Synthesis and evaluation of a series of homologues of lobelane at the vesicular monoamine transporter-2

Article abstract of DOI:10.1016/j.bmcl.2008.10.042

A series of lobelane homologues has been synthesized and evaluated for their [³H]DTBZ binding affinity at the vesicular monoamine transporter-2 (VMAT2). The structure-activity relationships (SAR) indicate that for retention of binding affinity at VMAT2, the lengths of the methylene linkers should be no shorter than one methylene unit at C-6 of the piperidine ring, and no shorter than two methylene units at C-2 of the piperidine ring. These results indicate that the intramolecular distances between the piperidine ring and two phenyl rings in lobelane analogues are an important criterion for retention of high affinity at VMAT2.

Full text of DOI:10.1016/j.bmcl.2008.10.042

Bioorganic & Medicinal Chemistry Letters 18 (2008) 6509–6512
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Bioorganic & Medicinal Chemistry Letters
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Synthesis and evaluation of a series of homologues of lobelane at the
vesicular monoamine transporter-2
*
Guangrong Zheng, Linda P. Dwoskin, Agripina G. Deaciuc, Peter A. Crooks
Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Rose Street, Lexington, KY 40536-0082, USA
a r t i c l e i n f o
a b s t r a c t
A series of lobelane homologues has been synthesized and evaluated for their [3H]DTBZ binding affinity
at the vesicular monoamine transporter-2 (VMAT2). The structure–activity relationships (SAR) indicate
that for retention of binding affinity at VMAT2, the lengths of the methylene linkers should be no shorter
than one methylene unit at C-6 of the piperidine ring, and no shorter than two methylene units at C-2 of
the piperidine ring. These results indicate that the intramolecular distances between the piperidine ring
and two phenyl rings in lobelane analogues are an important criterion for retention of high affinity at
VMAT2.
Article history:
Received 27 August 2008
Revised 7 October 2008
Accepted 10 October 2008
Available online 14 October 2008
Keywords:
Vesicular monoamine transporter
Lobelane
Ó 2008 Elsevier Ltd. All rights reserved.
Structure–activity relationship
The brain vesicular monoamine transporter-2 (VMAT2) is be-
lieved to play an important role in mediating the neurochemical
and behavioral effects of psychostimulants.1 And this transporter
is currently recognized as a valid target for the development of po-
tential treatments for methamphetamine abuse.2 (À)-Lobeline (1,
Fig. 1), the major alkaloid of Lobelia inflata, has been shown to in-
hibit dopamine uptake into synaptic vesicles via an interaction
with the tetrabenazine (TBZ) binding site on VMAT2.3 The des-oxy-
gen lobeline analogue, lobelane (2b, Fig. 1), has increased potency
and selectivity for interaction with the TBZ binding site on VMAT2
compared to lobeline.4 Moreover, lobelane dose dependently de-
creases methamphetamine self-administration without altering
sucrose-maintained responding, which is more specific compared
to lobeline, suggesting that it has potential as a novel treatment
for methamphetamine abuse.5
Extensive structure–activity relationship (SAR) studies on lobe-
lane have been carried out, including structural modifications of
both the piperidine ring and two phenyl rings; these have included
changing the chirality of piperidinyl C-2 and C-6 stereo centers,
and altering the positions of the two phenethyl substituents on
the piperidine ring.4,6 As part of a continuing effort to explore
the SAR for lobelane analogues, the goal of the present investiga-
tion was to determine the optimal methylene linker lengths be-
tween the piperidine ring and each of the two phenyl rings in
the lobelane molecule. Thus, we herein investigate a series of lobe-
lane homologues, in which the two ethylene linkers connecting
each of the two phenyl rings to the C-6 and C-2 positions of the
piperidine ring have been replaced by a range of methylene linkers
of different length (0–3 carbons), that is, combinations of 0,0-, 0,1-,
0,2-, 0,3-, 1,1-, 1,2-, 1,3-, 2,3-, and 3,3-carbons at C-6 and C-2 of the
piperidine ring, as new ligands for VMAT2 (Fig. 1 and Table 1).
