Ng-acylated imidazolylpropylguanidines as potent histamine H4 receptor agonists: selectivity by variation of the n g-substituent?

Article abstract of DOI:10.1021/jm9000693

3-(1H-Imidazol-4-yl)propylguanidine (SK&F 91486, 4) was identified as a potent partial agonist at the human histamine H3 receptor (hH₃R) and human histamine H₄ receptor (hH₄R). With the aim to increase selectivity for the

Full text of DOI:10.1021/jm9000693

J. Med. Chem. 2009, 52, 2623–2627
2623
NG-Acylated Imidazolylpropylguanidines as Potent Histamine H4 Receptor Agonists: Selectivity
by Variation of the NG-Substituent3
Patrick Igel,† Erich Schneider,‡ David Schnell,‡ Sigurd Elz,† Roland Seifert,§ and Armin Buschauer*,†
Department of Pharmaceutical/Medicinal Chemistry and Department of Pharmacology and Toxicology, Faculty of Chemistry and Pharmacy,
UniVersity of Regensburg, UniVersita¨tsstrasse 31, D-93053 Regensburg, Germany, and Institute of Pharmacology, Medical School of HannoVer,
D-30625 HannoVer, Germany
ReceiVed October 18, 2008
3-(1H-Imidazol-4-yl)propylguanidine (SK&F 91486, 4) was identified as a potent partial agonist at the human
histamine H3 receptor (hH3R) and human histamine H4 receptor (hH4R). With the aim to increase selectivity
for the hH4R, the guanidine group in 4 was acylated. N1-Acetyl-N2-[3-(1H-imidazol-4-yl)propyl]guanidine
(UR-PI288, 13) was a potent full agonist at the hH4R (pEC50 ) 8.31; R ) 1.00), possessing more than
1000- and 100-fold selectivity relative to the hH1R and hH2R, respectively, and possessing only low intrinsic
activity (R ) 0.27) at the hH3R.
Chart 1. Structures of Selective H4R Agonists 15 and 2,2
Introduction
Imidazolylpropylguanidines 37 and 4,9 Selective H4R Antagonist
5,10 and Novel NG-Acylated Imidazolylpropylguanidines 6-16
Presented in This Study
The biogenic amine histamine mediates its effects via four
histamine receptor subtypes, termed H1, H2, H3, and H4 receptors
(H1R, H2R, H3R, and H4R, respectively), all belonging to the
superfamily of G-protein-coupled receptors (GPCRsa).1 The H4R
is the most recently identified member of the family of histamine
receptors and was identified in 2000 and 2001, when several
research groups identified and cloned the gene encoding the
hH4R based on its high degree of sequence homology with the
hH3R. This rather high degree of homology (37% at the protein
level) explains the high affinity of many H3R ligands for the
hH4R, in particular imidazole-containing compounds such as
thioperamide [4-(1H-imidazol-4-yl)piperidin-1-yl)(piperidin-1-
yl)methanethione].2 The hH4R is mainly expressed on hemato-
poietic cells like mast cells, eosinophils, dendritic cells, T
lymphocytes, and monocytes and seems to play a crucial role
in inflammatory and immunological processes (for recent
reviews, see refs 3 and 4).
To further study the (patho)physiological functions of the
receptor, selective ligandssincluding agonistssare required as
pharmacological tools. At present, only a few selective H4R
agonists like 1 (OUP-16) or 2 (4-methylhistamine) are available
(Chart 1).2,3,5 Among a series of NG-acylated imidazolylpropyl-
guanidines6-8 originally developed by our group as potent H2R
agonists, several compounds were serendipitously found to be
even more potent at the hH3R and the hH4R [for example, 3
(UR-AK24) (Chart 1)].7,8 Most of the investigated compounds
displayed high intrinsic activity at the hH4R but low intrinsic
activity at the hH3R. Subsequent evaluation of the parent
structure, 3-(1H-imidazol-4-yl)propylguanidine [4 (SK&F 91486)
(Chart 1)], a weak partial H2R agonist,9 surprisingly revealed
this compound as a highly potent partial agonist at the hH3R
and hH4R (Table 1).
