Non-Imidazole Histamine H3 Ligands. Part VII. Synthesis, In Vitro and In Vivo Characterization of 5-Substituted-2-thiazol-4-n-propylpiperazines

H3 receptors present on histaminergic and non-histaminergic neurons, act as autoreceptors or heteroreceptors controlling neurotransmitter release and synthesis. Previous, studies have found that the compound N-methyl-N-3-phenylalkyl-2-[2-(4-n-propylpiperazin-1-yl)-1,3-thiazol-5-yl]ethan-1-amine (ADS-531, 2c) exhibits high in vitro potency toward H3 guinea pig jejunal receptors, with pA2 = 8.27. To optimize the structure of the lead compound ADS-531, a series of 5-substituted-2-thiazol-4-n-propylpiperazines 3 were synthesized and subjected to in vitro pharmacological characterization; the alkyl chain between position 2 of the thiazole ring and the terminal secondary N-methylamino function was elongated from three to four methylene groups and the N-methylamino functionality was substituted by benzyl-, 2-phenylethyl-, and 3-phenyl-propyl- moieties. SAR studies on novel non-imidazole, 5-substituted-2-thiazol-4-n-propyl-piperazines 3 showed that the most active compound 3a (pA2 = 8.38), additionally possessed a weak competitive H1-antagonistic activity. Therefore, compound ADS-531, which did not exhibit any H1-antagonistic activity, was chosen for further evaluation for its affinity to the recombinant rat and human histamine H3 receptors (rH3R and hH3R, respectively). ADS-531 exhibited nanomolar affinity for both rH3R and hH3R receptors. It was also shown that, ADS-531 given subchronically to rats (s.c. 3 mg/kg, 5 days) penetrated the brain, where it affected dopamine, noradrenaline and serotonin concentration; however, it did not affect histamine concentration nor feeding behavior.


Introduction
The H 3 receptors mediate the diverse biological effects of the neurotransmitter histamine [1] and they are widely expressed in the mammalian brain, particularly in areas involved in cognitive processes and arousal, i.e., the cerebral cortex, hippocampus, basal ganglia, and hypothalamus [2,3]. H 3 receptors are located on histaminergic or non-histaminergic neurons, respectively acting as autoreceptors or heteroreceptors, controlling the release and synthesis of histamine [4] and of multiple neurotransmitters such as acetylcholine [5], norepinephrine [6] and dopamine [7]. These data suggested that H 3 antagonists could affect a number of behaviors and be useful in the treatment of cognitive deficits Molecules 2018, 23, 326 2 of 20 associated with a variety of disease states including Alzheimer's disease (AD) [8], attention deficit hyperactivity disorder (ADHD) [9], schizophrenia [10], and obesity [11].
The first generation of active histamine H 3 receptor antagonists were designed on the basis of a structural modification of the endogenous ligand, histamine, wherein the imidazole ring plays an important role [12]. Widely known representatives are thioperamide [13], and clobenpropit [14], both containing an isothiourea group (Figure 1).
Molecules 2018, 23, x FOR PEER REVIEW 2 of 20 multiple neurotransmitters such as acetylcholine [5], norepinephrine [6] and dopamine [7]. These data suggested that H3 antagonists could affect a number of behaviors and be useful in the treatment of cognitive deficits associated with a variety of disease states including Alzheimer's disease (AD) [8], attention deficit hyperactivity disorder (ADHD) [9], schizophrenia [10], and obesity [11]. The first generation of active histamine H3 receptor antagonists were designed on the basis of a structural modification of the endogenous ligand, histamine, wherein the imidazole ring plays an important role [12]. Widely known representatives are thioperamide [13], and clobenpropit [14], both containing an isothiourea group (Figure 1). Many ligands of this type have found utility in experimental studies as pharmacological tools [15]. However, the presence of an imidazole ring with strong hydrogen bond acceptor and donor properties causes low bioavailability and greatly limits penetration of the blood-brain barrier [16,17]. Among others, these compounds bind to the heme iron in CYP enzymes [18], and when co-administrated with other interacting drugs, can lead to adverse side-effects through drug-drug interactions [19]. Following these discoveries, and the successful cloning of the H3 receptor by Lovenberg et al. [20] in 1999, the intensive search for non-imidazole-based compounds was The general synthetic procedure used in this study is illustrated in Scheme 1. The central building blocks of the title compounds (compounds 3a-c and 3d-f) were 3-[2-(4-propylpiperazin-1-yl)-1,3-thiazol-5-yl]propan-1-amine (10a) and 4-[2-(4-propylpiperazin-1-yl)-1,3-thiazol-5-yl]butan-1-amine (10b), respectively (Scheme 1). The phthalimidobutanols 5a and 5b were prepared from ω-chloroalkanols 4a and 4b and phthalimide to yield 70% and 62% of the desired compounds, respectively. Scheme 1. Synthetic routes to compounds 3a-f.
The ω-phthalimidoalkanals 6a and 6b were obtained from ω-phthalimidoalkanols 5a and 5b by reaction with oxalyl chloride/dimethyl sulfoxide, followed by proton abstraction with triethylamine and hydrolysis to the corresponding aldehydes. Bromination of the aldehydes was performed in carbon tetrachloride and after identification with NMR, the crude 2-bromo-ω-phtalimidoalkanals 7a and 7b were used in the cyclization reaction (Scheme 1). Ring closure of the crude 2-bromo-ω-phtalimidoalkanals 7a and 7b with 1-(4-n-propyl)piperazine thioamide (8) was performed in anhydrous DMF under an argon atmosphere giving high yields of the desired 5-(ω-phthalimidoalkyl)thiazoles 9a, 9b. Subsequent hydrazinolysis, basification with sodium hydroxide and extraction with chloroform led to the production of pure amines 10a, 10b. Derivatives 12a and 12b were prepared from compound 10a, 10b by two-step synthesis including formylation with methyl formate to compounds 11a, 11b and finally reduction with LiAlH4 in dry ethyl ether. Propan-1-amines 3a-c and butan-1-amines 3d-f were synthesized from compounds 12a, 12b by alkylation with the corresponding primary phenyloalkyl halides in the presence of K2CO3 in DMF followed by purification by column chromatography. All free bases were treated with methanolic HBr and the hydrobromides were precipitated with dry diethyl ether. The 1-(4-n-propyl)piperazine Scheme 1. Synthetic routes to compounds 3a-f.

