Fluoroethoxy-1,4-diphenethylpiperidine and piperazine derivatives: Potent and selective inhibitors of [3H]dopamine uptake at the vesicular monoamine transporter-2

Article abstract of DOI:10.1016/j.bmcl.2017.10.039

A small library of fluoroethoxy-1,4-diphenethyl piperidine and fluoroethoxy-1,4-diphenethyl piperazine derivatives were designed, synthesized and evaluated for their ability to inhibit [³H]dopamine (DA) uptake at the vesicular monoamine transporter-2 (VMAT2) and dopamine transporter (DAT), [³H]serotonin (5-HT) uptake at the serotonin transporter (SERT), and [³H]dofetilide binding at the human-ether-a-go-go-related gene (hERG) channel. The majority of the compounds exhibited potent inhibition of [³H]DA uptake at VMAT2, Ki changes in the nanomolar range (K_i = 0.014–0.073 μM). Compound 15d exhibited the highest affinity (K_i = 0.014 μM) at VMAT2, and had 160-, 5-, and 60-fold greater selectivity for VMAT2 vs. DAT, SERT and hERG, respectively. Compound 15b exhibited the greatest selectivity (>60-fold) for VMAT2 relative to all the other targets evaluated, and 15b had high affinity for VMAT2 (K_i = 0.073 μM). Compound 15b was considered the lead compound from this analog series due to its high affinity and selectivity for VMAT2.

Full text of DOI:10.1016/j.bmcl.2017.10.039

Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Fluoroethoxy-1,4-diphenethylpiperidine and piperazine derivatives:
Potent and selective inhibitors of [3H]dopamine uptake at the vesicular
monoamine transporter-2
Emily R. Hankosky a, Shyam R. Joolakanti b, Justin R. Nickell a, Venumadhav Janganati b, Linda P. Dwoskin a,
Peter A. Crooks b,
⇑
a Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, KY 40536, USA
b Department of Pharmaceutical Sciences, College of Pharmacy, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A small library of fluoroethoxy-1,4-diphenethyl piperidine and fluoroethoxy-1,4-diphenethyl piperazine
derivatives were designed, synthesized and evaluated for their ability to inhibit [3H]dopamine (DA)
uptake at the vesicular monoamine transporter-2 (VMAT2) and dopamine transporter (DAT), [3H]sero-
tonin (5-HT) uptake at the serotonin transporter (SERT), and [3H]dofetilide binding at the human-
ether-a-go-go-related gene (hERG) channel. The majority of the compounds exhibited potent inhibition
of [3H]DA uptake at VMAT2, Ki changes in the nanomolar range (Ki = 0.014–0.073 mM). Compound 15d
exhibited the highest affinity (Ki = 0.014 mM) at VMAT2, and had 160-, 5-, and 60-fold greater selectivity
for VMAT2 vs. DAT, SERT and hERG, respectively. Compound 15b exhibited the greatest selectivity (>60-
fold) for VMAT2 relative to all the other targets evaluated, and 15b had high affinity for VMAT2 (Ki =
0.073 mM). Compound 15b was considered the lead compound from this analog series due to its high
affinity and selectivity for VMAT2.
Received 29 August 2017
Revised 6 October 2017
Accepted 19 October 2017
Available online xxxx
Keywords:
Lobelane
Fluoroethoxy piperidine and piperazine
analogs
VMAT2
Dopamine uptake
hERG
Ó 2017 Elsevier Ltd. All rights reserved.
Psychostimulant use disorders are serious and escalating prob-
lems globally, and methamphetamine (METH) is one of the most
addictive psychostimulant drugs of abuse,1 causing dopaminergic
neuronal damage in humans following long-term METH use.2
METH use disorders result in severe health consequences, includ-
ing mood disturbances, infectious diseases (e.g., HIV and hepatitis
C), cardiovascular complications, oral disease, and death.3–7 Cur-
rently, there are no FDA-approved drugs for the treatment of METH
use disorder.
The vesicular monoamine transporter-2 (VMAT2) plays a major
role in mediating the neurochemical and behavioral effects of psy-
chostimulants.8 VMAT2 is recognized as an important target for
the discovery of therapeutics for METH use disorder.9 Small mole-
cules that modulate VMAT2 function and inhibit the pharmacolog-
ical effects of METH may prove to be efficacious
pharmacotherapies for METH use disorder. In this respect, lobeline
(1, Fig. 1), the major alkaloid of Lobelia inflata, has been shown to
inhibit dopamine (DA) uptake into synaptic vesicles via an interac-
tion with VMAT2.10,11 Evaluation of lobeline’s effects in clinical tri-
als revealed some minor side-effects, including bitter taste and
nausea, likely the result of an action as an antagonist at nicotinic
acetylcholine receptors (nAChRs).12,13 Another limitation was the
short plasma half-life of lobeline (ꢀ50 min in rodents),14 which
would require the administration of multiple doses per day to
maintain efficacy, and multiple dosing would likely result in
diminished compliance.
Our research program set out to identify analogs with enhanced
affinity and selectivity for VMAT2. The structure of lobeline was
modified to remove the oxygen functionalities, resulting in the
symmetrical molecule, lobelane (2, Fig. 1). Lobelane exhibited high
affinity (Ki = 0.045 mM) for inhibition of [3H]DA uptake at VMAT2
with 43-, 35-, and 2.5-fold greater selectivity for VMAT2 relative
to DAT, SERT, and hERG respectively.11,15,16 Moreover, lobelane
dose-dependently decreased METH self-administration in rats,
without decreasing sucrose-maintained responding, revealing
behavioral specificity and affording enhanced potential as a novel
treatment for METH use disorder.17
Lobelane was modified further to provide two families of ana-
logs, one based on a 2,6-disubstituted piperidine scaffold and
another incorporating a 1,4-disubstituted piperidine scaffold, both
of which afforded potent inhibitors of DA uptake at VMAT2.18–21
To further evaluate structure activity relationships around these
scaffolds, 1,4-diphenethylpiperidine and 1,4-diphenethyl-piper-
⇑
Corresponding author.
E-mail address: pacrooks@uams.edu (P.A. Crooks).
https://doi.org/10.1016/j.bmcl.2017.10.039
0960-894X/Ó 2017 Elsevier Ltd. All rights reserved.
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2
E.R. Hankosky et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
4
these molecules also may afford improved drug-likeness
properties.
