Structure-Activity Relationships for a Series of (Bis(4-fluorophenyl)methyl)sulfinylethyl-aminopiperidines and -piperidine amines at the Dopamine Transporter: Bioisosteric Replacement of the Piperazine Improves Metabolic Stability

Abstract

Despite considerable efforts to develop medications to treat psychostimulant use disorders, none have proven effective, leaving an underserved patient population and unanswered questions as to what mechanism(s) of action should be targeted for developing pharmacotherapies. Atypical dopamine transporter (DAT) inhibitors, based on (±)modafinil, have shown therapeutic potential in preclinical models of psychostimulant abuse. However, metabolic instability among other limitations to piperazine analogues 1–3 have impeded further development. Herein, bioisosteric substitutions of the piperazine ring were explored with a series of aminopiperidines (A) and piperidine amines (B) wherein compounds with either a terminal tertiary amine or amide were synthesized. Several lead compounds showed high to moderate DAT affinities and metabolic stability in rat liver microsomes. Aminopiperidines 7 (DAT K_i = 50.6 nM), 21b (DAT K_i = 77.2 nM) and 33 (DAT K_i = 30.0 nM) produced only minimal stimulation of ambulatory activity in mice, compared to cocaine, suggesting an atypical DAT inhibitor profile.

Graphical Abstract

INTRODUCTION

Psychostimulant use disorders continue to be a public health problem that, in the United States, has been further exacerbated by the opioid crisis.¹ In fact, overdose deaths involving either heroin or synthetic opioids in combination with cocaine, termed “speedballing” or “super speedballing”, rose 36% annually from 2010 to 2015,² and overdose deaths involving any opioid with abused psychostimulants rose 55%, from 2016 to 2017.² Morbidity and mortality rates reflect that cocaine and abused psychostimulants are mixed with opioids,² with 73% of cocaine-related deaths in 2017³ concomitant with opioid use, and 63% of opioid-related deaths in 2018⁴ connected with cocaine and/or psychostimulant use. Despite these alarming statistics, an FDA-approved pharmacotherapeutic remains an unmet medical need for the treatment of psychostimulant use disorders.

Cocaine disrupts canonical dopamine recycling by binding the outward facing conformation of the dopamine transporter (DAT), which oscillates between outward and inward facing conformations at the plasma membrane to allow for the cotransport of the endogenous ligand, dopamine, with Na⁺ and Cl⁻ ions down their respective concentration gradients.^{5, 6, 7} Subsequently, blockade of DAT results in dopamine accumulation in the synaptic cleft, which in the mesolimbic brain region produces feelings of stimulation and euphoria that can lead to abuse. In addition to inhibiting dopamine transport, methamphetamine, a substrate of DAT, is imported intracellularly where it interacts with the vesicular monoamine transporter (VMAT) to induce a rapid efflux of dopamine from intracellular vesicles.⁸ In addition, methamphetamine can reverse DAT to efflux dopamine into the synapse, resulting in rapid extrasynaptic accumulation of dopamine.^9–11 Although their mechanisms of action differ, their pharmacological actions and abuse liability is dependent on DAT function, which can be altered with chronic use.^{5, 12, 13}

While DAT is a molecular target implicated in psychostimulant use disorders, a class of “atypical” DAT inhibitors have been identified that bind to DAT with high affinity and selectivity but are predicted to have low abuse liability, based on results from numerous animal models.^14–16 Moreover, many cocaine-induced behaviors such as increased locomotor activity, conditioned place preference, self-administration and reinstatement to drug seeking behaviors have been mitigated by these atypical DAT inhibitors, suggesting promise for therapeutic development.

In general, the atypical DAT inhibitors prefer to bind a more occluded conformational state of DAT, and their behavioral profiles, which differ from those of typical DAT inhibitors, such as cocaine, appear to be related to this conformational difference, although why this is the case remains unclear. Compounds based on benztropine, GBR12909, and (±)-modafinil have been characterized as atypical DAT inhibitors, although none of these novel analogues has FDA approval for the treatment of psychostimulant use disorders.¹⁷

(±)-Modafinil (Provigil ®) and its R-enantiomer (Nuvigil ®) are FDA-approved treatments for narcolepsy and other sleep disorders, with additional off-label use in cognitive enhancement and mood improvement.¹⁸ Clinical trials with (±)-modafinil as a therapy for psychostimulant use disorders have provided mixed results, showing that (±)-modafinil may be a viable treatment option for a subpopulation of subjects suffering from cocaine use disorders that were not also abusing alcohol, or for patient populations suffering from methamphetamine use disorder and were not using other illicit substances. Noncompliance with treatment protocols was also suggested as a reason that statistical significance was not reached in these large clinical trials.^19–22

Due to its low affinity (DAT K_i = 2.52 μM)²³ and poor solubility, structural modifications of (±)-modafinil have been undertaken to investigate structure-activity relationships (SAR) at DAT and also provide higher affinity tools with which the atypical DAT inhibitor hypothesis could be further explored.^24–30 In Cao et al. 2016,²³ an extensive SAR study was conducted based on the modafinil scaffold, where the benzhydrol moiety was halogenated or unsubstituted, the terminal portion of the scaffold was functionalized with aliphatic and aryl piperazine moieties, and the oxidation state of the sulfide was explored. These structural modifications greatly improved the affinity and selectivity of novel analogues for DAT in comparison to the parent compound. Lead compounds JJC8–091 (1), JJC8–088 (2), and JJC8–089 (3a) had moderate to high affinities of 230 nM, 2.60 nM, and 37.8 nM for DAT, respectively, and had selectivities for DAT over the serotonin transporter (SERT) ranging from 180-fold to 4,885-fold (Figure 1 and Table 1). These leads were carried forward for additional evaluation both in vitro and in vivo,^{16, 31, 32} Behavioral characterization in models of short access and long access methamphetamine self-administration showed 1 was effective in attenuating this behavior in both groups, 3a was effective only in the long access group, and 2 was not effective in either rodent population.³¹ It was suggested that compound 2, the analogue with the highest affinity and selectivity for DAT, may have been ineffective due to its poor pharmacokinetic profile.³¹ However, more recently, computational studies showed that compound 2 exhibited a cocaine-like behavioral profile which was attributed to it preferring an open outward conformation of DAT, similar like cocaine.¹⁶

Figure 1.

Chemical structures of lead piperazine analogues of modafinil and the modified structural templates (A) and (B).

Table 1.

DAT, SERT, σ₁ binding and SAR^a

						Ki ± SEM (nM)
Compd	Temp	Y	Z	R1	R2	DAT	SERT	σ1	SERT/DAT	σ1/DAT
(±)-modafinil						8160 ± 3120	31300 ± 6570	NT	3.84	NC
1						230 ± 40.5	97800 ± 22600	454 ± 87.1	425.22	1.97
2						2.60 ± 0.445	12700 ± 3010	41.60 ± 2.530	4884.62	16.0
3a						37.8 ± 8.72	6800 ± 1870	2.24 ± 0.467	179.89	0.059
3b						23.1 ±1.81	14800 ± 2270	5.62 ± 1.21	640.69	0.243
6a	A	S	CH2	phenyl	H	32.9 ± 5.86	409 ± 58.2	3.81 ± 0.639	12.43	0.12
6b	A	S	CH2	4-fluorophenyl	H	25.5 ± 6.95	278 ± 5.96	13.3 ± 2.75	10.90	0.52
6c	A	S	CH2	4-chlorophenyl	H	39.6 ± 9.27	358 ± 0.928	45.5 ± 13.2	9.04	1.15
6d	A	S	CH2	4-CF3-phenyl	H	190 ± 47.9	1630 ± 176	186 ± 53.0	8.58	0.98
6e	A	S	CH2	benzyl	H	31.5 ± 5.32	144 ± 17.0	57.7 ± 6.63	4.57	1.83
7	A	S=O	CH2	4-fluorophenyl	H	50.6 ± 11.2	373 ± 23.8	26.5 ± 3.88	7.37	0.52
10	A	S	CH2	1-hydroxy-2-phenylethyl	H	31.4 ± 9.64	129 ± 33.4	309 ± 38.3	4.11	9.84
11	A	S=O	CH2	1-hydroxy-2-phenylethyl	H	91.8 ± 21.3	599 ± 54.4	351 ± 25.9	6.53	3.82
15a	B	S	CH2	phenyl	H	108 ± 17.5	331 ± 34.6	60.9 ± 8.90	3.06	0.56
15b	B	S	CH2	4-fluorophenyl	H	55.9 ± 6.08	268 ± 9.33	41.4 ± 10.9	4.79	0.74
15c	B	S	CH2	4-chlorophenyl	H	86.5 ± 35.6	397 ± 56.9	62.4 ± 6.95	4.59	0.72
15d	B	S	CH2	4-(trifluoromethyl) phenyl	H	128 ± 31.9	1050 ± 191	162 ± 32.5	8.20	1.27
16	B	S	CH2	1-hydroxy-2-phenylethyl	H	47.7 ± 2.62	66.8 ± 2.82	88 ± 4.02	1.40	1.84
19a	A	S	C=O	phenyl	H	4.51 ± 0.86	283 ± 25.9	2.03 ± 0.302	62.75	0.45
19b	A	S	C=O	4-fluorophenyl	H	7.04 ± 0.824	489 ± 32.3	32.1 ± 4.38	69.46	4.59
19c	A	S	C=O	benzofuran-2-yl	H	115 ± 19.5	97.0 ± 23.8	304 ± 23.1	0.84	2.64
19d	A	S	C=O	indol-2-yl	H	177 ± 36.7	944 ± 76.8	1130 ± 285	5.33	6.38
19e	A	S	CH2	indol-2-yl	H	179 ± 22.1	1730 ± 519	205 ± 42.2	9.66	1.15
20	A	S	CH2	benzofuran-2-yl	H	120 ± 24.3	695 ± 99.4	205 ± 1.02	5.79	1.71
21a	A	S=O	C=O	phenyl	H	79.1 ± 20.6	7780 ± 734	585 ± 21.5	98.36	7.40
21b	A	S=O	C=O	4-fluorophenyl	H	77.2 ± 4.54	4640 ± 381	1440 ±131	60.10	18.7
24a	A	S	C=O	t-butyl	H	352 ± 137	1010 ± 230	163 ± 8.77	2.87	0.46
24b	A	S	C=O	cyclopropyl	H	178 ± 35	1010 ± 94	46.4 ± 4.13	5.67	0.26
24c	A	S	CH2	cyclopropyl	H	407 ± 65	1600 ±164	269 ± 23	3.93	0.66
25	A	S	CH2	t-butyl	H	279 ± 39.4	947 ± 101	315 ± 76	3.39	1.13
26	A	S=O	C=O	t-butyl	H	10200±3610	32000 ± 6760	11200 ± 2540	3.14	1.10
27	A	S=O	CH2	t-butyl	H	431 ± 28.2	6990 ± 229	542 ± 224	16.22	1.26
32	A	S	C=O	2-fluorophenyl	H	46.1± 17.1	351± 25.0	3.57 ± 0.998	7.61	0.08
33	A	S	C=O	2,4-difluorophenyl	H	30.0 ± 8.25	296 ± 35.3	20.6 ± 2.38	9.87	0.69
34	A	S=O	C=O	2-fluorophenyl	H	604 ± 158	14200 ± 136	1310 ± 361	23.5	2.16
35	A	S=O	C=O	2,4-difluorophenyl	H	564 ± 223	4610 ± 228	171 ± 4.89	8.17	0.30
36	A	S	C=O	4-fluorophenyl	CH3	190 ± 28.2	2440 ± 266	7.11±2.39	12.8	0.04
37	A	S	C=O	2-fluorophenyl	CH3	222 ± 37.0	992 ± 36.5	5.87 ± 0.986	4.47	0.03
38	A	S	C=O	2-fluorophenyl	isopropyl	1530 ± 402	16300 ± 2370	126 ± 34.5	10.7	0.08
39	A	S=O	C=O	2-fluorophenyl	CH3	7380 ± 1780	251000 ± 172000	516 ± 161	34.0	0.07
40						4090 ± 781	48700 ± 5990	>100000	11.9	>24
41b						2970 ± 604	15000 ± 7440	>100000	5.05	>34
42b						4830 ± 1370	38900 ± 3630	>100000	8.05	>21

^aEquilibrium dissociation constants (K_i) were derived from IC₅₀ values using the Cheng-Prusoff equation⁴⁵. Each K_i value represents the arithmetic mean ± S.E.M of at least three independent experiments, each performed in triplicate. NT = Not Tested; NC = Not Calculated.

^bCompounds previously described^{42, 43}.

Thus far, pharmacological testing, determination of metabolic and pharmacokinetic profiles as well as behavioral studies have cumulatively indicated 1 as a lead candidate for further development.^{16, 31} Recently, additional modifications of this scaffold were reported, wherein a new lead, 3b (RDS3–94) was identified.³³ By replacing the piperazine ring with a rel-cis-2,6-dimethyl piperazine and replacing the sulfoxide of 1 with the sulfide of 3a, a new and potentially atypical DAT inhibitor was identified that is currently being evaluated in behavioral models of cocaine abuse.³³ Nevertheless, poor metabolic stability remained a problem with this compound and although the primary metabolite also had relatively high affinity for DAT, a more metabolically stable lead molecule was desirable.³³

Based on metabolite identification studies of previous leads 1, 2 and 3b, it became clear that the piperazine group in these molecules was metabolically unstable, and efforts to modify the piperazine or other substituents in the lead molecules were not successful in mitigating its instability in vivo.^{31, 33} Hence, in the current study, we have further extended SAR for this class of modafinil-based compounds with the goal of improving metabolic stability. We hypothesized that the introduction of bioisosteric moieties to replace the metabolically susceptible piperazine ring would improve the metabolic profiles of our previous leads, while retaining the preferred atypical binding conformation at DAT. Hence, a series of aminopiperidines (A) and piperidine amines (B) (Figure 1) were synthesized where benzylic, heterocyclic, and aliphatic moieties were terminally appended. The importance of the tertiary amine was explored through compounds with either a tertiary amine or amide, and the oxidation state of the sulfide was varied. Of note, the bis(4-F-phenyl)methyl moiety of the previous lead compounds (1-3) was retained in these new series. All final compounds were evaluated for their affinities at both DAT and SERT, due to the high degree of homology between these two transporters,^{6, 34} as well as at the σ₁ receptor due to preclinical literature precedent suggesting that a dual DAT/σ₁ mechanism of action might provide therapeutic efficacy.^{35, 36} A subset of these compounds, chosen for their favorable binding profiles and/or resemblance to previous leads, were evaluated for their off-target affinities at the norepinephrine transporter (NET) and across dopamine D₂, D₃, and D₄ receptor subtypes. Moreover, a subset of nine analogues was also characterized for metabolic stability profiles in rat liver microsomes. Three of the most stable compounds were also evaluated for metabolic stability in mouse liver microsomes and tested for locomotor activity in mice as compared to cocaine.

CHEMISTRY

The first series of aminopiperidines (A) and piperidine amines (B) were prepared as described in Scheme 1. Compound 4, previously reported in Cao et al. 2016,²³ served as the key starting intermediate for this synthesis. N-alkylation of 4 in a suspension of K₂CO₃ in acetonitrile was carried out using the appropriately substituted aminopiperidine analogues. Commercially available (5a), previously prepared (5b-5d),³⁷ or (5e) prepared by TFA deprotection of the corresponding tert-butyl (1-phenethylpiperidin-4-yl)carbamate,³⁸ yielded compounds 6a-6e in 19–69% yield. Further, compound 6b was oxidized at the sulfide moiety using H₂O₂ in acetic acid (AcOH) and methanol to afford the corresponding racemic sulfoxide analogue 7 in 45% yield. N-alkylation of 4 with 1-(4-aminopiperidin-1-yl)-3-phenylpropan-2-ol (8), which was prepared from 9, in a suspension of K₂CO₃ in acetonitrile and subsequently deprotected in TFA, gave compound 10 in 68% yield, which was likewise oxidized using H₂O₂ in AcOH and methanol to afford the corresponding racemic sulfoxide analogue 11 in 46% yield.

