Dopamine/serotonin receptor ligands. Part IV [1]: Synthesis and pharmacology of novel 3-benzazecines and 3-benzazonines as potential 5-HT2A and dopamine receptor ligands

Article abstract of DOI:10.1002/1521-4184(200212)335:9<443::AID-ARDP443>3.0.CO;2-U

LE 300 represents a structurally novel type of antagonist acting preferentially at the dopamine D₁/D₅ receptors and the serotonin 5-HT_2A receptor. The compound consists of a 10-membered central azecine ring fused to indole on one and to benzene on the other side. To estimate the importance of the indole moiety in this highly active benz-indolo-azepine, the indole has to be removed and the de-indolised analog reinvestigated pharmacologically. Accordingly, we synthesized 3-benzazecines and in addition some homologuous 3-benzazonines. Methoxylated β-phenylethylamines were treated with ethyl ω-bromo-butanoates and -pentanoates, respectively, to give the corresponding lactams which were cyclized (POCl₃) and reduced (NaBH₄), yielding the cis-annelated (X-ray) benzindolizines and -quinolizines. The 10- and 9-membered rings were obtained by cleavage of the central C-N bond, which was performed in the following two ways: Quarternisaion with methyl iodide and cleavage with sodium in liquid ammonia gave the NCH₃ derivatives, reaction with benzyloxycarbonyl chloride/NaBH₄ followed by catalytic debenzylation yielded a corresponding NH compound. Functional experiments on rat artery segments precontracted with ketanserin and radioligand binding experiments using human cloned dopamine receptor subtypes were conducted with all of the benzazecine and benzazonine derivatives. In contrast to the benz-indolo-compound LE 300 they did not show any significant affinity towards the D₁, D₂, D₄, and D₅ receptors and only moderate antagonistic activity at the 5-HT_2A receptor. It can be concluded from our study that an indole moiety or at least another second aromatic system at the central azecine ring is part of the pharmacophore and thus essential for high biological activity.

Full text of DOI:10.1002/1521-4184(200212)335:9<443::AID-ARDP443>3.0.CO;2-U

Arch. Pharm. Pharm. Med. Chem. 2002, 9, 443–448
4,5-Bis(4-hydroxyphenyl)imidazoles 443
Hussein El-Subbagha, b
Thomas Wittigb,
Michael Deckerb,
Sigurd Elzc,
,
Dopamine/Serotonin Receptor Ligands. Part IV [1]:
Synthesis and Pharmacology of Novel
3-Benzazecines and 3-Benzazonines as Potential
5-HT2A and Dopamine Receptor Ligands
Martin Niegerd,
Jochen Lehmannb
LE 300 represents a structurally novel type of antagonist acting preferentially at the
dopamine D1/D5 receptors and the serotonin 5-HT2A receptor.The compound con-
sists of a 10-membered central azecine ring fused to indole on one and to benzene
on the other side.To estimate the importance of the indole moiety in this highly active
benz-indolo-azepine, the indole has to be removed and the “de-indolised” analog
reinvestigated pharmacologically.Accordingly, we synthesized 3-benzazecines and
in addition some homologuous 3-benzazonines. Methoxylated β-phenylethyl-
amines were treated with ethyl ω-bromo-butanoates and -pentanoates, respec-
tively, to give the corresponding lactams which were cyclized (POCl3) and reduced
(NaBH4), yielding the cis-annelated (X-ray) benzindolizines and -quinolizines. The
10- and 9-membered rings were obtained by cleavage of the central C–N bond,
which was performed in the following two ways: Quarternisaion with methyl iodide
and cleavage with sodium in liquid ammonia gave the NCH3 derivatives, reaction
with benzyloxycarbonyl chloride/NaBH4 followed by catalytic debenzylation yielded
a corresponding NH compound. Functional experiments on rat artery segments
precontracted with ketanserin and radioligand binding experiments using human
cloned dopamine receptor subtypes were conducted with all of the benzazecine and
benzazonine derivatives. In contrast to the benz-indolo-compound LE 300 they did
not show any significant affinity towards the D1, D2, D4, and D5 receptors and only
moderate antagonistic activity at the 5-HT2A receptor. It can be concluded from our
study that an indole moiety or at least another second aromatic system at the central
azecine ring is part of the pharmacophore and thus essential for high biological ac-
tivity.
