Clozapine derived 2,3-dihydro-1H-1,4- and 1,5-benzodiazepines with D4 receptor selectivity: Synthesis and biological testing

Article abstract of DOI:10.1016/j.bmc.2004.03.023

Novel 4-arylpiperazin-1-yl-substituted 2,3-dihydro-1H-1,4- and 1H-1,5-benzodiazepines and their aza-analogues were synthesized as debenzoclozapine derivatives for evaluation as potential D4-ligands. While K_i values of some of the title compounds came within the range of clozapine, they showed an impressively greater selectivity over other dopamine receptor subtypes, especially D2. For the most promising compounds, intrinsic activity and binding properties to serotonin 5-HT1_A and 5-HT2 were also determined.

Full text of DOI:10.1016/j.bmc.2004.03.023

Bioorganic & Medicinal Chemistry 12 (2004) 2625–2637
Clozapine derived 2,3-dihydro-1H-1,4- and 1,5-benzodiazepines
with D4 receptor selectivity: synthesis and biological testing
*
€
Thomas Hussenether, Harald Hubner, Peter Gmeiner and Reinhard Troschutz
€
Department of Medicinal Chemistry, Emil Fischer Center, Friedrich-Alexander University, Schuhstrasse 19,
D-91052 Erlangen, Germany
Received 11 November 2003; accepted 10 March 2004
Available online 15 April 2004
Abstract—Novel 4-arylpiperazin-1-yl-substituted 2,3-dihydro-1H-1,4- and 1H-1,5-benzodiazepines and their aza-analogues were
synthesized as debenzoclozapine derivatives for evaluation as potential D4-ligands. While Ki values of some of the title compounds
came within the range of clozapine, they showed an impressively greater selectivity over other dopamine receptor subtypes, espe-
cially D2. For the most promising compounds, intrinsic activity and binding properties to serotonin 5-HT1A and 5-HT2 were also
determined.
Ó 2004 Elsevier Ltd. All rights reserved.
1. Introduction
Me
N
The neurotransmitter dopamine plays an important role
in the development of several neurological and psychi-
atric disorders such as schizophrenia,1;2 Huntington’s
disease, and Parkinson’s disease.3 Thus, the investiga-
tion of the dopaminergic system has become an impor-
tant target in research in order to understand the
etiology of these diseases and to find efficient drugs for
their treatment. Making use of modern molecular bio-
logical techniques, five dopamine receptor subtypes
(D1–D5) were characterized and divided into two main
families, the D1-like (including the D1 and D5 subtype)
and the D2-like (D2, D3, and D4).4;5
N
N
Cl
B
A
C
N
H
clozapine 1
Me
N
Me
N
N
N
N
N
Cl
B
B
A
C
N
H
N
H
3
2
Clozapine (1) (Fig. 1), an important atypical neuroleptic
agent, blocks preferentially the D4 receptor with sig-
nificant selectivity versus the D2 subtypes. This is con-
sidered to be one of the main reasons causing almost no
extra pyramidal motoric disturbance. In vivo, supported
by antagonistic effects on serotonin, muscarine, adren-
aline, and histamine receptors, amelioration of positive
and negative psychotic symptoms as well as of treat-
ment-resistant schizophrenia has been observed.6
Unfortunately the therapeutic use of clozapine is limited
by a particular incidence of agranulocytosis (0.05–2%).7
Figure 1.
In the course of our search for new D4 ligands,8 we
planned extensive structure modifications of the tricyclic
clozapine (1) by formal elimination of ring C as well as
ring A giving the bicyclic debenzoclozapines 2 and 3,
respectively.
As their biological testing showed only low D4 affinity
(about 17% displacement of radioligand at a concen-
tration of 10 lM for 2 and about 30% for 3), we decided
to attempt further structural variations. Kulagowski
et al.9 have reported that the attachment of a differently
substituted N-phenylpiperazinylmethyl group in indole
* Corresponding author. Tel.: +49-9131-8523923; fax: +49-9131-852-
2587; e-mail: troschuetz@pharmazie.uni-erlangen.de
0968-0896/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.03.023

2626
T. Hussenether et al. / Bioorg. Med. Chem. 12 (2004) 2625–2637
R1
Cl
R2
H
5a
N
N
Cl
5b CF
H
3
N
N
N
5c
F
H
H
allyl
Me
N
R1
N
N
Cl
5d OMe
5e
5f
H
H
N
N
N
R2
N
H
5a
FAUC 113 4
R3
Figure 2.
N
N
N
N
and 7-azaindole raises the affinity to D4 receptors
10
enormously. Lober et al. have confirmed this obser-
€
N
N
N
N
N
N
vation by synthesizing and testing FAUC 113 (4)
(Fig. 2), a pyrazolo[1,5-a]pyridine derivative with high
affinity for the D4 receptor and good selectivity over the
other dopamine receptors. Inspired by these results, we
replaced the methyl group in 2 with a phenyl group
giving the 7-chloro-4-(4-phenylpiperazin-1-yl)-2,3-dihy-
dro-1H-1,5-benzodiazepine (5a) (Fig. 2). Preliminary
calculations in a comparative molecular field analysis
(CoMFA) based on D4 ligands of the heteroarylmethyl-
piperazinylaryl type11 have predicted a pKi value of 7.28
for compound 5a.
N
H
N
H
6g
N
R3
R3
6Hh
6a
H
6d 4-Cl
6e 3-Cl
6b 4-F
6c 2-OMe 6f 2-Cl
R3
N
N
N
N
N
For further structure–activity relationship (SAR) studies
we prepared a series of 2,3-dihydro-1H-1,5-benzodiaze-
pines by altering substituents at ring A (R1 in 5a–d), at
the nitrogen atom in position 5 (R2 in 5e,f) as well as at
the phenyl group of the piperazine side chain (R3 in 6a–
f) or even by changing the phenyl to the benzyl (6g) or
the pyridin-2-yl (6h) group. Finally, we exchanged the
benzene ring A in 5a to a more polar pyridine moiety,
which we introduced in two different ways. The
8-chloro-4,5-dihydro-3H-pyrido[2,3-b][1,4]diazepine 7b
represents an aza-analogue of 5a. In the pyridodiazepine
8 the nitrogen atom at position 8 should seize the role of
the electronegative chloro residue as realized in 5a (Fig.
3).
R1
N
N
N
N
H
N
H
R1
7a H
7b Cl
7c H
R3
H
8
H
Cl
Figure 3.
R3
R3
N
N
In a similar manner, title compounds 9 and 10 were
3
deduced from 2,3-dihydro-1H-1,4-benzodiazepine
(Fig. 4).
N
N
N
N
2. Chemistry
N
N
H
After retro synthetic considerations, we planned the
synthesis of the title compounds 5–10 similar to that of
clozapine (1)12 by first preparing the lactams 15, 16a–f,
26a,b, 30, and 34, which should be connected to the
appropriate 4-phenylpiperazines in the final step.
R3
R3
H
9a
9b 4-F
9c
2-OMe
H
10a
10b 4-F
10c 2-OMe
Figure 4.
2.1. Preparation of lactams 15, 16a–f, 26a,b, 30, and 34
14c–f with zinc powder and concd H3PO4 in refluxing
dioxane and following ring closure6 gave rise to the
lactams 16c–f (Scheme 1).
Lactam 15 was prepared from o-phenylenediamine (11)
and acrylic acid (12) as described in literature.13 In
analogy to the synthesis of the known lactams 16a,b14
the nitro anilines 13c–f were treated with acrylic acid in
concd H2SO4 giving the b-alaninates 14c–f. N-Allylnit-
roaniline (13e) was prepared from 2-fluoronitrobenzol
and N-allylamine.15 The reduction of the nitro group in
In order to obtain the pyridodiazepinones 26a,b and 30,
we needed the N-(3-nitropyridin-2-yl)-b-alaninates
24a,16b as well as the N-(3-nitropyridin-4-yl)-b-alaninate
28 as starting material. Our synthesis of pyridodiazepi-

