Synthesis and Characterization of Selective Dopamine D₂ Receptor Antagonists. 2. Azaindole, Benzofuran, and Benzothiophene Analogs of L-741,626

Abstract

A series of indole, 7-azaindole, benzofuran, and benzothiophene compounds have been prepared and evaluated for affinity at D_2-like dopamine receptors. These compounds share structural elements with the classical D_2-like dopamine receptor antagonists haloperidol, N-methylspiperone and benperidol. Two new compounds, 4-(4-iodophenyl)-1-((4-methoxy-1H-indol-3-yl)methyl)piperidin-4-ol (6) and 4-(4-iodophenyl)-1-((5-methoxy-1H-indol-3-yl)methyl)piperidin-4-ol (7), were found to have high affinity to and selectivity for D₂ versus D₃ receptors. Changing the aromatic ring system from an indole to other heteroaromatic ring systems reduced the D₂ binding affinity and the D₂ versus D₃ selectivity.

1. Introduction

The neurotransmitter dopamine is synthesized by mesencephalic neurons of the substantia nigra and ventral tegmental area and by hypothalamic neurons of the arcuate and periventricular nuclei. Dopamine is present in the central nervous system and peripheral nervous system.¹ Dopamine is associated with fine movement coordination, emotion, affect, cognition and memory.² Regulation of dopamine plays a crucial role in mental and physical health. Abnormal activity of the dopamine system has been implicated in psychological and neurological disorders including Parkinson’s disease, schizophrenia, mood disorders, addiction, attention deficit hyperactivity disorder, and Tourette syndrome.^3–10

Dopamine receptors belong to a large superfamily of neurotransmitter and hormone receptors which are coupled to their specific effectors function via guanine nucleotide regulatory (G) proteins. Based upon genomic and cDNA cloning studies, it is thought that there are five functionally active dopamine receptor subtypes expressed in mammals. These five receptor subtypes have been classified into two major subtypes (D_1-like and D_2-like) based on their pharmacological, biochemical, and physiological characteristics. The D_1-like receptor subtypes consist of the D₁ (D_1a) and the D₅ (D_1b) dopamine receptors. The D_2-like receptor subtypes are known to include several receptor subtypes and isoforms, including D_2short (D_2S), D_2long (D_2L), D₃, and D₄ receptors.¹¹ Agonist stimulation of D_1-like receptors results in an activation of adenylyl cyclase with increased production of cAMP.¹² Stimulation of the D_2-like receptors results in an inhibition of adenylyl cyclase activity, a decrease in intracellular levels of cAMP, an increase in the release of arachidonic acid and an increase in phosphatidylinositol hydrolysis.¹⁰

The D₂ and D₃ dopamine receptors have approximately 46% amino acid homology. In contrast, the transmembrane spanning (TMS) regions of the D₂ and D₃ receptors, which are thought to construct the ligand binding site, share 78% homology.¹³ Despite their structural similarities, D₂ and D₃ receptors differ in their (a) neuroanatomical localization,¹⁴ (b) levels of receptor expression,^15,16 (c) efficacy in response to agonist stimulation,¹⁰ and (d) regulation and desensitization.¹⁷ Because of the high degree of homology between D₂ and D₃ receptor binding sites, the pharmacologic properties of these two receptor subtypes are similar, and it has been difficult to obtain compounds that can bind selectively to either the D₂ or the D₃ dopamine receptor subtype.^{10,15,16,18–20} Despite this difficulty, D₂ and D₃ dopamine receptor selective agonists and antagonists would be useful pharmacologic tools to precisely define the role of the two D_2-like receptor subtypes in a variety of experimental physiological and behavioral situations, including the reinforcing and toxic properties of cocaine,^21,22 socialization, memory, and the regulation of interneuronal activity in the basal ganglia.²³

Previously, we reported the in vitro binding properties of a series of indole derivatives based on haloperidol, N-methylspiperone, and benperidol (Figure 1). Several novel analogues of L-741,626 (1a), a D₂-selective antagonist,²⁴ were made and shown to have selectivity for D₂ over D₃ and D₄ receptors^25,26 (Figure 1). The current study involved the following strategies: (a) continuation of the previous study by making additional structural congeners of 1a; (b) replacement of the indole ring with other heteroaromatic ring systems such as 7-azaindole, benzofuran, and benzothiophene; and (c) the development of fluorine-containing analogs which could serve as potential radiotracers for imaging dopamine D₂ versus D₃ receptors with positron emission tomography (PET).

Figure 1.

Structures of the lead compounds for the development of D₂-selective radiotracers.

2. Chemistry

Compounds 5–15 were synthesized by treating the respective gramine derivatives 2a–f with the appropriate pyridine derivatives 3a–d and 4 in refluxing toluene (Method A, Scheme 1). Gramine and 5-methoxygramine are commercially available. The gramines 2d and 2e were obtained by alkylation of 4-hydroxyindole or 5-hydroxyindole with 1-bromo-2-fluoroethane, followed by reacting with N,N-dimethylmethyleneammonium iodide (Eschenmoser’s salt) (Scheme 2). The 4-methoxygramine, 2b, and 7-azagramine, 2f, were synthesized by treating 4-methoxyindole and 7-azagramine with N,N-dimethylmethylene-ammonium iodide, respectively.

Scheme 1.

Method A

Scheme 2.

Compounds 18–27 were synthesized by reacting the respective bromomethyl- benzofurans (17a–b) or benzo[b]thiophenes (17c–d) with the appropriate piperidines in the presence of potassium carbonate and potassium iodide in acetonitrile (Method B, Scheme 3). The required bromomethyl derivatives were made by refluxing benzofurans or benzo[b]thiophenes with N-bromosuccinimide in carbon tetrachloride. The piperidine 28b was prepared from 1-phenyl-1,3,8-triazaspiro[4.5]decan-4-one (30). First, the N-protected derivative 31 was synthesized and alkylated with 1-bromo-2-fluoroethane, followed by deprotection with trifluoroacetic acid to give the required piperidine 28b (Scheme 4). Compound 29 was synthesized via alkylation of 31 with propargyl bromide, followed by deprotection with trifluoroaetic acid to give 32. N-alkylation of 32 with freshly prepared 3-bromomethylbenzofuran 17b, followed by reaction with 1-azido-2-fluoroethane in the presence of sodium ascorbate and copper sulfate gave the triazole analog, 29 (Scheme 5).

Scheme 3.

Method B

Scheme 4.

Scheme 5.

3. Radioligand binding studies at dopamine receptors

Competitive radioligand binding studies were performed to determine the equilibrium dissociation constants of each compound at human D₂, D₃ and D₄ dopamine receptors (Table 1). For these studies, tissue homogenates from stably transfected HEK 293 cells were used in conjunction with the radioligand ¹²⁵I-IABN. We have previously reported that the benzamide ¹²⁵I-IABN binds with high affinity and selectively to D_2-like dopamine receptors, but that it binds non-selectively to D₂ versus D₃ dopamine receptor subtypes.²⁷

Table I.

