Solid-supported synthesis, molecular modeling, and biological activity of long-chain arylpiperazine derivatives with cyclic amino acid amide fragments as 5-HT7 and 5-HT1A receptor ligands

Article abstract of DOI:10.1016/j.ejmech.2014.03.005

A 47-membered library of novel long-chain arylpiperazines, which contained cyclic amino acid amides in the terminal fragment (pyrrolidine-2-carboxamide and 1,2,3,4-tetrahydroisoquinoline-3-carboxamide), was synthesized on Rink-amide resin and biologically evaluated for binding affinity for 5-HT₇ and 5-HT_1A receptors. Surprisingly, members of the designed series containing piperidine-2-carboxamide fragments underwent hydrolysis, which occurred during the acidic treatment for release from the solid-support, to their respective pipecolic acid analogs. Representative compounds from the library displayed high-to-low affinity for 5-HT₇ (K_i = 18-3134 nM) and 5-HT_1A (K_i = 0.5-6307 nM) sites. The possible interactions implicated in binding of the studied compounds to the 5-HT₇ receptor were supported by molecular modeling. Research was also applied to support the exploration of the influence of the amide fragment, the length of alkylene spacer, and arylpiperazine substituents on the receptor's affinity and selectivity.

Full text of DOI:10.1016/j.ejmech.2014.03.005

European Journal of Medicinal Chemistry 78 (2014) 10e22
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Solid-supported synthesis, molecular modeling, and biological activity
of long-chain arylpiperazine derivatives with cyclic amino acid amide
fragments as 5-HT7 and 5-HT1A receptor ligands
Vittorio Canale a, Pawe1 Guzik a, Rafa1 Kurczab b, Pascal Verdie c, Grzegorz Sata1a b,
Bart1omiej Kubica a, Maciej Paw1owski a, Jean Martinez c, Gilles Subra c,
,
Andrzej J. Bojarski b, Pawe1 Zajdel a
*
a Department of Medicinal Chemistry, Jagiellonian University Medical College, 9 Medyczna Street, 30-688 Kraków, Poland
b Department of Medicinal Chemistry, Institute of Pharmacology, Polish Academy of Sciences, 12 Sme˛tna Street, 31-343 Kraków, Poland
c Department of Amino Acids, Peptides and Proteins, Institute Biomolecules Max Mousseron, University Montpellier 1, 15 Avenue Charles Flahault,
34-093 Montpellier, France
a r t i c l e i n f o
a b s t r a c t
Article history:
A 47-membered library of novel long-chain arylpiperazines, which contained cyclic amino acid amides in
the terminal fragment (pyrrolidine-2-carboxamide and 1,2,3,4-tetrahydroisoquinoline-3-carboxamide),
was synthesized on Rink-amide resin and biologically evaluated for binding affinity for 5-HT7 and 5-HT1A
receptors. Surprisingly, members of the designed series containing piperidine-2-carboxamide fragments
underwent hydrolysis, which occurred during the acidic treatment for release from the solid-support, to
their respective pipecolic acid analogs. Representative compounds from the library displayed high-to-
low affinity for 5-HT7 (Ki ¼ 18e3134 nM) and 5-HT1A (Ki ¼ 0.5e6307 nM) sites. The possible in-
teractions implicated in binding of the studied compounds to the 5-HT7 receptor were supported by
molecular modeling. Research was also applied to support the exploration of the influence of the amide
fragment, the length of alkylene spacer, and arylpiperazine substituents on the receptor’s affinity and
selectivity.
Received 22 August 2013
Received in revised form
2 February 2014
Accepted 4 March 2014
Available online 5 March 2014
Keywords:
Long-chain arylpiperazines
Solid-phase synthesis
Prolinamides
Pipecolic acid
1,2,3,4-Tetrahydroisoquinoline-3-
carboxamides
Ó 2014 Elsevier Masson SAS. All rights reserved.
Hydrogen bonding
5-HT7 receptor ligands
5-HT1A receptor ligands
1. Introduction
could represent a new therapeutic target for the treatment of pain
[5,6].
The 5-HT7 receptor (5-HT7R) is the latest addition to a family of
serotonin receptors that are positively coupled to adenylyl cyclase
through a Gs protein [1]. The distribution of 5-HT7Rs in several
region of the brain, such as the hippocampus, hypothalamus, or
thalamus, has fortified interest in these sites as potential targets for
drug development. Notably, several antidepressant and antipsy-
chotic drugs display high affinity for 5-HT7Rs, and the antagonism
of 5-HT7Rs produces antidepressant and pro-cognitive effects [2e
4]. On the other hand, the activation of 5-HT7Rs produces anti-
nociceptive effects. Potential applications of 5-HT7R agonists as
co-adjuvants in opioid-mediated analgesia have been proposed and
In parallel to the acquisition of more detailed insight into the
function of 5-HT7R pharmacology, an academic and pharmaceutical
research was directed toward the development of 5-HT7R ligands
[7]. Several potent 5-HT7R agents belong to the group of long-chain
arylpiperazines (LCAP) [8]. Unfortunately, the fact that the arylpi-
perazine privileged core is easily recognized by other mono-
aminergic receptors (e.g., 5-HT1A, 5-HT2A, D2, D3, a1, H1) limits the
selectivity of the LCAPs [9,10].
Some of these obstacles were recently overcome by Leopoldo
et al. [11], who reported the generation of an amide series of potent
5-HT7R ligands (Fig. 1). The modification of the phenylpiperazine
pharmacophore and variation in the linker length were critical to
achieving 5-HT7/5-HT1A receptor selectivity.
Inspired by these findings, we continued our search for 5-HT7
ligands by designing a series of LCAPs functionalized with the
* Corresponding author.
E-mail address: pawel.zajdel@uj.edu.pl (P. Zajdel).
http://dx.doi.org/10.1016/j.ejmech.2014.03.005
0223-5234/Ó 2014 Elsevier Masson SAS. All rights reserved.

V. Canale et al. / European Journal of Medicinal Chemistry 78 (2014) 10e22
11
Fig. 1. LCAP examples of 5-HT7R ligands.
primary amides of cyclic amino acids in the terminal fragment
[12,13]. These compounds may be considered LP-44 analogs
because the exocyclic amide bond was replaced with an endocyclic
one and an additional primary amide group was introduced
concurrently (Fig. 2). On the other hand, these studies may be
regarded as an extension of our ongoing efforts toward the verifi-
cation of an effect caused by an amino acid-derived terminal frag-
ment on the affinity and selectivity of monoaminergic receptors
[14]. The latter issue was extensively evaluated in a series con-
taining N-acylated proline and cyclized aspartic acid moieties [15].
We present the design, which was directed by computational
modeling and simulations, and solid-supported synthesis of a 47-
membered library as well as the biological evaluation of selected
library members for binding to 5-HT7 and 5-HT1A receptors.
The structural modifications comprised the introduction of
three cyclic amino acids to the terminal fragment, giving proline
amides (Pro-amides, set I), piperidine-2-carboxamides (Pip-am-
ides, set II), and 1,2,3,4-tetrahydroisoquinoline-3-carboxamides
(Tic-amides, set III) as well as variation in the length of the alky-
lene spacer and the diversification of the aryl fragments of the N1-
piperazine moiety.
available Rink-amide resin, followed by coupling with Fmoc-
protected -amino acid 2{1e3} (Fig. 3) with HBTU.
After the removal of the Fmoc group, the resulting secondary
amines 3{1e3} were reacted with -bromo-acyl chlorides 4{1e4}
(Fig. 4) to furnish the corresponding amino acid derivatives 5{1e3,
1e4}.
The resin-bound intermediates were further submitted to a
nucleophile displacement with various substituted arylpiperazine
derivatives 6{1e10} (Fig. 5) in DMF at 75 ꢀC for 24 h.
The final compounds with general structures 8e24{1, 1e4, 1e
10}, 25e36{2, 1e3, 1e10} and 37e54{3, 1e4, 1e10} were released
from the resin via treatment with a mixture of TFA/CH2Cl2 (80/20,
v/v). All of the compounds were purified with a Waters preparative
LC/MS apparatus. The final compounds belonging to sets I (8e24)
and III (37e54) were obtained in moderate yields (33e45%) and
excellent purities (97e99%). In contrast, the designed pipecolic
amid derivatives (set II), when treated with acidic medium during
cleavage from solid support, underwent hydrolysis to form car-
boxylic acid analogs (Table 1).
Although a similar hydrolytic process was observed for an amide
bond in N-acylated pipecolic acid derivatives on a BAL linker
[16,17], no reports concerning the hydrolysis of pipecolic amides on
Rink-amide resin have been disclosed. Following our previous re-
ports, a detailed mechanism of the unexpected amide bond cleav-
age was proposed (Scheme 2). First, an intramolecular tetrahedral
intermediate was formed; the lone pair of electrons on the nitrogen
atom was no longer conjugated with the carbonyl bond in this
moiety. Consequently, a nitrogen atom became a proton acceptor,
allowing for the release of the cyclic structure from the support and
the formation of the oxazolinium intermediate (Muchnone). The
L
u
2. Results and discussion
2.1. Chemistry
Synthesis of the designed compounds was carried out on solid
support according to a six-step procedure (Scheme 1). Our work-
flow began with the base-mediated deprotection of a commercially
Fig. 2. General structure of the designed pyrrolidine-2-carboxamides (set I), piperidine-2-carboxamides (set II), and 1,2,3,4-tetrahydroisoquinoline-3-carboxamides (set III).

