Dual 5-HT1A agonists and 5-HT re-uptake inhibitors by combination of indole-butyl-amine and chromenonyl-piperazine structural elements in a single molecular entity

Article abstract of DOI:10.1016/j.bmc.2004.07.014

A series of compounds such as dual serotonin re-uptake inhibitor and 5-HT_1A receptor agonist was found to increase central serotonin levels in rat brain to a greater extent than established antidepressants. The dual serotonin (5-HT) re-uptake i

Full text of DOI:10.1016/j.bmc.2004.07.014

Bioorganic & Medicinal Chemistry 12 (2004) 4843–4852
Dual 5-HT1A agonists and 5-HT re-uptake inhibitors
by combination of indole-butyl-amine and chromenonyl-
piperazine structural elements in a single molecular entity
Timo Heinrich,* Henning Bottcher, Kai Schiemann, Gunter Holzemann, Michael Schwarz,
¨
¨
¨
Gerd D. Bartoszyk, Christoph van Amsterdam, Hartmut E. Greiner and
Christoph A. Seyfried
Preclinical Pharmaceutical Research, Merck KGaA, Frankfurter Str. 250, 64293 Darmstadt, Germany
Received 27 May 2004; accepted 6 July 2004
Available online 3 August 2004
Abstract—The dual serotonin (5-HT) re-uptake inhibitor and 5-HT1A receptor agonist vilazodone was found to increase central
serotonin levels in rat brain. In the course of structural modifications of vilazodone 3-{4-[4-(2-oxo-2H-1-benzopyran-6-yl)-1-pipera-
zinyl]-butyl}-1H-indole-5-carbonitrile 8i and its fluorine analogue 6-{4-[4-(5-fluor-3-indolyl)-butyl]-1-piperazinyl}-2H-1-benzopyran-
2-one have been identified. These unsubstituted chromenones are equally potent at the 5-HT1A receptor and 5-HT transporter. The
implementation of nitrogen functionalities in position 3 of the chromenones resulted in compounds acting as agonists at the 5-HT1A
receptor and as 5-HT re-uptake inhibitors like vilazodone. Ex vivo 5-HT re-uptake inhibition and in vitro 5-HT agonism were deter-
mined in the PCA- and GTPcS-assay, respectively. The potential of these chromenones to increase central 5-HT levels was measured
in microdialysis studies and especially the derivatives 3-{4-[4-(3-amino-2-oxo-2H-chromen-6-yl)-piperazin-1-yl]-butyl}-1H-indole-5-
carbonitrile 8f, ethyl (6-{4-[4-(5-cyano-1H-indol-3-yl)-butyl]-piperazin-1-yl}-2-oxo-2H-chromen-3-yl)-carbamate 8h and N-(6-{4-[4-
(5-cyano-1H-indol-3-yl)-butyl]-piperazin-1-yl}-2-oxo-2H-chromen-3-yl)-acetamide 8k give rise to rapid development of increased
serotonin levels in rat brain cortex, lasting longer than 3h.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
As a consequence, there have been intense efforts on op-
timising a single compound with dual activity to reduce
the time to on-set of action and to decrease side effects.
For example, Laboratorios Vita discussed VN-2222 as
5-HT1A antagonist and 5-HT re-uptake inhibitor exhib-
iting affinity for both targets with a Ki value of 20nM.10
Merck KGaA discovered vilazodone, a presynaptic 5-
HT1A agonist (0.2nM) and 5-HT re-uptake inhibitor
(0.5nM)11 and both compounds increase rat hippocam-
pal extra-cellular 5-HT level (Chart 1).10,12
Depression currently ranks as the worldÕs fourth greatest
cause of illness and is expected to rank second by the
year 2020 according to WHO studies.1 Tricyclic antide-
pressants, the monoamine oxidase inhibitors and the last
generation of selective serotonin re-uptake inhibitors
(SSRIs) although widely used lack sufficient influence
on this development. Treatment difficulties often are
compliance problems resulting from severe side effects,
dietary restrictions or slow onset of action, respectively.2
Hypothetical explanations of the SSRIÕs latency (time
taken to desensitise 5-HT autoreceptors on cell bodies)
are discussed in the literature3–5 and are supported
experimentally in compound combination approaches
(e.g., 5-HT1A ligand + SSRI).6–9
We knew from our previous work that 5-cyano-indole-
butyl-piperazines as potent 5-HT re-uptake inhibitors
tolerate a variety of substituents in the aryl moiety at-
tached to the piperazine. In addition, 2-carboxamido
benzofurane-5-yl as piperazine residue introduced the
desired 5-HT1A agonism, resulting in vilazodone that is
a clinical candidate as novel putative antidepressant
exhibiting both selective 5-HT re-uptake inhibition (5-
HT RUI) and 5-HT1A receptor agonism. In vivo micro-
dialysis revealed that a single treatment with vilazodone
Keywords: Serotonin; Microdialysis; Antidepressant.
*
Corresponding author. Tel.: +49-0-6151-726589; fax: +49-0-6151-
723129; e-mail: timo.heinrich@merck.de
0968-0896/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.07.014

4844
T. Heinrich et al. / Bioorg. Med. Chem. 12 (2004) 4843–4852
5-HT1A = 0.7; 5-HT RUI = 0.9; D2 > 100).11 The opti-
misation of the chromenone moiety to identify com-
pounds with a superior increase of the central 5-HT
level compared to vilazodone is described subsequently.
HO
N
N
S
MeO
VN-2222
NC
O
2. Results and discussion
2.1. Chemistry
CONH2
N
N
N
H
Founded on the above described re-uptake scaffold we
synthesised a variety of novel substituted chromenones
(Scheme 1).
Vilazodone
Chart 1. Dual 5-HT1A ligands and 5-HT re-uptake inhibitors.
Commercially available N-BOCpiperazine 1 was cou-
pled with 5-bromo-2-hydroxy-benzaldehyde 2 to the
protected 2-hydroxy-5-piperazin-1-yl-benzaldehyde 3 in
the presence of base and an appropriate palladium ca-
talyst in THF on a 10g scale with 97% yield. The subse-
quent tandem reactions, Knoevenagel and ring closure,
with different CH acids yielded the corresponding
chromenones 4, which were deprotected under acidic
conditions to the free amines 5a–d. The ester 4c was
transferred into the corresponding amide using 25%
aqueous ammonia and acidification of the crude pro-
duct yielded the free piperazine 5e in 54% over all.
The 3-nitro-chromenone 4d was used in further transfor-
mations as indicated in Scheme 2.
dose dependently increases extra-cellular 5-HT levels in
rat frontal cortex and ventral hippocampus.13 The selec-
tive serotonin re-uptake inhibitors fluvoxamine, fluoxe-
tine, citalopram and paroxetine were also shown to
significantly enhance 5-HT levels in certain brain areas
of the rat (Chart 2).
