Novel 4-phenylpiperidine-2,6-dione derivatives. Ligands for α1-adrenoceptor subtypes

Article abstract of DOI:10.1016/j.ejmech.2011.03.054

A number of new 4-phenylpiperidine-2,6-diones bearing at the 1-position an ω-[4-(substituted phenyl)piperazin-1-yl]alkyl moiety were designed and synthesized as ligands for the α₁-adrenergic receptor (α₁-AR) subtypes. Some synthesized compounds, tested in binding assays for the human cloned α_1A-, α_1B-, and α_1D-AR subtypes, displayed affinities in the nanomolar range. Highest affinity values were found in derivatives having a butyl connecting chain between the 4-phenylpiperidine-2,6-dione and the phenylpiperazinyl moieties. 1-[4-[4-(2-Methoxyphenyl)piperazin-1-yl]butyl]-4-phenylpiperidine-2,6- dione (34) showed the best affinity for the α_1A-AR (pK_i= 8.74) and 10-fold selectivity compared to the other two α₁-AR subtypes. Some representative compounds were also tested in order to evaluate their effects on the signal transduction pathway coupled to α₁-AR subtypes. They all blocked norepinephrine-induced stimulation of inositol phospholipid hydrolysis, thus behaving as antagonists. Binding data were used to refine a previously developed pharmacophoric model for α_1D-ARs. The revised model shows a highly predictive power and could be useful for the future design of high affinity α_1D-AR ligands.

Full text of DOI:10.1016/j.ejmech.2011.03.054

European Journal of Medicinal Chemistry 46 (2011) 2676e2690
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Novel 4-phenylpiperidine-2,6-dione derivatives. Ligands for a1-adrenoceptor
subtypes
,
a
a
a
a
Giuseppe Romeo a , Luisa Materia , Maria N. Modica , Valeria Pittalà , Loredana Salerno ,
*
Maria A. Siracusa a, Fabrizio Manetti b, Maurizio Botta b, Kenneth P. Minneman c
,
1
a Dipartimento di Scienze del Farmaco, Università di Catania, viale A. Doria 6, 95125 Catania, Italy
b Dipartimento Farmaco Chimico Tecnologico, Università degli Studi di Siena, via A. Moro, 53100 Siena, Italy
c Department of Pharmacology, Emory University, 1510 Clifton Road, Atlanta, GA 30322, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A number of new 4-phenylpiperidine-2,6-diones bearing at the 1-position an
piperazin-1-yl]alkyl moiety were designed and synthesized as ligands for the a1-adrenergic receptor (a1
u-[4-(substituted phenyl)
Received 13 January 2011
Received in revised form
16 March 2011
Accepted 25 March 2011
Available online 3 April 2011
-
AR) subtypes. Some synthesized compounds, tested in binding assays for the human cloned a1A-, a1B-,
and a1D-AR subtypes, displayed affinities in the nanomolar range. Highest affinity values were found in
derivatives having a butyl connecting chain between the 4-phenylpiperidine-2,6-dione and the phe-
nylpiperazinyl moieties. 1-[4-[4-(2-Methoxyphenyl)piperazin-1-yl]butyl]-4-phenylpiperidine-2,6-dione
(34) showed the best affinity for the a1A-AR (pKi ¼ 8.74) and 10-fold selectivity compared to the other
two a1-AR subtypes. Some representative compounds were also tested in order to evaluate their effects
on the signal transduction pathway coupled to a1-AR subtypes. They all blocked norepinephrine-induced
stimulation of inositol phospholipid hydrolysis, thus behaving as antagonists. Binding data were used to
refine a previously developed pharmacophoric model for a1D-ARs. The revised model shows a highly
predictive power and could be useful for the future design of high affinity a1D-AR ligands.
Ó 2011 Elsevier Masson SAS. All rights reserved.
Keywords:
a1-Adrenoceptor subtypes
4-Phenylpiperidine-2,6-dione
Ligands
Pharmacophoric model
1. Introduction
All three subtypes are also present in the central nervous system
(CNS) but their specific roles are still poorly understood [7];
a1-Adrenergic receptors (a1-ARsa) belong to the superfamily of
seven-transmembrane domain (7-TM) G protein coupled receptors
and mediate effects of the catecholamines epinephrine and
norepinephrine (NE).
Three different a1-AR subtypes, viz. a1A-, a1B-, and a1D-AR, have
been identified, cloned and characterized [1]. They are widely
distributed in peripheral tissues and organs and represent important
pharmacological targets because of their involvement in diseases such
as hypertension, myocardial hypertrophy, benign prostatic hyper-
plasia, and lower urinary tract symptoms (LUTS) [2e6].
a number of studies, however, suggest the involvement of a1-ARs in
modulation of stress-induced anxiety-like behavioural responses
and in control of motor activity [8,9]. A recent report demonstrated
that, in rat, a CNS-penetrating a1-AR antagonist can reduce L-
DOPA-induced dyskinesia [10].
In the last years genetically engineered strains of mice have
become available and provided clues to elucidating subtype-specific
physiological functions [11]. As an example, in a transgenic mouse
model that systematically overexpresses the a1B-AR subtype, animals
display symptoms and degenerative histology that share similarities
with a human Parkinsonian degenerative disease called Multiple
System Atrophy (MSA) which currently has no established therapy.
This phenotype was partially rescued with terazosin, a selective
a1-AR antagonist, indicating that a1-ARs directly participate in
the pathology. Thus, this model gives hints to the roles of a1B-ARs
in neurotransmission, locomotion, and in the mechanism of a
degenerative disease, as well as suggests the therapeutic potential
of a1-AR selective antagonists in the treatment of MSA [12]. In
another transgenic mouse model lacking the a1B-AR subtype (a1B-AR
knockout mice), it was observed that locomotor and rewarding
effects of psychostimulant drugs and opiates, such as morphine,
were greatly decreased with respect to wild-type mice [13e15].
Abbreviations: a1-ARs, a1-adrenergic receptors; NE, norepinephrine; LUTS,
lower urinary tract symptoms; CNS, central nervous system; MSA, multiple system
atrophy; PPD, 4-phenylpiperidine-2,6-dione; Epl, equatorial parallel; Epr, equatorial
perpendicular; Apr, axial perpendicular; Apl, axial parallel; IP, inositol phospolipid;
InsPs, inositol phosphates; PPZ, phenylpiperazinyl; RA, aromatic ring; HY, hydro-
phobic; HBA, hydrogen bond acceptor; PI, positive ionizable; SAR, structure-activity
relationship; TM, transmembrane domain; DMSO, dimethyl sulfoxide; HEPES, 4-(2-
hydroxyethyl)-1-piperazineethanesulfonic acid.
* Corresponding author. Tel.: þ39 095 738 4024; fax: þ39 095 222 239.
E-mail address: gromeo@unict.it (G. Romeo).
Present address: Division of Chemical and Life Sciences and Engineering, King
1
Abdullah University of Science and Technology, Thuwal, Saudi Arabia.
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.03.054

G. Romeo et al. / European Journal of Medicinal Chemistry 46 (2011) 2676e2690
2677
These data indicate that the a1B-AR subtype in the CNS could repre-
sent a pharmacological target in the treatment of addictive behaviour
induced by drugs of abuse. Very recently, it has also been reported
that a1A- and a1B-AR subtypes are differentially involved in regula-
tion of neurogenesis and gliogenesis [16]. This could be the basis for
a novel potential therapeutic option in the treatment of neurode-
generative diseases.
From the medicinal chemistry point of view, identification of the
three different a1-AR subtypes has, within the last decades,
directed research efforts towards the development of selective
ligands for each subtype [17,18]. Such compounds could, in fact,
represent very useful molecular probes in defining the functional
roles of each subtype as well as potential drugs with new thera-
peutic applications.
Among the subtype-selective a1-AR ligands, one example is
represented by 1 (BMY 7378) (Fig. 1) which has become the refer-
ence molecule for the characterization of the a1D-AR subtype (a1A
-
AR, pKi ¼ 7.37; a1B-AR, pKi ¼ 7.30; a1D-AR, pKi ¼ 8.98) [19].
However, the usefulness of this molecule is limited since its
selectivity does not exceed 100-fold. Moreover, 1 also binds with
high affinity to the serotonergic 5-HT1A receptor [20]. In order to
overcome the selectivity limits of 1, a number of modifications on
its structure have been made, leading to analogues having lower
affinity for the 5-HT1A receptor [21,22].
As an extension of our previous studies on a1-AR ligands
[23e25], in the present paper we report the synthesis and the
binding data of a series of new 4-phenylpiperidine-2,6-dione
derivatives (29e44) (Fig. 1) as ligands for the a1-AR subtypes. Their
structure is related to 1, the aforesaid a1D-AR selective ligand, and
to 2 (RN5) (Fig. 1), a pyrimido[5,4-b]indole derivative previously
discovered in our laboratories and characterized by high affinity for
all three a1-AR subtypes (a1A-AR, pKi ¼ 9.57; a1B-AR, pKi ¼ 8.74;
a1D-AR, pKi ¼ 9.44) [23,24]. In the new products (29e44) the 4-
phenylpiperidine-2,6-dione (PPD) moiety is connected, through an
alkyl chain of variable length, to a substituted phenylpiperazinyl
(PPZ) structure. Unlike the pyrimido[5,4-b]indole system charac-
terizing compound 2, in the new compounds the PPD moiety is not
planar and possesses a certain degree of conformational freedom.
The PPD moiety is structurally related to the dihydro-2H-pyran-
2,6(3H)-dione ring which had been previously studied by Altona
and coworkers from a conformational point of view [26e28]. It had
been shown that in 4-phenyldihydro-2H-pyran-2,6(3H)-dione 3
and in 4-(4-chlorophenyl)-4-methyldihydro-2H-pyran-2,6(3H)-
dione 4 (Fig. 2) the relative conformation of the phenyl group with
respect to the dihydro-2H-pyran-2,6(3H)-dione ring can be influ-
enced by the absence or the presence of a second substituent at the
4-position. The dihydro-2H-pyran-2,6(3H)-dione ring exists in
the so-called “sofa” conformation with the C4 atom being out of the
plane of the other ring atoms. In 3, the phenyl ring prefers to occupy
an equatorial position and the most likely minimum is represented
by the parallel conformation (Epl); that is the aromatic ring and the
C4-H bond axis lie in a plane (Fig. 2).
Insertion of a second substituent (i.e., a methyl) at C4 modifies
the conformation. In fact, in 4 the aryl group has no more a marked
preference for the equatorial position. Besides, both in the
Fig. 1. Structures of known a1-AR ligands 1, 2, and 60 along with new compounds
29e48 and 57e59.
Fig. 2. Conformations of dihydro-2H-pyran-2,6(3H)-dione derivatives
according to Altona [26e28].
3 and 4

