New 1,2,3,9-tetrahydro-4H-carbazol-4-one derivatives: Analogues of HEAT as ligands for the α1-adrenergic receptor subtypes

Article abstract of DOI:10.1016/j.bmc.2006.04.002

With the aim to develop new ligands able to discriminate among the three subtypes of α₁-adrenergic receptors (α_1A-AR, α_1B-AR, and α_1D-AR), a series of new 1,2,3,9-tetrahydro-4H-carbazol-4-ones bearing a 3-[[[2-(4-hydroxyphenyl)ethyl]amino]methyl] or a 3-[[4-(2-substitutedphenyl)piperazin-1-yl]methyl] side chain were synthesized. The general structure of the new compounds is reminiscent of HEAT and RN5, two potent α₁-AR antagonists which show high affinities for all three α₁-AR subtypes. Some derivatives in which one ring of the tetrahydrocarbazolone system was opened were also prepared. Compounds were tested in radioligand binding assays on human cloned α_1A-AR, α_1B-AR, and α_1D-AR subtypes stably expressed in HEK293 cells. They showed moderate to good affinities, although their selectivity among the receptor subtypes hardly reached one order of magnitude.

Full text of DOI:10.1016/j.bmc.2006.04.002

Bioorganic & Medicinal Chemistry 14 (2006) 5211–5219
New 1,2,3,9-tetrahydro-4H-carbazol-4-one derivatives: Analogues
of HEAT as ligands for the a -adrenergic receptor subtypes
1
a,
a
a
a
a
*
Giuseppe Romeo, Luisa Materia, Valeria Pittal a` , Maria Modica, Loredana Salerno,
a
a
b
Mariangela Siracusa, Filippo Russo and Kenneth P. Minneman
a
Dipartimento di Scienze Farmaceutiche, Universit a` di Catania, viale A. Doria 6, 95125 Catania, Italy
b
Department of Pharmacology, Emory University, 1510 Clifton Road, Atlanta, GA 30322, USA
Received 27 July 2005; revised 28 March 2006; accepted 4 April 2006
Available online 2 May 2006
Abstract—With the aim to develop new ligands able to discriminate among the three subtypes of a -adrenergic receptors (a1A-AR,
1
a1B-AR, and a1D-AR), a series of new 1,2,3,9-tetrahydro-4H-carbazol-4-ones bearing a 3-[[[2-(4-hydroxyphenyl)ethyl]amino]methyl]
or a 3-[[4-(2-substitutedphenyl)piperazin-1-yl]methyl] side chain were synthesized. The general structure of the new compounds is
reminiscent of HEAT and RN5, two potent a -AR antagonists which show high affinities for all three a -AR subtypes. Some deriv-
1
1
atives in which one ring of the tetrahydrocarbazolone system was opened were also prepared. Compounds were tested in radioligand
binding assays on human cloned a1A-AR, a1B-AR, and a1D-AR subtypes stably expressed in HEK293 cells. They showed moderate
to good affinities, although their selectivity among the receptor subtypes hardly reached one order of magnitude.
ꢀ
2006 Elsevier Ltd. All rights reserved.
1. Introduction
a1-ARs do not constitute a homogeneous receptor class
and three different subtypes, namely a1A-AR, a -AR
1B
Adrenergic receptors (ARs) play a key role in the mod-
ulation of sympathetic nervous system activity and are
classified into three different subfamilies (a -, a -, and
and a1D-AR, have been identified, cloned and character-
ized so far. They show preferential coupling to the Gq
family of G-proteins leading to the inositol(1,4,5)tri-
6
,7
1
2
2+
b-ARs), on the basis of their pharmacology, amino acid
phosphate-mediated increase of intracellular Ca . Each
subtype shows a discrete tissue distribution suggesting
1
,2
sequence and signalling mechanisms. They are mem-
brane proteins and all belong to the super-family of
8
specific physiological functions. Recent approaches to
G-protein-coupled receptors (GPCRs). a -ARs are ther-
apeutically relevant because of their primary role in the
cardiovascular system, notably in the control of the vas-
the study of a -AR pharmacology have been based on
1
1
9
10
transgenic knockout mice of a -AR, a -AR and
1A 1B
11
a1D-AR as well as on the use of oligonucleotide micro-
1
2
cular tone. a -ARs are also widely distributed outside
array techniques; such approaches have provided new
valuable insights into the comparative analysis of func-
tions mediated by each subtype. Thus, by using trans-
genic mice lacking a -AR and/or a1D-ARs, the
1
the cardiovascular system and are particularly abundant
in organs such as brain, liver, spleen and prostate.
Although the entire set of physiological roles in which
a -ARs are involved is still not completely clear, a -
1
B
differential contributions of these two subtypes in the
1
1
1
3,14
ARs represent pharmacological targets for a number
of drugs currently used in the treatment of two widely
occurring diseases, hypertension and benign prostatic
control of blood pressure have been clarified.
However, in the last two decades, the development of a1-
AR subtype-selective agonists and antagonists has also
been of great importance for the advancement in under-
3
–5
hyperplasia (BPH).
standing a -AR function. Their use has represented and
1
still remains an invaluable approach, complementary to
others, to the study of individual receptor subtypes.
Through the years, medicinal chemistry has provided
several selective molecules, especially for the a1A-AR,
which have been proved to be very useful experimental
Keywords:
subtypes; HEAT; 1,2,3,9-Tetrahydro-4H-carbazol-4-ones.
1 1
a -adrenergic receptor ligands; a -adrenergic receptor
*
0
Corresponding author. Tel.: +39 095 738 4027; fax: +39 095
22239; e-mail: gromeo@mbox.unict.it
1
5
2
tools and, in some cases, potentially effective drugs.
968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.04.002

5212
G. Romeo et al. / Bioorg. Med. Chem. 14 (2006) 5211–5219
Nevertheless, much research is still needed to discover
new and more selective ligands, particularly for the
a -AR and a1D-AR subtypes.
derivative SGB 1534 (4), one of the early a -AR ligands,
18
1
was used as template (Chart 1). In fact, in that case,
the pyrimido[5,4-b]indole-2,4-dione system in RN5 (2)
had been formally obtained by the insertion of a pyrrole
ring between the benzene and the pyrimidine rings of the
quinazoline-2,4-dione moiety of SGB 1534 (4).
1
B
HEAT (1) and RN5 (2) (Chart 1) are two known a -AR
1
antagonists with affinity values in the subnanomolar
1
6–19
range.
From a structural point of view, HEAT (1)
can be subdivided in a planar bicyclic system (a-tetr-
alone) and a tyramine moiety held together with a meth-
ylene bridge. The protonatable nitrogen of the tyramine
substructure represents the basic group which, in all
In addition to compounds 16–20, some other derivatives
(21–24) in which the protonatable nitrogen is part of a
4-(2-substitutedphenyl)piperazin-1-yl substructure in-
stead of a tyramine moiety were also prepared along
with derivatives 26–28 lacking one of the six-membered
rings of the TC system.
known a -AR ligands, is essential for interaction with
1
the receptor binding site. Similarly, in RN5 (2) a planar
tricyclic pyrimido[5,4-b]indole moiety is linked, through
an ethylene chain, to a phenylpiperazine (PP) portion
bearing the essential protonatable nitrogen.
