Targeting Dopamine D3 and Serotonin 5-HT1A and 5-HT2A

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Targeting Dopamine D3 and Serotonin 5‑HT1A and 5‑HT2A Receptors 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, Università degli Studi di Siena, via Aldo Moro 2, 53100 Siena, Italy § IRCCS-Istituto di Ricerche Farmacologiche Mario Negri, Via La Masa 19, 20156 Milano, Italy ∥ Dipartimento di Scienze della Vita, Università 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, Università degli Studi di Napoli “Federico II”, via D. Montesano 49, 80131 Napoli, Italy S Supporting Information *

ABSTRACT: Combination of dopamine D3 antagonism, serotonin 5-HT1A partial agonism, and antagonism at 5-HT2A leads to a novel approach to potent atypical antipsychotics. Exploitation of the original structure−activity relationships 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.

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INTRODUCTION Schizophrenia is a chronic, frequently disabling psychiatric disorder affecting about 1% of the world’s population.1 It 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 medication 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 (hyperdopaminergic tone in the mesolimbic pathway and hypodopaminergic signaling in the mesocortical pathway).2 On the basis of this model, antagonism toward D2 receptors (D2R) in the mesolimbic pathway reduces psychotic symptoms.3 Accord© XXXX American Chemical Society

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.4 The atypical antipsychotics (e.g., clozapine, 1, and olanzapine, 2, Chart 1), characterized by a multireceptor affinity profile which combines potent antagonism for serotonin 5-HT2A receptor (5HT2AR) to D2R, and D3R blockade,5 are 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 Received: August 21, 2014

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

Journal of Medicinal Chemistry

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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.14 Because 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. Recently, we identified a novel class of atypical antipsychotics, typified by 4.15 For 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 5HT2AR and partial agonism at serotonin 5-HT1AR, with a low affinity for D2R (no liability of EPS at antipsychotic doses) and 5-HT2CR (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-HT1AR and 5-HT2AR are particularly dense in PFC pyramidal neurons and GABA interneurons. In PFC N-methyl- D-aspartate (NMDA) receptors, channels are the target of 5-HT1AR and both receptors modulate the excitability of cortical neurons, thus affecting cognitive functions. Indeed, 5-HT1AR may be a therapeutic target for the development of improved antipsychotic drugs. Accordingly, 5-HT1AR 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-HT1AR activation, agonist properties at postsynaptic 5-HT1AR may be relevant to the therapeutic profile of atypical antipsychotic agents, improving negative

Chart 1. Reference Antipsychotics

Antipsychotic Trials of Intervention Effectiveness (CATIE) study6) and are accompanied by fewer EPS.7 Aripiprazole8 (3, Chart 1), also termed as generation III antipsychotic, has subnanomolar affinity for 5-HT2BR, D2LR, and D3R but also has significant affinity (5−30 nM) for several other 5-HT receptors (5-HT1A, 5-HT2A, 5-HT7) as well as for α1A-adrenergic and H1histamine receptors. Functionally, it behaves as an inverse agonist at 5-HT2BR and as partial agonist at 5-HT2AR, 5HT2CR, D3R, and D4R.9 Excluding 1, atypical antipsychotics are currently recommended as first-line agents for acute and maintenance therapy of schizophrenia.10 In the last 10 years, newer agents have been introduced (paliperidone, asenapine, iloperidone, and lurasidone).11 However, 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-HT2AR, inhibits neuronal activity in the substantia nigra and ventral tegmental area. 5-HT2AR 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-HT2AR antagonism may prevent or alleviate EPS induced by acute or long-term treatment with typical drugs such as haloperidol. D3R has been claimed as a potential target for antischizophrenic 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 proved15 that 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-HT1AR and 5-HT2AR, respectively. Accordingly,15 we selected the arylalkylpiperazine system as a flexible scaffold for achieving a fine balancing of dopamine D3R and serotonin 5-HT1AR and 5-HT2AR affinity, reducing D1, D2 and 5-HT2C receptor occupancy (4, Chart 2). Starting from the hit 4,15−20 the 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 (nanomolar 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-HT1AR, and 5-HT2AR and much lower affinity for D2R.

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

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 Scheme 2. Synthesis of Compound 5ha

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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 converted into the corresponding 4-hydroxybutylamides 7a−f by coupling with 4-aminobutanol, in the presence of 1hydroxybenzotriazole (HOBt) and N,N-dicyclohexylcarbodiimide (DCC) (or N-(3-(dimethylamino)propyl)-N′-ethylcarbodiimide hydrochloride (EDC) for the quinolin-6-yl carboxylic acid 6b). These latter amides after bromination of the hydroxylgroup (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.20 NArylation 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-bromobutanenitrile 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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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).23,24 Starting from the benzofuran-2carboxamide derivative 4, we explored the bioisosteric replacement of the benzofurancarboxamide oxygen with an NH, thus developing indole-2-carboxamide and indole-3carboxamide analogues (5g−i). We further explored the size of the nitrogen containing heterocycles23 by introducing various quinolines and isoquinolines (isoquinoline-2-carboxamides (5j−s), quinolin-2-carboxamides (5t−w), tetrahydroquinolin-2-carboxamides (5x−aa), a quinolin-6-carboxamide (5bb)) and pyridines (picolinamide (5gg−jj), a 6-methylpicolinamide (5kk), and a nicotinamide (5ll)). According to our pharmacological hypothesis, the novel arylpiperazines were tested on D2R, D3R, 5-HT1AR, and 5HT2AR. 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 Relationship (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,15 the dimethyl-substituted analogue 5b became a two-digit nM ligand for D2R and 5-HT2CR and a more potent ligand for the three receptors of interest (D3R, 5-HT1AR, and 5-HT2AR). Introduction of a polar 3-methoxy group (5c vs 5a) lowered D3R affinity by almost half, while the affinity against 5-HT2AR was increased by 10-fold. The presence of a 6-methylpyridine (5d) slightly decreased D3R affinity while increasing 5-HT2CR 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 characterized by an undesired profile (high affinity for D2R and 5-HT2CR of 5g and low affinity for 5-HT2AR of 5h). Compound 5i, with a 1-methylindole-3-carboxamide, was found equipotent at 5HT2AR and 5-HT2CR. Within the benzofurancarboxamide- and indolecarboxamidebased 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 compounds (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-HT2CR. Given that arylpiperazines are often metabolized by hydrox-