The syntheses of the lobelane homologues are outlined in
Schemes 1–3. Compound 3, which is devoid of methylene units be-
tween the central piperidine ring and two phenyl rings, was syn-
thesized by initial N-methylation of 2,6-biphenylpyridine (12)
with methyl p-tosylate, and reduction of the resulting quaternary
ammonium salt (13) with NaBH4, followed by catalytic hydrogena-
tion over Pd-C [Scheme 1(a)]. Analogues in which two phenyl rings
are linked to the piperidine ring by one (4a and 4b) or three meth-
ylene units (5a and 5b), were synthesized as illustrated in Scheme
1(b). Kumada coupling7 of 2,6-dibromopyridine (14) with the cor-
responding benzylmagnesium chloride or 3-phenyl-1-propylmag-
nesium bromide to afford intermediates 15 and 16, respectively,
OH
O
2
6
N
N
CH3
R
R = CH3 lobelane (2a)
R = H nor-lobelane (2b)
lobeline (1)
m = 0, 1, 2, or 3
n = 0, 1, 2, or 3
R = H or CH3
6
2
( )m
( )n
N
R
* Corresponding author. Tel.: +1 859 257 1718; fax: +1 859 257 7585.
E-mail address: pcrooks@email.uky.edu (P.A. Crooks).
Figure 1. Structures of (À)-lobeline (1), nor-lobelane (2a), lobelane (2b), and
lobelane homologues.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.10.042

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G. Zheng et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6509–6512
Table 1
coupling of 18 with phenylboronic acid was achieved using [1,3-
bis(2,6-diisopropylphenyl)imidazol-2-ylidene](3-chloropyridyl)-
palladium(II) dichloride (PEPPSI-IPr) as a catalyst and KOtBu as a
base in i-PrOH.8 The pyridine ring in the resulting coupled product
19 was reduced to the corresponding piperidine ring by catalytic
hydrogenation, as described above for the synthesis of compound
4a, to afford analogue 6a [Scheme 2(a)]. Compound 21, the com-
mon intermediate for the preparation of analogues 7a and 8a,
was synthesized by coupling of 2-bromo-6-methylpyridine (17)
with phenylboronic acid under Suzuki coupling conditions.9 Con-
densation of 21 with benzaldehyde in Ac2O under reflux afforded
compound 22. Compound 21 was also used to synthesize com-
pound 23 via deprotonation of the 2-methyl group followed by
an SN2 reaction with 2-iodoethylbenzene. Catalytic hydrogenation
of 22 and 23, as described above for the synthesis of compound 4a,
afforded compounds 7a and 8a, respectively [Scheme 2(b)]. Com-
pounds 6a, 7a, and 8a, were converted into 6b, 7b, and 8b, respec-
tively, by N-methylation, as described in Scheme 1.
Analogues 9a, 9b, 10a, and 10b, which contain a single methy-
lene linker at C-6 and an ethylene or a propylene linker at C-2 of
the piperidine ring interconnecting each of the two phenyl rings,
were synthesized as illustrated in Scheme 3(a). Compound 18 from
Scheme 2(a) was used as a starting material to synthesize these
analogues. Negishi coupling of 18 with 2-phenyl-1-ethylzinc io-
dide or 3-phenyl-1-propylzinc iodide, using PEPPSI-IPr as a catalyst
and LiBr as an additive in THF/DMF10, afforded compound 24 or 25,
respectively. Catalytic hydrogenation of 24 and 25, as described
above for the synthesis of compound 4a, afforded compounds 9a
and 10a, respectively, which were converted into 9b and 10b,
respectively, by N-methylation, as described in Scheme 1.
Inhibition constants (Ki) for lobelane homologues at the [3H]DTBZ binding site
(VMAT2) on rat synaptic vesicle membranes.
