Synthesis and Discovery of Arylpiperidinylquinazolines: New Inhibitors of the Vesicular Monoamine Transporter

Article abstract of DOI:10.1021/acs.jmedchem.8b00542

Methamphetamine, a human vesicular monoamine transporter 2 (VMAT2) substrate, releases dopamine, serotonin, and norepinephrine from vesicles into the cytosol of presynaptic neurons and induces reverse transport by the monoamine transporters to increase ex

Full text of DOI:10.1021/acs.jmedchem.8b00542

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Article
Synthesis and Discovery of Arylpiperidinylquinazolines:
new inhibitors of the vesicular monoamine transporter
Brian Provencher, Amy J. Eshleman, Robert A Johnson, Xiao Shi, Olga Kryatova,
Jared Nelson, Jianhua Tian, Mario Gonzalez, Peter C Meltzer, and Aaron Janowsky
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00542 • Publication Date (Web): 21 Sep 2018
Downloaded from http://pubs.acs.org on September 22, 2018
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Synthesis and Discovery of
Arylpiperidinylquinazolines: new inhibitors of the
vesicular monoamine transporter
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Brian A. Provencher,a,b,* Amy J. Eshleman,c,d Robert A. Johnson,c Xiao Shi, c,d Olga Kryatova,a
Jared Nelson,a Jianhua Tian,a Mario Gonzalez,a Peter C. Meltzer,a Aaron Janowskyc,d,e
a Organix Inc, 240 Salem St., Woburn, MA, 01801
b Department of Chemistry and Biochemistry, Merrimack College, North Andover, MA 01845
cResearch Service, VA Portland Health Care System
d Departments of Psychiatry and Behavioral Neuroscience, Oregon Health and Science
University,
e The Methamphetamine Abuse Research Center, Oregon Health and Science University,
Portland OR 97239
KEYWORDS APQ, VMAT2, DHTB, reserpine, vesicular transporter
Abstract: Methamphetamine, a human vesicular monoamine transporter 2 (VMAT2) substrate,
releases dopamine, serotonin and norepinephrine from vesicles into the cytosol of presynaptic
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neurons and induces reverse transport by the monoamine transporters to increase extracellular
neurotransmitters. Currently available radioligands for VMAT2 have considerable liabilities:
The binding of [3H]dihydrotetrabenazine ([3H]DHTB) to a site on VMAT2 is not dependent on
ATP and [3H]reserpine binds almost irreversibly to VMAT2. Herein we demonstrate that several
arylpiperidinylquinazolines (APQs) are potent inhibitors of [3H]reserpine binding at recombinant
human VMAT2 expressed in HEKꢀ293 cells. These compounds are biodiastereoselective and
bioenantioselective. The lead radiolabeled APQ is unique because it binds reversibly to VMAT2,
but does not bind the [3H]DHTB binding site. Furthermore, experimentation shows that several
novel APQ ligands have high potency for inhibition of uptake by both HEKꢀVMAT2 cells and
mouse striatal vesicles, and may be useful tools for characterizing drugꢀinduced effects on
human VMAT2 expression and function.
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Introduction
Methamphetamine is a highly addictive stimulant.1 The abuse of methamphetamine is
prevalent and escalates with time for many individuals.2 Stimulantꢀinduced psychosis and longꢀ
term neuronal loss are indications of methamphetamineꢀinduced neurotoxicity.3ꢀ5 Currently,
there are no approved pharmacotherapies for treatment of methamphetamine addiction and
behavioral therapy has had limited success.6
Among the primary initial neuronal targets for methamphetamine is the human vesicular
monoamine transporter 2 (VMAT2).7ꢀ10 Expressed in monoaminergic presynaptic neurons, as
well as in peripheral tissues, VMAT2 transports cytosolic dopamine (DA), serotonin (5ꢀHT) or
norepinephrine (NE) into storage vesicles.11 By transporting neurotransmitters into vesicles,
VMAT2 participates in the regulation of cytosolic levels and vesicular stores of biogenic amines.
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Methamphetamine competes with the endogenous neurotransmitters for binding sites and
uptake into VMAT2. When VMAT2 binds methamphetamine, vesicular degradation resulting in
dopamine efflux into the cytosol of presynaptic neurons is observed.12 This can cause reverse
transport by the dopamine transporter to increase dopamine in the extracellular space. Therefore,
VMAT2 is a possible target for therapeutics to treat methamphetamine abuse.6,8 The VMAT2,
while expressed in vivo on the vesicular membranes of neurons, is also functional when
expressed in mammalian cell lines, with the energy source for transport provided by the native
Vꢀtype H+ ꢀATPase.13
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Recent work reveals that in drosophila brain, amphetamine redistributes vesicle contents,
diminishes the vesicle pHꢀgradient needed for biogenic amine uptake, and requires VMAT2
function.14 Current therapy for methamphetamine abuse consists primarily of cognitive
behavioral interventions, however, pharmacological interventions could improve the treatment of
methamphetamine abuse.
Vocci and Appel, when reviewing possible targets for
methamphetamine pharmacotherapies, identified the VMAT2 as having an obligatory role in
methamphetamine activity.15
Some strategies for pharmacotherapy development have focused on altering psychostimulant
interaction with the VMAT2 or with altering VMAT2 function. Lobeline, a lipophilic alkaloid
of Indian tobacco, alters VMAT2ꢀmediated DA uptake and release and alters methamphetamineꢀ
induced DA release,16,17 and lobeline and its analogs are being investigated as possible
therapeutics for methamphetamine abuse.18ꢀ20 Reserpine, a high affinity VMAT2 blocker, has
been used to treat hypertension, but essentially irreversibly binds to VMAT2 which results in
depletion of biogenic amines including dopamine and epinephrine. Thus, recovery of biogenic
amine storage requires synthesis of new storage vesicles.21 In addition, reserpine binds to both
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the VMAT2 and the peripheral VMAT1, with side effects including sedation, inability to
concentrate or perform complex tasks and sometimes, psychotic depression that appears over
many weeks or months.22,23 Tetrabenazine (TBZ) and its analogue, dihydrotetrabenazine
(DHTB), inhibit VMAT2 function, bind reversibly to the VMAT2 at an allosteric binding site
and have shown effectiveness in treatment of hyperkinesias characterized by abnormal
involuntary movements as observed in Huntington’s disease, Tourette’s syndrome and tardive
dyskinesia.22,24 However, TBZ has side effects similar to reserpine including sedation,
depression, akathisia and Parkinsonism.24 Ketanserin binds to VMAT2 with relatively high
affinity (~6ꢀ45 nM),25 but is nonselective; displaying high affinity for the 5ꢀHT2A receptor, and
moderate affinities for the histamine H1 and 5ꢀHT2C receptors.26ꢀ28
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There are number of radioligands and drugs that bind to the VMAT2 and affect its function.
