Guanidine Derivatives: How Simple Structural Modification of Histamine H₃R Antagonists Has Led to the Discovery of Potent Muscarinic M₂R/M₄R Antagonists

Abstract

This article describes the discovery of novel potent muscarinic receptor antagonists identified during a search for more active histamine H₃ receptor (H₃R) ligands. The idea was to replace the flexible seven methylene linker with a semirigid 1,4-cyclohexylene or p-phenylene substituted group of the previously described histamine H₃R antagonists ADS1017 and ADS1020. These simple structural modifications of the histamine H₃R antagonist led to the emergence of additional pharmacological effects, some of which unexpectedly showed strong antagonist potency at muscarinic receptors. This paper reports the routes of synthesis and pharmacological characterization of guanidine derivatives, a novel chemotype of muscarinic receptor antagonists binding to the human muscarinic M₂ and M₄ receptors (hM₂R and hM₄R, respectively) in nanomolar concentration ranges. The affinities of the newly synthesized ADS10227 (1-{4-{4-{[4-(phenoxymethyl)cyclohexyl]methyl}piperazin-1-yl}but-1-yl}-1-(benzyl)guanidine) at hM₂R and hM₄R were 2.8 nM and 5.1 nM, respectively.

Introduction

In addition to mediating the inhibition of synthesis and release of histamine from histaminergic neurons via a negative feedback loop, the histamine H₃ receptor (H₃R) also exerts modulatory effects on numerous other neurotransmitter systems, including the cholinergic system, in both the central and peripheral nervous system. Stimulation of H₃ heteroreceptors in the central nervous system (CNS) by H₃R agonists (imetit, immepip) diminishes acetylcholine (ACh) release; however, blocking H₃ autoreceptors activity with the H₃R antagonist (thioperamide) increases ACh release.¹ Additionally, the ability of H₃R antagonists to improve cognition and to increase release of ACh in rats was described.² Activation of H₃R in CNS reduces ACh release in the rat cortex, hippocampus, nucleus accumbens, and basolateral amygdala.^1,3−6 In addition, the histaminergic neurons in the ventral striatum modulate the activity of neighboring cholinergic neurons. Histamine released from histaminergic nerve terminals inhibits dopamine release, which decreases γ-aminobutyric acid release, and, in turn, increases the release of acetylcholine. Disturbances in the CNS cholinergic system have been implicated in the pathophysiology of Alzheimer’s and Parkinson’s disease, schizophrenia, depression, or epilepsy.⁷⁻¹⁰ H₃R treatment has also been found to modulate cholinergic transmission in the peripheral nervous system.¹¹ H₃R activation reduces the release of [³H]-ACh induced by electrical stimulation in the longitudinal smooth muscle/myenteric plexus preparations.^12,13

Muscarinic M₂ and M₄ receptor (M₂R and M₄R, respectively) antagonists represent compounds of interest for potential drugs. Activation of M₂R and M₄R inhibits adenylyl cyclase via the stimulation of the G_i/o G-protein, whereas M₁R, M₃R, and M₅R mediate the stimulation of phospholipase C via G_q/11 G-protein.¹⁴ The cognitive deficits observed in aging and Alzheimer’s disease have been associated with brain cholinergic deficits. However, cognitive performance could be enhanced by selective blockade of presynaptically located M₂ autoreceptors, which could increase ACh release into the synaptic cleft.¹⁵ One of the acetylcholinesterase (AChE) inhibitors approved for use across the full spectrum of these cognitive disorders is donepezil; however, numerous potent M₂R antagonists including SCH-217443 (Chart 1) have shown efficacy in increasing ACh release and improving cognitive functions.¹⁶ The effective dose of SCH-217443 in the rodent cognition model was found to be 30-fold lower than that known to increase heart rate in rats.¹⁶ It is considered that ACh plays a crucial role in governing learning and memory processes.¹⁷ Therefore, AChE inhibitors constitute a major drug class in the treatment of dementia associated with Alzheimer’s disease, but provide only modest symptomatic benefit. Another approach to the therapy of Alzheimer’s disease is to inhibit the presynaptic activity of the muscarinic M₂R expressed on the cholinergic neurons, in regions involved in learning and memory processes.^18,19 As previously mentioned, the histamine H₃R exerts modulatory effects on the cholinergic system, and H₃R antagonist increases ACh release. Therefore, dual active H₃R/M₂R antagonists were invented and described.² Those dual active ligands (Chart 1) relates to the treatment of Alzheimer’s disease, attention deficit disorder, and autism.

Chart 1. Structures of Histamine H₃R (ADS1017, ADS1020), Muscarinic M₂R (SCH-217443), Muscarinic M₄R (PCS 1055), Muscarinic M₂R/M₄R (AF-DX 384), and H₃R/M₂R (8) Antagonists.

The muscarinic M₄R are expressed in the striatum, prefrontal cortex, and nucleus accumbens; these areas are all related to social behaviors and cognitive functions.²⁰⁻²² M₄R agonists and allosteric modulators may be useful for the therapy of Alzheimer’s disease, schizophrenia, cognitive disorders, or treatment of drug abuse. Many M₄R antagonists display limited selectivity between receptor subtypes. One recently described selective and potent M₄R competitive antagonist is PCS1055 (Chart 1).²³ M₄R antagonists may produce psychotic-like symptoms (ADHD, hallucinations); nevertheless, they may be useful pharmacological tools in elucidating the M₄R signaling mechanism. Experimental data suggest that M₄R autoreceptors located in cholinergic interneurons may be useful in increasing ACh release in the striatum. This approach may be a promising alternative therapeutic method in Parkinson’s disease therapy.²⁴

In contrast to the variety apparent in ligands, the binding sites of the H₃R and muscarinic receptors share many common features. Studies suggest that H₃R had a common ancestor with the muscarinic receptors.²⁵ A detailed comparison of the active site sequences is summarized in the Supporting Information (Table S1).

An analysis of the complexes formed between the hM₂R and hM₄R and their antagonists identifies 25 amino acids of key importance for ligand binding. As shown in Figure 1, 10 of these (40%) are identical to those of the hH₃R.²⁶ Most of the remaining amino acids retain the physicochemical properties of their histamine receptor analogs. It should be emphasized that the number and distribution of aromatic amino acids significantly modify the ligand available space at the binding site. In the case of the studied receptors, the conformation of aromatic amino acids 2.61, 2.64, 3.33, 6.51, 6.48, and 7.39 seems to be of particular importance. Earlier studies also indicate that phenylalanines F192, F193 located at the end of H₃R ECL2 and their analogs from M₂R (F180, F181) and M₄R (F189, L190) are involved in the binding of ligands.²⁷ Another significant similarity between the analyzed receptors is the presence of an extensive amino acid at position 7.42 (H₃R L7.42, M₂R and M₄R C7.42), which can modify the arrangement of W6.48 in the inactive state of the receptor.²⁸

Figure 1.

The amino acid sequence of the H₃R, specifying the amino acids involved in the binding of the ligands. Amino acids identical to the M₂R and M₄R are marked in green and differing in yellow.²⁶

The differences between the two pairs of amino acids seem particularly important due to their participation in the binding of endogenous ligands. The first is the glutamic acid present in the H₃R, E5.46, which is replaced by alanine in all muscarinic receptors. Mutagenesis studies carried out on the histamine H₃R within this site indicate that such a replacement significantly weakens or completely prevents the effective binding of both agonists and antagonists. The second pair is the T6.52 from the H₃R which replaces the N6.52 present in all muscarinic receptor subtypes. This limits the number of potential hydrogen bonds formed at this site while reducing the polarity of the surroundings. It is worth noting that mutagenesis studies indicate that the presence of asparagine at this position is essential for ligand binding at the muscarinic receptors binding site.^29,30 Such differences merit particular consideration when designing new ligands for both histamine H₃R and M₂R and M₄R; slight structural changes may significantly determine the activity profile of the new compounds.

Results and Discussion

This article describes the discovery of novel potent muscarinic receptor antagonists identified during a search for more active H₃R ligands. Our previous study clarified whether both nitrogen atoms of the piperazine ring are necessary to maintain a high activity in the H₃R antagonists, ADS1017 and ADS1020 (Chart 1), and determined the influence of moving the benzyl- and 4-trifluoromethylbenzyl substituents from the N¹ to the N³ position of the guanidine.³¹ Finally, two symmetrical compounds, 1,4-bis{4-[1-(4-trifluoromethylbenzyl)guandin-1-yl]but-1-yl}piperazine (ADS1030) and 1,4-bis(7-phenoxyheptyl)piperazine (ADS1031), were synthesized to identify the part of the parent compound that plays a key role in blocking H₃R. The most potent derivatives we found had piperazine as a central core with disubstitution to N¹ of guanidine. Compounds based on 1-[4-(piperazin-1-yl)but-1-yl]guanidine proved to be key to maintaining a high affinity at the histamine H₃R. Based on previously obtained data of the guanidine series, two representative of the lead compounds, that is, ADS1017 and ADS1020, were selected for further structural optimization.^31,32 In this paper, we have focused on the synthesis and pharmacological evaluation of a guanidine series where a flexible alkyl chain consisting of seven methylene groups, present in the lead compounds, is replaced by 1,4-cyclohexylene or p-phenylene group connected directly or by a methylene group to piperazine and phenoxy moieties. Additionally, for derivatives bearing a 1,4-disubstituted cyclohexylene group, the (E) and (Z) isomers were separated and pharmacologically tested independently (Chart 2).

Chart 2. Target Molecules of This Study.

The idea of synthesizing compounds in which the flexible alkyl chain was replaced by a semirigid aryl or cycloalkyl ring arose from our previous experiments and literature data.³³ Previous studies have demonstrated that replacement of the alkyl chain with more rigid moieties such as aryl or heterocyclic rings results in the formation of highly affine H₃R ligands.³⁴ Furthermore, a moiety with a more restricted conformation may be better fitted to the receptor-binding site due to reduced flexibility (i.e., degrees of freedom).²⁸ A key design element was to conserve the number of atoms between the phenoxy moiety and the basic nitrogen of piperazine and maintain an overall reduction in the number of rotatable bonds, thus producing more conformationally restricted compounds.

