Leveraging a Low-Affinity Diazaspiro Orthosteric Fragment to Reduce Dopamine D₃ Receptor (D₃R) Ligand Promiscuity across Highly Conserved Aminergic G‑Protein-Coupled Receptors (GPCRs)

Abstract

Previously, we reported a 3-(2-methoxyphenyl)-9-(3-((4-methyl-5-phenyl-4H-1,2,4-triazol-3-yl)thio)-propyl)-3,9-diazaspiro[5.5]undecane (1) compound with excellent dopamine D₃ receptor (D₃R) affinity (D₃R K_i = 12.0 nM) and selectivity (D₂R/D₃R ratio = 905). Herein, we present derivatives of 1 with comparable D₃R affinity (32, D₃R K_i = 3.2 nM, D₂R/D₃R ratio = 60) and selectivity (30, D₃R K_i = 21.0 nM, D₂R/D₃R ratio = 934). Fragmentation of 1 revealed orthosteric fragment 5a to express an unusually low D₃R affinity (K_i = 2.7 μM). Compared to piperazine congener 31, which retains a high-affinity orthosteric fragment (5d, D₃R K_i = 23.9 nM), 1 was found to be more selective for the D₃R among D₁- and D₂-like receptors and exhibited negligible off-target interactions at serotoninergic and adrenergic G-protein-coupled receptors (GPCRs), common off-target sites for piperazine-containing D₃R scaffolds. This study provides a unique rationale for implementing weakly potent orthosteric fragments into D₃R ligand systems to minimize drug promiscuity at other aminergic GPCR sites.

Graphical Abstract

INTRODUCTION

The dopamine (DA) D₃ receptor (D₃R) is a G-protein-coupled receptor (GPCR), viewed as a pharmacotherapeutic target for numerous neurological and psychiatric disorders as well as drug addiction.^1,2 Targeting this D₂-like receptor is a strategy for schizophrenia drug development due to the unwanted extrapyramidal side-effects elicited with many typical anti-psychotics.³ The appeal of D₃R for drug addiction therapeutics stems from the high expression of this protein in the mesolimbic pathway, a region of the brain implicated in reward and motivation.⁴ Human postmortem studies showing an enhanced expression of the D₃R in drug exposed brains have further validated the clinical importance of this receptor for substance abuse disorders.⁵ Selective engagement of this receptor is also of considerable interest for positron-emission tomography (PET) imaging applications to further our understanding and elucidate the complex molecular mechanisms of drug addiction.^6–9

The development of high-affinity D₃R selective ligands begins with a fragment-based approach in which the binding profiles of amino core synthons are examined at the orthosteric binding site (OBS) of the receptor. This region contains a highly conserved Asp110 [3.32] residue which forms a salt bridge interaction with the protonated nitrogen of the amino core in the ligand scaffold affording receptor recognintion.¹⁰ Due to the high degree of homology between the D₃R and the dopamine D₂ receptor (D₂R) in this domain, the ligand must then extend into a secondary binding pocket (SBP) containing residues unique to each receptor to confer D₃R selectivity.^10–12 However, the Asp110 [3.32] that forms the critical receptor−ligand interaction is also greatly conserved across other aminergic GPCRs.¹³ Thus, synthesizing D₃R selective compounds that concomitantly engage the Asp110 [3.32] residue and exhibit minimal off-target interactions continues to be problematic. As such, there has been no clinically viable D₃R selective therapeutic or PET imaging agent reported to date, preventing us from exploring the therapeutic potential of this receptor.

Recently, we reported a new class of D₃R selective compounds containing spirodiamine systems as an alternative to the piperazine amino core (Figure 1).¹⁴ In this study, we synthesized and evaluated analogs and fragments of 1 to identify the structural determinants responsible for ligand affinity and selectivity at the D₃R. Compared to the excellent binding profile of aryl piperazine orthosteric fragment 5d (K_i = 23.9 nM) of ligand 31, we found sython 5a of 1 to bind with lower affinity (K_i > 2 μM). Computational ligand docking studies were performed to elucidate the binding mode of 1, and other select compounds, within the D₃R crystal structure (3PBL). Finally, the pharmacological behavior of 1 and 31 was screened across all D₁ (D₁R and D₅R)- and D₂ (D₂R, D₃R and D₄R)-like receptors, and select aminergic GPCRs, revealing 1 to be a more selective ligand for the D₃R with less off-target interactions. These results provide rationale for utilizing primary pharmacophores (PP) with limited affinity for the highly conserved orthosteric binding pocket (OBP) of the receptor to develop selective D₃R ligands with minimized off-target interactions.

Figure 1.

Lead compounds identified with diazaspiro amino systems.

RESULTS AND DISCUSSION

Chemistry.

Synthesis of arylated diazaspiro and piperazine systems was achieved in excellent yields following our previously reported palladium (Pd) C−N cross-coupling reports outlined in Scheme 1.^15,16 Unsaturated diazaspiro[5.5]undec-1-ene motif 4a, a presumed cross-coupling β-hydride elimination side-product, was able to be obtained, albeit in low yields (<10%). Following removal of the BOC protecting groups from 4b, 4c, and 4e with trifluoroacetic acid (TFA) and basification, the corresponding free-amine intermediates 4b′, 4c′, and 4e′ were N-alkylated with 1-bromobutane at room temperature (rt) in acetone to afford desired synthons 5a−c. Similarly, S-alkylation of commercially available 4-methyl-5-phenyl-4H-1,2,4-triazole-3-thiol with the appropriate alkylating reagent yielded 6a,b in good yields. The formation of 1,2,4-triazole fragments 6c−e and 6g was readily accessed by reacting 6b with the desired amine in the presence of Cs₂CO₃ under refluxing conditions. BOC-deprotection with TFA, followed by base neutralization, afforded the final free-amine fragments 6f and 6h.

Scheme 1. Synthesis of Fragmented Synthons^a.

Reagents and conditions: (i) Pd₂(dba)₃, RuPhos, aryl halide, diazaspiro reagent, NaOt-Bu, dioxane, 100 °C, 20 min; (ii) TFA, CH₂Cl₂, rt, 3 h; (iii) 1-bromobutane, K₂CO₃, acetone, rt, 12 h; (iv) alkylating reagent, K₂CO₃, acetone, rt, 12 h; (v) amine, Cs₂CO₃, acetonitrile (ACN), 70 °C, 8 h.

Scheme 2 briefly illustrates the general synthesis, disclosed in our previous report,¹⁴ used to obtain ligands 1−3 and 7−34. Aryl amide scaffolds 35−37 were prepared by reacting 4b′ with 2-(4-bromobutyl)isoindoline-1,3-dione, to afford precursor E in a modest yield of 64%. Next, E was treated with hydrazine in refluxing EtOH to afford free-amine intermediate F in an excellent yield (97%). Finally, benzamide compounds 35−36 were synthesized by coupling F with the respective benzoic acid in the presence of 1-hydroxybenzotriazole (HOBt) hydrate and 1-ethyl-3-(3′-dimethylaminopropyl)carbodiimide (EDC) in dichloromethane at room temperature. Following our 2011 report,¹⁷ synthon 4b′ was N-alkylated with intermediate G to afford the aripiprazole analog 37 in a modest yield (62%).

Scheme 2. General Synthesis of Compounds 1−3 and 7–37^a.

Reagents and conditions: (i) (A) TFA, CH₂Cl₂, rt, 3 h; (ii) (B) alkylating reagent, K₂CO₃, acetone, rt, 12 h; (iii) (C, D) TEA, EtOH, 75 °C, 12 h; (iv) 4b′, 2-(4-bromobutyl)isoindoline-1,3-dione, KI and Cs₂CO₃, ACN, 75 °C for 3 h; (v) (E), hydrazine, EtOH, 75 °C for 2 h; (vi) (F), benzoic acid, HOBt, EDC, CH₂Cl₂, rt, 2 h; (vii) (G), KI, and K₂CO₃, ACN, 90 °C for 12 h.

Radioligand Binding Profiles of Compound 1 Analogs and Fragments.

Structural modifications were made to 1 in attempts to further enhance D₃R binding affinity and probe the structural determinants of this ligand template responsible for receptor selectivity. Compounds and synthons of select ligands were then evaluated in radioligand binding assays using [¹²⁵I]-N-benzyl-5-iodo-2,3-dimethoxy[3.3.1]azabicyclononan-3-β-yl-benzamide ([¹²⁵I]IABN) with human embryonic kidney 293 (HEK) cells stably expressing human D₃R and D_2L (Tables 1−5). Values of c log P and topological polar surface area (tPSA) are also included in Tables 1−4 to provide an estimate of lipophilicity and cell permeability, respectively, for each full-length ligand.

Table 1.

D₃R/D₂R Binding Profiles of 1 Analogs with Modifications to the 1,2,4-Triazole System^a

compound	R1	R2	Y	Ki ± SEM (nM)b		D2/D3 ratioe	clog P f	tPSAf
				D3Rc	D2Rd
1 g	phenyl	CH3	N	12.0 ± 2.8	10 895 ± 2069	905	4.88	43.67
7	4-N,N-dimethylaniline	CH3	N	138 ± 17.1	12 137 ± 2713	88	5.21	46.91
8	2-methoxyphenyl	CH3	N	63.0 ± 7.5	2112 ± 269	33.5	4.37	52.90
9	pyrimidin-3-yl	CH3	N	213 ± 21.4	48 584 ± 10 378	229	3.69	56.03
10	4-(thiophen-3-yl)phenyl	CH3	N	101 ± 32.3	3250 ± 782	32.2	6.41	43.67
11	4-(1,3-oxazol-2-yl)-phenyl	CH3	N	25.4 ± 2.7	1574 ±396	62.1	4.66	65.26
12	2-fluorophenyl	CH3	N	25.9 ± 3.7	19 655 ± 4441	759	5.03	43.67
13	4-fluorophenyl	CH3	N	39.8 ± 11.9	15 768 ± 1661	396	5.03	43.67
14	cyclohexyl	CH3	N	57.0 ± 13.6	12 584 ± 1732	221	5.17	43.67
15	phenyl	CH2CH3	N	48.7 ± 8.9	5650 ± 893	116	5.55	43.67
16	phenyl	NH2	N	75.0 ± 6.3	13 904 ± 453	185	4.90	69.69
17	phenyl	H	N	32.5 ± 2.6	1162 ±229	35.7	5.06	52.46
18	phenyl	H	CH	383 ± 53.7	5891 ± 267	15.4	6.24	40.1
19	phenyl	CH3	CH	418 ± 49.8	35 720 ± 5804	85.4	6.26	31.21

^aSpiperone assayed under the same conditions as a reference blocker: D₂R, 0.06 ± 0.001 nM; D₃R, 0.33 ± 0.02 nM; D₄R, 0.45 ± 0.01 nM.

^bMean ± standard error of the mean (SEM); K_i values were determined by at least three experiments.

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

^dK_i values for D₂ receptors were measured using human D₂ expressed in HEK cells with [¹²⁵I]IABN as the radioligand.

^e(K_i for D₂ receptors)/(K_i for D₃ receptors).

^fCalculated using ChemDraw Professional 15.1.

^gCompound reported in our initial work ref 14.

Table 5.

D₃R/D₂R Binding Profiles of Synthons^a

Compound	Synthon	Ki ± SEM (nM)b		D2R/D3R Ratioe	Compound	Synthon	Ki± SEM (nM)b		D2R/D3R Ratioe
		D3Rc	D2Rd				D3Rc	D2Rd
4b’		>39,319 ± NA	99,379 ± 11,752	>2.5	6a		>50,316 ±NA	180,375 ±70,719	>3.6
5a		2,773 ± 384	26,739 ± NA	>9.6	6c		1,098 ± 53.8	98,462 ± 18,965	89.6
5b		6,647 ± 4,559	25,823 ± 14,830	3.9	6d		631 ± 43.7	59,994 ± 22,704	95.1
5c		23,256 ± 9,615	26,050 ± NA	>1.1	6f		1,145 ± 97.1	>225,748 ± NA	>197
5d f		23.9 ±5.8	20.6 ±4.8	0.86	6h		72,753 ± 13,438	74,272 ± 17,815	1
5e		14.7 ± 1.2	22.3 ±4.9	1.5

^aSpiperone assayed under the same conditions as a reference blocker: D₂R, 0.06 ± 0.001 nM; D₃R, 0.33 ± 0.02 nM; D₄R, 0.45 ± 0.01 nM.

^bMean ± SEM; K_i values were determined by at least three experiments.

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

^dK_i values for D₂ receptors were measured using human D expressed in HEK cells with [¹²⁵I]IABN as the radioligand.

^e(K_i for D₂ receptors)/(K_i for D₃ receptors).

^fCompound and receptor binding data from ref 11.

Table 4.

