Novel Multitarget-Directed Ligands Aiming at Symptoms and Causes

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Novel multi-target-directed ligands aiming at symptoms and causes of Alzheimer’s disease Anna Wi#ckowska, Tomasz Wichur, Justyna Gody#, Adam Bucki, Monika Marcinkowska, Agata Siwek, Krzysztof Wi#ckowski, Paula Zar#ba, Damijan Knez, Monika G#uch-Lutwin, Grzegorz Kazek, Gniewomir Latacz, Kamil Mika, Marcin Ko#aczkowski, Jan Korabecny, Ondrej Soukup, Marketa Benkova, Katarzyna J. Kiec-Kononowicz, Stanislav Gobec, and Barbara Malawska ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.8b00024 • Publication Date (Web): 31 Jan 2018 Downloaded from http://pubs.acs.org on February 2, 2018

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ACS Chemical Neuroscience

ACS Paragon Plus Environment

ACS Chemical Neuroscience 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Novel multi-target-directed ligands aiming at symptoms and causes of Alzheimer’s disease Anna Więckowskaa*, Tomasz Wichura, Justyna Godyńa, Adam Buckib, Monika Marcinkowskab, Agata Siwekc, Krzysztof Więckowskid, Paula Zarębaa, Damijan Kneze, Monika Głuch-Lutwinc, Grzegorz Kazekf, Gniewomir Lataczg, Kamil Mikaa, Marcin Kołaczkowskib, Jan Korabecnyh,i, Ondrej Soukuph,i, Marketa Benkovah,i, Katarzyna Kieć-Kononowiczg, Stanislav Gobece, Barbara Malawskaa a

Department of Physicochemical Drug Analysis, Faculty of Pharmacy, Jagiellonian University

Medical College, 9 Medyczna Str. 30-688 Kraków, Poland b

Department of Medicinal Chemistry, Faculty of Pharmacy, Jagiellonian University Medical College,

9 Medyczna Str. 30-688 Kraków, Poland c

Department of Pharmacobiology, Faculty of Pharmacy, Jagiellonian University Medical College, 9

Medyczna Str. 30-688 Kraków, Poland d

Department of Organic Chemistry, Faculty of Pharmacy, Jagiellonian University Medical College, 9

Medyczna Str. 30-688 Kraków, Poland e

Faculty of Pharmacy, University of Ljubljana, Askerceva 7, 1000 Ljubljana, Slovenia

f

Department of Pharmacological Screening, Faculty of Pharmacy, Jagiellonian University Medical

College, 9 Medyczna Str. 30-688 Kraków, Poland g

Department of Technology and Biotechnology of Drugs, Faculty of Pharmacy, Jagiellonian

University Medical College , Medyczna 9, 30-688 Kraków, Poland h

Department of Toxicology and Military Pharmacy, Faculty of Military Health Sciences, Trebesska

1575, 500 01 Hradec Kralove, Czech Republic i

Biomedical Research Centre, University Hospital Hradec Kralove, Sokolska 581, 500 05 Hradec

Kralove, Czech Republic

*

Corresponding Author: tel: (+48 12) 620 54 50, fax: (+48 12) 657 02 62; e-mail:

[email protected]


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ABSTRACT Alzheimer’s disease (AD) is a major public health problem, which is due to its increasing prevalence and lack of effective therapy or diagnostics. Complexity of AD pathomechanism requires complex treatment e.g. multifunctional ligands targeting both causes and symptoms of the disease. Here, we present new multi-target-directed ligands combining pharmacophore fragments that provide blockade of serotonin 5-HT6 receptors, acetyl/butyrylcholinesterase inhibition, and amyloid β anti-aggregation activity. Compound 12 has displayed balanced activity as an antagonist of 5-HT6 receptors (Ki = 18 nM) and non-competitive inhibitor of cholinesterases (IC50hAChE = 14 nM, IC50eqBuChE = 22 nM). In further in vitro studies, compound 12 has shown amyloid β anti-aggregation activity (IC50 = 1.27 µM) and ability to permeate through the blood-brain barrier. The presented findings may provide and excellent starting point for further studies and facilitate efforts to develop new effective anti-AD therapy.

Keywords: Alzheimer’s disease, multi-target-directed ligands, acetylcholinesterase inhibitors, butyrylcholinesterase inhibitors, 5-HT6 receptor antagonists, inhibition of β-amyloid aggregation

1. INTRODUCTION Alzheimer’s disease (AD) is the leading cause of dementia and it affects 34 million people worldwide. With the aging of the population, the prevalence of AD continues to increase, and the World Health Organization (WHO) estimates that it can reach at least 95 million by 2050. Comprehensive care and treatment of dementia worldwide in 2015 generated costs that amounted to $818 billion.1 According to WHO, such numbers, along with a great burden on families and caregivers, make AD one of the leading social, medical and economic challenges faced by society today, and a global public health priority.2 AD is a neurodegenerative disease that is manifested by cognitive symptoms, e.g. decline in memory, language and speech impairment or time and space disorientation. In 80% cases these are accompanied by behavioural and psychological symptoms of dementia (BPSD), e.g. agitation, psychosis, depression



and anxiety.3–6 Neurodegeneration in AD causes neuronal and synaptic loss, and synaptic impairment, thus leading to neurochemical changes within a number of neurotransmitter systems, mainly cholinergic system.7–9 Adequately, currently available treatment includes acetylcholinesterase (AChE) or/and butyrylcholinesterase (BuChE) inhibitors: donepezil, rivastigmine, galantamine, which raise acetylcholine levels in the brain,10,11 and NMDA receptor antagonist memantine, which regulates the activity of glutamate in the brain.12 However, these drugs can only slow down the progress of the disease and they are effective only for a limited time. Despite making great efforts and growing investment in the pharmaceutical industry and in academia, no new drugs against AD have been developed in more than a decade.13 Among drug candidates in advanced stages of clinical trials, 5-HT6 receptor antagonists have been successfully evaluated in Phase II clinical trials. Two antagonists, idalopirdine and intepirdine, improved cognitive functions in donepezil-treated patients with mild to moderate AD. 14,15,16 Moreover, preclinical studies have shown that serotonergic 5-HT6 receptors are not only linked to cognitive dysfunction, but also to affective disorders, anxiety and depression, the very symptoms of BPSD observed in AD patients.17,18 There are also reports indicating disease-modifying potential of 5-HT6 receptor antagonists related to synaptic remodelling19 and neuronal hyperexcitability,20 which are regarded to be possible causes of AD. Hence, 5-HT6 antagonists may provide both causal and symptomatic treatment of AD. Disease-modifying treatment that could affect the underlying disease process, and therefore delay onset of the disease or slow its progress is the holy grail of medicine.21 However, it is a big challenge in the case of AD since its pathogenesis is not completely explained and it is not clear which of the mechanisms underlying the disease is the trigger. Compelling body of evidence supports amyloid cascade hypothesis22,23 according to which amyloid-β (Aβ) aggregation is the major and necessary factor that initiates other processes that lead up to neurodegeneration in AD. Amyloidlowering therapies are therefore in the limelight of research aimed at AD pathomechanism. There is a number of strategies undertaken to prevent Aβ aggregation, including inhibition or modulation of enzymes responsible for Aβ production (β-secretase and γ-secretase) and inhibition of Aβ aggregation itself. However, with respect to the mechanism-based side effects of the secretases inhibition,24,25 the inhibition of Aβ self-aggregation seems to be a more promising strategy. The direct association of Aβ


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with pathomechanism of AD makes amyloid β anti-aggregation activity a frequently used additional mechanism in designing new agents against AD.26–28 It is appetent that search for a drug that could act through only one of the potential biological targets may not be enough to address the problem of effective AD treatment. The currently available drugs that were developed in accordance with the ‘one-disease-one-target’ paradigm are not effective enough. This is most likely due to the complex nature of AD, and the multitude of factors involved in its pathogenesis. As a consequence, in the last few decades, a multi-target-directed ligands (MTDLs) strategy has gained much attention in making attempts to find the right treatment of complex neurodegenerative diseases like AD.29 MTDLs are specifically and rationally designed to modulate multiple targets simultaneously, which results in higher clinical efficiency of the therapy.30 Taking all the facts into consideration, we aimed at the synthesis of MTDLs that combine mechanism that would improve cognitive functions (ChE inhibition, 5-HT6 antagonism), alleviate psychological symptoms accompanying AD (5-HT6 antagonism), and affect causative processes (Aβ inhibition) (Figure 1).

Figure 1. Combination of the mechanisms providing disease modifying and symptomatic effects as the way to improved treatment of Alzheimer’s disease. 2. RESULTS AND DISCUSSION 2.1 Design



Recently, we reported on the first MTDLs that comprise cholinesterase inhibition and 5-HT6 receptor antagonism.31 A hit structure selected from the study (Cmp. III, Figure 2) has displayed well-balanced in vitro potencies (IC50 hAChE = 12 nM, IC50 hBuChE = 29 nM, Kb 5-HT6 = 27 nM) and the ability to cross BBB in in vitro and in vivo assays. Interesting biological activity of the compound is accompanied by poor physicochemical properties. That means it exceeds limits of Lipinski’s ‘rule of five’ that evaluates druglikeness of a molecule.32 This fact may result in a number of complications in further development of the molecule, mainly related to poor pharmacokinetics. To address this issue, we have expanded the library of compounds with multifunctional profile of activity and improved selected drug-like properties. As presented in Figure 2, in the design of the novel compounds we combined the pharmacophore fragments of cholinesterase inhibitors (donepezil and tacrine) and 5-HT6 antagonists (Cmp. I and Cmp. II). As fragments providing 5-HT6 antagonistic activity we have chosen two pharmacophores of proven strong interactions with 5-HT6 receptor: 1-(3-(benzyloxy)-2-methylphenyl)piperazine (Cmp. I, series I, Figure 2)33 and 1-benzyl-4-(piperazin-1-yl)-1H-indole (Cmp. II, series II, Figure 2).34,35 Introduction of these moieties instead of the previously used 1-(phenylsulfonyl)-4-(piperazin-1-yl)1H-indole resulted in the reduction of the molecular weight by about 50–60 Da. Since molecular weight is considered to be critical for optimal pharmacokinetic properties,36 its reduction was the first step in the gradual optimization of the compounds. Additionally, for some compounds of series I, we reduced aromatic ring count to the recommended three.37 As pharmacophore fragments targeting AChE and/or BuChE, we used tacrine (a former AD drug) and derivatives of N-benzylamine. Regardless of hepatotoxicity of tacrine itself, its derivatives are often devoid of toxic effects and tacrine is still the most commonly used pharmacophore fragment in the design of MTLDs against AD.38 N-Benzylamines were chosen as lower molecular weight pharmacophores and equally important fragments used as cholinesterase inhibitors in the design of new MTDLs against AD.39 The selected serotoninergic and cholinergic pharmacophore fragments were combined by different linkers to obtain potential MTDLs. In order to find the right distance between the pharmacophore fragments that would allow optimal interactions with their biological targets, we have chosen several flexible aliphatic chains of different lengths.


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Both donepezil and tacrine derivatives were reported before as inhibitors of amyloid β aggregation.39– 41

Taking this fact into consideration, we assume that the novel compounds may also inhibit amyloid β

aggregation and therefore gain another mechanism that is important for anti-AD agents.

Figure 2. The design of novel MTDLs - merging pharmacophores responsible for cholinesterase inhibition and 5-HT6 antagonism. 2.2. Chemistry The designed compounds were prepared in accordance with the previously developed and described synthetic procedures.31 Tacrine derivatives 9–18 were obtained by means of two methods shown in Scheme 1. The first one comprised alkylation of tacrine with suitable dibromoalkanes and subsequent use

of

the

resulting

bromoalkyl

tacrines

(5–8)

in

alkylation

of

(1-(3-(benzyloxy)-2-

methylphenyl)piperazine or 1-benzyl-4-(piperazin-1-yl)-1H-indole). These reactions were carried out in DMSO in the presence of KOH, or in acetonitrile in the presence of K2CO3/KI, and resulted in obtaining compounds 11–14, 17 and 18. Another method was used to synthesize tacrine derivatives 9, 10, 15, 16 with shorter, ethylene and propylene linkers (Scheme 1). Here, 9-chloro-1,2,3,4tetrahydroacridine reacted with the suitable amino alcohols in pentan-1-ol at 150 °C to give alcohols 1 and 2. Next, in a one-pot reaction the alcohols were converted into mesylates, which were used to



alkylate

1-(3-(benzyloxy)-2-methylphenyl)piperazine

or

1-benzyl-4-(piperazin-1-yl)-1H-indole

providing the desired compounds. Scheme 1. Synthesis of compounds 9–18.

Reagents and conditions: (a) suitable dibromoalkane, KOH, dry DMSO, rt, 24 h (for 5–8); (b) suitable amino alcohol, pentan-1-ol, 150 °C, 24 h (for 1, 2); (c) methanesulfonyl chloride, TEA, dry DCM, 0 °C, 30 min; (d) 1-(3-(benzyloxy)-2-methylphenyl)piperazine, DCM, 0 °C–rt, 24 h; (e) 1-(3(benzyloxy)-2-methylphenyl)piperazine, K2CO3, KI, acetonitrile, reflux, 24 h; (f) 1-benzyl-4(piperazin-1-yl)-1H-indole hydrochloride, DCM, 0 °C–rt, 24 h; (g) 1-benzyl-4-(piperazin-1-yl)-1Hindole, K2CO3, KI, acetonitrile, reflux, 24 h.

As outlined in the Scheme 2, compounds 25–31, 34 and 35 were prepared from commercially available amines in the following sequence of reactions: N-acylation – N’-alkylation – reduction. The N-acylation of benzylamine, N-methylbenzylamine, N-benzylpiperidine and N-benzylpyrrolidine with the appropriate haloacyl halides in dichloromethane (DCM) at 0 °C–rt yielded bromo- or chloroalkylamides 19–24, 32 and 33. These products were used in N’-alkylation of 1-(3-(benzyloxy)2-methylphenyl)piperazine and 1-benzyl-4-(piperazin-1-yl)-1H-indole in the presence of K2CO3 in acetonitrile. The resulting amides were reduced with lithium aluminium hydride (1 M solution in THF) to give final compounds 25–31, 34 and 35.

Scheme 2. Synthesis of compounds 25–31, 34 and 35.


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Reagents and conditions: (a) 2-bromoacetyl bromide (for 19, 20, 32 and 33), 3-bromopropanoyl chloride (for 21), 4-chlorobutanoyl chloride (for 22), 5-bromopentanoyl chloride (for 23 and 24), DCM, 0 °C–rt, 24 h; (b) 1-(3-(benzyloxy)-2-methylphenyl)piperazine, K2CO3, acetonitrile, 50 °C or reflux, 24 h (see Experimental section for further details); (c) 1 M LiAlH4 in THF, dry THF, 0 °C– reflux, 24 h; (d) 1-benzyl-4-(piperazin-1-yl)-1H-indole hydrochloride, K2CO3, DCM, 0 °C–rt, 24 h.

Compounds 37, 42–45 were prepared utilizing Gabriel synthesis and subsequent reductive amination as shown in Scheme 3. Firstly, 1-(3-(benzyloxy)-2-methylphenyl)piperazine and 1-benzyl-4(piperazin-1-yl)-1H-indole reacted with the appropriate bromoalkylphthalimides. The phthalimides were cleaved in the reaction with 40% aqueous solution of methylamine at 50 °C followed by hydrolysis with 1 M NaOH at room temperature yielding primary amines 36 and 38–41. The amines then reacted with benzaldehyde in the presence of sodium cyanoborohydride and glacial acetic acid in methanol to give the final compounds 37 and 42–45. Scheme 3. Synthesis of compounds 37 and 42–45.



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Reagents and conditions: (a) 2-(4-bromobutyl)isoindoline-1,3-dione (for 36 and 38) or 2-(5bromopentyl)isoindoline-1,3-dione (for 39), 2-(6-bromohexyl)isoindoline-1,3-dione (for 40), 2-(7bromoheptyl)isoindoline-1,3-dione (for 41), K2CO3, acetonitrile, reflux, 24 h; (b) methylamine (40% aq. sol.), 50 °C, 1 h, then 1 M NaOH, rt, 1 h; (c) benzaldehyde, glacial acetic acid, NaCNBH3, methanol, 0 °C–rt, 24 h.

Compounds 48, 49, 52 and 53 were prepared according to the synthetic route shown in Scheme 4. In the first step N-methyl-1-phenylmethanamine and 1-benzylpiperidin-4-amine were alkylated with 3chloropropyl acetate or 4-bromobutyl acetate respectively, in refluxing acetonitrile with K2CO3. The subsequent hydrolysis of the acetyl group in a methanol/water solution of K2CO3 at 50 °C yielded the desired alcohols. The alcohols were converted into mesylates and reacted with 1-(3-(benzyloxy)-2methylphenyl)piperazine and 1-benzyl-4-(piperazin-1-yl)-1H-indole. This led to obtaining the final products 48, 49 and 53. Compound 53 required additional acidolysis of BOC protecting group that was introduced to prevent side reactions of the secondary amine group. Compound 52 was synthesized by starting with reaction

of

1-(3-(benzyloxy)-2-methylphenyl)piperazine

with

1-bromo-3-

chloropropane in the presence of K2CO3 in acetonitrile at 50 °C. The resulting 50 was then used as alkylating agent in the reaction with 1-benzylpiperidin-4-amine, carried out by refluxing the reactants in acetonitrile in the presence of K2CO3 (Scheme 4).

Scheme 4. Synthesis of compounds 48, 49, 52 and 53.


