Dual acting norepinephrine reuptake inhibitors and 5-HT2A receptor antagonists: Identification, synthesis and activity of novel 4-aminoethyl-3-(phenylsulfonyl)-1H-indoles

Article abstract of DOI:10.1016/j.bmc.2009.09.023

The discovery of a series of 4-aminoethyl-3-(phenylsulfonyl)-1H-indoles, dual acting norepinephrine reuptake inhibitors (NRIs) and 5-HT_2A receptor antagonists, is described. The synthesis and structure-activity relationship (SAR) of this novel

Full text of DOI:10.1016/j.bmc.2009.09.023

Bioorganic & Medicinal Chemistry 17 (2009) 7802–7815
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Dual acting norepinephrine reuptake inhibitors and 5-HT2A receptor
antagonists: Identification, synthesis and activity of novel
4-aminoethyl-3-(phenylsulfonyl)-1H-indoles
a
a,
Gavin D. Heffernan a, , Richard D. Coghlan , Eric S. Manas , Robert E. McDevitt d, Yanfang Li d,
Paige E. Mahaney a,à, Albert J. Robichaud d, Christine Huselton c, Peter Alfinito b, Jenifer A. Bray b,
Scott A. Cosmi b, Grace H. Johnston b, Thomas Kenney b, Elizabeth Koury b, Richard C. Winneker b,
Darlene C. Deecher b, Eugene J. Trybulski a
*
a Chemical Sciences, Wyeth Research, 500 Arcola Road, Collegeville, PA 19426, USA
b Women’s Health and Musculoskeletal Biology, Wyeth Research, 500 Arcola Road, Collegeville, PA 19426, USA
c Drug Safety and Metabolism, Wyeth Research, 500 Arcola Road, Collegeville, PA 19426, USA
d Chemical Sciences, Wyeth Research, CN 8000, Princeton, NJ 08543, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 9 July 2009
Revised 10 September 2009
Accepted 12 September 2009
Available online 18 September 2009
The discovery of a series of 4-aminoethyl-3-(phenylsulfonyl)-1H-indoles, dual acting norepinephrine
reuptake inhibitors (NRIs) and 5-HT2A receptor antagonists, is described. The synthesis and structure–
activity relationship (SAR) of this novel series of compounds is also presented.
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Monoamine reuptake inhibitors
Norepinephrine reuptake inhibitors
NRI
5-HT2A receptor antagonists
4-Aminoethyl-3-(phenylsulfonyl)-1H-
indoles
1. Introduction
brain and in particular the cortical, hippocampal, and hypotha-
lamic regions.
Drugs that inhibit the reuptake of norepinephrine (NE) selec-
tively, or in combination with serotonin (5-HT), have shown clini-
cal efficacy in
a variety of neurological disorders. Selective
norepinephrine reuptake inhibitors (NRIs) are used for the
treatment of depression (e.g., reboxetine 1)1 and attention defi-
cit-hyperactivity disorder (e.g., atomoxetine 2).2 Dual 5-HT/NE
reuptake inhibitors (SNRIs) are marketed for the treatment of
depression (e.g., venlafaxine 3;3 desvenlafaxine 4;4 duloxetine 55)
and the management of pain associated with diabetic peripheral
neuropathy (e.g., duloxetine 56) ( Fig. 1). Neurological disorders
such as depression, pain, and vasomotor symptoms are thought
to result from dysregulation of the levels of NE and 5-HT in the
Me
O
N
H
O
NH
Me
OEt
O
1 ( )-reboxetine
2 atomoxetine
Me
N
S
Me
OH
Me
O
N
H
R
O
* Corresponding author. Tel.: +1 732 274 4420; fax: +1 732 274 4505.
E-mail address: hefferg@wyeth.com (G.D. Heffernan).
Present address: GlaxoSmithKline, 709 Swedeland Road, King of Prussia, PA
19406, USA.
3 venlafaxine (R = Me)
4 desvenlafaxine (R =H)
5 duloxetine
à
Present address: Boehringer Ingelheim Pharmaceuticals, Inc., 900 Ridgebury
Figure 1. Selective norepinephrine reuptake inhibitors (NRIs) and dual acting
Road, Ridgefield, CT 06877, USA.
serotonin/norepinephrine reuptake inhibitors (SNRIs).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.09.023

G. D. Heffernan et al. / Bioorg. Med. Chem. 17 (2009) 7802–7815
7803
NRIs exert their effects by binding to the presynaptic norepi-
nephrine transporter (NET), blocking the reuptake of NE from the
synaptic cleft. The resulting increase in extracellular levels of the
neurotransmitter NE leads to an increase in neurotransmission.
There is a growing interest in developing drugs that act specif-
ically on multiple targets, termed designed multiple ligands
(DMLs).7,8 Many complex CNS disorders, such as schizophrenia
and depression, benefit by treatment with drugs with a multi-fac-
eted pharmacological profile.9 5-HT2A antagonists have proven
beneficial in treating neurological disorders10 and there is evidence
to suggest that 5-HT2A antagonists could have utility in depres-
sion,11 pain,12,13 and vasomotor disorders.14 Additionally, Deecher
and Merchenthaler15 reported that the combination of a 5-HT2A
antagonist with a NRI achieves greater efficacy in animal models
of vasomotor symptoms than attained by the NRI alone.
Conversely, 5-HT2A antagonists encompass a large degree of
structural diversity.10,17 Rowley et al.18 suggested that 5-HT2A
antagonists can be divided into two pharmacophore classes: (a)
those with two aryl rings and a basic amine arranged in a triangu-
lar arrangement (e.g., mianserin 6, tefludazine 7), and (b) those
with a linear arrangement of an aromatic ring, basic amine and a
second aryl ring or heteroaromatic group (e.g., MDL-100907 8,
ketanserin 9) ( Fig. 2). The similarity between the monoamine
transporter inhibitor pharmacophore and the ‘triangular’ 5-HT2A
antagonist pharmacophore highlights the intriguing possibility
that
a single template could deliver both NRIs and 5-HT2A
antagonists.19
2.1. NRI pharmacophore model
Given the overlapping potential utilities of NRIs and 5-HT2A
receptor antagonists, a drug discovery program was initiated to
identify dual acting NRI/5-HT2A receptor antagonists, with selectiv-
ity over other amine transporters and 5-HT2 receptors. The identi-
fication of a selective dual acting NRI/5-HT2A antagonist would
allow for the determination of the pharmacological effects, and po-
tential therapeutic utility, of dual NET inhibition and 5-HT2A
antagonism.
A NRI pharmacophore model was derived from a diverse series
of five NRIs ((S,S)-reboxetine 10, nisoxetine 11, maprotiline 12,
desipramine 13, and indole 1420) (Fig. 3).
Monte Carlo conformational analysis (MMFF94 forcefield, GB/
SA solvation model21) was conducted on each compound and all
conformations within 5 kcal/mol energy from the global minimum
were chosen. The average inter-feature distances and tolerances
were calculated for each NRI and then assessed for commonalities
within the group. This approach identified a simple NRI pharmaco-
2. Identification of dual NRI/5-HT2A antagonists
One approach to identify dual acting compounds is to develop
hybrid molecules where two molecules possessing the desired sin-
gle activities are fused, or joined by a suitable linker.7,8 This ap-
proach has been used extensively in the synthesis of dual acting
D2/5-HT2A receptor antagonists.10 A downside to this approach is
large molecules with poor physicochemical properties are often
the result.
A potentially more attractive approach would be to identify a
single template that can lead to both desired activities. Enyedy
et al.16 proposed a simple pharmacophore model for monoamine
transporter inhibitors that consists of two aromatic rings and a ba-
sic amine in a triangular arrangement.
Me
O
NH
O
N
H
OEt
O
OMe
10 (S,S)-reboxetine
hNET Ki 0.3 nM
11 nisoxetine
hNET Ki 6 nM
Me
N
N
H
Me
Me
N
N
N
H
H
OH
Me
12 maprotiline
hNET Ki 12 nM
13 desipramine
hNET Ki 0.6 nM
14
hNET Ki 9 nM
"Triangular" 5-HT2A antagonists
Me
OH
Figure 3. NRIs used to derive the NRI pharmacophore model.
N
N
N
F3C
N
6 mianserin
7 tefludazine
F
"Linear" 5-HT2A antagonists
MeO OH
MeO
N
F
8 MDL-100907
O
O
N
N
F
N
H
O
9 ketanserin
Figure 2. Classes of 5-HT2A receptor antagonists.
Figure 4. NRI Pharmacophore model with (S,S)-reboxetine overlaid.

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G. D. Heffernan et al. / Bioorg. Med. Chem. 17 (2009) 7802–7815
Electrophilic sulfenylation23 of the bromoindoles 19a–b on
treatment with thiophenol and potassium triiodide afforded the
3-phenylsulfenyl indoles. Oxidation with OXONEÒ then gave the
bromo-3-(phenylsulfonyl)-1H-indoles 20a–b.
The 5-bromo-20c and 7-bromo-20d analogs were prepared
from the corresponding nitrobenzenes utilizing a vicarious nucleo-
philic substitution (VNS) approach described by Wojciechowski
and Makosza24 (Scheme 2).
VNS reaction of the bromo-nitrobenzenes 21a–b with the anion
derived from chloromethyl phenyl sulfone gave the 1-nitro-2-
[(phenylsulfonyl)methyl]benzenes 22a–b. Reduction of the nitro
group to an amine followed by ethyl formimidate formation and
base catalyzed cyclization afforded the 5- and 7-bromo-3-(phenyl-
sulfonyl)-1H-indoles 20c and 20d, respectively.
Me
Me
Me
Me
N
Me
O
N
N
N
S
H
N
N
Cl
S
N
H
15 olanzapine
h5-HT2A Ki 4 nM
16 zotepine
h5-HT2A Ki 0.5 nM
17 (S)-mianserin
h5-HT2A Ki 3 nM
Figure 5. 5-HT2A antagonists that fit the NRI pharmacophore model.
phore model consisting of two aryl rings and a basic amine ar-
ranged in a triangular arrangement (Fig. 4), similar to the mono-
amine transporter inhibitor pharmacophore model reported by
Enyedy.16
The bromo intermediates were then converted to the desired
aminoethyl analogs (Scheme 3). Stille reaction of bromoindoles
Analysis of a series of 5-HT2A antagonists indicated that olanza-
pine 15, zotepine 16 and (S)-mianserin 17 fit this NRI pharmaco-
phore model (Fig. 5). This result bears out the similarity of the
NRI and 5-HT2A antagonist pharmacophores and supports the use
of the NRI pharmacophore model to virtually screen for dual acting
NRI/5-HT2A antagonists.
O
O
S
4
Br
Br
6
a, b
N
H
N
H
19a, 4-isomer
19b, 6-isomer
20a, 4-isomer
20b, 6-isomer
2.2. Virtual screen results
Unity 3-d flex searches were used to search the corporate com-
pound inventory for matches to the NRI pharmacophore model.
Additional hit requirements were zero Lipinski violations, polar
surface area 6120, and no more than 10 rotatable bonds. The vir-
tual screen hits were then further refined to remove highly
strained matches and known NRI templates. The refined hits were
binned according to ring scaffold and representatives from each
scaffold profiled in hNET and h5-HT2A radioligand binding assays.
This process successfully identified a number of dual acting
compounds including the 4-dialkylaminoethyl-3-(phenylsulfo-
nyl)-1H-indoles 18a and 18b (Fig. 6) originally prepared as part
of a 5-HT6 program at Wyeth.22
Scheme 1. Reagents and conditions: (a) PhSH, KI3, EtOH, 92–99% yield; (b)
OXONEÒ, NaHCO3, aq acetone, 80–90% yield.
X
a
b
X
SO2Ph
NO2
NO2
Y
Y
21a, X = Br, Y = H
21b, X = H, Y = Br
22a, X = Br, Y = H
22b, X = H, Y = Br
O
O
S
Br
5
X
c, d
SO2Ph
NH2
N
H
7
Y
Me
Me
Et
Me
23a, X = Br, Y = H
23b, X = H, Y = Br
20c, 5-isomer
20d, 7-isomer
N
N
O
S
O
S
O
O
Scheme 2. Reagents and conditions: (a) ClCH2SO2Ph, KOtBu, À78 °C to rt, 72–73%
yield; (b) H2, Raney-Ni, EtOAc, EtOH, 99% yield; (c) pTsOH, HC(OEt)3, DCE, reflux; (d)
KOtBu, THF, rt, 72–95% yield (two steps).
N
N
H
H
18a
18b
hNET Ki 45 nM
hNET Ki 28 nM
O
S
O
S
h5-HT2A Ki 262 nM
h5-HT2A Ki 222 nM
O
O
Br
a
b, c, d
Figure 6. Dual acting NRI/5-HT2A antagonists identified from the virtual screen.
Values reported were generated from radioligand binding assays using the human
norepinephrine transporter and human 5-HT2A receptor.
N
N
20a, 4-isomer
20b, 6-isomer
20c, 5-isomer
20d, 7-isomer
24a, 4-isomer
24b, 6-isomer
24c, 5-isomer
24d, 7-isomer
H
H
R2
R1 N
3. Chemistry
TsO
O
S
O
S
O
O
4
To develop the structure–activity relationship (SAR) around the
lead molecules, 18a and 18b, chemical syntheses were designed to
explore the attachment point of the aminoethyl chain to the indole
nucleus, the amine portion of the molecule and the length of the
alkylamine chain.
Two methods were utilized to prepare the key bromo-3-(phen-
ylsulfonyl)-1H-indole intermediates. The 4-bromo-20a and 6-bro-
mo-20b analogs were prepared from commercially available 4-
bromoindole 19a and 6-bromoindole 19b, respectively (Scheme 1).
e
5
6
25a, 4-isomer
25b, 6-isomer
25c, 5-isomer
25d, 7-isomer
18a-l, 4-isomer
N
N
H
H
7
26a-b, 6-isomer
27a-b, 5-isomer
28a-b, 7-isomer
Scheme 3. Reagents and conditions: (a) tributyl(vinyl)tin, PdCl2(PPh3)2, PhMe,
reflux, 74–99% yield; (b) BH3-THF, THF, rt; (c) H2O2, aq NaOH, 53–76% yield (two
steps); (d) pTsCl, pyr, CH3CN, rt, 86–96% yield; (e) R1R2NH, THF, 70 °C, 62–99% yield.

