Approaches to protection against nerve agent poisoning. (Naphthylvinyl)pyridine derivatives as potential antidotes

Analogues of the potent inhibitor of choline acetyltransferase (CAT) (E)-4-(1-naphthylvinyl)pyridine methiodide were synthesized and evaluated for their ability to inhibit CAT and protect against nerve agent intoxication. Several compounds, notably (E)-1-(2-hydroxyethyl)-(1-naphthylvinyl)pyridinium bromide (3), (E)-1-methyl-4-(1-naphthylvinyl)-1,2,3,6-tetrahydropyridine hydrochloride (22), and (E)-1-methyl-4-(1-naphthylvinyl)piperidine hydrochloride (23), were found to afford significant protection against sarin in the mouse and against soman in the guinea pig. However, protection was apparently not related to CAT inhibition. Compound 23, our most effective compound in protecting against nerve agent, was without CAT inhibitory activity. Compound 22, which proved to be a potent CAT inhibitor, most likely owed this activity to being dehydrogenated back to the pyridinium quaternary salt by oxidative enzymes. Several of the (naphthylvinyl)pyridine quaternary salts, but not their tertiary amine analogues, were found to be effective in slowing the rate of aging of soman-inhibited acetylcholinesterase. Ability to slow the rate of aging was enhanced by introduction of methoxy substituents on the aryl moiety whereas the aging rate was actually accelerated by chloro substituents. To date, our most effective compound in slowing the rate of aging, (E)-4-[(4-methoxy-1-naphthyl)vinyl]pyridine methochloride (6), did not provide significant protection against soman in the mouse.     

Discovery of diaryl imidazolidin-2-one derivatives, a novel class of muscarinic M3 selective antagonists (Part 1)

Pharmacophore-based structural identification, synthesis, and structure-activity relationships of a new class of muscarinic M3 receptor antagonists, the diaryl imidazolidin-2-one derivatives, are described. The versatility of the discovered scaffold allowed for several structural modifications that resulted in the discovery of two distinct classes of compounds, specifically a class of tertiary amine derivatives (potentially useful for the treatment of overactive bladder by oral administration) and a class of quaternary ammonium salt derivatives (potentially useful for the treatment of respiratory diseases by the inhalation route of administration). In this paper, we describe the synthesis and biological activity of tertiary amine derivatives. For these compounds, selectivity for the M3 receptor toward the M2 receptor was crucial, because the M2 receptor subtype is mainly responsible for adverse systemic side effects of currently marketed muscarinic antagonists. Compound 50 showed the highest selectivity versus M2 receptor, with binding affinity for M3 receptor Ki = 4.8 nM and for M2 receptor Ki = 1141 nM. Functional in vitro studies on selected compounds confirmed the antagonist activity toward the M3 receptor and functional selectivity toward the M2 receptor.     

Tertiary 3- and 4-haloalkylamine analogues of oxotremorine as prodrugs of potent muscarinic agonists

A series of tertiary 3- and 4-haloalkylamines related to the muscarinic agent oxotremorine was synthesized. The compounds cyclized in neutral aqueous solution to quaternary ammonium salts, which, in contrast to the parent haloalkylamines, were potent muscarinic agonists in vitro. When administered systemically to mice, the haloalkylamines produced central (tremor and analgesia) and peripheral (salivation) muscarinic effects. Central potency was dependent on the rate of cyclization and on the route of administration. The N-methyl-N-(4-chlorobutyl)amine derivative 7 cyclized rapidly (t1/2 less than 0.4 min at 37 degrees C) and elicited tremor on iv but not on ip injection, whereas the N-methyl-N-(3-chloropropyl)amine 3 cyclized slowly (t1/2 = 436 min) and was not tremorogenic by either route of administration. The N-methyl-N-(3-bromopropyl)amine 4(t1/2 = 11 min) and its iodo analogue 5 (t1/2 = 14 min) were quite potent in eliciting central muscarinic effects on both iv and ip injection to mice. It is concluded that haloalkylamine analogues of oxotremorine may serve in vivo as prodrugs for potent quaternary ammonium salts and that they are capable of circumventing the blood-brain barrier to such salts.     

