4-Oxypiperidine Ethers as Multiple Targeting Ligands at Histamine H3 Receptors and Cholinesterases

This study examines the properties of a novel series of 4-oxypiperidines designed and synthesized as histamine H3R antagonists/inverse agonists based on the structural modification of two lead compounds, viz., ADS003 and ADS009. The products are intended to maintain a high affinity for H3R while simultaneously inhibiting AChE or/and BuChE enzymes. Selected compounds were subjected to hH3R radioligand displacement and gpH3R functional assays. Some of the compounds showed nanomolar affinity. The most promising compound in the naphthalene series was ADS031, which contained a benzyl moiety at position 1 of the piperidine ring and displayed 12.5 nM affinity at the hH3R and the highest inhibitory activity against AChE (IC50 = 1.537 μM). Eight compounds showed over 60% eqBuChE inhibition and hence were qualified for the determination of the IC50 value at eqBuChE; their values ranged from 0.559 to 2.655 μM. Therapy based on a multitarget-directed ligand combining H3R antagonism with additional AChE/BuChE inhibitory properties might improve cognitive functions in multifactorial Alzheimer’s disease.


INTRODUCTION
It has been more than 40 years since the seminal study by Arrang and co-workers, which demonstrated that histamine inhibits its own synthesis and release from cortical slices of the rat brain via a new histamine receptor that is pharmacologically distinct from both the histamine H 1 and H 2 receptors. 1The histamine H 3 receptor (H 3 R) was described as a presynaptic autoreceptor involved in the inhibition of histamine secretion in the brain and proved the neurotransmitter function of histamine. 2t was later discovered that H 3 Rs do not only control histamine release but also, functioning as heteroreceptors, modulate the other neurotransmitter systems, 3 e.g., the cholinergic, 4,5 dopaminergic, 6 noradrenergic, 7,8 and serotoninergic 9 systems, in both the central and peripheral nervous systems.H 3 R antagonists/inverse agonists demonstrated diverse pharmacological effects in preclinical and clinical studies, highlighting the importance of various central nervous system (CNS)-related therapeutic applications, including Parkinson's disease and Alzheimer's disease (AD), 10 narcolepsy, 11 obesity, 12 dementia, epilepsy, schizophrenia, 13 and attention deficit hyperactivity disorder (ADHD). 14Recently, many other CNSrelated disorders have also been suggested as candidates for treatment by H 3 R antagonist/inverse agonist ligands: Tourette syndrome, 15 Prader−Willi syndrome, 16 depression, 17 or autism. 18e most commonly prescribed drugs for the treatment of AD are cholinesterase inhibitors, which improve memory function and delay cognitive decline without altering the underlying pathology. 19However, these drugs show clear limitations concerning both efficacy and tolerability.Their efficacy on cognition enhancement is limited, with the preferred target patient population suffering mild to moderate cognitive impairment. 19From a neurochemical point of view, the decline in cognitive function associated with normal aging, mild cognitive impairment, AD, and related conditions is thought to be caused by a decrease in cortical cholinergic neurotransmission.Such neurotransmission can be improved and enhanced by both AChE/BuChE inhibitors and H 3 R antagonists, mediated by different neuronal mechanisms.For instance, the learning and memory-facilitating potential of plentiful H 3 R antagonists have been shown.−22 The arousal effect is cumulated with enhanced cortical fast rhythms that correspond to higher brain functions, including alertness, attention, and cognition. 21,22Moreover, H 3 R antagonists increase the synthesis and release of histamine and its associated memory enhancement.−25 This suggests that it may be possible to enhance cholinergic neurotransmission by combining the two activities in a single molecule as a multitargeting approach. 26,27Histamine can control the signaling effects induced by the activation of the cholinergic receptor through the H 3 R-controlled mechanism, regulating the excitatory effects of acetylcholine. 28H 3 Rs, modulating the cholinergic system, increase the level of acetylcholine in the synaptic cleft.Two main enzymes hydrolyze acetylcholine: AChE and butyrylcholinesterase (BuChE).AChE is a neuronal enzyme predominantly found in the neuronal cleft of a healthy brain, and its activity decreases when AD occur. 29,30When AChE activity in AD is reduced, AChE and BuChE progression is inverted, denoting the importance of both enzymes in cognitive dysfunction.Dual-acting compounds, inhibiting additionally AChE or BuChE, prolong the survival time of acetylcholine in the neuronal cleft. 31,32Co-administration of a H 3 R antagonist (MK-3134) and an AChE inhibitor (donepezil) showed a more pronounced, pro-cognitive outcome than the effects of each compound separately. 33,34Dual-acting drugs can be important for the treatment of Alzheimer's disease due to their lower toxicity, less drug−drug interactions, unified pharmacokinetic profile, and higher efficacy.By acting through H 3 R in the CNS, they lower peripheral AChE levels and may result in fewer side effects. 35By inhibiting AChE, these compounds may slow the β-amyloid peptide aggregation process promoted by AChE. 36n recent years, there has been the development of a novel class of procognitive dual-acting drugs, that exhibit both H 3 R antagonism/inverse agonism and inhibition of AChE and/or BuChE.Three such nonimidazole H 3 R antagonists with additional cholinesterase inhibition are shown in Figure 1.
Previously, our laboratory has described piperazine, 40 guanidine, 41 and 4-hydroxpiperidine-based H 3 R antagonists. 42,43The structure−activity relationship (SAR) of the 4-   2). 42,43Further in vivo evaluation of the impact of ADS003 on brain neurotransmitter systems showed its ability to cross the blood−brain barrier and potency at H 3 R similar to the reference compound, ciproxifan. 42

