Synthesis and structure-activity relationship in a class of indolebutylpiperazines as dual 5-HT1A receptor agonists and serotonin reuptake inhibitors

Article abstract of DOI:10.1021/jm040793q

Systematic structural modifications of indolealkylphenylpiperazines led to improved selectivity and affinity within this class of 5-HT_1A receptor agonists. Introduction of electron-withdrawing groups in position 5 on the indole raises serotonin transporter affinity, and the cyano group proved to be the best substituent here. 5-Fluoro and 5-cyano substituted indoles show comparable results in in vitro and in vivo tests, and bioisosterism between these substituents was supported by calculation of the molecular electrostatic potentials and dipole moments. Compounds showing promising in vitro data were further examined in ex vivo (p-chloroamphetamine assay) and in vivo (ultrasonic vocalization) tests. Optimization of the arylpiperazine moiety indicated that the 5-benzofuranyl-2-carboxamide was best suited to increase 5-HT transporter and 5-HT_1A receptor affinity and to suppress D₂ receptor binding. 5-{4-[4-(5-Cyano-3-indolyl)butyl]-1-piperazinyl}benzofuran-2- carboxamide 29 (vilazodone, EMD 68843) was identified as a highly selective 5-HT_1A receptor agonist [GTPγS, ED₅₀ = 1.1 nM] with subnanomolar 5-HT_1A affinity [IC₅₀ = 0.2 nM] and as a subnanomolar 5-HT reuptake inhibitor [RUI = 0.5 nM] showing a great selectivity to other GPCRs (e.g., D₂, IC₅₀ = 666 nM).

Full text of DOI:10.1021/jm040793q

4684
J . Med. Chem. 2004, 47, 4684-4692
Syn th esis a n d Str u ctu r e-Activity Rela tion sh ip in a Cla ss of
In d olebu tylp ip er a zin es a s Du a l 5-HT1A Recep tor Agon ists a n d Ser oton in
Reu p ta k e In h ibitor s
Timo Heinrich,* Henning Bo¨ttcher, Rolf Gericke, Gerd D. Bartoszyk, Soheila Anzali, Christoph A. Seyfried,
Hartmut E. Greiner, and Christoph van Amsterdam
Preclinical Pharmaceutical Research, Merck KGaA, Frankfurter Strasse 250, 64293 Darmstadt, Germany
Received February 23, 2004
Systematic structural modifications of indolealkylphenylpiperazines led to improved selectivity
and affinity within this class of 5-HT1A receptor agonists. Introduction of electron-withdrawing
groups in position 5 on the indole raises serotonin transporter affinity, and the cyano group
proved to be the best substituent here. 5-Fluoro and 5-cyano substituted indoles show
comparable results in in vitro and in vivo tests, and bioisosterism between these substituents
was supported by calculation of the molecular electrostatic potentials and dipole moments.
Compounds showing promising in vitro data were further examined in ex vivo (p-chloroam-
phetamine assay) and in vivo (ultrasonic vocalization) tests. Optimization of the arylpiperazine
moiety indicated that the 5-benzofuranyl-2-carboxamide was best suited to increase 5-HT
transporter and 5-HT1A receptor affinity and to suppress D2 receptor binding. 5-{4-[4-(5-Cyano-
3-indolyl)butyl]-1-piperazinyl}benzofuran-2-carboxamide 29 (vilazodone, EMD 68843) was
identified as a highly selective 5-HT1A receptor agonist [GTPγS, ED50 ) 1.1 nM] with
subnanomolar 5-HT1A affinity [IC50 ) 0.2 nM] and as a subnanomolar 5-HT reuptake inhibitor
[RUI ) 0.5 nM] showing a great selectivity to other GPCRs (e.g., D2, IC50 ) 666 nM).
Ch a r t 1. Selection of 5-HT1A Receptor Ligands
In tr od u ction
Selective 5-HT1A receptor full agonists did not prove
to be effective as antidepressants in the clinic so far,
although results from some animal models predictive
for antidepressive activity in man indicate that selective
presynaptic 5-HT1A agonists act as anxiolytics and that
postsynaptic 5-HT1A agonists show antidepressive ef-
fects.1 Clinical trials with quite selective 5-HT1A agonists
such as ipsapirone2 or eptapirone3 (Chart 1) could not
support this hypothesis. As a consequence, compounds
possessing simultaneous activity at serotonergic and/
or noradrenergic receptors and/or transporters are
under consideration.4 In particular, the combination of
simultaneous 5-HT1A receptor affinity and selective
serotonin reuptake inhibition is the focus of research
because addition of pindolol (5-HT1A autoreceptor an-
tagonist, Chart 1) to the current therapy with SSRIs
showed promising results in the clinic.5
Ch a r t 2. High Potential 5-HT1A Receptor Binding
Pharmacophore
From our previous work with the optimized 5-HT1A
receptor agonist class of indolebutylpiperazines, we
knew that such agonist with simultaneous inhibition
of serotonin reuptake (RUI) is able to increase the
serotonin level in important brain areas (e.g., frontal
cortex, ventral hypocampus). We have already reported
the optimization within the structural class of roxindole
toward selectivity of 5-HT1A binding, and Chart 2
summarizes the observed structure-related results:6-8
(1) In particular, hydroxy, methoxy, or carboxamide
in position 5 of the indole moiety yields high-affinity
5-HT1A ligands, tolerating a diversity of substituents on
the piperazine.6
(2) A chain of four saturated carbon centers between
the indole and the basic nitrogen is an optimum for
5-HT1A binding.8
(3) The introduction of a residue in the para position
of the aromatic system can reduce dopaminergic activ-
ity.6
Apparently the proper selection of substituents on the
two aromatic systems can be used to fine-tune receptor
affinity and selectivity.9 On this basis, further optimiza-
tion was performed to find a dual selective 5-HT1A
agonist and 5-HT reuptake inhibitor having both activi-
* To whom correspondence should be addressed. Phone: ++49 (0)
6151 72 65 89. Fax: ++49 (0) 6151 72 3129. E-mail: timo.heinrich@
merck.de.
