4-Hydroxypiperidines and Their Flexible 3-(Amino)propyloxy Analogues as Non-Imidazole Histamine H3 Receptor Antagonist: Further Structure–Activity Relationship Exploration and In Vitro and In Vivo Pharmacological Evaluation

Presynaptic histamine H3 receptors (H3R) act as auto- or heteroreceptors controlling, respectively, the release of histamine and of other neurotransmitters in the central nervous system (CNS). The extracellular levels of several neurotransmitters are enhanced by H3R antagonists, and there is a great interest for potent, brain-penetrating H3 receptor antagonists/inverse agonists to compensate for the neurotransmitter deficits present in various neurological disorders. We have shown that 1-[(benzylfuran-2-yl)methyl]piperidinyl-4-oxyl- and benzyl- derivatives of N-propylpentan-1-amines exhibit high in vitro potencies toward the guinea pig H3 receptor (jejunum), with pA2 = 8.47 and 7.79, respectively (the reference compound used was thioperamide with pA2 = 8.67). Furthermore, following the replacement of 4-hydroxypiperidine with a 3-(methylamino)propyloxy chain, the pA2 value for the first group decreased, whereas it increased for the second group. Here, we present data on the impact of elongating the aliphatic chain between the nitrogen of 4-hydroxypiperidine or 3-(methylamino)propan-1-ol and the lipophilic residue. Additionally, the most active compound in this series of non-imidazole H3 receptor antagonists/inverse agonists, i.e., ADS-003, was evaluated for its affinity to the recombinant rat and human histamine H3 receptors transiently expressed in HEK-293T cells. It was shown that ADS-003, given parenterally for 5 days, reduced the food intake of rats, as well as changed histamine and noradrenaline concentrations in the rats’ brain in a manner and degree similar to the reference H3 antagonist Ciproxifan.


Introduction
The histamine H 3 receptors were first identified in 1983 in the rat brain by J.-Ch. Schwartz and coworkers [1]. The presence of H 3 receptors was confirmed in the human brain a few years later [2]. The cDNA encoding the human H 3 receptor was successfully cloned and functionally expressed by Lovenberg et al. [3]. Histamine H 3 receptors predominantly have a presynaptic localization in histaminergic or other neurons and they modulate, through a negative feedback mechanism, the biosynthesis and release of histamine as autoreceptors [4] or the release of various other neurotransmitters, including norepinephrine [5], dopamine [6], serotonin [7], gamma-aminobutyric acid (GABA) [8], glutamate [9], and acetylcholine [10], acting as heteroreceptors. Consequently, it has been observed that administration of H 3 -antagonists to the CNS enhances neurotransmission and improves cognition and attention in relevant animal models of CNS diseases [11]. Therefore, H 3 -antagonists/inverse agonists have been proposed for the treatment of cognitive disorders, such as attention-deficit hyperactivity disorder (ADHD) [12] and Alzheimer's disease [13] as well as of memory and learning deficits [10]. They may also be useful in epilepsy [14], schizophrenia [15], and obesity [16].
The discovery of thioperamide [17], the prototype of the H 3 antagonist, led to the development of a wide range of imidazole-containing ligands [18]. Although many of them have found utility as pharmacological tools, the presence of an imidazole ring greatly limited brain penetration [19] and also introduced the potential for cytochrome P450 interactions [20]. For these reasons, efforts have been directed toward the design and synthesis of non-imidazole H 3 antagonists with a good binding affinity, CNS penetration ability, and reduced/no potential for cytochrome P 450 inhibition. A number of such antagonists with high selectivity and specificity have since been reported [21].
The marine natural product aplysamine-1 patented by the Harbor Branch Oceanographic Institution as a weak histamine H 3 receptor antagonist [22], possesses the characteristic 3-aminopropan-1-ol functionality in its structure [23]. This moiety has successfully been used by several laboratories for the development of non-imidazole histamine H 3 receptor antagonists and resulted in a number of highly potent and selective compounds, for example, JNJ-5207852 [24] and Pitolisant BF2.649 (Wakix) [25], the potent and selective H 3 R antagonist, which was approved by the European Medicine Agency (EMA) in March 2016 for the treatment of the orphan disease narcolepsy with and without cataplexy ( Figure 1). Later on, the successful replacement of the highly flexible 3-aminopropyloxy link with the 4-phenoxypiperidine moiety JNJ-7737782 [26] (Figure 1) or the partially rigid 2-aminoethylbenzofuran substructure ABT-239 [27] (Figure 1) was demonstrated.
In continuation of our search for new highly active and selective non-imidazole histamine H 3 receptor antagonists, the first step of this study aimed to clarify the significant difference between the potencies of the 4-hydroxypiperidine derivatives and their 3-(methylamino)propyloxy analogues, bearing ω-(benzofuran-2-yl)alkyl and ω-phenylakyl moieties, respectively. We synthesized and in vitro pharmacologically [32] evaluated a series of derivatives in which the aliphatic chain between the nitrogen of the 4-hydroxypiperidine or the 3-(methylamino)propan-1-ol and the lipophilic residue was elongated by two to three methylene groups (compounds 1b,c,e,f, and 2b,c,e,f; Figure 1, respectively). This was to check whether the earlier observed tendency would still be kept, or if it was an exceptional case only for short methyl chain. We also wanted to study how the elongation of the alkyl chain would affect the potency for the H 3 receptor. Additionally, the affinity of ADS-003-the most active compound that we have so far synthesized in this series-was estimated for the recombinant rH 3 R and hH 3 R (respectively), transiently expressed in HEK-293T cells. This derivative has also been proven to cross the blood-brain barrier; given parenterally for 5 days, ADS-003 reduced the food intake by rats as well as changed the cerebral histamine and noradrenaline concentrations in these animals in a manner and degree similar to the reference H 3 antagonist-Ciproxifan.