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G. Romeo et al. / European Journal of Medicinal Chemistry 46 (2011) 2676e2690
equatorial and in axial positions, perpendicular conformations (Epr
and Apr, respectively) are preferred over the parallel (Fig. 2).
With the idea that a second substituent at the 4-position could
similarly modify the conformation of the PPD moiety and thereby
influence the affinity and selectivity of new compounds for a1-AR
subtypes, we also synthesized compounds 45e48 (Fig.1) bearing an
additional 4-methyl group. Along with these molecules, a small
number of 2,3-dihydrospiro[1H-indene-1,40-piperidine]-20,60-dione
derivatives 57e59, in which the phenyl ring was frozen within
a spiro moiety, were also prepared (Fig. 1).
1(2H)-one ([125I]BE 2254) as radioligand. Their affinity values,
expressed as pKi, are summarized in Table 1.
In order to evaluate the effects of compounds 34, 47, and 59 on
the signal transduction pathway coupled to a1-ARs, we determined
their ability to block norepinephrine (NE)-induced stimulation of
inositol phospholipid (IP) hydrolysis by measurement of [3H]
inositol phosphate (InsPs) accumulation in HEK293 cells. Obtained
data, expressed as pKI values, are summarized in Table 2.
These new compounds were also used in refining a previously
developed ligand-based pharmacophoric model for the a1D-AR
subtype [24]. The new model, characterised by five different features,
is able to account for structure-affinity relationships seen in title
compounds and to predict affinities of a large set of a1D-AR ligands.
4. Results and discussion
4.1. Pharmacological activities at the three a1-AR subtypes
Inspection of binding data of title compounds suggests the
length of the connecting alkyl chain between the PPZ and PPD
2. Chemistry
moieties strongly affects their affinity values at a1A-, a1B,- and a1D-
ARs. Derivatives bearing an ethyl (C2) chain, the same as in 1 and 2,
invariably showed poor affinities for a1-ARs whereas longer chains
resulted in ligands with much higher affinities. As an example, 4-
phenylpiperidine-2,6-dione derivatives showed increasing affini-
ties in the order: 29 (C2) < 31 (propyl, C3) ꢁ 37 (pentyl, C5) < 34
(butyl, C4) (Fig. 3). This trend is evident for a1A-ARs and is
confirmed for the other two a1-AR subtypes. As shown in Fig. 3,
butyl compound 34 displayed the highest affinity for a1A-ARs
(pKi ¼ 8.74) coupled to a selectivity of one order of magnitude over
the other two subtypes.
In 34, introduction of a substituent (Cl or NO2) at the 4-position
of the phenyl ring of the PPD moiety was detrimental, since this led
to compounds (42 and 44) with lower affinities for all three
receptor subtypes. Moreover, the presence of the substituent in the
4-position of the phenyl ring modified the trend previously seen for
the unsubstituted compounds: C3 analogues (40 and 43) showed
affinities higher than or comparable to those of corresponding C4
compounds (42 and 44).
Insertion of a methyl group at the 4-position of the piperidine-
2,6-dione (done with the aim to change the preferential confor-
mation of the PPD moiety from Epl to Epr, vide infra) gave
compounds which showed differential trends according to the
receptor subtype. At a1B- and a1D-ARs, derivatives with a C4 con-
necting chain (47 and 48) displayed comparable or slightly higher
affinities than corresponding analogues 45 and 46 having a C3
chain. Conversely, at a1A-ARs, C3 derivatives behaved as more
potent ligands than the C4 ones (45 vs 47 and 46 vs 48).
The synthetic pathways to the final 4-arylpiperidine-2,6-diones
29e48 and 2,3-dihydrospiro[1H-indene-1,40-piperidine]-20,60-
dione derivatives 57e59 are shown in Schemes 1 and 2, respectively.
Starting 3-arylpentanedioic acids 5e8, commercially available or
prepared following literature methods [29e32], were caused to
react with urea, in absence of solvent at 160 ꢀC, to give 4-arylpi-
peridine-2,6-diones 9e12 in good yields. Reaction of 9e12
with 1-bromo-u-chloroalkanes provided the corresponding 1-(u-
chloroalkyl)-4-arylpiperidine-2,6-diones 17e25. Attempts to
directly obtain 2-chloroethyl-4-arylpiperidine-2,6-diones 15 and 16
respectively from 9 and 10 and 1-bromo-2-chloroethane failed.
However, when 1,2-dibromoethanewas used inplace of 1-bromo-2-
chloroethane, we were able to isolate alcohols 13 and 14 which, in
turn, were converted, by reaction with thionyl chloride, into
2-chloroethyl derivatives 15 and 16. Finally, reaction of 1-(2-
methoxyphenyl)piperazine 26, 1-(2-chlorophenyl)piperazine 27,
and 1-(5-chloro-2-methoxyphenyl)piperazine 28 with chloroalkyl
derivatives 15e25 gave the desired final compounds 29e48
(Scheme 1).
Preparation of final 2,3-dihydrospiro[1H-indene-1,40-piperi-
dine]-20,60-dione derivatives 57e59 was accomplished as outlined
in Scheme 2. Ethyl cyano(2,3-dihydro-1H-inden-1-ylidene)acetate
49, obtained from 1-indanone and ethyl cyanoacetate [33], was
treated with cyanoacetamide in the presence of sodium ethoxide to
give 2,3-dihydro-20,60-dioxospiro[1H-indene-1,40-piperidine]-30,50-
dicarbonitrile 50 in excellent yield. Hydrolysis, decarboxylation and
opening of the piperidine ring of compound 50 in strong acidic
medium afforded 1,1-indandiacetic acid 51. Its successive reaction
with urea at 160 ꢀC gave the 2,3-dihydrospiro[1H-indene-1,40-
piperidine]-20,60-dione 52 which was then caused to react with 1-
bromo-3-chloropropane or 1-bromo-4-chlorobutane to provide
In 2,3-dihydrospiro[1H-indene-1,40-piperidine]-20,60-dione
compounds 57e59 (in which the spiro moiety is constrained in an
Epl-like conformation), affinities at all three receptor subtypes
varied following the same trend observed in the 4-phenylpiperidine-
2,6-dione series, increasing from C2 (57) to C3 (58) to C4 derivative
(59) (at a1A-ARs; pKi ¼ 5.47, 7.20, 8.01, respectively).
corresponding 1-(u
-chloroalkyl)-2,3-dihydrospiro[1H-indene-1,40-
piperidine]-20,60-dione derivatives 55 and 56. As previously
observed in the synthesis of compounds 15 and 16, direct alkylation
of 52 with 1-bromo-2-chloroethane did not give the expected 2-
chloroethyl derivative 54. Thus, similarly to the preparation of 15
and 16, compound 54 was successfully synthesized via alcohol 53.
From the analysis of the binding data relative to C4 ligands 34, 47,
and 59 (belonging respectivelytothe 4-phenylpiperidine-2,6-dione,
4-methyl-4-phenylpiperidine-2,6-dione or 2,3-dihydrospiro[1H-
indene-1,40-piperidine]-20,60-dione series), it can be seen that 34 is
a better ligand than the other two for the a1A- and a1B-ARs; however,
the three compounds display similar affinities for a1D-ARs.
With regard to the PPZ moiety, 4-(2-methoxyphenyl)piperazinyl
derivatives generally showed higher affinities than corresponding
4-(2-chlorophenyl)piperazinyl and 4-(5-chloro-2-methoxyphenyl)
piperazinyl analogues (e.g.: 34 vs 35 and 36). The 5-chloro-2-
methoxy pattern of substitution, which in other series of
compounds had resulted in ligands with a preferential affinity for
a1D-ARs [25], in the present series did not give the expected results:
disubstituted compounds, such as 36 or 48, did not show any
significant selectivity among the receptor subtypes.
Subsequent reaction of u-chloroalkyl derivatives 54e56 with 1-(2-
methoxyphenyl)piperazine 26 afforded the final derivatives 57e59.
3. Pharmacology
4-Arylpiperidine-2,6-dione derivatives 29e48 and 2,3-dihy-
drospiro[1H-indene-1,40-piperidine]-20,60-dione derivatives 57e59
were tested in binding assays on human a1A-, a1B-, and a1D-AR
subtypes stably expressed in HEK293 cells using 2-[[2-(4-hydroxy-
3-[125I]-iodophenyl)ethylamino]methyl]-3,4-dihydronaphthalen-

Scheme 1. Synthesis of compounds 29e48. Reagents and conditions: (a) urea, 160 ꢀC, 1 h; (b) 1-bromo-
20 h; (c) 1,2-dibromoethane, potassium carbonate, potassium iodide, acetonitrile, reflux, 20 h; (d) SOCl2, toluene, reflux, 7 h; (e) 140 ꢀC, 30 min.
u-chloroalkane, potassium carbonate, potassium iodide, acetonitrile, reflux,

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G. Romeo et al. / European Journal of Medicinal Chemistry 46 (2011) 2676e2690
Scheme 2. Synthesis of compounds 57e59. Reagents and conditions: (a) cyanoacetamide, sodium ethoxyde, EtOH, 3 h, then HCl; (b) sulphuric acid, acetic acid, water, reflux, 3 days;
(c) urea, 160 ꢀC, 1 h; (d) 1-bromo-
u-chloroalkane, potassium carbonate, potassium iodide, acetonitrile, reflux, 20 h; (e) 1,2-dibromoethane, potassium carbonate, potassium iodide,
acetonitrile, reflux, 20 h; (f) SOCl2, toluene, reflux, 7 h; (g) 26, 140 ꢀC, 30 min.
Butyl ligands 34, 47, and 59, representative of the three different
series, were also tested for their functional activity at the three
a1-AR subtypes. As shown in Fig. 4, in HEK293 cells expressing
parameters and predictive power toward a large test set also con-
taining well known a1D-AR ligands (such as 1 and additional
compounds from the literature). Moreover, results from molecular
docking and dynamics simulations on an analogue of 1 [22],
the 8-[[2-(2,5-dichlorophenyl)piperazin-1-yl)ethyl]8-azaspiro[4,5]
decane-7,9-dione (60) (Fig. 1), showed two possible limitations of
the original pharmacophore. In fact, an analysis of the modeled
complex between 60 and the a1D-AR suggested that two hydrogen
bonds involving both the carbonyl groups of the dione moiety of the
ligand were unlikely to occur at the same time [22]. Furthermore,
the aromaticearomatic interaction between the 2,5-dichlorophenyl
group of 60 and the Phe365 of the modeled a1D-AR was one of the
most important ligand-receptor contact [22]. In disagreement with
that, the old pharmacophoric model showed the presence of two
hydrogen bond acceptor groups while the phenyl ring of the PPZ
moiety was codified by a generic hydrophobic region (HY2), instead
of a more appropriate aromatic ring feature (RA). These consider-
ations prompted us to retrace the steps performed to build the
original pharmacophoric model and gave us the suggestions to
optimize it into a second generation pharmacophore. In particular,
pharmacophoric models with only one hydrogen bond acceptor
group (corresponding to a carbonyl moiety of a pyrimidin-2,4-dione
ring) and an aromatic interaction (involving the phenyl ring of PPZ)
were already hypothesised during our previous calculations per-
formed to generate the first pharmacophoric model for a1D-AR
antagonists [24]. However, they have been no further investigated
single human a1A-, a1B- or a1D-AR subtypes, 34, 47, and 59 at 10
did not significantly modify IP hydrolysis with respect to basal
levels, whereas the agonist NE (10 M) induced, as expected,
a marked increase. Furthermore, these compounds were able to
completely antagonize 10 M NE-induced IP hydrolysis in HEK293
mM
m
m
cells; the concentrationeresponse curves for inhibition of IP
hydrolysis for each receptor subtype for compounds 34, 47, and 59
and the corresponding pKI values are shown in Fig. 5 and Table 2,
respectively. Thus, these results clearly indicate that compounds
34, 47, and 59 behave as antagonists at the three human a1-AR
subtypes.
4.2. Pharmacophoric model for the a1D-AR subtype
With our effort to identify the principal determinants respon-
sible for selectivity between a1-AR subtypes, we have previously
reported a six-feature pharmacophoric model able to rationalize, at
a quantitative level, the relationships between structural properties
and biological data of some pyrimido[5,4-b]indole derivatives with
high affinity toward a1D-ARs [24]. However, the pharmacophore
was unable to explain how changes in affinity were dependent
from the substituents at the 4-position of the phenyl ring of the
PPZ moiety, even if the model showed excellent statistical

