1,4-Diphenalkylpiperidines: A new scaffold for the design of potent inhibitors of the vesicular monoamine transporter-2

Article abstract of DOI:10.1016/j.bmcl.2016.05.025

A series of 1,4-diphenalkylpiperidine analogs were synthesized and evaluated for their affinity and inhibitory potency at the [³H]dihydrotetrabenazine (DTBZ) binding site and [³H]dopamine (DA) uptake site on the vesicular monoamine transporter-2 (VMAT2). Results revealed that translocation of the phenethyl side chains of lobelane from C2 and C6 to C1 and C4 around the central piperidine ring slightly reduces affinity and inhibitory potency at VMAT2 with respect to lobelane. However, methoxy and fluoro-substitution of either phenyl ring of these 1,4-diphenethyl analogs afforded VMAT2 inhibition comparable or higher (5-fold) affinity at the DTBZ binding and DA uptake sites relative to lobelane, whereas replacement of the 4-phenethyl moiety in these analogs with a 4-phenmethyl moiety markedly reduced affinity for the DTBZ binding and DA uptake sites by 3- and 5-fold, respectively. Among the twenty five 1,4-diphenethylpiperidine analogs evaluated, compounds containing a 4-(2-methoxyphenethyl) moiety exhibited the most potent inhibition of DTBZ binding and vesicular DA uptake. From this subgroup, analogs 8h, 8j and 8m exhibited K_i values of 9.3 nM, 13 nM and 13 nM, respectively, for inhibition of [³H]DA uptake by VMAT2, and represent some of the most potent inhibitors of VMAT2 function reported thus far.

Full text of DOI:10.1016/j.bmcl.2016.05.025

Bioorganic & Medicinal Chemistry Letters xxx (2016) xxx–xxx
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Bioorganic & Medicinal Chemistry Letters
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1,4-Diphenalkylpiperidines: A new scaffold for the design of potent
inhibitors of the vesicular monoamine transporter-2
Justin R. Nickell a,y, John P. Culver a,y, Venumadhav Janganati b, Guangrong Zheng a,b, Linda P. Dwoskin a,
Peter A. Crooks a,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 series of 1,4-diphenalkylpiperidine analogs were synthesized and evaluated for their affinity and inhi-
bitory potency at the [3H]dihydrotetrabenazine (DTBZ) binding site and [3H]dopamine (DA) uptake site
on the vesicular monoamine transporter-2 (VMAT2). Results revealed that translocation of the phenethyl
side chains of lobelane from C2 and C6 to C1 and C4 around the central piperidine ring slightly reduces
affinity and inhibitory potency at VMAT2 with respect to lobelane. However, methoxy and fluoro-substi-
tution of either phenyl ring of these 1,4-diphenethyl analogs afforded VMAT2 inhibition comparable or
higher (5-fold) affinity at the DTBZ binding and DA uptake sites relative to lobelane, whereas replacement
of the 4-phenethyl moiety in these analogs with a 4-phenmethyl moiety markedly reduced affinity for
the DTBZ binding and DA uptake sites by 3- and 5-fold, respectively. Among the twenty five 1,4-diphe-
nethylpiperidine analogs evaluated, compounds containing a 4-(2-methoxyphenethyl) moiety exhibited
the most potent inhibition of DTBZ binding and vesicular DA uptake. From this subgroup, analogs 8h, 8j
and 8m exhibited Ki values of 9.3 nM, 13 nM and 13 nM, respectively, for inhibition of [3H]DA uptake by
VMAT2, and represent some of the most potent inhibitors of VMAT2 function reported thus far.
Ó 2016 Elsevier Ltd. All rights reserved.
Received 15 April 2016
Revised 6 May 2016
Accepted 9 May 2016
Available online xxxx
Keywords:
Vesicular monoamine transporter-2
[3H]DTBZ binding
[3H]DA uptake inhibition at VMAT2
1,4 diphenethyl lobelane analogs
1-Phenethyl, 4-phenylmethylene
derivatives of lobelane
Methamphetamine (METH) is
a
highly addictive psycho-
abuse.5 Therefore, molecules that can modulate function and
prevent the pharmacological effects of METH may be suitable
candidates for the treatment of METH addiction.
