Discovery of Potential, Dual-Active Histamine H3 Receptor Ligands with Combined Antioxidant Properties

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Article Discovery of Potential, Dual-Active Histamine H3 Receptor Ligands with Combined Antioxidant Properties

Kamil J. Kuder 1,* , Magdalena Kota ńska 2 , Katarzyna Szczepa ńska 1, Kamil Mika 2, David Reiner-Link 3 , Holger Stark 3 and Katarzyna Kieć-Kononowicz 1

1 Department of Technology and Biotechnology of Drugs, Faculty of Pharmacy, Jagiellonian University Medical College, Medyczna 9, 30-699 Kraków, Poland; [email protected] (K.S.); [email protected] (K.K.-K.) 2 Department of Pharmacodynamics, Faculty of Pharmacy, Jagiellonian University Medical College, Medyczna 9, 30-699 Kraków, Poland; [email protected] (M.K.); [email protected] (K.M.) 3 Institute of Pharmaceutical and Medicinal Chemistry, Heinrich Heine University Düsseldorf, Universitaetsstr. 1, 40225 Duesseldorf, Germany; [email protected] (D.R.-L.); [email protected] (H.S.) * Correspondence: [email protected]; Tel.: +48-12-620-5583

Abstract: In an attempt to ﬁnd new dual acting histamine H3 receptor (H3R) ligands, we designed a series of compounds, structurally based on previously described in our group, a highly active and

selective human histamine H3 receptor (hH3R) ligand KSK63. As a result, 15 obtained compounds show moderate hH3R affinity, the best being the compound 17 (hH3R Ki = 518 nM). Docking to the histamine H3R homology model revealed two possible binding modes, with key interactions retained in both cases. In an attempt to find possible dual acting ligands, selected compounds were tested for antioxidant properties. Compound 16 (hH3R Ki = 592 nM) showed the strongest Citation: Kuder, K.J.; Kotańska,M.; antioxidant properties at the concentration of 10−4 mol/L. It significantly reduced the amount of Szczepańska,K.; Mika, K.; free radicals presenting 50–60% of ascorbic acid activity in the 2,2-diphenyl-1-picrylhydrazyl (DPPH) Reiner-Link, D.; Stark, H.; assay, as well as showed antioxidative properties in the ferric reducing antioxidant power (FRAP) Kieć-Kononowicz,K. Discovery of assay. Despite the yet unknown antioxidation mechanism and moderate hH3R affinity, 16 (QD13) Potential, Dual-Active Histamine H3 constitutes a starting point for the search of potential dual acting H R ligands-promising tools for the Receptor Ligands with Combined 3 Antioxidant Properties. Molecules treatment of neurological disorders associated with increased neuronal oxidative stress. 2021, 26, 2300. https://doi.org/ 10.3390/molecules26082300 Keywords: histamine H3 receptor; histamine H3 receptor ligands; piperazine derivatives; molecular modeling; antioxidative agents Academic Editor: Simona Rapposelli

Received: 12 March 2021 Accepted: 13 April 2021 1. Introduction Published: 15 April 2021 Histamine H3 receptor (H3R) is an outstanding member of the histamine receptor family. Since its discovery and gene cloning, it has served as a widely explored drug Publisher’s Note: MDPI stays neutral target due to its broad spectrum of neuromodulatory effects in the central nervous system with regard to jurisdictional claims in (CNS) [1]. On the one hand, activation of presynaptic receptors on histaminergic neurons published maps and institutional afﬁl- results in regulation of synthesis and release of the natural ligand histamine. On the other iations. hand, activation of H3R as a heteroreceptor inﬂuences the whole range of other neurotransmitters, including acetylcholine, dopamine, serotonin, norepinephrine, γ-aminobutyric acid, glutamate, and substance P, to name a few [2–4]. Anatomically speaking, H3Rs are prominently expressed in humans in the basal ganglia, globus pallidus, hippocampus, Copyright: © 2021 by the authors. and cortex–regions associated with cognition, sleep, wakefulness, and homeostatic regu- Licensee MDPI, Basel, Switzerland. lation [5]. Consequently, H3R ligands, either antagonist or inverse agonists, as they also This article is an open access article demonstrate constitutive activity, contribute to one of the largest groups of contempo- distributed under the terms and rary GPCR ligand research, with implications to treat a number of CNS-based diseases, conditions of the Creative Commons including Alzheimer’s (AD) and Parkinson’s disease (PD), schizophrenia, ADHD, obesity, Attribution (CC BY) license (https:// and narcolepsy [6,7]. However, while several big pharma companies were working on creativecommons.org/licenses/by/ 4.0/). development of H3R inhibiting ligand and reached the Phase III studies in these indications,

Molecules 2021, 26, 2300. https://doi.org/10.3390/molecules26082300 https://www.mdpi.com/journal/molecules Molecules 2021, 26, 2300 2 of 18

all of them failed. The only compound, pitolisant, reached the market under the name of Wakix® (Figure1), with an indication for narcolepsy treatment [ 8]. One exception worth mentioning is the LML134 compound (Figure1), which completed the Phase I study by means of safety and tolerability, and went for further trials, with promising results for the treatment of excessive sleep disorders. However, the studies were terminated due to business issues [9,10]. At the same time, increasing evidence indicates that oxidative stress plays an important role in the pathogenesis of many diseases. H3 receptor-induced activation of Gi/o-proteins seems to stimulate phospholipase A2 (PLA2) to induce the release of arachidonic acid, which, under pathological conditions, act as precursors for various inflammatory compounds, which in turn are crucial in mediating the oxidative and inflammatory responses in CNS pathologies, such as stroke, AD, PD and multiple sclerosis [11]. In fact, histamine H3 receptor ligands that might inhibit oxidative stress by reducing free radical concentrations have been shown to have potential therapeutic applications in several neurological disorders, such as Parkinson’s [12], Alzheimer’s disease [13], cerebral ischemia/stroke [14], depressive illness [15], epilepsy [16,17], schizophrenia [18], autism [19], and cardiovascular disorders, such as myocardial infarction [20]. All of these studies show that the antioxidant activities of histamine H3 receptor ligands play a significant role in reducing oxidative stress that accompany the above mentioned diseases. Throughout the years, an abundant number of H3R ligands have been synthesized, based on various heterocyclic moieties, starting from histamine’s native imidazole moiety to (substituted) piperidines, pyrrolidines, and piperazines, among others. Yet, the overall structure remained unchanged: basic cores connected via (rigidified) alkyl chain with polar moiety, which in turn is connected, either directly or via a second linker, with a so-called “eastern arbitrary region” [21]. While the latter ones are believed to modulate potency and pharmacokinetic properties of H3R antagonists/inverse agonists, the heterocyclic moiety maintains the necessary interactions with binding pocket amino acids, essential for the receptor binding. Recent publications from our group [22–24] allowed for the establishment of unique heterocyclic moiety, namely 4-pyridylpiperazine as a new bioisosteric replacement of piperidine. Molecular modeling studies confirmed its ability to form additional interactions, besides those of piperazine protonated nitrogen, with highly conserved H3R amino acids. These findings were tested in vitro, proving this scaffold to be a crucial element for high H3R affinity. In turn, bulky substituents of the so-called “eastern region”, such as benzophenones, have also shown to act as possible additional H-bond acceptor moiety, strengthening ligand-receptor interactions. It was again proved in vitro, resulting in one of the highest affine H R ligands from our group, KSK63 (hH R K = 3.12 nM, Figure1). Molecules 2021, 25, x FOR PEER REVIEW 3 3 i 3 of 18

Figure 1. Structure and properties of reference histamine H3 receptor ligands [9,24–26] (Ki-inhibi- Figure 1. Structure and properties of reference histamine H3 receptor ligands [9,24–26](Ki-inhibitory tory constant, IC50-half maximal inhibitory concentration) constant, IC50-half maximal inhibitory concentration). In light of the presented findings, we designed the series of ligands with modifications in the hydrophilic and distal regulatory regions, namely by introducing different substituents in the piperazine (R1) and benzophenone (R2) moieties, respectively, based on the general structure depicted in Figure 2. The high activity associated with the basic part of the molecule, prompts to look for new scaffolds, even if many attempts lead to inactive compounds. For this series, we have chosen five different (cyclo)alkyl carbonyl and 4-arylpiperazine substituents. Using direct (cyclo)alkyl carbonyl piperazine N4 nitrogen substituents, we wanted to test whether such reduction in piperazine N4 nitrogen negative charge would still be acceptable for the retention of H3R affinity [27]. On the other hand, in order to avoid the possible overlap of inductive and mesomeric effects of p-substituted methoxy group, we have chosen m-substituted derivatives, so as to take only the former one into account. On the eastern end, we tested whether the increase of the hydrophobicity, by introduction of halogen atoms to benzophenone moiety, is also tolerated. Hence, it is relevant to search for novel bioactive compounds able to combine in one molecule multiple action properties. The search for possible dual active H3R ligands, which might act as neuroprotectives, both directly (by expressing anti-oxidative properties) and indirectly (through the effect of H3R antagonism), seems necessary, as their applications might be beneficial in future therapies. Even though the obtained compounds lack the phenolic protons believed to be essential for the antioxidant activity, paying attention to, e.g., the structure of the known antioxidant melatonin [28], with respect to recent findings on the tetratargeting Contilisant [26,29], selected compounds were tested for possible antioxidative properties. Last, but not least, biological test results were complemented by the docking studies to the histamine H3 receptor homology model.

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In light of the presented findings, we designed the series of ligands with modifications in the hydrophilic and distal regulatory regions, namely by introducing different substituents in the piperazine (R1) and benzophenone (R2) moieties, respectively, based on the general structure depicted in Figure2. The high activity associated with the basic part of the molecule, prompts to look for new scaffolds, even if many attempts lead to inactive compounds. For this series, we have chosen five different (cyclo)alkyl carbonyl and 4-arylpiperazine substituents. Using direct (cyclo)alkyl carbonyl piperazine N4 nitrogen substituents, we wanted to test whether such reduction in piperazine N4 nitrogen negative charge would still be acceptable for the retention of H3R affinity [27]. On the other hand, in order to avoid the possible overlap of inductive and mesomeric effects of p-substituted methoxy group, we have chosen m-substituted derivatives, so as to take only the former one into account. On the eastern end, we tested whether the increase of the hydrophobicity,

Molecules 2021, 25, x FOR PEER REVIEWby introduction of halogen atoms to benzophenone moiety, is also tolerated. 4 of 18

FigureFigure 2. General structure structure of of designed designed ligands. ligands.

2. ResultsHence, and it isDiscussion relevant to search for novel bioactive compounds able to combine in one molecule2.1. Chemistry multiple action properties. The search for possible dual active H3R ligands, which might act as neuroprotectives, both directly (by expressing anti-oxidative properties) and The desired final compounds 4–18 were obtained through the synthetic route pre- indirectly (through the effect of H R antagonism), seems necessary, as their applications sented in Scheme 1, according to the3 previously reported methods [23,30]. Starting phe- mightnoxy-alkyl be beneficial bromides in were future obtained therapies. in one-step Even though O-alkylation the obtained of commercially compounds available lack the phenolic(substituted) protons p-hydroxy believed benzophenones to be essential with for 1, the3-dibromopropane, antioxidant activity, refluxed paying either attention in so- to, e.g.,dium the propanolate structure of (1 the) or known acetone antioxidant with powdered melatonin potassium [28], with carbonate, respect and to recent a catalytic findings onamount the tetratargeting of potassium iodide Contilisant (2, 3). Obtained [26,29], selected precursor compounds bromides were were then tested coupled for possible with antioxidativecommercially properties.available 4-substituted Last, but not piperazi least, biologicalnyl derivatives test results in the were mixture complemented of etha- by thenol/water docking with studies powdered to the potassium histamine Hcarbonate3 receptor and homology a catalytic model. amount of potassium iodide. Final products were obtained either as quick crystallizing free bases (5, 10–15), or 2. Results and Discussion were isolated as oxalic acid salts (4, 6–9, 16–18). For detailed information, please refer to 2.1.the Materials Chemistry and Methods section. The desired final compounds 4–18 were obtained through the synthetic route presented in Scheme1, according to the previously reported methods [ 23,30]. Starting phenoxyalkyl bromides were obtained in one-step O-alkylation of commercially available (substituted) p-hydroxy benzophenones with 1,3-dibromopropane, refluxed either in sodium propanolate (1) or acetone with powdered potassium carbonate, and a catalytic amount of potassium iodide (2, 3). Obtained precursor bromides were then coupled with commercially available 4-substituted piperazinyl derivatives in the mixture of ethanol/water with powdered potassium carbonate and a catalytic amount of potassium iodide. Final products were obtained either as quick crystallizing free bases (5, 10–15), or were isolated as oxalic acid salts (4, 6–9, 16–18). For detailed information, please refer to the Materials and Methods section.