Scheme 1: Synthesis of para-substituted and phenylalkyl compounds^a.

^aReagents and conditions: (a) appropriate piperidine analogue, K₂CO₃, acetonitrile, 3–4.5 h, reflux; (b) H₂O₂, AcOH/methanol, 40 °C, overnight; (c) tert-butyl piperidin-4-ylcarbamate, K₂CO₃, acetonitrile, reflux, 4.5 h; (d) TFA, RT, 1–1.5 h; (e) (i) appropriate benzaldehyde (14a–d), cat. AcOH, DCE, RT, 15 min; (ii) Na(AcO)₃BH, RT, overnight; (f) 2-benzyloxirane, n-butyllithium (1.15 M - 2.5 M in THF), THF, reflux, overnight.

The piperidine amine 12 was synthesized by N-alkylation of 4 using tert-butyl piperdin-4-yl carbamate in a suspension of K₂CO₃ in acetonitrile, as described above. Subsequent Boc-deprotection of 12 in TFA gave compound 13 in quantitative yield. Reductive amination of 13 in the presence of the appropriately substituted commercially available benzaldehyde (14a-14d) using sodium triacetoxyborohydride (Na(AcO)₃BH) in 1,2-dichloroethane (DCE) catalyzed with AcOH gave compounds 15a-15d in 41–71% yield. Finally, 13 was further reacted with 2-benzyloxirane in the presence of n-butyllithium in THF to afford the desired product 16 in 23% yield.

Based on preliminary binding data for the compounds prepared in Scheme 1, the aminopiperidine (A) template appeared to have somewhat higher binding affinities at DAT, hence all subsequent analogues were prepared in this series only. Seeking to further extend SAR at the N-terminus of this series, heterocycle-containing and amide-linked compounds were synthesized.

As described in Scheme 2, amide-linked aryl compounds were synthesized via amidation of commercially available benzoic acid (17a), 4-fluorobenzoic acid (17b), benzofuran-2-carboxylic acid (17c), or 1H-indole-2-carboxylic acid (17d) using 1,1’-carbonyldiimidazole (CDI), as the coupling reagent, and tert-butyl-piperidine-4-yl carbamate to respectively afford compounds 18a-18d in 77–99% yield. Further, compound 18e was synthesized via reductive amination using 1H-indole-2-carbaldehyde in 90% yield. Due to the volatility of intermediates 5a-5e (see Scheme 1), after the Boc-deprotection of compounds 18a-18e in TFA, the corresponding crude amine salts were immediately N-alkylated with 4 as described above to give compounds 19a-19e in 11–29% yield. Reduction of the amide in 19c was achieved using LiAlH₄ in THF to afford 20 in 45% yield. Oxidation of the sulfide in compounds 19a and 19b was performed as described in Scheme 1, using H₂O₂ in AcOH and methanol to give racemic compounds 21a and 21b in 60% and 85% yield, respectively.

Scheme 2: Synthesis of heterocycle-containing and amide-linked compounds^a.

^aReagents and conditions: (a) (i) CDI, THF, RT, 3h; (ii) tert-butyl piperidin-4-ylcarbamate, THF, RT, overnight; (b) (i) 1H-indole-2-carbaldehyde, cat. AcOH, DCE, RT, 15 min; (ii) Na(AcO)₃BH, RT, overnight; (c) (i) TFA, RT; (ii) 4, K₂CO₃, acetonitrile, 3 h, D; (d) LiAlH₄, THF, RT, 6 h; (e) 30% H₂O₂, AcOH/methanol, 40 °C, overnight

A series of compounds containing canonical and non-aryl bioisosteres^{39, 40} of the terminal phenyl moiety were synthesized to further explore the structural requirements of this region of the aminopiperidine (A) scaffold. As described in Scheme 3, amidation of pivalic acid (22a) or cyclopropanecarboxylic acid (22b) using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), N-hydroxybenzotriazole (HOBt), N,N-diisopropylethylamine (DIPEA or Hunig’s base), and tert-butyl-piperidine-4-yl carbamate in DCM afforded compounds 23a and 23b in 98% yield. Reductive amination using tert-butyl-piperidine-4-yl carbamate and cyclopropanecarbaldehyde gave compound 23c in 64% yield. Subsequent Boc-deprotection in TFA was followed by N-alkylation with 4 as described above to give compounds 24a-24c in 23–31% yield. Further reduction of the amide 24a using LiAlH₄ in THF afforded 25 in 46% yield. Finally, oxidation of the sulfides 24a and 25 was similarly performed using H₂O₂ in AcOH and methanol to afford racemic compounds 26 and 27, respectively (in 66% and 72% yield, respectively).

Scheme 3: Synthesis of Aliphatic Compounds^a.

^aReagents and conditions: (a) (i) EDC.HCl, HOBt, DIPEA, DCM, RT, 1 h; (ii) tert-butyl piperidin-4-ylcarbamate, RT, overnight; (b) (i) cyclopropanecarbaldehyde, cat. AcOH, DCE, RT, 15 min; (ii) Na(AcO)₃BH, RT, overnight; (c) (i) TFA, RT; (ii) 4, K₂CO₃, acetonitrile, Δ; (d) LiAlH₄, THF, RT, 6 h; (e) 30% H₂O₂, AcOH/methanol, 40 °C, overnight.

Terminal aryl substitution with fluorines was further explored based on computational data (unpublished) that suggested this substitution pattern may decrease activity at the human ether-à-go-go-related potassium channel (hERG). Numerous drugs have been withdrawn from the market due to cardiotoxicity related to hERG inhibition, and although not all hERG blockers lead to lethal cardiotoxicity, there are strong correlations.⁴¹ Hence, with the goal of reducing hERG activity as well attempting to improve metabolic stability while determining if N-alkylation was tolerated at DAT, a series of 2,4-difluoro-substituted aminopiperidine benzylamides were synthesized as described in Scheme 4. Compounds 28 and 29 were prepared from commercially available 2-fluorobenzoic acid or 2,4-difluorobenzoic acid and piperidin-4-one using CDI in 76–80% yield. Compound 4 was reacted with potassium phthalimide to give 30 which was deprotected with hydrazine to give the primary amine 31. Reductive amination with either 28 or 29 gave compounds 32 or 33, respectively. Oxidation of 32 or 33 was achieved as described above, in presence of 30% H₂O₂ in AcOH and methanol, to yield 34 and 35. Compounds 36, 37 and 38 were prepared via N-alkylation of 19b, 32 and 33, respectively. Meanwhile, H₂O₂ mediated oxidation of 37 yielded the desired sulfoxide 39.

Scheme 4: Synthesis of N-substituted aminopiperidines^a.

^aReagents and conditions: (a) appropriate acid, CDI, THF, RT, 3 h; (ii) piperidin-4-one, THF, RT, overnight; (b) phthalimide potassium salt, K₂CO₃, acetonitrile, reflux, overnight; (c) hydrazine, ethanol, reflux, overnight; (d) 28 or 29 or 37% formaldehyde in H₂O, Na(AcO)₃BH, DCE, overnight; (e) acetone, sodium cyanoborohydride, DCE, RT, 72 h; (f) 30% H₂O₂, AcOH/Methanol, 40 °C, overnight.

BIOLOGICAL RESULTS AND DISCUSSION

SAR at DAT, SERT, and σ₁.

Binding affinities for all lead compounds were evaluated at DAT, SERT and σ₁ receptors and were compared to reference compounds (±)-modafinil and its racemic JBG01–41 (40) and resolved bisfluoro-derivatives,^{42, 43} (S)-(+)-JBG01–48 (41) and (R)-(-)-JBG01–49 (42), as well as previously described leads 1, 2 and 3a. Characterization of these analogues at DAT and SERT was conducted in rat striatal and midbrain (brain stem) tissue preparations using [³H]WIN35,428 and [³H]citalopram as the labeling radioligands, respectively. σ₁ receptor binding experiments were conducted in guinea pig cortex tissue preparations using [³H](+)-pentazocine. The results of these experiments are reported in Table 1. These studies sought to determine (1) optimal regiochemistry of the aminopiperidine at DAT: template (A) vs. (B), (2) optimal terminal aryl or aliphatic substitution, (3) importance of a terminal tertiary amine vs. a tertiary amide, (4) the effect of oxidation state of the sulfide on DAT binding, (5) the effect of N-alkylation on selected template (A) compounds, and (6) structural features that optimized DAT selectivity over SERT and σ₁ or gave compounds with similar affinities across all three targets, which might also serve as interesting leads. Compounds with favorable binding profiles were carried on for additional in vitro testing.

Seeking to determine the orientation of the piperidine that provided the highest DAT binding affinities, aminopiperidine compounds 6a-6e and 10 and piperidine-amine compounds 15a-15d and 16 were synthesized with varying terminally appended alkyl-phenyl and para-substituted phenyl moieties. In general, both regiochemical orientations of the aminopiperidine moiety showed relatively similar SAR with little difference in DAT binding affinities between the para-substituents, and all analogues were modestly selective for DAT over SERT. When aminopiperidine compounds 6a-6c were compared to their piperidine-amine correlates 15a-15c, affinities were 2- to 3-fold higher for DAT in template (A). However, this trend was diminished when comparing the terminal 2-OH-propylphenyl-substituted analogues 10 (DAT K_i = 31.4 nM) and 16 (DAT K_i = 47.7 nM). Additionally, aminopiperidine compounds 6a and 6b had affinities of 3.81 nM and 13.3 nM at σ₁ respectively, lending themselves favorably to the possibility of a DAT/σ₁ dual activity profile.

To further extend aminopiperidine (A, Fig. 1) template SAR, compounds with terminally appended aryl and aliphatic moieties were characterized at DAT, SERT and σ₁ receptor. Heterocyclic analogues 19c-19e and 20 were not as well tolerated at DAT with affinities ranging from 115–179 nM. Similarly, cyclopropyl and tert-butyl analogues 24a-24b and 25–27 were not well tolerated with DAT affinity values decreasing to 178–10,200 nM. Interestingly, as evidenced by the relatively high DAT affinities of amide-linked compounds 19a (K_i = 4.51 nM) and 19b (K_i = 7.04 nM), we concluded that the tertiary terminal amine is not necessary for high binding affinity. Additionally, 19a and 19b showed high affinity at σ₁ receptor, with K_i values of 2.03 nM and 32.1 nM, respectively. Taken together these data indicate that the optimal terminal structural moiety is a simple phenyl ring, and that a tertiary amide is well-tolerated at DAT (and σ₁) when the terminal substituent is planar and monocyclic.

Oxidation to the sulfoxide analogues 7, 11, and 21a-21b resulted in a 2- to 3-fold loss in affinity at DAT when compared to their sulfide correlates, which has been noted for previously described modafinil analogues^23–25 (e.g. 1 vs. 3). In general, the sulfoxide derivatives maintained approximately the same selectivity profiles for DAT over SERT when compared to their sulfide correlates with the exception of 21a, for which a 1.6-fold improvement in selectivity was observed compared to the corresponding sulfide analogues 19a, to nearly 100-fold DAT over SERT selective.

N-alkylation (37, 38, 39, 40) and/or further, 2-fluoro-substitution (33, 35, 38, 39, 40) or 2,4-difluoro substitution (34, 36) on the terminal phenyl ring of compounds 7 and 19b was explored to determine tolerability at DAT, and to potentially prevent metabolic susceptibility.⁴⁴ N-alkylation uniformly decreased DAT binding affinities. However, although compound 33 showed lower DAT binding affinity than 19b (K_i = 30.0 vs. 7.04 nM), this affinity is still quite high. Coupled with its similar affinity at σ₁ (K_i = 20.6 nM), compound 33 was selected for additional testing.

Off-Target Binding Affinities at D₂, D₃, D₄ Receptors, and NET

A subset of analogues was selected based on their binding profiles at DAT, SERT, and σ₁ and their structural similarity to previous lead 2 for evaluation at dopamine D₂, D₃, and D₄ receptors and NET. Characterization of these analogues at the D₂-like receptors was conducted in HEK293 cells, stably expressing the receptor target of interest, using [³H]-N-methylspiperone as the radiolabeled antagonist. Evaluation of these analogues at NET was conducted in rat frontal cortex tissue preparations using [³H]-nisoxetine. The results of these experiments are shown in Table 2. (±)Modafinil’s off-target affinities are reported for comparison.

Table 2.

Off-target binding at NET and D₂-like receptors^a

	Ki ± SEM (nM)				MPOc scores
Compound	D2R	D3R	D4R	NET
(±)-Modafinil	>100,000b	39,000 ±1050b	>100,000b	>100,000	4.8
1	298 ± 61.1b	480 ± 184b	3820 ± 876b	>100,000	5.2
2	78.6 ± 16.6b	652 ± 105b	1750 ± 593b	8240 ± 1350	5.5
3a	693 ± 159b	424 ± 117b	6300 ± 1460b	11820 ± 217	3.8
3b	11900 ± 1620	1470 ± 205	NT	11500 ± 1650	NC
6b	2920 ± 307	911 ± 228	980 ± 89.3	NT	1.3
7	12400 ± 1330	12400 ± 1830	76800 ± 6220	11200 ± 1240	3.1
10	4020 ± 579	691 ± 104	2200 ± 84.3	585 ± 65	1.9
11	6610 ± 1250	5500 ± 1160	NT	NT	3.5
16	2270 ± 163	701 ± 97.3	2670 ± 415	NT	1.9
19a	2900 ± 604	2510 ± 243	4110 ± 293	920 ± 58	2.6
19b	8830 ± 2250	5230 ± 934	9020 ± 971	258 ± 14	2.3
21b	156000 ± 22500	28200 ± 6280	≫100,000	23000 ± 2120	4.1
24a	6070 ± 829	3420 ± 368	11900 ± 1090	NT	3.1
33	7440 ± 209	3260 ±168	NT	1619 ± 129	2.0

^aEquilibrium dissociation constants (K_i) were derived from IC₅₀ values using the Cheng-Prusoff equation⁴⁵. Each K_i value represents the arithmetic mean ± S.E.M of at least three independent experiments, each performed in triplicate.

^bBinding data previously reported in Cao et al. 2016.¹² NT = Not Tested;

^cMPO scores calculated using ChemDraw 15.1 and Chemicalize.

In general, low off-target activity was observed for this subset of analogues at the D₂-like receptor subtypes. For example, K_i values for this subset at the D₂-like receptors exceeded 2 μM at all subtypes with few exceptions. Compounds 6b, 10, and 16 had affinities at the D₃ receptor of 911 nM, 691 nM, and 701 nM, respectively. At the D₄ receptor, 6b had an affinity of 980 nM. Furthermore, low off-target activity was observed for selected lead molecules: 7, 21b, and 33 comparable to the off-target affinity of previous lead 2 at 8240 ± 1350 nM. In total, these data suggest that this subset of analogues interacts unfavorably with both the D₂-like receptors and NET, compared to DAT.

In addition, to off target binding, we calculated Multiparameter Optimization (MPO) scores⁴⁶ for all compounds in Table 2. The determination of MPO scores enables a better understanding and optimization of drug-like properties for compounds targeting the central nervous system (CNS). In particular, MPO analyses can be used for predicting compound’s CNS penetrability ranking them based on clogP, clogD, molecular weight (MW), total polar surface area (TPSA), total number of hydrogen bond donor atoms (HBD), and pKa.⁴⁶

Metabolic Stability in Rat and Mouse liver Microsomes

A subset of analogues (6b, 7, 10, 11, 16, 19a, 19b, 21b and 33) was tested for metabolic stability in rat liver microsomes following procedures previously described⁴⁷ to predict the susceptibility to phase I metabolism (Fig. 2A). Compounds 6b, 7, 10, 16, 21b and 33 were relatively metabolically stable with the sulfide analogues of 3a, 19a and 19b, and compound 11, an analogue of compound 2, being less stable than the other analogues tested.

Figure 2.

