Synthesis and pharmacological testing of 1,2,3,4,10,14b-hexahydro-6-methoxy-2-methyldibenzo[c,f]pyrazino[1,2-a]azepin and its enantiomers in comparison with the two antidepressants mianserin and mirtazapine

Article abstract of DOI:10.1021/jm010566d

The synthesis and resolution of 1,2,3,4,10,14b-hexahydro-6-methoxy-2-methy[dibenzo[c,f]pyrazino [1,2-α]azepin (6-methoxymianserin, 6) are described. Furthermore, the in vitro and in vivo effects of 6 and its enantiomers are presented. 6 displayed high aff

Full text of DOI:10.1021/jm010566d

3280
J . Med. Chem. 2002, 45, 3280-3285
Syn th esis a n d P h a r m a cologica l Testin g of
1,2,3,4,10,14b-Hexa h yd r o-6-m eth oxy-2-m eth yld iben zo[c,f]p yr a zin o[1,2-a ]a zep in
a n d Its En a n tiom er s in Com p a r ison w ith th e Tw o An tid ep r essa n ts Mia n ser in
a n d Mir ta za p in e
Ha˚kan V. Wikstro¨m,*,† Margue´rite M. Mensonides-Harsema,† Thomas I. F. H. Cremers,† Ejner K. Moltzen,‡ and
J ørn Arnt‡
Department of Medicinal Chemistry, University Center for Pharmacy, University of Groningen, A. Deusinglaan 1,
NL-9713 AV, Groningen, The Netherlands, and Department of Medicinal Chemistry and Pharmacology, H. Lundbeck A.S.,
Copenhagen, Denmark
Received December 20, 2001
The synthesis and resolution of 1,2,3,4,10,14b-hexahydro-6-methoxy-2-methyldibenzo[c,f]-
pyrazino[1,2-a]azepin (6-methoxymianserin, 6) are described. Furthermore, the in vitro and
in vivo effects of 6 and its enantiomers are presented. 6 displayed high affinity for the 5-HT2A/
2C receptors, only moderate affinity for the adrenoceptors, and no affinity for the NA reuptake
site. Surprisingly, 6 also showed moderate to high affinity for the dopamine D2 receptor, an
effect that resides in the (R)-(-)-enantiomer.
In tr od u ction
effects of 2 are known to reside in the (+)-enantiomer,
while the (-)-enantiomer is responsible for the 5-HT3
receptor antagonism.9-11 Both antidepressants have
negligible affinity for the dopamine D2 receptor.
In this paper, we report the synthesis and resolution
of the 6-methoxy analogue of mianserin (2) 1,2,3,4,10,14b-
hexahydro-6-methoxy-2-methyldibenzo[c,f]pyrazino[1,2-
a]azepin (6). It is noteworthy that the o-methoxyphen-
ylpiperazine moiety, inherent in the chemical structure
of compound 6, is present in several pharmacologically
active compounds that act through the serotonergic
system.12-15
Depression has long been associated with decreased
levels of both noradrenaline (NA) and serotonin (5-HT)
in the central nervous system.1,2 The marketed anti-
depressants mirtazapine (1, azamianserin, Remeron)
and mianserin (2, Tolvon) possess a tetracyclic structure
in which an arylpiperazine moiety is incorporated. The
The in vitro receptor affinities of 6-methoxymianserin
(6) and both its enantiomers at the different dopamin-
ergic, serotonergic, adrenergic, and histaminergic recep-
tor subtypes, as well as at the NA and the 5-HT
reuptake sites, were determined. In a microdialysis
study in the ventral hippocampus of awake, freely
moving rats, the in vivo effects of 5 mg/kg sc of 6 and
2.5 mg/kg sc of either of its enantiomers on the release
of NA and 5-HT and their metabolites dihydroxyphe-
nylacetic acid (DOPAC) and 5-hydroxyindoleacetic acid
(5-HIAA) were established. DOPAC is a metabolite
formed both in dopaminergic and in noradrenergic
neurons. In brain areas with no dopaminergic innerva-
tion, like the locus ceruleus and the ventral hippocam-
pus, DOPAC release is believed to reflect noradrenergic
activity.16,17 The result of this microdialysis study is
compared with previously reported effects of similar
doses of 1 and 2.4
mechanisms of action of these two antidepressants,
however, are believed to be different.