Starting from this model compound, we sought to explore
the potential to develop more selective hH4R agonists by
acylation of the guanidine group in 4. One strategy was to
introduce acyl residues containing structural motifs of the potent
and selective hH4R antagonist 5 (Chart 1).10 The resulting hybrid
molecules (6-12) consisting of an H4R agonistic and antago-
nistic moiety could be favorable for hH4R selectivity and
activity, provided that the acyl residue can additionally interact
with the binding site of 5 (JNJ 7777120). The second approach
focused on the size of the acyl residues with respect to selectivity
for the H4R over the H2R. The imidazolylpropylguanidine
portion of the NG-acylated imidazolylpropylguanidines is con-
sidered to be responsible for H2R agonistic activity, whereas
* To whom correspondence should be addressed. Phone: +49-941
9434827. Fax: +49-941 9434820. E-mail: armin.buschauer@chemie.uni-
regensburg.de.
3
Dedicated to Prof. Dr. Gottfried Märkl, Regensburg, on the occasion
of his 80th birthday.
†
Department of Pharmaceutical/Medicinal Chemistry, Faculty of Chem-
istry and Pharmacy, University of Regensburg.
‡
Department of Pharmacology and Toxicology, Faculty of Chemistry
and Pharmacy, University of Regensburg.
§
Medical School of Hannover.
a Abbreviations: gpH1R, guinea pig H1R; gpH2R, guinea pig H2R; GPCR,
G-protein-coupled receptor; Gꢀ1γ2, G-protein ꢀ1 and γ2 subunit; GRi, R
subunit of the Gi-protein; HMBC, heteronuclear multiple-bond correlation;
hH1R, hH2R, hH3R, and hH4R, human histamine receptor subtypes H1-4
,
respectively; hH2R-GsRS, fusion protein of the hH2R and the short splice
variant of GsR; hH4R-RGS19, fusion protein of the hH4R and RGS19; NG,
guanidine nitrogen; NOESY, nuclear Overhauser enhancement spectroscopy;
RGS, regulator of G-protein signaling proteins; TM, transmembrane domain
of a GPCR.
10.1021/jm9000693 CCC: $40.75
2009 American Chemical Society
Published on Web 03/24/2009

2624 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8
Brief Articles
Table 1. Activities of the Prepared Compounds at the hH1R, hH2R, hH3R, and hH4R in the Steady-State GTPase Assaya
hH1R
hH2R
hH3R
hH4R
compound
pEC50 (pKB)
R
n
pEC50
5.926
R
n
pEC50 (pKB)
R
n
pEC50 (pKB)
R
n
histamine
6.726
-
(<4.90)
(<5.00)
1.00
1.00
7.60 ( 0.05
7.01 ( 0.08
8.608
1.00
-0.71 ( 0.06
3
5
7.92 ( 0.09
6.96 ( 0.06
7.828
8.09 ( 0.04
7.72 ( 0.14
8.15 ( 0.17
1.00
-0.95 ( 0.07
8
6
thioperamide
-
-
-
36
4
0.357
7.177
0.877
0.248
0.848
ndb
2
2
2
2
5.59 ( 0.01 0.66 ( 0.02
7.44 ( 0.05 0.81 ( 0.02
7.09 ( 0.03 0.77 ( 0.02
6.54 ( 0.02 0.87 ( 0.00
6.02 ( 0.16 0.51 ( 0.01
7.08 ( 0.00 0.33 ( 0.04
6.46 ( 0.07 0.68 ( 0.04
7.52 ( 0.11 0.51 ( 0.01
6.14 ( 0.04 0.76 ( 0.02
6.43 ( 0.03 0.83 ( 0.08
6.85 ( 0.03 0.77 ( 0.02
6.96 ( 0.14 0.86 ( 0.01
3
2
2
2
3
2
3
2
2
2
2
2
8.12 ( 0.10
0.69 ( 0.04
3
2
2
2
2
2
2
2
3
3
3
3
0.83 ( 0.01
0.70 ( 0.12
0.45 ( 0.08
3
3
3
2
3
2
4
2
3
3
3
3
6
7
8
9
(5.68 ( 0.13) 0.00 ( 0.08
