Pyrrolidine analogs of GZ-793A: Synthesis and evaluation as inhibitors of the vesicular monoamine transporter-2 (VMAT2)

Article abstract of DOI:10.1016/j.bmcl.2013.03.092

Central heterocyclic ring size reduction from piperidinyl to pyrrolidinyl in the vesicular monoamine transporter-2 (VMAT2) inhibitor GZ-793A and its analogs resulted in novel N-propane-1,2(R)-diol analogs 11a-i. These compounds were evaluated for their affinity for the dihydrotetrabenazine (DTBZ) binding site on VMAT2 and for their ability to inhibit vesicular dopamine (DA) uptake. The 4-difluoromethoxyphenethyl analog 11f was the most potent inhibitor of [³H]-DTBZ binding (K_i = 560 nM), with 15-fold greater affinity for this site than GZ-793A (K_i = 8.29 μM). Analog 11f also showed similar potency of inhibition of [³H]-DA uptake into vesicles (K_i = 45 nM) compared to that for GZ-793A (K_i = 29 nM). Thus, 11f represents a new water-soluble inhibitor of VMAT function.

Full text of DOI:10.1016/j.bmcl.2013.03.092

Bioorganic & Medicinal Chemistry Letters 23 (2013) 3342–3345
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Pyrrolidine analogs of GZ-793A: Synthesis and evaluation
as inhibitors of the vesicular monoamine transporter-2 (VMAT2)
Narsimha Reddy Penthala a, Purushothama Rao Ponugoti b, Justin R. Nickell b, Agripina G. Deaciuc b,
Linda P. Dwoskin b, Peter A. Crooks a,
⇑
a Department of Pharmaceutical Sciences, College of Pharmacy, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA
b Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, KY 40536, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Central heterocyclic ring size reduction from piperidinyl to pyrrolidinyl in the vesicular monoamine
transporter-2 (VMAT2) inhibitor GZ-793A and its analogs resulted in novel N-propane-1,2(R)-diol analogs
11a–i. These compounds were evaluated for their affinity for the dihydrotetrabenazine (DTBZ) binding
site on VMAT2 and for their ability to inhibit vesicular dopamine (DA) uptake. The 4-difluoromethoxy-
phenethyl analog 11f was the most potent inhibitor of [3H]-DTBZ binding (Ki = 560 nM), with 15-fold
Received 5 March 2013
Revised 21 March 2013
Accepted 22 March 2013
Available online 2 April 2013
greater affinity for this site than GZ-793A (Ki = 8.29 lM). Analog 11f also showed similar potency of inhi-
Keywords:
Pyrrolidine analogs
GZ-793A
Dihydrotetrabenazine binding site
Vesicular monoamine transporter-2
N-Propane-1,2(R)-diols
bition of [3H]-DA uptake into vesicles (Ki = 45 nM) compared to that for GZ-793A (Ki = 29 nM). Thus, 11f
represents a new water-soluble inhibitor of VMAT function.
Ó 2013 Elsevier Ltd. All rights reserved.
Methamphetamine (METH) abuse is a growing problem in the
United States, especially in the rural West and Southwest. To date,
there is no approved therapeutic agent for treating METH addic-
tion. (ꢀ)-Lobeline (1, Fig. 1), the major alkaloid of Lobelia inflata,
has been shown to decrease the rewarding effects of methamphet-
amine in a rat behavioral model,1–4 and is believed to inhibit dopa-
mine (DA) uptake into synaptic vesicles via an interaction with the
tetrabenazine (TBZ) binding site on the vesicular monoamine
transporter-2 (VMAT2).5 METH is a substrate at the presynaptic
dopamine transporter (DAT), and within the presynaptic terminal,
METH interacts with VMAT2 located on synaptic vesicles, inhibit-
ing DA uptake and promoting vesicular DA release, leading to in-
creased cytosolic DA concentrations in the extracellular space.6,7
Hence, VMAT2 has been identified as a valid target for the develop-
ment of therapeutic agents that inhibit VMAT2 function, counter-
acting the effects of METH.
