Novel fused pyrrole heterocyclic ring systems as structure analogs of LE 300: Synthesis and pharmacological evaluation as serotonin 5-HT2A, dopamine and histamine H1 receptor ligands

Article abstract of DOI:10.1002/ardp.200900219

LE 300 represents a structurally novel type of antagonists acting preferentially at the dopamine D₁/D₅ receptors and the serotonin 5-HT_2A receptor. This compound consists of a ten-membered central azecine ring fused to an indole ring on one side and a benzene moiety on the other side. To estimate the importance of the indole and / or phenyl moieties in this highly active benzindolo-azecine, both rings were removed and replaced with a 1H-pyrrole counterpart. Accordingly, some new analogs of LE 300 namely, pyrrolo[2,3-g]indolizine, pyrrolo[3,2-a]quinolizine rings and their corresponding dimethylpyrrolo[2,3-d]azonine, and dimethylpyrrolo[2,3-d]azecine were synthesized to be evaluated for their activity at the 5-HT_2A and dopamine D₁, D_2L, D₄, D₅ receptors in relation to LE 300. In addition, their activity at the H1-histamine receptors was also determined. The results suggested that the rigid pyrrolo[2,3-g] indolizine 7 and pyrrolo[3,2-a]quinolizine 8 analogs lacked biological activity in the adopted three bioassays. However, their corresponding flexible pyrrolo[2,3-d]azonine 11 and pyrrolo[2,3-d]azecine 12 derivatives revealed weak partial agonistic activity and weak antagonistic potency at the serotonin 5-HT2A and histamine H1 receptors, respectively. Meanwhile, they showed no affinity to any of the four utilized dopamine receptors. Variation in ring size did not contribute to a significant influence on the three tested bioactivities. Removal of the hydrophobic moiety (phenyl ring) and replacement of the indole moiety with a 1H-pyrrole counterpart led to a dramatic alteration in the profile of activity of such azecine-type compounds.

Full text of DOI:10.1002/ardp.200900219

Arch. Pharm. Chem. Life Sci. 2010, 343, 73–80
S. A. F. Rostom
73
Full Paper
Novel Fused Pyrrole Heterocyclic Ring Systems as Structure
Analogs of LE 300: Synthesis and Pharmacological Evaluation
as Serotonin 5-HT , Dopamine and Histamine H Receptor
2
A
1
Ligands*
Sherif A. F. Rostom
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, King Abdulaziz University, Jeddah, Saudi
Arabia
LE 300 represents a structurally novel type of antagonists acting preferentially at the dopamine
D
1
5
/D receptors and the serotonin 5-HT2A receptor. This compound consists of a ten-membered
central azecine ring fused to an indole ring on one side and a benzene moiety on the other side.
To estimate the importance of the indole and / or phenyl moieties in this highly active benz-
indolo-azecine, both rings were removed and replaced with a 1H-pyrrole counterpart. Accord-
ingly, some new analogs of LE 300 namely, pyrrolo[2,3-g]indolizine, pyrrolo[3,2-a]quinolizine
rings and their corresponding dimethylpyrrolo[2,3-d]azonine, and dimethylpyrrolo[2,3-d]azecine
were synthesized to be evaluated for their activity at the 5-HT2A and dopamine D
receptors in relation to LE 300. In addition, their activity at the H -histamine receptors was also
1
, D2L, D
4
, D
5
1
determined. The results suggested that the rigid pyrrolo[2,3-g]indolizine 7 and pyrrolo[3,2-a]qui-
nolizine 8 analogs lacked biological activity in the adopted three bioassays. However, their corre-
sponding flexible pyrrolo[2,3-d]azonine 11 and pyrrolo[2,3-d]azecine 12 derivatives revealed
weak partial agonistic activity and weak antagonistic potency at the serotonin 5-HT2A and hista-
mine H receptors, respectively. Meanwhile, they showed no affinity to any of the four utilized
1
dopamine receptors. Variation in ring size did not contribute to a significant influence on the
three tested bioactivities. Removal of the hydrophobic moiety (phenyl ring) and replacement of
the indole moiety with a 1H-pyrrole counterpart led to a dramatic alteration in the profile of
activity of such azecine-type compounds.
Keywords: Azecine / Azonine / Indolizine / 1H-Pyrrole / Quinolizine /
Received: September 12, 2009; accepted: November 3, 2009
DOI 10.1002/ardp.200900219
Introduction
mitter in the central and peripheral nervous systems
CNS and PNS, respectively). 5-HT has been reported to
(
Over the past two decades, much evidence has been accu- modulate the regulation of a variety of important physio-
mulated on the biological importance of serotonin (5- logical functions including affective behavior, memory,
hydroxytryptamine, 5-HT) as an important neurotrans- and thermo-regulation via the interaction at serotonin
receptor subtypes [1]. At least 14 distinct serotonin recep-
tor subtypes are expressed in the mammalian CNS, each
of which is assigned to one of seven families, namely 5-
Correspondence: Sherif A. F. Rostom, Ph.D., Department of Pharma-
ceutical Chemistry, Faculty of Pharmacy, King Abdulaziz University, PO
Box 80260, Jeddah 21589, Saudi Arabia.
