Synthesis and SAR of 5-, 6-, 7- and 8-aza analogues of 3-aryl-4-hydroxyquinolin-2(1H)-one as NMDA/glycine site antagonists.

A series of 5-, 6-, 7- and 8-aza analogues of 3-aryl-4-hydroxyquinolin-2(1H)-one was synthesized and assayed as NMDA/glycine receptor antagonists. The in vitro potency of these antagonists was determined by displacement of the glycine site radioligand [(3)H]5,7-dicholorokynurenic acid ([(3)H]DCKA) in rat brain cortical membranes. Selected compounds were also tested for functional antagonism using electrophysiological assays in Xenopus oocytes expressing cloned NMDA receptor (NR) 1A/2C subunits. Among the 5-, 6-, 7-, and 8-aza-3-aryl-4-hydroxyquinoline-2(1H)-ones investigated, 5-aza-7-chloro-4-hydroxy-3-(3-phenoxyphenyl)quinolin-2-(1H)-one (13i) is the most potent antagonist, having an IC(50) value of 110 nM in [(3)H]DCKA binding and a K(b) of 11 nM in the electrophysiology assay. Compound 13i is also an active anticonvulsant when administered systemically in the mouse maximum electroshock-induced seizure test (ED(50)=2.3mg/kg, IP).

Characterization of the anxiolytic properties of a novel neuroactive steroid, Co 2-6749 (GMA-839; WAY-141839; 3alpha, 21-dihydroxy-3beta-trifluoromethyl-19-nor-5beta-pregnan-20-one), a selective modulator of gamma-aminobutyric acid(A) receptors.

The purpose of this study was to evaluate the effects of a novel neuroactive steroid, Co 2-6749 (GMA-839; WAY-141839; 3alpha, 21-dihydroxy-3beta-trifluoromethyl-19-nor-5beta-pregnan-20-one), on gamma-aminobutyric acid(A) receptors in vitro and to define its anxiolytic-like effects and side effect profile in vivo. Co 2-6749 fully inhibited [(35)S]t-butylbicyclophosphorothionate binding in rat brain cortical membranes with an IC(50) value of 230 nM and in human gamma-aminobutyric acid(A) receptor subunit combinations of alpha1beta2gamma2L, alpha2beta2gamma2L, alpha3beta2gamma2L, alpha4beta3gamma2L, alpha5beta2gamma2L, and alpha6beta3gamma2L receptors (IC(50) values of 200, 200, 96, 2300, 210, and 2000 nM). Rats were trained in a Geller-Seifter operant conflict paradigm. Co 2-6749 caused a dose-related increase in punished responding with a minimum effective dose of 1.6 mg/kg, p.o., a wide therapeutic index relative to a decrease in unpunished responding and relative to ataxia, and no tolerance. Additionally, ethanol caused less than a 2-fold shift to the left in the dose-response function of Co 2-6749 in the rotorod procedure in rats. In a pigeon conflict paradigm, punished responding was maximally increased to 784% of vehicle control by 30 mg/kg, p.o., with a 2-h duration and no effect on unpunished responding at this dose. Similarly, punished responding in squirrel monkeys was maximally increased to 1774% of control by 10 mg/kg, p.o., with no effect on unpunished responding at this dose. With robust anxiolytic-like activity across species, a large separation between anxiolytic-like effects and sedation/ataxia, a minimal interaction with ethanol, a lack of tolerance, and apparent oral bioavailability, Co 2-6749 makes an ideal candidate for development as a novel anxiolytic drug.

Synthesis of N-substituted 4-(4-hydroxyphenyl)piperidines, 4-(4-hydroxybenzyl)piperidines, and (+/-)-3-(4-hydroxyphenyl)pyrrolidines: selective antagonists at the 1A/2B NMDA receptor subtype.

Antagonists at the 1A/2B subtype of the NMDA receptor (NR1A/2B) are typically small molecules that consist of a 4-benzyl- or a 4-phenylpiperidine with an omega-phenylalkyl substituent on the heterocyclic nitrogen. Many of these antagonists, for example ifenprodil (1), incorporate a 4-hydroxy substituent on the omega-phenyl group. In this study, the position of this 4-hydroxy substituent was transferred from the omega-phenyl group to the benzyl or phenyl group located on the 4-position of the piperidine ring. Analogues incorporating pyrrolidine in lieu of piperidine were also prepared. Electrical recordings using cloned receptors expressed in Xenopus oocytes show that high-potency antagonists at the NR1A/2B subtype are obtained employing N-(omega-phenylalkyl)-substituted 4-(4-hydroxyphenyl)piperidine, 4-(4-hydroxybenzyl)piperidine, and (+/-)-3-(4-hydroxyphenyl)pyrrolidine as exemplified by 21 (IC(50) = 0.022 microM), 33 (IC(50) = 0.059 microM), and 40 (IC(50) = 0.017 microM), respectively. These high-potency antagonists are >1000 times more potent at the NR1A/2B subtype than at either the NR1A/2A or NR1A/2C subtypes. The binding affinities of 21 at alpha(1)-adrenergic receptors ([(3)H]prazosin, IC(50) = 0.54 microM) and dopamine D2 receptors ([(3)H]raclopride, IC(50) = 1.2 microM) are reduced by incorporating a hydroxy group onto the 4-position of the piperidine ring and the beta-carbon of the N-alkyl spacer to give (+/-)-27: IC(50) NR1A/2B, 0.026; alpha(1), 14; D2, 105 microM. The high-potency phenolic antagonist 21 and its low-potency O-methylated analogue 18 are both potent anticonvulsants in a mouse maximal electroshock-induced seizure (MES) study (ED(50) (iv) = 0.23 and 0.56 mg/kg, respectively). These data indicate that such compounds penetrate the blood-brain barrier but their MES activity may not be related to NMDA receptor antagonism.

