Structural modifications of N-(1,2,3,4-tetrahydronaphthalen-1-yl)-4-aryl-1- piperazinehexanamides: Influence on lipophilicity and 5-HT7 receptor activity. Part III

Article abstract of DOI:10.1021/jm800615e

Starting from the previously reported 5-HT₇ receptor agents 4-7 with N-(1,2,3,4-tetrahydronaphthalen-1-yl)-4-aryl-1-piperazinehexanamide structure, the 1-(2-methylthiophenyl)-, 1-(2-diphenyl)-, 1(2-isopropylphenyl)-, and 1-(2-methoxyphenyl)pipe

Full text of DOI:10.1021/jm800615e

J. Med. Chem. 2008, 51, 5813–5822
5813
Structural Modifications of N-(1,2,3,4-Tetrahydronaphthalen-1-yl)-4-Aryl-1-piperazinehexana-
mides: Influence on Lipophilicity and 5-HT7 Receptor Activity. Part III
Marcello Leopoldo,*,† Enza Lacivita,† Paola De Giorgio,† Claudia Fracasso,‡ Sara Guzzetti,‡ Silvio Caccia,‡
Marialessandra Contino,† Nicola A. Colabufo,† Francesco Berardi,† and Roberto Perrone†
UniVersita` degli Studi di Bari, Dipartimento Farmaco-Chimico, Via Orabona, 4, 70125 Bari, Italy, Istituto di Ricerche Farmacologiche Mario
Negri, Via La Masa 19, 20156 Milan, Italy
ReceiVed May 23, 2008
Starting from the previously reported 5-HT7 receptor agents 4-7 with N-(1,2,3,4-tetrahydronaphthalen-1-
yl)-4-aryl-1-piperazinehexanamide structure, the 1-(2-methylthiophenyl)-, 1-(2-diphenyl)-, 1-(2-isopropyl-
phenyl)-, and 1-(2-methoxyphenyl)piperazine derivatives 8-31 were designed with the primary aim to obtain
new compounds endowed with suitable physicochemical properties for rapid and extensive penetration into
the brain. The affinities for 5-HT7, 5-HT1A, and D2 receptors of compounds 8-31 were assessed, and several
compounds displayed 5-HT7 receptor affinities in the nanomolar range. Among these, N-(4-cyanophenyl-
methyl)-4-(2-diphenyl)-1-piperazinehexanamide (25) showed high 5-HT7 receptor affinity (Ki ) 0.58 nM),
high selectivity over 5-HT1A and D2 receptors (324- and 245-fold, respectively), and agonist properties
(maximal effect ) 82%, EC50 ) 0.60 µM). After intraperitoneal injection in mice, 25 rapidly reached the
systemic circulation and entered the brain. Its brain concentration-time profile paralleled that in plasma,
indicating that 25 rapidly and freely distributes across the blood-brain barrier. Compound 25 underwent
N-dealkylation to the corresponding 1-arylpiperazine metabolite.
Chart 1. Structures of 5-HT7 Receptor Agents
Introduction
Serotonin (5-hydroxytryptamine, 5-HT) controls a variety of
physiological functions in the central and peripheral nervous
system by interacting with a multitude of 5-HT receptor
subtypes. These subtypes can be divided into seven main classes
(5-HT1 to 5-HT7) on the basis of their pharmacological responses
to specific ligands, sequence similarities at the gene and amino
acid levels, gene organization, and second messenger coupling
pathways. Except for the 5-HT3 receptor, which is a ligand-
gated ion channel, all serotonin receptors are coupled to
G-proteins.1
The 5-HT7 receptor has been cloned from human, mouse,
rat, guinea pig, and pig cDNA. High sequence homology (90%)
has been observed between the 5-HT7 receptors from various
species, whereas a low degree of homology (40%) has been
evidenced between 5-HT7 receptor and the other G-protein
coupled 5-HT receptor subtypes.2 Studies in rodents on the
distribution of the 5-HT7 receptor protein in the central nervous
system (CNS)a revealed the highest abundance in thalamus,
hypothalamus, and hippocampus. Within the suprachiasmatic
nucleus of the hypothalamus, the 5-HT7 receptors are located
in both dendrites and axon terminals of mostly GABA contain-
ing neurons.3-6 Brain autoradiographic studies in human
indicated that these receptors are mainly localized in the anterior
thalamus and in the dentate gyrus.7 Irrespective of the experi-
mental method used, there is general agreement that 5-HT7
receptors are localized in regions of the brain involved in
affective behavior and cognition and recent studies support a
role for 5-HT7 receptors in the pathogenesis of depression. In
vitro binding studies in rat have also suggested that chronic
antidepressant treatment results in a functional downregulation
of 5-HT7-like binding sites in the hypothalamus.8 Studies on
5-HT7 receptor knockout mice have displayed “antidepressant-
like” phenotype in preclinical assay used to assess antidepressant
potential. Also pharmacological 5-HT7 receptor blockade by the
antagonists 1 (SB-258719) or 2 (SB-269970) (Chart 1) generated
robust antidepressant-like effects.9-13 It has been also suggested
that 5-HT7 receptor function plays a role in the control of
learning and memory processes.14-16 Finally, pharmacological
and genetic 5-HT7 blockade has shown to partially modulate
glutamatergic and dopaminergic function and therefore to be
clinically useful for the treatment of positive symptoms of
schizophrenia.17,18 In view of these outcomes, the 5-HT7
receptor can be considered an interesting target for drug
development.
* To whom correspondence should be addressed. Phone: +39 080
5442798. Fax: +39 080 5442231. E-mail: leopoldo@farmchim.uniba.it.
†
Universita` degli Studi di Bari.
Istituto di Ricerche Farmacologiche Mario Negri Milano.
Over the last 10 years, considerable efforts have been focused
on the development of novel and selective 5-HT7 receptor
agonists and antagonists.19,20 The first selective antagonist 1 was
identified in 1997 by GlaxoSmithKline and displayed an
‡
a Abbreviations: CNS, central nervous system; 5-CT, 5-carboxamido-
tryptamine; AUC, area under the curve; 8-OH-DPAT, 8-hydroxy-N,N-
dipropylaminotetraline.
10.1021/jm800615e CCC: $40.75
2008 American Chemical Society
Published on Web 08/23/2008

5814 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18
Leopoldo et al.
Table 1. Physicochemical and Binding Affinities of Reference
Compounds 4-7a
5 (ClogP ) 6.65) exceeds the guideline value and this could
limit its use in vivo. Keeping in mind that compound 5 was the
most selective 5-HT7 derivative, we selected also as reference
the compounds 4, 6, and 7 (Table 1) because they displayed
high affinity at 5-HT7 receptor and different selectivity profiles
over 5-HT1A and D2 receptors. Therefore, we targeted structural
modifications on compounds 4-5 to obtain the new derivatives
8-31, which showed ClogP values within the desired range
(Tables 2 and 3).
Ki, nM
Chemistry
compd
R
ClogPb log k′ 5-HT7 5-HT1A
D2L
7.3
224
15
4 (LP-44) SCH3
5 (LP-12) Ph
4.73
6.65
5.60
4.78
1.01
1.71
1.39
0.63
0.22
0.13
1.10
6.64
52.7
60.9
167
8.6
The synthesis of the final compounds 8-31 required the
starting amines 32a,b and 36a-d (Scheme 1). Among these,
only amine 36d was not commercially available; therefore, it
was prepared as follows: 4-hydroxybenzylamine hydrochloride32
was first N-BOC protected, then was reacted with methane-
sulfonyl chloride to give derivative 35. Deprotection of the latter
gave the benzyl amine 36d. The amines 32a,b and 36a-d
reacted with 6-bromohexanoyl chloride to give the correspond-
ing bromoalkyl derivatives 33a,b and 37a-d. Condensation of
33a,b and 37a-d with the appropriate 1-arylpiperazine gave
the desired compounds 8-31.
6
7
CH(CH3)2
OCH3
24.6
a Data taken from ref 28. b Calculated logP values (see ref 51).
arylsulfonamidoalkylamine structure, as well as compound 2,
which is considered to date the standard selective 5-HT7 receptor
antagonist.21 However, further studies with 2 have been
hampered by its rapid clearance in vivo in the rat. Years later,
GlaxoSmithKline reported on the identification of the antagonist
3 (SB-656104) (Chart 1), which showed an improved pharma-
cokinetic profile but displayed modest selectivity over 5-HT2A
,
5-HT2B, and 5-HT1D receptors.22 The considerable amount of
data on antagonists has allowed the generation of two receptor-
based pharmacophores: the first containing features necessary
for high 5-HT7 receptor affinity and the other defining selectivity
for this receptor subtype.23 Considering the 5-HT7 receptor
agonists, a pharmacophore model has been generated from a
set of 20 nonselective agonists.24 This set included also
5-carboxamidotryptamine (5-CT), which is to date used as
reference agonist.