T. Hussenether et al. / Bioorg. Med. Chem. 12 (2004) 2625–2637
2627
O
NMe2
H
N
R1
NO2
NH2
Cl
NO2
NH2
HOOC
a
NH2
NH2
Cl
a
+
+
H2N
N
+
N
R2
-
ClO4
Me2N
12
17
18
19
c
R1
H
R2
H
H
H
H
H
11
15
R1
NO2
1
R
NO2
16a Cl
b
b
b
COOH 16b CF3
16c
N
NH
R2
F
R2
Cl
Cl
NO2
NO2
Cl
Cl
16d OMe
d
c
16e
16f
H
H
allyl
Me
13a-f
14a-f
N
O
N
H
O
N
H
21
20
22
Scheme 1. Reagents and conditions: (a) EtOH, reflux; (b) acrylic acid
(12), concd H2SO4, 75–80 °C, 15 min; (c) Zn, dioxane, reflux, 2 h.
COOEt
e
H2N
23
none 26b started with the cyclocondensation of vina-
midinium perchlorate17 17 and 2-nitroethen-1,1-diamine
(18) in abs BuOH18 giving aminonitropyridine 19 in a
good yield. Thus, this new access can be considered as an
acceptable alternative to that reported in patent litera-
ture.19 Compound 19 was transformed to the known
chloronitropyridine 2020 in the next step. To obtain 20 we
also performed the nitration of commercially available
chloropyridone 21 and following chlorination by treat-
ing 22 with PCl5 in POCl3. However, this way was not
favored because of its low yield. According to a method
in literature8 the activated chloro moiety in position 2 of
20 was substituted with ethyl b-alaninate (23) in boiling
EtOH/Et3N leading to 24b. Intramolecular cyclization of
24a,b to the corresponding lactams 26a,b was easily
achieved by reducing the nitro group in abs EtOH/Pd–C
and refluxing in HOAc (96%) (Scheme 2).
R1
NH2
O
H
N
1
R
NO2
1
R
g
f
N
NH
N
NH
N
N
H
COOEt
COOEt
25a,b
R1
H
24b Cl
26a,b
24a
Scheme 2. Reagents and conditions: (a) BuOH, reflux, 4 h; (b) NaNO2,
concd HCl, ꢀ15 °C; (c) concd HNO3, concd H2SO4, 70 °C, 3 h; (d)
PCl5, POCl3, reflux; (e) ethyl b-alaninate HCl (23–HCl), NEt3, EtOH,
reflux, 2 h; (f) H2, Pd–C (5%), EtOH, 1500 hPa; (g) AcOH (96%),
reflux, 4 h.
NO2
In a similar manner, we prepared pyridodiazepinone 30
starting from the known 4-chloro-3-nitropyridine (27)21
and ethyl b-alaninate (23) in boiling EtOH/Et3N giving
28. After reduction of the nitro group, the 3-amino-
pyridine 29 surprisingly did not cyclize well. By varying
conditions, for use of numerous solvents and reaction
temperature, different kind of acid and base, the for-
mation of 30 succeeded finally in an unsatisfactory yield
of 26% using EtOH/concd HCl at 70 °C.
NH2
NH
NO2
Cl
N
N
a
b
N
NH
COOEt
COOEt
27
31
28
29
d
c
O
H
N
NH2
NH
NO2
NH
N
N
e
f
N
Thus, we thought about an alternative way22 to effect
closure of the seven-membered ring. Hydrolysis of the
ethyl ester 28 in 1 N NaOH/THF led to acid 31, which
was reduced in EtOH/Pd–C producing amino acid 32. In
the next step the carboxylic group was activated with
1,3-diisopropylcarbodiimide (DIC) and 1-hydroxy-
benzotriazole (HOBt) leading to ring closure to
1,2,3,5-tetrahydro-4H-pyrido[3,4-b][1,4]diazepin-4-one
(30) (Scheme 3).
N
H
32
COOH
30
COOH
Scheme 3. Reagents and conditions: (a) ethyl b-alaninate HCl (23–
HCl), NEt3, EtOH, reflux, 2h; (b) H2, Pd–C (5%), EtOH, 1500 hPa,
2 h; (c) EtOH, HCl, 70 °C, 12 h; (d) THF, 1 N NaOH, 50 °C, 4 h; (e) H2,
Pd–C (5%), EtOH, 3000 hPa, 6 h; (f) DIC, HOBt, DMF, rt, over night.
The preparation of lactam 34, a 1,4-benzodiazepine-5-
one, was easily performed by a regioselective reduction23
of dilactam 3324 with LiAlH4 in refluxing THF (Scheme
4).
O
O
H
N
H
N
a
N
H
N
H
2.2. Preparation of title compounds 5–10
O
33
34
Finally, the lactams 15 and 16a–f were converted into
the title compounds 5a–f, 6a–h by heating in toluene at
Scheme 4. Reagents and conditions: (a) LiAlH4, THF, reflux, 14 h.

2628
T. Hussenether et al. / Bioorg. Med. Chem. 12 (2004) 2625–2637
a
O
O
S
15,16a-f
5a-f, 6a-h
H
H
N
H
N
N
a
b
b, c
26a,b, 30
7a-c, 8
N
H
N
N
34
R2
36
37
N
N
N
c
N
HN
HN
HN
SMe
R2
R2
35g
35h
N
d
e
35a
H
35d 4-Cl
35e 3-Cl
10a-c
9a-c
35b 4-F
N
35c 2-OMe 35f 2-Cl
Scheme 5. Reagents and conditions: (a) 35a–f, TiCl4, toluene, 80 °C,
4 h; (b) PCl5/POCl3, pyridine, reflux, 4 h; (c) 35a–d, EtOH, reflux, 4 h.
38
Scheme 6. Reagents and conditions: (a) Allyl bromide, triethylamine,
benzene, reflux, 2 h; (b) P2S5, pyridine, reflux, 2.5 h; (c) NaOEt, MeI,
EtOH, reflux, 2 h; (d) 35a–c, AcOH, 120 °C, 12 h; (e) Pd/C (10%),
methanesulfonic acid, EtOH, reflux, 2 h.
80 °C after addition of excess of arylpiperazines 35a–g
and TiCl4.25 Due to the minor solubility in toluene,
lactams 26a,b and 30 could not be treated in the same
way. Hence, they were at first transformed to the reac-
tive imidoyl chlorides using PCl5 in POCl3/pyridine.
These intermediates subsequently reacted with the
appropriate phenylpiperazines 35a–d in boiling EtOH to
the pyridoazepines 7a–c and 8 in acceptable yields
(Scheme 5).26
value of 11 nM for the dopamine D4 subtype 6a comes
within the range of clozapine (1) and shows highest
affinity of all test compounds for this receptor subtype.
Small substituents, for example, a chloro (5a) and fluoro
group (5c) at position 8 in benzene ring A are tolerated
by dopamine D4 receptor, yet. A larger trifluoromethyl
(5b) or methoxy (5d) group at the same position effects
in a dramatic decrease of D4 affinity.
Lactam 34 did not react with any arylpiperazines 35
neither in the presence of TiCl4 nor after treating with
PCl5/POCl3. Thus, we activated the lactam group by
preparing the thiolactimmethylether 38.27 Therefore, the
secondary amine moiety in position 1 of 34 first was
protected with an allyl group to prevent from methyl-
ation by methyliodide. N-Allyllactam 36 was transformed
to thiolactam 37 by means of P2S5 in pyridine.28;29 De-
protonation with sodium ethoxide and treatment with
methyl iodide led to thiolactimmethylether 38,30 which
was stirred in a great excess of the phenylpiperazines
35a–c to exchange the methylsulfanyl group into a phen-
ylpiperazine side chain giving 9a–c.27 Cleavage of the N-
allyl protecting group was performed in almost quanti-
tative yield by heating 9a–c in ethanol with Pd/C (10%) in
the presence of equimolar amounts of methanesulfonic
acid31 yielding the title compounds 10a–c (Scheme 6).
A methyl or allyl group at the nitrogen atom in position
1 of the dihydrobenzo-1H-1,5-diazepine substructure
(5e,f) results in a moderate reduction of D4 binding in
comparison to 6a.
Substituents at the phenyl ring of the piperazine side
chain are accepted in ortho position (6c,f) with almost
no decline of affinity for D4. Substitution in meta- or
para position is more or less not tolerated by the
D4 receptor. Compounds carrying a benzyl (6g) or
pyridin-2-yl (6h) group instead of a phenyl group in the
piperazine side chain are less recognized by the D4
receptor.
While in comparison to clozapine there is nearly no
improvement in terms of affinity on D4 receptor, some
of the novel synthesized dihydro-1H-1,5-benzodiaze-
pines of type 5 and 6 show a remarkable increase in
selectivity for D4 especially over the D2 and D3 sub-
types. As specified in Table 1, D2long/4 and D2short/4
ratio of Ki values for clozapine (1) result approximately
in a factor 2 and D3/4 ratio in about factor 60. In
contrast, compounds 5c, 6a, and c show a 450–700-fold
higher binding on D4 than to D2 and 100–170-fold
higher binding on D4 than to D3 subtype. Best binding
properties were provided by compound 6a. (Ki ¼ 11 nM
for D4, about 740-fold selectivity over D2long). In this
context we like to mention, that compound 6g is the
most selective one tested (D2/4 ratio of Ki values >2700,
D3/4 ratio of Ki values ¼ 340).
3. Results and discussion
Dopamine D1 receptor binding was determined by
measuring the ability to displace [3H]SCH 23390 from
porcine D1 receptors.32 To assess D2long, D2short
33
,
D3,34
and D4.435 affinities cloned human dopamine receptor
subtypes stably expressed in Chinese hamster ovary cells
(CHO)32 and the radioligand [3H]spiperone were used
for competition experiments. Serotonin 5-HT1A and
5-HT2 receptor binding was determined utilizing [3H]8-
OH–DPAT, [3H]ketanserin and porcine cortical mem-
branes.36
Dopamine receptor binding data of the title compounds
5a–f, 6a–h, 7a–c, and 8 are depicted in Table 1. With a Ki

