Targeting dopamine D3 and serotonin 5-HT1A and 5-HT2A receptors for developing effective antipsychotics: Synthesis, biological characterization, and behavioral studies

Article

pubs.acs.org/jmc

Targeting Dopamine D₃and Serotonin 5‑HT_1Aand 5‑HT_2AReceptors

for Developing Eﬀective Antipsychotics: Synthesis, Biological

Characterization, and Behavioral Studies

Margherita Brindisi,^†,‡Stefania Butini,*^,†,‡Silvia Franceschini,^†,‡Simone Brogi,^†,‡Francesco Trotta,^†,‡

Sindu Ros,^†,‡Alfredo Cagnotto,^§Mario Salmona,^§Alice Casagni,^†,‡Marco Andreassi,^†,‡

Simona Saponara,^∥Beatrice Gorelli,^∥Pia Weikop,^⊥Jens D. Mikkelsen,^#Jorgen Scheel-Kruger,^∇

Karin Sandager-Nielsen,^∇,◆Ettore Novellino,^†,○Giuseppe Campiani,*^,†,‡and Sandra Gemma^†,‡

‡

^†European Research Centre for Drug Discovery and Development, and Dipartimento di Biotecnologie, Chimica e Farmacia,

Universita

^§IRCCS-Istituto di Ricerche Farmacologiche Mario Negri, Via La Masa 19, 20156 Milano, Italy

^∥Dipartimento di Scienze della Vita, Universita

degli Studi di Siena, via Aldo Moro 2, 53100 Siena, Italy

̀

degli Studi di Siena, via Aldo Moro 2, 53100 Siena, Italy

̀

^⊥Laboratory of Neuropsychiatry, Psychiatric Centre, University of Copenhagen, Blegdamsvej 3 DK-2100 Copenhagen, Denmark

^#Neurobiology Research Unit, University Hospital Rigshospitalet, Blegdamsvej 9, DK-2100 Copenhagen, Denmark

^∇NeuroSearch A/S, Pederstrupvej 93, DK-2750 Ballerup, Denmark

^○Dipartimento di Farmacia, Universita

̀

degli Studi di Napoli “Federico II”, via D. Montesano 49, 80131 Napoli, Italy

S

* Supporting Information

ABSTRACT: Combination of dopamine D₃antagonism, serotonin 5-HT_1A

partial agonism, and antagonism at 5-HT_2Aleads to a novel approach to potent

atypical antipsychotics. Exploitation of the original structure−activity relation-

ships resulted in the identiﬁcation of safe and eﬀective antipsychotics devoid of

extrapyramidal symptoms liability, sedation, and catalepsy. The potential

atypical antipsychotic 5bb was selected for further pharmacological

investigation. The distribution of c-fos positive cells in the ventral striatum

conﬁrmed the atypical antipsychotic proﬁle of 5bb in agreement with

behavioral rodent studies. 5bb administered orally demonstrated a biphasic

eﬀect on the MK801-induced hyperactivity at dose levels not able to induce

sedation, catalepsy, or learning impairment in passive avoidance. In

microdialysis studies, 5bb increased the dopamine eﬄux in the medial

prefrontal cortex. Thus, 5bb represents a valuable lead for the development of

atypical antipsychotics endowed with a unique pharmacological proﬁle for addressing negative symptoms and cognitive deﬁcits in

schizophrenia.

ingly, D₂R was accounted as the main target associated with

antipsychotic eﬃcacy as well as with side eﬀect liability

(extrapyramidal side eﬀects (EPS) and prolactin elevation).

The introduction of atypical antipsychotics (generation II

antipsychotics) represented an important advance in the

pharmacological treatment of neuropsychiatric disorders.⁴The

atypical antipsychotics (e.g., clozapine, 1, and olanzapine, 2,

Chart 1), characterized by a multireceptor aﬃnity proﬁle which

combines potent antagonism for serotonin 5-HT_2Areceptor (5-

HT_2AR) to D₂R, and D₃R blockade,⁵are endowed with

superior clinical eﬃcacy than generation I antipsychotics,

mainly against positive symptoms (this issue is still

controversial taking into account the results from the Clinical

INTRODUCTION

■

Schizophrenia is a chronic, frequently disabling psychiatric

disorder aﬀecting about 1% of the world’s population.¹It is

characterized by positive, negative, aﬀective, and cognitive

symptoms. It generally presents in late adolescence or early

adulthood and is associated with an increased risk of mortality

and social or occupational dysfunction. Antipsychotic medi-

cation is the main therapeutic intervention for neuropsychiatric

disorders, and most patients need life-long treatment. The

mechanism of action of the antipsychotics is based on the

dopaminergic hypothesis of schizophrenia, a disease that

involves a dysregulation of dopaminergic circuits (hyper-

dopaminergic tone in the mesolimbic pathway and hypodopa-

minergic signaling in the mesocortical pathway).²On the basis

of this model, antagonism toward D₂receptors (D₂R) in the

mesolimbic pathway reduces psychotic symptoms.³Accord-

Received: August 21, 2014

A

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and ziprasidone) and to an increased risk of sudden death.^12,13

Unmet clinical needs include the treatment of refractory

patients aﬀected by negative symptoms and neurocognition

dysfunction.¹⁴Because generation II antipsychotics do not oﬀer

major advantages in terms of eﬃcacy and range of therapeutic

activity, the development of more eﬀective and safer

antipsychotic agents is still a crucial issue.

Chart 1. Reference Antipsychotics

Recently, we identiﬁed a novel class of atypical anti-

psychotics, typiﬁed by 4.¹⁵For improving the structure−activity

relationships (SARs) of our arylpiperazine antipsychotics, we

further exploited the novel multireceptor aﬃnity proﬁle

approach, which combines antagonism at D₃R and at 5-

HT_2AR and partial agonism at serotonin 5-HT_1AR, with a low

aﬃnity for D₂R (no liability of EPS at antipsychotic doses) and

5-HT_2CR (reducing the risk of obesity under chronic

treatment). This pharmacological approach is based on several

considerations.

The serotoninergic system plays an important role in the

regulation of prefrontal cortex (PFC) functions (emotional

control, cognitive behavior, and working memory). 5-HT_1AR

and 5-HT_2AR are particularly dense in PFC pyramidal neurons

and GABA interneurons. In PFC N-methyl-^D-aspartate

(NMDA) receptors, channels are the target of 5-HT_1AR and

both receptors modulate the excitability of cortical neurons,

thus aﬀecting cognitive functions. Indeed, 5-HT_1AR may be a

therapeutic target for the development of improved anti-

psychotic drugs. Accordingly, 5-HT_1AR aﬃnity contributes to

the clinical eﬃcacy of most of the atypical antipsychotic drugs

(clozapine, olanzapine, and aripiprazole) and their low liability

for EPS. Furthermore, because glutamatergic transmission is

dysfunctional in schizophrenia and because glutamate release is

modulated by 5-HT_1AR activation, agonist properties at

postsynaptic 5-HT_1AR may be relevant to the therapeutic

proﬁle of atypical antipsychotic agents, improving negative

Antipsychotic Trials of Intervention Eﬀectiveness (CATIE)

study⁶) and are accompanied by fewer EPS.⁷Aripiprazole⁸(3,

Chart 1), also termed as generation III antipsychotic, has

subnanomolar aﬃnity for 5-HT_2BR, D_2LR, and D₃R but also has

signiﬁcant aﬃnity (5−30 nM) for several other 5-HT receptors

(5-HT_1A, 5-HT_2A, 5-HT₇) as well as for α_1A-adrenergic and H₁-

histamine receptors. Functionally, it behaves as an inverse

agonist at 5-HT_2BR and as partial agonist at 5-HT_2AR, 5-

HT_2CR, D₃R, and D₄R.⁹Excluding 1, atypical antipsychotics are

currently recommended as ﬁrst-line agents for acute and

maintenance therapy of schizophrenia.¹⁰In the last 10 years,

newer agents have been introduced (paliperidone, asenapine,

iloperidone, and lurasidone).¹¹However, the use of these drugs

is associated with potential long-term health risks for patients as

well as with decreased adherence to treatment regimens.⁴

Furthermore, extensive data link the use of antipsychotic drugs

to dose-dependent corrected QT (QTc) prolongation (e.g.,

haloperidol, mesoridazine, pimozide, thioridazine, sertindole,

Chart 2. Reference Compound 4 and Title Compounds 5a−ll

B

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symptoms, and cognitive deﬁcits. Serotonin, through its

interaction with 5-HT_2AR, inhibits neuronal activity in the

substantia nigra and ventral tegmental area. 5-HT_2AR

antagonists may increase the ﬁring rate of midbrain

dopaminergic neurons in a state-dependent manner and

potentiate the increase in the activity of nigrostriatal

dopamine-containing neurons in response to moderate D₂R

antagonism by antipsychotics. Therefore, 5-HT_2AR antagonism

may prevent or alleviate EPS induced by acute or long-term

treatment with typical drugs such as haloperidol.

Scheme 1. General Synthesis for Compounds 5a−g,i−w,bb−

a

ll

D₃R has been claimed as a potential target for antischizo-

phrenic drug development. In fact, dopamine through D₃R

modulates the cholinergic system at the prefrontal cortex level

and D₃R antagonists (devoid of muscarinic eﬀects) may

enhance acetylcholine release in frontal cortex. Although the

speciﬁc distribution of D₃Rs in the mesolimbic dopaminergic

system (no liability of EPS), the persistent concern is whether

selective D₃R blockade can relieve positive symptoms of

schizophrenia. We recently proved¹⁵that highly selective and

extremely potent D₃R antagonists and partial agonists are

devoid of behavioral eﬀects in rodent models of schizophrenia.

Thus, for eﬃcacy, D₃R antagonism must be associated with

partial agonism and antagonism at 5-HT_1AR and 5-HT_2AR,

respectively.

Accordingly,¹⁵we selected the arylalkylpiperazine system as a

ﬂexible scaﬀold for achieving a ﬁne balancing of dopamine D₃R

and serotonin 5-HT_1AR and 5-HT_2AR aﬃnity, reducing D₁, D₂

and 5-HT_2Creceptor occupancy (4, Chart 2). Starting from the

hit 4,¹⁵⁻²⁰the present article deals with the synthesis and

behavioral investigation of a set of arylpiperazines as novel

potential multitarget antipsychotics. Among the arylpiperazines

5a−ll, we have identiﬁed 5bb as a potent atypical antipsychotic

agent possessing the unique and predicted pharmacological

proﬁle. Despite its structural similarity to aripiprazole (nano-

molar aﬃnity for D₂R and D₃R), 5bb is endowed with a unique

pharmacological proﬁle for addressing negative symptoms and

cognitive deﬁcits, being characterized by high potency at D₃R,

5-HT_1AR, and 5-HT_2AR and much lower aﬃnity for D₂R.

a

Reagents and conditions: (a) 4-amino-1-butanol, DCC (or EDC for

7b), HOBt, dry DCM, rt, 12 h; (b) CBr₄, PPh₃, dry MeCN, rt, 2 h; (c)

TFA, DCM, rt, 1 h; (d) arylpiperazine, TEA, dry MeCN, reﬂux, 12 h;

(e) BBr₃, dry DCM, −78 °C to rt, 90 min.

while all attempts of deprotection by catalytic hydrogenation

failed also when performed under strong acidic conditions.

The bromination step in the synthesis of the 3-indolyl-based

analogue 5h reaction gave undesired byproducts. Consequently,

an alternative strategy (Scheme 2) was employed for its

a

Scheme 2. Synthesis of Compound 5h

CHEMISTRY

■

The synthesis of compounds 5a−ll is reported in Schemes 1−3.

Scheme 1 describes the general synthetic strategy followed to

obtain most of the compounds (namely 5a−g,i−w,bb−ll).

The acids 6a−f (6-quinolyl carboxylic acid, 6b, was

synthesized from 6-methylquinoline by means of a high

yielding chromium(VI)-based oxidation reaction) were con-

verted into the corresponding 4-hydroxybutylamides 7a−f by

coupling with 4-aminobutanol, in the presence of 1-

hydroxybenzotriazole (HOBt) and N,N-dicyclohexylcarbodii-

mide (DCC) (or N-(3-(dimethylamino)propyl)-N′-ethylcarbo-

diimide hydrochloride (EDC) for the quinolin-6-yl carboxylic

acid 6b). These latter amides after bromination of the hydroxyl-

group (8a−j,9a,b,d,e were previously described¹⁹) were treated

with the appropriate arylpiperazine in the presence of a base to

give the desired products. The synthesis of arylpiperazines

10a−f followed a previously described procedure.²⁰N-

Arylation of Boc-piperazine, through a classical palladium

catalyzed reaction, gave piperazines 9a−f in high yields.

Deprotection of 9a−f by triﬂuoroacetic acid (TFA) provided

the corresponding piperazines as triﬂuoroacetate salts 10a−f in

high overall yield. The arylpiperazine 11, necessary for the

synthesis of compound 5s, was treated with boron tribromide

to aﬀord the benzyl deprotected compound in very high yield,

a

Reagents and conditions: (a) 4-bromobutanenitrile, K₂CO₃, MeCN,

reﬂux, 12 h; (b) NaBH₄, NiCl₂, MeOH, rt, 90 min; (c) pyridine,

DCM, rt, 12 h.

synthesis. The N-alkylation of piperazine 12 by 4-bromobuta-

nenitrile aﬀorded the cyanoderivative 13, which was reduced

using sodium borohydride and nickel chloride. The resulting

poorly stable amine 15 was immediately used for the next

coupling reaction with the acyl chloride 14, thus aﬀording the

ﬁnal compound 5h.

C

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The synthesis of compounds (R)-5x,y and (S)-5z,aa

(Scheme 3) started from quinaldic acid (16), which was

cyclic) as “head” with various arylpiperazines as “tail” (Chart 2).

Structural modiﬁcations were conceived including at the “head”

diﬀerent heterocyclic systems bearing nitrogens and/or H-bond

donors/acceptors for improving the overall proﬁle (see

Supporting Information, Table 1SI for calculculated properties,

cLogS and cLogP).^23,24Starting from the benzofuran-2-

carboxamide derivative 4, we explored the bioisosteric

replacement of the benzofurancarboxamide oxygen with an

NH, thus developing indole-2-carboxamide and indole-3-

carboxamide analogues (5g−i). We further explored the size

of the nitrogen containing heterocycles²³by introducing

various quinolines and isoquinolines (isoquinoline-2-carbox-

amides (5j−s), quinolin-2-carboxamides (5t−w), tetrahydro-

quinolin-2-carboxamides (5x−aa), a quinolin-6-carboxamide

(5bb)) and pyridines (picolinamide (5gg−jj), a 6-methylpico-

linamide (5kk), and a nicotinamide (5ll)).

a

Scheme 3. Synthesis of Compounds (R)-5x,y and (S)-5z,aa

According to our pharmacological hypothesis, the novel

arylpiperazines were tested on D₂R, D₃R, 5-HT_1AR, and 5-

HT_2AR. The binding proﬁle of the tested molecules is reported

in Tables 1−3, while the in vivo characterization of a subset of

arylpiperazines is displayed in Table 4 and Figure 1. On the

basis of the reported data, 5bb was selected as the most

interesting analogue for further pharmacological investigation

in vitro and in vivo.

In Vitro Binding Assays on Dopaminergic and

Serotoninergic Receptors and Structure−Activity Rela-

tionship (SAR) Studies. Benzofurancarboxamide- and

Indolecarboxamide-Based Subseries of Compounds 5a−i.

As reported in Table 1, compound 5a (with a phenylpiperazine

“tail”) displayed and excellent in vitro proﬁle being potent and

selective for the receptors of interest. In agreement with our

previously described molecular modeling studies,¹⁵the

dimethyl-substituted analogue 5b became a two-digit nM

ligand for D₂R and 5-HT_2CR and a more potent ligand for the

three receptors of interest (D₃R, 5-HT_1AR, and 5-HT_2AR).

Introduction of a polar 3-methoxy group (5c vs 5a) lowered

D₃R aﬃnity by almost half, while the aﬃnity against 5-HT_2AR

was increased by 10-fold. The presence of a 6-methylpyridine

(5d) slightly decreased D₃R aﬃnity while increasing 5-HT_2CR

aﬃnity if compared to 5a. Replacement of the arylpiperazine of

5a by a benzylpiperazine (5e) determined a complete loss of

the desired in vitro pharmacological proﬁle. An improvement of

aﬃnity for the ﬁve receptors of the panel was obtained by

introducing an ortho-ﬂuorophenylpiperazine (5f). Replacement

of the benzofuran of 5a by a 2-indolecarboxamide (5g) or by a

3-indolecarboxamide (5h) provided two analogues character-

ized by an undesired proﬁle (high aﬃnity for D₂R and 5-HT_2CR

of 5g and low aﬃnity for 5-HT_2AR of 5h). Compound 5i, with

a 1-methylindole-3-carboxamide, was found equipotent at 5-

HT_2AR and 5-HT_2CR.

Within the benzofurancarboxamide- and indolecarboxamide-

based series of arylpiperazines, we selected compounds 5a and

5b for further in vivo evaluation as the best performing

compounds of the subseries.