10.1021/jm040793q CCC: $27.50 © 2004 American Chemical Society
Published on Web 08/10/2004

Indolebutylpiperazines
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 19 4685
Sch em e 1. Synthesis of
Sch em e 3. Reduction of Carboxamide 20 to Aldehyde
16 and Oxime 17a
3-(Piperazin-1-yl-butyl)-5-cyanoindolesa
a
(a) toluene, Vitride, room temp f 40 °C; (b) H2NOH‚HCl,
methanol, reflux.
contact in methanol to the corresponding amine 6. The
piperazine 7 was formed by reaction of 6 with bis(2-
chloroethyl)ammonium chloride, and subsequently 7
was alkylated by the indole derivative 3 to form ethyl
ester 8. The ester 8 was saponified with potassium
carbonate in methanol. The resulting acid 9 was acti-
vated with the Mukaiyama reagent13 and transferred
into the corresponding amide 10 with gaseous ammonia.
The synthesis of the 5-fluoro- 14 and the 5-chloroindole
15 has been published previously.6,7,14 The 5-carbalde-
hyde 16 was available by reduction of the corresponding
carboxamide with Vitride in 30% yield. The aldehyde
16 was transferred into the aldoxime 17 by reaction with
hydroxylammonium chloride in 80% yield (Scheme 3).
The chemical methodology for the syntheses of the acid
18, the ester 19, and the carboxamide 20 has already
been described by us.6
a
(a) 4-Chlorobutyryl chloride, isobutyl-AlCl2; (b) 2-(methoxy-
ethoxy)aluminum hydride; (c) R-piperazine, DMF, K2CO3; R ) 2,3-
dihydrobenzo[1,4]dioxin-6-yl for Table 1 and see Tables 2, 5, and
6.
Sch em e 2. Syntheses of Benzofuran-2-carboxamides 28
and 29a
Biology
The affinity of the compounds toward the dopamine
D2 receptor subtype was evaluated by their ability to
displace [3H]spiperone (dopamine antagonist)15,16 in rat
striatal membranes. Alternatively, cloned human recep-
tors were applied with spiperone as ligand. In all cases
there was no significant difference for one substance in
the described assays. Serotonergic activity of the com-
pounds was measured in rat striatal membranes with
[3H]-8-OH-DPAT as ligand.14 In vitro reuptake inhibi-
tion of [3H]-5-HT was evaluated in rat synaptosomes,17
and ex vivo after p-chloroamphetamine induced 5-HT
depletion (PCA assay) in rats.18 The intrinsic activity
of the compounds as 5-HT1A agonists or antagonists was
determined in functional GTPγS assays with recombi-
nant human 5-HT1A receptors stably expressed in
membranes of Chinese hamster ovary (CHO) cells.19 In
vivo the 5-HT1A agonistic activity was determined in the
rat ultrasonic vocalization test20,21 and the D2 antago-
nistic activity in the mouse climbing test.22,23
a
(a) Pd/C, H2; (b) bis(2-chloroethyl)ammonium chloride; (c)
5-CN- or 5-F-4-chlorobutylindole, K2CO3, NMP; (d) KOH, metha-
nol, 80 °C; (e) 1-methyl-2-chloropyridinium iodide, NH3 gas.
ties in the same order of magnitude within a nanomolar
range. We found that both the methyl ether (inductive
donor) and the carboxamide (mesomeric acceptor) in
position 5 of the indole resulted in reliable 5-HT1A
binding.6 Because we expected better metabolic stability
from an electron-deficient indole, we took electron-
withdrawing groups (EWG) under closer consideration.
Ch em istr y
The 5-cyanoindole derivatives were prepared as in-
dicated in Scheme 1. Acylation of commercially available
5-cyanoindole 110 in the presence of isobutyl-AlCl2 with
4-chlorobutyryl chloride led to the chlorobutanoylindole
2 (73%). After selective desoxygenation of the keto
function with sodium bis(2-methoxyethoxy)aluminum
hydride (Vitride/Red-Al) to building block 3 in moderate
yield, the chlorine was substituted with the appropriate
piperazines either in NMP or in DMF in the presence
of inorganic carbonates to yield scaffold 4 (55-60%). The
phenylpiperazines used are commercially available or
the synthesis is known from the literature.8,11,12 The
5-(piperazin-1-yl)benzofuran-2-carboxamides 28 and 29
were synthesized according to Scheme 2.
Resu lts a n d Discu ssion
As a starting point for our investigation, the benzo-
[1,4]dioxane derivatives 11-21 with different indole
5-residues were prepared to evaluate the influence of
these substituents on 5-HT reuptake inhibition in
relation to 5-HT1A and D2 receptor affinity (Table 1).
Compounds 12, 14, 16, 17, and 21 show nanomolar
or even subnanomolar binding to the 5-HT1A receptor
and equally potent 5-HT reuptake inhibition. The 5-HT
reuptake inhibition property does not seem to affect the
in vivo activity at 5-HT1A receptors in this series; e.g.,
13 and 20 had similar potency to inhibit ultrasonic
Commercially available ethyl 5-nitrobenzofuran-2-
carboxylate 5 was hydrogenated at a Raney nickel

4686 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 19
Heinrich et al.