T. Hussenether et al. / Bioorg. Med. Chem. 12 (2004) 2625–2637
2629
Table 1. Receptor binding data (Ki values [nM]) of compound 5a–f, 6a–h, 7a–c, and 8 employing porcine D1 and human D2, D3, and D4 receptorsa
Entry
[3H]SCH23390
D1
[3H]spiperone
D2short D3
3400
Ratio of Ki values
D2long
4700
D4
31
D2long/4
D2short/4
D3/4
5a
5b
5c
5d
5e
5f
1200
6900
530
3 000
30,000
4800
150
13
97
13
110w
4.2
110
3.7
110
37
30,000
5500
10,000
1300
2400
12
450
3.0
400
21
25,000
3500
2900
3400
4600
9200
5100
710
23,000
8200
7000
160,000
5600
9800
28,000
3600
2200
7600
33
250
120
740
360
440
2.2
170
170
530
400
610
1.8
59
11
6a
6b
6c
6d
6e
6f
8100
5900
1900
7000
170
81
31,000
5800
1300
34 000
8000
1100
86
13
2000
3000
150
5.0
1.1
41
600
1000
27
910
7400
690
6 600
1100
1100
0.89
270
>2100
18
0.67
240
>2100
13
1700
Ndb
6g
6h
7a
7b
7c
8
>100,000
63,000
18,000
37,000
25,000
33,000
>100,000
46,000
15,000
20,000
21,000
30,000
16,000
13,000
2300
47
340
3.7
7.2
100
14
26,000
6400
14,000
2200
19,000
3500
320
140
350
220
55
45
14,000
5000
3700
260
71
150
60
150
140
17
Clozapine (1)
420
41
28
960
16
2.6
1.8
60
a Ki values are the means of two independent experiments each carried out in triplicate.
b Nd ¼ not determined.
Bioisosteric exchange of benzene ring A for a pyridine
moiety as realized in 7a–c and 8 is accompanied by a loss
of D4 affinity and selectivity.
Table 2. Receptor binding data (Ki values [nM]) of compound 5a,c, 6a,
and 6c employing porcine 5-HT1A and 5-HT2 receptorsa
Entry
[3H]8-OH–DPAT
5-HT1A
[3H]ketanserin
5-HT2
In screening tests dihydro-1H-1,4-benzodiazepines 9a–c
and 10a–c already show no appreciable binding to
dopamine receptors. Therefore, no Ki values were
determined.
5a
5c
6a
6c
3700
48
9 050
58
52
105
250
2 550
Clozapine (1)
460
12
To investigate the intrinsic effects of representative
examples the compounds 5c, 6a, and 6c, which showed
the most promising binding properties, were taken a
mitogenesis assay.37 All of them had partial agonist effects
(5c: 37%, EC50 ¼ 12 nM; 6a: 22%, EC50 ¼ 13 nM; 6c: 45%,
EC50 ¼ 24 nM) in comparison to quinpirole (full agonist).
a Ki values are the means of two independent experiments each carried
out in triplicate.
chromatography was done on silica gel 60 (Merck).
Melting points were determined in open capillary tubes
on a Buechi 510 melting point apparatus and are
uncorrected. Elemental analyzes were performed by the
To get a more detailed binding profile the most prom-
ising compounds 5a,c and 6a,c were tested for affinity to
serotonin 5-HT1A and 5-HT2 receptors. The binding
results listed in Table 2 are quite different. Best binding
properties are achieved for 5c and 6a. In comparison to
clozapine (1), fluoro compound 5c shows a 10-fold high
affinity to 5-HT1A, whereas binding to the 5-HT2
receptor was reduced by factor 4. Larger substituents
like a chloro group on ring A in 5a or a methoxy group
in the phenylpiperazine side chain in 6c lead to a
decreased Ki value especially for 5-HT2.
€
Institut fur Organische Chemie (university of Erlangen/
Nuremberg) using Carlo Erba Elemental Analyzer 1108.
They are within ꢁ0.4% of the theoretical values if not
noted otherwise. 1H Nuclear magnetic resonance (1H
NMR) spectra were determined with a Bruker AM 360
(360 MHz) spectrometer in appropriate deuterated sol-
vents and are expressed in parts per million (d, ppm)
downfield from tetramethylsilane (internal standard).
NMR data are given as multiplicity (s, singlet; d, dou-
blet; t, triplet; q, quartet; m, multiplet), coupling con-
stants (J), and number of protons. Mass spectra (MS)
were taken with a Finnigan MAT TSQ 70 mass spec-
trometer in the electron impact mode (70 eV). Significant
infrared (IR) spectra were obtained on a Jasco FT/IR
410 or Perkin–Elmer 1740 spectrometer.
4. Experimental
Starting materials were obtained from commercial
sources and were used without further purification.
Solvents were dried by standard procedures. All anhy-
drous reactions were performed in oven-dried glassware.
Reaction progress was observed by thin layer chroma-
tography making use of commercial silica gel plates
(Merck, silica gel F254 on aluminum sheets). Column
4.1. N-Allyl-2-nitroaniline (13e)
2-Fluoronitrobenzene (11.29 g, 80 mmol) and allylamine
(4.56 g, 80 mmol) were refluxed in dry THF (50 mL) for

2630
T. Hussenether et al. / Bioorg. Med. Chem. 12 (2004) 2625–2637
12 h. Solvent and excess of educts were removed in
vacuum. The residue was purified by flash chromato-
graphy (cyclohexane/EtOAc ¼ 9:1) to obtain 13e as an
orange oil (10.0 g, 63%). 1H NMR (CDCl3) 3.97 (d, 2H,
J ¼ 6:3 Hz), 5.14–5.21 (m, 2H), 5.85–5.94 (m, 1H), 6.63
(m, 1H), 6.81 (m, 1H), 7.41 (m, 1H), 8.03–8.32 (m, 2H);
MS m=z 178 [Mþ]. Anal. C9H10N2O2 (178.07) C, H, N.
D2O); MS m=z 224 [Mþ]. Anal. C10H12N2O4 (224.22) C,
H, N.
4.6. 8-Fluoro-1,3,4,5-tetrahydro-2H-1,5-benzodiazepin-2-
one (16c)
Compound 14c (7.98 g, 35 mmol) was heated to 80 °C in
a mixture of dioxane (90 mL) and H3PO4 (85%, 20 mL).
Zinc powder (17.1 g, 262.5 mmol) was added portion-
wise within 1 h. The suspension was refluxed for another
hour. After cooling, firm components were filtered off
and washed profoundly with dioxane. The filtrate was
neutralized cautiously with concd NH3 (25%) under ice
cooling. Newly formed precipitate was filtered off
and washed profoundly with dioxane once again. The
filtrate was concentrated in vacuum. The residue
was slurred in 2 N NH3 solution. The crude product
was precipitated by cooling in the refrigerator over
night, filtered off, and washed with water. Recrystalli-
zation from EtOH and drying in vacuum gave pure
16c (4.37 g, 69%). Mp 144–146 °C (EtOH); IR 3321,
4.2. N-(4-Fluoro-2-nitrophenyl)-b-alanine (14c)
4-Fluoro-2-nitroaniline (13c) (7.80 g, 50 mmol) was
suspended in acrylic acid (10.8 g, 150 mmol). Concd
H2SO4 (98%, 5 mL) was added portion-wise and the
mixture heated to 75–80 °C and stirred for 15 min.
Water (50 mL) was added cautiously so that the tem-
perature did not rise over 80 °C. After cooling, the
precipitate was filtered off, washed well with water, and
dried in vacuum. The yielded crystals (10.7 g, 93%) were
used without further purification in the next step. For
analytical purposes crystallization from EtOH/water
(2:1) was performed. Mp 145–146 °C (154–155 °C)38
1
(EtOH/H2O); IR 3737, 3378, 3116, 1705, 1523 cmꢀ1; H
3217, 2924, 2854, 1670, 1608 cmꢀ1
;
1H NMR
NMR ([d6]DMSO) 2.61 (t, 2H, J ¼ 6:8 Hz), 3.58 (dt,
2H, J ¼ 6:8, 5.0 Hz), 7.17 (m, 1H), 7.54 (m, 1H), 7.86
(m, 1H), 8.15 (t, br, 1H, J ¼ 5:0 Hz, exchangeable with
D2O), 12.48 (s, br, 1H, exchangeable with D2O); MS
m=z 228 [Mþ]. Anal. C9H9FN2O4 (228.18) C, H, N.
([d6]DMSO) 2.49 (t, 2H, J ¼ 6:0 Hz), 3.40 (t, 2H,
J ¼ 2:8, 6.0 Hz), 5.56 (t, br, 1H, J ¼ 2:8 Hz, exchange-
able with D2O), 6.69–6.79 (m, 3H), 9.51 (s, br 1H,
exchangeable with D2O); MS m=z 180 [Mþ]. Anal.
C9H9FN2O (180.18) C, H, N.
4.3. N-(4-Methoxy-2-nitrophenyl)-b-alanine (14d)
4.7. 8-Methoxy-1,3,4,5-tetrahydro-2H-1,5-benzodia-
zepin-2-one (16d)
Preparation and purification according to 14c from 4-
methoxy-2-nitroaniline (13d) (8.41 g, 50 mmol) gave 14d
(10.1 g, 84%). Mp 130–131 °C (129–131 °C)39 (EtOH/
Preparation and purification according to 16c from 14d
(8.40 g, 35 mmol) gave 16d (4.03 g, 87%). Mp 137–
138 °C;40 1H NMR ([d6]DMSO) 2.43 (t, 2H, J ¼ 6:0 Hz),
3.40 (dt, 2H, J ¼ 3:0, 6.0 Hz), 3.64 (s, 3H), 5.12 (t, br,
1H, J ¼ 3:0 Hz, exchangeable with D2O), 6.48–6.79 (m,
3H), 9.37 (s, br, 1H, exchangeable with D2O); MS m=z
192 [Mþ]. Anal. C10H12N2O2 (192.22) C, H, N.
1
H2O); H NMR ([d6]DMSO) 2.61 (t, 2H, J ¼ 6:6 Hz),
3.57 (dt, 2H, J ¼ 6:6, 4.5 Hz), 3.75 (s, 3H), 7.10 (m, 1H),
7.29 (m, 1H), 7.50 (m, 1H), 8.10 (t, br, 1H, J ¼ 4:5 Hz,
exchangeable with D2O), 12.39 (s, br, 1H, exchangeable
with D2O); MS m=z 240 [Mþ]. Anal. C10H12N2O5
(240.22) C, H, N.
4.8. 5-Allyl-1,3,4,5-tetrahydro-2H-1,5-benzodiazepin-2-
one (16e)
4.4. N-Allyl-N-(2-nitrophenyl)-b-alanine (14e)
Preparation and purification according to 14c from N-
allyl-2-nitroaniline (13e) (8.90 g, 50 mmol) gave 14e
(9.5 g, 76%) as bright orange oil. H NMR ([d6]DMSO)
2.37 (t, 2H, J ¼ 7:3 Hz), 3.31 (d, 2H, J ¼ 7:3 Hz), 3.69
(d, 2H, J ¼ 6:0 Hz), 5.10–5.24 (m, 2H), 5.69–5.81 (m,
1H), 7.10 (m, 1H), 7.37 (m, 1H), 7.54 (m, 1H), 7.73 (m,
1H), 12.19 (s, br, 1H, exchangeable with D2O); MS m=z
250 [Mþ]. Anal. C12H14N2O4 (250.26) C, H, N.
Preparation and purification according to 16c from 14e
(8.76 g, 35 mmol) gave 16e(4.03 g, 87%). Mp 93–95 °C;
1H NMR ([d6]DMSO) 2.31 (t, 2H, J ¼ 6:7 Hz), 3.37 (t,
2H, J ¼ 6:7 Hz), 3.72 (d, 2H, J ¼ 6:1 Hz), 5.12–5.29 (m,
2H), 5.74–5.86 (m, 1H), 6.89–7.10 (m, 4H), 9.44 (s, br,
1H, exchangeable with D2O); MS m=z 202 [Mþ]. Anal.
C12H14N2O (202.26) C, H, N.
1
4.9. 5-Methyl-1,3,4,5-tetrahydro-2H-1,5-benzodiazepin-
2-one (16f)
4.5. N-Methyl-N-(2-nitrophenyl)-b-alanine (14f)
Preparation and purification according to 14c from N-
methyl-2-nitroaniline (13f) (7.90 g, 50 mmol) gave 14f
(9.7 g, 87%). Mp 188–189 °C (EtOH/H2O); 1H NMR
([d6]DMSO) 2.49 (t, 2H, J ¼ 7:3 Hz), 2.75 (s, 3H), 3.36
(t, 2H, J ¼ 7:3 Hz), 6.96 (m, 1H), 7.27 (m, 1H), 7.51 (m,
1H), 7.74 (m, 1H), 12.27 (s, br, 1H, exchangeable with
Preparation and purification according to 16c from 14f
(7.98 g, 35 mmol) gave 16f(3.63 g, 58%). Mp 122–123 °C;
1H NMR ([d6]DMSO) 2.29 (t, 2H, J ¼ 6:8 Hz), 2.73 (s,
3H), 3.38 (t, 2H, J ¼ 6:8 Hz), 6.89–7.13 (m, 4H), 9.45 (s,
br, 1H, exchangeable with D2O); MS m=z 176 [Mþ].
Anal. C10H12N2O (176.22) C, H, N.