Currently, available radioligands for labeling VMAT2, such as [3H]DHTB and [3H]reserpine
have considerable liabilities. [3H]DHTB binding is not ATPꢀdependent and thus mechanistically
is less similar to ATPꢀdependent VMAT2 function, and [3H]reserpine binds almost irreversibly
to VMAT2 and does not lend itself to highꢀthroughput screening.29,30 One model of the binding
sites on VMAT2 suggests that the reserpine site has high affinity for substrates and is directed
toward the cytosol; in contrast, after the binding of the substrate to VMAT2, a conformational
change results in the TBZꢀbinding conformation.31 Furthermore, it was found that TBZ does not
require a proton gradient for binding and that TBZ binding locks the transporter into a
confirmation which no longer allows the substrate to bind to VMAT2 by inhibiting the
cytoplasmic gate.32
With few available radioligands, the search for novel ligands to serve as chemical probes that
bind reversibly and specifically to the VMAT2, and are associated with VMAT2 function, can
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provide a springboard for the development of pharmacotherapies for symptoms related to
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methamphetamine
addiction.
Herein
we
describe
the
discovery
of
new
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arylpiperidinylquinazoline (APQ) ligands and their biological activity in VMAT2 assays.
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Chemistry
Novel APQ ligands (±)-5 were synthesized from commercially available ethyl 1ꢀbenzylꢀ4ꢀoxoꢀ
3ꢀpiperidinecarboxylate hydrochloride 1. Conversion from the ketone to triflate 2 was
accomplished using Nꢀphenyl bistrifluorsulfonamide. Suzuki coupling followed by reduction
afforded substituted arylꢀpiperidine (4). NꢀAlkylation with the known quinazoline 6 afforded
racemic, synꢀAPQ ligands (±)-5.33 To ensure water solubility, the APQs were converted to the
HCl salt with HCl in ether. The precipitate was filtered, dissolved in water and freeze dried to
afford the APQꢀHCl salt which was confirmed by elemental analysis. Enantiomers could be
resolved by crystalizing 4 with tartaric acid. Once purified, single enantiomers were subjected to
the same Nꢀalkylation and salt procedure as stated above.
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AntiꢀAPQ isomer 9, was prepared from intermediate 4b. Protection of the free amine with Boc
anhydride yielded 7. Heating 7 in the presence of sodium ethoxide converted the cisꢀ
diastereomer to the more stable transꢀdiastereomer. Deprotection followed by Nꢀalkylation with
the known quinazoline afforded (±)ꢀ9.
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Results and Discussion
To begin, the binding affinities of 13 APQ derivatives and reference compounds (ketanserin,
DHTB, methamphetamine, reserpine, Ro4ꢀ1284, serotonin, and lobeline) for VMAT2 were
assessed by inhibition of radioligand binding using [3H]reserpine and [3H]DHTB (Table 1).
Methamphetamine had micromolar affinity for the reserpine binding site but had 60 fold lower
affinity for the DHTB site. Ketanserin displays some affinity for the reserpine binding site
albeit fiveꢀfold lower than for the DHTB binding site (Table 1, Entry 1). Interestingly, APQs
displayed a wide range of binding affinities across both sites and were generally more selective
for the reserpine site. SynꢀAPQ (±)-5a bound to the reserpine site with a Ki=70 nM (Table 1,
entry 9). The paraꢀfluoro analog (±)-5b, which contains the same aryl moiety of ketanserin, was
found to bind with essentially the same Ki at the reserpine site (74 vs 70 nM). Neither compound
showed much affinity for the DHTB site. To explore the diastereoselectivity of the APQ ligand
on the receptors, antiꢀisomer (±)-9 was tested. APQ ligand 9 had less affinity for the reserpine
site than the synꢀanalog, (±)-5b. Additionally, (±)-9 was not as selective for the reserpine site
and competed at the DHTB site with a Ki=750 nM.
TABLE 1. Binding affinities for the VMAT2 binding sites of [3H]Reserpine and [3H]DHTBa
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a
The core APQ structure is given in Schemes 1 and 2. Experiments were conducted as detailed in the
methods. N ≥ 3, except when the Ki was greater than 10 µM, in which case n=2. If some experiments
yielded IC50 or Ki values less than 10 ꢀM and other experiments yielded IC50 or Ki values greater than 10
ꢀM, the latter experiments were assigned a value of 10 ꢀM and averages calculated. The actual value is
greater than that average and no standard error is reported. APQ compounds were tested at
concentrations ranging from 1 nM to 10 µM. Data are provided as Ki= nM ± sem unless otherwise
noted.
With the knowledge that the synꢀisomers (5) were more selective for reserpine over DHTB, we
explored the structureꢀactivity relationship of the APQ ligands. Compound (±)ꢀ5c lost affinity for
the reserpine site while 3ꢀtrifluorophenyl analog (±)ꢀ5d retained some affinity for the reserpine
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site albeit, much lower than (±)ꢀ5b. Other analogs in this series, did not bind at the reserpine or
DHTB sites (Table 1, entries 15ꢀ18). Thiophene analog, (±)ꢀ5i and dioxane analog, (±)ꢀ5j were
found to bind with moderate affinity to the reserpine site Ki= 450 and 652 nM respectively.
To explore the enantioselectivity of the ligands, (+)ꢀ5b and (ꢀ)ꢀ5b were isolated and subjected
to the reserpine and DHTB binding assays. (+)ꢀ5b had higher affinity than (±)ꢀ5b in the
reserpine binding assay with a Ki=62 nM (p<0.001, one way ANOVA followed by Tukey’s
multiple comparison test) and was essentially inactive in the DHTB binding assay. (ꢀ)ꢀ5b
displayed similar affinity as racemic (±)ꢀ5b (p>0.05) but lower affinity than the enantiomer (+)-
5b (p<0.01) (Ki = 179 nM) for the reserpine site while remaining inactive in the DHTB binding
assay. To further study the effect of the APQ ligand on VMAT2, compound (+)ꢀ5b, with the
highest affinity for the reserpine site, was selected for tritium radiolabeling.
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Figure 1: Effects of MgꢁATP on (a) [3H]reserpine, (b) [3H](+)ꢁ5b and (c) [3H]DHTB
The effects of MgꢁATP on radioligand binding were conducted as described in the methods.
Error bars (SEMs) were derived from multiple experiments conducted with duplicate
determinations.
The effect of MgꢀATP on radiolabeled [3H](+)-5b binding was determined. The binding of
[3H]reserpine and [3H](+)-5b, to VMAT2 was ATPꢀdependent. MgꢀATP (2 mM) enhanced
specific binding to recombinant VMAT2 approximately 8 and 15 fold for [3H]reserpine and
[3H](+)-5b, respectively (Fig 1A, 1B). In contrast, the binding of [3H]DHTB was not changed
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by the addition of 2 mM ATP (Fig 1C). Thus, ATP increases [3H](+)-5b and [3H]reserpine
binding, but not [3H]DHTB binding. The ATP enhancement of [3H](+)-5b binding suggests that
the binding is energyꢀdependent and may also be dependent on one or more proton(s) being
counter transported. ATP is required for the proton pump that regulates the vesicleꢀcytoplasm
protonꢀgradient that is required for substrate transport by various vesicular transporters.34
Additionally, this finding corroborates data suggesting that reserpine, and the APQs, but not
DHTB, bind to a site that is coupled to VMAT2 function.32
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Figure 2: Kinetics of [3H]reserpine and [3H](+)ꢁ5b binding to human VMAT2. (A) and (B)
[3H]Reserpine association and dissociation time curves, respectively. (C) and (D) [3H](+)ꢁ5b
association and dissociation time curves, respectively. Note the different X axis time ranges in
(B) and (D). Assays were conducted as described in Materials and Methods, with excess
concentration of respective nonꢁradiolabeled ligand.