Binding Affinities for Dopamine D₂/D₃ and Sigma σ₁/σ₂ Receptors.

Compound	Ki (nM)a
	D2b	D3c	D4d	D3:D2e	σ1f	σ2g	LogPh
5	3.8 ± 0.8	94.4 ± 1.2	53.4 ± 15.2	25	2.00 ± 0.52	2200 ± 437	1.98
6	0.9 ± 0.1	122 ± 2.0	60.4 ± 18.7	136	881 ± 154	970 ± 178	2.60
7	0.9 ± 0.1	100 ± 13	129 ± 5.1	112	197 ± 26	3262 ± 387	2.54
8	1396 ± 317	2927 ± 198	>10000	2.1	1527 ± 158	2761 ± 574	1.43
9	27.7 ± 6.6	849 ± 164	668 ± 22	31	3224 ± 479	9510 ± 1020	2.61
10	13.8 ± 2.5	188 ± 7.1	344 ± 49	14	227 ± 63	10420 ± 1512	2.54
11	23.3 ± 3.1	132 ± 32	1390 ± 108	5.7	4129 ± 382	16134 ± 2968	2.54
12	12.5 ± 2.0	68.2 ± 16	2230 ± 484	5.5	1768 ± 303	27230 ± 5205	2.45
13	212 ± 25	2983 ± 305	620 ± 72	14	15.1 ± 2.3	1.90 ± 0.15	2.20
14	61.0 ± 4.9	1585 ± 380	634 ± 85	26	1.20 ± 0.51	5.53 ± 2.37	2.46
15	254 ± 40	4938 ± 780	937 ± 145	20	8.07 ± 2.00	7.75 ± 1.24	1.85
18	17.3 ± 0.6	31.8 ± 4.1	254 ± 5.3	1.8	2.45 ± 0.78	1398 ± 98	3.19
19	14.3 ± 1.7	49.0 ± 5.6	245 ± 11	3.4	1.41 ± 0.41	2368 ± 52	3.45
20	46.8 ± 3.0	142 ± 27	377 ± 47	3.0	2.13 ± 0.44	1964 ± 177	4.97
21	6.6 ± 1.2	10.9 ± 0.5	94.5 ± 6.4	1.7	0.59 ± 0.15	377 ± 20	3.12
22	2.4 ± 0.7	11.2 ± 1.2	40.4 ± 10.0	4.7	0.51 ± 0.13	393 ± 54	3.38
23	13.6 ± 3.6	3.0 ± 0.7	163 ± 11.9	4.5	1779 ± 256	18054 ± 3531	3.39
24	37.8 ± 6.0	46.7 ± 4.8	72.6 ± 0.8	1.2	1879 ± 200	14575 ± 1168	4.73
25	7.1 ± 1.3	2.1 ± 0.5	492 ± 103	0.3	708 ± 141	24879 ± 4201	2.02
26	8.0 ± 1.9	9.3 ± 2.2	945 ± 162	1.2	28 ± 8	10164 ± 641	2.38
27	71.4 ± 14	60.1 ± 17	352 ± 72	0.8	331 ± 87	20248 ± 7658	3.36
29	2.0 ± 0.1	0.9 ± 0.2	978 ± 11.9	0.5	802 ± 227	22028 ± 3297	1.46
Haloperidol	1.1 ± 0.1	12.7 ± 3.9	NDi	12	1.45 ± 0.33	24.2 ± 3.0	4.50
1aj	10.0 ± 2.5	104 ± 19	449 ± 123	10	19.3 ± 3.6	1811 ± 569	1.54
1bj	4.2 ± 0.4	250 ± 6	>2000	59	>5000	1246 ± 234	2.10
1cj	4.2 ± 0.8	204 ± 60	>3500	49	242 ± 68	1900 ± 241	2.16
1dj	4.8 ± 0.3	110 ± 39	652 ± 7	28	12.1 ± 2.5	2134 ± 356	1.72
1ej	2.3 ± 0.7	190 ± 34	840 ± 197	82	2557 ± 334	943 ± 64	2.34
1fj	2.5 ± 0.7	96.9 ± 6.1	700 ± 80	39	108 ± 17	2702 ± 696	2.28

^aMean ± SEM, K_i values were determined by at least three experiments.

^bK_i values for D₂ receptors were measured on human D_2(long) expressed in HEK 293 cells using [¹²⁵I]ABN as the radioligand.

^cK_i values for D₃ receptors were measured on human D₃ expressed in HEK 293 cells using [¹²⁵I]ABN as the radioligand.

^dK_i values for D₄ receptors were measured on human D₄ expressed in HEK 293 cells using [¹²⁵I]ABN as the radioligand.

^eK_i for D₃ receptors/K_i for D₂ receptors.

^fK_i for inhibiting the binding of [³H](+)-pentazocine to guinea pig brain homogenates.

^gK_i for inhibiting the binding of [³H]DTG to rat liver homogenates.

^hCalculated value using the program Clog P.

ⁱNot determined.

^jData from reference ²⁵.

First, a comparison of indole analogs structurally related to haloperidol was made in which a substituent was added at the 4- or 5-position of the indolyl moiety. We found that the addition of the methoxy group resulted in a 4-fold increase in affinity at D₂ receptors but did not change the affinity at D₃ receptors, resulting in compounds that were 110- to 140-fold selective at D₂ receptors (7 and 6). The addition of 2-fluoroethoxy group resulted in a 3- to 6-fold decrease in affinity at D₂ receptors and a 2- to 8-fold decrease in affinity at D₃ receptors; thus compounds 10 and 9 resulted in no significant difference in selectivity compared to the unsubstituted analog (1d²⁵). The halide substituents on the phenyl-piperidinol moiety are pivotal for high affinity binding at the dopamine D₂ receptors and for selectivity over D₃ receptors. The iodide analogs (6 and 7) have higher affinity and selectivity at D₂ versus D₃ dopamine receptors than the chloride and bromide analogs (1b, 1c, 1e and 1f²⁵). In addition, the affinity at both D₂ and D₃ dopamine receptors decreases dramatically in the unsubstituted analog, 8. The 7-azaindole analogs (13–15) bind with 14- to 26-fold selectivity for D₂ over D₃ receptors. The benzofuran (18, 19, 21, 22, 23, 25, 26 and 29) and benzothiophene (20, 24, and 27) analogs bind non-selectively to the D₂ over D₃ dopamine receptors. Similar results for benzofuran analogs of L-741,626 were previously reported by Grundt et al.²⁶

4. Adenylyl cyclase studies with D_2-like receptors

The intrinsic efficacy of the two compounds exhibiting the greatest D₂ vs. D₃ receptor subtype binding selectivity (6 and 7) was evaluated using a forskolin-dependent adenylyl cyclase inhibition assay (Figure 2). The D_2-like receptor full agonist quinpirole was used as a reference compound. At a dose of 10 nM, quinpirole administration resulted in >70% inhibition of cAMP accumulation, whereas at the same dose of compounds 6 and 7 no appreciable increase or decrease in forskolin-dependent adenylyl cyclase activation was observed. In addition, both 6 and 7 were able to attenuate the effect of quinpirole. Based upon these results, we categorize compounds 6 and 7 as neutral antagonists at D₂ dopamine receptors. These results are consistent with our previous studies on this class of methoxy substituted indole piperidines.²⁵

Figure 2.