Fluoroethoxy-containing 1,4-diphenethylpiperidine derivatives
3
5
OH
O
2
6
N
CH3
N
CH3
1
were synthesized starting with benzaldehyde. As illustrated in
Scheme 1, a variety of variously substituted benzaldehydes (3)
were reacted with 4-picoline (4) via Aldol condensation in acetic
anhydride at reflux temperature to afford the corresponding (E)-
4-styrylpyridine (5). Compound 5 then was hydrogenated with
Adams catalyst (PtO2) in acetic acid under hydrogen gas (50 psi)
at room temperature to afford the corresponding 4-phenethylpi-
peridino intermediate (6). Intermediate 6 was alkylated with vari-
ous substituted phenethyl bromides (7) in the presence of K2CO3 in
acetonitrile at reflux temperature to yield the corresponding 1,4-
diphenethylpiperidine analogs 8a-8k; these compounds were fur-
ther O-alkylated utilizing ethylfluorotosylate and Cs2CO3 in DMF at
reflux temperature to yield the corresponding fluoroethoxy-substi-
tuted 1,4-diphenethyl-piperidine derivatives 9a-9k (Scheme 1).22
As illustrated in Scheme 2, the fluoroethoxy-substituted 1,4-
diphenethylpiperazine derivatives were synthesized starting from
1,4-piperazine. 1,4-Piperazine (10) was reacted with a variety of
substituted phenethylbromides (11) in toluene at reflux tempera-
ture to afford 1-phenethylpiperazine (12), which was alkylated
with various hydroxyphenethylbromides using K2CO3 in acetoni-
trile at reflux temperature, to yield the corresponding 1,4-
diphenethylpiperazine analogs 14a-14 f. These compounds were
O-alkylated utilizing ethylfluorotosylate and Cs2CO3 in DMF at
reflux temperature to yield the corresponding fluoroethoxy-substi-
tuted 1,4-diphenethylpiperazine derivatives 15a-15f (Scheme 2).22
All synthesized compounds were fully characterized by 1H
NMR, 13C NMR and high resolution mass spectral analysis.
Compounds 9a-9k and 15a-15f were evaluated for inhibition of
[3H]DA uptake at VMAT2 and DAT, [3H]5-HT uptake at the sero-
tonin transporter (SERT), and affinity for the human ether-a-go-
go related gene (hERG) channel to determine cardiotoxicity. The
results are provided in Table 1.23
2. Lobelane
1. Lobeline
2
3
3
2
4
1
1
4
N
N
N
5
6
5
6
A
B
Fig. 1. Structures of lobeline (1), lobelane (2), which incorporate a 2,6-disubstituted
piperidine scaffold. Structures A and B represent 1,4 disubstituted piperidine and
piperazine scaffolds, respectively.
azine analogs have been modified to incorporate aromatic fluo-
roethoxy moieties. Incorporation of fluorine atoms into strategic
positions in a drug molecule can improve electronic and metabolic
properties. In addition, fluorinated analogs incorporating an 18F
positron-emitting isotope have additional value as potential
ligands for positron emission tomography (PET) studies. Also, the
substitution of the piperidine moiety with a piperazine moiety in
R1
R1
CHO
a
b
R1
+
N
N
H
N
4
3
5
Br
6
c
R2
7
For inhibition of VMAT2 function, the [3H]DA uptake assay was
conducted using preparations of isolated synaptic vesicles from rat
brain.8,20 The majority of the analogs exhibited Ki values in the
nanomolar range (Ki = 0.014–0.073 mM) for inhibition of VMAT2
function. Compound 15d, 1-(2-chlorophenethyl)-4-(2-fluo-
roethoxyphenethyl)piperazine, was the most potent inhibitor of
[3H]DA uptake at VMAT2, exhibiting a Ki value of 0.014 mM and
exhibiting 3-fold greater affinity for VMAT2 compared to lobelane
(Ki = 0.045 mM). Compound 15d exhibited 160-, 5-, and 60-fold
greater selectivity for VMAT2 versus DAT, SERT, and hERG,
respectively.
HCl
R2
R3
R4
R1
d, e
N
N
R2
8a-8k
R1 = H or OH
R2 = H or OH or OCH3 or Cl or F
R3 = OCH2-CH2-F
9a-9k
R4 = H or OCH2-CH2-F
Scheme 1. Synthesis of fluoroethoxy-substituted 1,4-diphenethylpiperidine ana-
logs. Reagents and conditions: (a) Ac2O, reflux, 24 h, 36–48%; (b) 10% (w/v) PtO2/H2,
AcOH, 50 psi, rt, 12 h, 75%; (c) K2CO3/acetonitrile, 80 °C, 8 h, 75–80%; (d) F-CH2-
CH2-OTs Cs2CO3, DMF/reflux, 6 h, 65–70% (e) 2M HCl in diethyl ether.
Br
R1
Br
H
R2
N
N
N
N
13
a
R1
+
R2
R1
b
14a-14f
N
H
10
N
H
12
11
c, d
2HCl
N
N
R1
15a-15f
R1 = H or Cl or F
R2 = OH ; R3 = OCH2-CH2-F
R3
Scheme 2. Synthesis of fluoroethoxy-substituted 1,4-diphenethylpiperazine analogs. Reagents and conditions: (a) toluene/reflux, 8–12 h, 70–75% (b) K2CO3/acetonitrile, 80 °C,
8–10 h, 75–80%; (c) F-CH2-CH2-OTs, Cs2CO3, DMF/reflux, 6 h, 60–65% (d) 2M HCl in diethyl ether.
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E.R. Hankosky et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
3
Table 1
Fluoroethoxy-containing 1,4-diphenethylpiperidine and 1,4-diphenethylpiperazine analog inhibition of [3H]DA uptake at VMAT2 and DAT, [3H]5-HT uptake at SERT, and [3H]
dofetilide binding.
