N-(3-Fluoro-4-(4-(2-methoxy or 2,3-dichlorophenyl) piperazine-1-yl)-butyl)-aryl carboxamides as Selective Dopamine D3 Receptor Ligands: Critical Role of the Carboxamide Linker for D3 Receptor Selectivity

Abstract

N-(3-fluoro-4-(4-(2,3-dichloro- or 2-methoxyphenyl)piperazine-1-yl)-butyl)-aryl carboxamides were prepared and evaluated for binding and function at dopamine D3 (D3R) and D2 receptors (D2R). In this series, we discovered some of the most D3R selective compounds reported to date, (e.g. 8d and 8j >1000-fold D3R-selective over D2R.) In addition, chimeric receptor studies further identified the second extracellular (E2) loop as an important contributor to D3R binding selectivity. Further, compounds lacking the carbonyl group in the amide linker were synthesized and while these amine-linked analogues bound with similar affinities to the amides at D2R, this modification dramatically reduced binding affinities at D3R by >100-fold (e.g. D3RKi for 15b = 393 v. for 8j = 2.6 nM) resulting in compounds with significantly reduced D3R selectivity. This study supports a pivotal role for the D3R E2 loop and the carbonyl group in the 4-phenylpiperazine class of compounds and further reveals a point of separation between structure-activity relationships at D3R and D2R.

The dopamine D2-like receptor family, which is comprised of the D2, D3 and D4 receptor subtypes, has been a target for discovering medications for the treatment of neuropsychiatric and neurodegenerative disorders for decades. Following cloning and in situ studies, dopamine D3 receptors (D3R) were discovered to be primarily localized in limbic regions of the rat and human brain.^1,2 Studies have linked changes in the expression of D3R to drug addiction and this has led to an interest in the development of D3R-selective compounds as potential medications.^3,4 Because of the high degree of amino acid sequence homology (78% in the transmembrane regions) between D2R and D3R, it has been challenging to obtain subtype selective agents. However, both D2R and D3R selective agonists and antagonists have been described.^5–13

Recently, D3R selective agents with varying intrinsic activities have been identified.⁴ We have previously described novel substituted 4-phenylpiperazines, linked by 2-butenyl- or 3-hydroxy butyl linkers to an extended arylamide moiety, that are highly selective for the D3R.^5,6,11 Several of these compounds (e.g. PG 01037, 1) have provided excellent tools for investigating D3R in animal models of psychostimulant abuse.^14–20

Further, a recent extension of SAR at D3R has resulted in the discovery of some of the most D3R-selective compounds reported to date with D3R binding affinities in the low nanomolar range and ~400-fold selectivity over the D2R. Importantly, in that study, the first D3R antagonist (R-PG 648; 2) was reported wherein enantioselectivity was more pronounced at D3R than at D2R, and a binding region on the second extracellular loop (E2) was identified as playing a role in both enantioselectivity and D3R vs. D2R binding selectivity. Moreover, molecular models, based on SAR studies using site-directed mutagenesis and computational predictions, differentiate D2R from D3R and D4 receptor binding and also highlight the contributions of E2 to subtype selectivity.²¹

Structure-activity relationships (SAR) previously developed demonstrate the importance of the length and composition on the linking chain between the aryl amide and the 4-phenylpiperazine in this class of D3R ligands.^22,4 Herein, two new series of analogues were synthesized and evaluated for binding at hD3R and hD2R expressed in HEK 293 cells to 1) optimize D3R affinity and selectivity and to 2) further characterize the role of the linking chain in this class of compounds. Based on the success of previously reported 3-OH analogues (e.g. compound 2 in Fig. 1) and SAR that showed sterically bulky substituents in this position were not well tolerated at D3R,⁶ we replaced the 3-OH with a 3-F group. In addition, as Micheli and colleagues^12,13 reported that the amide function in their D3R antagonists, based on SB277011A (3), could be replaced with bioisosteric groups and had beneficial effects in vivo (e.g. 4), we compared analogues with and without the linking chain carbonyl group and further extended N-methyl and N-n-propyl substituents to potentially optimize for D3R affinity.

Figure 1.

Chemical Structures of Representative D3R-selective Ligands

Chemistry

Scheme 1 outlines the synthetic strategy used for the 3-F-substituted arylpiperazine butyl carboxamide derivatives. Compounds 5a and 5b were synthesized by employing an epoxide opening of 2-(2-(oxiran-2-yl)ethyl)isoindoline-1,3-dione with the corresponding arylpiperazines, under reflux conditions.⁶ These resulting alcohols were treated with N,N-diethylaminosulfur trifluoride (DAST) to give intermediates 6a and 6b, which were subjected to phthalimide deprotection using hydrazine to give the primary amine intermediates 7a and 7b. The desired 3-F-substituted carboxamides were synthesized by coupling these intermediates with the corresponding carboxylic acids or carboxylic acid chlorides.

Scheme 1. Synthesis of 3-F-amide Analogues^a.

^a Reagents and conditions: (a) DAST, CH₂Cl₂, −78 °C to RT, 12 h; (b) hydrazine, EtOH, reflux, 3 h; (c) CDI, THF (Method A); (d) SOCl₂ (Method B).

In Scheme 2, compounds 10a–b were prepared by deprotecting the corresponding phthalimide protected amines 9a–b and subjecting them to propionyl chloride in the presence of triethylamine, as the base, to give amides 11a–b. These amides were reduced to the corresponding amines 12a–b using LiAlH₄ and then treated with the appropriate aldehyde and subjected to a one-pot imine reduction with Na(OAc)₃BH, resulting in the desired n-propyl substituted tertiary amine compounds 13a–f. Compounds 14a–b were synthesized by stepwise imine reduction of 9H-fluorene-2-carbaldehyde with the corresponding amine. These secondary amines were converted into methyl substituted tertiary amines 15a–b by treating with formaldehyde and Na(OAc)₃BH. N-n-Propyl substituted tertiary amines were purified by preparative chromatography; all the remaining compounds were purified by flash column chromatography or recrystallization from 2-propanol. Reaction conditions and yields were not optimized, and spectroscopic data refer to the free base.

Scheme 2. Synthesis of Amine-linked Analogues^a.

^aReagents and conditions: (a) hydrazine, EtOH, reflux, 3 h; (b) propionylchloride, CH₂Cl₂, TEA, 0 °C to RT, 4 h; (c) LAH, THF, reflux, 4 h (d) aldehyde, Na(OAc)₃BH, DCE, RT, 24 h (Method C); (e) 9H-fluorene-2-carbaldehyde, MeOH, RT, 12h; (f) NaBH₄, 30 min; (g) HCHO, Na(OAc)₃BH, DCE, 12 h.

Pharmacological Results and Discussion

All compounds were evaluated in competition binding assays using stably transfected HEK 293 cells expressing either human D2_L, D3, or D4 dopamine receptors.⁵ The radioligand was the high-affinity D2-like receptor antagonist 2,3-dimethoxy-5-(¹²⁵I)-iodo-N-(9-benzyl-9-azabicyclo(3.3.1)nonan-3-yl)benzamide ([¹²⁵I]IABN).²³ In Table 1, binding data for the N-(3-fluoro-4-(4-(2-methoxy or 2,3-dichlorophenyl)piperazine-1-yl)-butyl)-aryl carboxamides are shown. None of the compounds tested showed high binding affinities for D4 receptors. In contrast, all compounds shown in Table 1 exhibited high binding affinities for D3R (K_i range = 1.5–28.6 nM) and selectivity over D2R. Notably, compounds 8d and 8j showed nanomolar binding affinities for D3R (K_i = 6.1 and 2.6 nM, respectively) and >1000-fold selectivity over D2R. Among the 2-OCH₃- series 8k and 8m showed >60-fold D3R-selectivity over D2R. In the 2,3-diCl-series 8h, 8l and 8n showed >130-fold D3R-selectivity over D2R. Indeed, in this series, all the 2,3-diClphenylpiperazine analogues exhibited greater D3R-selectivity compared to the corresponding 2-OCH₃-analogues, although their cLogP values of >5 may be a limitation for in vivo studies. There was no consistent effect on D2R or D3R binding affinities that was dictated by the nature of the terminal aryl amide.

Table 1.

Human D2R-Family Receptor Subtype Binding Data on N-(3-Fluoro-4-(4-(2-methoxy or 2,3-dichlorophenyl) piperazine-1-yl)-butyl)-aryl carboxamides^a

compd	R	Ar	clogPb	D2R	D3R	D4R	D2/D3	D4/D3
Ki±S.E.M, nM
8a	2-OCH3		3.9	390±42	20.5±4.3	ND	19	-
8b	2,3-diCl		5.5	378±42	6.20±1.5	3090±650	61	498
8c	2-OCH3		3.7	329±36	28.6±6.0	ND	11	-
8d	2,3-diCl		5.3	14600±5070 (9160±3730)	6.10±0.8 (5.40±0.6)	5340±2290	2400 (1700)c	875
8e	2-OCH3		4.0	274±13	13.9±1.1	ND	20	-
8f	2,3-diCl		4.9	279±63	8.00±1.4	ND	35	-
8g	2-OCH3		3.7	421±33	9.50±1.9	1490±39.6	44	157
8h	2,3-diCl		5.4	526±51	4.00±0.9	3360±484	131	841
8i	2-OCH3		5.1	2890±1040	10.1±1.9	3180±922	286	315
8j	2,3-diCl		6.1	4260±1990	2.60±0.7	ND	1640	-
8k	2-OCH3		3.7	241±38	3.80±0.8	ND	63	-
8l	2,3-diCl		5.4	472±76	2.90±0.4	ND	163	-
8m	2-OCH3		3.3	302±32	4.60±0.4	ND	66	-
8n	2,3-diCl		4.8	230±16	1.50±0.3	ND	153	-
8o	2,3-diCl		5.3	195±25	6.10±0.9	ND	32	-
1d	-	-	5.3	93.3±12.0	0.7±0.1	375±18	133	540
(±)2e	-	-	4.2	502±51.5	1.39±0.19	4900±1034	358	5300

^aBinding inhibition values determined using HEK 293 cells transfected with hD2_LR or hD3R and¹²⁵I-IABN radioligand as described.^5,6

^bPartition coefficients (clogP) were calculated using ChemDraw Ultra, Version 11.0, CambridgeSoft 2007;

^cD2/D3 ratio using K_i values from chimera study in Table 4;

^ddata previously reported in ref. 5;

^edata previously reported in ref. 11; ND = Not determined.

To determine the importance of the carboxamide moiety on D3R affinity and selectivity, we replaced the amide group linking the extended aryl ring system to the butyl-4-phenylpiperazine with a methylenamine, while preserving the linker length. Further, as the N-n-propyl substitution has demonstrated importance in D3R affinity and selectivity in previously reported and structurally similar compounds,²⁴ we compared this substituent effect to the unsubstituted and N-methyl substituted amines. These results are shown in Table 2. Reference amide-containing and D3R selective compounds 16–20 are included in Table 2 for comparison.

Table 2.