T. Hussenether et al. / Bioorg. Med. Chem. 12 (2004) 2625–2637
2631
4.10. 5-Chloro-3-nitropyridin-2-amine (19)
filtrate was concentrated in vacuum to obtain crude 21a,
which was directly refluxed in HOAc (96%, 100 mL) for
4 h. After cooling, the solvent was evaporated in vac-
uum. The residue was picked up in 2 N NH3. The crude
product was precipitated by cooling in the refrigerator
over night and was separated by filtration. Recrystal-
lization from EtOH/water and drying in vacuum gave
26a (1.99 g, 61%). Mp 253–255 °C (EtOH/H2O); IR
2-Nitroethene-1,1-diamine (18) (3.09 g, 30 mmol) and N-
[(2Z)-2-chloro-3-(dimethylamino)prop-2-en-1-ylidene]-N-
methyl-methanaminium perchlorate (17) (7.83 g,
30 mmol) were refluxed in dry n-BuOH for 4 h. After
cooling, the solvent was removed in vacuum. The resi-
due was separated between EtOAc and saturated
Na2CO3 solution. The organic layer was collected,
washed with saturated Na2CO3 solution, and dried over
Na2SO4. After evaporation of the solvent, the residue
was purified by flash chromatography (cyclohexane/
EtOAc ¼ 7:3) to obtain 19 (3.03 g, 58%). Mp 188–
3232, 3051, 3008, 1670, 1604 cmꢀ1
;
1H NMR
([d6]DMSO) 2.59 (t, 2H, J ¼ 4:9 Hz), 3.46 (dt, 2H,
J ¼ 4:9, 3.0 Hz), 6.58 (dd, 1H, J ¼ 7:7, 4.8 Hz), 6.62 (t,
br 1H, J ¼ 3:0 Hz, exchangeable with D2O), 7.18 (dd,
1H, J ¼ 7:8, 1.4 Hz), 7.74 (dd, 1H, J ¼ 4:8, 1.4 Hz), 9.52
(s, br, 1H, exchangeable with D2O); MS m=z 163 [Mþ].
Anal. C8H9N3O (163.18) C, H, N.
189 °C; IR 3467, 3278, 3140, 1651, 1554, 1512 cmꢀ1; H
1
NMR ([d6]DMSO) 8.06 (s, br, 2H, exchangeable with
D2O), 8.45 (d, 1H, J ¼ 2:5 Hz); MS m=z 173 [Mþ]. Anal.
C5H4ClN3O2 (173.56) C, H, N.
4.14. 8-Chloro-4,5-dihydro-1H-pyrido[2,3-b][1,4]diazepin-
2(3H)-one (26b)
4.11. 5-Chloro-3-nitro-1H-pyridin-2-one (22)
Preparation and purification according to 26a from 24b
(5.47 g, 20 mmol) gave 26b (1.54 g, 39%). Mp >270 °C
(EtOH/H2O); 1H NMR ([d6]DMSO) 2.62 (t, 2H,
J ¼ 4:6 Hz), 3.44 (dt, 2H, J ¼ 4:6 Hz, 3.5 Hz), 6.92 (t,
br, 1H, J ¼ 3:5 Hz, exchangeable with D2O), 7.24 (d,
1H, J ¼ 2:5 Hz), 7.74 (d, 1H, J ¼ 2:5 Hz), 9.63 (s, br,
1H, exchangeable with D2O); MS m=z 197 [Mþ]. Anal.
C8H8ClN3O (197.63) C, H, N.
While stirring oleum (65% SO3, d ¼ 1:99 g/cm3, 14 mL)
was dropped cautiously to ice-cooled HNO3 (100%,
d ¼ 1:52 g/cm3, 10 mL). 5-Chloro-1H-pyridin-2-one (21)
was added portion-wise so that the temperature did not
rise over 10 °C. The resulting suspension was allowed to
warm slowly to room temperature, and was stirred for
another 3 h. The mixture was poured onto crushed ice.
The precipitate was filtered off, washed well with water,
and dried at 50 °C in vacuum to get 22 (1.83 g, 17%). Mp
4.15. Ethyl N-(3-nitropyridin-4-yl)-b-alaninate (28)
225–226 °C; IR 3072, 1705, 1655, 1589, 1539, 1510 cmꢀ1
;
1H NMR ([d6]DMSO) 8.22 (s, 1H), 8.53 (s, 1H), 13.27
(s, br, 1H, exchangeable with D2O); MS m=z 174 [Mþ].
Anal. C5H3ClN2O3 (174.54) C, H, N.
Preparation and purification (cyclohexane/EtOAc/
MeOH ¼ 5:4:1) according to 24b from 27 (3.33 g,
21 mmol) gave 28 (3.11 g, 62%) as yellow powder. Mp
75–76 °C; IR 3311, 2755, 1721, 1622 cmꢀ1
;
1H NMR
4.12. Ethyl N-(5-chloro-3-nitro-pyridin-2-yl)-b-alaninate
(24b)
([d6]DMSO) 1.18 (t, 3H, J ¼ 7:1 Hz), 2.69 (t, 2H,
J ¼ 6:4 Hz), 3.67 (dt, 2H, J2 ¼ 6:4 Hz, J2 ¼ 5:4 Hz), 4.09
(q, 2H, J ¼ 7:1 Hz), 7.07 (d, 1H, J ¼ 6:4 Hz), 8.28 (d,
1H, J ¼ 6:4 Hz), 8.47 (t, br, 1H, J ¼ 5:4 Hz, exchange-
able with D2O), 9.03 (s, 1H); MS m=z 239 [Mþ]. Anal.
C10H13N3O4 (239.12) C, H, N.
2,5-Dichloro-3-nitropyridine (20) (4.05 g, 21 mmol),
ethyl 3-aminopropionate HCl (23–HCl) (5.83 g,
31.5 mmol), and triethylamine (5.83 g, 57.7 mmol) were
refluxed in dry EtOH (30 mL) for 2 h. After cooling, the
solvent was removed in vacuum. The residue was sepa-
rated between EtOAc and water. The organic layers
were collected, washed twice with water, and dried over
Na2SO4. After evaporation of the solvent the residue
was purified by flash chromatography (cyclohexane/
EtOAc ¼ 8:2) to obtain 24b (3.51 g, 61%) as yellow
powder. Mp 126–127 °C; IR 3390, 3082, 2981, 1732,
4.16. Ethyl N-(3-aminopyridin-4-yl)-b-alaninate (29)
Compound 28 (2.37 g, 10 mmol) was hydrogenated in
abs EtOH (100 mL) over Pd/C (5%, 200 mg) at 1500 hPa
(22 psi) and room temperature for 2 h. The catalyst was
filtered off and the filtrate was concentrated in vacuum
to get pure colorless crystals (1.92 g, 92%). Mp 116–
1616 cmꢀ1
;
1H NMR ([d6]DMSO) 1.18 (t, 3H,
118 °C (EtOH); IR 3211, 3016, 2966, 1721, 1607 cmꢀ1
;
J ¼ 7:1 Hz), 2.66 (t, 2H, J ¼ 6:9 Hz), 3.77 (q, 2H,
J ¼ 7:1 Hz), 3.81 (dt, 2H, J ¼ 6:9, 5.6 Hz), 8.47 (d, 1H,
J ¼ 2:5 Hz), 8.54 (d, 1H, J ¼ 2:5 Hz), 8.61 (t, br, 1H,
J ¼ 5:6 Hz, exchangeable with D2O); MS m=z 273 [Mþ].
Anal. C10H12ClN3O4 (273.68) C, H, N.
1H NMR ([d6]DMSO) 1.19 (t, 3H, J ¼ 7:2 Hz), 2.61 (t,
2H, J ¼ 6:6 Hz), 3.36 (dt, 2H, J ¼ 5:5, 6.6 Hz), 4.08 (q,
2H, J ¼ 7:2 Hz), 4.54 (s, br, 2H, exchangeable with
D2O), 5.40 (t, br, 1H, J ¼ 5:5 Hz, exchangeable with
D2O), 6.38 (d, 1H, J ¼ 5:4 Hz), 7.60 (d, 1H, J ¼ 5:4 Hz),
7.65 (s, 1H); MS m=z 209 [Mþ]. Anal. C10H15N3O2
(209.25) C, H, N.
4.13. 4,5-Dihydro-1H-pyrido[2,3-b][1,4]diazepin-2(3H)-
one (26a)
Ethyl 3-(3-nitropyridin-2-ylamino)-propionate (24a)
(4.78 g, 20 mmol) was hydrogenated in dry EtOH
(150 mL) with Pd/C (5%, 300 mg) at 1.5 bar and room
temperature for 2 h. The catalyst was filtered off and the
4.17. N-(3-Nitropyridin-4-yl)-b-alanine (31)
NaOH (1 N, 50 mL) was added to a solution of 28
(3.59 g, 15 mmol) in THF (50 mL). The mixture was