In this respect, lobeline, the principal alkaloidal constituent of
Lobelia inflata, has been shown to decrease amphetamine-induced
and METH-induced hyperactivity in rats and mice, and to inhibit
amphetamine-evoked and METH-evoked DA release from
superfused rat brain slices.6 Importantly, lobeline also decreases
METH self-administration in rats without acting as a substitute
reinforcer,7,8 indicating a lack of abuse liability.
The mechanism by which lobeline reduces the reinforcing and
rewarding effects of psychostimulants involves its ability to inter-
act with VMAT2.4,9 Lobeline inhibits VMAT2 with about 100-fold
higher affinity compared to its affinity for DAT, indicating that
the interaction with VMAT2 is essential for the observed decrease
in the behavioral effects of METH.4,9,10 Unlike METH, lobeline does
not inhibit MAO activity, allowing DA within the cytosol to be
metabolized to DOPAC.11 Thus, lobeline diminishes the cytosolic
DA pool available for METH-induced reverse transport by DAT.
Clinical evaluation of lobeline as a pharmacotherapy for METH
abuse revealed that the alkaloid is safe in METH addicted individ-
uals.12 However, bitter taste and nausea were noted as relatively
minor side-effects, likely the result of nicotinic acetylcholine
stimulant and its abuse produces severe, deleterious health effects,
including fatigue, dysphoric mood, anxiety, depression and symp-
toms of psychosis.1–3 Currently, there are no FDA-approved treat-
ments for METH abuse. The abuse liability of METH is, at least in
part, due to its interaction with the brain dopamine (DA) reward
system. METH reverses the normal function of the dopamine trans-
porter (DAT) and the vesicular monoamine transporter-2 (VMAT2)
greatly increasing DA concentrations in the cytosol of dopaminer-
gic presynaptic terminals as well as in the extracellular compart-
ment. METH also inhibits monoamine oxidase (MAO) activity,
preventing the metabolism of cytosolic DA to 3,4-dihydroxypheny-
lacetic acid (DOPAC), consequently increasing the availability of
cytosolic DA for METH-induced reverse transport by DAT and
increasing DA concentrations in the extracellular compartment,
resulting in subsequent stimulation of postsynaptic DA receptors.4
Since METH inhibits DA uptake at VMAT2 and promotes DA release
from vesicles to increase cytosolic DA, VMAT2 is considered an
important molecular target in the search for a treatment for METH
⇑
Corresponding author. Tel.: +1 501 686 6495; fax: +1 501 686 6057.
E-mail address: pacrooks@uams.edu (P.A. Crooks).
These authors contributed equally to this manuscript and are co-first authors.
y
http://dx.doi.org/10.1016/j.bmcl.2016.05.025
0960-894X/Ó 2016 Elsevier Ltd. All rights reserved.
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2
J. R. Nickell et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
receptor (nAChR) antagonism.13,14 Another limitation was the
short plasma half-life of lobeline, which would require multiple
doses as a therapeutic, likely diminishing compliance. In an
attempt to address these limitations, a significant drug discovery
effort was initiated in which the lobeline molecule was structurally
modified with the goal of improving potency and selectivity as an
inhibitor of VMAT2, while eliminating affinity for nAChRs and
other neurotransmitter transporters.9 Lobelane (1), a chemically
defunctionalized analog of lobeline, emerged as a lead analog that
exhibited a greater potency inhibiting VMAT2 function compared
to lobeline, while demonstrating little affinity for nAChRs.13
After carrying out comprehensive structure–activity relation-
ship (SAR) studies14 with lobelane analogs, it was determined that
relocation of the 2,6-phenethyl moieties in the lobelane molecule
(1) to the 1,4-positions on the central piperidine heterocycle to
afford 2 (R1 = R2 = H) (Fig. 1), resulted in no loss of affinity for the
[3H]DBTZ binding site on VMAT2 when compared with lobelane.