Scheme 1. General synthetic pathway for compounds 4–18.

2.2. Biological Evaluation

All compounds, in either basic or oxalate forms, were then tested in H3R binding studies in vitro, as described previously [31,32]. In brief, compounds were tested at five to eleven appropriate concentrations in a [3H]Nα-methylhistamine (KD = 3.08 nM) radiolig- and depletion assay to determine the affinity for the human recombinant histamine H3R

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Figure 2. General structure of designed ligands.

2. Results and Discussion 2.1. Chemistry The desired final compounds 4–18 were obtained through the synthetic route presented in Scheme 1, according to the previously reported methods [23,30]. Starting phenoxy-alkyl bromides were obtained in one-step O-alkylation of commercially available (substituted) p-hydroxy benzophenones with 1,3-dibromopropane, refluxed either in sodium propanolate (1) or acetone with powdered potassium carbonate, and a catalytic amount of potassium iodide (2, 3). Obtained precursor bromides were then coupled with commercially available 4-substituted piperazinyl derivatives in the mixture of ethanol/water with powdered potassium carbonate and a catalytic amount of potassium iodide. Final products were obtained either as quick crystallizing free bases (5, 10–15), or Molecules 2021, 26, 2300 4 of 18 were isolated as oxalic acid salts (4, 6–9, 16–18). For detailed information, please refer to the Materials and Methods section.

SchemeScheme 1. 1.GeneralGeneral synthetic synthetic pathway forfor compounds compounds4– 184–18. .

2.2.2.2. Biological Biological Evaluation Evaluation All compounds, in either basic or oxalate forms, were then tested in H R binding All compounds, in either basic or oxalate forms, were then tested in3 H3R binding studies in vitro, as described previously [31,32]. In brief, compounds were tested at five to studies in vitro, as described previously3 [31,32α ]. In brief, compounds were tested at five eleven appropriate concentrations in a [ H]N -methylhistamine (KD = 3.08 nM) radioli- to eleven appropriate concentrations in a [3H]Nα-methylhistamine (KD = 3.08 nM) radiolig- gand depletion assay to determine the affinity for the human recombinant histamine H3R andstably depletion expressed assay in HEK-293to determine cells. the For affini poorlyty soluble for the compounds, human recombinant16 and 17, an histamine addition H3R to 0.025% HCl in the binding test buffer was used. Human H3R binding characterization of the tested compounds are presented in Table1 . Most of the tested compounds show relatively low to no histamine H3 receptor affinity, with the exception of p-nitro- (14, 15) and m-methoxyphenyl derivatives (16–18), which display affinities at moderate levels, with compound 17 being the most affine (hH3R Ki = 518 nM) of the group. Regarding the piperazine substituents, one could observe that direct substitution of piperazine N4 nitrogen with simple (cyclo)alkyl carbonyl moieties has negative influence on hH3R affinity. Slightly higher values were demonstrated for p-acetylphenyl derivatives; however, still at an unacceptable level. It appears that such a group is not suitable for histamine H3 receptor binding. The reasoning behind these observations might be the low pKa value of N4 nitrogen of the obtained compounds. With, e.g., pKa = −6.0 for compounds 4–6, it might be assumed that the majority of the compounds at physiological pH are in non-ionized form. This might result in a lack of crucial interactions with binding pocket amino acids, resulting in very low or no affinity at all. It appears that only m-methoxy- (16–18) and p-nitrophenyl derivatives (14, 15) express the pKa at a level that allows for abundance of ionized forms. Calculated pKa values can be found in Table S1 of the Supplementary Material. Molecules 2021,, 25,, xx FORFOR PEERPEER REVIEWREVIEW 5 of 18 Molecules 2021, 25, x FOR PEER REVIEW 5 of 18 MoleculesMolecules 2021, 25 2021, x FOR, 25 ,PEER x FOR REVIEW PEER REVIEW 5 of 18 5 of 18 stably expressed in HEK-293 cells. For poorly soluble compounds, 16 andand 17,, anan additionaddition stably expressed in HEK-293 cells. For poorly soluble compounds, 16 and 17, an addition stablyto 0.025% stablyexpressed HCl expressed in inthe HEK-293 binding in HEK-293 cells.test buffer For cells. poorly wasFor poorlyused. soluble soluble compounds, compounds, 16 and 16 17 and, an 17addition, an addition to 0.025% HCl in the binding test buffer was used. Humanto 0.025% H3 RHCl binding in the characterization binding test buffer of the was test used.ed compounds are presented in Table 3 Human H3R binding characterization of the tested compounds are presented in Table 1. Most of theHuman tested H compounds3R binding characterization show relatively of low the to test noed histamine compounds H3 receptorreceptor are presented affinity,affinity, in Table 1. Most of the tested compounds show relatively low to no histamine H3 receptor affinity, 1.with Most the1. of Most exception the testedof the of testedcompounds p-nitro- compounds (14 show,, 15) relativelyandshow m relatively-methoxyphenyl low to low no histamine to no derivatives histamine H3 receptor ( 16H3– receptor18 ),affinity, which affinity, with the exception of p-nitro- (14,, 15) and m-methoxyphenyl derivatives (16–18), which displaywith affinities the exception at moderate of p-nitro- levels, (with14, 15 compound) and m-methoxyphenyl 17 beingbeing thethe mostmost derivatives affineaffine (hH(hH (163R–18 K),ii = =which display affinities at moderate levels, with compound 17 being the most affine (hH3R Ki = display518 nM)display affinities of the affinities group. at moderate Regarding at moderate levels, the levels, piperazinewith compound with substituents,substituents, compound 17 being 17 oneone thebeing couldcould most the observeobserve affine most affine(hH thatthat3 R directdirect (hHKi =3 R Ki = 518 nM) of the group. Regarding the piperazine substituents, one could observe that direct 518substitution nM)518 of nM) the of group.of piperazine the group. Regarding N Regarding4 nitrogen the piperazine withthe piperazine simple substituents, (cyclo)alkyl substituents, one carbonyl could one observecould moieties observe that has direct neg-that direct substitution of piperazine N4 nitrogen with simple (cyclo)alkyl carbonyl moieties has negative substitutioninfluence on of hHpiperazine3R affinity. N4 nitrogen Slightly with higher simple values (cyclo)alkyl were demonstrated carbonyl moieties for hasp- neg- ative influence on hH3R affinity. Slightly higher values were demonstrated for p- ativeacetylphenyl ativeinfluence influence derivatives; on hH on3R however,hHaffinity.3R affinity. Slightlystill at Slightly an higher unacceptable higher values values level.were Itweredemonstrated appears demonstrated that suchfor p a-for p- acetylphenyl derivatives; however, still at an unacceptable level. It appears that such a groupacetylphenyl is not suitable derivatives; for histamine however, H3 receptorreceptor still at binding.binding. an unacceptable TheThe reasoningreasoning level. behindbehindIt appears thesethese that ob-ob- such a 3 group is not suitable for histamine H3 receptorreceptor binding.binding. TheThe reasoningreasoning behindbehind thesethese ob-ob- servationsgroup might is not be suitable the low for pK histaminea valuevalue ofof HN34 receptornitrogen binding.of the obtained The reasoning compounds. behind With, these ob- a servations might be the low pKa valuevalue ofof N4 nitrogen of the obtained compounds. With, e.g., pKservationsa == −6.0 for might compounds be the low 4– pK6, itita valuemightmight of bebe N 4assumedassumed nitrogen thatthat of the thethe obtained majoritymajority compounds. ofof thethe com-com- With, e.g., pKa = −6.0 for compounds 4–6, it might be assumed that the majority of the com- e.g.,pounds pKe.g.,a at = physiologicalpK−6.0a =for −6.0 compounds for pH compounds are in4– 6,non-ionized it 4might–6, it mightbe form. assumed be This assumed mightthat the resultthat majority the in amajority lack of theof crucial ofcom- the compounds at physiological pH are in non-ionized form. This might result in a lack of crucial poundsinteractionspounds at physiological with at physiological binding pH pocket are pH in amino arenon-ionized in acids,non-ionized resulting form. Thisform. in might very This low mightresult or noresultin aaffinity lack in aof lack atcrucial all. of It crucial interactions with binding pocket amino acids, resulting in very low or no affinity at all. It interactionsappearsinteractions that with only binding withm-methoxy- binding pocket ( pocket16 amino–18) andamino acids, p-nitrophenyl acids,resulting resulting in derivativesvery in low very or low (no14, , affinity or15 )no express affinity at all. the Itat all. It appears that only m-methoxy- (16–18) and p-nitrophenyl derivatives (14,, 15) express the pKa atatappears aa levellevel thatthat allowsallowsonly m -methoxy-forfor abundanceabundance (16– 18ofof) ionized ionizedand p-nitrophenyl forms.forms. CalculatedCalculated derivatives pKa valuesvalues(14, 15 )cancan express bebe the pKa at a level that allows for abundance of ionized forms. Calculated pKa values can be pKfounda atpK ina level aTable at a that levelS1 ofallows thatthe Supplementaryallows for abundance for abundance Material. of ionized of ionized forms. forms. Calculated Calculated pKa values pKa values can be can be found in Table S1 of the Supplementary Material. foundThe foundin Tableoverall in S1Table affinity of the S1 ofSupplementaryrow the for Supplementary 4-piperaziny Material.l substituents Material. can be summarized as m-meth- The overall affinity row for 4-piperazinyl substituents can be summarized as m-meth- oxyphenylThe overall The> p-nitrophenyl overall affinity affinity row > forp-acetylphenyl row 4-piperaziny for 4-piperaziny >l cyclopropylmethasubstituentsl substituents can benone can summarized be > acyl summarized (best as tom- worst)meth- as m-methoxyphenyl > p-nitrophenyl > p-acetylphenyl > cyclopropylmethanone > acyl (best to worst) oxyphenylfor theoxyphenyl herein > p-nitrophenyl presented > p-nitrophenyl series. > p-acetylphenyl Moreover, > p-acetylphenyl no > cyclopropylmetha clear > cyclopropylmethastructure–affinitynone > nonerelationship acyl (best> acyl to (best amongworst) to worst) for the herein presented series. Moreover, no clear structure–affinity relationship among forthe thethreefor herein benzophenonethe herein presented presented series.modified series. Moreover, derivativesderivatives Moreover, no clear cancan no bebe structure–affinity clear drawn.drawn. structure–affinity relationship relationship among among the three benzophenone modified derivatives can be drawn. the threethe benzophenonethree benzophenone modified modified derivatives derivatives can be can drawn. be drawn. 2.3. Antioxidative Properties 2.3. Antioxidative Properties In2.3. order Antioxidative to determine Properties the possible antioxidant properties, selected compounds were In order to determine the possible antioxidant properties, selected compounds were testedIn inorder 2,2-diphenyl-1-picrylhydrazylIn toorder determine to determine the possible the possible antioxidant (DPP antioxidantH) and properties, ferric properties, reducing selected selected antioxidant compounds compounds power were were tested in 2,2-diphenyl-1-picrylhydrazyl (DPPH) and ferric reducing antioxidant power tested(FRAP) testedin assay 2,2-diphenyl-1-picrylhydrazyl in with 2,2-diphenyl-1-picrylhydrazyl ascorbic acid as a reference (DPPH) (DPP compoundandH) ferric and reducingferric(Table reducing 2). antioxidantFor details, antioxidant powerplease power (FRAP) assay with ascorbic acid as a reference compound (Table 2). For details, please (FRAP)refer to(FRAP) assaythe Materials assaywith ascorbic with and ascorbic Methods acid as acid Section.a reference as a referenceGraphical compound compoundrepresentation (Table (Table 2). Forof tests2). details, For results details, please can please refer to the Materials and Methods Section. Graphical representation of tests results can referbe found torefer the in to MaterialsSupplementary the Materials and Methods andMaterial. Methods Section. Section. Graphical Graphical representation representation of tests of results tests results can can be found in Supplementary Material. be foundAntioxidantbe foundin Supplementary in activitySupplementary at theMaterial. level Material. above 50% of ascorbic acid activity at a concentration Antioxidant activity at the level above 50% of ascorbic acid activity at a concentration of 10Antioxidant−4 mol/L might activity be considered at the level noteworthy. above 50% ofIn ascorbicthe DPPH acid assay, activity compound at a concentration 16 (QD13) of 10−4 mol/LAntioxidant might be consideredactivity at the noteworthy. level above In 50% the ofDPPH ascorbic assay, acid compound activity at 16 a concentration(QD13) of 10−4 mol/L might be considered noteworthy. In the DPPH assay, compound 16 (QD13) ofat 10a concentration mol/L−4 might of be 10 considered−4 mol/L caused noteworthy. a decrease In the in DPPHabsorbance assay, by compound 12.37 ± 1.31% 16 (QD13) when at a concentrationof 10 mol/L ofmight 10−4 mol/Lbe considered caused anoteworthy. decrease in In absorbance the DPPH by assay, 12.37 compound ± 1.31% when 16 (QD13) at a concentration of 10−4 mol/L caused a decrease in absorbance by 12.37 ± 1.31% when atcompared a concentrationat a concentrationto the sample of 10 containingof mol/L 10−4 mol/Lcaused the caused solventa decrease a +decrease reaction in absorbance in mixture absorbance by (blank, 12.37 by maximum ±12.37 1.31% ± 1.31%when con- when compared to the sample containing the solvent + reaction mixture (blank, maximum con- comparedcentration toof the the sample DPPH containingradical). Moreover, the solvent 16, + atreaction a concentration mixture (blank, of 10−4 maximum, had 60.28% con- of centrationcompared of the to DPPH the sample radical). containing Moreover, the 16, solvent at a concentration + reaction mixture of 10 −(blank,4 , had 60.28%maximum of concentration of the DPPH radical). Moreover, 16, at a concentration of 10−4, had 60.28% of Molecules 2021, 26, 2300 centrationthe ascorbic of acidthe DPPH activity radical). (10−4 mol/L, Moreover, calculated 16, at froma concentration Table 2). In of fact, 10 of, had all− 460.28%the tested of 5 of 18 the ascorbiccentration acid of activity the DPPH (10−4 radical). mol/L, calculatedMoreover, from 16, at Table a concentration 2). In fact, of 10all the, had tested 60.28% of the ascorbic acid activity (10−4 mol/L, calculated from Table 2). In fact, of all the tested thecompounds, ascorbicthe ascorbic acid16 expressedexpressed activity acid activity (10 thethe strongeststrongest mol/L, (10−4 mol/L, calculated antioxidantantioxidant calculated from properties.properties. Tablefrom Table2). In fact,2). In of fact, all theof all tested the tested compounds, 16 expressed the strongest antioxidant properties. compounds,compounds, 16 expressed 16 expressed the strongest the strongest antioxidant antioxidant properties. properties. Table 1. Structures of compounds 4–18 andand theirtheir inin vitrovitro humanhuman histaminehistamine HH3 receptorreceptor (hH(hH3R) 3 3 Table 1.Table Structures 1. Structures of compounds of compounds 4–18 and4 their–18 andin vitro their humanin vitro histaminehuman histamineH3 receptor H (hH3 receptor3R) (hH3R) Tableaffinities. 1.Table Structures Given 1. Structures data of representcompounds of compounds mean 4–18 values and 4–18 their withinwithin and in their vitrothethe 95%95%in human vitro confidenceconfidence human histamine histamineintervalinterval H receptor (CI).(CI). H3 receptor (hH R) (hH 3R) affinities. Given data represent mean values within the 95% confidence interval (CI). affinities.affinities.affinities. Given dataGiven Given represent data data represent represent mean values mean mean valueswithin values withinthe within 95% the the confidence 95% 95% confidence confidence interval interval (CI). (CI).