Effect of choline acetyltransferase inhibitors on mouse and guinea-pig brain choline and acetylcholine

These experiments measure the effect of two choline acetyltransferase (CAT) inhibitors, viz. 4-(1-naphthylvinyl) pyridine (NVP) and 4-(3-chlorophenylvinyl) pyridine (3'-chloro-4-stilbazole; CS), on mouse and guinea-pig brain acetylcholine (ACh) and choline. The intraperitoneal administration of NVP or CS appeared to inhibit CAT partially in both species, but both compounds were without effect on steadystate levels of ACh. In the mouse CS, but not NVP, increased brain choline. Both CS and NVP were shown to reduce, but not totally inhibit, the synthesis of mouse brain ACh. The inability of these compounds to decrease steady state levels, while apparently decreasing synthesis rates, suggests that CAT may not be the rate-limiting step in ACh synthesis.     

The effect of 4-(1-naphthylvinyl)-pyridine (NVP), a choline acetylase inhibitor, on autonomically innervated tissues of the rat

The effect of 4-(1-naphthylvinyl)-pyridine (NVP), an inhibitor of choline acetyltransferase, was investigated on responses to several cholinomimetics and to nervous stimulation of the rat bladder. NVP antagonised responses to acetylcholine (ACh) as well as responses to nervous stimulation suggesting that it has an inhibitory action on the smooth muscle. Furthermore, the reduction in the responses to nervous stimulation was independent of the frequency of stimulation suggesting that inhibition of choline acetyltransferase was not a factor in the effect of NVP on nervous stimulation. Another finding was that NVP produced more marked inhibition of responses to ACh and methacholine than of those to carbachol (CCh) or tetramethylammonium. Responses of the bladder to Ba²⁺ and bradykinin were also antagonised by high concentrations of NVP. Negative inotropic responses of driven rat atria to ACh were also inhibited by NVP whereas responses to CCh were not affected. On rat ileum however, both agonists were affected to a similar extent. The results indicate that NVP is not useful as a tool to investigate the effect of selective inhibition of choline acetyltransferase in the isolated parasympathetically innervated tissues investigated because of its inhibitory effects on directly acting agonists. However, the more marked inhibition of responses to ACh than to CCh in some tissues suggests that it may be of value in detecting differences between the binding of these two agonists to the muscarinic receptor.     

Effects of imidafenacin (KRP-197/ONO-8025), a new anti-cholinergic agent, on muscarinic acetylcholine receptors. High affinities for M3 and M1 receptor subtypes and selectivity for urinary bladder over salivary gland

Imidafenacin (CAS 170105-16-5, KRP-197, ONO-8025) is an antagonist for the muscarinic acetylcholine (ACh) receptor currently under development for the treatment of overactive bladder. Affinities of imidafenacin and other drugs for muscarinic ACh receptor subtypes were investigated by examining inhibitory effects on ACh release in the rat urinary bladder and K+ efflux in the rat salivary gland in functional and binding assays. In the functional assay, imidafenacin had higher affnities for M3 and M1 receptors than for the M2 receptor. In contrast, metabolites of imidafenacin (M-2, M-4 and M-9) had low affinities for muscarinic ACh receptor subtypes. Darifenacin had selectivity for the M3 receptor, while propiverine, tolterodine and oxybutynin had no selectivity for muscarinic ACh receptors. In carbamylcholine (CCh)-induced contraction in the urinary bladder, imidafenacin, propiverine, tolterodine and oxybutynin had affinities similar to those for the M3 receptor in the ileum. In the binding assay for human muscarinic ACh receptor subtypes, imidafenacin had higher affinities for m3 and m1 receptors than for m2 receptor, but tolterodine had no selectivity for m1, m2 and m3 receptors. In ACh release in the urinary bladder, inhibitory effects of imidafenacin, tolterodine, oxybutynin and darifenacin seemed to be partially mediated by the M1 receptor. In ACh-induced and electrical stimulation-induced K+ efflux from the salivary gland, inhibitory effects (IC50) of imidafenacin, propiverine, tolterodine, oxybutynin and darifenacin might be closely related to those for the M3 receptor in the ileum. These results suggest that imidafenacin more strongly antagonizes cholinomimetics on M3 and M1 receptors than on the M2 receptor. Moreover, imidafenacin seems to inhibit the contraction of the bladder smooth muscle by mediating antagonism to the M3 receptor and to regulate ACh release by mediating prejunctional facilitatory M1 receptor. Imidafenacin also inhibited K+ efflux from the salivary gland mainly by mediating the M3 receptor. Therefore, imidafenacin will have higher affinities for M3 and M1 receptors and higher selectivity for the urinary bladder than for the salivary gland.     

