Des-Formylflustrabromine (dFBr): A Structure-Activity Study on its Ability to Potentiate the Action of Acetylcholine at α4β2 Nicotinic Acetylcholine Receptors

Article abstract of DOI:10.1021/acschemneuro.8b00156

The naturally-occurring indole alkaloid des-formylflustrabromine (dFBr; 1) is one of the first agents shown to act as a selective positive allosteric modulator (PAM) at α4β2 nicotinic acetylcholine receptors (nAChRs). We previously deconstructed this agent to determine which of its structural features contribute to its actions and formulated a "working pharmacophore". Here, we elaborate the dFBr (1; EC50 = 0.6 μM) structure to identify how various structural modifications impact its actions. Electrophysiological studies with Xenopus laevis oocytes identified several compounds with dFBr-like potency and one, the 5-bromo analog of 1 (i.e., 5-bromo dFBr; 25; EC50 = 1.6 μM), with more than twice the efficacy of 1 as a PAM at α4β2 nAChRs.

Full text of DOI:10.1021/acschemneuro.8b00156

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Article
des-Formylflustrabromine (dFBr): A Structure-
Activity Study on its Ability to Potentiate the Action of
Acetylcholine at #4#2 Nicotinic Acetylcholine Receptors
Malgorzata Dukat, Atul D. Jain, Nadezhda German, Rossana Ferrara-Pontoriero,
Yanzhou Huang, Yilong Ma, Marvin K. Schulte, and Richard A. Glennon
ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.8b00156 • Publication Date (Web): 20 Jul 2018
Downloaded from http://pubs.acs.org on July 21, 2018
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des-Formylflustrabromine (dFBr): A StructureꢀActivity Study on its Ability
to Potentiate the Action of Acetylcholine at α4β2 Nicotinic Acetylcholine
Receptors
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Małgorzata Dukat, Atul Jain, Nadezhda German, Rossana FerraraꢀPontoriero,
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Yanzhou Huang, Yilong Ma, Marvin K. Schulte, and Richard A. Glennon
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Department of Medicinal Chemistry, School of Pharmacy, Virginia Commonwealth
University, Richmond, VA 23298ꢀ0540
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University of the Sciences, Philadelphia College of Pharmacy, Department of
Pharmaceutical Sciences, 600 S 43rd Street, Philadelphia, PA 19104ꢀ4495
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Institute of Arctic Biology, Department of Biology and Wildlife, University of Alaska
Fairbanks, Fairbanks, Alaska 99775ꢀ7000
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ABSTRACT
The naturallyꢀoccurring indole alkaloid desꢀformylflustrabromine (dFBr; 1) is one of the
first agents shown to act as a selective positive allosteric modulator (PAM) at α4β2
nicotinic acetylcholine receptors (nAChRs). We previously deconstructed this agent to
determine which of its structural features contribute to its actions and have identified an
agent that might serve as the basis for a “working pharmacophore”. Here, we elaborate
the dFBr (1; EC50 = 0.2 ꢁM) structure to identify how various structural modifications
impact its actions. Electrophysiological studies with Xenopus laevis oocytes identified
several compounds with dFBrꢀlike potency and one, the 5ꢀbromo analog of 1 (i.e., 5ꢀ
bromo dFBr; 25; EC50 = 0.4 ꢁM), with more than twice the efficacy of 1 as a PAM at
α4β2 nAChRs.
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Keywords: Positive allosteric modulator, electrophysiology, nAChRs, α4β2 PAM
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Introduction
The α4β2 subpopulation of nicotinic acetylcholine receptors (nAChRs) is implicated in
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drug and nicotine addiction, Parkinson’s disease, depression, and pain. However,
there are very few, if any, agents that show substantial selectivity for this receptor
subpopulation as agonists. Hence, there remains an unmet need for the development of
α4β2 nAChRꢀselective agents. The natural product desꢀformylflustrabromine (dFBr; 1)
has been identified as one of the first positive allosteric modulators (PAMs) of α4β2
7,8
nAChRs with little to no action at other populations of nAChRs. We “deconstructed”
the dFBr (1) molecule in a stepwise manner to determine the contribution of its various
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structural features to α4β2 PAM action. Found was that: i) conversion of the secondary
amine of dFBr to a primary amine (i.e., 2) reduced potency and efficacy, ii) reduction of
the unsaturated side chain of dFBr (i.e., 4) doubled its potency, with retention of
efficacy, iii) debromination of 1 and 4 (i.e., 3 and 5, respectively) reduced potency by at
least one order of magnitude, but did not detract from AChꢀpotentiating action, and that
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iv) a branched indole 2ꢀposition substituent was favored. Removal of the 2ꢀposition
substituent, and even the simple removal of both gem dimethyl groups (i.e., of 1 and 4),
resulted in loss of AChꢀpotentiating action. Although compound 6 was substantially less
potent than dFBr (1), it was the structurally simplest compound shown to potentiate the
action of ACh at α4β2 nAChRs. In contrast, compound 7 failed to potentiate the actions
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of ACh. Compound 6 would appear to represent the basis for a “working
pharmacophore” and is the most abbreviated agent that retains dFBrꢀlike PAM action.
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NHꢀR
NHꢀCH3
NHꢀCH3
CH3
CH3
CH3
CH3
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CH3
R
N
CH2
N
H
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H
CH3
CH
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X
H
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R = CH , X = Br (dFBr)
R = H, X = Br
3 R = CH , X = H
4 X = Br
X = H
6 R = CH3
7 R = H
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3
The above “deconstruction” investigation introduced no new substituents to the dFBr
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molecule; that is, dFBr substituents were simply “deleted” in a systematic fashion. In
the present study, we “elaborated” (for the most part) the structure of dFBr by adding
new substituents or by modifying various structural aspects of dFBr. An initial goal was
to confirm that a primary amine analog of dFBr (more specifically, of 3; i.e., 8) retains
action in the absence of the 6ꢀbromo group (and it might be noted that a decrease in
potency was expected based on our earlier studies). But, the major goal of the study
was to further explore the structureꢀactivity relationships of dFBr to identify what
structural features are required to retain or enhance its PAM actions on ACh at α4β2
nAChRs expressed in Xenopus laevis oocytes in an effort to identify those that might be
ultimately exploited for the development of agents with enhanced potency and/or
efficacy. At the time our work began, essentially nothing was known about the SAR of
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dFBr as an α4β2 nAChRs PAM other than what we have previously published.
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Results
Synthesis. The compounds examined in this study were prepared as described below.
