1H-indole-Based Multifunctional Ligands With Antipsychotic-like

Article pubs.acs.org/jmc

Novel 3‑(1,2,3,6-Tetrahydropyridin-4-yl)‑1H‑indole-Based Multifunctional Ligands with Antipsychotic-Like, Mood-Modulating, and Procognitive Activity † ́ Adam Bucki,*,† Monika Marcinkowska,† Joanna Sniecikowska, Krzysztof Więckowski,† Maciej Pawłowski,† Monika Głuch-Lutwin,† Anna Gryboś,† Agata Siwek,† Karolina Pytka,† Magdalena Jastrzębska-Więsek,† Anna Partyka,† Anna Wesołowska,† Paweł Mierzejewski,‡ and Marcin Kołaczkowski†,§ †

Faculty of Pharmacy, Jagiellonian University Medical College, 9 Medyczna Street, 30-688 Kraków, Poland Institute of Psychiatry and Neurology, 9 Sobieskiego Street, 02-957 Warsaw, Poland § Adamed Ltd., Pieńków 149, 05-152 Czosnów, Poland ‡

S Supporting Information *

ABSTRACT: The most troublesome aspects of behavioral and psychological symptoms of dementia (BPSD) are nowadays addressed by antidepressant, anxiolytic, and antipsychotic drugs, often administered off-label. Considering their modest effectiveness in dementia patients, the increased risk of adverse events and cognitive decline, there is an unmet need for welltolerated and effective therapy of BPSD. We designed and synthesized multifunctional ligands characterized in vitro as highaffinity partial agonists of D2R, antagonists of 5-HT6R, and blockers of SERT. Moreover, the molecules activated 5-HT1AR and blocked 5-HT7R while having no relevant affinity for off-target M1R and hERG channel. Compound 16 (N-{2-[4-(5-chloro-1Hindol-3-yl)-1,2,3,6-tetrahydropyridin-1-yl]ethyl}-3-methylbenzene-1-sulfonamide) exhibited a broad antipsychotic-, antidepressant-, and anxiolytic-like activity, not eliciting motor impairments in mice. Most importantly, 16 showed memory-enhancing properties and it ameliorated memory deficits induced by scopolamine. The molecule outperformed most important comparators in selected tests, indicating its potential in the treatment of both cognitive and noncognitive (behavioral and psychological) symptoms of dementia.

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deterioration, and mortality.7,8 Consequently, there is still a need for considerably more research on well-tolerated and effective therapy of BPSD.9 In the past years, the “one-target, one-drug” paradigm has been dominant in the drug discovery processes. This approach relies on finding a single compound that acts on a particular molecular target.10 For instance, so-called cognitive enhancers, frequently used in the treatment of memory and learning deficits mostly in dementia, act by inhibiting a single target, namely the enzyme acetylcholinesterase and thus increase both the level and duration of action of the neurotransmitter acetylcholine.11 They improve or delay cognitive decline and reduce behavioral changes, particularly apathy, depression, and aberrant motor behavior, being inefficient in the remaining

INTRODUCTION Dementia refers to cognitive issues such as memory loss and thinking inability of variable pathophysiology, the most common being Alzheimer’s disease (AD) and vascular dementia.1 Regardless of their origin, in 90% of patients, cognitive decline is complicated by combinations of behavioral and psychological symptoms of dementia (BPSD), such as agitation, restlessness, aggression, depression, anxiety, and psychosis. BPSD, also called neuropsychiatric symptoms (NPS),2 pose severe problems to patients, their families, and caregivers, in many cases more unbearable than the core cognitive decline.3,4 The most troublesome noncognitive aspects are addressed by antidepressant,5 anxiolytic,6 and antipsychotic drugs4 often administered off-label.4−6 Furthermore, antipsychotics have modest effectiveness in the treatment of agitation, aggression, and psychosis associated with dementia and increase risk of cerebrovascular adverse events, cognitive © 2017 American Chemical Society

Received: June 12, 2017 Published: August 1, 2017 7483

DOI: 10.1021/acs.jmedchem.7b00839 J. Med. Chem. 2017, 60, 7483−7501

Journal of Medicinal Chemistry

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Figure 1. Design of multifunctional ligands. Proposed binding mode of the prototype compound 14, the arylsulfonamide fragment of which mimics interactions of (1R)-3,N-dimethyl-N-[1-methyl-3-(4-methylpiperidin-1-yl)propyl]benzenesulfonamide (SB-258719)47 in the 5-HT7 receptor site (A) and the 3-(1,2,3,6-tetrahydropyridin-4-yl)-1H-indole moiety is responsible for SERT blocking properties (B). Structure of compound 14 (C) proved to satisfy steric and electrostatic requirements in docking studies to the homology models of the 5-HT6, 5-HT1A, and D2 receptors (D). Furthermore, the molecule was incompatible with structural representations of hERG channel and M1 receptor (E). The design stage resulted in a series of 36 multiple ligands characterized by potentially high affinity for the desirable biological targets and low risk of binding to the off-targets (F). Amino acid residues engaged in ligand binding (within 4 Å from the ligand atoms) are displayed as sticks, whereas those forming H-bonds (dotted yellow lines), π−π stacking (dotted blue lines), or cation−π (dotted green lines) are represented as thick sticks. M1 receptor structure displayed with antagonist tiotropium because docking of 14 resulted in no valid poses (E). For the sake of clarity, extracellular loop 2 (ECL2) of the given GPCRs was hidden (A,D). The detailed complexes are shown in Supporting Information, Figures S1−S6.

symptoms though.12,13 Consequently, to improve the pharmacotherapy of BPSD, atypical antipsychotics are often used in the combination with other drugs such as antidepressants (SSRIs)5 or anxiolytics (benzodiazepines).6 As a result, patients receive a pharmacological cocktail made up of several single drugs with potentially different and mutually intertwined bioavailability, pharmacokinetics, and metabolism as well as multiplied side

effects.14−16 Moreover, diversified dosing regimen in polypharmacy might be confusing and often cause drug−drug interactions, particularly in the case of elderly patients with coexisting diseases.17,18 Literature data suggests that it is possible to overcome those limitations by designing a multifunctional molecule which simultaneously modulates several targets and therefore being 7484



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amount of scientific data describes the research on 5-HT6 receptor ligands as effective procognitive agents in preclinical studies.39−41 Two antagonists, intepirdine and idalopirdine, have been successfully evaluated in phase II clinical trials, which revealed their potential as drug candidates for the symptomatic treatment of AD.42−45 The aforementioned facts have encouraged us to design multitarget ligands that will be able to precisely modulate the activity of several monoaminergic receptors and serotonin transporter, expecting that this multifunctional profile will contribute positively to the therapeutic potential in dementia patients. We have identified D2 and 5HT6 receptors as well as SERT as crucial molecular targets in the development of new potential anti-BPSD agents because drugs acting via these targets proved to be effective in the treatment of psychotic symptoms and mood disorders in patients with dementia. The pharmacological profile of the newly designed molecules is presumed to be improved compared to aripiprazole and enhanced with procognitive activity. Herein we present computer-aided design, synthesis, and in vitro activity characterization of a series of arylsulfonamide-based derivatives as multimodal agents. The most promising molecule of the whole series was further characterized in extended pharmacological studies.

beneficial in the treatment of various psychiatric diseases.14,19−21 In our search for a versatile molecule addressing the most common neuropsychiatric symptoms typical for dementia, we have recognized molecules that had been proven effective in the given single symptoms to inspire design of multiple ligands. Atypical antipsychotics have been extensively used to treat dementia patients, and the selected ones have been reported to improve neuropsychiatric symptoms. The American Psychiatric Association even recommended the use of antipsychotic medications, although only in emergency settings for the treatment of agitation or psychosis when symptoms are severe and dangerous.22 One of them, that was positively rated in terms of efficacy in the treatment of agitation, anxiety, and depression as well as safety and tolerance in Alzheimer’s disease patients, is aripiprazole.23−25 A molecular target that has gained our considerable attention is therefore dopamine D2 receptor, particularly effects of its partial activation. Aripiprazol, an atypical antipsychotic touted as a dopamine system stabilizer that acts primarily as D2 receptor partial agonist, possess also antidepressant and anxiolytic activity, which might result from its substantial affinity for serotonin 5-HT1A and 5-HT7 receptors, as well as dopamine D3 receptors.26 Furthermore, dopamine D2 receptor partial agonists demonstrated synergic mood modulating activity when administered together with selective serotonin reuptake inhibitors (SSRIs) in late-life depression.27,28 Serotonin transporter (SERT) inhibition itself is an attractive target for designing a molecule of therapeutic value in neuropsychiatric symptoms. Besides the clinically proven efficacy of SSRIs in major depressive disorder and other affective disorders, their favorable safety profile has been proven in dementia patients. Moreover, SSRIs show ability to reduce psychotic episodes and behavioral disturbances in patients with dementia and also depression.29,30 An example of single drug acting on multiple targets worth considering in the context of BPSD is vortioxetine, a serotonin modulator and stimulator approved for the treatment of major depressive disorder.31−33 Its antidepressant and additional anxiolytic effect result from the concomitant inhibition of SERT and modulation of a wide range of serotonergic receptors, such as 5-HT1A (agonist), 5-HT1B (partial agonist) as well as 5-HT1D, 5-HT3, and 5-HT7 (antagonist).34 It should be highlighted that the unique mechanism of action of vortioxetine, comparing to the currently available SSRIs, has largely contributed to the improvement of therapy of treatment-resistant patients.35 Another multifunctional ligand combining activity toward serotonin receptors and its transporter is lumateperone, a phase III investigational atypical antipsychotic capable of reducing negative symptoms of schizophrenia. It acts as a 5-HT2A receptor antagonist and D2 receptor partial agonist, having also SERT inhibitory properties.36 Interestingly none of the aforementioned drugs significantly influence 5-HT6 receptors. Degenerative changes in the CNS of dementia patients result in dysregulation of serotonergic system, which is in turn a source of cognitive dysfunction.37 A potential novel therapy of BPSD should include the effect on cognition. Undisputedly, cognitive functions mustn’t be disturbed to a greater extent in dementia patients.38 Given that the blockade of 5-HT6 receptors induces acetylcholine release, it became reasonable to consider that 5-HT6 receptor antagonism could be a promising approach for improving cognitive abilities. A large

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RESULTS AND DISCUSSION Design. In the course of discovery of novel designed multiple ligands (DML) targeting behavioral and psychological symptoms of dementia (BPSD), we have previously described a series of arylsulfonamide-based hybrid molecules.38,46 As a result of those studies, substantially supported by molecular modeling, we designed and pharmacologically characterized a group of compounds being the 5-HT6/5-HT7/5-HT2A and D2 receptor antagonists that displayed pronounced antipsychotic activity in the absence of cognitive or motor impairment. The design of these compounds was inspired by investigation into the binding mode of 5-HT7 receptor antagonists.47 On the basis of this, we constructed a series of multifunctional ligands by combining the 5-HT7 receptor blocking arylsulfonamide moiety, preferred by the 5-HT6 receptor as well, with the 3(piperidin-4-yl)-1,2-benzoxazole or 3-(piperazin-4-yl)-1,2-benzothiazole fragments responsible for the antagonism of the 5HT2A and D2 receptors.38,48 In the present study, we followed the same logic, but instead of the above-mentioned arylamine fragments we used the appropriately substituted 3-(1,2,3,6-tetrahydropyridin-4-yl)-1Hindole (THPI), a fragment that had previously been proven to introduce SERT inhibition (Figure 1A,B and Supporting Information, Figures S1, S2).49 This in turn might have implemented additional antidepressant activity besides the expected antipsychotic effect from the D2 receptors (partial agonism) and procognitive effect resulting from antagonism of the 5-HT6 receptors. Such a pharmacological profile would have enhanced the favorable activity, not causing excessive sedation or EPS resulting from D2 receptor blockade. The initially constructed hybrid molecule (Figure 1C, compound 14) was docked to the homology models of the main therapeutic targets, affinity for which we intended to incorporate in the novel multimodal ligands. Complementarity of interactions between the prototype compound and the active sites of 5-HT6, D2 as well as 5-HT1A/7 receptors, which was the main assumption of the design rationale, was therefore verified. The putative binding modes in the given receptor sites 7485



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Scheme 1. Synthesis of the Final Compounds 13−48a

a

Reagents and conditions: (a) K2CO3, Kl, CH3CN, 70 °C, 12 h; (b) 40% aq MeNH2, 50 °C, 4 h; (c) THF drv, 1 M KOH solution, rt, 12 h.

provided the basis to assess probability of binding to the given biological targets (Figure 1D, for details see Supporting Information, Figures S3−S5). The binding patterns of the prototype hybrid in complexes with the examined targets were encouraging enough to proceed with analysis of its potential interactions with the main antitargets. These included the M1 cholinergic receptor and the hERG potassium channel, blockade of which could lead to adverse effects such as memory disruption or cardiac arrhythmia, respectively. As expected, on the basis of the experiences with the previously described arylsulfonamide-based hybrids sharing the same general structure,38 also in the case of the 3-(1,2,3,6tetrahydropyridin-4-yl)-1H-indole derivatives the relevant affinity for both the antitargets occurred to be unlikely due to excessive volume of the molecule or lack of interaction complementarity (Figure 1E, Supporting Information, Figure S6). Subsequently, a first series of 18 hybrid molecules, varying in the substitution of the 3-(1,2,3,6-tetrahydropyridin-4-yl)-1Hindole moiety, length of the alkyl linker as well as structure of the arylsulfonamide portion, was designed and synthesized (Table 1). Having early in vitro results pointing out superior activity of the derivatives containing 5-chloro-3-(1,2,3,6tetrahydropyridin-4-yl)-1H-indole moiety, a further 18 modifications preserving the latter fragment were proposed in the structure optimization stage (Table 2). The presented project resulted in 36 original compounds (Figure 1F) which were scheduled for in-depth pharmacological characterization. Synthesis. The synthesis of the designed molecules 13−48 is outlined in Scheme 1. In the first step, the appropriately substituted 3-(1,2,3,6-tetrahydropyridin-4-yl)-1H-indole 1−3 was reacted with the corresponding 2-(bromoalkyl)-1Hisoindoline-1,3(2H)-dione in the presence of potassium carbonate and a catalytic amount of potassium iodide in acetonitryle at 70 °C to give key intermediates 4−12. In the next step, deprotection of phthalimides 4−12 with 40% solution of methylamine afforded free amines, which were reacted immediately with diverse commercially available sulfonyl chlorides to provide the final sulfonamides 13−48. The reaction was carried out in tetrahydrofuran in the presence of 1 M KOH solution. The structures of the final molecules are presented in Scheme 1. Structure−Activity Relationships. All the synthesized compounds were subjected for in vitro radioligand binding

assays to determine affinity for the serotonin 5-HT6, 5-HT7, 5HT1A, dopamine D2 receptors, and SERT. Series I. First, potent SERT-binding capabilities of the molecules containing the 3-(1,2,3,6-tetrahydropyridin-4-yl)-1Hindole fragment was proven. This conclusion is true for both 5F (Ki range 1.7 ± 0.9 to 8.5 ± 0.9 nM) and 5-Cl derivatives (2.7 ± 0.8 to 6.2 ± 2.2 nM). On the other hand, the 2-methyl-3(1,2,3,6-tetrahydropyridin-4-yl)-5-chloro-1H-indoles displayed considerably lower affinity for the transporter (Ki between 12 ± 4.6 and 40 ± 7.1 nM). The aromatic ring at the opposite end of the molecule proved to play a secondary role in binding to this target. Compounds containing 2-naphthalene, particularly those with a possibly short (2-unit) linker, performed slightly better than those having 3-methylphenyl, which in turn preferred a longer (4-unit) alkyl chain. Analysis of the affinity for the serotonin and dopamine receptors revealed poor performance of the 2-methyl-indole derivatives (Ki range 99 ± 5.6 to 610 ± 42 nM). This result was more surprising in that the analysis of the molecular surface of the given receptor models pointed to the possibility of introducing small alkyl group in position 2 of indole ring. Nevertheless, further development of methylated analogues was abandoned. Of the remaining combinations, the 3-(1,2,3,6tetrahydropyridin-4-yl)-5-chloro-1H-indoles displayed far better affinity for the tested serotonin receptors (Ki ≥ 0.6 ± 0.2 nM) than fluorine analogues (Ki ≥ 10 ± 1.6 nM), still maintaining reasonable activity toward dopamine receptor (Ki ≥ 2.5 ± 0.8 nM vs Ki ≥ 0.3 ± 0.1 nM, respectively). In this group, compound 16 was characterized by the most favorable in vitro profile, having one-digit nanomolar affinity for all the desired molecular targets (Table 1). The structure of this compound reflected preliminary requirements for potent DMLs of assumed activity: single-ring arylsulfonamide and short ethylene spacer were privileged in the binding sites of the receptors of interest. To explore in-depth the allowed chemical space around the most promising 3-(1,2,3,6-tetrahydropyridin4-yl)-5-chloro-1H-indole fragment, pharmacological characterization of a new series was performed. Series II. In the follow-up series of the 3-(1,2,3,6tetrahydropyridin-4-yl)-5-chloro-1H-indoles, the arylsulfonamide moiety and linker length were differentiated to optimize in vitro binding properties in the given chemical space. In the case of the 5-HT6 receptor affinity, superior properties were observed for derivatives possessing 2-unit linkers and 1-ring aryl 7486