conducted in duplicate.
n = 3ꢁ5 independent experiments
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The rates of association and dissociation for [3H](+)ꢀ5b and [3H]reserpine binding were
determined in the presence of 2 mM MgꢀATP. Kinetic analysis of [3H]reserpine (14ꢀ22 nM)
and [3H](+)ꢀ5b (24ꢀ28 nM) binding to VMAT2 indicates that the observed association rates
(kobs) were similar (0.028 ± 0.01, 0.051 ± 0.01, respectively, n=5, Fig 2A, 2B). By contrast, the
dissociation halfꢀlives for [3H]reserpine and [3H](+)ꢀ5b are significantly different (p < 0.05),
with [3H]reserpine having a dissociation halfꢀlife greater than 3 hrs. (data did not fit a one phase
exponential decay) and [3H](+)ꢀ5b having a dissociation halfꢀlife of less than 1 min, respectively
(Fig 2C, 2D). The kinetic characteristics of [3H](+)ꢀ5b binding to VMAT2 are quite unlike
[3H]reserpine, which binds essentially irreversibly (Fig. 2). Saturation binding of [3H]reserpine,
[3H](+)ꢀ5b and [3H]DHTB binding was also conducted, with the caveat that the nearly
irreversible [3H]reserpine binding does not obey the reversibility requirement for true
equilibrium. Saturation binding of [3H]reserpine, [3H](+)ꢀ5b and [3H]DHTB yielded Kd values
of 8.0 ± 1.2 nM, 93 ± 13 nM, and 71 ± 15 nM and Bmax values of 0.942 ± 0.085, 13.5 ± 1.4, and
41.2 ± 2.2 pmol/mg protein, respectively (Fig 3A, 3B, 3C). [3H]Reserpine binding to VMAT2
had a higher affinity but much lower density of binding sites compared to the other two ligands.
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Figure 3: Saturation binding of [3H]Reserpine, [3H](+)-5b and [3H]DHTB binding to HEKꢁ
VMAT2 cell membranes. (A) [3H]Reserpine, (B) [3H](+)-5b and (C) [3H]DHTB saturation
binding. Assays were conducted as described in Materials and Methods. Saturation curves are
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the average and SEM of 3ꢁ4 independent experiments each conducted in duplicate. A
representative Scatchard plot is shown for each radioligand. In the Scatchard analysis, the (ꢁ)
reciprocal of the slope of the line is an estimate of the Kd value for the radioligand.
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The fast off rate of [3H](+)ꢀ5b, compared to the offꢀrate for [3H]reserpine indicates that
binding is reversible, an important characteristic for screening possible therapeutics with high
throughput radioligand binding. Likewise, [3H]DHTB binding is reversible,35 with a relatively
fast offꢀrate, but the binding characteristics are not ATP dependent.
While APQ ligands did not appear to bind with great affinity for the reserpine or DHTB
binding sites, further experimentation with (+)ꢀ5b showed evidence that the ligands were binding
and affecting VMAT2 function. We lastly, screened the APQ ligands in a binding assay
competing with [3H](+)ꢀ5b (Table 2). Enantiomeric APQ ligand (ꢀ)ꢀ5b displayed moderate
affinity in this assay with a Ki=290 nM whereas the racemate, (±)ꢀ5b, was found to bind with a
Ki=161 nM.
Table 2: Binding affinities of APQ Ligands versus [3H](+)ꢀ5b at the human VMAT2, versus
[125I]DOI at the 5HT2A receptor and versus [125I]RTIꢀ55 at the DAT, and potencies for
inhibition of [3H]5ꢀHT uptake by the human VMAT2 expressed in HEK cells and by mouse
striatal VMAT2.a
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Mouse striatal
Human VMAT2
HEK-VMAT2
[3H]5HT Uptakeb
5HT2A
125I]DOI
hDAT
[
125I]RTI-55
Entry Compound
vesicular VMAT2
[3H]5HT Uptakeb
3
(+)-5b
[ H]
[
1
2
3
4
5
6
7
8
9
10
Ketanserin
DHTB
7
8
9
1190 210
>10
180 39
28.1 4.4
3620 560
13.2 1.5
154 31
100 35
19.80 0.46
2740 340
1.79 0.39
49.56 0.79
98 17
10.7 2.5
6.7 1.4
M
Methamphetamine
Reserpine
Ro4-1284
5-HT
24.5 3.1
410 100
1932 73
2290 330
1950 400
1150 280
287 46
101 14
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>1
M
>10
M
1430 320
4200 990
85.5 9.8
19.7 6.4
39.4 5.6
16.0 3.3
51.1 7.9
2900 1300
125 18
11.5 2.0
7400 1500
37.3 8.5
lobeline
381 56
24.7 6.3
9.1 3.1
3.46 0.95
3.39 0.95
6.6 2.2
ND
( )-9
>9.3
M
( )-5a, Ar= C6H5
>10
M
1610 180
4170 610
2440 390
9900 1800
( )-5b
, Ar= 4-FC6H4
161 70
93 13c
2380 600
11 (+)-5b
>10
>10
>10
>10
>10
>10
>10
>10
M
M
M
M
M
M
M
M
(-)-5b
( )-5c
12
13
14
15
16
17
18
19
290 110
720 120
350 21
, Ar=4-CF3-C6H4
>10
>10
M
M
( )-5d, Ar=3-CF3-C6H4
12.8 4.5
ND
( )-5e
, Ar=4-CH3-C6H4
( )-5f, Ar=4-NMe2-C6H4
( )-5g
452 33
2700 1300
5800 1000
7300 2100
1960 390
3770 210
ND
>10
>10
>10
>7.1
M
M
M
M
, Ar=4-Ph-C6H4
>10
>10
M
M
ND
( )-5h, Ar=4-CO2Et-C6H4 1660 120
ND
>9300
( )-5i
, Ar= 2-thienyl
900 230
101 17
31.5 6.4
O
O
20
( )-5j
, Ar=
700 220
142 36
ND
>10
M
>10
M
a Experiments were conducted as detailed in the methods. N ≥ 3, except when the Ki or IC50 was greater
than 10 µM, in which case n=2. If some experiments yielded IC50 or Ki values less than 10 ꢀM and other
experiments yielded IC50 or Ki values greater than 10 ꢀM, the latter experiments were assigned a value of
10 ꢀM and averages calculated. The actual value is greater than that average and no standard error is
reported. APQ compounds were tested at concentrations ranging from 0.01 nM to 10 µM. Data is
reported as Ki= nM ± sem unless otherwise noted. b IC50 = nM ± sem Kd= nM ± sem. ND= Not
c
Determined
The other racemic APQ ligands were tested against [3H](+)ꢀ5b as well. APQ ligand (±)ꢀ5a,
displayed moderate binding affinity albeit only two times less potent than (±)ꢀ5b. Other APQ
ligands with electron withdrawing substituents displayed moderate binding affinity with (±)ꢀ5d
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showing the best results out of the group (entries 13, 14 and 18; Table 2). SAR investigations
into electron donating groups also resulted in loss of potency in the [3H](±)ꢀ5b binding assay
(entries 15ꢀ17; Table 2).
