Andrew O. Stewart

Electrophysiological and in vivo characterization of A-317567, a novel blocker of acid sensing ion channels.

&NA; Acid Sensing Ion Channels (ASICs) are a group of sodium‐selective ion channels that are activated by low extracellular pH. The role of ASIC in disease states remains unclear partly due to the lack of selective pharmacological agents. In this report, we describe the effects of A‐317567, a novel non‐amiloride blocker, on three distinct types of native ASIC currents evoked in acutely dissociated adult rat dorsal root ganglion (DRG) neurons. A‐317567 produced concentration‐dependent inhibition of all pH 4.5‐evoked ASIC currents with an IC50 ranging between 2 and 30 μM, depending upon the type of ASIC current activated. Unlike amiloride, A‐317567 equipotently blocked the sustained phase of ASIC3‐like current, a biphasic current akin to cloned ASIC3, which is predominant in DRG. When evaluated in the rat Complete Freuds Adjuvant (CFA)‐induced inflammatory thermal hyperalgesia model, A‐317567 was fully efficacious at a dose 10‐fold lower than amiloride. A‐317567 was also potent and fully efficacious when tested in the skin incision model of post‐operative pain. A‐317567 was entirely devoid of any diuresis or natriuresis activity and showed minimal brain penetration. In summary, A‐317567 is the first reported small molecule non‐amiloride blocker of ASIC that is peripherally active and is more potent than amiloride in vitro and in vivo pain models. The discovery of A‐317567 will greatly help to enhance our understanding of the physiological and pathophysiological role of ASICs.

Synthesis and Evaluation of Benzothiazole-Based Analogues as Novel, Potent, and Selective Fatty Acid Amide Hydrolase Inhibitors

High-throughput screening (HTS) identified benzothiazole analogue 3 as a potent fatty acid amide hydrolase (FAAH) inhibitor. Structure-activity relationship (SAR) studies indicated that the sulfonyl group, the piperidine ring and benzothiazole were the key components to their activity, with 16j being the most potent analogue in this series. Time-dependent preincubation study of compound 3 was consistent with it being a reversible inhibitor. Activity-based protein-profiling (ABPP) evaluation of 3 in rat tissues revealed that it had exceptional selectivity and no off-target activity with respect to other serine hydrolases. Molecular shape overlay of 3 with a known FAAH inhibitor indicated that these compounds might act as transition-state analogues, forming putative hydrogen bonds with catalytic residues and mimicking the charge distribution of the tetrahedral transition state. The modeling study also indicated that hydrophobic interactions of the benzothiazole ring with the enzyme contributed to its extraordinary potency. These compounds may provide useful tools for the study of FAAH and the endocannabinoid system.

Analgesic and anti-inflammatory effects of A-286501, a novel orally active adenosine kinase inhibitor

&NA; Adenosine (ADO) is an inhibitory neuromodulator that can increase nociceptive thresholds in response to noxious stimulation. Inhibition of the ADO‐metabolizing enzyme, adenosine kinase (AK) increases extracellular ADO concentrations at sites of tissue trauma and AK inhibitors may have therapeutic potential as analgesic and anti‐inflammatory agents. N7‐((1′R,2′S,3′R,4′S)‐2′,3′‐dihydroxy‐4′‐amino‐cyclopentyl)‐4‐amino‐5‐bromo‐pyrrolo[2,3‐a]pyrimidine (A‐286501) is a novel and potent (IC50=0.47 nM) carbocyclic nucleoside AK inhibitor that has no significant activity (IC50>100 &mgr;M) at other sites of ADO interaction (A1, A2A, A3 receptors, ADO transporter, and ADO deaminase) or other (IC50 values>10 &mgr;M) neurotransmitter and peptide receptors, ion channel proteins, neurotransmitter reuptake sites and enzymes, including cyclooxygenases‐1 and ‐2. A‐286501 showed equivalent potency to inhibit AK from several mammalian species and kinetic studies revealed that A‐286501 was a reversible and competitive inhibitor with respect to ADO and non‐competitive with respect to MgATP2−. A‐286501 was orally effective to reduce nociception in animal models of acute (thermal), inflammatory (formalin and carrageenan), and neuropathic (L5/L6 nerve ligation and streptozotocin‐induced diabetic) pain. A‐286501 was particularly potent (ED50=1 &mgr;mol/kg, p.o.) to reduce carrageenan‐induced inflammatory thermal hyperalgesia as compared to its analgesic actions in other pain models (acute and neuropathic) and its ability to alter hemodynamic function and motor performance. A‐286501 was also effective to reduce carrageenan‐induced paw edema and myeloperoxidase activity, a measure of neutrophil influx (ED50=10 &mgr;mol/kg, p.o.), in the injured paw. The anti‐nociceptive effects of A‐286501 in the L5/L6 nerve injury model of neuropathic pain (ED50=20 &mgr;mol/kg, p.o.) were not blocked by the opioid antagonist naloxone, but were blocked by the ADO receptor antagonist, theophylline. Following repeated administration, A‐286501 showed less potential to produce tolerance as compared to morphine. Thus, A‐286501 is a structurally novel AK inhibitor that effectively attenuates nociception by a non‐opioid, non‐non‐steroidal anti‐inflammatory drug ADO, receptor mediated mechanism.

C-glycosidation of pyridyl thioglycosides

Abstract The pyridylthioglycosides 1 are efficiently transformed into the corresponding C-glycosides via reaction with silver(I) trinflate and a variety of carbon nucleophiles.

