Synthesis and structure-activity relationships of cyanoguanidine-type and structurally related histamine H4 receptor agonists

J. Med. Chem. 2009, 52, 6297–6313 6297

DOI: 10.1021/jm900526h

Synthesis and Structure-Activity Relationships of Cyanoguanidine-Type and Structurally Related

Histamine H₄Receptor Agonists^§

Patrick Igel,^†Roland Geyer,^†Andrea Strasser,^†Stefan Dove,^†Roland Seifert,^‡and Armin Buschauer*^,†

^†Department of Pharmaceutical/Medicinal Chemistry, Faculty of Chemistry and Pharmacy, University of Regensburg, Universitatsstrasse 31,

D-93053 Regensburg, Germany, and ^‡Institute of Pharmacology, Medical School of Hannover, D-30625 Hannover, Germany

Received April 24, 2009

Recently, we identified high-affinity human histamine H₃(hH₃R) and H₄receptor (hH₄R) ligands

among a series of N^G-acylated imidazolylpropylguanidines, which were originally designed as histamine

H₂receptor (H₂R) agonists. Aiming at selectivity for hH₄R, the acylguanidine group was replaced with

related moieties. Within a series of cyanoguanidines, 2-cyano-1-[4-(1H-imidazol-4-yl)butyl]-3-

[(2-phenylthio)ethyl]guanidine (UR-PI376, 67) was identified as the most potent hH₄R agonist

(pEC₅₀=7.47, R=0.93) showing negligible hH₁R and hH₂R activities and significant selectivity over

the hH₃R (pK_B= 6.00, R = -0.28), as determined in steady-state GTPase assays using membrane

preparations of hH_xR-expressing Sf9 cells. In contrast to previously described selective H₄R agonists,

this compound and other 3-substituted derivatives are devoid of agonistic activity at the other HR

subtypes. Modeling of the binding mode of 67 suggests that the cyanoguanidine moiety forms charge-

assisted hydrogen bonds not only with the conserved Asp-94 but also with the hH₄R-specific Arg-341

residue. 2-Carbamoyl-1-[2-(1H-imidazol-4-yl)ethyl]-3-(3-phenylpropyl)guanidine (UR-PI97, 88) was

unexpectedly identified as a highly potent and selective hH₃R inverse agonist (pK_B=8.42, >300-fold

selectivity over the other HR subtypes).

Introduction

subtype has attracted interest as a potential drug target for

the treatment of CNS disorders like attention-deficit hyper-

activity disorder, Alzheimer’s disease, Parkinson’s disease,

sleep disorders, or obesity.⁴Meanwhile, several H₃R antago-

nists, such as tiprolisant,⁷have been evaluated in the first

clinical trials.⁴

Histamine is a biogenic amine that influences a variety of

physiological and pathophysiological processes in the body

via stimulation of four histamine receptor (HR) subtypes, all

belonging to the class A of G-protein-coupled receptors.^1,2

H₁R^aand H₂R antagonists have been successfully used for

several decades in the treatment of allergic conditions and as

antiulcer drugs, respectively. The H₃R is mainly expressed in

the CNS and acts as a presynaptic auto- or heteroreceptor

modulating the release of several neurotransmitters.³This

The most recently identified fourth HR subtype, H₄R, was

discovered on the basis of its high sequence homology with the

H₃R in the years 2000 and 2001 independently by several

research groups.^5-12The H₄R is mainly expressed on hemato-

poietic cells such as mast cells, basophils, eosinophils, T-cells,

and dendritic cells.^5,7,8,12-15Up to now, little is known about

the biological functions of the H₄R. Mast cell chemotaxis and

calcium mobilization¹⁴as well as actin polymerization, shape

change, expression of adhesion proteins, and chemotaxis of

eosinophils are activated through this HR subtype.¹⁶Further-

more, the release of inflammatory mediators such as inter-

leukin-16,¹³leukotriene-B₄,¹⁷and chemokine ligand 2¹⁸is

modulated via the H₄R. In addition to such in vitro experi-

ments, several animal models were used to evaluate the role of

the H₄R. Blocking the H₄R with antagonists had protective

effects in the mouse zymosan-induced peritonitis model¹⁹and

in carrageenan-induced paw edemas in rats^20,21and decreased

lung inflammation in an asthma mouse model.²²Moreover,

the H₄R antagonist 1 (JNJ 7777120, Figure 1)²³proved to be

more effective in attenuation of pruritus than classic H₁R

antagonists.²⁴

^§Dedicated to Prof. Dr. Helmut Schonenberger, University Regens-

burg, on the occasion of his 85th birthday.

*To whom correspondence should be addressed. Phone: þ49-941

9434827. Fax: þ49-941 9434820. E-mail: armin.buschauer@chemie.

uni-regensburg.de.

^aAbbreviations: R, intrinsic activity; aq, aqueous; Boc, tert-butyloxy-

carbonyl; Cbz, benzyloxycarbonyl; CDI, 1,1⁰-carbonyldiimidazole;

DCM, dichloromethane; DMF, dimethylformamide; DMSO, dimethyl

sulfoxide; EDTA, ethylenediaminetetraacetic acid; EI-MS, electron-

impact ionization mass spectrometry; ES-MS, electrospray ionization

mass spectrometry; GTP, guanosine 5⁰-triphosphate; GPCR, G-protein-

coupled receptor; G_β1γ2, G protein β₁- and γ₂-subunit; G_Ri, R-subunit of

the G_iprotein that mediates inhibition of adenylyl cyclase; HMBC,

heteronuclear multiple bond correlation; H₁R, histamine H₁receptor;

H₂R, histamine H₂receptor; H₃R, histamine H₃receptor; H₄R, hista-

mine H₄receptor; hH₁R, human H₁R; hH₂R, human H₂R; hH₂R-G_sRS

,

fusion protein of the hH₂R and the short splice variant of G_sR; hH₃R,

human H₃R; hH₄R, human H₄R; hH₄R-RGS19, fusion protein of hH₄R

and RGS19; HR-MS, high resolution mass spectrometry; HSQC,

heteronuclear single quantum coherence; LSI-MS, liquid secondary

ion mass spectrometry; NOESY, nuclear Overhauser enhancement

spectrometry; RGS, regulator of G protein signaling proteins; SEM,

standard error of the mean; TM, transmembrane domain of a GPCR;

TFA, trifluoroacetic acid; THF, tetrahydrofuran.

The localizationof theH₄R on immune cellsand theinvitro

and in vivo findings suggest that the receptor plays a crucial

role in immunological and inflammatory processes. Hence,

r

2009 American Chemical Society

Published on Web 09/30/2009

pubs.acs.org/jmc

6298 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20

Igel et al.

Figure 1. Structural formulas of the H₄R antagonist 1 and the

selective H₄R agonists 2, 3, and 4.

Figure 3. Replacement of the central acylguanidine group with a

nonbasic cyanoguanidine or sulfonylguanidine group and a basic

carbamoylguanidine group.

aim to further increase selectivity for the H₄R relative to the

H₂R and H₃R, the present study was focused on the replace-

ment of the acylguanidine moiety. Unlike the N^G-acylated

imidazolylpropylguanidines (pK_a≈ 8),³⁶the first reported

selective H₄R agonist, compound 4,³⁰contains a nonbasic

cyanoguanidine moiety (pK_a≈ -0.4).³⁷Obviously, for H₄R

activation a second basic component besides the imidazole

ring is not essential. In contrast, when the strongly basic

guanidine group in the potent H₂R agonist impromidine (7)

is replaced, H₂R agonistic activity is abolished (Figure 2).³⁸

The antinociceptive drug improgan (6) (also containing a

cyanoguanidine moiety) displays low affinity for the H₄R

and lacks affinity for other HRs.⁷Moreover, in a series of

thiourea-type H₃R antagonists, compounds with partial ago-

nistic activity at the H₄R were identified²⁶and the thiourea

moiety is considered as a bioisostere of the cyanoguani-

dine group.³⁷Therefore, cyanoguanidine analogues of the

N^G-acylated imidazolylpropylguanidines^31-34,39were pre-

pared with the goal to obtain compounds that still exhibit

agonistic activity at the H₄R but are devoid of agonistic

activity at other HR subtypes. Besides the cyanoguanidines,

several compounds were synthesized containing a carbamoyl-

guanidine (pK_a≈ 8.0)³⁷or a sulfonylguanidine (pK_a≈ 0.7)³⁶

moiety instead of the acylguanidine group (Figure 3), since

central groups with different basicities may provide more

insight into the structure-activity relationships at the distinct

HR subtypes.

Figure 2. Imidazolylalkylguanidine derivatives reported as hista-

mine receptor ligands.

the H₄R may be a promising drug target for the treatment

of inflammatory and autoimmune diseases like allergic rhini-

tis, rheumatoid arthritis, bronchial asthma, and pruritus.²⁵To

further explore the biological function of this receptor, pharma-

cological tools (including selective agonists) are required.

Among the HR subtypes, the H₄R shows the highest degree

of homology with the H₃R. Therefore, it is not surprising that

many H₃R ligands also exhibit high affinity for the H₄R.²⁶In

particular for imidazole-based compounds such as thioper-

amide (N-cyclohexyl-4-(1H-imidazol-4-yl)piperidine-1-carbo-

thioamide)²⁷or proxyfan (5-[3-(benzyloxy)propyl]-1H-imi-

dazole)²⁸an overlap of H₃R- and H₄R-related effects is

observed.²⁶Only a limited number of selective H₄R agonists,

including 2, 4(5)-methylhistamine),²⁶3 (VUF 8430),²⁹and 4

(OUP-16),³⁰have been reported so far. However, these com-

pounds have limitations because, at least at higher concentra-

tions, they also activate other HR subtypes.

First approaches to the design of new selective H₄R ago-

nists were based on N^G-acylated imidazolylpropylguanidines,

originally developed as potent H₂R agonists.^31-33More

detailed pharmacological investigations revealed that several

of these compounds are also active at the H₃R and H₄R. For

example, compound 5 (UR-AK51)³²is highly potent at both

the H₃R and H₄R but exhibits lower efficacy at the H₃R

relative to the H₄R (Figure 2). Very recently, we reported that

imidazolylpropylguanidines bearing small N^G-acyl substitu-

ents show preference for H₄R and H₃R and considerably

improved selectivity for the H₄R versus the H₂R.^34,35With the

In this study the development of a new class of potent and

selective hH₄R agonists and the unexpected discovery of a

highly potent and selective carbamoylguanidine-type hH₃R

inverse agonist are reported.

Chemistry

The cyanoguanidines 48-84, 86, and 87 and were synthe-

sized by analogy to a previously described synthetic route

(Scheme 1).⁴⁰Aminolysis of diphenyl cyanocarbonimidate

(9)^41,42with the primary amines 10-26 at ambient tempera-

ture gave the isourea intermediates 27-43 that crystallized

from diethyl ether. Conversion of the isoureas to the cyano-

guanidines was performed by refluxing with histamine (44) or

the analogous primary amines 45-47 or 85 in acetonitrile.

Acidic hydrolysis of the cyanoguanidines resulted in the

carbamoylguanidines 88-91.³⁷

The sulfonylguanidines 94 and 95 were prepared employing

two different synthetic pathways (Scheme 2). 94 was synthe-

sized by reaction of imidazolylpropylguanidine 88³¹with

tosyl chloride (90) followed by detritylation under acidic

Article

Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20 6299

Scheme 1. Synthesis of the Isourea Precursors 27-43, the Cyanoguanidines 48-84, 86, and 87, and the Carbamoylguanidines 88-91^a

^aReagents and conditions: (i) DCM, 1 h, room temp; (ii) for 48-78, MeCN, reflux, overnight; (iii) HCl 1 M, 14 days, room temp; (iv) for 79-84,

MeCN, microwave, 140 °C, 15 min.

Scheme 2. Synthesis of the N^G-Sulfonated Imidazolylpropylguanidines 94 and 95^a

^aReagents and conditions: (i) NaH (60% dispersion in mineral oil) (2 equiv), NEt₃(2 equiv), THF, overnight, 0 °C f room temp; (ii) DIEA (3 equiv),

THF, overnight, 0 °C f room temp; (iii) TFA (20%), DCM, 5 h, room temp.

conditions. As several attempts to prepare 95 in the same

manner failed, 88 was replaced with the less basic tert-

butoxycarbonyl (Boc) protected analogue 89. This method

had already been successfully employed for the preparation of

N^G-acylated imidazolylpropylguanidines³⁴and turned out to

be also appropriate for the preparation of 95.

studies is that an identical, very proximal read-out in

G-protein-mediated signaling is used for any given HR sub-

type, namely, steady-state GTP hydrolysis. This read-out

avoids bias in data interpretation caused by limited availabil-

ity of downstream effectors.

Cyanoguanidines 48-84, 86, and 87 (Tables 1-4). Simple

replacement of the acylguanidine moiety in 5 with a cyano-

guanidine group should evaluate if this modification is

tolerated by the hH₄R with respect to agonistic activity.

