Synthesis of desformylflustrabromine and its evaluation as an α4β2 and α7 nACh receptor modulator

Article abstract of DOI:10.1016/j.bmcl.2007.06.047

Desformylflustrabromine (dFBr; 1) and desformylflustrabromine-B (dFBr-B; 2) have been previously isolated from natural sources, and the former has been demonstrated to be a novel and selective positive allosteric modulator of α4β2 nicotinic acetylcholine (nACh) receptors. The present study describes the synthesis of water-soluble salts of 1 and 2, and confirms and further investigates the actions of 1 and 2 using two-electrode voltage clamp recordings.

Full text of DOI:10.1016/j.bmcl.2007.06.047

Bioorganic & Medicinal Chemistry Letters 17 (2007) 4855–4860
Synthesis of desformylflustrabromine and its evaluation as an
a4b2 and a7 nACh receptor modulator
a
b
b
c
Jin-Sung Kim, Anshul Padnya, Maegan Weltzin, Brian W. Edmonds,
b
a,
*
Marvin K. Schulte and Richard A. Glennon
a
Department of Medicinal Chemistry, School of Pharmacy, Virginia Commonwealth University, Richmond, VA 23298, USA
Department of Chemistry and Biochemistry, Institute of Arctic Biology, University of Alaska Fairbanks, Fairbanks, AK 99775, USA
b
c
Department of Natural Sciences, University of Alaska Southeast, Juneau, AK 99801, USA
Received 11 June 2007; accepted 12 June 2007
Available online 14 June 2007
Abstract—Desformylflustrabromine (dFBr; 1) and desformylflustrabromine-B (dFBr-B; 2) have been previously isolated from nat-
ural sources, and the former has been demonstrated to be a novel and selective positive allosteric modulator of a4b2 nicotinic ace-
tylcholine (nACh) receptors. The present study describes the synthesis of water-soluble salts of 1 and 2, and confirms and further
investigates the actions of 1 and 2 using two-electrode voltage clamp recordings.
Ó 2007 Elsevier Ltd. All rights reserved.
Over the past decade or so, nicotinic acetylcholine
nACh) receptors have been targeted for the develop-
agents already have been proposed from docking studies
1
,10
(
using models of a3b4, a4b2, and a7 nACh receptors.
ment of agents with potential for the treatment of cer-
tain cardiovascular and neurological (e.g., Parkinson’s
disease, schizophrenia, Alzheimer’s disease) disorders,
Recently, desformylflustrabromine (dFBr; 1) was identi-
fied as a positive allosteric modulator of nACh recep-
tors. dFBr (1), desformylflustrabromine-B (dFBr-B; 2),
and at least a dozen additional indolic alkaloids have
been isolated and characterized from the marine bryo-
1
,2
memory and learning, appetite control, and pain.
These receptors are pentameric ion channel receptors
composed of heteromeric or homomeric assemblies
and, although numerous subtypes have been identified,
the most prevalent in brain are the a4b2 and the homo-
1
1–13
zoan Flustra foliacea.
1
meric a7 nACh receptors. A few recently developed li-
gands can differentiate between the latter two subtypes,
NH-CH3
NH-CH3
but few (if any) have been shown to selectively target
2
a4b2 or any other nACh receptor subtype. A novel
CH3
CH3
CH2
strategy for the modulation of nACh receptor subtypes
is the identification of allosteric modulators that interact
at receptors at a site distinct from that of the endoge-
nous ligand; they modulate the receptor protein via a
non-competitive mechanism. Negative allosteric modu-
lators of nACh receptors have received some attention
CH3
N
N
H
Br
Br
CH3
1
2
In particular, dFBr (1) and dFBr-B (2) (K = 3400 nM
i
1
,3–5
and >50,000 nM, respectively) lack appreciable affinity
for a4b2 receptors, and bind with >3500-fold lower
affinity at this receptor subtype than the standard ago-
(
published on positive nACh receptor allosteric modula-
reviewed
); however, relatively little work has been
1
,3,6,7
tors (or non-competitive agonists),
8
and it is not yet
known precisely how they work. Nevertheless, several
distinct binding domains for examples of the latter
,9
nist ligand (ꢀ)-nicotine (K < 1 nM); dFBr displays even
i
1
4
lower affinity (K > 50,000 nM) for a7 nACh receptors.
