Evaluation of N-Phenyl Homopiperazine Analogs as Potential Dopamine D₃ Receptor Selective Ligands

Abstract

A series of N-(2-methoxyphenyl)homopiperazine analogs was prepared and their affinities for dopamine D₂, D₃, and D₄ receptors were measured using competitive radioligand binding assays. Several ligands exhibited high binding affinity and selectivity for the D₃ dopamine receptor compared to the D₂ receptor subtype. Compounds 11a, 11b, 11c, 11f, 11j and 11k had K_i values ranging from 0.7–3.9 nM for the D₃ receptor with 30- to 170-fold selectivity for the D₃ vs. D₂ receptor. Calculated log P values (log P = 2.6–3.6) are within the desired range for passive transport across the blood brain barrier. When the binding and the intrinsic efficacy of these phenylhomopiperazines was compared to those of previously published phenylpiperazine analogues, it was found that a) affinity at D₂ and D₃ dopamine receptors generally decreased, b) the D₃ receptor binding selectivity (D₂:D₃ K_i value ratio) decreased and, c) the intrinsic efficacy, measured using a forskolin-dependent adenylyl cyclase inhibition assay, generally increased.

1. Introduction

The neurotransmitter dopamine has been implicated in a variety of physiological and pathophysiological processes. Dopamine’s effects are mediated by dopamine receptors, which belong to the family of G protein coupled receptors (GPCR) and share the characteristic structural architecture of seven transmembrane spanning regions. There are five different dopamine receptor subtypes that are classified into two protein families, the D₁-like (D₁ and D₅) and the D₂-like (D₂, D₃, and D₄) receptors, based upon related pharmacological and structural properties.^{1, 2} Abnormalities within the dopaminergic system are thought to play a role in psychiatric and neurological disorders, including Parkinson’s disease, substance abuse and schizophrenia.

The D₂ and D₃ receptor subtypes are highly homologous, especially within the helical transmembrane segments, which serve as the orthosteric binding site for dopamine. Therefore, while there has been interest in developing agonists, partial agonists and antagonists that are selective for the dopamine D₂ and D₃ receptor subtypes, this task has been difficult due to the high sequence homology for these two receptors.^3–5

Receptor autoradiography studies have shown that D₂ and D₃ receptors are widely distributed in striatal regions of human⁶ and nonhuman primate⁷ brain. The expression of D₃ receptors in limbic regions suggests that this receptor subtype may play an important role in the pathological abnormalities associated with many neuropsychiatric disorders. Autoradiography studies have revealed decreased D₃ receptor expression in the frontal cortex and increased expression in the ventral striatum of schizophrenics compared to normal individuals.^{8, 9} A selective dopamine D₃ receptor antagonist may provide antipsychotic properties in the absence of extrapyramidal side effects.¹⁰

Recent findings suggest that dopamine D₃ receptor agonists may also have beneficial effects for Parkinson’s patients.^{11, 12} Dopamine D₃ receptor partial agonists have also been reported to attenuate L-DOPA induced dyskinetic-like movements in animal models of Parkinson’s disease. D₃ receptor partial agonists normalize involuntary movement in MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine)-treated monkeys, a primate model for Parkinson’s disease.^{13, 14}

Finally, the activation of dopamine receptors in the nucleus accumbens is involved in the rewarding properties and/or the development of motivation for drug seeking behaviors for psychostimulants, such as cocaine. Therefore, partial agonists or antagonists that can reduce the interaction of psychostimulant-induced increases in synaptic dopamine levels with the D₃ receptor may be useful as pharmacotherapeutics for the treatment of cocaine abuse.^{4, 15–17}

A number of conformationally-flexible benzamide analogs displaying high affinity and binding selectivity for D₃ versus D₂ dopamine receptors have been reported in recent years. Examples include BP 897, NGB 2904, and the structural congeners 1–5 (Fig. 1).^18–20 A common structural feature of these conformationally-flexible benzamides is the N-arylpiperazine ring and the 4-carbon spacer group separating the benzamide and the piperazine moieties.^{18, 21–25} The lipophilic residue on the arylamide moiety permits diverse modifications including aryl, biphenyl, heteroaryl, or cycloalkyl substituents. However, the relatively high lipophilicity of these analogs is above the optimal range for passive transport across the blood brain barrier. For example, the calculated log P values of benzamide analogs BP 897, 1 and 2 (Fig. 1) are 4.7, 5.9 and 7.1, respectively, which are not within the range of log P values for compounds that can readily cross the blood brain barrier.^{26, 27}

Figure 1.

Structure and binding properties of D₃ receptor selective substituted N-phenylpiperazines as reported in the literature.

Previously, our group synthesized a series of N-phenylpiperazine analogs²² and evaluated their affinities and intrinsic activities at dopamine D₂ and D₃ receptors.³ These compounds share structural elements with the classic D₂-like dopamine receptor antagonists, including the 4-(2,3-dichlorophenyl)piperazino moiety. We have expanded upon those studies by synthesizing a new series of structurally related compounds and evaluating their binding affinities at D₂-like receptors. These modifications include: (a) replacement of the 2,3-dimethoxy-5-bromobenzene ring of 5 with other aromatic or heteroaromatic moieties to explore the structure activity relationship of the benzamide group, (b) replacement of the piperazine ring with homopiperazine and (c) comparison of the N-(2,3-dichlorophenyl)piperazine of NGB 2904 and compounds 1–5 with the N-(2-methoxyphenyl)piperazine analog of BP 897 to determine the effect of this substitution on dopamine receptor affinity and calculated log P values (Fig. 1).

The results of this study has led to the identification of a number of compounds possessing a high affinity (nM) and moderate selectivity (10 to 100-fold) for dopamine D₃ versus D₂ receptors with a log P value within the range desired for crossing the blood brain barrier through passive diffusion.

2. Chemistry

The syntheses of all target compounds (Fig. 2) are outlined in Scheme 1. The homopiperazine was protected to afford its N-Boc derivative 6. Compound 6 was reacted with 2-bromoanisole to obtain the N-phenyl homopiperazine, 7. Deprotection of 7 using trifluoroacetic acid yielded the deprotected N-phenylhomopiperazine 8. Finally, 8 was alkylated with N-(4-bromobutyl)phthalimide to give the corresponding phthalimido derivative 9, which was hydrolyzed with hydrazine hydrate to afford the amine 10. Condensation of this amine with the appropriate carboxylic acids in the presence of N,N′-dicyclohexylcarbodiimide (DCC) and 1-hydroxybenzotriazole (HOBt) gave the expected final compounds 11a–11t. Synthesis of 4-(2-hydroxyethyl)benzoic acid, 14 and 4-(2-fluoroethyl)benzoic acid, 16, was accomplished using the reaction sequence outlined in Scheme 2.

Figure 2.

General structure of target compounds 11a–11t.

Scheme 1.

Reagents: (a) di-tert-butyl dicarbonate, MeOH; (b) 2-bromoanisole, BINAP, Pd₂(dba)₃, KO^tBu/Et₃N, toluene, reflux; (c) trifluroacetic acid; (d) N-(4-bromobutyl)phthalimide, K₂CO₃, acetonitrile; (e) NH₂NH₂, EtOH; (f) ArCOOH, HOBt, DCC, dichloromethane.

Scheme 2.

Reagents: (a) (CH₃)₃OBF₄, CH₂Cl₂, N(C₂H₅)₃; (b) (1) BH₃/THF, (2) NaOH, 35% H₂O₂; (c) (1) NaOH, CH₃OH/H₂O, (2) HCl; (d) (C₂H₅)₂NSF₃, CH₂Cl₂;

3. Radioligand binding studies

Competitive radioligand binding studies were performed as previously described to determine the equilibrium dissociation constants of each compound at human D₂, D₃, and D₄ dopamine receptors (Table 1). Briefly, D₂-like receptors expressed in stably transfected HEK 293 cells were used in conjunction with the radioligand ¹²⁵I-IABN. We previously reported that the benzamide ¹²⁵I-IABN binds with high affinity and selectively to D_2-like dopamine receptors, but it binds non-selectively to the D₂ and D₃ dopamine receptor subtypes.²⁴ Measures were made of the affinity at D₂ and D₃ dopamine receptors of the new homopiperazine analogs, which have structural variations within the alkylamine moiety. When a comparison was made of the affinity of N-phenylhomopiperazine and the previously described corresponding N-phenylpiperazine analogues,²⁰ several differences between the two series of compounds became evident. First, the affinity of the homopiperazine analogs for D₂, there is a 2- to 6-fold lower, for D₃, 3.8- to 8-fold lower compared to the piperazine congener. Second, the D₂:D₃ affinity ratio is generally lower for the homopiperazine analogs compared to the corresponding piperazine compounds (e.g., reduced D₃ receptor selectivity).²⁰

Table 1.

