Design, synthesis, and: In vitro and in vivo characterization of 1-{4-[4-(substituted)piperazin-1-yl]butyl}guanidines and their piperidine analogues as histamine H3 receptor antagonists

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RESEARCH ARTICLE

Design, synthesis, and in vitro and in vivo

characterization of 1-{4-[4-(substituted)piperazin-

1-yl]butyl}guanidines and their piperidine

Cite this: DOI: 10.1039/c8md00527c

analogues as histamine H₃receptor antagonists†‡

a

*

Marek Staszewski,

Anna Stasiak,^bTadeusz Karcz,^cDaniel McNaught Flores,^d

Wiesława Agnieszka Fogel, ^bKatarzyna Kieć-Kononowicz,

c

Rob Leurs^dand Krzysztof Walczyński

a

*

Previously, we have shown that 1-substituted-[4-(7-phenoxyheptylpiperazin-1-yl)butyl]guanidine with

electron withdrawing substituents at position 4 in the benzyl moiety exhibits high in vitro affinities toward

the guinea pig jejunal histamine H₃receptor with pA₂ranging from 8.49 to 8.43. Here, we present data on

the impact of replacement of the piperazine scaffold by the piperidine ring (compounds 2a and 2b), mov-

ing benzyl- and 4-trifluoromethylbenzyl substituents from position 1 to 3 of the guanidine moiety (com-

pounds 2c and 2d), which decreases the guanidine basicity (compound 2e), and the influence of individual

synthons (compounds 2f–h), present in the lead compounds 1b and 1c, on the antagonistic activity against

the histamine H₃receptor. Additionally, the most active compounds 1a, 1c, and 1d were evaluated for their

affinity to the rat histamine H₃receptor and the human histamine H₃and H₄receptors. It was also shown

that compounds 1a, 1c and 1d, given parenterally for five days, reduced the food intake of rats and did not

influence the brain histamine or noradrenaline concentrations; however, significantly reduced serotonin

and dopamine concentrations were found in rats administered with compounds 1a and 1c, respectively.

Received 18th October 2018,

Accepted 29th November 2018

DOI: 10.1039/c8md00527c

rsc.li/medchemcomm

the H₄-receptor seems to be implicated in the inflammatory

process.²Histamine H₃receptors are located on histaminer-

1. Introduction

Histamine is a biogenic amine implicated in a wide range of

physiological processes through the activation of four

G-protein coupled receptors (H₁, H₂, H₃, and H₄).¹The post-

synaptic H₁- and H₂-receptors are mainly involved in the con-

trol of allergic response and gastric acid, respectively, while

gic or non-histaminergic neurons, respectively acting as

autoreceptors or heteroreceptors, controlling the release and

synthesis of histamine^3,4and of multiple neurotransmitters

such as acetylcholine,⁵norepinephrine⁶and dopamine.⁷

These data suggested that histamine H₃receptor antagonists

could affect a number of behaviors and be useful in the treat-

ment of cognitive deficits associated with a variety of disease

states including Alzheimer's disease (AD),⁸attention deficit

hyperactivity disorder (ADHD),⁹schizophrenia,¹⁰and

obesity.¹¹

The first generation of active histamine H₃receptor li-

gands was based on a structurally modified imidazole ring.¹²

Unfortunately, further pharmacological investigations have

proved that the imidazole derivatives show poor brain pene-

tration, CYP450 inhibition, drug–drug interactions, liver toxic-

ity, etc.^13,14For these reasons, and after the successful cloning

of the human histamine H₃receptor by Lovenberg,¹⁵efforts

have been directed toward the discovery of histamine H₃re-

ceptor antagonists without an imidazole moiety as these com-

pounds may offer improvements in binding affinity, CNS pen-

etration and reduced potential for cytochrome P₄₅₀enzyme

inhibition.^16,17

^aDepartment of Synthesis and Technology of Drugs, Medical University of Lodz,

ul. Muszyńskiego 1, 90-151 Łódź, Poland.

E-mail: marek.staszewski@umed.lodz.pl, krzysztof.walczynski@umed.lodz.pl;

Tel: +48 42 6779194, +48 42 6779196

^bDepartment of Hormone Biochemistry, Medical University of Lodz, ul.

Żeligowskiego 7/9, 90-752 Łódź, Poland. E-mail: anna.stasiak@umed.lodz.pl,

wieslawa.agnieszka.fogel@umed.lodz.pl

^cDepartment of Technology and Biotechnology of Drugs, Faculty of Pharmacy,

Jagiellonian UniversityMedical College, ul. Medyczna 9, 30-688 Kraków, Poland.

E-mail: t.karcz@uj.edu.pl, mfkonono@cyf-kr.edu.pl

^dAmsterdam Institute of Molecules, Medicines & Systems, Division of Medicinal

Chemistry, Vrije Universiteit Amsterdam, De Boelelaan 1108, 1081 HZ

Amsterdam, The Netherlands. E-mail: d.mcnaughtflores@vu.nl, r.leurs@vu.nl

† This article is dedicated to Professor Henk Timmerman on the occasion of his

80th birthday.

‡ Electronic supplementary information (ESI) available. See DOI: 10.1039/

c8md00527c

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Over the past fifteen years, the imidazole moiety has been

successfully replaced with pyrrolidine, piperidine, piperazine

and other basic tertiary amines.^18,19

phases.^25,26In addition to the search for histamine H₃re-

ceptor antagonists/inverse agonists for treatment of neuro-

degenerative diseases, efforts have been made to discover

histamine H₃receptor ligands for treatment of metabolic

diseases (e.g. obesity and dyslipidemia).²⁷Based on current

knowledge of the involvement of histamine in the regula-

tion of body weight, pharmacological manipulation of the

histaminergic system seems to be an obvious target for

anti-obesity drugs.²⁸

Previously, we have reported the synthesis and preliminary

pharmacological in vitro characterization of a series of potent

histamine H₃receptor non-imidazole antagonists belonging

to the class of 1-substituted-1-{ω-[4-(ω-phenoxyalkyl)piperazin-

1-yl]alkyl}guanidines.²⁹

Although one of the first known histamine H₃receptor an-

tagonists was a guanidine derivative, impromidine, which

was found to be a potent agonist of the H₂receptor, only a

small number of compounds containing this structure ele-

ment are described. However, it appears that the guanidine

derivatives, both imidazole, i.e. 1-cyclohexylmethyl-3-[3-(1H-

imidazol-4-yl)propyl]guanidinine²⁰(Chart 1), and non-imidaz-

ole, i.e. compound JB 98064 (Chart 1),²¹are potent and selec-

tive histamine H₃receptor antagonists.

Based on data reported in patent applications and chem-

istry papers, many efforts have been made to discover a

number of histamine H₃receptor ligands for treatment of

cognitive diseases/sleep disorders (e.g., Alzheimer, depres-

sion, anxiety, schizophrenia, and narcolepsy).^22–24Several of

these compounds have entered advanced clinical

The prominent members of this family are compounds

carrying electron-withdrawing substituents in position 1 of

the guanidine moiety or a chlorine atom at position 4 in the

benzene ring, 1a (ADS-1024; pA₂= 8.43), 1b (ADS-1020; pA₂=

Chart 1 Example structures of histamine H₃receptor antagonists carrying the guanidine moiety and target molecules of this study.

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8.49), 1d, (ADS-1022; pA₂= 7.80), respectively, and an

unsubstituted benzyl residue 1c (ADS-1017; pA₂= 8.21) ((Chart 1)

for reference thioperamide (pA₂= 8.72)). These compounds were

also routinely screened for potency towards the histamine H₁re-

ceptor and were found to possess weak but competitive H₁an-

tagonistic activity (pA₂= 6.65, pA₂= 6.46, pA₂= 6.62, and pA₂=

6.70, respectively) for this site in the functional assay on isolated

tissue preparations of the guinea-pig ileum.

tains seven and four methylene groups, respectively), which

is optimal according to our previous results.²⁹

In continuation of our search for new highly active and selec-

tive non-imidazole histamine H₃receptor antagonists, the first

part of this study aimed to clarify whether both nitrogen atoms

in the piperazine ring, present in the parent compounds 1a and

1b, are necessary to maintain a high activity or whether only one

of them (and which one) is absolutely necessary to retain the ac-

tivity. Therefore, two piperidine analogues, 2a (ADS-1025) and 2b

(ADS-1026) (Chart 1), of the lead compound 1b were synthesized

and pharmacologically evaluated (functionally on the in vitro test

Throughout the study, we choose to retain the length of

the linkers connecting the phenoxy and guanidine residues

with the piperazine and piperidine rings (the alkyl chain con-

Scheme 1 Synthesis of 1-{4-[4-(7-phenoxyheptyl)piperidin-1-yl]butyl}-1-[4-(trifluoromethyl)benzyl]guanidine dihydrochloride – 2a (ADS-1025).

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system using guinea pig jejunum preparations).³⁰Furthermore,

in the piperazine series, the influence of moving the benzyl- and

4-trifluoromethylbenzyl substituents from position 1 to 3 of the

guanidine moiety (compounds 2c (ADS-1029) and 2d (ADS-1028)

(Chart 1)), and introducing a nitrile group into the N²guanidine

residue (compound 2e (ADS-1027) (Chart 1)) on the histamine

H₃receptor antagonistic activity was studied. Finally, three com-

pounds i.e. 1,4-bis{4-[1-(4-trifluoromethylbenzyl)guandin-1-

yl]but-1-yl}piperazine 2f (ADS-1030), 1,4-bisĲphenoxyheptyl)-

piperazine 2g (ADS-1031) and 1-benzyl-1-(7-phenoxyhept-1-

yl)guanidine 2h (ADS-1032) (Chart 1) were synthesized in order

to explain which part of the parent compound 1b plays the key

role in blocking the histamine H₃receptor.

tor (PerkinElmer, Waltham, MA), respectively. Furthermore,

these derivatives were subjected to in vivo evaluation of their

impact on feeding behavior and brain neurotransmitter sys-

tems after repeated peripheral administration to rats. Post-

mortem analyses of the rat brain tissues were also carried out

to determine the activities of MAO-A, MAO-B, and HNMT.

2. Results and discussion

2.1. Chemistry

The general synthetic procedures used in this study are illus-

trated in Schemes 1–7.

1-{4-[4-(7-Phenoxyheptyl)piperidin-1-yl]butyl}-1-[4-

Furthermore, all the newly synthesized compounds were

also tested for H₁antagonistic effects in vitro, following stan-

dard methods, using the guinea pig ileum.³¹

(trifluoromethyl)benzyl]guanidine

dihydrochloride

2a

(Scheme 1) was prepared by an eleven-step synthesis includ-

ing deprotonation of the 4-methyl group in 4-methylpyridine

3, followed by alkylation with 2-(6-bromohexyloxy)tetrahydro-

2H-pyran 4 to 7-(pyridin-4-yl)heptan-1-ol hydrochloride 2a.1,

hydrogenation with a catalytic amount of platinum dioxide in

anhydrous ethanol to 7-(piperidin-4-yl)heptan-1-ol 2a.2, reac-

Additionally, the affinity of compounds 1a, 1c, and 1d –

the most active compounds that we have so far synthesized

in this series – was estimated to the recombinant rat hista-

mine H₃receptor (rH₃R) transiently expressed in HEK-293T

cells and human histamine H₃and H₄receptors (hH₃R,

hH₄R) on cell membrane preparations from HEK293 with sta-

ble expression of the human histamine H₃receptor and from

CHO cells stably expressing the human histamine H₄recep-

tion

with

di-tert-butyl

dicarbonate

to

7-[1-(tert-

butoxycarbonyl)piperidin-4-yl]heptan-1-ol 2a.3, replacement of

the hydroxyl group by bromine with tetrabromomethane in

the presence of triphenylphosphine to 1-(tert-butoxycarbonyl)-

Scheme 2 Synthesis of 1-{4-[1-(7-phenoxyheptyl)piperidin-4-yl]butyl}-1-[4-(trifluoromethyl)-benzyl]guanidine dihydrochloride 2b (ADS-1026).

