Design of 2,5-dimethyl-3-(6-dimethyl-4-methylpyridin-3-yl)-7-dipropylaminopyrazolo[1,5-a]pyrimidine (NBI 30775/R121919) and structure--activity relationships of a series of potent and orally active corticotropin-releasing factor receptor antagonists

We have previously shown that 3-phenylpyrazolo[1,5-a]pyrimidines exemplified by 8 were potent antagonists of the human corticotropin-releasing factor-1 receptor. A series of 3-pyridylpyrazolo[1,5-a]pyrimidines 15, 25-30, 34, and 35 containing a weakly basic pyridine ring at the 3-position of the bicyclic nucleus was designed to reduce lipophilicity from the initial leads such as 7. Here, we showed that these 3-pyridyl compounds exhibited potent antagonists at the human CRF(1) receptor. Moreover, the hydrophilic and weakly basic pyridine moiety increased the water solubility of some analogues. Compound 26 h exhibited good binding affinity at the human CRF(1) receptor with a K(i) value of 3.5 nM. As a functional antagonist, it dose-dependently inhibited CRF-stimulated cAMP production in cells expressing the CRF(1) receptor (IC(50) = 50 nM), and CRF-stimulated ACTH release from cultured rat pituitary cells (IC(50) = 20 nM). 26 h had a log P value of 4.9 and water solubility of greater than 10 mg/mL. Pharmacokinetic studies in rats showed that 26 h was orally bioavailable and able to penetrate into the brain. 26 h has been demonstrated in vivo efficacy in animal behavioral models that measure anxiolytic activity. These results suggest that analogues from this series were potent CRF(1) receptor antagonists with proper physicochemical properties and good pharmacokinetic profiles. 26 h was developed into a clinical compound and exhibited efficacy in patients with major depression.     

Synthesis of a novel series of tricyclic dihydrofuran derivatives: discovery of 8,9-dihydrofuro[3,2-c]pyrazolo[1,5-a]pyridines as melatonin receptor (MT1/MT2) ligands

Novel tricyclic dihydrofuran derivatives were designed, synthesized, and evaluated as melatonin receptor (MT(1)/MT(2)) ligands based on the previously reported 1,6-dihydro-2H-indeno[5,4-b]furan 1a. By screening the central tricyclic cores, we identified 8,9-dihydrofuro[3,2-c]pyrazolo[1,5-a]pyridine as a potent scaffold with a high ligand-lipophilicity efficiency (LLE) value. Subsequent optimization of the side chains led to identification of the potent MT(1)/MT(2) agonist 4d (MT(1), K(i) = 0.062 nM; MT(2), K(i) = 0.420 nM) with good oral absorption and blood-brain barrier (BBB) penetration in rats. The oral administration of compound 4d exhibited a sleep-promoting action in freely moving cats at 0.1 mg/kg.     

Stressin1-A, a potent corticotropin releasing factor receptor 1 (CRF1)-selective peptide agonist

The potencies and selectivity of peptide CRF antagonists is increased through structural constraints, suggesting that the resulting ligands assume distinct conformations when interacting with CRF1 and CRF2 receptors. To develop selective CRF receptor agonists, we have scanned the sequence -Gln-Ala-His-Ser-Asn-Arg- (residues 30-35 of [DPhe12,Nle21,38]Ac-hCRF4-41) with an i-(i+3) bridge consisting of the Glui-Xaa-Xbb-Lysi+3 scaffold, where residues i=30, 31, and 32. When i=31, stressin1-A, a potent CRF1 receptor-selective agonist was generated. In vitro, stressin1-A was equipotent to h/rCRF to release ACTH. Astressin1-A showed a low nanomolar affinity for CRF1 receptor (Ki=1.7 nM) and greater than 100-fold selectivity versus CRF2 receptor (Ki=222 nM). Stressin1-A released slightly less ACTH than oCRF in adult adrenal-intact male rats, with increased duration of action. Stressin1-A, injected intraperitoneally in rats, induced fecal pellet output (a CRF1 receptor-mediated response) and did not influence gastric emptying and blood pressure (CRF2 receptor-mediated responses).     