Dopamine D4 receptor signaling in the rat paraventricular hypothalamic nucleus: Evidence of natural coupling involving immediate early gene induction and mitogen activated protein kinase phosphorylation

The dopamine D4 receptor has been investigated for its potential role in several CNS disorders, notably schizophrenia and more recently, erectile dysfunction. Whereas studies have investigated dopamine D4 receptor-mediated signaling in vitro, there have been few, if any, attempts to identify dopamine D4 receptor signal transduction pathways in vivo. In the present studies, the selective dopamine D4 agonist PD168077 induces c-Fos expression and extracellular signal regulated kinase (ERK) phosphorylation in the hypothalamic paraventricular nucleus (PVN), a site known to regulate proerectile activity. The selective dopamine D4 receptor antagonist A-381393 blocked both c-Fos expression and ERK1/2 phosphorylation produced by PD168077. In addition, PD168077-induced ERK1/2 phosphorylation was prevented by SL327, an inhibitor of ERK1/2 phosphorylation. Interestingly, treatment with A-381393 alone significantly reduced the amount of Fos immunoreactivity as compared to basal expression observed in vehicle-treated controls. Dopamine D4 receptor and c-Fos coexpression in the PVN was observed using double immunohistochemical labeling, suggesting that PD168077-induced signaling may result from direct dopamine D4 receptor activation. Our results demonstrate functional dopamine D4 receptor expression and natural coupling in the PVN linked to signal transduction pathways that include immediate early gene and MAP kinase activation. Further, the ability of the selective dopamine D4 antagonist A-381393 alone to reduce c-Fos expression below control levels may imply the presence of a tonic dopamine D4 receptor activation under basal conditions in vivo. These findings provide additional evidence that the PVN may be a site of dopamine D4 receptor-mediated proerectile activity.

Synthesis and structure-activity relationships of 5-heteroatom-substituted pyridopyrimidines as adenosine kinase inhibitors

Under stressful conditions, many cells release adenosine to minimize tissue damage. Inhibition of intracellular adenosine kinase (AK) increases the local extracellular concentration of adenosine and its effect on traumatized tissue. The synthesis and SAR of a new series of pyridopyrimidines for the inhibition of AK are described. It was found that a range of analogs with position five substituted by an amine or ether functionality increased aqueous solubility while retaining the in vitro potency of initial leads. A narrower range of analogs was active in vivo in a rat inflammatory hyperalgesia model.

Structure-activity studies of 5-substituted pyridopyrimidines as adenosine kinase inhibitors.

The synthesis and SAR of a novel series of non-nucleoside pyridopyrimidine inhibitors of the enzyme adenosine kinase (AK) are described. It was found that pyridopyrimidines with a broad range of medium and large non-polar substituents at the 5-position potently inhibited AK activity. A narrower range of analogues was capable of potently inhibiting adenosine phosphorylation in intact cells indicating an enhanced ability of these analogues to penetrate cell membranes. Potent AK inhibitors were found to effectively reduce nociception in animal models of thermal hyperalgesia and persistent pain.

Adenosine kinase inhibitors: polar 7-substitutent of pyridopyrimidine derivatives improving their locomotor selectivity.

We have discovered that polar 7-substituents of pyridopyrimidine derivatives affect not only whole cell AK inhibitory potency, but also selectivity in causing locomotor side effects in vivo animal models. We have identified compound, 1o, which has potent whole cell AK inhibitory potency, analgesic activity and minimal reduction of locomotor activity.

Discovery and SAR of hydrazide antagonists of the pituitary adenylate cyclase-activating polypeptide (PACAP) receptor type 1 (PAC1-R)

Potent small molecule antagonists for the PAC(1)-R have been discovered. Previously known antagonists for the PAC(1)-R were slightly truncated peptide ligands. The hydrazides reported here are the first small molecule antagonists ever reported for this class B GPCR.

2-[4-(3,4-Dimethylphenyl)piperazin-1-ylmethyl]-1H benzoimidazole (A-381393), a selective dopamine D4 receptor antagonist

2-[4-(3,4-Dimethylphenlyl)piperazin-1-ylmethyl]-1H benzoimidazole (A-381393) was identified as a potent dopamine D4 receptor antagonist with excellent receptor selectivity. [3H]-spiperone competition binding assays showed that A-381393 potently bound to membrane from cells expressing recombinant human dopamine D4.4 receptor (Ki=1.5 nM), which was 20-fold higher than that of clozapine (Ki=30.4 nM). A-381393 exhibited highly selective binding for the dopamine D4.4 receptor (>2700-fold) when compared to D1, D2, D3 and D5 dopamine receptors. Furthermore, in comparison to clozapine and L-745870, A-381393 exhibits better receptor selectivity, showing no affinity up to 10 microM for a panel of more than 70 receptors and channels, with the exception of moderate affinity for 5-HT2A (Ki=370 nM). A-381393 potently inhibited the functional activity of agonist-induced GTP-gamma-S binding assay and 1 microM dopamine induced-Ca2+ flux in human dopamine D4.4 receptor expressing cells, but not in human dopamine D2L or D3 receptor cells. In contrast to L-745870, A-381393 did not exhibit any significant intrinsic activity in a D4.4 receptor. In vivo, A-381393 has good brain penetration after subcutaneous administration. A-381393 inhibited penile erection induced by the selective D4 agonist PD168077 in conscious rats. Thus, A-381393 is a novel selective D4 antagonist that will enhance the ability to study dopamine D4 receptors both in vitro and in vivo.