Compound
VMAT2
DAT
SERT
hERG [3H]Dofetilide Binding
IC50, mM
[3H]DA Uptake
Ki, mM
[3H]DA Uptake
Ki, mM
[3H]5-HT Uptake
Ki, mM
0.053 0.0059a
0.061 0.0052
0.067 0.0020
3.14 0.16 (60-fold)b
4.79 1.35 (79-fold)
3.70 0.92 (56-fold)
2.41 0.37 (46-fold)
0.47 0.09 (8-fold)
0.66 0.07 (10-fold)
0.75 0.31 (14-fold)
0.80 0.13 (13-fold)
0.28 0.06 (4-fold)
0.035 0.0043
0.025 0.0017
2.75 0.24 (78-fold)
2.31 0.39 (94-fold)
5.68 1.92 (161-fold)
1.16 0.32 (47-fold)
0.76 0.16 (22-fold)
0.43 0.15 (17-fold)
0.073 0.0058
0.062 0.0033
0.027 0.0028
5.58 0.87 (77-fold)
2.76 0.65 (45-fold)
2.14 0.26 (78-fold)
2.13 0.71 (29-fold)
1.45 0.10 (24-fold)
3.66 0.86 (134-fold)
0.35 0.07 (5-fold)
0.69 0.12 (11-fold)
0.38 0.09 (14-fold)
0.19 0.012
0.15 0.013
2.71 0.57 (15-fold)
1.06 0.04 (7-fold)
3.62 0.95 (20-fold)
0.39 0.09 (3-fold)
2.20 0.65 (12-fold)
0.35 0.16 (2-fold)
0.024 0.0047
0.035 0.0046
1.96 0.23 (81-fold)
4.57 0.66 (130-fold)
0.47 0.18 (19-fold)
0.26 0.04 (7-fold)
0.41 0.05 (17-fold)
5.49 2.23 (156-fold)
0.073 0.017
0.60 0.091
8.38 1.61 (115-fold)
2.74 0.13 (5-fold)
5.96 1.01 (82-fold)
3.37 0.81 (6-fold)
4.31 0.63 (59-fold)
3.05 0.93 (5-fold)
(continued on next page)
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E.R. Hankosky et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
Table 1 (continued)
Compound
VMAT2
DAT
SERT
hERG [3H]Dofetilide Binding
IC50, mM
[3H]DA Uptake
Ki, mM
[3H]DA Uptake
Ki, mM
[3H]5-HT Uptake
Ki, mM
0.014 0.0026
0.024 0.0027
0.45 0.026
2.24 0.10 (160-fold)
1.57 0.07 (65-fold)
5.36 0.12 (12-fold)
0.072 0.01 (5-fold)
0.30 0.01 (13-fold)
0.48 0.04 (1-fold)
0.85 0.15 (61-fold)
1.12 0.21 (47-fold)
1.40 0.34 (3-fold)
a
Mean SEM for n = 3–4/analog/assay.
Number within the parentheses represents fold selectivity relative to inhibition of VMAT2 function.
b
Other notably potent compounds in this series were 1-(2-fluo-
roethoxy)phenethyl)-4-(4-fluorophenethyl)piperazine (15e), 4-(2-
fluoroethoxy)phenethyl)-1-(2-ethoxyphenethyl)piperidine (9e),
4-(2-fluoroethoxy)phenethyl)-1-(4-fluorophenethyl)piperidine
(9h) and 1-(3-fluoroethoxy)phenethyl)-4-phenethyl piperidine
(9k), which exhibited inhibition of [3H]DA uptake at VMAT2 with
Ki values ranging from 0.024 to 0.027 mM. Two other compounds,
4-(2-fluoroethoxy)phenethyl-1-phenethyl-piperidine (9d) and 1-
(2-fluoroethoxy)phenethyl-4-phen-ethylpiperazine (15a), also
exhibited high affinities for VMAT2 (Ki’s = 0.035 mM), which were
similar to that for lobelane.
In the 1,4-substituted piperidine series, compounds 9j and 9 k
are positional isomers. Compound 9 k, a 1-(3-fluoroethoxy- phe-
nethyl) analog, exhibited 6-fold higher affinity (Ki = 0.024 mM)
when compared to 9j, the 1-(4-fluoroethoxyphenethyl) analog, in
the VMAT2 assay. Ortho- and meta-fluoroethoxylphenyl analogs
exhibited generally exhibited greater potency compared to para-
fluoroethoxyphenyl analogs. Similarly, in the 1,4-piperazine series,
compounds 15a, 15b, and 15c are all positional isomers involving
selectivity for VMAT2 relative to hERG. However, inclusion of a
piperazine ring, rather than a piperidine ring in the scaffold, while
having minimal impact on affinity for VMAT2 and DAT (as evi-
denced in comparing 9j with 15c, and 9 k with 15b), resulted in
markedly greater selectivity for VMAT2 over SERT and hERG. These
findings suggest that the piperazine-containing scaffold has impor-
tant advantages with respect to off-target activity (e.g., cardiotox-
icity) relative to the piperidine-containing scaffold in the pursuit of
potent and selective VMAT2 inhibitors as therapeutics for METH
use disorder.
We carried out in silico evaluation of several drug-like and
physicochemical properties of the above fluoroethoxy analogs
and compared them to those of their parent compounds (i.e. mole-
cules in which the fluoroethoxy moiety has been replaced with a
methoxy group) utilizing appropriate predictive algorithms (ACD
Profiler Suite); these properties included LogP, water-solubility,
pKa, Lipinski compliance, drug-likeness, and ADME profiling
(Caco-2, CNS penetration, and human intestinal absorption). The
fluoroethoxy-1,4-diphenethylpiperidine and piperazine analogs
generally exhibited good lipophilicity with logP values in the range
of 5.21–5.80 and 3.91–4.56, respectively, and had moderate to
the fluoroethoxyphenyl group. Compound 15a,
a
2-fluo-
roethoxyphenethyl-containing analog, and compound 15b, a 3-flu-
oroethoxyphenethyl-containing analog, exhibited high affinity for
VMAT2 (Ki = 0.035 and 0.073 mM, respectively), whereas com-
pound 15c, a 4-fluoroethoxyphenethyl-containing analog, was 8-
to 17-fold less potent than its positional isomeric analogs, 15a
and 15b. Compound 15d, containing a 2-fluoroethoxyphenethyl
moiety, had the highest affinity (Ki = 0.014 mM) for VMAT2 in this
series of analogs.
Interestingly, 15f, which contains a 4-fluoroethoxyphenethyl
moiety, was 32-fold less potent than 15d. Thus, regiospecificity
of the aromatic fluoroethoxy substituent plays an important role
in influencing the affinity of these inhibitors for VMAT2.
Evaluation of 9a-9k and 15a-15f as inhibitors of [3H]DA uptake
at DAT afforded Ki values in the range of 1.06–8.38 lM (Table 1),
indicating that these compounds have good selectivity for VMAT2
over DAT. In the SERT assay,20 Ki values were in the range of 0.070
mM to 6 lM (Table 1). Thus, for the majority of the compounds in
this series, good selectivity for VMAT2 over SERT also was found.
While 15d was the most potent (Ki = 0.014 mM) compound in the
series as an inhibitor of VMAT2, 15d exhibited 160-fold and 5-fold
selectivity for VMAT2 over DAT and SERT, respectively. Thus, 15d
was one of the least selective compounds in the series.