D₃R/D₂R Binding Profiles of 1 Analogs with Modifications to the Aryl Head Group^a

Compound	R	Ki ± SEM (nM)b		D2R/D3R Ratioe	cLogPf	tPSAf
		D3Rc	D2Rd
35		92.9 ± 20.5	2,766 ± 582	29.8	5.64	44.81
36		61.5 ±15.0	3,762 ± 776	61.2	4.43	48.05
37		14,587 ± 1,772	2,630 ± 400	0.1	4.50	54.04

^aSpiperone assayed under the same conditions as a reference blocker: D₂R, 0.06 ± 0.001 nM; D₃R, 0.33 ± 0.02 nM; D₄R, 0.45 ± 0.01 nM.

^bMean ± SEM; K_i values were determined by at least three experiments.

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

^dK_i values for D₂ receptors were measured using human D₂ expressed in HEK cells with [¹²⁵I]IABN as the radioligand.

^e(K_i for D₂ receptors)/(K_i for D₃ receptors).

^fCalculated using ChemDraw Professional 15.1.

We first modified the aryl 1,2,4-triazole ring system in 1 and evaluated several aryl moieties found to be tolerable in azaspirocyclic D₃R ligand architectures previously disclosed by Micheli and co-workers.^18–20 However, we observed a drastic decrease in receptor selectivity after installing the 4-(1,3-oxazol-2-yl)-phenyl (11) and cyclohexyl (14) substitutions (D₂R/D₃R ratio = 62 and 221, respectively), indicating that our 1,2,4-triazole ligand architecture follows a different structure−activity relationship (SAR) within the D₃R. When modifying the aryl ring with fluorine substitutions, we found the ortho position to be more preferred (12, K_i = 25.9 nM, D₂R/D₃R ratio = 759), as the 4-fluoro analog resulted in a 2-fold decrease in the receptor selectivity (13, K_i = 39.8 nM, D₂R/D₃R ratio = 396).

Next, binding profiles of ligands containing structural modifications to the 1,2,4-triazole ring system were evaluated (15−19). With respect to 1, replacing the −CH₃ group for a −CH₂CH₃ (15) or −NH₂ (16) substituent resulted in a 7-fold and 5-fold decrease in receptor selectivity, respectively. Similarly, the deletion of the −CH₃ group resulted in a drastic loss of receptor selectivity, affording a 35-fold D₂R/D₃R ratio for 17, combined with a moderate decrease in D₃R affinity (K_i = 32.5). Imidazole systems were also evaluated as triazole alternatives, however, we found a significant loss in receptor affinity and binding selectivity for ligands 18 and 19.

We then explored the effect on D₃R binding by manipulating the linker length in compounds 20 and 21 (Table 2). However, this modification afforded an ~8-fold reduction in D₃ R selectivity, demonstrating the N-propyl linker to be the more optimal spacer. Next, we probed the influence of the 2-methoxy substituent in compound 1 on D₃R ligand binding. When replacing the 2-methoxy with a 2-ethoxy in 22, receptor selectivity was diminished (D₂R/D₃R ratio = 114), along with a moderate decrease in binding affinity (K_i = 45.1 nM). Compared to 20, the 2-fluoroethoxy substituent showed to be more compatible, affording an increase in overall binding and selectivity (K_i = 33.3 nM, D₂R/D₃R ratio = 192). In our initial report, we found that moving the aryl methoxy substituent from the 2-position in 1 to the 4-position in 25 further enhanced the D₃R selectivity (D₂R/D₃R ratio = ~1000-fold) but also diminished ligand affinity for the receptor (K_i = 97.7 nM).¹⁴ Thus, we evaluated 26, a compound containing methoxy substituents in the ortho and para positions of the aryl ring, to determine if affinity could be enhanced while maintaining similar selectivity. Instead, we found the receptor affinity for 26 (K_i = 112 nM) to be comparable to 25, along with a ~2-fold reduction in D₃R selectivity (D₂R/D₃R ratio = 584).

Table 2.

D₃R/D₂R Binding Profiles of 1 Analogs with Modifications to the Linker and Aryl Ring^a

compound	n	R	Ki ± SEM (nM)b		D2/D3 ratioe	clog Pf	f
			D3Rc	D2Rd			tPSAf
20	0	2-methoxy	89.7 ± 22.5	9004 ± 1133	100	4.54		43.67
21	2	2-methoxy	59.2 ± 5.1	9248 ± 1745	156	5.06	43.67
22	1	2-ethoxy	45.1 ± 9.7	5165 ± 525	114	5.41	43.67
23	1	2-hydroxy	396 ± 17.3	17 595 ± 4185	44.4	4.33	54.67
24	1	2-fluorethoxy	33.3 ± 1.7	6379 ± 964	192	5.27	43.67
25 g	1	4-methoxy	97.7 ± 17.4	104 847 ± 29 076	1073	4.88	43.67
26	1	2,4-dimethoxy	112 ± 24.5	65 229 ± 37 252	584	4.89	52.90
27 g	1	4-fluoro	25.6 ± 5.6	9792 ± 1790	383	5.32	34.44
28	1	2-fluoro	208 ± 9.6	26 253 ± 6866	126	5.32	34.44
29	1	2-fluoro-4-trifluoromethyl	370 ± 17.8	21 172 ± 1499	57.2	6.38	34.44
30	1	2-methoxy-4-fluoro	21.0 ± 1.4	19 634 ± 3691	934	5.17	43.67

^aSpiperone assayed under the same conditions as a reference blocker: D₂R, 0.06 ± 0.001 nM; D₃R, 0.33 ± 0.02 nM; D₄R, 0.45 ± 0.01 nM.

^bMean ± SEM; K_i values were determined by at least three experiments.

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

^dK_i values for D₂ receptors were measured using human D₂ expressed in HEK cells with [¹²⁵I]IABN as the radioligand.

^e(K_i for D₂ receptors)/(K_i for D₃ receptors).

^fCalculated using ChemDraw Professional 15.1.

^gCompound reported in our initial work ref 14.

Next, we examined the binding profiles of fluorinated derivatives 28−30. When moving the fluorine atom from the 4-position (27) to the 2-position in 28, a loss in receptor affinity (K_i = 208 nM) and selectivity (D₂R/D₃R ratio = 126) is observed. Upon installing a −CF₃ functional group in the para position of the aryl ring in 29, we observed a further decrease in both D₃ R affinity (K_i = 370 nM) and selectivity (D₂ R/D₃ R ratio = 57). Due to the excellent D₃R binding properties observed with compounds 1 and 27, we evaluated the 4-fluoro-2-methoxyphenyl ring system in ligand 30. Compared to 1, compound 30 was found to have a comparable D₃R binding affinity (K_i = 21.0 nM) to 1, along with a modest improvement in receptor selectivity (D₂R/D₃R ratio = 934).

Next, we evaluated two additional analogs containing modified diazaspiro cores (Table 3). In contrast to 1 and 31, unsaturated ligand 32, containing a novel diazaspiro[5.5]undec-1-ene amino core, was found to bind with a higher affinity at the D₃R (K_i = 3.2 nM), although receptor selectivity was not maintained (D₂R/D₃R ratio = 60). Interestingly, when reversing the diazaspiro[4.5]decane moiety found in 33, a 2-fold increase in receptor selectivity was observed for ligand 34 (D₂R/D₃R ratio = 758), along with a similar D₃R binding affinity (K_i = 27.8 nM).

Table 3.

D₃R/D₂R Binding Profiles of 1 Analogs with Modifications to the Amino Core^a

Compound	Amino Core	Ki ± SEM (nM)b		D2/D3 Ratioe	cLogPf	IPSAf
		D3Rc	D2Rd
31 g		6.5 ±0.88	260 ± 44.2	40.2	4.09	43.67
32		3.2 ±0.71	197 ±24.4	60.7	5.38	43.67
33 g		19.6 ±4.7	6,168 ±939	315	4.32	43.67
34		27.8 ±4.4	21,098 ±3,069	758	4.32	43.67

^aSpiperone assayed under the same conditions as a reference blocker: D₂R, 0.06 ± 0.001 nM; D₃R, 0.33 ± 0.02 nM; D₄R, 0.45 ± 0.01 nM.

^bMean ± SEM; K_i values were determined by at least three experiments.

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

^dK_i values for D₂ receptors were measured using human D₂ expressed in HEK cells with [¹²⁵I]IABN as the radioligand.

^e(K_i for D₂ receptors)/(K_i for D₃ receptors).

^fCalculated using ChemDraw Professional 15.1.

^gCompound reported in our initial work ref 14.

Finally, we evaluated the diazaspiro[5.5]undecane amino core in two aryl amide templates that were previously identified as excellent D₃R-prefering scaffolds when conjugated to the 1-(2-methoxyphenyl)piperazine system (Table 4).^21,22 When main-taining the 2-methoxyphenyl aryl system, we investigated 4-(dimethylamino)- and 4-(thiophen-3-yl)-benzamides with a four-carbon spacer linked to the diazaspiro[5.5]undecane moiety. However, using this ligand framework, compounds 35 and 36 both exhibited poor D₃R receptor affinity and selectivity. This diazaspiro core was then evaluated in a D₂R-prefering aripiprazole ligand architecture disclosed in our previous work.¹⁷ Although 37 appears to be slightly D₂R-selective, receptor affinity was low.

Lead compound 1 was fragmented to identify synthons acting as primary (PP) or secondary pharmacophores (SP). Unexpectedly, fragments 4b′ and 5a were found to have a micromolar affinity for the receptor, with 5a exhibiting nominal D₃R selectivity (Table 5). Low receptor binding was also observed with synthons 5b and 5c, despite the K_i values of 19.6 nM and 24.2 nM obtained from the parent compounds 2 and 3, respectively. The binding affinities obtained with 5a−c are in contrast to those reported for alkylated aryl piperazine synthons (i.e., 5d¹¹ and 5e^21,23) of potent D₃R ligand scaffolds. These PP typically show K_i values of ~10−20 nM at the receptor by engaging the orthosteric binding pocket (OBP) of the protein. This allows the ionizable nitrogen of these PP to form an ionic interaction with the highly conserved Asp110 [3.32] residue within the OBP, affording the favorable receptor affinity of these orthosteric fragments.

Binding studies continued with the evaluation of synthons from the right-half of lead compound 1 to determine if receptor affinity was being engaged by an unexpected 1,2,4-triazole pharmacophore. Similar to the displacement observed with synthon 5a, low receptor binding was observed with fragments 6a, 6c, 6d, and 6f. Although minimal potency was observed with these 1,2,4-trizole synthons, a trend of increasing affinity was observed when elongating fragment 6a with piperdine (6c), and 4,4-dimethylpiperidine (6d). In addition, we found 6f to be ~197-fold selective for the D₃R over the D₂R, whereas piperazine analog 6h illustrated no receptor selectivity. We hypothesized that the 1,2,4-triazole system may be key in directing, and subsequently, stabilizing the bulky diazaspiro PPs of 1−3, within the OBP of the receptor. Once stabilized, these diazaspiro systems may be able to interact with residues within the OBP due to the large structural space these systems inhabit which are not accessible to the piperazine PP of 31.

Molecular Docking Studies.

To probe these potential protein−ligand interactions, selected compounds were modeled within the D₃R (3PBL) to identify residue contacts within orthosteric and SBP of the receptor (Figure 2A). A top docking pose of 1 was found to engage in multiple polar and nonpolar contacts between the arylated diazaspiro[5.5]undecane moiety, including π−π stacking interactions between the aryl ring and the Phe346^6.52 residue within the OBP of the receptor (Figure 2B). Similarly, ligand docking also predicted the diazaspiro[5.5]-undecane moiety of ligand 17 and diazaspiro[5.5]undec-1-ene system of high-affinity compound 32, to also engage in hydrophobic interactions with residues Phe346 [6.52] and Phe345 [6.51] (Figure 2C,F). When evaluating the potential salt bridge formation between residue Asp110 [3.32] and the ionizable nitrogens in the diazaspiro cores of scaffolds 1, 17, and 32, we identified distances of 3.4, 3.2, and 3.1 Å, respectively. Interestingly, these were all found to be greater than the predicted 2.7 Å distance observed with the binding pose of compound 31 (Figure 2E). These results could explain the contrasting receptor binding affinity observed between diazaspiro PP 5a and piperazine PP 5d. Thus, we surmise that the 1,2,4-triazole side of the molecule appropriately positions the bulky diazaspiro[5.5]undecane moiety within the hydrophobic cavity of the D₃R OBP, allowing the amino core to engage in nonpolar interactions with residues Phe346 [6.52] and Phe345 [6.51]. These contacts then assist in stabilizing this “weaker” ionic interaction with Asp110 [3.32], resulting in the observed receptor affinities for 1, 17, and 32. This analysis is also consistent with the poor binding affinity for compound 19, as the docking pose predicted minimal hydrophobic interactions with residues Phe345 [6.52] and Phe346 [6.51], and a distance of 4.2 Å between the protonated nitrogen of the ligand and the conserved Asp110 [3.32] side chain (Figure 2D).

Figure 2.

(A) Predicted binding modes of compound 1 derivatives in complex with the D₃R (3PBL). Predicted polar (yellow, 3.0 Å cut-off), weak hydrogen bonding (magenta, 3.6 Å cut-off), π−π stacking (purple, 5.5 Å cut-off), and hydrophobic (black, 4.0 Å cut-off) residue interactions with compounds 1 (B), 17 (C), 19 (D), 31 (E), and 32 (F) within the binding pocket of the receptor. Interaction between the Asp110 [3.32] residue and the ionizable nitrogen of the ligand scaffold is shown in red.

