Biomedical subjects
Publications and source records attributed to M A Cascieri.
L-770,644 (9c) is a potent and selective agonist of the human beta3 adrenergic receptor (EC50 = 13 nM). It shows good oral bioavailability in both dogs and rats (%F = 27), and is a full agonist for glycerolemia in the rhesus monkey (ED50 = 0.21 mg/kg). Based on its desirable in vitro and in vivo properties, L-770,644 was chosen for further preclinical evaluation.
We have identified a series of potent, orally bioavailable, non-peptidyl, triarylimidazole and triarylpyrrole glucagon receptor antagonists. 2-(4-Pyridyl)-5-(4-chlorophenyl)-3-(5-bromo-2-propyloxyphenyl)p yrr ole (L-168,049), a prototypical member of this series, inhibits binding of labeled glucagon to the human glucagon receptor with an IC50 = 3. 7 +/- 3.4 nM (n = 7) but does not inhibit binding of labeled glucagon-like peptide to the highly homologous human glucagon-like peptide receptor at concentrations up to 10 microM. The binding affinity of L-168,049 for the human glucagon receptor is decreased 24-fold by the inclusion of divalent cations (5 mM). L-168,049 increases the apparent EC50 for glucagon stimulation of adenylyl cyclase in Chinese hamster ovary cells expressing the human glucagon receptor and decreases the maximal glucagon stimulation observed, with a Kb (concentration of antagonist that shifts the agonist dose-response 2-fold) of 25 nM. These data suggest that L-168,049 is a noncompetitive antagonist of glucagon action. Inclusion of L-168, 049 increases the rate of dissociation of labeled glucagon from the receptor 4-fold, confirming that the compound is a noncompetitive glucagon antagonist. In addition, we have identified two putative transmembrane domain residues, phenylalanine 184 in transmembrane domain 2 and tyrosine 239 in transmembrane domain 3, for which substitution by alanine reduces the affinity of L-168,049 46- and 4. 5-fold, respectively. These mutations do not alter the binding of labeled glucagon, suggesting that the binding sites for glucagon and L-168,049 are distinct.
The cyclopentylpropylimidazolidinone L-766,892 is a potent beta3 AR agonist (EC50 5.7 nM, 64% activation) with 420- and 130-fold selectivity over binding to the beta1 and beta2 ARs, respectively. In anesthetized rhesus monkeys, L-766,892 elicited dose-dependent hyperglycerolemia (ED50 0.1 mg/kg) with minimal effects on heart rate.
Human beta3 adrenergic receptor agonists containing 5-membered ring ureas were shown to be potent partial agonists with excellent selectivity over beta1 and beta2 binding. L-760,087 (4a) and L-764,646 (5a) (beta3 EC50 = 18 and 14 nM, respectively) stimulate lipolysis in rhesus monkeys (ED50 = 0.2 and 0.1 mg/kg, respectively) with minimal effects on heart rate. Oral absorption in dogs is improved over other urea analogs.
The SAR of 2-pyridyl-3,5-diaryl pyrroles, ligands of the human glucagon receptor and inhibitors of p38 kinase, were investigated. This effort resulted in the identification of 2-(4-pyridyl)-5-(4-chlorophenyl)-3-(5-bromo-2-propyloxyphenyl)pyrr ole 49 (L-168,049), a potent (Kb = 25 nM), selective antagonist of glucagon.
The process of evaluating the in vivo efficacy of non-peptidyl receptor antagonists in animal models is frequently complicated by failure of compounds displaying high affinity against the human receptors to show measurable affinity at the corresponding rodent receptors. In order to generate a suitable animal model in which to evaluate the in vivo activity of non-peptidyl glucagon receptor antagonists, we have utilized a direct targeting approach to replace the murine glucagon receptor with the human glucagon receptor gene by homologous recombination. Specific expression of the human glucagon receptor (GR) in the livers of transgenic mice was confirmed with an RNase protection assay, and the pharmacology of the human GRs expressed in the livers of these mice parallels that of human GR in a recombinant CHO cell line with respect to both binding of 125I-glucagon and the ability of glucagon to stimulate cAMP production. L-168,049, a non-peptidyl GR antagonist selective for the human GR shows a 3.5 fold higher affinity for liver membrane preparations of human GR expressing mice (IC50 = 172 +/- 98 nM) in the presence of MgCl2 in marked contrast to the measured affinity of the murine receptor (IC50 = 611 +/- 197 nM) for this non-peptidyl antagonist. The human receptors expressed are functional as measured by the ability of glucagon to stimulate cAMP production and the selectivity of this antagonist for the human receptor is further verified by its ability to block glucagon-stimulated cyclase activity with 5 fold higher potency (IC50 = 97.2 +/- 13.9 nM) than for the murine receptor (IC50 = 504 +/- 247 nM). Thus we have developed a novel animal model for evaluating GR antagonists in vivo. These mice offer the advantage that the regulatory sequences which direct tissue specific and temporal expression of the GR have been unaltered and thus expression of the human gene in these mice remains in the normal chromosomal context.