a
Department of Medicinal
Chemistry,
Faculty of Pharmacy,
University of Mansoura,
Mansoura, Egypt
Institute of Pharmacy,
Pharmaceutical/Medicinal
Chemistry,
Friedrich-Schiller-
University Jena,
Jena, Germany
Institute of Pharmacy,
Regensburg University,
Regensburg, Germany
Institute of Inorganic
Chemistry, University
of Bonn,
b
c
d
Bonn, Germany
Keywords: 3-Benzazonine; 3-Benzazecine; 5-HT2A ligand; Dopamine receptor
ligand; LE 300
Received: March 12, 2002 [FP682]
quently, selective 5-HT2A receptor antagonists showed a
therapeutic efficacy for the treatment of mental and car-
Introduction
Serotonin (5-HT) plays a major role in the regulation of
numerous physiological functions including affective be-
havior, memory, and thermo-regulation [2], through the
interaction with a number of serotonin receptor subtypes
[3]. The 5-HT2A receptor subtype received particular at-
tention because it is well characterized in the human
body with respect to distribution and function. Stimula-
tion of 5-HT2A receptor would result in psychiatric symp-
toms, vasoconstriction, and platelet aggregation;conse-
diovascular illness [4]. On the other hand, various
dopamine subtype antagonists appear to offer promis-
ing approaches for the development of neuroleptic type
drugs [5–7].Several 5-HT2A blockers or antagonists such
as ketanserin and sarpogrelate [8] proved to be clinically
useful in treating hypertension and ischemia. Mean-
while, mixed D2/5-HT2A receptor antagonists such as
risperidone and clozapine proved to be atypical neuro-
leptics.
Correspondence: Jochen Lehmann, Institute of Pharmacy,
Recently, compound LE 300 was prepared in our labora-
tory [9]. LE 300 is a hybrid of tryptamine and phenethyl-
amine linked through the semi-rigid azecine ring, and
Pharmaceutical/Medicinal
Chemistry,
Friedrich-Schiller-
University Jena, Philosophenweg 14, D-07743 Jena,
Germany. Phone: +49 3641 949803, Fax: +49 3641 949802,
e-mail: j.lehmann@uni-jena.de
showed
a
subnanomolar affinity towards the rat
© 2002 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
0365-6233/09/0443 $ 17.50+.50/0

444 El-Subbagh et al.
Arch. Pharm. Pharm. Med. Chem. 2002, 335, 443–448
Results and discussion
Chemistry
The synthesis of the target compounds 20–24 is depict-
ed in Figure 2. The lactam 3 was prepared by refluxing
3-methoxyphenethylamine (1) and ethyl 4-bromobu-
tyrate in dioxane. The same procedure was adopted to
produce the reported compound 4 [11] in nearly quanti-
tative yield, using 3,4-dimethoxyphenethylamine (2) and
ethyl 4-bromobutyrate instead of butyrolactone [11]. 3
was cyclized using POCl3 in refluxing toluene to yield
the salt 7, which was subsequently reduced into its
corresponding hexahydropyrrolo[2,1-a]isoquinoline 11.
Treatment of 11 with CH3I/acetone afforded the quarter-
nary salt 15 which was subjected to ring opening using
Na/NH3 to produce the target compound 20. In order to
synthesize the 9,10-dimethoxy-3-benzazonines 21 and
Figure 1
striatal D1 receptor [9] and high functional activity at the
5-HT2A receptor [10]. In the present study, some new
analogs of LE 300 were synthesized from which the in-
dole moiety was omitted, in order to estimate its necessi-
ty for biological activity. In addition, methoxylation of the
remaining benzene part and ring constriction to azonine
analogs were performed.
Figure 2. Synthesis of target compounds.
a
(a) Dioxane, 120 °C, 12 h; (b) POCl3/toluene, reflux, 4 h; (c) NaBH4/MeOH; (d) CH3I/acetone; (f) Na,NH3(liq.); (e)
BnOCOCl, NaCNBH3; (g) H2,10 % Pd/C.

Arch. Pharm. Pharm. Med. Chem. 2002, 335, 443–448
4,5-Bis(4-hydroxyphenyl)imidazoles 445
Table 1. Percentage decrease of dopamine receptor (D1,
D2L, D4, and D5) bound radioactivity by 10 µM solutions
of compounds 11–14, 20–24, and LE 300.
Compounda
D1
D2L
D4
D5
11
12
13
14
20
21
22
23
24
–3
–14
–21
–8
–24
–8
–9
–0
–4
–96
–2
–6
–7
–3
–8
0
–5
–5
–4
–91
n.d.
0
n.d.
n.d.
n.d.
–9
n.d.
n.d.
0
n.d.
–14
n.d.
n.d.
n.d.