G. Romeo et al. / European Journal of Medicinal Chemistry 46 (2011) 2676e2690
2681
Table 1
Binding Properties of Piperidin-2,6-dione Derivatives.
Compound
pKi (M)a
a1A-AR
Res.c
a1B-AR
a1D-ARb
29d
30
31
32
33
34d
35
36
37
38
39d
40
41
42
43
44
45d
46
47
48
57
58
59d
2d
5.46 ꢂ 0.46
5.49 ꢂ 0.49
7.33 ꢂ 0.50
7.32 ꢂ 0.49
6.72 ꢂ 0.52
8.74 ꢂ 0.47
7.80 ꢂ 0.49
7.44 ꢂ 0.40
7.66 ꢂ 0.09
5.77 ꢂ 0.49
5.16 ꢂ 0.50
7.55 ꢂ 0.50
7.31 ꢂ 0.37
7.17 ꢂ 0.08
7.85 ꢂ 0.46
7.68 ꢂ 0.07
7.99 ꢂ 0.48
7.46 ꢂ 0.49
7.65 ꢂ 0.49
7.02 ꢂ 0.50
5.47 ꢂ 0.14
7.20 ꢂ 0.47
8.01 ꢂ 0.50
9.57 ꢂ 0.14
5.13 ꢂ 0.48
5.65 ꢂ 0.49
6.95 ꢂ 0.49
6.95 ꢂ 0.50
6.48 ꢂ 0.52
7.62 ꢂ 0.46
7.52 ꢂ 0.50
6.97 ꢂ 0.49
7.01 ꢂ 0.09
6.95 ꢂ 0.50
5.00 ꢂ 0.50
6.90 ꢂ 0.52
6.87 ꢂ 0.35
6.61 ꢂ 0.10
6.89 ꢂ 0.46
6.68 ꢂ 0.06
6.89 ꢂ 0.49
6.71 ꢂ 0.49
7.25 ꢂ 0.47
6.82 ꢂ 0.49
5.30 ꢂ 0.12
6.63 ꢂ 0.46
7.14 ꢂ 0.49
8.74 ꢂ 0.14
5.88 ꢂ 0.44 (5.96)
6.20 ꢂ 0.46 (6.09)
7.34 ꢂ 0.49 (7.34)
7.36 ꢂ 0.52 (7.37)
6.41 ꢂ 0.40 (7.42)
7.80 ꢂ 0.49 (8.06)
7.47 ꢂ 0.41 (7.89)
7.41 ꢂ 0.44 (7.43)
7.51 ꢂ 0.21 (7.70)
6.16 ꢂ 0.51 (6.28)
5.82 ꢂ 0.48 (6.25)
7.16 ꢂ 0.53 (7.11)
7.20 ꢂ 0.50 (7.10)
7.14 ꢂ 0.22 (7.23)
7.17 ꢂ 0.48 (7.31)
7.27 ꢂ 0.19 (7.48)
7.29 ꢂ 0.50 (7.02)
7.01 ꢂ 0.50 (7.01)
7.84 ꢂ 0.52 (8.04)
7.11 ꢂ 0.50 (7.01)
6.04 ꢂ 0.21 (6.77)
7.12 ꢂ 0.50 (6.96)
7.79 ꢂ 0.47 (7.62)
9.44 ꢂ 0.11 (9.40)
ꢃ0.08
0.11
0.00
ꢃ0.01
ꢃ1.01
ꢃ0.26
ꢃ0.42
0.02
ꢃ0.19
ꢃ0.12
ꢃ0.43
0.05
Fig. 3. Variation of pKi values at a1A-AR (A), a1B-AR (-), and a1D-AR (:) for
homologous compounds with increasing number of carbon atoms (from two to five;
compounds 29, 31, 34, and 37, respectively) in the n-alkyl chain connecting the PPZ and
PPD moieties.
0.10
ꢃ0.09
ꢃ0.14
ꢃ0.21
0.27
0.00
0.20
0.94 between experimental and estimated affinity (Table 1). The
new five-feature and the old six-feature pharmacophoric models
shared several common portions: the HY1, PI, HBA1, and HY3
regions of both models are located at almost the same spatial
positions (Fig. 6). Moreover, HY2 of the old model was replaced by
RA, while HBA2 disappeared, thus reducing the total number of
features from six to five.
0.10
ꢃ0.73
0.16
0.17
0.04
a
Each value is the mean ꢂ SE for data from three different experiments con-
ducted in duplicate.
b
Estimated and predicted affinity values calculated by Catalyst for the training set
The superposition pattern of 34, 47, and 59 (with the best
affinity for a1D-ARs among the compounds of each PPD subclass)
into the new pharmacophoric hypothesis showed a similar orien-
tation (Fig. 7): the 2-methoxyphenyl group filled the HY1eRA
features, the most basic nitrogen atom of the piperazine ring was
coded by the PI feature, and the remaining two features were
matched by the terminal heterocyclic portion of the new
compounds (the 4-substituted PPD). In particular, one of the
carbonyl moieties was superposed to HBA, and the terminal HY2
accommodated the phenyl ring. Importantly, the piperidine-2,6-
dione ring was found in the “sofa” conformation, and the C4
atom was out of the plane defined by the remaining atoms.
Moreover, the phenyl group at the 4-position assumed the so-called
Epl conformation in compound 34, differently from 47 where an
Epr conformation was found. This is in agreement with data
reported for 4-phenyldihydro-2H-pyran-2,6(3H)-dione derivatives
3 and 4 by Altona and coworkers [26e28]. An equatorial parallel-
like conformation was also found for the spiro derivative 59. In
such an orientation, pKi values of 34 and 59 were estimated 8.06
and test set, respectively, were reported in parentheses.
c
Difference between experimental and predicted/estimated pKi values for
a1D-ARs.
d
Compounds used to build the training set, together with additional fifteen 5H-
pyrimido[5,4-b]indole derivatives previously reported by our research group [24].
because of the lower quality of their statistical parameters, in
comparison to the other pharmacophoric hypotheses found during
the same calculation runs. Moreover, using the HipHop routine
implemented in Catalyst [34], similar pharmacophoric models were
also obtained, differing for the presence of an RA feature (belonging
to an RAeHYpair of features and representing the phenyl ring of the
PPZ and its substituent in 2-position) instead of one of the hydro-
phobics (HY) constituting the HY1eHY2 system of the final model
[24]. As previous calculations resulted in pharmacophoric models
partially in agreement with the results reported in the literature
cited above, we planned to use the training set of sixteen
compounds (from which the original pharmacophoric model was
derived), enlarged with five compounds belonging to the new
piperidine-2,6-dione series (namely, 29, 34, 39, 45, and 59), to re-
run the hypothesis generation (HypoGen) routine within Catalyst
[34]. Compounds of the new training set showed affinity toward
a1D-ARs (expressed as pKi values) in the range between 9.44 and
5.70. The best pharmacophore, constituted by two hydrophobics
(HY1 and HY2), a hydrogen bond acceptor (HBA), an aromatic ring
(RA), and a positively ionizable group (PI), showed a correlation of
Table 2
pKI Values of Compounds 34, 47, and 59 for a1-AR Subtypes.
Compound
pKI (M)a
a1A-AR
a1B-AR
a1D-AR
34
47
59
8.6 ꢂ 0.04
7.5 ꢂ 0.08
8.0 ꢂ 0.10
8.4 ꢂ 0.17
8.1 ꢂ 0.15
7.7 ꢂ 0.10
8.7 ꢂ 0.13
9.3 ꢂ 0.15
8.3 ꢂ 0.11
Fig. 4. IP hydrolysis in HEK293 cells expressing a1-AR subtypes in the presence of
10
recovered.
a
Values ꢂ SE determined from concentrationeresponse curves for inhibition of
M NE.
m
M 34, 47, 59 or NE. y-Axis is in count per minute of [3H]-inositol phosphates
InsPs formation stimulated by 10
m

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G. Romeo et al. / European Journal of Medicinal Chemistry 46 (2011) 2676e2690
Fig. 6. Comparison between the previous six-feature pharmacophoric hypothesis for
a1D-AR antagonists and the five-feature improved model described in the text. Black
and red labels are referred to the old and new pharmacophore, respectively. Phar-
macophoric features are color coded: blue for hydrophobics (HY), orange for aromatic
rings (RA), red for positively ionizable groups (PI), and green for hydrogen bond
acceptors (HBA). Four features of both the models are located in almost the same
regions of space: HY1eHY1, PIePI, HBA1eHBA, and HY3eHY2. The hydrophobic region
HY2 of the old model was replaced by an aromatic feature (RA), while one of the
hydrogen bond acceptor groups of the old model (HBA2) was lost. (For interpretation
of the references to colour in this figure legend, the reader is referred to the web
version of this article.)
1-yl]ethyl moiety and a six membered terminal heterocyclic ring
bearing two carbonyl groups, had very different pKi values (5.88 vs
9.44, respectively). However, the superposition pattern of
2
revealed a good complementarity between its planar tricyclic
moiety and the HBAeHY2 system of the pharmacophore, while
a poor matching was found between the non-planar 4-phenyl-
piperidine-2,6-dione group of 29 and the two terminal features. On
the basis of such considerations, the shape of the terminal moiety
appeared as a crucial key affecting affinity of these compounds for
a1D-ARs (vide infra). On the other hand, when the distance between
the PPZ moiety and the terminal heterocyclic group increased,
profitable interactions between the PPD moiety and the model
were found, as described above for 34, 47, and 59.
Fig. 5. Inhibition of 10 mM NE-stimulated formation of inositol phosphates (InsPs) by
34 (-), 59 (:), and 47 (B) in HEK293 cells stably expressing a1A-ARs (panel A),
a1B-ARs (panel B), and a1D-ARs (panel C).
and 7.62, in good agreement with experimental data (7.80 and 7.79,
respectively), as well as the affinity of 47 was well predicted by the
model (8.04 vs an experimental value of 7.84). The pharmacophoric
model was also able to account how a different alkyl spacer length
influenced affinity at a1D-ARs. In fact, shortening the poly-
methylene chain from pentyl, butyl or propyl to an ethyl spacer,
was detrimental for affinity. In general, compounds with a C2 spacer
(29, 30, 38, 39, and 57) showed the lowest affinity for a1D-ARs, their
pKi values ranging between 5.82 and 6.20. This was an unexpected
result, as 29 and 2, sharing the 2-[4-(2-methoxyphenyl)piperazin-
Fig. 7. Superposition of 34 (red), 47 (green), and 59 (black) (compounds with the
highest affinity in each subseries) into the new five-feature pharmacophoric model.
Compounds are aligned in a similar way and interact with all the pharmacophoric
features. The phenyl ring at the piperazine nucleus filled the region of the aromatic
ring (RA), while its methoxy substituent is accommodated within the hydrophobic
sphere HY1. The basic nitrogen atom of the piperazine is the positively ionizable group
(PI), while the hydrogen bond acceptor is represented by one of the carbonyl moieties
on the piperidine-2,6-dione ring. Finally, the terminal phenyl ring corresponds to the
second hydrophobic feature (HY2) of the model. Pharmacophoric features are color
coded as in Fig. 6. (For interpretation of the references to colour in this figure legend,
the reader is referred to the web version of this article.)

G. Romeo et al. / European Journal of Medicinal Chemistry 46 (2011) 2676e2690
2683
Replacement of the 2-methoxy group at the PPZ moiety with
a chlorine atom led to compounds with comparable (31, pKi ¼ 7.34
vs 32, pKi ¼ 7.36) or slightly lower a1D-AR affinity (34, pKi ¼ 7.80 vs
35, pKi ¼ 7.47), accounted by the fact that both the methoxy and
chlorine groups interacted in a similar way with HY1.
8-azaspiro [4,5]decane-7,9-dione moiety with a C2 spacer was more
likely to increase affinity compared to a PPD moiety linked to the
same spacer, thus suggesting that the smaller size of the first in
combination with a short chain was preferred for affinity. On the
other hand, lengthening the spacer increased affinity values for PPD
compounds while no significant increase in affinity was found for
8-azaspiro[4,5]decane-7,9-dione derivatives [22].
Leonardi and coworkers [22] described three-dimensional
theoretical models of complexes between 60 and the three a1-AR
subtypes, as determined by molecular dynamics calculations.
Having no structural information (i.e., distances between the key
To check the reliability of the model and to assess its predictive
power, a validation step was performed by predicting affinity data
for a large test set of compounds composed of piperidin-2,6-dione
and pyrimido[5,4-b]indole derivatives. Predicted values were in
high agreement with experimental data. In fact, differences
between actual and calculated pKi values were lower than 0.42 log
units, with only three exceptions in the range between 0.73 and
1.01 log units (Table 1 and Table 3). Furthermore, the major limi-
tation of the original pharmacophoric model, consisting in its
inability to account for the influence of substituents at the 4-
position of the PPZ nucleus on affinity data, was overcome by
a slightly different spatial position (with respect to the original
pharmacophoric model) of the features matched by the PPZ moiety
(RA and HY1). As an example, for previously reported pyrimido[5,4-
b]indole derivatives 61, 62, 63, and 64 [24], only a partial fit was
found for the phenyl ring and its 4-substituent on both RA and HY1,
respectively, while the basic nitrogen and the terminal tricyclic
moiety showed a precise superposition to PI, HBA and HY2,
respectively. This resulted in a good agreement between experi-
mental and predicted affinity values (Table 3).
In summary, the new pharmacophoric hypothesis for a1D-AR
antagonists was able to account for the major structure-activity
relationships (SARs) of compounds belonging to both the training
and the test sets. The model clearly showed that the distance
between the PPZ moiety and the terminal heterocyclic fragment
(PPD) was a crucial element in influencing affinity. The high affinity
of compounds belonging tothe class of 2 (with a C2 spacer) were also
rationalized in terms of the shape of the terminal moiety. Planar
systems (as in 2) allowed for good interactions with all the phar-
macophoric features also when the spacer was short (C2), while
compounds with the non-planar PPD moiety required at least a C3
spacer to have profitable interactions with the pharmacophoric
regions. However, in addition to the shape of the terminal hetero-
cyclic moiety, also its size and the length of the alkyl spacer played an
structural elements of these complexes), only
a qualitative
comparison between the cited model of the 60-a1D-AR complex
and the pharmacophoric model proposed in this paper can be
made. As a result, the PI feature of our pharmacophoric model
accounted for an expected strong electrostatic interaction between
the basic nitrogen atom of 60 and an aspartate (Asp176) of the third
transmembrane domain (TM3). Moreover, both the HY1 and RA
features of our pharmacophoric model corresponded to a hydro-
phobic pocket accommodating the substituted phenyl ring of the
PPZ moiety of 60. Within this region, the reciprocal orientation
between the dichlorophenyl moiety of 60 and the TM6 Phe364 gave
a profitable aromaticearomatic contact (possibly a T tilted inter-
action) perfectly reproduced by the RA feature of the model and the
phenyl substituent at the piperazine ring of our compounds.
Furthermore, hydrophobic residues (i.e., TM3 Val177) could corre-
spond to HY1 and interact with the methoxy or chloro group at the
PPZ moiety. An additional binding pocket was found that allows for
only one hydrogen bond between one of the carbonyl groups of the
dione moiety of 60 and TM3 Trp172 or/and TM7 Tyr392. The same
interaction occurred in the pharmacophoric model proposed by us,
where one of the carbonyl groups of the PPD moiety of new
compounds corresponded to HBA. Finally, a series of hydrophobic
amino acids were found surrounding the terminal heterocyclic
moiety of the ligands. In particular, the side chains of TM2 Glu157
and TM7 Lys385 contacted by van der Waals interactions the
cyclopentane ring at the edge of the ligand, similar to that found in
the pharmacophoric model where the HY3 feature was filled by the
phenyl ring attached to the PPD moiety.
important role in determining affinity of a compound toward a1D
-
All these considerations, together with the predictive power of
the pharmacophore for affinity values of a1D-AR inhibitors, lead to
the suggestion that the pharmacophoric model proposed in this
paper might incorporate the most relevant structural features of
a1D-ARs, as suggested by a qualitative comparisonwith a theoretical
model of a1D-ARs reported in the recent literature [22].
ARs, as already hypothesized by our and other groups on the basis of
SAR analyses. As an example, 60, bearing the 8-azaspiro[4,5]decane-
7,9-dione moiety as the terminal fragment bound to a C2 spacer,
showed an affinity value (pKi ¼ 9.89) [22] toward a1D-ARs compa-
rable to that of 2 and higher than those found for PPD derivatives
described here. This finding indicated that a combination of the
5. Conclusions
Table 3
Experimental and Predicted Binding Affinity of Selected Pyrimido[5,4-b]indole
Derivatives for the a1D-AR Subtype.
With the aim to develop new selective ligands for a1-AR
subtypes, a number of new 4-phenylpiperidine-2,6-dione deriva-
tives bearing at the 1-position an
u-[4-(substitutedphenyl)piper-
azin-1-yl]alkyl portion were prepared and tested in radioligand
binding assays.
The length of the connecting chain between the PPD and the PPZ
moieties seems to be the structural feature that most influences the
affinity values of these compounds. The highest affinities were dis-
played by compounds which possess a butyl (C4) chain; the best
ligand was 1-[4-[4-(2-methoxyphenyl)piperazin-1-yl]butyl]-4-phe-
nylpiperidine-2,6-dione (34) which displayed a pKi ¼ 8.74 at the
human cloned a1A-AR and a 10-fold selectivity over the other two
subtypes.
Structural modifications of the PPD moiety (insertion of a 4-
methyl group or conformational freezing within a spiro system),
meant to modify its conformation, did not notably influence ligands
affinity or selectivity for the three receptor subtypes. Nevertheless,
Compound
R
pKi (M)
Res.b
Experimentala
Predicted
61
62
63
64
CH3(CH3)CH
CH3(CH3)2C
CH3CH2(CH3)CH
CH3(CH2)2CH2
7.45 ꢂ 0.14
7.70 ꢂ 0.08
7.22 ꢂ 0.014
6.61 ꢂ 0.07
7.43
7.77
7.52
7.39
0.02
ꢃ0.07
ꢃ0.30
ꢃ0.78
a
Data from ref. [24].
Difference between experimental and predicted pKi values for a1D-ARs.
b