Ta ble 1. 5-HT1A and D2 Receptor Binding and 5-HT Reuptake
Inhibition of 3-{4-[4-(2,3-Dihydrobenzo[1,4]dioxin-6-yl)-
piperazin-1-yl]butyl}-1H-indol-5-yls with Different Residues on
Position 5 (IC50 ( SEM (nM))a
Ta ble 2. Comparison of 5-F and 5-CN Indolyl Derivatives (IC50
( SEM (nM))a
compd
R
5-HT1A
RUI
D2
>100
11
12
13
14
15
16
17
18
19
20
21
H
OH
8 ( 4
10 ( 11
0.5 ( 0.3
100 ( 29
1b ( 0.3
20 ( 21
1 ( 0.6
5 ( 2
0.2 ( 0.15
0.5 ( 0.2
5 ( 2.3
77 ( 36
120 ( 35
130 ( 61
>100
OCH3
F
Cl
40 ( 17
0.7 ( 0.4
0.4 ( 0.2
4 ( 3
0.6 ( 0.3
0.2 ( 0.2
1 ( 0.7
CHO
CdN-OH
CO2H
CO2CH3
CONH2
CN
<100
9.4 ( 4.9
>100
n.d.
a
50 ( 23
60 ( 22
0.4c ( 0.3
<100
IC50 values were obtained from 5-10 concentrations of the
40 ( 17
5.6 ( 0.6
compound, each in triplicate, and are defined as the concentration
(nM) resulting in 50% inhibition of binding. PCA: ED50 ) 3.8
mg/kg, po, 210 min.
b
a
IC50 values were obtained from 5-10 concentrations of the
compound, each in triplicate, and are defined as the concentration
Ta ble 3. In Vivo 5-HT1A Agonistic Activity in the Ultrasonic
Vocalization Test of Selected 5-F and 5-CN Indolyl Derivatives
(ID50 (mg/kg))
b
(nM) resulting in 50% inhibition of binding. PCA: 0.3 mg/kg po;
10% antagonist, 210 min. ED50 ) 0.44 mg/kg, 210 min, po. c PCA:
0.3 mg/kg po; 13% antagonist, 210 min.
24
25
30
18
n.d.a
31
vocalization (USV, ED50 ) 0.2 mg/kg sc for both).
Because of its potential for phase II metabolism, the
5-hydroxy derivative 12 was not examined further.6,8
The pharmaceutically unacceptable aldehyde 16 and the
strong dopamine D2 binding aldoxime 17 confirm that
EWGs contribute to improve 5-HT reuptake inhibition.
So consequently the fluorine (14) and the cyano (21)
scaffolds were subsequently evaluated in more detail.
Both derivatives show pronounced D2 receptor affinity;
the fluorinated compound 14 is a factor of 20 less potent.
In the p-chloroamphetamine assay (PCA)18 the results
suggest a very similar ex vivo profile for 14 and 21 (10%
or 13% antagonism, respectively, at 0.3 mg/kg po after
210 min). To further verify the assumption of bioisos-
terism of F and CN as substituents at the indole in
position 5 with the 5-HT receptor subtype 1A and the
5-HT transporter as target, the following series was
prepared and tested (Table 2).
po
sc
7
3
4
1
4
n.d.a
a
n.d. ) not determined.
Ta ble 4. Visualization of MEP and Dipole Moment for
Selected 5-Substituted Indoles
Though there is one exception (5-HT1A affinity of 22
is by a factor of 40 lower than that of 23), it is obvious
that F- and CN-indole derivatives behave much the
same way. The comparison of 24/25, 26/27, 28/29, and
30/31 revealed analogous properties in vitro. For ben-
zofuran derivatives 28 and 29, the inspection is also
extended to ex vivo experiments and both compounds
show the same result in the PCA (ED50 ) 3.8 mg/kg,
po). The analogy was also reflected in vivo in the
ultrasonic vocalization test (Table 3).
The benzodioxols 24 and 25 show comparable single-
digit nanomolar results independent from the applica-
tion, and the chromenones 30 and 31 give similar
inhibitory doses after po application. These results
substantiate the suggested bioisosterism between F and
CN substituents, and we were interested to see if
theoretical considerations might support this hypoth-
esis. Hardness has been discussed in the literature to
be an acceptable descriptor of bioisosteric activity or
even predictivity,24 but for quantification, calculation of
the energies of the highest occupied and lowest un-
occupied molecular orbital is problematic for larger
molecules. Therefore, we used the molecular electro-
static potentials (MEP) and dipole moments of indole
fragments not to quantify but rather to visualize pos-
sible comparability. The results are shown in Table 4.25
As can be seen from the illustrations in Table 4, the
MEP (two negative areas, red) and the dipole vector
(length and direction) are better comparable for CN and
F than for Cl and F as substituents. The chlorine
derivative shows properties similar to the unsubstituted
indole, which is in accord with the results for 11 and
15 from Table 1.
For further investigation, the 5-cyanoindole moiety
was chosen for closer examination with a series of
differently substituted arylpiperazines (Tables 5 and 6).
First, a series of monocyclic substituted piperazines was
examined for the 5-HT1A receptor binding and 5-HT
reuptake inhibition (Table 5).
Besides the unsubstituted phenyl compound 32, phen-
yl derivatives with electron-donating residues (33-35,
48) as well as with electron-withdrawing groups (36-
38, 45-47) were prepared. In addition to these, we

Indolebutylpiperazines
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 19 4687
Ta ble 5. 5-CN Indole Derivatives with Different Arylpiperazine Residues (IC50 ( SEM (nM))a
a
IC50 values were obtained from 5-10 concentrations of the compound, each in triplicate, and are defined as the concentration (nM)
resulting in 50% inhibition of binding. n.d. ) not determined.
synthesized further derivatives with a more sophisti-
cated substitution pattern (39-44). Results are in the
range of what we strive for (dual nanomolar activity);
however, the D2 binding affinity is too pronounced for
nearly all members of Table 5. Only the carboxamide
46 shows weak D2 data as desired, but for this com-
pound the 5-HT1A affinity is not strong enough (vide
infra). To optimize 5-HT1A binding and the selectivity
to the D2 receptor, bicyclic aryl substituents on the
piperazine were prepared and tested (Table 6).