E-mail: sherifrostom@yahoo.com
Fax: +966-2-6400000-22327
* Dedicated to Prof. Dr. Jochen Lehmann, Institute of Pharmacy, Pharma-
ceutical Medicinal Chemistry, Friedrich-Schiller-University Jena, Ger-
many, on the occasion of his 65 birthday.
th
Abbreviation: 5-Hydroxytryptamine (5-HT)
i
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74
S. A. F. Rostom
Arch. Pharm. Chem. Life Sci. 2010, 343, 73–80
HT
1
to 5-HT [2]. Among these, the 5-HT2A receptor subtype
7
is a member of the G-protein-coupled receptor (GPCR)
superfamily, and has been implicated in working mem-
ory, cognitive processes, affective disorders such as schiz-
ophrenia, and mediates the primary effects of hallucino-
genic drugs [3–5]. In addition, many peripheral tissues
also express the 5-HT2A receptor as they are involved in
platelet function, and induce contraction within the vas-
culature [6]. Stimulation of 5-HT2A receptor would result
in psychiatric symptoms, vasoconstriction, and platelet
aggregation. Consequently, selective 5-HT2A receptor
antagonists showed a therapeutic efficacy for the treat-
ment of mental and cardiovascular illness [7].
On the other hand, dopamine-receptor-mediated neu-
rotransmission is known to play an important role in reg-
ulating neuronal motor control, cognition, event predic-
tion, emotion, and vascular function. Neuropsychiatric
diseases such as schizophrenia, Parkinson's disease, or
addiction are strongly related to a disregulation of the
dopaminergic signal transduction [8, 9]. There are five
dopamine receptor subtypes that may be divided into
Figure 1. Structures of LE-300, relevant compounds A–D, and
targeted analogs E and F.
two subfamilies: the G
and the G -coupled D
Because of a lack of highly selective D
s
-coupled D
-like receptors (D
- and D
1
-like receptors (D
, D , D
-receptor 1H-pyrrole, respectively) and its location with respect to
1
, D
5
)
[20, 21] and changing one of the aromatic moieties
i
2
2
3
4
) [10]. (indole replaced by benzene, thiophene, and 1-methyl-
1
5
ligands, knowledge about the physiological impact of each other at the central alicyclic ring [22]. Such “aze-
activation or antagonism of these single receptors, espe- cine-type” dopamine receptor antagonists derived from
cially the D
11]. The D receptor is of special interest, because it is, in nanomolar, subnanomolar, and even picomolar [23]
contrast to the D receptor, directly coupled to the GABA
affinities predominantly for the D and D subtypes.
receptor, enabling inhibitory functional interactions
12].
5
receptor, is strictly limited or nonexistent LE 300 and its dibenzo-analogues were found to show
[
5
1
A
1
5
In the present study, and in continuation of the inter-
est in azecine-type compounds [20, 24], some new analogs
[
The observation that risperidone and clozapine can act of LE 300, namely pyrrolo[2,3-g]indolizine, pyrrolo[3,2-a]
as mixed D /5-HT2A receptor antagonists has led to the so- quinolizine ring systems and their corresponding dime-
2
called “atypical neuroleptics” which are distinguished thylpyrrolo[2,3-d]azonine, and dimethylpyrrolo[2,3-d]aze-
from the old traditional neuroleptics in their enhanced cine with general structures E and F, respectively, were
efficacy in treating the negative symptoms of schizophre- synthesized to be evaluated for their activity at the 5-HT2A
nia and related psychoses, and their decreased liability and dopamine D , D , D , D receptors. The newly
1
2L
4
5
for causing extra-pyramidal system (EPS) side effects [13, designed compounds were deprived of both the indole
4].
and aryl moieties present in LE 300, while replaced with
The lead compound LE 300 (7-methyl-6,7,8,9,14,15- a 1-methyl-1H-pyrrole counterpart together with a con-
1
hexahydro-5H-indolo[3,2-f][3]benzazecine (Fig. 1) intro- striction of the ten-membered azecine to the nine-mem-
duced by Lehmann et al. [15], revealed a subnanomolar bered azonine ring, in an attempt to estimate the influ-
affinity towards the rat striatal D receptor and high func- ence of such a structure variation on the anticipated bio-
1
tional activity at the 5-HT2A receptor [15–17]. The idea logical activities. The activities of the newly synthesized
behind the synthesis of such a heterocyclic system was to intermediate and target compounds at the 5-HT2A and
incorporate the substructures of tryptamine and b- dopamine D , D , D , D receptors were compared with
1
2L
4
5
phenylethylamine moieties into a moderately con- the previously reported structure analogs A–D [24]
strained ten-membered semi-rigid azecine ring [15]. (Fig. 1), whose dopaminergic binding profile was not
Intensive SAR studies were performed within this novel reported before. Furthermore, owing to the structure
class of dopamine receptor ligands, including variations similarity of such b-phenylethylamine-like compounds
in ring size [18], de-indolization of the structure [19], the with histamine, and the reported H -antihistaminic activ-
1
insertion of an additional oxygen atom into the alicyclus ity of the structure analogs A–D [24], it was considered
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Arch. Pharm. Chem. Life Sci. 2010, 343, 73–80
Novel Fused Pyrroles as Analogs of LE 300
75
worthwhile to investigate and compare the activity of
the target compounds at the H -histamine receptors with
1
those of their analogs A–D (Fig. 1).