G. D. Heffernan et al. / Bioorg. Med. Chem. 17 (2009) 7802–7815
7805
OTs
NH2
Allylic alcohol 30 was prepared from 4-formylindole 29 accord-
ing to the procedure of Kardos and Genet.25 Catalytic hydrogena-
tion afforded 31, which was subjected to the electrophilic
sulfenylation/oxidation protocol previously described to give 3-
phenylsulfonylindole 32. Conversion of the primary alcohol to
the p-toluenesulfonate followed by displacement of the tosylate
group with basic amines gave 4-aminopropyl-3-(phenylsulfonyl)-
1H-indoles 33a–c.
O
S
O
S
O
O
a, b
N
N
H
H
25a
18m
Scheme 4. Reagents and conditions: (a) NaN3, DMF, 100 °C, 93% yield; (b) H2, Pd/C,
EtOH, 86% yield.
4. Results and discussion
4.1. In vitro characterization
HO
HO
O
Compounds 18a–m, 26a–b, 27a–b, 28a–b, and 33a–c were
evaluated in vitro to determine their affinity for the hNET and abil-
ity to inhibit the uptake of NE into MDCK-Net6 cells stably trans-
fected with the human NET. Selected compounds (hNET IC50
<1 lM) were then evaluated to determine their affinity for the
h5-HT2A receptor and functional h5-HT2A activity.
The initial lead compounds, 18a and 18b, were potent inhibitors
of norepinephrine reuptake (hNET IC50 80 and 49 nM, respectively)
and possessed modest h5-HT2A antagonist activity (h5-HT2A IC50
459 and 832 nM, respectively).
a, b
c
N
H
N
H
N
H
29
30
31
R2
N
HO
R1
O
S
O
S
O
O
d, e
f, g
A systematic evaluation of attachment point of the aminoethyl
chain to the indole nucleus was first explored. It was found that
moving the aminoethyl chain from the 4-position to the 5-, 6-, or
7-position on the indole nucleus was clearly detrimental to NRI po-
tency (18a–b vs 27a-b, 26a–b, and 28a–b) (see Table 1). This is in
contrast to the behavior at the 5-HT6 receptor, where the 5- and 7-
aminoethyl-3-(phenylsulfonyl)-1H-indoles behave as potent full
antagonists and the 4-aminoethyl-3-(phenylsulfonyl)-1H-indoles
as low affinity, partial antagonists.
Exploration of the amine functionality within the 4-aminoethyl
series was then initiated (see Table 2). The isopropylamine 18f
(hNET IC50 51 nM), and piperidine 18i (hNET IC50 47 nM) analogs
possessed similar NRI potency to the lead 18b (hNET IC50 49 nM).
Secondary amines larger than isopropyl led to a reduction in NRI
potency (18f vs 18g) as did methyl substitution on the piperidine
analog (18i vs 18k and 18l).
N
H
N
H
32
33a-c
Scheme 5. Reagents and conditions: (a) EtO2CCH2P(O)(OEt)2, K2CO3, THF, reflux,
95% yield; (b) Dibal-H, THF, À78 °C to rt, 90% yield; (c) H2, Pd/C, EtOH, EtOAc, 93%
yield; (d) PhSH, KI, I2, EtOH, 65 °C, 71% yield; (e) OXONEÒ, NaHCO3, acetone, H2O, rt,
90% yield; (f) pTsCl, pyr, CH3CN, rt, 32% yield; (g) R1R2NH, THF, 65 °C, 43–69% yield.
20a–d with tributyl(vinyl)tin afforded styrenes 24a–d, which were
hydroborated and oxidized to give the primary alcohols. The alco-
hols were then converted to the p-toluenesulfonates 25a–d and
the tosylate group readily displaced by a variety of basic amines
to afford the desired targets 18a–l, 26a–b, 27a–b, and 28a–b.
The primary amine 18m was prepared via a standard two step
aziridination, reduction procedure (Scheme 4).
The 4-aminopropyl-3-(phenylsulfonyl)-1H-indoles 33a–c were
prepared from 4-formylindole 29 (Scheme 5).
Several analogs possessed improved h5-HT2A receptor antago-
nist activity over the initial leads (18a and 18b). The cyclopentyl-
Table 1
Characterization of 4-, 5-, 6- and 7-dialkylaminoethyl-3-(phenylsulfonyl)-1H-indoles 18a–b, 26a–b, 27a–b and 28a–b at the human norepinephrine transporter and human 5-
HT2A receptora
R1 R2
R2
N
O
S
O
S
N
O
O
O
S
O
S
R1
O
O
N
N
H
H
N
N
R1
H
H
N
R2
N
R1 R2
18a-b
28a-b
27a-b
26a-b
Compd
R1
R2
hNET Ki, nMb (SD)
hNET IC50, nMc (SD)
h5-HT2A Ki, nMd (SD)
h5-HT2A IC50, nMe (SD)
18a
18b
27a
27b
26a
26b
28a
28b
Me
Et
Me
Et
Me
Et
Me
Et
Me
Me
Me
Me
Me
Me
Me
Me
45 (2)
28 (9)
80 (12)
49 (11)
43%f
262 (34)
222 (145)
459 (158)
832 (435)
48%f
12%f
<10%f
<10%f
<10%f
a
b
c
d
e
f
Values are the mean of between 2 and 7 independent runs each in triplicate, unless otherwise indicated.
Inhibition of [3H] nisoxetine binding to MDCK-Net6 cells, stably transfected with the human norepinephrine transporter (hNET).
Inhibition of norepinephrine uptake in MDCK-Net6 cells, stably transfected with the human NET.
Inhibition of [3H] ketanserin binding to membranes from CHO cells, stably transfected with the human 5-HT2A receptor.
Inhibition of DOI induced intracellular calcium mobilization in CHO cells, stably transfected with the human 5-HT2A receptor, measured using FLIPR.
Percentage inhibition measured at a concentration of 1000 nM, data is the average of three triplicate runs.

7806
G. D. Heffernan et al. / Bioorg. Med. Chem. 17 (2009) 7802–7815
Table 2
Characterization of 4-aminoethyl-3-(phenylsulfonyl)-1H-indoles 18a–m at the human norepinephrine transporter and human 5-HT2A receptora
R1 R2
N
O
S
O
N
H
18a-m
Compd
R1
R2
hNET
hNET
h5-HT2A
h5-HT2A
Ki, nM (SD)
IC50, nM (SD)
Ki, nM (SD)
IC50, nM (SD)
18m
18c
18d
18e
18f
18g
18a
18b
18h
18i
H
Me
Et
nPr
iPr
cPent
Me
Et
H
H
H
H
H
H
Me
Me
792 (88)
306 (13)
78 (11)
487 (27)
25 (3)
428 (28)
45 (2)
28 (9)
73 (6)
27 (4)
208 (62)
125 (30)
1130 (424)
960 (234)
353 (50)
146 (27)
302 (112)
51 (8)
460 (82)
80 (12)
49 (11)
128 (47)
47 (22)
221 (67)
116 (32)
1092 (124)
32%b
0%b
273 (88)
389 (37)
367 (88)
110 (76)
19 (8)
262 (34)
222 (145)
117 (51)
59 (15)
33 (18)
89 (20)
46 (18)
1105c
1025 (259)
1078c
650 (284)
31 (18)
459 (158)
832 (435)
258 (102)
180 (83)
69 (63)
92 (29)
57 (15)
Pyrrolidine
Piperidine
Homopiperidine
2-Me piperidine
3-Me piperidine
18j
18k
18l
a
b
c
Values are the mean of between 2 and 7 independent runs each in triplicate, unless otherwise indicated.
Percentage inhibition measured at a concentration of 1000 nM.
Values are for a single experiment run in triplicate.
The piperidine analog 18i possessed a good balance of NRI (hNET
IC50 47 nM) and 5-HT2A antagonist activities (h5-HT2A IC50
180 nM) and was counter-screened against the human serotonin
transporter (hSERT) and the human dopamine transporter (hDAT)
and against h5-HT2B, h5-HT2C and h5-HT6 receptors (see Table 4).
Piperidine 18i was highly selective for the NE transporter over
the 5-HT and DA transporters, exhibiting poor functional activity
Table 3
Characterization of 4-dialkylaminopropyl-3-(phenylsulfonyl)-1H-indoles 33a–c at
the human norepinephrine transporter and human 5-HT2A receptora
R2
N
R1
O
S
O
at the hSERT (0% inhibition of 5-HT uptake at 1
affinity for the hDAT (3% inhibition of [3H]WIN-35,428 binding at
M). Additionally, 18i possessed 40-fold greater affinity for the
h5-HT2A receptor over the h5-HT2C receptor and little affinity for
the 5-HT2B receptor was observed. The 3-phenylsulfonylindole
template is a well established 5-HT6 scaffold,22,26 however, by
incorporating the aminoethyl chain at the 4-position of the indole
nucleus, 5-HT6 affinity was minimized and 18i possessed sevenfold
greater affinity for the 5-HT2A receptor over the 5-HT6 receptor.
lM) and weak
N
H
1
l
33a-c
Compd R1
R2
hNET
hNET
h5-HT2A
h5-HT2A
Ki, nM (SD) IC50, nM (SD) Ki, nM (SD) IC50, nM
33a
33b
33c
Et
iPr
Piperidine
Me
H
21 (9)
13 (2)
1030 (199)
29 (1)
12 (0.5)
707 (226)
713 (174)
619 (374)
854 (382)
477b
5979b
7972b
a
Values are the mean of between 2 and 3 independent runs each in triplicate,
unless otherwise indicated.
5. In vivo characterization
b
Values are for a single experiment run in triplicate.
To determine the brain penetration potential for 18i, plasma,
brain, and hypothalamus concentrations were determined follow-
ing a single sub-cutaneous dose of 30 mg/kg (see Table 5).
Compound 18i exhibited poor brain to plasma (0.05) and hypo-
thalamus to plasma ratios (0.12), obtained by comparing plasma
area-under-the-curve (AUC) to brain and hypothalamus AUCs,
respectively. However, the observed Cmax in the brain (348 ng/g,
amine 18g (h5-HT2A IC50 31 nM), homopiperidine 18j (h5-HT2A
IC50 69 nM), and 3-methyl piperidine 18l (h5-HT2A IC50 57 nM)
analogs were all potent h5-HT2A antagonists.
Finally, increasing the alkyl chain length between the basic
amine and the indole nucleus from 2 to 3 carbons was explored
(see Table 3). The ethyl methylamine 33a (hNET IC50 29 nM) and
isopropylamine 33b (hNET IC50 12 nM) analogs were potent NRIs
with improved NRI activity over the original leads (18a and 18b).
The ethyl methylamine analog 33a also retained similar h5-HT2A
receptor antagonist activity to the leads. In this series,
incorporation of a piperidine moiety was detrimental to both
NRI and h5-HT2A activity (33c, hNET IC50 707 nM, h5-HT2A IC50
7972 nM).
0.94
lM) and hypothalamus (782 ng/g, 2.12 lM) was significantly
above the hNET and h5-HT2A IC50 values.
NE has been shown to stimulate areas of the hypothalamus that
are important in temperature regulation27 and NRIs28 and SNRIs29
have proven successful in restoring normal thermoregulation in rat
models of vasomotor disorders. Compound 18i was assessed in a
telemetric rat model of ovariectomized (OVX)-induced thermoreg-
ulatory dysfunction.29,30
Intact cycling rats exhibit a diurnal rhythm during which, over a
24 h period, their tail-skin temperature (TST) decreases during the
dark (active) phase and remains elevated during the light (inactive)
4.2. Selectivity screening
A challenge in designing molecules with activity at more than
one target is in achieving selectivity over related biological targets.

