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

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Research Article

Guanidine Derivatives: How Simple Structural Modiﬁcation of

Histamine H₃R Antagonists Has Led to the Discovery of Potent

Muscarinic M₂R/M₄R Antagonists

́

Marek Staszewski,* Dominik Nelic, Jakub Jonczyk, Mariam Dubiel, Annika Frank, Holger Stark,

́

Marek Bajda, Jan Jakubik, and Krzysztof Walczynski

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ABSTRACT: This article describes the discovery of novel potent muscarinic receptor

antagonists identiﬁed during a search for more active histamine H₃receptor (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 M₂and 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.

KEYWORDS: Antagonists, histamine H₃receptor, muscarinic M₂receptor, muscarinic M₄receptor, structure−activity relationships,

guanidine derivatives

cholinergic transmission in the peripheral nervous system.¹¹

INTRODUCTION

■

H₃R activation reduces the release of [³H]-ACh induced by

In addition to mediating the inhibition of synthesis and release

electrical stimulation in the longitudinal smooth muscle/

of histamine from histaminergic neurons via a negative

feedback loop, the histamine H₃receptor (H₃R) also exerts

modulatory eﬀects on numerous other neurotransmitter

myenteric plexus preparations.^12,13

Muscarinic M₂and M₄receptor (M₂R and M₄R,

respectively) antagonists represent compounds of interest for

systems, including the cholinergic system, in both the central

potential drugs. Activation of M₂R and M₄R inhibits adenylyl

and peripheral nervous system. Stimulation of H₃hetero-

cyclase via the stimulation of the G_i/oG-protein, whereas M₁R,

receptors in the central nervous system (CNS) by H₃R

M₃R, and M₅R mediate the stimulation of phospholipase C via

agonists (imetit, immepip) diminishes acetylcholine (ACh)

G_q/11G-protein.¹⁴The cognitive deﬁcits observed in aging and

release; however, blocking H₃autoreceptors activity with the

Alzheimer’s disease have been associated with brain cholinergic

deﬁcits. However, cognitive performance could be enhanced

by selective blockade of presynaptically located M₂autor-

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

eceptors, which could increase ACh release into the synaptic

Activation of H₃R in CNS reduces ACh release in the rat

cleft.¹⁵One of the acetylcholinesterase (AChE) inhibitors

cortex, hippocampus, nucleus accumbens, and basolateral

approved for use across the full spectrum of these cognitive

amygdala.^1,3−6In addition, the histaminergic neurons in the

disorders is donepezil; however, numerous potent M₂R

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

Received: April 14, 2021

Accepted: May 25, 2021

Published: June 8, 2021

have been implicated in the pathophysiology of Alzheimer’s

and Parkinson’s disease, schizophrenia, depression, or

epilepsy.⁷⁻¹⁰H₃R treatment has also been found to modulate

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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

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

antagonists including SCH-217443 (Chart 1) have shown

eﬃcacy in increasing ACh release and improving cognitive

functions.¹⁶The 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.¹⁶It is considered that

ACh plays a crucial role in governing learning and memory

processes.¹⁷Therefore, 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

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disease is to inhibit the presynaptic activity of the muscarinic

M₂R expressed on the cholinergic neurons, in regions involved

in learning and memory processes.^18,19As 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.²Those dual active ligands (Chart 1)

relates to the treatment of Alzheimer’s disease, attention deﬁcit

disorder, and autism.

The muscarinic M₄R are expressed in the striatum,

prefrontal cortex, and nucleus accumbens; these areas are all

related to social behaviors and cognitive functions.²⁰⁻²²M₄R

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).²³M₄R 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

receptors binding site.^29,30Such 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

N¹to the N³position of the guanidine.³¹Finally, two

s y m m e t r i c a l c o m p o u n d s , 1 , 4 - b i s { 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 N¹of 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 optimiza-

tion.^31,32In 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-cyclo-

hexylene or p-phenylene group connected directly or by a

methylene group to piperazine and phenoxy moieties.

Additionally, for derivatives bearing a 1,4-disubstituted cyclo-

hexylene group, the (E) and (Z) isomers were separated and

pharmacologically tested independently (Chart 2).

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.²⁵A 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.²⁶Most 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.²⁷Another

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

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

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).²⁸A key design element

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a

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

a

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.

