Synthesis and dual histamine H1 and H2 receptor antagonist activity of cyanoguanidine derivatives

Article abstract of DOI:10.3390/molecules181114186

Premedication with a combination of histamine H1 receptor (H1R) and H2 receptor (H2R) antagonists has been suggested as a prophylactic principle, for instance, in anaesthesia and surgery. Aiming at pharmacological hybrids combining H1R and H2R antagonistic activity, a series of cyanoguanidines 14-35 was synthesized by linking mepyramine-type H1R antagonist substructures with roxatidine-, tiotidine-, or ranitidine-type H2R antagonist moieties. N-desmethylmepyramine was connected via a poly-methylene spacer to a cyanoguanidine group as the "urea equivalent" of the H2R antagonist moiety. The title compounds were screened for histamine antagonistic activity at the isolated ileum (H1R) and the isolated spontaneously beating right atrium (H2R) of the guinea pig. The results indicate that, depending on the nature of the H2R antagonist partial structure, the highest H1R antagonist potency resided in roxatidine-type compounds with spacers of six methylene groups in length (compound 21), and tiotidine-type compounds irrespective of the alkyl chain length (compounds 28, 32, 33), N-cyano-N'-[2-[[(2- guanidino-4-thiazolyl)methyl] thio]ethyl]-N″-[2-[N-[2-[N-(4-methoxybenzyl)-N-(pyridyl)-amino] ethyl]-N-methylamino]ethyl] guanidine (25, pKB values: 8.05 (H1R, ileum) and 7.73 (H2R, atrium) and the homologue with the mepyramine moiety connected by a six-membered chain to the tiotidine-like partial structure (compound 32, pKB values: 8.61 (H1R) and 6.61 (H2R) were among the most potent hybrid compounds. With respect to the development of a potential pharmacotherapeutic agent, structural optimization seems possible through selection of other H1R and H2R pharmacophoric moieties with mutually affinity-enhancing properties.

Full text of DOI:10.3390/molecules181114186

Molecules 2013, 18, 14186-14202; doi:10.3390/molecules181114186
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis and Dual Histamine H1 and H2 Receptor Antagonist
Activity of Cyanoguanidine Derivatives
Bassem Sadek 1,*, Rudi Alisch 2, Armin Buschauer 3 and Sigurd Elz 3
1
Department of Pharmacology and Therapeutics, College of Medicine and Health sciences,
UAE University, Al-Ain, P.O. Box 17666, UAE
2
Institute of Pharmacy, Free University of Berlin, Königin-Luise-Str. 2-4, Berlin D-14195, Germany
3
Institute of Pharmacy, University of Regensburg, University Str. 31, Regensburg D-93053, Germany
* Author to whom correspondence should be addressed; E-Mail: bassem.sadek@uaeu.ac.ae;
Tel.: +97-137-137-512; Fax: +97-137-672-033.
Received: 8 October 2013; in revised form: 10 November 2013 / Accepted: 11 November 2013/
Published: 15 November 2013
Abstract: Premedication with a combination of histamine H1 receptor (H1R) and H2
receptor (H2R) antagonists has been suggested as a prophylactic principle, for instance, in
anaesthesia and surgery. Aiming at pharmacological hybrids combining H1R and H2R
antagonistic activity, a series of cyanoguanidines 14–35 was synthesized by linking
mepyramine-type H1R antagonist substructures with roxatidine-, tiotidine-, or
ranitidine-type H2R antagonist moieties. N-desmethylmepyramine was connected via a
poly-methylene spacer to a cyanoguanidine group as the “urea equivalent” of the H2R
antagonist moiety. The title compounds were screened for histamine antagonistic activity
at the isolated ileum (H1R) and the isolated spontaneously beating right atrium (H2R) of the
guinea pig. The results indicate that, depending on the nature of the H2R antagonist partial
structure, the highest H1R antagonist potency resided in roxatidine-type compounds with
spacers of six methylene groups in length (compound 21), and tiotidine-type compounds
irrespective of the alkyl chain length (compounds 28, 32, 33), N-cyano-N'-[2-[[(2-
guanidino-4-thiazolyl)methyl]thio]ethyl]-N″-[2-[N-[2-[N-(4-methoxybenzyl)-N-(pyridyl)-amino]
ethyl]-N-methylamino]ethyl] guanidine (25, pKB values: 8.05 (H1R, ileum) and 7.73 (H2R,
atrium) and the homologue with the mepyramine moiety connected by a six-membered
chain to the tiotidine-like partial structure (compound 32, pKB values: 8.61 (H1R) and 6.61
(H2R) were among the most potent hybrid compounds. With respect to the development of a
potential pharmacotherapeutic agent, structural optimization seems possible through selection
of other H1R and H2R pharmacophoric moieties with mutually affinity-enhancing properties.

Molecules 2013, 18
Keywords: dual H1/H2 receptor antagonists; mepyramine; roxatidine; tiotidine; ranitidine
14187
1. Introduction
The biogenic amine histamine mediates its effects via four histamine receptor subtypes, termed H1,
H2, H3, and H4 receptors (HxR) [1–3]. The histamine receptors belong to class A of the superfamily of
G-protein-coupled receptors (GPCRs). As an autacoid and neurotransmitter, histamine is involved in
numerous physiological and pathophysiological processes. Antagonists of the H3R [4] and the most
recently discovered H4R [5] are still under investigation as potential drugs, e.g., for the treatment of
CNS disorders and inflammatory diseases, respectively [6]. By contrast, H1R and H2R antagonists are
well established therapeutic agents for decades. Numerous pathophysiological responses to histamine
released from mast cells and basophils through immunological or non-immunological mechanisms are
mediated by the H1R, for instance, vasodilatation via nitric oxide release, increase in capillary
permeability, contraction of smooth muscles, e.g., in gut and bronchi. The first H1R blockers [7] have
been described as “antihistamines” more than 70 years ago, and especially the newer non-sedating H1R
antagonists are widely used in the treatment of allergic conditions [7]. Stimulation of gastric acid
secretion is the most prominent physiological effect of H2R stimulation. In the 1970s the development
of the H2R antagonists revolutionized the treatment of gastric and duodenal ulcers [8]. Histamine can
also induce cardiovascular effects via H2Rs, e.g., increase in heart rate and cardiac contractility as well
as vasodilatation [9].
The combined administration of H1R and H2R antagonists has been suggested, for example, as a
prophylactic principle in anaesthesia, for the prevention of hypersensitivity reactions to drugs, e.g., in
cancer chemotherapy, or for the treatment of skin diseases [10–15]. Histamine release during
anaesthesia and surgery is still a widely known problem in clinical practice [10]. The ratio of
anaphylactic/anaphylactoid reaction incidents is about 20%–30%, and life threatening reactions are
observed in 0.1%–0.5% of all cases [16]. Cardiac arrhythmias induced by activation of H1 and H2
receptors are among the most serious consequences of mast cell degranulation. Several studies have
shown the effectiveness of a premedication with a combination of H1R and H2R antagonists, for
example dimetindene plus cimetidine [10,17–20].
The commercially available H1 and H2 antihistamines differ considerably concerning
physicochemical and pharmacokinetic properties. A combination of both qualities of action in one
hybrid drug could be of therapeutic advantage, for instance, in terms of pharmacokinetics. Such hybrid
antihistamines could also be of potential interest in other indications, e.g., in the therapeutic
management of atopic dermatitis. Icotidine (SK&F 93319), derived from the isocytosine series of H2R
antagonists, was the first substance reported to possess nearly equipotent antagonist activity at both
H1R and H2R [21] (Figure 1).