Indeed, the resulting compound 55 showed partial agonism

at the hH₄R (pEC₅₀= 6.12, R = 0.53). Moreover, 55 was

almost inactive at the hH₁R and exhibited only weak partial

agonism at the hH₂R (pEC₅₀= 4.85, R = 0.39). At the

hH₃R, 55 behaved as a moderate inverse agonist (pK_B=

5.72, R = -0.37). However, compared to the reference

compound 5 (hH₄R: pEC₅₀=7.77, R=0.74), efficacy and

in particular potency at the hH₄R drastically decreased

(almost 50-fold). With the aim of improving H₄R agonist

potency, the phenylpropyl portion was varied. Similar

residues as in the above-mentioned series of N^G-acylated

Results and Discussion

The prepared compounds were investigated for agonism

and antagonism at the four hHR subtypes in steady-state

GTPase assays measuring the enzymatic hydrolysis of radi-

olabeled GTP ([γ-³²P]GTP) by the G-protein coupled to the

respective HR subtype. These experiments were performed

using membrane preparations of Sf9 insect cells coexpressing

the hH₁R plus RGS4, expressing the hH₂R-G_sRSfusion

protein, coexpressing the hH₃R plus G_iR2plus G_β1γ2plus

RGS4, or coexpressing the hH₄R-RGS19 fusion protein plus

G_iR2plus G_β1γ2(Tables 1-5).^31,43,44The major advantage of

the well-proven test systemsapplied in this andinour previous

6300 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20

Igel et al.

Table 1. Potencies and Efficacies of the Prepared Cyanoguanidine-Type Compounds 48-54 at the hH₁R, hH₂R, hH₃R, and hH₄R in the Steady-State

GTPase Assay^a

^aSteady-state GTPase activity in Sf9 insect cell membranes expressing the hH₁R þ RGS4, hH₂R-G_sRSfusion protein, hH₃R þ G_iR2þ G_β1γ2þ RGS4,

or hH₄R-RGS19 fusion protein þ G_iR2þ G_β1γ2was determined as described under Pharmacological Methods. N gives the number of independent

experiments performed in duplicate each. n.d.: not determined. ^bNo agonistic activity up to a concentration of 1 mM.

imidazolylpropylguanidines were introduced, since most of

these compounds turned out to be potent hH₄R (partial)-

agonists.³¹Shortening (56) and elongation (57) of the carbon

chain resulted in considerably reduced potencies and effica-

cies. The branched diphenylpropyl residue in 58 caused a

complete loss of activity, and replacement of phenyl with

cyclohexyl (59) and 1H-indol-3-yl (61) eliminated hH₄R

agonism (R = -0.20 and -0.01, respectively). Only the

imidazolylpropylcyanoguanidine 60, bearing a phenylthio-

ethyl substituent, retained some degree of hH₄R agonistic

activity (pEC₅₀=5.89, R=0.33).

Since the hH₄R agonistic potency could not be increased

by modifying the N-substituent of 55, attention was paid to

optimization of the carbon chain connecting the imida-

zole ring with the cyanoguanidine group. This connecting

chain proved to be critical for hH₄R agonism. The lower

homologues of 55-61, compounds 48-54 (Table 1), were

substantially less potent and in all cases hH₄R antagonists.

For instance, 48 was almost inactive (pK_B< 5.00) compared

to 55. By contrast, increasing the chain length in order to

obtain higher similarity with the potent hH₄R agonist 4³⁰

turned out to be a key step in structural optimization. The

higher homologue of 55 (62) showed 5-fold higher potency

and essentially improved intrinsic activity at the hH₄R

(pEC₅₀=6.82, R=0.90). As expected from the impromidine

analogue 8³⁸(Figure 2), 62 exhibited only low potency and

no significant efficacy at the hH₂R (pEC₅₀=5.25, R=0.08).

Furthermore, 62 was found to be almost inactive at the hH₁R

(pK_B< 5.00) and only poorly active at the hH₃R (pK_B=

5.64, R=-0.03). Notably, 62 is a potent and selective hH₄R

agonist devoid of agonistic activity at the other HRs. Further

elongation to

a

five-membered carbon chain in

the compounds 79-84 (Table 4) was not tolerated with

respect to hH₄R agonism. Independent from the second

alkyl substituent, moderately potent hH₄R antagonists were

obtained.

On the basis of these results, 62 was used as new lead

structure to further improve hH₄R agonistic potency and

selectivity. Keeping the imidazolylbutyl portion cons-

tant, structural modifications were again focused on the

Article

Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20 6301

Table 2. Potencies and Efficacies of the Prepared Cyanoguanidine-Type Compounds 55-61 at the hH₂R, hH₃R, and hH₄R in the Steady-State GTPase

Assay^a

^aSteady-state GTPase activity in Sf9 insect cell membranes expressing the hH₂R-G_sRSfusion protein, hH₃R þ G_iR2þ G_β1γ2þ RGS4, or hH₄R-

RGS19 fusion protein þ G_iR2þ G_β1γ2was determined as described under Pharmacological Methods. N gives the number of independent experiments

performed in duplicate each. n.d.: not determined.

substituent “R” of the cyanoguanidine-type compounds.

Besides the residues employed in the homohistamine

(55-61, Table 2) and histamine (48-54, Table 1) series,

additional groups were introduced. Modifying the carbon

spacer between the phenyl ring and the cyanoguanidine

group was found to be critical. Reducing the chain length

by only one methylene group (63) clearly reduced potency

and efficacy (pEC₅₀= 5.96, R = 0.36). Elongation of the

spacer (64) even caused a loss of efficacy (R=0.05). Appar-

ently, a definite distance between the cyanoguanidine moiety

and the phenyl ring is required for hH₄R activation. Addi-

tionally, steric effects play a role. A bulky diphenylpropyl

residue (65) generated a weak inverse agonist at the hH₄R

(pK_B=5.60, R=-0.43). By contrast, a methyl group in R-

position to the phenyl ring (73) had no significant influence

on potency and efficacy compared to 62 (pEC₅₀=6.85, R=

0.79). Replacing the phenyl with a more bulky cyclohexyl

(66) or indole ring (68) was not tolerated in terms of hH₄R

activity. Notably, 68 was 10-fold more potent than 62 at the

hH₃R (pK_B= 6.77). Surprisingly, a fluorine substituent in

position 4 of the phenyl ring (69) was found to abolish hH₄R

agonistic efficacy (pK_B=6.59, R=-0.10). Replacement of

the phenyl with a 2-, 3-, or 4-pyridyl ring (70-72) also

resulted in lower potencies and efficacies at the hH₄R

compared to 62. Interestingly, potencies decreased in the

rank order from the 2-pyridyl to the 4-pyridyl analogue

(pEC₅₀= 6.24 f pEC₅₀= 5.59). In contrast, bioisosteric

replacement of a methylene group in the connecting chain

with a sulfur atom (67, UR-PI376) afforded a considerable

increase in potency and retained high efficacy at the hH₄R

(pEC₅₀=7.47, R=0.93) relative to compound 62. It is noted

that species-dependent discrepancies between affinities and

potencies are reported for numerous H₄R ligands.^26,45,46

Since preliminary investigations revealed a substantially

lower potency of compound 67 at the murine H₄R,⁴⁷more

detailed investigations on this aspect are the subject of on-

going work. At the hH₃R, 67 showed no agonism but

moderate inverse agonistic activity (pK_B= 6.00, R =

-0.30), and the (antagonistic) effects of 67 at the hH₁R

and hH₂R were negligible (pK_B< 5.00).

Thus minor structural modifications of “R” in the imid-

azolylbutylcyanoguanidines can drastically change potency

and efficacy. Since small alkyl substituents have already been

successfully applied in a series of acylguanidine-type com-

pounds to improve potency and intrinsic activity at the

hH₄R,³⁴a number of analogues bearing small alkyl residues

(75-78, Table 3) as well as the unsubstituted parent com-

pound (74) were prepared. Omitting the substituent (74) was

well tolerated with respect to hH₄R agonistic activity. 74

was essentially equipotent and equiefficacious with 62

(pEC₅₀=6.77, R=0.91). However, compound 74 possessed

similar potency at the hH₃R and, in contrast to the other

6302 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20

Igel et al.

Table 3. Potencies and Efficacies of the Prepared Cyanoguanidine-Type Compounds 62-78 at the hH₁R, hH₂R, hH₃R, and hH₄R in the Steady-State

GTPase Assay^a

^aSteady-state GTPase activity in Sf9 insect cell membranes expressing the hH₁R þ RGS4, hH₂R-G_sRSfusion protein, hH₃R þ G_iR2þ G_β1γ2þ RGS4,

or hH₄R-RGS19 fusion protein þ G_iR2þ G_β1γ2was determined as described under Pharmacological Methods. N gives the number of independent

experiments performed in duplicate each. n.d.: not determined.

Article

Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20 6303

Table 4. Potencies and Efficacies of the Prepared Cyanoguanidine-Type Compounds 79-84 at the hH₂R, hH₃R, and hH₄R in the Steady-State GTPase

Assay^a

^aSteady-state GTPase activity in Sf9 insect cell membranes expressing the hH₂R-G_sRSfusion protein, hH₃R þ G_iR2þ G_β1γ2þ RGS4, or hH₄R-

RGS19 fusion protein þ G_iR2þ G_β1γ2was determined as described under Pharmacological Methods. N gives the number of independent experiments

performed in duplicate each. n.d.: not determined. ^bN = 1.

imidazolylbutylcyanoguanidines, exhibited partial agonistic

activity at this subtype (pEC₅₀= 6.60, R = 0.42). The

substituent “R” appears to be indispensable to discriminate

between the hH₃R and hH₄R. This assumption is supported

by the data of the alkylated analogues 75-78. Introduction

of a methyl group (75) resulted in 10-fold lower potency at

the hH₃R (pK_B= 5.68, R = 0.16) but also hH₄R potency

decreased (pEC₅₀=6.02, R=0.75). However, enlarging the

alkyl substituent increased potency at the hH₄R (Me<Et<

i-Pr < i-Bu, pEC₅₀=6.02 f pEC₅₀=6.92) without remark-

ably affecting efficacy (R = 0.75-0.90). By contrast, at

the hH₃R, with increasing alkyl residues partial agonistic

activity was abolished whereas affinity was not markedly

affected (pK_B= 5.68 f pK_B= 5.47). In conclusion, the

pharmacological data show that larger alkyl substituents

are required for high hH₄R potency and selectivity relative to

the hH₃R. For example, an almost 30-fold selectivity in

combination with high potency and efficacy at the hH₄R

was achieved for compound 78, characterized by a bulky

isobutyl residue (pEC₅₀=6.92, R=0.90). The activities of

74-78 at the hH₁R and hH₂R were negligible (very weak

antagonism, pK_B< 5.00).

were prepared to evaluate if this modification can also

improve selectivity of cyanoguanidine-type hH₄R agonists.

Because of the availability of the corresponding amine, the

prepared compounds contain a sulfur atom in the chain

separating the imidazole ring from the cyanoguanidine

group. Unfortunately, both synthesized compounds (86

and 87) including the analogue of the lead compound 62

were almost inactive at the hH₄R (no agonism; very weak

antagonism, pK_B< 5.00).

The optimization steps from the acylguanidine-type com-

pound 5 toward the potent and selective cyanoguanidine-

type hH₄R agonist 67 are summarized in Figure 4 and the

structure-activity relationships of the cyanoguanidines

48-84, 86, and 87 in Figure 5.

Carbamoylguanidines 88-91 (Table 5). Although potent

and selective hH₄R agonists were obtained in the cyano-

guanidine series, the potencies of these compounds at the

hH₄R were lower than those of the corresponding acylgua-

nidine-type compounds.³⁴To study to which extent the lack

of basicity of the cyanoguanidine group accounts for the

differences in hH₄R potencies, some structurally related but

basic carbamoylguanidine-type compounds were prepared

(88-91; pK_a≈ 8.0).³⁷By analogy to the cyanoguanidine

series, compounds with a two- (88), three- (89 and 90), and

four-membered (91) carbon chain separating the imidazole

Recently, 5-methylhistamine was identified as a selective

hH₄R agonist.²⁶Hence, two compounds (86 and 87, Table 4)

bearing a methyl group at position 5 of the imidazole ring

6304 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20

Igel et al.

