Dopamine D3/D2 Receptor Ligands Based on Cariprazine for the Treatment of Psychostimulant Use Disorders that May be Dual Diagnosed with Affective Disorders

Emma S Gogarnoiu; Caleb D Vogt; Julie Sanchez; Alessandro Bonifazi; Elizabeth Saab; Anver Basha Shaik; Omar Soler-Cedeño; Guo-Hua Bi; Benjamin Klein; Zheng-Xiong Xi; J Robert Lane; Amy Hauck Newman

doi:10.1021/acs.jmedchem.2c01624

. Author manuscript; available in PMC: 2024 May 17.

Published in final edited form as: J Med Chem. 2023 Jan 20;66(3):1809–1834. doi: 10.1021/acs.jmedchem.2c01624

Dopamine D₃/D₂ Receptor Ligands Based on Cariprazine for the Treatment of Psychostimulant Use Disorders that May be Dual Diagnosed with Affective Disorders

Emma S Gogarnoiu ^†,^¶, Caleb D Vogt ^†,^¶, Julie Sanchez ^‡,^§,^¶, Alessandro Bonifazi ^†, Elizabeth Saab ^†, Anver Basha Shaik ^†, Omar Soler-Cedeño ^†, Guo-Hua Bi ^†, Benjamin Klein ^†, Zheng-Xiong Xi ^†, J Robert Lane ^‡,^§, Amy Hauck Newman ^†,^*

PMCID: PMC11100975 NIHMSID: NIHMS1989580 PMID: 36661568

Abstract

Highly selective dopamine D₃ receptor (D₃R) partial agonists/antagonists have been developed for the treatment of psychostimulant use disorders (PSUD). However, none have reached the clinic due to insufficient potency/efficacy or potential cardiotoxicity. Cariprazine, an FDA approved drug for the treatment of schizophrenia and bipolar disorder, is a high affinity D₃R partial agonist (K_i = 0.22 nM) with 3.6-fold selectivity over the homologous dopamine D₂ receptor (D₂R). We hypothesized that compounds that are moderately D₃R/D₂R selective partial agonists/antagonists, may be effective for treatment of PSUD. By systematically modifying the parent molecule, we discovered partial agonists/antagonists, as measured in BRET-based assays, with high D₃R affinities (K_i = 0.14–50 nM) and moderate selectivity (<100-fold) over D₂R. Cariprazine and two lead analogues, 13a and 13e, decreased cocaine self-administration (FR2; 1–10 mg/kg, i.p.) in rats, suggesting that partial agonists/antagonists with modest D₃R/D₂R-selectivity may be effective in treating PSUD and potentially comorbidities with other affective disorders.

Keywords: Dopamine D₃ receptors, Dopamine D₂ receptors, bitopic ligands, substance use disorders, psychostimulant use disorder, cocaine, affective disorders, schizophrenia, bipolar disorder, cariprazine, self-administration, reward

Graphical Abstarct

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INTRODUCTION

In the midst of the COVID-19 pandemic, drug overdose fatalities soared to >100,000 in 2021.¹ While opioids are involved with a majority of these deaths, polysubstance use is another contributing factor, leading to increases in morbidity that poses further challenges for prevention and treatment.² To further complicate matters, substance use disorders (SUDs) frequently occur with affective disorders, including anxiety, major depressive disorder, and bipolar disorder. Such dual disorders have also increased during the COVID-19 pandemic^3–5 and are particularly difficult to treat when comorbid with psychostimulant use disorder (PSUD),^{6, 7} for which there is currently no FDA-approved pharmacotherapeutic.

Development of highly selective dopamine D₃ receptor (D₃R) partial agonists/antagonists for the treatment of SUD has been of great interest due to their ability to attenuate drug reinforcement as well as inhibit cue- and stress-induced reinstatement for psychostimulants and opioids in animal models.^8–11 D₃R-selective antagonists, specifically, have been shown to block the expression of cocaine- or heroin-induced conditioned place preference and inhibit the rewarding effects of these drugs.^12–14 In addition to promising preclinical work, gaining D₃R selectivity over the dopamine D₂ receptor (D₂R) may avoid the extrapyramidal side effects, weight gain, metabolic disorders, and motor coordination deficits associated with D₂R antagonism.^15–20 To this end, highly selective compounds (Figure 1), such as SB277011A (>100-fold),²¹ PG01037 (>100-fold),²² (±)-VK4–116 (1,700-fold),^{23, 24} (±)-ABS-1–113 (>300-fold),²⁵ and BP1.4979 (~200-fold),²⁶ have been discovered in spite of the ~80% homology between the D₂R and D₃R subtypes.^27–29

Figure 1. — Structures of representative D₃R-selective partial agonists/antagonists.

While highly D₃R-preferential antagonists and low-efficacy partial agonists demonstrate potential as treatments for SUD,²⁹ there are two challenges that still need to be addressed. The first is that these compounds generally show favorable results in animal models of opioid use disorder but are often not as effective in mitigating cocaine or methamphetamine self-administration, especially at low fixed-ratio schedules (e.g., FR1 or FR2) of reinforcement.^{18, 29} Secondly, relatively high doses (e.g., 10–56 mg/kg, i.p.) are required to observe reductions in drug-seeking behaviors despite having very high affinities (K_i values in the low nM to pM range) for the desired target, D₃R. These challenges have led us to reconsider whether some D₂R agonist activity would improve both the efficacy and potency of our compounds. Bearing in mind that high affinity D₂R antagonists would likely cause negative side effects, including neuroleptic dysphoria³⁰ (especially in a patient population with SUD), we sought to design molecules with D₃R and D₂R agonist activities and binding selectivities in the 10- to 100-fold range. Ultimately, the goal of this study was to identify a pharmacological profile that would not only have promise for treating patients with PSUD but might also be efficacious in patients with dual diagnoses.

Of note, there is one D₃R-preferential agonist that is in clinical use for the treatment of schizophrenia and more recently, bipolar disorder. Cariprazine (see Figure 1), formerly described in the literature as RGH188, is a high affinity D₂R/D₃R agonist with preference for the D₃R (3- to 10-fold).^31–34 Most D₂-like antipsychotics on the market generally reduce positive symptoms (e.g., delusions, disorganized speech, and hallucinations) of schizophrenia and manic episodes of bipolar disorder but are not effective at ameliorating the spectrum of other debilitating symptoms.³⁴ However, cariprazine appears to be unique in that it also improves the negative (e.g., anhedonia, social and emotional withdrawal) and cognitive symptoms (e.g., attention deficit and executive function impairment) while significantly decreasing global illness severity of schizophrenia.³⁴ Cariprazine can effectively treat the manic and mixed episodes without induction of depression in bipolar 1 disorder (BD-I).^{35, 36} Most patients with BD-I take atypical antipsychotics in combination with an antidepressant to treat both poles of the disorder.³⁶ It is believed that these therapeutic benefits are associated with cariprazine’s profile as a relatively modest D₃R preferential agonist.^{34, 37, 38}

Herein, we report the design of cariprazine-based analogues resulting in a wide range of D₃R/D₂R selectivities while maintaining high (low nM to pM) binding affinities at D₃R. These ligands are considered bitopic, meaning they include a primary pharmacophore (PP), which binds to the orthosteric binding site, that is then linked to a secondary pharmacophore (SP), which binds to D₃R-unique secondary binding pocket (see Figure 1).³⁹ By changing the PP, SP, and linker portions of various bitopic ligands, we have previously altered their D₃R/D₂R selectivities and functional efficacies.^{17, 40–42} Recent work by Keserű and coworkers, in particular, suggests that adjustments to the aliphatic SP of cariprazine have the potential to “tune” efficacy by repositioning the molecule in the orthosteric binding site through its interactions with the secondary binding pocket.^{43, 44} Further investigation of this hypothesis with additional aliphatic SPs will be of interest to support these findings. Hence, using the bitopic ligand design, we explored different modifications to the 2,3-dichlorophenylpiperazine (PP), trans-1,4-cyclohexane ring system (linker), and N,N-dimethylurea (SP) of cariprazine. Analogues were tested for D₂R and D₃R binding affinities in transfected HEK293 cells, and a subset was selected for functional bioluminescence resonance energy transfer (BRET) assays to assess their intrinsic efficacy. Based on the results of these experiments and additional off-target studies, two analogues (13a and 13e) along with cariprazine were evaluated for effectiveness in reducing cocaine self-administration in rats.

RESULTS AND DISCUSSION

Chemistry

We modified the PP and SP of cariprazine following the synthetic routes outlined in Scheme 1. Initially, 2-(trans-4-((tert-butoxycarbonyl)amino)cyclohexyl)acetic acid (1) was reduced with borane-dimethyl sulfide (BMS) complex, and the resulting alcohol (2) was converted to aldehyde 3 via Swern oxidation. In the next step, primary pharmacophores were installed by reductive amination using various aryl piperazines (4a-e).⁴² Deprotection of these intermediates with trifluoroacetic acid (TFA) gave amines 5a-e, which were then functionalized with different SPs. To that end, cariprazine and substituted ureas 6b-7e were prepared from either N,N-dimethyl- or N-methylcarbamyl chloride. Other urea-containing analogues were accessed by treating a selection of the aforementioned amines (5a, 5b, and 5e) with potassium cyanate under acidic conditions (8a and 8e) or phosgene followed by 5,6,7,8-tetrahydro-1,6-naphthyridine (9a and 9b).⁴⁵ Lastly, amides 10a-13e were synthesized from a variety of carboxylic acids with either 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide•HCl (EDCI) or O-(1H-6-chlorobenzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU) serving as the coupling reagent.

Manipulations to the linker between the primary and secondary pharmacophores of cariprazine were additionally explored. As shown in Scheme 2, the cyclohexylamine was replaced with a piperidine ring using one of two aldehydes. The first (17a) retained the two-carbon chain found in cariprazine and was prepared from tert-butyl 4-oxopiperidine-1-carboxylate (14). Briefly, a Horner–Wadsworth–Emmons reaction furnished α,β-unsaturated ester 15, which was sequentially reduced by hydrogenation over palladium on carbon (16) and DIBALH to afford 17a. The second aldehyde (17b), possessing an extended carbon chain, was obtained using a Swern oxidation on commercially available tert-butyl 4-(3-hydroxypropyl)piperidine-1-carboxylate (18). With these materials in hand, we followed the same steps described in Scheme 1 to prepare analogues 21a-22b (i.e., reductive amination, Boc-deprotection of 19a and 19b, followed by functionalization of amines 20a and 20b).

Expanding on the previous set of compounds, we also introduced a hydroxyl group onto the linker, while simultaneously adjusting the ring size (Scheme 3). In this case, cariprazine analogues were constructed from building blocks 26a and 26b, which were accessed using a parallel sequence of transformations. Specifically, a three-carbon chain was introduced onto tert-butyl 3-oxopyrrolidine-1-carboxylate (23a) and tert-butyl 4-oxopiperidine-1-carboxylate (23b) through separate Grignard reactions with allyl magnesium bromide. The terminal alkenes of 24a and 24b were then subjected to hydroboration–oxidation reactions, and the corresponding alcohols (25a and 25b) were converted to hemiacetals 26a and 26b. Like before (see Schemes 1 and 2), the synthesis of analogues 29a-30b proceeded through intermediates 27a-28b.

Scheme 3. — Preparation of compounds bearing a hydroxyl group within the linker^a

^a Reagents and Conditions: (a) allyl magnesium bromide, Et₂O, 0 °C to rt; (b) BH₃•THF, THF, 0 °C to rt, then NaOH, H₂O₂, H₂O, 0 °C to rt; (c) DMSO, (COCl)₂, NEt₃, DCM, −78 °C to rt; (d) 1-(2,3-dichlorophenyl)piperazine•HCl, NaBH(OAc)₃, DCE, rt; (e) TFA, DCM, rt; (f) N,N-dimethylcarbamyl chloride, DIPEA, DCM, rt; (g) 3-methoxyproponic acid, HCTU, DIPEA, DCM, rt.

Binding affinities of cariprazine and its analogues at D₂R and D₃R

To investigate the structure-activity relationships (SAR) of our cariprazine analogues, we determined their affinities at D₂R and D₃R. Competitive binding experiments using membrane preparations from stably transfected HEK293 cells expressing human D_2L and D₃ receptors were performed with [³H]N-methylspiperone serving as the radioligand.⁴⁶ The K_i value for each compound is presented in Tables 1 and 2 along with the corresponding multiparameter optimization (MPO) scores, which are a prediction of brain penetrability.^{47, 48} For our purposes, derivatives with MPO scores >3 were considered as potentially drug-like.

Table 1.

D₃R and D₂R binding affinities of cariprazine and its analogues.^a


compound	Ar	R	MPO	K_i ± SEM (nM)		D₂R K_i / D₃R K_i
compound	Ar	R	MPO	D₂R	D₃R	D₂R K_i / D₃R K_i
4a			2.8	8.2 ± 2.6	2.13 ± 0.85	3.8
cariprazine			3.6	0.78 ± 0.17	0.22 ± 0.06	3.6
7a			3.5	1.21 ± 0.06	0.21 ± 0.02	5.7
8a			3.8	1.49 ± 0.03	0.26 ± 0.10	5.7
9a			2.8	1.86 ± 0.06	0.36 ± 0.05	5.1
10a			2.5	6.5 ± 1.9	0.31 ± 0.10	21
11a			2.8	29 ± 13	0.66 ± 0.28	44
12a			2.8	3.75 ± 0.65	0.59 ± 0.13	6.4
13a			3.6	2.85 ± 0.63	0.14 ± 0.05	20
6b			3.5	0.75 ± 0.07	0.26 ± 0.03	2.9
7b			3.4	0.48 ± 0.11	0.18 ± 0.01	2.6
9b			2.8	0.87 ± 0.08	0.29 ± 0.08	3.0
10b			2.5	7.8 ± 1.9	1.31 ± 0.46	6.0
11b			2.8	5.76 ± 0.93	0.34 ± 0.06	17
12b			2.8	1.80 ± 0.34	0.58 ± 0.07	3.1
13b			3.5	1.78 ± 0.18	0.25 ± 0.04	7.1
6c			5.2	134 ± 17	9.4 ± 1.2	14
13c			5.1	479 ± 26	1.31 ± 0.02	370
6d			4.7	26.7 ± 5.4	2.38 ± 0.54	11
11d			2.6	162 ± 48	1.00 ± 0.08	160
13d			4.6	127 ± 14	1.06 ± 0.08	120
4e			3.1	68.1 ± 4.3	38.8 ± 3.0	1.8
6e			3.4	4.12 ± 0.17	1.76 ± 0.11	2.3
7e			3.3	5.8 ± 1.3	0.64 ± 0.10	9.1
8e			3.5	5.3 ± 1.8	0.78 ± 0.28	6.8
10e			2.3	114 ± 36	1.03 ± 0.26	110
13e			3.3	15.2 ± 2.0	0.73 ± 0.16	21

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K_i values are derived from IC₅₀ values using the Cheng-Prusoff equation,⁵⁰ and calculated as the mean of at least three independent experiments. The radioligand used in these assays was [³H]-N-methylspiperone.

Table 2.

D₃R and D₂R binding affinities of linker modified compounds.^a


compound	linker	R	MPO	K_i ± SEM (nM)		D₂R K_i / D₃R K_i
compound	linker	R	MPO	D₂R	D₃R	D₂R K_i / D₃R K_i
21a			3.9	20.5 ± 2.1	1.02 ± 0.10	20
22a			4.4	86 ± 28	3.5 ± 1.3	25
21b			3.4	7.2 ± 1.1	1.86 ± 0.10	3.9
22b			3.7	19.4 ± 2.6	0.96 ± 0.05	20
29a			5.3	89.8 ± 5.9	29.5 ± 3.7	3.0
30a			5.1	175 ± 19	18.7 ± 2.5	9.4
29b			5.0	38.4 ± 5.2	50.7 ± 5.7	0.76
30b			5.1	125 ± 27	27.5 ± 3.3	4.5

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In the first series of compounds, we focused on modifying the SP (blue) of cariprazine (Table 1, 4a, cariprazine, and 7a-13a). Compared to the parent compound, similar D₃R binding affinities and selectivities were observed for the primary metabolites, 7a and 8a.⁴⁹ When the N,N-dimethylamino group of the SP was replaced with different heterocycles (9a-12a), D₃R affinity was retained (K_i = 0.31–0.66 nM), while selectivity over D₂R increased from 5- to 44-fold. Importantly, the analogue inspired by BP1.4979 (13a) exhibited similar affinity (K_i = 0.14 nM) but better selectivity (20-fold) for D₃R than cariprazine, while simultaneously maintaining a desirable MPO score (3.6).

The next several series of compounds explored different PPs (red) in varying combinations with the aforementioned SPs. As shown in Table 1, the 2,3-dichlorophenyl was substituted with a 2-chloro-3-ethylphenyl (6b, 7b, and 9b-13b) derived from our previously reported D₃R agonists.^{23, 25} Although high D₃R affinities were achieved (K_i = 0.18–1.31 nM), none of these analogues were substantially more selective than cariprazine, and most had poor MPO scores (<3). Conversely, the 2-fluoro-3-methoxyphenyl analogue (6c) noticeably decreased D₃R and D₂R affinities (K_i = 9.4 and 134 nM, respectively). Of note, compound 13c showed much greater D₃R/D₂R selectivity than any of the analogues in this series, which was a bit surprising. We have previously reported that D₂R agonists are sensitive to the radioligand they are displacing and typically show much lower affinities when tested against a D₂-like antagonist (e.g., [³H]-N-methylspiperone).^{51, 52} We considered that the 2-fluoro-3-methoxy-substituted phenyl piperazine PP might be conferring agonist actions and thus binding affinities using the D₂-like agonist [³H]7-OH-DPAT were tested (Supporting Information, Table S1). Indeed, we discovered that D₃R selectivities were much lower in this binding assay (D₂R K_i/D₃RK_i = 0.9 and 15 for 6c and 13c, respectively), suggesting these analogues were agonists at D₂R and possibly D₃R. The 2-fluoro-3-methoxy-containing PP also caused a loss in affinity when the N,N-dimethylurea was replaced with the 3-methoxypropanamide (13c), and further analogues were not pursued. The 2-trifluoromethyl substituted pyridine analogues (6d, 11d, and 13d) showed promising increases in D₃R selectivity. Specifically, SPs with the 4-quinoline (11d) and 3-methoxypropanamide (13d) showed a 160- and 120-fold D₃R/D₂R selectivity, respectively. However, replacing the 2,3-dichlorophenyl substituent with the 3-chloro-5-ethyl-2-methoxyphenyl group (4e, 6e-8e, 10e, and 13e) discovered in the highly D₃R-selective VK4–116²³ resulted in D₃R affinities and selectivities over D₂R that were only modestly different from the parent compound, unless the SP contained an indole (10e). Nevertheless, the 3-methoxypropanamide SP in this series gave compound 13e with a 21-fold D₃R/D₂R selectivity, subnanomolar affinity for D₃R (K_i = 0.73 nM), and potentially drug-like MPO score (3.3).

For the last series of analogues, the terminal urea of cariprazine was incorporated into the trans-1,4-cyclohexane ring system of the linker. As shown in Table 2, analogues 21a and 22a retained the two-carbon chain between the 6-membered ring and the PP, which resulted in ~20-fold D₃R selectivity over D₂R regardless of the SP. When the linking chain was increased by an additional methylene group, the D₃R binding affinity for 21b, which is a constitutional isomer of cariprazine, was reduced by ~9-fold compared to the parent drug. Interestingly, only the 3-methoxypropanamide containing analogue (22b) retained the ~20-fold D₃R selectivity. If a hydroxyl group was appended to the linking chain between the PP and SP (29a, 30a, 29b, and 30b), the MPO scores noticeably improved to ≥5, but the D₃R affinities were substantially reduced (K_i = 18.7–50.7 nM). The D₃R/D₂R selectivities for these analogues were also undesirable (<10-fold), so the synthesis of additional analogues containing this hydroxyl group was not pursued further.

Characterization of selected compounds in assays of D₂R and D₃R activation

With the binding studies and SAR established, we then characterized the abilities of 14 selected ligands to signal through both D₂R and D₃R along with quinpirole and dopamine as reference agonists and haloperidol as a reference antagonist.

To measure D₂R activation, we used two BRET assays. The first consisted of a relatively amplified and sensitive G protein activation (GPA) assay that measures Gβγ release from Gα_oA, which allows detection of signals from less efficacious agonists (Table 3, Figure 2A, C and E). The second measured recruitment of a truncated, soluble ‘mini’ Gi-protein fused with a Venus fluorescent protein (mGsi-Venus) to a D_2LR fused with a nanoluciferase (D_2LR-Nluc). This less amplified, proximal assay allows us to distinguish between full agonists and more efficacious agonists (miniG assay, Table 3, Figure 2B, D and F) that gave a maximal response relative to the reference agonist quinpirole in the GPA assay.^53–55

Table 3.

The maximal effects (E_max) and potencies (EC₅₀) of compounds determined in two assays of D₂R activation.^a

compound	D₂R GPA Gαo_A			D₂R mini G_si recruitment
compound	pEC₅₀ ± SEM	EC₅₀ (nM)	E_max ± SEM (%)	pEC₅₀ ± SEM	EC₅₀ (nM)	E_max ± SEM (%)
cariprazine	8.87 ± 0.08	1.35	94.2 ± 1.7	8.87 ± 0.28	1.34	22.5 ± 1.7
13a	8.77 ± 0.10	1.70	94.7 ± 2.2	8.65 ± 0.27	2.26	18.4 ± 1.4
11b	7.37 ± 0.19	42.60	41.4 ± 3.0	ND	ND	ND
13b	8.22 ± 0.10	5.98	75.0 ± 2.3	ND	ND	ND
6c	9.57 ± 0.15	0.27	102.3 ± 3.6	8.39 ± 0.06	4.10	92.4 ± 1.6
13c	9.13 ± 0.13	0.75	96.4 ± 3.3	7.96 ± 0.06	11.02	90.6 ± 2.0
11d	<7	>100	<60%	ND	ND	ND
13d	7.57 ± 0.15	27.12	64.3 ± 3.2	ND	ND	ND
6e	8.14 ± 0.14	7.18	74.3 ± 3.1	ND	ND	ND
7e	8.30 ± 0.11	5.00	95.9 ± 2.9	8.15 ± 0.43	7.08	11.1 ± 1.9
13e	8.28 ± 0.11	5.22	93.0 ± 2.9	7.58 ± 0.21	26.21	12.5 ± 0.9
21a	8.40 ± 0.10	3.97	95.9 ± 2.8	7.93 ± 0.12	11.86	36.7 ± 1.5
22a	8.20 ± 0.06	6.28	94.5 ± 1.9	7.66 ± 0.11	21.85	25.1 ± 0.9
22b	7.96 ± 0.08	10.89	86.7 ± 2.4	8.00 ± 0.18	9.92	15.4 ± 1.0
Quinpirole	9.03 ± 0.05	0.93	100.1 ± 1.3	7.40 ± 0.08	39.78	100.0 ± 2.7
Dopamine	-	-	-	7.18 ± 0.03	66.25	99.9 ± 1.1

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Compounds were tested in an amplified Gα_oA G protein (GPA Gα_oA) activation assay, shown on the left columns, and a less amplified miniG recruitment assay, shown on the right columns. ND indicates no agonist response detected, ‘-’ indicates compound not tested. Data represent the mean ± SEM of three independent experiments performed in duplicate.