Opioid activity profiles indicate similarities between the nociceptin/orphanin FQ and opioid receptors.

Nociceptin (orphanin FQ) is the recently discovered peptide agonist for the orphan receptor opioid receptor-like 1 (ORL1). Despite the high sequence homology between ORL1 and the opioid receptors, most opioids lack affinity for the nociceptin receptor. The affinity and functional profile of opioids possessing activity at the nociceptin receptor was determined using [3H]nociceptin and nociceptin-stimulated [35S]GTPgammaS binding. The mu-opioid receptor-selective agonist lofentanil potently and competitively displaced [3H]nociceptin at rat brain receptors (IC(50) 62 nM). Lofentanil exhibited full agonism for enhancement of [35S]GTPgammaS binding to human recombinant ORL1 receptors (EC(50) 50 nM). The related piperidines ohmefentanyl and sufentanil and the nonselective opioid receptor agonist etorphine were less potent nociceptin receptor agonists. The kappa(1)+kappa(3)-opioid receptor agonist/mu-opioid receptor antagonist naloxone benzoylhydrazone was a pure antagonist at both rat brain and human ORL1 receptors. The nonselective opioid receptor partial agonist buprenorphine and the nonselective opioid receptor antagonist (-)-quadazocine exhibited pure antagonism at rat brain receptors, but displayed partial agonism at human ORL1 receptors. Thus, opioids displaying full agonism at the nociceptin receptor are also opioid receptor agonists, whereas opioids that are antagonists or partial agonists at the nociceptin receptor show antagonism or partial agonism at opioid receptors. In addition, the stereospecificity required at opioid receptors appears to be retained at the nociceptin receptor, since (+)-quadazocine is inactive at both receptors. These findings illustrate the structural and functional homology of the opioid recognition site on these two receptor classes and suggest that opioids may provide leads for the design of nonpeptide nociceptin receptor agonists and antagonists lacking affinity for the classical opioid receptors.

4-Hydroxy-1-[2-(4-hydroxyphenoxy)ethyl]-4-(4-methylbenzyl)piperidine: a novel, potent, and selective NR1/2B NMDA receptor antagonist.

A structure-based search and screen of our compound library identified N-(2-phenoxyethyl)-4-benzylpiperidine (8) as a novel N-methyl-D-aspartate (NMDA) receptor antagonist that has high selectivity for the NR1/2B subunit combination (IC(50) = 0.63 microM). We report on the optimization of this lead compound in terms of potency, side effect liability, and in vivo activity. Potency was assayed by electrical recordings in Xenopus oocytes expressing cloned rat NMDA receptors. Side effect liability was assessed by measuring affinity for alpha(1)-adrenergic receptors and inhibition of neuronal K(+) channels. Central bioavailability was gauged indirectly by determining anticonvulsant activity in a mouse maximal electroshock (MES) assay. Making progressive modifications to 8, a hydroxyl substituent on the phenyl ring para to the oxyethyl tether (10a) resulted in a approximately 25-fold increase in NR1A/2B potency (IC(50) = 0.025 microM). p-Methyl substitution on the benzyl ring (10b) produced a approximately 3-fold increase in MES activity (ED(50) = 0.7 mg/kg iv). Introduction of a second hydroxyl group into the C-4 position on the piperidine ring (10e) resulted in a substantial decrease in affinity for alpha(1) receptors and reduction in inhibition of K(+) channels with only a modest decrease in NR1A/2B and MES potencies. Among the compounds described, 10e (4-hydroxy-N-[2-(4-hydroxyphenoxy)ethyl]-4-(4-methylbenzyl)piperid ine, Co 101244/PD 174494) had the optimum pharmacological profile and was selected for further biological evaluation.

Subtype-selective N-methyl-D-aspartate receptor antagonists: synthesis and biological evaluation of 1-(arylalkynyl)-4-benzylpiperidines.