All newly synthesized compounds were evaluated as antagonists at H₃R on guinea pig ileum (gpH₃R), following which their selectivity to histamine H₁R (gpH₁R) and muscarinic M₂R/M₃R (gpM₂R/M₃R) was investigated. During these bioactivity profiling studies, the tested compounds demonstrated high affinities at muscarinic receptor subtypes. Therefore, all compounds were subjected to radioligand displacement assay in membrane fractions of HEK-293 cells stably expressing human H₃R (hH₃R). Finally, hM₁–hM₅ radioligand binding experiments were carried out for selected ligands. For two compounds, the intracellular Ca²⁺ level was measured as a functional response to ACh. Further investigation of the potent H₃R antagonist ADS1017 also revealed additional moderate affinity to hM₂R and hM₄R, which may justify searching for other dual-active ligands in the guanidine group. The present paper describes the discovery of the novel potent muscarinic receptor antagonist ADS10227 (Chart 1), which demonstrates particular activity against the hM₂R and hM₄R. To understand the molecular basis of the unexpected muscarinic activity, in silico studies were also conducted.

Chemistry

Synthesis of (E)-1,4-Bis(bromomethyl)cyclohexane (3a) and (Z)-1,4-Bis(bromomethyl)cyclohexane (3b)

To synthesize 3a and 3b, we started with a commercially available mixture of (Z)- and (E)-1,4-cyclohexanedimethanol. The NMR spectrum indicated that the ratio of (Z) and (E) isomers was 35:65%. The E/Z 1,4-cyclohexanedimethanol mixture was reacted with benzoyl chloride to give a mixture of (E)-1,4-cyclohexanedimethanol dibenzoate (1a) and (Z)-1,4-cyclohexanedimethanol dibenzoate (1b). The isomers were separated by multiple recrystallizations from ethyl acetate. Pure (>99% purity) isomer (E) was isolated as transparent plaques by two recrystallizations. Isolation of isomer (Z) was more complicated. The filtrate obtained after the first recrystallization was evaporated and recrystallized again. The crystals were discarded, and the filtrate containing 87% of (Z)-isomer was evaporated and recrystallized. The resulting crystals contained high-purity isomer (Z) (>99% purity). 1,4-Cyclohexanedimethanol 2a (E-isomer) and 2b (Z-isomer) were obtained from 1a and 1b, respectively, by de-esterification with sodium hydroxide. 1,4-Bis(bromomethyl)cyclohexane 3a and 3b were obtained by bromination of 2a and 2b with phosphorus tribromide.

Synthesis of Guanidines ADS10207, ADS10239, ADS10183, ADS10210, ADS10283, ADS10227, and ADS10185

Further synthetic procedures were similar for all newly synthesized compounds and analogous to the previously described routes of synthesis.³² Etherification of 3a and 3b and commercially available 1,4-bis(bromomethyl)benzene with sodium phenoxide in anhydrous ethanol led to 4a, 4b, and 11. The 1-(substituted)piperazines 5a, 5b, 12, and 18 were obtained from 4a, 4b, and 11, commercially available 1-(bromomethyl)-4-phenoxybenzeneby alkylation with piperazine. N-alkylation of 5a, 5b, 12, and 18 with 4-bromobutyronitrile in the presence of potassium carbonate in acetonitrile led to formation of 4-[4-(substituted)piperazin-1-yl]butanenitrile 6a, 6b, 13, and 19. The 4-[4-(substituted)piperazin-1-yl]butan-1-amines 7a, 7b, 14, and 20 were reduced with LiAlH₄ in dry diethyl ether. N-acylation with benzoyl chloride or 4-(trifluoromethyl)benzoyl chloride in the presence of triethylamine led to the synthesis of N-{4-[4-(substituted)piperazin-1-yl]butyl}benzamides 8a, 8c, and 15a or N-{4-[4-(substituted)piperazin-1-yl]butyl}-4-(trifluoromethyl)benzamides 8b, 8d, 15b, and 21; subsequently reduced with LiAlH₄ in dry diethyl ether to 4-[4-(substituted)piperazin-1-yl]-N-(benzyl)butan-1-amines 9a, 9c, and 16a or 4-[4-(substituted)piperazin-1-yl]-N-[4-(trifluoromethyl)benzyl]butan-1-amines 9b, 9d, 16b, and 22. Guanylation with 1,3-bis(tert-butoxycarbonyl)-2-methylisothiourea in the presence of triethylamine and 10% excess of mercury(II) chloride resulted in 2,3-di(tert-butoxycarbonyl)-1-{4-[4-(substituted)piperazin-1-yl]but-1-yl}-1-(benzyl)guanidines 10a, 10c, and 17a or 2,3-di(tert-butoxycarbonyl)-1-{4-[4-(substituted)piperazin-1-yl]but-1-yl}-1-[4-(trifluoromethyl)benzyl]guanidines 10b, 10d, 17b, and 23.

The final compounds were obtained by acidic deprotection of Boc groups from the guanidine moiety, resulting in 1-{4-[4-(substituted)piperazin-1-yl]but-1-yl}-1-[4-(trifluoromethyl)benzyl]guanidines ADS10207, ADS10239, ADS10183, and ADS10210 or 1-{4-[4-(substituted)piperazin-1-yl]but-1-yl}-1-(benzyl)guanidines ADS10283, ADS10227, and ADS10185. An overview of the procedures is presented in the Methods section. For more details, see the Chemical synthesis and data analysis section in the Supporting Information. The structures and purity of the synthesized final products were confirmed by ¹H NMR, ¹³C NMR spectra and elemental analysis (see Supporting Information). The synthesis of the ADS10183, ADS10185, ADS10207, ADS10210, ADS10227, ADS10239, and ADS10283 compounds is depicted in Schemes 1, 2, and 3. ADS1017 was synthesized according to Staszewski et al.³²

Scheme 1. Synthesis of ADS10283, ADS10207, ADS10227, and ADS10239.

Reagents and conditions: (a) E/Z mixture 1,4-cyclohexanedimethanol (1.0 equiv), benzoyl chloride (2.0 equiv), triethylamine (2.0 equiv), DCM, 2.5 h, rt; (b) 1a/1b (1.0 equiv), NaOH (10 equiv), H₂O, MeOH, 24 h, 70 °C; (c) 2a/2b (1 equiv), PBr₃ (1.4 equiv), DMF, 90 min, 100 °C; (d) 3a/3b (1 equiv), sodium phenoxide (1 equiv), EtOH, 24 h, 80 °C; (e) 4a/4b (1 equiv), piperazine (5 equiv), THF, 24 h, reflux; (f) 5a/5b (1 equiv), 4-bromobutyronitrile (1.3 equiv), potassium carbonate (5 equiv), MeCN, 24 h, 80 °C; (g) 6a/6b (1 equiv), LiAlH₄ (4 equiv), diethyl ether, 24 h, rt; (h) 7a/7b (1 equiv), benzoyl chloride/4-(trifluoromethyl)benzoyl chloride (1.1 equiv), triethylamine (5 equiv), DCM, 3 h, rt (i) 8a/8b/8c/8d (1 equiv), LiAlH₄ (4 equiv), diethyl ether, 24 h, rt; (j) 9a/9b/9c/9d (1 equiv), 1,3-bis(tert-butoxycarbonyl)-2-methylisothiourea (1.1 equiv), HgCl₂ (1.1 equiv), triethylamine (5 equiv), DCM, 18 h, rt; (k) 10a/10b/10c/10d (1 equiv), 4 M solution HCl-dioxan (20 equiv), CHCl₃, 24 h, rt.

Scheme 2. Synthesis of ADS10183 and ADS10185.

Reagents and conditions: (a) 1,4-Bis(bromomethyl)benzene (1 equiv), sodium phenoxide (1 equiv), THF, 24 h, reflux; (b) 11 (1 equiv), piperazine (5 equiv), THF, 24 h, reflux; (c) 12 (1 equiv), 4-bromobutyronitrile (1.3 equiv), potassium carbonate (5 equiv), MeCN, 24 h, 80 °C; (d) 13 (1 equiv), LiAlH₄ (4 equiv), diethyl ether, 24 h, rt; (e) 14 (1 equiv), benzoyl chloride/4-(trifluoromethyl)benzoyl chloride (1.1 equiv), triethylamine (5 equiv), DCM, 3 h, rt; (f) 15a/15b (1 equiv), LiAlH₄ (4 equiv), diethyl ether, 24 h, rt; (g) 16a/16b (1 equiv), 1,3-bis(tert-butoxycarbonyl)-2-methylisothiourea (1.1 equiv), HgCl₂ (1.1 equiv), triethylamine (5 equiv), DCM, 18 h, rt; (h) 17a/17b (1 equiv), 4 M solution HCl-dioxan (20 equiv), CHCl₃, 24 h, rt.

Scheme 3. Synthesis of ADS10210.

Reagents and conditions: (a) 1-(Bromomethyl)-4-phenoxybenzene (1 equiv), piperazine (5 equiv), THF, 24 h, reflux; (b) 18 (1 equiv), 4-bromobutyronitrile (1.3 equiv), potassium carbonate (5 equiv), MeCN, 24 h, 80 °C; (c) 19 (1 equiv), LiAlH₄ (4 equiv), diethyl ether, 24 h, rt; (d) 20 (1 equiv), 4-(trifluoromethyl)benzoyl chloride (1.1 equiv), triethylamine (5 equiv), DCM, 3 h, rt; (e) 21 (1 equiv), LiAlH₄ (4 equiv), diethyl ether, 24 h, rt; (f) 22 (1 equiv), 1,3-bis(tert-butoxycarbonyl)-2-methylisothiourea (1.1 equiv), HgCl₂ (1.1 equiv), triethylamine (5 equiv), DCM, 18 h, rt; (h) 23 (1 equiv), 4 M solution HCl-dioxan (20 equiv), CHCl₃, 24 h, rt.