Figure 2. — Two differents BRET assays were used to assess D₂R activation: an amplified Gα_oA G protein (GPA Gα_oA) activation assay, shown on the left panels, and a less amplified miniG recruitment assay shown on the right panels. Each panel shows concentration response curves for a subset of the 14 selected ligands: 2,3-dichlorophenyl, 2-chloro-3-ethylphenyl, and 2-fluoro-3-methoxyphenyl compounds **(A)** GPA Gαo_A and **(B)** miniG_si; 2-trifluoromethyl substituted pyridine and 3-chloro-5-ethyl-2-methoxyphenyl compounds **(C)** GPA Gαo_A and **(D)** miniG_si; linker modified compounds **(E)** GPA Gα_oA and **(F)** miniG_si. Data points represent the mean ± SEM of three independent experiments performed in duplicate.

As expected, the reference agonist quinpirole displayed 43-fold lower potency in the miniG assay as compared to the GPA assay consistent with a lower degree of signal amplification. Quinpirole and dopamine displayed equivalent maximal responses in the miniG assay (Table 3). All ligands tested retained D₂R agonism to various levels of intrinsic efficacy. The 2,3-dichlorophenyl compounds (cariprazine and 13a) are D₂R agonists (Figure 2B, E_max value of 22.5% and 18.4% of the dopamine maximal response respectively in miniGsi recruitment assay) but displayed a maximal effect similar to quinpirole in the GPA assay. The 2-chloro-3-ethylphenyl compounds (11b and 13b) are weak agonists at D₂R displaying lower efficacy than cariprazine with submaximal responses in the GPA assay (41% and 75% of quinpirole response, respectively) and no detectable effect in the miniG assay.^{40, 42} The 2-fluoro-3-methoxyphenyl compounds (6c and 13c) displayed a maximal effect similar to that of dopamine and quinpirole in both assays indicating they are full D₂R agonists. Indeed, these data corroborated the binding data obtained using [³H]7-OH-DPAT as the radioligand, as described above (see Table S1). The 2-trifluoromethyl substituted pyridine compounds (11d and 13d) were both weak agonists displaying lower efficacy than cariprazine in both assays. The 3-chloro-5-ethyl-2-methoxyphenyl compounds (6e, 7e, and 13e) were also weak agonists displaying lower maximal effects than both quinpirole and cariprazine, but with higher potency than the 2-trifluoromethyl substituted pyridine compounds. Finally the modified linker compounds (21a, 22a, and 22b) all behaved as weak agonists in the miniG assay, displaying slightly higher (21a, 38% of dopamine E_max), similar (22a, 25%), and lower (22b, 15%) efficacy as compared to cariprazine (23%). Thus, apart from the compounds with the 2-fluoro-3-methoxyphenyl PP, all compounds exhibited weak agonism at the D₂R like cariprazine but with subtle differences in maximal effects across the compound set. As we have previously suggested,⁴¹ the efficacies of D₂R and D₃R compounds can be modified through modifications to PP, linker, and SP. As there is a range of potencies and efficacies in this series of analogues, we now have the tools necessary to determine the optimal pharmacological profile, including binding affinities, selectivities and functional efficacies at both D₂R and D₃R.

All 14 selected compounds were then tested for D₃R signaling profiles with the aim of selecting agonists or antagonists. The same GPA assay overexpressing Gα_oA was used to investigate D₃R agonism as the D₃R has been shown to selectively couple to this G protein α subunit (Table 4, Figure 3A, C and E).⁵⁶ The 2,3-dichlorophenyl compounds (cariprazine and 13a) both behaved as D₃R agonists in this assay with similar maximal effects (45% and 49% of quinpirole maximal effect), suggesting that this GPA assay would be suitable to detect D₃R agonism with a similar or lower level than that of cariprazine. The 2-fluoro-3-methoxyphenyl compounds did not display the desired weak agonist/antagonist properties at the D₃R but instead behaved as full agonists at D₃R in this assay, consistent with their action at the D₂R. The modified linkers compounds were all D₃R agonists with 21a exhibiting the strongest response with a higher efficacy than cariprazine (Figure 3E, E_max value of 73.2 %). Finally, no agonist response was detected for the 2-chloro-3-ethylphenyl (11b and 13b), the 2-trifluoromethyl substituted pyridine (11d and 13d), and the 3-chloro-5-ethyl-2-methoxyphenyl compounds (6e, 7e, and 13e).

Table 4.

The maximal effects (E_max) and potencies (EC₅₀ and IC₅₀) of compounds determined in an assay of D₃R activation.^a

compound	D₃R GPA Gα_oA agonism			D₃R GPA Gα_oA antagonism
compound	pEC₅₀ ± SEM	EC₅₀ (nM)	E_max ± SEM (%)	pIC₅₀ ± SEM	IC₅₀ (nM)
cariprazine	8.72 ± 0.14	1.89	45.1 ± 1.6	8.70 ± 0.32	2.02
13a	8.80 ± 0.20	1.60	49.3 ± 2.3	8.34 ± 0.23	4.62
11b	ND	ND	ND	7.44 ± 0.25	36.00
13b	ND	ND	ND	8.73 ± 0.16	1.87
6c	8.79 ± 0.14	1.64	95.4 ± 3.8	-	-
13c	8.54 ± 0.16	2.87	104.8 ± 4.8	-	-
11d	ND	ND	ND	7.47 ± 0.15	33.63
13d	ND	ND	ND	8.49 ± 0.24	3.21
6e	ND	ND	ND	7.87 ± 0.13	13.41
7e	ND	ND	ND	7.70 ± 0.19	19.85
13e	ND	ND	ND	7.76 ± 0.13	17.45
21a	8.22 ± 0.20	6.07	73.2 ± 4.9	-	-
22a	7.70 ± 0.13	19.91	41.5 ± 1.9	7.92 ± 0.35	12.12
22b	7.80 ± 0.28	16.02	33.0 ± 2.9	7.89 ± 0.37	12.93
Quinpirole	8.59 ± 0.06	2.59	100.1 ± 1.7	-	-
Haloperidol	-	-	-	8.60 ± 0.08	2.54

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Compounds were tested in an amplified Gα_oA G protein (GPA Gα_oA) activation assay as agonists and antagonists. ND indicates no agonist response detected, ‘-’ indicates compound not tested. Data represent the mean ± SEM of three independent experiments performed in duplicate.

Figure 3. — Using the Gα_oA assay, all ligands were tested for D₃R agonism, shown on the left panels and 11 out of the 14 selected ligands were tested for D₃R antagonism (compounds were added to cells together with and EC₅₀ concentration (3 nM) of quinpirole), shown on the right panels. 6c, **13c**, and **21a** signaled as robust D₃R agonists and were thus not tested as antagonists as they would not displace quinpirole signal. Each panel shows concentration response curves for a subset of the selected ligands: 2,3-dichlorophenyl, 2-chloro-3-ethylphenyl, and 2-fluoro-3-methoxyphenyl compounds **(A)** D₃R agonism and **(B)** D₃R antagonism; 2-trifluoromethyl substituted pyridine and 3-chloro-5-ethyl-2-methoxyphenyl compounds **(C)** D₃R agonism and **(D)** D₃R antagonism; linker modified compounds **(E)** D₃R agonism and **(F)** D₃R antagonism. Data points represent the mean ± SEM of three independent experiments performed in duplicate.

To quantify D₃R antagonism, a competitive assay was used where test compounds were added together with an EC₅₀ concentration of quinpirole to assess their ability to displace and antagonize the agonist response (Table 4, Figures 3B, 3D, and 3F). Haloperidol was used as a reference D₃R antagonist. Compounds that signaled as robust D₃R agonists (6c, 13c, and 21a) were not tested as antagonists. Out of the 11 remaining compounds, all showed some antagonist activity, with the agonists acting to reduce the quinpirole response down to their level of agonism (around 25% for cariprazine and 13a, around 30% for 22a and 22b). The most potent antagonists were 13b, 13d, and 13a, showing similar potencies to haloperidol. In addition, 13a showed a very similar signaling profile to cariprazine (weak agonist with similar efficacy at both the D₂R and D₃R) but with improved selectivity for D₃R. Thus, this compound, like 13e, is a agonist at D₂R (with 2-fold lower maximal effect than cariprazine) in the miniG assay but acted as a D₃R antagonist.

Off-target data for cariprazine and related derivatives

Eleven compounds with a range of selectivities for D₃R over D₂R (between 7.1- to 370-fold) were then evaluated for off-target binding affinities (Table 5) and functional potencies/efficacies (Supporting Information, Tables S2–S5) against cariprazine. All tested analogues were selective for D₃R over other dopamine receptors (i.e., D₁R and D₄R), exhibiting markedly lower affinities for these two subtypes (K_i ≥ 3 μM and 200 nM, respectively). Like cariprazine, several derivatives (e.g., 13a, 13b, and 13d) exhibited high binding affinities for the 5-HT_1A receptor (K_i <10 nM), which is a key target in the treatment of schizophrenia.^{57, 58} Interestingly, 5-HT_1A affinity decreased by 18-fold between 13a and 13e (K_i = 6.0 vs. 108 nM, respectively) when the classic 2,3-dichlorophenyl containing PP was replaced by a 3-chloro-5-ethyl-2-methoxyphenyl group. In general, compounds were far more selective for D₃R over 5-HT_2A and 5-HT_2C (≥216- and ≥253-fold, respectively). The only exceptions to this trend were 11b and 13b, which were not pursued further. The full subset of cariprazine analogues also displayed high affinities for 5-HT_2B (K_i ≤ 34.2 nM). Although activation of this serotonin receptor subtype has been associated with cardiotoxic effects,⁵⁹ all tested compounds behaved as 5-HT_2B antagonists in the functional assays, including cariprazine itself (see Table S5). Therefore, none of our analogues are expected to induce cardiotoxicity from 5-HT_2B stimulation.

Table 5.

Off-target binding affinities of cariprazine-based analogues.^a

compound	K_i ± SEM (nM)
compound	D₁R^b	D₄R^c	5-HT_1A^d	5-HT_2A^e	5-HT_2B^e	5-HT_2C^e
cariprazine	2920 ± 300	233 ± 20	1.81 ± 0.46	568 ± 72	0.151 ± 0.023	221.1 ± 8.0
11a	>3160	1390 ± 480	184 ± 25	1810 ± 640	1.99 ± 0.62	644 ± 58
13a	4600 ± 400	756 ± 63	6.0 ± 1.8	54 ± 15	1.47 ± 0.36	252 ± 64
11b	5390 ± 730	417 ± 40	75 ± 25	15.1 ± 4.8	1.27 ± 0.39	253 ± 86
13b	6800 ± 480	342 ± 15	7.7 ± 1.8	20.8 ± 6.9	1.71 ± 0.43	34.0 ± 8.1
6c	>10000	1257 ± 23	33.8 ± 5.9	>10000	34.2 ± 9.3	>10000
13c	>10000	734 ± 16	69.9 ± 1.7	>10000	29.6 ± 9.5	>10000
11d	>3160	1790 ± 430	22.3 ± 6.4	1450 ± 340	18.8 ± 6.2	5800 ± 1300
13d	>10000	2520 ± 170	9.6 ± 2.6	4420 ± 340	11.3 ± 2.2	3170 ± 200
6e	>10,000	2434± 87	45.4 ± 9.0	680 ± 200	7.3 ± 1.9	1062 ± 86
7e	8000 ± 1100	1346 ± 29	54.9 ± 4.2	207 ± 74	6.2 ± 1.5	162 ± 48
13e	>10,000	1870 ± 240	108 ± 11	158 ± 45	5.8 ± 1.7	800 ± 280

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Data were obtained through the NIDA Addiction Treatment Discovery Program Contract (ADA12013) with Oregon Health & Science University. The radioligands used in these assays were

[³H]-SCH23390,

[³H]-spiperone,

[³H]-8-OH-DPAT, and

[³H]5-HT.

Effects of cariprazine and selected analogues on cocaine self-administration in rats

Cariprazine was tested in a rat model of cocaine self-administration under a fixed-ratio 2 (FR2, i.e., two-active lever presses lead to one cocaine infusion) schedule of reinforcement. Figure 4A shows dose-dependent biphasic effects of cariprazine on cocaine self-administration, specifically an increase at a very low dose (0.3 mg/kg) and a decrease at the higher doses (1, 3 mg/kg). One-way ANOVA with repeated measures (RM) over drug doses revealed a significant treatment main effect (Figure 4A, F_{(3, 24)} = 9.616, p < 0.001). Dunnett’s post-hoc tests indicated a significant reduction in the number of cocaine infusions only after 3 mg/kg cariprazine administration compared to the vehicle control group (q^’ = 3.497, p = 0.005). In contrast, cariprazine failed to alter the number of inactive lever presses (Figure 4B, F_{(3, 24)} = 1.362, p > 0.05), suggesting that cariprazine did not produce sedation or locomotor impairment. Figure 4C shows the representative records of cocaine self-administration before and after the different doses of cariprazine administration, illustrating that at low doses (0.3mg/kg, i.p.), cariprazine produced an increase in drug taking, as previously reported,⁶⁰ which is possibly a compensatory response to a reduction in cocaine reward. However, at high doses (1, 3 mg/kg), cariprazine caused a typical extinction-like pattern of cocaine self-administration (i.e., an initial burst-like increase in drug intake followed by cessation of cocaine intake), indicating a robust reduction in cocaine reward after high dose cariprazine administration. In total, this classical pattern of drug self-administration suggests that cariprazine pretreatment produces a dose-dependent reduction in cocaine’s reinforcing effects (efficacy).

Figure 4. — **(A)** The dose-dependent effects of cariprazine on cocaine self-administration (infusions). **(B)** Effects of cariprazine on inactive lever presses. **(C)** Representative cocaine self-administration (infusions) records, illustrating the typical extinction-like patterns of drug taking and drug seeking—lower doses of cariprazine (0.3 mg/kg) increased, while at higher doses it produced an initial burst-like drug infusions followed by cessation of drug self-administration. *p < 0.05, **p < 0.01, compared to vehicle (0 mg/kg).

Based on D₃R binding affinities and selectivity profiles, as well as favorable MPO scores (>3) and agonist/antagonist profiles at both D₃R and D₂R, we selected 13a and 13e for behavioral testing in comparison to the parent compound. Similar to cariprazine (Figure 4), 13a also produced a dose-dependent biphasic effect on cocaine self-administration. That is, at 0.3 mg/kg, 13a produced a significant increase, while at higher doses (1, 3 mg/kg) it produced a dose-dependent reduction in the number of cocaine infusions (Figure 5A, F_{(3, 24)}= 23.822, p < 0.001; Dunnett’s post-hoc, q’= 2.556, p = 0.045 at 0.3 mg/kg; q’= 2.873, p = 0.022, at 1 mg/kg; q’= 5.395, p < 0.001, at 3 mg/kg). Like cariprazine, 13a pretreatment also failed to affect the number of inactive lever presses (Figure 5B; F_{(3, 24)}= 2.708, p > 0.05).

Figure 5. — **(A)** Mean numbers after different doses of **13a** administration. **(B)** Mean numbers of inactive lever presses after different doses of **13a** pretreatments. **(C)** Representative cocaine infusions behavior indicating an extinction-like pattern of cocaine self-administration after **13a** administration in a way similar to cariprazine. (*p < 0.05, **p < 0.01, ***p < 0.001, compared to the vehicle control group).

Next, we tested 13e and found that higher doses (10 mg/kg) of 13e are required to inhibit cocaine self-administration under FR2 reinforcement, in rats (Figure 6). One-way ANOVA revealed a significant treatment main effect on cocaine infusions (Figure 6A, F_{(4, 43)} = 4.130, p = 0.006; Dunnett’s post-hoc, q’= 3.283, p = 0.007, at 10 mg/kg compared to vehicle). As observed with cariprazine and 13a, there was no effect on the number of inactive lever presses (Figure 6B; F_{(4, 43)}= 0.736, p > 0.05).

Figure 6. — **(A)** Mean number of self-infusions after different doses of **13e**. **(B)** Mean numbers of inactive lever presses **13e** pretreatments. **(C)** Representative cocaine infusions illustrating the patterns of cocaine self-administration after vehicle or **13e** administration. (*p < 0.05; **p < 0.01, compared to the vehicle control group).

There are three important findings in this behavioral test. First, cariprazine and its analogs appear to be more potent than highly selective D₃R antagonists in reducing cocaine self-administration under low-cost high-payoff (FR2) reinforcement conditions. In particular, considerably lower doses (0.3, 1, 3 mg/kg) of cariprazine and 13a are required to alter cocaine self-administration. Second, cariprazine and 13a produced unique biphasic effects on cocaine self-administration—increasing the number of cocaine infusions at low doses and decreasing drug intake at higher doses. These biphasic effects are usually seen after pretreatment with non-selective dopamine receptor antagonists, such as pimozide and (+)-butaclamol.⁶¹ In contrast, highly selective D₃R antagonists usually produced a monophasic reduction in cocaine or opioid self-administration, and higher doses (10–30 mg/kg) are required to inhibit cocaine or oxycodone self-administration.^{8, 24} The increase in cocaine self-administration after low doses of cariprazine and 13a is a compensatory response to reduced cocaine reward, as the pattern of cocaine self-administration is comparable to the responses produced by lower doses of cocaine - evenly distributed drug intake with shorter infusion intervals.^{61, 62} Thus, the increased behavioral performance reveals a reduction in cocaine’s rewarding effects. Congruently, at higher doses, cariprazine, 13a and 13e all produced a classical extinction-like pattern of cocaine self-administration—initial burst-like increase in drug infusions followed by cessation of drug taking.

Taken together, these data show that cariprazine and its two analogs, 13a and 13e, produce a dose-dependent reduction in cocaine reward, which is consistent with their pharmacological profiles as D₃R agonists/antagonists with moderate D₃R/D₂R selectivities (cariprazine, 3.6-fold; 13a/13e, ~20-fold). We note that 13a displays similar pharmacological potency to cariprazine in attenuating cocaine reward, while 13e displays slightly lower potency as a higher dose (10 mg/kg) of 13e is required to inhibit cocaine self-administration. This behavioral potency difference is in line with their receptor binding profiles. Whereas 13a has a similar binding affinity to D₂R and D₃R as cariprazine (Table 1), 13e displays ~5-fold lower affinities for D₂R and D₃R compared to 13a (Table 3). Similarly, in vitro functional assays indicate that the EC₅₀ value of 13e in activating D₂R is ~10-fold higher than that of 13a. This is consistent with the effective dose of 13e (10 mg/kg, Figure 6) in attenuating cocaine self-administration that is also ~3-fold higher than that of 13a (3 mg/kg, Figure 5).

CONCLUSIONS

We have hypothesized that compounds that are moderately D₃R/D₂R selective and are partial agonists, especially at D₂R may be more effective than highly D₃R selective antagonists for treatment of PSUD. Previously, highly selective D₃R partial agonists/antagonists have been shown to block the reinforcing effects of opioids, such as oxycodone,^{24, 63, 64} but are often less effective for psychostimulants, such as cocaine or methamphetamine,^{18, 65} especially when the drug is readily available (low fixed ratio schedule of reinforcement e.g., FR1 or FR2). Nevertheless, compounds that are D₂R antagonists are generally not well tolerated in this patient population.³⁰ To test this hypothesis, we designed a series of compounds, based on cariprazine, with varying selectivities and functional efficacies at D₂R and D₃R, depending on their PP, SP, and/or linker. By systematically modifying each piece of the parent molecule, we have discovered compounds with high D₃R affinities (K_i < 1 nM) and improved selectivities (≥20-fold) over D₂R that have the desired D₂R partial agonist functional efficacies and are either low efficacy partial agonists or antagonists at D₃R, as measured in BRET-based assays for each receptor subtype. We have obtained off target activities (D₁R, D₄R, 5HT_1A, 5HT_2A, 5HT_2B, and 5HT_2C) on a subset of these compounds that had desirable D₃R/D₂R selectivity profiles as well as MPO scores (>3) that further demonstrated D₃R-selectivity.

Based on all the in vitro data obtained, we selected 13a and 13e as our initial lead compounds, which were tested side-by-side with cariprazine in a rat model of cocaine self-administration. The two new compounds had higher but still moderate D₃R/D₂R selectivities (~20-fold) as compared to cariprazine (3.6-fold) and were effective in blocking cocaine self-administration in the rats (FR2; 1–10 mg/kg, i.p.). Overall, these convincing behavioral data support the notion that moderately D₃R selective partial agonists/antagonists that are also partial agonists at D₂R, may be more effective than highly selective (>200-fold) D₃R antagonists in reducing drug-seeking behavior. Of course, additional testing will be needed to determine the suitability of 13a and 13e for further development. For example, the extrapyramidal side effects of our lead compounds will need to be examined as well as their efficacy in animal models of affective disorders, such as BD-I.^{66, 67} Nonetheless, the cariprazine analogs described herein demonstrate high potential for treating PSUD that may be dual diagnosed with affective disorders.

EXPERIMENTAL METHODS

Chemistry

General Information.