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For the purpose of this investigation, dFBr (1) was resynthesized as its hydrochloride
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salt as previously reported by us. Compound 8, the primary amine analog of 3, was
prepared in a similar manner (Scheme 1). That is, the primary amine of tryptamine (26)
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was protected by a Boc group (i.e., 27), prenylated to 28 using prenyl 9ꢀBBN, and
deprotected to afford 8. This sequence also was used to prepare several other targets.
a
Scheme 1. Synthesis of compound 8.
a
Reagents: (a) Boc O, Et N, DMF, room temperature, 20 h; (b) tꢀbutyl hypochlorite,
2
3
THF, ꢀ78 ºC, 45 min; prenyl 9ꢀBBN, 3 h, room temperature; (c) HCl, Et O, 0 ºC.
2
The free base of dFBr (1) was reductively alkylated using formaldehyde and NaCNBH3
to tertiary amine 9, which was subsequently methylated using MeI to give the N,N,Nꢀ
trimethyl quaternary amine 10. Alkylation of 5ꢀbromoꢀ2ꢀ(1,1ꢀdimethylallyl)indole (29)
with N,Nꢀdimethylaminoethyl chloride furnished isotryptamine 11. The free base of
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gramine 12 was prepared according to a literature procedure, and converted to a
waterꢀsoluble oxalate salt, and Nꢀmethylhomotryptamine (i.e., 3ꢀ(3ꢀmethylaminopropyl)ꢀ
12
indole) was Bocꢀprotected, prenylated, and deprotected to afford 13 using the same
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sequence of reactions shown in Scheme 1. Tetrahydroꢀβꢀcarboline 14 was obtained by
cyclization of 6ꢀbromotryptamine with acetone under PictetꢀSpengler conditions.
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Compound 15 was obtained as shown in Scheme 2. Condensation of 6ꢀbromoindole
(30) with 4ꢀpiperidone afforded tetrahydropyridine 31; catalytic reduction of 31 to
piperidine 32 followed by amine protection gave 33. Prenylation (as described in
Scheme 1) with subsequent deꢀprotection provided 15.
a
Scheme 2. Synthesis of compound 15.
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Reagents: (a) 4ꢀpiperidone⋅HCl, KOH, CH OH; (b) H , PtO , AcOH; (c) Boc O, Et N,
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CH Cl (d) i tꢀBuOCl, THF, ii prenyl 9ꢀBBN, THF; (e) HCl, EtOAC.
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2;
Compound 16, the αꢀmethyl analog of 2, was prepared in the same manner shown in
Scheme 1 using 1ꢀ(6ꢀbromoꢀ1Hꢀindolꢀ3ꢀyl)propanꢀ2ꢀamine (35) as starting material; 35
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was obtained by catalytic reduction of 3ꢀ[2ꢀnitropropꢀ1ꢀenꢀ1ꢀyl]ꢀ6ꢀbromoindole which had
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been previously reported, but not thoroughly characterized.
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Compounds 17 and 18 were synthesized via a common route (Scheme 3). Acylation of
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ꢀbromoaniline (38) with chloroacetyl chloride under FriedelꢀCrafts conditions afforded
9, which was subsequently cyclized by treatment with the appropriate Grignard
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reagents to yield compounds 40 and 41. The cyclization step involved a 1,2ꢀaryl
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migration as described by Pei et al.; although the latter investigators synthesized a
variety of 2ꢀsubstituted indoles, they never synthesized or utilized 39. The remainder of
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the syntheses involved a SpeeterꢀAnthony sequence where an indole is acylated with
oxalyl chloride, treated with an amine, and the resulting glyoxalyl amides reduced to the
corresponding amines.
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Scheme 3. Synthesis of 17 and 18.
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Reagents: (a) AlCl , BCl , ClCH CN, DCM, reflux, overnight; (b) THF, rt, 1.5h; (c) i
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COCl) , Et O, 0 ºC, 1h, ii CH NH (40% in H O), rt, overnight; (d) BH ⋅DMS, THF,
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reflux, overnight.
Compounds 19 and 20 were prepared from their corresponding 2ꢀsubstituted indoles
using a SpeeterꢀAnthony sequence. Likewise, this reaction scheme was also used in
the preparation of 22ꢀ25; however, for the latter four compounds, 2ꢀunsubstituted
indoles were used as starting materials and once the desired Nꢀmethyltryptamines were
obtained, the amine was Bocꢀprotected prior to prenylation, followed by deprotection.
N ꢀMethyl dFBr (21) was obtained by treatment of NꢀBocꢀprotected 1 with NaOH and
1
MeI, followed by deprotection.
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Electrophysiology. dFBr (1) was reꢀexamined in Xenopus oocytes in the same manner
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we described earlier, and its potency (EC = 0.2 ꢀM) and efficacy (265%, relative to
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ACh control: 100%) were consistent with what we have reported. Data obtained for
the present compounds are detailed in Table 1, and “maximal effect” is defined as the
Imax (calculated maximum current) for the potentiation doseꢀresponse curve. The
examined compounds also are shown in Figure 1 and their action and potency are
briefly summarized there for easy comparison (i.e., the figure provides their potency as
EC values, and maximal potentiation, or it is indicated that they failed to potentiate the
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actions of ACh – as noted by “NP” – no potentiation).
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Figure 1. Compounds examined in the present investigation. Values in parenthesis are
potencies for potentiation of ACh responses, followed by maximal potentiation effect;
NP = no potentiation. More detailed data are provided in Table 1. As comparator, the
EC value for dFBr (1) = 0.2 ꢀM, with 265% potentiation.
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Table 1. Potencies of dFBr (1) analogs to potentiate and/or inhibit the actions of 100 ꢁM
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ACh at α4β2 nAChRs.
c
EC , ꢂM
% change in
Imax (95% CL)
Maximal Effect
265%
IC , ꢂM
50
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(
pEC ± SEM)
(pIC ±SEM)
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1
(dFBr)
0.2 (6.7 ± 0.3)
6.3 (5.2 ± 0.2)
2.0 (5.7 ± 0.2)
350ꢀ510
50 (4.3 ± 0.4)
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250ꢀ310
360ꢀ610
160%
300%
160 (3.8 ± 0.1)
79 (4.1 ± 0.2)
2.5 (5.6 ± 0.1)
79 (4.1 ± 0.2)
5.0 (5.3 ± 0.1)
13 (4.9 ± 0.1)
5.0 (5.3 ± 0.1)
32 (4.5 ± 0.1)
250 (3.6 ± 0.1)
160 (3.8 ± 0.1)
25 (4.6 ± 0.1)
32 (4.5 ± 0.2)
13 (4.9 ± 0.1)
32 (4.5 ± 0.2)
100 (4.0 ± 0.1)
16 (4.8 ± 0.6)
32 (4.5 ± 0.2)
50 (4.3 ± 0.2)
d
9
e
10
11
12
NP
7.9 (5.1 ± 0.2)
240ꢀ540
313%
e
NP
e
13
14
15
NP
e
NP
13 (4.9 ± 0.1)
2.5 (5.6 ± 0.2)
0.8 (6.1 ± 0.4)
280ꢀ290
220ꢀ250
300ꢀ350
120%
120%
167%
d
16
d
17
e
18
NP
d
19
3.2 (5.5 ± 0.3)
150ꢀ390
123%
e
20
21
NP
4.0 (5.4 ± 0.2)
0.8 (6.1 ± 0.2)
210ꢀ650
250ꢀ330
343%
175%
d
22
e
2
3
4
5
NP
2
2
0.8 (6.1 ± 0.3)
0.4 (6.4 ± 0.1)
210ꢀ460
710ꢀ870
250%
620%
a
EC , I , and IC values were calculated using non linear regression to a bellꢀshaped dose
5
0
max
50
b
response model. Hill slopes were fixed at 1 both for potentiation and inhibition. I
values
max
represent the percent change of the ACh I
after fitting the data to a bellꢀshaped dose
max
c
response curve. Maximal effect; no value is provided where there was no potentiation.
