Design and Synthesis of D3R Bitopic Ligands with Flexible Secondary Binding Fragments: Radioligand Binding and Computational Chemistry Studies

A series of bitopic ligands based on Fallypride with a flexible secondary binding fragment (SBF) were prepared with the goal of preparing a D3R-selective compound. The effect of the flexible linker ((R,S)-trans-2a–d), SBFs ((R,S)-trans-2h–j), and the chirality of orthosteric binding fragments (OBFs) ((S,R)-trans-d, (S,R)-trans-i, (S,S)-trans-d, (S,S)-trans-i, (R,R)-trans-d, and (R,R)-trans-i) were evaluated in in vitro binding assays. Computational chemistry studies revealed that the interaction of the fragment binding to the SBF increased the distance between the pyrrolidine nitrogen and ASP1103.32 of the D3R, thereby reducing the D3R affinity to a suboptimal level.


Introduction
Dopamine receptors are a class of G protein-coupled receptors.Five subtypes of dopamine receptors have been identified, and they are divided into two classes based on their sequence and functional roles.The dopamine 1-like receptors include the dopamine 1 receptor (D 1 R) and the dopamine 5 receptor (D 5 R), and the dopamine 2-like receptors consist of the dopamine 2 receptor (D 2 R), the dopamine 3 receptor (D 3 R), and the dopamine 4 receptor (D 4 R) [1][2][3].Previous studies showed that the dysregulation of D 2 R and D 3 R is related to many central nervous system (CNS) diseases [4,5].These two dopamine receptors have been used as therapeutic targets for treating neurological and neuropsychiatric disorders, including schizophrenia, drug addiction, and Parkinson's disease (PD) [4,[6][7][8].
The D 2 R and D 3 R have a differential distribution in the human brain [9][10][11].For example, in the globus pallidus internal part, thalamus, red nucleus, and substantia nigra, the D 3 R has a higher density than the D 2 R [12,13].Moreover, an autoradiography study in chronic cocaine abuse showed that the density of the D 2 R and D 3 R changed differently (the D 2 R had no change vs. the D 3 R increased) [4,[14][15][16][17][18].An in vitro autoradiography study of PD brain samples yielded similar results as above [12,14].These results indicate that these two dopamine receptors play different roles in the CNS; the D 3 R is thought to play a key role in mediating L-DOPA-induced dyskinesia [16].
Positron emission tomography (PET) is a functional imaging technique that uses radiotracers to image receptors in the CNS.Over the past several years, [ 18 F]Fallypride [19][20][21][22], [ 11 C]Raclopride [23][24][25][26][27], [ 11 C]FLB 457 [20,26,28], and [ 11 C]-(+)-PHNO [29][30][31] have been used as radiotracers for PET imaging studies of the D 2 -like receptors in humans (Figure 1).Unfortunately, all four radiotracers cannot image the D 3 R independently of the D 2 R. The lack of highly selective D 3 R PET radiotracers has prevented the imaging of the D 3 R independently of the D 2 R. Therefore, developing a D 3 R selective radiotracer that can bind independently of the D 2 R is of high importance to further understand the behavior of the D 3 R in the CNS.
Molecules 2024, 29, x FOR PEER REVIEW 2 of 13 22], [ 11 C]Raclopride [23][24][25][26][27], [ 11 C]FLB 457 [20,26,28], and [ 11 C]-(+)-PHNO [29][30][31] have been used as radiotracers for PET imaging studies of the D2-like receptors in humans (Figure 1).Unfortunately, all four radiotracers cannot image the D3R independently of the D2R.The lack of highly selective D3R PET radiotracers has prevented the imaging of the D3R independently of the D2R.Therefore, developing a D3R selective radiotracer that can bind independently of the D2R is of high importance to further understand the behavior of the D3R in the CNS.The high structural similarity of the D2R and D3R (78% similar in the transmembrane spanning region) [4], has presented a challenge in the development of a D3R versus a D2R selective ligand [32].During the past decade, a number of D3R selective ligands with the phenylpiperazine structure have been reported.Our group has reported Fluortriopride (FTP; LS-3-134) [33] (Figure 2), which has high D3R affinity (Ki = 0.17 nM) and good selectivity versus the D2R (163-fold).The 1,2,4-triazole-based scaffold also has notable D3R versus D2R selectivity.For example, GSK598,809 (D3R Ki = 3.2 nM) [34] represents a D3R selective ligand with this scaffold and has a 670-fold D3R vs. D2R selectivity.Based on the structure of tranylcypromine, Chen et al. [35] reported a D3R ligand (CJ-1882) with 2.8 nM affinity for the D3R and 223-fold D3R versus D2R selectivity.Recently, we reported a flexible scaffold structure (HY-2-93) with 0.8 nM affinity for the D3R and 180-fold D3R versus D2R selectivity [36].This report describes the continuation of our effort to identify D3R antagonists having a high selectivity versus the D2R as a potential PET radiotracer for in vivo imaging studies.Our recent structure-activity relationship (SAR) studies based on Fallypride revealed some critical factors for designing a D3R versus D2R selective ligand [37].These results, together with the flexible D3R ligands we reported previously [36], inspired us to design a D3R selective antagonist by introducing a flexible secondary binding fragment (SBF) based on Fallypride.