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Figure 4: Concentrationꢁresponse curves of inhibition of [3H]5ꢁHT uptake into mouse striatal
vesicular membranes. Six APQ compounds and three standards are shown. N=3ꢁ6. All APQ
compounds were more potent than ketanserin in this assay.
The potency of the APQ compounds at inhibition of [3H]5ꢀHT uptake via human VMAT2
expressed in HEK cells was assessed. APQ ligands (±)ꢀ5a and all stereoisomers of 5b had
similar potencies, ranging from 16 to 51 nM (Table 2). The next most potent compounds ((±)ꢀ
5d, (±)ꢀ5i, (±)ꢀ5j) had potencies ranging from 101 nM to 142 nM. Interestingly, APQ (±)ꢀ5d
with a metaꢀtrifluormethyl group, had greater than 20 fold higher potency than (±)ꢀ5c with a
paraꢀtrifluormethyl. APQ ligands (±)ꢀ5e, (±)ꢀ5f, (±)ꢀ5g and (±)ꢀ5h demonstrated little potency in
the [3H]5ꢀHT uptake assay. The potencies of a subset of APQ compounds and standards for
inhibition of [3H]5ꢀHT uptake via endogenous VMAT2 in mouse striatal vesicular membranes
were assessed (Table 2, Figure 4). The potencies of all standard compounds were higher in the
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mouse vesicular preparations (Table 2). The subset of APQ ligands tested in this preparation had
higher potencies compared to potencies in the HEKꢀVMAT2 uptake assay. APQ ligands (±)ꢀ5a
and all stereoisomers of 5b were very potent, with IC50 values below 10 nM (Table 2). (±)ꢀ5d,
(+)ꢀ9, and (±)ꢀ5i had moderate potencies ranging from 12.8 to 31.5 nM. All APQ compounds
tested completely inhibited specific [3H]5ꢀHT uptake, defined with 1 µM reserpine.
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Figure 5: Correlations of binding affinities and uptake potencies for HEKꢁVMAT2 for select
compounds. Drugꢁinduced inhibition of (A) log (Ki) for inhibition of [3H](+)ꢁ5b binding versus
log (IC50) for inhibition of [3H]5ꢁHT uptake, (B) log (Ki) for inhibition of [3H]reserpine binding
versus log (IC50) for [3H]5ꢁHT uptake (C) log (Ki) for inhibition of [3H]DHTB binding versus
log (IC50) for [3H]5ꢁHT uptake. 14 APQ compounds and 7 standard compounds were included
in the analysis. Data were analyzed using Spearman’s nonparametric correlation analysis.
For the 14 APQ compounds and the seven standard compounds, a good correlation was
observed between the log IC50 value for [3H]5ꢀHT uptake and the log Ki value for inhibition of
[3H](+)ꢀ5b binding (Fig 5A, Spearman’s r= 0.63, p<0.01). With this same set of compounds,
there was an excellent correlation between the log IC50 value for [3H]5ꢀHT uptake and the log Ki
value for inhibition of [3H]reserpine binding (Fig 5B, Spearman’s r= 0.90, p<0.0001). There
was also a good correlation between the log IC50 value for [3H]5ꢀHT uptake and the log Ki value
for inhibition of [3H]DHTB binding (Fig 5C, Spearman’s r= 0.57, p<0.01), although the
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correlation was driven mainly by the control compounds, as the APQs generally had micromolar
to no measurable affinity for this site.
7
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9
Affinities for several other neuronal proteins were assessed (Table 2). At the 5ꢀHT2A
receptor, (±)ꢀ9 had nanomolar affinity similar to ketanserin. All other APQ ligands had little to
no measurable affinity for this receptor. Furthermore, (+)ꢀ5b and (±)ꢀ5a had Ki values of 2440
nM and 1610 nM, respectively, for displacement of [125I]RTIꢀ55 binding to the DAT; all other
APQs tested had Ki values greater than 4 µM (Table 2). Assessing the affinity of the lead
compound for other transporters, (+)ꢀ5b and the parent compound ketanserin had very low
affinities (>10 µM) for the [125I]RTIꢀ55 binding site on the human serotonin transporter and
human norepinephrine transporter while cocaine had Ki values of 632 ± 21 and 2,357 ± 39 nM,
respectively. No APQs had measurable affinity for 5ꢀHT1A receptor (data not shown).
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Figure 6: Effects of APQ compounds, standards and staurosporine (SP) on cell viability in the
MTT assay. APQ compounds, RO4ꢁ1284, ketanserin, METH and DHTB were tested at 10 µM,
and reserpine was tested at 1 µM. The positive control SP was tested at 0.1, 0.3, 1 and 3 µM.
Inset: test for linearity of HEKꢁhVMAT2 cell number vs OD at 590 nm.
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In addition, a subset of APQs was tested in the MTT cell viability assay. (±)ꢀ5a, (±)5b, (+)ꢀ5b,
(ꢀ)ꢀ5b, (±)ꢀ5d, (±)ꢀ5i, (±)ꢀ9, ketanserin, DHTB, RO4ꢀ1284, and methamphetamine at 10 µM and
reserpine at 1 µM had no significant effect on HEKꢀVMAT2 cell viability (one way ANOVA
followed by Tukey’s posthoc test, Figure 6). By contrast, staurosporine, the positive control,
decreased cell viability by 45% and 60% at 1 and 3 µM, respectively.
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Lastly, CNS MPO scores were calculated for the APQ compounds to determine viability of the
ligands moving forward.36,37 Ligands scored from 2.2 to 4.4 in the MPO calculation.38 Generally
scores greater than 4 are desired and of note, (±)ꢀ5b displayed one of the best MPO scores of 4.1.
The moderate MPO values obtained for the APQ ligands lends credence to the viability of APQs
as potential CNS therapeutics. The CNS MPO scores will help guide future investigations of the
APQ ligands.
There are numerous radioligands and drugs that bind to the VMAT2 and affect its function.
The results presented here suggest that the novel APQ compounds bind to a new, heretofore
unidentified site on the VMAT2. This site is associated with the region of VMAT2 involved in
transport activity, a claim supported by the correlation between binding affinity for this site and
inhibition of [3H]5ꢀHT uptake (Fig 4). The modifications of the ketanserin core structure have
eliminated affinity for the 5ꢀHT2A receptor and increased selectivity for the [3H]ꢀreserpine site
for most of the compounds reported. In addition, there is very low affinity for the DAT, an offꢀ
target site for some compounds that bind to the VMAT2, such as lobeline.39 The low affinities of
the compounds at the two offꢀtarget sites indicate that the APQs have high specificity for
VMAT2 compared to ketanserin, a ligand for VMAT2, as well as for 5HT receptors. Whether
the APQs also lack affinity for other receptors and transporters has not been determined. An
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important question for additional studies is whether the APQs interact with the vesicular
monoamine transporter 1 (VMAT1), expressed in the intestine, stomach and sympathetic
nervous system.40 Ketanserin has slightly higher potency for inhibition of transport by VMAT2
than VMAT1.41 An interesting finding is that the structural changes made to ketanserin have
modified the binding preference for the DHTB binding site; APQ ligands to prefer to bind to the
reserpine site. Additional detailed structureꢀactivity studies using APQs with varying affinities
for VMAT2 may help to characterize the relationships between radioligand binding sites and
VMAT2 function, an important precursor to the development of pharmacotherapeutic
compounds.