A) Rat liver microsomes. Compounds 11, 19a and 19b showed susceptibility to phase I metabolism in rat microsomes. Compounds 6b, 7, 10, 16, 21b and 33 were stable (70–95% remaining) after 60 min incubation. B) Mouse liver microsomes. Compounds 7, 21b, and 33 showed susceptibility to phase I metabolism in mouse liver microsomes, with 33 being the most stable with 33% remaining after 60 min incubation.

Based on their overall binding profiles and metabolic stability in rat liver microsomes, compounds 7, 21b and 33 were also tested for mouse microsomal metabolic stability and locomotor activity, in mice. All these compounds were significantly less stable in mouse liver microsomes (Figure 2B) vs. rat, although 33 was more stable than the other two (~30% at 60 min). Metabolic lability in mice is generally expected^{48, 49} and suggests that in order to see behavior, higher doses than DAT affinities predict may be required.

Locomotor activity of compounds 7, 21b and 33 compared to cocaine in mice

Despite relative metabolic instability in the mouse liver microsomes, based on the overall profiles in vitro, 7, 21b and 33 were chosen as the lead candidates for in vivo characterization, starting with locomotor activity studies in mice. Figure 3 shows the time course of effects of these analogues on stimulation of locomotor activity, expressed as distance travelled (cm/5min), compared to cocaine (see experimental methods for details). Systemic administration (i.p.) of the analogs 7, 21b, and 33 produced only minimal stimulation of ambulatory activity as shown in Figure 3 [Two-way ANOVA for repeated measures over time: Compound 7, Two-Way ANOVA: main effect Dose: F(3,20) = 1.84, NS; main effect Time: F(11,220) = 77.08, p<0.001; Time X Dose interaction: F(33,220) = 1.73, p<0.05; Compound 21b, Two-Way ANOVA: main effect Dose: F(3,20) = 1.14, NS; main effect Time: F(11,220) = 69.66, p<0.001; Time X Dose interaction: F(33,220) = 1.07, NS; Compound 33, Two-Way ANOVA: main effect Dose: F(3,19) = 5.59, p<0.05; main effect Time: F(11,209) = 51.31, p<0.001; Time X Dose interaction: F(33,209) = 1.35, NS). Administration of cocaine (3, 10 and 30 mg/kg) produced a large, significant increase in ambulatory activity [Two-way ANOVA for repeated measures over time, main effect Dose: F(3,20) = 15.76, p<0.01; main effect Time: F(11,220) = 85.49, p<0.001; Time X Dose interaction: F(33,220) = 5.02, p<0.01], that was also greater in magnitude (highest stimulation about 8,000 cm/5 min at the 30 mg/kg dose) compared to the effects produced by the aminopiperidine analogues (highest increase < 3,000 cm/5 min for all drugs at any dose tested).

Figure 3.

Panels A-D show the time course of the effects of administration of different doses of compounds 7, 21b, 33, and their vehicle (VEH), or cocaine on stimulation of ambulatory activity, expressed as distance traveled, cm/5 min, obtained in Swiss Webster mice. Panel A: Effects of 7 (VEH, 1, 10, 30 mg/kg i.p.; n=6 for all groups); Panel B: Effects of 21b (VEH, 1, 10, 30 mg/kg i.p.; n=6 for all groups); Panel C: Effects of 33 (VEH 3, 10, 30 mg/kg i.p.; n=5 for VEH, n=6 for all other groups); Panel D: Effects of cocaine (VEH, 1, 10, 30 mg/kg i.p.; n=6 for all groups). Each point represents the group mean ± S.E.M. (vertical bars) of the distance traveled (cm/5 min).

CONCLUSION

A series of aminopiperidine (A) and piperidine amine (B) analogues of previous lead compounds 1-3 were designed and synthesized in an attempt to improve metabolic stability and drug-like properties, identifying new leads for development as pharmacotherapies to treat psychostimulant use disorders. We hypothesized that the introduction of these bioisosteric moieties would result in improved metabolic profiles, while retaining binding affinity for DAT. Terminal N-benzylic, heterocyclic, and aliphatic moieties were appended and the importance of the terminal amine was explored through compounds with either a tertiary amine or amide. The oxidation state of the sulfur was also varied.

In general, most of the analogues in both (A) and (B) series showed moderately high binding affinities for DAT (K_i <200 nM), however a terminal aryl ring was required. Interestingly, replacing the piperazine resulted in much higher affinities at SERT, resulting in compounds that had a more balanced DAT/SERT profile than previous piperazine leads. Compound 21a was the exception with a DAT/SERT selectivity of ~100-fold. Most of the analogues showed σ1 binding affinities comparable to DAT affinities, with several analogues showing ~10-fold σ₁-selectivity (e.g., 32, 37-39). Importantly, we discovered that the terminal nitrogen in the (A)-series of aminopiperidines could be substituted with an amide function and retain high DAT affinity (e.g., 19a, K_i=4.51 nM and 19b, K_i=7.04 nM). As reported previously,^{23–25, 33} oxidizing the sulfide to a sulfoxide typically reduced DAT affinity (e.g., 19b vs. 21b), however the sulfoxide rendered these compounds more metabolically stable (Table 3) with higher MPO scores. None of the most interesting analogues showed appreciable binding affinities to D₂, D₃ D₄ receptors or NET and several showed MPO scores of >3, suggesting favorable CNS penetrability. A subset of nine analogues was also characterized for metabolic stability profiles in rat liver microsomes. Three of the most stable compounds (7, 21b and 33) were also evaluated for metabolic stability in mouse liver microsomes and tested for locomotor activity in mice, compared to cocaine. None of these analogues showed a cocaine-like profile as systemic administration produced only minimal stimulation of ambulatory activity as shown in Figure 3 at doses up to 30 mg/kg. Although additional in vivo testing is required, this behavioral profile is reminiscent of previously described atypical DAT inhibitors. Computational investigation is underway to determine if these compounds indeed bind DAT in an inward occluded conformation similarly to compound 1.

Table 3.

Phase I metabolic stability at 0 and 60 min, in rat liver microsome with NADPH.

Compound	0 min % remaining	60 min % remaining
6b	100 ± 6	70 ± 1
7	100 ± 4	95 ± 2
10	100 ± 5	74 ± 3
11	100 ± 4	22 ± 1
16	100 ± 3	69 ± 2
19a	100 ± 1	7 ± 0
19b	100 ± 5	24 ± 2
21b	100 ± 4	79 ± 1
33	100 ± 4	85 ± 1

EXPERIMENTAL METHODS

Synthesis.

All chemicals and solvents were purchased from chemical suppliers unless otherwise stated, and used without further purification. ¹H and ¹³C NMR spectra were acquired using a Varian Mercury Plus 400 spectrometer at 400 MHz and 100 MHz, respectively. Chemical shifts are reported in parts-per-million (ppm) and referenced according to deuterated solvent for ¹H NMR spectra (CDCl₃, 7.26 or acetone-d₆, 2.05) and ¹³C NMR spectra (CDCl₃, 77.2 or acetone-d₆, 29.8 and 206.0). Gas chromatography-mass spectrometry (GC/MS) data were acquired (where obtainable) using an Agilent Technologies (Santa Clara, CA) 7890B GC equipped with an HP-5MS column (cross-linked 5% PH ME siloxane, 30 m × 0.25 mm i.d. × 0.25 μm film thickness) and a 5977B mass-selective ion detector in electron-impact mode. Ultrapure grade helium was used as the carrier gas at a flow rate of 1.2 mL/min. The injection port and transfer line temperatures were 250 and 280 °C, respectively, and the oven temperature gradient used was as follows: the initial temperature (70°C) was held for 1 min and then increased to 300°C at 20°C/min over 11.5 min, and finally maintained at 300°C for 4 min. All column chromatography was performed using a Teledyne Isco CombiFlash RF flash chromatography system. HRMS (mass error within 5 ppm) and MS/MS fragmentation analysis were performed on a LTQ-Orbitrap Velos (Thermo-Scientific, San Jose, CA) coupled with an ESI source in positive ion mode. Combustion analyses were performed by Atlantic Microlab, Inc. (Norcross, GA) and agree with ± 0.4% of calculated values. All melting points were determined on an OptiMelt automated melting point system and are uncorrected. On the basis of NMR and combustion analysis or HRMS data, all final compounds are >95% pure.

General method for reductive amination:

The commercially available amine (1 eq) was dissolved in dichloroethane (0.03 M), and the reaction was permitted to stir until dissolved. Acetic acid (AcOH, catalytic) was added dropwise via syringe under an argon atmosphere. The appropriate aldehyde (1 eq) was added dropwise via syringe and was permitted to stir for 15 min. Sodium triacetoxyborohydride (1.5 eq) was added in one portion, and the reaction was stirred overnight at RT. Solvent was removed under reduced pressure, and the residue was resuspended in CH₂Cl₂. The combined organics were washed with NaHCO₃ then brine, and dried with MgSO₄. The organics were concentrated in vacuo and purified via flash column chromatography to afford the desired N-alkyl product.

General method for amidation:

To an appropriately size round bottom flask equipped with a stir bar was added the carboxylic acid (1 eq) in anhydrous THF (0.125 M) under an argon atmosphere, and the reaction mixture was permitted to stir until dissolved. CDI (1.2 eq) was added in one portion, and the reaction was stirred for 3 h at RT. The amine (1 eq) was dissolved in THF (0.2 M) and added dropwise via syringe, and the reaction was stirred overnight at RT. Solvent was removed, and the residue was purified via flash column chromatography to afford the desired amide product.

1-Phenethylpiperidin-4-amine (5e).

To a 15 mL round bottom flask equipped with a stir bar was added the tert-butyl (1-phenethylpiperidin-4-yl)carbamate (175 mg, 0.575 mmol)³⁸ and trifluoroacetic acid (TFA, 1.2 mL). The reaction was permitted to stir for 1 h at RT under an argon atmosphere. Solvent was removed under reduced pressure, and the residue was resuspended in CH₂Cl₂ (30 mL). The organics were washed with NaHCO₃ (3 × 10 mL, pH = 8) and rinsed with brine (3 × 10 mL). The combined aqueous layers were concentrated and extracted with CH₂Cl₂. The combined organics were dried with MgSO₄ and concentrated in vacuo to yield 5e (165 mg, quantitative yield) as a yellow amorphous solid, which was immediately used in the next step.

1-Benzyl-N-(2-((bis(4-fluorophenyl)methyl)thio)ethyl)piperidin-4-amine (6a).

To a 10 mL round bottom flask equipped with a stir bar and a condenser was added (bis(4-fluorophenyl)methyl)(2-bromoethyl)sulfane (200 mg, 0.583 mmol)^{23, 33} and dry K₂CO₃ (644 mg, 4.66 mmol). Anhydrous acetonitrile (2.33 mL) was added via syringe under an argon atmosphere, and the reaction mixture was permitted to stir. Commercially available 1-benzylpiperidin-4-amine (0.143 mL, 0.70 mmol) was added dropwise via syringe and was stirred for 4.5 hours at reflux. The reaction mixture was filtered to remove residual K₂CO₃, washed with cold acetonitrile, and the filtrate was concentrated under reduced pressure. The crude oil was purified by flash column chromatography (0–5% MeOH/0–0.125% NH₄OH in CH₂Cl₂) to afford 6a (181 mg, 0.400 mmol, 69% yield) as a yellow oil. The free base was converted to the corresponding HCl salt and recrystallized from hot isopropyl alcohol to give a colorless crystalline solid. Decomposition > 250 °C; ¹H NMR (400 MHz, CDCl₃) δ 7.41 – 7.22 (m, 9H), 7.00 (m, 4H), 5.17 (s, 1H), 3.51 (s, 2H), 2.85 (m, 2H), 2.77 (t, J = 6.6 Hz, 2H), 2.56 (t, J = 6.5 Hz, 2H), 2.42 (m, 1H), 2.03 (m, 2H), 1.98 – 1.86 (m, 1H), 1.86 – 1.77 (m, 2H), 1.47 – 1.34 (m, 2H); ¹³C NMR (100 MHz, CDCl₃) δ 163.11, 160.66, 138.35, 137.01, 136.97, 129.86, 129.83, 129.78, 129.75, 129.12, 128.20, 127.00, 115.62, 115.40, 63.00, 54.51, 52.56, 52.26, 45.11, 32.90, 32.57; Anal. (C₂₇H₃₀F₂N₂S·2HCl·0.5H₂O) C, H, N.

N-(2-((Bis(4-fluorophenyl)methyl)thio)ethyl)-1-(4-fluorobenzyl)piperidin-4-amine (6b).

Compound 6b was prepared as described for 6a using 1-(4-fluorobenzyl)piperidin-4-amine (67 mg, 0.322 mmol)³⁷ to give the product (940 mg, 93% yield) as a yellow oil. (53 mg, 0.11 mmol, 42% yield). The free base was converted to the corresponding HCl salt and was recrystallized from hot isopropyl alcohol to give a colorless crystalline solid. Decomposition > 215 °C; ¹H NMR (400 MHz, CDCl₃) δ 7.36 (m, 4H), 7.26 (m, 2H), 6.99 (m, 6H), 5.15 (s, 1H), 3.44 (s, 2H), 2.78 (m, 4H), 2.54 (m, 2H), 2.39 (m, 1H), 1.98 (m, 2H), 1.78 (m, 2H), 1.48 – 1.29 (m, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 163.12, 163.10, 160.69, 160.65, 136.98, 136.95, 134.28, 134.25, 130.47, 130.39, 129.82, 129.79, 129.74, 129.71, 115.65, 115.59, 115.44, 115.38, 115.02, 114.81, 62.18, 54.53, 52.55, 52.24, 45.12, 33.01, 32.72; FT-IR (ATR, υ, cm⁻¹) 2932, 2797, 1602, 1506, 1466, 1222, 1156, 1092,1015, 827; Anal. (C₂₇H₂₉F₃N₂S·2HCl·0.5H₂O) C, H, N.

N-(2-((Bis(4-fluorophenyl)methyl)thio)ethyl)-1-(4-chlorobenzyl)piperidin-4-amine (6c).

Compound 6c was prepared as described for 6a using 1-(4-chlorobenzyl)piperidin-4-amine (77 mg, 0.34 mmol) to afford 6c (75 mg, 0.15 mmol, 54% yield). The free base was converted to the corresponding HCl salt and was recrystallized from hot isopropyl alcohol to give a colorless crystalline solid. Decomposition > 215 °C; ¹H NMR (400 MHz, CDCl₃) δ 7.36 (m, 4H), 7.24 (m, 4H), 7.00 (m, 4H), 5.15 (s, 1H), 3.44 (s, 2H), 2.77 (m, 4H), 2.54 (t, J = 6.5 Hz, 2H), 2.39 (m, 1H), 1.98 (m, 2H), 1.78 (m, 2H), 1.54 – 1.29 (m, 3H); ¹³C NMR (101 MHz, CDCl₃) δ 163.10, 160.65, 137.19, 136.98, 136.95, 132.56, 130.26, 129.80, 129.72, 128.29, 115.60, 115.38, 62.21, 54.49, 52.56, 52.28, 45.13, 33.00, 32.70; FT-IR (ATR, υ, cm⁻¹) 2934, 2804, 1725, 1602, 1505, 1366, 1225, 1156, 1096, 835; Anal. (C₂₇H₂₉ClF₂N₂S·2HCl·0.5H₂O) C, H, N.

N-(2-((Bis(4-fluorophenyl)methyl)thio)ethyl)-1-(4-(trifluoromethyl)benzyl)piperidin-4-amine (6d).