The pharmacological profile of 1 is characterized by
R2, 5-HT2, 5-HT3, and histamine H1 receptor antago-
nist properties. The increase in rat hippocampal levels
of NA and 5-HT after administration of 1 are believed
to be due to R2-adrenoceptor antagonism.3-6 The R2-
autoreceptor and the 5-HT2 receptor blocking effects of
1 are known to reside predominantly in the (+)-
enantiomer, while the (-)-enantiomer is responsible for
blockage of the R2-heteroreceptor (located on the nerve
terminals of the 5-HT neurons) and the 5-HT3 receptor
antagonism.7,8 The pharmacological profile of 2 is
characterized by antagonistic properties at the 5-HT2,
5-HT3, H1 receptor, and the NA reuptake site. The
increase in rat hippocampal levels of NA after admin-
istration of 2 is believed to be due to blockage of the
NA reuptake site. The NA reuptake and 5-HT2 blocking
Ch em istr y
6 was synthesized as follows (Scheme 1). Styrene
oxide and 2-methylaminoethanol were reacted at 130
°C, yielding â-hydroxy-N-methyl-N-hydroxyethylphenyl
ethylamine (3), which was converted to the chlorinated
phenylethylamine 4 upon treatment with thionyl chlo-
ride in refluxing chloroform. 2-Amino-3-methoxybenzoic
acid was reduced to the corresponding alcohol in THF
* To whom correspondence should be addressed. Phone: +31-50-
3633297. Fax: +31-50-3636908. E-mail: h.v.wikstrom@farm.rug.nl.
† University of Groningen.
‡ H. Lundbeck A.S.
10.1021/jm010566d CCC: $22.00 © 2002 American Chemical Society
Published on Web 06/19/2002

Synthesis and Pharmacological Testing
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 15 3281
Sch em e 1a
serotonin 5-HT1A, 5-HT1D, 5-HT2A, 5-HT2C, 5-HT3,
and R1- and R2-adrenergic, histamine H1 and H2
receptors, and the 5-HT and NA reuptake sites were
determined.20-29 The extracellular levels of NA, DOPAC,
5-HT, and 5-HIAA in the ventral hippocampus were
measured by in vivo microdialysis after administration
of 5 mg/kg sc of the racemate of 6-methoxymianserin
or 2.5 mg/kg sc of either of the enantiomers. Levels were
measured every 15 min for a period of 2.5 h after
administration of the test compound. Before adminis-
tration of test compounds, four consecutive samples
were collected (baseline).30
a
Reagents: (a) 2-methylaminoethanol, 130 °C, 17 h; (b) SOCl2,
CH2Cl2, reflux, 3 h; (c) 2-amino-3-methoxybenzyl alcohol, N,N-
diisopropylethylamine, acetonitrile, reflux, 7 h; (d) PPA, 100 °C,
2 h.
Resu lts a n d Discu ssion
Sch em e 2a
The binding study (Table 1) shows that 6 has high
affinity for the 5-HT2A and 5-HT2C receptors, which
is found in both enantiomers. While both 1 and 2 have
high affinity for the 5-HT3 receptor, 6 is devoid of
affinity for this receptor subtype. 6 displays only moder-
ate affinity and no selectivity for the R1- and R2-
adrenoceptors and displays no affinity for the NA
reuptake site. The (S)-(+)-enantiomer of 6 shows re-
markable selectivity for the 5-HT2 receptor subtypes.
Surprisingly, the (R)-(-)-enantiomer showed a moderate
affinity for the dopamine D2 receptor. In antipsychotic
research, the ratio 5-HT2A/D2 affinity, the “Meltzer
ratio”, is used as a marker to classify atypical neuro-
leptics such as clozapine.31 The “Meltzer ratio” of (-)-6
to clozapine is highly favorable.32 The moderate affinity
of the (-)-enantiomer for the R2-adrenoceptor may add
to a possible positive antipsychotic profile, since it was
recently shown that R2-antagonists potentiate the corti-
cal output of dopamine.33 The two other 6-substituted
analogues 7 and 8 were only tested on a few receptors
(Table 1), and these data are not further discussed.
In a previous microdialysis study in the rat ventral
hippocampus, it was shown that 1 (5 mg/kg sc) causes
an increase in DOPAC levels up to 200% above baseline
(90-150 min) and a transient increase in 5-HT levels
up to 180% after 60 min.4 A similar dose of 2 induced
an increase in DOPAC levels up to 160% above baseline
(90-150 min) but had no effect on the 5-HT release.4
Administration of 1 had no effect on the 5-HIAA release.