R1
2
6
4
N
R2
R1
1
CH3
N
1
Lobelane (R1= R2= H)
R2
2
1, 4-diphenethylpiperidine analogs
Figure 1. Relocation of the 2,6-phenethyl moieties of lobelane (1, R1 = R2 = H) to the
1,4-positions on the central piperidine heterocycle to afford 1,4-diphenethylpiper-
idine (2, R1 = R2 = H).
This structural change also maintained the low affinity for
and 7 nAChRs that was exhibited by lobelane. An added advan-
tage was the achiral nature of 2.
In the current study, this new scaffold was exploited by building
a small library of aromatic substituted analogs of 2, and these com-
pounds exhibited inhibition of VMAT2 function with comparable
or higher affinity relative to lobelane. Herein, we present the syn-
thesis and evaluation of 1,4-diphenethylpiperidine and 1-phe-
nethyl-4-benzylpiperidine analogs as inhibitors of vesicular DA
uptake.
a4b2
a
R3
R3
R2
R1
R2
R1
CHO
R3
R1
R2
a
b
+
N
N
H
N
4
3
5
Br
6
The general synthetic approach adopted for the preparation of
the aromatic substituted 1,4-diphenethylpiperidine analogs is
illustrated in Scheme 1. The synthesis utilizes 4-picoline (4) which
is reacted with various substituted benzaldehydes (3) via Aldol
condensation in acetic anhydride at reflux temperature to afford
the corresponding (E)-4-styrylpyridine 5. Compound 5 is then sub-
jected to hydrogenation with Adams catalyst (PtO2) in acetic acid
under hydrogen gas (50 psi) at room temperature to afford the sat-
urated piperidino intermediate (6). Intermediate 6 is alkylated
with variously substituted phenethyl bromides (7) using K2CO3
in acetonitrile at reflux temperature to yield the corresponding
1,4-diphenethylpiperidine analogs 8a–8y, which were further con-
verted to hydrochloride salts with 2 M HCl in diethyl ether
(Scheme 1).15
c, d
R4
R5
7
R6
R2
R1
R6
HCl
N
R3
R4
R5
8a-8y
Scheme 1. Synthesis of 1,4-diphenethylpiperidine analogs. Reagents and condi-
tions: (a) Ac2O, reflux, 24 h, 48–60%; (b) 10% (w/v) PtO2/H2, AcOH, 50 psi, rt, 12 h,
75–80%; (c) K2CO3/acetonitrile, 80 °C, 8 h, 75–80%; (d) 2 M HCl in ether.
R2
R2
In addition, a small group of 1,4-diphenalkylpiperidine analogs
were synthesized in which the 4-phenethyl moiety was replaced
with a 4-benzyl moiety. The synthetic route for generating these
compounds is shown in Scheme 2 and utilized 4-bromopyridine
(9) as a starting point.
R1
R3
b
R1
R3
Br
a
Br
N
4-Bromopyridine was reacted with benzylmagnesium chloride
using NiCl2(dppp)/THF (Kumada coupling)16 at ambient tempera-
ture to obtain the 4-benzylpyridine intermediate 10. Compound
10 was then subjected to hydrogenation with Adams catalyst
(PtO2) in acetic acid under hydrogen gas (50 psi) at room temper-
ature to obtain the saturated benzylpiperidino intermediate, 11.
Compound 11 was then reacted with variously substituted phe-
nethyl bromides (7) in the presence of K2CO3 in acetonitrile at
reflux temperature to yield the corresponding 1-phenethyl-4-ben-
zylpiperidine derivatives 12a–12d, which were further converted
to hydrochloride salts with 2 M HCl in diethyl ether (Scheme 2).
All the synthesized compounds were evaluated for affinity for
the [3H]DTBZ binding site on VMAT2 and for affinity at the DA
translocation site on VMAT2, using the [3H]DA uptake assay in iso-
lated synaptic vesicles (Table 1). In the 1,4-diphen-ethylpiperidine
series of compounds (8a–8y, Table 1), the Ki values from the [3H]
N
10
N
H 11
9
R4
R5
c, d
7
R3
R1
R6
R4
R2
R5
HCl
N
R6
12a-12d
Scheme 2. Synthesis of 1-phenethyl-4-benzylpiperidine analogs. Reagents and
conditions: (a) benzylmagnesium chloride, NiCl2 (dppp), THF, rt 15 h, 85–90%; (b)
H2, PtO2, AcOH, 50 psi, rt, 12 h, 75–80%; (c) K2CO3/acetonitrile, 80 °C, 8 h, 75–80%;
(d) 2 M HCl in ether.