Next, we evaluated interactions between the nonconserved residues within the SBP of the D₃R, responsible for ligand selectivity,¹¹ and the predicted binding modes of 1 and its derivatives. In addition to the significant polar and nonpolar interactions between Val86 [2.61] and the 1,2,4-triazole system of lead compound 1, we also identified a potential weak hydrogen bond interaction between Glu90 [2.65] and the methyl substituent of the N-heterocycle (Figure 2B). Disruption of this C−H···O interaction may explain the reduction in receptor selectivity for compound 17 (Figure 2C), in which the only structural difference between this ligand and 1 is the removal of the −CH₃ group. When substituting the −CH₃ with −CH₂CH₃ (15) or −NH₂ (16), receptor selectivity is again diminished, while binding affinity is moderately maintained. Although the N−H···O hydrogen bond interactions of 17 and Glu90 [2.65] would appear to be more favorable than the C− H···O interactions of 1, previous studies have shown the latter to result in more favorable binding profiles due to the potential penalty associated with −NH₂ desolvation upon ligand binding.^24,25

Previous reports have discussed the critical role EL1 Gly residues play in determining D₂R/D₃R selectivity of ligands.^26,27 Glycine can act as an acceptor in weak hydrogen bond interactions in many protein−ligand complexes.²⁴ In our study, the predicted binding pose of 1 places the aryl ring off of the 1,2,4-triazole moiety in close proximity to the Gly93 [EL1] residue, affording potential weak C−H···O interactions. However, this interaction is found to be absent in the binding poses of less selective ligands 31 and 32. We hypothesize that the lack of receptor selectivity observed for compounds 10−11 and 13 may be a result of steric clashing between the para substituents of these ligands and the Gly93 [EL1] residue in the SBS. This steric clash could also explain the lack of receptor selectivity for compounds 21 and 35−36, which all contain a butyl spacer group, compared to the shorter propyl linker of 1. We observed this proposed SAR trend after installing a C−F bond in the para position of 1, resulting in a loss of receptor selectivity for 13 (D₂R/D₃R = 905 vs 396, respectively). Receptor selectivity was able to be slightly restored, however, after moving the C−F substitution to ortho site of the aryl ring in 12, potentially alleviating steric interactions afforded with Gly93 [EL1] and compound 13.

Compound 1 Competitively Inhibits DA at the OBP.

To confirm that the binding mode of 1 does indeed require occupancy within the OBP, we evaluated 1 (Figure 3A), 31 (Figure 3B), and known allosteric modulator SB269652^28,29 (Figure 3C) in a β-arrestin assay with endogenous ligand dopamine (DA) as the orthosteric ligand. Both 1 and 31 caused a limitless rightward shift in the dopamine−dose response curves, suggesting a competitive mode of action at the OBP.³⁰ This is in contrast to the limited dextral shift that is observed with SB269652, resulting from the allosteric pharmacology of the compound at the receptor. Furthermore, when plotting the concentration−response curves for 1 and SB269652 into a Schild plot, a linear regression slope near unity is obtained for 1, indicative of competitive antagonism,³¹ whereas a nonlinear regression slope is observed for SB269652, indicating an allosteric mode of the antagonism (see Supporting Information).^32–34 Overall, these data clearly indicate that 1 behaves in a competitive manner at the OBP with the orthosteric endogenous ligand DA, ruling out an allosteric binding mechanism for the diazaspiro ligand.

Figure 3.

Curve shift analysis of 1 (A), 31 (B), and SB269,625 (C) using a dopamine (DA)-mediated β-arrestin 2 recruitment assay. The receptor was stimulated with the indicated concentration of DA along with and without concentrations of the test compound. Data points represent the mean ± SEM of dopamine-induced luminescence response obtained from 7 to 16 replicates.

Psychoactive Drug Screening Program (PDSP) Pharmacological Evaluation.

Compounds 1 and 31 were then submitted to the Psychoactive Drug Screening Program (PDSP)^35,36 to evaluate the binding (Table 6) and functional behavior of the ligands at D₁- and D₂-like receptors (Figure 4). Compared to our initial report,¹⁴ the K_i values obtained by the PDSP for 1 and 31 differed slightly at the D₃R and D₂R. However, this is most likely due to the use of [³H]N-methylspiperone by the PDSP in their binding assay, compared to our use of [¹²⁵I]IABN, a high-affinity radioligand with negligible nonspecific binding.³⁷ Compound 1 showed no interaction at the D₄R- or D₁-like receptors, whereas 31 exhibited K_i values of 721 nM and ~3 μM at the D₄R and D₅R, respectively (Table 6). Lead compound 1 was found to behave as a preferential D₃R antagonist (IC₅₀ = 41.4 nM) with negligible functional potency at the D₂R (IC₅₀ > 1.5 μM) (Figure 4D). Although antagonist affinities are known to vary depending on the competing agonist used,³⁸ we are currently evaluating 1 in other cell-signaling cascade models to further assess the 39-fold D₂R/D₃R selectivity ratio observed in the PDSP Tango functional assays. Ligand 31, however, was found to be a weak agonist/antagonist at both the D₂R and D₃R (Figure 4C−F), a common nuance that has been disclosed with previously reported D₃R selective piperazine-based scaffolds.^11,39 This can be attributed to the lack of receptor selectivity that is engaged with the aryl piperazine orthosteric PP of the ligand scaffold. Finally, compound 31 was also identified as a weak partial agonist at the D₄R (IC₅₀ = 427 nM) (Figure 4G).

Table 6.

Binding Affnities of Compounds 1 and 31 at Select GPCRs^a

		Ki (nM)a or % inhibition at 10 μMb				Ki (nM)a or % inhibition at 10 μMb
GPCR		1	31	GPCR		1	31
dopamine	D1	N/A	N/A	muscarinic	M1	54%	N/A
	D2	N/A	577		M2	52	N/A
	D3	94	6		M3	514	N/A
	D4	N/A	721		M4	590	N/A
	D5	N/A	3016		M5	N/A	N/A
serotonin	5-HT1A	1297	0.9	adrenergic	α 1A	2178	49
	5-HT1B	N/A	2032		α 1B	2173	94
	5-HT1D	N/A	>50%		α 2A	N/A	221
	5-HT1E	N/A	N/A		α 2B	N/A	241
	5-HT2A	N/A	1415		α 2C	1557	433
	5-HT2B	2144	42		α 2 β 2	N/A	N/A
	5-HT2C	N/A	159		α 2 β 4	N/A	N/A
	5-HT3	N/A	N/A		α 3 β 2	N/A	N/A
	5-HT5A	N/A	N/A		α 3 β 4	8523	N/A
	5-HT6	N/A	N/A		α 4 β 2	N/A	N/A
	5-HT7A	N/A	128		α 4 β 4	N/A	N/A
histamine	H1	8	40		β 1	N/A	N/A
	H2	596	871		β 2	N/A	60%
	H3	509	N/A		β 3	N/A	N/A
	H4	N/A	N/A	dopamine active transporter (DAT)		N/A	N/A
opioid	μ	N/A	N/A	norepinephrine transporter (NET)		931	N/A
	κ	N/A	N/A	serotonin transporter (SERT)		295	N/A
	δ	N/A	N/A	GABAA		N/A	N/A

^aN/A, not active (<50% inhibition obtained in primary assay at 10 μM of the compound). K_i values determined by at least three experiments.

^b% of receptor inhibition at 10 μM of the compound, values determined by at least three experiments.

Figure 4.

Agonist and antagonist selectivity profiling with G-protein-independent β-arrestin recruitment Tango assays at human D₁- and D₂-like receptors. (A) Concentration−response to varying doses of D₁R agonists and control SKF81297. (B) Response to varying doses of D₁R antagonists and control SCH23390, added 30 min before the addition of a final EC₈₀ concentration (3 μM) of a reference agonist SKF81297. (C) Concentration−response to varying doses of D₂R agonists and control quinpirole. (D) Concentration−response to varying doses of D₂R antagonists and control haloperidol, added 30 min before the addition of a final EC₈₀ concentration (3 nM) of reference agonist quinpirole. (E) Concentration−response to varying doses of D₃R agonists and control quinpirole. (F) Concentration−response to varying doses of D₃R antagonists and control haloperidol, added 30 min before the addition of a final EC₈₀ concentration (3 nM) of the reference agonist quinpirole. (G) Concentration−response to varying doses of D₄R agonists and control lisuride. (H) Concentration−response to varying doses of D₄R antagonists and control nemonapride, added 30 min before the addition of a final EC₈₀ concentration (100 nM) of reference agonist lisuride. Data are shown as mean ± SEM of three independent experiments. N/A, not active. N/C, data not converged.

PDSP also evaluated compounds 1 and 31 for off-target interactions with other GPCRs (Table 6). Similar to the findings in our initial publication,¹⁴ ligand 31 was found to bind with high affinity at the 5-HT_1A (K_i = 0.9 nM) and 5-HT_2B (K_i = 42 nM) receptors and was also found to exhibit marginal inhibitions at the 5-HT_2C (K_i = 159 nM) and 5-HT_7A (K_i = 128 nM). Compound 1, however, exhibited negligible affinities (K_i > 1 μM) for all of the serotonin subclass receptors in the study. These findings show the potential of 1 as a D₃R PET agent, as serotoninergic interactions are known to hinder many piperazine-containing scaffolds.^23,40–43

Among the subclass of histamine GPCRs screened, both compounds bind at the H₁ histamine receptor, with a high affinity (1, K_i = 8 nM; 31, K_i = 40 nM). No appreciable opioid receptor inhibition was identified during the primary screen for 1 and 31 at 10 μM drug concentrations. Lead compound 1 was found to weakly interact at the muscarinic M₁ (54% inhibition at 10 μM of the compound) and M₃ (K_i = 514 nM) receptors as well.

Off-target interactions were discovered with compounds 1 and 31 at the subfamilies of the α₁- and α₂-adrenergic receptors, which are GPCRs known to play a role in vasoconstriction.⁴⁴ Although 1 showed no appreciable interactions at the examined adrenergic sites, compound 31 was found to be active at the α_1A (K_i = 49 nM) and the α_1B (K_i = 94 nM) receptors, with moderate inhibitive action at the α₂ subclass group. Adrenergic interactions have been observed with many piperazine-containing D₃R ligand scaffolds,⁴⁵ including BP 897.⁴⁶ This is consistent with a recent study indicating α₂-adrenoceptors are targets for known D₂-like receptor ligands including 7-OH-PIPAT, RO-105824, and dopamine.⁴⁷ Unsurprisingly, the liability of D₃R antagonists to induce hypertension, observed with GSK598809 in dog models,⁴⁸ has hindered these class of compounds from progressing in clinical trials as potential addiction therapeutics,⁴⁹ making the selective nature of 1 over the adrenergic receptors particularly encouraging.

CONCLUSIONS

In our previous report, we identified 1 as a new lead D₃R selective compound containing a novel diazaspiro[5.5]-undecane amino core.¹⁴ In this study, we imposed structural modification to 1, affording analogs with slightly enhanced D₃R selectivity (30, D₃R K_i = 21.0 nM, D₂R/D₃R ratio = 934) and affinity (32, D₃R K_i = 3.2 nM, D₂R/D₃R ratio = 60). The fragmentation of 1 revealed diazaspiro[5.5]undecane synthon 5a to be a low-affinity PP for the receptor (5a, D₃R K_i > 2.7 μM), despite the excellent potency observed with the full-length compound (1, D₃R K_i = 12.0 nM). Molecular docking studies predicted the 1,2,4-triazole half of 1 to favorably position the bulky diazaspiro within the OBP of the receptor, allowing the amino core to engage in hydrophobic interactions with the Phe345 [6.52] and Phe346 [6.51] residues, ultimately stabilizing the “weak” ionic interaction between the ionizable nitrogen of the PP and the highly conserved Asp110 [3.32]. To verify that 1 was not interacting at another receptor site (i.e., allosteric behavior), we utilized a dopamine-mediated β-arrestin 2 recruitment assay to show the competitive manner in which 1 behaves with the orthosteric ligand, revealing that the binding mode of 1 does indeed require OBP occupancy. Upon examining the functional behavior of compounds 1 and 31 at D₁- and D₂-like receptors, we found 1 to be a more D₃R selective ligand for both receptor binding and cell signaling. These results, combined with the minimal off-target interactions observed with 1 at other GPCRs, suggest that the unique binding contacts between the PP of 1 and the highly conserved residues within OBP of the D₃R receptor play a pivotal role in diminishing nonspecific ligand binding with other closely related serotoninergic and adrenergic sites. As such, installing low-affinity amino fragments, such as 5a, into D₃R ligand systems may be beneficial in overcoming drug promiscuity, commonly encountered with piperazine-based ligand scaffolds. This unorthodox approach could be exploited for the development of clinically viable D₃R selective compounds for addiction therapeutic and PET imaging applications.

EXPERIMENTAL SECTION

Chemistry.

