Deconstruction of the α4β2 nicotinic acetylcholine receptor positive allosteric modulator desformylflustrabromine

Article abstract of DOI:10.1021/jm200834x

Desformylflustrabromine (dFBr; 1), perhaps the first selective positive allosteric modulator of α4β2 neuronal nicotinic acetylcholine (nACh) receptors, was deconstructed to determine which structural features contribute to its actions on receptors expressed in Xenopus ooycytes using two-electrode voltage clamp techniques. Although the intact structure of 1 was found to be optimal, several deconstructed analogs retained activity. Neither the 6-bromo substituent nor the entire 2-position chain is required for activity. In particular, reduction of the olefinic side chain of 1, as seen with 6, not only resulted in retention of activity/potency but in enhanced selectivity for α4β2 versus α7 nACh receptors. Pharmacophoric features for the allosteric modulation of α4β2 nACh receptors by 1 were identified.

Full text of DOI:10.1021/jm200834x

ARTICLE
pubs.acs.org/jmc
Deconstruction of the α4β2 Nicotinic Acetylcholine Receptor Positive
Allosteric Modulator Desformylflustrabromine
||
Nadezhda German,† Jin-Sung Kim,† Atul Jain,† Malgorzata Dukat,† Anshul Pandya,‡, Yilong Ma,§
Maegan Weltzin,‡ Marvin K. Schulte,‡ and Richard A. Glennon*,†
†Department of Medicinal Chemistry, School of Pharmacy, Virginia Commonwealth University, Richmond, Virginia 23298,
United States
‡Department of Chemistry and Biochemistry, Institute of Arctic Biology, and §Department of Biology and Wildlife,
University of Alaska Fairbanks, Fairbanks, Alaska 99775-7000, United States
ABSTRACT: Desformylflustrabromine (dFBr; 1), perhaps the first selective positive allosteric
modulator of α4β2 neuronal nicotinic acetylcholine (nACh) receptors, was deconstructed to determine
which structural features contribute to its actions on receptors expressed in Xenopus ooycytes using two-
electrode voltage clamp techniques. Although the intact structure of 1 was found to be optimal, several
deconstructed analogs retained activity. Neither the 6-bromo substituent nor the entire 2-position chain is
required for activity. In particular, reduction of the olefinic side chain of 1, as seen with 6, not only resulted
in retention of activity/potency but in enhanced selectivity for α4β2 versus α7 nACh receptors.
Pharmacophoric features for the allosteric modulation of α4β2 nACh receptors by 1 were identified.
’ INTRODUCTION
receptors, are usually active only in the presence of the orthosteric
agonist, and might achieve selectivity of effect if different subtypes of
receptors possess different allosteric binding sites.3
The neurotransmitter acetylcholine (ACh) has been impli-
cated in several neurological and neuropsychiatric disorders, such
as Alzheimer’s disease, autism, and schizophrenia, and might play
a role in cognition, memory, and pain.1ꢀ3 Receptors for ACh are of
two broad types: muscarinic receptors and nicotinic receptors.1,4
Muscarinic receptors (m1ꢀm5) are G-protein coupled, whereas
nicotinic acetylcholine (nACh) receptors are associated with ion
channels and are composed of five membrane-spanning subunits.1,4
Neuronal nACh receptors, an object of the present study, consist of
either α or α and β subunits; multiple neuronal nACh receptor
types are possible because several isoforms of α and β subunits exist
(α2-α10 and β2-β4).4 The two most prevalent neuronal nACh
receptor subtypes in human brain are the heteromeric α4β2
receptors and the homomeric α7 receptors. A large number of
nACh receptor agonists and antagonists have been identified
(reviewed in ref 4), but a persistent problem is their low (or lack
of) selectivity among the various nACh receptor subtypes.
Several years ago, a positive allosteric modulator of α4β2 nACh
receptors was serendipitously discovered, desformylflustrabromine
(dFBr, 1).6,7 Initially isolated from a North Sea marine organism,6 1
was later synthesized as a water-soluble salt.8 In addition to
increasing whole cell current when coapplied with effective con-
centrations of ACh to α4β2-containing preparations, 1 failed to
potentiate the actions of ACh at α3β2, α3β4, α4β4, and α7 nACh
receptors expressed in Xenopus oocytes.7ꢀ9 As such, 1can now serve
as a template for the development of newer, more selective agents.
Apart from direct-acting agonists and competitive antagonists
that bind at the same (i.e., orthosteric) site as ACh, an alternate
approach to influence or modulate the actions of ACh is via allosteric
modulation of its receptors.2,3,5 Positive allosteric modulators, for
example, can enhance the actions of an endogenous ligand (e.g., a
neurotransmitter such as ACh) by interaction at a binding site
distinct from the orthosteric site. By definition, endogenous neuro-
transmitters and other orthosteric ligands normally do not interact
with high affinity at an allosteric binding site(s), and conversely,
allosteric ligands do not bind with high affinity at the orthosteric site.
Consequently, due to lack of suitable design templates, it is virtually
impossible, in the absence of any other available information (e.g.,
on some other allosteric agent that might bind in a similar fashion),
to design allosteric agents de novo. Yet, the availability of allosteric
agents can be advantageous, because they typically fail to desensitize
Because of its actions as a positive allosteric modulator, desformyl-
flustrabromine (1) represents a novel lead structure; however,
nothing is known about its structureꢀactivity relationships for
producing this effect. In order to understand which structural features
of desformylflustrabromine (1) are required for, or contribute to, this
action, the structure of 1 was “deconstructed”. Other goals are to
identify a pharmacophore for the action of 1 as a positive allosteric
modulator at α4β2 nACh receptors and, more long-term, to identify
structures that retain this action but more readily lend themselves to
convenient scale-up synthesis in order to have available sufficient
Received: June 27, 2011
Published: September 09, 2011
r
2011 American Chemical Society
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Journal of Medicinal Chemistry
ARTICLE
Scheme 1. Synthesis of Target Compounds 4ꢀ6 and 10a
a The synthesis of 1 has been previously reported and is shown here only for comparison of methods. Reagents: (a) (1) CO2Cl2, Et2O; (2) MeNH2;
(3) dimethylethylaminealane, THF. (b) (Boc)2O, Et3N, DMF. (c) (1) t-BuOCl, THF; (2) allyltributyltin, BF3 Et2O, THF. (d) NaBH4, CoCl2, EtOH.
3
(e) TFA, CH2Cl2. (f) (1) tBuOCl, THF; (2) prenyl 9-BBN, THF. (g) H2 10% Pd/C.
Scheme 2. Synthesis of Target Compounds 7 and 8 a
quantities of compound for in vivo behavioral (and other) studies.
We began our investigation by attempting to determine exactly what
it is, structurally, that makes desformylflustrabromine (1) a positive
allosteric modulator at α4β2 nACh receptors.
