E. Jon Jacobsen

Novel membrane localized iron chelators as inhibitors of iron-dependent lipid peroxidation.

Attachment of various iron chelating moieties to hydrophobic steroids greatly enhanced their abilities to inhibit iron-dependent lipid peroxidation. Using whole rat brain homogenates, lipid peroxidation initiated by the addition of 200 microM Fe2+ was assessed by the formation of thiobarbituric acid reactive products (TBAR). Under these conditions, 50% inhibitory concentrations of Fe3+ chelators such as desferrioxamine or N1,N8-bis(2,3-dihydroxybenzoyl) spermidine hydrobromide (compound II) were around 170 and 50 microM respectively. Coupling desferrioxamine or compound II to a steroid at the D ring increased their potency in lipid peroxidation assays by 5- to 10-fold. Evidence that inhibition of lipid peroxidation by the steroid-chelator adducts was due to iron chelation was suggested by the fact that methylation of the catechol oxygens of compound II, which are essential for chelation, completely eliminated activity of the steroid adduct. A series of 21-aminosteroids which complex Fe2+ iron and potently inhibit iron-dependent lipid peroxidation has also been synthesized. Coupling Fe2+ chelators to hydrophobic steroids increased their inhibitory potencies by as much as 10- to 100-fold. Some steroid-based Fe2+ chelators stimulated lipid peroxidation at low concentrations in the presence of Fe3+. The degree of stimulation was related to the affinity of a compound for Fe2+ with the stronger chelators causing greater stimulation. The most potent inhibitors of lipid peroxidation in the 21-aminosteroid series were found to be those compounds forming the weakest Fe2+ complexes. The findings suggest that it is iron at or near the membrane that is responsible for the catalysis of lipid peroxidation. The compounds described should provide useful tools for studies of the involvement of iron in the lipid peroxidation process.

Neuroprotective Properties of the Benzodiazepine Receptor, Partial Agonist PNU-101017 in the Gerbil Forebrain Ischemia Model

PNU-101017 is a novel, imidazoquinoline amide and benzodiazepine receptor partial agonist that has high affinity for the GABAA receptor subtypes containing the α1 and α3 or α5 subunits. At each of these receptors, the compound is a partial agonist with approximately 50% of the intrinsic activity of the full agonist diazepam. In view of the previously demonstrated anti-ischemic effects of some GABA agonists, the purpose of this study was to determine the ability of PNU-101017 to salvage selectively vulnerable neuronal populations in the gerbil forebrain ischemia model. In an initial set of experiments, male gerbils were pretreated 30 minutes before ischemia induction (5 minutes) with PNU-101017 (3, 10, or 30 mg/kg intraperitoneally) and again 2 hours after reperfusion. In vehicle (0.05 N HCl)-treated gerbils, the loss of hippocampal CA1 neurons at 5 days was 80%. PNU-101017 was shown to produce a dose-related increase in CA1 neuronal survival; at either 10 or 30 mg/kg, the loss of CA1 neurons was only 21% (P < 0.005 versus vehicle). A second experiment, examined the therapeutic window for PNU-101017 using the dose level of 30 mg/kg intraperitoneally. Administration of the first of two doses (2 hours apart) at the time of reperfusion resulted in an identical decrease in CA1 damage at 5 days to that seen with preischemic treatment (P < 0.003 versus vehicle). Even with a delay of the initial dosing until 4 hours after reperfusion, PNU-101017 reduced CA1 neuronal loss to only 32% (P < 0.01 versus vehicle). In a third experiment in which the duration of the ischemic insult was increased to 10 minutes and the brains were not analyzed until 28 days after ischemia, daily PNU-101017 dosing for the full 28 days still significantly preserved CA1 neurons, although less effectively than in the milder 5 minute-ischemia model. The loss of dopaminergic nigrostriatal neurons was also reduced. The neuroprotective effect of PNU-101017 was not associated with any overt CNS depression and it did not correlate with hypothermia. This benzodiazepine-receptor partial agonist may have potential for the treatment of global cerebral ischemia.