G. D. Heffernan et al. / Bioorg. Med. Chem. 17 (2009) 7802–7815
7807
Table 4
Selectivity screening of 18i versus serotonin and dopamine transporters and 5-HT2B, 5-HT2C and 5-HT6 receptorsa
N
O
S
O
N
H
18i
Compd
Amine transporters
hSERT uptake
5-HT2 receptors
5-HT6 receptor
hNET
IC50, nM (SD)
hDAT binding
% inhibition at 1
h5-HT2A
Ki, nM (SD)
h5-HT2B binding
% inhibition at 5
h5-HT2C
h5-HT6
% inhibition at 1
l
Mb
l
Mc
l
Md
Ki, nMf
Ki, nMg
18i
47 (22)
0%e
3%e
59 (15)
28%e
2400e
434e
a
b
c
d
e
f
Values are the mean of between 2 and 6 independent runs each in triplicate, unless otherwise indicated.
Inhibition of serotonin uptake in JAR cells, stably transfected with the human serotonin transporter (hSERT).
Inhibition of [3H] WIN-35,428 binding to membranes from CHO cells, stably transfected with the human dopamine transporter (hDAT).
Inhibition of [3H] 5-HT binding to membranes from CHO cells, stably transfected with the human 5-HT2B receptor.
Values are for a single experiment run in triplicate.
Inhibition of [125I] DOI binding to membranes from CHO cells, stably transfected with the human 5-HT2C receptor.
Inhibition of [3H] LSD binding to membranes from HeLa cells, stably transfected with the human 5-HT6 receptor.
g
Table 6
Table 5
Summary of the activity of 18i in a telemetric rat model of ovariectomized (OVX)-
Average pharmacokinetic parameters of 18i in female rats following a single sc dose
induced thermoregulatory dysfunctiona
of 30 mg/kga
Dose
(mg/kg)
Onset of
activity (h)
Duration of
action (h)
Mean reduction
in TST (°C)
Maximum
reduction in TST
(°C)
Matrix
Cmax (ng/mL Tmax AUClast (h ng/
Ratio AUClast
tissue/AUClast
plasma
or ng/g)
(h)
mL or h ng/g)
30
0.5
>12
À2.40
À3.85
Brain
Hypothalamus
Plasma
348
782
6142
2.5
2.5
1.5
986
2262
19,096
0.05
0.12
a
Compound dosed sc (n = 16 rats), 0.5% methylcellulose/2% Tween-80 in water
vehicle.
a
0.5% Methylcellulose/2% Tween-80 in water vehicle.
6. Conclusion
phase. Reduction in ovarian hormones as a result of ovariectomy
causes the TST of ovariectomized (OVX) rats to remain elevated
during both the dark and light phases. A drop in TST of 6 °C is ob-
served for intact rats during the dark (active) phase, but the drop in
TST is reduced to approximately 1 °C after ovariectomy. Treatment
of OVX-rats with estrogen has been reported to restore the diurnal
temperature pattern to that seen in an intact rat.30 At a dose of
30 mg/kg sc compound 18i significantly reduced the TST (maxi-
A pharmacophore model was derived from a diverse series of
five NRIs and was used to virtually screen the corporate compound
inventory for dual acting NRI/5-HT2A receptor antagonists. This ap-
proach successfully identified the dual acting 4-dialkylaminoethyl-
3-(phenylsulfonyl)-1H-indoles, 18a and 18b, a novel class of NRIs.
Exploration of the attachment point of the aminoethyl chain to the
indole nucleus, the amine portion of the molecule and the length of
the alkylamine chain identified piperidine 18i (hNET IC50 47 nM,
h5-HT2A IC50 180 nM), a balanced NRI and 5-HT2A antagonist. In
addition, 18i exhibited good selectivity for the hNET over the
hSERT and hDAT, and for the h5-HT2A receptor over the 5-HT2B
and 5-HT2C receptors. Compound 18i was assessed in a telemetric
rat model of ovariectomized (OVX)-induced thermoregulatory dys-
function and produced a significant and long lasting drop in TST at
a dose of 30 mg/kg sc.
mum
DTST – 3.85 °C) of OVX-rats in the active phase with a pro-
longed duration of action (>12 h) that lasted the entire dark
phase (see Fig. 7 and Table 6).
The 4-aminoalkyl-3-(phenylsulfonyl)-1H-indole scaffold could
also be utilized to access potent and selective NRIs (e.g., 33b, hNET
IC50 12 nM, >100-fold selective over the h5-HT2A receptor) and h5-
HT2A receptor antagonists (e.g., 18g, h5-HT2A IC50 31 nM, 15-fold
selective over the hNET).
7. Experimental
7.1. General methods: chemistry
Solvents were purchased as anhydrous grade and were used
without further purification. 1H NMR spectra were recorded on a
Varian INOVA 500 instrument, and chemical shifts are reported
in d values (parts per million, ppm) relative to an internal standard
tetramethylsilane in CDCl3 or DMSO-d6. Electrospray (ESI) mass
Figure 7. Effect of 18i (30 mg/kg sc) in a telemetric rat model of ovariectomized
(OVX)-induced thermoregulatory dysfunction. Error bars represent standard error
of the mean of each data point. Test compound administered 30 min prior to dark
phase.

7808
G. D. Heffernan et al. / Bioorg. Med. Chem. 17 (2009) 7802–7815
spectra were recorded using a Waters Alliance-ZMD mass spec-
trometer. Electron impact ionization (EI, EE = 70 eV) mass spectra
were recorded on a Finnigan Trace mass spectrometer. Analytical
thin layer chromatography (TLC) was performed on pre-coated
plates (Silica Gel 60 F-254) and were visualized using UV light
and/or staining with a phosphomolybdic acid solution in methanol.
In general, compound purity was assessed by 1H NMR and an LC/
UV/MS method.31 Biological results were obtained on compounds
of >95% chemical purity as determined by the above methods.
tetrahydrofuran (50 mL) at À65 °C under nitrogen was added a
solution of potassium tert-butoxide (1.0 M in tetrahydrofuran,
55 mL, 55 mmol). The deep purple reaction was allowed to warm
to 0 °C over 1.5 h and then treated with glacial acetic acid (4 mL).
The reaction was diluted with water (100 mL) and saturated aque-
ous NaHCO3 (100 mL), and then extracted with dichloromethane
(2 Â 200 mL). The combined organic extracts were dried over
MgSO4 and concentrated to afford a light orange solid. Trituration
with ethyl acetate and hexanes gave 5-bromo-2-nitro-1-(phen-
ylsulfonylmethyl)benzene 22a (6.45 g, 72%) as a pale yellow solid.
1H NMR (500 MHz, chloroform-d) d ppm 4.91 (s, 2H), 7.50–7.57 (m,
2H), 7.61 (d, J = 2.0 Hz, 1H), 7.65–7.71 (m, 2H), 7.74 (dd, J = 8.5,
1.1 Hz, 2H), 7.88 (d, J = 8.7 Hz, 1H); MS (ESI) m/z 354.0 [(MÀH)À].
7.1.1. 4-Bromo-3-(phenylthio)-1H-indole
To a solution of 4-bromoindole (19a) (5.68 g, 0.029 mol) and
thiophenol (2.97 mL, 0.029 mol) in ethanol (140 mL) at room tem-
perature under air was added a solution of potassium iodide
(4.81 g, 0.029 mol) and iodine (7.35 g, 0.029 mol) in 3:1 v/v water:
ethanol (100 mL) and the resulting black reaction mixture stirred
at 60 °C for 3 days. The reaction mixture was then diluted with
ethyl acetate (1 L), washed with 5% aq Na2S2O3 (800 mL), water
(1 L) and 1/2 saturated brine (1 L), dried over Na2SO4 and concen-
trated to afford a brown solid. The crude product was purified by
silica gel chromatography eluting with a hexanes–ethyl acetate
gradient (90/10 to 80/20) to give 4-bromo-3-(phenylthio)-1H-in-
dole (8.1 g, 92%) as a tan crystalline solid. 1H NMR (500 MHz,
DMSO-d6) d ppm 6.97 (d, J = 7.8 Hz, 2H), 7.02–7.11 (m, 2H), 7.19
(t, J = 7.8 Hz, 2H), 7.24 (d, J = 7.6 Hz, 1H), 7.51 (d, J = 8.1 Hz, 1H),
7.83 (s, 1H), 12.01 (br s, 1H); MS (ESI) m/z 301.9 ([MÀH]À).
7.1.6. 1-Bromo-2-nitro-3-[(phenylsulfonyl)methyl]benzene
(22b)
Prepared in essentially the same manner as 22a replacing 1-
bromo-4-nitrobenzene 21a with 1-bromo-2-nitrobenzene 21b.
Yield 73% of a tan solid. 1H NMR (500 MHz, DMSO-d6) d ppm
4.78 (s, 2H), 7.43 (d, J = 7.9 Hz, 1H), 7.53 (t, J = 7.9 Hz, 1H), 7.63
(t, J = 7.8 Hz, 2H), 7.69–7.73 (m, 2H), 7.75–7.80 (m, 1H), 7.91 (dd,
J = 8.2, 1.2 Hz, 1H); MS (ESI) m/z 354.0 ([MÀH]À).
7.1.7. {4-Bromo-2-[(phenylsulfonyl)methyl]phenyl}amine
(23a)
A solution of 5-bromo-2-nitro-1-(phenylsulfonylmethyl)ben-
zene 22a (0.23 g, 0.64 mmol) in ethyl acetate (30 mL) was hydroge-
nated in the presence of catalytic Raney nickel and hydrogen (45
psi) for 1 h. The reaction mixture was then filtered through
CeliteÒ and concentrated to give {4-bromo-2-[(phenylsulfo-
nyl)methyl]phenyl}amine 23a (0.21 g, 99%) as a light brown solid.
1H NMR (500 MHz, chloroform-d) d ppm 4.36 (s, 2H), 5.15 (br s,
2H), 6.71 (d, J = 2.3 Hz, 1H), 6.79 (d, J = 8.5 Hz, 1H), 7.25 (dd,
J = 8.5, 2.3 Hz, 1H), 7.56 (dd, J = 8.2, 7.5 Hz, 2H), 7.69 (t, J = 7.5 Hz,
1H), 7.79 (dd, J = 8.2, 1.0 Hz, 2H); MS (ESI) m/z 326.0 [(M+H)+].
7.1.2. 6-Bromo-3-(phenylthio)-1H-indole
Prepared in essentially the same manner replacing 4-bromoin-
dole (19a) with 6-bromoindole (19b). Yield 99% of an off-white
solid. 1H NMR (500 MHz, DMSO-d6) d ppm 6.97–7.03 (m, 2H),
7.03–7.10 (m, 1H), 7.14–7.24 (m, 3H), 7.31 (d, J = 8.5 Hz, 1H),
7.67 (d, J = 1.8 Hz, 1H), 7.79 (s, 1H), 11.80 (br s, 1H); MS (ESI) m/z
302.1 ([MÀH]À).
7.1.3. 4-Bromo-3-(phenylsulfonyl)-1H-indole (20a)
7.1.8. {2-Bromo-6-[(phenylsulfonyl)methyl]phenyl}amine
(23b)
To
a solution of 4-bromo-3-(phenylthio)-1H-indole (8.1 g,
0.0266 mol) in acetone (330 mL) was added 0.2 M aq NaHCO3
(330 mL) followed by OXONEÒ (40.92 g, 0.0666 mol) added over
ꢀ5 min and the reaction mixture stirred at room temperature for
63 h. The acetone was then removed under reduced pressure and
the resulting aqueous suspension partitioned between ethyl ace-
tate (750 mL) and water (750 mL). The organic phase was sepa-
rated, washed with water (750 mL) and 1/2 saturated brine
(500 mL), dried over Na2SO4 and concentrated to afford a cream so-
lid. The crude product was suspended in 2:1 v/v hexanes:ethyl ace-
tate (600 mL), the suspension stirred vigorously for 12 h then
filtered to afford 4-bromo-3-(phenylsulfonyl)-1H-indole 20a
(8.08 g, 90%) as an off-white solid. 1H NMR (400 MHz, DMSO-d6)
d ppm 7.13 (dd, J = 8.1 Hz, 1H), 7.33 (d, J = 7.6 Hz, 1H), 7.48–7.64
(m, 4H), 7.80–7.88 (m, 2H), 8.38 (s, 1H) 12.64 (br s, 1H); MS (ESI)
m/z 336.0 ([M+H]+).
Prepared in essentially the same manner as 23a replacing 5-bro-
mo-2-nitro-1-(phenylsulfonylmethyl)benzene 22a with 1-bromo-
2-nitro-3-[(phenylsulfonyl)methyl]benzene 22b. Yield 99% of a
white solid. 1H NMR (500 MHz, DMSO-d6) d ppm 4.70 (s, 2H), 5.25
(s, 2H), 6.40 (dd, J = 7.9, 7.6 Hz, 1H), 6.80 (dd, J = 7.6, 1.2 Hz, 1H),
7.34 (dd, J = 7.9, 1.2 Hz, 1H), 7.59 (dd, J = 7.5, 7.3 Hz, 2H), 7.72 (t,
J = 7.5 Hz, 1H), 7.78 (d, J = 7.3 Hz, 2H); MS (ESI) m/z 326.0 ([M+H]+).
7.1.9. 5-Bromo-3-(phenylsulfonyl)-1H-indole (20c)
A
stirred solution of {4-bromo-2-[(phenylsulfonyl)methyl]-
phenyl}amine 23a (3.26 g, 10.00 mmol, para-toluenesulfonic acid
monohydrate (0.20 g) and triethyl orthoformate (8.32 mL,
50 mmol) in 1,2-dichloroethane (70 mL) was heated at reflux un-
der nitrogen for 5 h and then stirred at room temperature for
16 h. The reddish reaction was concentrated under reduced pres-
sure to give a red oil. The resulting crude intermediate iminoether
was dissolved in tetrahydrofuran (50 mL) and a solution of potas-
sium tert-butoxide (1.0 M in tetrahydrofuran, 13 mL, 13 mmol)
added dropwise. After 5 min, a tan precipitate is evident. After
1 h, the reaction is treated with water (30 mL) and NH4Cl
(0.60 g), extracted with dichloromethane (150 mL), dried over
MgSO4 and concentrated to afford a light orange solid. Trituration
with ethyl acetate and hexanes gave 5-bromo-3-(phenylsulfonyl)-
1H-indole 20c (2.78 g, 72%) as a light orange solid. 1H NMR
(500 MHz, chloroform-d) d ppm 7.30 (d, J = 8.7 Hz, 1H), 7.39 (d,
J = 8.7 Hz, 1H), 7.47–7.58 (m, 3H), 7.89 (d, J = 3.1 Hz, 1H), 8.02 (d,
J = 8.1 Hz, 2H), 8.09 (s, 1H), 8.95 (br s, 1H); MS (ESI) m/z 334.0
[(MÀH)À].
7.1.4. 6-Bromo-3-(phenylsulfonyl)-1H-indole (20b)
Prepared in essentially the same manner as 20a replacing
4-bromo-3-(phenylthio)-1H-indole with 6-bromo-3-(phenylthio)-
1H-indole. Yield 80% of a white solid. 1H NMR (500 MHz, DMSO-
d6) d ppm 7.34 (dd, J = 8.5, 1.5 Hz, 1H), 7.51–7.64 (m, 3H), 7.68
(d, J = 1.5 Hz, 1H), 7.71 (d, J = 8.5 Hz, 1H), 7.91–8.00 (m, 2H), 8.21
(s, 1H), 12.37 (br s, 1H); MS (ESI) m/z 334.1 ([MÀH]À).
7.1.5. 5-Bromo-2-nitro-1-(phenylsulfonylmethyl)benzene
(22a)
To a stirred solution of 1-bromo-4-nitrobenzene 21a (5.05 g,
25 mmol) and chloromethylphenylsulfone (4.76 g, 25 mmol) in