i
However, it has been further demonstrated with several
heteromeric (i.e., a3b2, a3b4, a4b2, a4b4) and the
homomeric a7 nACh receptors expressed in Xenopus
oocytes, that dFBr (1), but not dFBr-B (2), selectively
increases the whole cell current obtained when
Keywords: Nicotinic cholinergic receptors; Allosteric modulators.
*
Corresponding author. E-mail: glennon@vcu.edu
0
960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.06.047

4856
J.-S. Kim et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4855–4860
co-applied with the endogenous agonist ACh in
alkylated with 1-chloro-3-methyl-2-butene to afford 2
after deprotection (Scheme 1). Both 1 and 2 were ob-
tained in moderately good yields and were isolated as
their water-soluble hydrochloride salts.
1
5
a4b2-containing preparations. Single channel studies
with dFBr are consistent with the hypothesis that it in-
creases the channel-opening probability, perhaps by
increasing the ratio of the rate constants for channel
1
5
opening and closing. As such, dFBr offers a novel tool
for the investigation of nACh receptors, and serves as a
lead structure for the development of selective agents
with potential therapeutic application.
Proton NMR spectra were obtained for dFBr (1) and
dFBr-B (2) as their free bases (for comparison with their
1
1,14
literature
spectra of the free bases were consistent with those re-
spectra) and as their salts. In general, the
ported except that the N-CH signal for 1 appeared as
3
1
8
11
To date, studies with dFBr (1) and dFBr-B (2) have em-
ployed DMSO solutions of their water-insoluble free
bases as isolated directly from the marine organism.
The purpose of the present study was (a) to identify a
convenient synthetic route to 1 and 2, and to prepare
their water-soluble salts, and (b) to confirm, and further
examine, their actions at a4b2 and a7 nACh receptors.
a singlet at d 2.43 rather than at d 3.39. A small sig-
nal at the latter position was evident in the spectra of 1
but appeared to be that from deuterated MeOH used as
solvent.
1
5
Sala et al. identified a potentiating action of dFBr on
b2-containing nACh receptors. We evaluated the actions
of synthetic dFBr (1) and dFBr-B (2) as their HCl salts
at concentrations ranging from 1 nM to 100 lM on hu-
man a7 and a4b2 nACh receptors expressed in Xenopus
Tryptamine 7 (Scheme 1) was a key intermediate in the
synthesis of both 1 and 2. Although 7 has been previ-
1
6
19
ously reported, a different method of preparation
was employed here. In the present study, 6-bromoindole
was used in a Speeter/Anthony reaction and the result-
ing glyoxylamide was conveniently reduced to amine 6
using DMEA-alane, and then converted to 7 by treat-
oocytes with two-electrode voltage clamp techniques.
Application of 100 lM ACh produced a near maximal
response in a7 receptors (Fig. 1). Co-application of
>0.1 lM dFBr (1) and 100 lM ACh inhibited the re-
sponse to ACh. No potentiation of a7 responses was ob-
served over the range of dFBr (1) concentrations tested.
A dose–response curve obtained from multiple experi-
ments identical to that shown in Figure 1A is plotted
ment with (Boc) O.
