Structural and thermodynamic characterization of the TYK2 and JAK3 kinase domains in complex with CP-690550 and CMP-6.

Janus kinases (JAKs) are critical regulators of cytokine pathways and attractive targets of therapeutic value in both inflammatory and myeloproliferative diseases. Although the crystal structures of active JAK1 and JAK2 kinase domains have been reported recently with the clinical compound CP-690550, the structures of both TYK2 and JAK3 with CP-690550 have remained outstanding. Here, we report the crystal structures of TYK2, a first in class structure, and JAK3 in complex with PAN-JAK inhibitors CP-690550 ((3R,4R)-3-[4-methyl-3-[N-methyl-N-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino]piperidin-1-yl]-3-oxopropionitrile) and CMP-6 (tetracyclic pyridone 2-t-butyl-9-fluoro-3,6-dihydro-7H-benz[h]-imidaz[4,5-f]isoquinoline-7-one), both of which bind in the ATP-binding cavities of both JAK isozymes in orientations similar to that observed in crystal structures of JAK1 and JAK2. Additionally, a complete thermodynamic characterization of JAK/CP-690550 complex formation was completed by isothermal titration calorimetry, indicating the critical role of the nitrile group from the CP-690550 compound. Finally, computational analysis using WaterMap further highlights the critical positioning of the CP-690550 nitrile group in the displacement of an unfavorable water molecule beneath the glycine-rich loop. Taken together, the data emphasize the outstanding properties of the kinome-selective JAK inhibitor CP-690550, as well as the challenges in obtaining JAK isozyme-selective inhibitors due to the overall structural and sequence similarities between the TYK2, JAK1, JAK2 and JAK3 isozymes. Nevertheless, subtle amino acid variations of residues lining the ligand-binding cavity of the JAK enzymes, as well as the global positioning of the glycine-rich loop, might provide the initial clues to obtaining JAK-isozyme selective inhibitors.

1-(2-Ethoxyethyl)-1H-pyrazolo[4,3-d]pyrimidines as potent phosphodiesterase 5 (PDE5) inhibitors.

1H-Pyrazolo[4,3-d]pyrimidines are a class of potent and selective second generation phosphodiesterase 5 (PDE5) inhibitors. This work explores the potency, selectivity and efficacy of 1-(2-ethoxyethyl)-1H-pyrazolo[4,5-d]pyrimidines as PDE5 inhibitors resulting in the advancement of a clinical candidate.

1-(2-(2,2,2-trifluoroethoxy)ethyl-1H-pyrazolo[4,3-d]pyrimidines as potent phosphodiesterase 5 (PDE5) inhibitors.

1H-Pyrazolo[4,3-d]pyrimidines were previously disclosed as a potent second generation class of phosphodiesterase 5 (PDE5) inhibitors. This work explores the advancement of more selective and potent PDE5 inhibitors resulting from the substitution of 2-(2,2,2-trifluoroethoxy)ethyl at the 1 position in the so-called alkoxy pocket.

Synthesis of a series of stromelysin-selective thiadiazole urea matrix metalloproteinase inhibitors.

The synthesis and enzyme inhibition data for a series of thiadiazole urea matrix metalloproteinase (MMP) inhibitors are described. A broad screening effort was utilized to identify several thiadiazoles which were weak inhibitors of stromelysin. Optimization of the thiadiazole leads to include an alpha-amino acid side chain with variable terminal amide substituents provided a series of ureas which were moderately effective stromelysin inhibitors, with Ki's between 0.3 and 1.0 microM. The most effective analogues utilized an L-phenylalanine as the amino acid component. In particular, unsubstituted 46 had a Ki of 710 nM, while the p-fluoro analogue 52 displayed increased potency (100 nM). Stromelysin inhibition was further improved using a pentafluorophenylalanine substituent which resulted in 70, a 14 nM inhibitor. While gelatinase inhibition was generally poor, the use of 1-(2-pyridyl)piperazine as the amide component usually provided for enhanced activity, with 71 inhibiting gelatinase with a Ki of 770 nM. The combination of this heterocycle with a p-fluorophenylalanine substituent provided the only analogue, 69, with collagenase activity (13 microM). The SAR for analogues described within this series can be rationalized through consideration of the X-ray structure recently attained for70 complexed to stromelysin. Uniquely, this structure showed the inhibitor to be completely orientated on the left side of the enzyme cleft. These results suggest that thiadiazole urea heterocycles which incorporate a substituted phenylalanine can provide selective inhibitors of stromelysin. Careful selection of the amide substituent can also provide for analogues with modest gelatinase inhibition.