12
V. Canale et al. / European Journal of Medicinal Chemistry 78 (2014) 10e22
Scheme 1. Solid-phase synthesis of LCAP-containing cyclic amino acid amide fragments. Reagents and conditions: (i) piperidine/DMF (20/80, v/v), 20 min, rt; (ii) Fmoc-protected
amino acid 2{1e3}, HBTU, DIEA, DMF, 2 ꢂ 2 h, rt; (iii) Acyl chloride 4{1e4}, DIEA, CH2Cl2, 4 h, rt; (iv) Arylpiperazine 6{1e10}, DMF, 24 h, 75 ꢀC; (v) TFA/CH2Cl2 (80/20, v/v), 2 h, rt.
latter intermediate reacted with traces of water to yield the pipe-
colic acid derivatives with concurrent lost of stereospecificity.
of an ionic interaction between the protonated amine group of the
ligand and Asp3.32 side chain. For each compound, 10 top-scored
complexes were considered, of which the best one was selected
by application of binding mode analysis model e SIFt-SVM, trained
and used previously [20].
2.2. Molecular modeling
Initially, recently released crystal structures of two serotonin
receptors, i.e. 5-HT1B (PDB ID:4IAR) and 5-HT2B (PDB ID:4IB4)
complexed with ergotamine, were used as templates to generate 5-
HT7 homology models. However, docking of reference ligands of
different structure, particularly various long-chain arylpiperazines,
showed incoherent binding modes. Hence, six different confor-
mations of modified rhodopsin-based 5-HT7R homology models
[18], successfully used in virtual screening of commercial databases
[19] and selection of compounds to synthesis from virtual combi-
natorial library [20], were used to perform analysis of binding
mechanism of the studied derivatives.
2.3. Pharmacology
2.3.1. In vitro evaluation
Radioligand binding assays were employed to determine the
affinity and selectivity profiles of the synthesized compounds for
human serotonin 5-HT1AR and 5-HT7BRs, which are both stably
expressed in HEK293 cells. This was accomplished by displacement
of [3H]-8-OH-DPAT (187 Ci/mmol) and [3H]-5-CT (39.2 Ci/mmol) for
5-HT1AR and 5-HT7R, respectively. Each compound was tested in
triplicate at 7e8 concentrations (10ꢁ11e10ꢁ4 M) [20]. The inhibition
constants (Ki) were calculated from the ChengePrusoff equation
[24]. The results were expressed as the mean of at least two
separate experiments.
The structures of the synthesized molecules were prepared
using LigPrep [21] and the appropriate ionization states at pH ¼ 7.4
were assigned using Epik [22]. Docking was performed by using
Glide at SP level [23]. The spatial constrain was imposed on creation
2.4. Structureeactivity relationship studies
A library of long-chain arylpiperazines based on LP-44 (a potent
5-HT7R ligand) and containing terminal cyclic amino acid amides
(pyrrolidine-2-carboxamide e Pro, 1,2,3,4-tetrahydroisoquinoline-
3-carboxamide e Tic) was synthesized on solid-support; the
members of the library were biologically evaluated for their affinity
for 5-HT7 and 5-HT1A receptors. Selected library members dis-
played high-to-low affinity for the tested receptors, ranging from 18
to 4151 nM and from 1 to 6307 nM for 5-HT7 and 5-HT1A receptors,
respectively (Table 2).
Fig. 3. Diverse Fmoc-protected amino acids, 2{1e3}.

V. Canale et al. / European Journal of Medicinal Chemistry 78 (2014) 10e22
13
Fig. 4. Diverse alkyl chlorides, 4{1e4}.
Fig. 5. Diverse secondary amines, 6{1e10}.
Our primary objective was to determine the effect of replacing
the 1,2,3,4-tetrahydronaphthalene system (present in LP-44) with
Subsequently, we investigated the contribution of the exocyclic
primary amide bond in the terminal amino acid fragment to 5-HT7R
binding. Because it contains both H-acceptor and H-donor func-
tionalities, the amide bond may interact with Tyr7.43 or/and
Arg7.36 (with C]O function), as well as with Glu7.35 (through NH2
function), by creating additional hydrogen bonds with the receptor
(Fig. 6B). Close examination of these two orientations revealed that,
for complexes of the same compound, the second orientation
usually scored higher (i.e., receptor conformations and com-
pounds). It was also observed that any interaction with Tyr7.43/
Arg7.36 shifted the ligand toward the top of the binding cavity,
which weakened the aromatic interaction between the aryl frag-
ment and Phe6.51/Phe6.52/Trp6.48.
(S)-pyrrolidine-2-carboxamide
and
(S)-1,2,3,4-
tetrahydroisoquinoline-3-carboxamide fragments. This modifica-
tion was derived from our previous studies, which indicated that N-
acylated and N-sulfonylated proline amides were moderate-to-low
affinity ligands of 5-HT7Rs [25]. Only
L-proline, which was previ-
ously demonstrated to preferentially interact with 5-HT7Rs over
D-
proline, was used in the current work. The results of docking of the
library members 9 and 38, as well as the reference compound LP-
44, were consistent with the accumulated knowledge of ligand
binding mode at 5-HT7R. Besides the key ionic interaction with
Asp3.32, the arylpiperazine moiety of the compounds had a specific
aromatic interaction (CHe
p
or
p
e
p
) with the Phe6.51/Phe6.52/
Based on SAR performed with direct Tic and Pro analogs (e.g., 23
and 53 (Ki ¼ 50 and 22 nM, respectively) or 8 and 37 (Ki ¼ 163 and
51 nM, respectively)), we hypothesized that the additional aromatic
system in the 1,2,3,4-tetrahydroisoquinoline-3-carboxamide moi-
ety is preferential for binding to the 5-HT7R. This proposal was
supported by molecular docking simulations; the Tic-amide may
Trp6.48 aromatic cluster [18]. Superimposition of the docked
compounds revealed the main differences between their respective
interactions at the terminal fragment (Fig. 6A). In compound 38,
this fragment created a CHe
Phe3.28, while LP-44 formed a
with the same amino acid.
p
interaction (edge-to-face) with
stacking aromatic interaction
pe
p
create CHep or pep interactions with Phe3.28, while the Pro
fragment displayed only weak hydrophobic interactions with
Phe3.28 or/and Ile 7.42 (Fig. 6C). On the other hand, the additional
aromatic systems present in Tic revealed no advantage over the
alicyclic proline during the interaction with 5-HT1ARs; Pro-amides
8, 19, and 21 (Ki ¼ 47, 573 and 4 nM, respectively) displayed com-
parable affinities to their Tic analogs, which were 37, 49, and 50
(Ki ¼ 18, 643 and 16 nM, respectively).
Table 1
Analytical data for the pipecolic acid derivatives 25e36 (set II).
To determine the influence of the linker type on 5-HT7R binding,
compounds containing tetra-, penta- and hexa-methylene group
spacers, as well as aryl fragments, were synthesized. Derivatives
containing an alkylene-type linker displayed similar affinities,
while the latter modification decreased the affinity for 5-HT7Rs
within the investigated compounds (e.g., 9 vs 11, and 50 vs 51).
These results indicated that a spacer with limited conformational
freedom is not suitable for binding the receptor pocket because the
H-bond interaction with the terminal amide fragment can no
longer occur. In contrast, analyzing the binding modes of the
structural analogs, which differ only in the type of spacer, did not
reveal any significant changes caused by using a polymethylene
group spacer or aryl moiety (Fig. 6D).
Compd
{BB1} Amino acid
{BB2} Spacer
{BB3} Ar
Yield [%]a
25
26
27
28
29
30
31
32
33
34
35
36
Pip
Pip
Pip
Pip
Pip
Pip
Pip
Pip
Pip
Pip
Pip
Pip
e(CH2)4e
e(CH2)3e
e(CH2)4e
e(CH2)3e
e(CH2)4e
e(CH2)3e
e(CH2)4e
e(CH2)3e
e(CH2)3e
e(CH2)4e
e(CH2)3e
e(CH2)4e
2-OCH3-Ph
2-OH-Ph
2-OH-Ph
2-Cl-Ph
2-Cl-Ph
3-Cl-Ph
3-Cl-Ph
4-Cl
4-F-Ph
4-F-Ph
2,3-diCl-Ph
2,3-diCl-Ph
47
32
33
36
32
32
27
22
37
40
33
31
a
The affinity for 5-HT1AR was more susceptible to modification
within the alkylene spacer relative to the affinity for 5-HT7Rs.
Yields are based on the weight of the purified products and are calculated
relative to the initial loading of the resin.

14
V. Canale et al. / European Journal of Medicinal Chemistry 78 (2014) 10e22
N
O
N
H+
N
N
n
n
O
O
N
N
N
H
N
H
O+
H
R
R
N+
N+
N
N
n
n
O
O
N
N
NH2
N
R
R
OH
O
H
N+
N
N
O
n
N
O
N
n
- H2O
O
N
R
HO
HO
R
Scheme 2. Unexpected acidolysis of an amide bond after the TFA-cleavage of the pipecolic acid derivatives (set II).
Elongation of the linker from four to five, or six methylene groups
for both series of compounds (Pro- and Tic-amides) increased up to
a 20-fold affinity for the 5-HT1A sites (e.g., 8 vs 9, and 10; 20 vs 21, 37
vs 38, and 39). Consequently, compounds containing an aryl frag-
ment in the spacer displayed the lowest affinities in the series.
Because many studies have revealed the critical role of the
arylpiperazine substitution pattern in determining the 5-HT7 and
5-HT1A receptor affinity, a more detailed study of the substituents
was undertaken. Generally, electron-donating substituents were
superior to electron-withdrawing groups for binding the above
receptors. Compounds containing 2-methylthio substituents,
which have a higher volume (e.g., 9, 38), displayed better selectivity
for 5-HT7R binding than their 2-methoxy analogs (e.g., 13, 42);
consequently, the 2-hydroxy counterparts (15, 44) were the least
potent. The same relationship was observed during the SAR anal-
ysis for 5-HT1AR binding. However, in contrast to LP-44, the intro-
duction of a 2-methylthio substituent was highly preferential for
the 5-HT1A sites over the 5-HT7 sites. Consequently, compound 38
displayed the highest affinity for 5-HT1ARs (Ki ꢃ 1 nM).
1312 nM, respectively). Interestingly, both series of 2,3-di-Cl ana-
logs of the Pro- and Tic-amides displayed a more pronounced
contribution of halogen bonds during the interactions with the 5-
HT1ARs.
To extend the structural modification of the arylpiperazine
moiety further, the phenyl ring was replaced with a benzisoxazole
system. This modification significantly increased the affinity of both
Pro-amides (22e24, Ki ¼ 108, 50 and 101 nM, respectively) and Tic-
amides (52e54, Ki ¼ 18, 22 and 28 nM, respectively) for the 5-HT7
receptor. On the other hand, it was found that the elongation of
alkylene spacer from four to five or six decrease the selectivity over
the 5-HT1A sites. Molecular modeling validated the presence of an
additional H-bond interaction with Ser5.42 or/and Thr3.37 (Fig. 6F).
3. Conclusions
In summary, we have designed and synthesized three series of
long-chain arylpiperazines, which contained cyclic amino acid
amides (Pro, Pip, Tic) in the terminal fragment, on a solid-support.
Surprisingly, the piperidine-2-carboxamides, after release from the
Rink-amide resin, were converted into pipecolic acid analogs.
Structureeactivity relationship studies within the library of com-
pounds were evaluated for 5-HT7Rs and 5-HT1ARs and supported
by molecular modeling; these studies demonstrated that the Tic-
amide set favorably interacted with the 5-HT7Rs by creating CHe
An interesting correlation between the binding modes and
biological data for 5-HT7R was found within the series of proline
amides containing halogen substituents (Fig. 6E) in the phenyl-
piperazine moiety, which included 16 (2-Cl analog),18 (3-Cl analog)
and 21 (2,3-diCl analog). In addition to the previously discussed
interactions, the potential for the halogenated derivatives to create
additional interactions with Cys3.36 and Thr3.37 has been detec-
ted. This type of ligand-protein contact was intensively studied
recently, especially in light of rational drug design [26]. The
strength of these interactions depends mainly on the distance be-
tween the halogen atom and any accessible Lewis base within the
p
or pep interactions with Phe3.28. Integrating an aromatic frag-
ment was not beneficial for binding with the 5-HT1A sites. In
contrast to LP-44, compounds containing methylsulfanyl sub-
stituents in the ortho-position were highly preferential for binding
with 5-HT1ARs. However, replacing the phenyl ring in the arylpi-
binding pocket, the
involved. According to these findings, a weak interaction with
Cys3.36 (only one halogen bond with the distance and -hole equal
s
-hole angle and the type of halogen atom
perazine moiety with a benzisoxazole system significantly
increased the compound’s affinity for 5-HT7Rs because an addi-
tional H-bond with Ser5.42 or/and Thr3.37 was formed. This study
permitted the identification of several compounds with significant
5-HT7 and 5-HT1A receptor affinities and provided insights into the
molecular basis for investigating 5-HT7 receptor agents.
s
ꢀ
ꢀ
to 3.5 A and 145 , respectively) was observed for the 2-Cl deriva-
tive, resulting in a low affinity for 5-HT7Rs (16, Ki ¼ 1312 nM). A
shift of chlorine atom from the ortho- to the meta-position allowed
the generation of a strong halogen bond with Cys3.36 (distance and
ꢀ
ꢀ
s
-hole were equal 3.4 A and 136 , respectively) and a weak halogen
4. Experimental protocols
ꢀ
ꢀ
bond (distance and
s
-hole were equal 4.0 A and 129 , respectively)
with Thr3.37. Additionally, a network of halogen bonds with
4.1. Chemistry
different strengths (distance and s-hole vary from 3.6 to 4.6 E and
122e154ꢀ, respectively) was revealed for the 2,3-di-Cl derivative.
The 2,3-di-Cl derivative 21 displayed higher affinity than both the
3-Cl derivative 18 and the ortho-derivative 16 (Ki ¼ 179, 478 and
Solid-phase organic transformations and resin washes were
carried out at ambient temperature, unless otherwise indicated.
Organic solvents (from Aldrich and Fluka) were of reagent grade