Quinolinecarboxamide-Based Subseries of Compounds

5j−bb. Among the isoquinolin-2-carboxamide-based analogues

(Table 2), the best performing “tails” were represented by

phenylpiperazine (5j), meta-substituted phenyl piperazines

(5k,l), and para-ﬂuorophenyl piperazine (5r). These com-

pounds (5j,k,l,r) displayed excellent in vitro proﬁles both in

terms of potency and selectivity. In contrast, a ﬂuorine at the

meta-position (5q) increased the potency at D₂R and 5-HT_2CR.

Given that arylpiperazines are often metabolized by hydrox-

a

Reagents and conditions: (a) PtO₂, H₂, MeOH, rt, 16 h; (b) SOCl₂,

MeOH, rt, 16 h; (c) α-chymotrypsin phosphate buﬀer; (d) CbzCl,

NaHCO₃2M, rt, 90 min; (e) NaOH, MeOH/H₂O, reﬂux, 2 h; (f) 4-

amino-1-butanol, DCC, HOBt, dry DCM, rt, 12 h; (g) CBr₄, PPh₃, dry

MeCN, rt, 2 h; (h) appropriate arylpiperazine, TEA, dry MeCN, reﬂux,

12 h; (i) 5% Pd/C, H₂, 60 psi MeOH/EtOAc, rt, 8 h.

partially hydrogenated to give acid ( )-17 as racemic mixture.

The acid ( )-17 was converted in the methyl ester ( )-19,²¹

which was submitted to a kinetic enzymatic resolution using α-

chymotrypsin that selectively hydrolyses the R-isomer of the

ester ( )-18,²¹giving a separate mixture of (S)-19 and (R)-

18.²²Both the acid and the ester, in their enantiomerically pure

forms (R)-17 and (S)-18, were N-Cbz protected and after

saponiﬁcation of the intermediate ester from (S)-18, the acids

(R)- and (S)-19 were obtained. For the synthesis of the ﬁnal

compounds, the acids (R)- and (S)-19 were subjected to the

classical protocol as described in Scheme 1, and after coupling

with aminobutanol ((R)- and (S)-20), bromination of the

alcoholic function ((R)- and (S)-21) and alkylation of the

appropriate arylpiperazine, the intermediates (R)-22a,b and

(S)-22a,b were obtained. Removal of the Cbz group under a

hydrogen atmosphere aﬀorded the ﬁnal products (R)-5x,y and

(S)-5z,aa in their enantiomerically pure form.

RESULTS AND DISCUSSION

■

With the aim of optimizing the multireceptor aﬃnity proﬁle

and the eﬃcacy of our recently identiﬁed atypical antipsychotic

hit 4 and improving SAR, we developed a set of compounds

(5a−ll, Chart 2 and Tables 1−3) based on the general structure

depicted in Chart 2. Speciﬁcally, the new compounds were

synthesized for improving the original multireceptor aﬃnity

proﬁle (binding to D₃R, 5-HT_1AR, and 5-HT_2AR) of 4 in order

to select a potent antipsychotic agent for further pharmaco-

logical investigation.^15,19,20A wide SAR analysis was performed

by merging speciﬁc heterocyclic systems (bicyclic or mono-

D

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Table 1. Binding Aﬃnities for D₂, D₃, 5-HT_1A, 5-HT_2A, and

a

5-HT_2CReceptors (K_inM) of Benzofuran-2-carboxamide-

Based and Indolecarboxamide-Based Analogues 5a−i, 4, and

Reference Compounds (Clozapine (1), Olanzapine (2),

Aripiprazole (3), and Risperidone)

a

Tests were performed at MDS, unless otherwise speciﬁed, and each

value is the mean of two determinations (all the compounds were

tested in ﬁve diﬀerent concentrations and twice at each single dose).

b

Tests were performed at Mario Negri Institute and were performed

as previously reported,¹⁵each value is the mean of three

determinations and SD were within 10% of the mean.

extra basic moiety at the “tail” of analogues was not tolerated;

the pyridin-2-yl (5m), the 6-methylpyridin-2-yl (5n), and the

pyrimidin-2-yl (5o) analogues lost the desired proﬁle at the

serotoninergic receptors. The introduction of a quinolin-3-yl as

in 5p led to a D₃R selective analogue. A trend comparable to

5j−m was observed with the quinolin-2-carboxamide analogues

5t−w. We also synthesized the tetrahydroquinolin-2-carbox-

amide analogues (5x−aa). By comparing (R)-tetrahydroquino-

lin-2-carboxamides vs the (S)-tetrahydroquinolin-2-carboxa-

mides (5x vs 5z, and 5y vs 5aa), as shown in Table 2, we

did not observe a noteworthy stereoselective interaction with

the D₃R, while sensitivity to stereochemistry was shown by 5-

HT_1AR. (R)-5x was 8.6 times more potent than the

corresponding (S)-isomer (5z); on the contrary, 5-HT_2AR

preferred the (S)-isomer, being this latter compound 2 times

more potent than 5x. The meta-tolyl derivatives (R)-5y and

(S)-5aa displayed a diﬀerent behavior and the 5-HTRs of the

panel preferred the (S)-enantiomer 5aa, this latter being 2.7,

15, and 6.1 times more potent than its (R)-counterpart at 5-

HT_1AR, 5-HT_2AR, and 5-HT_2CR, respectively. Within this

series, the quinolin-6-carboxamide analogue 5bb showed a

subnanomolar aﬃnity at D₃R combined with two-digit

nanomolar potency at 5-HT_1AR and 5-HT_2AR when tested

on the receptor panel (Table 2). Much lower potency was

found at D₂R and 5-HT_2CR. Notably, the binding proﬁle of 5bb

outﬂanked that of 4, 5a, and 5j.

Figure 1. Eﬀect of test compounds on MK801 and methamphetamine

(AMP) induced hyperactivity in mice. MK801 was administered at a

dose of 0.2 mg/kg, and AMP was administered at a dose of 2 mg/kg.

Both psychostimulants were administered ip in a dose volume of 10

mL/kg immediately before test start. Test compounds were

administered sc (5a, 5j, 5k, and 5l) or po (5bb) 30 min before test

start. Locomotor activity was evaluated in automated activity chambers

for 60 min. Statistical evaluations were performed by two-way

ANOVA, followed by Tukey test for post hoc comparisons. ###, p

< 0.001 as compared to vehicle treatment; */**/***, p < 0.05/0.01/

0.001 as compared to relevant psychostimulant alone.

ylation at the para-position, we hypothesized a similar

metabolic proﬁle for 5j and subsequently synthesized 5s. By

comparing the proﬁle of 5j and 5s, we could argue that the in

vivo hydroxylation would lead to a scarcely active compound at

5-HT_1AR, losing antipsychotic eﬃcacy.¹⁵Introduction of an

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Table 2. Binding Aﬃnities for D₂, D₃, 5-HT_1A, 5-HT_2A, and

5-HT_2CReceptors (K_inM) of Quinolinecarboxamide-Based

Table 3. Binding Aﬃnities for D₂, D₃, 5-HT_1A, 5-HT_2A, and

5-HT_2CReceptors (K_inM) of Benzoylamide-Based and

a

Analogues 5j−bb, 4, and Reference Compounds (Clozapine

(1), Olanzapine (2), Aripiprazole (3), and Risperidone)

Pyridincarboxamide-Based Analogues 5cc−ll, 4, and

Reference Compounds (Clozapine (1), Olanzapine (2),

Aripiprazole (3), and Risperidone)

a

Tests were performed at MDS, and each value is the mean of two

determinations (all the compounds were tested in ﬁve diﬀerent

concentrations and twice at each single dose).

compounds (5gg−jj) behaved nicely in the in vitro studies,

with the exception of 5ii, which lacked D₃R aﬃnity and

displayed poor 5-HT_2AR aﬃnity, being selective at the 5-HT_1AR

ligand. On the basis of the results displayed in Table 3,

5cc,dd,gg,jj were selected for in vivo studies.

Behavioral in Vivo Studies on a Set of Selected

Analogues 5a,b,j−l,r,u,bb−dd,gg,jj. Analysis of the data

obtained from the receptor binding assays allowed the selection

of 12 compounds (5a,b,j−l,r,u,bb−dd,gg,jj, Table 4) for testing

in vivo in several mice models sensitive to mesolimbic mediated

antipsychotic-like activity.

a

Tests were performed at MDS, unless otherwise speciﬁed, and each

value is the mean of two determinations (all the compounds were

tested in ﬁve diﬀerent concentrations and twice at each single dose).

b

Tests were performed at Mario Negri Institute and were performed

as previously reported;¹⁵each value is the mean of three

determinations and SD were within 10% of the mean.

The antagonism of hyperlocomotion induced by dopamine

receptor direct agonists (e.g., apomorphine) or compounds

which facilitate dopaminergic tone (e.g., amphetamine, AMP)

are murine models widely used for assessing antipsychotic

eﬃcacy. Compounds were also tested in the MK801-,

phencyclidine (PCP)-, or AMP-induced hyperactivity mod-

els.^15,25,26The choice was mainly dependent upon the binding

proﬁle shown. The observation that uncompetitive NMDA

receptor antagonists (e.g., PCP, MK801 and ketamine) induce

schizophrenic symptoms in healthy subjects and exacerbate

existing psychoses in schizophrenic patients has suggested that

endogenous dysfunction of NMDA receptor-mediated neuro-

transmission might contribute to the pathogenesis of

schizophrenia.^27,28It is well-known that systemic administration

of PCP or MK801 increases dopamine cell ﬁring rate in the

brain.²⁹In particular, hyperactivity produced by a low dose of

MK801 is dependent upon D₃R stimulation.³⁰Thus, the

hyperactivity induced by MK801 can be reduced in vivo by

Within the quinolinecarboxamide-based subseries, analogues

5j,k,l,r,u,bb (Table 4) were selected for further in vivo testing.

Benzoylamide- and Pyridincarboxamide-Based Subseries

of Compounds 5cc−ll. In general, the compounds bearing a

benzoylamide (5cc−ﬀ, Table 3) or a pyridinecarboxamide

(5gg−ll, Table 3) at the “head” displayed interesting in vitro

proﬁles.

Analogues 5ee and 5ﬀ bearing a 1-naphthyl and quinolin-3-yl

moiety at the “tail”, respectively, lost the desired in vitro

pharmacological proﬁle: 5ee became potent against the

monoaminergic receptors of the tested panel, while 5ﬀ lost

aﬃnity at 5-HTRs. The diﬀerent behavior of these two

analogues, as well as for 5p, is mostly due to the shape-

dependent eﬀect of the bicyclic system at the distal piperazine

nitrogen. The benzoyl amide of 5cc,dd was replaced by a

pycolinamide (5ii,jj, respectively). The picolinamide-based

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Table 4. Summary of Behavioral Eﬀects of Selected Compounds (5a,b,j−l,r,u,bb−dd,gg,jj) Compared to the Eﬀects of 1, 3, 4,

a

and Haloperidol Tested Under Identical Conditions

a

b

Tests performed as in previously described¹⁵and compounds were dosed subcutaneously (sc) unless otherwise noted (po: orally). EM:

c

d

spontaneous exploratory locomotor activity. MK801: MK801-induced locomotor activity. AMP: methamphetamine-induced locomotor activity.

e

f

PCP: PCP-induced locomotor activity. MED: minimal eﬀective dose; the minimal statistically signiﬁcant dose that reduce locomotor activity as

g

h

compared to vehicle or relevant psychostimulant. Data from ref 15. NT: not tested.

multitarget antipsychotic drugs characterized by high aﬃnity

and selectivity degree for D₃R.^27,30

decreased the PCP-induced hyperactivity only at the higher

10 mg/kg dose. The pyridinecarboxamide 5jj displayed a

superimposable proﬁle to that of 5cc,dd, while 5gg showed

some inhibition of PCP-induced hyperactivity at 3 mg/kg, but a

sedative eﬀect was preliminarily observed at the same dose in

the spontaneous explorative locomotor activity test.

Compound 5b, although displaying a decrease of MK801-

induced hyperactivity, also caused tremors at 30 mg/kg.

Accordingly, the evaluation of 5b was discontinued. The

quinolin-2-carboxamide 5u reduced MK801-induced hyper-

activity at 10 mg/kg, but at the same time this dose reduced

spontaneous exploratory activity. Also this compound increased

the response to PCP at 1, 3, and 10 mg/kg and caused tremors

at 30 mg/kg. On the other hand, compound 5r displayed a

decrease of MK801-induced hyperactivity, with marked eﬀects

at 60 mg/kg per os (po), (Table 4). Disappointingly,

spontaneous locomotor activity was also strongly and dose-

dependently diminished by 5r.

The best results were obtained with compounds 5a,j−l and

5bb. Compound 5a caused a reduction in MK801-, PCP-, and

AMP-induced hyperactivity in mice, reaching signiﬁcance at 3,

0.3, and ≤1 mg/kg, respectively (p < 0.05 against PCP-induced

hyperactivity, p < 0.001 against MK801- and AMP-induced

hyperactivity). At 0.3, 1, and 3 mg/kg, 5a reduced spontaneous

exploratory locomotor activity, but only marginally, suggesting

only mild sedative properties of this compound at eﬃcacious

doses. Tremors were observed at 10 mg/kg. Compound 5j

caused reductions in MK801-, PCP-, and AMP-induced

hyperactivity in mice, reaching signiﬁcance at 3, 3, and ≤1

mg/kg, respectively (p < 0.01, p < 0.01, and p < 0.001,

respectively). Very minor reduction of spontaneous exploratory

locomotor activity was observed at 10 mg/kg, with no eﬀect at

3 and 30 mg/kg. Compound 5k also caused a reduction in

MK801-, PCP-, and AMP-induced hyperactivity in mice,

The tests were performed according to our previously

reported protocols (female NMRI mice, Taconic, Denmark).¹⁵

Brieﬂy, the psychostimulants were administered immediately

before test start at the following doses: PCP, 4 mg/kg,

subcutaneously (sc), MK801:0.2 mg/kg, intraperitoneally (ip);

AMP, 2 mg/kg, ip. The tested compounds were mainly

administered sc (Table 4) at various doses 15−30 min prior to

administration of the psychostimulants. All compounds were

administered in a dose volume of 10 mL/kg. Locomotor

activity was assessed for 60 min in automated activity chambers

equipped with infrared photobeams. Because compounds that

cause sedation or induce motor disturbances can reduce

spontaneous locomotor activity, we preliminarily assessed this

parameter for the tested compounds.

To evaluate the liability for striatal-mediated side eﬀects and

for cataleptogenic potential, selected compounds were tested in

mice by the vertical grid test and the elevated bar test. Degree

of catalepsy was scored three times separated by 15 min each.

The compounds were administered 30 min before the ﬁrst

catalepsy assessment. All the results are reported in Table 4,

where the administration route is also indicated for all the

selected analogues. Figure 1 reports the data for the ﬁve best

performing compounds of the set (compounds 5a,j−l,bb) in

the MK801- and AMP-induced hyperactivity tests, while data

on the PCP-induced hyperactivity for compounds 5a,j−l are

given in Supporting Information, Figure 1SI.

Analysis of the data reported in Table 4 evidenced that the

selected benzoylamide and pyridinecarboxamide analogues

(5cc,dd,gg,jj) did not perform as expected in the in vivo

experiments. Both the benzoylamides 5cc and 5dd, which did

not produce motor disturbancies, were not eﬀective in the

PCP-hyperactivity model at 3 mg/kg (5cc), while 5dd

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reaching signiﬁcance at 3, 0.3, and ≤3 mg/kg, respectively (p <

0.001 for all doses). However, 5k caused dose-related

reductions of spontaneous exploratory locomotor activity at

3, 10, and 30 mg/kg (p < 0.001 for all doses tested). Despite

relatively high aﬃnity toward D₂R (K_i= 268 nM), 5k did not

show a cataleptic potential at doses up to 30 mg/kg (one-way

ANOVA, p > 0.05).

Pretreatment with 5l reduced MK801, PCP-, as well as AMP-

induced hyperactivity reaching statistical signiﬁcance at 10 mg/

kg against MK801- and PCP-induced hyperactivity and ≤1 mg/

kg for AMP induced hyperactivity (p < 0.001 for all assays).

Sedation was only registered at 10 and 30 mg/kg (p < 0.001 for

both doses against vehicle).

and twice at each single dose. 5bb showed a signiﬁcant to

moderate aﬃnity for the α₁and α₂adrenoreceptors (K_i= 50

and 150 nM, respectively) and histamine H1 receptor subtype

(K_i= 110 nM) (Table 5). In vivo, we observed a slight

Table 5. In Vitro Receptor Binding Proﬁle of 5bb on α₁, α₂,

and H₁Receptors, Functional Activity on D₃R, 5-HT_1AR, 5-

HT_2AR, and hERG Aﬃnity

a

b

c

d

α₁

α₂

H₁R

D₃R

(IC₅₀)

5-HT_1A

R

5-HT_2A

R

hERG

(K_i)

(EC₅₀)

(IC₅₀)

(μM)

compd (nM) (nM) (nM) (μM)

(μM)

5bb

50

150

110

0.258

1.8

2.6

∼10

a

[³⁵S]GTPγS binding at D₃R were performed in membranes prepared

b

from CHO-K1 cells expressing human D₃R. [³⁵S]GTPγS binding at

With respect to the other analogues tested, compound 5bb

(one of the best performing analogues in vitro in terms of

potency and selectivity, with an interesting cLogS, Supporting

Information, Table 1SI) was administered orally and it

produced remarkable results (data obtained after sc admin-

istration of 5bb are given in Supporting Information, Figure

2SI). In MK801-induced hyperactivity, predictive for atypical

antipsychotic activity, 5bb showed eﬃcacy already at 10 mg/kg

(p < 0.01), with a marked eﬀect at 30 mg/kg (p < 0.001). At

the same doses, 5bb caused statistically signiﬁcant, but only

minor, reduction in locomotor activity when tested in the

explorative spontaneous motility paradigm (p < 0.001 for all

doses tested). Catalepsy was not evident at 60 and 90 mg/kg (p

> 0.05 for both doses). Consistent with its in vitro

pharmacological proﬁle (characterized by a marked D₃R

preference exhibiting a D₂R/D₃R aﬃnity ratio >1666) 5bb

did not reduce, but rather moderately increased, AMP-induced

hyperactivity at 10 mg/kg after oral administration. This data is

in agreement with data reporting that preferential D₃R

antagonists fail to block agonist-induced hyperactivity at D₃R-

selective doses³¹but, rather, the D₃R-preferring antagonists

stimulate locomotor behavior.^26,32This suggests that post-

synaptic D₃R exert inhibitory actions on psychomotor

functions.³³Accordingly, after oral administration, 5bb is able

to exert eﬀects in animal models predicting antipsychotic

eﬃcacy (MK801-induced hyperactivity) with only marginal

eﬀects on spontaneous exploratory locomotory activity.