Piperazine imidazo[1,5-a]quinoxaline ureas as high-affinity GABAA ligands of dual functionality.

A series of imidazo[1,5-a]quinoxaline piperazine ureas appended with a tert-butyl ester side chain at the 3-position was developed. Analogues within this series have high affinity for the gamma-aminobutyric acid A (GABAA)/benzodiazepine receptor complex with efficacies ranging from inverse agonists to full agonists. Many analogues were found to be partial agonists as indicated by [35S]TBPS and Cl- current ratios. Uniquely, a number of these analogues were found to have a bell-shaped dose-response profile in the alpha1 beta2 gamma2 subtype as determined by whole cell patch-clamp technique, where in vitro efficacy was found to decrease with increasing drug concentration. Many of the compounds from this series were effective in antagonizing metrazole-induced seizures, consistent with anticonvulsant and possibly anxiolytic activity. Additionally, several analogues were also effective in lowering cGMP levels (to control values) after applied stress, also consistent with anxiolytic-like properties. The most effective compounds in these screens were also active in animal models of anxiety such as the Vogel and Geller assays. The use of the piperazine substituent allowed for excellent drug levels and a long duration of action in the central nervous system for many of the quinoxalines, as determined by ex vivo assay. Pharmacokinetic analysis of several compounds indicated excellent oral bioavailability and a reasonable half-life in rats. From this series emerged two partial agonists (55, 91) which had good activity in anxiolytic models, acceptable pharmacokinetics, and minimal benzodiazepine-type side effects.

Solution structures of stromelysin complexed to thiadiazole inhibitors.

Unregulated or overexpressed matrix metalloproteinases (MMPs), including stromelysin, collagenase, and gelatinase. have been implicated in several pathological conditions including arthritis and cancer. Small-molecule MMP inhibitors may have therapeutic value in the treatment of these diseases. In this regard, the solution structures of two stromelysin/ inhibitor complexes have been investigated using 1H, 13C, and 15N NMR spectroscopy. Both-inhibitors are members of a novel class of matrix metalloproteinase inhibitor that contain a thiadiazole group and that interact with stromelysin in a manner distinct from other classes of inhibitors. The inhibitors coordinate the catalytic zinc atom through their exocyclic sulfur atom, with the remainder of the ligand extending into the S1-S3 side of the active site. The binding of inhibitor containing a protonated or fluorinated aromatic ring was investigated using 1H and 19F NMR spectroscopy. The fluorinated ring was found to have a reduced ring-flip rate compared to the protonated version. A strong, coplanar interaction between the fluorinated ring of the inhibitor and the aromatic ring of Tyr155 is proposed to account for the reduced ring-flip rate and for the increase in binding affinity observed for the fluorinated inhibitor compared to the protonated inhibitor. Binding interactions observed for the thiadiazole class of ligands have implications for the design of matrix metalloproteinase inhibitors.

Two imidazoquinoxaline ligands for the benzodiazepine site sharing a second low affinity site on rat GABA(A) receptors but with the opposite functionality.

1. Imidazoquinoxaline PNU-97775 and imidazoquinoline PNU-101017 are benzodiazepine site ligands with a second low affinity binding site on GABA(A) 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. 2. 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. 3. These functional characteristics were monitored with Cl- currents measurements in cloned rat alphaxbeta2gamma2 subtypes of GABA(A) receptors expressed in human embryonic kidney 293 cells, and further confirmed in rat cerebrocortical membranes containing complex subtypes of GABA(A) receptors with binding of [35S]-TBPS, which is a high affinity ligand specific for GABA(A) receptors with exquisite sensitivity to allosteric modulations. 4. 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.