Evaluation of the intrinsic efficacy of compounds 6 and 7 using a Forskolin-Dependent Adenylyl Cyclase Inhibition Assay. The intrinsic efficacy of compounds 6 and 7 was evaluated by determining the percent inhibition of a forskolin-dependent adenylyl cyclase assay with human D_2long receptors expressed in stably transfected HEK 293 cells. The extent of inhibition of the two test compounds was compared to the percent inhibition obtained using the full agonist quinpirole (quin) at a concentration of 10 nM. At this concentration of quinpirole the mean ± S.E.M. inhibition of forskolin (fsk) stimulation was 71 ± 3.6 percent. The test drugs were tested at a final concentration equal to 10 nM, either in the absence or presence of quinpirole. The bar graph represents the mean ± the S.E.M. values obtained for n = 4.

5. Radioligand binding studies at sigma receptors

In vitro binding studies were conducted to determine the affinity of the target compounds at sigma-1 (σ₁) and sigma-2 (σ₂) receptors. The σ₁ binding studies were conducted using the σ₁-selective radioligand, [³H](+)-pentazocine in guinea pig brain membranes; σ₂ sites were assayed in rat liver membranes with [³H]DTG in the presence of 100 nM unlabeled (+)-pentazocine to mask σ₁ sites, or with the σ₂ selective ligand [³H]RHM-1, alone.^28,29 Although haloperidol is a dopaminergic antagonist that is used clinically as a neuroleptic, its high affinity at sigma receptors has precluded its usefulness as a radioligand for in vitro or in vivo radioligand binding studies. Almost all of the compounds in this study bound with low affinity (>350 nM) at σ₂ receptors with the exception of compound 13, which has a nanomolar (nM) affinity for σ₂ receptors and a 7-fold selectivity for σ₂ versus σ₁ receptors. The affinity at σ₁ receptors varied from 0.5 nM to >4000 nM. The 7-azaindole analogs have high affinity at both σ₁ and σ₂ receptors, whereas the benzofuran analogs 21 and 22 have a high affinity and selectivity for σ₁ versus σ₂ receptors.

6. Discussion

The current study is a continuation of our effort to develop ligands having a high affinity and selectivity for D₂ versus D₃ dopamine receptors. In previous studies, we prepared a number of structural analogs of the classical D_2-like dopamine receptor antagonist, haloperidol, having a moderate to high affinity at D₂ versus D₃ receptors.²⁵ We have expanded this initial structure-activity relationship (SAR) study and have identified two iodine-containing derivatives of the methoxy substituted indoles (6 and 7), which (a) bind at D₂ receptors with nanomolar affinity and (b) have >100-fold selectivity for human D₂ receptors compared to the human D₃ dopamine receptor subtype. These two analogs were also found to bind with low affinity at the σ₁ and σ₂ receptors. The binding profiles of 6 and 7 indicate that they are the most potent and selective D₂-antagonists reported to date. The lipophilicities (log P) of 6 and 7 (Table 1) also suggest that they will readily cross the blood-brain barrier and are good candidates for the development of D₂ receptor selective imaging agents for the functional imaging technique, PET. The iodo substituent on the phenyl-piperidinol moiety was found to be pivotal for high affinity binding at the dopamine D₂ receptors and for selectivity over D₃ receptors. The substitution at the 4- or 5-position on the indolyl moiety also affects the affinity binding and selectivity. The 2-fluoroethoxy substituted indoles (9 and 10) have lower affinity and selectivity for D₂ over D₃ compared to the methoxy substituted indoles (1e and 1f²⁵). However, the minor reduction in affinity in going from the methoxy group to the 2-fluoroethoxy group (compare 10 to 1f) suggests that this may be a useful strategy for preparing a fluorine-18 radiotracer for imaging D₂ versus D₃ receptors.

We have also examined heteroatom replacements in the indole ring. The 7-azaindole analogs have lower affinity at both D₂ and D₃ dopamine receptors than the corresponding indole analogs. The benzofuran and benzothiophene analogs bind non-selectively to both D₂ and D₃ dopamine receptors. The interesting SAR at σ₁ and σ₂ receptors of 13–15 and 18–22 may provide useful information for molecular modeling studies of sigma receptors.^30,31

7. Experimental

7.1 Chemical analysis

¹H NMR spectra were recorded on a Varian 300 MHz NMR spectrometer. Chemical shifts are reported in δ values (parts per million, ppm) relative to an internal standard of tetramethylsilane (TMS). The following abbreviations are used for multiplicity of NMR signals: br s = broad singlet, d = doublet, dd = doublet of doublets, dt = doublet of triplets, m = multiplet, s = singlet. Melting points were determined on an electrothermal melting point apparatus and are uncorrected. Elemental analyses were performed by Atlantic Microlab, Inc., Norcross, GA and were within ± 0.4% of the calculated values. Mass spectrometry was provided by the Washington University Mass Spectrometry Resource, an NIH Research Resource (Grant No. P41RR0954). All reactions were carried out under an inert atmosphere of nitrogen. Lipophilicity measurements of the compounds were estimated using the computational program, Clog P (Advanced Chemistry Development, Inc., Toronto, Canada).

7.2 General procedure: Method A

A mixture of gramine derivatives (4.0 mmol) and appropriate amines (4.8 mmol) in toluene (15 mL) was stirred at reflux overnight. The volatile components were evaporated and the resulting residue was purified by silica gel column chromatography (dichloromethane-methanol-ammonium hydroxide, 90:10:0.5) to afford the target compounds. The oxalate salt was prepared using one equivalent of oxalic acid in ethyl acetate.

7.3 General procedure : Method B

A mixture of methylbenzofurans, or methylbenzo[b]thiophenes (4.0 mmol) and N-bromosuccinimide (NBS, 4.4 mmol) in carbon tetrachloride was heated at reflux overnight. After cooling to room temperature, the reaction mixture was filtered and evaporated in vacuo to give the corresponding bromomethyl analogs. These crude bromomethyl analogs were treated with the appropriate amine (4.0 mmol) in the presence of K₂CO₃ and KI in acetonitrile at reflux overnight. After cooling to room temperature, the reaction mixture was filtered and evaporated in vacuo. Column chromatography (5% methanol in dichloromethane) of the resulting residue gave the desired compounds. The oxalate salt was prepared using one equivalent of oxalic acid in ethyl acetate.