’ CHEMISTRY
Compound 1 (as its HCl salt) was prepared exactly as previously
reported.8 That is, 6-bromoindole (13; Scheme 1) was subjected to a
Speeter/Anthony synthesis (i.e., reaction of an indole with oxalyl
chloride and treatment of the glyoxylyl chloride product with
methylamine, followed by reduction of the resulting glyoxylamide
with a hydride reducing agent, dimethylethylaminealane in the case
of 1) to afford tryptamine 3; protection of the basic amine of 3 with
a Boc group (i.e., 14) followed by prenylation (to 16) and acidic
deprotection provided 1. The primary amine counterpart of 1
(i.e., 2) was prepared in the same manner by prenylation of N-
Boc-protected 6-bromotryptamine, rather than 6-bromo-N-methyl-
a Reagents: (a) (1) t-BuOCl, THF; (2) prenyl 9-BBN, THF. (b) 10%
Pd/C, H2, 50 psi. (c) TFA, CH2Cl2.
tryptamine (14) used in the synthesis of 1, followed by deprotection.
Compound 14,8 however, was a useful intermediate for the
synthesis of several other desired targets (Scheme 1). Treatment of
14 with tert-butyl hypochlorite followed by allyl tributyltin and
boron trifluoride afforded intermediate15. Reduction of the double
bond of 15 using sodium borohydride and cobalt chloride,10
followed by deprotection, provided 4.
1H NMR signals indicated that two different positional isomers
with the same elemental composition were obtained, with 86% of
the product being the desired target 5 and 14% being the more
conjugated 10-alkenyl derivative. Neither isomer could be ob-
tained in pure form. Compound 15 was also employed in the
synthesis of 10 (Scheme 1); olefinic reduction of 15 and hydro-
genolysis of the halogen afforded 10 following deprotection.
The double bond of 168 was reduced using the same (i.e., sodium
borohydride and cobalt chloride) reduction procedure described
above (to yield 17), and deprotection of 17 afforded the desired 6
(Scheme 1).
Deprotection of 15 without the intervening reduction step should
have afforded 5. It appears, however, that some double-bond
migration occurred from the 20-position to the 10-position. That
1
is, although elemental analysis for 5 was correct, the H NMR
spectrum of the product showed several minor spurious peaks, such
as, for example, a small doublet of doublets at δ 1.85 (presumably
a CꢀCH3 signal). Elemental analysis and integration of the
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Journal of Medicinal Chemistry
ARTICLE
The desbromo analog of 1 (i.e., 7) was obtained from 18
(Scheme 2). Compound 18 was prepared in modest yield according
to a reported procedure.11 However, upon changing the reaction
solvent from dioxane to methylene chloride, the product could be
obtained in 80% yield as a white solid (mp 86ꢀ89 °C) with a 3-h
reaction time relative to that reported with dioxane (15 h). Com-
pound 8 was prepared from the same intermediate (i.e., 19) except
that the double bond was catalytically reduced prior to deprotection.
Compounds 9 and 12 were prepared in a similar fashion
(shown for 12 in Scheme 3). That is, the requisite indole (e.g.,
23) was treated with 1-dimethylamino-2-nitroethylene to afford
nitroalkene 24, which was readily reduced to primary amine 25;
acylation of 25 using ethyl chloroformate followed by reduction
of the resulting carbamate 26 using lithium aluminum hydride
provided the desired target 12. Compound 9 was prepared from
2-sec-butylindole (21) in like manner. The tert-butyl derivative
11 was prepared via a classical Speeter/Anthony synthesis from
2-tert-butylindole.
’ RESULTS AND DISCUSSION
A near-maximal effect in the present functional assays was
consistently observed at an ACh concentration of 10 μM. The decon-
structed analogs of 1 were examined using two-electrode voltage
clamp recordings with α4β2 nACh receptors expressed in Xenopus
oocytes in the presence and absence of this concentration of ACh.
None of the analogs produced an effect in the absence of ACh.
Several of the deconstructed analogs of 1 potentiated the effect
achieved by ACh (Table 1). As with 1 itself, all deconstructed
analogs that potentiated the action of ACh produced an inhibitory
effect at high concentrations (representative tracings are shown in
Figures 1 and 2). The mechanism underlying this action requires
further investigation; however, data obtained with 1 suggest that
the mechanism of the inhibitory component might be due to
open channel blockade.9
Scheme 3. Synthesis of Compound 12 a
At concentrations slightly higher than those required to poten-
tiate its ACh-enhancing action in the α4β2 receptor preparation,
1 inhibited (but failed to potentiate) the action of ACh in an α7
nACh receptor preparation.8 As an initial measure of selectivity,
the compounds prepared in this investigation were also examined
at α7 nACh receptors in functional assays (Table 2).
Effect of Deconstructed Analogs of 1 on α4β2 nACh
Receptor Function. The pEC50 for 1 (6.48; Table 1; EC50 ca.
0.32 μM) for the potentiation of 100 μM ACh was quite comparable
to what we originally reported (pEC50 ca. 6.8; EC50 = 0.12 μM);8 the
small difference likely reflects experimental variation and the much
a Reagents: (a) 1-dimethylamino-2-nitroethylene, TFA. (b) LiAlH4,
THF. (c) Cl-COOC2H5, CHCl3. The exact same method was employed
to synthesize 9 from 2-sec-butylindole (21).
Table 1. Effect of Deconstructed Analogs of Desformylflustrabromine (1) on the Action (potentiation or antagonism) of the
Functional Activity of ACh at α4β2 nACh Receptors Expressed in Xenopus Oocytes As Measured Using Two-Electrode Voltage
Clamp Methods
X
R1
R2
R3
agonist pEC50 ((SEM)
antagonist pIC50 ((SEM)
c
1
2
3a
ꢀBr
ꢀBr
ꢀBr
ꢀBr
ꢀBr
ꢀBr
ꢀH
ꢀH
ꢀH
ꢀH
ꢀH
ꢀH
ꢀCH3
ꢀCH3
ꢀ
ꢀCH3
ꢀCH3
ꢀ
ꢀCHdCH2
ꢀCHdCH2
ꢀ
6.48 (0.28)
6.29 (0.24)
NPb
NPb
NPb
ꢀ
c
ꢀ
4.40 (0.06)
4.14 (0.10)
5.45 (0.08)
4
ꢀH
ꢀH
ꢀCH2CH3
ꢀCHdCH2
ꢀCH2CH3
ꢀCHdCH2
ꢀCH2CH3
ꢀCH2CH3
ꢀCH2CH3
ꢀCH3
5
ꢀH
ꢀH
c
6
ꢀCH3
ꢀCH3
ꢀCH3
ꢀCH3
ꢀH
ꢀCH3
ꢀCH3
ꢀCH3
ꢀH
6.86 (0.16)
5.14 (3.45)
5.52 (1.03)
NPb
ꢀ
c
7
ꢀ
c
8
ꢀ
9
4.78 (0.55)
10
11
12
ꢀH
ꢀCH3
ꢀH
NPb
4.73 (0.06)
c
ꢀCH3
ꢀCH3
5.40 (1.50)
NPb
ꢀ
d
ꢀCH3
ꢀ
a N-Methyl-6-bromotryptamine. b NP = No potentiation of the action of ACh. c pIC50 could not be reliably determined. d Not determined.
