1,3-Dioxolane-based ligands incorporating a lactam or imide moiety: Structure-affinity/activity relationship at α1-adrenoceptor subtypes and at 5-HT1A receptors

Article abstract of DOI:10.1016/j.ejmech.2010.05.023

A series of 1,3-dioxolane-based compounds incorporating a lactam (2-4) or imide (5-7) moiety was synthesized and the pharmacological profile at α₁-adrenoceptor subtypes and 5-HT_1A receptor was assessed through binding and functional experiments. Starting from the 2,2-diphenyl-1,3-dioxolane derivative 1, previously shown to be a selective α_1a(A)/α_1d(D)-adrenoceptor subtype antagonist, over α_1b(B) subtype and 5-HT_1A receptor, and replacing one phenyl ring with lactam or imide moiety a reduction of α₁/5-HT_1A selectivity is observed, mainly due to the increase in 5-HT_1A affinity. In functional experiments lactam derivatives seems to favour 5-HT_1A receptor antagonism (pKb = 7.20-7.80) and α_1B-adrenoceptor antagonist selectivity (α_1B/α_1A and α_1B/ α_1D of about 10-fold). The most interesting of the various imide derivatives is compound 7t, which is a selective α_1D- adrenoceptor antagonist (pKb = 8.1 and α_1D/α_1A and α_1D/α_1B selectivity ratios of 16 and 11 respectively) whereas at 5-HT_1A receptor it is a potent partial agonist (pD2 = 7.98, E_max = 60%).]. Given that cis and trans diastereomer pairs for 2-7 are possible, a computational strategy based on molecular docking studies was used to elucidate the atomic details of the 5HT_1A/agonist and 5HT_1A/antagonist interaction. The lactam derivatives seems to favour 5-HT_1A receptor antagonism and α_1B-adrenoceptor antagonist selectivity. The imide derivative 7t is a selective α_1D-adrenoceptor antagonist whereas at 5-HT_1A receptor it is a potent partial agonist.

Full text of DOI:10.1016/j.ejmech.2010.05.023

European Journal of Medicinal Chemistry 45 (2010) 3740e3751
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
1,3-Dioxolane-based ligands incorporating a lactam or imide moiety:
Structureeaffinity/activity relationship at a1-adrenoceptor subtypes and at
5-HT1A receptors
Silvia Franchini a, Adolfo Prandi a, Annamaria Baraldi a, Claudia Sorbi a, Annalisa Tait a, Michela Buccioni b,
,
Gabriella Marucci b, Antonio Cilia c, Lorenza Pirona c, Paola Fossa d, Elena Cichero d, Livio Brasili a
*
a Dipartimento di Scienze Farmaceutiche, Università degli Studi di Modena e Reggio Emilia, Via Campi 183, 41100 Modena, Italy
b Dipartimento di Scienze Chimiche, Università degli Studi di Camerino, Via S. Agostino 1, 62032 Camerino, Italy
c Divisione Ricerca e Sviluppo, Recordati S.p.A., Via Civitali 1, 20148 Milano, Italy
d Dipartimento di Scienze Farmaceutiche, Università degli Studi di Genova, Viale Benedetto XV 3, 16132 Genova, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 1,3-dioxolane-based compounds incorporating a lactam (2e4) or imide (5e7) moiety was
synthesized and the pharmacological profile at a1-adrenoceptor subtypes and 5-HT1A receptor was
assessed through binding and functional experiments. Starting from the 2,2-diphenyl-1,3-dioxolane
Received 13 April 2010
Received in revised form
6 May 2010
Accepted 8 May 2010
Available online 15 May 2010
derivative 1, previously shown to be a selective a1a(A)/a1d(D)-adrenoceptor subtype antagonist, over a1b(B)
subtype and 5-HT1A receptor, and replacing one phenyl ring with lactam or imide moiety a reduction of
a1/5-HT1A selectivity is observed, mainly due to the increase in 5-HT1A affinity. In functional experiments
lactam derivatives seems to favour 5-HT1A receptor antagonism (pKb ¼ 7.20e7.80) and a1B-adrenoceptor
Keywords:
antagonist selectivity (a1B/a1A and a1B/a1D of about 10-fold). The most interesting of the various imide
Alpha1-adrenoceptor
5-HT1A receptor
1,3-Dioxolane
Agonist
derivatives is compound 7t, which is a selective a1D-adrenoceptor antagonist (pKb ¼ 8.1 and a1D
/a1A and
a1D a1B selectivity ratios of 16 and 11 respectively) whereas at 5-HT1A receptor it is a potent partial
/
agonist (pD2 ¼ 7.98, Emax ¼ 60%).]. Given that cis and trans diastereomer pairs for 2-7 are possible,
a computational strategy based on molecular docking studies was used to elucidate the atomic details of
the 5HT1A/agonist and 5HT1A/antagonist interaction.
Antagonist
Ó 2010 Elsevier Masson SAS. All rights reserved.
1. Introduction
the a1D receptor blockade may improve the irritative symptoms of
BHP caused by an involuntary contraction of the smooth muscle of
Since the discovery of a1-adrenoceptor subtypes, namely a1A
a1a), a1B a1b) and a1D a1d) medicinal chemists dedicated a great
the bladder [6]. Therefore the best selectivity profile for the treat-
ment of BHP appears to be a1A a1D a1B
Receptor selectivity is quite difficult to achieve and very
frequently a1 antagonists show a cross-reactivity also with 5-HT1A
(
(
(
¼
>
.
deal of effort to discovering selective ligands for one of the three
subtypes [1e3]. The quest for antagonists has been particularly
active. Initially introduced for hypertension management, they
have become increasingly common in the treatment of benign
prostatic hyperplasia (BPH). The a1A subtype has received a great
deal of attention as a potential target for BPH and several urose-
lective agents have been discovered [4]. Selective a1A antagonists,
particularly over a1B, would reduce the “dynamic” component of
the bladder outlet obstruction without the undesiderable side
effect of lowering blood pressure which would appear to be
controlled by the a1B subtype [5]. It has also been postulated that
receptor, probably as
a consequence of the high degree of
homology, approximately 45%, in the transmembrane amino acid
sequence of 5-HT1A subtype and a1-adrenoceptors [7].
5-HT1A receptor has been a major target for neurological
research and drug development. Agonists and partial agonists have
been seen to be effective in treating anxiety and depression [8e12].
In addition to therapeutic applications in the psychiatry field, more
recent preclinical studies have suggested that 5-HT1A receptor
agonists also have pronounced neuroprotective properties [13].
Potential therapeutic applications for 5-HT1A receptor antagonists
are evaluated, for example, in cognitive disorders [14].
We recently reported compound 1 (Fig. 1), as a 1,3-dioxolane
based a1-adrenoceptor antagonist. Binding and functional studies
* Corresponding author. Tel.: þ39 59 205 5139; fax: þ39 59 205 5131.
E-mail address: livio.brasili@unimore.it (L. Brasili).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.05.023

S. Franchini et al. / European Journal of Medicinal Chemistry 45 (2010) 3740e3751
3741
2. Results and discussion
2.1. Chemistry
O
H3CO
The synthesis ofcompounds 2e7ct isreported in Schemes 1 and 2.
The chloro derivatives 14e19ct were aminated with 1-(2-methoxy-
phenyl)-piperazine. For lactam series the diastereomeric mixtures
(8e10ct) were separated by flash chromatography at this stage. Each
diastereomeric pair was then transformed into the corresponding
oxalate salts 2e4c and 2e4t. Intheimideseries, dueto difficulties met
with the separation of amines (11e13ct), diastereomeric mixtures
were separated at chloroderivative stage and each diastereomeric
pair was reacted with 1-(2-methoxy-phenyl)-piperazine.
The chloro derivatives 14e16c,t were prepared as previously
reported [21]. Chloro derivatives 17e19 were obtained as depicted
in Scheme 2. The ketoimides 22e24, obtained from the appropriate
anhydride reacted with phenacylamide hydrochloride, by direct
treatment with 3-chloropropane-1,2-diol, (22) or by trans-
ketalization of ketals 20e21, gave the chloro derivatives 17e19. The
diastereomeric mixtures were separated by flash chromatography
to give 17e19c and 17e19t as diastereomeric pair.
O
N
N
1
Fig. 1. Structure of compound 1.
have shown that its pharmacological profile at a1-adrenoceptor
subtypes is that required for drugs that are therapeutically useful
for the treatment of benign prostatic hyperplasia (selectivity
a1D > a1B). In addition it binds significantly to the 5-HT1A
receptor (pKi ¼ 7.84) with a selectivity ratio a1/5-HT1A of about
a1A
¼
100-fold [15].
In this research, we aimed to study the effect of replacing one of
the phenyl ring in position 2 of the 1,3-dioxolane ring with a lactam
or an imide moiety which could potentially create discriminative
interactions within the a1-adrenoceptor subtypes and/or between
a1-adrenoceptors and 5-HT1A receptor. In doing so, a second ster-
eogenic centre of lead compound 1 is created (position 2), in
addition to that already present (position 4). A pair of diastereo-
mers (cis and trans) is therefore obtained for each derivative.
To support and guide the synthetic efforts, in silico docking
studies on compounds 2e7 were also performed. Up to now,
various models of ligand-5HT1A complexes have been calculated
and evaluated, one of the latest being the bovine rhodopsin-based
developed by Nowak [16]. With the aim at gaining a better
understanding of the 1,3-dioxolanes-5HT1A receptor interactions
and in particular to outline specific 5HT1A agonists binding mode,
a theoretical model of the human 5HT1A receptor was built using
the X-ray b2 adrenoreceptor coordinates (pdb code 2RH1) [17].
The model was used to simulate the binding mode of
compounds 2e7 diastereomer pairs (cis and trans). Our results
were in agreement with directed mutagenesis experiments
[18e20] and were consistent with the biological data here pre-
sented, thus providing indirect validation on both the homology
model and the docking protocol.
Accurate 1H NMR spectroscopy study was performed on each
diastereomeric pair (8e13) as free base. Considering the common
backbone structure, three pairs of methylene protons (C5, C6, C7)
(Chart 1) are easily recognized. Each pair has characteristic geminal
coupling constants (J2): 7.60e8.30 Hz (H5a,5b), 14.00e14.90 Hz
(H6a,6b) and 12.70e13.20 (H7a,7b) respectively. Also the vicinal
coupling constants (J3) have a similar trend: 6.1e8.3 Hz for H5a,b and
4.4e6.7 Hz for H7a,b with respect to H4.
The cis/trans stereochemistry was assigned by measurements of
1H NMR NOESY (Nuclear Overhauser Effect Spectroscopy). For the
cis isomers a correlation is seen between the H0 in ortho position of
the phenyl ring and the proton H4, whereas for the trans isomer
similar correlation is observed between the proton H4 and the
methylene protons in position 2 (H6a and H6b), albeit only in the
case of the five-membered lactam (8t) and the imide (11t). Further
support comes from the examination of proton chemical shifts.
According to previous studies on similar derivatives with 1,3-
dioxolane structure [22,23] the phenyl ring in position 2 has
a characteristic effect: the protons H4 and H5b are shielded in the cis
derivatives, whereas they are deshielded in the trans derivatives.
O
R
Cl
O
R =
O
O
O
N
14-16c,t ;
17-19c, 17-19t
N
N
;
;
i
14c,t
8c,t
15c,t
9c,t
16c,t
10c,t
4c,t
2c,t
3c,t
O
N
O
O
N
O
O
N
O
H3CO
N
H3CO
N
O
R
O
R
;
;
N
N
O
O
and enantiomer
and enantiomer
17c,t
11c,t
5c,t
18c,t
12c,t
6c,t
19c,t
13c,t
7c,t
cis
trans
8c, 9c, 10c
8t, 9t, 10t
11c, 12c, 13c
11t, 12t, 13t
ii
ii
2c, 3c, 4c
5c, 6c, 7c
2t, 3t, 4t
5t, 6t, 7t
Scheme 1. Reagents and conditions: (i) 1-(2-methoxy-phenyl)-piperazine, 2-methoxyethanol, KI, reflux, 20h; (ii) C2H2O4, Et2O or acetone.