Dihydrobenzopyran 49, dihydrobenzofuran 50 and its
2-carboxamide 58, 2,1,3-benzothiadiazoles 51 and 52,
indole 53 and its 2-carboxamide 59, benzothiophen-2-
carboxamide 60, and benzofuran 54 with a series of
corresponding carboxamides with a close relationship
to the benchmark 29 (55-57, 61) were synthesized and
analyzed for their receptor profiles.
reuptake inhibition. Comparable data were found for
the monocyclic derivatives (32-48) and the bicyclic
arylpiperazines (49-61). However, in most cases D2
receptor affinity is too strong. The exception is the
dihydrobenzofuran-2-carboxamide 58 showing low na-
nomolar in vitro data with a perfect selectivity to D2
dopamine receptor binding affinity (>1 µmol/L). Nev-
ertheless, compound 58 is still weaker than 29 on both
serotonin targets by a factor of 10. All other derivatives
and isomers do not differ significantly in their in vitro
profile and thus do not show advantages compared to
compound 29, and none of them reached the potent
activity of vilazodone 29.
The hypothesis that para-substituted arylpiperazines
in indolyl-5-carboxamides have reduced D2 affinity6 is
not valid for the 5-cyanoindoles. With regard to the
nanomolar dopaminergic receptor affinities found for the
set of compounds 21, 33, 34, 36, 40, 51, 53, 54, and 56
in comparison to the unsubstituted core structure 32,
it is obvious that for the 5-cyanoindole derivatives para
Summarizing the data in Tables 2, 5, and 6, com-
pounds containing the 5-cyanoindole-3-butylamine with
a basic nitrogen almost guarantee effective serotonin

4688 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 19
Heinrich et al.
Ta ble 7. Intrinsic 5-HT1A Receptor Activity of Some
Ta ble 6. 5-CN Indole Derivatives with Heterobicyclic
Arylpiperazine Substitution (IC50 ( SEM (nM))a
5-Cyanoindole Derivatives in GTPγS Binding (EC50 (nM))
23 29 30 35a 38 40a 42 51 52 54 55 56 57b 58 60
15.2 1.1 9.3 51 250 85 11
8
6
6.2 0.4 3.8 54 3.8 1.1
a
b
% control at 0.1 µM compound concentration. % control at 1
µM compound concentration.
Ta ble 8. In Vivo Activity of Selected 5-CN Indole Derivatives
(ID50 (mg/kg))
administration
route
23 25 29 31 36 51
inhibition of ultrasonic
vocalization
inhibition of climbing
po
sc
po
14
n.d.
n.d.
4
1
7
13
4
4
0.5
7
3
6
n.d.
3
n.d.
>30 n.d.
mining [35S]GTPγS binding (Table 7). The potential of
methyl ethers (35 and 40) to antagonize 5-HT induced
[35S]GTPγS binding (antagonism assay) was found to
be very low, indicating that the compounds act as
agonists. The 2,6-bis-cyan derivative 38 binds with an
IC50 of 40 nM at the 5-HT1A receptor and does not show
full agonism at the receptor up to 250 nM. The com-
pounds 23, 29, 30, 42, 51, 54-56, 58, and 60 show full
agonism at the 5-HT1A receptor with nanomolar or even
subnanomolar EC50 values. The 4-benzofuranyl deriva-
tive 57, a regioisomer of 29 (5-benzofuranyl), is the only
exception showing merely partial agonism in this func-
tional assay for 5-HT1A receptor activity.
In vivo 5-HT1A agonism was measured via inhibition
of ultrasonic vocalization (USV) in rats and D2 antago-
nism by inhibition of apomorphine-induced climbing in
mice. The results are shown in Table 8. Compatible with
the in vitro binding profile and the 5-HT1A agonistic
properties determined in the GTPγS assay, compounds
23, 25, 29, 31, 36, and 51 are active in vivo. The
electrically induced ultrasonic vocalization is inhibited
by these derivatives, in good correlation with the in vitro
results. A good bioavailability can be predicted from the
ratio of the po versus sc ID50 values. In line with the
determined affinity to D2 receptors, compounds 25 and
36 also inhibited apomorphine-induced climbing behav-
ior, indicating dopamine antagonistic properties, whereas
benzofurancarboxamide 29, without affinity for the
dopamine D2 receptor, was inactive.
In this report a series of indolealkylamines was tested
with emphasis on three targets (5-HT1A, D2, 5-HT
reuptake transporter). Other relevant G-protein-coupled
receptors (GPCRs) have been tested for those com-
pounds with promising profiles. For example, histamin-
ergic activity has to be precluded to avoid any sedative
side effects. In this context, the attractive bis-cyano
derivative 23 has been evaluated (5-HT1A, IC50 ) 1 nM;
USV, ID50 ) 14 mg/kg po; RUI, IC50 ) 1.3 nM; D2, IC50
> 250 nM) and was found to bind with two-digit
nanomolar affinity to the histaminergic receptor 1 (H1,
IC50 ) 55 nM).
Because of its outstanding affinity and selectivity
profile, compound 29 (vilazodone; 5-HT1A, IC50 ) 0.2 nM;
RUI, IC50 ) 0.5 nM; D2, IC50 ) 666 nM) was selected
for further development (Chart 3).