However, the increase induced by vilazodone is signifi-
cantly larger than that elicited by fluoxetine.13 In
the course of our research of vilazodone, we disco-
vered equally potent aromatic systems like the unsubsti-
tuted chromenones 3-{4-[4-(2-oxo-2H-1-benzopyran-
6-yl)-1-piperazinyl]-butyl}-1H-indole-5-carbonitrile 8i
([IC50 nM] 5-HT1A = 0.1; 5-HT RUI = 0.7; D2 = 740)
and its fluorine analogue 6-{4-[4-(5-fluor-3-indolyl)-but-
The nitro group was hydrogenated to the amine 4f at a
palladium charcoal contact in 70% yield. Deprotection
of 4f under acidic conditions yielded the 3-amino-6-pi-
perazinyl chromenone 5f. To generate the piperazines
5g and 5h, the amine 4f was acylated with pivaloyl
chloride or ethyl chloro formate, respectively, before
acidic deprotection.
yl]-1-piperazinyl}-2H-1-benzopyran-2-one
([IC50 nM]
NMe2
F
O
N
NH2
OMe
O
The chromenones 5i, 5j and 5k were accessible in two
steps (Scheme 3).
NC
F3C
F3C
Citalopram
Fluvoxamine
Commercially available 6-nitro chromenones (6i, 6j)
were hydrogenated to the corresponding amino deriva-
tives using Raney Nickel in 53% (7i) and 73% (7j) yield,
respectively. The piperazines were prepared in 48% (5i),
65% (5j) and 53% (5k)14 yield by stirring the amine inter-
mediate with bis-(2-chloro ethyl)-ammonium chloride in
NMP (7i, 7j) or chloro benzene (7k). The piperazines
were used to substitute the halogen of 3-(4-halo butyl)-
NH
O
O
O
NHMe
O
F
Fluoxetine
Paroxetine
Chart 2. Established 5-HT re-uptake inhibitors.
OH
O
R
O
OH
H
CHO
N
a.
BOC
b., c.
+
N
N
CHO
N
N
BOC
1
N
R'
d.
Br
4; R' = BOC 5; R' = H
4/5a; R = CN
2
3
4/5b; R = COMe
4/5c; R = CO2Et
4/5d; R = NO2
4/5e; R = CONH2
Scheme 1. Reagents and conditions: (a) Sodium tert-butylat; chloro-(di-2-norbornyl phosphino)-(20-dimethyl-amino-1,10-biphenyl-2-yl)-palla-
dium(II), toluene; (b) malononitrile or RCH2CO2Et piperidine, acetic acid; (c) HCl, EtOH; (d) Pd/C, H2, EtOH; (d) (i) 25% NH3/water, (ii) HCl, EtOH.

T. Heinrich et al. / Bioorg. Med. Chem. 12 (2004) 4843–4852
4845
a.
b., c.
HN
O
O
O
O
O
O
R
N
H
NO2
NH2
N
N
N
N
N
4f
BOC
4d
BOC
5f (R = H2; only c.)
5g (R = COtBu)
5h (R = CO2Et)
Scheme 2. Reagents and conditions: (a) 5% Pd/C, H2, EtOH; (b) RCOCl, base, dichloromethane; (c) HCl, EtOH.
O
O
O
O
R
R
O
O
R
N
a.
b.
R'
R'
O2N
H2N
R'
HN
7i (R = H; R' = H)
6i (R = H; R' = H)
5i (R = H; R' = H)
7j (R = OH; R' = H)
6j (R = OH; R' = H)
5j (R = OH; R' = H)
5k (R = H; R' = NHAc)
7k (R = H; R' = NHAc)
Scheme 3. Reagents and conditions: (a) Raney-Ni, H2, MeOH; (b) bis-(2-chloro ethyl)-ammonium chloride, NMP.
1H-indole-5-carbonitrile as previously described.15
Some piperazines (5e, 5i) reacted sufficiently with the
4-chloro derivative to the corresponding products 8e
and 8i, respectively. For the other piperazines 5a, 5b,
5f, 5g, 5h and 5k the 4-chloro building block had to
be transferred into its 4-iodo analogue to succeed in
the substitution reaction to 8a, 8b, 8f, 8g, 8h and 8k.
The trans-halogenation was done under Finkelstein con-
ditions.16 In this special case, it proved to be crucial to
add 6equiv of sodium iodide in portions in 24h intervals
to the solution of the chloro-derivative in acetone and to
complete the reaction quantitatively within 14 days.
Elevated temperature to accelerate the reaction progress
and attempts to purify the iodide caused product
degradation.
coupling the chromenone moiety with the indole build-
ing block.
Because of the insufficient reactivity of 5c with both of
the two 4-halo-butyl-indoles the synthesis of 8c was per-
formed as indicated in Scheme 5.
The iodide 9 was substituted with 10, the deprotected
piperazine of 3, to the intermediate 11 which was cy-
clised to the chromenone 8c under standard Knoevena-
gel conditions.
2.2. Biology
The affinity of the compounds towards the dopamine
D2-receptor subtype was evaluated by their ability to
displace [3H]spiperone (dopamine antagonist)17 in rat
striatal membranes or cloned human receptors. Seroton-
ergic activity (5-HT1A) of the compounds was measured
in rat hippocampal membranes with [3H]-8-OH-DPAT
as ligand.18 The intrinsic activity of the compounds such
The amine 8f was acylated with methyl chloroformate
to the carbamate 8l and the acetamide 8k was hydro-
lysed within 3h in refluxing 1N HCl to the hydroxy
chromenone 8j. Scheme 4 summarises how all the
chromenones but one (8c, vide infra) were prepared by
R'
R'
N
NC
NC
O
O
O
O
N
N
(CH2)4Cl/I
HN
+
R
R
N
N
H
H
8a,b,e-m
Scheme 4. A generic visualisation of the synthesis of compounds 8.
OH
N
CHO
OH
N
I
NC
NC
a.
b.
8c
N
CHO
+
HN
N
H
N
H
9
10
11
Scheme 5. Reagents and conditions: (a) acetonitrile, N-ethyl diisopropyl amine; (b) diethyl malonate, piperidine, glacial acetic acid.

4846
T. Heinrich et al. / Bioorg. Med. Chem. 12 (2004) 4843–4852
Table 2. Ex vivo 5-HT up-take and in vitro intrinsic activity at the
5-HT1A receptor
as 5-HT1A agonists was tested in GTPcS assays with
recombinant human 5-HT1A receptors stably expressed
in membranes of CHO cells.19 Re-uptake inhibition of
[3H]-5-HT was evaluated in rat synaptosomes in vitro20
and ex vivo after p-chloro amphetamine induced 5-HT
depletion (PCA assay) in rats.21 The measurement of
cortical serotonin levels in rat brain in vivo were done
via microdialysis.22 The 5-HT1A agonistic in vivo acti-
vity was determined in the rat ultrasonic vocalisation
test.12,23
No
PCA [ED50 mg/kg]a
GTPcS [EC50 SEM (n) (nM/L)]b
8e
8f
8g
8h
8i
>3*
1.7**
0.8 0.2 (4)
1
30
0.7 (4)
8 (4)
>1*
1.1**
18
2.5
11 (4)
0.9 (4)
0.05*/0.49**
>9*
8j
n.d.