DTBZ binding assay ranged from 0.15 to 2.8
l
M. However, 80% of
The two most potent compounds in this series were analogs 8j
and 8h (Fig. 2), which exhibited similar affinity in the [3H]DTBZ
the analogs evaluated had Ki values <1 M, notable exceptions
l
being the 1-(3,4-dimethoxyphenethyl) analog 8c (Ki = 2.8
and the 1,4-bis(4-methoxyphenethyl) analog 8w (Ki = 2.5
l
l
M),
M).
binding assay (Ki = 0.15
a 5 to 6-fold higher affinity than lobelane (Ki = 0.97
l
M, and 0.19
l
M, respectively), revealing
l
M)14 for the
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J. R. Nickell et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
3
Table 1
1,4-Diphenalkylpiperidine analogs inhibit [3H]DTBZ binding and [3H]DA uptake at VMAT2
Compound
R1
R2
R3
R4
R5
R6
[3H]DTBZ binding Ki (
lM)
[3H]DA uptake Ki (nM)
Lobelane (1)
8a
8b
8c
8d
8e
8f
8g
8h
8i
8j
8k
8l
8m
8n
8o
8p
8q
0.97 0.19a
1.87 0.25a
0.26 0.026
2.80 0.11
0.27 0.038
1.62 0.19
0.38 0.0058
1.82 0.079
0.19 0.0088
0.69 0.050
0.15 0.0058
0.50 0.10
0.19 0.020
0.40 0.097
0.32 0.015
0.32 0.055
0.23 0.019
0.45 0.13
0.46 0.098
0.96 0.047
0.51 0.087
0.42 0.019
0.23 0.047
2.5 0.27
45 8.0b
120 15
30 2.0
56 9.0
22 0.87
46 3.7
47 6.3
32 3.8
9.3 0.6
22 2.8
13 1.5
43 2.8
20 3.0
13 2.1
56 8.6
40 7.5
85 10
130 23
420 49
69 4.0
110 7.0
83 9.0
75 4.0
160 12
40 7.7
60 5.5
130 15
77 9.8
240 34
180 11
H
H
H
H
H
H
H
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
H
H
OCH3
H
H
H
Cl
H
OCH3
H
H
H
Cl
H
H
OCH3
H
H
Cl
H
H
OCH3
H
H
Cl
H
OCH3
H
H
H
H
H
OCH3
OCH3
H
H
H
H
H
OCH3
H
H
H
H
H
OCH3
H
H
H
H
H
OCH3
H
H
H
H
OCH3
OCH3
H
H
H
OCH3
H
OCH3
H
F
H
H
H
OCH3
H
F
H
H
H
OCH3
H
F
H
H
H
OCH3
H
F
H
H
OCH3
OCH3
H
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
H
H
H
H
H
8r
8s
8t
8u
8v
8w
8x
0.23 0.020
0.47 0.067
2.8 0.31
1.5 0.10
3.0 0.17
8y
H
H
H
H
12a
12b
12c
12d
H
H
H
H
H
H
H
1.9 0.43
a
Data taken from Ref. 17 (Zheng et al.).
Data taken from Ref. 18 (Ding et al.).
b
Figure 3. 1,4-Diphenethylpiperidines inhibit [3H]DA uptake at VMAT2 in rat
striatal vesicles. Concentration–response curves for inhibition of [3H]DA uptake
for compounds 8j, 8h and 8m, which exhibited highest affinity for the DA
translocation site on VMAT2. Control (CON) represents [3H]DA uptake in the
absence of compound. Data are mean ( SEM) specific [3H]DA uptake expressed as a
percentage of control (16.1 1.94 pmol/min/mg), n = 11 rats for control condition;
n = 3–4 rats/compound.