Chemistry
As shown in Scheme 1, we used a two-step route to prepare the Fallypride-based bitopic ligands.First, a mixture of (S,R)-trans-1 and 1,1-carbonyldiimidazole (CDI) in acetonitrile was stirred overnight.Second, the corresponding amines reacted with the crude The high structural similarity of the D 2 R and D 3 R (78% similar in the transmembrane spanning region) [4], has presented a challenge in the development of a D 3 R versus a D 2 R selective ligand [32].During the past decade, a number of D 3 R selective ligands with the phenylpiperazine structure have been reported.Our group has reported Fluortriopride (FTP; LS-3-134) [33] (Figure 2), which has high D 3 R affinity (K i = 0.17 nM) and good selectivity versus the D 2 R (163-fold).The 1,2,4-triazole-based scaffold also has notable D 3 R versus D 2 R selectivity.For example, GSK598,809 (D 3 R K i = 3.2 nM) [34] [20,26,28], and [ 11 C]-(+)-PHNO [29][30][31] have been used as radiotracers for PET imaging studies of the D2-like receptors in humans (Figure 1).Unfortunately, all four radiotracers cannot image the D3R independently of the D2R.The lack of highly selective D3R PET radiotracers has prevented the imaging of the D3R independently of the D2R.Therefore, developing a D3R selective radiotracer that can bind independently of the D2R is of high importance to further understand the behavior of the D3R in the CNS.The high structural similarity of the D2R and D3R (78% similar in the transmembrane spanning region) [4], has presented a challenge in the development of a D3R versus a D2R selective ligand [32].During the past decade, a number of D3R selective ligands with the phenylpiperazine structure have been reported.Our group has reported Fluortriopride (FTP; LS-3-134) [33] (Figure 2), which has high D3R affinity (Ki = 0.17 nM) and good selectivity versus the D2R (163-fold).The 1,2,4-triazole-based scaffold also has notable D3R versus D2R selectivity.For example, GSK598,809 (D3R Ki = 3.2 nM) [34] represents a D3R selective ligand with this scaffold and has a 670-fold D3R vs. D2R selectivity.Based on the structure of tranylcypromine, Chen et al. [35] reported a D3R ligand (CJ-1882) with 2.8 nM affinity for the D3R and 223-fold D3R versus D2R selectivity.Recently, we reported a flexible scaffold structure (HY-2-93) with 0.8 nM affinity for the D3R and 180-fold D3R versus D2R selectivity [36].This report describes the continuation of our effort to identify D3R antagonists having a high selectivity versus the D2R as a potential PET radiotracer for in vivo imaging studies.Our recent structure-activity relationship (SAR) studies based on Fallypride revealed some critical factors for designing a D3R versus D2R selective ligand [37].These results, together with the flexible D3R ligands we reported previously [36], inspired us to design a D3R selective antagonist by introducing a flexible secondary binding fragment (SBF) based on Fallypride.

Chemistry
As shown in Scheme 1, we used a two-step route to prepare the Fallypride-based bitopic ligands.First, a mixture of (S,R)-trans-1 and 1,1-carbonyldiimidazole (CDI) in acetonitrile was stirred overnight.Second, the corresponding amines reacted with the crude This report describes the continuation of our effort to identify D 3 R antagonists having a high selectivity versus the D 2 R as a potential PET radiotracer for in vivo imaging studies.Our recent structure-activity relationship (SAR) studies based on Fallypride revealed some critical factors for designing a D 3 R versus D 2 R selective ligand [37].These results, together with the flexible D 3 R ligands we reported previously [36], inspired us to design a D 3 R selective antagonist by introducing a flexible secondary binding fragment (SBF) based on Fallypride.