Compounds 9a-9k and 15a-15f were evaluated also for affinity
at the hERG channel to determine potential cardiotoxicity.21
Although the majority of the analogs exhibited moderate affinity
Fig. 2. Compound 15b selectively inhibits specific [3H]DA uptake into striatal
vesicles relative to inhibition of [3H]DA and [3H]5-HT uptake into striatal synap-
tosomes and [3H]dofetilide binding to the hERG channel protein in HEK-293 cells.
Data are the mean ( S.E.M.) specific [3H]DA or [3H]5-HT uptake or [3H]dofetilide
binding presented as a percentage of the control condition (VMAT2 [3H]DA uptake:
36.8 11.8 fmol/mg; DAT [3H]DA uptake: 13.4 2.71 pmol/mg/min; SERT [3H]5-HT
uptake: 0.87 0.25 fmol/mg; hERG [3H]dofetilide binding: 1215.8 353.0 fmol/mg;
n = 3–4 rats/uptake assay and 3 individual preparations of HEK-293 stock).
for hERG (Ki = 0.28–0.85 lM), most had at least 10-fold greater
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E.R. Hankosky et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
5
good water-solubility (0.41–6.14 and 0.37–3.70 mg/mL, respec-
A. Supplementary data
tively). The piperidine analogs exhibited pKa values in the range
9.1–9.3, whereas the piperazine analogs had pKa values in the
range 7.4–7.8. Lipinski compliance and drug-likeness were moder-
ate (1 violation) for the piperidine analogs and moderate to good
(0–1 violations) for the piperazine analogs. The ADME profiling
data indicated that both the piperidine and the piperazine analogs
were predicted to be highly absorbed (100%) from the human
intestine, to be highly permeable in the Caco-2 cell assay, and were
classified as CNS penetrants. These data are provided in Tables S1
and S2 in the Supporting Information. Comparison of the above
properties of the fluoroethoxy-1,4-diphenethylpiperidine and
piperazine analogs with those of their corresponding parent meth-
oxy analogs indicated that replacing the aromatic methoxy sub-
stituent with an aromatic fluoroethoxy substituent had little
overall effect on these properties.
Supplementary data associated with this article can be found, in
the online version, at https://doi.org/10.1016/j.bmcl.2017.10.039.
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Compound 15b was considered the lead compound based on its
high affinity for VMAT2 and due to it having the greatest selectivity
for VMAT2 in the compound series (Fig. 2, Table 1). Compound 15b
had a Ki value of 0.073 mM for VMAT2 and exhibited 115-, 82-, and
59-fold greater selectivity for VMAT2 compared to DAT, SERT and
hERG, respectively. Based on these characteristics, 15b selectively
interacts with VMAT2 and is predicted to have low abuse liability
and cardiotoxicity.
In Summary, a small library of fluoroethoxy-containing ana-
logs of 1,4-diphenethylpiperidine and 1,4-diphenethylpiperazine
were synthesized and evaluated for inhibition of VMAT2 function,
as well as for inhibition of DAT and SERT function and interaction
with the hERG channel. The parent compound, lobelane,
has a high affinity (Ki = 0.045 mM) for VMAT2 with 43-, 35-, and
2.5-fold greater selectivity relative to DAT, SERT, and hERG
respectively.
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Most of the compounds tested in this study exhibited affinity
for VMAT2 (Ki < 0.075
l
M), similar to lobelane. However, many
22. General synthetic procedure and analytical data for the fluoroethoxy-containing
1,4-diphenethylpiperidine and 1,4-diphenethylpiperazine analogs (9a-9k & 15a-
15f):
of the compounds exhibited improved selectivity for VMAT2 rela-
tive to DAT (45- to 160-fold), SERT (46- to 161-fold), and hERG (11-
to 156-fold). Generally, the piperazine and piperidine scaffolds
confer affinity at VMAT2 similar to lobelane, while improving
selectivity over off target proteins. Notably, the piperazine scaffold
may be beneficial for reducing cardiotoxicity.
To compounds of general structure
8 or 14 (0.61 mmol) in DMF (5 mL),
2-fluoroethyltosylate (0.91 mmol) and Cs2CO3 (0.91 mmol) were added at
ambient temperature. Reaction mixtures were heated at 120 °C for 6 h. After
completion of the reactions (monitored by TLC), reaction mixtures were diluted
with water and extracted with dichloromethane (2 Â 30 mL). Organic layers
were dried over Na2SO4 and concentrated under reduced pressure to afford
crude products. Crude products were purified by column chromatography
(silica gel, 3–5% methanol in dichloromethane) to afford pure products (yield
65–70%). These products were further converted to HCl salts by treatment with
a 2M HCl solution in diethyl ether.
Compound, 15d exhibited the highest affinity (Ki = 0.014 lM) at
VMAT2 relative to the other compounds in the series; however,
15d was not selective for VMAT2, exhibiting only 5-fold greater
selectivity for VMAT2 versus SERT. Among the compounds in this
series, 15b was the most selective compound for VMAT2, with
good affinity (Ki = 0.073 mM) at VMAT2 and 115-, 82-, and 59-fold
greater selectivity for VMAT2 relative to DAT, SERT, and hERG,
respectively. In particular, it was observed that the piperazino ana-
logs generally exhibited lower affinity for the hERG channel when
compared to their piperidino counterparts.
Compound 15b is considered the lead compound from this ser-
ies of analogs due to its nanomolar affinity at VMAT2, as well as its
> 60-fold selectivity for VMAT2 over other off target proteins.
Currently, 15b is being investigated further for in vivo activity in
preclinical models of METH self-administration as a potential
treatment for METH use disorder.
4-(3-(2-Fluoroethoxy)phenethyl)-1-phenethylpiperidine hydrochloride (9a): 1H
NMR (CD3OD, 400 MHz) d 7.30–7.15 (m, 6H), 6.80–6.74 (m, 3H), 4.76 (t, J =
4.0 Hz, 1H), 4.64 (t, J = 4.0 Hz, 1H), 4.21 (t, J = 4.0 Hz, 1H), 4.14 (J = 4.0 Hz, 1H),
3.17 (d, J = 11.6 Hz, 2H), 2.88–2.84 (m, 2H), 2.76- 2.72 (m, 2H), 2.64–2.60 (m,
2H), 2.27 (t, J = 11.2 Hz, 2H), 1.85 (d, J = 10 Hz, 2H), 1.61–1.56 (m, 2H), 1.39–
1.33 (m, 3H) ppm. 13C NMR (CD3OD, 100 MHz) d 160.1, 145.4, 140.4, 130.4,
129.6, 129.6, 127.4, 122.2, 115.8, 112.7, 84.0, 82.3, 68.5, 68.3, 61.2, 54.5, 39.1,
35.7, 33.9, 33.3, 32.2 ppm. HRMS (ESI) m/z calcd. for
C
23H31FNO (M+H)+
356.2384, found 356.2393.