In vitro Binding of Amine-linked Analogs at D2R and D3R^a

compound	temp	R	Ar	R′	R″	D2R Ki ± S.E.M., nM	D3R Ki ± S.E.M., nM	D2/D3
13a	B	2-OCH3		H	n-propyl	69.9±7.3	67.9±2.4	1
13b	B	2,3-diCl		H	n-propyl	162.0±1.2	101±27	2
13c	B	2-OCH3		H	n-propyl	196±30	71.5±2.0	3
13d	B	2,3-diCl		H	n-propyl	269±24	129.±30	2
13e	B	2-OCH3		H	n-propyl	119 ± 11	80.8±32	1.5
13f	B	2,3-diCl		H	n-propyl	313 ± 8.4	104±21	3
14a	B	2,3-diCl		OH	H	2090 ± 370	134±23	16
14b	B	2,3-diCl		F	H	5270±1900	467±60	11
15a	B	2,3-diCl		OH	Me	869±140	162±32	5
15b	B	2,3-diCl		F	Me	2060±320	393±38	5
16e	A	2-OCH3		H	-	33.6±5.9	0.30±0.06	112
17b,f	A	2,3-diCl		H	-	112 ± 22	2.00±0.4	56
18c,g	A	2-OCH3		H	-	55.4±5.3	0.30±0.1	185
19b,h	A	2,3-diCl		H	-	64.7±8.9	0.80±0.2	81
20b,i	A	2,3-diCl		H	-	44.8±11	0.80±0.3	56

^aBinding inhibition values determined using HEK 293 cells transfected with hD2_LR or hD3R dopamine receptors and¹²⁵I-IABN radioligand as described.^5,6

^bdata previously reported in ref. 5;

^cdata previously reported in ref. 6.

^eBP 897 (N-(4-(4-(2-methoxyphenyl)piperazin-1-yl)butyl)-2-naphthamide);

^fNGB 2904 (N-(4-(4-(2,3-dichlorophenyl)piperazin-1-yl)butyl)-9H-fluorene-2-carboxamide);

^gPG 576 (N-(4-(4-(2-methoxyphenyl)piperazin-1-yl)butyl)-9H-fluorene-2-carboxamide);

^hPG01032 (Benzo[b]thiophene-2-carboxylic acid {4-[4-(2,3-dichloro-phenyl)-piperazin-1-yl]-butyl}-amide);

ⁱPG01059 (N-(4-(4-(2,3-dichlorophenyl)piperazin-1-yl)butyl)benzofuran-2-carboxamide).

In general, when the amide was replaced by a methyleneamine, D2R binding was either unaffected or slightly changed, whereas D3R binding affinity was substantially decreased. For example, when the amide in 19 was replaced by an N-n-propyl amine linker (13b), D2R binding affinity was reduced by 2.5 fold, whereas D3R binding affinity was decreased by 126-fold. This resulted in decreasing D3R-selectivity from 81-fold to 2-fold. Another example is with compound 8j compared to 15b, wherein D3R binding affinity was reduced from 2.6 nM to 393 nM (150-fold), and D2R binding affinity was increased by a modest ~2-fold. Thus in this set of compounds, D3R-selectivity was reduced from 1640- to 5-fold by eliminating the carboxamide function and replacing it with a tertiary methyleneamine. This trend was apparent throughout the series. These results suggest that the amide group is necessary in this series of analogues for high affinity binding to D3R and provides a point of separation between the D2R and D3R pharmacophores. This SAR may appear to contrast with the recently described Glaxo D3R-selective antagonists wherein the amide group was not necessary for D3R binding.¹³ However, in the Glaxo compounds, an isosteric replacement of the amide group is present, in contrast to the amine analogues reported herein. The N-n-propyl substituted amines did not restore D3R binding affinity in this set of analogues.

In Table 3, additional in vitro binding data for selected compounds is shown. With few exceptions, the F-analogues did not bind appreciably to these off-target sites or dopamine D1 receptors (data not shown). Moreover, the only compounds that showed moderate binding affinities at 5HT1A (e.g. Kⁱ<100 nM, 8a, 8c, 8e) were less D3R selective over D2R, and therefore would not be candidates for further in vivo investigation. In contrast, the most D3/D2-selective compounds also showed selectivity over D1, 5HT1A and 5HT2A receptors (e.g. 8d, 8i and 8j).

Table 3.

Additional In Vitro Binding and Functional Data for Selected Compounds.

compd	D3 mitogenesis IC50±S.E.M., nMa	D2 mitogenesis IC50±S.E.M., nMa	D3 beta arrestin IC50 ± S.E.M., nM	5-HT1A [3H]-8-OH-DPAT Ki ± S.E.M., nMa	5HT1A [35S]GTPγS EC50 ± S.E.M., nM (% agonist)a	5-HT2A [125I]DOI Ki ± S.E.M., nMa
8a	390±130	NDe	699±18	49.0±13	71.6±1.3 (73)	2060±460
8b	256±79	NDe	IAf	159±37	1080±380 (123)	98±34
8c	440±130	NDe	NDe	37.9±9.4	134±44 (60)	4800±1300
8d	430±120	NDe	IAf	1150±340	NDe	>8800
8e	780±170	NDe	1240±220	73.0±17	600±170 (76)	3000±1100
8f	1430±480	1600±300	IAf	501±65	NDe	274±56
8g	820±300	NDe	IAf	101±19	1430±670 (83)	3240±840
8h	520±170	NDe	1220±310	223±60	2140±510 (88)	212±77
8i	214±47	NDe	IAf	299±30	NDe	>10,000
8j	800±310	NDe	IAf	6000±1400	ND	4800±1200
8k	83±30	421±47	3210±140	139±33	440±150 (57%)	3030±400
8l	17.0±5.2	NDe	3770±2900	2140±640	NDe	3290±480
8m	1140±310	NDe	2990±100	1170±190	NDe	3540±910
8n	320±100; 4.9±1.3 (14.9)b	NDe	IAf	IAf	NDe	272±79
8o	255±43	NDe	IAf	IAf	NDe	1910±350
15b	918±61	NDe	NDe	IAf	NDe	2580±400
(±)2	29.9±5.76c	4370±59.4 171.2±60.5 (22%)b,c	495 (n=2)	104±23.5c	NDe	ND
sulpiride	66.22±10.89	10.28±1.23	560±170	NDe	NDe	NDe

^aData were obtained through the NIDA Addiction Treatment Discovery Program contract with Oregon Health & Science University (Y1 DA 5007-05).

^bThe first value is the antagonist IC50, the second value is the agonist EC50 with % stimulation in parentheses.

^cdata previously reported in ref. 11.

^ddata previously reported in ref. 6.

^eND = Not determined;

^fIA= <50% inhibition at a concentration of 10 μM.

In addition to binding data at D2R and D3R, selected analogues were evaluated in the quinpirole-induced mitogenesis assay in hD2- or hD3-transfected CHOp cells and in a dopamine-induced beta-arrestin translocation assay in hD3-transfected CHO-K1 cells. These data are shown in Table 3 and compared to the D3R-selective antagonist (±)2 and the D2R-family nonselective antagonist sulpiride. Most of the analogues were antagonists in both the D3R mitogenesis and beta arrestin translocation assays although 8n was a more potent partial agonist than an antagonist in the mitogenesis assay. In general, functional potencies of these analogues were lower than their binding affinities and the compounds were less potent in the beta arrestin assay than in the mitogenesis assay resulting in some compounds showing <50% inhibition at a concentration of 10 μM. Unfortunately, due to solubility limitations, higher concentrations could not be tested. Although we cannot fully explain the disparity in absolute potencies across in vitro assays at this time, these functional assays were not run under steady state conditions as were the binding assays and were not optimized for this class of compounds. Further investigation into this is underway, as a consistent relationship between cell-based D3R functional assay potencies and behavior in vivo has yet to be established. ^{4 for review}

Compound 8d exhibited the greatest D3R versus D2R binding selectivity (2400-fold) of all the 4-phenylpiperazines that we have evaluated to date. Representative competitive radioligand binding curves for the inhibition of the binding of ¹²⁵I-IABN to human D2L and D3 receptors by compound 8d are shown in Figure 2. To begin to define the molecular basis for this receptor subtype binding selectivity, we performed a series of radioligand binding studies using D3/D2 chimeric receptor proteins to identify what binding domains in the D3R as compared to the D2R were interacting with compound 8d to achieve this magnitude of selectivity. Table 4 shows a schematic representation of the wild type human D2R (all black) and D3R (all grey) compared to the chimeric receptors. Table 4 also shows the affinity of the wild type and chimeric receptors for ¹²⁵I-IABN and 8d. IABN binds non-selectively at D2R (K_d = 0.03 nM) and D3R (K_d = 0.4 nM) and it binds with similar affinity at each of the chimeric proteins (K_i values range from 0.02 to 0.08 nM).

Figure 2.

Representative Radioligand Binding Competition Curve for the Binding of Compound 8d to human D2R and D3R

Table 4.

Binding Affinities at hD2R, hD3R and Chimeric Receptors for Compound 8d

		IABN	Compound 8d
	D2 Wild type	0.03 ± 0.004	9,163 ± 3731
	Chimera A	0.05 ± 0.009	2,369 ± 945
	Chimera B	0.02 ± 0.004	13.9 ± 1.8
	Chimera C	0.02 ± 0.006	20.8 ± 3.0
	Chimera D	0.01 ± 0.003	11.5 ± 2.1
	Chimera E	0.03 ± 0.001	1.7 ± 0.1
	Chimera F	0.03 ± 0.011	2.9 ± 0.3
	D2/D3 E2 loop	0.08 ± 0.007	245 ± 37
	D3/D2 E2 loop	0.03 ± 0.004	31.5 ± 3.0
	D3 Wild type	0.04 ± 0.003	5.4 ± 0.6

All of the dissociation constants are expressed as nM and are the mean± S.E.M. For IABN Kd values were obtained by Scatchard analysis of direct binding studies and values for the other compounds are Ki values obtained from competitive radioligand binding analysis. The number of independent experiments performed to obtain the mean values is n ≥ 3 for the chimeric receptors. For the binding of ¹²⁵I-IABN to D2R wild type n = 10 and for the D3R wild type n = 13. For the binding of 8d to D2R wild type n = 7 and for the D3R wild type n = 5.

A comparison of binding between the D2R with chimera A for 8d indicated that substitution of the first transmembrane spanning (TMS I) region from the D3R onto the D2R backbone resulted in a 4-fold increase in affinity. Previous studies have suggested that the extracellular portion of the TMS I region does not have H₂O accessible residues and is, therefore, unlikely to make direct contact with bound ligand.²⁵ Differences in amino acid sequence between the TMS I regions of the D2R and the D3R might contribute to topographical differences between the two binding sites. However, the observed low affinity of 8d at D2R (Figure 1) makes it problematic to determine the reliability of this difference in Ki values.

A more dramatic difference in the affinity (>400-fold) of 8d was observed between wild type D2R and chimeras B, C and D (Table 4). The affinities of 8d at these three chimeric receptors are similar. These results implicate TMS II and TMS III and the first extracellular (E1) loop as important structural elements in the binding selectivity of 8d at D2R and D3R. An additional 5- to 10-fold increase in binding selectivity was found for chimeras E and F compared to the wild type D2R suggesting that difference in the structure of TMS IV and V between D2R and D3R also contribute to the observed binding selectivity of 8d. It is interesting to note that the affinity of 8d for Chimera E was actually 3-fold higher than at the D3R wild type, emphasizing the importance of these regions to D3R binding affinities for the 4-phenylpiperazines.