Arylated amine synthons 4 and A−B were synthesized following our previously disclosed Pd-catalyzed C−N cross-coupling reports.^15,16 Compounds C, G, 6b, 1−3, 25, 27, 31, and 33 were obtained following the synthetic procedures outlined in our initial publication.¹⁴ Finally, 1,2,4-triazole compounds D and all other commercial reagents were purchased and used as without further purification. However, D analogs were also able to be obtained in good yield following the protocol described by Micheli and co-workers.¹⁸ NMR spectra were taken on a Bruker DMX 500 MHz. Compound structures and identity were confirmed by ¹H and ¹³C NMR, mass spectrometry. Compound purity greater than 95% was determined by liquid chromatography−mass spectrometry (LC−MS) analysis using a 2695 Alliance LC−MS. The purification of organic compounds was carried out on a Biotage Isolera One with a dual wavelength UV−vis. detector. Chemical shifts (δ) in the NMR spectra (¹H and ¹³C) were referenced by assigning the residual solvent peaks. Compounds were taken up with CH₂Cl₂ followed by the dropwise addition of a 2.0 M HCl solution in diethyl ether. After stirring at room temperature for 1 h, the solvent was removed under reduced pressure to afford the desired compound as a hydrochloride salt for in vitro studies.

General Method A: Synthesis of Fragmented Synthons 4−5.

The appropriate arylated diazaspiro and piperazine precursors (4), obtained following our previous reports,^15,16 were dissolved in CH₂Cl₂ (2 mL), followed by the dropwise addition of CF₃COOH (2 mL), and stirred at room temperature for 3 h. Volatiles were then removed under reduced pressure, and the crude product was neutralized with a saturated NaHCo_3(aq) solution (10 mL). The reaction mixture was extracted with CH₂Cl₂ (3 × 20 mL), and the organic layers were combined, dried, and concentrated to afford the free-amine intermediates (4′), which were used without further purification. A mixture of 4′ (1.0 mmol), 1-bromobutane (1.1 mmol), and K₂CO₃ (1.5 mmol) was stirred in acetone (5 mL) at room temperature for 5 h. The crude reaction mixture was then filtered, and the solvent was removed under reduced pressure. The residue was purified by flash chromatography on silica gel eluting with MeOH solution/CH₂Cl₂ (1:10) affording target compounds (5).

General Method B: Synthesis of Diazaspiro Analogs 1−3 and 7−34.

Target compounds can be obtained following the synthetic procedures outlined in our initial.¹⁴ Briefly, the appropriate arylated diazaspiro and piperazine precursors (A), obtained following our previous reports,^15,16 were dissolved in CH₂Cl₂ (2 mL), followed by the dropwise addition of CF₃COOH (2 mL), and stirred at room temperature for 3 h. Volatiles were then removed under reduced pressure, and the crude product was neutralized with a saturated NaHCo_3(aq) solution (10 mL). The reaction mixture was extracted with CH₂Cl₂ (3 × 20 mL), and the organic layers were combined, dried, and concentrated to afford the free-amine intermediates (B) which were used without further purification. A mixture of B (1 mmol), alkylating reagent (2 mmol), and K₂CO₃ (1.5 mmol) was stirred in acetone (5 mL) at room temperature for 12 h. The crude reaction mixture was then filtered, and the solvent was removed under reduced pressure. The residue was loaded onto a Biotage SNAP flash purification cartridge and eluded with 5% MeOH in CH₂Cl₂ affording intermediates C. Finally, a mixture of D (1 mmol), TEA (1.5 mmol), and ethanol (10 mL) was stirred at 75 °C for 15 min. The appropriate intermediate C (1 mmol) was then added, and the solution was stirred at 75 °C for 12 h. The solvent from the crude reaction mixture was then removed under reduced pressure. The residue was loaded onto a Biotage SNAP flash purification cartridge and eluded with 10% 7 N NH₃ in MeOH solution/CH₂Cl₂ to give the target compounds 1−3 and 7−34. The residue was loaded onto a Biotage SNAP flash purification cartridge and eluded with 10% 7 N NH₃ in MeOH solution/CH₂Cl₂ to give the target compounds 1−3 and 7−34.

2-(4-(9-(2-Methoxyphenyl)-3,9-diazaspiro[5.5]undecan-3-yl)-butyl)isoindoline-1,3-dione (E).

4b′ (1 mmol), 2-(4-bromobutyl)-isoindoline-1,3-dione (1.2 mmol), KI (1.0 mmol) and Cs₂CO₃ (2.5 mmol) were dissolved in acetonitrile (5 mL), and the reaction mixture was stirred at 75 °C for 3 h. The reaction mixture was then filtered, and the solvent was removed under reduced pressure. Crude residue was purified by silica gel column chromatography using ethyl acetate/hexane (1:1) as the mobile phase to afford E as a light-orange solid (Yield 64%). ¹H NMR (500 MHz, CDCl₃) δ 7.75−7.74 (m, 2H), 7.62−7.61 (m, 2H), 6.90−6.87 (m, 2H), 6.84−6.82 (m, 1H), 6.77 (d, J = 7.9 Hz, 1H), 3.77 (s, 3H), 3.63 (t, J = 6.9 Hz, 2H), 2.91−2.89 (m, 4H), 2.33−2.28 (m, 6H), 1.62−1.59 (m, 6H), 1.52−1.45 (m, 6H); ¹³C NMR (125 MHz, CDCl₃) δ 168.1, 152.0, 142.0, 133.7, 131.9, 122.9, 122.4, 120.7, 118.0, 110.8, 58.3, 55.1, 49.1, 46.5, 37.6, 35.6, 29.0, 26.5, 24.2; LC−MS (ESI) m/z: 462.64 [M + H].

4-(9-(2-Methoxyphenyl)-3,9-diazaspiro[5.5]undecan-3-yl)butan-1-amine (F).

E (1 mmol) and hydrazine hydrate (50−60%) were dissolved in ethanol (5 mL), and the reaction mixture was stirred at 75 °C for 2 h. The reaction mixture was then filtered, and the solvent was removed under reduced pressure. Crude residue was purified by silica gel column chromatography 10% 7 N NH₃ in MeOH solution/CH₂Cl₂ (1.5:1) to afford F as a clear oil (Yield 97%). ¹H NMR (500 MHz, CDCl₃) δ 6.89−6.86 (m, 2H), 6.82−6.78 (m, 1H), 6.75−6.73 (m, 2H), 3.75 (s, 3H), 2.89−2.87 (m, 4H), 2.62−2.59 (t, J = 6.7 Hz, 2H), 2.31 (bs, 4H), 2.26−2.23 (t, J = 7.0 Hz, 2H), 1.59−1.1.57 (m, 4H), 1.50−1.49 (m, 4H), 1.46−1.41 (m, 2H), 1.38−1.34 (m, 2H), 1.21 (bs, 2H); 13C NMR (125 MHz, CDCl₃) δ 152.0, 142.0, 122.3, 120.6, 117.9, 110.8, 58.8, 55.1, 49.1, 46.5, 41.9, 35.6, 31.7, 28.9, 24.3; LC−MS (ESI) m/z: 332.65 [M + H].

tert-Butyl 9-(2-methoxyphenyl)-3,9-diazaspiro[5.5]undec-7-ene-3-carboxylate (4a).

Compound 4a can be synthesized following the reaction conditions disclosed in Pd C−N cross-coupling report.¹⁵ The separation of 4a and the saturated major product 4b was achieved by flash chromatography on silica gel eluting with a 40% EtOAc/hexane gradient to afford a tan oil. (Yield 9%). ¹H NMR (500 MHz, CDCl₃) δ 7.02−6.98 (m, 1H), 6.95−6.93 (m, 1H), 6.91−6.86 (m, 2H), 6.28 (d, J = 8.1 Hz, 1H), 4.64 (d, J = 8.2 Hz, 1H), 3.83 (s, 3H), 3.53−3.50 (m, 2H), 3.46−3.43 (m, 2H), 3.41−3.35 (m, 2H), 1.74−1.72 (m, 2H), 1.54 (bs, 2H), 1.46 (s, 9H), 1.44−1.42 (m, 2H); ¹³C NMR (125 MHz, CDCl₃) δ 155.1, 152.4, 137.6, 132.0, 123.7, 122.2, 121.1, 112.1, 105.5, 79.3, 55.7, 44.2, 39.9, 38.5, 34.5, 30.3, 28.6; LC−MS (ESI) m/z: 303.47 [M − C(CH₃)₃ + H].

3-Butyl-9-(2-methoxyphenyl)-3,9-diazaspiro[5.5]undecane (5a).

Following general method A, 5a was obtained as a white semisolid. (Yield 38%). ¹H NMR (500 MHz, CDCl₃) δ 6.97−6.93 (m, 2H), 6.90−6.88 (m, 1H), 6.83−6.82 (m, 1H), 3.83 (s, 3H), 2.97−2.95 (m, 4H), 2.45 (bs, 4H), 2.38−2.35 (m, 2H), 1.68−1.65 (m, 4H), 1.61−1.59 (m, 4H), 1.52−1.46 (m, 2H), 1.34−1.28 (m, 2H) 0.90 (t, J = 7.3 Hz, 3H); ¹³C NMR (125 MHz, CDCl₃) δ 152.3, 142.2, 122.6, 120.9, 118.2, 111.0, 58.8, 55.3, 49.3, 46.7, 35.6, 29.1, 29.0, 20.9, 14.1; LC−MS (ESI) m/z: 317.68 [M + H].

8-Butyl-2-(2-methoxyphenyl)-2,8-diazaspiro[4.5]decane (5b).

Following general method A, compound 5b was obtained as an off-white semisolid. (Yield 59%). ¹H NMR (500 MHz, CDCl₃) δ 6.88−6.82 (m, 3H), 6.70 (d, J = 7.0 Hz, 1H), 3.80 (s, 3H), 3.37 (t, J = 6.9 Hz, 2H), 3.21 (s, 2H), 2.98 (bs, 2H), 2.81−2.78 (m, 2H), 1.99 (bs, 4H), 1.82 (t, J = 6.8 Hz, 2H), 1.76−1.73 (m, 2H), 1.38−1.34 (m, 2H), 0.94 (t, J = 7.3 Hz, 3H); ¹³C NMR (125 MHz, CDCl₃) δ 150.3, 139.2, 121.3, 120.0, 115.3, 112.0, 55.7, 50.5, 48.9, 38.9, 33.3, 26.5, 20.4, 13.7; LC−MS (ESI) m/z: 303.52 [M + H].

2-Butyl-7-(2-methoxyphenyl)-2,7-diazaspiro[4.4]nonane (5c).

Following general method A, 5c was obtained as an oil. (Yield 38%). 1H NMR (500 MHz, CDCl₃) δ 6.87−6.80 (m, 3H), 6.68−6.67 (m, 1H), 3.80 (s, 3H), 3.45−3.35 (m, 3H), 3.29 (d, J = 9.4 Hz, 1H), 3.12 (bs, 2H), 3.00 (bs, 2H), 2.78−2.75 (m, 2H), 2.07−1.91 (m, 4H), 1.65−1.58 (m, 2H), 1.38−1.33 (m, 2H), 0.92 (t, J = 7.3 Hz, 3H); ¹³C NMR (125 MHz, CDCl₃) δ 150.0, 139.1, 121.2, 119.6, 114.8, 112.0, 63.9, 61.8, 56.3, 55.6, 49.6, 47.6, 37.3, 35.7, 29.9, 20.4, 13.8; LC−MS (ESI) m/z: 289.93 [M + H].

1-Butyl-4-(2-(2-fluoroethoxy)phenyl)piperazine (5e).

Following general method A, 5e⁵⁰ was obtained as a colorless oil. (Yield 41%). 1H NMR (360 MHz, CDCl₃) δ 6.97−6.95 (m, 3H), 6.85 (m, 1H), 4.77 (dt, J = 47.5, 4.0 Hz, 2H), 4.25 (dt, J = 29.0, 4.0 Hz, 2H), 3.16 (s, 4H), 2.68 (s, 4H), 2.43 (t, J = 7.8 Hz, 3H), 1.54 (m, 2H), 1.35 (m, 2H) 0.93 (t, J = 7.3 Hz, 3H); ¹³C NMR (126 MHz, CDCl₃) δ 150.9, 141.9, 122.6, 122.1, 118.4, 113.7, 82.5, 81.2, 67.6, 67.4, 58.5, 53.4, 50.3, 28.6, 20.7, 11.9; LC−MS (ESI) m/z: 281.20 [M + H].

4-Methyl-3-phenyl-5-(propylthio)-4H-1,2,4-triazole (6a).

