Synthesis, docking studies, and pharmacological evaluation of 2-hydroxypropyl-4-arylpiperazine derivatives as serotoninergic ligands

Received: 3 November 2020

Revised: 17 December 2020

Accepted: 8 January 2021

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DOI: 10.1002/ardp.202000414

F U L L P A P E R

Synthesis, docking studies, and pharmacological evaluation

of 2‐hydroxypropyl‐4‐arylpiperazine derivatives as

serotoninergic ligands

Elisa Magli¹| Ewa Kędzierska²| Agnieszka A. Kaczor^3,4| Anna Bielenica⁵

Beatrice Severino¹| Ewa Gibuła‐Tarłowska²| Jolanta H. Kotlińska²

Angela Corvino¹| Rosa Sparaco¹| Giovanna Esposito¹| Stefania Albrizio¹

Elisa Perissutti¹| Francesco Frecentese¹| Anna Leśniak⁶

|

Magdalena Bujalska‐Zadrożny⁶| Marta Struga⁵| Raffaele Capasso¹

|

Vincenzo Santagada¹| Giuseppe Caliendo¹| Ferdinando Fiorino¹

¹Dipartimento di Farmacia, Università degli

Studi di Napoli “Federico II”, Napoli, Italy

Abstract

²Department of Pharmacology and

Pharmacodynamics, Faculty of Pharmacy with

Division of Medical Analytics, Medical

University of Lublin, Lublin, Poland

A new series of norbornene and exo‐N‐hydroxy‐7‐oxabicyclo[2.2.1]hept‐5‐ene‐2,3‐

dicarboximide derivatives was prepared, and their affinities to the 5‐HT_1A, 5‐HT_2A, and

5‐HT_2Creceptors were evaluated and compared with a previously synthesized series of

derivatives characterized by the same nuclei, to identify selective ligands for the sub-

type receptors. Arylpiperazines represent one of the most important classes of

5‐HT_1AR ligands, and the research of new derivatives has been focused on the mod-

ification of one or more portions of this pharmacophore. The combination of structural

elements (heterocyclic nucleus, hydroxyalkyl chain, and 4‐substituted piperazine),

known to be critical for the affinity to 5‐HT_1Areceptors, and the proper selection of

substituents resulted in compounds with high specificity and affinity toward ser-

otoninergic receptors. The most active compounds were selected for further in vivo

assays to determine their functional activity. Finally, to rationalize the obtained results,

molecular docking studies were performed. The results of the pharmacological studies

showed that 3e, 4j, and 4n were the most active and promising derivatives for the

serotonin receptor considered in this study.

³Department of Synthesis and Chemical

Technology of Pharmaceutical Substances

with Computer Modeling Laboratory, Faculty

of Pharmacy, Medical University of Lublin,

Lublin, Poland

⁴School of Pharmacy, University of Eastern

Finland, Kuopio, Finland

⁵Department of Biochemistry, Medical

University of Warsaw, Warsaw, Poland

⁶Department of Pharmacodynamics, Centre

for Preclinical Research and Technology,

Faculty of Pharmacy, Medical University of

Warsaw, Warsaw, Poland

Correspondence

Ferdinando Fiorino, Dipartimento di Farmacia,

Università degli Studi di Napoli “Federico II”,

Via D. Montesano, 49, 80131 Napoli, Italy.

Email: fefiorin@unina.it

K E Y W O R D S

5‐HT_1Areceptor ligands, arylpiperazine derivatives, exo‐N‐hydroxy‐7‐oxabicyclo[2.2.1]hept‐5‐

ene‐2,3‐dicarboximide, norbornene nucleus, serotonin

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1

INTRODUCTION

pharmacological properties, second messenger coupling, and signal

transduction characterization have allowed the identification of at

Serotonin (5‐hydroxytryptamine, 5‐HT) is an important neuromo-

dulator in the central and peripheral nervous systems (CNS and PNS,

respectively), which plays a critical role in a wide range of physio-

logical and pathophysiological processes. Molecular cloning techni-

ques, amino acid sequence determination, evaluation of its

least seven classes (5‐HT_1–7), with additional subclasses amounting

to 15 receptors.^[1]Whereas 5‐HT₃Rs are cation‐permeable ion

channels, all the others are G‐protein‐coupled receptors (GPCRs) and

are classified as rhodopsin‐like receptors. Serotonin receptors

(5‐HTRs) are the most widespread targets of drugs due to the

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Arch Pharm. 2021;354:e2000414.

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https://doi.org/10.1002/ardp.202000414

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MAGLI ET AL.

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numerous biological effects of the endogenous ligand; serotonin is

mainly involved in impulsivity and alcoholism, and in the different

phases of sleep, sexual behavior, appetite control, thermoregulation,

and cardiovascular function.^[2]In addition, it is already known that

5‐HT plays a fundamental role as a growth factor in several types of

nontumoral and tumoral cells, differentiation, and gene expression

also related to oncogenes.^[3]Consequently, pharmacological manip-

ulation of the 5‐HT system is assumed to have therapeutic potential,

and therefore it has been the subject of intense research.^[4]

disorders indicate the importance of serotonin and dopamine

target, besides 5‐HT₇, 5‐HT_1A, and D₂receptors. However, dif-

ferent studies demonstrate various dual‐ and multitarget acting

compounds that are useful against CNS disorders, which involve

serotoninergic receptors and other GPCRs, like muscarinic M4

receptors against schizophrenia.^[8]

Among different studies,^[9–17]performed in our laboratories,

that led to the synthesis of serotoninergic ligands characterized by

high affinity and selectivity, we described the synthesis and

pharmacological evaluation of a set of derivatives where the

piperazine‐N‐alkyl moiety has been linked via 2‐hydroxypropyl

spacing unit to norbornene and exo‐N‐hydroxy‐7‐oxabicyclo[2.2.1]

hept‐5‐ene‐2,3‐dicarboximide nuclei, respectively; the binding

data reported in these studies evidenced as the combination

of these structural elements afforded compounds with an inter-

esting affinity/selectivity profile toward 5‐HT_1Aand 5‐HT_2C

receptors.^[18]These results prompted us, in continuation of our

research program, to complete the norbornene and exo‐N‐

hydroxy‐7‐oxabicyclo[2.2.1]hept‐5‐ene‐2,3‐dicarboximide series

with some arylpiperazines that had not been considered pre-

viously (Tables 1 and 2). All the new compounds were tested for

their functional activity or affinity to 5‐HT_1A, 5‐HT_2A, and 5‐HT_2C

receptors, and their multireceptor profiles were also evaluated in

terms of functional activity for dopaminergic (D₂) and muscarinic

receptors. Moreover, compounds showing the best affinity and

selectivity binding profiles toward serotoninergic receptors, in-

cluding also compounds belonging to the previously synthetized

series, have been evaluated by in vivo assay (i.e., behavioral tests),

with the aim to discover novel pharmacological tools useful in

treating psychiatric and neurological disorders, such as schizo-

phrenia, depression, and anxiety. Therefore, we evaluated anti-

psychotic activity of the compounds in amphetamine‐induced

hyperactivity test, antidepressant‐like activity in the forced swim

test (FST), and anxiolytic‐like effects in the elevated plus‐maze

test (EPM). Moreover, we used additional tests—spontaneous lo-

comotor activity, rotarod, chimney test, and bar test—to assess

potential adverse effects of the compounds. Results obtained from

the tested compounds in the FST were compared with commonly

known antidepressant, fluoxetine, and those in EPM with the

clinically useful anxiolytic, buspirone.

Electrophysiological, pharmacological, and biochemical pieces of

evidence have demonstrated that 5‐HT_1ARs, localized in primary

afferent neurons, are involved in different neurological disorders and

are also known to be implicated in the proliferation of human tumor

cells; however, their function still remains poorly understood.

5‐HT_1AR antagonists inhibit the growth of different prostatic tumor

cell lines, such as PC‐3, DU‐145, and LNCaP, as well as the pro-

liferation of PC‐3 xenografted subcutaneously in athymic nude

mice.^[3]Concerning the 5‐HT₂receptor family (5‐HT_2A, 5‐HT_2B, and

5‐HT_2C), 5‐HT_2ARs activation stimulates the secretion of various

hormones and influences neuronal plasticity; peripheral 5‐HT_2Are-

ceptors mediate several processes such as vasoconstriction and

platelet aggregation.^[5]5‐HT_2Creceptor regulates physiological

functions such as locomotory activity, anxiogenesis, and neu-

roendocrine functions, besides being involved in sexual dysfunction

in males.^[6,7]

Arylpiperazines are one of the most important classes of

5‐HT_1AR ligands from which anxiolytics, including buspirone, anti-

psychotics, such as ziprasidone, perospirone, and aripiprazole, and

several other pharmacological tools originated. The general structure

of arylpiperazines consists of a terminal fragment containing an

amide, imide, alkyl, arylalkyl, heteroarylalkyl, or tetralin function

linked through a flexible aliphatic chain of variable length to the N‐1‐

arylpiperazine moiety. The research of new derivatives has been

focused on the modification of one or more portions of such a

pharmacophore. Two main interactions prove to be important for the

affinity of arylpiperazines for 5‐HT_1ARs: (a) an ionic bond between

the protonated nitrogen atom of the piperazine ring and the carboxyl

oxygen of the side chain of Asp3.32 and (b) an edge‐to‐face CH/π

interaction between the aromatic ring and the Phe6.52 residue,

which stabilizes the ligand binding. The basic pharmacophore of the

5‐HT_1AR ligands is the same for agonists and antagonists, and it

consists of an aromatic nucleus and a basic nitrogen atom, whose

optimal distance is 5.2 Å, but the nitrogen atom lies at 0.2 Å above

the plane defined by the reference ring.^[1]

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2

RESULTS AND DISCUSSION

Chemistry

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A limitation in the potential use of many 5‐HTR_1Areceptor

ligands as drugs or pharmacological tools of many 5‐HT_1Are-

ceptor ligands is their undesired high affinity for other receptors.

The dopaminergic D₂receptor and α₁‐adrenoceptor are two

other examples of receptors for which several 5‐HT_1Aligands

show high affinity. Nevertheless, polypharmacology is considered

an appropriate solution to achieve high‐efficacy complex therapy

for mood disorders and schizophrenia. Some new trends in

searching against the most common central nervous system

2.1

The general strategy for the synthesis of the target compounds

(Tables 1 and 2) is summarized in Scheme 1. The general procedure is

as follows: alkylation of the starting 4‐X‐substituted piperazines with

epichlorohydrin in absolute ethanol gave the corresponding

3‐chloro‐2‐hydroxypropyl‐4‐X‐substituted piperazines 2f–n. The

obtained intermediates were condensed with the desired hetero-

cycle endo‐N‐hydroxy‐5‐norbornene‐2,3‐dicarboximide or exo‐N‐

MAGLI ET AL.

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TABLE 1 Agonist activity of compounds 3f–n at the 5‐HT_1Areceptor

5‐HT_1Areceptor G‐protein stimulation

Compound^a

3f

X

pEC₅₀

EC₅₀SEM (nM)

E_maxSEM (%)

No activity

3g

3h

3i

5.9 0.44

No activity

1266 275

No activity

122.4 6.3*

No activity

3j

4.3 0.4

41360 2510

No activity

122 8.75*

No activity

3k

No activity

3l

No activity

3m

3n

8‐OH‐DPAT

7.5 0.11

27.2 0.13

154 2.3

^aAll the final compounds have been obtained and tested as racemic mixtures (R,S); [α]25D = 0.01° (c = 0.01, MeOH).

*p < .01 versus 8‐OH‐DPAT.

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Pharmacology

hydroxy‐7‐oxabicyclo[2.2.1]hept‐5‐ene‐2,3‐dicarboximide, in the

presence of NaOH pellets in absolute ethanol, to give the final

compounds 3f–n and 4f–n. Purification of each final product was

carried out by chromatography on silica gel column and further by

crystallization from the appropriate solvent. All new compounds

gave satisfactory elemental analysis results, and they were char-

acterized by ¹H NMR and mass spectrometry (API 2000 Applied

Biosystem). ¹H nuclear magnetic resonance (NMR), mass spectro-

metry (MS), and optical data for all final compounds obtained as

racemic mixtures were consistent with the proposed structures.

2.2

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2.2.1

Agonistic and antagonistic activity against

the 5‐HT_1Areceptor: Functional studies

Four newly synthesized arylpiperazines demonstrated an agonistic

activity at the 5‐HT_1Areceptor (Tables 3 and 4). Basal 5‐HT_1A

stimulation was set to 100%. When 2‐chlorophenylpiperazines

derivatives (3g, 4g) were compared, they both expressed similar

efficacies and equally low potencies. The E_max(baseline G‐protein

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MAGLI ET AL.

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TABLE 2 Agonist activity of

compounds 4f–n at the 5‐HT_1Areceptor

5‐HT_1Areceptor G‐protein stimulation

Compound^a

4f

X

pEC₅₀

EC₅₀SEM (nM)

E_maxSEM (%)

No activity

4g

4h

5.8 0.36

1388 229

No activity

138.6 10

No activity

4i

No activity

4j

6.4 0.4*

327.7 26.9

No activity

128 5.4**

No activity

4k

No activity

4l

No activity

4m

4n

8‐OH‐DPAT

7.5 0.11

27.2 0.13

154 2.3

^aAll the final compounds have been obtained and tested as racemic mixtures (R,S); [α]25D = 0.01°

(c = 0.01, MeOH).