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Reagents and conditions: (a) 3-chloropropyl acetate, tetra-N-butylammonium bromide, K2CO3, acetonitrile, reflux, 24 h; (b) K2CO3, methanol, H2O, 50 °C, 2.5 h; (c) methanesulfonyl chloride, TEA, dry DCM, 0 °C, 3 h; (d) 1-(3-(benzyloxy)-2-methylphenyl)piperazine hydrochloride (for 48) or 1benzyl-4-(piperazin-1-yl)-1H-indole hydrochloride (for 49), K2CO3, acetonitrile, reflux, 24 h; (e) 1bromo-3-chloropropane, K2CO3, acetonitrile, 50 °C, 24 h; (f) 1-benzylpiperidin-4-amine, K2CO3, acetonitrile, reflux, 24 h; (g) 4-bromobutyl acetate, K2CO3, acetonitrile, reflux, 24 h; (h) di-tert-butyl dicarbonate, TEA, dry THF, 0 °C–rt, 24 h; (i) 0.1 M HCl in ethyl acetate, ethyl acetate, rt, 24 h.

2.3. In vitro studies Affinity and functional activity on 5-HT6 receptor The affinity of all the novel compounds for recombinant human 5-HT6 receptor was evaluated in a radioligand binding assay42 and the results were presented as Ki values in Table 1 and 2. In series I, Ki values ranged from 10 nM to 845 nM, and from 72 nM to 916 nM in series II. The results have shown that the affinity of tacrine derivatives increased with the length of a linker up to five carbon atoms and reached Ki value of 10 nM for 11 and 72 nM for 17 and decreased for longer, seven (13) and eight (14) carbon atom linkers (Ki of 234 nM and 240 nM, respectively). Among non-tacrine derivatives, there were only a few compounds with Ki values lower than 100 nM: N-methyl-N-benzylamine derivatives 26 and 48, 1-benzylpiperidin-4-amine derivative 53, and N-benzylamine derivative 44. From both



series I and II we selected two representative derivatives for cell-based functional studies in order to verify their antagonistic mode of action. The Kb values of 132 nM and 510 nM for 12 and 44, respectively, proved that the compounds act as 5-HT6 receptor antagonists. Based on these results and the fact that both applied pharmacophore fragments aiming at 5-HT6 receptor are indeed strong antagonists of this receptor, we infer that their derivatives possess the same profile of functional activity. The above results indicate that the AChE-aiming fragment (tacrine or N-benzylamine) negatively affects, but does not exclude binding of the 5-HT6-aiming fragment in the orthosteric site of the 5-HT6 receptor. The binding mode of compounds with 5 and 6 carbon atoms linkers is disturbed the least. This might be due to the observed H-bonding of tacrine and N-benzylamine fragments with Asp7.36 in the accessory site that allows maintaining key interactions characteristic for the 5-HT6 receptor ligands in the orthosteric site (Figure 3).34,43 The latter include charge-reinforced hydrogen bond between piperazine moiety and Asp3.32, CH-π aromatic stackings of methylphenyl (12) or indole (44) with Phe6.52, and analogous interaction of benzyloxy (12) or benzyl (44) groups with Phe5.38 (Figure 3). Due to unfavorable arrangement of molecules in the binding site and lack of the stabilizing H-bonds described above, the compounds with shorter (i.e. 2–4 carbon atoms) and longer (i.e 7–8 carbon atoms) linkers display suboptimal binding mode in the 5-HT6 receptor and therefore show lower affinity.


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Figure 3. The predicted binding mode of compound 12 (orange) and 44 (green) in the binding site of 5-HT6 receptor. The interaction network is consistent within the high-affinity representatives of both series, which applies for the 5-HT6 receptor-aiming arylpiperazine fragment in the orthosteric site as well as the AChE-aiming tacrine and N-benzylamine moieties bound in the accessory site. Amino acid residues engaged in ligand binding (within 4 Å from the ligand atoms) are shown as sticks, whereas residues identified as crucial for ligand binding, e.g. forming H-bonds (dotted yellow lines) and CH-π stacking interactions (dotted cyan lines) are represented as thick sticks. ECL2 residues were hidden for clarity. Cholinesterase (AChE/BuChE) inhibitory activity The inhibitory activities of novel MTDLs were assessed in spectrophotometric Ellman’s assay44 using AChE form electric eel (eeAChE) and BuChE form horse serum (eqBuChE). First, the compounds were screened at the concentration of 10 µM and for those with inhibition exceeding 50%, IC50 values were determined. The most potent compounds were further tested on human AChE (hAChE) following the same procedure. The results determined for the test compounds as well as for tacrine and donepezil, used as references, are presented in Tables 1 and 2. Among all the compounds the most potent eeAChE inhibitors were tacrine derivatives 9–18. Their IC50 values have ranged from 5 to 44 nM, except for compounds 11 and 17 with 5 carbon atom linkers that displayed reduced activity with IC50 values of 176 nM and 250 nM, respectively. It is worth noting that the potencies of 13–16, and 18 were higher than tacrine itself (IC50 values of 5–17 nM, vs. 24 nM). This may be the result of additional interactions with the enzyme provided by the arylpiperazine 5-HT6-aiming fragments. Molecular docking studies predicted two distinctive binding modes for short-chain (2, 3 carbon atoms linker) compounds and long-chain (6–8 carbon atoms linker) compounds (Figure 4). The tacrine fragment of the short-chain compounds binds to the catalytic site of AChE and interacts with Trp84 and Phe330 (aromatic π-π interaction), whereas the 5-HT6 fragment is located in the peripheral site, forming cation-π interactions between piperazine and Tyr334 and π-π stacking between methylphenyl (series I) or indole (series II) fragment and Trp279 (Figure 4A). The



latter interactions are preserved in the long-chain compounds but piperazine forms cation-π interaction with Phe331 instead of Tyr334. Unlike in the short-chain compounds, tacrine takes a perpendicular position in the catalytic site and apart from aromatic π-π interaction with Trp84 and Phe330 it forms ππ stacking interactions with Tyr334. It is worth noting that the compounds with 5 carbon atoms linker (11 and 17) are less prone to form interactions with Trp279, Tyr334 or Phe331 that might explain their lower inhibitory potency (Table 1 and 2 and Figure 4B). The benzyloxy (series I) and benzyl (series II) fragments interact in nonspecific manner in the outer part of the enzyme's gorge, regardless of the length of the alkyl linker (Figure 4).

Figure 4. The predicted binding mode of a short-chain compound 10 (orange) and an alternative positioning of long-chain compound 14 (green) in the active site of AChE. The binding modes show equivalent interaction patterns, irrespective of the length of the alkyl linker (A). Compound 11 is unable to form aromatic interactions with Trp279 or Phe331/Tyr334 residues due to unfavorable 5unit spacer (B). Amino acid residues engaged in ligand binding (within 4 Å from the ligand atoms) are shown as sticks, whereas residues identified as crucial for ligand binding, e.g., forming π-π stacking interactions (dotted cyan lines) or cation-π (dotted green lines) are represented as thick sticks. In series I, among the non-tacrine derivatives, compound 29, with N-benzylamine substituent and the longest, 5 carbon atoms linker displayed IC50 value of 2.3 µM. Similar activities (IC50 values ranging from 3.1 µM to 8.2 µM) were recorded for N-benzylamine derivatives 42–45 from series II. Comparison of binding pattern predicted for donepezil (which mimics the one evidenced in the crystal structure 1EVE)45 and 29 unravels the possible reason for lower performance of N-benzylamine


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derivatives (Figure 5). The binding mode of donepezil includes π-π aromatic interactions of Nbenzylpiperidine with Trp84 and cation-π interactions with Phe330 and Tyr334, which allow the 2,3dihydroinden-1-on fragment for π-π stacking with Trp279. On the other hand, compound 29 interacts with Trp84 via cation-π interaction, reinforced by its highly basic N-benzylamine fragment (calculated pKA = 9.67 vs. 8.54 of donepezil), which draws the compound deeper in the catalytic site, preventing formation of the favorable aromatic interaction with Trp279 in the peripheral site. Although cation-π interactions with Phe330 and Tyr334 are preserved due to basic piperazine of 29, the overall binding mode of N-benzylamine derivatives appears to be disadvantageous (Figure 5).

Figure 5. The predicted binding mode of compound 29 (purple) vs. donepezil (grey) in the active site of AChE. Compound 29 is unable to reach Trp279 due to cation-π interaction with Trp84 provided by a high conformational flexibility of alkyl linker. Amino acid residues taking part in the ligand binding (within 4 Å from the ligand atoms) are shown as sticks, whereas residues identified as crucial for ligand binding, e.g., forming π-π stacking interactions (dotted cyan lines) or cation-π (dotted green lines) are represented as thick sticks. Additionally, we observed higher average activity of the compounds against eqBuChE than for eeAChE. Again, tacrine derivatives 9–18 were the most potent inhibitors with IC50 values ranging from 22 to 85 nM in series I and from 5 to 25 nM in series II. All except one (36) of the non-tacrine derivatives form series I and II, displayed inhibitory activities on eqBuChE in ranges from 0.196 µM



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for compound 49 to 7.78 µM for 34. It was demonstrated that BuChE, which plays rather supportive role in the healthy brain, in the course of AD takes over the function of AChE.46 Therefore search for both selective and non-selective BuChE inhibitors is important and may benefit AD patients. As the most active compounds from both series, the tacrine derivatives were tested against recombinant human AChE. Their IC50 values have ranged from 3 to 62 nM and were lower than the activity of unsubstituted tacrine (IC50 = 131 nM). We have tested two selected compounds against human BuChE, tacrine derivative – compound 12 (IC50 = 22 nM) and non-tacrine, selective BuChE inhibitor – compound 49 (IC50 = 620 nM). Table 1. Cholinesterase inhibitory potency and 5-HT6 receptor affinity of the hybrid compounds of series I.

IC50 [µM]a

Ki [µM] Cmpd.

n

R h5-HT6R

eeAChEb

hAChEc

eqBuChEd

9

2

0.232 ± 0.012

0.044 ± 0.0007

0.062 ± 0.0015

0.026 ± 0.0008

10

3

0.078 ± 0.002

0.025 ± 0.0007

0.010 ± 0.0003

0.045 ± 0.0007

11

5

0.010 ± 0.005

0.176 ± 0.003

0.024 ± 0.0003

0.085 ± 0.002

12

6

0.018 ± 0.001 0.132 ± 0.030e

0.043 ± 0.001

0.014 ± 0.0004

0.022 ± 0.0006 0.022 ± 0.0007f

13

7

0.234 ± 0.022

0.014 ± 0.0005

0.011 ± 0.0004

0.026 ± 0.0009

14

8

0.240 ± 0.006

0.005 ± 0.0001

0.003 ± 0.0001

0.024 ± 0.0007

25

2

0.096 ± 0.002

n.a

-

4.554 ± 0.269

27

3

0.157 ± 0.008

n.a

-

4.888 ± 0.242

37

4

0.242 ± 0.016

n.a

-

5.605 ± 0.202

29

5

0.140 ± 0.005

2.340 ± 0.042

-

3.733 ± 0.114

26

2

0.069 ± 0.002

n.a

-

4.497 ± 0.154

48

3

0.060 ± 0.001

n.a

-

4.814 ± 0.096

N N H


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28

4

0.845 ± 0.043

n.a

-

6.091 ± 0.154

30

5

0.300 ± 0.031

n.a

-

5.866 ± 0.181

34

2

0.225 ± 0.003

n.a

-

7.782 ± 0.074

36

2

0.320 ± 0.026

n.a

-

n.a

52

3

n.a.

n.a

-

4.990 ± 0.200

53

4

0.070 ± 0.002

n.a

-

4.461 ± 0.064

Cmp. I

0.003 ± 0.0002 0.007± 0.0002e

n.a.

n.a.

-

Donepe zil

-

0.010 ± 0.0002

1.830 ± 0.040

0.006 ± 0.0001

Tacrine

-

0.024 ± 0.0004

0.002 ± 0.0001

0.131 ± 0.002

a

IC50 values are expressed as the mean ± standard error of the mean (SEM) of at least three

experiments; bAChE from the electric eel; crecombinant human AChE; dBuChE from equine serum; e

Kb value; fhuman recombinant BuChE.

Table 2. Cholinesterase inhibitory potency and 5-HT6 receptor affinity of the hybrid compounds of series II.

IC50 [µM]a

Ki [µM] Cmpd.

n R h5-HT6R

eeAChEb

hAChEc

eqBuChEd

0.094 ± 0.002

0.011 ± 0.0003

0.026 ± 0.0007

0.005 ± 0.0002

0.148 ± 0.014

0.013 ± 0.0003

0.038 ± 0.0007

0.011 ± 0.0002

0.072 ± 0.002

0.25 ± 0.005

0.020 ±0.0001

0.025 ± 0.0005

15

2

16

3

17

5

18

6

0.130 ± 0.001

0.017 ± 0.0002

0.013 ± 0.0003

0.015 ± 0.0003

42

4

0.275 ± 0.028

7.285 ± 0.151

-

0.534 ± 0.006

43

5

0.407 ± 0.012

4.389 ± 0.070

-

1.333 ± 0.030

44

6

0.079 ± 0.007 0.510 ± 0.001e

8.217 ± 0.180

-

1.562 ± 0.039

45

7

0.165 ± 0.005

3.107 ± 0.075

-

1.115 ± 0.013

N N H



31

2

0.916 ± 0.071

n.a

-

1.027 ± 0.018

49

3

0.240 ± 0.013

n.a

-

0.196 ± 0.002 0.620 ± 0.012f

0.014 ± 0.003

n.a.

-

n.a.

Cmp. II a

IC50 values are expressed as the mean ± standard error of the mean (SEM) of at least three

experiments; bAChE from the electric eel; crecombinant human AChE; dBuChE from equine serum; e

Kb value; fhuman recombinant BuChE.

AChE/BuChE inhibition kinetic studies The mechanism of cholinesterase inhibition was determined for two potent inhibitors of both AChE and BuChE, compounds 12 and 18, the representatives of series I and II, respectively. As illustrated in Figure 6 by Lineweaver-Burk reciprocal plots, the compounds display non-competitive mode of action. This is indicated by increasing slopes (decreased Vmax) and preserved intercepts (unchanged Km) at increasing concentrations of the enzyme inhibitor. The nest of line converges at the x-axis that is characteristic for inhibitors displaying equal affinity for the free enzyme and for the enzymesubstrate complex. A

Compound 12

B

Compound 12 20

40

-20

-10

10

20

1/V

1/V

0 nM 7.5 nM 15 nM 30 nM

20

-20

0 nM 15 nM 25 nM 40 nM 75 nM

10

-10

10

1/ATC [mM]-1

20

30

1/BTC [mM]-1

-20 -10

Compound 18

C

D

Compound 18

20

-20

0 nM 7.5 nM 15 nM 30 nM 50 nM

10

-10

10

20

10

1/V

1/V

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 58

-20

0 nM 7.5 nM 15 nM 30 nM

5

-10

10

1/ATC [mM]-1

1/BTC [mM]-1

-10

-5


20

30

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Figure 6. Lineweaver – Burk plots illustrating hAChE and eqBuChE hydrolytic activity over a range of substrate concentrations and in the presence of increasing concentration of inhibitor 12 (A, B) and 18 (C, D). Inhibition of amyloid-β aggregation The inhibitory effect of the novel compounds presented herein (9–18, 25–31, 34, 36, 42–45, 49, 52, 53) on Aβ1-42 aggregation was determined in thioflavine T (ThT) fluorescence assay.47 ThT is a thioflavine dye that displays enhanced fluorescence emission at 482 nm when bound to amyloid fibrils. Compounds with anti-aggregation properties cause a reduction of the fluorescence emission in the assay. The results of the screening performed at a concentration of 10 µM of each compound and 1.5 µM of Aβ1–42 are presented in Figure 7 as a percentage of inhibition. Inhibitory activity of the compounds ranged from 48% to 95%. Ten compounds (9, 10, 12, 13, 15–18, 34, 45) exhibited equal or better inhibitory potency than resveratrol used as a reference (79%–95% vs. 79%). The most active compounds were tacrine derivatives 12, 13, 15–18 with inhibitory potency over 92%. Within tacrine derivatives from series I, we observed similar SAR when it comes to a length of a linker as for 5-HT6 receptor affinity. The activity increases with the linker elongation to reach maximum of 94.4% for compound 12 with 6 carbon atom linker and then decreases to 64.3% for 14 with 8 carbon atom linker. For compounds 12 and 15 we tested Aβ1–42 aggregation kinetics at the decreasing concentrations of each compound and determined IC50 values of 1.27 µM and 1.29 µM respectively.



Figure 7. The effect of the synthesized compounds on Aβ aggregation. The anti-aggregation activity was determined as reported in the experimental section in the presence of 10 µM of the synthesized compounds and 1.5 µM Aβ1–42. The activity of the new compounds was compared to that displayed by resveratrol used as the reference. The data is expressed as a mean percentage of inhibition ± standard deviation of at least three independent experiments; p < 0.05, statistically different compared to control experiments (Aβ1–42 + DMSO); one-way analysis of variance (ANOVA), followed by post hoc Bonferroni t-test (SigmaPlot v 12.0, GraphPad Prism 5).