6
N
2
( )m
( )n
R
Compound
R
m
n
[3H]DTBZ binding Ki, M, SEMa
l
Lobeline (1)
Nor-lobelane (2a)
Lobelane (2b)
3
4a
4b
5a
5b
6a
6b
7a
7b
8a
8b
9a
9b
10a
10b
11a
11b
—
H
—
2
2
0
1
1
3
3
0
0
0
0
0
0
1
1
1
1
2
2
—
2
2
0
1
1
3
3
1
1
2
2
3
3
2
2
3
3
3
3
2.76 0.64
2.31 0.21
0.97 0.19
>100
CH3
CH3
H
CH3
H
CH3
H
CH3
H
CH3
H
CH3
H
CH3
H
CH3
H
7.68 1.44
10.5 3.36
4.56 1.03
1.00 0.23
24.1 6.10
13.6 8.14
28.4 7.30
16.1 4.07
28.6 2.44
62.5 16.6
12.2 0.19
2.77 1.77
1.13 0.28
1.62 0.39
4.60 0.56
0.88 0.30
CH3
a
Each Ki value represents data from at least three independent experiments, each
performed in duplicate.
Analogues 11a and 11b, in which the two phenyl rings are
linked to the central piperidine ring by an ethylene moiety at C-6
and a propylene moiety at C-2, respectively, were synthesized by
initial mono-condensation of 2,6-lutidine (26) with benzaldehyde,
to afford the conjugated product 27, followed by a similar proce-
dure to that described in Scheme 2(b) for the synthesis of 8a and
8b [Scheme 3(b)]. The structures and purities of the analogues
were determined by 1H NMR, 13C NMR, GC-MS.11
The ability of the above lobelane homologues to interact with
VMAT2 was assessed by determining their ability to inhibit
[3H]dihydrotetrabenazine ([3H]DTBZ, Kd = 1.67 nM3b) binding to
rat brain vesicle membranes, utilizing a fast throughput 96-well
assay,12 and the results are summarized in Table 1.
followed by catalytic hydrogenation over Adams catalyst (PtO2)
yielded analogue 4a or 5a, respectively. N-Methylation of 4a and
5a were carried out utilizing NaBH3CN/(CH2O)n to afford analogues
4b and 5b, respectively.
Analogue pairs 6a and 6b, 7a and 7b, 8a and 8b, in which one
phenyl ring is attached directly to C-6 of the central piperidine
ring, while the other phenyl ring is linked to C-2 by one, two, or
three methylene units, were synthesized as illustrated in Scheme
2(a) and 2(b). Kumada coupling of 2-bromo-6-methoxypyridine
(17) with benzylmagnesium chloride, followed by treatment with
POCl3 in DMF afforded 2-chloro-6-benzyl-pyridine (18). Suzuki
a
a
b,c
6
N
2
N
N
CH3
p-tosylate
CH3
3
12
13
b
6
2
d
e
N
Br
N
14
Br
N
15
R
R = H 4a
f
R = CH3 4b
g
6
2
e
N
N
R = H 5a
R = CH3 5b
R
f
16
Scheme 1. Reagents and conditions: (a) Methyl p-tosylate, 180–200 °C; (b) NaBH4, MeOH, rt; (c) H2, Pd/C, MeOH, 10 psi, rt; (d) Benzylmagnesium chloride, NiCl2(dppp), THF,
rt; (e) H2, PtO2, HOAc, 50 psi, rt; (f) NaBH3CN, (CH2O)n, MeOH, rt; (g) (3-Bromopropyl)benzene, Mg, 1,2-dibromoethane, NiCl2(dppp), THF, rt.