*p < .05 versus 4j.

**p < .01 versus 8‐OH‐DPAT.

stimulation was set to 100%) value for compound 3g classified it as a

partial agonist (122.4% 6.3%), with potency (pEC₅₀equaling

pEC₅₀= 4.3 0.4; F_1,36= 4.7; p < .05). On the contrary, both

compounds expressed comparable partial agonist activity

)

a

5.9 0.44, whereas the corresponding values for its analog (4g) were

138.6% 10% and 5.8 0.36, respectively. Thus, the heterocyclic

scaffold does not influence pEC₅₀(F_1,36= 0.92; p > .05) or E_max

(F_1,36= 1.4; p > .05) parameters of the compound. In contrast,

the pyridyl piperazine derivative supporting an exo‐N‐hydroxy‐7‐

oxabicyclo[2.2.1]hept‐5‐ene‐2,3‐dicarboximide scaffold as a terminal

fragment (4j) was more potent (pEC₅₀= 6.4 0.4) than its analog (3j;

(128% 5.4% and 122% 8.75%; F_1,36= 0.8; p > .05). Instead, both

4‐methoxyphenylpiperazine derivatives (3f and 4f), the norbornene

derivative supporting a 3‐chlorophenylpiperazine moiety (3h), and

the derivative containing a naphthylpiperazine moiety (4n) acted as

antagonists at the 5‐HT_1Areceptor. In particular, the strongest an-

tagonistic profile was observed for 4n (pIC₅₀= 6.8 0.15;

F_4,85= 26.3; p < .001).

MAGLI ET AL.

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SCHEME 1 Reagents and conditions: (i) Cl–CH₂–CH(O)CH₂, EtOH abs; (ii) NaOH pellets, EtOH abs

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2.2.2

Binding affinity to the 5‐HT_2Aand 5‐HT_2C

(3i and 4i) derivatives. Finally, none of the tested compounds

expressed an antagonistic activity against the D₂receptor.

receptors: Competition studies

The difference in affinity observed between this new series of

derivatives (3f–n and 4f–n) and the previously described series,^[18]

characterized by the same norbornene or analog exo‐N‐hydroxy‐7‐

oxabicyclo[2.2.1]hept‐5‐ene‐2,3‐dicarboximide nucleus linked via

2‐hydroxypropyl spacing unit to 4‐substituted piperazines, further de-

monstrates that the isosteric substitution of a methylene group (3f–n)

with an oxygen atom (4f–n) does not represent a critical feature in

determining differences in binding with 5‐HTRs. Once again, also in

these novel derivatives, although they have a lower affinity profile

than the previously synthesized derivatives, the influence of the

2‐hydroxypropyl spacer associated with the appropriate substituents

on the phenylpiperazine ring and heterocyclic nucleus was particularly

profitable, not only in relation to 5‐HT_1Afunctional activity, but also

5‐HT_2Aand 5‐HT_2Creceptor affinity. However, to rationalize the

differential binding affinities/activities, molecular docking studies were

carried out on the complete series of derivatives.

All the new compounds were tested for their affinity to 5‐HT_2Aand

5‐HT_2Creceptors. Some of the newly synthesized derivatives

showed interesting affinity values in the nanomolar range

toward 5‐HT_2Areceptors and lower affinity toward 5‐HT_2Creceptors

(Tables 5 and 6). Besides the outstanding 5‐HT_2Areceptor affinity

and selectivity of compounds 3i (K_i= 32.7 nM with pK_i= 7.48 0.05

[F_1,38= 182.8; p < .001]) and 3n (K_i= 80 nM with pK_i= 7.1 0.06

[F_1,38= 36.7; p < .001]), other interesting K_ivalues were those of

compounds 3h (K_i= 371 nM with pK_i= 6.4 0.086 [F_1,38= 41.1;

p < .001]), 4n (K_i= 465 nM with pK_i= 6.3 0.097), 4i (K_i= 542 nM

with pK_i= 6.2 0.058), and 3g (K_i= 699 nM with pK_i= 6.16 0.11

[F_1,38= 22.1; p < .001]). Moreover, compound 3n showed an inter-

esting mixed 5‐HT_2A/5‐HT_2Cprofile with K_ivalues of 80/92.9 nM,

whereas compounds 4n, 3n, and 4l presented the most attractive

5‐HT_2Caffinity profile with K_ivalues of 69, 92.9, and 429 nM and pK_i

values of 7.1 0.16, 7.03 0.15, and 6.37 0.14, respectively.

As compared with the reference 5‐HT_2Areceptor ligand, ke-

tanserin (pK_i= 8.27 0.06), one can conclude that compounds 3i, 3n,

3h, 4n, 4i, and 3g expressed satisfactory affinities to the 5‐HT_2A

receptor. Simultaneously, the 3,4‐dichloro‐, naphthyl‐, 3‐chloro‐ and

2‐chlorophenylpiperazine substituents had the strongest influence

on the 5‐HT_2Areceptor binding affinity. When comparing the eval-

uated series of arylpiperazine derivatives with 5‐HT_2Creceptor‐

selective ligands, such as RS‐102221 (pK_i= 8.34 0.12), one can

point out 2‐hydroxypropoxyl derivatives supporting the naphthylpi-

perazine moiety as a terminal group (3n and 4n) as the most pro-

mising, with their pK_ivalue below 100 nM. The class of moderate

affinity 5‐HT_2Creceptor ligands (pK_ilower than 1 µM) consisted of

piperonylpiperazines (3l and 4l) and 3,4‐dichlorophenylpiperazine

|

2.2.3

In vitro evaluation of 5‐HT‐evoked

contractions

Successively, compounds 3n and 3i with better affinity/selectivity binding

profiles toward 5‐HT_2Areceptors have been tested by in vitro assay to

determine their activity concerning 5‐HT‐evoked contractions. In the rat

ileum, 5‐HT_2Areceptors are located on smooth muscles and their acti-

vation by 5‐HT is known to induce contraction. Consequently, 5‐HT_2A

antagonists depress 5‐HT‐induced contractions in the rat ileum.^[19]

According to Briejer and colleagues, we have shown that 5‐HT

contracted the rat ileum longitudinal muscle. In preliminary ex-

periments, we found that the neuronal blocker tetrodotoxin

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MAGLI ET AL.

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TABLE 3 Antagonistic activity of

compounds 3f–n at the 5‐HT_1Areceptor

5‐HT_1Areceptor G‐protein stimulation

Compound^a

3f

X

pIC₅₀SEM

IC₅₀SEM (nM)

pK_BSEM

4.5 0.19

29890 1458

4.95 0.16

3g

3h

No activity

4.6 0.4

No activity

4.96 0.3

26090 2511

3i

No activity

3j

No activity

3k

3l

No activity

3m

3n

WAY‐100635

8.4 0.12

4.3 1.4

8.78 0.15

^aAll the final compounds have been obtained and tested as racemic mixtures (R,S); [α]25D = 0.01°

(c = 0.01, MeOH).

(0.3 µM), the muscarinic receptor antagonist atropine (1 µM), the

adrenergic receptor antagonist phentolamine (10−6 M) plus

propranolol (10−6 M) did not affect the contractions by 5‐HT.

In contrast, ketanserin (0.1 µM), at a concentration that blocks

5‐HT_2Areceptors, depressed the contractions induced by 5‐HT.

Collectively, these results suggest that 5‐HT contracts the ileum

by acting on 5‐HT_2Areceptors located on smooth muscle, whereas

muscarinic or adrenergic receptors are not involved. Results show

the potency (expressed by the IC₅₀value) and the efficacy (ex-

pressed by the E_maxvalue) of the compounds under investigation

in inhibiting 5‐HT‐induced contractions in the rat ileum (a phar-

macological assay useful to detect activity toward 5‐HT_2Are-

ceptors). The compounds under investigation, 3n (E_max= 26.66%)

and 3i (E_max= 16.87%), did not significantly inhibit the contrac-

tions induced by 5‐HT. The rank order of efficacy was 3n > 3i.

Concerning the potency, these compounds displayed potency ap-

proximately in the 10⁻⁶to 10⁻⁵M range; specifically, the rank

order of potency was 3n (1.08 × 10⁻⁶M) > 3i (1.05 × 10⁻⁵M). Fi-

nally, none of the compounds under investigation contracted, per

se, the rat ileum.

MAGLI ET AL.

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TABLE 4 Antagonistic activity of

compounds 4f–n at the 5‐HT_1Areceptor

5‐HT_1Areceptor G‐protein stimulation

Compound^a

4f

X

pIC₅₀SEM

IC₅₀SEM (nM)

pK_BSEM

4.7 0.2

21310 1584

5.05 0.3

4g

4h

No activity

4i

No activity

4j

No activity

4k

4l

No activity

6.8 0.15

No activity

129.7 14.9

No activity

7.56 0.19

4m

4n

WAY‐100635

8.4 0.12

4.3 1.4

8.78 0.15

^aAll the final compounds have been obtained and tested as racemic mixtures (R,S); [α]25D = 0.01°

(c = 0.01, MeOH).

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2.3

Molecular docking studies

selection for the detailed description of molecular ligand–receptor in-

teractions was (i) their best receptor affinity at each receptor and (ii)

The analysis of molecular docking results of compounds 3f–n and 4f–n,

including also compounds previously synthetized (3a–e and 4a–e;

Figure 1),^[18]with all the studied receptors indicated that, in general, the

docking scores were comparable for both enantiomers. Slightly higher

values were obtained for R enantiomers, so the receptor complexes with

R enantiomers were used for further analysis. Figures 2 –4 present the

selection of the compound for in vivo studies. As the studied ligands

follow the classical pharmacophore model for aminergic GPCR

ligands,^[20]the electrostatic interaction between their protonatable ni-

trogen atoms and Asp3.32 is their main contact with the receptors.^[21]In

the case of all receptors and most active ligands, Trp6.46 and Phe6.52

are residues involved in π–π stacking interactions with N‐aryl groups of

the compounds, as reported previously for many similar ligand–receptor

complexes.^[22–26]Such a pattern of ligand–receptor interactions was

results of molecular docking of selected compounds to serotonin 5‐HT_1A

,

5‐HT_2A, and 5‐HT_2Creceptors, respectively. The rationale for compound

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MAGLI ET AL.

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TABLE 5 Affinities of compounds 3f–n toward the 5‐HT_2Aand 5‐HT_2Creceptors

Receptor binding affinity

Compound^a

3f

X

5‐HT_2ApK_i

5‐HT_2AK_iSD (nM)

5‐HT_2CpK_i

5‐HT_2CK_iSD (nM)

5.16 0.07

6880 1186

5.09 0.14

7995 403

3g

3h

6.16 0.11

6.4 0.086

699 126

371 122

5.57 0.25

4.99 0.13

2635 1380

10080 7413

3i

7.48 0.05

32.7 11.2

6.21 0.15

609 142

3j

5.2 0.045

4.96 0.11

5508 1109

5.26 0.26

5.3 0.19

5438 1819

4767 646

3k

10920 5959

3l

4.2 0.17

5.9 0.32

7.1 0.06

56560 14791

1034 209

80 11.6

6.26 0.14

5.8 0.31

7.03 0.15

544 266

1482 204.2

92.9 4.7

3m

3n

Ketanserin

RS‐102221

Serotonin

8.27 0.06

5.3 1.12

8.34 0.12

8.14 0.15

4.51 0.17

7.17 3.5

^aAll the final compounds have been obtained and tested as racemic mixtures (R,S); [α]25D = 0.01° (c = 0.01, MeOH).

found for ritanserin, an inverse agonist of serotonin 5‐HT_2Creceptor, in

the X‐ray structure of the respective ligand–receptor complex (PDB ID:

6BQH^[27]). This binding pose was verified by mutation of Phe5.47,

Phe6.44, and Trp6.48. Moreover, residues from extracellular loop 2 (ecl2)

are also important for ligand–receptor interactions, and they may be

responsible for subtype‐selective interactions,^[28,29]as they constitute a

differentiated receptor part involved in the recognition of the “address”

part of the ligands.^[30]

The ligands adopt an expanded docking conformation and are

situated parallel to the transmembrane helices. Their norbornene or

exo‐N‐hydroxy‐7‐oxabicyclo[2.2.1]hept‐5‐ene‐2,3‐dicarboximide

moiety is directed toward the extracellular vestibule, whereas their

N‐aryl group penetrates deeper into the cavities of the receptors,

which is also in accordance with the binding pose of ritanserin in its

abovementioned complex with serotonin 5‐HT_2Creceptor resolved

by X‐ray crystallography. The affinity of the studied compounds to

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TABLE 6 Affinities of compounds 4f–n for 5‐HT_2Aand 5‐HT_2Creceptors

Receptor binding affinity

Compound^a

4f

X

5‐HT_2ApK_i

5‐HT_2AK_iSD (nM)

5‐HT_2CpK_i

5‐HT_2CK_iSD (nM)