3742
S. Franchini et al. / European Journal of Medicinal Chemistry 45 (2010) 3740e3751
presence of a general strong deshielding effect (e5ppm) with respect
H3N
Cl
to the lactam series.
O
O
2.2. Modeling studies
O
N
N
iii
O
O
i
Since most of the GPCR key residues are conserved in 5HT1A
receptor sequence alignment of 5HT1A with human b2 adrenor-
eceptor (PDB code: 2RH1) was performed. The derived backbone
conformation was inspected by Ramachandran plot (showing
absence of outliers) and superimposed to the coordinates of the
template structure (Fig. 2a). Moreover, the reliability of the
obtained protein model was also verified by evaluation of an
appropriate distribution of the hydrophobic and hydrophilic
properties on the 5HT1A model surface. In fact, as shown in Fig. 2b,
the receptor transmembrane domain displays hydrophobic surface
properties (depicted in brown) while the portions extended
towards the extra-cellular (EC) or the intra-cellular (IC) environ-
ments are properly depicted in cyan (hydrophilic properties).
As concern docking calculations, the 1,3-dioxolane derivatives
bearing the lactam moiety cis isomers (compounds 2e4, showing
5HT1A neutral antagonist activity) seem to share the same putative
bindingmode. Onthecontrary, compounds2e4 transisomers display
dockingposes similartothoseof the1,3.dioxolanederivativesbearing
the imide moiety (compounds 5e7, showing 5HT1A agonist activity).
On this basis, in the following section only the selected docking
conformers of compound 2 have been discussed (Fig. 3).
O
+
O
O
O
O
O
22-24
20-21
ii
iv
O
O
N
O
O
N
O
O
O
O
N
;
;
N
O
Cl
O
17c,t
18c,t
19c,t
17-19c, 17-19t
Scheme 2. Reagents and conditions: (i) PhMe, DeaneStark, reflux, 23h; (ii) 3-chloro-
1,2-propandiol, PhMe, pTSA, DeaneStark, reflux, 24h; (iii) orthochloroformiate, pTSA,
MeOH, reflux, 23h; (iv) CoCl2, CH3CN, room temperature, 21h.
The compound 2 trans (taken as representative of the binding
mode for 5HT1A agonist, the S,S isomer showed the lowest S value)
shows H-bond interactions between the oxygen atom of the 2-
methoxy phenyl group and the Thr121 side chain and between the
protonated piperazine nitrogen and Asp116 (Fig. 3a). Van deer Waals
contacts are displayed between: (i) the 2-methoxy phenyl ring and
Cys120, Thr121;(ii)thepiperazinemoietyandVal117, Phe362;(iii)the
lactamgroupandIle189, Lys121;(iv)thephenylringandAla93,Ala94,
Asn386; (v) the 1,3-dioxolane moietyand Phe112, Phe 361. Finally, the
2-methoxy phenyl moiety and the phenyl group are also involved in
On the contrary H5a and H7a,b are shielded for the trans derivatives
and deshielded for the cis ones.
In the lactam series the analysis of the 13C chemical shifts
enables small but nevertheless significant differences between cis
and trans isomers to be observed. C-2 and C-7 are always shielded,
C-4, C-5 and C-6 are deshielded in the cis with respect to the trans
isomers. The behaviour of C-6 signals in imide series is the same of
that described in the lactam series. Cis isomers are deshielded in
pe
p
stacking with Trp258 and Tyr96, Tyr390, Trp387 respectively.
As shown in Fig. 2b, the compound 2 cis (taken as representative
of the binding mode for 5HT1A neutral antagonist the, S,R isomer
showed the lowest S value) shows H-bond interactions between
the nitrogen of the lactam moiety and the Lys191 3-amino group
and between the protonated piperazine nitrogen and Asp116. The
2-methoxy phenyl group displays Van deer Waals contacts with
O
O
H6a
H6a
H6b
H6b
H5a
H5a
N
N
O
O
H
H
5b
5b
X
X
N
N
O
O
H
oH4
H
oH4
Ala93, Ala94, Asn386 side chain and
pep stacking with the
H7b
H7b
H7a
TRANS
H7a
aromatic residues Tyr96, Tyr390, Trp387. The 1,3-dioxolane group
and the lactam portion of the ligand are oriented towards the
residues Leu173, Ile189, Ser190 and Lys191 (detecting hydrophobic
CIS
O
O
H6a
H6a
contacts), while the phenyl ring displays pep stacking with Tyr195.
H6b
O
H6b
N
N
In keeping with site-directed mutagenesis data and with the
computational results available in literature [16], all ligands display
a salt bridge between protonated piperazine nitrogen and Asp116
O
N
N
O
H
O
H
oH4
side chain and are engaged in Van deer Waals contacts or
pep
oH4
H7b
8 CIS
H7b
H7a
8 TRANS
stacking with the residues Ala93, Ala94, Tyr96, Asn386 and Trp387.
H-bond contacts with Thr121 or Lys191 seem to determine the
compounds agonist or neutral antagonist behaviour. Accordingly,
compounds 3e7 do not display H-bond with Lys191 since the
nitrogen of the compounds 5e7 imide moiety displays a lower
electron density in comparison with those of compounds 2e4
(bearing a lactam group on the 1,3-dioxolane ring).
H7a
O
H6a
O
H6a
H6b
O
H6b
O
N
N
The information obtained by the molecular docking studies
provide useful suggestions for the synthesis of new 1,3-dioxolane
derivatives bearing a lactam or a imide moiety as potential 5HT1A
neutral antagonist and agonist. Regarding the first series of
compounds, the orto and para position of the phenyl ring on the
dioxolane ring could be exploited to establish H-bond contacts with
O
O
N
N
O
O
H
oH4
H
oH4
H7b
H7b
H7a
11 TRANS
H7a
11 CIS
Chart 1.

S. Franchini et al. / European Journal of Medicinal Chemistry 45 (2010) 3740e3751
3743
Fig. 2. 5HT1A model (yellow) backbone conformation superimposed on the X-ray coordinates of 2RH1 (green). The carazolol X-ray structure is reported in ball and stick, coloured by
atom-type (a). Distribution of the hydrophobic (brown) and hydrophilic (cyan) properties on the 5HT1A model surface. The receptor transmembrane domain, the portions extended
towards the extra-cellular (EC) or the intra-cellular (IC) environments are delimited by grey lines (b). (For interpretation of the references to colour in this figure legend, the reader is
referred to the web version of this article.)
Ser199 and Tyr195 by introducing a H-bond acceptor group. In
addition, a naphthyl moiety on the piperazine N1 might allow the
beneficial for additional Van deer Waals and pep stacking with
residues Ala93, Ala94, Asn386 and Tyr96, Tyr390, Trp387.
establishment of
pep stacking with the aromatic residues Tyr96,
Tyr390, Trp387. As concern the 1,3-dioxolane derivatives bearing
a imide moiety, a bulky group on the imide ring would seem
particularly favourable detecting hydrophobic contacts with
Leu173, Ile189, Ser190 and Lys191. Instead of a phenyl ring, the
introduction of a naphthyl moiety on the dioxolane moiety could be
2.3. Pharmacological activity
The pharmacological profile of compounds 1, 2e7c/t, and BMY-
3748, 8-OH-DPAT, WAY100635 as reference compounds, was evalu-
ated by radioligand binding assay using [3H]prazosin to label cloned
Fig. 3. Selected docking pose of compound 2 S,S isomer (as representative 5HT1A agonist) into the 5HT1A putative binding site, H-bond interactions are depicted in black. The ligand
is reported in stick and coloured by atom-type (C, yellow) (a). Selected docking pose of compound 2 S,R isomer (as representative 5HT1A neutral antagonist) into the 5HT1A putative
binding site, H-bond interactions are depicted in black. The ligand is reported in stick and coloured by atom-type (C, green) (b). (For interpretation of the references to colour in this
figure legend, the reader is referred to the web version of this article.)