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Table 1. Structure and Receptor Binding Profile of Compounds 13−30

Data expressed as the mean ± SEM of three independent experiments carried out in duplicate. Affinities of reference compounds are as follows: 5HT6R Ki = 1.0 ± 0.1 nM and 5-HT7R Ki = 1.0 ± 0.1 nM (methiothepin), 5-HT1AR Ki = 1.3 ± 0.1 nM (serotonin), D2R Ki = 1.4 ± 0.4 nM (haloperidol), SERT Ki = 1.0 nM (imipramine). a

ring. Among them, m-CH3 derivative from series I subsided only the one containing m-OH (compound 40 of top Ki = 3.5 ± 0.6 nM). There are few examples of 3-unit spacer derivatives having moderate affinity, always inferior to those possessing shorter ones. The compounds having 4-unit spacer were able to compete only when coupled to the more extensive lipophilic rings (preferably 2-naphthyl, compound 27 of series I, Ki = 100 ± 7 nM). Similar relationships were observed for the 5-HT7 receptor, noting that propylene-linker derivatives had a slightly higher affinity than ethylene-based analogues (again preferred were m-CH3− and m-OH-phenyl, Ki = 0.6 ± 0.2 and 1.0 ± 0.3 nM, respectively) and definitely higher than those of butylene linker (Ki over 61 ± 9.3 nM). The relationships for the D2 receptor were reversed, favoring compounds with longer (3−4 unit) linkers. Nevertheless, there

were also examples of excellent binders within ethylene spacer derivatives (e.g., m-CH3− and m-OH-phenyl, Ki = 3.3 ± 1.2 and 1.5 ± 0.6 nM, respectively). Analysis of the 5-HT1A and SERT affinity suggested that there’s no certain linker length preferred. The compounds displayed very high affinities of usually one-digit nanomolar value, no matter which arylsulfonamide fragment was applied (Table 2). The novel ligands proved to have a wide safety window, showing an estimated affinity for the main antitargets of over 1 μM. The values were lower than 6.7% and 25.1% inhibition of control specific binding at the concentration of 1.0 × 10−6 M for M1 and hERG, respectively (Table 3). Screening for adverse targets was carried out for the most promising compounds of highest affinity for the main targets. 7487



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Table 2. Structure and Receptor Binding Profile of Compounds 31−48

Data expressed as the mean ± SEM of three independent experiments carried out in duplicate. Affinities of reference compounds are as follows: 5HT6R Ki = 1.0 ± 0.1 nM and 5-HT7R Ki = 1.0 ± 0.1 nM (methiothepin), 5-HT1AR Ki = 1.3 ± 0.1 nM (serotonin), D2R Ki = 1.4 ± 0.4 nM (haloperidol), SERT Ki = 1.0 nM (imipramine). a

In Vitro Functional Activity. Compounds of outstanding affinity were tested in functional in vitro assays toward the receptors of interest to pick up molecule of possibly most suitable pharmacological profile. Furthermore, serotonin (5HT) uptake reduction was determined. According to design assumptions, to exert the desirable pharmacological action, a molecule should act as a partial agonist at the D2 receptor (antipsychotic/antidepressant action resembling aripiprazole) and as antagonist at the 5-HT6 receptor (procognitive effect). Moreover, a molecule is supposed to efficiently block 5-HT uptake (antidepressant activity) and show agonistic properties for the 5-HT1A receptor (anxiolytic effect), while the 5-HT7 receptors should be antagonized. The tested compounds

proved to be efficient DMLs, which displayed partial agonist activity for the D2 receptor in the vast majority (up to 42% of agonist effect) and presented a prominent antagonist effect at the 5-HT6 receptor (92% inhibition of control response). The 5-HT uptake functional assay showed robust SERT inhibiting activity (IC50 ≤ 17 ± 1.7 nM). Additionally, the compounds demonstrated pronounced antagonist efficacy at the 5-HT7 receptor (even 91% inhibition of control agonist response) and were almost full agonists of the 5-HT1A receptor, reaching 100% of agonist control response (Table 4). Compound 16 demonstrated the highest agonist response toward the D2 receptor (42% in the concentration of 1.0 × 10−6 M), and thus functional profile of activity in multiple 7488



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bar test and the spontaneous locomotor activity test, respectively (Table 5).50,51 The main goal of the in vivo studies was to obtain an animal level proof-of-concept for the multimodal ligand activity in direct comparison to the reference compounds. Because all of the results for the reference drugs were previously obtained after intraperitoneal (ip) administration, we chose the same route of administration for the innovative compounds. Indeed, while testing the compounds by oral administration would provide an additional information, notably by suggesting favorable bioavailability, some potential confounds may prevent clear conclusions to be drawn. For example, the effects of the parent compound might be interfered by the effects of its metabolites (first pass effect), which would be undesired at this proof-of-concept level. Considering the above, at this stage, we chose the ip administration for the innovative compounds in all the in vivo studies. Compound 16 displayed antipsychotic-like activity, significantly reversing MK-801-induced hyperactivity at minimum effective dose (MED) 1.25 mg/kg. Moreover, in contrast to aripiprazole, it was also found to be active in classic models of antidepressant- and anxiolytic-like activity in the similar dose range (0.625−1.25 mg/kg). In fact, compound 16 was even more potent than the standard antidepressants imipramine and citalopram as well as the anxiolytic drug diazepam. It should be noted that, while being active in models of psychosis and mood deficits, compound 16 did not elicit catalepsy (ED50 > 100 mg/ kg) nor did it inhibit spontaneous locomotor activity (up to 10 mg/kg). Both the features related to motor impairments beneficially differentiated its activity profile from aripiprazole, which elicited sedation and catalepsy in the same experimental paradigm, presenting modest dose separation from the antipsychotic-like activity. Considering the rather benign safety profile of aripiprazole in clinic, it testifies for a particular safety of compound 16 in this respect.27,28 The effect of compound 16 on learning and memory was evaluated in the step-through passive avoidance test (a hippocampus-dependent memory task). It was shown that compound 16 administered at 0.3125 mg/kg displayed ̈ mice, as it significantly memory-enhancing properties in naive increased the latency time in the retention but not the acquisition trial. Moreover, the same dose of the compound 16 ameliorated memory deficits induced by the anticholinergic agent scopolamine, which is of particular importance in the

Table 3. Antitarget Activity Data for the Selected Compounds % activity at 1.0 × 10−6 M compd

M1Ra

hERGb

15 16 18 31 34 37 40 43 45 46

3.2 0.7 nd 0.0 6.2 6.7 3.7 0.0 1.1 6.4

25.1 8.6 19.0 6.2 11.9 7.8 0.0 10.9 2.6 12.1

% inhibition of control specific binding at the concentration of 1.0 × 10−6 M. b% inhibition of hERG-mediated potassium currents at the concentration of 1.0 × 10−6 M. Assays carried out in duplicate (n = 2). nd, not determined. Affinity of reference pirenzepine for M1R Ki = 20 nM and inhibition of hERG with E-4031, 96% at the concentration of 1.0 × 10−6 M. a

concentrations was determined to verify its performance indicated in screening tests. The studies confirmed a partial agonist function of compound 16 in the D2 receptor (EC50 = 56 ± 9.3 nM; Emax = 56 ± 2.9%; Kb = 2.1 ± 0.042 nM) as well as robust antagonist effect in the 5-HT6 receptor (KB = 5.6 ± 3.2 nM). Moreover, the functional 5-HT uptake assay resulted in favorable IC50 = 17 ± 1.7 nM. The above data satisfied the expected design criteria, and together with other advantageous pharmacological data, authorized its choice as a lead compound for in vivo behavioral studies. For safety reasons, concentration−response curve of hERG binding of compound 16 was evaluated in three independent experiments as well, resulting in IC50 > 1 μM each time. In Vivo Pharmacology. A set of behavioral tests were selected to challenge pharmacological effects of compound 16 against reference compounds. The compound was tested in mice for antipsychotic-like activity in the MK-801-induced hyperlocomotion test, anxiolytic-like effect in the four-plate test, and antidepressant-like capabilities in the forced swim test. Possible procognitive activity was assessed in the step-through passive avoidance task. Moreover, its potential adverse effects, i.e., the risk of causing catalepsy and sedation, were tested in the Table 4. Functional Data for the Selected Compounds D2R

5-HT6R

5-HT7R

5-HT1AR

5-HT uptake

compd

%AGOa

%ANTb

%AGOa

%ANTb

%AGOa

%ANTb

%AGOa

%ANTb

IC50 [nM]

15 16 18 31 34 37 40 43 45 46 ARI

8 42 36 15 5 1 10 12 28 14 49

84 106 94 26 60 28 100 73 92 44 96

nd 2 nd nd 1 1 3 0 1 3 0

nd 92 nd nd 87 63 86 76 7 50 40

nd 1 −1 nd 1 0 1 −2 0 1 0

nd 36 30 nd 25 25 91 50 16 18 43

24 92 102 98 90 40 92 96 99 73 91

51 −6 −37 −34 −6 57 −8 −22 −33 −13 0

nd 17 ± 1.7 nd nd 10 ± 2.4 11 ± 0.7 16 ± 2.1 12 ± 2.2 nd nd nd

Percent activity of agonist control response at 1.0 × 10−6 M. bPercent inhibition of control agonist response at 1.0 × 10−6 M. Assay carried out in duplicate (n = 2). ARI, aripiprazole; nd, not determined. 5-HT uptake for the reference imipramine IC50 = 61 nM. a

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Table 5. Characterization of Compound 16 in Behavioral in Vivo Pharmacological Tests in Comparison to Reference Drugsa test procedures

compd 16

aripiprazole

1. Catalepsy (bar test) ED50 [mg/kg] (dose range [mg/kg]) 2. Spontaneous locomotor activity MSD [mg/kg] (dose range [mg/kg]) 3. MK-801-induced hyperlocomotion MED [mg/kg] (dose range [mg/kg]) 4. Four-plate test MED [mg/kg] (dose range [mg/kg]) 5. Forced swim test MED [mg/kg] (dose range [mg/kg]) 6. Passive avoidance (PA) MED [mg/kg] (dose range [mg/kg]) 7. Scopolamine effect reversal in PA MED [mg/kg] (dose range [mg/kg])

>100 (10−100)

1.881 (1−10)

>10 (0.625−10)

1.0 (0.5−5)

1.25 (1−10)

0.5 (0.125−0.5)

0.625 (0.3125−10)

>0.5 (0.06−0.5)

1.25 (0.3125−5)

>0.5 (0.01−0.5)

0.3125 (0.15−0.625)

>2.0 (0.25−2)

0.3125 (0.15−0.625)

>2.0 (0.25−2)

diazepam

imipramine

ditalopram

2.5 (1.25−5)

>30 (10−30)

>10 (2.5−10)

1.25 (0.625−5)

30 (10−30)

5 (2.5−40)

10 (5−20)

2.5 (1.25−5)

a Tests carried out on CD-1 mice (tests 1, 3, 6, and 7) and Swiss albino mice (tests 2, 4, 5) after intraperitoneal (ip) administration. ED50, median effective dose; MED, minimum effective dose; MSD, minimum sedative dose.

The compound selected upon in vitro testing cascade, N-{2[4-(5-chloro-1H-indol-3-yl)-1,2,3,6-tetrahydropyridin-1-yl]ethyl}-3-methylbenzene-1-sulfonamide (16), proved to have a broad spectrum of antipsychotic-, antidepressant-, and anxiolytic-like activity in mice. Importantly, in the active dose range, it did not elicit catalepsy nor inhibit spontaneous locomotor activity, both features related to potential motor impairments. Most interestingly, it was shown that compound ̈ mice as 16 possessed memory-enhancing properties in naive well as it ameliorated memory disruption induced by cholinergic deficit. In the given experimental paradigm, compound 16 outperformed the most significant comparators, including aripiprazole. The overall effectiveness and expected safety margin implies therapeutic-like potential of the presented THPI derivatives and warrants their further evaluation as potential symptomatic treatment of dementia.

view of the cholinergic deficit in dementia patients. It is worth noting that the reference antipsychotic aripiprazole did not improve memory or ameliorate memory deficits caused by scopolamine. Similarly, our previous experiments demonstrated that the antidepressant fluoxetine (a selective serotonin reuptake inhibitor; 5−20 mg/kg) was also inactive in this test.52 Those results are even more significant, considering that most of the antipsychotic drugs elicit cognitive deficits in passive avoidance paradigm.53 Our previous efforts to develop designed multiple ligands with antipsychotic activity resulted in potent molecules with benign effect on cognition, however, not able to reverse the cholinergic deficits.38,46 This work constitutes therefore a step forward in search for novel candidates for treatment of both noncognitive and cognitive symptoms associated with dementia.

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CONCLUSIONS In search for new possibilities of dealing with symptoms of dementia, we designed multifunctional ligands of promising pharmacological activity, which address the main bothersome aspects: cognitive deficits, as well as noncognitive symptoms, e.g., psychosis, depression, and anxiety. Their therapeutic-like activity resulted from the unique structural features incorporated within the hybrid molecule, combining scaffolds of the 3(1,2,3,6-tetrahydropyridin-4-yl)-1H-indole (THPI) and (4methylpiperidin-1-yl)propyl]benzenesulfonamide. Such a combination of pharmacophore fragments allowed for specific interactions in nanomolar range with essentially matched biological targets, resulting in an adequate functional activity. These included partial agonist efficacy at the D2 dopamine receptor, which was supposed to exert antipsychotic/antidepressant effect resembling the one of aripiprazole, combined with antagonism of the 5-HT6 serotonin receptor, responsible for procognitive effect (vide intepirdine) and blockade of serotonin transporter, bringing antidepressant activity characteristic for SSRIs, e.g., escitalopram. The 5-HT1A receptors were considered secondary targets able to mediate anxiolytic effect upon their activation (like in the case of buspiron). Ultimately, the 5-HT7 receptors blockade have been anticipated to strengthen antidepressant effect. Along with optimizing affinity for the relevant receptor/transporter proteins, avoiding binding to antitargets (the M1 muscarinic receptor and hERG channel) was a major concern in order to unmask cholinomimetic action from the 5-HT6 receptors and avoid adverse cardiac effects, respectively.