(7.51 ( 0.12) -0.15 ( 0.02
(7.57 ( 0.15) -0.18 ( 0.02
(6.96 ( 0.04) -0.38 ( 0.01
(5.40 ( 0.00) 0.09 ( 0.02
5.77 ( 0.03
0.42 ( 0.00
(6.43 ( 0.02) 0.21 ( 0.03
ndb
ndb
ndb
ndb
ndb
ndb
7.80 ( 0.00
(7.17 ( 0.04) 0.10 ( 0.02
7.96 ( 0.08 0.30 ( 0.01
(7.17 ( 0.06) -0.18 ( 0.02
8.44 ( 0.14
8.80 ( 0.06
8.80 ( 0.07
8.85 ( 0.18
0.45 ( 0.03
7.55 ( 0.20
7.48 ( 0.12
6.82 ( 0.07
0.71 ( 0.05
0.37 ( 0.02
0.52 ( 0.06
10
11
12
13
14
15
16
(4.96 ( 0.01) 0.16 ( 0.08
2
2
2
2
2
(7.02 ( 0.05) -0.06 ( 0.14
4.89 ( 0.03
5.46 ( 0.15
5.66 ( 0.06
5.59 ( 0.04
0.24 ( 0.02
0.30 ( 0.01
0.33 ( 0.03
0.35 ( 0.01
0.27 ( 0.02
0.39 ( 0.03
0.37 ( 0.00
0.26 ( 0.02
8.31 ( 0.04
8.52 ( 0.04
8.59 ( 0.02
8.43 ( 0.02
1.00 ( 0.02
0.90 ( 0.02
0.96 ( 0.03
0.94 ( 0.04
a Steady-state GTPase activity in Sf9 membranes expressing the hH1R plus RGS4, hH2R-GsRS, hH3R plus GiR2 plus Gꢀ1γ2 plus RGS4 or hH4R-RGS19
fusion protein plus GiR2 plus Gꢀ1γ2 determined as described in Pharmacological methods in the Supporting Information. b Not determined.
Scheme 1. Synthesis of the NG-Acylated Imidazolylpropylguanidines 6-16 and 4a
a Reagents and conditions: (i) for 31, 33, and 35, CDI (1.2 equiv), NaH (60% dispersion in mineral oil) (2 equiv), THF, 5 h, room temperature; (ii) for
32, 34, and 36-41, EDC ·HCl (1.2 equiv), DMAP (1.1 equiv), DCM, 24 h, 0 °C f room temperature; (iii) for 31, 33, and 35, TFA (20%), DCM, 5 h, room
temperature; (iv) for 4, 32, 34, and 36-41, 1 M HCl, 30 min, reflux. b Compare to Scheme 2 in the Supporting Information. c Mixture of III and IV. d Isomer
III.
the NG-acyl residue is assumed to contribute to H2R affinity.8
However, the endogenous ligand histamine, lacking additional
affinity-conferring residues, is ∼50 times more potent at the
hH3R and the hH4R than at the hH2R (Table 1). As bulky NG-
acyl groups may be unnecessary or unfavorable for obtaining
highly potent compounds at the hH3R and hH4R, acylation of 4
with small alkanoyl groups (13-16) was explored to switch
selectivity toward the hH3R and hH4R and to gain more insight
into the structure-activity relationships of the NG-acylated
imidazolylpropylguanidines at the distinct histamine receptor
subtypes.
giving a six-membered ring; analyzed by mass spectrometry).
Therefore, a modified synthetic pathway was employed. To
avoid the strongly basic conditions, instead of the free guanidine
(pKa ≈ 13),11 Boc-protected derivative (pKa ≈ 5)11 18 or 19
(18, mixture of isomers I and II; 19, single isomer depending
on the synthetic procedure employed for the preparation; cf.
the Supporting Information) was used,12 yielding the trityl- and
Boc-protected intermediates 32, 34, and 36-41. This procedure
can be performed in a one-pot reaction without preactivation
of the carboxylic acids. Moreover, the isolation and purification
of the less polar and less basic 2-fold acylated intermediates
were facilitated. Fewer side reactions were observed, and
excessive acylation of the guanidine moiety was prevented.
