Towards novel 5-HT7 versus 5-HT1A receptor ligands among LCAPs with cyclic amino acid amide fragments: Design, synthesis, and antidepressant properties. Part II

Article abstract of DOI:10.1016/j.ejmech.2014.12.041

A 26-membered library of novel long-chain arylpiperazines, which contained primary and tertiary amides of cyclic amino acids (proline and 1,2,3,4-tetrahydroisoquinoline-3-carboxamide) in the terminal fragment was synthesized and biologically evaluated for binding affinity for 5-HT₇ and 5-HT_1A receptors. Docking studies confirmed advantages of Tic-amide over Pro-amide fragment for interaction with 5-HT₇ receptors. Selected compounds 32 and 28, which behaved as 5-HT₇Rs antagonist and 5-HT_1A partial agonist, respectively, produced antidepressant-like effects in the forced swim test in mice after acute treatment in doses of 10 mg/kg (32) and 1.25 mg/kg (28). Compound 32 reduced immobility in a manner similar to the selective 5-HT₇ antagonist SB-269970.

Full text of DOI:10.1016/j.ejmech.2014.12.041

European Journal of Medicinal Chemistry 92 (2015) 202e211
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Towards novel 5-HT7 versus 5-HT1A receptor ligands among LCAPs
with cyclic amino acid amide fragments: Design, synthesis, and
antidepressant properties. Part II
Vittorio Canale a, Rafał Kurczab b, Anna Partyka c, Grzegorz Satała b, Jagna Witek b,
Magdalena Jastrze˛bska-Wie˛sek c, Maciej Pawłowski a, Andrzej J. Bojarski b,
,
*
Anna Wesołowska c, Paweł Zajdel a
a
ꢀ
Department of Medicinal Chemistry, Jagiellonian University Medical College, 9 Medyczna Street, 30-688 Krakow, Poland
Department of Medicinal Chemistry, Institute of Pharmacology, Polish Academy of Sciences, 12 Sme˛tna Street, 31-343 Krakow, Poland
Department of Clinical Pharmacy, Jagiellonian University Medical College, 9 Medyczna Street, 30-688 Krakow, Poland
b
c
ꢀ
ꢀ
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 6 May 2014
Received in revised form
16 November 2014
Accepted 23 December 2014
Available online 24 December 2014
A 26-membered library of novel long-chain arylpiperazines, which contained primary and tertiary
amides of cyclic amino acids (proline and 1,2,3,4-tetrahydroisoquinoline-3-carboxamide) in the terminal
fragment was synthesized and biologically evaluated for binding affinity for 5-HT7 and 5-HT1A receptors.
Docking studies confirmed advantages of Tic-amide over Pro-amide fragment for interaction with 5-HT7
receptors. Selected compounds 32 and 28, which behaved as 5-HT7Rs antagonist and 5-HT1A partial
agonist, respectively, produced antidepressant-like effects in the forced swim test in mice after acute
treatment in doses of 10 mg/kg (32) and 1.25 mg/kg (28). Compound 32 reduced immobility in a manner
similar to the selective 5-HT7 antagonist SB-269970.
Keywords:
Homology modeling and docking to 5-HT1A
and 5-HT7Rs
© 2014 Published by Elsevier Masson SAS.
Prolinamides
1,2,3,4-Tetrahydroisoquinoline-3-
carboxamides
5-HT7 receptor antagonist
5-HT1A receptor partial agonist
Depression
1. Introduction
successfully explored as privileged structures for many subtypes of
5-HTRs [10,11]. Extending SAR studies around this class of com-
Among fourteen types of serotonin (5-HT) receptors, the 5-HT7
receptors (5-HT7Rs) are the latest addition to a subfamily of G-
protein-coupled receptors (GPCRs) [1]. It was found that blockade
of 5-HT7Rs produced an antidepressant-like effect in animal
models of depression [2e4], and may be responsible for improve-
ment of cognitive decline or neurological disorders [5,6]. On the
other hand, the activation of 5-HT7Rs is regarded as a potential
therapy for treatment of pain [7,8], and the Fragile X syndrome [9].
In view of aforementioned findings, the development of new 5-
HT7R agents seems critical for the acquisition of detailed insight
into the pharmacological function of 5-HT7Rs.
pounds, our group modified the amide fragment of LCAPs with N-
acylated amino acids to optimize potent 5-HT1A/5-HT2A receptor
ligands [12e14]. Recently, as our interest focused on high-end 5-
HTRs ligands, we investigated an influence of the primary amides
of cyclic amino acids (pyrrolidine-2-carboxamide e Pro-amide and
1,2,3,4-tetrahydroisoquinoline-3-carboxamide
e Tic-amide) on
compounds affinity for 5-HT7Rs and selectivity over 5-HT1A sites
[15] (Fig. 1).
As an extension of our ongoing efforts aimed at identification of
5-HT7 receptor ligands we designed a new series of LCAPs func-
tionalized with tertiary amides of amino acids in the terminal
fragment. Apart from diversification of the amide fragment, struc-
tural modifications comprised variation in the length of the alky-
lene spacer, and introduction of different substituents in ortho
position of phenylpiperazine (PhP). Herein we present the syn-
thesis of the designed compounds, determination of their affinity
For decades, long-chain arylpiperazines (LCAPs) have been
* Corresponding author.
E-mail address: pawel.zajdel@uj.edu.pl (P. Zajdel).
http://dx.doi.org/10.1016/j.ejmech.2014.12.041
0223-5234/© 2014 Published by Elsevier Masson SAS.

V. Canale et al. / European Journal of Medicinal Chemistry 92 (2015) 202e211
203
(187 Ci/mmol), [3H]-ketanserin (50.3 Ci/mmol), [3H]-LSD (81 Ci/
mmol) and [3H]-5-CT (39.2 Ci/mmol) for 5-HT1A, 5-HT2A, 5-HT6 and
5-HT7B receptors, respectively.
2.3.2. Functional in vitro evaluation
The functional activity of the selected compounds 28, 32, 35 and
37 on 5-HT7 and 5-HT1A receptors, was determined at Cerep (Le
Bois l'Eveque, 86,600 Celle L'Evescault, France), according to the
previously published methods [19,20]. Assays were carried out in
CHO and HEK-293 cells, which stably expressed the human 5-HT7
and 5-HT1A receptors, respectively.
Fig. 1. General structures of LCAP and L-Pro and L-Tic primary amide derivatives.
2.3.3. Behavioral evaluation
Potential antidepressant-like activity of selected compounds
with the highest affinities for 5-HT1A (28, 35) and 5-HT7 (32, 37)
receptors was evaluated in the forced swim test (FST) in mice [21]
and their activity was compared with that of the antidepressant
drug imipramine (28, 35) and the selective 5-HT7 antagonist SB-
269970 (32, 37). The influence of the effective doses recorded in
the FST on spontaneous locomotor activity in mice was studied in
order to exclude the possibility of competing behaviors such as
general locomotor activity.
for 5-HT7 and 5-HT1A receptors, and investigation of their binding
mode for 5-HT7 and 5-HT1ARs, followed with functional in vitro, as
well as behavioral evaluation in animal model of depression.
2. Results and discussion
2.1. Chemistry
The synthesis of target compounds, grouped in two series
22e34 (set I) and 35e47 (set II), was carried out by a multi-step
procedure according to Scheme 1. In order to obtain primary
amide derivatives 1e2, Boc-protected amino acids: (S)epyrrolidi-
nee2ecarboxylic acid (Pro) and (S)etetrahydroisoquinoline-3-
carboxylic acid (Tic) were subjected to the amidation reaction
with ammonia in the presence of isobutyl chloroformate and N-
methylmorpholine. Alternatively, tertiary amide derivatives 3e6,
were obtained from amino acids and alicyclic secondary amines e
piperidine or morpholine, under BOP-promoted coupling reaction.