7.4 1-((1H-Indol-3-yl)methyl)-4-(4-iodophenyl)piperidin-4-ol oxalate (5). Method A

Yield 56% from gramine (2a) and 4-(4-iodophenyl)-4-hydroxypiperidine (3b). Conversion to the oxalate salt gave 5 as an off-white powder, mp 149–150 °C (dec); ¹H NMR (free base, CDCl₃) δ 8.16 (br s, 1H), 7.79 (d, J = 7.9 Hz, 1H), 7.68 (d, J = 8.4 Hz, 2H), 7.40 (d, J = 7.9 Hz, 1H), 7.27 (d, J = 8.4 Hz, 2H), 7.14–7.24 (m, 3H), 3.82 (s, 2H), 2.90–2.94 (m, 2H), 2.47–2.55 (m, 2H), 2.08–2.18 (m, 2H), 1.69–1.74 (m, 2H). Anal. (C₂₀H₂₁IN₂O·C₂H₂O₄·H₂O) C, H, N.

7.5 4-(4-Iodophenyl)-1-((4-methoxy-1H-indol-3-yl)methyl)piperidin-4-ol oxalate (6). Method A

Yield 52% from 4-methoxygramine (2b) and 4-(4-iodophenyl)-4-hydroxypiperidine (3b). Conversion to the oxalate salt gave 6 as an off-white powder, mp 129–130 °C (dec); ¹H NMR (free base, CDCl₃ + CD₃OD) δ 7.63 (d, J = 8.5 Hz, 2H), 7.24 (d, J = 8.5 Hz, 2H), 6.96–7.08 (m, 3H), 6.49 (d, J = 7.5 Hz, 1H), 4.04 (s, 2H), 3.91 (s, 3H), 2.90–2.94 (m, 2H), 2.59–2.65 (m, 2H), 2.06–2.16 (m, 2H), 1.65–1.69 (m, 2H). Anal. (C₂₁H₂₃IN₂O₂·C₂H₂O₄·0.25 H₂O) C, H, N.

7.6 4-(4-Iodophenyl)-1-((5-methoxy-1H-indol-3-yl)methyl)piperidin-4-ol oxalate (7). Method A

Yield 45% from 5-methoxygramine (2c) and 4-(4-iodophenyl)-4-hydroxypiperidine (3b). Conversion to the oxalate salt gave 7 as a tan powder, mp 121–122 °C (dec); ¹H NMR (free base, CDCl₃) δ 8.30 (br s, 1H), 7.65 (d, J = 8.1 Hz, 2H), 7.27 (d, J = 8.1 Hz, 2H), 7.22 (d, J = 8.3 Hz, 2H), 7.15 (d, J = 2.3 Hz, 2H), 6.88 (dd, J = 9.0 and 2.3 Hz, 1H), 3.89 (s, 2H), 3.87 (s, 3H), 2.99–3.03 (m, 2H), 2.63–2.71 (m, 2H), 2.13–2.29 (m, 2H), 1.69–1.73 (m, 2H). Anal. (C₂₁H₂₃IN₂O₂·C₂H₂O₄·1.25 H₂O) C, H, N.

7.7 1-((5-Methoxy-1H-indol-3-yl)methyl)-4-phenylpiperidin-4-ol oxalate (8). Method A

Yield 93% from 5-methoxygramine (2c) and 4-hydroxy-4phenylpiperidine (3a). Conversion to the oxalate salt gave 8 as an off-white powder, mp 147–148 °C (dec). ¹H NMR (free base, CDCl₃) δ 8.17 (br s, 1H), 7.48–7.52 (m, 2H), 7.32–7.37 (m, 2H), 7.26 (d, J = 8.8 Hz, 2H), 7.22 (d, J = 2.4 Hz, 1H), 7.17 (d, J = 2.2 Hz, 1H), 6.87 (dd, J = 8.8 and 2.4 Hz, 1H), 3.88 (s, 3H), 3.79 (s, 2H), 2.90–2.94 (m, 2H), 2.51–2.60 (m, 2H), 2.14–2.24 (m, 2H), 1.73–1.78 (m, 2H), 1.66 (br s, 1H). Anal. (C₂₁H₂₄N₂O₂·C₂H₂O₄·0.25 H₂O) C, H, N.

7.8 4-(4-Bromophenyl)-1-((4-(2-fluoroethoxy)-1H-indol-3-yl)methyl)piperidin-4-ol oxalate (9). Method A

Yield 43% from 4-(2-fluoroethoxy)gramine (2d) and 4-(4-bromophenyl)-4-hydroxypiperidine (3c). Conversion to the oxalate salt gave 9 as an off-white powder, mp 179–180 °C (dec); ¹H NMR (free base, CDCl₃ + CD₃OD) δ 7.44 (d, J = 8.8 Hz, 2H), 7.37 (d, J = 8.8 Hz, 2H), 7.17 (s, 1H), 7.01–7.10 (m, 2H), 6.46–6.49 (m, 1H), 4.93–4.96 (m, 1H), 4.77–4.80 (m, 1H), 4.38–4.41 (m, 1H), 4.29–4.31 (m, 1H), 4.12(s, 2H), 2.96–3.00 (m, 2H), 2.69–2.76 (m, 2H), 2.11–2.21 (m, 2H), 1.68–1.72 (m, 2H). Anal. (C₂₂H₂₄BrFN₂O₂·C₂H₂O₄·0.5H₂O) C, H, N.

7.9 4-(4-Bromophenyl)-1-((5-(2-fluoroethoxy)-1H-indol-3-yl)methyl)piperidin-4-ol oxalate (10). Method A

Yield 27% from 5-(2-fluoroethoxy)gramine (2e) and 4-(4-bromophenyl)-4-hydroxypiperidine (3c). Conversion to the oxalate salt gave 10 as a tan powder, mp 163–164 °C (dec); ¹H NMR (free base, CDCl₃) δ 8.18 (br s, 1H), 7.45 (d, J = 8.8 Hz, 2H), 7.36 (d, J = 8.8 Hz, 2H), 7.17–7.29 (m, 3H), 6.91 (dd, J = 8.8 and 2.5 Hz, 1H), 4.79 (dt, J = 47.5 and 4.1 Hz, 2H), 4.28 (dt, J = 28.1 and 4.1 Hz, 2H), 3.77 (s, 2H), 2.88–2.92 (m, 2H), 2.48–2.56 (m, 2H), 2.08–2.18 (m, 2H), 1.68–1.72 (m, 2H). Anal. (C₂₂H₂₄BrFN₂O₂·C₂H₂O₄·0.5H₂O) C, H, N.