In Table 9 a more comprehensive binding data col-
lection is given for vilazodone 29, and as a resumption
and validation of the bioisosterism discussion above, the
data for the fluorine analogue 28 are given in paren-
theses if available.
a
IC50 values were obtained from 5-10 concentrations of the
compound, each in triplicate, and are defined as the concentration
(nM) resulting in 50% inhibition of binding.
substitution does not inherently reduce D2 binding. In
this context, the high dopaminergic activity of benzo-
dioxane 21 is surprising. Within this group of benzo-
dioxanepiperazines, the cyano group (21) and the cor-
responding oxime 17 show the strongest dopaminergic
D2 binding. The corresponding benzodioxole 25 shows
equally high potency to this receptor, and as for 17 or
21, this is astonishing because other comparable com-
pounds in Table 1 lack this D2 receptor affinity. It can
be speculated that these activities might be linked to
the different three-dimensional structures of the ligands
and their different orientations at the D2 receptor site26
because these compounds can form a multitude of
different conformations due to the flexible four-carbon
chain.
For a couple of compounds the intrinsic activity on
the 5-HT1A receptor was determined in vitro by deter-

Indolebutylpiperazines
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 19 4689
3-{4-[4-(2,3-Dih yd r oben zo[1,4]d ioxin -6-yl)p ip er a zin -1-
yl]bu tyl}-1H-in d ole-5-ca r ba ld eh yd e Oxim e (17). A sus-
pension of 1 g (2.4 mmol) of 16 in 20 mL of methanol was
treated with 170 mg (2.4 mmol) of hydroxylammonium chloride
and heated to reflux for 2 h. After the solution was cooled to
room temperature, some yellowish precipitate was formed. The
compound was filtered and washed with acetone, yielding 800
mg (80%) of 17 hydrochloride: mp 237-238 °C; 1H NMR
(DMSO-d6) δ 10.98 (s, 1H), 10.73 (s, 1H), 10.45 (br s, 1H), 8.15
(s, 1H), 7.67 (s, 1H), 7.48-7.30 (m, 2H), 7.18 (d, 1H, J ) 2.5
Hz), 6.73 (dd, 1H, J ) 2.2 and J ) 9.3 Hz), 6.46 (m, 2H), 4.17
(m, 4H), 3.50 (m, 4H), 3.03 (m, 6H), 2.74 (t, 2H, J ) 8.4 Hz),
1.72 (m, 4H). Anal. (C25H30N4‚HCl) C, H, N. Cl: calcd, 7.5;
found, 6.9.
Ch a r t 3
Ta ble 9. Receptor Binding Profile of Vilazodone 29 and Its
Fluorine Analogue 28a
target
IC50 (nM)
target
IC50 (nM)
5-HT1A
GTPγS
5-HT1B
5-HT1D
5-HT2A
5-HT2C
5-HT3
5-HT4
5-HT6
5-HT7
0.2 (3.0)
1.1
5000
4000 (>1000)
1510
2000 (>1000)
4300
252
3000
3900
RUI
PCA
D2
D3
D4
R1
0.5 (0.3)
3.8b (3.8b)
666 (>100)
71
3-[4-(4-Ben zo[1,3]d ioxol-5-ylp ip er a zin -1-yl)bu tyl]-1H-
in d ole-5-ca r bon itr ile (25). A solution of 14.2 g (0.1 mol) of
5-cyanoindole 110 and 23.1 mL (0.2 mol) of 4-chlorobutyric acid
chloride in 250 mL of dichloromethane was cooled to 15 °C,
and 31.9 g (0.2 mol) of isobutylaluminum chloride were added
drop by drop so that the temperature did not exceed 30 °C.
The precipitate was collected by filtration and stirred in 500
mL of water. After exhaustive extraction with ethyl acetate,
the combined organic phases were dried and cautiously
concentrated. The precipitate 2 was filtered, washed with
methyl tert-butyl ether, and dried: yield 17.9 g; 73%; mp 169-
3400
1980 (3000)
6000 (9000)
317
>1000
>1000
R2
H1
H2
κ
a
b
Data for 28 in parentheses. ED50 (mg/kg).
1
170 °C; H NMR (DMSO-d6) δ 12.47 (br s, 1H), 8.55 (s, 2H),
The pharmacological profile of 29 as dual subnano-
molar 5-HT reuptake inhibitor with a selective presyn-
aptic 5-HT1A receptor agonistic property was previously
described.20,27 Vilazodone was found to increase 5-HT
levels in rat brain frontal cortex to an extent not reached
with selective serotonin reuptake inhibitors28 and there-
fore is an interesting compound to further examine the
impact of increased serotonin levels in important brain
areas on mood and mood disorders.