V. Canale et al. / European Journal of Medicinal Chemistry 78 (2014) 10e22
15
Table 2
Analytical HPLC analyses were performed on an Alliance Waters
2695 Separations Module equipped with a Chromolith SpeedROD
Binding affinities of selected (S)-pyrrolidine-2-carboxamides and (S)-1,2,3,4-
tetrahydroisoquinoline-3-carboxamides for 5-HT7 and 5-HT1ARs.
RP 18.5
mm column (4.6 ꢂ 50 mm). Standard conditions were as
follows: eluent system A (water/0.1% TFA), system B (MeCN/0.1%
TFA). A flow rate of 5 mL/min with a gradient of (0e100)% B over
3 min was used. Detection was performed with a Waters 2998
Photodiode Array Detector.
LC/MS analyses were carried out on a system consisting of a
Waters Acquity UPLC coupled with a Waters TQD mass spectrom-
eter. All of the analyses were carried out with an Acquity UPLC BEH
C18, 50 ꢂ 2.1 mm column at 40 ꢀC. A flow rate of 0.3 mL/min and a
gradient of (5e95)% B over 5 min was used. The mobile phase
conditions were as follows: eluent A: water/0.1% HCO2H; eluent B:
MeCN/0.1% HCO2H. Retention times (Rt) are reported in minutes.
RP-preparative HPLC purification was performed on a Waters
Compd {BB1} {BB2}
Amino Spacer
acid
{BB3}
Ar
Ki [nM]a
5-HT7 5-HT1A 5-HT1A
Ratio Ki
/
5-HT7/1A
7
8
Pro
Pro
Pro
e(CH2)3e 2-SCH3-Ph
e(CH2)4e 2-SCH3-Ph
e(CH2)5e 2-SCH3-Ph
163
153
495
47
14
23
<1
<1
<1
3
11
21
9
10
HPLC 4000 instrument equipped with a UV detector 486 and a
11
Pro
2-SCH3-Ph
567
313
<1
2
ꢀ
Waters Delta-Pack 40 ꢂ 100 mm, 15 A, 100
mm, reversed-phase
column. Standard conditions were as follows: eluent system A
(water/0.1% TFA), system B (MeCN/0.1% TFA). A flow rate of 50 mL/
min and a gradient of (10e70)% B over 30 min were used with
detection at 214 nm.
12
13
Pro
Pro
e(CH2)3e 2-OCH3-Ph
e(CH2)4e 2-OCH3-Ph 1199
634
214
44
<1
<1
3
27
The resin washing steps occurred over 5 min and included three
washes with DMF, one wash with MeOH to shrink the resin beads
and two washes with DCM. The volume of solvent used for each
wash was at least 8 times the volume of the swollen beads. (i.e., for
1 g of resin, at least 40 mL of solvent was used). Unless otherwise
indicated, the yields of the final compounds were calculated based
on the initial loading of the starting resins and were the overall
yields of all reaction steps beginning from the resins. If the final
compounds were purified, these yields included the purification
yield.
14
Pro
2-OCH3-Ph 4151 6307
2
<1
15
16
17
18
19
20
21
22
23
24
37
38
39
Pro
Pro
Pro
Pro
Pro
Pro
Pro
Pro
Pro
Pro
Tic
e(CH2)3e 2-OH-Ph
e(CH2)4e 2-Cl-Ph
e(CH2)4e 2-F-Ph
e(CH2)4e 3-Cl-Ph
e(CH2)4e 4-F-Ph
1865
1312
3134
478
52
232
141
38
573
83
<1
<1
<1
<1
<1
<1
<1
27
4
36
6
22
13
2
1188
e(CH2)3e 2,3-diCl-Ph 113
e(CH2)4e 2,3-diCl-Ph 179
1
4
45
<1
<1
<1
3
e(CH2)3e BIOa
108 2908
e(CH2)4e BIOa
50
101
51
78
111
214
122
18
<1
2
e(CH2)5e BIOa
1
1H NMR and 13C NMR spectra were recorded at 300 MHz and
75 MHz (Varian BB 200 spectrometer) using TMS (0.00 ppm) as an
internal standard as well as CDCl3, and DMSO-d6 as solvents.
The J values are reported in Hertz (Hz), and the splitting patterns
are designated as follows: s (singlet), d (doublet), t (triplet), m
(multiplet), br s (broaded singlet).
e(CH2)3e 2-SCH3-Ph
e(CH2)4e 2-SCH3-Ph
e(CH2)5e 2-SCH3-Ph
<1
<1
<1
Tic
Tic
>78
55
40
Tic
2-SCH3-Ph
148
122
<1
1
Abbreviations used: DMF, Dimethylformamide; MeOH, Meth-
anol; DCM, Dichloromethane; Fmoc, Chloroformic acid 9H-fluo-
41
42
Tic
Tic
e(CH2)3e 2-OCH3-Ph
e(CH2)4e 2-OCH3-Ph
551
302
107
12
<1
<1
5
25
ren-9-ylmethyl
ester;
HBTU,
O-Benzotriazole-N,N,N’,N’-
tetramethyl-uronium-hexafluoro-phosphate; DIEA, N,N-Diiso-
propylethylamine; DMAP, 4-Dimethylaminopyridine; AcOH, Acetic
acid; TFA, Trifluoroacetic acid.
43
Tic
2-OCH3-Ph
579
77
<1
7
44
45
46
47
48
49
50
Tic
Tic
Tic
Tic
Tic
Tic
Tic
e(CH2)4e 2-OH-Ph
e(CH2)4e 2-Cl-Ph
e(CH2)4e 3-Cl-Ph
e(CH2)3e 4-Cl-Ph
e(CH2)3e 4-F-Ph
e(CH2)4e 4-F-Ph
e(CH2)4e 2,3-diCl-Ph
476
393
725
223
295 1914
107
95
72
236
499
211
<1
<1
<1
<1
6
7
2
1
4.2. Solid-supported library synthesis
4.2.1. Deprotection of rink amide resin and an amine group of the
amino acid
1
<1
<1
6
643
16
6
<1
The resin (2.25 g, 1.58 mmol, 0.7 mmol gꢁ1) was treated with a
20% solution of piperidine in DMF and allowed to rotate for 3 min.
Subsequently, the beads were drained and washed with DMF
(1 ꢂ 5 mL). The procedure described above was repeated for
another 15 min. Next, the resin was drained and washed with DMF
(4ꢂ), MeOH (1ꢂ), and DCM (4ꢂ) before being dried under low
vacuum.
51
Tic
2,3-diCl-Ph 1196
212
<1
6
52
53
54
Tic
Tic
Tic
e(CH2)3e BIOb
18
22
28
377
230
89
21
10
3
<1
<1
<1
e(CH2)4e BIOb
e(CH2)5e BIOb
a
4.2.2. Preparation of solid-supported carboxamides
Ki values (SEM ꢄ 22) based on three independent binding experiments.
BIO ¼ benzisoxazol-3-yl.
b
The amine resin was placed into 3 reactors for coupling with
different Fmoc-protected amino acids. The resin was swelled in
CH2Cl2 (10 mL) for 30 min and then washed with CH2Cl2eDMF (80/
20, v/v). Fmoc-amino acid 2{1e3} (5.4 mmol, 5 equiv), HBTU
(0.88 g, 5.4 mmol, 5 equiv) in DMF (8 mL), and DIEA (1.8 mL,
10.8 mmol, 10 equiv) were then added to the resin (0.6 g,
1.08 mmol, 1.8 mmol gꢁ1). The reaction mixture was agitated with
an orbital shaker for 2 h. The resin-bound amino acid was then
and were used without purification. Rink-amide-functionalized
resin with 1.3 mmol/g loading was purchased from Iris Biotech
(Germany). The resin-bound intermediates were air-dried after the
final CH2Cl2 washes unless they needed to be re-weighed; in the
latter case, the samples were treated for 24e36 h in a vacuum
desiccator.

16
V. Canale et al. / European Journal of Medicinal Chemistry 78 (2014) 10e22
Fig. 6. (AeF). Panel illustrating molecular modeling results of the designed library for the 5-HT7 receptor. (A) The comparison of the binding modes for reference ligand LP-44 (red)
and the closest analogs from the Pro (cyan) and Tic (green) series. (B) The two possible amide group interactions are shown for the two complexes of compound 8; in the first
orientation, the C]O group might form an H-bond with Tyr7.43 or/and Arg7.36 (cyan), but in the second, the NH2 can be H-bonded with Glu7.35 (green). (C) An aromatic
interaction between the terminal moiety of the Tic compounds (green) with Phe3.28 may cause their activity to outperform the activity of the Pro analogs (cyan). (D) The
introduction of linkers with different lengths ((CH2)4, 8 e green; (CH2)5, 9 e cyan, (CH2)6, 10 e yellow, p-C6H4, 11 e red) exhibited no significant changes to the binding mode. (E)
The correlation between the orientation of the different analogs with the halogenated phenyl ring (i.e., 16 (yellow), 18 (red), 21 (cyan)). Their activity might be caused by the creation
of halogen bonds with Cys3.36 or/and Thr3.37. (F) The most active compound in this series benzisoxazole derivative 52, might form additional H-bond contacts with Thr3.37 or/and
Ser5.42. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
drained and washed with DMF (4 ꢂ 5 mL), MeOH (1 ꢂ 5 mL), and
DCM (4 ꢂ 5 mL). The procedure described above was repeated. The
washed resin was dried under low vacuum for 12 h.
4.2.4. Nucleophilic displacement with arylpiperazine derivatives
The air-dried resin was placed into glass vials containing 1.5 mL
of 1 M solution of secondary amine 6{1e10} in DMF. The resulting
reaction mixture was heated to 75 ꢀC for 24 h. The resulting resin-
bound product was drained and sequentially washed with a 10%
solution of AcOH in DMF (2ꢂ), DMF (4ꢂ), MeOH (1ꢂ), and DCM
(4ꢂ) before being dried in open air.
4.2.3. Acylation using arylacyl chlorides
The deprotected resin (140 mg, 0.08 mmol, 0.57 mmol gꢁ1) was
swelled in 2 mL of DCM for 30 min; DIEA (0.08 mL, 0.45 mmol,
6 equiv) was added to the resin, followed by a solution of acyl
chloride 4{1e4} in 0.25 mL of DCM and DMAP (10.3 mg, 0.08 mmol,
1 equiv). The reaction mixture was allowed to rotate for 4 h. The
resin was drained and washed with DMF (4ꢂ), MeOH (1ꢂ), and
DCM (4ꢂ) before being allowed to dry in open air.
4.3. General procedure for the cleavage of the final products
Two milliliters of a TFA/DCM (80/20, v/v) mixture was added to
the resin, and the cleavage reaction was carried out for 2 h.