7.10 4-(2-Fluoroethoxy)gramine (2d)

A mixture of 4-hydroxyindole (1 equiv), 1-bromo-2-fluoroethane (5 equiv) and potassium carbonate (4 equiv) in acetone was heated at reflux overnight. The reaction mixture was cooled down, filtered and concentrated. Purification by silica gel column chromatography (hexane-ethyl acetate, 3:1) gave 4-(2-fluoroethoxy)indole (16a) (99%). ¹H NMR (CDCl₃) δ 8.16 (br s, 1H), 7.04–7.13 (m, 3H), 6.69–6.71 (m, 1H), 6.52 (d, J = 7.6 Hz, 1H), 4.90–4.93 (m, 1H), 4.74–4.77 (m, 1H), 4.41–4.44 (m, 1H), 4.31–4.34 (m, 1H). A solution of 16a (1 equiv) in acetronitrile was slowly added to a solution of N,N-dimethylmethyleneammonium iodide (1.2 equiv) in acetonitrile and acetic acid (2:1). The solution was stirred for 3 hours and partitioned between 2-propanol and chloroform (1:3) and 10% aqueous sodium hydroxide. The aqueous layer was extracted with the same organic solvent. The organic layers were combined, dried, and evaporated to give 2d as a tan solid (51%). This was used without further purification. ¹H NMR (CDCl₃ + CD₃OD) δ 7.07–7.17 (m, 3H), 6.48–6.51 (m, 1H), 4.94–4.97 (m, 1H), 4.78–4.81 (m, 1H), 4.38–4.41 (m, 1H), 4.29–4.31 (m, 1H), 4.23 (s, 2H), 2.53 (s, 6H).

7.11 5-(2-Fluoroethoxy)gramine (2e)

Preparation according to the method described for 2d afforded 2e (83% from 5-hydroxyindole) as a yellow solid which was used without further purification. ¹H NMR (CDCl₃ + CD₃OD) δ 7.27–7.33 (m, 1H), 7.13–7.17 (m, 2H), 6.87–6.91 (m, 1H), 4.85–4.88 (m, 1H), 4.69–4.72 (m, 1H), 4.32–4.35 (m, 1H), 4.22–4.25 (m, 1H), 3.63 (s, 2H), 2.29 (s, 6H).

7.12 1-(1-((4-Methoxy-1H-indol-3-yl)methyl)piperidin-4-yl)-1H-benzo[d]imidazol-2(3H)-one oxalate (11). Method A

Yield 60% from 4-methoxygramine (2b) and 4-(2-keto-1-benzimidazolinyl)piperidine (4). Conversion to the oxalate salt gave 11 as an off-white powder, mp 209–210 °C (dec); ¹H NMR (free base, CDCl₃) δ 8.73 (s, 1H), 8.31 (s, 1H), 6.97–7.35 (m, 7H), 6.53 (d, J = 7.6 Hz, 1H), 4.34–4.42 (m, 1H), 4.11 (s, 2H), 3.95 (s, 3H), 3.26–3.30 (m, 2H), 2.54–2.60 (m, 2H), 2.38–2.44 (m, 2H), 1.78–1.83 (m, 2H). Anal. (C₂₂H₂₄N₄O·C₂H₂O₄·H₂O) C, H, N.

7.13 1-(1-((5-Methoxy-1H-indol-3-yl)methyl)piperidin-4-yl)-1H-benzo[d]imidazol-2(3H)-one oxalate (12). Method A

Yield 87% from 5-methoxygramine (2c) and 4-(2-keto-1-benzimidazolinyl)piperidine (4). Conversion to the oxalate salt gave 12 as a tan powder, mp 197–198 °C (dec); ¹H NMR (free base, CDCl₃) δ 9.77 (s, 1H), 8.18 (s, 1H), 7.00–7.25 (m, 7H), 6.87 (dd, J = 8.8 and 2.4 Hz, 1H), 4.33–4.42 (m, 1H), 3.90 (s, 3H), 3.77 (s, 2H), 3.16–3.19 (m, 2H), 2.44–2.55 (m, 2H), 2.19–2.27 (m, 2H), 1.78–1.82 (m, 2H). Anal. (C₂₂H₂₄N₄O·C₂H₂O₄·H₂O) C, H, N.

7.14 1-((1H-pyrrolo[2,3-b]pyridine-3-yl)methyl)-4-(4-bromophenyl)piperidin-4-ol oxalate (13). Method A

Yield 75% from 7-azagramine (2f) and 4-(4-bromophenyl)-4-hydroxypiperidine (3c). Conversion to the oxalate salt gave 13 as a off-white powder, mp 208–209 °C; ¹H NMR (free base, CDCl₃ + CD₃OD) δ 8.22 (dd, J = 4.8 and 1.4 Hz, 1H), 8.11 (dd, J = 8.0 and 1.4 Hz, 1H), 7.45 (d, J = 8.8 Hz, 2H), 7.41 (s, 1H), 7.37 (d, J = 8.8 Hz, 2H), 7.13 (dd, J = 8.0 and 4.8 Hz, 1H), 3.88 (s, 2H), 2.92–2.96 (m, 2H), 2.66–2.74 (m, 2H), 2.10–2.20 (m, 2H), 1.73–1.78 (m, 2H). Anal. (C₁₉H₂₀BrN₃O·C₂H₂O₄·2H₂O) C, H, N.

7.15 1-((1H-pyrrolo[2,3-b]pyridine-3-yl)methyl)-4-(4-iodophenyl)piperidin-4-ol oxalate (14). Method A

Yield 48% from 7-azagramine (2f) and 4-(4-iodophenyl)-4-hydroxypiperidine (3b). Conversion to the oxalate salt gave 14 as a white powder, mp 187–188 °C; ¹H NMR (free base, CDCl₃ + CD₃OD) δ 8.26 (dd, J = 4.9 and 1.4 Hz, 1H), 8.14 (dd, J = 8.0 and 1.4 Hz, 1H), 7.66 (d, J = 8.7 Hz, 2H), 7.61 (s, 1H), 7.25 (d, J = 8.7 Hz, 2H), 7.18 (dd, J = 8.0 and 4.9 Hz, 1H), 4.17 (s, 2H), 3.00–3.22 (m, 4H), 2.30–2.42 (m, 2H), 1.78–1.83 (m, 2H). Anal. (C₁₉H₂₀IN₃O·C₂H₂O₄·2.5H₂O) C, H, N.