A search of our compound library for compounds with structural similarity to ifenprodil (5) and haloperidol (7) followed by in vitro screening revealed that 4-benzyl-1-(4-phenyl-3-butynyl)piperidine (8) was a moderately potent and selective antagonist of the NR1A/2B subtype of NMDA receptors. Substitution on the benzyl group of 8 did not significantly affect NR1A/2B potency, while addition of hydrogen bond donors in the para position of the phenyl group enhanced NR1A/2B potency. Addition of a hydroxyl moiety to the 4-position of the piperidine group slightly reduced NR1A/2B potency while reducing alpha-1 adrenergic and dopamine D2 receptor binding affinities substantially, resulting in improved overall selectivity for NR1A/2B receptors. Finally, the butynyl linker was replaced with propynyl or pentynyl. When the phenyl was para substituted with amine or acetamide groups, the NR1A/2B potency order was butynyl > pentynyl >> propynyl. For the para methanesulfonamide or hydroxyl groups, the order was butynyl approximately propynyl > pentynyl. The hydroxyl propyne (48) and butyne (23) were among the most potent NR1A/2B antagonists from this study. They both potentiated the effects of L-DOPA in the 6-hydroxydopamine-lesioned rat, a model of Parkinson's disease, dosed at 10 mg/kg ip, but 48 was not active at 30 mg/kg po.

Substituted 3beta-phenylethynyl derivatives of 3alpha-hydroxy-5alpha-pregnan-20-one: remarkably potent neuroactive steroid modulators of gamma-aminobutyric acidA receptors.

Neuroactive steroids are positive allosteric modulators of gamma-aminobutyric acidA (GABAA) receptor complexes. Synthetic modification generally does not increase neuroactive steroid potency beyond that of the naturally occurring progesterone metabolite, 3alpha-hydroxy-5alpha-pregnan-20-one (3alpha,5alpha-P). Recently, it has been shown that introduction of appropriately para-substituted phenylethynyl groups at the 3beta-position of 5beta steroids increases receptor potency. The present report presents the synthesis and pharmacological profile of an analogous series of 5alpha steroids. The most striking feature of this series is the further enhancement of in vitro and in vivo potency obtained. In particular, 3beta-(p-acetylphenylethynyl)-3alpha-hydroxy-5alpha-pr egnan-20-one (Co 152791) was 11-, 16- and 49-fold more potent than 3alpha, 5alpha-P in modulating the binding of [35S]TBPS, [3H]flunitrazepam and [3H]muscimol, respectively, in rat brain membranes (Co 152791 IC50 or EC50 = 2-7.5 nM). Similarly, Co 152791 was 3- to 20-fold more potent than 3alpha,5alpha-P as an inhibitor of [35S]TBPS binding in human recombinant receptor combinations containing alpha1, alpha2, alpha3 or alpha5 and beta2gamma2L subunits (Co 152791 IC50 1.4-5.7 nM). Co 152791 displayed low efficacy and 3alpha,5alpha-P had low potency at alpha4/6beta3gamma2L GABAA receptor complexes. Interestingly, Co 152791 demonstrated remarkable potency as a potentiator of GABA-evoked currents in Xenopus oocytes expressing alpha1beta2gamma2L receptors (EC50 0.87 nM), being 184-fold more potent than 3alpha,5alpha-P. High in vitro potency was also reflected in enhanced in vivo activity in that Co 152791 exhibited exceptional anticonvulsant potency, protecting mice from pentylenetetrazol-induced seizures at a approximately 5-fold lower dose than 3alpha,5alpha-P after i.p. administration (Co 152791 ED50 0.6 mg/kg). Moreover, Co 152791 was orally active (ED50 1.1 mg/kg) and exhibited a therapeutic index of 7 relative to rotorod impairment. The remarkable potency of Co 152791 as a positive allosteric modulator of GABAA receptors may be explained by its interaction with an auxiliary binding pocket in the neuroactive steroid binding site. In addition, modification at the 3beta-position probably hinders metabolism of the 3alpha-hydroxy group contributing to the exceptional anticonvulsant potency of this compound relative to other neuroactive steroids.

Anxiolytic and anticonvulsant activity of a synthetic neuroactive steroid Co 3-0593.

Endogeneously occurring neuroactive steroids, metabolites of progesterone and deoxycorticosterone, have been shown previously to interact with the GABAA receptor with great specificity in vitro and to have anticonvulsant, anxiolytic and sedative activity in vivo. However, these endogenously occurring steroids are not useful as therapeutic agents due to their potential metabolism to hormonally active steroids and their poor oral bioavailability. In an attempt to develop therapeutic agents which would maintain the pharmacological profiles of endogeneous neuroactive steroids but with increased oral bioavailability and reduced metabolic liability, we explored simple substitutions at the 3 beta-position of the endogenous neuroactive steroid, 3 alpha-hydroxy-5 alpha-pregnan-20-one (3 alpha, 5 alpha-P). This report describes part of the in vitro and in vivo pharmacological profile of a 3 beta-substituted analog, 3 beta-ethenyl-3 alpha-hydroxy-5 alpha-pregnan-20-one (Co 3-0593). The compound exhibited anticonvulsant activity against pentylenetrazol-induced seizures in mice and rats (ED50 = 5.6 and 11.5 mg/kg, i.p., respectively). Co 3-0593 showed robust anxiolytic effects, comparable to benzodiazepines in the Geller-Seifter test after both SC and oral administration. Furthermore, the anxiolytic activity was maintained after chronic administration suggesting an absence of tolerance. The compound did not affect the acquisition of a learned response at both anticonvulsant and anxiolytic doses. However, at higher doses the compound showed rotorod deficit which was further enhanced by ethanol. In summary, 3 beta-ethenyl-substituted 3 alpha, 5 alpha-P appeared to maintain the pharmacological activities of the endogenous neuroactive steroid with apparent oral activity.