EXPERIMENTAL SECTION

Molecular Modeling. Computer-aided ligand design consisted in ligand−receptor complex analysis resulting from docking to the previously developed homology models or refined crystal structures of the D2, 5-HT1A, 5-HT6, and 5-HT7 receptors as well as SERT. Protein optimization stage, common for all the evaluated structures, was based on induced-fit docking (IFD) from Schrödinger. General procedure for building ligand-optimized models of high predictive value has been characterized previously.54 Models of SERT and M1 receptor were derived from crystal structures, 5I7355 and 5CXV,56 respectively. Structure refinement stage using Protein Preparation Wizard was followed by docking (IFD) a reference inhibitor, s-citalopram to the SERT model, and antagonist, tiotropium in the case of the M1 receptor. Selection of the best complex was based on scoring function and visual analysis of binding mode. Protein models optimized that way served as grid for docking studies. The homology model of the hERG potassium channel and its induced fit structure applied for docking studies were acquired from http://www.schrodinger.com.57 For each of the remaining receptor types, the most appropriate structural template for homology modeling was chosen based on sequence identity and other structural characteristics, e.g., common construction of loops forming a binding site. hhsearch tool via GeneSilico Metaserver58 was applied for sequence identity prediction. A template that got the highest score and sequence identity index of all the crystal structures accessible (at the time of the given model preparation) was taken for homology modeling. Thus, the human 5HT7 receptor model was built using β2-adrenergic receptor (2RH1),38,59 the D2 receptor model was constructed on the D3 receptor structure (3PBL),60,61 while the 5-HT6 receptor model was based on the relatively novel 5-HT1B crystal structure 4IAR,62,63 similarly to the model of 5-HT1A receptor, which is presented for the 7490



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first time herein. The alignment of amino acid sequences (UniProt Data Base ID: P08908) of the 5-HT1A and 5-HT2B receptors was implemented from the hhsearch model, which established the overall amino acid sequence identity for 38%. Crude homology model was obtained via SwissModel platform.64 The artificial fragments replacing the third intracellular loop (ICL3) in the protein crystal structure were removed and short loops were created by joining Arg217 and Thr346. Then it was validated by processing in Protein Preparation Wizard. Ligand-based optimization of the binding site, which was performed using IFD protocol, as well as crossdocking-based (Glide XP) validation, were carried out using various chemical classes of high affinity 5-HT1AR ligands. The described procedure resulted in a variety of conformational models that served as molecular targets in docking studies. The binding mode of compound 2 (Figure 1 in the design section and Figure S4 in the Supporting Information) was presented in a model optimized using structure of agonist befiradol (3-chloro-4fluorophenyl-[4-fluoro-4-([(5-methylpyridin-2-yl)methylamino]methyl)piperidin-1-yl]methanone.65 Ligand structures were optimized using LigPrep tool. Glide XP flexible docking procedure was carried out using OPLS3 force field and default parameters. H-bond constraint, as well as centroid of a grid box for docking to 5-HT6 receptor were located on Asp3.32. Glide, induced fit docking, LigPrep, and Protein Preparation Wizard were implemented in the SmallMolecule Drug Discovery Suite (Schrödinger, Inc.), which was licensed for Jagiellonian University Medical College. Synthesis. General Chemistry Information. Unless otherwise indicated, all the starting materials were obtained from commercial suppliers and were used without further purification. Analytical thinlayer chromatography (TLC) was performed on Merck Kieselgel 60 F254 (0.25 mm) precoated aluminum sheets (Merck, Darmstadt, Germany). Visualization was performed with a 254 nm UV lamp. Column chromatography was performed using silica gel (particle size 0.063−0.200 mm; 70−230 mesh ATM) purchased from Merck. The UPLC-MS or UPLC-MS/MS analyzes were run on UPLC-MS/MS system comprising Waters ACQUITY UPLC (Waters Corporation, Milford, MA, USA) coupled with a Waters TQD mass spectrometer (electrospray ionization mode ESI with tandem quadrupole). Chromatographic separations were carried out using the Acquity UPLC BEH (bridged ethyl hybrid) C18 column: 2.1 mm × 100 mm and 1.7 μm particle size. The column was maintained at 40 °C and eluted under gradient conditions using 95% to 0% of eluent A over 10 min, at a flow rate of 0.3 mL/min. Eluent A, water/formic acid (0.1%, v/v); eluent B, acetonitrile/formic acid (0.1%, v/v). A total of 10 μL of each sample were injected, and chromatograms were recorded using a Waters eλ PDA detector. The spectra were analyzed in the range of 200−700 nm with 1.2 nm resolution and at a sampling rate of 20 points/s. MS detection settings of Waters TQD mass spectrometer were as follows: source temperature 150 °C, desolvation temperature 350 °C, desolvation gas flow rate 600 L/h, cone gas flow 100 L/h, capillary potential 3.00 kV, and cone potential 20 V. Nitrogen was used for both nebulizing and drying. The data were obtained in a scan mode ranging from 50 to 1000 m/z at 0.5 s intervals; 8 scans were summed up to obtain the final spectrum. Collision activated dissociation (CAD) analyzes were carried out with the energy of 20 eV, and all the fragmentations were observed in the source. Consequently, the ion spectra were obtained in the range from 50 to 500 m/z. MassLynx V 4.1 software (Waters) was used for data acquisition. Standard solutions (1 mg/mL) of each compound were prepared in a mixture comprising analytical grade acetonitrile/water (1/1, v/v). The UPLC/MS purity of all the test compounds and key intermediates was determined to be >95%. 1H NMR, 13C NMR, and 19 F NMR spectra were obtained in a Varian Mercury spectrometer (Varian Inc., Palo Alto, CA, USA) in CDCl3, CD3OD, DMSO, or acetone operating at 300 MHz (1H NMR), 75 MHz (13C NMR), and 282 MHz (19F NMR). Chemical shifts are reported in terms of δ values (ppm) relative to TMS δ = 0 (1H) as internal standard. The J values are expressed in hertz. Signal multiplicities are represented by the following abbreviations: s (singlet), br s (broad singlet), d (doublet), dd (doublet of doublets), dt (doublet of triplets), dtd (doublet of triplet of doublets), t (triplet), td (triplet of doublets), tdd

(triplet of doublet of doublets), q (quartet), quin (quintet), m (multiplet). Melting points (mp) were determined with a Büchi melting point B-540 apparatus using open glass capillaries and are uncorrected. Synthetic Procedures. General Procedure for the Synthesis of Phthalimide Derivatives 4−12. A mixture of 11.1 mmol (1.1 equiv) of appropriate tetrahydropyridinindole derivative 1−3 and 10 mmol (1 equiv) of appropriate 2-(bromoalkyl)-1H-isoindoline-1,3(2H)-dione, 20 mmol (2 equiv) of potassium carbonate, a catalytic amount of potassium iodide and 25 mL of CH3CN was stirred at 70 °C for 12 h. After that time, the solid was filtered and the solvent was evaporated under the reduced pressure. Next, the reaction mixture was purified by column chromatography over silica gel using chloroform/methanol 98:2 as eluent. 2-{2-[4-(5-Fluoro-1H-indol-3-yl)-1,2,3,6-tetrahydropyridin-1-yl]ethyl}-2,3-dihydro-1H-isoindole-1,3-dione (4). The title compound was prepared starting from 5-fluoro-3-(1,2,3,6-tetrahydropyridin-4-yl)1H-indole 1 (11.1 mmol, 1.1 equiv, 2.38 g) and 2-(2-bromoethyl)-2,3dihydro-1H-isoindole-1,3-dione (10 mmol, 1 equiv, 2.52 g). Yield 70%, yellowish solid. 1H NMR (300 MHz, DMSO-d6): δ 11.19 (br s, 1 H), 7.72−7.90 (m, 4 H), 7.47 (dd, J = 2.30, 10.80 Hz, 1 H), 7.42 (d, J = 2.31 Hz, 1 H), 7.33 (dd, J = 4.74, 8.85 Hz, 1 H), 6.92 (dt, J = 2.44, 9.04 Hz, 1 H), 6.00 (br s, 1 H), 3.75 (t, J = 6.28 Hz, 2 H), 3.14 (br s, 2 H), 2.56−2.73 (m, 4 H), 2.41 (br s, 2 H). LC-MS (ESI) calcd for C23H20FN3O2 390.16 [M + H+], found 390.32 [M + H+]. 2-{3-[4-(5-Fluoro-1H-indol-3-yl)-1,2,3,6-tetrahydropyridin-1-yl]propyl}-2,3-dihydro-1-H-isoindole-1,3-dione (5). The title compound was prepared starting from 5-fluoro-3-(1,2,3,6-tetrahydropyridin-4-yl)1H-indole 1 (11.1 mmol, 1.1 equiv, 2.38 g), 2-(3-bromopropyl)-2,3dihydro-1H-isoindole-1,3-dione (10 mmol, 1 equiv, 2.68 g). Yield 61%, yellowish solid. 1H NMR (300 MHz, CDCl3): δ 8.50 (br s, 1 H), 7.75−7.89 (m, 2 H), 7.58−7.67 (m, 2 H), 7.42 (dd, J = 2.18, 10.39 Hz, 1 H), 7.22−7.32 (m, 1 H), 7.11 (s, 1 H), 6.92 (dt, J = 2.31, 8.98 Hz, 1 H), 5.99 (br s, 1 H), 3.81 (t, J = 6.92 Hz, 2 H), 3.20 (br s, 2 H), 2.68− 2.78 (m, 2 H), 2.62 (t, J = 7.18 Hz, 2 H), 2.48 (br s, 2 H), 2.00 (t, J = 7.05 Hz, 2 H). LC-MS (ESI) calcd for C24H22FN3O2 404.18 [M + H+], found 404.34 [M + H+]. 2-{4-[4-(5-Fluoro-1H-indol-3-yl)-1,2,3,6-tetrahydropyridin-1-yl]butyl}-2,3-dihydro-1H-isoindole-1,3-dione (6). The title compound was prepared starting from 5-fluoro-3-(1,2,3,6-tetrahydropyridin-4-yl)1H-indole 1 (11.1 mmol, 1.1 equiv, 2.38 g), 2-(4-bromobutyl)-2,3dihydro-1H-isoindole-1,3-dione (10 mmol, 1 equiv, 2.82 g). Yield 64%, yellowish solid. 1H NMR (300 MHz, CDCl3): δ 8.72 (br s, 1 H), 7.78−7.89 (m, 2 H), 7.64−7.75 (m, 2 H), 7.45 (dd, J = 2.18, 10.39 Hz, 1 H), 7.25−7.34 (m, 1 H), 7.07−7.18 (m, 1 H), 6.91 (dt, J = 2.31, 8.98 Hz, 1 H), 6.03 (br s, 1 H), 3.74 (t, J = 6.67 Hz, 2 H), 3.30 (br s, 2 H), 2.81 (d, J = 11.28 Hz, 2 H), 2.50−2.70 (m, 4 H), 1.60−1.85 (m, 4 H). LC-MS (ESI) calcd for C25H24FN3O2 418.19 [M + H+], found 418.37 [M + H+]. 2-(2-(4-(5-Chloro-1H-indol-3-yl)-5,6-dihydropyridin-1(2H)-yl)ethyl)isoindoline-1,3-dione (7). The title compound was prepared starting from 5-chloro-3-(1,2,3,6-tetrahydropyridin-4-yl)-1H-indole 2 (11.1 mmol, 1.1 equiv, 2.56 g) and 2-(2-bromoethyl)isoindoline-1,3dione (10 mmol, 1 equiv, 2.52 g). Yield 65%, yellowish solid. 1H NMR (300 MHz, DMSO-d6): δ 11.28 (br s, 1 H), 7.87−7.77 (m, 4 H), 7.73 (d, J = 2.1 Hz, 1 H), 7.42 (d, J = 2.6 Hz, 1 H), 7.38−7.32 (m, 1 H), 7.07 (dd, J = 2.1, 8.5 Hz, 1 H), 6.02 (s, 1 H), 3.76 (t, J = 6.4 Hz, 2 H), 3.15 (d, J = 2.8 Hz, 2 H), 2.72−2.60 (m, 4 H), 2.41 (m, 2 H). LC-MS (ESI) calcd for C23H20ClN3O2 406.12 [M + H+], found 406.33 [M + H+]. 2-(3-(4-(5-Chloro-1H-indol-3-yl)-5,6-dihydropyridin-1(2H)-yl)propyl)isoindoline-1,3-dione (8). The title compound was prepared starting from 5-chloro-3-(1,2,3,6-tetrahydropyridin-4-yl)-1H-indole 2 (11.1 mmol, 1.1 equiv, 2.56 g) and 2-(3-bromopropyl)isoindoline-1,3dione (10 mmol, 1 equiv, 2.68 g). Yield 81%, yellowish solid. 1H NMR (300 MHz, DMSO-d6): δ 11.27 (br s, 1 H), 7.86−7.77 (m, 2 H), 7.75−7.66 (m, 3 H), 7.40−7.32 (m, 2 H), 7.08 (dd, J = 2.1, 8.5 Hz, 1 H), 5.96 (s, 1 H), 3.65 (t, J = 6.9 Hz, 2 H), 3.02 (d, J = 2.8 Hz, 2 H), 2.59−2.49 (m, 2 H), 2.42 (t, J = 6.7 Hz, 2 H), 2.36 (br s, 2 H), 1.80 7491



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N-{2-[4-(5-Fluoro-1H-indol-3-yl)-1,2,3,6-tetrahydropyridin-1-yl]ethyl}-3-methylbenzene-1-sulfonamide (13). The title compound was prepared starting from intermediate amine (0.5 mmol, 1 equiv, 0.130 g) and 3-methylbenzene-1-sulfonyl chloride (0.5 mmol, 1 equiv, 0.09 g). Yield 63%, yellowish solid, mp 118−122 °C. 1H NMR (300 MHz, CDCl3): δ 8.45 (br s, 1 H), 7.64−7.75 (m, 2 H), 7.47 (dd, J = 2.31, 10.26 Hz, 1 H), 7.36−7.43 (m, 2 H), 7.26−7.32 (m, J = 4.62 Hz, 1 H), 7.17 (s, 1 H), 6.93 (dt, J = 2.31, 8.98 Hz, 1 H), 5.97 (br s, 1 H), 3.07 (t, J = 5.77 Hz, 2 H), 2.98 (d, J = 2.56 Hz, 2 H), 2.49−2.61 (m, 4 H), 2.34−2.49 (m, 5 H), NH (sulfonamide) proton not detected. 13C NMR (75 MHz, CDCl3): δ 158.1 (d, J = 234.42 Hz), 139.4, 139.3, 133.4, 133.3, 129.5, 128.9, 127.4, 125.3 (d, J = 9.95 Hz), 124.2, 123.0, 118.3, 117.7 (d, J = 4.98 Hz), 111.9 (d, J = 9.95 Hz), 110.5 (d, J = 25.99 Hz), 105.6 (d, J = 24.33 Hz), 55.6, 52.3, 49.7, 39.6, 28.8, 21.3. LC-MS (ESI) calcd for C22H24FN3O2S 414.16 [M + H+], found 414.38 [M + H+]. N-{3-[4-(5-Fluoro-1H-indol-3-yl)-1,2,3,6-tetrahydropyridin-1-yl]propyl}-3-methylbenzene-1-sulfonamide (14). The title compound was prepared starting from intermediate amine (0.5 mmol, 1 equiv, 0.137 g) and 3-methylbenzene-1-sulfonyl chloride (0.5 mmol, 1 equiv, 0.09 g). Yield 57%, yellowish solid, mp 120−122 °C. 1H NMR (300 MHz, CDCl3): δ 8.38−8.53 (m, 1 H), 7.59−7.72 (m, 2 H), 7.46−7.56 (m, 1 H), 7.27−7.41 (m, 3 H), 7.18 (s, 1 H), 6.94 (d, J = 2.05 Hz, 1 H), 6.05 (br s, 1 H), 3.04−3.18 (m, 4 H), 2.65−2.73 (m, 2 H), 2.49− 2.62 (m, 4 H), 2.39 (s, 3 H), 1.73 (t, J = 5.64 Hz, 2 H), NH (sulfonamide) proton not detected. 13C NMR (75 MHz, CDCl3): δ 158.1 (d, J = 236.63 Hz), 139.8, 139.1, 133.3, 133.1, 129.8, 128.8, 127.2, 125.2 (d, J = 9.40 Hz), 124.0, 123.31, 117.8, 117.5 (d, J = 4.42 Hz), 112.0 (d, J = 9.95 Hz), 110.4 (d, J = 26.54 Hz), 105.5 (d, J = 24.88 Hz), 57.6, 53.4, 49.6, 44.2, 28.5, 24.4, 21.3. LC-MS (ESI) calcd for C23H26FN3O2S 428.18 [M + H+], found 428.34 [M + H+]. N-{4-[4-(5-Fluoro-1H-indol-3-yl)-1,2,3,6-tetrahydropyridin-1-yl]butyl}-2-methylbenzene-1-sulfonamide (15). The title compound was prepared starting from intermediate amine (0.5 mmol, 1 equiv, 0.144 g) and 3-methylbenzene-1-sulfonyl chloride (0.5 mmol, 1 equiv, 0.09 g). Yield 43%, yellowish solid, mp 117−120 °C. 1H NMR (300 MHz, DMSO-d6): δ 11.20 (br s, 1 H), 7.64 (t, J = 5.64 Hz, 1 H), 7.53−7.60 (m, 2 H), 7.50 (dd, J = 2.31, 10.77 Hz, 1 H), 7.39−7.45 (m, 3 H), 7.35 (dd, J = 4.87, 8.98 Hz, 1 H), 6.93 (dt, J = 2.31, 9.10 Hz, 1 H), 6.02 (br s, 1 H), 3.02 (br s, 2 H), 2.75 (d, J = 5.39 Hz, 2 H), 2.54 (d, J = 5.13 Hz, 2 H), 2.45 (br s, 2 H), 2.35 (s, 3 H), 2.23−2.32 (m, 2 H), 1.32−1.49 (m, 4 H). 13C NMR (75 MHz, DMSO-d6): δ 157.2 (d, J = 231.10 Hz), 140.5, 138.8, 133.5, 133.4, 132.8, 129.2, 128.9, 126.66, 124.6 (d, J = 11.06 Hz), 123.6, 117.7, 116.1 (d, J = 4.42 Hz), 112.6 (d, J = 9.95 Hz), 109.3 (d, J = 25.43 Hz), 104.8 (d, J = 24.33 Hz), 57.3, 52.6, 50.0, 42.5, 28.4, 27.1, 23.6, 20.8. LC-MS (ESI) calcd for C24H28FN3O2S 442.20 [M + H+], found 442.44 [M + H+]. N-(2-(4-(5-Chloro-1H-indol-3-yl)-5,6-dihydropyridin-1(2H)-yl)ethyl)-3-methylbenzenesulfonamide (16). The title compound was prepared starting from intermediate amine (0.5 mmol, 1 equiv, 0.131 g) and 3-methylbenzene-1-sulfonyl chloride (0.5 mmol, 1 equiv, 0.09 g). Yield 38%, yellowish solid, mp 127−130 °C. 1H NMR (300 MHz, DMSO-d6): δ 11.29 (br s, 1 H), 7.74 (d, J = 1.8 Hz, 1 H), 7.64−7.58 (m, 2 H), 7.49−7.45 (m, 1 H), 7.44−7.40 (m, 3 H), 7.37 (d, J = 9.0 Hz, 1 H), 7.08 (dd, J = 2.1, 8.5 Hz, 1 H), 5.99 (br s, 1 H), 3.02 (d, J = 2.3 Hz, 2 H), 2.91 (t, J = 6.7 Hz, 2 H), 2.57−2.50 (m, 2 H), 2.42 (t, J = 6.7 Hz, 4 H), 2.37 (s, 3 H). 13C NMR (75 MHz, DMSO-d6): δ 141.0, 139.3, 135.8, 133.3, 129.5, 127.2, 126.1, 124.9, 124.4, 124.1, 121.6, 119.5, 118.6, 116.2, 113.7, 57.3, 52.9, 50.4, 40.8, 28.8, 21.3. LC-MS (ESI) calcd for C22H24ClN3O2S 430.14 [M + H+], found 430.27 [M + H+]. N-(3-(4-(5-Chloro-1H-indol-3-yl)-5,6-dihydropyridin-1(2H)-yl)propyl)-3-methylbenzenesulfonamide (17). The title compound was prepared starting from intermediate amine (0.5 mmol, 1 equiv, 0.138 g) and 3-methylbenzene-1-sulfonyl chloride (0.5 mmol, 1 equiv, 0.09 g). Yield 31%, yellowish solid, mp 136−139 °C. 1H NMR (300 MHz, DMSO-d6): δ 11.35 (br s, 1 H), 7.75 (d, J = 2.1 Hz, 1 H), 7.58 (d, J = 8.5 Hz, 3 H), 7.46−7.35 (m, 3 H), 7.08 (dd, J = 1.9, 8.6 Hz, 1 H), 6.06−5.98 (m, 1 H), 2.99−2.90 (m, 2 H), 2.79 (t, J = 6.5 Hz, 2 H), 2.49 (d, J = 5.6 Hz, 2 H), 2.42−2.38 (m, 2 H), 2.37 (s, 3 H), 2.33−

