Rational Design, Synthesis and Pharmacological Evaluation of Chalcones as Dual-Acting Compounds—Histamine H₃ Receptor Ligands and MAO-B Inhibitors

Abstract

Chalcone-based derivatives were designed as dual-acting ligands targeting the histamine H₃ receptor (H₃R) and monoamine oxidase B (MAO-B), based on the lead compound DL76. Three series of compounds (1–18) were synthesised and characterised, including simple chalcones (1–9) and piperidinyl chalcones (10–18). All piperidinyl derivatives exhibited nanomolar affinity for human H₃R (hH₃R), with compounds 10–12 achieving K_i values ≤ 30 nM. Simple chalcones showed potent human MAO-B (hMAO-B) inhibition (IC₅₀: 0.85–337 nM), especially 3,4-dichloro derivatives. Compound 15 was the most active hybrid, with a K_i of 46.8 nM for hH₃R and an IC₅₀ of 212.5 nM for hMAO-B. Molecular docking and 250 ns simulations revealed stabilising interactions at both binding sites and clarified structural features behind dual activity. Preliminary ADMET profiling showed low Caco-2 permeability and rapid microsomal metabolism, mainly via hydroxylation. Compound 15 exhibited micromolar cytotoxicity in SH-SY5Y and HepG2 cells, induced G2/M arrest, disrupted mitochondrial homeostasis, and was genotoxic in Peripheral Blood Mononuclear Cells (PBMCs). Additionally, for H₃R ligands (15, DL76, pitolisant), the study reports the first use of Surface Plasmon Resonance Microscopy (SPRM) to assess their interactions with this receptor. Therefore, piperidinyl chalcones show promise as ligands with dual action on H₃R and MAO-B, useful in the treatment of neurodegeneration and/or CNS cancers.

1. Introduction

The histamine H₃ receptor (H₃R) belongs to the family of G protein-coupled receptors and, since its discovery in the 1980s, has continued to attract the interest of scientists [1,2]. This discovery changed the perception of histamine itself. Until then, it was considered only as a factor causing allergic and inflammatory effects, as well as influencing certain physiological functions, such as gastric juice secretion. This effect was associated with histamine-mediated activation of histamine H₁ and/or H₂ receptors [3]. The discovery revealed that histamine can also act as a neurotransmitter, and subsequent efforts to clone this receptor led to the identification of a new member of the histamine receptor family—the histamine H₄ receptor [4]. The H₃R is widely distributed throughout the brain, particularly in regions associated with memory and cognition. Its blockade increases the release of histamine and other neurotransmitters, such as acetylcholine, dopamine (DA), noradrenaline or serotonin [2]. The H₃R was cloned in 1999 by Lovenberg et al. [5]. Further research has shown that it can exist in 20 isoforms, which differ in the number of amino acids; of these, only 7 can perform pharmacological functions due to the presence of critical areas responsible for agonist binding and/or signalling [6]. In the 40 years since the discovery of this receptor, many of its ligands have been synthesised [7,8,9]. Research has primarily focused on the search for ligands that inhibit this receptor’s activity, i.e., antagonists and inverse agonists (as the H₃R exhibits high constitutive activity [10,11]). The obtained pharmacological studies suggested the utility of H₃R antagonists/inverse agonists in the treatment of various human disorders, e.g., Alzheimer’s Disease, ADHD, Parkinson’s Disease (PD), schizophrenia, narcolepsy or allergy [7,8,9]. So far, only one H₃R ligand, pitolisant, has entered the market as Wakix, a drug for the treatment of narcolepsy (EMA-2016, FDA-2019) [12] and as Ozawade (EMA-2021) for excessive daytime sleepiness associated with obstructive sleep apnea [13].

In recent years, H₃R antagonists/inverse agonists have been synthesised as multi-target, mostly dual-target ligands (DTLs) [14,15]. Compounds were designed as structures showing antagonism/inverse agonism at the H₃R while simultaneously affecting other biological targets.

Among these biological targets, monoamine oxidase B (MAO-B) enzyme is important due to its crucial role in the pathogenesis of PD. MAO-B belongs to the family of MAOs that catalyse the deamination of neurotransmitters (e.g., DA) and release reactive oxygen species as by-products. MAO-B dominates in the human brain (glia cells) and deaminates β-phenylethylamine (PEA). PEA increases the synaptic levels of DA and blocks its reuptake into neurons. MAO-B activity increases with age and is elevated in certain diseases, such as PD. Inhibitors of MAO-B stop the activity of this enzyme and block the breakdown of DA. Moreover, they exhibit neuroprotective effects and reduce oxidative stress [16]. Three of them are currently used in medicine: selegiline, rasagiline and safinamide (Figure 1) [17]. The first two are irreversible MAO-B inhibitors that form covalent bonds with the flavin cofactor of MAO-B. Safinamide is a reversible inhibitor, mainly forming hydrogen bonds with preserved water molecules and protein residues in the active site of the MAO-B enzyme [18]. Reversible inhibition seems to be more advantageous for the drug’s safety profile because de novo protein synthesis is not required to restore enzymatic activity, thereby minimising toxic side effects.

So far, only a limited number of compounds have been reported that simultaneously exhibit affinity for H₃R and inhibitory activity for MAO-B. Among them are indanone derivatives. The most potent compounds, A and B, are shown in Figure 1 [19,20]. They were characterised by very high affinity for human H₃R (hH₃R) and moderate inhibition of human MAO-B (hMAO-B). On the other hand, we obtained tert-butyl and tert-pentyl derivatives, with comparable activity for both biological targets, such as DL76 (Figure 1), or higher inhibitory activity for hMAO-B, e.g., compounds C and D (Figure 1) [21,22,23].

Continuing our work in this field, we chose compound DL76 as the lead structure for further modifications. Analysis of the literature directed our focus toward chalcones, which exhibit broad biological activity, acting on various biological targets, that potentially might be used in the treatment of PD. They have been reported to show MAO-B inhibition, adenosine receptor antagonism (A_2A and A₁), catechol-O-methyltransferase inhibition, and Nrf2 activation [24]. Various structurally diverse chalcones have been described in the literature as MAO-B inhibitors, exhibiting different strengths of inhibition towards this enzyme [24,25]. However, for this study, we were interested in structurally simple chalcones, including compounds 1 and 2 (Figure 1) [26,27]. These compounds were characterised by good hMAO-B inhibitory activity with an IC₅₀ below 200 nM. Moreover, we observed that the introduction of a chlorine atom into the molecule (position 4; compound 2) resulted in a 6-fold increase in hMAO-B inhibition capacity (compound 1). Thus, compound 2 was selected as the lead structure, and we decided to merge its core with our promising ligand DL76. Three series of compounds were designed based on these structures and the safinamide motif as described in Section 2.1. Compounds were synthesised and tested for both the hH₃R and hMAO-B. Based on the results obtained, the most active DTL was selected for further preliminary ADMET studies. Furthermore, molecular docking to H₃R and MAO-B was performed to characterise the possible interactions between these targets and the selected ligands.

Figure 1. Structures of market MAO-B drugs (top line), dual target ligands, i.e., histamine H₃ receptor ligands and MAO-B inhibitors (middle lines), and chalcones (bottom line). ^a data from Ref. [19]; ^b data from Ref. [20]; ^c data from Ref. [21]; ^d data from Ref. [23]; ^e data from Ref. [26]; ^f data from Ref. [27].

Figure 1. Structures of market MAO-B drugs (top line), dual target ligands, i.e., histamine H₃ receptor ligands and MAO-B inhibitors (middle lines), and chalcones (bottom line). ^a data from Ref. [19]; ^b data from Ref. [20]; ^c data from Ref. [21]; ^d data from Ref. [23]; ^e data from Ref. [26]; ^f data from Ref. [27].

2. Results and Discussion

2.1. Design of Compounds

As a result of our search for DTLs, we merged a motif characteristic of H₃R, i.e., piperidinylpropoxy, with a chalcone moiety as shown in Scheme 1 and designed a series 1 (Scheme 2). Then, we decided to add a fragment derived from safinamide, and depending on the attachment, two series were obtained: series 2 and series 3 (Scheme 2). Additionally, to compare the effect of a fragment characteristic of H₃R on MAO-B inhibition, corresponding simple chalcones (compounds 1–9) were obtained for each series. This also included the resynthesis of compounds 1 and 2 for better comparison with the compounds studied in our laboratory.

2.2. Synthesis of Compounds

Compounds were synthesised as shown in Scheme 3 and Scheme 4. Chalcones 2–18 were obtained according to Claisen-Schmidt condensation from proper aldehydes I–V and 4-hydroxyacetophenone (chalcones 2–9) or acyl derivative of piperidine VII (chalcones 10–18). Non-commercially available aldehydes were prepared by O-alkylation of 3- (IIa) or 4-hydroxybenzaldehyde (IIb) with proper benzyl chlorides (IIIa, IIIb) in basic conditions. Compounds VI and VII were synthesised as described previously by Honkisz-Orzechowska et al. [28] or by slight modification. Final compounds (2–18) were purified by crystallisation from ethanol or by flash chromatography. All chalcones (except 11) during synthesis were converted into hydrogen chloride. The final products were obtained with yields ranging from 6 to 32%. Differences in yield may be due to inadequately optimised reaction conditions that limited the progress of aldol condensation, such as insufficient reaction time or conducting the reaction at room temperature. Moreover, the purification step, particularly crystallisation, likely had a significant impact on the final yield due to the relatively high solubility of the compounds in ethanol.

The purity and identity of the compounds were confirmed by NMR spectroscopy (¹H and ¹³C) and mass spectrometry. ¹H NMR data proved that all chalcones are E isomers, which are more thermodynamically stable than the Z isomers. An α,β-unsaturated bond appeared as two doublets at about 7.6 and 7.9 ppm with coupling constant values of 15–16 Hz.

2.3. Preliminary In Vitro Pharmacological Studies of Compounds

2.3.1. Human Histamine H₃ Receptor Affinity

The affinity to hH₃R was evaluated only for compounds 10–18 as compounds 1–9 lack the essential structural elements required for interaction with H₃R, i.e. a moiety of basic character [15]. Compounds 10–18 were tested in a radioligand binding assay as described previously [28]. Results are shown in

Table 1. Structures and pharmacological results for evaluated chalcones 1–18 and reference compounds.

Comp.	X	hMAO-B a IC50 [nM]		Comp.	X	hH3R b Ki ± SEM [nM]	hMAO-B a IC50 ± SEM [nM] (% Inhibition) c	hMAO-A a (% Inhibition) d
1	H	96.0 ± 2.4 182 ± 5 f		10	H	17.0 ± 2.2	(18%)	nt e
2	4-Cl	2.67 ± 0.35 31 ± 1 g		11	4-Cl	17.0 ± 0.5	(36%)	nt e
3	3,4-diCl	0.85 ± 0.02		12	3,4-diCl	30.0 ± 0.9	500.0 ± 13.0	(3%)
4	H	85.2 ± 2.4		13	H	80.6 ± 9.0	(24%)	nt e
5	4-Cl	33.4 ± 1.7		14	4-Cl	54.0 ± 6.0	1121.4 ± 198.7	(8%)
6	3,4-diCl	20.6 ± 1.3		15	3,4-diCl	46.8 ± 1.9	212.5 ± 12.3	(22%)
7	H	287.5 ± 13.5		16	H	195.0 ± 13.0	(33%)	nt e
8	4-Cl	337.5 ± 17.1		17	4-Cl	89.0 ± 4.0	1114.3 ± 317.0	(0%)
9	3,4-diCl	70.3 ± 4.5		18	3,4-diCl	163.0 ± 12.0	355.9 ± 19.9	(13%)
safinamide		8 ± 1
rasagiline		25 ± 6

^a Fluorometric MAO-Assay; mean value ± SEM of 2–4 independent experiments. ^b [³H] N^α-Methylhistamine binding assay in CHO-K1 cells stably expressing the hH₃R; mean value of 2 independent experiments ± SEM. ^c %Inhibition of hMAO-B at 1 μM; mean values of 2 independent experiments. ^d %Inhibition of hMAO-A at 10 μM; mean values of 2 independent experiments. ^e Not tested. ^f Data from Ref. [26]. ^g Data from Ref. [27].

. All compounds had hH₃R affinity with K_i values below 170 nM. The best affinity had compounds 10–12 (series 1) with K_i ≤ 30 nM, whereas the weakest had compounds 16–18 (89 nM ≤ K_i ≤ 195 nM) (series 3). There is no correlation between the introduction of chlorine/chlorines into the phenyl ring and the hH₃R affinity. The highest hH₃R affinity was shown by compounds 10 and 11 with a K_i of 17 nM. In the safinamide derivatives (13–18), the compounds had lower affinity for hH₃R (K_i > 45 nM) compared with compounds 10–13. There is a clear correlation between the attachment of the benzyloxy substituent, meta (12–15) or para (16–18), and hH₃R affinity. Compounds with meta substitution (12–15) showed higher affinity than the corresponding analogues with para attachment (16–18). In this group, no visible relationship can be observed between the increase in affinity and the introduction of a chlorine atom/chlorine atoms into the benzyloxy substituent. Compound 15 had the highest affinity for hH₃R, with a K_i value of 46.8 nM.