Binding affinities for dopamine D₂-like receptors

Compound	Ki (nM)a			D2/D3e	Log Pf
	D2b	D3c	D4d
11a	128±8.8	0.7±0.10	187±21	183	3.50
11b	119±3.5	3.9±0.3	608±50	30	2.33
11c	101±15.5	2.9±0.5	372±7.1	35	3.62
11d	125±6.5	5.5±1.3	429 ±19	23	2.77
11e	108±7.5	9.0±0.7	522±51	12	2.19
11f	128±9.7	1.8±0.01	728±100	71	2.41
11g	111±9.2	3.7±0.3	350±47	30	2.64
11h	95.2±3.8	8.8±0.9	159±25	11	2.60
11i	56.5±5.9	7.5±0.7	188±43	8	2.16
11j	138±18.4	2.5±0.4	161±9.1	56	2.86
11k	125±7.0	1.5±0.2	297±35	82	4.01
11l	107±16.3	9.0±0.5	342±37	12	3.59
11m	151±11.2	13.2±1.7	748±36	11	1.01
11n	100±10.0	11.6±2.2	657±10	9	3.52
11o	43.7±7.0	4.8±1.0	177±19	9	2.81
11p	280±13.2	8.0±1.5	403±33	35	3.60
11q	119±7.5	15.0±0.7	641±57	8	1.89
11r	145±8.2	17.9±1.5	830±57	8	2.02
11s	152±25.6	44.2±3.7	441±98	3	1.23
11t	169±10.9	7.2±0.2	706±67	23	1.66
NGB-2904	112±22	2.0±0.4	NDg	56	6.94h

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

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

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

^dK_i values for D₄ receptors were measured using human D_4.4 expressed in HEK cells with [¹²⁵I]ABN as the radioligand.

^eK_i for D₃ receptors/ K_i for D₂ receptors.

^fCalculated C log P values using the program C log P by Advanced Chemistry Development, Inc. Toronto, Canada (ACD/Labs).

^gNot determined.

^hPublished data, Leopoldo et al, 2002. ²⁴

The substitution of the 4-position of the benzamide group with a 3-thiophene ring resulted in compound 11a. This analogue displayed both the highest D₃ binding affinity (0.7 nM) and greatest D₃ vs. D₂ receptor selectivity (187-fold) of the panel of compounds reported in this communication. Other potent and selective compounds included 11b, 11c, 11f, 11g, 11j and 11k (Table 1).

The phenylhomopiperazine compounds had uniformly low affinity at the D₄ dopamine receptor subtype (Table 1), with K_i values of >100 nM. The log P value for the homopiperazine analogs ranged from 1.0 to 4.0 (Table 1).

4. Adenylyl cyclase inhibition studies

D₂ and D₃ dopamine receptors are negatively coupled to adenylyl cyclase. Therefore, a forskolin-dependent adenylyl cyclase inhibition assay was used to determine the intrinsic efficacies of the new panel of homopiperazine compounds; these results were compared with the previously published values for the piperazine analogs (Table 2).²² The intrinsic efficacy of the homopiperazine compounds was generally found to be higher at D₂ dopamine receptors. The effect of this structural modification on efficacy appears to vary at D₃ receptors. The efficacy was comparable for some analogs (i.e., WC-26 vs. 11c, WC-28 vs. 11k and WC-34 vs. 11j) while the efficacy of the homopiperazine was higher for others (i.e., WC-10 vs. 11b, WC-21 vs. 11d and WC-23 vs. 11q) at D₃ dopamine receptors (Table 2). WC-44 was previously reported to be a full agonist at D₃ receptors but the homopiperazine analog, 11e, was found to be a strong partial agonist.

Table 2.

Comparison of the efficacy D₃ dopamine receptor for selective phenylhomopiperazine and phenylpiperazine (WC) analogues.

Compound	Ar	n	Percent Intrinsic Efficacy
			%IA D2	%IAD3
WC-10		n = 1	33.5±3.1	18.7±2.2
11b		n = 2	61.2±7.3	80.3±9.2
WC-26		n = 1	29.88±4.8	68.7±4.1
11c		n = 2	65.2±7.3	67.9±6.7
WC-21		n = 1	21.8±2.1	47.8±2.2
11d		n = 2	68.2±3.9	63.7±9.0
WC-44		n = 1	35.3±1.0	96.2±4.2
11e		n = 2	57.1±3.4	77.8±7.8
WC-28		n = 1	26.0±3.0	57.2±5.5
11k		n = 2	48.8±11.1	64.9±7.2
WC-34		n = 1	20.8±4.4	62.4±3.3
11j		n = 2	50.8±3.5	59.5±12.2
WC-23		n = 1	33.4±4.9	59.9±0.8
11q		n = 2	84.2±5.5	74.5±10.5

The intrinsic efficacy of the test compounds was evaluated by determining the percent inhibition of a forskolin-dependent whole cell adenylyl cyclase assay. The results were normalized to the percent inhibition obtained using the full agonist quinpirole at human D₂ (1 μM) and D₃ (100 nM) receptors expressed in stably transfected HEK 293 cells. The test drug was used at a concentration equal to approximately 10 × the K_i value that was determined from the radioligand binding analysis. The mean ± the S.E.M. values are reported for n ≥ 3.

Figure 3A shows a graph displaying the K_i values of the homopiperazine analogs at D₃ receptors versus their corresponding piperazine congeners. Figure 3B shows a similar representation between the homopiperazine/piperazine congeners with respect to intrinsic activity at the D₃ receptor. There was a linear correlation between the K_i values of the homopiperazine/piperazine congeners for binding to the D₃ receptor, but no such correlation was observed with respect to intrinsic activity (IA) at the D₃ receptor. These data suggest that although the homopiperazines and piperazines bind in a similar manner to the D₃ receptor, there is a fundamental difference in the ability of the structural congeners to activate D₃ receptor coupling to G proteins. This low correlation in IA is caused by the uniformly high intrinsic activity of the homopiperazine analogs at the D₃ receptor (ranging from 60–60%), whereas there was a large range in IA of the piperazine analogs at the D₃ receptor (ranging from 20–96%).

Figure 3.

(A) Comparison of the K_i values of the homopiperazine and piperazine analogs at D₃ receptors. (B) Similar representation for the Intrinsic Activity at D₃ receptors.

5. Modeling studies

In an attempt to better understand the structure-activity relationship of the homopiperazine analogs, we utilized the 3D-QSAR models previously built to predict the binding activities for this series of compounds. The ligand alignments were obtained following essentially the protocol previously described by our group.³ Specifically, a conformer library for each ligand was generated using the MCMM method available in MacroModel. ROCS (version 2.3.1, OpenEye Scientific Software, Santa Fe NM)²⁸ was used subsequently to retrieve the conformer from each library with the maximum shape alignment against a reference structure, the antagonist haloperidol which is bound to the orthosteric site of the refined homology models of D₂ and D₃.³ This procedure was applied to obtain two separate sets of ligand alignments for both the D₂ and D₃ binding sites. A salt bridge constraint between the highly conserved Asp carbonyl group in the third helical transmembrane spanning region and the protonated ligand amine was specified for both the piperazine and homopiperazine moieties.

Table 3 shows the data from both the a) experimental radioligand binding studies and b) the predicted binding affinities from the modeling studies. The residuals range from −0.41 to 1.24, indicating that our modeling studies are in good agreement with our experimental values. Figure 4 shows the phenylpiperazine WC-10 and the phenylhomopiperazine compound 11b as they occupy the human dopamine D₂ and D₃ receptor binding sites. We previously found that the orthosteric binding site, which is occupied by the substituted phenylpiperazine or phenylhomopiperazine moiety, is situated deeper in the D₃ receptor molecular than in the D₂ receptor. Therefore, the ligands assume a more linear conformation in the D₃ receptor binding site (Figure 4B). When situated in the D₂ receptor binding site, the 4-carbon chain assumes a bent conformation (Figure 4A). It is apparent from the modeling studies that the orientation of the piperazine andhomopiperazine moieties within the D₃ receptor binding site are very similar. However, the orientation of both the arylamide moiety and the substituted phenyl is slightly different between the piperazine and homopiperazine analogues when bound by the D₂ receptor. Our binding data indicates while the affinity of the homopiperazine analogs is lower for both the D₂ and the D₃ receptors, the difference in affinity is generally greater for the D₃ receptor than for D₂ receptor binding. Hence D₃ receptor selectivity decreases.

Table 3.

Comparison of predicted and experimental affinities values for the homopiperazine analogs.

