Des-keto lobeline analogs with increased potency and selectivity at dopamine and serotonin transporters

Article abstract of DOI:10.1016/j.bmcl.2006.07.070

A series of des-keto lobeline analogs has been synthesized and evaluated for their ability to inhibit the dopamine transporter (DAT) and serotonin transporter (SERT) function and for their affinity for the synaptic vesicle monoamine transporter (VMAT2), a

Full text of DOI:10.1016/j.bmcl.2006.07.070

Bioorganic & Medicinal Chemistry Letters 16 (2006) 5018–5021
Des-keto lobeline analogs with increased potency and selectivity
at dopamine and serotonin transporters
Guangrong Zheng, David B. Horton, Agripina G. Deaciuc,
Linda P. Dwoskin and Peter A. Crooks*
College of Pharmacy, University of Kentucky, Lexington, KY 40536-0082, USA
Received 12 June 2006; accepted 14 July 2006
Abstract—A series of des-keto lobeline analogs has been synthesized and evaluated for their ability to inhibit the dopamine trans-
porter (DAT) and serotonin transporter (SERT) function and for their affinity for the synaptic vesicle monoamine transporter
(VMAT2), as well as for a4b2* and a7* neuronal nicotinic acetylcholine receptors (nAChRs). The enantiomers 8R-hydroxylobel-
9-ene (3a) and 10S-hydroxylobel-7-ene (3c) exhibited high potency and selectivity at SERT and DAT, respectively.
Ó 2006 Elsevier Ltd. All rights reserved.
Monoamine neurotransmitter transporters such as the
dopamine transporter (DAT), the serotonin transporter
(SERT), the norepinephrine transporter (NET), and the
vesicular monoamine transporter (VMAT2) are consid-
ered valid targets for the development of therapeutic
agents aimed at treating a variety of neurological and
psychiatric diseases. For example, several antidepressant
drugs, such as fluoxetine, bupropion, and reboxetine, act
as SERT, DAT, and NET inhibitors, respectively. These
antidepressants increase the extracellular concentration
of the respective neurotransmitter by inhibiting trans-
porter function.1–7 Additionally, tetrabenazine, an
inhibitor of VMAT2 function, is used to treat Hunting-
ton’s Chorea.8,9 Recently, DAT has also been consid-
ered as a primary target for the development of
medications to treat cocaine abuse.10–12
over [3H]dopamine ([3H]DA) uptake, and also has in-
creased selectivity for these transporters as a result of re-
duced affinity for nAChRs.15 This intriguing result
prompted us to carry out a more detailed investigation
of the structure–activity relationships of various stereo-
isomeric forms of 3a and the double bond reduced ana-
log 4a. The pharmacological profile of these compounds
was expected to provide information on the importance
of the C-8/C-10 stereochemistry on the interaction with
DAT, SERT, and VMAT2. Thus, the present study
investigated the synthesis and pharmacological activities
of isomeric 8- and 10-hydroxy lobelenes, that is 8R-
hydroxylobel-9-ene (3a), 8S-hydroxylobel-9-ene (3b),
10S-hydroxylobel-7-ene (3c), and 10R-hydroxylobel-7-
ene (3d), as well as the isomeric 8- and 10-hydroxylobel-
anes, that is 8R-hydroxylobelane (4a), 8S-hydroxylobe-
lane (4b), 10S-hydroxylobelane (4c), and 10R-
hydroxylobelane (4d). These compounds were evaluated
for their ability to inhibit [3H]nicotine ([3H]NIC) bind-
ing (probing a4b2* nAChRs) and [3H]methyllycaconi-
tine ([3H]MLA) binding (probing a7* nAChRs) to rat
brain membranes, to inhibit [3H]5-HT and [3H]DA
uptake into rat hippocampal and striatal synaptosomes,
respectively, and to inhibit [3H]dihydrotetrabenazine
([3H]DTBZ) binding to rat synaptic vesicle membranes.