Pharmacology

Pharmacological results are assembled in Tables 1 and 2 including the previously described data for compound ADS1017.³² All graphs of the ex vivo assays including the inhibitory effect on the contraction of guinea pig ileum strips and hH₃, hM₁-hM₅ radioligand binding assays are presented in the Supporting Information.

Table 1. Ex Vivo Screening on the Isolated Guinea Pig Ileum^a.

^aValues are means ± sem from at least three independent experiments; sem: standard error of the mean; N: number of different animal preparations; caviae: number of animals; gpi: guinea pig ileum; 4-DAMP: 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide; RAMH: (R)(−)-α-methylhistamine.

^bDecrease of contractility in electrically stimulated guinea pig ileum.

Table 2. Radioligand Binding Results at Human Histamine H₃R and Muscarinic M₁R–M₅R^a.

^aInhibition constants K_i are expressed as negative logarithms. Values are means ± sem from at least three independent experiments; sem: standard error of the mean; h: human.

Ex Vivo Screening of Histamine H₃R Antagonist on Guinea Pig Ileum

The H₃R antagonist potency of the newly synthesized compounds was measured on the isolated guinea pig ileum electrically stimulated to the contractions according to Vollinga et al.³⁵ During the ex vivo assay, some additional effects were noticed. Concentrations of tested compounds of 0.3 μM and lower shifts the concentration–response curve very slightly to the right compared to the reference (R)(−)-α-methylhistamine (RAMH) curve, while the higher concentrations necessary to determine the pA₂ value significantly decreased electrically evoked tissue contractions. This effect prevented the applied functional assay from determining the pA₂ value for the H₃R. Nevertheless, the tested compounds were found to modify the contractility of the guinea pig ileum. This effect could be related to H₃R agonism as well as on the action on the muscarinic receptors present in the tested tissue. It is worth noting that M₂R and M₃R antagonists may decrease the electrically evoked contractility of ileum smooth muscles. In contrast to cell line methods, the functional ex vivo tests on the guinea pig ileum allows the effect on a single receptor to be tested, by blocking other receptors as well as interactions with other receptors due to the physiological complexity of animal tissues. In the next stage of the study, we evaluated the impact on the histamine and muscarinic receptors.

Decrease of Contractility in Electrically Stimulated Guinea Pig Ileum

A standard ex vivo assay based on the relaxant response of histamine H₃R agonists to electrically driven guinea pig ileum was used to test the influence of ADS compounds on the reduction of electrically evoked tissue contraction. This study, standardly used for screening H₃R agonists, was recruited to confirm or exclude ADS compounds as potential H₃R agonists. Testing agonists common measurement to quantify the potency is −log EC₅₀, defined as a molar concentration of an agonist required to produce 50% of the maximal response to the agonist.³⁶ The −log EC₅₀ value for RAMH and ADS compounds were evaluated. The results ranged from 5.51 (ADS10185) to 6.88 (ADS10227) and 7.70 for RAMH (Table 1). (Z)-Isomers (ADS10227, ADS10239) were the most effectively reduced contractility. Compounds with disubstituted p-phenylene group and (E)-isomers containing a 1,4-cyclohexylene group demonstrated a lower influence on contractility reduction. Another unique value that describes an agonist is intrinsic activity (the maximal response to an agonist expressed as a fraction of the maximal response for the entire system), where α = 1 indicates that the agonist produces the maximal response.³⁶ Based on the concentration–response curves, a significant difference was observed between the intrinsic activity of RAMH and ADS compounds (Supporting Information): The values were 0.82 for RAMH, 0.97 for ADS10227 (Supporting Information; Figure S44), and 0.98 for ADS10239 (Supporting Information; Figure S45). To confirm or exclude ADS compounds as H₃R agonists, the compound with the highest −log EC₅₀ was selected for further studies. Therefore, ADS10227 was used as a potential H₃R agonist and thioperamide as the H₃R antagonist. The study excluded ADS10227 as an H₃R agonist, as no shifts of the concentration–response curve of ADS10227 were observed, compared to those with or without thioperamide. The observed reduction of electrically evoked tissue contraction cannot be associated with H₃R agonism, and the values obtained for ADS compounds are not intrinsic activity of H₃R agonist. However, observed effects must be related to a different contractility reduction mechanism. At this stage, we associated observed effects with muscarinic receptors, which are able to decrease electrically evoked ileum to the contraction. Following this, we decided to evaluate the ADS series as potential M₂R/M₃R ligands on the isolated guinea pig ileum.

Ex Vivo Screening of Histamine H₁R Antagonist on Guinea Pig Ileum

The second histamine receptor located in the guinea pig ileum is H₁R. Because previously described data for compound ADS1017 showed weak, competitive H₁R antagonist potency, all newly synthesized compounds were also evaluated for H₁R. The H₁R antagonistic effect was measured on isolated guinea pig ileum stimulated to contract by histamine.³⁷ To assess and exclude the influence of the ADS compounds on muscarinic receptors, two test variations were used: one with the addition of 0.05 μM atropine and one without. The atropine method indicated an effect on the H₁R, while the atropine-free method demonstrated effects on H₁R and M₂R/M₃R. The highest pA₂ ratio between the two methods was observed for ADS10227, which further confirmed our presumption about the effect on muscarinic receptors.

Ex Vivo Screening of Muscarinic M₂R/M₃R Antagonist on Guinea Pig Ileum

Another group of guinea pig intestinal receptors able to regulate smooth muscle contraction is that of the muscarinic receptors, including M₂R and M₃R. All compounds were tested on the muscarinic receptors using the same animal model. This approach is similar to measuring histamine H₁R antagonist activity. Methacholine was used as a receptor agonist, while 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide (4-DAMP) was used as a reference M₃R antagonist. As methacholine is not selective according to M₂R and M₃R, it is not possible to precisely specify which one was responsible for the effect observed on the tissue preparations. Some authors often attribute the results to the minor M₃R subtype (gpM₂R:M₃R = 4:1 or 5:1), which is generally associated with the contractions,³⁸ but such an overinterpretation may be misleading, as indicated by our further hM₁R-hM₅R radioligand binding assay results shown in Table 2. The tested series demonstrates low to moderate affinities at the muscarinic receptors (pA₂ = 5.61–6.97) (Table 1). The two (Z)-isomers, ADS10227 and ADS10239, were the most potent muscarinic receptor antagonists in the series. Compounds with the highest pA₂ value against the muscarinic receptor demonstrated the greatest reduction of electrically evoked contraction as well (Table 1). As it was mentioned above, the highest pA₂ ratio between the two screening methods of histamine H₁R antagonist was observed for ADS10227, which was also the most potent muscarinic M₂R/M₃R antagonist. These findings partially explain the influence of ADS10227 on the contractility of guinea pig ileum following electrical stimulation. The potent H₃R antagonist ADS1017 also demonstrated moderate affinity (pA₂ = 6.36) to muscarinic receptors. This observation explains the effect of tested compounds on the isolated guinea pig ileum, but it does not identify the muscarinic receptor subtype the ADS compounds act on. In subsequent studies, we tried to clarify this.

hH₃ Radioligand Binding Assay

As it was not possible to determine the pA₂ value by measuring the potency of H₃R on electrically stimulated guinea pig ileum ex vivo, another research method was needed to evaluate the affinity of the ADS compounds for the histamine H₃R. Hence, the displacement binding assay was performed in membrane fractions of HEK-293 cells stably expressing hH₃R to determine the hH₃R binding affinities of the final compounds. [³H]-N^α-Methylhistamine was used as a radiolabeled ligand. Compounds with a 1,4-cyclohexylene or p-phenylene group incorporated into a 7-phenoxyheptyl residue showed at least a 10-fold decrease of affinity relative to ADS1017: The pK_i of ADS1017 was 6.80, while that of ADS10227 was 5.23, this being the lowest in the series (Table 2). Measuring the displacement curve obtained for [³H]-N^α-methylhistamine from the human histamine H₃R in HEK-293 cell membranes, we confirmed a decrease in affinity to the hH₃R.

hM₁R–hM₅R Radioligand Binding Assays

Unexpectedly, the tests carried out on the guinea pig ileum showed that the rigidity of the seven-carbon alkyl chain by 1,4-cyclohexylene or p-phenylene group not only decreases affinity at the histamine H₃R but also significantly increases activity at muscarinic receptors. However, the previously employed method does not strictly elucidate on which one of the muscarinic receptor subtypes the tested compounds act on. To clearly explain the obtained outcome, we engaged the radioligand binding experiment. The hM₁–hM₅ radioligand binding experiments were performed in the membrane fractions of Chinese hamster ovary cells (CHO) stably expressing human variants of muscarinic receptors. N-[³H]Methylscopolamine was used as the radiolabeled ligand.³⁹ Three compounds were selected from ex vivo screening on guinea pig ileum. The potent histamine H₃R antagonist, ADS1017, showed nanomolar affinities to the hM₂R (37 nM; −log K_i = 7.43) and hM₄R (68 nM; −log K_i = 7.17). This compound is also not completely selective for the remaining muscarinic receptor subtypes with −log K_i values over 6. ADS10227 demonstrated the highest affinity to hM₂R and hM₄R: 2.8 nM and 5.1 nM, respectively. It is also a potent muscarinic M₁R antagonist, and the M₁R/M₂R selectivity ratio is <10. It is worth noting that such nonspecific anticholinergics are often associated with neuropsychiatric and cognitive disturbances and that nonselective muscarinic M₁R/M₂R antagonists, such as scopolamine, can produce cognitive deficits. Further structural modifications of ADS10227 should be used to increase selectivity toward the M₂R, as these may present fewer potential side effects associated with the activation of the phospholipase C signaling pathway. The third tested compound ADS10239 showed affinity to hM₁R, hM₂R, and hM₄R: 45 nM, 52 nM, and 15 nM, respectively.

