Synthesis of a series of γ-amino alcohols comprising an N-methyl isoindoline moiety and their evaluation as NMDA receptor antagonists

We report a series of new stereoisomeric γ-amino alcohols comprising an N-methyl isoindoline moiety as ligands for the ifenprodil binding site of the NMDA receptor. Among the four series of stereoisomers, 8a-c, 9a-c, 10a-c, and 11a-c, synthesised, the highest potencies and NMDA-NR2B subtype selectivity was found for the methyl derivative 11a and the chloro derivative 11c, both possessing the [1S,1'S] configuration. However, additional moderate potency of 11a and 11c at the hERG channel with values of 2.6 ± 2.4% and 1.6 ± 2.0%, respectively, rendered them unsuitable for medical use.     

Characterisation of N-methyl-D-aspartate receptor-specific [(3)H]Ifenprodil binding to recombinant human NR1a/NR2B receptors compared with native receptors in rodent brain membranes

We have performed [(3)H]ifenprodil binding experiments under NMDA receptor-specific assay conditions to provide the first detailed characterisation of the pharmacology of the ifenprodil site on NMDA NR1/NR2B receptors, using recombinant human NR1a/NR2B receptors stably expressed in L(tk-) cells, in comparison with rat cortex/hippocampus membranes. [(3)H]Ifenprodil bound to a single, saturable site on both human recombinant NR1a/NR2B receptors and native rat receptors with B:(max) values of 1.83 and 2.45 pmol/mg of protein, respectively, and K:(D) values of 33.5 and 24.8 nM:, respectively. The affinity of various ifenprodil site ligands-eliprodil, (R:(*), R:(*))-4-hydroxy-alpha-(4-hydroxyphenyl)-beta-methyl-4-pehnyl-1-pi per idineethanol [(+/-)-CP-101,606], cis-3-[4-(4-fluorophenyl)-4-hydroxy-1-piperidinyl]-3, 4-dihydro-2H:-1-benzopyran-4,7-diol [(+/-)-CP-283,097], and (R:(*), S:(*))-alpha-(4-hydroxyphenyl)-beta-methyl-4-(phenylmethyl)-1-piperid inepropanol [(+/-)-Ro 25-6981] was very similar for inhibition of [(3)H]ifenprodil binding to recombinant human NR1a/NR2B and native rat receptors, whereas allosteric inhibition of [(3)H]ifenprodil binding by polyamine site ligands (spermine, spermidine, and arcaine) showed approximately twofold lower affinity for recombinant receptors compared with native receptors. Glutamate site ligands were less effective at modulating [(3)H]ifenprodil binding to recombinant NR1a/NR2B receptors compared with native rat receptors. The NMDA receptor-specific [(3)H]ifenprodil binding conditions described were also applied to ex vivo experiments to determine the receptor occupancy of ifenprodil site ligands [ifenprodil, (+/-)-CP-101,606, (+/-)-CP-283,097, and (+/-)-Ro 25-6981] given systemically.     

A2B adenosine receptor agonists: synthesis and biological evaluation of 2-phenylhydroxypropynyl adenosine and NECA derivatives

In the search for agonists for the elusive A2B adenosine receptor subtypes, 2-phenylhydroxypropynyl-5'-N-methylcarboxamido adenosine (PHPMECA, 14), 2-phenylhydroxypropynyl-5'-N-propylcarboxamido adenosine (PHPPECA, 15), and N6-ethyl-2-phenylhydroxypropynyl-5'-N-ethylcarboxamidoadenosine (19) were synthesized on the basis that introduction of alkynyl chains in 2-position of adenosine derivatives resulted in reasonably good A2B potency compared to NECA [see N6-ethyl-2-phenylhydroxypropynyl adenosine (5) EC50 = 1,700 nM and 2-phenylhydroxypropynyl-5'-N-ethylcarboxamido adenosine (PHPNECA, 8) EC50 = 1,100 nM, respectively]. Radioligand binding studies and adenylyl cyclase assays, performed with recently cloned human A1, A2A, A2B, and A3 adenosine receptors, showed that these modifications produced a decrease in potency at A2B receptor, as well as a general reduction in affinity at the other receptor subtypes. On the other hand, the contemporary presence of an ethyl substituent in N6-position and of a 4'-ethylcarboxamido group in the same compounds led to (R,S)-N6-ethyl-2-phenylhydroxypropynyl-5'-N-ethylcarboxamidoadenosine and (S)-N6-ethyl-2-phenylhydroxypropynyl-5'-N-ethylcarboxamidoadenosine, which did not show the expected increase in potency at A2B subtype. Hence, (S)-2-phenylhydroxypropynyl-5'-N-ethylcarboxamidoadenosine [(S)-PHPNECA] with EC50 A2B = 220 nM remains the most potent agonist at A2B receptor reported so far.     