3744
S. Franchini et al. / European Journal of Medicinal Chemistry 45 (2010) 3740e3751
human a1-adrenoceptors expressed in CHO cells, and [3H]8-OH-DPAT
to label cloned human 5-HT1A receptor expressed in HeLa cells [24].
An examination of the results reported in Table 1 shows that at
Table 2
Antagonist potency, expressed as pKb
compounds 1, 2-7c/t and BMY-7378 at a1 adrenoceptors in Isolated Prostatic Vas
Deferens (a1A), Spleen (a1B), and Thoracic Aorta (a1D) of rats.
ꢁ
SEM values,a and selectivitiesb of
a1-adrenoceptor subtypes,
a general reduction in affinity is
Compd
pKba1A
pKba1B
pKba1D
a1D/a1A
a1D
/a1B
a1A/a1B
observed compared to the lead compound 1. Conversely, at 5-HT1A
receptor, an increase in affinity is seen, with few exceptions
(compounds 5t and 6t). This results in a decrease of a1-adreno-
ceptor selectivity.
By examining each couple of diastereomeric pairs, it is evident
that stereochemistry plays a significant role. In all cases, the cis pair
is more potent at a1a subtype, whereas the counterpart, the trans
form, is more potent at a1d subtype.
1
2c
2t
3c
3t
4c
4t
5c
5t
6c
6t
7c
8.24
7.94
7.34
7.30
7.32
8.97
7.18
7.45
7.32
7.09
7.36
7.72
7.29
7.01
6.69
8.29
8.17
7.93
8.30
8.42
8.08
7.04
7.11
7.12
7.35
7.40
7.47
7.48
8.14
6.79
7.43
6.74
7.29
7.57
7.75
6.42
7.78
6.19
7.57
6.78
8.51
8.40
0.8
0.07
1
0.3
1
0.04
4
0.1
3
28
36
0.03
0.1
0.06
0.1
0.1
0.5
0.2
5
0.5
0.2
0.3
0.1
4
0.1
3
2
At a1b subtype the affinities, with the exception of 7t, are signif-
icantly lower than those found for the other subtypes. However
within the lactam series the trans isomer, similarly to the case
observed for the a1d subtype, is more active than the cis isomer,
whereas for the imide series the opposite is true for compounds 5
and 6. In the case of compound 7 cis and trans isomers are equiactive.
At 5-HT1A receptor the diastereoselectivity seems less important
or not important at all. The two diastereomers show similar affin-
ities to one another, the exception being imide derivative 6, for
which the diastereoselectivity ratio is about 14-fold, with the cis
isomer showing higher affinity with respect to the trans isomer.
Functional a1-adrenoceptor subtype selectivity was also deter-
mined ondifferentisolated tissues. Blockingactivity was assessed by
antagonism of (ꢀ)-noradrenaline-induced contraction of the pros-
tatic vas deferens (a1A) [25] or thoracic aorta (a1D)[26] in rats and by
antagonism of (ꢀ)-phenylephrine-induced contraction of rat spleen
0.1
2
0.1
2
1
1
0.1
0.2
3
7t
17
25
11
8
0.7
0.3
BMY-7378
a
pKb values were calculated according to van Rossum [38] at one or two
concentrations and agreed within 2%.
b
Antilog of the difference between the pKb values for a1A, a1B and a1D-
adrenoceptors.
they are consistent with a selectivity trend toward a1B subtype. In
this respect the best compound is compound 3t with selectivity
ratios a1B/a1A and a1B/a1D of about 10-fold. This is quite an inter-
esting discovery, since at present few a1B subtype antagonist are
known and the discovery of new and more selective ligands would
help to clearly define the role of a1B subtypes. For this purpose, the
lactam derivatives could be a new starting point.
(a1B) [27]. The antagonist potencies at a1-adrenoceptor subtypes are
Functional characterization at 5-HT1A receptor was performed
reported in Table 2. At a1A- and a1D- subtypes, with few exceptions,
the trend of activities parallel the affinities found in binding studies
as the potencies are lower than the lead compound 1. The exceptions
are compounds 4c and 7t, for which an enhancement of potency is
seen, for the former at a1A subtype (pKb ¼ 8.97) and for the latter at
a1D subtype (pKb ¼ 8.51). The diastereoselectivity trend is also
confirmed, the only exception being compounds 3 at a1A subtype,
where the trans and the cis form are equiactive.
Surprisingly, at a1B subtype enhancement of antagonist potency
is seen with respect to the lead compound 1. This enhancement is
greater for the lactam series (compounds 2e4) than for the imide
series, and with the exception of the derivative 4c the antagonist
potencies are higher than those found for both a1A and a1D
subtypes. Though the differences in potency are not considerable,
according to methods of Stanton and Beer [28], using [35S]GTP
gS
binding, in cell membrane from HeLa cells transfected with human
cloned 5-HT1A receptor. Stimulation of [35S]GTP
gS binding was
expressed as percent increase in binding above the basal value,
being the maximal stimulation observed with serotonin taken as
100%. The results are reported in Table 3. Lactam derivatives 2t and
4t and all the imide derivatives tested, 5c, 6c, 7c and 7t maintain the
partial agonist properties of the lead compound 1. The potencies,
expressed as pD2, are lower than that of compound 1, but in all
cases, efficacy (% Emax) is consistently higher. The best result is
obtained with compounds 7c and 7t, which show pD2 values of 7.80
and 7.98 with %Emax of 49 and 60, respectively.
On the contrary lactam derivatives 2c, 3c, 3t and 4c do not affect
the binding of [35S]GTP
gS, behaving therefore as neutral antagonists
(n.a.). Theywerethenstudied intheantagonistexperimentsdesigned
toevaluatetheirpotency. They behave ascompetitiveantagonist with
Table 1
Affinity constants, expressed as pKi valuesa and selectivitiesb of compounds 1,
2e7c/t, BMY-7378 and WAY10063 for human Recombinant
a1eAdrenoceptor
Subtypes and 5-HT1A receptors.
Table 3
Compds
pKia1a pKia1b pKia1d pKi 5-HT1A a1d
/
a1a a1d
/
a1b a1a
/
a1b 5-HT1A/a1
Agonist potency (pD2), relative effectiveness (Emax
)
a and antagonist potency (pKb) in
gS-binding assay at human 5-HT1A receptor.
1
9.58 8.17 9.09 7.64
8.86 6.76 7.87 8.16
7.90 7.15 8.28 7.95
9.06 6.89 7.85 8.28
7.94 7.29 8.83 8.29
9.20 7.14 8.39 8.25
7.57 7.74 8.60 8.14
8.54 7.07 7.40 8.29
7.39 6.65 7.79 7.59
8.18 7.33 7.49 8.50
7.67 6.88 8.07 7.35
8.69 8.03 8.32 8.58
8.00 8.00 8.86 8.90
0.3
0.1
2
0.1
8
0.2
11
0.1
3
0.2
3
8
13
14
9
35
18
7
2
14
2
15
2
7
550
14
26
126
6
148
0.9
115
0.7
30
6
7
0.01
0.2
0.5
the agonist-induced [35S]GTP
2c
2t
3c
3t
4c
4t
5c
5t
6c
6t
7c
7t
Compd
pD2
Emax
pKb
0.2
1
2c
2t
3c
3t
4c
4t
5c
8.30
n.a.b
7.14
n.a.b
n.a.b
n.a.b
6.57
7.05
7.23
7.80
7.98
9.27
7.83
n.a.b
16
e
e
0.3
0.2
0.4
0.6
0.6
2
0.2
0.8
1
7.16
e
34
e
7.38
7.39
7.25
e
e
e
39
45
34
49
60
26
100
e
6
5
1
2
e
0.4
7
6c
7c
7t
e
e
BMY-7378 6.42 6.15 8.89 8.90
WAY10063 7.73 7.18 8.34 9.48
295
4
1
14
e
3
BMY-7378
8-OH-DPTA
WAY100635
e
e
a
Ki values were derived from the ChengePrusoff equation [37] at one or two
9.33
concentrations and agreed within 10%.
b
a
Antilog of the difference between the pKi values for a1a
,
a1b,
a1d-adrenoceptors
Maximal stimulation expressed as a percentage of the maximal 5-HT response.
n.a.: neutral antagonist.
b
and 5-HT1A receptor.

