Discovery of novel α1-adrenoceptor ligands based on the antipsychotic sertindole suitable for labeling as PET ligands

Article abstract of DOI:10.1016/j.bmc.2012.10.049

The synthesis and in vitro preclinical profile of a series of 5-heteroaryl substituted analogs of the antipsychotic drug sertindole are presented. Compounds 1-(4-fluorophenyl)-3-(1-methylpiperidin-4-yl)-5-(pyrimidin-5-yl)-1H- indole (Lu AA27122, 3i) and 1-(4-fluorophenyl)-5-(1-methyl-1H-1,2,4-triazol-3- yl)-3-(1-methylpiperidin-4-yl)-1H-indole (3l) were identified as high affinity α_1A-adrenoceptor ligands with K_i values of 0.52 and 0.16 nM, respectively, and with a >100-fold selectivity versus dopamine D₂ receptors. Compound 3i showed almost equal affinity for α_1B- (K_i = 1.9 nM) and α_1D- adrenoceptors (K_i = 2.5 nM) as for α_1A, as well as moderate affinity for 5-HT_1B (K_i = 13 nM) and 5-HT ₆ (K_i = 16 nM) receptors, whereas 3l showed >40-fold selectivity toward all other targets tested. Based on in vitro assays for assessment of permeability rates and extent, it is predicted that both compounds enter the brain of rats, non-human primates, as well as humans, and as such are good candidates to be carried forward for further evaluation as positron emission tomography (PET) ligands.

Full text of DOI:10.1016/j.bmc.2012.10.049

Bioorganic & Medicinal Chemistry 21 (2013) 196–204
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Discovery of novel
a1-adrenoceptor ligands based on the antipsychotic
sertindole suitable for labeling as PET ligands
Morten Jørgensen a, Pernille N. Jørgensen a, Claus T. Christoffersen a, Klaus G. Jensen b, Thomas Balle c,d
,
Benny Bang-Andersen a,
⇑
a Neuroscience Drug Discovery Denmark, H. Lundbeck A/S, 9 Ottiliavej, DK-2500 Copenhagen-Valby, Denmark
b Drug ADME Research, H. Lundbeck A/S, 9 Ottiliavej, DK-2500 Copenhagen-Valby, Denmark
c Department of Drug Design and Pharmacology, The Faculty of Health and Medical Sciences, University of Copenhagen, 2 Universitetsparken, DK-2100 Copenhagen Ø, Denmark
d Faculty of Pharmacy, The University of Sydney, NSW 2006, Australia
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 26 June 2012
Revised 20 September 2012
Accepted 25 October 2012
Available online 15 November 2012
The synthesis and in vitro preclinical profile of a series of 5-heteroaryl substituted analogs of the antipsy-
chotic drug sertindole are presented. Compounds 1-(4-fluorophenyl)-3-(1-methylpiperidin-4-yl)-5-(pyr-
imidin-5-yl)-1H-indole (Lu AA27122, 3i) and 1-(4-fluorophenyl)-5-(1-methyl-1H-1,2,4-triazol-3-yl)-3-
(1-methylpiperidin-4-yl)-1H-indole (3l) were identified as high affinity
Ki values of 0.52 and 0.16 nM, respectively, and with a >100-fold selectivity versus dopamine D2 recep-
tors. Compound 3i showed almost equal affinity for a1B (Ki = 1.9 nM) and 1D-adrenoceptors
(Ki = 2.5 nM) as for 1A, as well as moderate affinity for 5-HT1B (Ki = 13 nM) and 5-HT6 (Ki = 16 nM) recep-
a1A-adrenoceptor ligands with
-
a
Keywords:
a
a1-Adrenoceptors
tors, whereas 3l showed >40-fold selectivity toward all other targets tested. Based on in vitro assays for
assessment of permeability rates and extent, it is predicted that both compounds enter the brain of rats,
non-human primates, as well as humans, and as such are good candidates to be carried forward for fur-
ther evaluation as positron emission tomography (PET) ligands.
PET
Antipsychotics
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
monkeys, but were not progressed due to low brain uptake.9 Here-
in, we report the identification of a series of novel a1-adrenoceptor
The
as three subtypes (a1A
in both the peripheral and the central nervous system (CNS).1,2 The
role of 1-adrenoceptors in the CNS is complex and not well under-
a
1-adrenoceptors are G protein-coupled receptors, existing
ligands (3a–3m, Table 1) that are predicted to enter the brain.
Moreover, the compounds are all N-methyl piperidines and
therefore suitable for 11C labeling as PET ligands.
,
a
1B, and 1D) with widespread distribution
a
a
stood,3 but a common feature of most antipsychotic drugs is their
high in vitro affinity for these receptors in addition to their in vitro
affinities for dopamine D2 and serotonin 5-HT2A receptors.4 Studies
with imaging ligands, for example, using positron emission tomo-
graphy (PET), have shown that antipsychotic drugs also bind to D2
and 5-HT2A receptors in vivo.5–7 However, no useful PET ligand or
2. Chemistry
The 1st generation a1-adrenoceptor selective sertindole analogs
were prepared via in situ conversion of the 5-bromoindole precur-
sors to the corresponding zinc species, after initial bromine-
lithium exchange, followed by a Negishi coupling.8 This synthetic
strategy could be hampered by the facile lithium migration to
the 2-position of the indole. The work reported herein was based
on the ‘inverted’ set of coupling partners for the construction of
the key bond connecting the indole nucleus at the 5-position to
the heteroaromatic substituent. The bromine in compounds 5a
and 5b would serve as a means for the Suzuki–Miyaura coupling10
to install the heteroaromatic substituent at the 5-position of the in-
dole, giving compounds 3a–3j (Scheme 1). Such a coupling reaction
is known to proceed most efficiently if the indole contains a bro-
mine atom rather than a boronic acid at the 5-position.11 Further-
more, boronic acids and esters, which would afford suitable
heteroaryl substituents, were readily available from commercial
sources.
imaging methodology is currently available for imaging central a1
-
adrenoceptors in vivo in rats or humans. As part of our continuing
interest in antipsychotic drugs, we have been seeking a PET ligand,
which would be selective for a1-adrenoceptors and therefore suit-
able for studying the occupancy of putative drugs targeting this
receptor. We previously reported our work on the discovery of
selective
a
1-adrenoceptor ligands based on sertindole (1).8 Two
of these sertindole derived analogs from the 1st generation (2a
and 2b, Fig. 1) were evaluated as PET ligands in cynomolgus
⇑
Corresponding author. Tel.: +45 3643 3247; fax: +45 3643 8237.
E-mail address: ban@lundbeck.com (B. Bang-Andersen).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.10.049

M. Jørgensen et al. / Bioorg. Med. Chem. 21 (2013) 196–204
197
NH
N
N
N
O
5% Pd(Ph3P)4
Na2CO3 (aq)
Br
HetAr
N
R = Cl
Sertindole (1)
toluene, 100°C
HetArB(OR)2
N
N
R
3a-3j
5a X=CF
5b X =N
N
X
X
N
N
N
N
N
N
R =
N
Substrate
HetArB(OR)2
Product
Yield
2a
2b
N
N
F
>95%
80%
5a
5b
3a
3b
O
B
4a
O
Figure 1. Sertindole and its 1st generation a1-adrenoceptor selective analogs.
N
N
O
75%
63%
5a
5b
3c
3d
4b
B
O
Table 1
In vitro data for compounds 3a–3m and reference compounds
16%
82%
5a
5b
3e
3f
N
B(OH)2
B(OH)2
B(OH)2
4c
4d
4e
N
N
58%
47%
5a
5b
3g
3h
N
N
29%
17%
5a
5b
3i
3j
HetAr
N
Scheme 1. Synthesis of 3a–3j using Suzuki–Miyaura coupling reactions.
R
3a-m
N
1) N-methyl-4-piperidone
Ki, nMa
D2/a1A
AcOH/TFA (2:1)
reflux
Compd
R
HetAr
2 ) PtO2 / H2, AcOH
a1A
D2
Br
Br
46% over two steps
Sertindole (1)
2a
2b
0.37
0.23
3.0
0.45
140
310
1.2
609
103
N
N
6
5a
F
F
N
N
3a
3b
0.32
19
15
47
46
F
F
N
N
N
880
N
N
N
N
3c
3d
3e
3f
2.7
38
10
24
2.3
22
150
6400
27
56
168
3
4 eq. CuI
4 eq. ZnO
4 eq. K2CO3
N
N
N
N
N
Br
Br
NMP, 110°C
I
N
F
F
N
H
N
1.1 eq.
N
N
N
260
76
11
33
50
7
5b
12%
3g
3h
N
N
N
1100
Scheme 2. Synthesis of building blocks 5a and 5b.
F
3i
3j
0.52
19
120
830
230
44
N
N
N
N
1) NaN3, Et3N.HCl
DME, reflux.
N
N
N
N
F
F
F
2) CH3I, KO(t-Bu), NMP
3) N-methyl-4-piperidone
AcOH/TFA, reflux
N
3k
3l
0.28
0.16
0.54
69
246
N
N
N
N
N
N
4) PtO2 / 3 bar H2, AcOH
220
160
1375
296
N
N
N
N
6% over four steps
3m
N
N
8
3k
a
Ki values are reported as the logarithmic average of at least two independent
determinations. In the experiments where N >2 the SD of the pKi were below 0.3.
F
F
Scheme 3. Synthesis of compound 3k.
The synthesis of the key building blocks 5a and 5b started from
known compounds 612 and 713 as shown in Scheme 2. Indole 6 was
condensed with N-methyl-4-piperidone, and the resulting tetrahy-
dropyridine was hydrogenated to the desired building block 5a. In-
dole 7 was N-arylated under classical conditions using a mixture of
CuI, ZnO, and 4-iodopyridine to give 5b in a relatively low yield.