2. Results and discussion
2.1. Synthesis
Although both 1 and 2 do not show appreciable selectiv-
ity among the a -AR subtypes, discrete modifications
1
on their structures could provide new potential
subtype-selective ligands.
The preparation of title compounds is outlined in
Schemes 1–3. 1,2,3,9-Tetrahydro-4H-carbazol-4-one 5,
obtained from phenylhydrazine and 1,3-cyclohexandi-
2
0
On this basis, and with the aim to obtain subtype-selec-
tive molecules, we now report the synthesis and the
one following the Fisher indole synthesis, was N-alkyl-
ated by the suitable alkyl halide in dimethylformamide
(DMF) in the presence of sodium hydride to give com-
pounds 6–9 in good yields (Scheme 1). Some attempts
to obtain 9-phenyl-1,2,3,9-tetrahydro-4H-carbazol-4-
one 12 by a direct arylation of 5 were made on the basis
of a previously reported N-arylation of the indole nucle-
us with bromobenzene in the presence of copper (I) bro-
binding properties to the three human cloned a -AR
1
subtypes of a number of new 1,2,3,9-tetrahydro-4H-car-
bazol-4-one (TC) derivatives 16–20. They are HEAT (1)
analogues in which the a-tetralone moiety has been
replaced by a tricyclic (TC) system which bears, at the
9
-position, groups of different nature and shape. The
2
1
larger aromatic plane of TC along with substituents at
the 9-position could be further sites of interaction with
the receptor binding sites and/or represent structural
features inducing subtype-selectivity.
mide and sodium carbonate. When we tried the same
experimental conditions on carbazolone 5, N-arylated
derivative 12 was obtained only in very low yields. An
alternative approach to the preparation of 12 had been
2
2
reported in literature in a 26% yield. It had been ob-
2
3
In these new compounds, the TC system can be consid-
ered an a-tetralone in which a pyrrole nucleus was
inserted between the benzene and the cyclohexenone
rings. A similar strategy had been successfully used in
the design of RN5 (2), where the quinazoline-2,4-dione
tained, along with N-methyl and N-benzyl derivatives
6 and 8, by means of dehydrogenation with chloranil of
the corresponding 9-substituted-1,2,3,4,5,6,7,8-octa-
hydrocarbazol-4-ones. However, we synthesized 12 in
comparable yields (23%) by ring closure of enehydrazine
1
1 in acetic acid and zinc chloride (Scheme 1).
Successively, compounds 6–9 and 12 were caused to
react with tyramine hydrochloride (13) in the presence
of paraformaldehyde to give final compounds 16–20 as
OH
OH
O
O
N
H
N
H
N
R
1
(HEAT)
1
6-20
R1
O
O
O
6
R = CH3
H
a
N
N
7 R = n-C3H7
N
O
X
N
N
R
8 R = CH2C6H5
9 R = CH2C6H11
N
R
N
H
N
N
N
H
21-24
O
5
2
3
(RN5) X = OCH3
X = Cl
OH
O
N
H
N
O
H
O
H
N
H
O
O
26
b
N
c
NH2
HCl
N
N
N
N
.
N
N
O
R1
O
N
N
4
(SGB 1534)
N
1
0
11
12
H
2
7-28
Scheme 1. Reagents: (a) NaH 80%, alkyl halides, dry DMF; (b) 1,3-
cyclohexanedione, EtOH/water, N ; (c) ZnCl , AcOH.
Chart 1.
2
2

G. Romeo et al. / Bioorg. Med. Chem. 14 (2006) 5211–5219
5213
OH
OH
H2N
.
O
HCl
1
3
N
.
H
HCl
a
N
R
1
1
1
1
6 R = CH3
7 R = n-C3H7
8 R = CH2C6H5
9 R = CH2C6H11
O
N
R
R1
20 R = C H
6
5
HN
N
.
5
-9, 12
HCl
1
1
4 R1 = OCH3
5 R1 = Cl
O
R1
N
N
a
N
R
. HCl
2
2
2
2
1 R = H; R1 = OCH3
2 R = CH3; R1 = OCH3
3 R = H; R1 = Cl
4 R = CH3; R1 = Cl
Scheme 2. Reagents and condition: (a) paraformaldehyde, EtOH, reflux.
OH
OH
H2N
.
O
HCl
1
3
N
.
H
HCl
a
N
H
2
6
O
N
R1
H
HN
N
.
25
HCl
1
1
4 R1 = OCH3
5 R1 = Cl
R1
O
N
N
a
N
H
. HCl
2
2
7 R1 = OCH3
8 R1 = Cl
Scheme 3. Reagents and condition: (a) paraformaldehyde, EtOH, reflux.
hydrochlorides. In the same experimental conditions,
reaction of carbazol-4-ones 5 and 6 with hydrochlorides
of 1-(2-methoxyphenyl)piperazine (14) or 1-(2-chloro-
phenyl)piperazine (15) afforded hydrochlorides of Man-
nich bases 21–24 in good yields (Scheme 2).
this context, however, some structure–affinity relation-
ships can be drawn.
Tyramine derivatives 16–20 displayed a slightly prefer-
ential affinity for the a1A-AR subtype. From a chemical
point of view, they differ from each other in the size of
the substituent at the 9-position of the TC system, going
from a simple methyl to the larger cyclohexyl or benzyl
groups. Nevertheless, these differences in the substituent
shape appear not to have great influence on affinity. In
fact, methyl derivative 16 and the analogous compound
Finally, reaction of 3-acetyl-1H-indole (25), commer-
cially available, with paraformaldehyde and amines 13,
1
4 or 15 led to final indole derivatives 26–28 (Scheme 3).
2
.2. Binding studies
18, bearing the bulkier benzyl moiety, displayed similar
Title compounds 16–24 and 26–28 were tested in binding
assays on human a1A-AR, a -AR and a1D-AR sub-
types stably expressed in HEK293 cells using
[ I]HEAT as radioligand. Affinities of tested com-
pounds for the three cloned receptor subtypes are
pK values at a -AR (7.71 and 7.65), a -AR (7.21 and
7.19) and a1D-AR (7.39 and 7.52), respectively.
i
1A
1B
1B
1
25
According to this trend, benzyl derivative 18 showed the
same affinity as its cyclohexylmethyl analogue 19 at the
expressed as pK values and summarized in Table 1.
i
a1A-AR. However, for the other two a -AR subtypes,
1
affinities of 19 were lower than those displayed by 18.