The synthesis of compounds (R)-5x,y and (S)-5z,aa (Scheme 3) started from quinaldic acid (16), which was Scheme 3. Synthesis of Compounds (R)-5x,y and (S)-5z,aaa

a

Reagents and conditions: (a) PtO2, H2, MeOH, rt, 16 h; (b) SOCl2, MeOH, rt, 16 h; (c) α-chymotrypsin phosphate buffer; (d) CbzCl, NaHCO3 2M, rt, 90 min; (e) NaOH, MeOH/H2O, reflux, 2 h; (f) 4amino-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 αchymotrypsin that selectively hydrolyses the R-isomer of the ester (±)-18,21 giving a separate mixture of (S)-19 and (R)18.22 Both 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.

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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-HT1AR, and 5-HT2AR) of 4 in order to select a potent antipsychotic agent for further pharmacological investigation.15,19,20 A wide SAR analysis was performed by merging specific heterocyclic systems (bicyclic or monoD



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Table 1. Binding Affinities for D2, D3, 5-HT1A, 5-HT2A, and 5-HT2C Receptors (Ki nM)a of Benzofuran-2-carboxamideBased 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, 15 each 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-carboxamide analogues (5x−aa). By comparing (R)-tetrahydroquinolin-2-carboxamides vs the (S)-tetrahydroquinolin-2-carboxamides (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 5HT1A R. (R)-5x was 8.6 times more potent than the corresponding (S)-isomer (5z); on the contrary, 5-HT2AR 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 5HT1AR, 5-HT2AR, and 5-HT2CR, respectively. Within this series, the quinolin-6-carboxamide analogue 5bb showed a subnanomolar affinity at D3R combined with two-digit nanomolar potency at 5-HT1AR and 5-HT2AR when tested on the receptor panel (Table 2). Much lower potency was found at D2R and 5-HT2CR. 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-HT1AR, losing antipsychotic efficacy.15 Introduction of an E



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Table 2. Binding Affinities for D2, D3, 5-HT1A, 5-HT2A, and 5-HT2C Receptors (Ki nM)a of Quinolinecarboxamide-Based Analogues 5j−bb, 4, and Reference Compounds (Clozapine (1), Olanzapine (2), Aripiprazole (3), and Risperidone)

Table 3. Binding Affinities for D2, D3, 5-HT1A, 5-HT2A, and 5-HT2C Receptors (Ki nM)a of Benzoylamide-Based and 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-HT2AR affinity, being selective at the 5-HT1AR 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. 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 models.15,25,26 The 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 neurotransmission might contribute to the pathogenesis of schizophrenia.27,28 It is well-known that systemic administration of PCP or MK801 increases dopamine cell firing rate in the brain.29 In particular, hyperactivity produced by a low dose of MK801 is dependent upon D3R stimulation.30 Thus, the hyperactivity induced by MK801 can be reduced in vivo by

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;15 each value is the mean of three determinations and SD were within 10% of the mean.

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 shapedependent 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 F



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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, and Haloperidol Tested Under Identical Conditionsa

a Tests performed as in previously described15 and compounds were dosed subcutaneously (sc) unless otherwise noted (po: orally). bEM: spontaneous exploratory locomotor activity. cMK801: MK801-induced locomotor activity. dAMP: methamphetamine-induced locomotor activity. e PCP: PCP-induced locomotor activity. fMED: minimal effective dose; the minimal statistically significant dose that reduce locomotor activity as compared to vehicle or relevant psychostimulant. gData from ref 15. hNT: not tested.

multitarget antipsychotic drugs characterized by high affinity and selectivity degree for D3R.27,30 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

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 MK801induced hyperactivity, also caused tremors at 30 mg/kg. Accordingly, the evaluation of 5b was discontinued. The quinolin-2-carboxamide 5u reduced MK801-induced hyperactivity 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 dosedependently 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, G



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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 (Ki = 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 AMPinduced 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). 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 administration 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 D3Rselective doses31 but, rather, the D3R-preferring antagonists stimulate locomotor behavior.26,32 This suggests that postsynaptic D3R exert inhibitory actions on psychomotor functions.33 Accordingly, 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 neuropsychiatric disorders. The intriguing profile that emerged for 5bb prompted us to further test this analogue both in vitro and in vivo. 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 Ki values are means of two independent determinations; 5bb was tested in five different concentrations

and twice at each single dose. 5bb showed a significant to moderate affinity for the α1 and α2 adrenoreceptors (Ki = 50 and 150 nM, respectively) and histamine H1 receptor subtype (Ki = 110 nM) (Table 5). In vivo, we observed a slight Table 5. In Vitro Receptor Binding Profile of 5bb on α1, α2, and H1 Receptors, Functional Activity on D3R, 5-HT1AR, 5HT2AR, and hERG Affinity compd 5bb

α2 α1 (Ki) (Ki) (nM) (nM) 50

150

H1R (Ki) (nM)

D3Ra (IC50) (μM)

5-HT1ARb (EC50) (μM)

5-HT2ARc (IC50) (μM)

hERGd (IC50) (μM)

110

0.258

1.8

2.6

∼10

a 35

[ S]GTPγS binding at D3R were performed in membranes prepared from CHO-K1 cells expressing human D3R. b[35S]GTPγS binding at 5-HT1AR expressed in CHO cells.; cRat aortic ring by measuring contraction response relative to 3 μM 5-HT. d5bb 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.