3 i 1 2 hH3R Ki [nM][nM] Compound R1 R2 3 i Compound R1 R2 hH3R Ki [nM] Compound R1 R2 hHx̅ ̅ [CIR KhH 95%] [nM]3R K i [nM] Compound 1 R 1 2 R 2 x [CI 95%] CompoundCompound R R R hHR 3R Ki [nM]x̅ ̅ [CI 95%] x¯ [CI 95%] 4 x [CI3653 95%]x ̅ [CI 95%] 4 H 3653 QD1144 4 H [2024,36533653 6594] 3653 QD11 H H [2024, 6594] QD11QD11 H [2024,[2024, 6594] 6594] 5 QD11 1257[2024, 6594] 5 Cl 1257 55 Cl 12571257 QD15 5 Cl Cl [599, 2638]1257 QD15QD15 Cl [599,[599, 2638] 2638] QD15 [599, 2638] 6 1100 66 F 11001100 QD46 6 F F [535,1100 2263] 1100 QD4QD4 F F [535,[535, 2263] 2263] MoleculesMolecules 20212021,, 2525,, xx FORFOR PEERPEER REVIEWREVIEW QD4 QD4 [535, 2263][535,66 ofof 2263]1818 Molecules 20212021,, 2525,, xx FORFOR PEERPEER REVIEWREVIEW 7 1334 66 ofof 1818 MoleculesMolecules 2021 2021, ,25 25, ,x x FOR FOR PEER PEER REVIEW REVIEW 77 H 133413346 6 ofof 1818 MoleculesMolecules 2021 2021, ,,25 25, ,,x xx FOR FORFOR PEER PEERPEER REVIEW REVIEWREVIEW 7 H H 13346 6 ofof 1818 QD14QD14 7 H [274,[274, 6505] 6505]1334 QD1488 H [274, 6505] 88 QD14 ClCl >1000>1000[274, 6505] QD19888 Cl >1000 QD1988 ClCl >1000>1000 QD19QD19 ClClCl >1000>1000>1000 QD19QD19QD1999 16691669 9 FF 1669 QD899 F [596,16691669 4676] QD8999 FF [596,166916691669 4676] QD8QD8 F FF [596,[596, 4676] 4676] QD8QD8QD8 [596,[596,[596,[596, 4676] 4676] 4676]4676] QD8QD8 [596,[596, 4676] 4676] 1010 633633 1010 HH 633633 QD101010 H [206,633633 1942] QD10101010 HH [206,633633633 1942] QD10QD10 H HH [206,[206, 1942]1942] QD10QD10QD10 [206,[206,[206,[206, 1942] 1942] 1942]1942]

1111 1111 ClCl >1000>1000 QD17QD171111 ClCl >1000>1000 QD17111111 ClCl >1000>1000 QD17QD17 ClClCl >1000>1000>1000 QD17QD17QD17