Characterization of U-101017 as a GABAA receptor ligand of dual functionality

Drugs acting on the benzodiazepine site of GABA(A) receptors are much safer than barbiturates, but are still liable to abuse. Recently, we have reported that a benzodiazepine site agonist, U-97775 (a dihydroimidazoquinoxaline analog), may have minimal abuse liability because of its interaction with a second, low-affinity site on GABA(A) receptors, the occupancy of which, at high drug concentrations, leads to a reversal of its agonistic activity on the benzodiazepine site and inhibition of GABA-induced Cl- currents [Br. J. Pharmacol. 115 (1995)19-24]. Here we report that U-101017 (7-chloro-5[(cis-3,5-dimethylpiperazine)carbonyl]imidazo[1,5a]quinoline- 3-carboxylate) is another similar benzodiazepine site agonist possessing the ability to reverse its agonistic activity at higher concentrations, but its ability to inhibit GABA currents is considerably milder than that of U-97775. In the alpha 6 beta 2 gamma 2 subtype where these drugs have no agonistic activity, for instance, U-101017 at concentrations up to 80 mu M, showed no appreciable effect on GABA currents, whereas U-97775 inhibited the currents with an IC(50) value of 10 mu M as measured with the whole cell patch clamp techniques in human embryonic kidney cells expressing recombinant receptors. Similar, milder inhibition of GABA currents by U-101017 was observed in the alpha 1 beta 2 gamma 2 and alpha 3 beta 2 gamma 2 subtypes. Furthermore, U-101017 was of higher efficacy in the alpha 1 beta 2 gamma 2 than alpha 3 beta 2 gamma 2 subtypes as compared to diazepam, although its binding affinity was not appreciably different in the two subtypes. We conclude that U-101017 is a partial benzodiazepine agonist, somewhat selective to the alpha 1 beta 2 gamma 2 subtype, and with the ability to limit its own agonistic activity over a wide range of doses through its interaction with the low affinity site, but without potential convulsant activity, inherent to agents which block GABA currents.

Characterization of U‐97775 as a GABAA receptor ligand of dual functionality in cloned rat GABAA receptor subtypes

1 U‐97775 (tert‐butyl 7‐chloro‐4,5‐dihydro‐5‐[(1‐(3,4,5‐trimethyl)piperazino)carbonyl]‐imidazo[1,5‐a])quinoxaline‐3‐carboxylate) is a novel GABAA receptor ligand of dual functionality and was characterized for its interactions with cloned rat GABAA receptors expressed in human embryonic kidney cells. 2 The drug produced a bell‐shaped dose‐response profile in the α1β2γ2 receptor subtype as monitored with GABA‐induced C1− currents in the whole cell patch‐clamp technique. At low concentrations (<0.5 μM), U‐97775 enhanced the currents with a maximal increase of 120% as normalized to 5 μM GABA response (control). An agonist interaction of U‐97775 with the benzodiazepine site is suggested, because Ro 15–1788 (an antagonist at the benzodiazepine site) abolished the current increase and [3H]‐flunitrazepam binding was inhibited by U‐97775 with a Ki of 1.2 nM. 3 The enhancement of GABA currents progressively disappeared as the U‐97775 concentration was raised above 1 μM, and the current amplitude was reduced to 40% below the control at 10 μM U‐97775. The current inhibition by U‐97775 (10 μM) was not affected by Ro 15–1788. It appears that U‐97775 interacts with a second site on GABA receptors, distinct from the benzodiazepine site, to reverse its agonistic activity on the benzodiazepine site and also to inhibit GABA currents. 4 U‐97775 at low concentrations reduced and at high concentrations enhanced [35S]‐TBPS binding. Ro 15–1788 selectively blocked the effect of U‐97775 at low concentrations. Analysis of the binding data in the presence of Ro 15–1788 yielded a single low affinity site with an estimated Kd of 407 nM. 5 In other αβγ receptor subtypes, U‐97775 at low concentrations enhanced C1− currents in the α3β2γ2 but not in the α6β2γ2 subtype. On the other hand, U‐97775 at high concentrations reduced C1− currents in all the receptor subtypes we examined, including those of two subunits, α1β2, β2γ2 and α1gamma;2 subtypes. 6 Therapeutically, U‐97775 could be unique among benzodiazepine ligands because of its ability to limit its own agonistic activity such that, at high doses the appearance of agonistic activity would be delayed until occupancy of its second site wanes. This property should make the total agonistic activity of U‐97775 relatively constant over a wide range of drug doses, and may minimize its liability to abuse.