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-HT2AR and partial agonism at 5-HT1AR to a low affinity for dopamine D2R and 5-HT2CR, 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 IC50 value 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-HT1AR 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 EC50 value at 5HT1AR was 1.8 μM (Table 5). 5-HT2AR 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-HT2AR 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 electrophysiological assays (test was performed as previously described 15). 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 IC50 value 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. Cardiotoxicity 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.34 Consequently, efforts to predict H



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Table 6. Effects of 5bb on HR, RR, PQ, QRS, QT, and QTc in Langendorff Perfused Rat Heartsa 5bb (μM) none 0.01 0.1 1 10

HR (BPM) 296.25 302.20 308.58 297.70 280.67

± ± ± ± ±

13.61 12.22 10.64 15.74 20.55

RR (ms) 203.61 199.43 194.96 204.53 218.07

± ± ± ± ±

9.17 7.38 6.46 11.10 14.74

PQ (ms) 41.98 41.95 42.32 41.51 44.51

± ± ± ± ±

QRS (ms)

2.55 2.34 1.82 2.18 1.67

12.33 13.17 12.70 13.38 14.24

± ± ± ± ±

0.29 0.28 0.27 0.35 0.36

QT (ms) 71.61 70.61 71.40 71.03 73.96

± ± ± ± ±

4.21 3.34 3.85 4.50 3.91

QTc (ms) 159.41 158.61 162.06 158.14 159.76

± ± ± ± ±

11.21 8.33 9.05 11.79 10.69

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.

a

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/mm2 in all regions. *P < 0.05, **P < 0.01 when compared to respective vehicle-treated group.

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-HT1AR agonist, can convert the “typical” pattern of haloperidol on c-fos expression into a pattern resembling that of 1.38 It 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-HT1AR 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 fearmotivated 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

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 Antipsychotic 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,36,37 5bb produced, 1 h after its administration,15 a 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 I



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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.42 First, 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 MK801induced 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 hydrochloride (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.43 Recent findings suggest that the 5HT1AR is involved in the marble-burying behavior.44 In fact, the atypical antipsychotic 3 was found to inhibit the marble-burying behavior in a 5-HT1AR-dependent fashion.45 In the marble burying test, treatment with 5bb dosedependently 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 antipsychotics (e.g., ziprasidone)46 in the treatment of cognitive impairment may be due to a profile based on D2R and 5-HT2AR antagonism associated with 5-HT1AR (partial) agonism.47 This profile, especially with regards to 5HT1AR 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.40 In 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,41 there 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-HT1AR involvement could be relevant for the J



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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 dopamine efflux with an effect which lasted for 90 min and with a maximum efflux measured after 90 min. Notably, 5bb did not induce an enhancement above the basal level of dopamine efflux measured during the time interval 0−90 min in the nucleus accumbens.

Figure 5. Effects of 5bb on marble burying in mice. Oral pretreatment 30 min before test start with 5bb in increasing doses reduced the number of buried marbles as compared to vehicle treatment, with 30 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.

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CONCLUSIONS In summary, we have herein described a further exploitation of the SAR of arylpiperazine-based antipsychotics. D3R potent antagonism combined to 5-HT1AR partial agonism and 5HT2AR antagonism was recently identified by us as a novel paradigm for the development of innovative antipsychotics devoid of D2R mediated side effects. Low 5-HT2CR 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.47 For 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



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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. N-(4-(4-(2,3-Dimethylphenyl)piperazin-1-yl)butyl)benzo[b]furan2-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) 151−152 °C. 1H 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). ESIMS 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-2carboxamide (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); mp (MeOH) 104−105 °C. 1H 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-2carboxamide (5d). Starting from 8a and arylpiperazine 10a and following the procedure described for 5a, the title compound was obtained as white solid (75% yield); mp (MeOH) 107−109 °C. 1H 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-carboxamide (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 (JC−F = 81.1 Hz), 119.1 (JC−F = 13.0 Hz), 122.5 (JC−F = 33.0 Hz), 122.9, 123.9, 124.7 (JC−F = 13.0 Hz), 127.0, 127.8, 140.2 (JC−F = 33.0 Hz), 149.3, 155.5 (JC−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-2carboxamide (5g). Starting from 8b and (3-trifluoromethyl)phenylpiperazine and following the procedure described for 5a, the title compound was obtained as yellow oil (56% yield). 1H 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-carboxamide (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 used in the following step without any further purification. 1H 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%

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-HT1AR partial agonism and 5-HT2AR 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.

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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 used for column chromatography. 1H 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 Electrothermal 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-carboxamide (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, L



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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. N-(4-(4-(3-Methoxyphenyl)piperazin-1-yl)butyl)-1-methyl-1H-indole-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) 141−142 °C. 1H 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-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 solid (76% yield); mp (MeOH) 153−154 °C. 1H 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-carboxamide (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-(3-Methoxyphenyl)piperazin-1-yl]butyl]isoquinoline-3carboxamide (5l). Starting from 8d (1.0 g, 2.39 mmol) and 1-(3methoxy)phenylpiperazine and following the procedure described for 5a, the title compound was obtained as colorless oil (54% yield). 1H 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-carboxamide (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); mp (MeOH) 108−109 °C. 1H 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-3carboxamide (5n). Starting from 8d and arylpiperazine 10a 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 1.71 (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). ESIMS m/z 426 [M + Na]+, 404 (100) [M + H]+. Anal. (C24H29N5O) C, H, N. N-(4-(4-(Pyrimidin-2-yl)piperazin-1-yl)butyl)isoquinoline-3-carboxamide (5o). Starting from 8d and 1-(2-pyrimidyl)piperazine dihydrochloride in dry MeCN and following the procedure described for 5a, the title compound was obtained as yellow oil (81% yield). 1H 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,