Actions of terazosin and its enantiomers at subtypes of α- and α2-Adrenoceptors in vitro

Abstract

Terazosin and its enantiomers, antagonists of α1-adrenoceptors, were studied in radioligand binding and functional assays to determine relative potencies at subtypes of α1- and α2-adrenoceptors in vitro. The racemic compound and its enantiomers showed high and apparently equal affinity for subtypes of α1-adrenoceptors with K values in the low nanomolar range, and showed potent antagonism of α1-adrenoceptors in isolated tissues, with the enantiomers approximately equipotent to the racemate at each α1-adrenoceptor subtype. At α2b sites, R(+) terazosin bound less potently than either the S(-) enantiomer or racemate. R(+) terazosin was also less potent than the S(-) enantiomer or the racemate at rat atrial α2B receptors. These agents were not significantly different in their potencies at α2a or α2A sites. Since the high affinity for α2B sites of quinazoline-type α-adrenoceptor antagonists has been used to differentiate α2-adrenoceptor subtypes, the low affinity of R(+) terazosin for these sites was unexpected. Because terazosin or its enantiomers are approximately equipotent at α1 -adrenoceptor subtypes, the lower potency of R(+) terazosin at α2B receptors indicates a somewhat greater selectivity for α1- compared to α2B adrenoceptor subtypes. The possible pharmacological significance of this observation is discussed.     

Establishment of CHO cell lines expressing four N-methyl-D-aspartate receptor subtypes and characterization of a novel antagonist PPDC

To develop an assay system that allows the N-methyl-D-aspartate (NMDA) receptor subtype-selective antagonistic potency of drugs, we have established Chinese hamster ovary cell lines expressing the four NMDA receptor subtypes (GluRepsilon1/zeta1-GluRepsilon4/zeta1) heat-indelibly. Using these clonal cells, we found that a novel antagonist, (1S,2R)-1-phenyl-2[(S)-1-aminopropyl]-N,N-diethylcyclopropanecarboxamide, was less selective for the GluRepsilon1/zeta1: the IC(50) values for the GluRepsilon1/zeta1-GluRepsilon4/zeta1 were 41.7, 13.3, 12.6 and 11.5 microM, respectively, while two well-known antagonists, DL-2-amino-5-phosphonovaleric acid and ifenprodil, showed the known potency and selectivity for each subtype. Thus, the established clonal cells are of use in characterizing the pharmacological properties of drugs that act on NMDA receptors.     

2-(N-acyl) and 2-N-acyl-N(6)-substituted analogues of adenosine and their affinity at the human adenosine receptors

A series of 2-(N-acyl) and 2-(N-acyl)-N(6)-alkyladenosine analogues have been synthesized from the intermediate 2-amino-6-chloroadenosine derivatives (2b and 7) and evaluated for their affinity at the human A(1), A(2A), and A(3) receptors. We found that 2-(N-acyl) derivatives of adenosine showed relatively low affinity at A(2A) and A(3) receptors, while the N(6)-cyclopentyl substituent in 4h and 4i induced high potency [A(1) (K(i))=20.7 and 31.8 nM respectively] at the A(1) receptor and resulted therefore in increased selectivity for this subtype. The general synthetic methods and their binding studies are presented herein.     