After the removal of Boc group using TFA in DCM, the resulting
2.4. Structureeactivity relationship studies
Continuing our studies aimed at verification of structural fea-
tures responsible for 5-HT7 and 5-HT1ARs affinity and selectivity in
a group of LCAPs with cyclic amino acids (Pro- and Tic-amides), we
focused our attention on a kind of amide group e primary, tertiary,
a length of an alkylene spacer, and a nature of substituent in ortho
position of phenylpiperazine.
The newly synthesized compounds displayed high-to-moderate
affinity for 5-HT7 and 5-HT1A receptors (5e135 nM and 1e66 nM,
respectively) (Table 1). Derivatives containing Tic-amides displayed
higher 5-HT7Rs affinity than the related Pro-amide compounds (22
vs 27, and 25 vs 31). On the other hand, a nature of amino acid
fragment has not impacted the affinity of compounds for 5-HT1ARs
(e.g. 26 vs 34, and 38 vs 45).
secondary amines were subsequently acylated with respective
u-
bromo-acyl chlorides to afford the corresponding intermediates
7e12 and 13e21. These were further reacted with appropriate
arylpiperazines in biphasic conditions to give final compounds
22e34 and 35e47.
The 1-arylpiperazines were obtained either commercially or
were prepared according to the details presented in Supporting
Information.
Investigation of a binding mode showed that interaction of the
Pro- and Tic-moieties with 5-HT7 and 5-HT1A Rs might be driven by
different mechanisms. The comparison of the binding modes for
structural analogs 22 and 27 (Fig. 2A), showed that the
teractions between an aromatic ring of the 1,2,3,4-
tetrahydroisoquinoline fragment and Phe3.28 was noticed for 5-
pep in-
2.2. Molecular modeling
Homology models of 5-HT1A and 5-HT7 receptors were built on
crystal structures of 5-HT1B (PDB ID: 4IAR), b2 (PDB ID: 3P0G), D3
(PDB ID: 3PBL) and H1 (PDB ID: 3RZE) receptors. Based on the
docking of a structurally diverse set of ligands it was found that
their fully coherent binding mode was obtained only in models
generated on the 5-HT1B templates. These models were next used
for the study of the binding mode of Tic and Pro derivatives for 5-
HT7 and 5-HT1ARs. Despite the fact that our previous 5-HT1A and 5-
HT7R models were limited only to transmembrane domain and
were built on different templates, the marked hot-spot residues
were the same [15e17].
HT1A, whereas CHe
the Tic-amide fragment created a catione
p
was detected in 5-HT7 model. Additionally,
interaction with
p
Arg7.36 and Thr2.64 in 5-HT7Rs. Furthermore, Pro- and Tic-amide
fragments created H-bond interaction with the Glu7.35, and thus
induced an orientation of these fragments to Phe2.38/Arg7.36/
Thr2.64 amino acids. Since, the pyrrolidine-2-carboxamide frag-
ment can form only weak (due to a distance) hydrophobic in-
teractions with the above-mentioned amino acids, Pro-derivatives
displayed lower affinity for 5-HT7Rs.
In the case of 5-HT1ARs, the Pro-amide moiety created hydrogen
bonds with Asn7.39 and Gln2.65, as well as hydrophobic in-
teractions with Tyr2.64, while for Tic-amide fragment an aromatic
interaction with Tyr2.64 and Trp78 from EL1 was identified.
Subsequently, following our previous findings that the primary
exocyclic amide bond localized in Pro- and Tic-moieties interacted
via an H-bond with Glu7.35 residue (through NH2 function) of the
5-HT7Rs, an influence of the H-bond acceptor and hydrophobic
properties coming from tertiary amides was evaluated.
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 in
competition experiments for human serotonin 5-HT1A, 5-HT2A, 5-
HT6 and 5-HT7B receptors, which were all stably expressed in
HEK293 cells. According to the previously published procedures
[18], the experiments were carried out using [3H]-8-OH-DPAT
It was notable that within compounds 35e47 an introduction of
the piperidinyl and morpholinyl moieties did not influence the
affinity for 5-HT7Rs; this modification decreased 5-HT7/5-HT1A

204
V. Canale et al. / European Journal of Medicinal Chemistry 92 (2015) 202e211
N
N
N
Boc
Br
N
R
iii, iv
v
N
n
n
Boc
Boc
O
O
O
N
H2N
H2N
H2N
O
O
O
HO
i
O
7-12
set I
22-34
1-2
ii
N
N
N
N
O
N
R
Boc
Br
iii, iv
v
n
n
O
N
N
N
N
O
O
O
X
X
X
HO
set II
35-47
3-6
13-21
n = 2, 3, 4
X = CH2, O
R = SCH3, isopropyl, phenyl
Scheme 1. The synthesis of L-cyclic amino acid amide derivatives 22e34 and 35e47. Reagents and conditions: (i) NH4 aq, IBCF, NMM, THF, 0 ꢀC / r.t., 4 h; (ii) Piperidine or
morpholine, BOP, TEA, DCM, r.t., 12 h; (iii) TFA/DCM 80:20, r.t., 2 h; (iv)
u
-Bromo-acyl chloride, TEA, DCM, 0 ꢀC, 12 h; (v) 1-Arylpiperazine, K2CO3, KI, Acetone, 60 ꢀC, 72 h.
selectivity (e.g. 25 vs 35, 36 or 32 vs 43, 44).
compounds 28 and 35 (containing ortho-methylthio and ortho-
The analysis of docking results indicated that an introduction of
tertiary amides switched off the ability of Pro- and Tic-amide
fragments to create hydrogen bonds with Glu7.35 and Asn7.39 in
5-HT7 and 5-HT1ARs, respectively (Fig. 2B). On the other hand, this
modification enabled creating of a set of an additional hydrophobic
interactions (mainly Phe6.51/Leu6.54 for 5-HT7R and Phe6.51/
Ile7.38 for 5-HT1AR).
Elongation of an alkylene spacer from four to five, or six
methylene groups within primary amides (22e34) decreased the
affinity for 5-HT7Rs (22 vs 23, 24 and 27 vs 28, 29), while among
tertiary amides 35e47 it slightly influenced the affinity for 5-HT7Rs
(37 vs 38, 39 and 43 vs 45 and 46). This was supported by docking
results where for direct analogs with four (22), five (23) and six (24)
methylene groups spacer any significant changes in the binding
mode for both 5-HT7 and 5-HT1ARs was observed (Fig. 2C). Inter-
estingly, a more detailed analysis of ligand complexes with 5-HT7
sites revealed that derivatives containing hexamethylene spacer
were the worst scored in Glide Score ranking. A careful analysis
showed that the minor influence of the type of an alkylene spacer
on the final score may result from a kind of substituent in the
arylpiperazine moiety and a type of cyclic amino acid in the ter-
minal fragment (Fig. 2D).
isopropyl substituent, respectively) and ortho-phenyl derivatives
32 and 37 were selected for extended in vitro and in vivo evaluation.
2.5. Functional and behavioral evaluation
Regarding many classes of 5-HT receptors involved in patho-
mechanisms of psychiatric disorders e.g. depression, much atten-
tion has been devoted to the role of 5-HT1A, 5-HT2A, 5-HT6 and 5-
HT7 subtypes. It has been commonly accepted that agonists/par-
tial agonists of 5-HT1A [24] and/or antagonists of 5-HT7 receptors
exhibit antidepressant-like effects [1,25].