Compound 6d was prepared as described for 6a using 1-(4-(trifluoromethyl)benzyl)piperidin-4-amine (140 mg, 0.542 mmol),³⁷ 5b to afford 6d (73 mg, 0.14 mmol, 31% yield). The free base was converted to the corresponding HCl salt and was recrystallized from hot isopropyl alcohol to give a colorless crystalline solid. Mp 234–236 °C; ¹H NMR (400 MHz, CDCl₃) δ 7.56 (m, 2H), 7.43 (m, 2H), 7.36 (m, 4H), 6.99 (m, 4H), 5.15 (s, 1H), 3.52 (s, 2H), 2.78 (m, 4H), 2.55 (t, J = 6.5 Hz, 2H), 2.40 (m, 1H), 2.02 (m, 2H), 1.79 (m, 2H), 1.61 (s, 1H), 1.46 – 1.31 (m, 2H); ¹³C NMR (100 MHz, CDCl₃) δ 163.11, 160.66, 142.98, 136.96, 136.93, 129.78, 129.70, 129.03, 125.15, 125.11, 125.08, 125.04, 115.60, 115.39, 62.42, 54.45, 52.57, 52.38, 45.10, 32.97, 32.67; FT-IR (ATR, υ, cm⁻¹) 2935, 2800, 1726, 1602, 1505, 1467, 1418, 1325, 1225, 1157, 824; Anal. (C₂₈H₂₉F₅N₂S·2HCl·0.5H₂O) C, H, N.

N-(2-((Bis(4-fluorophenyl)methyl)thio)ethyl)-1-phenethylpiperidin-4-amine (6e).

Compound 6e was prepared as described for 6a using 5e (118 mg, 0.578 mmol) to afford 6e (42 mg, 0.09 mmol, 19% yield). The free base was converted to the corresponding HCl salt and was recrystallized from hot isopropyl alcohol to give a colorless crystalline solid. Mp 239–243 °C; ¹H NMR (400 MHz, CDCl₃) δ 7.36 (m, 4H), 7.27 (m, 2H), 7.20 (d, J = 7.3 Hz, 3H), 7.00 (m, 4H), 5.16 (s, 1H), 2.96 (m, 2H), 2.86 – 2.72 (m, 4H), 2.63 – 2.51 (m, 4H), 2.42 (m, 1H), 2.07 (m, 2H), 1.93 – 1.74 (m, 3H), 1.48 – 1.33 (m, 2H); ¹³C NMR (100 MHz, CDCl₃) δ 163.11, 160.66, 140.36, 136.97, 136.94, 129.80, 129.75, 129.72, 128.68, 128.37, 126.01, 115.61, 115.39, 60.54, 54.42, 52.56, 52.37, 45.10, 33.77, 32.98, 32.63; FT-IR (ATR, υ, cm⁻¹) 2930, 1737, 1602, 1505, 1454, 1225, 1156, 1115, 835; Anal. (C₂₈H₃₂F₂N₂S·2HCl·0.25H₂O) C, H, N.

N-(2-((Bis(4-fluorophenyl)methyl)sulfinyl)ethyl)-1-(4-fluorobenzyl)piperidin-4-amine (7).

Compound 7 was prepared as previously described²³ using 6b (189 mg, 0.402 mmol) to afford product (87 mg, 0.179 mmol, 45% yield). The free base was converted to the corresponding HCl salt and recrystallized from hot methanol to give a colorless crystalline solid. Decomposition > 250 °C; ¹H NMR (400 MHz, CDCl₃) δ 7.41 (m, 4H), 7.25 (m, 2H), 7.08 (m, 4H), 6.97 (t, J = 8.6 Hz, 2H), 4.91 (s, 1H), 3.43 (s, 2H), 3.06 (t, J = 6.3 Hz, 2H), 2.78 (m, 2H), 2.59 (t, J = 6.4 Hz, 2H), 2.42 (m, 1H), 1.98 (t, J = 11.4 Hz, 2H), 1.86 – 1.75 (m, 2H), 1.66 (s, 1H), 1.34 (m, 2H); ¹³C NMR (100 MHz, CDCl₃) δ 163.99, 163.75, 163.10, 161.52, 161.27, 160.67, 134.21, 134.18, 131.62, 131.59, 131.05, 130.97, 130.50, 130.47, 130.44, 130.42, 130.36, 130.28, 116.42, 116.20, 115.86, 115.64, 115.01, 114.79, 70.34, 62.16, 54.83, 52.14, 51.55, 40.24, 32.66, 32.50; FT-IR (ATR, υ, cm⁻¹) 2936, 2799, 1602, 1505, 1467, 1415, 1221, 1159, 1042, 826; Anal. (C₂₇H₂₉F₃N₂OS·2HCl) C, H, N.

1-(4-Aminopiperidin-1-yl)-3-phenylpropan-2-ol (8).

To a 5 mL round bottom flask equipped with a stir bar and a condenser was added 9 (205 mg, 0.613 mmol) and trifluoroacetic acid (1.2 mL). The reaction was permitted to stir for 1.5 h at RT under an argon atmosphere. Solvent was removed under reduced pressure, and the residue was resuspended in CH₂Cl₂ (30 mL). The organics were washed with a 30% aqueous solution of NH₄OH (3 × 5 mL, pH = 9) and rinsed with brine (2 × 20 mL). The combined organics were dried with MgSO₄ and concentrated in vacuo to yield 8 (100 mg, 0.427 mmol, 70% yield). ¹H NMR (400 MHz, CDCl₃) δ 7.25 (m, 5H), 3.89 (m, 1H), 3.08 – 2.86 (m, 1H), 2.86 – 2.76 (m, 1H), 3.76 – 2.54 (m, 4H), 2.43 – 2.22 (m, 3H), 2.22 – 1.85 (m, 2H), 1.85 – 1.69 (m, 2H), 1.53 – 1.17 (m, 3H).

1-(2-Hydroxy-3-phenylpropyl)piperidin-4-yl)carbamate (9).

To a 15 mL round bottom flask equipped with a stir bar and a condenser was added the commercially available 2-benzyloxirane (263 mg, 1.86 mmol) and K₂CO₃ (2.17 g, 14.9 mmol). Anhydrous acetonitrile (7.8 mL) was added via syringe under an argon atmosphere, and the reaction mixture was permitted to stir. Commercially available tert-butyl piperidin-4-ylcarbamate (471 mg, 2.24 mmol) was added dropwise via syringe and was refluxed overnight. The reaction mixture was cooled to 0 °C and filtered to remove residual K₂CO₃, washed with cold acetonitrile, and the filtrate was concentrated under reduced pressure. The crude oil was purified by flash column chromatography (0–5% MeOH/0–0.125% NH₄OH in CH₂Cl₂) to afford 9 (205 mg, 0.613 mmol, 31% yield) as a colorless powder. ¹H NMR (400 MHz, CDCl₃) δ 7.40 – 7.15 (m, 5H), 4.42 (s, 1H), 3.89 (m, 1H), 3.45 (s, 2H), 3.01 – 2.84 (m, 2H), 2.84 – 2.79 (m, 2H), 2.43 – 2.22 (m, 3H), 2.11 – 1.81 (m, 3H), 1.44 (m, 11H).

1-(4-((2-((Bis(4-fluorophenyl)methyl)thio)ethyl)amino)piperidin-1-yl)-3-phenylpropan-2-ol (10).

Compound 10 was prepared as described for 6a using 8 (100 mg, 0.427 mmol) to afford product (120 mg, 0.242 mmol, 68% yield) as a yellow oil. The free base was converted to the corresponding HCl salt to give a colorless crystalline solid. Mp 169–171 °C; ¹H NMR (400 MHz, CDCl₃) δ 7.42 – 7.32 (m, 4H), 7.32 – 7.25 (m, 2H), 7.25 – 7.18 (m, 3H), 7.00 (m, 4H), 5.14 (s, 1H), 3.89 (m, 1H), 2.92 (m, 1H), 2.82 (dd, J = 13.7, 7.0 Hz, 1H), 2.74 (t, J = 6.5 Hz, 3H), 2.66 (dd, J = 13.7, 5.6 Hz, 1H), 2.62 – 2.49 (m, 2H), 2.48 – 2.21 (m, 4H), 1.92 (m, 1H), 1.78 (m, 2H), 1.44 – 1.22 (m, 2H); ¹³C NMR (100 MHz, CDCl₃) δ 163.11, 160.66, 138.36, 136.97, 136.94, 129.82, 129.78, 129.74, 129.70, 129.29, 128.31, 126.24, 115.60, 115.39, 67.38, 63.41, 54.35, 53.88, 52.61, 50.92, 45.14, 41.41, 33.04, 32.98, 32.68; FT-IR (ATR, υ, cm⁻¹) 3027, 2922, 2805, 1602, 1505, 1453, 1293, 1223, 1156, 1097, 1015, 835; Anal. (C₂₉H₃₄F₂N₂OS·2HCl·0.5H₂O) C, H, N.

1-(4-((2-((Bis(4-fluorophenyl)methyl)sulfinyl)ethyl)amino)piperidin-1-yl)-3-phenylpropan-2-ol (11).

Compound 11 was prepared as described for 7 using 10 (100 mg, 0.201 mmol) to afford product (47 mg, 0.092 mmol, 46% yield). The free base was converted to the corresponding HCl salt to give a yellow solid. Decomposition > 250 °C; ¹H NMR (400 MHz, CDCl₃) δ 7.40 (m, 4H), 7.28 (m, 2H), 7.21 (m, 3H), 7.08 (m, 4H), 4.89 (s, 1H), 3.93 (m, 1H), 3.07 – 3.03 (m, 2H), 2.97 – 2.94 (m, 1H), 2.85 – 2.71 (m, 2H), 2.70 – 2.56 (m, 3H), 2.48 (m, 1H), 2.41 – 2.30 (m, 3H), 2.10 – 1.96 (m, 1H), 1.85 (m, 2H), 1.50 – 1.17 (m, 2H); ¹³C NMR (100 MHz, CDCl3) δ 164.01, 163.78, 161.55, 161.31, 138.12, 131.47, 131.44, 131.03, 130.94, 130.41, 130.37, 130.34, 130.26, 129.28, 128.36, 126.32, 116.47, 116.26, 115.90, 115.78, 115.68, 70.54, 67.31, 63.35, 54.07, 53.38, 51.29, 51.28, 50.87, 41.44, 40.29, 32.28, 32.02, 31.76, 29.68; FT-IR (ATR, υ, cm⁻¹) 3384, 3062, 2924, 2808, 1603, 1506, 1454, 1226, 1159, 1040, 837; Anal. (C₂₉H₃₄F₂N₂O₂S·3HCl·H₂O·0.33NH₄OH) C, H, N; HRMS (ESI in positive mode) calculated 513.23818, found 513.23752 (+H⁺).

1-(2-((Bis(4-fluorophenyl)methyl)thio)ethyl)piperidin-4-amine (12).

Compound 12 was prepared as described for 6a using tert-butyl piperidin-4-ylcarbamate (1.05 g, 5.24 mmol) to afford 12 (1.79 g, 3.87 mmol, quantitative yield) as a yellow oil. ¹H NMR (400 MHz, CDCl₃) δ 7.35 (m, 4H), 6.99 (m, 4H), 5.18 (s, 1H), 4.39 (s, 1H), 3.50 – 3.34 (m, 1H), 2.72 (d, J = 11.6 Hz, 2H), 2.56 – 2.43 (m, 4H), 2.02 (td, J = 11.6, 2.6 Hz, 2H), 1.88 (d, J = 12.6 Hz, 2H), 1.50 – 1.31 (m, 11H).

1-(2-((Bis(4-fluorophenyl)methyl)thio)ethyl)piperidin-4-amine (13).

To a 10 mL round bottom flask equipped with a stir bar and a condenser was added 12 (0.150 g, 0.324 mmol) and trifluoroacetic acid (2.5 mL). The reaction was permitted to stir for 1 h at RT under an argon atmosphere. Solvent was removed under reduced pressure, and the residue was resuspended in CH₂Cl₂ (100 mL). The organics were washed with NaHCO₃ (3 × 50 mL, pH = 8) and rinsed with brine (2 × 50 mL). The combined organics were dried with MgSO₄ and concentrated in vacuo to yield 13 (117 mg, 0.323 mmol, quantitative yield) as a yellow oil. ¹H NMR (400 MHz, CDCl₃) δ 7.36 (m, 4H), 6.99 (m, 4H), 5.20 (s, 1H), 2.77 (d, J = 11.8 Hz, 2H), 2.64 (m, 1H), 2.59 – 2.44 (m, 4H), 1.99 (t, J = 11.3 Hz, 2H), 1.78 (d, J = 12.8 Hz, 2H), 1.56 (s, 2H), 1.44 – 1.28 (m, 2H).

N-Benzyl-1-(2-((bis(4-fluorophenyl)methyl)thio)ethyl)piperidin-4-amine (15a).

Compound 15a was prepared from 13 (165 mg, 0.455 mmol) and commercially available benzaldehyde (14a, 0.046 mL, 0.46 mmol) according to the general reductive amination procedure. The crude product was purified via flash chromatography (0–5% MeOH/0–0.125% NH₄OH in CH₂Cl₂) to afford 15a (131 mg, 0.289 mmol, 64% yield) as a yellow oil. The free base was converted to the corresponding HCl salt and was recrystallized with hot methanol to give a colorless crystalline solid. Decomposition > 250 °C; ¹H NMR (400 MHz, CDCl₃) δ 7.36 (m, 8H), 7.25 (m, 1H), 7.00 (m, 4H), 5.24 (s, 1H), 3.81 (s, 2H), 2.78 (m, 2H), 2.61 – 2.43 (m, 5H), 1.98 (m, 2H), 1.87 (m, 2H), 1.49 – 1.33 (m, 2H); ¹³C NMR (100 MHz, CDCl₃) δ 163.08, 160.63, 140.75, 137.12, 137.09, 129.85, 129.77, 128.41, 128.02, 126.86, 115.56, 115.34, 58.12, 54.04, 52.85, 52.35, 50.82, 32.69, 29.64; FT-IR (ATR, υ, cm⁻¹) 2933, 2802, 1602, 1505, 1224, 1156, 1113, 835, 745, 699; Anal. (C₂₇H₃₀F₂N₂S·2HCl·0.33H₂O) C, H, N.

1-(2-((Bis(4-fluorophenyl)methyl)thio)ethyl)-N-(4-fluorobenzyl)piperidin-4-amine (15b).

Compound 15b was prepared from 13 (0.117 g, 0.323 mmol) and commercially available 4-fluorobenzaldehyde (14b, 0.035 mL, 0.323 mmol) according to the general reductive amination procedure. The crude product was purified via flash chromatography (0–5% MeOH/0–0.125% NH₄OH in CH₂Cl₂) to afford 15b (62 mg, 0.132 mmol, 41% yield) as a yellow oil. The free base was converted to the corresponding HCl salt and was recrystallized with hot methanol to give a colorless crystalline solid. Decomposition > 250 °C; ¹H NMR (400 MHz, CDCl₃) δ 7.35 (m, 4H), 7.27 (m, 2H), 6.99 (m, 6H), 5.21 (s, 1H), 3.76 (s, 2H), 2.77 (m, 2H), 2.57 – 2.41 (m, 5H), 1.96 (m, 2H), 1.85 (m, 2H), 1.48 – 1.22 (m, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 163.07, 163.04, 160.63, 160.62, 137.06, 137.03, 136.43, 136.40, 129.81, 129.73, 129.52, 129.44, 115.54, 115.33, 115.24, 115.03, 58.05, 54.04, 52.86, 52.32, 50.05, 32.66, 29.63; FT-IR (ATR, υ, cm⁻¹) 2928, 2803, 1724, 1602, 1505, 1466, 1293, 1221, 1156, 1097, 825; Anal. (C₂₇H₂₉F₃N₂S·2HCl) C, H, N.

1-(2-((Bis(4-fluorophenyl)methyl)thio)ethyl)-N-(4-chlorobenzyl)piperidin-4-amine (15c).

