Article pubs.acs.org/jmc

Novel Arylsulfonamide Derivatives with 5‑HT6/5-HT7 Receptor Antagonism Targeting Behavioral and Psychological Symptoms of Dementia Marcin Kołaczkowski,*,†,‡ Monika Marcinkowska,‡ Adam Bucki,‡ Maciej Pawłowski,‡ Katarzyna Mitka,§ Jolanta Jaśkowska,§ Piotr Kowalski,§ Grzegorz Kazek,‡ Agata Siwek,‡ Anna Wasik,‡ Anna Wesołowska,‡ Paweł Mierzejewski,∥ and Przemyslaw Bienkowski∥ †

Adamed Ltd., Pieńków 149, 05-152 Czosnów, Poland Faculty of Pharmacy, Jagiellonian University Collegium Medicum, 9 Medyczna Street, 30-688 Cracow, Poland § Cracow University of Technology, 24 Warszawska Street, 31-155 Cracow, Poland ∥ Institute of Psychiatry and Neurology, 9 Sobieskiego Street, 02-957 Warsaw, Poland ‡

S Supporting Information *

ABSTRACT: In order to target behavioral and psychological symptoms of dementia (BPSD), we used molecular modelingassisted design to obtain novel multifunctional arylsulfonamide derivatives that potently antagonize 5-HT6/7/2A and D2 receptors, without interacting with M1 receptors and hERG channels. In vitro studies confirmed their antagonism of 5HT7/2A and D2 receptors and weak interactions with key antitargets (M1R and hERG) associated with side effects. Marked 5-HT6 receptor affinities were also observed, notably for 6fluoro-3-(piperidin-4-yl)-1,2-benzoxazole derivatives connected by a 3−4 unit alkyl linker with mono- or bicyclic, lipophilic arylsulfonamide moieties. N-[4-[4-(6-Fluoro-1,2-benzoxazol-3-yl)piperidin-1-yl]butyl]benzothiophene-2-sulfonamide (72) was characterized in vitro on 14 targets and antitargets. It displayed dual blockade of 5-HT6 and D2 receptors and negligible interactions at hERG and M1 receptors. Unlike reference antipsychotics, 72 displayed marked antipsychotic and antidepressant activity in rats after oral administration, in the absence of cognitive or motor impairment. This profile is particularly attractive when targeting a fragile, elderly BPSD patient population.

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INTRODUCTION Over 50% of patients with Alzheimer’s disease (AD) or with other dementias experience psychotic symptoms,1 and, together with other behavioral and psychological symptoms, their treatment constitutes a substantial unmet medical need. Indeed, effective pharmacological treatment of psychosis in elderly patients with cognitive deficits can be particularly intractable because psychotropic medications used in patients with dementia may themselves be a cause of further cognitive impairment.2 Moreover, the use of currently available antipsychotics in elderly patients with dementia has also been associated with other side effects, including motor, metabolic, or cardiac disturbances and, consequently, increased mortality.3 Despite “boxed warnings” issued by the Food and Drug Administration (FDA) for antipsychotics and their lack of approval for treatment of Behavioral and Psychological Symptoms of Dementia (BPSD),4,5 such drugs have been commonly used off-label.6 This is probably due to the fact that psychosis and physical aggression by dementia patients (rather than their cognitive impairment) are a major challenge for caregivers and are the most common cause of patient institutionalization.1,2 Therefore, the discovery of novel antipsychotic drugs with an improved safety profile and that © XXXX American Chemical Society

do not induce cognitive deficits would permit improved treatment of BPSD. At a pharmacological level, converging lines of evidence indicate that blockade of serotonin 5-HT6 receptors (5-HT6Rs) may be implicated in pro-cognitive effects due to the facilitation of cholinergic transmission,7,8 in antidepressant activity due to the increase in noradrenergic and dopaminergic tones, as well as in an anxiolytic effect, mediated by interaction with GABAergic transmission.9,10 These findings are further supported by the exclusive localization of 5-HT6 receptors in the central nervous system (CNS), especially in limbic and cortical brain areas involved in the control of mood and cognition.11 Recently, the selective 5-HT6R antagonists 3-phenylsulfonyl8-(piperazin-1-yl)quinoline (SB-742457) and [2-(6-fluoro-1Hindol-3-yl)-ethyl]-[3-(2,2,3,3-tetrafluoropropoxy)-benzyl]amine (Lu AE58054) showed pro-cognitive effects in phase II clinical trials with AD patients,12,13 confirming the therapeutic potential and relevance of 5-HT6R antagonists for dementia patients. Received: December 10, 2013

A

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Figure 1. Design of multifunctional ligands. Proposed binding modes presented for (1R)-3,N-dimethyl-N-[1-methyl-3-(4-methylpiperidin-1yl)propyl]benzenesulfonamide (SB-258719)24 in 5-HT7R (A) and compound 2 in the 5-HT6R (C) and hERG channel (D). Amino acid residues engaged in ligand binding (within 4 Å from the ligand atoms) are shown as thick sticks together with their van der Waals surfaces (wire frame). Distances between key amino acid moieties and ligand structural elements are shown in Supporting Information, Tables S1 and S2. The numbers in brackets next to the residues in the hERG channel indicate the subunit number. Docking of 2 in M1R resulted in no valid poses (E). Dotted yellow lines represent H-bonds with polar residues. For the sake of clarity, ECL2 was hidden (A and C). TMH, transmembrane helix; ECL, extracellular loop.

promising strategy for treating symptoms of BPSD while avoiding some of the side effects of current antipsychotic drugs. Nevertheless, due to the complex pathology of dementia and accompanying behavioral and psychological symptoms, it seems unlikely that focusing on a single therapeutic target would be sufficient to provide adequate clinical benefit, and it is likely that successful development of novel anti-BPSD agents should involve a “designed” multifactorial approach.17,18 Indeed, although some of the currently available antipsychotics block 5-HT6 or 5-HT7 receptors, they also interact with many other targets, including those that may elicit some side effects and/or limit (mask) their therapeutic efficacy. For example, these drugs also bind potently to muscarinic and histamine receptors, which are associated with cholinergic interference and sedation,

Another serotonin receptor subtype, 5-HT7, may play a role in the control of circadian rhythms, sleep, thermoregulation, pain, and migraine, as well as cognitive processes. Indeed, the high affinity and antagonistic activity of several antipsychotic and antidepressant drugs at 5-HT7 receptors suggest a potential role of these receptors in the pathophysiology of many neuropsychiatric disorders. Accordingly, selective 5-HT 7 receptor antagonists produce antidepressant and anxiolytic activity in rats and mice.14 Moreover, Galici et al. and Waters et al. showed that a selective 5-HT7 receptor antagonist (2R)-1[(3-hydroxyphenyl)sulfonyl]-2-(2-(4-methyl-1-piperidinyl)ethyl)pyrrolidine may also evoke antipsychotic-like effects.15,16 The above considerations suggest that targeting 5-HT6 and/ or 5-HT7 receptors with antagonist drugs could constitute a B

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Figure 2. Binding mode of compound 2 in (A) 5-HT7R, (B) 5-HT2AR, (C) D2R, and (D) 5-HT6R. Residues in the binding site (within 4 Å from the ligand atoms) are presented as thick sticks together with their van der Waals surfaces (wire frame). Distances between key amino acid moieties and ligand structural elements are shown in Supporting Information, Table S2. Dotted yellow lines represent H-bonds with polar residues. Where possible, ECL2 was partially or entirely hidden for the sake of clarity.