Nevertheless, as marked cleavage of one acyl group could be
detected by NMR after storage in solution for few days,
intermediates 32, 34, and 36-41 should be deprotected in a
timely manner. Detritylation of the imidazole ring was routinely
performed in a mixture of 20% TFA in dichloromethane (6, 8,
and 10).8 However, under these conditions, the deprotection of
compound 35 gave 10 in only 3% yield and produced a large
number of side products, whereas refluxing in hydrochloric acid
resulted in 10-20 times higher yields. In contrast to the TFA/
Chemistry
The NG-acylated imidazolylpropylguanidines 31, 33, and 35
were synthesized by acylation of the guanidine base 17 with
CDI-activated carboxylic acids (compounds 20, 22, 24) accord-
ing to the method described by Ghorai et al. (Scheme 1).8
However, preparation of the acylguanidines via this route
showed drawbacks. The synthesis of 34 failed since under the
strongly basic conditions (NaH) required for the deprotonation
of the guanidine moiety an intramolecular cyclization of the
carboxylic acid 23 occurred (acylation of the indole nitrogen

Brief Articles
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8 2625
dichloromethane mixture, side reactions were reduced in aque-
ous medium, presumably because the intermediate trityl cation
was trapped and precipitated as triphenylmethanol. This pro-
cedure was applied for the preparation of NG-acylated imida-
zolylpropylguanidines 7, 9, and 11-16 and for 4.9
In theory, acylation of the Boc-protected guanidine is possible
at the nitrogen adjacent to the carbon chain or at the unsubsti-
tuted nitrogen. However, two-dimensional NMR experiments
(HMBC and NOESY) with compound 15, synthesized from
building block 19, i.e., isomer I, bearing the Boc group at the
terminal nitrogen, confirmed the acylation at the designated third
guanidine nitrogen. The preparation of imidazolylpropyl-
guanidines 17-19, guanidinylation reagents 49-52, and car-
boxylic acids 23-26 is reported in the Supporting Information.
directly (8) or via amino acid linkers (9-12). The amino acid
spacers were considered as “ring-opened versions” of the
piperazine ring in 5, and the basic tertiary amine, which is
regarded as crucial for H4R affinity of 5,13 may be mimicked
by the acylguanidine group. Direct attachment of the indole-
2-carboxylic acid (8) significantly changed the pharmacological
profile at HxRs relative to reference compound 4. Compound 8
exhibited moderate partial agonism at hH1R (pEC50 ) 5.77; R
) 0.42). At the hH2R, 8 was ∼10 times more potent than 4 and
exhibited slightly elevated intrinsic activity (pEC50 ) 6.54; R
) 0.87). In contrast, at the hH3R and hH4R, 8 was >10 times
less potent and the intrinsic activity substantially decreased. The
incorporation of a glycine spacer (9) was not tolerated in terms
of hH2R activity; compared to that of 8, the agonstic activity
dropped remarkably (pEC50 ) 6.02; R ) 0.51). At the hH3R
and hH4R, 9 was more active than compound 8 and displayed
partial agonistic activity at both receptors. Replacing the glycine
spacer with ꢀ-alanine (10) and 4-aminobutyric acid (12)
increased activity at the hH2R by factors of 10 (pEC50 ) 7.08)
and 30 (pEC50 ) 7.52), respectively. Evidently, the increased
flexibility of the higher homologues favored high hH2R activity.
The hH3R and hH4R affinities remained largely unaffected,
whereas the intrinsic activities at these receptors were signifi-
cantly reduced. At all hHRs, especially at the hH2R, N-
methylation of the amide nitrogen (11) increased intrinsic
activities, but the affinities at the hH2R and hH4R (pEC50 values
of 6.46 and 6.82, respectively) were reduced, suggesting that
the amide NH group contributes to hH2R and hH4R binding,
although steric factors may also play a role. In contrast, at the
hH3R, 11 was more active as a partial agonist than unmethylated
analogue 10. Taken together, the hybrid approach, combining
hH4R agonistic (4) and antagonistic (5) structural elements, was
unsuccessful with respect to improving hH4R selectivity.