8k
8l
0.09*/0.39**
n.d.
4.3
6.8
0.2 (3)
0.5 (3)
The only compound with subnanomolar 5-HT1A bin-
ding affinity described in this work is the unsubstituted
chromenone 8i but most other derivatives show accept-
able single digit nanomolar affinity (Table 1).
*s.c.; **p.o.; n.d. = not determined.
a Results are the means of 4–8 rats per dose.
b Results are expressed as means SEM of (n) independent
determinations.
The bulky tert-butyl amide 8g shows the lowest 5-HT1A
receptor affinity. Concerning low D2 binding and very
strong re-uptake inhibition 8f, 8i and 8k are the most
interesting compounds. Of course the phenol 8m should
be considered cautiously because of its possible phase II
metabolism and no further work was initiated. Because
of their promising in vitro data a couple of compounds
were measured ex vivo in the PCA assay and the intrin-
sic activity was validated for the chromenones as shown
in Table 2.
5
Vehicle
(n=33)
Vilazodone,
1 mg/kg i.p. (n=10)
Compound 8i, 1 mg/kg i.p. (n= 8)
Compound 8k, 1 mg/kg i.p. (n= 8)
*
4
3
2
1
0
*
*p<0.05 vs. Vilazodone
*
*
*
*
*
Injection
*
Though the ethyl carbamate (8h) and the tert-butyl
amide (8g) are slightly less active all compounds con-
firmed expectations concerning their intrinsic activity
as full agonist. The unsubstituted chromenone 8i, the
amine 8f and its acetamide analog 8k were examined
in more detail. The microdialysis results shown in the
following figures indicate their potential to increase 5-
HT levels in the cortex of freely moving rats.
-50
0
50
100
150
200
Time (min)
Figure 1. Microdialysis of vilazodone in comparison to 8i and 8k. pg/
sample against time. * Marks statistical significance of these results.
pronounced as observed with vilazodone. In contrast,
the acetamide 8k enhanced 5-HT comparably to vilazo-
Figure 1 shows that at a similar dose, the increase in
extracellular cortical 5-HT levels induced by 8i is less
Table 1. 5-HT re-uptake inhibition (5-HT RUI) and receptor binding [IC50 SEM (nM)]
R
O
O
NC
N
N
N
H
R'
No
R
R0
5-HT1A
5-HT RUI
D2
75
8a
8b
8c
8e
8f
CN
COCH3
CO2Et
H
H
H
H
H
H
H
H
H
H
H
2.4
0.8
n.d.
5.7
3.9
3.3
2.9
6.5
19
3.1
1.4
0.3
0.7
0.4
7
37
n.d.
n.d.
46
3.9
2.9
3.1
56
0.4
1.1
1.2
32
CONH2
NH2
180
520
220
130
740
36
242
69
8g
8h
8i
NHCOC(CH3)3
NHCO2Et
H
9.1
0.1
5.3
2.5
2.4
3.1
2.9
0.05
2.5
0.6
0.8
1.2
6
4
17
0.7
2.1
2.9
4.5
0.2
0.5
0.6
1.6
1.5
0.1
364
15
8j
OH
8k
8l
NHCOCH3
NHCO2CH3
H
950
100
560
505
29
8m
OH
266
n.d. = not determined; IC50 values were obtained from 5 to 10 concentrations of the compound, each in triplicate, and are defined as the concen-
tration (nM) resulting in 50% inhibition of binding.

T. Heinrich et al. / Bioorg. Med. Chem. 12 (2004) 4843–4852
4847
lated that some pharmacokinetic issues are responsible
for the observed discrepancy.
5
4
3
2
1
0
Vehicle
(n=33)
Compound 8k, 0.3 mg/kg i.p. (n= 9)
Compound 8f, 0.3 mg/kg i.p. (n= 5)
*p<0.05 vs. 8k
To extend our knowledge about the chromenones, the
urethane 8h was tested in the microdialysis (Fig. 3).
*
*
The ethyl carbamate 8h does not cause such a fast sero-
tonin increase like vilazodone or the other nitrogen
derivatives discussed in Figures 1 and 2 but at a dose
of 1mg/kg it reaches a high (ꢀ280% bl) and long lasting
5-HT level (230% bl; 180min).
Injection
3. Conclusion
-50
0
50
100
150
200
With these findings we can conclude that the chrome-
nones, especially 3-piperazino chromenones linked to
the 3-butyl-1H-indole-5-carbonitrile scaffold, are able
to boost central serotonin to higher and longer lasting
levels in rat brain than it was possible with vilazodone.
Clinical evaluation of one of these compounds has to
verify if the property of increasing serotonin levels as
seen in certain rat brain areas results in a significant
antidepressant effect in humans and can cause a faster
onset of action compared to the established
antidepressants.
Time (min)
Figure 2. Microdialysis of 8f and 8k. pg/sample against time. * Marks
statistical significance of these results.
done. However, the duration of the effect is longer. In
comparison to vilazodone, 5-HT levels remain signifi-
cantly increased to ꢀ600% basal level at 180min post-
administration.
In line with the high 5-HT1A agonistic activity of 8i, the
unsubstituted chromenone potently inhibited ultrasonic
vocalisation in vivo after s.c. (ED50 0.5mg/kg) but was
much less potent after p.o. (ED50 9mg/kg) administra-
tion indicating poor bio-availability. The acetamide 8k
lacked any activity in the ultrasonic vocalisation test
up to 3mg/kg s.c. though the compound binds with sin-
gle digit nanomolar affinity to the 5-HT1A receptor. The
comparison in Figure 2 shows that at similar doses the
unsubstituted amine 8f is slightly more potent with re-
gard to peak 5-HT levels than the amide 8k already
mentioned in Figure 1. In these experiments 8f reached
ꢀ600% basal level 60min post-treatment. The contro-
versial results found for 8k in the ultrasonic vocalisation
assay and the microdialysis cannot be explained on the
basis of the available information. It can just be specu-
4. Experimental
4.1. General methods
Melting points were determined on a Buchi 535 melting
¨
point apparatus and are uncorrected. IR, H NMR and
1
mass spectra are in agreement with the structures and
were recorded on a Bruker IFS 48 IR spectrophoto-
meter, a Bruker AMX 300MHz NMR spectrometer
(TMS as an internal standard), and vacuum generators
VG 70-70 or 70-250 at 70eV, respectively. Elemental
analyses (obtained with a Perkin–Elmer 240 BCHN ana-
lyser) were within 0.4% of theoretical values. All reac-
tions were followed by TLCcarried out on Merck
KGaA F254 silica gel plates. Solutions were dried over
Na SO and concentrated with a Buchi rotary evapora-
2
¨
4
tor at low pressure. The obtained crystalline material
was recrystallised from EtOH.