Two imidazoquinoxaline ligands for the benzodiazepine site sharing a second low affinity site on rat GABAA receptors but with the opposite functionality

Imidazoquinoxaline PNU‐97775 and imidazoquinoline PNU‐101017 are benzodiazepine site ligands with a second low affinity binding site on GABAA receptors, the occupancy of which at high drug concentrations reverses their positive allosteric activity via the benzodiazepine site, and may potentially minimize abuse liability and physical dependence. In this study we discovered, with two imidazoquinoxaline analogues, that the functionality of the second site was altered by the nitrogen substituent on the piperazine ring moiety: PNU‐100076 with a hydrogen substituent on the position produced a negative allosteric effect via the second low affinity site, like the parent compounds, while PNU‐100079 with a trifluoroethyl substituent produced a positive allosteric response. These functional characteristics were monitored with Cl− currents measurements in cloned rat αxβ2γ2 subtypes of GABAA receptors expressed in human embryonic kidney 293 cells, and further confirmed in rat cerebrocortical membranes containing complex subtypes of GABAA receptors with binding of [35S]‐TBPS, which is a high affinity ligand specific for GABAA receptors with exquisite sensitivity to allosteric modulations. This structure‐functional relationship could be exploited to further our understanding of the second allosteric site of imidazoquinoxaline analogues, and to develop more effective benzodiazepine site ligands without typical side effects associated with those currently available on the market.

Asymmetric synthesis of (2R,6R) and (2S,6S)-2,6-dimethylpiperazine

Abstract The title compounds were prepared via two efficient routes. The first sequence utilized a diastereospecific triflate alkylation in the key bond forming step while the second method relied on a novel intramolecular Mitsunobu reaction to set the required stereochemistry.

Chapter 4. Acute Ischemic and Traumatic injury to the CNS

Publisher Summary Ischemic and traumatic injuries include post-traumatic injury (head and spinal cord), focal ischemia, focal ischemia with reperfusion (stroke), global ischemia (CPR), hemorrhagic injury (subarachnoid hemorrhage), and radiation injuries. Although ischemia itself can cause irreversible injury, reperfusion of the ischemic tissue is particularly damaging, perhaps because of iron release, mitochondrial leakage with superoxide anion formation, and increases in xanthine oxidase or neutrophil infiltration. Traumatic injury can lead to free radical damage because of local ischemia or iron release from the damaged cells or petechial hemorrhage. Although the initial events vary, the secondary, progressive processes that lead eventually to cell death and neurologic injury overlap for these diverse maladies. The mechanisms and agents through which these injuries develop include: lipid peroxidation, calcium influx, excitatory amino acids, and arachidonic acid metabolites. A variety of therapeutic interventions are being actively investigated because of the desperate clinical need for new agents. For example, inhibitors of lipid peroxidation, iron chelators, superoxide dismutase, calcium channel antagonists, magnesium, excitatory amino acid antagonists, prostanoid modulators, adenosine agonists, opioids, diuretics, mitochondrial stimulants, monogangliosides, anti-cholinergics, and hypothermics have all been evaluated in animal models of ischemia and trauma. This chapter describes such studies.