–13
n.d.
n.d.
–7
Figure 3. Stick and ball drawing obtained by X-ray from
crystals of the cis-annelated indolizinium compound 15.
LE 300
–95
–100
n.d.: not determined
24, reported procedures were adopted to prepare the re-
quired intermediates 8, 12, and 16 [11, 12]. Compound
16 was subjected to ring opening yielding the target 21.
Meanwhile, the dimethoxy intermediate 12 was reacted
with benzyl chloroformate and sodium cyanoborohy-
dride to give the 3-benzyloxycarbonyl-3-benzazonine
analog 19. Catalytic hydrogenolysis (H2, Pd/C) and de-
carboxylation produced the required secondary amine
24.
C,N cleavage using Na/NH3 to produce the target com-
pound 22.The synthesis of 23 necessitated the prepara-
tion of some reported intermediates such as 10, 14, and
18 [13]. Quinolizines and indolizines can be cis- or
trans-annelated. X-ray analysis of the quarternary salt
15 showed a cis-annelated indolizine (Figure 3). In ear-
lier studies we have confirmed by X-ray investigations
the same cis-annelated structure for benz-indolo-quino-
lizines [14].
In order to produce the homologous azecine series the
starting piperidones 5 and 6 [13] were prepared by re-
fluxing 1 and 2, respectively, with ethyl 5-bromovalerate
in dioxane, which gave better yields than the reported
procedure using δ-valerolactone [13]. 5 was cyclized us-
ing POCl3 in refluxing toluene to yield 9, which was sub-
sequently reduced into its quinolizine 13.Treatment of 13
with CH3I/acetone afforded 17 which was subjected to
Pharmacology
All of the “de-indolised” benzazonine and benzazecine
target compounds together with their precursors 11–14
Table 2. Affinity of compounds 19–21 and 23 towards 5-HT2A receptor at arbitrary concentration of 100 µM, using the
rat tail artery assay.
na
PA2
E
max(5-HT)c
c (µM)d
b
Compound
± SEM
± SEM (%)
19
20
21
23
5
5
5
5
22
36
<4
98 ± 2
85 ± 5
98 ± 4
97 ± 1
89–98
88–91
100
100
100
100
0.01–1
0.00047–0.047
5.09 ± 0.02
5.48 ± 0.05
4.13 ± 0.07
8.32 ± 0.02
9.55 ± 0.02
LE 300 [8]
Ketanserin [13]
a Number of experiments. b Absolute affinity of the tested compounds and positive controls. c Relative maximum effect
of 5-HT in presence of antagonist. d Micromolar concentrations of the tested compounds and positive controls.

446 El-Subbagh et al.
Arch. Pharm. Pharm. Med. Chem. 2002, 335, 443–448
(100 mL) was heated under reflux for 36 h.The reaction mixture
was cooled and evaporated in vacuo. EtOAc (50 mL) was add-
ed and the precipitated amine hydrobromide (6.6 g) was filtered
off.The filtrate was evaporated under reduced pressure and the
obtained residue was chromatographed on a silica gel column
and the “indolised” reference LE 300 were screened as
ligands for the human dopamine receptor family (D1, D2L,
D4, and D5) adopting the radioligand binding studies
described by Mierau et al. [15]. The results are given in
Table 1 and they clearly demonstrate the absence of any
affinity, with the expected exception of LE 300.In addition
the compounds were subjected to rat tail artery assay
[16–18] to evaluate their potency as 5-HT2A receptor lig-
ands. Only the benzazonine derivatives 20, 21 and the
benzazecine 23 showed moderate antagonistic activi-
ties toward 5-HT2A receptor with PA2 values of 5.09, 5.48,
and 4.13, respectively (Table 2).None of the tested com-
pounds showed activity comparable to the lead LE 300
(PA2 = 8.32) or the positive control ketanserin (PA2 =
9.55) [16]. Thus the replacement of the indole moiety in
LE 300 decreased the binding affinity not only for
dopamine receptors but also toward the 5-HT2A dramati-
cally.The results also show that the existence of either a
monomethoxy or a dimethoxy groups on the benzene
ring did not really contribute to the binding potential (20,
PA2 5.09 versus 21, PA2 5.48).The benzazonine ring sys-
tem in 20 and 21 favors the activity rather than the ben-
zazecine ring (23, PA2 4.13).The obtained results will set
the parameters for the future derivatization and modifi-
cation of the lead compound LE 300.