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Figure 1. Icotidine (SK&F 93319) and lead compound 1.
Previously, urea-type H1/H2 receptor antagonists such as compound 1 were synthesized and
pharmacologically investigated [22–24]. Lessons learned, in particular from the H4R and H3R, suggest
that differences between HxR species orthologues should to be taken into account [2,3]. Nevertheless,
isolated guinea-pig organs have been used as standard pharmacological models to characterize H1R
and H2R antagonists for many decades, and the data gained from these investigations provided a
reliable basis for numerous successfully marketed histamine receptor antagonists. Therefore, with
respect to potential studies in translational animal models, the synthesized compounds were
investigated on the guinea pig isolated ileum and the spontaneously beating right atrium. The urea
derivative 1, containing a three-membered carbon chain as a spacer and fluoropheniramine substructure,
had a pKB value of 8.21 at the isolated guinea pig ileum and proved to be more active than pheniramine at
the H1R. However, H2R antagonist activity (guinea pig atrium: pKB 6.68) was found to be moderate,
presumably due to introduction of a basic centre close to the polar group [23] (Figure 1).
As mepyramine (pA2 9.07 ± 0.03) [25] is more potent than pheniramine-like H1R antagonists, in the
present study, the mepyramine partial structure was used as a building block for the synthesis of hybrid
compounds. The H1R antagonist pharmacophoric moiety was connected via a carbon chain (spacer) of
variable length and a cyanoguanidine group with the H2R antagonist partial structure of roxatidine,
tiotidine, and ranitidine (Figure 2). Thereby, the cyanoguanidine moiety, a characteristic “urea
equivalent” [26,27] of H2R antagonists such as cimetidine, was kept unchanged as a bioisosteric
replacement of the polar moieties present in roxatidine, tiotidine and ranitidine, respectively.
Figure 2. General structure of spacer-linked combined H1/H2 receptor antagonists.

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2. Results and Discussion
2.1. Chemistry
The cyanoguanidines 14–35 were synthesized starting from triamines 4–13 which can be easily
prepared from the secondary amines 3a and 3b through alkylation with acrylonitrile or
ω-haloalkanenitriles of different chain lengths followed by reduction with LiAlH4 in diethyl ether
(Scheme 1). Secondary amines 3a and 3b were obtained by acylation of primary amines 2a and 2b
with ethyl chloroformate and subsequent reduction with LiAlH4 in tetrahydrofuran [23,24].
Scheme 1. Synthetic pathway for intermediates 4–13.
R1
H
Br
N
N
NH2
NH2
a
b
N
N
N
2-bromopyridine
2-(2-aminoethylamino)pyridine
R1
2a, 2b
R1
c
d
N
N
(CH2)y NH2
NH
N
CH3
N
CH3
N
3a, 3b
6
4-13
No.
R1
y
4
5
F
2
7
F
3
8
9
10
F
11
12
F
13
F
OCH3
2
OCH3
OCH3
4
F
OCH3
6
3
4
5
6
7
Reagents and Conditions: a Ethylenediamine, pyridine, reflux, 3 h; b NaH, 4-methoxybenzylchloride (for 2a),
c
4-fluorobenzylchloride (for 2b), DMSO, room temperature, 12 h; NaOH, ethyl chloroformate, THF, room
d
temperature; (i) Acrylonitrile or corresponding ω-chloroalkylnitrile, KI, excess Na2CO3, 60 °C, 2 h;
(ii) Lithium aluminum hydride (50% excess), diethyl ether, room temperature, 2 h.
The triamines 4–13 were allowed to react with the isourea intermediates A1–D1 (Scheme 2) to
yield the cyanoguanidines 14–35 [22–24,28–30]. Addition of the roxatidine-like primary amines A and
B resulted in 14–23 and 24, respectively, while addition of C and D afforded the compounds 25–34
and 35, respectively [24].
2.2. Pharmacology
The synthesized hybrid compounds 14–35 were investigated for histamine H1R and H2R
antagonism at the isolated guinea pig ileum (H1R) and the isolated spontaneously beating guinea pig
right atrium (H2R). The results are summarized in Table 1.

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Scheme 2. Synthetic pathway of cyanoguanidines 14–35.
Reagents and Conditions: a Diphenyl N-cyanocarbonimidate, room temperature, 2 h; b Respective diamine, A1-D1,
acetonitrile, reflux, 16 h.
Compounds 14–35 showed histamine H1R antagonistic activities on guinea pig ileum with pKB
values in the range from 6.8 (compound 14) to 8.6 (compounds 28, 32). Although the activity
increased with the length of the polymethylene spacer in case of the methoxy-substituted derivatives
bearing a roxatidine-like H2-antagonist moiety (compound 14 vs. 16, 18 and 21), a clear general
tendency was not obvious, neither for the corresponding fluorinated analogues nor for the
guanidinothiazoles 25–34. Highest H1R antagonistic activity resided in the latter group of compounds.
Though an affinity-increasing effect was not evident, it should be noted that the interactions with the
H1R were not dramatically affected in an adverse manner by the H2R antagonist moiety. The most
potent H1R antagonists achieved KB values in the one-digit nanomolar range corresponding to about 30%
of the activity of mepyramine at the guinea-pig ileum.