Furthermore, we did not observe cataleptogenic potential up

to the highest dose tested (90 mg/kg) and no reductions in

AMP hyperactivity, supporting a D₃R preference. These in vivo

experimental data further point out the unique and promising

proﬁle of 5bb as a potential antipsychotic agent. Diﬀerently

from aripiprazole (3), 5bb lacks signiﬁcant aﬃnity for D₂R and

further validates our pharmacological strategy to treat neuro-

psychiatric disorders. The intriguing proﬁle that emerged for

5bb prompted us to further test this analogue both in vitro and

in vivo.

c

5-HT_1AR expressed in CHO cells.; Rat aortic ring by measuring

contraction response relative to 3 μM 5-HT. 5bb was tested at a

d

single concentration on three diﬀerent cells and induced an inhibition

of 10% at 1 μM, 24% at 3 μM, and 47% at 10 μM.

reduction of blood pressure in anaesthetized rats after 10 mg/

kg intravenous (iv) and a corresponding minor increase in heart

rate (data not shown).

Because our original multireceptor aﬃnity proﬁle approach¹⁵

combines antagonism at dopamine D₃R and at 5-HT_2AR and

partial agonism at 5-HT_1AR to a low aﬃnity for dopamine D₂R

and 5-HT_2CR, we assessed the vitro functional activity for 5bb

at dopamine and serotonin receptors (MDS Pharma Services)

(Table 5). Furthermore, we investigated the capability of 5bb

to modulate ether-a-go-go realted gene (hERG) potassium

channels. [³⁵S]GTPγS binding at D₃R was performed in

membranes prepared from CHO-K1 cells expressing human

D₃R and the concentration-dependent increase in [³⁵S]GTPγS

binding produced by the agonist dopamine was suppressed by

5bb with an IC₅₀value of 0.258 μM (21% of agonist response

and 69% of antagonist response) (Table 5). A similar pattern of

data was acquired for [³⁵S]GTPγS binding at 5-HT_1AR

expressed in CHO cells and stimulated by 5-HT, where 5bb

behaved as a partial agonist (with 66% of agonist response and

19% of antagonist response). The measured EC₅₀value at 5-

HT_1AR was 1.8 μM (Table 5). 5-HT_2AR intrinsic activity

assessment was performed on a rat aortic ring by measuring

contraction response relative to 5-HT. 5bb behaved as a full

antagonist (94% of antagonist response) (the measured IC₅₀

value at 5-HT_2AR was of 2.6 μM, Table 5).

We also established the propensity of 5bb to block the

human hERG potassium channels in whole-cell based electro-

physiological assays (test was performed as previously

described¹⁵). Accordingly, 5bb was tested at a single

concentration on three diﬀerent cells and induced an inhibition

of 10% at 1 μM, 24% at 3 μM, and 47% at 10 μM, which

indicates an IC₅₀value close to 10 μM (Table 5), a result from

which we may expect a low incidence of cardiac eﬀects (such as

torsade des pointes). Molecular modeling on hERG is given as

Supporting Information (Figures 3SI−5SI). Investigation of the

hERG channel blockade is a signiﬁcant step along the drug

discovery trajectory of the pharmaceutical industries. Cardio-

toxicity is among the leading causes for drug attrition and is

therefore an essential subject in nonclinical and clinical safety

testing of new antipsychotic drugs. Drug-induced block of

hERG channels is commonly associated with prolongation of

the electrocardiographic QT interval, resulting in long QT

syndrome and an increased risk of cardiac arrhythmia in the

form of torsades de pointes.³⁴Consequently, eﬀorts to predict

Overall, the best performing analogues in vivo 5j,k,l and 5bb

were those characterized by improved cLogS while maintaining

a favorable cLogP. Among these derivatives, bearing quinoline

ring systems, still primarily hydrophobic in nature, the best

compromise was obtained with 5bb characterized by a

quinolin-6-carboxamide at the “head” and an unsubstituted

phenylpiperazine at the “tail”.

Further in Vitro Receptor Binding Proﬁle, Functional

Activity, and hERG Aﬃnity of 5bb. The in vitro proﬁle of

5bb was further explored with respect to adrenoceptors and

histamine receptors. Data were obtained at MDS Pharma

Services, and obtained K_ivalues are means of two independent

determinations; 5bb was tested in ﬁve diﬀerent concentrations

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a

Table 6. Eﬀects of 5bb on HR, RR, PQ, QRS, QT, and QTc in Langendorﬀ Perfused Rat Hearts

5bb (μM)

HR (BPM)

RR (ms)

PQ (ms)

QRS (ms)

QT (ms)

QTc (ms)

none

0.01

0.1

1

296.25 13.61

302.20 12.22

308.58 10.64

297.70 15.74

280.67 20.55

203.61 9.17

199.43 7.38

194.96 6.46

204.53 11.10

218.07 14.74

41.98 2.55

41.95 2.34

42.32 1.82

41.51 2.18

44.51 1.67

12.33 0.29

13.17 0.28

12.70 0.27

13.38 0.35

14.24 0.36

71.61 4.21

70.61 3.34

71.40 3.85

71.03 4.50

73.96 3.91

159.41 11.21

158.61 8.33

162.06 9.05

158.14 11.79

159.76 10.69

10

a

Each value represents mean

SEM (n = 5). HR, frequency; RR, cycle length; PQ, atrioventricular conduction time; QRS, intraventricular

conduction time; QT, duration of ventricular depolarization and repolarization, i.e., the action potential duration; QTc, corrected QT.

Figure 2. Eﬀect after an ip injection of 5bb on c-Fos in the nucleus accumbens shell and core subregions and in the dorsolateral part of the rostral

striatum. The results are expressed as means SEM of the number of c-Fos-immunoreactive cells/mm²in all regions. *P < 0.05, **P < 0.01 when

compared to respective vehicle-treated group.

long QT syndrome risk have been focused on assaying hERG

channel activities. To conﬁrm cardiac safety, the eﬀect of

compound 5bb was also assessed on cardiac mechanical

function and through electrocardiogram measurement (ECG)

in Langendorﬀ perfused rat hearts as previously described.³⁵

Under control conditions, left ventricle pressure (LVP) and

coronary perfusion pressure (CPP) values of 59.58 7.37 and

51.52 2.40 mmHg (n = 5), respectively, were obtained. At

concentrations up to 10 μM, 5bb did not aﬀect both LVP and

CPP (data not shown) as well as surface ECG (Table 6). Sinus

arrhythmia occurred, in the presence of 5bb, in 1 out of 5

hearts. In summary, 5bb did not aﬀect cardiac parameters up to

10 μM concentrations.

Mesocorticolimbic Selectivity and Atypical Antipsy-

chotic Proﬁle of 5bb: c-Fos Induction after Acute

Administration of 5bb. Immunochemical detection of the

Fos protein is useful in mapping single cells and circuits in the

CNS activated by various drugs. Increased number of c-Fos

neurons in the nucleus accumbens and not in the dorsal striatal

complex has been considered useful in discriminating between

atypical antipsychotics and typical neuroleptics with and

without motor side eﬀects. In fact, like other atypical

antipsychotics such as 1, which markedly enhanced the

expression of c-Fos in the nucleus accumbens,^36,375bb

produced, 1 h after its administration,¹⁵a dose-dependent

and site-selective induction of c-Fos in the nucleus accumbens

(Figure 2). The most marked eﬀect was found in the nucleus

accumbens shell region (ACCshell), where 5bb induced a

signiﬁcant eﬀect (P < 0.05) at 10 mg/kg and a marked eﬀect at

30 mg/kg (a modest eﬀect was found at 3 mg/kg). The nucleus

accumbens core region (ACCcore) was less sensitive because

the 10 mg/kg only induced a moderate but not signiﬁcant

eﬀect. On the contrary, a marked eﬀect was observed at 30

mg/kg (P < 0.05). In the motor related dorsolateral striatum

(DLstriatum) 5bb enhanced c-Fos induction but only at the

high dose of 30 mg/kg. Thus, 5bb enhanced c-Fos in the

DLstriatum at a signiﬁcant higher dose than that necessary for

inducing the expression in the ACCshell. The ACCshell region

belongs to the mesolimbic dopamine system, a key structure in

the neuronal network involved in schizophrenia. In contrast,

typical antipsychotic drugs like haloperidol all induce a very

strong increase in expression of c-Fos in the DLstriatum, which

is a brain region implicated in the control of motor function. It

is interesting to consider that 8-OH-DPAT, a 5-HT_1AR agonist,

can convert the “typical” pattern of haloperidol on c-fos

expression into a pattern resembling that of 1.³⁸It remains

therefore open to speculation whether the proﬁle of c-Fos

protein expression mediated by 5bb might be linked to partial

agonist properties at 5-HT_1AR or to its D₃R antagonism. These

data are in agreement with the negligible catalaptogenic

potential of 5bb.

Further Behavioral Studies on 5bb. After the positive

results obtained from the side eﬀect liability of 5bb (lack of

cardiac toxicity and low liability to induce EPS side eﬀects), the

proﬁle of the compound was further explored in vivo in models

for predicting its propensity to generate memory impairment

(passive avoidance test), for further testing its antipsychotic

potential (prepulse inhibition (PPI) test), for treating anxiety

(marble burying test), and the potential for ameliorating

cognitive impairment (microdyalisis studies). Neurocognition

deﬁcits are poorly treated by the conventional antipsychotics.¹⁴

Passive Avoidance. For evaluating if 5bb has the potential

to generate memory impairment in mice at the doses active in

reverting the MK801-induced hyperactivity, we performed the

passive avoidance test; for comparison, the reference

antipsychotic clozapine (1) was run under the same

experimental conditions. The passive avoidance test is a fear-

motivated test classically used to assess short-term or long-term

memory.³⁹

The latency to enter the dark compartment measured on the

training day for the animals treated with 5bb (Figure 3A) was

dose-relatedly increased with 60 mg/kg, reaching statistical

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modulation of the eﬃcacy on the reversal of PPI model.²⁵

Accordingly, 5bb being characterized by a multireceptor aﬃnity

proﬁle, we decided to investigate its performance in this

behavioral test. The test was performed as described.⁴²First,

the ability of 5bb to counteract impairment induced by PCP

was evaluated. As shown in Figure 4A, oral administration of 3

mg/kg of 5bb was able to ameliorate the PCP-induced

disruption of PPI (p < 0.05 as compared to PCP); 30 mg/kg

just failed to reach statistical signiﬁcance, most likely due to

enhanced variability in this group (p = 0.06 vs PCP). There was

no eﬀect at 3 and 30 mg/kg of 5bb on the startle reﬂex,

indicating lack of unspeciﬁc eﬀects at this dose range (Figure

4B). Furthermore, to investigate if 5bb was able to increase

PPI, the compound was dosed orally 30 min prior testing

without any pretreatment with PCP. As shown in Figure 4C,

5bb was able to increase PPI only at 30 mg/kg (p < 0.05

compared to vehicle). However, this dose (30 mg/kg) also

signiﬁcantly reduced the startle response, pointing to sedative

eﬀects at this dose when the mice were not pretreated with

PCP (p < 0.05 vs vehicle). The reason why this dose did not

aﬀect the startle response per se, when the animals were

pretreated with PCP (Figure 5B), could be that the stimulant

eﬀect of PCP may counteract the potential mild sedative eﬀects

of 30 mg/kg of 5bb. Together with the eﬀects on MK801-

induced hyperacticity, these data further underline the

antipsychotic proﬁle of 5bb.

Marble Burying. To evaluate the possible anxiolytic

properties of 5bb, the compound was tested in the marble

burying test, in comparison with chlordiazepoxide hydro-

chloride (CDP), as a standard anxiolytic reference. The marble

burying test is an unconditioned model of anxiety in mice in

which animals bury harmless novel objects such as glass

marbles placed on top of sawdust in an observation cage. The

marble burying test allows the evaluation of the potential

eﬃcacy of drugs for treatments of anxiety and or obsessive

compulsive disorders.⁴³Recent ﬁndings suggest that the 5-

HT_1AR is involved in the marble-burying behavior.⁴⁴In fact, the

atypical antipsychotic 3 was found to inhibit the marble-burying

behavior in a 5-HT_1AR-dependent fashion.⁴⁵

In the marble burying test, treatment with 5bb dose-

dependently reduced the number of marbles that the mice

buried, with 30 mg/kg reaching statistical signiﬁcance as

compared to vehicle treatment (p < 0.05) (Figure 5). The eﬀect

of this dose was in the same range as the eﬀect of the positive

control, CDP. As marble burying depends on the animal’s

ability to attend to and bury a number of marbles, the read out

can be confounded by compounds having sedative or motor

impairing eﬀects. The dose of CDP used in this study (10 mg/

kg, intraperitoneally) has repeatedly been shown in our

laboratory not to aﬀect spontaneous locomotor activity (data

now shown). However, 30 mg/kg of 5bb had signigicant, but

minor, eﬀects on spontaneous locomotor activity. This dose

also signiﬁcantly reduced the startle reﬂex (Figure 4D).

Consequently, it cannot be ruled out that mild sedative eﬀects

can contribute to the positive ﬁndings observed in this study.

Microdialysis Studies. A cardinal feature of schizophrenia is

cognitive impairment which is poorly treated by conventional

antipsychotics. The apparent eﬃcacy of the newer antipsy-

chotics (e.g., ziprasidone)⁴⁶in the treatment of cognitive

impairment may be due to a proﬁle based on D₂R and 5-HT_2AR

antagonism associated with 5-HT_1AR (partial) agonism.⁴⁷This

proﬁle, especially with regards to 5HT_1AR partial agonism, may

explain the increased release of dopamine in the PFC, which

Figure 3. Eﬀect of 5bb and atypical antipsychotic 1 on acquisition (A

and C, respectively) and retention (B and D, respectively) of passive

avoidance in mice. Oral administration of 5bb (A) or subcutaneous

administration of atypical antipsychotic 1 (C) 30 min prior to test start

resulted in dose-related increases in latency to enter the dark chamber,

reaching statistical signiﬁcance at 60 mg/kg (p < 0.05) and 3 mg/kg (p

< 0.05), respectively. Retention of information acquired during the

acquisition trial was evaluated by measuring latency to enter the dark

chamber 24 h after the acquisition trial. Oral administration of 5bb 24

h earlier resulted in a signiﬁcant decrease in latency to enter the dark

compartment at 60 mg/kg (p < 0.05) (B). Likewise, subcutaneous

administration of atypical antipsychotic 1 24 h earlier decreased the

latency to enter the dark compartment, reaching statistical signiﬁcance

at 10 mg/kg (p < 0.05). Results are expressed as means SEM latency

to enter the dark compartment during acquisition and retention

separated by 24 h. Statistical evaluation was performed by one-way

ANOVA on ranks followed by Dunn’s test for multiple comparisons.

signiﬁcance as compared to vehicle administration (p < 0.05),

indicating sedative eﬀects at this dose. When tested, drug free,

24 h later for retention of memory (Figure 3B), there was a

dose-related decrease in latency to enter the dark compartment,

again with 60 mg/kg reaching statistical signiﬁcance (p < 0.05).

This indicates that the doses conveying eﬀects against MK801

hyperactivity are not associated with liability for cognitive

impairment, and only doses 6-fold higher (60 mg/kg) impairs

the ability to acquire and/or retain memory for an adverse

event. These data are in line with the eﬀects observed with the

atypical antipsychotic 1 (Figure 3C,D), where impairments in

passive avoidance was evident at 10 mg/kg while reductions in

PCP induced hyperactivity was reached at 1 mg/kg (data not

shown).