Compound 15c was prepared from 13 (0.175 g, 0.483 mmol) and commercially available 4-chlorobenzaldehyde (14c, 68 mg, 0.48 mmol) according to the general reductive amination procedure. The crude product was purified via flash chromatography (0–5% MeOH/0–0.125% NH₄OH in CH₂Cl₂) to afford 15c (133 mg, 0.273 mmol, 57% yield) as a yellow oil. The free base was converted to the corresponding HCl salt and was recrystallized with hot methanol to give a colorless crystalline solid. Decomposition > 250 °C; ¹H NMR (400 MHz, CDCl₃) δ 7.35 (m, 4H), 7.26 (m, 4H), 6.98 (m, 4H), 5.21 (s, 1H), 3.75 (s, 2H), 2.76 (m, 2H), 2.56 – 2.40 (m, 5H), 1.95 (m, 2H), 1.84 (m, 2H), 1.45 – 1.32 (m, 2H); ¹³C NMR (100 MHz, CDCl₃) δ 163.07, 160.62, 139.20, 137.09, 137.06, 132.49, 129.83, 129.75, 129.35, 128.47, 128.45, 115.54, 115.33, 58.04, 54.02, 52.85, 52.29, 50.02, 32.62, 29.63; FT-IR (ATR, υ, cm⁻¹) 2925, 2802, 1712, 1601, 1504, 1466, 1358, 1221, 1156, 1095, 990, 825; Anal. (C₂₇H₂₉ClF₂N₂S·2HCl) C, H, N.

1-(2-((Bis(4-fluorophenyl)methyl)thio)ethyl)-N-(4-(trifluoromethyl)benzyl)piperidin-4-amine (15d).

Compound 15d was prepared from 13 (175 mg, 0.483 mmol) and commercially available 4-(trifluoromethyl)benzaldehyde (14d, 0.066 mL, 0.48 mmol) according to the general reductive amination procedure. The crude product was purified via flash chromatography (0–5% MeOH/0–0.125% NH₄OH in CH₂Cl₂) to afford 15d (179 mg, 0.344 mmol, 71% yield) as a yellow oil. The free base was converted to the corresponding HCl salt and was recrystallized with hot isopropyl alcohol to give a colorless crystalline solid. Mp 241–244 °C; ¹H NMR (400 MHz, CDCl₃) δ 7.56 (d, J = 8.1 Hz, 2H), 7.43 (d, J = 8.0 Hz, 2H), 7.35 (m, 4H), 6.98 (m, 4H), 5.22 (s, 1H), 3.85 (s, 2H), 2.77 (m, 2H), 2.57 – 2.40 (m, 5H), 1.96 (m, 2H), 1.85 (m, 2H), 1.39 (m, 2H), 1.31 – 1.22 (m, 1H); ¹³C NMR (100 MHz, CDCl₃) δ 163.07, 160.63, 145.03, 145.02, 137.10, 137.06, 129.83, 129.75, 128.16, 125.31, 125.27, 125.23, 125.20, 115.54, 115.33, 58.02, 54.13, 52.86, 52.28, 50.24, 32.69, 29.66; FT-IR (ATR, υ, cm⁻¹) 2936, 2904, 1713, 1602, 1505, 1323, 1222, 1157, 1119, 1017, 825; Anal. (C₂₈H₂₉F₅N₂S·2HCl) C, H, N.

1-((1-(2-((Bis(4-fluorophenyl)methyl)thio)ethyl)piperidin-4-yl)amino)-3-phenylpropan-2-ol (16).

To a 25 mL pear shaped flask equipped with a stir bar was added 13 (0.200 g, 0.552 mmol) and THF (2.75 mL) under an argon atmosphere, and the reaction was permitted to stir until dissolved. The solution was cooled to 0 °C. n-Butyllithium (0.43 mL, 1.15 M) was added dropwise via syringe and was permitted to stir for 15 minutes at 0 °C. A precooled solution of 2-benzyloxirane (0.066 mL, 0.49 mmol) in THF (0.2 mL) was added via cannula, and the reaction was stirred at 0 °C for 2 hours. The reaction was slowly warmed to RT and was stirred overnight, after which time it was heated to reflux for 5.5 hours. The reaction was cooled to 0 °C and quenched with a saturated solution of NH₄Cl (15 mL). THF was removed under reduced pressure, and deionized H₂O was added to the reaction (60mL). The aqueous layer was extracted with CH₂Cl₂ (3 × 30 mL). Combined organics were washed with brine (2 × 15 mL) and dried with MgSO₄. The crude oil was purified by flash column chromatography (0–5% MeOH/0–0.125% NH₄OH in CH₂Cl₂) to afford 16 (62 mg, 0.125 mmol, 23% yield) as a yellow oil. The free base was converted to the corresponding HCl salt and was recrystallized with hot isopropyl alcohol to give a yellow crystalline solid. Decomposition > 250 °C; ¹H NMR (400 MHz, CDCl₃) δ 7.35 – 7.17 (m, 9H), 6.98 (t, J = 8.4 Hz, 4H), 5.19 (s, 1H), 3.80 (m, 1H), 2.84 – 2.64 (m, 5H), 2.60 – 2.42 (m, 6H), 2.42 – 2.32 (m, 1H),1.93 (m, 2H), 1.85 – 1.73 (m, 2H), 1.32 (m, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 163.08, 160.63, 138.32, 137.06, 137.03, 129.82, 129.74, 129.32, 128.41, 126.33, 115.55, 115.34, 70.75, 57.95, 54.66, 52.86, 52.33, 51.53, 41.71, 32.94, 32.51, 29.64; FT-IR (ATR, υ, cm⁻¹) 3294, 2921, 1622, 1506, 1440, 1467, 1225, 1159, 1042, 1015, 844; Anal. (C₂₉H₃₄F₂N₂OS·2HCl·H₂O) C, H, N.

tert-Butyl (1-benzoylpiperidin-4-yl)carbamate (18a).

Compound 18a was prepared from commercially available benzoic acid (17a, 500 mg, 4.09 mmol) and commercially available tert-butyl piperidin-4-ylcarbamate (820 mg, 4.09 mmol) according to the general amidation procedure. The crude product was purified via flash chromatography (0–5% MeOH/0–0.125% NH₄OH in CH₂Cl₂) to afford 18a (1.27 g, 4.17 mmol, quantitative yield) as a colorless solid. ¹H NMR (400 MHz, CDCl₃) δ 7.45 – 7.33 (m, 5H), 4.55 (m, 2H), 3.71 (broad s, 2H), 3.03 (m, 2H), 2.00 (m, 2H), 1.52 – 1.21 (s, 11H).

tert-Butyl (1-(4-fluorobenzoyl)piperidin-4-yl)carbamate (18b).

Compound 18b was prepared from commercially available 4-fluorobenzoic acid (17b, 500 mg, 3.57 mmol) and commercially available tert-butyl piperidin-4-ylcarbamate (715 mg, 3.57 mmol) according to the general amidation procedure. The crude product was purified via flash chromatography (0–5% MeOH/0–0.125% NH₄OH in CH₂Cl₂) to afford 18b (982 mg, 3.05 mmol, 85% yield) as a colorless solid. ¹H NMR (400 MHz, CDCl₃) δ 7.39 (m, 2H), 7.09 (m, 2H), 4.51 (broad s, 2H), 3.71 (s, 2H), 3.03 (broad s, 2H), 2.11 – 1.85 (m, 2H), 1.45 (m, 11H).

1-(Benzofuran-2-carbonyl)piperidin-4-yl)carbamate (18c).

Compound 18c was prepared from commercially available benzofuran-2-carboxylic acid (17c, 500 mg, 3.08 mmol) and commercially available tert-butyl piperidin-4-ylcarbamate (618 mg, 3.08 mmol) according to the general amidation procedure. The crude product was purified via flash chromatography (0–5% MeOH/0–0.125% NH₄OH in CH₂Cl₂) to afford 18c (815 mg, 2.37 mmol, 77% yield) as a colorless solid. ¹H NMR (400 MHz, CDCl₃) δ 7.65 (m, 1H), 7.52 (dt, J = 8.4, 0.8 Hz, 1H), 7.40 (tt, J = 7.6, 0.9 Hz, 1H), 7.28 (m, 3H), 4.49 (s, 3H), 3.76 (s, 1H), 3.15 (s, 1H), 2.08 (m, 2H), 1.46 (s, 11H).

tert-Butyl (1-(1H-indole-2-carbonyl)piperidin-4-yl)carbamate (18d).

Compound 18d was prepared from commercially available 1H-indole-2-carboxylic acid (17d, 0.30 g, 1.86 mmol) and commercially available tert-butyl piperidin-4-ylcarbamate (0.373 g, 1.86 mmol) according to the general amidation procedure. The crude product was purified via flash chromatography (0–5% MeOH/0–0.125% NH₄OH in CH₂Cl₂) to afford 18d (0.528 mg, 1.54 mmol, 83% yield) as a colorless solid. ¹H NMR (400 MHz, CDCl₃) δ 9.29 (s, 1H), 7.65 (m, 1H), 7.43 (m, 1H), 7.38 (m, 2H), 7.14 (m, 1H), 6.77 (m, 1H), 4.63 (d, J = 13.7 Hz, 2H), 4.52 (s, 1H), 3.79 (s, 1H), 3.22 (s, 1H), 2.09 (d, J = 12.9 Hz, 2H), 1.58 – 1.35 (m, 11H).

tert-Butyl (1-((1H-indol-2-yl)methyl)piperidin-4-yl)carbamate (18e).

Compound 18e was prepared from commercially available tert-butyl piperidin-4-ylcarbamate (500 mg, 2.50 mmol) and commercially available 1H-indole-2-carbaldehyde (17e, 362 mg, 2.50 mmol) according to the general reductive amination procedure. The crude product was purified via flash chromatography (0–5% MeOH/0–0.125% NH₄OH in CH₂Cl₂) to afford 18e (738 mg, 2.24 mmol, 90% yield) as a purple amorphous solid. ¹H NMR (400 MHz, CDCl₃) δ 8.51 (s, 1H), 7.54 (m, 1H), 7.33 (d, J = 8.1 Hz, 1H), 7.14(m, 1H), 7.07 (m, 1H), 6.34 (d, J = 1.9 Hz, 1H), 4.43 (s, 1H), 3.65 (s, 2H), 3.49 (s, 1H), 2.82 (d, J = 11.5 Hz, 2H), 2.16 (t, J = 11.4 Hz, 2H), 1.93 (d, J = 12.7 Hz, 2H), 1.44 (s, 9H).

[(4-((2-((Bis(4-fluorophenyl)methyl)thio)ethyl)amino)piperidin-1-yl)(phenyl)methanone (19a).

To a 25 mL round bottom flask equipped with a stir bar was added 18a (200 mg, 0.657 mmol) and trifluoroacetic acid (1.3 mL). The reaction was permitted to stir for 1 h at RT under an argon atmosphere. Solvent was removed under reduced pressure, and the crude amine trifluoroacetic acid salt was used directly in the next step. To a 50 mL round bottom flask equipped with a stir bar and a condenser was added the crude amine salt and K₂CO₃ (1.04 g, 7.51 mmol). Anhydrous acetonitrile (1 mL) was added via syringe under an argon atmosphere, and the reaction mixture was permitted to stir at RT. Compound 4 (258 mg, 0.751 mmol)²³ was dissolved in anhydrous acetonitrile (2 mL) and was added dropwise via syringe at RT and was stirred for 3 hours at reflux. The reaction mixture was cooled to 0 °C and filtered to remove residual K₂CO₃, washed with cold acetonitrile, and the filtrate was concentrated under reduced pressure. The crude oil was purified by flash column chromatography (0–5% MeOH/0–0.125% NH₄OH in CH₂Cl₂) to afford 19a (74 mg, 0.159 mmol, 24% yield) over two steps. The free base was converted to the corresponding HCl salt and recrystallized from hot isopropyl alcohol to give a colorless crystalline solid. Mp 220–225 °C; ¹H NMR (400 MHz, CDCl₃) δ 7.41 – 7.33 (m, 9H), 7.00 (m, 4H), 5.14 (s, 1H), 4.54 (s, 1H), 3.82 – 3.60 (m, 1H), 2.99 (s, 2H), 2.77 (t, J = 6.5 Hz, 2H), 2.72 – 2.58 (m, 1H), 2.55 (t, J = 6.4 Hz, 2H), 2.01 – 1.67 (m, 2H), 1.57 – 1.16 (m, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 170.29, 163.11, 160.66, 136.91, 136.88, 136.18, 129.81, 129.76, 129.74, 129.68, 129.51, 128.44, 126.76, 115.66, 115.62, 115.44, 115.41, 54.35, 52.67, 45.12; FT-IR (ATR, υ, cm⁻¹) 2921, 2853, 1712, 1625, 1601, 1504, 1434, 1275, 1221, 1156, 1098, 1074, 826; Anal. (C₂₇H₂₈F₂N₂OS·HCl·0.25H₂O) C, H, N.

[(4-((2-((Bis(4-fluorophenyl)methyl)thio)ethyl)amino)piperidin-1-yl)(4-fluorophenyl)methanone (19b).

Compound 19b was prepared as described for 19a using 18b (200 mg, 0.620 mmol) to afford product (55 mg, 0.114 mmol, 18% yield) over two steps as a colorless solid. The free base was recrystallized from hot isopropyl alcohol to colorless crystalline solid. Mp 183–189 °C; ¹H NMR (400 MHz, CDCl₃/MeOD) δ 7.38 (m, 6H), 7.11 (m, 2H), 7.02 (m, 4H), 5.19 (s, 1H), 4.60 (s, 1H), 3.77 (s, 3H), 3.21 – 2.75 (m, 2H), 2.87 (t, J = 6.89 Hz, 2H), 2.63 (t, J = 6.9 Hz, 2H), 2.00 (m, 2H), 1.42 (m, 2H); ¹³C NMR (100 MHz, CDCl₃/MeOD) δ 170.18, 164.95, 163.37, 162.46, 160.92, 136.74, 136.71, 131.46, 131.42, 129.95, 129.87, 129.31, 129.22, 115.98, 115.83, 115.77, 115.61, 54.59, 52.80, 44.38, 30.73; FT-IR (ATR, υ, cm⁻¹) 2923, 1673, 1602, 1505, 1440, 1371, 1222, 1131, 1014, 908, 827; Anal. (C₂₇H₂₇F₃N₂OS·CH₂Cl₂·0.5H₂O) C, H, N.

Benzofuran-2-yl(4-((2-((bis(4-fluorophenyl)methyl)thio)ethyl)amino)piperidin-1-yl)methanone (19c).

Compound 19c was prepared as described for 19a using 18c (200 mg, 0.581 mmol) to afford 19c (86 mg, 0.17 mmol, 29% yield) over two steps as a yellow oil. The free base was converted to the corresponding HCl salt and recrystallized from hot isopropyl alcohol to give a to give a colorless crystalline solid. Mp 202–208 °C; ¹H NMR (400 MHz, CDCl₃) δ 7.64 (m, 1H), 7.52 (m, 1H), 7.42 – 7.32 (m, 5H), 7.31 – 7.23 (m, 2H), 7.05 – 6.96 (m, 4H), 5.15 (s, 1H), 4.42 (s, 2H), 3.40 – 2.89 (m, 2H), 2.81 – 2.78 (t, J = 6.4 Hz, 2H), 2.73 – 2.71 (m, 1H), 2.56 (t, J = 6.4 Hz, 2H), 1.92 (m, 2H), 1.50 – 1.28 (m, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 222.21, 163.13, 160.68, 159.82, 154.52, 149.19, 136.92, 136.89, 129.78, 129.70, 127.01, 126.29, 123.52, 122.15, 115.64, 115.42, 111.83, 111.38, 54.26, 52.69, 45.16, 33.04; FT-IR (ATR, υ, cm⁻¹) 2920, 2853, 1627, 1562, 1504, 1435, 1361, 1256, 1221, 1156, 1111, 826; Anal. (C₂₉H₂₈F₂N₂O₂S·HCl·H₂O) C, H, N.

(4-((2-((Bis(4-fluorophenyl)methyl)thio)ethyl)amino)piperidin-1-yl)(1H-indol-2-yl)methanone (19d).