Molecules 2013, 18
Table 1. Histamine H1 and H2 receptor antagonism of cyanoguanidines 14–35.
14191
H1 Receptor Antagonism
H2 Receptor Antagonism
rel.
rel.
a
a
Compound
R1
y
R2
pKB
conc. [M]
activity
pKB
conc. [M]
activity
Mepyramine
-
-
-
-
-
-
-
-
-
9.07 b
-
100
-
Cimetidine
6.40
7.49
6.68
6.63
5.97 e
6.37
6.0
100
Icotidine c
1 d
14
7.77
8.21
6.88
7.83
7.81
7.77 e
7.76
7.75
7.95
8.42
8.06
7.93
7.65
8.05
7.94
8.21
8.62
7.91
8.32
8.36
8.61
8.44
8.15
7.95
5.0
1230
14
1
0.3 × 10−6
0.3 × 10−6
190
170
37
10−6–0.3 × 10−5
OCH3
2
2
3
3
4
4
5
6
6
7
4
2
2
3
3
4
4
5
6
6
7
3
A
A
A
A
A
A
A
A
A
A
B
C
C
C
C
C
C
C
C
C
C
D
10−7–0.3 × 10−6
0.3 × 10−5
0.3 × 10−6
0.3 × 10−6–10−6
0.3 × 10−6
10−5–0.3 × 10−5
0.3 × 10−5
0.3 × 10−6
10−6
15
F
6
10−7
16
OCH3
6
10−7–0.3 × 10−6
10−7–0.3 × 10−6
10−8–10−7
10−7–0.3 × 10−7
10−7
10−7–0.3 × 10−7
10−7–0.3 × 10−7
10−7–0.3 × 10−7
10−7–0.3 × 10−6
10−7–0.3 × 10−7
10−8–0.3 × 10−7
10−8–0.3 × 10−7
10−8–0.3 × 10−8
0.3 × 10−7
93
17
F
5
40
18
OCH3
5
6.38
5.76 e
5.67
6.43
6.41
6.17
6.07
7.37
6.62
6.52
6.47
7.00
6.28
6.51
6.61
6.52
7.02
5.22 e
96
19
F
5
23
20
F
7
19
21
OCH3
22
10
7
107
102
59
22
F
23
F
10−6–0.3 × 10−5
10−6
24
F
OCH3
F
4
47
25
10
7
933
166
132
118
398
76
10−7
26
10−6
27
OCH3
F
14
36
7
10−6
28
0.3 × 10−6–0.3 × 10−5
10−7–0.3 × 10−6
10−6
29
OCH3
F
30
18
20
35
23
12
7
10−8–0.3 × 10−8
10−8–0.3 × 10−7
10−7–0.3 × 10−7
0.3 × 10−7
31
F
129
162
132
417
7
10−6–10−5
10−7–0.3 × 10−6
10−6
10−6–0.3 × 10−6
10−5
32
OCH3
F
33
34
F
10−8
10−8–10−7
35
OCH3
a
Determined at the isolated ileum (H1) and right atrium (H2) of the guinea pig; pKB mean values, S.E.M. for
ileum within ±0.2 by 16, 17, 18, and 19; ±0.1 by all other compounds; pKB mean values, S.E.M. for atrium
within ±0.2 by 14, and 17; ±0.1 by all other compounds; rel. activity, % activity relative to mepyramine,
b
c
d
icotidine or cimetidine, respectively; antag. conc., antagonist concentrations used; [25]; [21]; [23];
e
At 0.3 × 10−6 M, depression of the concentration response curve by 15%, 30%, 15%, and 20% for 15, 17,
and 19, respectively.
It is well known from numerous investigational and commercially available H2R antagonists
(e.g., cimetidine, ranitidine or tiotidine) that, comparing the numeric values, the highest antagonistic
activities achievable at the guinea pig right atrium (Table 1) are usually by one to two orders of

Molecules 2013, 18
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magnitude lower than at the guinea pig ileum. The same holds for the investigated hybrid compounds.
Highest H2R antagonistic activity resided in guanidinothiazole 25 with a pKB value of 7.37,
corresponding to nine-fold higher activity than cimetidine or three-fold lower activity than tiotidine,
respectively. Among the cyanoguanidines substituted with roxatidine-like partial structures
(compounds 14–23), lengthening of the spacer from ethylene to pentamethylene decreased both
antagonist activities on H1 and H2 receptors. However, cyanoguanidine derivative 21 (pKB ileum, 8.42,
pKB atrium, 6.43) with a hexamethylene spacer separating the basic center from the polar
cyanoguanidine moiety was approximately two times more active on H1 receptors, and the H2
antagonistic activity was in the same range as that of reference compounds 1 or cimetidine. Moreover,
the presence of a methoxy group at p-position of the mepyramine partial structure seemed to be of
advantage over fluorine substitution. Compared to 22, the H1R antagonist activity of 21 was
approximately twofold higher whereas the H2R antagonist remained unchanged. Further extension of
the spacer up to seven methylene groups in the H1R antagonist partial structure or up to four methylene
groups in the H2R antagonist moiety decreased both H1R and H2R antagonistic activities as
demonstrated by cyanoguanidine derivatives 23 (pKB ileum, 7.93, pKB atrium, 6.17) and 24
(pKB ileum, 7.65, pKB atrium, 6.07), respectively. Thus, compound 21 represents the optimum in terms
of combined H1R/H2R antagonistic activity among the piperidinomethylphenoxyalkylcyanoguanidines.
A further increase in potency was achieved by replacement of the roxatidine-like moiety with a
guanidinothiazole (compounds 25–34) as in tiotidine, resulting in the highest histamine H1R and H2R
antagonistic activities among this series of pharmacological hybrids. Compound 32 with a
hexamethylene spacer was found to be up to two times more potent at both H1R and H2R compared to
the roxatidine-like analog 21. In addition, the results revealed that a p-methoxy moiety (compound 32),
unlike p-fluoro substituent (compound 33), potentiates the antagonistic activities at both H1 and H2
receptors. Remarkably, further extension of the spacer up to 7 methylene groups resulted in
cyanoguanidine derivative 34, the most potent H2R antagonist amid the title compounds. However,
H1R antagonistic decreased up to 5-fold compared to compounds 32 and 33. The cyanoguanidine
derivative 35, bearing a ranitidine-like moiety, did not show an activity-enhancing effect compared to
the corresponding parent compounds. Compound 25 with an ethylene spacer and tiotidine partial
structure revealed the highest H2R antagonistic activity among the presented series of compounds.
In summary, the combination of the mepyramine substructure with a tiotidine-like guanidinothiazole
turned out to be most suitable to obtain pharmacological hybrid with activities comparable to those of
commercially available selective H1R and H2R antagonists.
3. Experimental
3.1. Chemistry
3.1.1. General Conditions
Melting points are uncorrected and determined in open capillaries in a Buechi 512 Dr. Tottoli
1
apparatus. H-NMR spectra were recorded on a Bruker WC 300 spectrometer with tetramethylsilane
(TMS) as internal standard. Chemical shifts are reported in ppm downfield from internal
1
tetramethylsilane as reference. H-NMR signals are reported in order: multiplicity (s, single;