d
Maximum inhibition was set at 0 to enable estimation of IC values and it was necessary to
50
e
fit the data using the bellꢀshaped dose response equation. NP: No potentiation.
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9
1
1
1
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2
2
2
2
2
2
2
2
2
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4
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4
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5
5
5
5
5
5
5
5
6
We had previously shown that Nꢀdesꢀmethyl dFBr (2) was nearly equipotent with dFBr
9
(1). Consistent with this finding, the primary amine counterpart of desꢀbromo dFBr (8;
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
EC = 6.3 ꢀM, Table 1) was nearly equipotent with its primary amine counterpart (i.e.,
50
9
desꢀbromo dFBr; 3, EC = 7.2 ꢀM). Nevertheless, 8 was substantially less potent than,
50
and only about half as efficacious as, 1. The N,Nꢀdimethyl tertiary amine analog of
dFBr, 9 (EC = 2.0 ꢂM), retained dFBrꢀlike action, was at least equiꢀefficacious, but
50
was 10ꢀfold less potent than dFBr (1) (Figure 2). In contrast, the N,N,Nꢀtrimethyl
quaternary amine analog of dFBr (i.e., 10) failed to potentiate the actions of ACh and
only produced an inhibitory effect. The isotryptamine isostere of 9 (i.e., 11; EC = 7.9
50
ꢀM) retained activity but was 4ꢀfold less potent than 9.
Representative concentrationꢀresponse curves (exemplified for ACh, 100 ꢀM, in
combination with compounds 1, 9, 15, and 24) showing enhancement of the AChꢀ
induced response at low concentrations followed by inhibition at higher concentrations
are shown in Figure 2.
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9
1
1
1
1
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2
2
2
2
2
2
2
2
2
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3
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4
4
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5
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5
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5
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6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 2. Typical responses and biphasic dose response curves for 1, 9, 15 and 24
showing potentiation and inhibition kinetics of analogs with different maximum observed
responses.
All potentiating compounds showed enhancement of AChꢀinduced
responses at low concentrations followed by inhibition at higher concentrations.
Inhibited responses typically displayed more rapid desensitization kinetics.
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9
1
1
1
1
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1
1
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2
2
2
2
2
2
2
2
2
3
3
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3
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3
4
4
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4
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5
6
The side chainꢀshortened and side chainꢀelongated analogs 12 and 13 (Table 1),
respectively, failed to potentiate the actions of ACh, as did the conformationallyꢀ
constrained analog of 4 where the amine was tethered to the 2ꢀposition substituent in
the form of a tetrahydroꢀβꢀcarboline (i.e., 14). Piperidine 15 (EC = 13 ꢀM) was a very
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
50
weak potentiating agent and only slightly (120%) enhanced the action of ACh.
Compound 16 (EC = 2.5 ꢀM), the racemic αꢀmethyl analog of 2, displayed
50
substantially reduced potency and efficacy compared to dFBr.
Compound 17 (EC = 0.8 ꢁM), the racemic mono desꢀmethyl counterpart of 4, retained
50
dFBrꢀlike action, but only half its efficacy; it might be noted that the individual optical
isomers of 17 were not examined. Other 2ꢀmodified compounds were either less
potent/efficacious (i.e., 19) or inactive (i.e., 18, 20). N ꢀMethyl dFBr (21; EC = 4.0 ꢁM)
1
50
was 20ꢀfold less potent than dFBr (1) but retained its efficacy (Table 1). Replacement of
the 6ꢀbromo substituent of dFBr (1) by a methyl group (i.e., 22) decreased potency by 4ꢀ
fold and halved efficacy, whereas replacement with a trifluoromethyl group (i.e., 23)
resulted in no potentiation. Moving the bromo substituent to the 5ꢀposition (i.e., 24)
reduced potency by 4ꢀfold but had relatively little effect on efficacy. 5ꢀBromo dFBr (i.e.,
2
5), although only half as potent as 1, displayed twice its efficacy.
Low concentrations of dFBr potentiated the actions of ACh whereas higher
8,9
concentrations had an inhibitory effect; for example, see Figure 2. In general, similar
actions were observed for all compounds in the present investigation, although several
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9
failed to enhance the action of ACh and simply displayed an inhibitory action (by a
mechanism that has yet to be fully elucidated).
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
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4
4
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5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Discussion
We previously examined “deconstructed” analogs of dFBr (1) to understand which of
(and to what extent) its various structural attributes impact its action as a PAM at α4β2
9
ACh receptors. A branched indole 2ꢀposition chain (although not chain unsaturation)
was a critical feature for activity, the simple 2ꢀnꢀpropyl counterpart of dFBr (i.e., dFBr
lacking both gemꢀdimethyl substituents) was inactive, but the 6ꢀposition bromo
9
substituent, although not required, was found important for efficacy. Compound 6,
although with 10ꢀfold reduced potency, was demonstrated to be the minimal structure
retaining dFBrꢀlike action. Here, we extend these structureꢀactivity findings.
9
Confirming a prior finding (i.e., with 2), a primary amine (i.e., 8) is tolerated for dFBrꢀlike
action. Lacking a 6ꢀbromo group, 8 was expected to be >10ꢀfold less potent than 1. This
was found to be the case. Table 1 shows that a tertiary amine, the simple N,Nꢀ
dimethylamine counterpart of 1, also was tolerated, but that 9 was 10ꢀfold less potent
than 1; however, a quaternary amine counterpart of dFBr, 10, was inactive. The latter
finding is compatible with the concept that dFBrꢀlike compounds do not bind at the α4β2
receptor orthosteric site (i.e., ACh, which is a quaternary amine, binds at the orthosteric
site by definition). Whereas introduction of an αꢀmethyl group also was tolerated (i.e.,
16), other changes to the indole 3ꢀposition substituent (e.g. chain shortening, chain
extension, conformational restriction; i.e., 12ꢀ14) resulted in loss of PAM action.