1212 10081008 1212 FF 10081008 QD3QD31212 FF [618,[618,10081008 1646]1646] QD3121212 FF [618,[618,100810081008 1646]1646] QD3QD3 F FF [618,[618, 1646] 1646] QD3QD3QD3 [618,[618,[618,[618, 1646] 1646] 1646]1646] 1313 1313 HH >1000>1000 QD121313 H >1000 QD12131313 HH >1000>1000 QD12QD12 HHH >1000>1000>1000 QD12QD12QD12 1414 622622 14 Cl 622 1414 Cl 622622 QD20QD20141414 ClCl [211,[211,622622622 1832]1832] QD20QD20 Cl ClCl [211,[211, 1832] 1832] QD20QD20QD20 Cl [211,[211,[211,[211, 1832] 1832] 1832]1832] QD20QD20 [211,[211, 1832] 1832] 1515 580580 151515 FF 580580580 QD6QD61515 F FF [177,[177,580580 1902]1902] QD6QD61515 FF [177,[177,[177,580580 1902] 1902]1902] QD6QD6 FF [177,[177, 1902] 1902] QD6QD6 [177,[177, 1902] 1902]aa QD61616 [177,592592 a1902] a 16 H 592592a a 1616 H 592592 a a QD13QD131616 H HH [394,[394,592592 889]889]a a a QD13QD1316 HH [394,[394,[394,592 889] 889]889] QD13QD13 HH [394,[394, 889]889]a QD13QD1317 [394,[394,518 889] 889] a QD13QD1317 [394,[394,518 a889] 889] a 1717 ClCl 518518a a 1717 Cl 518518 a a QD18QD181717 Cl ClCl [201,[201,518518 1336]1336] a a QD18QD18QD18 ClCl [201,[201,[201, 1336] 1336] 1336] QD18QD18 Cl [201,[201,[201, 1336] 1336]1336] QD18QD181818 [201,[201,715715 1336] 1336] 18 F 715715 1818 F F 715715 QD9QD91818 FF [352,[352,[352,715715 1453] 1453]1453] QD9QD9 FF [352,[352, 1453] 1453] a QD9QD9 F [352,[352, 1453] 1453] a ValuesQD9QD9 were obtained using 0.025% HCl in binding buffer. Values in square brackets[352, indicate[352,[352, 1453] 1453]1453] the confidence a ValuesValues werewere obtainedobtained usingusing 0.025%0.025% HClHCl inin bindingbinding buffer.buffer. ValuesValues inin squaresquare bracketsbrackets indicateindicate thethe aa Values were obtained using 0.025% HCl in binding buffer. Values in square brackets indicate the aa Values Valuesinterval were were obtained ofobtained 95%. K iusing -inhibitoryusing 0.025% 0.025% constant. HCl HCl in in binding binding buffer. buffer. Values Values in in square square brackets brackets indicate indicate the the aconfidence aValues Values were were interval obtained obtained of 95%. using using K i0.025% i-inhibitory0.025% HCl HCl constant. in in binding binding buffer. buffer. Values Values in in square square brackets brackets indicate indicate the the a confidenceValues ValuesValues were werewere interval obtained obtainedobtained of 95%.using usingusing K 0.025% i0.025%-inhibitory0.025% HCl HClHCl constant.in inin binding bindingbinding buffer. buffer. Values Values in in square square brackets brackets indicate indicate the the confidenceconfidence interval interval of of 95%. 95%. K Ki-inhibitoryi-inhibitory constant. constant. confidenceconfidence interval interval of of 95%. 95%. K Ki-inhibitoryi-inhibitory constant. constant. confidenceconfidence interval intervalThe overall of of 95%. 95%.affinity K Kii-inhibitoryii-inhibitory-inhibitory row constant. forconstant.constant. 4-piperazinyl substituents can be summarized as m- EightEightmethoxyphenyl ofof thethe otherother > compoundscompoundsp-nitrophenyl showedshowed > p-acetylphenyl aa slightlyslightly smallersmaller > cyclopropylmethanone butbut pronouncedpronounced decreasedecrease > acyl inin (best to EightEight of of the the other other compounds compounds showed showed a a slightly slightly smaller smaller but but pronounced pronounced−4 decrease decrease in in absorbanceEightEight ofof (antioxidant thethe otherother compoundscompounds activity >20% showedshowed of ascorbic a a slightlyslightly acid smallersmaller activity butbut at pronouncedpronounced 10 −4 mol/L); decrease decreasethus, scav- inin absorbanceEightEightworst) of of (antioxidantthe forthe other theother herein compounds compounds activity presented >20% showed showed series. of ascorbic a a slightly Moreover,slightly acid smaller smaller activity no clear but but at pronounced structure–affinitypronounced 10−4 mol/L); decrease decreasethus, relationship scav- in in absorbance (antioxidant activity >20% of ascorbic acid activity at 10−−44 mol/L); thus, scav- absorbanceabsorbance (antioxidant(antioxidant activityactivity >20%>20% ofof ascorbicascorbic acidacid activityactivity atat 1010−4− 4 mol/L);mol/L); thus,thus, scav-scav- absorbanceengingabsorbanceengingamong DPPHDPPH (antioxidant(antioxidant the freefree three radicalsradicals benzophenone activityactivity (Table(Table >20%>20% 2,2, leftleft modifiedof of panel).panel). ascorbicascorbic derivatives acidacid activityactivity can atat be 1010 drawn.−−44 mol/L); mol/L);mol/L); thus, thus,thus, scav- scav-scav- engingenging DPPHDPPH freefree radicalsradicals (Table(Table 2,2, leftleft panel).panel). engingengingOnOn DPPH DPPH thethe otherother free free hand,radicals hand,radicals inin (Table th(Tablethee FRAPFRAP 2, 2, left left assay,assay, panel). panel). compoundscompounds 1616––1818 showedshowed highesthighest activityactivity ofof OnOn thethe otherother hand,hand, inin ththee FRAPFRAP assay,assay, compoundscompounds 1616––1818 showedshowed − highest4highest activityactivity ofof allall thetheOnOn 2.3. testedtested thethe Antioxidative otherother compounds.compounds. hand,hand, Properties inin TheThe ththee FRAPlevel levelFRAP ofof assay,assay, absorbanceabsorbance compoundscompounds atat aa concentrationconcentration 1616––1818 showed showedshowed 1010 highest− highest4highest mol/Lmol/L activity waswasactivityactivity aboveabove of ofof all theOnOn tested thethe otherother compounds. hand,hand, inin The ththee FRAPlevelFRAP of assay,assay, absorbance compoundscompounds at a concentration 1616––1818 showedshowed 10− −4 highest4highest mol/L activitywasactivity above ofof allall the the tested tested compounds. compounds. The The level level of of absorbance absorbance at at− 4a a concentration concentration 10 10−−44 mol/L mol/L was was above above all8%all the the of tested ascorbictested compounds. compounds. acid activity The The at level alevel concentration of of absorbance absorbance of 10 at at −a 4a mol/Lconcentration concentration (Table 2, 10 10right−−4−4 4mol/L mol/L panel). was was above above allall8% the the of tested ascorbictestedIn compounds. compounds. order acid toactivity determine The The at level levela concentration the of of possible absorbance absorbance antioxidant of 10at at− a4 a mol/Lconcentration concentration properties, (Table 2, 10 10 right selected mol/L mol/Lmol/L panel). compoundswas waswas above aboveabove were 8% of ascorbic acid activity at a concentration of 10−−44 mol/L (Table 2, right panel). 8%8% ofof ascorbicascorbic acidacid activityactivity atat aa concentrationconcentration ofof 1010−4− 4 mol/Lmol/L (Table(Table 2,2, rightright panel).panel). 8%8% of ofConsidering Consideringascorbic testedascorbic in acid acid 2,2-diphenyl-1-picrylhydrazyl bothboth activity activity teststests at atresults,results, a a concentration concentration compoundcompound of of 16 (DPPH)16 10 10 showedshowed−−44 mol/L mol/Lmol/L and the the(Table (Table(Table ferric strongeststrongest 2, 2, reducing2, right rightright antioxidantantioxidant panel). panel).panel). antioxidant prop-prop- power ConsideringConsidering both both tests tests results, results,−4 compound compound 16 16 showed showed the the strongest strongest antioxidant antioxidant prop- prop- erties.erties.ConsideringConsidering AtAt aa concentrationconcentration both both tests tests ofof results, results,1010−4 mol/L,mol/L, compound compound itit significantlysignificantly 16 16 showed showedshowed reducedreduced the thethe strongest strongest strongest thethe numbernumber antioxidant antioxidantantioxidant ofof freefree radicalsradicals prop- prop-properties.ConsideringConsidering At a concentration both both tests tests of results, results,10−−44 mol/L, compound compound it significantly 16 16 showed showed reduced the the strongest strongest the number antioxidant antioxidant of free radicals prop- prop- erties.erties. At At a a concentration concentration of of 10 10−−44 mol/L, mol/L, it it significantly significantly reduced reduced the the number number of of free free radicals radicals erties.(DPPHerties.(DPPH At At test)test) a a concentration concentration andand exhibitedexhibited of of antioxidativeantioxidative10 10−−4−4 4mol/L, mol/L, it it significantly propertiessignificantlyproperties inin reduced reduced generalgeneral the(FRAP (FRAPthe number number test)test) ofdespite despiteof free free radicals radicals yetyet un-un- (DPPH(DPPH(DPPH test) test)test) and andand exhibited exhibitedexhibited antioxidative antioxidativeantioxidative properties propertiesproperties in inin general generalgeneral (FRAP (FRAP(FRAP test) test)test) despite despitedespite yet yetyet un- un-un- (DPPHknown(DPPH(DPPHknown test) test)test)mechanism.mechanism. and andand exhibited exhibitedexhibited Furthermore,Furthermore, antioxidative antioxidativeantioxidative otherother properties propertiesproperties compoundscompounds in inin general generalgeneral fromfrom thethe(FRAP (FRAP(FRAP mm-methoxyphenyl-methoxyphenyl test) test)test) despite despitedespite yet yetyet sub-sub- un- un-un- knownknown mechanism.mechanism. Furthermore,Furthermore, otherotherother compoundscompoundscompounds fromfromfrom thethethe mm-methoxyphenyl-methoxyphenyl-methoxyphenyl sub-sub-sub- knowngroup,knowngroup, 17mechanism.17mechanism. andand 1818,, alsoalso Furthermore,Furthermore, showedshowed antioxidantantioxidant otherotherother compoundscompoundscompounds activityactivity inin FRAPfromFRAPfromfrom theassay.theassay.the mm-methoxyphenyl -methoxyphenyl -methoxyphenylAmongAmong threethree variousvarious sub-sub-subgroup,group, 1717 and andand 18 18, ,, also alsoalso showed showedshowed antioxidant antioxidantantioxidant activity activityactivity in inin FRAP FRAPFRAP assay. assay.assay. Among AmongAmong three threethree various variousvarious group,phenylgroup,phenyl 17 17(Ph)(Ph) and andand substituents,substituents, 18 18,, , also alsoalso showed showedshowed onlyonly methoxymethoxy antioxidant antioxidantantioxidant derivaderiva activity activityactivitytivestives appearedin appearedinin FRAP FRAPFRAP assay. assay.toassay.to possesspossess Among AmongAmong antioxidantantioxidant three threethree various variousvarious prop-prop- phenylphenyl (Ph) (Ph) substituents, substituents, only only methoxy methoxy deriva derivativestivestives appeared appearedappeared to toto possess possesspossess antioxidant antioxidantantioxidant prop- prop-prop- phenylphenyl (Ph) (Ph) substituents, substituents, only only methoxy methoxy deriva derivativestives appeared appeared2 to to possess possess3 antioxidant antioxidant prop- prop- phenylerties.phenylerties. This(Ph)This (Ph) substituents, mightmightsubstituents, bebe duedue only only toto thethe methoxy methoxy factfact thatthat deriva deriva thethe tivesptivesptives-NO-NO appeared appearedappeared2–Ph,–Ph, p-COCHp-COCH to toto possess possesspossess3–Ph–Ph antioxidant areantioxidantantioxidantare capablecapable prop- prop-prop-ofof at-at- erties.erties. ThisThis mightmight bebe duedue toto thethe factfact thatthat thethe pp-NO-NO22–Ph,–Ph, p-COCHp-COCH33–Ph–Ph areare capablecapable ofof at-at- erties.erties. ThisThis mightmight bebe duedue toto thethe factfact thatthat thethe pp-NO-NO22–Ph,–Ph,3 p-COCHp-COCH33–Ph–Ph areare capablecapable ofof at-at- erties.tractingerties.tracting ThisThis electrons,electrons, mightmight be insteadbeinstead duedue to toofof the givingthegiving factfact them,them, thatthat the the whilewhile pp-NO-NO-NO pp-OCH-OCH22–Ph,2–Ph, 3–Ph p-COCH–Php-COCH areare proficientproficient33–Ph3–Ph areare capableinincapable givinggiving ofof elec-elec- at-attracting electrons, instead of giving them, while p-OCH33–Ph are proficient in giving elec- tractingtracting electrons,electrons, insteadinstead ofof givinggiving them,them, whilewhile pp-OCH-OCH33–Ph–Ph areare proficientproficient inin givinggiving elec-elec- tractingtronstractingtractingtrons ratherrather electrons, electrons,electrons, thanthan capturing capturing instead insteadinstead of ofof them, them, giving givinggiving whichwhich them, them,them, inin while while while turnturn is p isp-OCH -OCH -OCHanan indicationindication33–Ph3–Ph areare ofof proficientproficient theirtheir antioxidantantioxidant inin givinggiving activ-activ- elec-elec- tronstronstrons rather ratherrather than thanthan capturing capturingcapturing them, them,them, which whichwhich in inin turn turnturn is isis an anan indication indicationindication of ofof their theirtheir antioxidant antioxidantantioxidant activ- activ-activ- tronstrons rather rather than than capturing capturing them, them, which which in in turn turn is is an an indication indication of of their their antioxidant antioxidant3 activ- activ- tronsity.tronstronsity. However,However, rather ratherrather than thanthan withwith capturing capturingcapturing suchsuch anan them, them,electronthem,electron which whichwhich withdrawingwithdrawing in inin turn turnturn is isis an substituentanansubstituent indication indicationindication asas of ofof mm their -OCHtheirtheir-OCH antioxidant antioxidantantioxidant3,, antioxidantantioxidant activ- activ-activ- ac-ac- ity. However, with such an electron withdrawing substituent as m-OCH33, antioxidant ac- ity.ity. However,However, withwith suchsuch anan electronelectron withdrawingwithdrawing substituentsubstituent asas mm-OCH-OCH33,, antioxidantantioxidant ac-ac- ity.tivityity.ity.tivity However, However,However, appearsappears with withinwithin thethe such suchsuch studstud an ananiedied electron electronelectron groupgroup ofofwithdrawing withdrawingwithdrawing compounds.compounds. substituent substituentsubstituent as asas m m-OCH-OCH-OCH33,3 ,,antioxidant antioxidantantioxidant ac- ac-ac- tivitytivity appearsappears inin thethe studstudiedied groupgroup ofof compounds.compounds. tivitytivitytivity appears appearsappears in inin the thethe stud studstudiediedied group groupgroup of ofof compounds. compounds.compounds.

Molecules 2021, 26, 2300 6 of 18

(FRAP) assay with ascorbic acid as a reference compound (Table2). For details, please refer to the Materials and Methods Section. Graphical representation of tests results can be found in Supplementary Material.

Table 2. Absorbance changes in the 2,2-diphenyl-1-picrylhydrazyl (DPPH) and ferric reducing antioxidant power (FRAP) assays. The highest obtained values for the tested compounds were marked in bold.

DPPH Assay FRAP Assay (λ = 517 nm) (λ = 593 nm) Compound Absorbance Decrease in Absorbance Increase in 10−4 mol/L Concentration [%] 10−4 mol/L Concentration [%] Mean ± SEM Mean ± SEM Ascorbic acid 20.52 ± 0.51 344.20 ± 4.65 6 8.82 ± 0.12 0.79 ± 0.39 7 6.23 ± 0.53 1.43 ± 0.32 8 6.79 ± 0.51 0.48 ± 0.48 9 7.70 ± 0.41 1.75 ± 0.22 10 2.44 ± 0.97 3.66 ± 0.59 11 3.33 ± 0.45 3.34 ± 0.22 12 4.42 ± 0.33 2.70 ± 1.15 14 6.31 ± 1.14 0.22 ± 0.16 16 12.37 ± 1.31 32.91 ± 0.81 17 4.88 ± 0.18 40.86 ± 0.98 18 6.35 ± 0.55 29.73 ± 0.39

