Differential pharmacology between the guinea-pig and the gorilla 5-HT1D receptor as probed with isochromans (5-HT1D-selective ligands).

1. Both the 5-HT1D and 5-HT1B receptors are implicated in migraine pathophysiology. Recently isochromans have been discovered to bind primate 5-HT1D receptors with much higher affinity than 5-HT1B receptors. In the guinea-pig, a primary animal model for anti-migraine drug testing, however, isochromans bound the 5-HT1D receptor with lower affinity than the gorilla receptor. 2. This species-specific pharmacology was investigated, using site-directed mutagenesis on cloned guinea-pig receptors heterologously expressed in human embryonic kidney 293 cells. Mutations of threonine 100 and arginine 102 at the extracellular side of transmembrane II of the guinea-pig 5-HT1D receptor to the corresponding primate residues, isoleucine and histidine, respectively, enhanced its affinity for isochromans to that of the gorilla receptor, with little effects on its affinities for serotonin, sumatriptan and metergoline. Free energy change from the R102H mutation was about twice as much as that from the T100I mutation. 3. For G protein-coupling, serotonin marginally enhanced GTPgamma35S binding in membranes expressing the guinea-pig 5-HT1D receptor and its mutants, but robustly in membranes expressing the gorilla receptor. Sumatriptan enhanced GTPgamma35S binding in the latter nearly as much as serotonin, and several isochromans by 30-60% of serotonin. 4. We discovered key differences in the function and binding properties of guinea-pig and gorilla 5-HT1D receptors, and identified contributions of I100 and H102 of primate 5-HT1D receptors to isochroman binding. Among common experimental animals, only the rabbit shares I100 and H102 with primates, and could be useful for studying isochroman actions in vivo.

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.

PNU-96415E, a potential antipsychotic agent with clozapine-like pharmacological properties.

The atypical antipsychotic drug clozapine interacts with multiple transmitter systems, among them the D4 subtype of dopamine receptors. PNU-96415E is chemically unrelated to clozapine and has its highest binding affinity for the D4 and 5-HT2A receptors. In comparison to clozapine, PNU-96415E is weaker in binding to D1, D2, alpha1 and muscarinic receptors. PNU-96415E inhibited exploratory locomotor activity in mice and rats, and antagonized d-amphetamine-induced locomotor stimulation in rats. It antagonized apomorphine-induced cage climbing, and blocked head and body twitch produced by 5-HTP in mice. Like clozapine, but unlike haloperidol, PNU-96415E did not antagonize stereotypic behaviors produced by a high dose of d-amphetamine or methylphenidate in rats and mice. PNU-96415E blocked conditioned avoidance in rats but produced no catalepsy, a pattern similar to clozapine but different from haloperidol. In rats trained to discriminate clozapine from saline injections, the stimulus effect generalized completely with PNU-96415E, but not haloperidol. This profile of pharmacological activities is consistent with that of an atypical antipsychotic and, as in the case with clozapine, the behavioral effects of PNU-96415E cannot be ascribed to a single receptor mechanism.

Pharmacological characterization of U-101387, a dopamine D4 receptor selective antagonist.

Dopamine D2-like receptors play an important role in the pharmacotherapy of psychotic disorders. Molecular and cellular techniques have identified distinct gene products (D2-long, D2-short, D3 and D4) displaying the D2 receptor pharmacology. However, the contribution of each subtype in antipsychotic effects of or their physiological role remain unclear. Here we describe the pharmacological effects of a selective D4 antagonist, U-101387. U-101387 displayed moderately high affinity (Ki = 10 nM) and selectivity for the dopamine D4.2 receptor expressed in clonal cell lines. It lacked measurable affinity for not only other dopamine receptors but also noradrenalin, serotonin and histamine receptor families (Ki > 2000 nM). It fully and dose-dependently antagonized quinpirole-induced cAMP inhibition (without producing any effect by itself) in stably transfected cells. U-101387 also displayed excellent oral bioavailability, brain penetration and other pharmacokinetic characteristics. Unlike classical neuroleptics (e.g., haloperidol), U-101387 neither blocked acute behavioral effects of amphetamine or apomorphine nor did it alter spontaneous locomotion by itself. Additionally, U-101387 was without effect in behavioral and biochemical tests predictive of extrapyramidal and neuroendocrine side effects. Consistent with the lack of autoreceptor function of D4, acute administration of U-101387 failed to alter dopamine neuronal firing by itself or reverse the inhibition produced by dopamine agonists and to affect monoamine turnover in areas innervated by the mesencephalic or hypothalamic dopamine neurons. However, U-101387 potently induced c-fos mRNA expression in the infralimbic/ventral prelimbic cortex to a level similar to that produced by the atypical antipsychotic, clozapine. This is consistent with the predominantly cortical distribution of the D4 receptor. Taken together, these results demonstrate that the D4-selective antagonist, U-101387, produces effects that are distinct from those of the nonselective D2 antagonists as well as D3-preferring agents. U-101387 offers a unique tool to understand the role of dopamine D4 receptors in diseases involving central dopamine systems.