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Experimental
General Information and Materials. All reactions were magnetically stirred and monitored by
analytical thinꢀlayer chromatography (TLC) Silica gel 60 F254 plates using UV light to visualize
the compounds. Column chromatography was carried out on Teledyne ISCO Combiflash
automated chromatography units using standard Silica Gel cartridges. 1H and 13C NMR spectra
were recorded on a JEOL Eclipse 300 MHz spectrometer using tetramethylsilane (TMS) as an
internal reference. All target compounds were determined to be >95% pure by HPLC/MS
analysis using an Agilent 1200 HPLC apparatus with an ESI Mass Spectrum. Melting points
were obtained using a MelꢀTemp capillary meltingꢀpoint apparatus and are uncorrected.
Reagents and solvents were purchased from commercial suppliers and used without further
purification unless otherwise noted.
Ethyl 1-benzyl-4-(trifluoromethylsulfonyloxy)-1,2,5,6-tetrahydropyridine-3-carboxylate
(2). To a suspension of ethyl 1ꢀbenzylꢀ4ꢀoxoꢀ3ꢀpiperidinecarboxylate hydrochloride (3.32 g, 11.1
mmol) in H2O (30 mL) was added potassium carbonate (2.76 g, 20.0 mmol), and the mixture was
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extracted with CH2Cl2 (3 x 40 mL). The combined organic layers were dried (Na2SO4), filtered,
and concentrated to give the free base (100%) as an oil which was used without further
purification. This ketoꢀester was dissolved in anhydrous DMF (20 mL), fitted with a septum,
nitrogen inlet and cooled to 0 ºC. NaH (60% in mineral oil, 668 mg, 16.7 mmol) was added in
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one portion, and the solution was stirred at
0
ºC for 10 min. Nꢀphenylꢀ
bis(trifluoromethanesulfonimide) (4.36 g, 12.2 mmol) was added in one portion, and the solution
was stirred at 0 ºC for 1.5 h. Water (50 mL) was then added and the resulting mixture was
extracted with EtOAc (3 x 50 mL). The combined organic layers were washed with H2O (20
mL), dried (Na2SO4), filtered, and concentrated. The residue was purified by flash
1
chromatography eluting with 0ꢀ15% EtOAc/hexanes to give 2 (3.60 g, 83%) as a yellow oil: H
NMR (300 MHz, CDCl3) δ 7.34ꢀ7.25 (comp, 5H), 4.27 (q, J = 7.2 Hz, 2H), 3.67 (s, 2H), 3.42 (t,
J = 2.7 Hz, 2H), 2.71 (t, J = 5.4 Hz, 2H), 2.53ꢀ2.49 (m, 2H), 1.31 (t, J = 7.2 Hz, 3H).
General procedure for the synthesis Ethyl 1-benzyl-4-(aryl)-1,2,5,6-tetrahydropyridine-3-
carboxylate (3): A 250 mL round bottom flask was charged with triflate 2 (6.9 mmol), boronic
acid (15.1 mmol), PdCl2(PPh3)2 (0.21 mmol), LiCl (20.6 mmol), and K2CO3 (41.3 mmol). The
flask was evacuated and backꢀflushed with argon three times, anhydrous dioxane (100 mL) was
added, and the flask was heated to 95 °C overnight. The flask was cooled to room temperature
and CHCl3 (500 mL) was added. The mixture was then washed with water (2 x 150 mL), brine
(150 mL), dried (MgSO4), and concentrated. The residue was purified by flash chromatography
eluting with 0ꢀ30% EtOAc/hexanes to give pure 3.
General procedure for the synthesis of (±)-syn-Ethyl 4-arylpiperidine-3-carboxylate (4):
To a 50 mL round bottom flask was added 10% Pd/C (225 mg, 0.10 mmol, 50% wet) followed
by the addition of a solution of 3 (1 mmol) in EtOH (10 mL). The flask was evacuated and
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backꢀflushed with H2 three times, and the reaction mixture was stirred under a static atmosphere
of H2 at room temperature (unless otherwise noted) for 24ꢀ72 hrs. The suspension was filtered
through a pad of celite, washing with EtOH. The combined filtrate and washings were
concentrated and purified by flash chromatography eluting with 0ꢀ10% MeOH/EtOAc (1% Et3N)
to give pure 4.
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Synthesis of (±)-syn-Ethyl 1-(2-(2,4-dioxo-1,2-dihydroquinazolin-3(4H)-yl)ethyl)-4-
arylpiperidine-3-carboxylate (5): A 25 mL highꢀpressure tube was charged with 4 (0.4 mmol),
633 (0.42 mmol), and toluene or CH3CN (3 mL). The tube was sealed and heated to 110 ºC for 2
days. The reaction mixture was concentrated and the residue was purified by flash
chromatography eluting with 0ꢀ100% CH2Cl2/EtOAc [Rf ≈ 0.3 (50% CH2Cl2/EtOAc)] to give 5
as an offꢀwhite solid. The ligands were then converted to their hydrochloride salts prior to
biological testing. Compounds were dissolved in CHCl3 (1 ml) and 1 N HCl (2 eq) was then
added and the solution was stirred for 5 minutes. The solvent was removed under reduced
pressure and the crude solid was dissolved in water. After filtration and removal of the solvent
by lyophilization, pure salts could be obtained in good yields.
Synthesis of tritium labeled [3H]-(+)-syn-ethyl 1-(2-(2,4-dioxo-1,2-dihydroquinazolin-
3(4H)-yl) ethyl)-4-(4-fluoro phenyl)piperidine-3-carboxylate ([3H]-5b): The title compound
was prepared via direct tritiation of 5b by Morevek Bochemical. Specific Activity: 12.8
Ci/mmol, Radiochemical purity: 99.8%.
Synthesis of (±)-anti-Ethyl 1-tert-butyl 4-(4-fluorophenyl)piperidine-1,3-dicarboxylate
(7): A 50 mL round bottom flask was charged with 4b(215 mg, 0.86 mmol), MeOH (5mL),
triethylamine (240 uL, 1.72 mmol) and a magnetic stir bar. Boc2O (375 mg, 1.72 mmol) was
added with vigorous stirring and the mixture was heated to 60 ºC for 30 min. Volatiles were
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removed in vacuo and the residue was partitioned between CH2Cl2 (15 mL) and brine (5 mL).
The layers were separated, and the aqueous layer was further extracted with CH2Cl2 (2 x 10 mL).
The combined organic layers were dried (MgSO4), filtered, and concentrated, then purified by
flash chromatography eluting with 0ꢀ100% EtOAc/hexanes to give boc protected-4b (214 mg,
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1
71%): Rf = 0.75 (50% EtOAc/hexanes, 1% Et3N); H NMR (300 MHz, CDCl3) δ 7.19 (dd, J =
5.5, 8.8 Hz, 1H), 6.95 (t, J = 8.8 Hz, 2H), 4.09 ꢀ 4.64 (m, 2H), 3.92 (q, J = 7.2 Hz, 2H), 3.01 ꢀ
3.29 (m, 1H), 2.71 ꢀ 3.02 (m, 2H), 2.48 ꢀ 2.71 (m, 1H), 1.54 ꢀ 1.85 (m, 2H), 1.47 (s, 9H), 1.04 (t,
13
J = 7.2 Hz, 3H); C NMR (75 MHz, CDCl3) δ 171.6, 163.3, 160.0, 154.5, 138.3, 129.1, 129.0,
115.2, 115.0, 79.7, 60.2, 49.1, 45.7, 43.6, 42.5, 28.5, 25.9, 14.1.