S. Franchini et al. / European Journal of Medicinal Chemistry 45 (2010) 3740e3751
3745
moderate pKb values of 7.16, 7.38 7.39, and 7.25, respectively. Given
3.1.6. 1-(4-Chloromethyl-2-phenyl-[1,3]dioxolan-2-ylmethyl)-
pyrrolidine-2,5-dione (17)
the well known difficulty in discovering new antagonists for 5eHT1A
receptor, as antagonist activity would not appear to be conferred to
a given compound by a clear structural element (or elements), this
result is quite interesting, and it is tempting to speculate that a lactam
moiety, depending probably on stereochemistry (relative orientation
with respect to the piperazine basic portion) may do so.
In conclusion, replacement of one phenyl ring in 1 with lactam
or imide moiety, although detrimental for a1/5-HT1A selectivity,
favours antagonism at a1B-adrenoceptor subtype and 5-HT1A
receptor (lactams) and increases agonism efficacy (imides).
To a solution of 22 (4.56 g, 21 mmol) in 100 mL of toluene, (ꢁ)-3-
chloro-l,2-propanediol (2.68 mL, 31.5 mmol) and p-toluensulfonic
acid (0.88 g,4.6 mmol) were added. The reaction was heated at
reflux for 24 h with the use of a DeaneStark apparatus. After
cooling to room temperature the reaction mixture was washed
with NaHCO3 (saturated solution) (2 ꢂ 100 mL) and brine
(1 ꢂ70 mL) and dried over Na2SO4. Evaporation of the solvent gives
a residue that was purified on a silica gel column (cyclohexane :
ethyl acetate, 8.5:1.5), to give 1.83g (28.2%, 33% BORSM) of 17 as
diastereomeric pairs, 68/32 [(Z) pair]/[(E) pair].
[(Z) pair: SS and RR isomers] 17c. 1H NMR (200 MHz, CDCl3)
d 2.70
3. Experimental
(s, 4H, CH2-3 þ CH2-4), 3.54 (dd, J ¼ 3.7, 11.6, 1H, H7a), 3.63 (dd,
J ¼ 5.2,11.6,1H, H7b), 3.76 (dd, J ¼ 7.3, 8.4,1H, H5a), 3.89 (d, J ¼ 14.4,
H6a), 3.96 (d, J ¼ 14.4, H6b), 3.97 (dd, ¼ 4.3, 8.4, 1H, H5b), 4.22 (m,
1H, H4), 7.38 (m, 3H, H-arom), 7.56 (m, 2H, H-arom).
3.1. Chemistry
3.1.1. General procedures
[(E) pair: SR and RS isomers] 17t. 1H NMR (200 MHz, CDCl3)
d 2.66
Melting points were determined on a Büchi 510 capillary
melting point apparatus and are uncorrected. The microanalyses
were performed on a Carlo Erba 1106 Analyzer in the Microana-
lytical Laboratory of Dipartimento di Scienze Farmaceutiche of
Università degli Studi di Modena e Reggio Emilia and where indi-
cated by symbols, the results are within ꢁ0.4% of the theoretical
values. NMR (1H and 13C) spectra were recorded on Bruker FT NMR
DPX 200 and Avance400 spectrometers at a temperature of 300 K.
(s, 4H, CH2-3 þ CH2-4), 3.09 (dd, J ¼ 9.7, 10.7, 1H, H7a), 3.57 (dd,
J ¼ 4.5,10.7,1H, H7b), 3.76 (dd, J ¼ 6.1, 8.3,1H, H5a), 3.91 (s, 2H, CH2-
6), 4.32 (dd, J ¼ 6.2, 8.3, 1H, H5b), 4.39 (m, 1H, H4), 7.35 (m, 3H, H-
arom), 7.51 (m, 2H, H-arom).
3.1.7. General procedure for the preparation of compounds 20e21
To a solution of 23 or 24 (5.8 mmol) in anhydrous methanol
(10 mL), trimethyl orthoformate (6.35 mL, 58 mmol) (in two steps
portion) and p-toluensulfonic acid (0.165 g, 0.87 mmol) were
added. The reaction was refluxed under nitrogen for 23 h. After
cooling to room temperature the solvent was removed by evapo-
ration and the crude, dissolved in ethyl acetate, was washed with
Na2CO3 5% (2 ꢂ 100 mL) and brine (1 ꢂ 70 mL) and dried over
Na2SO4. Evaporation of the solvent gave the title compounds which
were used in the next step without further purification.
Chemical shifts are reported as
d (ppm) relative to tetramethylsi-
lane (s ¼ singlet, br s ¼ broad singlet, d ¼ doublet, dd ¼ double
doublet, quint ¼ quintept, t ¼ triplet, m ¼ multiplet). The obvious
signal assignments were made from 1H and 13C NMR spectra
according to the chemical shifts and multiplicities. Other remaining
1H and 13C signals and cis/trans stereochemistry were assigned with
the aid of 2D NMR spectra including 1H-1H COSY, HSQC, HMBC and
NOESY spectra acquired with standard pulse sequence. Proton
signals due to COOH and NHþ groups of oxalate salts 2e7 were not
detected. Silica gel TLC plates (Merck, Kieselgel 60, F254) were used
to monitor the progression of the reactions. Chromatographic
separations were performed on silica gel columns (Kieselgel 60,
0.040e0.063 mm, Merck) by flash chromatography. % Yield BORSM
are based on recovered starting material.
Analytical samples were purified on a silica gel column (cyclo-
hexane : ethyl acetate, 8.5:1.5) for 1H NMR characterization.
3.1.8. 1-(2,2-Dimethoxy-2-phenyl-ethyl)-piperidine-2,6-dione (20)
1H NMR (200 MHz, CDCl3)
d
1.63 (quint, J ¼ 6.4, 2H, CH2-40), 2.33
(t, J ¼ 6.4, 4H, CH2-30 þ CH2-50), 3.30 (s, 6H, OCH3), 4.29 (s, 2H, N-
CH2), 7.24e7.99 (m, 5H, H-arom).
3.1.2. General procedure for the preparation of compounds 22e24
To a solution of the appropriate anhydride (24 mmol) in 60 mL
of toluene, phenacylamine hydrochloride (4.12 g, 24 mmol) was
slowly added. The reaction was heated at reflux for 23 h with the
use of a DeaneStark apparatus. After cooling to room temperature
the solvent was removed by evaporation. The crude, dissolved in
ethyl acetate, was washed with NaOH 5% (2 ꢂ 100 mL) and brine
(1 ꢂ 100 mL) and dried over Na2SO4. Evaporation of the solvent
gave the title compounds as oils.
3.1.9. 8-(2,2-Dimethoxy-2-phenyl-ethyl)-8-aza-spiro[4.5]decane-
7,9-dione (21)
1H NMR (200 MHz, CDCl3)
d 1.70 (m, 8H, cyclopentane), 2.32 (s,
4H, CH2-3 þ CH2-5), 3.28 (s, 6H, OCH3), 4.29 (s, 2H, N-CH2),
7.25e7.98 (m, 5H, H-arom).
3.1.10. General procedure for the preparation of compounds 18e19
To the solution of 20 or 21 (1.0 mmol) in CH3CN (13 mL), CoCl2
(0.078 g, 0.6 mmol) and (ꢁ)-3-chloro-l,2-propanediol (0.17 mL,
2 mmol) and trimethylchlorosylane (0.13 mL, 1 mmol) were added.
The reaction was stirred at room temperature for 21 h. The solvent
was removed by evaporation. The crude, dissolved in chloroform,
was washed with Na2CO3 5% (2 ꢂ 100 mL) and brine (1 ꢂ 100 mL)
and dried over Na2SO4. Evaporation of the solvent gives the title
compounds that were purified and separated into the respective
diastereomeric pairs on a silica gel column (cyclohexane: ethyl
acetate, 6:4 and 9:1 respectively).
3.1.3. 1-(2-Oxo-2-phenyl-ethyl)-pyrrolidine-2,5-dione (22)
Yield 74%. 1H NMR (200 MHz, CDCl3)
4), 4.96 (s, 2H, N-CH2), 7.57 (m, 3H, H-arom), 8.01 (m, 2H, H-arom).
d
2.87 (s, 4H, CH2-3 þ CH2-
3.1.4. 1-(2-Oxo-2-phenyl-ethyl)-piperidine-2,6-dione (23)
Yield 64%. 1H NMR (200 MHz, CDCl3)
d
2.07 (quint, J ¼ 6.5, 2H,
CH2-40), 2.33 (t, J ¼ 6.5, 4H, CH2-30 þ CH2-50), 5.23 (s, 2H, N-CH2),
7.55 (m, 3H, H-arom), 7.97 (m, 2H, H-arom).
3.1.11. 1-(4-Chloromethyl-2-phenyl-[1,3]dioxolan-2-ylmethyl)-
piperidine-2,6-dione (18)
3.1.5. 8-(2-Oxo-2-phenyl-ethyl)-8-aza-spiro[4.5]decane-7,9-dione
(24)
Yield 0.607 g (29%, 36% BORSM), 63/37 [(Z) pair]/[(E) pair].
Red oil Yield 58%. 1H NMR (200 MHz, CDCl3)
d
1.70 (m, 8H, H-
[(Z) pair: SS and RR isomers] 18c. 1H NMR (200 MHz, CDCl3)
d 1.87
cyclopentane), 2.70 (s, 4H, CH2-3 þ CH2-5), 5.23 (s, 2H, N-CH2), 7.55
(quint, J ¼ 6.5, 2H, CH2-40), 2.62 (t, J ¼ 6.5, 4H, CH2-30 þ CH2-50), 3.56
(d, J ¼ 5.1, 2H, CH2-7), 3.74 (dd, J ¼ 7.2, 8.4,1H, H5a), 3.92 (dd, J ¼ 4.4,
(m, 3H, H-arom), 7.98 (m, 2H, H-arom).