The final step in the synthesis was the Suzuki–Miyaura coupling
of the indoles with the heteroaryl boronic acids or the correspond-
ing esters. A side-by-side comparison identified the ‘classical’
Pd(Ph3P)4 catalyst as superior to its ‘modern’ counterparts like

198
M. Jørgensen et al. / Bioorg. Med. Chem. 21 (2013) 196–204
the combination of Pd(OAc)2 or Pd2(dba)3 with either S-Phos or
P(tert-Bu)3-HBF4 under the conditions developed by Buchwald
and Fu, respectively.10,14–16 Intermediates 5a and 5b were each
coupled to five heteroaromatic boronic acids/esters (cf. Scheme 1).
In addition to compounds 3a–3j, we also prepared a number of
analogs from more advanced intermediates. More specifically, ni-
trile 812 was treated with sodium azide to yield the corresponding
tetrazole, which was subsequently methylated with methyl iodide
and condensed with N-methyl-4-piperidone, and finally reduced to
afford piperidine 3k as shown in Scheme 3.
Prior to developing the Suzuki–Miyaura protocol, two other
analogs were prepared as shown in Scheme 4. Compound 3l was
prepared according to the original Negishi-protocol8 from 5a and
3-bromo-1-methyl-1H-1,2,4-triazole17 in low yield, and previously
reported pyrazole 98 was methylated to give 3m.
attachment of the heteroaryl substituent to the indole nucleus, to
obtain high affinity for
1-adrenoceptors.18 Moreover, we have
a
previously suggested that bulk in the plane of the indole ring
would reduce affinity for D2 receptors when compared to sertin-
dole,19 and this is indeed further supported by the low D2 receptor
affinity of these compounds. The 4-fluorophenyl derivatives have
60- to 500-fold reduced affinities for D2 receptors compared to
sertindole, except for the 1-methylpyrazol-5-yl derivative 3a, for
which the decrease in D2 receptor affinity is only 30-fold and still
rather high (Ki = 15 nM). Compounds 3a and 3b are unique in the
sense that the 1-methyl group is expected to force the heteroaryl
substituent out of the plane of the indole, inducing bulk above
and below the plane instead of in the plane.8 Compounds 3i and
3l were selected for further preclinical testing in order to evaluate
their potential as PET ligands. The compounds were selected be-
cause both compounds showed high affinity for a1A-adrenoceptors
and good selectivity toward D2 receptors, as well as representing
compounds having either a 5- or a 6-membered ring at the 5-posi-
tion of the indole moiety. Compound 3i (Lu AA27122) was tested in
a broad panel of in vitro assays at MDS pharmaservices, as well as
in-house at H. Lundbeck A/S, against a total of 71 receptors, ion
channels, transporters, and enzymes. Compound 3i showed rela-
3. Structure–activity relationships
Compounds 3a–3m were evaluated in vitro for their affinity for
a
1A-adrenoceptors versus D2 receptors, as results from the evalua-
tion of the 1st generation analogs 2a and 2b indicated that the
affinity for 1A-adrenoceptors essentially were parallel to those of
1B- and
1D-adrenoceptors.8 The compounds were characterized
in receptor-binding assays at cloned bovine
a
tive high affinity (>50% inhibition at 1000 nM) for D2, 5-HT1B
,
a
a
5-HT2A, and 5-HT6 receptors as well as for the 1-adrenoceptor
a
a
1A-adrenoceptors by
displacement of [3H]prazosine and at cloned D2 receptors using
[3H]spiperone as radioligands. The results are shown in Table 1
together with the corresponding data for reference compounds
sertindole (1) and its heteroaryl derivatives 2a and 2b, which
subtypes. The 1,2,4-triazol-3-yl derivative 3l was tested in a more
limited number of assays, although in the assays relevant for direct
comparison with 3i. Ki values for 3i and 3l are reported herein on
key targets if they were below 100 nM in the broad screen. In short,
the 1,2,4-triazol-3-yl derivative 3l showed affinity below 100 nM
for 5-HT2A receptors (Ki = 89 nM), whereas the pyrimidin-5-yl
derivative 3i displayed affinities below 100 nM for 5-HT1B
(Ki = 13 nM) and 5-HT6 (Ki = 16 nM) receptors. Furthermore, com-
previously were investigated as potential
a1-adrenoceptor PET
ligands.9
The pyridyl derivatives 3b, 3d, 3f, 3h, and 3j, originally
designed to lower lipophilicity compared to the 4-fluorophenyl
derivatives, show generally one or two orders of magnitude lower
pound 3i showed almost equal affinity for all
types, whereas 3l showed selectivity for 1A-adrenoceptors as high
as 40- and 90-fold, respectively, compared to 1B and 1D, suggest-
1A-adrenoceptor selective ligand
a1-adrenoceptor sub-
a
affinity for
a1A-adrenoceptors when compared to the analogous
a
a
4-fluorophenyl counterparts as evident by comparing for example
compounds 3a and 3b (Table 1). Furthermore, the structure–
activity relationships of the 4-fluorophenyl-substituted derivatives
are parallel to the results described for the 1st generation
sertindole analogs 2a and 2b.8 All 4-fluorophenyl-substituted
derivatives, except the pyridin-4-yl derivative 3e (Table 1), display
ing that 3l may be termed an
a
(Table 2).
Compounds 3i and 3l were advanced for permeability and CNS
distribution assessment, since the brain exposure of a compound is
a function of both rate and extent of drug uptake into the CNS. The
rate of efflux due to transporters was examined by use of Caco-2
permeability studies, whereas the extend was examined by use
of equilibrium dialysis of rat brain homogenate and plasma in or-
der to estimate the steady state in vivo brain to plasma (B/P) ratio,
assuming passive distribution of compound. While the unbound
fractions in brain tissue are probably comparable across species,20
the unbound fractions in plasma can differ significantly between
species. This may potentially affect the distribution of drug to the
brain, that is higher protein binding of drug in plasma of a certain
species will result in reduced CNS exposure when compared to an-
other species where the degree of protein binding is lower (all
other things being equal). Thus, the unbound fractions of
single-digit nano- or sub-nanomolar affinity for
a1A-adrenocep-
tors. The slightly lower affinity of compound 3e agrees well with
previous observations, indicating the need for a hydrogen bond
acceptor in the ortho or meta position, relative to the point of
N
N
N
N
Br
5% Pd(PPh3)4
nBuLi then ZnCl2
THF, -78°C
N
N
N
N
N
5a
3l
N
Br
THF/DMF, 80°C, 8h
Table 2
3%
Further in vitro characterization of compounds 3i and 3l and reference compound
F
F
H
N
N
Compd
Ki, nMa
a1A
a1B
a1D
D2
N
N
N
CH3I, K2CO3
1
0.37b
0.23b
3.0c
0.52
0.16
0.33b
1.1b
6.0c
1.9
0.66b
0.45b
N
THF, rt, 6h
32%
2a
2b
3i
3l
2.0b
8.6c
2.5
140b
310b
120
N
N
9
3m
6.4
15
220
a
Ki values are reported as the logarithmic average of at least two independent
determinations.
F
F
b
Balle et al.8
c
Scheme 4. Synthesis of compounds 3l and 3m.
Balle et al.18

M. Jørgensen et al. / Bioorg. Med. Chem. 21 (2013) 196–204
199
Table 3
Summary of permeability and efflux ratios in Caco-2 cells, unbound drug fractions in plasma and brain homogenate, estimated brain/plasma (B/P) ratios at steady state and
measured rat B/P ratio at 1 h for compounds 3i and 3l and reference compounds
a
b
Papp (A–B),
Papp (B–A)/Papp
(A–B) ratio
fu plasma (%)
fu brain (%)
Estimated B/P ratio
at steady statec
Measured rat B/P ratio
at 1 hd
10ꢁ6 cm sꢁ1
Rat
Rat
Rat
Rat
Raclopride
WAY-100,635
2a
2b
3i
66
38
12
5
13
18
0.5
0.9
2.8
19
0.7
0.7
27 0.4
10 0.8
2.2 0.2
7.2 0.4
8.8 0.3
7.4 0.8
Rhesus
13 0.9
18 1.1
Cynomolgus
10 0.5
16 0.4
Human
8.0 0.4
8.4 0.4
0.35 0.02
0.81 0.07
0.25 0.01
0.20 0.01
3.4
1.2
6.3
8.9
35
37
6.4
1.7
0.26
>0.05
NT
NT
Rhesus
NT
3l
Rhesus
3i
3l
51e
87e
NT
Cynomolgus
Cynomolgus
NT
NT
Human
NT
NT
3i
3l
42e
82e
Human
3i
3l
9.6 0.6
17 0.5
38e
83e
a
b
c
Papp, apparent permeability (N = 2).
fu, Fraction unbound.