A 50 mL round bottom flask was charged with the previous product (194 mg, 0.55 mmol),
EtOH (5 mL), and NaOEt (56 mg, 0.83 mmol), then heated to 70 ºC for 3 h in an oil bath. The
solution was concentrated in vacuo and purified by flash chromatography eluting with 0ꢀ30%
1
EtOAc/hexanes to give pure 7. (137 mg, 71%): Rf = 0.87 (50% EtOAc/hexanes, +1% Et3N); H
NMR (300 MHz, CDCl3) δ 7.13 (dd, J = 5.2, 8.8 Hz, 2H), 6.96 (t, J = 8.8 Hz, 2H), 4.35 (br. s,
1H), 4.24 (br. s., 1H), 3.89 (q, J = 7.2 Hz, 2H), 2.72 ꢀ 3.04 (m, 3H), 2.47 ꢀ 2.71 (m, 1H), 1.69 ꢀ
13
1.87 (m, 1H), 1.56 ꢀ 1.69 (m, H), 1.48 (s, 9H), 0.95 (t, J = 7.2 Hz, 3H); C NMR (75 MHz,
CDCl3) δ 172.5, 163.4, 160.2, 154.5, 138.6, 129.0, 128.9, 115.5, 115.2, 80.2, 60.4, 49.1, 46.3,
45.1, 44.1, 32.9, 28.5, 14.0.
Synthesis of (±)-anti Ethyl 4-(4-fluorophenyl)piperidine-3-carboxylate (8): Compound 7
(126 mg, 0.36 mmol) was taken up in HCl (4M in dioxane, 6.3 mL) under N2. The solution was
stirred 45 min, and neutralized with solid NaHCO3 (4.60 g). The reaction mixture was diluted
with water (20 mL), and extracted with CH2Cl2 (3 x 20 mL). The combined organic layers were
dried (MgSO4), concentrated. The residue was purified by flash chromatography eluting with 0ꢀ
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10%MeOH/CH2Cl2 (1% Et3N) to give 9 (90 mg, 100%); 1H NMR (300 MHz, CDCl3) δ 7.15 (dd,
J = 5.2, 8.8 Hz, 2H), 6.94 (t, J = 8.8 Hz, 2H), 3.87 (q, J = 7.2 Hz, 2H), 3.33 (dd, J = 3.6, 11.8 Hz,
1H), 3.10 ꢀ 3.25 (m, 1H), 2.62 ꢀ 2.95 (m, 4H), 2.25 (br. s, 1H), 1.74 ꢀ 1.92 (m, 1H), 1.62 (dq, J =
4.1, 12.6 Hz, 1H), 0.95 (t, J = 7.2 Hz, 3H).
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(±)-anti-Ethyl-1-(2-(2,4-dioxo-1,2-dihydroquinazolin-3(4H)-yl)ethyl)-4-(4-fluorophenyl)
piperidine-3-carboxylate (9). The title compound was prepared from 8 and 6 as an offꢀwhite
solid (68 mg, 47%) using the general procedure described above for the synthesis of 5. mp =
195.0ꢀ197.0 ºC; Rf = 0.54 (10% MeOH/CH2Cl2); 1H NMR (300 MHz, DMSOꢀd6) δ 11.47 (s,
1H), 7.94 (dd, J = 1.1, 8.0 Hz, 1H), 7.66 (ddd, J = 1.4, 7.4, 8.0 Hz, 1H), 7.14 ꢀ 7.30 (m, 4H), 7.07
(t, J = 8.8 Hz, 2H), 4.04 (t, J = 6.9 Hz, 2H), 3.82 (qd, J = 1.7, 7.2 Hz, 2H), 3.18 (d, J = 10.7 Hz,
1H), 3.02 (d, J = 10.7 Hz, 1H), 2.65 ꢀ 2.78 (m, 2H), 2.60 (t, J = 6.9 Hz, 2H), 2.07 ꢀ 2.27 (m, 2H),
1.51 ꢀ 1.75 (m, 2H), 0.87 (t, J = 7.2 Hz, 3H); 13C NMR (75 MHz, DMSOꢀd6) δ 172.9, 163.0,
162.5, 159.8, 150.7, 140.2, 140.0, 135.6, 129.7, 129.6, 127.9, 123.1, 115.7, 115.6, 115.3, 114.3,
60.1, 56.2, 55.2, 53.9, 49.1, 44.7, 38.0, 33.3, 14.3; Anal. Calcd for C24H26N3O4F: C, 65.59; H,
5.96; N, 9.56; F, 4.32; Found: C, 65.52; H, 6.09; N, 9.42; F, 4.24; MS (APCI, [M+H]+, m/z)
440.2.
Cell culture. The human VMAT2 cDNA was generously supplied by Dr. Robert Edwards
(University of CA, San Francisco, CA) and subcloned into pcDNA3.1 with G418 resistance.
HEKꢀ293 cells were transfected with the human VMAT2 and characterized as described
previously.16,41 Cells were maintained in Dulbecco's modified Eagle's medium (DMEM)
supplemented with 10% FetalClone and 0.05 U of penicillin/streptomycin. Stock plates were
grown on 150 mm diameter tissue culture dishes in 10% CO2 at 37°C.
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[3H](+)-5b binding Assay. Conditions were optimized for [3H](+)-5b binding and [3H](+)-5b
binding to the human VMAT2 in HEKꢀVMAT2 cells was performed as follows. Cells were
grown to confluence and washed with Tris buffer (25 mM, pH 7.4). Cells were scraped from
plates in 5 ml Tris buffer, resuspended, and centrifuged at 30,900g for 20 min at 4°C. The
resulting pellet was resuspended in 2 ml of H2O and homogenized with a Polytron homogenizer
at setting 6 for 5ꢀ10 s. After 10 min incubation on ice, osmolarity was reestablished with the
addition of 338 ꢁl of 250 mM Tris (pH 7.4), 338 ꢁl of 1 M potassium tartrate, and 10 ꢁl of 0.9 M
MgSO4. The binding assays contained approximately 50 to 75 ꢁg membrane protein (less than
15% of the radioactivity added was bound) and [3H](+)-5b (50 ꢀ 100 nM final concentration) in a
final volume of 250 ꢁl. Tris assay buffer [25 mM Tris, 5 mM KCl, 2.5 mM MgSO4, 0.32 M
sucrose, 2 mM MgꢀATP, pH 7.4] was used for all assays, except as indicated. Specific binding
was defined as the difference in binding observed in the absence and presence of 10 ꢁM Oꢀ7443
(an analog of (+)-5b). The membranes were incubated for 60 min (equilibrium) at 4oC in the
dark, except where indicated. Assays were terminated by filtration through Filtermat A filters
(Perkin Elmer) using a 96ꢀwell Tomtec cell harvester. Scintillation fluid (50 ꢁl) was added to
each filtered spot, and the radioactivity remaining on the filters was determined using a Wallac
1205 Beta plate scintillation counter. Saturation binding experiments were conducted with
duplicate determinations at each ligand concentration, which included radiolabeled and nonꢀ
radiolabeled ligand for final concentrations ranging from 5 to 600 nM for (+)-5b. Protein
concentrations were determined using a modified bicinchoninic acid (BCA) protein assay.