Antioxidant activity at the level above 50% of ascorbic acid activity at a concentration of 10−4 mol/L might be considered noteworthy. In the DPPH assay, compound 16 (QD13) at a concentration of 10−4 mol/L caused a decrease in absorbance by 12.37 ± 1.31% when compared to the sample containing the solvent + reaction mixture (blank, maximum concentration of the DPPH radical). Moreover, 16, at a concentration of 10−4, had 60.28% of the ascorbic acid activity (10−4 mol/L, calculated from Table2). In fact, of all the tested compounds, 16 expressed the strongest antioxidant properties. Eight of the other compounds showed a slightly smaller but pronounced decrease in absorbance (antioxidant activity > 20% of ascorbic acid activity at 10−4 mol/L); thus, scavenging DPPH free radicals (Table2, left panel). On the other hand, in the FRAP assay, compounds 16–18 showed highest activity of all the tested compounds. The level of absorbance at a concentration 10−4 mol/L was above 8% of ascorbic acid activity at a concentration of 10−4 mol/L (Table2, right panel). Considering both tests results, compound 16 showed the strongest antioxidant properties. At a concentration of 10−4 mol/L, it significantly reduced the number of free radicals (DPPH test) and exhibited antioxidative properties in general (FRAP test) despite yet unknown mechanism. Furthermore, other compounds from the m-methoxyphenyl subgroup, 17 and 18, also showed antioxidant activity in FRAP assay. Among three various phenyl (Ph) substituents, only methoxy derivatives appeared to possess antioxidant properties. This might be due to the fact that the p-NO2–Ph, p-COCH3–Ph are capable of attracting electrons, instead of giving them, while p-OCH3–Ph are proficient in giving electrons rather than capturing them, which in turn is an indication of their antioxidant activity. However, with such an electron withdrawing substituent as m-OCH3, antioxidant activity appears in the studied group of compounds. Overall, these results prompt for future investigation of compounds 16–18 antioxidant properties in various disease entities. Despite its moderate affinity for histamine H3 receptors, 16 (QD13) could be investigated as an adjuvant to known therapies in many diseases, to which contributes an oxidative stress and where modulation of the brain levels of histamine and other neurotransmitters provide improvement. Molecules 2021, 26, 2300 7 of 18

2.4. Docking Studies Three out of four known and described histamine receptors still remain a Gordian knot by means of crystallography. To date, only histamine H1 receptor crystal structure was resolved (PDB ID: 3RZE [33]). Therefore, we used a previously described histamine H3R homology model in our study, which was constructed on the template of crystal structure of M2 muscarinic acetylcholine receptor (PDB ID: 3UON) [22]. Considering that, due to very low basicity, the abundance of compounds at physiological pH might appear in non-ionized forms, for docking studies, we prepared both protonated and non-protonated conformers. All of the compounds were docked and, independently of the form, ﬁt the H3R binding pocket. In case of non-protonated conformers, compounds were placed shallower Molecules 2021, 25, x FOR PEER REVIEWin the binding pocket, and mostly stabilized through π–π stacking or additional8 H-bonds of 18 with Y374 (3.51) or R381 (6.58). However, no interactions with either of the key amino acids,and W371 E206 (6.48), (5.46) andin the D114 case (3.32), most active were found.for this For series possible of m-methoxyphenyl non-ionized forms, derivatives, this might explainwas also very present. low or These no afﬁnity interactions for the might histamine explain H 3thereceptor. overall Calculatedhigher H3R bindingaffinity of poses the for non-protonatedligands from this conformers subgroup. can be found in the Supplementary Material (Table S2). Nonetheless,On the other hand, two different the UD mode binding was modes observed were for surprisingly p-acetylphenyl observed and p-nitrophenyl for protonated conformers:derivatives, with “standard the exception (ST)”, withof 18, the for easternwhich both region modes pointing were observed. to the extracellular Although, the space, andST mode “upside-down expressed slightly (UD)” (Figurelower glide3). The energy positions (−58.78 of vs protonated −56.08 kcal/mol). basic In moieties this mode, of the ligandsthe benzophenone were mostly fragments similar, independently reached deeper of towards the mode. the In narrow both cases, sub pocket a crucial localized histamine Hamong3R antagonist/inverse the transmembrane agonist regions interaction-salt (TM) 1, TM3, and bridge TM7. and/or The latter hydrogen is formed bond by formationlateral betweenL117 (3.35) protonated and W371 amine (6.48), nitrogenand S121 and(3.39) E206 and (5.46)A122 (3.40) was, however,from the bottom. preserved Additional due to the approximatepose stabilization location through of the cation– protonatedπ interactions nitrogen between (superscripts proton denoteated piperazine Ballesteros–Weinstein nitrogen numberingand F193 (ECL2), [34]). as well as the hydrogen bond between the carbonyl/nitro group and R381 (6.58) was found.

Figure 3. Putative binding poses of protonated conformers. Upper panel: KSK63 (left) 15 (middle) and 16 (right); lower panel:Figure17 (middle),3. Putative18 binding(right) poses in histamine of protonated H3 homology conformers. receptor. Upper panel: KSK63 (left) 15 (middle) and 16 (right); lower panel: 17 (middle), 18 (right) in histamine H3 homology receptor.

Along with the novel compounds, reference structure KSK63 was also docked. In this case the pyridyl moiety nitrogen is protonated instead of the piperazine one. This allows for a shift of the ligand towards TM3 and formation of ionic interaction with key aminergic GPCR anchor, D114 (3.32) (Figure 3). Lack of such interactions observed for the novel, described compounds, might also be responsible for their much lower affinity towards histamine H3 receptor. The stability of the calculated poses for this groups’ most affine compounds 15–17 and both orientations of 18 along with the reference compound KSK63 was further evaluated by means of molecular dynamics (MD) simulations. From each simulation, 7 poses

Molecules 2021, 26, 2300 8 of 18

In the ST mode, benzophenone fragments occupied the space fenced by aromatic features of F193, Y189 (ECL2), and Y394 (7.35) on the sides, and Y91 (2.61) and Y94 (2.64) on the top. In most cases, a hydrogen bond between the carbonyl group and R381(6.58) was found. The latter one was not observed for inactive compounds 4–9. Additional stabilization through halogen bond formation with Y91 (2.61) and π–π stacking with Y394 (7.35) and W371 (6.48), in the case most active for this series of m-methoxyphenyl derivatives, was also present. These interactions might explain the overall higher H3R affinity of the ligands from this subgroup. On the other hand, the UD mode was observed for p-acetylphenyl and p-nitrophenyl derivatives, with the exception of 18, for which both modes were observed. Although, the ST mode expressed slightly lower glide energy (−58.78 vs −56.08 kcal/mol). In this mode, the benzophenone fragments reached deeper towards the narrow sub pocket localized among the transmembrane regions (TM) 1, TM3, and TM7. The latter is formed by lateral L117 (3.35) and W371 (6.48), and S121 (3.39) and A122 (3.40) from the bottom. Additional pose stabilization through cation–π interactions between protonated piperazine nitrogen and F193 (ECL2), as well as the hydrogen bond between the carbonyl/nitro group and R381 (6.58) was found. Along with the novel compounds, reference structure KSK63 was also docked. In this case the pyridyl moiety nitrogen is protonated instead of the piperazine one. This allows for a shift of the ligand towards TM3 and formation of ionic interaction with key aminergic GPCR anchor, D114 (3.32) (Figure3). Lack of such interactions observed for the novel, described compounds, might also be responsible for their much lower affinity towards histamine H3 receptor. The stability of the calculated poses for this groups’ most affine compounds 15–17 and both orientations of 18 along with the reference compound KSK63 was further evaluated by means of molecular dynamics (MD) simulations. From each simulation, 7 poses were selected (starting pose, and after each 100ps up to 600ps). In the case of 15–17 (Figure4 ) and KSK63, complexes appeared stable through the whole 600ps simulation, retaining the key interactions, and the potential energy (U) of the atomic system at the level of ~1000 kcal/mol. In all cases, starting poses (marked in grey, Figure4) were very similar to its orientation at the end of simulation (marked yellow). However, a shift in the binding site with retained conformation appeared in the first 100ps of the simulation. On the other hand, the analysis of KSK63 behavior allowed for possible explanation of its’ high affinity. The conformation remained quite stable during the simulation with consistent set of interactions. What was not observed for the remaining structures, the protonated pyridyl nitrogen retained the interactions with not only the key ionic anchor D114 (3.32) but also with E206 (5.46), through the whole simulation. Detailed analysis of compounds behavior during MD simulations is provided by examining the changes in their interactions with H3R (Figure5 ). The results indicate a relatively consistent set of ligand-protein interactions occurring during the whole MD simulation. Most of the interactions occur within the TM3, TM5, TM6 and TM7 and ECL2, with consistent interactions with Y115 (3.33), Y189 (45.51), E206 (5.46), Y394 (7.35). In the case of 18, the standard orientation seems more stable during the simulation, with most of the starting key interactions retained in the last frame. Molecules 2021, 25, x FOR PEER REVIEW 9 of 18

were selected (starting pose, and after each 100ps up to 600ps). In the case of 15–17 (Figure 4) and KSK63, complexes appeared stable through the whole 600ps simulation, retaining Molecules 2021, 26, 2300 9 of 18 the key interactions, and the potential energy (U) of the atomic system at the level of ~1000 kcal/mol.

Figure 4.FigureOrientations 4. Orientations of: upper of: panel-KSK63 upper panel-KSK63 (left), 15 (left),(middle), 15 (middle), and 16 (right);and 16 lower(right); panel- lower17 panel-(middle),17 (middle), 18 (right) 18 during(right) during the the molecular dynamics simulations in 0ps–grey, 100ps–green, 200ps–teal, 300ps–green, 400ps–violet, 500ps–orange, molecular dynamics simulations in 0ps–grey, 100ps–green, 200ps–teal, 300ps–green, 400ps–violet, 500ps–orange, 600ps–yellow. Molecules 2021600ps–yellow., 25, x FOR PEER REVIEW 10 of 18

In all cases, starting poses (marked in grey, Figure 4) were very similar to its orientation at the end of simulation (marked yellow). However, a shift in the binding site with retained conformation appeared in the first 100ps of the simulation. On the other hand, the analysis of KSK63 behavior allowed for possible explanation of its’ high affinity. The conformation remained quite stable during the simulation with consistent set of interactions. What was not observed for the remaining structures, the protonated pyridyl nitrogen retained the interactions with not only the key ionic anchor D114 (3.32) but also with E206 (5.46), through the whole simulation. Detailed analysis of compounds behavior during MD simulations is provided by examining the changes in their interactions with H3R (Figure 5). The results indicate a relatively consistent set of ligand-protein interactions occurring during the whole MD simulation. Most of the interactions occur within the TM3, TM5, TM6 and TM7 and ECL2, with consistent interactions with Y115 (3.33), Y189 (45.51), E206 (5.46), Y394 (7.35). In the case of 18, the standard orientation seems more stable during the simulation, with most of the starting key interactions retained in the last frame.

FigureFigure 5. Interaction 5. Interaction ﬁngerprints fingerprints of compounds of compounds KSK63, KSK6315, 15–18–18with with H H3R3R model model duringduring molecular dynamics dynamics (MD) (MD) simula- simulation for particulartion for particular frames. frames. Colored Colored boxes boxes indicate indicate any any interaction interaction between between the the ligandligand and and corresponding corresponding amino amino acid. acid. Key Key interaction amino acids were marked violet bold. interaction amino acids were marked violet bold. 3. Materials and Methods 3.1. Chemistry All reagents were purchased from commercial suppliers (Alfa Aesar, Sigma-Aldrich, CHESS GmbH, Chempur) and were used without further purification. Melting points (mp) were determined on a Büchi Melting Point M560 apparatus (BÜCHI Labortechnik AG, Flawil, Switzerland) and were uncorrected. Liquid chromatography/mass spectra (LC/MS) were obtained using Waters TQ Detector mass spectrometer (Milford, MA, USA). 1H NMR spectra were recorded on a Varian Mercury 300 MHz PFG (Palo Alto, CA, USA) spectrometer in DMSO-d6. Chemical shifts were expressed in parts per million (ppm) using the solvent signal as an internal standard. Data have been reported in the following order: multiplicity (s, singlet; d, doublet; t, triplet; qi, quintet; m, multiplet; br, broad; Ac, acetyl; Prop, propionyl; t–b, tert–butyl; t–p, tert–pentyl; Ph, phenyl; Pip, piperazine), approximate coupling constants J expressed in Hertz (Hz), number of protons. 13C NMR spectra were recorded on Varian-Mercury-VX 300 MHz PFG or Bruker 400 MHz spectrometer (Berlin, Germany) at 75 MHz in DMSO-d6. LC–MS separation was carried out on a system consisting of a Waters ACQUITY UPLC, coupled to a Waters TQD mass spectrometer. Retention times (tR) were stated in minutes. For flash chromatography (FC) purification, silica gel 60 (0.063–0.20 mm; Merck) and the following solvent systems were used: I-petroleum ether:EtOAc (97.5:1.25); II-CH2Cl2:MeOH (95:5).