7.69-7.57 (m, 2H), 3.74 (t, 2H, J ) 6.9 Hz), 3.07 (t, 2H, J )
6.9 Hz), 2.12 (quint, 2H, J ) 6.9 Hz). An amount of 95 g (0.4
mol) of the previously prepared 3-(4-chlorobutyryl)-1H-indole-
5-carbonitrile 2 was suspended in 900 mL of THF and cooled
to 0 °C. A solution of 171 g of sodium bis(2-methoxyethoxy)-
aluminum hydride (245 mL/70% in toluene) in 250 mL of
toluene was added dropwise, and stirring was continued for
an additional 2 h. The reaction was terminated by the addition
of 200 mL of water. After phase separation and solvent
evaporation, the residue was filtered over silica gel and the
resulting material was washed with 2-propanol. The resulting
crystals 3 were purified by chromatography over silica gel:
yield 23.3 g; 26%; mp 99-99.5 °C; 1H NMR (DMSO-d6) δ 11.33
(br s, 1H), 8.06 (s, 1H), 7.50 (d, 1H, J ) 8.4 Hz), 7.38 (dd, 1H,
J ) 1.5 Hz and J ) 8.4 Hz), 7.32 (d, 1H, J ) 2.1 Hz), 3.67 (m,
2H), 2.75 (m, 2H), 1.78 (m, 4H). A suspension of 114 g (0.5
mol) of 3-(4-chlorobutyl)-1H-indole-5-carbonitrile 3, 119 g (0.5
mol) of 1-benzo[1,3]dioxol-5-yl-piperazine,29 135 g (1 mol) of
K2CO3, and 81 g (0.5 mol) of KI in 900 mL of DMF was refluxed
for 12 h. Subsequently the suspension was stirred with
charcoal at room temperature and filtered over silica gel. After
removal of the solvent, the residue was dissolved in acetone
and purified via chromatography. The resulting crude product
was recrystallized from methanol, yielding 115 g (60%) of 25:
mp 154-156 °C. A solution of 34 g (0.29 mol) of succinic acid
and 116 g (0.29 mol) of the previously prepared base 25 was
stirred at 35 °C until crystallization began. Crystallization was
continued at 10 °C and the crystals were filtered and washed
with ethyl ether and acetone, yielding 125 g (84%) of 25 as a
Exp er im en ta l Section
Melting points were determined on a Bu¨chi 535 melting
point apparatus and are uncorrected. IR, H NMR, and mass
1
spectra are in agreement with the structures and were
recorded on a Bruker IFS 48 IR spectrophotometer, a Bruker
AMX 300 MHz or DRX 500 MHz NMR spectrometer (TMS as
an internal standard), and Vaccum Generators VG 70-70 or
70-250 at 70 eV. Elemental analyses (obtained with a Perkin-
Elmer 240 BCHN analyzer) for the final products were within
0.4% of theoretical values if not otherwise stated. All reactions
were followed by TLC carried out on Merck KGaA F254 silica
gel plates. Solutions were dried over Na2SO4 and concentrated
with a Bu¨chi rotary evaporator at low pressure. Phenyl-
piperazines were available from EMKA.
Gen er al Meth ods. 3-{4-[4-(2,3-Dih ydr oben zo[1,4]dioxin -
6-yl)piper a zin -1-yl]bu tyl}-1H-in d ole-5-ca r ba ld eh yde (16).
A solution of sodium bis(2-methoxyethoxy)aluminum hydride
(Vitride, 23.6 mL/70% in toluene, 120 mmol) in 25 mL of
toluene was added dropwise to a suspension of 19.7 g (45
mmol) of 3-{4-[4-(2,3-dihydrobenzo[1,4]dioxin-6-yl)piperazin-
1-yl]butyl}indol-5-carboxamide in 400 mL of THF at room
temperature. After 12 h a second portion of 24 mL of Vitride
was added and stirring continued at 40 °C until the carbox-
amide had reacted completely (TLC). For workup, the batch
was cooled with ice and an amount of 30 mL of water was
added slowly. After filtration of the precipitate the solution
was concentrated in vacuo and purified by chromatography
on silica gel. The product fractions were collected and after
evaporation the product was crystallized from acetonitrile
giving 5.8 g (30%) of white crystals of 16: mp 131-135 °C; 1H
NMR (DMSO-d6) δ 9.97 (s, 1H), 8.18 (s, 1H), 7.63 (dd, 1H, J )
1.5 Hz and J ) 8.5 Hz), 7.49 (d, 1H, J ) 8.5 Hz), 7.31-7.16
(m, 2H), 6.71 (dd, 1H, J ) 1.0 Hz and J ) 8.0 Hz), 6.42 (m,
2H), 4.18 (m, 4H), 2.97 (m, 4H), 2.79 (t, 2H, J ) 7.3 Hz), 2.58-
2.32 (m, 6H), 1.79-1.66 (m, 2H), 1.62-1.52 (m, 2H). Anal.
(C25H29N3O3) H. C: calcd, 71.6; found, 71.1. N: calcd, 10.0;
found, 10.5.
1
succinate: mp 227-228 °C; H NMR (DMSO-d6) δ 11.32 (br
s, 1H), 8.05 (s, 1H), 7.51-7.31 (m, 3H), 6.73 (d, 1H, J ) 10.5
Hz), 6.63 (d, 1H, J ) 2.9 Hz), 6.30 (dd, 1H, J ) 2.9 Hz and J
) 10.5 Hz), 5.89 (s, 2H), 2.99 (m, 4H), 2.73 (t, 2H, J ) 8.9 Hz),
2.37-2.40 (m, 6H), 1.63 (m, 4H). Anal. (C24H26N4O2‚C4H6O4)
C, H, N.
5-{4-[4-(5-Cya n o-1H -in d ol-3-yl)b u t yl]p ip er a zin -1-yl}-
ben zofu r a n -2-ca r boxa m id e (29). A mixture of 812 g (3.45
mol) of ethyl 5-nitrobenzofuran-2-carboxylate 5 and 300 g of
wet Raney Nickel in 8 L of methanol were reacted with 232.1
L of H2 at 28 °C for 27 h. Filtration, evaporation, and
recrystallization from methanol yielded 664.6 g (93%) of the
amine 6: mp 59-60 °C; 1H NMR (DMSO-d6) δ 11.80 (br s,
2H), 7.87 (m, 3H), 7.55 (dd, 1H, J ) 2.1 Hz and J ) 8.9 Hz),
4.40 (q, 2H, J ) 7.1 Hz), 1.37 (t, 3H, J ) 7.1 Hz). A suspension
of 48.3 g (0.2 mol) of ethyl 5-aminobenzofuran-2-carboxylate
6, 37.7 g (0.2 mol) of bis(2-chloroethyl)ammonium chloride, and
15.2 g (0.1 mol) of potassium carbonate were heated to reflux
in 250 mL of 1-butanol for 48 h. The hot suspension was

4690 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 19
Heinrich et al.
decanted and filtered. After evaporation the crude product was
recrystallized from methanol, yielding 9.86 g (27%) of 7‚
hydrochloride: mp 242-244 °C; 1H NMR (DMSO-d6) δ 9.30
(br s, 2H), 7.63 (m, 2H), 7.28 (m, 2H), 4.36 (q, 2H, J ) 7.1 Hz),
3.37 (m, 4H), 3.24 (m, 1H), 1.34 (t, 3H, J ) 7.1 Hz). Anal.