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Figure 2. Concentration/response curves for representative decon-
structed analogs of desformylflustrabromine (1). Deconstructed com-
pounds 6 (A), 7 (B), and 8 (C) were coapplied with 100 μM ACh (i.e., a
concentration of ACh that produced the maximal effect or functio-
nal response = 1) on human α4β2 receptors. Peak amplitudes were
normalized to peak currents obtained from 100 μM ACh in the absence
of test compound. Curves were fit to a hormetic model. (A) For
compound 6, the half-maximal potentiation (EC50) is 0.10 ( 0.04 μM;
maximum potentiation was 290% of the controlled trace (100 μM
ACh). (B) For compound 7, EC50 = 6.77 ( 0.35 μM (250% of the
control trace). (C) For compound 8, EC50 = 1.22 ( 0.66 μM (260% of
the control trace).
Figure 1. Potentiation of ACh-evoked responses by representative
deconstructed analogs of desformylflustrabromine (1). Responses were
elicited by application of 100 μM ACh to Xenopus oocytes expressing
α4β2 nicotinic receptors. Test compounds 6ꢀ8 were coapplied with
ACh at the concentrations indicated. Each series of traces (A, B, and C
for compounds 6, 7, and 8, respectively) as shown here were elicited
from a single oocyte for each compound. Concentration/response
curves for these compounds are shown in Figure 2.
the saturated gem-dimethyl analog of 1, 6 (pEC50 = 6.86), was
at least as potent, if not twice as potent, as 1. Evidently, the
gem-dimethyl groups, but not the unsaturation associated with
the chain, is required for allosteric action.
greater number of determinations that now have been performed
with 1. The N-desmethyl primary amine counterpart of 1 (i.e., 2;
pEC50 = 6.29; Table 1) was found to be about half as potent as
1 as a positive allosteric modulator at α4β2 receptors. Hence,
all subsequent compounds retained the N-methyl group asso-
ciated with 1.
The next several compounds addressed the role of the
indole 2-position substituent. Complete elimination of the
2-position substituent (i.e., N-methyl-6-bromotryptamine, 3)
resulted in a compound that failed to enhance the action of
ACh at the highest concentrations at which it was evaluated
(Table 1). Similar results were obtained with the simple n-
propyl analog 4 (Table 1). Either unsaturation and/or at least
one of the gem-dimethyl groups is important for the ability of 1
to behave as a positive allosteric modulator at α4β2 nACh
receptors. The propenyl compound 5, lacking the gem-di-
methyl groups of 1, also lacked potentiating activity, whereas
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Table 2. Potency of Examined Analogs of Desformylflus-
trabromine (1), Relative to 1, To Inhibit the Effect of ACh on
Human α7 nACh Receptors Expressed in Xenopus Oocytes As
Measured Using Two-Electrode Voltage Clamp Methods
retained activity, that the intact structure of 1 seems optimal as a
positive allosteric modulator at α4β2 nACh receptors. The
reduced counterpart of 1 (i.e., 6), retains the action and potency
of 1; however, 6 offers no synthetic advantage in that it was
synthesized by reduction of 1. Also learned was that (a) N-
demethylation of 1 to its primary amine reduces potency by
50%, (b) the 6-bromo group of 1 (and 6) contributes to potency,
not action, (c) the presence of the gem-dimethyl groups plays an
important role in conferring activity, and (d) it is likely that the
quaternary nature of the branched carbon atom, not simply the
presence of gem-dimethyl groups, defines this action. Finally, as
with 1,8 analogs 2ꢀ11 were capable of attenuating the action of
ACh at α7 receptors; however, compound 6 was considerably
more selective than 1 as a positive allosteric modulator at α4β2
receptors. This might be an advantage for functional studies
where both receptor types are present. In summary, structural
features of 1 important for its action and potency as a positive
allosteric modulator at α4β2 nACh receptors were determined,
pharmacophoric features were identified, and 6 was found to be
at least as potent as, and more selective than, 1 in this regard.
pIC50
(SEM
1
5.73
5.24
4.96
4.68
4.53
4.59
5.11
4.69
5.01
4.35
5.32
NDa
0.05
0.14
0.32
0.10
0.14
0.24
0.14
0.20
0.10
0.19
0.11
2
3
4
5
6
7
8
9
10
11
12
a ND = Not determined.
Before continuing to investigate 2-position substitution, the
necessity of the 6-bromo group was examined. Desbromo 1 and 6
(i.e., 7 and 8; pEC50 = 5.14 and 5.52, respectively) (Table 1)
retained the allosteric action of their parents but were substan-
tially less potent. Apparently, the bromo group contributes to
potency, but its presence is not required for the compounds to
behave as positive allosteric modulators at α4β2 nACh receptors.
Further deconstruction of 7 and 8 provided analogs 9ꢀ12.
Again suggesting that the gem-dimethyl groups are important,
the racemic monomethyl counterpart of 8 (i.e., 9) (Table 1)
was inactive (as a positive allosteric modulator). This is further
supported by the inactivity of 10. Interestingly, although the
inactive compound 12 possesses a gem-dimethyl group, compound
11 (pEC50 = 5.40) is active (Table 1). It would seem that it is not
simply the presence of the gem-dimethyl group but, rather, the
quaternary nature of this carbon atom that contributes to activity.
Effect of Deconstructed Analogs of 1 on Blocking the
Functional Effects of ACh at α4β2 nACh Receptors. As
previously shown for 1,8 high concentrations of all the decon-
structed compounds blocked the action of 10 μM ACh at α4β2
nACh receptors. For those compounds acting as positive allos-
teric modulators, pIC50 values could not be determined with
accuracy. pIC50 values for compounds not acting as positive
allosteric modulators ranged from 5.45 for 5 to 4.14 for 4;
additional results are shown in Table 1. The mechanism(s)
whereby these analogs are able to functionally antagonize the
actions of ACh at α4β2 receptors remains to be elucidated.
Effect of deconstructed analogs of 1 on α7 nACh receptor
function. As an initial measure of selectivity, compounds were
examined for their ability to potentiate the electrophysiological
effects of ACh at α7 receptors. None proved to be active at the
highest concentrations examined. However, all of the examined
compounds inhibited the action of 10 μM ACh at α7 receptors
(Table 2). Interestingly, whereas 1 attenuated the action of ACh
at α7 receptors at a concentration of only 6 times greater than
that required for potentiation of ACh at α4β2 receptors, its
reduced counterpart 6 was 190-fold selective for the latter.