Results and Discussion
In view of the increasing interest in the development of 5-HT7
receptor agonists, we decided to expand our structure-activity
relationship studies on N-(1,2,3,4-tetrahydronaphthalen-1-yl)-
4-aryl-1-piperazinealkylamide derivatives in order to optimize
their 5-HT7 affinity and selectivity and to explore their potential
for in vivo pharmacological studies. Because a major determi-
nant of the activity of centrally acting drugs is the rate and extent
to which they enter the brain, we targeted structural modifica-
tions on compounds 4-7 to obtain the new derivatives 8-31,
which had suitable ClogP values for rapid penetration into the
brain. The structural modifications were performed taking into
account our previous structure-activity relationship findings.
We evidenced in this class of the compounds (Structure I, Chart
1) and also for other arylpiperazine-based 5-HT7 receptor
ligands33,34 that even limited structural changes on the 1-arylpip-
erazine moiety could reflect in a great variation of 5-HT7
receptor affinity. The highest values of affinity at 5-HT7 receptor
were shown by compounds with five methylenes. For these
reasons, we did not modify the arylpiperazine moiety and the
alkyl chain length of compounds 4-7. Therefore, we reasoned
on the possibility to reduce the lipophilicity of the terminal
fragment of compounds 4-7 by replacing the tetrahydronaph-
thalene nucleus with three structurally related moieties (isoin-
doline, tetrahydroisoquinoline, benzyl) that would have less
impact on the lipophilicity of the target molecules. As shown
in Tables 2 and 3, compounds 8-19 displayed lower ClogP
values than their corresponding tetrahydronaphthalene counter-
parts 4-7. However, the benzyl derivative 17 showed a ClogP
of 5.28, which is quite above the guideline value. With the aim
to obtain a reduction of the lipophilicity, the benzyl group
attached to the amide nitrogen was therefore replaced by more
polar groups such as 4-pyridinylmethyl, 4-cyanophenylmethyl,
and 4-methanesulfonylphenylmethyl. This structural modifica-
tion was performed on benzyl derivatives 16-19, leading to
compounds 20-31 (Table 3). The actual reduction of lipophi-
licity of the new molecules was confirmed by determining the
retention factor log k′ as lipophilicity index by using a reversed-
phase HPLC method.35 Binding affinities for the rat cloned
5-HT7 receptor were determined by radioligand displacement
assay using the condition summarized in the Experimental
More recently, several relatively selective 5-HT7 agonists have
been reported,25,26 including compounds 4 (LP-44) and 5 (LP-
12) (Table 1), which were identified during our structure-activity
relationship studies on a series of N-(1,2,3,4-tetrahydronaph-
thalen-1-yl)-4-aryl-1-piperazinealkylamides (structure I, Chart
1).27,28 Our studies indicated that for 5-HT7 affinity: (i) a
methoxy group on the 1,2,3,4-tetrahydronaphthalene ring was
not essential, (ii) five methylene units separating the amide
moiety and the piperazine ring were preferable, (iii) the position
and the nature of the substituent on the phenyl linked to the
piperazine ring was critical. In particular, we found that shifting
the R substituent from the 2-position to the 3- or 4-position
caused a significant loss in 5-HT7 receptor affinity. Examination
of a series of substituents in the 2-position, covering a wide
range of electronic, steric, and polar properties, revealed a key
role on both affinity and intrinsic activity at the 5-HT7 receptor.
Certain lipophilic substituents (R ) SCH3, CH(CH3)2, N(CH3)2,
CH3, Ph) led to high-affinity agonists, while the OH and NHCH3
substituents switched intrinsic activity toward antagonism. The
nature of this substituent was also relevant with respect to the
selectivity over 5-HT1A and D2 receptors. The most relevant
outcome from those studies was the identification of several
high-affinity agonists (compounds 4-7, Table 1). In particular,
compound 5 showed the highest selectivity among the studied
derivatives. However, while studying the structure-activity
relationships, we did not optimize the physicochemical proper-
ties required for penetration into the brain. It is widely accepted
that brain penetration by passive diffusion becomes poor when
molecular weight is larger than 450 Da, the logP is greater than
5.0, the number of hydrogen bond donors is greater than five,
or when the sum of all nitrogens and oxygens is greater than
10.29-31 As reported in Table 1, the lipophilicity of compound

Piperazinehexanamides. Part III
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18 5815
Table 2. Physicochemical and Binding Affinities of Compounds 8-15
Ki, nM ( SEMa
compd
n
R
ClogP
log k′
5-HT7
5-HT1A
D2
8
9
10
11
1
1
1
1
SCH3
Ph
CH(CH3)2
OCH3
2.89
4.41
3.76
2.35
1.12
1.65
1.55
0.76
42 ( 3.7
8.3 ( 2.1
54 ( 4
1.07 ( 0.05
124 ( 20
22 ( 0.5
96 ( 6
93 ( 2
65 ( 5
179 ( 15
3.2 ( 0.8
730 ( 50
12
13
14
15
2
2
2
2
SCH3
Ph
CH(CH3)2
OCH3
3.45
4.97
4.32
2.91
1.18
1.68
1.61
0.75
32.9 ( 2.1
3.81 ( 0.5
6 ( 0.4
2.04 ( 0.50
95 ( 5
3.7 ( 0.8
40 ( 4.5
40 ( 8
362 ( 75
1.3 ( 0.3
22 ( 4
31 ( 3.0
5-CT
8-OH-DPAT
haloperidol
0.50 ( 0.08
2.1 ( 0.4
4.2 ( 0.6
a The values are the means ( SEM from three independent experiments with samples in triplicate.
Table 3. Physicochemical and Binding Affinities of Compounds 16-31
a The values are the means ( SEM from three independent experiments with samples in triplicate. a Not detectable (see ref 34).
Section. Hill plot slope of all compounds (except 17) were close
to unity, indicating a competitive-type interaction between the
tested compounds and [3H]LSD. The analysis of the Kd shift
obtained by compound 17 resulted in a linear plot with a slope
significantly different from unity, suggesting an allosteric
interaction between compound 17 and [3H]LSD under the
experimental conditions used here.36
The 5-HT7 receptor affinities of target compounds 8-31 are
listed in Tables 2 and 3. Considering isoindolines 8-11,
isoquinolines 12-15, and benzyl derivatives 16-19, one can

5816 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18
Leopoldo et al.
Scheme 1a
a Reagents: (A) (i) (BOC)2O, Et3N; (ii) methanesulfonyl chloride, Et3N. (B) TFA. (C) 6-Bromohexanoyl chloride, 1.2% aqueous NaOH. (D) 1-Arylpiperazine.
Table 4. Relaxation Effect Induced by Selected Compounds and 5-CT
observe a reduction in 5-HT7 affinity as compared to the
on Substance P-Stimulated Guinea Pig Ileum Contracture
corresponding tetrahydronaphthalenyl derivatives 4-7. In par-
compd
% max activitya
EC50, µM
pA2 of compd 2b
ticular, the isoquinoline derivatives showed less marked lowering
than the isoindoline and benzyl derivatives. The reduction was
particularly evident for 2-methylthio derivatives 8, 12, and 16
and for derivative 18 (g100-fold), whereas compounds 9, 13,
and 14 retained affinity values in the nanomolar range. Plotting
the pKis against ClogP values of compounds 4-19 revealed a
low correlation coefficient (data not shown), suggesting that the
apolar nature of the terminal fragment is not the only feature
that modulates the interaction of this part of the ligand with the
5-HT7 receptor. To some extent, this observation was confirmed
by comparing 5-HT7 receptor affinity data of derivatives 16,
18, and 19 with those of their less lipophilic analogues 20,
22-24, 26-28, 30, 31 (Table 3). In fact, replacement of the
benzyl group attached to the amide nitrogen by more polar
groups had a limited impact on 5-HT7 affinity, except for 22
and 26, which were 42- and 25-fold more potent than 18. It is
interesting to note that the presence of 4-pyridinylmethyl,
4-cyanophenylmethyl, and 4-methanesulfonylphenylmethyl as
a terminal fragment was also well tolerated in the case of
2-phenyl derivatives 21, 25, and 29, which showed high 5-HT7
affinity (5.7 nM < Ki < 0.58 nM).
The affinities for 5-HT1A and D2 receptors of the target
compounds were also assessed because they may interfere with
the evaluation of pharmacological actions mediated by 5-HT7
receptors (i.e., body temperature, sleep patterns).37,38 The
structural simplification of the tetrahydronaphthalene nucleus
of 4-7 did not cause great variations in 5-HT1A affinity. The
isoquinoline, isoindoline, and benzyl derivatives 8-19 showed
Ki values that differed at least 10-fold from the parents 4-7,
except 12, which was about 26-fold more potent than 4. These
data indicated that, within this class of arylpiperazine-based
ligands, the affinity at 5-HT1A receptor is marginally affected
by the nature of the terminal fragment. A similar trend was
shown by affinity data at dopamine D2 receptor: Ki values of
isoindolines 8-11, isoquinolines 12-15, and benzyl derivatives
16-19 were within 1 order of magnitude as compared to
tetrahydronaphthalenyl counterparts 4-7. This binding profile
was replicated by derivatives 20-31, which showed even
smaller variation of affinity at both 5-HT1A and D2 receptors.