4-(3-(2-Fluoroethoxy)phenethyl)-1-(2-methoxyphenethyl)piperidine
hydrochloride (9b): 1H NMR (CD3OD, 400 MHz) d 7.14–7.06 (m, 3H), 6.85–6.79
(m, 2H), 6.73–6.68 (m, 3H), 4.69 (t, J = 3.6 Hz, 1H), 4.57 (t, J = 3.2 Hz, 1H), 4.12
(t, J = 4.0 Hz, 1H), 4.05 (t, J = 3.6 Hz, 1H), 3.73 (s, 3H), 3.01 (d, J = 10.8 Hz, 2H),
2.80–2.76 (m, 2H), 2.56–2.51 (m, 4H), 2.05 (t, J = 10.8 Hz, 2H), 1.71 (d, J = 8.8
Hz, 2H), 1.49 (s, 2H), 1.26 (s, 3H) ppm. 13C NMR (CD3OD, 100 MHz) d 160.0,
158.7, 145.4, 131.2, 130.4, 128.8, 128.5, 122.2, 121.5, 115.8, 112.6, 111.4, 84.0,
82.3, 68.4, 68.2, 59.7, 55.7, 54.5, 39.1, 35.8, 33.9, 32.3, 28.2 ppm. HRMS (ESI) m/z
calcd. for C24H33FNO2 (M+H)+ 386.2490, found 386.2504.
4-(3-(2-Fluoroethoxy)phenethyl)-1-(4-methoxyphenethyl)piperidine (9c): 1H
NMR (CD3OD, 400 MHz) d 7.17–7.11 (m, 3H), 6.83–6.71 (m, 5H), 4.73 (t, J =
4.0 Hz, 1H), 4.61 (t, J = 4.0 Hz, 1H), 4.17 (t, J = 4.0 Hz, 1H), 4.10 (t, J = 4.0 Hz, 1H),
3.71 (s, 3H), 3.17 (J = 11.2 Hz, 2H), 2.81–2.73 (m, 4H), 2.57 (t, J = 8.0 Hz, 2H),
2.31 (t, J = 10.8 Hz, 2H), 1.81 (d, J = 10 Hz, 2H), 1.56–1.51 (m, 2H), 1.35–1.33 (m,
3H) ppm. 13C NMR (CD3OD, 100 MHz) d 160.0, 159.7, 145.3, 131.7, 130.6, 130.4,
122.2, 115.8, 115.0, 112.7, 84.0, 82.3, 68.4, 68.2, 60.9, 55.6, 54.3, 38.8, 35.3,
33.8, 32.1, 31.8 ppm. HRMS (ESI) m/z calcd. for C24H33FNO2 (M+H)+ 386.2490,
found 386.2501.
Acknowledgements
This research was supported by DA013519, TR000117,
TR001998 and GM109005 NIH grants, and an Arkansas Research
Alliance (ARA) Scholar award. The University of Kentucky holds
patents on the novel compounds described herein. A potential roy-
alty stream to LPD and PAC may occur consistent with University
of Kentucky policy.
4-(2-(2-Fluoroethoxy)phenethyl)-1-phenethylpiperidine (9d): 1H NMR (CD3OD,
400 MHz) d 7.28–7.10 (m, 7H), 6.87 (t, J = 8.0 Hz, 2H), 4.75 (t, J = 3.6 Hz, 1H),
4.63 (t, J = 4.0 Hz, 1H), 4.17 (t, J = 4.0 Hz, 1H), 4.10 (J = 4.0 Hz, 1H), 3.11
Please cite this article in press as: Hankosky E.R., et al. Bioorg. Med. Chem. Lett. (2017), https://doi.org/10.1016/j.bmcl.2017.10.039

6
E.R. Hankosky et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
(d, J = 11.2 Hz, 2H), 2.86–2.82 (m, 2H), 2.72–2.62 (m, 4H), 2.23 (t, J = 10.8 Hz,
1-(2-Chlorophenethyl)-4-(2-(2-fluoroethoxy)phenethyl)piperazine dihydrochloride
(15d): 1H NMR (CDCl3, 400 MHz) d 13.87 (brs, 1H), 13.62 (brs, 1H), 7.34–7.24
(m, 6H), 6.94 (d, J = 6.8 Hz, 1H), 6.84 (d, J = 7.6 Hz, 1H), 4.88 (s, 1H), 4.76 (s, 1H),
4.27–4.09 (m, 6H), 3.64 (brs, 4H), 3.44–3.25 (m, 8H) ppm. 13C NMR (CDCl3, 100
MHz) d 156.0, 133.8, 132.7, 131.2, 130.9, 130.0, 129.4, 129.2, 127.7, 123.8,
121.8, 111.8, 82.8, 81.1, 67.4, 56.5, 56.1, 48.3, 28.3, 25.7 ppm. HRMS (ESI) m/z
calcd. for C22H29ClFN2O (M+H)+ 391.1947, found 391.1947.
2H), 1.82 (d, J = 10 Hz, 2H), 1.55–1.50 (m, 2H), 1.36–1.34 (m, 3H) ppm. 13C NMR
(CD3OD, 100 MHz) d 157.6, 140.2, 132.2, 130.9, 129.6, 129.5, 128.1, 127.4,
122.0, 112.6, 84.0, 82.3, 68.7, 68.5, 61.0, 54.4, 37.6, 35.7, 33.2, 32.0, 28.3 ppm.
HRMS (ESI) m/z calcd. for C23H31FNO (M+H)+ 356.2384, found 356.2390.