Chemistry
The general synthetic procedures used in this study are illustrated in Schemes 1 and 2.
The synthesis of the required intermediates 4a,b (Scheme 3) was carried out by first treating the 2-iodophenol with the appropriate ω-alkyn-1-ol in the presence of palladium acetate, copper(I) iodide, and triphenylphosphine in dry triethylamine [33], followed by mesylation of the hydroxyl group by methanesulfonyl chloride in pyridine to yield the corresponding methanesulfonate with a 2 and 3 methylene linker. The detailed synthetic procedure and analytical data for compounds 8a,b and 4a,b are shown in Supplementary Material A (Section 1). All newly synthesized compounds were converted into their dihydrogenoxolates salts and in vitro evaluated as H 3 receptor antagonists against H 3 agonist-induced inhibition of the electrically evoked contraction of the guinea pig jejunum [32].
The differences were observed within the 1a-c and 1d-f series. While the previously reported 4-hydroxypiperidine derivative bearing a 2-benzofuranylmethyl moiety 1a showed a high potency, the newly synthesized compounds 1b,c, where the aliphatic chain between the piperidine nitrogen and the benzofuranyl residue is elongated from 2 to 3 methylene groups, resulted in a decrease of potency (pA 2 = 7.26 and 7.11, respectively). A similar effect was observed when the 2-benzofuranylmethyl substituent was replaced by a ω-phenylakyl one (compounds 1d-f). In this series, derivative 1d (pA 2 = 7.79) showed a high potency, but an increase in the alkyl chain length to 2 methylene groups resulted in a decrease of antagonist activity for compound 1e (pA 2 = 6.67), and the activity increased again on further lengthening to 3 methylene groups (1f; pA 2 = 7.51).
In the series of derivatives 2a-c, bearing a ω-(benzofuran-2-yl)alkyl substituent-analogues of compounds 1a-c-only a weak activity (pA 2 ranging from 6.23 to 6.37), independent of the alkyl chain length, was observed. Elongation of the alkyl chain from 2 to 3 methylene groups in the analogue series of compounds 1e,f i.e., 2e,f resulted in a drastic reduction of potency (pA 2 = 6.72 and 6.79, respectively) in comparison to the parent compound 2d (pA 2 = 8.06).
Representative graphs of the antagonism by ADS-003 (1a) and thioperamide of the inhibitory effect of R-(−)-α-methylhistamine (R-α-MH) on the electrically induced contraction of guinea pig ileum strips are shown in Supplementary Material B (Section 2.2.1; Figure S2). The compounds 1b and 1f, possessing the highest potency for the H 3 receptors, were also tested for H 1 antagonistic effects in vitro, using standard methods [34]. Derivatives 1b and 1f did not show any antagonistic activity for the H 1 -receptor (pA 2 < 4; for pyrilamine pA 2 = 9.37).