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.

Antagonist, partial agonist, and full agonist imidazo[1,5-a]quinoxaline amides and carbamates acting through the GABAA/benzodiazepine receptor.

(4RS)-1-(5-Cyclopropyl-1,2,4-oxadiazol-3-yl)-12,12a-dihyd roimidazo[1,5- a]pyrrolo[2,1-c]quinoxalin-10(11H)-one (1a), 5-benzoyl-3-(5-cyclopropyl-1,2,4-oxadiazol-3-yl)-4,5- dihydroimidazo[1,5-a]quinoxaline (13b), and tert-butyl (4S)-12,12a-dihydroimidazo[1,5-a]pyrrolo[2,1- c]quinoxaline-1-carboxylate (1e), as well as other imidazo[1,5-a]quinoxaline amides and carbamates, represent a new series of compounds which bind with high affinity to the GABAA/benzodiazepine receptor. These compounds exhibit a wide range of intrinsic efficacies as measured by [35S]TBPS binding ratios. The synthesis of 1a begins with the addition of DL-glutamic acid to 1-fluoro-2-nitrobenzene, followed by reduction of the nitro group and subsequent ring closure to form 3-(carbethoxymethyl)-1,2,3,4-tetrahydroquinoxalin-2-one, followed by a second ring closure to afford (4RS)-1,5-dioxo-1,2,3,4,5,6-hexahydropyrrolo[1,2-a]quinoxali ne as the key intermediate. Appendage of a substituted imidazo ring via the anion of 5-cyclopropyl-1,2,4-oxadiazol-3-yl gives 1a. The (-)- and (+)-isomers of 1a were prepared from 1-fluoro-2-nitrobenzene and L- and D-glutamic acid, respectively. 1a and its enantiomers demonstrated affinity for the [3H]flunitrazepam binding site with Ki's of 0.87, 0.62, and 0.65 nM, respectively.

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.

Inhibitors of the protease from human immunodeficiency virus: synthesis, enzyme inhibition, and antiviral activity of a series of compounds containing the dihydroxyethylene transition-state isostere.

A number of potential HIV protease inhibitory peptides that contain the dihydroxyethylene isostere were prepared and evaluated for their enzyme binding affinity and antiviral activity in cell cultures. From the template of a previously reported active peptide A, modifications at the N- and C-terminal groups were assessed for potential maintenance of good inhibitory activity of the resulting peptides. Among the active peptides found, peptide XVIII exhibited potent enzyme inhibitory activity. Interestingly, the previously reported, effective 1(S)-amino-2(R)-hydroxyindan C-terminal group for the preparation of very active HIV protease inhibitory peptides could not be applied to the template of peptide XVIII. Molecular modeling of peptide XVIII was studied using the X-ray crystal structure of peptide A as a starting point in order to study the likely conformation of peptide XVIII in the active-site cleft. Relative binding conformations of peptide A and XVIII were obtained, although the reason for poor binding affinity for a number of congeneric peptides in this report was not straightforwardly apparent. More importantly, however, peptide XVIII was found to exhibit more effective antiviral activity in the HIV-1/PBMC assay than the reference peptide A which was previously reported to be approximately equal in efficacy to the reverse transcriptase inhibitor AZT in this assay.

Arylformamidines with antinociceptive properties.

A series of formamidines structurally related to clonidine were synthesized and investigated as potential nonopiate analgesics. Several of these compounds showed potent analgesic activity (ED50 on HCl writhing less than 1.0 mg/kg) with low potential for hypotensive effects. A qualitative description of the structure-activity relationship of this series reveals that the 2,4- and 2,6-dimethylphenyl compounds are more potent analgesics than are the corresponding dichlorophenyl compounds.

Renin inhibitors containing psi[CH2O] pseudopeptide inserts.

Renin inhibitors 2-4 with the D-Lys renin inhibitory peptide (RIP) sequence, but containing Leu psi[CH2O]Ala (2), Leu psi[CH2O]Val (3), and Leu psi[CH2O]Leu (4) at the P1-P1' site, were of a comparable potency to RIP. N-Terminal Boc-protected inhibitors containing Pro psi[CH2O]Phe in positions P4-P3 were potent inhibitors of renin, with Boc-Phe-Pro psi[CH2O]Phe-His-Leu psi[CH(OH)CH2]Val-Ile-(2-aminomethyl) pyridine (17) having an IC50 of 1.6 X 10(-9) M.

7-(Trifluoromethyl)-4-aminoquinoline hypotensives: novel peripheral sympatholytics.