Dopamine D4 receptors inhibit depolarization-induced [3H]GABA release in the rat subthalamic nucleus

We explored the role of dopamine D4 receptors on [3H]GABA release in the subthalamic nucleus. [3H]GABA release was evoked by high K+ in slices of the nucleus. The selective dopamine D4 receptor agonist PD168,077 (N-[[4-(2-cyanophenyl)-1-piperazynil]methyl]-3-methyl-benzamide) inhibited GABA release with greater potency (EC50=3.2 nM) than quinpirole (EC50=200 nM). SKF 21297 (6-chloro-7,8-dihydroxy-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrobromide), a dopamine D1-like receptor agonist, had no effect. L-745,870 (3-[[4-(4-chlorophenyl)piperazin-1-yl]methyl]-1-1H-pyrollo[2,3-b] pyridine), a selective dopamine D4 receptor antagonist, reverted the quinpirole inhibition with greater potency (IC50=8.7 nM) than that of the dopamine D2/D3 receptor antagonist sulpiride and raclopride (IC50=4804 and 788 nM, respectively). Both methylphenidate and methamphetamine, dopamine reuptake blockers, inhibited by 30% high K(+)-evoked GABA release; the inhibition was blocked by L-745,870. These results show that dopamine D4 receptors modulate GABA release in the subthalamic nucleus. The results would explain how agents that increase interstitial dopamine like methylphenidate and amphethamine might control locomotor hyperactivity seen in disorders of dopamine D4 receptors.     

Discovery of a series of imidazo[4,5-b]pyridines with dual activity at angiotensin II type 1 receptor and peroxisome proliferator-activated receptor-γ

Mining of an in-house collection of angiotensin II type 1 receptor antagonists to identify compounds with activity at the peroxisome proliferator-activated receptor-γ (PPARγ) revealed a new series of imidazo[4,5-b]pyridines 2 possessing activity at these two receptors. Early availability of the crystal structure of the lead compound 2a bound to the ligand binding domain of human PPARγ confirmed the mode of interaction of this scaffold to the nuclear receptor and assisted in the optimization of PPARγ activity. Among the new compounds, (S)-3-(5-(2-(1H-tetrazol-5-yl)phenyl)-2,3-dihydro-1H-inden-1-yl)-2-ethyl-5-isobutyl-7-methyl-3H-imidazo[4,5-b]pyridine (2l) was identified as a potent angiotensin II type I receptor blocker (IC(50) = 1.6 nM) with partial PPARγ agonism (EC(50) = 212 nM, 31% max) and oral bioavailability in rat. The dual pharmacology of 2l was demonstrated in animal models of hypertension (SHR) and insulin resistance (ZDF rat). In the SHR, 2l was highly efficacious in lowering blood pressure, while robust lowering of glucose and triglycerides was observed in the male ZDF rat.     

Design and synthesis of tricyclic imidazo[4,5-b]pyridin-2-ones as corticotropin-releasing factor-1 antagonists

The synthesis and SAR studies of tricyclic imidazo[4,5-b]pyridin-2-ones as human corticotropin-releasing factor receptor (CRF(1)) antagonists are discussed herein. Compound 16g was identified as a functional antagonist that inhibited CRF-stimulated cyclic adenosine monophosphate production and CRF-induced adrenocorticotrophic hormone release. Pharmacokinetics studies in rats showed that 16g was orally bioavailable, had good brain penetration, and had a moderate half-life. In our effort to identify CRF(1) antagonists with improved pharmacokinetics properties, 16g exhibited a favorably lower volume of distribution.     

Effects of T-82, a new quinoline derivative, on cholinesterase activity and extracellular acetylcholine concentration in rat brain

The effects of T-82 (2-[2-(1-benzylpiperidin-4-yl)ethyl]-2,3-dihydro-9-methoxy-1H-pyrrolo [3,4-b]quinolin-1-one hemifumarate), a new quinoline derivative, on acetylcholinesterase (AChE) activity and acetylcholine (ACh) release were compared with those of the well-known cholinesterase inhibitors tacrine and E2020. T-82, tacrine and E2020 all concentration-dependently inhibited AChE in rat brain homogenate (IC50 = 109.4, 84.2 and 11.8 nM, respectively). In addition, although tacrine strongly inhibited butyrylcholinesterase (BuChE), T-82 and E2020 showed only weak activity on BuChE in human plasma. In ex vivo experiments, intraperitoneal administration of T-82 at a dose of 30 mg/kg inhibited AChE activity in the hippocampus, frontal cortex and parietal cortex of rats. The effect of T-82 on the extracellular ACh concentration in rat brain was measured using in vivo microdialysis. T-82 at doses of 10 and 30 mg/kg, i.p. increased the extracellular ACh concentration in the hippocampus and striatum in a dose-dependent manner. These findings suggest that T-82 activates the central cholinergic system by selectively inhibiting AChE activity, while weakly affecting peripheral BuChE activity, and that T-82 increases the extracellular ACh concentration in the brain, which is followed by inhibited AChE activity.     