A mixture of 4-methyl-5-phenyl-4H-1,2,4-triazole-3-thiol (1.0 mmol), 1-bromobutane (1.1 mmol), and K₂CO₃ (1.5 mmol) was stirred in acetone (5 mL) at room temperature for 12 h. The crude reaction mixture was then filtered, and the solvent was removed under reduced pressure. The residue was purified by flash chromatography on silica gel eluting with 10% 7 N NH₃ in MeOH solution/CH₂Cl₂ (1:10) affording 6a as a white solid. (Yield 81%). ¹H NMR (500 MHz, CDCl₃) δ 7.58−7.56 (m, 2H), 7.44−7.41 (m, 3H), 3.53 (s, 3H), 3.19 (t, J = 7.2 Hz, 2H), 1.78−1.74 (quint, J = 7.2 Hz, 2H), 1.00 (t, J = 7.3 Hz, 3H); ¹³C NMR (125 MHz, CDCl₃ ) δ 155.8, 129.9, 128.8, 128.5, 127.1, 35.4, 31.6, 22.9, 13.2; LC−MS (ESI) m/z: 234.17 [M + H].

1-(3-((4-Methyl-5-phenyl-4H-1,2,4-triazol-3-yl)thio)propyl)-piperidine (6c).

A mixture of 6b¹⁴ (1.0 mmol), piperdine (1.1 mmol), and Cs₂ CO₃ (1.5 mmol) was stirred in acetonitrile (3 mL) at 70 °C for 12 h. The crude reaction mixture was then filtered, and the solvent was removed under reduced pressure. The residue was purified by flash chromatography on silica gel eluting with 10% 7 N NH₃ in MeOH solution/CH₂Cl₂ (1:10) affording 6c as a white solid. (Yield 20%). ¹H NMR (500 MHz, CDCl₃) δ 7.60−7.58 (m, 2H), 7.47−7.45 (m, 3H), 3.56 (s, 3H), 3.26 (t, J = 7.2 Hz, 2H), 2.51 (t, J = 7.3 Hz, 2H), 2.44 (bs, 4H), 2.04−1.98 (m, 2H), 1.61−1.57 (quint, J = 5.6 Hz, 4H), 1.41 (bs, 2H); ¹³C NMR (125 MHz, CDCl₃) δ 155.9, 151.8, 130.0, 128.9, 128.6, 127.1, 57.5, 54.5, 31.6, 31.5, 26.5, 25.5, 24.1; LC−MS (ESI) m/z: 317.15 [M + H].

4,4-Dimethyl-1-(3-((4-methyl-5-phenyl-4H-1,2,4-triazol-3-yl)-thio)propyl)piperidine (6d).

A mixture of 6b¹⁴ (1.0 mmol), 4,4-dimethylpiperdine (1.1 mmol), and Cs₂CO₃ (1.5 mmol) was stirred in acetonitrile (3 mL) at 70 °C for 8 h. The crude reaction mixture was then filtered, and the solvent was removed under reduced pressure. The residue was purified by flash chromatography on silica gel eluting with 10% 7 N NH₃ in MeOH solution/CH₂Cl₂ (1:10) affording 6d as a white solid. (Yield 26%).¹H NMR (500 MHz, CDCl₃) δ 7.59−7.58 (m, 2H), 7.46−7.44 (m, 3H), 3.57 (s, 3H), 3.25 (t, J = 6.7 Hz, 2H), 2.92−2.89 (m, 2H), 2.80 (bs, 4H), 2.24−2.20 (m, 2H), 1.60 (bs, 4H), 0.93 (s, 6H); ¹³C NMR (125 MHz, CDCl₃) δ 156.1, 151.3, 130.1, 128.9, 128.6, 126.9, 56.1, 49.6, 36.4, 31.7, 30.6, 28.0, 25.2; LC−MS (ESI) m/z: 345.18 [M + H].

tert-Butyl 9-(3-((4-methyl-5-phenyl-4H-1,2,4-triazol-3-yl)thio)-propyl)-3,9-diazaspiro[5.5]undecane-3-carboxylate (6e).

A mixture of 6b¹⁴ (1.0 mmol), tert-butyl 3,9-diazaspiro[5.5]undecane-3-carboxylate (1.1 mmol), and Cs₂ CO₃ (1.5 mmol) was stirred in acetonitrile (5 mL) at 70 °C for 12 h. The crude reaction mixture was then filtered, and the solvent was removed under reduced pressure. The residue was purified by flash chromatography on silica gel eluting with 10% 7 N NH₃ in MeOH solution/CH₂Cl₂ (1:10) affording 6e as an off-white solid (Yield 47%). ¹H NMR (500 MHz, CDCl₃) δ 7.55−7.54 (m, 2H), 7.42−7.40 (m, 3H), 3.52 (s, 3H), 3.28−3.26 (m, 4H), 3.21 (t, J = 7.0 Hz, 2H), 2.51 (t, J = 7.1 Hz, 2H), 2.42 (bs, 4H), 1.99−1.93 (quint, J = 7.1 Hz, 2H), 1.48 (t, J = 5.6 Hz, 4H), 1.36 (s, 9H), 1.34 (bs, 4H); ¹³C NMR (125 MHz, CDCl₃) δ 155.7, 154.8, 151.7, 129.9, 128.8, 128.4, 126.9, 79.1, 65.7, 56.9, 48.9, 39.6, 38.8, 34.8, 31.5, 30.9, 29.4, 28.3, 26.4, 15.2; LC−MS (ESI) m/z: 486.17 [M + H].

3-(2-((4-Methyl-5-phenyl-4H-1,2,4-triazol-3-yl)thio)propyl)-3,9-diazaspiro[5.5]undecane (6f).

The compound was dissolved in CH₂Cl₂ (2 mL), followed by the dropwise addition of CF₃COOH (2 mL), and stirred at room temperature for 3 h. Volatiles were then removed under reduced pressure, and the crude product was neutralized with a saturated NaHCO_{3 (aq)} solution (10 mL). The reaction mixture was extracted with CH₂Cl₂ (3 × 20 mL), and the organic layers were combined, dried, and concentrated to afford 6f as an off-white solid (Yield 97%). (HCl salt) ¹H NMR (500 MHz, (CD₃)₂SO) δ 10.97 (bs, 1H), 7.78−7.76 (m, 2H), 7.62−7.59 (m, 3H), 3.65 (s, 3H), 3.35 (t, J = 6.9 Hz, 2H), 3.29−3.26 (m, 2H), 3.21−3.16 (m, 2H), 2.99 (bs, 6H), 2.22−2.17 (m, 2H), 1.83−1.77 (m, 6H), 1.54−1.52 (m, 2H); ¹³C NMR (125 MHz, (CD₃)₂SO) δ 154.7, 151.2, 130.8, 128.9, 128.8, 125.0, 53.7, 47.0, 38.7, 38.6, 34.6, 32.2, 31.1, 30.1, 27.9, 26.7, 23.4; LC−MS (ESI) m/z: 386.68 [M + H].

1-(3-((4-Methyl-5-phenyl-4H-1,2,4-triazol-3-yl)thio)propyl)-piperazine (6h).

Following the procedure for 6f, compound 6h was obtained as a light tan oil. (Yield 12%). ¹H NMR (500 MHz, CDCl₃) δ 7.56−7.54 (m, 2H), 7.43−7.41 (m, 3H), 3.52 (s, 3H), 3.25−3.22 (m, 2H), 3.21 (s, 1H), 2.85−2.83 (t, J = 4.8 Hz, 4H), 2.42−2.38 (m, 6H), 1.95−1.89 (quint, J = 7.1 Hz, 2H); ¹³C NMR (125 MHz, CDCl₃) δ 155.7, 151.9, 130.0, 128.8, 128.5, 127.0, 57.2, 53.8, 45.6, 31.5, 31.0, 26.5; LC−MS (ESI) m/z: 318.66 [M + H].

4-(5-((3-(9-(2-Methoxyphenyl)-3,9-diazaspiro[5.5]undecan-3-yl)-propyl)thio)-4-methyl-4H-1,2,4-triazol-3-yl)-N,N-dimethylaniline (7).

Following general method B, 7 was obtained as a tan oil. (Yield 48%). ¹H NMR (500 MHz, CDCl₃) δ 7.46 (d, J = 8.8 Hz, 2H), 6.92−6.89 (m, 2H), 6.86−6.83 (m, 1H), 6.80−6.78 (m, 1H), 6.72 (d, J = 8.8 Hz, 2H), 3.79 (s, 3H), 3.54 (s, 3H), 3.20 (t, J = 7.1 Hz, 2H), 2.96 (s, 3H), 2.93−2.91 (m, 4H), 2.65 (t, J = 7.1 Hz, 2H), 2.57 (bs, 4H), 2.08−2.02 (quint, J = 7.1 Hz, 2H), 1.63 (bs, 8H); ¹³C NMR (125 MHz, CDCl₃) δ 156.4, 152.1, 151.1, 150.5, 141.9, 129.3, 122.6, 120.7, 118.1, 113.9, 111.7, 110.9, 56.7, 55.2, 48.9, 46.5, 45.8, 40.1, 35.8, 34.7, 31.6, 31.0, 28.9, 26.1; LC−MS (ESI) m/z: 535.32 [M + H].

3-(2-Methoxyphenyl)-9-(3-((5-(2-methoxyphenyl)-4-methyl-4H-1,2,4-triazol-3-yl)thio)propyl)-3,9-diazaspiro[5.5]undecane (8).

Following general method B, 8 was obtained as a clear oil. (Yield 39%). ¹H NMR (500 MHz, CDCl₃) δ 7.46−7.43 (m, 2H), 7.05−7.02 (m, 1H), 6.97−6.95 (m, 1H), 6.93−6.92 (m, 2H), 6.88−6.86 (m, 1H), 6.81 (d, J = 7.8 Hz, 1H), 3.82 (s, 3H), 3.78 (s, 3H), 3.35 (s, 3H), 3.28 (t, J = 6.9 Hz, 2H), 2.94 (t, J = 4.6 Hz, 4H), 2.54 (t, J = 7.1 Hz, 2H), 2.47 (bs, 4H), 2.03−2.00 (m, 2H), 1.66−164 (m, 4H), 1.60−1.58 (m, 4H); ¹³C NMR (125 MHz, CDCl₃) δ 157.2, 154.3, 152.2, 151.1, 142.1, 132.2, 132.0, 122.6, 121.0, 120.8, 118.2, 116.3, 111.0 (2 × CH), 57.2, 55.5, 55.3, 49.2, 46.6, 36.0, 35.5, 31.1, 30.9, 29.1, 26.7; LC−MS (ESI) m/z: 522.23 [M + H].

3-(2-Methoxyphenyl)-9-(3-((4-methyl-5-(pyridin-3-yl)-4H-1,2,4-triazol-3-yl)thio)propyl)-3,9-diazaspiro[5.5]undecane (9).

Following general method B, 9 was obtained as a light tan oil. (Yield 53%). ¹H NMR (500 MHz, CDCl₃) δ 8.85 (s, 1H), 8.69−8.68 (m, 1H), 7.98−7.96 (m, 1H), 7.43−7.40 (m, 1H), 6.94−6.90 (m, 2H), 6.86−6.83 (m, 1H), 6.80 (d, J = 7.7 Hz, 1H), 3.80 (s, 3H), 3.59 (s, 3H), 3.29 (t, J = 6.9 Hz, 2H), 2.94−2.92 (m, 4H), 2.56 (t, J = 6.3 Hz, 2H), 2.49 (bs, 4H), 2.06−2.00 (quint, J = 6.9 Hz, 2H), 1.64−1.62 (m, 4H), 1.60−1.58 (m, 4H); ¹³C NMR (125 MHz, CDCl₃) δ 153.1, 152.7, 152.2, 151.0, 148.8, 142.0, 136.0, 123.7, 123.5, 122.6, 120.8, 118.1, 110.9, 57.0, 55.3, 49.2, 46.6, 36.0, 35.3, 31.6, 31.1, 29.0, 26.5, 22.6; LC−MS (ESI) m/z: 493.24 [M + H].

3-(2-Methoxyphenyl)-9-(3-((4-methyl-5-(4-(thiophen-3-yl)-phenyl)-4H-1,2,4-triazol-3-yl)thio)propyl)-3,9-diazaspiro[5.5]-undecane (10).

Following general method B, 10 was obtained as a white solid. (Yield 15%) ¹H NMR (500 MHz, CDCl₃) δ 7.70 (d, J = 8.3 Hz, 2H), 7.65 (d, J = 8.3 Hz, 2H), 7.51−7.50 (m, 1H), 7.40−7.39 (m, 2H), 6.96−6.93 (m, 2H), 6.89−6.87 (m, 1H), 6.82 (d, J = 8.0 Hz, 1H), 3.82 (s, 3H), 3.59 (s, 3H), 3.29 (t, J = 7.2 Hz, 2H), 2.96−2.94 (m, 4H), 2.49 (t, J = 7.5 Hz, 2H), 2.42−2.40 (m, 4H), 2.02−1.96 (quint, J = 7.1 Hz, 2H), 1.66−1.64 (m, 4H), 1.57−1.55 (m, 4H); ¹³C NMR (125 MHz, CDCl₃) δ 155.5, 152.2, 152.0, 142.1, 141.1, 137.2, 128.9, 126.7, 126.6, 126.0, 125.6, 122.5, 121.3, 120.8, 118.1, 110.9, 57.3, 55.3, 49.2, 46.6, 36.1, 35.8, 31.6, 31.3, 29.1, 26.9; LC−MS (ESI) m/z: 574.19 [M + H].