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9
1
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5
5
5
5
5
5
5
6
9
A branched 2ꢀposition substituent was identified earlier as being important for activity.
Here, it was found that removal of one of the methyl groups is tolerated (i.e., 17); this
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
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8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
confirms the earlier suggestion that only one of the gem dimethyl groups might be
9
required, and this might be related to the lipophilicity of the substituent. Nevertheless,
1
7 displayed only half the ACh potentiating effect of dFBr. Compound 6 showed
9
potentiating action whereas its 2ꢀisopropyl counterpart, 7, was inactive. Likewise,
“
extending” the isopropyl group to a bulkier cyclopentyl group (i.e., 18) resulted in loss
of action. At this time, given the results with 17, we cannot explain this finding. However,
like 6, the cyclopropyl analog 19 was active but only weakly potentiated the action of
ACh (i.e., to 123%). The 2ꢀphenyl counterpart of dFBr (i.e., 20), which also might be
considered a “branched” substituent, was found to be inactive.
Because none of the above structural alterations resulted in enhanced potency or
efficacy, attention was directed to the indole nucleus; the 2ꢀ and 3ꢀposition substituents
of dFBr (1) were retained. Compound 11, the isotryptamine counterpart of 9, was about
4
0ꢀfold less potent than than 1, and 4ꢀfold less potent than 9, suggesting that the
location or presence of the indolic N or NH substituent plays a role in PAM potency.
Interestingly, the N ꢀmethyl counterpart of dFBr (i.e., 21) displayed 20ꢀfold reduced
1
potency relative to dFBr suggesting that the indolic 1ꢀposition hydrogen atom might be
involved in the potency of these compounds.
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9
Because removal of the 6ꢀbromo group of dFBr decreased its potency by at least 10ꢀ
fold (reference 9 and the present study), an attempt was made to determine if this was
related to the lipophilic or electronic character of the bromo substituent. Because the
bromo group of dFBr (1) and the methyl group of 22 are electronically opposite, and
because a bromo group is more lipophilic than a methyl group (π = 0.86 and 0.56,
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
16
respectively ), it would seem that its contribution might be related to lipophilicity. The
trifluoromethyl group of inactive 23 is equiꢀlipophilic (π = 0.88) with a bromo group;
17,18
however, it is sterically larger than a bromo or methyl group
and might not be
accommodated by the binding pocket. Indeed, several “size” indicies were examined
and are consistent with this concept (see Table S1, Supporting Information).
Translocation of the 6ꢀbromo group to the 5ꢀposition resulted in a compound (i.e., 24)
that was slightly less potent but equiꢀefficacious with dFBr. Thus, the position of the 6ꢀ
bromo group, although perhaps optimal, would not appear to be critical for AChꢀ
enhancing PAM action. The 5,6ꢀdibromo indolic compound 25 was only slightly less
potent than dFBr, but displayed twice its efficacy (620%). Additional 5ꢀsubstituted and
5
,6ꢀdisubstituted compounds remain to be examined.
Over the last few years attempts have been made, using siteꢀdirected mutagenesis, to
localize sites on α4β2 receptors where dFBr might act to elicit its allosteric effects;
19ꢀ21
21
several potential sites have been identified.
In particular, Deba et al. have
proposed two distinct sites and, additionally, have modeled possible dFBr interactions.
In one site, the protonated amine is proposed to form a hydrogen bond with the
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1
1
1
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1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
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3
3
3
3
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4
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5
5
5
5
6
carbonyl oxygen atom of a valine (V609) residue, and the 2ꢀposition alkyl group is
involved in hydrophobic interactions with lysine (L317) and phenylalanine (F312)
residues. This is consistent with our findings that the quaternary amine 10 is inactive,
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
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5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
and that a 2ꢀposition (branched) alkyl substituent contributes to activity. Furthermore,
the enhanced potency of 4 over dFBr might be related to the greater lipophilicity of the
ethyl group relative to its ethylene counterpart (π = 1.02 and 0.82 for CH CH and
2
3
1
6
CH=CH , respectively ). Docked in this site, the 6ꢀbromo group of dFBr is within 3 Å of
2
a phenylalanine (F316) residue, implicating a possible hydrophobic interaction. This is
also in accord with the action of 22. The second binding site involves, in addition to
hydrophobic interactions between the (branched) 2ꢀposition alkyl group and a leucine
moiety (L256), a hydrogen bond between the indole NH of dFBr and a cysteine (C259)
21
residue. The latter is consistent with the reduced potency of 21 (although it might be
noted that 21 retained dFBrꢀlike efficacy as a potentiating agent). Future homology
modelling/docking studies can be specifically informed by results such as those
provided here to validate and/or refine future models.
Thus far, the naturallyꢀoccurring 1 seems to be a nearly optimal structure (i.e., with
regard to potency and efficacy) for dFBrꢀlike compounds to potentiate the actions of
ACh at α4β2 nAChRs. But, it might be noted that potency and efficacy do not coꢀvary.
Apart from the saturated dFBr analog 4, which was about twice as potent as, but equiꢀ
9
efficacious with, dFBr (1), none of the compounds examined here displayed
substantially enhanced potency and efficacy. Potential structural modifications of 1
resulting in retention of potency and efficacy, thus far, appear limited. It is evident that
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9
manipulation of substituents on the indole nucleus (apart from the 2ꢀ and 3ꢀpositions)
can favorably impact activity and result in compounds with subꢀmicromolar potency and
even, as with 25, enhanced efficacy. Future studies will focus primarily on the indole
ring and substituents at the indole 4ꢀ – 7ꢀpositions. The ideal goal would be an agent
that enhances the actions of ACh without acting as an antagonist of ACh at higher
concentrations. This goal has yet to be achieved. But, the present findings provide
insight as to how dFBr substituents impact the action of ACh, provide the most complete
SAR study of dFBr (1) to date, and can inform future modeling/docking studies.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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9
0
1
2
3
4
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0
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9
1
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2
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2
2
2
2
2
2
3
3
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3
3
3
3
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4
4
4
4
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5
5
5
6
Experimental
Synthesis
Melting points (mp) were measured on a ThomasꢀHoover melting point apparatus using
1
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
glass capillary tubes and are uncorrected. H NMR spectra were recorded on a Varian
4
00 MHz spectrometer using tetramethylsilane (TMS) as an internal standard; peak
positions are given in parts per million (δ). UHPLCꢀMS were recorded on a Perkin Elmer
Flexar UHPLC with AxION 2 Time of Flight (TOF) mass spectrometer and the molecular
weight of the compounds was within 0.05% of calculated values. Elemental analysis
was performed on all target compounds by Atlantic Microlab Inc. (Norcross, GA) for the
elements indicated and the results were typically within 0.4% of calculated values.