Prepulse Inhibition (PPI). The PPI is deﬁned as the

attenuation of the acoustic startle reﬂex that occurs when the

eliciting stimulus is preceded by a weak, nonstartling

prestimulus. The PPI model of the acoustic startle reﬂex was

developed in rodents for mimicking the impairment in sensory

motor gating, which is frequently associated with several

neuropsychiatric disorders such as schizophrenia.⁴⁰In rats and

mice, deﬁcits in PPI can be induced by CNS stimulating drugs

such as amphetamine or PCP. Although D₃R have been

reported to play a pivotal role in PPI,⁴¹there is evidence that

D₃R antagonism alone does not reverse quinpirole or

apomorphine induced PPI in rats. It has been also raised the

hypothesis that 5-HT_1AR involvement could be relevant for the

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Figure 4. Eﬀects of 5bb on prepulse inhibition in mice. Subcutaneous administration of PCP (4 mg/kg, 10 min prior to test start) signiﬁcantly

impaired PPI (p < 0.01 compared to vehicle) (A). Oral administration of 5bb signiﬁcantly ameliorated the PCP-induced disruption of PPI in mice (p

< 0.05 vs PCP); 30 m/kg tended to counteract the PCP induced deﬁcit but did not reach statistical signiﬁcance (p = 0.06). There was no eﬀect of 3

and 30 mg/kg on startle response per se (B). Administering 5bb alone, without prior pretreatment with PCP, enhanced PPI as shown by a signiﬁcant

eﬀect of 30 mg/kg compared to vehicle treatment (p < 0.05) (D). This dose also signiﬁcantly reduced the startle reﬂex as compared to vehicle

treatment (p < 0.05). Results are expressed as means SEM of percentage of PPI/startle response. Statistical evaluation was performed by two-way

RM ANOVA (percentage of PPI, prepulse intensity and drug treatment as factors) or one-way ANOVA (startle response) followed by Fishers LSD

test for multiple comparisons; n = 9−10 per treatment group.

Figure 6. Eﬀect of 5bb (10 mg/kg, sc, n = 6) on dopamine eﬄux in

prefrontal cortex (open circles) and in nucleus accumbens (closed

circles). Extracellular concentration of dopamine is expressed as

percentage of the basal level.

release in the medial PFC reported as % with respect to basal

Figure 5. Eﬀects of 5bb on marble burying in mice. Oral pretreatment

dopamine eﬄux with an eﬀect which lasted for 90 min and with

30 min before test start with 5bb in increasing doses reduced the

a maximum eﬄux measured after 90 min. Notably, 5bb did not

number of buried marbles as compared to vehicle treatment, with 30

induce an enhancement above the basal level of dopamine

eﬄux measured during the time interval 0−90 min in the

nucleus accumbens.

mg/kg reaching statistical signiﬁcance (p < 0.05 vs vehicle). The

positive control, chlordiazepoxide (10 mg/kg intraperitoneally, 30 min

before test start), also resulted in signiﬁcantly reduced number of

buried marbles, conﬁrming the validity of the test (p < 0.05). Results

are expressed as means SEM of number of buried marbles. Statistical

evaluation was performed by one way ANOVA followed by Dunnett’s

test for multiple comparisons; n = 7 per treatment group.

CONCLUSIONS

■

In summary, we have herein described a further exploitation of

the SAR of arylpiperazine-based antipsychotics. D₃R potent

antagonism combined to 5-HT_1AR partial agonism and 5-

HT_2AR antagonism was recently identiﬁed by us as a novel

paradigm for the development of innovative antipsychotics

devoid of D₂R mediated side eﬀects. Low 5-HT_2CR aﬃnity and

reduced propensity to block hERG channels are additional and

pivotal features of the required multireceptor aﬃnity proﬁle.

On the basis of our previously identiﬁed hit 4, we have

may be beneﬁcial for improving cognition, because decreased

levels of dopamine in the PFC remain an issue of the cognitive

impairment in schizophrenia.⁴⁷For analyzing this problem, we

performed in vivo microdialysis studies in Wistar rats (Figure

6), and our data showed that administration of a single dose of

5bb (10 mg/kg sc) induced a sensible increment of dopamine

K

dx.doi.org/10.1021/jm501119j | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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3H), 7.02 (br s, 1H), 7.28 (m, 3H), 7.39 (m, 1H), 7.46 (m, 2H), 7.66

(m, 1H). ESI-MS m/z 400 [M + Na]⁺, 378 (100) [M + H]⁺. Anal.

(C₂₃H₂₇N₃O₂) C, H, N.

developed an initial set of 38 compounds which allowed the

selection of 12 arylpiperazines for in vivo studies. Among these

compounds, 5a,j−in l,bb proved to be eﬃcacious in behavioral

rodent models without or with only minor propensity for

inducing sedation. Among these latter, compound 5bb, which

showed a unique in vitro pharmacological proﬁle for the

selected receptors of interest, exhibited a signiﬁcant eﬃcacy,

after oral administration, in reverting the MK801-induced

hyperactivity accompanied by minor eﬀects on spontaneous

locomotor activity and no propensity to induce catalepsy.

Accordingly, 5bb was chosen for further in vitro and in vivo

characterization. Immunohistochemical data of c-fos expression

in mesocorticolimbic areas further conﬁrmed the atypical

antipsychotic proﬁle of 5bb. This compound showed a

promising therapeutic window when we compared the doses

eﬀective in behavioral tests for predicting antipsychotic

potential (MK801-induced hyperactivity and PCP−PPI) to

those eﬀective in tests for predicting side eﬀects (e.g., catalepsy

and passive avoidance tests). As a further feature, 5bb may have

beneﬁcial eﬀects against anxiety symptoms (marble burying)

and, inducing a signiﬁcant increase of dopamine eﬄux in the

PFC, may be endowed with procognitive eﬀects. To the best of

our knowledge, 5bb is the ﬁrst compound which combines

dopamine D₃R antagonism with 5-HT_1AR partial agonism and

5-HT_2AR antagonism which proved its eﬃcacy in various

rodent models of schizophrenia. Accordingly, 5bb represents an

excellent lead for further preclinical optimization strategy,

paving the way to the identiﬁcation of an industrial candidate.

N-(4-(4-(2,3-Dimethylphenyl)piperazin-1-yl)butyl)benzo[b]furan-

2-carboxamide (5b). Starting from 8a and 4-(2,3-dimethylphenyl)-

piperazine and following the procedure described for 5a, the title

compound was obtained as white solid (73% yield); mp (MeOH)

1

151−152 °C. H NMR 200 MHz (CDCl₃) δ 1.73 (m, 4H), 2.21 (s,

3H), 2.25 (s, 3H), 2.47 (m, 2H), 2.62 (m, 4H), 2.93 (m, 4H), 3.52 (m,

2H), 6.87 (m, 2H), 7.05 (m, 2H), 7.35 (m, 3H), 7.64 (m, 1H). ESI-

MS m/z 428 [M + Na]⁺, 406 [M + H]⁺. Anal. (C₂₅H₃₁N₃O₂) C, H, N.

N-(4-(4-(3-Methoxyphenyl)piperazin-1-yl)butyl)benzo[b]furan-2-

carboxamide (5c). Starting from 8a and 4-(3-methoxylphenyl)-

piperazine dihydrochloride and following the procedure described

for 5a, the title compound was obtained as white solid (70% yield);

1

mp (MeOH) 104−105 °C. H NMR 200 MHz (CDCl₃) δ 1.62 (m,

4H), 2.40 (m, 2H), 2.56 (m, 4H), 3.19 (m, 4H), 3.49 (m, 2H), 3.74 (s,

3H), 6.44 (m, 3H), 7.25 (m, 6H), 7.61 (m, 1H). ESI-MS m/z 430 [M

+ Na]⁺, 408 (100) [M + H]⁺. Anal. (C₂₄H₂₉N₃O₃) C, H, N.

N-(4-(4-(6-Methylpyridin-2-yl)piperazin-1-yl)butyl)benzofuran-2-

carboxamide (5d). Starting from 8a and arylpiperazine 10a and

following the procedure described for 5a, the title compound was

1

obtained as white solid (75% yield); mp (MeOH) 107−109 °C. H

NMR 200 MHz (CDCl₃) δ 1.70 (m, 4H), 2.39 (s, 3H), 2.45 (m, 2H),

2.58 (m, 4H), 3.54 (m, 6H), 6.45 (m, 2H), 7.02 (br s, 1H), 7.35 (m,

5H), 7.65 (m, 1H). ESI-MS m/z 415 [M + Na]⁺, 393 [M + H]⁺(100).

Anal. (C₂₃H₂₈N₄O₂) C, H, N.

N-(4-(4-Benzylpiperazin-1-yl)butyl)benzofuran-2-carboxamide

(5e). Starting from 8a and 1-benzylpiperazine and following the

procedure described for 5a, the title compound was obtained as yellow

oil (70% yield). ¹H NMR 200 MHz (CDCl₃) δ 1.64 (m, 4H), 2.39 (m,

2H), 2.50 (m, 8H), 3.49 (m, 4H), 7.04 (br s, 1H), 7.36 (m, 9H), 7.65

(m, 1H). ESI-MS m/z 414 [M + Na]⁺, 392 (100) [M + H]⁺. Anal.

(C₂₄H₂₉N₃O₂) C, H, N.

N-(4-(4-(2-Fluorophenyl)piperazin-1-yl)butyl)benzofuran-2-car-

boxamide (5f). Starting from 8a and 1-(2-ﬂuorophenyl)piperazine and

following the procedure described for 5a, the title compound was

obtained as white crystalline solid (70% yield); mp (MeOH) 104−105

°C. ¹H NMR 300 MHz (CDCl₃) δ 1.62 (m, 4H), 2.37 (t, 2H, J = 6.8

Hz), 2.56 (m, 4H), 3.06 (m, 4H), 3.48 (m, 2H), 6.91 (m, 4H), 7.30

(m, 4H), 7.57 (d, 1H, J = 7.5 Hz). ¹³C NMR 75 MHz (CDCl₃) δ 24.5,

27.7, 39.5, 50.6, 53.4, 58.1, 110.4, 111.9, 116.2 (J_C−F= 81.1 Hz), 119.1

EXPERIMENTAL SECTION

■

General Procedures. Unless otherwise speciﬁed, materials were

purchased from commercial suppliers and used without further

puriﬁcation. Reaction progress was monitored by TLC using silica

gel 60 F254 (0.040−0.063 mm) with detection by UV. Silica gel 60

(0.040−0.063 mm) or aluminum oxide 90 (0.063.0.200 mm) were

1

used for column chromatography. H NMR and ¹³C NMR spectra

were recorded on a Varian 300 MHz, Bruker 200 MHz, or Bruker 400

MHz spectrometer by using the residual signal of the deuterated

solvent as internal standard. Splitting patterns are described as singlet

(s), doublet (d), triplet (t), quartet (q), quintet (p), and broad (br);

the value of chemical shifts (δ) are given in ppm and coupling

constants (J) in hertz (Hz). Number of overlapping carbon signals are

reported in brackets (equivalent carbon atoms are always counted

once). ESI-MS spectra and analytical HPLC experiments were

performed by an Agilent 1100 series LC/MSD spectrometer. Melting

points were determined in Pyrex capillary tubes using an Electro-

thermal 8103 apparatus and are uncorrected. Optical rotation values

were measured at room temperature using a PerkinElmer model 343

polarimeter (operating at λ = 589 nm, corresponding to the sodium D

line). Yields refer to puriﬁed products and are not optimized. All

moisture-sensitive reactions were performed under argon atmosphere

using oven-dried glassware and anhydrous solvents. Elemental analyses

were performed in a PerkinElmer 240C elemental analyzer, and the

results were within 0.4% of the theoretical values unless otherwise

noted.

N-(4-(4-Phenylpiperazin-1-yl)butyl)benzo[b]furan-2-carboxa-

mide (5a). To a stirred solution of 8a (150.0 mg, 0.51 mmol) in dry

MeCN (20.0 mL) under argon, 1-phenylpiperazine (82.6 mg, 0.51

mmol) and TEA (156.6 μL, 1.11 mmol) were added; the solution was

reﬂuxed for 12 h under stirring. The solvent was removed under

reduced pressure, water was added, and the mixture was extracted with

DCM (3 × 30 mL). The organic layers were dried and concentrated,

and the crude product was chromatographed (10% MeOH in

chloroform) to give title compound as white solid (70% yield); mp

(MeOH) 149−150 °C. ¹H NMR 300 MHz (CDCl₃) δ 1.71 (m, 4H),

2.47 (m, 2H), 2.64 (m, 4H), 3.24 (m, 4H), 3.53 (m, 2H), 6.89 (m,

(J_C−F= 13.0 Hz), 122.5 (J_C−F= 33.0 Hz), 122.9, 123.9, 124.7 (J_C−F

13.0 Hz), 127.0, 127.8, 140.2 (J_C−F= 33.0 Hz), 149.3, 155.5 (J_C−F

=

193.5 Hz), 157.5, 159.2. ESI-MS m/z 396 [M + H]⁺. Anal.

(C₂₃H₂₆FN₃O₂) C, H, N.

N-(4-(4-(3-Triﬂuoromethylphenyl)piperazin-1-yl)butyl)indole-2-

carboxamide (5g). Starting from 8b and (3-triﬂuoromethyl)-

phenylpiperazine and following the procedure described for 5a, the

1

title compound was obtained as yellow oil (56% yield). H NMR 300

MHz (CDCl₃) δ 1.82 (m, 4H), 2.43 (m, 2H), 2.57 (m, 4H), 3.21 (m,

4H), 3.54 (m, 2H), 6.58 (m, 1H), 6.83 (s, 1H), 7.11 (m, 3H), 7.31 (m,

2H), 7.45 (d, 1H, J = 8.0 Hz), 7.61 (d, 1H, J = 7.9 Hz), 10.08 (br s,

1H). ESI-MS m/z 445 [M + H]⁺. Anal. (C₂₄H₂₇F₃N₄O) C, H, N.

N-(4-(4-(3-Methoxyphenyl)piperazin-1-yl)butyl)-1H-indole-3-car-

boxamide (5h). 1H-Indole-3-carbonyl chloride (15): To a solution of

1H-indole-3-carboxylic acid (100.0 mg, 0.60 mmol) in dry benzene

(2.0 mL), thionyl chloride (130.0 μL, 1.80 mmol) was added and the

mixture was reﬂuxed for 2 h. The crude was washed with benzene (2 ×

10 mL) and evaporated to give title compound (99% yield), which was

1

used in the following step without any further puriﬁcation. H NMR

200 MHz (CDCl₃) δ 7.03 (m, 1H), 7.19 (m, 1H), 7.30 (m, 1H), 7.48

(m, 1H), 8.20 (m, 1H), 10.85 (br s, 1H).

To a stirred solution of 14 (158.0 mg, 0.60 mmol) and 15 (100.0

mg, 0.60 mmol) in dry DCM (15.0 mL), pyridine (145 μL, 1.80

mmol) was added. The mixture was stirred at rt for 12 h. Then sodium

bicarbonate saturated solution was added, and the mixture was

extracted with EtOAc (3 × 15 mL), dried, and evaporated. The crude

product was puriﬁed by means of ﬂash chromatography (10% MeOH

in chloroform) to give pure title compound as a white solid (50%

L

dx.doi.org/10.1021/jm501119j | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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yield); mp (MeOH) 154−155 °C. ¹H NMR 200 MHz (CDCl₃) δ 1.65

(m, 4H), 2.41 (m, 2H), 2.56 (m, 4H), 3.13 (m, 4H), 3.50 (m, 2H),

3.77 (s, 3H), 6.45 (m, 2H), 7.19 (m, 3H), 7.38 (m, 1H), 7.66 (m, 1H),

7.95 (m, 1H), 9.79 (br s, 1H). ESI-MS m/z 429 [M + Na]⁺, 407 (100)

[M + H]⁺. Anal. (C₂₄H₃₀N₄O₂) C, H, N.

2H), 7.92 (m, 2H), 8.23 (d, 2H, J = 4.6 Hz), 8.31 (m, 1H), 8.55 (s,

1H), 9.08 (s, 1H). ESI-MS m/z 803 [2M + Na]⁺, 413 (100) [M +

Na]⁺, 391 [M + H]⁺. Anal. (C₂₂H₂₆N₆O) C, H, N.

N-(4-(4-(Quinolin-3-yl)piperazin-1-yl)butyl)isoquinoline-3-car-

boxamide (5p). Starting from 8d and arylpiperazine 10c and following

the procedure described for 5a, the title compound was obtained as

N-(4-(4-(3-Methoxyphenyl)piperazin-1-yl)butyl)-1-methyl-1H-in-

dole-2-carboxamide (5i). Starting from 8c and 4-(3-methoxylphenyl)-

piperazine and following the procedure described for 5a, the title

compound was obtained as white solid (60% yield); mp (MeOH)

1

white solid (70% yield); mp (MeOH) 153−154 °C. H NMR 200

MHz (CDCl₃) δ 1.80 (m, 4H), 2.37 (m, 2H), 2.67 (m, 4H), 3.32 (m,

4H), 3.75 (m, 2H), 7.30 (m, 1H), 7.45 (m, 2H), 7.67 (m, 4H), 7.93

(m, 2H), 8.33 (br t, 1H), 8.60 (m, 1H), 8.78 (m, 1H), 9.12 (m, 1H).

ESI-MS m/z 462 [M + Na]⁺, 440 (100) [M + H]⁺. Anal.

(C₂₇H₂₉N₅O) C, H, N.

1

141−142 °C. H NMR 200 MHz (CDCl₃) δ 1.69 (m, 4H), 2.48 (m,

2H), 2.65 (m, 4H), 3.08 (m, 4H), 3.47 (m, 2H), 3.84 (s, 3H), 4.03 (s,

3H), 6.90 (m, 6H), 7.16(m, 1H), 7.32 (m, 2H), 7.60 (m, 1H). ESI-MS

m/z 421 [M + H]⁺. Anal. (C₂₅H₃₂N₄O₂) C, H, N.

