Oral buspirone causes a shift in the dose-response curve between the elevated-plus maze and Vogel conflict tests in Long-Evans rats: relation of brain levels of buspirone and 1-PP to anxiolytic action.

Most studies concerning the effects of oral buspirone in the rat elevated plus-maze (EPM) test, spontaneous motor activity (SMA) test, and Vogel conflict (VC) test have used Sprague-Dawley or Wistar rats. Although it has been documented that the behavior of Long-Evans rats is more sensitive to detection of anxiolytics when compared to the aforementioned strains, the effects of oral buspirone have not been fully characterized in the Long-Evans strain in the EPM and VC tests. Thus, we studied the effects of orally administered buspirone (0.03-10.0 mg/kg) in the EPM, SMA, and VC (0.3-60.0 mg/kg) tests in Long-Evans rats. In a separate experiment, brain and plasma concentrations of buspirone and 1-(2-pyrimidinyl)-piperazine (1-PP) were determined after oral administration of buspirone (0.3 and 10 mg/kg) to relate the behavioral effects of buspirone with brain and plasma concentrations of buspirone and 1-PP. Our results showed that buspirone exhibited an inverted-U-shaped dose-response curve in both the EPM and the VC tests. In the EPM, buspirone produced anxiolytic activity in a low, narrow dose-range (0.03, 0.1, 0.3 mg/kg, p.o.) with maximum efficacy at 0.3 mg/kg, whereas in the VC test, significant anxiolytic activity was observed in a high, narrow dose-range (10, 30 mg/kg, p.o.) with maximum efficacy occurring at 10 mg/kg. In the SMA test, buspirone (10 mg/kg, p.o.) significantly decreased horizontal activity and vertical movements suggestive of sedation. Also, one hour following oral doses of buspirone (0.3 and 10 mg/kg), both buspirone and 1-PP concentrations were higher in brain when compared with those in plasma. Additionally, the concentrations of 1-PP were always higher in brain and in plasma compared with the concentrations of buspirone. Of particular interest is our finding of the shift in the dose-response curve between the EPM and VC tests. This shift in the dose-response curve is discussed in relation to brain levels of buspirone and 1-PP levels and their anxiolytic action.

Conformational analysis of the endogenous mu-opioid agonist endomorphin-1 using NMR spectroscopy and molecular modeling.

Endomorphin-1 (Tyr-Pro-Trp-Phe-NH2) is a highly selective and potent agonist of the mu-opioid receptor. To identify structural attributes unique to this opioid peptide and potential sites of recognition, a conformational analysis has been performed using multidimensional NMR and molecular modeling techniques. The spectroscopic results, derived from experiments in both DMSO and water, indicate that endomorphin-1 exists in the cis- and trans-configuration with respect to the Pro-omega bond in approximately 25% and 75% populations, respectively. In DMSO, the cis-configuration adopts a compact sandwich conformation in which the Tyr and Trp aromatic rings pack against the proline ring, whereas the trans-configuration adopts an extended conformation. Although non-random structure was not observed in water, condensed phase molecular dynamics calculations indicate that trans-isomers dominate the population in this higher dielectric medium. Structural comparison of the cis- and trans-configurations with morphine and selective mu-peptide ligands PL-017 and D-TIPP, as well as the delta-selective peptide ligands TIPP (delta-antagonist, mu-agonist) and DPDPE were also performed and suggest the trans-isomer is likely the bioactive form. A hypothesis is proposed to explain mu- and delta-selectivity based on the presence of spatially distinct selectivity pockets among these ligands.

Recent advances in traceless linkers.

The use of solid-phase organic synthesis for the preparation of small molecule compound libraries has been the driving force behind the development of new linkers and reaction technologies. Traceless linkers afford targets in which it is not readily apparent where the original point of attachment to the solid support had been. In many cases the site of linker attachment has been replaced with a hydrogen atom. This brief review summarizes some of the advances during the past several years involving traceless linkers in solid-phase organic synthesis.

Carbohydrate biguanides as potential hypoglycemic agents.