Compound	D2 (log Ki) (nM)			D3 (log Ki) (nM)
	Experiment	Predicted	Residual	Experiment	Predicted	Residual
11a	2.107	1.385	0.722	−0.155	0.256	−0.411
11b	2.074	1.524	0.550	0.591	0.204	0.387
11c	2.003	1.380	0.623	0.462	0.212	0.250
11d	2.097	1.839	0.258	0.740	0.145	0.595
11e	2.032	1.547	0.485	0.954	0.354	0.600
11f	2.106	1.533	0.573	0.255	0.359	−0.104
11g	2.043	1.249	0.794	0.568	0.403	0.165
11h	1.979	1.176	0.803	0.944	0.298	0.646
11i	1.752	1.607	0.145	0.875	0.422	0.453
11j	2.138	1.433	0.705	0.398	0.079	0.319
11k	2.096	1.927	0.169	0.176	0.186	−0.010
11l	2.029	1.965	0.064	0.954	0.216	0.738
11m	2.179	1.511	0.668	1.121	0.123	0.998
11n	1.998	1.256	0.742	1.064	−0.163	1.227
11o	1.640	1.391	0.249	0.681	0.438	0.243
11p	2.446	1.988	0.458	0.903	0.283	0.620
11q	2.073	1.573	0.500	1.176	0.279	0.897
11r	2.162	1.636	0.526	1.253	0.340	0.913
11s	2.180	1.455	0.725	1.645	0.401	1.244
11t	2.227	1.522	0.705	0.857	0.170	0.687

Figure 4. Structural alignment of 11b and WC-10 as they occupy the D₂ and D₃ dopamine receptor binding sites.

Compounds 11b (shown in cyan) and the piperazine analogue WC-10 (shown in magenta) as they occupy the binding sites of A) the human dopamine D₂ receptor and B) the human D₃ dopamine receptor. The compounds sit much deeper in D₃ than in D₂ and overall adopt a more linear orientation.

In an attempt to understand the interactions between the ligands and respective receptors, the “hybrid” docking program implemented in the OEdocking toolkit (version 3.0.0, OpenEye Scientific Software, Santa Fe NM)²⁹ was used to dock the conformer libraries of each homopiperazine compound into the orthosteric site into both D₂ and D₃. This process finds and scores the best fit of each conformer to the bound haloperidol in the orthosteric site. A custom constraint was imposed ensuring a conserved salt bridge between the protonated amine and conserved Asp (comparable with what was done for the QSAR alignment). The docked molecules were further optimized using the program “szybki” (version 1.7.0, OpenEye Scientific Software, Santa Fe NM) which performs a molecular mechanics optimization of the ligand within the binding site. This process produces protein ligand binding energies for each ligand. The average binding energies of the homopiperazines were found to be essentially equivalent between the two receptors; −12.81 kcal/mol with D₂, and −10.95 kcal/mol with D₃. Gratifyingly, the interaction of compound 11a was 18-fold better for D₃ than for D₂ (1.35 kcal/mol vs −24.67 kcal/mol) consistent with what was experimentally observed.

6. Discussion

The goal of the current study was to identify ligands having a high affinity and selectivity for D₃ versus D₂ dopamine receptors, with calculated log P values within the desired range for passive transport across the blood brain barrier. Although many potent and selective D₃ ligands have been reported in the literature, the majority of these ligands have calculated log P values > 5.0, which should limit their ability to cross the blood brain barrier. Previous studies with ¹¹C-labeled aliphatic alcohols have indicated that the optimal log P values for a compound to have a high brain uptake (i.e., % brain extraction > 85% at a cerebral blood flow of 100 mL/min) range from −0.32 to 3.2.²⁴ Given the structural requirements of the lead compounds for the current study (Figure 1), which consisted of a benzamide ring and an N-phenylhomopiperazine moiety separated by a 4-carbon spacer unit, it was unlikely that we would prepare an analog near the low end of the log P range described above. However, it should be possible to prepare analogs of the lead compounds that would fall within the upper limit of this log P range. Based on the data shown in Table 1, this goal was accomplished by a) replacing the N-(2,3-dichlorophenyl)piperazine group with an N-(2-methoxyphenyl) homopiperazine moiety, and b) attaching a heteroatom into the benzamide aromatic ring. The most promising analogs were achieved by substituting the para position of the benzamide ring with a 3-thiophene ring (i.e., 11a). This compound had a D₂:D₃ selectivity ratio >100 and a log P value of 3.5, which, based on the data presented by Dischino et al.,²⁴ should result in high brain uptake, barring other factors such as being substrates for P glycoprotein. This compound may be useful a probe in the study of the behavioral pharmacology of dopamine D₃ receptors. In addition, the presence of the 2-methoxy group of compound 11a indicates that the corresponding ¹¹C-labeled versions of the compound can be prepared via alkylation of the corresponding de-methyl precursor with [¹¹C]iodomethane. Therefore, [¹¹C]11a may be a useful radiotracer for studying the regulation of dopamine D₃ receptors in a variety of CNS disorders using Positron Emission Tomography (PET).

In an attempt to better understand the structure-activity relationship of the homopiperazine analogs, we utilized the 3D-QSAR models previously built for the piperazine based ligands to predict the binding activities of these homopiperazine based compounds. The predictions for the binding affinities of these homopiperazine analogs are in a reasonable range in comparison with the measured values by minimal residuals. It also further indicated they have moderate D₃ selectivity.

In addition to 3D-QSAR, docking studies of these compounds in both the D₂ and D₃ receptors were consistent with what was found in previous QSAR studies. The D₂ orthosteric site is shallower and closer to the solvent and favors a bent conformation in the ligands. The D₃ site is deeper and the ligands further from exposure to solvent.

This simplistic docking evaluation highlights one limitation of modeling studies. The protein models described above have been optimized using an antagonist (haloperidol) in the ligand binding site. Therefore, differences in the increased intrinsic efficacy of the homopiperazines compared to the piperazines were not captured by the current docking studies.

Over the last decade, it has become clear that ligand-dependent GPCR activation is likely a multi-state process. Therefore, each of these states result in a different orientation of the helices and loops which make up the binding site of the D₂ and D₃ receptors. Consequently, the use of a static structure is unable to assess this dynamic process. Differences in intrinsic activity between the piperazine and homopiperazine analogs result from unique interactions with each of the different conformations, and are not likely to be predicted in a model based on the binding of a D₂ and D₃ antagonist. This is illustrated in Figure 4 where the top graph shows an excellent correlation in K_i values for the homopiperazine and piperazine based compounds found in Table 2, while the lower graph shows no correlation in terms of the observed IA. Therefore, more advanced models are needed in order to better understand the interaction of ligands with the D₃ receptor leading to increased activation of second messenger systems.

In conclusion, we have completed a structure activity relationship study on a series of conformationally-flexible benzamides with the goal of identifying potential probes for studying the behavioral pharmacology and developing radiotracers for imaging dopamine D₃ receptors with PET. We also have compared the affinity and intrinsic efficacy D₃ dopamine receptor selective phenylpiperazine and phenylhomopiperazine analogues in Table 3. The conclusion is that D₂ and D₃ affinity decreases compare to piperazine analogs. The results of this study identified a number of compounds having a high affinity and selectivity for D₃ versus D₂ receptor with a log P value that may result in a high uptake in brain in vivo.

7. Experimental

7.1. Chemical analysis

Nuclear magnetic resonance (NMR) spectra were recorded on a Varian 300 MHz NMR spectrometer. Chemical shifts were reported in δ values (parts per million, ppm) relative to an internal standard of tetramethylsilane (TMS). The following abbreviations are used for multiplicity of NMR signals: br s = broad singlet, d = doublet, dd = doublet of doublets, dt = doublet of triplets, m = multiplet, s = singlet, t = triplet. Melting points were determined using MEL-TEMP 3.0 apparatus and uncorrected. Elemental analyses (C, H, N) were determined by Atlantic Microlab, Inc. and the analytical results were within ±0.4% of the theoretical values. HR-MS was performed on a Waters ZQ 4000 single quadrupole mass spectrometer equipped with an electrospray ionization (ESI) LC-MS interface. All reactions were carried out under an inert atmosphere of nitrogen. Lipophilicity measurements of the compounds were estimated using the computational program, Clog P (Advanced Chemistry Development, Inc., Toronto, Canada).