4.5 0.07

29070 11906

4.2 0.38

58830 24417

4g

4h

5.3 0.098

5955 1253

2220 210

4.7 0.21

17580 6115

2094 134

5.65 0.074

5.67 0.13

4i

6.2 0.058

542 114

6.15 0.10

708 235

4j

5.5 0.11

4.5 0.1

2812 1288

4.74 0.18

5.1 0.14

17960 6606

7626 323

4k

28630 13098

4l

4.5 0.13

5.8 0.25

6.3 0.097

31230 13489

1625 678

465 125

6.37 0.14

4.9 0.4

429 112

11010 2512

69.9 14.4

4m

4n

7.1 0.16

Ketanserin

RS‐102221

Serotonin

8.27 0.06

5.3 1.12

8.34 0.12

8.14 0.15

4.51 0.17

7.17 3.5

^aAll the final compounds have been obtained and tested as racemic mixtures (R,S); [α]25D = 0.01° (c = 0.01, MeOH).

serotonin receptor subtypes is mainly affected by the type of N‐aryl

substituents, and it is, to the lesser extent, driven by the presence of

norbornene or exo‐N‐hydroxy‐7‐oxabicyclo[2.2.1]hept‐5‐ene‐2,3‐

dicarboximide moiety, as previously reported by Zagórska et al.^[31]

for similar molecules. Following their results, ortho, meta, or para

substitutions on the phenyl ring seem to be generally preferred over

para substitution. This can be a consequence of the favorable pattern

of hydrogen bond interactions with ortho‐methoxy group

(compounds 3a and 4a) and/or most advantageous positions ortho‐

and meta‐substituted compounds can adopt in the binding site

(compounds 3b, 3d, 3g, 3h, 4b, 4d, 4g, and 4h). Good affinity of

compounds 3g, 3h, 3i, and 4i to serotonin 5‐HT_2Areceptor may be

explained by the possibility of halogen bond formation, as suggested

by Partyka et al.^[32]

Concerning serotonin receptor subtype selectivity of the in-

vestigated molecules, for the interactions of arylpiperazine group

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FIGURE 1 Norbornene and exo‐N‐hydroxy‐7‐oxabicyclo[2.2.1]hept‐5‐ene‐2,3‐dicarboximide series (3a–e and 4a–e) previously synthesized

with serotonin 5‐HT₂receptor subtypes, Asp3.32, Trp6.48, and one

or more aromatic residues at positions 5.47 and/or 6.52 are required.

For the serotonin 5‐HT_2Areceptor, Trp7.39 can be also important.^[33]

Our earlier molecular modeling studies allowed to conclude that for

the interactions with serotonin 5‐HT_2Creceptor, the ligands may

form additional hydrogen bonds with Ser2.60 or Asn7.35, which are

not found for interactions with 5‐HT_1Aand 5‐HT_2Areceptors.^[34]

In contrast, here we observed that Asn7.38 and Tyr7.42 seem to be

crucial for interactions with serotonin 5‐HT_1Areceptor. As an ex-

ample, when the selectivity profile of compound 3n versus 4n is

compared, the better affinity of compound 3n to 5‐HT_2Areceptor

can result from additional π–π stacking interaction with Phe6.51;

however, both ligands interact with Asp3.32, Trp6.48, and Phe6.52.

In the case of 5‐HT_2Creceptor, the affinity of both compounds is

comparable and results from interactions with Asp3.32, Phe5.47,

Trp6.48, and Phe6.52.

receptor can be attributed to the greater number of aromatic in-

teractions and additional hydrogen bond with Tyr7.42.

In conclusion, the molecular modeling results described above

may be useful to design serotonin receptor ligands with required

selectivity or polypharmacology.

|

2.4

In vivo behavioral tests

Compounds 3b, 3e, and 4a from the previously published series and

compounds 4j and 4n from this study were selected for further

functional in vivo studies. The first part of the experiments included

locomotor activity and motor coordination tests, generally accepted

as basic in central activity investigations of new agents.^[36]First,

none of the compounds at the dose of 30 mg/kg changed the beha-

vior of mice in the chimney test (Figure 6a) and impaired motor

coordination assessed in the rotarod test (Figure 6b). In the loco-

motor activity test, only compound 4j at a dose of 30 mg/kg de-

creased the spontaneous motility after 6 min (Figure 7a) and 20 min

(Figure 7b) of observation.

To study how a nonselective or a multitarget ligand 4n fits to the

binding pocket of all receptors, noncovalent interaction (NCI) maps

were generated. In the case of all receptors, compound 4n fits well to

the binding cavities, as depicted in Figure 5a –c. NCI is a visualization

index derived from the density and identification of NCIs. It is based

on the peaks that appear in the reduced density gradient at low

densities.^[35a]To further describe interactions of compound 4n with

the studied receptors, the most important ligand–receptor contacts

are shown in Table 7. It can be seen that π–π stacking interactions

between side chains of aromatic residues (in particular Trp6.48 and

Phe6.52) and the ligand naphthyl moiety are an important con-

tribution to interaction energy, as reported earlier.^[35b,c]Better af-

finity of compound 4n to serotonin 5‐HT_2Creceptor versus 5‐HT_2A

In the second stage of this study, we evaluated the antipsychotic

ability of the new compounds. Animal models of schizophrenia, com-

monly employed for preclinical studies of antipsychotic properties of

drugs, consider mainly amphetamine and MK‐801 models.^[37]The first

model is based on the manipulation of the dopaminergic system, and it

may primarily respond to drugs that affect this neurotransmitter system.

Many neuroleptics acting as dopaminergic antagonists reverse this ef-

fect.^[38]It is noteworthy that amphetamine‐induced hyperlocomotion is

sensitive to other classes of drugs, including mGluR2/3 agonists.^[39]

MAGLI ET AL.

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On the contrary, several preclinical tests have pointed to the role of

5‐HT_2Cligands in the modulation of monoaminergic systems, including

dopaminergic. Indeed, dysfunction in serotoninergic activity could con-

tribute to the alteration of dopaminergic function seen in schizo-

phrenia.^[40]In the amphetamine model, all tested compounds at a dose of

15 mg/kg reduced amphetamine‐induced hyperactivity of mice (Figure 8).

The observed positive antipsychotic effect could be related rather to

their interaction with other than dopaminergic receptors, because

the compounds evaluated are weak ligands of D₁and D₂receptors.

However, 5‐HT_2Cand/or 5‐HT_1Areceptors, through modulation of the

dopaminergic system, may play a significant role in this activity.

Another animal model used in this type of research is the MK‐801‐

induced hyperactivity test. Apart from the dopaminergic hypothesis,

numerous data also indicate the role of the glutamatergic system in the

development of schizophrenia. N‐Methyl‐^D‐aspartate (NMDA) receptor

ligands, for example, MK‐801, with antagonistic properties, increase

locomotor activity and disturb memory processes.^[41]Only compound

3e, used at the dose of 15 mg/kg, reduced the increased locomotor

activity of the animals in this test, which indicates that its mechanism of

action may be related to NMDA receptors (Figure 9).

Furthermore, due to the modulation of the central serotonin

neurotransmission, the new compounds may also show an anxiolytic

and/or antidepressant activity.^[42,43]Considering this premise, as

well as in vitro data obtained for the test compounds (mixed 5‐HT_1A

/

5‐HT_2Caffinity profile for all the compounds), we examined their

antidepressant and anxiolytic potential in behavioral models com-

monly used in mice, that is, FST and EPM.

The obtained results indicated that, except for 4j, all the tested

compounds (15 and 30 mg/kg) revealed antidepressant‐like properties,

observed as a shortening of the immobility time of mice to various ex-

tents in the FST (Figure 10). In addition, fluoxetine at the dose of 15 mg/

kg, used as a reference drug, caused a statistically significant decrease in

immobility time. Interestingly, compound 4j and two compounds active in

the FST test (3e and 4n) exhibited characteristics of anxiolytic drugs:

They increased in a statistically significant manner the percentage of

entries and the time spent in the open arms of the EPM. Compounds 3b

and 4a were inactive. This test is based on the hypothesis that exposure

to an elevated and open maze alley leads to an approach–avoidance

conflict that is considerably stronger in comparison to that evoked

by exposure to a closed maze alley.^[44]Fear‐induced inhibition of ex-

ploratory activity affects entries into open arms in this task. As a

FIGURE 2 Selected ligands in complex with serotonin 5‐HT_1A

receptor: (a) 3b, (b) 4a, and (c) 4j. Proteins are shown in wire

representation with cyan carbon atoms. The most important residues

are shown as sticks. Ligands are shown as sticks with gray carbon

atoms. Polar interactions are shown as red dashed lines. Nonpolar

hydrogen atoms are omitted for clarity

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MAGLI ET AL.

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significant increase in the percentage of time spent on the open arms and

the number of entries into open arms is observed only with drugs that

are clinically effective anxiolytics,^[45]this model has been used to assess

anxiolytic‐like activity of new putative anxiolytic compounds.^[46]Buspir-

one (5 mg/kg) used as a reference anxiolytic drug also prolonged time

and increased the percentage of entries into the open arms (Figure 11).

In this test, the new compounds 3e, 4j, and 4n were comparably effi-

cacious to buspirone.

In addition, we evaluated the potential adverse effects of

the compounds as the risk of catalepsy in the bar test. The motor

side effects of antipsychotic drugs are a main concern in clinics.

Therefore, various behavioral tests have been developed to determine

whether new antipsychotic agents or strategies generate motor side

effects in comparison to classical antipsychotics. Catalepsy in the bar

test is a frequently used model to predict the liability of drugs to

produce extrapyramidal side effects in humans.^[47]By themselves,

5‐HT_2Cligands do not induce catalepsy.^[48]Accordingly, any of the

here reported compounds exert cataleptogenic effects in the bar test.

In schizophrenia, there are additional cognitive disturbances in the

form of memory dysfunction, decreased concentration, or a decrease in

IQ. 5‐HT_1Areceptors play an important role in these processes.^[49]The

effect of the new compounds on memory impairment was assessed in

the passive avoidance (PA) test, which exploits the considerable pre-

ference of rodents for darkened versus illuminated places. An additional

aversive stimulus is an electrical impulse in the darkened zone. Memory

deficit in an animal model of cognitive impairment was caused by the

administration of MK‐801 at a dose of 0.3 mg/kg. The tested com-

pounds 3e, 4j, and 4n, used at the dose of 15 mg/kg, significantly im-

proved memory in mice treated with MK‐801 (Figure 12). The measure

of this activity was the lengthening of the transition time to the dark

room, compared with the control group and, at the same time, the

increase of the latency index (LI). Thus, these compounds clearly com-

pensate for memory deficits and stimulate consolidation processes.

These results may indicate the association of the activity of 3e, 4j, and

4n with their affinity for the 5‐HT_1Areceptor.^[50,51]

|

3

CONCLUSIONS

We have described the synthesis of a new series of arylpiperazines

as serotoninergic ligands (3f–n and 4f–n). The pyridyl piperazine

derivative supporting an exo‐N‐hydroxy‐7‐oxabicyclo[2.2.1]hept‐5‐

FIGURE 3 Selected ligands in complex with serotonin 5‐HT_2A

receptor: (a) 3d, (b) 3i, and (c) 3n. Proteins are shown in wire

representation with cyan carbon atoms. The most important residues

are shown as sticks. Ligands are shown as sticks with gray carbon

atoms. Polar interactions are shown as red dashed lines. Nonpolar

hydrogen atoms are omitted for clarity

MAGLI ET AL.

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ene‐2,3‐dicarboximide scaffold as a terminal fragment (4j) afforded a

favorable agonistic profile for 5‐HT_1Areceptors (pEC₅₀= 6.4 0.4),

whereas the derivative containing a naphthylpiperazine moiety (4n)

showed an interesting antagonist profile at the 5‐HT_1Areceptor

(pEC₅₀= 6.8 0.15). Due to their high potency at the 5‐HT_1Are-

ceptor affinity and selectivity, these compounds were selected to-

gether with compounds 3b, 3e, and 4a from the previously published

series for further in vivo studies to investigate their functional

activity.

The obtained results showed that, except for 4j, all tested

compounds exerted antidepressant‐like effects. Interestingly, com-

pounds 3n, 4j, and 4n revealed significant anxiolytic properties and,

in the EPM test, they were almost as efficacious as buspirone. Ad-

ditionally, no side effects, like catalepsy and motor impairment, were

observed after the injection of the tested compounds. Finally, the

derivatives 3e, 4j, and 4n showed a marked improvement of memory

in mice treated with MK‐801, indicating the association of activity of

these compounds with their affinity for the 5‐HT_1Areceptor.^[50,51]

However, further pharmacological studies are necessary to de-

termine their detailed mechanism of action and prospective clinical

usefulness.