Molecules 2021, 26, 2300 10 of 18

3. Materials and Methods 3.1. Chemistry All reagents were purchased from commercial suppliers (Alfa Aesar, Sigma-Aldrich, CHESS GmbH, Chempur) and were used without further purification. Melting points (mp) were determined on a Büchi Melting Point M560 apparatus (BÜCHI Labortechnik AG, Flawil, Switzerland) and were uncorrected. Liquid chromatography/mass spectra (LC/MS) were obtained using Waters TQ Detector mass spectrometer (Milford, MA, USA). 1H NMR spectra were recorded on a Varian Mercury 300 MHz PFG (Palo Alto, CA, USA) spectrometer in DMSO-d6. Chemical shifts were expressed in parts per million (ppm) using the solvent signal as an internal standard. Data have been reported in the following order: multiplicity (s, singlet; d, doublet; t, triplet; qi, quintet; m, multiplet; br, broad; Ac, acetyl; Prop, propionyl; t–b, tert–butyl; t–p, tert–pentyl; Ph, phenyl; Pip, piperazine), approximate coupling constants J expressed in Hertz (Hz), number of protons. 13C NMR spectra were recorded on Varian-Mercury-VX 300 MHz PFG or Bruker 400 MHz spectrometer (Berlin, Germany) at 75 MHz in DMSO-d6. LC–MS separation was carried out on a system consisting of a Waters ACQUITY UPLC, coupled to a Waters TQD mass spectrometer. Retention times (tR) were stated in minutes. For flash chromatography (FC) purification, silica gel 60 (0.063–0.20 mm; Merck) and the following solvent systems were used: I- petroleum ether:EtOAc (97.5:1.25); II-CH2Cl2:MeOH (95:5).

3.1.1. Procedure 1 To a solution of proper hydroxy benzophenone (0.01 mol) in acetone (60 mL), anhy- drous potassium carbonate (1.12 g, 0.008 mol) and catalytic amount of potassium iodide were added. The reaction mixture was stirred for 10 min at room temperature, after which 1,3-dibromopropane (12 g, 0.06 mol) was added. The reaction mixture was then heated at reflux for 24 h, after which the inorganic solids were filtered, and acetone was evaporated. Remaining oils were then purified by flash chromatography (system I). 4-(3-Bromopropoxyphenyl)(phenyl)methanone (1). Synthesis from (4-hydroxyphenyl) (phenyl) methanone (1.98 g, 0.01 mol). Obtained 2.78 g of white quickly crystalizing oil. C16H15BrO2, Molecular Weight (MW) = 319.20, Yield = 87%. 4-(3-Bromopropoxyphenyl)(4-chlorophenyl)methanone (2). Synthesis from (4-hydroxyphenyl)(4-chlorophenyl)methanone (2.32 g, 0.01 mol). Obtained 2.13 g of white quickly crystalizing oil. C16H14BrClO2, MW = 351.99, Yield = 60%)

3.1.2. Procedure 2 Procedure was performed according to the literature reference [30]. To a solution of freshly prepared sodium propanolate (25 mL propanol; 0.575 g, 0.025 mol Na), (4- fluorophenyl) (4-hydroxyphenyl)methanone (5.4 g, 0.025 mol) was added and stirred at room temperature (RT) for 15 min; 1,3-dibromopropane (15.14 g, 0.075 mol) was then added dropwise over one hour. The reaction mixture was stirred at 60 ◦C for 3 h, and then refluxed for another 3 h. After cooling down to RT reaction mixture was filtrated and evaporated. To a resulting brown oil, MeOH was added, and purified by flash chromatography (system II); obtaining 6 g of yellowish, quickly crystallizing oil.

4-(3-Bromopropoxyphenyl)(4-fluorophenyl)methanone (3). C16H14BrFO2, MW = 337.18, yield = 71%

3.1.3. Procedure 3 Final products were obtained according to the procedure described in [22] and puriﬁed by ﬂash chromatography (CH2Cl2/MeOH, 95:5). Resulting oils were either: transformed into oxalic acid salts, using 10% excess of oxalic acid solution in absolute ethanol in RT, and then precipitated by addition of ethyl ether or, in the case of self-crystalizing oils, remained free bases. Molecules 2021, 26, 2300 11 of 18

1-(4-(3-(4-Benzoylphenoxy)propyl)piperazin-1-yl)ethan-1-one hydrogen oxalate (4, QD11). Syn- thesis from 1 (0.47 g, 1.5 mmol) and 1-(piperazin-1-yl)ethan-1-one (0.47 g, 2.5 mmol). Purified by FC. Obtained 550 mg of white oil, transferred into oxalic acid salt. Yield = 63%; ◦ mp = 136.8–140 C; UPLC/MS purity 98.20%, tR = 5.05, C22H26N2O3 × C2H2O4, MW = 366.46, + 1 [M + H] 367.25. H NMR (500 MHz, DMSO-d6) δ ppm: 7.69–7.72 (m, 2H, Ph), 7.60–7.66 (m, 3H, Ph), 7.49–7.54 (m, 2H, Ph), 7.03–7.07 (m, 2H Ph), 4.11 (t, J = 6.01 Hz, 2H, CH2-O), 3.47–3.57 (m, 4H, Pip-2,6H), 2.75–2.83 (m, 4H, Pip-3,5H), 2.68–2.74 (m, 2H, NCH2), 1.98–2.04 13 (m, 2H, CH2), 1.97 (s, 3H, COCH3). C NMR (DMSO-d6): 194.96, 168.86, 164.29, 162.71, 138.29, 132.74, 129.78, 129.00, 114.89, 66.32, 54.13, 52.56, 52.14, 44.73, 25.18, 21.62. 1-(4-(3-(4-(4-Chlorobenzoyl)phenoxy)propyl)piperazin-1-yl)ethan-1-one (5, QD15). Synthesis from 2 (0.53 g, 1.5 mmol) and 1-(piperazin-1-yl)ethan-1-one (0.19 g, 1.5 mmol). Purified by FC. Obtained 330 mg of light yellow free-base solid. Yield = 37%; mp = 123.9–126.7 ◦C; + 1 UPLC/MS purity 100%, tR = 4.63, C22H25ClN2O3, MW = 400.90, [M + H] 401.14. H NMR (500 MHz, DMSO-d6) δ ppm: 7.65–7.72 (m, 4H, Ph), 7.57 (d, J = 8.02 Hz, 2H, Ph), 7.04 (d, J = 8.59 Hz, 2H, Ph), 4.09 (t, J=6.30 Hz, 2H, CH2-O), 3.34–3.41 (m, 4H, Pip-2,6H), 2.41 (t, J = 7.16 Hz, 2H, NCH2), 2.31–2.36 (m, 2H, Pip-3,5H), 2.27 (t, J = 4.87 Hz, 2H, Pip-3,5H), 13 1.94 (s, 3H, COCH3), 1.88 (quin, J = 6.73 Hz, 2H, CH2). C NMR (DMSO-d6): 193.80, 168.60, 163.08, 137.48, 137.00, 132.76, 131.68, 129.12, 114.94, 66.80, 54.68, 53.55, 53.02, 46.18, 41.36, 26.48, 21.71. 1-(4-(3-(4-(4-Fluorobenzoyl)phenoxy)propyl)piperazin-1-yl)ethan-1-one hydrogen oxalate (6, QD4). Synthesis from 3 (0.50 g, 1.5 mmol) and 1-(piperazin-1-yl)ethan-1-one (0.32 g, 2.5 mmol). Puri- fied by FC. Obtained 620 mg of yellow-green oil, transferred into oxalic acid salt. Yield = 70%; ◦ mp = 171.3–173.1 C; UPLC/MS purity 100%, tR = 4.26, C22H25FN2O3 × C2H2O4, MW = 384.45 + 1 (+90.04), [M + H] 385.19. H NMR (500 MHz, DMSO-d6) δ ppm: 7.69–7.75 (m, 4H, Ph), 7.32–7.37 (m, 2H, Ph), 7.04–7.07 (m, 2H, Ph), 4.12 (t, J = 6.01 Hz, 2H, CH2-O), 3.57–3.66 (m, 4H, Pip-3,5H), 3.01–3.07 (m, 4H, Pip-2,6H), 2.97 (br. s., 2H, NCH2), 2.05–2.14 (m, 2H, CH2), 13 2.00 (s, 3H, COCH3). C NMR (DMSO-d6): 193.63, 169.01, 163.88, 163.68, 162.59, 134.76, 132.68, 129.95, 116.16, 115.98, 114.93, 66.06, 53.77, 51.95, 51.59, 43.83, 24.37, 21.56. (4-(3-(4-Benzoylphenoxy)propyl)piperazin-1-yl)(cyclopropyl)methanone hydrogen oxalate (7, QD14). Synthesis from 1 (0.47 g, 1.5 mmol) and cyclopropyl(piperazin-1-yl)methanone (0.23 g, 1.5 mmol). Purified by FC. Obtained 520 mg of white oil, transferred into oxalic acid salt. ◦ Yield = 63%; mp = 167.3–168.6 C; UPLC/MS purity 100%, tR = 4.30, C24H28N2O3 × C2H2O4, + 1 MW = 392.21, [M + H] 393.41. H NMR (500 MHz, DMSO-d6) δ ppm: 7.69–7.74 (m, 2H, Ph), 7.59–7.66 (m, 3H, Ph), 7.49–7.54 (m, 2H, Ph), 7.03–7.08 (m, 2H, Ph), 4.12 (t, J = 6.30 Hz, 2H, CH2-O), 3.84 (br. s., 2H, Pip-3,5H), 3.60 (br. s., 2H, Pip-3,5H), 2.92–3.03 (m, 6H, Pip- 13 2,6H + NCH2), 2.03–2.11 (m, 2H, CH2), 1.93–1.99 (m, 1H, Cp), 0.66–0.74 (m, 4H, Cp). C NMR (DMSO-d6): 194.96, 171.76, 164.19, 162.63, 138.28, 132.73, 129.78, 129.00, 114.90, 66.18, 53.92, 24.69, 10.73, 7.70 (4-(3-(4-(4-Chlorobenzoyl)phenoxy)propyl)piperazin-1-yl)(cyclopropyl)methanone hydrogen oxalate (8, QD19). Synthesis from 2 (0.28 g, 0.78 mmol) and cyclopropyl(piperazin-1-yl)methanone (0.12 g, 0.78 mmol). Purified by FC. Obtained 220 mg of light-yellow oil solid, transferred ◦ into oxalic acid salt. Yield = 26%; mp = 176.9–179.2 C; UPLC/MS purity 100%, tR = 4.99, + 1 C24H27ClN2O3, MW = 426.94, [M + H] 427.41. H NMR (500 MHz, DMSO-d6) δ ppm: 7.69–7.73 (m, 2H, Ph), 7.64–7.69 (m, 2H, Ph), 7.56–7.60 (m, 2H, Ph), 7.03–7.08 (m, 2H, Ph), 4.12 (t, J = 6.01 Hz, 2H, CH2-O), 3.83 (br. s., 2H, Pip-3,5H), 3.60 (br. s., 2H, Pip-3,5H), 2.91–3.03 (m, 4H, Pip-2,6H), 2.88 (br. s., 2H, NCH2), 2.02–2.10 (m, 2H, CH2), 1.92–2.00 (m, 13 1H, Cp), 0.65–0.73 (m, 4H, Cp).). C NMR (DMSO-d6): 193.83, 171.75, 164.12, 162.79, 137.52, 136.95, 132.75, 131.69, 129.13, 114.98, 66.22, 53.91, 24.71, 10.73, 7.70 (4-(3-(4-(Cyclopropanecarbonyl)piperazin-1-yl)propoxy)phenyl)(4-fluorophenyl)methanone hydrogen oxalate (9, QD8). Synthesis from 3 (0.50 g, 1.5 mmol) and cyclopropyl(piperazin-1- yl)methanone (0.23 g, 1.5 mmol). Purified by FC. Obtained 530 mg of light oil, transferred ◦ into oxalic acid salt. Yield = 64%; mp = 181.1–182.7 C; UPLC/MS purity 100%, tR = 4.48, Molecules 2021, 26, 2300 12 of 18