7.2. tert-Butyl 1,4-diazepane-1-carboxylate (6)

A solution of di-tert-butyl dicarbonate (2.18 g, 10 mmol) in MeOH (25 mL) was slowly added to a stirring solution of homopiperazine (2.0 g, 20 mmol) in MeOH (50 mL) at 0 °C. The mixture was then stirred for 2 days at room temperature, and the solvent removed under reduced pressure. The crude solid was redissolved in diethyl ether (100 mL) with warming, and the white precipitate was removed by filtration. The product was extracted from the mother liquor with 1 M citric acid solution (3×50 mL), and the aqueous layer was washed with EtOAc (3×50 mL), basified with Na₂CO₃ (pH 11), and extracted with EtOAc (3×50 mL). The organic layer was dried over Na₂SO₄ and evaporated in vacuum to give tert-butyl 1-homopiperazinecarboxylate 6 as a waxy white liquid (crude, 1.61 g, 80%); ¹H NMR (300 MHz, CDCl₃) δ 3.44 (m, 4H), 2.87 (m, 4H), 2.04 (s, 1H), 1.77 (m, 2H), 1.46 (s, 9H).

7.3. tert-Butyl 4-(2-methoxyphenyl)-1,4-diazepane-1-carboxylate (7)

In a flask was added to degassed toluene (5 ml) in the following order 2-bromoanisole (929 mg, 5 mmol, 1.0 equiv), tert-butyl 1-homopiperazinecarboxylate (6) (1.10 g, 5.5 mmol, 1.1 equiv of the amine), Pd₂(dba)₃ (50 mg, 0.054 mmol, 1–2.5 mol% of the amine), (±)-BINAP (100 mg, 0.16 mmol, 1.5 equiv/Pd), Et₃N (0.30 mL, ~0.5 equiv), and KO^tBu (1.68 g, 15 mmol, 3.0 equiv). The resulting dark red mixture was heated to 110 °C (bath temperature) and stirred at this temperature for 21 hours. Once TLC indicated complete consumption of the aryl bromide, the brownish mixture was cooled to room temperature, then filtered over a thin layer of Celite, and the filter cake was thoroughly washed with EtOAc. The filtrate was concentrated in vacuo and the residue purified by flash chromatography using mixtures of hexanes and EtOAc (4/1) to give an oil (928 mg, 61%). TLC R_f 0.28 (hexanes/ethyl acetate 4/1); ¹H NMR (300 MHz, CDCl₃) δ 6.95-6.83 (m, 4H), 3.84 (s, 3H), 3.51 (m, 4H), 3.24 (m, 4H), 2.04 (s, 1H), 1.93 (m, 2H), 1.46 (s, 9H).

7.4. 1-(2-Methoxyphenyl)-1,4-diazepane (8)

tert-Butyl 4-(2-methoxyphenyl)homopiperazine-1-carboxylate (7) (570 mg, 1.86 mmol) was dissolved in a mixture of trifluoroacetic acid (10 mL) and water (2.5 mL). The solution was stirred at room temperature overnight, and then was evaporated to dryness in vacuo. The residue was dissolved in water (10 mL) and then basified with Na₂CO₃ (pH = 11), and extracted with EtOAc (3×50 mL). The organic layer was washed with brine (2×50 mL) and dried over Na₂SO₄ overnight. Evaporation under reduced pressure gave N-4-(2-methoxyphenyl)homopiperazine (8) as a yellow liquid (252 mg, 66%), which was used without further purification.

7.5. 2-(4-(4-(2-Methoxyphenyl)-1,4-diazepan-1-yl)butyl)isoindoline-1,3-dione (9)

To a solution of N-4-(2-methoxyphenyl)homopiperazine (8) (283 mg, 1.37 mmol) and N-(4-bromobutyl)phthalimide (387 mg, 1.37 mmol) in acetonitrile (20 mL) was added K₂CO₃ (567 mg, 4.11 mmol). The mixture was refluxed overnight under N₂, then filtered to remove the excess K₂CO₃, the filtrate was concentrated in vacuo and the residue purified with a mixture of hexanes and EtOAc (1:2) to give a yellow liquid (150 mg, 99%). TLC R_f 0.3 (hexanes/ethyl acetate 1:2); ¹H NMR (300 MHz, CDCl₃) δ 7.84 (d, J = 8.4 Hz, 2H), 7.72 (d, J = 8.4 Hz, 2H), 6.85–6.92 (m, 4H), 3.83 (s, 3H), 3.71 (m, 2H), 3.32 (m, 4H), 2.81 (m, 2H), 2.71 (m, 2H), 2.53 (m, 2H), 2.01 (m, 2H), 1.68 (m, 2H), 1.54 (m, 2H). ¹³C NMR (CDCl₃, 300 MHz): 167.2, 162.4, 141.3, 132.4, 123.6, 122.2, 121.6, 113.8, 59.6, 58.3, 56.8, 53.9, 52.2, 39.5, 26.9, 25.7. HRMS (ESI) Calcd for C₂₄H₂₉N₃O₃ (M+H)⁺: 408.2287. Found: 408.2275.

7.6. 4-(4-(2-Methoxyphenyl)-1,4-diazepan-1-yl)butan-1-amine (10)

Hydrazine hydrate (2 mL) was added into the solution of N-[4-[4-(2-methoxyphenyl)-1-homopiperazinyl)butyl]-phthalimide (9) ( 328 g, 0.80 mmol) in ethanol (10 mL). The reaction mixture was heated at reflux for 2 h. The reaction was cooled, filtered and concentrated in vacuo. The residue was dissolved in CH₂Cl₂ (20 mL) and washed with 1N NaOH (20 mL × 3) and brine solution. The organic phase was dried over Na₂SO₄ and concentrated in vacuo to afford target product as a light yellow oil (223 mg, 100%); ¹H NMR (300 MHz, CDCl₃) δ 6.92-6.85 (m, 4H), 3.83 (s, 3H), 3.33 (m, 4H), 2.83 (m, 2H), 2.71 (m, 4H), 2.51 (m, 2H), 1.96 (m, 2H), 1.54-1.44 (m, 4H). ¹³C NMR (CDCl₃, 300 MHz): 162.3, 141.3, 123.6, 121.9, 113.4, 59.6, 58.3, 56.8, 52.2, 41.5, 26.9, 26.2, 25.7. HRMS (ESI) Calcd for C₁₆H₂₇N₃O (M+H)⁺: 278.2232. Found: 278.2243.

7.7. Methyl 4-vinylbenzoate (12)

A solution of 4-vinylbenzoic acid (2.08 g, 14.0 mmol) and trimethyloxonium tetrafluoroborate (2.60 g,17.5 mmol) in CH₂Cl₂ (200 mL) was added triethylamine (1.56 g, 15.4 mmol) at ambient temperature. The reaction mixture was stirred for 20 hours at ambient temperature, then washed with saturated Na₂CO₃ (50 mL), saturated NaCl (50 mL) and dried over Na₂SO₄. After evaporation of the solvent in vacuo, the crude product was purified by silica gel column chromatography with hexane and ether (10/1) to afford 1.72 g (76%) of 12 as a white solid which was used directly in the next step. TLC R_f 0.25 (hexane and ether 10/1); ¹H NMR (300 MHz, CDCl₃) δ 8.01 (d, J = 8.4 Hz, 2H), 7.48 (d, J = 8.4 Hz, 2H), 6.76 (dd, J = 17.5 Hz, J = 10.8 Hz, 1H), 5.88 (d, J = 17.5 Hz, 1H), 5.40 (d, J = 10.8 Hz), 3.93 (s, 3H).

7.8. Methyl 4-(2-hydroxyethyl)benzoate (13)

Compound 12 (1.95 g, 12.0 mmol) in 1M BH₃ in THF (24 mL) was stirred 1h at 0 °C, then 1h at ambient temperature. A solution of 1N NaOH (36 mL) was added to the reaction mixture at 0 °C, then a solution of 35% H₂O₂ (20 mL) was added. The mixture was stirred 30 min at 0 °C, then 30 min at ambient temperature. Ethyl acetate (100 mL) was added, the organic layer was separated, washed with water (3×50 mL), saturated Na₂CO₃ (3×50 mL), saturated NaCl (3×50 mL) and dried over Na₂SO₄. After evaporation of the solvent in vacuo, the crude product was purified by chromatography with hexane ether (1:1) to afford 1.16 g (54%) of 13 as a colorless oil. TLC R_f 0.31 (hexane and ether 1:1); ¹H NMR (300 MHz, CDCl₃) δ 8.00 (d, J = 8.4 Hz, 2H), 7.32 (d, J = 8.4 Hz, 2H), 3.92 (s, 3H), 3.91 (t, J = 6.6 Hz, 2H), 2.95 (t, J = 6.6 Hz, 2H).