Inhibition of acetylcholine muscarinic M(1) receptor function by the M(1)-selective ligand muscarinic toxin 7 (MT-7)

MT-7 (1 - 30 nM), a peptide toxin isolated from the venom of the green mamba Dendroaspis angusticeps and previously found to bind selectively to the muscarinic M(1) receptor, inhibited the acetylcholine (ACh)-stimulated [(35)S]-guanosine-5'-O-(3-thio)triphosphate ([(35)S]-GTPgammaS) binding to membranes of Chinese hamster ovary (CHO) cells stably expressing the cloned human muscarinic M(1) receptor subtype. MT-7 failed to affect the ACh-stimulated [(35)S]-GTPgammaS binding in membranes of CHO cells expressing either the M(2), M(3) or M(4) receptor subtype. In N1E-115 neuroblastoma cells endogenously expressing the M(1) and M(4) receptor subtypes, MT-7 (0.3 - 3.0 nM) inhibited the carbachol (CCh)-stimulated inositol phosphates accumulation, but failed to affect the CCh-induced inhibition of pituitary adenylate cyclase activating polypeptide (PACAP) 38-stimulated cyclic AMP accumulation. In both CHO/M(1) and N1E-115 cells the MT-7 inhibition consisted in a decrease of the maximal agonist effect with minimal changes in the agonist EC(50) value. In CHO/M(1) cell membranes, MT-7 (0.05 - 25 nM) reduced the specific binding of 0.05, 1.0 and 15 nM [(3)H]-N-methylscopolamine ([(3)H]-NMS) in a concentration-dependent manner, but failed to cause a complete displacement of the radioligand. Moreover, MT-7 (3 nM) decreased the dissociation rate of [(3)H]-NMS by about 5 fold. CHO/M(1) cell membranes preincubated with MT-7 (10 nM) and washed by centrifugation and resuspension did not recover control [(3)H]-NMS binding for at least 8 h at 30 degrees C. It is concluded that MT-7 acts as a selective noncompetitive antagonist of the muscarinic M(1) receptors by binding stably to an allosteric site.     

Different Behavior Toward Muscarinic Receptor Binding Between Quaternary Anticholinergics and Their Tertiary Analogues

A number of corresponding tertiary and quaternary anticholinergic analogues were examined for their ability to inhibit specific ³H-dexetimide binding to calf brain muscarinic receptors. In all cases the tertiary antagonists (except pirenzepine) showed steep and monophasic inhibition curves, whereas those of the quaternary derivatives were shallow (thiazinamium, methylbenactyzine) or even biphasic (oxyphenonium, methylatropine, methylscopolamine). These observations show that the addition of a methyl group to the nitrogen atom changes the mode of interaction of the anticholinergics to muscarinic receptor binding sites. Whether there are separate binding sites present or differences in interaction mode for only the quaternary moiety is discussed.     