Histamine H 3 Receptor Affinity
The affinity, based on the SARs obtained for both series (compounds: 1a-f and 2a-f), of the most active compound 1a (ADS-003) was evaluated by measuring the displacement curve of [ 3 H]-N α -methylhistamine from the rat (rH 3 R) and human histamine H 3 receptor (hH 3 R) in HEK-293Tcell membranes, as described by Bonger [35].

Saturation of Rat and Human H 3 Receptors
The saturation of rat and human H 3 receptors were carried out as described previously [36]. A representative graph of saturation of rat and human H 3 R can be found in Supplementary Material B (Section 2.2.2; Figure S3).
The analysis of the [ 3 H]-N α -MH saturation binding yielded, at rH 3 R, a K D value of 2.72 ± 0.34 nM and a B max value of 2715 ± 445 fmol/mg protein and, at hH 3 R, a K D value of 0.9 ± 0.08 nM and a B max value of 632 ± 52 fmol/mg protein.

Competition Binding of H 3 Receptor Ligands
The affinity of ADS-003 (1a), histamine, and thioperamide was determined-the reference compounds were evaluated by measuring the displacement curves of [ 3 H]-N α -methylhistamine binding to rat and human histamine H 3 receptor expressed in HEK-293T membranes. Derivative ADS-003 (1a) possessed a high nanomolar affinity for the rat H 3 R, with a pK i value of 7.9 ± 0.1, similar to thioperamide (pK i value 7.9 ± 0.1) and slightly higher than histamine (pK i = 7.3 ± 0.1). A significantly lower affinity was observed for ADS-003 (1a) for the human H 3 R, with pK i 6.6 ± 0.1, in comparison with the pK i of thioperamide (7.2 ± 0.1) and the pK i of histamine (7.7 ± 0.1). Representative graphs of competition binding of H 3 R ligands to rat and human H 3 receptor are shown in Supplementary Material B (Section 2.2.2; Figures S4 and S5, respectively).

Verification of In Vivo Activity for Compound ADS-003 (1a)
Finally, compound ADS-003 (1a) was subjected to an in vivo evaluation of its impact on brain neurotransmitter systems. This assessment concerned: - The effects of the compound on the feeding behavior of rats after its repeated peripheral administration. Given that the compound enters the CNS and blocks the H 3 R, it should release histamine. Histamine, in turn, acting via H 1 R, would induce loss of appetite, i.e., the food intake of rats would decrease, - The influence on the cerebral amine neurotransmitter concentrations, as well as the activity of the monoamine oxidases A and B and histamine N-methyltransferase. The latter was accomplished by postmortem analyses of the brain tissues of the treated rats.
To evaluate the central effects of peripheral administration of ADS-003 (1a) to rats, the feeding behavior of rats was monitored after drug administration. The neuronal histaminergic system is known to be one of the regulatory systems in food intake. Studies in various animal models have convincingly shown that histamine H 1 R and H 3 R receptors play an important role in this respect. Activation of both histamine receptors is a critical part of the diurnal rhythm of the food consumption regulatory mechanism, as well as in energy intake and expenditure [37][38][39][40][41]. In a comprehensive study done in rats, it was demonstrated that centrally infused histamine or H 1 receptor agonists invariably decreased food intake, as did the strategies leading either to an enhanced release of hypothalamic histamine by the blocking the H 3 receptor or to an increase of available histamine by inhibiting its degradation. The opposite, i.e., hyperphagia, was seen for H 1 antagonists [38]. Compatible with the earlier reported experimental data, it was assumed that, if an H 3 receptor antagonist subcutaneously injected into rats crossed the blood-brain barrier (BBB), it would affect the animal food consumption [37]. As presented in Figure 2, the treatment of rats with either of the two H 3 R antagonists ADS-003 (3 mg/kg s.c.) and Ciproxifan (3 mg/kg s.c.) over a period of 5 days evoked a statistically significant reduction in the amount of consumed food by rats compared to the pre-treatment period. In in vivo studies, Ciproxifan was used as a reference instead of thioperamide, because the latter demonstrated lower bioavailability due to restricted brain penetration [42].
There was no difference in the efficacy of ADS-003 (1a) and Ciproxifan (the reference compound). These results suggest that ADS-003 crosses the BBB, and its potency at H 3 R is similar to that of Ciproxifan.