Administration of 6 (5.0 mg/kg sc) causes an increase
in NA levels up to 200% (45-150 min) and in DOPAC
levels up to 160% above baseline (60-150 min). Fur-
a
Reagents: (a) AlCl3, benzene, reflux, 17 h; (b) trifluoromethane-
sulfonyl anhydride, CH2Cl2, TEA, -78 °C to room temperature,
17 h; (c) Pd(OAc)2, PPh3, TEA, MeOH, reflux, 48 h.
using lithium aluminum hydride, and the resulting
compound was subsequently coupled to 4 in refluxing
acetonitrile in the presence of diisopropylethylamine,
yielding 1-methyl-3-phenyl-4-(2-methoxy-6-hydroxyethyl)-
phenylpiperazine‚HCl (5). Ring closure of 5 was ac-
complished upon treatment with PPA at 100 °C, afford-
ing 6.
The direct separation of the enantiomers of 6 was
achieved by means of HPLC using a semipreparative
Chiralcel OD column.18 Determination of the absolute
configuration was carried out indirectly by chemically
linking the levorotatory isomer (-)-6 to (R)-(-)-2 (Scheme
2).19
P h a r m a cology
The ability of 1, 2, 6, and its enantiomers to displace
the respective radioligands at the dopamine D2, D3, D4,
Ta ble 1. In Vitro Binding Profiles of (()-, (+)-, and (-)-6, (()-7, (()-8, (()-2, and (()-1 (Ki in nM)a
affinity Ki (nM)
affinity IC50 (nM)
receptor
(()-6
28 ( 9
260 ( 45
92 ( 6
345 ( 20
nt
0.70 ( 0.1
0.21 ( 0.03
2900 ( 35
84 ( 15
nt
(+)-6
(-)-6
(()-2
(()-1
(()-7
(()-8
D2
D3
D4.2
460 ( 120
3570 ( 410
160 ( 11
800 ( 130
1470 ( 150
0.55 ( 0.09
0.16 ( 0.01
>5000
20 ( 5
100 ( 6
47 ( 2
1000 ( 5
nt
1460 ( 430
>20
>25
>18
nt
nt
nt
nt
nt
12
2.0
nt
1000
1000
21
nt
nt
nt
nt
nt
277
nt
nt
nt
nt
5-HT1A
5-HT1D
5-HT2A
5-HT2C
5-HT3
alpha1
alpha2
H1
255 ( 61
80 ( 20
0.75 ( 0.18
0.39 ( 0.11
1650 ( 80
74 ( 12
37 ( 8
>500
nt
nt
1.5 ( 0.6
1.4 ( 0.1
70 ( 3
28 ( 5
nt
2.0 ( 0.4
5.5 ( 0.8
nt
205 ( 31
73 ( 24
13 ( 6
1050 ( 130
nt
1000
nt
nt
nt
56
14 ( 4
4.2 ( 0.3
5.1 ( 0.9
nt
H2
60
43
nt
nt
IC50 (nM)
2860 ( 630
NA-upt
5-HT-upt
1780 ( 100
2900 ( 100
1240 ( 70
1650 ( 80
22 ( 6
2900 ( 340
260 ( 99
>100
nt
nt
nt
nt
>10000
a
Affinities of compounds were determined using competition binding assays to determine IC50 values at the various receptors. See
text.

3282 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 15
Wikstro¨m et al.
atypical antipsychotic clozapine justifies future studies
of this enantiomer for its potential as an antipsychotic
agent.
Exp er im en ta l Section
Gen er a l. Melting points were determined with an electro-
thermal digital capillary melting point apparatus and are
uncorrected. NMR spectra were recorded at 200 and 300 MHz
on Varian Gemini and Varian VXR spectrometers, respec-
tively. CDCl3 was employed as the solvent unless otherwise
stated. Chemical shifts are given in δ units (ppm) and relative
to deuterated solvent. Infrared spectra were recorded with an
ATI-Mattson spectrometer. Elemental analyses were per-
formed in the labs of Merck KGaA Darmstadt (D) or in the
Microanalytical Department of the University of Groningen
(NL) and were within (0.4% of theoretical values unless
otherwise indicated. GC-MS (EI and CI) data were recorded
on a Unicam610 Automass 150 system (70 mV). HRMS spectra
were performed in the Microanalytical Department of the
University of Groningen (NL), and were recorded on a J EOL
The MS Route J MS 600H. For column chromatography, silica
gel 60 (70-230 mesh) (Merck) was used. Reactions were
carried out under a nitrogen atmosphere, solvents used were
distilled and/or dried by standard techniques immediately
prior to use, and in case of an aqueous workup, magnesium
sulfate monohydrate was used as the drying agent. HPLC
experiments were carried out using a semipreparative LC
system consisting of a Chiralcel OD column: Daicel Chemical
Industry Ltd. (Tokyo, J apan) 25 cm × 1.0 cm i.d., 10 µm
particle size silica gel; ISCO 2300 Waters HPLC pump
equipped with a Rheodyne injection valve and a 20 or 100 µL
injection loop; ISCO V4 absorbance detector with a 5 µL flow
cell (λ ) 250 nm, pathway 5 mm); Kipp and Zonen BD 40 two
channel recorder. The mobile phase used was n-hexane with
5% ethanol (both HPLC grade) and 0.1% triethylamine (ana-
lytical grade). Flow rate was set at 2.5-4.0 mL/min (ca. 0.15
psi). Optical rotations were measured in methanol (c ) 2.0)
at 21 °C on a Perklin-Elmer 241 polarimeter.