5-(N-oxyaza)-7-substituted-1,4-dihydroquinoxaline-2,3-diones: novel, systemically active and broad spectrum antagonists for NMDA/glycine, AMPA, and kainate receptors.

A group of 5-aza-7-substituted-1,4-dihydroquinoxaline-2,3-diones (QXs) and the corresponding 5-(N-oxyaza)-7-substituted QXs were prepared and evaluated as antagonists of ionotropic glutamate receptors. The in vitro potency of these QXs was determined by inhibition of [3H]-5,7-dichlorokynurenic acid ([3H]DCKA) binding to N-methyl-D-aspartate (NMDA)/glycine receptors, [3H]-(S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid ([3H]AMPA) binding to AMPA receptors, and [3H]kainate ([3H]KA) binding to KA receptors in rat brain membranes. 5-(N-Oxyaza)-QXs 12a-e all have low micromolar or submicromolar potency for NMDA/glycine receptors and low micromolar potencies for AMPA and KA receptors. QXs 12a-e display 2-12-fold selectivity for NMDA/glycine receptors compared to AMPA receptors, and approximately 2-fold difference between AMPA and KA potency. In contrast to other QXs that either show high selectivity for NMDA (such as ACEA 1021) or AMPA (such as NBQX) receptors, these molecules are broad spectrum antagonists of ionotropic glutamate receptors. 7-Nitro-5-(N-oxyaza)-QX (12e) is the most potent inhibitor among 12a-e, having IC50 values of 0.69, 1.3, and 2.4 microM at NMDA, AMPA, and KA receptors, respectively. In functional assays on glutamate receptors expressed in oocytes by rat cerebral cortex poly(A+) RNA, 7-chloro-5-(N-oxyaza)-QX (12a) and 7-nitro-5-(N-oxyaza)-QX (12e) have Kb values of 0.63 and 0.31 microM for NMDA/glycine receptors, and are 6- and 4-fold selective for NMDA over AMPA receptors, respectively. 5-(N-Oxyaza)-7-substituted-QXs 12a-e all have surprisingly high in vivo potency as anticonvulsants in a mouse maximal electroshock-induced seizure (MES) model. 7-Chloro-5-(N-oxyaza)-QX (12a), 7-bromo-5-(N-oxyaza)-QX (12b), and 7-methyl-5-(N-oxyaza)-QX (12c) have ED50 values of 0.82, 0.87, and 0.97 mg/kg i.v., respectively. The high in vivo potency of QXs 12a-e is particularly surprising given their low log P values (approximately -2.7). Separate studies indicate that QXs 12a and 12e are also active in vivo as neuroprotectants and also have antinociceptive activity in animal pain models. In terms of in vivo activity, these 5-(N-oxyaza)-7-substituted-QXs are among the most potent broad spectrum ionotropic glutamate antagonists reported.

CCD-3693: an orally bioavailable analog of the endogenous neuroactive steroid, pregnanolone, demonstrates potent sedative hypnotic actions in the rat.

An endogenous neuroactive steroid, pregnanolone, and an orally available synthetic analog, CCD-3693, were administered to rats at the middle of their circadian activity phase (6 hr after lights off). Electroencephalogram-defined sleep-wake states, locomotor activity and body temperature were concurrently measured 30 hr before and after treatment. Identical procedures were used to test triazolam and zolpidem. Triazolam (0.1-1.6 mg/kg), zolpidem (2.5-10 mg/kg) and the neuroactive steroids (10-30 mg/kg) produced dose-dependent increases in non-rapid eye movement (NREM) sleep. At this dose and time of day (in which the rats were predominantly awake during the 6 hr before treatment) the neuroactive steroids appeared more intrinsically efficacious in promoting NREM sleep than the benzodiazepine ligands. The neurosteroids did not, however, significantly interfere with rapid eye movement sleep and were more selective in reducing (EEG) wakefulness, with relatively less locomotor activity impairment during waking than triazolam and zolpidem. In addition, the benzodiazepine receptor ligands showed distinct "rebound" wakefulness after the NREM sleep-promoting effect subsided, although the neuroactive steroids did not. In addition, in vitro binding studies and in vivo pharmacological data confirmed that CCD-3693 was orally active in standard tests of anxiety, anticonvulsant, loss-of-righting and passive avoidance.

Effects of thiocyanate and AMPA receptor ligands on (S)-5-fluorowillardiine, (S)-AMPA and (R,S)-AMPA binding.