In conclusion, data presented in this study confirm that, as ob-

tained with the series previously synthesized,^[21,22]the novel syn-

thesized compounds display a general trend of affinity toward

5‐HT_1Areceptors. Molecular docking studies supported these

results, highlighting some selective and additional interactions of the

identified ligands with the investigated receptor subtype.

|

4

EXPERIMENTAL

Chemistry

|

4.1

|

4.1.1

General

All reagents and substituted piperazines were commercial pro-

ducts purchased from Sigma‐Aldrich. Melting points, determined

using a Buchi Melting Point B‐540 instrument, are uncorrected

and represent values obtained on recrystallized or chromato-

graphically purified material. ¹H NMR spectra were recorded on

a Varian Mercury Plus 400 MHz instrument. Unless otherwise

stated, all spectra were recorded in CDCl₃. Chemical shifts are

FIGURE 4 Selected ligands in complex with serotonin 5‐HT_2C

receptor: (a) 3b, (b) 3e, and (c) 4a. Proteins are shown in wire

representation with cyan carbon atoms. The most important residues

are shown as sticks. Ligands are shown as sticks with gray carbon

atoms. Polar interactions are shown as red dashed lines. Nonpolar

hydrogen atoms are omitted for clarity

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TABLE 7 Molecular interactions of nonselective compound 4n

with serotonin 5‐HT_1A, 5‐HT_2A, and 5‐HT_2creceptors

5HT_1Areceptor

5‐HT_2Areceptor

5‐HT_2Creceptor

Asp3.32 salt bridge

Tyr7.42

Trp6.48 π–π

Tyr7.42

hydrogen bond

stacking

hydrogen bond

Phe6.52 π–π stacking

Phe6.52 π–π

Phe5.47 π–π stacking

Trp6.48 π–π stacking

Phe6.52 π–π stacking

stacking

reported in ppm. The following abbreviations are used to de-

scribe peak patterns when appropriate: s (singlet), d (doublet), t

(triplet), m (multiplet), q (quartet), qt (quintet), dd (doublet of

doublet), bs (broad singlet), and mm (multiplet of multiplet). Mass

spectra of the final products were recorded on an API 2000

Applied Biosystems mass spectrometer. Optical rotation (α) of

the racemic mixture was evaluated by a JASCO P‐2000 optical

activity polarimeter. Elemental analyses were carried out on a

Carlo Erba model 1106; analyses indicated by the symbols of the

elements were within 0.4% of the theoretical values (see the

Supporting Information). All reactions were followed by thin‐

(a)

(b)

FIGURE 6 The influence of the tested compounds 3b, 3e, 4a, 4j,

and 4n (30 mg/kg) on motor coordination in mice evaluated in (a)

chimney and (b) rotarod tests. Investigated compounds were injected

intraperitoneally 60 min before the test. Data are expressed as

mean SEM values of the one independent experiment (n = 7–9

mice). One‐way analysis of variance did not show significant changes

in the chimney (F_5,38= 1.522; p = .2059) and the time spent on the

rotarod (F_5,40= 1.704; p = .1559)

FIGURE 5 Noncovalent interaction maps for compound 4n in

complex with serotonin (a) 5‐HT_1A, (b) 5‐HT_2A, and (c) 5‐HT_2C

receptors. Weak attractive interactions are shown in green. Proteins

are shown in ice‐blue as cartoon representation. Asp3.32 is shown in

yellow in stick representation. Compound 4n is shown in

Corey–Pauling–Koltun representation with cyan carbon atoms

MAGLI ET AL.

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(a)

FIGURE 9 The influence of the tested compounds 3b, 3e, 4a, 4j,

and 4n (15 mg/kg) on the hyperactivity of mice provoked by an acute

MK‐801 (0.3 mg/kg, ip). The tested compounds were injected 60 min

and MK‐801 immediately before the test. Locomotor activity was

measured for a period of 20 min. Data are expressed as mean SEM

values of the one independent experiment (n = 7–9 mice); ***p < .001

versus control; ^##p < .001 versus MK‐801. One‐way analysis of

variance showed significant changes in the locomotor activity of mice

(F_6,53= 11.58; p < .001). Dunnett's post hoc test confirmed a

significant increase in locomotor activity of mice after the

administration of MK‐801 (0.3 mg/kg; p < .001). Moreover,

compound3e at the dose of 15 mg/kg decreased MK‐801‐induced

hyperactivity of mice (p < .01)

(b)

FIGURE 7 The influence of the tested compounds 3b, 3e, 4a, 4j,

and 4n (7.5–30 mg/kg) on the spontaneous locomotor activity of

mice. Investigated compounds were injected intraperitoneally 60 min

before the test. Locomotor activity was measured after 6 and 20 min.

Data are expressed as mean SEM values of the one independent

experiment (n = 7–9 mice); *p < .05 versus control (Dunnett's test).

One‐way analysis of variance (ANOVA) showed significant changes

in locomotor activity of mice in 6 min after administration of the

compound 4j (F_3,31= 2.981; p < .05). Dunnett's post hoc test

confirmed a significant decrease in locomotor activity of mice after

the administration of compound 4j at the dose of 30 mg/kg (p < .05)

after 6 min of observation. One‐way ANOVA also revealed

significant changes in locomotor activity of mice in 20 min after

administration of the compound 3e (F_2,17= 4.234; p < .05) and 4j

(F_3,23= 3.795; p < .05). Dunnett's post hoc test confirmed a significant

decrease in locomotor activity of mice after the administration of

compound 3e at the doses of 30 (p < .05) and 4j at the dose of

30 mg/kg (p < .05) after 20 min of observation

layer chromatography, carried out on Merck silica gel 60 F₂₅₄

plates with a fluorescent indicator, and the plates were visualized

with UV light (254 nm). Preparative chromatographic purifica-

tions were performed using a silica gel column (Kieselgel 60).

Solutions were dried over Na₂SO₄and concentrated with a Buchi

R‐114 rotary evaporator at low pressure.

The InChI codes of the investigated compounds, together

with some biological activity data, are given as Supporting

Information.

FIGURE 10 The influence of the investigated compounds 3b, 3e,

4a, 4j, and 4n (7.5–30 mg/kg) and fluoxetine (15 mg/kg) on the total

duration in the forced swim test (FST) in mice. The investigated

compounds were administered intraperitoneally 60 min before the

test. The values represent mean SEM of the one independent

experiment (n = 7–9 mice); *p < .05; **p < .01 versus control

(Dunnett's test). One‐way analysis of variance showed significant

changes in immobility time after administration of the compound 3b

(F_2,21= 7.191; p < .01), 3e (F_3,23= 3.775; p < .05), 4a (F₂₁= 4.646;

p < .05), and 4j (F_3,28= 5.424; p < .01). Dunnett's post hoc test

confirmed a significant reduction in immobility time after the

administration of compounds 3b applied at doses of 15 and 30 mg/kg

(p < .01), 3e at doses of 15 and 30 mg/kg (p < .05), 4a at doses of 15

and 30 mg/kg (p < .05), and 4n applied at doses of 7.5 (p < .05), 15

(p < .05), and 30 mg/kg (p < .01). Also, fluoxetine (15 mg/kg) induced a

significant reduction in the immobility time (p < .001)

FIGURE 8 The influence of the tested compounds 3b, 3e, 4a, 4j,

and 4n (15 mg/kg) on the amphetamine‐induced hyperactivity of

mice. Compounds tested were injected 60 min and amphetamine

5 mg/kg 30 min before the test. Locomotor activity was measured for

a period of 20 min. Data are expressed as mean SEM values of the

one independent experiment (n = 7–9 mice); ^###p < .001 versus

control; *p < .05 vs control (Dunnett's test); ***p < .001 versus

amphetamine. One‐way analysis of variance showed significant

changes in the locomotor activity of mice (F_6,47= 20.91; p < .001).

Dunnett's post hoc test confirmed a significant increase in locomotor

activity of mice after the administration of amphetamine (5 mg/kg;

p < .001). Moreover, all tested compounds at the dose of 15 mg/kg

decreased amphetamine‐induced hyperactivity of mice (p < .001 for

compounds 3b, 3e, 4a, 4j, and p < .05 for compound 4n)

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MAGLI ET AL.

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4.1.2

General procedure for the synthesis of 1‐

was stirred overnight at room temperature. After evaporation, the

crude products were recrystallized from diethyl ether to give in-

termediates 2f–n as solids (yield ranging between 55% and 71%).

¹H NMR spectra for all intermediates were consistent with the

proposed structures.

chloro‐3‐(4‐substituted‐arylpiperazin‐1‐yl)propan‐2‐

ol (2f–n)

To a solution of the appropriate 4‐substituted arylpiperazine (1)

(1 g; 0.005 mol) in absolute ethanol (35 ml), epichlorohydrin

(0.462 g; 0.005 mol) was added dropwise, and the reaction mixture

|

4.1.3

General procedure for the reaction of endo‐

N‐hydroxy‐5‐norbornene‐2,3‐dicarboximide and exo‐N‐

hydroxy‐7‐oxabicyclo[2.2.1]hept‐5‐ene‐2,3‐

dicarboximide with derivatives 2f–n (3f–n; 4f–n)

A solution of absolute ethanol (35 ml) and 0.2 g (0.05 mol) of so-

dium hydroxide was reacted with 1 g (0.005 mol) of commercially

available endo‐N‐hydroxy‐5‐norbornene‐2,3‐dicarboximide or exo‐

N‐hydroxy‐7‐oxabicyclo[2.2.1]hept‐5‐ene‐2,3‐dicarboximide and

1.420 g (0.005 mol) of the appropriate 1‐chloro‐3‐(4‐substituted‐

arylpiperazin‐1‐yl)propan‐2‐ol (2f–n) at 70°C for 24 h. Afterward,

the mixture was cooled to room temperature, concentrated to

dryness, and the residue was diluted in water (40 ml). The solution

was extracted several times with CH₂Cl_2.The combined organic

layers were dried on anhydrous Na₂SO₄and concentrated in va-

cuo. The residue was purified by column chromatography (CH₂Cl₂/

methanol 9:1 v/v). The combined and evaporated product frac-

tions were crystallized from diethyl ether, yielding the desired

products (3f–n and 4f–n) as white solids.

FIGURE 11 The influence of the investigated compounds 3b, 3e, 4a,

4j, and 4n (7.5–30 mg/kg) on elevated plus‐maze (EPM) performance in

mice. (a) Percentage of time spent in open arms, (b) the percentage of the

open arm entries, and (c) total arm entries. Investigated compounds were

injected intraperitoneally 60 min before the test. The results are

expressed as mean SEM of the one independent experiment (n = 7–9

mice); ***p < .001; **p < .01; *p < .05 versus control (Dunnett's test). One‐

way analysis of variance showed significant changes in (a) the percentage

of time spent in open arms of EPM (3e F_3,30= 5.471; p < .01; 4j

FIGURE 12 The influence of the investigated compounds 3b,

3e, 4a, 4j, and 4n (15 mg/kg) on the memory impairment

provoked by an acute administration of MK‐801 (0.3 mg/kg, ip) in

the passive avoidance test in mice. The investigated compounds

were administered intraperitoneally, immediately after the

training. The values represent mean SEM of the one independent

experiment (n = 13 mice); *p < .05 versus control; ^##p < .01,

^###p < .001 versus MK‐801 (Dunnett's test). One‐way analysis of

variance revealed a statistically significant effect on the latency

index (LI) values for consolidation of long‐term memory

(F_6,84= 3.511; p < .01). Post hoc Dunnett's test indicated that

treatment with MK‐801 (0.3 mg/kg, ip) significantly decreased LI

values in mice as compared with the control group (p < .05). These

data showed that MK‐801, at the dose of 0.3 mg/kg, impaired

consolidation of long‐term memory. Moreover, an acute injection

of 3e (p < .001), 4j (p < .01), and 4n (p < .001) attenuated the

amnestic effect of MK‐801

F

_3,30= 7.961; p < .001; 4n F_3,31= 4.34; p < .05) and in (b) the percentage of

open arm entries (3e F_3,30= 3.009; p < .05; 4j F_3,30= 4.219; p < .05; 4n

_3,31= 3.215; p < .05). There were no significant changes in (c) the total

F

arm entries (F_14,89= 1.097; p = .3719). Dunnett's post hoc test confirmed

a significant increase in time spent in open arms after the administration

of compounds: 3e at doses of 7.5 (p < .01), 15 (p < .05), and 30 mg/kg

(p < .05); 4j at doses of 7.5 (p < .05) and 15 mg/kg (p < .001), and 4n at

doses of 7.5 and 15 mg/kg (p < .05). These compounds were also able to

increase the percentage of open arm entries: 3e at the dose of 7.5 mg/kg

(p < .05), 4j at the dose of 15 mg/kg (p < .05), and 4n at doses of 7.5 and

15 mg/kg (p < .05). Also, buspirone (5 mg/kg) induced a significant

increase in the percentage of time spent in open arms and in the

percentage of open arms entries (p < .01)

MAGLI ET AL.