Intracellular Ca²⁺ Measurement

The intracellular calcium level was determined fluorometrically to indicate the potency of ADS10227 to antagonize the functional response of muscarinic M₂R (pK_B = 8.17) and M₃R (pK_B = 7.43) expressed on CHO cells to ACh. Linear Schild plot indicates the competitive interaction (slope = 1) of the tested compound (Supporting Information, Figure S55).

In Silico Studies on Receptor Bindings and Selectivity

We conducted in silico molecular modeling studies to understand the molecular basis of the unexpected muscarinic activity of ADS1017 and its newly obtained derivatives. In this way, we wanted to determine the binding mode of the tested ligands to H₃R, M₂R, and M₄R and reveal the structural elements that are key to the interaction with these biological targets. From our point of view, it seems crucial to understand the effects of linker cyclization, which resulted in a strong shift of the activity profile of guanidine derivatives toward muscarinic receptors. Therefore, the next part of the study examined the binding mode of the analyzed compounds to the M₂R (PDB: 5ZKB) in an inactive state and to the remodeled M₄R and H₃R.

As the tested compounds were designed and optimized for the inhibition of the histamine H₃R, this was taken as our reference point. The comparison of the binding modes of the ADS1017 (−log K_i (H₃R) = 6.80) and ADS10227 (−log K_i (H₃R) = 5.23) provided some information that could explain the observed decrease in affinity. The final poses of the ADS1017 and ADS10227 at the histamine H₃R binding site are shown in Figure 2.

Figure 2.

Binding mode of (A) ADS1017 and (B) ADS10227 to the histamine H₃R. The ligands are shown as green sticks and the most important amino acids for interaction with the ligand as yellow sticks.

Docking studies indicated a very consistent binding mode. The locations of key ligand moieties were reproducible for all tested compounds. One of the most important features of this binding mode was the involvement of the entire orthosteric receptor binding site in the interaction with ligands.

The participation of two elements of the orthosteric binding site requires special emphasis. The first is the recognition site for the histamine imidazole fragment.¹³ It is composed of the amino acids from three transmembrane domains: TM3, TM5, and TM6. In the case of the histamine H₃R, E206^5.46 plays a very important role. The test compounds interact with this site via the benzyl-bound guanidino moiety. The positively charged guanidine forms an ionic bond with E206^5.46 and a cation−π with Y167^4.57. In addition, T119^3.37 stabilizes the system through hydrogen bonding. The benzyl fragment occupies a hydrophobic pocket built by F193, L199^5.39, W371^6.48, and M378^6.55. The second important element of the binding site is D114^3.32 which physiologically interacts with the protonated amine moiety of histamine.¹³ The tested compounds bind to D114^3.32 via the piperazine ring. According to our prediction, the piperazine ring of ADS1017 and ADS10227 was protonated at the nitrogen atom substituted by the 7-phenoxyheptyl or 4-(phenoxymethyl)cyclohexylmethyl substituent, respectively. This described binding mode indicates the participation of the first, protonated amine in the ionic interactions with D114^3.32 (salt bridge) and W110^3.28 (cation−π). The second, nonionized amino group was an acceptor of the hydrogen bond formed with the hydroxyl group Y374^6.51. Based on these observations, we can assume that both piperazine nitrogen atoms are essential, and removal of any of them may lead to weakening of ligand binding. This observation is consistent with the results of previous experiments.³¹

We found that the activity differences in the studied group of compounds are related to ligand alignment in the hydrophobic regions between TM2, TM3, and TM7. This area includes a number of aromatic amino acids such as Y91^2.61, Y94^2.64, W110^3.28, F398^7.39, and W402^7.43. The arrangement in this space determines the interaction of the phenoxy group in the so-called allosteric binding site. The long and flexible aliphatic linkage of ADS1017 allows the phenoxy group to create interactions with the amino acids of the outer part of the so-called allosteric site such as Y189^45.51 (π–π) and R381^6.58 (cation−π). Similar interactions were not observed in any of the newly synthesized compounds, which may be the reason for their much weaker interaction with H₃R.

In the case of muscarinic receptors, changes in the TM3, TM5, and TM6 regions responsible for selectivity for physiological ligands (ACh) alter the position of the guanidine fragment and benzyl group. The final binding poses of the ADS1017 and ADS10227 at the muscarinic M₂ receptor binding site are shown in Figure 3.

Figure 3.

Binding modes of (A) ADS1017 and (B) ADS10227 to the muscarinic M₂R. The ligands are shown as green sticks and the most important amino acids for interaction with the ligand as yellow sticks.

The key amino acids for both the muscarinic M₂R and M₄R (amino acid numeration M₂R/M₄R) are N404^6.52/N417^6.52 and A194^5.46/A203^5.46. They form a hydrogen-bond network with the guanidine group of the ligand. The associated aromatic ring was placed higher than during the binding of the same compound to the histamine H₃R, which strengthens the interactions with F181 and Y403^6.51 in the muscarinic M₂R. On this basis, we assumed that the switch from F181 in M₂R to L190 in M₄R may explain the slight difference between the activities at these receptors. The final binding poses of the ADS1017 and ADS10227 at the muscarinic M₄R binding site are shown in Figure 4.

Figure 4.

Binding modes of (A) ADS1017 and (B) ADS10227 to the muscarinic M₄R. The ligands are shown as green sticks and the most important amino acids for interaction with the ligand as yellow sticks.

The piperazine ring located in the middle of the ligand creates similar interactions in the active sites of the M₂R and M₄R to those observed in the H₃R. These regions are nearly identical between those receptors, which explains the affinity of the tested compounds against these biological targets. Again, the area between TM2 and TM7 contributes to ligand binding in this region and influences the position of the phenoxy group at the so-called allosteric binding site. 1,4-Cyclohexylene or p-phenylene group bind more strongly to the muscarinic receptor due to the slight differences in the structure of the outer part of the receptors. One of the key factors responsible for the stronger affinity of ADS1017 to H₃R seems to be the involvement of R381^6.58 in the creation of cation−π interactions. This interaction is possible thanks to a long flexible aliphatic chain between the piperazine ring and the p-phenylene group. Change to N410^6.58/N423^6.58 present in M₂R and M₄R weakens the binding of ADS1017 to these receptors. The binding mode of compounds with the cyclized linker indicates a preference for aromatic interactions with amino acids located on TM2 and TM7 of muscarinic receptors. In the compound ADS10227, Y80^2.61/Y89^2.61, Y83^2.64/Y92^2.64, W422^7.35/W435^7.35, and Y426^7.39/Y439^7.39 participate in the phenoxy group bonding. In this case, the change from H₃R Y394^7.35 to W422^7.35/W435^7.35 present in M₂R and M₄R allows a stronger binding of the aromatic system in ligands with cyclized linkers. A two-dimensional map of interactions between the ADS1017 and ADS10227 ligands and H₃, M₂, and M₄ receptors is presented in Supporting Information.

The in silico research yields two important observations. The first is the benzylguanidine fragment demonstrates a universal match for the recognition sites of specific ligands of the H₃R, M₂R, and M₄R located between TM3, TM5, and TM6. This is an important finding for scientists developing similar compounds because of their potential interaction with unintended biological targets (off-target determination). On-targeted optimization of such fragments can direct the affinity/activity profile of developed compounds. Second, the piperazine ring demonstrates a very stable position and may serve as a good core for new compounds with strong affinity to both the H₃R and muscarinic receptors. The key interactions created by ADS1017 and ADS10227 within H₃R, M₂R, and M₄R binding sites are presented in Table 3.

Table 3. Interactions Created by ADS1017 and ADS10227 within H₃, M₂, and M₄ Receptors Binding Sites^a.

^aThe amino acids with the strongest impact on the ligand binding are presented according to the Ballesteros–Weinstein convention. The strength of individual interactions was assessed using the Emodel evaluation function.⁴⁰ Stronger interactions are marked in green and the weaker ones in red. Blanks indicate no significant contribution to ligand binding. VdW: van der Waals force; SB: salt bridge; HB: hydrogen bonds.

Conclusion

A series of new guanidine derivatives was synthesized to identify new nonimidazole histamine H₃R antagonists. The seven-carbon chain present in the lead compounds ADS1017 and ADS1020 (Chart 1) was replaced by a semirigid moiety containing 1,4-cyclohexylene or p-phenylene group. In all cases, the substitution resulted in the significant decrease of H₃R antagonistic activity as well as the formation of potent muscarinic M₂R and M₄R antagonists which showed antagonist affinities in single-digit nanomolar concentration ranges. It is noteworthy that the histamine H₃R and muscarinic receptors have very similar binding sites (Table S1). The pharmacological profiling of the newly synthesized compounds led us to the identification of the most promising compound ADS10227, demonstrating the highest affinity to the hM₂R (2.8 nM; −log K_i = 8.55) and hM₄R (5.1 nM; −log K_i = 8.29) compared to the low affinity at H₃R and the other muscarinic receptors. This compound favoring muscarinic M₂R and M₄R may serve as a new lead structure for further structural modifications to develop a novel class of selective M₂R antagonists useful in the treatment of cognition deficit diseases such as Alzheimer’s disease, schizophrenia, or CNS learning disorders, such as autism or attention deficit disorder. ADS1017 also remains within the scope of interest as a dual-active H₃R/M₂R antagonist in relation to the treatment of cognition deficit disorders.

All the newly synthesized compounds were guanidine derivatives, which is the new chemotype of muscarinic receptor antagonists. Previous studies have described guanidine-containing compounds that can act as muscarinic receptor antagonists; however, the correct chemical nomenclature should classify them as carboximiamides.⁴¹ All muscarinic receptor subtypes share a high sequence homology in the binding site, which hinders the discovery of subtype-selective ligands. A small number of pharmacological agents that are selective to muscarinic receptors subtypes still remains challenging in the development of therapeutics that target muscarinic receptors. Such selective muscarinic ligands are needed to prevent undesired side-effects.