(quin, J = 6.9 Hz, 2 H). LC-MS (ESI) calcd for C24H22ClN3O2 420.14 [M + H+], found 420.32 [M + H+]. 2-(4-(4-(5-Chloro-1H-indol-3-yl)-5,6-dihydropyridin-1(2H)-yl)butyl)isoindoline-1,3-dione (9). The title compound was prepared starting from 5-chloro-3-(1,2,3,6-tetrahydropyridin-4-yl)-1H-indole 2 (11.1 mmol, 1.1 equiv, 2.56 g) and 2-(4-bromobutyl)isoindoline-1,3dione (10 mmol, 1 equiv, 2.82 g). Yield 65%, yellowish solid. 1H NMR (300 MHz, DMSO-d6): δ 11.29 (br s, 1 H), 7.88−7.78 (m, 4 H), 7.74 (d, J = 2.1 Hz, 1 H), 7.45−7.33 (m, 2 H), 7.08 (dd, J = 2.1, 8.7 Hz, 1 H), 6.02 (br s, 1 H), 3.59 (t, J = 6.9 Hz, 2 H), 3.04 (d, J = 2.8 Hz, 2 H), 2.60−2.52 (m, 2 H), 2.46−2.33 (m, 4 H), 1.70−1.56 (m, 2 H), 1.54−1.41 (m, 2 H). LC-MS (ESI) calcd for C25H24ClN3O2 434.16 [M + H+], found 434.39 [M + H+]. 2-{2-[4-(5-Chloro-2-methyl-1H-indol-3-yl)-1,2,3,6-tetrahydropyridin-1-yl]ethyl}-2,3-dihydro-1H-isoindole-1,3-dione (10). The title compound was prepared starting from 5-chloro-2-methyl-3-(1,2,3,6tetrahydropyridin-4-yl)-1H-indole 3 (5.5 mmol, 1.1 equiv, 1.357 g) and 2-(2-bromoethyl)-2,3-dihydro-1H-isoindole-1,3-dione (5 mmol, 1 equiv, 1.270 g). Yield 40%, yellowish solid. 1H NMR (300 MHz, CDCl3): δ 7.99 (br s, 1H), 7.82 (dd, J = 2.95, 5.51 Hz, 2H), 7.68 (dd, J = 3.08, 5.64 Hz, 2H), 7.45 (d, J = 1.80 Hz, 1H), 7.09−7.17 (m, 1H), 6.98−7.06 (m, 1H), 5.66 (br s, 1H), 3.93 (t, J = 6.80 Hz, 2H), 3.30 (d, J = 2.82 Hz, 2H), 2.77−2.89 (m, 4H), 2.50 (d, J = 1.54 Hz, 2H), 2.36 (s, 3H). LC-MS (ESI) calcd for C24H22ClN3O2 420.15 [M + H+], found 420.37 [M + H+]. 2-{3-[4-(5-Chloro-2-methyl-1H-indol-3-yl)-1,2,3,6-tetrahydropyridin-1-yl]propyl}-2,3-dihydro-1H-isoindole-1,3-dione (11). The title compound was prepared starting from 5-chloro-2-methyl-3-(1,2,3,6tetrahydropyridin-4-yl)-1H-indole 3 (5.5 mmol, 1.1 equiv, 1.357 g) and 2-(3-bromopropyl)-2,3-dihydro-1H-isoindole-1,3-dione (5 mmol, 1 equiv, 1.341 g). Yield 67%, yellowish solid. 1H NMR (300 MHz, CDCl3): δ 7.79−7.90 (m, 3H), 7.69 (dd, J = 3.08, 5.39 Hz, 2H), 7.43 (d, J = 1.54 Hz, 1H), 7.08−7.20 (m, 1H), 6.98−7.07 (m, 1H), 5.62 (br s, 1H), 3.82 (t, J = 6.92 Hz, 2H), 3.16 (d, J = 2.82 Hz, 2H), 2.64−2.74 (m, 2H), 2.59 (t, J = 7.18 Hz, 2H), 2.44 (d, J = 1.54 Hz, 2H), 2.37 (s, 3H), 1.98 (quin, J = 7.05 Hz, 2H). LC-MS (ESI) calcd for C25H24ClN3O2 434.16 [M + H+], found 434.39 [M + H+]. 2-{4-[4-(5-Chloro-2-methyl-1H-indol-3-yl)-1,2,3,6-tetrahydropyridin-1-yl]butyl}-2,3-dihydro-1H-isoindole-1,3-dione (12). The title compound was prepared starting from 5-chloro-2-methyl-3-(1,2,3,6tetrahydropyridin-4-yl)-1H-indole 3 (5.5 mmol, 1.1 equiv, 1.357 g) and 2-(4-bromobutyl)-2,3-dihydro-1H-isoindole-1,3-dione (5 mmol, 1 equiv, 1.411 g). Yield 67%, yellowish solid. 1H NMR (300 MHz, CDCl3): δ 7.93 (br s, 1H), 7.80−7.87 (m, 2H), 7.65−7.75 (m, 2H), 7.51 (d, J = 1.54 Hz, 1H), 7.11−7.18 (m, 1H), 6.98−7.07 (m, 1H), 5.67 (br s, 1H), 3.75 (t, J = 7.05 Hz, 2H), 3.18 (d, J = 2.82 Hz, 2H), 2.64−2.80 (m, 2H), 2.46−2.58 (m, 4H), 2.40 (s, 3H), 1.56−1.83 (m, 4H). LC-MS (ESI) calcd for C26H26ClN3O2 448.18 [M + H+], found 448.35 [M + H+]. General Procedure for the Synthesis of Intermediate Amines. The appropriate alkyltetrahydropyridine derivative 4−12 (5 mmol, 1 equiv) was dissolved in 40% aqueous solution of MeNH2 (50 mL), and the resulting reaction mixture was stirred at 50 °C for 4 h. Next, the reaction mixture was cooled to the room temperature and a solution of 10% KOH (25 mL) was added and the resulted mixture was stirred for additional 1 h. After that time, the reaction mixture was refrigerated overnight and the obtained oil was rinsed with water and dried in vacuum. The resulted intermediate was used directly to the next step without further purification. General Procedure for the Preparation of Sulfonamide Derivatives (13−48). To a solution of appropriate intermediate (0.5 mmol, 1 equiv) in 5 mL of THF dry, appropriate sulfonyl chloride (0.5 mmol, 1 equiv), 1 M KOH solution (0.5 mmol, 0.5 mL) was added. The resulted reaction mixture was stirred for 12 h at room temperature. After that time, THF was evaporated and EtOAc (10 mL) and water (10 mL) were added, the organic layer was separated and dried over sodium sulfate, and the crude mixture was purified by column chromatography over silica gel using chloroform/methanol 98:2 as eluent. 7492



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2.27 (m, 2 H), 1.56−1.53 (m, 2 H), NH protons not detected. 13C NMR (75 MHz, DMSO-d6): δ 143.8, 140.9, 139.3, 135.8, 133.3, 129.5, 127.2, 126.1, 124.9, 124.4, 124.1, 121.6, 119.6, 118.7, 116.2, 113.7, 55.4, 53.1, 50.8, 41.1, 29.0, 26.9, 21.3. LC-MS (ESI) calcd for C23H26ClN3O2S 444.14 [M + H+], found 444.39 [M + H+]. N-(4-(4-(5-Chloro-1H-indol-3-yl)-5,6-dihydropyridin-1(2H)-yl)butyl)-3-methylbenzenesulfonamide (18). The title compound was prepared starting from intermediate amine (0.5 mmol, 1 equiv, 0.145 g) and 3-methylbenzene-1-sulfonyl chloride (0.5 mmol, 1 equiv, 0.09 g). Yield 38%, yellowish solid, mp 120−123 °C. 1H NMR (300 MHz, DMSO-d6): δ 11.57 (br s, 1 H), 7.82 (s, 1 H), 7.68 (t, J = 5.9 Hz, 1 H), 7.63−7.55 (m, 3 H), 7.49−7.39 (m, 3 H), 7.12 (d, J = 8.7 Hz, 1 H), 6.07 (br s, 1 H), 3.26 (br s, 2 H), 3.02−2.93 (m, 2 H), 2.76 (d, J = 5.6 Hz, 4 H), 2.39−2.32 (m, 3 H), 2.29−2.21 (m, 2 H), 1.73−1.61 (m, 2 H), 1.43 (d, J = 14.4 Hz, 2 H). 13C NMR (75 MHz, DMSO-d6): δ 140.8, 139.3, 137.3, 135.9, 133.4, 129.7, 129.5, 128.0, 127.1, 126.5, 124.8, 124.1, 123.1, 122.0, 119.5, 114.7, 55.2, 50.4, 48.8, 42.5, 26.9, 25.5, 21.5, 21.3. LC-MS (ESI) calcd for C24H28ClN3O2S 458.17 [M + H+], found 458.32 [M + H+]. N-{2-[4-(5-Chloro-2-methyl-1H-indol-3-yl)-1,2,3,6-tetrahydropyridin-1-yl]ethyl}-3-methylbenzene-1-sulfonamide (19). The title compound was prepared starting from intermediate amine (0.5 mmol, 1 equiv, 0.145 g) and 3-methylbenzene-1-sulfonyl chloride (0.5 mmol, 1 equiv, 0.095 g). Yield 54%, yellowish solid, mp 90−93 °C. 1H NMR (300 MHz, CD3OD): δ 7.65−7.75 (m, 2 H), 7.45 (d, J = 4.36 Hz, 2 H), 7.39 (d, J = 1.80 Hz, 1 H), 7.18 (d, J = 8.46 Hz, 1 H), 6.95 (dd, J = 2.05, 8.46 Hz, 1 H), 5.60 (br s, 1 H), 3.17 (d, J = 2.82 Hz, 2 H), 3.09 (t, J = 6.80 Hz, 2 H), 2.69−2.77 (m, 2 H), 2.63 (t, J = 6.92 Hz, 2 H), 2.51 (d, J = 1.54 Hz, 2 H), 2.43 (s, 3 H), 2.37 (s, 3 H), NH protons not detected. 13C NMR (75 MHz, CD3OD): δ 141.7, 140.9, 135.4, 134.7, 134.6, 132.1, 130.3, 130.1, 128.5, 125.6, 125.4, 123.4, 121.6, 119.1, 114.8, 112.7, 58.3, 53.9, 51.6, 41.3, 31.1, 21.5, 12.8. LC-MS (ESI) calcd for C23H26ClN3O2S 444.15 [M + H+], found 444.36 [M + H+]. N-{3-[4-(5-Chloro-2-methyl-1H-indol-3-yl)-1,2,3,6-tetrahydropyridin-1-yl]propyl}-3-methylbenzene-1-sulfonamide (20). The title compound was prepared starting from intermediate amine (0.5 mmol, 1 equiv, 0.152 g) and 3-methylbenzene-1-sulfonyl chloride (0.5 mmol, 1 equiv, 0.09 g). Yield 58%, yellowish solid, mp 99−101 °C. 1H NMR (300 MHz, DMSO-d6): δ 11.39 (s, 1 H), 7.83 (t, J = 5.90 Hz, 1 H), 7.59−7.69 (m, 2 H), 7.42−7.55 (m, 3 H), 7.29 (d, J = 8.46 Hz, 1 H), 7.01 (dd, J = 2.05, 8.46 Hz, 1 H), 5.63 (br s, 1 H), 3.74 (br s, 2 H), 3.12 (br s, 4 H), 2.57−2.97 (m, 4 H), 2.40 (d, J = 1.80 Hz, 6 H), 1.80−2.03 (m, 2 H). 13C NMR (75 MHz, DMSO-d6): δ 140.1, 139.0, 134.4, 133.5, 133.1, 130.1, 129.1, 127.8, 126.8, 123.7, 123.5, 120.2, 117.5, 112.2, 111.7, 52.7, 49.8, 48.5, 30.7, 26.4, 24.0, 20.9, 12.68. LCMS (ESI) calcd for C24H28ClN3O2S 458.17 [M + H+], found 458.39 [M + H+]. N-{4-[4-(5-Chloro-2-methyl-1H-indol-3-yl)-1,2,3,6-tetrahydropyridin-1-yl]butyl}-2-methylbenzene-1-sulfonamide (21). The title compound was prepared starting from intermediate amine (0.5 mmol, 1 equiv, 0.159 g) and 3-methylbenzene-1-sulfonyl chloride (0.5 mmol, 1 equiv, 0.09 g). Yield 72%, yellowish solid, mp 102−105 °C. 1H NMR (300 MHz, CD3OD): δ 7.60−7.71 (m, 2 H), 7.40−7.48 (m, 3 H), 7.21 (d, J = 8.72 Hz, 1 H), 6.98 (dd, J = 1.80, 8.46 Hz, 1 H), 5.67 (br s, 1 H), 3.62 (d, J = 2.05 Hz, 2 H), 3.21 (t, J = 5.90 Hz, 2 H), 2.86−2.96 (m, 4 H), 2.73 (br s, 2 H), 2.41 (d, J = 2.56 Hz, 6 H), 1.77 (quin, J = 7.63 Hz, 2 H), 1.56 (quin, J = 6.99 Hz, 2 H), NH protons not detected. 13C NMR (75 MHz, CD3OD): δ 141.9, 140.9, 135.5, 135.3, 134.5, 132.5, 130.3, 129.8, 128.4, 126.0, 125.3, 121.9, 120.3, 119.0, 113.7, 112.9, 57.9, 52.8, 51.2, 43.6, 29.4, 28.3, 23.5, 21.5, 12.9. LC-MS (ESI) calcd for C25H30ClN3O2S 472.18 [M + H+], found 472.48 [M + H+]. N-{2-[4-(5-Fluoro-1H-indol-3-yl)-1,2,3,6-tetrahydropyridin-1-yl]ethyl}naphthalene-2-sulfonamide (22). The title compound was prepared starting from intermediate amine (0.5 mmol, 1 equiv, 0.130 g) and naphthalene-2-sulfonyl chloride (0.5 mmol, 1 equiv, 0.113 g). Yield 62%, yellowish solid, mp 124−128 °C. 1H NMR (300 MHz, CDCl3): δ 8.46 (s, 1 H), 8.31 (br s, 1 H), 7.79−8.04 (m, 4 H), 7.63 (dtd, J = 1.32, 7.17, 16.44 Hz, 2 H), 7.46 (dd, J = 2.42, 10.34 Hz, 1 H),