17 of 24

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4.1.4

Synthesis of 4‐{3‐[4‐(p‐methoxyphenyl)-

CH₂pip); 3.15 (bs, 4H, 2CH₂pip); 3.22 (s, 2H); 3.44 (s, 2H); 3.89–3.93

(m, 1H, CH–OH); 4.07 (dd, 2H, O–CH₂, J = 3.0, J = 7.0); 6.16 (s, 2H); 6.70

(dd, 1H); 6.93 (d, 1H); 7.25 (d, 1H, J = 7.3). ESI‐MS: 467.0 [M+H]⁺; 489.8

[M+Na]⁺; 505.8 [M+K]⁺(calcd: 466.36). Anal. (C₂₂H₂₅Cl₂N₃O₄), C, H, N.

piperazin‐1‐yl]propoxy‐2‐ol}‐4‐aza‐tricyclo[5.2.1.02,6]-

dec‐8‐ene‐3,5‐dione (3f)

From 2f and endo‐N‐hydroxy‐5‐norbornene‐2,3‐dicarboximide. Yield:

77%; mp: 124–125°C. ¹H NMR (400 MHz, CDCl₃) δː 1.50 (d, 1H,

J = 8.9); 1.76 (d, 1H, J = 8.9); 2.47 (dd, 1H, CH₂–N, J = 4.0, J = 8.8);

2.55 (dd, 1H, CH₂–N, J = 4.2, J = 8.1); 2.65 (bs, 2H, CH₂pip); 2.73 (bs,

2H, CH₂pip); 3.07 (bs, 4H, 2CH₂pip); 3.21 (s, 2H); 3.43 (s, 2H); 3.76

(s, 3H, −OCH₃); 3.89–3.93 (m, 1H, CH–OH); 4.06 (dd, 2H, O–CH₂,

J = 3.0, J = 7.0); 6.16 (s, 2H); 6.82 (d, 2H); 6.87 (d, 2H). Electrospray

ionization–mass spectrometry (ESI‐MS): 428.1 [M+H]⁺; 450.4 [M

+Na]⁺; 466.4 [M+K]⁺(calcd: 427.49). Anal. (C₂₃H₂₉N₃O₅), C, H, N.

|

4.1.8

Synthesis of 4‐{3‐[4‐(pyridin‐2‐yl)piperazin‐

1‐yl]propoxy‐2‐ol}‐4‐aza‐tricyclo[5.2.1.02,6]dec‐8‐ene‐

3,5‐dione (3j)

From 2j and endo‐N‐hydroxy‐5‐norbornene‐2,3‐dicarboximide. Yield:

61%; mp: 145–146°C; ¹H NMR (400 MHz, CDCl₃) δː 1.50 (d, 1H, J = 8.9);

1.76 (d, 1H, J = 8.9); 2.44 (dd, 1H, CH₂–N, J = 4.0, J = 8.8); 2.54 (dd, 1H,

CH₂–N, J = 4.2, J = 8.1); 2.59 (bs, 2H, CH₂pip); 2.66 (bs, 2H, CH₂pip);

3.21 (s, 2H); 3.43 (s, 2H); 3.52 (bs, 4H, 2CH₂pip); 3.89–3.93 (m, 1H,

CH–OH); 4.08 (dd, 2H, O–CH₂, J = 3.0, J = 7.0); 6.16 (s, 2H); 6.60–6.64

(m, 2H); 7.44–7.49 (m, 1H); 8.17 (dd, 1H). ESI‐MS: 399.2 [M+H]⁺; 421.3

[M+Na]⁺; 437.4 [M+K]⁺(calcd: 398.46). Anal. (C₂₁H₂₆N₄O₄), C, H, N.

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4.1.5

Synthesis of 4‐{3‐[4‐(o‐chlorophenyl)-

piperazin‐1‐yl]propoxy‐2‐ol}‐4‐aza‐tricyclo[5.2.1.02,6]-

dec‐8‐ene‐3,5‐dione (3g)

From 2g and endo‐N‐hydroxy‐5‐norbornene‐2,3‐dicarboximide.

Yield: 83%; mp: 125–127°C. ¹H NMR (400 MHz, CDCl₃) δː 1.50 (d,

1H, J = 8.9); 1.76 (d, 1H, J = 8.9); 2.46 (dd, 1H, CH₂–N, J = 4.0, J = 8.8);

2.56 (dd, 1H, CH₂–N, J = 4.2, J = 8.1); 2.67 (bs, 2H, CH₂pip); 2.75 (bs,

2H, CH₂pip); 3.06 (bs, 4H, 2CH₂pip); 3.22 (s, 2H); 3.44 (s, 2H);

3.89–3.94 (m, 1H, CH–OH); 4.08 (dd, 2H, O–CH₂, J = 3.0, J = 7.0);

6.17 (s, 2H); 6.97 (t, 1H); 7.02 (d, 1H); 7.21 (t, 1H); 7.34 (d, 1H). ESI‐

MS: 432.3 [M+H]⁺; 454.1 [M+Na]⁺; 470.1 [M+K]⁺(calcd: 431.91).

Anal. (C₂₂H₂₆ClN₃O₄), C, H, N.

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4.1.9

Synthesis of 4‐{3‐[4‐(pyrimidin‐2‐yl)-

piperazin‐1‐yl]propoxy‐2‐ol}‐4‐aza‐tricyclo[5.2.1.02,6]-

dec‐8‐ene‐3,5‐dione (3k)

From 2k and endo‐N‐hydroxy‐5‐norbornene‐2,3‐dicarboximide.

Yield: 65%; mp: 177–179°C; ¹H NMR (400 MHz, CDCl₃) δː 1.50 (d,

1H, J = 8.9); 1.76 (d, 1H, J = 8.9); 2.43 (dd, 1H, CH₂–N, J = 4.0, J = 8.8);

2.50 (dd, 1H, CH₂–N, J = 4.2, J = 8.1); 2.54 (bs, 2H, CH₂pip); 2.61 (bs,

2H, CH₂pip); 3.21 (s, 2H); 3.44 (s, 2H); 3.81 (bs, 4H, 2CH₂pip);

3.89–3.93 (m, 1H, CH–OH); 4.07 (dd, 2H, O–CH₂, J = 3.0, J = 7.0);

6.16 (s, 2H); 6.48 (t, 1H); 8.29 (d, 2H). ESI‐MS: 400.3 [M+H]⁺; 422.1

[M+Na]⁺; 438.0 [M+K]⁺(calcd: 399.44). Anal. (C₂₀H₂₅N₅O₄), C, H, N.

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4.1.6

Synthesis of 4‐{3‐[4‐(m‐chlorophenyl)-

piperazin‐1‐yl]propoxy‐2‐ol}‐4‐aza‐tricyclo[5.2.1.02,6]-

dec‐8‐ene‐3,5‐dione (3h)

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From 2h and endo‐N‐hydroxy‐5‐norbornene‐2,3‐dicarboximide.

Yield: 70%; mp: 100–102°C. ¹H NMR (400 MHz, CDCl₃) δː 1.51 (d,

1H, J = 8.9); 1.77 (d, 1H, J = 8.9); 2.46 (dd, 1H, CH₂–N, J = 4.0, J = 8.8);

2.55 (dd, 1H, CH₂–N, J = 4.2, J = 8.1); 2.63 (bs, 2H, CH₂pip); 2.70 (bs,

2H, CH₂pip); 3.18 (bs, 4H, 2CH₂pip); 3.21 (s, 2H); 3.44 (s, 2H);

3.89–3.93 (m, 1H, CH–OH); 4.07 (dd, 2H, O–CH₂, J = 3.0, J = 7.0);

6.17 (s, 2H); 6.76–6.81 (m, 2H, J = 7.6); 6.85 (s, 1H); 7.15 (t, 1H). ESI‐

MS: 432.1 [M+H]⁺; 454.1 [M+Na]⁺; 470.1 [M+K]⁺(calcd: 431.91).

Anal. (C₂₂H₂₆ClN₃O₄), C, H, N.

4.1.10

Synthesis of 4‐{3‐[4‐(piperonyl)piperazin‐1‐

yl]propoxy‐2‐ol}‐4‐aza‐tricyclo[5.2.1.02,6]dec‐8‐ene‐

3,5‐dione (3l)

From 2l and endo‐N‐hydroxy‐5‐norbornene‐2,3‐dicarboximide. Yield:

40%; mp: 110–111°C; ¹H NMR (400 MHz, CDCl₃) δː 1.50 (d, 1H,

J = 8.9); 1.75 (d, 1H, J = 8.9); 2.37–2.58 (m, 10H); 3.19 (s, 2H); 3.39 (s,

2H, −CH₂); 3.42 (s, 2H); 3.85–3.89 (m, 1H, CH–OH); 4.03 (dd, 2H,

O–CH₂, J = 3.0, J = 7.0); 5.93 (s, 2H); 6.14 (s, 2H); 6.73 (s, 2H); 6.83 (s,

1H). ESI‐MS: 455.9 [M+H]⁺; 478.2 [M+Na]⁺; 494.3 [M+K]⁺(calcd:

455.5). Anal. (C₂₄H₂₉N₃O₆), C, H, N.

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4.1.7

Synthesis of 4‐{3‐[4‐(3,4‐dichlorophenyl)-

piperazin‐1‐yl]propoxy‐2‐ol}‐4‐aza‐tricyclo[5.2.1.02,6]-

dec‐8‐ene‐3,5‐dione (3i)

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4.1.11

Synthesis of 4‐{3‐[4‐(thiophen‐2‐ylmethyl)-

piperazin‐1‐yl]propoxy‐2‐ol}‐4‐aza‐tricyclo[5.2.1.02,6]-

dec‐8‐ene‐3,5‐dione (3m)

From 2i and endo‐N‐hydroxy‐5‐norbornene‐2,3‐dicarboximide. Yield:

74%; mp: 160–162°C. ¹H NMR (400 MHz, CDCl₃) δː 1.51 (d, 1H,

J = 8.9); 1.77 (d, 1H, J = 8.9); 2.46 (dd, 1H, CH₂–N, J = 4.0, J = 8.8); 2.55

(dd, 1H, CH₂–N, J = 4.2, J = 8.1); 2.63 (bs, 2H, CH₂pip); 2.69 (bs, 2H,

From 2m and endo‐N‐hydroxy‐5‐norbornene‐2,3‐dicarboximide.

Yield: 55%; mp: 118–120°C; ¹H NMR (400 MHz, CDCl₃) δː 1.49 (d,

18 of 24

MAGLI ET AL.

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1H, J = 8.9); 1.75 (d, 1H, J = 8.9); 2.38–2.60 (m, 10H); 3.19 (s, 2H);

3.42 (s, 2H); 3.71 (s, 2H, –CH₂); 3.85–3.88 (m, 1H, CH–OH); 4.03

(dd, 2H, O–CH₂, J = 3.0, J = 7.0); 6.14 (s, 2H); 6.90–6.94 (m, 2H);

7.22 (d, 1H). ESI‐MS: 418.3 [M+H]⁺; 440.2 [M+Na]⁺; 456.2 [M+K]⁺

(calcd: 417.52). Anal. (C₂₁H₂₇N₃O₄S), C, H, N.

4.1.15

Synthesis of 4‐{3‐[4‐(m‐chlorophenyl)

piperazin‐1‐yl]propoxy‐2‐ol}‐10‐oxa‐4‐aza‐tricyclo-

[5.2.1.02,6]dec‐8‐ene‐3,5‐dione (4h)

From 2h and exo‐N‐hydroxy‐7‐oxabicyclo[2.2.1]hept‐5‐ene‐2,3‐

dicarboximide. Yield: 42%; mp: 113–115°C. ¹H NMR (400 MHz,

CDCl₃) δː 2.46 (dd, 1H, CH₂–N, J = 4.0, J = 8.8); 2.56 (dd, 1H, CH₂–N,

J = 4.2, J = 8.1); 2.63 (bs, 2H, CH₂pip); 2.70 (bs, 2H, CH₂pip); 2.79 (s,

2H); 3.17 (bs, 4H, 2CH₂pip); 4.01–4.05 (m, 1H, CH–OH); 4.21 (d, 2H,

O–CH₂, J = 8.1); 5.29 (d, 2H, J = 4.8); 6.52 (s, 2H); 6.76–6.81 (m, 2H,

J = 7.3); 6.85 (s, 1H); 7.15 (t, 1H). ESI‐MS: 434.3 [M+H]⁺; 456.2 [M

+Na]⁺; 472.2 [M+K]⁺(calcd: 433.89). Anal. (C₂₁H₂₄ClN₃O₅), C, H, N.

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4.1.12

Synthesis of 4‐{3‐[4‐(naphthalen‐1‐yl)-

piperazin‐1‐yl]propoxy‐2‐ol}‐4‐aza‐tricyclo[5.2.1.02,6]-

dec‐8‐ene‐3,5‐dione (3n)

From 2n and endo‐N‐hydroxy‐5‐norbornene‐2,3‐dicarboximide.

Yield: 77%; mp: 87–89°C. ¹H NMR (400 MHz, CDCl₃) δː 1.50 (d,

1H, J = 8.9); 1.77 (d, 1H, J = 8.9); 2.56 (dd, 1H, CH₂–N, J = 4.0,

J = 8.8); 2.63 (dd, 1H, CH₂–N, J = 4.2, J = 8.1); 2.78 (bs, 2H, CH₂

pip); 2.87 (bs, 2H, CH₂pip); 3.13 (bs, 4H, 2CH₂pip); 3.22 (s, 2H);

3.44 (s, 2H); 3.93–3.97 (m, 1H, CH–OH); 4.10 (dd, 2H, O–CH₂,

J = 3.0, J = 7.0); 6.18 (s, 2H); 7.07 (d, 1H); 7.39 (t, 1H); 7.46 (m, 2H);

7.54 (d, 1H); 7.81 (d, 1H); 8.17 (d, 1H). ESI‐MS: 448.1 [M+H]⁺;

470.2 [M+Na]⁺; 486.2 [M+K]⁺(calcd: 447.53). Anal. (C₂₆H₂₉N₃O₄),

C, H, N.