The major achievement of this study is the development of the ADS10227 nonselective muscarinic receptor antagonist. The separation of the E/Z-isomers of derivatives bearing a 1,4-cyclohexylene group provides a clearer picture of the spatial conformation of (Z)-isomers to fit the binding site of the M₂R and M₄R. The novel set of obtained ligands may constitute a promising toolbox to study the requirements of muscarinic receptors and could serve as starting points for further structural modifications, leading to the design of compounds with nanomolar affinity at muscarinic M₂R or M₄R.

Methods

Chemistry

All solvents were purchased from commercial suppliers (e.g., Avantor Performance Materials Poland S.A., PPH Stanlab Sp. z o.o. Lublin, Chempur Piekary Slaskie) and were used without further purification. The E/Z mixture of 1,4-cyclohexanedimethanol, phosphorus bromide, piperazine, phenol, sodium, 4-bromobutyronitrile,4-(trifluoromethyl)benzoyl chloride, benzyl bromide, 1,3-bis(tert-butoxycarbonyl)-2-methylisothiourea, 1,4-bis(bromomethyl)benzene, sodium, phenol, 1-(bromomethyl)-4-phenoxybenzene, and 4 M solution HCl in dioxane were purchased from commercial suppliers (Aldrich, TCI, Fluorochem, Fluka) and used without further purification. Nuclear magnetic resonance (NMR) spectra (¹H NMR, ¹³H NMR) were recorded on a Bruker Avance III 600 MHz (¹H NMR spectra were run at 600 MHz, while ¹³C NMR spectra were run at 150.95 MHz) spectrometer in CDCl₃, CD₃OD, and deuterium oxide. Chemical shifts were expressed in δ values, parts per million (ppm) using the solvent signal as an internal standard, and coupling constants (J) were given in hertz (Hz). Spectra obtained in deuterated chloroform were referenced to tetramethylsilane at 0.00 ppm for ¹H spectra and 77.02 ppm for ¹³C spectra. Spectra obtained in CD₃OD were referenced to residual CD₃OD at 3.31 ppm for ¹H spectra and 49.0 ppm for ¹³C spectra. Spectra obtained in deuterium oxide were referenced to residual deuterium oxide at 4.76 ppm for ¹H spectra. Signal multiplicities were characterized as br (broad), s (singlet), d (doublet), t (triplet), q (quartet), qt (quintet), m (multiplet), and * (exchangeable by deuterium oxide). Elemental analysis (C, H, and N) for all compounds were measured on PerkinElmer Series II CHNS/O analyzer 2400 and were within ±0.4% of the theoretical values. Reactions were monitored by thin-layer chromatography (TLC) on silica gel 60 F254 plates (Merck) and visualized using a UV Lamp (254 nm) and cerium molybdate stain. Flash column chromatography was performed using silica gel 60 Å 50 mm (J. T. Baker B. V.) and Normasil 60 silca gel 40–63 μm (VWR Chemicals), employing eluent indicated by TLC. Melting points (mp) were measured in open capillaries on an Electrothermal apparatus (Electrothermal, Southend, England) and are uncorrected.

Preparation of (E)-1,4-Cyclohexanedimethanol dibenzoate (1a) and (Z)-1,4-Cyclohexanedimethanol dibenzoate (1b)

A solution of benzoyl chloride (40.97 g, 0.29 mol) in 80 mL of DCM was added dropwise to an ice-cooled mixture of 1,4-cyclohexanedimethanol (E/Z mixture) (20.03 g; 0.14 mol) and triethylamine (84.00 g; 0.83 mol) in 200 mL of DCM. The reaction was stirred for 2.5 h at room temperature, then the mixture was washed sequentially twice with 200 mL of water. The water phase was washed four times with 50 mL of DCM, then the combined organic phases were dried over Na₂SO₄. The solvent was removed under vacuum, and the crude product was recrystallized twice from ethyl acetate to yield the pure products as a plaques (E-isomer). The filtrate obtained after the first recrystallization was collected, evaporated, and recrystallized twice from ethyl acetate collecting a (Z)-isomer-rich fraction (evaluated base on the NMR spectra) to yield the pure products as needles (Z-isomer).

(E)-1,4-Cyclohexanedimethanol dibenzoate (1a)

C₂₂H₂₄O₄. M = 352.43. Transparent plaques. 39.43% yield. Retardation factor (R_f) = 0.43 (hexane/EtOAc 9:1). Mp: 125.3–127.0 °C. ¹H NMR (600 MHz, CDCl₃) δ ppm 8.05–8.04 (m, 4H^arom., CH(CHCH)₂C), 7.56–7.54 (m, 2H^arom., CH(CHCH)₂C), 7.44–7.42 (m, 4C^arom., CH(CHCH)₂C), 4.18 (d, 4H, OCH₂, J = 6.42 Hz), 1.95–1.94 (m, 4H^cyclohexyl., CH₂), 1.81 (m, 2H ^cyclohexyl., OCH₂CH), 1.20–1.13 (m, 4H^cyclohexyl., CH₂). ¹³C NMR (150.95 MHz, CDCl₃) δ ppm 166.61 (2C, C=O), 132.84 (2C^arom., CH(CHCH)₂C), 130.50 (2C^quat./arom.., CO), 129.55 (4C^arom., CH(CHCH)₂C), 128.35 (4C^arom., CH(CHCH)₂C), 69.77 (2C, OCH₂), 37.32 (2C ^cyclohexyl., OCH₂CH), 29.02 (4C^cyclohexyl., CH₂).

(Z)-1,4-Cyclohexanedimethanol dibenzoate (1b)

C₂₂H₂₄O₄. M = 352.43. Transparent needles. 16.35% yield. R_f = 0.46 (hexane/EtOAc 9:1). Mp: 84.8–86.4 °C. ¹H NMR (600 MHz, CDCl₃) δ ppm 8.06–8.04 (m, 4H^arom., CH(CHCH)₂C), 7.56–7.54 (m, 2H^arom., CH(CHCH)₂C), 7.45–7.43 (m, 4H^arom., CH(CHCH)₂C), 4.28 (d, 4H, OCH₂, J = 7.26 Hz), 2.06–2.04 (m, 2H^cyclohexyl., OCH₂CH), 1.69–1.65 (m, 4H^cyclohexyl., CH₂), 1.62–1.56 (m, 4H^cyclohexyl., CH₂). ¹³C NMR (150.95 MHz, CDCl₃) δ ppm 166.60 (2C, C=O), 132.84 (2C^arom., CH(CHCH)₂C), 130.45 (2C^quat./arom.., CO), 129.54 (4C^arom., CH(CHCH)₂C), 128.34 (4C^arom., CH(CHCH)₂C), 67.59 (2C, OCH₂), 34.69 (2C ^cyclohexyl., OCH₂CH), 25.46 (4C^cyclohexyl., CH₂).

Preparation of (E)-1,4-cyclohexanedimethanol (2a)

A solution of sodium hydroxide (8.80 g; 0.22 mol) in 10.8 mL of water was added to a mixture of (E)-1,4-cyclohexanedimethanol dibenzoate (1a) (7.90 g; 2.2 × 10^–2 mol) in 250 mL of methanol. The reaction was stirred overnight at 70 °C. The solvents were removed under vacuum. The residue was diluted by 50 mL of water and extracted 5 × 50 mL with EtOAc. The organic phase was dried over anhydrous Na₂SO₄. The solvent was removed under vacuum to yield the pure product.

(E)-1,4-Cyclohexanedimethanol (2a)

C₈H₁₆O₂. M = 144.21. White waxy solid. 88.55% yield. R_f = 0.59 (EtOAc). Mp: 65.4–67.4 °C. ¹H NMR (600 MHz, CDCl₃) δ ppm 3.47 (d, 4H, HOCH₂, J = 6.27 Hz), 1.85–1.84 (m, 4H^cyclohexyl., CH₂), 1.46 (br, 4H: 2H^cyclohexyl., OCH₂CH; OH), 1.02–0.94 (m, 4H^cyclohexyl., CH₂). ¹³C NMR (150.95 MHz, CDCl₃) δ ppm 68.62 (2C, OCH₂), 40.65 (2C^cyclohexyl., CH), 28.91 (4C^cyclohexyl., CH₂).

Preparation of (Z)-1,4-Cyclohexanedimethanol (2b)

A solution of sodium hydroxide (6.00 g; 0.15 mol) in 9 mL of water was added to a mixture of (Z)-1,4-cyclohexanedimethanol dibenzoate (1b) (5.33 g; 1.5 × 10^–2 mol) in 200 mL of methanol. The reaction was stirred overnight at 70 °C. The solvents were removed under vacuum. The residue was diluted by 50 mL of water and extracted 5 × 50 mL with EtOAc. The organic phase was dried over anhydrous Na₂SO₄. The solvent was removed under vacuum, and the crude product was purified by column chromatography (EtOAc) to yield the pure product.

(Z)-1,4-Cyclohexanedimethanol (2b)

C₈H₁₆O₂. M = 144.21. Colorless sticky oil. 91.10% yield. R_f = 0.49 (EtOAc). Mp: 65.4–67.4 °C. ¹H NMR (600 MHz, CDCl₃) δ ppm 3.55 (d, 4H, HOCH₂), 1.70–1.69 (m, 2H^cyclohexyl., OCH₂CH), 1.57–1.53 (m, 4H^cyclohexyl., CH₂), 1.46–1.39 (m, 6H: m, 4H^cyclohexyl., CH₂, OH). ¹³C NMR (150.95 MHz, CDCl₃) δ ppm 66.09 (2C, CH₂OH), 38.12 (2C^cyclohexyl., CH), 25.31 (4C^cyclohexyl., CH₂).