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 alpha 1 and alpha 3 or alpha 5 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 HC1)-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.

Anxiolytic-like effects of PNU-101017, a partial agonist at the benzodiazepine receptor.

PNU-101017 is a chemically novel ligand at the benzodiazepine recognition site of cloned GABAA receptors. It was reported to potentiate GABA-mediated chloride current in cultured cells with a moderate intrinsic activity and a biphasic dose-response relationship. In this study, we confirmed that PNU-101017 has a partial agonist-like effect in the antagonism of metrazole-induced seizures in mice. It produced no sedation or ataxia, but did antagonize diazepam-induced motor deficit of mice in the rotarod test. PNU-101017 was weakly active in anti-conflict anxiolytic tests, but attenuated the plasma corticosteroid response to mild stress in rats. It also antagonized stress-induced elevation of cerebellar cGMP levels in mice. Like chlordiazepoxide, it increased drinking of saline solution in thirsty rats. PNU-101017 did not potentiate the CNS-depressant effects of ethanol, and produced no evidence of physical dependence when administered repeatedly. Agonists with low intrinsic activity at the benzodiazepine receptor, such as PNU-101017, should be further explored for therapeutic uses.

High-affinity alpha-aminobutyric acid A/benzodiazepine ligands: synthesis and structure-activity relationship studies of a new series of tetracyclic imidazoquinoxalines.

A series of tetracyclic imidazoquinoxaline analogs was developed which constrain the carbonyl group of the partial agonist 3-(5-cyclopropyl-1,2,4-oxadiazol-3-yl)-5-[(dimethylamino)carbonyl] - 4,5-dihydroimidazo[1,5-alpha]quinoxaline (2, U-91571) away from the benzene ring. These analogs orient the carbonyl group in the opposite direction of the previously reported full agonist 1-(5- cyclopropyl-1,2,4-oxadiazol-3-yl)-12,12a-dihydroimidazo[1,5- alpha]pyrrolo [2,1-c]quinoxalin-10(11H)-one (3, U-89267). A number of approaches were utilized to form the "bottom" ring of this tetracyclic ring system including intramolecular cyclizations promoted by Lewis acids or base, as well as metal-carbenoid conditions. The size and substitution pattern of the additional ring was widely varied. Analogs within this series had high affinity for the benzodiazepine receptor on the alpha-aminobutyric acid A chloride ion channel complex. From TBPS shift and Cl- current assays, the in vitro efficacy of compounds within this class ranged from antagonists to partial agonists with only 18a identified as a full agonist. Additionally, several analogs were quite potent at antagonizing metrazole-induced seizures indicating possible anticonvulsant or anxiolytic activity. Unlike 3, analogs in this series did not have high affinity for the diazepam insensitive alpha 6 beta 2 delta 2 subtype. These results suggest that either constraining the carbonyl group away from the benzene ring or the greater planarity that results from the additional cyclic structure provides analogs with partial agonist properties and prevents effective interaction with the alpha 6 beta 2 delta 2 subtype.

3-Phenyl-substituted imidazo[1,5-alpha]quinoxalin-4-ones and imidazo[1,5-alpha]quinoxaline ureas that have high affinity at the GABAA/benzodiazepine receptor complex.

A series of imidazo[1,5-alpha]quinoxalin-4-ones and imidazo[1,5-alpha]quinoxaline ureas containing substituted phenyl groups at the 3-position was developed. Compounds within the imidazo[1,5-alpha]quinoxaline urea series had high affinity for the GABAA/benzodiazepine receptor complex with varying in vitro efficacy, although most analogs were partial agonists as indicated by [35S]TBPS and Cl- current ratios. Interestingly, a subseries of piperazine ureas was identified which had biphasic efficacy, becoming more antagonistic with increasing concentration. Analogs within the imidazo[1,5-alpha]quinoxalin-4-one series had substantially decreased binding affinity as compared to the quinoxaline urea series. These compounds ranged from antagonists to full agonists by in vitro analysis, with several derivatives having roughly 4-fold greater intrinsic activity than diazepam as indicated by Cl- current measurement. Numerous compounds from both series were effective in antagonizing metrazole-induced seizures, consistent with anti-convulsant properties and possible anxiolytic activity. Most of the quinoxaline ureas and quinoxalin-4-ones were active in an acute electroshock physical dependence side effect assay in mice precluding further development.