Blood-brain barrier penetration In order to penetrate into the CNS and to reach their intended biological targets, the novel MTDLs must pass the blood-brain barrier (BBB). To estimate their ability to cross BBB by passive diffusion we have used parallel artificial membrane permeability assay for the BBB (PAMPA-BBB). The method was described by Di in 200348, and today it is commonly used as reliable, easy and fast in vitro permeability prediction method. By using PAMPA-BBB, we have determined effective permeability (Pe) for the selected compounds and for eight commercial drugs with known CNS penetration as references (Table 3). According to Di,48 the permeability ranges were set at Pe > 4.0 for compounds with high permeability, Pe < 2.0 for compounds with low permeability, and 4.0 > Pe > 2.0 for compounds with uncertain permeability. As the tested compounds 15, 42, 49 and 12 display Pe values higher than 4.0, we predict that they should cross the BBB by passive diffusion. Table 3. Permeability (Pe) of the selected compounds and commercial drugs in the PAMPA-BBB assay with prediction of their penetration in the CNS.

BBB penetration estimation Compound

Pe a

CNS (+/-)b

12

5.7 ± 1.4

CNS (+)

15

7.6 ± 1.1

CNS (+)

42

4.8 ± 0.3

CNS (+)

49

5.0 ± 0.5

CNS (+)

Donepezil

7.3 ± 0.9

CNS (+)


Page 20 of 58

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a

b

Rivastigmine

6.6 ± 0.5

CNS (+)

Tacrine

5.3 ± 0.2

CNS (+)

Testosterone

11.3 ± 1.6

CNS (+)

Chlorpromazine

5.6 ± 0.6

CNS (+)

Hydrocortisone

2.8 ± 0.1

CNS (+/-)

Theophylline

1.1 ± 0.2

CNS (-)

Atenolol

1.0 ± 0.4

CNS (-)

Data is expressed as a mean of four replicates (n = 4) ± SEM (10–6 cm s–1). CNS+, Pe > 4.0, high permeability (high BBB permeation predicted); CNS-, Pe < 2.0, low

permeability (low BBB permeation predicted); CNS±, 4.0 > Pe > 2.0, uncertain permeability. In silico and in vitro metabolic stability Biotransformation plays essential role in bioavailability and toxicity of drugs. The major site of biotransformation is liver and liver microsomal enzymes. Therefore, preliminary assessment of compounds’ metabolic stability in vitro is evaluated on liver microsomes. To predict metabolic stability of the most promising compound 12, we have used in silico MetaSite 5.1.1 tool, which shows metabolic sites and structures of the possible metabolites.49 As depicted in Figure 8, position 4 of tacrine moiety (marked in blue) was selected as the most sensitive site for metabolic biotransformations. The program predicted three possible reactions at this site: dehydrogenation, hydroxylation or oxidation. Other possible sites of metabolism were marked in red (for details see figure S1 in supplementary information).

Figure 8. In silico prediction of the most probable metabolic sites.



In vitro metabolic stability of compound 12 was evaluated using human liver microsomes (HLMs). After 120 minutes of incubation with HLMs compound 12 was metabolized in 50%. UPLC-MS analysis led to the identification of the main metabolite – M1, and small amounts of additional metabolites M2–M7 (Figure 9).

Figure 9. The UPLC spectrum after 120 minutes of incubation of 12 with HLMs. The MS analysis (see figure S2 in supporting information) allowed the determination of the molecular masses of metabolites’ quasimolecular ions [M+H]+: m/z = 473.46 (M1, main metabolite), m/z = 313.28 (M2), m/z = 579.47 (M3), m/z = 597.55 (M4), m/z = 199.18 (M5), m/z = 283.31 (M6) and m/z = 397.35 (M7). By using MetaSite, we have identified M1 as a product of ether bond hydrolysis (Figure 10).

Figure 10. The MS fragmentation analysis and the structure of the main metabolite M1. Other minor metabolites were analysed and identified as products of compound 12 degradation and hydroxylation (for further details see figures S3–S8 in supplementary information). We did not


Page 22 of 58

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identify any of known metabolites responsible for hepatotoxic effect of tacrine.50 Although, we have found traces of tacrine (M5) and metabolites M3 and M4 that contain hydroxylated tacrine fragments (figures S4–S6 in supporting information). Influence on CYP3A4 activity Alzheimer's disease affects mainly elderly patients who usually receive more than one drug at the time, and as a consequence they are exposed to drug-drug interactions (DDI). A major mechanism leading to DDI is an inhibition of cytochrome P450 (CYP). Among various CYP isoenzymes, CYP3A4 is responsible for metabolism of 40–50% of all marketed drugs, and therefore preliminary data is usually assessed by using this enzyme.51 We have tested the influence of compound 12 on cytochrome P450 CYP3A4 isoform activity in a luminescence method provided by Promega®.The method is based on the CYP3A4-catalysed conversion of the beetle D-luciferin derivative into Dluciferin.52 As shown in Figure 11, the inhibitory effect of compound 12 was 170-fold weaker than for

% of CYP3A4 activity

ketoconazole used as a reference (IC50 = 17.35 µM vs IC50 = 0.1 µM).

KE IC50 = 0.1 µM

100

12 IC50 = 17.35 µ

M

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


50

0 -9

-8

-7 -6 [log M]

-5

-4

Figure 11. Effect of ketoconazole (KE) and compound 12 on CYP3A4 activity. 3. CONCLUSIONS Last decade has shown how difficult it is to develop a new, safe and efficient drug to treat AD. One of the major obstacles is still incomplete understanding of pathomechanisms leading to the disease, and its complexity. Due to the latter, it is anticipated that we should be looking for a complex therapy consisting of drugs with different mechanisms of action. As an alternative to the combination therapy,



multi-target-directed ligands approach is considered to make a significant contribution to the development of new anti-AD treatment. In the pursuit of an effective strategy of AD treatment we have designed new multi-target-directed ligands aiming at both symptoms and causes of the disease. Pharmacophore fragments that were combined in these compounds provided them with significant in vitro cholinesterase inhibitory activity, 5-HT6 receptor antagonistic potential and amyloid β anti-aggregation activity. As one of the most potent compounds with well-balanced activity, we selected compound 12 with Ki = 18 nM and Kb= 132 nM against 5-HT6, and IC50 values of 14 nM and 22 nM against AChE and BuChE, respectively. The combination of pharmacophore fragments aiming at AChE and 5-HT6 receptor in this compound improved the hAChE inhibitory potency when compared to tacrine (IC50hAChE = 14 nM, vs. 131 nM for tacrine). Based on molecular modelling studies, we assume this might be a result of the additional interactions provided by 5-HT6-aiming fragment within AChE. We have also observed that AChE-aiming fragment decreases the interactions of 5-HT6-aiming fragment with 5-HT6 receptor resulting in decrease of affinity for the receptor. Therefore, it is important for further studies to choose carefully the 5-HT6 receptor pharmacophore fragment with the highest possible affinity for the receptor, most preferably of Ki value below 1 nM. Although compound 12 is tacrine derivative, in in vitro metabolic stability study on human liver microsomes, we did not detect any metabolites that are responsible for hepatotoxicity of tacrine. Another compound that attracted our attention is compound 49. This compound displays well-balanced activity against 5-HT6 receptor (Ki = 240 nM) and butyrylcholinesterase (IC50 = 196 nM), and, what is particularly interesting, it is benzylamine, not a tacrine derivative. This provides favourable physicochemical properties (lower molecular weight and aromatic ring count) and therefore better potential for further development of this compound as a pharmacological tool or anti-AD agent. A unique feature of the described compounds is that two synergistic mechanisms - cholinesterase inhibition and 5-HT6 receptor antagonism - were complemented with a significant inhibitory activity against self-induced amyloid β aggregation displayed by both compound 12 (94% at 10 µM, IC50 = 1.27 µM) and 49 (65% at 10 µM). Neuritic plaques formed from the aggregated amyloid β are considered to be a primary lesion in AD, and hence inhibition of aggregation is of a special interest in the search for new anti-AD agents.


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To sum up, the study has revealed new multi-target-directed ligands that interfere with the most crucial mechanisms underlying AD, that may serve as an excellent starting point for further development of a new anti-AD therapy.

4. 4.1.

METHODS Chemistry

4.1.1. General procedures. Column chromatography was performed on silica gel 60 (63–200 µm; Merck). Flash chromatography was performed on IsoleraTM Spectra (Biotage). Analytical thin layer chromatography was carried out using aluminium sheets precoated with silica gel 60 F254 (Merck). Compounds were visualized with UV light and by solution of ninhydrin. Analytical RPLC-MS was performed by tandem quadrupole mass spectrometry (Waters Acquity TQD), with detection by UV using a diode array detector with a UPLC BEH C18 column (1.7 µm, 2.1 × 100 mm, Aquity). An acetonitrile/H2O gradient with 0.1% HCOOH was used as the mobile phase, at a flow rate of 0.3 mL/min. 1H NMR and 13C NMR spectra were recorded on a Mercury 300 instrument (Varian), with 1H at 300 MHz, and 13C at 75 MHz. The chemical shifts are reported in ppm and were referenced to the residual solvent signals (CHLOROFORM-d 1H: 7.26 ppm, 1

13

C: 77.16 ppm; DEUTERIUM OXIDE

H: 4.79 ppm, DMSO-d6 1H: 2.50 ppm), coupling constants are reported in hertz (Hz). The purities of

the final compounds were determined using analytical RPLC-MS (Waters Acquity TQD) with a UPLC BEH C18 column (1.7 µm, 2.1 × 100 mm, Aquity), with detection at 214 nm and 254 nm. A acetonitrile/H2O gradient with 0.1% HCOOH was used as the mobile phase, at a flow rate of 0.3 mL/min. All of the compounds showed purities of >95%. All of the reagents were purchased from commercial suppliers and were used without further purification. Tetrahydrofuran (THF) and dichloromethane (DCM) were distilled under nitrogen immediately before use. The drying agent used for THF was sodium/ benzophenone ketyl, and for DCM, calcium hydride.

The following compounds: N-benzyl-2-bromoacetamide (19),31 N-benzyl-2-bromo-N-methylacetamide (20),31 N-benzyl-4-chloro-N-methylbutanamide (22),31 N-benzyl-5-bromopentanamide (23),31 N-



Page 26 of 58

benzyl-5-bromo-N-methylpentanamide (24),31 N-(1-benzylpyrrolidin-3-yl)-2-bromoacetamide (32),31 N-(1-benzylpiperidin-4-yl)-2-bromoacetamide (33),31 2-(1,2,3,4-tetrahydroacridin-9-ylamino)ethanol (1),31

3-(1,2,3,4-tetrahydroacridin-9-ylamino)propan-1-ol

(2),31

N-(5-bromopentyl)-1,2,3,4-

tetrahydroacridin-9-amine (5),31 N-(6-bromohexyl)-1,2,3,4-tetrahydroacridin-9-amine (6),31 N-(7bromoheptyl)-1,2,3,4-tetrahydroacridin-9-amine (7),31 N-(8-bromooctyl)-1,2,3,4-tetrahydroacridin-9amine (8),31 4-((1-benzylpiperidin-4-yl)(tert-butoxycarbonyl)amino)butyl methanesulfonate (51),31 1(3-(benzyloxy)-2-methylphenyl)piperazine,53 1-benzyl-4-(piperazin-1-yl)-1H-indole34,35 have been previously reported. 4.1.2. Synthesis of N-benzyl-3-bromopropanamide (21). To a solution of phenylmethanamine (535.8 mg, 5.0 mmol) in 40 mL of DCM cooled at 0–5 °C a solution of 3-bromopropanoyl chloride (985.7 mg, 5.75 mmol) in 20 mL of DCM was added dropwise. The reaction mixture was stirred at room temperature for 24 h, then cooled to 0–5 °C and 24 mL of saturated solution of NaHCO3 was added. The organic layer was washed with saturated solution of NaHCO3 (2 × 24 mL NaHCO3), dried over Na2SO4 and concentrated under reduced pressure to give a crude product that was purified using flash chromatography in 50% ethyl acetate in petroleum ether. Yield: 1,1181 g (97,6%). 1H NMR (300 MHz, CHLOROFORM-d) δ 7.28 - 7.39 (m, 5H), 5.81 (br. s, 1H), 4.50 (d, J=5.64 Hz, 2H), 3.69 (t, J=6.60 Hz, 2H), 2.80 (t, J=6.60 Hz, 2H). Formula: C10H12BrNO; MS: m/z 243 (M+H+). 4.1.3. General procedure for the synthesis of compounds 25–31, 34 and 35. A mixture of 1-(3-(benzyloxy)-2-methylphenyl)piperazine or 1-benzyl-4-(piperazin-1-yl)-1H-indole (in form of a hydrochloride salt or as a free base) (1 equiv), corresponding bromide/chloride (1 equiv) and K2CO3 (1–2 equiv) in acetonitrile was stirred at 50 °C for 24 h. After the mixture was cooled to room temperature, the solvent was removed, and the product was partitioned between saturated solution of NaHCO3 and DCM. The combined organic fractions were dried over Na2SO4 and concentrated under reduced pressure to give a crude product which was purified according to the methods described below. To an ice-bath cooled solution of the amide in anhydrous THF, 1M solution of lithium aluminium hydride in THF was added (2.5 equiv) dropwise. The mixture was stirred at room temperature for 12 h. When the reaction was completed, the mixture was ice-cooled, and diethyl


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ether (1 mL), water (10 mL) and 1 M solution of NaOH (0.1 mL) were added. The mixture then was extracted with ethyl acetate (3 × 10 mL). The combined organic fractions were dried over Na2SO4 and evaporated under reduced pressure to give a crude product that was purified according to the methods described below. 4.1.3.1. N-Benzyl-2-(4-(3-(benzyloxy)-2-methylphenyl)piperazin-1-yl)ethan-1-amine (25) Compound 25 was prepared using 1-(3-(benzyloxy)-2-methylphenyl)piperazine hydrochloride (159.4 mg, 0.50 mmol), N-benzyl-2-bromoacetamide (19) (114.0 mg, 0.50 mmol), K2CO3 (138.2 mg, 1.00 mmol) in 5 mL acetonitrile. Extraction: 10 mL NaHCO3 (sat.), 3 × 10 mL DCM. Purification: flash chromatography in 40–70% ethyl acetate in petroleum ether. Yield: 140 mg (65%). 1H NMR (300 MHz, CHLOROFORM-d) δ 7.56 (br. s., 1H), 7.25 - 7.49 (m, 10H), 7.11 (t, J = 8.21 Hz, 1H), 6.68 (dd, J = 3.98, 8.08 Hz, 2H), 5.07 (s, 2H), 4.52 (d, J = 5.90 Hz, 2H), 3.16 (s, 2H), 2.87 - 2.95 (m, 4H), 2.68 2.76 (m, 4H), 2.23 (s, 3H). Formula: C27H31N3O2; MS: m/z 430 (M+H+). The N-benzyl-2-(4-(3(benzyloxy)-2-methylphenyl)piperazin-1-yl)acetamide obtained (100.0 mg, 0.225 mmol) was dissolved in 1 mL THF and reduced using 1 M LiAlH4 in THF (0.56 mL, 0.56 mmol). Purification: column chromatography in DCM/methanol/NH3(aq) (9.5/0.5/0.05, v/v/v). Yield: 34.6 mg (37%). 1H NMR (300 MHz, CHLOROFORM-d) δ 7.23 - 7.48 (m, 10H), 7.08 - 7.15 (m, 1H), 6.70 (t, J = 8.46 Hz, 2H), 5.07 (s, 2H), 3.83 (s, 2H), 2.83 - 2.99 (m, 5H), 2.65 - 2.81 (m, 3H), 2.58 (t, J = 6.03 Hz, 5H), 2.23 (s, 3H). Formula: C27H33N3O; MS: m/z 416 (M+H+). 4.1.3.2. N-Benzyl-2-(4-(3-(benzyloxy)-2-methylphenyl)piperazin-1-yl)-N-methylethan-1-amine (26) Compound 26 was prepared using 1-(3-(benzyloxy)-2-methylphenyl)piperazine hydrochloride (159.4 mg, 0.50 mmol), N-benzyl-2-bromo-N-methylacetamide (20) (121.1 mg, 0.50 mmol), K2CO3 (276.4 mg, 1.00 mmol) in 5 mL acetonitrile. Extraction: 10 mL NaHCO3 (sat.), 3 × 10 mL DCM. Purification: flash chromatography in 40–70% ethyl acetate in petroleum ether. Yield: 146 mg (66%). Rotamers in ratio 1:0.9, rotamer 1: 1H NMR (300 MHz, CHLOROFORM-d) δ 7.19 - 7.49 (m, 10H), 7.11 (dt, J = 2.82, 8.08 Hz, 1H), 6.65 - 6.75 (m, 2H), 5.08 (d, J = 1.28 Hz, 2H), 4.62 (s, 2H), 3.32 (d, J = 1.28 Hz, 2H), 3.02 (s, 3H), 2.99 (t, J = 4.74 Hz, 4H), 2.73 (d, J = 15.64 Hz, 4H), 2.25 (s, 3H), rotamer 2: 1H NMR (300 MHz, CHLOROFORM-d) δ 7.19 - 7.49 (m, 10H), 7.11 (dt, J = 2.82, 8.08 Hz, 1H), 6.65 - 6.75 (m, 2H), 5.08 (d, J = 1.28 Hz, 2H), 4.73 (s, 2H), 3.32 (d, J = 1.28 Hz, 2H), 2.89 -



Page 28 of 58

2.96 (m, 7H), 2.73 (d, J = 15.64 Hz, 4H), 2.23 (s, 3H). Formula: C28H33N3O2; MS: m/z 444 (M+H+). The