(C15H18N2O3‚HCl) C, H, N. Cl: calcd, 11.4; found, 11.0. A
suspension of 4.9 g (16 mmol) of the previously prepared ethyl
5-(piperazin-1-yl)benzofuran-2-carboxylate hydrochloride 7‚
HCl, 3.7 g (16 mmol) of 3-(4-chlorobutyl)-1H-indole-5-carbo-
nitrile 3, 2.2 g (16 mmol) of potassium carbonate, and 2.8 mL
(16 mmol) of triethylamine in 120 mL of acetonitrile was
heated to reflux for 12 h. After the mixture was cooled and
evaporated, the residue was stirred in ethyl acetate and water.
After the organic phase was dried and evaporated, an amount
of 8 g of reddish oil was left. After chromatography an amount
of 2.6 g of a yellow oil was obtained, which was transformed
into the corresponding hydrochloride by dissolving the oil in
120 mL of acetone and adding 2 mL of HCl-saturated ethanol.
The resulting crystals were washed with acetone and ether
and dried, yielding 2.5 g (32%) of 8‚dihydrochloride as colorless
crystals: mp 221-223 °C; 1H NMR (DMSO-d6) δ 11.49 (s, 1H),
10.91 (br s, 1H), 8.10 (s, 1H), 7.64 (m, 2H), 7.51 (d, 1H, J )
8.4 Hz), 7.40 (m, 2H), 7.31 (m, 2H),4.35 (q, 2H, J ) 7.1 Hz),
3.74 (m, 2H), 3.59 (m, 2H), 3.18 (br s, 6H), 2.78 (t, 2H, J ) 7.2
Hz), 1.76 (m, 4H), 1.34 (t, 3H, J ) 7.1 Hz). Anal. (C28H30N4O3‚
2HCl) C, H, N, Cl. A suspension of 1.5 g (2.8 mmol) of ethyl
5-{4-[4-(5-cyano-3-indolyl)butyl]-1-piperazinyl}benzofuran-2-
carboxylate 8 and 0.63 g (11.2 mmol) of KOH in 100 mL of
methanol was heated to reflux for 3 h. The solvent was
evaporated, and the crude residue was dissolved in water. By
additio of 1 N HCl, the pH was adjusted to 7 and the
precipitate was filtered and dried, yielding 1 g (80%) of 5-{4-
[4-(5-cyano-1H-indol-3-yl)butyl]piperazin-1-yl}benzofuran-2-
incubated at 37 °C for 30 min (shaking water bath) in duplicate
in a total volume of 800 µL of buffer containing MgCl2 (3 mM),
NaCl (120 mM), EDTA (0.2 mM), GDP (10 µM), [35S]GTPγS
(0.1 nM), Tris (50 mM), and test compounds. Prior to addition
to the incubation mixture, the test compounds were dissolved
in twice distilled water. DMSO was used to aid in solubilizing
certain compounds. Nonspecific binding was defined with 0.1
µM GTPγS. 5-HT was tested as standard in each experiment
at concentrations of 100, 30, and 10 nM. Experiments were
terminated by rapid filtration through Whatman GF/B filters
using a Brandel cell harvester. Subsequently, the filters were
rinsed twice with 5 mL of ice-cold Tris-HCl and placed in
scintillation vials. Radioactivity was extracted in 4 mL of
scintillation fluid (Ultima Gold, Packard Instruments, Frank-
furt, Germany) and determined by liquid scintillation counting.
Binding isotherms were analyzed by nonlinear regression.
Agonist efficacy ()Emax) is expressed relative to that of 5-HT
()100%), which was tested at a maximally active concentration
(0.1 mM) in each experiment. EC50 values were defined as the
concentration of the compound at which 50% of its own
maximal stimulation was obtained.
[3H]-5-HT Reu p ta k e In h ibition Assa y. Crude synapto-
somal fraction (P2 fraction) of rat cerebral tissue was prepared
according to Whittaker et al.,31 giving a suspension enriched
in synaptosomes of 3 mg of protein/mL. The uptake was
determined in a total volume of 570 µL, which contained 12
nM [3H]-5-HT. Incubation was performed at 37 °C for 4 min
in Krebs-Ringer buffer (126 mM NaCl, 1.4 mM MgCl2, 4.8
mM KCl, 15.8 mM Na2HPO4, 11 mM glucose, 0.9 mM CaCl2,
pH 7.4).
Effects of Dr u gs on P CA (p-Ch lor oa m p h eta m in e)
In d u ced 5-HT Dep letion in Ra t Hyp oth a la m u s. The
experiments were essentially carried out as previously de-
scribed.32 Male rats (135-160 g body weight) were used. Brain
regions were dissected out on ice33 and immediately processed
for HPLC analysis. PCA (5 mg/kg ip in saline) was given 3 h
and drugs 3.5 or 5 h prior to decapitation. Brain tissue 5-HT
was determined by an automated reversed phase/ion pair,
direct injection HPLC method32 within a 25 min run. N-
Methyldopamine or N-ω-methylserotonin was used as an
internal standard and the recoveries were >95%.
1
carboxylic acid 9 as tetrahydrate: mp 189-192 °C; H NMR
(300 MHz, DMSO-d6) δ 11.37 (br s, 2H), 8.07 (s, 1H), 7.52 (m,
2H), 7.37 (m, 3H), 7.17 (m, 2H), 3.13 (br s, 8H), 2.75 (t, 2H, J
) 7.1 Hz), 2.61 (m, 8H), 3.24 (m, 4H), 1.62 (m, 4H). Anal.