Taking into account the intrinsic activity of the investigated
compounds (Table 2), we selected compounds 28, 35 (5-HT1A par-
tial agonists) and 32, 37 (5-HT7 antagonists) for further in vivo
studies towards potential antidepressant activity in FST. Our pre-
sent results showed that compounds 28 given in a dose of 1.25 mg/
kg and 32 given in a dose of 10 mg/kg produced a distinct
antidepressant-like effect in that test, significantly shortening the
immobility time of mice by 23% and 24%, respectively. Compound
35 did not influence the measured parameter, while compound 37,
administered in a dose of 2.5 mg/kg, reduced the immobility time
of animals; however, the results did not reach a statistically sig-
nificant level.
The effect of 28 was similar to that of the reference antide-
pressant drug, imipramine, that administered in a dose of 10 mg/kg
reduced the immobility time of mice by 28% (Fig. 4), while
antidepressant-like potency of 32 was similar to that of the selec-
tive 5-HT7 receptor antagonist SB-269970, which exerted an
antidepressant-like effect in one medium dose (10 mg/kg) signifi-
cantly shortening the immobility time of mice by 33% (Fig. 4). An-
tidepressant doses of derivatives 28 and 32 did not affect
spontaneous locomotor activity of mice during initial 2e6-min
session (i.e. at the time identical to the observation period in the
FST) (data not shown). The lack of antidepressant-like activity of
compounds 35 and 37 remains unclear. Further pharmacokinetic
studies may warrant explaining this phenomenon.
As demonstrated by other authors, introduction of highly lipo-
philic or sterically hindered substituents in an ortho-position of the
phenyl ring in arylpiperazine fragment increased the affinity for 5-
HT7Rs and selectivity over 5-HT1A sites [22].
Based on these findings, we previously synthesized ortho-
methylthio derivatives (22e24 and 27e29). Surprisingly, in
contrast to the observation described by Leopoldo et al. [23], this
modification shifted the receptor preference for 5-HT1ARs (Fig. 3).
To further extend chemical diversity in arylpiperazine fragment,
isopropyl, and phenyl substituents were introduced in the ortho
position of phenylpiperazine. Among both sets e primary and ter-
tiary amides, phenyl-substituted derivatives displayed higher af-
finities for 5-HT7Rs than their ortho-isopropyl analogs,
consequently, ortho-methylthio derivatives were the less potent
(i.e. 39 > 35, and 26 > 25 > 24 and 34 > 31 > 29). Additionally, it was
found that ortho-phenyl substituted compounds 32 and 37 dis-
played the highest 5-HT7/5-HT1A selectivity ratio.
3. Conclusions
Interestingly, analyzing an influence of the kind of substituent in
the ortho-position in PhP on compounds affinity for 5-HT1ARs, an
Extending our studies in identifying 5-HT7 receptors ligands, we
have designed and synthesized a novel series of long-chain aryl-
piperazines functionalized with cyclic amino acid (Pro and Tic)
primary or tertiary amide in the terminal fragment. Biological
evaluation, supported by molecular modeling, allowed for the
opposite
methylthio > isopropyl > phenyl.
Finally, according to the above-mentioned outcomes,
rank
order
was
established:

V. Canale et al. / European Journal of Medicinal Chemistry 92 (2015) 202e211
205
Table 1
chromatography (TLC) performed on Macherey-Nagel gel 60 F254
aluminum sheets (Düren, Germany). Spots were detected by their
Binding affinities of selected (S)-Pro and (S)- Tic primary and tertiary amide de-
rivatives for 5-HT7 and 5-HT1A receptors.
absorption under UV light (
l
¼ 254 nm).
Analytical HPLC analyses were run on an Alliance Waters 2695
Separations Module equipped with a Chromolith SpeedROD RP
18.5
m
m column (4.6 ꢁ 50 mm). Standard conditions were as fol-
lows: 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.
Compd Amino acid
X
n
R
Ki [nM]a
5-HT7 5-HT1A 5-HT1A
Ratio Ki
/
5-HT7/1A
LC/MS analyses were carried out on a system consisting of a
Waters Acquity UPLC coupled with a Waters TQD mass spec-
trometer (electrospray ionization mode ESI-tandem quadrupole).
All of the analyses were carried out with an Acquity UPLC BEH
7
22b
23b
24b
25
26
27b
28b
29b
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
Pro
Pro
Pro
Pro
Pro
Tic
Tic
Tic
Tic
Tic
Tic
Tic
Tic
Pro
Pro
Pro
Pro
Pro
Pro
Tic
Tic
Tic
Tic
Tic
Tic
Tic
e
2
3
4
4
4
2
3
4
2
4
2
3
4
4
4
2
3
4
4
3
4
2
2
3
4
4
SCH3
SCH3
SCH3
153
163
490
47
14
23
16
53
18
<1
2
34
20
66
26
61
1
17
35
18
9
35
16
14
47
36
19
40
16
<1
<1
<1
<1
3
<1
<1
<1
1
3
11
21
7
<1
3
>78
55
<1
2
<1
<1
<1
77
8
e
e
e
isopropyl 116
C
18, 50 ꢁ 2.1 mm column at 40 ꢀC. A flow rate of 0.3 mL/min and
e
phenyl
SCH3
SCH3
SCH3
isopropyl 30
isopropyl 43
phenyl
phenyl
phenyl
isopropyl 77
isopropyl 135
phenyl
phenyl
phenyl
phenyl
isopropyl 40
isopropyl 22
phenyl
phenyl
phenyl
phenyl
phenyl
18
51
78
a gradient of (5e95)% B over 13 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.
e
e
e
111
e
e
<1
6
3
1H NMR and 13C NMR spectra were recorded at 300 MHz and
75 MHz (Varian BB 200 spectrometer) using CDCl3, as solvent.
The J values are reported in Hertz (Hz), and the splitting patterns
are designated as follows: s (singlet), d (doublet), dd (doublet of
doublet), t (triplet), m (multiplet), br s (broad singlet).
Abbrevations used: BOC, tert-butyloxycarbonyl; BOP, (Benzo-
e
12
10
11
e
e
6
CH2
O
CH2
CH2
CH2
O
CH2
CH2
CH2
O
CH2
CH2
O
<1
<1
7
1
<1
1
<1
<1
5
4
2
5
16
15
30
<1
<1
2
triazol-1-yloxy)tris(dimethylamino)phosphonium
hexa-
<1
3
2
<1
<1
<1
<1
<1
fluorophosphate; IBCF, isobutyl chloroformate; MeOH, Methanol;
NMM, N-Methylmorpholine; DCM, Dichloromethane; TEA, Trie-
thylamine; TFA, Trifluoroacetic acid; THF, Tetrahydrofuran.
10
9
9
12
9
4.2. General procedure for preparation of final compounds
3
2
1-([1,1'-biphenyl]-2-yl)piperazine (60 mg, 0.24 mmol) was dis-
solved in acetone (5 ml). Then K2CO3 (66 mg, 0.48 mmol) and
catalytic amount of KI (4 mg, 0.024 mmol) were added followed by
dropwise addition of (S)-5-bromo-1-(2-(piperidine-1-carbonyl)
pyrrolidin-1-yl)pentan-1-one (13, 100 mg, 0.29 mmol). The reac-
tion was refluxed for 72 h. Inorganic residues were filtrated off and
organic mixture was concentrated under reduced pressure. The
obtained crude product was purified using silica gel column and
mixture DCM/MeOH ¼ 9/0.7 (v/v) as eluting system to yield 97 mg
(81%) of final compound 37.
a
Ki values (SEM 22) based on three independent binding experiments.