Molecules 2013, 18
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d, doublet; t, triplet; q, quintet; m, multiplet; br, broad; *, exchangeable by D2O), number of protons,
and approximate coupling constants in Hertz. Elemental analyses were performed on Perkin-Elmer
240B and 240C instruments. Analyses (C, H, N) indicated by the symbols of elements were within
±0.4% of the theoretical values. Chromatographic separations were done by rotation planar
chromatography (centrifugal layer chromatography) using a Chromatotron Model 7924 (Harrison
Research, Muttenz, Switzerland) with 4-mm layers of silica gel 60 PF254 containing gypsum (Merck).
To avoid tailing, a saturated ammonia atmosphere was produced by passing through a stream of
anhydrous gaseous ammonia via the “inert gas inlet” of the Chromatotron. EI-mass spectra were
recorded using Finnigan MAT CH7A (70 eV), Finnigan MAT 711 (80 eV), or Kratos MS 25 RF (70 eV).
+FAB-MS spectra were recorded on Finnigan MAT CH5DF instrument (xenon, DMSO)/glycerol). The
following abbreviations are used: DMF, N,N-dimethylformamide,; EtOH, ethanol; Et2O, diethyl ether;
MeOH, methanol; Me2SO, dimethyl sulfoxide; Ph, phenyl; * exchangeable with D2O; C2H2O4, oxalic
acid; Py, pyridyl; Pip, piperidyl; TA, C4H6O6, tartaric acid; C20H18O8, (−)-di-p-tolyltartaric acid;
decomp, decomposition.
3.1.2. Synthesis of Primary Amines 2a and 2b
2-Bromopyridine (109.30 mmol) and ethylenediamine (546 mmol) were refluxed in pyridine for 3 h.
Excess of ethylenediamine was evaporated under reduced pressure, water was added, the pH value was
adjusted to >11 using NaOH (1N), and the 2-(2-aminoethylamino)-pyridine was extracted with
chloroform [29]. After evaporation of chloroform, the product was purified by means of a
Chromatotron (chloroform/methanol, 97/3, V/V, ammonia atmosphere), and used in the following
reaction without further purifications. The primary amines 2a and 2b were synthesized by suspending
2-(2-aminoethylamino)pyridine (73.40 mmol) with sodium hydride (60% oil suspension, 64.50 mmol)
in DMSO under nitrogen atmosphere. The reaction temperature was increased gradually and kept at 85 °C
until hydrogen evolution ceased. For synthesis of 2a and 2b, 4-methoxybenzylchloride and
4-fluorobenzylchloride, respectively, were added, and the mixture was stirred for 12 h at room
temperature. Subsequently, water (500 mL) was added, 2a and 2b were extracted with diethyl ether at
pH > 11, combined extracts were dried over anhydrous potassium carbonate, and the solvent was
evaporated under reduced pressure yielding the oily products 2a and 2b, which were used in the
following reaction to prepare 3a and 3b without further purification.
3.1.3. Synthesis of Secondary Amines 3a and 3b
For synthesis of 3a and 3b, 43.64 mmol of the corresponding primary amine 2a and 2b were
dissolved in diethyl ether, NaOH (10%) was added and the solution was kept over ice-bath. Ethyl
chloroformate was added gradually to the two-phase system. After complete reaction, the diethyl ether
layer was separated, dried over sodium sulfate, and evaporated under reduced pressure to yield the oily
products 3a and 3b, respectively [22–24,28].

Molecules 2013, 18
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3.1.4. General Procedure for the Synthesis of the Diamines 4–13
As previously described, a mixture of the corresponding secondary amine 3a or 3b (10–30 mmol),
an equimolar amount of the pertinent ω-halonitrile, a catalytic amount of potassium iodide and a 100%
excess of sodium carbonate was stirred for 2 h at 60 °C in 20 mL of a mixture of acetonitrile and
dimethylformamide (1:1 (V/V) in case of chloroacetonitrile, 9:1 (V/V) in case of homologous
halonitriles) [22–24,28]. Subsequently, water was added, the mixture was extracted with toluene, the
combined extracts were dried over sodium sulfate and evaporated under reduced pressure yielding the
corresponding oily aminonitriles as intermediates, which were dissolved in 20 mL of anhydrous
diethyl ether and dropped to an ice-cold suspension of lithium aluminium hydride (50% excess). After
2 h stirring at room temperature the reaction mixture was hydrolyzed by addition of water followed by
3 mL of a 10% aqueous sodium hydroxide solution. The resulting diamines 4–13 were purified
chromatographically (Chromatotron, eluent: chloroform/methanol, gradient from 99:1 to 90:10 (V/V),
ammonia atmosphere), and were used in the following reactions for synthesis of cyanoguanidines
14–35 [22–24,28].
3.1.5. General Procedure for the Synthesis of Cyanoguanidines 14–35
The pertinent N-cyano O-phenyl isoureas (2–4 mmol) A1–D1, which were prepared by stirring of
equivalent amounts of A–D with diphenyl N-cyanocarbonimidate for 2 h, and equimolar amounts of
the respective diamines 4–13 in 30 mL of anhydrous acetonitrile, were heated under reflux for 16 h.
The mixture was evaporated to dryness and the cyanoguanidines 14–35 were isolated
chromatographically (Chromatotron, chloroform: methanol, gradient from 99:1 to 90:10, ammonia
atmosphere) [22–24,28].
N-Cyano-N'-[2-[N-[2-[N-(4-methoxybenzyl)-N-(2-pyridyl)amino]ethyl]-N-methylamino]ethyl]-N″-[3-
1
[3-(piperidinomethyl)phenoxy]propyl]guanidine (14). Yield: 65%, mp: 88–89 °C; H-NMR (CDCl3):
δ ppm = 1.41–1.42 (m, 2H, PipH); 1.53–1.57 (m, 4H, PipH), 1.98 (m, 2H, CH2); 2.25 (s, 3H, N-CH3);
2.34 (br, 4H, PipH); 2.57–2.62 (m, 4H, 2(CH2-NCH3)); 3.20–3.21 (m, 2H, CH2-NH); 3.30–3.32
(m, 2H, CH2-NH); 3.40 (s, 2H, Ph-CH2- Pip); 3.66 (t, J = 7, 2H, CH2-N); 3.76 (s, 3H, Ph-OCH3); 3.97
(t, J = 6, 2H, Ph-O-CH2); 4.60 (s, 2H, Ph-CH2-N); 5.85 (br, 1H*, NH); 6.40-6.43 (d, J = 9, 1H
aromatic, Py5-H); 6.51–6.55 (m, 1H aromatic, Py3-H); 6.73–6.75 (d, J = 8.5, 1H aromatic, 4-H);
6.81–6.84 (d, J = 8, 2H aromatic, 2-H); 6.87-6.90 (d, J = 8, 2H aromatic, 2-H); 7.10–7.13 (m, 2H
aromatic, 3-H); 7.10–7.13 (br, 1H*, NH); 7.17 (t, J = 8, 1H aromatic, 3-H); 7.36 (m, 1H aromatic,Py4-H);
8.12–8.13 (d, J = 3, 1H aromatic, Py6-H); MS: m/z (%) 612 (M+, 1), 121 (100); Anal. Calcd. for
C35H48N8O2·H2O: C, 66.60; H, 7.99; N, 17.80. Found: C, 66.70; H, 7.82; N, 17.45.
N-Cyano-N'-[2-[N-[2-[N-(4-fluorobenzyl)-N-(2-pyridyl)amino]ethyl]-N-methylamino]ethyl]-N″-[3-[3-
1
(piperidinomethyl)phenoxy]propyl]guanidine (15). Yield: 33%, mp: 106–108 °C; H-NMR (CDCl3):
δ ppm = 1.44 (m, 2H, PipH); 1.61 (m, 4H, PipH), 1.90 (m, 2H, CH2); 2.31 (s, 3H, N-CH3); 2.59–2.64
(br, 4H, PipH); 2.71 (m, 4H, 2(CH2-NCH3)); 3.24 (m, 2H, CH2-NH); 3.37–3.47 (m, 2H, CH2-NH);
3.63 (m, 2H, CH2-N); 3.83 (s, 2H, Ph-CH2-Pip); 3.96 (t, 2H, Ph-O-CH2); 4.18 (s, 6H, TA); 4.72 (s, 2H,
Ph-CH2-N); 5.20–5.60 (br, 6H*, TA-OH); 6.54 (m, 2H aromatic, Py5-H, Py3-H); 6.86–6.88 (m, 3H