2.3.2. Functional Characterisation in cAMP Accumulation Assay of Selected Compounds

The two most promising compounds from the tested series, i.e., compounds 12 and 15, were selected for a test to confirm their activity profile toward the H₃R. The intrinsic activity was evaluated in a 3′,5′-cyclic adenosine monophosphate (cAMP) accumulation assay in CHO-K1 cells stably transfected with the H₃R. Compounds were tested both in agonist and antagonist modes. Neither compound showed activity in agonist mode (E_max: 0% and 3%, respectively), whereas both tested in antagonist mode inhibited the activity of R-α-methyl histamine (H₃R agonist). This effect, in a concentration-dependent manner, led to an increase in cAMP levels in the cells. The equilibrium dissociation constant (K_b ± SD) of compound 12 was 3.94 ± 0.85 nM, whereas for compound 15, K_b was 34.20 ± 2.78 nM (Supplementary data).

2.3.3. Surface Plasmon Resonance Microscopy Kinetic Studies of Selected Compounds

Kinetic characteristics can act as key factors distinguishing and predicting a drug’s effectiveness and safety. Traditionally, drug candidates were chosen for their strong ligand–protein binding affinity, but high affinity alone does not ensure clinical success. Research indicates that drug efficacy often correlates more closely with the dissociation rate than with equilibrium binding. Binding kinetics—how fast a drug binds and how long it stays bound—are now seen as equally important. The kinetics of binding can be investigated using the SPRM (Surface Plasmon Resonance Microscopy) method [29]. SPRM combines high-resolution optical microscopy with Surface Plasmon Resonance (SPR) technology [30]. It can directly measure both equilibrium binding affinity and kinetics of ligand–membrane protein interactions, such as ligand–GPCR binding. Additionally, SPRM is a label-free method, eliminating the need for radioactive tracers in experimental assays. Here, we report the first use of SPRM technology to examine H₃R ligands, highlighting a novel approach in this field. Previously described studies have allowed the determination of binding kinetics parameters using fluorescence-based methods, such as calcium mobilisation [31], fluorescence polarisation [32] and NanoBRET assays [33]. For our studies, compound 15 was selected as the dual active ligand, while pitolisant (an approved H₃R antagonist/inverse agonist) and DL76 (our dual ligand) were included as reference compounds. Experiments were conducted on CHO-K1 cells overexpressing hH₃R. Stock solutions (10 mM) of the tested compounds were prepared in DMSO, with final assay concentrations ranging from 0.5 nM to 10 µM. Despite repeated trials, no kinetic binding data were obtained for compound 15. Analysis of the experimental workflow suggested that the lack of measurable response may be connected to the binding of compound 15 to bovine serum albumin (BSA), which was an essential component of the experimental conditions. Our assumptions were confirmed by LC-MS spectrum analysis and literature reports. Singh et al. [34] studied interactions of (E)-1-(2,4-dichlorophenyl)-3-(2-hydroxy-3-methoxyphenyl)prop-2-en-1-one with BSA. The study showed that this chalcone can bind to BSA without disturbing its natural structure or function. The interaction was found to be stable, as confirmed by NMR, high-resolution mass spectrometry, Fourier-transform infrared spectroscopy, UV spectroscopy, single-crystal X-ray diffraction, and computer analyses. For pitolisant and DL76, kinetic and equilibrium binding parameters were successfully determined (

Table 2. Affinity and kinetic values of DL-76 and pitolisant binding to the hH₃R ¹.

Compound	Affinity	Kinetics
	KD a ± SEM [nM]	KD b ± SEM [nM]	ka c ± SEM [×104 M−1 s −1]	kd d ± SEM [×10−3 s −1]	RT e ± SEM [min]
DL76	39.2 ± 13.6	43.6 ± 12.6	2.7 ± 0.6	1.2 ± 0.3	16.0 ± 3.6
Pitolisant	15.0 ± 4.3	15.3 ± 4.7	7.9 ± 2.3	1.2 ± 0.2	15.8 ± 3.0

¹ Mean values of 4–5 independence experiments. ^a Equilibrium constant from equilibrium binding experiment; ^b K_D = k_d/k_a; ^c association rate constant; ^d dissociation rate constant; ^e residence time (RT = 1/k_a).

). Histograms of the Gaussian distributions (red curves) for the respective kinetic parameters (k_a, k_d, K_D) of the tested compounds are shown in Figure 2. The equilibrium constant (K_D) values obtained from the ligand binding assay and SPRM kinetics tests are comparable. For DL76, they are as follows: 39.2 ± 13.6 nM vs. 43.6 ± 12.6 nM, whereas for pitolisant: 15.0 ± 4.3 nM vs. 15.3 ± 4.7 nM, respectively. The residence time (RT), which reflects the duration for which a compound remains bound to its target, is comparable for DL76 and pitolisant, with values of 16 min and 15.8 min, respectively. These results indicate comparable average interaction times of the compounds with the H₃R.

In the comparative kinetics study of non-equilibrium Ca²⁺ mobilisation assays by Riddy et al. [31], pitolisant was classified among H₃R antagonists as a “rapidly dissociating” ligand, with dissociation half-lives markedly shorter than those of the slowly dissociating class of compounds. Our SPRM measurements, however, yielded a dissociation rate constant k_off of 1.2 × 10⁻³ s⁻¹, corresponding to a RT of ≈15.8 min, which is significantly longer than the values commonly attributed to the “fast-off” group. Our results are consistent with those reported by Mocking et al. [33], who reported an RT of approximately 17 min. The extended RT observed under various experimental conditions suggests that system-specific factors (e.g., surface immobilisation, low flow rates, receptor construct, rebinding) may affect dissociation relative to other assay formats. This discrepancy highlights the importance of assay context when comparing residence times across studies.

2.3.4. Human MAO-B Inhibitory Activity

Pharmacological activity towards human MAO-B (hMAO-B) was evaluated in a spectrometric assay using Amplex Red, as previously described [21]. First, all compounds were tested at a concentration of 1 μM to assess their ability to inhibit MAO-B activity. Compounds that demonstrated more than 50% inhibition in two independent assays were further tested at different concentrations to determine IC₅₀ values. Results are presented in Table 1 and show that simple chalcones 2–9 have hMAO-B inhibitory activity with IC₅₀ below 340 nM. The most potent compounds had a 3,4-dichloro substituent in the phenyl ring, i.e., compounds 3, 6 and 9. The IC₅₀ value of compound 3 was even below 1 nM (IC₅₀ = 0.85 nM). Inhibitory activity of compounds 1 and 2 evaluated in our laboratory was higher than previously described by others (e.g., for 2: data from [27]—IC₅₀ = 31 nM vs. IC₅₀ = 2.67 nM). Therefore, we repeated the experiments that confirmed our results. In the piperidinyl series (10–18), compounds showed weaker activity than the corresponding chalcones 2–9. In this group, the inhibitory activity was higher in compounds with a safinamide motif (13–18). The same tendency was observed in all tested compounds (1–18), with the inhibitory activity increasing in the following order: unsubstituted ring < monochlorine substituted < dichlorine substituted. Among derivatives with the piperidinyl moiety (10–18), the most potent were compounds with a meta substitution of a benzyloxy moiety (13–15). In this group, the compound 15 exhibited the highest inhibitory activity, with an IC₅₀ of 212.5 nM; however, this activity was more than ten times weaker than that of simple chalcone 6 (IC₅₀ = 22.4 nM).

2.3.5. Reversibility of Human MAO-B

Reversibility tests were conducted to confirm the binding mode with the enzyme. The most active dual ligand, compound 15, was selected for this test. The test was performed using the rapid dilution method described by Copeland [35]. Safinamide (a reversible inhibitor) and rasagiline (an irreversible inhibitor) were used as reference substances. The enzyme was incubated with the tested inhibitors at a concentration corresponding to 10-fold the IC₅₀ value for 30 min, then the samples were diluted using a high concentration of substrate (p-tyramine) in assay buffer. To observe restoration of MAO-B activity after the dilution fluorescence measurements were taken every 5 min for 1 h. The results are shown in Figure 3. Compound 15 showed reversible inhibition, i.e., the activity of the enzyme after the dilution in excess of the enzyme’s substrate was similar to that of the non-inhibited enzyme. The curves indirectly represent the amount of product produced by MAO-B after dilution and show the enzyme’s ability to restore its activity after inhibition. Curves of compound 15 and safinamide are comparable. The activity of hMAO-B was restored after dilution of safinamide and compound 15, whereas rasagiline (an irreversible inhibitor), following the dilution of the inhibitor, was still able to inhibit most of the enzyme molecules, leading to a small amount of product released.

2.3.6. Modality of Reversible Inhibition of Human MAO-B

Compound 15 was chosen to investigate its mode of inhibition. Three concentrations of the inhibitor (corresponding to its IC₂₀, IC₅₀, and IC₈₀ values) were tested in conjunction with five substrate concentrations. As a substrate, p-tyramine was used at concentrations of 0.05, 0.1, 0.5, 1.0, 1.5 and 2.0 mM. Michaelis–Menten curves were fitted to the experimental data to obtain K_M and V_max values (

Table 3. Influence of different inhibition modalities on steady-state kinetic parameters and characteristic Lineweaver–Burk plot patterns of safinamide and compound 15.

Parameter	Safinamide	Compound 15
Affinity	Free enzyme > enzyme–substrate complex	Only free enzyme or free enzyme > enzyme–substrate complex
app. KM	↑ curvilinearly with ↑ [I]	↑ curvilinearly with ↑ [I]
app. Vmax	↓ curvilinearly with ↑ [I]	↓ curvilinearly with ↑ [I]
app. Vmax/app. KM	↓ curvilinearly with ↑ [I]	↓ curvilinearly with ↑ [I]
Lines on LB plot	Lines intersect to the left of the y-axis and above the x-axis	Lines intersect to the left of the y-axis and above the x-axis (almost on y-axis)
Mode of inhibition from kinetic values and LB plot	Mixed mode	Competitive/Mixed mode

[I]—concentration of the inhibitor, K_M—Michaelis constant, V_max—maximum velocity obtained at infinite substrate concentration, LB—Lineweaver-Burk double-reciprocal plot. ↑—increase, ↓—decrease.

). The data were then transformed into a double-reciprocal (Lineweaver–Burk) plot to illustrate the mode of inhibition more clearly (Figure 4).

The inhibition modality was proposed based on the analysis of changes in K_M and V_max, as well as on fitting the experimental data to multiple inhibition models. The inhibitor represents either competitive or a variant of mixed inhibition, very close to competitive. A competitive inhibitor binds exclusively to the free enzyme, preventing substrate binding. Increasing the substrate concentration decreases inhibition due to the competitive nature of the interaction. The mixed mode of inhibition is a broader term that includes cases where the inhibitor binds to both the free enzyme and the enzyme–substrate complex. In the case of compound 15, the mixed mode would be very close to competitive inhibition (sometimes referred to by some authors as mixed-competitive), meaning that the inhibitor shows affinity for both the free enzyme and the enzyme–substrate complex, but with a higher affinity for the free enzyme.

2.3.7. Human MAO-A Inhibitory Activity

Piperidinyl chalcones (compounds 12, 14, 15, 17 and 18) with inhibitory activity for hMAO-B were further tested to evaluate their selectivity activity for human MAO-A (hMAO-A). MAO-A and MAO-B are isoenzymes that differ in location, substrate preference and significance in the therapy of different diseases. Selective isoforms are considered safer for treatment [36]. Compounds were screened at a concentration of 10 μM in two independent experiments. The results (mean values) are shown in Table 1. None of them showed inhibition of hMAO-A higher than 25%, and they were not further tested.

2.4. In Silico Docking Studies

2.4.1. Docking Studies to Histamine H₃ Receptor

Published in 2022, the H₃R structure in complex with a H₃R antagonist (PF-03654746; PDB ID: 7F61 [37]), enabled validation of previously proposed ligand-receptor interactions and offered deeper insights into the receptor’s functional mechanisms. Based on these findings, compounds 11, 15 and 18 were selected for molecular docking studies to investigate how their structural differences might relate to the observed biological activity. The compounds were characterised by favourable MMGB–SA (Molecular Mechanics–Generalized Born Surface Area) binding free energies (ΔG_bind = −95.51, −99.69, and −78.51 kcal/mol, respectively) and moderate yet comparable ligand efficiencies derived from docking scores (glide LE = −0.294, −0.281, and −0.274, respectively). All ligands occupied the H₃R binding pocket in a similar manner, preserving the key antagonist/inverse agonist interaction, namely salt bridge and/or hydrogen bond formation between protonated amine nitrogen and ASP114^3.32 (superscripts denote Ballesteros–Weinstein numbering, as presented in GPCRdb [38]). Additional stabilisation of the basic part of the molecules was conducted through cation–π interactions with caging Y115^3.33 and F398^7.39. On top of that, compounds 11 and 15 formed a donor interaction via Y91^2.61, indicating a robust polar interaction network as well as π–cation contacts with R27^1.39, located near the extracellular face of the receptor, suggesting a possible role in initial ligand recognition or orientation. In contrast, the slightly different calculated orientation of compound 18 in the binding pocket did not allow for explicit hydrogen bond donor or acceptor interactions, suggesting reduced stabilisation through polar contacts (Figure 5).