Data taken from Ref. [15].
b
identification of several derivatives with significant 5-HT7 and 5-
HT1A receptor affinities. Selected compounds exhibited antagonist
properties at 5-HT7Rs (32 and 37) or partial agonist effects at 5-
HT1ARs (28 and 35) in vitro functional activity assays. The results
of pharmacological in vivo studies showed that compounds 32 and
28, produced specific antidepressant-like effects in FST in mice
after acute treatment in doses of 10 mg/kg (32) and 1.25 mg/kg (28),
whereas compounds 35 and 37 were not active. These preliminary
results are promising to provide further detailed studies aimed at
the investigation of 5-HT7R acting agents in the treatment of
depression.
4.2.1. (S)-1-(7-(4-(2-Isopropylphenyl)piperazin-1-yl)heptanoyl)
pyrrolidine-2-carboxamide (25)
Oil, 73 mg, (73% isolated yield) following chromatographic pu-
rification over silica gel with DCM/MeOH (9/1); LC/MS purity 99%,
tR ¼ 4.50 min C25H40N4O2, MW 428.61, Monoisotopic Mass 428.32,
[MþH]þ ¼ 429.5. 1H NMR (300 MHz, CDCl3):
1.20 (d, 6H, J ¼ 7 Hz),
d
1.31e1.40 (m, 4H), 1.51e1.69 (m, 4H), 1.81e2.23 (m, 4H), 2.29e2.33
(m, 2H), 2.46e2.59 (m, 2H), 2.66 (br s, 4H), 2.90e3.01 (m, 4H),
4. Experimental protocols
3.38e3.63 (m, 3H), 4.53 (d, 1H, J ¼ 8.03 Hz), 7.04e7.13 (m, 4H). 13
C
4.1. Chemistry
NMR (75 MHz, CDCl3): d 24.00, 24.60, 24.91, 26.35, 26.66, 27.25,
27.61, 29.16, 34.46, 47.51, 52.84, 53.79, 58.61, 59.39, 120.51, 124.55,
126.25, 126.32, 144.56, 150.41, 173.32, 174.02.
Organic transformations were carried out at ambient tempera-
ture, unless indicated otherwise indicated. Organic solvents used in
this study (from SigmaeAldrich and Chempur) were of reagent
grade and were used without purification. All other commercially
available reagents were of the highest purity (from SigmaeAldrich,
Alfa Aesar, Fluorochem).
All workup and purification procedures were carried out with
reagent-grade solvents under ambient atmosphere. Flash chroma-
tography was performed on MachereyeNagel silica gel 60 (70e230
mesh ASTM).
4.2.2. (S)-1-(7-(4-([1,1'-Biphenyl]-2-yl)piperazin-1-yl)heptanoyl)
pyrrolidine-2-carboxamide (26)
Oil, 77 mg (70% isolated yield) following chromatographic pu-
rification over silica gel with DCM/MeOH (9/1); LC/MS purity 99%,
tR ¼ 4.61 min C28H38N4O2, MW 462.63, Monoisotopic Mass 462.30,
[MþH]þ ¼ 463.4. 1H NMR (300 MHz, CDCl3)
d 1.31e1.40 (m, 4H),
1.51e1.69 (m, 4H), 1.81e2.23 (m, 4H), 2.29e2.33 (m, 2H), 2.46e2.59
(m, 6H), 2.90e3.01 (m, 4H), 3.36e3.45 (m, 1H), 3.52e3.59 (m, 1H),
4.53 (d, 1H, J ¼ 8.03 Hz), 7.05e7.10 (m, 2H), 7.18e7.30 (m, 3H),
The purity of the compounds were confirmed by the thin layer

206
V. Canale et al. / European Journal of Medicinal Chemistry 92 (2015) 202e211
Fig. 2. Panel illustrating molecular modeling results of the designed library for the 5-HT7 and 5-HT1ARs. (A) The comparison of the binding modes for Pro- (22 e cyan) and Tic-
amide (27 e green) derivatives. (B) The influence of the piperidinyl (43 e green) and morpholinyl (44 e cyan) moieties on the binding mode changes of corresponding primary
amide (32 e yellow). (C) The introduction of linkers of different length ((CH2)4, 22 e cyan; (CH2)5, 23 e yellow, (CH2)6, 24 e green) exhibited no significant changes to the binding
mode. (D) The orientation of different substituents in an ortho-position of the phenyl ring in arylpiperazine fragment (SCH3 e 24 (green), isopropyl e 25 (yellow), phenyl e 26
(cyan)). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
7.35e7.41 (m, 2H), 7.55e7.62 (m, 2H). 13C NMR (75 MHz, CDCl3):
24.42, 24.90, 26.08, 27.03, 27.57, 29.00, 34.39, 47.50, 50.38, 53.18,
4.2.3. (S)-2-(5-(4-(2-Isopropylphenyl)piperazin-1-yl)pentanoyl)-
1,2,3,4-tetrahydroisoquinoline-3-carboxamide (30)
d
58.41, 59.44, 118.22, 122.80, 126.77, 128.19, 128.32, 128.79, 131.44,
134.93, 141.02, 149.80, 173.36, 173.91.
Oil, 72 mg (65% isolated yield) following chromatographic pu-
rification over silica gel with DCM/MeOH (9/1); LC/MS purity 99%,
tR ¼ 4.85 min C28H38N4O2, MW 462.63, Monoisotopic Mass 462.30,
[MþH]þ ¼ 463.4. 1H NMR (300 MHz, CDCl3):
1.20 (d, 6H, J ¼ 7 Hz),
d
1.47e1.76 (m, 4H), 2.29e2.62 (m, 8H), 2.75e2.89 (m, 4H), 2.91 (dd,

V. Canale et al. / European Journal of Medicinal Chemistry 92 (2015) 202e211
207
120.50, 124.49, 125.58, 126.25, 126.33, 126.63, 127.69, 128.40,
132.49, 133.82, 144.59, 150.57, 173.19, 173.59.
4.2.5. (S)-2-(5-(4-([1,1'-Biphenyl]-2-yl)piperazin-1-yl)pentanoyl)-
1,2,3,4-tetrahydroisoquinoline-3-carboxamide (32)
Oil, 97 mg (89% isolated yield), following chromatographic pu-
rification over silica gel with DCM/MeOH (9/1); LC/MS purity 99%,
tR ¼ 5.19 min C31H36N4O2, MW 496.64, Monoisotopic Mass 496.28,
[MþH]þ ¼ 497.3 1H NMR (300 MHz, CDCl3):
d 1.31e1.43 (m, 2H),
1.43e1.55 (m, 2H), 1.61e1.73 (m, 2H), 2.28e2.52 (m, 6H), 2.82e2.90
Fig. 3. Structure and 5-HT7/5-HT1A binding profiles for LP-44 and 28.
Table 2
Receptor binding profile and functional properties for compounds 28, 32, 35, and 37, selected for behavioral evaluation in FST.
Compd
5-HT7
5-HT1A
5-HT2A Ki [nM]a
5-HT6 Ki [nM]a
Ki [nM]a
Agonistb
Antagonistc
Ki [nM]a
Agonistb
Antagonistc
28
32
35
37
78
12
77
5
NTe
6
NT
20
NT
94
NT
91
<1
66
1
19
NT
30
NT
98d
NT
282
594
603
376
2010
166
2148
1684
100d
NT
35
a
Ki values (SEM 22) based on three independent binding experiments.
% of control agonist response at 10ꢂ6 M.
b
c
% inhibition of control agonist response at 10ꢂ6 M.
d
e
The tested compound induced at least 25% agonist or agonist-like effect.