2
Compound 1 was subsequently obtained from 7 by reac-
1
7
tion with prenyl 9-BBN followed by deprotection,
whereas the anion generated from 7 using NaH was
Cl
NH-CH3
O
O
O
O
a
b
N
H
N
H
Br
Br
73% Br
N
H
3
4
5
c
64%
Boc
Boc
CH3
N
CH3
N
NH-CH3
g
d
CH3 98%
Br
N
H
86%
N
N
H
Br
Br
CH3
9
7
6
f
91%
e
43%
Boc
2
CH3
N
CH3
CH3
f
Figure 1. dFBr (1) effects on a7 nACh receptors. dFBr (1 nM to
100 lM) was co-applied with 100 lM ACh to Xenopus oocytes
expressing human a7 nACh receptors. Responses were obtained using
two-electrode voltage clamp at a ꢀ60 mV holding potential. Peak
amplitudes were normalized to the responses obtained using 100 lM
ACh in the absence of dFBr (1). (A) Currents elicited by 100 lM ACh
at dFBr (1) concentrations of 0, 0.1, and 1.0 lM. (B) Concentration/
response curve for dFBr inhibition of peak currents elicited by 100 lM
ACh (IC50 = 44 lM).
1
N
H
CH2
92%
Br
8
Scheme 1. Reagents and conditions: (a) oxalyl chloride, Et
MeNH /H O, rt; (c) DMEA-alane/THF, D; (d) (Boc) O, Et
e) i— BuOCl/THF, ꢀ78 °C; ii—prenyl 9-BBN, THF, ꢀ78 °C;
f) i—TFA, CH Cl , 30 min, rt; ii—HCl/Et O; (g) i—NaH, DMF,
°C; ii—(Me) C@CHACH
2
O, rt; (b)
2
t
2
2
3
N, DMF;
(
(
2
2
2
18
0
2
2
Cl.

J.-S. Kim et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4855–4860
4857
in Figure 1B. Peak amplitudes of currents obtained from
a7 receptors were inhibited by dFBr (1)
a binding assay and as a potentiator of ACh responses
1
5
in a functional assay. Unlike Sala et al., we observe
inhibition of 100 lM ACh responses at high (>10 lM)
dFBr concentrations. However, the current traces
obtained at these concentrations were not typical of
those resulting from displacement of agonist. Displace-
ment of agonist typically produces a decrease in overall
response amplitude with little change in response
profiles. In our study, high dFBr concentrations pro-
duced currents with a greater increase in the apparent
rate and extent of desensitization rather than a simple
decrease in overall response amplitudes (see Fig. 2A).
This is particularly evident when comparing equal
amplitude responses in the presence of dFBr (Fig. 2A,
c and d); trace c (1 lM dFBr—a sub-inhibiting dFBr
concentration) shows a relatively slow apparent rate of
desensitization, while trace d (10 lM—an inhibiting
dFBr concentration) shows a much sharper peak with
a faster apparent rate of desensitization. If inhibition
were due to displacement of ACh binding, these two
response profiles would be expected to be similar. We
also typically observed rebound currents on washout
of ACh/dFBr (1) (Fig. 3). These currents are commonly
observed with open-channel block due to removal of the
channel blocker prior to dissociation of the agonist from
the receptor. The rebound currents are particularly
evident in the 10 lM responses as shown in Figure 3.
Our data support the hypothesis that open-channel
block contributes to inhibition of ACh responses by
dFBr. Similar effects have been observed for other
potentiating ligands including physostigmine and levam-
(
tion is consistent with its low affinity for the receptors.
IC50 = 44 ± 1 lM). The low potency for dFBr inhibi-
11
Our data demonstrate an inhibitory action of dFBr (1)
5
1
on a7 receptors; Sala et al. also observed slight inhibi-
tion of a7 receptors by dFBr (1).