7.9. 4-(2-Hydroxyethyl)benzoic acid (14)

A solution of 13 (174 mg, 0.96 mmol) in methanol (3 mL) and water (1 mL) was added NaOH (58 mg, 1.43 mmol) at ambient temperature. The mixture was stirred for 2 days at ambient temperature, water (5 mL) was then added, and extracted with ether (3×20 mL). The aqueous layer was acidified with HCl (1:1) to pH = 1, the white solid was filtered out to afford 158 mg (100%) of 14 which was used directly in the next step. ¹H NMR (300 MHz, CDCl₃) δ 7.97 (d, J = 8.4 Hz, 2H), 7.27 (d, J = 8.4 Hz, 2H), 3.83 (t, J = 6.3 Hz, 2H), 2.88 (t, J = 6.3 Hz, 2H).

7.10. Methyl 4-(2-fluoroethyl)benzoate (15)

A solution of 13 (1.16 g, 6.44 mmol) in CH₂Cl₂ (20 mL) was added DAST (1.56 g, 9.66 mmol) at 0 °C. The mixture was warmed to ambient temperature and stirred overnight, then ethyl acetate (100 mL) was added, washed with water (50 mL), saturated Na₂CO₃ (50 mL), saturated NaCl (50 mL), and dried over Na₂SO₄. After evaporation of the solvent in vacuo, the crude product was purified by chromatography with hexane ether (10:1) to afford 1.06 g (91%) of 15 as a colorless oil. TLC R_f 0.25 (hexane ether 10:1); ¹H NMR (300 MHz, CDCl₃) δ 8.01 (d, J = 8.4 Hz, 2H), 7.33 (d, J = 8.1 Hz, 2H), 4.67 (dt, J = 47.1 Hz, J = 6.3 Hz, 2H), 3.93 (s, 3H), 3.08 (dt, J = 24.6 Hz, J = 6.3 Hz, 2H).

7.11. 4-(2-Fluoroethyl)benzoic acid (16)

Solid NaOH (58 mg, 1.43 mmol) was added to a solution of 15 (174 mg, 0.96 mmol) in methanol (3 mL) and water (1 mL) at ambient temperature. The mixture was stirred for 2 days at ambient temperature, water (5 mL) was added, extracted with ether (20 mL). The aqueous layer was acidified with HCl (1:1) to pH = 1, the white solid was filtered out to afford 160 mg (100%) of 16 which was used directly in the next step. ¹H NMR (300 MHz, CDCl₃) δ 8.07 (d, J = 8.4 Hz, 2H), 7.36 (d, J = 8.4 Hz, 2H), 4.68 (dt, J = 46.8 Hz, J = 6.3 Hz, 2H), 3.10 (dt, J = 24.6 Hz, J = 6.3 Hz, 2H).

7.12. General Procedure for Preparing the Substituted Benzamide Analogues N-(4-(4-(2-Methoxyphenyl)-1,4-diazepan-1-yl)butyl)-4-(thiophen-3-yl)benzamide (11a)

A mixture of compound 10 (135 mg, 0.49 mmol) and 4-(thiophen-3-yl)benzoic acid (99.4 mg, 0.49 mmol) in dichloromethane (10 mL) was stirred at 0 °C (ice-water bath). Dicyclohexylcarbodiimide (DCC) (121 mg, 0.59 mmol) and hydroxybenzotriazole (HOBt) (80 mg, 0.59 mmol) were added to the above solution. The ice bath was removed, and the reaction mixture was stirred at ambient temperature for 15 hours. Dichloromethane (20 mL) was added into the reaction mixture, and the solution was washed with saturated aqueous NaHCO₃ solution (3×10 mL). The organic layer was dried over Na₂SO₄, concentrated under reduced pressure, and the crude product was purified by silica gel column chromatography using dichloromethane methanol (20:1) as the mobile phase to give 11a as a white solid (194 mg, 86%). TLC R_f 0.20 (dichloromethane methanol 20:1); mp 125.5–126.6 °C. ¹H NMR (300 MHz, CDCl₃) δ 7.75 (d, J = 8.7 Hz, 2H), 7.54 (d, J = 8.7 Hz, 2H), 7.43 (s, 1H), 7.33 (s, 2H), 7.19 (s, 1H), 6.81–6.94 (m, 4H), 3.85 (s, 3H), 3.44 (q, J = 5.1 Hz, 2H), 3.25-3.20 (m, 4H), 2.83-2.76 (m, 4H), 2.57 (t, J = 5.2 Hz, 2H), 1.93 (t, J = 4.2 Hz, 2H), 1.61–1.69 (m, 4H). ¹³C NMR (CDCl₃, 300 MHz): 167.4, 151.4, 142.1, 141.2, 138.8, 133.4, 127.5, 126.6, 126.3, 126.1, 121.3, 121.0, 120.7, 117.9, 111.6, 57.1, 56.1, 55.3, 54.3, 52.5, 51.8, 39.4, 27.7, 27.3, 25.5. HRMS (ESI) Calcd for C₂₇H₃₃N₃O₂S (M+H)⁺: 464.2372. Found: 464.2379. Anal. (C₂₇H₃₃N₃O₂S·1.5H₂C₂O₄) C, H, N.

7.13. 4-(Dimethylamino)-N-(4-(4-(2-methoxyphenyl)-1,4-diazepan-1-yl)butyl)benzamide(11b)

Compound 11b was prepared according to the procedure for compound 11a except using 4-(dimethylamino)benzoic acid, which afford 102 mg (80%) of 11b as a white solid. TLC R_f 0.15 (dichloromethane methanol 20:1); mp 55–56 °C. ¹H NMR (300 MHz, CDCl₃) δ 7.89 (d, J = 9.0 Hz, 2H), 7.27 (m, 1H), 6.97 (m, 2H), 6.89 (m, 2H), 6.68 (d, J = 9.0 Hz, 2H), 3.82 (s, 3H), 3.54-3.48 (m, 8H), 3.28 (m, 2H), 3.12 (m, 2H), 3.07 (s, 6H), 2.46 (m, 2H), 2.04 (m, 2H), 1.74 (m, 2H). ¹³C NMR (CDCl₃, 300 MHz): 167.5, 162.4, 154.3, 141.1, 130.4, 123.5, 122.9, 121.4, 113.5, 111.7, 57.1, 56.4, 55.2, 54.3, 52.5, 51.8, 39.3, 27.7, 26.7, 25.4. HRMS (ESI) Calcd for C₂₅H₃₆N₄O₂ (M+H)⁺: 425.2917. Found: 425.2936. Anal. (C₂₅H₃₆N₄O₂·H₂C₂O₄·0.5H₂O) C, H, N.

7.14. N-(4-(4-(2-Methoxyphenyl)-1,4-diazepan-1-yl)butyl)-4-(methylthio)benzamide(11c)

Compound 11c was prepared according to the procedure for compound 11a except using 4-(methylthio)benzoic acid, which afford 71 mg (79%) of 11c as a white solid. TLC R_f 0.21 (dichloromethane methanol 20:1); mp 92.2–93 °C. ¹H NMR (300 MHz, CDCl₃) δ 7.63 (d, J = 7.5 Hz, 2H), 7.14 (d, J = 7.5 Hz, 2H), 6.85-6.77 (m, 5H), 3.87 (s, 3H), 3.53 (d, J = 4.2 Hz, 2H), 3.22 (m, 4H), 2.81-2.73 (m, 4H), 2.53 (t, J = 6.9 Hz, 2H), 2.41 (s, 3H), 1.91 (m, 2H), 1.57 (m, 4H). ¹³C NMR (CDCl₃, 300 MHz): 167.3, 162.2, 142.8, 141.1, 130.5, 127.8, 126.9, 123.0, 122.2, 113.5, 57.2, 56.2, 55.4, 54.4, 52.7, 51.8, 39.4, 27.7, 26.6, 25.4. 14.7. HRMS (ESI) Calcd for C₂₄H₃₃N₃O₂S (M+H)⁺: 428.2372. Found: 428.2369. Anal. (C₂₄H₃₃N₃O₂S·H₂C₂O₄) C, H, N.

7.15. N-(4-(4-(2-Methoxyphenyl)-1,4-diazepan-1-yl)butyl)benzofuran-2-carboxamide(11d)

Compound 11d was prepared according to the procedure for compound 11a except using 2-benzofurancarboxylic acid, which afford 65 mg (66%) of 11d as a white solid. TLC R_f 0.30 (Ethyl acetate methanol 10:1); mp 57.5–59 °C. ¹H NMR (300 MHz, CDCl₃) δ 7.67 (m, 1H), 7.48 (m, 1H), 7.40 (m, 1H), 7.23 (m, 1H), 7.19 (m, 1H), 6.85-6.77 (m, 5H), 3.87 (s, 3H), 3.47 (q, J = 6.0 Hz, 2H), 3.28 (m, 4H), 2.83-2.73 (m, 4H), 2.52 (t, J = 6.6 Hz, 2H), 1.93 (m, 2H), 1.63 (m, 4H). ¹³C NMR (CDCl₃, 300 MHz): 162.3, 157.2, 156.3, 150.1, 141.1, 128.8, 123.3, 123.0, 122.0, 120.9, 113.4, 111.5, 108.9, 57.2, 56.2, 55.4, 54.4, 52.7, 51.8, 39.3, 27.7, 26.7, 25.4. HRMS (ESI) Calcd for C₂₅H₃₁N₃O₃ (M+H)⁺: 422.2444. Found: 422.2427. Anal. (C₂₅H₃₁N₃O₃·H₂C₂O₄·0.25H₂O) C, H, N.