Results and Discussion
The synthesized compounds were investigated for agonism
and antagonism at the four human histamine receptor subtypes
in steady-state GTPase assays using membrane preparations of
Sf9 insect cells expressing the hH1R with RGS4 (regulator of
G-protein signaling 4), the hH2R-GsRS fusion protein, the hH3R
with GiR2, Gꢀ1γ2, and RGS4, or the hH4R-RGS19 fusion protein
with GiR2 and Gꢀ1γ2 (Table 1).8 A major advantage of the test
systems applied in this study is that for any given HxR subtype,
we used an identical readout, namely steady-state GTP hydroly-
sis. This is a very proximal readout in G-protein-mediated signal
transduction, reducing bias of agonist evaluation usually intro-
duced by downstream measurements of second-messenger
generation, changes in cell function, or gene transcription. Thus,
the receptor profiles described herein for acylguanidines reflect
true differences in pharmacology and not differences in readout
between various receptors. In addition, selected compounds were
investigated at the guinea pig ileum and the guinea pig right
atrium for activity at the gpH1R and gpH2R, respectively (see
the Supporting Information).
The second approach to increasing selectivity for the hH4R,
in particular over that of the hH2R, was focused on the
introduction of small NG-alkanoyl residues instead of the larger
arylalkanoyl groups found to be useful in H2R agonists.8 This
idea was stimulated by the fact that the natural agonist,
histamine, is 100 times more active at the hH4R than at the
hH2R (pEC50 values of 7.92 and 5.92, respectively). Obviously,
additional affinity-conferring substituents are not required to
achieve low nanomolar hH4R activities. Acetylation of the
guanidine group yielded 13 (UR-PI288) and provided a moder-
ate increase in agonistic activity (pEC50 ) 6.14; R ) 0.76)
relative to that of 4 at the hH2R. As expected, the activities of
the acylguanidines at the hH2R further increased (pEC50 ) 6.14
f pEC50 ) 6.96) with the extension of the alkanoyl residue
(14, UR-PI294, for the preparation and characterization of
tritiated 14 cf. ref 14; 15, UR-PI295; 16, UR-PI287), whereas
the intrinsic activities remained unaffected. However, with the
small NG-alkanoyl residues in 13-16, the hH2R agonistic
activity was kept considerably lower than with larger arylal-
kanoyl groups.8 The NG-alkanoylguanidines 13-16 were very
weak partial agonists at the hH1R. With regard to the hH3R,
acetylation of the guanidine group in 4 slightly increased affinity
but substantially decreased intrinsic activity (pEC50 ) 8.44; R
) 0.27). Likewise, substitution of the guanidine group with
larger alkanoyl residues was poorly tolerated in terms of hH3R
intrinsic activity. Obviously, acylation of the guanidine group
modifies the interaction of the imidazolylpropylguanidine moiety
with the hH3R in a manner that impedes the stabilization of an
active receptor conformation. Contrary to the structure-activity
relationships at the hH3R, the alkanoyl residue was beneficial
for the activation of the hH4R. The NG-alkanoyl imidazolyl-
Reference acylguanidine 3 was similar in potency to histamine
at the hH4R,8 had high affinity but low intrinsic activity at the
hH3R, was ∼35 times more potent than histamine at the hH2R,
and was a weak antagonist at the hH1R. 4, which is considered
an essential moiety for agonistic activity of such acylguanidine-
type H2R agonists,8 was approximately half as potent as
histamine at the hH2R and acted as a partial agonist (pEC50
)
5.59; R ) 0.66). This is in accordance with previous findings
at the guinea pig right atrium (H2R).9 At the hH3R and hH4R,
4 exhibited similar pEC50 values of ≈8.1 and intrinsic activities
of ≈0.7-0.8, whereas the compound was almost inactive at
the hH1R. These results suggest that the imidazolylpropylguani-
dine moiety is a suitable agonist structure not just at the hH2R,
but also at the hH3R and hH4R.
Most of the new acylguanidines (6-16) listed in Table 1,
except compounds 8 and 12, which had negligible intrinsic
activities if any, proved to be hH4R agonists achieving low
nanomolar activity. However, the two groups of compounds,
6-12 and 13-16, differed in particular in terms of intrinsic
activity and hH4R selectivity. In compounds 6-12, the guanidine
group of 4 was acylated with an indole-3-alkanoyl or indole-
2-carbonyl residue reminiscent of the core structure of selective
hH4R antagonist 5.10 Compared to that of 4, the acylation of
the guanidine group with indole-3-acetic or -propanoic acid (6
or 7, respectively) substantially increased agonistic activity at
the hH2R and thereby produced a diminished level of discrimi-
nation between the hH2R and hH4R. Intrinsic activity at the
hH3R was abolished. In compounds 8-12, indole-2-carboxylic
acid is attached to the imidazolylpropylguanidine portion either

2626 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8
Brief Articles
propylguanidines 13-16 were highly active (almost) full
agonists at the hH4R (pEC50 ) 8.31-8.59; R ) 0.90-1.00).