preferentially in the cavity between transmembrane helices (TMHs) 7−3 (Figure 1A). We also hypothesized that 5-HT7R ligands capable of forming strong interactions with this site may not be dependent on interactions with the cavity between TMHs 4−6, which is much more structurally conserved between monoaminergic GPCRs. Therefore, such ligands are likely to be more selective than those requiring specific interactions with the more conserved cavity between TMHs 4− 6. Considering the fact that our goal was to design multifunctional ligands acting on 5-HT7R, we found that the N-aminoalkylarylsulfonamide fragment is a suitable basis for structural modifications. Indeed, it provides strong 5-HT7R antagonism by anchoring between TMHs 7−3 (particularly Phe3.28 and Arg7.36) and allows for the introduction of additional moieties providing activity at other targets, such as the dopamine D2 receptor (D2R), serotonin 5-HT2A receptor (5-HT2AR), or serotonin transporter (SERT) (Figure 1B).22 In the present study, we focused on the moieties that block D2 and 5-HT2A receptors and exert potential antipsychotic-like activity. In view of this objective, we searched for commercially available fragments that could provide antagonism of D2 and 5-HT2A receptors and would be synthetically suitable for combination with the arylsulfonamide moiety by an alkyl linker. To this end, we analyzed structures of the D2/5-HT2A ligands available in the ChEMBL database (Ki < 10 nM for both receptors) and identified the arylamine moieties that could bind in the cavity between TMHs 4−6, which had been identified as a tolerant region for chemical expansion. Benzazolepiperazine/piperidine moieties were found to meet the above-mentioned criteria, and

respectively. In addition, they act as potent 5-HT2C receptor antagonists, a property which is a risk factor for metabolic dysfunction.19,20 Some authors proposed that an improved antipsychotic profile may be achieved by combining 5-HT6 antagonism with an absence of antimuscarinic activity, as is observed for sertindole, but not clozapine or olanzapine.21 However, clinical evaluation of sertindole was hampered by its arrhythmogenic potential, caused by potent hERG channel inhibition. On the basis of the above considerations, we designed a series of novel arylsulfonamide derivatives displaying high affinity for serotonin 5-HT6, 5-HT7, and 5-HT2A receptor subtypes, as well as dopamine D2 receptors, and devoid of significant interaction with muscarinic receptors or hERG channels.22 The present study describes computer-aided design and the synthesis, as well as the in vitro receptor profile of this new series of multimodal molecules. Moreover, the extensive pharmacological characterization of one of the most promising compounds, compound 72, is presented and discussed in relation to comparative data for 8 currently used reference antipsychotics.23

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RESULTS AND DISCUSSION Design of Multifunctional Ligands. In the course of our research into the structure of monoaminergic G proteincoupled receptors (GPCRs), we proposed a model of the binding mode of selective 5-HT 7 receptor (5-HT 7 R) antagonists.24 On the basis of molecular modeling studies, we concluded that the most selective 5-HT7 antagonists bind C

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Figure 3. General structures of hybrid molecules, benzazolepiperazines/piperidines I−III and arylsulfonyl fragments Ar (1−15).

(piperazin-1-yl)-1,2-benzothiazole moiety of compound 2 was found to form charge-reinforced H-bond interactions between the protonated nitrogen atom of piperazine and Asp3.32, as well as hydrophobic/aromatic interactions between the benzisothiazole moiety and the aromatic cluster of TMH6, showing very similar poses in this conserved pocket in all 4 receptors. The arylsulfonamide moiety occupies the second pocket, forming interactions between the aryl ring and particular aromatic residue (Phe/Trp3.28). Because of the relatively higher sequence diversity in the second pocket, there are some differences in the arrangement of the arylsulfonamide moiety between the receptors. For example, the aromatic residue in the 3.28 position determines the conformation of the latter. In the case of 5-HT6R and 5-HT2AR containing the Trp residue, the arylsulfonamide moiety expands between TMH1 and TMH2, while in 5-HT7R and D2R with the Phe residue, it kinks in between TMH2 and TMH3. Both of those geometries are additionally stabilized by H-bonds, formed between the sulfonamide group and Arg181, Arg7.36, or Ser226 of 5-HT6R, 5-HT7R, and 5-HT2AR, respectively. Encouraged by the promising in silico properties, which suggested high affinity for the most important therapeutic targets (5-HT6, 5-HT7, 5-HT2A, and D2 receptors), possible interactions with the undesired off-targets (called “antitargets”), hERG channels or M1 receptors, were also examined because blockade of these sites could negatively affect the compounds’ safety profile and mask potential therapeutic activity. In contrast to 5-HT6, 5-HT7, 5-HT2A, and D2 receptors, compound 2 was not optimally accommodated within the binding sites of the antitargets. In the case of the M1 receptor, the relatively long molecule could not freely expand throughout the binding site and form favorable interactions, similar to those observed in the targeted receptors, due to the

their drug-likeness was further supported by the fact that they could be found in some clinically used drugs.25 As a next step, a prototype hybrid molecule (compound 2) was designed by combining the 3-methyl-N-propylbenzenesulfonamide fragment with the 3-(piperazin-1-yl)-1,2-benzothiazole moiety (Figure 1C). The molecule was then docked to the models of 5-HT7, 5HT2A, and D2 receptors, showing good fit and favorable interactions that confirmed the assumed ligand binding mode at those targets (Figure 2A, B, and C, respectively). Because 5HT6R antagonism was considered a highly desirable feature of these ligands, the prototype, hybrid compound 2, was also docked to the 5-HT6R model, in order to assess the possibility of its interactions with this target or potential modifications required for this activity to occur. Surprisingly, compound 2 was very well accommodated by the 5-HT6 receptor, suggesting that it possessed marked affinity for this site, without particular modifications. The general orientation of the ligand in the binding site was similar to that observed for 5-HT7, 5-HT2A, or D2 receptors (Figures 1C and 2D). As mentioned above for 5-HT7R, the binding sites of 5HT2A, D2, and 5-HT6R are also divided into two pockets that extend to both sides of the main ligand-anchoring residue in monoaminergic receptors, Asp3.32. The first pocket is situated between TMHs 4−6, and its most important role in ligand binding lies in the formation of hydrophobic/aromatic interactions with aromatic clusters consisting of Phe6.51, Phe6.52, and Trp6.48, which are the conserved amino acid residues in these receptors. The second pocket is formed between TMHs 7−3, with the contribution of residues that ensure both aromatic (Phe3.28 in 5-HT7R and D2R or Trp3.28 in 5-HT6R and 5-HT2AR) and H-bond interactions (Arg7.36 in 5-HT7R, Asn7.36 in 5-HT2AR, and Thr7.39 in D2R). The 3D

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Table 1. Structure and Receptor Binding Profile of Compounds 1−81a

Data expressed as the mean ± SD of two independent experiments in duplicate. Affinities of reference compounds are as follows: 5-HT6 Ki = 4.1 ± 0.7 nM and 5-HT7 Ki = 0.9 ± 0.1 nM (methiothepin), 5-HT2A Ki = 1.0 ± 0.2 nM (risperidone), D2 Ki = 4.0 ± 0.3 nM (haloperidol), M1 Ki = 15 ± 3.1 nM (pirenzepine), hERG IC50 = 89 ± 18 nM (E-4031). a