As a result of the above-discussed trend of affinity at 5-HT7,
5-HT1A, and D2 receptors, the specificity for 5-HT7 receptor of
the new compounds 8-31 was, at best, unchanged as compared
to the reference compounds 4-7. From these data, it became
5c
13
21
25
74
89
87
82
100
1.77 ( 0.08
0.49 ( 0.03
0.31 ( 0.08
0.60 ( 0.04
0.63 ( 0.04
7.84 ( 0.20
7.80 ( 0.20
7.96 ( 0.20
7.69 ( 0.20
7.48 ( 0.12
5-CT
a Percentage of the relaxation effect mediated by 5-HT7 receptors. b pA2
values of the standard antagonist 2 determined by using the corresponding
agonist. c Data taken from ref 28.
apparent that maintaining potency and selectivity for the
biochemical target while optimizing the physicochemical prop-
erties is a complex challenge. Nonetheless, the 2-phenyl
derivatives 13, 21, and 25 showed appreciable selectivity (25-
to 324-fold over 5-HT1A and 95- to 245-fold over D2 receptors),
still retaining nanomolar affinity at 5-HT7 receptor and showing
lipophilicity within the target range. These ligands were further
characterized for their intrinsic activity at 5-HT7 receptor by
measuring the 5-HT7 agonist mediated relaxation of substance
P-induced contraction in an isolated guinea pig ileum assay
(Table 4). They showed full competitive agonism at 5-HT7
receptor, being that their effect abolished dose-dependently by
the selective 5-HT7 antagonist 2. In particular, compounds 21
and 25 demonstrated EC50 values comparable to that of the
standard 5-HT7 agonist 5-CT and higher potency than the
tetrahydronaphthalenyl derivative 5. It can be noted that the
modification of the terminal fragment of 5 left unchanged the
intrinsic activity of 13, 21, and 25, confirming the predominant
role of the arylpiperazine moiety on the intrinsic activity in this
class of compounds.
On the basis of its affinity for the 5-HT7 receptor (Ki ) 0.58
nM), selectivity over 5-HT1A and D2 receptors (324- and 245-
fold, respectively), and intrinsic activity profile (maximal effect
) 82%, EC50 ) 0.60 µM), compound 25 (LP-211) was injected
in male mice (10 mg/kg) to preliminarily determine its distribu-
tion between blood and brain and thus find out to what extent
it passes the blood-brain barrier and the concentrations reached
at the site of action. Moreover, the potential formation of 1-(2-
diphenyl)piperazine and its brain-to-plasma concentration ratio
were also examined because arylpiperazine derivatives generally
undergo N-dealkylation of the aliphatic chain attached to the
piperazine nitrogen.39 Similar studies were performed on
compound 4, which has a lipophilicity comparable with 25. As
shown in Figure 1, compound 25 rapidly reached the systemic

Piperazinehexanamides. Part III
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18 5817
yielding a mean brain-to-plasma distribution ratio (plasma/brain
AUC) of about 0.7. Compound 4 too peaked in brain at
approximately the same time as in plasma (Figure 2B), but mean
Cmax (0.58 ( 0.35 µg/g) was about twice that in plasma, looking
at the whole brain concentrations. However, the low and short-
lasting compound levels did not allow an accurate determination
of the terminal t1/2, although a rough estimate suggested that,
at least at this dose, compound 4 was less persistent in the brain
than compound 25 (elimination t1/2 about 20 and 69 min,
respectively). Unlike plasma concentrations, brain concentrations
of 1-(2-diphenyl)piperazine (Figure 2A) rose rapidly and reached
Cmax of 0.41 ( 0.06 µg/g within 120 min for compound 25,
then declined with an elimination t1/2 of 87 min. Mean AUC
was 94 µg/mL·min, yielding a metabolite-to-parent compound
ratio of about 1.6. Similarly, the metabolite of compound 4,
1-(2-methylthiophenyl)piperazine, appeared rapidly in mouse
brain (Cmax ) 0.50 ( 0.11 µg/g, at 60 min), disappearing
thereafter at a slower rate (elimination t1/2 about 82 min) than
the parent compound (Figure 2B). The AUC of 1-(2-methyl-
tiophenyl)piperazine (85 µg/mL·min) exceeded that of its parent
compound by about 4.1 times. This was not surprising, as
previous studies have shown that 1-arylpiperazines concentrate
in brain tissue, achieving concentrations several times those in
the blood.39 This is probably related to their lipophilicity, which
leads to the extensive uptake into lipid-containing tissue.40
Figure 1. Plasma concentration-time curves of compounds 25 (open
symbols) and 4 (closed symbols) after intraperitoneal injection in mice
(10 mg/kg; N ) 3).
Conclusions
In summary, we have described the synthesis of the new
1-arylpiperazine-based derivatives 8-31. These compounds
originated from the structural simplification of the previously
reported 5-HT7 receptor agents 4-7 with N-(1,2,3,4-tetrahy-
dronaphthalen-1-yl)-4-aryl-1-piperazinehexanamide structure.
The affinities of derivatives 8-31 for 5-HT7 receptor as well
as those for serotonin 5-HT1A and dopamine D2 were assessed
by radioligand binding assays. The proposed structural modi-
fications on the tetrahydronaphthalenyl ring of compounds 4-7
were, in most cases, detrimental for 5-HT7 receptor, whereas
they had limited impact on the affinity for 5-HT1A and D2
receptors. Nonetheless, the pursued strategy led to the identifica-
tion of the 1-(2-diphenyl)piperazine derivatives 13, 21, and 25,
which retained nanomolar affinity at 5-HT7 receptor, appreciable
selectivity (25- to 324-fold over 5-HT1A and 95- to 245-fold
over D2 receptors), still showing lipophilicity within the target
range. Compounds 13, 21, and 25 showed full competitive
agonism at 5-HT7 receptor in an isolated guinea pig ileum assay,
as well as their corresponding tetrahydronaphthalenyl derivative
5. Compound 25 rapidly crossed the blood-brain barrier and
equilibrated with plasma, achieving concentrations in the low
micromolar range after intraperitoneal doses in mice. Compound
25 undergoes N-dealkylation of the aliphatic chain attached to
the piperazine nitrogen, resulting in the formation of 1-(2-
diphenyl)piperazine, whose pharmacokinetic behavior is dif-
ferent from the parent compound. Future studies will focus on
characterization of the neuropharmacological profile of this
metabolite compared with compound 25.
Figure 2. Brain concentration-time curves of compounds 25 (A) and
4 (B) after intraperitoneal injection in mice (10 mg/kg). Each value is
the mean of 3 mice for the parent compound (closed symbols) and
their 1-aryl-piperazine metabolite (open symbols).
circulation in the mouse, achieving mean Cmax of 0.76 ( 0.32
µg/mL at 30 min, with quantifiable levels consistently evident
for up to 2 h. Under the assumption of complete absorption of
this derivative from the injection site into the systemic circula-
tion, the mean total body clearance approached liver blood flow.
In conjunction with a relatively high volume of distribution of
about 11 L/kg, this resulted in a mean elimination half-life of
65 min. In the same experimental conditions, compound 4
reached plasma Cmax (0.31 ( 0.07 µg/mL) at the first sampling
time (15 min). It was then cleared rapidly, with plasma
concentrations approximately halving after 30 min, and the
compound was no longer measurable in mouse plasma after
1 h (<0.1 µg/mL, using 0.1 mL of plasma). In both cases, the
plasma concentrations of their 1-arylpiperazine metabolite were
always below the detection limit of the analytical procedure, at
this dose of 25 and 4.
Experimental Section
Chemistry. Column chromatography was performed with 1:30
Merck silica gel 60A (63-200 µm) as the stationary phase. Melting
points were determined in open capillaries on a Gallenkamp
electrothermal apparatus. Elemental analyses (C, H, N) were
performed on Eurovector Euro EA 3000 analyzer; the analytical
results were within ( 0.4% of the theoretical values for the formula
Reflecting the time profile in the systemic circulation,
compound 25 achieved mean brain Cmax at approximately the
same time as in plasma, i.e., 30 min after dosing (Figure 2A).
Mean Cmax averaged 0.54 ( 0.06 µg/g. These concentrations
then declined almost parallel with plasma concentrations,
1
given. H NMR spectra were recorded at 300 MHz on a Varian
Mercury-VX spectrometer. All spectra were recorded on free bases.

5818 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18
Leopoldo et al.
