Salicylaldehyde-Promoted Cobalt-Catalyzed C-H/N-H Annulation of Indolyl Amides with Alkynes: Direct Synthesis of a 5-HT3 Receptor Antagonist Analogue.

A cobalt-catalyzed annulation of the C(sp2)-H/N-H bond of indoloamides with alkynes assisted by 8-aminoquinoline is reported for the synthesis of six-membered indololactams. The use of salicylaldehyde as the ligand is crucial for this transformation. The protocol has a broad scope for both alkynes and indoles. Preparing an active Co complex illustrates that salicylaldehyde plays a key role in the C-H activation step. The synthetic applications are proven by the gram-scale reaction and one-step construction of the multicyclic 5-HT3 receptor antagonist.

CSTI-300 (SMP-100); a Novel 5-HT3 Receptor Partial Agonist with Potential to Treat Patients with Irritable Bowel Syndrome or Carcinoid Syndrome.

The 5-hydroxytryptamine (5-HT) (serotonin) 5-HT3 receptor represents a clinical target for antagonists to deliver symptomatic relief to patients with diarrhea-predominant irritable bowel syndrome (IBS-d) or carcinoid syndrome. Unfortunately, this pharmacological strategy can present side effects (e.g., severe constipation). The present study investigates the potential of a novel 5-HT3 receptor partial agonist, CSTI-300, to treat patients with IBS-d and other conditions associated with discomfort from colonic distension, with a predicted reduced side-effect profile. The in vitro and in vivo preclinical pharmacology of the drug CSTI-300 was investigated to explore the potential to treat patients with IBS-d. CSTI-300 displayed selective high affinity for the human and rat 5-HT3 receptor (Ki approximately 2.0 nM) and acted as a partial agonist (approximately 30%-50% intrinsic efficacy) in vitro. In an in vivo model of IBS-d, the rat colon distension model, CSTI-300 displayed dose-dependent efficacy. In addition, oral administration of CSTI-300 to dogs that achieved plasma levels of the drug exceeding the Ki value for the 5-HT3 receptor failed to either evoke emesis or alter the state of feces. Pharmacokinetics for CSTI-300 in rat and dog identified high levels of oral availability with t 1/2 range of 1.6-4.4 hours. The preclinical pharmacology of the lead candidate drug, CSTI-300, supports the potential of this novel drug to offer symptomatic relief to patients with irritable bowel syndrome and carcinoid syndrome with a rationale for a reduced "on-target" side-effect profile relative to 5-HT3 receptor antagonists, such as alosetron. SIGNIFICANCE STATEMENT: There is a lack of effective current treatment for diarrhea-predominant irritable bowel syndrome and carcinoid syndrome, and in both conditions, overactivity of the 5-hydroxytryptamine (5-HT) 5-HT3 receptor is thought to be implicated in the pathophysiology. Because 5-HT3 receptor blockade with antagonists results in significant side effects, we present evidence that treatment with a suitable 5-HT3 receptor partial agonist will alleviate some symptoms associated with these conditions yet, without fully inhibiting the receptor, predict a less pronounced side-effect profile associated with this therapeutic strategy.

Superagonist, Full Agonist, Partial Agonist, and Antagonist Actions of Arylguanidines at 5-Hydroxytryptamine-3 (5-HT3) Subunit A Receptors.

Introduction of minor variations to the substitution pattern of arylguanidine 5-hydroxytryptamine-3 (5-HT3) receptor ligands resulted in a broad spectrum of functionally-active ligands from antagonist to superagonist. For example, (i) introduction of an additional Cl-substituent(s) to our lead full agonist N-(3-chlorophenyl)guanidine (mCPG, 2; efficacy % = 106) yielded superagonists 7-9 (efficacy % = 186, 139, and 129, respectively), (ii) a positional isomer of 2, p-Cl analog 11, displayed partial agonist actions (efficacy % = 12), and (iii) replacing the halogen atom at the meta or para position with an electron donating OCH3 group or a stronger electron withdrawing (i.e., CF3) group resulted in antagonists 13-16. We posit based on combined mutagenesis, crystallographic, and computational analyses that for the 5-HT3 receptor, the arylguanidines that are better able to simultaneously engage the primary and complementary subunits, thus keeping them in close proximity, have greater agonist character while those that are deficient in this ability are antagonists.

Unraveling mechanisms underlying partial agonism in 5-HT3A receptors.