Therapeutic efficacy of the adenosine A1 receptor agonist N6-cyclopentyladenosine (CPA) against organophosphate intoxication

The objective of the present study was to investigate whether reduction of central acetylcholine (ACh) accumulation by adenosine receptor agonists could serve as a generic treatment against organophosphate (OP) poisoning. The OPs studied were tabun ( O-ethyl- N-dimethylphosphoramidocyanidate), sarin (isopropylmethylphosphonofluoridate), VX ( O-ethyl- S-2-diisopropylaminoethylmethylphosphonothiolate) and parathion ( O, O-diethyl- O-(4-nitrophenyl)phosphorothioate). The efficacy of the adenosine A(1) receptor agonist N(6)-cyclopentyladenosine (CPA) against an OP intoxication was examined on the basis of the occurrence of clinical symptoms that are directly associated with such intoxication. CPA (1-2 mg/kg) effectively attenuated the cholinergic symptoms and prevented mortality in lethally tabun- or sarin-intoxicated rats. In contrast, CPA (2 mg/kg) proved to be ineffective against VX or parathion intoxication. Intracerebral microdialysis studies revealed that survival of sarin-poisoned and CPA-treated animals coincided with a minor elevation of extracellular ACh concentrations in the brain relative to the baseline value, whereas an 11-fold increase in transmitter levels was observed in animals not treated with CPA. In VX-intoxicated rats, however, the ACh amounts increased 18-fold, irrespective of treatment with CPA. The striatal acetylcholinesterase (AChE) activity following a lethal sarin intoxication was completely abolished in the vehicle-treated animals, whereas 10% and 60% AChE activity remained in animals treated with 2 mg/kg CPA 1 min after or 2 min prior to the poisoning, respectively. In VX-intoxicated animals the AChE activity in the brain was strongly reduced (striatum 10%, hippocampus 1%) regardless of the CPA treatment. These results demonstrate that CPA is highly effective against tabun or sarin poisoning, but fails to protect against VX or parathion. Survival and attenuation of clinical signs in tabun- or sarin-poisoned animals are associated with a reduction of ACh accumulation and with protection of AChE activity in the brain.     

Comparative effects of oral chlorpyrifos exposure on cholinesterase activity and muscarinic receptor binding in neonatal and adult rat heart

Organophosphorus (OP) pesticides elicit acute toxicity by inhibiting acetylcholinesterase (AChE), the enzyme responsible for inactivating acetylcholine (ACh) at cholinergic synapses. A number of OP toxicants have also been reported to interact directly with muscarinic receptors, in particular the M(2) muscarinic subtype. Parasympathetic innervation to the heart primarily regulates cardiac function by activating M(2) receptors in the sinus node, atrial-ventricular node and conducting tissues. Thus, OP insecticides can potentially influence cardiac function in a receptor-mediated manner indirectly by inhibiting acetylcholinesterase and directly by binding to muscarinic M(2) receptors. Young animals are generally more sensitive than adults to the acute toxicity of OP insecticides and age-related differences in potency of direct binding to muscarinic receptors by some OP toxicants have been reported. We thus compared the effects of the common OP insecticide chlorpyrifos (CPF) on functional signs of toxicity and cardiac cholinesterase (ChE) activity and muscarinic receptor binding in neonatal and adult rats. Dosages were based on acute lethality (i.e., 0.5 and 1x LD(10): neonates, 7.5 and 15 mg/kg; adults, 68 and 136 mg/kg). Dose- and time-related changes in body weight and cholinergic signs of toxicity (involuntary movements) were noted in both age groups. With 1x LD(10), relatively similar maximal reductions in ChE activity (95%) and muscarinic receptor binding (approximately 30%) were noted, but receptor binding reductions appeared earlier in adults and were more prolonged in neonates. In vitro inhibition studies indicated that ChE in neonatal tissues was markedly more sensitive to inhibition by the active metabolite of chlorpyrifos (i.e., chlorpyrifos oxon, CPO) than enzyme in adult tissues (IC(50) values: neonates, 17 nM; adults, 200 nM). Chelation of free calcium with EDTA had relatively little effect on in vitro cholinesterase inhibition, suggesting that differential A-esterase activity was not responsible for the age-related difference in cholinesterase sensitivity between age groups. Pre-incubation of neonatal and adult tissues with selective inhibitors of AChE and butyrylcholinesterase (BChE) indicated that a majority (82-90%) of ChE activity in the heart of both neonates and adults was BChE. The rapid onset (by 4h after dosing) of changes in muscarinic receptor binding in adult heart may be a reflection of the more potent direct binding to muscarinic receptors by chlorpyrifos oxon previously reported in adult tissues. The results suggest that ChE activity (primarily BChE) in neonatal heart may be inherently more sensitive to inhibition by some anticholinesterases and that toxicologically significant binding to muscarinic receptors may be possible with acute chlorpyrifos intoxication, potentially contributing to age-related differences in sensitivity.     