4-(2-(2-Fluoroethoxy)phenethyl)-1-(2-methoxyphenethyl)piperidine (9e): 1H
NMR (CD3OD, 400 MHz) d 7.29–7.14 (m, 4H), 6.99–6.87 (m, 4H), 4.81 (t, J =
3.6 Hz, 1H), 4.69 (t, J = 4.0 Hz, 1H), 4.25 (t, J = 4.0 Hz, 1H), 4.18 (t, J = 4.0 Hz, 1H),
3.87 (s, 3H), 3.65 (d, J = 13.2 Hz, 2H), 3.25–3.21 (m, 2H), 3.08–3.04 (m, 2H), 2.97
(t, J = 12.4 Hz, 2H), 2.71 (t, J = 7.6 Hz, 2H), 2.09 (d, J = 13.6 Hz, 2H), 1.63–1.49
(m, 5H) ppm. 13C NMR (CD3OD, 100 MHz) d 158.8, 157.7, 131.7, 131.5, 131.0,
129.9, 128.3, 125.4, 122.1, 121.9, 112.7, 111.7, 84.2, 82.5, 68.8, 68.6, 57.8, 55.8,
54.2, 37.4, 34.3, 30.8, 28.0, 26.6 ppm. HRMS (ESI) m/z calcd. for C24H33FNO2
(M+H)+ 386.2490, found 386.2499.
1-(2-(2-Fluoroethoxy)phenethyl)-4-(4-fluorophenethyl)piperazine dihydrochloride
(15e): 1H NMR (CDCl3, 400 MHz) d 13.87 (brs, 1H), 13.59 (brs, 1H), 7.29–7.20
(m, 4H), 7.02 (t, J = 8.4 Hz, 2H), 6.95 (t, J = 7.2 Hz, 1H), 6.86 (d, J = 8.4 Hz, 1H),
4.90 (s, 1H), 4.79 (s, 1H), 4.29–4.02 (7H), 3.68–3.59 (m, 4H), 3.31–3.24 (m, 7H)
ppm. 13C NMR (CDCl3, 100 MHz) d 163.5, 161.1, 156.1, 131.4, 130.4, 129.6,
123.8, 122.1, 116.4, 111.9, 82.9, 81.2, 67.6, 58.3, 56.7, 48.3, 29.5, 25.9 ppm.
HRMS (ESI) m/z calcd. for C22H29F2N2O (M+H)+ 375.2242, found 375.2215.
1-(2-Chlorophenethyl)-4-(4-(2-fluoroethoxy)phenethyl)piperazine (15f): 1H NMR
(CDCl3, 400 MHz) d 7.35–7.10 (m, 6H), 6.84 (d, J = 8.4 Hz, 2H), 4.78 (t, J = 4 Hz,
1H), 4.66 (t, J = 4 Hz, 1H), 4.20 (t, J = 4 Hz, 1H), 4.13 (t, J = 4 Hz, 1H), 2.95–2.91
(m, 3H), 2.76–2.72 (m, 3H), 2.62–2.54 (m, 10H) ppm. 13C NMR (CD3OD, 100
MHz) d 157.9, 133.6, 130.9, 129.6, 129.5, 129.0, 127.9, 127.4, 114.8, 82.5, 80.8,
67.3, 67.1, 29.0, 27.8 ppm. HRMS (ESI) m/z calcd. for C22H29ClFN2O (M+H)+
391.1947, found 391.1940.
4-(2-(2-Fluoroethoxy)phenethyl)-1-(4-methoxyphenethyl)piperidine (9f): 1H NMR
(CD3OD, 400 MHz) d 7.16–7.12 (m, 4H), 6.90–6.83 (m, 4H), 4.79 (t, J = 4.0 Hz,
1H), 4.67 (t, J = 4.0 Hz, 1H), 4.23 (t, J = 4.0 Hz, 1H), 4.16 (t, J = 4.0 Hz, 1H), 3.75 (s,
3H), 3.19 (d, J = 11.6 Hz, 2H), 2.84–2.65 (m, 6H), 2.32 (t, J = 10.8 Hz, 2H), 1.89 (d,
J = 9.6 Hz, 2H), 1.59–1.54 (m, 2H), 1.39–1.35 (m, 3H) ppm. 13C NMR (CD3OD,
100 MHz) d 159.6, 157.5, 132.0, 131.8, 130.7, 130.4, 127.9, 121.8, 114.8, 112.4,
83.9, 82.2, 68.6, 68.4, 61.0, 55.4, 54.3, 37.5, 35.6, 32.1, 31.9, 28.1 ppm. HRMS
(ESI) m/z calcd. for C24H33FNO2 (M+H)+ 386.2490, found 386.2489.
23. Assay procedures for Vesicular [3H]DA Uptake, Synaptosomal [3H]DA, [3H]5-HT
Uptake and hERG Binding.
1-(2-Chlorophenethyl)-4-(2-(2-fluoroethoxy)phenethyl)piperidine (9g): 1H NMR
(CD3OD, 400 MHz) d 7.44–7.42 (m, 2H), 7.33–7.29 (m, 2H), 7.18–7.15 (m, 2H),
6.93–6.88 (m, 2H), 4.81 (t, J = 4.0 Hz, 1H), 4.69 (t, J = 3.6 Hz, 1H), 4.26 (t, J = 4.0
Hz, 1H), 4.19 (t, J = 4.0 Hz, 1H), 3.70 (d, J = 12.8 Hz, 2H), 3.32–3.23 (m, 5H), 3.03
(t, J = 12 Hz, 2H), 2.72 (t, J = 7.6 Hz, 2H), 2.11 (d, J = 11.6 Hz, 2H), 1.64–1.54 (m,
4H) ppm. 13C NMR (CD3OD, 100 MHz) d 157.7, 135.2, 134.9, 132.3, 131.7, 131.0,
130.8, 130.2, 128.7, 128.3, 122.1, 112.7, 84.2, 82.5, 68.8, 68.6, 57.2, 54.2, 37.4,
34.3, 30.7, 29.2, 28.0 ppm. HRMS (ESI) m/z calcd. for C23H30ClFNO (M+H)+
390.1994, found 390.1993.
Vesicular [3H]DA Uptake Assays: Inhibition of [3H]DA (dihydroxyphenyl-
ethylamine,3,4-[7-3H], 27.8 Ci/mmol; Perkin-Elmer) uptake was conducted
using a preparation of isolated synaptic vesicles as previously described.10,20 In
brief, rat striata were homogenized with 10 up-and-down strokes of a Teflon
pestle homogenizer (clearance ꢀ0.003 inch) in 14 ml of 0.32
M sucrose
solution. Homogenates were centrifuged (2000 g for 10 min at 4 °C) and the
resulting supernatants were centrifuged again (10,000 g for 30 min at 4 °C).