5
Vehicle
Vilazodone,
(n=33)
mg/kg i.p. (n=10)
1
Compound 8h, 0.3 mg/kg i.p. (n= 6)
Compound 8h, 1 mg/kg i.p. (n= 9)
4
3
2
1
0
4.2. Building block 3
*p<0.05 vs. Vilazodone
*
*
*
*
Toluene (250mL) was degassed and charged with
725mg (3,2mmol) Pd(II)acetate and 445mg (2.2mmol)
tri tert-butyl phosphine. To the red suspension 25.5g
(125mmol) 5-bromo-2-hydroxy-benzaldehyde 2, 26.7g
(144mol) tert-butyl 1-piperazine carboxylate 1 and
26.5g (276mmol) sodium tert-butylate were added lea-
ding to a colour change to yellow. After 2h at 60ꢁC
500mL of water were poured into the brown suspension.
After adjusting the pH to 3 with acetic acid the organic
phase was extracted three times with ethyl acetate. The
combined organic phases were dried and after evapora-
tion of the solvent the residue was purified over silica gel
with ether/heptane to yield 19.3g (50%) yellow crystals 3
*
*
Injection
*
*
-50
0
50
100
150
200
Time (min)
Figure 3. Different doses of 8h. pg/sample against time. * Marks
statistical significance of these results.

4848
T. Heinrich et al. / Bioorg. Med. Chem. 12 (2004) 4843–4852
tert-butyl (4-(3-formyl-4-hydroxy-phenyl)-piperazine-1-
carboxylate acid ester, 99.6% purity, calibrated HPLC);
1H NMR (DMSO-d6) d 10.41 (s, 1H), 7.58 (br s, 1H),
7.32 (dd, 1H, J = 3.1Hz and J = 9.1Hz), 7.23 (d, 1H,
J = 3.1Hz), 7.06 (d, 1H, J = 9.1Hz), 3.46 (m, 4H),
3.03 (m, 4H), 1.42 (s, 9H).
yellow crystals 7i hydrochloride. Mp >280ꢁC. Anal.
(C9H7NO2*HCl) calcd C 54.7; H 4.1; N 7.1; Cl 17.9;
found: C55.1; H 4.2; N 7.2; Cl 17.3.
4.3.5. 6-Piperazin-1-yl-chromen-2-one (5i). A solution of
49.9g (0.28mol) bis-(2-chloro ethyl)-ammonium chlo-
ride in 50mL NMP was added dropwise at 130ꢁCto a
solution of 22.1g (0.11mol) 7i in 110mL NMP. After
60h, TLCindicated complete conversion and the
solvent was removed. The residue was washed with ethyl
ester and crystallised from 2-propanol. The green
crystals were washed with acetone and diethyl ether
and dried giving 14.4g (48%) 5i hydrochloride. Mp
293–295ꢁC. Anal. (C13H14N2O2*HCl) calcd C 58.5; H
5.7; N 10.5; Cl 13.3; found: C 58.4; H 5.9; N 12.7; Cl 13.7.
4.3. Chromenone intermediates
4.3.1. 2-Oxo-6-piperazin-1-yl-2H-chromene-3-carboni-
trile (5a). A suspension of 20g (65mmol) 3 and 4.7g
(71mmol) malononitrile in 325mL aqueous 0.05M
NaHCO3 were heated to 80ꢁCfor 15min. The resulting
crystals were filtered at room temperature, washed with
water and dried, yielding 21.8g (94%) tert-butyl 4-(3-
cyano-2-oxo-2H-chromen-6-yl)-piperazine-1-carboxylate
acid ester 4a. The urethane was deprotected without
further purification.
4.4. Indole intermediate
4.4.1. 3-(4-Iodo-butyl)-1H-indole-5-carbonitrile (9). Un-
der nitrogen 600g NaI was added to a solution
of 150g 3-(4-chloro-butyl)-1H-indole-5-carbonitrile11 in
500mL acetone in six portions in 24h intervals. The
insoluble part was filtered off and the solution evapo-
rated to dryness. The crude product was partitioned be-
tween water and ethyl acetate, the organic phase was
dried and again evaporated. The solid material was crys-
tallised from hexane/diethyl ether, washed with diethyl
ether and dried, yielding 184.1g (88%) 9. Mp 117–
119ꢁC.
A suspension of 1.78g (5mmol) 4a in 50mL ethanol and
2mL HCl saturated ethanol were heated to 80ꢁCfor 1h.
The resulting crystals were filtered, washed with ethanol
and dried, yielding 1.33g reddish crystals 5a hydrochlor-
ide. Mp 210ꢁC(decomp.) 1H NMR (DMSO-d6) d 9.45
(br s, 2H); 8.81 (s, 1H); 7.55 (dd, 1H, J = 2.8Hz,
J = 9.4Hz); 7.43 (d, 1H, J = 9.4Hz); 7.33 (d, 1H,
J = 2.8Hz); 3.42 (m, 4H); 3.24 (m, 4H); Anal.
(C14H13N3O2*HCl) calcd C 57.6; H 4.8; N 14.4; found:
C54.1; H 5.0; N 13.7.
4.3.2. tert-Butyl 4-(3-acetyl-2-oxo-2H-chromen-6-yl)-pip-
erazine-1-carboxylate (4b).
4.5. Indolyl-chromenones
A
solution of 6.13g
(20mmol) 3, 2.38mL (24mmol) piperidine, 1.37mL
glacial acid and 3.04mL (24mmol) 3-oxo-butyrate acid
ethyl ester in 100mL acetonitrile was refluxed for
30min when TLCindicated complete conversion. After
the suspension had cooled to room temperature, the
product was filtered and dried yielding 6.5g (87%) red
crystals 4b. Mp 187–189ꢁC; 1H NMR (DMSO-d6) d
8.54 (s, 1H), 7.47–7.32 (m, 3H), 3.48 (m, 4H), 3.13
(m, 4H), 2.54 (s, 3H), 1.43 (s, 9H); Anal.
(C20H24N2O5) calcd C64.5; H 6.4; N 7.5; found: C
64.2; H 6.3; N 7.5.
4.5.1. 3-{4-[4-(3-Cyano-2-oxo-2H-chromen-6-yl)-piper-
azin-1-yl]-butyl}-1H-indole-5-carbonitrile (8a). A solu-
tion of 2g (7mmol) 5a, 2.4g (7mmol) 9 and 3.1mL
(23mmol) triethyl amine in 100mL acetonitrile was
refluxed for 12h. The solvent was removed and the
residue partitioned between ethyl ester and water. After
the usual procedure, 2g crude product were purified by
chromatography yielding 0.7g red crystals. These were
dissolved in 120mL acetone and 1N HCl was added
dropwise until pH4 was reached. After 12h, yellow crys-
tals were filtered and washed with acetone and ether
yielding 0.7g (21%) 8a. Mp 283–285ꢁC; 1H NMR
(DMSO-d6) d 11.48 (br s, 1H), 10.86 (br s, 1H), 8.81
(s, 1H), 8.10 (s, 1H), 7.56 (dd, 1H, J = 2.8 and
J = 9.2Hz), 7.52 (d, 1H, J = 8.4Hz), 7.42 (m, 3H), 7.33
(d, 1H, J = 2.8Hz), 3.82 (br d, 2H, J = 11.0Hz), 3.58
(br d, 2H, J = 10.0Hz), 3.25–3.15 (m, 7H), 2.78 (t, 2H,
J = 7.4Hz) 1.82 (m, 2H), 1.71 (m, 2H). Anal.