The chemically defunctionalized lobeline analogue, lobelane
(2S,6R-1-methyl-2,6-diphenethylpiperidine) (2, Fig. 1), and its ana-
logues have recently been reported to have 10- to 15-fold more po-
tency and selectivity for inhibition of [3H]dopamine ([3H]DA)
uptake into synaptic vesicles when compared to lobeline.8–11
Additionally, an extensive study on the variation of methylene
linker length between C-6 and C-2 of the piperidine ring and each
of the two phenyl rings in the lobelane molecule confirmed that a
minimum of two methylene units in the alkane linker moiety is
essential for retention of binding affinity at the TBZ binding site
on VMAT2.12
Recently, we have identified a novel structurally optimized N-
propane-1,2-diol analogue of lobelane (GZ-793A; 3, Fig. 1) which
potently and selectively interacts with VMAT2 to inhibit DA up-
take.13,14 GZ-793A also blocked METH self-administration (SA)
and reward in rats, and had no effect on responding for food.15,16
The physicochemical properties of GZ-793A provide enhanced
water-solubility and drug-likeness relative to the majority of pre-
viously reported lobelane analogs.
In a previous communication, we have reported on a series of
pyrrolidino analogs of lobelane (2), and have shown that VMAT2
inhibition is increased when the piperidine ring of lobelane is re-
placed by a pyrrolidine ring.10 The main aim of the current study
was to determine [3H]DTBZ binding affinity and inhibition of
[3H]DA uptake by VMAT2 by, of a series of novel N-1,2(R)-dihydr-
oxylpropyl group (NDHP) substituted pyrrolidine analogs of GZ-
793A.
From previous SAR studies, it was found that lobelane ana-
logues containing either fluorophenethyl or methoxy-phenethyl
moieties were the most potent VMAT2 ligands.14 These observa-
tions prompted us to focus on the synthesis and evaluation of N-
1,2(R)-dihydroxylpropyl (NDHP) pyrrolidine analogues (11a–i) as
inhibitors of VMAT2 function.
The synthesis of analogues 11a–i was achieved via initial reac-
tion of benzotriazole, (S)-phenylglycinol (formed from LAH reduc-
⇑
Corresponding author. Tel.: +1 501 686 6495; fax: +1 501 686 6057.
E-mail address: pacrooks@uams.edu (P.A. Crooks).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.03.092

N. R. Penthala et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3342–3345
3343
Figure 1. Molecular structures of lobeline (1), lobelane (2), and GZ-793A (3).
tion of (S)-phenylglycine), and succindialdehyde at room tempera-
ture to afford the key synthon, (3S,5R,7aR)-5-(benzo-triazol-1-yl)-
3-phenyl[2,1-b]oxazolopyrrolidine (6),17 which upon reaction with
a variety of substituted phenethyl magnesium halides (7a–i) in tet-
rahydrofuran afforded a 2:1 mixture of the appropriate cis- and
trans-isomers, 8 and 9. These isomers were then separated by silica
gel chromatography (8:2 hexane: ethyl acetate) to afford the
appropriate 2R,5S- and 2R,5R-diastereomers 8a–i and 9a–i. Com-
pounds 8a–i were hydrogenolyzed by catalytic-transfer hydroge-
nation with palladium hydroxide-over-carbon, employing
ammonium formate as the hydrogen source in refluxing methanol.
These conditions afforded a quantitative conversion to the respec-
tive N-deprotected products 10a–i within 30 min, as opposed to
12 h utilizing previously reported conditions (i.e., H2/Pd–C).10 The
cis-meso-2,5-diarylethyl pyrrolidine free bases were each refluxed
with S-glycidol in ethanol to afford a series of substituted cis-
meso-2,5-diaryl ethylpyrrolidin-1-yl-propane-1,2(R)-diols (11a–i),
which were then converted to their respective HCl salts using ethe-
real HCl. The products were fully characterized by 1H and 13C NMR
spectroscopy and mass spectrometry; representative examples are
provided in the reference section.18
In the vesicular DA uptake assay, the 4-difluoromethoxy ana-
logue, 11f (Ki =45 nM) (Table 1, Fig. 3), and the 4-methoxy ana-
logue, 11d (Ki = 49 nM) (Table 1, Fig. 4), both exhibited similar
inhibition of [3H]DA uptake when compared to GZ-793A (3,
Ki = 29 nM). Thus, if one considers the binding affinity and func-
tional data for compounds 11d, 11f, and GZ-793A (3), ring size
reduction of the piperidino moiety in GZ-793A appears to lead gen-
erally to an improvement in affinity for the DTBZ binding site, but
does not alter inhibition of vesicular DA uptake (see Scheme 1).