F igu r e 1. Effects of 6-methoxymianserin and its enantiomers
on dopaminergic, noradrenergic, and serotonergic transmis-
sion. Effects were measured by on-line microdialysis of the
racemic mixture 6 (5.0 mg/kg sc, n ) 4) and the (R)-(-)-6 and
(S)-(+)-6 enantiomers (each 2.5 mg/kg sc, n ) 6) on NA,
DOPAC, 5-HT, and 5-HIAA release in the rat ventral hippo-
campus. Results are expressed as the area under the curve
and refer to cumulative percentage above baseline. Statistics
are from Mann-Whitney rank sum test: (/) p < 0.05 compared
to control; (#) p < 0.05 compared to 2.
â-H yd r oxy-N -m e t h yl-N -h yd r oxye t h ylp h e n yle t h yl-
a m in e (3). 2-Methylaminoethanol (4.90 g, 65.3 mmol) was
heated to 100 °C. To this, styrene oxide (5.30 g, 44.2 mmol)
was added dropwise. The temperature was raised to 130 °C,
and the mixture was stirred for 24 h, after which it was
purified by flash chromatography (CH2Cl2 with 10% EtOH)
yielding 7.5 g (57.5 mmol, 88%) of 3 as a light-yellow oil: 1H
thermore, it induces a concurrent, transient increase of
5-HT levels up to 200% above baseline at 60 min after
administration of 6, with stabilization at 140-150%
further during the course of the experiment. Adminis-
tration of (-)- or (+)-6 (2.5 mg/kg sc) also resulted in
an increase in NA levels up to 200% (45-150 min). The
DOPAC levels were elevated to 160% and 130% above
baseline for (-)- and (+)-6, respectively (60-150 min).
Both enantiomers induced a transient, concurrent in-
crease of the 5-HT release up to 200% above baseline
at 60 min after administration of the test compound.
The 5-HIAA release was elevated up to 120% after the
administration of the racemic mixture or of either of the
enantiomers (significant for the (-)-enantiomer). For a
graphical representation of the microdialysis data, see
Figure 1.
In summary, 6, a new tetracyclic azepine, showed
high affinity for the 5-HT2A/2C receptors, moderate
affinity for the R1- and R2-adrenoceptors, and no affinity
for the 5-HT3 receptor and for the NA reuptake sites.
In vivo, it increases the levels of NA and 5-HT to the
same extent as 1. The (R)-(-)-enantiomer of 6 appears
to be more potent in the microdialysis study than the
(S)-(+)-enantiomer (significant only for the DOPAC
release). This implies that at least the neurochemical
effects of 6 are similar to those of the antidepressant 1.
Future studies in animal models of depression will have
to prove whether 6 has prospective antidepressant
properties. The unexpected finding that (R)-(-)-6, in
vitro, shows moderate affinity for the D2 receptor
subtype and a favorable “Meltzer ratio” compared to the
NMR δ 7.40-7.16 (m, 5H), 4.80-0.4.73 (dd, J 1 ) 9.64, J 2
)
3.79, 1H), 3.69-3.60 (m, 2H), 3.50 (broad peak, OH), 2.79-
2.41 (m, 4H), 2.38 (s, 3H); 13C NMR δ 142.2, 128.3, 127.5, 125.9,
70.4, 61.4, 59.3, 54.9, 42.3; MS (EIPI) m/z 195 (M+).
â-Ch lor o-N-m eth yl-N-ch lor oeth ylph en yleth ylam in e (4).