Studies on the three-dimensional structure of bovine rhodopsin,²⁶ and more recently on the adrenergic receptors,^27,28 have indicated that the conserved disulfide bond, which joins the conserved cysteine residue located within the extracellular E2 loop with the top of the third (TMS III) region, brings the E2 loop in close proximity to the extracellular portion of the helical TMS regions of the G-protein coupled receptors. In addition, several studies have suggested that amino acid residues within the E2 loop of the dopamine receptors can interact with ligands positioned in the neurotransmitter (orthosteric) binding site, thereby influencing binding affinity.^{29, 30} Recently, this region of D3R was also implicated in the binding of a novel allosteric antagonist SB269,652 (N-((1R,4R)-4-(2-(7-cyano-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)cyclohexyl)-1H-indole-2-carboxamide), which bears structural similarity to compounds reported previously and herein.³¹

To further investigate the possible contribution of the D3R E2 loop in the binding selectivity of 8d, two additional chimeric receptors were constructed (D2/D3 E2 loop and D3/D2 E2 loop) and evaluated for affinity for 8d. The D2/D3 E2 loop chimera contains the wild type D2R structure but the E2 loop is identical to the amino acid sequence found in the D3R subtype. D3/D2 E2 loop chimera is the reciprocal mutant receptor. A 37-fold increase in affinity was observed by substituting the E2 loop of the D3R on the D2R scaffold and a >5-fold decrease in affinity was observed when the E2 loop of the D2 receptor was substituted for the E2 loop of the D3R on the D3R scaffold. The results of these binding studies suggest that 8d interacts with the E2 loop and that interaction contributes to the D3R binding selectivity. We hypothesize that the 3-F substitution on the four carbon linking chain of 8d is the moiety in contact with the E2 loop. These data further support SAR described in our previous study with the 3-OH-substituted phenylpiperazines.¹¹ However, the dramatic improvement in binding observed in chimera B, as compared to A, suggests that the E1 loop may also be involved in the differential binding of these compounds to D3R compared to D2R. Of note, while this manuscript was in preparation, the crystal structure of the human D3R binding in complex with the D2-like receptor antagonist eticlopride was published.³² In addition to characterizing the eticlopride binding domain, a second binding pocket was defined with compound 2 (R-22 = R-PG 648), wherein the 2,3-diCl-phenylpiperazine overlapped with eticlopride in the orthosteric site, and the 3-OH-butylamide-linked indole portion of the molecule occupied a second domain that was differentially engaged by nonconserved residues in D3R and D2R.³² These studies provided further evidence of differential binding domains that exist for D2R and D3R subtypes and highlight the importance of both the E1 and E2 loops that interface with TMS I, II and VII. Hence, additional chimera and single point mutation studies, along with extensive molecular dynamics studies are underway to further characterize the binding domains of the D3R and D2R subtypes and to further define these interactions of the D3R-selective ligands at the molecular level.

In summary, earlier SAR studies with prototypic 2,3-dichloro or 2-methoxy-4-phenylpiperazine analogues included varying substituents on the butyl amide linker that exhibited high affinity and D3R selectivity. Furthermore, enantioselectivity was achieved with the 3-OH-substituted analogue 2, with enantioselectivity being more pronounced (15-fold) at D3R than D2R (<2-fold).¹¹ In the present study, a series of 3-F-carboxamides also showed high D3R affinity and selectivity, including two of the most D3R selective compounds (8d and 8j) reported to date. Chimera studies confirmed the pivotal contribution of the E2 loop in the D3R binding selectivity of 8d suggesting that the 3-F, like the 3-OH group in compound 2, possesses physicochemical properties that are well tolerated in this region of D3R and likely influences how the rest of the molecule resides in the binding site. Further, the contribution of the E1 extracellular loop was also revealed and future chimera studies that explicitly test this are underway. In addition, the arylamide clearly extends beyond the orthosteric site that is indistinguishable in D2R and D3R, to a binding domain that only tolerates the D3R pharmacophore exemplified by compounds such as those reported herein and shown recently.³² Finally, the carbonyl of the carboxamide moiety in these molecules is essential for high affinity binding at D3R, but not D2R, further separating SAR between these homologous proteins. Structure-based protein modeling based on the D3R crystal structure will undoubtedly identify critical amino acid residues that are different in these regions between D3R and D2R and will provide further rationale for the design of novel D3R-selective agents.

Experimental Methods

¹H and ¹³C NMR spectra were acquired using a Varian Mercury Plus 400 spectrometer. Chemical shifts are reported and referenced according to deuterated solvent for ¹H spectra (CDCl₃, 7.26), ¹³C spectra (CDCl₃, 77.2), ¹⁹F spectra (CFCl₃, 0). Combustion analysis was performed by Atlantic Microlab, Inc. (Norcross, GA) and agrees within 0.4% of calculated values. Melting point determinations were conducted using a Thomas-Hoover melting point apparatus and are uncorrected. Anhydrous solvents were purchased from Aldrich were used without further purification, except for tetrahydrofuran, which was freshly distilled from sodium-benzophenone ketyl. All other chemicals and reagents were purchased from Aldrich Chemical Co., Combi-Blocks, TCI, America., Matrix Scientific; Lancaster Synthesis, Inc. (Alfa Aesar) and AK Scientific, Inc. Final compounds (free base) were purified by column chromatography (EMD Chemicals, Inc.; 230–400 mess, 60 Å) or preparative thin layer chromatography (silica gel, Analtech, 1000 μm). The final products were converted into oxalate salts, typically by treating the free base with 1:1 molar ratio of oxalic acid in acetone followed by precipitation from a combination of organic solvents. Yields and reaction conditions are not optimized. Generally, yields and spectroscopic data refer to the free base. On the basis of NMR, GC-MS (where obtainable), and combustion analysis data, all final compounds are >95% pure. The procedures to determine the binding affinities at the human D2-like receptors have been described.

Synthesis. 2-(3-fluoro-4-(4-(2,3-dichlorophenyl)piperazin-1-yl)butyl)isoindoline-1,3-dione (6a)

To a solution of compound 5a (1.8 g, 4.4 mmol) in dry CH₂Cl₂ (50 mL) cooled at −78 °C was added a solution of DAST (1.06 g, 6.6 mmol) in dry CH₂Cl₂ (25 mL) over a period of 30 min. This reaction mixture was continuously stirred for 12 h at room temperature. The reaction mixture was diluted with CH₂Cl₂ and washed with ice-H₂O and 5% NaHCO₃ solution, and the organic layer was dried over anhydrous Na₂SO_4. The solvent was removed under vacuum to afford the crude product. This was further purified by column chromatography (30% EtOAc-hexane) afford the compound 6a (1.09 g) in 60% yield. Mp 132–134 °C; ¹H NMR (400 MHz, CDCl₃) δ ppm 1.94–2.136 (m, 2H), 2.53–2.80 (m, 7H), 3.05 (brs, 4H), 3.86–3.91 (m, 1H), 6.94 (dd, J = 6.4, 2.8 Hz, 1H), 7.13–7.15 (m, 2H), 7.72 (dd, J = 5.6, 3.2 Hz, 2H), 7.86 (dd, J = 5.6, 3.2 Hz); ¹³C NMR (100 MHz, CDCl₃) δ 168.451, 151.43, 134.24, 132.31, 127.73, 127.67, 124.81, 123.52, 118.82, 90.76 (d, J = 170 Hz, 1C), 62.17 (d, J = 20.6 Hz, 1C), 54.10, 51.44, 34.72 (d, J = 4.6 Hz, 1C), 32.46 (d, J = 20.6 Hz, 1C); ¹⁹F NMR (376 MHz, CDCl₃/CFCl₃) δ −183.0 to −183.39 (m, 1F).

2-(3-Fluoro-4-(4-(2,3-dichlorophenyl)piperazin-1-yl)butyl)isoindoline-1,3-dione (6b)

This compound was prepared by the method described for compound 6a, employing 5b (1.6 g, 5 mmol) and DAST (870 mg, 5.4 mmol), afford 6b (940 mg) in 58% yield. Mp 97–99 °C; ¹H NMR (400 MHz, CDCl₃) δ ppm 1.95–2.13 (m, 2H), 2.50–2.78 (m, 7H), 3.07 (brs, 4H), 3.85 (m, 3H), 3.86–3.90 (m, 1H), 4.70–4.88 (m, 1H), 6.83–7.01 (m, 4H), 7.71 (dd, J = 5.6, 3.2 Hz), 7.85 (dd, J = 5.6, 2.8 Hz); ¹³C NMR (100 MHz, CDCl₃) δ 168.40, 152.37, 141.38, 134.11, 132.23, 123.41, 123.03, 121.08, 118.31, 111.23, 90.68 (d, J = 170 Hz, 1C), 62.27 (d, J = 20.6 Hz, 1C), 55.45, 54.16, 50.69, 34.64 (d, J = 5.3 Hz, 1C), 32.41 (d, J = 20.6 Hz, 1C); ¹⁹F NMR (376 MHz, CDCl₃/CFCl₃) δ − 182.85 to −183.30 (m, 1F).

3-Fluoro-4-(4-(2-methoxyphenyl)piperazin-1-yl)butan-1-amine (7a)

To a solution of 6a (1.14 g, 2.8 mmol) in EtOH was added hydrazine (270 mg, 8.4 mmol) and stirred at reflux for 3 h. The cooled reaction mixture was evaporated in vacuo, the solid residue was partitioned between CHCl₃ and 20% K₂CO₃ solution, and the organic layer was collected and dried over Na₂SO₄. The solvent was removed under vaccum to afford the 7a, which was used for next step without further purification. Yield: 95% (748 mg). ¹H NMR (400 MHz, CDCl₃) δ ppm 1.62–1.86 (m, 2H), 2.48–2.95 (m, 5H), 3.11 (brs, 4H), 3.86 (s, 3H), 4.76–4.96 (m, 1H), 6.84–7.02 (m, 4H); ¹³C NMR (100 MHz, CDCl₃) δ 152.48, 141.51, 123.15, 121.20, 118.42, 11.36, 91.06 (d, J = 168.4 Hz, 1C), 62.9 (d, J = 21.4 Hz, 1C), 55.57, 54.26, 50.81, 38.51 (d, J = 4.6 Hz, 1C), 37.63 (d, J = 19.8 Hz, 1C); ¹⁹F NMR (376 MHz, CDCl₃/CFCl₃) δ −182.5 to −182.9 (m, 1F); GC/MS R_T(RIMS): 14.1 min (281 [M]⁺ m/z).