In Vitro Pharmacological Studies H 3 Antagonistic Activity for Compounds 2a-c and 3a-f
The compounds were tested in vitro as H 3 receptor antagonists against H 3 agonist-induced inhibition of the electrically evoked contraction of the guinea-pig jejunum [33]. The potency of the newly synthesized compounds 3a-f are reported in Table 1, as well as the previously described data for compounds 2a,c [31] and 2b [32]. Derivatives 3a-f show moderate to pronounced antagonist activity at H 3 -receptor. Propan-1-amines 3a-c (Table 1) and butan-1-amines 3d-f (Table 1) were synthesized to optimize the structure of the lead compound ADS-531 and the complementary 2a-c derivatives series [31,32]. In this series, the alkyl chain between position 2 of the thiazole ring and the terminal secondary N-methylamino function was elongated from three to four methylene groups. The N-methylamino functionality was substituted by benzyl-, 2-phenylethyl-, and 3-phenylpropylsubstituents, with these showing the highest potency in previously described series 2a-c. A comparison of homologous triplets, carrying benzyl substituents (compounds 2a, 3a, 3d), found derivative 3a (pA 2 = 8.38) to have a higher potency than its analogs 2a, 3d (pA 2 = 7.76 and 7.46, respectively). In the case of derivatives bearing a 2-phenylethyl substituent (compounds 2b, 3b, and 3e) the potency increases slightly with increasing alkyl chain length (pA 2 = 7.61, 7.81 and 7.95, respectively). The highest potency, for the series of derivatives carrying of 3-phenylpropyl-substituent (compounds 2c, 3c, and 3f), is seen for 2c (pA 2 = 8.27), but an increase in the alkyl chain length to two methylene groups resulted in a decrease of antagonist activity for compound 3c (pA 2 = 7.46), while the activity increased again when the chain was further lengthened to three methylene groups (3f; pA 2 = 7.91).
Differences are observed within the 2a-c and 3a-c series. In the series of derivatives containing an ethyl linker between position 2 of the thiazole ring and the terminal secondary N-methylamino function (compounds 2a-c), the compound bearing a 3-phenylpropyl-residue (2c; pA 2 = 8.27) shows the highest potency at the H 3 -receptor. Shortening the alkyl chain to two methylenes (compound 2b) or one methylene group (compound 2a) leads to a compound with a lower potency (pA 2 = 7.61 and pA 2 = 7.76, respectively). These results are in contrast to the results obtained for a series containing a propyl linker (compounds 3a-c). The compound carrying a benzyl substituent (3a; pA 2 = 8.38) shows the highest potency at the H 3 receptor, while the derivative with a 3-phenylpropyl moiety (3c; pA 2 = 7.46) shows the lowest antagonist activity. Compounds 3d-f, containing a butyl linker, show moderate potency at H 3 -receptor, independent of the alkyl chain length in the ω-phenylalkyl substituent (pA 2 ranging from 7.91 to 7.97).
To summarize, the obtained results indicated that elongation of the alkyl chain from two to three methylene groups between position 2 of the thiazole ring and the terminal secondary N-methylamino function resulted in compound 3a (bearing a benzyl substituent, and propyl linker). This compound demonstrated slightly higher potency than the parent compound 2c (bearing a 3-phenylpropyl moiety, and an ethyl linker) but in contrast to 3a, 2c did not possess any activity at H 1 . H1 Antagonistic Activity for Compounds 3a, and 3d The final compounds showing the highest potency for the H3 receptors were also tested for H1 antagonistic effects in vitro, following standard methods, using the guinea pig ileum [34]. Compounds 3a, and 3d show weak, but competitive H1-antagonistic activity with pA2 = 5.5, and pA2 = 6.25 (Table 1), respectively (for pyrilamine pA2 = 8.66).