’ EXPERIMENTAL SECTION
Chemistry. Melting points were taken in glass capillary tubes on a
Thomas-Hoover melting point apparatus and are uncorrected. 1H NMR
spectra were recorded either with a Varian EM-390 or Bruker 400 MHz
spectrometer, and peak position are given in parts per million (δ)
downfield from tetramethylsilane as an internal standard. Purity of
compounds (>95%) was established by elemental analysis; microana-
lyses were performed by Atlantic Microlab (GA) for the indicated
elements, and the results are within 0.4% of theoretical values. Reactions
and product mixtures were routinely monitored by thin-layer chroma-
tography (TLC) on silica gel precoated F254 Merck plates, and chro-
matographic separations were performed using Aldrich Silica gel 60
columns unless otherwise stated.
6-Bromo-2-(1,1-dimethylallyl)tryptamine Hydrochloride
(2). Following the addition of tert-BuOCl (0.10 g, 0.9 mmol) to a stirred
solution of tert-butyl 2-(6-bromo-1H-indol-3-yl)ethylcarbamate12 (0.25 g,
0.7 mmol) and Et3N (0.90 g, 0.9 mmol) in THF (10 mL) at ꢀ78 °C,
stirring was allowed to continue for 45 min. Freshly prepared prenyl
9-BBN (1.5 mmol)13 was added in a dropwise manner over a 20-min
period while the temperature was maintained at ꢀ78 °C. The reaction
mixture was allowed to warm to room temperature and stirring was
continued for an additional 2 h. Aqueous NaOH (3 M, 3 mL) and H2O2
(30%, 3 mL) were added in a dropwise fashion and stirring continued for
another 1 h. The reaction mixture was diluted with Et2O (100 mL), and
the organic portion was washed with H2O (3 ꢁ 30 mL) and brine
(40 mL) and dried (Na2SO4). The solution was evaporated to dryness
and the residue was purified by chromatography using hexanes/EtOAc
(10:1) as eluent to afford 0.10 g (33%) of a white foam.
Gaseous HCl was bubbled into a stirred solution of the above
compound (0.09 g, 0.22 mmol) in dry EtOAc (10 mL) at 0 °C; stirring
was allowed to continue for 24 h, and the solvent was evaporated to yield
a white solid. Recrystallization from EtOAc/MeOH provided 0.03 g
(33%) of 2 as white crystals: mp 256ꢀ258 °C; 1H NMR (DMSO-d6) δ
1.49 (s, 6H, 2CH3), 2.85 (t, 2H, CH2), 3.05 (t, 2H, CH2), 5.05ꢀ5.10 (m,
2H, CH), 6.10ꢀ6.17 (m, 1H, CH), 7.12 (dd, 1H, ArH), 7.48ꢀ7.51 (m,
+
2H, ArH), 7.98 (br s, 3H, NH3 ), 10.76 (s, 1H, NH). Anal. Calcd for
(C15H19BrN2 HCl 0.25H2O) C, H, N.
3
3
N-Methyl-6-bromo-2-n-propyltryptamine Oxalate (4). Ac-
cording to the procedure described for preparation of 17, compound 15
(0.50 g, 1.27 mmol) was treated with NaBH4 (0.10 g, 2.54 mmol) and
’ CONCLUSION
Deconstruction of the structure of the positive allosteric
modulator 1 has shown, although several “deconstructed” analogs
CoCl2 6H2O (0.3 g, 1.27 mmol) to give 0.40 g (78%) of the reduced
3
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Journal of Medicinal Chemistry
ARTICLE
compound as a white solid: mp 157ꢀ159 °C; 1H NMR (CDCl3) δ 1.01
(t, J = 7.2 Hz, 3H, CH3), 1.42 (s, 9H, t-Bu), 1.62ꢀ1.77 (m, 2H, CH2),
2.63ꢀ2.78 (m, 2H, CH2), 2.80ꢀ2.96 (m, 2H, CH2), 2.88 (s, 3H,
NꢀCH3), 3.33ꢀ3.46 (m, 2H, CH2), 2.20 (dd, J = 1.5, 8.4 Hz, 1H, ArH),
7.35ꢀ7.47 (m, 2H, ArH), 7.86 (br s, 1H, NH).
manner to a stirred solution of 1811 (0.49 g, 1.8 mmol) and Et3N (0.30 mL,
2.2 mmol) in anhydrous THF (7.0 mL) at ꢀ78 °C. The clear solution was
allowed to stir for an additional 0.5 h before freshly prepared prenyl
9-BBN13 (0.68 g, 3.59 mmol) solution in THF was added in a dropwise
manner. After 30 min the reaction mixture was allowed to warm to room
temperature, and stirring was continued for 1 h. The addition of 3 M NaOH
(1.8 mL) and 30% H2O2 (1.8 mL) was followed by stirring for 1 h. The
reaction mixture was diluted with Et2O (30 mL), the organic layer was
washed with 3 M NaCl solution (3 ꢁ 50 mL) and dried (Na2SO4), and
solvent was removed under reduced pressure. The resultant residue was
subjected to flash chromatography (hexanes/EtOAc 10:1) to give 0.30 g
(40%) of 19 as an oil which crystallized upon standing: mp 141ꢀ143 °C;
1H NMR (CDCl3): δ 1.51 (s, 9H, Boc), 1.58 (s, 6H, 2CH3), 2.94 (s, 3H,
NꢀCH3), 2.93ꢀ3.10 (m, 2H, CH2), 3.38ꢀ3.52 (m, 2H, CH2), 5.18ꢀ5.22
(m, 2H, vinylic H), 6.16 (dd, J = 10.5, 17.1 Hz, 1H, vinylic H), 7.16 (m, 2H,
ArH), 7.30ꢀ7.35 (m, 1 H, ArH), 7.61 (m, 1H, ArH), 7.91 (br s, 1H, NH).