(ꢀ)-Lobeline (the 2R,6S,10S-stereoisomer, 1; Scheme 1),
the major alkaloid of Lobelia inflata, has high affinity for
several neuronal nicotinic acetylcholine receptor
(nAChR) subtypes,13–16 and interacts nonselectively
with monoamine transporters (DAT, SERT, NET, and
VMAT2).15–18 Structural modification of lobeline re-
vealed that the des-keto analog, 8R-hydroxylobel-9-ene
(3a; Scheme 1), has high potency and selectivity for inhi-
bition of [3H]5-hydroxytryptamine ([3H]5-HT) uptake
The synthetic routes to compounds 3a–3d19 and 4a–4d19
are illustrated in Scheme 1. Compound 2 was prepared
by dehydration of lobeline (1) with 85% H3PO4, to
afford the E-isomer exclusively, according to a previous-
ly reported method.20,21 Reduction of 2 gave a mixture
of two isomers, 3a and 3b, in a ratio of 9:20 (determined
Keywords: Lobeline; Dopamine transporter; Serotonin transporter;
Vesicular monoamine transporter.
*
Corresponding author. Tel.: +1 859 257 1718; fax: +1 859 257
7585; e-mail: pcrooks@email.uky.edu
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.07.070

G. Zheng et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5018–5021
5019
OH
c
OH
OH
N
N
CH3
CH3
O
3a
b
4a
N
+
CH3
2
OH
c
N
N
a
CH3
CH3
OH
10
O
8
3b
4b
6
2
N
CH3
1
d
OH
OH
OH
OH
c
c
3c
N
N
CH3
e
CH3
3c
4c
O
b
+
N
CH3
5
N
N
CH3
CH3
3d
4d
Scheme 1. Reagents and conditions: (a) 85% H3PO4, 60 °C; (b) NaBH4, EtOH, rt; (c) H2, 10% Pd/C, MeOH, 45 psi, rt; (d) Zn/Hg, HCl (5%), reflux;
(e) CrO3, H2SO4, acetone, 0 °C.
by GC–MS). The pure form of 3a was obtained by
fractional recrystallization of this isomeric mixture.
Compound 3b was obtained by silica gel chromatogra-
phy of the mother liquors from the crystallization of
3a. Compound 3c, which was prepared by Clemmensen
reduction of lobeline, as previously reported,20 was con-
verted into compound 5 by Jones oxidation. Compound
3d was obtained along with 3c, from 5, utilizing the same
procedure as that employed in the synthesis of 3a and 3b
from 2 (vide supra). Catalytic hydrogenation of the
unsaturated compounds 3a, 3b, 3c, and 3d afforded the
corresponding reduced compounds 4a, 4b, 4c, and 4d,
respectively.
The above lobeline analogs were evaluated as inhibitors
of [3H]NIC binding and [3H]MLA binding to rat brain
membranes, as inhibitors of [3H]DA uptake into rat stri-
atal synaptosomes to assess DAT function, as inhibitors
of [3H]5-HT uptake into rat hippocampal synaptosomes
to assess SERT function, and as inhibitors of [3H]DTBZ
binding to rat synaptic vesicle membranes to assess
interaction with VMAT2 (Table 1).17 Analog-induced
*
*
Table 1. Inhibition of [3H]NIC binding (probing a4b2* nAChRs) and the [3H]MLA binding (probing a7* nAChRs) on rat brain membranes,
[3H]DTBZ binding (probing VMAT2) on rat synaptic vesicle membranes, [3H]DA uptake into rat striatal synaptosomes, and [3H]5-HT uptake into
rat hippocampal synaptosomes by lobeline and its des-keto analogs
Ki, lM, SEMa
Ki ratiob
[3H]NIC (a4b2*)
[3H]MLA (a7*)
[3H]DA (DAT)
[3H]5-HT (SERT)
[3H]DTBZ (VMAT2)
DAT/SERT/VMAT2
Fluoxetine
GBR-12909
—
—
—
—
—
0.018c
—
0.041c
—
—
—
—
—
Ro 4-1284
Lobeline
—
—
—
46.8 3.7
0.044c
0.028 0.03
2.76 0.64
5.16 0.30
6.06 0.45
6.44 0.54
0.59 0.15
1.98 0.31
3.01 0.44
3.09 0.41
6.60 2.96
—
0.004 0.000
4.19 0.80
>100
6.26 1.30
1.70 0.32
>100
28.2 6.73
0.86c
10.2/17.0/1
19.5/1/117.3
1/3.9/6.3
1/173/58.5
1/26/2.0
13/1/13.2
1/1.8/2.4
1/3.1/2.2
1/12.8/11.6
3a
3b
3c
3d
4a
4b
4c
4d
0.96 0.11
0.11 0.003
0.29 0.02
1.88 0.12
1.26 0.17
1.39 0.08
0.57 0.04
3.75 0.75
19.0 3.9
7.50 1.80
0.15 0.02
2.22 0.36
4.27 1.00
7.30 0.50
9.75 0.91
>100
>100
>100
2.36 0.18
33.6 8.54
1.77 0.61
>100
1.21 0.09
> 100
39.3 12.9
>100
GBR-12909 (a specific DAT inhibitor), fluoxetine (a specific SERT inhibitor), and Ro 4-1284 (a specific VMAT2 inhibitor) were used as standards
for comparison.