7.16. 4-(2-Fluoroethyl)-N-(4-(4-(2-methoxyphenyl)-1,4-diazepan-1-yl)butyl)benzamide(11e)

Compound 11e was prepared according to the procedure for compound 11a except using 16, purified with ether methanol (10:1) to afford 112 mg (84%) of 11e as a white solid. TLC R_f 0.29 (dichloromethane methanol 15:1); mp 64.2–65.3 °C. ¹H NMR (300 MHz, CDCl₃) δ 7.67 (d, J = 8.4 Hz, 2H), 7.18 (d, J = 8.4 Hz, 2H), 6.85-6.77 (m, 5H), 4.62 (dt, J = 46.8 Hz, J = 6.3 Hz, 2H), 4.47 (dt, J = 46.8 Hz, J = 6.3 Hz, 2H), 3.86 (s, 3H), 3.49 (q, J = 6.0 Hz, 2H), 3.23 (m, 4H), 3.01-2.98 (m, 4H), 2.80-2.73 (m, 4H), 2.52 (m, 2H), 1.91 (m, 2H), 1.59 (m, 4H). ¹³C NMR (CDCl₃, 300 MHz): 167.4, 162.2, 142.8, 141.2, 131.4, 127.4, 123.0, 122.0, 113.5, 86.2, 57.5, 56.6, 55.4, 54.4, 52.6, 51.8, 39.3, 36.4, 27.7, 26.7, 25.4. HRMS (ESI) Calcd for C₂₅H₃₄FN₃O₂ (M+H)⁺: 428.2713. Found: 428.2730. Anal. (C₂₅H₃₄FN₃O₂· H₂C₂O₄) C, H, N.

7.17. N-(4-(4-(2-Methoxyphenyl)-1,4-diazepan-1-yl)butyl)-4-vinylbenzamide (11f)

Compound 11f was prepared according to the procedure for compound 11a except using 4-vinylbenzoic acid, purified with ether methanol (10:1) to afford 92 mg (81%) of 11f as a white solid. TLC R_f 0.31 (dichloromethane methanol 15:1); mp 72.5–74 °C. ¹H NMR (300 MHz, CDCl₃) δ 7.75 (d, J = 8.4 Hz, 2H), 7.43 (d, J = 8.4 Hz, 2H), 6.89-6.85 (m, 5H), 6.63 (dd, J = 17.7 Hz, J = 10.8 Hz, 1H), 5.74 (d, J = 17.7 Hz, 1H), 5.26 (d, J = 10.8 Hz, 1H), 3.86 (s, 3H), 3.49 (m, 2H), 3.31 (m, 4H), 2.88-2.73 (m, 4H), 2.59 (m, 2H), 1.99 (m, 2H), 1.67 (m, 4H). ¹³C NMR (CDCl₃, 300 MHz): 167.6, 162.2, 141.3, 136.1, 133.4, 129.2, 127.5, 123.0, 122.0, 114.4, 113.4, 57.2, 56.2, 55.4, 54.4, 52.7, 51.8, 39.3, 27.8, 26.6, 25.4. HRMS (ESI) Calcd for C₂₅H₃₃N₃O₂ (M+H)⁺: 408.2651. Found: 408.2638. Anal. (C₂₅H₃₃N₃O₂· H₂C₂O₄) C, H, N.

7.18. 4-Chloro-N-(4-(4-(2-methoxyphenyl)-1,4-diazepan-1-yl)butyl)benzamide(11g)

Compound 11g was prepared according to the procedure for compound 11a except using 4-chlorobenzoic acid, purified with ether methanol (10:1) to afford 83 mg (80%) of 11g as a white solid. TLC R_f 0.25 (dichloromethane methanol 20:1); mp 62–63.2 °C. ¹H NMR (300 MHz, CDCl₃) δ 7.88 (d, J = 8.4 Hz, 2H), 7.78 (s, 1H), 7.39 (d, J = 8.4 Hz, 2H), 6.95-6.85 (m, 4H), 3.82 (s, 3H), 3.53 (d, J = 4.2 Hz, 2H), 3.36-3.24 (m, 6H), 3.07 (m, 2H), 2.30 (m, 2H), 1.91 (m, 2H), 1.64 (m, 4H). ¹³C NMR (CDCl₃, 300 MHz): 167.5, 162.3, 141.1, 137.4, 132.4, 130.3, 128.8, 123.0, 122.0, 113.5, 57.2, 56.4, 55.4, 54.3, 52.7, 51.8, 39.5, 27.7, 26.6, 25.4. HRMS (ESI) Calcd for C₂₃H₃₀ClN₃O₂ (M+H)⁺: 416.2105. Found: 416.2129. Anal. (C₂₃H₃₀ClN₃O₂·H₂C₂O₄·H₂O) C, H, N.

7.19. 4-Fluoro-N-(4-(4-(2-methoxyphenyl)-1,4-diazepan-1-yl)butyl)benzamide(11h)

Compound 11h was prepared according to the procedure for compound 11a except using 4-fluorobenzoic acid, purified with ether methanol (10:1) to afford 75 mg (79%) of 11h as a white solid. TLC R_f 0.24 (dichloromethane methanol 20:1); mp 52.8–54 °C. ¹H NMR (300 MHz, CDCl₃) δ 7.95 (d, J = 8.1 Hz, 2H), 7.72 (s, 1H), 7.05 (d, J = 8.4 Hz, 2H), 6.97-6.88 (m, 4H), 3.82 (s, 3H), 3.50 (d, J = 4.2 Hz, 2H), 3.48-3.24 (m, 6H), 3.07 (m, 2H), 2.30 (m, 2H), 1.89 (m, 2H), 1.69 (m, 4H). ¹³C NMR (CDCl₃, 300 MHz): 167.7, 166.4, 162.5, 141.3, 130.0, 129.3, 123.1, 122.0, 115.7, 113.8, 57.2, 56.2, 55.4, 54.4, 52.7, 51.8, 39.4, 27.7, 26.7, 25.4. HRMS (ESI) Calcd for C₂₃H₃₀FN₃O₂ (M+H)⁺: 400.2400. Found: 400.2415. Anal. (C₂₃H₃₀FN₃O₂·H₂C₂O₄·0.5H₂O) C, H, N.

7.20. N-(4-(4-(2-Methoxyphenyl)-1,4-diazepan-1-yl)butyl)-5-methylthiophene-2-carboxamide(11i)

Compound 11i was prepared according to the procedure for compound 11a except using 5-methylthiophene-2-carboxylic acid, which afforded 100 mg (83%) of 11i as a white solid. TLC R_f 0.21 (dichloromethane methanol 20:1); mp 58.7–59.2 °C. ¹H NMR (300 MHz, CDCl₃) δ 7.40 (d, J = 3.6 Hz, 1H), 6.91-6.84 (m, 5H), 6.65 (d, J = 3.6 Hz, 1H), 3.85 (s, 3H), 3.45 (m, 2H), 3.35-3.26 (m, 4H), 3.06-2.98 (m, 4H), 2.77 (m, 2H), 2.46 (s, 3H), 2.14 (m, 2H), 1.74-1.66 (m, 4H). ¹³C NMR (CDCl₃, 300 MHz): 162.0, 151.7, 144.8, 142.7, 136.6, 128.5, 125.9, 120.9, 120.7, 117.9, 111.5, 57.6, 56.2, 55.6, 54.4, 52.7, 51.9, 39.5, 28.1, 27.6, 25.5. 15.5. HRMS (ESI) Calcd for C₂₂H₃₁N₃O₂S (M+H)⁺: 402.2215. Found: 402.2229. Anal. (C₂₂H₃₁N₃O₂S·H₂C₂O₄·0.5H₂O) C, H, N