Chemicals and solvents were purchased from commercial suppliers and used as received. Unless stated otherwise, reactions were performed under ambient conditions and monitored by thin-layer chromatography using Analtech silica gel GHLF (250 microns) coated glass plates, which were visualized with either phosphomolybdic acid, potassium permanganate, or vanillin stain. Normal phase column chromatography was conducted with a Teledyne Isco Combiflash Rf or EZ-Prep purification system (ELS detector associated). All nuclear magnetic resonance (NMR) spectra (¹H and ¹³C) were acquired in deuterated solvents (CDCl₃ or CD₃OD) on a Varian Mercury Plus 400 spectrometer. Chemical shifts are reported in parts per million (ppm) and were adjusted using the residual solvents (CDCl₃: 7.26 ppm for ¹H NMR, 77.2 ppm for ¹³C NMR; CD₃OD: 3.31 ppm for ¹H NMR, 49.0 ppm for ¹³C NMR) as an internal reference. Coupling constants are reported in Hertz (Hz) and peak multiplicities as either a singlet (s), doublet (d), triplet (t), quartet (q), or multiplet (m). Infrared (IR) spectra were acquired on a Perkin Elmer Spectrum Two FT-IR spectrometer. Melting points were determined on an Optimelt MPA100 instrument and are uncorrected. High-resolution mass spectrometry (HRMS) data were collected on a Thermo Scientific LTQ-Orbitrap Velos spectrometer using either electrospray ionization (ESI) or matrix-assisted laser desorption/ionization (MALDI). High-performance liquid chromatography (HPLC) was conducted on an Agilent Technologies 1260 Infinity system coupled to a diode-array detector. A Phenonmenex Gemini C18 100 Å LC column (50 × 4.6 mm, 3 μm particle size) was used as the stationary phase. For basic conditions, the mobile phase consisted of H₂O with 0.1% diethylamine (solvent A) and MeCN with 0.1% diethylamine (solvent B). For acidic conditions, solvents contained 0.1% TFA instead of diethylamine. All samples were prepared at a concentration of ca. 1 mg/mL in MeOH, and 20 μL of each solution was injected onto the column, which was maintained at 40 °C. Using a flow rate of 1.0 mL/min, the solvent gradient was as follows: 10% B held for 10 min, 10–40% B ramped over 10 min, 40% B held for 10 min, 40–80% B over 10 min, 80% B held for 20 min. Compound purity was determined based on peak integration (area under the curve) of the absorbance signals at 254 and 214 nm. Elemental analysis was performed by Atlantic Microlab, Inc. (Norcross, GA). All tested compounds were >95% pure by either HPLC or elemental analysis.

General Procedure A.

Using oven-dried glassware under an Ar atmosphere, a 0.60 M solution of (COCl)₂ in anhydrous DCM (1.5 equiv) was cooled to −78 °C. Then, anhydrous DMSO (3.0 equiv) was added over ca. 5 min, and the reaction was stirred for 30 min. Next, a 0.30 M solution of the alcohol in anhydrous DCM (1.0 equiv) was added over ca. 10 min, and the reaction was stirred for an additional 30 min. Finally, anhydrous NEt₃ (6.0 equiv) was added over ca. 5 min, and the mixture was allowed to warm to rt. When TLC analysis suggested the complete disappearance of starting material (ca. 2 h), the reaction was quenched with a 1.0 M aq solution of HCl, and the aq layer was extracted with DCM (2 ×). The combined organic layers were dried over Na₂SO₄, filtered, and concentrated. The crude product was purified by chromatography as described.

General Procedure B.

To a 0.08–0.13 M solution of 3 in anhydrous DCE (1.0 equiv) was added AcOH (1.0 equiv) followed by the appropriate aryl piperazine (1.1 equiv). The mixture was stirred for 30 min, and NaBH(OAc)₃ (1.5 equiv) was added in one portion. After stirring overnight, the reaction was quenched with a saturated aq solution of NaHCO₃, and the aq layer was extracted with DCM (2 ×). The combined organic layers were washed with brine, dried over Na₂SO₄, filtered, and concentrated. The crude product was purified by chromatography as described.

General Procedure C.

TFA (27–144 equiv) was added to a 0.03–0.15 M solution of the appropriate Boc-protected amine in DCM (1.0 equiv). The reaction mixture was stirred for ca. 4 h and then concentrated. The resulting residue was suspended in a 1.0 M aq solution of NaOH, and the aq layer was extracted with CHCl₃ (3 ×). The combined organic layers were dried over Na₂SO₄, filtered, and concentrated to afford the corresponding amine.

General Procedure D.

To a 0.07–0.10 M solution of the amine in DCM (1.0 equiv) was added DIPEA (1.5 equiv) followed by the appropriate carbamyl chloride (1.25 equiv) over 5 min. After stirring overnight, the reaction was quenched with a saturated aq solution of NaHCO₃, and the aq layer was extracted with CHCl₃ (3 ×). The combined organic layers were dried over Na₂SO₄, filtered, and concentrated. The crude product was purified by chromatography as described.

General Procedure E.

A 0.03–0.05 M mixture of the appropriate carboxylic acid (1.1 equiv) in CHCl₃ was cooled to 0 °C, and EDCI (1.3 equiv) followed by HOBt (1.2 equiv) were each added in one portion. After 30 min, the amine (1.0 equiv) was added followed by DIPEA (1.4 equiv). The mixture was allowed to warm to rt and stirred overnight. Then, a saturated aq solution of NaHCO₃ was added, and the aq layer was extracted with CHCl₃ (3 ×). The combined organic layers were dried over anhydrous Na₂SO₄, filtered, and concentrated. The crude product was purified by chromatography as described.

General Procedure F.

To a 0.10 M mixture of HCTU (1.2 equiv) in DCM was added 3-methoxypropanoic acid (1.1 equiv) in one portion. After stirring the mixture for 10 min, a solution of the amine (1.0 equiv) and DIPEA (1.2 equiv) in DCM (0.04 M with respect to the limiting reagent) was added over 5 min. The reaction was stirred overnight, quenched with a saturated aq solution of NaHCO₃, and the aq layer was extracted with DCM (3 ×). The combined organic layers were dried over anhydrous Na₂SO₄, filtered, and concentrated. The crude product was purified by chromatography as described to afford the corresponding amide.

tert-Butyl (trans-4-(2-hydroxyethyl)cyclohexyl)carbamate (2).

Using oven-dried glassware under an Ar atmosphere, a solution of 2-(trans-4-((tert-butoxycarbonyl)amino)cyclohexyl)acetic acid (6.01 g, 23.4 mmol) in anhydrous THF (30 mL) was cooled to 0 °C. Then, borane dimethylsulfide complex (3.4 mL, 36 mmol) was added dropwise over 5 min. The solution was allowed to warm to rt and stirred for 15 h. Afterward, the reaction was cooled back to 0 °C, quenched with a saturated aq solution of NaHCO₃ (60 mL), and the aq layer was extracted with EtOAc (2 × 60 mL). The organic layer was dried over anhydrous Na₂SO₄, filtered, and concentrated to afford 2 (5.18 g, 21.3 mmol, 91% yield) as a white solid. An analytical sample was prepared by recrystallization using Et₂O—hexanes (slow evaporation of solvent mixture). R_f = 0.4 (50% EtOAc/hexanes); ¹H NMR (400 MHz, CDCl₃) δ 4.44–4.27 (m, 1H), 3.71–3.64 (m, 2H), 3.43–3.28 (m, 1H), 2.04–1.96 (m, 2H), 1.81–1.73 (m, 2H), 1.51–1.26 (m, 4H), 1.43 (s, 9H), 1.15–0.96 (m, 4H); ¹³C NMR (101 MHz, CDCl₃) δ 155.4, 79.2, 60.9, 50.0, 39.8, 33.6 (3C), 32.0 (2C), 28.6 (3C); IR (neat) 3425, 3365, 1682, 1517 cm⁻¹; mp 102–103 °C (Et₂O—hexanes); HRMS (MALDI) m/z [M + Na]⁺ calcd for C₁₃H₂₅NNaO₃ 266.1727, found 266.1730.

tert-Butyl (trans-4-(2-oxoethyl)cyclohexyl)carbamate (3).

General Procedure A was followed using 2 (5.18 g, 21.3 mmol). After work-up, the crude product was purified by chromatography (120 g of silica gel, 0–40% EtOAc/hexanes) to afford 3 (3.04 g, 12.6 mmol, 59% yield) as a white solid. R_f = 0.5 (40% EtOAc/hexanes); ¹H NMR (400 MHz, CDCl₃) δ 9.78–9.73 (m, 1H), 4.47–4.29 (m, 1H), 3.45–3.26 (m, 1H), 2.35–2.29 (m, 2H), 2.06–1.95 (m, 2H), 1.90–1.74 (m, 3H), 1.44 (s, 9H), 1.20–1.03 (m, 4H); ¹³C NMR (101 MHz, CDCl₃) δ 202.3, 155.3, 79.3, 50.8, 49.6, 33.3 (2C), 31.9 (2C), 31.8, 28.6 (3C); IR (neat) 3377, 1715, 1686 cm⁻¹; mp 90–91 °C; HRMS (MALDI) m/z [M + Na]⁺ calcd for C₁₃H₂₃NNaO₃ 264.1570, found 264.1578.

tert-Butyl (trans-4-(2-(4-(2,3-dichlorophenyl)piperazin-1-yl)ethyl)cyclohexyl)carbamate (4a).

General procedure B was followed using 3 (2.00 g, 8.29 mmol) and 1-(2,3-dichlorophenyl)piperazine•HCl (2.44 g, 9.12 mmol) in DCE (56 mL). After work-up, the crude product was purified by chromatography (80 g of silica gel, 0–50% EtOAc/hexanes) to afford 4a (1.79 g, 3.92 mmol, 47% yield) as a white solid. R_f = 0.2 (50% EtOAc/hexanes); ¹H NMR (400 MHz, CDCl₃) δ 7.16–7.08 (m, 2H), 6.98–6.91 (m, 1H), 4.44–4.24 (m, 1H), 3.44–3.27 (m, 1H), 3.14–2.97 (m, 4H), 2.71–2.52 (m, 4H), 2.46–2.36 (m, 2H), 2.05–1.92 (m, 2H), 1.81–1.71 (m, 2H), 1.49–1.37 (m, 2H), 1.44 (s, 9H), 1.29–1.15 (m, 1H), 1.14–0.95 (m, 4H); ¹³C NMR (101 MHz, CDCl₃) δ 155.7, 151.8, 135.5, 128.0, 127.9, 125.0, 119.0, 79.5, 57.1, 53.9, 51.8, 50.4, 35.9, 34.4, 33.9 (2C), 32.5 (2C), 28.9 (3C); IR (neat) 3366, 1677 cm⁻¹; mp 147–148 °C; HRMS (MALDI) m/z [M + H]⁺ calcd for C₂₃H₃₆Cl₂N₃O₂ 456.2179, found 456.2174; t_R = 39.4 min (HPLC, basic). The oxalate salt was precipitated from a 0.03 M solution of the free base in 50% CHCl₃/acetone using oxalic acid (1.25 equiv). Dec >185 °C. Anal. calcd for C₂₃H₃₅Cl₂N₃O₂•1.25C₂H₂O₄: C, 53.83; H, 6.64; N, 7.39. Found: C, 54.06; H, 6.67; N, 7.48.

tert-Butyl (trans-4-(2-(4-(2-chloro-3-ethylphenyl)piperazin-1-yl)ethyl)cyclohexyl)carbamate (4b).

General procedure B was followed using 3 (0.710 g, 2.94 mmol) and 1-(2-chloro-3-ethylphenyl)piperazine (0.810 g, 3.60 mmol) in DCE (35 mL). After work-up, the crude product was purified by chromatography (silica gel, 0–70% EtOAc/hexanes) to afford 4b (0.990 g, 2.20 mmol, 75% yield) as a white solid. ¹H NMR (400 MHz, CDCl₃) δ 7.15 (t, J = 7.7 Hz, 1H), 6.96–6.91 (m, 2H), 4.41–4.31 (m, 1H), 3.45–3.29 (m, 1H), 3.16–2.95 (m, 4H), 2.77 (q, J = 7.6 Hz, 2H), 2.72–2.48 (m, 4H), 2.46–2.38 (m, 2H), 2.02–1.95 (m, 2H), 1.82–1.73 (m, 2H), 1.48–1.40 (m, 2H), 1.44 (s, 9H), 1.27–1.19 (m, 1H), 1.22 (t, J = 7.5 Hz, 3H), 1.13–0.99 (m, 4H).

tert-Butyl (trans-4-(2-(4-(2-fluoro-3-methoxyphenyl)piperazin-1-yl)ethyl)cyclohexyl)carbamate (4c).

General procedure B was followed using 3 (0.725 g, 3.00 mmol) and 1-(2-fluoro-3-methoxyphenyl)piperazine•HCl (0.816 g, 3.31 mmol) in DCE (25 mL). After work-up, the crude product was purified by chromatography (40 g of silica gel, 0–80% EtOAc/hexanes) to afford 4c (0.695 g, 1.60 mmol, 53% yield) as a light orange solid. R_f = 0.4 (80% EtOAc/hexanes); ¹H NMR (400 MHz, CDCl₃) δ 6.95 (dt, J = 8.3, 1.9 Hz, 1H), 6.66–6.53 (m, 2H), 4.43–4.26 (m, 1H), 3.86 (s, 3H), 3.43–3.25 (m, 1H), 3.19–2.98 (m, 4H), 2.68–2.47 (m, 4H), 2.43–2.35 (m, 2H), 2.03–1.92 (m, 2H), 1.82–1.69 (m, 2H), 1.48–1.36 (m, 2H), 1.43 (s, 9H), 1.29–1.15 (m, 1H), 1.12–0.95 (m, 4H); ¹³C NMR (101 MHz, CDCl₃) δ 155.4, 148.7 (d, J = 10 Hz), 145.8 (d, J = 245 Hz),141.2 (d, J = 6 Hz), 123.6 (d, J = 5 Hz), 111.2 (d, J = 2 Hz), 107.2, 79.2, 56.8, 56.6, 53.6 (2C), 50.8 (d, J = 3 Hz, 2C), 50.0, 35.6, 34.0, 33.6 (2C), 32.1 (2C), 28.6 (3C); IR (neat) 3370, 1685 cm⁻¹; mp 118–119 °C; HRMS (ESI) m/z [M + H]⁺ calcd for C₂₄H₃₉FN₃O₃ 436.2970, found 436.2964.

tert-Butyl (trans-4-(2-(4-(6-(trifluoromethyl)pyridin-2-yl)piperazin-1-yl)ethyl)cyclohexyl)carbamate (4d).

General procedure B was followed using 3 (1.00 g, 4.16 mmol) and 1-(6-(trifluoromethyl)pyridin-2-yl)piperazine (1.06 g, 4.58 mmol) in DCE (28 mL). Note: 3 was added to a solution of the aryl piperazine. After work-up, the crude product was purified by chromatography (40 g of silica gel, 0–80% EtOAc/hexanes) to afford 4d (1.23 g, 2.69 mmol, 65% yield) as a white solid. R_f = 0.3 (80% EtOAc/hexanes); ¹H NMR (400 MHz, CDCl₃) δ 7.55 (t, J = 8.0 Hz, 1H), 6.92 (d, J = 7.3 Hz, 1H), 6.75 (d, J = 8.7 Hz, 1H), 4.44–4.28 (m, 1H), 3.60 (t, J = 5.1 Hz, 4H), 3.43–3.27 (m, 1H), 2.51 (t, J = 5.1 Hz, 4H), 2.42–2.35 (m, 2H), 2.03–1.93 (m, 2H), 1.81–1.68 (m, 2H), 1.50–1.38 (m, 2H), 1.43 (s, 9H), 1.30–1.16 (m, 1H), 1.14–0.97 (m, 4H); ¹³C NMR (101 MHz, CDCl₃) δ 159.0, 155.4, 146.6 (q, J = 34 Hz), 138.3, 121.8 (q, J = 274 Hz), 109.5, 108.9 (q, J = 3 Hz), 79.2, 56.8, 53.1 (2C), 50.0, 44.9 (2C), 35.6, 34.0 (2C), 33.6, 32.1 (2C), 28.6 (3C); IR (neat) 3369, 1678 cm⁻¹; mp 131–132 °C; HRMS (ESI) m/z [M + H]⁺ calcd for C₂₃H₃₆F₃N₄O₂ 457.2785, found 457.2777.

tert-Butyl (trans-4-(2-(4-(3-chloro-5-ethyl-2-methoxyphenyl)piperazin-1-yl)ethyl)cyclohexyl)carbamate (4e).

General procedure B was followed using 3 (1.00 g, 4.16 mmol) and 1-(3-chloro-5-ethyl-2-methoxyphenyl)piperazine•HCl (1.33 g, 4.57 mmol) in DCE (27 mL). After work-up, the crude product was purified by chromatography (40 g of silica gel, 0–50% EtOAc/hexanes) to afford 4e (1.10 g, 2.29 mmol, 55% yield) as a yellow solid. R_f = 0.5 (50% MeOH/DCM); ¹H NMR (400 MHz, CDCl₃) δ 6.84 (s, 1H), 6.61 (s, 1H), 4.44–4.26 (m, 1H), 3.83 (s, 3H), 3.44–3.26 (m, 1H), 3.23–3.02 (m, 4H), 2.68–2.49 (m, 6H), 2.45–2.35 (m, 2H), 2.03–1.94 (m, 2H), 1.81–1.71 (m, 2H), 1.50–1.37 (m, 2H), 1.43 (s, 9H), 1.30–1.15 (m, 4H), 1.14–0.97 (m, 4H); ¹³C NMR (101 MHz, CDCl₃) δ 155.1, 146.2, 146.0, 140.6, 128.0, 122.0, 116.5, 78.90, 58.8, 56.5, 53.7 (2C), 50.1 (2C), 49.7, 35.3, 33.7, 33.3 (2C), 31.8 (2C), 28.30, 28.27 (3C), 15.2; IR (neat) 3364, 1681 cm⁻¹; mp 86–88 °C; HRMS (MALDI) m/z [M + H]⁺ calcd for C₂₆H₄₃ClN₃O₃ 480.2987, found 480.2985. The oxalate salt was precipitated from a 0.02 M solution of the free base in 50% CHCl₃/acetone using oxalic acid (1.25 equiv). mp 132–135 °C. Anal. calcd for C₂₆H₄₂ClN₃O₃•C₂H₂O₄: C, 58.99; H, 7.78; N, 7.37. Found: C, 58.70; H, 7.81; N, 7.30.

trans-4-(2-(4-(2,3-Dichlorophenyl)piperazin-1-yl)ethyl)cyclohexan-1-amine (5a).

General procedure C was followed using 4a (1.33 g, 2.91 mmol) and TFA (9.0 mL, 0.12 mol) in DCM (20 mL). After work-up, 5a (1.00 g, 2.81 mmol, 96% yield) was isolated as a white solid. R_f = 0.1 (10% MeOH/DCM); ¹H NMR (400 MHz, CDCl₃) δ 7.17–7.09 (m, 2H), 6.95 (dd, J = 6.4, 3.1 Hz, 1H), 3.15–2.96 (m, 4H), 2.72–2.51 (m, 5H), 2.47–2.37 (m, 2H), 1.84 (d, J = 12.2 Hz, 2H), 1.76 (d, J = 12.3 Hz, 2H), 1.48–1.32 (m, 4H), 1.32–1.17 (m, 1H), 1.14–0.92 (m, 4H); ¹³C NMR (101 MHz, CDCl₃) δ 151.5, 134.2, 127.7, 127.6, 124.6, 118.7, 56.9, 53.6 (2C), 51.5 (2C), 50.9, 36.9 (2C), 35.8, 34.2, 32.3 (2C); IR (neat) 1578 cm⁻¹; mp 72–74 °C; HRMS (MALDI) m/z [M + H]⁺ calcd for C₁₈H₂₈Cl₂N₃ 356.1655, found 356.1660.

trans-4-(2-(4-(2-Chloro-3-ethylphenyl)piperazin-1-yl)ethyl)cyclohexan-1-amine (5b).

General procedure C was followed using 4b (0.990 g, 2.20 mmol) and TFA (5.0 mL, 60 mmol) in DCM (15 mL). After work-up, 5b (0.775 g, 2,20 mmol, 100% yield) was isolated as a beige solid. ¹H NMR (400 MHz, CDCl₃) δ 7.15 (t, J = 7.8 Hz, 1H), 6.97–6.91 (m, 2H), 3.13–2.98 (m, 4H), 2.77 (q, J = 7.5 Hz, 2H), 2.72–2.54 (m, 5H), 2.47–2.40 (m, 2H), 1.88–1.81 (m, 2H), 1.80–1,73 (m, 2H), 1.48–1.40 (m, 2H), 1.28–1.18 (m, 1H), 1.22 (t, J = 7.5 Hz, 3H), 1.14–0.94 (m, 4H).

trans-4-(2-(4-(2-Fluoro-3-methoxyphenyl)piperazin-1-yl)ethyl)cyclohexan-1-amine (5c).

General procedure C was followed using 4c (0.251 g, 0.577 mmol) and TFA (1.8 mL, 24 mmol) in DCM (4.0 mL). After work-up, 5c (0.191 g, 0.569 mmol, 99% yield) was isolated as a tan solid. R_f = 0.1 (10% MeOH/DCM); ¹H NMR (400 MHz, CDCl₃) δ 6.95 (dt, J = 8.3, 1.9 Hz, 1H), 6.66–6.53 (m, 2H), 3.85 (s, 3H), 3.18–3.01 (m, 4H), 2.69–2.52 (m, 5H), 2.44–2.37 (m, 2H), 1.88–1.80 (m, 2H), 1.79–1.70 (m, 2H), 1.48–1.16 (m, 5H), 1.12–0.91 (m, 4H); ¹³C NMR (101 MHz, CDCl₃) δ 148.7 (d, J = 10 Hz), 145.8 (d, J = 245 Hz), 141.2 (d, J = 6 Hz), 123.6 (d, J = 5 Hz), 111.2 (d, J = 2 Hz), 107.1, 56.9, 56.6, 50.9, 50.8 (d, J = 3 Hz, 2C), 36.9, 35.8, 34.2, 32.3; IR (film) 1611, 1576 cm⁻¹; mp 59–60 °C; HRMS (ESI) m/z [M + H]⁺ calcd for C₁₉H₃₁FN₃O 336.2446, found 336.2442.

trans-4-(2-(4-(6-(Trifluoromethyl)pyridin-2-yl)piperazin-1-yl)ethyl)cyclohexan-1-amine (5d).