2-Phenylhydroxypropynyladenosine derivatives as high potent agonists at A2B adenosine receptor subtype

Adenosine derivatives bearing in 2-position the (R,S)-phenylhydroxypropynyl chain were evaluated for their potency at human A2B adenosine receptor, stably transfected on CHO cells, on the basis that (R,S)-2-phenylhydroxy-propynyl-5'-N-ethylcarboxyamidoadenosine [(R,S)-PHPNECA] was found to be a good agonist at the A2B receptor subtype. Biological studies demonstrated that the presence of small alkyl groups in N6-position of these molecules are well tolerated, whereas large groups abolished A2B potency. On the other hand, the presence of an ethyl group in the 4'-carboxamido function seems to be optimal, the (S)-PHPNECA resulting the most potent agonist at A2B receptor reported so far.     

Molecular modeling and mutagenesis of the ligand-binding pocket of the mGlu3 subtype of metabotropic glutamate receptor

A homology model of the extracellular domain of the mGlu3 subtype of metabotropic glutamate (mGlu) receptor was generated and tested using site-directed mutagenesis, a radioligand-binding assay using the Group II selective agonist (2S,2'R,3'R)-2-(2',3'-[3H]dicarboxycyclopropyl) glycine ([3H]DCG-IV), and in a fluorescence-based functional assay in live transiently transfected human embryonic kidney cells. Ten of the 12 mGlu3 mutants (R64A, R68A, Y150A, S151A, T174A, D194A, Y222A, R277A, D301A and K389) showed either no binding or a 90% or greater loss of specific [3H]DCG-IV binding. Several analogous mutations in mGlu2 supported the results obtained with mGlu3. These results demonstrate that the binding of [3H]DCG-IV to mGlu3 is exceptionally sensitive to mutagenesis-induced perturbations. In silico docking of DCG-IV into the agonist binding pocket of mGlu3 facilitated the interpretation the mutagenesis results. Tyrosines 150 and 222, and arginine 277 show close contacts with the third carboxylic acid group in DCG-IV, which is not present in glutamate or (2S,1'S,2'S)-2-(carboxycyclopropyl)glycine (L-CCG-I). Mutation of these three amino acids to alanine resulted in a near complete loss of receptor activation by DCG-IV and retention of near wild-type affinity for L-CCG-I. It is proposed that hydrogen bonding between this carboxylate and tyrosines 150 and 222 and arginine 277 provide a partial explanation for the high affinity and Group II selectivity of DCG-IV. These findings define the essential features of the ligand-binding pocket of mGlu3 and, together with other recent studies on mGlu receptors, provide new opportunities for structure-based drug design.     

N-4-t-Butylbenzyl 2-(4-methylsulfonylaminophenyl) propanamide TRPV1 antagonists: Structure-activity relationships in the A-region

Structure-activity relationships for the A-region in a series of N-4-t-butylbenzyl 2-(4-methylsulfonylaminophenyl) propanamides as TRPV1 antagonists have been investigated. Among them, the 3-fluoro analogue 54 showed high binding affinity and potent antagonism for both rTRPV1 and hTRPV1 in CHO cells. Its stereospecific activity was demonstrated with marked selectivity for the (S)-configuration (54S versus 54R). A docking study of 54S with our hTRPV1 homology model highlighted crucial hydrogen bonds between the ligand and the receptor contributing to its potency.     

Synthesis of pyrrolo[2,3-d]pyridazinones as potent, subtype selective PDE4 inhibitors

A series of pyrrolo [2,3-d]pyridazinones was synthesized and tested for their inhibitory activity on PDE4 subtypes A, B and D and selectivity toward Rolipram high affinity binding site (HARBS). New agents with interesting profile were reported; in particular compound 9e showed a good PDE4 subtype selectivity, being 8 times more potent (IC50 = 0.32 microM) for PDE4B (anti-inflammatory) than for PDE4D (IC50 = 2.5 microM), generally considered the subtype responsible for emesis. Moreover the ratio HARBS/PDE4B was particularly favourable for 9e (147), suggesting that the best arranged groups around the pyrrolopyridazinone core are an isopropyl at position-1, an ethoxycarbonyl at position-2, together with an ethyl group at position-6. For compounds 8 and 15a the ability to inhibit TNFalpha production in PBMC was evaluated and the results are consistent with their PDE4 inhibitory activity.     