On a4b2 receptors, co-application of 100 lM ACh and
dFBr (1) produced a biphasic response over the concen-
tration range tested (Fig. 2). Responses elicited by
1
1
00 lM ACh were potentiated by dFBr (1) (0.001–
00 lM), although an overlapping inhibition of the re-
sponse was observed at concentrations >10 lM dFBr
(
1), producing a bell shaped dose–response curve. Simi-
lar results were obtained when the slope of the rise time
of the response was plotted rather than peak amplitudes
(
data not shown). The IC50 value estimated for the
inhibitory component of this curve is 150 ± 0.2 lM. This
value is 44-fold higher than that reported by Peters
1
1
3
et al. (3.4 lM) for inhibition of [ H]epibatidine bind-
ing at rat a4b2 receptors. Similar to our data, Sala
et al. did not report inhibition of ACh responses at
1
5
these concentrations of dFBr but observed potentiation
at 3 lM. While Peters’ data showed displacement of
3
[
H]epibatidine binding it is not clear if this is due to
competitive or non-competitive effects. If dFBr binding
to an allosteric potentiation site reduces the affinity of
epibatidine, then dFBr could appear as an inhibitor in
2
0,21
isole.
concentrations may not be related to the inhibition of
Thus, the inhibition observed at high dFBr (1)
3
11
[ H]epibatidine binding observed by Peters et al. but
might occur via other mechanisms. Additional studies
will be necessary to fully explain inhibition of
[ H]epibatidine binding at potentiating dFBr (1) concen-
trations and the inhibition of response amplitudes at
higher dFBr (1) concentrations.
3
The potentiating component of the dose–response curve
obtained by co-application of dFBr (1) and 100 lM
ACh produced a half-maximal potentiation at a concen-
tration of 120 ± 0.6 nM dFBr (1) although the true
potency is likely obscured by the inhibitory component
of the dose–response curve (Fig. 2B). A maximum
Figure 2. dFBr (1) effects on a4b2 nACh receptors. (A) Responses
were obtained using two-electrode voltage clamp on Xenopus oocytes
expressing human a4b2 nACh receptors. The responses elicited by each
concentration of ACh/dFBr are indicated by the letters above the
current trace. The time of drug exposure is indicated by the horizontal
bar above the traces. Trace c (1 lM dFBr) produced a maximum
potentiation of the peak response. Reduced peak amplitudes are seen
at concentrations >1 lM dFBr (trace d). Peak amplitudes for each
trace are: a. 14 lA, b. 24 lA, c. 37 lA, d. 25 lA. (B) Dose/response
curve for dFBr applied in the presence of 100 lM ACh on human a4b2
receptors. Peak amplitudes were normalized to peak currents obtained
from 100 lM ACh in the absence of dFBr. Identical experimental
conditions were used in A and B. The half-maximal potentiation
observed is 120 nM (IC50 = 150 lM). Maximum potentiation was
Figure 3. Rebound currents evident on washout of ACh and dFBr.
Current traces were elicited by co-application of 100 lM ACh and
dFBr (10 and 30 lM). Experimental conditions are identical to those
shown in Figure 2. All three responses were obtained from a single
oocyte. The horizontal black bar above the traces indicates the
presence of ACh (a) or ACh + dFBr (b). Rebound currents are
particularly evident in trace b.
295% of the control trace (n > 4, 13 oocytes).

4858
J.-S. Kim et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4855–4860
potentiation of 295 ± 67% of the control trace was ob-
using holding potentials varying from ꢀ80 to ꢀ10 mV.
No effect of membrane potential on dFBr (1) potentia-
tion of a4b2 receptors was observed. A comparison of
ACh dose–response curves in the presence and absence
of 1 lM dFBr (Fig. 4) showed a decrease in the apparent
EC50 (24 ± 1 lM to 12 ± 1 lM) for ACh and a corre-
sponding increase in the maximal response obtained,
suggesting that potentiating effects may be due to bind-
ing of dFBr (1) to a site distinct from the agonist binding
site as has been proposed for other ACh potentiating
ligands.