Design.
In previous studies on 4-hydroxypiperidines, the two ligands ADS003 and ADS009 (Figure 2) were shown to be highly potent antagonists/inverse agonists at the H 3 R.They were used as lead compounds for the following structural modification: replacement of the flexible five-methylene group chain with a benzene, biphenyl, or naphthalene linker (Figure 2).The idea to replace the flexible alkyl linker with semirigid groups of the lead compounds is consistent with studies that have demonstrated that replacement of the alkyl chain with more rigid moieties results in the formation of higher affinity H 3 R ligands. 42A moiety with a more restricted conformation may be better fitted to the receptor-binding site due to reduced flexibility (i.e., degrees of freedom). 44n the first step, two compounds were synthesized (ADS021, ADS023; Scheme 1) in which the aliphatic linker was replaced by a benzene ring carrying an N-methyl-N-propylamine group in the para position.Following this, a series of derivatives were synthesized; these contained a methylene linker connected to the oxygen in position 4 of piperidine and a benzene ring carrying an N-methyl-N-propylaminomethyl substituent in the ortho-, meta-, or para-position (ADS027, ADS028, ADS025, ADS026, ADS022, and ADS024, respectively; Scheme 2).Finally, a series containing a biphenyl ring (ADS029, ADS030; Scheme 3) and a naphthalene ring (ADS031, ADS032; Scheme 4) in place of the aliphatic linker was prepared.
To synthesize the final compounds presented in Scheme 4, commercially available naphthalene-2,6-dicarboxylic acid (17)  was first treated with excess methyl iodide and lithium carbonate to obtain the diester derivative 18 (according to Benito and Meldal 45 ).Following this, 18 was reduced with LiAlH 4 to the relevant diol (19). 46Bromination of compound 19 led to derivative 20, 46 which was used as a substrate for the etherification process of tert-butyl 4-hydroxypiperidine-1carboxylate.The obtained ether 21 was treated with Nmethyl-N-propylamine in the presence of potassium carbonate, leading to compound 22. Acidic deprotection of Boc-groups from the piperidine moiety yielded 23.Finally, reductive amination of 23 and the corresponding aldehydes (benzaldehyde or benzofuran-2-carbaldehyde) in the presence of sodium triacetoxyborohydride allowed the final compounds ADS031 and ADS032 to be obtained.
All final ADS compounds were converted into their salts of oxalic or fumaric acid.