AMPA receptors can be labeled using the agonist radioligands [3H](R,S)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid ([3H](R,S)-AMPA), [3H](S)-AMPA or [3H](S)-5-fluorowillardiine. In the presence of KSCN, [3H](R,S)-AMPA and [3H](S)-AMPA bind to a single population of sites in rat brain membranes, whereas [3H](S)-5-fluorowillardiine binds with two affinity components. KSCN increased the affinity of the low affinity [3H](S)-5-fluorowillardiine component > 4-fold and increased the density of both components 1.5-1.7-fold, arguing against KSCN-induced interconversion of low to high affinity states. KSCN, which promotes receptor desensitization, increased the potency of AMPA isomers, (S)-5-fluorowillardiine, quisqualate and cyclothiazide for inhibition of [3H](S)-5-fluorowillardiine binding suggesting that these ligands discriminate desensitized and nondesensitized receptors. In contrast, KSCN did not greatly affect the potency of glutamate, kainate, or competitive antagonists suggesting that these ligands do not discriminate desensitized and nondesensitized receptors. In the presence of KSCN, the rank order potency for agonists and antagonists was similar or identical in all assays indicating that the three radioligands bind identical glutamate recognition sites, a conclusion supported by their identical total receptor density. However, AMPA isomers displayed 6-10-fold higher potency for displacement of [3H](S)- or (R,S)-AMPA relative to [3H](S)-5-fluorowillardiine binding. This finding, coupled with the marked two component binding by [3H](S)-5-fluorowillardiine but not [3H](S)- or (R,S)-AMPA, suggests qualitative differences between the interaction of these ligands with the agonist recognition site.

N-methyl-D-aspartate antagonists for stroke and head trauma.

The N-methyl-D-aspartate (NMDA) receptor is a ligand-gated ion channel which is widely distributed in the central nervous system (CNS), and which mediates most of the fast excitatory neuronal transmission in the CNS. As with other ligand-gated ion channels, the NMDA receptor is a macromolecular complex which possesses a number of intricate regulatory sites within and around a central ion channel. The key regulatory components for which prototypic antagonists have been developed are the competitive NMDA antagonist binding site, the non-competitive NMDA antagonist binding site within the ion channel, and the NMDA receptor-associated glycine antagonist site. The binding domains for each of these binding sites possess discrete and non-overlapping SAR with regard to the chemical series developed to date. The potential utility of NMDA antagonists in the treatment of stroke and traumatic brain injury was investigated soon after the synthesis of the first bioavailable NMDA antagonists. Efficacy in preclinical models was demonstrated with both competitive and non-competitive NMDA antagonists. However, preclinical testing also revealed potentially clinically-limiting side-effects which included phencyclidine (PCP)-like actions indicative of possible psychotomimetic activity, cerebral vacuolisation of limbic cortical neurones, low therapeutic indices relative to incapacitating motor side-effects and, in the case of non-competitive antagonists, hypertension. These limitations have led to the design of clinical trials that should define the therapeutic index for this type of compound in humans. Currently, the first competitive antagonist to enter clinical trials, selfotel, is on hold, while D-CPPene is still in development. The non-competitive antagonist, aptiganel, is currently in Phase III clinical trials and its therapeutic efficacy and index should be defined in 1997 and 1998. The well-defined limitations of competitive and non-competitive NMDA antagonists have been a key impetus in the investigation of alternative approaches to modulating the NMDA receptor complex. In the case of glycine site antagonists, these compounds have been shown in preclinical studies to be devoid of PCP-like actions and the neuronal vacuolisation associated with the competitive and non-competitive NMDA antagonists. This has induced the development of a number of chemical series with at least three compounds currently in Phase I and II clinical trials. These include ACEA 1021, GV150526A and ZD9379. Clinical efficacies and therapeutic indices of these compounds should be defined in 1998 and 1999. An alternative approach using a partial agonist of the glycine site (1-aminocyclopropane-carboxylic acid, ACPC) has been halted in Phase I. Another approach which has led to the development of NMDA receptor antagonists, selective for the NMDA receptor subunits 1A/2B (NR1A/2B subtype), was the discovery in early studies of the neuroprotective actions of ifenprodil. Structural analogues include eliprodil, CP-101,606 and lubeluzole. In the cases of eliprodil and lubeluzole, these compounds have demonstrated neuroprotection in preclinical models, but they possess the extremely dangerous side-effect of increasing cardiac repolarisation time (i.e., increased QTc interval). The therapeutic index for these compounds is low. This has led to the termination of eliprodil's development and has limited the current dosing strategy with lubeluzole. It has not been disclosed if CP-101,606 possesses this dose-limiting side-effect. In summary, strategies for drug design and development based on our knowledge of the NMDA receptor complex have led to the development of a new generation of compounds for the treatment of stroke and traumatic brain injury, which remain to be evaluated in the clinic. The success of this approach will be defined in the next two to three years.

Characterization of the anticonvulsant properties of ganaxolone (CCD 1042; 3alpha-hydroxy-3beta-methyl-5alpha-pregnan-20-one), a selective, high-affinity, steroid modulator of the gamma-aminobutyric acid(A) receptor.