Brain to plasma (B/P) ratio calculated as in vitro fu, plasma divided by fu, brain according to Summerfield et al.20
Dose of 5 mg/kg, s.c.
d
e
fu Brain, rat brain homogenate used; NT, not tested.
compounds 3i and 3l to rhesus and cynomolgus monkeys, and hu-
man plasma as well as rat plasma and brain were determined by
dialysis (Table 3). Based on these data, the estimated B/P ratio at
steady state can be calculated as the fraction of unbound com-
pound in plasma divided by the unbound fraction in rat brain.20
As shown in Table 3, compounds 3i and 3l display an apparent
labeling as PET ligands based on their high affinity for
ceptors as well as selectivity toward other targets. Furthermore,
compound 3l displayed >40-fold selectivity for 1A-adrenoceptors
when compared to a1B and 1D, suggesting that 3l may be termed
an 1A-adrenoceptor selective ligand. Both 3i and 3l showed good
in vitro brain permeability rates and brain distribution in dialysis
studies, and they are both predicted to enter the brain of non-
human primates as well as humans. Moreover, compounds 3i
and 3l are both N-methyl piperidines and therefore suitable for
11C labeling as PET ligands.
a1-adreno-
a
a
a
permeability (Papp
)
at 20
lM
concentration of 13 ꢀ 10ꢁ6 and
18 ꢀ 10ꢁ6 cm sꢁ1, respectively, and a non-polarized transport as
indicated by a ratio between basolateral to apical and apical to
basolateral transport close to 1 for both compounds. The Papp value
was not significantly different at 1 and 10 lM concentrations (data
not shown) for any of the compounds, indicating that the transport
rate was not concentration dependent. To put the apparent perme-
abilities into perspective, the two unsuccessful CNS PET ligands 2a
and 2b have Papp values of 12 ꢀ 10ꢁ6 and 5 ꢀ 10ꢁ6 cm sꢁ1, respec-
tively, and a highly polarized transport attributed to P-glycoprotein
efflux.9 In contrast, WAY-100,635 and raclopride, both well estab-
lished CNS PET ligands, have Papp values of 38 ꢀ 10ꢁ6 and
66 ꢀ 10ꢁ6 cm sꢁ1, respectively, and non-polarized transport. In
addition, the estimated rat B/P ratio at steady state showed a good
correlation with the measured in vivo rat B/P ratio at 1 h for raclo-
pride and WAY-100,635 (Table 3). In contrast, the estimated rat B/P
ratio at steady state for compounds 2a and 2b is over-predicting the
distribution to the brain when compared to the measured in vivo B/
P ratio in rat, that is 6.3 and 8.9 versus 0.26 and >0.05, respectively.
This is supporting that the active efflux is a probable course of the
poor brain distribution of these two compounds. However, com-
pounds 3i and 3l are expected to be superior to 2a and 2b based
on the permeability data and apparent lack of efflux measured in
Caco-2 cells. If the lack of active efflux means that estimates of
the B/P ratio at steady state are predictive for the in vivo situation,
both these compounds are expected to be very well distributed to
rat brain. The distribution to brain of 3i and 3l is predicted to be
even higher in rhesus and cynomolgus monkeys as well as human,
as the unbound fractions in plasma of 3i and 3l in these species are
considerably higher than in rats (Table 3).
5. Experimental
5.1. Chemistry
5.1.1. General
Reagents and solvents were obtained from commercial sources
and used as received. Thin layer chromatography was carried out
on Merck Silica gel 60 F254. Flash chromatography was performed
using Merck Silica Gel 60 (40–63 l
m). 1H and 13C nuclear magnetic
resonance (NMR) spectra were recorded at ambient temperature
on a Bruker Avance AV-500 at 500.13 and 125.77 MHz, respec-
tively, or a Bruker Avance AV-III-600 at 600.16 and 151.91 MHz,
respectively. Chemical shifts (d) are reported in parts per million
relative to tetramethylsilane (TMS) or residual solvent, and cou-
pling constants (J) are given in hertz. High-resolution mass spec-
trometry (HRMS) was recorded on a Bruker Daltonics MicrOTOF
operating with external calibration with lithium formate (for ESI)
and PEG200/PEG400/PEG600 (1:1:1) [for atmospheric pressure
photo ionization (APPI)], using an Agilent 1100 high performance
liquid chromatography (HPLC) system for inlet. LC/MS were run
on a Sciex API150EX equipped with APPI-source operating in posi-
tive ion mode. The HPLC consisted of Shimadzu LC10-ADvp LC
pumps, SPD-M20A PDA detector (operating at 254 nm), and
SCL-10A system controller. The autosampler was a Gilson 215,
the column oven was a Jones Chromatography 7990R, and the
ELS detector was a Sedere Sedex 55. The mobile phase was a gradi-
ent consisting of A: water + 0.05% trifluoro acetic acid (TFA) and B:
95% acetonitrile and 5% water + 0.035% TFA. Column temperature
was set to 60 °C. The methods used were one of the following:
4. Conclusion
We have prepared a series of selective a1-adrenoceptor ligands
derived from the antipsychotic drug sertindole (1). Compounds 3i
compounds 3a and 3b, column: Waters Symmetry C18 3.5
l,
(Lu AA27122) and 3l were identified as promising candidates for
4.6 ꢀ 30 mm. 10% B to 100% B in 1.6 min, 100% B to 10% B in

200
M. Jørgensen et al. / Bioorg. Med. Chem. 21 (2013) 196–204
0.05 min, 10% B for 0.05 min. Flow rate 5.2 mL/min; compounds 3c
and 3g, column: Waters Sunfire C18 3.5
100% B in 2.4 min, 100% B to 10% B in 0.4 min. Flow rate 3.3 mL/
14 h with fresh PtO2 (3.5 g). The catalyst was filtered off, and the
solvent was removed in vacuo. The residue was purified by chro-
matography (EtOAc/Et3N 96:4) to give slightly impure 5a. The
two reactions were repeated, and the combined product was re-
crystallized from heptane to give pure 5a (122 g, 23% over 2 steps).
1H NMR (500 MHz, DMSO-d6) 7.86 (d, J = 1.9 Hz, indole H-4), 7.68–
7.55 (m, 4-fluoro-phenyl 2 ꢀ H-2), 7.48 (s, indole H-2), 7.46–7.35
(m, 4-fluoro-phenyl 2 ꢀ H-3 and indole H-7), 7.30 (dd, J = 8.8,
1.9 Hz, indole H-6), 2.87 (d, J = 11.4 Hz, piperidine 2 ꢀ H-2a), 2.78
(ddd, J = 11.8, 8.2, 3.5 Hz, piperidine H-4), 2.22 (s, N-methyl),
2.10–2.00 (m, piperidine 2 ꢀ H-2b), 1.95 (d, J = 12.7 Hz, piperidine
2 ꢀ H-3a), 1.72 (qd, J = 12.7, 3.5 Hz, piperidine 2 ꢀ H-3b), 13C NMR
(126 MHz, CDCl3): 161.3 (d, J = 246.5 Hz), 135.8, 135.7, 135.3, 126.3
(d, J = 8.6 Hz, 2C), 125.5, 125.1, 122.3, 122.2, 116.8 (d, J = 22.9 Hz,
2C), 113.2, 112.0, 56.5 (2C), 46.8, 33.2 (2C), 33.1. HRMS calcd for
l, 4.6 ꢀ 30 mm. 10% B to
min; compounds 3d–3f, 3h, and 3j, column: Waters Atlantis
dC18 3
B in 0.4 min. Flow rate 3.3 mL/min; compound 3i, column: Waters
Sunfire C18 3.5
, 4.6 ꢀ 30 mm. 10% B to 100% B in 2.4 min, 100% B
for 2 min, 100% B to 10% B in 0.4 min. Flow rate 3.3 mL/min; or
compounds 3l and 3m column: Waters Symmetry C18 3.5
l, 4.6 ꢀ 30 mm. 2% B to 100% B in 2.4 min, 100% B to 2%
l
l,
4.6 ꢀ 30 mm. 10% B to 100% B in 4 min, 100% B to 10% B in
1 min. Flow rate 2 mL/min. All pharmacologically characterized
compounds were >95% pure according to ELS and >90% pure
according to UV besides from 3f, which was 89% on ELS and 78%
on UV (compounds 3a–3j, 3l, and 3m), or within 0.4% of theory
using elemental analysis (compound 3k). The compounds were
also characterized by 1H NMR and 13C NMR, as well as HRMS. All
analyses were conducted at H. Lundbeck A/S.
C
20H21BrFN2 (M+H+): 387.0867. Found: 387.0870.
5.1.4. 5-Bromo-3-(1-methylpiperidin-4-yl)-1-(pyridin-4-yl)-1H-
indole (5b)
5.1.2. (1-Methyl-1H-pyrazol-5-yl)boronic acid pinacol ester (4a)
A solution of 1-methylpyrazole (25.0 g, 305 mmol) in tetrahy-
drofuran (THF) (600 mL) was cooled to 0 °C. n-Butyl lithium
(1.6 M in hexanes, 209 mL, 335 mmol) was added drop-wise over
1 h, keeping the temperature below 7 °C. The solution was stirred
at room temperature (rt) for 3 h before cooling to ꢁ70 °C.
B(OMe)3 (44.4 mL, 0.40 mol) was slowly added, keeping the reac-
tion temperature below ꢁ65 °C. The resulting mixture was allowed
to warm to rt, before it was quenched with 15% aqueous (aq) NH4Cl
(450 mL). The mixture was extracted with THF (3 ꢀ 500 mL), and
the combined organic extracts were dried (Na2SO4) and evaporated
in vacuo to give a yellowish solid (15.6 g, 41%). The aq layer was
concentrated in vacuo, and the resulting solid was repeatedly ex-
tracted with THF (5 ꢀ 250 mL). The combined organic extracts
were dried (Na2SO4) and evaporated in vacuo, yielding a yellow so-
lid (20.3 g, 52%). The combined yield of (1-methyl-1H-pyrazol-5-
yl)boronic acid was 35.9 g (93%), and 20.3 g (161 mmol) of this
material was esterified with pinacole (28.4 g, 240 mmol) in THF
(200 mL). 4 Å molecular sieves (6.0 g dried in vacuo at 50 °C) were
added, and the resulting mixture was stirred at rt for 2 days. The
sieves were filtered off, and the filtrate was concentrated in vacuo.