General procedure C was followed using 4d (0.301 g, 0.659 mmol) and TFA (2.0 mL, 26 mmol) in DCM (4.0 mL). After work-up, 5d (0.234 g, 0.656 mmol, 100% yield) was isolated as a clear, colorless oil. R_f = 0.1 (10% MeOH/DCM); ¹H NMR (400 MHz, CDCl₃) δ 7.55 (t, J = 8.0 Hz, 1H), 6.92 (d, J = 7.3 Hz, 1H), 6.75 (d, J = 8.7 Hz, 1H), 3.60 (t, J = 5.1 Hz, 4H), 2.63–2.55 (m, 1H), 2.52 (t, J = 5.1 Hz, 4H), 2.42–2.35 (m, 2H), 1.88–1.81 (m, 2H), 1.79–1.71 (m, 2H), 1.47–1.38 (m, 2H), 1.34–1.16 (m, 3H), 1.13–0.93 (m, 4H); ¹³C NMR (101 MHz, CDCl₃) δ 159.0, 146.6 (q, J = 34 Hz), 138.3, 121.8 (q, J = 274 Hz), 117.7, 108.9 (q, J = 3 Hz), 56.9, 53.1 (2C), 50.9, 44.9 (2C), 36.5 (2C), 35.7, 34.1, 32.2 (2C); IR (neat) 1604 cm⁻¹; HRMS (ESI) m/z [M + H]⁺ calcd for C₁₈H₂₈F₃N₄ 357.2261, found 357.2255.

trans-4-(2-(4-(3-Chloro-5-ethyl-2-methoxyphenyl)piperazin-1-yl)ethyl)cyclohexan-1-amine (5e).

General procedure C was followed using 4e (1.10 g, 2.29 mmol) and TFA (7.0 mL, 91 mmol) in DCM (16 mL). After work-up, 5e (0.87 g, 2.3 mmol, 100% yield) was isolated as a clear, orange oil. R_f = 0.1 (10% MeOH/DCM); ¹H NMR (400 MHz, CDCl₃) δ 6.83 (d, J = 1.9 Hz, 1H), 6.61 (d, J = 2.0 Hz, 1H), 3.83 (s, 3H), 3.20–3.04 (m, 4H), 2.65–2.49 (m, 6H), 2.44–2.37 (m, 2H), 1.84 (d, J = 11.9 Hz, 2H), 1.76 (d, J = 12.0 Hz, 2H), 1.47–1.33 (m, 4H), 1.30–1.15 (m, 5H), 1.13–0.93 (m, 4H); ¹³C NMR (101 MHz, CDCl₃) δ 146.5, 146.3, 140.9, 128.3, 122.3, 116.8, 59.1, 57.0, 54.0 (2C), 50.9, 50.4 (2C), 36.9 (2C), 35.8, 34.2, 32.3 (2C), 28.6, 15.6; IR (neat) 1593, 1565 cm⁻¹; HRMS (MALDI) m/z [M + H]⁺ calcd for C₂₁H₃₅ClN₃O 380.2463, found 380.2467.

3-(trans-4-(2-(4-(2,3-Dichlorophenyl)piperazin-1-yl)ethyl)cyclohexyl)-1,1-dimethylurea (cariprazine).

General procedure D was followed using 5a (1.00 g, 2.81 mmol) and N,N-dimethylcarbamyl chloride (0.33 mL, 3.6 mmol) in DCM (28 mL). After work-up, the crude product was purified by chromatography (40 g of silica gel, 0–60% EtOAc/hexanes) to afford cariprazine (1.02 g, 2.39 mmol, 85% yield) as a white solid. R_f = 0.5 (10% MeOH/DCM); ¹H NMR (400 MHz, CDCl₃) δ 7.17–7.10 (m, 2H), 6.95 (dd, J = 6.3, 3.2 Hz, 1H), 4.11 (d, J = 7.6 Hz, 1H), 3.64–3.52 (m, 1H), 3.15–2.98 (m, 4H), 2.88 (s, 6H), 2.71–2.53 (m, 4H), 2.47–2.38 (m, 2H), 2.07–1.96 (m, 2H), 1.83–1.70 (m, 2H), 1.49–1.39 (m, 2H), 1.30–1.18 (m, 1H), 1.16–1.01 (m, 4H); ¹³C NMR (101 MHz, CDCl₃) δ 158.0, 151.5, 134.2, 127.7, 127.6, 124.6, 118.7, 56.8, 53.6 (2C), 51.5 (2C), 50.0, 36.3 (2C), 35.8, 34.2 (2C), 34.1, 32.3 (2C); IR (neat) 3338, 1622 cm⁻¹; mp 212–213 °C (dec); HRMS (MALDI) m/z [M + H]⁺ calcd for C₂₁H₃₃Cl₂N₄O 427.2026, found 427.2023; t_R = 32.1 min (HPLC, basic). The HCl salt was precipitated from 0.03 M solution of the free base in 50% CHCl₃/acetone using a 2.0 M solution of HCl in Et₂O (5.0 equiv). Dec >200 °C. Anal. calcd for C₂₁H₃₂Cl₂N₄O•2HCl: C, 50.41; H, 6.85; N, 11.20. Found: C, 50.43; H, 6.84; N, 11.01.

3-(trans-4-(2-(4-(2-Chloro-3-ethylphenyl)piperazin-1-yl)ethyl)cyclohexyl)-1,1-dimethylurea (6b).

General procedure D was followed using 5b (0.500 g, 1.43 mmol) and N,N-dimethylcarbamyl chloride (0.16 mL, 1.8 mmol) in DCM (20 mL). After work-up, the crude product was purified by chromatography (silica gel, 0–10% MeOH/CHCl₃) to afford 6b (0.414 g, 0.983 mmol, 69% yield) as a yellow solid. ¹H NMR (400 MHz, CDCl₃) δ 7.15 (t, J = 7.7 Hz, 1H), 6.97–6.89 (m, 2H), 4.11 (d, J = 7.6 Hz, 1H), 3.63–3.53 (m, 1H), 3.14–2.98 (m, 4H), 2.88 (s, 6H), 2.77 (q, J = 7.6 Hz, 2H), 2.72–2.54 (m, 4H), 2.46–2.38 (m, 2H), 2.07–1.97 (m, 2H), 1.83–1.72 (m, 2H), 1.48–1.40 (m, 2H), 1.30–1.18 (m, 1H), 1.22 (t, J = 7.5 Hz, 3H), 1.15–1.01 (m, 4H); ¹³C NMR (101 MHz, CDCl₃) δ 158.0, 149.8, 143.4, 128.8, 127.0, 124.1, 118.1, 56.9, 53.7 (2C), 51.7 (2C), 50.0, 36.3 (2C), 35.8, 34.2 (2C), 34.1, 32.3, 27.6, 14.2; t_R = 20.3 min (HPLC, acidic). The HCl salt was precipitated from 50% acetone/CHCl₃ using a 2.0 M solution of HCl in Et₂O. Dec >200 °C. Anal. calcd for C₂₃H₃₇ClN₄O•HCl•H₂O: C, 58.10; H, 8.48; N, 11.78. Found: C, 58.32; H, 8.24; N, 11.56.

3-(trans-4-(2-(4-(2-Fluoro-3-methoxyphenyl)piperazin-1-yl)ethyl)cyclohexyl)-1,1-dimethylurea (6c).

General procedure D was followed using 5c (0.189 g, 0.565 mmol) and N,N-dimethylcarbamyl chloride (66 μL, 0.72 mmol) in DCM (5.6 mL). After work-up, the crude product was purified by chromatography (12 g of silica gel, 0–10% MeOH/DCM) to afford 6c (0.179 g, 0.440 mmol, 78% yield) as a white solid. R_f = 0.5 (10% MeOH/DCM); ¹H NMR (400 MHz, CDCl₃) δ 6.95 (dt, J = 8.3, 1.8 Hz, 1H), 6.66–6.52 (m, 2H), 4.12 (d, J = 7.6 Hz, 1H), 3.85 (s, 3H), 3.63–3.50 (m, 1H), 3.18–3.01 (m, 4H), 2.87 (s, 6H), 2.68–2.50 (m, 4H), 2.44–2.35 (m, 2H), 2.05–1.94 (m, 2H), 1.82–1.70 (m, 2H), 1.47–1.38 (m, 2H), 1.30–1.16 (m, 1H), 1.14–0.99 (m, 4H); ¹³C NMR (101 MHz, CDCl₃) δ 157.9, 148.7 (d, J = 10 Hz), 145.8 (d, J = 245 Hz), 141.2 (d, J = 6 Hz), 123.6 (d, J = 5 Hz), 111.2 (d, J = 2 Hz), 107.1, 56.8, 56.6, 53.6 (2C), 50.8 (d, J = 3 Hz, 2C), 50.0, 36.3 (2C), 35.8, 34.2 (2C), 34.1, 32.2 (2C); IR (film) 3342, 1626 cm⁻¹; mp 159–160 °C (dec); HRMS (ESI) m/z [M + H]⁺ calcd for C₂₂H₃₆FN₄O₂ 407.2817, found 407.2811; t_R = 22.2 min (HPLC, basic). The HCl salt was precipitated from a 0.03 M solution of the free base in 50% CHCl₃/acetone using a 2.0 M solution of HCl in Et₂O (5.0 equiv). mp 175–177 °C (dec). Anal. calcd for C₂₂H₃₅FN₄O₂•2HCl•1.25H₂O: C, 52.64; H, 7.93; N, 11.16. Found: C, 52.50; H, 7.79; N, 11.01.

1,1-Dimethyl-3-(trans-4-(2-(4-(6-(trifluoromethyl)pyridine-2-yl)piperazin-1-yl)ethyl)cyclohexyl)urea (6d).

General procedure D was followed using 5d (0.232 g, 0.651 mmol) and N,N-dimethylcarbamyl chloride (80 μL, 0.81 mmol) in DCM (9.5 mL). After work-up, the crude product was purified by chromatography (silica gel, 0–10% MeOH/CHCl₃) to afford 6d (0.107 g, 0.249 mmol, 38% yield) as a white solid. ¹H NMR (400 MHz, CDCl₃) δ 7.55 (t, J = 8.2 Hz, 1H), 6.92 (d, J = 7.7 Hz, 1H), 6.75 (d, J = 8.5 Hz, 1H), 4.11 (d, J = 7.7 Hz, 1H), 3.65–3.52 (m, 5H), 2.88 (s, 6H), 2.57–2.46 (m, 4H), 2.43–2.35 (m, 2H), 2.05–1.96 (m, 2H), 1.83–1.72 (m, 2H), 1.47–1.39 (m, 2H), 1.32–1.18 (m, 1H), 1.16–1.00 (m, 4H); ¹³C NMR (101 MHz, CDCl₃) δ 159.0, 158.0, 146.6 (q, J = 34 Hz), 138.3, 121.8 (q, J = 274 Hz), 109.5, 108.9 (q, J = 3 Hz), 56.8, 53.1 (2C), 50.0, 44.9 (2C), 36.3 (2C), 35.8, 34.2 (2C), 34.0, 32.2 (2C); HRMS (MALDI) m/z [M + H]⁺ calcd for C₂₁H₃₃F₃N₅O 428.2632, found 428.2629;; t_R = 18.5 min (HPLC, acidic).

3-(trans-4-(2-(4-(3-Chloro-5-ethyl-2-methoxyphenyl)piperazin-1-yl)ethyl)cyclohexyl)-1,1-dimethylurea (6e).

General procedure D was followed using 5e (0.275 g, 0.723 mmol) and N,N-dimethylcarbamyl chloride (82 μL, 0.90 mmol) in DCM (10 mL). After work-up, the crude product was purified by chromatography (silica gel, 0–10% MeOH/CHCl₃) to afford 6e (0.215 g, 0.477 mmol, 66% yield) as an off-white solid. ¹H NMR (400 MHz, CDCl₃) δ 6.87 (d, J = 1.9 Hz, 1H), 6.63 (d, J = 2.0 Hz, 1H), 4.13 (d, J = 7.6 Hz, 1H), 3.82 (s, 3H), 3.64–3.49, (m, 1H), 3.46–3.21 (m, 4H), 3.06–2.77 (m, 4H), 2.87 (s, 6H), 2.76–2.60 (m, 2H), 2.54 (q, J = 7.6 Hz, 2H), 2.08–1.97 (m, 2H), 1.83–1.72 (m, 2H), 1.67–1.54 (m, 2H), 1.39–1.25 (m, 1H), 1.19 (t, J = 7.6 Hz, 3H), 1.16–1.02 (m, 4H); ¹³C NMR (101 MHz, CDCl₃) δ 158.0, 146.6, 146.4, 140.9, 128.4, 122.3, 116.8, 66.0, 59.1, 56.9, 54.0 (2C), 50.4 (2C), 50.0, 36.3 (2C), 35.8, 34.2 (2C), 32.2 (2C), 28.6, 15.4. The HCl salt was precipitated from 50% CHCl₃/acetone using a 2.0 M solution of HCl in Et₂O. Dec >189 °C. Anal. calcd for C₂₄H₃₉ClN₄O₂•2HCl: C, 55.02; H, 7.89; N, 10.69. Found: C, 54.96; H, 7.88; N, 10.54.

1-(trans-4-(2-(4-(2,3-Dichlorophenyl)piperazin-1-yl)ethyl)cyclohexyl)-3-methylurea (7a).

General procedure D was followed using 5a (0.250 g, 0.702 mmol) and N-methylcarbamyl chloride (83.0 mg, 0.456 mmol) in DCM (10 mL). After work-up, the crude product was purified by chromatography (silica gel, 0–10% MeOH/CHCl₃) to afford 7a (0.141 g, 0.341 mmol, 49% yield) as a white solid. ¹H NMR (400 MHz, CDCl₃) δ 7.19–7.13 (m, 2H), 6.97 (dd, J = 5.7, 3.9 Hz, 1H), 5.11–5.05 (m, 1H), 5.00 (d, J = 8.0 Hz, 1H), 3.53–3.40 (m, 1H), 3.18–2.94 (m, 4H), 2.72 (d, J = 4.7 Hz, 3H), 2.68–2.55 (m, 4H), 2.47–2.38 (m, 2H), 2.04–1.92 (m, 2H), 1.82–1.70 (m, 2H), 1.49–1.39 (m, 2H), 1.30–1.19 (m, 1H), 1.14–1.00 (m, 4H); ¹³C NMR (101 MHz, CDCl₃) δ 158.6, 151.1, 133.6, 127.3, 127.1, 124.3, 118.5, 56.4, 53.1 (2C), 51.1 (2C), 48.9, 35.3, 33.7 (3C), 31.9 (2C), 26.5. The HCl salt was precipitated from a 0.02 M solution of the free base in 50% CHCl₃/acetone using a 2.0 M solution of HCl in Et₂O (35 equiv). Dec >175 °C. Anal. calcd for C₂₀H₃₀Cl₂N₄O•HCl•1.25H₂O: C, 50.85; H, 7.15; N, 11.86. Found: C, 50.86; H, 6.86; N, 11.60.

1-(trans-4-(2-(4-(2-Chloro-3-ethylphenyl)piperazin-1-yl)ethyl)cyclohexyl)-3-methylurea (7b).

General procedure D was followed using 5b (0.172 g, 0.491 mmol) and N-methylcarbamyl chloride (0.139 g, 1.47 mmol) in DCM (15 mL). After work-up, the crude product was purified by chromatography (silica gel, 0–10% MeOH/CHCl₃) to afford 7b (0.150 g, 0.368 mmol, 75% yield) as a white solid. ¹H NMR (400 MHz, CDCl₃) δ 7.15 (t, J = 7.7 Hz, 1H), 6.96–6.90 (m, 2H), 4.28–4.21 (m, 1H), 4.15 (d, J = 8.0 Hz, 1H), 3.51–3.39 (m, 1H), 3.16–2.94 (m, 4H), 2.81–2.73 (m, 5H), 2.72–2.52 (m, 4H), 2.46–2.36 (m, 2H), 2.06–1.94 (m, 2H), 1.85–1.69 (m, 2H), 1.49–1.41 (m, 2H), 1.22 (t, J = 7.5 Hz, 3H), 1.31–1.17 (m, 1H), 1.15–1.01 (m, 4H); ¹³C NMR (101 MHz, CDCl₃) δ 158.3, 149.8, 143.4, 128.8, 127.0, 124.1, 118.1, 56.8, 53.7 (2C), 51.7 (2C), 49.8, 35.7, 34.09, 34.06 (2C), 32.2 (2C), 27.6, 27.4, 14.2. The HCl salt was precipitated from a 0.03 M solution of the free base in 50% CHCl₃/acetone using a 2.0 M solution of HCl in Et₂O (29 equiv). Dec >199 °C. Anal. calcd for C₂₂H₃₅ClN₄O•HCl•1.25H₂O: C, 56.71; H, 8.33; N, 12.02. Found: C, 56.54; H, 7.92; N, 11.94.

1-(trans-4-(2-(4-(3-Chloro-5-ethyl-2-methoxyphenyl)piperazin-1-yl)ethyl)cyclohexyl)-3-methylurea (7e).

General procedure D was followed using 5e (0.420 g, 1.11 mmol) and N-methylcarbamyl chloride (0.130 g, 1.38 mmol) in DCM (15 mL). After work-up, the crude product was purified by chromatography (silica gel, 0–10% MeOH/CHCl₃) to afford 7e (0.416 g, 0.952 mmol, 86% yield) as a white solid. ¹H NMR (400 MHz, CDCl₃) δ 6.84 (d, J = 2.0 Hz, 1H), 6.61 (d, J = 2.0 Hz, 1H), 4.16 (d, J = 5.1 Hz, 1H), 4.08 (d, J = 8.0 Hz, 1H), 3.83 (s, 3H), 3.52–3.40 (m, 1H), 3.22–3.05 (m, 4H), 2.77 (d, J = 4.9 Hz, 3H), 2.66–2.49 (m, 6H), 2.44–2.36 (m, 2H), 2.06–1.95 (m, 2H), 1.84–1.73 (m, 2H), 1.48–1.40 (m, 2H), 1.31–1.23 (m, 1H), 1.20 (t, J = 7.6 Hz, 3H), 1.15–1.01 (m, 4H); ¹³C NMR (101 MHz, CDCl₃) δ 158.3, 146.5, 146.3, 141.0, 128.4, 122.3, 116.8, 59.2, 56.9, 54.0 (2C), 50.4 (2C), 49.9, 35.7, 34.1 (2C), 34.0, 32.2 (2C), 28.6, 27.4, 15.6. The HCl salt was precipitated from a 0.04 M solution of the free base in 50% CHCl₃/acetone using a 2.0 M solution of HCl in Et₂O (27 equiv). Dec >220 °C. Anal. calcd for C₂₃H₃₇ClN₄O₂•2HCl•1.25H₂O: C, 51.88; H, 7.86; N, 10.52. Found: C, 51.89; H, 7.60; N, 10.36.

1-(trans-4-(2-(4-(2,3-Dichlorophenyl)piperazin-1-yl)ethyl)cyclohexyl)urea (8a).

To a solution of 5a (0.108 g, 0.303 mmol) and KOCN (0.256 g, 3.16 mmol) in H₂O (1.5 mL) and THF (3.0 mL) was added a 1.0 M aq solution of HCl (1.5 mL, 1.5 mmol) over 3 min. After stirring overnight, the reaction was quenched with a saturated aq solution of NaHCO₃ (20 mL), and the aq layer was extracted with EtOAc (2 × 20 mL). The combined organic layers were dried over Na₂SO₄, filtered, and concentrated. The crude product was purified by chromatography (12 g of silica gel, 0–10% MeOH/DCM) to afford 8a (71.3 mg, 0.179 mmol, 59% yield) as a white solid. R_f = 0.3 (10% MeOH/DCM); ¹H NMR (400 MHz, CDCl₃) δ 7.18–7.10 (m, 2H), 6.95 (dd, J = 6.5, 3.1 Hz, 1H), 4.38–4.23 (m, 3H), 3.50–3.37 (m, 1H), 3.16–2.97 (m, 4H), 2.72–2.52 (m, 4H), 2.42 (t, J = 8.0 Hz, 2H), 2.08–1.96 (m, 2H), 1.85–1.64 (m, 3H), 1.51–1.39 (m, 2H), 1.17–0.99 (m, 4H); ¹³C NMR (101 MHz, CDCl₃) δ 157.8, 151.5, 134.2, 127.7, 127.6, 124.7, 118.7, 56.7, 53.6 (2C), 51.5 (2C), 50.2, 35.6, 34.1, 33.8 (2C), 32.1 (2C); IR (neat) 3477, 3329, 1645 cm⁻¹; mp 212–213 °C (dec); HRMS (MALDI) m/z [M + H]⁺ calcd for C₁₉H₂₉Cl₂N₄O 399.1713, found 399.1711; t_R = 24.9 min (HPLC, basic).

1-(trans-4-(2-(4-(3-Chloro-5-ethyl-2-methoxyphenyl)piperazin-1-yl)ethyl)cyclohexyl)urea (8e).