Evidence for a M(1) muscarinic receptor on the endothelium of human pulmonary veins

1. To characterize the muscarinic receptors on human pulmonary veins associated with the acetylcholine (ACh)-induced relaxation, isolated venous and arterial preparations were pre-contracted with noradrenaline (10 microM) and were subsequently challenged with ACh in the absence or presence of selective muscarinic antagonists. 2. ACh relaxed venous preparations derived from human lung with a pD(2) value of 5.82+/-0.09 (n=16). In venous preparations where the endothelium had been removed, the ACh relaxations were abolished (n=4). ACh relaxed arterial preparations with a pD(2) value of 7. 06+/-0.14 (n=5). 3. Atropine (1 microM), the non selective antagonist for muscarinic receptors, inhibited ACh-induced relaxations in human pulmonary veins. The affinity value (pK(B) value) for atropine was: 8.64+/-0.10 (n=5). The selective muscarinic antagonists (darifenacin (M(3)), himbacine (M(2),M(4)), methoctramine (M(2)) and pFHHSiD (M(1),M(3))) also inhibited ACh-induced relaxations in venous preparations. The pK(B) values obtained for these antagonists were not those predicted for the involvement of M(2 - 5) receptors in the ACh-induced relaxation in human pulmonary veins. 4. The pK(B) value for darifenacin (1 microM) was significantly greater in human pulmonary arterial (8.63+/-0.14) than in venous (7.41+/-0.20) preparations derived from three lung samples. 5. In human pulmonary veins, the pK(B) values for pirenzepine (0.5 and 1 microM), a selective antagonist for M(1) receptors, were: 7.89+/-0.24 (n=7) and 8.18+/-0.22 (n=5), respectively. In the venous preparations, the pK(B) values derived from the functional studies with all the different muscarinic antagonists used were correlated (r=0.89; P=0.04; slope=0.78) with the affinity values (pK(i) values) previously published for human cloned m1 receptors in CHO cells. 6. These results suggest that the relaxations induced by ACh are due to the activation of M(1) receptors on endothelial cells in isolated human pulmonary veins.     

Desensitization of muscarinic M1 receptors of murine neuroblastoma cells (clone N1E-115) without receptor down-regulation and protein kinase C activity

Acute desensitization of M1 muscarinic receptor-mediated responses (cyclic GMP formation and inositol phosphate release) was studied in murine neuroblastoma cells (N1E-115 clone). After a 45-min incubation at 37 degrees of N1E-115 cells either in monolayer or in suspension, with the muscarinic agonist carbachol (1 mM), the receptor-mediated cyclic GMP response to carbachol was nearly completely lost. This loss was associated with greater than 80% loss of carbachol-mediated inositol phosphate release. The protein kinase C activator phorbol 12-myristate 13-acetate (PMA) inhibited both responses with similar potencies. Carbachol or PMA reduced by 30-40% the number of muscarinic receptor sites for antagonist and agonist on intact cells (determined in binding assays using [3H]N-methylscopolamine) only for cells in monolayer and not for those in suspension. PMA but not carbachol pretreatment of cells in monolayer or in suspension caused a translocation of [3H]phorbol 12,13-dibutyrate binding and protein kinase C activity. In addition, desensitization to carbachol occurred in cells largely depleted of protein kinase C by chronic exposure to PMA. Thus, agonist-mediated down-regulation is not needed for muscarinic M1 receptor desensitization, which may be a result of the activation of a receptor-activated kinase different from protein kinase C.     