2-Oxo-2H-Pyrimido[2,1-b]benzothiazoles inhibit brain benzodiazepine receptor binding in vitro

2-Oxo-2H-pyrimido[2,1-b]benzothiazole derivatives were found to inhibit the in vitro binding of (3)H-Ro 15-1788 ((3)H-flumazenil) to rat cortical benzodiazepine receptors with IC(50) values in the range of 0.7-13 micromol/l. The most potent compound, 2-oxo-4-phenyl-2H-pyrimido[2,1-b]- benzothiazole showed a similar potency to inhibit (3)H-Ro 15-1788 binding to membrane preparations of rat brain cortex, cerebellum and hippocampus as well as to various subunit combinations of recombinant human gamma-aminobutyric acid(A)/benzodiazepine receptors. Scatchard plot analysis showed that 2-oxo-4-phenyl-2H-pyrimido[2,1-b]benzothiazole is a competitive inhibitor of (3)H-Ro 15-1788 binding to rat brain cortical membrane preparations.     

Modulation of hypothalamic norepinephrine release by atrial natriuretic peptide: involvement of cyclic GMP.

The ability of atrial natriuretic peptide (ANP) to modulate K+-stimulated release of [3H]norepinephrine ([3H]NE) from rat hypothalamic slices was investigated. ANP-(1-28) significantly decreased K+-stimulated [3H]NE release in a concentration-dependent manner (maximal inhibition = 22% of control with 100 nM, ED50 = 70 pM). Pretreatment with pertussis toxin did not alter the response to ANP. 8Br-cGMP (10 microM), a cGMP analog, significantly decreased [3H]NE release and when combined with 10 nM ANP-(1-28), an additive effect was observed. Additionally, 3-isobutyl 1-methylxanthine (IBMX) (200 microM), a phosphodiesterase inhibitor, combined with ANP-(1-28) 10 nM, significantly decreased [3H]NE release. These results indicate that ANP-(1-28) modulated release of [3H]NE from rat hypothalamic slices and the effect is most likely mediated by elevation of intraneuronal cGMP.     

Regional CNS densities of serotonin 1A and dopamine D2 receptors in periadolescent alcohol-preferring P and alcohol-nonpreferring NP rat pups

The objective of the present study was to use quantitative autoradiography to determine binding densities of serotonin(1A) (5-HT(1A)) and dopamine (DA) D(2) receptors in alcohol-naive periadolescent P and NP rat pups. P (n=8) and NP (n=7) rat pups, 25 days of age, from different litters were used. Coronal brain sections were incubated with 2 nM [3H]8-OH-DPAT or 20 nM [3H]sulpiride for 5-HT(1A) or D(2) binding, respectively. Approximately 15-40% higher densities of [3H]8-OH-DPAT binding were observed in the anterior cortical regions of the periadolescent P rat compared with NP rat pups. Similar differences were also observed in posterior cortical regions with P rats having 25-40% higher [3H]8-OH-DPAT binding than NP rats. [3H]8-OH-DPAT binding was approximately 10-20% higher in posterior hippocampal regions of the P rat pups compared with the NP line. [3H]sulpiride binding was significantly different only in the ventral tegmental area (VTA), where binding was approximately 20% lower in the periadolescent P rats compared with the NP rat pups. Overall, these results are very similar to findings observed in adult alcohol-naive P and NP rats, and suggest that the innate differences in the neural systems implicated in high alcohol drinking behaviors may already be established in the periadolescent animal.     