Results and discussion
Chemistry
The synthetic strategies adopted to obtain the intermedi-
ate and target compounds are depicted in Scheme 1. The
key intermediate amine 2, namely 2-(2-aminoethyl)-1-
methyl-1H-pyrrole, was prepared according to a previ-
ously described procedure [24]. It involved condensation
of 1-methyl-1H-pyrrol-2-carbaldehyde with nitromethane
in the presence of piperidinium acetate to yield the 1-
methyl-2-(2-nitoethenyl)-1H-pyrrole 1, which, in turn, was
reduced to the amine 2 using lithium aluminium
hydride. The amine 2 was condensed under nitrogen
with either ethyl 4-bromobutanoate or ethyl 5-bromo-
pentanoate in the presence of potassium carbonate and
traces of potassium iodide to produce the lactams 3 and
4, respectively. Cyclization of these lactams to the corre-
sponding pyrrolo[2,3-g]indolizine 7 and pyrrolo[3,2-a]qui-
nolizine 8 ring systems was performed according to a
modified procedure of Lehmann et al. [25]. It comprised
Scheme 1. Synthesis of compounds 1–12.
internal cyclization of the lactams 3 and 4 with phospho- nal tract (GIT), and uterine smooth muscles, the tested
rous oxychloride according to Bischler–Napieralski reac- compounds were subjected to the rat-tail artery bioassay
tion conditions [26–28] followed by in-situ reduction of [30–32] to evaluate their potency as 5-HT2A receptor
the resulting indolizinium 5 and quinolizinium 6 salts ligands. The experiment aimed to assess the activity of
with sodium borohydride. The targeted dimethylpyr- the newly synthesized compounds (competitive antago-
rolo[2,3-d]azonine 11 and dimethylpyrrolo[2,3-d]azecine nists, full or partial agonists), in relation to the previ-
1
2 were obtained by initial quaternization of 7 and 8 ously reported analogs A–D [24], the structure lead LE-
using methyl iodide, followed by the reductive cleavage 300 (Fig. 1), and ketanserin (a reference 5-HT2A antago-
of the corresponding methiodides 9 and 10, with sodium nist). The results depicted in Table 1 revealed that the pyr-
metal in liquid ammonia at –788C, according to Birch- rolo[2,3-g]indolizine 7 and pyrrolo[3,2-a]quinolizine 8
reduction conditions for the cleavage of internal C-N derivatives were devoid of any activity at this receptor
bonds [29]. At this stage, it was considered worthwhile to site (pA
synthesize the secondary amine analogues 13 and 14 as sponding pyrrolo[2,3-d]azonine 11 and pyrrolo[2,3-d]aze-
interesting structure variants via quaternization of com- cine 12 compounds showed partial agonistic activity (pA
2
values a 4.00). On the other hand, the corre-
2
pounds 7 and 8 with benzyl chloroformate in the pres- values 4.08 and 4.01, respectively), when compared with
ence of sodium cyanoborohydride followed by catalytic their structurally relevant analogs C and D which
hydrogenolysis and decarboxylation according to a previ- showed remarkable antagonistic activity (pA
ously described procedure [19]. Unfortunately, trials to and 6.13, respectively), the lead LE 300, and ketanserin
isolate the expected intermediate carbamate salts were (the positive control) with pA
values of 8.32 and 9.55,
2
values 8.01
2
unsuccessful due to their high instability.
respectively.
Pharmacology
Dopamine-receptor ligand-binding activity
In-vitro serotonin 5-HT2A receptor-activity screening using
the rat-tail artery model
The newly synthesized target compounds 7, 8 and 11, 12
together with their structure analogs A–D (which were
Owing to the wide distribution of the serotonin 5-HT2A not previously investigated for this particular activity)
receptor subtype in many peripheral tissues mediating and the reference LE 300 were preliminarily screened as
contractile responses of various vascular, gastrointesti- ligands for the human dopamine receptor family (D
1
, D2L
,
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76
S. A. F. Rostom
Arch. Pharm. Chem. Life Sci. 2010, 343, 73–80
Table 1. Affinity of the tested compounds towards serotonin 5-HT2A receptor using the rat-tail artery assay.
a)
b)
c)
d)
Compound
c (lM)
N
pA
2
E
max (5-HT)
Description of
activity
l SEM
l SEM (%)
e)
7
8
100
100
100
100
100
1.0
0.01–1.0
10
0.1–1.0
3
3
4
4
4
6
20
6
22
36
a4.00
n.d.
inactive
inactive
partial agonist
partial agonist
partial agonist
antagonist
antagonist
antagonist
antagonist
reference antagonist
e)
a4.00
n.d.