Modulation of the N-Methyl-d-Aspartate Receptor by Haloperidol: NR2B-Specific Interactions

Abstract: The dopaminergic antagonist haloperidol has an eight- to 10-fold higher affinity for NMDA receptors containing the NR2B (ε2) subunit, showing the same subunit specificity as ifenprodil, polyamines, and magnesium. In the present study, we have compared the effects of mutations altering polyamine and ifenprodil sensitivity on haloperidol sensitivity of NMDA receptors. As seen for spermidine stimulation, high-affinity haloperidol inhibition is governed by the region around amino acid 198, based on results from chimeric murine NR2A/NR2B (ε1/ε2) receptors. Mutation of ε2E201 in this region to asparagine or arginine causes a 10-fold decrease in the ability of haloperidol to inhibit ¹²⁵I-MK-801 binding. ε2E201 does not govern the interactions of ifenprodil, because all of the mutants at ε2E201 exhibited wild-type affinity for ifenprodil. Mutation of ε2R337 causes a 400-fold loss in apparent affinity for ifenprodil but does not change the effects of haloperidol. The structural determinants of spermidine stimulation do not perfectly match those for haloperidol inhibition, as mutations of E200 remove haloperidol inhibition but do not alter polyamine stimulation. The present results thus demonstrate that although spermidine, haloperidol, and ifenprodil share subunit selectivity and overlapping pharmacology, they also have specific structural determinants.     

Dual antagonists of the bradykinin B1 and B2 receptors based on a postulated common pharmacophore from existing non-peptide antagonists

We have recently drawn attention to the fact that most non-peptide antagonists of the kinin B1 receptor reported so far are structurally related, possessing the core motif phenyl-SO2-NR-(spacer(2-4))-CO-NRR. This is found in compound A (N-[2-[4-(4,5-dihydro-1H-imidazol-2- yl)phenyl]ethyl] - 2- [(2R)-1-(2-napthylsulfonyl)-3-oxo-1,2,3,4-tetrahydroquinoxalin-2-yl]acetamide), a very potent and selective B1 receptor antagonist. A subset of specific bradykinin B2 receptor antagonists (LF16-0687, bradyzide and derivatives) possesses a similar 'scaffold' (phenyl-SO2-NR-CRR-CO-NRR). We investigated whether simple molecules mimicking the postulated pharmacophores could be identified in two public chemical databases. Receptor binding to B1 and B2 receptors expressed by rabbit cultured smooth-muscle cells was confirmed for some of these newly identified agents, with a loss of receptor subtype selectivity. For instance, compound 3[2-(3-oxo-1-(toluene-4-sulfonyl)-1,2,3,4-4H-quinoxalin-2-yl)-N-phenyl-acetamide] exhibits IC50 values of 2.13 and 126 microM in the radioligand competition assays for B1 and B2 receptors, respectively, and a pA2 of 6.27 at the rabbit B1 receptor in a functional test (Lys-des-Arg9-bradykinin-induced contractility of the isolated aorta). Compound 5 (a close analog of compound 3) is a more balanced dual antagonist of low potency (IC50 values of 30 and 117 microM, respectively). As predicted, compounds modeled on a postulated pharmacophore common to some non-peptide B1 or B2 receptor antagonists exhibit measurable binding with decreased receptor subtype selectivity. Dual B1/B(2) receptor antagonists are of possible therapeutic interest and should be developed.     

Synthesis of 2-(2,3-dimethoxyphenyl)-4-(aminomethyl)imidazole analogues and their binding affinities for dopamine D(2) and D(3) receptors.