2-(4-(5-((3-(9-(2-Methoxyphenyl)-3,9-diazaspiro[5.5]undecan-3-yl)propyl)thio)-4-methyl-4H-1,2,4-triazol-3-yl)phenyl)oxazole (11).

Following general method B, 11 was obtained as a beige solid. (Yield 17%) ¹H NMR (500 MHz, CDCl₃) δ 8.17 (d, J = 8.2 Hz, 2H), 7.75 (s, 1H), 7.74 (s, 2H), 7.26 (s, 1H), 6.94−6.93 (m, 2H), 6.89−6.87 (m, 1H), 6.83 (d, J = 7.8 Hz, 1H), 3.83 (s, 3H), 3.62 (s, 3H), 3.32−3.29 (t, J = 7.0 Hz, 2H), 2.96−2.94 (m, 2H), 2.56−2.53 (m, 2H), 2.47 (bs, 4h), 2.06−2.00 (quint, J = 7.0 Hz, 2H), 1.66−1.64 (m, 4H), 1.60−1.58 (m, 4H); ¹³C NMR (125 MHz, CDCl₃) δ 161.0, 155.1, 152.4, 152.3, 142.2, 139.2, 128.9, 128.8, 128.7, 126.8, 122.6, 120.9, 118.2, 111.0, 57.2, 55.3, 49.5, 49.3, 46.7, 36.1, 35.5, 31.8, 31.2, 29.1, 26.8; LC−MS (ESI) m/z: 559.23 [M + H].

3-(3-((5-(2-Fluorophenyl)-4-methyl-4H-1,2,4-triazol-3-yl)thio)-propyl)-9-(2-methoxyphenyl)-3,9-diazaspiro[5.5]undecane (12).

Following general method B, 12 was obtained as a beige solid. (Yield 44%) ¹H NMR (500 MHz, CDCl₃) δ 7.56−7.53 (m, 1H), 7.46−7.44 (m, 1H), 7.25−7.21 (m, 1H), 7.16−7.12 (m, 1H), 6.91−6.88 (m, 1H), 6.86−6.81 (m, 2H), 6.79−6.76 (m, 1H), 3.77 (s, 3H), 3.41 (s, 3H), 3.29−3.26 (t, J = 6.7 Hz, 2H), 2.99 (bs, 2H), 2.90 (bs, 8H), 2.28 (m, 2h), 1.81 (bs, 4H), 1.65 (m, 4H); ¹³C NMR (125 MHz, CDCl₃) δ 158.7 (d, J_C−F = 250.3 Hz), 152.2, 152.1, 151.5, 141.7, 132.7 (d, J_{C−C−C−F} = 8.1 Hz), 132.1, 124.9 (d, J_{C−C−C−F} = 3.0 Hz), 122.8, 120.8, 118.2, 116.1 (d, J_C−C−F = 21.1 Hz), 115.1 (d, J_C−C−F = 14.3 Hz), 111.0, 55.8, 55.3, 48.7, 46.5, 35.5 (bs), 33.2, 31.2 (d, J_CH3−F = 5.7 Hz), 30.4, 28.7, 24.8; LC−MS (ESI) m/z: 510.78 [M + H].

3-(3-((5-(4-Fluorophenyl)-4-methyl-4H-1,2,4-triazol-3-yl)thio)-propyl)-9-(2-methoxyphenyl)-3,9-diazaspiro[5.5]undecane (13).

Following general method B, 13 was obtained as a white solid. (Yield 24%) ¹H NMR (500 MHz, CDCl₃) δ 7.63−7.60 (m, 2H), 7.19−7.15 (m, 2H), 6.97−6.93 (m, 1H), 6.91−6.85 (m, 2H), 6.82−6.80 (m, 1H),3.82 (s, 3H), 3.59 (s, 3H), 3.30−3.28 (t, J = 6.9 Hz, 2H), 3.06−3.03 (m, 2H), 3.00 (bs, 2H), 2.95−2.93 (m, 6h), 2.36−2.32 (quint, J = 6.8 Hz, 2H), 1.88 (bs, 4H), 1.70 (bs, 4H); ¹³C NMR (125 MHz, CDCl₃) δ 162.8 (d, J_C−F = 250.7 Hz), 155.3, 152.2, 151.2, 141.6, 130.7 (d, J_{C−C−C−F} = 8.4 Hz), 123.1 (d, J_{C−C−C−F} = 3.8 Hz), 122.9, 120.9, 118.2, 116.3 (d, J_C−C−F = 22.0 Hz), 111.1, 55.8, 55.4, 48.7, 46.5, 33.2 (bs), 31.8, 30.4, 28.7, 24.7; LC−MS (ESI) m/z: 510.63 [M + H].

3-(3-((5-Cyclohexyl-4-methyl-4H-1,2,4-triazol-3-yl)thio)propyl)-9-(2-methoxyphenyl)-3,9-diazaspiro[5.5]undecane (14).

Following general method B, 14 was obtained as a white semisolid. (Yield 39%) 1H NMR (500 MHz, CDCl₃) δ 6.95−6.91 (m, 2H), 6.87−6.84 (m, 1H), 6.81−6.79 (m, 1H), 3.81 (s, 3H), 3.44 (s, 3H), 3.15 (t, J = 7.0 Hz, 2H), 2.94−2.92 (m, 4H), 2.60−2.54 (m, 3H), 2.50 (bs, 4H), 2.01−1.96 (quint, J = 7.0 Hz, 2H), 1.90−1.82 (m, 4H), 1.71−1.67 (m, 2H), 1.65−1.59 (m, 9H), 1.36−1.26 (m, 3H); ¹³C NMR (125 MHz, CDCl₃) δ 159.5, 152.2 149.9, 142.0, 122.6, 120.8, 118.1, 110.9, 57.0, 55.3, 49.1, 46.6, 35.9, 35.0, 31.2, 30.7, 29.8, 29.0, 26.4, 26.0, 25.6; LC−MS (ESI) m/z: 498.32 [M + H].

3-(3-((4-Ethyl-5-phenyl-4H-1,2,4-triazol-3-yl)thio)propyl)-9-(2-methoxyphenyl)-3,9-diazaspiro[5.5]undecane (15).

Following general method B, 15 was obtained as a white solid. (Yield 55%) ¹H NMR (500 MHz, CDCl₃) δ 7.56−7.54 (m, 2H), 7.46−7.44 (m, 3H), 6.93−6.89 (m, 2H), 6.87−6.85 (m, 1H), 6.81−6.79 (m, 1H), 3.96−3.92 (quart, J = 7.2 Hz, 2H), 3.80 (s, 3H), 3.30 (t, J = 7.1 Hz, 2H), 2.94−2.92 (m, 4H), 2.76 (t, J = 7.0 Hz, 2H), 2.69 (bs, 4H), 2.20−2.14 (quint, J = 7.1 Hz, 2H), 1.71−1.69 (m, 4H), 1.67−1.65 (m, 4H), 1.27 (t, J = 7.2 Hz, 3H); ¹³C NMR (125 MHz, CDCl₃) δ 155.5, 152.1, 150.9, 141.8, 130.0, 128.9, 128.5, 127.2, 122.7, 120.8, 118.1, 111.0, 56.6, 55.3, 48.9, 46.5, 39.6, 35.6, 34.3, 30.8, 28.9, 25.8, 15.4; LC−MS (ESI) m/z: 506.20 [M + H].

3-((3-(9-(2-Methoxyphenyl)-3,9-diazaspiro[5.5]undecan-3-yl)-propyl)thio)-5-phenyl-4H-1,2,4-triazol-4-amine (16).

Following general method B, 16 was obtained as a clear oil. (Yield 46%) ¹H NMR (500 MHz, CDCl₃) δ 8.03−8.01 (m, 2H), 7.42−7.41 (m, 3H), 6.97−6.91 (m, 2H), 6.89−6.88 (m, 1H), 6.82 (d, J = 8.0 Hz, 1H), 5.18 (s, 2H), 3.82 (s, 3H), 3.18 (t, J = 6.9 Hz, 2H), 2.94−2.92 (m, 4H), 2.53 (t, J = 7.0 Hz, 2H), 2.47−2.45 (m, 4H), 1.97−1.91 (quint, J = 7.2 Hz, 2H), 1.64−162 (m, 4H), 1.58−1.56 (m, 4H); ¹³C NMR (125 MHz, CDCl₃) δ 154.3, 152.8, 152.2, 142.0, 130.0, 128.5, 128.2, 126.5, 122.7, 120.9, 118.2, 111.0, 56.8, 55.3, 49.1, 46.6, 35.9, 35.2, 30.9, 29.0, 26.5; LC−MS (ESI) m/z: 493.24 [M + H].

3-(2-Methoxyphenyl)-9-(3-((5-phenyl-4H-1,2,4-triazol-3-yl)thio)-propyl)-3,9-diazaspiro[5.5]undecane (17).

Following general method B, 17 was obtained as a clear oil. (Yield 41%) ¹H NMR (500 MHz, CDCl₃) δ 11.1 (bs, 1H), 8.04 (d, J = 7.6 Hz, 2H), 7.36−7.30 (m, 3H), 6.96−6.92 (m, 2H), 6.89−6.86 (m, 1H), 6.81 (d, J = 8.0 Hz, 1H), 3.79 (s, 3H), 3.10 (t, J = 6.4 Hz, 2H), 2.96−2.94 (m, 4H), 2.57 (t, J = 6.3 Hz, 2H), 2.49 (bs, 4H), 1.98−1.92 (quint, J = 6.2 Hz, 2H), 1.69−1.66 (m, 8H); ¹³C NMR (125 MHz, CDCl₃) δ 161.1, 155.7, 152.0, 141.8, 130.0, 129.0, 128.3, 126.2, 122.6, 120.7, 118.1, 110.9, 55.1, 54.8, 48.8, 46.5, 35.6, 34.7, 30.3.28.9, 26.5; LC−MS (ESI) m/z: 478.15 [M + H].

3-(2-Methoxyphenyl)-9-(3-((5-phenyl-1H-imidazol-2-yl)thio)-propyl)-3,9-diazaspiro[5.5]undecane (18).

Following general method B, 18 was obtained as a light gray solid. (Yield 82%) ¹H NMR (500 MHz, CDCl₃) δ 10.92 (bs, 1H), 7.73 (d, J = 7.1 Hz, 2H), 7.31 (t, J = 7.7 Hz, 2H), 7.28 (s, 3H), 7.18 (t, J = 7.3 Hz, 1H), 6.99−6.88 (m, 3H), 6.84−6.83 (d, J = 7.8 Hz, 1H), 3.83 (s, 3H), 3.01 (t, J = 6.0 Hz, 2H), 2.97−2.95 (m, 4H), 2.67 (t, J = 6.6 Hz, 2H), 2.55 (bs, 4H), 1.93−1.88 (quint, J = 6.4 Hz, 2H), 1.68−1.64 (m, 8H); ¹³C NMR (125 MHz, CDCl₃) δ 152.2, 141.9, 141.8, 141.0, 133.4, 128.5, 126.6, 124.7, 122.7, 120.8, 118.2, 115.7, 111.1, 55.3, 54.8, 48.5, 46.5, 35.6, 35.0, 32.0, 29.0, 26.2; LC−MS (ESI) m/z: 477.22 [M + H].

3-(2-Methoxyphenyl)-9-(3-((1-methyl-5-phenyl-1H-imidazol-2-yl)thio)propyl)-3,9-diazaspiro[5.5]undecane (19).

Following general method B, 19 was obtained as a tan oil. (Yield 35%) ¹H NMR (500 MHz, CDCl₃) δ 7.39−7.36 (m, 2H), 7.32−7.31 (m, 3H), 7.05 (s, 1H), 6.92−6.90 (m, 2H), 6.86−6.85 (m, 1H), 6.80−6.79 (m, 1H), 3.80 (s, 3H), 3.54 (s, 3H), 3.12−3.10 (t, J = 6.9 Hz, 2H), 2.94−2.92 (m, 4H), 2.59−2.56 (t, J = 6.5 Hz, 2H), 2.51 (bs, 4H), 1.98−1.95 (m, 2H), 1.65−1.60 (m, 8H); ¹³C NMR (125 MHz, CDCl₃) δ 152.1, 142.7, 142.0, 129.9, 128.6, 128.3, 127.8, 122.5, 120.8, 118.1, 111.0, 57.0, 55.2, 49.0, 46.5, 35.8 (bs), 35.0, 32.0, 28.9, 26.5; LC−MS (ESI) m/z: 491.73 [M + H].

3-(2-Methoxyphenyl)-9-(2-((4-methyl-5-phenyl-4H-1,2,4-triazol-3-yl)thio)ethyl)-3,9-diazaspiro[5.5]undecane (20).