Not tested (only compounds with Ki below 50 nM were functionally evaluated).
Fig. 4. Effects of the active compounds 28 and 32, imipramine and SB-269970 on the immobility time in the forced swim test in Swiss albino mice.
1H, J1 ¼ 6.7 Hz, J2 ¼ 15.9 Hz), 3.24 (dd, 1H, J1 ¼ 5.2 Hz, J2 ¼ 14.8 Hz),
3.44e3.53 (m, 1H), 4.44e4.65 (m, 2H), 5.14 (t, 1H, J ¼ 5 Hz),
(m, 4H), 2.98 (dd, 1H, J1 ¼ 5 Hz, J2 ¼ 16 Hz), 3.24 (dd, 1H, J1 ¼ 5.2 Hz,
J2 ¼ 14.8 Hz), 4.50e4.75 (m, 2H), 5.14 (t, 1H, J ¼ 5 Hz), 7.04e7.16 (m,
7.06e7.27 (m, 8H). 13C NMR (75 MHz, CDCl3):
d
22.75, 24.07, 26.49,
2H), 7.18e7.31 (m, 9H), 7.33e7.41 (m,1H), 7.62 (d,1H, J ¼ 8.4 Hz). 13
C
27.75, 29.87, 33.83, 45.99, 52.63, 53.15, 53.83, 58.26, 121.58, 124.49,
125.53,126.25,126.33,127.63,127.89,128.40,132.49,134.82,144.59,
155.57, 173.19, 174.64.
NMR (75 MHz, CDCl3): d 22.70, 26.35, 29.83, 33.76, 45.97, 50.77,
52.65, 53.25, 58.11, 118.18, 122.26, 125.57, 126.63, 126.71, 127.69,
128.15, 128.29, 128.38, 128.83, 131.45, 132.45, 133.80, 134.91, 141.14,
150.08, 173.15, 173.58.
4.2.4. (S)-2-(7-(4-(2-Isopropylphenyl)piperazin-1-yl)heptanoyl)-
1,2,3,4-tetrahydroisoquinoline-3-carboxamide (31)
4.2.6. (S)-2-(6-(4-([1,1'-Biphenyl]-2-yl)piperazin-1-yl)hexanoyl)-
1,2,3,4-tetrahydroisoquinoline-3-carboxamide (33)
Oil, 93 mg (79% isolated yield) following chromatographic pu-
rification over silica gel with DCM/MeOH (9/1); LC/MS purity 97%,
tR ¼ 5.17 min C30H42N4O2, MW 490.68, Monoisotopic Mass 490.33,
Oil, 95 mg (78% isolated yield) following chromatographic pu-
rification over silica gel with DCM/MeOH (9/1); LC/MS purity 99%,
tR ¼ 5.26 min C32H38N4O2, MW 510.67, Monoisotopic Mass 510.30,
[MþH]þ ¼ 491.5. 1H NMR (300 MHz, CDCl3):
1.20 (d, 6H, J ¼ 7 Hz),
d
1.47e1.76 (m, 4H), 2.29e2.67 (m, 8H), 2.70e2.88 (m, 8H), 2.91 (dd,
1H, J1 ¼ 6.7 Hz, J2 ¼ 15.9 Hz), 3.24 (dd, 1H, J1 ¼ 5.2 Hz, J2 ¼ 14.8 Hz),
3.44e3.45 (m, 1H), 4.44e4.65 (m, 2H) 5.14 (t, 1H, J ¼ 5 Hz),
[MþH]þ ¼ 511.3. 1H NMR (300 MHz, CDCl3):
d 1.31e1.42 (m, 2H),
1.44e1.56 (m, 2H), 1.62e1.73 (m, 2H), 2.28e2.52 (m, 8H), 2.82e2.90
(m, 4H), 2.98 (dd, 1H, J1 ¼ 5 Hz, J2 ¼ 16 Hz), 3.24 (dd, 1H, J1 ¼ 5.2 Hz,
J2 ¼ 14.8 Hz), 4.50e4.75 (m, 2H), 5.14 (t, 1H, J ¼ 5 Hz), 7.04e7.16 (m,
2H), 7.18e7.31 (m, 9H), 7.33e7.41 (m, 1H), 7.62 (d, 1H, J ¼ 8.4 Hz).
7.06e7.27 (m, 8H). 13C NMR (75 MHz, CDCl3):
d 22.83, 24.01, 26.46,
26.67, 29.87, 33.83, 39.2, 43.91, 45.99, 52.63, 53.08, 53.83, 58.26,

208
V. Canale et al. / European Journal of Medicinal Chemistry 92 (2015) 202e211
13C NMR (75 MHz, CDCl3):
d
22.70, 26.35, 29.83, 32.19, 33.76,
purification over silica gel with DCM/MeOH (9/0.5); LC/MS purity
45.97, 50.77, 52.65, 53.25, 58.11, 118.18, 122.26, 125.57, 126.63,
126.71, 127.69, 128.15, 128.29, 128.38, 128.83, 131.45, 132.45, 133.80,
134.91, 141.14, 150.08, 173.15, 173.58.
98%, tR ¼ 5.10 min C32H44N4O2, MW 516.72, Monoisotopic Mass
516.35, [MþH]þ ¼ 517.4. 1H NMR (300 MHz, CDCl3):
d 1.20e1.74 (m,
14H), 1.76e1.85 (m, 2H), 2.20e2.31 (m, 2H), 2.48e2.62 (m, 2H),
2.81e2.92 (m, 4H), 3.41e3.72 (m, 8H), 4.19e4.22 (m, 2H), 4.85 (dd,
1H, J1 ¼ 3.6 Hz, J2 ¼ 7.9 Hz), 6.95e7.0 (m, 1H), 7.04e7.09 (m, 1H),
7.18e7.25 (m, 3H), 7.32e7.35 (m, 2H), 7.45e7.51 (m, 2H). 13C NMR
4.2.7. (S)-2-(7-(4-([1,1'-Biphenyl]-2-yl)piperazin-1-yl)heptanoyl)-
1,2,3,4-tetrahydroisoquinoline-3-carboxamide (34)
Oil, 90 mg (72% isolated yield) following chromatographic pu-
rification over silica gel with DCM/MeOH (9/1); LC/MS purity 97%,
tR ¼ 5.38 min C33H40N4O2, MW 524.70, Monoisotopic Mass 524.32,
(75 MHz, CDCl3): d 24.54, 24.57, 25.55, 26.35, 26.82, 28.90, 29.09,
33.44, 38.71, 43.26, 46.52, 47.33, 52.35, 56.59, 118.59, 123.75, 127.07,
128.36, 128.53, 128.74, 130.85, 131.49, 135.18, 140.60, 170.79, 171.74.
[MþH]þ ¼ 525.4. 1H NMR (300 MHz, CDCl3):
d 1.31e1.42 (m, 2H),
1.44e1.60 (m, 4H), 1.62e1.73 (m, 2H), 2.28e2.52 (m, 8H), 2.82e2.90
(m, 4H), 2.98 (dd, 1H, J1 ¼ 5 Hz, J2 ¼ 16 Hz), 3.24 (dd, 1H, J1 ¼ 5.2 Hz,
J2 ¼ 14.8 Hz), 4.50e4.75 (m, 2H), 5.14 (t, 1H, J ¼ 5 Hz), 7.04e7.16 (m,
4.2.12. (S)-7-(4-([1,1'-Biphenyl]-2-yl)piperazin-1-yl)-1-(2-
(piperidine-1-carbonyl)pyrrolidin-1-yl)heptan-1-one (39)
Oil, 98 mg (79% isolated yield) following chromatographic pu-
rification over silica gel with DCM/MeOH (9/0.5); LC/MS purity 98%,
tR ¼ 5.51 min C33H46N4O2, MW 530.74, Monoisotopic Mass 530.36,
2H), 7.18e7.31 (m, 9H), 7.33e7.41 (m,1H), 7.62 (d,1H, J ¼ 8.4 Hz). 13
C
NMR (75 MHz, CDCl3):
d 22.70, 26.32, 28.34, 29.79, 33.76, 45.97,
50.77, 52.65, 53.25, 58.11, 118.18, 122.26, 125.57, 126.63, 126.71,
127.69, 128.15, 128.29, 128.38, 128.83, 131.45, 132.45, 133.80, 134.91,
140.43, 150.12, 173.15, 173.65.