G. D. Heffernan et al. / Bioorg. Med. Chem. 17 (2009) 7802–7815
7809
7.1.10. 7-Bromo-3-(phenylsulfonyl)-1H-indole (20d)
24.88 mL, 24.88 mmol) and the reaction mixture stirred for
2 12 h. 10% aq NaOH (11.9 mL, 29.86 mmol) and 30% hydrogen per-
oxide (3.39 mL, 29.86 mmol) were added sequentially and the
mixture stirred at room temperature for 3 12 h. The reaction was
then quenched by the addition of saturated aq NH4Cl (140 mL),
stirred vigorously for 5 min and then partitioned between ethyl
acetate (1 L) and water (1 L). The organic phase was separated,
washed with 1/2 saturated brine (1 L), dried over Na2SO4 and
concentrated to afford an orange foam. The crude product was
purified by silica gel chromatography eluting with a hexanes–
ethyl acetate gradient (70/30 to 30/70) to give 2-[3-(phenylsulfo-
nyl)-1H-indol-4-yl]ethanol (4.0 g, 53%) as a cream foam. 1H NMR
(500 MHz, DMSO-d6) d ppm 3.03 (t, J = 7.0 Hz, 2H), 3.22–3.29 (m,
2H), 4.37 (t, J = 5.2 Hz, 1H), 6.97 (d, J = 7.3 Hz, 1H), 7.14 (dd,
J = 7.3, 8.2 Hz, 1H), 7.36 (d, J = 8.2 Hz, 1H), 7.52–7.65 (m, 3H),
7.82 (d, J = 7.3 Hz, 2H), 8.22 (s, 1H), 12.31 (br s, 1H); MS (ESI)
m/z 302.1 ([M+H]+).
Prepared in essentially the same manner as 20c replacing {4-
bromo-2-[(phenylsulfonyl)methyl]phenyl}amine 23a with {2-Bro-
mo-6-[(phenylsulfonyl)methyl]phenyl}amine 23b. Yield 95% of a
yellow foam. 1H NMR (500 MHz, DMSO-d6) d ppm 7.15 (dd,
J = 8.1, 7.6 Hz, 1H), 7.48 (d, J = 7.6 Hz, 1H), 7.52–7.64 (m, 3H),
7.78 (d, J = 8.1 Hz, 1H), 7.99 (d, J = 7.8 Hz, 2H), 8.21 (d, J = 3.2 Hz,
1H), 12.60 (br s, 1H); MS (ESI) m/z 334.0 ([MÀH]À).
7.1.11. 3-(Phenylsulfonyl)-4-vinyl-1H-indole (24a)
A
solution of 4-bromo-3-(phenylsulfonyl)-1H-indole 20a
(8.31 g, 0.0247 mol) and tributyl vinyl tin (10.11 mL, 0.0346 mol)
in toluene (210 mL) and dimethoxyethane (70 mL) was purged
with nitrogen for 2 h. trans-Dichlorobis(triphenylphosphine) palla-
dium(II) (1.73 g, 0.00247 mol) was then added and the reaction
mixture heated to reflux for 2 h. Additional trans-dichlorobis(tri-
phenylphosphine) palladium(II) (1.73 g, 0.00247 mol) was then
added and the mixture heated to reflux for 18 h. 1.0 M aq KF
(38.1 mL, 38.1 mmol) was then added to the cooled reaction and
mixture stirred at room temperature for 12 h. The mixture was
then filtered through silica and the filtrate concentrated under re-
duced pressure to afford an orange syrup. The crude product was
purified by silica gel chromatography eluting with a hexanes–ethyl
acetate gradient (70/30 to 60/40) to give 3-(phenylsulfonyl)-4-vi-
nyl-1H-indole 24a (6.93 g, 99%) as an orange oil. 1H NMR
(500 MHz, DMSO-d6) d ppm 5.13 (dd, J = 10.8, 1.5 Hz, 1H), 5.56
(dd, J = 17.1, 1.5 Hz, 1H), 7.23 (t, J = 7.8 Hz, 1H), 7.33 (d, J = 7.3 Hz,
1H), 7.46 (d, J = 7.9 Hz, 1H), 7.52 (m, 2H), 7.56–7.61 (m, 1H), 7.64
(dd, J = 17.1, 10.8 Hz, 1H), 7.81 (d, J = 7.6 Hz, 2H), 8.27 (s, 1H),
12.41 (br s, 1H); MS (ESI) m/z 284.1 ([M+H]+).
7.1.16. 2-[3-(Phenylsulfonyl)-1H-indol-6-yl]ethanol
Prepared in essentially the same manner replacing 3-(phenyl-
sulfonyl)-4-vinyl-1H-indole 24a with 3-(phenylsulfonyl)-6-vinyl-
1H-indole 24b. Yield 62% of a white solid. 1H NMR (400 MHz,
DMSO-d6) d ppm 2.77 (t, J = 7.0 Hz, 2H), 3.53–3.63 (m, 2H), 4.58
(t, J = 5.2 Hz, 1H), 7.05 (dd, J = 8.2, 1.2 Hz, 1H), 7.30 (br. s, 1H),
7.49–7.61 (m, 3H), 7.64 (d, J = 8.2 Hz, 1H), 7.94 (dd, J = 7.9,
1.6 Hz, 2H), 8.09 (s, 1H), 12.12 (br s, 1H); MS (ESI) m/z 300.1
([MÀH]À).
7.1.17. 2-[3-(Phenylsulfonyl)-1H-indol-5-yl]ethanol
Prepared in essentially the same manner replacing 3-(phenyl-
sulfonyl)-4-vinyl-1H-indole 24a with 3-(phenylsulfonyl)-5-vinyl-
1H-indole 24c. Yield 76% of a white solid. 1H NMR (500 MHz,
DMSO-d6) d ppm 2.79 (t, J = 7.0 Hz, 2H), 3.54–3.62 (m, 2H), 4.59
(t, J = 5.3 Hz, 1H), 7.10 (dd, J = 8.2, 1.2 Hz, 1H), 7.38 (d, J = 8.2 Hz,
1H), 7.50–7.62 (m, 4H), 7.96 (d, J = 8.5 Hz, 2H), 8.11 (d, J = 2.1 Hz,
1H), 12.15 (br s, 1H); MS (ESI) m/z 300.0 ([MÀH]À).
7.1.12. 3-(Phenylsulfonyl)-6-vinyl-1H-indole (24b)
Prepared in essentially the same manner as 24a replacing 4-
bromo-3-(phenylsulfonyl)-1H-indole 20a with 6-bromo-3-(phen-
ylsulfonyl)-1H-indole 20b. Yield 82% of a white solid. 1H NMR
(500 MHz, DMSO-d6) d ppm 5.21 (d, J = 11.0 Hz, 1H), 5.79 (d,
J = 17.7 Hz, 1H), 6.80 (dd, J = 17.7, 11.0 Hz, 1H), 7.39 (dd, J = 8.4,
1.1 Hz, 1H), 7.49 (s, 1H), 7.52–7.63 (m, 3H), 7.71 (d, J = 8.4 Hz,
1H), 7.95 (d, J = 7.3 Hz, 2H), 8.17 (s, 1H), 12.29 (br s, 1H); MS
(ESI) m/z 284.1 ([M+H]+).
7.1.18. 2-[3-(Phenylsulfonyl)-1H-indol-7-yl]ethanol
Prepared in essentially the same manner replacing 3-(phenyl-
sulfonyl)-4-vinyl-1H-indole 24a with 3-(phenylsulfonyl)-7-vinyl-
1H-indole 24d. Yield 66% of
a
pink semi-solid. 1H NMR
7.1.13. 3-(Phenylsulfonyl)-5-vinyl-1H-indole (24c)
(500 MHz, DMSO-d6) d ppm 2.98 (t, J = 6.7 Hz, 2H), 3.62–3.70 (m,
2H), 4.65 (br s, 1H), 7.06 (d, J = 7.5 Hz, 1H), 7.11 (dd, J = 7.5,
7.5 Hz, 1H), 7.50–7.64 (m, 4H), 7.96 (dd, J = 8.1, 1.4 Hz, 2H), 8.13
(s, 1H), 12.24 (br s, 1H); MS (ESI) m/z 302.1 ([M+H]+).
Prepared in essentially the same manner as 24a replacing 4-
bromo-3-(phenylsulfonyl)-1H-indole 20a with 5-bromo-3-(phen-
ylsulfonyl)-1H-indole 20c. Yield 74% of a white solid. 1H NMR
(500 MHz, DMSO-d6) d ppm 5.20 (d, J = 11.0 Hz, 1H), 5.75 (d,
J = 17.4 Hz, 1H), 6.84 (dd, J = 17.4, 11.0 Hz, 1H), 7.41–7.49 (m,
2H), 7.51–7.64 (m, 3H), 7.76 (s, 1H), 7.98 (d, J = 6.7 Hz, 2H), 8.16
(s, 1H), 12.28 (br s, 1H); MS (ESI) m/z 284.0 ([M+H]+).
7.1.19. 2-[3-(Phenylsulfonyl)-1H-indol-4-yl]ethyl 4-methyl-
benzenesulfonate (25a)
To a solution of 2-[3-(phenylsulfonyl)-1H-indol-4-yl]ethanol
(4.0 g, 13.27 mmol) in acetonitrile (100 mL) under nitrogen was
added pyridine (2.68 mL, 33.18 mmol) followed by para-toluene-
sulfonyl chloride (3.04 g, 15.93 mmol) and the reaction mixture
stirred at room temperature for 2 days. The mixture was then con-
centrated under reduced pressure to a small volume and parti-
tioned between ethyl acetate (200 mL) and 2.0 M aq HCl
(200 mL). The organic phase was separated, washed with 2.0 M
aq HCl (200 mL), water (200 mL), and 1/2 saturated brine
(200 mL), dried over MgSO4 and concentrated to afford a yellow
syrup. The crude product was purified by silica gel chromatogra-
phy eluting with a hexanes–ethyl acetate gradient (60/40 to 40/
60) to give 2-[3-(phenylsulfonyl)-1H-indol-4-yl]ethyl 4-methyl-
benzenesulfonate 25a (5.50 g, 91%) as a white foam. 1H NMR
(500 MHz, DMSO-d6) d ppm 2.36 (s, 3H), 3.17 (t, J = 6.3 Hz, 2H),
3.87 (t, J = 6.3 Hz, 2H), 6.86 (d, J = 7.3 Hz, 1H), 7.15 (dd, J = 7.3,
8.2 Hz, 1H), 7.29 (d, J = 8.2 Hz, 2H), 7.41 (d, J = 8.2 Hz, 1H), 7.43
(d, J = 8.2 Hz, 2H), 7.53 (dd, J = 7.6, 7.3 Hz, 2H), 7.58–7.63 (m, 1H),
7.1.14. 3-(Phenylsulfonyl)-7-vinyl-1H-indole (24d)
Prepared in essentially the same manner as 24a replacing 4-
bromo-3-(phenylsulfonyl)-1H-indole 20a with 7-bromo-3-(phen-
ylsulfonyl)-1H-indole 20d. Yield 78% of a yellow oil. 1H NMR
(500 MHz, DMSO-d6) d ppm 5.42 (d, J = 11.0 Hz, 1H), 5.94 (d,
J = 17.4 Hz, 1H), 7.16–7.25 (m, 2H), 7.48 (d, J = 7.3 Hz, 1H),
7.52–7.62 (m, 3H), 7.70 (d, J = 7.9 Hz, 1H), 7.97 (d, J = 6.7 Hz,
2H), 8.21 (d, J = 3.4 Hz, 1H), 12.46 (br s, 1H); MS (ESI) m/z
284.0 ([M+H]+).
7.1.15. 2-[3-(Phenylsulfonyl)-1H-indol-4-yl]ethanol
To
a solution of 3-(phenylsulfonyl)-4-vinyl-1H-indole 24a
(7.05 g, 24.88 mmol) in tetrahydrofuran (140 mL) in a room tem-
perature water bath under nitrogen was added slowly a solution
of borane–tetrahydrofuran complex (1.0 M in tetrahydrofuran,