Following general method B, 20 was obtained as a tan oil. (Yield 12%) ¹H NMR (500 MHz, CDCl₃) δ 7.62−7.60 (m, 2H), 7.48−7.46 (m, 3H), 6.96−6.93 (m, 2H), 6.89−6.86 (m, 1H), 6.83−6.81 (m, 1H), 3.83 (s, 3H), 3.58 (s, 3H), 3.44 (t, J = 6.7 Hz, 2H), 2.96−2.94 (m, 4H), 2.81 (t, J = 6.7 Hz, 2H), 2.52−2.49 (m, 4H), 1.65−1.63 (m, 4H), 1.57−1.55 (m, 4H); ¹³C NMR (125 MHz, CDCl₃) δ 155.8, 152.2, 152.1, 142.1, 130.0, 128.9, 128.5, 127.1, 122.6, 120.8, 118.2, 111.0, 57.8, 55.3, 49.0, 46.7, 36.0, 35.5, 31.7, 30.6, 29.1; LC−MS (ESI) m/z: 478.42 [M + H].

3-(2-Methoxyphenyl)-9-(4-((4-methyl-5-phenyl-4H-1,2,4-triazol-3-yl)thio)butyl)-3,9-diazaspiro[5.5]undecane (21).

Following general method B, 21 was obtained as a tan oil. (Yield 14%) ¹H NMR (500 MHz, CDCl₃) δ 7.61−7.59 (m, 2H), 7.48−7.46 (m, 3H), 6.96−6.91 (m, 2H), 6.88−6.87 (m, 1H), 6.82−6.81 (m, 1H), 3.82 (s, 3H), 3.58 (s, 3H), 3.27 (t, J = 6.9 Hz, 2H), 2.96−2.94 (m, 4H), 2.61−2.50 (m, 6H), 1.84−1.78 (m, 4H), 1.67−1.66 (m, 8H); ¹³C NMR (125 MHz, CDCl₃) δ 155.9, 152.3, 151.9, 142.0, 130.1, 128.9, 128.6, 127.1, 122.7, 120.9, 118.2, 111.1, 57.9, 55.3, 49.1, 46.6, 34.8, 32.8, 31.6, 29.0, 27.5, 25.1, 22.6; LC−MS (ESI) m/z: 506.20 [M + H].

3-(2-Ethoxyphenyl)-9-(3-((4-methyl-5-phenyl-4H-1,2,4-triazol-3-yl)thio)propyl)-3,9-diazaspiro[5.5]undecane (22).

Following general method B, 22 was obtained as a dark tan oil. (Yield 13%) ¹H NMR (500 MHz, CDCl₃) δ 7.60−7.58 (m, 2H), 7.47−7.45 (m, 3H), 6.90−6.88 (m, 2H), 6.87−6.85 (m, 1H), 6.84−6.78 (m, 1H), 4.03−3.99 (m, 2H), 3.55 (s, 3H), 3.28 (t, J = 7.1 Hz, 2H), 2.98−2.96 (m, 4H), 2.50 (t, J = 7.3 Hz, 2H), 2.43 (bs, 4H), 2.02−1.97 (quint, J = 7.0 Hz, 2H), 1.64−1.62 (m, 4H), 1.57−1.56 (m, 4H), 1.41 (t, J = 7.0 Hz, 3H); ¹³C NMR (125 MHz, CDCl₃) δ 155.7, 151.8, 151.5, 142.2, 129.9, 128.8, 128.5, 127.1, 122.3, 120.8, 118.1, 112.3, 63.4, 57.2, 49.2, 46.5, 36.1, 35.7, 31.5, 31.2, 29.1, 26.8, 14.9; LC−MS (ESI) m/z: 506.20 [M + H].

2-(9-(3-((4-Methyl-5-phenyl-4H-1,2,4-triazol-3-yl)thio)propyl)3,9-diazaspiro[5.5]undecan-3-yl)phenol (23).

Following general method B, 23 was obtained as a dark tan oil. (Yield 13%) ¹H NMR (500 MHz, CDCl₃) δ 7.63−7.60 (m, 2H), 7.50−7.48 (m, 3H), 7.15−7.13 (dd, J₁ = 1.4 Hz, J₂ = 1.4 Hz, 1H), 7.05−7.02 (dt, J₁ = 1.4 Hz, J₂ = 7.9 Hz, 1H), 6.93−6.91 (dd, J₁ = 1.4 Hz, J₂ = 8.0 Hz, 1H), 6.84−6.81 (dt, J₁ = 1.4 Hz, J₂ = 7.6 Hz, 1H), 3.59 (s, 3H), 3.32−3.29 (t, J = 7.0 Hz, 2H), 2.79−2.77 (m, 4H), 2.58−2.55 (t, J = 7.3 Hz, 2H), 2.50 (bs, 4H), 2.07−2.01 (m, 2H), 1.99 (s, 1H), 1.64−1.62 (m, 8H); ¹³C NMR (125 MHz, CDCl₃) δ 155.9, 151.9, 151.5, 139.9, 130.1, 129.0, 128.6, 127.2, 126.2, 121.2, 119.9, 113.9, 57.2, 49.2, 48.7, 36.7, 35.6, 31.7, 31.2, 29.1, 26.8, 22.7; LC−MS (ESI) m/z: 478.66 [M + H].

3-(2-(2-Fluoroethoxy)phenyl)-9-(3-((4-methyl-5-phenyl-4H-1,2,4triazol-3-yl)thio)propyl)-3,9-diazaspiro[5.5]undecane (24).

Following general method B, 24 was obtained as a white solid. (Yield 12%) ¹H NMR (500 MHz, CDCl₃) δ 7.61−7.59 (m, 2H), 7.47−7.45 (m, 3H), 6.92−6.89 (m, 3H), 6.81−6.79 (m, 1H), 4.78−4.77 (m, 1H), 4.69−4.67 (m, 1H), 4.24−4.22 (m, 1H), 4.18−4.16 (m, 1H), 3.57 (s, 3H), 3.27 (t, J = 7.0 Hz, 2H), 2.98−2.96 (m, 4H), 2.85−2.82 (m, 2H), 2.76 (bs, 4H), 2.21−2.16 (quint, J = 7.2 Hz, 2H), 1.75−1.73 (m, 4H), 1.66−164 (m, 4H); ¹³C NMR (125 MHz, CDCl₃) δ 156.0, 151.4, 150.9, 142.4, 130.1, 128.9, 128.5, 126.9, 122.5, 122.0, 118.5, 113.4, 82.7 (d, J_C−F = 170.1 Hz, CH₂F), 67.5 (d, J_C−F = 20.1 Hz, O−CH₂CH₂F), 56.4, 48.9, 46.4, 35.6, 34.0, 31.7, 30.7, 28.9, 25.5; LC−MS (ESI) m/z: 524.23 [M + H].

3-(2,4-Dimethoxyphenyl)-9-(3-((4-methyl-5-phenyl-4H-1,2,4-triazol-3-yl)thio)propyl)-3,9-diazaspiro[5.5]undecane (26).

Following general method B, 26 was obtained as an off-white solid. (Yield 8%) ¹H NMR (500 MHz, CDCl₃) δ 7.62−7.60 (m, 2H), 7.49−7.47 (m, 3H), 6.85−6.83 (m, 1H), 6.44−6.43 (m, 1H), 6.39−6.37 (m, 1H), 3.80 (s, 3H), 3.74 (s, 3H), 3.28 (t, J = 6.9 Hz, 2H), 2.88−2.87 (m, 4H), 2.75 (t, J = 7.0 Hz, 2H), 2.67 (bs, 4H), 2.17−2.11 (quint, J = 7.0 Hz, 2H), 1.69−1.66 (m, 8H); ¹³C NMR (125 MHz, CDCl₃) δ 156.0, 153.4, 151.6, 135.9, 130.1, 128.9, 128.6, 127.0, 118.5, 103.3, 99.8, 56.7, 55.5, 55.4, 49.1, 47.2, 36.0, 34.6, 31.7, 30.9, 28.9, 26.0; LC−MS (ESI) m/z: 522.09 [M + H].

3-(2-Fluorophenyl)-9-(3-((4-methyl-5-phenyl-4H-1,2,4-triazol-3yl)thio)propyl)-3,9-diazaspiro[5.5]undecane (28).

Following general method B, 28 was obtained as an off-white solid. (Yield 13%) ¹H NMR (500 MHz, CDCl₃) δ 7.62−7.60 (m, 2H), 7.49−7.46 (m, 3H), 7.02−6.95 (m, 1H), 6.94−6.90 (m, 1H), 6.89−6.87 (m, 1H), 3.58 (s, 3H), 3.29−3.27 (t, J = 7.0 Hz, 2H), 2.99−2.97 (m, 4H), 2.74 (bs, 2H), 2.67 (bs, 4H), 2.15−2.12 (m, 2H), 1.68−1.64 (m, 8H); ¹³C NMR (125 MHz, CDCl₃) δ 156.0, 154.8 (d, J_C−F = 245.4 Hz), 151.6, 140.7 (d, J_C−C−F = 8.3 Hz), 130.1, 128.9, 128.6, 127.1, 127.1, 124.4 (d, J_{C−C−C−F} = 3.5 Hz), 122.2 (d, J_{C−C−C−F} = 7.8 Hz), 119.1 (d, J_{C−C−C−C−F} = 2.7 Hz), 116.1 (d, J_C−C−F = 20.9 Hz), 56.7, 49.0, 46.0 (2 × CH), 35.6 (bs), 34.5, 31.7, 30.9, 28.9, 26.0; LC−MS (ESI) m/z: 480.16 [M + H].

3-(2-Fluoro-4-(trifluoromethyl)phenyl)-9-(3-((4-methyl-5-phenyl4H-1,2,4-triazol-3-yl)thio)propyl)-3,9-diazaspiro[5.5]undecane (29).

Following general method B, 29 was obtained as a white solid. (Yield 28%) ¹H NMR (500 MHz, CDCl₃) δ 7.62−7.60 (m, 2H), 7.49−7.46 (m, 3H), 7.28−7.26 (m, 1H), 7.22−7.19 (dd, J₁ = 1.8 Hz, J₂ = 12.7 Hz, 1H), 6.96−6.92 (t, J = 8.3 Hz, 1H), 3.58 (s, 3H), 3.30−3.27 (t, J = 7.0 Hz, 2H), 3.08−3.05 (m, 4H), 2.75 (bs, 2H), 2.67 (bs, 4H), 2.15−2.12 (m, 2H), 1.79−1.68 (m, 4H), 1.66−1.64 (m, 4H); ¹³C NMR (125 MHz, CDCl₃) δ 156.0, 153.6 (d, J_C−F = 249.6 Hz), 151.6, 143.5 (d, J_{C−C−C−F} = 8.5 Hz), 130.1, 129.0, 128.6, 127.1, 121.7 (q, J_C−C−CF3 = 3.5 Hz), 118.7 (d, J_{C−C−C−F} = 3.6 Hz), 113.4 (d, J_C−C−F = 21.0 Hz) and (quint, J_{C−C−C−CF}3 = 3.4 Hz), 56.7, 49.0, 46.0 (2 × CH), 35.4 (bs), 34.5, 31.7, 30.9, 29.0, 26.1; LC−MS (ESI) m/z: 548.14 [M + H].

3-(4-Fluoro-2-methoxyphenyl)-9-(3-((4-methyl-5-phenyl-4H-1,2,4-triazol-3-yl)thio)propyl)-3,9-diazaspiro[5.5]undecane (30).

Following general method B, 30 was obtained as an off-white solid. (Yield 24%) ¹H NMR (500 MHz, CDCl₃) δ 7.59−7.57 (m, 2H), 7.45−7.43 (m, 3H), 6.83−6.80 (m, 1H), 6.54−6.51 (m, 2H), 6.89−6.87, 3.78 (s, 3H), 3.54 (s, 3H), 3.28−3.25 (t, J = 7.1 Hz, 2H), 2.86−2.85 (m, 4H), 2.47−2.44 (t, J = 7.1 Hz, 2H), 2.38 (bs, 4H), 1.97−1.95 (quint, J = 7.1 Hz, 2H), 1.62−1.60 (m, 4H), 1.54−1.51 (m, 4H); ¹³C NMR (125 MHz, CDCl₃) δ 157.9 (d, J_C−F = 239.7 Hz), 155.7, 153.2 (d, J_{C−C−C−F} = 9.3 Hz), 151.9, 138.4 (d, J_{C−C−C−C−F} = 2.7 Hz), 129.9, 128.8. 128.5, 127.1, 118.4 (d, J_{C−C−C−F} = 9.6 Hz), 106.0 (d, J_C−C−F = 20.9 Hz), 99.6 (d, J_C−C−F = 26.5 Hz), 57.3, 55.6, 49.2, 47.0, 35.8 (bs), 31.5, 31.2, 29.0, 27.0; LC−MS (ESI) m/z: 510.63 [M + H].

3-(2-Methoxyphenyl)-9-(3-((4-methyl-5-phenyl-4H-1,2,4-triazol-3-yl)thio)propyl)-3,9-diazaspiro[5.5]undec-1-ene (32).