Pellets were re-suspended in 2 ml of 0.32 M sucrose solution and subjected to
osmotic shock by adding 7 ml ice-cold water, followed by restoration of
osmolality 5 min later by transferring suspensions to tubes containing 900 ml of
0.25 M HEPES buffer and 900 ml of 1.0 M potassium tartrate solution. Samples
were centrifuged (20,000 g for 20 min at 4 °C) and the resulting supernatants
were centrifuged again (55,000g for 1 h at 4 °C), followed by the addition of
100 ml of 1.0 M MgSO4, 100 ml of 0.25 M HEPES, and 100 ml of 1.0 M potassium
tartrate solution before the final centrifugation (100,000g for 45 min at 4 °C).
Final pellets were re-suspended in 2.4 ml of assay buffer (25 mM HEPES, 100
mM potassium tartrate, 50 mM EGTA, 100 mM EDTA, 1.7 mM ascorbic acid,
2 mM ATP-Mg2+, pH 7.4). Aliquots of the vesicular suspension (100 ml) were
added to tubes containing assay buffer, various concentration of analog (1 nM
to 0.1 mM) and 0.1 mM [3H]DA to afford a final volume of 500 ml. Nonspecific
uptake was determined in the presence of Ro4-1284 (10 mM). Reactions were
terminated by filtration using a cell harvester (MP-43RS; Brandel Inc.) and
radioactivity retained by Whatman GF/B filters (presoaked for 2 h in 0.5%
polyethyleneimine) was determined by scintillation spectrometry.
4-(2-(2-Fluoroethoxy)phenethyl)-1-(4-fluorophenethyl)piperidine (9h): 1H NMR
(CD3OD, 400 MHz) d 7.26–7.23 (m, 2H), 7.16–7.13 (m, 2H), 7.02 (t, J = 8.4 Hz,
2H), 6.92–6.86 (m, 2H), 4.80 (t, J = 4.0 Hz, 1H), 4.68 (t, J = 4.0 Hz, 1H), 4.25 (t, J =
4.0 Hz, 1H), 4.18 (t, J = 4.0 Hz, 1H), 3.22 (d, J = 11.2 Hz, 2H), 2.90–2.77 (m, 4H),
2.69 (t, J = 7.2 Hz, 2H), 2.37 (t, J = 10 Hz, 2H), 1.92 (d, J = 9.6 Hz, 2H), ⁄⁄1.60–
1.55 (m, 2H), 1.39–1.33 (m, 3H) ppm. 13C NMR (CD3OD, 100 MHz) d 164.3,
161.8, 157.7, 132.2, 131.4, 131.3, 131.0, 128.1, 122.0, 116.3, 116.1, 112.6, 84.1,
82.4, 68.8, 68.6, 60.9, 54.5, 37.7, 35.7, 32.3, 32.1, 28.3 ppm. HRMS (ESI) m/z
calcd. for C23H30F2NO (M+H)+ 374.2290, found 374.2300.
1-(2-Chlorophenethyl)-4-(4-(2-fluoroethoxy)phenethyl)piperidine (9i): 1H NMR
(CD3OD, 400 MHz) d 7.45–7.41 (m, 2H), 7.33–7.30 (m, 2H), 7.14–7.11 (m, 2H),
6.89–6.86 (m, 2H), 4.76 (t, J = 4.0 Hz, 1H), 4.64 (t, J = 4.0 Hz, 1H), 4.21 (t, J = 4.4
Hz, 1H), 4.14 (t, J = 4.4 Hz, 1H), 3.70 (d, J = 12.8 Hz, 2H), 3.32 (m, 4H), 3.03 (t, J =
12.4 Hz, 2H), 2.63 (t, J = 4.8 Hz, 2H), 2.08 (d, J = 12.4 Hz, 2H), 1.64–1.53 (m, 5H)
ppm. 13C NMR (CD3OD, 100 MHz) d 158.3, 135.6, 135.1, 134.9, 132.3, 130.8,
130.3, 130.2, 128.7, 115.6, 84.0, 82.3, 68.7, 68.5, 57.2, 54.2, 38.7, 34.1, 32.7, 30.7,
29.2 ppm. HRMS (ESI) m/z calcd. for C23H30ClFNO (M+H)+ 390.1994, found
390.2010.
Synaptosomal [3H]DA and [3H]5-HT Uptake Assays: Analog-induced inhibition of
[3H]DA and [3H]5-HT (5-hydroxytryptamine creatine sulfate 5-[1,2-3H[N]],
29.5 Ci/mmol; Perkin-Elmer) uptake into rat striatal synaptosomes was
determined using methods previously described.20 Briefly, striata were
homogenized in 20 ml of ice-cold 0.32 M sucrose solution containing 5 mM
NaHCO3, pH 7.4, with 16 up-and-down strokes of a Teflon pestle homogenizer
(clearance ꢀ0.005 inch). Homogenates were centrifuged at 2000g for 10 min at
4 °C, and resulting supernatants were centrifuged at 20,000g for 17 min at 4 °C.
Pellets were re-suspended in 1.5 ml of Krebs’ buffer, containing 125 mM NaCl,
1-(4-(2-Fluoroethoxy)phenethyl)-4-phenethylpiperidine (9j): 1H NMR (CD3OD,
400 MHz) d 7.24 (t, J = 4.0 Hz, 2H), 7.17–7.11 (m, 5H), 6.88 (d, J = 8.8 Hz, 2H),
4.74 (t, J = 4.0 Hz, 1H), 4.62 (t, J = 4.0 Hz, 1H), 4.19 (t, J = 4.0 Hz, 1H), 4.12 (t, J =
4.4 Hz, 1H), 3.11 (d, J = 11.2 Hz, 2H), 2.80–2.76 (m, 2H), 2.65–2.61 (m, 4H), 2.16
(t, J = 7.6 Hz, 2H), 1.82 (d, J = 10 Hz, 2H), 1.58–1.56 (m, 2H), 1.39–1.23 (m, 3H)
ppm. 13C NMR (CD3OD, 100 MHz) d 158.6, 143.7, 133.2, 130.7, 129.3, 129.3,
126.7, 115.7, 84.0, 82.3, 68.7, 68.5, 61.6, 54.6, 39.4, 35.9, 33.9, 32.7, 32.5 ppm.
HRMS (ESI) m/z calcd. for C23H31FNO (M+H)+ 356.2384, found 356.2395.