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-carboxamide (5p). Starting from 8d and arylpiperazine 10c and following the procedure described for 5a, the title compound was obtained as white solid (70% yield); mp (MeOH) 153−154 °C. 1H 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. N-(4-(4-(3-Fluorophenyl)piperazin-1-yl)butyl)isoquinoline-3-carboxamide (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 (JC−F = 24.6 Hz), 105.9 (JC−F = 21.4 Hz), 111.2 (JC−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 (JC−F = 241.9 Hz), 165.1. ESI-MS m/z 407 [M + H]+. Anal. (C24H27FN4O) C, H, N. N-(4-(4-(4-Fluorophenyl)piperazin-1-yl)butyl)isoquinoline-3-carboxamide (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 °C. 1H 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-3carboxamide (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-Methylphenyl)piperazin-1-yl)butyl)quinoline-2-carboxamide (5u). Starting from 8e and m-tolylpiperazine and following the procedure described for 5a, the title compound was obtained as yellow amorphous solid (51% yield). 1H 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-(3-Methoxyphenyl)piperazin-1-yl]butyl]quinoline-2-carboxamide (5v). Starting from 8e and 1-(3-methoxy)phenylpiperazine and following the procedure described for 5a, the title compound was obtained as white amorphous solid (55% yield). 1H NMR 200 MHz (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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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% yield). 1H NMR 200 MHz (CDCl3) δ 1.68 (m, 4H), 2.49 (m, 4H), 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. N-(4-(4-(Quinolin-3-yl)piperazin-1-yl)butyl)benzamide (5ff). Starting 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. 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 (82% yield). 1H 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. 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 obtained as yellow oil (72% yield). 1H 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). ESIMS m/z 369 [M + H]+. Anal. (C21H28N4O2) C, H, N. 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. 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-methylpyridine-2-carboxamide (5kk). Starting from 8i and 4-(3methoxyphenyl)piperazine and following the procedure described for 5a, the title compound was obtained as yellow oil (78% yield). 1H 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-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); mp (MeOH) 119−120 °C. 1H 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 6-Quinolinecarboxylic Acid (6b). To a solution of 6-methylquinoline (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

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. N-(4-(4-(Pyridin-2-yl)piperazin-1-yl)butyl)quinoline-2-carboxamide (5w). Starting from 8e and 1-(2-pyridin-2-yl)piperazine hydrochloride and following the procedure described for 5a, the title compound was obtained as white amorphous solid (64% yield). 1H 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. (R)-(+)-N-[4-(4-Phenylpiperazin-1-yl)butyl]-1,2,3,4-tetrahydroquinoline-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]20D = +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. (R)-(+)-N-[4-[4-(m-Tolyl)piperazin-1-yl]butyl]-1,2,3,4-tetrahydroquinoline-2-carboxamide (R)-(5y). Starting from (R)-22b and following the procedure described for (R)-5x, the title compound was obtained as colorless oil (85% yield); [α]20D = +42.1° (c 1.26, MeOH). 1H NMR 200 MHz (CDCl3) δ 1.20−1.58 (m, 4H), 1.86− 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. (S)-(−)-N-[4-(4-Phenylpiperazin-1-yl)butyl]-1,2,3,4-tetrahydroquinoline-2-carboxamide (S)-(5z). To a solution of (S)-22a and following the procedure described for (R)-5x, the title compound was obtained as colorless oil (67% yield); [α]20D = −41.7° (c 0.27, 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-tetrahydroquinoline-2-carboxamide (S)-(5aa). Starting from (S)-22b and following the procedure described for (R)-5x, the title compound was obtained as colorless oil (90% yield); [α]20D = −42.1° (c 1.26, 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 solid (65% yield); mp (MeOH) 151−152 °C. 1H 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. 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 solid (82% yield); mp (MeOH) 102−103 °C. 1H 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 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 solid (85% yield); mp (MeOH) 120−121 °C. 1H 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. ESIMS m/z 196 [M + Na]+, 174 (100) [M + H]+. Anal. (C10H7NO2) C, H, N. 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 as colorless oil (85% yield). 1H NMR 200 MHz (CDCl3) δ 1.51 (m, 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]+. 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. 1 H 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). ESIMS 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 obtained as yellow oil (99% yield). 1H NMR 200 MHz (CDCl3) δ 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]+. N-(4-Hydroxybutyl)-6-methylpicolinamide (7e). Starting from 6methylpicolinic acid 6e and following the procedure described for 7a, the title compound was obtained as yellow oil (80% yield). 1H 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]+. 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 (84% yield). 1H NMR 300 MHz (CDCl3) δ 1.96 (m, 4H), 3.56 (m,

4H), 3.66 (s, 3H), 7.28 (m, 4H), 7.60 (m, 1H), 9.80 (br s, 1H). ESIMS 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 obtained as amorphous yellow solid (75% yield). 1H 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 yellow oil (70% yield). 1H 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 obtained as amorphous white solid. 1H 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]+. ESIMS m/z 280 [M + Na]+, 258 (100) [M + H]+. N-(4-Bromobutyl)-6-methylpicolinamide (8i). Starting from 7e and following the procedure described for 8c, the title compound was obtained as amorphous white solid. 1H 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-Bromobutyl)nicotinamide (8j). Starting from 7f and following the procedure described for 8c, the title compound was obtained as yellow oil (60% yield). 1H 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 as colorless oil (78% yield). 1H NMR 300 MHz (CDCl3) δ 1.47 (s, 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 (JC−F = 24.7 Hz), 106.5 (JC−F = 21.4 Hz), 111.8 (2), 130.4 (JC−F = 9.9 Hz), 153.1 (JC−F = 9.9 Hz), 154.9, 164.0 (JC−F = 242.5 Hz). ESI-MS m/z 303 [M + Na]+, 281 (100) [M + H]+. 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 (MeOH) 114−115 °C. 1H NMR 200 MHz (CDCl3) δ 1.46 (s, 9H), 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). 1 H NMR 300 MHz (CDCl3) δ 5.16 (s, 2H), 7.08 (m, 2H), 7.64 (m, 7H). Starting from 1-(benzyloxy)-4-bromobenzene and following the procedure described for 9b, while heating at 90 °C for 15 h, the title compound was obtained as a colorless oil (30% yield). 1H NMR 300 MHz (CDCl3) δ 1.49 (s, 9H), 3.01 (m, 4H), 3.58 (m, 4H), 5.02 (s, 2H), 6.91 (m, 4H), 7.35 (m, 5H). ESI-MS m/z 369 [M + H]+. O