A series of 2-(2,3-dimethoxyphenyl)-4-(aminomethyl)imidazole derivatives was prepared and their affinity for dopamine D₂ and D₃ receptors was measured using in vitro binding assays. Several oxadiazole analogues were also prepared and tested for their affinity for dopamine D₂ and D₃ receptors. The results of receptor binding studies indicated that the incorporation of an imidazole moiety between the phenyl ring and the basic nitrogen did not significantly increase the selectivity for dopamine D₃ receptors, whereas the incorporation of an oxadiazole at the same region resulted in a total loss of affinity for both dopamine receptor subtype binding sites. The most selective compound in this series is 2-(5-bromo-2,3-dimethoxyphenyl)-4-(6,7-dimethoxy-1,2,3,4-tetrahydroisoquinolinomethyl)imidazole (5i), which has a D₃ receptor affinity of 21 nM and a 7-fold selectivity for D₃ versus D₂ receptors. The binding affinity for σ₁ and σ₂ receptors was also measured, and the results showed that several analogues were selective σ₁ receptor ligands.     

Differential responsiveness of CRF receptor subtypes to N-terminal truncation of peptidic ligands

The role of the N-terminal domains of corticotropin-releasing factor (CRF) and CRF-like peptides in receptor subtype selectivity, ligand affinity and biological potency was investigated. Therefore, human CRF(12-41), human URP(12-38) and antisauvagine-30 (aSvg) were N-terminally prolonged by consecutive addition of one or two amino acids. The peptides obtained were tested for their binding affinities to rat CRF1 and murine CRF(2beta) receptor, and their capability to stimulate cAMP-release by HEK cells producing either receptor.It was observed that human CRF N-terminally truncated by eight residues was bound with high affinity to CRF2 receptor (Ki=5.4nM), whereas affinity for CRF1 receptor was decreased (Ki=250 nM). A similar shift of affinity was found with sauvagine (Svg) analogs. Truncation of human URP analogs did not affect their preference for CRF(2beta) receptor, but reduced their affinity. Changes in affinity were positively correlated with changes in potency. These results indicated that CRF1 receptor was more stringent in its structural requirements for ligands to exhibit high affinity binding than CRF(2beta) receptor.     

Design, synthesis, and pharmacological evaluation of novel tetrahydroprotoberberine derivatives: selective inhibitors of dopamine D₁ receptor

A series of new tetrahydroprotoberberine (THPB) derivatives were designed, synthesized, and tested for their binding affinity towards dopamine (D(1) and D(2)) and serotonin (5-HT(1A) and 5-HT(2A)) receptors. Many of the THPB compounds exhibited high binding affinity and activity at the dopamine D(1) receptor, as well as high selectivity for the D(1) receptor over the D(2), 5-HT(1A), and 5-HT(2A) receptors. Among these, compound 19c exhibited a promising D(1) receptor binding affinity (K(i)=2.53nM) and remarkable selectivity versus D(2)R (inhibition=81.87%), 5-HT(1A)R (inhibition=61.70%), and 5-HT(2A)R (inhibition=24.96%). Compared with l-(S)-stepholidine (l-SPD) (D(1)K(i)=6.23nM, D(2)K(i)=56.17nM), compound 19c showed better binding affinity for the D(1) receptor (2.5-fold higher) and excellent D(2)/D(1) selectivity. Functional assays found compounds 18j, 18k, and 19c are pure D(1) receptor antagonists. These results indicate that removing the C10 hydroxy group and introducing a methoxy group at C11 of the pharmacophore of l-SPD can reverse the function of THPB compounds at the D(1) receptor. These results are in accord with molecular docking studies.     

Interaction of the nicotinic agonist (R,S)-3-pyridyl-1-methyl-2-(3-pyridyl)-azetidine (MPA) with nicotinic acetylcholine receptor subtypes expressed in cell lines and rat cortex