All chemical shift values are reported in ppm (δ). Recording of
mass spectra was done on an HP6890-5973 MSD gas chromato-
graph/mass spectrometer; only significant m/z peaks, with their
percentage of relative intensity in parentheses, are reported. ESI+/
MS/MS analysis were performed with an Agilent 1100 Series LC-
MSD trap System VL workstation. All spectra were in accordance
with the assigned structures. The purity of new compounds that
were essential to the conclusions drawn in the text were determined
by HPLC on a Perkin-Elmer Series 200 LC instrument using a
Phenomenex Prodigy ODS-3 RP-18 column, (250 mm × 4.6 mm,
5 µm particle size) and equipped with a Perkin-Elmer 785A UV/
vis detector setting λ ) 254 nm. Compounds 8-31 were eluted
with CH3OH/H2O/Et3N, 4:1:0.01, v/v, at a flow rate of 1 mL/min.
When necessary, a standard procedure was used to transform final
compounds into their hydrochloride salts. The following compounds
were synthesized according to published procedures: 4-hydroxy-
benzylamine,32 1-(2-isopropylphenyl)piperazine,41 1-(2-diphe-
nyl)piperazine.42
N-Benzyl-6-bromohexanamide (37a). Eluted with CHCl3; 81%
yield. 1H NMR (CDCl3): δ 1.42-1.53 (m, 2H), 1.64-1.74 (m, 2H),
1.82-1.92 (m, 2H), 2.25 (app. t, 2H), 3.40 (t, 2H, J ) 6.8 Hz),
4.44 (d, 2H, J ) 5.8 Hz), 5.76 (br t, 1H, D2O exchanged),
7.23-7.37 (m, 5H). GC/MS: m/z 285 (M+ +2, 28), 283 (M+, 28),
149 (99), 106 (54), 91 (100).
6-Bromo-N-(4-pyridinylmethyl)hexanamide (37b). Eluted with
1
CHCl3/MeOH 19:1; 84% yield. H NMR (CDCl3): δ 1.34-1.43
(m, 2H), 1.53-1.61 (m, 2H), 1.72-1.80 (m, 2H), 2.17 (app. t, 2H),
3.30 (t, 2H, J ) 6.8 Hz), 4.30 (d, 2H, J ) 5.8 Hz), 5.76 (br t, 1H,
D2O exchanged), 7.09-7.11 (m, 2H), 8.40-8.42 (m, 2H). GC/
MS: m/z 286 (M+ +2, 2), 284 (M+, 3), 150 (100), 92 (58).
6-Bromo-N-(4-Cyanophenylmethyl)hexanamide (37c). Eluted
with CHCl3/AcOEt 1:1; 78% yield. 1H NMR (CDCl3): δ 1.43-1.53
(m, 2H), 1.65-1.73 (m, 2H), 1.83-1.92 (m, 2H), 2.26 (app. t, 2H),
3.41 (t, 2H, J ) 6.6 Hz), 4.49 (d, 2H, J ) 6.05 Hz), 5.93 (br t, 1H,
D2O exchanged), 7.37 (d, 2H, J ) 8.5 Hz), 7.59-7.63 (m, 2H).
GC/MS: m/z 310 (M+ +2, 13), 308 (M+, 13), 174 (100), 131 (27),
116 (82).
N-t-Butoxycarbonyl-4-(methanesulfonyloxyphenyl)-metha-
namine (35). 4-Hydroxybenzylamine hydrochloride (3.18 g, 14.7
mmol) was solubilized in a mixture of H2O (25 mL) and THF (60
mL). Et3N (2.16 mL, 14.7 mmol) and di-t-butylcarbonate (3.4 g,
14.7 mmol) was added to the solution. The mixture was stirred at
room temperature overnight. Then THF was evaporated in vacuo
and the aqueous residue was extracted with AcOEt (3 × 20 mL).
The organic phases were collected, dried over Na2SO4, and
concentrated under reduced pressure. The crude residue was
chromatographed (CHCl3/ AcOEt 9:1 as eluent) to give the Boc
protected amine as a yellow solid. Triethylamine (2.5 mL, 17.9
mmol) and methanesulfonyl chloride (1.5 mL, 19.4 mmol) were
added to a solution of Boc protected amine (2.7 g, 12.1 mmol) in
CH2Cl2 cooled at -10 °C. The mixture was stirred at room
temperature for 6 h. Then, the reaction mixture was first washed
with a saturated aqueous solution of NaHCO3 and then with 3 N
HCl. The separated organic phase was dried over Na2SO4 and
concentrated under reduced pressure to yield pure 35 as a white
6-Bromo-N-(4-Methanesulfonyloxyphenylmethyl)hexanamide
(37d). Eluted with CHCl3/AcOEt 1:1; 30% yield. 1H NMR (CDCl3):
δ 1.43-1.53 (m, 2H), 1.62-1.75 (m, 2H), 1.83-1.92 (m, 2H), 2.24
(t, 2H, J ) 7.4 Hz), 3.14 (s, 3H), 3.40 (t, 2H, J ) 6.6 Hz), 4.44 (d,
2H, J ) 5.8 Hz), 5.82 (br t, 1H, D2O exchanged), 7.22-7.26 (m,
2H), 7.31-7.35 (m, 2H).
General Procedure for Preparation of Final Compounds 8-31.
A stirred mixture of alkylating agent 33a,b and 37a-d (8.0 mmol),
1-substituted piperazine (9.6 mmol), and K2CO3 (8.0 mmol) in
acetonitrile was refluxed overnight. After cooling, the mixture was
evaporated to dryness and H2O (20 mL) was added to the residue.
The aqueous phase was extracted with AcOEt (2 × 30 mL). The
collected organic layers were dried over Na2SO4 and evaporated
under reduced pressure. The crude residue was chromatographed
(CHCl3/MeOH, 19:1, as eluent) to afford pure compounds 8-31.
2-[6-[4-(2-Methylthiophenyl)-1-piperazinyl]-1-oxohexyl]isoindo-
line (8). Yield 73%. 1H NMR (CDCl3): δ 1.39-1.48 (m, 2H),
1.57-1.68 (m, 2H), 1.77 (quintet, 2H, J) 7.7 Hz), 2.37-2.40 (m,
2H), 2.40 (s, 3H), 2.47 (app. t, 2H), 2.68 (br s, 4H), 3.06 (br s,
4H), 4.81 (s, 4H), 7.05-7.11 (m, 4H), 7.26-7.31 (m, 4H). GC/
MS: m/z 424 (M+ +1, 3), 423 (M+, 15), 245 (94), 221 (100), 136
(33). The hydrochloride salt melted at 187-189 °C (from MeOH/
Et2O). Anal. (C25H33N3OS·HCl·0.3H2O) C, H, N.
1
semisolid (2.8 g, 77% yield). H NMR (CDCl3): δ 1.45 (s, 9H),
3.13 (s, 3H), 4.31 (br d, 2H), 7.21-7.25 (m, 2H), 7.33 (d, 2H, J)
8.5 Hz).
4-Methanesulfonyloxyphenylmethanamine (36d). A mixture of
35 (1.4 g, 4.65 mmol) and trifluoroacetic acid (1.04 mL, 14 mmol)
in CH2Cl2 (30 mL) was stirred at room temperature overnight. Then
the reaction mixture was washed twice with aqueous 20% Na2CO3
(2 × 20 mL). The organic phase was separated, dried over Na2SO4,
and evaporated under reduced pressure, giving pure 36d as a yellow
oil (0.78 g, 83% yield). 1H NMR (CDCl3): δ 2.12 (s, 2H, D2O
exchanged), 3.07 (s, 3H), 3.82 (s, 2H), 7.15-7.19 (m, 2H),
7.25-7.33 (m, 2H).
2-[6-[4-(2-Diphenyl)-1-piperazinyl]-1-oxohexyl]isoindoline (9).
1
Yield 82%. H NMR (CDCl3): δ 1.33-1.43 (m, 2H), 1.49-1.59
(m, 2H), 1.72 (quintet, 2H, J) 7.4 Hz), 2.32-2.39 (m, 8H), 2.88
(app t, 4H), 4.79 (s, 4H), 7.02-7.09 (m, 2H), 7.23-7.30 (m, 7H),
7.36-7.41 (m, 2H), 7.61-7.64 (m, 2H). GC/MS: m/z 454 (M+
+1, 5), 453 (M+, 7), 251 (71), 245 (100), 180 (48), 118 (30); mp
134-136 °C (from CHCl3/n-hexane). Anal. (C30H35N3O) C, H, N.