(C27H25N5O2*HCl*0.75 H2O) calcd C64.7; H 5.5; Cl
7.1; N 14.0; found: C64.7; H 5.2; Cl 7.1; N 13.8.
4.3.3. 3-Acetyl-6-piperazin-1-yl-chromen-2-one (5b). A
suspension of the above prepared urethane 4b (1.23g;
3mmol) in 30mL ethanol was treated with 1.23mL
HCl-saturated ethanol for 2h. The resulting crystals
were filtered, washed with ethanol and ether and dried,
yielding 0.8g (86%) 5b hydrochloride. Mp 308ꢁC(de-
1
comp.); H NMR (DMSO-d6) d 9.22 (br s, 2H); 8.57
(s, 1H); 7.51 (d, 1H, J = 2.0Hz); 7.49 (d, 1H,
J = 2.0Hz); 7.42 (s, 1H); 3.33 (m, 8H); 2.58 (s, 3H);
Anal. (C15H16N2O3*HCl) calcd C 58.4; H 5.5; N 9.1;
found: C57.9; H 5.8; N 8.8.
4.5.2. 3-{4-[4-(3-Acetyl-2-oxo-2H-chromen-6-yl)-piper-
azin-1-yl]-butyl}-1H-indole-5-carbonitrile (8b). A suspen-
sion of 1.54g (5mmol) 5b, 1.78g (5mmol) 9 and 2.9mL
(17mmol) triethyl amine in 150mL acetonitrile was ref-
luxed for 12h. The solution was concentrated in vacuo
and stirred with water and ethyl acetate. The organic
phase was evaporated to dryness and after chromatogra-
phy 0.8g (33%) of the free base was isolated. The base
was redissolved in 80mL acetone and 1N HCl was
added until pH4 was reached. The resulting yellow crys-
4.3.4. 6-Amino-chromen-2-one (7i). A solution of 41g
(0.21mol) 6i was dissolved in 450mL methanol and
added to 10g methanol–wet Raney Nickel. After 20h
and the addition of 3mol H2, TLCindicated complete
conversion. The solvent was partially removed and
HCl-saturated ethanol was added. The precipitating
salt was filtered and dried yielding 22.2g (53%)

T. Heinrich et al. / Bioorg. Med. Chem. 12 (2004) 4843–4852
4849
tals were filtered, washed with acetone and ether and
dried yielding 0.8g (95%) 8b hydrochloride; Mp 245–
247ꢁC; H NMR (DMSO-d6) d 11.46 (s, 1H), 10.61 (s,
1H), 8.57 (s, 1H), 8.11 (s, 1H), 7.51 (m, 3H), 7.41 (m,
3H), 3.83 (br d, 2H, J = 10.8Hz), 3.59 (br d, 2H,
J = 10.8Hz), 3.18 (m, 6H), 2.79 (t, 2H, J = 7.4Hz) 1.81
(m, 2H), 1.71 (m, 2H); Anal. (C28H28N4O3*HCl) calcd
C66.6; H 5.8; N 11.1; Cl 7.0; found: C65.4; H 5.7; N
11.1; Cl 7.4.
(m, 3H), 7.44–7.39 (m, 3H), 3.84 (br s, 4H), 3.16 (m,
6H), 2.78 (m, 2H), 1.80 (m, 2H), 1.70 (m, 2H); Anal.
(C27H27N5O3*HCl) calcd C 64.1; H 5.6; Cl 7.0; N
13.8; found: C63.7; H 5.1; Cl 7.5; N 13.1.
1
4.5.5. 3-{4-[4-(3-Amino-2-oxo-2H-chromen-6-yl)-piper-
azin-1-yl]-butyl}-1H-indole-5-carbonitrile (8f). A solution
of 100mg (0.31mmol) 5f dihydrochloride, 102mg
(0.31mmol) 9 and 122mg (0.94mmol) ethyl diisopro-
pyl-amine in 10mL acetonitrile was refluxed for 18h.
The mixture was evaporated to dryness and the crude
product was purified by chromatography directly to ob-
4.5.3. Ethyl 6-{4-[4-(5-cyano-1H-indol-3-yl)-butyl]-pip-
erazin-1-yl}-2-oxo-2H-chromene-3-carboxylate (8c). A
solution of 42.3g (0.14mol) 3 in 2-propanol (500mL)
was treated with saturated HCl solution in ethanol
(40mL) and refluxed overnight. The amount of solvent
was reduced to 100mL and the precipitating crude
2-hydroxy-5-piperazin-1-yl-benzaldehyde 10 hydrochlor-
ide was filtered off and dried. A solution of 33.4g
1
tain a colourless solid 8f (33mg, 24%). Mp 154ꢁC; H
NMR (DMSO-d6) d 11.35 (s, 1H), 8.07 (s, 1H), 7.49
(d, 1H, J = 8.4), 7.39 (d, 1H, J = 8.4Hz), 7.33 (s, 1H),
7.12 (d, 1H, J = 8.6Hz), 6.90–6.83 (m, 2H), 6.65 (s,
1H), 5.58 (s, 2H), 3.09 (br s, 4H), 2.74 (t, 2H,
J = 7.5Hz), 2.39 (br s, 4H), 2.36 (t, 2H, J = 7.5Hz),
1.68 (tt, 2H, J = 7.5Hz), 1.52 (tt, 2H, J = 7.5Hz); Anal.
(C26H27N5O2) calcd C70.7; H 6.2; N 15.9; found: C
70.2; H 6.4; N 15.5.
(0.14mol) 10, 44.7g (0.14mol)
9
and 58.7mL
(0.35mol) diethyl isopropyl-amine in 1.5L acetonitrile
was refluxed for 5h. The solvent was removed and the
residue partitioned between ethyl acetate and water.
After the usual procedure, the crude product was puri-
fied by chromatography and crystallised from ethyl ace-
tate and tert-butyl methyl ether yielding 23.1g (42%)
3-{4-[4-(3-formyl-4-hydroxy-phenyl)piperazin-1-yl]-but-
yl}-1H-indole-5-carbonitrile 11 as colourless crystals. 1H
NMR (DMSO-d6) d 11.36 (s, 1H), 10.21 (s, 1H), 10.18
(br s, 1H), 8.07 (s, 1H), 7.50 (d, 1H, J = 8.4), 7.40 (dd,
1H, J = 8.4Hz, J = 1.5Hz), 7.34 (d, 1H, J = 1.5Hz),
7.26 (dd, 1H, J = 9.0Hz, J = 3.0Hz), 7.12 (d, 1H,
J = 3.0Hz), 6.91 (d, 1H, J = 9.0Hz), 3.31 (br s, 2H),
3.04 (br s, 4H), 2.75 (t, 2H, J = 7.3Hz), 2.68–2.53 (m,
4H), 1.68 (tt, 2H, J = 7.3Hz), 1.53 (br s, 1H).
4.5.6. N-(6-{4-[4-(5-Cyano-1H-indol-3-yl)-butyl]-piper-
azin-1-yl}-2-oxo-2H-chromen-3-yl)-2,2-dimethyl-propion-
amide hydrochloride (8g).