V. Canale et al. / European Journal of Medicinal Chemistry 78 (2014) 10e22
17
Subsequently, the reaction was filtered and washed with a small
13C NMR (75 MHz, CDCl3):
d
23.23, 23.31, 24.83, 25.90, 28.07,
volume of DCM; the collected filtrates were concentrated in a
stream of argon to yield the target compounds.
33.54, 47.45, 47.61, 52.18, 55.44, 56.95, 59.65, 111.32, 118.88, 121.23,
124.53, 138.77, 152.04, 172.73, 174.45.
4.3.1. (S)-1-(5-(4-(2-(Methylthio)phenyl)piperazin-1-yl)pentanoyl)
pyrrolidine-2-carboxamide (8)
4.3.7. (S)-1-(4-((4-(2-Methoxyphenyl)piperazin-1-yl)methyl)
benzoyl)pyrrolidine-2-carboxamide (14)
Yellow oil, 20 mg (36% isolated yield) following preparative LC/
MS purification; LC/MS purity 99%, tR ¼ 1.02 min. C21H32N4O2S, MW
404.57, Monoisotopic Mass 404.22, [M þ H]þ ¼ 405.5. 1H NMR
Yellow oil, 16 mg (39% isolated yield) following preparative LC/
MS purification; LC/MS purity 99%, tR ¼ 1.00 min. C24H30N4O3, MW
422.52, Monoisotopic Mass 422.23, [M þ H]þ ¼ 423.7. 1H NMR
(300 MHz, CDCl3):
d 1.72e1.78 (m, 2H), 1.83e1.89 (m, 2H), 1.94e
(300 MHz, CDCl3):
d 1.81e1.92 (m, 2H), 2.01e2.19 (m, 2H), 2.32e
2.04 (m, 2H), 2.25e2.35 (m, 2H), 2.37e2.41 (m, 2H), 2.54 (s, 3H),
3.08e3.13 (m, 4H), 3.38e3.44 (m, 2H), 3.52e3.56 (m, 4H), 3.62e
3.68 (m, 2H), 4.48e4.51 (m, 1H), 6.85e6.9 (m, 1H), 6.92e6.94 (m,
2H), 7.07e7.12 (m, 1H).
2.45 (m, 2H), 3.44e3.53 (m, 4H), 3.55e3.63 (m, 4H), 3.85 (s, 3H),
4.27 (s, 2H), 4.7e4.74 (m, 1H), 5.50 (br. s., 1H), 5.75 (br. s., 1H), 6.86e
6.93 (m, 4H), 7.05e7.11 (m, 1H), 7.43 (s, 1H), 7.49e7.54 (m, 1H),
7.58e7.62 (m, 1H).
13C NMR (75 MHz, CDCl3):
d 25.38, 27.68, 47.54, 50.45, 51.81,
4.3.2. (S)-1-(6-(4-(2-(Methylthio)phenyl)piperazin-1-yl)hexanoyl)
pyrrolidine-2-carboxamide (9)
55.42, 59.77, 60.19, 111.30, 118.93, 121.24, 124.64, 128.16, 130.43,
131.32, 137.79, 138.62, 152.03, 162.03, 162.51, 169.81, 173.49.
Yellow oil, 22 mg (37% isolated yield) following preparative LC/
MS purification; LC/MS purity 98%, tR ¼ 1.06 min. C22H34N4O2S, MW
418.6, Monoisotopic Mass 418.24, [M þ H]þ ¼ 419.4. 1H NMR
4.3.8. (S)-1-(5-(4-(2-Hydroxyphenyl)piperazin-1-yl)pentanoyl)
pyrrolidine-2-carboxamide (15)
(300 MHz, CDCl3): d 1.43e1.49 (m, 2H),1.65e1.74 (m, 2H),1.78e1.86
Colorless oil, 13 mg (31% isolated yield) following preparative
LC/MS purification; LC/MS purity 99%, tR ¼ 0.88 min. C20H30N4O3,
MW 374.48, Monoisotopic Mass 374.23, [M þ H]þ ¼ 375.5. 1H NMR
(m, 4H), 1.95e2.04 (m, 2H), 2.49 (s, 3H), 3.03e3.09 (m, 4H), 3.22 (t,
2H), 3.36e3.44 (m, 2H), 3.56e3.61 (m, 4H), 3.62e3.69 (m, 2H), 4.5e
4.56 (m, 1H), 6.87e6.96 (m, 2H), 7.22e7.26 (m, 2H).
(300 MHz, CDCl3):
d 1.73e1.78 (m, 2H), 1.93e1.95 (m, 2H), 1.93e
2.02 (m, 2H), 2.06e2.12 (m, 2H), 2.39e2.44 (m, 2H), 3.09e3.19 (m,
4H), 3.39e3.44 (m, 2H), 3.55e3.6 (m, 4H), 3.65e3.68 (m, 2H), 4.43e
4.5 (m, 1H), 6.83e6.9 (m, 2H), 7.08e7.13 (m, 2H).
4.3.3. (S)-1-(7-(4-(2-(Methylthio)phenyl)piperazin-1-yl)heptanoyl)
pyrrolidine-2-carboxamide (10)
Yellow oil, 25 mg (38% isolated yield) following preparative LC/
MS purification; LC/MS purity 100%, tR ¼ 1.16 min. C23H36N4O2S,
MW 432.62, Monoisotopic Mass 432.26, [M þ H]þ ¼ 433.3. 1H NMR
4.3.9. (S)-1-(6-(4-(2-Chlorophenyl)piperazin-1-yl)hexanoyl)
pyrrolidine-2-carboxamide (16)
(300 MHz, CDCl3): d 1.21e1.33 (m, 4H), 1.39e1.43 (m, 2H), 1.62e1.71
(m, 2H), 1.78e1.84 (m, 4H), 1.93e2.01 (m, 2H), 2.47 (s, 3H), 3.03e
3.09 (m, 4H), 3.38e3.43 (m, 2H), 3.54e3.59 (m, 4H), 3.62e3.65 (m,
2H), 4.51e4.56 (m, 1H), 6.78e6.65 (m, 2H), 7.19e7.23 (m, 2H).
Pale oil, 17 mg (34% isolated yield) following preparative LC/MS
purification; LC/MS purity 99%, tR ¼ 1.12 min. C21H31ClN4O2, MW
406.95, Monoisotopic Mass 406.21, [M þ H]þ ¼ 407.3. 1H NMR
(300 MHz, CDCl3): d 1.46e1.51 (m, 2H), 1.63e1.68 (m, 2H), 1.7e1.75
4.3.4. (S)-1-(4-((4-(2-(Methylthio)phenyl)piperazin-1-yl)methyl)
benzoyl)pyrrolidine-2-carboxamide (11)
(m, 2H), 1.81e1.89 (m, 2H), 1.99e2.09 (m, 2H), 2.31e2.38 (m, 2H),
3.03e3.1 (m, 4H), 3.27e3.43 (m, 2H), 3.54e3.6 (m, 4H), 3.67e3.71
(m, 2H), 4.54e4.56 (m, 1H), 7.03e7.08 (m, 2H), 7.23e7.29 (m, 1H),
7.36e7.39 (m, 1H).
Brown oil, 19 mg (39% isolated yield) following preparative LC/
MS purification; LC/MS purity 98%, tR ¼ 1.1 min. C24H30N4O2S, MW
438.59, Monoisotopic Mass 438.21, [M þ H]þ ¼ 439.7. 1H NMR
(300 MHz, CDCl3):
d 1.86e1.88 (m, 2H), 2.03e2.15 (m, 2H), 2.35e
4.3.10. (S)-1-(6-(4-(2-Fluorophenyl)piperazin-1-yl)hexanoyl)
pyrrolidine-2-carboxamide (17)
2.39 (m, 2H), 2.6 (s, 3H), 3.48e3.51 (m, 4H), 3.55e3.62 (m, 4H), 4.29
(s, 2H), 4.71e4.75 (m, 1H), 6.87e6.93 (m, 4H), 7.05e7.1 (m, 1H), 7.28
(d, 1H), 7.42 (s, 1H), 7.51 (d, 1H).
Pale oil, 10 mg (33% isolated yield) following preparative LC/MS
purification; LC/MS purity 98%, tR ¼ 1.03 min. C21H31FN4O2, MW
390.49, Monoisotopic Mass 390.24, [M þ H]þ ¼ 391.6. 1H NMR
4.3.5. (S)-1-(5-(4-(2-Methoxyphenyl)piperazin-1-yl)pentanoyl)
pyrrolidine-2-carboxamide (12)
(300 MHz, CDCl3):
d 1.48e1.55 (m, 2H), 1.62e1.68 (m, 2H), 1.72e
1.75 (m, 2H), 1.81e1.89 (m, 2H), 1.99e2.06 (m, 2H), 2.3e2.38 (m,
2H), 3.03e3.14 (m, 4H), 3.27e3.46 (m, 2H), 3.54e3.6 (m, 4H), 3.67e
3.73 (m, 2H), 4.54e4.56 (m, 1H), 7.02e7.08 (m, 2H), 7.22e7.29 (m,
1H), 7.36e7.39 (m, 1H).
Yellow oil, 19 mg (39% isolated yield) following preparative LC/
MS purification; LC/MS purity 99%, tR ¼ 0.98 min. C21H32N4O3, MW
388.5, Monoisotopic Mass 388.25, [M þ H]þ ¼ 389.5. 1H NMR
(300 MHz, CDCl3/MeOD):
d 1.7e1.76 (m, 2H), 1.84e1.91 (m, 2H),
1.93e2.02 (m, 2H), 2.27e2.35 (m, 2H), 2.39e2.44 (m, 2H), 3.08e
3.13 (m, 4H), 3.38e3.44 (m, 2H), 3.5e3.56 (m, 4H), 3.62e3.68 (m,
2H), 3.86 (s, 3H), 4.48e4.5 (m, 1H), 6.87e6.9 (m, 1H), 6.92e6.94 (m,
2H), 7.05e7.1 (m, 1H).
4.3.11. (S)-1-(6-(4-(3-chlorophenyl)piperazin-1-yl)hexanoyl)
pyrrolidine-2-carboxamide (18)
Pale oil, 28 mg (33% isolated yield) following preparative LC/MS
purification; LC/MS purity 99%, tR ¼ 1.16 min. C21H31ClN4O2, MW
406.95, Monoisotopic Mass 406.21, [M þ H]þ ¼ 407.5. 1H NMR
4.3.6. (S)-1-(5-(4-(2-Methoxyphenyl)piperazin-1-yl)hexanoyl)
pyrrolidine-2-carboxamide (13)
(300 MHz, CDCl3):
d 1.43e1.50 (m, 2H), 1.68e1.75 (m, 2H), 1.78e
Yellow oil, 22 mg (37% isolated yield) following preparative LC/
MS purification; LC/MS purity 99%, tR ¼ 1.03 min. C22H34N4O3, MW
402.53, Monoisotopic Mass 402.26, [M þ H]þ ¼ 403.4. 1H NMR
1.86 (m, 4H), 1.89e1.94 (m, 2H), 2.94e3.02 (m, 4H), 3.03e3.08 (m,
2H), 3.38e3.46 (m, 2H), 3.53e3.57 (m, 2H), 3.60e3.67 (m, 4H),
4.54e4.57 (m, 1H), 6.72e6.74 (m, 2H), 6.79 (d, 1H, J ¼ 7.62 Hz),
7.24e7.30 (m, 1H).
(300 MHz, CDCl3): d 1.41e1.49 (m, 2H), 1.63e1.73 (m, 2H), 1.76e1.86
(m, 4H), 1.95e2.02 (m, 2H), 3.04e3.09 (m, 4H), 3.22 (t, 2H), 3.38e
3.46 (m, 2H), 3.56e3.61 (m, 4H), 3.62e3.68 (m, 2H), 3.92 (s, 3H),
4.49e4.53 (m, 1H), 6.87e6.96 (m, 2H), 7.23e7.26 (m, 2H).
13C NMR (75 MHz, CDCl3):
d 20.75, 23.17, 23.83, 25.20, 25.85,
26.31, 32.67, 43.66, 47.66, 52.12, 52.31, 55.44, 57.12, 111.35, 118.89,
118.95, 121.23, 124.64, 138.58, 152.05.