Histamine H3 Receptor Affinity
Additionally, the affinity of the most active compound ADS-531 was evaluated by measuring the displacement curve of [ 3 H]-N α -methylhistamine at the rat (rH3R) and human histamine H3 receptor (hH3R) in HEK-293Tcell membranes as described by Bongers [35].

Saturation of Rat and Human H3 Receptors
To determine the total and non-specific binding, membranes expressing rH3R or hH3R were incubated with different concentrations of [ 3 H]-N α -MH (0-20 nM) in the absence or presence of unlabeled thioperamide (10 μM) for two hours at 25 °C. The reaction was terminated by rapid filtration on GF/C 96 well plates and the levels of the bound radioligand were measured by scintillometry. Specific binding was defined as the difference between the total and non-specific binding conditions. A representative graph of the saturation of rat and human H3R can be found in the Supplementary Material. The final compounds showing the highest potency for the H 3 receptors were also tested for H 1 antagonistic effects in vitro, following standard methods, using the guinea pig ileum [34]. Compounds 3a, and 3d show weak, but competitive H 1 -antagonistic activity with pA 2 = 5.5, and pA 2 = 6.25 (Table 1), respectively (for pyrilamine pA 2 = 8.66).

Histamine H 3 Receptor Affinity
Additionally, the affinity of the most active compound ADS-531 was evaluated by measuring the displacement curve of [ 3 H]-N α -methylhistamine at the rat (rH 3 R) and human histamine H 3 receptor (hH 3 R) in HEK-293Tcell membranes as described by Bongers [35].

Saturation of Rat and Human H 3 Receptors
To determine the total and non-specific binding, membranes expressing rH 3 R or hH 3 R were incubated with different concentrations of [ 3 H]-N α -MH (0-20 nM) in the absence or presence of unlabeled thioperamide (10 µM) for two hours at 25 • C. The reaction was terminated by rapid filtration on GF/C 96 well plates and the levels of the bound radioligand were measured by scintillometry. Specific binding was defined as the difference between the total and non-specific binding conditions. A representative graph of the saturation of rat and human H 3 R can be found in the Supplementary Material.
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.