Synaptic vesicles were prepared as described previously.19
Briefly, fresh whole brain (excluding cerebellum) was homoge-
nized in 28 mL of ice-cold 0.32 M sucrose using a glass homoge-
nizer (7 up and down strokes of a Teflon pestle, clearance 0.003
in). Homogenates were centrifuged at 1000g for 12 min at 4 °C.
Resulting supernatants (S1) were centrifuged at 22,000g for
10 min. The resulting pellets (P2), containing the synaptosomes,
were resuspended in 18 mL of ice-cold Milli-Q water for 5 min
with 7 up and down strokes of the Teflon pestle homogenizer.
Osmolarity was restored by immediate addition of 2 mL of
25 mM HEPES and 100 mM K2-tartrate buffer (pH 7.5). Samples
were centrifuged at 20,000g for 20 min. MgSO4 (final concentra-
tion, 1 mM) was added to the resulting supernatants (S3). Final
centrifugations were performed at 100,000g for 45 min. Pellets
(P4) were resuspended immediately in ice-cold buffer providing
The hydrochloride salts of the pyrrolidino analogues (11a–i)
were evaluated for their affinity for the DTBZ binding site on
VMAT2 and for their ability to inhibit [3H]DA uptake into synaptic
vesicles (functional inhibition of VMAT2) (Table 1).
Nine GZ-793A analogues were evaluated. The binding affinity of
these compounds at the DTBZ binding site on VMAT2 ranged from
ꢁ15
lg of protein/100 lL.
[3H]DTBZ binding to synaptic vesicle membranes was per-
formed according to previously described procedures.20 Briefly,
560 nM to >100
lM. The most potent compound was the 4-difluo-
100 lL of vesicles suspension was incubated in assay buffer (in
romethoxy substituted 2,5-diphenethylpyrrolidin-1-yl-propane-
1,2(R)-diol analogue, 11f (Ki = 560 nM) (Table 1, Fig. 2), which
had a 15-fold greater affinity for this site than GZ-793A (3,
25 mM HEPES, 100 mM K2-tartrate, 5 mM MgSO4, 0.1 mM EDTA,
and 0.05 mM EGTA, pH 7.5, 25 °C) in the presence of 5 nM
[3H]DTBZ and 1 nM–1 mM lobelane analogs (final concentrations)
for 30 min at room temperature. Nonspecific binding was deter-
Ki = 8.29 lM). The binding affinity of the 2-4-methoxy analogues
11b–d also exhibited slightly higher affinity than GZ-793A at the
mined in the presence of 20 lM TBZ. Assays were performed in
DTBZ binding site on VMAT2.
duplicate using Unifilter 96-well GF/B filter plates (presoaked in
0.5% polyethylenimine) and terminated by harvesting using a Fil-
terMate harvester. After washing five times with 350 lL of the
ice-cold wash buffer (in 25 mM HEPES, 100 mM K2-tartrate,
5 mM MgSO4, and 10 mM NaCl, pH 7.5), filter plates were dried,
Table 1
Inhibition constants (Ki) for GZ-793A (3) analogs at the [3H]DTBZ binding site and
[3H]-DA uptake site on VMAT2
Compd
R
[3H]DTBZ bindinga VMAT2[3H]DA uptakec
Ki SEMb
M) Ki SEM (nM)
(
l
3
––
H
2-OCH3
3-OCH3
4-OCH3
3-F
8.29 2.79
5.91 1.57
2.35 0.44
2.28 0.44
3.94 1.12
6.76 1.68
0.56 0.23
29
279 35
270
160 10
49
170 15
45
8
11a
11b
11c
11d
11e
11f
11g
11h
11i
6
9
4-OCHF2
3,4-Methylenedioxy 8.47 5.19
3,4-Dimethoxy
3,5-Difluoro
7
269 28
3780 230
>100
4.06 0.81
200
1
a
b
c
All binding experiments have n = 3.