Gaseous HCl was bubbled through a solution of 19 (0.10 g, 0.29
mmol) in anhydrous EtOAc (10 mL). The salt was recrystallized from
MeOH/anhydrous Et2O to afford 0.04 g (50%) of 7 as an off-white
solid: mp 223ꢀ225 °C; 1H NMR (DMSO-d6) δ 1.56 (s, 6H, 2CH3),
2.61 (s, 3H, NꢀCH3), 2.90ꢀ3.00 (m, 2H, CH2), 3.02ꢀ3.12 (m, 2H,
CH2), 5.12 (dd, J = 1.2, 17.0 Hz, 2H, vinylic H), 6.16 (dd, J = 10.5, 17.0
Hz, 1H, vinylic H), 6.88ꢀ7.08 (m, 2H, ArH), 7.30 (d, J = 8.0 Hz, 1H,
ArH), 7.59 (d, J = 8.0 Hz, 1H, ArH), 10.61 (br s, 1H, NH). Anal. Calcd
The reduced product was deprotected as follows: TFA (0.8 mL) was
added to a solution of the above compound (0.13 g, 0.33 mmol) in
CH2Cl2 (5 mL) at room temperature, and the reaction mixture was
allowed to stir for 0.5 h. An additional 10 mL of CH2Cl2 was added; the
solution was cooled to 0 °C, treated with a saturated solution of NaHCO3,
and extracted with EtOAc (3 ꢁ 10 mL). The combined organic portion
was dried (Na2SO4) and the solvent was removed. The crude product
was purified by column chromatography (CH2Cl2/MeOH/NH4OH
1
9:1:0.1) to give 0.10 g (98%) of the free base of 4 as a brown oil: H
NMR (CDCl3) δ 0.96 (t, J = 7.2 Hz, 3H, CH3), 1.59ꢀ1.74 (m, 2H,
CH2), 2.52 (s, 3H, NꢀCH3), 2.63ꢀ2.73 (m, 2H, CH2), 2.86ꢀ3.03
(m, 4H, 2CH2), 4.65 (br s, 1H, amine NH), 7.16 (dd, J = 1.8, 8.4 Hz, 1H,
ArH), 7.35ꢀ7.43 (m, 2H, ArH), 8.25 (br s, 1H, NH). The free base in
Et2O (5 mL) was treated with ethereal oxalic acid. The precipitated
oxalate salt was collected by filtration, washed with anhydrous Et2O
(3 ꢁ 5 mL), and recrystallized from absolute EtOH/anhydrous Et2O to
afford 0.05 g (37%) of the salt as a off-white solid: mp 211ꢀ213 °C; 1H
NMR (DMSO-d6) δ 0.91 (t, J = 7.2 Hz, 3H, CH3), 1.58ꢀ1.72 (m, 2H,
CH2), 2.60 (s, 3H, NꢀCH3), 2.63ꢀ2.69 (m, 2H, CH2), 2.91ꢀ3.04 (m,
4H, 2CH2), 7.09 (dd, J = 1.5, 8.4 Hz, 1H, ArH), 7.41ꢀ7.48 (m, 2H, ArH),
for (C16H22N2 HCl) C, H, N.
3
11.09 (br s, 1H, NH). Anal. Calcd for (C14H19BrN2 C2H2O4) C, H, N.
N-Methyl-2-(1,1-dimethylpropyl)tryptamine Hydrochlor-
ide (8). A catalytic amount of 10% Pd/C (0.02 g) was added to 19 (0.09
g, 0.26 mmol) in MeOH (15 mL) and hydrogenated at ca. 50 psi for 3 h.
The catalyst was removed by filtration, and the solvent was removed
under reduced pressure. Compound 20 (0.09 g, 92%; mp 172ꢀ174 °C)
was obtained as an off-white solid and used without further purification.
Gaseous HCl was bubbled through a solution of 20 (0.09 g, 0.25
mmol) in dry EtOAc (10 mL). The salt was recrystallized from MeOH/
anhydrous Et2O to afford 0.04 g (50%) of 8 as an off-white solid: mp
234ꢀ235 °C; 1H NMR (DMSO-d6) δ 0.69 (t, J = 7.5 Hz, 3H, CH3), 1.41
(t, J = 12 Hz, 6H, 2CH3), 1.73 (q, J = 7.5 Hz, 2H, CH2), 2.58 (s, 3H,
NꢀCH3), 2.87ꢀ3.00 (m, 2H, CH2), 3.05ꢀ3.23 (m, 2H, CH2), 6.88ꢀ7.08
(m, 2H, ArH), 7.30 (d, J = 7.5 Hz, 1H, ArH), 7.59 (d, J = 7.5 Hz, 1H, ArH),
10.51(brs, 1H, NH). Anal. Calcdfor(C16H24N2 HCl 0.25H2O) C, H, N.
3
N-Methyl-2-allyl-6-bromotryptamine Oxalate (5). Accord-
ing to the procedure described for the preparation of 4, compound 15
(0.07 g, 0.17 mmol) was treated with TFA (0.5 mL) to give 0.04 g (74%)
of the free base 5 as a pale-yellow oil which was treated with ethereal
oxalic acid to afford the oxalate salt. Recrystallization from absolute
EtOH/anhydrous Et2O afforded 0.02 g (24%) of product as a pale-
yellow solid: mp 198ꢀ203 °C (dec); 1H NMR (DMSO-d6) δ 2.62 (s,
3H, NꢀCH3), 2.91ꢀ3.08 (m, 4H, 2CH2), 3.50 (d, J = 6.0 Hz, 2H, allylic
CH2), 5.08ꢀ5.20 (m, 2H, vinylic H), 5.99 (m, 1H, vinylic H), 7.13 (br d,
J = 8.7 Hz, 1H, ArH), 7.43ꢀ7.53 (m, 2H, ArH), 11.09 (br s, 1H, NH).
Anal. Calcd for (C14H17BrN2 C2H2O4) C, H, N. Note: subsequent
3
studies showed this product to be an isomeric mixture of positional
isomers (see the text).
3
3
N-Methyl-6-bromo-2-(1,1-dimethylpropyl)tryptamine
Oxalate (6). Sodium borohydride (0.02 g, 0.37 mmol) was added in
N-Methyl-2-sec-butyltryptamine Oxalate (9). Ethyl chloro-
formate (0.06 mL, 0.65 mmol) was added in a dropwise manner to a
solution of 22 (0.14 g, 0.65 mmol) in CHCl3 (5 mL) at 0 °C, followed by
addition of aqueous 4 M NaOH (0.17 mL, 0.65 mmol). The reaction
mixture was allowed to stir at room temperature for 2 h and diluted with
CHCl3 (20 mL). The organic portion was separated, washed with H2O
(20 mL), and dried (Na2SO4), and solvent was removed under reduced
pressure to afford 0.14 g (75%) of N-Boc-22 as a brown oil: 1H NMR
(CDCl3) δ 0.95 (t, J = 6 Hz, 3H, CH3), 1.21ꢀ1.61 (m, 6H, 2CH3),
1.62ꢀ2.12 (m, 2H, CH2), 3.03 (m, 3H, CH2, CH), 3.32ꢀ3.70 (m, 2H,
CH2), 3.95ꢀ4.30 (m, 2H, CH2), 7.13ꢀ7.21 (m, 2H, ArH), 7.38 (d, J = 9
Hz, 1H, ArH), 7.62 (d, J = 9 Hz, 1H, ArH), 8.33 (br s,1H, NH). The
crude reaction product was directly used in the next step.