Phe656(1) and Phe656(4) or in the vicinity of Phe656(3), where the sulfonamide moiety is placed. All those findings prompted us to synthesize a diverse set of multifunctional ligands that explored various parts of the prototype compound 2, including arylsulfonamide fragment, alkyl linker, and arylamine moiety (Figure 1F). The selection of particular building blocks was based primarily on their best fit to the binding site of 5-HT6R, which was considered the most important receptor target. Analysis of the composition of the binding site of 5-HT6R as well as the binding mode of compound 2 and its close analogues revealed that the aryl moiety connected with sulfonamide fragment penetrates the hydrophobic regions near TMHs 7 and 2. It forms van der Waals interactions with the hydrophobic residues of TMH2 (Ala2.61, Ala2.65), TMH3 (Trp3.28), or TMH7 (Trp7.40), and therefore, the arylsulfonyl moieties selected for the designed series were predominantly lipophilic, with a few exceptions to confirm this hypothesis (Figure 3). Moreover, the space available in the regions accommodating the arylsulfonamide moiety suggested that mono- or bicyclic aromatic fragments should be considered in the diverse set (Figure 2A). On the basis of the visual inspection of ligand− receptor complexes, the optimal length of the alkyl linker was assumed to be 3 or 4 units, and those lengths were selected to be thoroughly explored in the designed series. The longer linkers were not considered due to a high molecular weight of the resulting hybrids. However, considering their lower molecular weight, several combinations with a 2-unit alkyl linker were also obtained for comparison. All of the designed molecules were docked to 5-HT6, 5-HT7, 5-HT2A, D2, and M1

relatively limited volume of binding sites in muscarinic receptors (Figure 1E and Figure S1 in Supporting Information).26,27 In general, amino acid sequence homology in the region of the binding site between M1R and the receptors described above is low. There are numerous differences in sequence, which contribute to various dissimilarities in shape and properties. The binding site of M1R is substantially restricted spatially with higher contribution of hydrophilic residues. The reason for that is basically the presence of tyrosine residues, which replace several important amino acids present in previously discussed receptors. The first binding pocket (between TMHs 4−6) is confined by the presence of Tyr3.33 (instead of Val in the above-mentioned receptors) and Tyr6.51 (instead of Phe). Nevertheless, crucial for selectivity is in fact the absence of the second binding pocket (between TMHs 7−3). It is caused by the presence of Tyr7.39, Tyr2.61, and Tyr2.64, which respectively substitute Thr7.39, Ala2.61, and Asn2.64 in 5-HT6R, thus making the second binding cavity inaccessible for the ligand. Docking studies suggested the potentially poor blocking activity of compound 2 at the hERG channel as well. Although it could be accommodated in the hERG channel model, its binding mode was suboptimal, due to the lack of the hydrophobic/aromatic interactions in regions of four phenylalanine (Phe656) residues, which are considered important for channel blockade (Figure 1D and Figure S2 in Supporting Information).28,29 The prototype molecule binds to the model by a H-bond between the protonated nitrogen of piperazine and Ser624, although it seems to be insufficiently stabilized by aromatic interactions. In particular, there is no additional, required interaction in the region between E

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Scheme 1. General Synthesis of Hybrid Moleculesa

Reagents and conditions: (a) K2CO3, KI, DMF, rt, 48 h or DIPEA, KI, DMF/CH3CN 1:1, 70 °C, 3 days; (b) 40% aq MeNH2, rt, 76 h; (c) Et3N, DCM, 3 h, 10 °C then rt.

a

Figure 4. Binding mode of the compounds (A) 51 and (B) 72 in the 5-HT6 receptor. Residues in the binding site (within 4 Å from the ligand atoms) are presented as thick sticks together with their van der Waals surfaces (wire frame). Distances between key amino acid moieties and ligand structural elements are shown in Supporting Information, Tables S3 and S4. Dotted yellow lines represent H-bonds with polar residues. ECL2 was partially hidden for the sake of clarity. TMH, transmembrane helix; ECL, extracellular loop.

Moreover, numerous compounds possessed high affinity at the main targets (Ki < 10 nM), with some of them binding in the subnanomolar range, thus confirming the validity of the applied rational design approach. In general, the compounds displayed the highest affinity for 5-HT7 and 5-HT2A receptors as well as strong binding to D2 receptors, i.e., with the sites that were primarily targeted during the design process of the hybrid molecules. According to the molecular modeling studies, high affinity for 5-HT7 receptors is provided by the N-aminoalkylarylsulfonamide moiety, which strongly interacts with the part of the binding site between TMHs 7−3. This makes it relatively less sensitive to the influence of the arylamine part, accommodated in the region of TMHs 4−6. These properties of the N-aminoalkylarylsulfonamide moiety are complemented by those of the benzazolepiperazine/piperidine moiety, which provides high affinity for 5-HT2A and D2 receptors, the former being favored in this respect. However, 5-HT6R affinity varied widely, and some clear structure−activity relationships could be observed. First, the analogues containing the 6-fluoro-3-(piperidin-4yl)-1,2-benzoxazole III were somewhat more active at 5-HT6 sites than those containing 3-(piperazin-1-yl)-1,2-benzothiazole I, but both of these groups were much more active than the corresponding 3-(piperazin-1-yl)-1,2-benzoxazole II derivatives. Similar preferences were observed also for the other main

receptors as well as hERG channels, and their binding mode was analyzed, confirming in general the observations made for compound 2 and its close analogues. Consequently, the series of 185 combinations was selected for chemical synthesis.22 In this article, for the sake of clarity, we present 81 representative examples out of a total of 185 compounds synthesized and tested (Table 1). Synthesis. A series of hybrid molecules (1−81) was prepared in the three-step synthesis as presented in Scheme 1. In the first step, the alkylation of benzazolepiperazines/ piperidines I−III with 2-(bromoalkyl)-1H-isoindoline-1,3(2H)diones IV−VI afforded corresponding derivatives VII−XIV. Next, hydrolysis of the 1H-isoindoline-1,3(2H)-dione group with 40% methylamine aqueous solution afforded key intermediates XV−XXII, which were reacted with various commercially available sulfonyl chlorides (1−15) to give final arylsulfonamide derivatives (1−81). Structure−Affinity Relationships. The series of hybrid molecules that we had generated was evaluated in radioligand binding assays measuring their affinity for 5-HT6, 5-HT7, 5HT2A, D2, and M1 receptors as well as in the automated patchclamp assay for hERG channel blockade (Table 1). The majority of the compounds displayed marked affinity (Ki < 100 nM) for the targeted receptors and much lower activity at the antitargets (1000b >1000b >1000b >1000b >1000c 4000 ± 900c

Data are expressed as the mean ± SEM of three independent experiments performed in duplicate. Each cell line was transfected to stably express the indicated human recombinant receptor. bLess than 40% inhibition of binding observed at a concentration of 1 μM (n = 2). cIC50 value. h: human recombinant. a

Table 4. Extended Functional Profile of Compound 72a receptor h h h h h h h h h h h h

5-HT6 5-HT7 5-HT2A 5-HT1A 5-HT2C D2S D3 D4 D1 α1A α2C H1

functional test

agonist (conc, nM)

reference ligand (IC50, nM)

Kb ± SEM [nM]

cAMP cAMP IP1 cellular dielectric spectroscopy IP1 cAMP cAMP cAMP cAMP intracellular Ca2+ release cAMP intracellular Ca2+ release

serotonin (300) serotonin (300) serotonin (100) 8-OH-DPAT (100) serotonin (10) dopamine (300) dopamine (10) dopamine (100) dopamine (300) epinephrine (3.0) epinephrine (30) histamine (300)

methiothepin (11) methiothepin (1.5) ketanserin (14) WAY100635 (2.1) SB-206553 (9.4) butaclamol (5.0) butaclamol (16) clozapine (168) SCH23390 (1.7) WB4101 (1.0) rauwolscine (16) pyrilamine (3.0)

16 ± 4.7 4.5 ± 1.3 4.8 ± 0.87 >1000 N.C. 13 ± 8.2 2.3 ± 0.47 23 ± 6.3 37 ± 18 1.8 ± 0.17 25 ± 4.5 140 ± 20

Data are expressed as the mean ± SEM of three independent experiments performed in duplicate. cAMP, cyclic adenylyl monophosphate; IP1, inositol phosphate; h, human recombinant; N.C., noncalculable.