Corticotropin releasing factor stimulation of protein carboxylmethylation in mouse pituitary tumor cells

A putative role for the protein carboxylmethylase (PCM) enzyme has been suggested in exocytotic secretion. The involvement of 3H-methyl incorporation into protein carboxylmethyl esters during corticotropin releasing factor (CRF)-induced ACTH secretion from AtT-20/D16-16 mouse pituitary cells was investigated. Protein carboxylmethylation and ACTH secretion both increased as a function of extracellular CRF concentration, and both processes were temporally parallel up to 60 min incubation. The less potent [Met(O)21]-CRF also stimulated increases in protein carboxylmethylation and ACTH secretion. The free acid analogue of CRF did not alter either process. A combination of the PCM inhibitors, 3-deazaadenosine and L-homocysteine thiolactone, reduced both CRF-stimulated protein carboxylmethylation and ACTH release. Dexamethasone, known to inhibit ACTH secretion and synthesis, inhibited both CRF-stimulated protein carboxylmethylation and ACTH secretion.     

Synthesis and antiproliferative activity in vitro of new 3-substituted aminoisoxazolo[5,4-b]pyridines

The synthesis of 3-aminoisoxazolo[5,4-b]pyridine [III] and of several new 3-substituted aminoisoxazolo[5,4-b]pyridines is described. 3-Aminoisoxazlo[5,4-b]pyridine [III] was subjected to reactions with the acid halides and substituted aromatic aldehydes, leading to the production of the corresponding amides IV-VII, IX, XI-XVI and new tricyclic pyridoisoxazolopyrimidine VIII and Schiff bases XX-XXIV. 4-Chlorobutyroamide IX cyclized into 3-(pyrrolidinon-1-yl)isoxazolo[5,4-b]pyridine [X]. 3-Chloroacetylaminoisoxaxolo[5,4-b]pyridine [V] in reaction with secondary amines gave 3-aminoacetylaminoisoxazolo[5,4-b]pyridines XVII-XIX. The structures of the products II-XXIV were established on the basis of elemental analysis and spectral data IR, 1H NMR and MS. Selected compounds were tested for their antiproliferative activity in vitro. Two of them: 3-chloroacetyl-[V] and 3-2-bromo-propionylaminoisoxazolo[5,4-b]pyridine [VI] revealed cytotoxic activity against the cells of 8 various human or mouse tumor cell lines applied. Their ID50 (inhibitory dose 50%) values are in the range of the international activity criterion for synthetic agents (4 microg/ml).     

Effects of (2R)-(3-amino-2-fluoropropyl)sulphinic acid (AFPSiA) on transient lower oesophageal sphincter relaxation in dogs and mechanism of hypothermic effects in mice

The effects of the novel GABA analogue (2R)-(3-amino-2-fluoropropyl)sulphinic acid (AFPSiA) on transient lower oesophageal sphincter relaxations (TLOSRs) were studied in the dog. In addition, the GABA(A)/GABA(B) selectivity was determined in vitro and in vivo, and the pharmacokinetics and the metabolism of the compound were studied in the dog and rat. TLOSRs were reduced by 55 +/- 8% after intragastric administration of AFPSiA at 14 mumol kg(-1) and did not decrease further at higher doses. When evaluated 2 and 4 h after administration, the effect declined to 37 +/- 6 and 16 +/- 9%, respectively. Spontaneous swallowing was only significantly inhibited at 100 micromol kg(-1). The oral availability of AFPSiA was 52 +/- 17 and 71 +/- 4% in the dog and rat, respectively. A fraction of AFPSiA was oxidised to the corresponding sulphonate, (2R)-(3-amino-2-fluoropropyl)sulphonic acid (AFPSoA) after oral administration to the rat and dog.In rat brain membranes, AFPSiA was found to have ten times higher affinity for rat brain GABA(B) (K(i) =47 +/- 4.4 nM) compared to GABA(A) (K(i) = 430 +/- 46 nM) binding sites. The compound was a full agonist at human recombinant GABA(B(1a,2)) receptors (EC(50) = 130 +/- 10 nM). In contrast, the metabolite AFPSoA was considerably more selective for binding to rat brain GABA(A) (K(i) = 37 +/- 3.1 nM) vs GABA(B) (K(i) = 6800 +/- 280 nM) receptors. In the mouse, high doses (1-8 mmol kg(-1)) of AFPSiA induced a rapid and mild hypothermia followed by a profound and sustained hypothermia at the higher doses tested (6 and 8 mmol kg(-1)). This effect was unaffected by the selective GABA(B) receptor antagonist CGP62349. AFPSoA (1 and 2 mmol kg(-1)) produced transient and moderate hypothermia while the hypothermic response was considerably larger at 4 mmol kg(-1).It is concluded that AFPSiA inhibits but does not abolish TLOSRs in the dog. High doses of the compound induce hypothermia in the mouse, which probably is attributable to activation of the GABA(A) receptor. The latter effect may be caused both by AFPSiA and its oxidised sulphonic acid metabolite AFPSoA.     