1
1
1
2
4.08 l 0.06
4.01 l 0.10
4.49 l 0.10
6.74 l 0.06
8.01 l 0.03
6.13 l 0.05
8.32 l 0.02
9.55 l 0.02
94 l 4
99 l 7
70 l 4
93 l 2
98 l 2
99 l 7
89– 98
88–91
A*
B*
C*
D*
LE-300**
Ketanserin
**
– 4
– 2
4.7 N 10 to 4.7 N 10
a)
Micromolar concentrations of the tested compounds and positive controls.
Number of experiments.
Absolute affinity of the tested compounds and positive controls.
Relative maximum effect of 5-HT in the presence of the antagonist.
Not determined.
b)
c)
d)
e)
*
Reference [24].
** Reference [19].
Table 2. Percentage decrease of dopamine receptor (D
1
, D2L, D
4
Table 3. Affinity of the tested compounds towards histamine H
receptor using the guinea-pig ileum assay.
1
and D )-bound radioactivity by 10 lM solutions of the tested
5
compounds.
a)
b)
c)
Compound
c (lM)
N
pA
2
l SEM Description of
Compound
D
1
D
2L
D
4
D
5
activity
7
8
100
100
100
100
10–100
10
0.1–10
10
0.1–10
0.01–1.0
3
a4.00
a4.00
inactive
inactive
7
8
1
1
A
B
–5
–6
–6
0
–7
–5
–7
0
–10
–7
–9
3
4
4
8
4
9
5
–8
–17
–19
–11
1
1
1
2
4.01 l 0.10 weak antagonist
4.03 l 0.07 weak antagonist
5.49 l 0.04 antagonist
5.31 l 0.09 antagonist
8.01 l 0.03 antagonist
5.46 l 0.16 antagonist
1
2
–4
–11
A*
B*
C*
D*
–14
–14
–11
a)
a)
a)
a)
n.d.
–95
–87
–96
n.d.
–74
–46
–91
n.d.
–61
–51
–95
n.d.
C
D
–93
–56
–100
LE-300*
Ketanserin*
25 7.24 l 0.02 antagonist
23 8.85 l 0.08 antagonist
LE-300*
–
4
Mepyramine* 3.0 N 10 to 0.1 29 9.07 l 0.04 reference antagonist
a)
Not determined.
Reference [19].
a)
*
Micromolar concentrations of the tested compounds and positive con-
trols.
b)
c)
Number of experiments.
Absolute affinity of the tested compounds and positive controls.
Reference [24].
D
4
, and D ) adopting the radioligand binding assay
5
*
described by Mierau et al. [33].
The results are given in Table 2 and they clearly demon-
strate the absence of affinity of the target compounds 7, In-vitro histamine H
, 11, and 12 to all the four tested dopamine receptor The newly synthesized target compounds 7, 8 and 11, 12
types, when compared with the reference lead com- were further evaluated for their activity as histamine H
1
receptor activity
8
1
pound LE 300. However, structurally relevant, fused aze- receptor ligands using the guinea-pig ileum segments
cine analogs C and D revealed moderate to high affinity bioassay [32]. The experiment contributes to the contrac-
towards dopamine D
1
and D
receptors, whereas their tile response caused by histamine on the smooth muscles
5
fused quinolizine precursors A and B lacked affinity to of the ileum and aimed to assess the activity of the newly
all the tested dopamine receptor types. Out of the tested synthesized compounds (competitive antagonists, full or
compounds A–D, a potential high-activity profile was partial agonists), in relation to the previously reported
exhibited by the benzo[d]pyrrolo[3,2-g]azecine C which analogs A–D, the structure lead LE-300, and mepyr-
showed a remarkable high-affinity profile comparable amine, a reference H -histamine antagonist.
1
with that of the lead compound LE 300, particularly at
the D and D receptors (Table 2).
The results recorded in Table 3 declared that the pyr-
rolo[2,3-g]indolizine 7 and pyrrolo[3,2-a]quinolizine 8
1
5
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Arch. Pharm. Chem. Life Sci. 2010, 343, 73–80
Novel Fused Pyrroles as Analogs of LE 300
77
derivatives proved to be totally inactive at this receptor mine and/or serotonin receptors [19]. Consequently, the
site (pA value a 4.00). The target azonine 11 and azecine obtained results will set the parameters for the future
2 analogs, instead, showed weak antagonistic activity derivatization and modification of the lead compound
pA values 4.01 and 4.03, respectively), when compared LE 300.
with their structurally relevant analogs C and D which
showed remarkable antagonistic activity (pA values 8.01
2
1
(
2
2
Experimental
and 5.46, respectively). It should be noted that the ben-
zo[d]pyrrolo[3,2-g]azecine C showed distinctive antago-
Chemistry
nistic activity (pA
the reference lead compound LE 300 (pA
parable with that of mepyramine (pA : 9.07), the refer-
ence H -histamine antagonist used in this assay.