4-(4-(2,3-dichlorophenyl)piperazin-1-yl)-3-fluorobutan-1-amine (7b)

This compound was prepared by the method described for compound 7a, employing 6b (700 mg, 1.56 mmol) and hydrazine (164 mg, 4.7 mmol), afford 7b (480 mg) in 96% yield. ¹H NMR (400 MHz, CDCl₃) δ ppm 1.62–1.91 (m, 2H), 1.49–1.94 (m, 7H), 3.07 (brs, 4H), 4.75–4.94 (m, 1H), 6.95 (dd, J = 6.4, 2.8 Hz, 1H), 7.13–7.15 (m, 2H), 7.72 (dd, J = 5.6, 3.2 Hz, 2H), 7.11–7.16 (m, 2H); ¹³C NMR (100 MHz, CDCl₃) δ 151.36, 134.14, 127.64, 127.58, 124.71, 118.72, 90.9 (d, J = 168.5 Hz, 1C), 62.64 (d, J = 20.6 Hz, 1C), 53.99, 53.97, 51.38, 38.38 (d, J = 4.6 Hz, 1C), 37.45 (d, J = 19.8 Hz, 1C); ¹⁹F NMR (376 MHz, CDCl₃/CFCl₃) δ −182.61 to −183.01 (m, 1F); GC/MS R_T(RIMS): 15.5 min (299 [M-HF]⁺ m/z).

General Amidation Procedures

Procedure A

CDI (1 equiv) was added to a solution of the carboxylic acid (1 equiv) in THF (10 mL/mmol). The reaction mixture was stirred at room temperature for 2 h. The solution was cooled to 0 °C and the appropriate amine (1 equiv) was added dropwise. The reaction mixture was allowed to warm up to room temperature and stirred for 2–3 h. The solvent was removed in vacuo. The residue was diluted in CHCl₃ (30 mL) and washed with saturated aq NaHCO₃ solution (2 × 10 mL). The organic layer was dried with Na₂SO₄ and concentrated in vacuo. The crude product was purified by crystallization from 2-PrOH or flash column chromatography, as indicated.

Procedure B

Thionyl chloride (SOCl₂; 2 mL/mmol) was added to the carboxylic acid (1 equiv). The solution was stirred at reflux for 3h and concentrated in vacuo. Residual SOCl₂ was removed by azeotropic distillation in dry benzene. The resulting solid was dissolved in amylene stabilized CHCl₃ (5 mL). To a stirred solution of the amine (1 equiv) in amylene stabilized CHCl₃ (20 mL) and 0.5 M aq NaOH (8 mL) cooled to 0 °C, was added the acid chloride solution drop wise. The solution was stirred vigorously for 3 h at room temperature. The organic layer was separated, dried with Na₂SO₄ and concentrated in vacuo. The crude product was purified by crystallization from 2-PrOH or flash column chromatography, as indicated.

N-(3-fluoro-4-(4-(2-methoxyphenyl)piperazin-1-yl)butyl)-6-phenylnicotinamide (8a)

CDI (908 mg, 5.6 mmol) was added to a solution of 4-(pyridine-2-yl)benzoic acid hydrochloride (658 mg, 2.8 mmol) in pyridine (10 mL). The reaction mixture was stirred at room temperature for 2 h. The solution was cooled to 0 °C and the amine 7a (787 mg, 2.8 mmol) in pyridine (5 mL) was added dropwise. The reaction was allowed to warm to room temperature and stirred overnight. The resulting solution was evaporated, diluted in CHCl₃ (30 mL) and washed with saturated aq. NaHCO₃ (2 × 10 mL). The organic layer was dried over Na₂SO₄ and evaporated under reduced pressure. The crude product was purified by column chromatography using EtOAc/CHCl₃/MeOH (5:5:1) and 1% triethylamine afford 8a (470 mg) in 36% yield. The oxalate salt was precipitated from 2-PrOH. Mp 170–172 °C; ¹H NMR (400 MHz, CDCl₃) δ ppm 1.95–2.17 (m, 2H), 2.56–2.83 (m, 6H), 3.09 (brs, 4H), 3.59–3.76 (m, 2H), 3.86 (s, 3H), 4.82–5.0 (m, 1H), 6.7 (brs, 1H), 6.84–7.02 (m, 4H), 7.27–7.30 (m, 1H), 7.77–7.79 (m, 2H), 7.86–7.89 (m, 2H), 8.05–8.09 (m, 2H), 8.71–8.73 (m, 1H); ¹³C NMR (100 MHz, CDCl₃) δ 167.48, 156.45, 152.47, 150.01, 142.45, 141.38, 137.13, 134.91, 127.63, 127.27, 123.21, 122.98, 121.20, 121.08, 118.43, 111.35, 92.0 (d, J = 167.7 Hz, 1C), 62.37 (d, J = 21.3 Hz, 1C), 55.58, 54.30, 50.74, 37.05, 33.17(d, J = 19.8 Hz, 1C); ¹⁹F NMR (376 MHz, CDCl₃/CFCl₃) δ −181.38 to −181.82 (m, 1F); Anal. (C₂₇H₃₁FN₄O₂· C₂H₂O₄·1/2H₂O) C, H, N.

N-(4-(4-(2,3-Dichlorophenyl)piperazin-1-yl)-3-fluorobutyl)-6-phenylnicotinamide (8b)

This compound was prepared by the method described for compound 8a, employing 4-(pyridine-2-yl)benzoic acid hydrochloride (367 mg, 1.6 mmol), 7b (480 mg, 1.5 mmol), and CDI (243 mg, 1.5 mmol), afford 8b (406 mg) in 54% yield. Mp 163–165 °C; ¹H NMR (400 MHz, CDCl₃) δ ppm 1.95–2.17 (m, 2H), 2.56–2.84 (m, 6H), 3.07 (brs, 4H), 3.60–3.76 (m, 2H), 4.81–5.0 (m, 1H), 6.7 (brs, 1H), 6.93 (dd, J = 7.6, 2.0 Hz, 1H), 7.12–7.17 (m, 2H), 7.26–7.30 (m, 1H), 7.75–7.81 (m, 2H), 7.86–7.89 (m, 2H), 8.06–8.09 (m, 2H), 8.71–8.73 (m, 2H); ¹³C NMR (100 MHz, CDCl₃) δ 167.48, 156.41, 151.34, 150.09, 142.47, 137.15, 134.88, 134.24, 127.73, 127.68, 127.61, 127.28, 124.86, 122.99, 121.07, 118.82, 91.95 (d, J = 167.7 Hz, 1C), 62.24 (d, J = 21.4 Hz, 1C), 54.13, 51.41, 37.04 (d, J = 3.8 Hz, 1C), 33.18 (d, J = 19.9 Hz, 1C); ¹⁹F NMR (376 MHz, CDCl₃/CFCl₃) δ −181.6 to −182.06 (m, 1F); Anal. (C₂₆H₂₇Cl₂FN₄O) C, H, N.

N-(3-Fluoro-4-(4-(2-methoxyphenyl)piperazin-1-yl)butyl)benzofuran-2-carboxamide (8c)

Prepared from benzofuran-2-carboxylic acid and 7a according to general procedure B. The crude compound was purified by column chromatography using EtOAc/CHCl₃/MeOH (20:20:1) and 1% triethylamine to afford 8c in 73% yield. The oxalate salt was precipitated from EtOAc. Mp 168–170 °C; ¹H NMR (400 MHz, CDCl₃) δ ppm 1.99–2.12 (m, 2H), 2.56–2.83 (m, 6H), 3.12 (brs, 4H), 3.63–3.71 (m, 2H), 3.86 (s, 3H), 4.82–5.0 (m, 1H), 6.85–7.06 (m, 5H), 7.27–7.31 (m, 1H), 7.38–7.5 (m, 3H), 7.67 (d, J = 8.0 Hz, 1H); ¹³C NMR (100 MHz, CDCl₃) δ 159.26, 154.94, 152.48, 148.92, 141.39, 127.84, 127.11, 123.93, 123.24, 122.96, 121.20, 118.44, 111.98, 111.36, 110.63, 91.47 (d, J = 183.1 Hz, 1C), 62.35 (d, J = 21.3 Hz, 1C), 55.58, 54.30, 50.76, 36.12 (d, J = 4.6 Hz, 1C), 33.29 (d, J = 20.5 Hz, 1C); ¹⁹F NMR (376 MHz, CDCl₃/CFCl₃) δ −182.11 to −182.62 (m, 1F); Anal. (C₂₄H₂₈FN₃O₃· C₂H₂O₄· 1/2H₂O) C, H, N.

N-(4-(4-(2,3-Dichlorophenyl)piperazin-1-yl)-3-fluorobutyl)benzofuran-2- carboxamide (8d)

Prepared from benzofuran-2-carboxylic acid and 7b according to general procedure B. The crude compound was purified by column chromatography using EtOAc/CHCl₃/MeOH (20:20:1) and 1% triethylamine afford 8d in 82% yield. Mp 164–166 °C; ¹H NMR (400 MHz, CDCl₃) δ ppm 1.98–2.14 (m, 2H), 2.57–2.84 (m, 6H), 3.09 (brs, 4H), 3.63–3.73 (m, 2H), 4.81–5.0 (m, 1H), 6.94 (dd, J = 6.8, 2.4 Hz, 1H), 7.0 (brs, 1H), 7.11–7.18 (m, 2H), 7.28–7.50 (m, 3H), 7.66–7.69 (m, 1H); ¹³C NMR (100 MHz, CDCl₃) δ 159.26, 154.94, 151.38, 148.89, 134.27, 127.83, 127.75, 127.69, 127.15, 124.87, 123.96, 122.99, 118.82, 111.95, 110.67, 91.47 (d, J = 168.4 Hz, 1C), 62.23 (d, J = 20.6 Hz, 1C), 54.15, 51.46, 36.14 (d, J = 4.5 Hz, 1C), 33.29 (d, J = 20.5 Hz, 1C); ¹⁹F NMR (376 MHz, CDCl₃/CFCl₃) δ −182.36 to −182.75 (m, 1F); Anal. (C₂₃H₂₄Cl₂FN₃O₂· 1/5H₂O) C, H, N.

N-(3-Fluoro-4-(4-(2-methoxyphenyl)piperazin-1-yl)butyl)-5-methoxybenzofuran-2-carboxamide (8e)

Prepared from 5-methoxybenzofuran-2-carboxylic acid and 7a according to general procedure B. The crude compound was purified by column chromatography using EtOAc/CHCl₃/MeOH (20:20:1) and 1% triethylamine afford 8e in 82% yield. Mp 118–120 °C; ¹H NMR (400 MHz, CDCl₃) δ ppm 1.97–2.12 (m, 2H), 2.56–2.83 (m, 6H), 3.12 (brs, 4H), 3.62–3.72 (m, 2H), 3.85 (s, 3H), 3.86 (s, 3H), 4.82–5.0 (m, 1H), 6.85–7.09 (m, 7H), 7.35–7.41 (m, 2H); ¹³C NMR (100 MHz, CDCl₃) δ 159.25, 156.68, 152.48, 149.97, 141.41, 128.36, 123.22, 121.20, 118.43, 116.77, 112.55, 111.37, 110.71, 104.18, 91.48 (d, J = 167.6 Hz, 1C), 62.35 (d, J = 21.3 Hz, 1C), 55.58, 54.32, 50.77, 36.11, 33.29 (d, J = 20.6 Hz, 1C); ¹⁹F NMR (376 MHz, CDCl₃/CFCl₃) δ −182.1 to −182.6 (m, 1F); Anal. (C₂₅H₃₀FN₃O₄ ·1/2H₂O) C, H, N.