7810
G. D. Heffernan et al. / Bioorg. Med. Chem. 17 (2009) 7802–7815
7.74 (d, J = 7.3 Hz, 2H), 8.19 (s, 1H), 12.39 (br s, 1H); MS (ESI) m/z
7.2.2. N-Ethyl-N-methyl-N-{2-[3-(phenylsulfonyl)-1H-indol-4-
yl]ethyl}amine hydrochloride (18b)
455.9 ([M+H]+).
Yield: 97% of a tan solid; 1H NMR (400 MHz, DMSO-d6) d ppm
1.24 (t, J = 7.3 Hz, 3H), 2.75 (br s, 3H), 3.01–3.30 (br m, 6H), 7.08
(d, J = 7.3 Hz, 1H), 7.25 (dd, J = 8.1, 7.7 Hz, 1H), 7.47 (d, J = 8.1 Hz,
1H), 7.59 (m, 2H), 7.65 (m, 1H), 7.87 (m, 2H), 8.20 (d, J = 3.4 Hz,
1H), 10.09 (br s, 1H) 12.55 (br s, 1H); HRMS: m/z calcd for
C19H22N2O2S + H+, 343.1474. Found (ESI, [M+H]+), 343.1472.
7.1.20. 2-[3-(Phenylsulfonyl)-1H-indol-6-yl]ethyl 4-
methylbenzenesulfonate (25b)
Prepared in essentially the same manner as 25a replacing 2-[3-
(phenylsulfonyl)-1H-indol-4-yl]ethanol with 2-[3-(phenylsulfo-
nyl)-1H-indol-6-yl]ethanol. Yield 96% of a white solid. 1H NMR
(400 MHz, DMSO-d6) d ppm 2.29 (s, 3H), 2.94 (t, J = 6.3 Hz, 2H),
4.20 (t, J = 6.3 Hz, 2H), 6.98 (dd, J = 8.1, 1.3 Hz, 1H), 7.15 (d,
J = 8.2 Hz, 2H), 7.20 (s, 1H), 7.50 (d, J = 8.2 Hz, 2H), 7.52–7.61 (m,
3H), 7.64 (d, J = 8.1 Hz, 1H), 7.97 (dd, J = 7.8, 1.7 Hz, 2H), 8.13 (d,
J = 3.2 Hz, 1H), 12.14 (br s, 1H); MS (ESI) m/z 454.1 ([MÀH]À).
7.2.3. N-Methyl-N-{2-[3-(phenylsulfonyl)-1H-indol-4-
yl]ethyl}amine hydrochloride (18c)
Yield: 94% of a light brown solid; 1H NMR (500 MHz, DMSO-d6)
d ppm 2.14 (s, 3H), 2.27 (t, J = 7.6 Hz, 2H), 2.97 (t, J = 7.6 Hz, 2H),
6.93 (d, J = 7.3 Hz, 1H), 7.15 (dd, J = 8.2, 7.3 Hz, 1H), 7.36 (d,
J = 8.2 Hz, 1H), 7.55 (m, 2H), 7.62 (m, 1H), 7.82 (d, J = 7.6 Hz, 2H),
8.23 (s, 1H), 12.31 (br s, 1H); HRMS: m/z calcd for C17H18N2O2S +
H+, 315.1161. Found (ESI, [M+H]+), 315.1161.
7.1.21. 2-[3-(Phenylsulfonyl)-1H-indol-5-yl]ethyl 4-
methylbenzenesulfonate (25c)
Prepared in essentially the same manner as 25a replacing 2-[3-
(phenylsulfonyl)-1H-indol-4-yl]ethanol with 2-[3-(phenylsulfo-
nyl)-1H-indol-5-yl]ethanol. Yield 95% of a white solid. 1H NMR
(400 MHz, DMSO-d6) d ppm 2.33 (s, 3H), 2.96 (t, J = 6.3 Hz, 2H),
4.21 (t, J = 6.3 Hz, 2H), 6.99 (dd, J = 8.3, 1.6 Hz, 1H), 7.23 (d,
J = 7.8 Hz, 2H), 7.32 (d, J = 8.3 Hz, 1H), 7.46–7.64 (m, 6H), 7.97
(dd, J = 8.3, 1.5 Hz, 2H), 8.13 (d, J = 3.2 Hz, 1H), 12.19 (br s, 1H);
MS (ESI) m/z 454.1 ([MÀH]À).
7.2.4. N-Ethyl-N-{2-[3-(phenylsulfonyl)-1H-indol-4-
yl]ethyl}amine hydrochloride (18d)
Yield: 95% of a light brown solid; 1H NMR (400 MHz, DMSO-d6)
d ppm 1.19 (t, J = 7.2 Hz, 3H), 2.89 (br s, 2H), 2.99 (br s, 2H), 3.25
(m, 2H), 7.04 (d, J = 7.2 Hz, 1H), 7.23 (dd, J = 7.6, 7.2 Hz, 1H), 7.46
(d, J = 7.6 Hz, 1H), 7.58 (m, 2H), 7.64 (m, 1H), 7.91 (d, J = 7.0 Hz,
2H), 8.22 (d, J = 3.2 Hz, 1H), 8.70 (br s, 2H), 12.52 (br s, 1H); HRMS:
m/z calcd for C18H20N2O2S + H+, 329.1317. Found (ESI, [M+H]+),
329.1316.
7.1.22. 2-[3-(Phenylsulfonyl)-1H-indol-7-yl]ethyl 4-
methylbenzenesulfonate (25d)
Prepared in essentially the same manner as 25a replacing 2-[3-
(phenylsulfonyl)-1H-indol-4-yl]ethanol with 2-[3-(phenylsulfo-
nyl)-1H-indol-7-yl]ethanol. Yield 86% of a white solid. 1H NMR
(500 MHz, DMSO-d6) d ppm 2.33 (s, 3H), 3.16 (t, J = 6.4 Hz, 2H),
4.24 (t, J = 6.4 Hz, 2H), 6.97 (d, J = 7.3 Hz, 1H), 7.07 (dd, J = 7.9,
7.3 Hz, 1H), 7.17 (d, J = 8.0 Hz, 2H), 7.39 (d, J = 8.0 Hz, 2H), 7.52–
7.61 (m, 3H), 7.64 (d, J = 7.9 Hz, 1H), 7.97 (dd, J = 8.1, 1.4 Hz, 2H),
8.15 (d, J = 3.4 Hz, 1H), 12.25 (br s, 1H); MS (ESI) m/z 456.2
([M+H]+).
7.2.5. N-{2-[3-(Phenylsulfonyl)-1H-indol-4-yl]ethyl}-N-
propylamine hydrochloride (18e)
Yield: 96% of a white solid; 1H NMR (500 MHz, DMSO-d6) d ppm
0.92 (t, J = 7.3 Hz, 3H), 1.62 (m, 2H), 2.80 (br m, 2H), 3.01 (br m,
2H), 3.24 (m, 2H), 7.03 (d, J = 7.3 Hz, 1H), 7.23 (dd, J = 8.2, 7.3 Hz,
1H), 7.46 (d, J = 8.2 Hz, 1H), 7.58 (m, 2H), 7.64 (m, 1H), 7.90 (d,
J = 7.6 Hz, 2H), 8.22 (d, J = 2.7 Hz, 1H), 8.59 (br s, 2H), 12.50 (br s,
1H); HRMS: m/z calcd for C19H22N2O2S + H+, 343.1474. Found
(ESI, [M+H]+), 343.1473.
7.2. General Procedure 1: {2-[3-(phenylsulfonyl)-1H-indol-4-
yl]ethyl}amine hydrochlorides (18a–l), {2-[3-(phenylsulfonyl)-
1H-indol-6-yl]ethyl}amine hydrochlorides (26a–b), {2-[3-
(phenylsulfonyl)-1H-indol-5-yl]ethyl}amine hydrochlorides
(27a–b) and {2-[3-(phenylsulfonyl)-1H-indol-7-yl]ethyl}amine
hydrochlorides (28a–b)
7.2.6. N-{2-[3-(Phenylsulfonyl)-1H-indol-4-yl]ethyl}propan-2-
amine hydrochloride (18f)
Yield: 95% of a white solid; 1H NMR (400 MHz, DMSO-d6) d ppm
1.21 (d, J = 6.5 Hz, 6H), 3.00 (br m, 2H), 3.22 (br m, 1H), 3.27 (br m,
2H), 7.06 (d, J = 7.0 Hz, 1H), 7.23 (dd, J = 7.5, 7.0 Hz, 1H), 7.46 (d,
J = 7.5 Hz, 1H), 7.58 (m, 2H), 7.64 (m, 1H), 7.93 (m, 2H), 8.22 (s,
1H), 8.65 (br s, 2H), 12.52 (br s, 1H); HRMS: m/z calcd for
C19H22N2O2S + H+, 343.1474. Found (ESI, [M+H]+), 343.1474.
To a solution of the appropriate 4-methyl-benzenesulfonate
25a–25d (116 mg, 0.25 mmol) in THF (2 mL) was added the appro-
priate amine (1.25 mmol) and mixture heated to 71 °C for 24 h. The
mixture was then partitioned between ethyl acetate (20 mL) and
water (20 mL), and the aqueous layer was extracted with ethyl ace-
tate (10 mL). The combined organic layers were washed with 2.0 M
aq NaOH, (2 Â 15 mL), and saturated brine (30 mL), dried over
MgSO4, and concentrated. The crude product was purified by silica
gel chromatography eluting with 2.0 M ammonia in ethanol solu-
tion–dichloromethane (10/90). The product was dissolved in
diethyl ether and treated with 1 N HCl in diethyl ether (1.05 equiv)
and then filtered to afford the amine hydrochloride.
7.2.7. N-{2-[3-(Phenylsulfonyl)-1H-indol-4-
yl]ethyl}cyclopentanamine hydrochloride (18g)
Yield: 99% of a white solid; 1H NMR (400 MHz, DMSO-d6) d ppm
1.46–1.77 (m, 6H), 1.93 (m, 2H), 3.00 (br m, 2H), 3.29 (br m, 2H),
3.37 (br m, 1H), 7.06 (d, J = 7.3 Hz, 1H), 7.23 (dd, J = 7.5, 7.3 Hz,
1H), 7.46 (d, J = 7.5 Hz, 1H), 7.58 (m, 2H), 7.64 (m, 1H), 7.93 (m,
2H), 8.21 (d, J = 2.3 Hz, 1H), 8.87 (br s, 2H), 12.53 (br s, 1H); HRMS:
m/z calcd for C21H24N2O2S + H+, 369.1630. Found (ESI, [M+H]+),
369.1630.
7.2.1. N,N-Dimethyl-N-{2-[3-(phenylsulfonyl)-1H-indol-4-
yl]ethyl}amine hydrochloride (18a)
7.2.8. 3-(Phenylsulfonyl)-4-(2-pyrrolidin-1-ylethyl)-1H-indole
hydrochloride (18h)
Yield: 85% of a tan solid; 1H NMR (500 MHz, DMSO-d6) d ppm
2.63 (br s, 6H), 3.01 (br s, 2H), 3.17 (br m, 2H), 7.03 (d, J = 7.0 Hz,
1H), 7.23 (dd, J = 8.2, 7.0 Hz, 1H), 7.44 (d, J = 8.2 Hz, 1H), 7.58 (m,
2H), 7.64 (m, 1H), 7.84 (d, J = 7.3 Hz, 2H), 8.21 (d, J = 2.7 Hz, 1H),
9.81 (br s, 1H), 12.49 (br s, 1H); HRMS: m/z calcd for C18H20N2O2S
+ H+, 329.1317. Found (ESI, [M+H]+), 329.1316.
Yield: 98% of a white solid; 1H NMR (400 MHz, DMSO-d6) d ppm
1.84–2.07 (m, 4H), 3.02 (br s, 2H), 3.27 (br s, 4H), 3.50 (br s, 2H),
7.05 (d, J = 7.2 Hz, 1H), 7.24 (dd, J = 8.1, 7.2 Hz, 1H), 7.47 (d,
J = 8.1 Hz, 1H), 7.56–7.68 (m, 3H), 7.88 (d, J = 7.2 Hz, 2H), 8.21 (d,
J = 3.2 Hz, 1H), 10.39 (br s, 1H), 12.53 (br s, 1H); HRMS: m/z calcd
for C20H22N2O2S + H+, 355.1474. Found (ESI, [M+H]+), 355.1473.