G. Zheng et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6509–6512
6511
a
b
a,b
c
N
O
N
17
Br
Cl
N
18
19
d
6
N
2
R = H 6a
R = CH3 6b
e
R
f
g
N
N
Br
N
22
21
20
d
h
6
2
N
R
N
23
R = H 7a
R = CH3 7b
e
d
6
2
N
R
R = H 8a
R = CH3 8b
e
Scheme 2. Reagents and conditions: (a) Benzylmagnesium chloride, NiCl2(dppp), THF, rt; (b) POCl3, DMF, 80 °C; (c) Phenylboronic acid, PEPPSI-IPr, KO-tBu, iPrOH; (d) H2,
PtO2, HOAc, 50 psi, rt; (e) NaBH3CN, (CH2O)n, MeOH, rt; (f) Phenylboronic acid, Pd(PPh3)4, K2CO3, toluene; (g) Benzaldehyde, Ac2O, reflux; (h) i—LDA, THF, À78 °C; ii—(2-
Iodoethyl)benzene, THF, À78 °C–rt.
a
6
2
a
b
18
N
N
R
24
R = H 9a
R = CH3 9b
d
c
b
6
2
N
R
N
R = H 10a
R = CH3 10b
c
25
b
f
e
N
N
N
26
27
28
b
6
2
R = H 11a
R = CH3 11b
N
R
c
Scheme 3. Reagents and conditions: (a) Phenethylmagnesium chloride, Zn, I2, PEPPSI-IPr, LiBr, THF/DMF; (b) H2, PtO2, HOAc, 50 psi, rt; (c) NaBH3CN, (CH2O)n, MeOH, rt; (d)
(3-bromopropyl)benzene, Mg, 1,2-dibromoethane, Zn, I2, PEPPSI-IPr, LiBr, THF/DMF; (e) Benzaldehyde, Ac2O, reflux; (f) i—LDA, THF, À78 °C; ii—(2-iodoethyl)benzene, THF,
À78 °C–rt.
The cis-lobelane/nor-lobelane homologues 3, 4a, 4b, 5a, and 5b
are structurally symmetrical and isomerically pure meso-homo-
logues. The removal of the ethylene linker on both sides of the
piperidine ring of lobelane to afford homologue 3 resulted in a
complete loss of affinity for the DTBZ binding site on VMAT2. Re-
moval of one methylene unit on each side of the piperidine ring
of lobelane, to afford homologue 4b, also had a detrimental effect
on VMAT2 affinity, resulting in a 10-fold loss compared to lobelane.
On the other hand, adding one methylene unit to each side of the
piperidine ring of lobelane to produce homologue 5b (Ki = 1.00 lM)
resulted in a retention of affinity at VMAT2.
The non-symmetrical racemic homologues 6b, 7b, and 8b, in
which one phenyl ring is directly attached to the piperidine ring
at C-6, while the second phenyl ring is connected to the piperidine
ring at C-2 via a one-, two, or three methylene linker unit, respec-
tively, exhibited 14–64 fold less affinity at VMAT2 compared to
lobelane. Increasing the length of the C-2 methylene linker in 4b
by one or two additional methylene units to afford racemic homo-

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G. Zheng et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6509–6512
1.46 (m, 2H), 1.60–1.95 (m, 4H), 2.66 (ddd, J = 13.2, 8.4, 1.8 Hz, 1H), 2.78 (ddd,
logues 9b and 10b (Ki = 2.77 and 1.62 lM, respectively), respec-
J = 13.2, 8.4, 1.8 Hz, 1H), 2.89 (m, 1H), 3.55 (brd, J = 10.2 Hz, 1H), 7.12–7.37 (m,