N-(4-(4-(2,3-Dichlorophenyl)piperazin-1-yl)-3-fluorobutyl)-5-methoxybenzofuran-2-carboxamide (8f)

Prepared from 5-methoxybenzofuran-2-carboxylic acid and 7b according to general procedure B. The crude compound was recrystallized from 2-PrOH. Yield: 65%. The oxalate salt was precipitated from CHCl₃. Mp 128–130 °C; ¹H NMR (400 MHz, CDCl₃) δ ppm 1.96–2.14 (m, 2H), 2.56–2.83 (m, 6H), 3.08 (brs, 4H), 3.62–3.72 (m, 2H), 3.85 (s, 3H), 4.79–4.99 (m, 1H), 6.92–6.98 (m, 2H), 7.02 (dd, J = 8.8, 2.8 Hz, 1H), 7.08 (d, J = 2.4 Hz, 1H), 7.11–7.17 (m, 2H), 7.35–7.41 (m, 2H); ¹³C NMR (100 MHz, CDCl₃) δ 159.26, 156.70, 151.39, 149.96, 149.55, 134.27, 128.37, 127.69, 124.87, 118.82, 116.80, 112.53, 110.74, 104.20, 91.47 (d, J = 168.4 Hz, 1C), 62.23 (d, J = 20.6 Hz, 1C), 56.09, 54.17, 51.47, 36.11 (d, J = 4.6 Hz, 1C), 36.97, 33.3(d, J = 19.8 Hz, 1C); ¹⁹F NMR (376 MHz, CDCl₃/CFCl₃) δ −181.53 to − 181.92 (m, 1F); Anal. (C₂₄H₂₆Cl₂FN₃O· C₂H₂O₄·3/4H₂O) C, H, N.

N-(3-Fluoro-4-(4-(2-methoxyphenyl)piperazin-1-yl)butyl)-1H-indole-2-carboxamide (8g)

Prepared from 1H-indole-2-carboxylic acid and 7a according to general procedure A. The crude compound was purified by column chromatography using EtOAc/CHCl₃/MeOH (20:20:1) and 1% triethylamine afford 8g in 62% yield. Mp 148–150 °C; ¹H NMR (400 MHz, CDCl₃) δ ppm 1.90–2.15 (m, 2H), 2.56–2.84 (m, 6H), 3.11 (brs, 4H), 3.61–3.77 (m, 2H), 3.86 (s, 3H), 4.81–5.02 (m, 1H), 6.60 (brs, 1H), 6.83–7.04 (m, 5H), 7.14 (t, J = 7.6 Hz, 1H), 7.29 (t, J = 7.6 Hz, 1H), 7.44 (d, J = 8.0 Hz, 1H), 7.65 (d, J = 8.0 Hz, 1H), 9.31 (brs, 1H); ¹³C NMR (100 MHz, CDCl₃) δ 161.94, 152.48, 141.40, 136.47, 130.86, 127.87, 124.75, 123.24, 122.18, 121.22, 120.91, 118.43, 112.17, 111.37, 102.19, 91.9 (d, J = 167.7 Hz, 1C), 62.24 (d, J = 21.3 Hz, 1C), 55.58, 54.31, 50.77, 36.65, 33.3 (d, J = 19.8 Hz, 1C); ¹⁹F NMR (376 MHz, CDCl₃/CFCl₃) δ −181.8 to − 181.26 (m, 1F); Anal. (C₂₄H₂₉FN₄O₂· 1/4H2O) C, H, N.

N-(4-(4-(2,3-Dichlorophenyl)piperazin-1-yl)-3-fluorobutyl)-1H-indole-2-carboxamide (8h)

Prepared from 1H-indole-2-carboxylic acid and 7b according to general procedure A. The crude compound was purified by column chromatography using EtOAc/CHCl₃/MeOH (20:20:1) and 1% triethylamine afford 8h in 65% yield. The oxalate salt was precipitated from CHCl₃. Mp 203–205°C; ¹H NMR (400 MHz, CDCl₃) δ ppm 1.94–2.10 (m, 2H), 2.56–2.82 (m, 6H), 3.07 (brs, 4H), 3.61–3.76 (m, 2H), 4.81–5.0 (m, 1H), 6.60 (t, J = 5.2 Hz, 1H), 6.83–6.92 (m, 1H), 6.93 (dd, J = 7.2, 2.4 Hz, 1H), 7.11–7.26 (m, 3H), 7.27–7.31 (m, 2H), 7.45 (d, J = 8.0 Hz, 1H), 7.64 (d, J = 8.4 Hz, 1H), 9.47 (brs, 1H); ¹³C NMR (100 MHz, CDCl₃) δ 161.97, 151.36, 136.49, 134.27, 130.83, 127.86, 127.70, 124.88, 124.78, 122.17, 120.94, 118.12, 112.19, 102.20, 91.85 (d, J = 168.4 Hz, 1C), 62.26 (d, J = 21.4 Hz, 1C), 54.14, 51.43, 36.62, 33.31 (d, J = 19.8 Hz, 1C); ¹⁹F NMR (376 MHz, CDCl₃/CFCl₃) δ −182.0 to −182.5 (m, 1F); Anal. (C₂₃H₂₅Cl₂FN₄O· C₂H₂O₄) C, H, N.

N-(3-Fluoro-4-(4-(2-methoxyphenyl)piperazin-1-yl)butyl)-9H-fluorene-2-carboxamide (8i)

Prepared from 9H-fluorene-2-carboxylic acid and 7a according to general procedure A. The crude compound was purified by column chromatography using EtOAc/CHCl₃/MeOH (20:20:1) and 1% triethylamine afford 8i in 75% yield. Mp 175–178 °C; ¹H NMR (400 MHz, CDCl₃) δ ppm 1.96–2.13 (m, 2H), 2.58–2.84 (m, 6H), 3.10 (brs, 4H), 3.59–3.76 (m, 2H), 3.85 (s, 3H), 3.93 (s, 2H), 4.84–5.03 (m, 1H), 6.75 (brs, 1H), 6.84–6.91 (m, 3H), 6.97–7.02 (m, 1H), 7.34–7.42 (m, 2H), 7.57 (d, J = 7.2 Hz, 1H), 7.79–7.83 (m, 3H), 7.97 (s, 1H); ¹³C NMR (100 MHz, CDCl₃) δ 168.15, 152.46, 145.13, 144.25, 143.67, 141.31, 140.88, 133.01, 127.89, 127.22, 126.03, 125.44, 124.05, 123.27, 121.20, 120.79, 119.97, 118.41, 111.37, 91.96 (d, J = 167.7 Hz, 1C), 62.34 (d, J = 21.4 Hz, 1C), 55.58, 54.27, 50.67, 37.07 (d, J = 10.7 Hz, 1C), 33.2 (d, J = 19.8 Hz, 1C); ¹⁹F NMR (376 MHz, CDCl₃/CFCl₃) δ −181.04 to −181.54 (m, 1F); Anal. (C₂₉H₃₂FN₃O2) C, H, N.

N-(4-(4-(2,3-Dichlorophenyl)piperazin-1-yl)-3-fluorobutyl)-9H-fluorene-2-carboxamide (8j)

Prepared from 9H-fluorene-2-carboxylic acid and 7b according to general procedure B. The crude product was recrystallized from 2-PrOH afford 8j in 87% yield. The oxalate salt was precipitated from EtOAc. Mp 189–191 °C; ¹H NMR (400 MHz, CDCl₃) δ ppm 1.95–2.17 (m, 2H), 2.58–2.85 (m, 6H), 3.07 (brs, 4H), 3.60–3.77 (m, 2H), 3.94 (s, 2H), 4.82–5.02 (m, 1H), 6.55 (brs, 1H), 6.90 (dd, J = 7.6, 1.6 Hz, 1H), 7.08–7.16 (m, 2H), 7.34–7.43 (m, 2H), 7.56–7.59 (m, 1H), 7.76–7.84 (m, 3H), 7.97 (s, 1H); ¹³C NMR (100 MHz, CDCl₃) δ 168.12, 151.33, 145.17, 144.23, 143.69, 140.86, 134.27, 133.0, 127.92, 127.74, 127.67, 127.24, 125.99, 125.46, 124.88, 124.04, 120.80, 119.97, 118.79, 92.0 (d, J = 167.6 Hz, 1C), 62.26 (d, J = 20.6 Hz, 1C), 54.14, 51.39, 37.10 (d, J = 6.1 Hz, 1C), 33.24 (d, J = 19.9 Hz, 1C); ¹⁹F NMR (376 MHz, CDCl₃/CFCl₃) δ −181.52 to −181.9 (m, 1F); Anal. (C₂₈H₂₈Cl₂FN₃O· C₂H₂O₄) C, H, N.

N-(3-Fluoro-4-(4-(2-methoxyphenyl)piperazin-1-yl)butyl)benzo[b]thiophene-2-carboxamide (8k)

Prepared from benzo[b]thiophene-2-carboxylic acid and 7a according to general procedure B. The crude product was purified by column chromatography using EtOAc/CHCl₃/MeOH (20:20:1) and 1% triethylamine afford 8k in 81% yield. The oxalate salt was precipitated from CHCl₃. Mp 150–153 °C; ¹H NMR (400 MHz, CDCl₃) δ ppm 1.94–2.14 (m, 2H), 2.54–2.82 (m, 6H), 3.1 (brs, 4H), 3.58–3.74 (m, 2H), 3.86 (s, 3H), 4.81–5.0 (m, 1H), 6.69 (brs, 1H), 6.85 (d, J = 7.6 Hz, 1H), 6.91 (d, J = 7.6 Hz, 2H), 6.98–7.02 (m, 1H), 7.36–7.44 (m, 2H), 7.76 (s, 1H), 7.80–7.86 (m, 2H); ¹³C NMR (100 MHz, CDCl₃) δ 162.69, 152.48, 141.40, 141.03, 139.30, 138.66, 126.55, 125.38, 125.25, 125.16, 123.22, 122.96, 121.21, 118.41, 111.38, 91.94 (d, J = 168.4 Hz, 1C), 62.36 (d, J = 20.6 Hz, 1C), 55.58, 54.31, 50.77, 37.13 (d, J = 3.8 Hz, 1C), 33.15 (d, J = 20.6 Hz, 1C); ¹⁹F NMR (376 MHz, CDCl₃/CFCl₃) δ −181.68 to −182.08 (m, 1F); Anal. (C₂₄H₂₈FN₃O₂S· C₂H₂O₄) C, H, N.