To an ice-cooled solution of 3 (1.30 g, 6.70 mmol) in CHCl3 (20
mL) a solution of thionyl chloride (10 mL) in CHCl3 (20 mL)
was slowly added. The reaction mixture was refluxed for 2 h,
after which it was quenched with H2O (10 mL). The aqueous
layer was basified with a 2 N aqueous NaOH solution and
extracted with CHCl3. The combined organic layers were
washed with brine, dried, and concentrated in vacuo, yielding
1.00 g (4.49 mmol, 67%) of 4 as a brown oil. The intermediate
was used without further purification: 1H NMR δ 7.43-7.32
(m, 5H), 4.93 (t, J ) 7.08, 1H), 3.48 (t, J ) 7.10, 2H), 3.18-
2.94 (m, 2H), 2.83 (t, J ) 7.05, 2H), 2.38 (s, 3H); 13C NMR δ
140.2, 128.6, 128.4, 127.3, 65.6, 60.8, 59.3, 42.6, 41.3; MS
(EIPI) m/z 231 (M+).
2-Am in o-3-m eth oxyben zyl Alcoh ol. To an ice-cooled
solution of 2-amino-3-methoxybenzoic acid (2.50 g, 15.0 mmol)
in THF (150 mL) lithium aluminum hydride (2.30 g, 60.5
mmol) was added in small portions. The reaction mixture was
stirred at room temperature for 17 h. The reaction was
quenched subsequently with H2O (2.3 mL), a solution of 10%
aqueous NaOH (2.3 mL), and three more portions of H2O (2.3
mL). The mixture was stirred for 2 h at room temperature after
which the solvent was removed in vacuo. The residue was
purified using flash chromatography (CH2Cl2 with 2.5% metha-
nol), yielding 1.90 g (12.3 mmol, 82%) of 2-amino-3-methoxy-

Synthesis and Pharmacological Testing
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 15 3283
benzyl alcohol (dark-brown solid): mp 38 °C; 1H NMR δ 6.81-
6.68 (m, 3H), 4.60 (s, 2H), 3.86 (s, 3H); 13C NMR δ 147.5, 135.5,
121.2, 117.4, 110.2, 63.7, 55.7; IR (NaCl) 3367 cm-1 (OH); MS
(EIPI) m/z 153 (M+).
Ltd. (Tokyo, J apan) 25 cm × 1.0 cm i.d., 10 µm particle size
silica gel; ISCO 2300 HPLC pump equipped with a Rheodyne
injection valve and 1 mL injection loop; ISCO V4 absorbance
detector with a 5 µL flow cell (λ ) 250 nm, pathway 5 mm);
Kipp and Zonen BD 40 two-channel recorder; mobile phase,
n-hexane with 5% ethanol (HPLC grade) and 0.1% triethyl-
amine (analytical grade); flow rate of 4.0 mL/min at ca. 0.5
psi. The favorable separation of the two enantiomers allowed
for injecting 5 mg of racemic 6 per run. The direct separation
by means of HPLC using a Chiralcel OD column afforded 50
1-Meth yl-3-p h en yl-4-(2-m eth oxy-6-h yd r oxyeth yl)p h en -
ylp ip er a zin e (5). To a solution of 2-amino-3-methoxybenzyl
alcohol (2.70 g, 11.6 mmol) in acetonitrile (20 mL) was added
a solution of 4 (1.20 g, 7.80 mmol) in acetonitrile (10 mL). The
reaction mixture was stirred at room temperature for 30 min.
N,N-Diisopropylethylamine (1.00 g, 7.80 mmol) was added, and
the reaction mixture was refluxed for 7 h, after which it was
stirred at room temperature for 16 h. The formed hydrochlo-
ride salt was filtered and recrystallized from acetonitrile to
give 1.80 g (5.77 mmol, 74%) of 5 as white crystals (HCl salt):
mp 222 °C; 1H NMR (300 MHz, CD3OD) δ 6.71-6.32 (m, 7H),
4.47 (s, 3H), 4.42-4.38 (m, 1H), 3.96-3.91 (m, 1H), 3.48 (m,
1H), 3.35 (s, 2H), 2.92-2.85 (m, 1H), 2.72-2.68 (m, 1H), 2.50-
2.45 (m, 1H), 2.35-2.32 (m, 1H); 13C NMR (CD3OD) δ 157.8,
140.7, 137.0, 133.2, 128.0, 127.8, 127.5, 126.9, 119.6, 110.0,
60.8, 60.5, 59.2, 53.9, 53.8, 48.5, 42.2; MS (EIPI) m/z 312 (M+).