[3H]Reserpine binding assay. The [3H]reserpine binding assay was modified from the
literature procedure.42 [3H]Reserpine binding to the human VMAT2 in HEKꢀVMAT2 cells was
performed as follows. Membranes were prepared as for [3H](+)-5b binding (above). The assays
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contained approximately 200ꢀ300 ꢁg of membrane protein for [3H]reserpine binding (less than
15% of the radioactivity added was bound) and [3H]reserpine (6 ꢀ 8 nM final concentration) in a
final volume of 1 ml. Tris assay buffer was used for all assays, except as indicated. Specific
binding was defined as the difference in binding observed in the absence and presence of 1 ꢁM
reserpine. The membranes were incubated for 60 min (equilibrium) at 30oC. Assays were
terminated by addition of 1ml iceꢀcold Tris buffer, containing 2 µM nonꢀradiolabeled reserpine
and filtration over Whatman 25 mm GF/C filters presoaked in 0.05% polyethylenimine through a
12ꢀchannel Millipore filtration chamber. Filters were washed three times with 10 ml iceꢀcold Tris
buffer. Remaining radioactivity on the filters was counted on a Beckman LS6500 scintillation
counter. Saturation binding experiments were conducted with duplicate determinations at each
ligand concentration, which included radiolabeled and nonꢀradiolabeled ligand for final
concentrations ranging from 1 to 22 nM for Reserpine. Protein concentrations were determined
using a modified BCA protein assay.
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[3H]DHTB binding assay. [3H]DHTB binding to the human VMAT2 in HEKꢀVMAT2 cells
was performed as described previously, with minor modifications.42 Cells were grown to
confluence and washed with calciumꢀmagnesiumꢀfree phosphateꢀbuffered saline (cmfꢀPBS).
Cells were scraped from plates in 0.32 M sucrose (4 ml), homogenized with a Polytron
homogenizer at setting 6 for 5ꢀ10 s and centrifuged at 30,900g for 20 min at 4°C. The resulting
pellet was resuspended in 2 ml H2O, homogenized with a Polytron homogenizer, incubated for
10 min on ice, and osmolarity was reestablished by addition of 338 ꢁl of 250 mM Tris, 338 ꢁl 1
M potassium Tartrate, and 10 ꢁl 0.9 M MgSO4. The assays contained approximately 75ꢀ150 ꢁg
of membrane protein for [3H]DHTB binding (less than 15% of the radioactivity added was
bound) and [3H]DHTB (6 ꢀ 8 nM final concentration) in a final volume of 250 ꢁl. Tris assay
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buffer without ATP was used for all assays, except as indicated. Specific binding was defined as
the difference in binding observed in the absence and presence of 2 ꢁM DHTB. The membranes
were incubated for 90 min, unless otherwise indicated, at room temperature. Assays were
terminated by filtration through Filtermat A filters using a 96ꢀwell Tomtec cell harvester and
radioactivity determined as for [3H](+)-5b using a Perkin Elmer 1405 microBeta scintillation
counter. Saturation binding experiments were conducted with duplicate determinations at each
ligand concentration, which included radiolabeled and nonꢀradiolabeled ligand for final
concentrations ranging from 6 to 200 nM for DHTB. Protein concentrations were determined
using a modified BCA protein assay.
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[3H]5HT uptake assay, HEK- Human VMAT2. The HEKꢀVMAT2 [3H]5HT uptake assay
was performed as described previously, with minor modifications.43 [3H]5HT was used for the
uptake assay because it had slightly higher potency than DA or NE in preliminary uptake
experiments (data not shown) and has been used as [3H]substrate to characterize VMAT2
activity.44ꢀ46 HEKꢀhuman VMAT2 cells were grown until confluent. After removal of medium,
cells were scraped from plates and homogenized with 12 strokes of a glass/glass homogenizer in
sucrose (0.32M supplemented with protease inhibitors). After centrifugation at 800 x g for 10
min, the supernatant was removed and centrifuged at 10,000 x g for 20 min. The pellet was
resuspended in sucrose (0.32M with protease inhibitors). The membranes were osmotically
shocked by addition of 3.5 volumes of water (4oC) and homogenized with a glass/Teflon
homogenizer. Osmolarity was reestablished with addition of Tris (25 mM final), potassium
tartrate (100 mM), and MgSO4 (0.9 mM) and the membranes were centrifuged at 20,000xg for
20 min. After centrifugations, the pellet was resuspended in VMAT2 uptake buffer (100 mM
potassium tartrate, 25 mM Tris, 4 mM KCl, 2 mM MgSO4, 1.7 mM ascorbic acid, 100 µM
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tropolone and 1 µM pargyline, pH 7.4 at 25oC with MgATP, 2 mM) using 2 gentle strokes of a
Teflon/glass homogenizer. The uptake assay was conducted in duplicate and included
membrane preparation, drug, [3H]5ꢀHT (40 nM), and VMAT2 uptake buffer in a final volume of
0.25 ml. [3H]5ꢀHT uptake by HEKꢀVMAT2 membranes is ATP dependent (data not shown).
Membranes were preincubated with drugs for 10 min at 30oC. After addition of [3H]5HT, uptake
was conducted at 30oC for 6 min and terminated by filtration as described for [3H]DHTB
binding, using filters presoaked in 0.05% polyethylenimine. Specific uptake was defined as the
difference in uptake observed in the absence and presence of reserpine (1 µM).
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[3H]5HT uptake assay, vesicular preparation of mouse striata. The method was
adapted from the literature.47, 48 C57Bl/6J mice (males and females, 44 total, ages 121ꢀ152 days)
were used. All procedures were performed in accordance with the National Institutes of Health
(NIH) Guide for the Care and Use of Laboratory Animals, and were approved by the Institutional
Animal Care and Use Committee of the VA Portland Health Care System. Mice were sacrificed
by cervical dislocation, striata were dissected, placed on ice, and homogenized with 12 strokes of
a glass/Teflon homogenizer in sucrose (0.32M supplemented with protease inhibitors). After
centrifugation at 800 x g for 10 min, the supernatant was decanted, the pellet was resuspended in
sucrose solution, and centrifuged at 800xg for 10 min. The supernatants were combined and
centrifuged at 10,000 x g for 20 min. The pellet was resuspended in sucrose (0.32M with
protease inhibitors). The membranes were osmotically shocked by addition of 3.5 volumes of
water (4oC) and homogenized with a glass/Teflon homogenizer. Osmolarity was reestablished
with addition of Tris (25 mM final), potassium tartrate (100 mM), and MgSO4 (0.9 mM) and the
membranes were centrifuged at 20,000xg for 20 min. The supernatant was decanted, and
centrifuged at 100,000xg for 60 min in a Beckman Optima TLX Ultracentrifuge. Uptake was
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conducted at 30oC for 8 min and terminated as described above. Specific uptake was defined as
the difference in uptake observed in the absence and presence of the VMAT2ꢀselective
compound reserpine (1 µM) and was 81ꢀ84% of total [3H]5ꢀHT uptake.49 Because reserpine does
not bind to the DAT, NET or SERT, uptake by those transporters in the presence of the VMAT2
blocker, reserpine, is nonspecific uptake.50ꢀ52
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[125I]RTI-55 binding to hDAT and [125I]DOI binding to the human serotonin receptor
(5HT2A). [125I]RTIꢀ55 binding to the hDAT stably expressed in HEKꢀ293 cells was conducted
as previously described using a total particulate membrane preparation.43 The binding assay
included test compound or buffer, membranes, [125I]RTIꢀ55 (40ꢀ60 pM) and buffer in a final
volume of 250 µl. Specific binding was defined as the difference between total binding and
binding in the presence of 5 µM mazindol. [125I]RTIꢀ55 binding to the human norepinephrine
transporter and the human serotonin transporter were conducted as previously described.43
[125I]DOI binding to the h5ꢀHT2A receptor stably expressed in HEKꢀ293 cells was conducted as
previously described.53 The binding assay included test compound or buffer, wellꢀwashed cell
homogenate, [125I]DOI (50 pM) and buffer in a final volume of 250 µl. Specific binding was
defined as the difference between total binding and binding in the presence of 10 µM 5HT.