[MþH]þ ¼ 531.4. 1H NMR (300 MHz, CDCl3):
d 1.20e1.75 (m, 14H),
1.76e1.85 (m, 2H), 2.02e2.18 (m, 2H), 2.20e2.35 (m, 2H), 2.41e2.62
(m, 2H), 2.90e3.02 (m, 4H), 3.40e3.70 (m, 8H), 4.21 (m, 2H), 4.85
(dd,1H, J1 ¼3.6 Hz, J2 ¼ 7.9 Hz), 6.95e7.0 (m, 1H), 7.04e7.09 (m, 1H),
7.18e7.25 (m, 3H), 7.32e7.35 (m, 2H), 7.45e7.51 (m, 2H). 13C NMR
4.2.8. (S)-7-(4-(2-Isopropylphenyl)piperazin-1-yl)-1-(2-
(piperidine-1-carbonyl)pyrrolidin-1-yl)heptan-1-one (35)
(75 MHz, CDCl3): d 22.96, 23.72, 24.55, 24.59, 25.53, 26.32, 26.96,
Oil, 86 mg (73% isolated yield) following chromatographic pu-
rification over silica gel with DCM/MeOH (9/0.7); LC/MS purity
100%, tR ¼ 5.32 min C40H48N4O2, MW 496.73, Monoisotopic Mass
28.94, 34.33, 47.20, 50.17, 53.05, 56.21, 58.32, 68.11, 118.24, 122.84,
126.81, 128.22, 128.26, 128.32, 128.76, 131.42, 134.92, 140.97, 149.69,
170.07, 171.62.
496.38, [MþH]þ ¼ 497.4. 1H NMR (300 MHz, CDCl3):
d 1.20 (d, 6H,
J ¼ 7 Hz), 1.28e1.40 (m, 4H), 1.44e1.76 (m, 8H), 1.78e2.20 (m, 6H),
2.30e2.35 (m, 2H), 2.46e2.56 (m, 2H), 2.70 (br s, 4H), 2.90e3.01 (m,
4H), 3.38e3.56 (m, 5H), 3.60e3.72 (m, 2H), 4.88 (dd, 1H, J1 ¼ 3.6 Hz,
J2 ¼ 7.9 Hz), 6.98e7.15 (m, 3H), 7.22e7.27 (m,1H). 13C NMR (75 MHz,
4.2.13. (S)-7-(4-([1,1'-Biphenyl]-2-yl)piperazin-1-yl)-1-(2-
(morpholine-4-carbonyl)pyrrolidin-1-yl)heptan-1-one (40)
Oil, 102 mg (80% isolated yield) following chromatographic
purification over silica gel with DCM/MeOH (9/0.5); LC/MS purity
99%, tR ¼ 4.91 min C32H44N4O3, MW 532.72, Monoisotopic Mass
CDCl3): d 23.99, 24.42, 24.51, 24.76, 25.94, 26.76, 27.15, 29.12, 34.37,
42.44, 46.17, 47.23, 52.47, 53.67, 55.75, 58.48, 66.90, 120.73, 124.80,
126.36, 126.38, 144.59, 150.19, 170.79, 171.74.
532.34, [MþH]þ ¼ 533.3. 1H NMR (300 MHz, CDCl3):
d 1.30e1.49
(m, 8H), 1.51e1.70 (m, 4H), 1.81e2.05 (m, 2H), 2.08e2.20 (m, 2H),
2.25e2.35 (m, 2H), 2.48e2.60 (m, 2H), 2.90e3.01 (m, 4H),
3.48e3.80 (m, 8H), 4.18e4.21 (m, 2H), 4.85 (dd, 1H, J1 ¼ 3.6 Hz,
J2 ¼ 7.9 Hz), 6.95e7.0 (m, 1H), 7.04e7.09 (m, 1H), 7.18e7.25 (m, 3H),
7.32e7.35 (m, 2H), 7.45e7.51 (m, 2H). 13C NMR (75 MHz, CDCl3):
4.2.9. (S)-7-(4-(2-Isopropylphenyl)piperazin-1-yl)-1-(2-
(morpholine-4-carbonyl)pyrrolidin-1-yl)heptan-1-one (36)
Oil, 88 mg (71% isolated yield) following chromatographic pu-
rification over silica gel with DCM/MeOH (9/0.7); LC/MS purity
100%, tR ¼ 4.70 min C40H46N4O3, MW 498.70, Monoisotopic Mass
d
22.96, 23.72, 24.59, 26.32, 28.94, 34.33, 47.20, 49.65, 50.17, 53.05,
56.21, 58.32, 66.87, 68.16, 118.24, 122.84, 126.81, 128.22, 128.26,
128.32, 128.76, 131.42, 134.92, 140.97, 149.69, 170.07, 171.62.
498.36, [MþH]þ ¼ 499.4. 1H NMR (300 MHz, CDCl3):
d 1.18 (d, 6H,
J ¼ 7 Hz), 1.28e1.40 (m, 4H), 1.52e1.72 (m, 4H), 1.78e2.20 (m, 6H),
2.32e2.36 (m, 2H), 2.44e2.54 (m, 2H), 2.70 (br s, 4H), 2.90e3.02
(m, 4H), 3.38e3.84 (m, 9H), 4.84 (dd, 1H, J1 ¼ 3.6 Hz, J2 ¼ 7.9 Hz),
6.98e7.15 (m, 3H), 7.22e7.27 (m, 1H). 13C NMR (75 MHz, CDCl3):
4.2.14. (S)-6-(4-(2-Isopropylphenyl)piperazin-1-yl)-1-(3-
(piperidine-1-carbonyl)-1,2,3,4-tetrahydroisoquinolin-2-yl)hexan-
1-one (41)
d
23.99, 24.42, 24.76, 26.76, 27.15, 29.12, 34.37, 42.44, 46.17, 47.23,
Oil, 97 mg (70% isolated yield) following chromatographic pu-
rification over silica gel with DCM/MeOH (9/0.7); LC/MS purity 96%,
tR ¼ 6.0 min C34H48N4O2, MW 544,77, Monoisotopic Mass 544.38,
52.47, 53.67, 55.75, 58.48, 66.62, 66.90, 120.73, 124.80, 126.36,
126.38, 144.59, 150.19, 170.79, 171.74.