Pyrimido[5,4-b]indole derivatives. 1. A new class of potent and selective alpha 1 adrenoceptor ligands.

A number of 3-substituted pyrimido[5,4-b]indole-2,4-diones (7-23) were evaluated for their in vitro alpha 1 adrenoceptor affinity by radioligand receptor binding assays. Some compounds bearing a (phenylpiperazinyl)alkyl side chain were potent alpha 1 adrenoceptor ligands. The most active derivative in displacement of [3H]prazosin from rat cortical membranes was 3-[2-[4-(2-methoxyphenyl)piperazin-1-yl]ethyl]pyrimido[5,4-b]indol e- 2,4-dione (10) (Ki = 0.21 nM). Discrete modifications in the structure resulted in higher selectivity (greater than 10,000 times) for alpha 1 than alpha 2, beta 2, and 5HT1A receptors. Some compounds also had affinity for the 5HT1A receptor. The most selective alpha 1 ligand was 3-[2-[4-(2-chlorophenyl)piperazin-1-yl]ethyl]pyrimido[5,4-b)indole - 2,4-dione (13).     

Angiotensin II-induced responses in vascular smooth muscle cells: inhibition by non-peptide receptor antagonists

The present study investigates the effect of angiotensin II and LR-B/081 (-methyl 2-[[4-butyl-2-methyl-6-oxo-5-[[2′-(1H-tetra-zol-5-yl) [1,1′-biphenyl]-4-yl] methyl]-1(6H)-pyrimidinyl] methyl]-3-thiophenecarboxylate), a novel non-peptide angiotensin II receptor antagonist, on both early and late responses in rat vascular smooth muscle cells. Angiotensin II induced a rapid and transient elevation of inositol trisphosphate intracellular levels, triggered the release of both prostaglandin E₂ and prostaglandin I₂ (EC₅₀ = 21 ± 3 and 16 ± 2 nM, respectively), and, in long-term studies, increased leucine and thymidine incorporation. All angiotensin II effects were antagonized by LR-B/081 and losartan, the reference non-peptide angiotensin AT₁-selective receptor antagonist, whereas they were unaffected by PD123177 (1-(4-amino-3-methylphenyl)methyl-5-diphenylacetyl-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine carboxylic acid), a non-peptide angiotensin AT₂-selective receptor antagonist. LR-B/081 displayed a much higher potency than losartan in inhibiting angiotensin II-induced prostaglandin E₂ (IC₅₀ = 0.15 ± 0.02 and 39 ± 9 nM, respectively) and prostaglandin I₂ release (IC₅₀ = 0.18 ± 0.04 and 134 ± 40 nM, respectively) and was also more potent in blocking the increase in protein synthesis (IC₅₀ = 242 ± 119 nM and 1221 ± 687 nM, respectively). Moreover, LR-B/081 and losartan blocked the response to angiotensin III but failed to inhibit the prostaglandin release stimulated by vasopressin or the mitogenic effect of serum. LR-B/081 and losartan were devoid of intrinsinc agonistic properties in the experimental conditions employed. The present results describe LR-B/081 as a novel, highly specific and potent, non-peptide angiotensin AT₁-selective receptor antagonist, that is capable of blocking angiotensin II-proliferative responses, which may be of relevance for cardiovascular diseases.     

Effect of the tachykinin NK1 receptor antagonists, RP 67580 and SR 140333, on electrically-evoked substance P release from rat spinal cord.