N-(4-(4-(3-Fluorophenyl)piperazin-1-yl)butyl)isoquinoline-3-car-

boxamide (5q). Starting from 8d 1-(3-ﬂuorophenyl)piperazine 10b

and following the procedure described for 5a, the title compound was

obtained as colorless solid (76% yield); mp (MeOH) 165−166 °C. ¹H

NMR 300 MHz (CDCl₃) δ 1.71 (m, 4H), 2.44 (t, 2H, J = 7.2 Hz),

2.58 (t, 4H, J = 5.1 Hz), 3.19 (t, 4H, J = 4.8 Hz), 3.57 (q, 2H, J = 6.3

Hz), 6.67−6.47 (m, 3H), 7.16 (m, 1H), 7.71 (m, 2H), 7.98 (m, 2H),

8.35 (t, 1H, J = 5.4 Hz), 8.60 (s, 1H), 9.13 (s, 1H). ¹³C NMR 75 MHz

(CDCl₃) δ 24.6, 27.9, 39.6, 48.8 (2), 53.3 (2), 58.3, 102.7 (J_C−F= 24.6

Hz), 105.9 (J_C−F= 21.4 Hz), 111.2 (J_C−F= 2.2 Hz), 120.5, 127.8,

128.3, 129.0, 129.9, 130.2, 130.4, 131.3, 136.2, 144.0, 151.3, 153.1,

153.3, 164.0 (J_C−F= 241.9 Hz), 165.1. ESI-MS m/z 407 [M + H]⁺.

Anal. (C₂₄H₂₇FN₄O) C, H, N.

N-(4-(4-Phenylpiperazin-1-yl)butyl)isoquinoline-3-carboxamide

(5j). Starting from 8d and 1-phenylpiperazine and following the

procedure described for 5a, the title compound was obtained as white

1

solid (76% yield); mp (MeOH) 153−154 °C. H NMR 300 MHz

(CDCl₃) δ 1.71 (m, 4H), 2.46 (m, 2H), 2.62 (m, 4H), 3.21 (m, 4H),

3.57 (m, 2H), 6.88 (m, 3H), 7.26 (m, 2H), 7.72 (m, 2H), 8.00 (m,

2H), 8.36 (br s, 1H), 8.61 (m, 1H), 9.14 (m, 1H). ESI-MS m/z 411

[M + Na]⁺, 389 (100) [M + H]⁺. Anal. (C₂₄H₂₈N₄O) C, H, N.

N-(4-(4-(3-Methylphenyl)piperazin-1-yl)butyl)isoquinoline-3-car-

boxamide (5k). Starting from 8d and m-tolylpiperazine and following

the procedure described for 5a, the title compound was obtained as

yellow solid (48% yield); mp (MeOH) 152−153 °C. ¹H NMR

(CDCl₃) δ 1.68 (m, 4H), 2.30 (s, 3H), 2.46 (t, 2H, J = 6.8 Hz), 2.60

(t, 4H, J = 5.0 Hz), 3.20 (t, 4H, J = 4.9 Hz), 3.58 (q, 2H, J = 6.4 Hz),

6.70 (m, 3H), 7.14 (t, 1H, J = 8.1 Hz), 7.72 (m, 2H), 8.00 (t, 2H, J =

8.0 Hz), 8.33 (br s, 1H), 8.61 (s, 1H), 9.14 (s, 1H). ¹³C NMR 75 MHz

(DMSO-d₆) 21.3, 22.0, 27.1, 39.1, 46.2, 51.1, 55.7, 114.1, 117.5, 121.1,

121.9, 128.7, 129.0, 129.7, 130.2, 133.0, 136.5, 139.0, 143.0, 150.0,

151.7, 164.1, 170.0. ESI-MS m/z 403 [M + H]⁺. Anal. (C₂₅H₃₀N₄O)

C, H, N.

N-(4-(4-(4-Fluorophenyl)piperazin-1-yl)butyl)isoquinoline-3-car-

boxamide (5r). Starting from 8d and 1-(4-ﬂuorophenyl)piperazine

and following the procedure described for 5a, the title compound was

obtained as white crystalline solid (95% yield); mp (MeOH) 155−156

1

°C. H NMR 300 MHz (CDCl₃) δ 1.71 (m, 4H), 2.37 (m, 2H), 2.62

(m, 4H), 3.14 (m, 4H), 3.58 (m, 2H), 7.27 (m, 4H), 7.77 (m, 2H),

8.01 (m, 2H), 8.35 (br s, 1H), 8.62 (s, 1H), 9.14 (s, 1H). ESI-MS m/z

407 [M + H]⁺. Anal. (C₂₄H₂₇FN₄O) C, H, N.

N-(4-(4-(4-Hydroxyphenyl)piperazin-1-yl)butyl)isoquinoline-3-

carboxamide (5s). To a solution of compound 11 (100.0 mg, 0.20

mmol) in dry DCM (10.0 mL), boron tribromide was added dropwise

at −78 °C. The reaction was allowed to warm to rt in a period of 60

min and then stirred for additional 30 min. The reaction mixture was

then quenched by the dropwise addition of sodium carbonate and

extracted with DCM (3 × 10 mL). The organic layer were collected,

dried over sodium sulfate, and concentrated under reduced pressure.

The crude was puriﬁed by means of ﬂash chromatography (10%

MeOH in chloroform) to give the title compound as a white solid

(95% yield); mp (MeOH) 220−221 °C. ¹H NMR 300 MHz (CDCl₃)

δ 1.70 (m, 4H), 2.45 (m, 2H), 2.60 (m, 4H), 3.07 (m, 4H), 3.58 (m,

2H), 6.80 (m, 4H), 7.73 (m, 2H), 8.01 (m, 2H), 8.38 (br t, 1H), 8.61

(s, 1H), 9.14 (s, 1H). ESI-MS m/z 427 [M + Na]⁺, 405 (100) [M +

H]⁺. Anal. (C₂₄H₂₈N₄O₂) C, H, N.

N-(4-(4-Phenylpiperazin-1-yl)butyl)quinoline-2-carboxamide (5t).

Starting from 8e and 1-phenylpiperazine and following the procedure

described for 5a, the title compound was obtained as white solid (70%

yield); mp (MeOH) 120−121 °C. ¹H NMR 200 MHz (CDCl₃) δ 1.69

(m, 4H), 2.46 (m, 2H), 2.61 (m, 4H), 3.20 (m, 4H), 3.56 (m, 2H),

6.86 (m, 3H), 7.24 (m, 2H), 7.59 (m, 1H), 7.75 (m, 1H), 7.85 (m,

1H), 8.09 (m, 1H), 8.29 (m, 2H). ESI-MS m/z 411 [M + Na]⁺, 389

(100) [M + H]⁺. Anal. (C₂₄H₂₈N₄O) C, H, N.

N-[4-[4-(3-Methoxyphenyl)piperazin-1-yl]butyl]isoquinoline-3-

carboxamide (5l). Starting from 8d (1.0 g, 2.39 mmol) and 1-(3-

methoxy)phenylpiperazine and following the procedure described for

1

5a, the title compound was obtained as colorless oil (54% yield). H

NMR (CDCl₃) 300 MHz δ 1.64−1.75 (m, 4H), 2.44 (t, J = 7.2 Hz,

2H), 2.59 (t, J = 4.8 Hz, 4H), 3.20 (t, J = 4.5 Hz, 4H), 3.57 (m, 2H),

3.77 (s, 3H), 6.40 (dd, J₁= 1.8 Hz, J₂= 8.1 Hz, 1H), 6.45 (m, 1H),

6.53 (dd, J₁= 1.8 Hz, J₂= 8.1 Hz, 1H), 7.15 (t, J = 8.1 Hz, 1H), 7.70

(m, 2H), 7.97 (m, 2H), 8.35 (m, 1H), 8.60 (s, 1H), 9.12 (s, 1H). ¹³C

NMR (CDCl₃) 75 MHz 24.6, 27.9, 39.6, 49.2, 53.5, 55.4, 58.4, 102.6,

104.6, 109.0, 120.4, 127.8, 128.3, 129.0, 129.9, 130.0, 131.2, 136.2,

144.0, 151.3, 153.0, 160.8, 165.1. ESI-MS m/z 441 [M + Na]⁺, 419

(100) [M + H]⁺. Anal. (C₂₅H₃₀N₄O₂) C, H, N.

N-(4-(4-(Pyridin-2-yl)piperazin-1-yl)butyl)isoquinoline-3-carboxa-

mide (5m). Starting from 8d and 1-(2-pyridin-2-yl)piperazine

hydrochloride in dry MeCN and following the procedure described

for 5a, the title compound was obtained as white solid (93% yield);

1

mp (MeOH) 108−109 °C. H NMR 200 MHz (CDCl₃) δ 1.62 (m,

4H), 2.38 (m, 2H), 2.49 (m, 4H), 3.52 (m, 6H), 6.54 (m, 2H), 7.40

(m, 1H), 7.65 (m, 2H), 7.91 (t, 2H, J = 8.6 Hz), 8.11 (d, 1H, J = 4.7

Hz), 8.32 (br s, 1H), 8.54 (s, 1H), 9.06 (s, 1H). ESI-MS m/z 412

(100) [M + Na]⁺, 390 [M + H]⁺. Anal. (C₂₃H₂₇N₅O) C, H, N.

N-(4-(4-(6-Methylpyridin-2-yl)piperazin-1-yl)butyl)isoquinoline-3-

carboxamide (5n). Starting from 8d and arylpiperazine 10a and

following the procedure described for 5a, the title compound was

N-(4-(4-(3-Methylphenyl)piperazin-1-yl)butyl)quinoline-2-car-

boxamide (5u). Starting from 8e and m-tolylpiperazine and following

the procedure described for 5a, the title compound was obtained as

1

obtained as white solid (70% yield); mp (MeOH) 124−125 °C. H

1

NMR 200 MHz (CDCl₃) δ 1.71 (m, 4H), 2.38 (s, 3H), 2.47 (m, 2H),

2.56 (m, 4H), 3.55 (m, 6H), 6.44 (m, 2H), 7.30 (m, 1H), 7.72 (m,

2H), 8.00 (m, 2H), 8.33 (br s, 1H), 8.60 (m, 1H), 9.14 (m, 1H). ESI-

MS m/z 426 [M + Na]⁺, 404 (100) [M + H]⁺. Anal. (C₂₄H₂₉N₅O) C,

H, N.

yellow amorphous solid (51% yield). H NMR 200 MHz (CDCl₃) δ

1.68 (m, 4H), 2.31 (s, 3H), 2.48 (m 2H), 2.61 (m, 4H), 3.19 (m, 4H),

3.47 (m, 2H), 6.69 (m, 4H), 7.11 (m, 1H), 7.59 (m, 1H), 7.75 (m,

1H), 7.85 (m, 1H), 8.09 (m, 1H), 8.33 (m, 2H). Anal. (C₂₅H₃₀N₄O)

C, H, N.

N-(4-(4-(Pyrimidin-2-yl)piperazin-1-yl)butyl)isoquinoline-3-car-

boxamide (5o). Starting from 8d and 1-(2-pyrimidyl)piperazine

dihydrochloride in dry MeCN and following the procedure described

N-[4-[4-(3-Methoxyphenyl)piperazin-1-yl]butyl]quinoline-2-car-

boxamide (5v). Starting from 8e and 1-(3-methoxy)phenylpiperazine

and following the procedure described for 5a, the title compound was

1

for 5a, the title compound was obtained as yellow oil (81% yield). H

obtained as white amorphous solid (55% yield). H NMR 200 MHz

NMR 200 MHz (CDCl₃) δ 1.59−1.72 (m, 4H), 2.34−2.47 (m, 6H),

3.52 (m, 2H), 3.79 (m, 4H), 6.39 (t, 1H, J = 4.6 Hz), 7.57−7.71 (m,

(CDCl₃) δ 1.63 (m, 4H), 2.41 (m, 2H), 2.56 (m, 4H), 3.15 (m, 4H),

3.54 (m, 2H), 3.73 (s, 3H), 6.43 (m, 4H), 7.13 (m, 1H), 7.55 (m, 1H),

M

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7.70 (m, 1H), 7.81 (m, 1H), 8.06 (m, 1H), 8.26, (m, 1H), 8.31 (m,

1H). ESI-MS m/z 441 [M + Na]⁺, 419 (100) [M + H]⁺. Anal.

(C₂₅H₃₀N₄O₂) C, H, N.

ESI-MS m/z 391 [M + Na]⁺, 369 (100) [M + H]⁺. Anal.

(C₂₁H₂₈N₄O₂) C, H, N.

N-(4-(4-(Naphthalen-1-yl)piperazin-1-yl)butyl)benzamide (5ee).

Starting from 8g and arylpiperazine 10f and following the procedure

described for 5a, the title compound was obtained as yellow oil (70%

N-(4-(4-(Pyridin-2-yl)piperazin-1-yl)butyl)quinoline-2-carboxa-

mide (5w). Starting from 8e and 1-(2-pyridin-2-yl)piperazine

hydrochloride and following the procedure described for 5a, the title

1

yield). H NMR 200 MHz (CDCl₃) δ 1.68 (m, 4H), 2.49 (m, 4H),

1

compound was obtained as white amorphous solid (64% yield). H

2.70 (m, 4H), 3.34 (m, 4H), 7.01 (m, 2H), 7.43 (m, 7H), 7.78 (m,

3H), 8.18 (m, 1H). ESI-MS m/z 410 [M + Na]⁺, 388 (100) [M + H]⁺.

Anal. (C₂₅H₂₉N₃O) C, H, N.

NMR 200 MHz (CDCl₃) δ 1.82 (m, 4H), 2.39 (m, 2H), 2.50 (m,

4H), 3.52 (m, 6H), 6.55 (m, 2H), 7.40 (m, 1H), 7.53 (m, 1H), 7.69,

(m, 1H), 7.86 (m, 1H), 8.17 (m, 5H). ESI-MS m/z 412 [M + Na]⁺,

390 (100) [M + H]⁺. Anal. (C₂₃H₂₇N₅O) C, H, N.

N-(4-(4-(Quinolin-3-yl)piperazin-1-yl)butyl)benzamide (5ﬀ). Start-

ing from 8g and arylpiperazine 10c and following the procedure

described for 5a, the title compound was obtained as white solid (70%

yield); mp (MeOH) 124−125 °C. ¹H NMR 200 MHz (CDCl₃) δ 1.68

(m, 4H), 2.45 (m, 2H), 2.65 (m, 4H), 3.27 (m, 4H), 3.53 (m, 2H),

6.63 (br s, 1H), 7.38 (m, 6H), 7.65 (m, 1H), 7.75 (m, 2H), 7.97 (m,

1H), 8.76 (m, 1H). ESI-MS m/z 389 [M + H]⁺. Anal. (C₂₄H₂₈N₄O)

C, H, N.

(R)-(+)-N-[4-(4-Phenylpiperazin-1-yl)butyl]-1,2,3,4-tetrahydroqui-

noline-2-carboxamide (R)-(5x). To a solution of (R)-22a (50.0 mg,

0.15 mmol) in MeOH and EtOAc (1:1), catalytic Pd on carbon 5%

was added under argon and the suspension was hydrogenated at 60 psi

for 8 h. The mixture was then ﬁltered through Celite, and the ﬁltrate

was evaporated. The crude product was chromatographed (10%

MeOH in CHCl₃) to aﬀord pure title compound as colorless oil (92%

yield); [α]²⁰_D= +41.6° (c 0.24, CHCl₃). ¹H NMR 200 MHz (CDCl₃)

δ 151 (m, 4H), 1.79−1.95 (m, 2H), 2.24−2.46 (m, 2H), 2.48−2.78

(m, 6H), 3.15 (m, 4H), 3.29 (m, 2H), 3.96 (m, 1H), 4.12 (m, 1H),

6.59−6.74 (m, 2H), 6.80−7.06 (m, 5H), 7.24 (m, 2H). ESI-MS m/z

393 [M + H]⁺. Anal. (C₂₄H₃₂N₄O) C, H, N.

N-(4-(4-Phenylpiperazin-1-yl)butyl)picolinamide (5gg). Starting

from 8h and 1-phenylpiperazine and following the procedure

described for 5a, the title compound was obtained as yellow oil

1

(82% yield). H NMR 400 MHz (CDCl₃) δ 1.63 (m, 4H), 2.39 (m,

2H), 2.56 (m, 4H), 3.17 (m, 4H), 3.47 (m, 2H), 6.80 (m, 1H), 6.88

(m, 2H), 7.21 (m, 2H), 7.35 (m, 1H), 7.78 (m, 1H), 8.16 (m, 1H),

8.48 (m, 1H). ESI-MS m/z 361 [M + Na]⁺, 339 (100) [M + H]⁺.

Anal. (C₂₀H₂₆N₄O) C, H, N.