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yield). 1H 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 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 NaHCO3 and 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]20D +41.3 (c 0.58, CHCl3); spectroscopic data were identical to those previously reported for the racemate and were consistent with the literature.22 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. (S)-(−)-1-(Benzyloxycarbonyl)-1,2,3,4-tetrahydroquinoline-2-carboxylic 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 NaHCO 3 benzyl 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 compound as colorless oil (80%yield); [α]20D = −50.0° (c 0.94, MeOH). 1H 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]20D = −50.0° (c 0.98, MeOH). 1H 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),

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 (80% yield). 1H 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). 13 C NMR 75 MHz (CD3OD) δ 43.4 (2), 46.1 (2), 103.4 (JC−F = 25.3 Hz), 107.0 (JC−F = 21.4 Hz), 112.0 (2), 130.4 (JC−F = 9.9 Hz), 152.2 (JC‑F = 9.9 Hz), 164.0 (JC−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]+. 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 10b, the title compound was obtained as viscous oil (98% yield). 1H 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-3carboxamide (11). Starting from 8d and 1-(4-(benzyloxy)phenyl)piperazine 10d and following the procedure described for 5a, the title compound was obtained as colorless oil (55% yield). 1H NMR 300 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-bromobutanenitrile (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% nhexane in EtOAc) to give pure title compound as yellow oil (60% P



Article

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 Università 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, spontaneously 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, CaCl2 2.5, MgSO4 1.2, NaHCO3 25, KH2PO4 1.2, glucose 11.5, Na pyruvate 2, and EDTA 0.5, bubbled with a 95% O2−5% CO2 gas mixture (pH 7.4), and kept at 37 °C, as described elsewhere.48 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). 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). 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. Behavioral Tests and in Vivo Tests. Catalepsy tests,15 PCP- and AMP-induced hyperactivity tests,15 PPI tests,42 and in vivo microdialysis studies50 were performed as previously described. 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

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. (R)-(+)-1-(Benzyloxycarbonyl)-1,2,3,4-tetrahydroquinoline-2-carboxylic 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 KHSO4 while cooling at 0 °C), title compound was obtained as amorphous solid (81% yield); [α]20D = +47.6° (c 0.42, MeOH); spectroscopic data were identical to those already reported for (S)-19. (R)-(+)-N-(4-Hydroxybutyl)-1-(benzyloxycarbonyl)-1,2,3,4-tetrahydroquinoline-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]20D = +41.9° (c 1.94, CHCl3). 1H 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. (S)-(−)-N-(4-Hydroxybutyl)-1-(benzyloxycarbonyl)-1,2,3,4-tetrahydroquinoline-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]20D −50.0° (c 1.56, CHCl3); spectroscopic data were identical to those reported for (R)20. (R)-(+)-N-(4-Bromobutyl)-1-(benzyloxycarbonyl)-1,2,3,4-tetrahydroquinoline-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]20D = +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-tetrahydroquinoline-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]20D = −50.9° (c 0.53, CHCl3); spectroscopic data were identical to those reported for (R)-22. (R)-(+)-N-[4-(4-Phenylpiperazin-1-yl)butyl]-1-(benzyloxycarbonyl)-1,2,3,4-tetrahydroquinoline-2-carboxamide (R)-(22a). Starting from (R)-21 and 1-phenylpiperazine and following the procedure described for 5a, the title compound was obtained as colorless oil (80% yield); [α]20D = +30.7° (c 0.19, CHCl3). 1H NMR 200 MHz (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]+. (S)-(−)-N-[4-(4-Phenylpiperazin-1-yl)butyl]-1-(benzyloxycarbonyl)-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]20D = −31.0° (c 0.19, CHCl3); spectroscopic data were identical to those reported for (R)-22a. (R)-(+)-N-[4-[4-(m-Tolyl)piperazin-1-yl]butyl]-1-(benzyloxycarbonyl)-1,2,3,4-tetrahydroquinoline-2-carboxamide (R)-(22b). Starting 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]20D = +34.3° (c 0.19, CHCl3). 1 H 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). (S)-(−)-N-(4-(4-m-Tolylpiperazin-1-yl)butyl)-1-(benzyloxycarbonyl)-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 yellow oil (41% yield); [α]20D = −34.2° (c 1.75, CHCl3); spectroscopic data were identical to those reported for (R)-22b. Q



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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.

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. 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% CO2 and 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 CaCl2 and 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. Analysis of Neurotransmitters. The concentrations of dopamine were determined by HPLC with electrochemical detection. The column was a Prodigy 3 μ ODS (3) C18 (2 mm × 100 mm, particle

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ASSOCIATED CONTENT

S Supporting Information *

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.

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AUTHOR INFORMATION

Corresponding Authors

*For G.C.: phone, 0039 0577 234172; fax, 0039 0577 234254; E-mail, [email protected]. *For S.B.: phone, 0039 0577 234161; fax, 0039 0577 234254; E-mail, [email protected]. Present Address

◆ For K.S.N.: Saniona Aps, Baltorpvej 154, DK2750 Ballerup, Denmark.

Notes

The authors declare no competing financial interest.

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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.