1,2,3,4,10,14b-Hexa h yd r o-6-m eth oxy-2-m eth yld iben zo-
[c,f]p yr a zin o[1,2-a ]a zep in (6). Compound 5 (1.30 g, 4.20
mmol) was suspended in polyphosphoric acid (14 g), and the
mixture was stirred at a 100 °C for 2 h. The reaction was
quenched with ice (140 mL), after which CH2Cl2 (140 mL) was
added. The mixture was basified with 2 N aqueous NaOH. The
organic layer was separated from the aqueous layer, which
was subsequently extracted with CH2Cl2. The combined or-
ganic layers were washed with brine, dried, and concentrated
in a vacuum. The residue was purified by flash chromatogra-
phy (CH2Cl2 with 5% EtOH), yielding 0.89 g (3.07 mmol, 73%)
of 6 as a pinkish solid. Of the obtained product, 0.29 g was
converted to the oxalate salt and recrystallized from ethanol,
yielding 0.31 g (2.45 mmol, 80%) of 6 as off-white crystals; mp
205 °C (monooxalate salt); 1H NMR (300 MHz, CD3OD) δ
6.77-6.68 (m, 4H), 6.36-6.21 (m, 3H), 4.47 (s, 3H), 4.44-4.39
(m, 1H), 4.20-4.10 (m, 1H), 3.75-3.65 (m, 1H), 3.02-2.68 (m,
4H), 2.91 (s, 3H), 2.43 (s, 2H); 13C NMR (CD3OD) δ 154.0,
143.3, 140.4, 137.0, 128.7, 127.8, 126.6, 126.1, 123.3, 118.6,
111.2, 63.4, 62.9, 55.8, 54.5, 50.9, 48.2, 47.8, 44.5, 38.2; MS
(EIPI) m/z 294 (M+); HRMS calc (obsd) for C19H22N2O, 294.173
(294.172).
mg each of the base form of the enantiomers (-)-6 ([R]21
D
-403.0°) and (+)-6 ([R]21 +407.6°), both rotations measured
D
at c ) 2.0 mg/mL MeOH.
In d ir ect Deter m in a tion of th e Absolu te Con figu r a tion
of (-)-6. O-Demethylation of an analytical amount of (-)-6
(20 mg) was effected, as descibed for the preparation of racemic
7 above, upon treatment with 6 equiv of AlCl3 in freshly
distilled, refluxing benzene, providing (-)-6-hydroxymianserin
((-)-7), which was triflated without further purification with
trifluoromethanesulfonyl anhydride in CH2Cl2 and in the
presence of triethylamine. This yielded 6-trifluoromethane-
sulfonyloxymianserin ((-)-8), which was detriflated, without
further purification, with palladium acetate in refluxing
methanol in the presence of triphenylphospine and triethyl-
amine to afford, after purification, 10 mg of (-)-2 (Scheme 2),
as determined by measuring the k′, which under the present
condition was measured to be 0.77, which is the same as that
measured for an authentic sample of (-)-2 ((+)-2 shows a k′
) 1.00).19
Biologica l Assa ys. For the microdialysis experiments male
Wistar rats (280-350 g) were used. The animals were housed
in groups (four rats per cage) in a temperature- and humidity-
controlled colony room (20 ( 2 °C; 50-60%) on a natural day-
night cycle (light period 6:30-18:30) until surgery. Food and
water were available ad libitum at all times. Testing was done
between 10:00 and 18:00 during the light phase of the day-
night cycle. Procedures were conducted in accordance with
guidelines published in the NIH Guide for the Care and Use
of Laboratory Animals, and protocols were approved by the
Groningen University Institutional Animal Care and Use
Committee.