Pharmacology. 2.3.1. Ex Vivo gpH 3 R Screening on Isolated Guinea Pig Ileum and hH 3 R Radioligand Displacement Assays.
All newly synthesized compounds were evaluated ex vivo as H 3 R antagonists/inverse agonists on guinea pig ileum (gpH 3 R) stimulated electrically for contraction. 47Two of the most active pairs (containing benzyl and benzofuran groups) of compounds ADS022 and ADS024 as well as ADS031 and ADS032, all exceeded a pA 2 of 7, were subjected to a radioligand displacement assay in membrane fractions of HEK-293 cells stably expressing human H 3 R (hH 3 R). 48The affinity for all compounds is summarized in Table 1.
In this series, derivatives ADS021 and ADS023 showed the lowest potency (pA 2 < 4).The introduction of an additional methylene group between 4-oxypiperidine and the aromatic ring resulted in a significant increase in activity, viz., ADS022 pA 2 = 7.42 and ADS024 pA 2 = 7.57, carrying the N-methyl-N-propylaminomethyl substituent at position 4 of the benzene ring.Those with N-methyl-N-propyl-aminomethyl substituted at position 3 exhibited lowered activity, viz.ADS025 (pA 2 = 6.89) and ADS026 (pA 2 = 7.03).In addition, by altering the Nmethyl-N-propyl-aminomethyl substituent at position 2 of the aromatic ring, synthesized compounds (ADS027 and ADS028) demonstrated a further decrease in activity.By looking at the drug-likeness properties of all compounds calculated in the SwissADME web tool, 49 we have observed that only ADS021 of the benzyl derivatives has two H-bond acceptors, while the rest of the derivatives contain three H-bond acceptors.A similar observation applies to benzofuranyl derivatives, where ADS023 contains three H-bond acceptors while the other benzofuranyl derivatives have four H-bond acceptors.As we show later in the manuscript, the lack of an additional fragment engaged in hydrogen bond formation could be related to the low affinity of AD021 and ADS023.Moreover, both compounds consist of only seven rotatable bonds, while the others have nine or even ten (ADS029 and ADS030), which in this case may make it difficult to fit into the binding site.Anyway, all compounds do not exceed ten rotatable bonds, which is consistent with the drug-likeness Veber assumptions. 50AR showed that substitution at position 4 of the aromatic ring enhances H 3 R antagonistic activity.Following this, derivatives with biphenyl and naphthalene linkers in place of the 1,4-disubstituted aromatic ring were synthesized to increase structural rigidity and lipophilicity (Schemes 3 and 4).ADS029 and ADS030 containing the biphenyl linker showed lower affinity to gpH 3 R than the parent compounds ADS022 and ADS024.Compounds with a naphthalene linker, viz., ADS029 and ADS030, showed slightly higher activity than those with a biphenyl moiety.Among all derivatives, the most potent compound in the benzene series was ADS024, which contained a benzofuranyl substituent at position 1 of the piperidine ring, while the most potent compound in the naphthalene series was ADS031, which contained a benzyl moiety at position 1 of the piperidine ring.
Derivatives with the highest potency at gpH 3 R (ADS022, ADS024, ADS031, and ADS032) were subjected to the hH 3 R radioligand displacement assay, which demonstrated a nanomolar affinity among all tested compounds (Table 1).The lowest K i value (12.5 nM; pK i = 7.90) was observed for ADS031.The benzofuranyl derivative ADS032 showed K i = 44.1 nM (pK i = 7.36).H 3 R ligands are known to have a distinct affinity profile and species-dependent pharmacology. 51The affinity toward the guinea pig and human H 3 R differed, which can be explained by the species-dependent isoforms of the receptor. 52,53−56 Results with higher affinities were observed for naphthalene derivatives, including K i values evaluated at hH 3 R, but only lead compound ADS031 did not exceed a log P of 6 in all predictive methods.All data determined by the SwissADME web tool can be found in the Supporting Information.