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ABBREVIATIONS USED EPS, extrapyramidal side effects; D2R, dopamine D2 receptors; 5-HT2AR, serotonin 5-HT2A receptor; D3R, dopamine D3 receptor; 5-HT1AR, serotonin 5-HT1A receptor; 5-HT2CR, serotonin 5-HT2C receptor; CATIE, Clinical Antipsychotic Trials of Intervention Effectiveness; 5-HT2BR, serotonin 5HT2B receptor; D4R, dopamine D4 receptor; hERG, human ether-à-go-go related gene; Cbz, carboxybenzyl; DCC, N,N1dicyclohexylcarbodiimide; EDC, N-(3-(dimethylamino)propyl)-N′-ethylcarbodiimide hydrochloride; HOBt, 1-hydroxybenzotriazole; 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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REFERENCES

(1) Insel, T. R. Rethinking schizophrenia. Nature 2010, 468, 187− 193. (2) Davis, K. L.; Kahn, R. S.; Ko, G.; Davidson, M. Dopamine in schizophrenia: a review and reconceptualization. Am. J. Psychiatry 1991, 148, 1474−1486. R



Article

(3) Seeman, P. Dopamine receptor sequences. Therapeutic levels of neuroleptics occupy D2 receptors, clozapine occupies D4. Neuropsychopharmacology 1992, 7, 261−284. (4) Miyamoto, S.; Miyake, N.; Jarskog, L. F.; Fleischhacker, W. W.; Lieberman, J. A. Pharmacological treatment of schizophrenia: a critical review of the pharmacology and clinical effects of current and future therapeutic agents. Mol. Psychiatry 2012, 17, 1206−1227. (5) Campbell, M.; Young, P. I.; Bateman, D. N.; Smith, J. M.; Thomas, S. H. The use of atypical antipsychotics in the management of schizophrenia. Br. J. Clin. Pharmacol. 1999, 47, 13−22. (6) Lieberman, J. A.; Stroup, T. S.; McEvoy, J. P.; Swartz, M. S.; Rosenheck, R. A.; Perkins, D. O.; Keefe, R. S.; Davis, S. M.; Davis, C. E.; Lebowitz, B. D.; Severe, J.; Hsiao, J. K. Effectiveness of antipsychotic drugs in patients with chronic schizophrenia. N. Engl. J. Med. 2005, 353, 1209−1223. (7) Leucht, S.; Corves, C.; Arbter, D.; Engel, R. R.; Li, C.; Davis, J. M. Second-generation versus first-generation antipsychotic drugs for schizophrenia: a meta-analysis. Lancet 2009, 373, 31−41. (8) Buckley, P. F. Aripiprazole: efficacy and tolerability profile of a novel-acting atypical antipsychotic. Drugs Today 2003, 39, 145−151. (9) Shapiro, D. A.; Renock, S.; Arrington, E.; Chiodo, L. A.; Liu, L. X.; Sibley, D. R.; Roth, B. L.; Mailman, R. Aripiprazole, a novel atypical antipsychotic drug with a unique and robust pharmacology. Neuropsychopharmacology 2003, 28, 1400−1411. (10) Buchanan, R. W.; Kreyenbuhl, J.; Kelly, D. L.; Noel, J. M.; Boggs, D. L.; Fischer, B. A.; Himelhoch, S.; Fang, B.; Peterson, E.; Aquino, P. R.; Keller, W. The 2009 schizophrenia PORT psychopharmacological treatment recommendations and summary statements. Schizophr. Bull. 2010, 36, 71−93. (11) Miyake, N.; Miyamoto, S.; Jarskog, L. F. New serotonin/ dopamine antagonists for the treatment of schizophrenia: are we making real progress? Clin. Schizophr. Relat. Psychoses 2012, 6, 122− 133. (12) Glassman, A. H.; Bigger, J. T., Jr. Antipsychotic drugs: prolonged QTc interval, torsade de pointes, and sudden death. Am. J. Psychiatry 2001, 158, 1774−1782. (13) Ray, W. A.; Chung, C. P.; Murray, K. T.; Hall, K.; Stein, C. M. Atypical antipsychotic drugs and the risk of sudden cardiac death. N. Engl. J. Med. 2009, 360, 225−235. (14) Millan, M. J.; Fone, K.; Steckler, T.; Horan, W. P. Negative symptoms of schizophrenia: clinical characteristics, pathophysiological substrates, experimental models and prospects for improved treatment. Eur. Neuropsychopharmacol. 2014, 24, 645−692. (15) Butini, S.; Gemma, S.; Campiani, G.; Franceschini, S.; Trotta, F.; Borriello, M.; Ceres, N.; Ros, S.; Coccone, S. S.; Bernetti, M.; De Angelis, M.; Brindisi, M.; Nacci, V.; Fiorini, I.; Novellino, E.; Cagnotto, A.; Mennini, T.; Sandager-Nielsen, K.; Andreasen, J. T.; Scheel-Kruger, J.; Mikkelsen, J. D.; Fattorusso, C. Discovery of a new class of potential multifunctional atypical antipsychotic agents targeting dopamine D3 and serotonin 5-HT1A and 5-HT2A receptors: design, synthesis, and effects on behavior. J. Med. Chem. 2009, 52, 151−169. (16) Campiani, G.; Butini, S.; Gemma, S.; Nacci, V.; Fattorusso, C.; Catalanotti, B.; Giorgi, G.; Cagnotto, A.; Goegan, M.; Mennini, T.; Minetti, P.; Di Cesare, M. A.; Mastroianni, D.; Scafetta, N.; Galletti, B.; Stasi, M. A.; Castorina, M.; Pacifici, L.; Ghirardi, O.; Tinti, O.; Carminati, P. Pyrrolo[1,3]benzothiazepine-based atypical antipsychotic agents. Synthesis, structure−activity relationship, molecular modeling, and biological studies. J. Med. Chem. 2002, 45, 344−359. (17) Campiani, G.; Butini, S.; Fattorusso, C.; Catalanotti, B.; Gemma, S.; Nacci, V.; Morelli, E.; Cagnotto, A.; Mereghetti, I.; Mennini, T.; Carli, M.; Minetti, P.; Di Cesare, M. A.; Mastroianni, D.; Scafetta, N.; Galletti, B.; Stasi, M. A.; Castorina, M.; Pacifici, L.; Vertechy, M.; Di Serio, S.; Ghirardi, O.; Tinti, O.; Carminati, P. Pyrrolo[1,3]benzothiazepine-based serotonin and dopamine receptor antagonists. Molecular modeling, further structure−activity relationship studies, and identification of novel atypical antipsychotic agents. J. Med. Chem. 2004, 47, 143−157. (18) Campiani, G.; Butini, S.; Fattorusso, C.; Trotta, F.; Gemma, S.; Catalanotti, B.; Nacci, V.; Fiorini, I.; Cagnotto, A.; Mereghetti, I.;