This demonstrates that acylation of the guanidine group in 4 is
a successful way to shift the agonistic activity to the hH4R at
the expense of hH3R agonism.
imidazolylpropylguanidines can be obtained by variation of the
NG-acyl substituent.
Experimental Section
Chemical Synthesis and Analytical Methods. See the Sup-
porting Information. The purity of all pharmacologically investi-
gated compounds was >95% as determined by RP-HPLC.
Summary and Conclusion
Preparation of the Boc/Trityl-Protected NG-Acylated
Imidazolylpropylguanidines (32, 34, and 36-41). General
Procedure A. To a solution of the pertinent carboxylic acid (1
equiv) and Boc-protected guanidine 18 or 19 (1 equiv) in DCM
(20 mL) were added EDC ·HCl (1.2 equiv) and DMAP (1.1 equiv)
at 0 °C. After being stirred for 4 h at 0 °C, the solution was allowed
to warm to ambient temperature and stirred for an additional 20 h.
DCM (20 mL) was added, and the organic phase was washed with
water and brine and dried over Na2SO4. The solvent was evaporated
and the crude product purified by flash chromatography.
Starting from the potent nonselective acylguanidine-type
hH4R agonist 3, which was initially designed and synthesized
as an H2R agonist,8 we identified parent compound 4 (weak
partial agonist at the H2R9) as a highly active hH3R and hH4R
partial agonist. With the aim of increasing the selectivity of the
acylguanidine-type compounds for the hH4R, two distinct
strategies were explored.
In the first approach, the guanidine group in 4 was acylated
with indolealkanoic and indole-2-carboxylic acid moieties as
structural motifs derived from the selective, high-affinity hH4R
antagonist 5. Depending on the residues and amino acid spacers,
compounds with varying activities (GTPase assay) at the
different histamine receptor subtypes were obtained. Clearly,
the compounds containing the indole substructure were not
suitable for conferring additional affinity by interaction with
the binding site of 5 as hH4R activity was not substantially
improved. An explanation therefore may be provided by an
hH4R homology model, suggesting that both histamine and 5
mainly interact with Asp-94 of TM3 and Glu-182 of TM5.13
Presumably, as previously described for the H2R,8,15 histamine
and NG-acylated imidazolylpropylguanidines also predominantly
interact with identical amino acid residues at the hH4R, resulting
in overlapping binding sites of the imidazolylpropylguanidine
group and 5.
N1-Acetyl-N2-(tert-butoxycarbonyl)-N3-[3-(1-trityl-1H-imida-
zol-4-yl)propyl]guanidine (38). The title compound was prepared
from acetic acid 27 (60 mg, 1.0 mmol), 19 (510 mg, 1.0 mmol),
EDC·HCl (230 mg, 1.2 mmol), and DMAP (134 mg, 1.1 mmol)
according to general procedure A. Purification by flash chroma-
tography (CHCl3/MeOH, 97.5/2.5, v/v) yielded a colorless oil (360
mg, 65%): 1H NMR (300 MHz, CDCl3) δ 1.50 (s, 9H), 1.85-1.99
3
(m, 2H), 2.16 (s, 3H), 2.59 (t, 2H, J ) 7.6 Hz), 3.39-3.49 (m,
4
2H), 6.53 (d, 1H, J ) 1.3 Hz), 7.08-7.17 (m, 6H), 7.29-7.39
(m, 10H), 8.96 (t, 1H, 3J ) 5.2 Hz), 12.41 (brs, 1H); ES-MS (DCM/
MeOH + NH4OAc) m/z (%) 552 (100) [M + H]+. C33H37N5O3
(551.68).
Compounds 32, 34, 36, 37, and 39-41 were prepared by analogy
(see the Supporting Information).
Preparation of NG-Acylated Imidazolylpropylguanidines 7,
9, and 11-16. General Procedure B. The pertinent Boc-trityl-
protected NG-acylated imidazolylpropylguanidine was refluxed for
30 min in HCl (1 M, 20 mL). After the precipitated trityl alcohol
had been removed by filtration, the solvent was removed in vacuo.