A solution of 244mg
(0.74mmol) 5g, 360mg (1.11mmol) 9 and 287mg
(2.22mmol) ethyl diisopropyl-amine in 15mL NMP
was heated at 110ꢁCfor 2days. The mixture was poured
into ice water and the precipitate was filtered off. The
crude product was purified by chromatography. The ob-
tained colourless solid was transferred to the hydrochlo-
ride salt using a hydrochloric acid isopropanol solution,
yielding 36mg (9%) 8g hydrochloride as colourless solid.
1
Mp 100ꢁC; H NMR (DMSO-d6) d 11.43 (s, 1H), 9.98
(br s, 1H), 8.57 (s, 1H), 8.49 (s, 1H), 8.10 (s, 1H), 7.51
(d, 1H, J = 8.4), 7.41 (dd, 1H, J = 8.4Hz, J = 2.9Hz),
7.38 (d, 1H, J = 2.0Hz), 7.35 (d, 1H, J = 9.0Hz), 7.30
(d, 1H, J = 2.8Hz), 7.23 (dd, 1H, J = 9.2Hz,
J = 2.8Hz), 3.84 (d, 2H, J = 12.0Hz), 3.57 (d, 2H,
J = 12.0Hz), 3.22–3.04 (m, 6H), 2.78 (t, 2H,
J = 7.2Hz) 1.81–1.66 (m, 4H), 1.25 (s, 9H); Anal.
(C31H35N5O3*HCl*H2O) calcd C64.2; H 6.6; N 12.1;
Cl 6.1; found: C 64.1; H 6.7; N 12.0.
A mixture of 100mg (0.25mmol) 11, 38mL (0.25mmol)
diethyl malonate, 25mL (0.25mmol) piperidine and
14mL (0.25mmol) acetic acid in ethanol (5mL) was ref-
luxed for 18h. Water (5mL) was added and the formed
precipitate was filtered off and dried. 110mg (90%) of
pure colourless solid of 8c were obtained. Mp 103ꢁC;
1H NMR (DMSO-d6) d 11.35 (s, 1H), 8.65 (s, 1H),
8.07 (s, 1H), 7.49 (d, 1H, J = 8.4), 7.43–7.35 (m, 3H),
7.33 (d, 1H, J = 1.7Hz), 7.30 (d, 1H, J = 9.0Hz), 4.29
(q, 2H, J = 7.1Hz), 3.16 (m, 4H), 2.74 (t, 2H,
J = 7.3Hz), 2.54–2.48 (m, 4H), 2.38 (t, 2H, J = 7.3Hz),
1.68 (tt, 2H, J = 7.2Hz), 1.53 (tt, 2H, J = 7.2Hz), 1.31
(t, 3H, J = 7.1Hz); Anal. (C29H30N4O4) calcd C69.9;
H 6.1; N 11.2; found: C69.6; H 6.3; N 11.0.
4.5.7. Ethyl (6-{4-[4-(5-cyano-1H-indol-3-yl)-butyl]-pip-
erazin-1-yl}-2-oxo-2H-chromen-3-yl)-carbamate, (8h). A
solution of 162mg (0.51mmol) 5h (the dihydrochloride
salt was dissolved in 2N NaOH solution and extracted
with ethyl acetate three times, the combined organic lay-
ers were dried with sodium sulfate, filtered and evapo-
rated to dryness), 166mg (0.51mmol) 9 and 200mg
(1.54mmol) ethyl diisopropyl-amine in 10mL acetonitri-
le was refluxed for 2days. The solvent was removed and
the residue partitioned between ethyl ester and
water. Theorganiclayerwaspurifiedbychromatography.
The obtained colourless solid was transferred to the
hydrochloride salt using a hydrochloric acid isopropa-
nol solution, yielding 181mg (64%) 8h hydrochloride
4.5.4. 6-{4-[4-(5-Cyano-1H-indol-3-yl)-butyl]-piperazin-1-
yl}-2-oxo-2H-chromene-3-carboxamide (8e). A suspen-
sion of 6.36g (16.4mmol) 5e, 7.64g (32.84mmol) 3-(4-
chloro-butyl)-1H-indole-5-carbonitrile11
and
6.4g
(49.2mmol) ethyl diisopropyl-amine in 200mL acetonit-
rile was refluxed for 12h. The resulting precipitate was
filtered off and the liquid phase evaporated. The crude
product was purified via chromatography and the result-
ing colourless foam was transferred into the correspond-
ing hydrochloride with HCl saturated ethanol yielding
1
as colourless solid. Mp 220ꢁC; H NMR (DMSO-d6) d
11.45 (s, 1H), 10.41 (br s, 1H), 8.89 (s, 1H), 8.20 (s,
1H), 8.10 (s, 1H), 7.51 (d, 1H, J = 8.4), 7.41 (dd, 1H,
J = 8.4Hz, J = 1.5Hz), 7.39 (d, 1H, J = 1.5Hz), 7.32
(d, 1H, J = 9.0Hz), 7.28 (d, 1H, J = 2.8Hz), 7.20 (dd,
1
800mg (10%) of 8e hydrochloride. Mp 280–282ꢁC; H
NMR (DMSO-d6) d 11.48 (br s, 1H), 10.50 (br s, 1H),
8.81 (s, 1H), 8.10 (s, 2H), 7.89 (br s, 1H), 7.52–7.49

4850
T. Heinrich et al. / Bioorg. Med. Chem. 12 (2004) 4843–4852
1H, J = 9.0Hz, J = 2.8Hz), 4.17 (q, 2 H. J = 7.1Hz),
3.82 (d, 2H, J = 8.5Hz), 3.55 (d, 2H, J = 8.5Hz), 3.21–
3.10 (m, 6H), 2.78 (t, 2H, J = 7.2Hz) 1.83–1.66 (m,
4H), 1.25 (t, 3H, J = 7.1Hz); Anal. (C29H31N5O4*HCl)
calcd C63.3; H 5.9; N 12.7; Cl 6.4; found: C63.1; H
6.1; N 12.8; Cl 6.4.
4.5.11. Methyl (6-{4-[4-(5-cyano-1H-indol-3-yl)-butyl]-
piperazin-1-yl}-2-oxo-2H-chromen-3-yl)-carbamate (8l).
To a solution of 518mg (1.17mmol) 8f and 0.11mL
(1.41mmol) pyridine in 10mL dichloromethane was
added 0.11mL (1.41mmol) methyl chloroformate drop-
wise at 0ꢁC. The mixture was stirred at 0ꢁCfor 1h and
12h at rt, poured into ice water and the colourless pre-
cipitate was filtered off yielding 57mg (10%) 8l. Mp
4.5.8. 3-{4-[4-(2-Oxo-2H-1-benzopyran-6-yl)-1-pipera-
zinyl]-butyl}-indol-5-carbonitrile (8i). A suspension of
2.33g (10mmol) 3-(4-chloro-butyl)-1H-indole-5-carbo-
nitrile,11 66g (10mmol) 5i, 2.8mL (20mmol) triethyl
amine and 1.38g (10mmol) K2CO3 in 1L acetonitrile
was refluxed for 36h. The solvent was removed and
the residue partitioned between ethyl ester and water.