portions to a solution of 168 (0.08 g, 0.18 mmol) and CoCl2 6H2O
3
(0.04 g, 0.18 mmol) in EtOH (2 mL) at 0 °C. The reaction mixture was
allowed to stir under a N2 atmosphere at room temperature for 1 h and
quenched by addition of H2O (5 mL). The aqueous solution was
extracted with Et2O (3 ꢁ 10 mL). The combined organic portion was
dried (Na2SO4) and solvent was removed under reduced pressure. The
crude product was purified by column chromatography (hexane/EtOAc
5:1) to give 0.06 g (76%) of 17 as a white solid: mp 154ꢀ157 °C; 1H
NMR (CDCl3) δ 0.79 (t, J = 7.2 Hz, 3H, CH3), 1.47 (s, 6H, 2CH3), 1.52
(s, 9H, t-Bu), 1.78 (q, J = 7.2 Hz, 2H, CH2), 2.93 (s, 3H, NꢀCH3),
3.02ꢀ3.09 (m, 2H, CH2), 3.36ꢀ3.46 (m, 2H, CH2), 7.19 (dd, J = 1.5,
8.4 Hz, 1H, ArH), 7.41ꢀ7.48 (m, 2H, ArH), 7.92 (br s, 1H, NH).
The reduced product (0.08 g, 0.18 mmol) was treated with TFA
(0.45 mL) to give the free base of 6 (0.06 g, 96%) as a brown oil which
was converted to the oxalate salt: mp 192ꢀ194 °C following recrys-
tallization from absolute EtOH/anhydrous Et2O; 1H NMR (DMSO-d6)
δ 0.68 (t, J = 7.2 Hz, 3H, CH3), 1.39 (s, 6H, 2CH3), 1.70 (q, J = 7.2 Hz,
2H, CH2), 2.63 (s, 3H, NꢀCH3), 2.90ꢀ3.01 (m, 2H, CH2), 3.06ꢀ3.16
(m, 2H, CH2), 7.10 (dd, J = 1.5, 8.7 Hz, 1H, ArH), 7.44 (d, J = 1.5 Hz,
1H, ArH), 7.48 (d, J = 8.7 Hz, 1H, ArH), 10.70 (br s, 1H, NH). Anal.
A solution of the above product (0.14 g, 0.48 mmol) in anhydrous
THF (5 mL) was added in a dropwise manner to a stirred suspension of
LiAlH4 (0.11 g, 3 mmol) in dry THF (10 mL) at 0 °C. The stirred
mixture was heated at reflux under a N2 atmosphere for 2 h; cooled to
0 °C; successively quenched with MeOH (1 mL), H2O (1.5 mL), and
NaOH (3 M, 1 mL); and diluted with CH2Cl2 (10 mL). The organic
portion was dried (Na2SO4) and concentrated under reduced pressure.
The crude residue was purified on a silica gel column using CH2Cl2/
MeOH (9:1) f CH2Cl2/MeOH/Et3N (9:1:0.1) as eluent to afford the
amine as a brown oil. The oxalate salt was prepared and recrystallized
from MeOH/anhydrous Et2O to give 0.08 g (48%) of 9 as a beige solid:
mp 180ꢀ181 °C; 1H NMR (DMSO-d6) δ 0.79 (t, J = 6.9 Hz, 3H, CH3),
Calcd for (C16H23BrN2 C2H2O4) C, H, N.
3
N-Methyl-2-(1,1-dimethylallyl)tryptamine Hydrochloride
(7). tert-Butyl hypochlorite (0.19 mL, 2.15 mmol) was added in a dropwise
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Journal of Medicinal Chemistry
ARTICLE
1.29 (d, J = 6.9 Hz, 3H, CH3), 1.57ꢀ1.72 (m, 2H, CH2), 2.57ꢀ2.67
(m, 4H, 2CH2), 2.62 (s, 3H, CH3), 2.93ꢀ3.00 (m, 1H, CH), 6.95ꢀ7.08
(m, 2H, ArH), 7.29 (d, J = 7.5 Hz, ArH), 7.50 (d, J = 7.5 Hz, 1H, ArH),
amine as a light-yellow oil: 1H NMR (DMSO-d6) δ 1.28 (d, J = 9 Hz, 6H,
2CH3), 2.51 (s, 3H, CH3), 2.60ꢀ2.70 (m, 2H, CH2), 2.74ꢀ2.81 (m,
2H, CH2), 3.22 (m, 1H, CH), 6.90ꢀ7.00 (m, 2H, ArH), 7.25 (d, J = 9
Hz, 1H, ArH), 7.48 (d, J = 9 Hz, 1H, ArH).
+
10.79 (s, 1H, NH3 ). Anal. Calcd for (C15H22N2 C2H2O4 0.25H2O)
3
3
C, H, N.
Gaseous HCl was bubbled through a solution of the amine in dry
EtOAc (10 mL) to obtain 0.04 g (50%) of 12 as white crystals after
N-Methyl-2-(n-propyl)tryptamine Oxalate (10). Asuspension
of 4(0.13 g, 0.33 mmol) in absolute EtOH (10 mL) was hydrogenated in the
presence of 10% Pd/C (0.03 g) under a H2 atmosphere (45 psi) at room
temperature for 1 h. The catalyst was removed by filtration, and the filtrate
was evaporated to give 0.10 g (100%) of a cream-colored solid: mp
142ꢀ144 °C; 1H NMR (CDCl3) δ 1.01 (t, J = 7.5 Hz, 3H, CH3), 1.44
(s, 9H, 3CH3), 1.64ꢀ1.80 (m, 2H, CH2), 2.74 (t, J = 7.8 Hz, 2H, CH2),
2.82ꢀ3.00 (m, 2H, CH2), 2.90 (s, 3H, NꢀCH3), 3.43 (t, J = 7.8 Hz, 2H,
CH2), 7.10(dt, J = 1.5, 6.9 Hz, 1H, ArH), 7.14 (dt, J= 1.5, 6.9 Hz, 1H, ArH),
7.30 (dd, J = 1.5, 6.9 Hz, 1H, ArH), 7.56 (m, 1H, ArH), 7.83 (br s, 1H, NH).