served at approximately 3 lM dFBr (1). This is similar
1
5
to the 250% potentiation observed by Sala et al. To
determine the effect of holding potential on potentiation
by dFBr (1), voltage clamp experiments were performed
2
2
1
1
0
0
.5
.0
.5
.0
.5
.0
No dFBr
1μM dFBr
dFBr-B (2) was evaluated for its effects on human a7
and a4b2 nACh receptors (Fig. 5). Co-application of 2
with 100 lM ACh produced responses that were inhib-
ited when compared to those obtained using 100 lM
ACh alone (IC50 = 14 ± 1 lM). Inhibited response pro-
files were similar to those resulting from co-application
of dFBr (1) on a7 receptors; currents were decreased
in amplitude with no alteration in the apparent rate of
desensitization. In contrast, co-application of dFBr-B
-
9.0
-7.5
-6.0
-4.5
-3.0
Log [Ach(M)]
Figure 4. Acetylcholine concentration/response curve in the presence
and absence of dFBr (1). Responses were obtained using two-electrode
voltage clamp on Xenopus oocytes expressing human a4b2 receptors.
Peak amplitudes were normalized to peak currents obtained in the
presence of 100 lM ACh in the absence of dFBr. A comparison of
EC50 values with and without dFBr shows a shift in potency of ACh
for a4b2 receptors from 24 lM (without dFBr) to 12 lM (with dFBr).
dFBr increased the maximal response by 225% when compared to the
maximal peak amplitudes in the absence of dFBr. The data shown
reflect at least four repeated evaluations for each point on a minimum
of six oocytes.
(
2) with 100 lM ACh produced a slight potentiation
of responses (although the observed increases were not
statistically significant) followed by inhibition of the
responses (IC50 = 209 ± 4 lM). Inhibiting concentra-
tions of dFBr-B (2) produced responses showing
increased apparent desensitization rates and decreased
peak currents similar to those obtained for high
Figure 5. dFBr-B (2) effects on (A) a4b2 and (B) a7 nACh receptors. Currents elicited by 100 lM ACh in the presence of dFBr-B (2) using two-
electrode voltage clamp. dFBr-B (2) was co-applied at varying concentrations with 100 lM ACh to Xenopus oocytes expressing human a4b2 or
human a7 nACh receptors. (C) Concentration/response curve showing the normalized response to co-application of dFBr-B (2) and 100 lM ACh on
human a7 nACh receptors. (D) Concentration/response curve of the normalized response to co-application of dFBr-B (2) and 100 lM ACh on
human a4b2 nACh receptors. Peak amplitudes were normalized to peak currents elicited by 100 lM ACh in the absence of dFBr-B (2). Data reflect at
least four repeated evaluations for each point on a minimum of five oocytes.

J.-S. Kim et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4855–4860
4859
concentrations of dFBr (1) and dFBr-B (2) on a4b2
receptors (Fig. 5B). These data suggest that 1 and 2
may inhibit a4b2 responses by a similar mechanism.
Since both 1 and 2 alter apparent desensitization rates
on a4b2 but not a7 receptors their mechanism of action
might be different on each receptor subtype.
modulation is due to an increase in efficacy of ACh
and not simply a shift in EC50 value. Effects are
observed at dFBr concentrations ranging from 30 nM
to 100 lM although inhibition of peak amplitudes and
alterations in desensitization rates are observed at con-
centrations >3 lM. dFBr-B (2) inhibited ACh-induced
responses both on a7 and a4b2 receptors. Although
dFBr-B (2) does not significantly potentiate responses
to ACh on a4b2 receptors, our data show that it pos-
sesses a similar capacity for inhibiting response ampli-
tudes and altering apparent desensitization rates at
higher concentrations. Hence, similar effects produced
by 1 and 2 might be due to structural features associated
with the parent N-methyl-6-bromotryptamine moiety
common to both, whereas the potentiating effects of
dFBr (1) appear related to structural features not shared
by these two agents. The selectivity of dFBr (1) for
b2-containing nACh receptors makes it an ideal lead
molecule for future studies involving selective modula-
tion of a4b2 nACh receptors and a possible candidate
for the treatment of diseases such as autism or
Alzheimer’s disease in which a4b2 nACh receptors are
decreased in some brain regions. Selective, potentiating
ligands would produce increased response amplitudes
at nicotinic synapses without the loss of synaptic control
or desensitization that results from the use of nicotinic
agonists.