7.29 (dd, J = 4.84, 8.80 Hz, 1 H), 7.16 (d, J = 2.64 Hz, 1 H), 6.96 (dt, J = 2.20, 9.02 Hz, 1 H), 5.92 (t, J = 3.30 Hz, 1 H), 3.05−3.16 (m, 2 H), 2.93 (d, J = 3.08 Hz, 2 H), 2.54 (q, J = 5.72 Hz, 4 H), 2.40−2.48 (m, 2 H), NH (sulfonamide) proton not detected. 13C NMR (75 MHz, CDCl3): δ 158.1 (d, J = 234.42 Hz), 136.3, 134.7, 133.3, 132.1, 129.4, 129.4, 129.2, 128.8, 128.4, 127.9, 127.6, 125.2 (d, J = 9.95 Hz), 123.0, 122.3, 118.3, 117.7 (d, J = 4.42 Hz), 111.9 (d, J = 9.40 Hz), 110.5 (d, J = 25.43 Hz), 105.62 (d, J = 25.43 Hz), 55.5, 52.2, 49.8, 39.6, 28.7. LCMS (ESI) calcd for C25H24FN3O2S 450.16 [M + H+], found 450.41 [M + H+]. N-{3-[4-(5-Fluoro-1H-indol-3-yl)-1,2,3,6-tetrahydropyridin-1-yl]propyl}naphthalene-2-sulfonamide (23). The title compound was prepared starting from intermediate amine (0.5 mmol, 1 equiv, 0.137 g) and naphthalene-2-sulfonyl chloride (0.5 mmol, 1 equiv, 0.113 g). Yield 65%, yellowish solid, mp 179−180 °C. 1H NMR (300 MHz, CDCl3): δ 8.59 (br s, 1 H), 8.40 (s, 1 H), 7.75−7.98 (m, 4 H), 7.46− 7.67 (m, 3 H), 7.30 (dd, J = 4.49, 8.85 Hz, 1 H), 7.15 (s, 1 H), 6.94 (dt, J = 2.31, 8.98 Hz, 1 H), 6.03 (br s, 1 H), 3.15 (t, J = 5.64 Hz, 2 H), 3.06 (d, J = 2.56 Hz, 2 H), 2.61−2.71 (m, 2 H), 2.43−2.60 (m, 4 H), 1.71 (quin, J = 5.64 Hz, 2 H), NH (sulfonamide) proton not detected. 13 C NMR (75 MHz, CDCl3): δ 158.1 (d, J = 232.76 Hz), 136.8, 134.6, 133.3, 132.1, 129.8, 129.3, 129.1, 128.6, 128.1, 127.8, 127.4, 125.2 (d, J = 9.40 Hz), 123.4, 122.2, 117.7, 117.3 (d, J = 4.98 Hz), 112.1 (d, J = 9.40 Hz), 110.4 (d, J = 26.54 Hz), 105.5 (d, J = 24.88 Hz), 57.5, 53.3, 49.56, 44.2, 28.5, 24.3. LC-MS (ESI) calcd for C26H26FN3O2S 464.18 [M + H+], found 464.31 [M + H+]. N-{4-[4-(5-Fluoro-1H-indol-3-yl)-1,2,3,6-tetrahydropyridin-1-yl]butyl}naphthalene-1-sulfonamide (24). The title compound was prepared starting from intermediate amine (0.5 mmol, 1 equiv, 0.144 g) and naphthalene-2-sulfonyl chloride (0.5 mmol, 1 equiv, 0.113 g). Yield 74%, yellowish solid, mp 176−177 °C. 1H NMR (300 MHz, CD3OD): δ 8.39 (s, 1 H), 7.98 (d, J = 8.72 Hz, 1 H), 7.92 (d, J = 7.69 Hz, 2 H), 7.83 (dd, J = 1.67, 8.59 Hz, 1 H), 7.52−7.67 (m, 2 H), 7.45 (dd, J = 2.31, 10.52 Hz, 1 H), 7.32 (dd, J = 4.62, 8.98 Hz, 1 H), 7.28 (s, 1 H), 6.89 (dt, J = 2.44, 9.04 Hz, 1 H), 5.95 (br s, 1 H), 2.89−3.04 (m, 4 H), 2.46−2.63 (m, 4 H), 2.32 (t, J = 7.05 Hz, 2 H), 1.46 (br s, 4 H), NH protons not detected. 13C NMR (75 MHz, CD3OD): δ 159.4 (d, J = 232.21 Hz), 139.2, 136.2, 135.4, 133.7, 131.4, 130.6, 130.3, 129.89, 129.1, 129.0, 128.7, 126.6 (d, J = 9.95 Hz), 125.3, 123.7, 118.1, 117.9 (d, J = 4.42 Hz), 113.4 (d, J = 9.40 Hz), 110.8 (d, J = 26.54 Hz), 106.1 (d, J = 24.33 Hz), 59.0, 53.7, 51.5, 44.1, 29.4, 29.0, 24.9. LC-MS (ESI) calcd for C27H28FN3O2S 478.2 [M + H+], found 478.47 [M + H+]. N-(2-(4-(5-Chloro-1H-indol-3-yl)-5,6-dihydropyridin-1(2H)-yl)ethyl)naphthalene-2-sulfonamide (25). The title compound was prepared starting from intermediate amine (0.5 mmol, 1 equiv, 0.131 g) and naphthalene-2-sulfonyl chloride (0.5 mmol, 1 equiv, 0.113 g). Yield 43%, yellowish solid, mp 145−148 °C. 1H NMR (300 MHz, CD3OD): δ 8.47−8.43 (m, 1 H), 8.03−7.98 (m, 2 H), 7.95−7.83 (m, 2 H), 7.71−7.68 (m, 2 H), 7.66−7.57 (m, 2 H), 7.33−7.27 (m, 1 H), 7.08−7.03 (m, 1 H), 5.92 (td, J = 1.9, 3.3 Hz, 1 H), 3.11 (t, J = 6.9 Hz, 2 H), 3.06−3.00 (m, 2 H), 2.64−2.58 (m, 2 H), 2.56−2.50 (m, 2 H), 2.47 (dd, J = 1.8, 5.1 Hz, 2 H), NH protons not detected. 13C NMR (75 MHz, CD3OD): δ 137.3, 135.6, 134.8, 132.2, 129.7, 129.1, 128.7, 128.4, 127.7, 127.6, 127.3, 125.9, 124.8, 123.4, 122.0, 121.3, 119.1, 117.0, 116.0, 112.3, 56.7, 52.3, 50.0, 39.9, 27.9. LC-MS calcd for C25H24ClN3O2S 466.13 [M + H+], found 466.33 [M + H+]. N-(3-(4-(5-Chloro-1H-indol-3-yl)-5,6-dihydropyridin-1(2H)-yl)propyl)naphthalene-2-sulfonamide (26). The title compound was prepared starting from intermediate amine (0.5 mmol, 1 equiv, 0.138 g) and naphthalene-2-sulfonyl chloride (0.5 mmol, 1 equiv, 0.113 g). Yield 32%, yellowish solid, mp 132−135 °C. 1H NMR (300 MHz, acetone-d6): δ 8.45 (d, J = 1.8 Hz, 1 H), 8.12−8.08 (m, 2 H), 8.04− 7.99 (m, 1 H), 7.92−7.80 (m, 3 H), 7.68−7.59 (m, 2 H), 7.43−7.37 (m, 1 H), 7.11 (dd, J = 1.9, 8.6 Hz, 1 H), 6.12−6.05 (m, 1 H), 3.11− 2.99 (m, 4 H), 2.63−2.54 (m, 2 H), 2.53−2.49 (m, 2 H), 2.44 (t, J = 6.5 Hz, 2 H), 1.70 (t, J = 6.5 Hz, 2 H), NH protons not detected. 13C NMR (75 MHz, acetone-d6): δ 138.0, 134.7, 132.3, 129.5, 129.3, 129.1, 128.5, 127.9, 127.8, 127.5, 127.3, 126.2, 124.7, 123.8, 122.6, 121.5, 119.5, 118.4, 116.6, 112.9, 56.0, 53.0, 49.9, 42.3, 31.7, 25.8. LC7493



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MS calcd for C26H26ClN3O2S 480.14 [M + H+], found 480.30 [M + H+]. N-(4-(4-(5-Chloro-1H-indol-3-yl)-5,6-dihydropyridin-1(2H)-yl)butyl)naphthalene-2-sulfonamide (27). The title compound was prepared starting from intermediate amine (0.5 mmol, 1 equiv, 0.145 g) and naphthalene-2-sulfonyl chloride (0.5 mmol, 1 equiv, 0.113 g). Yield 36%, yellowish solid, mp 186−189 °C. 1H NMR (300 MHz, DMSO-d6): δ 11.31 (br s, 1 H), 8.40 (s, 1 H), 8.14−8.05 (m, 2 H), 8.00 (d, J = 7.7 Hz, 1 H), 7.85−7.73 (m, 3 H), 7.71−7.57 (m, 2 H), 7.44−7.34 (m, 2 H), 7.09 (dd, J = 2.1, 8.7 Hz, 1 H), 5.98 (br s, 1 H), 2.94 (br s, 2 H), 2.80 (br s, 2 H), 2.40 (br s, 2 H), 2.24 (br s, 2 H), 1.39 (br s, 4 H), 1.29−1.18 (m, 2 H). 13C NMR (75 MHz, DMSOd6): δ 138.2, 135.8, 134.5, 132.2, 129.7, 129.6, 129.5, 129.0, 128.2, 127.9, 127.6, 126.1, 124.9, 124.4, 122.8, 121.6, 119.6, 118.6, 116.2, 113.7, 57.7, 53.0, 50.4, 43.0, 28.9, 27.6, 24.1. LC-MS (ESI) calcd for C27H28ClN3O2S 494.17 [M + H+], found 494.36 [M + H+]. N-{2-[4-(5-Chloro-2-methyl-1H-indol-3-yl)-1,2,3,6-tetrahydropyridin-1-yl]ethyl}naphthalene-2-sulfonamide (28). The title compound was prepared starting from intermediate amine (0.5 mmol, 1 equiv, 0.145 g) and naphthalene-2-sulfonyl chloride (0.5 mmol, 1 equiv, 0.113 g). Yield 56%, yellowish solid, mp 103−107 °C. 1H NMR (300 MHz, CD3OD): δ 8.45 (s, 1 H), 7.96−8.07 (m, 2 H), 7.83−7.95 (m, 2 H), 7.53−7.68 (m, 2 H), 7.36 (d, J = 1.80 Hz, 1 H), 7.16 (d, J = 8.46 Hz, 1 H), 6.93 (dd, J = 1.80, 8.46 Hz, 1 H), 5.50 (br s, 1 H), 3.10 (t, J = 6.92 Hz, 2 H), 3.03 (d, J = 2.82 Hz, 2 H), 2.48−2.65 (m, 4 H), 2.40 (br s, 2 H), 2.31 (s, 3 H), NH protons not detected. 13C NMR (75 MHz, CD3OD): δ 137.8, 135.4, 134.4, 133.7, 132.8, 131.0, 129.8, 129.4, 129.1, 129.0, 128.4, 128.2, 127.9, 124.7, 122.6, 120.7, 118.1, 113.9, 111.7, 57.4, 52.9, 50.6, 40.5, 30.2, 11.9. LC-MS (ESI) calcd for C26H26ClN3O2S 480.15 [M + H+], found 480.39 [M + H+]. N-{3-[4-(5-Chloro-2-methyl-1H-indol-3-yl)-1,2,3,6-tetrahydropyridin-1-yl]propyl}naphthalene-2-sulfonamide (29). The title compound was prepared starting from intermediate amine (0.5 mmol, 1 equiv, 0.152 g) and naphthalene-2-sulfonyl chloride (0.5 mmol, 1 equiv, 0.113 g). Yield 64%, yellowish solid, mp 112−114 °C. 1H NMR (300 MHz, CD3OD): δ 8.46 (s, 1 H), 8.02−8.13 (m, 2 H), 7.98 (d, J = 7.44 Hz, 1 H), 7.87 (dd, J = 1.67, 8.59 Hz, 1 H), 7.66 (tt, J = 5.39, 7.31 Hz, 2 H), 7.44 (d, J = 1.54 Hz, 1 H), 7.22 (d, J = 8.46 Hz, 1 H), 7.00 (dd, J = 1.80, 8.46 Hz, 1 H), 5.63 (br s, 1 H), 3.69 (br s, 2 H), 3.08− 3.17 (m, 2 H), 3.05 (t, J = 6.28 Hz, 2 H), 2.73 (br s, 2 H), 2.41 (s, 3 H), 1.88−2.02 (m, 2 H), NH protons not detected, signals from two aliphatic protons overlap with solvent peak (MeOH: 3.36−3.26 ppm). 1 H NMR (300 MHz, DMSO-d6): δ 11.34 (s, 1 H), 8.47 (s, 1 H), 8.10−8.24 (m, 2 H), 8.05 (d, J = 7.44 Hz, 1 H), 7.91−8.00 (m, 1 H), 7.79−7.90 (m, 1 H), 7.69 (t, J = 6.54 Hz, 2 H), 7.43−7.55 (m, 1 H), 7.28 (d, J = 8.46 Hz, 1 H), 7.01 (dd, J = 1.80, 8.72 Hz, 1 H), 5.59 (br s, 1 H), 3.43−3.89 (m, 2 H), 2.94−3.29 (m, 4H signals partially overlap with water peak), 2.90 (q, J = 5.64 Hz, 2 H), 2.54−2.78 (m, 2 H), 2.38 (s, 3 H), 1.89 (d, J = 12.57 Hz, 2 H). 13C NMR (75 MHz, CD3OD): δ 138.7, 136.4, 135.5, 133.8, 132.6, 130.9, 130.4, 130.1, 129.7, 129.3, 129.2, 128.9, 126.0, 123.5, 122.0, 119.3, 118.9, 113.3, 112.9, 106.6, 55.6, 52.6, 51.2, 41.7, 28.9, 26.4, 12.9. LC-MS (ESI) calcd for C27H28ClN3O2S 494.17 [M + H+], found 494.42 [M + H+]. N-{4-[4-(5-Chloro-2-methyl-1H-indol-3-yl)-1,2,3,6-tetrahydropyridin-1-yl]butyl}naphthalene-1-sulfonamide (30). The title compound was prepared starting from intermediate amine (0.5 mmol, 1 equiv, 0.159 g) and naphthalene-2-sulfonyl chloride (0.5 mmol, 1 equiv, 0.113 g). Yield 59%, yellowish solid, mp 113−116 °C. 1H NMR (300 MHz, CD3OD): δ 8.42 (s, 1 H), 7.90−8.08 (m, 3 H), 7.85 (dd, J = 1.80, 8.72 Hz, 1 H), 7.61 (ddd, J = 1.54, 5.39, 7.44 Hz, 2 H), 7.44 (d, J = 1.80 Hz, 1 H), 7.21 (d, J = 8.46 Hz, 1 H), 6.99 (dd, J = 1.92, 8.59 Hz, 1 H), 5.56 (br s, 1 H), 3.53 (d, J = 2.05 Hz, 2 H), 3.12 (t, J = 5.77 Hz, 2 H), 2.96 (t, J = 6.41 Hz, 2 H), 2.81−2.92 (m, 2 H), 2.66 (br s, 2 H), 2.38 (s, 3 H), 1.62−1.78 (m, 2 H), 1.47−1.61 (m, 2 H), NH protons not detected. 13C NMR (75 MHz, CD3OD): δ 139.0, 136.3, 135.4, 135.3, 133.7, 132.5, 130.7, 130.3, 130.0, 129.8, 129.1, 129.1, 128.9, 126.0, 123.6, 121.9, 119.8, 119.0, 113.5, 112.9, 57.6, 52.5, 51.0, 43.6, 29.1, 28.2, 23.3, 12.9. LC-MS (ESI) calcd for C28H30ClN3O2S 508.18 [M + H+], found 508.45 [M + H+].