2-[6-[4-(2-(1-Methylethyl)phenyl)-1-piperazinyl]-1-oxohexyl-
General Procedure for Preparation of Alkylating Agents 33a,b
and 37a-d. A cooled solution of amine 32a,b and 36a-d (4.0
mmol) in CH2Cl2 was stirred vigorously with 2% aqueous NaOH
(9.6 mL, 4.8 mmol) while 6-bromohexanoyl chloride (4.8 mmol)
in CH2Cl2 was added dropwise. The same NaOH solution was then
used to maintain pH at 9, and at constant pH the layers were
separated. The organic phase was washed with 3 N HCl, with H2O,
and then dried over Na2SO4 and evaporated under reduced pressure.
The crude residue was chromatographed as detailed below to give
compounds 33a,b and 37a-d as white semisolids.
1
]isoindoline (10). Yield 56%. H NMR (CDCl3): δ 1.18 (s, 3H),
1.21 (s, 3H), 1.42-1.49 (m, 2H), 1.61-1.67 (m, 2H), 1.77 (quintet,
2H, J ) 7.4 Hz), 2.40 (t, 2H, J ) 7.4 Hz), 2.48 (app. t, 2H), 2.66
(br s, 4H), 2.93 (app t, 4H), 3.45-3.51 (m, 1H), 4.81 (s, 4H),
7.08-7.16 (m, 3H), 7.25-7.31 (m, 5H). GC/MS: m/z 420 (M+
+1, 3), 419 (M+, 7), 245 (100), 217 (45), 118 (28); mp 114-116
°C (from CHCl3/n-hexane). Anal. (C27H37N3O) C, H, N.
2-[6-[4-(2-Methoxyphenyl)-1-piperazinyl]-1-oxohexyl]isoindo-
line (11). Yield 74%. 1H NMR (CDCl3): δ 1.37-1.47 (m, 2H),
1.55-1.65 (m, 2H), 1.76 (quintet, 2H, J ) 7.4 Hz), 2.39 (t, 2H, J
) 7.4 Hz), 2.42-2.46 (m, 2H), 2.66 (br s, 4H), 3.10 (br s, 4H),
3.86 (s, 3H), 4.80 (s, 4H), 6.84-7.02 (m, 4H), 7.25-7.33 (m, 4H).
GC/MS: m/z 408 (M+ +1, 5), 407 (M+, 18), 392 (39), 245 (74),
205 (100), 118 (50). The hydrochloride salt melted at 198-200 °C
(from MeOH/Et2O). Anal. (C25H33N3O2 ·2HCl·0.2H2O) C, H, N.
2-[6-[4-(2-Methylthiophenyl)-1-piperazinyl]-1-oxohexyl]-1,2,3,4-
tetrahydroisoquinoline (12). Yield 60%. 1H NMR (CDCl3): δ
1.38-1.46 (m, 2H), 1.58-1.61 (m, 2H), 1.66-1.76 (m, 2H), 2.41
(s, 3H), 2.42-2.47 (m, 4H), 2.64 (br s, 4H), 2.85 (app t, 1H), 2.90
(t, 1H J ) 5.8 Hz), 3.06 (br s, 4H), 3.68 (app t, 1H), 3.83 (t, 1H,
J ) 6.05 Hz), 4.62 (s, 1H), 4.73 (s, 1H), 7.04-7.10 (m, 4H),
N-(6-Bromo-1-oxohexyl)isoindoline (33a). Eluted with CHCl3/
1
AcOEt, 9:1; 80% yield. H NMR (CDCl3): δ 1.48-1.59 (m, 2H),
1.71-1.81 (m, 2H), 1.87-1.97 (m, 2H), 2.40 (app t, 2H), 3.43 (t,
2H, J ) 6.8 Hz), 4.80 (s, 4H), 7.24-7.31 (m, 4H). GC/MS: m/z
297 (M+ +2, 32), 295 (M+, 32), 216 (32), 161 (68), 160 (64), 146
(43), 118 (100).
N-(6-Bromo-1-oxohexyl)-1,2,3,4-tetrahydroisoquinoline (33b). Elut-
ed with CHCl3/AcOEt 9:1; 90% yield. 1H NMR (CDCl3): δ
1.46-1.56 (m, 2H), 1.66-1.76 (m, 2H), 1.85-1.95 (m, 2H), 2.42
(app t, 2H), 2.83-2.92(m, 2H), 3.39-3.44 (m, 2H), 3.38 (t, 1H, J
) 5.8 Hz), 3.83 (app t, 1H), 4.62 (s, 1H), 4.73 (s, 1H), 7.09-7.23
(m, 4H). GC/MS: m/z 311 (M+ +2, 91), 309 (M+, 93), 203 (54),
188 (43), 174 (93), 132 (100), 117 (51), 104 (42).

Piperazinehexanamides. Part III
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18 5819
7.12-7.22 (m, 4H). GC/MS: m/z 438 (M+ +1, 4), 437 (M+, 8),
259 (83), 221 (100), 206 (30), 132 (54). The hydrochloride salt
melted at 112-114 °C dec (from MeOH/Et2O). Anal.
(C26H35N3OS·HCl·H2O) C, H, N.
2-[6-[4-(2-Diphenyl)-1-piperazinyl]-1-oxohexyl]-1,2,3,4-tetrahy-
droisoquinoline (13). Yield 48%. 1H NMR (CDCl3): δ 1.30-1.43
(m, 2H), 1.47-1.52 (m, 2H), 1.61-1.73 (m, 2H), 2.36-2.42 (m,
8H), 2.82-2.88 (m, 6H), 3.66 (app t, 1H), 3.82 (app t, 1H), 4.60
(s, 1H), 4.72 (s, 1H), 7.02-7.10 (m, 2H), 7.12-7.31 (m, 7H),
7.36-7.41 (m, 2H), 7.61-7.64 (m, 2H). GC/MS: m/z 468 (M+
+1, 4), 467 (M+, 10), 259 (100), 251 (78), 180 (65). The
hydrochloride salt melted at 70-72 °C dec (from MeOH/Et2O).
Anal. (C31H37N3O·HCl·H2O) C, H, N.
2.40 (s, 3H), 2.53 (app, t, 2H), 2.75 (br s, 4H), 3.10 (br t, 4H),
4.46 (d, 2H, J ) 6.1 Hz), 6.23 (br t, 1H, D2O exchanged),
7.03-7.12 (m, 4H), 7.20 (dd, 2H, J ) 1.65, 4.4 Hz), 8.55 (dd, 2H,
J ) 1.65, 4.4 Hz). GC/MS: m/z 413 (M+ +1, 3), 412 (M+, 16),
397 (39), 234 (72), 221 (100), 206 (59); mp 50-52 °C (from
n-hexane). Anal. (C23H32N4OS) C, H, N.
4-(2-Diphenyl)-N-(4-pyridinylmethyl)-1-piperazinehexana-
1
mide (21). Yield 64%. H NMR (CDCl3): δ 1.28-1.38 (m, 2H),
1.43-1.54 (m, 2H), 1.68 (q, 2H, J ) 7.4 Hz), 2.25 (t, 2H, J ) 7.4
Hz), 2.31-2.33 (m, 2H), 2.35 (br s, 4H), 2.86 (app t, 4H), 4.44 (d,
2H, J ) 6.1 Hz), 6.00 (br t, 1H, D2O exchanged), 7.01-7.09 (m,
2H), 7.16-7.18 (m, 2H), 7.22-7.31 (m, 3H), 7.36-7.41 (m, 2H),
7.60-7.64 (m, 2H), 8.54 (dd, 2H, J ) 1.7, 4.4 Hz). GC/MS: m/z
443 (M+ +1, 1), 442 (M+, 10), 251 (82), 234 (78), 180 (100). The
hydrochloride salt melted at 50 °C dec (from MeOH/Et2O). Anal.
(C28H34N4O·2HCl·1.5H2O) C, H, N.
2-[6-[4-(2-(1-Methylethyl)phenyl)-1-piperazinyl]-1-oxohexyl]-
1,2,3,4-tetrahydroisoquinoline (14). Yield 95%. 1H NMR (CDCl3):
δ 1.19 (s, 3H), 1.21 (s, 3H), 1.38-1.46 (m, 2H), 1.55-1.62 (m,
2H), 1.66-1.77 (m, 2H), 2.40-2.47 (m, 4H), 2.67 (br s, 4H), 2.85
(app t, 1H), 2.88-2.94 (m, 5H), 3.44-3.53 (m, 1H), 3.67 (app t,
1H), 3.83 (t, 1H, J ) 6.05 Hz), 4.62 (s, 1H), 4.73 (s, 1H), 7.06-7.20
(m, 6H), 7.21-7.26 (m, 2H). GC/MS: m/z 434 (M+ +1, 2), 433
(M+, 5), 259 (100), 217 (38). The oxalate salt melted at 210-212
°C (from MeOH/Et2O). Anal. (C28H39N3O·(COOH)2) C, H, N.