Mennini, T.; Minetti, P.; Di Cesare, M. A.; Stasi, M. A.; Di Serio, S.; Ghirardi, O.; Tinti, O.; Carminati, P. Novel atypical antipsychotic agents: rational design, an efficient palladium-catalyzed route, and pharmacological studies. J. Med. Chem. 2005, 48, 1705−1708. (19) Campiani, G.; Butini, S.; Trotta, F.; Fattorusso, C.; Catalanotti, B.; Aiello, F.; Gemma, S.; Nacci, V.; Novellino, E.; Stark, J. A.; Cagnotto, A.; Fumagalli, E.; Carnovali, F.; Cervo, L.; Mennini, T. Synthesis and pharmacological evaluation of potent and highly selective D3 receptor ligands: inhibition of cocaine-seeking behavior and the role of dopamine D3/D2 receptors. J. Med. Chem. 2003, 46, 3822−3839. (20) Butini, S.; Campiani, G.; Franceschini, S.; Trotta, F.; Kumar, V.; Guarino, E.; Borrelli, G.; Fiorini, I.; Novellino, E.; Fattorusso, C.; Persico, M.; Orteca, N.; Sandager-Nielsen, K.; Jacobsen, T. A.; Madsen, K.; Scheel-Kruger, J.; Gemma, S. Discovery of bishomo(hetero)arylpiperazines as novel multifunctional ligands targeting dopamine D(3) and serotonin 5-HT(1A) and 5-HT(2A) receptors. J. Med. Chem. 2010, 53, 4803−4807. (21) Nagata, R.; Tanno, N.; Kodo, T.; Ae, N.; Yamaguchi, H.; Nishimura, T.; Antoku, F.; Tatsuno, T.; Kato, T.; Tanaka, Y.; Nakamura, M. Tricyclic quinoxalinediones: 5,6-dihydro-1H-pyrrolo[1,2,3-de] quinoxaline-2,3-diones and 6,7-dihydro-1H,5H-pyrido[1,2,3de] quinoxaline-2,3-diones as potent antagonists for the glycine binding site of the NMDA receptor. J. Med. Chem. 1994, 37, 3956− 3968. (22) Katayama, S.; Ae, N.; Nagata, R. Enzymatic resolution of 2substituted tetrahydroquinolines. Convenient approaches to tricyclic quinoxalinediones as potent NMDA−glycine antagonists. Tetrahedron: Asymmetry 1998, 9, 4295−4299. (23) Chen, J.; Ding, K.; Levant, B.; Wang, S. Design of novel hexahydropyrazinoquinolines as potent and selective dopamine D3 receptor ligands with improved solubility. Bioorg. Med. Chem. Lett. 2006, 16, 443−446. (24) Grundt, P.; Carlson, E. E.; Cao, J.; Bennett, C. J.; McElveen, E.; Taylor, M.; Luedtke, R. R.; Newman, A. H. Novel heterocyclic trans olefin analogues of N-{4-[4-(2,3-dichlorophenyl)piperazin-1-yl]butyl}arylcarboxamides as selective probes with high affinity for the dopamine D3 receptor. J. Med. Chem. 2005, 48, 839−848. (25) Reavill, C.; Taylor, S. G.; Wood, M. D.; Ashmeade, T.; Austin, N. E.; Avenell, K. Y.; Boyfield, I.; Branch, C. L.; Cilia, J.; Coldwell, M. C.; Hadley, M. S.; Hunter, A. J.; Jeffrey, P.; Jewitt, F.; Johnson, C. N.; Jones, D. N.; Medhurst, A. D.; Middlemiss, D. N.; Nash, D. J.; Riley, G. J.; Routledge, C.; Stemp, G.; Thewlis, K. M.; Trail, B.; Vong, A. K.; Hagan, J. J. Pharmacological actions of a novel, high-affinity, and selective human dopamine D(3) receptor antagonist, SB-277011-A. J. Pharmacol. Exp. Ther. 2000, 294, 1154−1165. (26) Manzanedo, C.; Aguilar, M. A.; Minarro, J. The effects of dopamine D2 and D3 antagonists on spontaneous motor activity and morphine-induced hyperactivity in male mice. Psychopharmacology 1999, 143, 82−88. (27) Javitt, D. C.; Zukin, S. R. Recent advances in the phencyclidine model of schizophrenia. Am. J. Psychiatry 1991, 148, 1301−1308. (28) Lahti, A. C.; Weiler, M. A.; Tamara Michaelidis, B. A.; Parwani, A.; Tamminga, C. A. Effects of ketamine in normal and schizophrenic volunteers. Neuropsychopharmacology 2001, 25, 455−467. (29) Murase, S.; Mathe, J. M.; Grenhoff, J.; Svensson, T. H. Effects of dizocilpine (MK-801) on rat midbrain dopamine cell activity: differential actions on firing pattern related to anatomical localization. J. Neural. Transm.: Gen. Sect. 1993, 91, 13−25. (30) Leriche, L.; Schwartz, J. C.; Sokoloff, P. The dopamine D3 receptor mediates locomotor hyperactivity induced by NMDA receptor blockade. Neuropharmacology 2003, 45, 174−181. (31) Waters, N.; Svensson, K.; Haadsma-Svensson, S. R.; Smith, M. W.; Carlsson, A. The dopamine D3-receptor: a postsynaptic receptor inhibitory on rat locomotor activity. J. Neural. Transm.: Gen. Sect. 1993, 94, 11−19. (32) Sautel, F.; Griffon, N.; Sokoloff, P.; Schwartz, J. C.; Launay, C.; Simon, P.; Costentin, J.; Schoenfelder, A.; Garrido, F.; Mann, A. Nafadotride, a potent preferential dopamine D3 receptor antagonist, S