Reduction in acetylcholine release in the hippocampus of dopamine D5 receptor-deficient mice.

Activation of the dopamine D1-like receptor stimulates acetylcholine (ACh) release in the hippocampus, apparently through the molecularly defined d5 receptor. In the present study, we used a transgenic mouse completely deprived of functional d5 receptor (d5-/-) to confirm the role and elucidate the possible function of the d5 receptor subtype on hippocampal cholinergic neurotransmission. ACh release was measured using in vivo microdialysis in the mouse dorsal hippocampus of 4 months old homozygous (d5-/-), heterozygous (d5+/-), and the wild-type (d5+/+) littermates. Using the no net flux technique, a significant reduction in basal hippocampal ACh level was found in the d5-/- compared to d5+/- and d5+/+ mice. Moreover, the administration of SKF 38393, a D1-like receptor agonist, systemically (2.0 and 10.0 mg/kg ip), or locally through the dialysis probe (10 and 50 microM), produced a dose-dependent enhancement of ACh release in the d5+/+, a moderate stimulation in the d5+/- but had no effect in the d5-/- mice. Quantitative receptor autoradiography revealed significant increases in M1-like but not in M2-like muscarinic receptor binding sites in the hippocampal formation. These results confirm and extend the role of the d5 receptor in the modulation of hippocampal ACh release and provide evidence for long-term alteration of hippocampal cholinergic neurotransmission resulting from the absence of the d5 receptors including chronically reduced ACh release and change in M1-like receptor levels.     

[3H]-(+)-N-methyl-4-methyldiphenhydramine, a quaternary radioligand for the histamine H1-receptor.

1. A series of derivatives of 4-methyldiphenhydramine have been examined as potential quaternary radioligands for the histamine H1-receptor. 2. [3H]-(+)-N-methyl-4-methyldiphenhydramine ([3H]-QMDP), 83 Ci mmol-1, was synthesized by methylation of the tertiary analogue and purified by high-voltage electrophoresis. 3. [3H]-QMDP bound to H1-receptors in a washed homogenate from guinea-pig cerebellum with an affinity constant, Ka, of 1.14 +/- 0.03 x 10(9) M-1. The proportion of non-specific binding of 0.3-0.6 nM [3H]-QMDP, defined by 0.4 microM mepyramine, was usually in the range 15-45%, depending on the method of measurement of binding. The affinity of [3H]-QMDP was similar in guinea-pig cerebellum, cerebral cortex and hippocampus, but was lower, 1.4 x 10(8) M-1, in rat cerebral cortex. 4. Evidence was obtained for the presence of a secondary, non-muscarinic, binding site for [3H]-QMDP in guinea-pig cerebellum, approximate Ka 1.5 x 10(7) M-1, accounting for circa 4% of the total binding of 1 nM [3H]-QMDP. 5. There was a very good correlation between the affinities of 15 compounds for the H1-receptor determined from inhibition of [3H]-QMDP binding and from inhibition of [3H]-mepyramine binding. 6. The potential utility of [3H]-QMDP for studies of H1-receptors in the plasma membrane of cells in culture is discussed.     