Infrared spectra were obtained on a Thermo Nicolet iS10 FTꢀIR. Flash chromatography
was performed on a CombiFlash Companion/TS instrument (Telodyne Isco Inc.,
Lincoln, NE) using packed silica gel (Silica Gel 230ꢀ400 mesh) columns (RediSep Rf
Normalꢀphase Silica Flash Column, Teledyne Isco Inc., Lincoln, NE). All reactions were
monitored by thinꢀlayer chromatography (TLC) on silica gel GHLF plates (250 ꢀ, 2.5 x
1
0 cm; Analtech Inc., Newark, DE).
2
ꢀ(1,1ꢀDimethylallyl)tryptamine Hydrochloride (8). Gaseous HCl was bubbled into a
solution of tertꢀbutylꢀ2ꢀ(2ꢀ(1,1ꢀdimethylallyl)ꢀ1Hꢀindolꢀ3ꢀyl)ethylcarbamate 28 (0.4 g) in
anhydrous Et O (20 mL) at 0 °C. The reaction mixture was allowed to stir for 24 h and
2
the solvent was evaporated to yield a purple solid which was recrystallized from iꢀ
1
PrOH/Et O to yield 0.1 g (28%) of 8 as brown crystals: mp 231ꢀ232 °C; H NMR
2
(
DMSOꢀd ) δ 1.50 (s, 6H, CH ), 2.86 (t, 2H, CH ), 3.07 (t, 2H, CH ), 5.06ꢀ5.11 (m, 2H,
6
3
2
2
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CH ), 6.12ꢀ6.18 (m, 1H, CH), 6.95ꢀ7.06 (m, 2H, Ar), 7.34 (d, J = 7.9 Hz, 1H, Ar), 7.51 (d,
2
+
3
J = 7.8 Hz, 1H, Ar), 8.05 (br s, 3H, NH aliphatic), 10.59 (br s, 1H, indolic NH). Anal.
Calcd for (C H N —HCl) C, H, N.
15
20
2
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
6
ꢀBromoꢀ2ꢀ(1,1ꢀdimethylallyl)ꢀN,Nꢀdimethyltryptamine Oxalate (9). Formaldehyde
(
0.09 mL, 1.25 mmol) and NaBH CN (0.03 g, 0.5 mmol) were added to a stirred solution
3
of 1 (free base, 0.08 g, 0.25 mmol) in CH CN (15 mL) under a N atmosphere. The
3
2
reaction mixture was allowed to stir for 1 h at room temperature, and then 2N HCl (8
mL) was added in a dropwise manner. After stirring for 2 h, the reaction mixture was
diluted with Et O (10 mL), and the organic layer was separated and washed with H O (2
2
2
x 10 mL). The aqueous portions were combined, basified by addition of NaHCO (pH
3
8
), and extracted with CH Cl (3 x 10 mL). The organic portions were combined, dried
2 2
(Na SO ), and concentrated under reduced pressure. The residue was purified by
2
4
column chromatography (Aldrich silica gel 60) using DCM/MeOH (10:1) as eluent to
1
afford the free base of 9 (0.06 g, 60% yield) as an oil: H NMR (CDCCl ): δ 1.53 (s, 6H,
3
2
5
1
x CH ), 2.34 (s, 6 H, 2 x CH ), 2.48ꢀ2.52(m, 2H, CH ), 2.96ꢀ3.00 (m, 2H, CH ), 5.14ꢀ
3 3 2 2
.18 (m, 2H, CH ), 6.07ꢀ6.14 (m, 1H, CH), 7.15 (d, J = 1.6 Hz, 1H, ArꢀH), 7.36 (d, J =
2
.6, 1H, ArꢀH), 7.42 (s, 1H, 2 x ArꢀH), 7.83 (b s,1H, NH). A solution of the free base
(
0.05 g, 0.15 mmol) and oxalic acid (0.01 g, 0.15 mmol) in CHCl (3 mL) was allowed to
3
stir for 0.5 h and the precipitate was collected by filtration. The solid product was
recrystallized from iꢀPrOH to afford 9 (0.02 g, 36% yield) as a white solid: mp 172ꢀ173
1
ºC; H NMR (DMSOꢀd ) δ 1.58 (s, 6H, 2 x CH ), 2.73 (s, 6H, 2 x NꢀCH ), 4.39 (br.s, 2H,
6
3
3
CH ), 5.25ꢀ5.29 (m, 2H, CH ), 6.31ꢀ6.38 (m,1H, CH), 7.25 (d, J = 8.44 Hz, 1H, ArH),
2
2
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9
1
1
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1
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1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
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5
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5
5
5
5
6
7
.59 (d, J = 1.60 Hz, 1H, ArH), 7.70 (d, J = 8.44 Hz, 1H, ArH), 11.27 (s, oxalic acid).
Anal. Calcd for (C H BrN — C H O ) C, H, N.
17
23
2
2
2
4
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
6
ꢀBromoꢀ2ꢀ(1,1ꢀdimethylallyl)ꢀN,N,Nꢀtrimethyltryptamine Methiodide (10). A
solution of 6ꢀbromoꢀ2ꢀ(1,1ꢀdimethylallyl)ꢀN,Nꢀdimethyltryptamine (9) (0.05g, 0.2 mmol)
in anhydrous iꢀPrOH (4 mL) was cooled to 0 °C (iceꢀbath). Iodomethane (0.06 g, 0.5
mmol) was added and the reaction mixture was allowed to stir for 22 h at room
temperature under a N atmosphere. The precipitate was collected by filtration and
2
washed with anhydrous Et O (3 x 15 mL), air dried, and recrystallized from MeOH/iꢀ
2
1
PrOH to afford 0.02 g (30%) of 10 as yellow crystals: mp 208ꢀ210 °C; H NMR (CD CN)
3
δ 1.54 (s, 6H, (CH ) ), 3.15 (s, 9H, N(CH ) ), 3.22ꢀ3.38 (m, 4H CH ), 5.25 (m, 2H, CH ),
3
2
3 3
2
2
6
.22 (m, 1H, CH), 7.22 (dd, J = 8.4, 1.7 Hz, 1H, Ar), 7.46 (d, J = 8.5 Hz, 1H, Ar), 7.59
(d, J = 1.5 Hz, 1H, Ar), 9.35 (br s, 1H, indolic NH). Anal. Calcd for (C H BrIN —C H O)
18
26
2
3
8
C, H, N.
5
ꢀBromoꢀ2ꢀ(1,1ꢀdimethylallyl)ꢀN,Nꢀdimethylisotryptamine Hydrogen Oxalate (11).