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4-(7-bromoheptyl)piperidine 2a.4, etherification with sodium

phenoxide 5 in anhydrous ethanol to 1-(tert-butoxycarbonyl)-

4-(7-phenoxyheptyl)piperidine 2a.5, removal of the N-Boc

protecting group in the presence of hydrochloric acid in diox-

ane to 4-(7-phenoxyheptyl)piperidine 2a.6, N-alkylation with

4-bromobutyronitrile 6 in the presence of potassium carbon-

ate in acetonitrile to 4-[4-(7-phenoxyheptyl)piperidin-1-

yl]butanenitrile 2a.7, reduction with LiAlH₄in dry ethyl ether

to 4-[4-(7-phenoxyheptyl)piperidin-1-yl]butan-1-amine 2a.8,

N-acylation with 4-(trifluoromethyl)benzoyl chloride 7 in the

presence of triethylamine to N-{4-[4-(7-phenoxyheptyl)-

piperidin-1-yl]butyl}-4-(trifluoromethyl) benzamide 2a.9, reduc-

tion with LiAlH₄in dry ethyl ether to 4-[4-(7-

phenoxyheptyl)piperidin-1-yl]-N-[4-(trifluoromethyl)benzyl]-

ride

7

in the presence of triethylamine to N-{4-[1-(7-

phenoxyheptyl)piperidin-4-yl]butyl}-4-(trifluoromethyl)-

benzamide 2b.5, reduction with LiAlH₄in dry ethyl ether to

4-[1-(7-phenoxyheptyl)piperidin-4-yl]-N-[4-(trifluoromethyl)-

benzyl]butan-1-amine 2b.6, guanylation with 1,3-bisĲtert-

butoxycarbonyl)-2-methyl-2-thiopseudourea in the presence of

triethylamine and 10% excess of mercuryĲII) chloride to 2,3-

diĲtert-butoxycarbonyl)-1-[4-[1-(7-phenoxyheptyl)piperidin-4-yl]-

butyl]-1-[4-(trifluoromethyl)benzyl]-guanidine 2b.7, and finally

acid deprotection of the product from the guanylation to

1-{4-[1-(7-phenoxyheptyl)piperidin-4-yl]butyl}-1-[4-

(trifluoromethyl)benzyl]-guanidine dihydrochloride 2b.

1-Benzyl-3-{4-[4-(7-phenoxyheptyl)piperazin-1-yl]butyl}-

guanidine trihydrochloride 2c and 1-[4-[4-(7-phenoxyheptyl)-

piperazin-1-yl]butyl]-3-[4-(trifluoromethyl)benzyl]guanidine tri-

hydrochloride 2d were prepared as outlined in Scheme 3.

Treatment of the appropriate benzyl bromide 10 and 11 with

1,3-bisĲtert-butoxycarbonyl)-2-methyl-2-thiopseudourea in the

presence of 60% sodium hydride oil dispersion in DMF

afforded 1,3-diĲtert-butoxycarbonyl)-1-benzyl-2-methyl-2-thio-

butan-1-amine

2a.10,

guanylation

with

1,3-bisĲtert-

butoxycarbonyl)-2-methyl-2-thiopseudourea in the presence of

triethylamine and 10% excess of mercuryĲII) chloride to 2,3-

diĲtert-butoxycarbonyl)-1-{4-[4-(7-phenoxyheptyl)piperidin-1-yl]-

butyl}-1-[4-(trifluoromethyl)benzyl]guanidine 2a.11, and finally

acid deprotection of the product from the guanylation to

1-{4-[4-(7-phenoxyheptyl)piperidin-1-yl]butyl}-1-[4-

(trifluoromethyl)benzyl]guanidine dihydrochloride 2a.

pseudourea

2c.1

and

1,3-diĲtert-butoxycarbonyl)-1-[4-

(trifluoromethyl)benzyl]-2-methyl-2-thiopseudourea 2d.1, re-

spectively. Compounds 2c.1 and 2d.1 were reacted with 4-[4-

(7-phenoxyheptyl)piperazin-1-yl]butan-1-amine 12 in a mixture

of THF–water to give compounds 2,3-diĲtert-butyloxycarbonyl)-

3-benzyl-1-{4-[4-(7-phenoxyheptyl)piperazin-1-yl]butyl}guanidine

2c.2 and 2,3-diĲtert-butyloxycarbonyl)-1-{4-[4-(7-phenoxyheptyl)-

piperazin-1-yl]butyl}-3-[4-(trifluoromethyl)-benzyl]guanidine

2d.2, respectively. Finally, compounds 2c.2 and 2d.2 were

treated with HCl in dioxane to cleave the Boc moieties respec-

tively, affording compounds 2c and 2d in quantitative yields.

The synthesis of 1-benzyl-3-cyano-1-{4-[4-(7-phenoxyheptyl)-

piperazin-1-yl]but-1-yl}guanidine trihydrochloride 2e was

1-{4-[1-(7-Phenoxyheptyl)piperidin-4-yl]butyl}-1-[4-

(trifluoromethyl)benzyl]guanidine

dihydrochloride

2b

(Scheme 2) was prepared by eight-step synthesis including

conversion of 4-piperidine butyric acid hydrochloride 8 to

methyl 4-(piperidin-4-yl)butanoate 2b.1, formation of

4-(piperidin-4-yl)butanamide 2b.2 by the reaction of the ester

2b.1 with aqueous ammonium hydroxide, N-alkylation of 2b.2

with 7-phenoxyheptyl bromide to 4-[1-(7-phenoxyheptyl)-

piperidin-4-yl]butanamide 2b.3, reduction with LiAlH₄in dry

ethyl ether to 4-[1-(7-phenoxyheptyl)piperidin-4-yl]butan-1-

amine 2b.4, N-acetylation with 4-(trifluoromethyl)benzoyl chlo-

Scheme

3 Synthesis of 1-benzyl-3-{4-[4-(7-phenoxyheptyl)piperazin-1-yl]butyl}guanidine trihydrochloride 2c (ADS-1029) and 1-[4-[4-(7-

phenoxyheptyl)piperazin-1-yl]butyl]-3-[4-(trifluoromethyl)-benzyl]guanidine trihydrochloride 2d (ADS-1028).

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Scheme 4 Synthesis of 1-benzyl-3-cyano-1-{4-[4-(7-phenoxyheptyl)piperazin-1-yl]but-1-yl}guanidine trihydrochloride 2e (ADS-1027).

Scheme 5 Synthesis of 1,4-bis{4-[1-(4-trifluoromethylbenzyl)guandin-1-yl]but-1-yl}piperazine tetrahydrochloride 2f (ADS-1030).

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Scheme 6 Synthesis of 1,4-bisĲ7-phenoxyheptyl)piperazine dihydrochloride 2g (ADS-1031).

carried out as outlined in Scheme 4. Treatment of N-cyano-

diphenylimidocarbonate 13 with a 10% ammonia solution in

methanol afforded 1-cyano-2-phenylisourea 2e.1. The reaction

of N-benzyl-4-[4-(7-phenoxyheptyl)piperazin-1-yl]butan-1-amine

14 with guanylating reagent 2e.1 in acetonitrile yielded 2e.2

which was converted into trihydrochloride salt 2e in 16% hy-

drogen chloride–methanol solution.

2f.4, guanylation with 1,3-bisĲtert-butoxycarbonyl)-2-methyl-2-

thiopseudourea in the presence of triethylamine and 10%

excess

of

mercuryĲII)

chloride

to

1,4-bis{4-[1-(4-

trifluoromethylbenzyl)-2,3-diĲtert-butyloxycarbonyl)guandin-1-

yl]but-1-yl}piperazine 2f.5, and finally acid deprotection of

the product from the guanylation to 1,4-bis{4-[1-(4-

trifluoromethylbenzyl)guandin-1-yl]but-1-yl}piperazine

tetra-

1,4-Bis{4-[1-(4-trifluoromethylbenzyl)guandin-1-yl]but-1-yl}-

piperazine tetrahydrochloride 2f (Scheme 5) was prepared by

six-step synthesis including bis-N-alkylation of piperazine 15

with 4-bromobutyronitrile 6 in the presence of potassium

carbonate in acetonitrile to 1,4-bisĲ4-nitrilobutyl)piperazine

2f.1, reduction with LiAlH₄in dry ethyl ether to 1,4-bisĲ4-

hydrochloride 2f.

1,4-BisĲ7-phenoxyheptyl)piperazine dihydrochloride 2g

(Scheme 6) was obtained by condensing moles of

7-phenoxyheptylbromide 9 with piperazine 15 in acetonitrile

with the presence of K₂CO₃. The free base was treated with a

4 M solution of hydrogen chloride–dioxane and dihydro-

chloride was precipitated with dry methanol.

2

aminobutyl)piperazine

2f.2,

bis-N-acylation

with

4-(trifluoromethyl)benzoyl chloride 7 in the presence of tri-

ethylamine to 1,4-bisĳN-(4-trifluoromethylbenzoil)aminobut-4-

yl]piperazine 2f.3, reduction with LiAlH₄in dry ethyl ether

to 1,4-bisĳN-(4-trifluoromethylbenzyl)aminobut-4-yl]piperazine

1-Benzyl-1-(7-phenoxyhept-1-yl)guanidine

(Scheme 7) was prepared by six-step synthesis including

etherification of sodium phenoxide with 7-bromo-

heptanenitrile 16 to 7-phenoxyheptanenitrile 2h.1, reduction

fumarate

2h

5

Scheme 7 Synthesis of 1-benzyl-1-(7-phenoxyhept-1-yl)guanidine fumarate 2h (ADS-1032).

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with LiAlH₄in dry ethyl ether to 7-phenoxyheptan-1-amine

2h.2, N-acylation with benzoyl chloride 17 in the presence of

triethylamine to N-(7-phenoxyheptyl)benzamide 2h.3, reduc-

tion with LiAlH₄in dry ethyl ether to N-benzyl-7-

phenoxyheptan-1-amine 2h.4, guanylation with 1,3-bisĲtert-

butoxycarbonyl)-2-methyl-2-thiopseudourea in the presence of

triethylamine and 10% excess of mercuryĲII) chloride to

1-benzyl-1-(7-phenoxyhept-1-yl)-2,3-diĲtert-butyloxycarbonyl)-

guanidine 2h.5, and finally acid deprotection of the product

from the guanylation to 1-benzyl-1-(7-phenoxyhept-1-

yl)guanidine fumarate 2h.