The synthetic HCl salts of dFBr (1) and dFBr-B (2) eval-
uated in this study behaved similarly to the compounds
obtained from F. foliacea. Two primary differences were
1
5
observed. First, while Sala et al. observed only poten-
tiation, we observed both potentiation and inhibition of
peak amplitudes by dFBr (1) on a4b2 receptors and
inhibition only on a7 receptors. IC50 values for inhibi-
tion of acetylcholine responses on a7 receptors agree
1
1
with the K values determined by Peters et al. in com-
i
petition binding experiments. IC50 values obtained on
a4b2 receptors were significantly higher than those
obtained by Peters for inhibition of [ H]epibatidine
3
binding and did not produce the decrease in overall
responses typical of competitive antagonists. The change
in response profiles, particularly the apparent loss of the
slow desensitizing phase and the presence of rebound
currents on washout of acetylcholine/dFBr, supports
the hypothesis that inhibition is due to open-channel
block at higher dFBr concentrations. In addition to
the inhibitory effects of dFBr, we also observed an in-
creased potency for potentiation and a slight increase
in the maximal amount of potentiation obtained
1
5
compared to data presented by Sala et al. While these
differences are not easily explained they could be due to
a difference in solubility between the synthetic HCl salt
used in our study and the compound purified from
Note added in proof
While our manuscript was being reviewed, Lindel et al.,
Organic Lett., 2007, 9, 283, published an alternative syn-
thesis of desformylflustrabromine.
15
F. foliacea used by Sala. Sala et al. utilized DMSO to sol-
ubilize dFBr due to its low solubility in buffer, while we
used a more soluble HCl salt that may have permitted
better dissolution of the compound. Decreased avail-
ability of dFBr due to DMSO solubilization or de-
creased concentrations of dFBr due to solubility
problems would be reflected in an apparent decrease in
potency for potentiation and may have prevented obser-
vation of inhibition. This conclusion is consistent with
the higher potency and maximal potentiation we
observed. In our preparation, dFBr showed potentiating
effects at concentrations as low as 30 nM, whereas Sala
References and notes
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. Glennon, R. A. In Progress in Medicinal Chemistry;
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4
15
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et al. reported no potentiation below 3 lM. The rapid
perfusion system used in the present study may also
have contributed to these differences. We use a modified
Xenopus oocytes recording chamber that permits solu-
tion changes that are significantly faster than those used
6
. Albuquerque, E. X.; Santos, M. D.; Alkondon, M.;
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001, 15, S19.
7
. Hurst, R. S.; Hajos, M.; Raggenbass, M.; Wall, T. M.;
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1
5
by Sala et al. This system more accurately detects peak
amplitudes prior to desensitization since solution ex-
change takes place within 200 ms. Slower perfusion
and exchange rates would result in smaller response
amplitudes due to receptor desensitization, and less
potentiation would be observed at lower dFBr
concentrations.
8
1
024.
The water-soluble HCl salt of dFBr (1) examined in this
study provides a novel, subtype selective modulator of
nACh receptors. We have confirmed that dFBr (1),
but not dFBr-B (2), is capable of increasing responses
on a4b2 receptors when co-applied with ACh. This
9
. Smulders, C. J. G. M.; Zwart, R.; Bermudez, I.; van Kleef,
R. G. D. M.; Groot-Kormelink, P. J.; Vijerberg, H. P. M.
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1
8
1
1
1
1
1
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by addition of prenyl 9-BBN, was purified by column
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foam.
Compound 9, from the anion of 7 (NaH/DMF) at 0 °C
under N , treated with 1-chloro-3-methyl-2-butene in
2
DMF; the resulting oil was purified by a column chroma-
tography (hexane/EtOAc = 5:1) to give a homogeneous,
colorless oil.
2002, 1056. www.znaturforsch.com.