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

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a

Scheme 2. Synthesis of ADS10183 and ADS10185

a

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.

by two recrystallizations. Isolation of isomer (Z) was more

complicated. The ﬁltrate obtained after the ﬁrst recrystalliza-

tion 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-Cyclohexanedime-

thanol 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.³²Etheriﬁcation 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 pipera-

zine. N-alkylation of 5a, 5b, 12, and 18 with 4-bromobutyr-

onitrile 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 LiAlH₄in 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

LiAlH₄in 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

Pharmacology. Pharmacological results are assembled in

Tables 1 and 2 including the previously described data for

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a

Scheme 3. Synthesis of ADS10210

a

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.

compound ADS1017.³²All graphs of the ex vivo assays

including the inhibitory eﬀect on the contraction of guinea pig

ileum strips and hH₃, hM₁-hM₅radioligand binding assays are

presented in the Supporting Information.

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 measure-

ment 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.³⁶The −log EC₅₀value 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.³⁶Based on the concen-

tration−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 EC₅₀was 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 con-

traction cannot be associated with H₃R agonism, and the

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.³⁵During 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 concen-

trations necessary to determine the pA₂value signiﬁcantly

decreased electrically evoked tissue contractions. This eﬀect

prevented the applied functional assay from determining the

pA₂value 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

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a

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

a

Values 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-

b

methylhistamine. Decrease of contractility in electrically stimulated guinea pig ileum.

a

Table 2. Radioligand Binding Results at Human Histamine H₃R and Muscarinic M₁R−M₅R

a

Inhibition constants K_iare expressed as negative logarithms. Values are means sem from at least three independent experiments; sem: standard

error of the mean; h: human.

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.³⁷To assess and exclude the inﬂuence of the ADS

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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 pA₂ratio between the two

methods was observed for ADS10227, which further

conﬁrmed our presumption about the eﬀect on muscarinic

receptors.

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−hM₅radioligand 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.³⁹Three 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_ivalues 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 non-

selective muscarinic M₁R/M₂R antagonists, such as scopol-

amine, 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.

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-diphenylacetoxypiper-

idinium 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,³⁸but such an over-

interpretation 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. Com-

pounds with the highest pA₂value against the muscarinic

receptor demonstrated the greatest reduction of electrically

evoked contraction as well (Table 1). As it was mentioned

above, the highest pA₂ratio 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.

hH₃Radioligand Binding Assay. As it was not possible to

determine the pA₂value 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 displace-

ment 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-Methylhist-

amine 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_iof 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.

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.

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

hM₁R−hM₅R Radioligand Binding Assays. Unexpectedly,

the tests carried out on the guinea pig ileum showed that the

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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 M₂receptor

binding site are shown in Figure 3.

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.

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.¹³It is composed of the

amino acids from three transmembrane domains: TM3, TM5,

and TM6. In the case of the histamine H₃R, E206^5.46plays 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 E206^5.46and a cation−π

with Y167^4.57. In addition, T119^3.37stabilizes 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

D114^3.32which physiologically interacts with the protonated

amine moiety of histamine.¹³The tested compounds bind to

D114^3.32via 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 sub-

stituent, 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

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

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interactions with F181 and Y403^6.51in 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.

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 R381^6.58in 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/N423^6.58present 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/Y439^7.39participate in the

phenoxy group bonding. In this case, the change from H₃R

Y394^7.35to W422^7.35/W435^7.35present 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 M₄receptors 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.

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.

CONCLUSION

■

The piperazine ring located in the middle of the ligand

creates similar interactions in the active sites of the M₂R and

A series of new guanidine derivatives was synthesized to

identify new nonimidazole histamine H₃R antagonists. The

a

Table 3. Interactions Created by ADS1017 and ADS10227 within H₃, M₂, and M₄Receptors Binding Sites

a

The 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.⁴⁰Stronger 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.

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standard, and coupling constants (J) were given in hertz (Hz). Spectra

obtained in deuterated chloroform were referenced to tetramethylsi-

lane 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.

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.⁴¹All 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.

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 recrystalliza-

tion 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

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.

(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

1

9:1). Mp: 84.8−86.4 °C. H 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

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-cyclohexanedi-

methanol, phosphorus bromide, piperazine, phenol, sodium, 4-

bromobutyronitrile,4-(triﬂuoromethyl)benzoyl chloride, benzyl bro-

mide, 1,3-bis(tert-butoxycarbonyl)-2-methylisothiourea, 1,4-bis-

(bromomethyl)benzene, sodium, phenol, 1-(bromomethyl)-4-phe-

noxybenzene, 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

(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 × 10⁻²mol) 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

1

°C. H NMR (600 MHz, CDCl₃) δ ppm 3.47 (d, 4H, HOCH₂, J =

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6.27 Hz), 1.85−1.84 (m, 4H^cyclohexyl., CH₂), 1.46 (br, 4H: 2H^cyclohexyl.