2.2. Pharmacology

2.2.1. In vitro pharmacological studies

2.2.1.1. Histamine H₃receptor antagonistic activity of

compounds 1a–d and 2a–h. The compounds were tested

in vitro as histamine H₃receptor antagonists against

histamine H₃receptor agonist-induced inhibition of the elec-

trically evoked contraction of the guinea-pig jejunum.³⁰The

potencies of the newly synthesized compounds 2a–e and 2f–h

are reported in Table 1 including the previously described

data for compounds 1a–d (ref. 29) (Table 1). Derivatives 2a–h

show weak to pronounced antagonist activity against the his-

tamine H₃receptor.

Detailed synthetic procedure and analytical data for com-

pounds 2a.1–2a.11; 2b.1–2b.7; 2c.1, 2c.2; 2d.1, 2d.2; 2e.1,

2e.2; 2f.1–2f.5, and 2h.1–2h.5 are shown in Appendix A (sub-

section 1): ESI.‡

Recently, we described a novel 1-{4-[4-(7-phenoxyheptyl)-

piperazin-1-yl]butyl}-1-[4-(substituted)benzyl]guanidine series²⁹

Table 1 Histamine H₃- and H₁-receptor antagonistic potency in the in vitro test system in the Guinea pig jejunum and radioligand binding results on

human histamine H₃and H₄receptors for compounds 1a–d and 2a–h

gp pA₂(sem)

N (caviae)

gp pA₂(sem)

N (caviae)

rH₃R pK_isem

hH₃R

hH₃R pK_i

sem

hH₄R pK_i

sem

selectivity

(H₄/H₃)

Cpd.

H₃

H₁

1a (ref. 29)

1b (ref. 29)

1c (ref. 29)

1d (ref. 29)

2a

2b

2c

2d

2e

2f

2g

2h

8.43 (0.11)

8.49 (0.05)

8.21 (0.05)

7.80 (0.1)

7.90 (0.05)

8.35 (0.08)

8.10 (0.06)

7.97 (0.02)

7.30 (0.08)

7.99 (0.08)

5.78 (0.13)

5.78 (0.03)

8.98 (0.08)

10 (3)

13 (4)

12 (4)

9 (3)

12 (4)

9 (3)

12 (4)

9 (3)

16 (5)

6.65 (0.08)

6.46 (0.06)

6.70 (0.10)

6.62 (0.15)

7.19 (0.05)

6.90 (0.06)

7.10 (0.11)

7.18 (0.01)

6.97 (0.16)

7.22 (0.10)

NA

9 (3)

7.8 0.1

6.84 0.02

5.92 0.01

8.3

12 (4)

15 (5)

8 (2)

12 (4)

8 (2)

12 (3)

7.9 0.1

7.6 0.1

6.80 0.04

6.56 0.03

5.49 0.07

5.97 0.03

20.9

3.9

6.80 (0.25)

8 (2)

Thioperamide

Pyrilamine

7.9 0.1

6.75 0.11

7.34 2.55

0.3

9.18 (0.05)

20 (5)

sem: standard error of the mean; N: number of different animal preparations; caviae: number of animals. gp: guinea pig; r: rat; h: human.

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with various substituents at position 4 in the benzyl moiety.

The introduction of strong electron-withdrawing substituents

led to compounds 1a (pA₂= 8.43) and 1b (pA₂= 8.49) show-

ing the highest potency for that series. Slightly lower potency

the parent compound 1b (pA₂= 6.46). Taking into consider-

ation the obtained results, we chose to retain the 1-butyl-4-(7-

phenoxyheptyl)piperazine moiety as an optimal fragment for

further structural modification. Therefore, the influence of

moving the benzyl- and 4-trifluoromethylbenzyl substituents

from N¹to the N³position of the guanidine moiety in com-

pounds 2c and 2d (Table 1) on the antagonistic activity

against the histamine H₃receptor was investigated. As seen

in the case of the piperidine analogues, both 1,3-disubstituted

guanidines (2c and 2d) were less active (pA₂= 8.10 and pA₂=

7.97) than the corresponding 1,1-disubstituted guanidines 1c

and 1b (pA₂= 8.21 and pA₂= 8.49, respectively). Slightly

higher potency was demonstrated by derivative 2c with an

unsubstituted benzyl ring than its 4-trifluoromethylbenzyl an-

alogue 2d. Additionally, in order to decrease the guanidine

basicity, a nitrile group was introduced into the N²nitrogen

of derivative 1c which resulted in compound 2e showing the

lowest potency (pA₂= 7.3) within the series of compounds

2a–e (Table 1). Because of the lower potency of 2e compared

to 1c, we might conclude that the decreasing basicity of the

N²nitrogen has a negative effect on blocking the histamine

H₃receptor.

was shown by derivative 1c (pA₂

= 8.21) bearing an

unsubstituted benzyl moiety. Additionally, all the compounds

(1a–1d) showed weak, but competitive H₁-antagonistic activ-

ity, with pA₂ranging from 6.46 to 6.70.

With the aim of optimizing the structure of the lead com-

pound 1b, we wanted to clarify whether both nitrogen atoms

in the piperazine ring, present in the parent compound 1b,

are necessary to maintain a high activity or whether only one

of them (and which one) is absolutely necessary to retain the

activity. Therefore, two piperidine analogues, 2a – where the

butyl-[4-(trifluoromethyl)benzyl]guanidine residue was directly

bonded to the piperidine nitrogen, and 2b – where the

7-phenoxyheptyl moiety was attached to the piperidine nitro-

gen, were synthesized and pharmacologically evaluated. On

comparing the potency of 2a (pA₂= 7.90) and 2b (pA₂= 8.35)

with that of the parent compound 1b (pA₂= 8.49; Table 1), it

was shown that the piperidine analogues possessed slightly

lower antagonistic activity than the lead compound 1b

containing the piperazine scaffold, but derivative 2b pos-

sessed higher potency to the H₁receptor (pA₂= 6.90) than

Finally, in order to explain which synthon of the parent

compounds 1b and 1c plays the key role in blocking the

Fig. 1 Daily food consumption during pre-test (three days) and five days of treatment with the newly synthesized histamine H₃receptor antago-

nists 1a, 1c, and 1d, and ciproxifan. Ciproxifan, a histamine H₃-receptor antagonist/inverse agonist, was used as a reference compound. The me-

dian (the line in the middle of the box) and the range of values (whiskers) are given for eight rats.

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histamine H₃receptor, three compounds were synthesized

and pharmacologically evaluated in vitro (Table 1): 1,4-bis{4-

[1-(4-trifluoromethylbenzyl)guandin-1-yl]but-1-yl}piperazine

tetrahydrochloride 2f, 1,4-bisĲ7-phenoxyheptyl)piperazine 2g,

and 1-benzyl-1-(7-phenoxyhept-1-yl)guanidine 2h. Bisguanidine

derivative 2f showed potency (pA₂= 7.99; Table 1) on the

same level as compound 2d (pA₂= 7.97; Table 1). In the case

of bisĲ7-phenoxyheptyl) derivative 2g, a drastic reduction of

affinity (pA₂= 5.78; Table 1) was observed. Removal of the

1-butylpiperazine fragment from derivative 1c resulted in

compound 2h, with only a weak activity (pA₂= 5.78; Table 1)

against the H₃receptor. Based on the obtained results, it is

seen that the trifluoromethylbenzylguandinbutyl moiety plays

the key role in blocking the histamine H₃receptor in the pi-

perazine series.

All the graphs of antagonism by 2a–h and thioperamide

and the inhibitory effect of R-(−)-α-methyl-histamine (RAMH)

on the electrically induced contraction of guinea-pig ileum

strips are shown in Appendix B (subsection 2.2.1; Fig. 1):

ESI.‡

2.2.1.2. H₁antagonistic activity of compounds 2a–h. Due to

the fact that the most active previously obtained compounds

(1a–d) showed weak, competitive H₁-antagonistic activity, all

the newly synthesized compounds (2a–h) were also tested for

H₁antagonistic effects in vitro, following standard methods,

using the guinea pig ileum.³¹The highest potency to the H₁

membranes expressing rH₃R were incubated with different

concentrations of [³H]-N^α-MH (0–20 nM) in the absence or

presence of unlabeled thioperamide (10 μM) to determine

the total and non-specific binding, respectively, for 2 h at 25

°C. The reaction was terminated by rapid filtration on GF/C

96 well plates and the bound radioligand was measured by

scintillometry. Specific binding was defined as the difference

between the total and non-specific binding conditions. A rep-

resentative graph of the saturation of the rat histamine H₃re-

ceptor can be found in Appendix B (subsection 2.2.3; Fig. S3):

ESI.‡

Analysis of the [³H]-N^α-MH saturation binding yielded on

the rat histamine H₃receptor a K_Dvalue of 2.72 0.34 nM

and a B_maxvalue of 2715 445 fmol mg⁻¹protein and on the

human histamine H₃receptor a K_Dvalue of 0.9 0.08 nM

and a B_maxvalue of 632 52 fmol mg⁻¹protein.

Competitive radioligand binding – the affinity of 1a, 1c,

1d, histamine, and thioperamide – the reference compound,

was determined by measuring the displacement curves of

[³H]-N^α-methylhistamine binding on the rat histamine H₃re-

ceptor expressed in HEK-293T membranes. Derivatives 1c

and 1a possess a high nanomolar affinity for the rat hista-

mine H₃receptor with a pK_ivalue of 7.9 0.1 and 7.8 0.1,

respectively. Slightly lower affinity is seen for compound 1d

with a pK_ivalue of 7.6 0.1 in comparison with the pK_iof

thioperamide (pKi value = 7.9 0.1). All the investigated com-

pounds show slightly higher affinity than histamine (pK_i=

7.3 0.1). A representative graph of the competitive binding

of histamine H₃receptor ligands on the rat histamine H₃re-

ceptor can be found in Appendix B (subsection 2.2.2.1; Fig.

S4): ESI.‡

2.2.2.2. Competitive binding of human H₃and H₄receptor

ligands. Previously, compounds 1a, 1c, and 1d, and

thioperamide – the reference compound – were investigated

for human histamine H₃and H₄receptor in vitro binding

studies.³³[³H]N^α-methylhistamine was used as the

radioligand in binding assays to test the affinity to the human

recombinant histamine H₃receptor stably expressed in

HEK293 cells³⁴(the experimentally determined radioligand

K_Dvalue of 0.34 nM was used). [³H]histamine was used as the

radioligand in binding assays to test the affinity on

membrane preparations from CHO cells, expressing the

recombinant human histamine H₄receptor³⁵(the

experimentally determined radioligand K_Dvalue of 9.70 nM

was used). Pharmacological results are presented in Table 1.