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aromatic, 4-H, 2-H); 6.86–6.88 (br, 3H*, 3(H-(N+)); 6.92 (m, 2H aromatic, 2-H); 6.92 (br, 1H*, NH);
7.11 (m, 2H aromatic, 3-H); 7.25 (m, 2H aromatic, 3-H); 7.25 (br, 1H*, NH); 7.45 (m, 1H aromatic,
Py4-H); 8.06 (d, 1H aromatic, Py6-H); 10.50 (br, 3H*, TA-COOH); MS: m/z (%) 601 (M+, 7), 76
(100); Anal. Calcd. for C34H45FN8O·3C4H6O6: C, 52.60; H, 6.04; N, 10.70. Found: C, 52.50; H, 6.27;
N, 10.60.
N-Cyano-N'-[3-[N-[2-[N-(4-methoxybenzyl)-N-(2-pyridyl)amino]ethyl]-N-methylamino]propyl]-N″-
[3[3-piperidinomethyl)phenoxy]propyl]guanidine (16). Yield: 51%, mp: 62–65 °C; 1H-NMR (CDCl3):
δ ppm = 1.41–1.42 (m, 2H, PipH); 1.53–1.57 (m, 4H, PipH), 1.66 (m, 2H, CH2); 2.25 (s, 3H, N-CH3);
2.34 (br, 4H, PipH); 2.57–2.62 (m, 4H, 2(CH2-NCH3)); 3.20–3.21 (m, 2H, CH2-NH); 3.30–3.32 (m,
2H, CH2-NH); 3.40 (s, 2H, Ph-CH2- Pip); 3.66 (t, J = 7, 2H, CH2-N); 3.76 (s, 3H, Ph-OCH3); 3.97
(t, J = 6, 2H, Ph-O-CH2); 4.60 (s, 2H, Ph-CH2-N); 5.85 (br, 1H*, NH); 6.40–6.43 (d, J = 9, 1H
aromatic, Py5-H); 6.51–6.55 (m, 1H aromatic, Py3-H); 6.73–6.75 (d, J=8.5, 1H aromatic, 4-H);
6.81–6.84 (d, J = 8, 2H aromatic, 2-H); 6.87–6.90 (d, J = 8, 2H aromatic, 2-H); 7.10–7.13 (m, 2H
aromatic, 3-H); 7.10–7.13 (br, 1H*, NH); 7.17 (t, J = 8, 1H aromatic, 3-H); 7.36 (m, 1H aromatic, Py4-H);
8.12–8.13 (d, J = 3, 1H aromatic, Py6-H); MS: m/z (%) 627 (M+, 6), 241 (11), 121 (100); Anal. Calcd.
for C36H50N8O2: C, 69.0; H, 8.04; N, 17.90. Found: C, 68.80; H, 8.27; N, 17.70.
N-Cyano-N'-[3-[N-[2-[N-(4-fluorobenzyl)-N-(2-pyridyl)amino]ethyl]-N-methylamino]propyl]-N″-[3-
1
[3-(piperidinomethyl)phenoxy]propyl]guanidine (17). Yield: 57%, mp: 65–68 °C; H-NMR (CDCl3):
δ ppm = 1.43 (m, 2H, PipH); 1.57–1.61 (m, 4H, PipH); 1.68 (m, 2H, CH2); 1.99–2.05 (m, 2H, CH2);
2.28 (s, 3H, N-CH3); 2.40 (br, 4H, PipH); 2.51 (t, J = 7, 2H, CH2-NCH3); 2.58 (t, J = 7, 2H, CH2-N-
CH3); 3.25–3.26 (m, 2H, CH2-NH); 3.39 (m, 2H, CH2-NH); 3.45 (s, 2H, Ph-CH2-Pip); 3.70 (t, J = 7.5,
2H, CH2-N); 4.03 (t, J = 6, 2H, Ph-O-CH2); 4.63 (s, 2H, Ph-CH2-N); 6.41 (d, J = 9, 1H aromatic, Py5-
H); 6.55 (t, J = 6, 1H aromatic, Py3-H); 6.75–6.78 (d, J = 8, 1H aromatic, 4-H); 6.89-6.92 (d, J = 6, 2H
aromatic, 2-H); 6.89–6.92 (br, 1H*, NH); 6.97 (t, J = 8, 2H aromatic, 2-H), 7.13–7.21 (m, 2H
aromatic, 3-H, 3-H); 7.13–7.21 (br, 1H*, NH); 7.38 (t, J = 7, 1H aromatic, Py4-H); 8.12–8.13 (d, J = 5,
1H aromatic, Py6-H); MS: m/z (%) 615 (M+, 14), 229 (100), 109 (96); Anal. Calcd. for
C35H47FN8O·3C4H6O6: C, 68.80; H, 8.27; N, 17.70. Found: C, 68.53; H, 8.55; N, 17.21.
N-Cyano-N'-[4-[N-[2-[N-(4-methoxybenzyl)-N-(2-pyridyl)amino]ethyl]-N-methylamino]butyl]-N″-[3-
1
[3-(piperidinomethyl)phenoxy]propyl]guanidine (18). Yield: 47%, mp: 106–108 °C; H-NMR
(CDCl3): δ ppm = 1.44 (m, 4H, PipH); 1.59 (m, 6H, PipH); 1.91 (m, 2H, CH2); 2.50 (s, 3H, N-CH3);
2.64 (m, 4H, PipH); 2.86 (m, 2H, CH2-N-CH3); 3.11 (m, 2H, CH2-NCH3); 3.28 (m, 4H, CH2-NH,
Ph-CH2-Pip); 3.44 (m, 2H, CH2-NH); 3.71 (s, 3H, Ph-OCH3); 3.75 (m, 2H, CH2-N), 3.97 (m, 2H, Ph-
O-CH2); 4.12 (s, 6H, TA); 4.65 (s, 2H, Ph-CH2-N); 6.00 (br, 6H*), TA-OH); 6.55–6.58 (m, 2H
aromatic, Py5-H, Py3-H); 6.86–6.88 (m, 3H aromatic, 4-H, 2-H); 6.86–6.88 (br, 4H*), 3 (H-(N+)),
NH); 6.92–7.00 (m, 2H aromatic, 2-H); 7.06 (br, 1H*, NH); 7.13 (m, 2H aromatic, 3-H); 7.26 (m, 1H
aromatic, 3-H); 7.43–7.46 (d, J = 7, 1H aromatic, Py4-H); 8.08 (m, 1H aromatic, Py6-H); 10.50 (br,
3H*), TA-COOH); MS: m/z (%) 641 (M+, 1), 241 (7), 121 (100); Anal. Calcd. for
C37H52N8O2·3C4H6O6: C, 51.60; H, 6.29; N, 10.20. Found: C, 52.01; H, 6.47; N, 10.30.