Although the functional presence of beta(3)-adrenergic receptors (beta(3)-AR) in rodents is well established, its significance in human adipose tissue has been controversial. One of the issues confounding the experimental data has been the lack of potent and selective human beta(3)-AR ligands analogous to the rodent-specific agonist BRL37344. Recently, we described a new class of aryloxypropanolamine beta(3)-AR agonists that potently and selectively activate lipolysis in rhesus isolated adipocytes and stimulate the metabolic rate in rhesus monkeys in vivo. In this article, we describe novel and selective beta(3)-AR antagonists with high affinity for the human receptor. L-748,328 and L-748,337 bind the human cloned beta(3)-AR expressed in Chinese hamster ovary (CHO) cells with an affinity of 3.7 +/- 1.4 and 4.0 +/- 0.4 nM, respectively. They display an affinity of 467 +/- 89 and 390 +/- 154 nM for the human beta(1)-AR. Their selectivity for human beta(3)-AR versus beta(2)-AR is greater than 20-fold (99 +/- 43 nM) and 45-fold (204 +/- 75 nM), respectively. These compounds are competitive antagonists capable of inhibiting the functional activation of agonists in a dose-dependent manner in cells expressing human cloned beta(3)-AR. Moreover, both L-748,328 and L-748,337 inhibit the lipolytic response elicited by the beta(3)-AR agonist L-742,791 in isolated nonhuman primate adipocytes. The aryloxypropanolamine benzenesulfonamide ligands illustrated here and elsewhere demonstrate high-affinity human beta(3)-AR binding. In addition, we describe specific 3'-phenoxy substitutions that transform these compounds from potent agonists into selective antagonists.
Structural modifications requiring novel synthetic chemistry were made to the morpholine acetal human neurokinin-1 (hNK-1) receptor antagonist 4, and this resulted in the discovery of 2-(R)-(1-(R)-3, 5-bis(trifluoromethyl)phenylethoxy)-3-(S)-(4-fluoro)phenyl-4-(3-ox o-1 ,2,4-triazol-5-yl)methyl morpholine (17). This modified compound is a potent, long-acting hNK-1 receptor antagonist as evidenced by its ability to displace [125I]Substance P from hNK-1 receptors stably expressed in CHO cells (IC50 = 0.09 +/- 0.06 nM) and by the measurement of the rates of association (k1 = 2.8 +/- 1.1 x 10(8) M-1 min-1) and dissociation (k-1 = 0.0054 +/- 0.003 min-1) of 17 from hNK-1 expressed in Sf9 membranes which yields Kd = 19 +/- 12 pM and a t1/2 for receptor occupancy equal to 154 +/- 75 min. Inflammation in the guinea pig induced by a resiniferatoxin challenge (with NK-1 receptor activation mediating the subsequent increase in vascular permeability) is inhibited in a dose-dependent manner by the oral preadmininstration of 17 (IC50 (1 h) = 0.008 mg/kg; IC90 (24 h) = 1.8 mg/kg), indicating that this compound has good oral bioavailbility and peripheral duration of action. Central hNK-1 receptor stimulation is also inhibited by the systemic preadministration of 17 as shown by its ability to block an NK-1 agonist-induced foot tapping response in gerbils (IC50 (4 h) = 0.04 +/- 0.006 mg/kg; IC50 (24 h) = 0.33 +/- 0.017 mg/kg) and by its antiemetic actions in the ferret against cisplatin challenge. The activity of 17 at extended time points in these preclinical animal models sets it apart from earlier morpholine antagonists (such as 4), and the piperidine antagonists 2 and 3 and could prove to be an advantage in the treatment of chronic disorders related to the actions of Substance P. In part on the basis of these data, 17 has been identified as a potential clinical candidate for the treatment of peripheral pain, migraine, chemotherapy-induced emesis, and various psychiatric disorders.