N-benzyl-2-(4-(3-(benzyloxy)-2-methylphenyl)piperazin-1-yl)-N-methylacetamide

obtained

(105.0 mg, 0.24 mmol) was dissolved in 1 mL THF and reduced using 1 M LiAlH4 in THF (0.6 mL, 0.60 mmol). Purification: column chromatography in DCM/methanol (9.5/0.5, v/v). Yield: 32.0 mg (31%). 1H NMR (300 MHz, CHLOROFORM-d) δ 7.22 - 7.48 (m, 10H), 7.11 (t, J = 8.21 Hz, 1H), 6.70 (dd, J = 8.21, 10.52 Hz, 2H), 5.08 (s, 2H), 3.57 (s, 2H), 2.95 (t, J = 4.74 Hz, 4H), 2.53 - 2.76 (m, 8H), 2.28 (s, 3H), 2.23 (s, 3H), 13C NMR (75 MHz, CHLOROFORM-d) δ 157.7, 152.6, 138.7, 137.6, 129.1, 128.5, 128.2, 127.7, 127.1, 127.1, 126.3, 121.2, 111.9, 107.0, 70.1, 62.8, 56.5, 54.5, 54.1, 51.8, 42.7, 11.0. Formula: C28H35N3O; MS: m/z 430 (M+H+). 4.1.3.3. N-Benzyl-3-(4-(3-(benzyloxy)-2-methylphenyl)piperazin-1-yl)propan-1-amine (27) Compound 27 was prepared using 1-(3-(benzyloxy)-2-methylphenyl)piperazine (141.2 mg, 0.50 mmol), N-benzyl-3-bromopropanamide (21) (121.1 mg, 0.50 mmol), K2CO3 (69.1 mg, 0.50 mmol) in 5 mL acetonitrile. Extraction: 10 mL NaHCO3 (sat.), 3 × 10 mL DCM. Purification: column chromatography in 1–5% methanol in DCM with 0.25% NH3(aq). Yield: 220 mg (50%). 1H NMR (300 MHz, CHLOROFORM-d) δ 8.82 (br. s., 1H), 7.24 - 7.48 (m, 10H), 7.07 - 7.15 (m, 1H), 6.68 - 6.71 (m, 1H), 6.52 - 6.59 (m, 1H), 5.07 (s, 2H), 4.45 (d, J = 5.39 Hz, 2H), 2.76 (br. s., 4H), 2.70 (t, J = 6.03 Hz, 2H), 2.62 (br. s, 4H), 2.47 (t, J = 6.03 Hz, 2H), 2.20 (s, 3H). Formula: C28H33N3O2; MS: m/z 444 (M+H+). The N-benzyl-3-(4-(3-(benzyloxy)-2-methylphenyl)piperazin-1-yl)propanamide obtained (180.0 mg, 0.40 mmol) was dissolved in 1 mL THF and reduced using 1 M LiAlH4 in THF (1.0 mL, 1.0 mmol). Purification: column chromatography in DCM/methanol/NH3(aq) (9.5/0.5/0.05, v/v/v). Yield: 78.0 mg (45%). 1H NMR (300 MHz, CHLOROFORM-d) δ 7.23 - 7.48 (m, 10H) 7.12 (t, J = 8.08 Hz, 1H), 6.69 (dd, J = 7.82, 5.77 Hz, 2H), 5.08 (s, 2H), 3.82 (s, 2H), 2.93 (t, J = 4.74 Hz, 4H), 2.74 (t, J = 6.80 Hz, 2H), 2.62 (br. s., 4H), 2.49 (t, J = 7.44 Hz, 2H), 2.24 (s, 3H), 2.06 (br. s., 1H), 1.77 (quin, J = 6.99 Hz, 2H), 13C NMR (75 MHz, CHLOROFORM-d) δ 157.6, 152.6, 140.4, 137.6, 128.4, 128.3, 128.0, 127.6, 127.0, 126.8, 126.2, 121.1, 111.7, 106.9, 70.0, 57.1, 53.9, 53.7, 51.9, 48.0, 24.1, 11.0. Formula: C28H35N3O; MS: m/z 430 (M+H+). 4.1.3.4. N-Benzyl-4-(4-(3-(benzyloxy)-2-methylphenyl)piperazin-1-yl)-N-methylbutan-1-amine (28)


Page 29 of 58 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Compound 28 was prepared using 1-(3-(benzyloxy)-2-methylphenyl)piperazine (141.2 mg, 0.50 mmol), N-benzyl-4-chloro-N-methylbutanamide (22) (112.9 mg, 0.50 mmol), K2CO3 (69.1 mg, 0.50 mmol) in 5 mL acetonitrile. Extraction: 10 mL NaHCO3 (sat.), 3 × 10 mL DCM. Purification: column chromatography in DCM/methanol/NH3(aq) (9.5/0.5/0.025, v/v/v). Yield: 118 mg (50%). Rotamers in ratio 1:0.8, rotamer 1: 1H NMR (300 MHz, CHLOROFORM-d) δ 7.15 - 7.48 (m, 10H), 7.07 - 7.14 (m, 1H), 6.65 - 6.74 (m, 2H), 5.07 (s, 2H), 4.61 (s, 2H), 2.87 - 2.99 (m, 7H), 2.55 - 2.74 (m, 4H), 2.40 2.53 (m, 4H), 2.24 (s, 3H),1.86 - 2.00 (m, 2H), rotamer 2: 1H NMR (300 MHz, CHLOROFORM-d) δ 7.15 - 7.48 (m, 10H), 7.07 - 7.14 (m, 1H), 6.65 - 6.74 (m, 2H), 5.07 (s, 2H), 4.58 (s, 2H), 2.87 - 2.99 (m, 7H), 2.55 - 2.74 (m, 4H), 2.40 - 2.53 (m, 4H), 2.23 (s, 3H), 1.86 - 2.00 (m, 2H). Formula: C30H37N3O2; MS: m/z 472 (M+H+). The N-benzyl-4-(4-(3-(benzyloxy)-2-methylphenyl)piperazin-1yl)-N-methylbutanamide obtained (118.0 mg, 0.25 mmol) was dissolved in 1 mL THF and reduced using 1 M LiAlH in THF (0.625 mL, 0.625 mmol). Purification: column chromatography in 10% methanol in chloroform with 0.25% NH3(aq). Yield: 66.0 mg (58%).

1

H NMR (300 MHz,

CHLOROFORM-d) δ 7.22 - 7.48 (m, 10H), 7.12 (t, J = 8.08 Hz, 1H), 6.72 (d, J = 12.82 Hz, 1H), 6.70 (d, J = 13.08 Hz, 1H), 5.08 (s, 2H), 3.51 (s, 2H), 2.96 (t, J = 4.74 Hz, 4H), 2.63 (br. s., 4H), 2.37 2.48 (m, 4H), 2.25 (s, 3H), 2.21 (s, 3H), 1.53 - 1.63 (m, 4H), 13C NMR (75 MHz, CHLOROFORM-d) δ 157.7, 152.7, 139.0, 137.6, 129.1, 128.5, 128.2, 127.7, 127.1, 126.9, 126.3, 121.2, 111.8, 107.0, 70.1, 62.3, 58.6, 57.2, 53.7, 51.9, 31.5, 25.4, 24.7, 11.1. Formula: C30H39N3O; MS: m/z 458 (M+H+). 4.1.3.5. N-Benzyl-5-(4-(3-(benzyloxy)-2-methylphenyl)piperazin-1-yl)pentan-1-amine (29) Compound 29 was prepared using 1-(3-(benzyloxy)-2-methylphenyl)piperazine hydrochloride (159.4 mg, 0.50 mmol), N-benzyl-5-bromopentanamide (23) (135.1 mg, 0.50 mmol), K2CO3 (138.2 mg, 1.0 mmol) in 5 mL acetonitrile. Extraction: 10 mL NaHCO3 (sat.), 3 × 10 mL DCM. Purification: column chromatography in DCM/methanol/NH3(aq) (9.5/0.5/0.05, v/v/v). Yield: 112 mg (48%). 1H NMR (300 MHz, CHLOROFORM-d) δ 7.24 - 7.48 (m, 10H), 7.11 (t, J = 8.21 Hz, 1H), 6.68 (d, J = 8.21 Hz, 2H), 6.00 (br. s., 1H), 5.07 (s, 2H), 4.45 (d, J = 5.64 Hz, 2H), 2.90 - 2.93 (m, 4H), 2.59 (br. s., 4H), 2.39 2.47 (m, 2H), 2.27 (t, J = 7.18 Hz, 2H), 2.23 (s, 3H), 1.66 - 1.79 (m, 2H) ,1.52 - 1.65 (m, 2H). Formula:

C30H37N3O2;

MS:

m/z

472

(M+H+).

The

N-benzyl-5-(4-(3-(benzyloxy)-2-

methylphenyl)piperazin-1-yl)pentanamide obtained (112.0 mg, 0.24 mmol) was dissolved in 1 mL



THF and reduced

using 1 M

Page 30 of 58

LiAlH4 in THF (0.59 mL, 0.59 mmol). Purification: column

chromatography in DCM/methanol/NH3(aq) (9.5/0.5/0.05, v/v/v). Yield: 70.0 mg (64%). 1H NMR (300 MHz, CHLOROFORM-d) δ 7.21 - 7.50 (m, 10H), 7.11 (t, J = 8.21 Hz, 1H), 6.70 (dd, J = 12.18, 8.08 Hz, 2H), 5.08 (s, 2H), 3.80 (s, 2H), 2.95 (t, J=4.74 Hz, 4H), 2.54 - 2.72 (m, 6H), 2.41 (dd, J = 8.72, 6.67 Hz, 2H), 2.24 (s, 3H), 1.68 (br. s., 1H), 1.49 - 1.63 (m, 4H), 1.32 - 1.44 (m, 2H), 13C NMR (75 MHz, CHLOROFORM-d) δ 157.7, 152.7, 140.4, 137.6, 128.5, 128.4, 128.2, 127.7, 127.1, 126.9, 126.3, 121.2, 111.8, 106.9, 70.1, 58.8, 54.1, 53.8, 51.9, 49.3, 30.0, 26.8, 25.4, 11.0. Formula: C30H39N3O; MS: m/z 458 (M+H+). 4.1.3.6. N-Benzyl-5-(4-(3-(benzyloxy)-2-methylphenyl)piperazin-1-yl)-N-methylpentan-1-amine (30) Compound 30 was prepared using 1-(3-(benzyloxy)-2-methylphenyl)piperazine hydrochloride (159.4 mg, 0.50 mmol), N-benzyl-5-bromopentanamide (23) (142.1 mg, 0.50 mmol), K2CO3 (138.2 mg, 1.0 mmol) in 5 mL acetonitrile. Extraction: 10 mL NaHCO3 (sat.), 3 × 10 mL DCM. Purification: column chromatography in DCM/methanol/NH3(aq) (9.5/0.5/0.05, v/v/v). Yield: 74 mg (30%). Rotamers in ratio 1:0.6, rotamer 1: 1H NMR (300 MHz, CHLOROFORM-d) δ 7.22 - 7.49 (m, 9H), 7.07 - 7.21 (m, 2H), 6.65 - 6.75 (m, 2H), 5.08 (s, 2H), 4.61 (s, 2H), 2.91 - 3.00 (m, 7H), 2.53 - 2.73 (m, 4H), 2.34 2.51 (m, 4H), 2.25 (m, 3H), 1.53 - 1.81 (m, 4H), rotamer 2: 1H NMR (300 MHz, CHLOROFORM-d) δ 7.22 - 7.49 (m, 9H), 7.07 - 7.21 (m, 2H), 6.65 - 6.75 (m, 2H), 5.08 (s, 2H), 4.56 (s, 2H), 2.91 - 3.00 (m, 7H), 2.53 - 2.73 (m, 4H), 2.34 - 2.51 (m, 4H), 2.24 (s, 3H), 1.53 - 1.81 (m, 4H). Formula: C31H39N3O2; MS: m/z 486 (M+H+). The N-benzyl-5-(4-(3-(benzyloxy)-2-methylphenyl)piperazin-1yl)-N-methylpentanamide obtained (74.0 mg, 0.15 mmol) was dissolved in 1 mL THF and reduced using 1 M LiAlH4 in THF (0.38 mL, 0.38 mmol). Purification: column chromatography in DCM/methanol/NH3(aq) (9.0/1.0/0.05, v/v/v). Yield: 37.0 mg (52%).

1

H NMR (300 MHz,

CHLOROFORM-d) δ 7.21 - 7.49 (m, 10H), 7.12 (t, J = 8.08 Hz, 1H), 6.71 (dd, J = 12.95, 8.08 Hz, 2H), 5.08 (s, 2H), 3.49 (s, 2H), 2.96 (t, J = 4.74 Hz, 4H), 2.61 (br. s., 4H), 2.35 - 2.44 (m, 4H), 2.25 (s, 3H), 2.20 (s, 3H), 1.49 - 1.64 (m, 4H), 1.32 - 1.43 (m, 2H), 13C NMR (75 MHz, CHLOROFORM-d) δ 157.7, 152.7, 139.2, 137.6, 129.0, 128.5, 128.2, 127.7, 127.1, 126.9, 126.3, 121.2, 111.8, 106.9, 70.1, 62.4, 58.9, 57.4, 53.8, 51.9, 42.3, 27.4, 26.9, 25.5, 11.1. Formula: C31H41N3O; MS: m/z 472 (M+H+). 4.1.3.7. N-Benzyl-2-(4-(1-benzyl-1H-indol-4-yl)piperazin-1-yl)-N-methylethan-1-amine (31)


Page 31 of 58 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Compound 31 was prepared using 1-benzyl-4-(piperazin-1-yl)-1H-indole hydrochloride (229.5 mg, 0.70 mmol), N-benzyl-2-bromo-N-methylacetamide (20) (169.5 mg, 0.70 mmol), K2CO3 (193.5 mg, 1.40 mmol) in 7 mL acetonitrile. Extraction: 10 mL NaHCO3 (sat.), 3 × 10 mL DCM. Purification: column chromatography in 30–90% ethyl acetate in petroleum ether. Yield: 160 mg (50%). Rotamers in ratio 1:0.9, rotamer 1: 1H NMR (300 MHz, CHLOROFORM-d) δ 7.19 - 7.42 (m, 8H), 7.04 - 7.14 (m, 4H), 6.93 - 6.99 (m, 1H), 6.52 - 6.63 (m, 2H), 5.30 (s, 2H), 4.63 (s, 2H), 3.22 - 3.39 (m, 6H), 3.04 (s, 3H), 2.82 (td, J = 4.71, 15.20 Hz, 4H), rotamer 2: 1H NMR (300 MHz, CHLOROFORM-d) δ 7.19 7.42 (m, 8H), 7.04 - 7.14 (m, 4H), 6.93 - 6.99 (m, 1H), 6.52 - 6.63 (m, 2H), 5.30 (s, 2H), 4.75 (s, 2H), 3.22 - 3.39 (m, 6H), 2.95 (s, 3H), 2.82 (td, J = 4.71, 15.20 Hz, 4H). Formula: C29H32N4O; MS: m/z 453 (M+H+). The N-benzyl-2-(4-(1-benzyl-1H-indol-4-yl)piperazin-1-yl)-N-methylacetamide obtained (140.0 mg, 0.31 mmol) dissolved in 1 mL THF and reduced using 1 M LiAlH4 in THF (0.77 mL, 0.77 mmol). Purification: flash chromatography in DCM/methanol (9.5/0.5, v/v). Yield: 92.0 mg (68%). 1H NMR (300 MHz, CHLOROFORM-d) δ 7.21 - 7.39 (m, 8H), 7.05 - 7.13 (m, 4H), 6.96 (d, J = 8.21 Hz, 1H), 6.58 - 6.62 (m, 1H), 6.55 (dd, J = 0.77, 3.08 Hz, 1H), 5.30 (s, 2H), 3.58 (s, 2H), 3.26 - 3.34 (m, 4H), 2.70 - 2.78 (m, 4H), 2.61 - 2.69 (m, 4H), 2.30 (s, 3H), 13C NMR (75 MHz, CHLOROFORM-d) δ 145.8, 138.8, 137.6, 137.5, 129.1, 128.7, 128.3, 127.5, 127.0, 126.7, 122.4, 121.9, 106.6, 104.5, 100.3, 62.8, 56.6, 54.5, 54.0, 51.2, 50.2, 42.7. Formula: C29H34N4; MS: m/z 439 (M+H+). 4.1.3.8. 1-Benzyl-N-(2-(4-(3-(benzyloxy)-2-methylphenyl)piperazin-1-yl)ethyl)pyrrolidin-3-amine (34) Compound 34 was prepared using 1-(3-(benzyloxy)-2-methylphenyl)piperazine hydrochloride (159.4 mg, 0.50 mmol), N-(1-benzylpyrrolidin-3-yl)-2-bromoacetamide hydrobromide (32) (189.1 mg, 0.50 mmol), K2CO3 (207.3 mg, 1.5 mmol) in 5 mL acetonitrile. Extraction: 10 mL NaHCO3 (sat.), 3 × 10 mL DCM. Purification: flash chromatography in DCM/methanol (9.5/0.5, v/v). Yield: 123 mg (49%). 1

H NMR (300 MHz, CHLOROFORM-d) δ 7.20 - 7.55 (m, 11H), 7.15 (t, J = 8.08 Hz, 1H), 6.73 (t, J =

8.46 Hz, 2H), 5.09 (s, 2H), 4.41 - 4.54 (m, 1H), 3.54 - 3.72 (m, 2H), 3.04 (d, J = 1.80 Hz, 2H), 2.84 3.01 (m, 5H), 2.63 - 2.75 (m, 4H), 2.53 - 2.61 (m, 2H), 2.26 - 2.39 (m, 2H), 2.24 (s, 3H), 1.58 - 1.69 (m,

1H).

Formula:

C31H38N4O2;

MS:

m/z

499

(M+H+).