Ganaxolone (CCD 1042) is a 3beta-methyl-substituted analog of the endogenous neuroactive steroid 3alpha-hydroxy-5alpha-pregnan-20-one. Ganaxolone inhibited binding of the gamma-aminobutyric acid (GABA)A receptor-chloride channel ligand t-[35S]butylbicyclophosphorothionate (IC50 of 80 nM) and enhanced binding of the benzodiazepine site ligand [3H]flunitrazepam (EC50 of 125 nM) and the GABA site ligand [3H]muscimol (EC50 of 86 nM), consistent with activity as a positive allosteric modulator of the GABA(A) receptor. Electrophysiological recordings showed that, whereas nanomolar concentrations of ganaxolone potentiated GABA-evoked chloride currents in Xenopus oocytes expressing the human GABA(A) receptor subunits alpha1beta1gamma2L, alpha2beta1gamma2L or alpha3beta1gamma2L, direct activation of chloride flux occurred to a limited extent only at micromolar concentrations. Ganaxolone was effective in nontoxic doses against clonic convulsions induced by s.c. pentylenetetrazol administration in mice and rats (ED50 values of 4.3 and 7.8 mg/kg i.p., respectively). Ganaxolone also exhibited potent anticonvulsant activity against seizures induced by s.c. bicuculline (ED50 of 4.6 mg/kg i.p.), i.p. TBPS (ED50 of 11.7 mg/kg i.p.) and i.p. aminophylline (ED50 of 11.5 mg/kg i.p.) in mice. Although ganaxolone effectively blocked tonic seizures induced by maximal electroshock in mice (ED50 of 29.7 mg/kg i.p.), it did so only at doses that produced ataxia on the Rotorod (TD50 of 33.4 mg/kg i.p.). Conversely, ganaxolone was a potent anticonvulsant against fully kindled stage 5 seizures induced by corneal kindling in rats (ED50 of 4.5 mg/kg i.p.), producing these effects at doses well below those that resulted in ataxia (TD50 of 14.2 mg/kg i.p.). The seizure threshold, as determined by an increase in the dose of i.v. infused pentylenetetrazol required to induce clonus, was also significantly elevated by nontoxic doses of ganaxolone in mice. In summary, these data indicate that ganaxolone is a high-affinity, stereoselective, positive allosteric modulator of the GABA(A) receptor complex that exhibits potent anticonvulsant activity across a range of animal procedures. The profile of anticonvulsant activity obtained for ganaxolone supports clinical evaluation of this drug as an antiepileptic therapy with potential utility in the treatment of generalized absence seizures as well as simple and complex partial seizures.

Structure-activity relationships of alkyl- and alkoxy-substituted 1,4-dihydroquinoxaline-2,3-diones: potent and systemically active antagonists for the glycine site of the NMDA receptor.

We report on a series of alkyl- and alkoxy-substituted 1,4-dihydroquinoxaline-2,3-diones (QXs), prepared as a continuation of our structure-activity relationship (SAR) study of QXs as antagonists for the glycine site of the N-methyl-D-aspartate (NMDA) receptor. The in vitro potency of these antagonists was determined by displacement of the glycine site radioligand [3H]-5,7-dichlorokynurenic acid ([3H]DCKA) in rat brain cortical membranes. In general, methyl is a good replacement for chloro or bromo in the 6-position, and alkoxy-substituted QXs have lower potencies than alkyl- or halogen-substituted QXs. Ethyl-substituted QXs are generally less potent than methyl-substituted QXs, especially in the 6-position of 5,6,7-trisubstituted QXs. Fusion of a ring system at the 6,7-positions results in QXs with low potency. Several methyl-substituted QXs are potent glycine site antagonists that have surprisingly high in vivo activity in the maximal electroshock (MES) test in mice. Among these, 7-chloro-6-methyl-5-nitro QX (14g) (IC50 = 5 nM) and 7-bromo-6-methyl-5-nitro QX (14f) (IC50 = 9 nM) are comparable in potency to 6,7-dichloro-5-nitro QX (2) (ACEA 1021) as glycine site antagonists. QX 14g has an ED50 value of 1.2 mg/kg iv in the mouse MES assay. Interestingly, alkyl QXs with log P values of 0.5 or less tend to be more bioavailable than QXs with higher log P values. QX 14g has 440-fold selectivity for NMDA vs alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, as determined electrophysiologically under steady-state conditions in oocytes expressing rat cerebral cortex poly(A)+ RNA. Overall, 14g was found to have the best combination of in vitro and in vivo potency of all the compounds tested in this and previous studies on the QX series.

Synthesis and in vitro activity of 3 beta-substituted-3 alpha-hydroxypregnan-20-ones: allosteric modulators of the GABAA receptor.

Two naturally occurring metabolites of progesterone, 3 alpha-hydroxy-5 alpha- and 5 beta-pregnan-20-one (1 and 2), are potent allosteric modulators of the GABAA receptor. Their therapeutic potential as anxiolytics, anticonvulsants, and sedative/hypnotics is limited by rapid metabolism. To avoid these shortcomings, a series of 3 beta-substituted derivatives of 1 and 2 was prepared. Small lipophilic groups generally maintain potency in both the 5 alpha- and 5 beta-series as determined by inhibition of [35S]TBPS binding. In the 5 alpha-series, 3 beta-ethyl, -propyl, -trifluoromethyl and -(benzyloxy)methyl, as well as substituents of the form 3 beta-XCH2, where X is Cl, Br, or I or contains unsaturation, show limited efficacy in inhibiting [35S]TBPS binding. In the 5 beta-series, the unsubstituted parent 2 is a two-component inhibitor, whereas all of the 3 beta-substituted derivatives of 2 inhibit TBPS via a single class of binding sites. In addition, all of the 3-substituted 5 beta-sterols tested are full inhibitors of [35S]TBPS binding. Electrophysiological measurements using alpha 1 beta 2 gamma 2L receptors expressed in oocytes show that 3 beta-methyl- and 3 beta-(azidomethyl)-3 alpha-hydroxy-5 alpha-pregnan-20-one (6 and 22, respectively) are potent full efficacy modulators and that 3 alpha-hydroxy-3 beta-(trifluoromethyl)-5 alpha-pregnan -20-one (24) is a low-efficacy modulator, confirming the results obtained from [35S]TBPS binding. These results indicate that modification of the 3 beta-position in 1 and 2 maintains activity at the neuroactive steroid site on the GABAA receptor. In animal studies, compound 6 (CCD 1042) is an orally active anticonvulsant, while the naturally occurring progesterone metabolites 1 and 2 are inactive when administered orally, suggesting that 3 beta-substitution slows metabolism of the 3-hydroxyl, resulting in orally bioavailable steroid modulators of the GABAA receptor.