The resulting crude product was dissolved in heptane (500 mL) and
washed with water (2 ꢀ 250 mL). The organic layer was dried
(Na2SO4) and concentrated in vacuo to afford a white solid. This
material was recrystallized from acetonitrile yielding white crys-
tals (22.5 g, 67%). Mp 71.0–71.6 °C (Ivachtchenko et al.,21 74–
76 °C). 1H NMR (500 MHz, DMSO-d6): d, 7.46 (d, J = 1.9 Hz, pyrazole
H-3), 6.62 (d, J = 1.9 Hz, pyrazole H-4), 3.98 (s, N-methyl), 1.30 (s,
pinacol methyl groups, 12H). 13C NMR (126 MHz, DMSO-d6): d,
138.3, 115.9, 84.4 (2C), 41.9, 24.9 (4C). Anal. calcd for C10H17BN2O2:
C, 57.73; H, 8.24; N, 13.46. Found: C, 57.72; H, 8.08; N, 13.40.
Compound 713 (15.2 g, 51.2 mmol), 4-iodopyridine (21.0 g,
102 mmol), K2CO3 (28.3 g, 205 mmol), CuI (39.0 g, 205 mmol),
and ZnO (16.7 g, 205 mmol) were added to a 500 mL round-bottom
flask, and the flask was closed, evacuated, and back-filled three
times with argon. NMP (200 mL) was introduced, and the mixture
was stirred at 110 °C for 24 h. The mixture was diluted with EtOAc
(200 mL) and filtered through celite. Water (200 mL) and 25% aq
NH3 (50 mL) were added, and the mixture was filtered again. The
organic layer was washed with saturated aq NaCl (3 ꢀ 200 mL),
dried (Na2SO4), and concentrated in vacuo. The residue was puri-
fied by column flash chromatography (EtOAc/Et3N 95:5, 0–10%
MeOH gradient) yielding a yellowish foam (2.26 g, 12%). 1H NMR
(500 MHz, DMSO-d6): d, 8.69 (d, J = 6.1 Hz, pyridine 2 ꢀ H2), 7.90
(d, J = 1.9 Hz, indole H-4), 7.75 (d, J = 8.5 Hz, indole H-7), 7.70–
7.67 (m, indole H-2 and pyridine 2 ꢀ H-3), 7.37 (dd, J = 1.9 Hz,
8.5 Hz, indole H-6), 2.88 (broad d, J = 11.8 Hz, piperidine 2 ꢀ H-
2a), 2.78 (tt, J = 3.5 Hz, 11.8 Hz, piperidine H-4), 2.23 (s, N-methyl),
2.07 (t, J = 11.3 Hz, piperidine 2 ꢀ H-2b), 1.95 (d, J = 12.7 Hz, piper-
idine 2 ꢀ H-3a), 1.78–1.69 (m, piperidine 2 ꢀ H–3b). 13C NMR
(126 MHz, DMSO-d6): d, 151.6 (2C), 145.7, 133.8, 131.2, 125.9,
124.9, 124.0, 122.3, 117.2 (2C), 113.7, 113.5, 56.0 (2C), 46.6, 32.5,
32.2 (2C). HRMS calcd for C19H21BrN3 (M+H+): 370.0913. Found:
370.0907.
5.1.5. General procedure for Suzuki–Miyaura couplings
The boronic acid or ester (2 equiv), aryl bromide 5a/5b (1 equiv,
0.5–2.1 mmol) and Pd(Ph3P)4 (5 mol % based on 5a/5b) were vigor-
ously mixed in a 5 mL microwave vial. The vial was capped, evac-
uated and back-filled with argon three times. Toluene (3 mL) and
2 M aq Na2CO3 (2 mL) were added, and the reaction was stirred
at 100 °C overnight. The reaction mixture was diluted with EtOAc
(5 mL) and extracted with water (3 ꢀ 5 mL). The organic layer
was dried (Na2SO4), evaporated in vacuo, and purified by column
flash chromatography (EtOAc/Et3N 95:5, 0–10% MeOH gradient
as eluent, unless stated otherwise).
5.1.3. 5-Bromo-1-(4-fluorophenyl)-3-(1-methylpiperidin-4-yl)-
1H-indole (5a)
N-Methyl-4-piperidone (460 g, 3.00 mol) was boiled under re-
flux in a mixture of AcOH (625 mL) and TFA (1150 mL). A solution
of 612 (200 g, 0.69 mol) in a mixture of AcOH (625 mL) and TFA
(125 mL) was added drop-wise over 1 h. The reaction was boiled
under reflux for an additional 2 h before it was cooled to rt. The
crude mixture was partitioned between EtOAc and dilute aq NH3.
The aq layer was extracted with EtOAc, and the combined organic
extracts were dried over MgSO4, filtered, and concentrated in va-
cuo. The residue was purified by flash chromatography (EtOAc/
Et3N 96:4) to give 195 g of 5-bromo-1-(4-fluoro-phenyl)-3-(1-
5.1.6. 1-(4-Fluorophenyl)-5-(1-methyl-1H-pyrazol-5-yl)-3-(1-
methylpiperidin-4-yl)-1H-indole (3a)
Prepared according to the general procedure from (1-methyl-
1H-pyrazol-5-yl)boronic acid pinacol ester 4a (425 mg, 2.04 mmol)
and 5a (402 mg, 1.04 mmol). Isolated as a brownish oil (432 mg,
>95%.). 1H NMR (500 MHz, DMSO-d6): d, 7.78 (broad s, indole H-
4), 7.66–7.64 (m, 4-fluoro-phenyl 2 ꢀ H-2), 7.56 (d, J = 8.5 Hz, in-
dole H-7), 7.50 (s, indole H-2) 7.47 (broad d, pyrazole H-3), 7.43
(broad t, J = 8.5 Hz, 4-fluoro-phenyl 2 ꢀ H-3), 7.30 (broad d,
J = 8.5 Hz, indole H-6), 6.37 (broad s, pyrazole H-4), 3.85 (s,
pyrazole N-methyl), 2.92–2.87 (m, piperidine 2 ꢀ H-2a and H-4),
methyl-1,2,3,6-tetrahydro-pyridin-4-yl)-1H-indole
sufficiently
pure for the next step. This material was hydrogenated (3 bar H2
pressure) over PtO2 (3 g) in AcOH (1 L) for 52 h. The catalyst was
filtered off, and the filtrate was hydrogenated for an additional

M. Jørgensen et al. / Bioorg. Med. Chem. 21 (2013) 196–204
201
2.21 (s, piperidine N-methyl), 2.14–2.05 (m, piperidine 2 ꢀ H-2b),
5.1.10. 1-(4-Fluorophenyl)-3-(1-methylpiperidin-4-yl)-5-
(pyridin-4-yl)-1H-indole (3e)
2.02–1.97 (m, piperidine 2 ꢀ H-3a), 1.82–1.72 (m, piperidine
2 ꢀ H-3b). 13C NMR (126 MHz, DMSO-d6): d, 160.7 (d,
J = 243.6 Hz), 144.1, 138.1, 135.7, 135.6, 128.3, 126.3 (d,
J = 8.3 Hz, 2C), 125.6, 123.5, 122.7, 122.3, 120.1, 116.9 (d,
J = 22.9 Hz, 2C), 110.9, 105.9, 56.2 (2C), 46.7, 37.6, 32.8 (2C), 32.5.
HRMS calcd for C24H26FN4 (M+H+): 389.2136. Found: 389.2141.
Prepared according to the general procedure from pyridin-4-
ylboronic acid (246 mg, 2.00 mmol) and 5a (396 mg, 1.02 mmol).
Isolated as a colorless oil (65 mg, 16%). 1H NMR (500 MHz,
DMSO-d6): d, 8.61 (d, J = 6.1 Hz, pyridine 2 ꢀ H-2), 8.11 (s, indole
H-4), 7.75 (d, J = 6.1 Hz, pyridine 2 ꢀ H-3), 7.62–7.55 (m, 4-flu-
oro-phenyl 2xH-2 and indole H-6 and H-7), 7.45 (s, indole H-2),
7.40 (m, 4-fluoro-phenyl 2 ꢀ H-3), 2.89–2.83 (m, piperidine
2 ꢀ H-2a and piperidine H-4), 2.22 (s, N-methyl), 2.10–2.02 (m,
piperidine 2 ꢀ H-2b), 2.01 (broad d, J = 12.7 Hz, piperidine 2 ꢀ H-
3a), 1.82–1.37 (m, piperidine 2 ꢀ H-3b). 13C NMR (126 MHz,
DMSO-d6): d, 160.6 (d, J = 243.6 Hz), 150.5 (2C), 148.5, 136.4,
135.7, 129.2, 128.8, 126.1 (d, J = 8.2 Hz, 2C), 125.6, 123.3, 121.7,
121.6 (2C), 118.3, 166.9 (d, J = 22.5 Hz, 2C), 111.3, 56.2 (2C), 46.7,
32.9 (2C), 32.5. HRMS calcd for C25H25FN3 (M+H+): 386.2027.
Found: 386.2023.