7.16 1-((1H-pyrrolo[2,3-b]pyridine-3-yl)methyl)-4-(4-(methylthio)phenyl)piperidin-4-ol oxalate (15). Method A

Yield 55% from 7-azagramine (2f) and 4-(4-methylthiophenyl)-4-hydroxypiperidine (3d). Conversion to the oxalate salt gave 15 as a tan powder, mp 191–192 °C (dec); ¹H NMR (free base, CDCl₃ + CD₃OD) δ 8.21 (dd, J = 4.8 and 1.4 Hz, 1H), 8.10 (dd, J = 8.0 and 1.4 Hz, 1H), 7.42 (d, J = 8.4 Hz, 2H), 7.41 (s, 1H), 7.24 (d, J = 8.4 Hz, 2H), 7.13 (dd, J = 8.0 and 4.8 Hz, 1H), 3.84 (s, 2H), 2.86–2.92 (m, 2H), 2.60–2.70 (m, 2H), 2.47 (s, 3H), 2.05–2.18 (m, 2H), 1.74–1.80 (m, 2H). Anal. (C₂₀H₂₃N₃OS·C₂H₂O₄·H₂O) C, H, N.

7.17 1-(Benzofuran-2-ylmethyl)-4-(4-bromophenyl)piperidin-4-ol oxalate (18). Method B

Yield 16% from 2-methylbenzofuran and 4-(4-bromophenyl)-4-hydroxypiperidine (3c). Conversion to the oxalate salt gave 18 as an off-white powder, mp 174–175 °C (dec); ¹H NMR (free base, CDCl₃) δ 7.49–7.54 (m, 2H), 7.47 (d, J = 8.8 Hz, 2H), 7.37 (d, J = 8.8 Hz, 2H), 7.18–7.29 (m, 2H), 6.63 (s, 1H), 3.76 (s, 2H), 2.87–2.91 (m, 2H), 2.52–2.61 (m, 2H), 2.14–2.24 (m, 2H), 1.69–1.75 (m, 2H). Anal. (C₂₀H₂₀BrNO₂·C₂H₂O₄·H₂O) C, H, N.

7.18 1-(Benzofuran-2-ylmethyl)-4-(4-iodophenyl)piperidin-4-ol oxalate (19). Method B

Yield 61% from 2-methylbenzofuran and 4-(4-iodophenyl)-4-hydroxypiperidine (3b). Conversion to the oxalate salt gave 19 as an off-white powder, mp 199–200 °C (dec). ¹H NMR (free base, CDCl₃) δ 7.67 (d, J = 8.7 Hz, 2H), 7.48–7.55 (m, 2H), 7.26 (d, J = 8.7 Hz, 2H), 7.18–7.29 (m, 2H), 6.62 (s, 1H), 3.76 (s, 2H), 2.87–2.90 (m, 2H), 2.51–2.60 (m, 2H), 2.13–2.23 (m, 2H), 1.69–1.74 (m, 2H). Anal. (C₂₀H₂₀INO₂·C₂H₂O₄·H₂O) C, H, N.

7.19 1-(Benzo[b]thiophen-2-ylmethyl)-4-(4-bromophenyl)piperidin-4-ol oxalate (20). Method B

Yield 69% from 2-methylbenzo[b]thiophene and 4-(4-bromophenyl)-4-hydroxypiperidine (3c). Conversion to the oxalate salt gave 20 as a white powder, mp 225–226 °C (dec); ¹H NMR (free base, CDCl₃) δ 7.78–7.81 (m, 1H), 7.68–7.71 (m, 1H), 7.48 (d, J = 8.8 Hz, 2H), 7.40 (d, J = 8.8 Hz, 2H), 7.27–7.34 (m, 2H), 7.17 (s, 1H), 3.86 (s, 2H), 2.87–2.91 (m, 2H), 2.49–2.58 (m, 2H), 2.10–2.20 (m, 2H), 1.69–1.75 (m, 2H). Anal. (C₂₀H₂₀BrNOS·C₂H₂O₄·0.5H₂O) C, H, N.

7.20 1-(Benzofuran-3-ylmethyl)-4-(4-bromophenyl)piperidin-4-ol oxalate (21). Method B

Yield 22% from 3-methylbenzofuran and 4-(4-bromophenyl)-4-hydroxypiperidine (3c). Conversion to the oxalate salt gave 21 as an off-white powder, mp 202–203 °C (dec); ¹H NMR (free base, CDCl₃) δ 7.72–7.76 (m, 1H), 7.57 (s, 1H), 7.48–7.50 (m, 1H), 7.47 (d, J = 8.8 Hz, 2H), 7.38 (d, J = 8.8 Hz, 2H), 7.23–7.33 (m, 2H), 3.72 (s, 2H), 2.85–2.89 (m, 2H), 2.46–2.55 (m, 2H), 2.06–2.16 (m, 2H), 1.69–1.74 (m, 2H). Anal. (C₂₀H₂₀BrNO₂·C₂H₂O₄) C, H, N.

7.21 1-(Benzofuran-3-ylmethyl)-4-(4-iodophenyl)piperidin-4-ol oxalate (22). Method B

Yield 33% from 3-methylbenzofuran and 4-(4-iodophenyl)-4-hydroxypiperidine (3b). Conversion to the oxalate salt gave 22 as a white powder, mp 198–199 °C; ¹H NMR (free base, CDCl₃) δ 7.72–7.75 (m, 1H), 7.67 (d, J = 8.7 Hz, 2H), 7.57 (s, 1H), 7.46–7.50 (m, 1H), 7.25 (d, J = 8.7 Hz, 2H), 7.24–7.33 (m, 2H), 3.72 (s, 2H), 2.84–2.88 (m, 2H), 2.45–2.54 (m, 2H), 2.05–2.16 (m, 2H), 1.68–1.74 (m, 2H). Anal. (C₂₀H₂₀INO₂·C₂H₂O₄) C, H, N.

7.22 1-(1-(Benzofuran-3-ylmethyl)piperidin-4-yl)-1H-benzo[d]imidazol-2-(3H)-one oxalate (23). Method B

Yield 22% from 3-methylbenzofuran and 4-(2-keto-1-benzimidazolinyl)piperidine (4). Conversion to the oxalate salt gave 23 as a white powder, mp 244–245 °C; ¹H NMR (free base, CDCl₃) δ 9.72 (s, 1H), 7.76–7.79 (m, 1H), 7.58 (s, 1H), 7.48–7.51 (m, 1H), 7.28–7.35 (m, 3H), 7.03–7.12 (m, 3H), 4.33–4.41 (m, 1H), 3.72 (s, 2H), 3.12–3.16 (m, 2H), 2.42–2.54 (m, 2H), 2.20–2.27 (m, 2H), 1.80–1.84 (m, 2H). Anal. (C₂₁H₂₁N₃O₂·C₂H₂O₄·0.25H₂O) C, H, N.