2-[6-[4-(2-Methoxyphenyl)-1-piperazinyl]-1-oxohexyl]-1,2,3,4-tet-
rahydroisoquinoline (15). Yield 90%. 1H NMR (CDCl3): δ
1.35-1.45 (m, 2H), 1.55-1.61 (m, 2H), 1.65-1.77 (m, 2H),
2.39-2.46 (m, 4H), 2.67 (br s, 4H), 2.85 (t, 1H, J ) 6.05 Hz),
2.90 (app. t, 1H), 3.11 (br s, 4H), 3.68 (app t, 1H), 3.83 (t, 1H, J
) 6.05 Hz), 3.86 (s, 3H), 4.62 (s, 1H), 4.73 (s, 1H), 6.84-7.03
(m, 4H), 7.10-7.22 (m, 4H). GC/MS: m/z 422 (M+ +1, 5), 421
(M+, 16), 406 (46), 259 (83), 205 (100), 190 (32). The hydrochlo-
ride salt melted at 164-166 °C (from MeOH/Et2O). Anal.
(C26H35N3O2 ·2HCl·0.7H2O) C, H, N.
4-[2-(1-Methylethyl)phenyl]-N-(4-pyridinylmethyl)-1-piperazine-
1
hexanamide (22). Yield 95%. H NMR (CDCl3): δ 1.18 (s, 3H),
1.21 (s, 3H), 1.35-1.45 (m, 2H), 1.55-1.65 (m, 2H), 1.73 (quintet,
2H, J ) 7.4 Hz), 2.29 (t, 2H, J ) 7.4 Hz), 2.44 (app. t, 2H), 2.64
(br s, 4H), 2.94 (app t, 4H), 3.43-3.52 (m, 1H), 4.46 (d, 2H, J )
6.1 Hz), 6.10 (br t, 1H, D2O exchanged), 7.06-7.16 (m, 4H),
7.18-7.20 (m, 1H), 7.24-7.25 (m, 1H), 8.55 (dd, 2H, J ) 1.4, 4.4
Hz). GC/MS: m/z 409 (M+ +1, 1), 408 (M+, 6), 234 (100), 217
(58), 160 (30). The hydrochloride salt melted at 163-166 °C dec
(from MeOH/Et2O). Anal. (C25H36N4O·3HCl·H2O) C, H, N.
4-(2-Methoxyphenyl)-N-(4-pyridinylmethyl)-1-piperazinehex-
anamide (23). Yield 70%. 1H NMR (CDCl3): δ 1.34-1.43 (m, 2H),
1.53-1.63 (m, 2H), 1.67-1.77 (m, 2H), 2.28 (t, 2H, J ) 7.4 Hz),
2.42 (app. t, 2H), 2.66 (br s, 4H), 3.10 (br s, 4H), 3.85 (s, 3H),
4.45 (d, 2H, J ) 6.1 Hz), 6.10 (br t, 1H, D2O exchanged),
6.84-7.03 (m, 4H), 7.18 (dd, 2H, J ) 1.6, 4.4 Hz), 8.54 (dd, 2H,
J ) 1.6, 4.4 Hz). GC/MS: m/z 397 (M+ +1, 2), 396 (M+, 10), 234
(36), 205 (100), 190 (38); mp 93-94 °C (from n-hexane). Anal.
(C23H32N4O2) C, H, N.
N-(4-Cyanophenylmethyl)-4-(2-methylthiophenyl)-1-piperazine-
hexanamide (24). Yield 66%. 1H NMR (CDCl3): δ 1.33-1.43 (m,
2H), 1.52-1.62 (m, 2H), 1.66-1.76 (m, 2H), 2.27 (t, 2H, J ) 7.4
Hz), 2.40 (s, 3H), 2.42-2.45 (m, 2H), 2.63 (br s, 4H), 3.03 (app t,
4H), 4.50 (d, 2H, J ) 4.5 Hz), 6.00 (br t, 1H, D2O exchanged),
7.02-7.13 (m, 4H), 7.36-7.39 (m, 2H), 7.60-7.63 (m, 2H). GC/
MS: m/z 437 (M+ +1, 5), 436 (M+, 16), 221 (100), 206 (19), 116
(23). The hydrochloride salt melted at 100-103 °C dec (from
MeOH/Et2O). Anal. (C25H32N4OS·HCl·H2O) C, H, N.
N-Benzyl-4-(2-methylthiophenyl)-1-piperazinehexanamide (16).
1
Yield 90%. H NMR (CDCl3): δ 1.34-1.42 (m, 2H), 1.52-1.62
(m, 2H), 1.71 (quintet, 2H, J ) 7.4 Hz), 2.23 (t, 2H, J ) 7.4 Hz),
2.41 (s, 3H), 2.42-2.45 (m, 2H), 2.64 (br s, 4H), 3.03 (br t, 4H),
4.44 (d, 2H, J ) 5.5 Hz), 5.80 (br t, 1H, D2O exchanged),
7.04-7.11 (m, 4H), 7.26-7.36 (m, 5H). GC/MS: m/z 442 (M+
+1, 3), 441 (M+, 10), 251 (100), 233 (38), 180 (61). The
hydrochloride salt melted at 150-152 °C (from MeOH/Et2O). Anal.
(C24H33N3OS·HCl·H2O) C, H, N.
N-Benzyl-4-(2-diphenyl)-1-piperazinehexanamide (17). Yield
90%. 1H NMR (CDCl3): δ 1.27-1.37 (m, 2H), 1.44-1.54 (m, 2H),
1.66 (quintet, 2H, J ) 7.4 Hz), 2.20 (t, 2H, J ) 7.4 Hz), 2.30 (app.
t, 2H), 2.35 (br s, 4H), 2.85 (app t, 4H), 4.43 (d, 2H, J ) 5.8 Hz),
5.79 (br t, 1H, D2O exchanged), 7.02-7.09 (m, 2H), 7.23-7.41
(m, 10H), 7.61-7.64 (m, 2H). GC/MS: m/z 412 (M+ +1, 3), 411
(M+, 6), 233 (42), 221 (100), 206 (27). The hydrochloride salt
melted at 212-214 °C (from MeOH/Et2O). Anal. (C29H35N3O·HCl)
C, H, N.
N-(4-Cyanophenylmethyl)-4-(2-diphenyl)-1-piperazinehexana-
1
mide (25). Yield 90%. H NMR (CDCl3): δ 1.27-1.37 (m, 2H),
1.44-1.54 (m, 2H), 1.66 (quintet, 2H, J ) 7.4 Hz), 2.23 (t, 2H, J
) 7.4 Hz), 2.28-2.32 (m, 2H), 2.35 (br s, 4H), 2.85 (app t, 4H),
4.48 (d, 2H, J ) 6.1 Hz), 5.97 (br t, 1H, D2O exchanged),
7.00-7.10 (m, 2H), 7.22-7.31 (m, 3H), 7.33-7.41 (m, 4H),
7.58-7.64 (m, 4H). GC/MS: m/z 467 (M+ +1, 9), 466 (M+, 20),
251 (100), 180 (51), 116 (26); mp 50 °C dec (from n-hexane). Anal.
(C30H34N4O) C, H, N.
N-Benzyl-4-[2-(1-methylethyl)phenyl]-1-piperazinehexana-
1
mide (18). Yield 90%. H NMR (CDCl3): δ 1.19 (s, 3H), 1.21 (s,
3H), 1.33-1.43 (m, 2H), 1.53-1.63 (m, 2H), 1.71 (quintet, 2H, J
) 7.4 Hz), 2.24 (app t, 2H), 2.43 (app. t, 2H), 2.62 (br s, 4H), 2.92
(app t, 4H), 3.41-3.55 (m, 1H), 4.45 (d, 2H, J ) 5.8 Hz), 5.80 (br
t, 1H, D2O exchanged), 7.06-7.18 (m, 4H), 7.24-7.37 (m, 5H).
GC/MS: m/z 408 (M+ +1, 2), 407 (M+, 4), 233 (100), 217 (73),
160 (21). The hydrochloride salt melted at 116-119 °C (from
MeOH/Et2O). Anal. (C26H37N3O·HCl·1.2H2O) C, H, N.
N-(4-Cyanophenylmethyl)-4-[2-(1-methylethyl)phenyl]-1-pipera-
zinehexanamide (26). Yield 83%. 1H NMR (CDCl3): δ 1.18 (s,
3H), 1.21 (s, 3H), 1.34-1.43 (m, 2H), 1.53-1.64 (m, 2H), 1.72
(quintet, 2H, J ) 7.4 Hz), 2.28 (t, 2H, J ) 7.4 Hz), 2.43 (app. t,
2H), 2.62 (br s, 4H), 2.92 (app t, 4H), 3.43-3.52 (m, 1H), 4.50 (d,
2H, J ) 6.1 Hz), 6.03 (br t, 1H, D2O exchanged), 7.07-7.18 (m,
3H), 7.24-7.27 (m, 1H), 7.38 (d, 2H, J ) 8.3 Hz), 7.60-7.63 (m,
2H). GC/MS: m/z 433 (M+ +1, 3), 432 (M+, 8), 258 (100), 217
(99), 160 (34), 132 (31). The hydrochloride salt melted at 83-85
°C dec (from MeOH/Et2O). Anal. (C27H36N4O·2HCl) C, H, N.