A series of monosaccharides containing a biguanide functionality was prepared and evaluated for hypoglycemic activity. Among the analogues prepared were those involving D-glucose substituted on the 6- or 1-position (19 and 24), D-galactose substituted on the 6-position (7), and D-arabinose (31). The target compounds were evaluated in a modified rat glucose-tolerance test (oral glucose load/oral drug, 100 mg/kg). Compounds 8 [6-biguanidino-1,2:3,5-bis-O-(1-methylethylidene)-6-deoxy-al pha-D- glucofuranose] and 23 [methyl 6-biguanidino-6-deoxy-2,3,4-O-tribenzyl-alpha-D-glucopyra nos ide] were the most active, exhibiting nearly equivalent hypoglycemic activity to that of phenformin (1) and metformin (2), as measured by the inhibition of the rise of blood glucose. Compound 31 was somewhat less active with 26% inhibition, as compared to 64% inhibition with 1 and 41% inhibition with 2.

Conjugates of catecholamines. 6. Synthesis and beta-adrenergic activity of N-(hydroxyalkyl)catecholamine derivatives.

A new series of catecholamines has been prepared in which the N-alkyl substituent of dl-epinephrine or dl-isoproterenol has been extended by a methylene chain terminated by a hydroxyl group or derived functionality (e.g., carbamate or ester). These functionalized catecholamines (congeners) and model compounds were prepared with the goal of eventual attachment to polymeric carrier molecules. The beta-adrenergic agonist activity of the derivatives was evaluated in vitro by measuring the intracellular accumulation of cyclic AMP in S49 mouse lymphoma cells and by the displacement of iodocyanopindolol (ICYP). A n-butylcarbamate derivative (compound 15) was the most active compound in this series with a potency 190 times greater than dl-isoproterenol in the S49 assay. The biological results indicate that minor modifications in structure in the N-alkyl substituent of the catecholamine can influence the pharmacologic activity.

Conjugates of catecholamines. 5. Synthesis and beta-adrenergic activity of N-(aminoalkyl)norepinephrine derivatives.

A novel series of N-aminoalkyl congeners and model derivatives of norepinephrine has been synthesized. Compounds that were structurally related to epinephrine were prepared from fully protected intermediates. Alternatively, isoproterenol-related compounds were synthesized via reductive amination of preformed methyl ketone derivatives with norepinephrine. The beta-adrenergic activities of these new compounds were assessed through measurement of intracellular cyclic AMP accumulation in S49 mouse lymphoma cells and displacement of iodocyanopindolol (ICYP) from membrane preparations. Congeners that contained an underivatized primary amine function exhibited virtually no activity in these assays. However, when this amine function was acylated (e.g., to an amide, carbamate, urea, sulfonamide, etc.), the products exhibited generally increased beta-adrenergic activity, which was, however, strongly dependent on the nature of the acylating group and also the length of the spacer. In particular, a benzyl carbamate derivative containing a branched, seven-carbon spacer group was 40 times more potent than isoproterenol in the in vitro S49 assay.

Small-molecule immunostimulants. Synthesis and activity of 7,8-disubstituted guanosines and structurally related compounds.

A series of 7,8-disubstituted guanosine derivatives was designed and prepared as potential B-cell-selective activators of the humoral immune response. These compounds were evaluated for their ability to act as B-cell mitogens and to augment the antibody response of B cells to sheep red blood cell (SRBC) challenge (adjuvanticity). In addition, they were tested for their ability to stimulate the natural killer (NK) cell response in murine in vitro cell assays. Certain of the compounds demonstrated in vivo activity when administered either intravenously, subcutaneously, or orally. Analogues with a medium-length alkyl chain (2-4 carbons, 5-7) on the 7-position of 7-alkyl-8-oxoguanosines were found to be particularly potent. Compounds bearing hydroxyalkyl, aminoalkyl, or substituted aminoalkyl substituents on this 7-position were weakly active. However, benzyl groups, including those substituted with heteroatoms (e.g., p-nitrobenzyl, 14), were active. Oxo, thioxo, and seleno groups on C-8 of the guanosine ring all imparted strong activity, whereas other larger substituents did not (e.g., N = CN). Stereochemical inversion of the 2'-hydroxyl on the ribose ring in this series, giving arabinose analogue 70, lessened activity. However, removal of the 2'-hydroxyl, either with (64) or without (73) removal of the 3'-hydroxyl, resulted in excellent activity and improved solubility; 64 also displayed good oral in vivo activity as well. A series of ketals involving the 2',3'-hydroxyls were prepared; certain of the nonpolar ketals (e.g., 48) were remarkably active, pointing to an ancillary hydrophobic binding region that can augment activity. 5'-Phosphate derivative 57 was fairly active, and acyclovir analogue 90 displayed good NK-selective activity: other N-9 sugar mimetics were also active (97-104), although this activity did not carry over into the human B-cell assay. A total of 80 compounds were prepared and evaluated for their immunostimulating activity. Within this group, compounds could be divided into those that were active in all three assays, those that displayed some measure of selectivity for the adjuvanticity assay, and those that preferentially activated NK responses. Because of its overall biological profile and ease of synthesis, 7-allyl-8-oxoguanosine (6; loxoribine, RWJ-21757) was chosen for further development. It is among the most potent compounds evaluated in the three biological assays.