The benzyl and the cyclohexylmethyl groups have simi-
lar steric bulk but differ in aromaticity, which could be
the discriminant feature. Thus, a nonaromatic cyclo-
alkyl moiety seems to be less tolerated than an aromatic
As a general trend, all tested compounds showed lower
affinities than HEAT (1) and RN5 (2) for all three
a1-AR subtypes. In addition, their subtype-selectivity
was generally less than one order of magnitude. Within

5214
G. Romeo et al. / Bioorg. Med. Chem. 14 (2006) 5211–5219
Table 1. Binding properties of 1,2,3,9-tetrahydro-4H-carbazol-4-one (16–24) and 1H-indole derivatives (26–28)
a
Compound
i
pK (M)
a1A-AR
a
1B-AR
a1D-AR
1
1
1
1
2
2
2
2
2
2
2
2
1
2
6
7
8
9
0
1
2
3
4
6
7
8
7.71 ± 0.11
7.87 ± 0.08
7.65 ± 0.06
7.64 ± 0.24
7.92 ± 0.17
7.51 ± 0.06
7.22 ± 0.08
7.08 ± 0.20
6.63 ± 0.17
7.10 ± 0.08
8.23 ± 0.10
7.87 ± 0.18
9.74
7.21 ± 0.09
7.19 ± 0.13
7.19 ± 0.14
6.69 ± 0.20
7.78 ± 0.19
7.10 ± 0.21
6.76 ± 0.06
6.75 ± 0.16
6.37 ± 0.16
6.76 ± 0.16
7.39 ± 0.14
7.59 ± 0.19
9.70
7.39 ± 0.10
7.43 ± 0.10
7.52 ± 0.06
6.57 ± 0.17
7.51 ± 0.21
7.88 ± 0.09
7.37 ± 0.14
7.30 ± 0.14
6.97 ± 0.16
7.10 ± 0.10
8.48 ± 0.09
7.95 ± 0.17
9.27
b
(HEAT)
c
(RN5)
9.57 ± 0.14
8.74 ± 0.14
9.44 ± 0.11
a
b
c
Each value is the mean ± SE for data from three different experiments conducted in duplicate.
Data from Ref. 7.
Data from Ref. 19.
benzyl moiety by the a -AR and a1D-AR binding sites
suggest that at physiologic pH (7.4) a reasonable frac-
tion of both ligands should be in the positively ionized
form which is supposed to interact with the receptor
binding site.
1
B
and, on the other hand, at the a1A-AR subtype, this
structural difference appears to have no influence on
affinity. As a consequence of this behaviour, compound
19, unlike 18, shows a slight selectivity for the a1A-AR
subtype.
In compounds 26–28, a tyramine, a 2-MeOPP or a 2-
ClPP moiety was linked, through a propanone chain,
to the 3-position of an 1H-indole nucleus. These three
compounds can be considered the ‘open’ analogues of
derivatives 16, 21 and 23 inasmuch as they lack part
of the cyclohexenone ring of the TC system. Among
them, tyramine derivative 26 showed lower affinity val-
Derivative 20, bearing a phenyl ring linked directly to
the TC nitrogen, displayed comparable affinities at the
three binding sites and, for the a -AR receptor, pre-
1
B
sented the best pK value in the series.
i
In compounds 21–24, a 4-(2-methoxyphenyl)piperazinyl
(
ues than its analogue 16 for all three a -ARs, whereas
the opposite was observed for the PP derivatives 27
and 28. In fact, both these compounds displayed higher
1
2-MeOPP) or a 4-(2-chlorophenyl)piperazinyl (2-ClPP)
moiety was inserted in place of the tyramine moiety be-
cause the PP substructure represents the pharmaco-
phoric portion in RN5 (2), in its analogue 3 (Chart 1),
affinity for all a -AR subtypes than their TC analogues
1
21 and 23.
1
5
and in other a -AR antagonists. Since in tyramine
1
derivatives 16–20, the nature of the substituent at the
In particular, 3-[4-(2-methoxyphenyl)piperazin-1-yl]-
1-(indol-3-yl)propan-1-one 27 displayed the best affinity
for the a1A-AR (pK = 8.23) and the a -AR (pK =
9
-position of the TC system had not greatly influenced
affinity values, only phenylpiperazine derivatives with
22 and 24) or without (21 and 23) a simple methyl
i
1D
i
(
8.48) in the series and possesses an a1A/1D-AR selectivity
group were then synthesized.
over the a -AR subtype of about one order of
1
B
magnitude.
When tested in binding assays, no sign of an appreciable
subtype-selectivity emerged for any of them. In general,
the lack of the substituent at the 9-position and the
presence of a 2-methoxy group on the PP moiety gave
compounds showing higher affinity values for all three
a1-ARs than analogues bearing a 9-methyl group or a
Recently, a homology model of a1A-AR based on struc-
2
4
ture of bovine rhodopsin was described. In GPCRs for
biogenic amines, it is generally accepted that the ligand
binding site is located within the bundle formed by seven
transmembrane domains (TM 1–7) and that an acidic
residue in TM 3 is involved in binding the protonatable
amine group of the ligand. In the case of human a1A-AR
sequence, this residue is represented by Asp106. Accord-
ing to the above-quoted model, Asp106 in TM 3 can be
considered the central anchoring point for ligands,
dividing the receptor binding site into two different sub-
pockets. One is defined by TM 4–7 and the other by TM
1–3 and 7. It was also reported that a1A-AR ligands con-
taining the 2-MeOPP moiety probably fit the binding
site arranging the 2-methoxyphenyl group in the sub-
2
-ClPP moiety (21 vs 22; 21 vs 23; 22 vs 24).
In order to have a rough idea of the fraction of positive-
ly ionized form of these ligands at the physiologic pH, a
potentiometric measure of pK was performed for com-
a
pounds 21 and 16, taken as representatives of 2-MeOPP
and tyramine derivatives prepared in this study, respec-
tively. A pK value of 7.21 was estimated for 21, whereas
a
two different pK values (8.81 and 11.29) were obtained
a
for 16 due to the presence of a phenolic group in
addition to the protonated amine function. These data
2
4
pocket defined by TM 4–7.

G. Romeo et al. / Bioorg. Med. Chem. 14 (2006) 5211–5219
5215
On these bases, a putative binding mode of 2-MeOPP
derivative 27 at the a1A-AR can be supposed to take
place in a similar way: an electrostatic interaction occurs
between the central ionized amine group of the ligand
and Asp106 side chain, with the 2-methoxyphenyl group
addressing the subpocket defined by TM 4–7 and the 1-
for C, H and N were within ±0.4% of theoretical
values and were performed on a Carlo Erba Elemental
Analyzer Mod. 1108 apparatus. H NMR spectra
1
were recorded on a Varian Inova Unity 200 spectro-
1
meter (200 MHz for H NMR and 50 MHz for
13
C
NMR) in DMSO-d solution. Chemical shifts are giv-
6
(
indol-3-yl)propan-1-one moiety extending to the other.
en in d values (ppm), using tetramethylsilane as the
internal standard; coupling constants (J) are given in
Hz. Signal multiplicities are characterized as s (sin-
glet), d (doublet), t (triplet), q (quartet), m (multiplet),
or br (broad signal). All the synthesized compounds
were tested for purity on TLC (aluminium sheet coat-
ed with silica gel 60 F254, Merck) and visualized by
UV (k = 254 and 366 nm). All chemicals and solvents
were of reagent grade and were purchased from com-
mercial vendors.
It is noteworthy that 1,2,3,9-tetrahydro-4H-carbazol-2-
one derivative 21, the analogue of 27 in which part of
the propanone chain has been incorporated into a ring,
showed 5-fold lower a1A-AR affinity than 27. It could be
argued that, in 21, the presence of an additional ring or
the reduced flexibility of the 1,2,3,9-tetrahydro-4H-car-
bazol-2-one system compared to the 1-(indol-3-yl)pro-
pan-1-one moiety induces a less favourable fit with the
subpocket defined by the TM 1–3 and 7.
4.1.1. 9-Propyl-1,2,3,9-tetrahydro-4H-carbazol-4-one (7).