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G. Romeo et al. / European Journal of Medicinal Chemistry 46 (2011) 2676e2690
at the a1A-AR subtype only, a different trend was noted for
compounds bearing an additional methyl group at the 4-position of
the piperidine-2,6-dione ring with respect to the non-methylated
or spiro analogues. In this case, C3 derivatives behaved as better
ligands than the C4 ones.
6.1.3. 4-(4-Nitrophenyl)piperidine-2,6-dione (11) [36]
The same procedure, as described for the synthesis of 9, was
followed using 3-(4-nitrophenyl)pentanedioic acid (7). Recrystal-
lization from ethanol afforded 11 (61%) as a yellow powder: mp
241e243 ꢀC; IR (KBr) cmꢃ1 3274 (NH), 1701(CO); 1H NMR (DMSO-
Some representative compounds of the series (34, 47, and 59),
when tested to assess their functional activity at the three a1-AR
subtypes, demonstrated a dose-related block of norepinephrine-
induced stimulation of IP hydrolysis, thus revealing their antago-
nist properties.
d6) d 10.94 (s,1H, NH which exchanges with D2O), 8.28e8.18 (m, 2H,
aromatic), 7.68e7.58 (m, 2H, aromatic), 3.78e3.55 (m, 1H, CH), 2.87
(dd, 2J ¼ 16.5 Hz, 3J ¼ 11.2 Hz, 2H, CHAHB), 2.68 (dd, 2J ¼ 16.5 Hz,
3J ¼ 4.4 Hz, 2H, CHAHB). Anal. (C11H10N2O4) calcd: C 56.41, H 4.30, N
11.96; found: C 56.25, H 4.37, N 12.01.
Title compounds were also used to refine a ligand-based pharma-
cophoric model for a1D-ARs previously reported by us. The new phar-
macophore, constituted by five different features, is qualitatively in
agreement with a literature receptor-based model for a1D-ARs. This
model was able to account for differences in binding affinity of a large
set of ligands, pointing out the importance of the length of the con-
necting chain and of the shape of the terminal heterocyclic moiety on
affinity. Thus, the new pharmacophoric model could be useful in the
design of novel molecules for binding a1D-ARs.
6.1.4. 4-Methyl-4-phenylpiperidine-2,6-dione (12) [35]
The same procedure, as described for the synthesis of 9, was
followed using 3-methyl-3-phenylpentanedioic acid (8). Recrys-
tallization from toluene afforded 12 (44%) as a pure product: mp
150e151 ꢀC; IR (KBr) cmꢃ1 3186 (NH), 1733, 1675 (CO); 1H NMR
(DMSO-d6)
d 10.70 (br s, 1H, NH which exchanges with D2O),
7.45e7.18 (m, 5H, aromatic), 3.04 (d, J ¼ 16.4 Hz, 2H, CHAHB), 2.80
(d, J ¼ 16.4 Hz, 2H, CHAHB), 1.29 (s, 3H, CH3). Anal. (C12H13NO2)
calcd: C 70.92, H 6.45, N 6.89; found: C 70.88, H 6.41, N 6.70.
6. Experimental protocols
6.1.5. 1-(2-Hydroxyethyl)-4-phenylpiperidine-2,6-dione (13)
Compound 9 (0.50 g, 2.64 mmol) was dissolved in acetonitrile
(7 mL). Then, potassium carbonate (1.09 g, 7.92 mmol), 1,2-dibro-
moethane (1.24 g, 6.60 mmol) and potassium iodide (0.05 g,
0.30 mmol) were added. The reaction mixture was heated under
reflux for 20 h. After being cooled, it was concentrated under
reduced pressure. Then, water and ethyl acetate were added. The
organic layer was separated and dried over anhydrous sodium
sulphate. After filtration, solvent was eliminated at reduced pres-
sure and residue was purified by column chromatography, using
ethyl acetate/cyclohexane (7/3, v/v) as eluent. The homogeneous
fractions were evaporated at reduced pressure to give 13 as a pure
product (0.30 g, 48%): mp 101e102 ꢀC; IR (KBr) cmꢃ1 3516 (OH),
6.1. Chemistry
Melting points were determined in a Gallenkamp apparatus
with a digital thermometer MFB-595 in glass capillary tubes and
are uncorrected. Infrared spectra were recorded on a Perkin Elmer
FTIR 1600 spectrometer in KBr disks or NaCl crystal windows.
Elemental analyses for C, H, and N were within ꢂ0.4% of theoretical
values and were performed on a Carlo Erba Elemental Analyzer
Mod. 1108 apparatus. 1H NMR spectra were recorded on a Varian
Inova Unity 200 spectrometer in DMSO-d6 solution. Chemical shifts
are given in d values (ppm), using tetramethylsilane as the internal
standard; coupling constants (J) are given in Hz. Signal multiplici-
ties are characterized as s (singlet), d (doublet), t (triplet), q
(quartet), m (multiplet), br (broad signal). All the synthesized
compounds were tested for purity on TLC (aluminium sheet coated
1718, 1660 (CO); 1H NMR (DMSO-d6)
d 7.40e7.20 (m, 5H, aromatic),
4.76 (t, J ¼ 6.0 Hz, 1H, OH which exchanges with D2O), 3.76
(t, J ¼ 6.4 Hz, 2H, CONCH2), 3.52e3.32 (m, 1H þ 2H, CH þ CH2OH),
2.91 (dd, 2J ¼ 16.2 Hz, 3J ¼ 10.6 Hz, 2H, CHCHAHB), 2.79 (dd,
2J ¼ 16.2 Hz, 3J ¼ 5.0 Hz, 2H, CHCHAHB). Anal. (C13H15NO3) calcd: C
66.94, H 6.48, N 6.00; found: C 67.11, H 6.32, N 5.85.
with silica gel 60 F254, Merck) and visualized by UV (
l
¼ 254 and
366 nm). Purification of synthesized compounds by column chro-
matography was performed using silica gel 60 (Merck). All chem-
icals and solvents were reagent grade and were purchased from
commercial vendors.
6.1.6. 4-(4-Chlorophenyl)-1-(2-hydroxyethyl)piperidine-
2,6-dione (14)
The same procedure, as described for the synthesis of 13, was
followed using compound 10. Purification by column chromatog-
raphy using a mixture of ethyl acetate/cyclohexane (7/3, v/v) as
eluent gave 14 (45%) as a pure product: mp 105e106 ꢀC; IR (KBr)
6.1.1. 4-Phenylpiperidine-2,6-dione (9) [35]
3-Phenylpentanedioic acid (5) (5.00 g, 24.01 mmol) and urea
(3.60 g, 59.94 mmol) were mixed and heated at 160 ꢀC for 1 h. After
being cooled, the reaction mixture was poured into water (20 mL).
The solid was filtered off, washed with water and dried. Recrys-
tallization from toluene gave 9 (3.26 g, 72%) as a white powder: mp
173e174 ꢀC; IR (KBr) cmꢃ1 3184 (NH), 1734, 1686 (CO); 1H NMR
cmꢃ1 1663 (CO); 1H NMR (DMSO-d6)
d 7.45e7.28 (m, 4H, aromatic),
4.76 (t, J ¼ 6.0 Hz, 1H, OH which exchanges with D2O), 3.75 (t,
J ¼ 6.4 Hz, 2H, CONCH2), 3.55e3.38 (m, 1H þ 2H, CH þ CH2OH),
3.00e2.71 (m, 4H, CHCH2). Anal. (C13H14ClNO3) calcd: C 58.32, H
5.27, N 5.23; found: C 58.31, H 5.17, N 5.31.
(DMSO-d6)
d 10.86 (br s, 1H, NH which exchanges with D2O),
7.42e7.18 (m, 5H, aromatic), 3.50e3.35 (m, 1H, CH), 2.90e2.43 (m,
4H, CH2). Anal. (C11H11NO2) calcd: C 69.83, H 5.86, N 7.40; found: C
69.71, H 5.83, N 7.29.
6.1.7. 1-(3-Chloropropyl)-4-phenylpiperidine-2,6-dione (17)
Compound 9 (1.00 g, 5.28 mmol) was dissolved in acetonitrile
(14 mL). Successively, potassium carbonate (2.19 g, 15.85 mmol),
1-bromo-3-chloropropane (2.08 g, 13.21 mmol) and potassium
iodide (0.10 g, 0.60 mmol) were added. The reaction mixture was
heated under reflux for 20 h. After being cooled, it was concen-
trated under reduced pressure. Then, water and ethyl acetate were
added. The organic layer was separated and dried over anhydrous
sodium sulphate. After filtration, solvents were eliminated under
reduced pressure to give 17 (0.80 g, 57%) as a white solid. This crude
product was used without further purification: mp 83e85 ꢀC; IR
6.1.2. 4-(4-Chlorophenyl)piperidine-2,6-dione (10)
The same procedure, as described for the synthesis of 9, was fol-
lowed using 3-(4-chlorophenyl)pentanedioic acid (6). Recrystalliza-
tion from toluene afforded 10 (81%) as a pure product: mp 160 ꢀC; IR
(KBr) cmꢃ13318 (NH),1665 (CO); 1H NMR (DMSO-d6)
d11.00 (s, 1H, NH
which exchanges with D2O), 7.60e7.20 (m, 4H, aromatic), 3.68e3.40
(m, 1H, CH), 2.94 (dd, 2J ¼ 16.5 Hz, 3J ¼ 11.0 Hz, 2H, CHAHB), 2.76 (dd,
2J ¼ 16.5 Hz, 3J ¼ 4.8 Hz, 2H, CHAHB). Anal. (C11H10ClNO2) calcd: C 59.07,
H 4.51, N 6.26; found: C 58.96, H 4.70, N 6.39.
(KBr) cmꢃ11724,1667 (CO); 1H NMR (DMSO-d6)
d 7.40e7.20 (m, 5H,