Affinities of compounds were determined using competition
binding assays to determine IC50 values at the various recep-
tors: dopamine D2 receptors, displacement of 0.50 nM [3H]-
spiperone at membranes from rat corpus striatum;20 h-dopam-
ine D3 receptor, displacement of 0.30 nM [3H]-spiperone at
human-cloned D3 receptors expressed in CHO-cells;21 h-
dopamine D4.2 receptor, displacement of 0.06 nM [3H]-YM-
09151-2 at human-cloned D4.2 receptors expressed in CHO-
cells (method modified from NEN Life Science Products, Inc.,
technical data certificate PC2533-10/96); h-serotonin 5-HT1A
receptor, displacement of 2 nM [3H]-5-CT at human-cloned
5-HT1A receptors expressed in HeLa cells.22 Additional pa-
rameters are as follows: serotonin 5-HT1D receptor, displace-
ment of 2 nM [3H]-5-CT at membranes from bovine caudate
tissue;23 serotonin 5-HT2A receptor, displacement of 0.50 nM
[3H]-ketanserin at membranes from rat cortex tissue;24 sero-
tonin 5-HT2C receptor, displacement of 0.50 nM [3H]-me-
sulergine from rat cloned 5-HT2C receptors expressed in NIH/
3T3 cells (method available from H. Lundbeck A/S, Copenhagen);
serotonin 5-HT3 receptor, displacement of [3H]-LY-278584 at
membranes of rat brain tissue;25 R1-adrenoceptor, displace-
ment of 0.25 nM [3H]-prazosin at membranes of rat brain
tissue;20 R2-adrenoceptor, displacement of 0.50 nM [3H]-RX-
821002 at membranes of rat brain tissue;26 histamine H1
receptor, displacement of 1.0 nM [3H]-pyrilamine at mem-
branes of guinea pig lung tissue;27 histamine H2 receptor,
displacement of 0.10 nM [3H]-APT at membranes of guinea
pig striatum tissue.28 Inhibition of 5-HT and NA uptake into
rat brain synaptasomes was described by K. P. Bøgesø.29 Data
calculations are as follows: Results on receptor binding studies
are given as Ki values (nM). Two complete concentration-
response curves were determined by using five concentrations
of the test drugs in triplicate (covering 3 decades). IC50 values
1,2,3,4,10,14b-Hexa h yd r o-6-h yd r oxy-2-m eth yld iben zo-
[c,f]p yr a zin o[1,2-a ]a zep in (7). To a solution of 6 (300 mg,
1.02 mmol) in freshly distilled benzene (30 mL) aluminum
trichloride (1.00 g, 7.59 mmol) was added. The reaction was
refluxed for 48 h, after which the solvent was removed in a
vacuum. The residue was purified by flash chromatography
(CH2Cl2 with 5% ethanol basified with 25% NH4OH(aq)),
yielding 150 mg (0.53 mmol, 53%) of 7 as a white foam and
100 mg (33%) of starting material: 1H NMR (300 MHz, CD3-
OD) δ 6.89-6.28 (m, 7H), 4.69-4.23 (m, 3H), 3.44 (s, 3H),
3.32-2.84 (m, 4H), 2.58 (s, 2H); 13C NMR δ 129.1, 127.9, 125.6,
118.9, 116.6, 63.0, 61.6, 56.3, 49.5, 44.4, 39.7; MS (EIPI) m/z
280 (M+); HRMS calc (obsd) for C18H20N2O, 280.158 (280.158).
1,2,3,4,10,14b-Hexah ydr o-2-m eth yl-6-tr iflu or om eth an e-
su lfon oxyd iben zo[c,f]p yr a zin o[1,2-a ]a zep in (8). A solu-
tion of 7 (150 mg, 0.53 mmol) and triethylamine (1 mL) in
CH2Cl2 (15 mL) was cooled to -78 °C, after which trifluo-
romethanesulfonic anhydride (90 µL) was added. The reaction
mixture was subsequently stirred at room temperature for 4
h. The solvent was removed in a vacuum, and the residue was
purified by flash chromatography (CH2Cl2 with 5% ethanol),
yielding 220 mg (0.53 mmol, 100%) of 8 as a brown oil: 1H
NMR δ 7.28-6.82 (m, 7H), 4.72-4.48 (dd, J 1 ) 0.18, J 2 ) 0.06,
2H), 3.74 (t, J ) 0.05, 1H), 3.49 (d, J ) 0.07, 1H), 3.15 (d, J )
0.06, 1H), 2.94-2.73 (dd, J 1 ) 0.15, J 2 ) 0.06, 2H), 2.55-2.25
(m, 2H), 2.36 (s, 3H); 13C NMR δ 128.2, 126.5, 126.1, 125.7,
125.2, 121.1, 119.6, 57.9, 54.5, 50.1, 44.5, 37.7; MS (EIPI) m/z
412 (M+); HRMS calc (obsd) for C19H19N2O3F3S, 412.107
(412.108).
Ch r om a togr a p h ic (HP LC) Resolu tion of Ra cem ic 6 on
a Ch ir a l Sta tion a r y P h a se. The semipreparative LC system
consisted of a Chiralcel OD column: Daicel Chemical Industry

3284 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 15
Wikstro¨m et al.
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Pharmacol. 1994, 253, R5-R6.