Table 5. Potencies and Efficacies of the Prepared Carbamoylguanidine-Type Compounds 88-91 and Sulfonylguanidine-Type Compounds 94 and 95 at

the hH₁R, hH₂R, hH₃R, and hH₄R in the Steady-State GTPase Assay^a

^aSteady-state GTPase activity in Sf9 insect cell membranes expressing the hH₁R þ RGS4, hH₂R-G_sRSfusion protein, hH₃R þ G_iR2þ G_β1γ2þ RGS4,

or hH₄R-RGS19 fusion protein þ G_iR2þ G_β1γ2was determined as described under Pharmacological Methods. N gives the number of independent

experiments performed in duplicate each. n.d.: not determined.

ring from the carbamoylguanidine group were investigated

for their pharmacological activities at the hHRs. In contrast

to the cyanoguanidine 55 that showed partial agonistic

activity at the hH₄R (R = 0.53), the carbamoylguanidine

analogue 89 exerted considerable inverse agonistic activity

(R=-0.75). Potency of 89 at the hH₄R was moderate (pK_B=

6.77). The phenylbutyl analogue 90 exhibited comparable

potency and intrinsic activity. Like at the hH₄R, 89 and 90

were moderately potent inverse agonists at the hH₃R. Inter-

estingly, both carbamoylguanidines showed moderate par-

tial agonistic activity at hH₂R (R ≈ 0.60). As expected from

48, the histamine analogue 88 (UR-PI97) exhibited weak

inverse agonistic activity at the hH₄R (pK_B= 5.89, R =

-0.51). Analogous to 89 and 90, 88 behaved as weak partial

agonist at the hH₂R (pEC₅₀=5.70, R=0.26) and showed

negligible activity at the hH₁R (pK_B< 5.00). Surprisingly,

this compound turned out to be a highly potent hH₃R inverse

agonist (pK_B=8.42, R=-0.97). Compared to the cyano-

guanidine analogue 48, introduction of the carbamoylgua-

nidine moiety resulted in a more than 1500-fold increase in

potency. In addition, 88 is highly selective over the hH₁R,

hH₂R, and hH₄R (>2500-fold, > 500-fold, and >300-fold

selectivity, respectively). The high selectivity of 88 relative to

the hH₄R is not typical, as many imidazole containing H₃R

ligands such as thioperamide or iodophenpropit (1-[3-(1H-

imidazol-5-yl)propylthio]-N⁰-[2-(4-iodophenyl)ethyl]formami-

dine)⁴⁸also have high affinity for the H₄R.²⁶In the cyano-

guanidine series highest hH₄R agonistic activity resided in

compounds having a tetramethylene chain connecting the

imidazole ring and the cyanoguanidine group. By contrast,

the imidazolylbutylcarbamoylguanidine 91 showed no ago-

nistic activity at the hH₄R and had similar pharmacological

activities as the carbamoylguanidines 89 and 90 at the other

HR subtypes.

Although the cyanoguanidine- and carbamoylguanidine-

type compounds have closely related structures, the com-

pounds have distinct pharmacological profiles. In contrast to

the cyanoguanidines, the basicity of the carbamoylguani-

dines is sufficiently high to allow protonation under physio-

logical conditions. However, the discrepancies in the

pharmacological activities at the HRs cannot only be ex-

plained by differences in basicity. Ionic interactions with the

conserved Asp^3.32of TM3 that are crucial for histamine

binding at all HR subtypes may be replaced by charge-

assisted hydrogen bonds in the case of neutral groups.

Obviously, variations in the bulk and the binding conforma-

tions of the central groups are the main reasons for the

observed differences in HR subtype activity.

Sulfonylguanidines 94 and 95 (Table 5). Additional inves-

tigations were carried out to evaluate the influence of the

basicity on HR subtype activity and selectivity using the

sulfonylguanidines 94 and 95. Unlike the acylguanidine

group (pK_a≈ 8),³⁶the sulfonylguanidine group (pK_a

≈

0.7)³⁶is virtually uncharged at physiological pH. The sulfo-

nylguanidine analogue of 5, 95, showed a more than 50-fold

decrease in potency at the hH₄R (pK_B=5.85) and, contrary

to 5, had no agonistic activity. The same was observed for the

sulfonylguanidine 94 (acylguanidine analogue at the hH₄R:

pEC₅₀=8.21, R=0.40). 94 and 95 acted as moderate inverse

agonists at the hH₃R and showed partial agonism at the

hH₂R (R ≈ 0.50). However, potencies were reduced by 2

orders of magnitude relative to the acylated guanidines at the

Article

Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20 6305

moiety is docked as proposed by Jongejan et al.⁵²and

corresponding to results on hH₄R mutants. In this binding

mode, Glu-182^5.46is assumed to be protonated and serves as

hydrogen bond donor for the π nitrogen. (Superscripts

indicate the generic numbering scheme of amino acids in

TMs 1-7 proposed by Ballesteros and Weinstein⁵⁵). The τ

nitrogen forms another H bond with the side chain oxygen of

Thr-178^5.42. However, a similar bidentate interaction is

possible with the couple Ser-179^5.43/Glu-182^5.46if the imida-

zole ring is assumed to be coplanar with the butyl chain. Both

the Thr-178 and the Ser-179 alanine mutations lead to an

only 3- to 4-fold reduction of histamine affinity and pote-

ncy.⁵¹Thus, no definitive conclusion about the presence and

the partner of a second hydrogen bond can be drawn. The

given mode is more likely, since a nearly perpendicular

conformation of the imidazolyl ring with respect to an alkyl

chain is energetically favorable and present in the crystal

structure of histamine monohydrobromide, too.⁵⁶

Figure 4. Optimization steps from the acylguanidine-type com-

pound 5 toward the potent and selective cyanoguanidine-type

hH₄R agonist 67.

Figure 6C shows that the binding mode of the semirigid

cyanoguanidine-type agonist 4 is very similar to that of

compound 67. The butyl chain of 67 aligns in a staggered

conformation with the furanylmethyl spacer of 4, leading to

˚

the same imidazole-guanidine distance of 6.0 A and to

closely corresponding positions of the imidazole and the

cyanoguanidine moieties in both structures. A rather roomy

binding pocket in the region of the spacer enables the fit of

different folds and bulks. Interactions occur with Tyr-95^3.34

,

Cys-98^3.37, Trp-316^6.48, Tyr-319^6.51, and Phe-344^7.39. The

inactivity of the 5-methylhistamine derivatives 86 and 87

may be due to sterical hindrance of the 5-methyl group by

Tyr-95^3.34or, in the case of interactions with the Ser-179^5.43

/

Glu-182^5.46couple, by Tyr-319^6.51

.

In both models (Figure 6B,C), the cyanoguanidine moiety

is stacked with the phenyl ring of Phe-344^7.39and forms two

charge-assisted hydrogen bonds with the carboxylate oxy-

Figure 5. H₄R agonistic potency of the cyanoguanidines 48-84,

86, and 87: summary of structure-activity relationships.

gens of Asp-94^3.32(distances 2.0-2.1 A), an amino acid

˚

proven to be essential for histamine binding by in vitro

mutagenesis.⁵¹This arrangement allows the arylthioalkyl

substituent of compound 67 to point outward like the

isopropyl group of carazolol in the crystal structure of the

β₂-adrenoceptor.⁴⁹In the given Z configuration, the cyano

group forms two additional charge-assisted hydrogen bonds

with the guanidine moiety of Arg-341^7.36(distances 2.1-

H₂R subtype (pEC₅₀=4.92). Again, these findings do not

indicate that basicity is a necessary condition for interaction

with HR subtypes. Steric and electronic effects, as for

example the different orientation of the oxygen atoms and

the influence of the sulfonyl group on the conformation of

the chain may play a role in altering the pharmacological

activities.

˚

2.2 A) which is also involved in a salt bridge with Glu-165

Model of the hH₄R in Complex with Compound 67 and

Other hH₄R Agonists. To explore the putative binding site of

the cyanoguanidines, a homology model of the hH₄R was

developed based on the crystal structures of the human

β₂-adrenoceptor.^49,50Compound 67 was manually docked

in an energetically favorable conformation, considering

results from in vitro mutagenesis⁵¹and modeling appro-

aches.⁵²Figure 6A,B shows the complex, minimized sub-

sequent to MD simulations in a membrane environment (see

Experimental Section). The cyanoguanidine moiety is pre-

sented as the “cyanoimine” tautomer which is, according to

ab initio calculations,⁵³about 12 kcal mol^-1lower in energy

than the most favorable conformation of the “cyanoamine”

tautomer. The given Z isomer may slowly topomerize at

room temperature into the E configuration by inversion at

the planar imine nitrogen (calculated topomerization barrier

of ∼23 kcal mol^-1).⁵⁴

(E2). This arginine is species specific (rat and mouse H₄R:

serine) and replaced by a glutamate in the hH₃R. Therefore,

the suggested interactions with Arg-341^7.36may contribute

to the hH₄R subtype and species selectivity of the cyanogua-

nidines. Except His-75^2.64(hH₃R, Tyr), Thr-178^5.42(Ala),

and Gln-347^7.42(Leu), all other transmembrane amino acids

of the binding site in Figure 6 are identical in hH₃R and

hH₄R. Interestingly, the species selectivity of agonistic imi-

dazolylpropylguanidines for the gpH₂R versus the hH₂R is

also related to a mutation in position 7.36 (gpH₂R, Asp;

hH₂R, Ala) as shown by in vitro mutagenesis.^43,57For the

carbamoyl derivatives representing inverse agonists at the

hH₄R (Table 5), another binding mode of the central proto-

nated guanidine moiety must be supposed. One reason is the

additional bulk of the carbamoyl NH₂group. Furthermore,

the guanidinium cation may adopt another position with

respect to Asp-94^3.32by forming a salt bridge.

The binding site of compound 67, consisting of 20 amino

Also the considerably less potent imidazolylpropyl anal-

ogue of 67, compound 60, may form the suggested key

interactions of the imidazole and the cyanoguanidine moiety

˚

acids with side chains not more than 3 A distant from the

ligand, is located between TM 2 and TM 7. The imidazole

6306 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20

Igel et al.

Figure 6. Model of the hH₄R binding site for the agonists 67, 4, and 60. The backbone, C atoms, and some essential hydrogens of the amino

acids are individually drawn in spectral colors: TM1, red; TM2, orange; TM3, yellow; TM4, green; E2, cyan; TM5, green-blue; TM6, blue;

TM7, violet; nitrogens, blue; oxygens, red; sulfur, yellow; C and H atoms of the ligands, gray. (A) Space fill representation of compound 67

within a tube model of the backbone. Side chains probably forming key interactions with the cyanoguanidines are drawn as sticks, the

interacting atoms as balls. (B-D) Detailed model of the binding modes of (B) compound 67, (C) compound 4, and (D) compound 60. Drawn

˚

are the CR trace (lines), CR atoms, and side chains (sticks) of all amino acids within 3 A around the ligands, and the ligands (balls and sticks).

Atoms forming key interactions are marked as balls. For clarity of presentation, the labeling of amino acids in panels C and D is restricted to

key and extra contacts.

with the hH₄R (Figure 6D). However, the conformational

strain of the propyl spacer is higher than in the case of the

butyl chain, and the guanidine is not ideally face to face with

Asp-94^3.32. The position of the imidazole plane differs from

that in compound 67 by ∼30°, leading to an additional weak

contact with Thr-323^6.55. Taken together, the model in

Figure 6D provides possible reasons for the lower potency

of compound 60.

The outstanding potency of compound 67 is probably due

to the close fit of the phenylthioethyl substituent R into a

predominantly hydrophobic binding pocket at the top of

TMs 2 and 3 (Figure 6B), leading to interactions that may

even compensate for the entropically and conformationally

more favorable binding of compound 4. The phenylthio

moiety is surrounded by the side chains of Leu-71^2.60, Tyr-

72^2.61, His-75^2.64, and Trp-90^3.28. In the model, this pocket is

quite narrow, suggesting why even slight variations of the

geometry and the bulk of R (phenylpropyl, 62) reduce

potency and why a cyclohexylpropyl group (inverse hH₄R

agonist 66) may not fit in the given binding mode. In

contrast, it becomes obvious that small alkyl chains and

methyl branches in R-, β-, and γ-position are well tolerated

(73, 75-78). For instance, the methyl group of 4 is centered

to the π-plane of Tyr-72^2.61(Figure 6C). In the case of a

diphenylpropyl substituent (inverse hH₄R agonist 65), the

second phenyl group clashes with Tyr-72^2.61. Interestingly,

this compound is inactive at the hH₂R, indicating that the

binding mode of the cyanoguanidines at this subtype differs

from that proposed for corresponding H₂R agonistic N^G

acylated imidazolylpropylguanidines.³¹In this mode, diphe-

nylpropyl groups are favorable, since the hH₂R contains a

serine in position 2.61 which provides enough space to

accommodate the second aryl moiety into a binding pocket

between TM 2 and TM 7.

The model in Figure 6 does not directly explain why p-F

substitution changes the agonism of 62 into antagonism

(compound 69). Also, the hH₄R antagonist 64, the phenyl-

butyl analogue of 67, may principally form the key interac-

tions of the imidazole and the cyanoguanidine moiety with

the receptor described above. However, docking approaches

with 64 (not shown) indicate that the long phenylbutyl chain

affects the conformation of E2, thereby possibly preventing

the stabilization of the active state. Compound 69 may

behave in a similar manner. Since a single amino acid in

the E2 loop, Phe-169, seems to account for the differences in

agonist binding between human and mouse H₄receptors,⁴⁵

a

Article

Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20 6307

specific conformational change of E2 bringing Phe-169 close

to the binding pocket could be necessary for the active state

formation.