3746
S. Franchini et al. / European Journal of Medicinal Chemistry 45 (2010) 3740e3751
8.4, 1H, H5b), 4.15 (m, 1H, H4), 4.29 (s, 2H, CH2-6), 7.33 (m, 3H, H-
arom), 7.50 (m, 2H, H-arom).
8t 1H NMR (200 MHz, CDCl3) 1.92 (m, 2H, CH2-40), 2.27 (m, 2H,
d
CH2-30), 2.44 (dd, J ¼ 4.8, 13.1, 1H, H7a), 2.54 (dd, J ¼ 6.4, 13.1, 1H,
H7b), 2.68 (m, 2H, H piperazine), 2.83 (m, 2H, H piperazine), 3.08
(m, 4H, H piperazine), 3.43 (m, 3H, H5a þ CH2-50), 3.59 (d, J ¼ 14.3,
1H, H6a), 3.69 (d, J ¼ 14.3, 1H, H6b), 3.86 (s, 3H, OCH3), 4.27 (dd,
J ¼ 6.3, 8.3, 1H, H5b), 4.46 (m, 1H, H4), 6.93 (m, 4H, H-arom), 7.32
[(E) pair: SR and RS isomers] 18t. 1H NMR (200 MHz, CDCl3)
d 1.84
(quint, J ¼ 6.5, 2H, CH2-40), 2.58 (t, J ¼ 6.5, 4H, CH2-30 þ CH2-50), 3.07
(dd, J ¼ 8.9, 10.7, 1H, H7a), 3.56 (dd, J ¼ 4.8, 10.7, 1H, H7b), 3.67 (dd,
J ¼ 5.9, 8.2, 1H, H5a), 4.29 (s, 2H, CH2-6), 4.31 (m, 1H, H4), 4.34 (dd,
J ¼ 5.1, 8.2, 1H, H5b), 7.34 (m, 3H, H-arom), 7.51 (m, 2H, H-arom).
(m, 3H, H-arom), 7.53 (m, 2H, H-arom). 13C NMR (CDCl3):
d 18.47(C-
40), 30.61 (C-30), 48.85 (C-50), 50.16 (C-6), 50.40 (C-2, C-6 pipera-
zine), 53.91 (C-3, C-5 piperazine), 55.39 (OCH3), 61.01 (C-7), 68.95
(C-5), 75.95 (C-4), 110.15 (C-2), 111.22 (C-6 methoxyphenyl), 118.25
(C-3 methoxyphenyl), 121.02 (C-4 methoxyphenyl), 123.05 (C-5
methoxyphenyl), 125.94 (C-2,C-6phenyl), 128.11 (C-3, C-5 phenyl),
128.44 (C-4 phenyl), 141.10 (C-1 phenyl), 141.17 (C-1 methox-
yphenyl), 152.29 (C-2 methoxyphenyl), 175.42 (CO).
3.1.12. 8-(4-Chloromethyl-2-phenyl-[1,3]dioxolan-2-ylmethyl)-8-
aza-spiro[4.5]decane-7,9-dione (19)
Yield 0.350 g (9%, 14% BORSM), 47/53 [(Z) pair]/[(E) pair]
[(Z) pair: SS and RR isomers] 19c. 1H NMR (200 MHz, CDCl3)
d 1.48
(m, 4H, CH2-10 þ CH2-40), 1.71 (m, 4H, CH2-20 þ CH2-30), 2.60 (s, 4H,
CH2-60 þ CH2-100), 3.53 (dd, J ¼ 6.2, 10.9, 1H, H7a), 3.57 (dd, J ¼ 4.0,
10.9, 1H, H7b), 3.77 (dd, J ¼ 7.2, 8.5, 1H, H5a), 3.92 (dd, J ¼ 4.6, 8.5,
1H, H5b), 4.16 (m, 1H, H4), 4.31 (s, 2H, CH2-6), 7.37 (m, 3H, H-arom),
7.55 (m, 2H, H-arom).
The free amine was transformed into the corresponding oxalate
salt which was crystallized from methanol/diethyl ether to give
compound 2t: m.p. 155e158 ꢃC; 1H NMR (200 MHz, DMSO-d6):
[(E) pair: SR and RS isomers] 19t. 1H NMR (200 MHz, CDCl3)
d
1.50
d
1.82 (m, 2H, CH2-40), 2.09 (m, 2H, CH2-30), 2.71 (dd, J ¼ 6.9, 13.2,
(m, 4H, CH2-10 þ CH2-40), 1.71 (m, 4H, CH2-20 þ CH2-30), 2.57 (s, 4H,
CH2-60 þ CH2-100), 3.01 (dd, J ¼ 9.1, 10.6, 1H, H7a), 3.57 (dd, J ¼ 4.9,
10.6, 1H, H7b), 3.64 (dd, J ¼ 6.1, 8.2, 1H, H5a), 4.26 (s, 2H, CH2-6),
4.33 (dd, J ¼ 6.1, y13.11, 1H, H5b), 4.33 (m, 1H, H4), 7.35 (m, 3H, H-
arom), 7.52 (m, 2H, H-arom).
1H, H7a), 2.82 (dd, J ¼ 4.1, 13.2, 1H, H7b), 2.87e3.00 (m, 4H, H
piperazine), 3.05 (m, 4H, H piperazine), 3.39 (m, 3H, H5a þ CH2-50),
3.52 (s, 2H, H6), 3.77 (s, 3H, OCH3), 4.28 (dd, J ¼ 6.2, 8.3, 1H, H5b),
4.53 (m, 1H, H4), 6.92 (m, 4H, H-arom), 7.39 (m, 5H, H-arom).
C28H35N3O8. Anal. (C, H, N).
3.1.13. General procedure for the synthesis of amines 8e13 and
their oxalate salts 2e7 (Scheme 2)
3.1.15. 1-{4-[4-(2-methoxy-phenyl)-piperazin-1-ylmethyl]-2-
phenyl-[1,3]dioxolan-2-ylmethyl}-piperidin-2-one (9c,t) and
oxalate salts 3c,t
0.61 g (1.97 mmol) of 15c,t gives 0.758 g of 9: 0.584 g (63.5%) of
[(Z) pair: SR and RS isomers] 9c and 0.174 g (18.9%) of [(E) pair: SS
and RR isomers] 9t. Flash chromatography: ethyl acetate / ethyl
acetate/methanol, 99:1.
A solution of the appropriate chloromethyl derivative (14-19) in
2-methoxy-ethanol with a large excess (5e10 equiv) of 1-(2-
methoxy-phenyl)-piperazine and with a catalytic amount of KI was
refluxed for 18e24 h. The solvent was evaporated under vacuum.
CHCl3 was added, and the residue was washed with a solution of 5%
NaOH (3ꢂ) and then with brine (2ꢂ). The organic layer was dried
over Na2SO4 and the solvent was evaporated under vacuum to give
the desired amine as oil. Purification and diastereomeric separations
were accomplished by flash chromatography. The free amines were
quantitatively transformed into the corresponding oxalate salts.
9c 1H NMR (400 MHz, CDCl3)
d
1.76 (m, 4H, CH2-40 þ CH2-50),
2.31 (m, 2H, CH2-30), 2.65 (dd, J ¼ 5.4, 12.9, 1H, H7a), 2.72 (m, 2H, H-
piperazine), 2.77 (dd, J ¼ 6.3, 12.9, 1H, H7b), 2.86 (m, 2H, H-piper-
azine), 3.23 (m, 4H, H-piperazine), 3.43 (m, 1H, H60a); , 3.55 (m, 1H,
H60b), 3.77 (dd, J ¼ 6.5, 7.9, 1H, H5a), 3.81 (d, J ¼ 14.9, 1H, H6a), 3.90
(s, 3H, OCH3), 3.92 (d, J ¼ 14.9, 1H, H6b), 3.94 (dd, J ¼ 7.8, 7.9, 1H,
H5b), 4.21 (m, 1H, H4), 6.94 (m, 4H, H-arom), 7.33 (m, 3H, H-arom),
3.1.14. 1-{4-[4-(2-methoxy-phenyl)-piperazin-1-ylmethyl]-2-
phenyl-[1,3]dioxolan-2-ylmethyl}-pyrrolidin-2-one (8c,t) and
oxalate salts 2c,t
7.51 (m, 2H, H-arom). 13C NMR (400 MHz, CDCl3):
d
21.45 (C-40),
23.46 (C-50), 32.26 (C-30), 49.63 (C-60), 50.59 (C-2, C-6 piperazine),
52.88 (C-6), 54.07 (C-3, C-5 piperazine), 55.39 (OCH3), 61.53 (C-7),
68.74 (C-5), 73.84 (C-4), 110.74 (C-2), 111.24 (C-6 methoxyphenyl),
118.23 (C-3 methoxyphenyl), 121.02 (C-4 methoxyphenyl), 122.97
(C-5 methoxyphenyl), 126.09 (C-2,C-6phenyl), 128.03 (C-3, C-5
phenyl), 128.35 (C-4 phenyl), 140.36 (C-1 phenyl), 141.29 (C-1
methoxyphenyl), 152.30 (C-2 methoxyphenyl), 169.94 (CO).
The free amine was transformed into the corresponding oxalate
salt which was crystallized from methanol/diethyl ether to give
compound 3c: m.p. 126e129 ꢃC; 1H NMR (200 MHz, DMSO-d6)
1 g (3.38 mmol) of 14c,t gives 1.01 g of 8: 0.78 g (51.4%) of [(Z)
pair: SR and RS isomers] 8c and 0.23 g (15.1%) of [(E) pair: SS and RR
isomers] 8t. Flash chromatography: ethyl acetate / ethyl acetate/
methanol, 98:2.
8c 1H NMR (200 MHz, CDCl3) 1.96 (m, 2H, CH2-40), 2.25 (m, 2H,
d
CH2-30), 2.62 (dd, J ¼ 5.3, 13.0, 1H, H7a), 2.74 (dd, J ¼ 6.2, 13.0, 1H,
H7b), 2.75 (m, 4H, H piperazine), 3.09 (m, 4H, H-piperazine), 3.51
(m, 2H, CH2-50), 3.65 (d, J ¼ 14.5,1H, H6a), 3.70 (d, J ¼ 14.5,1H, H6b),
3.74 (dd, J ¼ 6.4, 7.8, 1H, H5a), 3.87 (s, 3H, OCH3), 3.94 (m, 1H, H5b),
4.22 (m, 1H, H4), 6.94 (m, 4H, H-arom), 7.31 (m, 3H, H-arom), 7.52
d
1.62 (m, 4H, CH2-40 þ CH2-50), 2.14 (m, 2H, CH2-30), 3.20 (m, 12H,
(m, 2H, H-arom). 13C NMR (400 MHz, CDCl3):
d
18.45 (C-40), 30.62
8H-piperazine þ CH2-7 þ CH2-60), 3.54 (s, 2H, CH2-6), 3.77 (m, 2H,
CH2-5), 3.79 (s, 3H, OCH3), 4.32 (m, 1H, H4), 6.93 (m, 4H, H-arom),
7.40 (m, 5H, H-arom). C29H37N3O8. Anal. (C, H, N).
(C-30), 48.94 (C-50), 49.88 (C-6), 50.52 (C-2, C-6 piperazine), 54.03
(C-3, C-5 piperazine), 55.39 (OCH3), 61.37 (C-7), 68.83 (C-5), 74.05
(C-4), 110.24 (C-2), 111.24 (C-6 methoxyphenyl), 118.24 (C-3
methoxyphenyl), 121.02 (C-4 methoxyphenyl), 123.01 (C-5
methoxyphenyl), 125.96 (C-2, C-6phenyl), 128.20 (C-3, C-5 phenyl),
128.51 (C-4 phenyl), 140.15 (C-1 phenyl), 141.23 (C-1 methox-
yphenyl), 152.30 (C-2 methoxyphenyl), 175.29 (CO).
9t 1H NMR (400 MHz, CDCl3)
d
1.74 (m, 4H, CH2-40 þ CH2-50),
2.29 (m, 2H, CH2-30), 2.43 (dd, J ¼ 4.4, 13.0, 1H, H7a), 2.56 (dd,
J ¼ 6.7, 13.0, 1H, H7b), 2.68 (m, 2H, H-piperazine), 2.86 (m, 2H, H-
piperazine), 3.12 (m, 4H, H-piperazine), 3.36 (m, 1H, H60a), 3.46 (m,
1H, H60b), 3.47 (pseudot, J ¼ 8.3,1H, H5a), 3.72 (d, J ¼ 14.2,1H, H6a),
3.90 (s, 3H, OCH3), 3.95 (d, J ¼ 14.2, 1H, H6b), 4.30 (dd, J ¼ 6.3, 8.3,
1H, H5b), 4.48 (m, 1H, H4), 6.92 (m, 4H, H-arom), 7.33 (m, 3H, H-
The free amine was transformed into the corresponding oxalate
salt which was crystallized from methanol/diethyl ether to give
compound 2c: m.p. 144e148 ꢃC; 1H NMR (200 MHz, DMSO-d6)
arom), 7.51 (m, 2H, H-arom). 13C NMR (400 MHz, CDCl3):
d 21.39 (C-
d
1.85 (m, 2H, CH2-40), 2.13 (m, 2H, CH2-30), 3.15 (m, 10H, 8H-
40), 23.45 (C-50), 32.25 (C-30), 49.52 (C-60), 50.56 (C-2, C-6 pipera-
zine), 53.26 (C-6), 53.99 (C-3, C-5 piperazine), 55.39 (OCH3), 61.16
(C-7), 68.97 (C-5), 75.91 (C-4), 110.59 (C-2), 111.22 (C-6 methox-
piperazine þ CH2-7), 3.37 (m, 2H, CH2-50), 3.58 (s, 2H, CH2-6), 3.82
(m, 2H, CH2-5), 3.87 (s, 3H, OCH3), 4.31 (m, 1H, H4), 6.92 (m, 4H, H-
arom), 7.36 (m, 5H, H-arom). C28H35N3O8. Anal. (C, H, N).
yphenyl),
118.23
(C-3
methoxyphenyl),
121.01
(C-4