The

2-(4-(3-(benzyloxy)-2-

methylphenyl)piperazin-1-yl)-N-(1-benzylpyrrolidin-3-yl)acetamide obtained (83.0 mg, 0.17 mmol) was dissolved in 1 mL THF and reduced

using 1 M LiAlH4 in THF (0.42 mL, 0.42 mmol).



Purification: column chromatography in DCM/methanol/NH3(aq) (9.5/0.5/0.05, v/v/v). Yield: 13.3 mg (16%). 1H NMR (300 MHz, CHLOROFORM-d) δ 7.21 - 7.48 (m, 10H), 7.11 (t, J = 8.08 Hz, 1H), 6.70 (t, J = 8.34 Hz, 2H), 5.06 - 5.10 (m, 2H), 3.56 - 3.68 (m, 2H), 3.28 - 3.38 (m, 1H), 2.92 (t, J = 4.62 Hz, 4H), 2.50 - 2.81 (m, 11H), 2.34 - 2.41 (m, 1H), 2.23 (s, 3H), 2.06 - 2.20 (m, 2H), 1.55 - 1.67 (m, 1H), 13C NMR (75 MHz, CHLOROFORM-d) δ. 157.7, 152.7, 138.9, 137.6, 128.9, 128.5, 128.2, 127.7, 127.1, 127.0, 126.3, 121.2, 111.8, 106.9, 70.1, 60.5, 60.5, 57.9, 57.4, 53.7, 53.0, 51.9, 44.9, 32.0, 11.1. Formula: C31H40N4O; MS: m/z 485 (M+H+). 4.1.3.9. 1-Benzyl-N-(2-(4-(3-(benzyloxy)-2-methylphenyl)piperazin-1-yl)ethyl)piperidin-4-amine (35) Compound 35 was prepared using 1-(3-(benzyloxy)-2-methylphenyl)piperazine (141.2 mg, 0.50 mmol), N-(1-benzylpiperidin-4-yl)-2-bromoacetamide (33) (155.6 mg, 0.50 mmol), K2CO3 (69.1 mg, 0.50 mmol) in 5 mL acetonitrile. Extraction: 10 mL NaHCO3 (sat.), 3 × 10 mL DCM. Purification: column chromatography in DCM/methanol/NH3(aq) (9.5/0.5/0.025, v/v/v). Yield: 116 mg (45%). 1H NMR (300 MHz, CHLOROFORM-d) δ 7.26 - 7.47 (m, 10H), 7.13 (t, J = 8.21 Hz, 1H), 6.70 (d, J = 8.21 Hz, 2H), 5.08 (s, 2H), 3.79 - 3.93 (m, 1H), 3.50 (s, 2H), 3.05 (s, 2H), 2.87 - 2.96 (m, 4H), 2.78 (d, J = 11.03 Hz, 2 H), 2.63 - 2.71 (m, 4H), 2.22 (s, 3H), 2.09 - 2.19 (m, 2H), 1.86 - 1.97 (m, 2H),1.46 1.54 (m, 2H), NH signal not detected. Formula: C32H40N4O2; MS: m/z 513 (M+H+). The 2-(4-(3(benzyloxy)-2-methylphenyl)piperazin-1-yl)-N-(1-benzylpiperidin-4-yl)acetamide obtained (77.0 mg, 0.15 mmol) was dissolved in 1 mL THF and reduced using 1 M LiAlH4 in THF (0.37 mL, 0.37 mmol). Purification: column chromatography in DCM/methanol/NH3(aq) (9.0/1.0/0.05, v/v/v). Yield: 35.0 mg (47%). 1H NMR (300 MHz, CHLOROFORM-d) δ 7.20 - 7.51 (m, 10H), 7.11 (t, J = 8.08 Hz, 1H), 6.70 (t, J = 8.46 Hz, 2H), 5.07 (s, 2H), 3.51 (s, 2H), 2.82 - 3.00 (m, 6H), 2.73 - 2.81 (m, 2H), 2.55 2.67 (m, 4H), 2.48 (ddd, J = 3.85, 10.39, 14.49 Hz, 1H), 2.35 (br. s., 2H), 2.23 (s, 3H), 1.98 - 2.09 (m, 2H), 1.88 (d, J = 12.05 Hz, 2H), 1.35 - 1.52 (m, 2H), NH signal not detected, 13C NMR (75 MHz, CHLOROFORM-d) δ 157.7, 152.7, 138.5, 137.6, 129.1, 128.4, 128.1, 127.6, 127.1, 126.9, 126.2, 121.2, 111.8, 106.9, 70.1, 63.0, 57.9, 55.1, 53.6, 52.4, 52.0, 43.2, 32.6, 11.0. Formula: C32H42N4O; MS: m/z 499 (M+H+). 4.1.4. General procedure for the synthesis of compounds 36, 38–41.


Page 32 of 58

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A mixture of 2-(4-bromobutyl)isoindoline-1,3-dione, 2-(5-bromopentyl)isoindoline-1,3-dione, 2-(6bromohexyl)isoindoline-1,3-dione, 2-(7-bromoheptyl)isoindoline-1,3-dione (1 equiv) and 1-(3(benzyloxy)-2-methylphenyl)piperazine or 1-benzyl-4-(piperazin-1-yl)-1H-indole (1 equiv) with K2CO3 (1.2–2 equiv) in acetonitrile was refluxed overnight. After the mixture was cooled to room temperature, the solvent was evaporated, and the residue was treated with saturated solution of NaHCO3, extracted with DCM, dried (Na2SO4) and concentrated under reduced pressure to give a crude product that was purified according to the methods described below. To the product obtained, methylamine (40% aq. sol.) was added (10 mL for 1 mmol), and the mixture was heated in 50 °C for 1.5 h. After cooling to ambient temperature, 1 M NaOH was added (10 mL for 1 mmol) and the mixture was stirred at room temperature for 1 h. Then the mixture was extracted with DCM and used without further purification. 4.1.4.1. 4-(4-(3-(Benzyloxy)-2-methylphenyl)piperazin-1-yl)butan-1-amine (36) Compound 36 was prepared using 2-(4-bromobutyl)isoindoline-1,3-dione (423.2 mg, 1.50 mmol), 1benzyl-4-(piperazin-1-yl)-1H-indole hydrochloride (478.3 mg, 1.50 mmol), K2CO3 (414.0 mg, 3.00 mmol) in 10 mL acetonitrile. Purification: flash chromatography in 1–10% methanol in DCM. 2-(4-(4(3-(benzyloxy)-2-methylphenyl)piperazin-1-yl)butyl)isoindoline-1,3-dione (710.0 mg, 1.47 mmol), methylamine (15 mL), 1 M NaOH (15 mL). Yield: 432 mg (overall 83%). 1H NMR (300 MHz, CHLOROFORM-d) δ 7.28 - 7.48 (m, 5H), 7.11 (t, J = 8.08 Hz, 1H), 6.71 (d, J = 12.31 Hz, 1H), 6.68 (d, J=12.31 Hz, 1H), 5.07 (s, 2H), 2.95 (t, J = 4.74 Hz, 4H), 2.73 (t, J = 6.80 Hz, 2H), 2.62 (br. s., 4H), 2.36 - 2.46 (m, 2H), 2.23 (s, 3H), 1.40 - 1.65 (m, 6H). Formula: C22H31N3O; MS: m/z 354 (M+H+). 4.1.4.2. 4-(4-(1-Benzyl-1H-indol-4-yl)piperazin-1-yl)butan-1-amine (38) Compound 38 was prepared using 2-(4-bromobutyl)isoindoline-1,3-dione (141.0 mg, 0.50 mmol), 1benzyl-4-(piperazin-1-yl)-1H-indole (146.0 mg, 0.50 mmol), K2CO3 (83.0 mg, 0.60 mmol) in 5 mL acetonitrile. Purification: flash chromatography in 40–92% ethyl acetate in petroleum ether. 2-(4-(4(1-Benzyl-1H-indol-4-yl)piperazin-1-yl)butyl)isoindoline-1,3-dione

(138.0

mg,

0.28

mmol),

methylamine (2.8 mL), 1 M NaOH (2.8 mL). Yield: 102 mg (overall 69%). 1H NMR (300 MHz, CHLOROFORM-d) δ 7.23 - 7.29 (m, 3H), 7.05 - 7.12 (m, 4H), 6.95 (d, J = 8.46 Hz, 1H), 6.60 (d, J =



Page 34 of 58

6.92 Hz, 1H), 6.55 (dd, J = 0.77, 3.08 Hz, 1H), 5.28 - 5.32 (m, 2H), 3.25 - 3.35 (m, 4H), 2.68 - 2.77 (m, 6H), 2.42 - 2.50 (m, 2H), 1.45 - 1.70 (m, 6H). Formula: C23H30N4; MS: m/z 363 (M+H+). 4.1.4.3. 5-(4-(1-Benzyl-1H-indol-4-yl)piperazin-1-yl)pentan-1-amine (39) Compound 39 was prepared using 2-(5-bromopentyl)isoindoline-1,3-dione (148.0 mg, 0.50 mmol), 1benzyl-4-(piperazin-1-yl)-1H-indole (146.0 mg, 0.50 mmol), K2CO3 (83.0 mg, 0.60 mmol) in 5 mL acetonitrile. Purification: flash chromatography in 40–92% ethyl acetate in petroleum ether. 2-(5-(4(1-benzyl-1H-indol-4-yl)piperazin-1-yl)pentyl)isoindoline-1,3-dione

(153.0

mg,

0.30

mmol),

methylamine (3.0 mL), 1 M NaOH (3.0 mL). Yield: 109 mg (overall 72%). 1H NMR (300 MHz, CHLOROFORM-d) δ 7.23 - 7.29 (m, 3H), 7.04 - 7.13 (m, 4H), 6.95 (d, J = 8.21 Hz, 1H), 6.60 (d, J = 7.18 Hz, 1H), 6.55 (d, J = 2.56 Hz, 1H), 5.30 (s, 2H), 3.26 - 3.35 (m, 4H), 2.67 - 2.76 (m, 6H), 2.40 2.49 (m, 2H), 1.44 - 1.65 (m, 6H), 1.33 - 1.42 (m, 2H). Formula: C24H32N4; MS: m/z 377 (M+H+). 4.1.4.4. 6-(4-(1-Benzyl-1H-indol-4-yl)piperazin-1-yl)hexan-1-amine (40) Compound 40 was prepared using 2-(6-bromohexyl)isoindoline-1,3-dione (155.0 mg, 0.50 mmol), 1benzyl-4-(piperazin-1-yl)-1H-indole (146.0 mg, 0.50 mmol), K2CO3 (83.0 mg, 0.60 mmol) in 5 mL acetonitrile. Purification: flash chromatography in 40–88% ethyl acetate in petroleum ether. 2-(6-(4(1-Benzyl-1H-indol-4-yl)piperazin-1-yl)hexyl)isoindoline-1,3-dione

(163.0

mg,

0.31

mmol),

methylamine (3.1 mL), 1 M NaOH (3.1 mL). Yield: 120 mg (overall 81%). 1H NMR (300 MHz, CHLOROFORM-d) δ 7.24 - 7.28 (m, 3H), 7.05 - 7.12 (m, 4H), 6.95 (d, J = 8.21 Hz, 1H), 6.60 (dd, J = 0.64, 7.57 Hz, 1H), 6.55 (dd, J = 0.77, 3.33 Hz, 1H), 5.30 (s, 2H), 3.26 - 3.35 (m, 4H), 2.66 - 2.75 (m, 6H), 2.39 - 2.49 (m, 2H), 1.79 (br. s., 2H), 1.53 - 1.63 (m, 2H), 1.44 - 1.51 (m, 2H), 1.32 - 1.41 (m, 4H). Formula: C25H34N4; MS: m/z 391 (M+H+). 4.1.4.5. 7-(4-(1-Benzyl-1H-indol-4-yl)piperazin-1-yl)heptan-1-amine (41) Compound 41 was prepared using 2-(7-bromoheptyl)isoindoline-1,3-dione (162.0 mg, 0.50 mmol), 1benzyl-4-(piperazin-1-yl)-1H-indole (146.0 mg, 0.50 mmol), K2CO3 (83.0 mg, 0.60 mmol) in 5 mL acetonitrile. Purification: flash chromatography in 40–88% ethyl acetate in petroleum ether. 2-(7-(4(1-Benzyl-1H-indol-4-yl)piperazin-1-yl)heptyl)isoindoline-1,3-dione

(169.0

mg,

0.32

mmol),

methylamine (3.2 mL), 1 M NaOH (3.2 mL). Yield: 121 mg (overall 70%). 1H NMR (300 MHz, CHLOROFORM-d) δ 7.22 - 7.28 (m, 3H), 7.04 - 7.12 (m, 4H), 6.94 (d, J = 8.21 Hz, 1H), 6.60 (d, J =


Page 35 of 58 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


7.44 Hz, 1H), 6.53 - 6.56 (m, 1H), 5.29 (s, 2H), 3.26 - 3.34 (m, 4H), 2.65 - 2.74 (m, 6H), 2.39 - 2.47 (m, 2H), 1.66 (br. s., 2H), 1.51 - 1.60 (m, 2H), 1.41 - 1.49 (m, 2H), 1.32 - 1.37 (m, 6H). Formula: C26H36N4; MS: m/z 405 (M+H+). 4.1.5. General procedure for the synthesis of compounds 37 and 42–45. To the solution of amine 36, 38, 39, 40 or 41 (1.3 equiv) in methanol, benzaldehyde (1 equiv), glacial acetic acid (10 µL for 0.1 mmol of benzaldehyde) and NaCNBH3 (3 equiv) were added at 0 °C, and stirring was continued overnight at room temperature. After that time, the mixture was quenched with water and extracted with DCM. The product obtained was purified by column chromatography in DCM/methanol/NH3 (9.5/0.5/0.05, v/v/v). 4.1.5.1. N-Benzyl-4-(4-(3-(benzyloxy)-2-methylphenyl)piperazin-1-yl)butan-1-amine (37) Compound 37 was prepared using 4-(4-(3-(benzyloxy)-2-methylphenyl)piperazin-1-yl)butan-1-amine 36 (100.0 mg, 0.28 mmol), benzaldehyde (22.3 mg, 0.21 mmol), glacial acetic acid (20 µL) and NaCNBH3 (39.6 mg, 0.63 mmol) in 3 mL MeOH. Yield: 75.0 mg (81%). 1H NMR (300 MHz, CHLOROFORM-d) δ 7.27 - 7.48 (m, 10H), 7.11 (t, J = 8.08 Hz, 1H), 6.69 (d, J = 4.10 Hz, 1H), 6.66 (d, J = 3.85 Hz, 1H), 5.07 (s, 2H), 3.85 (s, 2H), 3.18 (br. s., 1H), 2.88 (t, J = 4.49 Hz, 4H), 2.72 (t, J = 6.41 Hz, 2H), 2.64 (br. s., 4H), 2.46 (t, J = 7.05 Hz, 2H), 2.22 (s, 3H), 1.59 - 1.68 (m, 4H), 13C NMR (75 MHz, CHLOROFORM-d) δ 157.7, 152.4, 138.8, 137.6, 128.6, 128.5, 128.4, 127.7, 127.4, 127.1, 126.3, 121.2, 111.8, 107.0, 70.1, 58.5, 53.6, 53.6, 51.7, 49.0, 27.7, 24.7 11.0. Formula: C29H37N3O; MS: m/z 444 (M+H+). 4.1.5.2. N-Benzyl-4-(4-(1-benzyl-1H-indol-4-yl)piperazin-1-yl)butan-1-amine (42) Compound 42was prepared using 4-(4-(1-benzyl-1H-indol-4-yl)piperazin-1-yl)butan-1-amine (38) (102.0 mg, 0.28 mmol), benzaldehyde (23.0 mg, 0.22 mmol), glacial acetic acid (24 µL) and NaCNBH3 (41.0 mg, 0.66 mmol) in 3.5 mL MeOH. Yield: 71.0 mg (71%). 1H NMR (300 MHz, CHLOROFORM-d) δ 7.25 - 7.41 (m, 8H), 7.04 - 7.13 (m, 4H), 6.94 - 6.99 (m, 1H), 6.49 - 6.56 (m, 2H), 5.30 (s, 2H), 3.88 (s, 2H), 3.14 - 3.25 (m, 4H), 2.71 - 2.79 (m, 6H), 2.52 (t, J = 6.41 Hz, 2H), 1.64 - 1.78 (m, 4H), NH signal not detected,

13

C NMR (75 MHz, CHLOROFORM-d) δ 145.3, 137.5,

137.5, 128.8, 128.7, 128.6, 127.9, 127.5, 126.8, 126.7, 122.4, 121.9, 106.7, 104.7, 100.1, 58.3, 53.4, 53.1, 50.9, 50.2, 48.7, 27.4, 24.6. Formula: C30H36N4; MS: m/z 453 (M+H+).