18
V. Canale et al. / European Journal of Medicinal Chemistry 78 (2014) 10e22
4.3.12. (S)-1-(6-(4-(4-Fluorophenyl)piperazin-1-yl)hexanoyl)
pyrrolidine-2-carboxamide (19)
1H), 5.46 (br. s.,1H), 7.04 (br. s.,1H), 7.21 (ddd, J ¼ 8.05, 6.26,1.78 Hz,
1H), 7.41e7.48 (m, 2H), 7.69 (dt, J ¼ 8.06, 0.91 Hz, 1H).
Pale oil, 20 mg (35% isolated yield) following preparative LC/MS
purification; LC/MS purity 98%, tR ¼ 1.03 min. C21H31FN4O2, MW
390.49, Monoisotopic Mass 390.24, [M þ H]þ ¼ 391.4. 1H NMR
13C NMR (75 MHz, CDCl3):
d 22.67, 24.94, 26.51, 27.21, 28.65,
29.22, 34.51, 46.79, 47.52, 48.15, 52.51, 58.65, 59.30, 63.90, 110.45,
116.16, 122.18, 122.28, 129.50, 161.26, 163.96, 173.69, 174.52.
(300 MHz, CDCl3): d 1.38e1.47 (m, 2H),1.58e1.62 (m, 2H),1.69e1.72
(m, 2H), 1.78e1.86 (m, 2H), 1.81e1.84 (m, 2H), 3.06e3.11 (m, 4H),
3.19e3.26 (m, 4H), 3.48e3.49 (m, 2H), 3.69e3.73 (m, 4H), 4.53e
4.58 (m, 1H), 6.86e6.92 (m, 2H), 6.95e7.01 (m, 2H).
d 21.46, 23.17, 23.83, 25.24, 25.85,
26.31, 33.67, 43.66, 47.66, 52.12, 52.31, 55.75, 57.12, 111.85, 119.28,
129.35, 130.23, 145.72, 152.15.
4.3.18. 1-(6-(4-(2-Methoxyphenyl)piperazin-1-yl)hexanoyl)-
piperidine-2-carboxylic acid (25)
Yellow oil, 22 mg (47% isolated yield) following preparative LC/
MS purification; LC/MS purity 100%, tR ¼ 1.13 min. C23H35N3O4, MW
417.54, Monoisotopic Mass 417.26, [M þ H]þ ¼ 418.6. 1H NMR
13C NMR (75 MHz, CDCl3):
(300 MHz, CDCl3): d 1.39e1.41 (m, 2H),1.43e1.45 (m, 2H),1.62e1.64
(m, 2H), 1.66e1.7 (m, 2H), 1.77e1.8 (m, 2H), 2.26e2.41 (m, 2H),
2.44e2.51 (m, 2H), 3.04e3.14 (m, 4H), 3.2e3.25 (m, 4H), 3.5e3.54
(m, 2H), 3.69e3.76 (m, 2H), 3.85 (s, 3H), 5.28e5.3 (m, 1H), 6.87e6.9
(m, 1H), 6.93e6.97 (m, 2H), 7.05e7.11 (m, 1H), 11.1 (br s, 1H).
d 20.75, 20.76, 23.17, 23.83, 25.20,
25.85, 26.31, 32.67, 43.66, 47.66, 52.12, 52.31, 55.44, 57.12, 111.35,
118.89, 118.95, 121.23, 124.64, 138.58, 152.05.
4.3.13. (S)-1-(5-(4-(2,3-Dichlorophenyl)piperazin-1-yl)pentanoyl)
pyrrolidine-2-carboxamide (20)
Pale oil, 19 mg (31% isolated yield) following preparative LC/MS
purification; LC/MS purity 98%, tR ¼ 1.24 min. C20H28Cl2N4O2, MW
427.37, Monoisotopic Mass 426.16, [M þ H]þ ¼ 427.2. 1H NMR
13C NMR (75 MHz, CDCl3):
(300 MHz, CDCl3): d 1.73e1.77 (m, 2H), 1.9e2.02 (m, 2H), 2.27e2.31
(m, 2H), 2.39e2.43 (m, 2H), 3.04e3.21 (m, 4H), 3.28 (t, 2H), 3.4 (t,
2H), 3.57e3.64 (m, 2H), 3.68e3.71 (m, 4H),4.51e4.54 (m, 1H),
6.98e7.01 (m, 1H), 7.16e7.28 (m, 2H).
4.3.19. 1-(5-(4-(2-Hydroxyphenyl)piperazin-1-yl)pentanoyl)-
piperidine-2-carboxylic acid (26)
Yellow oil, 15 mg (32% isolated yield) following preparative LC/
MS purification; LC/MS purity 99%, tR ¼ 1 min. C21H31N3O4, MW
389.49, Monoisotopic Mass 389.23, [M þ H]þ ¼ 390.5. 1H NMR
4.3.14. (S)-1-(6-(4-(2,3-Dichlorophenyl)piperazin-1-yl)hexanoyl)
pyrrolidine-2-carboxamide (21)
Pale oil, 11 mg (33% isolated yield) following preparative LC/MS
purification; LC/MS purity 97%, tR ¼ 1.26 min. C21H30Cl2N4O2, MW
441.39, Monoisotopic Mass 440.17, [M þ H]þ ¼ 441.2. 1H NMR
(300 MHz, CDCl3): d 1.39e1.48 (m, 2H),1.58e1.61 (m, 2H),1.64e1.71
(m, 2H), 1.74e1.77 (m, 2H), 2.28e2.38 (m, 2H), 2.43e2.51 (m, 2H),
3.07e3.12 (m, 4H), 3.19e3.27 (m, 2H), 3.49e3.61 (m, 2H), 3.69e3.74
(m, 4H), 5.23e5.24 (m,1H), 5.53 (br s,1H), 6.83e6.88 (m,1H), 6.93e
6.95 (m, 1H), 7.05e7.12 (m, 2H), 11.1 (br s, 1H).
(300 MHz, CDCl3): d 1.43e1.5 (m, 2H), 1.67e1.74 (m, 2H), 1.78e1.86
(m, 2H), 1.93e2.02 (m, 2H), 2.27e2.38 (m, 4H), 3.00e3.11 (m, 4H),
3.27e3.43 (m, 4H), 3.54e3.61 (m, 2H), 3.67e3.71 (m, 2H)), 4.52e
4.55 (m, 1H), 6.98e7.01 (m, 1H), 7.23e7.26 (m, 2H).
4.3.20. 1-(6-(4-(2-Hydroxyphenyl)piperazin-1-yl)hexanoyl)-
piperidine-2-carboxylic acid (27)
4.3.15. (S)-1-(5-(4-(Benzo[d]isoxazol-3-yl)piperazin-1-yl)
pentanoyl)pyrrolidine-2-carboxamide (22)
Yellow oil, 17 mg (33% isolated yield) following preparative LC/
MS purification; LC/MS purity 95%, tR ¼ 0.98 min. C22H33N3O4, MW
403.52, Monoisotopic Mass 403.25, [M þ H]þ ¼ 404.5. 1H NMR
Colorless Oil, 30 mg (39% isolated yield) following preparative
LC/MS purification; LC/MS purity 98%, tR ¼ 0.98 min. C21H29N5O3,
MW 399.49, Monoisotopic Mass 399.23, [M þ H]þ ¼ 400.3. 1H NMR
(300 MHz, CDCl3):
d 1.41e1.44 (m, 2H), 1.46e1.58 (m, 2H), 1.6e1.63
(m, 2H), 1.65e1.67 (m, 2H), 1.72e1.74 (m, 2H), 1.77e1.79 (m, 2H),
2.27e2.41 (m, 2H), 2.46e2.54 (m, 2H), 3.06e3.17 (m, 4H), 3.25e
3.29 (m, 2H), 3.49e3.66 (m, 2H), 3.72e3.77 (m, 4H), 5.26e5.27 (m,
1H), 5.55 (br s, 1H), 6.84e6.9 (m, 1H), 6.92e6.95 (m, 1H), 7.07e7.15
(m, 2H), 11.4 (br s, 1H).
(300 MHz, CDCl3): d 1.21e1.34 (m, 2H), 1.31e1.47 (m, 4H), 1.73e1.85
(m, 2H), 1.92e1.98 (m, 2H), 2.22e2.39 (m, 2H), 3.02e3.07 (m, 4H),
3.38e3.42 (m, 2H), 3.59e3.66 (m, 4H), 4.46e4.49 (m,1H), 7.12 (br. s,
2H), 7.24e7.29 (m, 1H), 7.46e7.56 (m, 2H), 7.65 (br. s, 1H).
4.3.16. (S)-1-(6-(4-(Benzo[d]isoxazol-3-yl)piperazin-1-yl)
hexanoyl)pyrrolidine-2-carboxamide (23)
4.3.21. 1-(5-(4-(2-Chlorophenyl)piperazin-1-yl)pentanoyl)-
piperidine-2-carboxylic acid (28)
Colorless Oil, 28 mg (40% isolated yield) following preparative
LC/MS purification; LC/MS purity 96%, tR ¼ 1.01 min. C22H31N5O3,
MW 413.51, Monoisotopic Mass 413.24, [M þ H]þ ¼ 414.5. 1H NMR
Yellow oil, 15 mg (36% isolated yield) following preparative LC/
MS purification; LC/MS purity 99%, tR ¼ 1.2 min. C21H30ClN3O3, MW
407.93, Monoisotopic Mass 407.2, [M þ H]þ ¼ 408.7. 1H NMR
(300 MHz, CDCl3):
d
1.25e1.48 (m, 2H), 1.61e1.71 (m, 2H), 1.73e1.85
(300 MHz, CDCl3): d 1.4e1.43 (m, 2H), 1.61e1.63 (m, 2H), 1.7e1.73
(m, 2H), 1.94e2.02 (m, 2H), 2.05e2.16 (m, 2H), 2.22e2.39 (m, 2H),
3.03e3.09 (m, 4H), 3.37e3.46 (m, 4H), 3.55e3.64 (m, 4H), 4.46e
4.49 (m, 1H), 7.06 (br. s, 2H), 7.24e7.29 (m, 1H), 7.46e7.56 (m, 2H),
7.61 (br. s, 1H).
(m, 2H), 1.78e1.83 (m, 2H), 2.28e2.38 (m, 2H), 2.41e2.54 (m, 2H),
3.08e3.13 (m, 4H), 3.24e3.28 (m, 2H), 3.39e3.43 (m, 2H), 3.69e
3.74 (m, 4H), 5.27e5.28 (m, 1H), 7.02e7.07 (m, 2H), 7.23e7.28 (m,
1H), 7.35e7.38 (m, 1H), 11.4 (br s, 1H).
13C NMR (75 MHz, CDCl3):
d
23.92, 24.24, 25.61, 27.81, 30.22,
13C NMR (75 MHz, CDCl3):
d 20.82, 21.48, 22.67, 25.15, 26.38,
33.91, 47.59, 48.15, 49.78, 54.69, 56.51, 62.56, 63.90, 109.65, 116.16,
122.18, 123.80, 130.85, 161.26, 163.96, 173.51, 174.23.
31.45, 44.66, 46.50, 51.33, 51.53, 52.62, 56.61, 56.79, 113.54, 115.82,
120.13, 134.7, 137.11, 150.30, 175.64, 175.83.
4.3.17. (S)-1-(7-(4-(Benzo[d]isoxazol-3-yl)piperazin-1-yl)
heptanoyl)pyrrolidine-2-carboxamide (24)
4.3.22. 1-(6-(4-(2-Chlorophenyl)piperazin-1-yl)hexanoyl)-
piperidine-2-carboxylic acid (29)
Colorless Oil, 27 mg (38% isolated yield) following preparative
LC/MS purification; LC/MS purity 97%, tR ¼ 1.1 min. C23H33N5O3,
MW 427.54, Monoisotopic Mass 427.26, [M þ H]þ ¼ 428.4. 1H NMR
Pale oil, 13 mg (32% isolated yield) following preparative LC/MS
purification; LC/MS purity 99%, tR ¼ 1.24 min. C22H32ClN3O3, MW
421.96, Monoisotopic Mass 421.21, [M þ H]þ ¼ 422.3. 1H NMR
(300 MHz, CDCl3):
d
1.21e1.33 (m, 4H), 1.34e1.46 (m, 4H), 1.49e1.69
(300 MHz, CDCl3): d 1.4e1.43 (m, 2H), 1.46e1.56 (m, 2H), 1.61e1.63
(m, 4H), 2.29e2.46 (m, 4H), 2.64 (t, J ¼ 5.20 Hz, 4H), 3.14 (q,
(m, 2H), 1.7e1.73 (m, 2H), 1.78e1.83 (m, 2H), 2.28e2.38 (m, 2H),
2.41e2.54 (m, 2H), 3.08e3.13 (m, 4H), 3.24e3.28 (m, 2H), 3.39e
J ¼ 7.30 Hz, 2H), 3.59 (t, J ¼ 5.20 Hz, 4H), 4.58 (dd, J ¼ 8.03, 2.00 Hz,