N-(4-Cyanophenylmethyl)-4-(2-methoxyphenyl)-1-piperazine-
hexanamide (27). Yield 95%. 1H NMR (CDCl3): δ 1.33-1.43 (m,
2H), 1.53-1.63 (m, 2H), 1.71 (quintet, 2H, J ) 7.4 Hz), 2.27 (t,
2H, J ) 7.4 Hz), 2.43 (app. t, 2H), 2.67 (br s, 4H), 3.10 (app t,
4H), 3.86 (s, 3H), 4.49 (d, 2H, J ) 6.1 Hz), 6.03 (br t, 1H, D2O
exchanged), 6.84-6.89 (m, 1H), 6.916.96 (m, 2H), 6.97-7.03 (m,
1H), 7.37 (d, 2H, J ) 8.5 Hz), 7.59-7.62 (m, 2H). GC/MS: m/z
N-Benzyl-4-(2-methoxyphenyl)-1-piperazinehexanamide (19).
1
Yield 82%. H NMR (CDCl3): δ 1.32-1.42 (m, 2H), 1.52-1.62
(m, 2H), 1.71 (quintet, 2H, J ) 7.4 Hz), 2.23 (t, 2H, J ) 7.4 Hz),
2.42 (app. t, 2H), 2.66 (br s, 4H), 3.10 (br s, 4H), 3.86 (s, 3H),
4.44 (d, 2H, J ) 5.5 Hz), 5.79 (br t, 1H, D2O exchanged),
6.84-7.03 (m, 4H), 7.27-7.37 (m, 5H). GC/MS: m/z 396 (M+
+1, 4), 395 (M+, 15), 233 (27), 205 (100), 190 (29). The
hydrochloride salt melted at 174-176 °C (from MeOH/Et2O). Anal.
(C24H33N3O2 ·2HCl·0.5H2O) C, H, N.
4-(2-Methylthiophenyl)-N-(4-pyridinylmethyl)-1-piperazinehex-
anamide (20). Yield 87%. 1H NMR (CDCl3): δ 1.36-1.46 (m, 2H),
1.61-1.69 (m, 2H), 1.71-1.79 (m, 2H), 2.31 (t, 2H, J ) 7.4 Hz),

5820 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18
Leopoldo et al.
421 (M+ +1, 7), 420 (M+, 24), 205 (100), 190 (20); mp 123-125
°C dec (from CHCl3/n-hexane). Anal. (C25H32N4O2) C, H, N.
2. Radioligand Binding Assay at Rat Cloned 5-HT7Rs. Binding
of [3H]-LSD at rat cloned 5-HT7 receptor was performed according
to Jasper et al.43 with minor modifications. In 1 mL of incubation
buffer (50 mM Tris, 10 mM MgCl2 and 0.5 mM EDTA, pH 7.4)
were suspended 30 µg of membranes, 2.5 nM [3H]-LSD, the drugs,
or reference compound (6-9 concentrations). The samples were
incubated for 60 min at 37 °C. The incubation was stopped by rapid
filtration on GF/A glass fiber filters (presoaked in 0.5% polyeth-
ylenimine for 30 min). The filters were washed with 3 × 53 mL of
ice-cold buffer (50 mM Tris, pH 7.4). Nonspecific binding was
determined in the presence of 10 µM 5-CT. Approximately 90%
of specific binding was determined under these conditions.
N-(4-Methanesulfonyloxyphenylmethyl)-4-(2-methylthiophenyl)-
1-piperazinehexanamide (28). Yield 95%. 1H NMR (CDCl3): δ
1.32-1.42 (m, 2H), 1.53-1.63 (m, 2H), 1.70 (quintet, 2H, J )
7.4 Hz), 2.24 (t, 2H, J ) 7.4 Hz), 2.41 (s, 3H), 2.43-2.46 (m,
2H), 2.65 (br s, 4H), 3.04 (app t, 4H), 3.13 (s, 3H), 4.45 (d, 2H, J
) 5.8 Hz), 5.94 (br t, 1H, D2O exchanged), 7.03-7.16 (m, 4H),
7.23-7.25 (m, 2H), 7.32-7.35 (m, 2H). ESI+/MS m/z 506 (MH+).
ESI+/MS/MS m/z 341 (42), 298 (100), 249 (49), 185 (53). The
hydrochloride salt melted at 118-120 °C (from MeOH/Et2O). Anal.
(C25H35N3O4S2 ·HCl·0.7H2O) C, H, N.
3. Radioligand Binding Assay at Human Cloned 5-HT1A Recep-
tor. Human 5-HT1A serotonin receptors stably expressed in HEK-
293 cells were radiolabeled with 1.0 nM [3H]-8-OH-DPAT.44
Samples containing 40 µg of membrane protein and different
concentrations of each compound ranging from 0.1 nM to 10 µM
were incubated in a final volume of 500 µL of 50 mM Tris-HCl
pH 7.4, 5 mM MgSO4 for 120 min at 37 °C. After this incubation
time, samples were filtered through GF/C presoaked in polyethyl-
enimine 0.5% for at least 30 min prior to use. The filters were
washed twice with 1 mL of ice-cold buffer (50 mM Tris-HCl, pH
7.4). Nonspecific binding was determined in the presence of 10
µM 5-HT.
N-(4-Methanesulfonyloxyphenylmethyl)-4-(2-diphenyl)-1-pipera-
zinehexanamide (29). Yield 95%. 1H NMR (CDCl3): δ 1.27-1.36
(m, 2H), 1.44-1.54 (m, 2H), 1.66 (quintet, 2H, J ) 7.7 Hz), 2.21
(t, 2H, J ) 7.7 Hz), 2.28-2.33 (m, 2H), 2.36 (br s, 4H), 2.86 (app
t, 4H), 3.13 (s, 3H), 4.43 (d, 2H, J ) 6.1 Hz), 5.89 (br t, 1H, D2O
exchanged), 7.01-7.10 (m, 2H), 7.22-7.41 (m, 9H), 7.61-7.64
(m, 2H). ESI+/MS m/z 536 (MH+). ESI+/MS/MS m/z 298 (100),
251 (32), 185 (56). The hydrochloride salt melted at 160-163 °C
(from MeOH/Et2O). Anal. (C30H37N3O4S·HCl·H2O) C, H, N.
N-(4-Methanesulfonyloxyphenylmethyl)-4-[2-(1-methylethyl)phe-
nyl]-1-piperazinehexanamide (30). Yield 88%. 1H NMR (CDCl3):
δ 1.18 (s, 3H), 1.21 (s, 3H), 1.33-1.43 (m, 2H), 1.53-1.63 (m,
2H), 1.71 (quintet, 2H, J ) 7.7 Hz), 2.25 (t, 2H, J ) 7.7 Hz), 2.42
(app. t, 2H), 2.61 (br s, 4H), 2.92 (app t, 4H), 3.13 (s, 3H),
3.43-3.53 (m, 1H), 4.45 (d, 2H, J ) 4.5 Hz), 5.94 (br t, 1H, D2O
exchanged), 7.06-7.18 (m, 4H), 7.22-7.26 (m, 2H), 7.32-7.35
(m, 2H). ESI+/MS m/z 502 (MH+). ESI+/MS/MS m/z 298 (100),
185 (56). The hydrochloride salt melted at 83-85 °C dec (from
MeOH/Et2O). Anal. (C27H39N3O4S·HCl·1.5H2O) C, H, N.
N-(4-Methanesulfonyloxyphenylmethyl)-4-(2-methoxyphenyl)-
1-piperazinehexanamide (31). Yield 80%. 1H NMR (CDCl3): δ
1.32-1.42 (m, 2H), 1.52-1.62 (m, 2H), 1.70 (quintet, 2H, J )
7.4 Hz), 2.24 (t, 2H, J ) 7.4 Hz), 2.40 (app. t, 2H), 2.65 (br s,
4H), 3.09 (br s, 4H), 3.13 (s, 3H), 3.86 (s, 3H), 4.44 (d, 2H, J )
6.1 Hz), 5.89 (br t, 1H, D2O exchanged), 6.84-7.03 (m, 4H),
7.21-7.27 (m, 2H), 7.31-7.36 (m, 2H). ESI+/MS m/z 490 (MH+).
ESI+/MS/MS m/z 396 (65), 298 (100), 185 (56). The hydrochloride
salt melted at 146-149 °C dec (from MeOH/Et2O). Anal.
(C25H35N3O5S·2HCl·H2O) C, H, N.