a Each Ki value represents data from at least four independent experiments, each performed in duplicate.
b For ratios between three monoamine transporters (DAT, SERT, and VMAT2), the highest affinity value was taken as 1.
c Data as reported in Ref. 15.

5020
G. Zheng et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5018–5021
inhibition was compared with that induced by lobeline
and the selective DAT, SERT, and VMAT2 transporter
inhibitors GBR-12909, fluoxetine, and Ro 4-1284,
respectively.11,22,23 Lobeline potently inhibited [3H]NIC
binding with a Ki value of 4 nM, and had low affinity
(Ki = 6.26 lM) for a7* nAChRs.24 des-Keto lobeline
analogs exhibited diminished affinity at a4b2* and a7*
nAChRs, except for compounds 3a and 4a, which had
slightly higher potency than lobeline at a7* nAChRs.
These results indicate the importance of the keto group
in lobeline for a4b2* binding. Lobeline exhibited moder-
ate selectivity for VMAT2 (Ki = 2.76 lM) over DAT
(Ki = 28.2 lM) and SERT (Ki = 46.8 lM), and had rela-
tively low affinity for the latter two transporters. How-
ever, most of the des-keto analogs exhibited higher
potency as well as selectivity for DAT or SERT when
compared to lobeline. None of these analogs exhibited
high affinity and selectivity for VMAT2. All the des-keto
analogs were generally equipotent with lobeline (within
one order of magnitude of each other), and compound
3d exhibited the highest affinity (Ki = 0.59 lM). These
results are consistent with earlier results obtained with
previously reported defunctionalized lobeline analogs.15
In the current des-keto series, all analogs exhibited
increased potency for inhibition of DAT and SERT
compared to lobeline. Within this series, compound 3c
exhibited the highest affinity for DAT (Ki = 0.11 lM)
and compound 3a, the enantiomer of 3c, exhibited the
highest affinity for SERT (Ki = 0.044 lM).
In summary, a series of des-keto lobeline analogs has
been synthesized, in which the oxygen of the keto group
of lobeline has been eliminated. Pharmacological evalu-
ation shows that all the analogs have diminished affinity
at a4b2* nAChRs and most of them also have dimin-
ished affinity at a7* nAChRs. In addition, all the ana-
logs are equipotent with lobeline at VMAT2.
Moreover, some of these analogs have high potency
and selectivity at either DAT or SERT. The current
study indicates that the stereochemistry at C-8/C-10 in
these molecules is important for inhibition of SERT,
but not for inhibition of DAT. In contrast, the double
bond in these analogs is more important for inhibition
of DAT than for inhibition of SERT function. Further
structural modification based on this series of analogs
may reveal important information about the DAT and
SERT pharmacophores.
Acknowledgment
This research was supported by NIH Grant DA 13519.
References and notes
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1095.