(R)-(+)-N-[4-[4-(m-Tolyl)piperazin-1-yl]butyl]-1,2,3,4-tetrahydro-

quinoline-2-carboxamide (R)-(5y). Starting from (R)-22b and

following the procedure described for (R)-5x, the title compound

N-(4-(4-(3-Methoxyphenyl)piperazin-1-yl)butyl)picolinamide

(5hh). Starting from 8h and 4-(3-methoxylphenyl)piperazine and

following the procedure described for 5a, the title compound was

was obtained as colorless oil (85% yield); [α]²⁰= +42.1° (c 1.26,

D

1

MeOH). H NMR 200 MHz (CDCl₃) δ 1.20−1.58 (m, 4H), 1.86−

1

1.95 (m, 4H), 2.19−1.37 (m, 3H), 2.39−2.46 (m, 2H), 2.57−2.77 (m,

4H), 3.27−3.33 (m, 6H), 3.96 (m, 1H), 4.19 (m, 1H), 6.68 (m, 5H),

7.00 (m, 2H), 7.12 (m, 1H). ESI-MS m/z 407 [M + H]⁺. MS/MS

(407) m/z 300, 276, 258, 248, 231, 189, 177, 161, 132. Anal.

(C₂₅H₃₄N₄O) C, H, N.

obtained as yellow oil (72% yield). H NMR 400 MHz (CDCl₃) δ

1.57 (m, 4H), 2.33 (m, 2H), 2.49 (m, 4H), 3.11 (m, 4H), 3.43 (m,

2H), 3.68 (s, 3H), 6.32 (m, 2H), 6.38 (m, 1H), 6.45 (m, 1H), 7.07 (m,

1H), 7.30 (m, 1H), 7.72 (m, 1H), 8.11 (m, 1H), 8.43 (m, 1H). ESI-

MS m/z 369 [M + H]⁺. Anal. (C₂₁H₂₈N₄O₂) C, H, N.

(S)-(−)-N-[4-(4-Phenylpiperazin-1-yl)butyl]-1,2,3,4-tetrahydroqui-

noline-2-carboxamide (S)-(5z). To a solution of (S)-22a and

following the procedure described for (R)-5x, the title compound

N-(4-(4-(6-Methylpyridin-2-yl)piperazin-1-yl)butyl)picolinamide

(5ii). Starting from 8h and arylpiperazine 10a and following the

procedure described for 5a, the title compound was obtained as yellow

oil (90% yield). ¹H NMR 300 MHz (CDCl₃) δ 1.58 (m, 4H), 2.31 (m,

5H), 2.45 (m, 4H), 3.43 (m, 6H), 6.36 (m, 2H), 7.29 (m, 2H), 7.72

(m, 1H), 8.11 (m, 2H), 8.43 (m, 1H). ESI-MS m/z 376 [M + Na]⁺,

354 (100) [M + H]⁺. Anal. (C₂₀H₂₇N₅O) C, H, N.

was obtained as colorless oil (67% yield); [α]²⁰= −41.7° (c 0.27,

D

CHCl₃); spectroscopic data were identical to those reported for (R)-

5x. Anal. (C₂₄H₃₂N₄O) C, H, N.

(S)-(−)-N-(4-(4-m-Tolylpiperazin-1-yl)butyl)-1,2,3,4-tetrahydro-

quinoline-2-carboxamide (S)-(5aa). Starting from (S)-22b and

following the procedure described for (R)-5x, the title compound

N-(4-(4-(6-Methoxypyridin-2-yl)piperazin-1-yl)butyl)picolinamide

(5jj). Starting from 8h and arylpiperazine 10e and following the

procedure described for 5a, the title compound was obtained as yellow

oil (80% yield). ¹H NMR 300 MHz (CDCl₃) δ 1.63 (m, 4H), 2.37 (m,

2H), 2.48 (m, 4H), 3.47 (m, 6H), 3.80 (m, 3H), 6.05 (m, 2H), 7.34

(m, 2H), 7.77 (m, 1H), 8.14 (m, 2H), 8.47 (m, 1H). ¹³C NMR

(CDCl₃); 24.5, 27.8, 39.5 45.3, 53.2 (2C), 58.3, 98.2 (2C), 122.3,

126.3, 137.5, 140.2, 148.2, 150.2, 158.5, 163.2, 164.5. ESI-MS m/z 392

[M + Na]⁺, 370 (100) [M + H]⁺. Anal. (C₂₀H₂₇N₅O₂) C, H, N.

N-(4-(4-(3-Methoxyphenyl)piperazin-1-yl)butyl)-6-methylpyri-

dine-2-carboxamide (5kk). Starting from 8i and 4-(3-

methoxyphenyl)piperazine and following the procedure described for

was obtained as colorless oil (90% yield); [α]²⁰= −42.1° (c 1.26,

D

MeOH); spectroscopic data were identical to those reported for (R)-

5y. Anal. (C₂₅H₃₄N₄O) C, H, N.

N-(4-(4-Phenylpiperazin-1-yl)butyl)quinoline-6-carboxamide

(5bb). Starting from 8f and 1-phenylpiperazine, and following the

procedure described for 5a, the title compound was obtained as white

1

solid (65% yield); mp (MeOH) 151−152 °C. H NMR 300 MHz

(CDCl₃) δ 1.70 (m, 4H), 2.45 (m, 2H), 2.58 (m, 4H), 3.14 (m, 4H),

3.54 (m, 2H), 6.84 (m, 3H), 7.07 (br s, 1H), 7.23 (m, 2H), 7.41 (m,

1H), 8.03 (m, 1H), 8.13 (m, 2H), 8.26 (m, 1H), 8.95 (m, 1H). ¹³C

NMR (CDCl₃) δ 24.7, 27.6, 40.4, 49.2, 53.4, 58.1, 116.2, 120.0, 122.1,

127.5, 127.8, 129.3, 130.1, 133.1, 137.1, 149.5, 151.4 (2C), 125.1,

167.3. ESI-MS m/z 411 [M + Na]⁺, 389 [M + H]⁺. Anal.

(C₂₄H₂₈N₄O) C, H, N.

1

5a, the title compound was obtained as yellow oil (78% yield). H

NMR 200 MHz (CDCl₃) δ 1.64 (m, 4H), 2.41 (m, 2H), 2.53 (s, 3H),

2.59 (m, 4H), 3.17 (m, 4H), 3.49 (m, 2H), 3.75 (s, 3H), 6.45 (m, 3H),

7.17 (m, 2H), 7.67 (m, 1H), 7.97 (m, 1H), 8.14 (br s, 1H). ESI-MS

m/z 405 [M + Na]⁺, 383 (100) [M + H]⁺. Anal. (C₂₂H₃₀N₄O₂) C, H,

N.

N-(4-(4-(6-Methylpyridin-2-yl)piperazin-1-yl)butyl)benzamide

(5cc). Starting from 8g and arylpiperazine 10a and following the

procedure described for 5a, the title compound was obtained as white

N-(4-(4-Phenylpiperazin-1-yl)butyl)nicotinamide (5ll). Starting

from 8j and 1-phenylpiperazine and following the procedure described

for 5a, the title compound was obtained as white solid (80% yield);

1

solid (82% yield); mp (MeOH) 102−103 °C. H NMR 200 MHz

(CDCl₃) δ 1.59 (m, 4H), 2.34 (m, 5H), 2.46 (m, 4H), 3.46 (m, 6H),

6.39 (m, 2H), 7.07 (br s, 1H), 7.33 (m, 4H), 7.73 (m, 2H). ESI-MS

m/z 375 [M + Na]⁺, 353 (100) [M + H]⁺. Anal. (C₂₁H₂₈N₄O) C, H,

N

1

mp (MeOH) 119−120 °C. H NMR 400 MHz (CDCl₃) δ 1.65 (m,

4H), 2.42 (m, 2H), 2.56 (m, 4H), 3.12 (m, 4H), 3.46 (m, 2H), 6.84

(m, 3H), 7.11 (br s, 1H), 7.23 (m, 2H), 7.31 (m, 1H), 8.07 (m, 1H),

8.65 (m, 1H), 8.94 (m, 1H). ESI-MS m/z 339 [M + H]⁺. Anal.

(C₂₀H₂₆N₄O) C, H, N

N-(4-(4-(6-Methoxypyridin-2-yl)piperazin-1-yl)butyl)benzamide

(5dd). Starting from 8g and arylpiperazine 10e and following the

procedure described for 5a, the title compound was obtained as white

6-Quinolinecarboxylic Acid (6b). To a solution of 6-methylquino-

line (100.0 mg, 0.70 mmol) in H₂O (1.0 mL) and H₂SO₄(0.25 mL),

chromium trioxide (272.0 mg, 2.72 mmol) was added in portions at 0

°C and reﬂuxing for 21 h. The crystalline precipitate of the

1

solid (85% yield); mp (MeOH) 120−121 °C. H NMR 200 MHz

(CDCl₃) δ 1.61 (m, 4H), 2.37 (m, 2H), 2.47 (m, 4H), 3.43 (m, 6H),

3.81 (s, 3H), 6.06 (m, 2H), 6.85 (m, 1H), 7.38 (m, 4H), 7.72 (m, 2H).

N

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hydrosulfate, which separated upon cooling, was removed by ﬁltration,

dissolved in 10% sodium hydroxide water solution and, after washing

with hexane, was precipitated with acetic acid to give title compound

(70% yield) that was used in the following step without further

puriﬁcation.¹H NMR 300 MHz (DMSO-d₆) δ 7.61 (dd, 1H, J₁= 8.3

Hz, J₂= 4.2 Hz), 8.08 (d, 1H, J = 8.8 Hz), 8.20 (dd, 1H, J₁= 8.8 Hz, J₂

= 1.7 Hz), 8.56 (d, 1H, J = 8.2 Hz), 8.67 (m, 1H), 9.00 (dd, 1H, J₁=

4.1 Hz, J₂= 1.5 Hz), 13.20 (br s, 1H). ¹³C NMR 75 MHz (DMSO-d₆)

122.9, 127.9, 129.2, 129.5, 130.0, 131.7, 138.2, 150.0, 153.4, 167.7. ESI-

MS m/z 196 [M + Na]⁺, 174 (100) [M + H]⁺. Anal. (C₁₀H₇NO₂) C,

H, N.

4H), 3.66 (s, 3H), 7.28 (m, 4H), 7.60 (m, 1H), 9.80 (br s, 1H). ESI-

MS m/z 331 [M + Na]⁺, 309 [M + H]⁺.

N-(4-Bromobutyl)quinoline-6-carboxamide (8f). Starting from 7b

and following the procedure described for 8c, the title compound was

1

obtained as amorphous yellow solid (75% yield). H NMR 300 MHz

(CDCl₃) δ 1.66 (m, 2H), 1.77 (m, 2H), 3.26 (m, 2H), 3.36 (m, 2H),

7.22 (dd, 1H, J₁= 8.2 Hz, J₂= 4.4 Hz), 7.79 (br s, 1H), 7.88 (m, 2H),

7.97 (dd, 1H, J₁= 8.9 Hz, J₂= 1.9 Hz), 8.17 (d, 1H, J = 1.5 Hz), 8.75

(dd, 1H, J₁= 4.3 Hz, J₂= 1.6 Hz). ESI-MS m/z 637 [2M + Na]⁺, 330

[M + Na]⁺, 308 [M + H]⁺.

N-(4-Bromobutyl)benzamide (8g). Starting from 7c and following

the procedure described for 8c, the title compound was obtained as

N-(4-Hydroxybutyl)-1-methyl-1H-indole-2-carboxamide (7a). To

a solution of 1-methyl-1H-indole-2-carboxylic acid (150.0 mg, 0.86

mmol) 6a in dry dichloromethane (DCM, 10.0 mL), HOBt (145.5

mg, 0.95 mmol) and DCC (196.0 mg, 0.95 mmol) were added at 0 °C

under argon; the suspension was warmed to rt and stirred for 1 h.

Then 4-amino-1-butanol (79.3 μL, 0.86 mmol) was added and the

mixture was stirred for 12 h at rt. The resulting suspension was ﬁltered

through Celite, washed with chloroform (3 × 10 mL), and the ﬁltrate

evaporated. The crude product was puriﬁed by means of ﬂash

chromatography (10% MeOH in chloroform) to give title compound

1

yellow oil (70% yield). H NMR 200 MHz (CDCl₃) δ 1.84 (m, 4H),

3.44 (m, 4H), 6.37 (br s, 1H), 7.43 (m, 3H), 7.75 (m, 2H). ESI-MS

m/z 279 [M + Na]⁺, 257 (100) [M + H]⁺.

N-(4-Bromobutyl)picolinamide (8h). Starting from 7d and

following the procedure described for 8c, the title compound was

1

obtained as amorphous white solid. H NMR 200 MHz (CDCl₃) δ

1.68 (m, 4H), 3.27 (m, 4H), 7.21 (m, 1H), 7.65 (m, 1H), 7.99 (m,

2H), 8.29 (m, 1H). ES-MS m/z: 280 [M + Na]⁺, 258 [M + H]⁺. ESI-

MS m/z 280 [M + Na]⁺, 258 (100) [M + H]⁺.

1

as colorless oil (85% yield). H NMR 200 MHz (CDCl₃) δ 1.51 (m,

N-(4-Bromobutyl)-6-methylpicolinamide (8i). Starting from 7e

and following the procedure described for 8c, the title compound was

4H), 2.96 (m, 2H), 3.53 (m, 2H), 3.60 (s, 3H), 4.78 (br s, 1H), 7.08

(m, 2H), 7.42 (m, 2H), 7.60 (m, 1H), 8.56 (br s, 1H). ESI-MS m/z

247 [M + H]⁺.

1

obtained as amorphous white solid. H NMR 300 MHz (CDCl₃) δ

1.68 (m, 4H), 2.60 (s, 3H), 3.27 (m, 4H), 7.21 (m, 1H), 7.65 (m, 1H),

7.99 (m, 1H), 8.29 (m, 1H). ESI-MS m/z 293 [M + Na]⁺, 271 [M +

H]⁺.

N-(4-Hydroxybutyl)quinoline-6-carboxamide (7b). To a solution

of 6-quinolinecarboxylic acid (6b) (200.0 mg, 1.16 mmol) in dry

DCM (20.0 mL), triethylamine (TEA; 162.0 μL, 1.16 mmol), HOBt

(171.0 mg, 1.27 mmol), and EDC (243.0 mg, 1.27 mmol) were added

at 0 °C under argon atmosphere; the suspension was warmed to rt and

stirred for 1 h. Then 4-amino-1-butanol (117.0 μL, 1.27 mmol) was

added and the mixture was stirred for 12 h at rt. The resulting

suspension was evaporated, and the crude product was puriﬁed by

means of ﬂash chromatography (10% MeOH in chloroform) to give

title compound as white solid (97% yield); mp (MeOH) 121−122 °C.

¹H NMR 300 MHz (CDCl₃) δ 1.75 (m, 4H), 2.13 (br s, 1H), 3.48−

3.59 (m, 2H), 3.76 (m, 2H), 7.02 (br s, 1H), 7.43 (m, 1H), 8.01−8.12

(m, 2H), 8.20 (d, 1H, J = 8.5 Hz), 8.30 (m, 1H), 8.94 (m, 1H). ESI-

MS m/z 511 [2M + Na]⁺, 267 [M + Na]⁺, 245 [M + H]⁺.

N-(4-Hydroxybutyl)benzamide (7c). Starting from benzoic acid 6c,

and following the procedure described for 7a, the title compound was

N-(4-Bromobutyl)nicotinamide (8j). Starting from 7f and following

the procedure described for 8c, the title compound was obtained as

1

yellow oil (60% yield). H NMR 400 MHz (CDCl₃) δ 1.65 (m, 2H),

1.79 (m, 2H), 3.33 (m, 4H), 7.21 (m, 1H), 7.94 (m, 1H), 8.54 (m,

1H), 8.81 (m, 1H). ESI-MS m/z 279 [M + Na]⁺.

tert-Butyl 4-(3-Fluorophenyl)piperazine-1-carboxylate (9b). In a

sealed tube, 1-bromo-3-ﬂuorobenzene (469 mg, 2.68 mmol),

Pd₂(dba)₂(2%), ( )-BINAP (4%), and sodium t-butoxide (500.0

mg, 2.59 mmol) were added to N-Boc-piperazine (322.0 mg, 1.73

mmol) in dry toluene (4 mL). The mixture was stirred at 70 °C for 90

min, ﬁltered over Celite, washed with EtOAc, and evaporated under

reduced pressure. The crude was puriﬁed by means of ﬂash

chromatography (40% EtOAc in n-hexane) to give title compound

1

as colorless oil (78% yield). H NMR 300 MHz (CDCl₃) δ 1.47 (s,

1

obtained as yellow oil (99% yield). H NMR 200 MHz (CDCl₃) δ

9H), 3.11 (m, 4H), 3.55 (m, 4H), 6.58 (m, 3H), 7.19 (m, 1H). ¹³C

NMR 75 MHz (CDCl₃) δ 28.6 (3), 43.6 (2), 49.0 (2), 80.2, 103.3

(J_C−F= 24.7 Hz), 106.5 (J_C−F= 21.4 Hz), 111.8 (2), 130.4 (J_C−F= 9.9

Hz), 153.1 (J_C−F= 9.9 Hz), 154.9, 164.0 (J_C−F= 242.5 Hz). ESI-MS

m/z 303 [M + Na]⁺, 281 (100) [M + H]⁺.

1.57 (m, 4H), 3.33 (m, 2H), 3.58 (m, 2H), 6.42 (br s, 1H), 7.31 (m,

2H), 7.47 (m, 1H), 7.64 (m, 2H). ESI-MS m/z 216 [M + Na]⁺.