7.21. N-(4-(4-(2-Methoxyphenyl)-1,4-diazepan-1-yl)butyl)-1H-indole-2-carboxamide(11j)

Compound 11j was prepared according to the procedure for compound 11a except using indole-2-carboxylic acid, which afford 87 mg (75%) of 11j as a white solid. TLC R_f 0.25 (ethyl acetate methanol 15:1); mp 72.9–74 °C. ¹H NMR (300 MHz, CDCl₃) δ 9.83 (br, 1H), 7.66 (d, J = 8.4 Hz, 1H), 7.46 (d, J = 8.4 Hz, 1H), 7.30 (t, J = 7.5 Hz, 1H), 7.16 (t, J = 7.5 Hz, 1H), 7.02 (m, 1H), 6.9-6.82 (m, 5H), 3.88 (s, 3H), 3.55 (m, 2H), 3.32 (m, 4H), 2.72 (m, 4H), 2.62 (t, J = 6.9 Hz, 2H), 2.02 (m, 2H), 1.73 (m, 4H). ¹³C NMR (CDCl₃, 300 MHz): 162.2, 160.7, 141.3, 139.4, 138.6, 131.1, 123.1, 122.0, 120.7, 119.8, 114.9, 113.5, 111.2, 57.9, 56.2, 55.5, 54.4, 52.7, 51.9, 39.3, 27.7, 26.7, 24.6. HRMS (ESI) Calcd for C₂₅H₃₂N₄O₂ (M+H)⁺: 421.2604. Found: 421.2621. Anal. (C₂₅H₃₂N₄O₂·H₂C₂O₄·0.25H₂O) C, H, N

7.22. N-(4-(4-(2-Methoxyphenyl)-1,4-diazepan-1-yl)butyl)benzo[b]thiophene-2-carboxamide(11k)

Compound 11k was prepared according to the procedure for compound 11a except using thianaphthene-2-carboxylic acid, which afford 81 mg (78%) of 11k as a white solid. TLC R_f 0.32 (ethyl acetate methanol 10:1); mp 66–67 °C. ¹H NMR (300 MHz, CDCl₃) δ 7.86-7.78 (m, 3H), 7.44-7.36 (m, 2H), 7.02-6.84 (m, 5H), 3.85 (s, 3H), 3.51 (q, J = 6.3 Hz, 2H), 3.1 (m, 4H), 2.86 (m, 4H), 2.58 (t, J = 6.9 Hz, 2H), 1.98 (m, 2H), 1.67 (m, 4H). ¹³C NMR (CDCl₃, 300 MHz): 162.6, 161.4, 146.6, 145.7, 141.1, 139.8, 124.3, 123.1, 122.8, 113.7, 57.6, 56.4, 55.3, 54.4, 52.7, 51.9, 39.6, 27.6, 26.8, 25.4. HRMS (ESI) Calcd for C₂₅H₃₁N₃O₂S (M+H)⁺: 438.2215. Found: 438.2236. Anal. (C₂₅H₃₁N₃O₂S·H₂C₂O₄·0.5H₂O) C, H, N.

7.23. N-(4-(4-(2-Methoxyphenyl)-1,4-diazepan-1-yl)butyl)-5-phenylthiophene-2-carboxamide(11l)

Compound 11l was prepared according to the procedure for compound 11a except using 4-phenyl-thiophene-2-carboxylic acid, which afford 80 mg (81%) of 11l as a white solid. TLC R_f 0.22 (dichloromethane methanol 15:1); mp 77–78 °C. ¹H NMR (300 MHz, CDCl₃) δ 7.79 (m, 1H), 7.57-7.53 (m, 3H), 7.40-7.25 (m, 3H), 6.87-6.75 (m, 5H), 3.80 (s, 3H), 3.47 (q, J = 6.3 Hz, 2H), 3.29 (m, 4H), 2.86 (m, 4H), 2.58 (m, 2H), 1.96 (m, 2H), 1.66 (m, 4H). ¹³C NMR (CDCl₃, 300 MHz): 162.4, 151.3, 142.5, 141.1, 140.2, 135.0, 128.9, 127.5, 127.3, 126.2, 124.4, 122.3, 120.9, 117.9, 111.5, 110.0, 57.2, 56.9, 55.3, 54.4, 52.7, 51.9, 38.7, 27.5, 26.4, 25.3. HRMS (ESI) Calcd for C₂₇H₃₃N₃O₂S (M+H)⁺: 464.2372. Found: 464.2358. Anal. (C₂₇H₃₃N₃O₂S·H₂C₂O₄·0.25H₂O) C, H, N.

7.24. 4-(2-Hydroxyethyl)-N-(4-(4-(2-methoxyphenyl)-1,4-diazepan-1-yl)butyl)benzamide(11m)

Compound 11m was prepared according to the procedure for compound 11a except using 14, purified with ether methanol (10:1) to afford 102 mg (84%) of 11m as a white solid. TLC R_f 0.27 (dichloromethane methanol 12:1); mp 52–53 °C. ¹H NMR (300 MHz, CDCl₃) δ 7.77 (d, J = 6.9 Hz, 2H), 7.26 (m, 3H), 6.86 (m, 4H), 3.89-3.82 (m, 5H), 3.48 (q, J = 5.4 Hz, 2H), 3.26 (m, 4H), 3.06-2.98 (m, 4H), 2.89 (m, 2H), 2.76 (m, 2H), 2.09 (m, 2H), 1.77-1.69 (m, 4H). ¹³C NMR (CDCl₃, 300 MHz): 167.5, 152.4, 141.8, 141.2, 131.6, 127.8, 127.4, 123.0, 122.1, 121.9, 113.7, 61.3, 57.5, 56.2, 55.6, 54.4, 52.7, 51.7, 39.4, 38.9, 27.7, 26.5, 25.0. HRMS (ESI) Calcd for C₂₅H₃₅N₃O₃ (M+H)⁺: 426.2757. Found: 426.2742.Anal. (C₂₅H₃₅N₃O₃·H₂C₂O₄) C, H, N.

7.25. N-(4-(4-(2-Methoxyphenyl)-1,4-diazepan-1-yl)butyl)-9-oxo-9H-fluorene-4-carboxamide(11n)

Compound 11n was prepared according to the procedure for compound 11a except using 9-oxofluorene-4-carboxylic acid, which afford 80 mg (81%) of 11n as a white solid. TLC R_f 0.30 (ethyl acetate methanol 10:1); mp 61–62 °C. ¹H NMR (300 MHz, CDCl₃) δ 7.98 (m, 1H), 7.77 (d, J = 7.2 Hz, 1H), 7.69 (d, J = 7.2 Hz, 2H), 7.57 (m, 3H), 7.35-7.25 (m, 3H), 6.87-6.75 (m, 5H), 3.80 (s, 3H), 3.47 (m, 2H), 3.14-3.04 (m, 4H), 2.72 (m, 2H), 2.63 (m, 2H), 2.55 (m, 2H), 1.77-1.69 (m, 6H). ¹³C NMR (CDCl₃, 300 MHz): 193.8, 162.3, 144.6, 141.5, 139.5, 137.0, 134.2, 133.0, 131.8, 130.2, 127.4, 126.8, 123.1, 122.1, 121.9, 113.8, 57.6, 56.5, 55.6, 54.4, 52.7, 51.8, 39.4, 27.5, 25.4, 24.4. HRMS (ESI) Calcd for C₃₀H₃₃N₃O₃ (M+H)⁺: 484.2600. Found: 484.2612. Anal. (C₃₀H₃₃N₃O₃·H₂C₂O₄·0.25H₂O) C, H, N.

7.26. 5-Bromo-N-(4-(4-(2-methoxyphenyl)-1,4-diazepan-1-yl)butyl)thiophene-2-carboxamide(11o)

Compound 11o was prepared according to the procedure for compound 11a except using 5-methyl-thiophene-2-carboxylic acid, which afford 103 mg (81%) of 11o as a white solid. TLC R_f 0.21 (dichloromethane methanol 15:1); mp 69–70 °C. ¹H NMR (300 MHz, CDCl₃) δ 7.35 (d, J = 4.2 Hz, 1H), 7.01-6.87 (m, 6H), 3.85 (s, 3H), 3.44 (m, 2H), 3.22 (m, 4H), 2.85 (m, 2H), 2.77 (m, 2H), 2.60 (m, 2H), 2.00 (m, 2H), 1.69 (m, 4H). ¹³C NMR (CDCl₃, 300 MHz): 162.7, 161.3, 141.4, 140.1, 138.8, 132.5, 124.6, 123.4, 122.1, 121.8, 113.7, 57.6, 56.2, 55.2, 54.4, 52.7, 51.7, 38.6, 26.9, 25.5, 24.5. HRMS (ESI) Calcd for C₂₁H₂₈BrN₃O₂S (M+H)⁺: 466.1164. Found: 466.1182. Anal. (C₂₁H₂₈BrN₃O₂S·H₂C₂O₄·0.5H₂O) C, H, N.