10H); 13C NMR (75 MHz, CDCl3) d 25.55, 32.40, 35.23, 44.09, 59.20, 62.45,
126.28, 126.29, 127.00, 128.38, 128.51, 129.30, 129.27, 145.57 ppm; MS m/z
250 (MÀ1)+.Compound 6b, 1H NMR (300 MHz, CDCl3) d 0.86 (m, 1H), 1.18–1.40
(m, 2H), 1.45–1.78 (m, 3H), 2.18 (s, 3H), 2.25 (m, 1H), 2.53 (dd, J = 13.2, 9.3 Hz,
1H), 2.97 (dd, J = 11.1, 2.4 Hz, 1H), 3.27 (dd, J = 13.2, 3.6 Hz, 1H), 7.15–7.42 (m,
10H); 13C NMR (75 MHz, CDCl3) d 24.91, 31.41, 36.59, 41.09, 41.15, 66.49,
71.52, 125.96, 126.83, 127.51, 128.24, 128.48, 129.74, 140.74, 146.16 ppm; MS
m/z 264 (MÀ1)+. Compound 7a, 1H NMR (300 MHz, CDCl3) d 1.13–1.27 (m, 2H),
1.39–1.58 (m, 2H), 1.64–1.94 (m, 5H), 2.60–2.75 (m, 5H), 3.61 (dd, J = 10.5,
2.4 Hz, 1H), 7.13–7.40 (m, 10H); 13C NMR (75 MHz, CDCl3) d 25.57, 32.25,
32.54, 34.89, 39.23, 57.47, 62.55, 125.83, 126.83, 127.10, 128.41, 128.44,
142.36, 145.55 ppm; MS m/z 265 (M+).Compound 7b, 1H NMR (300 MHz,
CDCl3) d 1.35–1.90 (m, 6H), 1.98 (m, 1H), 2.02 (s, 3H), 2.13 (m, 1H), 2.59 (m,
1H), 2.80 (m, 1H), 2.97 (dd, J = 9.9, 4.5 Hz, 1H), 7.12–7.38 (m, 10H); 13C NMR
(75 MHz, CDCl3) d 25.04, 31.16, 31.87, 35.59, 35.85, 40.02, 64.70, 71.46, 125.80,
127.06, 127.57, 128.41, 128.53, 142.68, 145.04 ppm; MS m/z 279
(M+).Compound 8a, 1H NMR (300 MHz, CDCl3) d 1.15 (m, 1H), 1.36–1.57 (m,
4H), 1.60–1.94 (m, 6H), 2.55–2.70 (m, 5H), 3.61 (dd, J = 10.8, 2.4 Hz, 1H), 7.10–
7.40 (m, 10H); 13C NMR (75 MHz, CDCl3) d 25.61, 28.24, 32.28, 34.86, 36.38,
37.23, 58.00, 62.74, 125.81, 126.89, 127.13, 128.38, 128.45, 128.50, 142.52,
145.49 ppm; MS m/z 278 (MÀ1)+.Compound 8b, 1H NMR (300 MHz, CDCl3) d
1.23–1.87 (m, 9H), 1.93 (s, 3H), 2.02 (m, 1H), 2.51–2.66 (m, 4H), 2.90 (dd,
J = 10.5, 2.4 Hz, 1H), 7.10–7.38 (m, 10H); 13C NMR (75 MHz, CDCl3) d 25.16,
26.98, 31.52, 34.04, 36.56, 36.70, 40.39, 64.41, 71.20, 125.70, 126.66, 127.42,
128.30, 128.35, 128.45, 142.68, 146.19 ppm; MS m/z 293 (M+). Compound 9a,
1H NMR (300 MHz, CDCl3) d 1.00–1.38 (m, 4H), 1.50–1.82 (m, 5H), 2.28–2.77
(m, 6H), 6.92–7.35 (m, 10H); 13C NMR (75 MHz, CDCl3) d 24.88, 32.16, 32.46,
32.81, 38.75, 43.82, 56.21, 58.63, 125.70, 126.31, 128.26, 128.32, 128.48,
129.24, 139.21, 141.83 ppm; MS m/z 278 (MÀ1)+.Compound 9b, 1H NMR (300
MHz, CDCl3) d 1.14–1.56 (m, 5H), 1.60–1.78 (m, 2H), 1.92 (m, 1H), 2.32 (s, 3H),
2.30–2.74 (m, 5H), 3.13 (dd, J = 12.0, 2.7 Hz, 1H), 7.15–7.35 (m, 10H); 13C NMR
(75 MHz, CDCl3) d 24.76, 26.93, 27.56, 32.48, 32.87, 36.59, 41.41, 63.05, 65.39,
125.72, 125.95, 128.23, 128.35, 128.47, 129.44, 140.24, 142.64 ppm; MS m/z
292 (MÀ1)+.Compound 10a, 1H NMR (300 MHz, CDCl3) d 0.96–1.82 (m, 11H),