Post-Mortem Biochemical Analysis of the Brain Tissues of ADS-003 (1a)-Treated Rats
Postmortem biochemical analysis of the brain tissues of ADS-003-treated rats quantified the brain concentration of histamine, serotonin, dopamine, noradrenaline, and the activities of monoamine oxidase (MAO)-A, MAO-B, and HNMT. As shown in Figure 3, the histamine concentration in the hypothalamus, where histaminergic cell bodies are located, showed a tendency to increase-which could be explained by the stimulation of the amine synthesis following its release by H 3 R blockade with 1a (ADS-003) or Ciproxifan to replenish vesicular stores. Yet, one-way ANOVA and Tukey's multiple comparisons test showed no statistically significant differences. Similarly, no changes were found in the histamine levels in the cerebral cortex of the treated rats ( Figure 3). Paired t-test: p < 0.05 versus "before treatment" for eight rats. Paired t-test, p < 0.05, p < 0.01 versus "before treatment".
On the other hand, both H 3 R antagonists caused a significant increase in noradrenaline levels in the cerebral cortex ( Figure 4). There were no changes in serotonin and dopamine concentration. The increase in tissue NA is compatible with previous data reporting an inhibitory control exerted by H 3 histamine receptors on NA neuronal function in the cortex [43,44]. The fact that both histamine H 3 receptor antagonists, Ciproxifan and ADS-003, enhanced the tissue levels of NA in a similar manner strengthens this idea.
Using sensitive isotopic assays, neither changes in monoamine oxidase A and B nor in histamine N-methyltransferase in the brain tissues of rats were observed (Table 2). The values are given as means ± SEM for four-eight rats. The drugs were administered subcutaneously (s.c.) at a dose of 3 mg/kg of body mass for 5 consecutive days. MAO-monoamine oxidase, HNMT-histamine N-methyltransferase, CTX-cerebral cortex, HTH-hypothalamus. one-way ANOVA and Tukey's multiple comparisons test showed no statistically significant differences.

General Methods
All melting points (mp) were measured in open capillaries in an electrothermal apparatus and are uncorrected. The 1 H NMR spectra were recorded in CDCl 3 as a solvent in a 600 MHz spectrometer, a Bruker Avance III spectrometer at ambient temperature. The chemical shifts are reported in ppm on scale downfield from tetramethylsilane (TMS ) as internal standard, and the signal patterns are indicated as follows: s = singlet, d = doublet, t = triplet, m = multiplet, br = broad; * exchangeable by D 2 O; a number of protons, and J approximate coupling constant in Hertz. The 13 C NMR spectra were recorded in a 600 MHz spectrometer, a Bruker Avance III (150 MHz). Elemental analyses (C, H, N) for all compounds were measured in Perkin Elmer Series II CHNS/O Analyzer 2400 and agreed with the theoretical values within ±0.4%. TLC data were obtained with Merck silica gel 60F 254 aluminum sheets. For flash column chromatography using silica gel, 60 Å, 50 µm (J. T. Baker B. V.), the same solvent system as for TLC, was used. All obtained final free bases were treated with methanolic oxalic acid, and the dihydrogenoxolates were precipitated with dry diethyl ether and crystallized twice from ethanol. All dihydrogenoxolates were obtained as white crystalline solids.

General Procedure for the Preparation of Compounds 1b,c and 2b,c
To a solution of the corresponding amines 3 or 6 (1.26 mmol) in acetonitrile (5 mL), the appropriate methanesulfonate 4a or 4b (1.05 mmol) was added. The reaction mixture was stirred at 40 • C for 24 h. After completion of the reaction, water was added. The mixture was extracted with dichloromethane (3 × 20 mL), and the organic layer was dried over MgSO 4 and filtered. The solvent was evaporated, and the residue was purified by silica gel-flash column chromatography to yield a sticky oil.

General Procedure for the Preparation of Compounds 5a,b and 7a,b
The corresponding amines 3 or 6 (2.6 mmol) and Et 3 N (3.12 mmol) were dissolved in dichloromethane and cooled to 0 • C. After flushing with argon, 2-phenylacetyl chloride (3.12 mmol) or 3-phenylpropanoyl chloride (3.12 mmol) was slowly added to the mixture, and the reaction was stirred at room temperature for 12 h. After completion of the reaction, an aqueous solution of K 2 CO 3 was added. The reaction mixture was extracted with dichloromethane (3 × 20 mL), and the organic layer was dried over MgSO 4 and filtered. The solvent was evaporated, and the residue was purified by silica gel-flash column chromatography to yield a sticky oil. To a vigorous stirred solution of the corresponding amides 5a,b or 7a,b (1.0 mmol) in 250 mL of anhydrous diethyl ether, LiAlH 4 (2.5 mmol) was added portionwise. The mixture was stirred at reflux for 2 h, cooled to room temperature, and quenched by a dropwise addition of water (0.5 mL). The suspension was stirred for 30 min and filtered. The filter cake was washed with diethyl ether. The solvent was evaporated, and the residue was purified by silica gel-flash column chromatography (eluent: CH 2 Cl 2 /MeOH/NH 3(aq) 8:1:1%) to yield a sticky oil.  37 (m, 2H, H-3