3
. Conclusions
This procedure is presented as an example for the syn-
thesis of compounds 6–8. To a mixture of 1,2,3,9-tetra-
hydro-4H-carbazol-4-one (5) (1.00 g, 5.39 mmol) in dry
DMF (8 mL), NaH (80% m/m suspension in mineral
oil) (0.24 g, 8.00 mmol) was added in a single portion
under stirring. After the evolution of hydrogen ceased,
1-iodopropane (0.91 g, 5.36 mmol) was added and the
reaction mixture was stirred at room temperature for
With the aim to develop new ligands able to discriminate
among the three a -AR subtypes, a series of new com-
pounds structurally related to the potent antagonist
HEAT (1) was prepared and tested in binding assays
on human cloned a1A-AR, a -AR and a1D-AR sub-
types stably expressed in HEK293 cells.
1
1B
24 h. Then the mixture was poured in ice water
The replacement, in the HEAT structure, of the bicyclic
a-tetralone moiety with a larger tricyclic TC system led
to derivatives 16–20 which showed reduced affinity for
all three subtypes. A slight preference for the a1A-AR
over the other two receptors was displayed by some of
them; however, this subtype-selectivity reached, at the
most, one order of magnitude in 9-cyclohexylmethyl
derivative 19.
(50 mL) and stirred for 2 h. The solid was filtered off,
washed with water and dried. Recrystallization from
EtOH/water (1:1, v/v) gave 7 (0.90 g, 73%) as a pure sol-
ꢀ
1
id: mp 149–151 ꢁC; IR (KBr, selected lines) cm 3111,
2954, 1701, 1662, 1549, 1485, 1234, 973, 743, 611; H
1
NMR (DMSO-d ) d 8.06–7.97 (m, 1H, aromatic),
6
7.61–7.52 (m, 1H, aromatic), 7.30–7.12 (m, 2H, aromat-
ic), 4.16 (t, J = 7.2 Hz, 2H, NCH ), 3.00 (t, J = 6.2 Hz,
2
2H, COCH ), 2.44 (t, J = 6.0 Hz, 2H, COCH CH CH ),
2 2 2 2
These results indicate that the a-tetralone moiety is a
critical determinant for the high affinity of HEAT at
a1-ARs and its enlargement to the TC system is detri-
mental for the stability of the receptor–ligand complex.
2.22–2.02 (m, 2H, COCH CH ), 1.84–1.61 (m, 2H,
2 2
NCH CH ), 0.88 (t, J = 7.4 Hz, 3H, NCH CH CH ).
2
2
2
2
3
Anal. (C H NO) C, H, N.
15 17
4.1.2.
9-Methyl-1,2,3,9-tetrahydro-4H-carbazol-4-one
The successive replacement of the tyramine moiety with
a PP substructure, the pharmacophoric portion of RN5
(6). The same procedure, as described for the synthesis
of 7, was followed using iodomethane. Recrystallization
from EtOH gave 6 (65%) as a pure solid: mp 192–
(
which displayed moderate affinity and no appreciable
2) and other a -AR ligands, gave derivatives 21–24
1
ꢀ
1
193 ꢁC; IR (KBr, selected lines) cm
1631, 1605, 1473, 1317, 1131, 1090, 956, 747, 548; H
3048, 2947,
1
subtype-selectivity.
NMR (DMSO-d ) d 8.04–7.96 (m, 1H, aromatic),
6
Finally, opening of the TC system afforded the more
flexible indole derivatives 26–28. Among them, com-
pound 27, bearing the 2-MeOPP moiety, was endowed
with good affinity in the nanomolar range and a prefer-
7.60–7.48 (m, 1H, aromatic), 7.30–7.11 (m, 2H, aromat-
ic), 3.72 (s, 3H, NCH ), 2.98 (t, J = 6.2 Hz, 2H,
3
COCH ), 2.42 (t, J = 5.6 Hz, 2H, COCH CH CH ),
2
2
2
2
2.22–2.02 (m, 2H, COCH CH ). Anal. (C H NO) C,
2
2
13 13
ential a1A/1D-AR affinity over the a -AR subtype.
1B
H, N.
4.1.3. 9-Phenylmethyl-1,2,3,9-tetrahydro-4H-carbazol-4-
4
. Experimental
one (8). The same procedure, as described for the synthe-
sis of 7, was followed using chloromethylbenzene.
Recrystallization from EtOH/water (1:1, v/v) gave 8
4
.1. Chemistry: General methods
(
39%) as a pure solid: mp 148–150 ꢁC; IR (KBr, selected
ꢀ
1
lines) cm 3033, 2937, 1635, 1528, 1463, 1357, 1130,
Melting points were determined using a Gallenkamp
apparatus with a digital thermometer MFB-595 in
glass capillary tubes and are uncorrected. Infrared
spectra were recorded on a Perkin-Elmer FT IR
1
953, 744, 696, 556; H NMR (DMSO-d ) d 8.10–7.99
6
(m, 1H, aromatic), 7.61–7.46 (m, 1H, aromatic), 7.43–
7.08 (m, 2H + 5H, aromatic), 5.50 (s, 2H, NCH ), 2.99
2
1
600 spectrometer in KBr disks. Elemental analyses
(t, J = 6.0 Hz, 2H, COCH ), 2.46 (t, J = 6.0 Hz, 2H,
2

5216
G. Romeo et al. / Bioorg. Med. Chem. 14 (2006) 5211–5219
COCH CH CH ), 2.22–2.04 (m, 2H, COCH CH ).
2
cooled, 6 (0.50 g, 2.50 mmol), and paraformaldehyde
(0.07 g, 2.50 mmol) were added to the mixture which
was then refluxed for 10 h. After the reaction mixture
was cooled, the precipitate was filtered off, washed with
diethyl ether and triturated in acetone. The solid was fil-
tered off, dried and recrystallized from EtOH/diethyl
ether (1/2, v/v) to give 16 (0.20 g, 21%) as a pure prod-
2
2
2
2
Anal. (C H NO) C, H, N.
17
1
9
4
.1.4.
9-Cyclohexylmethyl-1,2,3,9-tetrahydro-4H-car-
bazol-4-one (9). To a mixture of 5 (1.00 g, 5.39 mmol)
in dry DMF (8 mL), NaH (80% m/m suspension in min-
eral oil) (0.24 g, 8.00 mmol) was added in a single por-
tion under stirring. After the evolution of hydrogen
ceased, cyclohexylmethyl bromide (0.95 g, 5.39 mmol)
was added and the reaction mixture was stirred at room
temperature for 24 h. Then the mixture was poured in
ice water (50 mL) and extracted with ethyl acetate (3·
ꢀ
1
uct: mp 216–218 ꢁC; IR (KBr, selected lines) cm
3242, 2939, 2787, 1613, 1516, 1481, 1468, 1262, 833,
1
756; H NMR (DMSO-d ) d 9.41 (s, 1H, OH which
6
exchanges with D O), 8.06–7.94 (m, 1H, aromatic),
2
7.62–7.51 (m, 1H, aromatic), 7.34–7.18 (m, 2H, aromat-
ic), 7.17–7.02 (m, 2H, aromatic), 6.81–6.67 (m, 2H, aro-
matic), 3.75 (s, 3H, NCH ), 3.55–3.39 (m, 1H, COCH),
2
5 mL). The combined organic layers were dried over
anhydrous sodium sulfate and the solvent evaporated
in vacuo. The resulting crude oil (0.30 g), containing 9,
was successively used for the synthesis of 19 without fur-
ther purification.