2.34–2.76 (m, 6H), 7.03–7.35 (m, 10H); 13C NMR (75 MHz, CDCl3) d 24.96,
28.01, 32.48, 32.78, 36.18, 37.02, 43.96, 57.08, 58.75, 62.45, 125.75, 126.30,
128.32, 128.42, 128.51, 129.29, 139.25, 142.44 ppm; MS m/z 292
(MÀ1)+.Compound 10b, 1H NMR (300 MHz, CDCl3) d 1.13–1.82 (m, 10H),
2.45 (s, 3H), 2.40–2.80 (m, 4H), 2.90 (m, 1H), 3.24 (dd, J = 12.6, 3.0 Hz, 1H),
7.10–7.35 (m, 10H); 13C NMR (75 MHz, CDCl3) d 23.59, 25.10, 25.99, 28.04,
30.67, 33.02, 35.94, 39.83, 64.43, 66.05, 125.90, 126.65, 128.39, 128.58, 129.44,
137.78, 141.77 ppm; MS m/z 306 (MÀ1)+.Compound 11a, 1H NMR (300 MHz,
CDCl3) d 1.00–1.20 (m, 2H), 1.28 (m, 1H), 1.40–1.83 (m, 9H), 2.44–2.68 (m, 6H),
7.10–7.30 (m, 10H); 13C NMR (75 MHz, CDCl3) d 24.84, 28.20, 32.42, 32.63,
36.30, 36.94, 38.76, 57.08, 57.32, 125.81, 125.90, 128.36, 128.41, 128.47,
142.07, 142.45 ppm; MS m/z 306 (MÀ1)+.Compound 11b, 1H NMR (300 MHz,
CDCl3) d 1.22–1.50 (m, 6H), 1.55–1.78 (m, 5H), 1.87 (m, 1H), 2.13 (s, 3H), 2.35
(m, 1H), 2.54–2.70 (m, 3H), 7.14–7.32 (m, 10H); 13C NMR (75 MHz, CDCl3) d
25.19, 27.38, 28.38, 31.52, 32.54, 34.38, 36.47, 63.03, 63.49, 125.75, 128.36,
128.39, 128.51, 142.73 ppm; MS m/z 321 (M+).
tively, resulted in increased affinity at VMAT2 compared to 4b;
these homologues were only 2–3 times less potent than lobelane.
Interestingly, the addition of an additional methylene unit to the
C-2 ethylene linkers in the lobelane molecule, to produce racemic
homologue 11b, had no significant effect on affinity at VMAT2
compared to lobelane. In fact, 11b (Ki = 0.88
lM) was slightly more
potent than lobelane (Ki = 0.97 M). The nor analogues 6a–11a
l
exhibited affinities at VMAT2 which were within an order of mag-
nitude difference compared to their corresponding N-methylated
analogues 6b–11b, which is consistent with previous data from
the lobelane analogue series.6a
The results of this study indicate that for retention of binding
affinity at VMAT2, structurally related lobelane homologues should
contain a C-6 methylene linker that is no shorter than one methy-
lene unit, and a C-2 methylene linker that is no shorter than two
methylene units between the piperidine ring and the phenyl ring.
The optimal methylene linker lengths appear to be a combination
of a two methylene unit linker at C-6 and a three methylene unit
linker at C-2.
In conclusion, a series of lobelane homologues has been synthe-
sized. These results indicate that the intramolecular distances be-
tween the piperidine ring and two phenyl rings in lobelane
analogues are important for retention of high potency at VMAT2.
Acknowledgment
This research was supported by NIH Grant DA 13519.
