, I.p.) and p-MPPF (10 mg/Kg, I.p.) antagonized the hypothermia induced by the 5-HT_1A receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) (0.5 mg/Kg, S.c.). Neither p-MPPI nor p-MPPF administered alone at a dose of 10 mg/kg (i.p.) induced hypothermia. Similarly, both p-MPPI (10 mg/Kg, I.p.) and p-MPPF (2.5 mg/Kg, I.p.) completely antagonized 8-OH-DPAT (2 mg/Kg, S.c.)-induced forepaw treading in rats pretreated with reserpine (1 mg/Kg, S.c., 18–24 hours prior to the experiment). p-MPPI and p-MPPF, at doses of 10 mg/kg (i.p.) did not induce forepaw treading in reserpine pretreated animals. The results of the present study demonstrate that p-MPPI and p-MPPF act as 5-HT_1A receptor antagonists in these measures of postsynaptic 5-HT_1A a receptor activation.     

Discovery of a potent and selective agonist of the prostaglandin EP4 receptor

Analogues of PGE(2) wherein the hydroxycyclopentanone ring has been replaced by a lactam have been prepared and evaluated as ligands for the EP(4) receptor. An optimized compound (19a) shows high potency and agonist efficacy at the EP(4) receptor and is highly selective over the other seven known prostaglandin receptors.     

Identification of a potent and selective 5-HT1B receptor antagonist

An SAR study around the mixed 5-HT1ABD receptor antagonist SB-272183 found that introduction of cis-2,6-dimethyl substitution onto the piperazine ring was a key structural change, which imparted a combination of both excellent selectivity over the 5-HT1A and 5-HT1D receptors and low intrinsic activity. This led to the identification of the selective 5-HT1B receptor antagonist SB-616234.