Taken together, the model shown in Figure 6, although

based only on the putative inactive state of the receptor,

provides some information about which amino acids and

interactions could account for the hH₄R agonistic potency

and selectivity of the cyanoguanidines. A reliable model of

the fully active state cannot be obtained without appropriate

crystal structures. However, the docking poses in Figure 6

contain direct contacts of the spacers with Trp-316^6.48and

interactions with Tyr-319^6.51probably participating in cor-

related rotamer changes by which Trp-316^6.48may serve as

toggle switch, leading to subsequent modification of the

proline kink in TM6.⁵⁸A pocket of amino acids in TMs 6

and 7, consisting of Tyr-319^6.51, Phe-344^7.39(in contact with

the cyanoguanidine moiety and with Arg-341^7.36), Gln-

347^7.42, and Trp-348^7.43, may contribute to the conforma-

tional change of Trp-316^6.48, suggesting that flexibility of

TM7 could affect the toggle switch. Thus, hH₄R-selective

potency and efficacy may indeed be due to specific interac-

tions of the cyanoguanidines with TM7. Perhaps different

conformations and flexibilities of the E2 loop⁴⁵with indivi-

dual transient interactions and energy gradients during

binding may contribute to affinity, potency, efficacy, and

selectivity.

Affinities of 67 for the hH₁R, hH₂R, hH₃R, and hH₄R

Subtypes in Radioligand Binding Experiments. 67 was identi-

fied as the most potent and selective hH₄R agonist in the

cyanoguanidine series by using steady-state GTPase assays

(pEC₅₀= 7.46, R = 0.93, ∼30-fold selectivity toward the

hH₃R). Nevertheless, agonist potencies determined in func-

tional assays depend on different factors such as receptor/G-

protein stoichiometry, compartmentation of signaling pro-

teins, and G-protein subtype.^33,59This can cause discrepan-

cies between potencies obtained in functional experiments

and binding studies. For this reason inhibition constants

(pK_Ivalues) of 67 at the different hHR subtypes were

determined. 67 displaced [³H]mepyramine from the hH₁R,

[³H]tiotidine from the hH₂R, [³H]N^R-methylhistamine from

the hH₃R, and [³H]histamine from the hH₄R, yielding

monophasic competition binding isotherms (Figure 7). In

accordance with the results from the GTPase assay, 67

bound with the highest affinity to the hH₄R (pK_I= 7.24)

and, as expected, showed a remarkably lower affinity for the

hH₃R (pK_I=6.28). However, the affinity of 67 for the hH₄R

was slightly reduced compared to the pEC₅₀value deter-

mined in the GTPase activity assay (pEC₅₀=7.46), while the

contrary was observed for the affinity of 67 to the hH₃R

(pEC₅₀=6.00). Therefore, selectivity of 67 for the hH₄R over

the hH₃R is about 3 times lower than in the functional

experiments. At the hH₁R and hH₂R, the compound dis-

played low affinity, resulting in a 450- and 70-fold selectivity

for the hH₄R, respectively. Overall, the determined pK_I

values and their rank orders are in agreement with the

potencies evaluated in the functional GTPase assays and

confirm 67 to be a high-affinity and selective ligand for the

hH₄R.

Figure 7. Displacement of [³H]mepyramine (5 nM), [³H]tiotidine

(10 nM), [³H]N^R-methylhistamine (3 nM), and [³H]histamine

(10 nM) with 67 from Sf9 insect cell membranes expressing the

hH₁R þ RGS4, hH₂R-G_sRSfusion protein, hH₃R þ G_iR2þ G_β1γ2

þ

RGS4, or hH₄R-RGS19 fusion protein þ G_iR2þ G_β1γ2. Radio-

ligand binding was determined as described under Pharmacological

Methods. Data were analyzed for best fit to one site (monophasic)

competition curves. Data points shown are the means values of n

independent experiments each performed in duplicate.

G-proteins are directly accessible to the evaluated com-

pounds. Therefore, the possibility of direct, receptor-inde-

pendent G-protein activation has to be taken into account.

For many compounds like the wasp venom mastoparan,

local anesthetics, β-adrenoceptor antagonists, and cationic-

amphiphilic HR ligands, direct G-protein activation has

been reported.^60-6267 with its polar and basic imidazole

ring and more lipophilic side chain possesses cationic-

amphiphilic properties. To exclude a direct G-protein acti-

vation, GTPase activity was stimulated with 67 (1 μM) and

the effect of increasing concentrations of the H₄R antago-

nists (inverse agonists) thioperamide, iodophenpropit, and 1

was evaluated. As shown in Figure 8, all compounds inhi-

bited the 67-induced GTP hydrolysis in a concentration-

dependent manner. Thioperamide was more efficacious in

suppressing GTP hydrolysis than the other standard H₄R

ligands because of its pronounced inverse agonistic activity

at the hH₄R (Table 1). The pK_Bvalues determined for

thioperamide, iodophenpropit, and 1 against 67 in the func-

tional assay were in good accordance with pK_Ivalues from

binding studies reported in the literature (Figure 8). These

results indicate the H₄R ligands to compete with 67 for the

same binding site at the hH₄R and suggest the GTPase

activation through 67 to be receptor-mediated. Direct

G-protein activation by HR receptor ligands was reported

to occur at >10 μM.^60-62By contrast, 67 stimulated GTPase

activation in membranes expressing the hH₄R was detectable

at g10 nM. The inverse agonistic activity of 67 at the hH₃R

also supports a receptor-dependent effect, as both the hH₃R

and hH₄R were coexpressed with G_iR2. Finally, 67 was found

to displace [³H]histamine from the hH₄R (Figure 7). These

data confirm 67 to act as a hH₄R agonist in the GTPase

assay, whereas direct G-protein stimulation can definitely be

ruled out.

Inhibition of the 67-Stimulated GTP Hydrolysis by Stan-

dard H₄R Antagonists. The functional pharmacological acti-

vities of all compounds were determined in steady-state

GTPase activity assays using membrane preparations of

Sf9 insect cells expressing the respective HR subtype. When

membrane preparations are used instead of intact cells,

6308 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20

Igel et al.

activity at the hH₄R, whereas partial agonism was observed at

the hH₂R. With 88 unexpectedly a new highly potent carba-

moylguanidine-type hH₃R inverse agonist was discovered

that is 25 times more potent than the standard H₃R ligand

thioperamide. Incontrast tothioperamide, whichisessentially

equipotent at the hH₃R and hH₄R, for 88, a more than 300-

fold selectivityover the hH₄R was found. Since many reported

imidazole-type H₃R antagonists/inverse agonists also bind

with high affinity to the H₄R,²⁶88 belongs to the most

selective imidazole containing hH₃R inverse agonists known

so far.

Furthermore, new potent and selective cyanoguanidine-

type hH₄R agonists were obtained. 67 turned out to be the

most potent hH₄R agonist in this series. Compared to the lead

compound 5, 67 is devoid of agonistic activities at the hH₂R

and hH₃R, and the selectivity for the hH₄R toward the hH₂R

and hH₃R subtype considerably increased (approximately

300- and 30-fold in the GTPase assay, respectively). In con-

trast to 67 other selective hH₄R agonists such as 5-methylhist-

amine²⁶(Table 1), S-(2-guanidinoethyl)isothiourea,²⁹or 4³⁰

have agonistic activities at other HR subtypes. Moreover, 67

has negligible activity at the hH₁R whereas the hH₄R agonist

clozapine shows more than 100-fold higher affinity for this

histamine receptor subtype relative to the hH₄R.^26,63There-

fore, the new cyanoguanidine-type H₄R agonist 67 will be a

valuable additional pharmacological tool to study the biolo-

gical functions of the hH₄R.

Figure 8. Inhibition of the 67-stimulated GTP hydrolysis at the

hH₄R by the H₄R antagonists thioperamide, iodophenpropit, and

1. Steady-state GTPase activity in Sf9 insect cell membranes ex-

pressing the hH₄R-RGS19 fusion protein þ G_iR2þ G_β1γ2was

determined as described under Pharmacological Methods. Reaction

mixtures contained 1 μM 67. Data were analyzed by nonlinear

regression and were best-fit to sigmoid concentration-response

curves. Data points shown are the mean values of two independent

experiments each performed in duplicate. Footnote “a” indicates

from ref 26.

Experimental Section

Summary and Conclusion

Chemistry. General Conditions. Commercial reagents and

chemicals were purchased from Acros Organics (Geel,

Belgium), IRIS Biotech GmbH (Marktredwitz, Germany), Alfa

Aesar GmbH & Co. KG (Karlsruhe, Germany), Merck KGaA

(Darmstadt, Germany), Sigma-Aldrich Chemie GmbH

(Munich, Germany), TCI Europe (Zwijndrecht, Belgium) and

used without further purification. Deuterated solvents for

NMR spectroscopy were from Deutero GmbH (Kastellaun,

Germany). All solvents were of analytical grade or distilled prior

to use. If moisture-free conditions were required, reactions were

performed in dried glassware under inert atmosphere (argon or

nitrogen). Anhydrous DMF was purchased from Sigma-Al-

drich Chemie GmbH. 3-(1-Trityl-1H-imidazol-4-yl)propan-1-

ol (96) was a gift from Prof. Dr. Sigurd Elz, Department of

Pharmaceutical/Medicinal Chemistry I, University of Regens-

burg. Flash chromatography was performed on silica gel

(Merck silica gel 60, 40-63 μM). Reactions were monitored

by TLC on aluminum plates coated with silica gel (Merck silica

gel 60 F₂₅₄, thickness 0.2 mm). The compounds were detected by

UV light (254 nm), a 0.3% solution of ninhydrine in n-butanol

(amines), or a 1.0% solution of Fast Blue B salt (imidazole

containing compounds) in EtOH/H₂O=30/70 (v/v). All melting

points are uncorrected and were measured on a Buchi 530

(Buchi GmbH, Essen, Germany) apparatus. Microwave as-

sisted reactions were performed on an Initiator 2.0 synthesizer

(Biotage, Uppsala, Sweden).

Starting from the N^G-acylated imidazolylpropylguanidine

5, which is lacking HR subtype selectivity and shows high

activities at hH_2/3/4Rs, several classes of compounds with

structurally related polar central groups were prepared. The

aim was to obtain potent hH₄R agonists with improved

selectivity compared to 5 and information about structure-

activity relationships of the compounds at the distinct HR

subtypes.

Replacing the acylguanidine moiety in 5 with a cyanogua-

nidine group produced 55 that exhibited moderate partial

agonism at the hH₄R. Altering the chain length between the

imidazole ring and the cyanoguanidine group revealed a

tetramethylene spacer to be optimal for high potency and

efficacy at the hH₄R. By contrast, two- and five-membered

carbon chains were not tolerated with respect to hH₄R

agonism. The substituent “R” of the imidazolylbutylcyano-

guanidines was found tobesensitive toward variations. Minor

modifications, for example, altering the chain length or

fluorine substitution of the phenyl ring, substantially reduced

hH₄R activity. Introduction of small alkyl residues was well

accepted, but larger substituents increased selectivity over the

hH₃R. At the hH₁R and hH₂R, most cyanoguanidines

showed negligible efficacies and just low potencies. Compared

to the hH₁R and hH₂R, higher activities were observed for

the hH₃R. Almost all imidazolylbutylcyanoguanidines were

potent agonists or partial agonists at the hH₄R but were

antagonists at the other hHR subtypes. Modeling of their

binding mode suggests that the cyanoguanidine moiety

forms charge-assisted hydrogen bonds not only with the

conserved Asp-94^3.32but also with the hH₄R-specific

Arg-341^7.36residue.

Nuclear magnetic resonance spectra (¹H NMR and ¹³C

NMR) were recorded with Bruker Avance 300 (¹H, 300.1

MHz; ¹³C, 75.5 MHz), Avance 400 (¹H, 400.1 MHz; ¹³C,

100.6 MHz) or Bruker Avance 600 (¹H, 600.1 MHz; ¹³C,

150.9 MHz) NMR spectrometers (Bruker BioSpin GmbH,

Rheinstetten, Germany). Chemical shifts are given in δ (ppm)

relative to external standards. Abbreviations for the multipli-

cities of the signals are as follows: s (singlet), d (doublet), t

(triplet), q (quartet), quin (quintet), sep (septet), m (multiplet),

brs (for broad singlet), and combinations thereof. The multi-

plicity of carbon atoms (¹³C NMR) was determined by DEPT

135 (distortionless enhancement by polarization transfer): “þ”,

Exchange of the cyanoguanidine with a carbamoylguani-

dine group drastically changed the pharmacological acti-

vities at the HRs. All compounds displayed inverse agonistic

Article

Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20 6309

primary and tertiary carbon atom (positive DEPT 135 signal);

“-”, secondary carbon atom (negative DEPT 135 signal);

“quat”, quaternary carbon atom. In certain cases 2D-NMR

techniques (COSY, HMQC, HSQC, HMBC, NOESY) were

used to assign ¹H and ¹³C chemical shifts. Infrared spectra (IR)

were measured on a Bruker Tensor 27 spectrometer equipped

with an ATR (attenuated total reflection) unit from Harrick

Scientific Products Inc. (Ossining, NY). Mass spectra (MS) were

recorded on a Finnigan MAT 95 (EI-MS 70 eV, HR-MS),

Finnigan SSQ 710A (CI-MS (NH₃)), or a Finnigan Thermo-

Quest TSQ 7000 (ES-MS) spectrometer. The peak intensity in %

relative to the strongest signal is indicated in parentheses.