1-(3-(2-Fluoroethoxy)phenethyl)-4-phenethylpiperidine (9k): 1H NMR (CD3OD,
400 MHz) d 7.27–7.14 (m, 6H), 6.91–6.84 (m, 3H), 4.76 (t, J = 4.0 Hz, 1H), 4.64
(t, J = 4.0 Hz, 1H), 4.24 (t, J = 3.6.0 Hz, 1H), 4.17 (t, J = 4.0 Hz, 1H), 3.64 (d, J =
12.8 Hz, 2H), 3.32–3.28 (m, 3H), 3.07–2.93 (m, 4H), 2.66 (t, J = 7.6 Hz, 2H), 2.05
(d, J = 13.2 Hz, 2H), 1.64–1.51 (m, 4H) ppm. 13C NMR (CD3OD, 100 MHz) d 160.5,
143.1, 139.3, 131.1, 129.4, 129.3, 126.9, 122.4, 116.3, 114.2, 84.0, 82.3, 68.6,
68.4, 59.1, 54.1, 38.6, 34.2, 33.6, 31.3, 30.7 ppm. HRMS (ESI) m/z calcd. for
5 mM KCl, 1.5 mM MgSO4, 1.25 mM CaCl2, 1.5 mM KH2PO4, 10 mM
a-d-
glucose, 25 mM HEPES, and 0.1 mM EDTA, with 0.1 mM pargyline and 0.1 mM
ascorbic acid, saturated with 95% O2/5% CO2, pH 7.4. Synaptosomal suspensions
(20 mg of protein/50 ml) were added to duplicate tubes containing 50 ml of
analog (1 nM to 0.1 mM, final concentration) and 350 ml of buffer and
incubated at 34 °C for 5 min in a total volume of 450 ml. Samples were placed
on ice, and 50 ml of [3H]DA or [3H]5-HT (10 nM, final concentration) was added
to each tube for a final volume of 500 ml. Reactions proceeded for 10 min at
34 °C and were terminated by the addition of 3 ml of ice-cold Krebs’ buffer.
Nonspecific [3H]DA and [3H]5-HT uptake were determined in the presence of 10 mM
GBR 12909 and 10 mM fluoxetine, respectively. Samples were rapidly filtered
through Whatman GF/B filters. Filters were washed three times with 4 ml of ice-
cold Krebs’ buffer containing catechol (1 mM). Complete counting cocktail was
added to the filters, and radioactivity was determined by scintillation spectrometry.
hERG Binding Assays: Binding assays were conducted as described previously
using membranes from HEK-293 cells, which specifically and stably express
hERG channel protein (Millipore, Billerica, MA).21,24 Briefly, cell membrane
C
23H31FNO (M+H)+ 356.2384, found 356.2394.
1-(2-(2-Fluoroethoxy)phenethyl)-4-phenethylpiperazine (15a): 1H NMR (CD3OD,
400 MHz) d 7.37–7.26 (m, 7H), 7.03–6.95 (m, 2H), 4.91–4.89 (m, 1H), 4.79–4.77
(m, 1H), 4.36–4.34 (m, 1H), 4.29–4.26 (m, 1H), 4.09–3.58 (m, 7H), 3.54–3.47
(m, 5H), 3.20–3.14 (m, 4H) ppm. 13C NMR (CD3OD, 100 MHz) d 157.7, 137.1,
131.8, 130.2, 130.0 129.8, 128.4, 125.2, 122.6, 113.1, 84.2, 82.6, 68.9, 68.7, 31.2,
26.5 ppm. HRMS (ESI) m/z calcd. for C22H30FN2O (M+H)+ 357.2337, found
357.2307.
1-(3-(2-Fluoroethoxy)phenethyl)-4-phenethylpiperazine (15b): 1H NMR (CD3OD,
400 MHz) d 7.37–7.24 (m, 7H), 6.96–6.93 (m, 2H), 4.76 (t, J = 4.0 Hz, 1H), 4.64
(t, J = 4.0 Hz, 1H), 4.23 (t, J = 4.0 Hz, 1H), 4.16 (t, J = 4.0 Hz, 1H), 4.09–3.59 (m,
7H), 3.54–3.47 (m, 5H), 3.18–3.08 (m, 4H) ppm. 13C NMR (CD3OD, 100 MHz) d
158.0, 135.7, 129.5, 128.6, 128.4, 127.9, 127.0, 114.7, 82.5, 80.8, 67.3, 67.1, 33.9,
29.7, 28.9 ppm. HRMS (ESI) m/z calcd. for C22H30FN2O (M+H)+ 357.2337, found
357.2301.
suspension (5
lg) was added to duplicate tubes containing assay buffer (50
mM Tris, 10 mM KCl, and 1 mM MgCl2, pH 7.4), 25 ml of a single concentration
(10 nM to 100 mM) of analog or amitriptyline (positive control),25,26 and 25 ml
(5 nM, final concentration) of [3H]dofetilide (dofetilide [N-methyl-3H], 80 Ci/mmol;
American Radiolabeled Chemicals) for a final assay volume of 250 ml. Samples
were incubated for 60 min at 25 °C and reactions terminated by rapid filtration
through Whatman GF/B filters pre-soaked for 2 h in 0.5% polyethyleneimine.
Filters were washed 3 times with 1 ml of ice-cold assay buffer. Radioactivity
retained by the filters was determined by scintillation spectrometry.
1-(4-(2-Fluoroethoxy)phenethyl)-4-phenethylpiperazine (15c): 1H NMR (CD3OD,
400 MHz) d 7.38–7.26 (m, 6H), 6.96–6.87 (m, 3H), 4.78–4.76 (m, 1H), 4.66–4.64
(m, 1H), 4.27–4.20 (m, 1H), 4.20–4.18 (m, 1H), 4.11–3.59 (m, 7H), 3.55–3.48
(m, 5H), 3.17–3.11 (m, 4H) ppm. 13C NMR (CDCl3, 100 MHz) d 159.1, 137.3,
135.6, 129.7, 129.6, 128.6, 128.4, 127.0, 121.0, 119.3, 115.2, 114.9, 113.0, 82.5,
80.8, 67.2, 67.0, 29.7 ppm. HRMS (ESI) m/z calcd. for C22H30FN2O (M+H)+
357.2337, found 357.2348.
24. Sviripa VM, Zhang W, Balia AG, et al. J Med Chem. 2014;57:6083.
25. Jo S-H, Youm JB, Lee CO, Earm YE, Ho W-K. Br J Pharmacol. 2000;129:1474.
26. Teschemacher AG, Seward EP, Hancox JC, Witchel HJ. Br
J Pharmacol.
1999;128:479.
Please cite this article in press as: Hankosky E.R., et al. Bioorg. Med. Chem. Lett. (2017), https://doi.org/10.1016/j.bmcl.2017.10.039