N-(2-(4-(5-Chloro-1H-indol-3-yl)-5,6-dihydropyridin-1(2H)-yl)ethyl)naphthalene-1-sulfonamide (31). The title compound was prepared starting from intermediate amine (0.5 mmol, 1 equiv, 0.131 g) and naphthalene-1-sulfonyl chloride (0.5 mmol, 1 equiv, 0.113 g). Yield 25%, yellowish solid, mp 98−103 °C. 1H NMR (300 MHz, DMSO-d6): δ 11.28 (br s, 1 H), 8.66 (d, J = 7.7 Hz, 1 H), 8.18 (dd, J = 2.6, 7.8 Hz, 2 H), 8.06 (d, J = 8.0 Hz, 1 H), 7.74−7.56 (m, 5 H), 7.41− 7.33 (m, 2 H), 7.08 (dd, J = 1.9, 8.5 Hz, 1 H), 5.90 (br s, 1 H), 3.02− 2.87 (m, 4 H), 2.46−2.38 (m, 2 H), 2.37−2.27 (m, 4 H). 13C NMR (75 MHz, DMSO-d6): δ 136.3, 135.8, 134.3, 134.0, 129.4, 129.3, 128.8, 128.2, 128.1, 127.2, 126.1, 125.2, 124.9, 124.8, 124.4, 121.6, 119.5, 118.5, 116.2, 113.7, 57.3, 52.8, 50.3, 31.1, 28.7. LC-MS (ESI) calcd for C25H24ClN3O2S 466.14 [M + H+], found 466.37 [M + H+]. N-(3-(4-(5-Chloro-1H-indol-3-yl)-5,6-dihydropyridin-1(2H)-yl)propyl)naphthalene-1-sulfonamide (32). The title compound was prepared starting from intermediate amine (0.5 mmol, 1 equiv, 0.138 g) and naphthalene-1-sulfonyl chloride (0.5 mmol, 1 equiv, 0.113 g). Yield 40%, yellowish solid, mp 127−130 °C. 1H NMR (300 MHz, DMSO-d6): δ 11.33 (br s, 1 H), 8.64 (d, J = 9.0 Hz, 2 H), 8.14−8.02 (m, 4 H), 7.69−7.60 (m, 3 H), 7.44−7.31 (m, 2 H), 7.16−7.02 (m, 1 H), 6.02−5.88 (m, 1 H), 2.95−2.90 (m, 2 H), 2.89−2.77 (m, 3 H), 2.40 (d, J = 5.4 Hz, 3 H), 2.26 (t, J = 6.8 Hz, 2 H), 1.58−1.41 (m, 2 H). 13C NMR (75 MHz, DMSO-d6): δ 136.0, 135.8, 134.3, 134.1, 129.5, 129.0, 128.2, 128.0, 127.2, 127.3, 126.0, 125.9, 125.8, 125.1, 125.0, 124.4, 121.7, 119.5, 116.0, 113.7, 55.0, 52.7, 50.0, 31.6, 30.3. LC-MS (ESI) calcd for C26H26ClN3O2S 480.14 [M + H+], found 480.33 [M + H+]. N-(4-(4-(5-Chloro-1H-indol-3-yl)-5,6-dihydropyridin-1(2H)-yl)butyl)naphthalene-1-sulfonamide (33). The title compound was prepared starting from intermediate amine (0.5 mmol, 1 equiv, 0.145 g) and naphthalene-1-sulfonyl chloride (0.5 mmol, 1 equiv, 0.113 g). Yield 43%, yellowish solid, mp 202−205 °C. 1H NMR (300 MHz, DMSO-d6): δ 11.31 (br s, 1 H), 8.66 (d, J = 8.2 Hz, 1 H), 8.19 (d, J = 8.2 Hz, 1 H), 8.13−8.01 (m, 3 H), 7.75 (d, J = 2.1 Hz, 1 H), 7.68− 7.59 (m, 3 H), 7.45−7.35 (m, 2 H), 7.09 (dd, J = 2.1, 8.5 Hz, 1 H), 5.98 (s, 1 H), 2.89−2.78 (m, 4 H), 2.40 (s, 4 H), 2.13 (br s, 2 H), 1.32−1.24 (m, 4 H). 13C NMR (75 MHz, DMSO-d6): δ 136.3, 135.8, 134.3, 134.0, 129.4, 129.4, 128.9, 128.2, 128.0, 127.2, 126.1, 125.2, 124.9, 124.9, 124.4, 121.6, 119.6, 118.7, 116.2, 113.7, 57.6, 53.0, 50.3, 42.8, 28.9, 27.5, 23.9. LC-MS (ESI) calcd for C27H28ClN3O2S 494.17 [M + H+], found 494.36 [M + H+]. N-(2-(4-(5-Chloro-1H-indol-3-yl)-5,6-dihydropyridin-1(2H)-yl)ethyl)-3-fluorobenzenesulfonamide (34). The title compound was prepared starting from intermediate amine (0.5 mmol, 1 equiv, 0.131 g) and 3-fluorobenzene-1-sulfonyl chloride (0.5 mmol, 1 equiv, 0.09 g). Yield 35%, yellowish solid, mp 149−150 °C. 1H NMR (300 MHz, DMSO-d6): δ 11.59 (d, J = 2.1 Hz, 1 H), 10.81 (br s, 1 H), 8.40 (s, 1 H), 7.82 (d, J = 1.8 Hz, 1 H), 7.75−7.63 (m, 2 H), 7.63−7.49 (m, 2 H), 7.42 (d, J = 8.7 Hz, 1 H), 7.13 (dd, J = 2.1, 8.7 Hz, 1 H), 6.08 (s, 1 H), 4.08−3.92 (m, 1 H), 3.81 (m, 1 H), 3.69−3.53 (m, 1 H), 3.27 (br s, 5 H), 2.85 (br s, 1 H), 2.78−2.57 (m, 1 H). 13C NMR (75 MHz, DMSO-d6): δ 142.2 (d, J = 7.2 Hz), 135.9, 132.3 (d, J = 8.3 Hz), 129.7, 126.2, 125.7, 124.9, 123.4 (d, J = 8.3 Hz), 122.0, 120.5, 120.3, 119.5, 115.6, 114.4, 114.0 (d, J = 8.3 Hz), 112.3, 54.5, 50.3, 48.8, 37.8, 24.8. LC-MS (ESI) calcd for C21H21ClFN3O2S 434.10 [M + H+], found 434.26 [M + H+]. N-(3-(4-(5-Chloro-1H-indol-3-yl)-5,6-dihydropyridin-1(2H)-yl)propyl)-3-fluorobenzenesulfonamide (35). The title compound was prepared starting from intermediate amine (0.5 mmol, 1 equiv, 0.138 g) and 3-fluorobenzene-1-sulfonyl chloride (0.5 mmol, 1 equiv, 0.09 g). Yield 44%, yellowish solid, mp 175−177 °C. 1H NMR (300 MHz, DMSO-d6): δ 11.54 (br s, 1 H), 10.06 (br s, 1 H), 7.97 (t, J = 5.9 Hz, 1 H), 7.83 (d, J = 1.8 Hz, 1 H), 7.67−7.57 (m, 3 H), 7.42 (d, J = 8.7 Hz, 2 H), 7.16−7.11 (m, 1 H), 6.10 (s, 1 H), 4.04−3.90 (m, 1 H), 3.73 (d, J = 13.8 Hz, 1 H), 3.58 (d, J = 10.5 Hz, 1 H), 3.32−3.09 (m, 3 H), 2.92−2.67 (m, 4 H), 1.89 (dd, J = 5.8, 9.9 Hz, 2 H). 13C NMR (75 MHz, DMSO-d6): δ 142.7 (d, J = 6.6 Hz), 135.9, 132.2 (d, J = 8.3 Hz), 130.3, 129.7, 126.3, 125.7, 124.9, 123.2 (d, J = 3.3 Hz), 122.1, 119.4, 115.9, 115.6, 114.4, 113.9 (d, J = 8.3 Hz), 112.9, 53.1, 50.2, 48.8, 25.0, 7494



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24.5. LC-MS (ESI) calcd for C22H23ClFN3O2S 448.12 [M + H+], found 448.29 [M + H+]. N-(4-(4-(5-Chloro-1H-indol-3-yl)-5,6-dihydropyridin-1(2H)-yl)butyl)-3-fluorobenzenesulfonamide (36). The title compound was prepared starting from intermediate amine (0.5 mmol, 1 equiv, 0.145 g) and 3-fluorobenzene-1-sulfonyl chloride (0.5 mmol, 1 equiv, 0.09 g). Yield 40%, yellowish solid, mp 97−101 °C. 1H NMR (300 MHz, DMSO-d6): δ 11.31 (br s, 1 H), 7.85 (br s, 1 H), 7.76 (d, J = 2.1 Hz, 1 H), 7.66−7.59 (m, 2 H), 7.55 (td, J = 1.6, 8.8 Hz, 1 H), 7.52−7.45 (m, 1 H), 7.43 (d, J = 2.3 Hz, 1 H), 7.38 (d, J = 8.7 Hz, 1 H), 7.09 (dd, J = 2.1, 8.5 Hz, 1 H), 6.03 (br s, 1 H), 3.01 (d, J = 2.6 Hz, 2 H), 2.79 (br s, 2 H), 2.59−2.50 (m, 2 H), 2.44 (br s, 2 H), 2.28 (br s, 2 H), 1.49− 1.34 (m, 4 H). 13C NMR (75 MHz, DMSO-d6): δ 160.6, 143.3, 135.8, 132.0, 129.5, 126.1, 124.9, 124.4, 123.1, 121.6, 120.0, 119.6, 118.7, 116.2, 114.0, 113.7, 57.7, 53.1, 50.4, 43.0, 29.0, 27.5, 24.1. LC-MS (ESI) calcd for C23H25ClFN3O2S 462.14 [M + H+], found 462.38 [M + H+]. 3-Chloro-N-(2-(4-(5-chloro-1H-indol-3-yl)-5,6-dihydropyridin1(2H)-yl)ethyl)benzenesulfonamide (37). The title compound was prepared starting from intermediate amine (0.760 mmol, 1 equiv, 0.200 g) and 3-chlorobenzene-1-sulfonyl chloride (0.760 mmol, 1 equiv, 0.160 g). Yield 47%, yellowish solid, mp 236−238 °C. 1H NMR (300 MHz, DMSO-d6): δ 11.63 (d, J = 1.5 Hz, 1 H), 10.95 (br s, 1 H), 8.47 (s, 1 H), 7.93−7.71 (m, 3 H), 7.69−7.55 (m, 2 H), 7.42 (d, J = 8.7 Hz, 1 H), 7.12 (dd, J = 1.8, 8.7 Hz, 1 H), 6.07 (s, 1 H), 4.07−3.90 (m, 1 H), 3.85−3.71 (m, 1 H), 3.67−3.53 (m, 1 H), 3.28 (m, 5 H), 2.87−2.86 (m, 1 H), 2.77−2.62 (m, 1 H). 13C NMR (75 MHz, DMSO-d6): δ 142.1, 135.9, 134.4, 133.2, 131.9, 129.7, 126.7, 126.2, 125.9, 125.7, 124.9, 122.0, 119.5, 114.4, 113.9, 112.2, 54.5, 50.2, 48.7, 37.8, 24.8. LC-MS (ESI) calcd for C21H21Cl2N3O2S 450.07 [M + H+], found 450.21 [M + H+]. 3-Chloro-N-(3-(4-(5-chloro-1H-indol-3-yl)-5,6-dihydropyridin1(2H)-yl)propyl)benzenesulfonamide (38). The title compound was prepared starting from intermediate amine (0.5 mmol, 1 equiv, 0.138 g) and 3-chlorobenzene-1-sulfonyl chloride (0.5 mmol, 1 equiv, 0.105 g). Yield 63%, yellowish solid, mp 139−140 °C. 1H NMR (300 MHz, DMSO-d6): δ 11.64 (d, J = 2.1 Hz, 1 H), 10.82 (br s, 1 H), 8.11 (t, J = 6.0 Hz, 1 H), 7.86−7.69 (m, 3 H), 7.67−7.56 (m, 2 H), 7.43 (d, J = 8.5 Hz, 1 H), 7.12 (dd, J = 2.1, 8.7 Hz, 1 H), 6.08 (s, 1 H), 4.01−3.85 (m, 1 H), 3.70 (dd, J = 5.5, 16.0 Hz, 1 H), 3.53 (d, J = 11.3 Hz, 1 H), 3.29−3.09 (m, 3 H), 2.85 (q, J = 6.4 Hz, 3 H), 2.77−2.64 (m, 1 H), 1.93 (dd, J = 6.7, 11.3 Hz, 2 H). 13C NMR (75 MHz, DMSO-d6): δ 142.5, 135.9, 134.3, 132.9, 131.8, 129.7, 126.7, 126.7. 126.2, 125.8, 124.9, 122.0, 119.5, 114.4, 114.0, 112.3, 52.9, 49.9, 48.6, 24.8, 24.3. LC-MS (ESI) calcd for C22H23Cl2N3O2S 464.09 [M + H+], found 464.24 [M + H+]. 3-Chloro-N-(4-(4-(5-chloro-1H-indol-3-yl)-5,6-dihydropyridin1(2H)-yl)butyl)benzenesulfonamide (39). The title compound was prepared starting from intermediate amine (0.5 mmol, 1 equiv, 0.145 g) and 3-chlorobenzene-1-sulfonyl chloride (0.5 mmol, 1 equiv, 0.105 g). Yield 38%, yellowish solid, mp 190−193 °C. 1H NMR (300 MHz, DMSO-d6): δ 11.34 (s, 1 H), 7.79 (d, J = 1.5 Hz, 1 H), 7.75 (td, J = 1.4, 7.6 Hz, 1 H), 7.67 (ddd, J = 1.3, 2.1, 8.0 Hz, 1 H), 7.63−7.54 (m, 2 H), 7.49 (s, 1 H), 7.39 (d, J = 8.7 Hz, 1 H), 7.34 (d, J = 4.1 Hz, 1 H), 7.09 (dd, J = 2.1, 8.7 Hz, 1 H), 6.08 (br s, 1 H), 4.00 (br s, 2 H), 2.99 (br s, 1 H), 2.81 (t, J = 6.7 Hz, 2 H), 2.74−2.61 (m, 3 H), 2.26 (s, 1 H), 1.84 (s, 1 H), 1.64−1.52 (m, 2 H), 1.46 (dd, J = 7.2, 13.8 Hz, 2 H). 13C NMR (75 MHz, DMSO-d6): δ 142.9, 135.7, 134.3, 132.6, 131.5, 130.0, 129.7, 128.7, 126.5, 125.9, 125.5, 124.7, 121.7, 119.4, 115.3, 113.6, 56.4, 51.6, 49.6, 42.6, 29.4, 27.1, 22.6. LC-MS (ESI) calcd for C23H25Cl2N3O2S 478.11 [M + H+], found 478.27 [M + H+]. N-(2-(4-(5-Chloro-1H-indol-3-yl)-5,6-dihydropyridin-1(2H)-yl)ethyl)-3-hydroxybenzenesulfonamide (40). The title compound was prepared starting from intermediate amine (0.5 mmol, 1 equiv, 0.131 g) and 3-hydroxybenzene-1-sulfonyl chloride (0.5 mmol, 1 equiv, 0.09 g). Yield 31%, yellowish solid, mp 122−124 °C. 1H NMR (300 MHz, DMSO-d6): δ 11.41 (br s, 1 H), 10.22 (br s, 1 H), 9.34 (br.s., 1H), 7.80 (d, J = 2.1 Hz, 1 H), 7.49−7.39 (m, 3 H), 7.29−7.23 (m, 2 H), 7.13 (dd, J = 2.1, 8.7 Hz, 1 H), 7.06 (ddd, J = 1.0, 2.4, 8.1 Hz, 1 H), 6.07−6.05 (m, 1 H), 3.09−3.01 (m, 2 H), 3.17−3.10 (m, 2 H), 2.97 (t,