Lipophilicity. Lipophilicity indices were measured by a reversed-
phase HPLC method consisting in a Perkin-Elmer series 200 LC
apparatus equipped with a Perkin-Elmer 785A UV/vis detector set
at 254 nm. UV signals were monitored and obtained peaks
integrated using a personal computer running Perkin-Elmer Tur-
bochrom Software. The capacity factors were measured with a
Phenomenex Prodigy ODS-3 RP-18 (250 mm × 4.6 mm, 5 µm
particle size) as nonpolar stationary phase and with MeOH/0.01 M
phosphate buffer pH 6 (70:30 v/v) as mobile phase. This ratio was
found to be the best compromise for a large number of derivatives.
All compounds were dissolved in methanol, and the measurements
were made at a flow rate of 1 mL/min. Capacity factors were
calculated as: k′ ) (tR - t0)/t0, where tR is the retention time of the
solute and t0 is the column dead time, measured as the solvent front.
Biological Methods. 1. General. Male albino Dunkin-Hartley
guinea pigs (300-350 g) were from Harlan (S. Pietro al Natisone,
Italy). The animals were handled according to internationally
accepted principles for care of laboratory animals (EEC Council
Directive 86/609, O. J. no. L358, Dec 18, 1986). Rat recombinant
serotonin 5-HT7 receptors and human recombinant serotonin 5-HT1A
receptors expressed in HEK-293 cells were purchased from Perkin-
Elmer (Zaventem, Belgium). Human recombinant D2L dopamine
receptors expressed in rat C6 glioma cells were gifted by Prof.
Roberto Maggio (Universita` di L’Aquila, Italy). [3H]-LSD, [3H]-
8-OH-DPAT, and [3H]-spiroperidol were obtained from Perkin-
Elmer-NEN (Zaventem, Belgium). Compound 2, 5-CT, and sub-
stance P were purchased from Tocris Bioscience (Bristol, UK).
8-OH-DPAT hydrobromide was from RBI. Haloperidol were
purchesed from Sigma-Aldrich (Milan, Italy).
4. Radioligand Binding Assay at Human Cloned D2L Dopa-
minergic Receptors. Binding of [3H]-spiroperidol at human cloned
receptors was performed according to Scarselli et al.45 with minor
modifications. The incubation buffer (120 mM NaCl, 5.0 mM KCl,
5.0 mM MgCl2, 1 mM EDTA, 50 mM Tris, pH 7.4) contained 100
µg of dopamine dilute membranes, 0.30-0.50 nM [3H]-spiroperidol
(Kd ) 0.093 nM), and 6-9 concentrations of drug solution in a
final volume of 500 µL. The samples were incubated for 120 min
at 25 °C, then the incubation was stopped by rapid filtration through
Whatman GF/C glass fiber filters (presoaked in 0.5% polyethyl-
enimine for 60 min). The filters were washed 3 × 1 mL of ice-
cold 50 mM Tris, 0.9% NaCl, pH 7.4. Non specific binding was
determined in the presence of 10 µM haloperidol. The radioactivity
bound to the filters was measured by liquid scintillation using
LS6500 multipurpose scintillation Counter, Beckman.
5. Isolated Guinea-Pig Ileum Assay. guinea pigs were anes-
thetized and then decapitated and the proximal ileum removed. The
intestinewascarefullyflushedseveraltimeswithwarmKrebs-Henseleit
solution (118 mM NaCl, 25 mM NaHCO3, 4.7 mM KCl, 0.6 mM
MgSO4, 1.2 mM KH2PO4, 1.2 mM CaCl2, 11.2 mM glucose, pH
7.4). Whole ileal segments, of about 3 cm in length, were suspended
under 1.0 g tension in Krebs solution gassed with 95% O2 and 5%
CO2 and maintained at 37 °C. According to Carter et al.46 with
minor modification, the bathing medium contained 1 µM atropine
to antagonize cholinergically mediated contractions due to activation
of 5-HT3 and 5-HT4 receptors, 1 µM ketanserin to block 5-HT2A
receptors, and 1 µM pyrilamine to block H1 receptors. Changes in
tension of the tissue were recorded by Fort 10 Original WPI
isometric transducer (2Biological Instruments, Italy) connected to
a PowerLab/400 workstation.47 Tissue responses were recorded as
gram changes in isometric tension and expressed as percentage of
reduction in the height of the contraction. Tissue was contracted
by 100 nM substance P. This value was preliminary determined
by concentration-response curves (1 nM to 200 nM). 100 nM
substance P elicited 80% of maximum contraction. The reference
agonist 5-CT was added 5 min before substance P addition and
noncumulative concentration-response curves were constructed
(0.001-µM). We have determined that 5-CT induced relaxation
with maximal response (39%) at 3 µM concentration. Analogously,
each compound tested (0.001-10 µM) was added 5 min before
substance P addition and noncumulative concentration-response
curve was constructed. Relaxant responses to full agonists 5-CT
13, 21, and 25 were surmountably antagonized by 2 (0.1-3 µM).
The isolated guinea pig ileum was equilibrated for 15 min with 2
before constructing concentration-response curves of tested com-
pounds. The corresponding pA2 values of 2 versus each studied

Piperazinehexanamides. Part III
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 18 5821
agonist are listed in Table 4. Desensitization was then used to
discriminate if the studied compounds were acting as 5-HT7 receptor
agonists or nonspecific smooth muscle relaxant. The agonist studies
were consequently repeated in the presence of 3 µM 5-CT,
following equilibration with 5-CT for 60 min, during which time
the bathing solution was changed at 15 min intervals. This
concentration of 5-CT abolished relaxant responses emanating from
5-HT7 receptor stimulation.
6. Disposition Studies. Male CD1 albino mice, 25-30 g
(Charles River, Italy) were used. Procedures involving animals and
their care were conducted in conformity with the institutional
guidelines that are in compliance with national (D.L., no. 116, G.U.,
suppl. 40, 18 Febbraio 1992, Circolare no. 8, G.U., 14 Luglio 1994)
and international laws and policies (EEC Council Directive 86/
609, O. J. L358, 1, Dec 12, 1987; Guide for the Care and Use of
Laboratory, U.S. National Research Council, 1996).
Mice were given the test compounds intraperitoneally (10 mg/
kg) and were killed by decapitation at various times thereafter.
Mixed arteriovenous trunk blood was collected in heparinized tubes,
centrifuged at 3000g for 10 min, and the plasma was stored at -20
°C. Brain was removed immediately, blotted with paper to remove
surface blood, and quickly frozen in dry ice. Compounds and their
1-arylpiperazine metabolites were extracted from plasma and brain
homogenate and quantified by reversed-phase HPLC with UV
detection (230 nm). Briefly: to 0.1 mL of plasma, 0.2 mL of 20
mM of ammonium bicarbonate and 0.02 mL of a methanolic
solution of the internal standard (100 µg/mL) were added; samples
were then extracted twice with 1.5 mL of hexane containing 1%
of isoamyl alcohol, and the combined extracts were evaporated to
dryness and reconstituted in 0.15 mL of the mobile phase, which
was injected into the chromatographic column. Brain tissue was
homogenized in distilled water (1 g/10 mL), and 1 mL of the
homogenate was extracted twice with 1.5 mL of hexane/isoamyl
alcohol as described for plasma. Then, the organic phase was shaken
with 0.2 mL of the mobile phase (compound 25 only); after
centrifugation, 0.1 mL of the aqueous phase was injected into the
chromatographic column.
Separation was done on a Brownlee Laboratory Spheri 5 RP 8
(4.6 mm × 250 mm; 5 µm), with 0.01 M KH2PO4/CH3CN/CH3OH
(50:45:5 v/v, compound 25; 50:40:10 v/v, compound 4), at pH 3.5,
as the mobile phase. A derivative of the present series was used as
internal standard.
Standard curves were prepared by spiking blank mouse plasma
and brain homogenate. The lower limit for quantification was about
0.1 µg/mL in plasma and 0.1 µg/g in brain tissue, using respectively
0.1 mL of plasma and 1 mL of brain homogenate (or approximately
100 mg of brain tissue). At these concentrations the coefficient of
variation for the precision of the assay was 10-20%. Higher
concentrations generally gave coefficients of variation less than
10%, regardless of the compound and tissue analyzed. Brain
concentrations were not corrected for compounds 4 and 25
contributed by the residual blood, because this generally amounted
to only 1-3% of the measured brain concentrations, assuming a
mean value of 20 µL per gram of brain.48
7. Statistical and Pharmacokinetic Analysis. The inhibition
curves on the different binding sites of the compounds reported in
Tables 2 and 3 were analyzed by nonlinear curve fitting utilizing
the GraphPad Prism program.49 The value for the inhibition
constant, Ki, was calculated by using the Cheng-Prusoff equation.50
Agonist potencies, expressed as EC50, were obtained from nonlinear
iterative curve fitting by GraphPad Prism.
Supporting Information Available: Elemental analysis data of
target compounds. This material is available free of charge via the
Internet at http://pubs.acs.org.
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