Purification of the crude product was performed by preparative
HPLC. The solvent was removed by lyophilization, and the
compounds were obtained as trifluoroacetates.
In the second approach, small alkanoyl residues were attached
to 4 (13-16). With the increasing size of the alkanoyl residues,
activity at the hH2R also increased, confirming the importance
of the acyl group as an affinity-conferring moiety at the hH2R.
At the hH3R, acylation drastically lowered efficacy, whereas
the same compounds turned out to be highly potent full (or
nearly full) hH4R agonists. Thus, although the imidazolylpro-
pylguanidine portion is capable of stimulating the hH2R, hH3R,
and hH4R, selectivity can be achieved by appropriate NG-
acylation.
N1-Acetyl-N2-[3-(1H-imidazol-4-yl)propyl]guanidinium Ditri-
fluoroacetate (13). The title compound was prepared from 38 (350
mg, 0.63 mmol) according to general procedure B. Purification by
preparative HPLC [MeCN/0.1% TFA (aqueous), 4/96] yielded a
colorless semisolid compound (150 mg, 54%): 1H NMR (300 MHz,
D2O, trifluoroacetate) δ 1.84-1.97 (m, 2H), 2.08 (s, 3H), 2.69 (t,
2H, 3J ) 7.6 Hz), 3.26 (t, 2H, 3J ) 6.9 Hz), 7.12 (d, 1H, 4J ) 1.3
Hz), 8.46 (d, 1H, 4J ) 1.3 Hz); 13C NMR (75 MHz, D2O,
trifluoroacetate) δ 21.06, 23.69, 26.04, 40.38, 115.48, 132.44,
133.03, 152.98, 174.73; IR (cm-1) 3139, 3035, 2854, 1662, 1629,
1179, 1124; HRMS (EI-MS) calcd for C9H15N5O [M+•] 209.1277,
found 209.1275. C9H15N5O·2TFA (437.30).
hH4R agonists 13-16 are among the most active hH4R
agonists reported to date and may become useful experimental
tools in addition to previously described H4R ligands like
selective hH4R agonist 22 or selective H4R antagonist 510 to
analyze the as yet incompletely understood (patho)physiological
functions of the H4R. In most immune cells like mast cells or
eosinophils, where the H4R is mainly located, H3Rs are not
expressed.4,16,17 Therefore, on the basis of the >100-fold
selectivity relative to the hH1R and hH2R subtypes, NG-
alkanoylimidazolylpropylguanidines like 13 may also be useful
for investigating the function of the hH4R in such native cells
devoid of the hH3R. Moreover, these potent hH4R agonists may
be suitable pharmacological probes for desensitization studies
with the hH4R.
Compounds 7, 9, 11, 12, and 14-16 were prepared by analogy
(see the Supporting Information).
Acknowledgment. We are grateful to Mrs. Kerstin Fisch,
Mrs. Karin Schadendorf, Mrs. Gertraud Wilberg, Mrs. Christine
Braun, and Mrs. Kerstin Ro¨hrl for expert technical assistance.
This work was supported by the Graduate Training Program
(Graduiertenkolleg) GRK 760, “Medicinal Chemistry: Molecular
Recognition-Ligand-Receptor Interactions”, of the Deutsche
Forschungsgemeinschaft.
Supporting Information Available: Synthesis and analytical
data of 4, 6-12, 14-16, 23-26, 31-37, 39-41, 43-52, 56, and
58-61; HPLC purity data of target compounds 6-16; pharmaco-
logical activities of 6, 7, and 11-16 at the guinea pig ileum (gpH1R)
and guinea pig right atrium (gpH2R); and pharmacological methods.
This material is available free of charge via the Internet at http://
pubs.acs.org.
Altogether, as previously observed, from the medicinal
chemistry perspective, the imidazolylpropylguanidine scaffold
may be considered a “privileged structure” providing ligands
for several histamine receptor subtypes.8 However, as demon-
strated in this study, activities and receptor selectivities of the

Brief Articles
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8 2627
A partial agonist at histamine H2-receptors. Agents Actions 1975, 5,
464.
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