[MþH]þ ¼ 545.5. 1H NMR (300 MHz, CDCl3):
1.20 (d, 6H, J ¼ 7 Hz),
d
4.2.10. (S)-5-(4-([1,1'-Biphenyl]-2-yl)piperazin-1-yl)-1-(2-
(piperidine-1-carbonyl)pyrrolidin-1-yl)pentan-1-one (37)
1.31e1.44 (m, 4H), 1.48e1.69 (m, 8H), 2.38e2.48 (m, 4H), 2.62e2.68
(m, 4H), 2.88e2.94 (m, 4H), 3.02e3.06 (m, 2H), 3.26e3.64 (m, 5H),
4.69 (s, 2H), 5.46 (t, 1H, J ¼ 5.4 Hz), 7.04e7.81 (m, 8H). 13C NMR
Oil, 98 mg (81% isolated yield) following chromatographic pu-
rification over silica gel with DCM/MeOH (9/0.5); LC/MS purity 98%,
tR ¼ 5.30 min C31H42N4O2, MW 502.69, Monoisotopic Mass 502.33,
(75 MHz, CDCl3):
d 23.98, 24.54, 24.59, 25.55, 26.81, 28.90, 30.39,
30.68, 33.99, 38.71, 43.40, 46.52, 49.25, 53.29, 66.81, 120.65, 124.61,
125.50, 126.31, 126.35, 126.50, 127.24, 128.18, 133.16, 133.29, 144.61,
150.49, 169.50, 171.97.
[MþH]þ ¼ 503.4. 1H NMR (300 MHz, CDCl3):
d 1.20e1.75 (m, 12H),
1.76e1.85 (m, 2H), 2.18e2.33 (m, 2H), 2.41e2.62 (m, 2H), 3.10 (br s,
4H), 3.40e3.70 (m, 8H), 4.21e4.28 (m, 2H), 4.85 (dd, 1H, J1 ¼ 3.6 Hz,
J2 ¼ 7.9 Hz), 6.95e7.0 (m, 1H), 7.04e7.09 (m, 1H), 7.18e7.25 (m, 3H),
7.32e7.35 (m, 2H), 7.45e7.51 (m, 2H). 13C NMR (75 MHz, CDCl3):
4.2.15. (S)-7-(4-(2-Isopropylphenyl)piperazin-1-yl)-1-(3-
(piperidine-1-carbonyl)-1,2,3,4-tetrahydroisoquinolin-2-yl)heptan-
1-one (42)
d
24.54, 24.57, 25.55, 26.35, 28.90, 29.09, 33.44, 38.71, 43.26, 46.52,
47.33, 52.35, 56.59, 118.59, 123.75, 127.07, 128.36, 128.53, 128.74,
130.85, 131.49, 135.18, 140.60, 170.79, 171.74.
Oil, 89 mg (67% isolated yield) following chromatographic pu-
rification over silica gel with DCM/MeOH (9/0.7); LC/MS purity 97%,
tR ¼ 6.10 min C35H50N4O2, MW 558.80, Monoisotopic Mass 558.39,
4.2.11. (S)-6-(4-([1,1'-Biphenyl]-2-yl)piperazin-1-yl)-1-(2-
(piperidine-1-carbonyl)pyrrolidin-1-yl)hexan-1-one (38)
Oil, 91 mg (73% isolated yield) following chromatographic
[MþH]þ ¼ 559.4. 1H NMR (300 MHz, CDCl3):
1.20 (d, 6H, J ¼ 7 Hz),
d
1.31e1.44 (m, 4H), 1.48e1.71 (m, 10H), 2.38e2.48 (m, 4H), 2.62 (m,
4H), 2.88e2.94 (m, 4H), 3.02e3.06 (m, 2H), 3.26e3.64 (m, 5H), 4.69

V. Canale et al. / European Journal of Medicinal Chemistry 92 (2015) 202e211
209
(s, 2H), 5.46 (t, 1H, J ¼ 5.4 Hz), 7.04e7.81 (m, 8H). 13C NMR (75 MHz,
4.2.20. (S)-7-(4-([1,1'-Biphenyl]-2-yl)piperazin-1-yl)-1-(3-
(morpholine-4-carbonyl)-1,2,3,4-tetrahydroisoquinolin-2-yl)
CDCl3):
d
23.98, 24.54, 24.59, 25.55, 26.81, 28.90, 30.39, 30.68,
33.46, 33.99, 38.71, 43.40, 46.52, 49.25, 53.29, 66.81, 120.77, 125.52,
126.42, 126.46, 126.56, 127.25, 128.08, 144.56, 150.41, 133.02, 133.27,
144.44, 169.50, 171.97.
heptan-1-one (47)
Oil, 99 mg (71% isolated yield) following chromatographic pu-
rification over silica gel with DCM/MeOH (9/0.5); LC/MS purity 99%,
tR ¼ 5.65 min C37H46N4O3, MW 594.79, Monoisotopic Mass 594.35,
4.2.16. (S)-5-(4-([1,1'-Biphenyl]-2-yl)piperazin-1-yl)-1-(3-
(piperidine-1-carbonyl)-1,2,3,4-tetrahydroisoquinolin-2-yl)pentan-
1-one (43)
[MþH]þ ¼ 595.5. 1H NMR (300 MHz, CDCl3):
d 1.20e1.39 (m, 8H),
1.41e1.52 (m, 2H), 1.59e1.69 (m, 2H), 2.29e2.50 (m, 8H), 2.81e2.92
(m, 4H), 3.05 (d, 2H, J ¼ 6 Hz), 3.50e3.82 (m, 8H), 4.69 (d, 2H,
J ¼ 4 Hz), 5.49 (t, 1H, J ¼ 5 Hz), 7.15e7.35 (m, 5H), 7.35e7.42 (m, 2H),
Oil, 103 mg (76% isolated yield) following chromatographic
purification over silica gel with DCM/MeOH (9/0.5); LC/MS purity
99%, tR ¼ 6.09 min C36H44N4O3, MW 564.76, Monoisotopic Mass
7.62e7.67 (m, 2H). 13C NMR (75 MHz, CDCl3):
d 22.77, 25.90, 26.57,
29.64, 30.68, 33.48, 46.17, 46.52, 49.19, 50.31, 53.03, 58.00, 66.87,
118.29, 122.83, 125.50, 126.54, 126.76, 127.25, 128.09, 128.18, 128.33,
128.81, 131.44, 133.02, 133.25, 134.95, 141.04, 149.80, 169.49, 171.81.
564.35, [MþH]þ ¼ 565.3. 1H NMR (300 MHz, CDCl3):
d 1.40e1.69
(m, 12H), 2.30e2.45 (m, 6H), 2.82e2.91 (m, 4H), 3.05e3.09 (m, 2H),
3.50e3.82 (m, 8H), 4.69 (s, 2H), 5.49 (t, 1H, J ¼ 5 Hz), 7.07e7.33 (m,
5H), 7.35e7.42 (m, 2H), 7.60e7.64 (m, 2H). 13C NMR (75 MHz,
CDCl3)
d
22.77, 24.60, 25.70, 25.90, 26.57, 30.68, 33.48, 46.17, 49.19,
4.3. Molecular modeling
50.31, 53.03, 58.00, 118.29, 122.83, 125.50, 126.54, 126.76, 127.25,
128.09, 128.18, 128.33, 128.81, 131.44, 133.02, 133.25, 134.95, 141.04,
149.80, 169.49, 171.81.
Homology models of 5-HT1A and 5-HT7 receptors were built on a
crystal structures of 5-HT1B (PDB ID: 4IAR), b2 (PDB ID: 3P0G), D3
(PDB ID: 3PBL) and H1 (PDB ID: 3RZE) receptors, retrieved from the
Protein Data Bank [26]. Sequences of the modeled receptor and
selected templates were aligned manually using Accelrys Discovery
Studio v3.0 [27], making sure that the most conserved amino acid
in each helix, and motifs characteristic for class A GPCRs are on
equivalent positions. Ranges of helices were determined on the
basis of crystal structures; loop regions were modeled, but not
refined. For each template, 20 models were generated using
MODELLER 9v8 software [28], and underwent validation by dock-
ing of active and inactive compounds, and enrichment calculation.