1
(20 % hexane/EtOAc) to produce 6.4 g (97 % yield) of 3. H
NMR (CDCl3):δ 1.9–2.15 (m, 2 H, CH2), 2.32–3.5 (m, 2 H, CH2),
2.72–2.95 (t, J Hz, 2 H, CH2), 3.24–3.41 (t, J Hz, 2 H, CH2),
3.47–3.69 (m, 2 H, CH2), 3.85 (s, 3 H, OCH3), 6.72–6.96 (m,
3 H, ArH), 7.42 (s, 1 H, ArH). m/z (219.1; 45 %). Anal.
(C13H17NO2) C, H, N.
8-Methoxy-1,2,3,5,6,10b-hexahydropyrrolo[2,1-a]isoquinoline
(11)
A solution of 3 (3 g, 13.7 mmol) and POCl3 (4 mL) in dry toluene
(50 mL) was heated under reflux for 4 h, and then allowed to
cool. Dry ether (100 mL) was added and the obtained oily pro-
duct (7) was separated and used for the next reaction without
further purification. The oily salt was dissolved in methanol
(50 mL), then 7 g of NaBH4 was added in portions with stirring
for 2 h at room temperature. Methanol was evaporated under
reduced pressure and water (50 mL) was added. The resulted
emulsion was extracted with ether, separated, dried, and evap-
1
orated to give 2.4 g (86 % yield) of 11. H NMR (CDCl3): δ
0.8–3.5 (m, 11 H, 5CH2 + CH), 3.85 (s, 3 H, OCH3), 6.5–7.3 (m,
3 H, ArH). m/z (203.1; 51 %). Anal. (C13H17NO) C, H, N.
4-Methyl-8-methoxy-1,2,3,5,6,10b-hexahydropyrrolo[2,1-a]-
isoquinolinium iodide (15)
A solution of 11 (2.4 g, 11.8 mmol) in dry acetone (30 mL) was
treated with iodomethane (4 mL) and allowed to stand at room
temperature for 12 h. The precipitate obtained was recrystal-
lized from ethanol to afford 15, the methiodide salt of 11 (3 g,
74 % yield), mp 234–235 °C.1H NMR (DMSO-d6):δ 1.9–4.0 (m,
10 H 5CH2), 3.3 (s, 3 H, NCH3), 3.88 (s, 3 H, OCH3), 4.6–4.9 (m,
1 H, CH), 6.85–7.3 (m, 3 H, ArH). Anal. (C14H20INO) C, H, N.
Acknowledgements
The Alexander von Humboldt foundation fellowship
awarded to Professor Dr. Hussein El-Subbagh is greatly
appreciated.
10-Methoxy-3-methyl-2,3,4,5,6,7-hexahydro-1H-3-benzazo-
nine (20)
Experimental
Chemistry
A mixture of 15 (1.0 g, 2.9 mmol) and liquified NH3 (30 mL) was
stirred at –40 °C for 1 h during which Na metal (2.0 g) was add-
ed portionwise.The reaction was finally quenched by the addi-
tion of a saturated aqueous solution of NH4Cl and stirring was
continued at room temperature for 12 h. H2O (20 mL) was then
added and the resulting emulsion was extracted with ether
(100 mL).The ethereal extract was washed with 5 % NaOH so-
lution (30 mL) and then H2O (30 mL), separated, dried, and
evaporated to give 20 (0.5 g, 78 % yield). 1H NMR (CDCl3): δ
0.9–1.3 (m, 2 H, CH2), 1.5–1.9 (m, 2 H, CH2), 2.1–3.2 (m, 8 H,
CH2), 2.3 (s, 3 H, NCH3), 3.85 (s, 3 H, OCH3), 6.5–7.1 (m, 3 H,
ArH). m/z (219.2, 83 %). Anal. (C14H21NO) C, H, N.
Melting points were determined in open capillaries on a Gal-
lenkamp melting point apparatus and are uncorrected.1H NMR
spectra were recorded on Bruker AC 400 (400 MHz) spectrom-
eter, chemical shifts are given in δ (ppm) values downfield from
Me4Si as an internal standard. Elemental analyses (C, H, N)
were carried out at the Institute of Pharmacy, University of
Bonn, Germany, results were within –0.4 % of the theoretical
values. Mass spectrometry were carried on MS-30 and MS-50
instruments of A.E.I., Manchester, England.Thin layer chroma-
tography (TLC) was performed on Merck 5 × 10 cm plates, pre-
coated with silica gel GF254. Chromatographic separations
were carried out either on gravity column or on a Chromatotron
model No. 7924T, Harrison Research, Palo Alto, CA, USA.