Article

activates locomotion in rodents. J. Pharmacol. Exp. Ther. 1995, 275, 1239−1246. (33) Watson, D. J.; Marsden, C. A.; Millan, M. J.; Fone, K. C. Blockade of dopamine D(3) but not D(2) receptors reverses the novel object discrimination impairment produced by post-weaning social isolation: implications for schizophrenia and its treatment. Int. J. Neuropsychopharmacol. 2012, 15, 471−484. (34) Hancox, J. C.; McPate, M. J.; El Harchi, A.; Zhang, Y. H. The hERG potassium channel and hERG screening for drug-induced torsades de pointes. Pharmacol. Ther. 2008, 119, 118−132. (35) Gemma, S.; Camodeca, C.; Sanna Coccone, S.; Joshi, B. P.; Bernetti, M.; Moretti, V.; Brogi, S.; Bonache de Marcos, M. C.; Savini, L.; Taramelli, D.; Basilico, N.; Parapini, S.; Rottmann, M.; Brun, R.; Lamponi, S.; Caccia, S.; Guiso, G.; Summers, R. L.; Martin, R. E.; Saponara, S.; Gorelli, B.; Novellino, E.; Campiani, G.; Butini, S. Optimization of 4-aminoquinoline/clotrimazole-based hybrid antimalarials: further structure−activity relationships, in vivo studies, and preliminary toxicity profiling. J. Med. Chem. 2012, 55, 6948−6967. (36) Deutch, A. Y.; Lee, M. C.; Iadarola, M. J. Regionally specific effects of atypical antipsychotic drugs on striatal Fos expression: The nucleus accumbens shell as a locus of antipsychotic action. Mol. Cell. Neurosci. 1992, 3, 332−341. (37) Robertson, G. S.; Fibiger, H. C. Neuroleptics increase c-fos expression in the forebrain: contrasting effects of haloperidol and clozapine. Neuroscience 1992, 46, 315−328. (38) Tremblay, P. O.; Gervais, J.; Rouillard, C. Modification of haloperidol-induced pattern of c-fos expression by serotonin agonists. Eur. J. Neurosci. 1998, 10, 3546−3555. (39) Vianna, M. R.; Izquierdo, L. A.; Barros, D. M.; Ardenghi, P.; Pereira, P.; Rodrigues, C.; Moletta, B.; Medina, J. H.; Izquierdo, I. Differential role of hippocampal cAMP-dependent protein kinase in short- and long-term memory. Neurochem. Res. 2000, 25, 621−626. (40) Braff, D. L.; Geyer, M. A.; Swerdlow, N. R. Human studies of prepulse inhibition of startle: normal subjects, patient groups, and pharmacological studies. Psychopharmacology 2001, 156, 234−258. (41) Park, W. K.; Jeong, D.; Cho, H.; Lee, S. J.; Cha, M. Y.; Pae, A. N.; Choi, K. I.; Koh, H. Y.; Kong, J. Y. KKHA-761, a potent D3 receptor antagonist with high 5-HT1A receptor affinity, exhibits antipsychotic properties in animal models of schizophrenia. Pharmacol., Biochem. Behav. 2005, 82, 361−372. (42) Sandager-Nielsen, K.; Andersen, M. B.; Sager, T. N.; Werge, T.; Scheel-Kruger, J. Effects of postnatal anoxia on striatal dopamine metabolism and prepulse inhibition in rats. Pharmacol., Biochem. Behav. 2004, 77, 767−774. (43) Joel, D. Current animal models of obsessive compulsive disorder: a critical review. Prog. Neuropsychopharmacol. Biol. Psychiatry 2006, 30, 374−388. (44) Matsushita, M.; Egashira, N.; Harada, S.; Okuno, R.; Mishima, K.; Iwasaki, K.; Nishimura, R.; Fujiwara, M. Perospirone, a novel antipsychotic drug, inhibits marble-burying behavior via 5-HT1A receptor in mice: implications for obsessive-compulsive disorder. J. Pharmacol. Sci. 2005, 99, 154−159. (45) Egashira, N.; Okuno, R.; Matsushita, M.; Abe, M.; Mishima, K.; Iwasaki, K.; Nishimura, R.; Oishi, R.; Fujiwara, M. Aripiprazole inhibits marble-burying behavior via 5-hydroxytryptamine (5-HT)1A receptorindependent mechanisms. Eur. J. Pharmacol. 2008, 592, 103−108. (46) Harvey, P. D.; Meltzer, H.; Simpson, G. M.; Potkin, S. G.; Loebel, A.; Siu, C.; Romano, S. J. Improvement in cognitive function following a switch to ziprasidone from conventional antipsychotics, olanzapine, or risperidone in outpatients with schizophrenia. Schizophr. Res. 2004, 66, 101−113. (47) Ichikawa, J.; Ishii, H.; Bonaccorso, S.; Fowler, W. L.; O’Laughlin, I. A.; Meltzer, H. Y. 5-HT(2A) and D(2) receptor blockade increases cortical DA release via 5-HT(1A) receptor activation: a possible mechanism of atypical antipsychotic-induced cortical dopamine release. J. Neurochem. 2001, 76, 1521−1531. (48) Saponara, S.; Ferrara, A.; Gorelli, B.; Shah, A.; Kawase, M.; Motohashi, N.; Molnar, J.; Sgaragli, G.; Fusi, F. 3,5-D-4-(3phenoxyphenyl)-1,4-dihydro-2,6-dimethylpyridine (DP7): a new

multidrug resistance inhibitor devoid of effects on Langendorffperfused rat heart. Eur. J. Pharmacol. 2007, 563, 160−163. (49) Ferrara, A.; Fusi, F.; Gorelli, B.; Sgaragli, G.; Saponara, S. Effects of freeze-dried red wine on cardiac function and ECG of the Langendorff-perfused rat heart. Can. J. Physiol. Pharmacol. 2014, 92, 171−174. (50) Weikop, P.; Egestad, B.; Kehr, J. Application of triple-probe microdialysis for fast pharmacokinetic/pharmacodynamic evaluation of dopamimetic activity of drug candidates in the rat brain. J. Neurosci. Meth. 2004, 140, 59−65.

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