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Differences in modulation of noradrenergic and serotonergic
transmission by the alpha-2 adrenoceptor antagonists, mirtaza-
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were estimated from hand-drawn log-logit analysis. In a
series of n determinations, the variance of the log ratio (VARR)
between the double determinations is determined according
to the formula VARR ) Σ(log R1)2/(2n), where R1 is the ratio
and n is the number of observations. The VARR is equivalent
to the square of the standard deviation of the log ratio (SDR2).
In case the ratio is greater than that corresponding to 2 ×
SDR (95% confidence interval), further determinations were
performed and outliers discarded. For the binding analysis,
the following SDRs were obtained: D2, 1.5 (n ) 100); D3, 1.4
(n ) 26); D4.2, 1.4 (n ) 26); 5-HT1A, 1.5 (n ) 24); 5-HT1D,
see ref 23; 5-HT2A, 1.4 (n ) 100); 5-HT2C, 1.3 (n ) 24); R1,
1.5 (n ) 100), see ref 26; H1, see ref 27; H2, see ref 28; 5-HT
and NA uptake, 95% confidence ratios of 2.04 and 2.68,
respectively.3
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Microdialysis30 rats were anaesthetized with ketamine/
xylazine (50/8 mg/kg ip) and atropine (0.1 mg/kg sc) after a
premedication of midazolam (5 mg/kg sc). Preceding insertion
of the microdialysis probe, rats were placed in a stereotaxic
frame (Kopf). Following local lidocaine application (10% m/v),
a homemade I-shaped probe, made of polyacrylonitrile/sodium
methyl sulfonate copolymer dialysis fiber (i.d. 0.22 mm, o.d.
0.31 mm, AN 69, Hospal, Italy) was implanted in the ventral
hippocampus (coordinates: (IA) +3.7 mm; (lateral) +4.8 mm;
(ventral) -8.0 mm from the Dura Mater, Paxinos and Watson,
1982) and secured with stainless steel screws and dental
cement. The exposed length of the membrane was 4 mm. After
surgery, rats were allowed a recovery period of at least 24 h
before starting the experiments. Probes were perfused with
artificial CSF containing 147 mM NaCl, 3.0 mM KCl, 1.2 mM
CaCl2, and 1.2 mM MgCl2, at a speed of 1.5 µL/min (Harvard
apparatus, South Natick, MA). Rats were housed individually
in Plexiglas cages (25 cm × 25 cm × 35 cm) with food and
water ad libitum. Samples were collected on-line in a 20 µL
loop and injected automatically onto the column every 15 min.
5-HT was analyzed using an HPLC pump (Shimadzu LC-10
AD liquid chromatograph) in combination with a reversed-
phase column (Phenomenex hypersil 3 C18, 100 mm × 2.0 mm,
3 µm, Bester, Amstelveen, The Netherlands) and an EC
detector with a carbon electrode (Intro, Antec Leyden, Leiden,
The Netherlands) working at a potential of 500 mV vs Ag/
AgCl reference electrode. The mobile phase consisted of 5.0
g/L (NH4)2SO4, 0.5 g/L EDTA, 50.0 mg/L heptanesulfonic acid,
and 30 µL/L triethylamine in 4.5% methanol (v/v) at pH 4.65
and 30 °C. The flow of the mobile phase was set at 400 µL/
min. The detection limit of 5-HT was 0.5 fmol per 20 µL of
sample (signal-to-noise ratio of 2). NA, DOPAC, and HIAA
were analyzed using an HPLC pump (Shimadzu LC-10 AD
liquid chromatograph) in combination with a reversed-phase
column (C18, 15 mm × 0.46 mm, 3 m, Supelco, Zwijndrecht,
The Netherlands). Quantification was performed using an
electrochemical detector (ESA, Bedford, MA). Upon oxidation
at 175 mV, molecules were reduced at -250 mV. The mobile
phase consisted of 4.1 g/L sodiumacetate, 140.0 mg/L octane-
sulfonic acid, 50.0 mg/L EDTA, and 7% methanol (v/v (pH )
4.4.). At a flow of 1 mL/min, the detection limit of the assay
was 2 fmol/sample (signal-to-noise ratio of 2). Four consecutive
microdialysis samples with less than 20% difference were
considered as control and set at 100%. Data are presented as
percentages of control level (mean + SEM). Statistical analysis
was performed using Sigmastat for Windows (J andel Corpora-
tion). Treatment values were compared using two-way ANOVA
for repeated measurements, followed by Student Newman-
Keuls posthoc analysis. For the area under the curves, a
Mann-Whitney rank sum test was performed. Significance
levels were set at p < 0.05.
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