Compounds 4 [9], 8, 12 [9, 10], 16 [10], 6, 10, 14, 18 [11] were
previously reported. [3H]-Spiperone was purchased from Am-
ersham, UK, with a specific activity of 97.0 Ci/mmol. [3H]-SCH
23390 was also purchased from Amersham, UK, with a specific
activity of 83.0 Ci/mmol. Prazosin, ketanserin, haloperidol, and
fluphenazine were purchased from Sigma-Aldrich, Germany.
9,10-Dimethoxy-3-methyl-2,3,4,5,6,7-hexahydro-1H-3-benz-
azonine (21)
A mixture of 16 (1.0 g, 2.6 mmol) and liquified NH3 (g) (30 mL)
was stirred at –40 °C for 1 h during which Na metal (2.0 g) was
added in portions and the procedure continued as mentioned
under 20 to give 21 (0.4 g, 62 %).1H-NMR (CDCl3):δ 1.05–1.12
(m, 2 H, CH2), 1.64–1.71 (m, 2 H, CH2), 2.30 (s, 3 H, N-CH3),
2.35–2.4 (m, 2 H, CH2), 2.44–2.47 (m, 2 H, CH2), 2.61–2.64 (m,
2 H, CH2), 2.90–2.94 (m, 2 H, CH2), 3.78 (s, 6 H, OCH3), 6.47 (s,
1 H, ArH), 6.56 (s, 1 H, ArH). m/z (249.2, 50 %). Anal.
(C15H23NO2) C, H, N.
N-[2-(3-Methoxyphenyl)ethyl]-2-pyrrolidone (3)
A mixture of 3-methoxyphenethylamine (1, 9.83 g, 65 mmol)
and ethyl 4-bromobutyrate (5.85 g, 30 mmol) in dioxane

Arch. Pharm. Pharm. Med. Chem. 2002, 335, 443–448
4,5-Bis(4-hydroxyphenyl)imidazoles 447
11-Methoxy-3-methyl-1,2,3,4,5,6,7,8-octahydro-3-benzaze-
cine (22)
3-Benzyloxycarbonyl-9,10-dimethoxy-2,3,4,5,6,7-hexahydro-
1H-3-benzazonine (19)
A mixture of 17 (1.0 g, 2.8 mmol) and liquified NH3 (30 mL) was
stirred at –40 °C for 1h during which Na metal (2.0 g) was added
portionwise and the procedure continued as mentioned under
20 to give 22 (0.4 g, 61 %). 1H NMR (CDCl3): δ 1.18–2.23 (m,
10 H, CH2), 2.05 (s, 3 H, N-CH3), 2.6–2.86 (m, 4 H, CH2), 3.78
(s, 3 H, OCH3), 6.64–6.78 (m, 2 H, ArH), 7.02–7.1 (m, 1 H, ArH).
m/z (233.2, 100 %). Anal. (C15H23NO) C, H, N.
A solution of 12 (2.3 g, 10 mmol) in dryTHF (30 mL) was cooled
to –70 °C. Benzyl chloroformate (3.4 g, 2.8 mL, 20 mmol) was
added dropwise and stirring continued for 1 h at –70 °C, then
NaCNBH3 (0.94 g, 15 mmol) was added at the same tempera-
ture and stirring continued for another 1 h.The reaction mixture
warmed up to room temperature and stirred for 12 h, then
poured into NaOH solution (20 %, 30 mL) and extracted with
EtOAc (100 mL). The organic layer was separated, dried, and
evaporated.The oily product was chromatographed on a silica
gel column (1 : 1 CH2Cl2/hexane) to give 19 (1.2 g, 32 % yield).
1H NMR (CDCl3):δ 0.88–0.95 (m, 2 H, CH2), 1.02–1.16 (m, 2 H,
CH2), 1.25–1.45 (m, 2 H, CH2), 1.64–1.85 (m, 2 H, CH2), 2.55–
2.65 (m, 2 H, CH2), 2.80–2.95 (m, 2 H, CH2), 3.84 (s, 3 H,
OCH3), 3.88 (s, 3 H, OCH3), 5.20 (d, 2 H, J Hz, CH2-Ph), 6.59 (s,
1 H, ArH), 6.62 (s, 1 H, ArH), 7.30–7.41 (m, 5 H, ArH). Anal.
(C22H27NO4) C, H, N.