2632
T. Hussenether et al. / Bioorg. Med. Chem. 12 (2004) 2625–2637
stirred vigorously for 4 h. The aqueous layer was sepa-
rated. The organic layer was extracted twice with
aqueous 1 N NaOH. Aqueous layers were combined,
cooled in an ice bath, and cautiously adjusted to pH 3–4
by adding concd HCl. The precipitate was filtered off
and washed with water. Recrystallization from EtOH
gave pure 31 (2.25 g, 71%) as colorless crystals. Mp 144–
stirring for 10 min a solution of 16a (99 mg, 0.5 mmol) in
dry toluene (10 mL) was added. While stirring the mix-
ture was heated under a nitrogen atmosphere to 80 °C
for 4 h. After cooling water (10 mL) was added cau-
tiously and pH was adjusted to 7–8 with concd NH3.
The precipitate was filtered off and washed well with
CHCl3. The aqueous layer of the filtrate was separated
and extracted with CHCl3. Combined organic layers
were washed with 2 N NH3, dried over Na2SO4, and
concentrated in vacuum. The residue was purified by
column chromatography (CH2Cl2/MeOH ¼ 97:3) to
yield 5a (59 mg, 42%) as light yellow powder. Mp 170–
172 °C; IR 3429, 2954, 2819, 1597, 1500 cmꢀ1; 1H NMR
([d6]DMSO) 2.62 (t, 2H, J ¼ 6:0 Hz), 3.19 (t, 4H,
J ¼ 4:6 Hz), 3.55 (dt, 2H, J ¼ 3:0, 6.0 Hz), 3.68 (t, 4H,
J ¼ 4:6 Hz), 5.09 (t, 1H, NH, J ¼ 3:0 Hz, exchangeable
with D2O), 6.68–7.29 (m, 8H); MS m=z 340 [Mþ]. Anal.
C15H21ClN4 (340.86) C, H, N.
146 °C (EtOH); IR 3350, 3114, 1698, 1633, 1560 cmꢀ1
;
1H NMR ([d6]DMSO) 2.62 (t, 2H, J ¼ 6:4 Hz), 3.62 (dt,
2H, J ¼ 5:6, 6.4 Hz), 7.07 (d, 1H, J ¼ 6:4 Hz), 8.27 (d,
1H, J ¼ 6:4 Hz), 8.47 (t, br, 1H, J ¼ 5:6 Hz, exchange-
able with D2O), 9.03 (s, 1H); MS m=z 211 [Mþ]. Anal.
C8H9N3O4 (211.18) C, H, N.
4.18. N-(3-Aminopyridin-4-yl)-b-alanine (32)
Compound 31 (2.37 g, 10 mmol) was hydrogenated in
dry EtOH (150 mL) over Pd–C (5%, 300 mg) at 3000 hPa
(44 psi) and room temperature for 6 h. The catalyst was
filtered off and the filtrate was concentrated in vacuum
to get pure colorless crystals (2.25 g, 81%). Mp 159–
4.21. 4-(4-Phenylpiperazin-1-yl)-7-trifluoromethyl-2,3-di-
hydro-1H-1,5-benzodiazepine (5b)
161 °C (EtOH); IR 3267, 3099, 2765, 1703 cmꢀ1
;
1H
NMR ([d6]DMSO) 2.63 (t, 2H, J ¼ 6:4 Hz), 3.49 (t, 2H,
J ¼ 6:4 Hz), 5.61 (s, br, 2H, exchangeable with D2O),
6.71 (d, 1H, J ¼ 6:0 Hz), 7.21 (s, br, 1H, exchangeable
with D2O), 7.61 (s, 1H), 7.78 (d, 1H, J ¼ 6:0 Hz); MS
m=z 181 [Mþ]. Anal. C8H11N3O (181.20) C, H, N.
Preparation and purification (CH2Cl2/MeOH ¼ 96:4)
according to 5a from 16b (115 mg, 0.5 mmol) gave 5b
(103 mg, 55%) as light yellow powder. Mp 190–192 °C;
1H NMR ([d6]DMSO) 2.71 (t, 2H, J ¼ 5:5 Hz), 3.19 (t,
4H, J ¼ 4:9 Hz), 3.57 (dt, 2H, J ¼ 2:7, 5.5 Hz), 3.69 (t,
4H, J ¼ 4:9 Hz), 5.80 (t, br, 1H, J ¼ 2:7 Hz, exchange-
able with D2O), 6.73–7.32 (m, 8H); MS m=z 374 [Mþ].
Anal. C20H21F3N4 (374.41) C, H, N.
4.19. 1,2,3,5-Tetrahydro-4H-pyrido[3,4-b][1,4]diazepin-4-
one (30)
Method A. Compound 29 (1.67 g, 8 mmol) was heated to
70 °C in a mixture of dry EtOH (50 mL) and concd HCl
(36%, 5 mL) for 12 h. The resulting solution was cooled
in an ice bath and pH was adjusted to 6–7 by adding
aqueous concd NH3 solution. After evaporation to
dryness, the residue was sonicated with MeOH. Firm
components were filtered off and washed well with
methanol. The filtrate was concentrated in vacuum and
purified by flash chromatography (CH2Cl2/MeOH ¼ 3:1)
gives 30 (235 mg, 18%) as colorless powder.
4.22. 7-Fluoro-4-(4-phenylpiperazin-1-yl)-2,3-dihydro-
1H-1,5-benzodiazepine (5c)
Preparation and purification (CH2Cl2/MeOH ¼ 96:4)
according to 5a from 16c (90 mg, 0.5 mmol) gave 5c
(122 mg, 38%) as light yellow powder. Mp 164–165 °C;
1H NMR ([d6]DMSO) 2.58 (t, 2H, J ¼ 5:5 Hz), 3.19 (t,
4H, J ¼ 4:9 Hz), 3.55 (dt, 2H, J ¼ 2:9, 5.5 Hz), 3.69 (t,
4H, J ¼ 4:9 Hz), 4.77 (t, br, 1H, J ¼ 2:9 Hz, exchange-
able with D2O), 6.45–7.39 (m, 8H); MS m=z 324 [Mþ].
Anal. C19H21FN4 (324.40) C, H, N.
Method B. 1,3-Diisopropylcarbodiimide (3.53 g,
28 mmol) and 1-hydroxybenzotriazolÆH2O were added to
a solution of 32 (1.27 g, 7 mmol) in abs DMF (30 mL).
The mixture was stirred over night at room temperature.
After evaporation to dryness the residue was extracted
with MeOH and purified as described under method A to
get 30 (639 mg, 56%) as colorless powder. Mp >300 °C
4.23. 7-Methoxy-4-(4-phenylpiperazin-1-yl)-2,3-dihydro-
1H-1,5-benzodiazepine (5d)
Preparation and purification (CH2Cl2/MeOH ¼ 95:5)
according to 5a from 16d (115 mg, 0.5 mmol) gave 5d
(86 mg, 51%) as light yellow powder. Mp 168–170 °C; 1H
NMR ([d6]DMSO) 2.44 (t, 2H, J ¼ 5:8 Hz), 3.20 (t, 4H,
J ¼ 2:8, 5.0 Hz), 3.52 (t, 2H, J ¼ 5:3 Hz), 3.64 (s, 3H),
3.66 (t, 4H, J ¼ 5:3 Hz), 5.29 (t, br, 1H, J ¼ 2:8 Hz,
exchangeable with D2O), 6.67–7.27 (m, 8H); MS m=z
336 [Mþ]. Anal. C20H24N4O (336.44) C, H, N.
(decomposition); IR 3221, 2924, 1694, 1643, 1570 cmꢀ1
;
1H NMR ([d6]DMSO) 2.64 (t, 2H, J ¼ 4:6 Hz), 3.49 (dt,
2H, J ¼ 4:6, 4.6 Hz), 6.74 (d, 1H, J ¼ 6:0 Hz), 7.79 (d,
1H, J ¼ 6:0 Hz), 7.84 (t, br, 1H, J ¼ 4:6 Hz, exchange-
able with D2O), 7.95 (s, 1H), 9.71 (s, 1H); MS m=z 163
[Mþ]. Anal. C8H8N3O (163.18) C, H, N.
4.20. 7-Chloro-4-(4-phenylpiperazin-1-yl)-2,3-dihydro-
1H-1,5-benzodiazepine (5a)
4.24. 1-Allyl-4-(4-phenylpiperazin-1-yl)-2,3-dihydro-1H-
1,5-benzodiazepine (5e)
N-Phenylpiperazine (35a) (0.5 g) was added to a solution
of TiCl4 (189 mg, 1 mmol) in dry toluene (10 mL). After
Preparation and purification (CH2Cl2/MeOH ¼ 97:3)
according to 5a from 16e (88 mg, 0.5 mmol) gave 5e