Each Ki value represents mean SEM.
All uptake experiments represent n = 4.
Figure 2. Affinity of analogue 11f at the TBZ binding site on VMAT2 in the [3H]DTBZ
binding assay.

3344
N. R. Penthala et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3342–3345
a Packard Top Count NXT scintillation counter and a Packard Win-
dows NT-based operating system.
Inhibition of [3H]DA uptake was conducted as previously de-
scribed,20 utilizing a striatal synaptic vesicle preparation. Briefly,
rat striata were homogenized with 10 up and down strokes of a
Teflon pestle homogenizer (clearance ꢁ0.00300) in 14 ml of 0.32 M
sucrose solution. Homogenate was centrifuged (2000g for 10 min
at 4 °C), and the resulting supernatant was centrifuged again
(10,000g for 30 min at 4 °C). The pellet was resuspended in 2 ml
of 0.32 M sucrose solution and subjected to osmotic shock by add-
ing 7 ml of ice-cold water to the preparation, followed by the
immediate restoration of osmolarity by adding 900
ll of 0.25 M
HEPES buffer and 900 L of 1.0 M potassium tartrate solution.
l
Samples were centrifuged (20,000g for 20 min at 4 °C), and the
resulting supernatant was centrifuged again (55,000g for 1 h at
4 °C), followed by the addition of 100
lL of 10 mM MgSO4,
Figure 3. Inhibition of [3H]DA uptake into rat synaptic vesicles by analogue 11d.
100 L of 0.25 M HEPES and 100 L of 1.0 M potassium tartrate
l
l
solution prior to the final centrifugation (100,000g for 45 min at
4 °C). The final pellet was resuspended in 2.4 mL of assay buffer
(25 mM HEPES, 100 mM potassium tartrate, 50
100
M EDTA, 1.7 mM ascorbic acid, 2 mM ATP-Mg2+, pH 7.4). Ali-
quots of the vesicular suspension (100 L) were added to tubes
containing assay buffer, various concentrations of inhibitor
(0.1 nM–10 mM) and 0.1 L.
M [3H]DA in a final volume of 500
Nonspecific uptake was determined in the presence of Ro4-1284
(10 M). Reactions were terminated by filtration, and radioactivity
lM EGTA,
l
l
l
l
l
retained by the filters was determined by liquid scintillation spec-
troscopy (Liquid scintillation analyzer; PerkinElmer Life and Ana-
lytical Sciences, Boston, MA).
In summary, reduction in ring size of the central heterocyclic
piperidine ring of GZ-793A has led to the discovery of the 4-meth-
oxy and 4-difluoromethoxy cis-meso-2,5-disubstitued pyrrolidine
analogues 11d and 11f. These two analogues showed similar inhib-
itory potency of [3H]DA uptake into vesicles (Ki = 49 and 45 nM,
respectively) when compared to that for GZ-793A (Ki = 29 nM),
and were the most potent analogues in the series in this assay.
Analogue 11f was also the most potent inhibitor of [3H]DTBZ bind-
ing in the series (Ki = 560 nM), and had a 15-fold greater affinity for
Figure 4. Inhibition of [3H]DA uptake into rat synaptic vesicles by analogue 11f.
bottoms were sealed, and each well was filled with 40
lL of Pack-
ard’s MicroScint 20 cocktail. Bound [3H]DTBZ was measured using
this site than GZ-793A (Ki = 8.29 lM). Thus, 11d and 11f represent
Scheme 1. Reagents and conditions: (a) LiAlH4, THF, reflux, 24 h; (b) 2,5 dimethoxy tetrahydrofuran, benzotriazole, 10% aq HCl, rt, 12 h; (c) substituted phenethyl magnesium
bromides (7a–i), THF, rt, 12 h; (d) Pd(OH)2, NH4HCO2, MeOH, reflux, 30 min; (e) (S)-glycidol, EtOH, reflux, 24 h.