Competitive Binding of H 3 Receptor Ligands
The affinity of ADS-531, histamine and thioperamide-the reference compound-were determined by measuring the displacement curves of [ 3 H]-N α -methylhistamine binding to the rat and human histamine H 3 receptor expressed in HEK-293T membranes. Derivative ADS-531 possesses a slightly lower nanomolar affinity for the rat H 3 R (pK i 7.5 ± 0.1) than thioperamide (pK i 7.9 ± 0.1), and slightly higher than histamine (pK i = 7.3 ± 0.1). A significantly higher affinity is observed for ADS-531 at the human H 3 R (pK i 8.5 ± 0.1) than of thioperamide (pK i 7.2 ± 0.1) and pK i of histamine (7.7 ± 0.1). Representative graphs of competition binding of H 3 R ligands on the rat and human H 3 receptors are shown in the Supplementary Material (Section 2.2.2; Figures S2 and S3, respectively).

Verification of In Vivo Activity of Compound ADS-531
The brain histaminergic system participates in the regulation of feeding behavior, and of the four histamine receptors, H 1 and H 3 play an important role. Their activation is a critical part of the regulatory mechanism behind the diurnal rhythm of food consumption, as well as energy intake and expenditure [36][37][38][39]. Studies have shown that the central administration of histamine and likewise H 1 receptor agonists, lowered food intake. Also, the strategies leading to enhanced synaptic histamine availability-i.e., the blockade of the H 3 receptor or inhibition of histamine catabolism-caused hypophagia while the administration of H 1 antagonists resulted in hyperphagia [37]. An in vivo evaluation was therefore performed on the impact of compound ADS-531 on brain neurotransmitter systems. Given that the compound enters the CNS and blocks H 3 R, its peripheral administration should result in neuronal histamine release. The released histamine, in turn, acting via H 1 R, would induce loss of appetite, resulting in a decrease of food intake. To ensure conclusive results, a five-day course of treatment was chosen with daily monitoring of consumption at 9 a.m. Any influence of subchronic administration of ADS-531 on cerebral amine neurotransmitters concentrations and/or the activities of catabolic enzymes, monoamine oxidases A and B and histamine N-methyltransferase would be disclosed by post-mortem analyses of the brain tissues of the treated rats. In our in vivo studies, Lewis rats were used as subjects, and ciproxifan was used as a reference instead of thioperamide, because the latter demonstrated lower bioavailability due to restricted brain penetration [40].
As can be seen in Figure 2, five-day treatment with ADS-531 did not influence food intake by the rats, whereas treatment with ciproxifan caused a significant decrease in consumption. It is important to note that the treatment was preceded by an adaptive period to experimental conditions. No significant changes were observed in CNS histamine concentration (Figure 3), nor in the enzyme activities related to histamine catabolism in the brain tissue of the sacrificed animals ( Table 2). In the hypothalamus ( Figure 3B), where the histamine cell bodies are located, the amine concentration in the ciproxifan-treated rats tended to be higher than in the untreated controls and was significantly higher than in ADS-531 injected rats. This could suggest some enhancement of histamine synthesis by ciproxifan, following the presumed enhanced release of histamine to the synapses with its anorectic effects; this observation agrees closely with the observed intravital decrement of food intake ( Figure 2B). Histamine is metabolized in the mammalian brain exclusively by the N-methylation pathway, involving histamine N-methyltransferase at the first step, followed by monoamine oxidase B, which catalyzes the oxidative deamination of N-telemethylhistamine. Neither of the two drugs used affected this pathway ( Table 2).      The values are given as means ± SEM for seven to eight rats; The drugs were administered subcutaneously at a dose of 3 mg/kg of body mass for five consecutive days; MAO-monoamine oxidase, HNMT-histamine N-methyltransferase, CTX-cerebral cortex, HTH-hypothalamus.  The values are given as means ± SEM for seven to eight rats; The drugs were administered subcutaneously at a dose of 3 mg/kg of body mass for five 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. The apparent lack of the effects of the tested compound on the histaminergic system was by no means caused by the lack of its ability to cross the blood-brain barrier. The data presented in Figure 4 clearly indicates that ADS-531 caused alterations in the concentrations of dopamine, noradrenaline, and serotonin.
An increase of 5HT and NA level throughout the brain with the exception of the hypothalamus indicates decreased serotonergic and noradrenergic activity and a concomitant increase in DA system activity. These findings may suggest that ADS-531 is likely to show some agonistic activity to serotonin autoreceptors, thereby modifying dopamine and noradrenaline release [41].