7.27. 5-(4-Fluorophenyl)-N-(4-(4-(2-methoxyphenyl)-1,4-diazepan-1-yl)butyl)thiophene-2-carboxamide(11p)

Compound 11p was prepared according to the procedure for compound 11a except using 5-(4-fluorophenyl)thiophene-2-carboxylic acid, which afford 93 mg (75%) of 11p as a white solid. TLC R_f 0.23 (dichloromethane methanol 15:1); mp 130–131 °C. ¹H NMR (300 MHz, CDCl₃) δ 7.50 (m, 3H), 7.08-6.94 (m, 4H), 6.85-6.76 (m, 4H), 3.85 (s, 3H), 3.34 (m, 2H), 3.20 (m, 4H), 2.99 (m, 2H), 2.93 (m, 2H), 2.67 (m, 2H), 2.03 (m, 2H), 1.67 (m, 4H). ¹³C NMR (CDCl₃, 300 MHz): 163.0, 162.2, 161.3, 148.1, 141.1, 138.2, 137.3, 129.4, 129.1, 123.1, 121.9, 121.1, 116.0, 113.5, 57.6, 56.2, 55.6, 54.4, 52.7, 51.9, 49.1, 39.9, 33.9, 27.5, 26.4, 25.3. HRMS (ESI) Calcd for C₂₇H₃₂FN₃O₂S (M+H)⁺: 482.2278. Found: 482.2259. Anal. (C₂₇H₃₂FN₃O₂S·H₂C₂O₄·0.5H₂O) C, H, N.

7.28. 6-Chloro-N-[4-[4-(2-methoxyphenyl)-1,4-diazepan-1-yl)butyl)nicotinamide (11q)

Compound 11q was prepared according to the procedure for compound 11a except using 6-chloronicotinic acid, which afford 95 mg (82%) of 11q as a white solid. TLC R_f 0.21 (dichloromethane methanol 15:1); mp 43–44 °C. ¹H NMR (300 MHz, CDCl₃) δ 9.00 (d, J = 2.4 Hz, 1H), 8.45 (dd, J = 8.6 Hz, J = 2.4 Hz, 1H), 8.32 (d, J = 8.4 Hz, 1H), 7.35 (d, J = 8.4 Hz, 1H), 7.03-6.84 (m, 4H), 3.85 (s, 3H), 3.49 (m, 2H), 3.39-3.26 (m, 4H), 3.07 (m, 2H), 2.34 (m, 2H), 2.00 (m, 2H), 1.71 (m, 4H). ¹³C NMR (CDCl₃, 300 MHz): 165.7, 162.4, 154.7, 150.4, 141.1, 140.4, 128.4, 126.1, 123.0, 122.1, 121.9, 113.5, 57.6, 56.2, 55.6, 54.4, 52.7, 51.9, 39.9, 27.6, 25.6, 24.4. HRMS (ESI) Calcd for C₂₂H₂₉ClN₄O₂ (M+H)⁺: 417.2057. Found: 417.2049. Anal. (C₂₂H₂₉ClN₄O₂·H₂C₂O₄) C, H, N.

7.29. 6-Bromo-N-[4-[4-(2-methoxyphenyl)-1,4-diazepan-1-yl)butyl)nicotinamide (11r)

Compound 11r was prepared according to the procedure for compound 11a except using 6-bromonicotinic acid, which afford 91 mg (83%) of 11r as a white solid. TLC R_f 0.21 (dichloromethane methanol 15:1); mp 50–51 °C. ¹H NMR (300 MHz, CDCl₃) δ 8.95 (d, J = 2.4 Hz, 1H), 8.36 (dd, J = 8.6 Hz, J = 2.4 Hz, 1H), 8.20 (d, J = 8.4 Hz, 1H), 7.53 (d, J = 8.4 Hz, 1H), 7.03-6.86 (m, 4H), 3.85 (s, 3H), 3.53 (m, 2H), 3.38-3.31 (m, 4H), 3.04 (m, 2H), 2.33 (m, 2H), 1.97 (m, 2H), 1.72 (m, 4H). ¹³C NMR (CDCl₃, 300 MHz): 167.5, 162.3, 141.1, 137.4, 132.4, 130.3, 128.8, 123.0, 122.0, 113.5, 57.6, 56.2, 55.6, 54.4, 52.7, 51.9, 39.5, 27.7, 26.6, 25.4. HRMS (ESI) Calcd for C₂₃H₃₀ClN₃O₂ (M+H)⁺: 416.2105. Found: 416.2129. Anal. (C₂₂H₂₉BrN₄O₂·H₂C₂O₄·0.5H₂O) C, H, N.

7.30. 2,3-Dimethoxy-N-[4-[4-(2-Methoxyphenyl)-1,4-diazepan-1-yl)butyl)benzamide (11s)

Compound 11s was prepared according to the procedure for compound 11a except using 2,3-dimethoxybenzoic acid, which afford 70 mg (79%) of 11s as a white solid. TLC R_f 0.26 (dichloromethane methanol 15:1); mp 43–44 °C. ¹H NMR (300 MHz, CDCl₃) δ 8.05 (m, 1H), 7.67 (d, J = 7.8 Hz, 1H), 7.14 (t, J = 8.1 Hz, 1H), 7.02 (d, J = 7.8 Hz, 1H), 6.91-6.83 (m, 4H), 3.90 (s, 3H), 3.83 (s, 3H), 3.51 (d, J = 4.2 Hz, 2H), 3.33 (m, 4H), 2.99-2.91 (m, 4H), 2.71 (t, J = 6.9 Hz, 2H), 2.01 (m, 2H), 1.67 (m, 4H). ¹³C NMR (CDCl₃, 300 MHz): 169.3, 162.7, 154.0, 149.8, 141.1, 128.5, 123.0, 122.1, 120.8, 118.7, 113.8, 61.0, 57.6, 56.2, 55.6, 54.4, 52.7, 51.9, 39.6, 27.7, 26.8, 24.4. HRMS (ESI) Calcd for C₂₅H₃₅N₃O₄ (M+H)⁺: 442.2706. Found: 442.2718. Anal. (C₂₅H₃₅N₃O₄·H₂C₂O₄·0.25H₂O) C, H, N.

7.31. N-[4-[4-(2-Methoxyphenyl)-1,4-diazepan-1-yl)butyl)-4-(methylamino)benzamide (11t)

Compound 11t was prepared according to the procedure for compound 11a except using 4-(methylamino)benzoic acid, which afford 102 mg (81%) of 11t as a white solid. TLC R_f 0.19 (dichloromethane methanol 15:1); mp 58–59 °C. ¹H NMR (300 MHz, CDCl₃) δ 7.72 (d, J = 6.9 Hz, 2H), 6.94-6.81 (m, 5H), 6.55 (d, J = 6.9 Hz, 2H), 3.82 (s, 3H), 3.45 (m, 2H), 3.34 (m, 2H), 3.28 (m, 2H), 3.07-3.00 (m, 4H), 2.85 (s, 3H), 2.78 (m, 2H), 2.16 (m, 2H), 1.79-1.65 (m, 4H). ¹³C NMR (CDCl₃, 300 MHz): 167.9, 162.1, 154.0, 141.1, 130.4, 123.2, 122.6, 121.4, 113.9, 112.7, 57.6, 56.2, 55.6, 54.4, 52.7, 51.9, 41.4, 39.5, 29.8, 26.7, 25.6, 24.9. HRMS (ESI) Calcd for C₂₄H₃₄N₄O₂ (M+H)⁺: 411.2760. Found: 411.2749. Anal. (C₂₄H₃₄N₄O₂·H₂C₂O₄·0.5H₂O) C, H, N.

7.32. Dopamine receptor binding assays

A filtration binding assay was used to characterize the binding properties of membrane-associated receptors.²⁶ For human D_{2 long}, D₃, and D₄ dopamine receptors expressed in HEK 293 cells, membrane homogenates (50 μL) 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. Non-specific binding was defined using 20 μM (+)-butaclamol. For competition experiments the radioligand concentration is generally equal to 0.5 times the K_d value and the concentration of the competitive inhibitor ranges over 5 orders of magnitude. Each competition curve was performed using two concentrations of inhibitor per decade and all assays are performed in triplicate. Binding was terminated by the addition of cold wash buffer (10 mM Tris HCl/150 mM NaCl, pH 7.5) and filtration over a glass-fiber filter (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.³⁰ Data from competitive inhibition experiments were modeled using nonlinear regression analysis to determine the concentration of inhibitor that inhibits 50% of the specific binding of the radioligand. Competition curves were modeled for a single site and the IC₅₀ values will be converted to equilibrium dissociation constants (K_i values) using the Cheng and Prusoff equation.³¹ Mean K_i values ± S.E.M are reported for at least three independent experiments.