4.1.5.3. N-Benzyl-5-(4-(1-benzyl-1H-indol-4-yl)piperazin-1-yl)pentan-1-amine (43) Compound 43 was prepared using 5-(4-(1-benzyl-1H-indol-4-yl)piperazin-1-yl)pentan-1-amine (39) (109.0 mg, 0.29 mmol), benzaldehyde (23.0 mg, 0.22 mmol), glacial acetic acid (24 µL) and NaCNBH3 (41.0 mg, 0.66 mmol) in 3.5 mL MeOH. Yield: 69.0 mg (67%). 1H NMR (300 MHz, CHLOROFORM-d) δ 7.32 - 7.35 (m, 4H), 7.24 - 7.29 (m, 4H), 7.06 - 7.12 (m, 4H), 6.95 (d, J = 8.46 Hz, 1H), 6.60 (d, J = 7.44 Hz, 1H), 6.55 (dd, J = 0.51, 3.08 Hz, 1H), 5.30 (s, 2H), 3.80 (s, 2H), 3.27 3.34 (m, 4H), 2.63 - 2.74 (m, 6H), 2.41 - 2.48 (m, 2H), 1.65 (br. s., 1H), 1.52 - 1.63 (m, 4H), 1.34 1.43 (m, 2H),

13

C NMR (75 MHz, CHLOROFORM-d) δ 145.9, 140.5, 137.6, 137.5, 128.7, 128.4,

128.1, 127.5, 126.9, 126.7, 126.7, 122.4, 121.9, 106.5, 104.4, 100.3, 58.8, 54.1, 53.7, 51.3, 50.2, 49.4, 30.0, 26.9, 25.4. Formula: C31H38N4; MS: m/z 467 (M+H+). 4.1.5.4. N-Benzyl-6-(4-(1-benzyl-1H-indol-4-yl)piperazin-1-yl)hexan-1-amine (44) Compound 44 was prepared using 6-(4-(1-benzyl-1H-indol-4-yl)piperazin-1-yl)hexan-1-amine (40) (100.0 mg, 0.26 mmol), benzaldehyde (21.0 mg, 0.20 mmol), glacial acetic acid (20 µL) and NaCNBH3 (38.0 mg, 0.60 mmol) in 3.5 mL MeOH. Yield: 50.0 mg (52%). 1H NMR (300 MHz, CHLOROFORM-d) δ 7.32 - 7.36 (m, 4H), 7.24 - 7.30 (m, 4H), 7.06 - 7.14 (m, 4H), 6.96 (d, J = 8.21 Hz, 1H), 6.62 (d, J = 7.18 Hz, 1H), 6.57 (dd, J = 0.64, 3.21 Hz, 1H), 5.30 (s, 2H), 3.81 (s, 2H), 3.27 3.36 (m, 4H), 2.69 - 2.75 (m, 4H), 2.66 (t, J = 7.18 Hz, 2H), 2.40 - 2.49 (m, 2H), 1.78 (br. s., 1H), 1.49 - 1.65 (m, 4H), 1.33 - 1.43 (m, 4H), 13C NMR (75 MHz, CHLOROFORM-d) δ 145.9, 140.5, 137.6, 137.5, 128.7, 128.4, 128.1, 127.5, 126.9, 126.7, 126.7, 122.5, 121.9, 106.5, 104.5, 100.4, 58.9, 54.1, 53.7, 51.4, 50.2, 49.4, 30.1, 27.6, 27.4, 26.9. Formula: C32H40N4; MS: m/z 481 (M+H+). 4.1.5.5. N-Benzyl-7-(4-(1-benzyl-1H-indol-4-yl)piperazin-1-yl)heptan-1-amine (45) Compound 45 was prepared using 7-(4-(1-benzyl-1H-indol-4-yl)piperazin-1-yl)heptan-1-amine (41) (96.0 mg, 0.24 mmol), benzaldehyde (19.0 mg, 0.18 mmol), glacial acetic acid (20 µL) and NaCNBH3 (34.0 mg, 0.54 mmol) in 3 mL MeOH. Yield: 46.0 mg (52%). 1H NMR (300 MHz, CHLOROFORMd) δ 7.31 - 7.37 (m, 4H), 7.25 - 7.30 (m, 4H), 7.05 - 7.14 (m, 4H), 6.96 (d, J = 8.46 Hz, 1H), 6.61 (d, J = 6.92 Hz, 1H), 6.56 (dd, J = 0.64, 3.21 Hz, 1H), 5.30 (s, 2H), 3.81 (s, 2H), 3.27 - 3.36 (m, 4H), 2.69 2.76 (m, 4H), 2.65 (t, J = 7.44 Hz, 2H), 2.40 - 2.49 (m, 2H), 1.78 (br. s., 1H), 1.48 - 1.61 (m, 4H), 1.32 - 1.38 (m, 6H), 13C NMR (75 MHz, CHLOROFORM-d) δ 145.9, 140.5, 137.6, 137.5, 128.7, 128.4,


Page 36 of 58

Page 37 of 58 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


128.1, 127.5, 126.9, 126.7, 126.7, 122.5, 121.9, 106.5, 104.5, 100.4, 59.0, 54.1, 53.7, 51.4, 50.2, 49.5, 30.1, 29.5, 27.6, 27.3, 26.9. Formula: C33H42N4; MS: m/z 495 (M+H+). 4.1.6. General procedure for the synthesis of compounds 9, 10, 15 and 16. To the solution of alcohol 1 or 2 (1 equiv) and anhydrous TEA (1.1 equiv) in anhydrous DCM, methanesulfonyl chloride (1.1 equiv) was added dropwise at 0 °C, and the reaction mixture was stirred at this temperature for 30 min. Then 1-(3-(benzyloxy)-2-methylphenyl)piperazine or 1-benzyl-4(piperazin-1-yl)-1H-indole (in a form of a free base or as a hydrochloride salt, 1.1 equiv) was added and the mixture was stirred at ambient temperature for 24 h. After that time, saturated solution of NaHCO3 was added and the mixture was extracted with DCM. The combined organic fractions were dried (Na2SO4) and concentrated under reduced pressure to give a crude product that was purified according to the methods described below. 4.1.6.1. N-(2-(4-(3-(Benzyloxy)-2-methylphenyl)piperazin-1-yl)ethyl)-1,2,3,4-tetrahydroacridin-9amine (9) Compound 9 was prepared using 2-(1,2,3,4-tetrahydroacridin-9-ylamino)ethanol (1) (109.0 mg, 0.45 mmol), TEA (50.6 mg, 0.50 mmol), methanesulfonyl chloride (57.2 mg, 0.50 mmol), 1-(3(benzyloxy)-2-methylphenyl)piperazine hydrochloride (159.4 mg, 0.50 mmol) in 1.5 mL DCM. Extraction: 5 mL NaHCO3 (sat.), 3 × 5 mL DCM. Purification: column chromatography in DCM/methanol/NH3(aq) (9.5/0.5/0.05, v/v/v). Yield: 27.0 mg (12%).

1

H NMR (300 MHz,

CHLOROFORM-d) δ 8.07 (dd, J = 0.77, 8.46 Hz, 1H), 7.95 (d, J = 7.69 Hz, 1H), 7.57 (ddd, J = 1.28, 6.99, 8.40 Hz, 1H), 7.28 - 7.48 (m, 6H), 7.11 - 7.19 (m, 1H), 6.73 (dd, J = 8.08, 11.93 Hz, 2H), 5.40 (br. s., 1H), 5.08 (s, 2H), 3.64 (br. s., 2H), 3.06 - 3.15 (m, 2H), 2.93 - 3.04 (m, 4H), 2.60 - 2.85 (m, 8H), 2.25 (s, 3H), 1.93 (t, J = 3.21 Hz, 4H), 13C NMR (75 MHz, CHLOROFORM-d) δ 157.9, 157.8, 152.5, 151.4, 137.6, 128.6, 128.5, 128.1, 127.7, 127.1, 126.4, 123.7, 122.9, 121.2, 120.0, 115.7, 111.7, 107.1, 70.1, 57.6, 53.1, 52.2, 45.1, 33.5, 24.9, 23.1, 22.7, 11.1. Formula: C33H38N4O; MS: m/z 507 (M+H+). 4.1.6.2. N-(3-(4-(3-(Benzyloxy)-2-methylphenyl)piperazin-1-yl)propyl)-1,2,3,4-tetrahydroacridin-9amine (10)



Page 38 of 58

Compound 10 was prepared using 3-(1,2,3,4-tetrahydroacridin-9-ylamino)propan-1-ol (2) (100.0 mg, 0.39 mmol), TEA (43.5 mg, 0.43 mmol), methanesulfonyl chloride (49.3 mg, 0.43 mmol), 1-(3(benzyloxy)-2-methylphenyl)piperazine (76.2 mg, 0.27 mmol) in 1.5 mL DCM. Extraction: 5 mL NaHCO3 (sat.), 3 × 5 mL DCM. Purification: column chromatography in DCM/methanol/NH3(aq) (9.5/0.5/0.05, v/v/v). Yield: 25.0 mg (12%). 1H NMR (300 MHz, CHLOROFORM-d) δ 8.07 (dd, J = 8.46, 1.03 Hz, 1H), 7.91 (dd, J = 8.46, 1.03 Hz, 1H), 7.55 (ddd, J = 8.40, 6.86, 1.41 Hz, 1H), 7.28 7.48 (m, 6H), 7.14 (t, J = 8.46 Hz, 1H), 6.72 (t, J = 8.08 Hz, 2H), 5.20 (br. s., 1H), 5.08 (s, 2H), 3.62 (t, J = 6.16 Hz, 2H), 3.04 - 3.11 (m, 2H), 3.00 (t, J = 4.62 Hz, 4H), 2.65 - 2.81 (m, 6H), 2.62 (t, J = 6.16 Hz, 2H), 2.25 (s, 3H), 1.86 - 1.99 (m, 6H),

13

C NMR (75 MHz, CHLOROFORM-d) δ 158.4,

157.7, 152.5, 151.1, 147.4, 137.6, 128.7, 128.5, 128.23, 127.7, 127.1, 126.4, 123.4, 123.1, 121.3, 120.2, 115.9, 111.7, 107.1, 70.1, 57.6, 54.1, 51.8, 49.1, 34.0, 27.3, 25.7, 23.1, 22.8, 11.0. Formula: C34H40N4O; MS: m/z 521 (M+H+). 4.1.6.3. N-(2-(4-(1-Benzyl-1H-indol-4-yl)piperazin-1-yl)ethyl)-1,2,3,4-tetrahydroacridin-9-amine (15) Compound 15 was prepared using 2-(1,2,3,4-tetrahydroacridin-9-ylamino)ethanol (1) (109.0 mg, 0.45 mmol), TEA (50.6 mg, 0.50 mmol), methanesulfonyl chloride (57.3 mg, 0.50 mmol), 1-benzyl-4(piperazin-1-yl)-1H-indole (145.7 mg, 0.50 mmol) in 1.5 mL DCM. Extraction: 5 mL NaHCO3 (sat.), 3 × 5 mL DCM. Purification: column chromatography in DCM/methanol/NH3(aq) (9.5/0.5/0.05, v/v/v). Yield: 35.0 mg (15%). 1H NMR (300 MHz, CHLOROFORM-d) δ 8.09 (dd, J = 0.90, 8.59 Hz, 1H), 7.98 (d, J = 7.95 Hz, 1H), 7.57 (ddd, J = 1.28, 6.99, 8.40 Hz, 1H), 7.36 (ddd, J = 1.28, 6.99, 8.40 Hz, 1H), 7.23 - 7.32 (m, 3H), 7.06 - 7.16 (m, 4H), 6.99 (d, J = 8.21 Hz, 1H), 6.64 (d, J = 7.18 Hz, 1H), 6.55 (dd, J = 0.77, 3.08 Hz, 1H), 5.48 (br. s., 1H), 5.31 (s, 2H), 3.62 - 3.72 (m, 2H), 3.30 - 3.40 (m, 4H), 3.05 - 3.14 (m, 2H), 2.69 - 2.84 (m, 8H), 1.87 - 1.98 (m, 4H),

13

C NMR (75 MHz,

CHLOROFORM-d) δ 157.8, 151.5, 145.6, 137.5, 137.5, 128.7, 128.0, 127.6, 126.9, 126.7, 123.7, 123.0, 122.4, 121.9, 119.9, 115.6, 106.5, 104.7, 100.2, 57.5, 53.0, 51.6, 50.2, 45.0, 33.4, 24.8, 23.0, 22.7. Formula: C34H37N5; MS: m/z 516 (M+H+). 4.1.6.4. N-(3-(4-(1-Benzyl-1H-indol-4-yl)piperazin-1-yl)propyl)-1,2,3,4-tetrahydroacridin-9-amine (16)


Page 39 of 58 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Compound 16 was prepared using 3-(1,2,3,4-tetrahydroacridin-9-ylamino)propan-1-ol (2) (100.0 mg, 0.39 mmol), TEA (44.0 mg, 0.43 mmol), methanesulfonyl chloride (49.0 mg, 0.43 mmol), 1-benzyl-4(piperazin-1-yl)-1H-indole (125.0 mg, 0.43 mmol) in 2 mL DCM. Extraction: 10 mL NaHCO3 (sat.), 3 × 10 mL DCM. Purification: column chromatography in DCM/methanol/NH3(aq) (9.5/0.5/0.05, v/v/v). Yield: 66.0 mg (32%). 1H NMR (300 MHz, CHLOROFORM-d) δ 8.09 (dd, J = 0.77, 8.46 Hz, 1H), 7.95 (d, J = 8.21 Hz, 1H), 7.55 (ddd, J = 1.28, 6.99, 8.40 Hz, 1H), 7.25 - 7.37 (m, 4H), 7.06 - 7.15 (m, 4H), 6.98 (d, J = 8.21 Hz, 1H), 6.59 - 6.64 (m, 1H), 6.55 (dd, J = 0.77, 3.33 Hz, 1H), 5.30 (s, 2H), 3.67 (t, J = 6.03 Hz, 2H), 3.31 - 3.40 (m, 4H), 3.04 - 3.13 (m, 2H), 2.73 - 2.85 (m, 6H), 2.67 (t, J = 6.16 Hz, 2H), 1.86 - 1.99 (m, 6H), NH signal not detected, 13C NMR (75 MHz, CHLOROFORM-d) δ 158.03, 151.62, 145.79, 137.66, 128.84, 128.65, 128.27, 127.69, 126.95, 126.86, 126.74, 123.69, 123.28, 122.61, 122.08, 120.08, 115.69, 106.63, 104.82, 100.35, 57.91, 54.23, 51.38, 50.35, 49.36, 33.73, 27.31, 25.86, 23.10, 22.84. Formula: C35H39N5; MS: m/z 530 (M+H+).

4.1.7. General procedure for the synthesis of compounds 11-14, 17 and 18. A solution of 1-(3-(benzyloxy)-2-methylphenyl)piperazine or 1-benzyl-4-(piperazin-1-yl)-1H-indole (in a form of a free base or as a hydrochloride salt, 1 equiv), an appropriate ω-(bromoalkylamino)1,2,3,4-tetrahydroacridine (1 equiv), K2CO3 (1–3 equiv) and KI (0.9–1 equiv) in acetonitrile was heated under reflux for 24 hours. After cooling to ambient temperature, the solvent was evaporated and the residue was treated with saturated solution of NaHCO3, extracted with DCM, dried (Na2SO4) and concentrated under reduced pressure to give a crude product that was purified according to the methods described below. 4.1.7.1. N-(5-(4-(3-(Benzyloxy)-2-methylphenyl)piperazin-1-yl)pentyl)-1,2,3,4-tetrahydroacridin-9amine (11) Compound 11 was prepared using 1-(3-(benzyloxy)-2-methylphenyl)piperazine (25.0 mg, 0.09 mmol), N-(5-bromopentyl)-1,2,3,4-tetrahydroacridin-9-amine (5) (30.0 mg, 0.09 mmol), K2CO3 (12.0 mg, 0.09 mmol), KI (15.0 mg, 0.09 mmol) in 2 mL acetonitrile. Extraction: 5 mL NaHCO3 (sat.), 3 × 5 mL DCM. Purification: column chromatography in DCM/Et2O/methanol/NH3(aq) (7.5/2/0.5/0.05, v/v/v/v). Yield: 15.4 mg (31%). 1H NMR (300 MHz, CHLOROFORM-d) δ δ 7.97 - 8.05 (m, 2H), 7.58 (ddd, J



Page 40 of 58

= 1.28, 6.86, 8.27 Hz, 1H), 7.42 - 7.47 (m, 2H), 7.30 - 7.41 (m, 4H), 7.06 - 7.15 (m, 1H), 6.69 (t, J = 8.08 Hz, 2H), 5.07 (s, 2H), 3.58 (t, J = 7.18 Hz, 2H), 3.12 (br. s., 2H), 3.00 - 3.08 (m, 2H), 2.86 - 2.98 (m, 5H), 2.70 (br. s., 2H), 2.60 (br. s., 2H), 2.35 - 2.46 (m, 2H), 2.23 (s, 3H), 1.93 (td, J = 3.43, 6.22 Hz, 4H), 1.73 (td, J = 7.28, 14.43 Hz, 2H), 1.52 - 1.64 (m, 2H), 1.40 - 1.52 (m, 2H). Formula: C36H44N4O; MS: m/z 549 (M+H+). 4.1.7.2. N-(6-(4-(3-(Benzyloxy)-2-methylphenyl)piperazin-1-yl)hexyl)-1,2,3,4-tetrahydroacridin-9amine (12) Compound 12 was prepared using 1-(3-(benzyloxy)-2-methylphenyl)piperazine dihydrochloride (103.1 mg, 0.29 mmol), N-(6-bromohexyl)-1,2,3,4-tetrahydroacridin-9-amine (5) (104.6 mg, 0.29 mmol), K2CO3 (120.2 mg, 0.87 mmol), KI (43.0 mg, 0.26 mmol) in 3 mL acetonitrile. Extraction: 10 mL

NaHCO3

(sat.),

3

×

10

mL

DCM.