Conclusions
ADS-531 was found to exhibit the highest in vitro affinity toward the H 3 guinea pig jejunal receptors with pA 2 = 8.27. In competition radioligand binding studies at the rat histamine H 3 receptor, compound ADS-531 (pK i 7.5 ± 0.1) showed slightly lower nanomolar affinity than the reference compound-thioperamide (pK i = 7.9), and slightly higher than histamine (pK i = 7.3 ± 0.1). Significantly higher affinity was observed for ADS-531 at the human H 3 R (pK i = 8.5 ± 0.1) than thioperamide (pK i = 7.2 ± 0.1) and pK i of histamine (7.7 ± 0.1). ADS-531 given parenterally for five days did not influence the food intake in rats. No significant changes were observed in histamine concentration, nor in the enzyme activities related to histamine metabolism examined in the brain. The apparent lack of the effects of the tested compound on the histaminergic system was by no means caused by the lack of its ability to cross the blood-brain barrier. The presented data leaves no doubt that ADS-531 caused alterations in the concentrations of dopamine, noradrenaline, and serotonin. The high potency and affinity for H 3 receptors and in vivo activity suggest that further study on ADS-531 is merited.

General Information
All melting points (m.p.) were measured in open capillaries on an electrothermal apparatus and are uncorrected. Infrared spectra (IR) were measured on a FT-IR vegus spectrophotometer (Thermo Nicolet, city, state abbrev if USA, country). For all compounds, 1 H-NMR spectra were recorded on a Mercury VX 300 MHz spectrometer (Varian, city, state abbrev if USA, country). Chemical shifts are expressed in ppm downfield from internal TMS as a reference. 1 H-NMR data are reported in order: multiplicity (br, broad; s, singlet; d, doublet; t, triplet; m, multiplet; * exchangeable by D 2 O) number of protons, and approximate coupling constant in Hertz. 13 C-NMR spectra were recorded on an Avance III 600 MHz spectrometer (Bruker, city, state abbrev if USA, country). Elemental analysis (C, H, N) for all compounds was measured on Series II CHNS/O Analyzer 2400 (Perkin Elmer, city, state abbrev if USA, country) and were within ±0.4% of theoretical values. TLC was performed on silica gel 60 F 254 plates (Merck, city, state abbrev if USA, country). Flash column chromatography was carried out using silica gel 60Å 50 µm (J. T. Baker B. V., Phillipsburg, NJ, USA), employing the same eluent as was indicated by TLC. All obtained final free bases were treated with methanolic HBr, the hydrobromide was precipitated with dry diethyl ether and crystallized twice from ethanol.
The residue was dissolved in 150 mL of ethyl acetate, and after standing overnight at 5 • C, the solution was filtered and concentrated in vacuo. The residue was taken up in 80 mL of CHCl 3 . After extraction with 60 mL of 5% aqueous solution of NaHCO 3 and 3 × 60 mL of H 2 O, the solvent was dried over Na 2 SO 4 and removed in vacuo, giving:

General Procedure for the Preparation of Compounds 6a,b
To a well-stirred solution of oxalyl chloride (6.08 mL, 0.07 mol) in dry CH2Cl2 (120 mL), a solution of anhydrous DMSO (5.69 mL, 0.08 mol) in CH 2 C1 2 (200 mL) was added under an argon atmosphere at −70 • C at such a rate that the temperature was maintained at −70 • C. After the addition was completed, stirring was continued for 15 min, and then, a solution of 5a (7.7 g, 0.033 mol) or 5b (8.16 g, 0.033 mol) in dry CH 2 C1 2 (70 mL) was added while keeping the temperature at −70 • C. The reaction mixture was stirred for another 30 min at −70 • C, and Et 3 N (17.1 mL, 0.122 mol) was added. The mixture was allowed to warm to ambient temperature, and 100 mL of 5% aqueous solution of HCl was added and stirring was continued for 20 min. The organic layer was separated and extracted with H 2 O to the almost neutral reaction, dried over anhydrous Na 2 SO 4 , filtered, and concentrated in vacuo to yield the crude product 6a and 6b. The obtained viscous oils were stored under argon and used without further purification for the preparation of 7a and 7b:

General Procedure for the Preparation of Compounds 7a,b
Small portions of a solution of Br 2 (4.8 g, 0.03 mol) in acetonitrile (10 mL) were added to a stirred solution of 6a (6.93 g, 0.03 mol) or 6b (7.36 g, 0.03 mol) in CCl 4 (130 mL) under an argon atmosphere. The reaction mixture was stirred for two hours at ambient temperature, and then 5% aqueous solution of NaHCO 3 (200 mL) and CHCl 3 (50 mL) were added followed by stirring for 15 min. The organic phase was separated and extracted under an argon atmosphere with H 2 O till neutral reaction. After drying over anhydrous Na 2 SO 4 and filtration, the solvent was removed in vacuo. The resulting viscous oils were used without further purification for the preparation of 9a and 9b.