Partial agonists have emerged as attractive therapeutic molecules. 2-Me-5HT and tryptamine have been defined as partial agonists of 5-HT3 receptors on the basis of macroscopic measurements. Because several mechanisms may limit maximal responses, we took advantage of the high-conductance form of the mouse serotonin type 3A (5-HT3A) receptor to understand their molecular actions. Individual 5-HT-bound receptors activate in long episodes of high open probability, consisting of groups of openings in quick succession. The activation pattern is similar for 2-Me-5HT only at very low concentrations since profound channel blockade takes place within the activating concentration range. In contrast, activation episodes are significantly briefer in the presence of tryptamine. Generation of a full activation scheme reveals that the fully occupied receptor overcomes transitions to closed preopen states (primed states) before opening. Reduced priming explains the partial agonism of tryptamine. In contrast, 2-Me-5HT is not a genuine partial agonist since priming is not dramatically affected and its low apparent efficacy is mainly due to channel blockade. The analysis also shows that the first priming step is the rate-limiting step and partial agonists require an increased number of priming steps for activation. Molecular docking suggests that interactions are similar for 5-HT and 2-Me-5HT but slightly different for tryptamine. Our study contributes to understanding 5-HT3A receptor activation, extends the novel concept of partial agonism within the Cys-loop family, reveals novel aspects of partial agonism, and unmasks molecular actions of classically defined partial agonists. Unraveling mechanisms underlying partial responses has implications in the design of therapeutic compounds.

Functional probes of drug-receptor interactions implicated by structural studies: Cys-loop receptors provide a fertile testing ground.

Structures of integral membrane receptors provide valuable models for drug-receptor interactions across many important classes of drug targets and have become much more widely available in recent years. However, it remains to be determined to what extent these images are relevant to human receptors in their biological context and how subtle issues such as subtype selectivity can be informed by them. The high precision structural modifications enabled by unnatural amino acid mutagenesis on mammalian receptors expressed in vertebrate cells allow detailed tests of predictions from structural studies. Using the Cys-loop superfamily of ligand-gated ion channels, we show that functional studies lead to detailed binding models that, at times, are significantly at odds with the structural studies on related invertebrate proteins. Importantly, broad variations in binding interactions are seen for very closely related receptor subtypes and for varying drugs at a given binding site. These studies highlight the essential interplay between structural studies and functional studies that can guide efforts to develop new pharmaceuticals.

5-Chloroindole: a potent allosteric modulator of the 5-HT3 receptor.

BACKGROUND AND PURPOSE: The 5-HT3 receptor is a ligand-gated ion channel that is modulated allosterically by various compounds including colchicine, alcohols and volatile anaesthetics. However the positive allosteric modulators (PAMs) identified to date have low affinity, which hinders investigation because of non-selective effects at pharmacologically active concentrations. The present study identifies 5-chloroindole (Cl-indole) as a potent PAM of the 5-HT3 receptor. EXPERIMENTAL APPROACH: 5-HT3 receptor function was assessed by the increase in intracellular calcium and single-cell electrophysiological recordings in HEK293 cells stably expressing the h5-HT3A receptor and also the mouse native 5-HT3 receptor that increases neuronal contraction of bladder smooth muscle. KEY RESULTS: Cl-indole (1-100 µM) potentiated agonist (5-HT) and particularly partial agonist [(S)-zacopride, DDP733, RR210, quipazine, dopamine, 2-methyl-5-HT, SR57227A, meta chlorophenyl biguanide] induced h5-HT3A receptor-mediated responses. This effect of Cl-indole was also apparent at the mouse native 5-HT3 receptor. Radioligand-binding studies identified that Cl-indole induced a small (≈ twofold) increase in the apparent affinity of 5-HT for the h5-HT3A receptor, whereas there was no effect upon the affinity of the antagonist, tropisetron. Cl-indole was able to reactivate desensitized 5-HT3 receptors. In contrast to its effect on the 5-HT3 receptor, Cl-indole did not alter human nicotinic α7 receptor responses. CONCLUSIONS AND IMPLICATIONS: The present study identifies Cl-indole as a relatively potent and selective PAM of the 5-HT3 receptor; such compounds will aid investigation of the molecular basis for allosteric modulation of the 5-HT3 receptor and may assist the discovery of novel therapeutic drugs targeting this receptor.

Discriminating between 5-HT3A and 5-HT3AB receptors.

The 5-HT3B subunit was first cloned in 1999, and co-expression with the 5-HT3A subunit results in heteromeric 5-HT3AB receptors that are functionally distinct from homomeric 5-HT3A receptors. The affinities of competitive ligands at the two receptor subtypes are usually similar, but those of non-competitive antagonists that bind in the pore often differ. A competitive ligand and allosteric modulator that distinguishes 5-HT3A from 5-HT3AB receptors has recently been described, and the number of non-competitive antagonists identified with this ability has increased in recent years. In this review, we discuss the differences between 5-HT3A and 5-HT3AB receptors and describe the possible sites of action of compounds that can distinguish between them.