10,11-Dimethoxy-3-methyl-1,2,3,4,5,6,7,8-octahydro-3-benz-
azecine (23)
A mixture of 18 (1.0 g, 2.7 mmol) and liquified NH3 (30 mL) was
stirred at –40 °C for 1h during which Na metal (2.0 g) was added
in portions and the procedure continued as mentioned under
20 to give 23 (0.4 g, 56.2 %). 1H-NMR (CDCl3): δ 0.84–2.03 (m,
10 H, CH2), 2.10 (s, 3 H, N-CH3), 2.30–2.85 (m, 4 H, CH2), 3.85
(s, 6 H, OCH3), 6.68 (s, 2 H, ArH). m/z (263.3, 88 %). Anal.
(C16H25NO2) C, H, N.
9,10-Dimethoxy-2,3,4,5,6,7-hexahydro-1H-3-benzazonine (24)
X-Ray structure analysis of compound 15
A slurry of 19 (0.5 g, 1.4 mmol) and 250 mg of 10 % Pd/C in
methanol (200 mL) was subjected to hydrogenation using a
Parr hydrogenator at 50 psi for 3 h. The catalyst was then re-
moved by filtration and the filtrate was evaporated in vacuo to
give crude 24.The oily product was chromatographed on a sili-
ca gel column (2 : 1 CH2Cl2/hexane) to give 24 as a colourless
oil (0.2 g, 63 % yield). 1H NMR (CDCl3): δ 0.88–0.97 (m, 2 H,
CH2), 1.0–1.2 (m, 2 H, CH2), 1.25–1.5 (m, 2 H, CH2), 1.65–1.76
(m, 2 H, CH2), 2.45–2.72 (m, 4 H, CH2), 3.0–3.15 (m, 1 H, NH),
3.85 (s, 6 H, OCH3), 6.52 (s, 1 H, ArH), 6.62 (s, 1 H, ArH), Anal.
(C14H21NO2) C, H, N.
Compound 15 (C14H20INO), colorless crystals, crystal dimen-
sion 0.20 × 0.35 × 0.45 mm3; M = 663.5; monoclinic, space
group P21/c (no. 14), a = 7.5156(2), b = 15.7478(5), c =
12.5268(3) Å, β = 105.342(2)°, V = 1.42978(7) nm3, Z = 4,
µ(MoKα) = 2.226 mm–1, T = 123(2) K, F(000) = 688. 16347 re-
flection up to 2θmax. = 56.6° were measured on an Nonius-
Kappa CCD diffractometer with MoKα radiation, 3315 of which
were independent and used for all calculations. The structure
was solved by direct methods and refined to F 2 anisotropically,
the H atoms were refined with a riding model. The final quality
coefficient wR2(F 2) was 0.0475, with a conventional R(F ) =
0.0192 for 156.An empirical absorption correction was applied.
Crystallographic data (excluding structure factors) for the
structures reported in this paper have been deposited with the
Cambridge Crystallographic Data Centre as supplementary
publication no. CCDC 178049. Copies of the data can be ob-
tained free of charge on application to The Director,
CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (Fax:
+44 1223336033; e-mail: deposit@ccdc.cam.ac.uk).
N-[2-(3-Methoxyphenyl)ethyl]-2-piperidone (5)
A mixture of 3-methoxyphenethylamine (1, 9.8 g, 65 mmol) and
ethyl 5-bromovalerate (6.3 g, 30 mmol) in dioxane (100 mL)
was heated under reflux for 36 h and continued as mentioned
under 3. The crude product was chromatographed on a silica
gel column (20 % hexane/EtOAc) to produce 6.8 g (97 % yield)
of oily 5. 1H NMR (CDCl3): δ 1.6–1.95 (m, 4 H, CH2), 2.25–2.5
(m, 2 H, CH2), 2.65–3.34 (m, 4 H, CH2), 3.43–3.7 (m, 2 H, CH2),
3.85 (s, 3 H, OCH3), 6.62–6.87 (m, 3 H, ArH), 7.13–7.38 (m,
1 H, ArH). m/z (233.2, 31 %). Anal. (C14H19NO2) C, H, N.
Biological testing
A) 5-HT2A receptor ligand activity
2-Methoxy-1,3,4,6,7,11b-hexahydro-2H-benzo[a]quinolizine
(13)
In brief, cylindrical segments of 3–4 mm length were mounted
isometrically (initial tension 5 mN) by means of two stainless L-
shaped steel hooks (diameter 0.15 mm) in a modified Krebs-
Henseleit solution (37 °C) of composition (mM): NaCl 118.1,
KCl 4.7, CaCl2 2.5, MgSO4 1.2, KH2PO4 1.2, NaHCO3 25.0, and
D-glucose 10.0. The solution was aerated with 95 % O2/5 %
CO2 and contained prazosin (30 nM) to block α1 adrenocep-
tors, and cocaine (6 µM) to block neuronal uptake of amines.