T. Hussenether et al. / Bioorg. Med. Chem. 12 (2004) 2625–2637
2633
(66 mg, 41%) as light yellow powder. Mp 117–119 °C; 1H
4.29. 4-[4-(4-Chlorophenyl)piperazin-1-yl]-2,3-dihydro-
1H-1,5-benzodiazepine (6d)
NMR (CDCl3) 2.75 (t, 4H, J ¼ 5:0 Hz), 2.99–3.14 (m,
4H), 3.23(t, 4H, J ¼ 5:0 Hz), 4.76–4.81 (m, 2H), 4.97 (d,
1H, J ¼ 8:6 Hz), 5.24 (d, 2H, J ¼ 5:3 Hz), 5.90–6.02 (m,
1H), 6.82–9.97 (m, 3H), 7.20–7.31 (m, 4H), 7.69–7.78
(m, 1H). MS m=z 346 [Mþ]. Anal. C22H26N4 (346.48) C,
H, N.
Preparation and purification (CH2Cl2/MeOH ¼ 95:5)
according to 5a from 15 (81 mg, 0.5 mmol) and N-(4-
chlorophenyl)piperazine (35d) (0.5 g) gave 6d (84 mg,
49%) as light yellow powder. Mp 149–150 °C; 1H NMR
([d6]DMSO) 2.59 (t, 2H, J ¼ 5:7 Hz), 3.19 (t, 4H,
J ¼ 4:7 Hz), 3.55 (t, 2H, J ¼ 2:8, 5.7 Hz), 3.65 (t, 4H,
J ¼ 4:7 Hz), 4.90 (s, br, 1H, J ¼ 2:8 Hz, exchangeable
with D2O), 6.50–7.33 (m, 8H); MS m=z 340 [Mþ]. Anal.
C19H21ClN4 (340.86) C, H, N.
4.25. 1-Methyl-4-(4-phenylpiperazin-1-yl)-2,3-dihydro-
1H-1,5-benzodiazepine (5f)
Preparation and purification (CH2Cl2/MeOH ¼ 97:3)
according to 5a from 16f (88 mg, 0.5 mmol) gave 5f
(66 mg, 41%) as light yellow powder. Mp 161–163 °C; 1H
NMR ([d6]DMSO) 2.49 (t, 2H, J ¼ 6:4 Hz), 2.63 (s, 3H),
3.21 (t, 4H, J ¼ 5:0 Hz), 3.37 (t, 2H, J ¼ 6:4 Hz), 3.68 (t,
4H, J ¼ 5:0 Hz), 6.72–7.29 (m, 9H); MS m=z 320 [Mþ].
Anal. C20H24N4 (320.44) C, H, N.
4.30. 4-[4-(3-Chlorophenyl)piperazin-1-yl]-2,3-dihydro-
1H-1,5-benzodiazepine (6e)
Preparation and purification (CH2Cl2/MeOH ¼ 95:5)
according to 5a from 15 (81 mg, 0.5 mmol) and N-(3-
chlorophenyl)piperazine (35e) (0.5 g) gave 4h (78 mg,
46%) as light yellow powder. Mp 173–175 °C; 1H NMR
([d6]DMSO) 2.59 (t, 2H, J ¼ 6:0 Hz), 3.24 (t, 4H,
J ¼ 5:2 Hz), 3.56 (t, 2H, J ¼ 6:0 Hz), 3.64 (t, 4H,
J ¼ 5:2 Hz), 4.91 (s, br, 1H, exchangeable with D2O),
6.65–7.27 (m, 8H); MS m=z 340 [Mþ]. Anal. C19H21N4O
(340.86) C, H, N.
4.26. 4-(4-Phenylpiperazin-1-yl)-2,3-dihydro-1H-1,5-
benzodiazepine (6a)
Preparation and purification (CH2Cl2/MeOH ¼ 95:5)
according to 5a from 1,3,4,5-tetrahydro-2H-1,5-ben-
zodiazepin-2-one (15) (81 mg, 0.5 mmol) and N-phenyl-
piperazine (35a) (0.5 g) gave 6a (64 mg, 42%) as light
yellow powder. Mp 185–187 °C; 1H NMR (CDCl3) 2.67
(t, 2H, J ¼ 6:3 Hz), 3.29 (t, 4H, J ¼ 5:3 Hz), 3.77 (t, 2H,
J2 ¼ 2:9 Hz, J2 ¼ 6:3 Hz), 3.81 (t, 4H, J ¼ 5:3 Hz), 6.71–
7.23 (m, 9H), 7.09 (t, br, 1H, J ¼ 2:9 Hz, exchangeable
with D2O); MS m=z 306 [Mþ]. Anal. C19H22N4 (306.41)
C, H, N.
4.31. 4-[4-(2-Chlorophenyl)piperazin-1-yl]-2,3-dihydro-
1H-1,5-benzodiazepine (6f)
Preparation and purification (CH2Cl2/MeOH ¼ 97:3)
according to 5a from 15 (81 mg, 0.5 mmol) and N-(2-
chlorophenyl)piperazine (35f) (0.5 g) gave 6f (8 5 mg,
50%) as light yellow powder. Mp 148–149 °C; 1H NMR
([d6]DMSO) 2.61 (t, 2H, J ¼ 5:8 Hz), 3.03 (t, 4H,
J ¼ 4:8 Hz), 3.57 (t, 2H, J ¼ 2:7, 5.8 Hz), 3.68 (t, 4H,
J ¼ 4:8 Hz), 4.94 (s, br, 1H, J ¼ 2:7 Hz, exchangeable
with D2O), 6.65–7.48 (m, 8H); MS m=z 340 [Mþ]. Anal.
C19H21ClN4 (340.86) C, H, N.
4.27. 4-[4-(4-Fluorophenyl)piperazin-1-yl]-2,3-dihydro-
1H-1,5-benzodiazepine (6b)
Preparation and purification (CH2Cl2/MeOH ¼ 97:3)
according to 5a from 15 (81 mg, 0.5 mmol) and N-(4-
fluorophenyl)piperazine (35b) (0.5 g) gave 6b (83 mg,
1
51%) as light yellow powder. Mp 115–116 °C; H NMR
4.32. 4-(4-Benzylpiperazin-1-yl)-2,3-dihydro-1H-1,5-
benzodiazepine (6g)
([d6]DMSO) 2.59 (t, 2H, J ¼ 6:0 Hz), 3.13 (t, 4H,
J ¼ 5:0 Hz), 3.55 (t, 2H, J ¼ 2:9, 6.0 Hz), 3.65 (t, 4H,
J ¼ 5:0 Hz), 4.90 (s, br, 1H, J ¼ 2:9 Hz, exchangeable
with D2O), 6.65–7.16 (m, 8H); MS m=z 324 [Mþ]. Anal.
C19H21FN4 (324.40) C, H, N.
Preparation and purification (CH2Cl2/MeOH ¼ 97:3)
according to 5a from 15 (81 mg, 0.5 mmol) and N-benz-
ylpiperazine (35g) (0.6 g) gave 6g (75 mg, 44%) as light
1
yellow powder. Mp 162–164 °C; H NMR ([d6]DMSO)
2.20–2.57 (m, 8H), 2.75 (t, 2H, J ¼ 7:6 Hz), 2.95 (t, 2H,
J ¼ 7:6 Hz), 3.44 (s, 2H), 4.91 (s, br, 1H, exchangeable
with D2O), 7.06–7.14 (m, 2H), 7.20–7.36 (m, 5H), 7.39–
7.51 (m, 2H); MS m=z 320 [Mþ]. Anal. C20H24N4
(320.44) C, H, N.
4.28. 4-[4-(2-Methoxyphenyl)piperazin-1-yl]-2,3-dihydro-
1H-benzodiazepine (6c)
Preparation and purification (CH2Cl2/MeOH ¼ 96:4)
according to 5a from 15 (81 mg, 0.5 mmol) and N-(2-
methoxyphenyl)piperazine (35c) (0.5 g) gave 6c (73 mg,
43%) as light yellow powder. Mp 170–171 °C; 1H
NMR ([d6]DMSO) 2.58 (t, 2H, J ¼ 6:0 Hz), 3.01 (t,
4H, J ¼ 4:7 Hz), 3.56 (t, 2H, J ¼ 2:8, 6.0 Hz, H-2),
3.64 (t, 4H, J ¼ 4:7 Hz), 3.81 (s, 3H), 4.90 (t, br,
1H, J ¼ 2:8 Hz, exchangeable with D2O), 6.66–7.00 (m,
8H); MS m=z 340 [Mþ]. Anal. C19H21ClN4 (340.86) C,
H, N.
4.33. 4-(4-Pyridin-2-ylpiperazin-1-yl)-2,3-dihydro-1H-
1,5-benzodiazepine (6h)
Preparation and purification (CH2Cl2/MeOH ¼ 95:5)
according to 5a from 15 (81 mg, 0.5 mmol) and N-
(pyridin-2-yl)piperazine (35h) (0.5 g) gave 6h (85 mg,
50%) as light yellow powder. Mp 148–149 °C; 1H NMR