3 alpha-Hydroxy-3 beta-(phenylethynyl)-5 beta-pregnan-20-ones: synthesis and pharmacological activity of neuroactive steroids with high affinity for GABAA receptors.

Neuroactive steroids that allosterically modulate GABAA receptors have potential uses as anticonvulsants, anxiolytics, and sedative-hypnotic agents. Recently, a series of pregnanes substituted with simple alkyl groups at the 3 beta-position were synthesized and found to be active in vitro. The present report describes the synthesis of a series of substituted 3 alpha-hydroxy-3 beta-(phenylethynyl)pregnan-20-ones and their in vitro structure-activity relationship determined by their potency for inhibition of [35S]TBPS binding in rat brain membranes. Appropriate substitution of the phenyl group results in ligands with particularly high affinity for the neuroactive steroid site on GABAA receptors (e.g., 4-acetyl 28, IC50 10 nM). The potency of selected steroids was confirmed electrophysiologically in oocytes expressing cloned human GABAA alpha 1 beta 2 gamma 2L receptors (e.g., compound 28, EC50 6.6 nM). Consistent with their in vitro activity, some of the 3 beta-(phenylethynyl)-substituted steroids displayed anticonvulsant activity in the pentylenetetrazol (PTZ) and maximal electroshock (MES) tests following ip administration in mice. Notably, the 3 beta-[(4-acetylphenyl)ethynyl]-19-nor derivative 36 demonstrated an attractive anticonvulsant profile (PTZ and MES ED50 values of 2.8 and 9.2 mg/kg, respectively). A new pharmacophore for the neuroactive steroid site of GABAA receptors is proposed based upon the high affinity of certain substituted 3 beta-(phenylethynyl) steroids.

The N-methyl-D-aspartate (NMDA) receptor glycine site antagonist ACEA 1021 does not produce pathological changes in rat brain.

ACEA 1021 is a potent, selective N-methyl-D-aspartate (NMDA) receptor glycine site antagonist under clinical evaluation as a neuroprotectant for stroke and head trauma. The potential of ACEA 1021 to produce morphologic changes in cerebrocortical neurons of the rat was assessed since it is known that noncompetitive (e.g., MK-801) and competitive (e.g., CGS 19755)NMDA receptor antagonists produce neuronal vacuolization and necrosis in the rat posterior cingulate/retrosplenial cortex. Male and female adult rats were treated intravenously with either vehicle (Tris) or 10 mg/kg or 50 mg/kg ACEA 1021. MK-801 (5 mg/kg, s.c.) served as positive control. Whereas MK-801 produced characteristic neuronal vacuolization and necrosis in the posterior cingulate/retrosplenial cortex, neither dose of ACEA 1021 had any effect on neuronal morphology. The absence of neuropathological changes in rats supports the further clinical evaluation of ACEA 1021 for stroke and head trauma, and suggests that glycine site antagonists may be devoid of neurotoxic potential.

Novel phosphoserine phosphatase inhibitors.

Phosphoserine phosphatase (EC 3.1.1.3) catalyzes the final step in the major pathway of L-serine biosynthesis in brain. This enzyme may also regulate the levels of glycine and D-serine, the known and putative co-agonists for the glycine site of the N-methyl-D-aspartate receptor in caudal and rostral brain regions, respectively. Using L-phosphoserine as substrate, the rank order potency for inhibition of phosphoserine phosphatase was p-chloromercuriphenylsulfonic acid (CMPSA) > glycerophosphorylcholine >> hexadecylphosphocholine > or = phosphorylcholine > N-ethylmaleimide > or = L-serine > fluoride > D-2-amino-3-phosphonopropionic acid (D-AP3). Glycerylphosphorylcholine (IC50 18 microM) was found to be an uncompetitive inhibitor of phosphoserine phosphatase. Glycerylphosphorylcholine probably binds a novel site on the enzyme since the known allosteric inhibitor L-serine is highly selective for its feedback regulatory site, indicated by the inactivity of 25 L-serine analogs. Fluoride ion (IC50 770 microM) may bind the active site as has been shown for other Mg2+-dependent enzymes. The sulfhydryl reagent CMPSA is a potent, noncompetitive inhibitor of the enzyme using L-phosphoserine as substrate (IC50 9 microM) but is > 300-fold less potent using D-phosphoserine as substrate. Substrate-dependent differences are also observed with the sulfhydryl alkylator N-ethylmaleimide, which inhibits L-phosphoserine, but stimulates D-phosphoserine hydrolysis. These sulfhydryl reagents may dissociate multimeric forms of the enzyme to form monomers; the multimeric forms and monomers may preferentially cleave L- and D-phosphoserine, respectively. Phosphorylcholine esters and sulfhydryl reagents may prove useful in determining the contribution of phosphoserine phosphatase to the biosynthesis of glycine and D-serine in neuronal tissue in vitro.

