[3-(1H-Imidazol-4-yl)propyl]guanidines containing furoxan moieties: A new class of H3-antagonists endowed with NO-donor properties

Article abstract of DOI:10.1016/S0968-0896(02)00651-X

Synthesis and pharmacological characterisation of a series of products obtained by coupling the H₃-antagonist SKF 91486 through appropriate spacers with the NO-donor 3-phenylfuroxan-4-yloxy and 3-benzenesulfonylfuroxan-4-yloxy moieties, as well as with the corresponding furazan substructures, devoid of NO-donating properties, are reported. All the products were tested for their H₃-antagonistic and H₂-agonistic properties on electrically-stimulated guinea-pig ileum segments and guinea-pig papillary muscle, respectively. The whole series of compounds displayed good H₃-antagonist behaviour and feeble partial H₂-agonist activity. Among furoxan derivatives, the benzenesulfonyl hybrid 28, a good NO-donor, triggered a dual NO-dependent muscle relaxation and H₃-antagonistic effect on guinea-pig intestine.

Full text of DOI:10.1016/S0968-0896(02)00651-X

Bioorganic & Medicinal Chemistry 11 (2003) 1197–1205
[3-(1H-Imidazol-4-yl)propyl]guanidines Containing Furoxan
Moieties: A New Class of H3-Antagonists Endowed with
NO-Donor Properties
Massimo Bertinaria,a Antonella Di Stilo,a Paolo Tosco,a Giovanni Sorba,b Enzo Poli,c
Cristina Pozzoli,c Gabriella Coruzzi,c Roberta Frutteroa,* and Alberto Gascoa
a
`
Dipartimento di Scienza e Tecnologia del Farmaco, Universita degli Studi di Torino, Via Pietro Giuria 9, I-10125 Turin, Italy
Dipartimento di Scienze Chimiche, Alimentari, Farmaceutiche e Farmacologiche, Universita degli Studi del Piemonte Orientale,
b
`
Viale Ferrucci 33, I-28100 Novara, Italy
cUniversity of Parma, School of Medicine, Dipartimento di Anatomia Umana, Farmacologia e Scienze Medico-Forensi,
via Volturno 39, I-43100 Parma, Italy
Received 25October 2002; accepted 9 December 2002
Abstract—Synthesis and pharmacological characterisation of a series of products obtained by coupling the H3-antagonist SKF
91486 through appropriate spacers with the NO-donor 3-phenylfuroxan-4-yloxy and 3-benzenesulfonylfuroxan-4-yloxy moieties, as
well as with the corresponding furazan substructures, devoid of NO-donating properties, are reported. All the products were tested
for their H3-antagonistic and H2-agonistic properties on electrically-stimulated guinea-pig ileum segments and guinea-pig papillary
muscle, respectively. The whole series of compounds displayed good H3-antagonist behaviour and feeble partial H2-agonist activity.
Among furoxan derivatives, the benzenesulfonyl hybrid 28, a good NO-donor, triggered a dual NO-dependent muscle relaxation
and H3-antagonistic effect on guinea-pig intestine.
# 2003 Elsevier Science Ltd. All rights reserved.
Introduction
involved. In our laboratories furoxan moieties have
been used to create different classes of hybrids, such as
ibuprofen-like anti-inflammatory drugs7 or lamtidine-
like H2 receptor antagonists,8 obtaining prototypical
compounds which combine anti-inflammatory or gastric
antisecretory activities with NO-dependent gastro-
protective effects. As further development in the field of
hybrid design we now propose a series of NO–H3-
antagonists. Histamine, through H3 receptors,9ꢀ11 and
NO12 display a variety of effects in different body com-
partments including central and peripheral nervous sys-
tem. While the role of H3 receptors in the periphery has
still to be established, their role in different brain areas,
such as the hippocampus and the hypothalamus, has
been demonstrated. Accordingly, a number of potential
therapeutic applications for H3 receptor antagonists in
several CNS pathologies, including memory and learn-
ing disorders, have been suggested.10 Since NO too is
implicated in learning and memory formation,13 the
simultaneous block of histamine H3 receptors and NO
release in specific areas of the brain could synergistically
contribute to a curative effect in such pathological
The synthesis of molecular hybrids is a strategy to
design novel products endowed with multiple pharma-
cological properties. In recent years, several classes of
hybrid compounds, obtained combining appropriate
pharmacophoric groups with NO-releasing functions,
have been described.1 A number of them, such as NO-
aspirin,2 NO-steroids3 and NO-ursodeoxycholic acid4
are now under clinical investigations. There is strong
evidence that the furoxan system (1,2,5-oxadiazole 2-
oxide) is able to release NO under the action of thiol
cofactors.5 By introducing appropriate substituents at
the ring, it is possible to modulate the rate and amount
of NO-production.6 The overall reaction mechanism is
complex, and different NO-redox forms could be
*Corresponding author. Tel.: +39-011-670-7850; fax: +39-011-670-
7972; e-mail: roberta.fruttero@unito.it
0968-0896/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0968-0896(02)00651-X