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19. The cDNAs for human a7 and a4b2 receptors were
obtained from Dr. Jon Lindstrom’s laboratory. Subunit
cDNA was cloned into a pBud-CE4.1 (Invitrogen, CA)
vector prior to mRNA synthesis. Xenopus laevis frogs
and frog food were purchased from Xenopus Express
(Homosassa, FL). Ovarian lobes were surgically removed
1
1
6. Burm, B. E. A.; Meijler, M. M.; Korver, J.; Wanner, M. J.;
Koomen, G.-J. Tetrahedron 1998, 54, 6135.
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Depew, K. M.; Danishefsky, J. J. J. Am. Chem. Soc. 1999,
+2
from X. laevis frogs and washed twice in Ca -free
Barth’s buffer (82.5 mM NaCl/2.5 mM KCl/1 mM
121, 11964.
1
8. Compounds analyzed within 0.4% of theory for C, H, N
where indicated. The free base of dFBr (1) was obtained as
2
MgCl /5 mM Hepes, pH 7.4) then gently shaken with
1.5 mg/ml collagenase (Sigma type II, Sigma–Aldrich) for
1 h at 20–25 °C. Stage IV oocytes were selected for
microinjection. Synthetic cRNA transcripts for human a7
and a4b2 were prepared using the mMESSAGE mMA-
1
a pale yellow oil by deprotection of 8 with TFA at rt: H
NMR (CD
CH ), 2.71–2.80 (m, 2H, CH ), 2.95–3.04 (m, 2H, CH ),
3 3
OD) d 1.55 (s, 6H, 2CH ), 2.43 (s, 3H, N-
3
2
2
TM
5
.11 (dd, J = 1.2, 10.5 Hz, 1H, vinylic H), 5.13 (dd,
J = 1.2, 17.4 Hz, 1H, vinylic H), 6.18 (dd, J = 10.5,
7.4 Hz, 1H, vinylic H), 7.08 (dd, J = 1.8, 8.4 Hz, 1H,
ArH), 7.39 (d, J = 8.4 Hz, 1H, ArH), 7.47 (d, J = 1.8 Hz,
CHINE High Yield Capped RNA Transcription Kit
(Ambion, TX). Oocytes were injected with a total of
50 nL cRNA at a concentration of 0.2 ng/nL in appro-
priate subunit ratios then incubated at 19 °C for 24 to
72 h prior to their use in voltage clamp experiments.
Recordings were made using an automated two-electrode
voltage-clamp system incorporating an OC-725C oocyte
clamp amplifier (Warner Instruments, CT) coupled to a
computerized data acquisition (Datapac 2000, RUN
technologies) and autoinjection system (Gilson). Record-
ing and current electrodes with resistance 1–4 MX were
filled with 3 M KCl. Details of the chambers and
methodology employed for electrophysiological record-
1
1
1
H, ArH). 1ÆHCl (off-white solid; mp 211–214 °C);
H
), 2.61 (s, 3H, N-
NMR (DMSO-d
6
) d 1.49 (s, 6H, 2CH
3
CH ), 2.90–3.01 (m, 2H, CH
3
2 2
), 3.01–3.11 (m, 2H, CH ),
5.10 (dd, J = 1.2, 17.7 Hz, 1H, vinylic H), 5.11 (dd,
J = 1.2, 10.8 Hz, 1H, vinylic H), 6.14 (dd, J = 10.8, 17.7,
1
7
1
H, vinylic H), 7.13 (dd, J = 1.8, 8.4 Hz, 1H, ArH), 7.46–
.54 (m, 2H, ArH), 8.54 (br s, 2H, H O, HCl), 10.79 (br s,
2
H, NH); Anal. for C H Br N ÆHClÆH O. dFBr-B was
1
6
21
2
2
1
obtained as a pale yellow oil from 9; H NMR (CD
3
OD) d
2
1
1.79 (br s, 3H, CH ), 1.86 (br s, 3H, CH ), 2.42 (s, 3H, N-
CH ), 2.83–2.89 (m, 2H, CH ), 2.90–2.97 (m, 2H, CH ),
3
3
ings have been described earlier. Oocytes were held in a