Purification:

column

chromatography

in

DCM/Et2O/methanol/NH3(aq) (7.5/2/0.5/0.05, v/v/v/v). Yield: 37.7 mg (23%). 1H NMR (300 MHz, CHLOROFORM-d) δ 7.92 - 8.00 (m, 2H), 7.56 (ddd, J = 1.41, 6.86, 8.40 Hz, 1H), 7.28 - 7.47 (m, 6H), 7.11 (t, J = 8.34 Hz, 1H), 6.69 (dd, J = 8.21, 10.26 Hz, 2H), 5.07 (s, 2H), 4.06 (br. s, 1H), 3.53 (t, J = 7.05 Hz, 2H), 3.04 - 3.12 (m, 2H), 2.94 (t, J = 4.74 Hz, 4H), 2.53 - 2.74 (m, 6H), 2.39 (dd, J = 6.67, 8.72 Hz, 2H), 2.23 (s, 3H), 1.87 - 1.97 (m, 4H), 1.69 (quin, J = 7.18 Hz, 2H), 1.49 - 1.60 (m, 2H), 1.35 - 1.48 (m, 4H), 13C NMR (75 MHz, CHLOROFORM-d) δ 157.9, 157.7, 152.7, 151.1, 146.8, 137.6, 128.6, 128.5, 128.1, 127.7, 127.1, 126.3, 123.7, 123.0, 121.2, 119.9, 115.5, 111.8, 106.9, 70.1, 58.7, 53.8, 51.9, 49.4, 33.6, 31.7, 27.4, 26.9, 26.8, 24.7, 23.0, 22.6, 11.1. Formula: C37H46N4O; MS: m/z 563 (M+H+). 4.1.7.3. N-(7-(4-(3-(Benzyloxy)-2-methylphenyl)piperazin-1-yl)heptyl)-1,2,3,4-tetrahydroacridin-9amine (13) Compound 13 was prepared using 1-(3-(benzyloxy)-2-methylphenyl)piperazine (125.7 mg, 0.45 mmol), N-(7-bromoheptyl)-1,2,3,4-tetrahydroacridin-9-amine (7) (167.1 mg, 0.45 mmol), K2CO3 (142.8 mg, 1.04 mmol), KI (68.0 mg, 0.41 mmol) in 5 mL acetonitrile. Extraction: 10 mL NaHCO3 (sat.), 3 × 10 mL DCM. Purification: column chromatography in DCM/Et2O/methanol/NH3(aq) (7.5/2/0.5/0.05, v/v/v/v). Yield: 139.2 mg (54%). 1H NMR (300 MHz, CHLOROFORM-d) δ 7.92 8.00 (m, 2H), 7.56 (ddd, J = 1.28, 6.99, 8.40 Hz, 1H), 7.27 - 7.48 (m, 6H), 7.05 - 7.14 (m, 1H), 6.68


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(d, J = 8.21 Hz, 1H), 6.71 (d, J = 7.95 Hz, 1H), 5.07 (s, 2H), 3.52 (t, J = 7.18 Hz, 2H), 3.05 - 3.13 (m, 2H), 2.95 (t, J = 4.74 Hz, 4H), 2.67 - 2.76 (m, 3H), 2.53 - 2.66 (m, 4H), 2.35 - 2.44 (m, 2H), 2.23 (s, 3H), 1.92 (td, J = 3.46, 6.41 Hz, 4H), 1.61 - 1.75 (m, 2H), 1.47 - 1.60 (m, 2H), 1.29 - 1.46 (m, 6H), 13C NMR (75 MHz, CHLOROFORM-d) δ 157.7, 152.7, 151.1, 137.6, 128.6, 128.5, 128.2, 127.7, 127.1, 126.3, 123.7, 122.9, 121.2, 119.9, 115.5, 111.8, 106.9, 70.1, 58.8, 53.8, 51.9, 49.5, 33.6, 31.7, 29.3, 27.5, 26.9, 24.7, 23.0, 22.6, 11.1. Formula: C38H48N4O; MS: m/z 577 (M+H+).

4.1.7.4. N-(8-(4-(3-(Benzyloxy)-2-methylphenyl)piperazin-1-yl)octyl)-1,2,3,4-tetrahydroacridin-9amine (14) Compound 14 was prepared using 1-(3-(benzyloxy)-2-methylphenyl)piperazine dihydrochloride (100.7 mg, 0.29 mmol), N-(8-bromooctyl)-1,2,3,4-tetrahydroacridin-9-amine (8) (112.9 mg, 0.29 mmol), K2CO3 (92.2 mg, 0.66 mmol), KI (43.0 mg, 0.26 mmol) in 5 mL acetonitrile. Extraction: 10 mL

NaHCO3

(sat.),

3

×

10

mL

DCM.

Purification:

column

chromatography

in

DCM/Et2O/methanol/NH3(aq) (7.5/2/0.5/0.05, v/v/v/v). Yield: 80.0 mg (47%). 1H NMR (300 MHz, CHLOROFORM-d) δ 7.98 (d, J = 8.46 Hz, 2H), 7.56 (ddd, J = 1.28, 6.99, 8.40 Hz, 1H), 7.25 - 7.47 (m, 6H), 7.10 (t, J = 8.08 Hz, 1H), 6.67 (d, J = 8.21 Hz, 1H), 6.71 (d, J = 7.95 Hz, 1H), 5.06 (s, 2H), 3.53 (t, J = 7.18 Hz, 2H), 3.47 (s, 1H), 3.09 (br. s., 2H), 2.95 (t, J = 4.62 Hz, 4H), 2.69 (br. s., 2H), 2.61 (br. s., 3H), 2.35 - 2.43 (m, 2H), 2.23 (s, 3H), 1.91 (td, J = 3.37, 6.35 Hz, 4H), 1.68 (quin, J = 7.18 Hz, 2H), 1.53 (td, J = 6.92, 13.85 Hz, 2H), 1.24 - 1.44 (m, 9H). Formula: C39H50N4O; MS: m/z 591 (M+H+). 4.1.7.5. N-(5-(4-(1-Benzyl-1H-indol-4-yl)piperazin-1-yl)pentyl)-1,2,3,4-tetrahydroacridin-9-amine (17) Compound 17 was prepared using 1-benzyl-4-(piperazin-1-yl)-1H-indole (61.0 mg, 0.21 mmol), N-(5bromopentyl)-1,2,3,4-tetrahydroacridin-9-amine (5) (73.0 mg, 0.21 mmol), K2CO3 (67.0 mg, 0.48 mmol), KI (32.0 mg, 0.19 mmol) in 3.5 mL acetonitrile. Extraction: 10 mL NaHCO3 (sat.), 3 × 10 mL DCM. Purification: column chromatography in DCM/Et2O/methanol/NH3(aq) (7.5/2/0.5/0.05, v/v/v/v). Yield: 21.0 mg (18%). 1H NMR (300 MHz, CHLOROFORM-d) δ 7.97 (dd, J = 1.03, 8.46 Hz, 1H), 7.91 (dd, J = 0.77, 8.46 Hz, 1H), 7.55 (ddd, J = 1.41, 6.86, 8.40 Hz, 1H), 7.35 (ddd, J = 1.28, 6.99,



8.40 Hz, 1H), 7.24 - 7.29 (m, 3H), 7.04 - 7.12 (m, 4H), 6.95 (d, J = 8.21 Hz, 1H), 6.60 (dd, J = 0.64, 7.57 Hz, 1H), 6.55 (dd, J = 0.77, 3.08 Hz, 1H), 5.30 (s, 2H), 3.51 (t, J = 7.18 Hz, 2H), 3.25 - 3.33 (m, 4H), 3.03 - 3.11 (m, 2H), 2.65 - 2.77 (m, 6H), 2.40 - 2.49 (m, 2H), 1.89 - 1.97 (m, 4H), 1.72 (quin, J = 7.31 Hz, 2H), 1.54 - 1.66 (m, 2H), 1.42 - 1.52 (m, 2H), NH signal not detected, 13C NMR (75 MHz, CHLOROFORM-d) δ 158.5, 150.7, 147.6, 145.8, 137.6, 137.5, 128.8, 128.7, 128.3, 128.1, 127.5, 126.7, 126.6, 123.6, 122.8, 122.4, 121.9, 120.2, 115.9, 106.5, 104.5, 100.3, 58.6, 53.7, 51.3, 50.2, 49.5, 34.1, 31.8, 26.7, 25.0, 24.8, 23.1, 22.8. Formula: C37H43N5; MS: m/z 558 (M+H+). 4.1.7.6. N-(6-(4-(1-Benzyl-1H-indol-4-yl)piperazin-1-yl)hexyl)-1,2,3,4-tetrahydroacridin-9-amine (18) Compound 18 was prepared using 1-benzyl-4-(piperazin-1-yl)-1H-indole (85.0 mg, 0.29 mmol), N-(6bromohexyl)-1,2,3,4-tetrahydroacridin-9-amine (6) (105.0 mg, 0.29 mmol), K2CO3 (93.0 mg, 0.67 mmol), KI (43.0 mg, 0.26 mmol) in 4 mL acetonitrile. Extraction: 10 mL NaHCO3 (sat.), 3 × 10 mL DCM. Purification: column chromatography in DCM/Et2O/methanol/NH3(aq) (7.5/2/0.5/0.05, v/v/v/v). Yield: 84.0 mg (51%). 1H NMR (300 MHz, CHLOROFORM-d) δ 7.94 (ddd, J = 0.77, 8.46, 16.16 Hz, 2H), 7.55 (ddd, J = 1.41, 6.86, 8.40 Hz, 1H), 7.35 (ddd, J = 1.28, 6.92, 8.46 Hz, 1H), 7.23 - 7.29 (m, 3H), 7.04 - 7.13 (m, 4H), 6.92 - 6.98 (m, 1H), 6.60 (dd, J = 0.64, 7.57 Hz, 1H), 6.54 (dd, J = 0.77, 3.33 Hz, 1H), 5.30 (s, 2H), 3.50 (t, J = 7.18 Hz, 2H), 3.25 - 3.34 (m, 4H), 3.03 - 3.11 (m, 2H), 2.65 - 2.77 (m, 6H), 2.39 - 2.46 (m, 2H), 1.90 - 1.96 (m, 4H), 1.63 - 1.75 (m, 2H), 1.51 - 1.63 (m, 2H), 1.38 - 1.49 (m, 4H), NH signal not detected,

13

C NMR (75 MHz, CHLOROFORM-d) δ 158.5, 150.7, 147.5,

145.8, 137.6, 137.5, 128.8, 128.7, 128.3, 127.5, 126.7, 123.6, 122.8, 122.4, 121.9, 120.3, 115.9, 106.5, 104.5, 100.3, 58.7, 53.7, 51.3, 50.2, 49.5, 34.1, 31.8, 29.7, 27.4, 26.9, 24.8, 23.1, 22.8. Formula: C38H45N5; MS: m/z 572 (M+H+). 4.1.8. Synthesis of 3-(benzyl(methyl)amino)propan-1-ol (46) The solution of N-methyl-1-phenylmethanamine (1500.0 mg, 12.4 mmol), 3-chloropropyl acetate (1407.0 mg, 10.3 mmol), K2CO3 (1424.0 mg, 10.3 mmol) and TBAB (322.0 mg, 1.0 mmol) in 100 mL acetonitrile was refluxed for 24 h. The solvent was then evaporated, 25 mL saturated solution of NaHCO3 was added and extracted with 3 × 25 mL DCM. The combined organic fractions were dried


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over Na2SO4 and concentrated under reduced pressure to give a crude product that was purified by flash chromatography in 50% ethyl acetate in petroleum ether. Yield: 1111.0 mg (49%). Formula: C13H19NO2; MS: m/z 222 (M+H+). The obtained 3(benzyl(methyl)amino)propyl acetate (1107.0 mg, 5.0 mmol) was dissolved in a solution containing 50.0 mL methanol, 7.5 mL H2O and K2CO3 (1382.0 mg, 10 mmol) and the mixture was stirred at 50 °C for 2.5 h. After that time, solvents were evaporated and the residue was treated with saturated solution of NaHCO3, extracted with DCM, dried (Na2SO4) and concentrated under reduced pressure to give a crude product which was used without further purification. Yield: 887.0 mg (99%). Formula: C11H17NO; MS: m/z 180 (M+H+). 4.1.9. General procedure for the synthesis of compounds 48 and 49. To the solution of 3-(benzyl(methyl)amino)propan-1-ol (1 equiv) and anhydrous TEA (1.1 equiv) in anhydrous DCM, methanesulfonyl chloride (1.1 equiv) was added dropwise at 0 °C, and the reaction mixture was stirred at this temperature for 30 min. Then 1-(3-(benzyloxy)-2-methylphenyl)piperazine hydrochloride or 1-benzyl-4-(piperazin-1-yl)-1H-indole was added and the mixture was stirred at ambient temperature for 24 h. After that time, saturated solution of NaHCO3 was added and the mixture was extracted with DCM. The combined organic fractions were dried (Na2SO4) and concentrated under reduced pressure to give a crude product that was purified using flash chromatography in 1–10% methanol in DCM. 4.1.9.1. N-Benzyl-3-(4-(3-(benzyloxy)-2-methylphenyl)piperazin-1-yl)-N-methylpropan-1-amine (48) Compound 48 was prepared using 3-(benzyl(methyl)amino)propan-1-ol (46) (100.0 mg, 0.56 mmol), TEA (61.7 mg, 0.61 mmol), methanesulfonyl chloride (69.9 mg, 0.61 mmol), 1-(3-(benzyloxy)-2methylphenyl)piperazine hydrochloride (194.5 mg, 0.61 mmol) in 1.5 mL DCM. Yield: 128.0 mg (52%). 1H NMR (300 MHz, CHLOROFORM-d) δ 7.23 - 7.48 (m, 10H), 7.12 (t, J = 8.21 Hz, 1H), 6.70 (dd, J = 8.08, 10.64 Hz, 2H), 5.08 (s, 2H), 3.54 (s, 2H), 2.96 (t, J = 4.74 Hz, 4H), 2.56 - 2.76 (m, 4H), 2.44 - 2.53 (m, 4H), 2.24 (s, 3H), 2.24 (s, 3H), 1.75 - 1.87 (m, 2H),

13

C NMR (75 MHz,

CHLOROFORM-d) δ 157.7, 152.6, 138.6, 137.6, 129.1, 128.5, 128.3, 127.7, 127.1, 126.3, 121.2, 111.9, 107.0, 70.1, 62.2, 56.6, 55.3, 53.7, 51.7, 42.2, 24.5, 11.1. Formula: C29H37N3O; MS: m/z 444 (M+H+).



Page 44 of 58

4.1.9.2. N-Benzyl-3-(4-(1-benzyl-1H-indol-4-yl)piperazin-1-yl)-N-methylpropan-1-amine (49) Compound 49 was prepared using 3-(benzyl(methyl)amino)propan-1-ol (46) (80.7 mg, 0.45 mmol), TEA (50.6 mg, 0.50 mmol), methanesulfonyl chloride (57.3 mg, 0.50 mmol), 1-benzyl-4-(piperazin-1yl)-1H-indole (145.7 mg, 0.50 mmol) in 1.5 mL DCM. Yield: 101.8 mg (50%). 1H NMR (300 MHz, CHLOROFORM-d) δ 7.23 - 7.36 (m, 8H), 7.07 - 7.13 (m, 4H), 6.96 (d, J = 8.46 Hz, 1H), 6.58 - 6.63 (m, 1H), 6.55 (dd, J = 0.64, 3.21 Hz, 1H), 5.30 (s, 2H), 3.54 (s, 2H), 3.27 - 3.35 (m, 4H), 2.70 - 2.78 (m, 4H), 2.44 - 2.56 (m, 4H), 2.25 (s, 3H), 1.77 - 1.89 (m, 2H), 13C NMR (75 MHz, CHLOROFORMd) δ 145.8, 138.7, 137.6, 137.5, 129.1, 128.7, 128.3, 127.5, 127.1, 126.7, 122.4, 121.9, 106.6, 104.5, 100.3, 62.2, 56.7, 55.4, 53.6, 51.2, 50.2, 42.2, 24.6. Formula: C30H36N4; MS: m/z 453 (M+H+). 4.1.10. Synthesis

of

1-benzyl-N-(3-(4-(3-(benzyloxy)-2-methylphenyl)piperazin-1-

yl)propyl)piperidin-4-amine (52) The solution of 1-(3-(benzyloxy)-2-methylphenyl)piperazine hydrochloride (318.8 mg, 1.0 mmol), 1bromo-3-chloropropane (472.3 mg, 3.0 mmol), K2CO3 (207.3 mg, 1.5 mmol) in 20 mL acetonitrile was stirred at 50 °C for 24 h. The solvent was then evaporated, 20 mL saturated solution of NaHCO3 was added and extracted with 3 × 20 mL DCM. The combined organic fractions were dried over Na2SO4 and concentrated under reduced pressure to give a crude product which was used without further purification. Yield: 105.0 mg (29%). Formula: C21H27ClN2O; MS: m/z 359 (M+H+). The obtained 1-(3(benzyloxy)-2-methylphenyl)-4-(3-chloropropyl)piperazine (105.0 mg, 0.29 mmol) was added to the solution of 1-benzylpiperidin-4-amine (55.2 mg, 0.29 mmol) and K2CO3 (40.1 mg, 0.29 mmol) in 10 mL acetonitrile and refluxed overnight. After that time, solvents were evaporated and the residue was treated with saturated solution of NaHCO3, extracted with DCM, dried (Na2SO4) and concentrated under reduced pressure to give a crude product which was by column chromatography in chloroform/methanol/NH3 (9/1/0.05, v/v/v). Yield: 56.0 mg (38%).