G. Romeo et al. / European Journal of Medicinal Chemistry 46 (2011) 2676e2690
2685
aromatic), 3.79 (t, J ¼ 6.8 Hz, 2H, CONCH2), 3.60 (t, J ¼ 6.4 Hz, 2H,
6.1.14. 1-(3-Chloropropyl)-4-methyl-4-phenylpiperidine-2,6-dione
(24)
CH2Cl), 3.58e3.30 (m, 1H, CH), 2.92 (dd, 2J ¼ 16.2 Hz, 3J ¼ 10.6 Hz,
2H, CHCHAHB), 2.80 (dd, 2J ¼ 16.2 Hz, 3J ¼ 4.6 Hz, 2H, CHCHAHB),
1.98e1.81 (m, 2H, CH2CH2Cl).
The same procedure, as described for the synthesis of 17, was
followed using 12 and 1-bromo-3-chloropropane. Obtained crude
24 (90%) was used without further purification: mp 74e77 ꢀC; IR
6.1.8. 1-(4-Chlorobutyl)-4-phenylpiperidine-2,6-dione (18)
(KBr) cmꢃ1 1723, 1675 (CO); 1H NMR (DMSO-d6)
d 7.40e7.19 (m, 5H,
The same procedure, as described for the synthesis of 17, was
followed using 1-bromo-4-chlorobutane. Obtained crude 18 (77ꢃ%1)
was used without further purification: mp 53e54 ꢀC; IR (KBr) cm
aromatic), 3.65 (t, J ¼ 6.8 Hz, 2H, CONCH2), 3.30e3.18 (m, 2H þ 2H,
CCHAHB þ CH2Cl), 2.96 (d, J ¼ 16.4 Hz, 2H, CCHAHB), 1.70e1.50
(m, 2H, CH2CH2Cl), 1.29 (s, 3H, CH3).
1728, 1667 (CO); 1H NMR (DMSO-d6)
d 7.40e7.20 (m, 5H, aromatic),
3.75e3.59 (m, 2H þ 2H, CONCH2 þ CH2Cl), 3.49e3.34 (m, 1H, CH),
2.95 (dd, 2J ¼ 16.1 Hz, 3J ¼ 10.6 Hz, 2H, CHCHAHB), 2.81 (dd,
2J ¼ 16.1 Hz, 3J ¼ 5.2 Hz, 2H, CHCHAHB), 1.78e1.45 (m, 2H þ 2H,
CH2CH2CH2Cl).
6.1.15. 1-[2-[4-(2-Methoxyphenyl)piperazin-1-yl]ethyl]-4-phe-
nylpiperidine-2,6-dione (29)
Compound 13 (0.10 g, 0.43 mmol) was dissolved in toluene
(5 mL) and thionyl chloride (0.10 g, 0.86 mmol) was added. The
reaction mixture was heated under reflux for 7 h; then, it was
concentrated under reduced pressure. The resulting crude oil, 1-(2-
chloroethyl)-4-phenylpiperidine-2,6-dione (15) (0.11 g), was
successively used for the synthesis of 29 without further
purification.
A mixture of compound 15 (0.11 g, 0.44 mmol) and 1-(2-
methoxyphenyl)piperazine (26) (0.42 g, 2.20 mmol) was heated in
an oil bath at 140 ꢀC for 30 min. After being cooled, ethyl acetate
(5 mL) was added to the reaction mixture. Solids were filtered off
and the filtrate was, successively, evaporated under reduced pres-
sure. The residue was purified by column chromatography using
a mixture of ethyl acetate/cyclohexane (1/1, v/v) as eluent. The
homogeneous fractions were evaporated in vacuo to afford 29
(0.03 g, 17%) as a white solid: mp 179e180 ꢀC; IR (KBr) cmꢃ1 1714,
6.1.9. 1-(5-Chloropentyl)-4-phenylpiperidine-2,6-dione (19)
The same procedure, as described for the synthesis of 17, was
followed using 1-bromo-5-chloropentane. Obtained crude 19 (84ꢃ%1)
was used without further purification: mp 84e85 ꢀC; IR (KBr) cm
1728, 1665 (CO); 1H NMR (DMSO-d6)
d
7.40e7.20 (m, 5H, aromatic),
3.70e3.58 (m, 2H þ 2H, CONCH2 þ CH2Cl), 3.50e3.25 (m, 1H, CH),
2.95 (dd, 2J ¼ 16.3 Hz, 3J ¼ 10.4 Hz, 2H, CHCHAHB), 2.84 (dd,
2J ¼ 16.3 Hz, 3J ¼ 5.0 Hz, 2H, CHCHAHB), 1.80e1.60 (m, 2H,
CH2CH2Cl), 1.50e1.20 (m, 2H þ 2H, CH2CH2CH2CH2Cl).
6.1.10. 4-(4-Chlorophenyl)-1-(3-chloropropyl)piperidine-2,6-dione
(20)
The same procedure, as described for the synthesis of 17, was
followed using 10 and 1-bromo-3-chloropropane. Obtained crude
20 (79%) was used without further purification: mp 92e93 ꢀC; IR
1669 (CO); 1H NMR (DMSO-d6)
7.01e6.90 (m, 4H, aromatic), 3.90e3.70 (m, 2H
CONCH2 þ CH3), 3.50e3.40 (m, 1H, CH), 3.15e2.76 (m, 4H þ 4H,
CHCH2 þ piperazine), 2.68e2.49 (m, 4H, piperazine), 2.40 (t,
J ¼ 6.6 Hz, 2H, CONCH2CH2). Anal. (C24H29N3O3) calcd: C 70.74, H
7.17, N 10.31; found: C 70.74, H 7.06, N 10.46.
d
7.41e7.20 (m, 5H, aromatic),
3H,
þ
(KBr) cmꢃ11726, 1665 (CO); 1H NMR (DMSO-d6)
d
7.42e7.30 (m, 4H,
aromatic), 3.79 (t, J ¼ 6.6 Hz, 2H, CONCH2), 3.60 (t, J ¼ 6.6 Hz, 2H,
CH2Cl), 3.50e3.40 (m,1H, CH), 3.08e2.70 (m, 4H, CHCH2),1.90e1.80
(m, 2H, CH2CH2Cl).
6.1.11. 1-(4-Chlorobutyl)-4-(4-chlorophenyl)piperidine-2,6-dione
(21)
6.1.16. 1-[2-[4-(2-Chlorophenyl)piperazin-1-yl]ethyl]-4-phenyl-
piperidine-2,6-dione (30)
The same procedure, as described for the synthesis of 17, was
followed using 10 and 1-bromo-4-chlorobutane. Obtained crude 21
(88%) was used without further purification: mp 96 ꢀC; IR (KBr)
Compound 30 was prepared according to the procedure presented
for 29. Reaction between 15 and 1-(2-chlorophenyl)piperazine (27)
gave crude 30. Purification by column chromatography using a mixture
of ethyl acetate/cyclohexane (1/1, v/v), afforded 30 as a white product
(23%): mp 162e163 ꢀC; IR (KBr) cmꢃ11725,1666 (CO); 1H NMR (DMSO-
cmꢃ1 1721, 1661 (CO); 1H NMR (DMSO-d6)
d 7.45e7.25 (m, 4H,
aromatic), 3.75e3.57 (m, 2H þ 2H, CONCH2 þ CH2Cl), 3.53e3.38
(m, 1H, CH), 2.96 (dd, 2J ¼ 16.0 Hz, 3J ¼ 10.4 Hz, 2H, CHCHAHB), 2.80
(dd, 2J ¼ 16.0 Hz, 3J ¼ 5.0 Hz, 2H, CHCHAHB), 1.60e1.50 (m, 2H þ 2H,
CH2CH2CH2Cl).
d6) d 7.45e7.20 (m, 5H, aromatic), 7.20e6.90 (m, 4H, aromatic), 3.83 (t,
J ¼ 7.0 Hz, 2H, CONCH2), 3.38e3.25 (m,1H, CH), 3.05e2.75 (m, 4H þ 4H,
CHCH2 þ piperazine), 2.63e2.50 (m, 4H, piperazine), 2.42 (t, J ¼ 7.0 Hz,
2H, CONCH2CH2). Anal. (C23H26ClN3O2) calcd: C 67.06, H 6.36, N 10.20;
found: C 67.01, H 6.58, N 10.23.
6.1.12. 1-(3-Chloropropyl)-4-(4-nitrophenyl)piperidine-2,6-dione
(22)
The same procedure, as described for the synthesis of 17, was
followed using 11 and 1-bromo-3-chloropropane. Recrystallization
from ethanol gave 22 (45%) as a yellow solid: mp 119e120 ꢀC; IR
6.1.17. 1-[3-[4-(2-Methoxyphenyl)piperazin-1-yl]propyl]-4-phenyl
piperidine-2,6-dione (31)
A mixture of compound 17 (0.20 g, 0.75 mmol) and 1-(2-
methoxyphenyl)piperazine (26) (0.72 g, 3.75 mmol) was heated in
an oil bath at 140 ꢀC for 30 min. After being cooled, ethyl acetate
(10 ml) was added to the reaction mixture. Solids were filtered off
and, successively, the filtrate was evaporated under reduced pres-
sure. The residue was purified by column chromatography using
ethyl acetate as eluent. The homogeneous fractions were evapo-
rated in vacuo to afford 31 as a pure product (0.05 g, 16%): mp
81e82 ꢀC; IR (KBr) cmꢃ1 1726, 1664 (CO); 1H NMR (DMSO-d6)
(KBr) cmꢃ1 1727, 1673 (CO); 1H NMR (DMSO-d6)
d 8.24e8.18 (m, 2H,
aromatic), 7.64e7.58 (m, 2H, aromatic), 3.80 (t, J ¼ 7.0 Hz, 2H,
CONCH2), 3.65e3.58 (m, 1H þ 2H, CH þ CH2Cl), 3.09e2.78 (m, 4H,
CHCH2), 1.98e1.81 (m, 2H, CH2CH2Cl). Anal. (C14H15ClN2O4) calcd: C
54.11, H 4.87, N 9.02; found: C 53.88, H 4.82, N 9.14.
6.1.13. 1-(4-Chlorobutyl)-4-(4-nitrophenyl)piperidine-2,6-dione
(23)
The same procedure, as described for the synthesis of 17, was fol-
lowed using 11 and 1-bromo-4-chlorobutane. Obtained crude 23 (36%)
was obtained as a yellow oily product and used without further purifi-
d 7.40e7.20 (m, 5H, aromatic), 7.00e6.90 (m, 4H, aromatic), 3.77 (s,
3H, CH3), 3.72 (t, J ¼ 8.0 Hz, 2H, CONCH2), 3.50e3.39 (m, 1H, CH),
3.03e2.74 (m, 4H þ 4H, CHCH2 þ piperazine), 2.53e2.41 (m, 4H,
piperazine), 2.31 (t, J ¼ 7.6 Hz, 2H, CONCH2CH2CH2), 1.72e1.53 (m,
2H, CONCH2CH2). Anal. (C25H31N3O3) calcd: C 71.23, H 7.41, N 9.97;
found: C 71.40, H 7.28, N 10.08.
cation: 1H NMR (DMSO-d6)
d8.34e8.12 (m, 2H, aromatic), 7.70e7.50 (m,
2H, aromatic), 3.80e3.53 (m, 2H þ 1H þ 2H, CONCH2 þ CH þ CH2Cl),
3.15e2.80 (m, 4H, CHCH2), 1.80e1.50 (m, 2H þ 2H, CH2CH2CH2Cl).