During an equilibration period of 120 min the preparations were
primed once (after 60 min) with 5-HT (1 and 10 µM) to monitor
tissue viability. Two cumulative concentration-effect curves for
5-HT (0.01–30 µM for the first curve) were determined in the
absence and presence of potential antagonists which were
usually incubated for 30 min or 120 min (LE 300, ketanserin at
0.47 and 1 nM, respectively). Control experiments in the ab-
sence of antagonist revealed that for the 30-min protocol, two
successive concentration-effect curves for 5-HT were superim-
posable (data not shown).When the 120-min protocol was ap-
plied, a leftward shift of the control curves of approximately
0.1–0.3 logarithmic units was usually observed. The daily
mean sensitisation was used to correct the dextral shift meas-
ured for treated organs [13–15]. Results are expressed as
A solution of 5 (6.9 g, 29.6 mmol) and POCl3 (10 mL) in dry tolu-
ene (100 mL) was heated under reflux for 4 h and continued as
mentioned under 11 to produce 9 which was used for next reac-
tion without further purification.The oily salt 9 was dissolved in
methanol (100 mL), then 14 g of NaBH4 were added in portions
with stirring over a period of 2 h at room temperature and con-
tinued as mentioned under 11 to produce 5.5 g (84 % yield) of
13. 1H NMR (CDCl3): δ 1.20–3.30 (m, 13 H, CH2 & CH), 3.85 (s,
3 H, OCH3), 6.56–6.94 (m, 2 H, ArH), 7.15 (m, 1 H, ArH). m/z
(217.3, 58 %). Anal. (C14H19NO) C, H, N.
9-Methoxy-5-methyl-1,3,4,6,7,11b-hexahydro-2H-benzo[a]-
quinolizinium iodide (17)
A solution of 13 (4.8 g, 22.0 mmol) in dry acetone (20 mL) was
treated with iodomethane (5 mL) and continued as mentioned
under 15 to produce 6.0 g (75 % yield) of 17. 1H-NMR (DMSO-
d6): δ 1.52–2.1 (m, 2 H, CH2), 2.96–4.3 (m, 10 H, CH2), 3.42 (s,
3 H, NCH3), 3.80 (s, 3 H, OCH3), 4.35–4.66 (m, 1 H, CH), 6.8–
7.05 (m, 2 H, ArH), 7.3 (m, 1 H, ArH).Anal.(C15H22INO) C, H, N.

448 El-Subbagh et al.
Arch. Pharm. Pharm. Med. Chem. 2002, 335, 443–448
the filters was counted using a Beckman LS 6000 SC scintilla-
tion counter.The competition binding data was analysed by the
software GraphPad PrismTM using non-linear least squares fit.
mean ± standard error (SEM or SE) unless otherwise indicat-
ed.Antagonist affinity was calculated as apparent pA2 value ac-
cording to equation (1) when only one or two antagonist con-
centrations were used [14]. ([c] = mol/L, r is the ratio of agonist
concentrations in the presence and absence of antagonist that
elicit 50 % of the respective maximum effect.)
References
pA2 = –log10 c(antagonist) + log10 (r – 1)
(1)
Full pA2 values were calculated according to the method of
Schild [15] when a set of different antagonist concentrations
over at least 1.5 logarithmic units was studied. Single organ
preparations were from at least three (5-HT2A) animals.
[1] Part III:A.H.Abadi, S.Lankow, B.Höfgen, M.Decker, M.U.
Kassack, J. Lehmann, Arch. Pharm. Pharm. Med. Chem.
2002, 335, 367–373.
[2] E. Zifa, G. Fillion, Pharmacol. Rev. 1992, 44, 401–457.
B) Dopamine receptor ligand activity
i) Cell culture
[3] L. J. Siever, R. S. Khan, B. A. Lawlor, R. L. Trestman, T. L.
Lawrence, E. F. Corraco, Pharmacol. Rev. 1991, 43, 509–
525.
Human D1, D2L, D4, and D5 receptors were stably expressed in
Chinese hamster ovary (CHO) cells as previously described
[5]. The density of receptors measured with [3H]-SCH 23390
was 307.15 fmol/mg protein for D1 and 679.44 fmol/mg for D5.