+ 1 C24H27FN2O3 × C2H2O4, MW = 410.48 (+90.04), [M + H] 411.18. H NMR (500 MHz, DMSO-d6) δ ppm: 7.68–7.76 (m, 4H, Ph), 7.32–7.37 (m, 2H, Ph), 7.03–7.07 (m, 2H, Ph), 4.12 (t, J = 6.01 Hz, 2H, CH2-O), 3.83 (br. s., 2H, Pip-2,6H), 3.59 (br. s., 2H, Pip-2,6H), 2.90–3.00 (m, 4H, Pip-3,5H), 2.87 (br. s., 2H, NCH2), 2.03–2.11 (m, 2H, CH2), 1.96 (tt, J = 7.73, 4.87 Hz, 13 1H, Cp), 0.66–0.74 (m, 4H, Cp). C NMR (DMSO-d6): 193.62, 171.75, 165.87, 164.08, 162.65, 134.80, 132.69, 129.90, 116.15, 115.98, 114.93, 66.20, 53.94, 24.74, 10.72, 7.70. 1-(4-(4-(3-(4-Benzoylphenoxy)propyl)piperazin-1-yl)phenyl)ethan-1-one (10, QD10). Synthesis from 1 (0.47 g, 1.5 mmol) and 1-(4-(piperazin-1-yl)phenyl)ethan-1-one (0.51 g, 2.5 mmol). Pu- rified by FC. Obtained 300 mg of yellow free-base solid. Yield = 62%; mp = 148.4–151.4 ◦C; + 1 UPLC/MS purity 100%, tR = 4.94, C28H30N2O3, MW = 442.56, [M + H] 443.22. H NMR (500 MHz, DMSO-d6) δ ppm: 7.76 (d, J = 9.17 Hz, 2H, Ph), 7.69–7.72 (m, 2H, Ph), 7.59–7.66 (m, 3H, Ph), 7.49–7.53 (m, 2H, Ph), 7.03–7.07 (m, 2H, Ph), 6.92 (d, J = 9.17 Hz, 2H, Ph), 4.10 (t, J = 6.30 Hz, 2H, CH2-O), 3.26–3.31 (m, 6H, Pip-2,6H + NCH2), 2.45–2.47 (m, 4H, 13 Pip-3,5H), 2.41 (s, 3H, COCH3), 1.91 (quin, J = 6.73 Hz, 2H, CH2). C NMR (DMSO-d6): 196.08, 194.94, 162.95, 154.38, 138.33, 132.75, 130.60, 129.77, 128.98, 114.88, 113.58, 66.81, 54.77, 52.99, 47.18, 26.62. 1-(4-(4-(3-(4-(4-Chlorobenzoyl)phenoxy)propyl)piperazin-1-yl)phenyl)ethan-1-one (11, QD17). Syn- thesis from 2 (0.53 g, 1.5 mmol) and 1-(4-(piperazin-1-yl)phenyl)ethan-1-one (0.3 g, 1.5 mmol). Purified by FC. Obtained 260 mg of yellowish free-base solid. Yield = 35%; mp = 180.6–183.4 ◦C; + 1 UPLC/MS purity 100%, tR = 5.49, C28H29ClN2O3, MW = 477.00, [M + H] 477.18. H NMR (500 MHz, DMSO-d6) δ ppm: 7.76 (m, J = 8.88 Hz, 2H, Ph), 7.65–7.71 (m, 4H, Ph), 7.56–7.59 (m, 2H, Ph), 7.04–7.08 (m, 2H, Ph), 6.93 (d, J = 9.16 Hz, 2H, Ph), 4.11 (t, J = 6.30 Hz, 2H, CH2-O), 3.29–3.31 (m, 10H, Pip-3,5H + Pip-2,6H + NCH2), 2.41 (s, 3H, COCH3), 1.88–1.95 13 (m, 2H, CH2). C NMR (DMSO-d6): 196.30, 163.10, 158.53, 132.79, 131.70, 130.61, 129.45, 129.13, 114.96, 113.59, 63.87, 53.00, 26.64. 1-(4-(4-(3-(4-(4-Fluorobenzoyl)phenoxy)propyl)piperazin-1-yl)phenyl)ethan-1-one (12, QD3). Syn- thesis from 3 (0.50 g, 1.5 mmol) and 1-(4-(piperazin-1-yl)phenyl)ethan-1-one (0.51 g, 2.5 mmol). Purified by FC. Obtained 230 mg of yellowish free-base solid. Yield = 31%; ◦ mp = 162–163.6 C; UPLC/MS purity 98.12%, tR = 5.11, C28H29FN2O3, MW = 460.55, + 1 [M + H] 461.23. H NMR (500 MHz, DMSO-d6) δ ppm: 7.69–7.78 (m, 6H, Ph), 7.34 (t, J = 8.88 Hz, 2H, Ph), 7.06 (m, J = 9.17 Hz, 2H, Ph), 6.93 (m, J = 9.16 Hz, 2H, Ph), 4.11 (t, J = 6.30 Hz, 2H, CH2-O), 2.44–2.47 (m, 10H, Pip-3,5H + Pip-2,6H + NCH2), 2.41 (s, 3H, 13 COCH3), 1.92 (m, J = 6.87 Hz, 2H, CH2). C NMR (DMSO-d6): 196.12, 162.97, 132.72, 130.60, 129.70, 116.14, 115.97, 114.91, 113.59, 66.83, 54.77, 53.00, 47.18, 26.63, 26.54. (4-(3-(4-(4-Nitrophenyl)piperazin-1-yl)propoxy)phenyl)(phenyl)methanone (13, QD12). Synthesis from 1 (0.47 g, 1.5 mmol) and 1-(4-nitrophenyl)piperazine (0.31 g, 1.5 mmol). Purified by FC. Obtained 130 mg of yellow, quick crystalizing oil. Yield = 18%; mp = 182.3–183.9 ◦C; + 1 UPLC/MS purity 100%, tR = 5.28, C26H27N3O4, MW = 445.51, [M + H] 446.40. H NMR (500 MHz, DMSO-d6) δ ppm: 8.00–8.03 (m, 2H, Ph), 7.69–7.72 (m, 2H, Ph), 7.63–7.66 (m, 2H, Ph), 7.60–7.62 (m, 1H, Ph), 7.49–7.53 (m, 2H, Ph), 7.04–7.07 (m, 2H, Ph), 7.04–7.07 (m, 2H, Ph), 4.11 (t, J = 6.44 Hz, 2H, CH2-O), 3.40–3.44 (m, 8H, Pip-3,5H + Pip-2,6H), 3.27 (m, 2H, 13 NCH2), 1.92 (m, J = 7.02 Hz, 2H, CH2). C NMR (DMSO-d6): 196.19, 168.03, 164.95, 156.22, 132.77, 129.78, 129.00, 126.26, 114.89, 113.13, 66.80, 52.87, 46.84, 25.76. (4-Chlorophenyl)(4-(3-(4-(4-nitrophenyl)piperazin-1-yl)propoxy)phenyl)methanone (14, QD20). Syn- thesis from 2 (0.53 g, 1.5 mmol) and 1-(4-nitrophenyl)piperazine (0.31 g, 1.5 mmol). Purified by FC. Obtained 500 mg of yellow, quick crystallizing oil. Yield = 67%; mp = 154.5–155.8 ◦C; + 1 UPLC/MS purity 100%, tR = 5.83, C26H27ClN3O4, MW = 479.96, [M + H] 480.39. H NMR (500 MHz, DMSO-d6) δ ppm: 8.01 (d, J = 9.74 Hz, 2H, Ph), 7.66 (d, J = 8.02 Hz, 2H, Ph), 7.70 (d, J = 9.17 Hz, 2H, Ph), 7.57 (d, J = 8.59 Hz, 2H, Ph), 7.05 (d, J = 8.59 Hz, 2H, Ph), 6.99 (d, J = 9.17 Hz, 2H, Ph), 4.11 (t, J = 6.01 Hz, 2H, CH2-O), 3.41 (br. s., 4H, Pip-3,5H), 13 3.23–3.37 (m, 6H, Pip-2,6H + NCH2), 1.88–1.96 (m, 2H, CH2). C NMR (DMSO-d6): 193.81, Molecules 2021, 26, 2300 13 of 18

163.09, 155.28, 137.48, 137.37, 137.00, 132.77, 131.68, 129.12, 126.24, 114.95, 113.12, 66.83, 54.65, 52.86, 46,85, 26.51. (4-Fluorophenyl)(4-(3-(4-(4-nitrophenyl)piperazin-1-yl)propoxy)phenyl)methanone (15, QD6). Syn- thesis from 3 (1 g, 3 mmol) and 1-(4-nitrophenyl)piperazine (0.62 g, 3 mmol). Purified by FC. Obtained 245 mg of yellow, quick crystallizing oil. Yield = 34%; mp = 173.3–174.2 ◦C; + 1 UPLC/MS purity 100%, tR = 5.52, C26H27FN3O4, MW = 463.51, [M + H] 464.15. H NMR (500 MHz, DMSO-d6) δ ppm: 8.01 (m, J = 9.74 Hz, 2H, Ph), 7.67–7.76 (m, 4H, Ph), 7.34 (t, J = 8.88 Hz, 2H, Ph), 7.06 (m, J = 9.16 Hz, 2H, Ph), 6.99 (d, J = 9.74 Hz, 2H, Ph), 4.11 (t, J = 6.30 Hz, 2H, CH2-O), 3.39–3.44 (m, 4H, Pip-3,5H), 3.29 (s, 6H, Pip-2,6H + NCH2), 1.92 13 (quin, J = 6.59 Hz, 2H, CH2). C NMR (DMSO-d6): 193.62, 162.96, 155.28, 137.36, 132.71, 129.70, 126.25, 116.14, 115.97, 114.91, 113.12, 66.80, 54.66, 52.86, 46.85, 26.51. (4-(3-(4-(3-Methoxyphenyl)piperazin-1-yl)propoxy)phenyl)(phenyl)methanone hydrogen oxalate (16, QD13). Synthesis from 1 (0.47 g, 1.5 mmol) and 1-(3-methoxyphenyl)piperazine (0.28 g, 1.5 mmol). Purified by FC. Obtained 650 mg of yellow oil, transferred into ◦ oxalic acid salt. Yield = 87%; mp = 143.6–146.1 C; UPLC/MS purity 100%, tR = 5.32, + 1 C27H30N2O3 × C2H2O4, MW = 430.55 (+ 90.04), [M + H] 431.44. H NMR (500 MHz, DMSO-d6) δ ppm: 7.70–7.73 (m, 2H, Ph), 7.60–7.66 (m, 3H, Ph), 7.50–7.54 (m, 2H, Ph), 7.05–7.12 (m, 3H, Ph), 6.53 (dd, J = 8.02, 2.00 Hz, 1H, Ph), 6.47 (t, J = 2.29 Hz, 1H, Ph), 6.38 (dd, J = 8.16, 1.86 Hz, 1H, Ph), 4.13 (t, J=6.01 Hz, 2H, CH2-O), 3.68 (s, 3H, CH3), 3.31 (br. s., 4H, Pip-3,5H), 3.10 (br. s., 4H, Pip-2,6H), 3.02–3.07 (m, 2H, NCH2), 2.08–2.14 (m, 2H, CH2). 13 C NMR (DMSO-d6): 194.98, 164.54, 162.63, 160.80, 160.78, 151.87, 138.29, 132.75, 132.73, 129.80, 129.01, 114.93, 108.81, 105.45, 102.47, 66.18, 55.49, 53.79, 51.78, 46.69, 24.54. (4-Chlorophenyl)(4-(3-(4-(3-methoxyphenyl)piperazin-1-yl)propoxy)phenyl)methanone hydrogen oxalate (17, QD18). Synthesis from 2 (0.53 g, 1.5 mmol) and 1-(3-methoxyphenyl)piperazine (0.28 g, 1.5 mmol). Purified by FC. Obtained 350 mg of yellow oil, transferred into ◦ oxalic acid salt. Yield = 46%; mp = 100.2–102.4 C; UPLC/MS purity 100%, tR = 5.95, + 1 C27H29ClN2O3, MW = 464.98, [M + H] 465.44. H NMR (500 MHz, DMSO-d6) δ ppm: 7.65–7.71 (m, 4H, Ph), 7.55–7.59 (m, 2H, Ph), 7.03–7.07 (m, 3H, Ph), 6.47 (dd, J = 8.02, 1.72 Hz, 1H, Ph), 6.39 (t, J = 2.29 Hz, 1H, Ph), 6.39 (t, J = 2.29 Hz, 1H, Ph), 4.10 (t, J = 6.30 Hz, 2H, CH2- O), 3.30 (s, 3H, CH3), 3.05–3.11 (m, 4H, Pip-3,5H), 2.42–2.47 (m, 6H, Pip-2,6H + NCH2), 1.90 13 (quin, J = 6.73 Hz, 2H, CH2). C NMR (DMSO-d6): 193.80, 163.11, 160.71, 152.94, 137.48, 137.00, 132.76, 131.68, 129.11, 114.95, 108.52, 104.58, 101.94, 66.88, 55.37, 53.29, 48.70, 26.58. (4-Fluorophenyl)(4-(3-(4-(3-methoxyphenyl)piperazin-1-yl)propoxy)phenyl)methanone hydrogen oxalate (18, QD9). Synthesis from 3 (0.4 g, 1 mmol) and 1-(3-methoxyphenyl)piperazine (0.19 g, 1 mmol). Purified by FC. Obtained 430 mg of light oil, transferred into oxalic acid ◦ salt. Yield = 57%; mp = 127.4–129.7 C; UPLC/MS purity 100%, tR = 5.47, C27H29FN2O3, + 1 MW = 448.54, [M + H] 449.20. H NMR (500 MHz, DMSO-d6) δ ppm: 7.69–7.75 (m, 4H, Ph), 7.32–7.37 (m, 2H, Ph), 7.05–7.12 (m, 3H, Ph), 6.53 (dd, J = 8.31, 2.00 Hz, 1H, Ph), 6.47 (t, J = 2.00 Hz, 1H, Ph), 6.38 (dd, J = 8.02, 2.29 Hz, 1H, Ph), 4.13 (t, J = 6.30 Hz, 2H, CH2-O), 3.68 (s, 3H, CH3), 3.31 (br. s., 4H, Pip-3,5H), 3.09 (br. s., 4H, Pip-2,6H), 3.01–3.06 (m, 2H, 13 NCH2), 2.07–2.14 (m, 2H, CH2). C NMR (DMSO-d6): 193.63, 165.87, 164.60, 162.64, 160.78, 151.90, 134.80, 132.69, 130.33, 129.92, 116.16, 114.94, 108.79, 105.42, 102.45, 66.20, 55.47, 53.81, 51.83, 46.77, 24.61