Compound 19d was prepared as described for 19a using 18d (125 mg, 0.364 mmol)) to afford 19d (20 mg, 0.040 mmol, 11% yield) over two steps as a yellow oil. The free base was converted to the corresponding HCl salt and recrystallized from hot isopropyl alcohol to give a colorless crystalline solid. Decomposition > 250 °C; ¹H NMR (400 MHz, CDCl₃) δ 9.89 (s, 1H), 7.62 (d, J = 8.1 Hz, 1H), 7.43 (d, J = 8.3 Hz, 1H), 7.29 (m, 5H), 7.12 (t, J = 7.5 Hz, 1H), 6.98 (td, J = 8.7, 2.0 Hz, 4H), 6.70 (s, 1H), 5.11 (s, 1H), 4.56 (d, J = 13.5 Hz, 2H), 3.09 (s, 2H), 2.84 (q, J = 8.7, 6.6 Hz, 3H), 2.59 (t, J = 6.8 Hz, 2H), 1.93 (d, J = 12.9 Hz, 2H), 1.53 – 1.33 (m, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 163.16, 162.66, 160.71, 136.66, 136.62, 135.91, 129.78, 129.69, 129.01, 127.27, 124.38, 121.73, 120.57, 115.67, 115.46, 111.91, 105.07, 54.52, 52.76, 44.68, 31.41; FT-IR (ATR, υ, cm⁻¹) 3259, 1676, 1599, 1505, 1442, 1223, 1014, 828, 747, 572; Anal. (C₂₉H₂₉F₂N₃OS·2HCl·H₂O) C, H, N.

1-((1H-Indol-2-yl)methyl)-N-(2-((bis(4-fluorophenyl)methyl)thio)ethyl)piperidin-4-amine (19e).

Compound 19e was prepared as described for 19a using 18e (200 mg, 0.607 mmol) afford 19e (47 mg, 0.0956 mmol, 16% yield) over two steps as a brown semi-solid. The free base was converted to the corresponding HCl salt to give a colorless solid. Decomposition > 250 °C; ¹H NMR (400 MHz, CDCl₃) δ 8.84 (s, 1H), 7.54 (d, J = 7.5 Hz, 1H), 7.33 (m, 5H), 7.13 (m, 1H), 7.06 (m, 1H), 6.99 (m, 4H), 6.32 (d, J = 2.0 Hz, 1H), 5.12 (s, 1H), 3.61 (d, J = 7.2 Hz, 2H), 2.83 (m, 2H), 2.74 (t, J = 6.5 Hz, 2H), 2.66 – 2.48 (m, 2H), 2.40 (m, 1H), 2.12 – 1.93 (m, 2H), 1.87 – 1.72 (m, 2H), 1.45 – 1.17 (m, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 172.80, 163.11, 160.66, 136.97, 136.94, 136.20, 136.16, 129.79, 129.71, 128.33, 121.42, 120.06, 119.49, 115.61, 115.40, 110.76, 101.32, 55.84, 54.48, 52.58, 52.47, 45.14, 33.00, 32.70, 31.96, 22.66; FT-IR (ATR, υ, cm⁻¹) 3187, 2920, 2808, 1670, 1602, 1504, 1456, 1415, 1456, 1223, 1156, 1097, 826; Anal. (C₂₉H₃₁F₂N₃S·2HCl·2H₂O) C, H, N.

1-(Benzofuran-2-ylmethyl)-N-(2-((bis(4-fluorophenyl)methyl)thio)ethyl)piperidin-4-amine (20).

To an oven-dried 25 mL round bottom flask equipped with a stir containing a suspension of LiAlH₄ (24 mg, 0.63 mmol) in anhydrous THF (2.5 mL) was added a solution of 19c (112 mg, 0.221 mmol) in anhydrous THF (2.5 mL) at 0 °C. The reaction was permitted to slowly warm to RT and stirred for 6 hours, upon which time the reaction was quenched with a solution of MeOH/2N NaOH (1:1, 2 mL). The reaction mixture was filtered over a pad of celite, and the filtrate was concentrated under reduced pressure. The crude oil was purified by flash column chromatography (0–5% MeOH/0–0.125% NH₄OH in CH₂Cl₂) to afford 20 (49 mg, 0.099 mmol, 45% yield) as a colorless oil. The free base was converted to the corresponding HCl salt and recrystallized from hot isopropyl alcohol to give a colorless crystalline solid. Mp 191–195 °C; ¹H NMR (400 MHz, CDCl₃) δ 7.51 (d, J = 7.5 Hz, 1H), 7.47 (d, J = 8.0 Hz, 1H), 7.41 – 7.29 (m, 4H), 7.22 (m, 2H), 6.99 (m, 4H), 6.57 (s, 1H), 5.14 (s, 1H), 3.67 (s, 2H), 2.92 (d, J = 11.2 Hz, 2H), 2.74 (t, J = 6.6 Hz, 2H), 2.52 (t, J = 6.5 Hz, 2H), 2.38 (m, 1H), 2.12 (t, J = 11.4 Hz, 2H), 1.88 – 1.74 (m, 2H), 1.42 (m, 3H); ¹³C NMR (100 MHz, CDCl₃) δ 163.10, 160.65, 155.03, 154.86, 136.96, 136.93, 129.79, 129.71, 128.30, 123.84, 122.61, 120.64, 115.64, 115.60, 115.43, 115.38, 111.28, 105.45, 55.46, 54.29, 52.53, 52.52, 52.29, 45.11, 32.98, 32.60; FT-IR (ATR, υ, cm⁻¹) 2936, 2807, 1602, 1505, 1454, 1371, 1224, 1156, 1098, 829, 751; Anal. (C₂₉H₃₀F₂N₂OS·2HCl·H₂O) C, H, N.

[(4-((2-((Bis(4-fluorophenyl)methyl)sulfinyl)ethyl)amino)piperidin-1-yl)(phenyl)methanone (21a).

Compound 21a was prepared as described for 7 using 19a (431 mg, 0.924 mmol) to afford product (268 mg, 0.555 mmol, 60% yield). The free base was converted to the corresponding HCl salt to give a colorless solid. Mp 86–89 °C; ¹H NMR (400 MHz, CDCl₃) δ 7.40 (m, 9H), 7.09 (m, 4H), 4.88 (s, 1H), 4.50 (s, 1H), 3.70 (s, 1H), 3.16 – 2.89 (m, 4H), 2.72 (tt, J = 9.8, 3.9 Hz, 1H), 2.59 (m, 2H), 2.03 – 1.14 (m, 5H); ¹³C NMR (100 MHz, CDCl₃) δ 170.27, 164.00, 163.77, 161.54, 161.30, 136.13, 131.52, 131.49, 131.01, 130.93, 130.44, 130.41, 130.32, 130.24, 129.51, 128.42, 126.74, 116.45, 116.24, 115.87, 115.65, 70.58, 54.54, 51.49, 40.34; FT-IR (ATR, υ, cm⁻¹) 3294, 3060, 2921, 1621, 1506, 1445, 1362, 1225, 1159, 1042, 1015, 837; Anal. (C₂₇H₂₈F₂N₂O₂S·HCl·1.25H₂O) C, H, N.

(4-((2-((Bis(4-fluorophenyl)methyl)sulfinyl)ethyl)amino)piperidin-1-yl)(4-fluorophenyl)methanone (21b).

Compound 21b was prepared as described for 21a using 19b (230 mg, 0.475 mmol) to afford product (201 mg, 0.402 mmol, 85% yield). The free base was converted to the corresponding HCl salt to give a colorless solid. Mp 110–116 °C; ¹H NMR (400 MHz, CDCl₃) δ 7.41 (m, 6H), 7.08 (m, 6H), 4.88 (s, 1H), 4.46 (s, 1H), 3.70 (s, 1H), 3.22 – 2.90 (m, 4H), 2.74 (m, 1H), 2.60 (m, 2H), 2.14 – 1.12 (m, 5H); ¹³C NMR (100 MHz, CDCl₃) δ 169.38, 164.52, 164.02, 163.79, 162.04, 161.56, 161.32, 132.11, 132.07, 131.47, 131.44, 130.99, 130.91, 130.42, 130.38, 130.31, 130.23, 129.12, 129.04, 116.47, 116.26, 115.89, 115.68, 115.59, 115.38, 54.47, 51.49, 40.37; FT-IR (ATR, υ, cm⁻¹) 3294, 2921, 2855, 1622, 1604, 1506, 1440, 1467, 1363, 1225, 1159, 1042, 844; Anal. (C₂₇H₂₇F₃N₂O₂S·HCl·2H₂O) C, H, N.

tert-Butyl (1-pivaloylpiperidin-4-yl)carbamate (23a).

To a 100 mL round bottom flask equipped with a stir bar was added commercially available pivalic acid (22a, 2.5 g, 24.5 mmol) and anhydrous CH₂Cl₂ (100 mL) under an argon atmosphere, and the reaction was permitted to stir until dissolved. EDC (7.04 g, 36.7 mmol) and HOBt (3.74 g, 27.7 mmol) were added in one portion. N,N-Diisopropylethylamine (12.8 mL) was added dropwise via syringe, and the reaction was permitted to stir for 1 h at RT. Commercially available tert-butyl piperidin-4-ylcarbamate (2.0 g, 10 mmol) was added in one portion, and the reaction was stirred overnight at RT. The reaction was washed with NaHCO₃ (2 × 150 mL, pH = 10) and rinsed with brine (1 × 150 mL). The organics were dried with MgSO₄, and the crude product was purified by flash column chromatography (0–30% ethyl acetate in hexanes) to afford 23a (2.78 g, 9.775 mmol, 98% yield) as a colorless powder. ¹H NMR (400 MHz, CDCl₃) δ 4.47 (s, 1H), 4.32 (d, J = 13.7 Hz, 2H), 3.68 (s, 1H), 2.92 (t, J = 12.8 Hz, 2H), 1.99 (m, 2H), 1.45 (s, 9H), 1.27 (m, 11H).

tert-Butyl (1-(cyclopropanecarbonyl)piperidin-4-yl)carbamate (23b).

Compound 23b was prepared as described for 23a using cyclopropanecarboxylic acid (0.158 mL, 2.00 mmol) to afford 23b (535 mg, 2.00 mmol, quantitative yield) as a colorless crystalline solid. ¹H NMR (400 MHz, CDCl₃) δ 4.47 (s, 2H), 4.15 (d, J = 13.1 Hz, 1H), 3.69 (s, 1H), 3.20 (t, J = 12.9 Hz, 1H), 2.76 (t, J = 12.4 Hz, 1H), 2.06 (dd, J = 16.4, 3.5 Hz, 2H), 1.93 (d, J = 12.9 Hz, 1H), 1.73 (q, J = 7.8, 6.5 Hz, 1H), 1.45 (m, 9H), 1.25 (s, 1H), 1.03 – 0.91 (m, 2H), 0.75 (dt, J = 7.6, 2.7 Hz, 2H).

tert-Butyl (1-(cyclopropylmethyl)piperidin-4-yl)carbamate (23c).

Compound 23c was prepared from commercially available tert-butyl piperidin-4-ylcarbamate (400 mg, 2.00 mmol) and commercially available cyclopropanecarbaldehyde (0.149 mL, 2.00 mmol) according to the general reductive amination procedure. The crude product was purified via flash column chromatography (0–100% ethyl acetate in hexanes) to afford 23c (326 mg, 1.28 mmol, 64%). ¹H NMR (400 MHz, CDCl₃) δ 4.43 (s, 1H), 3.46 (s, 1H), 2.97 (d, J = 11.3 Hz, 2H), 2.23 (d, J = 6.6 Hz, 2H), 2.07 (t, J = 11.6 Hz, 2H), 1.94 (d, J = 12.7 Hz, 2H), 1.65 (s, 1H), 1.45 (s, 10H), 0.51 (d, J = 7.7 Hz, 2H), 0.09 (d, J = 5.0 Hz, 2H).

1-(4-((2-((Bis(4-fluorophenyl)methyl)thio)ethyl)amino)piperidin-1-yl)-2,2-dimethylpropan-1-one (24a).

Compound 24a was prepared as described for 19a using 23a (258 mg, 0.907 mmol) to afford product (119 mg, 0.266 mmol, 29% yield) over two steps as a colorless oil. The free base was converted to the corresponding HCl salt to give a yellow solid. Mp 170–171 °C; ¹H NMR (400 MHz, CDCl₃) δ 7.37 (m, 4H), 7.00 (t, J = 8.6 Hz, 4H), 5.16 (s, 1H), 4.32 (d, J = 13.5 Hz, 2H), 3.25 (s, 1H), 2.95 – 2.73 (m, 4H), 2.67 (m, 1H), 2.58 (t, J = 6.6 Hz, 2H), 1.85 (d, J = 12.8 Hz, 2H), 1.26 (s, 11H); ¹³C NMR (100 MHz, CDCl₃) δ 176.14, 163.09, 160.63, 136.89, 136.86, 129.77, 129.69, 115.59, 115.37, 54.52, 52.63, 44.84, 43.70, 38.64, 32.55, 32.42, 28.36; FT-IR (ATR, υ, cm⁻¹) 293, 1621, 1505, 1479, 1421, 1364, 1272, 1223, 1157, 1183, 1098, 1014, 835; Anal. (C₂₅H₃₂F₂N₂OS·HCl·H₂O) C, H, N.

(4-((2-((Bis(4-fluorophenyl)methyl)thio)ethyl)amino)piperidin-1-yl)(cyclopropyl)methanone (24b).

Compound 24b was prepared as described for 19a using 23b (535 mg, 1.99 mmol) to afford 24b (213 mg, 0.495 mmol, 31% yield) over two steps as a pale yellow oil. The free base was converted to the corresponding oxalate salt. Mp 167–172 °C; ¹H NMR (400 MHz, CDCl₃) δ 7.36 (dd, J = 8.3, 5.4 Hz, 4H), 7.01 (t, J = 8.4 Hz, 4H), 5.15 (s, 1H), 4.43 (d, J = 13.3 Hz, 1H), 4.14 (d, J = 13.7 Hz, 1H), 3.15 (t, J = 12.8 Hz, 1H), 2.78 (t, J = 6.5 Hz, 3H), 2.65 (tt, J = 10.2, 4.2 Hz, 1H), 2.56 (t, J = 6.5 Hz, 2H), 1.84 (d, J = 18.0 Hz, 3H), 1.74 (tt, J = 8.3, 4.8 Hz, 1H), 1.50 (s, 1H), 1.38 – 1.14 (m, 1H), 0.95 (dd, J = 5.3, 2.3 Hz, 2H), 0.74 (dq, J = 7.1, 4.0 Hz, 2H); ¹³C NMR (100 MHz, CDCl₃) δ 171.74, 163.12, 160.67, 136.92, 136.89, 129.76, 129.68, 115.61, 115.39, 77.33, 77.22, 77.02, 76.70, 54.50, 52.67, 45.13, 44.08, 40.89, 33.21, 33.00, 32.18, 10.99, 7.17; FT-IR (ATR, υ, cm⁻¹) 2922, 2853, 1630, 1504, 1437, 1220, 1156, 1129, 1014, 825, 572; Anal. (C₂₄H₂₈F₂N₂OS·C₂H₂O₄·0.25H₂O) C, H, N.

N-(2-((Bis(4-fluorophenyl)methyl)thio)ethyl)-1-(cyclopropylmethyl)piperidin-4-amine (24c).

Compound 24c was prepared as described for 19a using 23c (326 mg, 1.28 mmol) to afford 24c (124 mg, 0.298 mmol, 23% yield) over two steps as a pale yellow oil. The free base was converted to the corresponding oxalate salt to give a colorless solid. Mp 213–218 °C; ¹H NMR (400 MHz, Chloroform-d) δ 7.35 (m, 4H), 7.00 (m, 4H), 5.15 (s, 1H), 3.02 (dd, J = 11.6, 4.3 Hz, 2H), 2.76 (t, J = 6.5 Hz, 2H), 2.54 (t, J = 6.5 Hz, 2H), 2.39 (tt, J = 10.3, 4.1 Hz, 1H), 2.24 (d, J = 6.5 Hz, 2H), 2.01 (m, 2H), 1.90 – 1.76 (br m, 2H), 1.48 – 1.33 (m, 2H), 1.26 (s, 1H), 0.87 (dddd, J = 9.6, 8.1, 5.7, 2.5 Hz, 1H), 0.51(m, 2H), 0.10 (m, 2H); ¹³C NMR (100 MHz, cdcl₃) δ 163.10, 160.65, 136.95, 136.92, 129.78, 129.70, 115.58, 115.37, 63.71, 54.44, 52.52, 52.39, 45.06, 32.98, 32.76, 32.57, 29.68, 8.43, 4.02, 3.97; FT-IR (ATR, υ, cm⁻¹) 3001, 2922, 1602, 1505, 1466, 1330, 1292, 1223, 1156, 1098, 1015, 826, 782, 572; Anal. (C₂₄H₃₀F₂N₂S·2C₂H₂O₄·0.5H₂O) C, H, N.