Based on preliminary functional pharmacological studies,

subsection 2.2.1.1, the radioligand binding studies confirmed

the affinity of the investigated derivatives 1a, 1c, and 1d with

the human histamine H₃receptor (Table 1). All the investi-

gated compounds showed good affinities for the human his-

tamine H₃receptor in a pK_irange of 6.84–6.56 in comparison

with the pK_iof thioperamide (K_ivalue 6.75). The competitive

radioligand binding assay on hH₄R revealed weak selectivity

towards the human histamine H₃receptor for derivatives 1a

and 1d carrying a substituent at position 4 in the N-benzyl-

guanidine moiety. Significant selectivity towards the

receptor was seen for compounds 2a, 2c, 2d and 2f (pA₂

=

7.19, pA₂= 7.10, pA₂= 7.18, and pA₂= 7.22, respectively;

Table 1). A similar potency for the histamine H₁receptors

and histamine H₃receptors was observed for compound 2e

(H₁pA₂= 6.97, H₃pA₂= 7.30) (Table 1). In the case of

compound 2h (where the butylpiperazine fragment was

removed) the antagonistic activity against the H₁receptor

was higher (pA₂= 6.8) than that against the histamine H₃

receptor (pA₂= 5.78; Table 1). No activity for the H₁receptor

was observed for derivative 2g only, but a drastic reduction of

potency was observed for the histamine H₃receptor (pA₂

=

5.78), compared to the parent compound 1c (pA₂= 8.21). All

the graphs of antagonism by 2a–h and pyrilamine and the

inhibitory effect of histamine on the contraction of guinea-

pig ileum strips are shown in Appendix B (subsection 2.2.2;

Fig. S2): ESI.‡

2.2.2. Histamine H₃and H₄receptor affinity. Taking into

consideration the potency of the newly synthesized com-

pounds 2a–h, the parent compounds 1a, 1c, and 1d demon-

strated significantly higher potency. For this reason, deriva-

tives 1a, 1c, and 1d were evaluated for their affinity to the

recombinant rat histamine H₃receptor (rH₃R) and human

histamine H₃and H₄receptors (hH₃R and hH₄R).

2.2.2.1. Histamine H₃receptor affinity studies. The affinity

of the aforementioned compounds was evaluated by

measuring

the

displacement

curve

of

[³H]-N^α-

methylhistamine on the rat (rH₃R) histamine H₃receptor in

HEK-293T cell membranes as described by Bongers.³²

2.2.2.1.1. Competitive binding of rat histamine H₃receptor

ligands. Saturation of the rat histamine H₃receptor –

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histamine H₃receptor was shown for unsubstituted N-benzyl-

guanidine derivative 1c (Table 1). This compound also showed

higher affinity to the human histamine H₃receptor with a pK_i

value of 6.80 compared with thioperamide (pK_ivalue = 6.75).

2.2.3. Verification of in vivo activity for compounds 1a, 1c

and 1d. Furthermore, derivatives 1a, 1c, and 1d were

subjected to preliminary in vivo evaluation of their impact on

brain neurotransmitter systems. This assessment concerned:

- The effects of the compounds on the feeding behavior of

rats after their repeated peripheral administration.

- The influence on the cerebral amine neurotransmitter

concentrations, as well as the activity of monoamine oxidases

A and B and histamine N-methyltransferase. The latter was

accomplished by postmortem analyses of the brain tissue of

the treated rats.

Derivatives 1a, 1c and 1d were chosen for the aforemen-

tioned studies and not one of the latest compounds, as for

this class of non-imidazole antagonists so far no proof of in

vivo activity has been reported. As such, we have chosen the

best compounds available for the in vivo studies.

In accordance with the earlier reported experimental

data,^36–38it was assumed that if a histamine H₃receptor an-

tagonist subcutaneously injected into rats crossed the blood–

brain barrier (BBB), it would affect the animal food consump-

tion. To evaluate the central effects of peripheral administra-

tion of 1a, 1c, and 1d to rats, the feeding behavior of rats was

monitored after each drug administration.

As presented in Fig. 2, treatment of rats with either of the

four histamine H₃receptor antagonists 1a (3 mg kg⁻¹s.c.), 1c

(3 mg kg⁻¹s.c.) and 1d (3 mg kg⁻¹s.c.) and ciproxifan (3 mg

kg⁻¹s.c. – based on the literature data³⁹the dose 3 mg kg⁻¹

was selected to evaluate the central effects of peripheral ad-

ministration of derivatives 1a, 1c, and 1d to rats on their

feeding behavior) over a period of five days evoked a statisti-

cally significant reduction in the amount of consumed food

by rats compared to the pre-treatment period.

Fig. 2 The effect of the newly synthesized histamine H₃receptor

antagonists 1a (s.c. 3 mg kg⁻¹daily for 5 days), 1c (s.c. 3 mg kg⁻¹daily

for 5 days), and 1d (s.c. 3 mg kg daily for 5 days) on food consumption.

Ciproxifan (s.c. 3 mg kg⁻¹daily for 5 days), a histamine H₃-receptor

antagonist/inverse agonist, was used as a reference compound. The

values are mean sem. All white bars correspond respectively to food

consumed during the 3-day pre-test, whereas bars with patterns cor-

respond to food consumed during the 5-day treatment with appropri-

ate drugs. Paired t-test, *p < 0.05, **p < 0.01, ***p < 0.001 vs. before

treatment.

In Fig.

2 the effect of five-day treatment versus

pretreatment is presented as related to the consumed food. It

can be inferred from these data that all the tested histamine

H₃receptor ligands have entered the brain for they signifi-

cantly modified the feeding pattern, with compound 1d

exhibiting a potency similar to that of ciproxifan.

A statistically significant decline in food consumption was

noted in rats treated with all the compounds examined

(Fig. 1 and 2); 1d, being the most effective, caused a total of

13.5% reduction in food consumption compared with the re-

sults recorded before administration of the drug (7.54 0.17

vs. 6.52 0.13 g per 100 g of body weight) (Fig. 2, p < 0.001).

This effect was most noticeable on the 5th day (Fig. 1) and

was similar to that caused by ciproxifan (Fig. 1 and 2). 1a de-

creased the pellet consumption to a lesser extent by 10%, i.e.

7.42 0.22 vs. 6.73 0.17 g per 100 g of body weight, P =

0.0205 (Fig. 2). The lowest reduction in food consumption

was noted for compound 1c.

2.2.3.1. Post-mortem biochemical analysis of the brain

tissues of 1a, 1c, and 1d treated rats. To find out whether

there are any post-treatment alterations in the cerebral amine

neurotransmitter systems, after the last record was taken in

metabolic cages the rats were sacrificed and their brains were

collected to measure the amine concentrations by RIA or

ELISA (DIAsource ImmunoAssays S.A., Nivelles, Belgium), as

well as the histamine N-methyltransferase and monoamine

oxidase (MAO) activities by radioisotopic assays.

None of the tested compounds influenced the histamine

or noradrenaline concentrations in the rat cortex. However,

cortical concentrations of serotonin and dopamine were sig-

nificantly lower in rats administered with compounds 1a and

1c, respectively, when compared to the concentrations mea-

sured in ciproxifan-treated rats. The serotonin concentration

Based on in vitro and in vivo studies in the literature,⁴⁰

ciproxifan was shown as a pure competitive antagonist of the

histamine H₃receptor. Therefore, ciproxifan was used for the

in vivo studies, instead of thioperamide because the in vivo

potency of thioperamide is rather low compared with it's

in vitro potency, which can result in restricted drug bioavail-

ability, particularly its brain penetration.

Male Wistar or Lewis rats were used to verify the in vivo ef-

ficacy of the selected histamine H₃receptor ligands. Four

days before the experiment, the rats were placed individually

in metabolic cages (Tecniplast Gazzada, Italy) with free access

to feed and water. After a one-day adaptation period to the

new conditions, food consumptions were monitored daily,

between 8:00–8:30 a.m., for the next three days and the

means related to the pretreatment were calculated. Each of

the tested compounds was given in a dose of 3 mg kg⁻¹per

day, subcutaneously, for five days. Ciproxifan was employed

as the reference. Every morning, the consumed food was

recorded. Fig. 1 illustrates a decrease in the daily food intake

upon the repeated drug administration.

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was roughly one third and the dopamine concentration two-

thirds of those obtained in ciproxifan-treated rats. A repre-

sentative graph of cerebral amine neurotransmitter concen-

tration in rats subchronically treated with the newly synthe-

sized 1a, 1c, and 1d histamine H₃receptor antagonists and

the reference compound ciproxifan can be found in Appendix

B (subsection 2.2.3.1; Fig. S5): ESI.‡

The activities of the brain catabolic enzymes have not

been affected by the tested histamine H₃receptor ligands.

Clearly, the MAO enzymes do not contribute to the changes

in serotonin or dopamine concentrations resulting from the

treatment with 1a, 1c, and 1d. The effect of subchronic ad-

ministration of 1a, 1c and 1d (s.c. 3 mg kg⁻¹daily for 5 days)

and ciproxifan (s.c. 3 mg kg⁻¹daily for 5 days) on cerebral

MAO and HNMT activities can be found in Appendix B (sub-

section 2.2.3.1; Table S1): ESI.‡

highest histamine H₃receptor selectivity was seen for com-

pound 1c; ratio K_iH₄/K_iH₃= 20.9.

Compounds 1a, 1c and 1d, given parenterally for five days,

reduced the food intake of rats. Of the tested compounds,

the best turned out to be 1d which had a similar potency to

ciproxifan in terms of the food consumption decrease; a

somewhat lower effect was found for 1c and a lower one for

1a. Post-mortem biochemical analysis of the brain tissues of

1a, 1c, and 1d treated rats indicates that none of the tested

compounds influenced the brain histamine or noradrenaline

concentrations. However, significantly reduced serotonin and

dopamine concentrations were found in the rats adminis-

tered with compounds 1a and 1c, respectively.

The high potency and affinity for H₃receptors and the

in vivo activity warrant further study on compounds 1a and

1c.

3. Experimental section

General methods

Conclusions

Of both series (1a–d and 2a–h), the 1-substituted-1-{4-[4-(7-

phenoxyalkyl)piperazin-1-yl]butyl}guanidine derivatives 1a (pA₂

= 8.43) and 1b (pA₂= 8.49) carrying electron-withdrawing sub-

stituents at position 4 in the benzene ring demonstrated the

highest in vitro potency as histamine H₃receptor antagonists.