Using the procedure described for 4, the amine (0.1 g, 0.31 mmol)
was treated with TFA (0.8 mL) to give 0.06 g (93%) of a pale-yellow oil:
1H NMR (CDCl3) δ 1.01 (t, J = 7.5 Hz, 3H, CH3), 1.64ꢀ1.78 (m, 2H,
CH2), 2.47 (s, 3H, NꢀCH3), 2.75 (t, J = 7.5 Hz, 2H, CH2), 2.83ꢀ3.00
(m, 4H, 2CH2), 7.10 (dt, J = 1.5, 6.9 Hz, 1H, ArH), 7.15 (dt, J = 1.5, 6.9
Hz, 1H, ArH), 7.30 (m, 1H, ArH), 7.58 (dd, J = 1.5, 6.9 Hz, 1H, ArH),
8.00 (br s, 1H, NH). The free base in Et2O (5 mL) was treated with an
ethereal solution of oxalic acid to afford0.07 g (69%) of10: mp 177ꢀ179 °C
after recrystallization from absolute EtOH/anhydrous Et2O; 1H NMR
(DMSO-d6) δ 0.92 (t, J = 7.2 Hz, 3H, CH3), 1.56ꢀ1.74 (m, 2H, CH2),
2.60 (s, 3H, NꢀCH3), 2.67 (t, J = 7.5 Hz, 2H, CH2), 2.88ꢀ3.06 (m, 4H,
2CH2), 6.91ꢀ7.04 (m, 2H, ArH), 7.25 (br d, J = 7.8 Hz, 1H, ArH),
7.48 (br d, J = 7.8 Hz, 1H, ArH), 10.85 (br s, 1H, NH). Anal. Calcd for
1
recrystallization from MeOH/anhydrous Et2O: mp 212ꢀ213 °C; H
NMR (DMSO-d6) δ 1.30 (d, J = 6.9 Hz, 6H, 2 ꢁ CH3), 2.59 (s, 3H,
CH3), 2.92ꢀ3.05 (m, 4H, 2CH2), 3.24 (m, 1H, CH), 6.95ꢀ7.08 (m,
2H, ArH), 7.29 (d, J = 7.5 Hz, 1H, ArH), 7.53 (d, J = 7.5 Hz, 1H, ArH),
+
9.00 (s, 1H, NH), 10.87 (br br s, 1H, NH3 ). Anal. Calcd for
(C14H20N2 HCl) C, H, N.
3
N-[2-(2-Allyl-6-bromo-1H-indol-3-yl)ethyl]-N-methylcar-
bamic Acid tert-Butyl Ester (15). tert-Butyl hypochlorite (0.58 mL,
5.10 mmol) was added in a dropwise manner to a stirred solution of 148
(1.50 g, 4.25 mmol) and Et3N (0.71 mL, 5.10 mmol) in THF (25 mL) at
ꢀ78 °C under a N2 atmosphere. After 1.5 h at ꢀ78 °C, allyltributyltin
(2.61 mL, 8.49 mmol) was added followed by addition of BF3 Et2O
3
(1.1 mL, 8.49 mmol). The reaction mixture was allowed to stir for 0.5 h
at the same temperature, and then quenched by addition of a saturated
aqueous solution of NaHCO3 (10 mL). Ethyl acetate (100 mL) and
H2O (100 mL) were added, and the aqueous portion was extracted
with EtOAc (3 ꢁ 50 mL). The combined organic portion was dried
(Na2SO4) and solvent was removed under reduced pressure. The
residue was purified by column chromatography (hexane/EtOAc 7:1)
to give 0.74 g (44%) of a white solid: mp 138ꢀ140 °C. The product was
immediately used in the synthesis of 4.
2-sec-Butyltryptamine (22). A solution of 2-sec-butylindole
(21)14 (0.35 g, 2 mmol) in CH2Cl2 (2 mL) was added to a stirred
solution of 1-dimethylamino-2-nitroethylene (0.23 g, 2 mmol) in TFA
(1.2 mL) at 0 °C. The reaction mixture was allowed to stir for 2 h, poured
into ice-cold H2O (50 mL), and extracted with EtOAc (2 ꢁ 20 mL). The
combined organic portion was washed with brine (20 mL) and dried
(Na2SO4), and solvent was removed under reduced pressure. The residue
was recrystallized from EtOAc to yield the corresponding nitroalkene
(0.27 g, 75% based on recovered starting material) as orange crystals: mp
141 °C;1H NMR(CDCl3) δ 0.94 (t, J = 7.5 Hz, 3H, CH3), 1.43 (d, J = 7.0
Hz, 3H, CH3), 1.72ꢀ1.84 (m, 2H, CH2), 3.28 (m, 1H, CH), 7.30ꢀ7.34
(m, 2H, ArH), 7.42ꢀ7.48 (m, 1H, CH), 7.72ꢀ7.78(m, 1H, CH), 7.87 (d,
J = 13 Hz, 1H, ArH), 8.40 (d, J = 13 Hz, 1H, ArH), 8.74 (br s, 1H, NH).
A solution of the alkene (0.27 g, 1.1 mmol) in dry THF (5 mL) was
added in a dropwise manner at 0 °C to a stirred suspension of LiAlH4
(0.26 g, 6.9 mmol) in anhydrous THF (10 mL). The reaction mixture
was heated overnight at reflux under a N2 atmosphere; cooled to 0 °C;
quenched with MeOH (1 mL), H2O (1.5 mL), NaOH (1 mL); and
diluted with CH2Cl2 (30 mL). After acid (2 N HCl, 30 mL)/base (3 N
NaOH, 30 mL) extraction, the organic portion was separated and dried
(Na2SO4), and solvent was removed under reduced pressure to give 0.14
g (62%) of 22 as a light-brown oil: 1H NMR (CDCl3) δ 0.91 (t, J = 7.5
Hz, 3H, CH3), 1.33 (d, J = 6.0 Hz, 3H, CH3), 1.62ꢀ1.79 (m, 2H, CH2),
2.84ꢀ2.97 (m, 2H, CH2), 2.98ꢀ3.12 (m, 3H, CH, CH2), 7.03ꢀ7.25
(m, 2H, ArH), 7.33 (d, J = 8.0 Hz, 1H, ArH), 7.82 (br s, 1H, NH).
2-Isopropyl-1H-indole (23). A solution of nBuLi (1.6 M in
hexane, 14 mL, 22.4 mmol) was added to a stirred solution of N-(o-
tolyl)isobutyramide15 (2.0 g, 11 mmol) in anhydrous THF (20 mL) at
0 °C. After stirring for 1 h, the reaction mixture was allowed to warm
gradually to room temperature. After an additional 8 h, EtOAc (15 mL)
and then saturated aqueous NH4Cl (5 mL) were added. The organic
portion was separated and dried (Na2SO4) and solvent was removed
under reduced pressure. The crude oily residue was purified by column
chromatography using hexanes/EtOAc (20:1) as eluent to afford 0.70 g
(66% based on recovered material) of 23 as an off-white solid: mp
75ꢀ76 °C (lit.16 mp 73ꢀ74 °C); 1H NMR (CDCl3) δ 1.41 (d, J = 6 Hz,
6H, 2 ꢁ CH3), 3.09ꢀ3.16 (m, 1H, CH), 6.31 (s, 1H, CH), 7.10ꢀ7.16
(C14H20N2 C2H2O4) C, H, N.
3
N-Methyl-2-(tert-butyl)tryptamine Hydrogen Oxalate (11).
Oxalyl chloride (0.2 mL, 2.308 mmol) was added in a dropwise manner
at ꢀ5 °C to a stirred solution of 2-tert-butyl-1H-indole14 (0.02 g, 1.154
mmol) in anhydrous Et2O (10 mL). The reaction mixture was allowed
to stir for 6 h at 0 °C; after evaporation of solvent under reduced
pressure, methylamine (40% aqueous solution, 5 mL) was added at
room temperature, and the reaction mixture was allowed to stir over-
night. The precipitate was collected by filtration and recrystallized from
MeOH to afford 0.17 g (56%) of the corresponding glyoxylamide as
brown solid: mp 190ꢀ191 °C; 1H NMR (CDCl3) δ 1.46 (s, 9H, 3CH3),
3.1 (s, 3H, NꢀCH3), 6.77 (s, 1H, NH), 7.07ꢀ7.45 (m, 4H, ArH), 7.76
(br s, 1H, NH).