,

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 23.⁴²1,3-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 ADS10210.⁴²4 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

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 × 10⁻²mol) 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

1

°C. H 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 × 10⁻²mol) 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 chromatog-

raphy (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:

1

54.2−55.2 °C. H 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 × 10⁻²mol) 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

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Research Article

temperature (pH 7.4) containing (mM) NaCl (136.9), KCl (2.6),

KH₂PO₄(1.47), Na₂HPO₄(9.58), and indomethacin (1 × 10⁻⁶mol/

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 × 10⁻⁷mol/L). The solution

was continuously bubbled with a 95% O₂:5% CO₂mixture 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 × 10⁻⁵mol/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 pA₂values

were calculated according to Arunlakshana and Schild.³⁷Statistical

analysis was carried out with the Students’ t test. In all tests, a p < 0.05

was considered statistically signiﬁcant. The pA₂values were compared

with the aﬃnity of thioperamide.

Ex Vivo Assay for Screening Histamine H₃R Agonists:

Determination of the −log EC₅₀Coeﬃcient 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 × 10⁻⁷mol/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 × 10⁻⁵mol/L concentration in organ bath) and pyrilamine (1 ×

10⁻⁵mol/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 pD₂value

glucose (5.55), and indomethacin (3 × 10⁻⁷mol/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% CO₂mixture 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 pA₂values 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 ×

10⁻⁶mol/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 × 10⁻⁷mol/L). The solution was

continuously bubbled with a 95% O₂:5% CO₂mixture 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

pA₂values 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 pA₂values 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.⁴³For 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 (Perki-

nElmer). 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_ivalues were calculated from the IC₅₀values using the Cheng−

Prusoﬀ equation.⁴⁴The statistical calculations were performed on

−log K_i. The mean values and 95% conﬁdence intervals were

transformed to nanomolar concentrations.

(−log EC₅₀)

sem. The −log EC₅₀diﬀerences 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 × 10⁻⁶mol/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),

Cell Culture and Membrane Preparation. The culture was derived

from CHO cells stably transfected with the genes of human variants of

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Research Article

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.⁴⁵The cells were

grown to conﬂuence in 75 cm²ﬂasks 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.

where y is speciﬁc binding at free concentration x, B_MAXis maximum

binding capacity, and K_Dis 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 normal-

ization to binding in the absence of the tested compound, eq 2 was

ﬁtted to the data:

100 × x^nH

y = 100 −

nH

IC + x^nH

(2)

50

where y is a speciﬁc radioligand binding at concentration x of the

tested compound expressed as a percent of binding in its absence,

IC₅₀is the concentration of tested compound inhibiting 50% of

[³H]NMS binding, and nH is the Hill coeﬃcient.

IC₅₀

K_i=

[D]

1 +

K_D

(3)

Determination of hM₁R−hM₅R Aﬃnities. All radioligand binding

experiments were optimized and carried out according to general

guidelines.³⁹Brieﬂy, 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

IC₅₀value was computed according to eq 2, and the inhibition

constant K_iaccording 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.

where K_iis the inhibition constant of the tested compound, K_Dis 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:

(E_MAX− 1) × x^nH

y = 1 +

x^nH+ EC₅₀

(4)

where y is the normalized response at ACh concentration x, E_MAXis

the maximal eﬀect, EC₅₀is the concentration of ACh causing half-

maximal eﬀect, and nH is Hill coeﬃcient. From a series of EC₅₀values

of apparent inhibition constant K_Bwas calculated by ﬁtting eq 5 to

dose ratio (DR) induced by tested compound at a given

concentration:

log(DR − 1) = log[B] − log K_B

(5)

where DR is ratio of EC₅₀in the presence of tested compound at

concentration [B] to EC₅₀in its absence.

In Silico Studies. All tested ligands were prepared with the

̈

appropriate spatial conﬁguration in the Maestro program (Schro-

dinger) and then charged at pH 7.4 0.2 using the LigPrep program

̈

(Schrodinger Release 2017−1; Maestro-Schrodinger, LLC, New

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:

York).