The density of receptors measured with [3H]-Spiperone was
2021 fmol/mg protein for D2L and 137.21 fmol/mg for D4.These
cells were grown at 37 °C under a humidified atmosphere of
5 % CO2: 95 % air in HAM/F12-medium (Sigma-Aldrich), sup-
plemented with 10 % fetal bovine serum, 1 mM l-glutamine,
20 U/mL penicillinG, 20 µg/mL streptomycin, and 0.2 µg/mL G
418 (all from Sigma-Aldrich).
[4] D. Hoyer, D. E. Clarke, J. R. Fozard, P. R. Hartig, G. R. Mar-
tin, E. J. Mylecharane, P. R. Saxena, P. P. A. Humphrey,
Pharmacol. Rev. 1994, 46, 157–203.
[5] H. Anderson, B. Nielsen, Antipsychotics. DN&P 1991, 4,
150–157.
[6] P. Sokoloff, B. Giros, M. Martres, M. Bouthenet, J.
Schwartz, Nature 1990, 347, 146–151.
[7] H.VanTol, J. Bunzow, H. Guan, R. Sunahara, P. Seemann,
H. Niznik, O. Civelli, Nature 1991, 350, 610–614.
ii) Preparation of whole-cell-suspension
[8] H. Hara, M. Osakabe, A. Kitajima, Y. Tamao, R. Kikumoto,
Human D1, D2L, D4, and D5 receptor cell lines (CHO) were
grown on T 175 culture dishes (Nunc) to 85 % confluency, the
medium was removed and the cells were incubated with 6 mL
trypsine-EDTA-solution (Sigma-Aldrich) to remove the cells
from the culture dish.The resulting suspension was centrifuged
(1000 rot/min, 4 °C, 4 min), the pellet resuspended in 10 mL
PBS (ice-cooled, calcium- and magnesium-free), pelleted, and
this procedure repeated. The resulting pellet was then resus-
pended in 12 mL buffer (5 mM magnesium chloride, 50 mM
TRIS-HCl pH = 7.4) and the resulting suspension was directly
used for the radioligand binding assay.
Thromb. Haemost. 1991, 65, 415–420.
[9] T. Witt, F. J. Hock, J. Lehmann, J. Med. Chem. 2000, 43,
2079–2081.
[10] S. A. F. Rostom, A. M. Farghaly, F. S. G. Solimann, M. M.
El-Semary, S.Elz, J.Lehmann, Arch.Pharm.Pharm.Med.
Chem. 2001, 334, 241–247.
[11] K.Wiesner, Z.Valenta, A. J. Manson, F.W. Stonner, J. Am.
Chem. Soc. 1955, 77, 675–83.
[12] J.B.Bremner, K. N.Winzenberg, Austr.J.Chem.1984, 37,
iii) Radioligand binding assay
1203–15.
For the binding studies a procedure according to Mierau et al.
was used [16]. The binding assays with the whole-cell-sus-
pension were carried out in triplicate in a volume of 1.1 mL
(final concentration): TRIS-Mg2+-buffer (690 µL), [3H]-ligand
(100 µL), whole-cell-suspension (200 µL) and appropriate
drugs (110 µL).Non-specific binding was determined using flu-
phenazine (100 µM) in D1 and D5 tests and haloperidol (10 µM)
in D2 and D4 tests.For a fast screening’the drugs were used in a
concentration of 100 µM, and the percentage of removed radio-
ligand determined. The incubation was initiated by addition of
the radioligand. The incubations were carried out at 27 °C for
2 h, and stopped by rapid filtration through a glass fiber filter
(Schleicher and Schüll, Germany), previously treated with
0.25 % polyethyleneimine solution (Sigma-Aldrich), which was
washed twice with ice-cold water.The radioactivity retained on
[13] J.B.Bremner, K. N.Winzenberg, Austr.J.Chem.1985, 38,
1591–612.
[14] J. Lehmann, M. Nieger, T. Witt, Heterocycles 1994, 38,
511–528.
[15] J. Mierau, F. J. Schneider, H. A. Ensinger, C. L. Chio, M. E.
Lajiness, R. M. Huff, Mol. Pharmacol. 1995, 290, 29–36.
[16] H. Pertz, S. Elz, J. Pharm. Pharmacol. 1995, 47, 310–316.
[17] R. F. Furchgott in Catecholamines, Handbook of Experi-
mental Pharmacology, Vol. 33 (Eds.: H. Blaschko, E.
Muscholl), Springer-Verlag, Berlin, 1972, 283–335.
[18] O. Arunlakshana, H. O. Schild, Br. J. Pharmacol. Chemo-
ther. 1959, 14, 48–58.