a

without significant sedation or hypotension.36 Furthermore, in patients with Alzheimer’s disease, α1 receptor deregulation has been reported in the frontal cortex of the post-mortem brain,37 suggesting that targeting this receptor may correct a neurobiological imbalance associated with the disorder. However, antagonism of peripheral α1 receptors is associated with hypotensive activity, which would need to be addressed in elderly patients with potentially fragile cardiac function. In spite of significant activity at H1 receptors, the separation between the antagonist properties at those sites and the main therapeutic targets is an order of magnitude (Kb = 140 nM vs 4.5−16 nM). In contrast, antipsychotic compounds with known, strong antihistaminic properties, like olanzapine or clozapine, show the opposite trend, with higher affinity at H1 receptors than at the principal therapeutic targets.32 Since the blockade of H1 receptors has been associated with sedative effects,38 the relatively weak antihistaminic activity of compound 72 may contribute to its low propensity to elicit sedation (see the next section). Moreover, the low affinity and

25 nM, respectively). Significant binding affinity for D4 and H1 receptors (Ki = 14 and 69 nM, respectively) was in line with the antagonistic effects at those sites (Kb = 23 and 136 nM, respectively). However, compound 72 only weakly bound to 5HT1A and 5-HT2C receptors or to the serotonin transporter, resulting in very weak antagonist properties (Kb or IC50 > 1000 nM). Of the above properties of compound 72, strong blockade of D3 receptors may be considered as a desirable attribute since it elicits procognitive effects via the facilitation of acetylcholine release.34 Moreover, antagonism of α2C adrenergic receptors could also positively influence the mood-modulating properties of compound 72 due to the elevation of prefrontal cortical monoamine outflow.35 Antagonism of central α1 receptors may also be beneficial for a compound targeting behavioral and psychological symptoms of dementia, especially agitation and aggression. Indeed, the α1 receptor antagonist, prazosin, elicited pronounced antiagitation and antiaggressive effects in patients with Alzheimer’s disease H

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enabled us to generate a series of compounds that display the desired in vitro receptor profile. Namely, we managed to obtain multimodal ligands that exert simultaneous blockade of 5-HT6 and D2 receptors and without deleterious interactions with key antitargets (muscarinic receptors or hERG channels) and distinct from currently available antipsychotics. Such a molecular interaction pattern, enriched with antagonism of other important targets, including 5-HT7 and 5-HT2A receptors, should elicit potentially beneficial psychotropic effects with an improved side effect profile. In addition, in vivo characterization of a representative molecule from the series, compound 72, revealed its exceptional activity in behavioral tests. Unlike reference antipsychotics, compound 72 achieved marked therapeutic-like activity in rat models of psychosis and depression without interfering with cognitive function or motor control and indicates that further investigation of this chemical series is warranted.

absence of antagonist properties of compound 72 at 5-HT2C receptors avoids concerns associated with this site related to increased appetite and body weight or metabolic disturbances.39 In Vivo Activity of Compound 72. In view of the encouraging in vitro profile described above, compound 72 was subjected to pharmacological experiments in rats. Specifically, the antipsychotic-like and antidepressant-like activity of compound 72 was compared with its influence on cognition. Psychotic disturbances in dementia may have different neurobiological substrates than in schizophrenia, so nondopaminergic models of psychosis were employed. Indeed, MK-801-induced hyperlocomotion and DOI-induced head twitches represent experimental paradigms based on the disruption of glutamate and serotonin transmission, respectively. Antidepressant-like activity was measured using Porsolt’s forced swim test (FST), a well validated procedure for primary screening, while the influence of compound 72 on cognition was tested using the passive avoidance test. Moreover, given that potential motor impairment is an important consideration in elderly patients, induction of catalepsy and changes in spontaneous locomotor activity were also quantified. The results showed that compound 72 dose-dependently reversed hyperlocomotion and head-twitches induced by MK-801 and DOI, with minimum effective doses (MED) of 10 and 3 mg/kg p.o. respectively, thus demonstrating a pronounced antipsychotic-like effect. Furthermore, it decreased immobility time in the FST by 20% over a relatively broad dose range, from 0.3 to 3 mg/kg p.o., consistent with a prominent antidepressant-like effect. The effect size of compound 72 in the FST was substantial and similar to that of the tricyclic antidepressant, imipramine, tested under the same conditions. In contrast, among the 8 clinically used antipsychotic drugs tested as references, only clozapine was able to show activity in the FST at more than one dose, with a maximal effect size >11% of control value, whereas the other drugs had little or no activity in the FST (Supporting Information, Table S5).23 In addition to a robust, oral activity in the therapeutic-like models, compound 72 did not disrupt memory in the passive avoidance test even at the highest dose tested (100 mg/kg p.o.). This is a striking observation because dementia patients with BPSD also suffer from core, cognitive deficits that should not be further exacerbated by pharmacotherapy. In contrast, the reference antipsychotic drugs dose-dependently disrupted cognition in the passive avoidance test at antipsychotic-like doses, in accordance with clinical findings on the cognitive impairments elicited by these medications (Supporting Information, Table S5).2,23 The only exception was aripiprazole, which did not disrupt passive avoidance response but also failed to exhibit robust antipsychotic-like effects. This observation is in line with clinical studies on aripiprazole, showing a lack of satisfactory antipsychotic efficacy in dementia patients.40 Finally, compound 72 did not significantly modify spontaneous locomotor activity or induce catalepsy up to a dose of 100 mg/kg p.o., suggesting that it does not interfere with normal motor control. In contrast, most of the reference antipsychotics tested produced dose-dependent catalepsy, and all of them produced significant sedation at doses in the therapeutic-like range (Supporting Information, Table S5).23

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EXPERIMENTAL SECTION

Molecular Modeling. The homology models of the human D2 dopamine and 5-HT7 serotonin receptors used herein were the same as those generated and described in our previously published papers.41,42 The additional homology models of other GPCRs presented in the current article (i.e., serotonin 5-HT6, 5-HT2A and muscarinic M1 receptors) were derived in accordance with the same, well validated method. It should be noted that the models used in the present work differ from most of previously published models of the GPCRs in question,43−47 in at least one of the two main aspects. First, they were prepared using templates of recently characterized highresolution crystal structures of human β 2 adrenergic and D 3 dopaminergic receptors, displaying much higher structural identity with the modeled receptors than the previously used templates, e.g., bovine rhodopsin. Second, the optimization of the binding sites of the new models was performed using the recently introduced methodology, based on the ligand-induced fit, which has been shown to result in significantly more robust models.41 The specific characteristics of the receptor models used in the present study are described in detail below. The homology model of the hERG potassium channel and its induced fit structure applied for docking studies were acquired from http://www.schrodinger.com.48 The novel models used for docking experiments were built on the template of β2 adrenergic receptor (β2R) crystal structure (Protein Data Bank ID: 2RH1).49 Reasonable sequence similarity between modeled receptors and the adrenergic β2 receptor justified the choice of the latter structure as a template for homology modeling. The crystal structure containing the heptahelical bundle and all of the originally occurring loops was used, with the exception of artificial fragments replacing the third intracellular loop (ICL3) in the crystallized receptor, which were removed. Short loops were created then to avoid modeling of the long ICL3, which is distant from the binding site and therefore beyond the scope of our experiments. Sequence alignment was performed via GeneSilico Metaserver,50 which is a gateway to multiple fold recognition servers. The results proposed by a profile−profile comparison tool, hhsearch, were selected as the starting alignments between the sequences of 5HT6, 5-HT2A, and M1 receptors (UniProt accession numbers P50406, P28223, and P11229, respectively)51 and the template. The alignments were manually adjusted in Swiss Pdb Viewer and submitted to the SwissModel server. Using the SwissModel algorithm, energyminimized (GROMOS96) homology models were obtained.52−54 Further improvements of the models were applied using software implemented in Schrödinger Suite 2009. All of the operations engaging Schrödinger’s tools were executed with default settings, unless stated otherwise. The homology models were validated by processing in Protein Preparation Wizard, which consisted of optimizing the hydrogen-bonding network and restrained minimization of the whole system with the OPLS_2005 force field. The least