Chemistry
As shown in Scheme 1, we used a two-step route to prepare the Fallypride-based bitopic ligands.First, a mixture of (S,R)-trans-1 and 1,1-carbonyldiimidazole (CDI) in acetonitrile was stirred overnight.Second, the corresponding amines reacted with the crude product from the previous step in toluene at 100 • C, delivering the designed bitopic ligands (R,S)-trans-2a-j in good yield.
product from the previous step in toluene at 100 °C, delivering the designed bitopic ligands (R,S)-trans-2a-j in good yield.product from the previous step in toluene at 100 °C, delivering the designed bitopic ligands (R,S)-trans-2a-j in good yield.

SAR Study of Flexible Bitopic Ligands towards D 2 R and D 3 R
The compounds synthesized in Schemes 1 and 2 were submitted for in vitro binding assays measuring their affinity for D 3 R and D 2 R. The results of the binding assays are shown in Tables 1 and 2.  Ligands with a flexible aliphatic linker (i.e., (R,S)-trans-2a-d) have a high affinity for the D 3 R; as the length of the aliphatic linkers increased, their affinity for the D 2 R decreased.These results suggest that bitopic ligands with longer aliphatic linkers increase the selectivity for the D 3 R versus the D 2 R. Bitopic ligands with a polyethylene glycol (PEG) as a flexible linker were also evaluated ((R,S)-trans-2e-g).Although these bitopic ligands maintained high affinity for the D 3 R, they did not have the expected higher D 3 R versus D 2 R selectivity.The ligands (R,S)-trans-2h-j, which have a flexible linker containing a thiophene in the SBF, were evaluated for their affinity for the D 2 R and D 3 R.The 2-thiophene analog with a 6-carbon spacer, (R,S)-trans-2h, had a sub-nanomolar affinity for the D 3 R (K i = 0.6 ± 0.1 nM); increasing the spacer group to 8 carbon atoms did not affect the D 3 R affinity (K i = 0.7 ± 0.1 nM).The analog having a 6-carbon spacer with a 3-thiophene had lower D 3 R affinity relative to its 2-thiophene congener, (R,S)-trans-2h.
We next evaluated the effect of the chirality of OBFs (Table 2).The enantiomers and diastereomers of (R,S)-trans-2d and (R,S)-trans-2i were utilized for this as these two ligands have comparatively better affinity for the D 3 R. Changing the chiral centers of OBFs has a dramatic effect on their affinity for the D 2 R and D 3 R. (S,R)-trans-2d is an enantiomer of (R,S)-trans-2d.The affinity for the D 2 R and the D 3 R decreased one thousand-fold to 1945 nM and 2628 nM, respectively.Meanwhile, the D 2 R and D 3 R affinity of (S,S)-cis-2d and (R,R)-cis-2d, the diastereomers of (R,S)-trans-2d, were decreased by one hundred to one thousand-fold.These results were also observed for the enantiomer and diastereomers of (R,S)-trans-2i.

β-Arrestin Competition Assay
A β-arrestin competition assay was conducted to determine the potency of the abovementioned compounds for competing with dopamine (30 nM) for the D 3 R. High potency in this assay (EC50 ~2 nM) is needed in order to compete with synaptic dopamine for binding to the D 3 R in vivo [39].As shown in Table 1, (R,S)-trans-2a has high potency in the β-arrestin assay.Compounds having a longer aliphatic linker ((R,S)-trans-2b-d) have a similar IC 50 value in this assay.As expected, the IC 50 value of β-arrestin assay parallels the K i value in the D 3 R binding assay.It is of interest to note that the potency of (R,S)-trans-2a in the β-arrestin assay is higher than its potency in the in vitro binding assay for the D 3 R.