Characterization of U-101017 as a GABA(A) 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.

High-affinity partial agonist imidazo[1,5-a]quinoxaline amides, carbamates, and ureas at the gamma-aminobutyric acid A/benzodiazepine receptor complex.

A series of imidazo[1,5-a]quinoxaline amides, carbamates, and ureas which have high affinity for the gamma-aminobutyric acid A/benzodiazepine receptor complex was developed. Compounds within this class have varying efficacies ranging from antagonists to full agonists. However, most analogs were found to be partial agonists as indicated by [35S]TBPS and Cl- current ratios. Many of these compounds were also effective in antagonizing metrazole-induced seizures in accordance with anticonvulsant and possible anxiolytic activity. Selected quinoxalines displayed limited benzodiazepine-type side effects such as ethanol potentiation and physical dependence in animal models. Dimethylamino urea 41 emerged as the most interesting analog, having a partial agonist profile in vitro while possessing useful activity in animal models of anxiety such as the Vogel and Geller assays. In accordance with its partial agonist profile, 41 was devoid of typical benzodiazepine side effects.

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 alpha 1 beta 2 gamma 2 receptor subtype as monitored with GABA-induced Cl- currents in the whole cell patch-clamp technique. At low concentrations (< 0.5 microM), U-97775 enhanced the currents with a maximal increase of 120% as normalized to 5 microM 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 microM, and the current amplitude was reduced to 40% below the control at 10 microM U-97775. The current inhibition by U-97775 (10 microM) 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 alpha beta upsilon receptor subtypes, U-97775 at low concentrations enhanced Cl- currents in the alpha 3 beta 2 upsilon 2,but not in the alpha 6 beta 2 upsilon 2 subtype. On the other hand, U-97775 at high concentrations reduced Cl- currents in all the receptor subtypes we examined, including those of two subunits, alpha 1 beta 2, beta 2 upsilon 2 and alpha 1 upsilon 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.

Differential affinity of dihydroimidazoquinoxalines and diimidazoquinazolines to the alpha 1 beta 2 gamma 2 and alpha 6 beta 2 gamma 2 subtypes of cloned GABAA receptors.

1. In this study, we compared two series of newly discovered ligands for their selectivity to benzodiazepine sites in the alpha 1 beta 2 gamma 2 and the alpha 6 beta 2 gamma 2 subtypes of cloned gamma-aminobutyric acidA (GABAA) receptors, the latter being unique in not interacting with classical benzodiazepines. 2. The prototype compounds, U-85575 (12-chloro-5-(5-cyclopropyl-1',2',4'- oxadiazol-3'-yl)-2,3-dihydro-diimidazo [1,5-a;1,2-c]quinazoline), and U-92330 (5-acetyl-3-(5'-cyclopropyl-1',2',4'-oxadiazole-3'-yl)-7-chloro-4,5-d ihy dro [1,5-a]quinoxaline), appear to share an overlapping recognition site with classical benzodiazepines on the GABAA receptor, because their potentiation of GABA-mediated Cl- currents in both subtypes were sensitive to Ro 15-1788, a classical benzodiazepine antagonist. 3. Minor changes in the ring substituents of the drugs reduced their affinity to the alpha 6 beta 2 gamma 2 subtype more pronouncedly than to the alpha 1 beta 2 gamma 2 subtype. The diimidazoquinazoline containing a 2-methyl group which projected below the plane of the rigid ring showed a markedly lower affinity to the alpha 6 beta 2 gamma 2 subtype as compared to its stereoisomer having the methyl group above the plane of the ring. Also, the dihydroimidazoquinoxalines containing the 5-benzoyl group showed a lower affinity to the alpha 6 beta 2 gamma 2 subtype than the 5-acetyl counterpart. In particular, the 5-benzoyl analogue containing a 6-fluoro group showed no interaction with the alpha 6 beta 2 gamma 2 subtype even at the concentration of 10 microM, probably due to stabilization of the benzoyl group in the out-of-plane region by the steric and electrostatic effects of the 6-fluoro group.4. We propose that the benzodiazepine site of the alpha 6 beta 2 gamma 2 subtype shares overlapping regions with that of the alpha 1 beta 2 gamma 2 subtype, but has a sterically restricted out-of-plane region, which may be also incompatible with the 5-phenyl group of classical benzodiazepines.