22
3,5ꢀDibromoꢀ1Hꢀindole (0.5 g, 1.8 mmol) was added in one portion to freshly prepared
10
prenyl 9ꢀBBN (5.4 mmol) and Et N (0.9 mL, 6.3 mmol) in anhydrous THF (10 mL) at
3
room temperature. The reaction mixture was allowed to stir at room temperature for 4 h
and then quenched with a saturated solution of NaHCO (20 mL). The organic layer was
3
separated and the aqueous portion was extracted with Et O (2 x 20 mL). The combined
2
organic portion was washed with H O (2 x 30 mL), brine (30 mL), dried (Na SO ), and
2
2
4
evaporated to dryness under reduced pressure to yield a crude, darkꢀyellow oil. The
residue was purified by column chromatography (silica gel; hexanes/EtOAc 100:1 to
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8
9
1
3
0:1) to afford 0.3 g (78%) of 5ꢀbromoꢀ2ꢀ(1,1ꢀdimethylallyl)indole (29) as a yellow oil: H
NMR (CDCl ) δ 1.47 (s, 6H, CH ), 5.10 (m, 2H, CH ), 6.01 (m, 1H, CH), 6.24 (s, 1H, Ar),
3
3
2
7.20 (dd, 2H, Ar), 7.89 (br s, 1H, indolic NH).
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
In a dry, roundꢀbottom flask NaH (60% oil dispersion) (0.06 g, 1.4 mmol) was allowed
to stir with anhydrous toluene in an ice bath for 10 min. After removal of the toluene, a
solution of 29 (0.15 g, 0.6 mmol) in anhydrous DMF (5 mL) was added and stirring was
continued in an ice bath (0 °C) for another 20 min. Prepared in a separate beaker, a
solution of N,Nꢀdimethylaminoethyl chloride hydrochloride (0.2 g, 1.1 mmol), KtBuO (0.2
g, 1.4 mmol) and KI (0.08 g, 0.6 mmol) in cold anhydrous DMF (3 mL) was then added
to the roundꢀbottom flask and the reaction mixture was heated at reflux for 17 h. The
reaction mixture was quenched with H O (20 mL) and extracted with EtOAc (2 x 25 mL)
2
and CH Cl (2 x 25 mL). The combined organic portions were washed with H O (2 x 50
2
2
2
mL), brine (50 mL), dried (Na SO ), and evaporated to dryness to yield 0.1 g (63%) of
2
4
free base as a brownꢀcolored oil. Saturated oxalic acid solution in anhydrous Et O (5
2
mL) was added to a solution of the free base (0.1 g) in anhydrous Et O (5 mL) at 0 °C
2
and allowed to stir overnight. The precipitate formed was collected by filtration and
recrystallized from MeOH/Et O to afford 0.04 g (32%) of 11 as white flakes: mp 185ꢀ188
2
1
°
C; H NMR (DMSOꢀd ) δ 1.49 (s, 6H, (CH ) ), 2.69 (s, 6H N(CH ) ), 3.03 (t, 2H, CH ),
6
3 2
3 2
2
4.42 (t, 2H, CH ), 5.17 (m, 2H, CH ), 6.09 (m, 1H, CH), 6.37 (s,1H, CH), 7.25 (d, 1H, J
2
2
=
8.7 Hz, Ar), 7.43 (s, 1H, J = 8.7 Hz, Ar), 7.70 (d, 1H, Ar). Anal. Calcd for
(C H BrN —C H O ) C, H, N.
17
23
2
2
2
4
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2
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2
2
2
2
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6
2
ꢀ(1,1ꢀDimethylallyl)gramine Hydrogen Oxalate (12). Dimethylamine (40%, 0.1 mL,
0
.8 mmol) and HCHO (37%, 0.7 mL, 0.8 mmol) were added to a solution of 2ꢀ(1,1ꢀ
11
dimethylallyl)ꢀ1Hꢀindole (0.10 g, 0.5 mmol) in glacial HOAc (3 mL) maintained at 5 °C.
When the vapors ceased, MeOH (4 mL) was added to make a clear solution and the
reaction mixture was allowed to stir overnight at room temperature. The organic solvent
was removed by evaporation under reduced pressure and the solution was basified with
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
NaOH (3M, to pH 10). The aqueous portion was extracted with Et O (3 x 30 mL) and
2
the combined organic portion was washed with H O (3 x 50 mL), brine (50 mL), dried
2
(Na SO ), and the solvent was removed under reduced pressure to yield the gramine as
2 4
a free base which was converted to an oxalate salt by addition of a saturated oxalic acid
solution in anhydrous Et O (5 mL) to a solution of crude 2ꢀ(1,1ꢀdimethylallyl)gramine in
2
anhydrous Et O (5 mL) at 0 °C. The precipitate was collected by filtration, dried and
2
recrystallized from iꢀPrOH to afford 0.04 g (22%) of 12 as white crystals: mp 155ꢀ158
1
°
C; H NMR (DMSOꢀd ) δ 1.54 (s, 6H, (CH ) ), 2.72 (s, 6H, N(CH ) ), 4.39 (s, 2H, CH ),
6
3 2
3 2
2
5
.22 (m, 2H, CH ), 6.33 (m, 1H, CH), 7.04ꢀ7.68 (m, 4H, Ar), 11.13 (br s, 1H, COOH).
2
Anal. Calcd for (C H N —C H O —0.5 H O) C, H, N.
16 22
2
2
2
4
2
NꢀMethylꢀ3ꢀ(2ꢀ(1,1ꢀdimethylallyl)ꢀ1Hꢀindolꢀ3ꢀyl)propanamine Hydrogen Oxalate
13). Triethylamine (0.4 mL, 2.6 mmol) and diꢀtertꢀbutyl dicarbonate (0.6 g, 2.6 mmol)
(
12
were added to a CH Cl (15 mL) solution of 3ꢀ(3ꢀmethylaminopropyl)indole (0.5 g, 2.6
2
2
mmol) and allowed to stir at room temperature for 19 h. The reaction mixture was
quenched with H O (50 mL) and extracted with CH Cl (2 x 30 mL). The combined
2
2
2
organic portion was washed with H O (3 x 50 mL), dried (Na SO ) and evaporated
2
2
4
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under reduced pressure to obtain a yellow oil. The crude product was purified by
column chromatography (silica gel; hexanes/EtOAc; 100:1 to 25:1) to afford 0.4 g (58%)
1
of tertꢀbutylꢀ3ꢀ(1Hꢀindolꢀ3ꢀyl)propylꢀNꢀmethylcarbamate as a white foam: H NMR
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
CDCl ) δ 1.37 (s, 9H, (CH ) ), 1.83ꢀ1.90 (m, 2H, CH ), 2.68 (t, 2H, CH ), 2.78 (s, 3H,
3 3 3 2 2
CH ), 3.24 (m, 2H, CH ), 6.93 (s, 1H, Ar), 7.01ꢀ7.03 (dt, 1H, Ar), 7.09ꢀ7.13 (dt, 1H, Ar),
3
2
7
.28 (d, J = 8.0 Hz, 1H, Ar), 7.52 (d, J = 7.8 Hz, 1H, Ar), 7.86 (br s, 1H, indolic NH).