7.23 1-(1-(Benzo[b]thiophen-3-ylmethyl)piperidin-4-yl)-1H-benzo[d]imidazol-2-(3H)-one oxalate (24). Method B

Yield 63% from 3-methylbenzo[b]thiophene and 4-(2-keto-1-benzimidazolinyl)piperidine (4). Conversion to the oxalate salt gave 24 as a pale yellow powder, mp 242–243 °C; ¹H NMR (free base, CDCl₃) δ 9.92 (s, 1H), 8.01–8.04 (m, 1H), 7.86–7.89 (m, 1H), 7.34–7.46 (m, 3H), 7.23–7.26 (m, 1H), 7.03–7.12 (m, 3H), 4.36–4.44 (m, 1H), 3.82 (s, 2H), 3.12–3.16 (m, 2H), 2.43–2.54 (m, 2H), 2.21–2.29 (m, 2H), 1.79–1.84 (m, 2H). Anal. (C₂₁H₂₁N₃OS·C₂H₂O₄) C, H, N.

7.24 8-(Benzofuran-3-ylmethyl)-3-methyl-1-phenyl-1,3,8-triazaspiro[4.5]decan-4-one oxalate (25). Method B

Yield 34% from 3-methylbenzofuran and 3-methyl-1-phenyl-1,3,8-triazaspiro[4.5]decan-4-one (28a). Conversion to the oxalate salt gave 25 as an off-white powder, mp 209–210 °C; ¹H NMR (free base, CDCl₃) δ 7.81–7.84 (m, 1H), 7.57 (s, 1H), 7.46–7.49 (m, 1H), 7.27–7.33 (m, 4H), 6.82–6.92 (m, 3H), 4.66 (s, 2H), 3.72 (s, 2H), 2.99 (s, 3H), 2.87–2.90 (m, 4H), 2.65–2.73 (m, 2H), 1.62–1.67 (m, 2H). Anal. (C₂₃H₂₅N₃O₂·C₂H₂O₄·0.25H₂O) C, H, N.

7.25 8-(Benzofuran-3-ylmethyl)-3-(2-fluoroethyl)-1-phenyl-1,3,8-triazaspiro[4.5]decan-4-one oxalate (26). Method B

Yield 31% from 3-methylbenzofuran and 3-(2-fluoroethyl)-1-phenyl-1,3,8-triazaspiro[4.5]decan-4-one (28b). Conversion to the oxalate salt gave 26 as a white powder, mp 175–176 °C; ¹H NMR (free base, CDCl₃) δ 7.80–7.83 (m, 1H), 7.56 (s, 1H), 7.45–7.49 (m, 1H), 7.27–7.33 (m, 4H), 6.84–6.94 (m, 3H), 4.79 (s, 2H), 4.64 (dt, J = 47.2 and 4.6 Hz, 2H), 3.73 (dt, J = 28.6 and 4.6 Hz, 2H), 3.71 (s, 2H), 2.80–2.89 (m, 4H), 2.62–2.73 (m, 2H), 1.65–1.70 (m, 2H). Anal. (C₂₄H₂₆FN₃O₂·C₂H₂O₄·0. 5H₂O) C, H, N.

7.26 8-(Benzo[b]thiophen-3-ylmethyl)-3-methyl-1-phenyl-1,3,8-triazaspiro[4.5]decan-4-one oxalate (27). Method B

Yield 37% from 3-methylbenzo[b]thiophene and 3-methyl-1-phenyl-1,3,8-triazaspiro[4.5]decan-4-one (28a). Conversion to the oxalate salt gave 27 as a white powder, mp 199–200 °C; ¹H NMR (free base, CDCl₃) δ 8.08–8.11 (m, 1H), 7.85–7.87 (m, 1H), 7.28–7.44 (m, 5H), 6.81–6.91 (m, 3H), 4.66 (s, 2H), 3.83 (s, 2H), 2.99(s, 3H), 2.88–2.91 (m, 4H), 2.65–2.76 (m, 2H), 1.62–1.66 (m, 2H). Anal. (C₂₃H₂₅N₃OS·C₂H₂O₄) C, H, N.

7.27 3-(2-Fluoroethyl)-1-phenyl-1,3,8-triazaspiro[4.5]decan-4-one (28b)

A solution of ditert-butyl dicarbonate ((BOC)₂O, 7.0 mmol) in dichloromethane was added in a solution of 1-phenyl-1,3,8-triazaspiro[4.5]decan-4-one (30, 6.5 mmol) and triethylamine (7.0 mmol) in dichloromethane. The reaction mixture was stirred at room temperature for 3 hours. After the work-up, the N-protected compound 31 was obtained. Sodium hydride (15.8 mmol) and 1-bromo-2-fluoroethane (7.0 mmol) were added in a cold solution of 31 (4.5 mmol) in anhydrous N,N-dimethylformamide. The reaction mixture was stirred at room temperature for 4 hours. The obtained product was treated with trifluoroacetic acid to give 28b, which was used without further purification.

7.28 8-(Benzofuran-3-ylmethyl)-3-((1-(2-fluoroethyl)-1H-1,2,3-triazol-4-yl)methyl)-1-phenyl-1,3,8-triazaspiro[4.5]decan-4-one oxalate (29)

Sodium hydride (2.5 equiv) and propagyl bromide (1.5 equiv) were added into a cold solution of 31 (1.0 equiv) in anhydrous tetrahydrofuran. The reaction mixture was stirred at ambient temperature for 4 hours and washed with water. Evaporation of the organic layer gave the product which was treated with trifluoroacetic acid to give 32. Using the general method B, 33 was made from 32 and 3-methylbenzofuran. To a solution of 33 (1 equiv) and 1-azido-2-fluoroethane (2 equiv) in anhydrous N,N-dimethylformamide was added sodium ascorbate (5 equiv) and copper sulfate pentahydrate (0.5 equiv). The reaction was stirred for 3 hours and TLC analysis indicated complete consumption of the reactants. Water was added and the product was extracted with ethyl acetate. The combined organic layers were washed with water, dried, and concentrated. Column chromatography (5% methanol in dichloromethane) of the resulting residue gave the product (4% overall yield from 3-methybenzofuran). Conversion to the oxalate salt gave 29 as a white powder, mp 160–161 °C; ¹H NMR (free base, CDCl₃) δ 7.80–7.83 (m, 1H), 7.69 (s, 1H), 7.56 (s, 1H), 7.46–7.49 (m, 1H), 7.24–7.33 (m, 3H), 6.81–6.91 (m, 3H), 4.85–4.88 (m, 1H), 4.76 (s, 2H), 4.71 (s, 2H), 4.69–4.72 (m, 2H), 4.60–4.63 (m, 1H), 3.72 (s, 2H), 2.82–2.88 (m, 4H), 2.62–2.73 (m, 2H), 1.61–1.65 (m, 2H). Anal. (C₂₇H₂₉FN₆O₂·C₂H₂O₄·H₂O) C, H, N.