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4.1.16

Synthesis of 4‐{3‐[4‐(3,4‐dichlorophenyl)-

piperazin‐1‐yl]propoxy‐2‐ol}‐10‐oxa‐4‐aza‐tricyclo-

[5.2.1.02,6]dec‐8‐ene‐3,5‐dione (4i)

From 2i and exo‐N‐hydroxy‐7‐oxabicyclo[2.2.1]hept‐5‐ene‐2,3‐

dicarboximide. Yield: 24%; mp: 141–143°C. ¹H NMR (400 MHz,

CDCl₃) δː 2.46 (dd, 1H, CH₂–N, J = 4.0, J = 8.8); 2.56 (dd, 1H, CH₂–N,

J = 4.2, J = 8.1); 2.62 (bs, 2H, CH₂pip); 2.69 (bs, 2H, CH₂pip); 2.79 (s,

2H); 3.15 (bs, 4H, 2CH₂pip); 4.00–4.05 (m, 1H, CH–OH); 4.21 (d, 2H,

O–CH₂, J = 8.1); 5.29 (d, 2H, J = 4.8); 6.52 (s, 2H); 6.70 (dd, 1H,

J = 7.3); 6.93 (d, 1H, J = 7.3); 7.25 (d, 1H). ESI‐MS: 469.3 [M+H]⁺

(calcd: 468.33). Anal. (C₂₁H₂₃Cl₂N₃O₅), C, H, N.

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4.1.13

Synthesis of 4‐{3‐[4‐(p‐methoxyphenyl)-

piperazin‐1‐yl]propoxy‐2‐ol}‐10‐oxa‐4‐aza‐tricyclo-

[5.2.1.02,6]dec‐8‐ene‐3,5‐dione (4f)

From 2f and exo‐N‐hydroxy‐7‐oxabicyclo[2.2.1]hept‐5‐ene‐2,3‐

dicarboximide. Yield: 53%; mp: 141–143°C. ¹H NMR (400 MHz,

CDCl₃) δː 2.49 (dd, 1H, CH₂–N, J = 4.0, J = 8.8); 2.61 (dd, 1H,

CH₂–N, J = 4.2, J = 8.1); 2.64 (bs, 2H, CH₂pip); 2.73 (bs, 2H, CH₂

pip); 2.78 (s, 2H); 3.07 (bs, 4H, 2CH₂pip); 3.76 (s, 3H, –OCH₃);

4.01–4.03 (m, 1H, CH–OH); 4.20 (d, 2H, O–CH₂, J = 8.1); 5.28 (d,

2H, J = 4.8); 6.51 (s, 2H); 6.81 (d, 2H); 6.87 (d, 2H). ESI‐MS: 430.2

[M+H]⁺; 452.2 [M+Na]⁺; 468.2 [M+K]⁺(calcd: 429.47). Anal.

(C₂₂H₂₇N₃O₆), C, H, N.

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4.1.17

Synthesis of 4‐{3‐[4‐(pyridin‐2‐yl)piperazin‐

1‐yl]propoxy‐2‐ol}‐10‐oxa‐4‐aza‐tricyclo[5.2.1.02,6]-

dec‐8‐ene‐3,5‐dione (4j)

From 2j and exo‐N‐hydroxy‐7‐oxabicyclo[2.2.1]hept‐5‐ene‐2,3‐

dicarboximide. Yield: 27%; mp: 145–146°C. ¹H NMR (400 MHz,

CDCl₃) 2.45 (dd, 1H, CH₂–N, J = 4.0, J = 8.8); 2.55 (dd, 1H, CH₂–N,

J = 4.2, J = 8.1); 2.60 (bs, 2H, CH₂pip); 2.67 (bs, 2H, CH₂pip); 2.79

(s, 2H); 3.52 (bs, 4H, 2CH₂pip); 4.00–4.05 (m, 1H, CH–OH); 4.21

(d, 2H, O–CH₂, J = 8.2); 5.29 (d, 2H, J = 4.4); 6.52 (s, 2H); 6.60–6.64

(m, 2H); 7.44–7.49 (m, 1H); 8.17 (dd, 1H). ESI‐MS: 401.3 [M+H]⁺;

423.3 [M+Na]⁺; 439.0 [M+K]⁺(calcd: 400.43). Anal. (C₂₀H₂₄N₄O₅),

C, H, N.

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4.1.14

Synthesis of 4‐{3‐[4‐(o‐chlorophenyl)-

piperazin‐1‐yl]propoxy‐2‐ol}‐10‐oxa‐4‐aza‐tricyclo-

[5.2.1.02,6]dec‐8‐ene‐3,5‐dione (4g)

From 2g and exo‐N‐hydroxy‐7‐oxabicyclo[2.2.1]hept‐5‐ene‐2,3‐

dicarboximide. Yield: 30%; mp: 121–122°C. ¹H NMR (400 MHz,

CDCl₃) δː 2.48 (dd, 1H, CH₂–N, J = 4.0, J = 8.8); 2.57 (dd, 1H,

CH₂–N, J = 4.2, J = 8.1); 2.63–2.67 (m, 4H, CH₂pip); 2.79 (bs, 4H,

2CH₂pip); 3.06 (s, 2H); 4.02–4.06 (m, 1H, CH–OH); 4.21 (d, 2H,

O–CH₂, J = 8.1); 5.29 (d, 2H, J = 4.8); 6.52 (s, 2H); 6.96 (t, 1H);

7.02 (d, 1H); 7.21 (t, 1H); 7.34 (d, 1H). ESI‐MS: 434.3 [M+H]⁺;

456.2 [M+Na]⁺; 472.2 [M+K]⁺(calcd: 433.89). Anal.

(C₂₁H₂₄ClN₃O₅), C, H, N.

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4.1.18

Synthesis of 4‐{3‐[4‐(pyrimidin‐2‐yl)-

piperazin‐1‐yl]propoxy‐2‐ol}‐10‐oxa‐4‐aza‐tricyclo-

[5.2.1.02,6]dec‐8‐ene‐3,5‐dione (4k)

From 2k and exo‐N‐hydroxy‐7‐oxabicyclo[2.2.1]hept‐5‐ene‐2,3‐

dicarboximide. Yield: 58%; mp: 149–151°C; ¹H NMR (400 MHz,

CDCl₃) δː 2.43 (dd, 1H, CH₂–N, J = 4.0, J = 8.8); 2.49 (dd, 1H, CH₂–N,

J = 4.2, J = 8.1); 2.53 (bs, 2H, CH₂pip); 2.61 (bs, 2H, CH₂pip); 2.79 (s,

MAGLI ET AL.

19 of 24

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2H); 3.81 (bs, 4H, 2CH₂pip); 4.02–4.05 (m, 1H, CH–OH); 4.22 (d, 2H,

O–CH₂, J = 3.0, J = 7.0); 5.29 (d, 2H); 6.48 (t, 1H); 6.52 (s, 2H); 8.29 (d,

2H). ESI‐MS: 402.4 [M+H]⁺; 424.2 [M+Na]⁺; 440.1 [M+K]⁺(calcd:

401.42). Anal. (C₁₉H₂₃N₅O₅), C, H, N.

(50 mM Tris‐HCl, 1 mM EDTA, 1 mM dithiothreitol, pH 7.4). Next,

the homogenate was centrifuged at 21,000g for 30 min at 4°C. The

pellet was suspended in 30 vol TED buffer (pH 7.4) and incubated in

a water bath for 10 min at 37°C to remove endogenous ligands. The

suspension was centrifuged again at 21,000g for 30 min at 4°C. The

pellet was resuspended in 30 vol TED buffer (pH 7.4) and the cen-

trifugation step was repeated. The final pellet was suspended in

10 vol 50 mM Tris‐HCl (pH 7.4) and stored at −80°C until use. In the

agonist mode, 15 μg/ml of hippocampus homogenate was incubated

in triplicate with 0.8 nM [³⁵S]GTPγS in an assay buffer (50 mM Tris‐

HCl, pH 7.4, 1 mM EGTA, 3 mM MgCl₂, 100 mM NaCl, 30 µM GDP)

in the presence of increasing concentrations of the tested com-

pounds (10⁻¹⁰to 10⁻⁵M). In the antagonist mode, compounds were

additionally incubated with 100 nM 8‐OH‐DPAT. Nonspecific binding

was determined with 100 µM of unlabeled GTPγS. The reaction

mixture was incubated for 90 min at 37°C in a volume of 250 µl.

Next, 96‐well Unifilter® Plates (PerkinElmer) were presoaked for 1 h

with 50 mM Tris‐HCl (pH 7.4) before harvesting. The reaction was

terminated by vacuum filtration on filter plates with the FilterMate

Harvester® (PerkinElmer). The samples were then rapidly washed

with 2 ml of 50 mM Tris‐HCl (pH 7.4) buffer. Filter plates were dried

for 2 h at 50°C. After drying, 45 µl of EcoScint‐20 scintillant (Perki-

nElmer) was added to every well. Radioactivity was counted in a

Trilux MicroBeta²counter (PerkinElmer). Data were analyzed with

GraphPad Prism 5.0 software (GraphPad Software; www.graphpad.

com). Curves were fitted with a one‐site nonlinear regression model.

Efficacy (E_max) and potency (pEC₅₀for agonists; pIC₅₀and pK_Bfor

antagonists) were calculated from the Cheng–Prusoff and Gaddum/

Schild models and expressed as mean SEM. Differences in com-

pound potency and efficacy were evaluated with the extra sum‐of‐

squares F test. Baseline G‐protein stimulation was set to 100%. One,

two, or three symbols represent statistical significance of 0.05, 0.01,

and 0.001, respectively. Differences in potency and efficacy between

the ligands were analyzed with the extra sum‐of‐squares F test.

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4.1.19

Synthesis of 4‐{3‐[4‐(piperonyl)piperazin‐1‐

yl]propoxy‐2‐ol}‐10‐oxa‐4‐aza‐tricyclo[5.2.1.02,6]dec‐

8‐ene‐3,5‐dione (4l)

From 2l and exo‐N‐hydroxy‐7‐oxabicyclo[2.2.1]hept‐5‐ene‐2,3‐

dicarboximide. Yield: 70%; mp: 144–146°C; ¹H NMR (400 MHz,

CDCl₃) δː 2.37–2.59 (m, 10H); 2.77 (s, 2H); 3.39 (s, 2H, –CH₂);

3.96–4.02 (m, 1H, CH–OH); 4.15 (dd, 2H, O–CH₂, J = 3.0, J = 7.0);

5.28 (d, 2H); 5.93 (s, 2H); 6.51 (s, 2H); 6.73 (s, 2H); 6.83 (s, 1H). ESI‐

MS: 458.1 [M+H]⁺; 480.2 [M+Na]⁺; 496.2 [M+K]⁺(calcd: 457.48).

Anal. (C₂₃H₂₇N₃O₇), C, H, N.

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4.1.20

Synthesis of 4‐{3‐[4‐(thiophen‐2‐ylmethyl)-

piperazin‐1‐yl]propoxy‐2‐ol}‐10‐oxa‐4‐aza‐tricyclo-

[5.2.1.02,6]dec‐8‐ene‐3,5‐dione (4m)

From 2m and exo‐N‐hydroxy‐7‐oxabicyclo[2.2.1]hept‐5‐ene‐2,3‐

dicarboximide. Yield: 60%; mp: 122–124°C; ¹H NMR (400 MHz,

CDCl₃) δː 2.39–2.62 (m, 10H); 2.78 (s, 2H); 3.71 (s, 2H, –CH₂);

3.95–4.02 (m, 1H, CH–OH); 4.15 (d, 2H, O–CH₂, J = 3.0, J = 7.0); 5.28

(d, 2H); 6.51 (s, 2H); 6.90–6.95 (m, 2H); 7.22 (d, 1H). ESI‐MS: 420.2

[M+H]⁺; 442.3 [M+Na]⁺; 458.3 [M+K]⁺(calcd: 419.49). Anal.

(C₂₀H₂₅N₃O₅S), C, H, N.

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4.1.21

Synthesis of 4‐{3‐[4‐(naphthalen‐1‐yl)-

piperazin‐1‐yl]propoxy‐2‐ol}‐10‐oxa‐4‐aza‐tricyclo-

[5.2.1.02,6]dec‐8‐ene‐3,5‐dione (4n)

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From 2n and exo‐N‐hydroxy‐7‐oxabicyclo[2.2.1]hept‐5‐ene‐2,3‐

dicarboximide. Yield: 53%; mp: 70–71°C. ¹H NMR (400 MHz, CDCl₃)

δː 2.57 (dd, 1H, CH₂–N, J = 4.0, J = 8.8); 2.64 (dd, 1H, CH₂–N, J = 4.2,

J = 8.1); 2.79 (bs, 4H); 2.87 (bs, 2H, CH₂pip); 3.13 (bs, 4H, 2CH₂pip);

4.06–4.09 (m, 1H, CH–OH); 4.25 (d, 2H, O–CH₂, J = 8.1); 5.30 (d, 2H,

J = 4.8); 6.52 (s, 2H); 7.07 (d, 1H); 7.39 (t, 1H); 7.46 (m, 2H); 7.54 (d,

1H); 7.81 (d, 1H); 8.17 (d, 1H). ESI‐MS: 450.5 [M+H]⁺; 474.6 [M

+Na]⁺; 488.4 [M+K]⁺(calcd: 449.5). Anal. (C₂₅H₂₇N₃O₅), C, H, N.