MTT cell viability assay. Compounds were tested for effects on cell viability using a MTT
cell proliferation assay kit (Abcam, Cambridge, MA), HEKꢀVMAT2 cells (5x104) were plated
in 96ꢀwell plates in 100 µl DMEM supplemented with 10% FetalClone, 0.05 U of
penicillin/streptomycin and G418. This density of cells was in the linear response range for the
assay (data not shown). On day 2, drugs (100 µl) in unsupplemented DMEM or DMEM were
added to triplicate wells. The medium was removed 24 hours later, and unsupplemented media
and MTT reagent were added. The plate was incubated for 2 hrs. at 37oC, medium was removed,
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and MTT solvent was added. After 15 min on a shaker, the plate was read at 590 nm. Data was
normalized to the response of control cells. Data was analyzed with oneꢀway ANOVA followed
with Dunnett’s test. All conditions had 0.1% DMSO. As a positive control, staurosporine,
which induces programmed cell death, decreased cell viability at 1 and 3 µM (data not shown).
Statistical Analysis. GraphPad Prism 7.02 Software (GraphPad Inc., La Jolla, CA) was used
to analyze all kinetic, saturation and competition binding data, and was used for analysis of
variance (ANOVA) with Bonferroni postꢀtests. Association experiments with a single
concentration of radioligand were analyzed using one phase exponential association nonlinear fit
to determine kobs. Dissociation experiments were analyzed using a one phase exponential decay
nonlinear fit to determine the dissociation rate constant and halfꢀlife. Data shown are mean ±
S.E.M., except as indicated. t tests (twoꢀtailed, unpaired) were performed using Microsoft Excel
(Microsoft, Redmond, WA). Correlations were assessed with Spearman’s nonparametric
correlation.
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AUTHOR INFORMATION
Corresponding Author
* Brian Provencher email: ProvencherB@merrimack.edu Phone: (978) 837ꢀ5296
Author Contributions
The manuscript was written through contributions of all authors. All authors have given approval
to the final version of the manuscript. The authors contributed equally.
ACKNOWLEDGMENT
The authors would like to thank John F. Reed for technical assistance. This work was funded
by the Department of Veterans Affairs Merit Review Program on Novel Pharmacotherapies for
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Psychostimulant Addiction (I01 BX000939), and Research Career Scientist Program, the
National Institute on Drug Abuse (NIDA)ꢀfunded Methamphetamine Abuse Research Center
(P50 DA018165), and an NIHꢀVA interagency agreement (ADA15001). The contents do not
represent the views of the U.S. Department of Veterans Affairs or the United States Government.
ABBREVIATIONS
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APQ, arylpiperidinylquinazoline; CCR2, CC chemokine receptor 2; CCL2, CC chemokine
ligand 2; CCR5, CC chemokine receptor 5; DAT, dopamine transporter; DHTB,
Dihydrotetrabenazine; TLC, thin layer chromatography; VMAT1, vesicular monoamine
transporter 1; VMAT2, vesicular monoamine transporter 2.
Supporting Information
The Supporting Information is available free of charge on the ACS Publications website.
Molecular formula strings (.CSV), detailed experimental procedures and spectroscopic data (1H
NMR) and MPO calculations for the APQ ligands. (PDF)
REFERENCES
1. Abbruscato, T. J.; Tripper, P.C. DARK classics in chemical neuroscience:
methamphetamine. ACS Chem. Neurosci. [Online Early Access] DOI:
10.1021/acschemneuro.8b00123.
Published
Online:
March
30,
2018.
https://pubs.acs.org/doi/pdf/10.1021/acschemneuro.8b00123 (Accessed July 15, 2018).
2. The DAWN Report: Emergency Department Visits Involving Methamphetamine: 2007 to
2011. Substance Abuse and Mental Health Services Administration, Center for Behavioral
Health Statistics and Quality. Rockville, MD. June 19, 2014.
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3. Harris, D.; Battki, S. L. Stimulant psychosis: symptom profile and acute clinical course.
5
6
Am J Addict. 2000. 9, 28ꢀ37.
7
8
9
4. McCann, U. D.; Wong, D. F.; Yokoi, F.; Villemagne, V.; Dannals, R. F.; Ricaurte, G. A.
Reduced striatal dopamine transporter density in abstinent methamphetamine and
methcathinone users: evidence from positron emission tomography studies with
[11C]WINꢀ35,428. J. Neurosci. 1998, 18, 8417ꢀ8422.
10
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5. Sitte, H.; Freissmuth, M. Amphetamines, new psychoactive drugs and the monoamine
transporter cycle. Trends in Pharmacological Sciences. 2015, 36, 41ꢀ50.
6. Nickell, J. R.; Siripurapu, K. B.; Vartak, A.; Crooks, P. A.; Dwoskin, L. P. The vesicular
monoamine transporterꢀ2: an important pharmacological target for the discovery of novel
therapeutics to treat methamphetamine abuse. Adv. Pharmacol. 2014, 69, 71ꢀ106.
7. Eiden, L. E.; Weihe, E. VMAT2: A dynamic regulator of brain monoaminergic neuronal
function interacting with drugs of abuse. Ann. N.Y. Acad. Sci. 2011, 1216, 86ꢀ98.
8. Bernstein, A. I.; Stout, K. A.; Miller, G. W. The vesicular monoamine transporter 2: an
underexplored pharmacological target. Neurochem. Int. 2014, 73, 89ꢀ97.
9. Sora,
Ichiro.
Monoamine
transporter
as
a
target
molecule
for
psychostimulants. International Review of Neurobiology. 2009, 85, 29ꢀ33.
10. Lee, N.ꢀR.; Zheng, G.; Crooks, P. A.; Bardo, M. T.; Dwoskin, L. P. New scaffold for lead
compounds to treat methamphetamine use disorders. AAPS Journal. 2018, 20, 1ꢀ10.
11. Grouleff, J.; Ladefoged, K.; Koldso, H.; Schiott, B. Monoamine transporters: insights
from molecular dynamics simulations. Front Pharmacol. 2015, 6, 235.
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