3.2. Pharmacology 3 α [ H]N -Methylhistamine hH3R Displacement Assay The displacement binding assay was carried out as described by Kottke et al. with slight modiﬁcations [30,31]. Frozen crude membrane preparations of HEK-293 cells stably expressing the recombinant full length hH3R were thawed, homogenized, and incubated for 90 min at RT (continuously shaking) with [3H]Nα-Methylhistamine (2 nM) and different concentrations of the test compounds (ﬁve to eleven appropriate concentrations between 10−11 and 10−4 M, dilutions prepared robot-assisted with a Tecan Freedom EVO Molecules 2021, 26, 2300 14 of 18

(Männedorf, Switzerland) or HP D300 Dispenser (Männedorf, Switzerland) in a final assay volume of 200 µL per well. Non-specific binding was determined in the presence of pitolisant (10 µM). The bound radioligand was separated from the free radioligand by filtration through GF/B filters (pretreated with 0.3% (m/v) polyethylenimine) using an Inotech cell harvester (Dottikin, Switzerland). Unbound radioligand was removed by three washing steps with ice-cold water. Scintillation cocktail was added, and the liquid scintillation counting was performed with a Perkin Elmer TriluxBeta counter (Perkin Elmer, Rodgau, Germany). Scintillation data (c.p.m.) corrected for non-specific binding were analyzed using GraphPad Prism (V6.01, San Diego, CA, USA) software, using non-linear least squares/regr- ession fit. Ki values were calculated from IC50 values according to the Cheng–Prusoff equation [35]. Statistical analyses were performed on pKi values from at least three experiments, each performed, at least, in duplicates. Mean affinity values (Ki) were transferred into nanomolar concentrations (with 95% confidence interval).

3.3. In Vitro Antioxidant Activity Antioxidant properties of compounds were tested in vitro in two different assays: the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay [36,37] and the FeCl3 reduction activity assay (FRAP, ferric reducing antioxidant power) [37]. The tested compounds and ascorbic acid were dissolved in DMSO at the 10−2 mol/L concentration, and in ethanol at the concentration of 10−4 mol/L respectively. Moreover, 30% and 50% ascorbic acid activity cut-off for DPPH and FRAP assays respectively were set ad hoc.

3.3.1. DPPH Assay DPPH is a molecule containing a stable free radical. The reduction of DPPH (purple) in ethanol solution takes place in the presence of a hydrogen-donating antioxidant due to the formation of the non-radical form of DPPH-H (yellow). This transformation results in color change from purple to yellow, which can be measured spectrophotometrically. Thus, the decrease in absorbance is proportional to the decrease in concentration of DPPH (free radical) and discoloration of reaction mixture indicates the scavenging of free radicals by a tested antioxidant. In order to determine antioxidant capacity, 20 µL of the compound solutions (dissolved in 96% ethanol) was mixed with 180 µL of 0.3 mM ethanolic DPPH solution. The change in the absorbance was detected at 517 nm after 30 min of incubation. Results were expressed as percentage decrease in absorbance of the tested sample, compared to the sample containing the solvent + reaction mixture (blank with maximum concentration of the DPPH radical). L-ascorbic acid was used as a reference compound. Calculations were made using MS Excel, from the proportion where the change of absorbance in the vitamin C (vit. C) group was treated as 100%, according to Equation (1):

∆A × 100 % activity of Vit.C = b (1) ∆AVit. C

Calculation of % Vit. C activity: ∆Ab-the change in the absorbance of the test sample and ∆AVit. C is change in the absorbance of reference sample, or with vit. C.

3.3.2. FRAP Assay A modiﬁed method of Benzie and Strain [38] was adopted for the FRAP assay. The stock solutions included 300 mM acetate buffer (3.1 g of C2H3NaO2 × 3H2O and 16 mL of C2H4O2), pH 3.6, 10 mM TPTZ (2,4,6-tripyridyl-s-triazine) solution in 40 mM HCl, and 20 mM FeCl3 × 6H2O solution. The working solution was prepared by mixing 10 parts of acetate buffer, 1 part of TPTZ solution, and 1 part of FeCl3 × 6H2O solution. A portion of 180 µL of the FRAP solution was mixed with 20 µL of the tested compound solution and incubated at room temperature for 10 min in the dark. Readings of the color product (ferrous tripyridyl triazine complex) were then taken at 593 nm against ethanol. Results are Molecules 2021, 26, 2300 15 of 18

expressed as an increase in absorbance of the test sample compared to a sample containing the solvent. L-ascorbic acid was used as reference compound.

3.4. Molecular Modeling For docking purposes, Schrödinger Maestro Suite (v. 11.5.011, Release 2019-01) was used [39]. Ligands were built in either their forms (protonated N1 piperazine nitrogen, structure charge +1) or non-ionized forms. Bioactive conformations were generated using ConfGen module [40,41] (force field: OPLS3, water environment; minimization method: Polak-Ribier Conjugate Gradient (PRCG) with maximum iterations of 2500 and a conver- gence threshold of 0.05; conformational search of 100 steps per rotational bond). For all compounds, the five lowest energy conformers were selected for docking studies. Possi- ble binding pocket adaptation in the presence of certain ligands was examined using an induced fit refinement protocol [42] with 17. In order to validate the methods used, the ligand was then re-docked with high confidence. The site was then centered on the ligand. Docking to rigid the form of the receptor was performed using the Glide module [43,44] (extra precision, flexible ligand sampling, and a maximum of five poses per conformer). Ligands were rated according to their position in the binding pocket, interactions with the binding pocket amino acids, as well as the docking score value. Ligand–receptor visualizations were generated using Schrödinger Maestro. Theoretical pKa values were calculated using the Jaguar module [45,46]. Dynamic simulations (in time of 600 ps, T = 300 K) were performed using the Nosé– Poincaré–Andersen equations of motion, forcefield AMBER10: EHT; R-Field 1:80, cutoff (8,10), and performed in MOE v. 2019.1 [47].

4. Conclusions Looking for a new, active structures, is not an easy task. It even becomes harder when the reference structure is of one of the highest affinity hH3R ligands obtained to date; thus, increasing the possibility of ending up with lower affinity compounds. However, obtaining such compounds teaches us a lesson on structural uniqueness. In an attempt to find new possibly dual acting H3R ligands, in this study we obtained a series of compounds of moderate hH3R affinity, ranging around Ki of 500 nM (17), on the one hand, and on the other hand, expressing antioxidative properties (e.g., 16, −4 hH3R Ki = 592 nM, showing 50–60% of vitamin C activity at a 10 mol/L). To our knowl- edge, alongside a recently published Tetratarget ligand Contilisant and its predecessor ASS234 [27], obtained herein derivatives are some of the few histamine H3R ligands with described antioxidative properties. From a structural point of view, it appeared that simple (cyclo)alkyl substituents at the 4-position of piperazine negatively impact the ligand’s activity at the desired target, when compared to KSK63 (Figure1). Nonetheless, in the case of simple acyl moiety, a benzene ring separating the acyl from the basic core, through additional interactions, might force slightly higher affinity within our novel series (e.g., 10 vs. 4), yet still on the low level. Interestingly, docking into the histamine H3R homology model revealed two putative binding modes, with ionic key interactions retained in both cases. The stability of such obtained complexes was demonstrated by MD simulations. This also allowed for possible explanation of KSK63 high affinity. Last, but not least, in an attempt to find possible dual acting ligands, selected compounds were tested for antioxidant properties. Among others, compound 16 (hH3R Ki = 592 nM) showed the strongest antioxidant properties at a 10−4 mol/L concentration. Compound 16 not only significantly reduced the amount of free radicals showing 50–60% of AA activity, but had also exhibited antioxidative properties in general. Despite yet unknown mechanisms and moderate hH3R affinity, compound 16 (QD13) constitutes a novel, potential dual acting H3R ligand-promising starting point towards treatment of neurologic disorders associated with increased neuronal oxidative stress. In conclusion, although the obtained series of compounds appeared to be weak histamine H3 receptors ligands, they might be valuable pharmacological tools in the Molecules 2021, 26, 2300 16 of 18

search for novel molecules, targeting oxidative stress-based diseases. Identiﬁcation of strong histamine H3 receptor ligands, with antioxidative properties, will drive our further, planned studies.

Supplementary Materials: The following are available online, The Supplementary Material contain Table S1: Calculated pKa values for piperazine N1 and N4 nitrogen, Table S2: Putative binding poses for non-protonated conformers, Table S3: Putative binding poses for protonated conformers, Table S4: Molecular dynamics frames alignment for selected ligands and Figure S1: Antioxidant activity of the tested compounds in DPPH and FRAP assay. Author Contributions: Conceptualization, K.J.K. and M.K.; methodology, K.J.K., M.K. and D.R.-L.; validation, K.S., K.M. and D.R.-L.; formal analysis, K.S.; investigation, K.J.K., K.M. and D.R.-L.; data curation, K.J.K., M.K. and K.S.; writing—original draft preparation, K.J.K.; writing—review and editing, K.K.-K., M.K., D.R.-L., H.S. and K.S.; visualization, K.K.-K. and M.K.; supervision, K.K.-K. and H.S.; project administration, M.K.; funding acquisition, M.K. All authors have read and agreed to the published version of the manuscript. Funding: This study was partly supported by statutory funds from the Faculty of Pharmacy Jagiel- lonian University Medical College, Krakow, Poland (No: N42/DBS/000041 and N42/DBS/000068). This work was partly supported by National Science Center, Poland, granted on the basis of decision no: 2016/23/B/NZ7/01063. Further support by COST Actions CM1103, CA15135, CA18133, and CA18240 as well as DFG INST 208/664e1 FUGG (Germany) is also kindly acknowledged (to HS). Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The data presented in this study are available on request from the corresponding author. Conﬂicts of Interest: The authors declare no conﬂict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

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