1

H NMR (300 MHz,

CHLOROFORM-d) δ 7.22 - 7.47 (m, 10H), 7.11 (t, J = 8.08 Hz, 1H), 6.65 - 6.74 (m, 2H), 5.07 (s, 2H), 3.50 (s, 2H), 2.94 (t, J = 4.49 Hz, 4H), 2.86 (d, J = 12.05 Hz, 2H), 2.73 (t, J = 6.80 Hz, 2H), 2.63 (br. s., 4H), 2.45 - 2.54 (m, 3H), 2.23 (s, 3H), 1.97 - 2.08 (m, 3H), 1.83 - 1.93 (m, 2H), 1.75 (quin, J = 6.92 Hz, 2H), 1.39 - 1.50 (m, 2H),

13

C NMR (75 MHz, CHLOROFORM-d) δ 157.7, 152.6, 138.6,


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137.6, 129.1, 128.5, 128.1, 127.7, 127.1, 126.9, 126.3, 121.2, 111.8, 107.0, 70.1, 63.0, 57.3, 55.0, 53.8, 52.4, 52.0, 45.7, 32.5, 29.7, 11.1. Formula: C33H44N4O; MS: m/z 513 (M+H+). 4.1.11. Synthesis

of

1-benzyl-N-(3-(4-(3-(benzyloxy)-2-methylphenyl)piperazin-1-

yl)propyl)piperidin-4-amine (53) The solution of 4-((1-benzylpiperidin-4-yl)(tert-butoxycarbonyl)amino)butyl methanesulfonate (118.0 mg, 0.27 mmol), 1-(3-(benzyloxy)-2-methylphenyl)piperazine dihydrochloride (96.0 mg, 0.27 mmol), K2CO3 (111.9 mg, 0.81 mmol) in 2 mL acetonitrile was stirred at reflux for 24 h. The solvent was then evaporated, 5 mL saturated solution of NaHCO3 was added and extracted with 3 × 5 mL DCM. The combined organic fractions were dried over Na2SO4 and concentrated under reduced pressure to give a crude product that was purified using flash chromatography in 1–10% methanol in DCM. Yield: 72.5 mg (43%). 1H NMR (300 MHz, CHLOROFORM-d) δ 7.22 - 7.48 (m, 10H), 7.11 (t, J = 8.21 Hz, 1H), 6.70 (dd, J = 8.46, 9.49 Hz, 2H), 5.08 (s, 2H), 3.49 (s, 2H), 3.02 - 3.16 (m, 2H), 2.89 - 2.98 (m, 6H), 2.61 (br. s., 3H), 2.41 (t, J = 7.05 Hz, 2H), 2.24 (s, 3H), 1.97 - 2.10 (m, 3H), 1.48 - 1.78 (m, 9H), 1.46 (s, 9H). Formula: C39H54N4O3; MS: m/z 627 (M+H+). The obtained tert-butyl (4-(4-(3-(benzyloxy)-2methylphenyl)piperazin-1-yl)butyl)(1-benzylpiperidin-4-yl)carbamate (66.4 mg, 0.11 mmol) was dissolved in 1 mL EtOAc and 1 M HCl solution in EtOAc was added and stirred at ambient temperature for 24 h. After that time, solvents were evaporated and the residue was treated with saturated solution of NaHCO3, extracted with DCM, dried (Na2SO4) and concentrated under reduced pressure to give final product. Yield: 38.0 mg (38%). 1H NMR (300 MHz, CHLOROFORM-d) δ 6.74 - 7.18 (m, 10H), 6.28 - 6.57 (m, 3H), 4.56 (br. s., 2H), 4.14 (s, 2H), 3.24 - 3.52 (m, 6H), 2.68 - 3.02 (m, 12H), 2.21 (d, J = 12.82 Hz, 2H), 1.81 (br. s., 3H), 1.55 - 1.76 (m, 6H). Formula: C34H46N4O; MS: m/z 527 (M+H+).

4.2.

In vitro AChE and BuChE inhibition assay. To measure the inhibitory activities of the

synthesized compounds against the cholinesterases we used the spectrophotometric method described by Ellman et al.,44 as modified for 96-well microplates. AChE from Electrophorus electricus (eeAChE), human recombinant AChE (hAChE), BuChE from equine serum (eqBuChE),

5,5'-

dithiobis-(2-nitrobenzoic acid) (DTNB), acetylthiocholine iodide (ATC) and butyrylthiocholine iodide



(BTC) were purchased from Sigma–Aldrich. Human recombinant butyrylcholinesterase (hBuChE) was from Vivonics (Bedford, MA, USA). 500 U of eeAChE, hAChE or eqBuChE was dissolved in water to give stock solutions of 5 U/mL. For studies with animal enzymes aqueous solutions of 0.0025 M DTNB and 0.00375 M ATC/BTC were prepared. For assays with hAChE we used 0.0042 M DTNB and 0.00625 M ATC aqueous solutions. At first, 25 µL of the test compound (or water; i.e. blank samples) was incubated (5 min) in 200 µL of 0.1 M phosphate buffer (pH 8.0) with DTNB (20 µL) and the enzyme (20 µL) at 25 °C (eeAChE/eqBuChE) or 36 °C (hAChE). After 5 min of incubation 20 µL of acetylthiocholine iodide (ATC) or butyrylthiocholine iodide (BTC) solutions (depending on the enzyme used) were added to start the reaction. Finally after 5 min of the reaction, changes in absorbance were measured at 412 nm, using the microplate reader (EnSpire Multimode; PerkinElmer). All the compounds were tested at screening concentration of 10 µM. Percent of inhibition was calculated from 100 - (S/B) × 100, where S and B represent enzyme activities with and without the test sample, respectively. For compounds with at least 50% inhibitory activity at 10 µM, IC50 values were determined. To determine IC50 value, seven different concentrations of each compound were used to obtain enzyme activities between 5% and 95%. Each concentration was measured in triplicate. The IC50 values were calculated using nonlinear regression (GraphPad Prism 5 [GraphPad Software, San Diego California USA 5.00]) by plotting the residual enzyme activities against the applied inhibitor concentration. Donepezil and tacrine were used as the reference compounds. Data are expressed as means ± SEM.

4.3.

Kinetic studies of AChE and BuChE inhibition. The kinetic studies were performed, using

Ellman’s method,44 modified for 96-well microplates. A stock solution of an appropriate enzyme (5U/mL) was diluted before use to a final concentration of 0.639 U/mL (hAChE) or 0.384 U/mL (eeBuChE). A stock solution (0.02125 M) of the substrate (ATC or BTC) was prepared in demineralized water and diluted before use. Six different concentrations of inhibitors were used to obtain enzyme activities between 30% and 80%. For each concentration of the test compounds, ATC was used at the concentration of 0.5, 0.4, 0.3, 0.2, 0.1 and 0.067 mM in a well, while BTC was used at concentrations of 0.3, 0.24, 0.18, 0.12, 0.06, and 0.04 mM in the wells. Assays were performed


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according to the protocol described above (paragraph 4.2.). Each experiment was performed in triplicate. Vmax and Km values of the Michaelis−Menten kinetics were calculated by nonlinear regression from substrate−velocity curves. Lineweaver-Burk and Cornich-Bowden plots were calculated using linear regression in GraphPad Prism 5.

4.4.

Radioligand binding assay for 5-HT6 receptors. The assay was performed according to the

protocol reported before.31 10 mM stock solutions of the 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) or CyBi Felix (CyBio AG). Each compound was tested in 10 concentrations ranging from 10–6 to 10–10 M (final concentration). Radioligand binding was performed using membranes from CHO-K1 cells stably transfected with the human 5-HT6 receptor. All assays were carried out in duplicates. 50 µL working solution of the tested compounds, 50 µL [3H]-LSD (final concentration 1,2 nM, Kd = 0.6 nM) and 150 µL diluted membranes prepared in assay buffer (50 mM Tris, pH 7.4, 10 mM MgCl2, 0.1 mM EDTA) were transferred to polypropylene 96-well microplate using 96-wells pipetting station Rainin Liquidator (MettlerToledo). Methiotepin (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 GF/B filter mate presoaked with 0.5% polyethyleneimine for 30 minutes. Ten rapid washes with 200 µL of 50 mM Tris buffer (pH 7.4) were performed at 4 °C using automated harvester system Harvester-96 MACH III FM (Tomtec). The filter mates were dried at 37 °C in forced air fan incubator and then solid scintillator MeltiLex was melted on filter mates at 90 °C for 5 minutes. Radioactivity was counted in MicroBeta2 scintillation counter (PerkinElmer) at approximately 30% efficiency. 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. 4.5. Functional assay for 5-HT6 receptors. Test and reference compounds were dissolved in DMSO at a concentration of 1 mM. Serial dilutions of 8 to 10 concentrations were prepared in 96-well microplate using assay buffer. A cellular aequorin-based functional assay was performed with



recombinant CHO-K1 cells expressing mitochondrially targeted aequorin, human GPCR and the promiscuous G protein α16 for 5-HT6. Assay was executed according to previously described protocol 42

. After thawing, cells were transferred to assay buffer (DMEM/HAM’s F12 with 0.1% protease free

BSA) and centrifuged. The cell pellet was resuspended in assay buffer and coelenterazine h was added at the final concentrations of 5 µM. The cells suspension was incubated at 16 °C, protected from light with constant agitation for 16 h and then diluted with assay buffer to the concentration of 5000 cells/mL. After 1 h of incubation, 50 µL of the cells suspension was dispensed using automatic injectors built into the radiometric and luminescence plate counter MicroBeta2 LumiJET (PerkinElmer, USA) into white opaque 96-well microplates preloaded with test compounds. Immediate light emission generated following calcium mobilization was recorded for 60 s. In antagonist mode, after 30 min of incubation the reference agonist was added to the above assay mix and light emission was recorded again. Final concentration of the reference agonist was equal to EC80 (40 nM serotonin). 4.6. Thioflavin-T (ThT) fluorometric assay.47 The inhibition of Aβ1–42 aggregation was measured fluorimetrically as described previously.54 Briefly, HFIP-pretreated Aβ1–42 (Merck Millipore, Darmstadt, Germany) at 1.5 µM, the test compound (10 µM final concentration) and Thioflavin-T (10 µM final concentration) were incubated at room temperature in 96-well microplate (covered with aluminum foil to prevent evaporation) with continuous shaking for 48 h. The fluorescence intensity (λex = 440 nm; λem = 490 nm) was measured every 3 min (SynergyTM H4 plate reader, BioTek Instruments, Inc. Winooski, VT, USA). The assay was run in quadruplicates. For the IC50 determination, the dilutions of compounds were prepared covering concentrations from 30 nM to 30 µM (final concentrations). The percentage of inhibition was calculated as described above. The IC50 values were calculated using nonlinear regression (GraphPad Prism 6 [GraphPad Software, San Diego California USA 5.00]) by plotting the percentage of inhibition against the applied inhibitor concentration.

4.7. In vitro blood-brain barrier permeability assay. Penetration across the BBB is an essential property for compounds targeting the CNS. In order to predict passive blood-brain penetration of the


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novel compounds, modified parallel artificial membrane permeation assay (PAMPA) has been used based on reported protocol.48,55 The filter membrane of the donor plate was coated with PBL (Polar Brain Lipid, Avanti, USA) in dodecane (4 µL of 20 mg/ml PBL in dodecane) and the acceptor well was filled with 300 µl of PBS pH 7.4 buffer (VD). The tested compounds were dissolved first in DMSO and then diluted with PBS pH 7.4 to reach the final concentration of 100 µM in the donor well. Concentration of DMSO did not exceed 0.5% (V/V) in the donor solution. 300 µL of the donor solution was added to the donor wells (VA) and the donor filter plate was carefully put on the acceptor plate so that the coated membrane was “in touch” with both donor solution and acceptor buffer. Test compound diffused from the donor well through the lipid membrane (Area = 0.28 cm2) to the acceptor well. The concentration of the drug in both donor and the acceptor wells was assessed after 3, 4, 5 and 6 hours of incubation in quadruplicate using the UV plate reader Synergy HT (Biotek, USA) at the maximum absorption wavelength of each compound. Concentration of the compounds was calculated from the standard curve and expressed as the effective permeability (Pe) according the equation (1):56,57

log = log × − 1 −

!"

#$ %ℎ'(' = )-*

*+ ×*,

+ .*, /×01×234

5 (1)

4.8. Metabolic stability. The in vitro evaluation of the metabolic stability of 12 was performed using human liver microsomes (HLMs) (Sigma-Aldrich, St. Louis, MO, USA). The reaction mixture consisted of 50 µM of tested compounds with microsomes (1 mg/ml) in 10 mM tris-HCl buffer. After 5 min of preincubation the 50 µL of NADPH Regeneration System (Promega, Madison, WI, USA) was added to initiate the reaction. The reaction mixture was incubated for 120 min at 37 °C. In order to terminate the reaction, 200 µL of cold extra pure methanol was added. Then, the mixture was centrifuged 14000 rpm for 15 min and the supernatant was analysed using LC/MS Waters ACQUITY™ TQD system with the TQ Detector (Waters, Milford, USA). 4.9. The influence on CYP3A4 activity. The luminescent CYP3A4 P450-Glo™ assay and protocol were provided by Promega (Madison, WI, USA). The experiments were performed in white polystyrene, flat-bottom Nunc™ MicroWell™ 96-Well Microplates (Thermo Scientific, Waltham, MA USA). The luminescence signal was measured with a microplate reader (EnSpire) in



luminescence mode. The signal produced by CYPs without the presence of compound 12 was considered as a 100% of CYP activity. The IC50 value of the reference inhibitor ketoconazole was calculated according to the manufacturer’s recommendations as reported previously.58 Compound 12 was tested in triplicate at the final concentrations in range from 0.025 µM to 25 µM. GraphPad Prism™ software (version 5.01, San Diego, CA, USA) was used to calculate IC50 value of compound 12. 4.10. Molecular modeling. Ligand structures were prepared in Maestro 2D Sketcher and were optimized using LigPrep tool. Glide XP flexible docking procedure was carried out using OPLS3 force field. Homology model of serotonin 5-HT6 receptor and 2 crystal structure-based AChE models served as molecular targets. Homology modeling procedure was reported previously59 and was applied for preparation of a model based on the serotonin 5-HT1B receptor crystal structure (PDB ID: 4IAR).31,60 H-bond constraints, as well as centroid of a grid box for docking studies were located on Asp3.32. The AChE models were developed on the basis of experimental structures of the enzyme. To capture characteristic interactions of diverse ligand structures and mimic conformational flexibility of the protein, 2 individual structural representations were prepared. For studies describing tacrine derivatives, a model based on crystal structure complexed with alkylene-linked bis-tacrine dimer (PDB ID: 2CMF) was used.61 All the other derivatives, containing N-benzylamine moiety, were docked to the model based on donepezil-complexed crystal structure (PDB ID: 1EVE).45 The initial structures were refined using Protein Preparation Wizard. Water molecules and hetero groups other than the ligand were deleted, the missing protein side chain atoms were predicted using Prime and the energy of the whole system was minimized (OPLS3). Grid boxes for docking in AChE models were placed in a centroid of co-crystallized ligands. The models were tested throughout docking studies involving AChE inhibitors of experimentally proven affinity. The obtained consistent binding modes of the reference compounds verified accuracy of the crystal-based models. The aforementioned software was implemented in Small-Molecule Drug Discovery Suite (Schrödinger, Inc.), which was licensed for Jagiellonian University Medical College.


Page 50 of 58

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Abbreviations Used: AD, Alzheimer’s disease; BPSD, behavioural and psychological symptoms of dementia; ACh, acetylcholine; AChE, acetylcholinesterase; BuChE, butyrylcholinesterase; Aβ, amyloid

β;

MTDLs,

butyloxycarbonyl

multi-target-directed

protecting

group;

TEA,

ligands;

BBB,

triethylamine;

blood-brain-barrier; BOC, THF,

tetrahydrofuran;

tertDCM,

dichloromethane; DMSO, dimethyl sulfoxide; EtOAc, ethyl acetate; eeAChE, AChE from the electric eel; eqBuChE, BuChE from horse serum; hAChE, human AChE; ATC, acetylthiocholine; BTC, butyrylthiocholine; CNS, central nervous system; PAMPA-BBB, parallel artificial membrane permeability assay for the blood-brain barrier Acknowledgments: this study received financial support from the Polish Ministry for Science and Higher

Education

(grant

No

IP2012063272),

National

Science

Centre

Poland

(2016/23/D/NZ7/01328), by Jagiellonian University Medical College (K/ZDS/007216),and the Slovenian Research Agency (research program P1-0208 and research project L1-8157). Author Contributions: A.W. designed and coordinated the study and analysed the data; A.W., T.W. wrote the manuscript; A.W., T.W., K.W., M.M., P.Z. synthesized the compounds; J.G. performed Ellman’s assay; A.B., M.K. performed molecular modelling; A.S., G.K., M.G-L. performed radioligand binding assays and functional assays on 5-HT6 receptors; D.K., S.G. performed thioflavine-T fluorescence assay; G.L., K.K-K. performed in silico and in vitro metabolic stability assay; J.K., O.S., M.B. performed PAMPA-BBB assay; B.M., K.M., K.W., D.K. corrected the manuscript. All the authors contributed to and approved the final manuscript.

Supporting Information: Metabolites of compound 12 predicted by MetaSite; MS spectra of compound 12 and its metabolites; Compound 12 - MS fragmentation analysis; fragmentation and in silico analysis of metabolites of compound 12.

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Page 58 of 58

5-HT6 RECEPTOR Page 59 of 58 ACS ANTAGONISM

1 2 3 4 5

MULTI-TARGET DIRECTED

CHOLINESTERASE INHIBITION

LIGANDS Chemical Neuroscience

Cmp. 12

INHIBITION OF Aβ

ACS Paragon Plus Environment AGGREGATION

Ki 5-HT6 = 18 nM

IC50 Aβ aggreg.= 1.3 μM

IC50 hAChE = 14 nM IC50 eqBuChE = 22 nM

Novel Multitarget-Directed Ligands Aiming at Symptoms and Causes

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