Recent developments in 5-HT3 receptor pharmacology.

Three decades ago the development of 5-hydroxytryptamine(3) (5-HT(3)) receptor antagonists had a major impact on the treatment of nausea and vomiting, and the cloning of the receptor a decade later enabled researchers to better understand its physiology. In the last decade we have seen the publication of detailed molecular structures of closely related proteins, allowing us to reconcile the locations of binding-site residues with earlier pharmacological and biochemical studies. There are more than 500 5-HT(3) receptor ligands. The majority are competitive antagonists resulting from screens of structurally related analogues, but several non-competitive antagonists have also been described. Some ligands are noteworthy because they distinguish between receptor subtypes or have allosteric mechanisms. They will help us to further probe the physiological role of these receptors and could ultimately find applications in clinical and veterinary research. In this review, I consider recently identified ligands with an emphasis on their pharmacology and ligand-receptor interactions.

Discovery of a novel allosteric modulator of 5-HT3 receptors: inhibition and potentiation of Cys-loop receptor signaling through a conserved transmembrane intersubunit site.

The ligand-gated ion channels in the Cys-loop receptor superfamily mediate the effects of neurotransmitters acetylcholine, serotonin, GABA, and glycine. Cys-loop receptor signaling is susceptible to modulation by ligands acting through numerous allosteric sites. Here we report the discovery of a novel class of negative allosteric modulators of the 5-HT(3) receptors (5-HT(3)Rs). PU02 (6-[(1-naphthylmethyl)thio]-9H-purine) is a potent and selective antagonist displaying IC(50) values of ~1 µM at 5-HT(3)Rs and substantially lower activities at other Cys-loop receptors. In an elaborate mutagenesis study of the 5-HT(3)A receptor guided by a homology model, PU02 is demonstrated to act through a transmembrane intersubunit site situated in the upper three helical turns of TM2 and TM3 in the (+)-subunit and TM1 and TM2 in the (-)-subunit. The Ser(248), Leu(288), Ile(290), Thr(294), and Gly(306) residues are identified as important molecular determinants of PU02 activity with minor contributions from Ser(292) and Val(310), and we propose that the naphthalene group of PU02 docks into the hydrophobic cavity formed by these. Interestingly, specific mutations of Ser(248), Thr(294), and Gly(306) convert PU02 into a complex modulator, potentiating and inhibiting 5-HT-evoked signaling through these mutants at low and high concentrations, respectively. The PU02 binding site in the 5-HT(3)R corresponds to allosteric sites in anionic Cys-loop receptors, which emphasizes the uniform nature of the molecular events underlying signaling through the receptors. Moreover, the dramatic changes in the functional properties of PU02 induced by subtle changes in its binding site bear witness to the delicate structural discrimination between allosteric inhibition and potentiation of Cys-loop receptors.

Novel serotonin type 3 receptor partial agonists for the potential treatment of irritable bowel syndrome.

Serotonin type 3 (5-HT(3)) receptor partial agonists are being targeted as potential new drugs for the treatment of irritable bowel syndrome (IBS). Two new chemical series bearing indazole and indole cores have exhibited nanomolar binding affinity for the h5-HT(3)A receptor. A range of partial agonist activities in HEK cells heterologously expressing the h5-HT(3)A receptor were measured for the indazole series. Excellent 5-HT(3) receptor selectivity, favorable in vitro metabolic stability and CYP inhibition properties, and good oral in vivo potency in the murine von Bezold-Jarisch reflex model is exemplified thereby indicating the series to have potential utility as improved IBS agents.

Regulation of the 5-HT3A receptor-mediated current by alkyl 4-hydroxybenzoates isolated from the seeds of Nelumbo nucifera.

Four known alkyl 4-hydroxybenzoates, i.e., methyl 4-hydroxybenzoate (1), ethyl 4-hydroxybenzoate (2), propyl 4-hydroxybenzoate (3), and butyl 4-hydroxybenzoate (4), were isolated from the seeds of Nelumbo nucifera Gaertner (Nymphaeaceae) for the first time. The structures of the isolates were identified by 1D- and 2D-NMR spectroscopy and comparison with published values. The compounds were evaluated for their effects on the 5-HT-stimulated inward current (I(5-HT)) mediated by the human 5-HT(3)A receptors expressed in Xenopus oocytes. Compounds 1 and 2 enhanced the I(5-HT), but 4 reduced it. These results indicate that 4 is an inhibitor of the 5-HT(3)A receptors expressed in Xenopus oocytes.