2686
G. Romeo et al. / European Journal of Medicinal Chemistry 46 (2011) 2676e2690
6.1.18. 1-[3-[4-(2-Chlorophenyl)piperazin-1-yl]propyl]-4-phenyl
piperidine-2,6-dione (32)
3.50e3.35 (m,1H, CH), 3.05e2.75 (m, 4H þ 4H, CHCH2 þ piperazine),
2.55e2.40 (m, 4H, piperazine), 2.30 (t, 6.6 Hz, 2H,
CONCH2CH2CH2CH2),1.55e1.30 (m, 2H þ 2H, CONCH2CH2CH2). Anal.
(C26H32ClN3O3) calcd: C 66.44, H 6.86, N 8.94; found: C 66.20, H 7.03,
N 8.74.
J
¼
Compound 32 was prepared according to the procedure pre-
sented for 31. Reaction between 17 and 1-(2-chlorophenyl)pipera-
zine (27) gave crude 32. Purification by column chromatography
using ethyl acetate as eluent afforded 32 as a pure product (62%):
mp 68e70 ꢀC; IR (KBr) cmꢃ1 1724, 1665 (CO); 1H NMR (DMSO-d6)
6.1.23. 1-[5-[4-(2-Methoxyphenyl)piperazin-1-yl]pentyl]-4-phe-
nylpiperidine-2,6-dione (37)
d
7.42e7.20 (m, 5H, aromatic), 7.20e6.98 (m, 4H, aromatic), 3.73 (t,
J ¼ 7.2 Hz, 2H, CONCH2), 3.51e3.45 (m, 1H, CH), 3.10e2.70 (m,
4H þ 4H, CHCH2 þ piperazine), 2.63e2.40 (m, 4H, piperazine), 2.34
(t, J ¼ 6.8 Hz, 2H, CONCH2CH2CH2), 1.75e1.50 (m, 2H, CONCH2CH2).
Anal. (C24H28ClN3O2) calcd: C 67.67, H 6.63, N 9.87; found: C 67.61, H
6.45, N 8.70.
Compound 37 was prepared according to the procedure pre-
sented for 31. Reaction between 19 and 1-(2-methoxyphenyl)
piperazine (26) gave crude 37. Purification by column chromatog-
raphy using ethyl acetate as eluent afforded 37 as a pure product
(62%): mp 85e86 ꢀC; IR (KBr) cmꢃ11722,1671 (CO); 1H NMR (DMSO-
d6) d 7.40e7.20 (m, 5H, aromatic), 7.00e6.90 (m, 4H, aromatic), 3.76
6.1.19. 1-[3-[4-(5-Chloro-2-methoxyphenyl)piperazin-1-yl]propyl]-
4-phenylpiperidine-2,6-dione (33)
(s, 3H, CH3), 3.66 (t, J ¼ 6.8 Hz, 2H, CONCH2), 3.50e3.35 (m, 1H, CH),
3.05e2.70 (m, 4H þ 4H, CHCH2 þ piperazine), 2.60e2.40 (m, 4H,
piperazine), 2.28 (t, J ¼ 7.0 Hz, 2H, CONCH2CH2CH2CH2CH2),
1.55e1.35 (m, 2H þ 2H, CONCH2CH2CH2CH2), 1.30e1.20 (m, 2H,
CONCH2CH2CH2). Anal. (C27H35N3O3) calcd: C 72.13, H 7.85, N 9.35;
found: C 72.24, H 7.71, N 9.36.
Compound 33 was prepared according to the procedure presented
for 31. Reaction between 17 and 1-(5-chloro-2-methoxyphenyl)
piperazine (28) gave crude 33. Purification by column chromatography
using ethyl acetate as eluent afforded 33 as a pure product (87%): mp
103e104 ꢀC; IR (KBr) cmꢃ1 1721, 1669 (CO); 1H NMR (DMSO-d6)
d
7.40e7.20 (m, 5H, aromatic), 7.00e6.80 (m, 3H, aromatic), 3.77 (s, 3H,
6.1.24. 4-(4-Chlorophenyl)-1-[2-[4-(2-methoxyphenyl)piperazin-1-
yl]ethyl]piperidine-2,6-dione (38)
CH3), 3.71 (t, J ¼ 7.0 Hz, 2H, CONCH2), 3.50e3.39 (m,1H, CH), 3.10e2.75
(m, 4H þ 4H, CHCH2 þ piperazine), 2.59e2.40 (m, 4H, piperazine), 2.31
(t, J ¼ 7.6 Hz, 2H, CONCH2CH2CH2), 1.75e1.58 (m, 2H, CONCH2CH2).
Anal. (C25H30ClN3O3) calcd: C 65.85, H 6.63, N 9.22; found: C 65.73, H
6.65, N 9.27.
Compound 14 (0.20 g, 0.75 mmol) was dissolved in toluene (8 mL)
and thionyl chloride (0.18 g, 1.5 mmol) was added. The reaction
mixture was heated under reflux for 7 h. Then, it was concentrated
under reduced pressure. The resulting crude oil, 1-(2-chloroethyl)-4-
(4-chlorophenyl)piperidine-2,6-dione (16) (0.20 g), was successively
used for the synthesis of 38 without further purification.
A mixture of compound 16 (0.107 g, 0.37 mmol) and 1-(2-
methoxyphenyl)piperazine (26) (0.36 g, 1.87 mmol) was heated in
an oil bath at 140 ꢀC for 30 min. After being cooled, ethyl acetate
(5 mL) was added to the reaction mixture. Solids were filtered off
and the filtrate was, successively, evaporated under reduced pres-
sure. The residue was purified by column chromatography using
a mixture of ethyl acetate/cyclohexane (7/3, v/v) as eluent. The
homogeneous fractions were evaporated in vacuo to afford 38
(0.060 g, 36%) as a pure solid: mp 131e132 ꢀC; IR (KBr) cmꢃ1 1724,
6.1.20. 1-[4-[4-(2-Methoxyphenyl)piperazin-1-yl]butyl]-4-phenyl-
piperidine-2,6-dione (34)
Compound 34 was prepared according to the procedure pre-
sented for 31. Reaction between 18 and 1-(2-methoxyphenyl)
piperazine (26) gave crude 34. Purification by column chromatog-
raphy using ethyl acetate as eluent aꢃff1orded 34 as a pure product
(60%): mp 105e106 ꢀC; IR (KBr) cm 1720, 1668 (CO); 1H NMR
(DMSO-d6)
d 7.58e7.20 (m, 5H, aromatic), 7.15e6.80 (m, 4H,
aromatic), 3.90e3.70 (m, 3H þ 2H, CH3 þ CONCH2), 3.70e3.25 (m,
1H, CH), 3.20e2.78 (m, 4H þ 4H, CHCH2 þ piperazine), 2.70e2.40
(m, 4H, piperazine), 2.40e2.25 (m, 2H, CONCH2CH2CH2CH2),
1.60e1.30 (m, 2H þ 2H, CONCH2CH2CH2). Anal. (C26H33N3O3) calcd:
C 71.70, H 7.64, N 9.65; found: C 71.56, H 7.54, N 9.73.
1677 (CO); 1H NMR (DMSO-d6)
7.00e6.90 (m, 4H, aromatic), 3.90e3.70 (m, 3H
CH3 þ CONCH2), 3.55e3.40 (m, 1H, CH), 3.01e2.71 (m, 4H þ 4H,
CHCH2 þ piperazine), 2.61e2.49 (m, 4H, piperazine), 2.40 (t,
J ¼ 6.6 Hz, 2H, CONCH2CH2). Anal. (C24H28ClN3O3) calcd: C 65.22, H
6.39, N 9.51; found: C 64.97, H 6.50, N 9.35.
d
7.45e7.25 (m, 4H, aromatic),
2H,
þ
6.1.21. 1-[4-[4-(2-Chlorophenyl)piperazin-1-yl]butyl]-4-phenyl-
piperidine-2,6-dione (35)
Compound 35 was prepared according to the procedure pre-
sented for 31. Reaction between 18 and 1-(2-chlorophenyl)pipera-
zine (27) gave crude 35. Purification by column chromatography
using ethyl acetate as eluent afforded 35 as a pure product (62%): mp
118e119 ꢀC; IR (KBr) cmꢃ1 1723, 1665 (CO); 1H NMR (DMSO-d6)
6.1.25. 4-(4-Chlorophenyl)-1-[2-[4-(2-chlorophenyl)piperazin-1-yl]
ethyl]piperidine-2,6-dione (39)
Compound 39 was prepared according to the procedure presented
for 38. Reaction between 1-(2-chloroethyl)-4-(4-chlorophenyl)piperi-
dine-2,6-dione (16) and 1-(2-chlorophenyl)piperazine (27) gave crude
39. Purification by column chromatography using ethyl acetate/
cyclohexane (7/3, v/v) as eluent afforded 39 as a pure product (24%):
d
7.50e6.99 (m, 5H þ 4H, aromatic), 3.65 (t, J ¼ 6.8 Hz, 2H, CONCH2),
3.58e3.30 (m,1H, CH), 3.10e2.78 (m, 4H þ 4H, CHCH2 þ piperazine),
2.70e2.40 (m, 4H, piperazine), 2.32 (t, 6.8 Hz, 2H,
J
¼
1
CONCH2CH2CH2CH2),1.60e1.30 (m, 2H þ2H, CONCH2CH2CH2). Anal.
(C25H30ClN3O2) calcd: C 68.25, H 6.87, N 9.55; found: C 68.48, H 6.73,
N 9.52.
mp 174e175 ꢀC; IR (KBr) cmꢃ1 1724, 1664 (CO); H NMR (DMSO-d6)
d
7.45e7.20 (m, 4H, aromatic), 7.20e6.90 (m, 4H, aromatic), 3.82
(t, J ¼ 6.8 Hz, 2H, CONCH2), 3.52e3.39 (m, 1H, CH), 3.06e2.77 (m,
4H þ 4H, CHCH2 þ piperazine), 2.63e2.52 (m, 4H, piperazine), 2.42 (t,
J ¼ 6.8 Hz, 2H, CONCH2CH2). Anal. (C23H25Cl2N3O2) calcd: C 61.89, H
5.65, N 9.41; found: C 61.85, H 5.75, N 9.51.
6.1.22. 1-[4-[4-(5-Chloro-2-methoxyphenyl)piperazin-1-yl]butyl]-
4-phenylpiperidine-2,6-dione (36)
Compound 36 was prepared according to the procedure pre-
sented for 31. Reaction between 18 and 1-(5-chloro-2-methox-
yphenyl)piperazine (28) gave crude 36. Purification by column
chromatography using ethyl acetate as eluent afforded 36 as a pure
product (62%): mp 93e94 ꢀC; IR (KBr) cmꢃ11720,1668 (CO); 1H NMR
6.1.26. 4-(4-Chlorophenyl)-1-[3-[4-(2-methoxyphenyl)piperazin-1-
yl]propyl]piperidine-2,6-dione (40)
Compound 40 was prepared according to the procedure pre-
sented for 31. Reaction between 20 and 1-(2-methoxyphenyl)
piperazine (26) gave crude 40. Purification by column chromatog-
raphy using ethyl acetate afforded 40 as a pure product (20%): mp
(DMSO-d6)
d 7.40e7.20 (m, 5H, aromatic), 7.02e6.78 (m, 3H,
aromatic), 3.77 (s, 3H, CH3), 3.64 (t, J ¼ 6.8 Hz, 2H, CONCH2),