S. Franchini et al. / European Journal of Medicinal Chemistry 45 (2010) 3740e3751
3747
methoxyphenyl), 122.97 (C-5 methoxyphenyl), 126.09 (C-2,C-6
phenyl), 127.91 (C-3, C-5 phenyl), 128.26 (C-4 phenyl), 141.29 (C-1
methoxyphenyl), 141.37 (C-1 phenyl), 152.31 (C-2 methoxyphenyl),
170.02(CO).
H-piperazine), 3.35 (pseudot, 1H, J ¼ 8.2, H5a), 3.39 (m, 2H, CH2-70),
3.65 (d, J ¼ 14.1, 1H, H6a), 3.75 (d, J ¼ 14.1, 1H, H6b), 3.77 (s, 3H,
OCH3); 4.26 (dd, dd, J ¼ 6.1, 8.2, 1H, H5b); 4.54 (m, 1H, H4), 6.92 (m,
4H, H-arom), 7.36 (m, 5H, H-arom). C30H39N3O8. Anal. (C, H, N).
The free amine was transformed into the corresponding oxalate
salt which was crystallized from methanol/diethyl ether to give
3.1.17. [(Z) pair: SR and RS isomers] 1-{4-[4-(2-methoxy-phenyl)-
piperazin-1-ylmethyl]-2-phenyl-[1,3]dioxolan-2-ylmethyl}-
pyrrolidine-2,5-dione (11c) and oxalate salt 5c
compound 3t: m.p. 165e168 ꢃC 1H NMR (200 MHz, DMSO-d6)
d 1.61
(m, 4H, CH2-40 þ CH2-50), 2.08 (m, 2H, CH2-30), 2.73 (dd, J ¼ 7.1, 13.5,
1H, H7a), 2.81e3.08 (m, 10H, 8H-piperazine þ H7b), 3.30 (m, 2H,
CH2-60), 3.36 (pseudot, J ¼ 8.3, 1H, H5a), 3.67 (d, J ¼ 14.2.1H, H6a),
3.75 (d, J ¼ 14.2.1H, H6b), 3.78 (s, 3H, OCH3), 4.28 (dd, J ¼ 6.2, 8.3,
1H, H5b), 4.54 (m, 1H, H4), 6.92 (m, 4H, H arom), 7.37 (m, 5H, H
arom). C29H37N3O8. Anal. (C, H, N).
0.360 g (1.16 mmol) of 17c gives 0.352 g (64.9%) of 11c. Flash
chromatography: cyclohexane/ethyl acetate 40:60. 1H NMR
(200 MHz, CDCl3)
d
2.37 (m, 2H, H-piperazine), 2.49 (dd, J ¼ 5.6,
12.9, 1H, H7a), 2.64 (dd, J ¼ 5.9, 12.9, 1H, H7b), 2.68 (s, 4H, CH2-
30 þ CH2-40), 2.79 (m, 2H, H-piperazine), 3.08 (m, 4H, H-piperazine),
3.71 (dd, J ¼ 6.1, 7.9,1H, H5a), 3.87 (s, 3H, OCH3), 3.88 (dd,J ¼ 6.6, 7.9,
1H, H5b), 3.95 (s, 2H, CH2-6), 4.16 (m,1H, H4), 6.96 (m, 4H, H-arom),
7.38 (m, 3H, H-arom), 7.56 (m, 2H, H-arom). 13C NMR (400 MHz,
3.1.16. 1-4-[4-(2-methoxy-phenyl)-piperazin-1-ylmethyl]-2-
phenyl-[1,3]dioxolan-2-ylmethyl}-azepan-2-one (10c,t) and oxalate
salts 4c,t
1.00 g (3.06 mmol) of 16c,t gives 0.698 of 10: 0.356 g (24.4%) of
[(Z) pair: SR and RS isomers] 10c and 0.342 g (23.4%) of [(E) pair: SS
and RR isomers] 10t. Flash chromatography: ethyl acetate / ethyl
acetate/methanol, 95:5.
CDCl3):
d
28.12(C-30 þ C-40), 44.95 (C-6), 50.46 (C-2, C-6 pipera-
zine), 54.02 (C-3, C-5 piperazine), 55.39 (OCH3), 61.45 (C-7), 68.89
(C-4), 73.88 (C-5), 109.39 (C-2), 111.20 (C-6 methoxyphenyl), 118.26
(C-3 methoxyphenyl), 121.02 (C-4 methoxyphenyl), 123.03 (C-5
methoxyphenyl), 126.08 (C-2,C-6 phenyl), 128.34 (C-3, C-5 phenyl),
128.79 (C-4 phenyl), 139.64 (C-1 phenyl), 141.17 (C-1 methoxy-
phenyl), 152.28 (C-2 methoxyphenyl), 176.36 (CO).
10c 1H NMR (200 MHz, CDCl3)
d
1.65 (m, 6H, CH2-40 þ CH2-
50 þ CH2-60), 2.45 (m, 2H, CH2-30), 2.59 (dd, J ¼ 5.3, 12.8, 1H, H7a),
2.67 (m, 4H, H-piperazine), 2.74 (dd, J ¼ 6.4, 12.8, 1H, H7b), 3.08 (m,
4H, H-piperazine), 3.52 (m, 2H, CH2-70), 3.71 (dd, J ¼ 6.7, 7.6, 1H,
H5a), 3.78 (d, J ¼ 14.5, 1H, H6a), 3.85 (d, J ¼ 14.5, 1H, H6b), 3.85 (s,
3H, OCH3), 3.91 (t, J ¼ 7.6, 1H, H5b), 4.16 (m, 1H, H4), 6.93 (m, 4H, H-
arom), 7.32 (m, 3H, H-arom), 7.50 (m, 2H, H-arom). 13C NMR
The free amine was transformed into the corresponding oxalate
salt which was crystallized from methanol/diethyl ether to give
compound 5c: m.p. 157e160 ꢃC; 1H NMR (200 MHz, DMSO-d6)
d
2.64 (s, 4H, CH2-30 þ CH2-40), 2.70e3.27 (m, 10H, 8H-piper-
azine þ CH2-7), 3.63 (m, 1H, H5a), 3.72 (s, 2H, CH2-6), 3.79 (s, 3H,
OCH3), 3.80 (m, 1H, H5b),4.24 (m, 1H, H4), 6.92 (m, 4H, H arom),
7.43 (m, 5H, H arom). C28H33N3O9.2H2O. Anal. (C, H, N).
(400 MHz, CDCl3): d
23.45 (C-40), 28.39 (C-50), 29.97 (C-60), 37.13 (C-
30), 50.61 (C-2, C-6 piperazine), 50.90 (C-70), 53.72 (C-6), 54.06 (C-3,
C-5 piperazine), 55.38 (OCH3), 61.44 (C-7), 68.77 (C-5), 73.90 (C-4),
110.62 (C-2), 111.25 (C-6 methoxyphenyl), 118.21 (C-3 methoxy-
phenyl), 121.02 (C-4 methoxyphenyl), 122.95 (C-5 methoxyphenyl),
126.17 (C-2,C-6phenyl), 128.04 (C-3, C-5 phenyl), 128.30 (C-4-
phenyl), 140.55 (C-1 phenyl), 141.30 (C-1 methoxyphenyl), 152.30
(C-2 methoxyphenyl), 176.01 (CO).
3.1.18. [(E) pair: SS and RR isomers] 1-{4-[4-(2-methoxy-phenyl)-
piperazin-1-ylmethyl]-2-phenyl-[1,3]dioxolan-2-ylmethyl}-
pyrrolidine-2,5-dione (11t) and oxalate salt 5t
0.493 g (1.59 mmol) of 17t gives 0.263 g (35.4%) of 11t. Flash
chromatography: cyclohexane/ethyl acetate 20:80. 1H NMR
The free amine was transformed into the corresponding oxalate
salt which was crystallized from methanol/tert-butylmethyl ether
to give compound 4c: m.p. 130e132 ꢃC; 1H NMR (200 MHz, DMSO-
(200 MHz, CDCl3)
d
2.41 (dd, J ¼ 4.7, 13.1, 1H, H7a), 2.53 (dd, J ¼ 6.4,
13.1,1H, H7b), 2.64 (m, 2H, H-piperazine), 2.67 (s, 4H, CH2-30 þ CH2-
40), 2.83 (m, 2H, H-piperazine), 3.10 (m, 4H, H-piperazine), 3.44 (t,
J ¼ 8.2, 1H, H5a), 3.87 (s, 3H, OCH3), 3.91 (s, 2H, CH2-6), 4.26 (dd,
J ¼ 6.2, 8.2, 1H, H5b), 4.41 (m, 1H, H4); 6.90 (m, 4H, H-arom), 7.35
(m, 3H, H-arom), 7.55 (m, 2H, H-arom). 13C NMR (400 MHz, CDCl3):
d6)
d
1.55 (m, 6H, CH2-40 þ CH2-50 þ CH2-60), 2.34 (m, 2H, CH2-30),
2.95e3.10 (m, 10H, 8H-piperazine þ CH2-7), 3.42 (m, 2H, CH2-70),
3.70 (d, J ¼ 14.3, 1H, H6a), 3.78(d, J ¼ 14.3, 1H, H6b), 3.78 (s, 3H,
OCH3), 4.25 (m, 1H, H4), 6.92 (m, 4H, H arom), 7.38 (m, 5H, H arom).
C30H39N3O8. Anal. (C, H, N).
d
28.11 (C-30 þ C-40), 45.46(C-6), 50.31 (C-2, C-6 piperazine), 53.90
(C-3, C-5 piperazine), 55.40 (OCH3), 61.48 (C-7), 68.93 (C-4), 71.18
(C-5), 109.72 (C-2), 111.18 (C-6 methoxyphenyl), 118.30 (C-3
methoxyphenyl), 121.03 (C-4 methoxyphenyl), 123.26 (C-5
methoxyphenyl), 125.99 (C-2,C-6 phenyl), 128.23 (C-3, C-5 phenyl),
128.71 (C-4 phenyl), 141.54 (C-1 methoxyphenyl), 141.57 (C-1
phenyl), 152.26 (C-2 methoxyphenyl), 176.53 (CO).
10t 1H NMR (200 MHz, CDCl3)
d
1.64 (m, 6H, CH2-40 þ CH2-
50 þ CH2-60) 2.38 (dd, J ¼ 4.6, 13.2, 1H, H7a), 2.47 (m, 2H, CH2-30),
2.53 (dd, J ¼ 6.7, 13.2, 1H, H7b), 2.67 (m, 2H, H-piperazine), 2.81 (m,
2H, H-piperazine), 3.09 (m, 4H, H-piperazine), 3.42 (pseudot, 1H,
J ¼ 8.3, H5a), 3.47 (m, 2H, CH2-70), 3.72 (d, J ¼ 14.4,1H, H6a), 3.84 (d,
J ¼ 14.4, 1H, H6b), 3.89 (s, 3H, OCH3), 4.26 (dd, ¼ 6.5, 8.3, 1H, H5b),
4.44 (m, 1H, H4), 6.95 (m, 4H, H-arom), 7.35 (m, 3H, H-arom), 7.54
The free amine was transformed into the corresponding oxalate
salt which was crystallized from acetone to give compound 5t: m.p.
(m, 2H, H-arom). 13C NMR (400 MHz, CDCl3):
d
23.46 (C-40), 28.36
158e162 ꢃC; 1H NMR (200 MHz, DMSO-d6)
d 2.62 (s, 4H, CH2-
(C-50), 29.96 (C-60), 37.19 (C-30), 50.60 (C-2, C-6 piperazine), 50.72
(C-70), 54.00 (C-3, C-5 piperazine þ C-6), 55.38 (OCH3), 61.27 (C-7),
68.93 (C-5), 76.05 (C-4), 110.55 (C-2), 111.22 (C-6 methoxyphenyl),
118.21 (C-3 methoxyphenyl), 121.01 (C-4 methoxyphenyl), 122.95
(C-5 methoxyphenyl), 126.21 (C-2,C-6phenyl), 128.05 (C-3, C-5
phenyl), 128.23 (C-4-phenyl), 141.31 (C-1 methoxyphenyl), 141.62
(C-1 phenyl), 152.31 (C-2 methoxyphenyl), 176.14 (CO).
30 þ CH2-40), 2.94 (m, 6H, 4H-piperazine þ CH2-7), 3.13 (m, 4H, H-
piperazine), 3.34 (m, 1H, H5a), 3.73 (s, 2H, CH2-6), 3.79 (s, 3H,
OCH3), 4.23 (m, 1H, H5b), 4.50 (m, 1H, H4), 6.92 (m, 4H, H-arom),
7.43 (m, 5H, H-arom). C28H33N3O9.H2O. Anal. (C, H, N).
3.1.19. [(Z) pair: SR and RS isomers] 1-{4-[4-(2-methoxy-phenyl)-
piperazin-1-ylmethyl]-2-phenyl-[1,3]dioxolan-2-ylmethyl}-
piperidine-2,6-dione (12c) and oxalate salt 6c
The free amine was transformed into the corresponding oxalate
salt which was crystallized from methanol/diethyl ether to give
compound 4t: m.p. 158e160 ꢃC; 1H NMR (200 MHz) (DMSO-d6):
0.270 g (0.84 mmol) of 18c gives 0.270 g (67.5%) of 12c. Flash
chromatography: ethyl acetate. 1H NMR (200 MHz, CDCl3)
d 1.87
d
1.41 (m, 6H, CH2-40 þ CH2-50 þ CH2-60), 2.31 (m, 2H, CH2-30) 2.71
(quint, J ¼ 6.4, 2H, CH2-40), 2.55 (dd, J ¼ 5.7, 12.8, 1H, H7a), 2.62 (t,
J ¼ 6.4, 4H, CH2-30 þ CH2-50), 2.62 (m, 2H, H-piperazine), 2.66 (dd,
J ¼ 6.2, 12.8, 1H, H7b), 2.83 (m, 2H, H-piperazine), 3.09 (m, 4H, H-
(dd, J ¼ 7.2, 13.2, 1H, H7a), 2.83 (dd, J ¼ 3.2, 13.2, 1H, H7b), 2.88 (m,
2H, H-piperazine), 3.02 (m, 2H, H-piperazine), 3.06 (m, 4H,