N-(4-(4-(2,3-Dichlorophenyl)piperazin-1-yl)-3-fluorobutyl)benzo[b]thiophene -2-carboxamide (8l)

Prepared from benzo[b]thiophene-2-carboxylic acid and 7b according to general procedure B. The crude product was recrystallized from 2-PrOH afford 8l in 80% yield. The oxalate salt was precipitated from EtOAc. Mp 185–187 °C; ¹H NMR (400 MHz, CDCl₃) δ ppm 1.94–2.16 (m, 2H), 2.56–2.83 (m, 6H), 3.07 (brs, 4H), 3.59–3.76 (m, 2H), 4.81–5.0 (m, 1H), 6.57 (brs, 1H), 6.92 (dd, J = 7.6, 2.4 Hz, 1H), 7.11–7.17 (m, 2H), 7.37–7.45 (m, 2H), 7.76 (s, 1H), 7.81–7.87 (m, 2H); ¹³C NMR (100 MHz, CDCl₃) δ 162.65, 151.36, 141.01, 139.28, 138.53, 134.27, 127.75, 127.69, 126.59, 125.43, 125.26, 125.19, 124.88, 122.96, 118.80, 91.95 (d, J = 168.5 Hz, 1C), 62.23 (d, J = 20.6 Hz, 1C), 54.14, 51.44, 37.15 (d, J = 3.8 Hz, 1C), 33.15 (d, J = 19.8 Hz, 1C); ¹⁹F NMR (376 MHz, CDCl₃/CFCl₃) δ −181.8 to −182.3 (m, 1F); Anal. (C₂₃H₂₄Cl₂FN₃OS· C₂H₂O₄) C, H, N.

N-(3-Fluoro-4-(4-(2-methoxyphenyl)piperazin-1-yl)butyl)-5-methoxy-1H-indole-2-carboxamide (8m)

Prepared from 5-methoxy-1H-indole-2-carboxylic acid and 7a according to general procedure A. The crude product was purified by column chromatography using EtOAc/CHCl₃/MeOH (20:20:1) and 1% triethylamine afford 8m in 64% yield. The oxalate salt was precipitated from CHCl₃. Mp 140–142 °C; ¹H NMR (400 MHz, CDCl₃) δ ppm 1.96–2.13 (m, 2H), 2.55–2.83 (m, 6H), 3.11 (brs, 4H), 3.60–3.76 (m, 2H), 3.84 (s, 3H), 3.86 (s, 3H), 4.81–5.0 (m, 1H), 6.57 (brs, 1H), 6.74–6.76 (m, 1H), 6.85–7.04 (m, 5H), 7.33 (d, J = 8.8 Hz, 1H), 9.32 (brs, 1H); ¹³C NMR (100 MHz, CDCl₃) δ 161.67, 154.67, 152.25, 141.17, 131.57, 131.06, 128.0, 123.01, 120.98, 118.20, 115.81, 112.82, 111.14, 102.30, 101.60, 91.65 (d, J = 167.7 Hz, 1C), 62.16 (d, J = 20.6 Hz, 1C), 55.71, 55.35, 54.08, 50.54, 36.35, 33.08 (d, J = 19.8 Hz, 1C); ¹⁹F NMR (376 MHz, CDCl₃/CFCl₃) δ −181.84 to −182.29 (m, 1F); Anal. (C₂₅H₃₁FN₄O₃· C₂H₂O₄· H2O) C, H, N.

N-(4-(4-(2,3-Dichlorophenyl)piperazin-1-yl)-3-fluorobutyl)-5-methoxy-1H-indole-2-carboxamide (8n)

Prepared from 5-methoxy-1H-indole-2-carboxylic acid and 7b according to general procedure A. The crude product was purified by column chromatography using EtOAc/CHCl₃/MeOH (20:20:1) and 1% triethylamine afford 8n in 46% yield. The oxalate salt was precipitated from CHCl₃. Mp 170–172 °C; ¹H NMR (400 MHz, CDCl₃) δ ppm 1.96–2.15 (m, 2H), 2.55–2.83 (m, 6H), 3.07 (brs, 4H), 3.61–3.77 (m, 2H), 3.84 (s, 3H), 4.81–5.0 (m, 1H), 6.56 (t, J = 5.6 Hz, 1H), 6.75 (d, J = 2.0 Hz, 1H), 6.91–6.98 (m, 2H), 7.03 (d, J = 2.4 Hz, 1H), 7.11–7.17 (m, 2H), 7.34 (d, J = 9.2 Hz, 1H), 9.48 (brs, 1H); ¹³C NMR (100 MHz, CDCl₃) δ 161.98, 154.90, 151.37, 134.27, 131.88, 131.25, 128.21, 127.75, 127.69, 124.88, 118.82, 116.06, 113.10, 102.51, 101.84, 91.84 (d, J = 168.4 Hz, 1C), 62.27 (d, J = 20.6 Hz, 1C), 55.94, 54.14, 51.44, 36.62 (d, J = 4.6 Hz, 1C), 33.33 (d, J = 20.6 Hz, 1C); ¹⁹F NMR (376 MHz, CDCl₃/CFCl₃) δ −182.10 to − 182.54 (m, 1F); Anal. (C₂₄H₂₇Cl₂FN₄O₂· C₂H₂O₄·1/2H2O) C, H, N.

N-(4-(4-(2,3-Dichlorophenyl)piperazin-1-yl)-3-fluorobutyl)-2-naphthamide (8o)

Prepared from 2-naphthoic acid and 7b according to general procedure B. The crude compound was recrystallized from 2-PrOH. Yield: 72%. The oxalate salt was precipitated from CHCl₃. Mp 171–173 °C; ¹H NMR (400 MHz, CDCl₃) δ ppm 1.98–2.20 (m, 2H), 2.58–2.85 (m, 6H), 3.05 (brs, 4H), 3.63–3.79 (m, 2H), 4.84–5.01 (m, 1H), 6.76 (brs, 1H), 6.88 (dd, J = 7.6, 2.0 Hz, 1H), 7.01–7.17 (m, 2H), 7.52–7.59 (m, 2H), 7.81–7.93 (m, 4H), 8.28 (s, 1H); ¹³C NMR (100 MHz, CDCl₃) δ 167.92, 151.35, 134.97, 134.27, 132.85, 131.99, 129.14, 128.75, 128.01, 127.89, 127.75, 127.67, 127.56, 127.04, 124.88, 123.77, 118.79, 92.05 (d, J = 167.7 Hz, 1C), 62.24 (d, J = 21.3 Hz, 1C), 54.15, 51.42, 37.12 (d, J = 3.8 Hz, 1C), 36.97, 33.2(d, J = 19.8 Hz, 1C); ¹⁹F NMR (376 MHz, CDCl₃/CFCl₃) δ −181.53 to −181.92 (m, 1F); Anal. (C₂₅H₂₆Cl₂FN₃O· C₂H₂O₄· 1/2H₂O) C, H, N.

N-(4-(4-(2-Methoxyphenyl)piperazin-1-yl)butyl)propionamide (11a)

Propionyl chloride (0.45 g, 4.8 mmol) was added to a solution of compound 10a (1.07 g, 4 mmol) and triethylamine (1.7 mL, 12mmol) in anhydrous CH₂Cl₂ (10 mL) at 0 °C under argon atmosphere and then stirred at room temperature for 4 h. The reaction was diluted with CH₂Cl₂ and washed with H₂O and brine, and the organic layer was dried over Na₂SO₄. The solvent was removed under vacuum afford compound 11a (0.76 g) in 59% yield. ¹H NMR (400 MHz, CDCl₃) δ ppm 1.15 (t, J = 7.2 Hz, 3H), 1.55–1.61 (m, 4H), 2.18 (q, J = 8 Hz, 2H), 2.43 (t, J = 7.2 Hz, 2H), 2.65 (brs, 4H), 3.10 (brs, 4H), 3.25–3.30 (m, 2H), 3.86 (s, 3H), 6.04 (brs, 1H), 6.84–7.02 (m, 4H); ¹³C NMR (100 MHz, CDCl₃) δ 173.78, 152.33, 141.28, 123.07, 121.06, 118.23, 111.23, 58.12, 55.43, 53.47, 50.07, 39.43, 29.91, 27.60, 24.47, 10.10.

N-(4-(4-(2,3-Dichlorophenyl)piperazin-1-yl)butyl)propionamide (11b)

This compound was prepared by the method described for compound 11a, employing 10b (2.3 g, 7.6 mmol), propionyl chloride (0.84 g, 4.8 mmol) and triethylamine (3.2 mL, 23 mmol), afford 11b (1.5 g) in 56% yield. ¹H NMR (400 MHz, CDCl₃) δ ppm 1.15 (t, J = 7.2 Hz, 3H), 1.56–1.60 (m, 4H), 2.19 (q, J = 8 Hz, 2H), 2.44 (t, J = 7.2 Hz, 2H), 2.64 (brs, 4H), 3.07 (brs, 4H), 3.25–3.31 (m, 2H), 5.88 (brs, 1H), 6.93–6.96 (m, 1H), 7.11–7.17 (m, 2H); ¹³C NMR (100 MHz, CDCl₃) δ 173.81, 151.31, 134.19, 127.64, 127.61, 124.77, 118.67, 58.09, 53.40, 51.39, 39.46, 29.98, 27.68, 24.46, 10.12.

4-(4-(2-Methoxyphenyl)piperazin-1-yl)-N-propylbutan-1-amine (12a)

Compound 11a (0.76 g, 2.4 mmol) in anhydrous THF (6 mL) was added dropwise into a suspension of lithium aluminum hydride (LiAlH₄; 0.46 g, 12 mmol) in anhydrous THF (20 mL) at 0 °C under an argon atmosphere. The reaction mixture was stirred at reflux for 4 h, cooled to room temperature, and then cooled further to 0 °C. Saturated NaOH/H₂O (5 mL) was added drop wise to quench the excess LiAlH₄. The mixture was filtered, and the reaction mixture was dried over Na₂SO₄. The solvent was removed under vacuum afford compound 12a (0.52 g) in 71 % yield. ¹H NMR (400 MHz, CDCl₃) δ ppm 0.92 (t, J = 7.6 Hz, 3H), 1.49–1.57 (m, 6H), 2.42 (t, J = 7.2 Hz, 2H), 2.56–2.66 (m, 6H), 3.09 (brs, 4H), 6.84–7.01 (m, 4H); ¹³C NMR (100 MHz, CDCl₃) δ 152.38, 141.45, 123.02, 121.10, 118.31, 111.23, 58.75, 55.46, 53.59, 51.93, 50.77, 49.91, 28.19, 24.92, 23.21, 11.94.

4-(4-(2,3-Dichlorophenyl)piperazin-1-yl)-N-propylbutan-1-amine (12b)

This compound was prepared by the method described for compound 12a, employing 11b (1.4 g, 3.9 mmol) and lithium aluminum hydride (LiAlH₄) (0.74 g, 19.5 mmol), afford 12b (1.1 g) in 82% yield. ¹H NMR (400 MHz, CDCl₃) δ ppm 0.93 (t, J = 7.6 Hz, 3H), 1.50–1.60 (m, 6H), 2.39–2.46 (m, 2H), 2.58–2.66 (m, 6H), 3.07 (brs, 4H), 6.94–6.98 (m, 1H), 7.13–7.16 (m, 2H).

General procedure C for imine reduction

4-(4-(2-methoxy or 2,3-dichlorophenyl)piperazin-1-yl)-N-propylbutan-1-amine (1 equiv) and appropriate aldehyde (1 equiv) were mixed in 1,2-dichloroethane (5 mL) and then treated with sodium triacetoxyborohydride (1.5 equiv) and AcOH (0.2 g). The mixture was stirred at room temperature under an argon atmosphere for 24 h. The reaction mixture was quenched by adding 1 N NaOH, and the product was extracted with EtOAc. The EtOAc extract was washed with brine, dried over Na₂SO₄ and concentrated in vacuo. The crude product was purified by preparative thin layer chromatography using EtOAc/CHCl₃/MeOH (5:5:1).