G. Romeo et al. / European Journal of Medicinal Chemistry 46 (2011) 2676e2690
2687
89e90 ꢀC; IR (KBr) cmꢃ1 1724, 1666 (CO); 1H NMR (DMSO-d6)
7.50e7.25 (m, 4H, aromatic), 7.05e6.79 (m, 4H, aromatic), 3.76
6.1.31. 1-[3-[4-(2-Methoxyphenyl)piperazin-1-yl]propyl]-4-methyl-
4-phenylpiperidine-2,6-dione (45)
d
(s, 3H, CH3), 3.71 (t, J ¼ 7.2 Hz, 2H, CONCH2), 3.55e3.30 (m, 1H, CH),
3.15e2.76 (m, 4H þ 4H, CHCH2 þ piperazine), 2.60e2.40 (m, 4H,
piperazine), 2.30 (t, J ¼ 6.8 Hz, 2H, CONCH2CH2CH2), 1.70e1.50
(m, 2H, CONCH2CH2). Anal. (C25H30ClN3O3) calcd: C 65.85, H 6.63, N
9.22; found: C 65.90, H 6.87, N 9.27.
Compound 45 was prepared according to the procedure pre-
sented for 31. Reaction between 24 and 1-(2-methoxyphenyl)
piperazine (26) gave crude 45. Purification by column chromatog-
raphy using ethyl acetate as eluent afforded 45 as a pure product
(11%): mp 78e79 ꢀC; IR (KBr) cmꢃ1 1719, 1672 (CO); 1H NMR
(DMSO-d6)
aromatic), 3.76 (s, 3H, OCH3), 3.58 (t, J ¼ 7.2 Hz, 2H, CONCH2), 3.20
(d, 16.0 Hz, 2H, CCHAHB), 2.90e2.82 (m, 2H 4H,
CCHAHB þ piperazine), 2.43e2.30 (m, 4H, piperazine), 2.06 (t,
J ¼ 7.0 Hz, 2H, CONCH2CH2CH2), 1.48e1.30 (m, 2H, CONCH2CH2),
1.29 (s, 3H, CH3). Anal. (C26H33N3O3) calcd: C 71.70, H 7.64, N 9.65;
found: C 71.68, H 7.60, N 9.52.
d 7.50e7.18 (m, 5H, aromatic), 7.00e6.78 (m, 4H,
6.1.27. 4-(4-Chlorophenyl)-1-[3-[4-(2-chlorophenyl)piperazin-1-yl]
propyl]piperidine-2,6-dione (41)
J
¼
þ
Compound 41 was prepared according to the procedure presented
for 31. Reaction between 20 and 1-(2-chlorophenyl)piperazine (27)
gave crude 41. Purification by column chromatography using ethyl
acetate as eluent afforded 41 as a pure product (33%): mp 81e82 ꢀC; IR
(KBr) cmꢃ1 1722, 1666 (CO); 1H NMR (DMSO-d6)
d 7.55e7.25 (m, 4H,
aromatic), 7.25e7.00 (m, 4H, aromatic), 3.72 (t, J ¼ 7.2 Hz, 2H, CONCH2),
3.52e3.40 (m, 1H, CH), 3.02e2.75 (m, 4H þ 4H, CHCH2 þ piperazine),
2.60e2.42 (m, 4H, piperazine), 2.33 (t, J ¼ 7.0 Hz, 2H, CONCH2CH2CH2),
1.70e1.49(m, 2H, CONCH2CH2). Anal. (C24H27Cl2N3O2) calcd: C 62.61, H
5.91, N 9.13; found: C 62.56, H 5.90, N 9.29.
6.1.32. 1-[3-[4-(5-Chloro-2-methoxyphenyl)piperazin-1-yl]propyl]-
4-methyl-4-phenylpiperidine-2,6-dione (46)
Compound 46 was prepared according to the procedure
presented for 31. Reaction between 24 and 1-(5-chloro-2-
methoxyphenyl)piperazine (28) gave crude 46. Purification by
column chromatography using ethyl acetate as eluent afforded 46
as a pure product (87%): mp 103e104 ꢀC; IR (KBr) cmꢃ1 1720, 1676
6.1.28. 4-(4-Chlorophenyl)-1-[4-[4-(2-methoxyphenyl)piperazin-1-yl]
butyl]piperidine-2,6-dione (42)
(CO); 1H NMR (DMSO-d6)
d 7.42e7.20 (m, 5H, aromatic), 6.90e6.80
Compound 42 was prepared according to the procedure pre-
sented for 31. Reaction between 21 and 1-(2-methoxyphenyl)
piperazine (26) gave crude 42. Purification by column chromatog-
raphy using ethyl acetate as eluent aꢃff1orded 42 as a pure product
(57%): mp 114e115 ꢀC; IR (KBr) cm 1725, 1672 (CO); 1H NMR
(m, 3H, aromatic), 3.76 (s, 3H, OCH3), 3.61 (t, J ¼ 7.2 Hz, 2H,
CONCH2), 3.20 (d, J ¼ 15.8 Hz, 2H, CCHAHB), 3.10e2.85 (m, 2H þ 4H,
CCHAHB þ piperazine), 2.40e2.28 (m, 4H, piperazine), 2.05 (t,
J
¼
7.2 Hz, 2H, CONCH2CH2CH2), 1.70e1.40 (m, 2H
þ
3H,
CONCH2CH2 þ CH3). Anal. (C26H32ClN3O3) calcd: C 66.44, H 6.86, N
(DMSO-d6)
aromatic), 3.76 (s, 3H, CH3), 3.64 (t, J ¼ 6.4 Hz, 2H, CONCH2),
3.50e3.25 (m, 1H, CH), 3.05e2.70 (m, 4H 4H,
d
7.45e7.25 (m, 4H, aromatic), 7.00e6.80 (m, 4H,
8.94; found: C 66.44, H 6.85, N 8.77.
þ
6.1.33. 1-[4-[4-(2-Methoxyphenyl)piperazin-1-yl]butyl]-4-methyl-
4-phenylpiperidine-2,6-dione (47)
CHCH2 þ piperazine), 2.55e2.40 (m, 4H, piperazine), 2.29 (t,
J ¼ 6.4 Hz, 2H, CONCH2CH2CH2CH2), 1.48e1.36 (m, 2H þ 2H,
CONCH2CH2CH2). Anal. (C26H32ClN3O3) calcd: C 66.44, H 6.86, N
8.94; found: C 66.41, H 6.82, N 9.15.
Compound 12 (0.55 g, 2.71 mmol) was dissolved in acetonitrile
(5 mL). Successively, potassium carbonate (1.12 g, 8.12 mmol), 1-
bromo-4-chlorobutane (1.16 g, 6.77 mmol) and potassium iodide
(0.05 g, 0.30 mmol) were added. The reaction mixture was heated
under reflux for 8 h. After being cooled, it was concentrated under
reduced pressure; then, water and ethyl acetate were added. The
organic layer was separated and dried over anhydrous sodium
sulphate. After filtration, solvents were eliminated under reduced
pressure. The resulting crude oil, 1-(4-chlorobutyl)-4-methyl-4-
phenylpiperidine-2,6-dione (25) (0.78 g), was successively used
without further purification.
A mixture of compound 25 (0.39 g, 1.33 mmol) and 1-(2-
methoxyphenyl)piperazine (26) (1.28 g, 6.64 mmol) was heated in
an oil bath at 140 ꢀC for 30 min. After being cooled, ethyl acetate
(15 mL) was added to the reaction mixture. Solids were filtered off
and the filtrate was, successively, evaporated under reduced pres-
sure. The residue was purified by column chromatography using
a mixture of ethyl acetate/methanol (95/5, v/v) as eluent. The
homogeneous fractions were evaporated in vacuo to give 47 (0.13 g,
22%) as a pure oil: IR (NaCl) cmꢃ1 1724, 1672 (CO); 1H NMR (DMSO-
6.1.29. 1-[3-[4-(2-Methoxyphenyl)piperazin-1-yl]propyl]-4-(4-
nitrophenyl)piperidine-2,6-dione (43)
Compound 43 was prepared according to the procedure pre-
sented for 31. Reaction between 22 and 1-(2-methoxyphenyl)
piperazine (26) gave crude 43. Purification by column chromatog-
raphy using ethyl acetate/methanol (9/1, v/v) as eluent afforded 43
as a yellow solid (20%): mp 124e126 ꢀC; IR (KBr) cmꢃ1 1726, 1671
(CO); 1H NMR (DMSO-d6)
d
8.30e8.18 (m, 2H, aromatic), 7.70e7.58
(m, 2H, aromatic), 6.99e6.81 (m, 4H, aromatic), 3.77 (s, 3H, CH3),
3.76e3.60 (m, 2H þ 1H, CONCH2 þ CH), 3.18e2.80 (m, 4H þ 4H,
CHCH2
þ
piperazine), 2.60e2.41 (m, 4H, piperazine), 2.31
(t, J ¼ 6.8 Hz, 2H, CONCH2CH2CH2), 1.78e1.58 (m, 2H, CONCH2CH2).
Anal. (C25H30N4O5) calcd: C 64.36, H 6.48, N 12.01; found: C 64.09,
H 6.56, N 12.22.
6.1.30. 1-[4-[4-(2-Methoxyphenyl)piperazin-1-yl]butyl]-4-(4-nitro-
phenyl)piperidine-2,6-dione (44)
d6) d 7.45e7.20 (m, 5H, aromatic), 7.00e6.90 (m, 4H, aromatic), 3.77
Compound 44 was prepared according to the procedure pre-
sented for 31. Reaction between 23 and 1-(2-methoxyphenyl)
piperazine (26) gave crude 44. Purification by column chromatog-
raphy using a mixture of ethyl acetate/methanol (95/5, v/v) affor-
ded 44 as a light yellow powder (14%): mp 143e144 ꢀC; IR (KBr)
(s, 3H, OCH3), 3.57 (t, J ¼ 6.6 Hz, 2H, CONCH2), 3.20 (d, J ¼ 16.2 Hz,
2H, CCHAHB), 3.10e2.80 (m, 2H þ 4H, CCHAHB þ piperazine),
2.50e2.30 (m, 4H, piperazine), 2.16 (t,
J
¼
6.8 Hz, 2H,
CONCH2CH2CH2CH2), 1.30 (s, 3H, CH3), 1.25e1.05 (m, 2H þ 2H,
CONCH2CH2CH2). Anal. (C27H35N3O3) calcd: C 72.13, H 7.85, N 9.35;
found: C 71.95, H 8.01, N 9.31.
cmꢃ1 1723, 1671 (CO); 1H NMR (DMSO-d6)
d 8.26e8.17 (m, 2H,
aromatic), 7.67e7.58 (m, 2H, aromatic), 7.00e6.80 (m, 4H,
aromatic), 3.77 (s, 3H, CH3), 3.75e3.55 (m, 2H þ 1H, CONCH2 þ CH),
3.16e2.78 (m, 4H þ 4H, CHCH2 þ piperazine), 2.55e2.40 (m, 4H,
piperazine), 2.30 (t, J ¼ 6.6 Hz, 2H, CONCH2CH2CH2CH2), 1.55e1.30
(m, 2H þ 2H, CONCH2CH2CH2). Anal. (C26H32N4O5) calcd: C 64.98, H
6.71, N 11.66; found: C 64.70, H 6.88, N 11.83.
6.1.34. 1-[4-[4-(5-Chloro-2-methoxyphenyl)piperazin-1-yl]butyl]-
4-methyl-4-phenylpiperidine-2,6-dione (48)
Compound 48 was prepared according to the procedure pre-
sented for 47. Reaction between 25 and 1-(5-chloro-2-methox-
yphenyl)piperazine (28) gave crude 48. Purification by column