3
3.28–2.81 (m, 8H, CH ), 2.55–2.29 (m, 1H, COCH-
2
CH H CH ), 2.12–1.86 (m, 1H, COCHCH H CH );
A
B
2
A
B
2
1
3
C NMR (DMSO-d ) d 192.30, 156.06, 153.36,
6
1
37.55, 129.62, 127.21, 124.12, 122.84, 122.38, 120.06,
0
0
4
(
(
.1.5. 3-(N ,N -Diphenylhydrazino)-2-cyclohexen-1-one
115.43, 115.34, 110.48, 48.81, 47.25, 42.38, 30.54,
29.92, 26.93, 20.97. Anal. (C H ClN O ) C, H, N.
11). A solution of 1,1-diphenylhydrazine hydrochloride
10) (10.0 g, 45.3 mmol) in EtOH (175 mL) was added,
2
2
25
2
2
dropwise under nitrogen, to a well-stirred solution of
,3-cyclohexandione (5.08 g, 45.3 mmol) in water
700 mL) at room temperature. Successively, the mix-
4.1.8. 3-[[[2-(4-Hydroxyphenyl)ethyl]amino]methyl]-9-(1-
propyl)-1,2,3,9-tetrahydro-4H-carbazol-4-one hydrochlo-
ride (17). The same procedure, as described for the syn-
thesis of 16, was followed using compound 7 as starting
material. Recrystallization from EtOH gave 17 (10%) as
a pure product: mp 198–200 ꢁC; IR (KBr, selected lines)
1
(
ture was stirred for 30 min and the solid was filtered
off, washed with water and dried. Recrystallization from
EtOH/water (1:2, v/v) afforded 11 as a pure solid (9.00 g,
ꢀ
1
ꢀ1
7
3
8%): mp 165–167 ꢁC; IR (KBr, selected lines) cm
cm 3293, 2952, 2814, 1627, 1516, 1436, 1268, 1209,
1
176, 1584, 1491, 1363, 1187, 1141, 749, 694; H NMR
1
831, 751; H NMR (DMSO-d ) d 9.39 (s, 1H, OH which
6
(
D O), 7.50–7.22 (m, 4H, aromatic), 7.22–6.91 (m, 6H,
DMSO-d ) d 9.70 (br s, 1H, NH which exchanges with
2
exchanges with D O), 8.09–7.97 (m, 1H, aromatic),
6
2
7.70–7.56 (m, 1H, aromatic), 7.35–7.17 (m, 2H, aromat-
ic), 7.17–7.00 (m, 2H, aromatic), 6.84–6.68 (m, 2H, aro-
matic), 4.20 (t, J = 6.8 Hz, 2H, NCH ), 3.55–3.39 (m,
aromatic), 5.13 (s, 1H, COCH), 2.43 (t, J = 5.6 Hz,
2
H, COCH ), 2.12 (t, J = 6.4 Hz, 2H, COCH CH CH ),
2
2
2
2
2
2
H, N.
.01–1.82 (m, 2H, COCH CH ). Anal. (C H N O) C,
18 18
1H, COCH), 3.29–2.82 (m, 8H, CH ), 2.46–2.28 (m,
2
2
2
2
1H, COCHCH H CH ), 2.12–1.88 (m, 1H, COC-
A
B
2
HCH H CH ), 1.87–1.65 (m, 2H, NCH CH ), 0.90 (t,
2
A
B
2
2
1
3
4
(
.1.6.
12). Compound 11 (9.00 g, 32.33 mmol) was added to
9-Phenyl-1,2,3,9-tetrahydro-4H-carbazol-4-one
J = 7.2 Hz, 3H, NCH CH CH ); C NMR (DMSO-
2 2 3
d6) d 192.55, 156.06, 152.95, 136.93, 129.64, 127.21,
124.23, 122.89, 122.35, 120.18, 115.34, 110.78, 110,56,
48.79, 47.26, 44.57, 42.39, 30.60, 27.10, 22.54, 21.10,
11.09. Anal. (C H ClN O ) C, H, N.
a hot solution of zinc chloride (32.30 g, 237 mmol) in
glacial acetic acid (40 mL). The mixture was stirred at
1
00 ꢁC for 4 h. After the mixture was cooled, the precip-
2
4
29
2
2
itate was filtered off, washed with water and dried. The
crude solid was purified by flash chromatography using
ethyl acetate/cyclohexane (1:1, v/v) as eluent. The homo-
geneous fractions were evaporated in vacuo to afford 12
as a pure solid (1.94 g, 23%): mp 141–143 ꢁC (litera-
4.1.9.
3-[[[2-(4-Hydroxyphenyl)ethyl]amino]methyl]-9-
phenylmethyl-1,2,3,9-tetrahydro-4H-carbazol-4-one hydro-
chloride (18). The same procedure, as described for the
synthesis of 16, was followed using compound 8 as start-
ing material. Recrystallization from 2-propanol gave 18
(9%) as a pure product: mp 208–210 ꢁC; IR (KBr, selected
lines) cm 3100, 2930, 2681, 1615, 1516, 1457, 1262,
1127, 1075, 832; H NMR (DMSO-d ) d 9.40 (s, 1H,
OH which exchanges with D O), 8.11–7.98 (m, 1H, aro-
2
2
ꢀ1
ture mp 171 ꢁC); IR (KBr, selected lines) cm 1718,
1
1
654, 1498, 1455, 751, 697; H NMR (DMSO-d )
6
ꢀ1
d 8.14–8.07 (m, 1H, aromatic), 7.72–7.52 (m, 5H, aromat-
ic), 7.33–7.12 (m, 3H, aromatic), 2.84 (t, J = 5.8 Hz, 2H,
COCH ), 2.51 (t, J = 6.4 Hz, 2H, COCH CH CH ),
1
6
2
2
2
2
2
1
2
.23–2.05 (m, 2H, COCH CH ); H NMR (CDCl3)
2
matic), 7.63–7.49 (m, 1H, aromatic), 7.44–7.14 (m,
2H + 5H, aromatic), 7.14–7.02 (m, 2H, aromatic), 6.82–
6.67 (m, 2H, aromatic), 5.53 (s, 2H, NCH ), 3.55–3.39
2
d 8.40–8.25 (m, 1H, aromatic), 7.65–7.10 (m, 7H, aromat-
ic), 2.82 (t, J = 6.2 Hz, 2H, COCH ), 2.63 (t, J = 6.0 Hz,
2
2
2
Anal. (C H NO) C, H, N.
H, COCH CH CH ), 2.31–2.12 (m, 2H, COCH CH ).
2
(m, 1H, COCH), 3.29–2.83 (m, 8H, CH ), 2.46–2.30 (m,
2
2
2
2
2
1H, COCHCH H CH ), 2.15–1.90 (m, 1H, COC-
A
1
8
15
B
2
1
HCH H CH ). Anal. ðC28
H
29ClN
2
O
2
ꢁ H
2
2
OÞ C, H, N.
A
B
2
4
.1.7.
3-[[[2-(4-Hydroxyphenyl)ethyl]amino]methyl]-9-
methyl-1,2,3,9-tetrahydro-4H-carbazol-4-one hydrochlo-
ride (16). This procedure is presented as an example
for the synthesis of compounds 16–20. Tyramine hydro-
chloride (13) (0.43 g, 2.50 mmol) was dissolved in abso-
lute EtOH (10 mL) by gentle heating. After being
4.1.10.