4.2.2

5‐HT_2Acompetition binding assay

Male SD rats were decapitated and their brains removed and placed

on ice. Frontal cortices were homogenized with a glass homogenizer

in 30 vol ice‐cold homogenization buffer (50 mM Tris‐HCl, 1 mM

EDTA, 5 mM MgCl₂, pH 7.4). Next, the homogenate was centrifuged

at 20,000g for 15 min at 4°C. The pellet was suspended in 30 vol

50 mM Tris‐HCl (pH 7.4) and incubated in a water bath for 15 min at

37°C to remove endogenous serotonin. The suspension was again

centrifuged at 20,000g for 15 min at 4°C. The pellet was re-

suspended in 10 vol 50 mM Tris‐HCl (pH 7.4) and the centrifugation

step was repeated. The final pellet was suspended in 10 vol 50 mM

Tris‐HCl (pH 7.4) and stored at −80°C. For the 5‐HT_2Aassay, frontal

cortex homogenates (160 µg protein/ml) were incubated in triplicate

with 1 nM [³H]ketanserin for 60 min at 36°C in a 50 mM Tris‐HCl

(pH 7.4) buffer containing 0.1% ascorbate, 3 mM CaCl₂, and 10 µM

pargyline, and increasing concentrations (10⁻⁹to 10⁻⁵M) of the

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4.2

In vitro receptor assays

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4.2.1

Functional 5‐HT_1Areceptor assay

Male Sprague–Dawley (SD) rats were decapitated and their brains

removed and placed on ice. Hippocampi were dissected and homo-

genized with a glass homogenizer in 30 vol ice‐cold TED buffer

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MAGLI ET AL.

|

compound of interest. Nonspecific binding was determined in the pre-

sence of 10 μM mianserin. After incubation, the reaction mixture was

deposited on UniFilter‐96 GF/B plates with the aid of a FilterMate‐96

Harvester. Filter plates were presoaked beforehand with 0.4% poly-

ethylenimine (PEI) for 1 h. Next, each filter well was washed with

1.75 ml of 50 mM Tris‐HCl (pH 7.4) and left to dry on a heating block

set to 50°C for 2 h. Then, 45 µl of Microscint‐20 scintillation fluid was

added to each filter well and left to equilibrate overnight. Filter‐bound

radioactivity was counted in a MicroBeta²Microplate Counter. Binding

curves were fitted with a one‐site nonlinear regression model. Inhibition

curves were fitted with a one‐site nonlinear regression model. Affinity

was presented as the inhibitory constant (pK_iand K_iSEM) from two or

three separate experiments. Differences in K_ivalues were evaluated

with the extra sum‐of‐squares F test. One, two, or three symbols re-

present statistical significance of 0.05, 0.01, and 0.001, respectively.

glass homogenizer in 30 vol ice‐cold TED buffer (50 mM Tris‐HCl,

1 mM EDTA, 1 mM dithiothreitol, pH 7.4). Next, the homogenate was

centrifuged at 21,000g for 30 min at 4°C. The pellet was suspended

in 30 vol TED buffer (pH 7.4) and incubated in a water bath for

10 min at 37°C to remove endogenous ligands. The suspension was

centrifuged again at 21,000g for 30 min at 4°C. The pellet was re-

suspended in 30 vol TED buffer (pH 7.4) and the centrifugation step

was repeated. The final pellet was suspended in 10 vol 50 mM Tris‐

HCl (pH 7.4) and stored at −80°C until use. For the D₂receptor

antagonist [³⁵S]GTPγS assay, 15 μg/ml of striatal homogenate was

incubated in triplicate with 0.8 nM [³⁵S]GTPγS in an assay buffer

(50 mM Tris‐HCl, pH 7.4, 1 mM EGTA, 3 mM MgCl₂, 100 mM NaCl,

0.1 mM dithiothreitol, 500 µM ascorbic acid, 20 µM GDP, and

100 µM dopamine) in the presence of increasing concentrations of

the tested compounds (10⁻⁹to 10⁻⁵M). The effect on the basal

G‐protein activation threshold was determined in assay buffer

deprived of dopamine. The final dimethyl sulfoxide (DMSO)

concentration in the assay was 5%. Dopamine was dissolved in

50 mM Tris buffer (pH 7.4) supplemented with 500 µM ascorbic acid

to prevent oxidation. Nonspecific binding was determined with

100 µM of unlabeled GTPγS. The reaction mixture was incubated for

60 min at 30°C at a volume of 250 µl. Next, 96‐well Unifilter Plates

were presoaked for 1 h with 50 mM Tris‐HCl (pH 7.4) before

harvesting. The reaction was terminated by vacuum filtration on

filter plates with the FilterMate Harvester. The samples were then

rapidly washed with 2 ml of 50 mM Tris‐HCl (pH 7.4) buffer. Filter plates

were dried for 2 h at 50°C. After drying, 45 µl of EcoScint‐20 scintillant

was added to the wells. Radioactivity was counted in a Trilux MicroBeta²

counter. Data were analyzed with GraphPad Prism 5.0 software. Curves

were fitted with a one‐site nonlinear regression model. Potency (pIC₅₀

and pK_B) and efficacy (E_max) were calculated from the Cheng–Prusoff and

Gaddum/Schild models and expressed as mean SEM.

|

4.2.3

5‐HT_2Ccompetition binding assay

Male SD rats were decapitated and their brains removed and placed on

ice. Frontal cortices were homogenized with a glass homogenizer in 30

vol ice‐cold homogenization buffer (50 mM Tris‐HCl, 10 mM MgCl₂, pH

7.4). Next, the homogenate was centrifuged at 20,000g for 15 min at

4°C. The pellet was suspended in 30 vol 50 mM Tris‐HCl (pH 7.4) and

incubated in a water bath for 15 min at 37°C to remove endogenous

serotonin. The suspension was again centrifuged at 20,000g for 15 min

at 4°C. The pellet was resuspended in 10 vol 50 mM Tris‐HCl (pH 7.4)

and the centrifugation step was repeated. The final pellet was sus-

pended in 10 vol 50 mM Tris‐HCl (pH 7.4) and stored at −80°C. For the

5‐HT_2Cassay, frontal cortex homogenates (250 µg protein/ml) were

incubated in triplicate with 1 nM [³H]mesulergine for 60 min at 36°C in

a 50 mM Tris‐HCl (pH 7.4) buffer containing 0.1% ascorbate, 10 mM

MgCl_2,10 µM pargyline, 100 nM spiperone, and increasing concentra-

tions (10⁻⁹to 10⁻⁵M) of the compound tested. Nonspecific binding was

determined in the presence of 10 μM mianserin. After incubation, the

reaction mixture was deposited on UniFilter‐96 GF/B plates with the

aid of a FilterMate‐96 Harvester. Filter plates were presoaked be-

forehand with 0.4% PEI for 1 h. Next, each filter well was washed with

1.75 ml of 50 mM Tris‐HCl (pH 7.4) and left to dry for 2 h on a heating

block set to 50°C. Then, 45 µl of Microscint‐20 scintillation fluid was

added to each filter well and left to equilibrate overnight. Filter‐bound

radioactivity was counted in a MicroBeta²Microplate Counter. Binding

curves were fitted with a one‐site nonlinear regression model. Affinity

was presented as the inhibitory constant (pK_iand K_iSEM) from two or

three separate experiments. Differences in K_ivalues were evaluated

with the extra sum‐of‐squares F test. One, two, or three symbols re-

present statistical significance of 0.05, 0.01, and 0.001, respectively.

|

4.3

Computational methods

|

4.3.1

Compound preparation

The studied compounds 3a–n and 4a–n were modeled using the

LigPrep module^[52]of Schrödinger suite of software, v. 2019‐4 as

previously reported.^[22–26]Both enantiomers regarding the config-

uration of the hydroxyl group were modeled, if applicable. To identify

the protonation state, the Epik module^[53]of Schrödinger suite of

software, v. 2019‐4 was applied.

|

4.3.2

Receptor structures

In the cases when receptor X‐ray structures were available, they

were taken for molecular docking after necessary mutations: ser-

otonin 5‐HT_2Areceptor in complex with the antagonist risperidone

(PDB ID: 6A93^[54]) and serotonin 5‐HT_2Creceptor in complex with

the inverse agonist ritanserin (PDB ID: 6BQH^[27]). In the case of

|

4.2.4

Functional D₂receptor assay

Male SD rats were decapitated and their brains removed and placed

on ice. The striatal tissue was dissected and homogenized with a

MAGLI ET AL.

21 of 24

|

serotonin 5‐HT_1Areceptor, the previously published homology

model was used.^[23]All receptor models were in an inactive con-

formation. The structures of the biomolecules were preprocessed

using the Protein Preparation Wizard of Maestro Release 2019.4^[55]

to optimize the hydrogen bonding network and to remove any pos-

sible artifacts as reported previously.^[56]

administered intraperitoneally diluted in saline and injected 30 min

(amph), 60 min (buspirone, fluoxetine), or immediately (MK‐801 for

hyperactivity test) before the tests. All compounds were given in a

volume of 10 ml/kg to mice. The control animals received an

equivalent volume of the solvent at the respective time before the

tests. In the PA test, MK‐801 was administered 15 min after ad-

ministration of the compounds.

All the experiments were conducted in the light phase between

09.00 a.m. and 14.00 p.m. The experiments were performed by an

observer unaware of the treatment administered.

|

4.3.3

Molecular docking

Standard Precision (SP) approach of Glide^[57]from Schrödinger re-

lease 2019‐4 was used for molecular docking of the studied com-

pounds to receptor models, as reported previously.^[22–26]The grid

files were generated on the basis of co‐crystallized or co‐modeled

ligand. The hydroxyl groups of the following residues of the active

sites were made flexible: Tyr96 (2.63), Thr121 (3.37), Ser168 (4.57),

Thr188 (extracellular loop 2, ecl2), Ser190 (ecl2), Tyr195 (5.39),

Thr196 (5.40), Ser199 (5.43), Thr200 (5.44), Tyr390 (7.42) for ser-

otonin 5‐HT_1Areceptor; Ser131 (2.60), Thr134 (2.63), Ser159 (3.36),

Ser207 (4.57), Ser226 (ecl2), Tyr370 (7.42) for serotonin 5‐HT_2A

receptor; and Ser 110 (2.60), Tyr118 (ecl1), Thr 206 (ecl2), Tyr358

(7.42) for serotonin 5‐HT_2Creceptor (numbers in parenthesis in-

dicate GPCRdb generic numbers^[58]). Twenty poses were generated

for each receptor and each compound. The final poses were selected

based on Glide docking scores and visual inspection among the poses

where the protonatable nitrogen atom of the ligand interacted with

conserved Asp 3.32. Visualization of molecular modeling results was

achieved with Maestro Release 2019.4^[55]and PyMol 2.0.4^[59]soft-

ware. NCI maps of ligand–receptor interactions were computed with

NCIPlot v. 3.0^[60]at the distance below 4 Å from the ligand and vi-

sualized with VMD v. 1.9.1,^[61]as reported earlier.^[62]

|

4.4.2

Spontaneous locomotor activity,

amphetamine‐ and MK‐801‐induced hyperactivity

The locomotor activity of mice was measured using an animal activity

meter Opto‐Varimex‐4 Auto‐Track (Columbus Instruments). This

automatic device consists of eight transparent cages with a lid, set of

four infrared emitters (each emitter has 16 laser beams), and eight

detectors monitoring animal movements. To assess the spontaneous

activity of mice, the compounds or vehicles (as a control) were ad-

ministered 60 min before the test. In another set of experiments, the

influence of tested compounds on amph‐ and MK‐801‐induced hy-

peractivity in mice was evaluated. The study was conducted in the

same apparatus, but each mouse received amph (5 mg/kg, sc) 30 min

after injection of vehicle or tested compounds, or MK‐801 (0.3 mg/

kg, ip) 60 min after or immediately before the test. The animals were

placed in the cage individually, 50 min after the administration of

the tested compounds, for a period of 10 min for acclimatization.

After this time, their activity was noted after 6 min (corresponded

with the time duration of the FST and close [5 min] to the EPM test,

respectively) and after 20 min, to observe the dynamics of changes.

The distance traveled in centimeters was measured. The cages were

cleaned up with 10% ethanol after each mouse trial.

|

4.4

In vivo behavioral tests

|

4.4.1

General procedures

|

4.4.3

Motor coordination

The studies were conducted on male Albino Swiss mice (18–23 g).

The mice were housed in cages, five individuals per cage in

environmentally controlled rooms (ambient temperature 22 1°C;

relative humidity 50–60%; 12‐h light/dark cycle, lights on at 8:00).

Standard laboratory food (LSM; Agropol‐Motycz) and filtered water

were available ad libitum. All the experimental procedures were

carried out according to the National Institute of Health Guidelines

for the Care and Use of Laboratory Animals and the European

Community Directive for the Care and Use of Laboratory Animals of

November 24, 1986 (86/609/EEC), and approved by the Local Ethics

Committee for Animal Experimentation.

The effects of investigated compounds on motor coordination were

evaluated in the rotarod^[63]and chimney^[64]tests. In the first test, motor

impairments were measured, defined as the inability to main-

tain balance on a rotating rod (at a constant speed of 18 rpm) for 1 min.