The same procedure as the one described for 8a was followed starting from 5e (0.203 g, 0.534 mmol). After work-up, the crude product was purified by chromatography (12 g of silica gel, 0–10% MeOH/DCM) to afford 8e (93.0 mg, 0.220 mmol, 41% yield) as a tan solid. R_f = 0.3 (10% MeOH/DCM); ¹H NMR (400 MHz, CDCl₃) δ 6.83 (d, J = 2.0 Hz, 1H), 6.61 (d, J = 2.0 Hz, 1H), 4.58–4.33 (m, 3H), 3.83 (s, 3H), 3.49–3.34 (m, 1H), 3.26–2.99 (m, 4H), 2.68–2.48 (m, 6H), 2.45–2.34 (m, 2H), 2.06–1.94 (m, 2H), 1.85–1.70 (m, 2H), 1.48–1.38 (m, 2H), 1.30–1.22 (m, 1H), 1.19 (t, J = 7.6 Hz, 3H), 1.16–1.00 (m, 4H); ¹³C NMR (101 MHz, CDCl₃) δ 158.2, 146.5, 146.3, 140.9, 128.3, 122.3, 116.8, 59.1, 56.8, 54.0 (2C), 50.4 (2C), 50.0, 35.6, 34.0, 33.8 (2C), 32.1 (2C), 28.6, 15.6; IR (neat) 3492, 3348, 1647 cm⁻¹; mp 163–166 °C (dec); HRMS (MALDI) m/z [M + H]⁺ calcd for C₂₂H₃₆ClN₄O₂ 423.2521, found 423.2518; t_R = 32.0 min (HPLC, basic).

N-(trans-4-(2-(4-(2,3-Dichlorophenyl)piperazin-1-yl)ethyl)cyclohexyl)-7,8-dihydro-1,6-naphthyridine-6(5H)-carboxamide (9a).

Using oven-dried glassware under an Ar atmosphere, a solution of NEt₃ (0.21 mL, 1.5 mmol) in anhydrous THF (3.5 mL) was cooled to 0 °C, and a 15 wt% solution of phosgene in toluene (0.40 mL, 0.56 mmol) was added over 3 min. Next, a solution of 5a (0.179 g, 0.502 mmol) in anhydrous THF (3.5 mL) was added over 3 min, and the reaction was stirred for 1 h. Then, a solution of 5,6,7,8-tetrahydro-1,6-naphthyridine (80.5 mg, 0.600 mmol) in anhydrous THF (3.5 mL) was added over 3 min, and the reaction was allowed to warm to rt. After stirring for 19 h, the reaction was quenched with a saturated aq solution of NaHCO₃ (40 mL), and the aq layer was extracted with EtOAc (40 mL). The organic layer was dried over anhydrous Na₂SO₄, filtered, and concentrated. The crude product was purified by chromatography (12 g of silica gel, 0–10% MeOH/DCM) to afford 9a (35.1 mg, 68.0 μmol, 14% yield) as a white solid. R_f = 0.4 (10% MeOH/DCM); ¹H NMR (400 MHz, CDCl₃) δ 8.42 (d, J = 4.2 Hz, 1H), 7.42 (d, J = 7.6 Hz, 1H), 7.17–7.10 (m, 3H), 6.95 (dd, J = 6.4, 3.2 Hz, 1H), 4.57 (s, 2H), 4.32 (d, J = 7.6 Hz, 1H), 3.71–3.57 (m, 3H), 3.14–2.96 (m, 6H), 2.76–2.52 (m, 4H), 2.47–2.39 (m, 2H), 2.08–2.00 (m, 2H), 1.83–1.75 (m, 2H), 1.48–1.39 (m, 2H), 1.30–1.20 (m, 1H), 1.18–1.03 (m, 4H); ¹³C NMR (101 MHz, CDCl₃) δ 156.9, 155.0, 151.4, 148.0, 134.2, 134.1, 129.1, 127.62, 127.56, 124.6, 121.7, 118.7, 56.8, 53.5 (2C), 51.5 (2C), 50.1, 45.0, 41.6, 35.8, 34.08 (2C), 34.05, 32.2 (3C); IR (neat) 3326, 1620 cm⁻¹; mp 163–164 °C (dec); HRMS (MALDI) m/z [M + H]⁺ calcd for C₂₇H₃₆Cl₂N₅O 516.2291, found 516.2283; t_R = 33.8 min (HPLC, basic).

N-(trans-4-(2-(4-(2-Chloro-3-ethylphenyl)piperazin-1-yl)ethyl)cyclohexyl)-7,8-dihydro-1,6-naphthyridine-6(5H)-carboxamide (9b).

The same procedure as the one described for 9a was followed starting from 5b (0.205 g, 0.586 mmol). After work-up, the crude product was purified by chromatography (12 g of silica gel, 0–10% MeOH/DCM) to afford 9b (0.192 g, 0.376 mmol, 64% yield) as a white solid. R_f = 0.4 (10% MeOH/DCM); ¹H NMR (400 MHz, CDCl₃) δ 8.42 (d, J = 4.7 Hz, 1H), 7.43 (d, J = 7.7 Hz, 1H), 7.18–7.10 (m, 2H), 6.93 (t, J = 7.2 Hz, 2H), 4.57 (s, 2H), 4.32 (d, J = 7.5 Hz, 1H), 3.71–3.59 (m, 3H), 3.12–2.98 (m, 6H), 2.77 (q, J = 7.5 Hz, 2H), 2.71–2.54 (m, 4H), 2.47–2.39 (m, 2H), 2.10–2.01 (m, 2H), 1.84–1.74 (m, 2H), 1.50–1.40 (m, 2H), 1.30–1.18 (m, 1H), 1.22 (t, J = 7.5 Hz, 3H), 1.15–1.04 (m, 4H); ¹³C NMR (101 MHz, CDCl₃) δ 156.9, 155.1, 149.8, 148.0, 143.4, 134.2, 129.1, 128.8, 127.0, 124.1, 121.7, 118.1, 56.9, 53.7 (2C), 51.7 (2C), 50.2, 45.0, 41.6, 35.8, 34.1 (3C), 32.2 (3C), 27.6, 14.4; IR (neat) 3323, 1619 cm⁻¹; mp 58–60 °C; HRMS (MALDI) m/z [M + H]⁺ calcd for C₂₉H₄₁ClN₅O 510.2994, found 510.2988; t_R = 35.2 min (HPLC, basic).

N-(trans-4-(2-(4-(2,3-Dichlorophenyl)piperazin-1-yl)ethyl)cyclohexyl)-1H-indole-2-carboxamide (10a).

General procedure E was followed using 5a (0.101 g, 0.283 mmol) and 1H-indole-2-carboxylic acid (62.0 mg, 0.385 mmol) in CHCl₃ (8.0 mL). After work-up, the crude product was product was purified by chromatography (12 g of silica gel, 0–10% MeOH/DCM) to afford 10a (20.2 mg, 40.2 μmol, 14% yield) as a white solid. R_f = 0.5 (10% MeOH/DCM); ¹H NMR (400 MHz, CDCl₃) δ 9.22 (s, 1H), 7.64 (d, J = 8.0 Hz, 1H), 7.43 (d, J = 8.3 Hz, 1H), 7.32–7.28 (m, 1H), 7.19–7.10 (m, 3H), 6.97 (dd, J = 6.5, 3.1 Hz, 1H), 6.80 (s, 1H), 5.95 (d, J = 8.2 Hz, 1H), 4.03–3.89 (m, 1H), 3.17–2.00 (m, 4H), 2.76–2.55 (m, 4H), 2.47 (t, J = 7.9 Hz, 2H), 2.13 (d, J = 11.8 Hz, 2H), 1.86 (d, J = 12.4 Hz, 2H), 1.54–1.42 (m, 2H), 1.37–1.10 (m, 5H); ¹³C NMR (101 MHz, CDCl₃) δ 160.8, 151.4, 136.2, 134.2, 131.2, 127.9, 127.7, 127.6, 124.7, 124.6, 122.0, 120.8, 118.7, 112.0, 101.6, 56.7, 53.6 (2C), 51.5 (2C), 49.1, 35.6, 34.0, 33.4 (2C), 32.1 (2C); IR (neat) 3616, 3268, 1624 cm⁻¹; mp 240–241 °C (dec); HRMS (MALDI) m/z [M + H]⁺ calcd for C₂₇H₃₃Cl₂N₄O 499.2026, found 499.2025; t_R = 37.8 min (HPLC, basic).

N-(trans-4-(2-(4-(2-Chloro-3-ethylphenyl)piperazin-1-yl)ethyl)cyclohexyl)indole-2-carboxamide (10b).

General procedure E was followed using 5b (0.100 g, 0.286 mmol) and 1H-indole-2-carboxylic acid (58.0 mg, 0.360 mmol) in CHCl₃ (10 mL). After work-up, the crude product was purified by chromatography (silica gel, 0–50% EtOAc/hexanes) to afford 10b (70 mg, 0.142 mmol, 50% yield) as an off-white solid. ¹H NMR (400 MHz, CDCl₃) δ 9.16 (s, 1H), 7.64 (d, J = 8.0 Hz, 1H), 7.43 (d, J = 8.4 Hz, 1H), 7.31–7.26 (m, 1H), 7.18–7.11 (m, 2H), 6.97–6.92 (m, 2H), 6.80 (dd, J = 2.1, 0.9 Hz, 1H), 5.94 (d, J = 8.2 Hz, 1H), 4.00–3.89 (m, 1H), 3.16–2.98 (m, 4H), 2.78 (q, J = 7.5 Hz, 2H), 2.73–2.55 (m, 4H), 2.50–2.41 (m, 2H), 2.17–2.08 (m, 2H), 1.90–1.82 (m, 2H), 1.53–1.45 (m, 2H), 1.38–1.10 (m, 7H); ¹³C NMR (101 MHz, CDCl₃) δ 160.8, 149.8, 143.4, 136.2, 131.2, 128.9, 127.9, 127.0, 124.6, 124.1, 122.0, 120.8, 118.2, 112.0, 101.6, 56.8, 53.8 (2C), 51.8 (2C), 49.1, 35.7, 34.1, 33.4 (2C), 32.1 (2C), 27.6, 14.3. The HCl salt was precipitated from 50% CHCl₃/acetone using a 2.0 M solution of HCl in Et₂O. Anal. calcd for C₂₉H₃₇ClN₄O•HCl•0.75H₂O: C, 64.14; H, 7.33; N, 10.32. Found: C, 64.11; H, 7.09; N, 10.04.

N-(trans-4-(2-(4-(3-Chloro-5-ethyl-2-methoxyphenyl)piperazin-1-yl)ethyl)cyclohexyl)indole-2-carboxamide (10e).

General procedure E was followed using 5e (0.125 g, 0.329 mmol) and 1H-indole-2-carboxylic acid (58.3 mg, 0.362 mmol) in CHCl₃ (8.0 mL). After work-up, the crude product was purified by chromatography (silica gel, 0–10% MeOH/CHCl₃) to afford 10e (0.133 g, 0.254 mmol, 77% yield) as a white solid. ¹H NMR (400 MHz, CDCl₃) δ 9.33 (s, 1H), 7.64 (d, J = 8.0 Hz, 1H), 7.44 (d, J = 8.3 Hz, 1H), 7.33–7.24 (m, 1H), 7.14 (t, J = 7.5 Hz, 1H), 6.85 (s, 1H), 6.81 (s, 1H), 6.62 (s, 1H), 5.97 (d, J = 8.2 Hz, 1H), 4.02–3.90 (m, 1H), 3.85 (s, 3H), 3.25–3.06 (m, 4H), 2.69–2.51 (m, 6H), 2.47–2.40 (m, 2H), 2.17–2.08 (m, 2H), 1.90–1.81 (m, 2H), 1.53–1.44 (m, 2H), 1.39–1.09 (m, 8H). The HCl salt was precipitated from a 0.06 M solution of the free base in 50% CHCl₃/acetone using a 2.0 M solution of HCl in Et₂O (20 equiv). Dec >220 °C. Anal. calcd for C₃₀H₃₉ClN₄O₂•2HCl•0.25H₂O: C, 60.00; H, 6.97; N, 9.33. Found: C, 60.16; H, 7.10; N, 9.12.

N-(trans-4-(2-(4-(2,3-Dichlorophenyl)piperazin-1-yl)ethyl)cyclohexyl)quinoline-4-carboxamide (11a).

General procedure E was followed using 5a (0.221 g, 0.620 mmol) and quinoline-4-carboxylic acid (0.118 g, 0.680 mmol) in CHCl₃ (15 mL). Note: The reaction mixture was washed with a 1.0 M aq solution of NaOH. After work-up, the crude product was product was purified by chromatography (24 g of silica gel, 0–10% MeOH/DCM) to afford 11a (0.231 g, 0.451 mmol, 73% yield) as a white solid. R_f = 0.5 (10% MeOH/DCM); ¹H NMR (400 MHz, CDCl₃) δ 8.89 (d, J = 4.3 Hz, 1H), 8.18 (d, J = 8.4 Hz, 1H), 8.12 (d, J = 8.4 Hz, 1H), 7.75 (ddd, J = 8.4, 6.8, 1.2 Hz, 1H), 7.60 (ddd, J = 8.3, 6.9, 1.2 Hz, 1H), 7.38 (d, J = 4.3 Hz, 1H), 7.17–7.11 (m, 2H), 6.96 (dd, J = 6.4, 3.1 Hz, 1H), 5.96 (d, J = 8.2 Hz, 1H), 4.10–3.98 (m, 1H), 3.18–2.94 (m, 4H), 2.75–2.51 (m, 4H), 2.50–2.38 (m, 2H), 2.24–2.12 (m, 2H), 1.92–1.81 (m, 2H), 1.54–1.43 (m, 2H), 1.37–1.11 (m, 5H); ¹³C NMR (101 MHz, CDCl₃) δ 166.7, 151.4, 150.0, 148.8, 142.5, 134.2, 130.1, 130.0, 127.8, 127.64, 127.57, 125.3, 124.7, 124.6, 118.7, 118.4, 56.7, 53.6 (2C), 51.5 (2C), 49.6, 35.6, 34.0, 33.2 (2C), 32.0 (2C); IR (neat) 3274, 1633 cm⁻¹; mp 222–223 °C (dec); HRMS (ESI) m/z [M + H]⁺ calcd for C₂₈H₃₃Cl₂N₄O 511.2026, found 511.2024; t_R = 36.0 min (HPLC, basic). The HCl salt was precipitated from 1 0.03 M solution of the free base in 50% CHCl₃/acetone using a 2.0 M solution of HCl in Et₂O (14 equiv). Dec >270 °C. Anal. calcd for C₂₈H₃₂Cl₂N₄O•2HCl•0.5H₂O: C, 56.67; H, 5.95; N, 9.44. Found: C, 56.68; H, 5.81; N, 9.34.

N-(trans-4-(2-(4-(2-Chloro-3-ethylphenyl)piperazin-1-yl)ethyl)cyclohexyl)quinoline-4-carboxamide (11b).

General procedure E was followed using 5b (0.125 g, 0.358 mmol) and quinoline-4-carboxylic acid (68.0 mg, 0.393 mmol) in CHCl₃ (9.0 mL). After work-up, the crude product was purified by chromatography (silica gel, 0–8% MeOH/CHCl₃) to afford 11b (0.185 g, mmol, ~100% yield) as a white solid, which was purified further by HCl salt formation. ¹H NMR (400 MHz, CDCl₃) δ 8.90 (d, J = 4.3, 1H), 8.19 (d, J = 8.5 Hz, 1H), 8.13 (d, J = 8.5 Hz, 1H), 7.75 (t, J = 7.7 Hz, 1H), 7.60 (t, J = 7.7 Hz, 1H), 7.39 (d, J = 4.3, 1H), 7.18–1.7.12 (m, 1H), 6.94 (t, J = 7.6 Hz, 2H), 5.94 (d, J = 8.3 Hz, 1H), 4.11–3.98 (m, 1H), 3.19–2.95 (m, 4H), 2.77 (q, J = 7.3 Hz, 2H), 2.72–2.51 (m, 4H), 2.50–2.41 (m, 2H), 2.23–2.15 (m, 2H), 1.91–1.83 (m, 2H), 1.54–1.45 (m, 2H), 1.41–1.11 (m, 8H); ¹³C NMR (101 MHz, CDCl₃) δ 166.7, 150.0, 149.8, 148.8, 143.4, 142.5, 130.2, 130.1, 128.8, 127.8, 127.0, 125.3, 124.6, 124.1, 118.5, 118.1, 56.8, 53.7 (2C), 51.7 (2C), 49.6, 35.6, 34.0, 33.2 (2C), 32.0 (2C), 27.6, 14.3. The HCl salt was precipitated from 50% CHCl₃/acetone using a 2.0 M solution of HCl in Et₂O. Dec >250 °C. Anal. calcd for C₃₀H₃₇ClN₄O•2HCl•2H₂O: C, 58.68; H, 7.06; N, 9.12. Found: C, 58.74; H, 6.97; N, 8.94.

N-(trans-4-(2-(4-(6-(Trifluoromethyl)pyridin-2-yl)piperazin-1-yl)ethyl)cyclohexyl)quinoline-4-carboxamide (11d).

General procedure E was followed using 5d (0.234 g, 0.656 mmol) and quinoline-4-carboxylic acid (0.125 g, 0.720 mmol) in CHCl₃ (16 mL). Note: The reaction mixture was washed with a 1.0 M aq solution of NaOH. After work-up, the crude product was product was purified by chromatography (24 g of silica gel, 0–10% MeOH/DCM) to afford 11d (0.234 g, 0.458 mmol, 70% yield) as a white solid. R_f = 0.2 (5% MeOH/DCM); ¹H NMR (400 MHz, CDCl₃) δ 8.88 (d, J = 4.3 Hz, 1H), 8.18 (d, J = 8.5 Hz, 1H), 8.11 (d, J = 8.4 Hz, 1H), 7.74 (ddd, J = 8.4, 6.9, 1.4 Hz, 1H), 7.63–7.52 (m, 2H), 7.37 (d, J = 4.3 Hz, 1H), 6.93 (d, J = 7.3 Hz, 1H), 6.76 (d, J = 8.7 Hz, 1H), 5.98 (d, J = 8.2 Hz, 1H), 4.10–3.97 (m, 1H), 3.61–3.53 (m, 4H), 2.60–2.47 (m, 4H), 2.46–2.35 (m, 2H), 2.23–2.14 (m, 2H), 1.91–1.82 (m, 2H), 1.53–1.43 (m, 2H), 1.38–1.10 (m, 5H); ¹³C NMR (101 MHz, CDCl₃) δ 166.7, 159.0, 150.0, 148.8, 146.5 (q, J = 34 Hz), 142.5, 138.4, 130.1, 130.0, 127.8, 125.3, 124.6, 121.8 (q, J = 274 Hz), 118.4, 109.5, 109.0 (q, J = 3 Hz), 56.7, 53.1 (2C), 49.6, 44.9 (2C), 35.5, 33.9, 33.2 (2C), 32.0 (2C); IR (neat) 3328, 1635 cm⁻¹; mp 187–188 °C (dec); HRMS (ESI) m/z [M + H]⁺ calcd for C₂₈H₃₃F₃N₅O 512.2632, found 512.2627; t_R = 34.3 min (HPLC, basic). The HCl salt was precipitated from a 0.03 M solution of the free base in 50% CHCl₃/acetone using a 2.0 M solution of HCl in Et₂O (14 equiv). mp 292–294 °C (dec). Anal. calcd for C₂₈H₃₂F₃N₅O•2HCl•0.25H₂O: C, 57.10; H, 5.90; N, 11.89. Found: C, 57.06; H, 5.93; N, 11.78.

N-trans-4-(2-(4-(2,3-Dichlorophenyl)piperazin-1-yl)ethyl)cyclohexyl)-4-(pyridin-3-yl)benzamide (12a).

General procedure E was followed using 5a (0.101 g, 0.284 mmol) and 4-(pyridin-3-yl)benzoic acid•HCl (73.1 mg, 0.310 mmol) in CHCl₃ (8.0 mL). After work-up, the crude product was product was purified by chromatography (12 g of silica gel, 0–10% MeOH/DCM) to afford 12a (0.120 g, 0.224 mmol, 79% yield) as a white solid. R_f = 0.5 (10% MeOH/DCM); ¹H NMR (400 MHz, CDCl₃) δ 8.88–8.84 (m, 1H), 8.63 (d, J = 4.9 Hz, 1H), 7.92–7.83 (m, 3H), 7.67–7.61 (m, 2H), 7.39 (dd, J = 7.9, 4.8 Hz, 1H), 7.18–7.10 (m, 1H), 6.96 (dd, J = 6.3, 3.2 Hz, 1H), 5.98 (d, J = 8.1 Hz, 1H), 4.02–3.90 (m, 1H), 3.16–2.97 (m, 4H), 2.74–2.53 (m, 4H), 2.49–2.40 (m, 2H), 4.02–3.90 (m, 2H), 2.18–2.09 (m, 2H), 1.89–1.80 (m, 2H), 1.54–1.42 (m, 2H), 1.37–1.00 (m, 5H); ¹³C NMR (101 MHz, CDCl₃) δ 166.3, 151.5, 149.3, 148.5, 140.9, 135.7, 134.7, 134.5, 134.5, 134.2, 127.8 (2C), 127.7, 127.6, 127.4 (2C), 123.8, 118.7, 56.7, 53.6 (2C), 51.5 (2C), 49.4, 35.7, 34.1 (2C), 33.3, 32.1 (2C); IR (neat) 3269, 1629 cm⁻¹; mp 257–258 °C (dec); HRMS (MALDI) m/z [M + H]⁺ calcd for C₃₀H₃₅Cl₂N₄O 537.2182, found 537.2185; t_R = 36.9 min (HPLC, basic).

N-(trans-4-(2-(4-(2-Chloro-3-ethylphenyl)piperazin-1-yl)ethyl)cyclohexyl)-4-(yridine-3-yl)benzamide (12b).

General procedure E was followed using 5b (101 mg, 0.289 mmol) and 4-(pyridine-3-yl)benzoic acid•HCl (74.8 mg, 0.317 mmol) in CHCl₃ (8.0 mL). After work-up, the crude product was purified by chromatography (silica gel, 0–10% MeOH/CHCl₃) to afford 12b (0.150 g, 0.282 mmol, 98% yield) as a white solid. ¹H NMR (400 MHz, CDCl₃) δ 8.86 (s, 1H), 8.63 (s, 1H), 7.94–7.81 (m, 3H), 7.69–7.61 (m, 2H), 7.43–7.34 (m, 1H), 7.19–7.11 (m, 1H), 6.98–6.90 (m, 2H), 5.95 (d, J = 8.0 Hz, 1H), 4.03–3.90 (m, 1H), 3.19–2.96 (m, 4H), 2.78 (q, J = 7.6 Hz, 2H), 2.73–2.52 (m, 4H), 2.51–2.40 (m, 2H), 2.19–2.08 (m, 2H), 1.92–1.81 (m, 2H), 1.54–1.45 (m, 2H), 1.37–1.11 (m, 8H); ¹³C NMR (101 MHz, CDCl₃) δ 166.3, 149.8, 149.3, 148.5, 143.4, 140.9, 135.8, 134.7, 134.6, 128.9, 127.8, 127.4, 127.0, 124.1, 123.8, 118.2, 56.8, 53.8 (2C), 51.7 (2C), 49.4, 35.7, 34.1, 33.4 (2C), 32.1 (2C), 27.6, 14.3. The HCl salt was precipitated from a 0.02 M solution of the free base in 50% CHCl₃/acetone using a 2.0 M solution of HCl in Et₂O (44 equiv). Dec >223 °C. Anal. calcd for C₃₂H₃₉ClN₄O•2HCl•2.25H₂O: C, 59.63; H, 7.12; N, 8.69. Found: C, 59.24; H, 6.82; N, 8.54.