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N-Cyano-N'-[4-[N-[2-[N-(4-fluorobenzyl]-N-(2-pyridyl)amino]ethyl]-N-methylamino]butyl]-N″-[3-[3-
1
(piperidinomethyl)phenoxy]propyl]guanidine (19). Yield: 48%, mp: 84–86 °C; H-NMR (CDCl3):
δ ppm = 1.37 (m, 2H, CH2); 1.46 (m, 2H, CH2); (see 15); MS: m/z (%) 629 (M+, 5), 2229 (84), 109 (100);
Anal. Calcd. for C36H49FN8O·3C4H6O6: C, 53.90; H, 6.73; N, 10.40. Found: C, 51.30; H, 6.46; N, 9.97.
N-Cyano-N'-[4-[N-[2-[N-(4-methoxybenzyl)-N-(2-pyridyl)amino]ethyl]-N-methylamino]pentyl]-N″-
1
[3-[3-(piperidinomethyl)phenoxy]propyl]guanidine (20). Yield: 17%, mp: 86–88 °C; H-NMR
(CDCl3): δ ppm = 1.22 (m, 4H, 2(CH2)); (see 17); MS: m/z (%) 643 (M+, 7), 229 (54), 154 (100); Anal.
Calcd. for C37H51FN8O·3C4H6O6: C, 53.80; H, 6.36; N, 10.30. Found: C, 54.30; H, 6.83; N, 10.30.
N-Cyano-N'-[6-[N-[2-[N-(4-methoxybenzyl)-N-(2-pyridyl)amino]ethyl]-N-methylamino]hexyl]-N″-[3-
1
[3-(piperidinomethyl)phenoxy]propyl]guanidine (21) Yield: 66%, mp: 79–81 °C; H-NMR (CDCl3):
δ ppm = 1.23–1.25 (m, 4H, 2(CH2)); 1.42–1.43 (m, 4H, 2(CH2)); (see 14); MS: m/z (%) 669 (M+, 5),
121 (100); Anal. Calcd. for C39H56N8O2·3C4H6O6: C, 54.70; H, 6.66; N, 10.10. Found: C, 54.60; H,
7.01; N, 9.79.
N-Cyano-N'-[6-[N-[2-[N-(4-fluorobenzyl)-N-(2-pyridyl)amino]ethyl]-N-methylamino]hexyl]-N″-[3-
[3-(piperidinomethyl)phenoxy]propyl]guanidine (22). Yield: 35%, mp: >58 °C decomposed; 1H-NMR
(CDCl3): δ ppm = 1.25 (m, 4H, 2(CH2)); 1.42 (m, 2H, CH2); (see 25); MS: m/z (%) 657 (M+, 11), 229
(100); Anal. Calcd. for C38H53FN8O·3C4H6O6: C, 52.50; H, 6.61; N, 9.80. Found: C, 52.70; H, 6.80;
N, 9.80.
N-Cyano-N'-[7-[N-[2-[N-(4-fluorobenzyl)-N-(2-pyridyl)amino]ethyl]-N-methylamino]heptyl]-N″-[3-
1
[3-(4-(piperidin-1-ylmethyl)phenoxy]propyl]guanidine (23). Yield: 41%, mp: 96–98 °C; H-NMR
(CDCl3): δ ppm = 1.21-1.25 (m, 6H, 3(CH2)); 1.42 (m, 2H, CH2); (see 17); MS: m/z (%) 671 (M+, 3),
229 (100); Anal. Calcd. for C39H55FN8O·1.5C20H18O8: C, 65.30; H, 6.62; N, 8.83. Found: C, 65.24;
H, 6.90; N, 8.65.
N-Cyano-N'-[4-[N-[2-(4-fluorobenzyl)-N-(2-pyridyl)amino]ethyl]-N-methylamino]butyl)-N″-[3-[4-(4-
(piperidin-1-ylmethyl)phenoxy]butyl]guanidine (24). Yield: 37%, mp: 102–104 °C; 1H-NMR (CDCl3):
δ ppm = 1.68-1.75(m, 2H, CH2); (see 17); MS: m/z (%) 629 (M+, 18), 229 (98), 109 (100); Anal.
Calcd. for C36H49FN8O·1.5C20H18O8: C, 64.60; H, 6.36; N, 9.13. Found: C, 64.40; H, 6.51; N, 8.77.
N-Cyano-N'-[2-[[(2-guanidino-4-thiazolyl)methyl]thio]ethyl]-N″-[2-[N-[2-[N-(4-methoxybenzyl)-N-
1
(2-pyridyl)amino]ethyl]-N-methylamino]ethyl]guanidine (25). Yield: 58%, mp: 56–58 °C; H-NMR
(CDCl3): δ ppm = 2.23 (s, 3H, N-CH3); 2.50 (m, 4H, CH2-S, CH2-NCH3); 2.56 (m, 2H, CH2-NCH3);
3.16–3.17 (m, 2H, CH2-NH); 3.26–3.28 (m, 2H, CH2-NH); 3.37 (s, 2H, Thi-CH2-S); 3.58 (m, 2H,
CH2-N); 3.70 (s, 3H, Ph-OCH3); 4.66 (s, 2H, Ph-CH2-N); 6.45 (s, 1H aromatic., Thi5-H); 6.50–6.54
(m, 2H aromatic, Py5-H, Py3-H);, 6.84–6.87 (m, 2H aromatic, 2-H); 6.84–6.87 (br, 2H*, 2(NH)); 6.94
(br, 4H*, NC(NH2)2); 7.14 (d, J = 8, 2H aromatic, 3-H); 7.41 (m, 1H aromatic, Py4-H); 8.05–8.07
(d, J = 4, 1H aromatic, Py6-H); MS: m/z (%) 596 (M+, 2), 121 (100); Anal. Calcd. for
C27H37N11OS2·1.5C2H2O4: C, 54.30; H, 6.75; N, 24.0. Found: C, 54.21; H, 6.63; N, 24.11.