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CONCLUSIONS Taken together, the present study indicates that the applied drug design and structure−activity relationship (SAR) strategy I

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General Procedure for the Deprotection of the Phthalimide Group (XV−XXII). A mixture of 6 mmol of an appropriate compound VII−XIV and 30 mL of 40% aqueous solution of methylamine was stirred at room temperature for 3 days. To the resulting solution, 30 mL of 20% aqueous solution of sodium hydroxide was added, and the resulted mixture was stirred for 1.5 h. Then 4 g of sodium chloride was added, and the solution was extracted with methylene chloride (30 mL). The organic layer was washed with water (30 mL) and then dried over anhydrous magnesium sulfate. The product was obtained by evaporation of methylene chloride from dry solution. If necessary, the product was purified using column chromatography on silica gel using chloroform/methanol 95:5 as eluent. The structure was confirmed by mass spectrometry. General Procedure for the Preparation of Sulfonamide Derivatives (1−81). Triethylamine (3.6 mmol (7.2 equiv)) and 0.5 mmol (1 equiv) of suitable arylsulfonyl chloride (1−15) were added to the solution of 0.5 mmol (1 equiv) of amine, XV−XXII, in 10 mL of methylene chloride at 10 °C. The reaction mixture was left at room temperature for 3 h, then the solvent and excess of triethylamine were evaporated. The resulting precipitate was then dissolved in 10 mL of methylene chloride and washed subsequently with 5% solution of sodium hydrogen carbonate (10 mL) and water (10 mL). The organic layer was dried over anhydrous magnesium sulfate, and the solvent was evaporated. Crude sulfonamides were purified by crystallization from methanol and some of them were by using column chromatography on silica gel using chloroform/methanol 9:1 as eluent. The structure and purity of prepared compounds was confirmed by LCMS data, and for selected compounds, structure identification was confirmed by NMR analysis. Chemical information for the 9 structurally most representative examples is presented below, while the data for all the other compounds are available in Supporting Information. N-(2-(4-(1,2-Benzothiazol-3-yl)piperazin-1-yl)ethyl)-3-methylbenzenesulfonamide (1). The title compound was prepared starting from amine (XV, R = H, X = N, Y = S, n = 0; 0.5 mmol, 131.19 mg) and 3methylbenzenesulfonyl chloride (1, 0.5 mmol, 95.32 mg). Yield: 49% (102 mg), yellowish oil. 1H NMR (300 MHz, CDCl3): δ 7. 79−7. 86 (m, 2H), 7.66−7.72 (m, 2H), 7.31−7.49 (m, 4H), 5.28 (s, 1H), 3.43− 3.48 (m, 4H), 3.01−3.08 (m, 2H), 2.46−2.53 (m, 6H), 2.43 (s, 3H). 13 C NMR (75 MHz, CDCl3): δ 163.59, 152.76, 139.37, 139.30, 133.48, 129.00, 127.88, 127.61, 127.50, 124.16, 123.96, 123.72, 120.61, 55.81, 52.28, 49.81, 39.17, 21.39. MS: 417 [M + H+]. N-(3-(4-(1,2-Benzothiazol-3-yl)piperazin-1-yl)propyl)-3-methylbenzenesulfonamide (2). The title compound was prepared starting from the amine (XVI, R = H, X = N, Y = S, n = 1; 0.5 mmol, 138.20 mg) and 3-methylbenzenesulfonyl chloride (1, 0.5 mmol, 95.32 mg). Yield: 49% (105 mg), yellowish oil. 1H NMR (300 MHz, DMSO-d6) δ: 8.03 (t, 2H, J = 7.2 Hz), 7.60−7.39 (m, 6H), 3.35−3.39 (m, 4H), 2.82−2.94 (m, 2H), 2.46−2.50 (m, 4H), 2.38 (s, 3H), 2.30 (t, 2H, J = 7.2 Hz), 1.54 (quintet, 2H, J = 6.9 Hz). 13C NMR (75 MHz, CDCl3): δ 163.78, 152.18, 139.96, 139.85, 132.42, 128.89, 127.63, 127.57, 127.51, 124.04, 123.98, 123.94, 120.61, 58.18, 52.89, 49.91, 43.96, 23.96, 20.73. MS: 431 [M + H+]. N-(4-(4-(1,2-Benzothiazol-3-yl)piperazin-1-yl)butyl)-3-methylbenzenesulfonamide (3). The title compound was prepared starting from the amine (XVII, R = H, X = N, Y = S, n = 2, 0.5 mmol, 145.21 mg) and 3-methylbenzenesulfonyl chloride (1, 0.5 mmol, 95.32 mg). Yield: 51% (113 mg), yellowish oil. 1H NMR (300 MHz, DMSO-d6) δ: 8.05 (d, 2H, J = 7.2 Hz), 7.41−7.60 (m, 6H), 3.35−3.40 (m, 4H), 2.72− 2.79 (m, 2H), 2.48−2.50 (m, 4H), 2.38 (s, 3H), 2.26−2.28 (m, 2H), 1.44−1.36 (m, 4H). 13C NMR (75 MHz, CDCl3): δ 163.70, 152.61, 139.81, 139.75, 133.21, 128.89, 127.51, 127.31, 126.91, 124.20, 124.07, 123.67, 120.61, 58.21, 48.76, 42.77, 21.34. MS: 445 [M + H+]. N-(3-(4-(Benzo[d]isoxazol-3-yl)piperazin-1-yl)propyl)-3-methylbenzenesulfonamide (27). The title compound was prepared starting from amine (XIX, R = H, X = N, Y = O, n = 1, 0.5 mmol, 130.17 mg) and 3-methylbenzenesulfonyl chloride (1, 0.5 mmol, 95.32 mg). Yield: 47% (120 mg), yellowish oil. 1H NMR (300 MHz, CDCl3): δ 7.63− 7.68 (m, 2H), 7.32−7.41 (m, 2H), 7.24−7.27 (m, 2H), 7.13−7.18 (m, 1H), 6.99−7.08 (m, 1H), 3.58−3.76 (m, 4H), 3.08−3.14 (m, 2H),