N. R. Penthala et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3342–3345
3345
13. Horton, D. B.; Kiran, B. S.; Zheng, G.; Crooks, Peter A.; Dwoskin, L. P. J.
Pharmacol. Exp. Ther. 2011, 339, 286.
14. Zheng, G.; David, B. H.; Reddy, P. N.; Nickell, J. R.; Culver, J. P.; Deaciuc, A. G.;
Dwoskin, L. P.; Crooks, P. A. Med. Chem. Commun. 2013, 4, 564.
new water-soluble analogues of GZ-793A that inhibit VMAT2
function.
15. Alvers, K. M.; Beckmann, J. S.; Zheng, G.; Crooks, P. A.; Dwoskin, L. P.; Bardo, M.
T. Psychopharmacology (Berl.) 2012, 224, 255.
Acknowledgments
16. Beckmann, J. S.; Denehy, E. D.; Zheng, G.; Crooks, P. A.; Dwoskin, L. P.; Bardo, M.
T. Psychopharmacology (Berl.) 2012, 220, 395.
This research was supported by NIH grant U01 DA13519. The
University of Kentucky holds patents on lobeline and the analogues
described in the current work. A potential royalty stream to L.P.D.
and P.A.C. may occur consistent with University of Kentucky policy.
17. Katritzky, A. R.; Cui, X.; Baozhen, Y.; Steel, P. J. J. Org. Chem. 1999, 64, 1979.
18. Spectral data of some potent compounds; Compound 11a: (Free base), 1H NMR
(300 MHz, CDCl3): d 7.12–7.40 (m, 10H), 3.90–3.95 (m, 1H), 3.80–3.46 (m, 3H),
3.40–2.95 (m, 4H), 2.72–2.40 (m, 4H), 2.38–1.95 (m, 6H), 1.90–1.82 (m, 2H)
ppm; 13C NMR (75 MHz, CDCl3): d 140.0, 128.8, 128.5, 126.6, 72.1, 69.7, 67.6,
56.1, 32.7, 32.3, 27.8 ppm; MS (EI) m/z 353 (M+). Compound 11c: 1H NMR
(300 MHz, CDCl3): d 10.38 (br s, 1H), 7.16 (t, 2H, J = 7.5 Hz), 6.74–6.70 (m, 6H),
4.15 (m, 1H), 3.75 (s, 6H), 3.58–3.35 (m, 2H), 3.13–2.78 (m, 4H), 2.77–2.74 (m,
2H), 2.50–2.38 (m, 6H), 2.16–1.99 (m, 4H); 13C NMR (75 MHz, CDCl3): d 159.9,
141.2, 129.9, 120.8, 114.3, 112.0, 70.5, 69.32, 67.6, 64.3, 55.5, 32.7, 31.5, 27.6;
MS (EI) m/z; 413 (M+). Compound 11d: 1H NMR (300 MHz, CDCl3): d 9.86 (br s,
1H), 7.05–6.95 (m, 4H), 6.95–6.82 (m, 4H), 4.05 (br s, 1H), 3.80–3.42 (m, 12H),
3.28–2.95 (m, 2H), 2.80–1.79 (m, 10H) ppm; 13C NMR (75 MHz, CDCl3): d
158.2, 131.7, 129.5, 114.2, 70.3, 69.0, 67.7, 64.4, 55.6, 32.1, 31.9, 27.8 ppm; MS
(EI) m/z; 413 (M+). Compound 11f: 1H NMR (300 MHz, CDCl3): d 10.85 (br s, 1H),
7.35–7.15 (m, 4H), 6.95–7.10 (m, 4H), 6.47 (t, 2H, J = 72.0 Hz), 4.19 (br s, 1H),
3.60–3.32 (m, 6H), 3.15–2.85 (m, 4H), 2.75–1.70 (m, 8H) ppm; 13C NMR
(75 MHz, CDCl3): d 149.7, 137.2, 129.7, 119.7, 115.9, 67.9, 65.9, 64.0, 48.6, 32.9,