2
: 8.01), which was more potent than
Melting points were determined in open capillaries on a Gallen-
kamp melting point apparatus (Weiss-Gallenkamp, London, UK)
and are uncorrected. The infrared (IR) spectra were recorded on
Perkin-Elmer FTIR “Paragor 1000” infrared spectrophotometer
2
: 7.24) and com-
2
1
(
Perkin-Elmer, Norwalk, CT, USA) using the KBr disc technique.
Collectively, the obtained data showed that the rigid
pyrrolo[2,3-g]indolizine 7 and pyrrolo[3,2-a]quinolizine 8
analogs lacked biological activity in the adopted three
bioassays. However, their corresponding flexible pyr-
rolo[2,3-d]azonine 11 and pyrrolo[2,3-d]azecine 12 deriv-
atives revealed weak partial agonistic activity and weak
antagonistic potency at the serotonin 5-HT2A and hista-
1
H-NMR spectra were recorded on Bruker AC400 (400 MHz) spec-
trometer (Bruker Bioscience, USA), chemical shifts are given in d
ppm) using tetramethylsilane as an internal standard and
CDCl as a solvent. Splitting patterns were designated as follows:
(
3
s: singlet; d: doublet; m: multiplet. Elemental analyses (C, H, N)
were carried out at the Institute of Pharmacy, University of
Bonn, Germany, results were within l 0.4% of the theoretical val-
ues. Follow up of the reactions and checking the homogeneity of
the compounds were made by TLC on silica gel-protected alumi-
num sheets (Type 60 F254, Merck, Germany) and the spots were
detected by exposure to UV-lamp at k 254. Chromatographic sep-
arations were carried out on gravity column using silica gel S
mine H receptors, respectively. Meanwhile, they showed
1
no affinity to any of the four utilized dopamine recep-
tors.
Compared with the previously described analogs A–D
(
0.2–0.5 mm) for column chromatography. The synthesis of the
(
Fig. 1), while the target rigid structures 7 and 8 were
starting 1-methyl-2-(2-nitoethenyl)-1H-pyrrole 1 and 2-(2-amino-
ethyl)-1-methyl-1H-pyrrole 2, was performed according to a
reported literature procedure [24].
deprived of any biological activity, their structure conge-
ners benzo[a]pyrrolo[2,3-h]quinolizine A and indolo[2,3-a]
pyrrolo[2,3-h]quinolizine B exhibited an observable pro-
file of bioactivities at the serotonin 5-HT2A and histamine N-[2-(1-Methyl-1H-pyrrol-2-yl)ethyl]-2-pyrrolidone and N-
H
1
receptors. Meanwhile, the flexible analogs 11 and 12 [2-(1-methyl-1H-pyrrol-2-yl)ethyl]-2-piperidone 3, 4
Under nitrogen atmosphere, a mixture of 2-(2-aminoethyl)-1-
exhibited a weaker activity profile at the same receptor
sites when compared with their structure analogs
methyl-1H-pyrrole (2, 3.73 g, 30 mmol) and ethyl 4-bromobuta-
noate or ethyl 5-bromopentanoate (20 mmol), anhydrous potas-
sium carbonate (4.1 g, 30 mmol) and few crystals of potassium
iodide in absolute ethanol (100 mL) was heated under reflux for
48 h. After evaporating the solvent to dryness in vacuo, the resi-
due was treated with water and extracted several times with
chloroform. The organic extract was washed three times with a
saturated solution of sodium chloride, then thoroughly washed
with water, dried over anhydrous sodium sulphate, and filtered.
Rotary evaporation of chloroform afforded a creamy white solid
residue which was crystallized from toluene/petroleum ether
benzo[d]pyrrolo[3,2-g]azecine
rolo[3,2-g]azecine D and the structure lead LE 300 (Tables
and 3). On the other hand, none of the tested com-
pounds was able to show affinity to any of the utilized
dopamine receptors (D , and D ) in the radioligand
C and indolo[3,2-d]pyr-
1
1
, D2L, D
4
5
binding assay, except for the analog C which showed a sig-
nificant dopamine receptor binding affinity comparable
with that of the reference lead compound LE 300, particu-
(
60:80) (3:1).
larly at the D
1
and D receptor sites (Table 2).