Aromatic and hydrophobic interactions were broadly conserved across all three ligands, primarily involving Y91^2.61, Y94^2.64, W110^3.28, Y115^3.33, Y189(EL2), F193(EL2), Y374^6.51, Y394^7.35, F398^7.39, and W402^7.43, consistent with the π-rich character of the orthosteric site. However, compound 15 itself established additional contact with Y194(EL2), expanding its aromatic interface relative to compounds 11 and 18. Notably, compound 18 did not exhibit interaction with Y94^2.64. This absence may be attributed to the para-substitution of the proximal 3,4-dichlorobenzyl moiety in compound 18, which orients the aromatic ring more distally from the core binding pocket. In comparison, the meta-substitution pattern observed in compound 15 appears to promote a more optimal spatial alignment with Y94^2.64, thereby enabling favourable π–π stacking or edge-to-face aromatic interactions. This structural difference may lead to weaker hydrophobic stabilisation in compound 18, which could partly explain its distinct interaction profile and slightly lower H₃R affinity.

The stability of the calculated poses was further evaluated using 250 ns molecular dynamics (MD) simulations. All data used for the analysis, as described in the paragraphs below (for both H₃R and MAO-B), are provided in the Supplementary data.

In the recorded trajectory of the 15-H₃R complex, the ligand maintained a generally stable binding pose, evidenced by stable interactions and favourable trends in calculated binding energy. Throughout the trajectory, D114^3.32 remained a persistent polar interaction partner, present in over 95% of simulation frames, with additional stabilisation contributed by cation–π interactions involving Y115^3.33 and F398^7.39. An initial hydrogen bond donor interaction from Y91^2.61 was also observed; however, the role of this residue shifted toward supporting the ligand’s first aromatic ring via π–π stacking and aromatic hydrogen bonding between the distal, extracellular-facing substituted aromatic ring of compound 15 and residues R27 and H187 (EL2) were transiently observed, suggesting a dynamic but non-essential contribution from these residues. At the same time, its surface exposure and flexibility may enhance binding entropy (Figure 6).

The binding free energy (expressed by ΔG Bind values) remained consistently favourable across trajectory snapshots, averaging approximately −155.5 kcal/mol during equilibrated frames, with only minor variation observed in early frames before system relaxation. Detailed Prime energy values for each simulated complex, including the corresponding MM–GBSA ligand efficiencies, are available in the Supplementary data. Hydrophobic interactions constituted the primary energetic contribution, complemented by favourable hydrogen bonding and packing interactions, which together support the thermodynamic feasibility of high-affinity binding under membrane-mimicking conditions. Root Mean Square Deviation (RMSD) analysis revealed moderate ligand fluctuation relative to the starting pose, followed by convergence, indicating no significant displacement from the orthosteric pocket. Residues forming the aromatic cage—Y94^2.64, W110^3.28, Y115^3.33, and Y374^6.51—maintained stable contact distances throughout the simulation, underscoring the role of π–π and hydrophobic interactions in anchoring the ligand. Across the three simulated ligand–receptor complexes, several shared and divergent interaction patterns were observed, highlighting the structural determinants of ligand stability and binding efficiency in the H₃R orthosteric pocket. All three ligands maintained stable polar anchoring via D114^3.32 and hydrophobic interactions with conserved aromatic residues. However, differences emerged in how each compound engaged the extracellular vestibule through its substituted aromatic extensions. Similar to compound 15, compound 11 occupied a well-defined cleft between TM2 and TM7, forming stable π–π interactions with Y91^2.61 and Y94^2.64, as well as with F398^7.39 and W402^7.43. This conformation contributed to consistently calculated binding-free energy profiles and low RMSD fluctuations, indicating a high degree of structural and energetic stability. In contrast, in compound 18 the para-substituted 3,4-dichlorobenzyl group of compound 18, promoted the position of the distal ring farther from TM2, limiting interactions with Y94^2.64 and redirecting engagement toward TM7, where the contacts were observed to be more transient. This repositioning corresponded with increased RMSD variability and more fluctuant ΔG Bind values across frames, indicating a less constrained binding mode. The highest-energy state corresponds to reduced π-stacking and the loss of polar contacts. RMSD analysis revealed greater structural deviation and delayed convergence compared to compounds 11 and 15, consistent with a more flexible or less tightly bound ligand conformation. Thus, while all three ligands exploit a shared pharmacophoric core, the geometry and dynamics of the distal fragment strongly influence binding stability and entropic behaviour, and might contribute to differences in receptor interactions.

Molecular dynamics simulations of compounds 11, 15, and 18 were evaluated relative to the reference histamine H₃ receptor antagonist PF03654746 co-resolved within the H₃R complex [37]. The analysis of the native ligand simulation revealed a rigid, thermodynamically favourable ligand pose anchored by a stable ionic interaction with D114^3.32, π–cation engagement with Y115^3.33 and C118^3.36, and aromatic stacking with Y91^2.61 and F398^7.39. RMSD remained at low values, and the ligand maintained minimal internal strain across the trajectory, confirming its suitability as a reference conformation for antagonist binding. All three ligands in question retained the key ionic anchor with D114^3.32 and engaged conserved aromatic cage residues (Y91^2.61_, Y115^3.33, F398^7.39) through π–π or cation–π interactions, sharing the core pharmacophoric anchor with the PF03654746 ligand, but differed in distal orientation, receptor engagement patterns, and dynamic stability. Compound 15 most closely reproduced the reference pose, exhibiting stable binding energy and extended polar interactions at the extracellular face. Compound 11 showed a similarly stable, though more compact, pose with high ligand efficiency. In contrast, compound 18, due to its para-substituted dichlorobenzyl group, deviated from the optimal alignment, losing interaction with Y94^2.64 and displaying increased RMSD fluctuations and less stable binding, consistent with a more flexible and less tightly bound profile.

Last but not least, H₃R inactive state interaction in the D^3.49-R^3.50-F^3.51 motif, with the salt bridge and/or H-bond formed between D131^3.49 and R132^3.50 and the postulated H₃R inactive state 3–7 lock between D114^3.32 and W402^7.43 [39], was maintained for the majority of the simulation time, which may give additional confirmation of the presumed stabilisation of the inactive state of the receptor by the tested ligands.

2.4.2. Docking Studies to Human MAO-B

An additional in silico analysis with a possible explanation for the hMAO-B inhibition of compound 15 has also been performed, using the well-described 2V5Z structure [18]. The active site comprises a characteristic aromatic cage, formed by Y398 and Y435, which stabilises ligands through π–π stacking, adjacent to a polar cavity lined with Q206 and structured water molecules that facilitate hydrogen bonding. The flavin adenine dinucleotide (FAD) cofactor, located at the bottom of the pocket, constitutes the redox centre and contributes to the pocket’s conformation and polarity. Throughout the recorded trajectory, compound 15 formed consistent interactions with key aromatic cage residues identified in the 2V5Z structure, including π–π stacking with Y398 and Y435 and hydrogen bonding to the polar cavity Q206. The distal moiety of compound 15 occupied the substrate cavity and engaged the FAD cofactor region transiently (Figure 7). Calculated binding free energy averaged approximately ΔG Bind of −232.5 kcal/mol (min −263.8, max −203.8) with low frame-to-frame fluctuation, indicative of relatively stable binding. RMSD analysis showed modest deviations, further confirming structural stability during the simulation.

For comparative purposes, compound 6, a simple chalcone precursor of compound 15, which displays a high hMAO-B inhibition with an IC₅₀ of 20.6 nM, has also been used. Compound 6 lacks the piperidinylpropoxy motif and, consequently, features an unsubstituted 4-hydroxyphenyl group, introducing the potential for additional hydrogen bonding with the polar cavity and surrounding water molecules. This modification shifts the balance between hydrophobic and polar interactions within the binding site, accounting for the observed differences in potency and dynamic behaviour between the two compounds. Compound 6 exhibited notable differences in binding behaviour within the MAO-B active site. While both ligands maintained core interactions, compound 6 displayed stronger and more persistent engagement of the distal pocket and FAD region, exhibiting more sustained π–π stacking with aromatic cage residues and water-mediated bridges.

Under the simulation conditions, both ligands stabilised quickly, with the protein RMSD levelling off at approximately ~1.5 Å and the ligand RMSD remaining low across the recorded trajectories, reflecting stable complex formation. Notably, compound 6 exhibited a lower average ligand RMSD and a narrower fluctuation range compared to compound 15 (Figure 8), suggesting tighter anchoring and reduced conformational mobility. Root-mean-square fluctuation (RMSF) analysis confirmed that both ligands maintained the rigidity of the aromatic cage, but compound 6 induced slightly lower flexibility in adjacent loop regions. Consistent with its hydroxyl-substituted aromatic ring, halogen contacts were diminished in compound 6, and the ligand torsion profiles revealed a more restricted conformation with limited rotational flexibility compared to compound 15. However, the calculated binding free energy of compound 6 was less negative than that of compound 15, averaging ΔG Bind of –187.6 kcal/mol (min = −199.9, max = −174.9). Together, these findings demonstrate that the structural specificity of compound 6 enhances its complementarity and enthalpic stabilisation within the MAO-B active site, accounting for its improved binding affinity and inhibitory activity.

To understand the structural determinants of MAO-B inhibition for compounds 6 and 15, their molecular dynamics trajectories were evaluated in direct comparison with the native binding mode of the co-crystallised ligand Safinamide (SAF) of the 2V5Z structure. Both ligands preserved the core SAF pharmacophore, including π–π stacking with Y398 and Y435 and polar interactions with Q206 near the FAD cofactor. These conserved contacts were maintained throughout 250 ns molecular dynamics simulations, indicating stable anchoring of the ligands within the substrate cavity. Compound 15 mimicked the SAF pose while adding pharmacophoric bulk without compromising anchoring; however, it extended beyond the substrate cavity via its piperidinylpropoxy moiety, maintaining favourable binding energy and interaction coverage. This extension introduced moderate conformational flexibility, reflected in slightly elevated ligand RMSD and broader torsional distributions, yet the average MM–GBSA binding energy remained strongly favourable (−232.5 kcal/mol), exceeding that of SAF. In contrast, compound 6 adopted a more compact, deeply inserted pose with improved polar complementarity and lower RMSD. Although π–π stacking with Y398 and Y435 remained consistent, the absence of halogen substitution reduced hydrophobic packing, and torsional profiles showed a more conformationally restricted ligand.

2.5. Preliminary In Vitro ADMET Studies of Selected Compounds

Two compounds, 12 and 15, the most potent dual-target ligands, were selected for preliminary ADMET (Absorption, Distribution, Metabolism, Excretion, Toxicity) studies. Both compounds were evaluated in complementary in vitro assays to characterise their permeability, microsomal stability, CYP inhibition potential, and cellular toxicity.

2.5.1. Permeability Evaluation

First, an absorption study was performed using the Parallel Artificial Membrane Permeability Assay (PAMPA), which is used to determine the ability of molecules to passively diffuse through biological membranes [40]. Both compounds precipitated under used assay conditions (100 μM solution of compounds; PBS, pH 7.4, 20% methanol, 5 h), leaving no detectable analyte in acceptor compartments as well as no (compound 15) or trace amount (compound 12) in donor ones. Membrane adsorption was also considered, preventing a reliable determination of P_e values. These results indicate poor solubility and/or instability in the PAMPA setup, suggesting limited passive diffusion potential. Therefore, the PAMPA test appeared to be insufficient for determining the permeability parameter of these compounds; thus, an assay with Caco-2 cells was used. The Caco-2 cell line, originating from human colorectal carcinoma, is widely employed as an in vitro model to predict intestinal drug absorption [41]. The results of the studies are summarised in

Table 4. Results from the Caco-2 permeability assay.

Compound	Apparent Permeability (Papp)		Efflux Ratio (ER) a
	Papp (A − B) ×10−6 cm/s ± SD	Papp (B − A) ×10−6 cm/s ± SD
12	0.75 ± 0.05	0.52 ± 0.03	0.70
15	0.14 ± 0.02	0.06 ± 0.03	0.43
Caffeine	30.16 ± 2.94	34.80 ± 10.88	1.08

^a Efflux ratio calculation: ER = P_app (B − A)/P_app (A − B). An efflux ratio less than 2 suggests passive diffusion is the primary mode of transport. An efflux ratio greater than 2 indicates active transport, likely involving efflux pumps.

and expressed as the apparent permeability coefficient (P_app), which indicates the potential of a drug candidate to be absorbed across the human intestinal epithelium, and as the efflux ratio (ER), which provides insight into the mechanisms of drug transport across biological membranes. Caffeine was used as the well-permeable reference (P_app(A→B) = 30.16 ± 2.94; P_app(B→A) = 34.80 ± 10.88 × 10⁻⁶ cm/s). Both tested compounds (12 and 15) showed very low permeability with ERs below unity, indicating no active efflux involvement. Compound 12 exhibited P_app(A→B) = 0.75 ± 0.05 and P_app(B→A) = 0.52 ± 0.03 × 10⁻⁶ cm/s (ER ≈ 0.70), while compound 15 showed P_app(A→B) = 0.14 ± 0.02 and P_app(B→A) = 0.06 ± 0.03 × 10⁻⁶ cm/s (ER ≈ 0.43).