V. Canale et al. / European Journal of Medicinal Chemistry 78 (2014) 10e22
19
3.43 (m, 2H), 3.69e3.74 (m, 4H), 5.27e5.28 (m, 1H), 7.02e7.07 (m,
2H), 7.23e7.28 (m, 1H), 7.35e7.38 (m, 1H), 11.4 (br s, 1H).
MW 442.38, Monoisotopic Mass 441.16, [M þ H]þ ¼ 442.5. 1H NMR
(300 MHz, CDCl3):
d 1.4e1.48 (m, 2H), 1.59e1.61 (m, 2H), 1.63e1.7
(m, 2H), 1.78e1.84 (m, 2H), 2.28e2.41 (m, 2H), 2.44e2.57 (m, 2H),
3.06e3.14 (m, 4H), 3.23e3.28 (m, 2H), 3.38e3.41 (m, 2H), 3.69e
3.73 (m, 4H), 5.26e5.28 (m, 1H), 6.97e7.01 (m, 1H), 7.15e7.18 (m,
1H), 7.21e7.26 (m, 1H), 11.3 (br s, 1H).
4.3.23. 1-(5-(4-(3-Chlorophenyl)piperazin-1-yl)pentanoyl)-
piperidine-2-carboxylic acid (30)
Pale oil, 15 mg (22% isolated yield) following preparative LC/MS
purification; LC/MS purity 99%, tR ¼ 1.2 min. C21H30ClN3O3, MW
407.93, Monoisotopic Mass 407.2, [M þ H]þ ¼ 408.7. 1H NMR
4.3.29. 1-(6-(4-(2,3-Dichlorophenyl)piperazin-1-yl)hexanoyl)-
piperidine-2-carboxylic acid (36)
(300 MHz, CDCl3):
d 1.41e1.43 (m, 2H), 1.61e1.64 (m, 2H), 1.7e1.73
(m, 2H), 1.78e1.83 (m, 2H), 2.28e2.38 (m, 2H), 2.41e2.54 (m, 2H),
3.07e3.13 (m, 4H), 3.24e3.28 (m, 2H), 3.39e3.44 (m, 2H), 3.69e
3.74 (m, 4H), 5.27e5.28 (m, 1H), 6.81e6.84 (m, 2H), 7.01e7.05 (m,
1H), 7.2e7.24 (m, 1H), 11.4 (br s, 1H).
Yellow oil, 14 mg (31% isolated yield) following preparative LC/
MS purification; LC/MS purity 99%, tR ¼ 1.35 min. C22H31Cl2N3O3,
MW 456.41, Monoisotopic Mass 455.17, [M þ H]þ ¼ 456.4. 1H NMR
(300 MHz, CDCl3): d 1.39e1.41 (m, 2H),1.44e1.46 (m, 2H),1.58e1.62
(m, 2H), 1.67e1.7 (m, 2H), 1.72e1.81 (m, 2H), 2.27e2.41 (m, 2H),
2.44e2.54 (m, 2H), 3.07e3.13 (m, 4H), 3.21e3.32 (m, 2H), 3.38e
3.42 (m, 2H), 3.68e3.76 (m, 4H), 5.28e5.29 (m, 1H), 6.98e7.01 (m,
1H), 7.16e7.21 (m, 1H), 7.23e7.26 (m, 1H), 11.5 (br s, 1H).
4.3.24. 1-(6-(4-(3-Chlorophenyl)piperazin-1-yl)hexanoyl)-
piperidine-2-carboxylic acid (31)
Pale oil, 13 mg (27% isolated yield) following preparative LC/MS
purification; LC/MS purity 99%, tR ¼ 1.25 min. C22H32ClN3O3, MW
421.96, Monoisotopic Mass 421.21, [M þ H]þ ¼ 422.3. 1H NMR
(300 MHz, CDCl3):
d 1.4e1.43 (m, 2H), 1.46e1.56 (m, 2H), 1.61e1.63
4.3.30. (S)-2-(5-(4-(2-(Methylthio)phenyl)piperazin-1-yl)
(m, 2H), 1.7e1.73 (m, 2H), 1.78e1.83 (m, 2H), 2.28e2.38 (m, 2H),
2.41e2.54 (m, 2H), 3.08e3.13 (m, 4H), 3.24e3.28 (m, 2H), 3.39e
3.43 (m, 2H), 3.69e3.74 (m, 4H), 5.27e5.28 (m, 1H), 6.8e6.84 (m,
2H), 7.02e7.05 (m, 1H), 7.21e7.24 (m, 1H), 11.4 (br s, 1H).
pentanoyl)-1,2,3,4-tetrahydroisoquinoline-3-carboxamide (37)
Yellow oil, 20 mg (32% isolated yield) following preparative LC/
MS purification; LC/MS purity 98%, tR ¼ 1.32 min. C26H34N4O2S, MW
466.64, Monoisotopic Mass 466.24, [M þ H]þ ¼ 467.4. 1H NMR
(300 MHz, CDCl3):
d 1.44e1.47 (m, 2H), 1.62e1.66 (m, 2H), 2.33 (s,
4.3.25. 1-(5-(4-(4-Chlorophenyl)piperazin-1-yl)pentanoyl)-
piperidine-2-carboxylic acid (32)
3H), 2.45e2.49 (m, 4H), 2.87e2.95 (m, 2H), 3.09e3.13 (m, 2H),
3.33e3.41 (m, 6H), 4.57e4.77 (m, 2H), 4.94e4.98 (m, 1H), 7.05e7.10
(m, 4H), 7.14e7.19 (m, 4H).
Pale oil, 12 mg (22% isolated yield) following preparative LC/MS
purification; LC/MS purity 99%, tR ¼ 1.21 min. C21H30ClN3O3, MW
407.93, Monoisotopic Mass 407.2, [M þ H]þ ¼ 408.6. 1H NMR
4.3.31. (S)-2-(6-(4-(2-(Methylthio)phenyl)piperazin-1-yl)
(300 MHz, CDCl3): d 1.39e1.43 (m, 2H),1.59e1.63 (m, 2H),1.71e1.73
hexanoyl)-1,2,3,4-tetrahydroisoquinoline-3-carboxamide (38)
Yellow oil, 22 mg (34% isolated yield) following preparative LC/
MS purification; LC/MS purity 97%, tR ¼ 1.35 min. C27H36N4O2S, MW
480.67, Monoisotopic Mass 480.26, [M þ H]þ ¼ 481.4. 1H NMR
(m, 2H), 1.81e1.85 (m, 2H), 2.27e2.31 (m, 2H), 2.41e2.54 (m, 2H),
2.98e3.02 (m, 4H), 3.06e3.1 (m, 2H), 3.23e3.26 (m, 2H), 3.58e3.73
(m, 4H), 5.23e5.25 (m, 1H), 6.83e6.87 (m, 2H), 7.21e7.27 (m, 2H),
11.2 (br s, 1H).
13C NMR (75 MHz, CDCl3):
d 20.66, 21.40, 22.98, 25.05, 26.26,
(300 MHz, DMSO-d6): d 1.32e1.35 (m, 2H), 1.45e1.47 (m, 2H), 1.62e
1.66 (m, 2H), 2.36 (s, 3H), 2.47e2.49 (m, 4H), 2.87e2.95 (m, 2H),
3.09e3.15 (m, 2H), 3.33e3.41 (m, 6H), 4.57e4.77 (m, 2H), 4.95e4.98
(m, 1H), 7.05e7.10 (m, 4H), 7.14e7.18 (m, 4H).
32.15, 43.66, 46.50, 51.44, 51.80, 52.40, 56.61, 56.79, 114.92, 116.98,
121.38, 130.45, 135.17, 150.50, 173.20, 173.54.
4.3.26. 1-(5-(4-(4-Fluorophenyl)piperazin-1-yl)pentanoyl)-
piperidine-2-carboxylic acid (33)
4.3.32. (S)-2-(7-(4-(2-(Methylthio)phenyl)piperazin-1-yl)
Yellow oil, 20 mg (35% isolated yield) following preparative LC/
MS purification; LC/MS purity 99%, tR ¼ 1.12 min. C21H30FN3O3, MW
391.48, Monoisotopic Mass 391.23, [M þ H]þ ¼ 392.7. 1H NMR
heptanoyl)-1,2,3,4-tetrahydroisoquinoline-3-carboxamide (39)
Yellow oil, 26 mg (37% isolated yield) following preparative LC/
MS purification; LC/MS purity 98%, tR ¼ 1.41 min. C28H38N4O2S, MW
494.69, Monoisotopic Mass 494.27, [M þ H]þ ¼ 495.5. 1H NMR
(300 MHz, CDCl3): d 1.37e1.43 (m, 2H), 1.59e1.66 (m, 2H), 1.7e1.75
(m, 2H), 1.81e1.85 (m, 2H), 2.28e2.31 (m, 2H), 2.41e2.54 (m, 2H),
2.98e3.04 (m, 4H), 3.06e3.11 (m, 2H), 3.23e3.26 (m, 2H), 3.6e3.73
(m, 4H), 5.23e5.25 (m, 1H), 6.83e6.87 (m, 2H), 7.22e7.27 (m, 2H),
11.2 (br s, 1H).
(300 MHz, CDCl3):
d 1.32e1.42 (m, 4H), 1.48e1.59 (m, 2H), 1.60e
1.72 (m, 2H), 2.33e2.54 (m, 4H), 2.64e2.71 (m, 4H), 2.94e3.12 (m,
1H), 3.21e3.38 (m, 1H), 3.53e3.62 (m, 4H), 4.50e4.70 (m, 2H), 5.11
(t, J ¼ 5.56 Hz, 1H), 5.73 (br. s., 1H), 6.35 (br. s., 1H), 7.08e7.23 (m,
5H), 7.39e7.50 (m, 2H), 7.68 (d, J ¼ 8.08 Hz, 1H).
4.3.27. 1-(6-(4-(4-Fluorophenyl)piperazin-1-yl)hexanoyl)-
piperidine-2-carboxylic acid (34)
13C NMR (75 MHz, CDCl3):
d 24.66, 26.26, 27.12, 29.10, 30.16,
33.84, 45.99, 46.83, 47.94, 52.43, 52.93, 58.54, 110.41, 116.08, 122.19,
122.36, 125.70, 126.71, 127.67, 128.28, 129.56, 132.54, 133.71, 161.15,
163.91, 173.55, 173.91.
Yellow oil, 18 mg (33% isolated yield) following preparative LC/
MS purification; LC/MS purity 100%, tR ¼ 1.14 min. C22H32FN3O3,
MW 405.51, Monoisotopic Mass 405.24, [M þ H]þ ¼ 406.4. 1H NMR
(300 MHz, CDCl3): d 1.37e1.43 (m, 2H),1.45e1.49 (m, 2H),1.59e1.68
(m, 2H), 1.72e1.75 (m, 2H), 1.83e1.87 (m, 2H), 2.28e2.31 (m, 2H),
2.44e2.54 (m, 2H), 2.95e3.03 (m, 4H), 3.06e3.11 (m, 2H), 3.23e
3.26 (m, 2H), 3.62e3.73 (m, 4H), 5.23e5.26 (m, 1H), 6.83e6.87 (m,
2H), 7.23e7.27 (m, 2H), 11.5 (br s, 1H).
4.3.33. (S)-2-(4-((4-(2-(Methylthio)phenyl)piperazin-1-yl)methyl)
benzoyl)-1,2,3,4-tetrahydroisoquinoline-3-carboxamide (40)
Brown oil, 23 mg (35% isolated yield) following preparative LC/
MS purification; LC/MS purity 97%, tR ¼ 1.34 min. C29H32N4O2S, MW
500.65, Monoisotopic Mass 500.22, [M þ H]þ ¼ 501.5. 1H NMR
4.3.28. 1-(5-(4-(2,3-Dichlorophenyl)piperazin-1-yl)pentanoyl)-
piperidine-2-carboxylic acid (35)
Yellow oil, 15 mg (33% isolated yield) following preparative LC/
MS purification; LC/MS purity 99%, tR ¼ 1.31 min. C21H29Cl2N3O3,
(300 MHz, CDCl3): d 2.43 (s, 3H), 2.03e2.17 (m, 2H), 2.33e2.37 (m,
2H), 3.49e3.51 (m, 4H), 3.55e3.6 (m, 4H), 4.57e4.77 (m, 2H), 4.95e
4.98 (m, 1H), 7.05e7.1 (m, 4H), 7.14e7.18 (m, 4H), 7.43e7.46 (m, 2H),
7.5e7.53 (m, 2H).