General Procedure for the Preparation of Compounds 9a,b
A solution of 4-propylpiperazine-1-carbothioamide (8, 4.12 g, 0.022 mol) in anhydrous DMF (250 mL) was added to a stirred solution of 7a (7.06 g, 0.022 mol) or 7b (7.2 g, 0.022 mol) in anhydrous DMF (250.0 mL) under an argon atmosphere. The reaction mixture was heated at 95 • C for nine hours. After cooling, the solvent was removed in vacuo. The residue was dissolved in n-propanol and cooled to 5 • C. The precipitate was filtered off and washed with ether. The hydrobromide product was obtained as a brown solid. The free base was obtained as follows: the hydrobromide was mixed with saturated aqueous potassium carbonate solution over night at room temperature. The solid was filtered, washed with water, ether and air dried to leave a light brown solid. The crude product was purified by silica gel flash-column chromatography, giving:

General Procedure for the Preparation of Compounds 10a,b
A solution of 9a (3.19 g, 0.008 mol) or 9b (3.30 g, 0.008 mol) and of N 2 H 4· H 2 O (0.8 g, 0.016 mol) in MeOH (50 mL) was refluxed for nine hours. The solvent was evaporated and the remaining material was dissolved in 30 mL of methylene chloride. After cooling, the crystallized phthalhydrazide was filtered off. Concentration in vacuo provided a white sticky semi-solid, which was purified by column chromatography on silica gel, giving:  To a vigorous stirred suspension of 11a (0.98 g, 0.0033 mol) or 11b (1.02 g, 0.0033 mol) in anhydrous ethyl ether (200 mL), LiAlH 4 (0.35 g, 0.009 mol) was added. The mixture was stirred at room temperature for two hours, and quenched by dropwise addition of water (0.7 mL), 5% of NaOH solution (0.6 mL), and water (0.2 mL). The suspension was stirred for 30 min, and filtered. The filter cake was washed with ether (2 × 50 mL). The combined organic extracts were washed with water (3 × 50 mL), dried (Na 2 SO 4 ), and filtered. The solvent was evaporated and remaining material was purified by column chromatography on silica gel, giving:

General Procedure for the Preparation of Compounds 3a-f
The corresponding phenylalkyl bromide (0.001 mol) was added to a solution of 12a (0.283 g, 0.001 mol) or 12b (0.296 g, 0.001 mol) in the presence of potassium carbonate (0.002 mol) in acetonitrile (20.0 mL). The reaction mixture was stirred for ninety-six hours at room temperature. The solvent was evaporated and water (25 mL) was added to the residue the mixture was extracted with dichloromethane (3 × 25 mL). The water layer was discarded and the solvent was dried over Na 2 SO 4 and filtered. The solvent was evaporated and the crude product was purified by column chromatography on silica gel, giving: N-Benzyl-N-methyl-3-[2-(4-n-piperazin-1-yl)-1,3-thiazol-5-yl]propan-1-amine (3a). A sticky oil, yield 74.0% (0.28 g); R f = 0. 16