Following general method B, 32 was obtained as a dark red oil. (Yield 18%) ¹H NMR (500 MHz, CDCl₃) δ 7.64−7.62 (m, 2H), 7.51−7.49 (m, 3H), 7.02−6.98 (m, 1H), 6.94−6.92 (m, 1H), 6.90−6.86 (m, 2H), 6.27−6.26 (d, J = 8.1 Hz, 1H), 4.62 (d, J = 7.9 Hz, 1H), 3.83 (s, 3H), 3.60 (s, 3H), 3.43−3.41 (m, 2H), 3.32 (t, J = 7.0 Hz, 2H), 2.80−2.74 (m, 4H), 2.73−2.67 (bs, 2H), 2.20−2.14 (quint, J = 7.5 Hz, 2H), 1.75−1.72 (m, 6H); ¹³C NMR (125 MHz, CDCl₃) δ 156.1, 152.4, 151.7, 137.5, 132.2, 130.2, 129.0, 128.7, 127.1, 123.8, 122.3, 121.1, 112.0, 56.9, 55.7, 49.5, 44.3, 37.7, 31.7, 31.0, 29.7, 26.1; LC−MS (ESI) m/z: 490.17 [M + H].

8-(2-Methoxyphenyl)-2-(3-((4-methyl-5-phenyl-4H-1,2,4-triazol-3-yl)thio)propyl)-2,8-diazaspiro[4.5]decane (34).

Following general method B, 34 was obtained as a tan semisolid. (Yield 40%) ¹H NMR (500 MHz, CDCl₃) δ 7.58−7.57 (m, 2H), 7.45−7.43 (m, 3H), 6.94−6.91 (m, 1H), 6.86−6.83 (m, 2H), 6.79−6.76 (m, 1H), 3.18 (s, 3H), 3.57 (s, 3H), 3.29−3.26 (m, 2H), 3.21−3.10 (m, 4H), 3.00 (bs, 2H), 2.91 (bs, 4H), 2.30−2.27 (m, 2H), 1.93 (bs, 2H), 1.84 (bs, 4H); ¹³C NMR (125 MHz, CDCl₃) δ 156.1, 152.1, 151.0, 141.4, 130.1, 128.9, 128.5, 126.8, 122.9, 120.8, 118.3, 111.0, 55.3, 54.4, 52.9, 48.4, 40.3, 36.7, 35.2, 31.7, 30.2, 26.2; LC−MS (ESI) m/z: 478.81 [M + H].

N-(4-(9-(2-Methoxyphenyl)-3,9-diazaspiro[5.5]undecan-3-yl)-butyl)-4-(thiophen-3-yl)benzamide (35).

Compound F (1.0 mmol), 4-(thiophen-3-yl)benzoic acid (1.1 mmol), 1-hydroxybenzotriazole (HOBt) hydrate (1.0 mmol), 1-ethyl-3-(3′-dimethylaminopropyl)-carbodiimide (EDC)·HCl (1.0 mmol) were stirred in 15 mL of CH₂Cl₂ at room temperature for 2 h. The reaction mixture was then washed with a saturated NaHCO_3(aq) solution (10 mL). The reaction mixture was extracted with CH₂Cl₂ (3 × 20 mL), and the organic layers were combined, dried, and concentrated to afford a crude white sold. The residue was purified by flash chromatography on silica gel eluting with MeOH/CH₂Cl₂ (1:10) to afford 35 as a white solid. (Yield 31%) ¹H NMR (500 MHz, (CD₃)₂SO) δ 8.66 (m, 1H), 8.00 (bs, 1H), 7.95 (d, J = 7.7 Hz, 2H), 7.82 (d, J = 8.2 Hz, 2H), 7.67−7.65 (m, 1H), 7.63−7.62 (m, 1H), 6.91−6.88 (m, 3H), 6.85−6.83 (m, 1H), 3.75 (s, 3H), 3.31 (quint, J = 5.9 Hz, 2H), 2.96−2.87 (m, 9H), 1.69 (bs, 6H), 1.57−1.56 (m, 6H); ¹³C NMR (125 MHz, (CD₃)₂SO) δ 165.7, 152.0, 141.8, 140.5, 137.5, 132.8, 127.9, 127.3, 126.2, 125.7, 122.3, 122.2, 120.7, 118.1, 111.7, 55.2, 47.5, 45.8, 38.5, 32.6, 28.4, 26.5, 21.5; LC−MS (ESI) m/z: 518.22 [M + H].

4-(Dimethylamino)-N-(4-(9-(2-methoxyphenyl)-3,9-diazaspiro-[5.5]undecan-3-yl)butyl)benzamide (36).

Compound F (1.0 mmol), 4-dimethylamino benzoic acid (1.1 mmol), 1-hydroxybenzotriazole (HOBt) hydrate (1.0 mmol), 1-ethyl-3-(3′-dimethylaminopropyl)-carbodiimide (EDC)·HCl (1.0 mmol) were stirred in 15 mL of CH₂Cl₂ at room temperature for 2 h. The reaction mixture was then washed with a saturated NaHCO_{3 (aq)} solution (10 mL). The reaction mixture was extracted with CH₂Cl₂ (3 × 20 mL), and the organic layers were combined, dried, and concentrated to afford a crude white sold. The residue was purified by flash chromatography on silica gel eluting with MeOH/CH₂Cl₂ (1:10) to afford 36 as a white solid. (Yield 40%) ¹H NMR (500 MHz, CDCl₃) δ 7.85 (d, J = 8.8 Hz, 2H), 7.33 (bs, 1H), 6.99−6.96 (m, 1H), 6.92−6.88 (m, 2H), 6.84−6.83 (m, 1H), 6.65 (d, J = 8.9 Hz, 2H), 3.84 (s, 3H), 3.47−3.44 (m, 2H), 3.10−2.95 (m, 16H), 1.91−1.90 (m, 6H), 1.71−1.67 (m, 6H); ¹³C NMR (125 MHz, CDCl₃) δ 167.8, 152.4, 152.2, 141.5, 128.8, 123.1, 121.0, 120.9, 118.3, 111.2, 111.1, 56.3, 55.4, 48.4, 46.5, 40.2, 38.0, 32.4, 28.7,26.5, 21.1; LC−MS (ESI) m/z: 479.35 [M + H].

7-(3-(9-(2-Methoxyphenyl)-3,9-diazaspiro[5.5]undecan-3-yl)-propoxy)-3,4-dihydroquinolin-2(1H)-one (37).

4b′ (1.5 mmol), G¹⁷ (1.5 mmol), KI (1.5 mmol), and K₂CO₃ (5.0 mmol) were dissolved in acetonitrile (15 mL), and the reaction mixture was stirred at 90 °C for 12 h. The reaction mixture was then filtered, and the solvent was removed under reduced pressure. The crude residue was purified by silica gel column chromatography eluting with 10% 7 N NH₃ in MeOH solution/CH₂Cl₂ (1:10) to afford 37 as a white solid (Yield 62%). ¹H NMR (500 MHz, CDCl₃) δ 8.88 (s, 1H), 7.02−7.00 (d, J = 8.4 Hz, 1H), 6.98−6.95 (m, 2H), 6.91−6.88 (m, 1H), 6.84−6.83 (d, J = 8.0 Hz, 1H), 6.52−6.50 (dd, J₁ = 2.1 Hz, J₂ = 8.2 Hz, 1H), 6.37−6.36 (m, 1H), 3.97−3.95 (t, J = 6.1 Hz, 2H), 3.84 (s, 3H), 2.99−2.97 (m, 4H), 2.89−2.86 (t, J = 7.4 Hz, 2H), 2.61−2.58 (t, J = 7.9 Hz, 2H), 2.53−2.50 (t, J = 7.0 Hz, 2H), 2.45 (bs, 4H), 2.01−1.95 (m, 2H), 1.69−1.67 (m, 4H), −1.59 (m, 4H); ¹³C NMR (125 MHz, CDCl₃) δ 172.2, 158.7, 152.3, 142.3, 138.3 (2 × CH), 128.6, 122.6, 120.9, 118.3, 115.7, 111.1, 108.7, 102.4, 66.7, 55.7, 55.4, 49.4, 46.8, 36.2 (bs), 35.8, 31.1, 29.2, 26.9, 24.6; LC−MS (ESI) m/z: 464.68 [M + H].

Receptor Binding Assays.

Receptor K_i values were measured using human D₂ (long) and D₃ expressed in HEK cells with [¹²⁵I]IABN as the radioligand. The binding properties of membrane-associated receptors were characterized by a filtration binding assay.³⁷ Membrane homogenates were suspended in 50 mM Tris−HCl/150 mM NaCl/10 mM EDTA buffer, pH 7.5, and incubated with [¹²⁵I]IABN³⁷ at 37 °C for 60 min, using 20 μM (+)-butaclamol to define the nonspecific binding.

The radioligand concentration was equal to approximately 0.5 (D_2/3R) times the K_d, and the concentration of the competitive inhibitor ranged over 5 orders of magnitude. For each competition curve, two concentrations of inhibitor per decade were used, and triplicates were performed. Binding was terminated by the addition of ice cold wash buffer (D_2/3R, 10 mM Tris−HCl, 150 mM NaCl, pH 7.5; 5-HT_1AR, 10 mM Tris−HCl, pH 7.4) and filtration over a glass-fiber filter (D_3/2R, Schleicher and Schuell No. 32; 5-HT_1AR, Whatman grade 934-AH, GE Healthcare Bio-Sciences, Pittsburgh, PA). A Packard Cobra scintillation counter was used to measure the radioactivity. The equilibrium dissociation constant and maximum number of binding sites were generated using unweighted nonlinear regression analysis of data modeled according to the equation describing mass R-binding. The concentration of inhibitor that inhibits 50% of the specific binding of the radioligand (IC₅₀) was determined by using nonlinear regression analysis to analyze the data of competitive inhibition experiments. Competition curves were modeled for a single site, and the IC₅₀ values were converted to equilibrium dissociation constants (K_i) using the Cheng and Prusoff⁵¹ correction. Mean K_i ± SEM values are reported for at least three independent experiments.

β-Arrestin Recruitment Assay.

β-arrestin 2 recruitment to D₃R was assayed using the DiscoverX Pathhunter kit according to the manufacturer’s instructions, with some minor modifications. In brief, CHO-K1 cells expressing human β-arrestin 2 tagged with a β-galactosidase enzyme lacking a part of the catalytic domain, and human D₃R C-terminally tagged with a complementary fragment of β-galactosidase, were grown in culture medium (DiscoverX) supplemented with G418 and hygromycin. Cells were seeded into white plastic 96-well plates at a density of 2 × 10⁴ cells/well, 24 h prior to the assay, in a volume of 50 μL/well of Cell Plating Reagent 2 (CP2; DiscoverX).

Test compounds were dissolved in DMSO and diluted to the appropriate concentrations in CP2, added the cells in a volume of 30 μL/well, and preincubated with the cells at 37 °C for 30 min. Next, dopamine (dissolved and diluted in 30 μL CP2 so as to obtain the various concentrations used to construct concentration−response curves) was applied, followed by another 90 min of incubation at 37 °C.

Finally, to detect functional complementation of β-galactosidase upon dopamine-induced β-arrestin 2 recruitment to D₃R, cells were treated with a detection cocktail containing a coelenterazine-based β-galactosidase substrate (as described by the manufacturer), and incubated for another 60 min at room temperature, prior to assay read-out using a PerkinElmer Enspire plate reader (luminescence mode; read time 1 s/well).

Molecular Docking Studies.

In silico molecular docking studies were performed following by the previous study.⁵² Compounds 1, 17, 19, 31, and 32 were drawn on ChemDraw Profession 15.1 (PerkinElmer Informatics, Inc.), then imported to Chem3D Ultra 15.1 (PerkinElmer Informatics, Inc.) to minimize individual structures by MMFF94 force field for preparation of molecular docking. Molecular docking studies were performed via AutoDock 4.2⁵³ plugin on PyMOL (pymol.org). X-ray structure of dopamine 3 receptor (D₃R) (PDB ID 3PBL, Resolution 2.89 Å) was obtained from RCSB Protein Data Bank (www.rcsb.org). Waters and other heteroatoms were removed from the structure, followed by adding polar hydrogens. Nonpolar hydrogens were removed from every compound. A grid box with a dimension of 30 × 30 × 28.2 ų was applied to the D₃R X-ray structure, covering orthosteric and secondary binding sites. The Lamarckian Genetic Algorithm with a maximum of 2 500 000 energy evaluations was used to calculate 100 protein−ligand binding poses for each compound to each protein. The protein−ligand complex reported for each compound exhibited the most ligand−protein contacts with the lowest free binding energy.

Data Analysis.

Statistical analyses from pharmacological assays were conducted on a GraphPad Prism 7.04 software.

ABBREVIATIONS

ACN

acetonitrile

AMP

adenosine monophosphate

CNS

central nervous system

DA

dopamine

DCM

dichloromethane

D₂R

dopamine D₂ receptor

D₃R

dopamine D₃ receptor

GPCR

G-protein-coupled receptor

HEK

human embryonic kidney 293

[¹²⁵I]IABN

[¹²⁵I]-N-benzyl-5-iodo-2,3-dimethoxy[3.3.1]azabicyclononan-3-β-ylbenzamide

Pd

palladium

PET

positron-emission tomography

TFA

trifluoroacetic acid