vertical flow chamber of 200-lL volume and perfused
with ND-96 recording buffer (96 mM NaCl/2 mM KCl/
3
2
2
4.68 (br d, J = 6.9 Hz, 2H, allylic CH
2
), 5.35 (m, 1H,
vinylic H), 7.05 (br s, 1H, ArH), 7.14 (dd, J = 1.8, 8.7 Hz,
H, ArH), 7.47 (d, J = 8.7 Hz, 1H, ArH), 7.49 (d,
J = 1.8 Hz, 1H, ArH). 2ÆHCl (creamy-white solid; mp
2 2
1.8 mM CaCl /1 mM MgCl /5 mM Hepes; pH 7.4) at a
1
rate of 20 mL/min. Test compounds were dissolved in
ND-96 buffer and injected into the chamber at a rate of
20 mL/min using the Gilson auto-sampler injection sys-
tem. For dFBr (1) and dFBr-B (2) experiments, com-
pounds were co-applied with 100 lM ACh. Data
analysis: Concentration/response curves were fit by non-
linear curve fitting and GraphPad Prism Software (San
Diego, CA) using standard built-in algorithms. For IC50
determinations, data were fit to a single site competition
model. For the potentiation/inhibition curves obtained
for a4b2 modulation by dFBrÆHCl, the data were fit to a
bell shaped dose–response equation. This equation simul-
taneously fits both the potentiation and inhibitory effects
to give an EC50 for the potentiation component and an
IC50 for the inhibitory component. The accuracy by
which this algorithm separates these two constants is
dependent on the amount of difference between them. In
the case of dFBrÆHCl and dFBr-BÆHCl, this difference is
about 33-fold making it difficult to fully separate the two
values.
1
1
1
CH
61–163 °C); H NMR (DMSO-d
.82 (s, 3H, CH ), 2.57 (s, 3H, N-CH
), 3.07–3.18 (m, 2H, CH ), 4.73 (br d, J = 6.6 Hz, 2H,
allylic CH ), 5.32 (m, 1H, vinylic H), 7.18 (dd, J = 1.8,
6
) d 1.73 (s, 3H, CH
3
),
3
3
), 2.98–3.07 (m, 2H,
2
2
2
8
1
.7 Hz, 1H, ArH), 7.26 (s, 1H, ArH), 7.58 (d, J = 8.7 Hz,
H, ArH), 7.67 (d, J = 1.8 Hz, 1H, ArH), 8.82 (br s, 2H,
NH, HCl). Anal. for C H N ÆHCl.
1
6
21
2
Compound 5, prepared by reaction of oxalyl chloride with
3
followed by stirring with H NMe (40% in water) (off-
2
white solid; mp 249–252 °C, dec), has been prepared by a
1
6
different method, but no melting point was reported.
Compound 6, prepared by DMEA-alane (0.5 M in toluene)
reduction of 5 and purified by column chromatography
(
9
CH
2
Cl
2
/MeOH = 9:1 ! CH
2
Cl
2
/
4
MeOH/NH OH =
:1:0.1) (off-white solid, mp 112–114 °C), although reported
16
as a salt obtained by a different synthetic route, has not
been previously isolated as its free base.
Compound 7: Reaction of di-tert-butyl dicarbonate, Et N,
3
and 6 in DMF gave a crude product which was purified by
column chromatography (hexane/EtOAc = 2:1) to afford 7
20. Rauniyar, V.; Hall, D. G. J. Am. Chem. Soc. 2004, 126,
4518.
21. Joshi, P. R.; Suryanarayanan, A.; Schulte, M. K.
J. Neurosci. Methods 2004, 132, 69.
t
(
white solid, mp 120–124 °C).Compound 8, from BuOCl in
THF with 7 and Et N in THF at ꢀ78 °C under N , followed
3
2