N-(4-Hydroxybutyl)picolinamide (7d). Starting from picolinic acid

6d and following the procedure described for 7a, the title compound

was obtained as yellow oil (86% yield). ¹H NMR 200 MHz (CDCl₃) δ

1.59 (m, 4H), 3.00 (br s, 1H), 3.41 (m, 2H), 3.57 (m, 2H), 7.28 (m,

1H), 7.71 (m, 1H), 8.08 (m, 2H), 8.40 (m, 1H). ESI-MS m/z 217 [M

+ Na]⁺.

tert-Butyl-4-(quinolin-3-yl)piperazine-1-carboxylate (9c). Starting

from 3-bromoquinoline and following the procedure described for 9b,

the title compound was obtained as a white solid (80% yield); mp

1

(MeOH) 114−115 °C. H NMR 200 MHz (CDCl₃) δ 1.46 (s, 9H),

N-(4-Hydroxybutyl)-6-methylpicolinamide (7e). Starting from 6-

3.18 (m, 4H), 3.57 (m, 4H), 7.28 (m, 1H), 7.43 (m, 2H), 7.63 (m,

1H), 7.96 (m, 1H), 8.74 (m, 1H). ESI-MS m/z 336 [M + Na]⁺.

tert-Butyl-4-(4-(benzyloxy)phenyl)piperazine-1-carboxylate (9d).

1-(Benzyloxy)-4-bromobenzene: To a solution of 4-bromophenol (2.0

g, 11.6 mmol) in DMF (10 mL), benzyl chloride (1.73 μL, 15.0 mmol)

and potassium carbonate (2.1 g, 15.0 mmol) were added and the

reaction mixture was stirred for 12 h at rt. After this time, the reaction

was put in ice−water and extracted with EtOAc/toluene (5:1,3 × 10

mL), and the collected organic layers were dried over sodium sulfate,

ﬁltered, and concentrated under reduced pressure. The crude was

puriﬁed by means of ﬂash chromatography (7% EtOAc in n-hexane),

and pure title compound was obtained as a colorless oil (99% yield).

¹H NMR 300 MHz (CDCl₃) δ 5.16 (s, 2H), 7.08 (m, 2H), 7.64 (m,

7H).

methylpicolinic acid 6e and following the procedure described for 7a,

the title compound was obtained as yellow oil (80% yield). H NMR

300 MHz (CDCl₃) δ 1.59 (m, 4H), 2.55 (s, 3H), 3.00 (br s, 1H), 3.41

(m, 2H), 3.57 (m, 2H), 7.28 (m, 1H), 7.71 (m, 1H), 8.08 (m, 1H),

8.40 (m, 1H). ESI-MS m/z 209 [M + H]⁺.

1

N-(4-Hydroxybutyl)nicotinamide (7f). Starting from nicotinic acid

7f and following the procedure described for 7a, the title compound

was obtained as yellow oil (70% yield). ¹H NMR 400 MHz (CDCl₃) δ

1.55 (m, 4H), 3.05 (m, 2H), 3.53 (m, 2H), 7.55 (m, 1H), 8.13 (m,

1H), 8.76 (m, 1H), 8.93 (m, 1H). ESI-MS m/z 217 (100) [M + Na]⁺.

N-(4-Bromobutyl)-1-methyl-1H-indole-2-carboxamide (8c). To a

solution of 7a (504.9 mg, 2.05 mmol) in dry MeCN (30.0 mL),

triphenylphosphine (808.0 mg, 3.08 mmol) and carbon tetrabromide

(1021.0 mg, 3.08 mmol) were added under vigorous stirring at rt.

After 2 h, the mixture was quenched with 15% NaOH and extracted

with EtOAc (3 × 10 mL). The organic layers were dried and

evaporated. The residue was chromatographed (10% MeOH in

chloroform) to give the title compound was achieved as yellow oil

Starting from 1-(benzyloxy)-4-bromobenzene and following the

procedure described for 9b, while heating at 90 °C for 15 h, the title

1

compound was obtained as a colorless oil (30% yield). H NMR 300

MHz (CDCl₃) δ 1.49 (s, 9H), 3.01 (m, 4H), 3.58 (m, 4H), 5.02 (s,

1

(84% yield). H NMR 300 MHz (CDCl₃) δ 1.96 (m, 4H), 3.56 (m,

2H), 6.91 (m, 4H), 7.35 (m, 5H). ESI-MS m/z 369 [M + H]⁺.

O

dx.doi.org/10.1021/jm501119j | J. Med. Chem. XXXX, XXX, XXX−XXX

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1

tert-Butyl 4-(6-Methoxypyridin-2-yl)piperazine-1-carboxylate

(9e). Starting from 2-bromo-6-methoxypyridine and following the

procedure described for 10b, the title compound was obtained as

colorless oil (75% yield). ¹H NMR 200 MHz (CDCl₃) δ 1.42 (s, 9H),

3.19 (m, 4H), 3.73 (s, 3H), 3.79 (m, 4H), 5.70 (m, 1H), 5.90 (m, 1H),

7.44 (m, 1H). ESI-MS m/z 316 [M + Na]⁺, 294 [M + H]⁺, 283 (100).

tert-Butyl 4-(Naphthalen-1-yl)piperazine-1-carboxylate (9f).

Starting from 1-bromonaphthalene and following the procedure

described for 9b, the title compound was obtained as a colorless oil

yield). H NMR 200 MHz (CDCl₃) δ 1.54 (m, 4H), 2.40 (m, 4H),

2.58 (m, 4H), 3.19 (m, 4H), 3.76 (s, 3H), 6.46 (m, 3H), 7.16 (m, 1H).

ESI-MS m/z 286 [M + Na]⁺264 (100) [M + H]⁺.

1,2,3,4-Tetrahydroquinoline-2-carboxylic Acid (17). To a solution

of quinaldic acid 16 (2.00 g, 11.55 mmol) in dry MeOH (20.0 mL),

platinum dioxide (0.11 mmol) was added under argon and the

resulting mixture was taken under hydrogen atmosphere for 16 h.

Then the suspension was ﬁltered through Celite and ﬁltrate

evaporated. The title compound was obtained in quantitative yield

and was used in the next step without further puriﬁcation. ESI-MS m/z

176 [M − H]⁻. Spectroscopic data are identical to those reported in

the literature.²¹

Methyl 1,2,3,4-Tetrahydroquinoline-2-carboxylate (18). To a

solution of acid 17 (2.00 g, 11.30 mmol) in dry MeOH (20.0 mL)

cooled at 0 °C, thionyl chloride (1.87 g, 15.82 mmol) was added

dropwise and the mixture was stirred at rt for 16 h. Thionyl chloride

and MeOH were then removed by distillation, the residue was

dissolved in water (20.0 mL), and the pH was adjusted to 8 with a

saturated solution of NaHCO₃. The aqueous layer was extracted with

EtOAc (3 × 25 mL), and the collected organic layers were dried and

evaporated. The crude product was puriﬁed by means of ﬂash

chromatography (15% EtOAc in n-hexane) to aﬀord title compound

(racemate) as a yellow oil (60% yield). ¹H NMR 200 MHz (CDCl₃) δ

1.95−2.17 (m, 1H), 2.22−2.36 (m, 1H), 2.68−2.89 (m, 2H), 3.78 (s,

3H), 4.05 (m, 1H), 4.36 (br s, 1H), 6.57−6.70 (m, 2H), 7.01 (m, 2H).

ESI-MS m/z 214 [M + Na]⁺, 192 [M + H]⁺, 132 (100), data were

consistent with the literature.²¹

1

(80% yield). H NMR 200 MHz (CDCl₃) δ 1.55 (s, 9H), 3.05 (m,

4H), 3.72 (m, 4H), 7.05 (m, 1H), 7.49 (m, 4H), 7.82 (m, 1H), 8.22

(m, 1H). ESI-MS m/z 335 [M + Na]⁺, 313 (100) [M + H]⁺.

4-(3-Fluorophenyl)piperazin-1-ium 2,2,2-triﬂuoroacetate (10b).

TFA (4 mL) was added to a solution of 9b (315 mg, 1.07 mmol) in

DCM (4 mL) while cooling in an ice bath, and the mixture was stirred

for 1 h at rt. The mixture was then concentrated and washed with

Et₂O until the solid became colorless. The compound was obtained as

a slight brownish solid (67% yield) and was used in the following step

without any further puriﬁcation. ¹H NMR 200 MHz (CD₃OD) δ 3.33

(m, 4H), 3.42 (m, 4H), 6.62 (m, 1H), 6.78 (m, 2H), 7.26 (m, 1H).

¹³C NMR 75 MHz (CD₃OD) δ 43.4 (2), 46.1 (2), 103.4 (J_C−F= 25.3

Hz), 107.0 (J_C−F= 21.4 Hz), 112.0 (2), 130.4 (J_C−F= 9.9 Hz), 152.2

(J_C‑F= 9.9 Hz), 164.0 (J_C−F= 241.3 Hz).

4-(Quinolin-3-yl)piperazin-1-ium 2,2,2-triﬂuoroacetate (10c).

Starting from 9c and following the procedure described for 10b, the

title compound was obtained as viscous oil (97% yield). ESI-MS m/z

214 [M + H]⁺.

Enzymatic Kinetic Resolution of the Racemate 18. The racemate

18 was transformed in the corresponding hydrochloride salt by a

standard procedure. To a suspension of ( )-18 (1.83 g, 8.07 mmol)

phosphate buﬀer 0.1 M (50.0 mL, pH = 7.5), α-chymotrypsin (3.05

mmol, suspended in the same buﬀer inside a dialysis tube) were added

and the system was vigorously stirred at rt. The reaction was

monitored via HPLC until the resolution was complete. When the

resolution was complete, the enzyme was removed and the aqueous

solution was extracted with EtOAc (3 × 40 mL). The organic layers

were washed with a saturated solution of NaHCO₃and brine, then

collected and evaporated. The crude product was puriﬁed by means of

ﬂash chromatography (10% EtOAc in n-hexane) to aﬀord isomer (S)-

18 (50% yield); [α]²⁰_D+41.3 (c 0.58, CHCl₃); spectroscopic data were

identical to those previously reported for the racemate and were

consistent with the literature.²²

1-(4-(Benzyloxy)phenyl)piperazin-1-ium 2,2,2-triﬂuoroacetate

(10d). Starting from 9d and following the procedure described for

10b, the title compound was obtained as colorless oil (97% yield). ¹H

NMR 300 MHz (MeOD) δ 3.32 (m, 8H), 5.08 (s, 2H), 7.04 (m, 2H),

7.25 (m, 2H), 7.37 (m, 5H).

4-(6-Methoxypyridin-2-yl)piperazin-1-ium 2,2,2-triﬂuoroacetate

(10e). Starting from 9e and following the procedure described for

1

10b, the title compound was obtained as viscous oil (98% yield). H

NMR 200 MHz (CDCl₃) δ 3.16 (m, 4H), 3.69 (m, 4H), 3.80 (s, 3H),

6.10 (m, 2H), 6.58 (m, 2H), 7.37 (m, 1H). ESI-MS m/z 194 [M +

H]⁺.

4-(Naphthalen-1-yl)piperazin-1-ium 2,2,2-triﬂuoroacetate (10f).

Starting from 9f and following the procedure described for 10b, the

title compound was obtained as colorless oil (99% yield). ESI-MS m/z

213 [M + H]⁺.

N-(4-(4-(4-(Benzyloxy)phenyl)piperazin-1-yl)butyl)isoquinoline-3-

carboxamide (11). Starting from 8d and 1-(4-(benzyloxy)phenyl)-

piperazine 10d and following the procedure described for 5a, the title

The water phase, containing the amino acid (R)-17, was evaporated

to give crude (R)-17, which was used in the next step without further

puriﬁcation.

1

compound was obtained as colorless oil (55% yield). H NMR 300

(S)-(−)-1-(Benzyloxycarbonyl)-1,2,3,4-tetrahydroquinoline-2-car-

boxylic Acid (S)-(19). 2-Methyl (S)-(−)-1-(Benzyloxycarbonyl)-

1,2,3,4-tetrahydroquinoline-2-carboxylate: To a solution of (S)-18

(160.0 mg, 0.84 mmol) in aqueous 2 M NaHCO₃benzyl

chloroformate (158.2 mg, 0.92 mmol) was added dropwise in 30

min. The mixture was stirred for 1.5 h at rt then evaporated. The

residue was extracted with EtOAc (3 × 20 mL), and the organic layers

were dried and evaporated. The crude product was puriﬁed by means

of ﬂash chromatography (20% acetone in n-hexane) to give title

MHz (CDCl₃) δ 1.71 (m, 4H), 2.46 (t, 2H, J = 7.0 Hz), 2.62 (m, 4H),

3.11 (m, 4H), 3.58 (m, 2H), 5.00 (s, 2H), 6.90 (s, 4H), 7.35 (m, 5H),

7.71 (m, 2H), 7.99 (m, 2H), 8.37 (br t, 1H), 8.62 (s, 1H), 9.13 (s,

1H). ESI-MS m/z 517 [M + Na]⁺, 495 (100) [M + H]⁺.

4-(4-(3-Methoxyphenyl)piperazin-1-yl)butanenitrile (13). To a

stirred solution of 4-(3-methoxylphenyl)piperazine dihydrochloride

(12) (133.1 mg, 0.52 mmol) in MeCN (10.0 mL), 4-bromobutaneni-

trile (84.7 mg, 0.57 mmol) and potassium carbonate (107.6 mg, 0.78

mmol) were added at rt. The mixture was reﬂuxed for 12 h, and then it

was ﬁltered and evaporated. The crude product was puriﬁed by means

of ﬂash chromatography (10% MeOH in chloroform) to give pure title

compound as a yellow oil (73% yield). ¹H NMR 200 MHz (CDCl₃) δ

1.83 (m, 2H), 2.49 (m, 8H), 3.17 (m, 4H), 3.77 (s, 3H), 6.48 (m, 3H),

7.16 (m, 1H). ESI-MS m/z 282 [M + Na]⁺260 (100) [M + H]⁺.

4-(4-(3-Methoxyphenyl)piperazin-1-yl)butan-1-amine (14). To a

stirred solution of 13 (300.0 mg, 1.16 mmol) in dry MeOH (15.0 mL),

nickel(II) chloride hexahydrate (28.0 mg, 0.12 mmol) and sodium

borohydride (307.2 mg, 8.12 mmol) were added while cooling at 0 °C.

The mixture was stirred at rt for 90 min, then it was ﬁltered over Celite

and washed with MeOH, and the ﬁltrate was evaporated under

reduced pressure. The residue was extracted with EtOAc (3 × 30 mL),

the organic layers were ﬁltered and concentrated, and the crude

product was puriﬁed by means of ﬂash chromatography (15% n-

hexane in EtOAc) to give pure title compound as yellow oil (60%

compound as colorless oil (80%yield); [α]²⁰= −50.0° (c 0.94,

D

1

MeOH). H NMR 200 MHz (CDCl₃) δ 1.81−1.99 (m, 1H), 2.31−

2.43 (m, 1H), 2.43−2.69 (m, 2H), 3.61 (s, 3H), 4.96 (t, 1H, J = 7.6

Hz), 5.24 (s, 2H), 7.02 (m, 2H), 7.19 (m, 1H), 7.29 (m, 5H), 7.81 (d,

1H, J = 7.4 Hz). ESI-MS m/z 673 [2M + Na]⁺, 348 (100) [M + Na]⁺,

281, 192, 132. To a solution of this ester (218.5 mg, 0.67 mmol) in a

3:2 MeOH/water mixture, NaOH (27.0 mg, 0.67 mmol) was added

and the mixture was reﬂuxed for 2 h. Then the solvents were

evaporated, water was added to the residue, and the mixture was

acidiﬁed with 1 N HCl. The aqueous layer was extracted with

chloroform (3 × 15 mL), and the collected organic layers were dried

and evaporated. The crude product was puriﬁed by means of ﬂash

chromatography (CHCl₃/MeOH/AcOH 9:1:0.1) to aﬀord title

compound as amorphous solid (99% yield); [α]²⁰= −50.0° (c

D

1

0.98, MeOH). H NMR 200 MHz (CDCl₃) δ 1.85−1.99 (m, 1H),

2.35−2.50 (m, 1H), 2.58−2.79 (m, 2H), 4.99 (t, 1H, J = 7.7 Hz),

P

dx.doi.org/10.1021/jm501119j | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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5.34−5.19 (m, 2H), 7.05 (m, 2H), 7.21 (m, 1H), 7.35 (m, 5H), 7.78

(d, 1H, J = 7.9 Hz), 9.94 (br s, 1H). ESI-MS m/z 310 [M − H]⁻

(100), 266, 202.