Elemental analyses (C, H, N, Heraeus Elementar Vario EL III)

were performed by the Analytical Department of the University

Regensburg and are within (0.4% unless otherwise noted.

Preparative HPLC was performed with a pump model

K-1800 (Knauer, Berlin, Germany). The column was Euro-

sphere-100 (250 mm ꢀ 32 mm) (Knauer), which was attached

to a UV detector model K-2000 (Knauer). UV detection of the

compounds was done at 210 nm. The temperature was 25 °C and

the flow rate 37 mL/min. The mobile phase was 0.1% TFA in

Millipore water and MeCN. Analytical HPLC was performed

on a system from Thermo Separation Products (TSP, Egels-

bach, Germany) equipped with a SN 400 controller, P4000

pump, an AS3000 autosampler, and a Spectra Focus UV/vis

detector. Stationary phase was a Eurosphere-100 C-18 (250 mm

ꢀ 4.0 mm, 5 μm) column (Knauer) thermostatted at 30 °C. The

flow rate was 0.8 mL/min, and the dead time (t₀) was 3.32 min.

For some compounds analytical HPLC was performed on a

Merck-Hitachi system equipped with a L-5000 LC controller,

655A-12 LC pump, a 655A-40 autosampler, and a L-4250 UV/

vis detector. Stationary phase was a Eurosphere-100 C-18

(250 mm ꢀ 4.0 mm, 5 μm) column (Knauer) thermostatted at

25 °C. The flow rate was 0.7 mL/min, and the dead time (t₀) was

2.54 min. Mobile phase gradients of MeCN/0.05% TFA (aq)

were used, and the absorbance was detected at 210 nm. Com-

pound purities were calculated as the percentage peak area of

the analyzed compound by UV detection at 210 nm. HPLC

conditions, retention times (t_R), capacity factors (k⁰=(t_R- t₀)/

t₀), and purities of the synthesized compounds are listed in the

Supporting Information.

a small amount of most compounds was converted to the

hydrogen oxalate by addition of a saturated solution of oxalic

acid in Et₂O to a solution of the cyanoguanidine in EtOH.

2-Cyano-1-[4-(1H-imidazol-4-yl)butyl]-3-(2-phenylthioethyl)-

guanidine (67). The title compound was prepared from 32 (0.60

g, 2.0 mmol) and 46 (0.31 g, 2.2 mmol) in MeCN (50 mL)

according to the general procedure. The crude product was

purified by flash chromatography (CHCl₃/MeOH/7 M NH₃in

MeOH, 92.5/5.5/2 v/v/v), yielding a colorless foamlike solid

1

(0.42 g, 62%), mp (hydrogenoxalate) 130-133 °C. H NMR

(300 MHz, CD₃OD): δ [ppm]=1.45-1.70 (m, 4H, Im-4-CH₂-

CH₂-CH₂), 2.58 (t, 2H, ³J=7.1 Hz, Im-4-CH₂), 3.07 (t, 2H, ³J=

3

7.0 Hz, S-CH₂-CH₂), 3.13 (t, 2H, J = 6.9 Hz, Im-4-(CH₂)₃-

CH₂), 3.39 (t, 2H, ³J=7.0 Hz, S-CH₂), 6.76 (d, 1H, ⁴J=1.1 Hz,

1H, Im-5-H), 7.10-7.41 (m, 5H, Ph-H), 7.53 (d, 1H, ⁴J=1.1 Hz,

Im-2-H). ¹³C NMR (75 MHz, CD₃OD): δ [ppm]=27.17 (-, Im-

4-CH₂), 27.67 (-, Im-4-CH₂-CH₂), 29.80 (-, Im-4-(CH₂)₂-

CH₂), 33.53 (-, S-CH₂), 42.27 (-, Im-4-(CH₂)₃-CH₂), 42.61

(-, S-CH₂-CH₂), 117.88 (þ, Im-C-5), 120.17 (C_quat, CtN),

127.30 (þ, Ph-C-4), 130.16 (þ, 2 Ph-C), 130.35 (þ, 2 Ph-C),

135.76 (þ, Im-C-2), 136.94 (C_quat, Ph-C-1), 138.10 (C_quat, Im-C-

4), 161.02 (C_quat, CdN). IR (cm^-1)=3267 (N-H), 2989 (C-H),

2901 (C-H), 2159 (CtN), 1576 (CdN). ES-MS (MeCN þ

TFA) m/z (%): 343 (100) [M þ H]^þ. HRMS (EI-MS) calcd

for C₁₇H₂₂N₆S [M^þ•

] 342.1627; found 342.1625. Anal.

(C₁₇H₂₂N₆S 0.75C₂H₂O₄) C, H, N. C₁₇H₂₂N₆S (342.46).

3

Compounds 48-66, 68-78, 86, and 87 were prepared by

analogy (see Supporting Information).

Preparation of the Carbamoylguanidines 88-91. General

Procedure. The pertinent cyanoguanidine was dissolved in 1 M

HCl (25 mL) and left for 14 days at room temperature. After

removing the solvent in vacuo, the crude product was purified

by preparative HPLC, followed by flash chromatography under

basic conditions to remove possible impurities with the corre-

sponding guanidine.

2-Carbamoyl-1-[2-(1H-imidazol-4-yl)ethyl]-3-(3-phenylpropyl)-

guanidine (88). The title compound was prepared from 48 (0.41

g, 1.38 mmol) according to the general procedure. Purification

by preparative HPLC (MeCN/0.1% TFA (aq), 20/80) followed

by flash chromatography (CHCl₃/MeOH/7 M NH₃in MeOH,

90/8/2 v/v/v) yielded a colorless semisolid compound (0.35 g,

81%). ¹H NMR (300 MHz, CD₃OD, trifluoroacetate):

Preparation of the Isoureas 27-43. General Procedure. A

solution of the pertinent primary amine (1 equiv) and diphenyl

cyanocarbonimidate (9, 1 equiv) in DCM was stirred for 1 h.

After evaporation of the solvent, the product was crystallized

from Et₂O.

3

δ [ppm]=1.89-2.04 (m, 2H, Ph-CH₂-CH₂), 2.70 (t, 2H, J=

7.6 Hz, Ph-CH₂), 3.07 (t, 2H, ³J=6.9 Hz, Im-4-CH₂), 3.24-3.37

(m, 2H, overlap with solvent, Ph-CH₂-CH₂-CH₂), 3.65 (t, 2H,

³J=6.9 Hz, Im-4-CH₂-CH₂), 7.14-7.32 (m, 5H, Ph-H), 7.43 (d,

1-Cyano-2-phenyl-3-[2-(phenylthio)ethyl]isourea (32). The title

compound was prepared from 15⁶⁴(1.53 g, 10.0 mmol) and 9

(2.38 g, 10.0 mmol) in DCM (50 mL) according to the general

1H, ⁴J=1.2 Hz, Im-5-H), 8.84 (d, 1H, ⁴J=1.4 Hz, Im-2-H). ¹³

C

NMR (75 MHz, CD₃OD, trifluoroacetate): δ [ppm]=25.27 (-,

Im-4-CH₂), 31.20 (-, Ph-CH₂-CH₂), 33.69 (-, Ph-CH₂), 41.37

(-, Im-4-CH₂), 42.45 (-, Ph-(CH₂)₂-CH₂), 118.19 (þ, Im-C-5),

127.34 (þ, Ph-C-4), 129.47 (þ, 2 Ph-C), 129.67 (þ, 2 Ph-C),

131.56 (C_quat, Im-C-4), 135.37 (þ, Im-C-2), 142.07 (C_quat, Ph-C-

1), 154.71 (C_quat, CdN), 156.86 (C_quat, CdO). IR (cm^-1)=3153,

2930, 1662, 1603, 1181, 1128. ES-MS (H₂O/MeCN) m/z (%):

315 (100) [M þ H]^þ. HRMS (EI-MS) calcd for C₁₆H₂₂N₆O

[M^þ•] 314.1855; found 314.1860. C₁₆H₂₂N₆O (314.39).

1

procedure, yielding a white solid (2.6 g, 86%); mp 110 °C. H

NMR (300 MHz, CDCl₃): δ [ppm]=3.18 (t, 2H, ³J=7.0 Hz, Ph-

S-CH₂), 3.55-3.69 (m, 2H, Ph-S-CH₂-CH₂), 7.05 (d, 2H, ³J=7.8

3

Hz, Ph-H), 7.15-7.50 (m, 8H, Ph-H), 7.57 (t, 1H, J=6.1 Hz,

N-H). ¹³C NMR (75 MHz, CDCl₃): δ [ppm]=33.22 (-, Ph-S-

CH₂), 41.68 (-, Ph-S-CH₂-CH₂), 115.55 (C_quat, CtN), 121.44

(þ, 2 Ph-C), 126.71 (þ, 1 Ph-C), 126.79 (þ, 1 Ph-C), 129.26 (þ, 2

Ph-C), 128.62 (þ, 2 Ph-C), 129.92 (þ, 2 Ph-C), 134.60 (C_quat, 1

Ph-C), 150.98 (C_quat, 1 Ph-C), 163.88 (C_quat, CdN). IR (cm^-1)=

3252 (N-H), 3212, 3065, 2190 (CtN), 1635 (CdN), 1433, 1207.

ES-MS (DCM/MeOH þ NH₄OAc) m/z (%): 298 (100) [M þ

H]^þ. Anal. (C₁₆H₁₅N₃OS) C, H, N. C₁₆H₁₅N₃OS (297.37).

Compounds 27-31 and 33-43 were prepared by analogy (see

Supporting Information).

Compounds 89, 90, and 91 were prepared by analogy (see

Supporting Information).

Preparation of the Trityl-Protected Sulfonylguanidines 92 and

93. 1-(4-Tosyl)-2-[3-(1-trityl-1H-imidazol-4-yl)propyl]guanidine

(92). Amixtureof88³¹(0.41 g, 1.0 mmol) and NaH (60% dispersion

in mineral oil) (0.08 g, 2.0 mmol) in THF_abs(15 mL) was stirred

for 45 min at 50 °C. After the mixture was cooled to

0 °C, NEt₃(0.28 mL, 2.0 mmol) and 90 (0.19 g, 1.0 mmol) in

THF_abs(15 mL) were subsequently added. Stirring was continued

overnight at ambient temperature. After removal of the solvent in

vacuo, the crude product was purified by flash chromatography

(CHCl₃/MeOH/NH₃(aq) 32%, 80/9/1 v/v/v), yielding a colorless

foamlike solid (0.22 g, 39%). ¹H NMR (300 MHz, CDCl₃):

δ [ppm]=1.73-1.94 (m, 2H, Im-4-CH₂-CH₂), 2.36 (s, 3H, CH₃),

Preparation of the Cyanoguanidines 48-78, 86, and 87. Gen-

eral Procedure. Hydrochlorides of 44 and 45 were converted into

the bases by passing a basic ion exchanger (Merck, ion exchan-

ger III, mobile phase MeOH). The respective isourea (1 equiv)

and the pertinent amine (1.1 equiv) were refluxed in MeCN for

12 h. After removal of the solvent in vacuo, the crude product

was purified by flash chromatography. For analytical purposes

6310 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20

Igel et al.

2.44-2.79 (m, 2H, Im-4-CH₂), 3.25-3.42 (m, 2H, Im-4-(CH₂)₂-

CH₂), 6.72 (s, 1H, Im-5-H), 7.06-7.15 (m, 6H, Ph-H), 7.19 (d, 2H,

³J=8.0 Hz, Ph-3,5-H), 7.30-7.45 (m, 10H, Ph-H þ Im-2-H), 7.74

(d, 2H, ³J=8.0 Hz, Ph-2,6-H). ES-MS (DCM/MeOH þ NH₄OAc)

m/z (%): 564 (100) [M þ H]^þ. C₃₃H₃₃N₅O₂S (563.71).

from PerkinElmer Life Sciences (Boston, MA). All unlabeled

nucleotides, glycerol 3-phosphate dehydrogenase, triose phosphate

isomerase, glyceraldehyde 3-phosphate dehydrogenase, and lactate

dehydrogenase were from Roche (Mannheim, Germany). 3-Phos-

phoglycerate kinase and ^L-R-glycerol phosphate were from Sigma.

GF/C filters were from Whatman (Maidstone, U.K.).

Data Analysis and Pharmacological Parameters. All data are

presented as the mean of n independent experiments ( SEM.

Agonist potencies were given as pEC₅₀values (negative decadic

logarithm of the molar concentration of the agonist causing

50% of the maximal response). Maximal responses (intrinsic

activities) were expressed as R values. The R value of histamine

was set to 1.00, and R values of other compounds were refer-

enced to this value.

IC₅₀values were converted to K_Iand K_Bvalues using the

Cheng-Prussoff equation.⁶⁷pK_Ivalues were analyzed by non-

linear regression and best-fit to one-site (monophasic) competi-

tion isotherms. pEC₅₀and pK_Bvalues from the functional

GTPase assays were analyzed by nonlinear regression and

best-fit to sigmoidal dose-response curves (GraphPad Prism,

version 4.02 software, San Diego, CA).