20
V. Canale et al. / European Journal of Medicinal Chemistry 78 (2014) 10e22
4.3.34. (S)-2-(5-(4-(2-Methoxyphenyl)piperazin-1-yl)pentanoyl)-
1,2,3,4-tetrahydroisoquinoline-3-carboxamide (41)
454.99, Monoisotopic Mass 454.21, [M þ H]þ ¼ 455.2. 1H NMR
(300 MHz, CDCl3):
d 1.82e1.87 (m, 2H), 1.93e1.97 (m, 2H), 2.49e
Yellow oil, 15 mg (36% isolated yield) following preparative LC/
MS purification; LC/MS purity 99%, tR ¼ 1.15 min. C26H34N4O3, MW
450.57, Monoisotopic Mass 450.26, [M þ H]þ ¼ 451.3. 1H NMR
2.54 (m, 2H), 2.63e2.69 (m, 2H), 3.02e3.06 (m, 4H), 3.25e3.27 (m,
2H), 3.34e3.61 (m, 4H), 4.54e4.68 (m, 2H), 5.07e5.11 (m, 1H),
6.75e6.79 (m, 2H), 6.91e6.94 (m, 2H), 7.13e7.3 (m, 4H).
(300 MHz, CDCl3):
d 1.79e1.83 (m, 2H), 1.96e2.01 (m, 2H), 2.43e
2.71 (m, 2H), 3.01e3.08 (m, 4H), 3.2e3.33 (m, 2H), 3.51e3.55 (m,
4H), 3.62e3.65 (m, 2H), 3.86 (s, 3H), 4.55e4.69 (m, 2H), 5.1e5.13
(m, 1H), 6.88e7.06 (m, 4H), 7.22e7.26 (m, 4H).
4.3.41. (S)-2-(5-(4-(4-Fluorophenyl)piperazin-1-yl)pentanoyl)-
1,2,3,4-tetrahydroisoquinoline-3-carboxamide (48)
Pale oil, 13 mg (33% isolated yield) following preparative LC/MS
purification; LC/MS purity 100%, tR ¼ 1.19 min. C25H31FN4O2, MW
438.54, Monoisotopic Mass 438.24, [M þ H]þ ¼ 439.5. 1H NMR
4.3.35. (S)-2-(6-(4-(2-Methoxyphenyl)piperazin-1-yl)hexanoyl)-
1,2,3,4-tetrahydroisoquinoline-3-carboxamide (42)
(300 MHz, CDCl3):
d 1.85e1.97 (m, 4H), 2.49e2.69 (m, 2H), 3.01e
Yellow oil, 20 mg (37% isolated yield) following preparative LC/
MS purification; LC/MS purity 98%, tR ¼ 1.19 min. C27H36N4O3, MW
464.6, Monoisotopic Mass 464.28, [M þ H]þ ¼ 465.3. 1H NMR
3.07 (m, 4H), 3.17e3.28 (m, 2H), 3.35e3.47 (m, 4H), 3.62e3.73 (m,
2H), 4.6e4.68 (m, 2H), 5.08e5.11 (m, 1H), 6.86e6.95 (m, 2H), 6.96e
7.02 (m, 2H), 7.13e7.26 (m, 4H).
(300 MHz, DMSO-d6): d 1.21e1.24 (m, 2H), 1.28e1.35 (m, 2H), 1.52e
1.56 (m, 2H), 2.47e2.49 (m, 4H), 2.71e2.75 (m, 2H), 2.93e2.95 (m,
2H), 3.01e3.11 (m, 2H), 3.35e3.59 (m, 4H), 3.75 (s, 3H), 4.57e4.77
(m, 2H), 4.94e4.98 (m, 1H), 6.85e6.95 (m, 4H), 7.14e7.20 (m, 4H).
4.3.42. (S)-2-(6-(4-(4-Fluorophenyl)piperazin-1-yl)hexanoyl)-
1,2,3,4-tetrahydroisoquinoline-3-carboxamide (49)
Pale oil, 19 mg (38% isolated yield) following preparative LC/MS
purification; LC/MS purity 99%, tR ¼ 1.21 min. C26H33FN4O2, MW
452.56, Monoisotopic Mass 452.26, [M þ H]þ ¼ 453.3. 1H NMR
4.3.36. (S)-2-(4-((4-(2-Methoxyphenyl)piperazin-1-yl)methyl)
benzoyl)-1,2,3,4-tetrahydroisoquinoline-3-carboxamide (43)
Brown oil, 27 mg (34% isolated yield) following preparative LC/
MS purification; LC/MS purity 97%, tR ¼ 1.32 min. C29H32N4O3, MW
484.59, Monoisotopic Mass 484.25, [M þ H]þ ¼ 485.5. 1H NMR
(300 MHz, CDCl3):
d 1.48e1.54 (m, 2H), 1.69e1.85 (m, 4H), 2.36e
2.65 (m, 2H), 2.96e3.07 (m, 4H), 3.28e3.35 (m, 2H), 3.47e3.55 (m,
4H), 3.67e3.7 (m, 2H), 4.64e4.79 (m, 2H), 5.1e5.13 (m, 1H), 6.87e
6.93 (m, 2H), 6.97e7.03 (m, 2H), 7.15e7.26 (m, 4H).
(300 MHz, CDCl3): d 2.03e2.17 (m, 2H), 3.49e3.51 (m, 4H), 3.55e3.6
(m, 4H), 3.33e3.37 (m, 2H), 3.85 (s, 3H), 4.57e4.77 (m, 2H), 4.95e
4.98 (m, 1H), 7.05e7.1 (m, 4H), 7.14e7.18 (m, 4H), 7.43e7.46 (m, 2H),
7.5e7.53 (m, 2H).
4.3.43. (S)-2-(6-(4-(2,3-Dichlorophenyl)piperazin-1-yl)hexanoyl)-
1,2,3,4-tetrahydroisoquinoline-3-carboxamide (50)
Pale oil, 29 mg (32% isolated yield) following preparative LC/MS
purification; LC/MS purity 98%, tR ¼ 1.41 min. C26H32Cl2N4O2, MW
503.46, Monoisotopic Mass 502.19, [M þ H]þ ¼ 503.2. 1H NMR
4.3.37. (S)-2-(6-(4-(2-Hydroxyphenyl)piperazin-1-yl)hexanoyl)-
1,2,3,4-tetrahydroisoquinoline-3-carboxamide (44)
(300 MHz, DMSO-d6): d 1.31e1.36 (m, 2H), 1.51e1.56 (m, 2H), 1.62e
Colorless oil, 12 mg (34% isolated yield) following preparative
LC/MS purification; LC/MS purity 99%, tR ¼ 1.14 min. C26H34N4O3,
MW 450.57, Monoisotopic Mass 450.26, [M þ H]þ ¼ 451.5. 1H NMR
1.65 (m, 2H), 2.33e2.41 (m, 2H), 2.45e2.49 (m, 2H), 2.61e2.72 (m,
4H), 2.91e2.98 (m, 2H), 3.01e3.11 (m, 4H), 4.49e4.69 (m, 2H), 5.10e
5.14 (m, 1H), 5.89 (s, 1H), 6.41 (s, 1H), 6.89e6.95 (m, 1H), 7.07e7.11
(m, 2H), 7.13e7.26 (m, 4H).
(300 MHz, CDCl3): d 1.51e1.56 (m, 2H),1.81e1.86 (m, 2H), 2.14e2.37
(m, 4H), 3.04e3.09 (m, 4H), 3.28e3.35 (m, 2H), 3.33e3.41 (m, 4H),
3.67e3.71 (m, 2H), 4.61e4.74 (m, 2H), 5.11e5.15 (m, 1H), 6.88e6.96
(m, 2H), 7.1e7.19 (m, 2H), 7.21e7.26 (m, 4H).
4.3.44. (S)-2-(4-((4-(2,3-Dichlorophenyl)piperazin-1-yl)methyl)
benzoyl)-1,2,3,4-tetrahydroisoquinoline-3-carboxamide (51)
Pale oil, 16 mg (38% isolated yield) following preparative LC/MS
purification; LC/MS purity 97%, tR ¼ 1.42 min. C28H28Cl2N4O2, MW
523.45, Monoisotopic Mass 522.16, [M þ H]þ ¼ 523.3. 1H NMR
4.3.38. (S)-2-(6-(4-(2-Chlorophenyl)piperazin-1-yl)hexanoyl)-
1,2,3,4-tetrahydroisoquinoline-3-carboxamide (45)
Pale oil, 10 mg (32% isolated yield) following preparative LC/MS
purification; LC/MS purity 99%, tR ¼ 1.31 min. C26H33ClN4O2, MW
469.02, Monoisotopic Mass 468.23, [M þ H]þ ¼ 469.5. 1H NMR
(300 MHz, CDCl3): d 2.33e2.37 (m, 2H), 3.49e3.51 (m, 4H), 3.55e
3.6 (m, 4H), 3.36e3.39 (m, 2H), 4.57e4.77 (m, 2H), 4.95e4.98 (m,
1H), 5.89 (s, 1H), 6.41 (s, 1H), 6.89e6.95 (m, 1H), 7.13e7.26 (m, 4H),
7.42e7.48 (m, 2H), 7.55e7.58 (m, 2H).
(300 MHz, CDCl3):
d 1.5e1.53 (m, 2H), 1.71e1.84 (m, 2H), 2.17e2.21
(m, 4H), 3.04e3.06 (m, 4H), 3.21e3.33 (m, 2H), 3.4e3.44 (m, 4H),
3.67e3.7 (m, 2H), 4.5e4.75 (m, 2H), 5.13e5.17 (m, 1H), 7.04e7.16
(m, 2H), 7.21e7.29 (m, 4H), 7.37e7.4 (m, 2H).
4.3.45. (S)-2-(5-(4-(Benzo[d]isoxazol-3-yl)piperazin-1-yl)
pentanoyl)-1,2,3,4-tetrahydroisoquinoline-3-carboxamide (52)
Colorless oil, 18 mg (38% isolated yield) following preparative
LC/MS purification; LC/MS purity 98%, tR ¼ 1.16 min. C26H31N5O3,
MW 461.56, Monoisotopic Mass 461.24, [M þ H]þ ¼ 462.2. 1H NMR
4.3.39. (S)-2-(6-(4-(3-Chlorophenyl)piperazin-1-yl)hexanoyl)-
1,2,3,4-tetrahydroisoquinoline-3-carboxamide (46)
Pale oil, 15 mg (37% isolated yield) following preparative LC/MS
purification; LC/MS purity 99%, tR ¼ 1.33 min. C26H33ClN4O2, MW
469.02, Monoisotopic Mass 468.23, [M þ H]þ ¼ 469.5. 1H NMR
(300 MHz, CDCl3): d 1.28e1.57 (m, 4H), 2.11e2.15 (m, 2H), 2.22e
2.37 (m, 2H), 2.77 (t, 4H), 3.10e3.13 (m, 2H), 3.57 (t, 4H), 4.61e4.79
(m, 2H), 5.13e5.17 (m, 1H), 7.15e7.21 (m, 1H), 7.22e7.3 (m, 4H),
7.48e7.54 (m, 2H), 7.57e7.61 (m, 1H).
(300 MHz, CDCl3):
d 1.48e1.53 (m, 2H), 1.69e1.84 (m, 2H), 2.04e
2.17 (m, 4H), 2.98e3.05 (m, 4H), 3.29e3.35 (m, 2H), 3.49e3.55 (m,
4H), 3.66e3.75 (m, 2H), 4.59e4.65 (m, 2H), 5.11e5.15 (m, 1H),
6.81e6.86 (m, 2H), 7.18e7.22 (m, 4H), 7.23e7.25 (m, 1H), 7.27e7.31
(m, 1H).
13C NMR (75 MHz, CDCl3):
d 25.33, 27.12, 32.25, 33.87, 43.85,
45.99, 46.83, 48.34, 52.43, 52.98, 58.54, 111.48, 116.08, 122.19,
122.36, 125.70, 126.71, 127.67, 128.45, 129.56, 132.54, 134.31, 161.15,
163.91, 173.55, 174.12.
4.3.40. (S)-2-(5-(4-(4-Chlorophenyl)piperazin-1-yl)pentanoyl)-
1,2,3,4-tetrahydroisoquinoline-3-carboxamide (47)
Pale oil, 14 mg (34% isolated yield) following preparative LC/MS
purification; LC/MS purity 99%, tR ¼ 1.29 min. C25H31ClN4O2, MW
4.3.46. (S)-2-(6-(4-(Benzo[d]isoxazol-3-yl)piperazin-1-yl)
hexanoyl)-1,2,3,4-tetrahydroisoquinoline-3-carboxamide (53)
Colorless oil, 21 mg (36% isolated yield) following preparative
LC/MS purification; LC/MS purity 99%, tR ¼ 1.26 min. C27H33N5O3,