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N-Cyano-N'-[2-[[(2-guanidino-4-thiazolyl)methyl]thio]ethyl]-N″-[2-[N-[2-[N-(4-fluoro-benzyl)-N-(2-
pyridyl)amino]ethyl]-N-methylamino]ethyl]guanidine (26). Yield: 50%, mp: >109 °C decomposed;
1H-NMR (CDCl3): δ ppm = 2.23 (s, 3H, N-CH3); 2.50 (m, 2H, CH2-S); 2.55 (m, 4H, 2(CH2-NCH3));
3.15 (m, 2H, CH2-NH); 3.27 (m, 2H, CH2-NH); 3.58 (m, 4H, Thi-CH2-S, CH2-N); 4.72 (s, 2H, Ph-
CH2-N); 6.46 (s, 1H, Thi5-H); 6.51–6.54 (m, 2H aromatic, Py5-H, Py3-H); 6.80 (br, 6H*, 2(NH),
NC(NH2)2); 7.11 (t, J=9, 2H aromatic, 2-H); 7.22–7.24 (d, J=6, 2H aromatic, 3-H); 7.44 (m, 1H
aromatic, Py4-H); 8.06 (d, 1H aromatic, Py6-H); MS: m/z (%) 584 (M+, 3), 121 (100); Anal. Calcd. for
C26H34FN11S2·2C4H6O6·1.5H2O: C, 44.80; H, 5.42; N, 16.90. Found: C, 44.61; H, 4.91; N, 17.11.
N-Cyano-N'-[2-[[(2-guanidino-4-thiazolyl)methyl]thio]ethyl]-N″-[3-[N-[2-[N-(4-methoxybenzyl)-N-
1
(2-pyridyl)amino]ethyl]-N-methylamino]propyl]guanidine (27). Yield: 45%, mp: 54–56 °C; H-NMR
(CDCl3): δ ppm = 1.66 (m, 2H, CH2); 2.26 (s, 3H, N-CH3); 2.48 (m, 2H, CH2-S); 2.58 (t, J = 7, 2H,
CH2-NCH3); 2.68 (t, J = 7, 2H, CH2-NCH3); 3.21 (m, 2H, CH2-NH); 3.29 (m, 2H, CH2-NH); 3.58
(s, 2H, Thi-CH2-S); 3.72 (t, J = 8, 2H, CH2-N); 3.76 (s, 3H, Ph-OCH3); 4.60 (s, 2H, Ph-CH2-N); 5.74
(br, 2H*, 2(NH)); 6.33 (S, 1H aromatic, Thi5-H); 6.43–6.44 (d, J = 8.5, 2H aromatic, Py5-H, Py3-H);
6.54 (br, 4H*, NC(NH2)2); 6.81–6.84 (m, 2H aromatic, 2-H); 7.10–7.13 (d, J=8,5, 2H aromatic, 3-H);
7.34–7.39 (m, 1H aromatic, Py4-H); 8.11–8.12 (d, J=3, 1H aromatic, Py6-H); MS: m/z (%) 624 (M+,
1), 121 (100); Anal. Calcd. for C28H39N11OS2·H2O: C, 53.60; H, 6.58; N, 24.50. Found: C, 53.81; H,
6.43; N, 24.41.
N-Cyano-N'-[3-[N-[2-[N-(4-fluorobenzyl)-N-(2-pyridyl)amino]ethyl]-N-methylamino]propyl]-N″-[2-
1
[[2-guanidino-4-thiazolyl)methyl]thio]ethyl]guanidine (28). Yield: 35%, mp: oil; H-NMR (CDCl3):
δ ppm = 1.67 (m, 2H ,CH2); 2.27 (s, 3H, N-CH3); 2.49 (t, 2H, CH2-S); 2.58 (t, 2H, CH2, NCH3); 2.73
(t, 2H, CH2-NCH3); 3.21 (m, 2H, CH2-NH); 3.32 (m, 2H, CH2-NH); 3.60 (s, 2H, Thi-CH2-S); 3.73 (m,
2H, CH2-N); 4.66 (s, 2H, Ph-CH2-N); 6.35 (s, 1H aromatic, Thi5-H); 6.41–6.43 (d, J = 9, 1H aromatic,
Py5-H); 6.57 (t, J = 5, 1H aromatic, Py3-H); 6.80 (br, 6H*, 2(NH), NC(NH2)2); 6.99 (t, J=8.5, 2H
aromatic, 2-H); 7.17 (m, 2H aromatic, 3H); 7.37–7.39 (m, 1H aromatic, Py4-H); 8.14 (d, 1H, Py6-H);
MS: m/z (%) 598 (M+, 2), 121 (100); Anal. Calcd. for C27H36FN11S2: C, 53.80; H, 6.58; N, 24.50.
Found: C, 53.81; H, 6.43; N, 24.40.
N-Cyano-N'-[2-[[(2-guanidino-4-thiazolyl)methyl]thio]ethyl]-N″-[4-[N-[2-[N-(4-methoxybenzyl)-N-
(2-pyridyl)amino]ethyl]-N-methylamino]butyl]guanidine (29). Yield: 60%, mp: hygroscopic foam;
1H-NMR (CDCl3): δ ppm = 1.36–1.44 (m, 4H, 2(CH2)); (see 25); MS: m/z (%) 624 (M+, 1), 121 (100);
Anal. Calcd. for C29H41N11OS2·1.5H2O: C, 53.50; H, 6.81; N, 23.70. Found: C, 53.42; H, 6.51; N, 24.01.
N-Cyano-N'-[4-[N-[2-[N-(4-fluorobenzyl)-N-(2-pyridyl)amino]ethyl]-N-methylamino]butyl]-N″-[2-
[[2-guanidino-4-thiazolyl)methyl]thio]ethyl]guanidine (30). Yield: 60%, mp: >137 °C decomposed;
1H-NMR (CDCl3): δ ppm = 1.49–1.58 (m, 2H, CH2); (see 28); MS: m/z (%) 612 (M+, 2), 229 (100),
109 (38); Anal. Calcd. for C28H38FN11S2·1.5C20H18O8·2H2O: C, 56.80; H, 5.66; N, 12.60. Found: C,
56.40; H, 5.30; N, 12.51.
N-Cyano-N'-[5-[N-[2-[N-(4-fluorobenzyl)-N-(2-pyridyl)amino]ethyl]-N-methylamino]pentyl]-N″-[2-
1
[[2-guanidino-4-thiazolyl)methyl]thio]ethyl]guanidine (31). Yield: 29%, mp: 103–105 °C; H-NMR