5
Based on the above-mentioned results, it could be
clearly concluded that variation in ring size (9- or 10-
membered rings) did not contribute to a significant influ-
ence on the three tested bioactivities. Moreover, removal
of the hydrophobic moiety (phenyl ring) and replace-
ment of the indole moiety with a 1H-pyrrole counterpart
led to a dramatic decrease in the profile of activity of
such azecine-type compounds. These results are concord-
ant with the previous findings of Lehmann et al., who
stated that the presence of two aromatic rings annulated
to a central 9- or 10-membered azonine or azecine ring
systems are indispensable for high affinities at the dopa-
Compound 3
–
1
1
M.p.: 92–948C; yield: 2.15 g (56%); IR (cm ) m: 1630 (C=O); H-
NMR d: 1.84–2.16 (m, 2H, CH ), 2.36 (t, J = 7 Hz, 2H, CH ), 2.80 (t, J
7 Hz, 2H, CH ), 3.20–3.50 (m, 4H, 2 CH ), 3.60 (s, 3H, CH ), 5.92–
2
2
=
2
2
3
6.12 (m, 2H, pyrrole C -H and C -H), 6.60 (d, J = 7 Hz, 1H, pyrrole
3
4
C
5
16 2
-H). Anal. calculated for C11H N O (192.26): C, 68.72; H, 8.39; N,
1
4.57. Found: C, 68.59; H, 8.47; N, 14.41.
Compound 4
–
1
1
M.p.: 105–1078C; yield: 3.5 g (85%); IR (cm ) m: 1630 (C=O); H-
NMR d: 1.60–2.04 (m, 4H, 2 CH ), 2.20–2.50 (m, 2H, CH ), 2.84 (t, J
7 Hz, 2H, CH ), 3.04–3.28 (m, 2H, CH ), 3.36–3.72 (m, 5H, CH
2
2
2
=
2
2
and CH ), 5.84–6.12 (m, 2H, pyrrole C -H and C -H), 6.50 (d, J = 7
3
3
4
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78
S. A. F. Rostom
Arch. Pharm. Chem. Life Sci. 2010, 343, 73–80
Hz, 1H, pyrrole C
5
-H). Anal. calculated for (C12
H
18
N
2
O) (206.28): C,
Compound 10
1
69.87; H, 8.80; N, 13.58. Found: C, 70.04; H, 8.67; N, 13.42.
M.p.: 183–1858C; yield: 1.56 g (47%); H-NMR d: 1.72–1.92 (m,
4
3
CH
3
H, 2 CH
.28 (m, 2H, CH
), 4.08 (m, 1H, CH), 6.04 (d, J = 7 Hz, 1H, pyrrol-H), 6.56 (d, J = 7
Hz, 1H, pyrrol-H). Anal. calculated for C13 (332.22): C,
7.00; H, 6.37; N, 8.43. Found: C, 46.89; H, 6.44; N, 8.29.
2
), 2.24–2.48 (m, 2H, CH
2
), 2.68–2.92 (m, 2H, CH
2
), 3.08–
2
), 3.40–3.68 (m, 5H, CH
2
and CH ), 3.76 (s, 3H,
3
4
,5,7,8,9,9a-Hexahydro-3-methyl-3H-pyrrolo[2,3-g]
H21IN
2
indolizine and 3,4,5,7,8,9,10,10a-octahydro-3-
methylpyrrolo[3,2-a]quinolizine 7, 8
4
Under anhydrous conditions, a mixture of the appropriate lac-
tam 3 or 4 (20 mmol) and phosphorous oxychloride (10 mL) was
heated under reflux for 4 h and then allowed to cool. Dry ether
1,4,5,6,7,8,9,10-Octahydro-1,8-dimethylpyrrolo[2,3-d]
azonine and 4,5,6,7,8,9,10,11-octahydro-1,9-dimethyl-
(100 mL) was added where an oily product was separated. Excess
1
H-pyrrolo[2,3-d]azecine 11, 12
solvent was evaporated under reduced pressure to afford the
intermediate oily salts (5, 6) which were used for the next reac-
tion in situ without further purification. The residue was dis-
solved in methanol (50 mL) and then 7 g of sodium borohydride
were added in portions with stirring for 2 h while keeping cool
at 08C. Stirring was continued for further 18 h at room temper-
ature, then, methanol was evaporated in vacuo and the residue
was treated with water (100 mL). The resulting emulsion was
extracted thoroughly with ether, and the ethereal extract was
washed with a saturated solution of sodium chloride, then thor-
oughly washed with water, dried over anhydrous sodium sul-
phate, and filtered. Evaporation of excess ether under reduced
pressure afforded a yellow solid residue which was crystallized
from ethanol/petroleum ether (60:80) (4:1).
To a stirred suspension of the appropriate methiodide salt 9 or
0 (5 mmol) in absolute ethanol (5 mL) in a three-necked flask
immersed in a liquified nitrogen bath, liquified ammonia (30
mL) was introduced. To the reaction mixture (maintained at
1
–
408C with methanol-dry ice), sodium metal (2 g) was added por-
tionwise over a period of 45 min and the resulting deep blue sol-
ution was stirred for further 45 min. The reaction was finally
quenched by the addition of a few crystals of ammonium chlor-
ide, and stirring was continued at room temperature for further
1
8 h. Water (20 mL) was then added and the resulting emulsion
was extracted thoroughly with methylene chloride (5625 mL).