Preparation of (E)-1,4-Bis(bromomethyl)cyclohexane (3a)

A solution of (E)-1,4-cyclohexanedimethanol (2a) (3.20 g; 2.22 × 10^–2 mol) in 10 mL of DMF was added dropwise to an ice-cooled mixture of phosphorus bromide (8.45 g; 3.12 × 10^–2 mol) in 10 mL of toluene. Then, the reaction was stirred for 90 min at 100 °C. The mixture was cooled, and 50 mL of crushed ice was added and then extracted 3 × 20 mL with DCM. The organic phases were combined and dried over anhydrous Na₂SO₄. The solvent was removed under vacuum, and the crude product was purified by column chromatography (hexane) to yield the pure product.

(E)-1,4-Bis(bromomethyl)cyclohexane (3a)

C₈H₁₄Br₂. M = 270.00. Transparent crystals. 71.28% yield. R_f = 0.61 (hexane). Mp: 54.2–55.2 °C. ¹H NMR (600 MHz, CDCl₃) δ ppm 3.29 (d, 4H, BrCH₂, J = 6.28 Hz), 1.95–1.94 (m, 4H^cyclohexyl., CH₂), 1.61 (m, 2H^cyclohexyl., BrCH₂CH), 1.08–1.05 (m, 4H^cyclohexyl., CH₂). ¹³C NMR (150.95 MHz, CDCl₃) δ ppm 39.89 (2C, BrCH₂), 39.81 (2C^cyclohexyl., CH), 31.06 (4C^cyclohexyl., CH₂).

Preparation of (Z)-1,4-Bis(bromomethyl)cyclohexane (3b)

A solution of (Z)-1,4-cyclohexanedimethanol (2b) (1.92 g; 1.33 × 10^–2 mol) in 6 mL of DMF was added dropwise to an ice-cooled mixture of phosphorus bromide (4.33 g; 1.60 × 10^–2 mol) in 5 mL of toluene. Then, the reaction was stirred for 90 min at 100 °C. The mixture was cooled, and 30 mL of crushed ice was added and then extracted 3 × 15 mL with DCM. The organic phases were combined and dried over anhydrous Na₂SO₄. The solvent was removed under vacuum, and the crude product was purified by column chromatography (hexane) to yield the pure product.

(Z)-1,4-Bis(bromomethyl)cyclohexane (3b)

C₈H₁₄Br₂. M = 270.00. Colorless liquid. 84.58% yield. R_f = 0.72 (hexane). ¹H NMR (600 MHz, CDCl₃) ppm 3.40 (d, 4H, BrCH₂), 1.94–1.82 (m, 2H^cyclohexyl., BrCH₂CH), 1.69–1.58 (m, 4H^cyclohexyl., CH₂), 1.56–1.49 (m, 4H^cyclohexyl., CH₂). ¹³C NMR (150.95 MHz, CDCl₃) δ ppm 38.06 (2C, BrCH₂), 37.64 (2C^cyclohexyl., CH), 27.05 (4C^cyclohexyl, CH₂).

General Procedure for the Preparation of Compounds 4a, 4b, and 11

Phenol (1 equiv) was added to sodium (1.05 equiv) dissolved in anhydrous ethanol and stirred for 30 min at room temperature, yielding sodium phenoxide solution. A freshly prepared sodium phenoxide solution (or evaporated sodium phenoxide solution dissolved in THF) was added dropwise to a solution of the corresponding bromide (1 equiv) (3a, 3b) in anhydrous ethanol and heated to 65 °C (or in anhydrous THF heated to 66 °C for compounds 1,4-bis(bromomethyl)benzene). The reaction was stirred overnight at 80 °C. The solvent was removed under vacuum, and the mixture was purified by column chromatography to yield the pure product.

General Procedure for the Preparation of Compounds 5a, 5b, 12, and 18

A mixture of corresponding bromide (1 equiv) (4a, 4b, 11, and 1-(bromomethyl)-4-phenoxybenzene) in anhydrous THF was added dropwise to a solution of piperazine (5 equiv) in THF heated to 66 °C. The reaction was stirred overnight at 66 °C. The precipitate was discarded. The solvent was removed under vacuum, and the residue was diluted by water, alkalized by 5% NaOH solution, and extracted with DCM. The combined organic phases were dried over anhydrous Na₂SO₄. The solvent was removed under vacuum, and the crude product was purified by column chromatography to yield the pure product.

General Procedure for the Preparation of Compounds 6a, 6b, 13, and 19

Potassium carbonate (5 equiv) and 4-bromobutyronitrile (1.3 equiv) were added to a solution of the corresponding 1-substituted piperazine (1 equiv) (5a, 5b, 12, and 18) in acetonitrile. The reaction was stirred overnight at 80 °C and then filtered. The precipitate was discarded. The solvent was removed under vacuum, and the crude product was purified by column chromatography to yield the pure product.

General Procedure for the Preparation of Compounds 7a, 7b, 14, and 20

LiAlH₄ (4 equiv) was slowly added to a solution of the corresponding nitrile (1 equiv) (6a, 6b, 13, and 19) in 50 mL of anhydrous diethyl ether. The reaction was stirred overnight at room temperature, and the mixture was quenched by dropwise addition of water (16 equiv) and 10% NaOH solution (16 equiv) stirred for 2 h and then filtered. The precipitate was discarded. The organic layer was dried over Na₂SO₄, the solvent was removed under vacuum, and the crude product was purified by column chromatography to yield the pure product.

General Procedure for the Preparation of Compounds 8a, 8b, 8c, 8d, 15a, 15b, and 21

4-(Trifluoromethyl)benzoyl chloride (1.1 equiv) or benzoyl chloride (1.1 equiv) in DCM was added dropwise to a solution of the corresponding primary amine (1 equiv) (7a, 7b, 14, and 20) and triethylamine (5 equiv) in DCM. The reaction was stirred for 3 h at room temperature, and then the mixture was washed three times with water and dried over Na₂SO₄. The solvent was removed under vacuum, and the crude product was purified by column chromatography to yield the pure product.

General Procedure for the Preparation of Compounds 9a, 9b, 9c, 9d, 16a, 16b, and 22

LiAlH₄ (4 equiv) was added to a solution of the corresponding amide (1 equiv) in anhydrous diethyl ether (8a, 8b, 8c, 8d, 15a, 15b, and 21) or THF (8b). The reaction was stirred overnight at room temperature, and the mixture was quenched by dropwise addition of water (16 equiv) and 10% NaOH solution (16 equiv) stirred for 2 h and then filtered. The precipitate was discarded. The organic layer was dried over Na₂SO₄, the solvent was removed under vacuum, and the crude product was purified by column chromatography to yield the pure product.

General Procedure for the Preparation of Compounds 10a, 10b, 10c, 10d, 17a, 17b, and 23(42)

1,3-Bis(tert-butoxycarbonyl)-2-methylisothiourea (1.1 equiv) and mercury II chloride (1.1 equiv) were sequentially added to an ice-cooled mixture of the corresponding secondary amine (1 equiv) (9a, 9b, 9c, 9d, 16a, 16b, and 22) and triethylamine (5 equiv) in DCM. The ice bath was removed, and the reaction was stirred 18 h at room temperature and then filtered. The precipitate was discarded. The filtrate was washed sequentially twice with H₂O and twice with brine. The combined organic phases were dried over Na₂SO₄, the solvent was removed under vacuum, and the crude product was purified by column chromatography to yield the pure product.

General Procedure for the Preparation of Compounds ADS10207, ADS10239, ADS10283, ADS10227, ADS10183, ADS10185, and ADS10210(42)

4 M solution HCl in 1,4-dioxane (20 equiv) was added dropwise to a solution of the corresponding Boc-protected guanidine (1 equiv) (10a, 10b, 10c, 10d, 17a, 17b, and 23) in chloroform. The reaction was stirred overnight at room temperature, and the solvent was removed under vacuum. The crude product was evaporated twice from chloroform and twice from EtOAc and then recrystallized from anhydrous ethanol or 2-propanol to yield the pure product.

Biological Evaluation

Ex Vivo Assay for Screening Histamine H₃R Antagonists on Guinea Pig Ileum

Male guinea pigs, weighing 300–400 g, were euthanized by a blow to the neck. Following this, a 20–30 cm length of the distal ileum, apart from the terminal 5 cm, was rapidly removed and placed in phosphate buffer at room temperature (pH 7.4) containing (mM) NaCl (136.9), KCl (2.6), KH₂PO₄ (1.47), Na₂HPO₄ (9.58), and indomethacin (1 × 10^–6 mol/L)). The intraluminal content was rinsed, and the isolated intestine was cut into 1.5–2 cm segments. The preparations were mounted between two platinum electrodes isotonically in a 20 mL organ bath filled with Krebs buffer: composition (mM) NaCl (118), KCl (5.6), MgSO₄ (1.18), CaCl₂ (2.5), NaH₂PO₄·H₂O (1.28), NaHCO₃ (25), glucose (5.55), and indomethacin (3 × 10^–7 mol/L). The solution was continuously bubbled with a 95% O₂:5% CO₂ mixture and maintained at 37 °C under a constant load of 1.0 g (Hugo Sachs Hebel–Messvorsatz (Tl-2)/HF-modem; Hugo Sachs Elektronik, Hugstetten, Germany) connected to a pen recorder (Kipp & Zonen BD41, Delft, Holland). During an equilibration period of 60 min, the Krebs buffer was changed every 10 min. The preparations were then continuously stimulated at 15–20 V at a frequency of 0.1 Hz for a duration of 0.5 ms, with rectangular-wave electrical pulses (Grass Stimulator S-88; Grass Instruments Co., Quincy, Massachusetts, USA). After about 30 min, the twitches were recurrent. Five min before RAMH administration, pyrilamine (1 × 10^–5 mol/L concentration in organ bath) was added. The first cumulative concentration–response curve was determined for RAMH (10 nM – 10 mM) at increasing concentrations spaced by 3- or 3.3-fold. The second to the fourth curves were measured against increasing antagonist concentrations (incubation time 20 min). The pA₂ values were calculated according to Arunlakshana and Schild.³⁷ Statistical analysis was carried out with the Students’ t test. In all tests, a p < 0.05 was considered statistically significant. The pA₂ values were compared with the affinity of thioperamide.