In the second test, motor impairments were assessed by the inability of

the mouse to climb up the tube backward (3 cm in inner diameter,

25 cm long) within 60 s. Before the tests, the animals were trained once

a day for 3 days. The animals that were able to stay on the rotating rod

or to leave the chimney for 60 s were approved for experiments.

The investigated compounds (3b, 3e, 4a, 4j, 4n) in all tests were

administered intraperitoneally, dissolved in DMSO (final concentra-

tion of 0.1%), and then diluted by aqueous solution of 0.5% me-

thylcellulose (tylose) and injected 60 min before the tests. Other

drugs (amphetamine [amph], MK‐801, buspirone, fluoxetine) were

|

4.4.4

FST (Porsolt's test) in mice

The experiment was carried out according to the method of Porsolt

et al.^[65]Mice were individually placed in a glass cylinder (25 cm high;

22 of 24

MAGLI ET AL.

|

10 cm in diameter) containing water maintained at 23–25°C, and

they were left there for 6 min. The total duration of immobility was

recorded during the last 4 min of a 6‐min test session. A mouse was

regarded as immobile when it remained floating on the water,

making only small movements to keep its head above the water.

compartment of the apparatus and allowed to explore the light box.

After 30 s, the guillotine door was opened, and the mouse was allowed

to enter the dark compartment. When the mice entered the dark

compartment, the guillotine door was closed and an electric foot shock

(0.2 mA) of 2 s duration was delivered immediately to the animal via the

grid floor of the dark room by an insulated stimulator. The latency with

which the animal crossed into the dark compartment was recorded

(TL1). After 20 s, the animal was removed from the apparatus and

placed temporarily in its home cage. If the mouse failed to enter the

dark box within 300 s, it was placed into this dark box, the door was

closed, and electric foot shock was delivered to the animal. In this case,

TL1 value was recorded as 300 s. After 24 hours, in the subsequent trial

(retention), the same mice were again placed individually in the light

compartment of the PA apparatus. After a 30 s adaptation period in the

light (safe) chamber, the guillotine door was opened and the time taken

to re‐enter the dark compartment was recorded (TL2). No foot shock

was applied in this trial. If the animal did not enter the dark chamber

within 300 s, the retention test was terminated and TL2 was recorded

as 300 s. No foot shock was applied in this trial.

|

4.4.5

EPM test

The EPM studies were carried out on mice according to the method

of Lister.^[66]The EPM apparatus was made of plexiglass and con-

sisted of two open (30 × 5 cm) and two enclosed (30 × 5 × 15 cm)

arms. The arms extended from a central platform of 5 × 5 cm. The

apparatus was mounted on a plexiglass base, raising it 38.5 cm above

the floor, and illuminated by a red light. The test consisted of placing

a mouse at the center of the apparatus (facing an open arm) and

allowing it to freely explore. The number of entries into the open

arms and the time spent in these arms were scored for a 5‐min test

period. An entry was defined as placing all four paws within the

boundaries of the arm. The following measures were obtained from

the test: the total number of arm entries; the percentage of arm

entries into the open arms; and the time spent in the open arms

expressed as a percentage of the time spent in both the open and

closed arms. Anxiolytic activity was indicated by increases in the

time spent in open arms and in the number of open arm entries. The

total number of entries into either type of arm was used additionally

as a measure of the overall motor activity.

In this experiment, the effect of 3b, 3e, 4a, 4j, and 4n on the

consolidation of the passive avoidance response in mice was ex-

amined. All compounds were given immediately after the first trial,

and 15 min later, MK‐801 (0.3 mg/kg, ip) was administered.

For the memory‐related behaviors, the changes in PA perfor-

mance were expressed as the difference between retention and

training latencies, which was taken as LI. LI was calculated for each

animal as the ratio: LI = (TL2 − TL1)/TL1. TL1 is the time taken to

enter the dark compartment during the training and TL2 is the time

taken to re‐enter the dark compartment during the retention.^[51]

|

4.4.6

Bar test

|

Statistical analysis

Catalepsy was measured using the bar test 60 min after drug ad-

ministration: the front paws of each subject were placed on a cy-

lindrical metal bar (0.75 cm diameter) that was elevated 4.5 cm

above the table. The time during which both forelimbs remained on

the bar was recorded up to a maximum of 30 s. The test was re-

peated three times (intertrial interval: 1 min). Animals were put back

in their home cage after each measurement of catalepsy. Mice that

remained motionless with their paws on the bar for 10 s (with the

exception of respiratory movements) were scored as cataleptic.^[67]

4.4.8

The results were calculated by the one‐way analysis of variance,

followed by Dunnett's post hoc test. The results are presented as

mean SEM. The level of p < .05 was considered as statistically sig-

nificant. All the figures were prepared by the GraphPad Prism ver-

sion 5.00 for Windows.

|

4.5

Ex vivo assays

|

General procedures

4.4.7

PA test

4.5.1

The PA apparatus consists of a two‐compartment acrylic box with a

lighted compartment (10 × 13 × 15 cm) and a darkened compartment

(25 × 20 × 15 cm). The light chamber was illuminated by a fluorescent

light (8 W) and was connected to the dark chamber that was

equipped with an electric grid floor. Entry of animals to the dark box

was punished by an electric foot shock (0.2 mA for 2s).

Male rats (SD, 160–200 g; Harlan Laboratories) were manipulated

and cared for in strict compliance with the Principles of Laboratory

Animal Care (NIH publication no. 86–23, revised 1985) and the Italian

D.L. no. 116 of January 27, 1992, and associated guidelines in the

European Communities Council Directive of November 24, 1986

(86/609/ECC). Animal housing complied with recent pharmacological

guidance.^[68]All animals weighing 160–200 g were used after a 1‐week

acclimation period (temperature 23 2°C; humidity 60%, free access to

water, and standard food).

On the first day of training (pre‐test), all mice were allowed to

habituate in the experimental room for at least 30 min before the

experiments. Each animal was then gently placed in the light

MAGLI ET AL.

23 of 24

|

4.5.2

Ileum preparation and evaluation of

[2] A. Ju, B. Fernandez‐Arroyo, Y Wu, D. Jacky, A. Beyeler, Mol. Brain

2020, 13, 99. https://doi.org/10.1186/s13041-020-00605-5

[3] A. Corvino, F. Fiorino, B. Severino, I. Saccone, F. Frecentese,

E. Perissutti, P. Di Vaio, V. Santagada, G. Caliendo, E. Magli, Curr.

Med. Chem. 2018, 25(27), 3214.

5‐HT‐evoked contractions

Rats were asphyxiated using CO₂, and segments (1–1.5 cm) of ileum

were removed, flushed of luminal contents, and placed in Krebs solution

(119 mM NaCl, 4.75 mM KCl, 1.2 mM KH₂PO₄, 25 mM NaHCO₃, 2.5 mM

CaCl₂, 1.5 mM MgSO₄, and 11 mM glucose). The segments were pre-

pared as previously described^[51]: the segments were set up in such a way

as to record contractions mainly from the longitudinal axis, in an organ

bath containing 20 ml of Krebs solution, bubbled with 95% O₂and 5%

CO₂, and maintained at 37°C. The tissues were connected to an isotonic

transducer (load: 0.5 g), connected to a PowerLab system (Ugo Basile).

Ileal segments were equilibrated for 60 min,^[19]followed by three

repeated additions of submaximal concentration of 5‐HT (10⁻⁵M) to

record stable control contractions. To evaluate the inhibitory activity, the

responses were observed in the presence of increasing concentrations

(10⁻⁸to 10⁻⁵M). In preliminary experiments, the effect of 5‐HT was

observed in the presence of the neuronal blocker tetrodotoxin (0.3 µM),

the muscarinic receptor antagonist atropine (1 µM), the adrenergic re-

ceptor antagonist phentolamine (10⁻⁶M) plus propranolol (10⁻⁶M), and

the 5‐HT_2Aantagonist ketanserin (0.1 µM). The contact time for each

concentration was 10 min. The compounds were dissolved in DMSO.

DMSO (<0.01%) did not modify 5‐HT‐induced contractions. Results are

expressed as mean (SEM). The concentration of the compounds that

produced 50% inhibition of 5‐HT‐induced contractions (IC₅₀) or maximal

inhibitory effect (E_max) was used to characterize compounds' potency and

efficacy, respectively. The IC₅₀and E_maxvalues were calculated with the

aid of a computer program (GraphPad Prism 5).

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2002, 71, 533.

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92(4), 1523.

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483(1), 37.

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G. Latacz, G. Satala, W. Nitek, K. Kiec‐Kononowicz, J. Handzlik,

MedChemComm 2018, 9,1033.

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V. Santagada, Eur. J. Pharm. Sci. 2002, 16, 15.

[11] F. Fiorino, E. Perissutti, B. Severino, V. Santagada, D. Cirillo,

S. Terracciano, P. Massarelli, G. Bruni, E. Collavoli, C. Renner,

G. Caliendo, J. Med. Chem. 2005, 48, 5495.

[12] F. Fiorino, B. Severino, F. De Angelis, E. Perissutti, F. Frecentese,

P. Massarelli, G. Bruni, E. Collavoli, V. Santagada, G. Caliendo, Arch.

Pharm. (Weinheim) 2008, 341, 20.

[13] F. Fiorino, B. Severino, F. De Angelis, E. Perissutti, E. Magli,

F. Frecentese, A. Esposito, P. Massarelli, C. Nencini, B. Viti,

V. Santagada, G. Caliendo, Pharmazie 2009, 64(9), 555.

[14] F. Fiorino, B. Severino, F. De Angelis, E. Perissutti, E. Magli,

F. Frecentese, A. Esposito, P. Massarelli, C. Nencini, V. Santagada,

G. Caliendo, Bioorg. Med. Chem. Lett. 2010, 20(9), 2978.

[15] F. Fiorino, B. Severino, E. Magli, E. Perissutti, F. Frecentese,

A. Esposito, G. M. Incisivo, A. Ciano, P. Massarelli, C. Nencini,

V. Santagada, G. Caliendo, Eur. J. Med. Chem. 2012, 47, 520.

[16] (a) F. Fiorino, A. Ciano, E. Magli, B. Severino, A. Corvino,

E. Perissutti, F. Frecentese, P. Di Vaio, A. A. Izzo, R. Capasso,

P. Massarelli, C. Nencini, I. Rossi, E. Kedzierska, J. Orzelska‐Gòrka,

A. Bielenica, V. Santagada, G. Caliendo, Eur. J. Med. Chem. 2016,

110, 133.; (b) Bielenica, A., E. Kedzierska, M. Kolinski, S. Kmiecik,

A. Kolinski, F. Fiorino, B. Severino, E. Magli, A. Corvino, I. Rossi,

P. Massarelli, A. E. Koziol, A. Sawczenko, M. Struga, Eur. J. Med.

Chem. 2016, 116, 173.

ACKNOWLEDGMENTS

The NMR spectral data were provided by Centro di Ricerca Inter-

dipartimentale di Analisi Strumentale, Università degli Studi di Na-

poli “Federico II.” The assistance of the staff is gratefully appreciated.

Calculations were partially performed under a computational grant

by the Interdisciplinary Center for Mathematical and Computational

Modeling (ICM), Warsaw, Poland (Grant Number G30‐18), and under

resources and licenses from CSC, Finland. The authors sincerely

thank Agata Pawłowska for the technical performance of the radi-

oligand binding assay. Radioligand binding studies were carried out

with the use of the Center for Preclinical Research and Technology,

Medical University of Warsaw infrastructure.

[17] F. Fiorino, E. Magli, E. Kedzierska, A. Ciano, A. Corvino, B. Severino,

E. Perissutti, F. Frecentese, P. Di Vaio, I. Saccone, A. A. Izzo,

R. Capasso, P. Massarelli, I. Rossi, J. Orzelska‐Gòrka, J. H. Kotlinska,

V. Santagada, G. Caliendo, Bioorg. Med. Chem. 2017, 25(20), 5820.

[18] F. Fiorino, E. Magli, B. Severino, A. Corvino, A. Ciano, E. Perissutti,

F. Frecentese, P. Massarelli, C. Nencini, V. Santagada, G. Caliendo,

Arch. Pharm. (Weinheim) 2014, 347(10), 698.

CONFLICTS OF INTERESTS

The authors declare that there are no conflicts of interests.

[19] M. R. Briejer, C. Mathis, J. A. Schuurkes, Neurogastroenterol. Motil.

1997, 9(4), 231.

ORCID

[20] A. A. Kaczor, E. Rutkowska, D. Bartuzi, K. M. Targowska‐Duda,

D. Matosiuk, J. Selent, Methods Cell Biol. 2016, 132, 359.

[21] D. Bartuzi, A. A. Kaczor, K. M. Targowska‐Duda, D. Matosiuk,

Molecules 2017, 22, 340.

Ferdinando Fiorino

http://orcid.org/0000-0001-7357-5751

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How to cite this article: Magli E, Kędzierska E, Kaczor AA,

et al. Synthesis, docking studies, and pharmacological

evaluation of 2‐hydroxypropyl‐4‐arylpiperazine derivatives as

serotoninergic ligands. Arch Pharm. 2021;354:e2000414.

https://doi.org/10.1002/ardp.202000414

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Synthesis, docking studies, and pharmacological evaluation of 2-hydroxypropyl-4-arylpiperazine derivatives as serotoninergic ligands

DOI: 10.1002/ardp.202000414

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