Figure 2. 1,4-Diphenethylpiperidines inhibit [3H]DTBZ binding to VMAT2 in rat
brain vesicles. Concentration–response curves for inhibition of [3H]DTBZ binding
for compounds 8j, 8h and 8m. Control (CON) represents [3H]DTBZ binding in the
absence of compound. Data are mean ( SEM) specific [3H]DTBZ binding expressed
as a percentage of control (1250 50 fmol/mg), n = 11 rats for control condition;
n = 3–5 rats/compound.
DTBZ binding site on VMAT2. It should be noted that these com-
pounds contain a 4-(2-methoxyphenethyl) moiety.
were less potent inhibitors of [3H]DA uptake, with Ki values in
the range 40–420 nM and 40–160 nM, respectively. Interestingly,
further analysis of the data for compounds 8a–8y revealed a posi-
tive correlation between affinity for the DTBZ binding and DA
uptake sites on VMAT2 (Spearman r value = 0.443, p <0.05), sug-
gesting that these compounds may inhibit VMAT2 function via
an allosteric interaction with the DTBZ binding site; or alterna-
tively, they may interact with both the binding and substrate
translocation sites on VMAT2.
Evaluation of compounds 8a–8y in the [3H]DA uptake assay
revealed Ki values in the range 9.3–420 nM. The most potent com-
pounds in the series were analogs containing a 4-(2-methoxyphe-
nethyl) moiety (8h–8m). Three compounds in this group exhibited
high affinity (Fig. 3), i.e., Ki values of 9.3 nM (8h), 13 nM (8j) and
13 nM (8m), and were 4 to 5-fold more potent than lobelane
(Ki = 45 nM)15 inhibiting [3H]DA uptake at VMAT2. Analogs incor-
porating a 4-(3-methoxyphenethyl) or a 4-(4-methoxyphenethyl)
moiety (i.e., compounds 8n–8s and 8t–8y, respectively) generally
A subset of compounds (12a–12d) in which the 4-phenethyl
moiety was replaced with a 4-benzyl moiety, also was evaluated
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J. R. Nickell et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
in the [3H]DTBZ binding and [3H]DA uptake assays. While these
Supplementary data
compounds exhibited affinities (Ki = 1.5–3.0
binding site comparable to lobelane (Ki = 0.97
l
M) for the DTBZ
lM), they exhibited
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2016.05.
025.
lower potency than their corresponding 4-phenethyl analogs in the
[3H]DA uptake assay, with Ki values in the range 77–240 nM. In
contrast to the positive correlation between affinity for the DTBZ
binding and DA uptake sites for compounds 8a–8y, Spearman
analysis revealed no correlation for compounds 12a–12d bearing
a 4-benzyl moiety; however, this may be due to the limited
number of compounds in the 4-benzyl series.
In summary, translocation of the phenethyl side chains of
lobelane from the 2,6- to the 1,4-position on the central piperidine
ring has afforded a new scaffold that has been utilized to discover
novel inhibitors of VMAT2 function with inhibitory potency com-
parable to, or greater than that of lobelane. Among the twenty five
1,4-diphenthylpiperidine analogs tested, compounds containing a
1-(2-methoxyphenethyl) moiety exhibited the most potent inhibi-
tion of vesicular DA uptake. From this subgroup, analogs 8h, 8j and
8m exhibited the highest affinity with Ki values of 9.3 nM, 13 nM
and 13 nM, respectively, for inhibition of [3H]DA uptake by VMAT2.
These compounds represent some of the most potent inhibitors of
VMAT2 function reported thus far, and were considered worthy of
further investigation as potential clinical candidates for treatment
of METH abuse.
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This research was supported by NIH (United States) grants
DA13519, TR000117 and GM109005, and an Arkansas Research
Alliance (ARA) (United States) Scholar award. Methods for the
[3H]DTBZ binding assay and [3H]DA uptake assay, and full
characterization data for all synthetic products can be found in
Supporting information.
Please cite this article in press as: Nickell, J. R.; et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.05.025