N-(trans-4-(2-(4-(2,3-Dichlorophenyl)piperazin-1-yl)ethyl)cyclohexyl)-3-methoxypropanamide (13a).

General procedure F was followed using 5a (0.85 g, 2.4 mmol). After work-up, the crude product was purified by chromatography (40 g of silica gel, 0–5% MeOH/DCM) to afford 13a (1.04 g, 2.35 mmol, 99% yield) as a white solid. R_f = 0.5 (10% MeOH/DCM); ¹H NMR (400 MHz, CDCl₃) δ 7.17–7.11 (m, 2H), 6.95 (dd, J = 6.4, 3.2 Hz, 1H), 5.95 (d, J = 8.2 Hz, 1H), 3.77–3.66 (m, 1H), 3.62 (t, J = 5.8 Hz, 2H), 3.36 (s, 3H), 3.17–2.98 (m, 4H), 2.73–2.52 (m, 4H), 2.47–2.37 (m, 4H), 2.04–1.92 (m, 2H), 1.82–1.72 (m, 2H), 1.47–1.38 (m, 2H), 1.31–1.18 (m, 1H), 1.17–1.01 (m, 4H); ¹³C NMR (101 MHz, CDCl₃) δ 170.7, 151.4, 134.2, 127.65, 127.57, 124.7, 118.7, 69.0, 58.9, 56.7, 53.6 (2C), 51.5 (2C), 48.6, 37.4, 35.6, 34.0, 33.2 (2C), 32.0 (2C); IR (neat) 3279, 1635 cm⁻¹; mp 180–181 °C (dec); HRMS (MALDI) m/z [M + H]⁺ calcd for C₂₂H₃₄Cl₂N₃O₂ 442.2023, found 442.2023; t_R = 30.3 min (HPLC, basic). The HCl salt was precipitated from a 0.03 M solution of the free base in 50% CHCl₃/acetone using a 2.0 M solution of HCl in Et₂O (5.0 equiv). Dec >235 °C. Anal. calcd for C₂₂H₃₃Cl₂N₃O₂•HCl•0.75H₂O: C, 53.66; H, 7.27; N, 8.53. Found: C, 53.54; H, 6.95; N, 8.49.

N-(trans-4-(2-(4-(2-Chloro-3-ethylphenyl)piperazin-1-yl)ethyl)cyclohexyl)-3-methoxypropanamide (13b).

General procedure F was followed using 5b (0.197 g, 0.563 mmol). After work-up, the crude product was purified by chromatography (silica gel, 0–5% MeOH/CHCl₃) to afford 13b (0.245 g, 0.562 mmol, 100% yield) as an off-white solid. ¹H NMR (400 MHz, CDCl₃) δ 7.15 (t, J = 7.8 Hz, 1H), 6.96–6.91 (m, 2H), 5.93 (d, J = 8.2 Hz, 1H), 3.77–3.66 (m, 1H), 3.63 (t, J = 5.8 Hz, 2H), 3.36 (s, 3H), 3.13–2.98 (m, 4H), 2.77 (q, J = 7.5 Hz, 2H), 2.71–2.53 (m, 4H), 2.46–2.39 (m, 4H), 2.03–1.95 (m, 2H), 1.82–1.73 (m, 2H), 1.48–1.41 (m, 2H), 1.32–1.25 (m, 1H), 1.22 (t, J = 7.5 Hz, 3H), 1.17–1.02 (m, 4H); ¹³C NMR (101 MHz, CDCl₃) δ 170.7, 149.8, 143.4, 128.9, 127.0, 124.1, 118.2, 69.0, 58.9, 56.8, 53.7 (2C), 51.8 (2C), 48.6, 37.4, 35.7, 34.1, 33.2 (2C), 32.0 (2C), 27.6, 14.3. The HCl salt was precipitated from a 0.04 M solution of the free base in 50% CHCl₃/acetone using a 2.0 M solution of HCl in Et₂O (22 equiv). Dec >240 °C. Anal. calcd for C₂₈H₃₈ClN₃O₂•HCl: C, 61.01; H, 8.32; N, 8.89. Found: C, 60.82; H, 8.21; N, 8.81.

N-(trans-4-(2-(4-(2-Fluoro-3-methoxyphenyl)piperazin-1-yl)ethyl)cyclohexyl)-3-methoxypropanamide (13c).

General procedure F was followed using 5c (0.243 g, 0.724 mmol). After work-up, the crude product was purified by chromatography (24 g of silica gel, 0–5% MeOH/DCM) to afford 13c (0.159 g, 0.377 mmol, 52% yield) as a tan solid. R_f = 0.2 (5% MeOH/DCM); ¹H NMR (400 MHz, CDCl₃) δ 6.96 (td, J = 8.3, 1.8 Hz, 1H), 6.66–6.54 (m, 2H), 5.95 (d, J = 8.2 Hz, 1H),3.86 (s, 3H), 3.76–3.65 (m, 1H), 3.61 (t, J = 5.8 Hz, 2H), 3.35 (s, 3H), 3.15–3.03 (m, 4H), 2.66–2.53 (m, 4H), 2.45–2.35 (m, 4H), 2.03–1.92 (m, 2H), 1.81–1.72 (m, 2H), 1.48–1.38 (m, 2H), 1.30–1.18 (m, 1H), 1.16–1.00 (m, 4H); ¹³C NMR (101 MHz, CDCl₃) δ 170.6, 148.7 (d, J = 10 Hz), 145.8 (d, J = 245 Hz), 141.2 (d, J = 6 Hz), 123.6 (d, J = 5 Hz), 111.2 (d, J = 2 Hz), 107.2, 69.0, 58.9, 56.8, 56.6, 53.6 (2C), 50.8 (d, J = 3 Hz, 2C), 48.5, 37.4, 35.6, 34.0, 33.2 (2C), 32.0 (2C); IR (neat) 3275, 1635 cm⁻¹; mp 156–157 °C (dec); HRMS (ESI) m/z [M + H]⁺ calcd for C₂₃H₃₇FN₃O₃ 422.2813, found 422.2807; t_R = 21.6 min (HPLC, basic). The HCl salt was precipitated from a 0.03 M solution of the free base in 50% CHCl₃/acetone using a 2.0 M solution of HCl in Et₂O (5.0 equiv). mp 211–213 °C (dec). Anal. calcd for C₂₃H₃₆FN₃O₃•2HCl•2H₂O: C, 52.07; H, 7.98; N, 7.92. Found: C, 51.96; H, 7.64; N, 7.78.

3-Methoxy-N-(trans-4-(2-(4-(6-(trifluoromethyl)pyridin-2-yl)piperazin-1-yl)ethyl)cyclohexyl)propenamide (13d).

General procedure F was followed using 5d (0.424 g, 1.19 mmol). After work-up, the crude product was purified by chromatography (40 g of silica gel, 0–5% MeOH/CHCl₃) to afford 13d (0.289 g, 0.654 mmol, 55% yield) as a white solid. R_f = 0.2 (5% MeOH/DCM); ¹H NMR (400 MHz, CDCl₃) δ 7.56 (t, J = 8.1 Hz, 1H), 6.93 (d, J = 6.8 Hz, 1H), 6.76 (d, J = 8.6 Hz, 1H), 5.95 (d, J = 8.2 Hz, 1H), 3.77–3.66 (m, 1H), 3.65–3.54 (m, 6H), 3.36 (s, 3H), 2.58–2.47 (m, 4H), 2.44–2.33 (m, 4H), 2.04–1.92 (m, 2H), 1.83–1.73 (m, 2H), 1.48–1.39 (m, 2H), 1.32–1.21 (m, 1H), 1.17–1.01 (m, 4H); ¹³C NMR (101 MHz, CDCl₃) δ 170.6, 159.0, 146.6 (q, J = 35 Hz), 138.4, 121.8 (q, J = 274 Hz), 109.5, 108.9, 69.0, 58.9, 56.8, 53.1 (2C), 48.5, 44.9 (2C), 37.4, 35.6, 33.9, 33.2 (2C), 32.0 (2C); IR (film) 3277, 1634 cm⁻¹; mp 167–168 °C (dec); HRMS (ESI) m/z [M + H]⁺ calcd for C₂₂H₃₄F₃N₄O₂ 443.2628, found 443.2625; t_R = 14.5 min (HPLC, acidic). The HCl salt was precipitated from a 0.04 M solution of the free base in 50% CHCl₃/acetone using a 2.0 M solution of HCl in Et₂O (3.0 equiv). Note: Et₂O was added to initiate precipitation. Dec >229 °C. Anal. calcd for C₂₂H₃₃F₃N₄O₂•HCl•0.5H₂O: C, 54.15; H, 7.23; N, 11.48. Found: C, 53.96; H, 6.99; N, 11.50.

N-(trans-4-(2-(4-(3-Chloro-5-ethyl-2-methoxyphenyl)piperazin-1-yl)ethyl)cyclohexyl)-3-methoxypropanamide (13e).

General procedure F was followed using 5e (0.87 g, 2.3 mmol). After work-up, the crude product was purified by chromatography (40 g of silica gel, 0–5% MeOH/DCM) to afford 13e (0.89 g, 1.9 mmol, 83% yield) as a white solid. R_f = 0.2 (5% MeOH/DCM); ¹H NMR (400 MHz, CDCl₃) δ 6.85 (d, J = 2.0 Hz, 1H), 6.61 (d, J = 2.0 Hz, 1H), 6.02 (d, J = 8.1 Hz, 1H), 3.83 (s, 3H), 3.75–3.65 (m, 1H), 3.62 (t, J = 5.8 Hz, 2H), 3.36 (s, 3H), 3.24–3.10 (m, 4H), 2.77–2.62 (m, 4H), 2.58–2.46 (m, 4H), 2.41 (t, J = 5.8 Hz, 2H), 1.97 (d, J = 9.7 Hz, 2H), 1.78 (d, J = 10.2 Hz, 2H), 1.15–1.42 (m, 2H), 1.33–1.22 (m, 1H), 1.19 (t, J = 7.6 Hz, 3H), 1.15–1.01 (m, 4H); ¹³C NMR (101 MHz, CDCl₃) δ 170.6, 146.4, 146.0, 140.8, 128.2, 122.3, 116.7, 68.8, 59.0, 58.7, 56.6, 53.8 (2C), 50.0 (2C), 48.4, 37.2, 35.4, 33.6, 33.0 (2C), 31.8 (2C), 28.4, 15.4; IR (neat) 3303, 1634 cm⁻¹; mp 118–120 °C; HRMS (MALDI) m/z [M + H]⁺ calcd for C₂₅H₄₁ClN₃O₃ 466.2831, found 466.2824; t_R = 35.1 min (HPLC, basic). The HCl salt was precipitated from a 0.03 M solution of the free base in 50% CHCl₃/acetone using a 2.0 M solution of HCl in Et₂O (5.0 equiv). Dec >240 °C. Anal. calcd for C₂₅H₄₀ClN₃O₃•2HCl•0.25H₂O: C, 55.25; H, 7.88; N, 7.73. Found: C, 55.06; H, 7.69; N, 7.62.

tert-Butyl 4-(2-methoxy-2-oxoethylidene)piperidine-1-carboxylate (15).

Using oven-dried glassware under an Ar atmosphere, a mixture of 90 wt% NaH (0.521 g, 19.5 mmol) in anhydrous THF (76 mL) was cooled to 0 °C, and methyl 2-(dimethoxyphosphoryl)acetate (3.2 mL, 20 mmol) was added over 10 min. After stirring the mixture vigorously for 30 min, a solution of tert-butyl 4-oxopiperidine-1-carboxylate (3.01 g, 15.1 mmol) in anhydrous THF (38 mL) was added over 10 min. The reaction was allowed to warm to rt and stirred for an additional 18 h. Then, H₂O (50 mL) was added, and the aq layer was extracted with EtOAc (2 × 50 mL). The combined organic layers were washed with brine (50 mL), dried over anhydrous Na₂SO₄, filtered, and concentrated. The crude product was purified by chromatography (80 g of silica gel, 0–20% EtOAc/hexanes) to afford 15 (3.69 g, 14.5 mmol, 96% yield) as a white solid. R_f = 0.4 (20% EtOAc/hexanes); ¹H NMR (400 MHz, CDCl₃) δ 5.71 (s, 1H), 3.69 (s, 3H), 3.54–3.43 (m, 4H), 2.97–2.89 (m, 2H), 2.31–2.23 (m, 2H), 1.47 (s, 9H); ¹³C NMR (101 MHz, CDCl₃) δ 166.9, 158.4, 154.7, 115.0, 80.0, 51.2, 44.7 (2C), 36.6, 29.7, 28.6 (3C); IR (neat) 1711, 1680, 1655 cm⁻¹; mp 64–65 °C; HRMS (MALDI) m/z [M + Na]⁺ calcd for C₁₃H₂₁NNaO₄ 278.1363, found 278.1367.

tert-Butyl 4-(2-methoxy-2-oxoethyl)piperidine-1-carboxylate (16).

Using a Parr shaker, a mixture of 15 (2.00 g, 7.84 mmol), 10 wt% Pd/C (0.839 g, 0.787 mmol), and MeOH (60 mL) was agitated under H₂ (50 psi) for 5 h. Afterward, the mixture was passed through a pad of Celite (ca. 30 g) using EtOAc (3 × 50 mL), and the filtrate was concentrated. The crude product was purified by chromatography (40 g of silica gel, 0–20% EtOAc/hexanes) to afford 16 (1.81 g, 7.03 mmol, 90% yield) as a clear, colorless oil. R_f = 0.3 (20% EtOAc/hexanes); ¹H NMR (400 MHz, CDCl₃) δ 4.21–3.96 (m, 2H), 3.67 (s, 3H), 2.71 (t, J = 12.9 Hz, 2H), 2.24 (d, J = 7.1 Hz, 2H), 1.98–1.86 (m, 1H), 1.68 (d, J = 13.1 Hz, 2H), 1.44 (s, 9H), 1.22–1.07 (m, 2H); ¹³C NMR (101 MHz, CDCl₃) δ 173.0, 154.9, 79.5, 51.6, 43.8 (2C), 41.0, 33.2, 32.0 (2C), 28.6 (3C); IR (neat) 1737, 1688 cm⁻¹; HRMS (MALDI) m/z [M + Na]⁺ calcd for C₁₃H₂₃NNaO₄ 280.1519, found 280.1525.

tert-Butyl 4-(2-oxoethyl)piperidine-1-carboxylate (17a).

Using oven-dried glassware under an Ar atmosphere, a solution of 16 (1.10 g, 4.27 mmol) in anhydrous DCM (33 mL) was cooled to −78 °C, and a 1.0 M solution of DIBALH in toluene (5.4 mL, 5.4 mmol) was added over 5 min. The reaction was stirred for 3 h and allowed to warm to rt. Then, a saturated aq solution of Rochelle salt (60 mL) was added, and the aq layer was extracted with DCM (3 × 30 mL). The combined organic layers were dried over Na₂SO₄, filtered, and concentrated. The crude product was purified by chromatography (80 g of silica gel, 0–40% EtOAc/hexanes) to afford 17a (0.492 g, 2.17 mmol, 51% yield) as a clear, colorless oil. R_f = 0.5 (40% EtOAc/hexanes); ¹H NMR (400 MHz, CDCl₃) δ 9.80–9.76 (m, 1H), 4.21–3.96 (m, 2H), 2.73 (t, J = 12.8 Hz, 2H), 2.38 (d, J = 6.7 Hz, 2H), 2.11–1.98 (m, 1H), 1.68 (d, J = 13.2 Hz, 2H), 1.45 (s, 9H), 1.24–1.09 (m, 2H); ¹³C NMR (101 MHz, CDCl₃) δ 201.6, 154.9, 79.6, 50.3, 43.9 (2C), 32.1 (2C), 30.8, 28.6 (3C); IR (neat) 1722, 1686 cm⁻¹; HRMS (MALDI) m/z [M + Na]⁺ calcd for C₁₂H₂₁NNaO₃ 250.1414, found 250.1416.

tert-Butyl 4-(3-oxopropyl)piperidine-1-carboxylate (17b).

General procedure A was followed using tert-butyl 4-(3-hydroxypropyl)piperidine-1-carboxylate (1.02 g, 4.18 mmol). After work-up, the crude product was purified by chromatography (40 g of silica gel, 0–30% EtOAc/hexanes) to afford 17b (0.769 g, 3.19 mmol, 76% yield) as a clear, colorless oil. R_f = 0.4 (30% EtOAc/hexanes); ¹H NMR (400 MHz, CDCl₃) δ 9.77 (t, J = 1.3 Hz, 1H), 4.22–3.94 (m, 2H), 2.65 (t, J = 13.1 Hz, 2H), 2.46 (td, J = 7.5, 1.7 Hz, 2H), 1.68–1.54 (m, 4H), 1.44 (s, 9H), 1.42–1.33 (m, 1H), 1.15–1.02 (m, 2H); ¹³C NMR (101 MHz, CDCl₃) δ 202.4, 154.9, 79.4, 44.0 (2C), 41.3, 35.6, 32.0 (2C), 28.58 (3C), 28.55; IR (neat) 1724, 1686 cm⁻¹; HRMS (MALDI) m/z [M + Na]⁺ calcd for C₁₃H₂₃NNaO₃ 264.1570, found 264.1575.

tert-Butyl 4-(2-(4-(2,3-dichlorophenyl)piperazin-1-yl)ethyl)piperidine-1-carboxylate (19a).

General procedure B was followed using 17a (0.486 g, 2.14 mmol) and 1-(2,3-dichlorophenyl)piperazine•HCl (0.688 g, 3.22 mmol) in DCE (14 mL). After work-up, the crude product was purified by chromatography (40 g of silica gel, 0–80% EtOAc/hexanes) to afford 19a (0.61 g, 1.4 mmol, 64% yield) as a white solid. R_f = 0.3 (80% EtOAc/hexanes); ¹H NMR (400 MHz, CDCl₃) δ 7.18–7.10 (m, 2H), 6.95 (dd, J = 6.5, 3.1 Hz, 1H), 4.18–3.94 (m, 2H), 3.15–2.96 (m, 4H), 2.78–2.52 (m, 6H), 2.49–2.40 (m, 2H), 1.67 (d, J = 13.0 Hz, 2H), 1.52–1.39 (m, 3H), 1.45 (s, 9H), 1.20–1.05 (m, 2H); ¹³C NMR (101 MHz, CDCl₃) δ 155.0, 151.4, 134.2, 127.7, 127.6, 124.7, 118.7, 79.4, 56.3, 53.6 (2C), 51.5 (2C), 44.2 (2C), 34.7, 33.7, 32.4 (2C), 28.6 (3C); IR (neat) 1682 cm⁻¹; mp 116–117 °C; HRMS (MALDI) m/z [M + H]⁺ calcd for C₂₂H₃₄Cl₂N₃O₂ 442.2023, found 442.2021.

tert-Butyl 4-(3-(4-(2,3-dichlorophenyl)piperazin-1-yl)propyl)piperidine-1-carboxylate (19b).

General procedure B was followed using 17b (0.764 g, 3.17 mmol) and 1-(2,3-dichlorophenyl)piperazine•HCl (0.933 g, 3.49 mmol) in DCE (31 mL). After work-up, the crude product was purified by chromatography (40 g of silica gel, 0–100% EtOAc/hexanes) to afford 19b (0.884 g, 1.94 mmol, 61% yield) as a clear, orange oil. R_f = 0.2 (EtOAc); ¹H NMR (400 MHz, CDCl₃) δ 7.19–7.07 (m, 2H), 6.95 (dd, J = 6.4, 3.1 Hz, 1H), 4.19–3.95 (m, 2H), 3.17–2.96 (m, 4H), 2.78–2.48 (m, 6H), 2.43–2.35 (m, 2H), 1.71–1.62 (m, 2H), 1.61–1.49 (m, 2H), 1.45 (s, 9H), 1.42–1.33 (m, 1H), 1.30–1.22 (m, 2H), 1.15–1.01 (m, 2H); ¹³C NMR (101 MHz, CDCl₃) δ 155.0, 151.4, 134.2, 127.7, 127.6, 124.7, 118.7, 79.3, 59.0, 53.5 (2C), 51.5 (2C), 44.2 (2C), 36.2, 34.5, 32.4 (2C), 28.6 (3C), 24.2; IR (film) 1692 cm⁻¹; HRMS (ESI) m/z [M + H]⁺ calcd for C₂₃H₃₆Cl₂N₃O₂ 456.2179, found 456.2175.

1-(2,3-Dichlorophenyl)-4-(2-(piperidin-4-yl)ethyl)piperazine (20a).

General procedure C was followed using 19a (0.250 g, 0.566 mmol) and TFA (7.0 mL, 91 mmol) in DCM (37 mL). After work-up, 20a (0.191 g, 0.558 mmol, 99% yield) was isolated as a tan solid. R_f = 0.1 (10% MeOH/DCM); ¹H NMR (400 MHz, CDCl₃) δ 7.16–7.10 (m, 2H), 6.95 (dd, J = 6.4, 3.2 Hz, 1H), 3.14–2.94 (m, 6H), 2.73–2.49 (m, 6H), 2.47–2.38 (m, 2H), 1.73–1.55 (m, 3H), 1.51–1.34 (m, 3H), 1.00–1.06 (m, 2H); ¹³C NMR (101 MHz, CDCl₃) δ 151.5, 134.1, 127.64, 127.56, 124.6, 118.7, 56.3, 53.6 (2C), 51.5 (2C), 47.0 (2C), 35.0, 34.5, 34.0 (2C); IR (neat) 1577 cm⁻¹; mp 78–80 °C; HRMS (MALDI) m/z [M + H]⁺ calcd for C₁₇H₂₆Cl₂N₃ 342.1498, found 342.1500.