H 3 Antagonistic Activity for Compounds 3a-f
In the first step, all obtained compounds were tested for H 3 antagonistic effects in vitro, according to standard methods, based on electrically contracting guinea pig jejunum [33]. 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 and 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. Contractions were recorded isotonically under 1.0 g tension with Hugo Sachs Hebel-Messvorsatz (Tl-2)/HF-modem (Hugo Sachs Elektronik, Hugstetten, Germany) connected to a pen recorder. The equilibration took place for one hour with washings every 10 min. The muscle segments were then stimulated at a maximum between 15 and 20 V, continuously at a frequency of 0.1 Hz for a duration of 0.5 ms, with rectangular-wave electrical pulses, delivered by a Grass Stimulator S-88 (Grass Instruments Co., Quincy, MA, USA). After 30 min of stimulation, pyrilamine (1 × 10 −5 mol/L concentration in organ bath) was added, followed by (R)-α-methylhistamine five minutes later, and then cumulative concentration-response curves (half-log increments) of (R)-α-methylhistamine, H 3 -agonist, were recorded until no further change in response was found.
Five minutes before adding the tested compounds, pyrilamine (1 × 10 −5 mol/L concentration in an organ bath) was added, after another 20 min cumulative concentration-response curves (half-log increments) of (R)-α-methylhistamine, an H 3 -agonist, were recorded until no further change in response was found. Statistical analysis was carried out with the Students' t-test. In all tests, p < 0.05 was considered statistically significant. The potency of an antagonist is expressed by its pA 2 value, calculated from the Schild [33] regression analysis where at least three concentrations were used. The pA 2 values were compared with the potency of thioperamide.

H 1 Antagonistic Activity for Compounds 3a,d
Selected final compounds were tested for H 1 antagonistic effects in vitro, following standard methods, using the guinea pig ileum [34]. The donors were male guinea pigs (300-400 g) as mentioned above. The excised ileum was placed in phosphate buffer at room temperature (pH 7.4) containing (mM) NaCl (136.9); KCl (2.68); NaHPO 4 (7.19). The intraluminal content was flushed, and segments about 2 cm in lenght were cut and mounted for isotonic contractions in water mixed with 20 mL organ baths filled with oxygenated (O 2 :CO 2 = 95:5, v/v) Krebs buffer containing (mM) NaCl (117.5); KCl (5.6); MgSO 4 (1.18); CaCl 2 (2.5); NaH 2 PO 4 (1.28); NaHCO 3 (25); glucose (5.5) and indomethacin (1 × 10 −6 mol/L) at 37 • C under a constant load of 0.5 g. After a 30 min equilibration period with washings every 10 min, a submaximal priming dose of histamine (1 mM) was given and washed out (standard washing procedure: 3 changes of buffer during 30 min). After washing out, the antagonistic activity of given compounds was measured by recording a Concentration Response Curve (CRC) for histamine in the presence of the tested compounds 3a and 3d, which were added 5 min before histamine. This procedure was repeated with higher concentrations of the compounds. The antagonism was of a competitive nature causing a parallel shift of the CRC. The pA 2 -values were calculated according to Arunlakshana and Schild [34]. The pA 2 values were compared with the affinity of pyrilamine.

Antagonist Binding to the 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 and 100 IU·mL −1 penicillin and 100 µg·mL −1 streptomycins at 37 • C and 5% CO 2 . The day prior transfection two million cells were seeded in 10 cm dishes. Approximately four 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, medium from 10 cm dishes was replaced with fresh culture medium and the transfection mix was subsequently added drop-wise to the cells and incubated for 48 h at 37 • C and 5% CO 2 .

Crude Membrane Extracts
Forty-eight hours after transfection cells were washed with ice-cold phosphate buffered saline (PBS) scrapped and the homogenate centrifuged for 10 min at~2000 g, 4 • C. The supernatant was aspirated and cell pellets were resuspended in 1 mL ice-cold PBS and centrifuged again under same conditions, the supernatant aspirated and the membranes stored at −20 • C until further use. Total and non-specific binding was determined in the absence or presence of excess non-labeled thioperamide (10 µM), respectively. For the competition binding assay, homogenates were incubated with increasing concentrations of receptor ligands (10 −11 to 10 −4 M) and~2.5 nM of [ 3 H]-NαMH. All assays were incubated at 25 • C for two hours on a shaking table (600 rpm). The reaction was terminated by rapid filtration into 0.5% polyethyleneimine pre-soaked glass fiber C plates (GF/C Perkin Elmer) followed by three washes with ice-cold Tris-HCl buffer (pH 7.4 at 4 • C). The plates were dried for one hour at 50 • C and scintillation liquid were added to each well (25 µL). Retained radioactivity was determined by liquid scintillation counting in a Wallac Microbeta (Perkin Elmer). Protein determination for B max estimation was performed with a Pierce BCA protein assay kit and measured by spectrophotometry in Power Wave X340 (Bio-Tek Instruments Inc., BioTek, Winooski, VT, USA).