J = 6.9 Hz, 2 H), 2.64 (d, J = 5.1 Hz, 2 H), 2.53 (td, J = 1.7, 3.7 Hz, 2 H). 13C NMR (75 MHz, DMSO-d6): δ 158.2, 141.9, 135.8, 130.7, 129.5, 126.0, 124.9, 124.4, 121.6, 119.8, 119.5, 118.2, 117.4, 116.1, 113.7, 113.5, 57.1, 52.8, 50.3, 32.1, 28.6. LC-MS (ESI) calcd for C21H22ClN3O3S 432,11 [M + H+], found 432.25 [M + H+]. N-(3-(4-(5-Chloro-1H-indol-3-yl)-5,6-dihydropyridin-1(2H)-yl)propyl)-3-hydroxybenzenesulfonamide (41). The title compound was prepared starting from intermediate amine (0.5 mmol, 1 equiv, 0.138 g) and 3-hydroxybenzene-1-sulfonyl chloride (0.5 mmol, 1 equiv, 0.09 g). Yield 30%, yellowish solid, mp 141−143 °C. 1H NMR (300 MHz, DMSO-d6): δ 11.63 (d, J = 2.3 Hz, 1 H), 10.28 (br s, 1 H), 9.53 (br s, 1 H), 7.84 (d, J = 2.1 Hz, 1 H), 7.59 (d, J = 2.6 Hz, 1 H), 7.48−7.35 (m, 2 H), 7.26−7.19 (m, 2 H), 7.16−7.01 (m, 2 H), 6.10− 6.07 (m, 1 H), 3.79−3.75 (m, 2 H), 3.54−3.41 (m, 2 H), 3.15−3.05 (m, 2 H), 2.81 (d, J = 15.1 Hz, 4 H), 1.97−1.83 (m, 2 H). 13C NMR (75 MHz, DMSO-d6): δ 158.3, 141.6, 135.9, 130.8, 129.7, 126.1, 125.7, 124.9, 122.0, 120.0, 119.5, 117.4, 116.7, 114.5, 114.0, 113.6, 53.2, 50.2, 49.0, 48.8, 25.2, 24.5. LC-MS (ESI) calcd C22H24ClN3O3S for 446.12 [M + H+] found 446.36 [M + H+]. N-(4-(4-(5-Chloro-1H-indol-3-yl)-5,6-dihydropyridin-1(2H)-yl)butyl)-3-hydroxybenzenesulfonamide (42). The title compound was prepared starting from intermediate amine (0.5 mmol, 1 equiv, 0.145 g) and 3-hydroxybenzene-1-sulfonyl chloride (0.5 mmol, 1 equiv, 0.09 g). Yield 19%, yellowish solid, mp 99−102 °C. 1H NMR (300 MHz, DMSO-d6): δ 11.33 (br s, 1 H), 10.10 (s, 1 H), 7.77 (d, J = 1.8 Hz, 1 H), 7.64−7.54 (m, 1 H), 7.46 (d, J = 2.6 Hz, 1 H), 7.41−7.31 (m, 2 H), 7.21−7.13 (m, 2 H), 7.09 (dd, J = 2.1, 8.5 Hz, 1 H), 6.98 (ddd, J = 0.9, 2.5, 8.1 Hz, 1 H), 6.04 (br s, 1 H), 3.16 (br s, 2 H), 2.80−2.61 (m, 4 H), 2.50 (br s, 2 H), 2.42 (br s, 2 H), 1.53−1.34 (m, 4 H). 13C NMR (75 MHz, DMSO-d6): δ 158.6, 158.2, 142.1, 135.8, 130.7, 129.6, 126.0, 125.1, 124.5, 121.7, 119.7, 119.5, 117.4, 115.9, 113.7, 113.5, 57.2, 55.3, 52.6, 50.1, 42.9, 27.3, 23.6. LC-MS (ESI) calcd for C23H26ClN3O3S 460.15 [M + H+], found 460.32 [M + H+]. N-(2-(4-(5-Chloro-1H-indol-3-yl)-5,6-dihydropyridin-1(2H)-yl)ethyl)-4-fluorobenzenesulfonamide (43). The title compound was prepared starting from intermediate amine (0.5 mmol, 1 equiv, 0.131 g) and 4-fluorobenzene-1-sulfonyl chloride (0.5 mmol, 1 equiv, 0.09 g). Yield 72%, yellowish solid, mp 117−120 °C. 1H NMR (300 MHz, DMSO-d6): δ 11.30 (br s, 1 H), 7.88 (dd, J = 5.3, 8.6 Hz, 2 H), 7.74 (s, 1 H), 7.64 (br s, 1 H), 7.48−7.30 (m, 4 H), 7.08 (d, J = 6.9 Hz, 1 H), 6.00 (br s, 1 H), 3.02 (br s, 2 H), 2.92 (t, J = 6.5 Hz, 2 H), 2.53 (d, J = 5.1 Hz, 2 H), 2.46−2.37 (m, 4 H). 13C NMR (75 MHz, DMSOd6): δ 137.5, 135.8, 130.0, 129.5, 126.1, 124.9, 124.4, 121.6, 119.5, 118.5, 116.8, 116.5, 116.2, 113.7, 57.3, 52.9, 50.4, 28.8. LC-MS (ESI) calcd for C21H21ClFN3O2S 434.11 [M + H+], found 434.33 [M +H+]. N-(3-(4-(5-Chloro-1H-indol-3-yl)-5,6-dihydropyridin-1(2H)-yl)propyl)-4-fluorobenzenesulfonamide (44). The title compound was prepared starting from intermediate amine (0.5 mmol, 1 equiv, 0.138 g) and naphthalene-1-sulfonyl chloride (0.5 mmol, 1 equiv, 0.113 g). Yield 48%, yellowish solid, mp 127−128 °C. 1H NMR (300 MHz, DMSO-d6): δ 11.55 (br s, 1 H), 10.25 (br s, 1 H), 7.92−7.79 (m, 3 H), 7.61 (d, J = 2.3 Hz, 1 H), 7.50−7.35 (m, 3 H), 7.20−7.08 (m, 1 H), 6.14−6.05 (m, 1 H), 4.04−3.90 (m, 1 H), 3.78−3.64 (m, 1 H), 3.58 (m, 2 H), 3.21−3.09 (m, 2 H), 2.83 (d, J = 6.2 Hz, 4 H), 1.93− 1.81 (m, 1 H), 1.74 (m, 1 H). 13C NMR (75 MHz, DMSO-d6): δ 138.2, 137.0, 135.7, 130.1, 129.9, 129.6, 126.2, 125.7, 124.9, 122.1, 119.4, 117.1, 116.7, 114.4, 114.0, 112.5, 66.9, 53.7, 50.3, 25.5, 23.5. LC-MS (ESI) calcd for C22H23ClFN3O2S 448.12 [M + H+], found 448.35 [M + H+]. N-(4-(4-(5-Chloro-1H-indol-3-yl)-5,6-dihydropyridin-1(2H)-yl)butyl)-4-fluorobenzenesulfonamide (45). The title compound was prepared starting from intermediate amine (0.5 mmol, 1 equiv, 0.145 g) and 4-fluorobenzene-1-sulfonyl chloride (0.541 mmol, 1 equiv, 0.09 g). Yield 38%, yellowish solid, mp 129−132 °C. 1H NMR (300 MHz, acetone-d6): δ 8.78−8.57 (m, 1 H), 7.96 (d, J = 7.4 Hz, 1 H), 7.91− 7.82 (m, 3 H), 7.49−7.42 (m, 2 H), 7.22 (t, J = 8.8 Hz, 2 H), 7.12 (dd, J = 1.9, 8.6 Hz, 1 H), 6.15 (br s, 1 H), 3.13 (d, J = 3.3 Hz, 2 H), 2.93 (t, J = 6.3 Hz, 2 H), 2.71−2.65 (m, 2 H), 2.63−2.58 (m, 2 H), 2.45− 2.40 (m, 2 H), 1.59 (td, J = 3.2, 5.9 Hz, 4 H). 13C NMR (75 MHz, acetone-d6): δ 137.6, 137.6, 135.7, 129.8, 129.7, 129.7, 126.2, 124.8, 7495



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123.9, 121.5, 119.5, 118.3, 116.5, 116.0, 115.7, 113.0, 57.7, 52.5, 50.4, 42.9, 31.7, 24.3, 22.4. LC-MS (ESI) calcd for C23H25ClFN3O2S 462.14 [M + H+], found 462.38 [M + H+]. 4-Chloro-N-(2-(4-(5-chloro-1H-indol-3-yl)-5,6-dihydropyridin1(2H)-yl)ethyl)benzenesulfonamide (46). The title compound was prepared starting from intermediate amine (0.5 mmol, 1 equiv, 0.131 g) and 3-chlorobenzene-1-sulfonyl chloride (0.5 mmol, 1 equiv, 0.105 g). Yield 47%, yellowish solid, mp 122−125 °C. 1H NMR (300 MHz, DMSO-d6): δ 11.30 (br s, 1 H), 7.82 (d, J = 8.7 Hz, 2 H), 7.74 (d, J = 2.1 Hz, 1 H), 7.64 (d, J = 8.7 Hz, 3 H), 7.44−7.34 (m, 2 H), 7.08 (dd, J = 2.1, 8.5 Hz, 1 H), 6.00 (br s, 1 H), 3.02 (d, J = 2.3 Hz, 2 H), 2.93 (t, J = 6.7 Hz, 2 H), 2.58−2.50 (m, 2 H), 2.42 (t, J = 6.5 Hz, 4 H). 13C NMR (75 MHz, DMSO-d6): δ 140.1, 137.6, 135.8, 129.7, 129.5, 128.9, 126.1, 124.9, 124.4, 121.6, 119.5, 118.5, 116.2, 113.7, 57.3, 52.9, 50.4, 28.8. LC-MS (ESI) calcd for C21H21Cl2N3O2S 450.08 [M + H+], found 450.28 [M + H+]. 4-Chloro-N-(3-(4-(5-chloro-1H-indol-3-yl)-5,6-dihydropyridin1(2H)-yl)propyl)benzenesulfonamide (47). The title compound was prepared starting from intermediate amine (0.5 mmol, 1 equiv, 0.138 g) and naphthalene-1-sulfonyl chloride (0.5 mmol, 1 equiv, 0.113 g). Yield 48%, yellowish solid, mp 142−144 °C. 1H NMR (300 MHz, DMSO-d6): δ 11.55 (br s, 1 H), 10.25 (br s, 1 H), 7.92−7.79 (m, 3 H), 7.61 (d, J = 2.3 Hz, 1 H), 7.50−7.35 (m, 3 H), 7.20−7.08 (m, 1 H), 6.14−6.05 (m, 1 H), 4.04−3.90 (m, 1 H), 3.78−3.64 (m, 1 H), 3.58 (m, 2 H), 3.21−3.09 (m, 2 H), 2.83 (d, J = 6.2 Hz, 4 H), 1.93− 1.81 (m, 1 H), 1.74 (m, 1 H). 13C NMR (75 MHz, DMSO-d6): δ 138.2, 137.0, 135.7, 130.1, 129.9, 129.6, 126.2, 125.7, 124.9, 122.1, 119.4, 117.1, 116.7, 114.4, 114.0, 112.5, 66.9, 53.7, 50.3, 25.5, 23.5. LC-MS (ESI) calcd for C22H23Cl2N3O2S 464.09 [M + H+], found 464.31 [M + H+]. 4-Chloro-N-(4-(4-(5-chloro-1H-indol-3-yl)-5,6-dihydropyridin1(2H)-yl)butyl)benzenesulfonamide (48). The title compound was prepared starting from intermediate (0.5 mmol, 1 equiv, 0.145 g) and 4-chlorobenzene-1-sulfonyl chloride (0.5 mmol, 1 equiv, 0.113 g). Yield 40%, yellowish solid, mp 195−196 °C. 1H NMR (300 MHz, DMSO-d6): δ 11.33 (br s, 1 H), 7.89−7.71 (m, 4 H), 7.62 (d, J = 8.7 Hz, 2 H), 7.47−7.34 (m, 2 H), 7.09 (dd, J = 1.9, 8.6 Hz, 1 H), 6.04 (br s, 1 H), 3.01 (br s, 2 H), 2.77 (d, J = 3.1 Hz, 2 H), 2.58−2.50 (m, 2 H), 2.45 (br s, 2 H), 2.28 (br s, 2 H), 1.40 (br s, 4 H). 13C NMR (75 MHz, DMSO-d6): δ 140.0, 137.5, 135.8, 129.7, 129.7, 129.5, 128.8, 128.8, 126.1, 124.9, 124.4, 121.6, 119.6, 118.6, 116.2, 113.7, 57.7, 53.0, 50.4, 43.0, 28.9, 27.5, 24.1. LC-MS (ESI) calcd for C23H25Cl2N3O2S 478.11 [M + H+], found 478.33 [M + H+]. In Vitro Studies. The tested compounds were examined for known classes of assay interference compounds. According to SwissADME tool,66 none of the compounds contain substructural features recognized as pan assay interference compounds (PAINS).67 Radioligand Binding Assays for D2R, 5-HT6R, 5-HT7R, 5HT1AR, M1R, and SERT. The radioligand binding assays for D2R, 5HT6R, 5-HT7R, 5-HT1AR, and SERT were carried out at the Faculty of Pharmacy, Jagiellonian University Medical College. The remaining in vitro studies (binding to M1R and functional activity assays) were performed by Cerep SA (Le Bois l’Evêque, Poitiers, France). Inhibition of hERG was tested by ChanTest (USA). Preparation of Solutions of Test and Reference Compounds. One mM stock solutions of tested compounds were prepared in DMSO. Serial dilutions of compounds were prepared in 96-well microplate in assay buffers using automated pipetting system epMotion 5070 (Eppendorf). Each compound was tested in 10 concentrations from 1.0 × 10−6 to 1.0 × 10−11 M (final concentration). 5-HT 1A Receptor Binding Assay. Radioligand binding was performed using membranes from CHO-K1 cells stably transfected with the human 5-HT1A receptor (PerkinElmer). All assays were carried out in duplicates. First, 50 μL working solutions of the tested compounds, 50 μL of [3H]-8-OH-DPAT (final concentration 1 nM), and 150 μL of diluted membranes (10 μg protein per well) prepared in assay buffer (50 mM Tris, pH 7.4, 10 mM MgSO4, 0,5 mM EDTA, 0.1% ascorbic acid) were transferred to a polypropylene 96-well microplate using a 96-well pipetting station Rainin Liquidator (MettlerToledo). Serotonin (10 μM) was used to define nonspecific

binding. Microplate was covered with a sealing tape, mixed, and incubated for 60 min at 27 °C. The reaction was terminated by rapid filtration through a GF/C filter mate presoaked with 0.3% polyethylenimine for 30 min. Ten rapid washes with 200 μL of 50 mM Tris buffer (4 °C, pH 7.4) were performed using an automated harvester system Harvester-96 MACH III FM (Tomtec). The filter mates were dried at 37 °C in a forced air fan incubator, and then solid scintillator MeltiLex was melted on filter mates at 90 °C for 4 min. Radioactivity was counted in MicroBeta2 scintillation counter (PerkinElmer). Data were fitted to a one-site curve-fitting equation with Prism 6 (GraphPad Software), and Ki values were estimated from the Cheng−Prusoff equation. 5-HT2A Receptor Binding Assay. Radioligand binding was performed using membranes from CHO-K1 cells stably transfected with the human 5-HT2A receptor (PerkinElmer). All assays were carried out in duplicates. First, 50 μL of working solution of the tested compounds, 50 μL of [3H]-ketanserin (final concentration 1 nM), and 150 μL of diluted membranes (7 μg protein per well) prepared in assay buffer (50 mM Tris, pH 7.4, 4 mM CaCl2, 0.1% ascorbic acid) were transferred to a polypropylene 96-well microplate using a 96-well pipetting station Rainin Liquidator (MettlerToledo). Mianserin (10 μM) was used to define nonspecific binding. Microplate was covered with a sealing tape, mixed, and incubated for 60 min at 27 °C. The reaction was terminated by rapid filtration through a GF/B filter mate presoaked with 0.5% polyethylenimine for 30 min. Ten rapid washes with 200 μL of 50 mM Tris buffer (4 °C, pH 7.4) were performed using an automated harvester system Harvester-96 MACH III FM (Tomtec). The filter mates were dried at 37 °C in a forced air fan incubator, and then solid scintillator MeltiLex was melted on filter mates at 90 °C for 5 min. Radioactivity was counted in MicroBeta2 scintillation counter (PerkinElmer). Data were fitted to a one-site curve-fitting equation with Prism 6 (GraphPad Software), and Ki values were estimated from the Cheng−Prusoff equation. 5-HT6 Receptor Binding Assay. Radioligand binding was performed using membranes from CHO-K1 cells stably transfected with the human 5-HT6 receptor (PerkinElmer). All assays were carried out in duplicates. First, 50 μL of working solution of the tested compounds, 50 μL of [3H]-LSD (final concentration 1 nM), and 150 μL of diluted membranes (8 μg protein per well) prepared in assay buffer (50 mM Tris, pH 7.4, 10 mM MgCl2, 0.1 mM EDTA) were transferred to a polypropylene 96-well microplate using a 96-well pipetting station Rainin Liquidator (MettlerToledo). Methiothepin (10 μM) was used to define nonspecific binding. Microplate was covered with a sealing tape, mixed, and incubated for 60 min at 37 °C. The reaction was terminated by rapid filtration through a GF/A filter mate presoaked with 0.5% polyethylenimine for 30 min. Ten rapid washes with 200 μL of 50 mM Tris buffer (4 °C, pH 7.4) were performed using an automated harvester system Harvester-96 MACH III FM (Tomtec). The filter mates were dried at 37 °C in a forced air fan incubator, and then solid scintillator MeltiLex was melted on filter mates at 90 °C for 5 min. Radioactivity was counted in MicroBeta2 scintillation counter (PerkinElmer). Data were fitted to a one-site curve-fitting equation with Prism 6 (GraphPad Software), and Ki values were estimated from the Cheng−Prusoff equation. 5-HT7 Receptor Binding Assay. Radioligand binding was performed using membranes from CHO-K1 cells stably transfected with the human 5-HT7 receptor (PerkinElmer). All assays were carried out in duplicates. First, 50 μL of working solution of the tested compounds, 50 μL of [3H]-LSD (final concentration 3 nM), and 150 μL of diluted membranes (17 μg protein per well) prepared in assay buffer (50 mM Tris, pH 7.4, 10 mM MgSO4, 0.5 mM EDTA) were transferred to a polypropylene 96-well microplate using a 96-well pipetting station Rainin Liquidator (MettlerToledo). Methiothepin (10 μM) was used to define nonspecific binding. Microplate was covered with a sealing tape, mixed, and incubated for 120 min at 27 °C. The reaction was terminated by rapid filtration through GF/A filter mate presoaked with 0.3% polyethylenimine for 30 min. Ten rapid washes with 200 μL of 50 mM Tris buffer (4 °C, pH 7.4) were performed using an automated harvester system Harvester-96 MACH III FM (Tomtec). The filter mates were dried at 37 °C in a forced air fan incubator, and then solid 7496