For 1a and 1b, the in vitro potency was found to slightly de-

crease when the piperazine scaffold was replaced by a piperi-

dine ring (2a – pA₂= 7.90 and 2b – pA₂= 8.35). In the pipera-

zine series, transfer of the benzyl- and 4-trifluoromethylbenzyl

substituents from the N¹to the N³position of the guanidine

All melting points (mp) were measured in open capillaries on

electrothermal apparatus and are uncorrected. All ¹H NMR

and ¹³H NMR spectra were recorded on Varian Mercury VX

300 MHz and Bruker Avance III 600 MHz (¹H NMR spectra

were run at 600 MHz, while ¹³C NMR spectra were run at

150.95 MHz) spectrometers in deuterated dimethyl sulfoxide

(DMSO-d₆), chloroform-d and deuterium oxide. Chemical

shifts are expressed in ppm downfield from internal TMS as

a reference. ¹H NMR data are reported in the order: multi-

plicity (br, broad; s, singlet; d, doublet; t, triplet; q, quartet;

qt, quintet; m, multiplet; *, exchangeable by deuterium ox-

ide), number of protons, and approximate coupling constant

in Hertz. Elemental analysis (C, H, and N) for all compounds

was conducted on a Perkin Elmer Series II CHNS/O Analyzer

2400 and results are 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 on silica gel

60 Å 50 mm (J. T. Baker B. V.) and Normasil 60 silica gel 40–

63 μm (VWR Chemicals), employing the eluent indicated by

TLC.

moiety resulted in further decreases in potency (2c – pA₂

=

8.10 and 2d – pA₂= 7.97). Decreasing the guanidine basicity

following the introduction of a nitrile substituent into the N²

nitrogen derivative 1c resulted in compound 2e, showing the

lowest potency (pA₂= 7.3) within the series of compounds

2a–e. In the series of compounds 2f–h, where the synthons:

phenoxyheptyl-,

trifluoromethylbenzylguandinbutyl-,

and

1-piperazinynylobutyl- were removed from the structure of the

parent compound 1b, only bisguanidine derivative 2f showed

a similar potency (pA₂= 7.99) to 2d (pA₂= 7.97). Compounds

2g and 2h showed weak potency (pA₂= 5.78, pA₂= 5.78, re-

spectively) for the histamine H₃receptor. All the newly

obtained compounds 2a–h (pA₂from 6.90 to 7.22) exhibited

higher potency for the H₁receptor than the lead compounds

1a–d (pA₂from 5.85 to 6.7). Competitive radioligand binding

studies on the rat histamine H₃receptors for compounds 1a,

1c and 1d showed an affinity (pK_ifrom 7.9 to 7.6, respec-

tively) on a comparable level to the reference compound –

thioperamide (pK_i= 7.9), confirming the high potency shown

by the tested compounds in the functional assay in the

guinea pig jejunum. Additionally, competitive radioligand

binding studies on the human histamine H₃receptor also

showed good affinity for these compounds with a pK_ivalue

ranging from 6.84 to 6.56 while a pK_ivalue of 6.75 was found

for thioperamide. The competitive radioligand binding assay

on the H₄receptor revealed weak selectivity towards the hu-

man histamine H₃receptor for compounds 1a and 1d. The

2-(6-Bromohexyloxy)tetrahydro-2H-pyran,

di-tert-butyl

dicarbonate, tetrabromomethane, 4-bromobutyronitrile, 4 M

solution hydrogen chloride in dioxane, 4-(trifluoromethyl)-

benzoyl chloride, 4-piperidine butyric acid hydrochloride,

benzyl bromide, 4-(trifluoromethyl)benzyl bromide, and piper-

azine were purchased from commercial suppliers (Aldrich,

TCI, Fluorochem, Fluka) and used without further

purification.

N-Benzyl-4-(4-(7-phenoxyheptyl)piperazin-1-yl)butan-1-amine

was prepared according to Staszewski and Walczyński.²⁹

3.1. Chemistry

General procedure for the preparation of guanidines 2a,

2b, 2c, 2d, 2f and 2h. To a solution of the appropriate 2,3-

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bisĲtert-butoxycarbonyl)guanidine (2a.11, 2b.7, 2c.2, 2d.2, 2f.5

and 2h.5) (1 eq.) in chloroform, a 4 M solution of hydrogen

chloride–dioxane (20 eq.) was added dropwise. The reaction

was stirred overnight at room temperature and then the sol-

vent was removed under vacuum. The crude product was

evaporated twice with chloroform and twice with EtOAc, then

recrystallized from dry ethanol or methanol to yield the pure

product. In the case of compound 2h, the following proce-

dure was used: the crude product was evaporated twice from

chloroform and twice from EtOAc then evaporated, dissolved

in methanol, alkalized by 5% sol. of sodium hydroxide,

extracted with DCM and dried over Na₂SO₄. The crude prod-

uct was dissolved in methanol and 1 eq. fumaric acid was

added. After 30 minutes the solvent was evaporated, then the

product was recrystallized from dry 2-propanol to yield the

pure product.

1.26 (m, 13H: 3H^Piperidine, 10H^alif., CHCH₂, CH₂CH₂CH₂). ¹³C

NMR (600 MHz, DMSO) δ ppm 158.36 (1C^{quat./phenoxy.}, CO), 156.79

(1C^quat., CNH), 139.63 (1C^quat./arom., CCH₂), 129.97 (2C^phenoxy.

,

C(CHCH)₂CH), 129.77, 129.55, 129.34, 129.11 (1C^arom, CCF₃),

127.43 (2C^arom., (CHCH)₂CCF₃), 126.98, 125.18, 123.38, 121.75

(1C, CF₃), 125.92, 125.89 (2C^arom., (CH)₂CCF₃), 121.75 (1C^phenoxy.

,

C(CHCH)₂CH), 115.03 (2C^phenoxy., C(CHCH)₂CH), 68.55 (1C,

PhOCH₂), 57.07 (1C, CH₂N^piperidine), 52.94 (2C^piperidine

,

CHĲCH₂CH₂)₂N), 51.81 (1C, CH₂Ph), 49.56 (1C, CH₂CH₂NC(N)),

34.43 (1C, CH₂CH₂CH₂), 32.74 (1C, CH₂CH₂CH₂), 29.20

(2C^piperidine, CHĲCH₂CH₂)₂N), 28.34 (1C^piperidine, CHĲCH₂CH₂)₂N),

27.86 (1C, CH₂CH₂CH₂), 26.61 (1C, CH₂CH₂CH₂), 25.74 (1C,

CH₂CH₂CH₂), 24.94 (1C, CH₂CH₂CH₂), 23.27 (1C, CH₂CH₂CH₂),

22.79 (1C, CH₂CH₂CH₂). Anal. calcd: C, 59.23; H, 7.70; N, 8.91.

Found: C, 59.47; H, 7.87; N, 8.89.

2c – 1-benzyl-3-{4-[4-(7-phenoxyheptyl)piperazin-1-yl]butyl}-

2a

–

1-{4-[4-(7-phenoxyheptyl)piperidin-1-yl]butyl}-1-(4-

dihydrochloride.

guanidine trihydrochloride. C₂₉H₄₅N₅O·3HCl·0.5H₂O. M =

(trifluoromethyl)-benzyl)guanidine

C₃₁H₄₅F₃N₄O·2HCl. M = 619.65. White solid. 89.1% yield. mp:

598.10. White solid. 95.3% yield. mp: 218.6–220.6 °C with

decomposition. ¹H NMR (600 MHz, D₂O) δ ppm 7.52–7.50 (m,

1

159.6–161.5 °C. H NMR (600 MHz, D₂O) δ ppm 7.82–7.81 (m,

2H^arom.), 7.46–7.42 (m, 5H: 2H^phenoxy., C(CHCH)₂CH, 3H^arom.

,

2H^arom., (CH)₂CCF₃), 7.52–7.51 (m, 2H^arom., (CHCH)₂CCF₃),

C(CHCH)₂CH), 7.12–7.07 (m, 3H^phenoxy., C(CHCH)₂CH), 4.52 (s,

2H, CH₂Ph), 4.15–4.13 (t, 2H, OCH₂, J = 6.48 Hz), 3.70 (br,

8H^piperazine), 3.34–3.31 (m, 6H, CH₂N^piperazine, CH₂CH₂NC(N)),

1.84–1.76 (m, 6H, CH₂CH₂CH₂), 1.72–1.67 (qt, 2H, CH₂CH₂CH₂),

1.53–1.46 (m, 6H, CH₂CH₂CH₂). ¹³C NMR (600 MHz, DMSO) δ

ppm 158.54 (1C^{quat./phenoxy.}, CO), 155.86 (1C^quat., CNH), 137.24

(1C^quat./arom., CCH₂), 129.35 (2C^phenoxy., C(CHCH)₂CH), 128.41

(2C^arom., C(CHCH)₂CH), 127.28 (1C^arom., C(CHCH)₂CH), 127.06

(2C^arom., C(CHCH)₂CH), 120.25 (1C^phenoxy., C(CHCH)₂CH), 114.32

7.44–7.41 (m, 2H^phenoxy., C(CHCH)₂CH), 7.10–7.07 (m, 3H^phenoxy.

,

C(CHCH)₂CH), 4.73 (s, 2H, CH₂Ph), 4.15–4.13 (t, 2H, OCH₂, J =

6.52 Hz), 3.52–3.47 (m, 4H: 2H^alif.CH₂CH₂NC(N), 2H^piperidine

CHĲCH₂CH₂)₂N), 3.06–3.04 (m, 2H, CH₂N^piperidine), 2.88–2.84

(m, 2H^piperidine, CHĲCH₂CH₂)₂N), 1.99–1.97 (m, 2H^piperidine

,

CHĲCH₂CH₂)₂N), 1.83–1.78 (qt, 2H, OCH₂CH₂), 1.71–1.70 (m,

4H, NCH₂CH₂CH₂CH₂N), 1.57–1.55 (m,

1H^piperidine

,

CHĲCH₂CH₂)₂N), 1.51–1.46 (m, 2H, OCH₂CH₂CH₂), 1.43–1.30

(m, 10H^alif.: CHCH₂, CH₂CH₂CH₂). ¹³C NMR (600 MHz, DMSO)

δ ppm 158.54 (1C^{quat./phenoxy.}, CO), 156.55 (1C^quat., CNH),

140.79 (1C^quat./arom., CCH₂), 129.33 (2C^phenoxy., C(CHCH)₂CH),

(2C^phenoxy.

, C(CHCH)₂CH), 67.14 (1C, PhOCH₂), 55.52 (1C,

CH₂N^piperazine), 55.12 (1C, CH₂N^piperazine), 47.83 (4C^piperazine),

43.89 (1C, NCH₂Ph), 40.15 (1C, CH₂CH₂NC(N)), 28.47 (1C,

CH₂CH₂CH₂), 28.09 (1C, CH₂CH₂CH₂), 25.81 (1C, CH₂CH₂CH₂),

25.53 (1C, CH₂CH₂CH₂), 25.19 (1C, CH₂CH₂CH₂), 22.79 (1C,

CH₂CH₂CH₂), 20.15 (1C, CH₂CH₂CH₂). Anal. calcd: C, 58.24; H,

8.26; N, 11.71. Found: C, 58.58; H, 8.61; N, 11.63.

128.35, 128.14, 127.93, 127.72 (1C^arom, CCF₃), 127.36 (2C^arom.

,

(CHCH)₂CCF₃), 126.81, 125.01, 123.20, 121.40 (1C, CF₃), 125.43,

125.41 (2C^arom., (CH)₂CCF₃), 120.27 (1C^phenoxy., C(CHCH)₂CH),

114.29 (2C^phenoxy., C(CHCH)₂CH), 67.15 (1C, PhOCH₂), 55.32

(1C, CH₂N^piperidine), 51.71 (1C^piperidine, CHĲCH₂CH₂)₂N), 51.70

(1C^piperidine, CHĲCH₂CH₂)₂N), 50.29 (1C, NCH₂Ph), 47.86 (1C,

2d

–

1-{4-[4-(7-phenoxyheptyl)piperazin-1-yl]butyl}-3-[4-

trihydrochloride.