3748
S. Franchini et al. / European Journal of Medicinal Chemistry 45 (2010) 3740e3751
piperazine), 3.68 (dd, J ¼ 6.1, 7.9, 1H, H5a), 3.87 (s, 3H, OCH3), 3.87
(t, J ¼ 7.9, 1H, H5b), 4.17 (m, 1H, H4), 4.29 (d, J ¼ 14.0, 1H, H6a), 4.37
(d, J ¼ 14.0, 1H, H6b), 6.96 (m, 4H, H-arom), 7.34 (m, 3H, H-arom),
methoxyphenyl), 123.01 (C-5 methoxyphenyl), 126.22 (C-2,C-6
phenyl), 128.23 (C-3, C-5 phenyl), 128.55 (C-4 phenyl), 140.16 (C-1
phenyl), 141.10 (C-1 methoxyphenyl), 152.28 (C-2 methoxyphenyl),
171.70 (CO).
7.53 (m, 2H, H-arom). 13C NMR (400 MHz, CDCl3):
d
17.00 (C-40),
32.93 (C-30 þ C-40), 44.14 (C-6), 50.49 (C-2, C-6 piperazine), 54.02
(C-3, C-5 piperazine), 55.38 (OCH3), 61.63 (C-7), 68.76 (C-5), 73.63
(C-4), 109.96 (C-2), 111.21 (C-6 methoxyphenyl), 118.26 (C-3
methoxyphenyl), 121.02 (C-4 methoxyphenyl), 123.01 (C-5
methoxyphenyl), 126.17 (C-2,C-6 phenyl), 128.16 (C-3, C-5 phenyl),
128.53 (C-4 phenyl), 140.13 (C-1 phenyl), 141.18 (C-1 methox-
yphenyl), 152.29 (C-2 methoxyphenyl), 171.94 (CO).
The free amine was transformed into the corresponding oxalate
salt which was crystallized from methanol/diethyl ether to give
compound 6c: m.p. 165e173 ꢃC, 1H NMR (200 MHz, DMSO-d6)
The free amine was transformed into the corresponding oxalate
salt which was crystallized from methanol/diethyl ether to give
compound 7c: m.p. 162e167 ꢃC; 1H NMR (200 MHz, DMSO-d6)
d
1.39 (m, 4H, CH2-10 þ CH2-40), 1.62 (m, 4H, CH2-20 þ CH2-30), 2.60
(s, 4H, CH2-60 þ CH2-100), 3.05 (m, 6H, 4H piperazine þ CH2-7), 3.11
(m, 4H, H piperazine), 3.62 (m, 1H, H5a), 3.79 (s, 3H, 3H, OCH3), 3.79
(m, 1H, H5b), 4.15 (br s, 2H, CH2-6), 4.18 (m, 1H, H4), 6.94 (m, 4H, H-
arom), 7.41 (m, 5H, H-arom). C29H35N3O9. Anal. (C, H, N).
3.1.22. [(E) pair: SS and RR isomers] 8-{4-[4-(2-methoxy-phenyl)-
piperazin-1-ylmethyl]-2-phenyl-[1,3]dioxolan-2-ylmethyl}-8-aza-
spiro[4.5]decane-7,9-dione (13t) and oxalate salt 7t
d
1.76 (quint, J ¼ 6.4, 2H, CH2-40), 2.57 (t, J ¼ 6.4, 4H, CH2-30 þ CH2-
50), 2.86e3.33 (m, 10H, 8H-piperazine þ CH2-7), 3.61 (m, J ¼ 6.5, 8.1,
1H, H5a), 3.80 (m, 1H, H5b), 3.80 (s, 3H, OCH3), 4.14 (s, 2H, CH2-6),
4.20 (m, 1H, H4), 6.94 (m, 4H, H-arom), 7.39 (m, 5H, H-arom).
C33H41N3O9. Anal. (C, H, N).
0.228 g (0.60 mmol) of 19t gives 0.210 g (71.3%) of 13t. Flash
chromatography: cyclohexane/ethyl acetate 60:40 / 50:50. 1H
NMR (200 MHz, CDCl3)
d
1.50 (m, 4H, CH2-10 þ CH2-40), 1.71 (m, 4H,
CH2-20 þ CH2-30), 2.33 (dd, J ¼ 4.5, 13.2, 1H, H7a), 2.49 (dd, J ¼ 6.5,
13.2, 1H, H7b), 2.57 (s, 4H, CH2-60 þ CH2-100), 2.62 (m, 2H, H
piperazine), 2.82 (m, 2H, H piperazine), 3.08 (m, 4H, H piperazine),
3.40 (pseudot, J ¼ 8.2, 1H, H5a), 3.87 (s, 3H, OCH3), 4.23 (dd, J ¼ 6.3,
8.2, 1H, H5b), 4.28 (s, 2H, CH2-6), 4.36 (m, 1H, H4), 6.97 (m, 4H, H-
arom), 7.35 (m, 3H, H-arom), 7.56 (m, 2H, H-arom). 13C NMR
3.1.20. [(E) pair: SS and RR isomers] 1-{4-[4-(2-methoxy-phenyl)-
piperazin-1-ylmethyl]-2-phenyl-[1,3]dioxolan-2-ylmethyl}-
piperidine-2,6-dione (12t) and oxalate salt 6t
0.224 g (0.69 mmol) of 18t gives 0.270 g (81.7%) of 12t. Flash
chromatography: ethyl acetate/methanol 95:5. 1H NMR (200 MHz,
CDCl3)
d
1.84 (quint, J ¼ 6.4, 2H, CH2-40), 2.37 (dd, J ¼ 4.6, 12.7, 1H,
(400 MHz, CDCl3):
d
24.35 (C-20 þ C-30), 37.69 (C-10 þ C-40), 39.08
H7a), 2.56 (dd, J ¼ 6.3, 12.7, 1H, H7b), 2.58 (t, J ¼ 6.4, 4H, CH2-
30 þ CH2-50), 2.62 (m, 2H, H-piperazine), 2.82 (m, 2H, H-piperazine),
3.09 (m, 4H, H-piperazine), 3.42 (dd, J ¼ 8.1, 8.3,1H, H5a), 3.87 (s, 3H,
OCH3), 4.24 (dd, J ¼ 6.4, 8.1, 1H, H5b), 4.26 (d, J ¼ 13.8,1H, H6a), 4.34
(d, J ¼ 13.8,1H, H6b), 4.39 (m,1H, H4), 6.97 (m, 4H, H-arom), 7.32 (m,
3H, H-arom), 7.54 (m, 2H, H-arom). 13C NMR (400 MHz, CDCl3):
(C-50), 44.73 (C-6), 44.94 (C-60 þ C-100), 50.32 (C-2, C-6 piperazine),
53.89 (C-3, C-5 piperazine), 55.39 (OCH3), 61.14 (C-7), 68.68 (C-5),
77.25 (C-4), 109.77 (C-2), 111.19 (C-6 methoxyphenyl), 118.26 (C-3
methoxyphenyl), 121.02 (C-4 methoxyphenyl), 123.05 (C-5
methoxyphenyl), 126.20 (C-2,C-6 phenyl), 128.07 (C-3, C-5 phenyl),
128.43 (C-4 phenyl), 141.16(C-1 methoxyphenyl), 141.35 (C-1
phenyl), 152.28 (C-2 methoxyphenyl), 171.80 (CO).
d
17.00 (C-40), 32.91 (C-30 þ C-40), 44.71 (C-6), 50.38 (C-2, C-6
piperazine), 54.02 (C-3, C-5 piperazine), 55.39 (OCH3), 61.63 (C-7),
68.76 (C-5), 73.63 (C-4), 109.46 (C-2), 111.21 (C-6 methoxyphenyl),
118.27 (C-3 methoxyphenyl), 121.03 (C-4 methoxyphenyl), 123.09
(C-5 methoxyphenyl), 126.08 (C-2,C-6 phenyl), 128.26 (C-3, C-5
phenyl), 128.55 (C-4 phenyl), 140.99 (C-1 phenyl), 141.13 (C-1
methoxyphenyl), 152.28 (C-2 methoxyphenyl), 172.08 (CO).
The free amine was transformed into the corresponding oxalate
salt which was crystallized from methanol/diethyl ether to give
compound 7t: m.p.103e110 ꢃC; 1H NMR (200 MHz, DMSO-d6)
d 1.38
(m, 4H, CH2-10 þ CH2-40), 1.61 (m, 4H, CH2-20 þ CH2-30), 2.56 (s, 4H,
CH2-60 þ CH2-100), 2.63e2.98 (m, 6H, 4H piperazine þ CH2-7),3.06
(m, 4H, H piperazine),3.31 (pseudot, J ¼ 8.2, 1H, H5a), 3.78 (s, 3H,
OCH3), 4.10 (s, 2H, CH2-6), 4.21 (m, 1H, H5b),4.43 (m, 1H, H4), 6.93
(m, 4H, H-arom), 7.37 (m, 5H, H-arom). C29H35N3O9. Anal. (C, H, N).
The free amine was transformed into the corresponding oxalate
salt which was crystallized from methanol/diethyl ether to give
compound 6t: m.p. 165e171 ꢃC; 1H NMR (200 MHz, DMSO-d6)
d
1.73 (quint, J ¼ 6.4, 2H, CH2-40), 2.53 (t, J ¼ 6.4, 4H, CH2-30 þ CH2-
3.2. Modeling studies
50), 2.62e3.17 (m, 6H, 4H-piperazine þ CH2-7), 3.06 (m, 4H, H-
piperazine), 3.33 (m, 1H, H-5a), 3.78 (s, 3H, OCH3), 4.07 (d, J ¼ 13.7,
1H, H6a), 4.15 (d, J ¼ 13.7, 1H, H6b) 4.22 (dd, J ¼ 6.3,8.3, 1H,
H5b),4.43 (m, 1H, H4), 6.92 (m, 4H, H-arom), 7.38 (m, 5H, H-arom).
C33H41N3O9. Anal. (C, H, N).
The R,R; R,S; S,R; S,S isomer of 2e7 were built, parameterised
(Gasteiger-Huckel method) and energy minimized within MOE
using MMFF94 forcefield [29].
Starting from four databases, comprising the R,R; R,S; S,R; S,S
isomer of 2e7, a docking procedure was performed. In absence of
crystallographic data for a 1,3-dioxolane/5HT1A complex, a theo-
retical model of the human 5HT1A receptor was built. Based on the
assumption that GPCRs share similar transmembrane (TM)
boundaries and topology, the human 5HT1A receptor homology
model was generated starting from the X-ray structure of human b2
adrenoreceptor (PDB code: 2RH1) as template [17]. The amino acid
sequence of 5HT1A was retrieved from the SWISSPROT database
[30] while the three-dimensional structure coordinates file of
human b2 adrenoreceptor (PDB code: 2RH1) was obtained from the
Protein Data Bank [31].
Since the 5HT1A receptor contains many of the conserved motifs
associated with GPCRs, the amino acid sequences of 5HT1A TM
helices were aligned with the corresponding residues of the human
b2 adrenoreceptor. The connecting loops were constructed by the
loop search method implemented in MOE. The protein structure
was minimized with MOE using the AMBER94 forcefield [32]. The
3.1.21. [(Z) pair: SR and RS isomers] 8-{4-[4-(2-methoxy-phenyl)-
piperazin-1-ylmethyl]-2-phenyl-[1,3]dioxolan-2-ylmethyl}-8-aza-
spiro[4.5]decane-7,9-dione (13c) and oxalate salt 7c
0.276 g (0.73 mmol) of 19c gives 0.290 g (74.6%) of 13c. Flash
chromatography: cyclohexane/ethyl acetate 50:50. 1H NMR
(200 MHz, CDCl3)
d
1.52 (m, 4H, CH2-10 þ CH2-40), 1.72 (m, 4H, CH2-
20 þ CH2-30), 2.59 (s, 4H, CH2-60 þ CH2-100), 2.55 (dd, J ¼ 5.4, 13.0,
1H, H7a), 2.62 (m, 2H, H piperazine), 2.70 (dd, J ¼ 6.2,13.0,1H, H7b),
2.85 (m, 2H, H piperazine), 3.08 (m, 4H, H piperazine), 3.64 (m, 1H,
H5a), 3.92 (m, 1H, H5b), 3.88 (s, 3H, OCH3), 4.11 (m, 1H, H4), 4.31 (s,
2H, CH2-6), 6.96 (m, 4H, H-arom), 7.36 (m, 3H, H-arom), 7.57 (m,
2H, H-arom). 13C NMR (400 MHz, CDCl3):
d
24.23 (C-20 þ C-30), 37.83
(C-10 þ C-40), 39.25 (C-50), 44.24 (C-6), 44.96 (C-60 þ C-100), 50.37
(C-2, C-6 piperazine), 54.02 (C-3, C-5 piperazine), 55.38 (OCH3),
61.42 (C-7), 68.68 (C-5), 77.26 (C-4), 109.96 (C-2), 111.20 (C-6
methoxyphenyl), 118.28 (C-3 methoxyphenyl), 121.03 (C-4