Inhibition of electric eel AChE and equine serum BuChE.
All compounds were tested for their ability to inhibit acetylcholinesterase (eeAChE from electric eel) and butyrylcholinesterase (eqBuChE from equine serum) using spectroscopic Ellman's protocol, 57 modified for 96-well microplates.Target compounds were tested at a screening concentration of 10 μM.Four compounds (ADS022, ADS025, ADS029, and ADS031) showed over 60% eeAChE inhibition, and hence the IC 50 value was determined.highest inhibitory activity was demonstrated by compound ADS031 (IC 50 = 1.537 μM).Moreover, a comparison of the structures of ADS022, ADS025, ADS029, and ADS031 found their AChE inhibitory activity to increase according to the aromatic moiety, i.e., from benzene through biphenyl to naphthalene derivatives.Eight compounds showed over 60% eqBuChE inhibition and were qualified for the determination of the IC 50 value (Table 1).Of these, the IC 50 values ranged from 0.559 to 2.655 μM, with the lowest IC 50 value (IC 50 = 0.559 μM) being observed for ADS021, one of the least active compounds at gpH 3 R. ADS031 showed micromolar inhibitory activity for both AChE and BuChE enzymes and demonstrated higher inhibitory activity against BuChE (IC 50 = 1.353 μM) than AChE (IC 50 = 1.537 μM).Selectivity is claimed as a therapeutic advantage, but therapy based on a multitargetdirected ligand combining H 3 R antagonism with additional AChE/BuChE inhibitory properties might improve cognitive functions in multifactorial Alzheimer's disease.Nanomolar affinity at H 3 R of ADS031 and micromolar inhibition of both cholinesterases could be more helpful, as BuChE takes over the task if AChE is inhibited.

Molecular Docking Studies.
The obtained compounds were docked to the recently published crystal structure of H 3 R to determine their binding modes. 58The induced fit docking procedure revealed similar arrangements in the receptor binding pocket for all derivatives, although they sometimes differed from one another in observed interactions.They were stretched from an orthosteric binding site where the crucial interaction was found with Asp114 from transmembrane domain TM3 toward the extracellular loop ECL2 (Figure 3).Compound ADS031, which showed the highest affinity to H 3 R (K i = 12.5 nM) as well as other compounds with high pA 2 values, demonstrated significant interactions within the receptor.The aromatic benzyl or benzofuranyl moiety provided π−π stacking interactions with Trp402.The protonated nitrogen atom from the piperidine ring created a salt bridge with Asp114 and cation−π interactions with Tyr115 and Phe398.The ether oxygen atom could form a hydrogen bond with the hydroxyl group of Tyr115.The second aromatic fragment (benzene, naphthalene, and biphenyl) was engaged in π−π stacking with Phe193 and Tyr189.The terminal protonated amino group formed a salt bridge with Glu395.Contrary to this, compounds with shorter ether linkers, a single-second aromatic ring, and an aromatic nonprotonated amine group, such as ADS021 and ADS023, were characterized by reduced π−π stacking interactions and a lack of a salt bridge, which resulted in low pA 2 values (below 4).