2.5.2. Metabolic Stability

The metabolic stability studies in vitro were conducted using rat liver microsomes (RLMs) for compounds 12 and 15. Results are shown in

Table 5. In vitro metabolic stability and the most probable metabolic pathways of compounds 12 and 15 after incubation for 2 h with rat liver microsomes.

Substrate		Metabolite
Molecular Mass (m/z)	Retention Time (min)	Molecular Mass (m/z)	Retention Time (min)	Metabolic Pathway
12 (418.36)	7.25	434.14	7.27	hydroxylation
15 (524.19)	8.22	540.22	8.22	hydroxylation

. Incubation with RLMs (2 h) resulted in the appearance of new chromatographic peaks absent in controls lacking microsomes, confirming the metabolic transformation of both compounds. UPLC-MS analyses showed the formation of the main metabolite as a product of hydroxylation (m/z + 16). For both compounds, the retention times of the product and the starting substance were very similar, and in the case of compound 15, they coincided exactly. Hydroxylation most likely occurs in the piperidine ring or propyl chain. Such sites of metabolism are suggested by the MetaSite 6.0.1 in silico tool (Supplementary data).

For compound 15 in mouse liver microsomes (MLMs), pharmacokinetic parameters were determined experimentally, i.e., intrinsic clearance (Cl_int) and half-life (t_0.5). The results were compared to the unstable drug Verapamil and are summarised in

Table 6. In vitro Metabolic stability of compound 15 and Verapamil (reference unstable drug) after incubation for 2 h in mouse liver microsomes.

Compound	Clint a (mL/min/kg)	t0.5 b (min)	Metabolic Stability
15	285.75	18.99	weak/unstable
Verapamil	239.5	22.60	unstable

^a Intrinsic clearance; ^b half-life.

.

Compound 15 displayed weak metabolic stability, similar to the reference unstable drug Verapamil. The calculated parameters t_0.5 and Cl_int for compound 15 were 18.99 min and 287.75 mL/min/kg, respectively, while for Verapamil, they were 22.60 min and 239.50 mL/min/kg.

Compounds 12 and 15 were also tested for their influence on two major cytochrome (CYP) P450 isoforms (CYP3A4 and CYP2D6) at concentrations ranging from 0.1 to 25 µM. The results are shown in Figure 9. Each compound exhibited a slightly different behaviour: compound 12 caused a slight activation of CYP3A4 at concentrations of 1 µM and 10 µM, whereas compound 15 inhibited CYP3A4 activity at 10 µM. In the case of CYP2D6, both compounds inhibited enzyme activity by approximately 70% at a concentration of 10 µM. The selective reference inhibitors ketoconazole and quinidine almost completely inhibited their respective CYP isoforms at a concentration of 1 µM (Figure 9).

2.5.3. Preliminary Cell Toxicity

The toxicity of compounds 12 and 15 was evaluated in three cell lines: SH-SY5Y (human neuroblastoma cell line), HepG2 (human liver cancer cell line) and Caco2 (human colorectal cancer cell line). Both compounds exhibited hepatotoxicity and neurotoxicity. Neurotoxicity was observed at 5 µM for compound 12 and at 10 µM for compound 15 (Figure 10A). Compound 12 induced complete death of HepG2 cells at a concentration of 5 µM, whereas compound 15 caused the same effect only at 25 µM (Figure 10B). The compounds showed the lowest toxicity in the Caco-2 cell line; although some toxicity was observed at 25 µM, it did not result in complete cell death (Figure 10C). Furthermore, microscopy images of neuroblastoma and HepG2 cells revealed compound precipitation at concentrations of 25–100 µM for compound 12 and at 50 and 100 µM for compound 15 (Supplementary data).

2.6. Activity Profile of Compound 15—In Vitro Studies

2.6.1. Effect of Compound 15 on Human Peripheral Blood Mononuclear Cell (PBMC) Viability

In the subsequent step, we examined the effect of compound 15 on the viability of human PBMCs. The results are shown in Figure 11 and

Table 7. IC₅₀ values obtained for compound 15 against PBMCs and SH-SY5Y (neuroblastoma) cells.

Incubation Time	IC50 (μM)
	PBM Cells	SH-SY5Y Cells
2 h	80.30	(-)
24 h	20.99	4.46
48 h	14.25	(-)
72 h	11.97	3.44

IC₅₀ values were determined after 2–72 h of incubation of PBMCs or SH-SY5Y cells with compound 15 at concentrations ranging from 0.195 to 100 μM (4 experiments) and 0.39 to 25 μM (6 experiments), respectively. (-)—not analysed.

(left column).

The cytotoxic effect of compound 15 on PBMCs depends on its concentration and incubation time. According to the data obtained, a 2 h incubation at concentrations ranging from 0.195 to 25 µM did not induce cytotoxicity (Figure 11a). For the two highest concentrations (50 and 100 µM), a statistically significant reduction in PBMCs compared to the negative control (NC) was observed (One-way ANOVA and Dunnett’s multiple comparisons test, p < 0.001), although at 50 µM, it did not exceed 12% (Figure 11a). At longer incubation times (24 h, 48 h, and 72 h), a significant decrease in PBMC viability compared to the control was observed at 6.25 μM and above, as shown by One-way ANOVA and Dunnett’s multiple comparisons test (p < 0.001). Notably, the highest concentrations of compound 15 (50 and 100 μM) reduced PBMC number by over 98% (Figure 11b–d). Additionally, during 48 h and 72 h incubation, treating cells with 25 μM of compound 15 produced a similar reduction.

In summary, the resazurin reduction assay on PBMCs demonstrated the toxicity of compound 15 within the micromolar range, as indicated by the calculated IC₅₀ values (Table 7, left column). The IC₅₀ values for compound 15 against PBMCs range from 11.97 to 80.30 μM and decrease with incubation time.

2.6.2. Effect of Compound 15 on the Viability and Proliferation of the Human Neuroblastoma Cell Line (SH-SY5Y)

The SH-SY5Y cells were also cultured with compound 15 at seven different concentrations ranging from 0.39 to 25 μM for 24 and 72 h. Pitolisant, the H₃R antagonist/inverse agonist, was used as a reference drug. The results showed that compound 15, only at the lowest concentration used (0.39 μM), did not affect cell viability (Figure 12a,c). The study revealed a significant decrease at p < 0.001 in cell number starting from a concentration of 3.125 μM. The most notable reduction in neuroblastoma viability (over 90% compared to control cells) at both incubation times was observed at concentrations of 12.5 and 25 μM. The IC₅₀ values for compound 15 against SH-SY5Y cells, depending on incubation time, are 4.46 and 3.44 μM (Table 7, right column).

Pitolisant—unlike compound 15—showed only a minor effect on neuroblastoma cells, causing a maximum decrease in their viability of about 20% at the two highest concentrations: 12.5 and 25 μM (Figure 12b,d). The viability of SH-SY5Y cells, assessed by IC₅₀ values after 24 h and 72 h treatment with pitolisant, is 425 and 64.97 μM, respectively.

Besides cell viability, the growth patterns of SH-SY5Y cells in the presence of compound 15 were also examined. As in previous experiments, neuroblastoma cells were treated with increasing concentrations of the tested compound (0.39–6.25 µM) for 24 or 72 h. Using image cytometry, the DNA content index was calculated as the 2N/4N DNA ratio to analyse cell cycle distribution. In this study, pitolisant was also utilised as a reference compound (concentration range: 0.39–25 µM). Figure 13a,c demonstrate that incubation with compound 15 at a concentration of 6.25 µM caused cell cycle arrest in the G2/M (4N) phase, as evidenced by a statistically significant decrease in the 2N/4N DNA ratio compared to cells treated with the vehicle. The inhibitory effect of compound 15 on cell cycle progression directly reduced the number of SH-SY5Y cells, as shown in complementary experiments (Figure 13a,c). Pitolisant, at the concentrations used for 24 h and 72 h, did not affect the cell cycle (Figure 13b,d).

Figure 14 shows changes in mitochondrial mass in SH-SY5Y cells (measured as mean total fluorescence intensity) incubated with compound 15 or pitolisant at concentrations of 0.39, 0.78, 1.56, 3.125, and 6.25 µM for 24 and 72 h. Treatment of SH-SY5Y cells with compound 15 for either 24 or 72 h led to an increase in mitochondrial biogenesis, probably as an adaptive response to metabolic demands and cellular stress. These effects were significant and noticeable at 3.125–6.25 µM of compound 15 compared to untreated cells. No changes in mitochondrial mass were observed following incubation with the same concentrations of pitolisant.

2.6.3. The Genotoxic Potential of Compound 15—Results from the Comet Assay

The genotoxic potential of compound 15 was assessed using human PBMCs through the comet assay (Figure 15). This technique detects DNA damage at the single-cell level following exposure to genotoxic agents. It acts as a key indicator of pathogenicity, demonstrating antiproliferative and, consequently, anticancer effects. Generally, the extent of DNA damage in PBMCs treated with compound 15 correlates with its concentration and incubation duration. Four concentrations of compound 15 were chosen to evaluate DNA damage: 0.39, 1.56, 6.25, and 12.5 μM. The compound induces statistically significant DNA damage in PBMCs compared to untreated cells at all tested concentrations (One-way ANOVA and Dunnett’s multiple comparisons test, p < 0.01 or p < 0.001), even after just 2 h of incubation (Figure 15a). At the highest concentration of 12.5 μM, compound 15 results in DNA damage levels of 36.52 ± 3.96% and 45.37 ± 3.79% after 2 and 24 h of incubation, respectively. The DNA damage caused by compound 15 at 12.5 μM over 2 h and at 6.25 and 12.5 μM over 24 h exceeds that of the positive control, which is 20 μM H₂O₂ for 15 min on ice (Figure 15). These findings suggest that compound 15 exhibits genotoxic activity against PBMCs at all tested concentrations. Figure 16 presents example images from the comet assay.

These results, along with the antiproliferative effect observed at higher micromolar concentrations (Figure 10), suggest promising avenues for future research into the therapeutic potential of compound 15 for central nervous system cancers, using cancer cell lines and animal models.

3. Materials and Methods

3.1. Chemistry

Reagents and solvents were purchased from relevant companies (Chemat Gdańsk, Poland; Sigma Aldrich: Darmstadt, Germany) and used without purification. Purity and identity of synthesised compounds were confirmed by performing thin-layer chromatography (TLC) on Merck silica gel 60F₂₅₄ aluminium sheets (UV light detection and/or stained with Dragendorff’s reagent), and measuring NMR (¹H and ¹³C) spectra in DMSO-d₆ on spectrometers (Mercury 300 MHz PFG spectrometer: Varian, Palo Alto, CA, USA; or FT-NMR 500 MHz spectrometer: Jeol Ltd., Akishima, Tokyo, Japan). Mass spectra (LC/MS) were performed on a Waters TQ Detector (Waters Corporation, Milford, CT, USA) mass spectrometer. Retention times (t_R) are presented in minutes. The UPLC/MS purity of all final compounds was determined (%). All compounds (except 3 and 12) had purity above 97%.

3.1.1. Synthesis of Starting Compounds

Synthesis of Benzyloxyaldehydes—General Method

To a 3- or 4-hydroxybenzaldehyde (12.5 mmol; 1.52 g) in 30 mL of ethanol, potassium carbonate (6.25 mmol; 0.86 g) was added, followed by a proper benzyl chloride (12.5 mmol). The mixture was refluxed for 18 h. The precipitate was then filtered, washed with ethanol, and evaporated. The residue was purified by extraction (DCM: 1% NaOH; DCM: NaCl saturated) or Flash Chromatography (FC) (solvent: DCM).

3-(benzyloxy)benzaldehyde (IVa). Synthesis from benzyl chloride (1.58 g). Purified by extraction. Yield: 33% (white oily solid).

3-((4-chlorobenzyl)oxy)benzaldehyde (IVb). Synthesis from 4-chlorobenzyl chloride (2.01 g). Purified by extraction. Yield: 80%.

3-((3,4-dichlorobenzyl)oxy)benzaldehyde (IVc). Synthesis from 3,4-dichlorobenzyl chloride (2.44 g). Purified by FC. Yield: 21%.

4-(benzyloxy)benzaldehyde (Va). Synthesis from benzyl chloride (1.58 g). Purified by FC. Yield: 33%.

4-((4-chlorobenzyl)oxy)benzaldehyde (Vb). Synthesis from 4-chlorobenzylchloride (2.01 g). Purified by extraction. Yield: 89%.

4-((3,4-dichlorobenzyl)oxy)benzaldehyde (Vc). Synthesis from 3,4-dichlorobenzyl chloride (2.44 g). Purified by extraction. Yield: 80%.