5.1.7. 5-(1-Methyl-1H-pyrazol-5-yl)-3-(1-methylpiperidin-4-yl)-
1-(pyridin-4-yl)-1H-indole (3b)
Prepared according to the general procedure from (1-methyl-
1H-pyrazol-5-yl)boronic acid pinacol ester 4a (208 mg, 1.00 mmol)
and 5b (192 mg, 0.519 mmol). Purified by column flash chromatog-
raphy (EtOAc/Et3N 95:5, 0–10% MeOH gradient) to yield an yellow
oil (155 mg, 80%). 1H NMR (500 MHz, DMSO-d6): d, 8.75–8.72 (m,
pyridine 2 ꢀ H-2), 7.78 (d, J = 8.4 Hz, indole H-7), 7.72 (d,
J = 1.5 Hz, indole H-4), 7.55 (d, J = 1.9 Hz, pyrazole H-3), 7.49-7.46
(m, pyridine 2 ꢀ H-3), 7.32 (dd, J = 1.5 Hz, 8.4 Hz, indole H-6),
7.24 (broad s, indole H-2), 6.34 (d, J = 1.9 Hz, pyrazole H-4), 3.89
(s, pyrazole N-methyl), 3.85–3.75 (m, piperidine 2 ꢀ H-2a), 3.20–
3.12 (m, piperidine H-4), 3.02–2.90 (m, piperidine 2 ꢀ H-2b and
N-methyl), 2.50–2.40 (m, piperidine 2 ꢀ H-3a), 1.82–1.72 (m,
piperidine 2 ꢀ H-3b). 13C NMR (126 MHz, CDCl3): d, 151.9 (2C),
146.9, 144.6, 138.9, 135.5, 129.8, 125.4, 124.6, 124.0, 123.6,
120.8, 117.5 (2C), 114.5, 106.5, 56.6 (2C), 47.0, 37.8, 33.5, 33.3
5.1.11. 3-(1-Methylpiperidin-4-yl)-1,5-di(pyridin-4-yl)-1H-
indole (3f)
Prepared according to the general procedure from pyridin-4-
ylboronic acid (123 mg, 1.00 mmol) and 5b (179 mg, 0.483 mmol).
Isolated as a colourless oil (75 mg, 42%). 1H NMR (500 MHz, DMSO-
d6): d, 8.70 (broad d, J = 4.7 Hz, C-bound pyridine 2 ꢀ H-2), 8.61
(broad d, J = 4.7 Hz, N-bound pyridine 2 ꢀ H-2), 8.12 (d, J = 1.4 Hz,
indole H-4), 7.87 (d, J = 9.0 Hz, indole H-7), 7.77 (dd, J = 1.4 Hz,
4.7 Hz, C-bound pyridine 2 ꢀ H-3), 7.68 (dd, J = 1.4 Hz, 4.7 Hz, N-
bound pyridine 2 ꢀ H-3), 7.66–7.64 (m, indole H-6 and H-1),
2.88–2.85 (m, piperidine 2 ꢀ H-2a and H-4), 2.20 (s, N-methyl),
2.09–2.02 (m, piperidine 2 ꢀ H-2b), 2.02–1.96 (m, piperidine
2 ꢀ H-3a), 1.82–1.72 (m, piperidine 2 ꢀ H-3b). 13C NMR
(126 MHz, DMSO-d6): d, 151.6 (2C), 150.4 (2C), 148.0, 145.9,
153.5, 130.4, 130.1, 125.3, 124.4, 122.3, 121.7 (2C), 118.5, 117.0
(2C), 112.3, 56.1 (2C), 46.7, 32.6, 32.4 (2C). HRMS calcd for
(2C). HRMS calcd for
372.2174.
C
23H26N5 (M+H+): 372.2183. Found:
5.1.8. 1-(4-Fluorophenyl)-5-(1-methyl-1H-pyrazol-4-yl)-3-(1-
methylpiperidin-4-yl)-1H-indole (3c)
Prepared according to the general procedure from (1-methyl-
1H-pyrazol-4-yl)boronic acid pinacol ester 4b (416 mg, 2.01 mmol)
and 5a (420 mg, 1.08 mmol). Purified by column flash chromatog-
raphy (EtOAc/MeOH/Et3N (90:5:5) to yield a colorless oil (317 mg,
75%). Crystallized as white crystals from acetonitrile. Mp 158.8–
159.4 °C. 1H NMR (500 MHz, DMSO-d6): d, 8.14 (s, pyrazole H-3),
7.87 (s, pyrazole H-5), 7.83 (broad s, indole H-4), 7.63–7.59 (m,
4-fluoro-phenyl 2 ꢀ H-2), 7.46 (d, J = 8.5 Hz, indole H-7), 7.42–
7.38 (m, 4-fluoro-phenyl 2 ꢀ H-3 and indole H-2 and H-6), 3.88
(s, pyrazole N-methyl), 2.95–2.85 (m, piperidine 2 ꢀ H-2a), 2.81
(broad t, J = 11.5 Hz, piperidine H-4), 2.23 (s, piperidine N-methyl),
2.07 (broad t, J = 11.5 Hz, piperidine 2 ꢀ H-2b), 2.03–1.97 (m,
piperidine 2 ꢀ H-3a), 1.81–1.71 (m, piperidine 2 ꢀ H-3b). 13C
NMR (126 MHz, DMSO-d6): d, 160.4 (d, J = 243.9 Hz), 136.2,
136.0, 134.6, 128.7, 127.6, 125.8 (d, J = 8.8 Hz, 2C), 125.0, 124.8,
123.3, 122.5, 120.8, 116.7 (d, J = 22.8 Hz, 2C), 115.7, 110.9, 56.4
C
24H25N4 (M+H+): 369.2074. Found: 369.2076.
5.1.12. 1-(4-Fluorophenyl)-3-(1-methylpiperidin-4-yl)-5-
(pyridin-3-yl)-1H-indole (3g)
Prepared according to the general procedure using pyridin-3-
ylboronic acid (246 mg, 2.00 mmol) and 5a (399 mg, 1.03 mmol).
Purified by column flash chromatography (EtOAc/MeOH/Et3N
90:5:5) to give a white foam (232 mg, 58%). Crystallized as a white
solid from acetonitrile. Mp 148.6–150.0 °C. 1H NMR (500 MHz,
DMSO-d6): d, 8.96 (d, J = 1.4 Hz, pyridine H-2), 8.54 (dd,
J = 1.4 Hz, 4.7 Hz, pyridine H-4), 8.12 (d, J = 8.0 Hz, pyridine H-6),
8.02 (d, J = 1.4 Hz, indole H-4), 7.67–7.63 (m, 4-fluoro-phenyl
2 ꢀ H-2), 7.59 (d, J = 8.5 Hz, indole H-7), 7.54 (dd, J = 1.4 Hz,
8.5 Hz, indole H-6), 7.50–7.46 (m, indole H-2 and pyridine H-5),
7.45–7.40 (m, 4-fluoro-phenyl 2 ꢀ H-3), 2.93–2.86 (m, piperidine
2 ꢀ H-2a and H-4), 2.22 (s, N-methyl), 2.10–2.01 (m, piperidine
2 ꢀ H-2b and 2 ꢀ H-3a), 1.82–1.72 (m, piperdine 2 ꢀ H-3b). 13C
NMR (126 MHz, DMSO-d6): 160.5 (d, J = 243.2 Hz), 148.2, 147.9,
137.0, 135.8, 135.7, 134.5, 129.3, 128.9, 126.1 (d, J = 8.6 Hz, 2C),
125.4, 124.2, 123.1, 121.9, 118.2, 116.9 (d, J = 22.8 Hz, 2C), 111.3,
(2C), 46.8, 38.9, 32.8 (2C), 32.6. HRMS calcd for
C24H26FN4
(M+H+): 389.2136. Found: 389.2138.
5.1.9. 5-(1-Methyl-1H-pyrazol-4-yl)-3-(1-methylpiperidin-4-yl)-
1-(pyridin-4-yl)-1H-indole (3d)
Prepared according to the general procedure from (1-methyl-
1H-pyrazol-4-yl)boronic acid pinacol ester 4b (208 mg, 1.00 mmol)
and 5b (193 mg, 0.521 mmol). Isolated as a colorless oil (122 mg,
63%). 1H NMR (500 MHz, DMSO-d6): d, 8.65 (d, J = 6.1 Hz, pyridine
2 ꢀ H-2), 8.14 (s, pyrazole H-5), 7.92 (s, pyrazole H-3), 7.87 (broad
s, indole H-4), 7.75 (d, J = 8.5 Hz, indole H-7), 7.63 (d, J = 6.1 Hz,
pyridine 2 ꢀ H-3), 7.54 (s, indole H-2), 7.45 (dd, J = 1.4 Hz, 8.5 Hz,
indole H-6), 3.88 (s, pyrazole N-methyl), 2.86 (d, J = 11.2 Hz, piper-
idine 2 ꢀ H-2a), 2.79 (tt, J = 3.3 Hz, 11.8 Hz, piperidine H-4), 2.19 (s,
piperidine N-methyl), 2.07–2.00 (m, piperidine 2 ꢀ H-2b), 2.00–
1.94 (m, piperidine 2 ꢀ H-3a), 1.81–1.71 (m, piperidine 2 ꢀ H-
3b). 13C NMR (126 MHz, DMSO-d6): d, 151.5 (2C), 146.1, 136.3,
133.7, 130.1, 127.8, 126.1, 124.8, 123.5, 123.0, 121.4, 116.6 (2C),
116.0, 111.9, 56.2 (2C), 46.7, 38.9, 32.6 (2C), 32.5. HRMS calcd for
56.3 (2C), 46.8, 32.9 (2C), 32.5. HRMS calcd for
C25H25FN3
(M+H+): 386.2027. Found: 386.2030.
5.1.13. 3-(1-Methylpiperidin-4-yl)-5-(pyridin-3-yl)-1-(pyridin-
4-yl)-1H-indole (3h)
Prepared according to the general procedure from pyridin-3-
ylboronic acid (123 mg, 1.00 mmol) and 5b (179 mg, 0.483 mmol).