N-(2-((Bis(4-fluorophenyl)methyl)thio)ethyl)-1-neopentylpiperidin-4-amine (25).

Compound 25 was prepared as described for 20 using 24a (316 mg, 0.708 mmol) to afford product (142 mg, 0.328 mmol, 46% yield) as a pale yellow oil. The free base was converted to the corresponding oxalate salt to give a colorless solid. Mp 215–218 °C; ¹H NMR (400 MHz, CDCl₃) δ 7.36 (m, 4H), 6.99 (m, 4H), 5.15 (s, 1H), 2.83 – 2.68 (m, 4H), 2.54 (t, J = 6.5 Hz, 2H), 2.33 (m, 1H), 2.21 (m, 2H), 2.02 (s, 2H), 1.79 – 1.64 (m, 2H), 1.50 (s, 1H), 1.34 (m, 2H), 0.84 (s, 9H); ¹³C NMR (100 MHz, CDCl₃) δ 163.09, 160.64, 137.02, 136.98, 129.82, 129.79, 129.71, 115.57, 115.36, 69.72, 55.12, 54.38, 52.55, 45.10, 33.23, 33.11, 33.05, 27.70; FT-IR (ATR, υ, cm⁻¹) 2949, 1603, 1505, 1466, 1381, 1225, 1156, 1108, 1015, 833, 793; Anal. (C₂₅H₃₄F₂N₂S·2C₂H₂O₄·0.25H₂O) C, H, N.

1-(4-((2-((Bis(4-fluorophenyl)methyl)sulfinyl)ethyl)amino)piperidin-1-yl)-2,2-dimethylpropan-1-one (26).

Compound 26 was prepared as described for 7 using 24a (200 mg, 0.448 mmol) to afford product (137 mg, 0.296 mmol, 66% yield) as a yellow oil. The free base was converted to the corresponding oxalate salt and recrystallized from hot acetone to give a colorless crystalline solid. Mp 162–165 °C; ¹H NMR (400 MHz, CDCl₃) δ 7.41 (m, 4H), 7.09 (m, 4H), 4.90 (s, 1H), 4.27 (d, J = 13.4 Hz, 2H), 3.18 – 3.01 (m, 2H), 2.90 (t, J = 12.5 Hz, 2H), 2.74 – 2.66 (m, 1H), 2.65 – 2.54 (m, 2H), 1.88 – 1.84 (m, 3H), 1.84 – 1.62 (m, 1H), 1.26 (s, 11H); ¹³C NMR (100 MHz, CDCl₃) δ 176.12, 163.98, 163.75, 161.51, 161.28, 131.50, 131.47, 131.00, 130.92, 130.43, 130.40, 130.31, 130.23, 116.43, 116.22, 115.85, 115.63, 70.55, 54.75, 51.48, 43.68, 43.56, 40.30, 38.63, 32.77, 32.56, 28.36; FT-IR (ATR, υ, cm⁻¹) 3297, 2933, 1604, 1506, 1480, 1423, 1364, 1225, 1160, 1042, 1015, 836; Anal. (C₂₅H₃₂F₂N₂O₂S·C₂H₂O₄) C, H, N.

N-(2-((Bis(4-fluorophenyl)methyl)sulfinyl)ethyl)-1-neopentylpiperidin-4-amine (27).

Compound 27 was prepared as described for 7 using 25 (97 mg, 0.22 mmol) to afford 27 (72 mg, 0.16 mmol, 72% yield). The free base was converted to the corresponding oxalate salt and recrystallized from hot methanol to give a colorless crystalline solid. Mp 192–195 °C; ¹H NMR (400 MHz, CDCl₃) δ 7.42 (m, 4H), 7.09 (m, 4H), 4.92 (s, 1H), 3.07 (t, J = 6.4 Hz, 2H), 2.74 (d, J = 11.6 Hz, 2H), 2.60 (t, J = 6.4 Hz, 2H), 2.37 (m, 1H), 2.21 (t, J = 11.6 Hz, 2H), 2.01 (s, 2H), 1.84 – 1.64 (m, 3H), 1.45 – 1.21 (m, 2H), 0.83 (s, 9H); ¹³C NMR (100 MHz, CDCl₃) δ 164.00, 163.76, 161.53, 161.29, 131.65, 131.62, 131.05, 130.96, 130.48, 130.45, 130.36, 130.28, 116.40, 116.18, 115.84, 115.62, 70.33, 69.69, 55.01, 55.00, 54.65, 51.55, 40.16, 33.14, 33.08, 32.98, 27.68; FT-IR (ATR, υ, cm⁻¹) 2950, 1604, 1508, 1359, 1230, 1160, 1106, 1043, 837, 792, 573; Anal. (C₂₅H₃₄F₂N₂OS·2C₂H₂O₄) C, H, N.

1-(2-Fluorobenzoyl)piperidin-4-one (28).

Compound 28 was prepared from commercially available 2-fluorobenzoic acid (3.5 g, 25 mmol) and commercially available piperidin-4-one (2.48 g, 25 mmol) according to the general amidation procedure. The crude product was purified via flash chromatography (100% ethyl acetate) to afford 28 (4.2 g, 19 mmol, 76% yield) as a colorless oil. ¹H NMR (400 MHz, CDCl₃):δ7.40–7.45 (m, 2H), 7.21–7.25 (m, 1H), 7.09–7.14 (m, 1H), 4.05–4.13 (m, 2H), 3.62–3.63 (m, 2H), 2.58–2.61 (m, 2H), 2.45–2.48 (m, 2H); GC/MS (EI): m/z 221 (M⁺).

1-(2,4-Difluorobenzoyl)piperidin-4-one (29).

Compound 29 was prepared from commercially available 2,4-difluorobenzoic acid (3.16 g, 20 mmol) and commercially available piperidin-4-one (1.98 g, 20 mmol) according to the general amidation procedure. The crude product was purified via flash chromatography (100% ethyl acetate) to afford 29 (3.83 g, 16 mmol, 80% yield) as a colorless oil. ¹H NMR (400 MHz, CDCl₃):δ7.44–7.46 (m, 1H), 6.88–6.98 (m, 2H), 4.05–4.13 (m, 2H), 3.63–3.64 (m, 2H), 2.59–2.61 (m, 2H), 2.45–2.48 (m, 2H).

(4-((2-((Bis(4-fluorophenyl)methyl)thio)ethyl)amino)piperidin-1-yl)(2-fluorophenyl)methanone (32).

Compound 4²³ (12.01 g, 34.96 mmol) was dissolved in DMF (200 mL) followed by addition of phthalimide potassium salt (7.77 g, 41.95 mmol). The reaction mixture stirred at room temperature overnight. Ice-water was added, the resulting precipate was collected by fitration. The solid was washed (H₂O), dried and dissolved in ethanol (250 mL). Hydrazine (3 mL) was added. The reaction mixture stirred at reflux overnight. Solvent was removed in vacuo to give the crude amine 31. Compound 32 was prepared using 31 (5.05 g, 18 mmol) and 28 (4 g, 18 mmol) according to the general reductive amination procedure. The crude product was purified via flash chromatography (0–5% MeOH/0–0.125% NH₄OH in CH₂Cl₂) to afford product (6.0 g, 12.4 mmol, three steps 69% yield) as a yellow oil. The free base was converted to the corresponding HCl salt as a white foam. ¹H NMR (400 MHz, CDCl₃)δ7.33–7.37(m, 6H), 7.17–7.20 (m, 1H), 7.06–7.11 (m, 1H), 6.98–7.02 (m, 4H), 5.14 (s, 1H), 4.56–4.59 (d, J=13.6 Hz, 1H), 3.53–3.56 (d, J=12.8 Hz, 2H), 2.94–3.04 (m, 2H), 2.55–2.77 (m, 5H), 1.92–1.95 (m, 2H), 1.75–1.78 (d, J=13.2 Hz, 1H), 1.35–1.40 (m, 2H); ¹³C NMR (100 MHz, CDCl₃)δ165.1, 163.2, 160.7, 159.4, 156.9, 136.9, 131.2, 131.1, 129.8, 129.7, 129.0, 128.9, 124.7, 124.6, 124.5, 124.3, 115.8, 115.6, 115.4, 54.3, 52.7, 45.6, 45.0, 40.4, 32.8, 31.9; Anal. (C₂₇H₂₇F₃N₂OS·HCl·H₂O) C, H, N.

(4-((2-((Bis(4-fluorophenyl)methyl)thio)ethyl)amino)piperidin-1-yl)(2,4-difluorophenyl)methanone (33).

Compound 33 was prepared from 31 (2.96 g, 10.6 mmol) and 29 (2.53 g, 10.6 mmol) according to the general reductive amination procedure. The crude product was purified via flash chromatography (0–5% MeOH/0–0.125% NH₄OH in CH₂Cl₂) to afford product (4.0 g, 8.0 mmol, 75% yield) as a yellow oil. The free base was converted to the corresponding HCl salt as a white foam. ¹H NMR (400 MHz, CDCl₃)δ7.30–7.35(m, 6H), 6.98–7.02 (m, 6H), 5.14 (s, 1H), 4.51–4.54 (d, J=13.2 Hz, 1H), 3.51–3.54 (d, J=13.6 Hz, 1H), 2.76–3.05 (m, 2H), 2.55–2.76 (m, 5H), 1.90–1.94 (d, J=13.2 Hz, 1H), 1.75–1.78 (d, J=12.4 Hz, 1H), 1.27–1.41 (m, 2H); ¹³C NMR (100 MHz, CDCl₃)δ164.8, 164.7, 164.2, 163.2, 162.3, 162.2, 160.7, 159.9, 159.7, 157.4, 157.3, 136.9, 130.4, 130.3, 130.2, 129.8, 129.7, 120.8, 120.7, 120.6, 115.6, 115.4, 112.2, 112.0, 104.4, 104.1, 103.9, 54.1, 52.7, 45.7, 45.2, 40.6, 33.0, 32.9, 32.1; Anal. (C₂₇H₂₆F₄N₂OS·HCl·0.75H₂O) C, H, N.

(4-((2-((Bis(4-fluorophenyl)methyl)sulfinyl)ethyl)amino)piperidin-1-yl)(2-fluorophenyl)methanone (34).

Compound 32 (1.06 g, 2.19 mmol) was dissolved in a solution of 2.19 mL AcOH in 21.9 mL MeOH, followed by the addition of 30% H₂O₂ (0.66 mL). The reaction mixture was stirred at RT overnight. The reaction was quenched with Na₂SO₃ and basified with NH₄OH to pH 9. Methanol was removed in vacuo. The aqueous mixture was extracted with chloroform (3 × 100 ml). The organic layer was dried over MgSO₄, the solvent was removed in vacuo, and the crude product was purified by flash column chromatography [CH₂Cl₂/CH₃OH/NH₄OH= 95:5:0.5] to give the product as a yellow oil (1.0 g, 91%). The free base was converted to the HCl salt as a yellow foam. ¹H NMR (400 MHz, CDCl₃)δ7.32–7.42 (m, 6H), 7.05–7.20 (m, 6H), 4.85–4.88 (d, J=11.6 Hz, 1H), 4.49–4.55 (t, J=12.2 Hz, 1H), 3.51–3.54 (d, J=13.2 Hz, 1H), 2.99–3.08 (m, 4H), 2.59–2.72 (m, 3H), 1.93–1.96 (d, J=12.0 Hz, 1H), 1.78–1.81 (d, J=12.4 Hz, 1H), 1.25–1.42 (m, 2H); ¹³C NMR (100 MHz, CDCl₃)δ165.1, 164.1, 163.8, 161.6, 161.4, 159.3, 156.9, 131.5, 131.4, 131.1, 131.0, 130.4, 130.3, 128.9, 124.6, 124.5, 124.3, 116.5, 116.3, 115.9, 115.8, 115.7, 115.6, 70.7, 54.5, 51.5, 51.3, 45.5, 40.4, 40.3, 32.9, 32.6, 32.1, 31.9; Anal. (C₂₇H₂₇F₃N₂O₂S·HCl·1.25H₂O) C, H, N.

(4-((2-((Bis(4-fluorophenyl)methyl)sulfinyl)ethyl)amino)piperidin-1-yl)(2,4-difluorophenyl)methanone (35).

Compound 35 was prepared as described for 34 using 33 (1.1 g, 2.19 mmol) to afford product (800 mg, 1.53 mmol, 70 % yield). The free base was converted to the corresponding HCl salt as a light yellow foam. ¹H NMR (400 MHz, CDCl₃)δ7.34–7.42 (m, 5H), 6.81–7.12 (m, 6H), 4.85–4.87 (d, J=10.4 Hz, 1H), 4.46–4.52 (t, J=12.8 Hz, 1H), 3.50–3.53 (d, 1H, J=12.4 Hz), 3.00–3.08 (m, 4H), 2.73 (s, 1H), 2.54 (s, 2H), 1.92–1.96 (d, J=12.8 Hz, 1H), 1.79–1.82 (d, J=12.0 Hz, 1H), 1.26–1.39 (m, 2H); ¹³C NMR (100 MHz, CDCl₃)δ164.2, 164.7, 164.2, 164.1, 163.8, 162.3, 162.2, 161.6, 161.4, 159.9, 159.7, 157.4, 157.2, 131.4, 131.0, 130.9, 130.3, 130.2, 120.7, 120.5, 116.5, 116.3, 115.9, 115.7, 112.2, 112.0, 104.4, 104.1, 103.9, 54.5, 54.4, 51.6, 51.4, 45.6, 40.4, 32.9, 32.6, 32.1; Anal. (C₂₇H₂₆F₄N₂O₂S·HCl ·H₂O) C, H, N.

(4-((2-((Bis(4-fluorophenyl)methyl)thio)ethyl)(methyl)amino)piperidin-1-yl)(4-fluorophenyl)methanone (36).

Compound 36 was prepared from 19b (242 mg, 0.5 mmol) and 37% formaldehyde in H₂O (0.12 mL, 1.5 mmol) according to the general reductive amination procedure. The crude product was purified via flash chromatography (ethyl acetate/triethylamine = 95:5) to give product (220 mg, 88% yield) as a yellow oil. The free base was converted to the oxalate salt and recrystallized from methanol to give a white solid. Mp 152–153 °C ¹H NMR (400 MHz, CDCl₃)δ7.34–7.41 (m, 6H), 6.98–7.11 (m, 6H), 5.18 (s, 1H), 4.69 (s, 1H), 3.77 (s, 1H), 2.77–2.92 (m, 2H), 2.45–2.63 (m, 5H), 2.19–2.25 (m, 3H), 1.25–2.00 (m, 4H); ¹³C NMR (100 MHz, CDCl₃)δ169.4, 164.6, 163.1, 162.1, 160.7, 137.0, 132.0, 129.8, 129.7, 129.2, 129.1, 115.6, 115.4, 61.1, 53.4, 53.0, 52.9, 37.8, 30.7; Anal. (C₂₈H₂₉F₃N₂OS·C₂H₂O₄·0.5H₂O) C, H, N.

(4-((2-((Bis(4-fluorophenyl)methyl)thio)ethyl)(methyl)amino)piperidin-1-yl)(2-fluorophenyl)methanone (37).