(C26H26N4O3‚4H2O) H, N. C: calcd, 60.7; found, 59.7. After
exhaustive drying, 1 g (2.2 mmol) of the previously prepared
acid 9 and 1.4 g (5.5 mmol) of 1-methyl-2-chloropyridinium
iodide were suspended in 20 mL of NMP. While introducing
NH3 gas via a cannula, 2.6 mL of ethyldiisopropylamine were
added drop by drop. The temperature rose to 41 °C and
dropped to room temperature again after 15 min. The reaction
mixture was poured onto water and extracted exhaustively
with ethyl acetate, yielding 0.7 g (72%) of the free base 29.
The compound was dissolved in 30 mL of hot 2-propanol. At
room temperature, HCl-saturated 2-propanol was added slowly
until complete precipitation occurred, yielding 0.6 g (79%) of
29 as the hydrochloride: mp 277-279 °C; 1H NMR (500 MHz,
DMSO-d6) δ 11.45 (d, 1H, J ) 1.9 Hz), 10.80 (br s, 1H), 8.07
(d, 1H, J ) 1.5 Hz), 7.57 (br s, 1H), 7.50 (d, 1H, J ) 8.4 Hz),
7.49 (d, 1H, J ) 8.4 Hz), 7.42 (d, 1H, J ) 1 Hz), 7.37 (dd, 2H,
J ) 1.5 Hz and J ) 8.4 Hz), 7.24 (d, 1H, J ) 2.5 Hz), 7.18 (dd,
1H, J ) 2.5 Hz and J ) 9.1 Hz), 3.71 (m, 2H), 3.53 (m, 4H),
3.12 (m, 4H), 2.75 (t, 2H, J ) 7.2 Hz), 1.79 (m, 2H), 1.67 (m,
2H). Anal. (C26H27N5O2‚HCl) C, H, N, Cl.
Ultr a son ic Voca liza tion Test. Male Sprague Dawley rats
(180-280 g) from Charles River (Sulzfeld, Germany) were
used. Ultrasonic vocalization was measured in
a sound-
attenuated test chamber (width of 24 cm, length of 22 cm,
height of 22 cm) with a grid floor for delivery of foot shock
(scrambled shock of 0.2 mA for 0.5 s, shocker Getra BN 2002).
Ultrasonic vocalization was recorded (microphone 4004, Bruel
and Kjær) and processed by an interface (developed at Merck,
Darmstadt) to select 22 ( 4 kHz signals and to digitize the
resulting signals for automatic processing in
a personal
computer. In a priming phase, each rat was placed in the test
chamber. After a 2 min time period, a series of at most 10
shocks (trials), 1.8 mA for 0.3 s separated by 20 s shock-free
intervals, was delivered via the grid floor of the test chamber.
In the shock-free intervals, the occurrence of ultrasonic
vocalization was automatically recorded and the duration of
ultrasonic vocalization was calculated immediately. The prim-
ing session was terminated either when the rat constantly
vocalized at least for 10 s on three consecutive trials or after
the tenth trial. Rats that did not respond with ultrasonic
vocalization on three consecutive trials were excluded from
further testing. In the actual test performed on the next day,
each rat received five initial shocks (1.8 mA for 0.3 s, separated
by 20 s shock-free intervals) in the test chamber, and the
duration of ultrasonic vocalization was recorded during the
following 3 min period. Animals were tested 2 h after admin-
istration of compounds. ID50 values ()calculated dose for half-
maximal inhibition of ultrasonic vocalization) were determined
from the dose-response curves.
P h a r m a cologica l Meth od s. Ra d ioliga n d Bin d in g As-
sa y a t Ra t Hip p oca m p u s 5-HT1A Ser oton er gic Recep tor s.
The method was adapted from Cossery et al.30 Rat hippocam-
pus membranes (0.2 mg of protein/tube) were incubated with
0.5 nM [3H]-8-OH-DPAT in a total volume of 0.5 mL at 25 °C
for 30 min. Nonspecific binding was determined in the pres-
ence of 1 µM serotonin.
Stim u la tion of [35S]GTP γS bin d in g a t Clon ed 5-HT1A
Recep tor s. The effects of different compounds tested on [35S]-
GTPγS binding were evaluated according to the method of
Newman-Tancredi et al.19 with modifications. Membranes of
Chinese hamster ovary (CHO) cells stably expressing the
recombinant human 5-HT1A receptor were obtained from NEN
(catalog no. CRM035, GenBank no. X13556). The membranes
were stored at -70 °C. Prior to use, membranes were thawed
and rehomogenized in assay buffer (MgCl2, NaCl, and EDTA
in Tris-HCl, pH 7.4). Membranes (∼10 µg of protein) were
Clim bin g Test. Male NMRI mice (24-34 g) from Charles
River (Sulzfeld, Germany) were used. Climbing was induced
by injection of 1.25 mg/kg apomorphine sc and 2 min later
assessed in cylindrical wire mesh cages (11.5 cm diameter, 18

Indolebutylpiperazines
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 19 4691
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cm height) by scoring every 2 min for a period of 20 min as
follows: (0) no climbing behavior, (1) at least two forelimbs
on the wire mesh, (2) all four forelimbs on the wire mesh and
climbing for at least 30 s (maximal score was 20). Drugs were
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Ack n ow led gm en t. We thank Horst Hochsta¨tter,
Marion Sturm, Claudia Daum, Bernd Arzt, Beate Opelt,
Andreas J onke, Uwe Eckert, and Kurt Schuster for the
preparation of the compounds, and we thank Christl
Roos and Herbert Ziegler for performing pharmacologi-
cal tests.
Su p p or tin g In for m a tion Ava ila ble: Spectral data, el-
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11-61. This material is available free of charge via the
Internet at http://pubs.acs.org.
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