Ex Vivo Assay for Screening Histamine H₃R Agonists: Determination of the −log EC₅₀ Coefficient on Guinea Pig Ileum

Male guinea pigs, weighing 300–400 g, were euthanized by a blow to the neck. A 20–30 cm length of the distal ileum, apart from the terminal 5 cm, was rapidly removed and placed in phosphate buffer at room temperature (pH 7.4) containing (mM) NaCl (136.9), KCl (2.6), KH₂PO₄ (1.47), Na₂HPO₄ (9.58), and indomethacin (1 × 10^–6 mol/L)). The intraluminal content was rinsed, and the isolated intestine was cut into 1.5–2 cm segments. The preparations were mounted between two platinum electrodes isotonically in a 20 mL organ bath filled with Krebs buffer: composition (mM) NaCl (118), KCl (5.6), MgSO₄ (1.18), CaCl₂ (2.5), NaH₂PO₄·H₂O (1.28), NaHCO₃ (25), glucose (5.55), and indomethacin (3 × 10^–7 mol/L). The solution was continuously bubbled with a 95% O₂:5% CO₂ mixture and maintained at 37 °C under a constant load of 1.0 g (Hugo Sachs Hebel–Messvorsatz (Tl-2)/HF-modem; Hugo Sachs Elektronik, Hugstetten, Germany) connected to a pen recorder (Kipp & Zonen BD41, Delft, Holland). During an equilibration period of 60 min, Krebs buffer was changed every 10 min. Following this, the preparations were continuously stimulated at 15–20 V, at a frequency of 0.1 Hz for a duration of 0.5 ms, with rectangular-wave electrical pulses (Grass Stimulator S-88; Grass Instruments Co., Quincy, Massachusetts, USA). After about 30 min, twitches were recurrent. Thirty min before RAMH or tested agonist administration, famotidine (1 × 10^–5 mol/L concentration in organ bath) and pyrilamine (1 × 10^–5 mol/L concentration in organ bath) was added. Cumulative concentration–response curve was determined to RAMH or tested agonist (10 nM to 10 mM) at increasing concentrations spaced by 3- or 3.3-fold. The agonist potency is expressed as pD₂ value (−log EC₅₀) ± sem. The −log EC₅₀ differences were not corrected since three successive curves were superimposable.

Ex Vivo Assay for Screening Histamine H₁R Antagonists on Guinea Pig Ileum

Male guinea pigs, weighing 300–400 g, were euthanized by a blow to the neck. A 20–30 cm length of the distal ileum, apart from the terminal 5 cm, was rapidly removed and placed in phosphate buffer at room temperature (pH 7.4) containing (mM) NaCl (136.9), KCl (2.6), KH₂PO₄ (1.47), Na₂HPO₄ (9.58), and indomethacin (1 × 10^–6 mol/L). The intraluminal content was rinsed, and the isolated intestine was cut into 1.5–2 cm segments. The preparations were mounted isotonically in a 20 mL organ bath filled with Krebs buffer: composition (mM) NaCl (118), KCl (5.6), MgSO₄ (1.18), CaCl₂ (2.5), NaH₂PO₄·H₂O (1.28), NaHCO₃ (25), glucose (5.55), and indomethacin (3 × 10^–7 mol/L). Depending on the type of assay, Krebs buffer additionally contained or did not contain 0.05 μM of atropine. The solution was continuously bubbled with a 95% O₂:5% CO₂ mixture and maintained at 37 °C under a constant load of 0.5 g (Hugo Sachs Hebel–Messvorsatz (Tl-2)/HF-modem; Hugo Sachs Elektronik, Hugstetten, Germany) connected to a pen recorder (Kipp & Zonen BD41, Delft, Holland). During an equilibration period of 40 min, Krebs buffer was changed every 10 min. The first cumulative concentration–response curve was determined for histamine (10 nM to 10 mM) at increasing concentrations spaced by 3- or 3.3-fold. The second to the fourth (or fifth) curves were measured in the presence of an increasing concentrations of antagonist (incubation time 10 min). The pA₂ values were calculated according to Arunlakshana and Schild.³⁷ Statistical analysis was carried out with the Students’ t test. In all tests, a p < 0.05 was considered statistically significant. The pA₂ values were compared with the affinity of pyrilamine.

Ex Vivo Assay for Screening M₂R/M₃R Antagonists on Guinea Pig Ileum

Male guinea pigs, weighing 300–400 g, were euthanized by a blow to the neck. A 20–30 cm length of the distal ileum, apart from the terminal 5 cm, was rapidly removed and placed in phosphate buffer at room temperature (pH 7.4) containing (mM) NaCl (136.9), KCl (2.6), KH₂PO₄ (1.47), Na₂HPO₄ (9.58), and indomethacin (1 × 10^–6 mol/L)). The intraluminal content was rinsed, and the isolated intestine was cut into 1.5–2 cm segments. The preparations were mounted isotonically in a 20 mL organ bath filled with Tyrode’s buffer: composition (mM) NaCl (137), KCl (2.7), MgCl₂·6H₂O (1.0), CaCl₂ (1.8), NaH₂PO₄·H₂O (0.4), NaHCO₃ (11.9), glucose (5.61), and indomethacin (3 × 10^–7 mol/L). The solution was continuously bubbled with a 95% O₂:5% CO₂ mixture and maintained at 37 °C under a constant load of 0.5 g (Hugo Sachs Hebel–Messvorsatz (Tl-2)/HF-modem; Hugo Sachs Elektronik, Hugstetten, Germany) connected to a pen recorder (Kipp & Zonen BD41, Delft, Holland). During an equilibration period of 40 min, Tyrode’s buffer was changed every 10 min. The first cumulative concentration–response curve was determined for methacholine (10 nM to 3 mM) at increasing concentrations spaced by 3- or 3.3-fold. The second to the fourth (or fifth) curves were measured in the presence of an increasing concentration of antagonist (incubation time 20 min). The pA₂ values were calculated according to Arunlakshana and Schild.³⁷ Statistical analysis was carried out with the Students’ t test. In all tests, a p < 0.05 was considered statistically significant. The pA₂ values were compared with the affinity of 4-DAMP.

Determination of hH₃R Affinity

The radioligand displacement binding assay was performed in membrane fractions of HEK-293 cells stably expressing hH₃R. Cell cultivation and membrane preparation were performed according to Kottke et al.⁴³ For the radioligand displacement assay, radioactively labeled [³H]N^α-methylhistamine was used at a final concentration of 2 nM (K_D = 3.08 nM). The total assay volume was set to 200 μL. The compounds were tested in several appropriate concentrations between 100 μM and 0.1 nM. Pipetting was partly done by Freedom Evo (Tecan). Pitolisant was used to determine nonspecific binding at a concentration of 10 μM. The membrane fraction (20 μg/well), test compounds, and radiolabeled ligand were incubated for 90 min at 25 °C while shaking. The bound radioligand was separated from free radioligand by filtration through GF/B filters pretreated with 0.3% (m/v) polyethylenimine using a cell harvester. Radioactivity was determined by liquid scintillation counting using a MicroBeta Trilux (PerkinElmer). The data were obtained in duplicates in at least three independent experiments. Nonspecific binding was subtracted from the raw data to calculate specific-binding values. The evaluation was performed with GraphPad Prism 6.1 (San Diego, CA, USA) using nonlinear regression (one-site competition with a logarithmic scale). The K_i values were calculated from the IC₅₀ values using the Cheng–Prusoff equation.⁴⁴ The statistical calculations were performed on −log K_i. The mean values and 95% confidence intervals were transformed to nanomolar concentrations.

Cell Culture and Membrane Preparation

The culture was derived from CHO cells stably transfected with the genes of human variants of muscarinic receptors. These were purchased from Missouri S&T cDNA Resource Center (Rolla, MO, USA). Cell cultures and crude membranes were prepared as described previously.⁴⁵ The cells were grown to confluence in 75 cm² flasks in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, and 2 million cells were subcultured to 100 mm Petri dishes. The medium was supplemented with 5 mM butyrate for the last 24 h of cultivation to increase receptor expression. Cells were detached by mild trypsinization on day 5 after subculture. Detached cells were washed twice in 50 mL of phosphate-buffered saline and 3 min centrifugation at 250g. Washed cells were suspended in 20 mL of ice-cold incubation medium (100 mM NaCl, 20 mM Na-HEPES, 10 mM MgCl₂, pH = 7.4) supplemented with 10 mM EDTA and homogenized on ice by two 30 s strokes using a Polytron homogenizer (Ultra-Turrax; Janke & Kunkel GmbH & Co. KG, IKA-Labortechnik, Staufen, Germany) with a 30 s pause between strokes. Cell homogenates were centrifuged for 30 min at 30,000g. Supernatants were discarded, and pellets suspended in the fresh incubation medium, incubated on ice for 30 min, and centrifuged again. The resulting membrane pellets were kept at −80 °C until assay within a maximum of 10 weeks.