Compound 37 was prepared as described for 36 using 33 (968 mg, 2.0 mmol) to give the product (940 mg, 93% yield) as a yellow oil. The free base was converted to the HCl salt as a yellow foam. ¹H NMR (400 MHz, CDCl₃)δ7.34–7.52 (m, 6H), 6.98–7.20 (m, 6H), 5.18 (s, 1H), 4.75–4.79 (d, J=14.8 Hz, 1H), 3.56–3.59 (d, J=12.0 Hz, 1H), 2.99 (s, 1H), 2.71–2.77 (t, , J=12.0 Hz, 1H), 2.45–2.63 (m, 5H), 2.19–2.25 (m, 3H), 1.80–1.83 (d, J=12.4Hz, 1H), 1.45–1.65 (m, 3H); ¹³C NMR (100 MHz, CDCl₃)δ165.0, 163.1, 160.7, 159.4, 156.9, 137.0, 131.2, 131.1, 129.8, 129.7, 129.0, 124.7, 124.6, 124.5, 124.3, 115.8, 115.6, 115.4, 61.0, 53.0, 46.6, 41.4, 37.9, 30.6, 28.8, 27.5; Anal. (C₂₈H₂₉F₃N₂OS·HCl·1.75H₂O ·C₄H₈O₂) C, H, N.

(4-((2-((Bis(4-fluorophenyl)methyl)thio)ethyl)(isopropyl)amino)piperidin-1-yl)(2-fluorophenyl)methanone (38).

A mixture of 32 (800 mg, 1.65 mmol), acetone (2 mL), sodium cyanoborohydride (400 mg, 6.36 mmol) in dichloroethane (50 mL) was stirred at RT for 3 days. The solvent was removed, H₂O (100 mL) was added to the residue, and the aqueous mixture was extracted with chloroform (3 × 100 mL). The organic layer was dried over MgSO₄, the solvent was removed in vacuo, and the crude product was purified by flash column chromatography [hexane/ethyl acetate/triethylamine = 50:50:2] to give product (680 mg, 78% yield) as a colorless oil. The free base was converted to the HCl salt as a white foam. ¹H NMR (400 MHz, CDCl₃) δ 7.34–7.38 (m, 6H), 6.96–7.21 (m, 6H), 5.16 (s, 1H), 4.72–4.74 (d, J=11.6 Hz, 1H), 3.50–3.53 (d, J=12.8 Hz, 1H), 2.93–2.97 (m, 2H), 2.57–2.69 (m, 4H), 2.34–2.38 (m, 2H), 1.69–1.72 (d, J=12.0Hz, 1H), 1.43–1.54 (m, 3H), 0.91–0.97 (m, 6H); ¹³C NMR (100 MHz, CDCl₃)δ171.1, 164.9, 163.1, 160.6, 159.4, 156.9, 137.1, 137.0, 131.1, 129.8, 129.7, 129.0, 124.7, 124.6, 124.5, 124.3, 115.8, 115.6, 115.4, 115.3, 60.4, 56.6, 52.6, 48.7, 46.8, 45.5, 41.7, 33.7, 31.3, 30.4, 21.0, 20.8, 20.5, 14.2; Anal. (C₃₀H₃₃F₃N₂OS·HCl·1.25H₂O) C, H, N.

(4-((2-((Bis(4-fluorophenyl)methyl)sulfinyl)ethyl)(methyl)amino)piperidin-1-yl)(2-fluorophenyl)methanone (39).

Compound 39 was prepared as described for 34 using 37 (660 mg, 1.33 mmol) to afford product (400 mg, 1.53 mmol, 59 % yield). The free base was converted to the corresponding HCl salt to give a yellow ¹H NMR (400 MHz, CDCl₃) δ 7.31–7.42 (m, 6H), 7.03–7.18 (m, 6H), 4.88(s, 1H), 4.72–4.80 (t, J=16.2 Hz 1H), 3.55–3.58 (d, J=13.6 Hz, 1H),2.77–3.01 (m, 2H), 2.47–2.76 (m, 5H), 2.16–2.23 (m, 3H), 1.80–1.87 (d, J=14.2 Hz, 1H), 1.63–1.70 (d, J=14.6 Hz, 1H), 1.46–1.56 (m, 2H); ¹³C NMR (100 MHz, CDCl₃) δ 165.0, 164.0, 163.7, 161.5, 161.3, 159.3, 156.9, 131.8, 131.2, 131.1, 131.0, 130.9, 130.7, 130.6, 130.3, 130.2, 129.0, 128.9, 124.7, 124.6, 124.4124.2, 116.4, 116.2, 115.8, 115.6, 69.9, 61.7, 61.4, 53.4, 49.6, 46.9, 46.6, 46.1, 41.4, 41.3, 37.8, 37.3, 29.1, 28.7, 27.7, 26.9; Anal. (C₂₈H₂₉F₃N₂O₂S·HCl·H₂O) C, H, N.

Radioligand Binding Studies

DAT Binding Assay.

Frozen striatum membranes³³ dissected from male Sprague-Dawley rat brains (supplied on ice by Bioreclamation, Hicksville, NY) were homogenized in 20 volumes (w/v) of ice cold modified sucrose phosphate buffer (0.32 M sucrose, 7.74 mM Na₂HPO₄, and 2.26 mM NaH₂PO₄, pH adjusted to 7.4) using a Brinkman Polytron (Setting 6 for 20 s) and centrifuged at 48,400 × g for 10 min at 4°C. The resulting pellet was resuspended in buffer, recentrifuged, and suspended in ice cold buffer again to a concentration of 20 mg/mL, original wet weight (OWW). Experiments were conducted in 96-well polypropylene plates containing 50 μL of various concentrations of the inhibitor, diluted using 30% DMSO vehicle, 300 μL of sucrose phosphate buffer, 50 μL of [³H]-WIN35,428 (final concentration 1.5 nM; PerkinElmer Life Sciences, Waltham, MA), and 100 μL of tissue (2.0 mg/well OWW). All compound dilutions were tested in triplicate and the competition reactions started with the addition of tissue, and the plates were incubated for 120 min at 0–4°C. Nonspecific binding was determined using 10 μM indatraline.

SERT Binding Assay.

Frozen stem membranes³³ dissected from male Sprague-Dawley rat brains (supplied on ice by Bioreclamation, Hicksville, NY) were homogenized in 20 volumes (w/v) of 50 mM Tris buffer (120 mM NaCl and 5 mM KCl, adjusted to pH 7.4) at 25°C using a Brinkman Polytron (Setting 6 for 20 s) and centrifuged at 48,400 × g for 10 min at 4°C. The resulting pellet was resuspended in buffer, recentrifuged, and suspended in buffer again to a concentration of 20 mg/mL, OWW. Experiments were conducted in 96-well polypropylene plates containing 50 μL of various concentrations of the inhibitor, diluted using 30% DMSO vehicle, 300 μL of Tris buffer, 50 μL of [³H]-citalopram (final concentration 1.5 nM; PerkinElmer Life Sciences, Waltham, MA), and 100 μL of tissue (2.0 mg/well OWW). All compound dilutions were tested in triplicate and the competition reactions started with the addition of tissue, and the plates were incubated for 60 min at RT. Nonspecific binding was determined using 10 μM fluoxetine.

NET Binding Assay.

Frozen frontal cortex membranes³³ dissected from male Sprague-Dawley rat brains (supplied on ice from Bioreclamation, Hicksville, NY) were homogenized in 20 volumes (w/v) of 50 mM Tris buffer (300 mM NaCl and 5 mM KCl, adjusted to pH 7.4) at 25 °C using a Brinkman Polytron (Setting 6 for 20 s). The tissue was centrifuged at 48,400 × g for 10 min at 4 °C. The resulting pellet was suspended in fresh buffer and centrifuged again. The final pellet was resuspended in cold binding buffer to a concentration of 80 mg/mL OWW. Experiments were conducted in glass assay tubes containing 50 μL of various concentrations of the inhibitor, diluted using 30% DMSO vehicle, 300 μL of Tris buffer. 50 μL of [³H]nisoxetine (final concentration 0.5 nM; Perkin-Elmer Life Sciences), and 100 μL of tissue (8.0 mg/tube OWW). The reaction was started with the addition of the tissue, and the tubes were incubated for 180 min at 0–4 °C. Nonspecific binding was determined using 10 μM desipramine.

σ1 Receptor Binding Assay.

Frozen cortex membranes⁵⁰ dissected from male guinea pig brains (supplied on ice by Bioreclamation, Hicksville, NY) were homogenized in 10 volumes (w/v) of ice cold modified sucrose Tris buffer (10 mM Tris-HCl with 0.32 M sucrose, adjusted to pH 7.4) with a glass and Teflon homogenizer. The homogenate was centrifuged at 1,240 × g for 10 min at 4 °C. The supernatant was collected into a clean centrifuge tube, and the remaining pellet was resuspended in 10 volumes (w/v) of buffer and centrifuged again at 48,400 × g for 15 min at 4 °C. The resulting pellet was resuspended in ice cold buffer to 50 mg/mL, OWW. Experiments were conducted in 96-well polypropylene plates containing 50 μL of various concentrations of the inhibitor, diluted using 30% DMSO vehicle, 300 μL of modified sucrose Tris buffer, 50 μL of [³H]-(+)-pentazocine (final concentration 3 nM; PerkinElmer Life and Analytical Sciences, Waltham, MA) and 100 μL of tissue (5.0 mg/well OWW). All compound dilutions were tested in triplicate and the competition reactions started with the addition of tissue, and the plates were incubated for 120 min at RT. Nonspecific binding was determined using 10 μM of either PRE084 or (+)-pentazocine.

D₂-like Receptor Binding Assay.

HEK293 cells stably expressing human D_2LR, D₃R or D_4.4R^47,51 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 pre-mixed Earle’s Balanced Salt Solution (EBSS) with 5 mM EDTA (Life Technologies) and centrifuged at 3,000 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 14,500 rpm (~25,000 g) for 30 min at 4 °C. The pellet was then resuspended in fresh binding buffer. A Bradford protein assay (Bio-Rad, Hercules, CA) was used to determine the protein concentration. On test day, each test compound was diluted into half-log serial dilutions using 30% 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 diluted test compound, 100 μl of membranes (20 μg/well total protein for hD_2LR and hD₃R, and 30 μg/well total protein for hD_4.4R), and 50 μl of radioligand diluted in binding buffer ([³H]-N-methylspiperone: 0.4 nM final concentration; Perkin Elmer, Waltham, MA). Nonspecific binding was determined using 10 μM (+)-butaclamol (Sigma-Aldrich, St. Louis, MO) and total binding was determined with 30% DMSO vehicle. All compound dilutions were tested in triplicate and the reaction incubated for 60 min at RT.

For all binding assays, incubations were terminated by rapid filtration through Perkin Elmer Uni-Filter-96 GF/B (DAT, SERT, σ1R and D₂-like) or Whatman GF/B filters (NET), presoaked in either 0.5% (D₂-like), 0.3% (SERT and NET) or 0.05% (DAT and σ1R) polyethylenimine, using a Brandel 96-Well Plates Harvester Manifold or Brandel R48 filtering manifold (Brandel Instruments, Gaithersburg, MD). The filters were washed a total of 3 times with 3 mL (3 × 1 mL/well or 3 × 1 mL/tube) of ice cold binding buffer. For DAT, SERT, σ1R and D₂-like binding experiment 65 μL Perkin Elmer MicroScint 20 Scintillation Cocktail was added to each filter well. For NET binding experiment, the filters were transferred in 24-well scintillation plates and 600 μL of CytoScint was added to each well. All the plates/filters were counted using a Perkin Elmer MicroBeta Microplate Counter. IC₅₀ values for each compound were determined from inhibition curves and K_i values were calculated using the Cheng-Prusoff equation⁴⁵. 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 non-linear regression analysis. These analyses were performed using GraphPad Prism version 8.00 for Macintosh (GraphPad Software, San Diego, CA). K_d values for the radioligands were determined via separate homologous competitive binding or radioligand binding saturation experiments. Aliquots of radioligand solutions were also quantified accurately to determine how much radioactivity was added in each experiements, taking in account the experimentally determined counter efficiency for each radioligand. K_i values were determined from at least 3 independent experiments performed in triplicate and are reported as mean ± SEM, and the results were rounded to the third significant figure.

Phase I Metabolism in Liver Microsomes

The phase I metabolic stability assay was conducted in liver microsomes as previously described with minor modifications.⁵² In brief, the reaction was carried out with 100 mM potassium phosphate buffer, pH 7.4, in the presence of NADPH regenerating system, (compound final concentration was 10 μM; and 0.5 mg/mL microsomes). Positive controls for phase I metabolism (buprenorphine) were also evaluated. Compound disappearance was monitored over time using a liquid chromatography and tandem mass spectrometry (LC/MS/MS) method. All reactions were performed in triplicate.

Chromatographic analysis was performed using an Accela™ ultra-high-performance system consisting of an analytical pump and an autosampler coupled with a TSQ Vantage mass spectrometer (Thermo Fisher Scientific Inc., Waltham, MA). Chromatographic separation was achieved at ambient temperature using Agilent Eclipse Plus column (100 × 2.1mm i.d.) packed with a 1.8 μm C18 stationary phase. The mobile phase consisted of 0.1% formic acid in acetonitrile and 0.1% formic acid in H₂O with gradient elution, starting with 10% (organic) linearly increasing to 99% (0–2 min), maintaining at 99% (2–2.5 min) and re-equilibrating to 10% by 2.7 min. The total run time for each analyte was 4.5 min. The mass transitions used for compounds for LC/MS/MS analysis are given in Supporting Information.

Locomotor Activity Studies in Mice

Mice were transferred from the animal facility vivarium to the behavioral testing room about 2h before the administration of drugs or vehicle, in order to avoid potential stress related to change of environment. After the acclimatization period, mice were administered the test compounds, cocaine or vehicle by the i.p. route, and then immediately placed into the open field chambers (clear acrylic testing chambers, 40 cm³, Med Associates, St. Albans, VT). The chambers had infrared light beam sources spaced 2.5 cm apart along two perpendicular walls directed at light sensitive detectors mounted on the opposing walls. Detection of ambulatory activity was obtained by interruption of the light beams, which occurrence during a 2 h session was automatically recorded every 5 min, and then transformed in distance traveled (cm/5 min). The data obtained for each animal and dose of the different compounds, cocaine, and vehicle (10% DMSO, 15% Tween-80, 75% sterile water) was averaged to obtain the group mean and correspondent SEM. In Figure 3, data have been expressed as the distance traveled in cm/5 min over a 2 h session. Data collected during the first hour after drug administration were analyzed by two-way ANOVA with “Dose” and “Drug” as factors. Tuckey’s post-hoc test was employed to compare the activity produced by administration of different doses of test compounds and vehicle. Mice were used only once.

Highlights:

• Atypical DAT inhibitors, based on (±)modafinil, have demonstrated therapeutic potential in animal models of psychostimulant use disorders

• A series of aminopiperidine and piperidine amine analogues of previously described piperazines (e.g., JJC8–091) showed high to moderate DAT binding affinities and metabolic stability in rat liver microsomes

• Compounds 7 (DAT K_i = 50.6 nM), 21b (DAT K_i = 77.2 nM) and 33 (DAT K_i = 30.0 nM) were identified as lead molecules for further testing in vivo

• Minimal stimulation of ambulatory activity compared to cocaine in mice suggests these compounds have potential as atypical DAT inhibitors

ABBREVIATIONS

ANOVA

Analysis of variance

Boc

tert-Butyloxycarbonyl

CDI

Carbonyldiimidazole

DAT

Dopamine transporter

DCE

Dichloroethane

DCM

Dichloromethane

dec.

Decomposed

EI

Electron ionization

FDA

Food and Drug Administration

GC

Gas chromatography

hERG

Human Ether-a-go-go-related Gene

HESI

Heated electrospray ionization

i.p.

Intraperitoneal

IC₅₀

Half maximal inhibitory concentration

K_d

Dissociation constant

K_i

Inhibitor constant

LC

Liquid chromatography

MeOH

Methanol

MS

Mass spectrometry

NADPH

Nicotinamide adenine dinucleotide phosphate

NET

Norepinephrine transporter

NMR

Nuclear magnetic resonance

NS

Not significant

NT

Not tested

NC

Not calculated

OWW

Original wet weight

ppm

parts-per-million

rDAT

Rat dopamine transporter

RT

room temperature

σ1R

Sigma 1 receptor

SEM

Standard error of the mean

SERT

Serotonin transporter

t_1/2

Half-life

VEH

Drug vehicle