conserved fragments of the models, i.e., extracellular loops (ECLs), among which ECL2 is known to be involved in ligand binding, were additionally refined using the Prime Refinement tool. Binding pockets of the resulting homology models were optimized using modified induced fit docking (IFD) workflow.55,56 This method of ligand-based structure optimization proved to be useful in homology modeling of GPCRs.41 It combines flexible ligand docking (Glide algorithm, which uses the GlideScore scoring function)57,58 with protein structure prediction and side chain refinement (Prime protein prediction program).59,60 Modification of the IFD procedure consisted of appending another cycle of optimization of the receptor structure and docking the ligand using Glide XP (extra precision). The modified procedure allowed the models to improve in terms of assessing the proper ligand binding mode. For each optimized receptor, a group of several bioactive compounds were selected for the purpose of ligand-steered binding site optimization. Two-dimensional structures of the ligands were sketched or sourced from the ChEMBL database61 and then converted to 3D and optimized using LigPrep (OPLS_2001 force field was applied). If available, ring conformations for cyclic ligands were taken from their crystal structures deposited in the Cambridge Crystalographic Data Centre (CCDC) database and retained in the process of flexible docking.62 The top-scoring ligand−receptor complexes obtained by way of IFD were inspected visually to check the compliance with the common binding mode for monoaminergic receptor ligands. That procedure resulted in models that served as molecular targets in further docking studies. Parameters of Glide docking procedure were set to defaults, with the exception of docking precision set to XP (extra precision) and the flexible docking option retaining original conformations of amide bonds. H-bond constraints were applied on Asp3.32 since its interaction with the protonated nitrogen atom of the ligand is considered crucial for the monoaminergic GPCRs.63 The centroid of a grid box (22 × 22 × 22 Å) was located on Asp3.32. Glide, induced fit docking, LigPrep, Prime, and Protein Preparation Wizard were implemented in Schrödinger Suite 2009, which was licensed for Adamed Ltd. Chemistry. General Methods. All the reagents were purchased from Aldrich, Alfa Aesar, and Fluorochem. 1 HNMR and 13CNMR spectra were obtained with a Varian BB 200 spectrometer using TMS (0.00 ppm) in chloroform-d1 and DMSO-d6 and were recorded at 300 and 75 MHz, respectively. J values are in hertz (Hz), and splitting patterns are designated as follows: s (singlet), bs (broad singlet), d (doublet), t (triplet), and m (multiplet). The UPLC/MS system consisted of a Waters Aquity UPLC coupled to a Waters SQD mass spectrometer. Chromatographic separations were carried out using the Acquity UPLC BEH C18 column, 2.1 × 100 mm and 1.7 μm particle size. The column was maintained at 60 °C and eluted under gradient conditions: 0−4 min, a linear gradient from 80−0.1% of eluent A, at a flow rate of 0.5 mL/ min. Eluent A, water/formic acid (0.02%, v/v); and eluent B, methanol−acidic gradient. Eluent A, water/formic acid/ammonia solution (0.01%:0.1%, v/v/v); and eluent B, methanol−alkaline gradient. The UPLC/MS purity of all compounds except 58 and 60 was confirmed to be >95%. The table with the specific purity values for all compounds in 2 gradients is available in Supporting Information. Synthetic Procedures. General Procedure for the Synthesis of Phthalimide Derivatives VII−XIV. A mixture of 10 mmol of appropriate benzazole/I−III and 10 mmol of appropriate 2(bromoalkyl)-1H-isoindoline-1,3(2H)-dione IV−VI, 30 mmol of potassium carbonate, catalytic amount of potassium iodide, and 20 mL of N,N-dimethylformamide was stirred at room temperature until the disappearance of starting materials (TLC monitoring). The reaction mixture was subsequently poured into 50 mL of water, and the precipitate thus formed was isolated by filtration. The crude product was suspended in 20 mL of methanol, and then the solid was filtered off and dried to constant weight. Alternatively, the reaction was carried out in the mixture of acetonitrile and N,N-dimethylformamide stirring at 70 °C for 3 days in the presence of 30 mmol of DIPEA and a catalytic amount of potassium iodide. J

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Table 5. Radioligand Binding Assay Conditions for 5 HT6, 5 HT7, 5 HT2A, and D2 Receptors radioligand (final concentration/Kd)

assay buffer

incubation condition

50 mM Tris, 10 mM MgCl2,0.5 mM EDTA, pH 7.4

60 min, 37 °C

50 mM Tris, 10 mM MgSO4, 0.5 mM EDTA, pH 7.4

120 min, 27 °C

[ H]ketanserin (1.0/0.5 nM)

10 μM methiothepin 10 μM methiothepin 100 μM serotonin

50 mM Tris, 4 mM CaCl2, 0.1% ascorbic acid, pH 7.4

60 min, 27 °C

[3H]N-methylspiperone (0.4/0.2 nM)

10 μM (+)-butaclamol

50 mM HEPES, 50 mM NaCl, 5 mM MgCl2, 0.5 mM EDTA, pH 7.4

60 min, 37 °C

receptor (source) 5-HT6 (human recombinant, HEK293 cells) 5-HT7 (human recombinant, CHOK1 cells) 5-HT2A (human recombinant, CHO-K1 cells) D2 (human recombinant, CHO-K1 cells)

[3H]LSDm (2.5/2.0 nM) [3H]LSD (3.0/2.4 nM) 3

nonspecific binding

2.40−2.55 (m, 6H), 1.64−1.72 (m, 2H). 13C NMR (75 MHz, CDCl3): δ 163.28, 152.70, 143.28, 139.96, 139.28, 133.28, 130.58, 127.28, 124.03, 118.42, 116.36, 108.78. MS: 515 [M + H+]. N-(4-(4-(Benzo[d]isoxazol-3-yl)piperazin-1-yl)butyl)-3-methylbenzenesulfonamide (28). The title compound was prepared starting from the amine (XVIII, R = H, X = N, Y = O, n = 2, 0.5 mmol, 137.18 mg) and 3-methylbenzenesulfonyl chloride (1, 0.5 mmol, 95.32 mg). Yield: 53% (117 mg), pale yellow solid. 1H NMR (300 MHz, DMSOd6): δ 10.61 (bs, 1H), 8.21−8.00 (m, 1H), 7.55−7.65 (m, 4H), 7.42− 7.50 (m, 2H), 7.30−7.36 (m, 1H), 4.08−4.31 (m, 2H), 3.42−3.58 (m, 4H), 3.00−3.25 (m, 4H), 2.70−2.78 (m, 2H), 2.30 (s, 3H), 1.60−1.72 (m, 2H), 1.35−1.48 (m, 2H). 13C NMR (75 MHz, DMSO-d6) δ 163.77, 161.10, 140.30, 139.38, 133.47, 130.91, 129.58, 127.12, 124.05, 117.25, 110.70, 55.82, 50.42, 45.18, 40.77, 21.33, 20.97, 26.30. MS: 445 [M + H+]. N-[2-[4-(6-Fluoro-1,2-benzoxazol-3-yl)piperidin-1-yl]ethyl]-3methylbenzenesulfonamide (50). The title compound was prepared starting from the amine (XXII, R = F, X = C, Y = O, n = 0, 0.5 mmol, 131.66 mg) and 3-methylbenzenesulfonyl chloride (1, 0.5 mmol, 95.32 mg). Yield: 41% (85 mg), yellowish oil. 1H NMR (300 MHz, CDCl3) δ: 7.66−7.71 (m, 3H), 7.38−7.64 (m, 2H), 7.26−7.36 (m, 1H), 7.08 (t, 1H, J = 9 Hz), 5.27 (s, 1H), 2.98−3.08 (m, 3H), 2.73−2.77 (m, 2H), 2.42−2.48 (m, 5H), 2.07−2.16 (m, 2H), 1.96−2.03 (m, 4H). 13C NMR (75 MHz, CDCl3): δ 165.62, 163.91, 162.40, 160.27, 139.25, 139.92 132.90, 128.71, 127.20, 123.90, 122.12 (d, J = 11 Hz), 112.62 (d, J = 25.4 Hz), 97.30 (d, J = 26.5 Hz), 55.23, 52.81, 39.53, 34.12, 30.41, 21.30. MS: 418 [M + H+]. N-[3-[4-(6-Fluoro-1,2-benzoxazol-3-yl)piperidin-1-yl]propyl]-3methylbenzenesulfonamide (51). The title compound was prepared starting from the amine (XXI, R = F, X = C, Y = O, n = 1, 0.5 mmol, 138.67 mg) and 3-methylbenzenesulfonyl chloride (1, 0.5 mmol, 95.32 mg). Yield: 55% (118 mg), white solid. 1H NMR (300 MHz, DMSOd6): δ 7.80−7.85 (m, 1H), 7.63−7.69 (m, 2H), 7.34−7.41 (m, 2H), 7.20−7.25 (m, 1H), 7.04−7.12 (m, 1H), 3.08−3.12 (m, 3H), 2.96− 3.04 (m, 2H), 2.44−2.50 (m, 2H), 2.40 (s, 3H), 2.04−2.18 (m, 6H), 1.62−1.70 (m, 2H). 13C NMR (75 MHz, CDCl3): δ 165.77, 164,14 (d, J = 13.8 Hz), 162.45, 160.71, 139.90, 139.19, 133.15, 128.90, 127.31, 124.02, 123.10 (d, J = 11 Hz), 112.78 (d, J = 24.8 Hz), 97.53 (d, J = 26.5 Hz), 58.23, 53.19, 44.28, 34. 25, 30.41, 23.95, 21.36. MS: 432 [M + H+]. N-(4-(4-(6-Fluorobenzo[d]isoxazol-3-yl)piperidin-1-yl)butyl)benzo[b]thiophene-2-sulfonamide (72). The title compound was prepared starting from the amine (XX, R = F, X = C, Y = O, n = 2, 0.5 mmol, 145.68 mg) and benzo[b]thiophene-2-sulfonyl chloride (10, 0.5 mmol, 116.35 mg). Yield: 87% (211 mg), yellowish oil. 1H NMR (300 MHz, CDOD3) δ: 7.90−7.98 (m, 4H), 7.45−7.51 (m, 2H), 7.38−7.44 (m, 1H), 7.15−7.23 (m, 1H), 3.46−3.70 (m, 3H), 3.04−3.28 (m, 6H), 2.22−2.42 (m, 4H), 1.82−1.90 (m, 2H), 1.68−1.56 (m, 2H). 19FNMR (300 MHz, CDOD3): δ −109.30−109.45 (1F, m). 13C NMR (75 MHz, CDOD3): δ 166.15, 163.86 (d, J = 13.8 Hz), 162.84, 159.43, 141.59 (d, J = 15 Hz), 137.82, 128.5, 126.9, 125.3, 125.25, 122.9 (d, J = 11 Hz), 122.3, 116.7, 112.41 (d, J = 25 Hz), 96.81 (d, J = 27 Hz). MS: 488 [M + H+]. Radioligand Binding Assays for 5-HT6, 5-HT7, 5-HT2A, and D2 Receptors. One millimolar stock solutions of the compounds to be tested were prepared in DMSO. Serial dilutions of compounds were prepared in 96-well microplates in assay buffers using an automated pipetting system (epMotion 5070; Eppendorf). Radioligand binding