A family of 7-(trifluoromethyl)-4-aminoquinolines that are hypotensive agents and that act by a novel sympatholytic mechanism is described. Structure-activity relationships in this series have been elucidated. Some of the more potent hypotensives were evaluated for safety in the mouse. A candidate, 1-[(4-fluorophenyl)sulfonyl]-4-[4-[[7-(trifluoromethyl)-4- quinolinyl]amino]benzoyl]piperazine hydrochloride (losulazine hydrochloride) has been selected for clinical development. Losulazine hydrochloride is a hypotensive agent in the rat, cat, and dog. At acute effective hypotensive doses, it does not block the response of the sympathetic nervous system to stimuli. Both animal pharmacology and clinical experience suggest that losulazine hydrochloride may be free of the clinically limiting side effects that often plague compounds that decrease blood pressure by interfering with autonomic neurogenic function.

1-(alkylamino)isochromans: hypotensives with peripheral and central activities.

A series of 1-[1-(3,4-dimethoxy-1H-2-benzopyran-1-yl)alkyl]-4-arylpiperazines that shows hypotensive activity in the conscious rat has been investigated. Structure-activity relationships are described. A typical example that was investigated in greater detail is 1-[2-(3,4-dihydro-6,7-dimethoxy-1H-2-benzopyran-1-yl)ethyl]-4-(4-fluorophenyl)piperazine. This compound decreases sympathetic nerve activity recorded from the external carotid and splanchnic nerves of baroreceptor-denervated cats and, therefore, has a central component to its mechanism of action. It also blocks pressor effects of norepinephrine and phenylephrine and is thus an alpha-adrenergic antagonist. Binding data characterize this as alpha 1-adrenergic receptor blockade.

Tetrahydro-2-benzoxepins: a novel family of hypotensives.

A novel series of 1-(alkylamino)-1,3,4,5-tetrahydro-7,8-dimethoxy-2-benzoxepins shows hypotensive activity. A typical example is 1-[2-(1,3,4,5-tetrahydro-7,8-dimethoxy-2-benzoxepin-1-yl)ethyl]-4-(4-fluorophenyl)piperazine. This compound is an alpha blocker with peripheral and central activities.

1-[Ethoxyamino)methyl]-1,3,4,5-tetrahydro-7,8-dimethoxy-2-benzoxepins: a new class of antianaphylactic agents.

The synthesis and biological activity in the rat passive cutaneous anaphylaxis (PCA) test of a new class of compounds, 1-[(ethoxyamino)methyl]-1,3,4,5-tetrahydro-7,8-dimethoxy-2-benzoxepins, are reported. These compounds are synthesized from the adduct of 1-(bromomethyl)-1,3,4,5-tetrahydro-7,8-dimethoxy-2-benzoxepins, are reported. These compounds are synthesized from the adduct of 1-(bromomethyl)-1,3,4,5-tetrahydro-7,8-dimethoxy-2-benzoxepin and ethylene glycol or chloroethanol. The influence of the amine function on activity in the rat PCA is discussed. Aryl- or heteroarylpiperazines favor activity with this class of compounds.

Hypobetalipoproteinemic agents. 2. Compounds related to 4-(1-adamantyloxy)aniline.

While the previously used displacement reaction of sodim 1-adamantyl oxide on 4-fluoronitrobenzene was applicable to the preparation of 4-(1-adamantyloxy)aniline and several related compounds, certain derivatives were not easily accessible by this route. Thus the recently reported ortho alkylation of anilines and the dicyclohexylcarbodiimide-promoted coupling of 1-adamantanol with phenols were useful in the preparation of aromatic-substituted derivatives. Furthermore, addition of phenylmagnesium bromide to 1-cyanoadamantane provided entry to the 4-(1-adamantylmethyl)aniline series. 4-(1-Adamantyloxy)aniline (3) is herein reported to be a more potent hypobetalipoproteinemic agent than the previously reported bicyclooctyloxy analogue. Replacement of the oxygen atom of 3 with sulfur (74) or methylene (62), but not nitrogen (71), results in active compounds. In the oxygen series derived from 3, the widest scope of substitution on nitrogen resulting in activity is found. The N-ethoxycarbonyl (5), acetyl (6), methyl (12), ethyl (13), N-methyl-N-(2-hydroxyethyl) (19), N-methyl-N-formyl (22), N,N-dimethyl (26), pyrrolidine (14), and piperidine (15) derivatives are active. Aromatic ring substitution also provided the active 3-chloro (44b), 2-fluoro (41b, 42, and 43), and 2-methylthiomethyl (48) compounds. Thus these active compounds are identified for further development as hypobetalipoproteinemic agents.

Hypo-beta-lipoproteinemic agents. 1. Bicyclo[2.2.2]octyloxyaniline and its derivatives.

A new assay for agents which normalize beta-lipoproteins in cholesterol-cholic acid fed rats is described. Both lowering of serum cholesterol and of serum heparin precipitable lipoproteins (HPL) were measured at the end of the treatment period. Compounds which shifted the ratio of the decrease in favor of HPL are considered hypo-beta-lipoproteinemic. p-(1-Bicyclo[2.2.2]octyloxy)aniline and several of its derivatives proved active in this assay. The synthesis of these compounds is described.