2634
T. Hussenether et al. / Bioorg. Med. Chem. 12 (2004) 2625–2637
([d6]DMSO) 2.61 (t, 2H, J ¼ 6:0 Hz), 3.53–3.60 (m, 6H),
3.64 (m, 4H), 4.92 (t, br, J ¼ 2:9 Hz, exchangeable with
D2O), 6.63–6.77 (m, 4H), 6.80 (m, 1H), 6.86 (d, 1H,
J ¼ 8:5 Hz), 7.56 (m, 1H), 8.14 (dd, 1H, J ¼ 4:6, 1.1 Hz);
MS m=z 307 [Mþ]. Anal. C18H21N5 (307.40) C, H, N.
ylpiperazine (35a) (0.5 g) gave 8 (60 mg, 39%). Mp 193–
1
194 °C; H NMR ([d6]DMSO) 2.61 (t, 4H, J ¼ 4:8 Hz),
2.81 (d, 2H, J ¼ 6:9 Hz), 3.03 (t, 2H, J ¼ 6:9 Hz), 3.19
(t, 4H, J ¼ 4:8 Hz), 6.73 (s, br, 1H, exchangeable with
D2O), 6.93–6.99 (m, 2H), 7.11–7.21 (m, 3H), 7.66 (d,
1H, J ¼ 6:2 Hz), 8.83 (d, 1H, J ¼ 6:2 Hz), 9.02 (s, 1H);
MS m=z 307 [Mþ]. Anal. C18H21N5 (307.40) C, H, N.
4.34. 2-(4-Phenylpiperazin-1-yl)-4,5-dihydro-3H-pyr-
ido[2,3-b][1,4]diazepine (7a)
4.38. 1,2,3,4-Tetrahydro-5H-1,4-benzodiazepin-5-one
(34)
A mixture of 26a (82 mg, 0.5 mmol), PCl5 (105 mg,
0.5 mmol), POCl3 (5 mL) and pyridine (two drops) was
heated to 135 °C under a nitrogen atmosphere for 4 h.
After cooling to room temperature volatile components
were evaporated. The residue was dried in vacuum over
night and refluxed in EtOH. Without further purifica-
tion 1-phenylpiperazine (35a) (0.5 g) was added and the
resulting mixture was refluxed in dry EtOH (10 mL) for
another 4 h. The solvent was removed in vacuum. The
residue was purified by column chromatography
(CH2Cl2/MeOH ¼ 94:6) to obtain 7a (74 mg, 48%). Mp
176–178 °C. 1H NMR ([d6]DMSO) 2.61 (t, 4H,
J ¼ 4:6 Hz), 2.86 (t, 2H, J ¼ 7:5 Hz), 3.05 (t, 2H,
J ¼ 7:5 Hz), 3.12 (t, 4H, J ¼ 4:6 Hz), 6.76 (m, 1H, H-8),
6.89–6.95 (m, 2H), 7.13–7.23 (m, 3H), 7.88 (d, 1H,
J ¼ 7:4 Hz), 8.25 (d, 1H), 12.76 (s, br, 1H, exchangeable
with D2O); MS m=z 307 [Mþ]. Anal. C18H21N5 (307.40)
C, H, N.
3,4-Dihydro-1H-1,4-benzodiazepin-2,5-dione (33) (8.8 g,
50 mmol) was added in small portions to a slurry of
LiAlH4 (3.91 g, 115 mmol) in dry THF (100 mL) at a
rate, which causes mild reflux. Refluxing was continued
for 14 h, and then kept at room temperature over night.
The mixture was treated carefully with water (20 mL),
2 N NaOH (15%, 10 mL) and finally with water (50 mL).
The formed slurry was filtered through a Celite pad and
washed with hot THF several times. The filtrate and
washings were combined and extracted with CHCl3.
Combined organic layers were washed with brine, dried
over Na2SO4, and concentrated in vacuum. The residue
was purified by column chromatography (CH2Cl2/
MeOH ¼ 97:3) to yield 34 (2.98 g, 37%) as colorless
powder. Mp 143–145 °C.; IR 2924, 2852, 1660, 1631,
1
1599, 1495 cmꢀ1; H NMR ([d6]DMSO) 3.27–3.43 (m,
4H), 6.39–6.58 (m, 2H), 6.66 (d, 1H, J ¼ 8:4 Hz), 7.09–
7.17 (m, 1H), 7.68–7.75 (m, 1H), 7.93 (t, br, 1H,
J ¼ 5:2 Hz, exchangeable with D2O); MS m=z 162 [Mþ].
Anal. C9H10N2O (162.19) C, H, N.
4.35. 8-Chloro-2-(4-phenylpiperazin-1-yl)-4,5-dihydro-
3H-pyrido[2,3-b][1,4]diazepine (7b)
Preparation and purification (CH2Cl2/MeOH ¼ 97:3)
according to 7a from 26b (99 mg, 0.5 mmol) and
1-phenylpiperazine (35a) (0.5 g) gave 7b (70 mg, 41%).
Mp 153–155 °C; 1H NMR ([d6]DMSO) 2.56 (t, 2H,
J ¼ 6:8 Hz,), 2.72 (t, 4H, J ¼ 5:0 Hz), 3.16 (t, 2H,
J ¼ 6:8 Hz), 3.28 (t, 4H, J ¼ 5:0 Hz), 6.56 (s, br, 1H),
6.83–7.00 (m, 2H), 7.14 (s, 1H), 7.23–7.52 (m, 3H), 7.64
(s, 1H, exchangeable with D2O); MS m=z 341 [Mþ].
Anal. C18H20ClN5 (341.85) C, H, N.
4.39. 1-Allyl-1,2,3,4-tetrahydro-5H-1,4-benzodiazepin-5-
one (36)
To a solution of 34 (2.43 g, 15 mmol) in dry benzene
(10 mL), allyl bromide (1.82, 15 mmol) and triethyl-
amine (0.2 mL) were added and the mixture was refluxed
with stirring for 2 h and allowed to stand over night at
room temperature. The solvent and excess of allyl bro-
mide was removed in vacuum. The residue was treated
with ice water (20 mL) and extracted three times with
diethyl ether (50 mL). Combined ether layers were dried
over Na2SO4 and concentrated in vacuum. The residue
was purified by flash chromatography (cyclohexane/
EtOAc ¼ 6:4) to yield 36 (1.61 g, 53%) as colorless
4.36. 2-[(4-(4-Chlorophenyl)piperazin-1-yl]-4,5-dihydro-
3H-pyrido[2,3-b][1, 4]diazepine (7c)
Preparation and purification (CH2Cl2/MeOH ¼ 96:4)
according to 5a from 26a (82 mg, 0.5 mmol) and 1-(4-
chlorophenyl)piperazine (35d) (0.5 g) gave 7c (70 mg,
41%). Mp 196–198 °C; 1H NMR 2.59 (t, 4H,
J ¼ 4:5 Hz), 2.85 (t, 2H, J ¼ 7:5 Hz), 3.04 (t, 2H,
J ¼ 7:5 Hz), 3.12 (t, 4H, J ¼ 4:5 Hz), 6.93 (d, 2H), 7.14–
7.23 (m, 3H), 7.88 (d, 1H, J ¼ 7:6 Hz), 8.24 (d, 1H,
J ¼ 3:3 Hz), 12.78 (s, br, 1H, exchangeable with D2O);
MS m=z 341 [Mþ]. Anal. C18H20ClN5 (341.85) C, H, N.
1
powder. Mp 92–94 °C; H NMR ([d6]DMSO) 3.20 (m,
4H), 3.80 (d, 2H, J ¼ 5:7 Hz), 5.14–5.27 (m, 2H), 5.77–
5.90 (m, 1H), 6.90 (m, 1H), 7.33 (m, 1H), 7.47 (m, 1H),
8.15 (s, br, 1H, exchangeable with D2O); MS m=z 202
[Mþ]. Anal. C12H14N2O (202.26) C, H, N.
4.40. 1-Allyl-1,2,3,4-tetrahydro-5H-1,4-benzodiazepine-
5-thione (37)
4.37. 4-(4-Phenylpiperazin-1-yl-)-2,3-dihydro-1H-pyr-
ido[3,4-b][1,4]diazepine (8)
Compound 36 (1.52 g, 7.5 mmol) and P2S5 (2.22 g,
10 mmol) were refluxed for 2.5 h in dry pyridine. The
solvent was removed in vacuum. The brown, resinous
residue was suspended in a Na2CO3 solution (10%,
50 mL) and extracted with CH2Cl2. Combined organic
Preparation and purification (CH2Cl2/MeOH ¼ 95:5)
according to 7a from 30 (82 mg, 0.5 mmol) and 1-phen-