tertꢀButyl hypochlorite (0.2 g, 1.7 mmol) was added to a solution of the above
carbamate (0.4 g, 1.4 mmol) and Et N (0.2 g, 1.7 mmol) in THF (15 mL) at ꢀ78 °C and
3
10
allowed to stir for 45 min. Freshly prepared prenylꢀ9ꢀBBN (2.8 mmol) was added in a
dropwise manner over 10 min while maintaining temperature at ꢀ78 °C. The reaction
mixture was allowed to warm to room temperature and stirred for additional 2 h. Sodium
hydroxide (3M, 5 mL) and H O (30% v/v, 5 mL) were added in a dropwise manner and
2
2
the solution was allowed to stir for 1 h and diluted with Et O (100 mL). The organic layer
2
was washed with H O (3 x 60 mL), brine (80 mL), dried (Na SO ), and evaporated
2
2
4
under reduced pressure to yield a yellow oil which was subjected to purification by
column chromatography (silica gel; hexanes/EtOAc; 100:1 to 25:1) to afford 0.2 g (40%)
of tertꢀbutylꢀ3ꢀ(2ꢀ(1,1ꢀdimethylallyl)ꢀ1Hꢀindolꢀ3ꢀyl)propylꢀNꢀmethylcarbamate as a glassy
1
solid: mp 116ꢀ118 °C; H NMR (CDCl ) δ 1.38 (s, 9H, (CH ) ), 1.45 (m, 8H, CH , CH ),
3
3 3
2
3
2.72 (t, 2H, CH ), 2.81 (s, 3H, CH ), 3.25 (m, 2H, CH ), 5.05ꢀ5.10 (m, 2H, CH ), 6.01ꢀ
2
3
2
2
6
1
.08 (m, 1H, CH), 6.97ꢀ7.06 m, 2H, Ar), 7.20 (d, J = 7.8 Hz, 1H, Ar), 7.41 (d, J = 7.6 Hz,
H, Ar), 7.74 (br s, 1H, indolic NH).
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Gaseous HCl was bubbled into a solution of tertꢀbutylꢀ3ꢀ(2ꢀ(1,1ꢀdimethylallyl)ꢀ1Hꢀindolꢀ
3
ꢀyl)propylꢀNꢀmethylcarbamate (0.1 g, 0.3 mmol) in dry EtOAc (10 mL) at 0 °C. The
reaction mixture was allowed to stir at room temperature overnight. The solvent was
evaporated under reduced pressure to yield a brown solid which was converted to the
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
oxalate salt and recrystallized from iꢀPrOH to yield 0.03 g (28%) of 13 as a yellow solid:
1
mp 135ꢀ138 °C; H NMR (DMSOꢀd ) δ 1.49 (s, 6H, (CH ) ), 1.83 (m, 2H, CH ), 2.55 (s,
6
3 2
2
3
H, CH ), 2.76 (t, 2H, CH ), 2.94 (t, 2H, CH ), 5.09 (m, 2H, CH ), 6.16 (m, 1H, CH), 6.95
3 2 2 2
(t, 1H, Ar), 7.21 (t, 1H, Ar), 7.32 (d, J = 8.0 Hz, 1H, Ar), 7.44 (d, J = 7.8 Hz, 1H, Ar),
1
0.47 (s, 1H, COOH). Anal. Calcd for (C H N —1.3C H O ) C, H, N: 62.95, 7.56,
17 24
2
2
2
4
+
8.09%. Found: 63.04, 7.18, 7.50%. UHPLCꢀMS; [M 1] calculated for C H N 257.15,
17
24
2
found 257.15.
7ꢀBromoꢀ1,1ꢀdimethylꢀ1,2,3,4ꢀtetrahydroꢀβꢀcarboline oxalate (14). A mixture of 6ꢀ
13
bromotryptamine (2.0 g, 8.4 mmol), acetone (0.6 mL, 8.8 mmol) and 2N HCl (8.40 mL,
1
6.80 mmol) in 30 mL of H O was heated at reflux for 5 h. The reaction mixture was
2
washed with Et O (30 mL), basified with 1N NaOH and extracted with CH Cl (2 x 30
2
2
2
mL). The combined organic portion was dried (Na SO ) and the solvent was removed
2
4
under reduced pressure. The oily residue was purified by column chromatography
silica gel) using CH Cl /MeOH (8:2) to afford 0.40 g (17%) of the desired product as a
(
2
2
1
yellow solid: mp 188ꢀ192 °C; H NMR (CDCl ) δ 1.40 (s, 6H, CH x 2), 1.48 (br s, 1H,
3
3
NH), 2.61 (t, J = 5.76 Hz, 2H, CH ), 3.13 (t, J = 5.76 Hz, 2H, CH ), 7.11 (dd, J = 8.36,
2
2
1
.68 Hz, 1H, ArH), 7.25 (d, J = 8.36 Hz, 1H, ArH), 7.38 (s, J = 1.68 Hz, 1H, ArH), 7.61
(br s, 1H, ArNH).
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The free base (0.40 g, 1.43 mmol) was dissolved in Et O and an ethereal solution of
2
oxalic acid (0.13 g, 1.43 mmol) was added to afford 0.37 g (70%) of 14 as a white
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
powder following recrystallization from abs EtOH/anhydrous Et O: mp 249ꢀ250 °C (with
2
1
dec.); H NMR (DMSOꢀd ) δ 1.53 (s, 6H, CH x 2), 2.73 (t, J = 5.76 Hz, 2H, CH ), 3.24
6
3
2
(
t, J = 5.6 Hz, 2H, CH ), 7.11 (dd, J = 8.36, 1.56 Hz, 1H, ArH), 7.37 (d, J = 8.36 Hz, 1H,
2
ArH), 7.48 (s, J = 1.56 Hz, 1H, ArH), 11.86 (br s, 1H, ArNH). Anal. Calcd for
(C H BrN ) —C H O —H O)] C, H, N.
[
13
15
2 2
2
2
4
2
6ꢀBromoꢀ2ꢀ(1,1ꢀDimethylallyl)ꢀ3ꢀ(piperidinꢀ4ꢀyl)ꢀ1Hꢀindole Hydrochloride (15).