9-Cyclohexylmethyl-3-[[[2-(4-hydroxyphenyl)-
ethyl]amino]methyl]-1,2,3,9-tetrahydro-4H-carbazol-4-one
hydrochloride (19). The same procedure, as described for
the synthesis of 16, was followed using compound 9 as
starting material. Recrystallization from 2-propanol gave

G. Romeo et al. / Bioorg. Med. Chem. 14 (2006) 5211–5219
5217
1
selected lines) cm 3245, 2927, 2850, 1611, 1516, 1453,
9 (10%) as a pure product: mp 194–195 ꢁC; IR (KBr,
(21%) as a pure product: mp 188–190 ꢁC; IR (KBr,
ꢀ
1
ꢀ1
selected lines) cm
1643, 1500, 1475, 1232, 1121, 1026, 755; H NMR
3448, 3044, 2938, 2827, 2625,
1
1
1
OH which exchanges with D O), 8.10–7.95 (m, 1H, aro-
263, 832, 759; H NMR (DMSO-d ) d 9.41 (s, 1H,
6
(DMSO-d ) d 10.53 (br s, 1H, NH which exchanges with
2
6
matic), 7.66–7.53 (m, 1H, aromatic), 7.35–7.16 (m, 2H,
aromatic), 7.15–6.98 (m, 2H, aromatic), 6.85–6.68 (m,
D O), 8.10–7.95 (m, 1H, aromatic), 7.65–7.49 (m, 1H,
aromatic), 7.35–7.16 (m, 2H, aromatic), 7.12–6.86 (m,
4H, aromatic), 3.81 (s, 3H, OCH ), 3.76 (s, 3H,
2
2
3
2
H, aromatic), 4.07 (d, J = 7.0 Hz, 2H, NCH ), 3.58–
2
.45 (m, 1H, COCH), 3.28–2.85 (m, 8H, CH ), 2.46–
2
3
NCH ), 3.74–3.58 (m, 2H, CH ), 3.58–3.42 (m, 2H,
3
2
.29 (m, 1H, COCHCH H CH ), 2.13–1.92 (m, 1H,
A
CH ), 3.42–2.95 (m, 1H + 8H, COCH + CH ), 2.67–
2 2
B
2
COCHCH H CH ) 1.92–1.40 (m, 6H, cyclohexyl),
2
2.51 (m, 1H, COCHCH H CH ), 2.20–1.92 (m, 1H,
A B 2
A
B
1
.34–0.91
(m,
1
2
5H,
cyclohexyl).
Anal.
COCHCH H CH ). Anal. (C H ClN O ) C, H, N.
A B 2 25 30 3 2
ðC H ClN O ꢁ H OÞ C, H, N.
28
35
2
2
2
4.1.14.
3-[[4-(2-Chlorophenyl)piperazin-1-yl]methyl]-
4
.1.11. 3-[[[2-(4-Hydroxyphenyl)ethyl]amino]methyl]-9-
1,2,3,9-tetrahydro-4H-carbazol-4-one hydrochloride (23).
The same procedure, as described for the synthesis of 21,
was followed using 1-(2-chlorophenyl)piperazine hydro-
chloride 15. Recrystallization from MeOH gave 23
(13%) as a pure product: mp 204–205 ꢁC; IR (KBr,
phenyl-1,2,3,9-tetrahydro-4H-carbazol-4-one hydrochlo-
ride (20). The same procedure, as described for the syn-
thesis of 16, was followed using compound 12 as starting
material. Recrystallization from 2-propanol/diethyl
ether (1:1, v/v) gave 20 (10%) as a pure product: mp
ꢀ1
selected lines) cm
3446, 3069, 2940, 2846, 2579,
ꢀ
1
1
1647, 1582, 1468, 1228, 1176, 1038, 766; H NMR
2
1
15 ꢁC; IR (KBr, selected lines) cm
3292, 2940,
650, 1614, 1509, 1437, 1214, 759; H NMR (DMSO-
1
(DMSO-d ) d 12.21 (br s, 1H, carbazole NH which
6
d ) d 9.39 (s, 1H, OH which exchanges with D O),
exchanges with D O), 10.30 (br s, 1H, NH which
exchanges with D O), 8.07–7.91 (m, 1H, aromatic),
6
2
2
8
.16–8.05 (m, 1H, aromatic), 7.75–7.53 (m, 5H, aromat-
2
ic), 7.37–7.13 (m, 3H, aromatic), 7.13–6.98 (m, 2H, aro-
matic), 6.81–6.68 (m, 2H, aromatic), 3.59–3.40 (m, 1H,
COCH), 3.27–2.72 (m, 8H, CH ), 2.44–2.24 (m, 1H,
7.58–7.05 (m, 7H, aromatic), 3.89–3.62 (m, 2H, CH2),
3.60–3.01 (m, 1H + 10H, COCH + CH ), 2.50–2.38 (m,
2
1H, COCHCH H CH ), 2.22–1.94 (m, 1H, COC-
A
2
B
2
COCHCH H CH ), 2.14–1.90 (m, 1H, COC-
2
HCH H CH ). Anal. (C H Cl N O) C, H, N.
3
A
B
A
B
2
23 25
2
HCH H CH ). Anal. (C H ClN O ÆH O) C, H, N.
A
B
2
27 27
2
2
2
4.1.15.
3-[[4-(2-Chlorophenyl)piperazin-1-yl]methyl]-9-
4
1
.1.12. 3-[[4-(2-Methoxyphenyl)piperazin-1-yl]methyl]-
,2,3,9-tetrahydro-4H-carbazol-4-one hydrochloride (21).
methyl-1,2,3,9-tetrahydro-4H-carbazol-4-one hydrochlo-
ride (24). The same procedure, as described for the syn-
thesis of 21, was followed using compounds 6 and 15 as
starting materials. Recrystallization from MeOH gave
24 (25%) as a pure product: mp 193 ꢁC; IR (KBr, select-
This procedure is presented as an example for the syn-
thesis of compounds 21–24. 1-(2-Methoxyphenyl)piper-
azine hydrochloride (14) (0.37 g, 1.62 mmol) was
solubilized in absolute EtOH (7 mL) by gentle heating.
After being cooled, 5 (0.30 g, 1.62 mmol), and parafor-
maldehyde (0.05 g, 1.62 mmol) were added to the mix-
ture which was then refluxed for 10 h. After the
reaction mixture was cooled, the precipitate was filtered
off, washed with cold EtOH and dried. Recrystallization
from EtOH/diethyl ether (1/5, v/v) gave 21 (0.22 g, 32%)
as a pure product: mp 206–208 ꢁC; IR (KBr, selected
ꢀ1
ed lines) cm 3045, 2925, 2625, 1646, 1477, 1126, 1040,
1
759; H NMR (DMSO-d ) d 10.37 (br s, 1H, NH which
6
exchanges with D O), 8.09–7.95 (m, 1H, aromatic),
2
7.65–7.52 (m, 1H, aromatic), 7.51–7.05 (m, 6H, aromat-
ic), 3.77 (s, 3H, NCH ), 3.74–3.58 (m, 2H, CH ), 3.56–
2.96 (m, 1H + 10H, COCH + CH ), 2.64–2.50 (m, 1H,
3
2
2
COCHCH H CH ), 2.20–1.92 (m, 1H, COC-
A
B
2
HCH H CH ). Anal. (C H Cl N O) C, H, N.