(trifluoromethyl)-benzyl]guanidine

CH₂CH₂NC(N)), 35.19 (1C, OĲCH₂)₃CH₂), 32.83 (1C^piperidine

CHĲCH₂CH₂)₂N), 28.94 (1C^piperidine, CHĲCH₂CH₂)₂N), 28.67 (1C,

CH₂CH₂CH₂), 28.57 (3C: 1C^piperidine, CHĲCH₂CH₂)₂N, 2C^alif.

CH₂CH₂CH₂), 25.69 (1C, OĲCH₂)₂CH₂), 25.38 (1C, CH₂CH₂CH₂),

24.05 (1C, CH₂CH₂NC(N)), 20.02 (1C, CH₂CH₂CH₂). Anal. calcd:

C, 60.09; H, 7.65; N, 9.04. Found: C, 59.69; H, 8.05; N, 9.06.

,

C₃₀H₄₄F₃N₅O·3HCl. M = 657.10. Light yellow solid. 90.9% yield.

mp: 227.4–229.4 °C with decomposition. ¹H NMR (600 MHz,

D₂O) δ ppm 7.79–7.78 (m, 2H^arom.), 7.57–7.55 (m, 2H^arom.), 7.44–

,

7.41 (m, 2H^phenoxy., C(CHCH)₂CH), 7.11–7.08 (m, 1H^phenoxy.

,

C(CHCH)₂CH), 7.06–7.05 (m, 2H^phenoxy., C(CHCH)₂CH), 4.59 (s,

2H, CH₂Ph), 4.11–4.09 (t, 2H, OCH₂, J = 6.50 Hz), 3.72 (br,

8H^piperazine), 3.36–3.31 (m, 6H: CH₂N^piperazine, CH₂CH₂NC(N)),

1.85–1.76 (m, 6H, CH₂CH₂CH₂), 1.73–1.68 (qt, 2H,

CH₂CH₂CH₂), 1.50–1.44 (m, 6H, CH₂CH₂CH₂). ¹³C NMR (600

2b

(trifluoro

–

1-{4-[1-(7-phenoxyheptyl)piperidin-4-yl]butyl}-1-[4-

methyl)-benzyl]guanidine dihydrochloride.

C₃₁H₄₅F₃N₄O·2HCl·0.5H₂O. M = 628.66. White solid. 96.6% yield.

1

mp: 88.2–90.2 °C. H NMR (600 MHz, D₂O) δ ppm 7.81–7.79 (m,

MHz, DMSO) δ ppm 158.52 (1C^{quat./phenoxy.}, CO), 155.94 (1C^quat.

,

2H^arom., (CHCH)₂CCF₃), 7.53–7.52 (m, 2H^arom., (CHCH)₂CCF₃),

CNH), 142.28 (1C^quat./arom.

,

CCH₂), 129.31(2C^phenoxy.

,

7.43–7.40 (m, 2H^phenoxy., C(CHCH)₂CH), 7.09–7.05 (m, 3H^phenoxy.

,

C(CHCH)₂CH), 128.15, 127.94, 127.73, 127.52 (1C^arom., CCF₃),

127.70 (2C^arom., (CHCH)₂CCF₃), 126.83, 125.03, 123.23, 121.42

C(CHCH)₂CH), 4.78 (s, 2H, CH₂Ph), 4.12–4.09 (t, 2H, OCH₂, J =

6.46 Hz), 3.52–3.50 (m, 2H^Piperidine, CHĲCH₂CH₂)₂N), 3.46–3.44

(m, 2H, CH₂CH₂NC(N)), 3.07–3.04 (m, 2H, CH₂N^piperidine), 2.82–

(1C, CF₃), 125.22, 125.20 (2C^arom., (CH)₂CCF₃), 120.21 (1C^phenoxy.

,

C(CHCH)₂CH), 114.29 (2C^phenoxy.

, C(CHCH)₂CH), 67.11(1C,

2.78 (m, 2H^Piperidine, CHĲCH₂CH₂)₂N), 1.91–1.88 (m, 2H^Piperidine

,

PhOCH₂), 55.48 (1C, CH₂N^piperazine), 55.06 (1C, CH₂N^piperazine),

47.78 (4C^pip.), 43.36 (1C, NCH₂Ph), 40.16 (1C, CH₂CH₂NC(N)),

28.44 (1C, CH₂CH₂CH₂), 28.06 (1C, CH₂CH₂CH₂), 25.79 (1C,

CHĲCH₂CH₂)₂N), 1.83–1.79 (qt, 2H, OCH₂CH₂), 1.75–1.70 (m,

2H, CH₂CH₂N^piperidine), 1.66–1.61 (m, 2H, CH₂CH₂NC(N)), 1.52–

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CH₂CH₂CH₂), 25.52 (1C, CH₂CH₂CH₂), 25.16 (1C, CH₂CH₂CH₂),

22.72 (1C, CH₂CH₂CH₂), 20.10 (1C, CH₂CH₂CH₂). Anal. calcd: C,

54.84; H, 7.21; N, 10.66. Found: C, 54.91; H, 7.37; N, 10.61.

2f – 1,4-bis{4-[1-(4-trifluoromethylbenzyl)guandin-1-yl]but-1-

yl}piperazine tetrahydrochloride. C₃₀H₄₂F₆N₈·4HCl·1.5H₂O. M =

801.58. White solid. 91.60% yield. mp: 261.2–263.4 °C with

decomposition. ¹H NMR (600 MHz, D₂O) δ ppm 7.82–7.81

(m, 4H^arom.), 7.52–7.51 (m, 4H^arom.), 4.80 (s, 4H, CH₂Ph),

3.69–3.68 (m, 8H^piperazine), 3.54–3.53 (m, 4H, CH₂CH₂N),

3.31–3.29 (m, 4H, CH₂CH₂N), 1.79–1.78 (m, 8H, CH₂CH₂CH₂).

¹³C NMR (600 MHz, D₂O) δ ppm 156.67 (2C^quat., CNH),

139.04 (2C^quat./arom., CCH₂), 129.68, 129.47, 129.26, 129.05

6H, CH₂CH₂CH₂). ¹³C NMR (600 MHz, D₂O) δ ppm 158.19

(1C^{quat./phenoxy.}, CO), 155.89 (1C^quat., CNH), 153.94 (1C^quat.

,

CN), 133.78 (1C^quat./arom.

,

CCH₂), 129.98 (2C^phenoxy.

C(CHCH)₂CH), 128.44

C(CHCH)₂CH), 129.26 (1C^arom.

,

(2C^arom.), 126.88 (2C^arom.), 121.53 (1C^phenoxy., C(CHCH)₂CH),

115.07 (2C^phenoxy., C(CHCH)₂CH), 68.65 (1C, PhOCH₂), 57.07

(1C, CH₂N^piperazine), 56.27 (1C, CH₂N^piperazine), 52.15 (1C,

NCH₂Ph), 48.68, 48.63 (5C: 4C^piperazine., CH₂CH₂NC(N)), 28.22

(1C, CH₂CH₂CH₂), 27.81 (1C, CH₂CH₂CH₂), 25.49 (1C,

CH₂CH₂CH₂), 24.92 (1C, CH₂CH₂CH₂), 23.39 (1C,

CH₂CH₂CH₂), 23.26 (1C, CH₂CH₂CH₂), 20.72 (1C,

CH₂CH₂CH₂). Anal. calcd: C, 56.20; H, 7.86; N, 13.11. Found: C,

56.37; H, 8.11; N, 13.05.

(2C^arom.

, , (CHCH)₂CCF₃), 126.75,

CCF₃), 126.93 (4C^arom.

124.96, 123.16, 121.36 (2C, CF₃), 125.86, 125.84 (4C^arom.

,

Synthesis of 1,4-bisĲ7-phenoxyheptyl)piperazine dihydro-

chloride 2g. A 4 M solution of hydrogen chloride–dioxane (3

(CH)₂CCF₃), 56.24 (2C, CH₂CH₂N), 56.23 (2C, CH₂CH₂N),

51.46 (2C, CH₂PhCF₃), 48.59, 48.56 (4C^piperazine), 23.51 (2C,

CH₂CH₂CH₂), 20.63 (2C, CH₂CH₂CH₂). Anal. calcd: C, 44.95;

H, 6.16; N, 13.98. Found: C, 45.28; H, 6.34; N, 13.65.

eq.) was added dropwise to

a solution of 1,4-bisĲ7-

phenoxyheptyl)piperazine (2g.1) (1 eq.) in chloroform. The re-

action was stirred overnight at room temperature, then the

solvent was removed under vacuum. The crude product was

evaporated twice with chloroform and twice with EtOAc, then

recrystallized from dry methanol to yield the pure product.

2h

– 1-benzyl-1-(7-phenoxyhept-1-yl)guanidine fumarate.

C₂₁H₂₉N₃O·C₄H₄O₄. M = 455.54. White solid. 40.6% yield. mp:

103.4–105.0 °C. ¹H NMR (600 MHz, CD₃OD) δ ppm 7.40–7.38

(m, 2H^arom.

,

C(CHCH)₂CH), 7.33–7.31 (m, 1H^arom.

,

2g – 1,4-bisĲ7-phenoxyheptyl)piperazine dihydrochloride.

C(CHCH)₂CH), 7.26–7.22 (m, 4H: 2H^phenoxy., C(CHCH)₂CH),

2H^arom., C(CHCH)₂CH, 6.90–6.87 (m, 3H^phenoxy., C(CHCH)₂CH),

6.69 (s, 2H^fum., CH), 4.62 (s, 2H, PhCH₂), 3.96–3.94 (t, 2H, OCH₂,

J = 6.37 Hz), 3.34–3.32 (t, 2H, CH₂CH₂C(N)N), 1.77–1.72 (qt, 2H,

CH₂CH₂CH₂), 1.65–1.59 (qt, 2H, CH₂CH₂CH₂), 1.49–1.44 (qt,

2H, CH₂CH₂CH₂), 1.40–1.30 (m, 4H, CH₂CH₂CH₂). ¹³C NMR

C₃₀H₄₆N₂O₂·2HCl. M = 539.64. White solid. 82.6% yield. mp:

244.0–246.0 °C with decomposition. ¹H NMR (600 MHz, CD₃OD)

δ ppm 7.26–7.23 (m, 4H^phenoxy., C(CHCH)₂CH), 6.91–6.88 (m,

6H^phenoxy., C(CHCH)₂CH), 3.99–3.97 (t, 4H, OCH₂, J = 6.33 Hz),

3.90–3.31 (br, 8H^piperazine), 3.19 (m, 4H, CH₂N^piperazine), 1.81–

1.77 (qt, 8H, CH₂CH₂CH₂), 1.56–1.51 (qt, 4H, CH₂CH₂CH₂),

1.49–1.46 (m, 8H, CH₂CH₂CH₂). ¹³C NMR (600 MHz, CD₃OD) δ

(600 MHz, CD₃OD)

δ ppm 171.17 (2C, CO), 160.55

(1C^{quat./phenoxy.}, CO), 158.38 (1C^quat., CN), 136.42 (1C^quat./arom.),

136.12 (2C, CC), 130.39, 130.05 (4C^arom., C(CHCH)₂CH),

ppm 160.54 (2C^{quat./phenoxy.}

,

CO), 130.41 (4C^phenoxy.