1. The effects of the non-peptide tachykinin NK1 receptor antagonists, RP 67580, SR 140333, CP-96,345 and CP-99,994 have been investigated on electrically-evoked release of substance P-like immunoreactivity (SP-LI) from rat spinal cord slices. 2. RP 67580 (10 nM) and SR 140333 (1 nM), perfused 5 min prior to and during 8 min stimulation of the dorsal roots (20 V, 0.5 ms, 1 Hz), significantly enhanced SP-LI release by 213 +/- 43 (n = 8) and 203 +/- 31 (n = 5) % of control evoked release (187 +/- 16% of basal outflow, n = 22) respectively. Neither compound modified basal outflow of SP-LI (15.3 +/- 2.5 fmol/8 ml, n = 10). 3. RP 67580 (10 nM) did not modify electrically-evoked release of calcitonin gene-related peptide-LI from rat spinal cord slices. 4. CP-96,345 (10 nM) and CP-99,994 (1 and 10 nM) did not alter electrically-evoked SP-LI release; however, they both inhibited release at 1 microM. Inhibition was also induced by 1 microM RP 67580 but not 1 microM SR 140333. 5. The effect of the NK1 receptor agonists, [Sar9Met (O2)11]SP and [Sar9]SP, could not be tested on SP-LI release due to interference with the substance P radioimmunoassay (RIA). The other NK1 receptor agonists used, GR 73632, [Pro9]SP and septide, which did not interfere with the RIA, increased SP-LI basal outflow by 1807 +/- 713% (n = 3), 1259 +/- 160% (n = 3) and 620 +/- 69% (n = 3) at 10 nM, 1 nM and 1 microM, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Substituent effects of N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamides on positive allosteric modulation of the metabotropic glutamate-5 receptor in rat cortical astrocytes

CDPPB [3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide] was recently described as the first centrally active, positive allosteric modulator of rat and human metabotropic glutamate receptor (mGluR) mGluR5 subtype. We explored the structural requirements for potentiation of glutamate-induced calcium release in naturally expressed mGluR5 in cultured rat astrocytes and increasing affinity for the allosteric antagonist binding site by evaluating 50 analogues of CDPPB. In the fluorometric calcium assay, CDPPB exhibited an EC50 value of 77 +/- 15 nM in potentiating mGluR5-mediated responses in cortical astrocytes and a Ki value of 3760 +/- 430 nM in displacing [3H]methoxyPEPy binding in membranes of cultured HEK-293 cells expressing rat mGluR5. The structure-activity relationships showed that electronegative aromatic substituents in the para-position of the benzamide moiety of CDPPB increase potency. Both binding and functional activities were further increased with a halogen atom in the ortho-position of the 1-phenyl ring. These effects of substitution do not match those of either aromatic ring of MPEP [2-methyl-6-(phenylethynyl)pyridine] for the antagonist allosteric binding site. Combination of the optimal substituents and aromatic positions resulted in 4-nitro-N-(1-(2-fluorophenyl)-3-phenyl-1H-pyrazol-5-yl)benzamide (VU-1545) showing Ki = 156 +/- 29 nM and EC50 = 9.6 +/- 1.9 nM in the binding and functional assays, respectively.     

Synthesis and pharmacological characterization of nicotinic acetylcholine receptor properties of (+)- and (-)-pyrido-[3,4-b]homotropanes

(+/-)-Pyrido[3,4-b]homotropane [(+/-)-1] is a conformationally rigid analogue of nicotine (2) or nornicotine (3) that showed high affinity for nicotinic acetylcholine receptors. Even though the synthesis and potent activity of this highly interesting compound was originally reported in 1986 (Kanne, D. B.; Ashworth, D. J.; Cheng, M. T.; Mutter, L. C.; Abood, L. G. Synthesis of the first highly potent bridged nicotinoid. 9-Azabicylo[4.2.l]nona[2,3-c]pyridine (pyrido[3,4-b]homotropane). J. Am. Chem. Soc. 1986, 108, 7864-7865), the individual optical isomers have not been prepared and studied. In this study, we report the synthesis of (+)- and (-)-1 and show that (+)-1 has Ki = 1.29 nM at the alpha4beta2* nAChR and has over 260 times higher affinity than (-)-1. Single-crystal X-ray analysis of an intermediate used to prepare the isomers established the absolute stereochemistry as (1S,6S)-(+)-1 and (1R,6R)-(-)-1. Surprisingly, both isomers failed to produce antinociception in the mouse tail-flick and hot-plate assays, engender nicotine-like responding in rat drug discrimination, or alter current amplitude in alpha4beta2- and alpha3beta4-containing cells. However, (-)-1 antagonized nicotine-induced antinociception with an ED50 of 0.07 microg/kg in the tail-flick assay. The reason for this unusual pharmacology is unknown, but it is possible that (-)-1 is acting at a non-epibatidine-sensitive receptor subtype to antagonize nicotine's effects in the tail-flick assay.     