2688
G. Romeo et al. / European Journal of Medicinal Chemistry 46 (2011) 2676e2690
chromatography using a mixture of ethyl acetate/cyclohexane (9/1,
6.1.39. 10-(3-Chloropropyl)-2,3-dihydrospiro[1H-indene-1,40-
piperidine]-20,60-dione (55)
The same procedure, as described for the synthesis of 17, was
followed starting from 52. Crude oil 55 (75%) was used without
further purification: IR (NaCl) cmꢃ1 1725, 1671 (CO); 1H NMR
v/v) afforded 48 as a pure oil (24%): IR (NaCl) cmꢃ1 1724, 1672 (CO);
1H NMR (DMSO-d6)
d
7.40e7.20 (m, 5H, aromatic), 7.00e6.80 (m,
3H, aromatic), 3.76 (s, 3H, OCH3), 3.52 (t, J ¼ 6.4 Hz, 2H, CONCH2),
3.20 (d, J ¼ 16.4 Hz, 2H, CCHAHB), 3.05e2.82 (m, 2H þ 4H,
CCHAHB þ piperazine), 2.45e2.30 (m, 4H, piperazine), 2.14 (t,
J ¼ 7.0 Hz, 2H, CONCH2CH2CH2CH2), 1.28 (s, 3H, CH3), 1.22e1.00 (m,
2H þ 2H, CONCH2CH2CH2). Anal. (C27H34ClN3O3) calcd: C 67.00, H
7.08, N 8.68; found: C 67.21, H 7.23, N 8.64.
(DMSO-d6)
d
7.38e7.10 (m, 4H, aromatic), 3.84 (t, J ¼ 7.0 Hz, 2H,
CONCH2), 3.70e3.60 (t, J ¼ 6.6 Hz, 2H, CH2Cl), 3.06 (d, J ¼ 16.5 Hz,
2H, CCHAHBCO), 2.90 (t, J ¼ 7.2 Hz, 2H, ArCH2), 2.69 (d, J ¼ 16.5 Hz,
2H, CCHAHBCO), 2.12e1.80 (m, 2H þ 2H, ArCH2CH2 þ CH2CH2Cl).
6.1.35. 2,3-Dihydro-20,60-dioxospiro[1H-indene-1,40-piperidine]-
30,50-dicarbonitrile (50)
6.1.40. 2,3-Dihydrospiro-10-[2-[4-(2-methoxyphenyl)piperazin-
1-yl]ethyl][1H-indene-1,40-piperidine]-20,60-dione (57)
Cyanoacetamide (6.62 g, 78.77 mmol) was slowly added to
a solution of sodium ethoxide (5.36 g, 78.77 mmol) in dry ethanol
(110 mL). Successively, ethyl cyano(2,3-dihydro-1H-inden-1-yli-
dene)acetate (49) (17.90 g, 78.77 mmol) was added. The reaction
mixture was stirred for 3 h at room temperature; then, water
(100 mL) and concentrated HCl (15 mL) were added. The obtained
suspension was first stirred for 2 h at room temperature and then
stored at 4 ꢀC for further 2 h. Solids were filtered off, washed with
water (30 mL) and then with cold ethanol (10 mL). Recrystallization
from ethanol/water (2/1, v/v) afforded 50 (33.23 g, 95%) as
a yellowish solid: mp 270e271 ꢀC; IR (KBr) cmꢃ1 3223 (NH), 2265
Compound 53 (0.14 g, 0.54 mmol) was dissolved in toluene
(5 mL) and thionyl chloride (0.128 g, 1.08 mmol) was added. The
reaction mixture was heated under reflux for 4 h; then, it was
concentrated under reduced pressure. The resulting crude oil,10-(2-
chloroethyl)-2,3-dihydrospiro[1H-indene-1,40-piperidine]-20,60-
dione (54) (0.15 g, 99%), was successively used for the synthesis of
57 without further purification.
A mixture of compound 54 (0.15 g, 0.54 mmol) and 1-(2-
methoxyphenyl)piperazine (26) (0.52 g, 2.70 mmol) was heated in
an oil bath at 140 ꢀC for 30 min. After being cooled, ethyl acetate
(10 mL) was added to the reaction mixture that was gently heated
for 10 min. Then, the mixture was allowed to cool to room
temperature, solids were filtered off and the filtrate was, succes-
sively, evaporated under reduced pressure. The residue was puri-
fied by column chromatography using a mixture of ethyl acetate/
cyclohexane (1/1, v/v) as eluent. The homogeneous fractions were
evaporated in vacuo to give 57 (0.080 g, 34%) as a pure product: mp
78e80 ꢀC, IR (NaCl) cmꢃ1 1725, 1672 (CO); 1H NMR (DMSO-d6)
(CN), 1746, 1707 (CO); 1H NMR (DMSO-d6)
d 12.40 (br s, 1H, NH
which exchanges with D2O), 7.60e7.20 (m, 4H, aromatic), 5.28 (s,
2H, CH), 3.17 (t, J ¼ 7.2 Hz, 2H, ArCH2), 2,31 (t, J ¼ 7.2 Hz, 2H,
ArCH2CH2). Anal. (C15H11N3O2) calcd: C 67.92, H 4.18, N 15.84;
found: C 67.92, H 4.29, N 15.66.
6.1.36. 1,1-Indandiacetic acid (51)
Compound 50 (20.00 g, 75.40 mmol) was added to a mixture of
concentrated sulphuric acid (52 mL) and glacial acetic acid (76 mL).
Reaction mixture was refluxed for 2 h without stirring; then, reflux was
continued for 80 h under stirring. After being cooled, water (500 mL)
was added to the reaction mixture which was stored at 4 ꢀC overnight.
The precipitate was filtered off and washed with ice water. Recrystal-
lization from water afforded 51 (4.50 g, 25%): mp 120e121 ꢀC; IR (KBr)
d 7.36e7.15 (m, 4H, aromatic), 7.00e6.80 (m, 4H, aromatic), 3.89 (t,
J ¼ 6.4 Hz, 2H, CONCH2), 3.77 (s, 3H, CH3), 3.10e2.85 (m,
4H þ 2H þ 2H, piperazine þ CCHAHBCO þ ArCH2), 2.73 (d,
J
¼
16.6 Hz, 2H, CCHAHBCO), 2.65e2.40 (m, 4H
þ
2H,
piperazine þ CONCH2CH2), 1.98 (t, J ¼ 7.0 Hz, 2H, ArCH2CH2). Anal.
(C26H31N3O3) calcd: C 72.03, H 7.21, N 9.69; found: C 71.87, H 7.06, N
9.54.
cmꢃ1 1712 (CO); 1H NMR (DMSO-d6)
d 12.03 (s, 2H, COOH which
exchanges with D2O), 7.15e7.00 (m, 4H, aromatic), 2.85 (t, J ¼ 7.2 Hz,
2H, ArCH2), 2.80 (d, J ¼ 15.4 Hz, 2H, CHAHBCOOH), 2.65 (d, J ¼ 15.4 Hz,
2H, CHAHBCOOH), 2.17 (t, J ¼ 7.2 Hz, 2H, ArCH2CH2). Anal. (C13H14O4)
calcd: C 66.66, H 6.02; found: C 66.51, H 6.22.
6.1.41. 2,3-Dihydrospiro-10-[3-[4-(2-methoxyphenyl)piperazin-1-yl]
propyl][1H-indene-1,40-piperidine]-20,60-dione (58)
Compound 58 was prepared according to the procedure
presented for 31. Reaction between 55 and 1-(2-methoxyphenyl)
piperazine (26) gave crude 58. Purification by column chromatog-
raphy using a mixture of ethyl acetate/cyclohexane (4/1, v/v) as
eluent afforded 58 as a pure product (50%): mp 97e98 ꢀC; IR (KBr)
6.1.37. 2,3-Dihydrospiro[1H-indene-1,40-piperidine]-20,60-dione (52)
The same procedure, as described for the synthesis of 9, was
followed using 51. Recrystallizaꢃti1on from toluene afforded 52 (46%):
mp 182e183 ꢀC; IR (KBr) cm 3193 (NH), 1686 (CO); 1H NMR
cmꢃ1 1724, 1672 (CO); 1H NMR (DMSO-d6)
d 7.26e7.14 (m, 4H,
aromatic), 6.90e6.80 (m, 4H, aromatic), 3.85e3.70 (m,
3H þ 2H, CH3 þ CONCH2), 3.15e2.80 (m, 4H þ 2H þ 2H,
piperazine þ CCHAHBCO þ ArCH2), 2.69 (d, J ¼ 16.4 Hz, 2H,
CCHAHBCO), 2.55e2.42 (m, 4H, piperazine), 2.34 (t, J ¼ 6.8 Hz,
2H, CONCH2CH2CH2), 1.93 (t, J ¼ 7.2 Hz, 2H, ArCH2CH2), 1.75e1.55
(m, 2H, CONCH2CH2). Anal. (C27H33N3O3) calcd: C 72.46, H 7.43, N
9.39; found: C 72.46, H 7.40, N 9.43.
(DMSO-d6)
d
10.96 (br s, 1H, NH which exchanges with D2O),
2H,
7.30e7.18 (m, 4H, aromatic), 2.95e2.85 (m, 2H
þ
CCHAHBCO þ ArCH2), 2.52 (d, J ¼ 16.2 Hz, 2H, CCHAHBCO), 1.92 (t,
J ¼ 7.0 Hz, 2H, ArCH2CH2). Anal. (C13H13NO2) calcd: C 72.54, H 6.09,
N 6.51; found: C 72.30, H 6.23, N 6.37.
6.1.38. 2,3-Dihydrospiro-10-(2-hydroxyethyl)[1H-indene-1,40-
piperidine]-20,60dione (53)
6.1.42. 2,3-Dihydrospiro-10-[4-[4-(2-methoxyphenyl)piperazin-1-
yl]butyl][1H-indene-1,40-piperidine]-20,60-dione (59)
The same procedure, as described for the synthesis of 13, was
followed using 52. Purification by column chromatography using
a mixture of ethyl acetate/cyclohexane (1/1, v/v) afforded 53 (34%)
Compound 52 (0.25 g, 1.16 mmol) was dissolved in acetonitrile
(5 mL). Successively, potassium carbonate (0.48 g, 3.47 mmol),
1-bromo-4-chlorobutane (0.50 g, 2.92 mmol) and potassium iodide
(0.05 g, 0.30 mmol) were added. The reaction mixture was heated
under reflux for 7 h. After being cooled, it was concentrated under
reduced pressure. Then, water and ethyl acetate were added. The
organic layer was separated and dried over anhydrous sodium
sulphate. After filtration, solvents were eliminated under reduced
pressure. The resulting crude oil (0.35 g, 100%), 1-(4-chlorobutyl)-
as a pure oil: 1H NMR (DMSO-d6)
d 7.30e7.15 (m, 4H, aromatic),
4.79 (t, J ¼ 5.6 Hz, 1H, OH which exchanges with D2O), 3.82
(t, J ¼ 6.8 Hz, 2H, CONCH2), 3.51e3.37 (m, 2H, CH2OH), 3.00 (d,
J ¼ 16.6 Hz, 2H, CCHAHBCO), 2.90 (t, J ¼ 7.2 Hz, 2H, ArCH2), 2.70 (d,
J ¼ 16.6 Hz, 2H, CCHAHBCO), 1.93 (t, J ¼ 7.2 Hz, 2H, ArCH2CH2).
Anal. (C15H17NO3) calcd: C 69.48, H 6.61, N 5.40; found: C 69.67, H
6.74, N 5.34.

G. Romeo et al. / European Journal of Medicinal Chemistry 46 (2011) 2676e2690
2689
2,3-dihydrospiro[1H-indene-1,40-piperidine]-20,60-dione (56), was
successively used without further purification.
were calculated using GraphPad Prism taking into account EC50
values previously determined for NE [43].
A mixture of compound 56 (0.35 g, 1.16 mmol) and 1-(2-
methoxyphenyl)piperazine (26) (1.11 g, 5.80 mmol) was heated in
an oil bath at 140 ꢀC for 30 min. After being cooled, ethyl acetate
(15 mL) was added to the reaction mixture. Solids were filtered off
and the filtrate was, successively, evaporated under reduced pres-
sure. The residue was purified by column chromatography using
a mixture of ethyl acetate/methanol (95/5, v/v) as eluent. The
homogeneous fractions were evaporated in vacuo to give 59 (0.30 g,
54%) as a pure product: mp 89e90 ꢀC; IR (KBr) cmꢃ1 1721, 1671
6.4. Computational methods
Calculations and graphic manipulations were performed on SGI
workstations and an SGI Origin300 server by means of the software
MacroModel 8.0 [44] and Catalyst 4.9 [34].
Considering the presence of two conformationally flexible rings
(the piperazine and the piperidine-2,6-dione moieties) in title
compounds, together with the known limitation of the poling
algorithm of Catalyst in treating rings (i.e., in piperazine containing
compounds, the software generated many chair conformations
with axial substituents), we decided to submit such compounds to
an exhaustive conformational search. For this purpose, compounds
reported in the present paper were built using the sketcher menu
of MacroModel and submitted to a conformational search using the
Monte Carlo Multiple Minimum method (10,000 steps) and
a continuum solvation treatment with water as the solvent. Among
various force fields available, OPLSeAA [45] was selected, being the
sole force field providing high quality parameters for the
compounds studied. Minimization of the structures was performed
by the PolakeRibiere conjugate gradient method.
(CO); 1H NMR (DMSO-d6)
(m, 4H, aromatic), 3.80e3.65 (m, 3H þ 2H, CH3 þ CONCH2), 3.05 (d,
16.4 Hz, 2H, CCHAHBCO), 3.00e2.82 (m, 4H 2H,
piperazine þ ArCH2), 2.69 (d, J ¼ 16.4 Hz, 2H, CCHAHBCO),
2.55e2.40 (m, 4H, piperazine), 2.30 (t, 6.6 Hz, 2H,
d 7.30e7.19 (m, 4H, aromatic), 6.93e6.88
J
¼
þ
J
¼
CONCH2CH2CH2CH2), 1.92 (t, J ¼ 7.0 Hz, 2H, ArCH2CH2), 1.60e1.40
(m, 2H þ 2H, CONCH2CH2CH2). Anal. (C28H35N3O3) calcd: C 72.86, H
7.64, N 9.10; found: C 72.65, H 7.75, N 8.99.
6.2. Binding experiments on human cloned a1-AR subtypes
6.2.1. Transfection and cell culture
Sixteen pyrimido[5,4-b]indole derivatives taken from our
previous work [24], and five compounds among the new synthe-
sized derivatives (viz., 29, 34, 39, 45, and 59) along with their
associated conformational models and a1D-AR affinity values, were
submitted to Catalyst HypoGen with the aim of building a potential
pharmacophore model for a1D-AR subtype antagonists.
HEK293 cells were transfected with the constitutively active
pRSVICAT vectors containing the human a1A-AR [37], a1B-AR [38],
or a1D-AR [39] cDNAs by calcium phosphate transfection [40]. Cells
were propagated for several weeks in the presence of 400 mg/mL
geneticin, and subclones screened by radioligand binding for high
receptor expression. Transfected HEK293 cells were propagated in
75 cm2 flasks at 37 ꢀC in a humidified 5% CO2 incubator in Dul-
becco’s modified Eagle’s medium containing 4.5 g/L glucose, 1.4%
glutamine, 20 mM HEPES, 100 mg/L streptomycin, 105 units/L
penicillin, and 10% calf serum. The cells were detached by trypsi-
nization and subcultured at a ratio 1:4 upon reaching confluency.
Acknowledgement
This study was supported by Università di Catania and Italian
MIUR. We thank Dr M. Sanfilippo for technical assistance in the
synthesis of some compounds. 1-(5-Chloro-2-methoxyphenyl)
piperazine was a kind gift from Clariant Life Science Molecules.
6.2.2. Radioligand binding
Confluent 100-mm plates were washed with phosphate buffered
saline (18 mM Na2HPO4, 2 mM NaH2PO4, 154 mM NaCl, pH 7.6)
and harvested by scraping. Cells were collected by centrifugation
and homogenized with a Polytron. Cell membranes were collected
by centrifugation at 30,000 ꢄ g for 10 min and resuspended by
homogenization. Receptor density was determined by saturation
analysis of the non-subtype-selective a1-AR specific antagonist
radioligand [125I]BE 2254 (20e800 pM) [41]. For analysis of
competition by selective drugs, 50 pM radioligand was used. Curves
were analyzed by nonlinear regression analysis using GraphPad
Prism [42]. Nonspecific binding was determined in the presence of
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