All docking experiments were performed with the Glide program

46

̈

(Maestro-Schrodinger) with the SP level of calculation accuracy.

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.⁴⁹The 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

47

aromatic amino acids Y104^3.33and Y426^7.39and W99^3.28

.

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

H₃R²⁸and the structure of the M₄R (PDB: 5DSG). The key

diﬀerences in the position of the most important H₃amino acids are

given in Figure S56.

B_MAX× x

y =

x + K_D

(1)

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Research Article

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

Jan Jakubik − Department of Neurochemistry, Institute of

Physiology CAS, CZ142 20 Prague, Czech Republic;

orcid.org/0000-0002-1737-1487

́

Krzysztof Walczynski − Department of Synthesis and

̈

(Maestro-Schrodinger). Such optimized complexes were used for the

Technology of Drugs, Medical University of Lodz, 90-151

ﬁnal analyzes, and all tested ligands were docked to them. The ﬁnal

results were visualized using PyMol 0.99 rc6 software (DeLano

Scientiﬁc LLC).

́

Łódz, Poland

Complete contact information is available at:

https://pubs.acs.org/10.1021/acschemneuro.1c00237

ASSOCIATED CONTENT

■

sı

* Supporting Information

Author Contributions

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acschemneuro.1c00237.

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 EC₅₀coeﬃcient), data analysis, elaboration and

description of the results, wrote the manuscript. D.N.:

Determination of human muscarinic M₁−M₅receptors aﬃnity

at radioligand binding experiments, intracellular Ca²⁺measure-

ment, data analysis, and elaboration and description of the

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 EC₅₀coeﬃcient. 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 inter-

actions between the ADS1017 and ADS10227 ligands

and H₃, M₂and M₄receptors (PDF)

H3_ADS1017 (PDB)

H3_ADS10227 (PDB)

M2_ADS1017 (PDB)

M2_ADS10227 (PDB)

M4_ADS1017 (PDB)

M4_ADS10227 (PDB)

Molecular formula strings (CSV)

́

results. J. Jonczyk.: 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 manu-

script. 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−M₅receptors aﬃnity at

radioligand binding experiment and intracellular Ca²⁺measure-

ment, interpretation of the obtained results, and discussion and

extensive commenting on manuscript. K.W.: Supervision and

discussion and extensive commenting on manuscript.

Funding

AUTHOR INFORMATION

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

■

Corresponding Author

Marek Staszewski − Department of Synthesis and Technology

of Drugs, Medical University of Lodz, 90-151 Łódz, Poland;

́

orcid.org/0000-0003-2783-522X;

Email: marek.staszewski@umed.lodz.pl

́

Science Center in Poland (J. Jonczyk). We kindly acknowledge

partial support from EU COST Actions CA18133 (H.S. and

M.S.), CA15135, and CA18240 (H.S.).

Authors

Dominik Nelic − Department of Neurochemistry, Institute of

Physiology CAS, CZ142 20 Prague, Czech Republic

Jakub Jonczyk − Department of Physicochemical Drug

Analysis, Faculty of Pharmacy, Jagiellonian University

Medical College, 30-688 Kraków, Poland

Mariam Dubiel − Institute of Pharmaceutical and Medicinal

Chemistry, Heinrich Heine University Dusseldorf, Duesseldorf

̈

40225, Germany

Annika Frank − Institute of Pharmaceutical and Medicinal

Chemistry, Heinrich Heine University Dusseldorf, Duesseldorf

40225, Germany

Holger Stark − Institute of Pharmaceutical and Medicinal

Chemistry, Heinrich Heine University Dusseldorf, Duesseldorf

40225, Germany; orcid.org/0000-0003-3336-1710

Marek Bajda − Department of Physicochemical Drug Analysis,

Faculty of Pharmacy, Jagiellonian University Medical College,

30-688 Kraków, Poland; orcid.org/0000-0001-6032-

0354

Notes

́

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.

ABBREVIATIONS USED

̈

■

CNS, Central nervous system; ACh, acetylcholine; H₃R,

histamine H₃receptor; H₁R, histamine H₁receptor; gpH₃R,

guinea pig histamine H₃receptor; gpH₁R, guinea pig histamine

H₁receptor; gpM₂R/M₃R, guinea pig muscarinic M₂R/M₃R;

hH₃R, human histamine H₃receptor; hM₁₋₅, human

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Article Doi

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

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