CONCLUSIONS
Among the tested compounds, ADS031 (pA 2 = 7.54) and ADS024 (pA 2 = 7.57) demonstrated the highest ex vivo potency as antagonist/inverse agonist at the gpH 3 R. Comparing the obtained pA 2 values presented in Table 1, it was found that the ex vivo potency increases when the N-methyl-N-propylaminomethyl substituent is present in the para position of the aromatic moiety.In the radioligand displacement binding assay studies at the hH 3 R, compound ADS031 showed the highest affinity (K i = 12.5 nM), while the benzofuranyl derivative ADS032 showed a lower nanomolar affinity (K i = 44.1 nM).The lead compound ADS031 was classified as a potent H 3 R antagonist/inverse agonist in the cAMP accumulation assay.Molecular docking studies in the hH 3 R binding pocket indicate that the lead compound ADS031 creates significant interactions, including π−π stacking interactions between Trp402 for the benzyl moiety, a salt bridge with Asp114, and cation−π interactions with Tyr115 and Phe398 and the protonated nitrogen from the piperidine ring: it also forms a hydrogen bond between the Tyr115 and ether oxygen atoms, and π−π stacking between Phe193 and Tyr189 for the naphthalene fragment.
The inhibitory activity of the novel compounds their second target was assessed by an AChE/BuChE spectrophotometric assay.All compounds were tested at a concentration of 10 μM and four of them: ADS022, ADS025, ADS029, and ADS031 demonstrated over 60% inhibition of eeAChE and were hence tested for their IC 50 value.Compound ADS031 showed the highest inhibitory activity (IC 50 = 1.537 μM).
In contrast, eight compounds were qualified for IC 50 testing against eqBuChE.The lowest IC 50 value was noticed for ADS021 (IC 50 = 0.559 μM), one of the least active compounds against gpH 3 R, while a lower value was demonstrated by ADS031.The most potent compound in the study toward hH 3 R (IC 50 = 1.353 μM).
Therapy incorporating H 3 R antagonists with additional AChE/BuChE inhibitory effects may improve cognitive functions in Alzheimer's disease.two or more pharmacophore motifs in a single molecule is one of the strategies for designing multiple targeting ligands.One of the most popular motifs of AChE inhibitors is tacrine.Analogues of tacrine easily lead to potent AChE inhibitors; however, tarcrine has poor therapeutic efficacy and a high prevalence of detrimental effects.Although our lead compounds do not appear to be more active than the known dual-targeting ligands shown in Figure 1, they represent a good basis for structure optimization and promising pharmacological tools for further biological evaluation.Therefore, the assessment of their pharmacokinetic properties will be the subject of our further studies.
Nuclear magnetic resonance spectra, 1 H NMR and 13 C NMR, were recorded on a Bruker Avance III 600 MHz spectrometer (Bruker, Billerica, MA, USA) in CDCl 3 and CD 3 OD.The 1 H NMR spectra were run at 600 MHz, and 13 C NMR spectra were run at at 150.95 MHz.The chemical shifts are reported in ppm on scale downfield from tetramethylsilane (TMS) as the internal standard, and the signal patterns are indicated as follows: s = singlet, d = doublet, t = triplet, m = multiplet, and br = broad; the number of protons, and J approximate coupling constant in Hertz.Elemental analyses (C, H, and N) for all compounds were measured in a PerkinElmer Series II CHNS/O Analyzer 2400 (PerkinElmer, Waltham, MA, USA); the results agreed with the theoretical values within ±0.4%.TLC data were obtained with Merck silica gel 60F254 aluminum sheets.Flash column chromatog-raphy using silica gel, 60 Å, 50 μm (J.T. Baker B. V.) used the same solvent system as for TLC.All obtained final free bases were treated with a methanolic solution of oxalic acid or fumaric acid, and the salts were precipitated with dry diethyl ether and crystallized twice from ethanol.All oxalic (ADS021, ADS022, ADS023, ADS024, ADS025, ADS026, ADS027, ADS028, ADS029, ADS031, and ADS032) and fumaric (ADS030) acid salts were obtained as white crystalline solids.ADS021, ADS022, ADS023, ADS024, ADS025,  ADS026, ADS027, ADS028, and ADS030.LiAlH 4 (2 equiv) was slowly added to a solution of the corresponding amide (1 equiv) (5.1, 5.2, 12a.1, 12b.1, 12c.1, 12a.2, 12b.2, 12c.2, and 16) in 50 mL of anhydrous diethyl ether.The reaction mixture was stirred at 36 °C for 3 h.After completing the reaction, the mixture was quenched by the dropwise addition of water (8 equiv) and 10% NaOH solution (8 equiv), stirred for 2 h, and then filtered by Celite.The precipitate was discarded.The solvent was removed under vacuum, and the crude product was purified by column chromatography to yield the pure product.