G. D. Heffernan et al. / Bioorg. Med. Chem. 17 (2009) 7802–7815
7811
7.2.9. 3-(Phenylsulfonyl)-4-(2-piperidin-1-ylethyl)-1H-indole
hydrochloride (18i)
1H); HRMS: m/z calcd for C18H20N2O2S + H+, 329.1317. Found
(ESI, [M+H]+), 329.1318.
Yield: 95% of a white solid; 1H NMR (400 MHz, DMSO-d6) d ppm
1.40 (m, 1H), 1.65–1.89 (m, 5H), 2.85 (m, 2H), 3.18 (m, 2H), 3.29 (br
m, 2H), 3.39 (br m, 2H), 7.06 (d, J = 7.0 Hz, 1H), 7.25 (dd, J = 7.5,
7.0 Hz, 1H), 7.47 (d, J = 7.5 Hz, 1H), 7.56–7.69 (m, 3H), 7.86 (d,
J = 7.0 Hz, 2H), 8.19 (d, J = 3.1 Hz, 1H), 10.12 (br s, 1H), 12.54 (br
s, 1H); HRMS: m/z calcd for C21H24N2O2S + H+, 369.1630. Found
(ESI, [M+H]+), 369.1629.
7.2.16. N-Ethyl-N-methyl-N-{2-[3-(phenylsulfonyl)-1H-indol-5-
yl]ethyl}amine hydrochloride (27b)
Yield: 91% of a white solid; 1H NMR (500 MHz, DMSO-d6) d ppm
1.23 (t, 3H), 2.79 (s, 3H), 3.01–3.16 (br m, 3H), 3.17–3.28 (br m,
3H), 7.18 (dd, J = 8.3, 1.5 Hz, 1H), 7.47 (d, J = 8.3 Hz, 1H), 7.52–
7.63 (m, 3H), 7.72 (s, 1H), 7.98 (d, J = 7.0 Hz, 2H), 8.17 (d,
J = 3.4 Hz, 1H), 9.79 (br s, 1H), 12.31 (br s, 1H); HRMS: m/z calcd
for C19H22N2O2S + H+, 343.1474. Found (ESI, [M+H]+), 343.1472.
7.2.10. 4-(2-Azepan-1-ylethyl)-3-(phenylsulfonyl)-1H-indole
hydrochloride (18j)
7.2.17. N,N-Dimethyl-N-{2-[3-(phenylsulfonyl)-1H-indol-7-
yl]ethyl}amine hydrochloride (28a)
Yield: 62% of a white solid; 1H NMR (400 MHz, DMSO-d6) d ppm
1.52–1.73 (m, 4H), 1.77–1.90 (m, 4H), 3.06–3.19 (m, 2H), 3.20–3.48
(m, 6H), 7.09 (d, J = 7.5 Hz, 1H), 7.25 (dd, J = 8.1, 7.5 Hz, 1H), 7.47
(d, J = 8.1 Hz, 1H), 7.57–7.70 (m, 3H), 7.85 (d, J = 7.5 Hz, 2H), 8.19
(d, J = 3.1 Hz, 1H), 10.05 (br s, 1H), 12.53 (br s, 1H); HRMS: m/z
Yield: 99% of a yellow solid; 1H NMR (500 MHz, DMSO-d6) d
ppm 2.80 (s, 6H), 3.27 (s, 4H), 7.11–7.20 (m, 2H), 7.52–7.62 (m,
3H), 7.68 (dd, J = 7.8, 1.1 Hz, 1H), 7.97 (d, J = 6.7 Hz, 2H), 8.26 (s,
1H), 10.16 (br s, 1H), 12.64 (br s, 1H); HRMS: m/z calcd for
C18H20N2O2S + H+, 329.1317. Found (ESI, [M+H]+), 329.1316.
calcd for C22H26N2O2S
383.1787.
+
H+, 383.1787. Found (ESI, [M+H]+),
7.2.18. N-Ethyl-N-methyl-N-{2-[3-(phenylsulfonyl)-1H-indol-7-
yl]ethyl}amine hydrochloride (28b)
7.2.11. 4-[2-(2-Methylpiperidin-1-yl)ethyl]-3-(phenylsulfonyl)-
1H-indole hydrochloride (18k)
Yield: 90% of a white solid; 1H NMR (500 MHz, DMSO-d6) d ppm
1.22 (t, 3H), 2.81 (s, 3H), 3.09 (m, 2H), 3.19–3.29 (m, 4H), 7.14–7.21
(m, 2H), 7.52–7.63 (m, 3H), 7.69 (dd, J = 7.2, 1.4 Hz, 1H), 7.97 (d,
J = 6.7 Hz, 2H), 8.29 (d, J = 3.0 Hz, 1H), 9.81 (br s, 1H), 12.55 (br s,
1H); HRMS: m/z calcd for C19H22N2O2S + H+, 343.1474. Found
(ESI, [M+H]+), 343.1475.
Yield: 69% of a white solid; 1H NMR (400 MHz, DMSO-d6) d ppm
1.18 (d, J = 6.7 Hz, 1H), 1.28 (d, J = 6.3 Hz, 2H), 1.38–1.91 (m, 6H),
2.78–2.92 (m, 1H), 3.03–3.57 (m, 6H), 7.05 (d, J = 7.0 Hz, 0.66 H),
7.08 (d, J = 7.0 Hz, 0.34 H), 7.22 (m, 1H), 7.44 (m, 1H), 7.52–7.66
(m, 3H), 7.82 (m, 2H), 8.14 (m, 1H), 10.07 (2 Â br s, 1H), 12.50
(br s, 1H); HRMS: m/z calcd for C22H26N2O2S + H+, 383.1787. Found
(ESI, [M+H]+), 383.1787.
7.2.19. 4-(2-Azidoethyl)-3-(phenylsulfonyl)-1H-indole
7.2.12. 4-[2-(3-Methylpiperidin-1-yl)ethyl]-3-(phenylsulfonyl)-
1H-indole hydrochloride (18l)
To a solution of 2-[3-(phenylsulfonyl)-1H-indol-4-yl]ethyl 4-
methyl-benzenesulfonate 25a (171 mg, 0.30 mmol) in anhydrous
DMF (2.5 mL) was added sodium azide (0.14 g, 2.2 mmol) and
the mixture heated to 100 °C for 6 h. The cooled reaction mixture
was then partitioned between ethyl acetate (20 mL) and water
(20 mL), and the aqueous layer extracted with ethyl acetate
(10 mL). The combined organic layers were washed with brine
(15 mL), dried over MgSO4, and concentrated to afford the crude
product. Purification by silica gel chromatography eluting with
hexanes–ethyl acetate (60/40) gave 4-(2-azidoethyl)-3-(phenylsul-
fonyl)-1H-indole (120 mg, 93%) as clear waxy solid. MS (ESI) m/z
327.1 ([M+H]+).
Yield: 75% of a white solid; 1H NMR (400 MHz, DMSO-d6) d
ppm 0.92 (d, J = 6.5 Hz, 3H), 1.68–2.02 (m, 4H), 2.75 (m, 1H),
3.08–3.22 (m, 2H), 3.24–3.33 (m, 3H), 3.36–3.48 (m, 3H), 7.06
(d, J = 7.3 Hz, 1H), 7.26 (dd, J = 7.8, 7.3 Hz, 1H), 7.47 (d,
J = 7.8 Hz, 1H), 7.60 (m, 2H), 7.66 (m, 1H), 7.85 (d, J = 7.0 Hz,
2H), 8.20 (d, J = 3.4 Hz, 1H), 10.13 (br s, 1H), 12.54 (br s, 1H);
HRMS: m/z calcd for C22H26N2O2S + H+, 383.1787. Found (ESI,
[M+H]+), 383.1786.
7.2.13. N,N-Dimethyl-N-{2-[3-(phenylsulfonyl)-1H-indol-6-
yl]ethyl}amine hydrochloride (26a)
Yield: 80% of a yellow solid; 1H NMR (400 MHz, DMSO-d6) d
ppm 2.78 (s, 6H), 3.05 (m, 2H), 3.26 (m, 2H), 7.12 (dd, J = 8.3,
1.5 Hz, 1H), 7.38 (s, 1H), 7.51–7.62 (m, 3H), 7.73 (d, J = 8.3 Hz,
1H), 7.96 (dd, J = 8.1, 1.5 Hz, 2H), 8.17 (d, J = 2.9 Hz, 1H), 9.84 (br
s, 1H), 12.33 (br s, 1H); HRMS: m/z calcd for C18H20N2O2S + H+,
329.1317. Found (ESI, [M+H]+), 329.1317.
7.2.20. {2-[3-(Phenylsulfonyl)-1H-indol-4-yl]ethyl}amine
hydrochloride (18m)
To a suspension of 10% palladium on carbon (30 mg) in ethanol
(10 mL) was added a solution of 4-(2-azidoethyl)-3-(phenylsulfo-
nyl)-1H-indole (100 mg, 0.30 mmol) in ethanol (20 mL) and the
mixture hydrogenation at 40 psi for 1 h. The reaction mixture was
then filtered through CeliteÒ and concentrated in vacuo to give a
white solid (0.1 g, 86%). This solid was dissolved in diethyl ether,
treated with 1 N HCl in diethyl ether (0.16 mL, 0.16 mmol) and then
concentrated to afford {2-[3-(phenylsulfonyl)-1H-indol-4-yl]ethy-
l}amine hydrochloride 18m as a light brown foam. 1H NMR
(500 MHz, DMSO-d6) d ppm 2.88 (br s, 2H), 3.17 (m, 2H), 7.00 (d,
J = 7.3 Hz, 1H), 7.21 (dd, J = 8.2, 7.3 Hz, 1H), 7.45 (d, J = 8.2 Hz, 1H),
7.57 (m, 2H), 7.63 (m, 1H), 7.90 (br s, 3H), 7.93 (d, J = 7.3 Hz, 2H),
8.24 (d, J = 3.0 Hz, 1H), 12.52 (br s, 1H); HRMS: m/z calcd for
C16H16N2O2S + H+, 301.1004. Found (ESI, [M+H]+), 301.1006.
7.2.14. N-Ethyl-N-methyl-2-[3-(phenylsulfonyl)-1H-indol-6-
yl]ethanamine hydrochloride (26b)
Yield: 90% of a white solid; 1H NMR (400 MHz, DMSO-d6) d ppm
1.21 (t, J = 7.2 Hz, 3H) 2.75 (br s, 3H) 2.99–3.12 (m, 3H) 3.15–3.30
(m, 3H) 7.13 (d, J = 8.1 Hz, 1H) 7.40 (s, 1H) 7.49–7.63 (m, 3H) 7.72
(d, J = 8.1 Hz, 1H) 7.96 (d, J = 6.7 Hz, 2H) 8.17 (d, J = 3.0 Hz, 1H)
10.03 (br s, 1H) 12.35 (br s, 1H); HRMS: calcd for C19H22N2O2S +
H+, 343.1475; found (ESI, [M+H]+), 343.1477.
7.2.15. N,N-Dimethyl-N-{2-[3-(phenylsulfonyl)-1H-indol-5-
yl]ethyl}amine hydrochloride (27a)
Yield: 77% of a white solid; 1H NMR (500 MHz, DMSO-d6) d ppm
2.82 (s, 6H), 3.06 (m, 2H), 3.28 (m, 2H), 7.16 (dd, J = 8.5, 1.5 Hz, 1H),
7.47 (d, J = 8.5 Hz, 1H), 7.52–7.63 (m, 3H), 7.71 (s, 1H), 7.98 (d,
J = 7.0 Hz, 2H), 8.17 (d, J = 3.0 Hz, 1H), 9.75 (br s, 1H), 12.30 (br s,
7.2.21. 3-(1H-Indol-4-yl)propan-1-ol (31)
To a suspension of 10% palladium on carbon (200 mg) in abso-
lute ethanol (30 mL) was added a solution of (2E)-3-(1H-indol-4-