(−)Tertatolol is a potent antagonist at pre- and postsynaptic serotonin 5-HT1A receptors in the rat brain

Summary The potential 5-HT_1A antagonist properties of the ß-antagonist tertatolol were assessed using biochemical and electrophysiological assays in the rat. (±) Tertatolol bound with high affinity (K_i = 38 nM) to 5-HT_1A sites labelled by [³H]8-OH-DPAT in hippocampal membranes. The (–)stereoisomer (K_i = 18 nM) was about 50-fold more potent than the (+)stereoisomer (K_i = 864 nM) to inhibit the specific binding of [³H]-8-OHDPAT. As expected of a 5-HT_1A antagonist, (–)tertatolol prevented in a concentration-dependent manner (K_i = 24 nM) the inhibitory effect of 8-OH-DPAT on forskolin-stimulated adenylate cyclase activity in rat hippocampal homogenates. Furthermore in vivo pretreatment with (–)tertatolol (5 mg/kg s.c.) significantly reduced the inhibitory influence of 8-OH-DPAT (0.3 mg/ kg s.c.) on the accumulation of 5-hydroxytryptophan in various brain areas after the blockade of aromatic L-amino acid decarboxylase by NSD-1015 (100 mg/kg i.p.). In vitro (in brainstem slices; K_i 50 nM) and in vivo (in chloral hydrate anaesthetized rats; ID₅₀ 0.40 mg/kg i.v.), (–)tertatolol prevented the inhibitory effects of the 5-HT_1A receptor agonists 8-OH-DPAT, ipsapirone and lesopitron on the firing rate of serotoninergic neurones within the dorsal raphe nucleus. In about 25% of these neurones, the basal firing rate was significantly increased by (–)tertatolol (up to +47% in vitro, and +30% in vivo). These data indicate that (-)tertatolol is a potent competitive antagonist at both pre (in the dorsal raphe nucleus) - and post (in the hippocampus) - synaptic 5-HT_1A receptors in the rat brain.Abbreviations ACSF artificial cerebrospinal fluid - BMY-7378 8-[2-[4(2-methoxy-phenyl)-1-piperazinyl]ethyl]-8-azaspiro-[4,5]-decane-7,9dione - DRN dorsal raphe nucleus - 5-HIAA 5-hydroxyindole acetic acid - 5-HT 5-hydroxytryptamine (serotonin) - 5-HTP 5-hydroxytryptophan - MDL 73005 EF 8-[2-[(2,3-dihydro-1,4-benzodioxin-2-yl)-methylamino]ethyl]-8-azaspiro-[4,5]-decane-7,9-dione, methyl sulfonate - NAN-190 1-(2-methoxyphenyl)-4-[4-(2-phthalimido)butyl]piperazine - 8-OH-DPAT 8-hydroxy-2-(di-n-propylamino) tetralin - (S)-UH-301 (S)-5-fluoro-8-hydroxy-2-(dipropylamino) tetralin - WAY-100,135 N-tert-butyl-3,4-(2-methoxyphenyl)-piperazin-1-yl-2-phenylpropanamide Correspondence to T. Jolas at the above address     

sigma receptors in rat brain and testes show similar reductions in response to chronic haloperidol.

The effect of haloperidol upon [3H]1,3-di-o-tolylguanidine ([3H]DTG) binding sites was assessed in rat brain and testes. An acute injection (10 mg/kg), in rats culled 2 h later, changed Kd values from 18 +/- 2 to 108 +/- 26 nM (brain) and 14 +/- 1 to 116 +/- 35 nM (testes), with unchanged Bmax values. Rats were injected with 4 mg/kg per day for 7, 14 and 21 days and culled 4 days after the last injection. By day 21, there was an average fall in Bmax for brain of 30% and for testes of 38%. Kd values remained unchanged. Thus peripheral and central [3H]DTG binding sites were reduced by chronic haloperidol in a similar way.