The active (Ki < 100 nM) and inactive (Ki > 1000 nM) sets of
compounds towards each target, were retrieved from ChEMBL v11
database [29]. The whole set of inactive compounds (1194 for 5-
HT1AR and 1594 for 5-HT7R) was used for docking, while actives
were hierarchically clustered using Moldprint2D, Tanimoto metric
and Complete Cluster Linking Method implemented in Canvas v2.0
[30]. From each cluster containing more than two members, the
centroid was selected. Finally, all centroids of active (229 for 5-
HT1AR and 118 for 5-HT7R) and all sets of inactive compounds
constituted an input for docking. The 3-dimensional structures of
the synthesized compounds were prepared using LigPrep v3.0 [31]
and the appropriate ionization states at pH ¼ 7.4 were assigned
using Epik version 2.8 [32]. The Protein Preparation Wizard was
used to assign the bond orders, check the steric clashes, and assign
appropriate amino acid ionization states. The receptor grids were
generated (the OPLS_2005 force field) by centered the grid box of
the size of 15 Å on the Asp3.32. Automated docking was performed
by using Glide v6.3 [33] at SP level with the flexible docking option
turned on.
The docking procedure was validated by calculating the ROC
curve, based on the Glide Score values of docked compounds. Here,
the undocked actives and inactives were assigned as false negatives
and true negatives, respectively. The final model quality was
determined by the Area Under ROC curve (AUROC). Three models
per template with the highest AUROC value were selected for
further studies. The final validation of the selected receptor models
was performed by docking (Glide SP mode) the all set of studied
analogs. The spatial constrain was imposed on creation of an ionic
interaction between the protonated amine group of the ligand and
Asp3.32 side chain. Only those models were kept for which
coherent, for the whole set of compounds, and closest compliance
with common binding mode for monoaminergic receptor ligands
were observed [34]. The Ramachandran plots for both homology
models, generated by the service RAMPAGE, were added to the
Supplementary Materials [35].
4.2.17. (S)-5-(4-([1,1'-Biphenyl]-2-yl)piperazin-1-yl)-1-(3-
(morpholine-4-carbonyl)-1,2,3,4-tetrahydroisoquinolin-2-yl)
pentan-1-one (44)
Oil, 100 mg (74% isolated yield) following chromatographic
purification over silica gel with DCM/MeOH (9/0.5); LC/MS purity
99%, tR ¼ 5.28 min C35H42N4O3, MW 566.73, Monoisotopic Mass
566.33, [MþH]þ ¼ 567.4. 1H NMR (300 MHz, CDCl3):
d 1.44e1.53
(m, 6H), 1.56e1.63 (m, 2H), 2.29e2.50 (m, 8H), 2.81e2.92 (m, 4H),
3.05 (d, 2H, J ¼ 6 Hz), 3.50e3.82 (m, 8H), 4.69 (d, 2H, J ¼ 4 Hz), 5.49
(t,1H, J ¼ 5 Hz), 7.05e7.33 (m, 5H), 7.35e7.42 (m, 2H), 7.52e7.60 (m,
2H). 13C NMR (75 MHz, CDCl3):
d 22.86, 26.23, 29.69, 30.58, 33.59,
46.13, 49.05, 50.63, 53.16, 58.09, 66.83, 118.20, 122.68, 125.50,
126.67, 126.71, 127.43, 128.06, 128.16, 128.30, 128.82, 131.45, 132.90,
133.18, 134.90, 141.11, 150.01, 170.03, 172.01.
4.2.18. (S)-6-(4-([1,1'-Biphenyl]-2-yl)piperazin-1-yl)-1-(3-
(piperidine-1-carbonyl)-1,2,3,4-tetrahydroisoquinolin-2-yl)hexan-
1-one (45)
Oil, 113 mg (82% isolated yield) following chromatographic pu-
rification over silica gel with DCM/MeOH (9/0.5); LC/MS purity 98%,
tR ¼ 6.12 min C37H46N4O3, MW 578.79, Monoisotopic Mass 578.36,
[MþH]þ ¼ 579.5. 1H NMR (300 MHz, CDCl3):
d 1.42e1.53 (m, 6H),
1.54e1.70 (m, 8H), 2.30e2.45 (m, 6H), 2.82e2.91 (m, 4H), 3.05 (m,
2H), 3.50e3.82 (m, 8H), 4.69 (m, 2H), 5.49 (t, 1H, J ¼ 5 Hz),
7.09e7.31 (m, 5H), 7.35e7.42 (m, 2H), 7.60 (m, 2H). 13C NMR
(75 MHz, CDCl3):
d 22.77, 24.60, 25.70, 25.90, 26.57, 29.64, 30.68,
33.48, 46.17, 49.19, 50.31, 53.03, 58.00, 118.29, 122.83, 125.50,
126.54, 126.76, 127.25, 128.09, 128.18, 128.33, 128.81, 131.44, 133.02,
133.25, 134.95, 141.04, 149.80, 169.49, 171.81.
4.2.19. (S)-7-(4-([1,1'-Biphenyl]-2-yl)piperazin-1-yl)-1-(3-
(piperidine-1-carbonyl)-1,2,3,4-tetrahydroisoquinolin-2-yl)heptan-
1-one (46)
Oil, 112 mg (79% isolated yield) following chromatographic pu-
rification over silica gel with DCM/MeOH (9/0.5); LC/MS purity 95%,
tR ¼ 6.23 min C38H48N4O2, MW 592.81, Monoisotopic Mass 592.38,
[MþH]þ ¼ 593.5. 1H NMR (300 MHz, CDCl3):
d 1.42e1.51 (m, 4H),
1.53e1.76 (m, 10H), 2.29e2.50 (m, 8H), 2.81e2.92 (m, 4H),
3.05e3.11 (m, 2H), 3.50e3.82 (m, 8H), 4.69 (m, 2H), 5.49 (t, 1H,
J ¼ 5 Hz), 7.12e7.32 (m, 5H), 7.35e7.42 (m, 2H), 7.59e7.64 (m, 2H).
13C NMR (75 MHz, CDCl3):
d 22.77, 24.51, 25.70, 25.90, 26.57, 29.64,
30.68, 33.48, 46.17, 46.52, 49.19, 50.31, 53.03, 58.00, 118.29, 122.83,
125.50, 126.54, 126.76, 127.25, 128.09, 128.18, 128.33, 128.81, 131.44,
133.02, 133.25, 134.95, 141.04, 149.80, 169.49, 171.81.

210
V. Canale et al. / European Journal of Medicinal Chemistry 92 (2015) 202e211
4.4. In vitro pharmacology
p < 0.05 was considered statistically significant.
4.4.1. Radioligand binding assays
Acknowledgments
Following incubation, the receptor preparations were rapidly
filtered under vacuum through GF/B or GF/C glass fiber filters; the
filters were washed extensively with an ice cold buffer using a
harvester. Bound radioactivity was measured by scintillation
counting using a liquid scintillation cocktail.
Inhibition constants (Ki) were calculated from the Chenge-
Prusoff equation [36]. Results are expressed as means of at least
three separate experiments, each performed for 7e8 concentra-
tions (10ꢂ11e10ꢂ4 M) of a compound, run in triplicate.
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).
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.12.041.
4.4.2. Effects on adenylate cyclase activity
Adenylate cyclase activity was expressed as percentage of the
maximal effect obtained with 300 nM serotonin. cAMP concen-
tration was measured using HTRF method. The compounds were
tested in one concentration at 10ꢂ6 M, run in duplicate. Reference
agonists e serotonin and 8-OH-DPAT, and antagonists e methio-
thepin and WAY-100635, were used for 5-HT7 and 5-HT1A assays,
respectively.
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