7812
G. D. Heffernan et al. / Bioorg. Med. Chem. 17 (2009) 7802–7815
yl)prop-2-en-1-ol 30 (prepared according to the procedure of
Kardos, N.; Genet, J.-P. Tetrahedron: Asymmetry 1994, 5, 1525)
(0.98 g, 5.66 mmol) in ethyl acetate (30 mL) and the mixture
hydrogenated at 50 psi for 15 min. The reaction mixture was fil-
tered through celite and concentrated under reduced pressure to
afford a light purple syrup. The crude product was purified by silica
gel chromatography eluting with a hexanes–ethyl acetate gradient
(80/20 to 50/50) to give 3-(1H-indol-4-yl)propan-1-ol 31 (0.92 g,
93%) as a colorless syrup. 1H NMR (400 MHz, DMSO-d6) d ppm
1.72–1.87 (m, 2H), 2.82 (t, J = 7.8 Hz, 2H), 3.44 (m, 2H), 4.43 (t,
J = 5.2 Hz, 1H), 6.44 (m, 1H), 6.77 (d, J = 7.3 Hz, 1H), 6.96 (dd,
J = 8.1, 7.3 Hz, 1H), 7.20 (d, J = 8.1 Hz, 1H), 7.27 (m, 1H), 11.00 (br
s, 1H); MS (ESI) m/z 176.2 [(M+H)+].
acetate gradient (100/0 to 60/40) afforded 3-phenylsulfonyl-4-(3-
chloro-propyl)-1H-indole (0.51 g, 48%) as a colorless foam. MS
(ESI) m/z 334.3 [(M+H)+]. Further elution afforded 3-[3-(phenylsul-
fonyl)-1H-indol-4-yl]propyl 4-methylbenzenesulfonate (0.48 g,
32%) as a white foam. 1H NMR (400 MHz, DMSO-d6) d ppm 1.58
(m, 2H), 2.42 (s, 3H), 2.77 (m, 2H), 3.90 (t, J = 6.3 Hz, 2H), 6.78 (d,
J = 7.2 Hz, 1H), 7.11 (dd, J = 7.4, 7.2 Hz, 1H), 7.36 (d, J = 7.4 Hz,
1H), 7.44–7.54 (m, 4H), 7.59 (m, 1H), 7.70–7.81 (m, 4H), 8.22 (s,
1H), 12.36 (br s, 1H); MS (ESI) m/z 469.7 [(M+H)+].
7.3. General procedure 2: 3-[3-(phenylsulfonyl)-1H-indol-4-
yl]propan-1-amine hydrochlorides (33a–c)
To a solution of 3-[3-(phenylsulfonyl)-1H-indol-4-yl]propyl 4-
methylbenzenesulfonate (0.21 g, 0.447 mmol) in tetrahydrofuran
(5 mL) was added the appropriate amine (8.944 mmol) and the
reaction mixture heated to 65 °C in a sealed vessel for 16 h. The
cooled reaction mixture was then diluted with 1.0 M aq NaOH
(50 mL) and the resulting milky suspension extracted with ethyl
acetate (50 mL). The organic phase was separated, washed with
water (50 mL) and brine (50 mL), dried over Na2SO4, and concen-
trated. The product was dissolved in absolute ethanol, 1.25 M
HCl in ethanol (1.3 equiv) added and the resulting suspension fil-
tered to afford the amine hydrochloride.
7.2.22. 3-[3-(Phenylthio)-1H-indol-4-yl]propan-1-ol
To
a solution of 3-(1H-indol-4-yl)propan-1-ol 31 (0.91 g,
5.19 mmol) and thiophenol (0.53 mL, 5.19 mmol) in absolute etha-
nol (30 mL) was added a solution of potassium iodide (0.862 g,
5.19 mmol) and iodine (1.318 g, 5.19 mmol) in ethanol (7.5 mL)
and water (22.5 mL) over 5 min. The reaction was then heated at
65 °C for 5 12 h. The cooled reaction mixture was diluted with ethyl
acetate (250 mL), washed with 5% aq Na2S2O3 solution (250 mL),
water (250 mL) and brine (250 mL), dried over Na2SO4, and con-
centrated to afford an off-white solid. The crude product was puri-
fied by silica gel chromatography eluting with a hexanes–ethyl
acetate gradient (75/25 to 25/75) to afford a white solid. The prod-
uct was recrystallized from 3:1 v/v hexanes:ethyl acetate (60 mL)
to afford 3-[3-(phenylthio)-1H-indol-4-yl]propan-1-ol (1.048 g,
71%) as a white crystalline solid. 1H NMR (400 MHz, DMSO-d6) d
ppm 1.63 (m, 2H), 2.90 (m, 2H), 3.29 (m, 2H), 4.30 (t, J = 5.1 Hz,
1H), 6.80 (d, J = 7.3 Hz, 1H), 6.95 (d, J = 7.3 Hz, 2H), 7.00–7.10 (m,
2H), 7.19 (m, 2H), 7.30 (d, J = 7.3 Hz, 1H), 7.67 (d, J = 2.9 Hz, 1H),
11.67 (br s, 1H); MS (ESI) m/z 284.1 [(M+H)+].
7.3.1. N-Ethyl-N-methyl-3-[3-(phenylsulfonyl)-1H-indol-4-
yl]propan-1-amine hydrochloride (33a)
Yield: 69% as white needles; 1H NMR (400 MHz, DMSO-d6) d
ppm 1.18 (t, 3H), 1.80 (m, 2H), 2.64 (s, 3H), 2.82 (m, 2H), 2.89 –
3.08 (br m, 4H), 6.99 (d, J = 6.7 Hz, 1H), 7.20 (dd, J = 7.2, 6.7 Hz,
1H), 7.41 (d, J = 7.2 Hz, 1H), 7.54–7.68 (m, 3H), 7.82 (m, 2H), 8.21
(d, J = 3.3 Hz, 1H), 9.88 (br s, 1H), 12.46 (br s, 1H); HRMS: m/z calcd
for C20H24N2O2S + H+, 357.1630. Found (ESI, [M+H]+), 357.1628.
7.2.23. 3-[3-(Phenylsulfonyl)-1H-indol-4-yl]propan-1-ol (32)
To a solution of 3-[3-(phenylthio)-1H-indol-4-yl]propan-1-ol
(1.024 g, 3.61 mmol) in acetone (45 mL) was added a solution of
sodium hydrogen carbonate (0.759 g, 9.03 mmol) in water
(45 mL) followed by OXONEÒ (supplied by DuPont, potassium per-
oxymonosulfate as active ingredient) (5.55 g, 9.03 mol) and the
reaction mixture stirred at room temperature for 26 h. The acetone
was removed under reduced pressure and the resulting suspension
partitioned between ethyl acetate (120 mL) and water (100 mL).
The organic phase was separated, washed with water (100 mL)
and brine (100 mL), dried over Na2SO4, and concentrated to afford
a white foam. Ethyl acetate (30 mL) was added to the crude prod-
uct and the mixture stirred vigorously for 2 h then filtered to afford
3-[3-(phenylsulfonyl)-1H-indol-4-yl]propan-1-ol 32 (1.027 g, 90%)
as a white solid. 1H NMR (400 MHz, DMSO-d6) d ppm 1.39 (m, 2H),
2.82 (m, 2H), 3.28 (m, 2H), 4.33 (t, J = 5.1 Hz, 1H), 6.91 (d, J = 7.3 Hz,
1H), 7.16 (dd, J = 7.5, 7.3 Hz, 1H), 7.34 (d, J = 7.5 Hz, 1H), 7.49–7.65
(m, 3H), 7.80 (m, 2H), 8.22 (s, 1H), 12.31 (br s, 1H); MS (ESI) m/z
314.0[(MÀH)À].
7.3.2. N-Isopropyl-3-[3-(phenylsulfonyl)-1H-indol-4-yl]propan-
1-amine hydrochloride (33b)
Yield: 57% of a white crystalline solid; 1H NMR (400 MHz,
DMSO-d6) d ppm 1.20 (d, J = 6.7 Hz, 6H), 1.78 (m, 2H), 2.78 (m,
2H), 2.87 (m, 2H), 3.22 (m, 1H), 6.98 (d, J = 7.2 Hz, 1H), 7.20 (dd,
J = 7.7, 7.2 Hz, 1H), 7.40 (d, J = 7.7 Hz, 1H), 7.53–7.67 (m, 3H),
7.82 (d, J = 6.9 Hz, 2H), 8.21 (s, 1H), 8.52 (br s, 2H), 12.44 (br s,
1H); HRMS: m/z calcd for C20H24N2O2S + H+, 357.1630. Found
(ESI, [M+H]+), 357.1629.
7.3.3. 3-(Phenylsulfonyl)-4-(3-piperidin-1-ylpropyl)-1H-indole
hydrochloride (33c)
Yield: 43% of a white solid; 1H NMR (400 MHz, DMSO-d6) d ppm
1.35 (m, 1H), 1.59–1.89 (m, 7H), 2.72–2.87 (m, 4H), 2.93 (br s, 2H),
3.33 (br s, 2H), 6.99 (d, J = 7.2 Hz, 1H), 7.20 (dd, J = 8.2, 7.2 Hz, 1H),
7.41 (d, J = 8.2 Hz, 1H), 7.54–7.69 (m, 3H), 7.82 (d, J = 7.4 Hz, 2H),
8.22 (d, J = 3.1 Hz, 1H), 9.58 (br s, 1H), 12.44 (br s, 1H); HRMS:
m/z calcd for C22H26N2O2S + H+, 383.1787. Found (ESI, [M+H]+),
383.1789.
7.2.24. 3-[3-(Phenylsulfonyl)-1H-indol-4-yl]propyl 4-
methylbenzenesulfonate
7.4. General procedure 3: whole cell radioligand binding assay
in cells expressing human norepinephrine transporter (hNET)
To a solution of 3-[3-(phenylsulfonyl)-1H-indol-4-yl]propan-1-
ol 32 (1.006 g, 3.19 mmol) in acetonitrile (25 mL) under nitrogen
was added pyridine (0.65 mL, 7.97 mmol) followed by para-tolu-
enesulfonyl chloride, and the reaction mixture stirred at room tem-
perature for 11 days. The reaction mixture was then concentrated
to a small volume, and the mixture partitioned between ethyl ace-
tate (100 mL) and 1.0 M aq HCl (100 mL). The organic phase was
separated, washed with water (2 Â 100 mL) and brine (200 mL),
dried over Na2SO4, and concentrated to afford a yellow syrup. Puri-
fication by silica gel chromatography eluting with a hexanes–ethyl
Twenty-four hours prior to assay, MDCK-Net6 cells, stably
transfected with human NET {Pacholczyk et al. Nature 1991,
6316, 350} were plated in 96-well plates at 3000–5000 cells/well
in growth medium and maintained in a cell incubator (37 °C, 5%
CO2). On day 2, growth medium was replaced with 75 lL of as-
say buffer (25 mM HEPES; 120 mM NaCl; 5 mM KCl; 2.5 mM
CaCl2; 1.2 mM MgSO4; 2 mg/mL glucose (pH 7.4, 37 °C)) contain-
ing 0.2 mg/mL ascorbic acid and 1
lM pargyline. Five microliter
aliquots of test compound in assay buffer were added directly

G. D. Heffernan et al. / Bioorg. Med. Chem. 17 (2009) 7802–7815
7813
to triplicate wells to yield final test concentrations of 10–
10,000 nM. Data from wells containing desipramine (1 M) were
used to define non-specific hNET binding (minimum hNET bind-
from the cell incubator 2 h prior to assay and maintained at room
temperature to equilibrate the growth medium to ambient oxygen
concentration. Subsequently, the growth medium was replaced
l
ing in the presence of an NRI). Total radioligand bound is defined
with 200
KCl; 2.5 mM CaCl2; 1.2 mM MgSO4; 2 mg/mL glucose (pH 7.4,
37 °C)) containing 0.2 mg/mL ascorbic acid and 1 M pargyline
and the cells were incubated for 5 min at 37 °C. A stock solution
of paroxetine was prepared in DMSO (10 mM) and delivered to
triplicate wells containing cells for a final test concentration of
lL of assay buffer (25 mM HEPES; 120 mM NaCl; 5 mM
by addition of 5
[3H]nisoxetine (Perkin–Elmer). The radioligand binding reaction
was initiated by addition of [3H]nisoxetine in 25
L of assay buf-
lL of binding buffer alone in the presence of
l
l
fer to each well for a final concentration of 3 nM. The KD value
estimated for [3H]nisoxetine was 10 nM using intact whole cells.
The cells in the assay buffer with test compound and radioligand
were incubated for 2 h at 37 °C. The cells were washed twice
1 lM. Data from these wells were used to define non-specific 5-
HT uptake (minimum 5-HT uptake in the presence of a SRI). Test
compounds were prepared in DMSO (10 mM) and diluted in assay
buffer according to test range (1–1000 nM). Twenty-five microli-
ters of assay buffer (maximum 5-HT uptake) or test compound
with 200
bound radioligand. The plates were inverted and left to dry for
10 min. Microscint20 scintillation fluid (100 L) (Perkin–Elmer
lL assay buffer at room temperature to remove un-
l
Life and Analytical Sciences, Boston, MA) was added to each well
and the plates were sealed. The plates were then placed on an
orbital shake table (Bellco) for 10 min to ensure adequate mixing.
The plates were counted in the Wallac Microbeta counter
(Perkin–Elmer).
were added directly to triplicate wells containing cells in 200 lL
of assay buffer. The cells were incubated with the compound for
10 min (37 °C). To initiate the reaction, [3H]hydroxytryptamine
creatinine sulfate (Perkin–Elmer) diluted in assay buffer was deliv-
ered in 25 lL aliquots to each well for a final test concentration of
15 nM. The cells were incubated with the reaction mixture for
7.5. General procedure 4: norepinephrine (NE) uptake assay in
cells expressing human norepinephrine transporter (hNET)
9 min at 37 °C. Decanting the supernatant from the plates termi-
nated the reaction. The cells were washed twice with 200 lL assay
buffer (37 °C) to remove free radioligand. The plates were inverted
On day 1, MDCK-Net6 cells, stably transfected with human NET,
{Pacholczyk et al. Nature 1991, 6316, 350} were plated at 3000
cells/well in a 96-well plate in growth medium and maintained
in a cell incubator (37 °C, 5% CO2). On day 2, growth medium
and left to dry for 2 min, then re-inverted and air-dried for an addi-
tional 10 min. Subsequently, the cells were lysed in 25
NaOH (4 °C) then placed on a shaker table and shaken vigorously
for 5 min. After cell lysis, 75 L of scintillation cocktail was added
lL of 0.25 N
l
was replaced with 200
120 mM NaCl; 5 mM KCl; 2.5 mM CaCl2; 1.2 mM MgSO4; 2 mg/
mL glucose [pH 7.4, 37 °C]) containing 0.2 mg/mL ascorbic acid
and 10 lM pargyline. Cells were equilibrated in assay buffer for
l
L
of assay buffer (25 mM HEPES;
to the wells, the plates were sealed with film tape and replaced on
the shake table for a minimum of 10 min. The plates were counted
in a Wallac Microbeta counter (Perkin–Elmer) to collect the raw
cpm data.
10 min at 37 °C prior to addition of compounds. A stock solution
of desipramine was prepared in DMSO (10 mM) and delivered to
triplicate wells containing cells for a final test concentration of
7.7. General procedure 6: dopamine transporter (hDAT)
membrane binding assay
1 lM. Data from these wells were used to define non-specific NE
uptake (minimum NE uptake). Test compound stock solutions
were prepared in DMSO or DMSO:water (1:1) (10 mM) and diluted
Frozen membrane samples from CHO cells expressing recombi-
nant hDAT, purchased from Perkin–Elmer, Boston, MA (Cat. No.
RBHDATM, Lot #2227), were diluted to 7.5 mL in binding buffer
(50 mM Tris–HCl; pH 7.4, 100 mM NaCl), homogenized with a tis-
sue-tearer (Polytron PT 1200C, Kinematica AG) and delivered at a
volume of 75 lL to each well of a polypropylene 96-well plate.
The binding reaction was carried out in polypropylene 96-well
plates. Homogenized membrane preparation was delivered at a
volume of 75 lL to each well of a reaction plate. A stock solution
of mazindol was prepared in DMSO (10 mM) and delivered to trip-
licate wells containing membranes for a final test concentration of
10 lM. Mazindol has been reported to be a dopamine transporter
inhibitor (DRI) {Pristupa et al. Mol. Pharmacol. 1994, 45, 125} with
in assay buffer according to test range (1–10,000 nM). A 25-
quot of vehicle or various concentrations of test compound were
added directly to triplicate wells containing cells in 200 L of assay
lL ali-
l
buffer. The cells were preincubated with test compound for 10 min
at 37 °C. To initiate transport, [3H]NE (DL-[7-3H]norepinephrine)
(Perkin–Elmer) was diluted in assay buffer (120 nM final assay
concentration) and delivered in 25
the plates were incubated for 10 min at 37 °C. The cells were
washed twice with 200 L assay buffer to remove unincorporated
[3H]NE label. The plates were inverted and left to dry for 10 min.
The cells were lysed in 25 L of 0.25 N NaOH (4 °C), placed on an
orbital shake table (Bellco) and vigorously shaken for 5 min. After
cell lysis, 75 L of scintillation cocktail was added to each well
lL aliquots to each well and
l
l
an IC50 value of 20.5 nM in our assays. Data from wells containing
l
mazindol (10
binding (minimum hDAT binding in the presence of a DRI). Total
radioligand bound is defined by addition of 5 L of binding buffer
alone in the presence of [3H]WIN-35,428. Stock solutions of test
compounds were prepared in DMSO at concentration of
lM) were used to define non-specific (NSB) hDAT
and the plates were sealed with film tape. The plates were returned
to the shake table and vigorously shaken for a minimum of 10 min
to ensure adequate partitioning of organic and aqueous solutions.
The plates were counted in a Wallac Microbeta counter (Perkin–El-
mer) to collect the raw cpm data.
l
a
10 mM. On day of assay, the test compound stock solution was di-
luted in assay buffer according to test range (3000–100,000 nM)
ensuring a maximal DMSO concentration of less than 0.5% in the
assay reaction wells. Homogenized membranes were preincubated
with test compound for 20 min at 4 °C before the initiation of the
binding reaction. The binding reaction was initiated by addition
7.6. General procedure 5: serotonin (hSERT) uptake assay in
cells expressing human serotonin transporter
On day 1, JAR cells (human placental choriocarcinoma), pur-
chased from ATCC (Cat. No. HTB-144), were plated at 15,000
cells/well in 96-well plates containing growth medium (RPMI
1640 with 10% FBS) and maintained in a cell incubator (37 °C, 5%
CO2). On day 2, cells were stimulated with staurosporine (40 nM)
to increase the expression of the 5-HT transporter {Ramamoorthy
et al. J. Biol. Chem. 1995, 270, 17189}. On day 3, cells were removed
of 25 l
L of [3H]WIN-35,428 diluted in binding buffer. The final con-
centration of [3H]WIN-35,428 delivered was 32 nM. The KD value
estimated for [3H]WIN-35,428 (Lot# 2227) in hDAT membranes
was 29.7 nM. The plates containing the radioligand binding reac-
tions were incubated for 2 h at 4 °C on a shaking table (Bellco) at