was performed using cryopreserved membranes from cells stably expressing the relevant human receptor under conditions as indicated in Table 5. Fifty microliters of working solution of the tested compounds, 50 μL radioligand solution, and 150 μL of diluted membranes prepared in assay buffer were transferred to 96-well microplates. These were covered with sealing tape, mixed, and incubated. The reaction was terminated by rapid filtration through a UniFilter 96 GF/B filter microplate, and 10 rapid washes with 200 μL of 50 mM Tris buffer (4 °C, pH 7.4) were performed using a vacuum manifold and 96-well pipettor. The UniFilter microplates were dried overnight at 37 °C in a dry incubator. The UniFilter bottoms were sealed, and 30 μL of liquid scintillator Betaplate Scint (PerkinElmer) was added to each well. The plates were allowed to equilibrate for 1 h, and then radioactivity was counted in a MicroBeta TriLux 1450 scintillation counter (PerkinElmer) at approximately 30% efficiency. Data were fitted to a one-site curve-fitting equation with Prism 5 (GraphPad Software), and Ki values were calculated using the Cheng− Prusoff equation. A large number of data points were generated in the screening experiments: in total, 185 compounds were tested on 4 receptors; of these, 81 representative compounds, tested on 4 receptors, are shown in the present article. The Ki values shown in Table 1 are the means of two independent experiments. Each compound was tested in 10 concentrations from 1 × 10−06 M to 1 × 10−10 M (final concentration). All of the assays were carried out in duplicate (n = 2). The assays presented in Table 1 were performed at the Faculty of Pharmacy, Jagiellonian University Collegium Medicum, except for M1R and hERG, which were tested by Cerep and ChanTest, respectively (see below). Functional Assays for 5 HT 6, 5 HT 7, 5 HT 2A, and D2 Receptors. One millimolar stock solutions of the tested compounds were prepared in DMSO. Serial dilutions were prepared in 96-well microplate in assay buffers using an automated pipetting system epMotion 5070 (Eppendorf). Two independent experiments in duplicate were performed, and 6 to 10 concentrations were tested. A cellular aequorin-based functional assay was performed with γirradiated recombinant CHO-K1 cells expressing mitochondrially targeted Aequorin, human GPCR, and the promiscuous G protein α16 for 5-HT 6 and 5-HT 2A receptors, Gαqi/5 for D 2 receptor (PerkinElemer). The assay was performed according to the standard protocol provided by the manufacturer. After thawing, cells were transferred to assay buffer (DMEM/HAM’s F12 with 0.1% proteasefree BSA) and centrifuged. The cell pellet was resuspended in assay buffer and coelenterazine h was added at final concentrations of 5 μM. The cell suspension was incubated at 21 °C, protected from light with constant agitation for 4 h, and then diluted with assay buffer to a concentration of 250,000 cells/mL. After 1 h of incubation, 50 μL of the cell suspension was dispensed using automatic injectors built into the radiometric and luminescence plate counter MicroBeta2 LumiJET (PerkinElmer, USA) into white opaque 96-well microplates preloaded with test compounds. Immediate light emission generated following calcium mobilization was recorded for 30−60 s. In antagonist mode, after 15−30 min of incubation the reference agonist was added to the above assay mix, and light emission was recorded again. Final concentration of the reference agonist was equal to EC80: 40 nM serotonin for the 5-HT6 receptor, 30 nM α-methylserotonin for the 5HT2A receptor, and 30 nM apomorphine for the D2 receptor. K