V. Canale et al. / European Journal of Medicinal Chemistry 78 (2014) 10e22
21
MW 475.58, Monoisotopic Mass 475.26, [M þ H]þ ¼ 476.4. 1H NMR
available in on-line version at http://dx.doi.org/10.1016/j.ejmech.
2014.03.005.
(300 MHz, CDCl3): d 1.39e1.47 (m, 2H),1.53e1.58 (m, 2H),1.67e1.77
(m, 2H), 2.39e2.44 (m, 2H), 2.47e2.54 (m, 2H), 2.63 (t, 4H), 2.98
(dd, 1H, J1 ¼ 10 Hz, J2 ¼ 6 Hz), 3.14 (dd, 1H, J1 ¼ 10 Hz,, J2 ¼ 6 Hz),
3.58 (t, 4H), 4.48e4.67 (m, 2H), 5.15e5.19 (m, 1H), 7.11 (d, 1H,
J ¼ 6 Hz), 7.18e7.26 (m, 4H), 7.27e7.47 (m, 2H), 7.68 (d, 1H).
References
13C NMR (75 MHz, CDCl3):
d 26.29, 27.12, 29.10, 32.25, 34.56,
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122.36, 124.70, 125.73, 127.67, 128.28, 129.56, 132.54, 134.62, 161.15,
163.91, 173.55, 173.85.
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d 1.32e1.42 (m, 4H), 1.49e1.58 (m,
2H), 1.60e1.69 (m, 2H), 2.40e2.56 (m, 4H), 2.68 (t, J ¼ 6.05 Hz, 4H),
2.96e3.11 (m, 2H), 3.22e3.33 (m, 1H), 3.57 (t, J ¼ 6.1 Hz, 4H), 4.64e
4.70 (m, 1H), 5.06e5.13 (m, 1H), 7.10e7.22 (m, 5H), 7.40e7.49 (m,
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13C NMR (75 MHz, CDCl3):
d 26.26, 27.12, 29.10, 30.16, 32.25,
33.90, 43.85, 45.99, 46.83, 47.94, 52.43, 52.93, 58.54, 110.41, 116.08,
122.19, 122.36, 125.70, 126.71, 127.67, 128.28, 129.56, 132.54, 133.71,
161.15, 163.91, 173.55, 173.91.
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4 mM MgCl2, 10 M pargyline and 0.1% ascorbate.
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mM
m
m
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HT1AR; 0.6 nM [3H]-5-CT (39.2 Ci/mmol) for the 5-HT7bR.
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Acknowledgments
This study was partially supported by the Polish Ministry of
Science and Higher Education (MNiSW), Grant No. N N405 671540
and by the project POIG.01.01.02-12-004/09-00 (De-Me-Ter), which
was co-financed by the European Union from the European Fund of
Regional Development (EFRD).
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Appendix A. Supplementary data
Supplementary data available: representative LC/MS, 1H NMR,
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