Previously reported studies from these laboratories described the design of a novel series of high-affinity NK1 antagonists based on the 4,4-disubstituted piperidine ring system. Further structure-activity studies have now established that for high NK1 affinity the benzyl ether side chain must be 3,5-disubstituted and highly lipophilic, the optimal side chain being the 3, 5-bis(trifluoromethyl)benzyl ether, 12 (hNK1 IC50 = 0.95 nM). Additional studies have shown that this class of NK1 antagonist tolerates a wider range of substituents on the piperidine nitrogen, including acyl (38) (hNK1 IC50 = 5.3 nM) and sulfonyl (39) (hNK1 IC50 = 5.7 nM) derivatives. Following preliminary pharmacokinetic analysis, two compounds (32 and 43) were selected for in vivo study in the resiniferotoxin-induced vascular leakage model, both showing excellent profiles (ID50 = 0.22 and 0.28 mg/kg, respectively).
The 3-pyridylethanolamine L-757,793 is a potent beta 3 AR agonist (EC50 6.3 nM, 70% activation) with 1,300- and 500-fold selectivity over binding to the beta 1 and beta 2 ARs, respectively. L-757,793 stimulated lipolysis in rhesus monkeys (ED50 0.2 mg/kg) with a maximum response equivalent to that elicited by isoproterenol.
The localization of substance P in brain regions that coordinate stress responses and receive convergent monoaminergic innervation suggested that substance P antagonists might have psychotherapeutic properties. Like clinically used antidepressant and anxiolytic drugs, substance P antagonists suppressed isolation-induced vocalizations in guinea pigs. In a placebo-controlled trial in patients with moderate to severe major depression, robust antidepressant effects of the substance P antagonist MK-869 were consistently observed. In preclinical studies, substance P antagonists did not interact with monoamine systems in the manner seen with established antidepressant drugs. These findings suggest that substance P may play an important role in psychiatric disorders.
Pyridyloxypropanolamines L-749,372 (8, beta 3 EC50 = 3.6 nM) and L-750,355 (29, beta 3 EC50 = 13 nM) are selective partial agonists of the human receptor, with 33% and 49% activation, respectively. Both stimulate lipolysis in rhesus monkeys (ED50 = 2 and 0.8 mg/kg, respectively), with minimal effects on heart rate. Oral bioavailability in dogs, 41% for L-749,372 and 47% for L-750,355, is improved relative to phenol analogs.
We report that a systematic modification of an NK1 receptor selective antagonist resulted in the identification of novel compounds, 4c and 4d, with high affinity for both NK1 and NK2 receptors.
Modifications to the spirocyclic aryl sulfonamide portion of serine derived NK1 antagonists allow a partial pharmacophore model to be developed.
A series of novel serine derived NK1 antagonists is described. The effect of variations in the N-benzyl, O-benzyl and serine groups are used to define the elements which are necessary for binding.
High affinity, selective hNK2 or hNK3 ligands can be prepared from the common template 1 in a few simple chemical operations. The hNK3 ligands 3 antagonise the calcium mobilisation caused by activation of hNK3 receptors expressed in CHO cells as measured using fura-2 microspectrofluorimetry.
Activation of beta3 adrenergic receptors on the surface of adipocytes leads to increases in intracellular cAMP and stimulation of lipolysis. In brown adipose tissue, this serves to up-regulate and activate the mitochondrial uncoupling protein 1, which mediates a proton conductance pathway that uncouples oxidative phosphorylation, leading to a net increase in energy expenditure. While chronic treatment with beta3 agonists in nonprimate species leads to uncoupling protein 1 up-regulation and weight loss, the relevance of this mechanism to energy metabolism in primates, which have much lower levels of brown adipose tissue, has been questioned. With the discovery of L-755,507, a potent and selective partial agonist for both human and rhesus beta3 receptors, we now demonstrate that acute exposure of rhesus monkeys to a beta3 agonist elicits lipolysis and metabolic rate elevation, and that chronic exposure increases uncoupling protein 1 expression in rhesus brown adipose tissue. These data suggest a role for beta3 agonists in the treatment of human obesity.
Heterocyclic replacements for the carboxamido group of the previously disclosed phenylglycinol-based human NK1 (hNK1) receptor antagonists have been investigated, ultimately leading to acyclic compounds with sub-nanomolar affinity for the hNK1 receptor.