Compound 3a was 20-fold more selective in inhibiting
SERT over DAT15 and was 117-fold more selective
for SERT over VMAT2. Compound 3b, which has
the antipodal chirality at the C8-hydroxyl group com-
pared to compound 3a, showed similar affinity for
DAT and VMAT2 as 3a, but was 2 orders of magni-
tude less potent than 3a for SERT. Interestingly, com-
pound 3c, the enantiomer of 3a, exhibited 220-fold
greater selectivity in inhibiting DAT over SERT, which
is the reverse of the selectivity observed with 3a. How-
ever, this reversal of selectivity did not occur in other
pairs of enantiomers, that is, compounds 3b and 3d,
both of which showed greater potency in inhibiting
DAT over SERT (4- and 25-fold, respectively), and
3d was more selective than 3b at DAT. All four of
these compounds inhibited DAT with Ki values all
within one order of magnitude of each other. Thus,
the binding site on SERT is more sensitive to stereo-
chemical changes at the C-8/C-10 hydroxyl group than
is the binding site on DAT.
15. Miller, D. K.; Crooks, P. A.; Zheng, G.; Grinevich, V. P.;
Norrholm, S.; Dwoskin, L. P. J. Pharmacol. Exp. Ther.
2004, 310, 1035.
16. Dwoskin, L. P.; Crooks, P. A. Biochem. Pharmacol. 2002,
63, 89.
Compounds 4a–4d generally exhibited a similar potency
and selectivity profile as their corresponding double
bond unsaturated congeners, 3a–3d. The four com-
pounds 4a–4d were slightly less potent than their corre-
sponding precursors (3a–3d) in inhibiting DAT
function. Moreover, compounds 4a–4d exhibited similar
potency as their corresponding precursors (3a–3d) in
inhibiting SERT function. Thus, compounds 4a–4d are
less selective for DAT and SERT, compared to their
corresponding double bond analogs. This indicates the
double bond in these compounds is more important
for the binding at DAT than at SERT.
17. Teng, L.; Crooks, P. A.; Sonsalla, P. K.; Dwoskin, L. P. J.
Pharmacol. Exp. Ther. 1997, 280, 1432.
18. Teng, L.; Crooks, P. A.; Dwoskin, L. P. J. Neurochem.
1998, 71, 258.
25
19. Compound 3a: ½aꢁD 41.5° (c 1.0, CHCl3); mp 104–105 °C;
1H NMR (300 MHz, CDCl3) d 1.42–1.72 (m, 6H), 1.87 (m,
1H), 2.04 (m, 1H), 2.32 (s, 3H), 2.93 (m, 1H), 3.28 (m, 1H),
4.98 (dd, J = 10.5, 3.3 Hz, 1H), 6.23 (dd, J = 16.2, 6.3 Hz,
1H), 6.45 (d, J = 16.2 Hz, 1H), 7.20–7.42 (m, 10H); 13C

G. Zheng et al. / Bioorg. Med. Chem. Lett. 16 (2006) 5018–5021
5021
NMR (75 MHz, CDCl3) d 24.6, 26.7, 33.0, 41.5, 63.4, 65.5,
74.6, 125.6, 126.3, 127.2, 127.5, 128.4, 128.7, 130.4, 133.1,
137.2, 145.4 ppm; MS m/z 321 (M+); Anal. Calcd for
C22H27NOÆHClÆ1.0H2O: C, 70.29; H, 8.04; N, 3.73.
Found: C, 70.20; H,8.28; N, 4.01.
62.0, 65.0, 76.4, 125.6, 125.9, 127.1, 128.4, 128.5, 128.6,
142.3, 145.2 ppm; MS m/z 323 (M+); Anal. Calcd for
C22H29NOÆHClÆ2/3H2O: C, 71.04; H, 8.49; N, 3.77.
Found: C, 71.00; H, 8.62; N, 4.09.
25
Compound 4b: ½aꢁD ꢀ75.4° (c 1.0, CHCl3); 1H NMR
25
Compound 3b: ½aꢁD ꢀ131.0° (c 1.0, CHCl3); mp 117–
(300 MHz, CDCl3) d 1.26–1.44 (m, 2H), 1.44–1.57 (m,
2H), 1.67–2.00 (m, 6H), 2.36 (m, 1H), 2.39 (s, 3H), 2.60–
2.72 (m, 3H), 5.14 (t, J = 6.0 Hz, 1H), 7.15–7.40 (m, 10H);
13C NMR (75 MHz, CDCl3) d 24.7, 26.7, 31.5, 33.7, 36.1,
39.1, 61.8, 62.8, 72.5, 125.7, 125.9, 126.8, 128.3, 128.5,
142.4, 145.7 ppm; MS m/z 323 (M+); Anal. Calcd for
C22H29NOÆHClÆ2/3H2O: C, 71.04; H, 8.49; N, 3.77.