Determination of hM₁R–hM₅R Affinities

All radioligand binding experiments were optimized and carried out according to general guidelines.³⁹ Briefly, membranes were incubated in 96-well plates at 30 °C in the incubation medium described above. Incubation volume was 400 μL or 800 μL for competition and saturation experiments with [³H]NMS, respectively. Approximately 30 μg of membrane proteins per sample were used. N-Methylscopolamine binding was measured directly in saturation experiments using six concentrations (30–1000 pM) of [³H]NMS during incubation for 1 h (M₂R), 3 h (M₁R, M₃R, and M₄R), or 5 h (M₅R). For calculations of the equilibrium dissociation constant (K_D), concentrations of free [³H]NMS were calculated by subtraction of bound radioactivity from total radioactivity in the sample and fitting eq 1 (Experimental Data analysis section). The binding of the tested ligands was determined in competition experiments with 100 pM [³H]NMS. The IC₅₀ value was computed according to eq 2, and the inhibition constant K_i according to eq 3. Samples were first preincubated for 1 h with [³H]NMS. Then the tested compound was added and incubation continued for an additional 5 h. Nonspecific binding was determined in the presence of 1 μM unlabeled atropine. Incubations were terminated by filtration through Whatman GF/C glass fiber filters (Whatman) using a Brandel cell harvester (Brandel, Gaithersburg, MD, USA). The filters were dried in a microwave oven, and then solid scintillator Meltilex A was melted on filters (105 °C, 70 s) using a hot plate. The filters were cooled and counted in the Wallac Microbeta scintillation counter.

Intracellular Ca²⁺ Measurement

Intracellular Ca²⁺ level was taken as a functional response to ACh. Black 96-well plates were seeded with 12,000 CHO cells per well. After two days of cultivation in DMEM at 37 °C under a humidified atmosphere containing 5% CO₂, cells were washed with Krebs-HEPES buffer (KHB) (composition: (mM) NaCl (138), KCl (4), CaCl₂ (1.3), MgCl₂ (1), NaH₂PO₄ (1.2), HEPES (20), glucose (10), pH adjusted to 7.4) KHB was loaded with 5 μM Fura-2 (Sigma-Aldrich) for 1 h. The cells were washed with fresh KHB and preincubated with tested compounds for 1 h. Then plates were placed in a Cytation 3 reader. The first basal level (fluorescence dual excitation at 340 and 380 nm, emission at 510 nm) was measured. Following this, ACh was added to the desired concentration (ranging from 10 pM to 100 μM) by instrument injectors, and fluorescence was measured immediately. Intracellular Ca²⁺ level was calculated as a ratio of 340 to 380 nm excitation fluorescence. The changes in intracellular Ca²⁺ level were compared as a fold increase over the basal level of the corresponding well.

Experimental Data Analysis

Saturation of [³H]NMS Binding

The binding of [³H]NMS at various concentrations was measured. After subtraction of nonspecific binding and calculation of free radioligand concentration, eq 1 was fitted to the data:

1

where y is specific binding at free concentration x, B_MAX is maximum binding capacity, and K_D is the equilibrium dissociation constant of [³H]NMS.

Binding Parameters of Tested Compounds

Tested compounds are competitive antagonists of [³H]NMS binding. [³H]NMS binding was determined in the presence of tested compounds at various concentrations. After subtraction of nonspecific binding and normalization to binding in the absence of the tested compound, eq 2 was fitted to the data:

2

where y is a specific radioligand binding at concentration x of the tested compound expressed as a percent of binding in its absence, IC₅₀ is the concentration of tested compound inhibiting 50% of [³H]NMS binding, and nH is the Hill coefficient.

3

where K_i is the inhibition constant of the tested compound, K_D is the equilibrium dissociation constant, and [D] is the concentration of [³H]NMS.

Concentration Response to Acetylcholine

The intracellular level of calcium at various concentrations of ACh was measured. After subtraction of background values and normalization to the level in the absence of ACh, eq 4 was fitted to the data:

4

where y is the normalized response at ACh concentration x, E_MAX is the maximal effect, EC₅₀ is the concentration of ACh causing half-maximal effect, and nH is Hill coefficient. From a series of EC₅₀ values of apparent inhibition constant K_B was calculated by fitting eq 5 to dose ratio (DR) induced by tested compound at a given concentration:

5

where DR is ratio of EC₅₀ in the presence of tested compound at concentration [B] to EC₅₀ in its absence.

In Silico Studies

All tested ligands were prepared with the appropriate spatial configuration in the Maestro program (Schrödinger) and then charged at pH 7.4 ± 0.2 using the LigPrep program (Schrödinger Release 2017–1; Maestro-Schrödinger, LLC, New York).

All docking experiments were performed with the Glide program (Maestro-Schrödinger) with the SP level of calculation accuracy.⁴⁶ For the docking purposes, grids centered on the AF-DX 384 (Chart 1) ligand position, sized to dock ligands with length ≤25 Å, were prepared.

The in silico research used the M₂R (PDB: 5ZKB) and M₄R (PDB: 5DSG) complexes available in the Protein Data Base (PDB) and the previously published homology model of the histamine H₃R.^28,47,48 All proteins used in the study were prepared with the ProteinPrepare (PlayMolecule-Acellera) website.⁴⁹ The structure of the ligand has a significant influence on the conformation of the amino acids in the binding site of GPCRs. Being aware of the large differences in the size of the studied ligands and compounds present in the crystallized complexes, it was decided to optimize the structure of the M₄R and H₃R based on the M₂R complex with the compound AF-DX 384 (PDB: 5ZKB) which demonstrated conformational changes in the aromatic amino acids Y104^3.33 and Y426^7.39 and W99^3.28.⁴⁷ Shifting these residues opens up the space needed for larger ligands to interact. This pattern was used to remodel the arrangement of the amino acids in the previously published homologous model of the histamine H₃R²⁸ and the structure of the M₄R (PDB: 5DSG). The key differences in the position of the most important H₃ amino acids are given in Figure S56.

The ligands ADS1017 and ADS10227 were docked to the receptors prepared in this way. Complexes showing consistent binding modes were optimized using the Refine Protein–Ligand Complex function with the Monte Carlo minimization method (Maestro-Schrödinger). Such optimized complexes were used for the final analyzes, and all tested ligands were docked to them. The final results were visualized using PyMol 0.99 rc6 software (DeLano Scientific LLC).

Glossary

Abbreviations Used

CNS

Central nervous system

ACh

acetylcholine

H₃R

histamine H₃ receptor

H₁R

histamine H₁ receptor

gpH₃R

guinea pig histamine H₃ receptor

gpH₁R

guinea pig histamine H₁ receptor

gpM₂R/M₃R

guinea pig muscarinic M₂R/M₃R

hH₃R

human histamine H₃ receptor

hM_1–5

human muscarinic M_1–5 receptors

GPCR

G protein-coupled receptor

ECL2

the second extracellular loop

RAMH

(R)-(−)-α-methylhistamine

4-DAMP

1,1-dimethyl-4-diphenylacetoxypiperidinium iodide

NMR

nuclear magnetic resonance

[³H]NMS

N-[³H] methylscopolamine

rt

room temperature

EtOAc

ethyl acetate

DCM

dichloromethane

THF

tetrahydrofuran

Boc

tert-butoxycarbonyl

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acschemneuro.1c00237.

• Chemical synthesis and data analysis. NMR spectra. Pharmacological assay results. Ex vivo assay for histamine H₁R antagonists (variant with 0.05 μM atropine addition). Ex vivo assay for histamine H₁R antagonists (variant without atropine addition). Ex vivo assay for histamine M₂R/M₃R antagonists. Decrease of contractility in electrically stimulated guinea pig ileum - determination of the −log EC₅₀ coefficient. hH₃R radioligand binding assay. hM₁R-hM₅R radioligand binding assay. Intracellular Ca²⁺ measurement. In silico assay results: Comparison of active sites of histamine and muscarinic receptors to H₃R. Change of amino acid conformation in the H₃R binding site on the example of the homology model. Two-dimensional map of interactions between the ADS1017 and ADS10227 ligands and H₃, M₂ and M₄ receptors (PDF)

• H3_ADS1017 (PDB)

• H3_ADS10227 (PDB)

• M2_ADS1017 (PDB)

• M2_ADS10227 (PDB)

• M4_ADS1017 (PDB)

• M4_ADS10227 (PDB)

• Molecular formula strings (CSV)

Author Contributions

M.S.: Conceptualization, synthesis of chemical compounds, ex vivo pharmacological studies on the guinea pig ileum (histamine H₃R, H₁R, muscarinic M₂R/M₃R, determination of the −log EC₅₀ coefficient), data analysis, elaboration and description of the results, wrote the manuscript. D.N.: Determination of human muscarinic M₁–M₅ receptors affinity at radioligand binding experiments, intracellular Ca²⁺ measurement, data analysis, and elaboration and description of the results. J. Jończyk.: Molecular modeling, data analysis, visualization, and elaboration and description of the results. M.D.: Determination of human histamine H₃R affinity at radioligand binding experiment, data analysis, and elaboration and description of the results. A.F.: Determination of human histamine H₃R affinity at radioligand binding experiment, data analysis, and elaboration and description of the results. H.S.: Coordination of the human histamine H₃R affinity at radioligand binding experiment, interpretation of the obtained results, and discussion and extensive commenting on manuscript. M.B.: Molecular modeling project administration, interpretation of the obtained results, and discussion and extensive commenting on manuscript. J. Jakubik: Coordination of the human muscarinic M₁–M₅ receptors affinity at radioligand binding experiment and intracellular Ca²⁺ measurement, interpretation of the obtained results, and discussion and extensive commenting on manuscript. K.W.: Supervision and discussion and extensive commenting on manuscript.

This study was supported by the following sources: The Medical University of Lodz grant number 503/3-016-01/503-31-001 (M.S.), the Grant Agency of the Czech Republic grant 19-05318S (J. Jakubik), the Institutional support of the Czech Academy of Sciences RVO:67985823 (J. Jakubik), research grant (UMO-2014/15/N/NZ7/02965) from the National Science Center in Poland (J. Jończyk). We kindly acknowledge partial support from EU COST Actions CA18133 (H.S. and M.S.), CA15135, and CA18240 (H.S.).

We confirm that all animal experiments performed in the manuscript were conducted according to the current law on the protection of animals used for scientific or educational purposes and in compliance with institutional guidelines. Marek Staszewski possesses individual permission - certificate no. 279/2017, issued by the University of Lodz, Faculty of Biology and Environmental Protection.

The authors declare no competing financial interest.