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Table 6. Outline of Methods Used in Functional Activity Assays assay D2S agonist effect69 D2S antagonist effect69 5-HT6 agonist effect70 5-HT6 antagonist effect70 5-HT7 agonist effect71 5-HT7 antagonist effect71 5-HT1A agonist effect72 5-HT1A antagonist effect72 5-HT uptake73

source human recombinant (HEK-293 cells) human recombinant (HEK-293 cells) human recombinant (CHO cells) human recombinant (CHO cells) human recombinant (CHO cells) human recombinant (CHO cells) human recombinant (HEK-293 cells) human recombinant (HEK-293 cells) rat brain synaptosomes

substrate/stimulus

incubation

measured component

detection method

none (3 μM DA for control)

28 °C

impedance

DA (30 nM)

28 °C

impedance

none (10 μM 5-HT for control) 5-HT (300 nM)

45 min 37 °C 30 min 37 °C 30 min 37 °C 30 min 37 °C 37 °C

cAMP

cellular dielectric spectroscopy cellular dielectric spectroscopy HTRF

cAMP

HTRF

cAMP

HTRF

cAMP

HTRF

impedance

37 °C

impedance

15 min 37 °C

[3H]5-HT incorporation into synaptosomes

cellular dielectric spectroscopy cellular dielectric spectroscopy scintillation counting

none (10 μM 5-HT for control) 5-HT (300 nM) none (10 μM 8-OH-DPAT for control) 8-OH-DPAT (100 nM) [3H]5-HT (0.2 μCi/mL)

scintillator MeltiLex was melted on filter mates at 90 °C for 5 min. Radioactivity was counted in MicroBeta2 scintillation counter (PerkinElmer). Data were fitted to a one-site curve-fitting equation with Prism 6 (GraphPad Software), and Ki values were estimated from the Cheng−Prusoff equation. SERT Binding Assay. Radioligand binding was performed using membranes from HEK-293 cells stably transfected with the human serotonin transporter (PerkinElmer). All assays were carried out in duplicates. First, 50 μL of working solution of the tested compounds, 50 μL of [3H]-imipramine (final concentration 2 nM), and 150 μL of diluted membranes (9 μg protein per well) prepared in assay buffer (50 mM Tris, pH 7.4, 120 mM NaCl, 5 mM KCl) were transferred to a polypropylene 96-well microplate using a 96-well pipetting station Rainin Liquidator (MettlerToledo). Imipramine (10 μM) was used to define nonspecific binding. Microplate was covered with a sealing tape, mixed, and incubated for 30 min at 27 °C. The reaction was terminated by rapid filtration through a GF/A filter mate presoaked with 0.5% polyethylenimine for 30 min. Ten rapid washes with 200 μL 50 mM Tris buffer (4 °C, pH 7.4) were performed using an automated harvester system Harvester-96 MACH III FM (Tomtec). The filter mates were dried at 37 °C in a forced air fan incubator,, and then solid scintillator MeltiLex was melted on filter mates at 90 °C for 5 min. Radioactivity was counted in MicroBeta2 scintillation counter (PerkinElmer). Data were fitted to a one-site curve-fitting equation with Prism 6 (GraphPad Software), and Ki values were estimated from the Cheng−Prusoff equation. D2 Receptor Binding Assay. Radioligand binding was performed using membranes from CHO-K1 cells stably transfected with the human D2 receptor (PerkinElmer). All assays were carried out in duplicates. First, 50 μL of working solution of the tested compounds, 50 μL of [3H]-methylspiperone (final concentration 0.4 nM), and 150 μL of diluted membranes (3 μg protein per well) prepared in assay buffer (50 mM Tris, pH 7.4, 50 mM HEPES, 50 mM NaCl, 5 mM MgCl2, 0.5 mM EDTA) were transferred to a polypropylene 96-well microplate using a 96-well pipetting station Rainin Liquidator (MettlerToledo). Haloperidol (10 μM) was used to define nonspecific binding. Microplate was covered with a sealing tape, mixed, and incubated for 60 min at 37 °C. The reaction was terminated by rapid filtration through a GF/B filter mate presoaked with 0.5% polyethylenimine for 30 min. Ten rapid washes with 200 μL of 50 mM Tris buffer (4 °C, pH 7.4) were performed using an automated harvester system Harvester-96 MACH III FM (Tomtec). The filter mates were dried at 37 °C in a forced air fan incubator, and then solid scintillator MeltiLex was melted on filter mates at 90 °C for 5 min. Radioactivity was counted in MicroBeta2 scintillation counter (PerkinElmer). Data were fitted to a one-site curve-fitting equation with Prism 6 (GraphPad Software), and Ki values were estimated from the Cheng−Prusoff equation.

M1 Receptor Binding Assay. Binding affinity for human recombinant M1 receptor was tested using CHO cells and [3H]pyrilamine radioligand at concentration of 2 nM (Kd 13 nM). Atropine (1 μM) was used to assess nonspecific binding. Incubation time was 60 min at room temperature and the signal level was detected by scintillation counting.68 The experiment was performed in duplicate in tested ligand concentration 1.0 × 10−6 M. All the radioligand binding assays shown in Tables 1 and 2 were performed in three independent experiments in duplicate (n = 2). Functional Activity Assays for D2R, 5-HT6R, 5-HT7R, 5-HT1AR, and 5-HT Uptake. EC50, IC50, and KB values were determined from six concentrations (1.0 × 10−6 to 1.0 × 10−10 M). Screening assays from Table 4 were performed in duplicate at concentration 1.0 × 10−6 M. The results are expressed as a percent of control specific agonist response ((measured specific response/control specific agonist response) × 100) and as a percent inhibition of control specific agonist response (100 − ((measured specific response/control specific agonist response) × 100)) obtained in the presence of the test compounds for agonist and antagonist effect, respectively. An outline of methodologies and references for further methodological details are shown in Table 6. Inhibition of hERG Potassium Channel. Blockade of hERGmediated potassium currents was carried out by ChanTest (Cleveland, Ohio) and expressed as mean % of inhibition at 1.0 × 10−6 M (experiment performed in duplicate, Table 3) or IC50 value determined from six concentrations (1.0 × 10−6 to 1.0 × 10−10 M), performed in three experiments in duplicate. 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 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 was performed using the IonWorks QuattroTM 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 are the currents elicited by the test pulse in control and in the presence of a test compound, respectively. hERG inhibition of reference E-4031 in this assay was determined, EC50 = 89 nM. In Vivo Pharmacological Studies. Substances. Compound 16, aripiprazole, and diazepam were prepared as a suspension in 1% aqueous solution of Tween 80, while MK-801, citalopram, imipramine, and scopolamine were dissolved in distilled water. An injection volume 7497



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30−59.9 s, and a maximum score of 3 for staying on the bar for >60 s. The minimum cataleptogenic dose was defined as the lowest dose inducing a mean catalepsy score of >1 at 30, 60, or 120 min posttreatment. Spontaneous Locomotor Activity in CD-1 Mice. The locomotor activity was recorded with an Opto M3 multichannel activity monitor (MultiDevice Software v.1.3, Columbus Instruments). The mice were individually placed in plastic cages (22 cm × 12 cm × 13 cm), and then the crossings of each channel (ambulation) were counted during 2 h with data recording every 5 min. The cages were cleaned up with 70% ethanol after each mouse. Drugs were administered to 10 mice per treatment group. MK-801-Induced Locomotor Hyperactivity in CD-1 Mice. The locomotor activity was recorded according to method described above. MK-801 was injected at a dose of 0.2 mg/kg 15 min before the test. Drugs were administered to 10 mice per treatment group. Passive Avoidance Task in CD-1 Mice. Animals. Adult male mice (stock’s name: CD-1, 18−21 g), purchased from Animal House at the Faculty of Pharmacy, Jagiellonian University Medical College, Krakow, Poland, were used in the experiments. The groups of 15 mice were kept to a plastic cage (60 cm × 38 cm × 20 cm) at a room temperature (22 ± 2 °C), on 12 h light/dark cycles (the lights turned on at 7:00 and off at 19:00). Animals had free access to standard laboratory food and tap water. Behavioral experiments were performed between 09:00 am and 14:00, and each experimental group consisted of 8−10 randomly selected animals. Mice were used only once in each test and then killed by cervical dislocation. All experimental procedures were carried out in accordance with EU Directive 2010/63/EU and approved by the I Local Ethics Committee for Experiments on Animals of the Jagiellonian University in Krakow, Poland (approval no. 103/2016). Step-Through Passive Avoidance Task in Mice after Acute Administration. Step-through passive avoidance task was performed according to the method previously described.77 The apparatus for step-through passive avoidance task consisted of two compartments, separated by an automated sliding door (LE872, Bioseb, France). For acquisition session, mice were placed individually in an illuminated white compartment (20 cm × 21 cm × 20 cm, 1000 lx) with the closed door to a smaller dark compartment (7.3 cm × 7.5 cm × 14 cm, 10 lx) equipped with an electric grid floor (stainless steel rods through which an electric footshock is delivered). After 30 s, the door to a smaller compartment was opened. Immediately after the mouse entered the smaller dark compartment, the door closed and the rodent was punished by an inescapable electric foot shock (0.2 mA for 2 s). The mice which did not enter the dark compartment within 50 s were excluded from the study. On the following day (24 h later), the pretrained animals were placed again into the illuminated compartment and observed up to 300 s (retention session). The experimental procedure was similar to acquisition session, but this time mice did not receive the electric shock after the entrance to the smaller dark compartment. Mice which avoided the dark compartment for 300 s were considered to remember the task. ̈ Mice. Evaluation of Cognitive-Enhancing Properties in Naive Compound 16 (0.15−5 mg/kg) and aripiprazole (0.25−2 mg/kg) was injected (ip) 30 min before the acquisition trial. Control groups were administered (ip) with saline. The Effect on Scopolamine-Induced Cognitive Dysfunction. Compound 16 (0.15−0.625 mg/kg), aripiprazole (0.25−2 mg/kg), and scopolamine (1 mg/kg) were administered (ip) 30 min before the acquisition trial. Control groups received (ip) saline. Data Analysis. Results are presented as means ± SEM. The comparisons between experimental and control groups were performed by one-way ANOVA followed by Tukey’s test post hoc. A value of p < 0.05 was considered to be significant.

10 mL/kg of all compounds was administered intraperitoneally (ip) for 30 min (for compound 4, aripiprazole, citalopram, imipramine, and scopolamine), 60 min (for diazepam), and 15 min (for MK-801) before appropriate experiment. All drugs, except for scopolamine (Sigma-Aldrich, Germany), imipramine (Sigma-Aldrich, UK), and MK-801 (Sigma-Aldrich, UK), were synthesized by Adamed Ltd. (Pieńków, Poland). Animals. Swiss albino mice weighing 21−22 g upon arrival from a licensed breeder (Staniszewska; Ilkowice, Poland) or CD-1 mice from the animal facility localized at Medical College of Jagiellonian University were group-housed for a 3−4 day period in polycarbonate Makrolon type 3 cages (dimensions 26.5 cm × 15 cm × 42 cm) in an environmentally controlled, experimental room (ambient temperature 22 ± 2 °C; relative humidity 50−60%; 12:12 light:dark cycle, lights on at 8:00), in groups of 15. Standard laboratory food (Ssniff M-Z) and filtered water were freely available. On the day before experiments, equipment producing “white noise” was turned on for 30 min and mice were weighted exact to 1 g. Animals were assigned randomly to treatment groups. All the experiments were performed by two observers unaware of the treatment applied between 9:00 and 14:00 on separate groups of animals. All animals were used only once and were killed immediately after the experiment. All the experimental procedures were approved by the IV Local Ethics Commission in Warszawa. Forced Swim Test in Swiss Albino Mice. The experiment was carried out according to the method of Porsolt et al.74 Briefly, mice were individually placed in a glass cylinder (25 cm high; 10 cm in diameter) containing 6 cm of water maintained at 23−25 °C and were left there for 6 min. A mouse was regarded as immobile when it remained floating on the water, making only small movements to keep its head above it. The total duration of immobility was recorded during the last 4 min of a 6 min test session. Drugs were administered to nine mice per treatment group. Four-Plate Test in Swiss Albino Mice. The four-plate apparatus (BIOSEB, France) consisted of a cage (25 cm × 18 cm × 16 cm) floored by four identical rectangular metal plates (8 cm × 11 cm) separated from one another by a gap of 4 mm. The top of the cage was covered by a transparent Perspex lid that prevents escape behavior. The plates were connected to a device that can generate electric shocks. Following a 15 s habituation period, the animal’s motivation to explore a novel environment was suppressed by an electric foot shock (0.8 mA, 0.5 s) every time it moved from one plate to another during a 1 min test session.75 This action is referred to as a “punished crossing”, and was followed by a 3 s shock interval, during which the animal could move across plates without receiving a shock. Drugs were administered to 10 mice per treatment group. Spontaneous Locomotor Activity in Swiss Albino Mice. The locomotor activity was recorded with an Opto M3 multichannel activity monitor (MultiDevice Software v.1.3, Columbus Instruments). Swiss mice were individually placed in plastic cages (22 cm × 12 cm × 13 cm) for a 30 min habituation period, and then the crossings of each channel (ambulation) were counted from 2 to 6 min with data recording every 2 min (i.e., the time equal to the observation period in the forced swim test) or during the first 5 min with data recording every 1 min (i.e., the time equal to the observation period in the fourplate test). The cages were cleaned up with 70% ethanol after each mouse. Drugs were administered to 6 or 10 mice per treatment group. The influence of effective doses only recorded in the forced swim and the four-plate tests was studied in spontaneous locomotor activity in order to exclude the possibility of competing behaviors such as general locomotor activity. Catalepsy (Bar Test) in CD-1 Mice. Compounds were administered to 10 mice per treatment group. Animal’s forelimbs were draped over a thin, cylindrical horizontal rod elevated 4 cm above the tabletop at 30, 60, and 120 min after administration of a test compound. The length of time the animal touched the bar with both front paws was measured up to a preset cutoff time of 60 s. A maximum of three trials was used for each animal. A scoring system used by Ö gren et al. was employed.76 Results of each trial were scored as follows: 0 for holding the position for

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