The interaction of the nicotinic agonist (R,S)-3-pyridyl-1-methyl-2-(3-pyridyl)-azetidine (MPA) with different nicotinic acetylcholine receptor (nAChR) subtypes was studied in cell lines and rat cortex. MPA showed an affinity (Ki = 1.21 nM) which was higher than anatoxin-a > (−)-nicotine > (+)-[R]nornicotine > (−)-[S]nornicotine > and (+)-nicotine, but lower than cytisine (Ki = 0.46 nM) in competing for (−)-[³H]nicotine binding in M10 cells, which stably express the recombinant ₄β₂ nAChR subtype. A one-binding site model was observed in all competing experiments between (−)-[³H]nicotine binding and each of the agonists studied in M10 cells. MPA showed a 13-fold higher affinity for (−)-[³H]nicotine binding sites compared to the [³H]epibatidine binding sites in rat cortical membranes. In human neuroblastoma SH-SY5Y cells, which predominantly express the ₃ nAChR subunit mRNA, MPA displaced [³H]epibatidine binding from a single population of the binding sites with an affinity in the same nM range as that observed MPA in displacing [³H]epibatidine binding in rat cortical membranes. Chronic treatment of M10 cells with MPA significantly up-regulated the number of (−)-[³H]nicotine binding sites in a concentration dependent manner. Thus MPA appears to have higher affinity to 4-subunit containing receptor subtype than ₃-subunit containing receptor subtype of nAChRs. Furthermore MPA binds to ₄β₂ receptor subtype with higher affinity than (−)-nicotine and behaves, opposite to cytisine, as a full agonist in up-regulating the number of nAChRs. © 1998 Elsevier Science Ltd. All rights reserved.     

QSAR of adenosine receptor antagonists. Part 3: Exploring physicochemical requirements for selective binding of 1,2,4-triazolo[5,1-i]purine derivatives with human adenosine A3 receptor subtype

Considering potential of selective adenosine A3 receptor antagonists in the development of prospective therapeutic agents, an attempt has been made to explore selectivity requirements of 1,2,4-triazolo[5,1-i]purine derivatives for binding with cloned human adenosine A3 receptor subtype. In this study, partition coefficient (logP) values of the molecules (calculated by Crippen's fragmentation method) and Wang-Ford charges of the common atoms of the triazolopurine nucleus (calculated from molecular electrostatic potential surface of energy minimized geometry using AM1 technique) were used as independent variables along with suitable dummy parameters. The best equation describing A3 binding affinity [n=29, Q2=0.796, Ra2=0.853, R2=0.874, R=0.935, s=0.342, F=41.5 (df 4,24), SDEP=0.396] showed parabolic relation with logP (optimum value being 4.134). Further, it was found that an aromatic substituent conjugated with the triazole nucleus should be present at R2 position for A3 binding affinity. Again, high negative charges on N2 and N4 are conducive to the binding affinity. While exploring selectivity requirements of the compounds for binding with A3 receptor over that with A2A receptor, the selectivity relation [n=23, Q2=0.909, Ra2=0.918, R2=0.933, R=0.966, s=0.401, F=62.4 (df 4,18), SDEP=0.412] showed that an aromatic R2 substituent conjugated with the triazole nucleus contributes significantly to the selectivity. Again, presence of a 4-substituted-phenyl ring (except 4-OH-phenyl and 4-CH3-phenyl) at R2 position also increases selectivity. Further, charge difference between N2 and N11 (negative charge on the former should be higher and that on the latter should be less) contributes significantly to the selectivity. In addition, negative charge on N7 is conducive while presence of substituents like propyl, butyl, pentyl or phenyl at R1 position is detrimental for the A3 selectivity.     

1-, 3- and 8-substituted-9-deazaxanthines as potent and selective antagonists at the human A2B adenosine receptor