C(CHCH)₂CH), 115.53

,

C(CHCH)₂CH), 121.57 (2C^phenoxy.

,

129.14

(1C^arom.

,

C(CHCH)₂CH),

128.06

(2C^phenoxy.

,

(4C^phenoxy., C(CHCH)₂CH), 68.74 (2C, OCH₂), 57.62 (2C^piperazine),

57.48 (2C,CH₂N^piperazine), 57.33 (2C^piperazine), 30.25 (2C,

CH₂CH₂CH₂), 29.84 (2C, CH₂CH₂CH₂), 27.49 (2C, CH₂CH₂CH₂),

26.93 (2C, CH₂CH₂CH₂), 25.12 (2C, CH₂CH₂CH₂). Anal. calcd: C,

66.77; H, 8.97; N, 5.19. Found: C, 66.38; H, 9.35; N, 5.16.

C(CHCH)₂CH), 121.55 (1C^phenoxy.

,

C(CHCH)₂CH), 115.53

(2C^phenoxy., C(CHCH)₂CH), 68.78 (1C, OCH₂), 52.67 (1C, PhCH₂),

49.87 (1C, CH₂CH₂C(N)N), 30.25 (1C, CH₂CH₂CH₂), 30.04 (1C,

CH₂CH₂CH₂), 28.07 (1C, CH₂CH₂CH₂), 27.39 (1C, CH₂CH₂CH₂),

26.99 (1C, CH₂CH₂CH₂). Anal. calcd: C, 64.64; H, 7.38; N, 9.05.

Found: C, 64.86; H, 7.09; N, 9.12.

Synthesis of 1-benzyl-3-cyano-1-{4-[4-(7-phenoxyheptyl)-

piperazin-1-yl]but-1-yl}guanidine trihydrochloride 2e. To a so-

lution of 1-benzyl-3-cyano-1-[4-[4-(7-phenoxyheptyl)piperazin-1-

yl]butyl]guanidine (2e.2) (1 eq.) in methanol, 16% w/

w solution of hydrogen chloride–methanol (4 eq.) was added

dropwise. The reaction was stirred for 1 hour at room tem-

perature and then the solvent was removed under vacuum.

The crude product was evaporated twice from EtOAc, then

recrystallized from dry ethanol to yield the pure product.

3.2. In vitro pharmacology

All experiments with animals (guinea pigs and rats) were

performed in compliance with the Polish legislation

concerning the protection and welfare of animals used for

scientific purposes and followed the university guidelines. All

animal procedures have been approved by the 9 Local Ethical

Committee for Animal Experiments in Lodz, approval no. 60/

ŁB681/2013 and 86/ŁB696/2013, respectively. The HEK293T

cell line was kindly provided by Prof. Dr Rob Leurs, Vrije

Universiteit Amsterdam; the HEK-hH3R cell line was kindly

provided by Prof. Dr. Jean-Charles Schwartz, Bioprojet,

France; CHO cells stably expressing hH₄R were obtained from

PerkinElmer, Waltham, MA, U.S.

3.2.1. Histamine H₃receptor antagonistic activity of com-

pounds 2a–h. In the first step, all the newly synthesized com-

pounds were tested for histamine H₃receptor antagonistic ef-

fects in vitro, following standard methods, using an

electrically-contracting guinea pig jejunum.³⁰

2e

–

1-benzyl-3-cyano-1-{4-[4-(7-phenoxyheptyl)piperazin-1-

₃₀H₄₄N₆O·3HCl

yl]but-1-yl}-guanidine trihydrochloride.

C

·1.5H₂O. M = 641.13. White solid. 64.2% yield. mp: 124.4–126.4

°C. ¹H NMR (600 MHz, D₂O) δ ppm 7.47–7.44 (m, 2H^arom.),

7.42–7.39 (m, 1H^arom.), 7.37–7.35 (2H^phenoxy., C(CHCH)₂CH),

7.29–7.28 (m, 2H^arom), 7.04–6.99 (m, 3H^phenoxy., C(CHCH)₂CH),

4.75 (s, 2H, CH₂Ph), 4.07–4.05 (t, 2H, OCH₂, J = 6.49 Hz), 3.59–

3.51 (m, 10H: 8H^pip

, CH₂CH₂NC(N)), 3.25–3.19 (m, 4H:

CH₂N^piperazine), 1.76–1.71 (m, 8H, CH₂CH₂CH₂), 1.45–1.38 (m,

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Male guinea pigs weighing 300–400 g were sacrificed and

a portion of the small intestine, 20–50 cm proximal to the

ileocaecal valve (jejunum), was removed and placed in Krebs

buffer (composition (mM) NaCl 118; KCl 5.6; MgSO₄1.18;

CaCl₂2.5; NaH₂PO₄1.28; NaHCO₃25; glucose 5.5 and indo-

methacin (1 × 10⁻⁶mol L⁻¹)). Whole jejunum segments (2

cm) were prepared and mounted between two platinum

electrodes (4 mm apart) in 20 mL Krebs buffer, continuously

gassed with 95% O₂: 5% CO₂and maintained at 37 °C. Con-

tractions were recorded isotonically under 1.0 g tension with

a Hugo Sachs Hebel-Messvorsatz (Tl-2)/HF-modem (Hugo

Sachs Elektronik, Hugstetten, Germany) connected to a pen

recorder. The equilibration was performed for one hour with

washings every 10 minutes. The muscle segments were then

stimulated at a maximum between 15 and 20 volts, continu-

ously at a frequency of 0.1 Hz for a duration of 0.5 ms, with

rectangular-wave electrical pulses, delivered by a Grass Stimu-

lator S-88 (Grass Instruments Co., Quincy, USA). After 30 min

of stimulation and five minutes before adding (R)-α-

methylhistamine, pyrilamine (1 × 10⁻⁵mol L⁻¹concentration

in an organ bath) was added, and then cumulative concentra-

tion–response curves (half-log increments) of (R)-α-

methylhistamine, a histamine H₃receptor agonist, were

recorded until no further change in response was found. Five

minutes before adding the tested compounds, pyrilamine (1

× 10⁻⁵mol L⁻¹concentration in an organ bath) was added,

and after 20 minutes cumulative concentration–response

curves (half-log increments) of (R)-α-methylhistamine, an his-

tamine H₃receptor agonist, were recorded until no further

change in response was found. Statistical analysis was carried

out with Student's t-test. In all the tests, a p < 0.05 was con-

sidered statistically significant. The potency of an antagonist

is expressed by its pA₂value, calculated from the Schild³¹re-

gression analysis where at least three concentrations were

used. The pA₂values were compared with the potency of

thioperamide.

3.2.2. H₁antagonistic activity of compounds 2a–h. All the

final compounds were tested for H₁antagonistic effects

in vitro, following standard methods, using the guinea pig il-

eum.³⁰The donors were male guinea pigs (300–400 g) as men-

tioned above. The excised ileum was placed in phosphate

buffer at room temperature (pH 7.4) containing (mM) NaCl

(136.9), KCl (2.68), and NaHPO₄(7.19). The intraluminal con-

tent was flushed and segments of about 2 cm long were cut

and mounted for isotonic contractions in water mixed with 20

mL organ baths filled with oxygenated (O₂: CO₂= 95 : 5, v/v)

Krebs buffer containing (mM) NaCl (117.5), KCl (5.6), MgSO₄

(1.18), CaCl₂(2.5), NaH₂PO₄(1.28), NaHCO₃(25), glucose (5.5)

and indomethacin (1 10⁻⁶mol L⁻¹) at 37 °C under a constant

load of 0.5 g. After a 30 min equilibration period with wash-

ings every 10 min, a submaximal priming dose of histamine

(1 mM) was given and washed out (standard washing proce-

dure: 3 changes of buffer for 30 min). After washing out, the

antagonistic activity of the given compounds was measured

by recording a concentration–response curve (CRC) for hista-

mine in the presence of the tested compounds which were

added 10 min before histamine. This procedure was repeated

with higher concentrations of the compounds. The antago-

nism has a competitive nature causing a parallel shift in the

CRC. The pA₂-values were calculated according to

Arunlakshana and Schild.³¹The pA₂values were compared

with the affinity of pyrilamine.

3.2.3. Antagonist binding to the rat histamine H₃receptor

(rH₃R), human histamine H₃receptor (hH₃R), and human

histamine H₄receptor (hH₄R)

3.2.3.1. Antagonist binding to the rat histamine H₃receptor

(rH₃R). The affinities of the most active compounds 1a, 1c and

1d were evaluated by measuring the displacement curve of [³H]-

N^α-methylhistamine on the rat (rH₃R) histamine H₃receptor in

HEK-293Tcell membranes as described by Bongers.³²

Cell culture and transfection

Human embryonic kidney cells (HEK293T) were cultured in

DMEM supplemented with 10% fetal bovine serum and 100 IU

mL⁻¹penicillin and 100 μg mL⁻¹streptomycin at 37 °C and 5%

CO₂. The day prior to transfection, two million cells were

seeded in 10 cm dishes. Approximately four million cells were

transfected by the polyethyleneimine (PEI) method with 5 μg of

cDNA at a ratio of 1 : 4 (DNA : PEI). Briefly, 0.5 μg of pcDNA3-

rH₃R and 4.5 μg of empty plasmid (pcDNA3.1) were mixed with

20 μg of 25 kDa linear PEI in 500 μl of 150 mM NaCl and incu-

bated for 30 minutes at 22 °C. Meanwhile, the medium from

10 cm dishes was replaced with fresh culture medium and the

transfection mix was subsequently added dropwise to the cells

and incubated for 48 hours at 37 °C and 5% CO₂.

Crude membrane extracts

Forty-eight hours after transfection, cells were washed with

ice-cold phosphate-buffered saline (PBS) and scrapped and

the homogenate was centrifuged for 10 minutes at ∼2000g, 4

°C. The supernatant was aspirated and cell pellets were

resuspended in 1 ml of ice-cold PBS and centrifuged again

under the same conditions; the supernatant was aspirated

and the membranes were stored at −20 °C until further use.

[³H]-N^α-methylhistamine binding

Frozen cell pellets were dissolved in 50 mM Tris-HCl buffer

(pH 7.4), homogenized by sonication (40 Watt Labsonic 1510)

for 3 to 5 s and kept on ice until use. Cell homogenates were

incubated in the presence of increasing concentrations of

[³H]-NαMH (0–20 nM) in 50 mM Tris-HCl binding buffer for

saturation binding. Total and non-specific binding was deter-

mined in the absence or presence of excess non-labeled thio-

peramide (10 μM), respectively. For the competitive binding

assay, the homogenates were incubated with increasing con-

centrations of receptor ligands (10⁻¹¹to 10⁻⁴M) and ∼2.5 nM

[³H]-NαMH. All assays were incubated at 25 °C for two hours

on a shaking table (600 rpm). The reaction was terminated by

rapid filtration into 0.5% polyethyleneimine pre-soaked glass

fiber C plates (GF/C Perkin Elmer) followed by three washes

with ice-cold Tris-HCl buffer (pH 7.4 at 4 °C). The plates were

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dried for one hour at 50 °C and scintillation liquid was added

to each well (25 μl). The retained radioactivity was deter-

mined by liquid scintillation counting in a Wallac Microbeta

(Perking Elmer). Protein determination for B_maxestimation

was performed with a Pierce BCA protein assay kit and mea-

sured by spectrophotometry in a Power Wave X340 (Biot-Tek

Instruments Inc.).

post-mortem analyses of neurotransmitter systems in the

brain tissues from the treated rats.