Steady-State GTPase Activity Assay. GTPase activity assays

were performed as previously described.^31,43,44,68For H₁R

assays, Sf9 insect cell membranes coexpressing the hH₁R and

RGS4 were employed. For H₂R assays, Sf9 insect cell mem-

branes expressing the hH₂R-G_sRSfusion protein were used. For

H₃R assays, Sf9 insect cell membranes coexpressing the hH₃R,

mammalian G_iR2, G_β1γ2, and RGS4 were employed. For H₄R

assays, Sf9 insect cell membranes coexpressing the hH₄R-

RGS19 fusion protein and mammalian G_iR2and G_β1γ2were

used. The respective membranes were thawed and sedimented

by centrifugation at 4 °C and 13000g for 10 min. Membranes

were resuspended in 10 mM Tris-HCl, pH 7.4. Each assay tube

contained Sf9 membranes expressing the respective HR subtype

1-(tert-Butoxycarbonyl)-2-(2-phenylethylsulfonyl)-1-[3-(1-tri-

tyl-1H-imidazol-4-yl)propyl]guanidine (93). To a solution of 89³⁴

(0.51 g, 1.0 mmol) and DIEA (0.39 g, 3.0 mmol) in DCM (20

mL), 91⁶⁵(0.21 g, 1.0 mmol) dissolved in DCM (10 mL) was

added dropwise at 0 °C. After 1 h, stirring was continued

overnight at room temperature and the solvent was evaporated.

The crude product was purified by flash chromatography

(CHCl₃/MeOH, 97.5/2.5 v/v), yielding the title compound as

colorless oil (0.34 g, 50%). ¹H NMR (300 MHz, CDCl₃)

isomers: δ [ppm]=1.47 (s, 3H, C(CH₃)₃), 1.49 (s, 6H, C(CH₃)₃),

1.86-1.91 (m, 2H, Im-4-CH₂-CH₂), 2.51-2.63 (m, 2H, Im-4-

CH₂), 3.04-3.17 (m, 2H, Ph-CH₂), 3.23-3.35 (m, 2H, Ph-CH₂-

CH₂), 3.76-3.85 (m, 2H, Im-4-(CH₂)₂-CH₂), 6.53 (d, 0.7H, ⁴J=

1.3 Hz, Im-5-H), 6.55 (d, 0.3H, ⁴J=1.3 Hz, Im-5-H), 7.06-7.38

(m, 21H, Ph-H þ Im-2-H). ES-MS (DCM/MeOH þ NH₄OAc)

m/z (%): 678 (100) [M þ H]^þ. C₃₉H₄₃N₅O₄S (677.85).

Preparation of the Sulfonylguanidines 94 and 95. 1-[3-(1H-

Imidazol-4-yl)propyl]-2-(4-tosyl)guanidine (94). A mixture of 92

(0.19 g, 0.34 mmol) and TFA (5 mL) in DCM (20 mL) was

stirred for 5 h. After evaporation of the solvent, the crude

product was purified by preparative HPLC (MeCN/0.1%

TFA (aq), 25/75), yielding the title compound as a colorless

semisolid (0.12 g, 82%). ¹H NMR (300 MHz, CD₃OD,

trifluoroacetate): δ [ppm] = 1.76-1.92 (m, 2H, Im-4-CH₂-

3

CH₂), 2.38 (s, 3H, CH₃), 2.70 (t, 2H, J=7.3 Hz, Im-4-CH₂),

3.23 (t, 2H, ³J=6.7 Hz, Im-4-(CH₂)₂-CH₂), 7.29 (d, 2H, ³J=8.3

Hz, Ph-3-H), 7.33 (s, 1H, Im-5-H), 7.72 (d, 2H, ³J=8.3 Hz, Ph-2-

4

H), 8.76 (d, 1H, J = 1.4 Hz, Im-2-H). ¹³C NMR (75 MHz,

CD₃OD, trifluoroacetate): δ [ppm]=21.46 (þ, CH₃), 22.35 (-,

Im-4-CH₂), 29.64 (-, Im-4-CH₂-CH₂), 40.67 (-, Im-4-(CH₂)₂-

CH₂), 117.02 (þ, Im-C-5), 127.21 (þ, 2 Ph-C-2), 130.41 (þ, 2 Ph-

(10-20 μg protein/tube), MgCl₂(H_1,2R assays, 1.0 mM; H_3,4

R

assays, 5.0 mM), 100 μM EDTA, 100 μM ATP, 100 nM GTP,

100 μM adenylyl imidophosphate, 5 mM creatine phosphate,

40 μg of creatine kinase, and 0.2% (w/v) bovine serum albumin

in 50 mM Tris/HCl, pH 7.4, and the investigated ligands at

various concentrations. All H₄R assays additionally contained

100 mM NaCl. For the determination of pK_Bvalues (antagonist

mode of the GTPase activity assay) histamine was added to the

reaction mixtures (final concentrations: H₁R, 200 nM; H₂R,

1 μM; H_3,4R, 100 nM).

C-3), 134.67 (þ, Im-C-2), 134.85 (C_quat, Im-C-4), 141.99 (C_quat

,

Ph-C-1), 143.83 (C_quat, Ph-C-4), 158.70 (C_quat, CdN). IR

(cm^-1)=3159, 2989, 2901, 1663, 1624, 1548, 1166, 1128, 1081.

HRMS (EI-MS) calcd for C₁₄H₁₉N₅O₂S [M^þ•] 321.1259; found

321.1257. C₁₄H₁₉N₅O₂S TFA (435.42).

3

1-[3-(1H-Imidazol-4-yl)propyl]-2-(2-phenylethylsulfonyl)gua-

nidine (95). A mixture of 93 (0.30 g, 0.44 mmol) and TFA (5 mL)

in DCM (20 mL) was stirred for 5 h. After evaporation of the

solvent, the crude product was purified by preparative HPLC

(MeCN/0.1% TFA (aq), 25/75), yielding the title compound as

colorless semisolid (0.14 g, 71%). ¹H NMR (300 MHz, CD₃OD,

trifluoroacetate): δ [ppm]=1.82-1.96 (m, 2H, Im-4-CH₂-CH₂),

2.76 (t, 2H, ³J=7.3 Hz, Im-4-CH₂), 3.01-3.11 (m, 2H, Ph-CH₂),

3.19 (t, 2H, ³J=6.9 Hz, Im-4-(CH₂)₂-CH₂), 3.23-3.29 (m, 2H,

Reaction mixtures (80 μL) were incubated for 2 min at 25 °C.

After the addition of 20 μL of [γ-³²P]GTP (0.1 μCi/tube),

reaction mixtures were incubated for 20 min at 25 °C. Reactions

were terminated by the addition of 900 μL of slurry consisting of

5% (w/v) activated charcoal and 50 mM NaH₂PO₄, pH 2.0.

Charcoal absorbs nucleotides but not P_i. Charcoal-quenched

reaction mixtures were centrifuged for 7 min at room tempera-

ture at 13000g. An amount of 600 μL of the supernatant was

removed, and ³²P_iwas determined by liquid scintillation count-

ing. Spontaneous [γ-³²P]GTP degradation was determined in

tubes containing all components described above, plus a high

concentration of unlabeled GTP (1 mM) that due to competi-

tion with [γ-³²P]GTP prevents [γ-³²P]GTP hydrolysis by enzy-

matic activities present in Sf9 membranes. Spontaneous

[γ-³²P]GTP degradation was <1% of the total amount of

radioactivity added. The experimental conditions chosen

ensured that not more than 10% of the total amount of

[γ-³²P]GTP added was converted to ³²P_i.

Radioligand Binding Assays. For the binding experiments, the

Sf9 insect cell membranes described above were employed. The

respective membranes were thawed and sedimented by centri-

fugation at 4 °C and 13000g for 10 min. Membranes were

resuspended in binding buffer (12.5 mM MgCl₂, 1 mM EDTA,

and 75 mM Tris/HCl, pH 7.4). Each tube (total volume 500 μL)

contained 50 μg (hH₁R, hH₃R), 120 μg (hH₄R), or 250 μg

(hH₂R) of membrane protein. Competition binding experiments

4

Ph-CH₂-CH₂), 7.15-7.31 (m, 5H, Ph-H), 7.37 (s, 1H, J=1.3

Hz, Im-5-H), 8.76 (d, 1H, ⁴J=1.3 Hz, Im-2-H). ¹³C NMR (75

MHz, CD₃OD, trifluoroacetate): δ [ppm] = 22.42 (-, Im-4-

CH₂), 29.48 (-, Im-4-CH₂-CH₂), 31.42 (-, Ph-CH₂), 40.63 (-,

Im-4-(CH₂)₂-CH₂), 56.24 (-, Ph-CH₂-CH₂), 117.07 (þ, Im-C-

5), 127.66 (þ, Ph-C-4), 129.44 (þ, 2 Ph-C), 129.77 (þ, 2 Ph-C),

134.70 (þ, Im-C-2), 134.89 (C_quat, Im-C-4), 140.27 (C_quat, Ph-C-

1), 158.95 (C_quat, CdN). IR (cm^-1)=3149, 2972, 2901, 1666,

1625, 1554, 1186, 1131, 1101. HRMS (EI-MS) calcd for

C₁₅H₂₁N₅O₂S [M^þ•] 335.1416; found 335.1421. C₁₅H₂₁N₅-

O₂S TFA (449.45).

3

Pharmacological Methods. Materials. Histamine dihydro-

chloride was purchased from Alfa Aesar GmbH & Co. KG

(Karlsruhe, Germany). Thioperamide maleate and iodophenpropit

dihydrobromide were from Tocris Bioscience (Ellisville, MO). The

H₄R antagonist 1 was synthesized according to ref 23. [³H]-

Mepyramine, [³H]tiotidine, [³H]N^R-methylhistamine, and [³H]-

histamine were from PerkinElmer Life Sciences (Boston, MA).

[γ-³²P]GTP was synthesized according to a previously described

method.⁶⁶[³²P]P_i(8500-9100 Ci/mmol orthophosphoric acid) was

Article

Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20 6311

were performed in the presence of 5 nM [³H]mepyramine

(hH₁R), 10 nM [³H]tiotidine (hH₂R), 3 nM [³H]N^R-methylhista-

mine (hH₃R), or 10 nM [³H]histamine (hH₄R) and increasing

concentrations of unlabeled ligands. Incubations were con-

ducted for 60 min at 25 °C and shaking at 250 rpm. Bound

radioligand was separated from free radioligand by filtration

through 0.3% polyethyleneimine-pretreated GF/C filters, fol-

lowed by three washes with 2 mL of cold binding buffer (4 °C)

using a Brandel harvester. Filter-bound radioactivity was

determined after an equilibration phase of at least 12 h by liquid

scintillation counting.

Molecular Modeling. First, a rough model of the hH₄R was

directly constructed from the PDB file 2RH1 of the β₂-adreno-

ceptor crystal structure,⁴⁹using the software suite SYBYL 7.3

(Tripos, L.P., St. Louis, MO) on a SGI Octane workstation. The

backbone coordinates of TMs 1-7, helix 8, the C1 loop, and

identical side chains were retained. All other cocrystallized

substructures and the lysozyme domain were deleted. The C3

loop was truncated; only the first two and the last five positions

were transferred, and the gap was closed by five alanine residues.

All other loop regions were constructed by Sybyl loop searches.

The side chains differing between the β₂-adrenoceptor and the

hH₄R were mutated.

The resulting model was prepared for optimization by adding

hydrogens and providing atoms with Amber-FF99 charges.⁶⁹

Bad contacts of side chains were removed using the Lovell

rotamer library.⁷⁰Subsequently, the model was roughly mini-

mized (25 cycles of steepest descent with fixed TM backbone,

100 cycles of Powell conjugated gradient, Amber-FF99 force

field,⁶⁹distant dependent dielectric constant ε=4) and checked

for inconsistencies of the local geometry with Sybyl ProTable.

To test the influence of alternative side chain rotamer confor-

mations, molecular dynamics (MD) simulations were per-

formed following a protocol published in detail elsewhere.⁷¹In

brief, the model was embedded into an environment consisting

of 93 1-palmitoyl-2-oleoylphosphatidylcholine molecules,

14 429 intracellular and extracellular water molecules, and 8

Na^þand 24 Cl^-ions to achieve electroneutrality, and the whole

complex was energetically minimized. The 1.35 ns equilibrium

phase of the MD was divided into one 250 ps and eleven 100 ps

cycles with gradually descending constraints for the backbone

and the side chain atoms. Then, a 1 ns productive phase without

constraints was performed. The simulations were carried out at

310 K and 1 atm with Berendsen, temperature, and pressure

coupling using the software package GROMACS 3.2⁷²with the

ffG53A6 force field.⁷³

Immunology, Johnson & Johnson Pharmaceutical R&D, San

Diego, CA) for hH₃R cDNA, and to David Schnell for

providing the hH₃R baculovirus. This work was supported by

the Graduate Training Program (Graduiertenkolleg) GRK

760, “Medicinal Chemistry: Molecular Recognition, Ligand-

Receptor Interactions”, of the Deutsche Forschungsge-

meinschaft and the COST program BM0806 (H₄R network)

of the European Union.