N-(Benzo[b]thiophen-2-ylmethyl)-4-(4-(2-methoxyphenyl)piperazin-1-yl)-N-propylbutan-1-amine (13a)

Prepared from 4-(4-(2-methoxyphenyl)piperazin-1-yl)-N-propylbutan-1-amine(12a) and benzo[b]thiophene-2-carbaldehyde according to general procedure C in 29% yield. The oxalate salt was precipitated from EtOAc. Mp 122–126 °C; ¹H NMR (400 MHz, CDCl₃) δ ppm 0.87 (t, J = 7.2 Hz, 3H), 1.46–1.58 (m, 6H), 2.33 (t, J = 7.2 Hz, 2H), 2.42–2.50 (m, 4H), 2.57 (brs, 4H), 3.07 (brs, 4H), 3.78 (d, J = 1.2 Hz, 2H), 3.86 (s, 3H), 6.84–7.02 (m, 4H), 7.28 (s, 1H), 7.32–7.38 (m, 2H), 7.82–7.85 (m, 2H), 7.94–7.98 (m, 2H); ¹³C NMR (100 MHz, CDCl₃) δ 152.4, 141.5, 140.8, 139.1, 135.2,124.3, 123.8, 123.7, 123.0, 122.9, 122.8, 121.1, 118.4, 111.3, 58.7, 56.5, 56.5, 55.5, 54.0, 53.5, 53.3, 50.7, 25.2, 24.8, 20.4, 12.1; Anal. (C₂₇H₃₇N₃OS· 2C₂H₂O₄·3/2H₂O) C, H, N.

N-(Benzo[b]thiophen-2-ylmethyl)-4-(4-(2,3-dichlorophenyl)piperazin-1-yl)-N-propylbutan-1-amine (13b)

Prepared from 4-(4-(2,3-dichlorophenyl)piperazin-1-yl)-N-propylbutan- 1-amine (12b) and benzo[b]thiophene-2-carbaldehyde according to general procedure C in 22 % yield. The oxalate salt was precipitated from EtOAc. Mp 168–171 °C; ¹H NMR (400 MHz, CDCl₃) δ ppm 0.88 (t, J = 7.2 Hz, 3H), 1.40–1.60 (m, 6H), 2.31 (t, J = 7.2 Hz, 2H), 2.42–2.45 (m, 4H), 2.52 (brs, 4H), 3.03 (brs, 4H), 3.78 (s, 2H), 6.92–6.95 (m, 1H), 7.28 (s, 1H), 7.31–7.38 (m, 2H), 7.82–7.85 (m, 1H), 7.95–7.98 (m, 1H); ¹³C NMR (100 MHz, CDCl₃) δ 151.5, 140.8, 139.1, 135.2, 134.1, 127.6, 124.6, 124.3, 123.8, 123.7, 122.9, 122.8, 118.7, 116.1; Anal. (C₂₃H₃₅Cl₂N₃· 2C₂H₂O₄) C, H, N.

N-((9H-Fluoren-2-yl)methyl)-4-(4-(2-methoxyphenyl)piperazin-1-yl)-N-propyl butan-1-amine (13c)

Prepared from 4-(4-(2-methoxyphenyl)piperazin-1-yl)-N-propylbutan-1-amine (12a) and 9H-fluorene-2-carbaldehyde according to general procedure C in 56 % yield. The oxalate salt was precipitated from EtOAc. Mp 112–114 °C; ¹H NMR (400 MHz, CDCl₃) δ ppm 0.87 (t, J = 7.2 Hz, 3H), 1.48–1.58 (m, 6H), 2.36–2.50 (m, 6H), 2.62 (brs, 4H), 3.07 (brs, 4H), 3.62 (s, 2H), 3.85 (s, 3H), 3.88 (s, 2H), 6.84–7.02 (m, 4H), 7.21–7.38 (m, 3H), 7.50–7.54 (m, 2H), 7.71 (d, J = 8 Hz, 1H), 7.76 (d, J = 7.6 Hz, 1H); ¹³C NMR (100 MHz, CDCl₃) δ 152.4, 143.4, 141.8, 141.5, 127.7, 126.8, 126.5, 125.7, 125.1, 123.0, 121.1, 119.8, 119.5, 118.3, 111.3, 59.0, 58.8, 56.1, 55.5, 53.8, 53.6, 50.7, 37.0, 24.8, 20.3, 12.1; Anal. (C₃₂H₄₁N₃O· 9/4C₂H₂O₄· 3/4H₂O) C, H, N.

N-((9H-Fluoren-2-yl)methyl)-4-(4-(2,3-dichlorophenyl)piperazin-1-yl)-N-propylbutan-1-amine (13d)

Prepared from 4-(4-(2,3-dichlorophenyl)piperazin-1-yl)-N-propylbutan-1-amine (12b) and 9H-fluorene-2-carbaldehyde according to general procedure C in 40% yield. The oxalate salt was precipitated from EtOAc. Mp 136–140 °C; ¹H NMR (400 MHz, CDCl₃) δ ppm 0.88 (t, J = 7.2 Hz, 3H), 1.49–1.55 (m, 6H), 2.35–2.47 (m, 6H), 2.59 (brs, 4H), 3.03 (brs, 4H), 3.62 (s, 2H), 3.89 (s, 2H), 6.91–6.94 (m, 1H), 7.12–7.15 (m, 2H), 7.27–7.38 (m, 3H), 7.51–7.54 (m, 2H), 7.71 (d, J = 8.0 Hz, 1H), 7.76 (d, J = 7.8 Hz, 1H); ¹³C NMR (100 MHz, CDCl₃) δ 151.5, 143.4, 141.8, 140.5, 134.1, 127.7, 127.6, 126.8, 126.5, 125.7, 125.2, 124.6, 119.8, 119.5, 118.7, 59.0, 58.7, 56.1, 53.8, 53.4, 51.4, 37.0, 25.2, 24.8, 20.3, 12.1; Anal. (C₃₁H₃₇Cl₂N₃· 2C₂H₂O₄· 1/2H₂O) C, H, N.

4-(4-(2-Methoxyphenyl)piperazin-1-yl)-N-(naphthalen-2-ylmethyl)-N-propylbutan-1-amine (13e)

Prepared from 4-(4-(2-methoxyphenyl)piperazin-1-yl)-N-propylbutan-1-amine (12a) and 2-naphthaldehyde according to general procedure C in 17% yield. The oxalate salt was precipitated from EtOAc. Mp 151–154 °C; ¹H NMR (400 MHz, CDCl₃) δ ppm 0.90 (t, J = 7.2 Hz, 3H), 1.50–1.60 (m, 6H), 2.39 (t, J = 6.8 Hz, 2H), 2.45 (t, J = 7.2 Hz, 2H), 2.50 (t, J = 6.2 Hz, 2H), 2.63 (brs, 4H), 3.10 (brs, 4H), 3.73 (s, 2H), 3.87 (s, 3H), 6.85–7.04 (m, 4H), 7.43–7.50 (m, 2H), 7.50 (dd, J = 10.4, 1.6 Hz, 1H), 7.76 (s, 1H), 7.80–7.85 (m, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 152.3, 141.4, 138.0, 133.4, 132.7, 127.7, 127.6, 127.4, 127.1, 125.8, 125.4, 122.9,121.0, 118.2, 111.2, 59.0, 58.8, 56.0, 55.4, 53.8, 53.5, 50.7, 25.2, 24.8, 20.3, 12.0; Anal. (C₂₉H₂₉N₃O· 2C₂H₂O₄·H₂O) C, H, N.

4-(4-(2,3-Dichlorophenyl)piperazin-1-yl)-N-(naphthalen-2-ylmethyl)-N-propylbutan-1-amine (13f)

Prepared from 4-(4-(2,3-dichlorophenyl)piperazin-1-yl)-N-propylbutan-1-amine (12b) and 2-naphthaldehyde according to general procedure C in 21% yield. The oxalate salt was precipitated from EtOAc. Mp 158–161 °C; ¹H NMR (400 MHz, CDCl₃) δ ppm 0.88 (t, J = 7.2 Hz, 3H), 1.49–1.56 (m, 6H), 2.36 (t, J = 7.2 Hz, 2H), 2.41–2.48 (m, 4H), 2.58 (brs, 4H), 3.04 (brs, 4H), 3.70 (s, 2H), 3.86 (s, 3H), 6.92–6.95 (m, 1H), 7.13–7.15 (m, 2H), 7.32–7.38 (m, 2H), 7.42–7.46 (m, 2H), 7.50 (dd, J = 10.0, 1.6 Hz, 1H), 7.74 (s, 1H), 7.78–7.83 (m, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 151.6, 138.2, 134.2, 133.6, 132.9, 127.9, 127.8, 127.6, 127.3, 126.0, 125.6, 124.7, 118.8, 59.2, 58.8, 56.2, 53.9, 53.4, 51.5, 25.3, 24.9, 20.5, 12.2; Anal. (C₂₃H₃₅Cl₂N₃· 5/2C₂H₂O₄· H₂O) C, H, N.

4-(((9H-Fluoren-2-yl)methyl)amino)-1-(4-(2,3-dichlorophenyl)piperazin-1-yl)butan-2-ol (14a)

To a solution of compound 10c (318 mg, 1 mmol) in MeOH (10 mL) was added 9H-fluorene-2-carbaldehyde (194 mg, 1 mmol) and the reaction mixture was stirred under argon for 12 h. NaBH₄ (75 mg, 2 mmol) was added to the reaction mixture and stirred for 30 min. The reaction was quenched by adding 1 N NaOH (15 mL). The resulting mixture was filtered through celite, and the residue was washed with CHCl₃ (50 mL). The organic layer was separated and dried over Na₂SO₄. The solvent was removed under reduced pressure. The crude product was purified by column chromatography using CHCl₃/MeOH (9:1) to afford the compound 14a (455 mg) in 92% yield. The oxalate salt was precipitated from CHCl₃. Mp 168–170 °C; ¹H NMR (400 MHz, CDCl₃) δ ppm 1.58–1.71 (m, 2H), 2.36–3.0 (m, 8H), 3.07 (brs, 4H), 3.88 (s, 4H), 3.91–3.98 (m, 1H), 6.94 (dd, J = 6.8, 2.8 Hz, 1H), 7.11–7.16 (m, 1H), 7.27–7.39 (m, 3H), 7.51–7.55 (m, 2H), 7.71–7.78 (m, 2H); ¹³C NMR (100 MHz, CDCl₃) δ 151.38, 143.71, 143.41, 141.62, 140.88, 138.69, 134.14, 127.61, 127.57, 127.03, 126.86, 126.70, 125.16, 125.06, 124.69, 119.92, 119.87, 118.72, 67.81, 64.71, 54.39, 53.68, 51.49, 47.50, 36.96, 34.34; Anal. (C₂₈H₃₁Cl₂N₃O· 2C₂H₂O₄· 4/5H₂O) C, H, N.