Subtype-selective inhibition of [methyl-3H]-N-methylscopolamine binding to muscarinic receptors by α-truxillic acid esters

Seven esters of alpha-truxillic acid have been synthesized: bis-3-piperidylpropyl ester and its quaternary bis-N-ethyl derivative, bis-N-diethylaminopropyl ester and its quaternary bis-N-methyl derivative, and bis-4-piperidylbutyl ester and its quaternary bis-N-methyl and bis-N-ethyl derivatives. All esters inhibited the specific binding of muscarinic receptor antagonist [methyl-3H]-N-methylscopolamine ([3H]-NMS) to muscarinic receptors in membranes of CHO cell lines stably expressing the human gene for the M1, M2, M3 or M4 subtype of muscarinic receptors. All esters displayed the highest potency at the M2 and the lowest potency at the M3 receptor subtype. In experiments performed on the M2 muscarinic receptor subtype, the affinity between the receptors and the esters was greatly increased when the concentration of ions was diminished. The highest affinities were found for the tertiary bis-3-piperidylpropyl and bis-4-piperidylbutyl aminoesters (equilibrium dissociation constants of 52 and 179 pM, respectively, in the low ionic strength medium). All investigated esters slowed down the dissociation of [3H]-NMS from the M2 muscarinic receptor subtype. [3H]-NMS dissociation from the M1, M3 and M4 muscarinic receptor subtypes was investigated in experiments with the bis-4-piperidylbutyl aminoester and also found to be decelerated. It is concluded that the esters of alpha-truxillic acid act as M2-selective allosteric modulators of muscarinic receptors and that, by their potency, the tertiary bis-3-piperidylpropyl and bis-4-piperidylbutyl aminoesters surpass the other known allosteric modulators of these receptors.     

Muscarinic responses and binding in a murine neuroblastoma clone (N1E-115). Selective loss with subculturing of the low-affinity agonist site mediating cyclic GMP formation

Cells of the murine neuroblastoma clone N1E-115 possess muscarinic receptors that influence the intracellular level of cyclic nucleotides. The stimulation of [3H]cyclic GMP levels occurs only with intact cells and has an EC50 near the "low-affinity" agonist equilibrium dissociation constant (KL) determined by radioligand binding assays. The inhibition of prostaglandin E1-stimulated [3H]cyclic AMP formation has an EC50 close to the value for the "high-affinity" agonist equilibrium dissociation constant (KH). During sequential subculturing in medium supplemented with newborn bovine serum, the inhibition of [3H]cyclic AMP was maintained, but the [3H]cyclic GMP response declined dramatically, and after 7 subculturings it was essentially absent. The time course for [3H]cyclic GMP formation in a late subculture with an 88% loss of the response was identical with the time course in early subcultures. A normal [3H]cyclic GMP response to bradykinin and histamine was demonstrated to be present in cells that had lost the [3H]cyclic GMP response to carbachol. The EC50 and KD values for the two muscarinic responses and binding sites increased 3- to 4-fold after several subculturings. A 90% loss of low-affinity binding sites was closely correlated with a similar loss of the [3H]cyclic GMP response. High-affinity binding sites did not decline significantly in concentration until the 11th subculture, where the total number of muscarinic sites was only 6% of the earliest subculture. In all subcultures, however, the ability of the muscarinic receptor to decrease [3H]cyclic AMP levels was maintained. These data, which show that the subculturing of N1E-115 cells in medium supplemented with newborn calf serum results in a selective loss of one muscarinic function, strongly support the hypothesis that these cells contain two separate muscarinic receptor-effector systems. One receptor subtype or conformation has a low affinity for the agonist and mediates cyclic GMP formation. The other receptor subtype or conformation has a higher affinity for the agonist and mediates an inhibition of prostaglandin E1-stimulated cyclic AMP formation.     

A new class of high affinity ligands for the neurokinin A NK2 receptor: psi (CH2NR) reduced peptide bond analogues of neurokinin A4-10.