Gaseous HCl was bubbled through a solution of 34 (0.10 g, 0.70 mmol) in anhydrous
EtOAc (10 mL). The solid product was recrystallized from iꢀPrOH to afford 15 (0.04 g,
1
41% yield) as a white solid: mp > 300 ºC; H NMR (DMSOꢀd ) δ 1.41 (s, 6H, 2xCH ),
6
3
1
1
1
.60ꢀ1.63 (m, 2H, CH ), 2.21ꢀ2.30 (m, 2H, CH ), 2.79ꢀ2.86 (m, 2H, CH ), 3.09ꢀ3.21 (m,
2 2 2
H, CH), 3.27ꢀ3.31 (m, 2H, CH ), 5.05ꢀ5.10 (m, 2H, CH ), 6.08ꢀ6.15 (dd, J = 10.5, J =
2
2
7.3 Hz, 1H, CH), 7.04ꢀ7.07 (dd, J = 1.8, J = 8.5 Hz, 1H, ArꢀH), 7.51 (d, J = 1.8 Hz, 1H,
+
2
ꢀ
ArꢀH), 7.71 (d, J = 8.5 Hz, 1H, ArꢀH), 10.63 (s, 1H, R NH Cl ). Anal. Calcd for
2
(
C H BrN ꢃHClꢃ2H O ) C, H, N.
18 23 2 2
1ꢀ(6ꢀBromoꢀ2ꢀ(1,1ꢀdimethylallyl)ꢀ1Hꢀindolꢀ3ꢀyl)propanꢀ2ꢀamine Hydrochloride (16).
Gaseous HCl was bubbled into a solution of 37 (300 mg, 0.2 mmol) in anhydrous Et O
2
(20 mL) at 0 °C. The reaction mixture was allowed to stir at room temperature for 24 h
and the solvent was evaporated to yield a white solid which was recrystallized from iꢀ
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2
2
2
2
2
2
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4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
PrOH/Et O to yield 60 mg (27%) of 16 as white crystals: mp 229ꢀ230 °C; H NMR
2
(
DMSOꢀd ) δ 1.09 (d, 3H, CH ), 1.51 (d, 6H, (CH ) ), 2.95ꢀ3.14 (m, 2H, CH ), 3.43 (m,
6 3 3 2 2
1H, CH), 5.08ꢀ5.15 (m, 2H, CH), 6.14ꢀ6.21 (m, 1H, CH), 7.14 (dd, 1H, Ar), 7.51 (m, 2H,
+
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Ar), 8.07 (br s, 3H, NH aliphatic), 10.86 (br s, 1H, indolic NH). Anal. Calcd for
(
C H BrN —HCl) C, H, N.
16 21 2
NꢀMethylꢀ6ꢀbromoꢀ2ꢀsecꢀbutyltryptamine Oxalate (17). Oxalyl chloride (0.2 mL, 2.4
mmol) was added in a dropwise manner to a stirred solution of 40 (0.5 g, 1.98 mmol) in
anhydrous Et O (20 mL) at 0 °C. The reaction mixture was allowed to stir for 30 min at 0
2
°C. The solvent was removed under reduced pressure and the residual yellow oil was
washed with Et O (2 x 5 mL) to remove excess oxalyl chloride. A solution of 40% aq.
2
MeNH (25 mL), cooled to 0 °C, was slowly added to 6ꢀbromoꢀ3ꢀylꢀ2ꢀsecꢀbutylꢀglyoxyl
2
chloride and the reaction mixture was allowed to stir overnight at room temperature. The
reaction mixture was concentrated in vacuo and the crude product, 42, (0.5 g) was used
in the next step.
A solution of 42 (0.50 g, 1.5 mmol) in anhydrous THF (12 mL) was heated to 60 °C.
Then BH ⋅DMS (0.42 mL, 4.5 mmol) was added and the reaction mixture was heated at
3
reflux with stirring overnight. The reaction mixture was cooled in an ice bath, quenched
with 2N HCl (10 mL), heated at reflux for 30 min, cooled and extracted with Et O (2 x 20
2
mL). The aqueous portion was treated with 10% NaOH to pH 14, and extracted with
CH Cl (2 x 20 mL). The combined organic portion was washed with H O (2 x 15 mL),
2
2
2
dried (Na SO ), and the solvent removed under reduced pressure. The solution of crude
2
4
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1
2
3
4
5
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7
8
9
residue (0.23 g, 0.74 mmol) and oxalic acid (0.07 g, 0.74 mmol) in CHCl (3 mL) was
3
allowed to stir for 0.5 h and the precipitate was collected by filtration. The solid product
was recrystallized from iꢀPrOH to afford 17 (0.12 g, 43% yield) as a white solid: mp 126ꢀ
1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
29 ºC; H NMR (DMSOꢀd ) δ 0.70 (t, J = 7.0 Hz, 3H, CH ), 0.85ꢀ0.87 (d, J = 7 Hz, 3H,
6
3
CH ), 1.42ꢀ1.50 (m, 2H, CH ), 2.42 (s, 3H, CH ), 2.76ꢀ2.81 (m, 5H, 2 x CH , CH ), 6.92
3
2
3
2
3
(d, J=4Hz, 1H, ArH), 7.24ꢀ7.28 (m, 2H, ArH,), 10.78 (s, 1H, oxalic acid). Anal. Calcd. For
(
C H BrN —C H O —0.5H O) C, H, N.
15
21
2
2
2
4
2
NꢀMethylꢀ6ꢀbromoꢀ2ꢀcyclopentyltryptamine Hydrochloride (18). A solution of 43
(
0.40 g, 1.15 mmol) in anhydrous THF (12 mL) was heated to 60 °C. Then BH ⋅DMS
3
(0.33 mL, 3.44 mmol) was added and the reaction mixture was heated at reflux with
stirring overnight. The reaction mixture was cooled in an ice bath, quenched with 2N
HCl (10 mL), heated at reflux for 30 min, cooled and extracted with Et O (2 x 20 mL).
2
The aqueous portion was treated with 10 % NaOH until pH 14, and extracted with
CH Cl (2 x 20 mL). The combined organic portion was washed with H O (2 x 15 mL),
2
2
2
dried (Na SO ), and the solvent removed under reduced pressure. The crude product
2
4
was collected by filtration through a small amount of silica gel using CH Cl /MeOH
2
2
(
9:1/1% TEA) to give 0.24 g of a yellow oil. The oil was dissolved in CH Cl (20 mL) and
2 2
washed with 10% HCl (10 mL); the organic portion was dried (Na SO ) and the solvent
2
4
was removed to provide 0.03 g (6%) of 18 as white crystals following recrystallization
1
from absolute EtOH/Et O: mp 262ꢀ264 °C (dec.); H NMR (DMSOꢀd ) δ 1.67ꢀ2.01 (m,
2
6
8
H, CH ), 2.58 (s, 4H, CH ꢀCH ꢀN), 2.98 (s, 3H, CH ), 3.24 (m, 1H, CH ), 7.10 (dd,
2 2 2 3 2
J=8.4, 1.8 Hz, 1H, ArH), 7.43 (d, J=1.8 Hz, 1H, ArH,), 7.45 (d, J=8.4, 1H, ArH,), 8.61 (br
29
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