Synthesis of 1-(4-(3-Bromo propoxy)phenyl)ethan-1-one (VI)

Synthesised from 4-hydroxyacetophenone (0.1 mol; 13.62 g) and 1,3-dibromopropane (0.11 mol; 11.2 mL) in acetone as described by Honkisz-Orzechowska et al. [27].

Synthesis of 1-(4-(3-Piperidin-1-yl)propoxy)phenyl)ethan-1-one (VII) (CAS256952-65-5)

Synthesised from 1-(4-(3-bromo propoxy)phenyl)ethan-1-one (VI) (0.6 mol; 11.49 g), and piperidine (0.6 mol; 4.40 mL) in acetonitrile as described by Honkisz-Orzechowska et al. [27].

3.1.2. General Method of Synthesis of Chalcones

Synthesised from 4-hydroxyacetophenone (1 equivalent) and an appropriate aldehyde (1 equivalent) in absolute ethanol with 15% potassium hydroxide solution as described by Honkisz-Orzechowska et al. [27] or with 40% potassium hydroxide solution. Products were purified by crystallisation from ethanol or by FC (eluent: DCM: methanol; 9:1) and then converted into hydrogen chloride.

(E)-1-(4-hydroxyphenyl)-3-phenylprop-2-en-1-one (1) CAS2657-25-2. Synthesis from benzaldehyde (5 mmol, 0.53 g) with 40% KOH (4 mL). Purified by crystallisation from ethanol. Yield 34% (380 mg). C₁₅H₁₂O₂ (MW 224.25). ¹H NMR (500 MHz, DMSO-d₆) δ: 10.40 (br. s., 1H), 8.04 (d, J = 8.59 Hz, 2H), 7.88 (d, J = 15.47 Hz, 1H), 7.83 (dd, J = 1.86, 7.31 Hz, 2H), 7.64 (d, J = 15.76 Hz, 1H), 7.36–7.46 (m, 3H), 6.86 (d, J = 8.59 Hz, 2H). UPLC-MS (m/z): 225.21 ([M + H]⁺). Purity: 98.85%, t_R = 6.93 min.

(E)-3-(4-chlorophenyl)-1-(4-hydroxyphenyl)prop-2-en-1-one (2) CAS19152-38-6. Synthesis from 4-chlorobenzaldehyde (5 mmol, 0.70 g) with 40% KOH (4 mL). Purified by crystallisation from ethanol. Yield 10% (130 mg). C₁₅H₁₁ClO₂ (MW 258.70). ¹H NMR (500 MHz, DMSO-d₆) δ: 10.42 (br. s., 1H), 8.04 (d, J = 8.88 Hz, 2H), 7.84–7.94 (m, 3H), 7.63 (d, J = 15.76 Hz, 1H), 7.48 (d, J = 8.31 Hz, 2H), 6.86 (d, J = 8.88 Hz, 2H). UPLC-MS (m/z): 258.70 ([M]⁺). Purity: 99.72%, t_R = 7.61 min.

(E)-3-(3,4-dichlorophenyl)-1-(4-hydroxyphenyl)prop-2-en-1-one (3) CAS 204974-31-2. Synthesis from 3,4-dichlorobenzaldehyde (2.5 mmol, 0.44 g) with 40% KOH (2 mL). Purified by crystallisation from ethanol. Yield 6% (45 mg). C₁₅H₁₀Cl₂O₂ (MW 293.14). ¹H NMR (500 MHz, DMSO-d₆) δ: 10.44 (s, 1H), 8.24 (d, J = 1.72 Hz, 1H), 8.07 (d, J = 8.60 Hz, 2H), 7.99 (d, J = 15.47 Hz, 1H), 7.82 (dd, J = 2.01, 8.31 Hz, 1H), 7.65–7.70 (m, 1H), 7.60 (d, J = 15.76 Hz, 1H), 6.86 (d, J = 8.59 Hz, 2H). UPLC-MS (m/z): 293.03 ([M + H]⁺). Purity: 93.88%, t_R = 8.22 min.

(E)-3-(3-(benzyloxy)phenyl)-1-(4-hydroxyphenyl)prop-2-en-1-one (4). Synthesis from 3-(benzyloxy)benzaldehyde (5 mmol, 1.06 g) with 40% KOH (4 mL). Purified by crystallisation from ethanol. Yield 24% (390 mg). C₂₂H₁₈O₃ (MW 330.38). ¹H NMR (500 MHz, DMSO-d₆) δ: 10.40 (br. s., 1H), 8.05 (d, J = 8.88 Hz, 2H), 7.89 (d, J = 15.47 Hz, 1H), 7.61 (d, J = 15.76 Hz, 1H), 7.55 (s, 1H), 7.46 (d, J = 7.45 Hz, 2H), 7.36–7.41 (m, 3H), 7.32 (d, J = 7.45 Hz, 2H), 7.05 (dd, J = 1.72, 8.02 Hz, 1H), 6.86 (d, J = 8.59 Hz, 2H), 5.14 (s, 2H). UPLC-MS (m/z): 331.18 ([M + H]⁺). Purity: 98.22%; t_R = 8.36 min.

(E)-3-(3-((4-chlorobenzyl)oxy)phenyl)-1-(4-hydroxyphenyl)prop-2-en-1-one (5). Synthesis from 3-((4-chlorobenzyl)oxy)benzaldehyde (5 mmol, 1.23 g) with 40% KOH (4 mL). Purified by crystallisation from ethanol. Yield 18% (330 mg). C₂₂H₁₇ClO₃ (MW 364.83). ¹H NMR (500 MHz, DMSO-d₆) δ: 10.41 (br. s., 1H), 8.05 (d, J = 8.60 Hz, 2H), 7.89 (d, J = 15.76 Hz, 1H), 7.61 (d, J = 15.47 Hz, 1H), 7.54 (s, 1H), 7.46–7.51 (m, 2H), 7.41–7.46 (m, 2H), 7.35–7.40 (m, 1H), 7.30–7.35 (m, 1H), 7.04 (dd, J = 2.01, 8.02 Hz, 1H), 6.86 (d, J = 8.88 Hz, 2H), 5.14 (s, 2H). UPLC-MS (m/z): 365.12 ([M + H]⁺). Purity: 99.11%; t_R = 8.84 min.

(E)-3-(3-((3,4-dichlorobenzyl)oxy)phenyl)-1-(4-hydroxyphenyl)prop-2-en-1-one (6). Synthesis from 3-((3,4-dichlorobenzyl)oxy)benzaldehyde (6.4 mmol, 1.80 g) with 40% KOH (5 mL). Purified by FC. Yield 13% (340 mg). Yellow solid. C₂₂H₁₆Cl₂O₃ (MW 399.27). ¹H NMR (500 MHz, DMSO-d₆) δ: 10.41 (br. s., 1H), 8.05 (d, J = 8.88 Hz, 2H), 7.89 (d, J = 15.76 Hz, 1H), 7.74 (d, J = 1.72 Hz, 1H), 7.58–7.68 (m, 2H), 7.55 (s, 1H), 7.45 (dd, J = 1.72, 8.31 Hz, 1H), 7.37–7.42 (m, 1H), 7.29–7.37 (m, 1H), 7.05 (dd, J = 1.86, 7.88 Hz, 1H), 6.86 (d, J = 8.60 Hz, 2H), 5.16 (s, 2H). UPLC-MS (m/z) ([M]⁺): 399.09. Purity: 97.87%; t_R = 9.38 min.

(E)-3-(4-(benzyloxy)phenyl)-1-(4-hydroxyphenyl)prop-2-en-1-one (7). Synthesis from 4-(benzyloxy)benzaldehyde (2.5 mmol, 0.53 g) with 40% KOH (2 mL). Purified by crystallisation from ethanol. Yield 28% (230 mg). C₂₂H₁₈O₃ (MW 330.38). ¹H NMR (500 MHz, DMSO-d₆) δ: 10.35 (br s, 1H), 8.02 (d, J = 8.59 Hz, 2H), 7.79 (d, J = 8.88 Hz, 2H), 7.71–7.77 (m, 1H), 7.58–7.66 (m, 1H), 7.41–7.47 (m, 2H), 7.37 (t, J = 7.45 Hz, 2H), 7.27–7.33 (m, 1H), 7.05 (d, J = 8.88 Hz, 2H), 6.85 (d, J = 8.88 Hz, 2H), 5.14 (s, 2H). UPLC-MS (m/z): 330.92 ([M]⁺). Purity: 100%; t_R = 8.25 min.

(E)-3-(4-((4-chlorobenzyl)oxy)phenyl)-1-(4-hydroxyphenyl)prop-2-en-1-one (8). Synthesis from 4-((4-chlorobenzyl)oxy)benzaldehyde (2.5 mmol, 0.62 g) with 40% KOH (2 mL). Purified by crystallisation from ethanol. Yield 29% (260 mg). C₂₂H₁₇ClO₃ (MW 364.83). ¹H NMR (500 MHz, DMSO-d₆) δ: 10.35 (br. s., 1H), 8.02 (d, J = 8.59 Hz, 2H), 7.79 (d, J = 8.88 Hz, 2H), 7.71–7.77 (m, 1H), 7.57–7.65 (m, 1H), 7.39–7.49 (m, 4H), 7.04 (d, J = 8.59 Hz, 2H), 6.85 (d, J = 8.88 Hz, 2H), 5.14 (s, 2H). UPLC-MS (m/z): 364.87 ([M]⁺). Purity: 98.82%; t_R = 8.77 min.

(E)-3-(4-((3,4-dichlorobenzyl)oxy)phenyl)-1-(4-hydroxyphenyl)prop-2-en-1-one (9). Synthesis from 4-((3,4-dichlorobenzyl)oxy)benzaldehyde (2.5 mmol, 0.71 g) with 40% KOH (2 mL). Purified by FC. Yield 30% (300 mg). C₂₂H₁₆Cl₂O₃ (MW 399.27). ¹H NMR (500 MHz, DMSO-d₆) δ: 10.35 (br. s., 1H), 8.02 (d, J = 8.88 Hz, 2H), 7.80 (d, J = 8.88 Hz, 2H), 7.73–7.77 (m, 1H), 7.71 (d, J = 1.72 Hz, 1H), 7.57–7.67 (m, 2H), 7.43 (dd, J = 1.72, 8.31 Hz, 1H), 7.05 (d, J = 8.60 Hz, 2H), 6.85 (d, J = 8.60 Hz, 2H).

(E)-3-(phenyl)-1-(4-(3-(piperidin-1-yl)propoxy)phenyl)prop-2-en-1-one hydrogen chloride (10). Synthesis from benzaldehyde (2 mmol, 0.212 g) with 15% KOH (1 mL). Purified by crystallisation (ethanol). Yield 11% (75 mg). C₂₃H₂₇NO₂ × HCl (MW 385.93). ¹H NMR (500 MHz, DMSO-d₆) δ: 8.13 (d, J = 9.2 Hz, 2H), 7.91 (d, J = 15.5 Hz, 1H), 7.84 (dd, J = 2.3, 6.9 Hz, 2H), 7.67 (d, J = 15.5 Hz, 1H), 7.48–7.36 (m, 3H), 7.05 (d, J = 8.6 Hz, 2H), 4.16–4.03 (m, 2H), 3.33 (br. s., 2H), 2.60 (br. s., 4H), 1.97 (br. s., 2H), 1.55 (br. s., 4H), 1.39 (br. s., 2H). UPLC-MS (m/z): 350.30 ([M + H]⁺). Purity: 100%; t_R = 5.11 min.

(E)-3-(4-chlorophenyl)-1-(4-(3-(piperidin-1-yl)propoxy)phenyl)prop-2-en-1-one hydrogen oxalate (11). Synthesis from 4-chlorobenzaldehyde (2 mmol, 0.281 g) with 15% KOH (1 mL). Purified by crystallisation (ethyl acetate). Yield 9% (78 mg). C₂₃H₂₆ClNO₂ × HCl (MW 420.37). ¹H NMR (300 MHz, DMSO-d₆) δ: 8.16 (d, J = 8.8 Hz, 1H), 8.02–7.85 (m, 2H), 7.76 (d, J = 15.82Hz, 1H), 7.46 (m, 1H), 7.29 (t, J = 8.8 Hz, 1H), 7.18–6.91 (m, 3H), 6.78 (d, J = 12.9 Hz, 1H), 4.30–3.99 (m, 2H), 3.27–2.78 (m, 6H), 2.23-2.00 (m, 2H), 1.70 (br s, 4H), 1.50 (br. s., 2H). ¹³C NMR (126 MHz, DMSO-d₆) δ: 187.8, 165.3, 162.9, 142.3, 135.5, 134.3, 131.6, 131.1, 131.0, 129.5, 123.2, 115.0, 66.0, 53.8, 52.6, 23.9, 23.1, 22.0. UPLC-MS (m/z): 384.06 ([M + H]⁺). Purity: 99.51%; t_R = 5.65 min.