Isolated Rat Heart Preparation and Perfusion. All animal care

and experimental procedures complied with the Guide for the Care

and Use of Laboratory Animals published by the U.S. National

Institutes of Health (NIH Publication no. 85-23, revised 1996) and

were approved by the Animal Care and Ethics Committee of the

(R)-(+)-1-(Benzyloxycarbonyl)-1,2,3,4-tetrahydroquinoline-2-car-

boxylic Acid (R)-(19). Starting from (R)-17 and following the

procedure above-described for Cbz-protection (the work up was

done by adding water and adjusting at pH = 2 with KHSO₄while

cooling at 0 °C), title compound was obtained as amorphous solid

̀

Universita di Siena, Italy (08-02-2012). Male Sprague−Dawley rats

(300−350 g; Charles River Italia, Calco, Italy) were anaesthetized (ip)

with a mixture of Ketavet (30 mg/kg ketamine; Intervet, Aprilia, Italy)

and Xilor (8 mg/kg xylazine; Bio 98, San Lazzaro, Italy) containing

heparin (5000 U/kg), decapitated, and bled. The hearts, sponta-

neously beating, were rapidly explanted and mounted on a

Langendorﬀ apparatus for retrograde perfusion via the aorta at a

constant ﬂow rate of 10 mL/min with a Krebs−Henseleit solution of

the following composition (mM): NaCl 118, KCl 4.7, CaCl₂2.5,

MgSO₄1.2, NaHCO₃25, KH₂PO₄1.2, glucose 11.5, Na pyruvate 2,

and EDTA 0.5, bubbled with a 95% O₂−5% CO₂gas mixture (pH

7.4), and kept at 37 °C, as described elsewhere.⁴⁸

(81% yield); [α]²⁰= +47.6° (c 0.42, MeOH); spectroscopic data

D

were identical to those already reported for (S)-19.

(R)-(+)-N-(4-Hydroxybutyl)-1-(benzyloxycarbonyl)-1,2,3,4-tetra-

hydroquinoline-2-carboxamide (R)-(20). Starting from the acid (R)-

20 and following the procedure described for 7a, the title compound

was obtained as white oil (72% yield); [α]²⁰= +41.9° (c 1.94,

D

1

CHCl₃). H NMR 200 MHz (CDCl₃) δ 1.21−1.40 (m, 4H), 2.01−

2.28 (m, 2H), 2.56−2.74 (m, 2H), 2.82 (br s, 1H), 3.11 (m, 2H), 3.42

(m, 2H), 4.90 (m, 1H), 5.11−5.26 (m, 2H), 6.51 (m, 1H), 6.95−7.17

(m, 3H), 7.30 (m, 5H), 7.64 (d, 1H, J = 8.1 Hz). ESI-MS m/z 405 [M

+ Na]⁺. MS/MS (405) m/z 361, 270.

The hearts were allowed to equilibrate for at least 20 min before

drug exposure. Heart contractility was measured as left ventricle

pressure (LVP) by means of latex balloon, inserted into the left

ventricle via the mitral valve and connected to a pressure transducer

(BLPR, WPI, Berlin, Germany). The balloon was inﬂated with

deionized water from a microsyringe until a left ventricular end

diastolic pressure of 10 mmHg was obtained.

Alteration in coronary perfusion pressure (CPP), arising from

changes in coronary vascular resistance, were recorded by another

pressure transducer (BLPR, WPI, Berlin, Germany) placed in the

inﬂow line.

A surface electrocardiogram (ECG) was recorded at a sampling rate

of 1 kHz by means of two steel electrodes, one placed on the apex and

the other on the left atrium of the heart. The ECG analysis included

the following measurements: RR (cycle length), HR (frequency), PQ

(atrioventricular conduction time), QRS (intraventricular conduction

time), and QT (overall action potential duration).⁴⁹

LVP, CPP, and ECG were recorded with a digital PowerLab data

acquisition system (PowerLab 8/30; ADInstruments, Castle Hill,

Australia) and analyzed by using Chart Pro for Windows software

(PowerLab; ADInstruments, Castle Hill, Australia).

(S)-(−)-N-(4-Hydroxybutyl)-1-(benzyloxycarbonyl)-1,2,3,4-tetra-

hydroquinoline-2-carboxamide (S)-(20). Starting from the acid (S)-

19 and following the procedure described for 7a, the title compound

was obtained as colorless oil (83% yield); [α]²⁰−50.0° (c 1.56,

D

CHCl₃); spectroscopic data were identical to those reported for (R)-

20.

(R)-(+)-N-(4-Bromobutyl)-1-(benzyloxycarbonyl)-1,2,3,4-tetrahy-

droquinoline-2-carboxamide (R)-(21). Starting from (R)-20 and

following the procedure described for 8c, the title compound was

obtained as white oil (33% yield); [α]²⁰_D= +46.7° (c 0.15, CHCl₃). ¹H

NMR 200 MHz (CDCl₃) δ 1.34−1.68 (m, 4H), 2.15−2.26 (m, 2H),

2.57−2.78 (m, 2H), 3.02−3.30 (m, 4H), 5.00 (t, 1H, J = 6.7 Hz),

5.14−5.30 (m, 2H), 6.07 (m, 1H), 6.99−7.21 (m, 3H), 7.33 (m, 5H),

7.61 (d, 1H, J = 8.0 Hz). ESI-MS m/z 912 [2M + Na]⁺, 467 [M +

Na]⁺.

(S)-(−)-N-(4-Bromobutyl)-1-(benzyloxycarbonyl)-1,2,3,4-tetrahy-

droquinoline-2-carboxamide (S)-(21). Starting from (S)-20 and

following the procedure described for 8c, the title compound was

obtained as yellow oil (30% yield); [α]²⁰= −50.9° (c 0.53, CHCl₃);

D

spectroscopic data were identical to those reported for (R)-22.

(R)-(+)-N-[4-(4-Phenylpiperazin-1-yl)butyl]-1-(benzyloxycarbon-

yl)-1,2,3,4-tetrahydroquinoline-2-carboxamide (R)-(22a). Starting

from (R)-21 and 1-phenylpiperazine and following the procedure

LVP was calculated by subtracting the left ventricular diastolic

pressure from the left ventricular systolic pressure. As the QT interval

is aﬀected by heart rate (shortened with more rapid heart rates),

correction factors are routinely used to avoid confounding eﬀects of

heart rate changes. Bazett’s formula was used for any heart rate

changes (QTc = QT/(RR)^1/2).

described for 5a, the title compound was obtained as colorless oil

1

(80% yield); [α]²⁰= +30.7° (c 0.19, CHCl₃). H NMR 200 MHz

D

(CDCl₃) δ 1.32 (m, 4H), 2.20−2.45 (m, 4H), 2.52 (m, 4H), 2.60−

2.82 (m, 2H), 3.17 (m, 4H), 4.99 (t, 1H, J = 6.7 Hz), 5.16−5.32 (m,

2H), 6.08 (m, 1H), 6.81 (m, 3H), 7.01−7.35 (m, 5H), 7.42 (m, 5H),

7.63 (d, 1H, J = 7.8 Hz). ESI-MS m/z 548 [M + Na]⁺, 527 (100) [M +

H]⁺.

Compound 5bb, administered as hydrochloride salt, was dissolved

in water.

Statistical Analysis. Data are reported as mean

SEM; n

(indicated in parentheses) represents the number of rat hearts.

Analysis of data was accomplished using GraphPad Prism version 5.04

(GraphPad Software, U.S.A.). Statistical analyses and signiﬁcance as

measured by ANOVA (followed by Dunnett’s post test) were obtained

using GraphPad InStat version 3.06 (GraphPad Software, U.S.A.). In

all comparisons, P < 0.05 was considered signiﬁcant.

(S)-(−)-N-[4-(4-Phenylpiperazin-1-yl)butyl]-1-(benzyloxycarbon-

yl)-1,2,3,4-tetrahydroquinoline-2-carboxamide (S)-(22a). Starting

from (S)-21 and 1-phenylpiperazine and following the procedure

described for 5a, the title compound was obtained as white oil (40%

yield); [α]²⁰= −31.0° (c 0.19, CHCl₃); spectroscopic data were

D

Behavioral Tests and in Vivo Tests. Catalepsy tests,¹⁵PCP- and

AMP-induced hyperactivity tests,¹⁵PPI tests,⁴²and in vivo micro-

dialysis studies⁵⁰were performed as previously described.

identical to those reported for (R)-22a.

(R)-(+)-N-[4-[4-(m-Tolyl)piperazin-1-yl]butyl]-1-(benzyloxycar-

bonyl)-1,2,3,4-tetrahydroquinoline-2-carboxamide (R)-(22b). Start-

ing from (R)-21 and 1-(m-tolyl)piperazine dihydrochloride and

following the procedure described for 5a, the title compound was

obtained as colorless oil (40% yield); [α]²⁰_D= +34.3° (c 0.19, CHCl₃).

¹H NMR 200 MHz (CDCl₃) δ 1.25−1.46 (m, 4H), 2.16−2.28 (m,

4H), 2.31 (s, 3H), 2.53 (m, 4H), 2.64−2.80 (m, 2H), 3.12−3.25 (m,

6H), 4.98 (m, 1H), 5.16−5.32 (m, 2H), 6.05 (m, 1H), 6.69 (m, 3H),

7.04−7.21 (m, 3H), 7.25 (s, 1H), 7.35 (m, 5H), 7.63 (d, 1H, J = 8.1

Hz).

Spontaneous Exploratory Locomotor Activity. Drug eﬀects on

spontaneous locomotor activity in female NMRI mice (Taconic

Denmark) was measured in automated activity frames (TSE home

cage Activity Monitoring System, MoTil, TSE Technical and Scientiﬁc

Equipment GmbH, Germany) equipped with infrared photobeam

emitters and sensors. To assess the drug eﬀect on exploratory

locomotor activity, the mice were transferred to new home cages

immediately before test start and the activity was measured for 30 min

with activity counts each 5 min.

Psychostimulant-Induced Hyperactivity. PCP-, MK801-, or

methamphetamine (AMP)-induced hyperactivity in mice (female

NMRI mice, Taconic, Denmark) was measured in automated activity

frames as described above. PCP was administered at a dose of 4 mg/kg

(sc), MK801 was administered at a dose of 0.2 mg/kg (ip), and AMP

(S)-(−)-N-(4-(4-m-Tolylpiperazin-1-yl)butyl)-1-(benzyloxycarbon-

yl)-1,2,3,4-tetrahydroquinoline-2-carboxamide (S)-(22b). Starting

from (S)-21 and 1-(m-tolyl)piperazine dihydrochloride and following

the procedure described for 5a, the title compound was obtained as

D

yellow oil (41% yield); [α]₂₀= −34.2° (c 1.75, CHCl₃);

spectroscopic data were identical to those reported for (R)-22b.

Q

dx.doi.org/10.1021/jm501119j | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

was administered at a dose of 2 mg/kg (ip). All compounds were

dosed immediately before test start. The tested compounds were

mainly administered sc (unless otherwise indicated) at various doses

15−30 min prior to administration of the psychostimulant. All

compounds were administered in a dose volume of 10 mL/kg.

Locomotor activity was then assessed for 60 min.

Passive Avoidance. Mice were tested in a step-through passive

avoidance task using a two-compartment chamber (ENV-307, MED

Associates Inc., USA). The light and the dark compartment consists of

plexiglass boxes of equal size (15 × 17 × 13 cm³; width × length ×

height) with metal grid ﬂoors. A sliding guillotine door is located at the

aperture (4 × 4 cm²) connecting the two compartments. A manual

grid scrambler (ENV-412, MED-Associates, US) is used to provide the

0.6 mA foot-shock.

On the acquisition day, mice were introduced individually to the lit

compartment and allowed to habituate for 60 s before access to the

dark compartment was granted when its central door openedd. When

a mouse entered the dark compartment with all doue paws, the latency

time to enter was recorded and then two mild foot shocks (0.6 mA, 0.5

s, 5 s between foot shocks) were delivered through the grid ﬂoor. The

animal was then kept in the dark compartment for 60 s, after which it

were removed and returned to its home cage. Twenty-four h after this

training session, the mouse was again introduced to the lit

compartment for 60 s, and thereafter, the latency to enter the dark

chamber was taken as a measure of memory retention. If a mouse did

not cross into the dark compartment within 180 s cut-oﬀ time, it was

assigned this value and removed from the apparatus. The tested drugs

were administered to mice 30 min before training (5bb po 10, 30, and

60 mg/kg dissolved in 5% cremophor; clozapine (0.3, 1.0, 3.0, and 10

mg/kg sc dissolved in 10% Tween80 diluted to appropriate volume in

NaCl). Both compounds were administered in a dose volume of 10

mL/kg.

size 3 μm, Phenomenex). The mobile phase consisted of 55 mM

sodium acetate, 1 mM octanesulfonic acid, 0.1 mM Na₂EDTA, and 8%

acetonitrile, adjusted to pH 3.2 with 0.1 M acetic acid, and was

degassed using an online degasser. Then 20 μL of the samples were

injected and the ﬂow rate was 0.15 mL/min. The electrochemical

detection was accomplished using an amperometric detector (Antec

Decade from Antec, Leiden, The Netherlands) with a glassy carbon

electrode set at 0.8 V, with an Ag/AgCl as reference electrode. The

output was recorded on a computer program LC solution from

Shimadzu, which also was used to calculate the peak areas.

ASSOCIATED CONTENT

* Supporting Information

■

S

Predicted physicochemical properties and elemental analysis

results for ﬁnal compounds. This material is available free of

charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION

Corresponding Authors

*For G.C.: phone, 0039 0577 234172; fax, 0039 0577 234254;

E-mail, campiani@unisi.it.

■

*For S.B.: phone, 0039 0577 234161; fax, 0039 0577 234254;

E-mail, butini3@unisi.it.

Present Address

^◆For K.S.N.: Saniona Aps, Baltorpvej 154, DK2750 Ballerup,

Denmark.

Notes

The authors declare no competing ﬁnancial interest.

Marble Burying. Mice were placed individually in novel home cages

containing 20 glass marbles (15 mm in diameter) placed in four rows

of ﬁve marbles on top of 5 cm of sawdust bedding. The mean number

of glass marbles buried after 30 min was taken as an index of “anxiety”,

i.e., the more marbles buried the more anxious the mouse. A marble

was classiﬁed as buried by an experimenter blind to treatment when at

least two-thirds was covered by sawdust. Compound 5bb was

administered po at doses of 3 and 30 mg/kg dissolved in 5%

cremophor in a dose volume of 10 mL/kg, 30 min prior to test start.

Microdyalisis. Surgery. The rats were placed in a stereotaxic

instrument under servoﬂurane anesthesia (2%) in a mixture of 20%

CO₂and 80% oxygen. The anesthesia was maintained during the

entire surgery. Two small holes were drilled to allow a microdialysis

probe to be placed into ventral striatum and PFC. The 2 mm probe

(CMA/12 from CMA/Microdialysis AB, Stockholm, Sweden) was

implanted at the following coordinates in mm: AP, +1.8; ML, + or −

1.4; DV, −8 (relative to bregma) for ventral striatum (relative to

bregma) and AP, +2.6; ML, + or − 1.2; DV, −3.6 (relative to bregma)

for PFC.

To maintain the body temperature at 37.1 °C, the rat was placed on

a thermoregulatory heating pad with a rectal probe (CMA/150).

After termination of the experiments, the rats were sacriﬁced and

their brains taken out and sliced. They were examined for correct

probe placement. Only results from rats with probes veriﬁed to be

located in the ventral striatum and in PFC are reported in this study.

Probe Perfusion and Sampling. All microdialysis probes were

perfused with Ringer solution, a physiological solution (containing 147

mM NaCl, 4.0 mM KCl, and 2.3 mM CaCl₂and adjusted to pH 6.5)

at a ﬂow rate of 2 μL/min during all experiments. The samples were

collected every 20 min. The samples collected during the ﬁrst 60 min

after starting the perfusion were discarded. The following two samples

were collected and analyzed to determine the basal level. The samples

following these initial 200 min were analyzed in order to determine the

drug eﬀects on neurotransmitter concentration as compared to the

basal level.

ACKNOWLEDGMENTS

■

We thank NeuroSearch A/S Ballerup, Denmark, for ﬁnancial

support. We also acknowledge COST Action CM1103. ACD

Inc. and S-IN are also acknowledged for providing the trial

license for evaluating the Percepta PhysChem Suite.

ABBREVIATIONS USED

■

EPS, extrapyramidal side eﬀects; D₂R, dopamine D₂receptors;

5-HT_2AR, serotonin 5-HT_2Areceptor; D₃R, dopamine D₃

receptor; 5-HT_1AR, serotonin 5-HT_1Areceptor; 5-HT_2CR,

serotonin 5-HT_2Creceptor; CATIE, Clinical Antipsychotic

Trials of Intervention Eﬀectiveness; 5-HT_2BR, serotonin 5-

HT_2Breceptor; D₄R, dopamine D₄receptor; hERG, human

ether-a-

̀

go-go related gene; Cbz, carboxybenzyl; DCC, N,N¹-

dicyclohexylcarbodiimide; EDC, N-(3-(dimethylamino)-

propyl)-N′-ethylcarbodiimide hydrochloride; HOBt, 1-hydrox-

ybenzotriazole; DCM, dichloromethane; TEA, triethylamine;

DMF, dimethylformamide; TFA, triﬂuoroacetic acid; SARs,

structure−activity relationships; SD, standard deviation; PCP,

phencyclidine; AMP, methamphetamine; sc, subcutaneously;

ip, intraperitoneally; po, orally; MED, minimal eﬀective dose;

NT, not tested; ( )-BINAP, ( )-2,2′-bis(diphenylphosphino)-

1,1′-binaphtyl; ECG, electrocardiogram; LVP, left ventricle

pressure; CPP, coronary perfusion pressure; HR, frequency;

RR, cycle length; PQ, atrioventricular conduction time; QRS,

intraventricular conduction time; QT, duration of ventricular

depolarization and repolarization

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