After extraction of the aqueous phase the organic phase
was dried and concentrated. The residue was purified by
chromatography and crystallised from diethyl ether
yielding 1.1g yellow crystals 8i. Mp 135–137ꢁC; 1H
NMR (DMSO-d6) d 10.64 (br s, 1H), 9.91 (br s, 1H),
7.35 (s, 1H), 7.24 (d, 1H, J = 8.7Hz), 6.83 (d, 1H,
J = 7.6Hz), 6.72 (m, 2H), 6.69 (s, 2H), 6.62 (s, 1H),
5.87 (d, 1H, J = 8.7Hz), 3.45 (br d, 2H, J = 10.3Hz),
3.23 (br d, 2H, J = 10.3Hz), 2.88 (m, 6H), 2.52 (t, 2H,
J = 6.4Hz), 1.74–1.52 (m, 4H); Anal. (C26H24N4O2) cal-
cd C73.3; H 6.1; N 13.1; found: C73.4; H 6.1; N 13.1.
1
107ꢁC; H NMR (DMSO-d6) d 11.35 (s, 1H), 8.97 (s,
1H), 8.17 (s, 1H), 8.07 (s, 1H), 7.49 (d, 1H, J = 8.4),
7.40 (d, 1H, J = 8.4Hz), 7.33 (s, 1H), 7.25 (d, 1H,
J = 9.0Hz), 7.17–7.10 (m, 2H), 3.71 (s, 3H), 3.19–3.12
(m, 4H), 2.74 (t, 2H, J = 7.5Hz), 2.52–2.46 (m, 4H),
2.40–2.33 (m, 2H), 1.68 (tt, J = 7.5Hz), 1.53 (tt,
J = 7.5Hz); Anal. (C28H29N5O4) calcd C67.3; H 5.9;
N 14.0; found: C66.9; H 6.3; N 13.7.
4.6. Pharmacological methods
4.6.1. Receptor binding assays. The affinity of com-
pounds for dopamine receptors were determined in a
total volume of 2mL containing 2–3nM [3H]ADTN or
0.1nM [3H]spiperone resp., and 0.-3-1.0mg of protein
of rat striatal membranes per mL. Assay buffer was
50mol/L Tris/HCl, pH7.1, 120mmol/L NaCl, 5mmol/
L KCl, 1mmol/L CaCl2, 1mmol/L MgCl2, and 0.1%
ascorbic acid in the case of ADTN and 50mmol Tris/
HCl, pH7.7 and 0.1% ascorbic acid in the case of spip-
erone. Incubations were carried out at 37ꢁCfor 15min
and terminated by rapid filtration (Whatman GF/B)
and three washes with ice-cold buffer. Nonspecific bind-
ing was determined in the presence of 1mmol/L (+)-
butaclamol.17,24
4.5.9. 3-{4-[4-(3-Hydroxy-2-oxo-2H-chromen-6-yl)-pip-
erazin-1-yl]-butyl}-1H-indole-5-carbonitrile (8j). One
hundred milligrams (0.19mmol) of 8k were treated with
1N HCl solution (5mL) and refluxed for 3h. The solu-
tion was neutralised with aqueous ammonia, the precip-
itate was collected and purified by chromatography
directly. A colourless solid 8j was obtained (20mg,
24%). Mp 160ꢁC; 1H NMR (DMSO-d6) d 11.39 (s,
1H), 8.07 (s, 1H), 7.49 (d, 1H, J = 8.4), 7.39 (dd, 1H,
J = 8.4Hz, J = 1.3Hz), 7.33 (d, 1H, J = 1.3Hz), 7.13
(d, 1H, J = 9.5Hz), 6.93–6.87 (m, 2H), 6.82 (s, 1H),
3.09 (t, 4H, J = 4.6Hz), 2.74 (t, 2H, J = 7.4Hz), 2.50–
2.46 (m, 4H), 2.36 (t, 2H, J = 7.4Hz), 1.67 (tt, 2H,
J = 7.3Hz), 1.52 (tt, 2H, J = 7.3Hz); Anal.
(C26H26N4O3) calcd C70.6; H 5.9; N 12.7; found: C
70.2; H 6.2; N 12.5.
4.7. Microdialysis
Male rats (250–300g body weight) were anesthetised
with pentobarbital (50mg/kg intraperitoneally (i.p.))
and placed in a stereotaxic frame (Stoelting Co., IL,
USA). Body temperature was maintained at 37ꢁCwith
a heated pad and a temperature controller (CMA/105;
CMA/Microdialysis, Stockholm, Sweden). A CMA/11
guide cannula plugged with an obturator was implanted
into the frontal cortex (AP + 3.2, L + 2.5 from bregma,
Vꢁ5.0; Paxinos and Watson, 1986). By using dental
cement and anchoring screws, the guide was fixed to
the skull. Post-operatively, the rats were left to recover
from surgery 24h with free access to food and water.
Then the obturator was removed from the guide cannula
and a microdialysis probe (CMA/11, membrane length
4mm, O.D. 0.25mm) was inserted.
4.5.10. N-(6-{4-[4-(5-Cyano-1H-indol-3-yl)-butyl]-piper-
azin-1-yl}-2-oxo-2H-chromen-3-yl)-acetamide (8k).
A
solution of 12.4g (43mmol) 5k, 13.9g (43mmol) 9 and
11.1g (86mmol) ethyl diisopropyl amine in 150mL
NMP was heated at 120ꢁCfor 3days. The mixture
was poured into ice water and the precipitate was fil-
tered off. The crude product was purified by chromato-
graphy. The obtained colourless solid was transferred to
the hydrochloride salt using a hydrochloric acid isoprop-
anol solution, yielding 3.4g (15%) 8k hydrochloride as
colourless solid. Mp 206ꢁC; 1H NMR (DMSO-d6) d
11.44 (s, 1H), 10.35 (br s, 1H), 9.69 (s, 1H), 8.59 (s,
1H), 8.10 (s, 1H), 7.51 (d, 1H, J = 8.4), 7.41 (dd, 1H,
J = 8.4Hz, J = 1.5Hz), 7.39 (d, 1H, J = 1.5Hz), 7.30
(d, 1H, J = 9.0Hz), 7.28 (d, 1H, J = 2.5Hz), 7.20 (dd,
1H, J = 9.0Hz, J = 2.5Hz), 3.85 (br s, 2H), 3.55 (br s,
2H), 3.21–3.10 (m, 6H), 2.78 (t, 2H, J = 7.2Hz), 2.17,
(s, 3H), 1.82–1.66 (m, 4H); Anal. (C28H29N5O3*HCl*2
H2O) calcd C60.6; H 6.2; N 12.6; Cl 6.4; found: C
61.0; H 6.1; N 12.4; Cl 6.6.
The day after inserting of the microdialysis probe, the
rats were placed in a CMA/120 microdialysis system.