Isolated as a colorless oil (87 mg, 47%). 1H NMR (500 MHz, DMSO-
d6): d, 8.98 (d, J = 1.9 Hz, C-bound pyridine H-2), 8.70 (d, J = 6.1 Hz,
N-bound pyridine 2 ꢀ H-2), 8.56 (dd, J = 1.4 Hz, 4.7 Hz, C-bound
pyridine H-6), 8.14 (dt, J = 1.9 Hz, 8.0 Hz, C-bound pyridine H-4),
8.04 (d, J = 1.4 Hz, indole H-4), 7.90 (d, J = 8.5 Hz, indole H-7),
C
23H26N5 (M+H+): 372.2183. Found: 372.2171.

202
M. Jørgensen et al. / Bioorg. Med. Chem. 21 (2013) 196–204
7.71 (d, J = 6.1 Hz, N-bound pyridine 2 ꢀ H-3), 7.66 (s, indole H-2),
7.61 (dd, J = 1.4 Hz, 8.5 Hz, indole H-6), 7.48 (dd, J = 4.7 Hz, 8.0 Hz,
C-bound pyridine H-5), 2.90–2.86 (m, piperidine 2 ꢀ H-2a and H-
4), 2.22 (s, N-methyl), 2.08 (broad t, J = 11.5 Hz, piperidine 2 ꢀ H-
2b), 2.01 (broad d, J = 12.3 Hz, piperidine 2 ꢀ H-3a), 1.82–1.73
(m, piperidine 2 ꢀ H-3b). 13C NMR (126 MHz, DMSO-d6): d, 151.6
(2C), 148.2, 148.1, 146.0, 136.7, 134.9, 134.6, 130.4, 130.2, 125.2,
124.2, 124.2, 122.6, 118.4, 116.9 (2C), 112.3, 56.2 (2C), 46.7, 32.6
(2C), 32.4. HRMS calcd for C24H25N4 (M+H+): 369.2074 Found:
369.2065.
umn flash chromatography (CH2Cl2 and then EtOAc) to give 8.7 g of
1-(4-fluorophenyl)-5-(2-methyl-2H-tetrazol-5-yl)-1H-indole as a
solid material sufficiently pure for the next step. A portion of this
material (4.0 g, 13.6 mmol) was added portion-wise to a boiling
suspension of N-methyl-4-piperidone hydrochloride hydrate
(12.5 g, 81.6 mmol) in a mixture of AcOH (70 mL) and TFA
(70 mL) under reflux. The resulting mixture was boiled under re-
flux for 1.5 h. After cooling to rt, the crude mixture was concen-
trated in vacuo, and the residue was partitioned between water
(250 mL) and EtOAc (2 ꢀ 100 mL) after basification with NaOH.
The combined organic extracts were washed with sat. aq NaCl
(2 ꢀ 250 mL), dried (Na2SO4), filtered, and concentrated in vacuo
to afford 1-(4-fluorophenyl)-3-(1-methyl-1,2,3,6-tetrahydropyri-
5.1.14. 1-(4-Fluorophenyl)-3-(1-methylpiperidin-4-yl)-5-
(pyrimidin-5-yl)-1H-indole (3i)
Prepared according to the general procedure from pyrimidin-5-
ylboronic acid (248 mg, 2.00 mmol) and 5a (398 mg, 1.03 mmol).
Purified by column flash chromatography (EtOAc/MeOH/Et3N
90:5:5) to give a white solid (117 mg, 29%). Re-crystallized from
acetonitrile. Mp 219.8–220.5 °C. 1H NMR (500 MHz, DMSO-d6): d,
9.21 (s, pyrimidine 2 ꢀ H-3), 9.15 (s, pyrimidine H-1), 8.14 (broad
s, indole H-4), 7.66–7.60 (m, 4-fluoro-phenyl 2 ꢀ H-2 and indole H-
6 and H-7), 7.49 (s, indole H-2), 7.45–7.42 (m, 4-fluoro-phenyl
2 ꢀ H-3), 2.92–2.87 (m, piperidine 2 ꢀ H-2a and H-4), 2.22 (s, N-
methyl), 2.10–2.02 (m, piperidine 2 ꢀ H-2b and 2 ꢀ H-3a), 1.81–
1.72 (m, piperidine 2 ꢀ H-3b). 13C NMR (126 MHz, DMSO-d6): d,
160.6 (d, J = 243.9 Hz), 150.4, 148.5 (2C), 136.4, 135.7 (d,
J = 1.8 Hz), 129.3, 128.8, 126.1 (d, J = 8.4 Hz, 2C), 125.6, 123.3,
121.6, 121.6, 118.3, 116.8 (d, J = 23.2 Hz, 2C), 111.4, 56.2 (2C),
din-4-yl)-5-(2-methyl-2H-tetrazol-5-yl)-1H-indole
phenyl)-3-(1-methyl-1,2,3,6-tetrahydro-pyridin-4-yl)-5-(2-methyl-
2H-tetrazol-5-yl)-1H-indole (3.7 g) sufficiently pure for the next
1-(4-fluoro-
step.
A portion of this material (2.2 g, 5.7 mmol) and PtO2
(150 mg) were added to AcOH (50 mL), and the resulting mixture
was treated with H2 (3 bar) for 10 h at rt. The catalyst was filtered
off, and the filtrate was concentrated in vacuo. The residue was
partitioned between water (50 mL) and EtOAc (2 ꢀ 100 mL) after
basification with NaOH. The combined organic layers were washed
with sat. aq NaCl (2 ꢀ 200 mL), dried (Na2SO4), filtered, and con-
centrated in vacuo. The residue was purified by column flash
chromatography (EtOAc/EtOH/Et3N 100:25:4) to afford the title
compound as a solid material, which was re-crystallized from ace-
tone to give pure 3k (0.34 g, 16%). Mp 148–150 °C. 1H NMR
(500 MHz, DMSO-d6): d, 8.36 (d, J = 1.6 Hz, indole H-4), 7.89 (dd,
J = 1.6 Hz, 8.7 Hz, indole H-6), 7.68–7.63 (m, 4-fluoro-phenyl
2 ꢀ H-2), 6.62 (d, J = 8.7 Hz, indole H-7), 7.51 (s, indole H-2), 7.42
(app. t, J = 8.7 Hz, 4-fluoro-phenyl 2 ꢀ H-3), 4.41 (s, tetrazole-N-
methyl), 2.94–2.80 (m, piperidine 2 ꢀ H-2a and H-4), 2.22 (s,
piperidine-N-methyl), 2.08 (app. dt, J = 1.7 Hz, 11.4 Hz, piperidine
2 ꢀ H-2b), 2.01–1.95 (m, piperidine 2 ꢀ H-3a), 1.81 (app. dq,
J = 3.6 Hz, 12.9 Hz, piperidine 2 ꢀ H-3b), 13C NMR (126 MHz,
DMSO-d6): d, 165.8, 161.0 (d, J = 242.5 Hz), 137.3, 135.8, 128.6,
126.7 (d, J = 8,5 Hz, 2C), 126.5, 123.3, 121.4, 119.4, 118.3, 117.2
(d, J = 23.0 Hz, 2C), 111.8, 56.5 (2C), 47.0, 33.2 (2C), 33.1. HRMS
calcd for C22H24FN6 (M+H+): 391.2041. Found: 391.2050.
46.7, 32.8 (2C), 32.5. HRMS calcd for
387.1980. Found: 387.1979.
C
24H24FN4 (M+H+):
5.1.15. 3-(1-Methylpiperidin-4-yl)-1-(pyridin-4-yl)-5-
(pyrimidin-5-yl)-1H-indole (3j)
Prepared according to the general procedure from pyrimidin-5-
ylboronic acid (159 mg, 1.28 mmol) and 5b (183 mg, 0.494 mmol).