Alpha(2) adrenoceptor agonists as potential analgesic agents. 3. Imidazolylmethylthiophenes.

A series of imidazolylmethylthiophenes has been prepared and evaluated as ligands for the alpha(2) adrenoceptor. These compounds were tested in two animal models that are predictive of analgesic activity in humans. The 3-thienyl compounds were generally the most potent, particularly those with substitution in the 4-position. A subset of the most active compounds was further evaluated for adverse cardiovascular effects in the anesthetized rat model. In addition to excellent binding at the alpha(2D) adrenoceptor, the 4-bromo analogues 20e and 21e were very active in the rat abdominal irritant test (RAIT) with ED(50) doses of 0.38 and 0.31 mg/kg, respectively. We constructed a pharmacophore model based on the biological activity of the present series, dexmedetomidine (1), and conformationally restrained analogues 3 and 4.

N-aryl-N'-benzylpiperazines as potential antipsychotic agents.

N1-(2-Alkoxyphenyl)piperazines additionally containing an N4-benzyl group bearing alcohol, amide, imide, or hydantoin functionalities were prepared and evaluated in the conditioned avoidance response (CAR) test predictive of clinical antipsychotic activity and in in vitro receptor-binding assays. Certain of the compounds display high affinity for the D2, 5-HT1A, and alpha 1-adrenergic receptors. Structures bearing acyclic amide, lactam, and imide functionalities display good biological activity, with a preference for the 1,3-disubstituted phenyl ring relative to the 1,4- and 1,2-congeners (7 vs 10 and 12). Every possible position of hydantoin attachment was investigated (e.g., substitution at N1, N3, and C5). The hydantoin involving attachment to N1 (24) was found to have good biological activity, whereas those hydantoins with attachment to N3 or C5 (22, 23, and 25) were inactive. Several of the smaller acetylated derivatives (30 and 33) have fair in vivo activity, which was lost in the case of the larger benzoyl analog 31. Uracil congener 34 had modest affinity for the D2 receptor (65 nM) as well as excellent in vivo activity. Benzylamino compounds display (viz. 27 and 35-38) moderate CAR activity but have surprising receptor affinity, often greater than those of comparable structures bearing a carbonyl (36 vs 7). Benzyl and benzhydryl alcohol compounds 40-48 are more active than amino structures 27 and 35-38 and also exhibit excellent in vivo activity in the CAR test with modest D2 and 5-HT1A receptor binding.

Orally active benzamide antipsychotic agents with affinity for dopamine D2, serotonin 5-HT1A, and adrenergic alpha1 receptors.

New antipsychotic drugs are needed because current therapy is ineffective for many schizophrenics and because treatment is often accompanied by extrapyramidal symptoms and dyskinesias. This paper describes the design, synthesis, and evaluation of a series of related (aminomethyl)benzamides in assays predictive of antipsychotic activity in humans. These compounds had notable affinity for dopamine D2, serotonin 5-HT1A, and alpha1-adrenergic receptors. The arylpiperazine 1-[3-[[4-[2-(1-methylethoxy)phenyl]-1-piperazinyl]methyl]benzoyl]p ipe ridine (mazapertine, 6) was chosen because of its overall profile for evaluation in human clinical trials. The corresponding 4-arylpiperidine derivative 67 was also highly active indicating that the aniline nitrogen of 6 is not required for activity. Other particularly active structures include homopiperidine amide 14 and N-methylcyclohexylamide 31.