N-((9H-Fluoren-2-yl)methyl)-4-(4-(2,3-dichlorophenyl)piperazin-1-yl)-3-fluorobutan-1-amine (14b)

This compound was prepared by the method described for compound 14a, employing 7b (320 mg, 1 mmol), 9H-fluorene-2-carbaldehyde (194 mg, 1 mmol) and NaBH₄ (75 mg, 2 mmol), afford 14b (448 mg) in 90% yield. The oxalate salt was precipitated from CHCl₃. Mp 202–204 °C; ¹H NMR (400 MHz, CDCl₃) δ ppm 1.74–1.98 (m, 2H), 2.49–2.87 (m, 8H), 3.05 (brs, 4H), 3.87 (s, 2H), 3.89 (s, 2H), 4.76–4.95 (m, 1H), 6.92 (dd, J = 8.0, 2.4 Hz, 1H), 7.09–7.17 (m, 2H), 7.27–7.39 (m, 3H), 7.52–7.55 (m, 2H), 7.72–7.78 (m, 2H); ¹³C NMR (100 MHz, CDCl₃) δ 151.36, 143.71, 143.41, 141.66, 140.82, 139.14, 134.14, 127.63, 127.57, 126.97, 126.87, 126.69, 125.17, 125.01, 124.70, 119.91, 119.87, 118.71, 91.25 (d, J = 168.4 Hz, 1C), 62.56 (d, J = 21.3 Hz, 1C), 54.46, 53.98, 51.37, 45.49 (d, J = 4.6 Hz, 1C), 36.97, 34.18 (d, J = 10.2 Hz, 1C); ¹⁹F NMR (376 MHz, CDCl₃/CFCl₃) δ −181.98 to −181.59 (m, 1F); Anal. (C₂₈H₃₀Cl₂FN₃· 2C₂H₂O₄) C, H, N.

4-(((9H-Fluoren-2-yl)methyl)(methyl)amino)-1-(4-(2,3-dichlorophenyl)piperazin-1-yl)butan-2-ol (15a)

14a (124 mg, 0.25 mmol) and formaldehyde 37 % in H₂O (61μL) were mixed in 1,2-dichloroethane (5 mL) and then treated with sodium triacetoxyborohydride (159 mg, 0.75 mmol). The mixture was stirred at room temperature for 12 h. The reaction mixture was quenched by adding 1N NaOH (5 mL), and the product was extracted with EtOAc. The organic extract was washed with brine, dried over Na₂SO₄ and concentrated in vacuo. The crude product was purified by column chromatography using CHCl₃/MeOH (9:1) to afford 15a (103 mg) in 81 % yield. The oxalate salt was precipitated from CHCl₃. Mp 155–157 °C; ¹H NMR (400 MHz, CDCl₃) δ ppm 1.58–1.78 (m, 2H), 2.25 (s, 3H), 2.30–2.80 (m, 8H), 3.04 (brs, 4H), 3.47–3.72 (m, 2H), 3.87 (s, 2H), 3.91–3.98 (m, 1H), 6.90–6.95 (m,1H), 7.09–7.16 (m, 2H), 7.25–7.37 (m, 3H), 7.49–7.53 (m, 2H), 7.71–7.80 (m, 2H); ¹³C NMR (100 MHz, CDCl₃) δ 151.40, 143.57, 143.39, 141.53, 140.99, 136.96, 134.06, 127.98, 127.50, 126.81, 126.69, 125.86, 125.12, 124.56, 119.88, 119.71, 118.66, 68.94, 64.79, 63.0, 56.10, 53.79, 51.38, 42.05, 36.92, 31.34; Anal. (C₂₉H₃₃Cl₂N₃O· 2C₂H₂O₄· H₂O) C, H, N.

N-((9H-Fluoren-2-yl)methyl)-4-(4-(2,3-dichlorophenyl)piperazin-1-yl)-3-fluoro-N-methylbutan-1-amine (15b)

This compound was prepared by the method described for compound 15a, employing 14b (160 mg, 0.32 mmol), formaldehyde 37 % in H₂O (78μL) and sodium triacetoxyborohydride (203 mg, 0.96 mmol), afford 15b (121 mg) in 74% yield. The oxalate salt was precipitated from CHCl₃. Mp 173–175 °C; ¹H NMR (400 MHz, CDCl₃) δ ppm 1.78–1.98 (m, 2H), 2.25 (s, 3H), 2.48–2.77 (m, 8H), 3.05 (brs, 4H), 3.57 (s, 2H), 3.89 (s, 2H), 4.75–4.94 (m, 1H), 6.92 (dd, J = 7.6, 2.4 Hz, 1H), 7.10–7.17 (m, 2H), 7.26–7.39 (m, 3H), 7.51–7.55 (m, 2H), 7.71–7.78 (m, 2H); ¹³C NMR (100 MHz, CDCl₃) δ 151.37, 143.54, 143.42, 141.68, 140.86, 137.83, 134.13, 127.86, 127.62, 127.57, 126.85, 126.67, 125.83, 125.16, 124.67, 119.88, 119.66, 118.70, 91.05 (d, J = 168.4 Hz, 1C), 62.80, 62.45 (d, J = 20.6 Hz, 1C), 53.97, 53.24 (d, J = 4.6 Hz, 1C), 51.37, 42.42, 36.96, 31.65 (d, J = 20.6 Hz, 1C); ¹⁹F NMR (376 MHz, CDCl₃/CFCl₃) δ −181.67 to −181.28 (m, 1F); Anal. (C₂₉H₃₂Cl₂FN₃· 2C₂H₂O₄·5/4H2O) C, H, N.

Radioligand Dopamine Receptor Binding Assays

A filtration binding assay was used to characterize the binding properties of membrane-associated receptors. For human D2L, D3, and D4 dopamine receptors expressed in HEK 293 cells, tissue homogenates (50 mL) were suspended in 50 mM Tris-HCl/150 mM NaCl/10 mM EDTA buffer pH 7.5 and incubated with 50 μL of 125I-IABN at 37 °C for 60 min. Nonspecific binding was determined using 25 μM (+)-butaclamol. For competition experiments, the radioligand concentration was generally equal to 0.5 times the Kd value, and the concentration of the competitive inhibitor ranged over 5 orders of magnitude. Binding was terminated by the addition of cold wash buffer (10 mM Tris-HCl/150 mM NaCl, pH 7.5) and filtration over glass-fiber filters (Schleicher and Schuell No. 32). Filters were washed with 10 mL of cold buffer, and the radioactivity was measured using a Packard Cobra gamma counter. Estimates of the equilibrium dissociation constant and maximum number of binding sites were obtained using unweighted nonlinear regression analysis of data modeled according to the equation describing mass action binding. Data from competitive inhibition experiments were modeled using nonlinear regression analysis to determine the concentration of inhibitor that displaced 50% of the specific binding of the radioligand. Competition curves were modeled for a single site, and the IC50 values were converted to equilibrium dissociation constants (K_i values) using the Cheng-Prusoff correction. Mean K_i values SEM are reported for at least three independent experiments.

Preparation of chimeric receptors

Four methods were utilized to construct the chimeras discussed in this paper. The first method exploits a Pst1 restriction site in the first intracellular loop (I1) of the receptor that is common to both the human D2 and human D3R cDNAs. Digested fragments were gel purified, ligated into the pIRESneo2 expression vector and transformed into competent DH5-alpha E. coli cells. Individual colonies were picked and plasmid DNA was purified. Constructs were sequenced to verify the chimeras and then transfected into HEK 293 cells. The second method involved making chimeric D2/D3Rs using the method of Moore and Blakely (1994). A human D3R cDNA and a human D2 receptor cDNA were cloned in tandem into the pIRESneo2 vector with a unique restriction site (Hpa1) located between the two receptor cDNAs. The construct was digested at this site and the linear construct was transformed into competent cells. DNA was prepared from individual colonies and sequenced to verify the chimera and then transfected into HEK-293 cells for expression. The third method was devised to create chimeras that the previous methods had not produced, specifically in TMS4 and TMS5. Site-directed mutagenesis (Quick-Change Site-Directed Mutagenesis Kit, Stratagene) was performed on D2 and D3 clones in pGEM7Z to create a unique restriction site to both cDNAs at the two different TMS regions (Xho1 at TMS4 and Xba1 at TMS5). Clones were then digested with the appropriate enzyme, fragments were gel purified and ligated to create chimeras that transitioned at TMS4 and TMS5 locations. Site-directed mutagenesis was performed on these new chimeras to delete the previously added restriction sites and the DNA sequence was verified for each mutant. Chimeric receptors were then cloned into the pIRES vector and were transfected into HEK-293 cells. Expression of the receptor construct was verified using a radioligand binding assay. Finally, The D2/D3E2 and D3/D2E2 receptor chimeras were prepared using a PCR based site-directed mutagenesis (Quick-Change Site-Directed Mutagenesis Kit, Stratagene) strategy with synthetic oligonucleotides encoding the E2 loop with the appropriate 5′ and 3′ flanking regions. Both wild type receptor genes were in the pIRES expression vector (Clontech). The size of the oligonucleotide for preparation of the D2/D3E2 chimera was 69 bases and for the D3/D2E2 loop was 66 bases. The chimeric receptors were transfected into HEK-293 cells. The authenticity of the chimeric receptor was verified by DNA sequencing and the expression of the receptor construct in HEK 293 was verified by radioligand binding using [¹²⁵I]IABN. Tables S2 and S3 in the Supplementary Information provide the sequence transition points for the chimeric receptor proteins and amino acid sequence of wild type and chimeric human D3/D2 dopamine receptors, respectively.

Beta Arrestin Assay

The ability of the agonist-activated D3R to recruit beta-arrestin-2 is based on the DiscoverRx PathHunter technology which involves complementation of beta-galactosidase. The amino-terminal fragment of beta-galactosidase is fused to the D3R while the carboxyl-terminal fragment of the enzyme is fused to beta-arrestin-2. Upon agonist activation of the receptor, beta-arrestin is recruited to the receptor resulting in the formation of an active beta-galactosidase. Beta-galactosidase activity is then detected through the addition of a luminogenic substrate to the transfected cells as follows. CHO-K1 D3R cells (DiscoveRx) were seeded at a density of 5,000 cells/well in 384-well black, clear-bottom plates. Following 24-hour incubation, the cells were treated with multiple concentrations of the antagonist under study (0 – 10 μM) in Hanks’ Balanced Buffer Solution containing 2% DMSO. Following a 10 minute preincubation, the cells were next treated with an EC95 dose of Dopamine (100 nM) and then incubated at 37°C for 90 minutes. DiscoveRx reagent (2.5X) was then added to cells followed by a 60 minute incubation at room temperature. Luminescence was measured on a Hamamatsu FDSS μ-cell reader.

Abbreviations

DAST

N,N-diethylaminosulfur trifluoride

D2R

dopamine D2 receptor

D3R

dopamine D3 receptor

E2

second extracellular loop

BBB

blood-brain barrier

TMS

transmembrane spanning

D2/D3E2

a human D2 receptor with the E2 loop of the D3 receptor

D3/D2E2

a human D3 receptor with the E2 loop of the D2 receptor

hD2 or hD3

human D2 or D3 receptor proteins