3
A
B
2
24 27
2
ꢀ
1
lines) cm 3145, 2918, 2837, 2572, 1614, 1500, 1452,
1
1
242, 1176, 1023, 744; H NMR (DMSO-d ) d 12.20
4.1.16. 3-[[2-(4-Hydroxyphenyl)ethyl]amino]-1-(indol-3-
yl)propan-1-one hydrochloride (26). This procedure is
presented as an example for the synthesis of compounds
26–28. Tyramine hydrochloride (13) (0.54 g, 3.14 mmol)
was dissolved in absolute EtOH (15 mL) by gentle heat-
ing. After being cooled, 3-acetyl-1H-indole (25) (0.50 g,
3.14 mmol) and paraformaldehyde (0.09 g, 3.14 mmol)
were added to the mixture which was then refluxed for
10 h. After the reaction mixture was cooled, the precip-
itate was filtered off, washed with EtOH and triturated
twice in acetone. The solid was filtered off, dried and
recrystallized from DMF/ethyl acetate (1/3, v/v) to give
26 (0.13 g, 12%) as a pure product: mp 206–207 ꢁC; IR
6
(
br s, 1H, carbazole NH which exchanges with D O),
2
1
8
0.18 (br s, 1H, NH which exchanges with D O),
2
.06–7.93 (m, 1H, aromatic), 7.52–7.36 (m, 1H, aromat-
ic), 7.26–7.10 (m, 2H, aromatic), 7.09–6.85 (m, 4H, aro-
matic), 3.81 (s, 3H, OCH ), 3.76–3.60 (m, 2H, CH ),
3
3
3
2
.58–3.44 (m, 2H, CH ), 3.42–3.32 (m, 1H, COCH),
2
.32–2.98 (m, 8H, CH ), 2.50–2.36 (m, 1H, COCH-
2
CH H CH ), 2.20–1.92 (m, 1H, COCHCH H CH );
A
B
2
A
B
2
1
3
C NMR (DMSO-d ) d 192.22, 152.51, 151.85,
6
1
1
5
39.44, 136.11, 124.48, 123.47, 122.85, 121.94, 120.90,
20.03, 118.28, 111.98, 111.83, 110.88, 56.44, 55.42,
1.91, 46.69, 40.96, 28.53, 22.17. Anal. (C H ClN O )
2
4
28
3
2
ꢀ
1
C, H, N.
(KBr, selected lines) cm
1
3262, 3093, 2929, 2784,
1
615, 1516, 1439, 1242, 1141, 742; H NMR (DMSO-
4
.1.13. 3-[[4-(2-Methoxyphenyl)piperazin-1-yl]methyl]-9-
d6) d 12.21 (br s, 1H, indole NH which exchanges with
methyl-1,2,3,9-tetrahydro-4H-carbazol-4-one hydrochlo-
ride (22). The same procedure, as described for the syn-
thesis of 21, was followed using compound 6 as starting
material. Recrystallization from 2-propanol gave 22
D O), 9.42 (s, 1H, OH which exchanges with D O),
9.10 (br s, 2H, NH which exchanges with D O), 8.41
(s, 1H, indole), 8.46–8.35 (m, 1H, indole), 7.60–7.43
(m, 1H, indole), 7.34–7.14 (m, 2H, indole), 7.13–6.98
2
2
2

5218
G. Romeo et al. / Bioorg. Med. Chem. 14 (2006) 5211–5219
3
0
(
3
(
m, 2H, aromatic), 6.84–6.65 (m, 2H, aromatic), 3.40–
.22 (m, 4H, CH ), 3.21–3.05 (m, 2H, CH ), 2.97–2.76
Prism. Nonspecific binding was determined in the
presence of 10 lM phentolamine.
2
2
1
m, 2H, CH ). Anal. ðC H ClN O ꢁ H OÞ C, H, N.
2
19 21
2
2
2
2
4
.3. pK determination
a
4
.1.17. 3-[4-(2-Methoxyphenyl)piperazin-1-yl]-1-(indol-3-
yl)propan-1-one hydrochloride (27). The same procedure,
as described for the synthesis of 26, was followed using
compound 14 as starting material. Recrystallization
from DMF/ethyl acetate (1/3, v/v) gave 27 (63%) as a
pure product: mp 220–222 ꢁC; IR (KBr, selected lines)
pK values for compounds 16 and 21 (as hydrochlorides)
a
were evaluated by potentiometric determination follow-
3
1
ing a literature method and using an automatic titrator
(Crison Compact Titrator) equipped with a combined
glass pH electrode (Crison 52-02). Briefly, titrations
were performed with standardised 0.1 mol/L sodium
hydroxide by constant volume addition of 0.03 mL at
25 ± 1 ꢁC under a nitrogen atmosphere and magnetic
stirring. Each compound (40 mg) was dissolved in
40 mL of a methanol/water (1:3) mixture containing
ꢀ
1
cm 3128, 2926, 2672, 1647, 1496, 1439, 1242, 1148,
1
7
NH which exchanges with D O), 10.97 (br s, 1H, NH
54; H NMR (DMSO-d ) d 12.22 (br s, 1H, indole
6
2
which exchanges with D O), 8.48 (s, 1H, indole), 8.26–
2
8
7
.12 (m, 1H, indole), 7.57–7.43 (m, 1H, indole), 7.32–
.14 (m, 2H, indole), 7.10–6.84 (m, 4H, aromatic), 3.80
0.1 M potassium chloride. A pK value was calculated
a
(
4
s, 3H, OCH ), 3.74–3.42 (m, 8H, CH ), 3.40–2.95 (m,
3
from several points (8–10) of the titration curve using
the following relationship: pK = pH + log[(V ꢀ V )/
2
H, CH ). Anal. (C H ClN O ) C, H, N.
3
2
22 26
2
a
d
eq
d
V ], where V is the volume titrant used at the equiva-
d
eq
4
.1.18. 3-[4-(2-Chlorophenyl)piperazin-1-yl]-1-(indol-3-
lence point and V and pH are the volume titrant and
d d
yl)propan-1-one hydrochloride (28). The same procedure,
as described for the synthesis of 26, was followed using
compound 15 as starting material. Recrystallization
from DMF gave 28 (22%) as a pure product: mp 227–
pH value at the datapoint, respectively. pK values from
a
different datapoints of each titration curve were aver-
aged and the standard deviation was estimated at 0.02.
ꢀ
1
2
28 ꢁC; IR (KBr, selected lines) cm
674, 2578, 1651, 1522, 1438, 1242, 1148, 940, 754; H
3129, 2922,
Acknowledgments
1
2
NMR (DMSO-d ) d 12.22 (br s, 1H, indole NH which
6
This work was supported by grants from the Italian
MIUR and Universit a` di Catania.
exchanges with D O), 11.02 (br s, 1H, NH which
exchanges with D O), 8.48 (s, 1H, indole), 8.28–8.13
2
2
(
m, 1H, indole), 7.60–7.05 (m, 3H + 4H, indole + aro-
matic), 3.83–3.06 (m, 12H, CH ). Anal. (C H Cl N O)
2
21 23
2
3
Supplementary data
C, H, N.
.2. Binding experiments on human cloned a -AR
subtypes
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.bmc.2006.