Alpha(2) adrenoceptor agonists as potential analgesic agents. 1. (Imidazolylmethyl)oxazoles and -thiazoles.

A series of (imidazolylmethyl)oxazoles and -thiazoles were prepared and evaluated as alpha(2) adrenoceptor agonists. These compounds were also tested in in vivo paradigms that are predictive of analgesic activity. Variations in both the imidazole and thiazole portions of the molecule were investigated. Some of the more potent compounds such as 22, 26, 45, and 53 displayed alpha(2) receptor binding in the 10-20 nM range and also had significant antinociceptive activity in the mouse abdominal irritant test (MAIT).

Synthesis of an eight-membered cyclic pseudo-dipeptide using ring closing metathesis.

Ring closing metathesis of diallylglycine 6 provided cyclic Z-olefin 7 in 80% yield. The reaction was promoted by substitution of the amide nitrogen with the 2,4-dimethoxybenzyl group allowing for the required cis diallylglycine amide rotamer. Removal of the protecting groups provided cyclic dipeptide 2, a constrained scaffold useful in peptidomimetic research.

New directions in anxiolytic drug research.

Agents to treat anxiety have gained in acceptance and importance in the fast pace of life in the second half of this century. The discovery and refinement of the benzodiazepines represented a quantum leap in therapy from early compounds which were essentially sedatives. With the advent of molecular biology, an understanding of the basic mechanism by which the benzodiazepines exert their effects was revealed through the discovery and isolation of the GABAA receptor and its benzodiazepine binding site. This, in turn, has enabled benzodiazepines to be classified into a broad spectrum of pharmacological types ranging from agonist to inverse agonist, thus allowing fine tuning with respect to side-effects. Consequently, newer, more promising agents have emerged which bind at the GABAA BZD site and have reduced side-effects. An example of this is RWJ-51204 (92), a member of a novel structural type which is superior to several marketed benzodiazepines in animals in terms of efficacy and side-effects. The cost-conscious environment of managed health care presents continuing challenges to the discovery and development of safe, highly efficacious, and cost-effective anxiolytic agents.

In vitro biotransformation of the new antipsychotic agent, RWJ-46344 in rat hepatic S9 fraction: API-MS/MS/MS identification of metabolites.

The in vitro biotransformation of the antipsychotic agent, RWJ-46344 was studied after incubation with rat hepatic S9 fraction in the presence of an NADPH-generating system. Unchanged RWJ-46344 (approximately 37% of the sample) plus 12 metabolites were profiled, quantified, and tentatively identified on the basis of API (ionspray)-MS/MS/MS data. The proposed metabolic pathways for RWJ-46344 are proposed, and the six metabolic pathways are 1, O-dealkylation; 2, piperidinyl oxidation; 3, N-debenzylation; 4, phenyl hydroxylation; 5, dehydration; and 6, reduction. Pathways 1 to 3 formed O-desisopropyl RWJ-46344 (M3, approximately 13% of the sample) and its hydroxy-metabolite (M5, approximately 8%), hydroxypiperidinyl RWJ-46344 (M1, approximately 5%) and a phenylpiperidinyl metabolite (M8, approximately 24%) as major and moderate metabolites. Eight minor metabolites (each < 2%) were formed via a combination of six steps. RWJ-46344 is metabolized substantially by this rat hepatic system.

Hepatic metabolism of the new antipsychotic agent, mazapertine, in rat--API-MS/MS identification of metabolites.

The In vitro biotransformation of the antipsychotic agent, mazapertine was studied after incubation with rat hepatic S9 fraction in the presence of an NADPH-generating system. Unchanged mazapertine (42% of the sample) plus 12 metabolites were profiled, quantified, and tentatively identified on the basis of API (ionspray)-MS/MS data. The proposed metabolic pathways for mazapertine are proposed, and the 6 metabolic pathways are: (1) phenylhydroxylation, (2) piperidyl oxidation, (3) O-dealkylation, (4) N-dephenylation, (5) oxidative N-debenzylation, and (6) dehydration. Pathways 1 to 3 formed 4-OH-phenyl (M1, 10%) and 4-OH-piperidyl (M2, 9%)-mazapertine, O-desisopropyl mazapertine (M3, 17%), and N-desbenzoylpiperidine-mazapertine (M8, 14%) as 4 major metabolites. Mazapertine is extensively metabolized in rat hepatic S9 fraction.