Isolated as a white solid (32 mg, 17%). 1H NMR (500 MHz, DMSO-
d6): d, 9.23 (s, pyrimidine 2 ꢀ H3), 9.17 (s, pyrimidine H-1), 8.70
(broad d, J = 4.7 Hz, pyridine 2 ꢀ H-2), 8.18 (d, J = 1.9 Hz, indole
H-4), 7.94 (d, J = 9.0 Hz, indole H-7), 7.73 (broad d, J = 4.7 Hz, pyr-
idine 2 ꢀ H-3), 7.70–7.68 (m, indole H-2 and H-6), 2.91–2.89 (m,
piperidine 2 ꢀ H-2a+H-4), 2.24 (s, N-methyl), 2.15–2.06 (m, piper-
idine 2 ꢀ H-2b), 2.06–2.02 (m, piperidine 2ꢀH-3a), 1.82–1.72 (m,
piperidine 2 ꢀ H-3b). 13C NMR (126 MHz, DMSO-d6): d, 155.1
(2C), 151.6 (2C), 145.9, 135.2, 134.3, 130.3, 126.9, 125.3, 124.5,
122.4, 118.8, 117.1, 117.1 (2C), 112.5, 56.2 (2C), 46.7, 32.6 (2C),
32.3. HRMS calcd for C23H24N5 (M+H+): 370.2026 Found: 370.2023.
5.1.17. 1-(4-Fluorophenyl)-5-(1-methyl-1H-1,2,4-triazol-3-yl)-3-
(1-methylpiperidin-4-yl)-1H-indole (3l)
Prepared following the general procedure described in Balle
et al.8 starting from 5a (4.2 g, 10.9 mmol) and 3-bromo-1-
methyl-1H-1,2,4-triazole17 (1.75 g, 10.8 mmol). Compound 3l was
obtained as an off-white solid (150 mg, 3%). Mp 286–288 °C. 1H
NMR (500 MHz, DMSO-d6): d, 8.50 (s, triazole H-5), 8.35 (broad s,
indole H-4), 7.89 (dd, J = 1.4 Hz, 8.6 Hz, indole H-6), 7.68–7.63
(m, 4-fluoro-phenyl 2 ꢀ H-2), 7.57–7.52 (m, indole H-2 and H-7),
7.46–7.42 (m, 4-fluoro-phenyl 2 ꢀ H-3), 3.93 (s, triazole-N-
methyl), 3.55 (broad s, piperidine 2 ꢀ H-2a), 3.17 (broad s, piperi-
dine 2 ꢀ H-2b and H-4), 2.81 (s, piperidine-N-methyl), 2.21 (broad
d, J = 13.2 Hz, piperidine 2 ꢀ H-3a), 2.10–1.98 (m, piperidine 2 ꢀ H-
3b). 13C NMR (151 MHz, DMSO-d6): d, 162.4, 160.8 (d, J = 243.9 Hz),
145.9, 136.5, 135.7, 127.9, 126.5 (d, J = 8.9 Hz, 2C), 126.0, 123.9,
121.4, 120.9, 117.2, 117.1 (d, J = 23.1 Hz, 2C), 111.1, 54.3 (2C),
43.3, 36.4, 30.8, 30.3 (2C). HRMS calcd for C23H25FN5 (M+H+):
390.2089. Found: 390.2097.
5.1.16. 1-(4-Fluorophenyl)-5-(2-methyl-2H-tetrazol-5-yl)-3-(1-
methylpiperidin-4-yl)-1H-indole (3k)
Nitrile 812 (20 g, 84.6 mmol) was dissolved in 1,2-dimethoxy-
ethane (250 mL), and triethyl amine hydrochloride (35.0 g,
254 mmol) and NaN3 (20.0 g, 769 mmol) were added. The reaction
was boiled under reflux overnight, cooled to rt, filtered, and con-
centrated in vacuo. The residue was partitioned between EtOAc
(250 mL), water (250 mL), and AcOH (30 mL). The aq layer was ex-
tracted with EtOAc (400 mL), and the combined organic layers
were washed quickly with water (400 mL), sat. aq NaCl (400 mL),
dried (Na2SO4), filtered, and concentrated to afford 17.2 g of 1-(4-
fluorophenyl)-5-(2H-tetrazol-5-yl)-1H-indole as a solid sufficiently
pure for the next step.
A portion of this material (11.7 g,
41.9 mmol) was dissolved in NMP (150 mL), and KO(tBu) (5.20 g,
46.1 mmol) was added at 10-20 °C under a N2 atmosphere. CH3I
(6.0 mL, ꢂ96 mmol) was added, and the mixture was stirred at rt
for 2 h. The crude mixture was poured into a mixture of water
(200 mL) and EtOAc (200 mL). The organic layer was washed with
water (200 mL) and sat. aq NaCl (2 ꢀ 200 mL), dried (Na2SO4), fil-
tered, and concentrated in vacuo. The residue was purified by col-
5.1.18. 1-(4-Fluorophenyl)-5-(1-methyl-1H-pyrazol-3-yl)-3-(1-
methylpiperidin-4-yl)-1H-indole (3m)
Compound 98 (0.94 g, 2.5 mmol) was dissolved in THF (45 mL),
and K2CO3 (0.52 g, 3.7 mmol), Et3N (1 mL), and CH3I (0.19 mL,
3.0 mmol) were added. The mixture was stirred at rt for 6 h. The
crude mixture was filtered, and the filtrate was concentrated in va-
cuo. The residue was purified by column flash chromatography

M. Jørgensen et al. / Bioorg. Med. Chem. 21 (2013) 196–204
203
(EtOAc/EtOH/Et3N 70:25:5) to give a semisolid, which was crystal-
lized as a white product from Et2O/(iPr)2O (0.31 g, 32%). Mp 137–
139 °C. 1H NMR (500 MHz, DMSO-d6): d, 8.05 (d, J = 1.4 Hz, pyra-
zole H-5), 7.70 (d, J = 2.2 Hz, indole H-4), 7.65–7.59 (m, 4-fluoro-
phenyl 2 ꢀ H-2 and indole H-6), 7.46 (d, J = 8.7 Hz, indole H-7),
7.43–7.38 (m, 4-fluoro-phenyl 2 ꢀ H-3 and indole H-2), 6.69 (d,
J = 1.4 Hz, pyrazole H-4), 3.90 (s, pyrazole-N-methyl), 2.92–2.78
(m, piperidine 2 ꢀ H-2a and H-4), 2.21 (s, piperidine-N-methyl),
2.12–1.92 (m, piperidine 2 ꢀ H-2b and 2 ꢀ H-3a), 1.85–1.72 (m,
piperidine 2 ꢀ H-3b). 13C NMR (126 MHz, DMSO-d6): d, 160.5 (d,
J = 243.1 Hz), 151.3, 135.8, 135.8, 135.4, 132.4, 128.4, 126.1 (d,
J = 8.5 Hz, 2C), 126.0, 125.0, 122.7, 120.8, 116.9 (d, J = 22.9 Hz,
2C), 110.7, 102.5, 56.2 (2C), 46.8, 38.8, 32.9 (2C), 32.8. Anal. calcd
for C24H25FN4: C, 74.20; H, 6.49; N, 14.42. Found C, 74.05; H,
6.61; N, 14.23.
predicted from the in vitro free fraction data under the assumption
of steady-state conditions and that passive diffusion govern the
brain distribution. Under these assumptions, unbound plasma con-
centrations, expressed as Ctotal,plasma ꢀ fu,plasma, equals unbound
brain concentrations, expressed as Ctotal,brain ꢀ fu,brain. Thus, in
theory, the in vivo brain/plasma ratio (Ctotal,brain/Ctotal,plasma) equals
20,25
fu,plasma/fu,brain
,
which was how the estimated B/P ratios were
calculated.
5.3. In vitro receptor binding assays
5.3.1. Assays performed in-house
CHO cell lines expressing the rat
a1D-adrenoceptor and the hu-
man D2 receptor, and BHK cell lines expressing the bovine a1A
-
adrenoceptor were generated in-house at H. Lundbeck A/S using
standard stable transfection techniques. Rat-1 cells expressing
hamster
a1B-adrenoceptors were obtained from the University of
5.2. ADME assays
Utah, Salt Lake City, UT. Hela cells expressing human 5-HT1B recep-
tors were obtained from Seattle Veterans Affairs Medical Center.
Hela cells expressing human 5-HT6 receptors were obtained from
NIH. CHO cells expressing human 5-HT2C receptors (VSV) were
purchased from Euroscreen, Brussel, Belgium.
The cells were grown to 90% confluence, detached and homog-
enized in ice-cold 50 mM Tris, pH 7.4, using an Ultra-Turrax, and
the homogenates were either kept on ice or stored at ꢁ80 °C until
use.
5.2.1. Efflux in Caco-2 cells
The polarized transport of the compounds was studied in Caco-
2 cells grown on permeable filters (TranswellÒ, Corning Costar) for
21–28 days. Culture conditions were as published elsewhere.22 For
the experiment, a 2 mM DMSO stock solution was diluted to the
appropriate concentrations (1–20 lM) with Hanks-Hepes buffer,
pH 7.4. The test solution was added to either the upper chamber
(apical-to-basolateral transport, A–B) or the lower chamber (baso-
lateral-to-apical transport, B–A), and from the chamber opposite to
the test solution, samples were removed during 240 min and re-
placed with an equal amount of fresh buffer.23 The stop plates were
centrifuged for 10 min at 3300 rpm and 4 °C, before being analyzed
by liquid-chromatography coupled to a tandem mass spectrometer
(LC–MS/MS, Waters QuattroMicro, Manchester, UK). The apparent
permeability coefficient, Papp was calculated as:
In all
used as assay buffer, and [3H]prazosine was used as radioligand
at a concentration of 0.3 nM (a1A and 1D) or 0.5 nM ( 1B). Non-
specific binding was defined as the binding in the presence of
2-(2,6-dimethoxyphenoxyethyl)aminomethyl-1,4-benzodi-
a1-adrenoceptor binding assays, 50 mM Tris, pH 7.7 was
a
a
1 lM
oxane (WB-4101), and mixtures were incubated at 22 °C for
20 min.
In the 5-HT6 receptor binding assay, 50 mM Tris, pH 7.7 was
used as assay buffer, [3H]LSD was used as radioligand at a concen-
tration of 1 nM. Non-specific binding was defined as the binding in
the presence of 10 lM 1-(2-{4-[5-fluoro-1-(4-fluoro-phenyl)-1H-
indol-3-yl]-piperidin-1-yl}-ethyl)-imidazolidin-2-one, and the
mixtures were incubated at 22 °C for 60 min.
Papp ¼ dQ =dt ꢀ ð1=ðA ꢀ C0ÞÞ
where dQ/dt is the steady state flux across the monolayer, A is the
area of the monolayer (1.1 cm2) and C0 is the initial concentration
in the test solution.24 A Papp (B–A)/Papp (A–B) ratio of one is indica-
tive of pure passive diffusion, whereas a ratio significantly higher
than one indicates polarized transport in favour of the B–A direc-
tion, suggesting involvement of an efflux system such as P-
glycoprotein.
In the D2 receptor binding assay, 50 mM Tris, pH 7.4 was used
as assay buffer, and [3H]spiperone was used as radioligand at a
concentration of 0.1 nM. Non-specific binding was defined as the
binding in the presence of 10 lM haloperidol, and the mixtures
were incubated at 37 °C for 30 min.