31.3, 27.5 ppm; MS (EI) m/z; 485 (M+). Compound 11g: 1H NMR (300 MHz,
CDCl3): d 10.42 (br s, 1H), 6.70–6.59 (m, 6H), 5.89 (s, 4H), 5.32 (br s, 1H), 4.20
(m, 1H), 3.73–3.50 (m, 2H), 2.33–3.98 (m, 3H), 2.82–2.62 (m, 2H), 2.60–2.28
(m, 6H), 2.16–2.00 (m, 4H); 13C NMR (75 MHz, CDCl3): d 147.9, 146.2, 133.3,
121.3, 108.6, 101.1, 70.6, 69.3, 67.7, 64.3, 57.5, 32.4, 31.8, 27.6; MS (EI) m/z 441
(M+). Compound 11i: 1H NMR (300 MHz, CDCl3): d 10.92 (br s, 1H), 6.91–6.64
(m, 6H), 4.30–4.21 (m, 1H), 3.65–3.55 (m, 2H), 3.25–2.95 (m, 4H), 2.92–2.83
(m, 4H), 2.62–2.25 (m, 4H), 2.25–1.90 (m, 4H); 13C NMR (75 MHz, DMSO-d6): d
163.2, 141.3, 112.3, 102.1, 69.3, 67.7, 65.3, 49.5, 32.4, 31.8, 27.6; MS (EI) m/z;
425(M+).
References and notes
1. Miller, D. K.; Crooks, P. A.; Teng, L.; Witkin, J. M.; Munzar, P.; Goldberg, S. R.;
Acri, J. B.; Dwoskin, L. P. J. Pharmacol. Exp. Ther. 2001, 296, 1023.
2. Harrod, S. B.; Dwoskin, L. P.; Crooks, P. A.; Klebaur, J. E.; Bardo, M. T. J.
Pharmacol. Exp. Ther. 2001, 298, 172.
3. Miller, D. K.; Harrod, S. B.; Green, T. A.; Wong, M. Y.; Bardo, M. T.; Dwoskin, L. P.
Pharmacol. Biochem. Behav. 2002, 74, 279.
4. Harrod, S. B.; Dwoskin, L. P.; Green, T. A.; Gehrke, B. J.; Bardo, M. T.
Psychopharmacology 2003, 165, 397.
5. (a) Teng, L.; Crooks, P. A.; Sonsalla, P. K.; Dwoskin, L. P. J. Pharmacol. Exp. Ther.
1997, 280, 1432; (b) Teng, L.; Crooks, P. A.; Sonsalla, P. K.; Dwoskin, L. P. J.
Neurochem. 1998, 71, 258.
6. Sulzer, D.; Rayport, S. Neuron 1990, 5, 797.
7. Brown, J. M.; Hanson, G. R.; Fleckenstein, A. E. J. Neurochem. 2000, 74, 2221.
8. Zheng, G.; Dwoskin, L. P.; Deaciuc, A. G.; Norrholm, S. D.; Crooks, P. A. J. Med.
Chem. 2005, 48, 5551.
9. Nickell, J. R.; Krishnamurthy, S.; Norrholm, S.; Deaciuc, G.; Zheng, G.; Crooks, P.
A.; Dwoskin, L. P. J. Pharmacol. Exp. Ther. 2009, 332, 612.
10. Vartak, A. P.; Nickell, J. R.; Chagkutip, J.; Dwoskin, L. P.; Crooks, P. A. J. Med.
Chem. 2009, 52, 7878.
11. Vartak, A. P.; Deaciuc, A. G.; Dwoskin, L. P.; Crooks, P. A. Bioorg. Med. Chem. Lett.
2010, 20, 3584.
12. Zheng, G.; Dwoskin, L. P.; Deaciuc, A. G.; Crooks, P. A. Bioorg. Med. Chem. Lett.
2008, 18, 6509.
19. Nickell, J. R.; Zheng, G.; Deaciuc, G.; Crooks, P. A.; Dwoskin, L. P. J. Pharmacol.
Exp. Ther. 2011, 336, 724.
20. Teng, L.; Crooks, P. A.; Dwoskin, L. P. J. Neurochem. 1998, 71, 258.