Analogs of 3-hydroxy-1H-1-benzazepine-2,5-dione: structure-activity relationship at N-methyl-D-aspartate receptor glycine sites.

A series of aromatic and azepine ring-modified analogs of 3-hydroxy-1H-1-benzazepine-2,5-dione (HBAD) were synthesized and evaluated as antagonists at NMDA receptor glycine sites. Aromatic ring-modified HBADs were generally prepared via a Schmidt reaction with substituted 2-methoxynaphthalene-1,4-diones followed by demethylation. Electrophilic aromatic substitution of benzazepine 3-methyl ethers gave 7-substituted analogs. The preparation of multiply substituted 2-methoxynaphthalene-1,4-diones was effected via Diels-Alder methodology utilizing substituted butadienes with 2-methoxybenzoquinones followed by aromatization. Structural modifications, such as elimination of the aromatic ring, removal of the 3-hydroxyl group, and transfer of the hydroxyl group from C-3 to C-4, were also studied. An initial evaluation of NMDA antagonism was performed using a [3H]MK801 binding assay. HBADs demonstrating NMDA antagonist activity as indicated by inhibition of [3H]MK801 binding were further evaluated employing a [3H]-5,7-dichlorokynurenic acid (DCKA) glycine site binding assay. Selected HBADs were characterized for functional antagonism of NMDA and AMPA receptors using electrophysiological assays in Xenopus oocytes and cultured rat cortical neurons. Antagonist potency of HBADs showed good correlation between the different assay systems. HBADs substituted at the 8-position possessed the highest potency with the 8-methyl (5), 8-chloro (6), and 8-bromo (7) analogs being the most active. For HBAD 6, the IC50 in [3H]-DCKA binding assays was 0.013 microM and the Kb values for antagonism of NMDA receptors in oocytes (NR1a/2C) and cortical neurons were 0.026 and 0.048 microM, respectively. HBADs also antagonized AMPA-preferring non-NMDA receptors expressed in oocytes but at a lower potency than corresponding inhibition of NMDA receptors. HBADs demonstrating a high potency for NMDA glycine sites showed the highest steady-state selectivity index relative to AMPA receptors. Substitution at the 6-, 7-, and 9-positions generally reduced or eliminated glycine site affinity. Moving the hydroxyl group from C-3 to C-4 reduced receptor affinity, and potency was eliminated by the removal of the aromatic ring or the hydroxyl group. These data indicate that the HBAD series has specific structural requirements for high receptor affinity. With the exception of substitution at C-8, modified HBADs generally have a lower affinity at NMDA receptor glycine sites than the parent compound 3. Mouse maximum electroshock-induced seizure studies show that the three HBADs selected for testing have in vivo potency with the 6,8-dimethyl analog (52) being the most potent (ED50 = 3.9 mg/kg, iv).

Structure-activity relationships of 4-hydroxy-3-nitroquinolin-2(1H)-ones as novel antagonists at the glycine site of N-methyl-D-aspartate receptors.

A series of 4-hydroxy-3-nitroquinolin-2(1H)-ones (HNQs) was synthesized by nitration of the corresponding 2,4-quinolinediols. The HNQs were evaluated as antagonists at the glycine site of NMDA receptors by inhibition of [3H]DCKA binding to rat brain membranes. Selected HNQs were also tested for functional antagonism by electrophysiological assays in Xenopus oocytes expressing either 1a/2C subunits of NMDA receptors or rat brain AMPA receptors. The structure-activity relationships (SAR) of HNQs showed that substitutions in the 5-, 6-, and 7-positions in general increase potency while substitutions in the 8-position cause a sharp reduction in potency. Among the HNQs tested, 5,6,7-trichloro HNQ (8i) was the most potent antagonist with an IC50 of 220 nM in [3H]DCKA binding assay and a Kb of 79 nM from electrophysiological assays. Measured under steady-state conditions HNQ 8i is 240-fold selective for NMDA over AMPA receptors. The SAR of HNQs was compared with those of 1,4-dihydroquinoxaline-2,3-diones (QXs) and 1,2,3,4-tetrahydroquinoline-2,3,4-trione 3-oximes (QTOs). In general, HNQs have similar potencies to QXs with the same benzene ring substitution pattern but are about 10 times less active than the corresponding QTOs. HNQs are more selective for NMDA receptors than the corresponding QXs and QTOs. The similarity of the SAR of HNQs, QXs, and QTOs suggested that these three classes of antagonists might bind to the glycine site in a similar manner. With appropriate substitutions, HNQs represent a new class of potent and highly selective NMDA receptor glycine site antagonists.