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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.
Resulting pellets (P2), containing the synaptosomes, were resuspended in
18 mL ice-cold Milli-Q water for 5 min with 7 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 concentration, 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
11. Selected spectra data: compound 3, 1H NMR (300 MHz, CDCl3) d 1.42–1.86 (m,
6H), 1.79 (s, 3H), 3.05 (dd, J = 10.5, 2.7 Hz, 2H), 7.15–7.50 (m, 10H); 13C NMR
(75 MHz, CDCl3)
d 25.43, 37.19, 42.47, 71.05, 126.80, 127.43, 128.45,
146.24 ppm; MS m/z 251 (M+).Compound 4a, 1H NMR (300 MHz, CDCl3) d
1.10–1.35 (m, 3H), 1.53–1.82 (m, 4H), 2.50–2.70 (m, 6H), 7.05–7.25 (m, 10H);
13C NMR (75 MHz, CDCl3) d 24.82, 32.52, 43.78, 58.48, 126.05, 128.27, 129.03,
138.89 ppm; MS m/z 264 (MÀ1)+.Compound 4b, 1H NMR (300 MHz, CDCl3) d
1.08 (m, 1H), 1.17–1.43 (m, 4H), 1.58 (m, 1H), 2.50 (s, 3H), 2.35–2.58 (m, 4H),
3.21 (m, 2H), 7.10–7.35 (m, 10H); 13C NMR (75 MHz, CDCl3) d 24.34, 27.83,
35.06, 41.73, 65.70, 126.04, 128.33, 129.52, 140.27 ppm; MS m/z 278
(MÀ1)+.Compound 5a, 1H NMR (300 MHz, CDCl3) d 0.85–1.07 (m, 2H), 1.20–
1.46 (m, 1H), 1.50–1.80 (m, 7H), 2.37–2.52 (m, 2H), 2.60 (t, J = 7.5 Hz, 4H),
7.10–7.32 (m, 10H); 13C NMR (75 MHz, CDCl3) d 25.04, 28.22, 32.83, 36.36,
37.34, 57.20, 125.77, 128.33, 128.47, 142.49 ppm; MS m/z 321 (M)+.Compound
5b, 1H NMR (300 MHz, CDCl3) d 1.20–1.78 (m, 14H), 2.12 (s, 3H), 2.22–2.38 (m,
2H), 2.50–2.68 (m, 4H), 7.05–7.32 (m, 10H); 13C NMR (75 MHz, CDCl3) d 24.97,
27.48, 28.48, 32.05, 34.22, 36.41, 63.78, 125.77, 128.35, 128.48, 142.58 ppm;
MS m/z 335 (MÀ1)+.Compound 6a, 1H NMR (300 MHz, CDCl3) d 1.28 (m, 1H),
buffer (see below) providing ꢀ15
lg protein/100 l
L. [3H]DTBZ binding assay.
One hundred microliters of vesicle suspension was incubated in assay buffer
(in mM: 25 HEPES, 100 K2-tartrate, 5 MgSO4, 0.1 EDTA and 0.05 EGTA, pH 7.5,
25 °C) in the presence of 5 nM [3H]DTBZ and 1 nM–1 mM lobelane analogues
(final concentrations) for 30 min at room temperature. Nonspecific binding
was determined in the presence of 10 lM Ro4-12084. Assays were performed
in duplicate using UnifilterÀ96 96-well GF/B filter plates (presoaked in 0.5%
polyethylenimine) and terminated by harvesting using a FilterMate harvester.
After washing 5 times with 350
lL of the ice-cold wash buffer (in mM: 25
HEPES, 100 K2-tartrate, 5 MgSO4 and 10 NaCl, pH 7.5), filter plates were dried,
bottom-sealed and each well filled with 40 lL Packard’s MicroScint 20 cocktail.
Bound [3H]DTBZ was measured using a Packard TopCount NXT scintillation
counter and a Packard Windows NT-based operating system.