H 3 Antagonistic Activity for Compounds 1a-f and 2a-f
In the first step, all the obtained compounds were tested for their H 3 antagonistic effects in vitro, following standard methods, using the electrically contracting guinea pig jejunum [32].
Male guinea pigs weighing 300-400 g were sacrificed, and a portion of the small intestine, 20-50 cm proximal to the ileocaecal valve (jejunum), was removed and placed in Krebs buffer (composition (mM) NaCl 118; KCl 5.6; MgSO 4 1.18; CaCl 2 2.5; NaH 2 PO 4 1.28; NaHCO 3 25; glucose 5.5; indomethacin (1 × 10 −6 mol/L)). Whole jejunum segments (2 cm) were prepared and mounted between two platinum electrodes (4 mm apart) in 20 mL Krebs buffer, continuously gassed with 95% O 2 :5% CO 2 , and maintained at 37 • C. the contractions were recorded isotonically under 1.0 g tension with a Hugo Sachs Hebel-Messvorsatz (Tl-2)/HF-modem (Hugo Sachs Elektronik, Hugstetten, Germany) connected to a pen recorder. The equilibration lasted for one hour with washings every 10 min. The muscle segments were then stimulated at a maximum between 15 and 20 Volts, continuously at a frequency of 0.1 Hz for a duration of 0.5 msec, with rectangular wave electrical pulses, delivered by a Grass Stimulator S-88 (Grass Instruments Co., Quincy, MA, USA). After 30 min of stimulation and 5 minutes before adding (R)-α-methylhistamine, pyrilamine (1× 10 −5 mol/L concentration in organ bath) was added, and then cumulative concentration-response curves (half-log increments) of (R)-α-methylhistamine, an H 3 -agonist, were recorded until no further change in the responses was found. Five minutes before adding the tested compounds, the pyrilamine (1 × 10 −5 mol/L concentration in an organ bath) was added. The antagonists were preincubated for 20 min during the stimulation, before the preparation were challenged with (R)-α-methylhistamine. Antagonist potency was determined by the construction of a Schild plot [34], using three different concentrations of the antagonist. The potency of an antagonist is expressed by its pA 2 value. The pA 2 values were compared with those indicating the potency of thioperamide.

H 1 Antagonistic Activity for Compounds 1b and 1f
In addition, the compounds 1b and 1f with the highest potency at the H 3 receptors were also tested for H 1 antagonistic effects in vitro, using standard methods [34]. The potencies of the aforementioned ligands were determined for the guinea pig ileum histamine H 3 receptors as described previously [36], using histamine as a competing agonist.
The pA 2 values were calculated according to Arunlakshana and Schild [34]. The pA 2 values were compared with those of pyrilamine.

Antagonist Binding to Rat rH 3 R and Human hH 3 R Cell Culture and Transfection
Human Embryonic Kidney cells (HEK293T) were cultured in DMEM supplemented with 10% Fetal Bovine Serum, 100 IU·mL −1 penicillin, and 100 µg·mL −1 streptomycin at 37 • C and 5% CO 2 . The day prior to transfection, 2 million cells were seeded in 10 cm dishes. Approximately 4 million cells were transfected by the polyethyleneimine (PEI) method with 5 µg of cDNA in a ratio of 1:4 (DNA/PEI). Briefly, 0.5 µg of pcDNA3-rH 3 R or pcDNA3.1-hH 3 R and 4.5 µg of empty plasmid (pcDNA3.1) were mixed with 20 µg of 25 kDa linear PEI in 500 µL of 150 mM NaCl and incubated for 30 min at 22 • C. Meanwhile, the medium in the 10 cm dishes was replaced with fresh culture medium, and the transfection mix was subsequently added dropwise to the cells, which were incubated for 48 h at 37 • C and 5% CO 2 .