Molecular Dynamic Simulation (MDS) Studies
The representative binding poses for the D 3 R or D 2 R from the MDS production runs for four compounds with different lengths of aliphatic groups in the SBFs ((R,S)-trans-2a, (R,S)-trans-2b, (R,S)-trans-2c, and (R,S)-trans-2d) are shown in Figure 3.The "Fallypride" fragment of all four compounds posed in the orthosteric binding pocket interacted with the amino acid residues at transmembrane (TM) 3, 5, and 6 in both the D 2 R and the D 3 R.The SBF of all four compounds interacted with the amino acid residues in TM 2 and 7 in the secondary binding site for the D 3 R.For the D 2 R, the SBF of the four compounds interacted with the secondary binding pocket (TM 2 and 7) or the loop region, EC1 or EC2.Hydrogen bonds formed between ASP110 3.32 and the protonated nitrogen in the pyrrolidine ring, the key interaction that has been reported for the D 3 R [39,40], were observed in all four compounds in the D 3 R (Figure 3A-D).The benzene rings of (R,S)-trans-2a, (R,S)-trans-2b, and (R,S)-trans-2c formed a π-stacking interaction with PHE345 6.51 in the orthosteric binding pocket of the D 3 R (Figure 3A-C).For the D 2 R, a hydrogen bond or salt bridge formed between ASP114 3.32 and the protonated nitrogen in the pyrrolidine ring (the key interaction that has been reported for D 2 R [41,42]) was observed in the MDS for all four compounds (Figure 3F-I).The absence of a π-stacking interaction in the orthosteric binding site of the D 2 R may partially explain the higher affinity of the four compounds for the D 3 R.
The distance between the protonated nitrogen in the pyrrolidine ring of six ligands ((R,S)-trans-2a-d, (R,S)-trans-2h, and (R,S)-trans-2i) and ASP110 3.32 in the D 3 R was in the range of 3.28 to 3.79 Å (Table 3); this distance is greater than that with Fallypride (3.17 ± 0.21 Å) [39].This indicates that these compounds have weaker interactions with ASP110 3.32 in the D 3 R compared to Fallypride, resulting in a reduction in the binding affinity for the D 3 R. Furthermore, the distance between ASP114 3.32 in the D 2 R and the protonated nitrogen in the pyrrolidine ring of the six ligands was greater (3.45 to 9.32 Å; Table 3) and had a higher standard deviation (0.94 to 2.20 Å; Table 3); these results are consistent with the low stability of these compounds in the binding pocket and are reflected in the lower binding affinity of the six compounds for the D 2 R.
Another method for studying ligand-protein interactions is to measure the frequency of the contact between the ligand and amino acid residues in the ligand binding site.The summary of the frequency of contacts for each compound in the D 3 R or D 2 R is shown in Figure 4.A stable salt bridge or hydrogen bound was formed between ASP110 3.32 in the D 3 R and the pyrrolidine nitrogen in all six compounds (frequency of contact > 0.8; Figure 4A).Consequently, (R,S)-trans-2a-d, (R,S)-trans-2h, and (R,S)-trans-2i all had high affinity for the D 3 R (0.6-3.4 nM).For the D 2 R, (R,S)-trans-2a-d, (R,S)-trans-2h, and (R,S)-trans-2i displayed a poor to moderate probability of forming a hydrogen bound with ASP114 3.32  (frequency of contact = 0.01-0.81; Figure 4B).There was also a wide range in the probability of forming hydrophobic interactions (0.15-0.87) in the orthosteric binding pocket (TM 3 and 5; Figure 4B).The low interaction with ASP114 3.32 and variable hydrophobic interactions likely explain the high range in the D 2 R affinity for the six compounds.3. The distance between the protonated nitrogen in the pyrrolidine ring of ligand and ASP110 3.32 in the D3R and ASP114 3.32 in the D2R.

Compound
Distance to D3R ASP110 Another method for studying ligand-protein interactions is to measure the frequency of the contact between the ligand and amino acid residues in the ligand binding site.The summary of the frequency of contacts for each compound in the D3R or D2R is shown in Figure 4.A stable salt bridge or hydrogen bound was formed between ASP110 3.32 in the D3R and the pyrrolidine nitrogen in all six compounds (frequency of contact > 0.8; Figure 4A).Consequently, (R,S)-trans-2a-d, (R,S)-trans-2h, and (R,S)-trans-2i all had high affinity for the D3R (0.6-3.4 nM).For the D2R, (R,S)-trans-2a-d, (R,S)-trans-2h, and (R,S)-trans-2i displayed a poor to moderate probability of forming a hydrogen bound with ASP114 3.32  (frequency of contact = 0.01-0.81; Figure 4B).There was also a wide range in the probability of forming hydrophobic interactions (0.15-0.87) in the orthosteric binding pocket (TM 3 and 5; Figure 4B).The low interaction with ASP114 3.32 and variable hydrophobic interactions likely explain the high range in the D2R affinity for the six compounds.
Although there were differences in the interaction of (R,S)-trans-2a-d, (R,S)-trans-2h, and (R,S)-trans-2i with VAL86 2.61 in the secondary binding pocket of the D3R, this interaction was inconsequential since all six compounds had similar affinity for the D3R.On the other hand, the interaction of the SBF with the D2R may play a role in the D2R affinity of the six ligands.(R,S)-trans-2c, (R,S)-trans-2d, (R,S)-trans-2h, and (R,S)-trans-2i, which have a longer length of the SBF (8 and 10 carbon spacer group), displayed a higher frequency of contact of TRP100 EC1 in the loop between TM 2 and 3 (EC1) and ILE184 EC2 in the