(E)-3-(3,4-dichlorophenyl)-1-(4-(3-(piperidin-1-yl)propoxy)phenyl)prop-2-en-1-one hydrogen chloride (12). Synthesis from 3,4-dichlorobenzaldehyde (2 mmol, 0.35 g) with 15% KOH (1 mL). Purified by crystallisation from ethanol. Yield 26% (0.235 g). C₂₃H₂₅Cl₂O₂ × HCl (MW 454.82). ¹H NMR (300 MHz, DMSO-d₆) δ: 10.35 (br. s., 1H), 8.27 (d, J = 1.76 Hz, 1H), 8.19 (d, J = 8.79 Hz, 2H), 8.05 (d, J = 15.82 Hz, 1H), 7.86 (dd, J = 1.76, 8.79 Hz, 1H), 7.70 (d, J = 8.21 Hz, 1H), 7.65 (d, J = 15.24 Hz, 1H), 7.08 (d, J = 8.79 Hz, 2H), 4.18 (t, J = 6.15 Hz, 2H), 3.38–3.54 (m, 2H), 3.15 (d, J = 4.69 Hz, 2H), 2.88 (d, J = 7.62 Hz, 2H), 2.12–2.32 (m, 2H), 1.61–1.89 (m, 5H), 1.39 (br. s., 1H). ¹³C NMR (126 MHz, DMSO-d₆) δ: 187.6, 163.0, 141.0, 136.2, 133.1, 132.3, 131.7, 131.5, 130.9, 130.6, 129.7, 124.5, 115.0, 66.1, 53.8, 52.5, 23.7, 22.8, 21.9. UPLC-MS (m/z): 418.22 ([M]⁺). Purity: 94.80%; t_R = 6.37 min.

(E)-3-(3-(benzyloxy)phenyl)-1-(4-(3-(piperidin-1-yl)propoxy)phenyl)prop-2-en-1-one hydrogen chloride (13). Synthesis from 3-(benzyloxy)benzaldehyde (1 mmol, 0.22 g) with 15% KOH (0.5 mL). Purified by FC. Yield 8% (40 mg). C₃₀H₃₃NO₃ × HCl (MW 492.05). ¹H NMR (500 MHz, DMSO-d₆) δ: 10.40 (br. s., 1 H), 8.16 (d, J = 8.6 Hz, 2H), 7.93 (d, J = 15.8 Hz, 1H), 7.64 (d, J = 15.5 Hz, 1H), 7.56 (br. s., 1H), 7.46 (d, J = 7.2 Hz, 2H), 7.42–7.23 (m, 5H), 7.06 (d, J = 8.6 Hz, 3H), 5.15 (s, 2H), 4.15 (t, J = 5.7 Hz, 2H), 3.41 (d, J = 11.2 Hz, 2H), 3.14 (d, J = 4.9 Hz, 2H), 2.93–2.72 (m, 2H), 2.29–2.07 (m, 2H), 1.75 (br. s., 4H), 1.67 (d, J = 12.9 Hz, 1H), 1.34 (d, J = 7.2 Hz, 1H). ¹³C NMR (126 MHz, DMSO-d₆) δ: 187.9, 162.8, 159.3, 143.7, 137.5, 136.8, 131.5, 131.1, 130.5, 129.0, 128.5, 128.4, 122.8, 122.5, 117.7, 115.0, 114.7, 69.9, 66.0, 53.8, 52.5, 23.7, 22.9, 21.9. UPLC-MS (m/z): 456.41 ([M + H]⁺). Purity: 100%; t_R = 6.46 min.

(E)-3-(3-((4-chlorobenzyl)oxy)phenyl)-1-(4-(3-(piperidin-1-yl)propoxy)phenyl)prop-2-en-1-one hydrogen chloride (14). Synthesis from 3-((4-chlorobenzyl)oxy)benzaldehyde (0.5 mmol, 0.13 g) with 15% KOH (0.25 mL). Purified by FC. Yield 23% (60 mg). C₃₀H₃₂NO₃Cl × HCl (MW 526.48). ¹H NMR (500 MHz, DMSO-d₆) δ: 10.35 (br. s., 1H), 8.15 (d, J = 8.59 Hz, 2H), 7.93 (d, J = 15.47 Hz, 1H), 7.64 (d, J = 15.75 Hz, 1H), 7.56 (s, 1H), 7.42–7.52 (m, 4H), 7.40 (d, J = 7.73 Hz, 1H), 7.31–7.37 (m, 1H), 7.06 (d, J = 8.59 Hz, 3H), 5.15 (s, 2H), 4.15 (t, J = 5.87 Hz, 2H), 3.41 (d, J = 11.74 Hz, 2H), 3.09–3.19 (m, 2H), 2.76–2.91 (m, 2H), 2.12–2.28 (m, 2H), 1.75 (br. s., 4H), 1.66 (d, J = 13.17 Hz, 1H), 1.35 (dd, J = 7.45, 13.17 Hz, 1H). ¹³C NMR (126 MHz, DMSO-d₆) δ: 187.8, 162.8, 159.1, 143.6, 136.8, 136.5, 133.0, 131.6, 131.1, 130.5, 130.3, 129.0, 122.8, 122.6, 117.7, 115.0, 114.8, 69.1, 66.0, 53.8, 52.6, 23.7, 22.9, 21.9. UPLC-MS (m/z): 490.37 ([M]⁺). Purity: 100%; t_R = 6.87 min.

(E)-3-(3-((3,4-dichlorobenzyl)oxy)phenyl)-1-(4-(3-(piperidin-1-yl)propoxy)phenyl)prop-2-en-1-one hydrogen chloride (15). Synthesis from 3-((3,4-dichlorobenzyl)oxy)benzaldehyde (1 mmol, 0.28 g) with 15% KOH (0.5 mL). Purified by FC. Yield 23% (130 mg). C₃₀H₃₁NO₃Cl₂ × HCl (MW 560.94). ¹H NMR (500 MHz, DMSO-d₆) δ: 10.55 (br. s., 1H), 8.16 (d, J = 8.59 Hz, 2H), 7.94 (d, J = 15.75 Hz, 1H), 7.74 (d, J = 1.72 Hz, 1H), 7.65 (d, J = 2.29 Hz, 1H), 7.61–7.64 (m, 1H), 7.57 (s, 1H), 7.45 (dd, J = 2.00, 8.31 Hz, 1H), 7.39–7.43 (m, 1H), 7.34 (t, J = 7.88 Hz, 1H), 7.02–7.11 (m, 3H), 5.17 (s, 2H), 4.15 (t, J = 6.01 Hz, 2H), 3.41 (d, J = 11.74 Hz, 2H), 3.06–3.20 (m, 2H), 2.75–2.93 (m, 2H), 2.13–2.28 (m, 2H), 1.70–1.92 (m, 4H), 1.66 (d, J = 12.89 Hz, 1H), 1.28–1.42 (m, 1H). ¹³C NMR (126 MHz, DMSO-d₆) δ: 187.8, 162.8, 158.9, 143.6, 138.7, 136.8, 131.7, 131.6, 131.3, 131.1, 131.0, 130.6, 130.2, 128.6, 122.9, 122.8, 117.6, 115.0, 114.8, 68.3, 65.9, 53.8, 52.6, 23.8, 23.0, 21.9. HR-MS (ESI-QTOF) calcd for C₃₀H₃₁NO₃Cl₂ [M + H]⁺: 524.1761; found: 524.1724. UPLC-MS (m/z): Purity: 95.82%; t_R = 7.98 min.

(E)-3-(4-(benzyloxy)phenyl)-1-(4-(3-(piperidin-1-yl)propoxy)phenyl)prop-2-en-1-one hydrogen chloride (16). Synthesis from 4-(benzyloxy)benzaldehyde (1 mmol, 0.21 g) with 15% KOH (0.5 mL). Purified by crystallisation from ethanol. Yield 22% (110 mg). C₃₀H₃₃NO₃ x HCl (MW 492.05). ¹H NMR (500 MHz, DMSO-d₆) δ: 10.65 (br. s., 1H), 8.13 (d, J = 8.59 Hz, 2H), 7.75–7.84 (m, 3H), 7.61–7.68 (m, 1H), 7.40–7.46 (m, 2H), 7.37 (t, J = 7.45 Hz, 2H), 7.27–7.33 (m, 1H), 7.01–7.11 (m, 4H), 5.15 (s, 2H), 4.15 (t, J = 6.01 Hz, 2H), 3.40 (d, J = 10.88 Hz, 2H), 3.12 (br. s., 2H), 2.83 (d, J = 10.31 Hz, 2H), 2.10–2.25 (m, 2H), 1.71–1.89 (m, 4H), 1.66 (d, J = 12.89 Hz, 1H), 1.27–1.43 (m, 1H). ¹³C NMR (126 MHz, DMSO-d₆) δ: 187.7, 162.6, 160.8, 143.7, 137.3, 131.4, 131.2, 129.0, 128.5, 128.3, 128.2, 120.1, 115.7, 115.0, 69.9, 66.0, 53.8, 52.5, 23.7, 22.8, 22.0. UPLC-MS (m/z): 456.40 ([M + H]⁺). Purity: 100%; t_R = 6.32 min.

(E)-3-(4-((4-chlorobenzyl)oxy)phenyl)-1-(4-(3-(piperidin-1-yl)propoxy)phenyl)prop-2-en-1-one hydrogen chloride (17). Synthesis from 4-((4-chlorobenzyl)oxy)benzaldehyde (2 mmol, 0.49 g) with 15% KOH (1 mL). Purified by FC. Yield 32% (340 mg). C₃₀H₃₂NO₃Cl x HCl (MW 526.49). ¹H NMR (500 MHz, DMSO-d₆) δ: 10.81 (br. s., 1H), 8.16 (d, J = 8.59 Hz, 2H), 7.94 (d, J = 15.47 Hz, 1H), 7.64 (d, J = 15.47 Hz, 1H), 7.57 (br. s., 1H), 7.46–7.52 (m, 2H), 7.37–7.45 (m, 3H), 7.33 (t, J = 7.88 Hz, 1H), 7.06 (d, J = 8.59 Hz, 3H), 5.15 (s, 2H), 4.15 (t, J = 5.87 Hz, 2H), 3.38–3.47 (m, 2H), 3.12 (br. s., 2H), 2.83 (d, J = 10.31 Hz, 2H), 2.14–2.28 (m, 2H), 1.59–1.91 (m, 5H), 1.25–1.43 (m, 1H). ¹³C NMR (126 MHz, DMSO-d₆) δ: 187.8, 162.8, 159.1, 143.6, 136.8, 136.5, 133.0, 131.6, 131.1, 130.5, 130.2, 129.0, 122.8, 115.0, 114.7, 69.1, 66.1, 53.8, 52.5, 23.6, 22.8, 22.0. LC-MS (m/z): 490.35 [M + H]+; C₃₀H₃₂NO₃Cl (calculated MW: 490.21). Purity (UPLC-MS): 100%; t_R = 6.81. UPLC-MS (m/z): 490.04 ([M]⁺). Purity: 97.08%; t_R = 6.81 min.

(E)-3-(4-((3,4-dichlorobenzyl)oxy)phenyl)-1-(4-(3-(piperidin-1-yl)propoxy)phenyl)prop-2-en-1-one hydrogen chloride (18). Synthesis from 4-((3,4-dichlorobenzyl)oxy)benzaldehyde (1 mmol, 0.28 g) with 15% KOH (0.5 mL). Purified by FC. Yield 32% (340 mg). C₃₀H₃₁NO₃Cl₂ x HCl (MW 560.94). ¹H NMR (500 MHz, DMSO-d₆) δ: 10.54 (br. s., 1H), 8.12 (d, J = 8.59 Hz, 2H), 7.76–7.87 (m, 3H), 7.71 (d, J = 0.86 Hz, 1H), 7.59–7.67 (m, 2H), 7.42 (d, J = 7.16 Hz, 1H), 7.05 (dd, J = 6.59, 8.02 Hz, 4H), 5.16 (s, 2H), 4.14 (t, J = 5.73 Hz, 2H), 3.40 (d, J = 10.31 Hz, 2H), 3.13 (br. s., 2H), 2.83 (br. s., 2H), 2.20 (dd, J = 5.87, 9.02 Hz, 2H), 1.59–1.86 (m, 5H), 1.27–1.41 (m, 1H). ¹³C NMR (126 MHz, DMSO-d₆) δ: 187.7, 162.6, 160.4, 143.6, 138.5, 131.7, 131.4, 131.3, 131.3, 131.3, 131.0, 130.1, 128.5, 120.3, 115.8, 115.0, 68.3, 66.0, 53.8, 52.5, 23.7, 22.9, 21.9. UPLC-MS (m/z): 524.24 ([M]⁺). Purity: 97.99%; t_R = 7.97 min.