4
1
0
4.002.
4.2.1. Transfection and cell culture. HEK293 cells were
transfected with the constitutively active pRSVICAT
References and notes
2
5
26
vectors containing the human a1A-AR, a1B-AR, or
28
a1D-AR cDNAs by calcium phosphate transfection.
2
7
1. Hieble, J. P.; Bondinell, W. E.; Ruffolo, R. R., Jr. J. Med.
Chem. 1995, 38, 3415.
2. Ruffolo, R. R., Jr.; Bondinell, W. E.; Hieble, J. P. J. Med.
Chem. 1995, 38, 3681.
3. Grassi, G. Curr. Hypertens. Rep. 2003, 5, 277.
. Schwinn, D. A. BJU International 2000, 86(Suppl. 2), 11.
. Nickel, Curtis J. Urol. 2003, 62(Suppl. 3A), 34.
. Hieble, J. P.; Bylund, D. B.; Clarke, D. E.; Eikenburg, D.
C.; Langer, S. Z.; Lefkowitz, R. J.; Minneman, K. P.;
Ruffolo, R. R., Jr. Pharmacol. Rev. 1995, 47, 267.
. Hancock, A. A. Drug Dev. Res. 1996, 39, 54.
Cells were propagated for several weeks in the presence
of 400 lg/mL geneticin, and subclones screened by radi-
oligand binding for high receptor expression. Transfec-
2
ted HEK293 cells were propagated in 75 cm flasks at
3
4
5
6
7 ꢁC in a humified 5% CO incubator in Dulbecco’s
2
modified Eagle’s medium containing 4.5 g/L glucose,
.4% glutamine, 20 mM HEPES, 100 mg/L streptomy-
1
5
cin, 10 U/L penicillin and 10% calf serum. The cells
were detached by trypsinization and subcultured at a
ratio of 1:4 upon reaching confluency.
7
8
. Price, D. T.; Lefkowitz, R. J.; Caron, M. G.; Berkowitz,
D.; Schwinn, D. A. Mol. Pharmacol. 1994, 45, 171.
4
.2.2. Radioligand binding. Confluent 100-mm plates
9. Rokosh, D. G.; Simpson, P. C. Proc. Natl. Acad. Sci.
U.S.A. 2002, 99, 9474.
10. Cavalli, A.; Lattion, A. L.; Hummler, E.; Nenniger, M.;
Pedrazzini, T.; Aubert, J.-F.; Michel, M. C.; Yang, M.;
Lembo, G.; Vecchione, C.; Mostardini, M.; Schmidt, A.;
Beermann, F.; Cotecchia, S. Proc. Natl. Acad. Sci. U.S.A.
were washed with phosphate-buffered saline (18 mM
Na HPO , 2 mM NaH PO and 154 mM NaCl, pH
2
4
2
4
7
.6) and harvested by scraping. Cells were collected by
centrifugation and homogenized with a Polytron. Cell
membranes were collected by centrifugation at 30,000g
for 10 min and resuspended by homogenization. Recep-
tor density was determined by saturation analysis of the
1
997, 94, 11589.
1
1. Tanoue, A.; Nasa, Y.; Koshimizu, T.; Shinoura, H.;
Oshikawa, S.; Kawai, T.; Sunada, S.; Takeo, S.; Tsuji-
moto, G. J. Clin. Invest. 2002, 109, 765.
2. Gonzalez-Cabrera, P. J.; Gaivin, R. J.; Yun, J.; Ross, S.
A.; Papay, R. S.; McCune, D. F.; Rorabaugh, B. R.;
Perez, D. A. Mol. Pharmacol. 2003, 63, 1104.
1
25
a -AR specific antagonist radioligand [ I]HEAT (20–
1
2
9
8
00 pM). For analysis of competition by selective
1
drugs, 50 pM radioligand was used. Curves were ana-
lyzed by nonlinear regression analysis using GraphPad

G. Romeo et al. / Bioorg. Med. Chem. 14 (2006) 5211–5219
5219
1
1
3. Chalothorn, D.; McCune, D. F.; Edelmann, S. E.; Tobita,
K.; Keller, B. B.; Lasley, R. D.; Perez, D. M.; Tanoue, A.;
Tsujimoto, G.; Post, G. L.; Piascik, M. T. J. Pharmacol.
Exp. Ther. 2003, 305, 1045.
4. Hosoda, C.; Koshimizu, T. A.; Tanoue, A.; Nasa, Y.;
Oikawa, R.; Tomabechi, T.; Fukuda, S.; Shinoura, H.;
Oshikawa, S.; Takeo, S.; Kitamura, T.; Cotecchia, S.;
Tsujimoto, G. Mol. Pharmacol. 2005, 67, 912.
22. Sucrow, W.; Slopianka, M.; Mentzel, C. Chem. Ber. 1973,
106, 745.
23. Sucrow, W.; Wiese, E. Chem. Ber. 1970, 103, 1767.
24. Evers, A.; Klabunde, T. J. Med. Chem. 2005, 48, 1088.
25. Hirasawa, A.; Horie, K.; Tanaka, T.; Takagaki, K.;
Murai, M.; Yano, J.; Tsujimoto, G. Biochem. Biophys.
Res. Commun. 1993, 195, 902.
26. Ramarao, C. S.; Denker, J. M.; Perez, D. M.; Gaivin, R.
J.; Riek, R. P.; Graham, R. M. J. Biol. Chem. 1992, 267,
21936.
27. Esbenshade, T. A.; Hirasawa, A.; Tsujimoto, G.; Tanaka,
T.; Yano, J.; Minneman, K. P.; Murphy, T. J. Mol.
Pharmacol. 1995, 47, 591.
28. Minneman, K. P.; Theroux, T. L.; Hollinger, S.;
Han, C.; Esbenshade, T. A. Mol. Pharmacol. 1994,
46, 929.
29. Theroux, T. L.; Esbenshade, T. A.; Peavy, R. D.;
Minneman, K. P. Mol. Pharmacol. 1996, 50, 1376.
30. GraphPad Prism; GraphPad Software Inc., San Diego,
CA.
31. van Steen, B. J.; van Wijngaarden, I.; Tulp, M. T. M.;
Soudijn, W. J. Med. Chem. 1993, 36, 2751.
1
1
1
1
1
5. Romeo, G.; Materia, L.; Salerno, L.; Russo, F.; Minn-
eman, K. P. Exp. Opin. Ther. Patents 2004, 14, 619.
6. Heinz, N.; Hofferber, E. Arzneimittelforschung 1980, 30,
135.
7. G o¨ thert, M.; Nolte, J.; Weinheimer, G. Eur. J. Pharmacol.
981, 70, 35.
8. Russo, F.; Romeo, G.; Guccione, S.; De Blasi, A. J. Med.
Chem. 1991, 34, 1850.
9. Romeo, G.; Materia, L.; Manetti, F.; Cagnotto, A.;
Mennini, T.; Nicoletti, F.; Botta, M.; Russo, F.; Minn-
eman, K. P. J. Med. Chem. 2003, 46, 2877.
2
1
2
2
0. Lauer, K.; Kiegel, E.U.S. Patent 4,273,711, 1981.
1. Pozharskii, A. F.; Martsokha, B. K.; Simonov, A. M. Zh.
Obshch. Chim. 1963, 33, 1005.