1-(2,3-Dichlorophenyl)-4-(3-(piperidin-4-yl)propyl)piperazine (20b).

General procedure C was followed using 19b (0.244 g, 0.535 mmol) and TFA (1.6 mL, 21 mmol) in DCM (3.7 mL). After work-up, 20b (0.188 g, 0.527 mmol, 99% yield) was isolated as a clear, yellow oil. R_f = 0.1 (10% MeOH/DCM); ¹H NMR (400 MHz, CDCl₃) δ 7.16–7.08 (m, 2H), 6.98–6.90 (m, 1H), 2.17–2.92 (m, 6H), 2.74–2.49 (m, 6H), 2.42–2.31 (m, 2H), 1.95–1.77 (m, 1H), 1.72–1.60 (m, 2H), 1.59–1.43 (m, 2H), 1.42–1.16 (m, 3H), 1.15–0.98 (m, 2H); ¹³C NMR (101 MHz, CDCl₃) δ 151.5, 134.2, 127.7, 127.6, 124.7, 118.7, 59.0, 53.5 (2C), 51.5 (2C), 46.0 (2C), 35.8, 34.7, 32.2 (2C), 24.1; IR (neat) 1577 cm⁻¹; HRMS (ESI) m/z [M + H]⁺ calcd for C₁₈H₂₈Cl₂N₃ 356.1655, found 356.1652.

4-(2-(4-(2,3-Dichlorophenyl)piperazin-1-yl)ethyl)-N,N-dimethylpiperidine-1-carboxamide (21a).

General procedure D was followed using 20a (0.184 g, 0.538 mmol) and N,N-dimethylcarbamyl chloride (65 μL, 0.71 mmol) in DCM (5.4 mL). After work-up, the crude product was purified by chromatography (12 g of silica gel, 0–10% MeOH/DCM) to afford 21a (0.159 g, 0.385 mmol, 71% yield) as a tan solid. R_f = 0.5 (10% MeOH/DCM); ¹H NMR (400 MHz, CDCl₃) δ 7.17–7.10 (m, 2H), 6.95 (dd, J = 6.4, 3.1 Hz, 1H), 3.68–3.60 (m, 2H), 3.14–2.94 (m, 4H), 2.80 (s, 6H), 2.72 (td, J = 12.7, 2.3 Hz, 2H), 2.67–2.52 (m, 4H), 2.48–2.40 (m, 2H), 1.73–1.65 (m, 2H), 1.53–1.40 (m, 3H), 1.28–1.13 (m, 2H); ¹³C NMR (101 MHz, CDCl₃) δ 165.3, 151.4, 134.1, 127.64, 127.57, 124.7, 118.7, 56.3, 53.6 (2C), 51.5 (2C), 47.3 (2C), 38.7 (2C), 34.9, 33.8, 32.4 (2C); IR (neat) 1640 cm⁻¹; mp 96–97 °C; HRMS (MALDI) m/z [M + H]⁺ calcd for C₂₀H₃₁Cl₂N₄O 413.1869, found 413.1870; t_R = 32.6 min (HPLC, basic).

4-(3-(4-(2,3-Dichlorophenyl)piperazin-1-yl)propyl)-N,N-dimethylpiperidine-1-carboxamide (21b).

General procedure D was followed using 20b (0.188 g, 0.527 mmol) and N,N-dimethylcarbamyl chloride (65 μL, 0.71 mmol) in DCM (5.5 mL). After work-up, the crude product was purified by chromatography (12 g of silica gel, 0–10% MeOH/DCM) to afford 21b (0.176 g, 0.410 mmol, 78% yield) as a tan, amorphous solid. R_f = 0.5 (10% MeOH/DCM); ¹H NMR (400 MHz, CDCl₃) δ 7.17–7.10 (m, 2H), 6.95 (dd, J = 6.3, 3.3 Hz, 1H), 3.68–3.59 (m, 2H), 3.16–2.96 (m, 4H), 2.80 (s, 6H), 2.75–2.51 (m, 6H), 2.43–2.34 (m, 2H), 1.73–1.69 (m, 2H), 1.60–1.48 (m, 2H), 1.45–1.34 (m, 1H), 1.31–1.22 (m, 2H), 1.21–1.08 (m, 2H); ¹³C NMR (101 MHz, CDCl₃) δ 165.3, 151.4, 134.1, 127.63, 127.56, 124.7, 118.7, 59.0, 53.5 (2C), 51.5 (2C), 47.3 (2C), 38.7 (2C), 36.4, 34.6, 32.3 (2C), 24.2; IR (film) 1643 cm⁻¹; mp 71–73 °C; HRMS (ESI) m/z [M + H]⁺ calcd for C₂₁H₃₃Cl₂N₄O 427.2026, found 427.2021; t_R = 35.5 min (HPLC, basic).

1-(4-(2-(4-(2,3-Dichlorophenyl)piperazin-1-yl)ethyl)piperidin-1-yl)-3-methoxypropan-1-one (22a).

General procedure F was followed using 20a (0.192 g, 0.560 mmol). After work-up, the crude product was purified by chromatography (12 g of silica gel, 0–10% MeOH/DCM) to afford 22a (0.121 g, 0.283 mmol, 50% yield) as a tan solid. R_f = 0.5 (10% MeOH/DCM); ¹H NMR (400 MHz, CDCl₃) δ 7.19–7.11 (m, 2H), 6.96 (dd, J = 7.0, 2.6 Hz, 1H), 4.61 (d, J = 13.3 Hz, 1H), 3.87 (d, J = 13.5 Hz, 1H), 3.70 (t, J = 6.7 Hz, 2H), 3.36 (s, 3H), 3.18–2.95 (m, 5H), 2.82–2.64 (m, 4H), 2.60 (t, J = 6.7 Hz, 2H), 2.57–2.44 (m, 3H), 1.74 (t, J = 12.1 Hz, 2H), 1.64–1.45 (m, 3H), 1.23–1.07 (m, 2H); ¹³C NMR (101 MHz, CDCl₃) δ 169.2, 151.3, 134.2, 127.7, 127.6, 124.8, 118.7, 69.0, 59.0, 56.2, 53.5 (2C), 51.3 (2C), 46.1, 42.0, 34.7, 33.7, 33.4, 33.0, 32.1; IR (neat) 1644 cm⁻¹; mp 88–90 °C; HRMS (MALDI) m/z [M + H]⁺ calcd for C₂₁H₃₂Cl₂N₃O₂ 428.1866, found 428.1860; t_R = 27.4 min (HPLC, basic).

1-(4-(3-(4-(2,3-Dichlorophenyl)piperazin-1-yl)propyl)piperidin-1-yl)-3-methoxypropan-1-one (22b).

General procedure F was followed using 20b (0.183 g, 0.513 mmol). After work-up, the crude product was purified by chromatography (24 g of silica gel, 0–5% MeOH/DCM) to afford 22b (0.175 g, 0.396 mmol, 77% yield) as a clear, orange oil. R_f = 0.2 (5% MeOH/DCM); ¹H NMR (400 MHz, CDCl₃) δ 7.21–7.11 (m, 2H), 7.01–6.94 (m, 1H), 4.69–4.55 (m, 1H), 3.95–3.83 (m, 1H), 3.76–3.64 (m, 2H), 3.36 (s, 3H), 3.21–3.04 (m, 4H), 3.03–2.93 (m, 1H), 2.85–2.66 (m, 4H), 2.65–2.42 (m, 5H), 1.82–1.68 (m, 2H), 1.66–1.42 (m, 3H), 1.36–1.21 (m, 2H), 1.18–1.01 (m, 2H); ¹³C NMR (101 MHz, CDCl₃) δ 169.2, 151.1, 134.2, 127.7, 127.6, 125.0, 118.8, 69.0, 59.0, 58.9, 53.5 (2C), 51.1 (2C), 46.1, 42.1, 36.2, 34.2, 33.7, 32.9, 32.0, 23.8; IR (neat) 1634 cm⁻¹; HRMS (ESI) m/z [M + H]⁺ calcd for C₂₂H₃₄Cl₂N₃O₂ 442.2023, found 442.2019; t_R = 17.8 min (HPLC, acidic). The HCl salt was prepared from a 0.03 M solution of the free base in 50% CHCl₃/acetone using a 2.0 M solution of HCl in Et₂O (15 equiv). The solution was concentrated, and the resulting residue was sonicated with hexanes to obtain a yellow solid, which was recrystallized from CHCl₃—hexanes (slow evaporation of solvent mixture). Mp 193–194 °C (dec). Anal. calcd for C₂₂H₃₃Cl₂N₃O₂•HCl•0.25H₂O: C, 54.66; H, 7.19; N, 8.69. Found: C, 54.37; H, 7.04; N, 8.52.

tert-Butyl 3-allyl-3-hydroxypyrrolidine-1-carboxylate (24a).

Using oven-dried glassware under an Ar atmosphere, a solution of tert-butyl 3-oxopyrrolidine-1-carboxylate (1.52 g, 8.21 mmol) in anhydrous Et₂O (50 mL) was cooled to 0 °C. Then, a 1.0 M solution of allyl magnesium bromide in Et₂O (9.8 mL, 9.8 mmol) was added over 5 min, and the mixture was allowed to warm to rt. After stirring for 4 h, the reaction was quenched with a saturated aq solution of NH₄Cl (10 mL), and the aq layer was extracted with Et₂O (3 × 10 mL). The combined organic layers were washed with brine, dried over Na₂SO₄, filtered, and concentrated. The crude product was purified by chromatography (40 g of silica gel, 0–30% EtOAc/hexanes) to afford 24a (1.11 g, 4.88 mmol, 60% yield) as a clear, yellow oil. R_f = 0.2 (30% EtOAc/hexanes); ¹H NMR (400 MHz, CDCl₃, mixture of rotamers) δ 5.94–5.79 (m, 1H), 5.25–5.15 (m, 2H), 3.59–3.42 (m, 2H), 3.41–3.20 (m, 2H), 2.39 (d, J = 7.5 Hz, 2H), 1.90–1.80 (m, 3H), 1.46 (s, 9H); ¹³C NMR (101 MHz, CDCl₃, mixture of rotamers) δ 154.8, 133.0, 119.9, 79.5, 79.0, 78.2, 57.7, 57.5, 44.9, 44.4, 43.5, 37.8, 37.3, 28.7 (3C); IR (film) 3412, 1697, 1670 cm⁻¹; mp 110–112 °C; HRMS (ESI) m/z [M + Na]⁺ calcd for C₁₂H₂₁NO₃Na 250.1414, found 250.1414.

tert-Butyl 4-allyl-4-hydroxypiperidine-1-carboxylate (24b).

The same procedure as the one described for 24a was followed starting from tert-butyl 4-oxopiperidine-1-carboxylate (2.00 g, 10.0 mmol). After work-up, the crude product was purified by chromatography (silica gel, 0–40% EtOA/hexanes) to afford 24b (1.44 g, 6.00 mmol, 60% yield) as a clear, colorless oil. ¹H NMR (400 MHz, CDCl₃) δ 5.93–5.79 (m, 1H), 5.25–5.10 (m, 2H), 4.00–3.60 (m, 2H), 3.28–3.04 (m, 2H), 2.23 (d, J = 7.6 Hz, 2H), 1.74–1.34 (m, 5H), 1.45 (s, 9H).

tert-Butyl 3-hydroxy-3-(3-hydroxypropyl)pyrrolidine-1-carboxylate (25a).

A 1.0 M solution of BH₃•THF in THF (15 mL, 15 mmol) was added over 5 min to a solution of 24a (1.75 g, 7.72 mmol) in THF (15 mL) at 0 °C. The reaction mixture was stirred for 3 h, and a 2.0 M aq solution of NaOH (24 mL, 47 mmol) was added followed by 30 wt% H₂O₂ in H₂O (24 mL, 0.23 mol). The resulting mixture was stirred for an additional 3 h and allowed to warm to rt. The mixture was diluted with H₂O (30 mL) and extracted with EtOAc (3 × 150 mL). The combined organic extracts were dried over anhydrous Na₂SO₄, filtered, and concentrated. The crude product was purified by chromatography (120 g of silica gel, 0–100% hexanes/EtOAc) to afford 25a (1.34 g, 5.48 mmol, 71% yield) as a clear, colorless oil. R_f = 0.3 (EtOAc); ¹H NMR (400 MHz, CDCl₃, mixture of rotamers) δ 3.79–3.61 (m, 2H), 3.55–3.32 (m, 3H), 3.26–3.16 (m, 1H), 1.97–1.89 (m, 1H), 1.86–1.66 (m, 5H), 1.45 (s, 9H); ¹³C NMR (101 MHz, CDCl₃, mixture of rotamers) δ 155.1, 154.9, 79.5, 79.4, 78.3, 63.1, 58.3, 57.9, 45.1, 44.5, 38.4, 37.7, 36.9, 36.6, 28.7 (3C), 28.1, 27.8; IR (film) 3374, 1668 cm⁻¹; HRMS (ESI) m/z [M + Na]⁺ calcd for C₁₂H₂₃NO₄Na 268.1519, found 268.1517.

tert-Butyl 4-hydroxy-4-(3-hydroxypropyl)piperidine-1-carboxylate (25b).

The same procedure as the one described for 25a was followed starting from 24b (2.60 g, 10.8 mmol). After work-up, the crude product was purified by chromatography (silica gel, 0–100% hexanes/EtOAc) to afford 25b (1.14 g, 4.40 mmol, 41% yield) of a clear, colorless oil. ¹H NMR (400 MHz, CDCl₃) δ 3.80 (d, J = 12.9 Hz, 2H), 3.70 (t, J = 6.1 Hz, 2H), 3.16 (t, J = 12.3 Hz, 2H), 2.06–1.80 (m, 2H), 1.73–1.65 (m, 2H), 1.63–1.48 (m, 6H), 1.45 (s, 9H).

tert-Butyl 2-hydroxy-1-oxa-7-azaspiro[4.4]nonane-7-carboxylate (26a).

General procedure A was followed using 25a (1.34 g, 5.46 mmol). After work-up, the crude product was purified by chromatography (80 g of silica gel, 0–50% EtOAc/hexanes) to afford 26a (0.958 g, 3.94 mmol, 72% yield) as a white solid. R_f = 0.3 (50% EtOAc/hexanes); ¹H NMR (400 MHz, CDCl₃, mixture of rotamers and diastereomers) δ 5.53 (s, 1H), 3.67–2.99 (m, 5H), 2.27–1.67 (m, 6H), 1.44 (s, 9H); ¹³C NMR (101 MHz, CDCl₃, mixture of rotamers and diastereomers) δ 154.7, 98.9, 89.2, 89.1, 88.4, 88.3, 79.4, 57.9, 57.5, 57.0, 56.4, 45.3, 44.8, 38.6, 38.1, 37.8, 37.1, 33.7, 33.6, 32.1, 32.0, 31.9, 31.4, 28.7 (3C); IR (film) 3406, 1695, 1675 cm⁻¹; HRMS (ESI) m/z [M + Na]⁺ calcd for C₁₂H₂₁NO₄Na 266.1363, found 266.1362.

tert-Butyl 2-hydroxy-1-oxa-8-azaspiro[4.5]decane-8-carboxylate (26b).

General procedure A was followed using 25b (1.14 g, 4.40 mmol). After work-up, the crude product was purified by chromatography (silica gel, 0–40% EtOAc/hexanes) to afford 26b (0.450 g, 1.75 mmol, 40% yield) as a white solid. ¹H NMR (400 MHz, CDCl₃) δ 5.55–5.48 (m, 1H), 3.69–3.48 (m, 2H), 3.44–3.26 (m, 2H), 2.56–2.50 (m, 1H), 2.01–1.85 (m, 2H), 1.85–1.64 (m, 3H), 1.62–1.39 (m, 3H), 1.45 (s, 9H).

tert-Butyl 3-(3-(4-(2,3-dichlorophenyl)piperazin-1-yl)propyl)-3-hydroxypyrrolidine-1-carboxylate (27a).

General procedure B was followed using 26a (0.958 g, 3.94 mmol) and 1-(2,3-dichlorophenyl)piperazine•HCl (1.16 g, 4.33 mmol) in DCE (38 mL). After work-up, the crude product was purified by chromatography (silica gel, 0–10% MeOH/DCM) to afford 27a (0.754 g, 1.64 mmol, 42% yield) as a yellow solid. ¹H NMR (400 MHz, CDCl₃, mixture of rotamers) δ 7.19–7.10 (m, 2H), 6.98–6.93 (m, 1H), 3.60–3.38 (m, 2H), 3.35–3.18 (m, 2H), 3.17–2.97 (m, 4H), 2.90–2.58 (m, 4H), 2.56–2.47 (m, 2H), 1.92–1.69 (m, 6H), 1.48–1.41 (m, 9H).

tert-Butyl 4-(3-(4-(2,3-Dichlorophenyl)piperazin-1-yl)propyl)-4-hydroxypiperidine-1-carboxylate (27b).

General procedure B was followed using 26b (0.445 g, 1.73 mmol) and 1-(2,3-dichlorophenyl)piperazine•HCl (0.509 g, 1.90 mmol) in DCE (18 mL). After work-up, the crude product was purified by chromatography (silica gel, 0–10% MeOH/DCM) to afford 27b (0.810 g, 1.71 mmol, 99% yield) as a clear, yellow oil. ¹H NMR (400 MHz, CDCl₃) δ 7.19–7.12 (m, 2H), 6.96 (dd, J = 7.1, 2.5 Hz, 1H), 3.89–3.64 (m, 2H), 3.24 (t, J = 11.8 Hz, 2H), 3.16–2.96 (m, 4H), 2.83–2.57 (m, 4H), 2.53–2.46 (m, 2H), 1.74–1.63 (m, 4H), 1.56–1.38 (m, 5H), 1.45 (s, 9H).

3-(3-(4-(2,3-Dichlorophenyl)piperazin-1-yl)propyl)pyrrolidin-3-ol (28a).

General procedure C was followed using 27a (0.754 g, 1.64 mmol) and TFA (2.5 mL, 33 mmol) in DCM (12 mL). After work-up, 28a (0.422 g, 1.18 mmol, 72% yield) was isolated and used immediately.

4-(3-(4-(2,3-Dichlorophenyl)piperazin-1-yl)propyl)piperidin-4-ol (28b).

General procedure C was followed using 27b (0.814 g, 1.72 mmol) and TFA (2.6 mL, 34 mmol) in DCM (12 mL). After work-up, 28b (0.500 g, 1.34 mmol, 78% yield) was isolated and used immediately.

3-(3-(4-(2,3-Dichlorophenyl)piperazin-1-yl)propyl)-3-hydroxy-N,N-dimethylpyrrolidine-1-carboxamide (29a).

General procedure D was followed using 28a (0.150 g, 0.419 mmol) and N,N-dimethylcarbamyl chloride (48 μL, 0.52 mmol) in DCM (6.0 mL). After work-up, the crude product was purified by chromatography (silica gel, 0–5% MeOH/DCM) to afford 29a (68.0 mg, 0.158 mmol, 38% yield) as a clear, yellow oil. ¹H NMR (400 MHz, CDCl₃) δ 7.16–7.08 (m, 2H), 6.97–6.91 (m, 1H), 3.70–3.60 (m, 1H), 3.45–3.35 (m, 2H), 3.24–3.18 (m, 1H), 3.15–2.95 (m, 4H), 2.82–2.58 (m, 4H), 2.81 (s, 6H), 2.53–2.46 (m, 2H), 1.87–1.67 (m, 6H); HRMS (MALDI) m/z [M + H]⁺ calcd for C₂₀H₃₁Cl₂N₄O₂ 429.1819, found 429.1802. The HCl salt was precipitated from a 0.06 M solution of the free base in 50% acetone/CHCl₃ using a 2.0 M solution of HCl in Et₂O (4.0 equiv). Mp 184–186 °C. Anal. calcd for C₂₀H₃₀Cl₂N₄O₂•1.25HCl: C, 50.58; H, 6.63; N, 11.80. Found: C, 50.64; H, 6.39; N, 11.60.

4-(3-(4-(2,3-Dichlorophenyl)piperazin-1-yl)propyl)-4-hydroxy-N,N-dimethylpiperidine-1-carboxamide (29b).

General procedure D was followed using 28b (0.804 g, 2.16 mmol) and N,N-dimethylcarbamyl chloride (0.25 mL, 2.7 mmol) in DCM (42 mL). After work-up, the crude product was purified by chromatography (silica gel, 0–5% MeOH/DCM) to afford 29b (0.272 g, 0.613 mmol, 28% yield) as a clear, yellow oil. ¹H NMR (400 MHz, CDCl₃) δ 7.18–7.11 (m, 2H), 6.99–6.92 (m, 1H), 3.45–3.37 (m, 2H), 3.31–3.19 (m, 2H), 3.18–2.96 (m, 4H), 2.87–2.57 (m, 4H), 2.80 (s, 6H), 2.53–2.44 (m, 2H), 1.74–1.63 (m, 4H), 1.58–1.48 (m, 4H); HRMS (MALDI) m/z [M + H]⁺ calcd for C₂₁H₃₃Cl₂N₄O₂ 443.1975, found 443.1971. The HCl salt was precipitated from a 0.06 M solution of the free base in 50% acetone/CHCl₃ using a 2.0 M solution of HCl in Et₂O (2.5 equiv). mp 189–192 °C. Anal. calcd for C₂₁H₃₂Cl₂N₄O₂•HCl•0.25H₂O: C, 52.07; H, 6.97; N, 11.57. Found: C, 51.98; H, 7.01; N, 11.22.

1-(3-(3-(4-(2,3-Dichlorophenyl)piperazin-1-yl)propyl)-3-hydroxypyrrolidin-1-yl)-3-methoxypropan-1-one (30a).