Effect of the 21-aminosteroid, U-74389F, on hyperoxic lung injury in rats.

Hyperoxia (> 95% oxygen) in rats caused an increase in lung weight and an accumulation of fluid in the thorax. The mean lung wet weight of air-breathing controls at 60 h was 1.2 +/- 0.01 g, and that of vehicle-treated, oxygen-exposed animals was 2.45 +/- 0.05 g. Treatment with the 21-aminosteroid U-74389F, 3, 10, and 30 mg/kg twice daily throughout oxygen exposure, produced 8, 42, and 18% inhibition of the oxygen-induced increase in lung weight, respectively. However, U-74389F did not inhibit the hyperoxia-induced accumulation of neutrophils in bronchoalveolar lavage fluid. No pleural fluid could be aspirated from the thorax of air-breathing controls. The volume of pleural fluid in oxygen-exposed, vehicle-treated animals and animals treated with 3, 10, and 30 mg/kg U-74389F b.i.d. was 6.5 +/- 0.9, 2.6 +/- 0.6, 0.8 +/- 0.3, and 1.3 +/- 0.5 ml, respectively. U-74389F or its biologs are of potential value for the treatment of lung diseases in which oxidant damage has been implicated.

2-(Aminomethyl)chromans that inhibit iron-dependent lipid peroxidation and protect against central nervous system trauma and ischemia.

A series of 2-(aminomethyl)chromans was developed as potent inhibitors of iron-dependent lipid peroxidation. Compounds within this class are extremely effective at inhibiting lipid peroxidation with IC50's as low as 0.2 microM. Selected members were found to enhance early neurological recovery and survival in a mouse head injury model. In this assay, improvement in the 1-h post-head-injury neurological status (grip test score) by as much as 230% of control was observed. One of the most efficacious compounds (35) was evaluated in two models of cerebral ischemia where significant neuroprotection was observed. These results provide further support for the importance of cerebroprotective antioxidants for the treatment of traumatic and ischemic injury as well as additional evidence for the role of oxygen radicals in postischemic brain damage.

Characterization of functional interactions of imidazoquinoxaline derivatives with benzodiazepine-gamma-aminobutyric acidA receptors.

U-78875 [imidazo[1,5-a]quinoxalin-4(5H)-one, 3-(5-cyclopropyl-1,2,4-oxadiazol-3-yl)-5-(1-methylethyl)] belongs to a series of imidazoquinoxaline derivatives, recently discovered ligands with high affinity for benzodiazepine receptors. In this study, we have examined the drug and its analogs for their modes of interaction with the receptors, with a particular emphasis on finding molecular determinants for their functional properties. Changes in the substituents on N5 and C6 of the heterocyclic ring produced no major effects on binding characteristics but yielded drugs of widely varying efficacy (antagonist to full agonist), measured as gamma-aminobutyric acid (GABA)-mediated 36Cl- uptake and t-butylbicyclophosphoro[35S]thionate binding in rat cerebrocortical membranes. The relative binding affinity and efficacy of the analogs measured in brain membranes were similar to those in cloned GABAA receptors of the alpha 1 beta 2 gamma 2 (type I) and alpha 3 beta 2 gamma 2 (type II) subtypes. The imidazoquinoxalines showed no marked subtype selectivity. Their Ki value against [3H]flunitrazepam binding for type I was only 2-3 times lower than that for type II, and their rank order for agonistic activity was the same in the two subtypes, measured as GABA-mediated Cl- currents in human kidney cells (A293) expressing the subtypes of GABAA receptors. According to computational modeling of the drugs using both molecular and quantum mechanics, the agonistic activity of the imidazoquinoxaline derivatives depends on the presence of a bulky alkyl substituent at N5 and the deformation of the substituted portion of the otherwise planar ring system induced by a bulky moiety at N5 or C6. With a fixed N5 substituent (isopropyl), the relative efficacy in the brain membranes, as well as in the cloned receptors, appeared to be dependent on the degree of the ring deformation. This out-of-plane portion of the imidazoquinoxalines can be assigned to the general region occupied by the 5-phenyl group of diazepam and other agonistic functional groups of several nonbenzodiazepine ligands. It seems that this region, apparently common to various agonistic ligands, interacts with an agonistic pocket in type I and type II subtypes of the benzodiazepine receptors in the brain. Our results also provide direct support for the view that the agonists and nonagonists share largely overlapping binding regions in the benzodiazepine receptor, which has been proposed earlier from in vivo efficacy measurements of other series of ligands.