3.2. In Vitro Biological Studies

3.2.1. Radioligand Binding Assay to Human Histamine H₃ Receptor

The affinity for the hH₃R was assessed using a radioligand binding assay in CHO K1 cells, employing N^α-methylhistamine as the radioligand, following the procedure described by Łażewska et al. [20]. 10 mM stock solutions of the tested compounds were prepared in DMSO. Serial dilutions of compounds were prepared in a 96-well microplate using assay buffers with an automated pipetting system, epMotion 5070 (Eppendorf, Hamburg, Germany). Each compound was tested in 8 concentrations from 10⁻⁵ to 10⁻¹² M (final concentration). All assays were performed in duplicate. 50 µL working solution of the tested compounds, 50 µL [³H]-N^α-methylhistamine (spec. act. 82.9 Ci/mmol, final concentration 1.0 nM) and 150 µL diluted membranes (15 µg protein per well) prepared in assay buffer (50 mM Tris, pH 7.4, 5 mM MgCl₂) were transferred to polypropylene 96-well microplate using 96-well pipetting station, Rainin Liquidator (Mettler Toledo Inc., Greifensee, Switzerland ). (R)(-)-α-methylhistamine (100 μM) was used to define nonspecific binding. The microplate was covered with sealing tape, mixed, and incubated for 60 min at 27 °C. The reaction was terminated by rapid filtration through a GF/B filter mate presoaked with 0.5% polyethyleneimine for 30 min. Five rapid washes with 300 µL 50 mM Tris buffer (4 °C, pH 7.4) were performed using a 96-well FilterMate harvester (PerkinElmer, Shelton, CT, USA). The filter mates were dried at 37 °C in a forced air fan incubator, and then the solid scintillator MeltiLex was melted on the filter mates at 90 °C for 4 min. Radioactivity was counted in a MicroBeta2 scintillation counter (PerkinElmer, Shelton, CT, USA). Data were fitted to a one-site curve-fitting equation with Prism 5 (GraphPad Software 8.0), and K_i values were estimated from the Cheng−Prusoff equation [43].

3.2.2. Functional Assays for Histamine H₃ Receptor

Stock solutions (10 mM) of the reference and tested compounds (12 and 15) were prepared in DMSO. Serial dilutions were performed in 96-well assay plates using the assay buffer (5 mM HEPES, 0.5 mM IBMX, pH 7.4), resulting in 8 concentrations for each compound. The adenylyl cyclase activity mediated by the hH₃R was measured in cryopreserved CHO-K1 cells stably transfected with the receptor (Revvity, Waltham, MA, USA). After thawing, cells were resuspended in stimulation to a final density of 1.7 × 10⁵ cells/mL. For each assay condition, 6 µL of the cell suspension was combined with 3 µL of the compound dilution in white, opaque 384-well plates. In antagonist mode, α-methylhistamine (100 nM) was used as the reference agonist and was added simultaneously with the test compound. The plates were incubated for 1 h at room temperature. After incubation, intracellular cAMP levels were determined using the homogeneous TR-FRET LANCE Ultra cAMP assay kit (Revvity, Waltham, MA, USA). Six microliters each of Eu-cAMP tracer and ULight-anti-cAMP solutions were added per well, mixed, and incubated for an additional 1 h. TR-FRET signals were measured using an EnVision multimode plate reader (PerkinElmer, Shelton, CT, USA). Concentration–response data were fitted by nonlinear regression using GraphPad Prism 8.0 software.

3.2.3. Surface Plasmon Resonance Microscopy (SPRM) Kinetic Studies

Cell culture conditions. Cells were cultured according to the manufacturer’s protocol provided by Revvity (Waltham, MA, USA). CHO-K1 cells overexpressing the hH₃R were cultured in Ham’s F12 Nutrient Mix (ThermoFisher, Waltham, MA, USA) containing 10% foetal bovine serum (ThermoFisher, Waltham, MA, USA), 500 units/mL penicillin, and 500 μg/mL streptomycin (Sigma Aldrich, Darmstadt, Germany) at 37 °C in a humidified incubator (5% CO₂). Geneticin (400 µg/mL) was used as a selective antibiotic for receptor expression selection.

Preparation of solutions of test and reference compounds. 10 mM stock solutions of the tested compounds were prepared in DMSO. Serial dilutions were prepared in glass tubes using buffer (Phosphate-Buffered Saline with 0.01% BSA). The final concentrations of the test and reference compounds ranged from 10 µM to 0.5 nM, specifically: 10 µM, 5 µM, 1 µM, 500 nM, 100 nM, 50 nM, 10 nM, 5 nM, 1 nM and 0.5 nM.

SPRM Analysis. CHO-K1 cell line transfected with hH₃R was seeded at a density of 20,000 cells on a sterile gold SPRm chip, which was pre-coated with 100 µg/mL Poly-D-Lysine as an adhesion factor. After 48 h of incubation, the cells were washed with DPBS and subsequently fixed with 4% paraformaldehyde (PFA) for 10 min. After PFA removal and subsequent washing with DPBS, cells were incubated with a 3% BSA solution for 1h to reduce nonspecific binding. Following the application of a running buffer (PBS with 0.01% BSA), the chip was mounted onto the SPRm device (Biosensing Instruments Inc., Tempe, AZ, USA). The tested compounds were then applied at six to seven increasing concentrations for analysis. Data were analysed using the Biosensing Instruments SPRm Data Analysis Program. The association rate constant (k_a), dissociation rate constant (k_d), and equilibrium dissociation constant (K_D) were obtained from affinity analyses of ROI sensograms.

3.2.4. Human MAO-B and MAO-A Inhibitory Activity

The compounds were evaluated by the spectrophotometric method described earlier [20]. The enzymes were purchased from Sigma-Aldrich (Steinheim, Germany). Screening was carried out at a concentration of 1 μM at the MAO-B and 10 μM at the MAO-A. The assay was carried out in a 96-well plate. 2 µL of an appropriate concentration of tested compounds in DMSO were added to wells that contained 98 µL of enzyme dilution (0.52 U/well) in phosphate buffer (50 mM, pH 7.4). After the 30 min of preincubation at room temperature, 50 µL of the solution of 800 μM 10-acetyl-3,7-dihydroxyphenoxazine (Cayman Chemical, Ann Arbor, MI, USA) and 4 U/mL horse radish peroxidase (HRP) was added, and the enzymatic reaction was started by the addition of 50 µL of 800 µM p-tyramine solution. The signal was measured after 1 h (excitation at 570 nm and emission at 585 nm) using Spark^® multimode microplate reader (Tecan; Männedorf, Switzerland). The compounds that showed more than 50% inhibition were subjected to further testing to determine the IC₅₀ value. Data are expressed as mean values ± SEM of at least two independent experiments.

3.2.5. Human MAO-B Reversibility Studies

Enzyme was incubated with inhibitors (compound 15, safinamide and rasagiline) in a concentration equivalent to 10 × IC₅₀ for 30 min. The mixture was diluted 100× in the buffer containing MAO-B substrate (p-tyramine) and detection reagent (horseradish peroxidase and 10-Acetyl-3,7-dihydroxyphenoxazine). Fluorescence signal was measured every 5 min for 1 h. Baseline (fluorescence signal of reaction mixture without p-tyramine) was subtracted from all samples.

3.2.6. Human MAO-B Kinetic Studies

To determine the inhibitory mode of human MAO-B inhibition, substrate-dependent kinetic experiments were performed. The data allowed the generation of progress curves and Lineweaver–Burk plots. Measurements of the enzyme’s initial catalytic rates were conducted using six different p-tyramine concentrations (0.05–2.0 mM), both without inhibitor and with compound 15 added at three concentrations corresponding to its IC₂₀, IC₅₀, and IC₈₀ values. These experiments followed the assay conditions for hMAO-B described earlier.

3.3. Molecular Modelling Studies of the Histamine H₃ Receptor and MAO-B

For docking purposes, Schrodinger 2022-4 [44] was used. Ligand structures in their E isomeric forms were built using Maestro 2D sketcher, and their 3D conformations were obtained using the LigPrep module (OPLS4, target pH = 7.4 ± 0.5). Protein structures of the histamine H₃ receptor in its inactive form, and MAO-B complexed with an inhibitor, were obtained from the PDB database (ID codes: 7F61 and 2V5Z, respectively). In the original 7F61 structure publication [36], a cholesterol molecule was resolved at an allosteric site, where it was suggested to contribute to receptor inactive-state stabilisation; accordingly, the cholesterol molecule was retained during modelling to account for its potential modulatory role in receptor conformation and ligand binding. An initial relaxation protocol into a local energy minimum using Brownian motion simulation (in time of 100 ps) was performed with Desmond [45]. To validate the methods used, the native ligands were redocked with high confidence (glide RMSD to input of 0.181 and 0.207, respectively). Tested ligands were then docked using Glide XP (7F61) and Induced Fit (2V5Z, since no poses for compound 15 were returned in standard XP protocol) protocols with grid box centred on co-resolved ligand [46,47], followed by ligand energy calculation using Prime MM–GBSA [48]. Binding pose metadynamics on the top 5 scoring complexes was then performed (10 trials per pose) so as to predict the correct pose. Dynamics simulations (time 250 ns, T = 300 K) were run in Desmond [44]. For each run, 1000 frames were produced. The obtained trajectories were then analysed visually, as well as using the Simulation Interaction Analysis tool of Desmond/Maestro. Membrane orientations of the 7F61 protein complexes were computed with the PPM 3.0 server [49], applying the TIP3P solvent model [50] and embedding them in a POPC membrane. For the 2V5Z system, solvation was performed using the TIP3P model within an orthorhombic box extending 10 Å beyond the solute in each dimension. Per-residue RMSD and MM–GBSA ΔG Bind values were calculated using Schrödinger Maestro command line scripts. All of the figure’s components come from the Schrödinger package and were prepared using freely available graphics software.

3.4. Preliminary ADMET Evaluation of Compounds 12 and 15

3.4.1. Permeability Evaluation

The passive permeability assessment was performed by the Pre-coated PAMPA Plate System Gentest^TM Corning (Tewksbury, MA, USA) according to the procedure provided by the manufacturer. In brief, the tested compounds 12 and 15 and the reference permeable caffeine were diluted first in the PBS buffer (pH 7.4). In order to increase the unsatisfying solubility of compounds 12 and 15, 20% of methanol was added to PBS. The compounds were applied next to the PAMPA donor plate at a final concentration of 100 μM. After 5 h of incubation at RT, UPLC-MS spectrometry with internal standard was used to estimate the quantity of compounds that penetrated from donor to acceptor wells. The permeability coefficients (P_e, cm/s) were calculated using the formulas provided by Corning. The Caco-2 cell line HTB-37^TM (ATCC, Manassas, VA, USA) was cultivated in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% foetal bovine serum (FBS) (both obtained from ThermoFisher Scientific, Waltham, MA, USA) until the appropriate confluence (70–80%) was achieved. Next, the cells were seeded into Corning^® 3413 Transwell^® 6.5 mm polycarbonate membrane inserts with 0.4 µm pores purchased from Sigma-Aldrich (Saint Louis, MO, USA). Cells were seeded at a 2 × 10⁻⁴ concentration per insert in the apical compartment, and 600 µL of media was added to the basolateral one. TEER (transepithelial electrical resistance) measurements were performed by Millicell ERS-2 Volt-Ohm Metre (Merck Millipore, Burlington, MA, USA) in order to confirm the membrane integrity, ready for the experiment. Around 20–21 days post-seeding, the monolayer was rinsed with HBSS (Hank’s balanced salt solution), and compounds 12 and 15, as well as reference (caffeine), were tested at 10 µM concentration in HBSS. Compounds were added together with the integrity marker lucifer yellow either into the apical chambers (A-B direction) or basolateral chambers (B-A direction). The plate was placed in the orbital shaker (60 rpm) for 2 h at 37 °C. The samples were collected, and the compounds’ concentrations were analysed using the UPLC-MS method with an internal standard. The fluorescence of lucifer yellow was measured by using a Synergy H1 microplate reader (BioTek, Winooski, VT, USA) to confirm the membrane integrity. The apparent permeability P_app was calculated according to the following formula: P_app = dc/dt × V/(A × C₀).

3.4.2. Metabolic Stability Studies

The most probable metabolic pathways of compounds 12 and 15 were determined by incubation with rat liver microsomes (RLMs, Sigma-Aldrich, St. Louis, MO, USA) in potassium phosphate buffer (100 mM, pH 7.4, 37 °C). The reactions were terminated at 2 h by the addition of cold methanol and centrifuged. The UPLC-MS analyses were performed on the supernatants. The determination of metabolites from the obtained MS spectra was supported by MetaSite 6.0.1 software (Molecular Discovery Ltd., Hertfordshire, UK). The PK in vitro parameters of 15 were estimated in mouse liver microsomes (MLMs) obtained from Sigma-Aldrich. The reaction mixtures consisted of potassium phosphate buffer (100 mM, pH 7.4), mouse liver microsomes (0.5 mg/mL), tested compound (1 µM) and NADPH (1 mM). The five independent reactions were terminated at 0, 5, 15, 30 and 45 min by the addition of cold methanol containing IS and centrifuged. The UPLC-MS analyses were performed next on the supernatants. Verapamil was used as the reference unstable drug. The t_1/2 value and intrinsic clearance CL_int were calculated using the protocols and formulas proposed by Obach [51]. The microsomal protein/g of liver weight was considered as 45 mg, and the liver weight was 88 g/kg of body weight in the mouse.