General procedure F was followed using 28a (0.422 g, 1.18 mmol). After work-up, the crude product was purified by chromatography (silica gel, 0–5% MeOH/DCM) to afford 30a (0.137 g, 0.309 mmol, 26% yield) as a clear, yellow oil. ¹H NMR (400 MHz, CD₃OD) δ 7.31–7.24 (m, 2H), 7.16–7.10 (m, 1H), 3.76–3.60 (m, 4H), 3.59–3.45 (m, 2H), 3.35–3.32 (m, 3H), 3.28–3.15 (m, 4H), 3.14–2.96 (m, 4H), 2.94–2.82 (m, 2H), 2.68–2.51 (m, 2H), 2.04–1.70 (m, 6H); HRMS (MALDI) m/z [M + H]⁺ calcd for C₂₁H₃₂Cl₂N₃O₃ 444.1815, found 444.1814; t_R = 16.9 min (HPLC, acidic).

1-(4-(3-(4-(2,3-Dichlorophenyl)piperazin-1-yl)propyl)-4-hydroxypiperidin-1-yl)-3-methoxypropan-1-one (30b).

General procedure F was followed using 28b (0.500 g, 1.34 mmol). After work-up, the crude product was purified by chromatography (silica gel, 0–5% MeOH/CHCl₃) to afford 30b (0.313 g, 0.309 mmol, 50% yield) as a yellow solid. ¹H NMR (400 MHz, CDCl₃) δ 7.22–7.11 (m, 2H), 7.00–6.94 (m, 1H), 4.38–4.29 (m, 1H), 3.74–3.67 (m, 2H), 3.66–3.58 (m, 1H), 3.43 (t, J = 12.6 Hz, 1H), 3.35 (s, 3H), 3.27–3.03 (m, 5H), 2.95–2.69 (m, 4H), 2.61 (t, J = 6.9 Hz, 4H), 1.84–1.53 (m, 6H), 1.50–1.36 (m, 2H); HRMS (MALDI) m/z [M + H]⁺ calcd for C₂₂H₃₄Cl₂N₃O₃ 458.1972, found 458.1962. The HCl salt was precipitated from a 0.08 M solution of the free base in 50% acetone/CHCl₃ using a 2.0 M solution of HCl in Et₂O (10 equiv). Dec >160 °C. Anal. calcd for C₂₂H₃₃Cl₂N₃O₃•HCl•H₂O: C, 51.52; H, 7.08; N, 8.19. Found: C, 51.62; H, 6.81; N, 8.14.

hD₂R and hD₃R Radioligand Binding Studies

Radioligand binding assays were conducted similarly as previously described.⁴⁶ HEK293 cells stably expressing human D₂LR or D₃R were grown in a 50:50 mix of DMEM and Ham’s F12 culture media, supplemented with 20 mM HEPES, 2 mM L-glutamine, 0.1 mM non-essential amino acids, 1X antibiotic/antimycotic, 10% heat-inactivated fetal bovine serum, and 200 μg/mL hygromycin (Life Technologies, Grand Island, NY) and kept in an incubator at 37 °C and 5% CO₂. Upon reaching 80−90% confluence, cells were harvested using premixed Earle’s balanced salt solution with 5 mM EDTA (Life Technologies) and centrifuged at 3000 rpm for 10 min at 21 °C. The supernatant was removed, and the pellet was resuspended in 10 mL hypotonic lysis buffer (5 mM MgCl₂, 5 mM Tris, pH 7.4 at 4 °C) and centrifuged at 14500 rpm (~25000 g) for 30 min at 4 °C. The pellet was then resuspended in binding buffer. Bradford protein assay (Bio-Rad, Hercules, CA) was used to determine the protein concentration. For [³H]-N-methylspiperone binding studies membranes were diluted to 500 μg/mL, in fresh EBSS binding buffer made from 8.7 g/L Earle’s Balanced Salts without phenol red (US Biological, Salem, MA), 2.2 g/L sodium bicarbonate, pH to 7.4, and stored in a −80 °C freezer for later use. For [³H]-(R)-(+)-7- OH-DPAT binding studies,⁵¹ membranes were harvested and used fresh; the binding buffer was made from 50 mM Tris, 10 mM MgCl₂, 1 mM EDTA, pH 7.4. On the test day, each test compound was diluted into half-log serial dilutions using the 30% dimethyl sulfoxide (DMSO) vehicle. When it was necessary to assist solubilization of the drugs at the highest tested concentration, 0.1% AcOH (final concentration v/v) was added alongside the vehicle. Membranes were diluted in fresh binding buffer. Radioligand competition experiments were conducted in 96-well plates containing 300 μL fresh binding buffer, 50 μL of the diluted test compound, 100 μL of membranes (for [³H]-N-methylspiperone assays: 10−20 μg/well total protein for hD_2LR and hD₃R; for [³H]-(R)-(+)-7- OH-DPAT assays: 40−80, and 20−40 μg/well total protein for hD_2LR, and hD₃R, respectively), and 50 μL of radioligand diluted in binding buffer ([³H]-N-methylspiperone: 0.4 nM final concentration for all the hD₂-like receptor subtypes; [³H]-(R)-(+)-7-OH-DPAT: 1.5 nM final concentration for hD_2L, and 0.5 nM final concentration for hD₃; Perkin Elmer). Aliquots of radioligands solution were also quantified accurately in each experiment replicate, to determine how much radioactivity was added, taking in account the experimentally determined counter efficiency. Nonspecific binding was determined using 10 μM (+)-butaclamol (Sigma-Aldrich, St. Louis, MO), and total binding was determined with the 30% DMSO vehicle. All compound dilutions were tested in triplicate, and the reaction incubated for 60 min ([³H]-N-methylspiperone assays) or 90 min ([³H]-(R)-(+)-7-OH-DPAT assays) at RT. The reaction was terminated by filtration through PerkinElmer Uni-Filter-96 GF/B, presoaked for the incubation time in 0.5% polyethylenimine, using a Brandel 96-Well Plates Harvester Manifold (Brandel Instruments, Gaithersburg, MD). The filters were washed thrice with 3 mL (3 X ~1 mL/well) of ice-cold binding buffer. PerkinElmer MicroScint 20 Scintillation Cocktail (65 μL) was added to each well, and filters were counted using a PerkinElmer MicroBeta Microplate Counter. IC₅₀ values for each compound were determined from dose−response curves, and K_i values were calculated using the Cheng−Prusoff equation.⁵⁰ K_d values were determined via separate homologous competitive binding experiments. When a complete inhibition could not be achieved at the highest tested concentrations, K_i values have been extrapolated by constraining the bottom of the dose−response curves (=0% residual specific binding) in the nonlinear regression analysis. These analyses were performed using GraphPad Prism version 8 for Macintosh (GraphPad Software, San Diego, CA). All results were rounded to the third significant figure. K_i values were determined from at least three independent experiments, each performed in triplicate, and are reported as the mean ± standard error of the mean (SEM).

BRET Studies of D₂R and D₃R Signaling

All reagents were purchased from Sigma Aldrich-Merck unless otherwise stated. BRET experiments were performed in transiently transfected human embryonic kidney 293 T (HEK 293T) cells, as described previously.^{53, 68} Briefly, cells were grown and maintained at 37 °C in 5% CO₂ in Dulbecco’s modified eagle medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS). Cells were seeded in 10 cm Petri dishes (2.5 × 10⁶ cells per dish) and allowed to grow overnight in media at 37 °C, 5% CO₂. The following day, cells were transiently transfected in full media supplemented with antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin, Gibco) using a 1:6 total DNA to PEI (PolySciences Inc) ratio. BRET constructs were as follows: 2 μg of WT-Gαo_A, 1 μg of Gβ1-Venus(156–239), 1 μg of Gγ2-Venus(1–155), 1 μg of masGRK3ct-Rluc8 and 1 μg of receptor (D_2LR or D₃R) for GPA assays and 4 μg of Venus-mG_si and 1 μg of D₂LR-Rluc8 for mini G recruitment assays. Cells were then allowed to grow overnight at 37 °C, 5% CO₂. The next day, cells were plated in Greiner poly-D-lysine-coated 96-well plates (SLS) in media and allowed to grow overnight. On the day of the assay (48h post-transfection), cells were washed once with D-PBS (Lonza, SLS) and incubated in D-PBS for 30 min at 37 °C, 5% CO₂. The Rluc substrate coelenterazine h (NanoLight) was added to each well (final concentration of 5 μM) and cells were incubated for 5 minutes at 37 °C. After 5 minutes, ligands (final concentration from 10 μM to 0.01 nM in D-PBS) were added to the plate and cells were incubated for a further 10 minutes at 37 °C. For the antagonist-mode assays that require co-addition, quinpirole was added together with the ligands (final concentration of 3 nM) to generate 50–70% of the signal that can then be displaced by an antagonist. The plate was then read in a PHERAstar FSX microplate reader (Venus and Rluc emission signals at 535 and 475 nm respectively, BMG Labtech). The ratio of Venus:Rluc counts was used to quantify the BRET signal in each well. Data were normalized to the maximal response of dopamine/quinpirole or no drug for the 100% or 0% response, respectively and as indicated in the figure axes titles. All experiments were performed in duplicate and at least three times independently. All data points represent the mean value and error bars represent the standard error of the mean (SEM). Data were fitted using the built-in log(agonist) vs. response (three parameters) model in Prism 9.0 (GraphPad software Inc., San Diego, CA). For agonist-mode assays, data were fitted to a three-parameter concentration-response model where EC₅₀ is the concentration of the agonist needed to elicit half the maximal response of the particular agonist, defined as E_max. For the antagonist-mode assays, data points were fitted using a three-parameter concentration-response model where IC₅₀ is the concentration required to inhibit half the maximum response of the agonist used at a particular concentration. Values of pEC₅₀ or pIC₅₀ plus/minus error are given as the error has a gaussian distribution whereas the error associated with the antilog value does not.

Cocaine Self Administration

Animals.

Male Long–Evans rats 275–325 g (Charles River Laboratories) were used in the experiments. All animals were housed individually in a climate-controlled animal room on a reversed light–dark cycle with free access to food and water. All experimental procedures were conducted in accordance with the Guide for the Care and Use of Laboratory Animals of the United States National Research Council and were approved by the National Institute on Drug Abuse Animal Care and Use Committee.

Surgery.

Rats used in cocaine self-administration experiments were implanted with a microrenathane intravenous (i.v.) catheter (Braintree Scientific Inc., Braintree, MA, USA). Each rat was first anaesthetized with ketamine (100 mg/kg i.p.) and xylazine (10 mg/kg, i.p.) and then a small incision was made to the right of the midline of the neck to expose the external jugular vein. One end of the i.v. catheter was next inserted into the vein with the catheter tip reaching the right atrium. The catheter was then secured to the vein with silk suture and the other end fed subcutaneously around the back of the neck to exit near the back of the skull, connected to a bent 24-gauge stainless steel cannula (Plastics One Inc., Roanoke, VA, USA) with a threaded head used to secure a dummy cannula and, during experimentation, an infusion line. The catheter and the guide cannula were secured to the skull with four stainless steel screws threaded into the skull and dental cement. After surgery, the catheters were flushed daily with gentamicin– and heparin–saline solutions (0.1 mg/ml gentamicin and 30 IU/ml heparin; ICN Biochemicals, Cleveland, OH, USA) as precaution against catheter clogging and infection. The animals were allowed to recover for at least 7 days before behavioral training started.

Apparatus.

Standard MED associates operant test chambers were used for self-administration experiments. Each box was equipped with two levers (active and inactive) and a house light set to on at the beginning of each session. Presses on the active lever triggered a compound light and tone cue. Data were collected and analyzed using MED PC software.

Cocaine self-administration under Fixed Ratio (FR) reinforcement.

A total of 48 animals underwent intravenous catheterization surgery. Following recovery, subjects were trained to lever press for cocaine (1 mg/kg/infusion) under an FR1 reinforcement schedule for five sessions. Then cocaine self-administration continued under FR2 reinforcement at 0.5 mg/kg per infusion for approximately two weeks until stable responding was observed (more than 20 infusions, <20% variability in responding across three consecutive sessions and a ratio of at least 2:1 active to inactive lever presses). Each session was 3 h long with a cap of 50 infusions at a dose of 1 mg/kg/infusion to prevent overdose and a cap of 100 infusions at a dose of 0.5 mg/kg/infusion. Rats were randomly assigned to receive intraperitoneal injections of vehicle (25% 2-hydroxypropyl-beta-cyclodextrin; Onbio Inc., Ontario, Canada) or cariprazine (0.3, 1, and 3 mg/kg), 13a (0.3, 1, and 3 mg/kg), 13e (0.3, 1, 3, and 10 mg/kg), 30 min before cocaine self-administration testing. Each animal received 4 or 5 injections for the different drug doses. The sequence of the drug doses was counterbalanced. Between tests, subjects underwent an additional three to five sessions of cocaine self-administration until the baseline response was re-established.

Data analysis.

Behavioral experiments were analyzed with SigmaPlot 12.5 Software (Systat, Palo Alto, CA). All dose treatments groups were compared to vehicle group using One-way Repeated Measures ANOVA or One-way ANOVA tests followed by Dunnet’s post-hoc test.

Supplementary Material

Gogarnoiu et al JMC 2023 SI

NIHMS1989580-supplement-Gogarnoiu_et_al_JMC_2023_SI.docx^{(1.4MB, docx)}

ACKNOWLEDGMENTS

This project was supported by the National Institute on Drug Abuse – Intramural Research Program (ZIADA000424) and the NIDA-IRP Medications Development Program. The authors thank Dr. Ning Sheng Cai from the Integrative Neurobiology Section at NIDA-IRP for assistance with cell lines and tissue culture protocols. We also acknowledge Dr. Shelley Jackson (Translational Analytical Core, NIDA-IRP) for providing all HRMS data. The ACUC approved animal protocol number is 22-BNRB-48.

ABBREVIATIONS

BD-I: bipolar 1 disorder
BMS: borane-dimethyl sulfide
BRET: bioluminescence resonance energy transfer
D₂R: dopamine D₂ receptor
D₃R: dopamine D₃ receptor
DIPEA: N,N-diisopropylethylamine
EDCI: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide•HCl
GPA: G-protein Activation
HCTU: O-(1H-6-chlorobenzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate
HEK 293 cells: human embryonic kidney 293 cells
HOBt: hydroxybenzotriazole
MPO: multiparameter optimization
PP: primary pharmacophore
PSUD: psychostimulant use disorders
SP: secondary pharmacophore
SUD: substance use disorders

Footnotes

The Supporting Information is available free of charge on the ACS Publications website.

Supplementary Tables and representative HPLC traces (pdf)

SMILES strings (excel)

The authors declare no competing financial interest.

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Gogarnoiu et al JMC 2023 SI

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PERMALINK

Dopamine D3/D2 Receptor Ligands Based on Cariprazine for the Treatment of Psychostimulant Use Disorders that May be Dual Diagnosed with Affective Disorders

Emma S Gogarnoiu

Caleb D Vogt

Julie Sanchez

Alessandro Bonifazi

Elizabeth Saab

Anver Basha Shaik

Omar Soler-Cedeño

Guo-Hua Bi

Benjamin Klein

Zheng-Xiong Xi

J Robert Lane

Amy Hauck Newman

Abstract

Graphical Abstarct

INTRODUCTION

Figure 1.

RESULTS AND DISCUSSION

Chemistry

Scheme 1.

Scheme 2.

Scheme 3.

Binding affinities of cariprazine and its analogues at D2R and D3R

Table 1.

Table 2.

Characterization of selected compounds in assays of D2R and D3R activation

Table 3.

Figure 2. Agonism profiles at D2R.

Table 4.

Figure 3. Functional profiles at the D3R.

Off-target data for cariprazine and related derivatives

Table 5.

Effects of cariprazine and selected analogues on cocaine self-administration in rats

Figure 4. Dose-dependent effects of cariprazine on cocaine self-administration in rats.

Figure 5. Dose-dependent effects of 13a on cocaine self-administration.

Figure 6. Dose-dependent effects of 13e on cocaine self-administration.

CONCLUSIONS

EXPERIMENTAL METHODS

Chemistry

General Information.

General Procedure A.

General Procedure B.

General Procedure C.

General Procedure D.

General Procedure E.

General Procedure F.

tert-Butyl (trans-4-(2-hydroxyethyl)cyclohexyl)carbamate (2).

tert-Butyl (trans-4-(2-oxoethyl)cyclohexyl)carbamate (3).

tert-Butyl (trans-4-(2-(4-(2,3-dichlorophenyl)piperazin-1-yl)ethyl)cyclohexyl)carbamate (4a).

tert-Butyl (trans-4-(2-(4-(2-chloro-3-ethylphenyl)piperazin-1-yl)ethyl)cyclohexyl)carbamate (4b).

tert-Butyl (trans-4-(2-(4-(2-fluoro-3-methoxyphenyl)piperazin-1-yl)ethyl)cyclohexyl)carbamate (4c).

tert-Butyl (trans-4-(2-(4-(6-(trifluoromethyl)pyridin-2-yl)piperazin-1-yl)ethyl)cyclohexyl)carbamate (4d).

tert-Butyl (trans-4-(2-(4-(3-chloro-5-ethyl-2-methoxyphenyl)piperazin-1-yl)ethyl)cyclohexyl)carbamate (4e).

trans-4-(2-(4-(2,3-Dichlorophenyl)piperazin-1-yl)ethyl)cyclohexan-1-amine (5a).

trans-4-(2-(4-(2-Chloro-3-ethylphenyl)piperazin-1-yl)ethyl)cyclohexan-1-amine (5b).

trans-4-(2-(4-(2-Fluoro-3-methoxyphenyl)piperazin-1-yl)ethyl)cyclohexan-1-amine (5c).

trans-4-(2-(4-(6-(Trifluoromethyl)pyridin-2-yl)piperazin-1-yl)ethyl)cyclohexan-1-amine (5d).

trans-4-(2-(4-(3-Chloro-5-ethyl-2-methoxyphenyl)piperazin-1-yl)ethyl)cyclohexan-1-amine (5e).

3-(trans-4-(2-(4-(2,3-Dichlorophenyl)piperazin-1-yl)ethyl)cyclohexyl)-1,1-dimethylurea (cariprazine).

3-(trans-4-(2-(4-(2-Chloro-3-ethylphenyl)piperazin-1-yl)ethyl)cyclohexyl)-1,1-dimethylurea (6b).

3-(trans-4-(2-(4-(2-Fluoro-3-methoxyphenyl)piperazin-1-yl)ethyl)cyclohexyl)-1,1-dimethylurea (6c).

1,1-Dimethyl-3-(trans-4-(2-(4-(6-(trifluoromethyl)pyridine-2-yl)piperazin-1-yl)ethyl)cyclohexyl)urea (6d).

3-(trans-4-(2-(4-(3-Chloro-5-ethyl-2-methoxyphenyl)piperazin-1-yl)ethyl)cyclohexyl)-1,1-dimethylurea (6e).

1-(trans-4-(2-(4-(2,3-Dichlorophenyl)piperazin-1-yl)ethyl)cyclohexyl)-3-methylurea (7a).

1-(trans-4-(2-(4-(2-Chloro-3-ethylphenyl)piperazin-1-yl)ethyl)cyclohexyl)-3-methylurea (7b).

1-(trans-4-(2-(4-(3-Chloro-5-ethyl-2-methoxyphenyl)piperazin-1-yl)ethyl)cyclohexyl)-3-methylurea (7e).

1-(trans-4-(2-(4-(2,3-Dichlorophenyl)piperazin-1-yl)ethyl)cyclohexyl)urea (8a).

1-(trans-4-(2-(4-(3-Chloro-5-ethyl-2-methoxyphenyl)piperazin-1-yl)ethyl)cyclohexyl)urea (8e).

N-(trans-4-(2-(4-(2,3-Dichlorophenyl)piperazin-1-yl)ethyl)cyclohexyl)-7,8-dihydro-1,6-naphthyridine-6(5H)-carboxamide (9a).

N-(trans-4-(2-(4-(2-Chloro-3-ethylphenyl)piperazin-1-yl)ethyl)cyclohexyl)-7,8-dihydro-1,6-naphthyridine-6(5H)-carboxamide (9b).

N-(trans-4-(2-(4-(2,3-Dichlorophenyl)piperazin-1-yl)ethyl)cyclohexyl)-1H-indole-2-carboxamide (10a).

N-(trans-4-(2-(4-(2-Chloro-3-ethylphenyl)piperazin-1-yl)ethyl)cyclohexyl)indole-2-carboxamide (10b).

N-(trans-4-(2-(4-(3-Chloro-5-ethyl-2-methoxyphenyl)piperazin-1-yl)ethyl)cyclohexyl)indole-2-carboxamide (10e).

N-(trans-4-(2-(4-(2,3-Dichlorophenyl)piperazin-1-yl)ethyl)cyclohexyl)quinoline-4-carboxamide (11a).

N-(trans-4-(2-(4-(2-Chloro-3-ethylphenyl)piperazin-1-yl)ethyl)cyclohexyl)quinoline-4-carboxamide (11b).

N-(trans-4-(2-(4-(6-(Trifluoromethyl)pyridin-2-yl)piperazin-1-yl)ethyl)cyclohexyl)quinoline-4-carboxamide (11d).

N-trans-4-(2-(4-(2,3-Dichlorophenyl)piperazin-1-yl)ethyl)cyclohexyl)-4-(pyridin-3-yl)benzamide (12a).

N-(trans-4-(2-(4-(2-Chloro-3-ethylphenyl)piperazin-1-yl)ethyl)cyclohexyl)-4-(yridine-3-yl)benzamide (12b).

N-(trans-4-(2-(4-(2,3-Dichlorophenyl)piperazin-1-yl)ethyl)cyclohexyl)-3-methoxypropanamide (13a).

Dopamine D₃/D₂ Receptor Ligands Based on Cariprazine for the Treatment of Psychostimulant Use Disorders that May be Dual Diagnosed with Affective Disorders

Binding affinities of cariprazine and its analogues at D₂R and D₃R

Characterization of selected compounds in assays of D₂R and D₃R activation

Figure 2. Agonism profiles at D₂R.

Figure 3. Functional profiles at the D₃R.

hD₂R and hD₃R Radioligand Binding Studies

BRET Studies of D₂R and D₃R Signaling