A solution of the glyoxylamide (0.17 g, 0.65 mmol) in dioxane (5 mL)
was added to a stirred suspension of LiAlH4 (0.25 g, 6.5 mmol) in
dioxane (10 mL) at 60 °C and then heated at reflux overnight. The
reaction mixture was cooled to room temperature and diluted with THF
(10 mL), MeOH (3 mL), and NaOH (3N, 3 mL). The mixture was
filtered and the filtrate was heated at reflux with THF (15 mL). The
combined organic portion was evaporated to dryness under reduced
pressure, and the residue was purified by column chromatography using
CH2Cl2/MeOH (10:1) as eluent to afford the desired amine. The
oxalate salt was prepared to give 0.03 g (56%) of 11 as white flakes after
recrystallization from 2-PrOH: mp 194ꢀ195 °C; 1H NMR (DMSO-d6)
δ 1.43 (s, 9H, 3CH3), 2.64 (s, 3H, NꢀCH3), 2.94 (d, 2H, CH2+), 3.15 (d,
2H, CH2), 6.96ꢀ7.51 (m, 4H, ArH), 10.55 (s, 1H, R2NH2 COOꢀ).
Anal. Calcd for (C15H22N2 (C2H2O4) 2-PrOH) C, H, N.
3
3
N-Methyl-2-isopropyltryptamine Hydrochloride (12). A
solution of 26 (0.17 g, 0.61 mmol) in dry THF (5 mL) was added to
a stirred suspension of LiAlH4 (0.14 g, 3.7 mmol) in anhydrous THF
(10 mL) at 0 °C, and the reaction mixture was heated at reflux for 2 h,
cooled in an ice bath, and quenched with MeOH (1 mL), NaOH (15%,
1.5 mL) and H2O (1 mL). The organic portion was separated and dried
(Na2SO4), and solvent was evaporated under reduced pressure to yield
an oily residue. Purification by column chromatography using CH2Cl2/
MeOH (9:1) f CH2Cl2/MeOH/Et3N (9:1:0.1) afforded 0.07 g of the
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Journal of Medicinal Chemistry
ARTICLE
(m, 2H, ArH), 7.35 (d, J = 9 Hz, 1H, ArH), 7.60 (d, J = 9 Hz, 1H, ArH),
7.85 (br s, 1H, NH).
20 mL/min using the Gilson autosampler injection system. Compounds
were coapplied with the EC75 concentration (100 μM) of 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 α4β2 modulation, the data were fit to a bell-shaped doseꢀ
response equation as previously described.9
2-Isopropyltryptamine (25). A solution of 23 (0.30 g, 1.9 mmol)
in CH2Cl2 (2 mL) was added to a stirred solution of 1-dimethylamino-2-
nitroethylene (0.22 g, 1.9 mmol) in TFA (1.2 mL) at 0 °C. The reaction
mixture was allowed to stir at room temperature for 2 h, poured into ice-
cold water (20 mL), and extracted with EtOAc (20 mL). The organic
portion was washed with brine (15 mL) and dried (Na2SO4), and the
solvent was removed under reduced pressure. The crude product was
purified recrystallized from EtOAc to afford 0.18 g (60%) of 24 as orange
crystals: mp 159 °C; 1H NMR (CDCl3) δ 1.44 (d, J = 6.0 Hz, 6H, 2 ꢁ
CH3), 3.51ꢀ3.62 (m, 1H, CH), 7.28ꢀ7.35 (m, 2H, ArH), 7.42ꢀ7.46
(m, 1H, CH), 7.72ꢀ7.75 (m, 1H, CH), 7.84 (d, J = 13 Hz, 1H, ArH),
8.42 (d, J = 13 Hz, 1H, ArH), 8.74 (br s, 1H, NH).
’ AUTHOR INFORMATION
Corresponding Author
*Phone: (804)-828-8487. E-mail: glennon@vcu.edu.
Present Addresses
A solution of 24 (0.26 g, 1.1 mmol) in anhydrous THF (5 mL) was
added in a dropwise manner to a stirred suspension of LiAlH4 (0.26 g,
6.9 mmol) in THF (10 mL) at 0 °C. The reaction mixture was heated at
reflux overnight, cooled in an ice bath, and quenched with MeOH
(1 mL), NaOH (15%, 1.5 mL), and H2O (1 mL). After acid (3 N HCl,
10 mL)/base (3 N NaOH, 15 mL) extraction, the organic portion was
dried (Na2SO4) and solvent was removed under reduced pressure to
afford 0.14 g (61%) of 25 as a light-yellow oil: 1H NMR (DMSO-d6) δ
1.28 (d, J = 7.5 Hz, 6H, 2CH3), 1.73 (br s, 2H, NH2), 2.73ꢀ2.84 (m, 4H,
2 ꢁ CH2), 3.21 (m, 1H, CH), 6.89ꢀ7.01 (m, 2H, ArH), 7.27 (d, J = 7.5
Hz, 1H, ArH), 7.44 (d, J = 7.5 Hz, 1H, ArH), 8.78 (br s, 1H, NH).
Compound 25 was used immediately in the synthesis of 26.
Laboratory of Neurobiology, National Institute of Environmen-
tal Health Sciences, National Institutes of Health, Department of
Health and Human Services, Research Triangle Park, NC 27709.
’ ACKNOWLEDGMENT
This study was funded, in part, by the National Institutes of
Health, National Institute of Neurological Disorders and Stroke
[Grant 1R01NS066059] and by a Virginia Center on Aging. N.G.
was partially supported by TA grant DA 007027.
’ ABBREVIATIONS USED
Ethyl [2-(2-Isopropyl-1H-indol-3-yl)ethyl]carbamate (26).
Ethyl chloroformate (0.07 mL, 0.70 mmol) was added in a dropwise
manner at 0 °C to a stirred solution of 25 (0.14 g, 0.7 mmol) in CHCl3
(5 mL), followed by addition of a 4 N aqueous solution of NaOH
(0.20 mL, 0.70 mmol). The reaction mixture was allowed to stir for 2 h at
room temperature and diluted with CHCl3 (15 mL). The organic
portion was dried (Na2SO4), and solvent was evaporated under reduced
pressure to afford 0.17 g (88%) of 26 as an oily residue which was used in
the preparation of 12 without further purification.
ACh, acetylcholine; dFBr, desformylflustrabromine; nACh, nicotinic
acetylcholine; TFA, trifluoroacetic acid
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