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dibenzo[a,d]cyclohepten-5,10-imine (MK-801).64 Briefly, groups of drug-naive rats (n = 7−8) were transferred in their home cages to the experimental room 24 h prior to testing and allowed to habituate for 60 min and returned to the colony room. The next day, locomotor activity was assessed in black octagonal open fields (80 cm in diameter, 30 cm high) under dim light and continuous white noise (65 dB). Each animal was placed in the center of the open field and allowed to explore the whole area for 30 min. Subjects did not have visual contact with other rats. Forward locomotion (cm/30 min) was recorded and analyzed with the aid of a computerized video tracking system (Videomot, TSE, Bad Homburg, Germany). Rats were preadministered the drug or vehicle p.o. 60 min before the start of the locomotor activity test. Fifteen minutes before the start of the test, rats were administered MK-801 (0.3 mg/kg i.p.). When assessing spontaneous locomotor activity, rats were administered saline 15 min prior to the test. DOI-Induced Head Twitches. All tests were carried out in a soundattenuated experimental room between 10:00 a.m. and 4:00 p.m. DOIinduced head twitches were scored as described by Schreiber et al.65 Rats were injected with 2,5-dimethoxy-4-iodoamphetamine (DOI 2.5 mg/kg, i.p.) and placed in glass observation cages (25 × 25 × 40 cm3, W × H × L) with wood chip bedding on the floor. Five minutes later, head twitches were counted for 5 min by a trained observer. The test compound or its vehicle was injected p.o. 60 min before the start of the observation period. Forced Swimming Test. The procedure used to determine antidepressant-like activity was based on the technique described previously.66 Briefly, rats were individually placed in glass cylinders (40 cm in height, 17 cm in diameter) filled with water (temperature: 23 ± 1 °C) at a height that made it impossible to reach the bottom with hind paws (25 cm) There were two swimming sessions separated by 24 h: an initial 15 min pretest and a 5 min test. The duration of immobility (s) in the test session was recorded by a blind observer located in an adjacent room with the aid of a video camera. A rat was considered immobile when it floated without moving except to keep its head above the water surface. The test compound or its vehicle was administered p.o. 60 min before the experiment. Step-through Passive Avoidance Test. Effects of antipsychotics on memory function were evaluated as previously described.23,31 Briefly, the passive avoidance apparatus (PACS-30, Columbus Instruments, Columbus, OH, USA) comprised four identical stainless-steel cages with black Plexiglas covers. Each cage consisted of lighted and dark compartments (23 × 23 × 23 cm3) and a stainless-steel grid floor. The compartments were separated by an automated sliding door. In the training (acquisition) session, animals were individually placed in the lighted compartment and allowed to explore it for 10 s. The sliding door was then opened, and the step-through latency for animals to enter the dark compartment was measured (300-s cutoff time). As soon as the animals entered the dark compartment, the door was closed. An inescapable foot-shock (0.5 mA pulse constant current for 3 s) was delivered 3 s later through the grid floor with a constant current shock generator (EACS-30 Columbus Instruments). The test compound or its vehicle was administered p.o. 60 min before the start of the training session. All vehicle-treated animals entered the dark compartment during the training session and received a footshock. Drug-treated animals that did not enter the dark compartment in the training session were not subjected to the test session. In the present study, all of the tested animals entered the dark compartment. The test session was performed 24 h after the training session using the same paradigm but without the foot-shock. Step-through latencies for animals to enter the dark compartment were measured with a 300-s cutoff time. Drug-induced decreases in step-through latencies to enter the dark compartment in the test session were taken as a measure of the drug’s “amnesic” effects. Catalepsy Bar Test. Catalepsy was assessed using the bar test. Each rat was placed on a clean, smooth table with a wooden bar (2 × 3 × 25 cm3, H × W × L) suspended 10 cm above the working surface. The animal’s hindlimbs were freely placed on the table, the tail laid out to the back, and the forelimbs gently placed over the bar. The length of time the animal touched the bar with both front paws was measured

For the 5-HT7 receptor, adenylyl cyclase activity was monitored using cryopreserved CHO-K1 cells, expressing the human serotonin 5HT7 receptor. Thawed cells were resuspended in stimulation buffer (HBSS, 5 mM HEPES, 0.5 IBMX, and 0.1% BSA at pH 7.4) at 200,000 cells/mL. Ten microliters of cell suspension was added to 10 μL of tested compounds loaded onto a white opaque half area 96-well microplate. An antagonist response experiment was performed with 10 nM serotonin as the reference agonist, and the agonist and antagonist were added simultaneously. Cell stimulation was performed for 60 min at room temperature. After incubation, cAMP measurements were performed with homogeneous TR-FRET immunoassay using the LANCE Ultra cAMP kit (PerkinElmer, USA). Ten microliters of EucAMP Tracer Working Solution and 10 μL of ULight-anti-cAMP Tracer Working Solution were added, mixed, and incubated for 1 h. The TR-FRET signal was read on an EnVision microplate reader (PerkinElmer, USA). IC50 and EC50 were determined by nonlinear regression analysis using GraphPad Prism 6.0 software. The log IC50 was used to obtain the Kb by applying the Cheng−Prusoff approximation. The assays presented in Table 2 were performed at Faculty of Pharmacy, Jagiellonian University Collegium Medicum. Other in Vitro Studies. The extended pharmacological profile in vitro for compound 72, with respect to GPCRs and SERT, was outsourced to Cerep (Le Bois l’Evêque, Poitiers, France). An outline of methodologies is shown in Tables 3 and 4. Further methodological details are available on the company Web site (www.cerep.fr). Data from all experiments were analyzed using nonlinear curve fitting programs, and the results are given as Ki values for binding affinity or Kb values for antagonist potency. Blockade of hERG-mediated potassium currents was carried out by ChanTest (Cleveland, Ohio) and expressed as the mean % of inhibition at 1.0 × 10−06 M (Table 1) or IC50 value (Table 3). Ability to block hERG potassium channels was determined using the electrophysiological method and cloned hERG potassium channels (KCNH2 gene, expressed in CHO cells) as biological material. The effects were evaluated using an IonWorks Quattro system (Molecular Devices Corporation, Union City CA). hERG current was elicited using a pulse pattern with fixed amplitudes (conditioning prepulse, −80 mV for 25 ms; test pulse, +40 mV for 80 ms) from a holding potential of 0 mV. hERG current was measured as a difference between the peak current at 1 ms after the test step to +40 mV and the steady-state current at the end of the step to +40 mV. Data acquisition and analyses were performed using the IonWorks Quattro system operation software (version 2.0.2; Molecular Devices Corporation, Union City, CA). Data were corrected for leak current. The hERG block was calculated as % block = (1 − I TA/IControl) × 100%, where IControl and ITA were the currents elicited by the test pulse in control and in the presence of a test compound, respectively. All assays for the lead compound 72 (shown in Tables 3 and 4) were performed in 3−4 independent experiments. The Ki and Kb values were determined from 6 concentrations from 1 × 10−06 M to 1 × 10−10 M. All of the assays were carried out in duplicate (n = 2). In Vivo Studies. Animals. Drug-naive male Wistar rats (Charles River, Sulzfeld, Germany) weighing 200−225 g on arrival were used for behavioral experiments. Animals were supplied by the breeder 2−3 weeks before experimentation. Rats were housed four per plastic cage and kept in a room with constant environmental conditions (22 ± 1 °C, relative humidity 60%, a 12:12 light-dark cycle with lights on at 7:00 a.m.). During this time, the rats were weighed and handled several times. Tap water and standard laboratory chow (Labofeed H, WPIK, Kcynia, Poland) were available ad libitum. All tests were carried out in a sound-attenuated experimental room between 09:00 a.m. and 3:00 p.m. All procedures for the treatment of rats in the present study were as humane as possible and were reviewed and approved by the institutional ethics committee. Procedures conformed to animal care standards laid down in Polish regulations and complied with international European guidelines (Directive no. 86/609/EEC). MK-801-Induced Hyperlocomotion. Antipsychotic-like activity was assessed by the inhibition of hyperactivity elicited by the NMDA receptor antagonist, [5R,10S]-[+]-5-methyl-10,11-dihydro-5HL

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up to a preset cutoff time of 180 s. Results of each trial were scored as follows: 0 for holding the position for 60 s. The minimum cataleptogenic dose was defined as the lowest dose inducing a mean catalepsy score of >1.67 Catalepsy was scored 30, 60, and 120 min after administration of vehicle or a test drug (p.o.).

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ASSOCIATED CONTENT

* Supporting Information S

Additional binding site and ligand binding mode descriptions, extended chemical information and compound characterization, and detailed purity data for all compounds. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: 48-697064058. Fax: 48-126205458. E-mail: marcin. [email protected]; [email protected]. Notes

The authors declare the following competing financial interest(s): Marcin Kolaczkowski is an employee of Adamed Ltd.

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ACKNOWLEDGMENTS This study was financially supported by Adamed Ltd. and the National Centre for Research and Development (NCBR) Grant No. KB/88/12655/IT1-C/U/08.

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ABBREVIATIONS USED BPSD, behavioral and psychological symptoms of dementia; 5HT2AR, 5-HT2A serotonin receptor; 5-HT6R, 5-HT6 serotonin receptor; 5-HT 7 R, 5-HT 7 serotonin receptor; D 2 R, D 2 dopamine receptor; M1R, M1 muscarinic receptor; β2R, β2 adrenergic receptor; TMH, transmembrane helix; ECL, extracellular loop; ICL, intracellular loop; IFD, induced fit docking

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REFERENCES

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