The probes were perfused with filtered DulbeccoÕs phos-
phate buffered saline (composition in mM: CaCl2 · 2
H2O 0.9, MgCl2 · 6 H2O 0.5, KCl 2.7, K2HPO4 1.5,
NaCl 138 and Na2HPO4 8.1, pH7.4) and ascorbic acid
(10lM) at a rate of 1.2lL/min using a microdialysis
pump (CMA/100). The samples were automatically col-
lected by a CMA/140 microfraction collector in 300lL
borosilicate vials and frozen until HPLCanalysis. A
2h stabilisation period was allowed following start of

T. Heinrich et al. / Bioorg. Med. Chem. 12 (2004) 4843–4852
4851
the microdialysis procedure. A 20min sampling regimen
was used throughout the experimental period. After
three basal samples were collected, drugs were adminis-
tered i.p. at a volume of 0.2mL/100g body weight and
the response was followed for another 3h. At the end
of the experiment, brains were removed and cut into
slices following formalin fixation. Probe placement was
verified by visual inspection.
ber. After a 2min time period, a series of at most 10
shocks (trials), 1.8mA for 0.3s, separated by 20s
shock-free intervals, was delivered via the grid floor of
the test chamber. In the shock-free intervals, the occur-
rence of ultrasonic vocalisation was automatically re-
corded, and the duration of ultrasonic vocalisation
was calculated immediately. The priming session was
terminated either when the rat constantly vocalised at
least for 10s on three consecutive trials or after the
10th trial. Rats which did not respond with ultrasonic
vocalisation on three consecutive trials were excluded
from further testing. In the actual test performed on
the next day, each rat received 5 initial shocks (1.8mA
for 0.3s, separated by 20s shock-free intervals) in the
test chamber, and the duration of ultrasonic vocalisa-
tion was recorded during the following 3min period.
Animals were tested 30min after s.c. or p.o. administra-
tion of compounds, respectively. The given values are
the mean of 6–10 animals per dose. The half-maximum
effect was determined from the dose–response curve.
Dialysate concentration of 5-HT was determined using a
reversed phase/ion pair HPLCmethod with electro-
chemical detection. The dialysate was injected automat-
ically into the HPLC system by a HTC-PAL (CTC
Analytics AG, Zwingen, Switzerland) refrigerated auto-
sampler programmed to inject 20lL of dialysate. The
degassed (CMA/260 degasser) mobile phase consisting
of 50mM sodium citrate, 27lM disodium-EDTA,
10mM diethylamine–HCl, 10mM NaCl, 2mM de-
cane–sulfonic acid (sodium salt) and 17.5% (v/v) aceto-
nitrile was delivered at a flow rate of 60lL/min by a
Rheos 2000 LC/MS (Flux Instruments AG, Basel, Swit-
zerland) pump. Analytes were separated on a Ultracarb
RP-18 ODS 30 5lm column (150 · 1mm, Phenomenex,
CA, USA), maintained at ambient temperature. For
electrochemical detection, a BAS LC4C detector (Bioan-
alytical Systems, IN, USA) with a glassy carbon elec-
trode set at a potential of +650mV versus a Ag/AgCl
electrode was used. Retention time (9–11min for 5-
HT), peak areas and peak heights were measured with
a data management software system (ChemServer Tar-
get Software, Falcon Integrator, Hewlett–Packard Com-
pany, NE, USA). Dialysate concentrations of 5-HT
were calculated by comparing peak areas with those of
a range of standard concentrations. Data are presented
as pg/sample (mean S.E.M.). Values were compared
using two-way ANOVA followed by Bonferroni post-
hoc analysis. Significance levels were set at p < 0.05.
4.7.3. Stimulation of [35S]GTPcS binding at cloned
5-HT1A receptors. The effects of different compounds
tested on [35S]GTPcS binding were evaluated according
to a method of Newman-Tancredi et al.27 with modifica-
tions. Membranes of 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 ꢁ70ꢁC. Prior
to use, membranes were thawed and rehomogenised in
assay buffer (MgCl2, NaCl and EDTA in Tris–HCl,
pH7.4). Membranes (ꢀ10lg protein) were incubated
at 37ꢁCfor 30min (shaking water bath) in duplicate in
a total volume of 800lL of buffer containing MgCl2
(3mM), NaCl (120mM), EDTA (0.2mM), GDP
(10lM), [35S]GTPcS (0.1nM), Tris (50mM) and test
compounds. Prior to adding to the incubation mixture,
the test compounds were dissolved in bi-distilled water.
DMSO was used to aid in solubilising certain com-
pounds. Nonspecific binding was defined with 0.1lM
GTPcS. 5-HT was tested as standard in each experiment
at concentrations of 100, 30 and 10nM. Experiments
were terminated by rapid filtration through Whatman
GF/B filters using a Brandel cell harvester. Subse-
quently, the filters were rinsed twice with 5mL ice cold
Tris–HCl and placed in scintillation vials. Radioactivity
was extracted in 4mL scintillation fluid (Ultima GoldR,
Packard Instruments, Frankfurt, Germany) and deter-
mined by liquid scintillation counting. Binding iso-
therms were analysed by nonlinear regression. Agonist
efficacy (=Emax) is expressed relative to that of 5-HT
(=100%), which was tested at a maximally active con-
centration (0.1mM) in each experiment. EC50 values
were defined as the concentration of the compound at
which 50% of its own maximal stimulation was
obtained.
4.7.1. PCA assay. Effects of drugs on PCA (p-chloroam-
phetamine) induced 5-HT depletion in rat hypothalamus
were carried out as previously described.25 Male rats
(135–160g body weight) were used. Brain regions were
dissected out on ice26 and immediately processed for
HPLCanalysis. PCA (5mg/kg i.p. in saline) was given
3h and drugs 3.5 or 5h prior to decapitation. Brain
tissue 5-HT was determined by an automated reversed
24
phase/ion pair, direct injection HPLCmethod
within a 25min run. N-Methyldopamine or N-x-methyl-
serotonin was used as internal standard and the recovery
was >95%.
4.7.2. Ultrasonic vocalisation test. Male rats weighing
180–280g from Charles River (Sulzfeld, Germany) were
used. Ultrasonic vocalisation was measured in a sound-
attenuated test chamber (W 24cm, L 22cm, H 22cm)
with a grid floor for delivery of foot-shock (scrambled
shock of 0.2mA for 0.5s, shocker Getra BN 2002).
Ultrasonic vocalisation was recorded (microphone
4004, Bruel and Kjær) and processed by an interface
(developed at Merck, Darmstadt) to select
22kHz 4kHz signals and to digitise the resulting sig-
nals for automatic processing in a personal computer.
In a priming phase, each rat was placed in the test cham-
Acknowledgements
We thank Jurgen Eckstein, Uwe Eckert and Kurt Schus-
¨
ter for the preparation of the compounds and Herbert
Ziegler for performing pharmacological tests.

4852
T. Heinrich et al. / Bioorg. Med. Chem. 12 (2004) 4843–4852
14. N-(6-Amino-2-oxo-2H-chromen-3-yl)-acetamide 5k is
commercially available (Maybridge).
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