Supporting Information Available: Synthetic procedures and

analytical data for compounds 27-31, 33-43, 45, 46, 48-66,

68-84, 86, 87, and 89-91; purity data (elemental analysis

results, HPLC data) of all target compounds. This material is

available free of charge via the Internet at http://pubs.acs.org.

References

(1) Hill, S. J.; Ganellin, C. R.; Timmerman, H.; Schwartz, J.-C.;

Shankley, N. P.; Young, J. M.; Schunack, W.; Levi, R.; Haas,

H. L. International Union of Pharmacology. XIII. Classification of

histamine receptors. Pharmacol. Rev. 1997, 49, 253–278.

(2) Hough, L. B. Genomics meets histamine receptors: new subtypes,

new receptors. Mol. Pharmacol. 2001, 59, 415–419.

(3) Schlicker, E.; Kathmann, M. Modulation of in vitro neurotrans-

mission in the CNS and in the retina via H₃heteroreceptors.

Pharmacochem. Libr. 1998, 30, 13–26.

(4) Sander, K.; Kottke, T.; Stark, H. Histamine h(3) receptor antago-

nists go to clinics. Biol. Pharm. Bull. 2008, 31, 2163–2181.

(5) Oda, T.; Morikawa, N.; Saito, Y.; Masuho, Y.; Matsumoto, S.

Molecular cloning and characterization of a novel type of hista-

mine receptor preferentially expressed in leukocytes. J. Biol. Chem.

2000, 275, 36781–36786.

(6) Nakamura, T.; Itadani, H.; Hidaka, Y.; Ohta, M.; Tanaka, K.

Molecular cloning and characterization of a new human histamine

receptor, hH₄R. Biochem. Biophys. Res. Commun. 2000, 279, 615–620.

(7) Zhu, Y.; Michalovich, D.; Wu, H.-L.; Tan, K. B.; Dytko, G. M.;

Mannan, I. J.; Boyce, R.; Alston, J.; Tierney, L. A.; Li, X.; Herrity,

N. C.; Vawter, L.; Sarau, H. M.; Ames, R. S.; Davenport, C. M.;

Hieble, J. P.; Wilson, S.; Bergsma, D. J.; Fitzgerald, L. R. Cloning,

expression, and pharmacological characterization of a novel

human histamine receptor. Mol. Pharmacol. 2001, 59, 434–441.

(8) Morse, K. L.; Behan, J.; Laz, T. M.; West, R. E., Jr.; Greenfeder,

S. A.; Anthes, J. C.; Umland, S.; Wan, Y.; Hipkin, R. W.;

Gonsiorek, W.; Shin, N.; Gustafson, E. L.; Qiao, X.; Wang, S.;

Hedrick, J. A.; Greene, J.; Bayne, M.; Monsma, F. J., Jr. Cloning

and characterization of a novel human histamine receptor.

J. Pharmacol. Exp. Ther. 2001, 296, 1058–1066.

(9) O’Reilly, M.; Alpert, R.; Jenkinson, S.; Gladue, R. P.; Foo, S.;

Trim, S.; Peter, B.; Trevethick, M.; Fidock, M. Identification of a

histamine H₄receptor on human eosinophils. Role in eosinophil

chemotaxis. J. Recept. Signal Transduction Res. 2002, 22, 431–448.

(10) Coge, F.; Guenin, S.-P.; Rique, H.; Boutin, J. A.; Galizzi, J.-P.

Structure and expression of the human histamine H₄-receptor gene.

Biochem. Biophys. Res. Commun. 2001, 284, 301–309.

(11) Nguyen, T.; Shapiro, D. A.; George, S. R.; Setola, V.; Lee, D. K.;

Cheng, R.; Rauser, L.; Lee, S. P.; Lynch, K. R.; Roth, B. L.;

O’Dowd, B. F. Discovery of a novel member of the histamine

receptor family. Mol. Pharmacol. 2001, 59, 427–433.

(12) Liu, C.; Ma, X.-J.; Jiang, X.; Wilson, S. J.; Hofstra, C. L.; Blevitt,

J.; Pyati, J.; Li, X.; Chai, W.; Carruthers, N.; Lovenberg, T. W.

Cloning and pharmacological characterization of a fourth hista-

mine receptor (H₄) expressed in bone marrow. Mol. Pharmacol.

2001, 59, 420–426.

(13) Gantner, F.; Sakai, K.; Tusche, M. W.; Cruikshank, W. W.;

Center, D. M.; Bacon, K. B. Histamine H₄and H₂receptors

control histamine-induced interleukin-16 release from human

CD8^þT cells. J. Pharmacol. Exp. Ther. 2002, 303, 300–307.

(14) Hofstra, C. L.; Desai, P. J.; Thurmond, R. L.; Fung-Leung, W.-P.

Histamine H₄receptor mediates chemotaxis and calcium mobiliza-

tion of mast cells. J. Pharmacol. Exp. Ther. 2003, 305, 1212–1221.

(15) Lippert, U.; Artuc, M.; Grutzkau, A.; Babina, M.; Guhl, S.; Haase,

I.; Blaschke, V.; Zachmann, K.; Knosalla, M.; Middel, P.; Kruger-

Krasagakis, S.; Henz, B. M. Human skin mast cells express H₂and

H₄, but not H₃receptors. J. Invest. Dermatol. 2003, 123, 116–123.

(16) Buckland, K. F.; Williams, T. J.; Conroy, D. M. Histamine induces

cytoskeletal changes in human eosinophils via the H₄receptor.

Br. J. Pharmacol. 2003, 140, 1117–1127.

The final snapshot of the MD simulation was roughly mini-

mized (SYBYL 7.3, Amber-FF99 force field). Compound 67

was manually docked into the model in an energetically favor-

able conformation, considering results of previous in vitro

mutagenesis⁵¹and theoretical studies.⁵²Then the same MD

simulation protocol as described above was applied to the

ligand-hH₄R complex. Parameters for compound 67 were

adopted from the ffG53A6 force field. The ligand-hH₄R com-

plex from the final (1 ns) snapshot of the productive phase was

extracted and gradually minimized using SYBYL 7.3: (1) ligand

fixed and provided with Gasteiger-Huckel charges, Amber-

FF99 force field, distant dependent ε=4, 25 cycles of steepest

descent, then Powell algorithm; (2) compound 67 and a region of

6 A around, Tripos force field,⁷⁴distant dependent ε=1, Powell

˚

method, and rms gradient of 0.05 kcal mol^-1; (3) as in step 1,

Powell method, and rms gradient 0.01 kcal mol^-1. The rms

deviation of the TM backbones between the resulting model and

˚

the β₂-adrenoceptor crystal structure 2RH1 amounts to 1.07 A.

Acknowledgment. The authors are grateful to Kerstin Fisch,

Karin Schadendorf, Gertraud Wilberg, and Astrid Seefeld

for expert technical assistance, to Dr. Erich Schneider for help-

ful discussions, to Dr. Robin Thurmond (Department of

(17) Takeshita, K.; Sakai, K.; Bacon, K. B.; Gantner, F. Critical role

of histamine H₄receptor in leukotriene B₄production and mast

6312 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20

Igel et al.

cell-dependent neutrophil recruitment induced by zymosan in vivo.

J. Pharmacol. Exp. Ther. 2003, 307, 1072–1078.

(18) Dijkstra, D.; Leurs, R.; Chazot, P.; Shenton, F. C.; Stark, H.;

Werfel, T.; Gutzmer, R. Histamine downregulates monocyte

CCL2 production through the histamine H₄receptor. J. Allergy

Clin. Immunol. 2007, 120, 300–307.

(19) Thurmond, R. L.; Desai, P. J.; Dunford, P. J.; Fung-Leung, W.-P.;

Hofstra, C. L.; Jiang, W.; Nguyen, S.; Riley, J. P.; Sun, S.;

Williams, K. N.; Edwards, J. P.; Karlsson, L. A potent and selective

histamine H₄receptor antagonist with anti-inflammatory proper-

ties. J. Pharmacol. Exp. Ther. 2004, 309, 404–413.

(20) Coruzzi, G.; Adami, M.; Guaita, E.; de Esch, I. J. P.; Leurs, R.

Antiinflammatory and antinociceptive effects of the selective hista-

mine H₄-receptor antagonists JNJ7777120 and VUF6002 in a rat

model of carrageenan-induced acute inflammation. Eur. J. Phar-

macol. 2007, 563, 240–244.

guanidine ([(3)H]UR-PI294), a high-affinity histamine H(3) and

H(4) receptor radioligand. ChemMedChem 2009, 4, 225–231.

(36) pK_avalue was calculated with the following: ACD Labs 9.0;

Advanced Chemistry Development: Toronto, Canada.

(37) Durant, G. J.; Emmett, J. C.; Ganellin, C. R.; Miles, P. D.; Parsons,

M. E.; Prain, H. D.; White, G. R. Cyanoguanidine-thiourea

equivalence in the development of the histamine H₂-receptor

antagonist, cimetidine. J. Med. Chem. 1977, 20, 901–906.

(38) Durant, G. J.; Ganellin, C. R.; Hills, D. W.; Miles, P. D.; Parsons,

M. E.; Pepper, E. S.; White, G. R. The histamine H₂receptor

agonist impromidine: synthesis and structure-activity considera-

tions. J. Med. Chem. 1985, 28, 1414–1422.

(39) Kraus, A. Highly Potent, Selective Acylguanidine-Type Histamine

H2 Receptor Agonists: Synthesis and Structure-Activity Rela-

tionships. Ph.D. Thesis, University of Regensburg, Regensburg,

Germany, 2007; http://www.opus-bayern.de/uni-regensburg/front-

door.php?source_opus=904&la=de.

(40) Buschauer, A. Synthesis and pharmacological activity of aryl-

methylthioethylguanidines. Arzneim.-Forsch. 1987, 37 (II), 1008–

1012.

(21) Smits, R. A.; Lim, H. D.; Hanzer, A.; Zuiderveld, O. P.; Guaita, E.;

Adami, M.; Coruzzi, G.; Leurs, R.; de Esch, I. J. P. Fragment based

design of new H₄receptor-ligands with anti-inflammatory proper-

ties in vivo. J. Med. Chem. 2008, 51, 2457–2467.

(22) Dunford, P. J.; O’Donnell, N.; Riley, J. P.; Williams, K. N.;

Karlsson, L.; Thurmond, R. L. The histamine H₄receptor mediates

allergic airway inflammation by regulating the activation of CD4^þ

T cells. J. Immunol. 2006, 176, 7062–7070.

(41) Webb, R. L.; Eggleston, D. S.; Labaw, C. S.; Lewis, J. J.; Wert, K.

Diphenyl cyancarbonimidate and dichlorodiphenoxymethane as

synthons for the construction of heterocyclic systems of medicinal

interest. J. Heterocycl. Chem. 1987, 24, 275–278.

(23) Jablonowski, J. A.; Grice, C. A.; Chai, W.; Dvorak, C. A.; Venable,

J. D.; Kwok, A. K.; Ly, K. S.; Wei, J.; Baker, S. M.; Desai, P. J.;

Jiang, W.; Wilson, S. J.; Thurmond, R. L.; Karlsson, L.; Edwards,

J. P.; Lovenberg, T. W.; Carruthers, N. I. The first potent and

selective non-imidazole human histamine H₄receptor antagonists.

J. Med. Chem. 2003, 46, 3957–3960.

(24) Dunford, P. J.; Williams, K. N.; Desai, P. J.; Karlsson, L.;

McQueen, D.; Thurmond, R. L. Histamine H₄receptor antago-

nists are superior to traditional antihistamines in the attenuation of

experimental pruritus. J. Allergy Clin. Immunol. 2007, 119, 176–

183.

(42) Webb, R. L.; Labaw, C. S. Diphenyl cyanocarbonimidate. a

versatile synthon for the construction of heterocyclic systems.

J. Heterocycl. Chem. 1982, 19, 1205–1206.

(43) Kelley, M. T.; Burckstummer, T.; Wenzel-Seifert, K.; Dove, S.;

Buschauer, A.; Seifert, R. Distinct interaction of human and guinea

pig histamine H₂-receptor with guanidine-type agonists. Mol.

Pharmacol. 2001, 60, 1210–1225.

(44) Seifert, R.; Wenzel-Seifert, K.; Burckstummer, T.; Pertz, H. H.;

Schunack, W.; Dove, S.; Buschauer, A.; Elz, S. Multiple differences

in agonist and antagonist pharmacology between human and

guinea pig histamine H₁-receptor. J. Pharmacol. Exp. Ther. 2003,

305, 1104–1115.

(45) Lim, H. D.; Jongejan, A.; Bakker, R. A.; Haaksma, E.; de Esch,

I. J. P.; Leurs, R. Phenylalanine 169 in the second extracellular loop

of the human histamine H-4 receptor is responsible for the differ-

ence in agonist binding between human and mouse H-4 receptors.

J. Pharmacol. Exp. Ther. 2008, 327, 88–96.

(46) Altenbach, R. J.; Adair, R. M.; Bettencourt, B. M.; Black, L. A.;

Fix-Stenzel, S. R.; Gopalakrishnan, S. M.; Hsieh, G. C.; Liu, H.;

Marsh, K. C.; McPherson, M. J.; Milicic, I.; Miller, T. R.;

Vortherms, T. A.; Warrior, U.; Wetter, J. M.; Wishart, N.; Witte,

D. G.; Honore, P.; Esbenshade, T. A.; Hancock, A. A.; Brioni,

J. D.; Cowart, M. D. Structure-activity studies on a series of a

2-aminopyrimidine-containing histamine H4 receptor ligands.