Analogues of [Leu10]NKA4-10 were synthesized in which each of the amide bonds was sequentially replaced with the reduced amide psi (CH2NH) bond to determine the effect of this structural modification on the antagonism of NKA binding to the HUB NK2 receptor. [psi (CH2-NH)9,Leu10]NKA4-10 (6) retained significant affinity for the NK2 receptor (IC50 = 115 nM) and showed weak partial stimulation of PI turnover (approximately 10-15% of NKA maximum). 6 behaves as a competitive antagonist of NKA-stimulated PI turnover with a pA2 = 6.7. The secondary amine of the psi (CH2NH) moiety of 6 was converted to a tertiary amine by alkylation. This modification was found to have a small effect upon receptor affinity but did result in attenuation of partial agonist activity. A combination of amino acid substitutions and psi (CH2NH) alkylation yielded [beta Ala8,psi (CH2N(CH2)2CH3)9,Phe10]NKA4-10 (21) which has very high affinity for the HUB NK2 receptor. This compound inhibited [125I]NKA binding with an IC50 = 1 nM which is equal to the receptor affinity of NKA. Compound 21 also shows very weak partial agonism of PI turnover (< or = 5% of NKA maximum) which makes this the most potent member of a new class of NKA ligands: psi(CH2NR)9-NKA4-10 analogues which potently antagonize NKA binding and possess minimal partial agonist activity.     

(+/-)alpha-Phenyl-beta-(3,4-dimethoxy)- and (+/-)alpha-phenyl-beta -(3,4-dihydroxy)phenethylamines: potential probes for nicotinic acetylcholine receptor-ion channel molecule from torpedo electric organ

The synthesis of some N-methyl, N-alkyl derivatives of (+/-)alpha-phenyl-beta-(3,4-dimethoxy)- and (+/-)alpha-phenyl-beta-(3,4-dihydroxy)-phenethylamines was achieved. These compounds were shown to bear certain structural features of acetylcholine (ACh), as well as phencyclidine (PCP). The latter was reported to act as a specific probe for the nicotinic ACh receptor-ion channel molecule from Torpedo electric organ. Biochemical binding studies revealed that for the nicotinic ACh receptor, the 3,4-dimethoxy derivatives behaved as blockers for the binding interaction of [3H]ACh, whereas the 3,4-dihydroxy analogues stimulated such binding. On the other hand, all of the tested phenethylamines exhibited potent blockade towards [3H]PCP binding interactions. The results indicated that the tested compounds might be applied as potential probes for the ACh receptor-ion channel molecule.     

Chemical synthesis and molecular pharmacology of hydroxylated 1-(1-phenylcyclohexyl-piperidine derivatives

The following monohydroxy derivatives of 1-(1-phenylcyclohexyl)piperidine (phencyclidine, PCP) were synthesized: o-, m-, and p-phenols of PCP, 1-(1-phenylcyclohexyl)-4-piperidinol, and two stereoisomeric pairs of 3-phenyl-3-(1-piperidinyl)cyclohexanol and 4-phenyl-4-(1-piperidinyl)cyclohexanol. Inhibition of specific binding of tritiated PCP, morphine, or quinuclidinyl benzylate (QNB) in rat brain homogenates was measured for these compounds. Inhibition of PCP binding for selected compounds correlated with mouse rotarod assay activity. The most characteristic effects of hydroxylation of PCP on the cyclohexyl, piperidine, or phenyl moieties are the following: (i) it generally decreases its activity in inhibiting [3H]PCP binding by a factor of 10 to 80; (ii) it does not produce a large variation in the affinity for the morphine receptor; (iii) it produces a considerable decrease of the affinity for the muscarinic receptor. An important exception to these general observations was the metaphenolic derivative of PCP. This PCP derivative has an affinity for the [3H]PCP binding sites that is 8 times higher than that of PCP itself; its affinity for the muscarinic receptor is only twice lower than that of PCP, but its affinity for the morphine receptor is 430 times higher than that of PCP and only one order of magnitude lower than that of morphine itself.