5.2.2. Equilibrium dialysis measurements
In the 5-HT2C receptor binding assay, 50 mM Tris, pH 7.7 was
used as assay buffer, and [3H]Mesulergine was used as radioligand
at a concentration of 0.5 nM. Non-specific binding was defined as
the binding in the presence of 100 lM 3-(2-aminoethyl)-5-hydrox-
yindole creatinin sulphate, and the mixtures were incubated at
The methodology employed in this study was a modification of
a previously reported method.25 Briefly, a 96-well equilibrium dial-
ysis apparatus was used to determine the free-fraction in the plas-
ma and brain for each drug (HT Dialysis LLC, Gales Ferry, CT, USA).
Membranes (3 kDa cut-off) were conditioned in deionized water
for 40 min, followed by conditioning in 80:20 deionised water:eth-
anol for 20 min, and then rinsed in deionised water before use.
Plasma was diluted 1:1 with phosphate-buffered saline (PBS),
while the brain tissue was homogenised with PBS to a final compo-
sition of 1:2 brain:PBS, by means of ultrasonication (Tomtec Autog-
iser, Receptor Technologies, Adderbury, Oxon, UK) in an ice bath.
Plasma and brain homogenate were spiked with the test com-
pound (1000 ng/g), and 99 aliquots (n = 6 replicate determinations)
were loaded into the 96-well equilibrium dialysis plate. Dialysis
37 °C for 60 min.
In the a1, D2, 5-HT6 and 5-HT2C assays, bound and free radioac-
tivity were separated by vacuum filtration on GF/B filters. The
filters were dried, scintillation fluid (Optiphase Supermix, Perkin-
Elmer) was added, and the samples were counted in a scintillation
counter (Trilux, Wallac).
In the 5-HT1B binding assay, 50 mM Tris, pH 7.7 with 0.8 mg/mL
WGA SPA beads were used as assay buffer, and [3H]CT (TRK 1038)
was used as radioligand at a concentration of 1.5 nM. Non-specific
binding was defined as the binding in the presence of 10 lM 3-(2-
versus PBS (100 lL) was carried out for 5 h in a temperature-con-
aminoethyl)-5-hydroxyindole creatinin sulphate, and the mixtures
were incubated at room temperature for 60 min and subsequently
counted in a scintillation counter (Trilux, Wallac).
Data shown in the tables from the above assays are means of a
minimum of two full concentration-response curves using 10 con-
centrations of drugs (covering 4 decades). The results are given as
Ki values (nM) derived from computer-fitted IC50 values converted
trolled incubator at 37 °C. The samples were analyzed by liquid-
chromatography coupled to a tandem mass spectrometer (LC–
MS/MS, Waters QuattroMicro, Manchester, UK). The free fraction
in plasma (fu,plasma) and brain tissue (fu,brain) was calculated accord-
ing to Kalvass and Maurer25 with their dilution factor of the brain
taken into account. Brain/plasma (B/P) distribution ratios were

204
M. Jørgensen et al. / Bioorg. Med. Chem. 21 (2013) 196–204
to Ki values using the Cheng–Prusoff equation ((Ki = IC50/(1 + (L/
KD))). Absolute deviation from the mean for the pKi values were be-
low 0.3 for all reported compounds.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.10.049.
These data include MOL files and InChiKeys of the most important
compounds described in this article.
5.3.2. Assays performed externally at Cerep and MDS
pharmaservices
External testing in human 5-HT2A receptor binding was done at
Cerep (www.cerep.fr, catalog Ref. 808-2ah). Compounds were
References and notes
tested at two concentrations (1 lM and 100 nM), and Ki values
1. Zhong, H.; Minneman, K. P. Eur. J. Pharmacol. 1999, 375, 261.
2. Gross, G.; Hanft, G.; Mehdorn, H. M. Eur. J. Pharmacol. 1989, 169, 325.
3. Docherty, J. R. Cell. Mol. Life Sci. 2010, 67, 405.
4. Arnt, J.; Skarsfeldt, T. Neuropsychopharmacology 1998, 18, 63.
5. Olsen, C. K.; Brennum, L. T.; Kreilgaard, M. Eur. J. Pharmacol. 2008, 584, 318.
6. Nord, M.; Farde, L. CNS Neurosci. Ther. 2011, 17, 97.
7. Paterson, L. M.; Kornum, B. R.; Nutt, D. J.; Pike, V. W.; Knudsen, G. M. Med. Res.
Rev. 2011. http://dx.doi.org/10.1002/med.20245.
8. Balle, T.; Perregaard, J.; Ramirez, M. T.; Larsen, A. K.; Søby, K. K.; Liljefors, T.;
Andersen, K. J. Med. Chem. 2003, 46, 265.
9. Balle, T.; Halldin, C.; Andersen, L.; Hjorth, A. L.; Badolo, L.; Jensen, K. G.; Chou, Y.
W.; Andersen, K.; Perregaard, J.; Farde, L. Nucl. Med. Biol. 2004, 31, 327.
10. Barder, T. E.; Walker, S. D.; Martinelli, J. R.; Buchwald, S. L. J. Am. Chem. Soc.
2005, 127, 4685.
were subsequently estimated from computer-fitted IC50 values
converted to Ki values using the Cheng–Prusoff equation
((Ki = IC50/(1 + (L/KD))). The compounds were only tested once, so
the standard errors are not known.
Broad in vitro profiling was performed by MDS pharmaservices
(Taiwan), and compounds were assayed for their ability to displace
radioligand binding to the assayed targets at a concentration of
1000 nM. The following targets were tested at MDS Pharmaservic-
es: Adenosine A1, adenosine A2A, adenosine A3, adrenergic a1A
,
adrenergic 1B, adrenergic 1D, adrenergic 2A, adrenergic b1,
a
a
a
adrenergic b2, transporter: norepinephrine (NET), bradykinin B1,
bradykinin B2, calcium channel L-type: benzodiazepine, calcium
channel L-type: dihydropyridine, calcium channel N-type, dopa-
mine D1, dopamine D2S, dopamine D3, dopamine D4.2, transporter:
dopamine (DAT), endothelin ETA, endothelin ETB, epidermal
growth factor (EGF), estrogen ERalpha, transporter: GABA, GABAA:
agonist site, GABAA: benzodiazepine: central, GABAB1A, glucocorti-
coid, Glutamate: Kainate, Glutamate: NMDA: Agonism, Glutamate:
NMDA: Glycine, glutamate: NMDA: phencyclidine, histamine H1,
histamine H2, histamine H3, imidazoline I2: central, interleukin
IL-1, leukotriene: cysteinyl cysLT1, melatonin MT1, muscarinic
M1, muscarinic M2, muscarinic M3, tachykinin NK1, neuropeptide
Y Y1, neuropeptide Y Y2, nicotinic acetylcholine, nicotinic acetyl-
choline: bungarotoxin-sensitive: neuromuscular, opiate delta
(OP1: DOP), opiate kappa (OP2: KOP), opiate mu (OP3: MOP), phor-
bol ester, platelet activating factor (PAF), potassium channel
11. Prieto, M.; Zurita, E.; Rosa, E.; Muñoz, L.; Lloyd-Williams, P.; Giralt, E. J. Org.
Chem. 2004, 69, 6812.
12. Perregaard, J.; Arnt, J.; Bøgesø, K. P.; Hyttel, J.; Sànchez, C. J. Med. Chem. 1992,
35, 1092.
13. Borghese, A.; Antoine, L.; Stephenson, G. Tetrahedron Lett. 2002, 43, 8087.
14. Walker, S. D.; Barder, T. E.; Martinelli, J. R.; Buchwald, S. L. Angew. Chem., Int. Ed.
2004, 43, 1871.
15. Littke, A. F.; Fu, G. C. Angew. Chem., Int. Ed. 1998, 37, 3387.
16. Littke, A. F.; Dai, C.; Fu, G. C. J. Am. Chem. Soc. 2000, 122, 4020.
17. Bernardini, A.; Viallefont, P.; Daunis, J.; Roumestant, M. L.; Soulami, A. B. Bull.
Soc. Chim. Fr. 1975, 647.
18. Balle, T.; Andersen, K.; Søby, K. K.; Liljefors, T. J. Mol. Graphics Modell. 2003, 21,
523.
19. Balle, T.; Perregaard, J.; Larsen, A. K.; Ramirez, M. T.; Søby, K. K.; Liljefors, T.;
Andersen, K. Bioorg. Med. Chem. 2003, 11, 1065.
20. Summerfield, S. G.; Stevens, A. J.; Cutler, L.; del Carmen Osuna, M.; Hammond,
B.; Tang, S. P.; Hersey, A.; Spalding, D. J.; Jeffrey, P. J. Pharmacol. Exp. Ther. 2006,
316, 1282.
21. Ivachtchenko, A. V.; Kravchenko, D. V.; Zheludeva, V. I.; Pershin, D. G. J.
Heterocycl. Chem. 2004, 41, 931.
22. Artursson, P.; Karlsson, J. Biochem. Biophys. Res. Commun. 1991, 175, 880.
23. Burton, P. S.; Conradi, R. A.; Hilgers, A. R.; Ho, N. F. H. Biochem. Biophys. Res.
Commun. 1993, 190, 760.
24. Hovgaard, L.; Brøndsted, H.; Buur, A.; Bundgaard, H. Pharm. Res. 1995, 12, 387.
25. Kalvass, J. C.; Maurer, T. S. Biopharm. Drug Dispos. 2002, 23, 327.
[KATP], potassium channel HERG, prostanoid EP4, purinergic P2X
,
purinergic P2Y, rolipram (PDE4), serotonin 5-HT1A, serotonin 5-
HT3, transporter: serotonin (SERT), sigma1, sigma2, sodium chan-
nel: site 2, testosterone, thyroid hormone.