N-((6-(((
4.2.Biological Activity.The compounds were tested against H 3 R by an ex vivo assay using isolated guinea pig ileum electrically stimulated to contraction, according to Vollinga et al. 47 The pA 2 -values were calculated according to Arunlakshana and Schild. 59The radioligand-displacement assay was performed in membrane fractions of HEK-293 cells stably expressing hH 3 R. Cell cultivation and membrane preparation were performed according to Kottke et al. 48he K i values were calculated from the IC 50 values using the Cheng− Prusoff equation. 60The statistical calculations were performed on −log(K i ).The mean values and 95% confidence intervals were transformed to nanomolar concentrations.The inhibitory activities toward AChE and BuChE of novel ADS compounds were assessed in a spectrophotometric Ellman's assay using electric eel AChE (eeAChE) and equine BuChE (eqBuChE). 57Intracellular cAMP accumulation was measured with a homogeneous TR-FRET immunoassay using the LANCE Ultra cAMP kit (PerkinElmer) and HEK293 cells stably expressing the human histamine H 3 receptor. 61A more detailed description of the biological methods is presented in the Supporting Information.
4.3.Molecular Docking Studies.Ligand structures were prepared with LigPrep (Schrodinger Suite) from SMILES strings.Protonation states were predicted with Epik at pH 7.4 ± 0.2.The recently obtained crystal structure of H 3 R was used for the studies (PDB code: 7F61). 58he binding modes of the analyzed compounds were predicted by the Induced Fit Docking procedure (Schrodinger Suite) based on the standard protocol and the OPLS_2005 force field.The binding site was defined as a box with the center at Asp3.32 and size adjusted to allow docking of ligands with length ≤25 Å.The obtained ligand poses were assessed by IFDScore and GlideScore.The interaction networks were visualized with Maestro (Schrodinger Suite) and PyMOL 0.99rc6 (DeLano Scientific LLC).
Chemical synthesis and NMR spectra; description of the biological methods, including an ex vivo assay for histamine H 3 R receptor antagonists on guinea pig ileum, an hH 3 R radioligand displacement binding assay, inhibition of electric eel AChE and equine serum BuChE, and H 3 R intrinsic activity, and physiochemical parameters determined at the SwissADME web tool (PDF) ■ AUTHOR INFORMATION Corresponding Authors

Figure 1 .
Figure 1.Representative structures of nonimidazole H 3 R antagonists with an AChE/BuChE inhibitory effect.(a) Data from ref 37. (b) Data from ref 38.(c) Data from ref 39.

Figure 2 .
Figure 2. Target molecules for this study.

2 . 3 . 3 . H 3 R
Intrinsic Activity.For the lead compound ADS031, intrinsic activity at H 3 R was tested in HEK293 cells expressing recombinant human receptors using a cAMP accumulation assay.The tested compound was able to counteract (R)-(−)-α-methylhistamine-driven inhibition of cAMP production in forskolin-stimulated cells, which was indicative of its H 3 R antagonist/inverse agonist properties.The IC 50 value obtained in the H 3 R functional assay was 41.7 ± 8.5 [nM] (K b = 0.75 ± 0.15 [nM]).

Figure 3 .
Figure 3. Predicted binding mode for the compound ADS031 with the highest affinity to the histamine H 3 receptor (general view�left panel, 2D interaction diagram�right panel).

Table 1 .
Results of ex vivo gpH 3 R Screening on the Isolated Guinea Pig Ileum; hH 3 R Radioligand Displacement Assays; Inhibition of electric eel AChE and equine serum BuChE a pA 2 , K i , eeAChE%inh.and eqBuChE%inh.are means from at least three independent experiments.b IC 50 inhibitory concentration of electric eel AChE, mean value ± sem of triplicate independent experiments.c IC 50 inhibitory concentration of BuChE from equine serum, mean value ± sem of triplicate independent experiments.
sem: standard error of the mean, h: human; gp: guinea pig.