For the first stage, food intakes were measured daily dur-

ing subchronic drug administration. The histamine H₃recep-

tor antagonists show the ability to inhibit appetite.

Male rats aged between 9 and 10 weeks old were used. In ex-

periments with compound 1a, Wistar rats were used; the Lewis

strain was employed with compounds 1c and 1d due to a short-

age of Wistar rats of the required age. Although these rat

strains differ somewhat in the cerebral amine concentrations

and enzyme activities, both responded in the same way to

ciproxifan as checked. Metabolic cages (Tecniplast, Italy) were

used to measure feed consumption. The cages have a standard-

ized size that allows the animal to move inside freely. Through-

out the adaptive period and the experimental one, access to

feed and fluid was unlimited. The illumination cycle was stable

at 12 h light on, 12 h light off. The rats were individually placed

in the cages four days before the onset of treatment to adapt to

a new environment and conditions of housing. The consump-

tion and excretion were recorded every morning and the moni-

toring continued as long as the rats stayed in metabolic cages.

The treatment ran for five days from the fifth day.

Subcutaneous injections of either 1a, 1c, 1d or ciproxifan,

which was used as a reference (3 mg kg⁻¹per day to n = 8 rats),

in the study group and physiological saline (0.2 ml per day to n =

8 rats) in the case of the control group were conducted. Records

of consumption were taken as g of feed per 100 g body mass.

3.3.1. Post-mortem biochemical analyses. Post-mortem

brain analyses included estimation of the amine neurotrans-

mitter concentrations as well as the activities of the degrada-

tive enzymes HNMT, MAO-A and MAO-B.

The rats were sacrificed 24 hours after the last injection.

The brains were collected, cerebral cortex separated

according to the Glowinski and Iversen method,³⁸immedi-

ately frozen in liquid nitrogen and kept at −80 °C until

assayed. The thawed tissue was homogenized in 3 volumes of

bi-distilled water and aliquots were further processed to esti-

mate the concentrations of the amines and activities of the

enzymes. The amine concentrations: dopamine, noradrena-

line, and serotonin were measured by RIA and histamine by

Research ELISA kits (DIAsource ImmunoAssays S.A. Louvain-

la-Neuve, Belgium). The enzyme activities of histamine

N-methyltransferase and monoamine oxidase A and B were

estimated by radioisotopic assays according to Taylor and

Snyder⁴¹and Fowler and Tipton⁴²with modifications by

Gómez et al.,⁴³respectively. For MAO-A, serotonin (5-[2-14C]-

hydroxytryptamine binoxalate) was used as a substrate and

for MAO-B, β-[ethyl-1-¹⁴C]-phenylethylamine hydrochloride

was used as a substrate; clorgyline (MAO-A) and deprenyl

(MAO-B) (fin. conc. 10⁻⁹M) were used as enzyme inhibitors.

The protein was measured by the Lowry method.⁴⁴

Chemicals

Dubelcco's modified Eagle's medium (DMEM), phosphate

buffered saline (PBS) trizma base, and polyethyleneimine so-

lution (50%, PEI) were purchased from Sigma Aldrich. Fetal

bovine serum (FBS, Bodinco BV, the Netherlands), penicillin/

streptomycin (streptomycin 10 000 IU mL⁻¹; penicillin and

10 000 μg mL⁻¹, Thermo Fischer Scientific), linear 25 kDa

polyethyleneimine (PEI, Polysciences, Warrington, PA, U.S.),

[³H]-N-α-methylhistamine (specific activity 79.7 Ci mmol⁻¹,

Perkin Elmer), thioperamide, (Abcam), and histamine (TCI).

3.2.3.2. Antagonist binding to the human histamine H₃

receptor (hH₃R). Competitive radioligand binding to cell

membranes of HEK293 cells stably expressing the human

histamine H₃receptor was conducted as presented in detail

elsewhere.³⁴[³H]-N^α-methylhistamine (1 nM) was incubated

with the cell membrane preparation in 50 mM Tris/HCl, 5

mM MgCl₂assay buffer (35 μg of protein in a single assay

tube) and serial dilutions of the tested compounds for 1 h at

25 °C with continuous shaking (400 rpm). Total radioligand

binding was measured in the presence of 2.5% DMSO and

nonspecific binding was assessed by addition of unlabeled

(R)Ĳ−)-α-methylhistamine (20 μM). The bound radioligand was

detected after filtration of the incubation mixture through

GF/B filters presoaked with 0.5% (m/v) polyethyleneimine.

For the calculation of K_ivalues, the Cheng–Prusoff equation

and experimentally determined radioligand K_Dvalue of 0.34

were used.

3.2.3.3. Antagonist binding to human hH₄R. Histamine H₄

receptor affinity was evaluated on membrane preparations

from CHO cells, expressing the recombinant human

histamine H₄receptor. Experiments were performed as

described before.³⁵In brief: cell membranes (15 μg protein

per sample) were incubated for 1 h at 25 °C with tritiated

histamine (10 nM) and serial dilutions of the evaluated

compounds in assay buffer (50 mM Tris/HCl and 5 mM

EDTA in ultrapure water, pH 7.4). Non-specific binding was

determined in the presence of 100 μM un-labeled histamine.

The bound radioligand was collected on GF/B filters

pretreated with 0.3% (m/v) polyethyleneimine and the cap-

tured radioactivity was measured by liquid scintillation

counting. Affinity values (pK_i) were calculated according to

the Cheng–Prusoff equation (K_Dfor [³H]histamine: 9.7 nM).

3.3. Verification of in vivo activity for compounds 1a, 1c, and 1d

Author contributions

The histamine H₃receptor antagonistic activity of 1a, 1c, and

1d toward the brain histamine receptors was assessed in vivo

by the intravital study of the feeding behavior and then by

K. Walczyński was responsible for the supervision and devel-

opment of the whole project.

Med. Chem. Commun.

View Article Online

MedChemComm

Research Article

M. Staszewski performed the chemical syntheses of the

newly synthesized compounds and the preliminary pharmaco-

logical studies in vitro, both on the histamine H₃receptor and

histamine H₁receptor, elaborated and described the results.

A. Stasiak performed the extended pharmacological stud-

ies in vivo, elaborated and described the results.

T. Karcz performed the human histamine H₃receptor and

human histamine H₄receptor binding affinity tests, elabo-

rated and described the results.

10 C. Pillot, J. Ortiz, A. Héron, S. Ridray, J.-C. Schwartz and

J.-M. Arrang, J. Neurosci., 2002, 22, 7272–7280.

11 K. Takahashi, H. Suwa, T. Ishikawa and H. Kotani, J. Clin.

Invest., 2002, 110, 1791–1799.

12 H. Stark, M. Kathmann, E. Schlicker, W. Schunack, B.

Schlegel and W. Sippl, Mini-Rev. Med. Chem., 2004, 4,

965–977.

13 C. Papp-Jambor, U. Jaschinski and H. Forst, Anaesthesist,

2002, 51, 2–15.

D. McNaught Flores performed the rat histamine H₃re-

ceptor binding affinity test, elaborated and described the

results.

W. A. Fogel coordinated the advanced pharmacological

studies in vivo and interpreted the obtained results.

K. Kieć-Kononowicz coordinated the human histamine H₃

receptor and human histamine H₄receptor binding affinity

tests and interpreted the obtained results.

14 M. Zhang, M. E. Ballard, L. Pan, S. Roberts, R. Faghih, M.

Cowart, T. A. Esbenschade, G. B. Fox, M. W. Decker, A. A.

Hancock and L. E. Rueter, Brain Res., 2005, 1045, 142–149.

15 T. W. Lovenberg, B. L. Roland, S. J. Wilson, X. Jiang, J. Pyati,

A. Huvar, M. R. Jackson and M. G. Erlander, Mol.

Pharmacol., 1999, 55, 1101–1107.

16 S. Celanire, M. Wijtmans, P. Talaga, R. Leurs and I. J. P. de

Esch, Drug Discovery Today, 2005, 10, 1613–1627.

17 M. Cowart, R. Altenbach, L. Black, R. Faghih, C. Zhao and

A. A. Hancock, Mini-Rev. Med. Chem., 2004, 4, 979–992.

18 M. A. Khanfar, A. Affini, K. Lutsenko, K. Nikolic, S. Butini

and H. Stark, Front. Neurosci., 2016, 10, 1–17.

19 H. Stark, M. Kathmann, E. Schlicker, W. Schunack, B.

Schlegel and W. Sippl, Mini-Rev. Med. Chem., 2004, 4,

965–977.

20 H. Stark, M. Krause, J.-M. Arrang, X. Ligneau, J.-C. Schwartz and

W. Schunack, Bioorg. Med. Chem. Lett., 1994, 4, 2907–2912.

21 I. D. Linney, I. M. Buck, E. A. Harper, S. B. Kalindjian, M. J.

Pether, N. P. Shankley, G. F. Watt and P. T. Wright, J. Med.

Chem., 2000, 43, 2362–2370.

22 M. Berlin, C. W. Boyce and M. De Lera Ruiz, J. Med. Chem.,

2011, 54, 26–53.

23 T. A. Esbenshade, K. E. Browman, R. S. Bitner, M. Strakhova,

M. D. Cowart and J. D. Brioni, Br. J. Pharmacol., 2008, 154,

1166–1181.

R. Leurs coordinated the rat histamine H₃receptor bind-

ing affinity test and interpreted the obtained results.

Conflicts of interest

The authors have declared no conflict of interest.

Acknowledgements

This study was supported by departmental sources: Medical

University of Lodz grant numbers 503/3-016-01/503-31-001;

503/5-087-02/503-01 and COST Action CA 15135.

The authors would like to thank Mr. Mieczyslaw Wośko,

president of the pharmaceutical company Polfarmex SA, for

providing financial support for the purchase of the necessary

reagents for in vivo studies and also to thank Prof. H. Stark,

from Heinrich-Heine-Universität Düsseldorf, Germany for

kindly donating ciproxifan for use as the reference com-

pound for the in vivo study.

24 M. J. Gemkow, A. J. Davenport, S. Harich, B. A. Ellenbroek,

A. Cesura and D. Hallett, Drug Discovery Today, 2009, 14,

509–515.

25 K. Nikolic, F. Slavica, D. Agbaba and H. Stark, CNS Neurosci.

Ther., 2014, 20, 613–623.

26 M. A. Khanfar, A. Affini, K. Lutsenko, K. Nikolic, S. Butini

and H. Stark, Front. Neurosci., 2016, 10, 1–17.

27 T. A. Esbenshade, G. B. Fox and M. D. Cowart, Mol.

Interventions, 2006, 6, 77–88.

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Med. Chem. Commun.

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Design, synthesis, and: In vitro and in vivo characterization of 1-{4-[4-(substituted)piperazin-1-yl]butyl}guanidines and their piperidine analogues as histamine H3 receptor antagonists

DOI: 10.1039/c8md00527c

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