The organic extract was washed twice with 5% sodium hydrox-
ide solution, then thoroughly washed with water, dried over
anhydrous sodium sulphate, filtered, and evaporated to dryness
under reduced pressure. The remaining light orange oil was
chromatographed on a silica gel column using a mixture ethyl
acetate/methanol (8:2) as eluent to yield a deep yellow oily prod-
uct.
Compound 7
1
M.p.: 134–1368C; yield: 1.62 g (46%); H-NMR d: 1.60–2.00 (m,
4
H, 2 CH
Hz, 1H, CH), 5.88 (d, J = 7 Hz, 1H, pyrrol-H), 6.50 (d, J = 7 Hz, 1H,
pyrrol-H). Anal. calculated for C11 (176.26): C, 74.96; H, 9.15;
2 2 3
), 2.60–3.0 (m, 6H, 3 CH ), 3.44 (s, 3H, CH ), 3.72 (t, J = 7
Compound 11
16 2
H N
1
Yield: 0.54 g (56%); H-NMR d: 1.0–1.50 (m, 4H, 2 CH
2
), 1.72–2.0
), 2.36–2.80 (m, 4H, 2 CH ), 2.96–
), 3.50 (s, 3H, CH ), 6.12 (d, J = 7 Hz, 1H, pyrrol-H),
.50 (d, J = 7 Hz, 1H, pyrrol-H). Anal. calculated for C12
N, 15.89. Found: C, 75.09; H, 9.02; N, 15.67.
(
3
6
m, 2H, CH
2
), 2.16 (s, 3H, CH
3
2
.32 (m, 2H, CH
2
3
H
20
N
2
Compound 8
1
(192.3): C, 74.95; H, 10.48; N, 14.57. Found: C, 74.81; H, 10.62; N,
4.38.
M.p.: 151–1538C; yield: 2.1 g (55%); H-NMR d: 1.24–1.96 (m, 4H,
1
2
CH
CH), 3.44 (s, 3H, CH
Hz, 1H, pyrrol-H). Anal. calculated for C12
H, 9.53; N, 14.72. Found: C, 75.81; H, 9.48; N, 14.81.
2
), 2.20–2.50 (m, 4H, 2 CH
), 5.88 (d, J = 7 Hz, 1H, pyrrol-H), 6.44 (d, J = 7
(190.28): C, 75.74;
2 2
), 2.72–3.12 (m, 5H, 2 CH and
3
H
18
N
2
Compound 12
1
Yield: 0.51 g (49%); H-NMR d: 1.24–1.88 (2 m, 6H, 3 CH ), 2.08 (s,
2
3
7
H, CH
Hz, 1H, pyrrol-H), 6.08 (d, J = 7 Hz, 1H, pyrrol-H). Anal. calcu-
(206.33): C, 75.68; H, 10.75; N, 13.58. Found: C,
5.52; H, 10.88; N, 13.41.
3 2 3
), 2.28–3.16 (4 m, 8H, 4 CH ), 3.50 (s, 3H, CH ), 5.88 (d, J =
Methyl 4,5,7,8,9,9a-hexahydro-3-methyl-3H-pyrrolo[2,3-g] lated for C13
22 2
H N
7
indolizinim iodide and methyl 3,4,5,7,8,9,10,10a-octa-
hydro-3-methylpyrrolo[3,2-a]quinolizinium iodide 9, 10
Under anhydrous conditions, a solution of the appropriate
cyclized compound 7 or 8 (10 mmol) in dry acetone (30 mL) was
treated with iodomethane (4 mL) while stirring for 2 h and then
allowed to stand at room temperature overnight. The precipitate
obtained was filtered, washed with cold acetone, dried, and
recrystallized from ethanol.
Biological testing
In-vitro serotonin 5-HT2A receptor ligand activity
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 (378C) of composition (mM): NaCl 118.1, KCl
4
1
.7, CaCl
2
2.5, MgSO
4
1.2, KH
2
PO
4
1.2, NaHCO
3
25.0, and D-glucose
/5% CO and con-
0.0. The solution was aerated with 95% O
2
2
Compound 9
tained prazosin (30 nM) to block a1 adrenoceptors, and cocaine
(6 lM) to block neuronal uptake of amines. During an equilibra-
tion period of 120 min, the preparations were primed once
(after 60 min) with 5-HT (1 and 10 lM) to monitor tissue viability.
Two cumulative concentration-effect curves for 5-HT (0.01 to 30
lM for the first curve) were determined in the absence and pres-
ence of potential antagonists which were usually incubated for
1
M.p.: 175–1778C; yield: 1.52 g (48%); H-NMR d: 1.72–1.92 (m,
2
H, CH
CH ), 4.08–4.32 (m, 4H, 2 CH
7 Hz, 1H, pyrrol-H), 6.60 (d, J = 7 Hz, 1H, pyrrol-H). Anal. calcu-
lated for C12 (318.2): C, 45.30; H, 6.02; N, 8.80. Found: C,
5.18; H, 6.17; N, 8.64.