Crude Membrane Extracts
Forty-eight hours after transfection, the cells were washed with ice-cold phosphate buffered saline (PBS) and scrapped, and the homogenate was centrifuged for 10 min at~2000× g, 4 • C. The supernatant was aspirated, and the cell pellets were resuspended in 1 ml ice-cold PBS and centrifuged again under the same conditions, aspirating the supernatant. The membranes were stored at −20 • C until further use.
The affinities of the derivative ADS-003 were determined for rat and human histamine H 3 receptors (445 isoforms) as described previously [36]

Verification of In Vivo Activity of Compound ADS-003 (1a)
All animal experimental procedures were in accordance with EU directives and local ethical regulations. Male Wistar rats (260-300 g) were used. The animals were maintained under standard laboratory conditions (liquid and food available ad libitum, 12 h light-dark cycle). For feeding behavior examination, the rats were placed individually in metabolic cages (TecniplastGazzada, Buguggiate, Italy) and kept there throughout the entire test. Pharmacotherapy was preceded by a control period of 4 days aimed to determine the basal feed and water consumptions as well as urine excretion. The rats (n = 8 per group) were randomly given the H 3 R antagonists ADS-003 or Ciproxifan, the latter serving as a reference compound [40]. Control rats were treated with an equivalent volume of physiological saline. The compounds, dissolved in distilled water with DMSO (7%, v/v), were administered subcutaneously at a dose of 3 mg/kg of body mass for 5 consecutive days, always during the morning hours, following the record of feeding parameters. The dose of ADS-003 had been determined on the basis of a thorough characterization in vitro. The recorded volumes of consumed food are expressed in g or ml per 100 g of body weight or in ml per 24 h, respectively. The final results are given as means, with SEM calculated for each 24 h period, computed from a four-day (before treatment) or a five-day (treatment) monitoring

Post-Mortem Biochemical Analyses
Following the behavioral study, the rats were sacrificed, and their brains were collected. From each brain, the cerebral cortex and hypothalamus were quickly dissected according to the Glowinski and Iversen method [37]. The samples were immediately frozen in liquid nitrogen and kept at −70 • C until assayed.
The enzyme activities are expressed as pmol/min/mg protein. Protein concentration was analyzed according to Lowry's method [47].

Conclusions
The highest in vitro potency as an H 3 receptor antagonist for both series was observed for the (benzylfuran-2-yl)methyl derivative of 4-hydroxypiperidine 1a (ADS-003) and the benzyl derivative of 3-(methylamino)propan-1-ol 2d. Compared to 1a and 2d, the in vitro potency was found to decrease with increasing chain length (1a-f; 2d-f), with the exception of the series 2a-c, where only weak potency was observed, independent of the alkyl chain length.
In competition radioligand binding studies to the rat histamine H 3 receptor, compound ADS-003 (pK i = 7.9 ± 0.1) showed nanomolar affinity at the same level as the reference compound thioperamide (pK i = 7.9 ± 0.1) and a slightly higher affinity than histamine (pK i = 7.3 ± 0.1). A significantly lower affinity was observed for ADS-003 for the human H 3 R, with pK i 6.6 ± 0.1, in comparison with the pK i of thioperamide (7.2 ± 0.1) and the pK ii of histamine (7.7 ± 0.1).
ADS-003, given parenterally for 5 days, reduced the food intake of rats as well as changed rats' brain amine concentrations in a manner and degree similar to those observed for the reference H 3 antagonist Ciproxifan. These results indicate that the compound crosses the blood-brain barrier and acts also as an H 3 R antagonist in vivo in the rat brain.
Supplementary Materials: Supplementary materials can be found at http://www.mdpi.com/1422-0067/19/4/ 1243/s1. financial support for the purchase of the necessary reagents for in vivo studies and also to thank H. Stark, from Heinrich-Heine-Universität Düsseldorf, Germany, for kindly donating Ciproxifan, the reference compound for in vivo study.
Author Contributions: Krzysztof Walczyński was responsible for the supervision and development of the whole project. Beata Olszewska performed the chemical syntheses of the newly synthesized compounds and performed preliminary pharmacological studies in vitro, both at H 3 and H 1 receptor. AnnaStasiak performed the extended pharmacological studies in vivo, elaborated and described the results. Daniel McNaught Flores performed the hH 3 and rH 3 binding affinity test, elaborated and described the results. Agnieszka Fogel coordinated the advanced pharmacological studies in vivo and interpreted the obtained results. Rob Leurs coordinated the hH 3 and rH 3 binding affinity test and interpreted the obtained results.

Conflicts of Interest:
The authors have declared no conflict of interest.