S. Franchini et al. / European Journal of Medicinal Chemistry 45 (2010) 3740e3751
3749
energy minimization was carried out by the 1000 steps of steepest
descent followed by conjugate gradient minimization until the rms
gradient of the potential energy was less than 0.1 kcal mol-1 Å-1. The
stereochemistry of the model was validated through the analysis of
Ramachandran plot, chi plot and clash contacts reports (Molecular
Operating Environment suite).
receptor were evaluated according to the method of Stanton and
Beer [28] with minor modifications.
Stimulation experiments: Cell membranes were resuspended in
buffer containing 20 mM HEPES, 3 mM MgSO4, and 120 mM NaCl
(pH 7.4). The membranes were incubated with 30 mM GDP and
various concentrations (from 0.1 nM to 100 mM) of test drugs or 5-
HT (reference curve) for 20 min at 30 ꢃC in a final volume of 0.5 mL.
Subsequently, a 100 ps molecular dynamics simulation at 300 K
(step length: 0.001 ps) involving only the side chain atoms was
carried out. The obtained structure was then subjected to another
5000 steps of energy minimization. Structural evaluation of the
overall model was accomplished using the program PROCHECK
[33]. Docking studies were subsequently performed, according to
the following protocol. Each isomer was docked into the putative
ligand binding site using the flexible docking module implemented
in MOE. The binding site of the ligand in the 5HT1A receptor was
determined starting from the fact that, for the ligand activity,
formation of the salt bridge between the protonated piperazine
nitrogen on the ligand and Asp116 is necessary (see mutagenesis
data [18e20]). For all compounds, the best docking geometries,
evaluated in terms of “London dG”, were refined by energy mini-
mization (MMFF94) and rescored according to “Affinity dG” (Kcal/
mol of total estimated binding energy). Following this procedure
already described [34], on the basis of the final docking scoring
function (S), we identified the most probable 2e7 isomer inter-
acting with 5HT1A (lowest mean S value).
Samples were transferred to ice, [35S]GTP
gS (200 pM) was added,
and samples were incubated for another 30 min at 30 ꢃC.
Antagonism experiments: Cell membranes were resuspended in
buffer containing 20 mM HEPES, 3 mM MgSO4, and 120 mM NaCl
(pH 7.4). The membranes were incubated with 30 mM GDP and
various concentrations of 5-HT (from 0.1 nM to 100 mM) in the
absence or presence of antagonist, or with 30 mM GDP and various
concentrations (from 0.1 nM to 100 mM) of test drugs in the
presence of 5-HT (1 mM) for 20 min at 30 ꢃC in a final volume of
0.5 mL. Samples were transferred to ice, [35S]GTP
gS (200 pM) was
added, and samples were incubated for a further 30 min at 30 ꢃC.
The pre-incubation with both agonist and antagonist, before initi-
ating the [35S]GTP
are at equilibrium.
For both procedures, nonspecific binding was determined in the
presence of 10 mM GTP S. Incubation was stopped by the addition
gS binding, ensures that agonist and antagonist
g
of ice-cold HEPES buffer and rapid filtration on Schleicher & Schuell
GF52 filters using a Brandel cell harvester. The filters were washed
with ice-cold buffer, and the radioactivity retained on the filters
was determined by liquid scintillation counting at 90% efficiency.
3.3. Biological assays
3.3.3. Functional antagonism in isolated tissues
3.3.1. Radioligand binding assay at human recombinant 5-HT1A
receptors and a1 adrenoceptor subtypes
Male Wistar rats (275e300 g) were killed by cervical disloca-
tion, and the organs required were isolated, freed from adhering
connective tissues, and set up rapidly under a suitable resting
tension in 20 mL organ baths containing a physiological salt solu-
tion kept at 37 ꢃC and aerated with 5% CO2 and 95% O2 at pH 7.4.
Concentrationeresponse curves were constructed by cumulative
addition of agonist. The concentration of agonist in the organ bath
was increased approximately threefold at each step, with each
addition being made only after the response to the previous addi-
tion had attained a maximal level and remained steady. Contrac-
tions were recorded by means of a force displacement transducer
connected to the Mac Lab system PowerLab/800 and to a polygraph
channel recorder (Gemini). In addition, parallel experiments were
carried out in which tissues did not receive any antagonist in order
to check any variation in sensitivity. All animal testing was carried
out according to European Community Council Directive 86/609/
EEC (November 1986).
A human cell line (HeLa) stably transfected with genomic clone
G-21 coding for the human 5-HT1A serotoninergic receptor was
used. Cells were grown as monolayersin Dulbecco’s modified Eagle’s
medium supplemented with 10% fetal calf serum and gentamicin
(100 mg mLꢀ1) under 5% CO2 at 37 ꢃC. Cells were detached from the
growth flask at 95% confluence by a cell scraper and were lysed in
ice-cold Tris (5 mM) and EDTA buffer (5 mM, pH 7.4). Homogenates
were centrifuged for 20 min at 40,000 g, and pellets were resus-
pended in a small volume of ice-cold Tris/EDTA buffer (above) and
immediately frozen and stored at ꢀ70 ꢃC until use. On the day of
experiment, cell membranes were resuspended in binding buffer
(50 mM Tris, 2.5 mM MgCl2, and 10 mM pargiline, pH 7.4).
Membranes were incubated in a final volume of 1 mL for 30 min at
30 ꢃC with 1 nM [3H]8-OH-DPAT, in the absence or presence of
various concentrations of the competing drugs (1 pMe10 mM); each
experimental condition was performed in triplicate. Nonspecific
binding was determined in the presence of 10 mM 5-HT [24].
Binding to recombinant human a1 adrenoceptor subtypes was per-
formed in membranes from Chinese hamster ovary (CHO) cells
transfected by electroporation with DNA expressing the gene
encoding each a1 adrenoceptor subtype. Cloning and stable
expression of the human a1 adrenoceptor genes were performed as
described [35]. CHO cell membranes were incubated in 50 mM Tris
(pH 7.4) with 0.2 nM [3H]prazosin, in a final volume of 1.02 mL for
30 min at 25 ꢃC, in the absence or presence of competing drugs (1
pMe10 mM). Nonspecific binding was determined in the presence
of 10 mM phentolamine. The incubation was stopped by addition of
ice-cold Tris buffer and rapid filtration through 0.2% poly-
ethyleneimine-pretreated Whatman GF/B or Schleicher & Schuell
GF52 filters.
3.3.3.1. Vas deferens prostatic portion. This tissue was used to assess
the antagonism toward a1A adrenoceptors [25]. Prostatic portions
of 2 cm length were mounted under 0.5 g tension at 37 ꢃC in tyrode
solution of the following composition (in mM): NaCl, 130; KCl, 1;
CaCl2, 1.8; MgCl2, 0.89; NaH2PO4, 0.42; NaHCO3, 25; glucose, 5.6.
Cocaine hydrochloride (0.1 mM) was added to the tyrode to prevent
neuronal uptake of (ꢀ)-norepinephrine. The preparations were
equilibrated for 60 min with washing every 15 min. After the
equilibration period, tissues were primed twice by addition of
10
mM norepinephrine. After another washing and equilibration
period of 60 min, a norepinephrine concentrationeresponse curve
was constructed (basal response). The antagonist was allowed to
equilibrate for 30 min before constructing
a new concen-
trationeresponse curve to the agonist. (ꢀ)-Norepinephrine solu-
tions contained 0.05% Na2S2O5 to prevent oxidation.
3.3.2. [35S]GTPgS binding
The effects of the various compounds tested on [35S]GTP
gS
3.3.3.2. Spleen. This tissue was used to assess the antagonism
toward a1B adrenoceptors [27]. The spleen was removed and
binding in HeLa cells expressing the recombinant human 5-HT1A

3750
S. Franchini et al. / European Journal of Medicinal Chemistry 45 (2010) 3740e3751
bisected longitudinally into two strips, which were suspended in
tissue baths containing Krebs solution of the following composition
(in mM): NaCl, 120; KCl, 4.7; CaCl2, 2.5; MgSO4, 1.5; KH2PO4, 1.2;
NaHCO3, 20; glucose, 11; K2EDTA, 0.01. Propranolol hydrochloride
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Acknowledgements
This work was supported by grants from the University of
Modena and Reggio Emilia, and MIUR.

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