Bioorganic Chemistry 91 (2019) 103071 Contents lists available at ScienceDirect Bioorganic Chemistry journal homepage: www.elsevier.com/locate/bioorg Structural modifications and in vitro pharmacological evaluation of 4- T pyridyl-piperazine derivatives as an active and selective histamine H3 receptor ligands Katarzyna Szczepańskaa, Tadeusz Karcza, Agata Siwekb, Kamil J. Kudera, Gniewomir Latacza, Marek Bednarskic, Małgorzata Szafarzd, Stefanie Hagenowe, Annamaria Lubelskaa, Agnieszka Olejarz-Macieja, Michał Sobolewskia, Kamil Mikac, Magdalena Kotańskac, ⁎ Holger Starke, Katarzyna Kieć-Kononowicza, a Department of Technology and Biotechnology of Drugs, Faculty of Pharmacy, Jagiellonian University Medical College, Medyczna 9, Kraków 30-688, Poland b Department of Pharmacobiology, Faculty of Pharmacy, Jagiellonian University Medical College, Medyczna 9, Kraków 30-688, Poland c Department of Pharmacodynamics, Faculty of Pharmacy, Jagiellonian University Medical College, Medyczna 9, Kraków 30-688, Poland d Department of Pharmacokinetics and Physical Pharmacy, Jagiellonian University Medical College, Medyczna 9, Kraków 30-688, Poland e Institute of Pharmaceutical and Medicinal Chemistry, Heinrich Heine University Düsseldorf, Universitaetsstr. 1, 40225 Duesseldorf, Germany ARTICLE INFO ABSTRACT Keywords: A novel series of 4-pyridylpiperazine derivatives with varying alkyl linker length and eastern part substituents Histamine H3 receptor proved to be potent histamine H3 receptor (hH3R) ligands in the nanomolar concentration range. While paying Histamine H3 receptor ligands attention to their alkyl linker length, derivatives with a six methylene linker tend to be more potent than their Non-imidazole histamine H3R ligands five methylene homologues. Moreover, in the case of both phenoxyacetyl- and phenoxypropionyl- derivatives, Piperazine derivatives an eight methylene linkers possess lower activity than their seven methylene homologues. However, in global Selective ligands analysis of collected data on the influence of alkyl linker length, a three methylene homologues appeared tobeof Molecular docking Metabolic stability highest hH3R affinity among all described 4-pyridylpiperazine derivatives from our group up to date. Inthecase of biphenyl and benzophenone derivatives, compounds with para- substituted second aromatic ring were of higher affinity than their meta analogues. Interestingly, benzophenone derivative 18 showed the highest affinity among all tested compounds (hH3R Ki = 3.12 nM). The likely protein-ligand interactions, responsible for their high affinity were demonstrated using molecular modeling techniques. Furthermore, selectivity, intrinsic ac- tivity at H3R, as well as drug-like properties of selected ligands were evaluated using in vitro methods. 1. Introduction observed. Involvement in cognition, sleep-wake status, energy homeo- static regulation, inflammation etc. has attracted pharmaceutical re- Function of histamine as neurotransmitter has been proven with the search for numerous so far unmet therapeutic approaches in different discovery of the H3 receptor [1,2]. It is presynaptically located as au- peripheral, but mainly central diseases [5,6]. The wide spectrum of toreceptor controlling the synthesis and release of histamine. As het- possible therapeutic implications makes H3Rs one of the most re- eroreceptor it modulates with presynaptical localization the release of searched areas in the vast field of GPCR ligands – starting from imi- numerous other neurotransmitters, e.g. acetylcholine, norepinephrine, dazole containing ligands, through various non-imidazole derivatives, dopamine, serotonin, glutamate, γ-aminobutyric acid [3]. The hista- with recent introduction of successful Wakix® to pharmaceutical mine H3 receptor is anatomically localized primarily to the Central market. Nervous System (CNS) with prominent expression in basal ganglia, One such replacement for an imidazole is a piperazine moiety, an hippocampus, cortex and striatal area [4]. In the periphery H3R can be important versatile scaffold in rational drug design for most ofthe found with low density in gastrointestinal, bronchial and cardiovas- GPCR ligands. It is one of the most inherent structural elements in drug cular system. As H3 autoreceptor activation stimulates the negative finding, marked with a large variety of successes in biological appli- feed-back mechanism, reduced central histaminergic activity is cations. Appropriate physicochemical properties of the piperazine ⁎ Corresponding author. E-mail address: [email protected] (K. Kieć-Kononowicz). https://doi.org/10.1016/j.bioorg.2019.103071 Received 13 May 2019; Received in revised form 12 June 2019; Accepted 14 June 2019 Available online 20 June 2019 0045-2068/ © 2019 Published by Elsevier Inc. K. Szczepańska, et al. Bioorganic Chemistry 91 (2019) 103071 template make this molecular subunit a useful and well-positioned modifications [12,13]. Considered compounds represent promising, system in the search for new drugs. Furthermore, it