Discussion
We prepared a panel of bitopic D3R ligands based on Fallypride with the goal of developing a D3R selective antagonist.The most D3R selective compounds were (R,S)-trans-2c and (R,S)-trans-2d, which had a ~3 nM affinity for the D3R and 5-10-fold selectivity versus the D2R.This selectivity was attributed to their reduced affinity for the D2R, which was likely caused by the lower interactions in the orthosteric binding site of the D2R.(R,S)trans-2a had higher potency (IC50 = 0.8 ± 0.5 nM) to compete with dopamine than Fallypride (IC50 = 1.7 ± 0.8 nM) in the β-arrestin recruitment assay, which was unexpected given its lower affinity for the D3R relative to Fallypride.The reason for this discrepancy be- Table 3.The distance between the protonated nitrogen in the pyrrolidine ring of ligand and ASP110 3.32  in the D 3 R and ASP114 3.32 in the D 2 R.

Compound
Distance to D 3 R ASP110 Although there were differences in the interaction of (R,S)-trans-2a-d, (R,S)-trans-2h, and (R,S)-trans-2i with VAL86 2.61 in the secondary binding pocket of the D 3 R, this interaction was inconsequential since all six compounds had similar affinity for the D 3 R.On the other hand, the interaction of the SBF with the D 2 R may play a role in the D 2 R affinity of the six ligands.(R,S)-trans-2c, (R,S)-trans-2d, (R,S)-trans-2h, and (R,S)-trans-2i, which have a longer length of the SBF (8 and 10 carbon spacer group), displayed a higher frequency of contact of TRP100 EC1 in the loop between TM 2 and 3 (EC1) and ILE184 EC2 in the loop between TM 4 and 5 (EC2; Figure 4B).The favorable interaction of (R,S)-trans-2c and (R,S)-trans-2d with the loop region of EC2 interferes with the formation of the hydrogen bond with ASP114 3.32 , and this likely explains the lower D 2 R affinity of (R,S)-trans-2c and (R,S)-trans-2d.

Discussion
We prepared a panel of bitopic D 3 R ligands based on Fallypride with the goal of developing a D 3 R selective antagonist.The most D 3 R selective compounds were (R,S)trans-2c and (R,S)-trans-2d, which had a ~3 nM affinity for the D 3 R and 5-10-fold selectivity versus the D 2 R.This selectivity was attributed to their reduced affinity for the D 2 R, which was likely caused by the lower interactions in the orthosteric binding site of the D 2 R. (R,S)-trans-2a had higher potency (IC 50 = 0.8 ± 0.5 nM) to compete with dopamine than Fallypride (IC 50 = 1.7 ± 0.8 nM) in the β-arrestin recruitment assay, which was unexpected given its lower affinity for the D 3 R relative to Fallypride.The reason for this discrepancy between the receptor affinity and potency in the β-arrestin competition assay is not clear.(R,S)-trans-2d had modest D 3 R versus D 2 R selectivity (~12.7-fold) and a much better ability to compete with endogenous dopamine (IC 50 = 21.2 ± 9.8 nM) than the highly D 3 R-selective radiotracer FTP (IC 50 = 611.7 ± 101.3 nM).Increasing the length and steric bulk of the flexible linker in the SBF did not improve the D 3 R versus D 2 R selectivity.This structural modification also reduced the affinity of the bitopic compounds for both the D 3 R and the D 2 R relative to that of Fallypride, which is largely due to the effect of the substituent in the SBF increasing the distance between the pyrrolidine nitrogen and the key ASP 3.32 residues in the orthosteric binding site of the D 3 R and the D 2 R. Consequently, the SAR studies described above indicate that the modification of Fallypride to contain an SBF does not improve the selectivity of this scaffold that is needed to generate a D 3 R-selective PET radiotracer.