J. Med. Chem. 2008, 51, 6571–6580.

(25) Jablonowski, J. A.; Carruthers, N. I.; Thurmond, R. L. The

histamine H₄receptor and potential therapeutic uses for H₄

ligands. Mini-Rev. Med. Chem. 2004, 4, 993–1000.

(26) Lim, H. D.; van Rijn, R. M.; Ling, P.; Bakker, R. A.; Thurmond, R.

L.; Leurs, R. Evaluation of histamine H₁-, H₂-, and H₃-receptor

ligands at the human histamine H₄receptor: identification of

4-methylhistamine as the first potent and selective H₄receptor

agonist. J. Pharmacol. Exp. Ther. 2005, 314, 1310–1321.

(27) Arrang, J. M.; Garbarg, M.; Lancelot, J. C.; Lecomte, J. M.;

Pollard, H.; Robba, M.; Schunack, W.; Schwartz, J. C. Highly

potent and selective ligands for histamine H3-receptors. Nature

1987, 327, 117–123.

(28) Morisset, S.; Rouleau, A.; Ligneau, X.; Gbahou, F.; Tardivel-

Lacombe, J.; Stark, H.; Schunack, W.; Ganellin, C. R.; Schwartz,

J. C.; Arrang, J. M. High constitutive activity of native H3

receptors regulates histamine neurons in brain. Nature 2000, 408,

860–864.

(29) Lim, H. D.; Smits, R. A.; Bakker, R. A.; van Dam, C. M. E.; de

Esch, I. J. P.; Leurs, R. Discovery of S-(2-guanidylethyl)-isothio-

urea (VUF 8430) as a potent nonimidazole histamine H₄receptor

agonist. J. Med. Chem. 2006, 49, 6650–6651.

(30) Hashimoto, T.; Harusawa, S.; Araki, L.; Zuiderveld, O. P.; Smit,

M. J.; Imazu, T.; Takashima, S.; Yamamoto, Y.; Sakamoto, Y.;

Kurihara, T.; Leurs, R.; Bakker, R. A.; Yamatodani, A. A selective

human H₄-receptor agonist: (-)-2-cyano-1-methyl-3-{(2R,5R)-

5-[1H-imidazol-4(5)-yl]tetrahydrofuran-2-yl}methylguanidine.

J. Med. Chem. 2003, 46, 3162–3165.

(31) Ghorai, P.; Kraus, A.; Keller, M.; Gotte, C.; Igel, P.; Schneider, E.;

Schnell, D.; Bernhardt, G.; Dove, S.; Zabel, M.; Elz, S.; Seifert, R.;

Buschauer, A. Acylguanidines as bioisosteres of guanidines:

N^G-acylated imidazolylpropylguanidines, a new class of histamine

H₂receptor agonists. J. Med. Chem. 2008, 51, 7193–7204.

(32) Xie, S.-X.; Kraus, A.; Ghorai, P.; Ye, Q.-Z.; Elz, S.; Buschauer, A.;

Seifert, R. N¹-(3-Cyclohexylbutanoyl)-N²-[3-(1H-imidazol-4-yl)-

propyl]guanidine (UR-AK57), a potent partial agonist for the

human histamine H₁- and H₂-receptors. J. Pharmacol. Exp. Ther.

2006, 317, 1262–1268.

(33) Xie, S.-X.; Ghorai, P.; Ye, Q.-Z.; Buschauer, A.; Seifert, R. Probing

ligand-specific histamine H₁- and H₂-receptor conformations with

N^G-acylated imidazolylpropylguanidines. J. Pharmacol. Exp.

Ther. 2006, 317, 139–146.

(34) Igel, P.; Schneider, E.; Schnell, D.; Elz, S.; Seifert, R.; Buschauer,

A. N^G-Acylated imidazolylpropylguanidines as potent histamine

H₄receptor agonists: selectivity by variation of the N^G-substituent.

J. Med. Chem. 2009, 52, 2623–2627.

(47) Schnell, D. Department of Pharmacology and Toxicology, Uni-

versity of Regensburg, Germany. Personal communication, 2009.

(48) Jansen, F. P.; Rademaker, B.; Bast, A.; Timmerman, H. The first

radiolabeled histamine H3 receptor antagonist, [

¹²⁵I]iodo-

phenpropit: saturable and reversible binding to rat cortex mem-

branes. Eur. J. Pharmacol. 1992, 217, 203–205.

(49) Cherezov, V.; Rosenbaum, D. M.; Hanson, M. A.; Rasmussen,

S. G.; Thian, F. S.; Kobilka, T. S.; Choi, H. J.; Kuhn, P.; Weis,

W. I.; Kobilka, B. K.; Stevens, R. C. High-resolution crystal

structure of an engineered human beta2-adrenergic G protein-

coupled receptor. Science 2007, 318, 1258–1265.

(50) Rosenbaum, D. M.; Cherezov, V.; Hanson, M. A.; Rasmussen,

S. G.; Thian, F. S.; Kobilka, T. S.; Choi, H. J.; Yao, X. J.; Weis,

W. I.; Stevens, R. C.; Kobilka, B. K. GPCR engineering yields

high-resolution structural insights into beta2-adrenergic receptor

function. Science 2007, 318, 1266–1273.

(51) Shin, N.; Coates, E.; Murgolo, N. J.; Morse, K. L.; Bayne, M.;

Strader, C. D.; Monsma, F. J., Jr. Molecular modeling and site-

specific mutagenesis of the histamine-binding site of the histamine

H4 receptor. Mol. Pharmacol. 2002, 62, 38–47.

(52) Jongejan, A.; Lim, H. D.; Smits, R. A.; de Esch, I. J. P.; Haaksma,

E.; Leurs, R. Delineation of agonist binding to the human hista-

mine H-4 receptor using mutational analysis, homology modeling,

and ab initio calculations. J. Chem. Inf. Model. 2008, 48, 1455–

1463.

(53) Arbuznikov, A. V.; Sheludyakova, L. A.; Burgina, E. B. Ab-initio

study of cyanoguanidine isomers. Chem. Phys. Lett. 1995, 240,

239–244.

(54) Bach, R. D.; Mcdouall, J. J. W.; Owensby, A. L.; Schlegel, H. B.;

Holubka, J. W.; Ball, J. C. Structure and dynamics of dicyandi-

amide: a theoretical study. J. Phys. Org. Chem. 1991, 4, 125–134.

(55) Ballesteros, J. A.; Weinstein, H. Integrated methods for the con-

struction of three-dimensional models and computational probing

(35) Igel, P.; Schnell, D.; Bernhardt, G.; Seifert, R.; Buschauer, A.

Tritium-labeled N(1)-[3-(1H-imidazol-4-yl)propyl]-N(2)-propionyl-

Article

Journal of Medicinal Chemistry, 2009, Vol. 52, No. 20 6313

of structure-function relations in G protein-coupled receptors.

(64) Pathak, U.; Raza, S. K.; Kulkarni, A. S.; Vijayaraghvan, R.;

Kumar, P.; Jaiswal, D. K. Novel S-substituted aminoalkylamino

ethanethiols as potential antidotes against sulfur mustard toxicity.

J. Med. Chem. 2004, 47, 3817–3822.

Methods Neurosci. 1995, 25, 366–428.

(56) Prout, K.; Critchle, Sr.; Ganellin, C. R. 2-(4-Imidazolyl)ethyl-

ammonium bromide (histamine monohydrobromide). Acta Crys-

tallogr. B 1974, 30, 2884–2886.

(57) Preuss, H.; Ghorai, P.; Kraus, A.; Dove, S.; Buschauer, A.; Seifert,

R. Mutations of Cys-17 and Ala-271 in the human histamine H2

receptor determine the species selectivity of guanidine-type ago-

nists and increase constitutive activity. J. Pharmacol. Exp. Ther.

2007, 321, 975–982.

(65) Hannedouche, J.; Clarkson, G. J.; Wills, M. A new class of

“tethered” ruthenium(II) catalyst for asymmetric transfer hydro-

genation reactions. J. Am. Chem. Soc. 2004, 126, 986–987.

(66) Walseth, T. F.; Johnson, R. A. The enzymatic preparation of

[r-³²P] nucleoside triphosphates, cyclic [³²P]AMP, and cyclic

[

³²P]GMP. Biochim. Biophys. Acta 1979, 562, 11–31.

(58) Lei, S.; Liapakis, G.; Xu, R.; Guarnieri, F.; Ballesteros, J. A.;

Javitch, J. A. Beta(2) adrenergic receptor activation. Modulation

of the proline kink in transmembrane 6 by a rotamer toggle switch.

J. Biol. Chem. 2002, 277, 40989–40996.

(59) Seifert, R.; Wenzel-Seifert, K.; Kobilka, B. K. GPCR-Galpha

fusion proteins: molecular analysis of receptor-G-protein coupling.

Trends Pharmacol. Sci. 1999, 20, 383–389.

(67) Cheng, Y.-C.; Prusoff, W. H. Relationship between the inhibition

constant (K_I) and the concentration of inhibitor which causes 50%

inhibition (I₅₀) of an enzymatic reaction. Biochem. Pharmacol.

1973, 22, 3099–3108.

(68) Schneider, E. H.; Schnell, D.; Papa, D.; Seifert, R. High constitu-

tive activity and a G-protein-independent high-affinity state of the

human histamine H(4)-receptor. Biochemistry 2009, 48, 1424–

1438.

(69) Wang, J.; Wolf, R. M.; Caldwell, J. W.; Kollman, P. A.; Case, D. A.

Development and testing of a general amber force field. J. Comput.

Chem. 2004, 25, 1157–1174.

(70) Lovell, S. C.; Word, J. M.; Richardson, J. S.; Richardson,

D. C. The penultimate rotamer library. Proteins 2000, 40, 389–

408.

(71) Strasser, A.; Striegl, B.; Wittmann, H. J.; Seifert, R. Pharmacolo-

gical profile of histaprodifens at four recombinant histamine H-1

receptor species isoforms. J. Pharmacol. Exp. Ther. 2008, 324,

60–71.

(72) Van der Spoel, D.; Lindahl, E.; Hess, B.; Groenhof, G.; Mark, A.

E.; Berendsen, H. J. C. GROMACS: fast, flexible, and free.

J. Comput. Chem. 2005, 26, 1701–1718.

(73) Oostenbrink, C.; Villa, A.; Mark, A. E.; Van Gunsteren, W. F. A

biomolecular force field based on the free enthalpy of hydration

and solvation: the GROMOS force-field parameter sets 53A5 and

53A6. J. Comput. Chem. 2004, 25, 1656–1676.

(60) Klinker, J. F.; Hageluken, A.; Grunbaum, L.; Heilmann, I.;

Nurnberg, B.; Harhammer, R.; Offermanns, S.; Schwaner, I.;

Ervens, J.; Wenzel-Seifert, K. Mastoparan may activate GTP

hydrolysis by Gi-proteins in HL-60 membranes indirectly through

interaction with nucleoside diphosphate kinase. Biochem. J. 1994,

304 (Part 2), 377–383.

(61) Hageluken, A.; Grunbaum, L.; Klinker, J. F.; Nurnberg, B.;

Harhammer, R.; Schultz, G.; Leschke, C.; Schunack, W.; Seifert, R.

Histamine receptor-dependent and/or -independent activation of gua-

nine nucleotide-binding proteins by histamine and 2-substituted hista-

mine derivatives in human leukemia (HL-60) and human

erythroleukemia (HEL) cells. Biochem. Pharmacol. 1995, 49, 901–914.

(62) Seifert, R.; Hageluken, A.; Hoer, A.; Hoer, D.; Grunbaum, L.;

Offermanns, S.; Schwaner, I.; Zingel, V.; Schunack, W.; Schultz, G.

The H₁receptor agonist 2-(3-chlorophenyl)histamine activates G_i

proteins in HL-60 cells through a mechanism that is independent of

known histamine receptor subtypes. Mol. Pharmacol. 1994, 45,

578–586.

(63) Adachi, N.; Liu, K.; Motoki, A.; Nishibori, M.; Arai, T. Suppres-

sion of ischemia/reperfusion liver injury by histamine H4 receptor

stimulation in rats. Eur. J. Pharmacol. 2006, 544, 181–187.

(74) Clark, M.; Cramer, R. D. I.; Van Opdenbosch, N. Validation of the

general purpose Tripos 5.2 force field. J. Comput. Chem. 1989, 10,

982–1012.

Article Doi

Synthesis and structure-activity relationships of cyanoguanidine-type and structurally related histamine H4 receptor agonists

DOI: 10.1021/jm900526h

Source and publish data:

Authors:

Article abstract of DOI:10.1021/jm900526h

Full text of DOI:10.1021/jm900526h

Products guided by the article

R&D Labs maybe for 1192559-80-0

Relevant to this article

Hot Product