acts as efficient and very high H3R antagonist potency, as well as interesting anticonvulsant, particular subject for a range of miscellaneous scientific objectives in precognitive and anorectic pharmacological profile in several rodent medicinal chemistry and thus, piperazine moiety is regarded as privi- models. leged framework [7]. This scaffold is mainly observed in molecules Structural modifications of leads performed within this work were targeting various CNS receptors, such as different subtypes of adre- divided into two phases. Phase 1 included: nergic, dopaminergic and serotoninergic receptors [8–10]. Therefore, paying attention to its versatile properties and pointing out that pi- • extension of the alkyl chain to 5–6 methylene groups, perazine is also observed as a key structural feature in many histamine • as well as (in the case of acetyl and propionyl derivatives generally H3R ligands (as a successful imidazole replacement), we focused on more active than tert-butyl and tert-pentyl analogues) both: exten- such derivatives in this work. sion of linker up to 8 and subtraction to 2 methylene groups. In general, all compounds presented herein fit the traditional pharmacophore model for histamine H3R blocking compounds, that After the selection of optimal linker length (based on in vitro stu- contain a basic moiety linked by a spacer to a central, mostly aromatic dies), in phase 2, various moieties (methyl, ethyl, phenyl, benzoyl) were core structure which then is connected to further affinity enhancing introduced to the aromatic ring in eastern part of compounds. elements, e.g. another basic moiety or hydrophilic/lipophilic groups or Paying attention to structural similarity of concerned compounds to a combination thereof [11]. other GPCR ligands, determination of affinity to histamine1 H , dopa- Structure-activity studies of previously described ligands allowed mine D2, muscarinic M1 and α1 adrenergic receptors was also carried for the establishment of the 4-pyridyl-piperazine moiety a new bioi- out. sosteric piperidine replacement in H3R ligands [12]. The results of the in vitro tests proved this scaffold as a crucial element for high hH3R affinity. Furthermore, modeling studies showed its role in the formation 2. Results and discussion of suitable interactions with the hH3R. Therefore, we selected eight previously described compounds 2.1. Chemistry KSK29, KSK19, KSK30, KSK25, KSK3, KSK31, KSK9 and KSK32, pre- sented in the Fig. 1, that served as lead structures for further The synthesis of desired final compounds 2–21 was achieved through the synthetic route presented in Scheme 1. According to the Fig. 1. KSK29, KSK19, KSK30, KSK25, KSK3, KSK31, KSK9, KSK32 and general structures of described ligands; hH3R – human histamine H3 receptor. 2 K. Szczepańska, et al. Bioorganic Chemistry 91 (2019) 103071 Scheme 1. General synthetic pathway for compounds 2–21. procedure [14] modified by us [12,13], the phenoxy alkyl bromides studies, using slightly modified methods to those described previously 1a–u were obtained by one-step alkylation of commercially available [15]. Briefly, compounds were tested at five to eleven appropriate 3 α phenols with α,ω-dibromoalkanes in propan-1-ol under reflux condi- concentrations in a [ H]N –Methylhistamine (KD = 3.08 nM) radi- tions. Obtained precursor bromides were then coupled with 1-(pyridin- oligand depletion assay to determine the affinity at human recombinant 4-yl)piperazine in the mixture of ethanol/water with powdered po- histamine H3R stably expressed in HEK-293 cells. tassium carbonate and a catalytic amount of potassium iodide. Final In vitro affinity data are assembled in Table 1. Paying attention to products were obtained as free bases and isolated as oxalic acid salts. the alkyl linker length, derivatives with either a five or a six methylene linker show similar affinity with a Ki value below 100 nM with the 2.2. Pharmacology exception of compound 7 and 10 (hH3R Ki = 115 and 397 nM, re- spectively). According to our assumptions, acetyl and propionyl deri- vatives tend to show a higher affinity at3 H R than tert-butyl and tert- 2.2.1. Histamine H3 receptor affinity pentyl analogues (6, 10, 7, 11 vs. 4, 8, 5 and 9). All compounds (as oxalate salts) were tested in H3R in vitro binding Table 1 Structures of compounds 2–21 and their in vitro histamine H3 receptor (hH3R) affinities. Given data represent mean values within the 95% confidence interval (CI). 1 2 1 2 No. N R R hH3R Ki [nM] x̅ [CI 95%] No. n R R hH3R Ki [nM] x̅ [CI 95%] 2 1 H 1957 [1212, 3158] 12 6 H 20.0 [13.6, 29.4] 3 1 H 1007 [622, 1630] 13 6 H 19.3 [6.57, 56.4] 4 4 H 29.9 [1.26, 708] 14 7 H 40.5 12.3, 134] 5 4 H 26.7 [15.9, 44.7] 15 7 H 38.9 [9.52, 159] 6 4 H 62.1 [7.02, 549] 16 2 H 21.1 [3.83, 116] 7 4 H 115 [26.8, 493] 17 2 H 53.6 [16.6, 174] 8 5 H 12.7 [4.35, 36.9] 18 2 H 3.12 [0.66, 14.6] 9 5 H 16.9 [7.95, 36.0] 19 2 H 22.2 [3.30, 149] 10 5 H 397 [220, 715] 20 2 CH2CH3 H 40.4 [17.1, 95.9] 11 5 H 69.2 [29.6, 162] 21 2 CH3 H 42.7 [12.4, 147] 3 K.