T. Hussenether et al. / Bioorg. Med. Chem. 12 (2004) 2625–2637
2635
layers were washed with brine, dried over Na2SO4, and
concentrated in vacuum. The residue was purified by
1 mmol) and 1-(2-methoxyphenyl)piperazine (35c)
(960 mg, 5 mmol) gave 9c (140 mg, 37%) as light yellow
oil. H NMR (CDCl3) 3.10 (s, 3H), 3.22–3.56 (m, 6H),
3.73 (d, 2H, J ¼ 5:1 Hz), 3.80–3.91 (m,, 4H), 5.10–5.28
(m, 2H), 5.74–5.84 (m, 1H), 6.83–7.07 (m, 5H), 7.24–
7.40 (m, 3H); MS m=z 376 [Mþ]. Anal. C23H28N4O
(376.51) C, H, N.
1
flash chromatography (cyclohexane/EtOAc ¼ 7:3) to
1
obtain 37 (1.37 g, 84%) as yellow oil;
H NMR
([d6]DMSO) 3.21–3.30 (m, 4H), 3.75 (d, 2H, J ¼ 5:8 Hz),
5.13–5.29 (m, 2H), 5.73–5.86 (m, 1H), 6.92–7.01 (m,
2H), 7.35 (t, 1H, J ¼ 7:8 Hz), 7.69 (d, 1H), J ¼ 3:9 Hz),
10.68 (s, br, 1H). MS m=z 218 [Mþ]. Anal. C12H14N2S
(218.32) C, H, N.
4.45. 5-(4-Phenylpiperazin-1-yl)-2,3-dihydro-1H-1,4-
benzodiazepine (10a)
4.41. 1-Allyl-5-methylsulfanyl-2,3-dihydro-1H-1,4-benzo-
diazepine (38)
A mixture of 9a (104 mg, 0.3 mmol), Pd/C (10%, 55 mg)
and methanesulfonic acid (29 mg, 0.3 mmol) in dry eth-
anol (5 mL) was refluxed for 2 h under a nitrogen
atmosphere. The reaction mixture was filtered through a
Celite pad. The residue was washed with ethanol and the
filtrate was concentrated in vacuum. The residue was
purified by column chromatography (cyclohexane/
EtOAc/TEA ¼ 50:45:5) to obtain 10a (74 mg, 71%) as
light yellow powder. Mp 129–131 °C. 1H NMR (CDCl3)
3.22 (t, 4H, J ¼ 4:8 Hz), 3.37 (t, 4H, J ¼ 4:8 Hz), 3.56 (t,
2H, J ¼ 5:2 Hz), 3.68 (t, 2H, J ¼ 5:2 Hz), 6.68–6.74 (m,
1H), 6.82–6.99 (m, 4H), 7.18–7.38 (m, 5H); MS m=z 306
[Mþ]. Anal. C19H22N4 (306.41) C, H, N.
Compound 37 (1.31 g, 6 mmol) was dissolved in a solu-
tion of sodium ethoxide (408 mg, 6 mmol) in ethanol
(20 mL). MeI (851 mg, 6 mmol) was added. The reaction
mixture was heated under reflux for 2 h. After cooling,
water (40 mL) was added. The mixture was extracted
with CHCl3. Combined organic layers were washed with
water, dried over Na2SO4, and concentrated in vacuum.
The residue was purified by flash chromatography
(cyclohexane/EtOAc ¼ 9:1) to yield 38 (909 mg, 65%) as
light yellow oil. 1H NMR ([d6]DMSO) 2.35 (s, 3H), 3.43
(t, 2H, J ¼ 5:8 Hz), 3.73 (d, 2H, J ¼ 3:0 Hz), 5.14–5.25
(m, 2H), 5.73–5.87 (m, 1H), 6.90–6.96 (m, 2H), 7.30–
7.41 (m, 2H); MS m=z 232 [Mþ]. Anal. C13H16N2S
(232.35) C, H, N.
4.46. 5-[4-(4-Fluorophenyl)piperazin-1-yl]-2,3-dihydro-
1H-1,4-benzodiazepine (10b)
4.42. 1-Allyl-5-(4-phenylpiperazin-1-yl)-2,3-dihydro-1H-
1,4-benzodiazepine (9a)
Preparation and purification (cyclohexane/EtOAc/
TEA ¼ 50:45:5) according to 10a from 9b (107 mg,
0.3 mmol) gave 10b (67 mg, 69%) as light yellow powder.
Mp 150–151 °C. 1H NMR (CDCl3) 3.16 (t, 4H,
J ¼ 4:6 Hz), 3.41 (t, 4H, J ¼ 4:6 Hz), 3.58 (t, 2H,
J ¼ 4:8 Hz), 3.71 (t, 2H, J ¼ 4:8 Hz), 6.70–6.76 (m, 1H),
6.83–7.01 (m, 5H), 7.20–7.36 (m, 3H); MS m=z 324 [Mþ].
Anal. C19H21FN4 (324.40) C, H, N.
A stirred mixture of 38 (232 mg, 1 mmol), 1-phenylpipe-
razine (35a) (810 mg, 5 mmol), and acetic acid (two
drops) was heated to 120 °C for 12 h. Excess of amine
was removed under reduced pressure. The residue was
purified by column chromatography (cyclohexane/
EtOAc/TEA ¼ 50:47:3) to yield 9a (164 mg, 48%) as light
1
yellow oil. H NMR (CDCl3) 3.20 (m, 4H), 3.39 (m,
8H), 3.72 (d, 2H, J ¼ 5:7 Hz), 5.09–5.29 (m, 2H), 5.73–
5.88 (m, 1H), 6.81–7.04 (m, 5H), 7.21–7.39 (m, 4H); MS
m=z 346 [Mþ]. Anal. C22H26N4 (346.48) C, H, N.
4.47. 5-[4-(2-Methoxyphenyl)piperazin-1-yl]-2,3-dihydro-
1H-1,4-benzodiazepine (10c)
Preparation and purification (cyclohexane/EtOAc/
TEA ¼ 50:45:5) according to 10a from 9c (112 mg,
0.3 mmol) gave 10c (75 mg, 75%) as light yellow powder.
Mp 191–193 °C. 1H NMR (CDCl3) 3.14 (t, 4H,
J ¼ 4:8 Hz), 3.48 (t, 4H, J ¼ 4:8 Hz), 3.58 (t, 2H,
J ¼ 5:3 Hz), 3.70 (t, 2H, J ¼ 5:3 Hz), 3.84 (s, 3H) 6.70–
6.76 (m, 1H), 6.83–7.01 (m, 5H), 7.20–7.36 (m, 3H); MS
m=z 336 [Mþ]. Anal. C20H24N4O (336.44) C, H, N.
4.43. 1-Allyl-5-[4-(4-fluorophenyl)piperazin-1-yl]-2,3-di-
hydro-1H-1,4-benzodiazepine (9b)
Preparation and purification (cyclohexane/EtOAc/
TEA ¼ 50:47:3) according to 9a from 38 (232 mg,
1 mmol) and 1-(4-fluorophenyl)piperazine (35b) (900 mg,
1
5 mmol) gave 9b (154 mg, 43%) as light yellow oil. H
NMR (CDCl3) 3.12–3.53 (m, 12H), 3.86 (d, 2H,
J ¼ 5:2 Hz), 5,16–5.29 (m, 2H), 5.68–5.83 (m, 1H), 6.83–
6.93 (m, 3H), 6.95–7.03 (m, 4H), 7.11–7.20 (m, 1H); MS
m=z 364 [Mþ]. Anal. C22H25FN4 (364.47) C, H, N.
5. Receptor binding studies
Receptor binding assays at the dopamine D1 receptor
were carried out using porcine striatal membranes with a
final protein concentration of 25 lg/assay tube and a Kd
value of 0.27–0.32 nM considering the radioligand
[3H]SCH23390 as previously described.32 Preparations
of membranes from CHO cells expressing human
dopamine D2long, D2short, D3, and D4.4 receptors were
employed for competition binding analysis displacing
4.44. 1-Allyl-5-[4-(2-methoxyphenyl)piperazin-1-yl]-2,3-
di-hydro-1H-1,4-benzodiazepine (9c)
Preparation and purification (cyclohexane/EtOAc/
TEA ¼ 50:47:3) according to 9a from 38 (232 mg,

2636
T. Hussenether et al. / Bioorg. Med. Chem. 12 (2004) 2625–2637
the radioligand [3H]spiperone according to literature.32
The assays were run with a protein concentration of 5–
20 lg/assay tube, with Kd values being 0.10–0.20, 0.10,
0.20, and 0.10–0.30 nM for the D2long, D2short, D3, and
D4.4 receptors, respectively. Protein concentration was
established by the method of Lowry using bovine serum
albumin as standard.41 Screening of receptor binding to
the different dopamine receptors was achieved by mea-
suring the displacement of radio ligands by the test
compounds of representative concentrations of 10,
100 lM, and 1 nM. Compounds reflecting interesting
binding affinities have been further investigated.
Receptor binding studies were carried out as described
in literature.35 Mitogenesis experiments were done
employing CHO 10001 stably expressing the human
dopamine D4.2 receptor as described previously.37;42 In
brief, the 5-HT1A and 5-HT2 receptor assays were done
with porcine cortical membranes at a final protein
concentration of 400 mg/assay tube and 200 lg/assay
tube, respectively. The radioligand [3H]8-OH–DPAT
was used for experiments with 5-HT1A receptors
(Kd ¼ 2:2 nM) and [3H]ketanserin was established for
the 5-HT2 receptor experiments (Kd ¼ 2:7 nM). Both
assays were run with the radioligands at a final con-
centration of 0.5 nM.
2. Davis, K. L.; Kahn, R. S.; Ko, G.; Davidson, M. Am. J.
Psychiatry 1991, 148, 1474.
3. Drogen und Psychopharmaka; Julien, R. M., Ed.; Spek-
trum Akad.: Heidelberg, Berlin, Oxford, 1996; p 287.
4. Sibley, D. R.; Monsma, F. J. Trends. Pharmacol. Sci.
1992, 13, 61.
5. Neve, A. K.; Neve, R. L. In The Dopamine Receptors
Neve; Neve, K. A., Neve, R. L., Eds.; Humana: Totowa,
New Jersey, 1997; pp 27–76.
6. Wagstaff, A. J.; Bryson, H. M. CNS Drugs 1995, 4, 370.
7. Arzneimittelwirkungen; Mutschler, E., Geisslinger, G.,
€
Kroemer, H. K., Schafer-Korting, M., Eds.; Wissenscha-
fliche Verlagsgesellschaft mbH: Stuttgart, 2001; pp 157–
170.
€
€
8. Moll, A.; Hubner, H.; Gmeiner, P.; Troschutz, R. Bioorg.
Med. Chem. 2002, 10, 1671.
9. Kulagowski, J. J.; Broughton, H. B.; Curtis, N. R.;
Mawer, I. M.; Ridgill, M. P.; Baker, R.; Emms, F.;
Freedman, S. B.; Marwood, R.; Patel, S.; Patel, S.; Ragan,
C. J.; Leeson, P. D. J. Med. Chem. 1996, 39, 1941.
€
€
10. Lober, S.; Hubner, H.; Gmeiner, P. Bioorg. Med. Chem.
Lett. 1999, 9, 97.
11. Lanig, H.; Utz, W.; Gmeiner, P. J. Med. Chem. 2001, 44,
1151.
€
12. Hunziker, F.; Kunzle, O.; Schindler, O.; Schmutz, J. Helv.
Chim. Acta 1964, 47, 1163.
13. Bachmann, G. B.; Heisey, L. V. J. Am. Chem. Soc. 1949,
71, 1985.
14. Bauer, A.; Weber, K.-H.; Unruh, M. Arch. Pharm.
(Weinheim, Ger.) 1972, 305, 557.
15. Forlani, L.; Boga, C.; Mazza, M.; Cavrini, V.; Andrisano,
V. Tetrahedron 1998, 54, 4647–4654.
6. Data analysis
16. Tomczuk, B. E.; Taylor, C. R., Jr.; Moses, L. M.;
Sutherland, D. B.; Lo, Y. S.; Johnson, D. N.; Kinnier,
W. B.; Kilpatrick, B. F. J. Med. Chem. 1991, 34, 2993.
17. Jutz, C.; Kirchlechner, R.; Seidel, H. J. Chem. Ber. 1969,
102, 2301.
€
18. Troschutz, R.; Karger, A. J. Heterocycl. Chem. 1997, 34,
1147.
19. Shen, T. Y.; Clark, R. L; Pessolano, A. A.; Witzel, B. E.;
Lanza; T. J. U.S. Patent 4,038,396, 1977; Chem. Abstr.
1977, 87, 152185.
The resulting competition curves of the receptor binding
experiments were analyzed by nonlinear regression using
the algorithms in PRISM (Graph Pad Software, San
Diego, CA). The data were fit in accordance to a sig-
moid model to provide an IC50 value, representing the
concentration corresponding to 50% of maximal inhi-
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Acknowledgements
The authors are indebted to Dr. H. H. M. Van Tol
(Clarke Institute of Psychiatry, Toronto), Dr. J.-C.
Schwartz, and Dr. P. Sokoloff (INSERM, Paris) as well
as Dr. J. Shine (The Garvan Institute of Medical Re-
search, Sydney) for providing dopamine D4, D3, and
D2 receptor expressing cell lines, respectively. Thanks
are also due to Mr. Dr. R. Waibel for NMR data and
interpretation as well as Dr. W. Utz for computer cal-
culations. We thank Mrs. B. Maar, Mrs. M. Unzeitig,
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Hubner for helpful technical assistance.
€
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