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(CDCl3): δ ppm = 1.23 (m, 2H, CH2); 1.35 (m, 4H, 2(CH2)); (see 26); MS: m/z (%) 626 (M+, 3), 154
(100); Anal. Calcd. for C29H40FN11S2·3C4H6O6·0.5H2O: C, 45.40; H, 5.48; N, 14.20. Found: C, 45.12;
H, 5.32; N, 14.19.
N-Cyano-N'-[2-[[(2-guanidino-4-thiazolyl)methyl]thio]ethyl]-N″-[6-[N-[2-[N-(4-methoxybenzyl)-N-
(2-pyridyl)amino]ethyl]-N-methylamino]hexyl]guanidine (32). Yield: 29%, mp: >52 °C decomp.;
1H-NMR (CDCl3): δ ppm = 1.27 (m, 4H, 2(CH2)); 1.42 (m, 2H, CH2); (see 27); MS: m/z (%) 652
(M+, 12), 121 (100); Anal. Calcd. for C31H45N11OS2·0.5H2O: C, 56.31; H, 7.01; N, 23.30. Found: C,
56.42; H, 7.17; N, 23.31.
N-Cyano-N'-[6-[N-[2-[N-(4-fluorobenzyl)-N-(2-pyridyl)amino]ethyl]-N-methylamino]hexyl]-N″-[2-
[[2-guanidino-4-thiazolyl)methyl]thio]ethyl]guanidine (33). Yield: 61%, mp: 75–77 °C decomp.;
1H-NMR (CDCl3): δ ppm = 1.26 (m, 4H, 2(CH2)); 1.43 (m, 2H, CH2); (see 28); MS: m/z (%) 639 (M+,
9), 229 (100), 109 (88); Anal. Calcd. for C30H42FN11S2·3C4H6O6·0.25H2O: C, 46.10; H, 5.57; N,
14.10. Found: C, 45.81; H, 5.62; N, 14.62.
N-Cyano-N'-[7-[N-[2-[N-(4-fluorobenzyl)-N-(2-pyridyl)amino]ethyl]-N-methylamino]heptyl]-N″-[2-
1
[[2-guanidino-4-thiazolyl)methyl]thio]ethyl]guanidine (34). Yield: 24%, mp: 118–120 °C; H-NMR
(CDCl3): δ ppm = 1.24 (m, 6H, 3(CH2)); 1.42 (m, 2H, CH2); (see 28); MS: m/z (%) 654 (M+, 2), 91
(100); Anal. Calcd. for C31H44FN11S2·1.5C20H18O8·2H2O: C, 57.71; H, 5.96; N, 12.11. Found: C,
57.61; H, 5.51; N, 12.42.
N-Cyano-N'-[3-[N-[2-[N-(4-methoxybenzyl)-N-(2-pyridyl)amino]ethyl]-N-methylamino]propyl]-N″-
[2-[5-(dimethylaminomethyl)furfuryl]thio]-ethyl]guanidine (35). Yield: 38%, mp: 74–76 °C; 1H-NMR
(CDCl3): δ ppm = 1.68 (q, 2H, CH2); 2.23 (s, 6H, N(CH3)2); 2.28 (s, 3H, N-CH3); 2.49 (t, 2H, CH2-S);
2.56–2.65 (m, 4H, 2(CH2-NCH3)); 3.23–3.30 (m, 4H, 2(CH2-NH)); 3.40 (s, 2H, Fur-CH2-S); 3.67 (s,
2H, Fur-CH2-N); 3.75 (t, J = 8, 2H, CH2-N); 3.78 (s, 3H, Ph-OCH3); 4.63 (s, 2H, Ph-CH2-N);
6.11–6.12 (q, J = 4, 2H aromatic, Fur3-H, Fur4-H); 6.44–6.47 (d, J = 8.5, 1H aromatic, Py5-H); 6.55
(m, 1H aromatic, Py3-H); 6.55 (br, 1H*, NH); 6.83–6.85 (d, J = 8.5, 2H aromatic, 2-H); 6.83–6.85 (br,
1H*); 7.12–7.15 (d, J = 8.5, 2H aromatic, 3-H); 7.38 (m, 1H aromatic, Py4-H); 8.12–8.13 (d,
J = 3, 1H aromatic, Py6-H); MS: m/z (%) 595 (M+, 4), 121 (100); Anal. Calcd. for
C31H44N8O2S·3C4H6O6·0.5H2O: C, 49.10; H, 6.03; N, 10.71. Found: C, 48.71; H, 6.26; N, 11.22.
3.2. Pharmacology
3.2.1. Histamine H1 Receptor Antagonist Activity on the Isolated Guinea Pig Ileum
H1 receptor antagonist activity was determined from isotonically recorded (load 10 mN) cumulative
concentration-response curves as described using histamine dihydrochloride (0.01–3 µM) as the
reference agonist [31]. Whole segments of proximal ileum were mounted in an organ bath containing
20 mL of Tyrode solution ([mM] NaCl 137, KCl 2.7, NaH2PO4 4.2, NaHCO3 11.9, CaCl2 1.8, MgCl2
1.0, D-glucose 5.6) containing 0.1 µM atropine, aerated with 95% O2/5% CO2, bath temperature 37 °C.
The compounds were tested at different concentrations with at least four experiments for each
concentration. Determination of H1 receptor antagonism at low concentrations (≤10−8 M) of the

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potential antagonists required a longer incubation period up to 20 min in order to achieve equilibrium
states; at higher concentrations periods of 10–15 min were sufficient. pKB values (mean of at least
three independent experiments) were calculated from the expression pKB = −log [antagonist] + log
(concentration ratio–1) as the compounds produced a dose-dependent depression of the concentration-
response curves [32].
3.2.2. Histamine H2 Receptor Antagonist Activity on the Isolated Guinea Pig Right Atrium
The investigations for H2 receptor antagonism followed the procedure described by Black et al. [33]
with minor modifications. In brief, male guinea pigs (350–400 g) were killed by a blow on the head and
exsanguinated. Right atria were rapidly removed, attached to a tissue holder in an organ bath (32.5 °C)
containing 20 mL of Krebs-Henseleit solution, containing [mM] NaCl 118, NaHCO3 25, KCl 4.7, KH2PO4
1.2, CaCl2 2.5, MgSO4 1.6, glucose 6.2, gassed with 95% O2/5% CO2, bath temperature 32.5 °C. The
antagonistic potency was determined from isometrically recorded cumulative concentration-response
curves using histamine dihydrochloride (0.1–10 µM) as the reference substance [34]. The incubation
period was generally 30 min for the antagonists. For pharmacological screening, 2–5 experiments were
carried out for each antagonist at one or two concentrations (Table 1) and pKB values were calculated
as above.
4. Conclusions
Starting from lead compound 1 according to the working hypothesis that the incorporation of a
mepyramine-like structure linked via a six-membered-spacer with a cyanoguanidine instead of urea as
a polar group and combined with a tiotidine-like partial structure, significantly improved H1R
antagonist activity and restored pronounced H2R antagonism. However, the affinity at both receptors
should be further increased with respect to the development of combined H1R/H2R antihistamine as
potential pharmacotherapeutic agents. Generally, pharmacological hybrids harbour the potential of
improving the pharmacokinetic properties compared to single drugs with different half lives. Further
progress in structural optimization seems possible through selection of other H1R and H2R
pharmacophoric moieties known to possess histamine receptor subtype affinity. Nevertheless, this has
to be proven experimentally, because the mutual affinity-conferring or -enhancing contribution of the
respective substructures cannot be predicted on the basis of the structure-activity relationships
available so far. Different routes of administration are conceivable, for example, topical applications of
combined H1R/H2R antagonists in dermatology or systemic administration for premedication in
anaesthesia and surgery or for the prevention of hypersensitivity reactions to drug treatment, e.g., in
cancer chemotherapy. This has to be taken into consideration with respect to optimization of the
physicochemical properties.
Acknowledgments
The authors are very grateful to Walter Schunack (deceased April 2011), Berlin, for mentoring,
provision of laboratory capacity and funds, and for long-standing excellent collaboration. The financial

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support provided in form of Faculty Grant NP/13/17 by College of Medicine and Health Sciences,
United Arab Emirates University of Science and Technology is gratefully acknowledged.
Conflicts of Interest
The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 25 and 32 are available from the authors.
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