3.4.3. Drug–Drug Interaction Studies

The prediction of drug–drug interactions was performed using the CYP3A4 and CYP2D6 P450-Glo^TM assays purchased from Promega (Madison, WI, USA). The influence of compounds on CYP’s activity was tested in white polystyrene, flat-bottom Nunc^TM MicroWell^TM 96-well microplates (Thermo Scientific, Waltham, MA, USA). The bioluminescence signal was measured with a microplate reader, Synergy H1 (BioTek, Winooski, VT, USA) in luminescence mode. Compounds 12 and 15 were tested in triplicate in two independent experiments in the range of 0.1–25 µM. The 1 µM of selective CYP inhibitor ketoconazole (3A4) and quinidine (2D6) was used as the positive control.

3.4.4. Preliminary Cell Toxicity

Cytotoxicity tests were performed in hepatoma HepG2 (ATCC^® HB-8065 ^TM; ATCC; Manassas, VA, USA), neuroblastoma SH-SY5Y (ATCC^® CRL-2266^TM; ATCC; Manassas, VA, USA) and colorectal adenocarcinoma Caco-2 (ATCC^® HTB-37^TM; ATCC; Manassas, VA, USA) cell lines. The viability of cells was assessed using the colourimetric MTS assay (CellTiter96^® Aqueous One Solution Cell Proliferation Assay kit, Promega, Madison, WI, USA). All cell culture media were purchased from ThermoFisher Scientific (Waltham, MA, USA). Cells were cultured under standard conditions (37 °C, 5% CO₂) in the recommended ATCC media supplemented with 10% (HepG2, SH-SY5Y) or 20% (Caco-2) of FBS. Before the assay, HepG2 cells were seeded into 96-well plates in 1.5 × 10⁴ cells/well, whereas Caco-2 and SH-SY5Y cells were seeded in 0.5 × 10⁴ cells/well. After 24 h of incubation, the medium was replaced, and cells were treated with the tested compounds 12 and 15. Doxorubicin was also added as a positive control. Following 72 h (HepG2, SH-SY5Y) or 48 h (Caco-2) exposure, the medium was removed, and 100 μL of fresh medium containing MTS reagent was added. After 2 h of incubation, absorbance at 490 nm was recorded using a Synergy H1 microplate reader (BioTek, Winooski, VT, USA). Wells without cells served as blanks, while 1% DMSO-treated wells represented controls for cell viability.

3.5. Activity Profile of Compound 15—In Vitro Studies

3.5.1. Cell Culture

The neuroblastoma SH-SY5Y cell line was obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA). The cells were cultured in DMEM/F-12 (Biowest, Nuaillé, France) supplemented with 10% foetal bovine serum (PAN-Biotech, Aidenbach, Germany), non-essential amino acids (Biowest, Nuaillé, France), and antibiotics (Life Technologies, Carlsbad, CA, USA) at 37 °C in a humidified atmosphere containing 5% CO₂. Peripheral blood mononuclear cells (PBMCs) were isolated from a leucocyte buffy coat collected from the blood of healthy non-smoking donors at the Blood Bank in Lodz, Poland. These cells include lymphocytes, monocytes, and other white blood cells with a round nucleus. The study protocol was approved by the Committee for Research on Human Subjects at the University of Lodz, Poland (12/KEBNUŁ/I/2024-2025) on 17 December 2024.

3.5.2. Cytotoxicity Assessment of Compound 15 and Its Effect on Cell Proliferation

The cytotoxicity of compound 15 was evaluated using PBMCs and SH-SY5Y cells. Additionally, in studies on SH-SY5Y cells, the effect of the compound on the cell cycle (by determining the 2N/4N DNA ratio index), as well as mitochondrial potential and mass, was checked (High Content Analysis Image Cytometry).

Effect of Compound 15 on Human PBMCs Viability

A portion of the leucocyte buffy coat was diluted in 1% phosphate-buffered saline (PBS), centrifuged, and cells were prepared as previously described in detail [52,53]. The cell pellet was resuspended in RPMI 1640 medium (Lonza, Basel, Switzerland), and the resazurin reduction assay was performed in the same manner as described previously by O’Brien et al. [54]. Resazurin salt powder was dissolved in sterile PBS. Cells were seeded on the 96-well plates at 5 × 10⁴ for PBMCs per well. Compound 15 was added to wells to achieve ten final concentrations within a range of 0.195–100 µM. Subsequently, the plates were incubated at 37 °C in 5% CO₂ for 2, 24, 48, and 72 h. Afterwards, 10 µL of resazurin salt was added to each well, and the plates were incubated again under the same conditions for 2 h. Next, fluorescence was measured with a microplate reader Synergy HT (BioTek Instruments, Winooski, VT, USA) using an excitation wavelength of 530/25 nm and an emission wavelength of 590/35 nm. The viability of PBMCs was assessed after 2, 24, 48, and 72 h of incubation with compound 15. The viability for each sample was calculated relative to the negative control (NC, untreated cells), which was set at 100%. The vehicle (Veh) consisted of PBMCs incubated with 0.1% DMSO. The results, expressed as means with SEM from four independent experiments, were given as percentages of untreated control cells. Values were plotted against all tested concentrations of compound 15 to determine the viability inhibition concentration at 50% (IC₅₀) using GraphPad Prism 6.07 (GraphPad Software, Inc., San Diego, CA, USA).

Assessment of the Effect of Compound 15 on SH-SY5Y Cell Proliferation, Mitochondrial Membrane Potential, and Mitochondrial Mass (High Content Analysis Image Cytometry)

The effect of compound 15 on cell proliferation and the cellular mitochondrial mass was assessed using image cytometry as described previously [53,55,56]. SH-SY5Y cells were initially seeded in 96-well plates at a density of 5000 cells per well. Twenty-four hours later, the cells were treated with various concentrations of compound 15 (range: 0.39–6.25 μM) or pitolisant (concentration range: 0.39–25 μM) for 24 or 72 h. Then, after adding MitoTracker Orange CMTMRos (500 nM) (Invitrogen, Eugene, Oregon, USA), the cells were incubated for 30 min, washed with PBS, fixed with 4% formaldehyde for 20 min and stained with 1 µg/mL Hoechst 33,342 (Life Technologies, Eugene, OR, USA) for 30 min at room temperature. Images of the cells were taken in two runs using an ArrayScan VTI HCS Reader (ThermoFisher Scientific, Inc., Waltham, MA, USA). First, a series of detailed images was taken with an ArrayScan VTI HCS Reader equipped with a 10x objective, and single cells were analysed with Cell Health Profiling Bioapplication V3 software (200 cells per well).

The mitochondrial mass was expressed as average total fluorescence (average of 200 cells). Cell number was calculated as the sum of the Hoechst-33342-stained cell nuclei using Cell Cycle Bioapplication V3 software and expressed as a percentage of control. The remaining adherent cells in culture wells indicated the cytotoxic potential of the tested compound. IC₅₀ values were calculated with a nonlinear fit to a sigmoidal dose—response curve (log compound vs. normalised response) via GraphPad Prism 6.07. Next, the DNA content of single SH-SY5Y cells was examined. A series of detailed images was captured using the ArrayScan VTI HCS Reader equipped with a 10× objective, collecting at least 500 nuclei per replicate. The DNA content of single cells was analysed based on Hoechst-33342 fluorescence intensity of their nuclei using Cell Cycle Bioapplication V3 software, and expressed as a DNA 2N/4N ratio. This index indicates the ratio of cells in the G0/G1 phase (DNA content = 2N, where N represents haploid DNA content) to those in the G2/M phase (DNA content = 4N). All experiments were conducted three times, each with four replicates. IC₅₀ values were determined by fitting a nonlinear model to a sigmoidal dose–response curve (log compound versus normalised response) using GraphPad Prism 6.07. The results are presented as mean ± SEM from at least three independent experiments. Statistical significance was evaluated using one-way ANOVA followed by Dunnett’s test. * p < 0.05, ** p < 0.01, and *** p < 0.001 indicate significant differences compared to control counterparts.

3.5.3. The Comet Assay—DNA Damage

The genotoxic potential of compound 15 was evaluated using the comet assay. The alkaline comet assay is a sensitive and straightforward method for detecting DNA damage, including single- and double-strand breaks, as well as alkaline-labile sites, in living cells. PBMCs were used in this study. Compound 15 was added to the PBM cell suspension to achieve final concentrations ranging from 0.39 to 12.5 µM. PBMCs were incubated for 2 h or 24 h at 37 °C in 5% CO₂. The experiment included, in addition to the Negative Control (NC, untreated cells) and Vehicle (Veh, PBMCs in 0.1% DMSO), a positive control (PC), which was a cell sample incubated with 25 µM H₂O₂ for 15 min on ice. The comet assay was performed under alkaline conditions according to the procedure described by Tokarz et al. [57]. A freshly prepared PBMC suspension in 0.75% LMP agarose dissolved in PBS was layered onto microscope slides (Superior, Germany), which were pre-coated with 0.5% NMP agarose. Then, the cells were lysed for 1 h at 4 °C in a buffer containing 2.5 M NaCl, 0.1 M EDTA, 10 mM Tris, 1% Triton X-100, pH = 10. After lysis, electrophoretic separation was performed in the solution containing 30 mM NaOH and 1 mM EDTA under alkaline conditions (pH > 13), at an ambient temperature of 4 °C (the temperature of the running buffer did not exceed 12 °C), for 20 min at an electric field strength of 0.73 V/cm (28 mA). Then, the slides were washed in water, drained, stained with 2 µg/mL DAPI, and covered with coverslips. To prevent additional DNA damage, the procedure was conducted under dark conditions. The comets were observed at 200× magnification using an Eclipse fluorescence microscope (Nikon, Japan) connected to a COHU 4910 video camera (Cohu, Inc., San Diego, CA, USA), which was equipped with a UV-1 A filter block and linked to a personal computer-based image analysis system, Lucia-Comet v. 7.3 (Laboratory Imaging, Praha, Czech Republic). One hundred images (comets) were randomly selected from each sample. A separate nucleoid consists of the undamaged part (the head) and the damaged part of DNA (the tail). The average DNA value in the comet tail was used as an indicator of DNA damage (expressed as a percentage). The longer and brighter the tail, the more severe the DNA damage.

4. Conclusions

In general, structural fusion of the chalcone core with the typical piperidinylpropoxy-H₃R ligand motif resulted in dual ligands: H₃R antagonists and MAO-B inhibitors. All hybrid compounds (10–18) had good affinity for hH₃R with K_i values < 170 nM, while only three of them showed inhibitory activity for hMAO-B in the (sub)micromolar range (200 nM < IC₅₀ < 1200 nM). Structure–activity relationship analysis indicated that a 3,4-dichloro substitution in the phenyl ring strongly enhanced hMAO-B inhibition, whereas a meta-benzyloxy attachment (opposed to a para-benzyloxy) in the piperidinyl chalcones favoured hH₃R affinity. Among the synthesised hybrids, compound 15 was the best DTL with a K_i of 46.8 nM for hH₃R and an IC₅₀ of 212.5 nM for hMAO-B. Additional pharmacological studies of this compound confirmed its antagonist profile in the cAMP assay (K_d = 34.2 nM) and a competitive/mixed mode of reversible hMAO-B inhibition which makes possible the use of drugs such as compound 15 in the treatment of PD.

The preliminary ADMET properties highlighted limited permeability and rapid metabolism, compound 15 also displayed marked antiproliferative (SH-SY5Y–72 h: IC₅₀ = 3.44 μM) and genotoxic effects in cell-based assays, further suggesting its value for evaluation in anticancer studies. In many types of cancer (including colorectal cancer, glioblastoma, breast cancer, lung cancer, pancreatic cancer, and liver cancer), MAO-B overexpression is observed, suggesting its connection with cancer development [58,59]. Several MAO-B inhibitors with anticancer potential have been identified. In preclinical studies, they affected cell proliferation and apoptosis. Similarly, H₃R overexpression occurs in cancer tissues compared to healthy tissues, i.e., in breast cancer, lung cancer, glioma, prostate cancer, and ovarian cancer. Some of the described H₃R ligands inhibited cancer cell proliferation and affected their growth [60]. Thus, such dual activity may provide a beneficial effect in the treatment of cancer. Furthermore, many chalcones have demonstrated anticancer activity against various tumour cell lines by affecting different molecular processes associated with tumour formation and development [61]. Thus, the potential of compound 15 in CNS tumours should be further investigated in detail. Currently, therapeutic strategies are available that aim to destroy or inhibit tumour cells, including those of the nervous system, while minimising damage to normal neurons and other healthy cells. An example of such a drug is vorasidenib, which selectively inhibits only the mutated isocitrate dehydrogenase enzymes present in certain gliomas (affecting tumour growth) while sparing healthy cells that have the wild-type enzyme [62].

In this study, we described, for the first time, the use of the SPRM technique to study the kinetics of H₃R ligands. Although it was not possible to perform such a study for compound 15 (due to its interactions with BSA), the other reference ligands (DL76 and pitolisant) were evaluated. The calculated residence times for them were similar, around 16 min, suggesting that these compounds could be classified as not rapidly dissociating.

In conclusion, our research confirmed that hybrid chalcones are a promising starting scaffold for DTLs, as H₃R antagonists, and MAO-B inhibitors, with potential utility not only in neurodegenerative diseases but also in anticancer therapy.