U-78517F: a potent inhibitor of lipid peroxidation with activity in experimental brain injury and ischemia.

U-78517F (2-[4-[2,6-di-(1-pyrrolidinyl)-4-pyridinyl)-1-piperazinyl] methyl]-3,4-dihydro-2,5,7,8-tetramethyl-2H-1-benzopyran-6-ol, dihydrochloride), which combines the antioxidant ring portion of alpha-tocopherol together with the amine of the previously described 21-aminosteroids (e.g., U-74006F), is a novel inhibitor of iron-catalyzed lipid peroxidation. U-78517F was found to have a 50% inhibitory concentration (IC50) of 0.6 microM against 200 microM ferrous chloride-initiated lipid peroxidation in rat brain homogenates, compared to 8 microM for U-74006F, 28 microM for alpha-tocopherol and 43 microM for the ring portion of alpha-tocopherol (i.e., trolox). Both stereoisomers of the racemic U-78517F proved to be equally active antioxidants. Against lipid peroxidation initiated by xanthine/xanthine oxidase, U-78517F was even more potent, with an IC50 of 0.01 microM. U-78517F was also observed to protect cultured mouse spinal neurons against iron-induced damage, with an IC50 of approximately 0.5 microM. When administered to male CF-1 mice i.v. at 5 min after a severe concussive head injury. U-78517F produced a dose-related improvement in the 1-hr neurological recovery. The minimum effective i.v. dose was 1.0 micrograms/kg. Measurement of U-78517 concentrations in the brains of mice after administration of a 10-mg/kg i.v. dose revealed effective antioxidant levels for as long as 2 hr. Evidence of an in vivo antioxidant action was provided by the attenuation of iron-induced blood-brain barrier disruption (i.e., Evans' blue extravasation) in rats pretreated with U-78517F.(ABSTRACT TRUNCATED AT 250 WORDS)

Novel inhibitors of pulmonary eosinophil accumulation.

The effects of representatives of three classes of compounds were investigated on antigen-induced bronchopulmonary eosinophilia in sensitized Brown-Norway rats. Rats were sensitized by 3 weekly inhalation provocations with aerosols of ovalbumin. Twenty-four hours after a fourth weekly antigen provocation, cell populations were enumerated following bronchoalveolar lavage (BAL) in animals treated with test compounds or the appropriate vehicle. A marked eosinophil-rich influx of inflammatory cells into the bronchial lumen followed the antigen provocation in sensitized animals. Dose-related inhibitions of antigen-induced lung eosinophilia were demonstrated with: 1) glucocorticoids, given po (methylprednisolone acetate, U-8210) or by inhalation (methylprednisolone suleptanate, U-67590A); 2)the non-glucocorticoid 21-amino steroid, U-75412E, and 3) the leukotriene B4 antagonist, U-75302. The steroids methylprednisolone and U-75412E were tested for glucocorticoid activity using phorbol ester-differentiated U937 (human macrophage) cells. Methylprednisolone but not U-75412E produced a dose-dependent inhibition of lipopolysaccharide-stimulated thromboxane synthesis by the U937 cells. Leukotriene B4 antagonists and the novel 21-aminosteroid, U-75412E, which lacks glucorticoid activity, provide leads for the development of compounds which inhibit the chronic airway inflammation associated with asthma in man.