Found: C, 70.84; H, 8.27; N, 4.06.
118 °C; 1H NMR (300 MHz, CDCl3) d 1.35–1.64 (m, 3H),
1.66–1.80 (m, 2H), 1.87 (m, 1H), 2.02 (m, 1H), 2.45 (m,
1H), 2.47 (s, 3H), 2.65 (m, 1H), 5.20 (dd, J = 11.1, 3.3 Hz,
1H), 6.13 (dd, J = 16.2, 8.4 Hz, 1H), 6.47 (d, J = 16.2 Hz,
1H), 7.20–7.42 (m, 10H); 13C NMR (75 MHz, CDCl3) d
24.0, 30.0, 33.7, 40.2, 41.6, 63.3, 68.6, 72.1, 125.6, 126.3,
127.0, 127.5, 128.3, 128.7, 130.7, 134.1, 137.0, 145.4 ppm;
MS m/z 321 (M+); Anal. Calcd for C22H27NOÆHClÆ1/
3H2O: C, 72.61; H, 7.94; N, 3.85. Found: C, 72.43; H,
8.02; N, 3.97.
25
Compound 4c: ½aꢁD ꢀ77.9° (c 1.0, CHCl3); mp 83–84 °C;
1H, 13C NMR and MS are same as those of compound 4a;
Anal. Calcd for C22H29NOÆHClÆ1.0H2O: C, 69.91; H, 8.53;
N, 3.71. Found: C, 69.63; H, 8.36; N, 3.88.
25
Compound 3c: ½aꢁD ꢀ44.8° (c 1.0, CHCl3); mp 107–
25
108 °C; 1H, 13C NMR and MS are same as those of
compound 3a; Anal. Calcd for C22H27NOÆHClÆ0.5H2O: C,
72.01; H, 7.97; N, 3.82. Found: C, 71.96; H, 8.12; N, 3.77.
Compound 3d: ½aꢁ2D5 128.8° (c 1.0, CHCl3); mp 118–119 °C;
1H, 13C NMR and MS are same as those of compound 3b;
Anal. Calcd for C22H27NOÆHClÆ0.1H2O: C, 73.46; H, 7.90;
N, 3.89. Found: C, 73.47; H, 7.78; N, 4.24.
Compound 4d: ½aꢁD 74.4° (c 1.0, CHCl3); H, 13C NMR
and MS are same as those of compound 4b; Anal. Calcd
for C22H29NOÆHClÆ1/3H2O: C, 72.21; H, 8.45; N, 3.83.
Found: C, 72.05; H, 8.71; N, 4.06.
1
20. Flammia, D.; Malgorzata, D.; Damaj, M. I.; Martin, B.;
Glennon, R. A. J. Med. Chem. 1999, 42, 3726.
21. Ebno¨ther, A. Helv. Chim. Acta 1958, 41, 386.
22. Fuller, R. W.; Wong, D. T.; Robertson, D. W. Med. Res.
Rev. 1991, 11, 17.
23. Lee, L. C.; Vander Borght, T.; Sherman, P. S.; Frey, K.
A.; Kilbourn, M. R. J. Med. Chem. 1996, 39, 191.
24. Zheng, G.; Dwoskin, L. P.; Deaciuc, A. G.; Norrholm, S.
D.; Crooks, P. A. J. Med. Chem. 2005, 48, 5551.
25
Compound 4a: ½aꢁD 75.5° (c 1.0, CHCl3); mp 83–84 °C; 1H
NMR (300 MHz, CDCl3) d 1.14 (m, 1H), 1.25 (m, 1H),
1.45–1.70 (m, 5H), 1.76–2.04 (m, 3H), 2.30 (s, 3H), 2.57–
2.79 (m, 2H), 2.87 (m, 1H), 3.20 (m, 1H), 5.01
(dd,J = 10.8, 3.0 Hz, 1H), 7.15–7.43 (m, 10H); 13C NMR
(75 MHz, CDCl3) d 23.7, 23.9, 25.4, 25.7, 32.6, 36.0, 40.2,