2
), 2.96–3.20 (m, 4H, 2 CH
2 3
), 3.48 (s, 3H, CH ), 3.60 (s, 3H,
), 4.88 (t, J = 7 Hz, 1H, CH), 5.94 (d, J
3
2
=
H19IN
2
4
i
2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2010, 343, 73–80
Novel Fused Pyrroles as Analogs of LE 300
79
3
0 or 120 min (LE 300, ketanserin at 0.47 and 1 nM, respectively).
viously treated with a 0.25% polyethylene imine solution
(Sigma-Aldrich), which was washed twice with ice-cold water.
The radioactivity retained on the filters was counted using a
Beckman LS 6000 SC scintillation counter (Beckman Instru-
ments, USA). The competition binding data was analyzed by the
Control experiments in the absence of antagonist revealed that
for the 30-min protocol, two successive concentration-effect
curves for 5-HT were superimposable (data not shown). When
the 120-min protocol was applied, a leftward shift of the control
curves of approximately 0.1 to 0.3 logarithmic units was usually
observed. The daily mean sensitization was used to correct the
dextral shift measured for treated organs. Results are expressed
as mean l standard error (SEM or SE) unless otherwise indicated.
TM
TM
software GraphPad Prism (GraphPad Prism , San Diega, CA,
USA) using non-linear least squares fit.
In-vitro histamine H receptor activity
1
2
Antagonist affinity was calculated as apparent pA value accord-
Whole segments of guinea-pig ileum (1.5 to 2.0 cm in length)
were mounted isotonically (preload 0.5 g) in Tyrode solution
ing to equation (1) when only one or two antagonist concentra-
tions were used. ([c] = mol/L, r is the ratio of agonist concentra-
tions in the presence and absence of antagonist that elicit 50% of
the respective maximum effect).
(
378C) of the composition (mM): NaCl 136.9, KCl 2.7, CaCl
MgCl 1.0, KH PO 0.4, NaHCO 11.9, and D-glucose 5.1. The solu-
tion was aerated with 95% O /5% CO and contained atropine
0.1 lM) to block ileal M receptors. During an equilibrium
2
1.8,
2
2
4
3
2
2
(
3
pA
2
= – log10 c (antagonist) + log10 (r – 1)
(1)
period of 80 min, the organs were primed three times with hista-
mine (1 and 10 lM every time). Up to four cumulative concentra-
tion-effect curves for histamine (0.1 to 30 lM for first curve) were
determined in the absence and presence of potential antagonists
which were incubated for 10 min. Control experiments in the
absence of antagonist revealed that four successive concentra-
tion-effect curves for histamine were superimposable.
Full pA
2
values were calculated according to the method of
Schild [30] when a set of different antagonist concentrations
over at least 1.5 logarithmic units was studied. Single organ
preparations were obtained from at least three animals.
Dopamine receptor ligand activity
Cell culture
Human D , D2L, D , and D receptors were stably expressed in Chi-
1 4 5
nese hamster ovary (CHO) cells as previously described [34]. The
density of receptors measured with [ H]-SCH 23390 was 307.15
fmol/mg protein for D
of receptors measured with [ H]-Spiperone was 2021 fmol/mg
protein for D2L and 137.21 fmol/mg for D . These cells were
grown at 378C under a humidified atmosphere of 5% CO /95%
Results are expressed as mean l standard error (SEM or SE)
unless otherwise indicated. Antagonist affinity was calculated as
2
apparent pA value as previously described under equation (1).
Full pA values were calculated according to the method of
2
3
Schild [30] when a set of different antagonist concentrations
over at least 1.5 logarithmic units was studied. Single organ
preparations were from at least two animals.
1 5
and 679.44 fmol/mg for D . The density
3
4
2
The author is extremely grateful to Prof. Dr. Jochen Lehmann,
Institute of Pharmacy, Pharmaceutical Medicinal Chemistry,
Friedrich-Schiller-University Jena, Jena, Germany, for his gener-
ous guidance during the course of this investigation. His great
efforts and sincere assistance in managing the biological stud-
ies in this work are highly appreciated.
air in HAM/F12-medium (Sigma-Aldrich, Germany), supple-
mented with 10% fetal bovine serum, 1 mM L-glutamine, 20 U/
mL penicillin G, 20 lg/mL streptomycin, and 0.2 lg/mL G 418 (all
from Sigma-Aldrich).
Preparation of whole-cell-suspension
Human D , D2L, D , and D receptor cell lines (CHO) were grown
1 4 5
on T 175 culture dishes (Nunc, Denmark) 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
The author has declared no conflict of interest.
(
(
1000 rot/min, 48C, 4 min), the pellet resuspended in 10 mL PBS
ice-cooled, calcium- and magnesium-free), pelleted, and this
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