A large series of piperazin-, piperidin- and tetrahydroisoquinolinamides of 4-(1,3-dialkyl-9-deazaxanthin-8-yl)phenoxyacetic acid were prepared through conventional or multiple parallel syntheses and evaluated for their binding affinity at the recombinant human adenosine receptors, chiefly at the hA(2B) and hA(2A) receptor subtypes. Several ligands endowed with high binding affinity at hA(2B) receptors, excellent selectivity over hA(2A) and hA(3) and a significant, but lower, selectivity over hA(1) were identified. Among them, piperazinamide derivatives 23 and 52, and piperidinamide derivative 69 proved highly potent at hA(2B) (K(i)=11, 2 and 5.5 nM, respectively) and selective towards hA(2A) (hA(2A)/hA(2B) SI=912, 159 and 630, respectively), hA(3) (hA(3)/hA(2B) SI=>100, 3090 and >180, respectively) and hA(1) (hA(1)/hA(2B) SI=>100, 44 and 120, respectively), SI being the selectivity index. A number of selected ligands tested in functional assays in vitro showed very interesting antagonist activities and efficacies at both A(2A) and A(2B) receptor subtypes, with pA(2) values close to the corresponding pK(i)s. Structure-affinity and structure-selectivity relationships suggested that the binding potency at the hA(2B) receptor may be increased by lipophilic substituents at the N4-position of piperazinamides and that an ortho-methoxy substituent at the 8-phenyl ring and alkyl groups at N1 larger than the ones at N3, in the 9-deazaxanthine ring, may strongly enhance the hA(2A)/hA(2B) SI.     

Spontaneous mobility of GABAA receptor M2 extracellular half relative to noncompetitive antagonist action

The gamma-aminobutyric acid type A receptor beta(3) homopentamer is spontaneously open and highly sensitive to many noncompetitive antagonists(NCAs) and Zn(2+). Our earlier study of the M2 cytoplasmic half (-1' to 10') established a model in which NCAs bind at pore-lining residues Ala(2)', Thr(6)', and Leu(9)'. To further define transmembrane 2 (M2) structure relative to NCA action, we extended the Cys scanning to the extra cellular half of the beta(3) homopentamer (11' to 20'). Spontaneous disulfides formed with T13'C, L18'C, and E20'C from M2/M2 cross-linking and with I14'C (weak), H17'C, and R19'Con bridging M2/M3 intersubunits, based on single (M2 Cys only) and dual (M2 Cys plus M3 C289S) mutations. Induced disulfides also formed with T16'C, but there were few or none with M11'C, T12'C, and N15'C. These findings show conformational flexibility/mobility in the M2 extracellular half 17' to 20' region interpreted as a deformed beta-like conformation in the open channel. The NCA radioligands used were [(3)H]1-(4-ethynylphenyl)-4-n-propyl-2,6,7-trioxabicyclo[2.2.2]octane ([(3)H]EBOB) and [(3)H]3,3-bis-trifluoromethylbicyclo[2.2.1]heptane-2,2-dicarbonitrile with essentially the same results. NCA binding was disrupted by individual Cys substitutions at 13',14',16',17', and 19'. The inactivity of T13'C/T13'S may have been due to disturbance of the channel gate; I14'S and T16'S showed much better binding activity than their Cys counterparts, and the low activities of H17'C and R19'C were reversed by dithiothreitol. Zn(2+) potency for inhibition of [(3)H]EBOB binding was lowered 346-fold by the mutation H17'A. We propose that NCAs enter their binding site both directly, through the channel pore, and indirectly, through the water cavity of adjacent subunits.     

Synthesis of 2-(2,3-dimethoxyphenyl)-4-(aminomethyl)imidazole analogues and their binding affinities for dopamine D2 and D3 receptors

A series of 2-(2,3-dimethoxyphenyl)-4-(aminomethyl)imidazole derivatives was prepared and their affinity for dopamine D₂ and D₃ receptors was measured using in vitro binding assays. Several oxadiazole analogues were also prepared and tested for their affinity for dopamine D₂ and D₃ receptors. The results of receptor binding studies indicated that the incorporation of an imidazole moiety between the phenyl ring and the basic nitrogen did not significantly increase the selectivity for dopamine D₃ receptors, whereas the incorporation of an oxadiazole at the same region resulted in a total loss of affinity for both dopamine receptor subtype binding sites. The most selective compound in this series is 2-(5-bromo-2,3-dimethoxyphenyl)-4-(6,7-dimethoxy-1,2,3,4-tetrahydroisoquinolinomethyl)imidazole (5i), which has a D₃ receptor affinity of 21 nM and a 7-fold selectivity for D₃ versus D₂ receptors. The binding affinity for σ₁ and σ₂ receptors was also measured, and the results showed that several analogues were selective σ₁ receptor ligands.