8. Radioligand binding and functional assays

8.1 Dopamine receptor binding assays

The method for the iodination of ¹²⁵I-IABN using peracetic acid has been previously described.²⁷ For radioligand binding studies, membrane homogenates from stably transfected HEK 293 cells expressing either the human D₂, D₃ or D₄ receptors were prepared using a polytron tissue homogenizer (Brinkman Instruments, Westbury, NY). The tissue was suspended in 50 mM Tris-HCl, 150 mM NaCl and 1 mM EDTA at pH 7.5 to approximately 5 to 20 µg of protein per 50 µl prior to the assay. Assays were performed in a total volume of 150 µl. Binding reactions were carried out for 60 min at 37°C and the reaction was terminated by rapid filtration over Schleicher and Schuell No 32 glass fiber filters (Whatman plc, Maidstone, England,). After washing filters with buffer, the radioactivity of the ¹²⁵I-labeled ligand was quantitated using a Packard Cobra gamma counter with an efficiency of 75%. Protein concentrations were determined using a BCA reagent (Pierce, Rockford, Illinois) with bovine serum albumin as the protein standard.

For competition curves using a transfected cell line expressing D₂, D₃ or D₄ dopamine receptors, experiments were performed in triplicate with two concentrations of inhibitor per decade over at least 5 orders of magnitude. The concentration of the radioligand was approximately equal to the K_d values. Controls containing either no inhibitor or 2 µM (+)-butaclamol were used to define total binding and nonspecific binding, respectively. Competition data for D_2-like dopamine receptors were modeled for a single-site fit using the TableCurve program (Jandel Scientific Software, San Rafael, California); the IC₅₀ values for the competitive inhibitors were converted to K_i values using the Cheng and Prusoff corrections.³²

8.2 Sigma receptor binding assays

Test compounds were dissolved in N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO) or ethanol and then diluted in 50 mM Tris-HCl buffer, pH 7.4, containing 150 mM NaCl and 100 mM EDTA. Membrane homogenates were made from guinea pig brain for σ₁ binding assay and rat liver for σ₂ binding assay. Membrane homogenates were diluted with 50 mM Tris-HCl buffer, pH 8.0, and incubated at 25°C in a total volume of 150 µL in 96-well plates with the radioligand and test compounds with concentrations ranging from 0.1 nM to 10 µM. After incubation was completed, the reactions were terminated by the addition of 150 µL of ice-cold wash buffer (10 mM Tris-HCl, 150 mM NaCl, pH 7.4) using a 96-channel transfer pipette (Fisher Scientific, Pittsburgh, PA), and the samples harvested and filtered rapidly through 96-well fiber glass filter plate (Millipore, Billerica, MA) that had been presoaked with 100 µL of 50 mM Tris-HCl buffer, pH 8.0, for 1 h. Each filter was washed three times with 200 µL of ice-cold wash buffer. A Wallac 1450 MicroBeta liquid scintillation counter (Perkin-Elmer, Boston, MA) was used to quantitate the bound radioactivity.

The σ₁ receptor binding assay was conducted using guinea pig brain membrane homogenates (~300 µg protein) and ~5 nM [³H](+)-pentazocine (34.9 Ci/mmol, Perkin-Elmer, Boston, MA). The incubation time was 90 min. Nonspecific binding was determined from samples that contained 10 µM of cold haloperidol.

The σ₂ receptor binding assays were conducted using rat liver membrane homogenates (~300 µg protein) and ~1 nM [³H]RHM-1 (80 Ci/mmol, American Radiolabeled Chemicals Inc., St. Louis, MO) alone or ~5 nM [³H]DTG (58.1 Ci/mmol, Perkin-Elmer, Boston, MA) in the presence of 1 µM (+)-pentazocine to block σ₁ sites. The incubation time was 60 min for [³H]RHM-1 and 120 min for [³H]DTG. Nonspecific binding was determined from samples that contained 10 µM of cold haloperidol.

Data from the competitive inhibition experiments were modeled using nonlinear regression analysis to determine the concentration of inhibitor that inhibits 50% of the specific binding of the radioligand (IC₅₀ value). Competitive curves were best fit to a one-site fit and gave pseudo-Hill coefficients of 0.6 – 1.0. K_i values were calculated using the method of Cheng and Prusoff³⁰ and represent mean values ± SEM. The K_d value used for [³H](+)-pentazocine with guinea pig brain homogenates was 7.89 nM, 30.73 nM was used for [³H]DTG with rat liver, while 0.66 nM was used for [³H]RHM-1 with rat liver.²⁹

8.3 Whole cell adenylyl cyclase assay

The accumulation of ³H-cyclic AMP in HEK cells was measured by a modification of the method of Shimizu et al.³³ as previously described.²⁷ Transfected HEK cells were treated with serum-free medium containing 2,8-³H-adenine (ICN) and cells were incubated at 37 °C for 75 min. Cells and drugs diluted in serum-free media containing 0.1 mM 3-isobutyl-1-methylxanthine (Sigma) were mixed to give a final volume of 500 µL and cells were incubated for 20 min at 37 °C. The reaction was stopped by addition of 500 µL of 10% trichloroacetic acid and 1 mM cyclic AMP. After centrifugation, the supernatants were fractionated using Dowex AG1-X8 and neutral alumina to separate the ³H-ATP and the ³H-cyclic AMP. Individual samples were corrected for column recovery by monitoring the recovery of the cyclic AMP using spectrophotometric analysis at OD 259 nm.^27,33

Appendix.

Elemental Analyses

Compounds	%C		%H		%N
	Calc’d	Found	Calc’d	Found	Calc’d	Found
5	48.90	48.50	4.66	4.44	5.18	4.76
6	49.61	49.92	4.62	4.98	5.03	4.63
7	48.05	47.95	4.82	4.55	4.87	4.46
8	64.10	64.07	6.20	6.08	6.50	6.37
9	52.76	52.96	4.98	5.03	5.13	5.09
10	52.76	53.13	4.98	5.25	5.13	5.25
11	61.53	61.13	6.02	5.74	11.96	11.77
12	61.53	61.50	6.02	5.72	11.96	11.75
13	49.23	48.83	5.11	4.80	8.20	7.81
14	44.38	44.32	4.79	4.39	7.39	7.39
15	57.25	57.63	5.90	5.62	9.10	9.31
18	53.45	53.66	4.89	5.18	2.83	2.76
19	48.81	49.17	4.47	4.63	2.59	2.51
20	52.70	52.97	4.62	4.57	2.79	2.78
21	55.48	55.25	4.66	4.69	2.94	3.00
22	50.49	50.31	4.24	4.28	2.68	2.71
23	62.51	62.61	5.36	5.22	9.51	9.53
24	60.91	61.05	5.11	5.21	9.27	9.10
25	63.89	63.93	5.90	5.75	8.94	8.93
26	61.65	61.84	5.77	5.62	8.30	8.29
27	62.35	62.22	5.65	5.64	8.73	8.54
29	58.38	58.16	5.58	5.41	14.09	13.69