Chemistry
The starting materials and anhydrous solvents were purchased from Sigma-Aldrich, TCI America, Alfa Aesar, and Ambeed and were used without further purification.5-(3fluoropropyl)-2,3-dimethoxybenzoic acid was prepared using a reported method.The NMR spectra were taken on a Bruker DMX 400 MHz.The chemical shifts (δ) in the NMR spectra ( 1 H and 13 C) were referenced by assigning the residual solvent peaks.The purification of organic compounds was carried out on a Biotage Isolera One with a dual-wavelength UV−vis detector (silica gel: 230−400 mesh, 60 Å).The compound structures and identity were confirmed by 1 H-and 13 C NMR and mass spectrometry (Supplementary Materials).The procedure for the synthesis of 1 can be found in our previous report [37].

General Methods for the Synthesis of 2
A mixture of 1 (0.2 mmol) and 1,1 ′ -Carbonyldiimidazole (CDI) (0.2 mmol) in acetonitrile (4 mL) was stirred at room temperature overnight.The solvent was removed under a vacuum, and the crude product was used for the next step directly.
A solution of the crude product mentioned above and the corresponding amine (0.4 mmol) in toluene was heated to 100 • C overnight.The solvent was removed under vacuum, and the crude product was purified by flash silica chromatography (CH 2 Cl 2 /MeOH = 95:5) to afford 2.

Receptor Binding and β-Arrestin Assays
The receptor binding assay for the D 2 R and the D 3 R and the β-arrestin recruitment assay for the D 3 R were performed by following the previously reported methods [37].

Figure 1 .
Figure 1.Structure and data of radiotracers for imaging D2-like receptors.

Figure 1 .
Figure 1.Structure and data of radiotracers for imaging D 2 -like receptors.
represents a D 3 R selective ligand with this scaffold and has a 670-fold D 3 R vs. D 2 R selectivity.Based on the structure of tranylcypromine, Chen et al. [35] reported a D 3 R ligand (CJ-1882) with 2.8 nM affinity for the D 3 R and 223-fold D 3 R versus D 2 R selectivity.Recently, we reported a flexible scaffold structure (HY-2-93) with 0.8 nM affinity for the D 3 R and 180-fold D 3 R versus D 2 R selectivity [36].22], [ 11 C]Raclopride [23-27], [ 11 C]FLB 457

Figure 1 .
Figure 1.Structure and data of radiotracers for imaging D2-like receptors.

Figure 2 .
Figure 2. The D 3 R versus D 2 R selective ligands.

Figure 4 .
Figure 4. Summary of frequency of interactions between ligands and the residues in the binding pocket of (A) D3R and (B) D2R.

Figure 4 .
Figure 4. Summary of frequency of interactions between ligands and the residues in the binding pocket of (A) D 3 R and (B) D 2 R.

Table 1 .
The results of D 3 R vs. D 2 R affinity and β-arrestin assay IC 50 a .All compounds were tested as HCl salts; b Data from reference [38]; c K i values for D 2 R were determined by at least three experiments and measured using human D 2 expressed in HEK cells with [ 125 I]IABN as the radioligand; [39]Ki values for D 3 R were determined by at least three experiments and measured using human D 3 expressed in HEK cells with [ 125 I]IABN as the radioligand; e (K i for D 2 R)/(K i for D 3 R); f IC 50 values were determined by at least three experiments, mean ± standard error of the mean (SEM); g Data from reference[39]; h Calculated using ChemDraw Professional 15.1; ND: not detected.

Table 2 .
The results of D 3 R vs. D 2 R affinity and β-arrestin assay IC 50 a .
[39]l compounds were tested as HCl salts; b Data from reference [38]; c K i values for D 2 R were determined by at least three experiments and measured using human D 2 expressed in HEK cells with [ 125 I]IABN as the radioligand; d Ki values for D 3 R were determined by at least three experiments and measured using human D 3 expressed in HEK cells with [ 125 I]IABN as the radioligand; e (K i for D 2 R)/(K i for D 3 R); f IC 50 values were determined by at least three experiments, mean ± standard error of the mean (SEM); g Data from reference[39]; h Calculated using ChemDraw Professional 15.1; ND: not detected.