Human hepatic metabolism of the anxiolytic agent, RWJ-51521--API-MS/MS identification of metabolites.

The In vitro metabolism of the anxiolytic agent, RWJ-51521 was conducted after incubation with human hepatic S9 fraction in the presence of an NADPH-generating system. Unchanged RWJ-51521 (30% of the sample) and a total of 11 metabolites were profiled, quantified, and tentatively identified on the basis of API (ionspray)-MS/MS data. The 4 proposed metabolic pathways for RWJ-51521 are: (1) N/O-dealkylation, (2) phenylhydroxylation, (3) pyrido-oxidation, and (4) dehydration. Pathway 1 formed 2 major and 3 minor N/O-desalkyl metabolites (M1 & M3, 50%) and in conjunction with pathway 4, formed 2 moderate dehydrated metabolites (M4 & M5, 14%). Pathways 2 and 3 alone, and in conjunction with pathway 4, produced 4 minor metabolites (each < or =2%). RWJ-51521 is extensively metabolized in human hepatic S9 fraction.

Metabolism of the new nonbenzodiazepine anxiolytic agent, RWJ-51204, in mouse, rat, dog, monkey and human hepatic S9 fractions, and in rats, dogs and humans.

The in vitro and in vivo metabolism of the nonbenzodiazepine anxiolytic agent, RWJ-51204 was investigated after incubation with mice, rat, dog, monkey, and human hepatic S9 fractions in the presence of NADPH-generating system, and a single oral dose administration to rats (100 mg/kg), dogs (5 mg/kg), and humans (2.5 mg/subject). Plasma and red blood cells (2 h, rat) and urine samples (0-24 h, rat, dog and human) were obtained postdose. Unchanged RWJ-51204 (39-93% of the sample in vitro; < or =5% of the sample in vivo) plus 14 metabolites were profiled, quantified and tentatively identified on the basis of API-MS and MS/MS data, and by comparison of synthetic samples. The in vitro and in vivo metabolic pathways for RWJ-51204 are proposed, and the metabolite formations are via the following five pathways: 1. phenyl oxidation, 2. pyrido-oxidation, 3. N-deethoxymethylation, 4. dehydration, and 5. glucuronidation. Pathway 1 formed 4-hydroxy-2-fluoro-phenyl-RWJ-51204 (M1, 7-24% in vitro; 5-60% in vivo) in major amounts, OH-benzimidazole-RWJ-51204 (M2, 5-8% in vitro and in vivo) and diOH-phenyl-RWJ-51204 (< or =5-16% in vitro and in vivo); in conjunction with pathway 5 produced M1 glucuronide (60% in rat & dog; 17% in human), M2 glucuronide (16% in human). Pathways 2-4 formed minor/trace oxidized, and dehydrated metabolites. RWJ-51204 is extensively metabolized in vitro (except dog) and in vivo in rats, dogs and humans.

Mephedrone ("bath salt") pharmacology: insights from invertebrates.

Psychoactive bath salts (also called meph, drone, meow meow, m-CAT, bounce, bubbles, mad cow, etc.) contain a substance called mephedrone (4-methylcathinone) that may share psychostimulant properties with amphetamine and cocaine. However, there are only limited studies of the neuropharmacological profile of mephedrone. The present study used an established invertebrate (planarian) assay to test the hypothesis that acute and repeated mephedrone exposure produces psychostimulant-like behavioral effects. Acute mephedrone administration (50-1000 µM) produced stereotyped movements that were attenuated by a dopamine receptor antagonist (SCH 23390) (0.3 µM). Spontaneous discontinuation of mephedrone exposure (1, 10 µM) (60 min) resulted in an abstinence-induced withdrawal response (i.e. reduced motility). In place conditioning experiments, planarians in which mephedrone (100, 500 µM) was paired with the non-preferred environment during conditioning displayed a shift in preference upon subsequent testing. These results suggest that mephedrone produces three behavioral effects associated with psychostimulant drugs, namely dopamine-sensitive stereotyped movements, abstinence-induced withdrawal, and environmental place conditioning.