7814
G. D. Heffernan et al. / Bioorg. Med. Chem. 17 (2009) 7802–7815
3 rpm. The MultiScreen-FB opaque 96-well filtration plates con-
taining Millipore glass fiber filters were used to terminate the
binding reactions and to separate bound from free radioligand.
The plates were presoaked with 0.5% polyethylenimine (PEI) in
water for a minimum of 2 h at room temperature to reduce non-
specific binding of [3H]WIN-35,428 during the harvest procedure.
Prior to harvesting the reaction plates, the PEI solution was aspi-
rated from the filter plates using a vacuum manifold. Aliquots of
vices) is used to separate bound from free radioactivity. The drug
reaction is aspirated directly from the reaction plate onto a glass
fiber mat, and rinsed four times with 200 lL wash buffer (50 mM
Tris–HCl, pH 7.4, 4 °C). The individual filter disks are transferred
to scintillation vials. Scintillation fluid (Packard Ultima Gold) is
added to the vials, and data (dpm) are collected from the Packard
2200CA scintillation counter (Perkin–Elmer).
each reaction (90
l
L of each 100
l
L reaction well) were transferred
7.9. General procedure 8: 5-HT2A calcium mobilization FLIPR
assay
from the reaction plates to the filter plates using a Zymark Rapid
Plate-96 automated pipet station. The binding reaction was termi-
nated by vacuum filtration through the glass fiber filters. The filter
plates were aspirated at 5–10 in. Hg, and the wells are washed nine
times with 200 lL wash buffer (50 mM Tris–HCl, 0.9% NaCl, pH 7.4;
4 °C) using a 12-channel aspiration/wash system. Plastic bottom
Chinese hamster ovary cells (CHO-K1) were stably transfected
with the human 5-HT2A receptor. The CHO cells were grown in cul-
ture medium containing Dulbecco’s modified Eagle’s Medium
(Invitrogen) supplemented with 10% fetal bovine serum (Invitro-
gen), 1% penicillin–streptomycin (Invitrogen), 1% non-essential
amino acids (Invitrogen), and 1% GlutaMax (Invitrogen). The cells
supports were removed from the filter plates and the plates were
placed in plastic liners. A 100-lL aliquot of scintillation fluid was
added to each well and the top of each plate was sealed with adhe-
sive film. The plates were vigorously shaken on an orbital shake ta-
ble at 5 rpm for 10–15 min to ensure adequate equilibration of
aqueous to solvent partitioning. The collection of raw cpm data
was done using a Wallac Microbeta counter (Perkin–Elmer).
were propagated in selection media containing 800
lg/mL of
G418 (Invitrogen) and 500 g/mL of Zeocin (Invitrogen) and main-
l
tained at 37 °C in a 5% CO2 incubator at 95% humidity. Cells were
plated at a density of 0.4 Â 106 in a tissue culture flask (T75 cm2)
(Falcon) and cultured twice weekly using a final concentration of
0.05% Trypsin–EDTA (Invitrogen). For the calcium mobilization
bioassay, cells were plated at 40,000 cells per well in sterile black
7.8. General procedure 7: Human Serotonin 2A (5-HT2A
membrane binding assay
)
96-well plates (Costar) in 200 lL culture medium containing 1.5%
FBS for 24 h. The cells were utilized for the functional bioassay be-
Frozen cell pellets from CHO cells expressing the human 5-HT2A
receptor were provided by Wyeth BioPharma, Andover. Cells are
grown in suspension in a 10L bioreactor until they reach a density
of 1.5–1.9 Â 106 cells/mL. Cells are decanted, pelleted in 1 L ali-
quots, and the pellets are frozen at À80 °C. Each 1 L pellet is re-sus-
pended in 48 mL of binding buffer (50 mM Tris–HCl, 4 mM CaCl2,
pH 7.4), aliquotted into 16 Â 3 mL samples and homogenized with
a tissue-tearer (Polytron PT 1200C) for 10 strokes. The homogenate
from all 16 tubes is combined, then aliquoted into eight tubes and
centrifuged at 4 °C at low speed (600g) for 30 min. The supernatant
is collected and re-centrifuged at high speed (40,000g) for 30 min,
and the resulting pellets are stored at À80 °C. On day of use, pellets
are thawed on ice and homogenized in 2–2.5 mL binding buffer for
tween passages 5 and 25.
On day of assay, cells were plated in 96-well plates and visually
assessed for cell density and validity. The assay buffer contained
Hanks balanced salt solution without phenol red (Invitrogen) and
20 mM HEPES (Invitrogen). The Calcium3 dye (Molecular Devices)
was dissolved in 100 mL of assay buffer containing 2.5 mM pro-
benecid (Sigma) prepared fresh on the day of assay. Prior to assay,
the cell culture media was decanted and 180 lL of the dye solution
was added to cells. The cells were incubated for 60 min at 37 °C in a
5% CO2 incubator. Two 96-well drug plates were prepared for each
cell plate. Plate 1 contained test compounds and 1 lM 5-HT2A/2C
receptor agonist, (R)-(À)-DOI hydrochloride (Sigma). Plate 2 con-
tained 3 nM DOI that was chosen based on the EC80 value of the
calcium response. All compounds were diluted in assay buffer from
a protein concentration of 0.5 mg/mL (50 lg/100 lL). Protein con-
centration is determined prior to assay using a BCA Protein Assay
1 mM stock in DMSO with a final concentration of 1 lM in with a
Kit (Pierce).
final concentration of 0.1% DMSO present in the wells. The 0.1%
DMSO concentration showed no effect alone on cell viability or re-
sponse (unpublished data). Cell, compound, and DOI plates were
loaded into the Fluorometric Imaging Plate Reader (FLIPR, Molecu-
lar Devices). The laser intensity was set to a suitable level to obtain
basal values of approximately 12,000 fluorescence units. The vari-
ation in basal fluorescence units across the plate was usually less
than 10%. The baseline fluorescence reading was obtained every
second for 1 min and every second for 10 s before and after test
compound addition, respectively. Ten microliters of compound
were transferred from the compound plate to the cells at a speed
Binding reactions are run in polypropylene 96-well plates using
a total reaction volume of 200
in a final volume of 160 L to each well of a reaction plate. A stock
solution of mianserin was prepared in DMSO (10 mM) and deliv-
lL. Membrane (50 lg) is delivered
l
ered to triplicate wells containing membranes for a final test con-
centration of 10
(10 M) were used to define non-specific (NSB) 5-HT2A-receptor
binding. Total radioligand bound is defined by addition of 5 L of
lM. Data from wells containing mianserin
l
l
binding buffer alone in the presence of [3H]ketanserin. Compound
solutions at several concentrations are generated by serial dilution
of 10 mM stocks made from powder. The resulting solutions are
further diluted to delivery concentration (10Â assay concentra-
tion) in binding buffer. This procedure for compound dilution is
followed to ensure that the concentration of solvent (i.e., DMSO)
used for the stock solutions is constant across all treatments. Com-
pounds that have been diluted to appropriate delivery concentra-
of 50 lL per second to ensure rapid equilibrium of compound with-
out dislodging cells. The drug addition was made from a height
that would ensure no air bubbles in the wells. To minimize inter-
ference with fluorescence measurements, the black tips were not
removed at the end of compound addition. The fluorescence signal
recorded every 6 s for 2 min to determine if agonist activity was
tion are added in 20
membrane in binding buffer. The binding reaction is initiated by
addition of 25
L of [3H]ketanserin-HCl (diluted to 4 nM in binding
l
L aliquots to reaction wells containing
noted. For antagonist determination, 10
ferred from plate 2 to the cells and the fluorescence signal recorded
every 6 s for 5 min. The final assay volume was 200 L per well.
lL of 3 nM DOI was trans-
l
l
buffer) and incubated 1 h at room temperature. Skatron glass fiber
mats (#11731) are incubated in 0.5% polyethylenimine (PEI; Sigma
Cat. No. P-3143) in water for 15–30 min at room temperature to re-
duce non-specific binding of the radioligand prior to harvesting the
reaction plates. A Skatron Micro96 cell harvester (Molecular De-
Fluorescence measurements were captured by a cooled CCD cam-
era and integrated to an on-line computer. A negative control
was defined by assay buffer containing 0.1% DMSO. The maximum
fluorescent signal obtained by 1
lM DOI was typically 25,000 fluo-
rescent units and expressed as 100% signal to determine the per-

G. D. Heffernan et al. / Bioorg. Med. Chem. 17 (2009) 7802–7815
7815
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DOI stimulation was expressed as a percentage of the response ob-
served with 3 nM DOI alone. A compound would be considered an
active antagonist if it inhibited 3 nM DOI mediated calcium stimu-
lation by 60% or greater.
20. Kim, C. Y.; Mahaney, P. E.; Trybulski, E. J.; Zhang, P.; Terefenko, E. A.; McComas,
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Acknowledgements
The authors thank Drs. Ron Magolda and Magid Abou-Gharbia
for their support. The authors are also grateful to Scott Brecker, Re-
becca Dooley, Lisa Nogle, Christopher Petucci and Ani Sarkahian for
analytical support and to Guo Ming Zhang and Michael Olsen for
the 5-HT2B and 5-HT2C binding studies, respectively.
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column (2 Â 50 mm, 5
l
m). All compounds were run using a linear gradient
from 100% mobile phase
A
(95:5 water/CH3CN with 10 mM ammonium
14. Waldinger, M. D.; Berendsen, H. H. G.; Schweitzer, D. H. Maturitas 2000, 36, 165.
15. Deecher, D. C.; Merchenthaler, I. J. U.S. Patent Application 0180879, 2004;
Chem. Abstr. 2004, 140, 350603.
acetate) to 100% mobile phase B (95:5 CH3CN/water with 10 mM ammonium
acetate) over 4 min at a rate of 0.8 mL/min.