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M. Bertinaria et al. / Bioorg. Med. Chem. 11 (2003) 1197–1205
conditions. In this paper we discuss synthesis and pre-
liminary ‘in vitro’ pharmacological characterisation of
novel hybrids obtained by coupling, through appro-
priate spacers, furoxan groups (3-phenylfuroxan-4-
yloxy and 3-benzenesulfonylfuroxan-4-yloxy) to the
compound SKF 91486. Such compound is the [3-(1H-
imidazol-4-yl)propyl]guanidino portion of the histamine
H2-agonist/H3-antagonist impromidine, endowed with
feeble activity at the histamine H2 receptor.14 The use of
SKF 91486 as a pharmacophore could allow the gen-
eration of novel H3 receptor ligands able to release NO
(der.s 24, 26, 28). The corresponding furazan models
(der.s 23, 25, 27), devoid of NO-donating properties,
were also synthesised and tested as control compounds.
The combined NO-donating properties and the activity
at histamine H3 receptors of all the synthesised com-
pounds were evaluated ‘in vitro’ using the isolated,
electrically-driven, guinea-pig ileum, while the activity
Scheme 1. (a) EtMgBr, DMF, rt; (b) NaNH2, (EtO)2P(O)CH2CN, THF, reflux; (c) H2, Pd/C, THF; (d) LiAlH4/AlCl3, Et2O/THF/(2:1), rt; (e) PPh3,
Phtalimide, DIAD, THF, 0 ꢁC; (f) 40% aq CH3NH2, EtOH, rt; (g) 9, CH2Cl2; (h) 2-aminoethanol or 3-aminopropan-1-ol, THF, reflux; (i) 50% aq
NaOH, 20–25 ꢁC.

M. Bertinaria et al. / Bioorg. Med. Chem. 11 (2003) 1197–1205
1199
at histamine H2 receptors was evaluated on the isolated
guinea-pig papillary muscle.
good yields the expected amino derivative 6. This pro-
duct was not isolated and purified but used directly for
further transformation. Nucleophilic substitution of the
phenoxy group in 10 by the appropriate aminoalcohol
in THF at reflux gave the intermediate alcohols 11, 12.
It is known that bis(benzenesulfonyl)furoxan (16) when
treated with ethanol under basic conditions in distilled
THF at room temperature, selectively affords 3-benze-
nesulfonyl-4-ethoxyfuroxan.17 Under the same conditions,
also the nucleophilic displacement of 4-benzenesulfonyl
group from the 3-phenylfuroxan 14 occurs. Related
furazans 13, 15 behave similarly. Therefore, to prepare
the protected derivatives 17–22 we allowed the appro-
priate furoxan and furazan derivative to react with
alcohols 11, 12 in the above reported conditions. 13C
Results
Chemistry
According to our strategy, synthesis of the final pro-
ducts 23–28 requires the preparation of the common
intermediate N-benzoyl-O-phenylisourea 10 (Scheme 1).
This compound can be easily obtained by nucleophilic
displacement at room temperature in CH2Cl2 solution
of one of the two phenoxy groups present in N-ben-
zoyldiphenylimidocarbonate (9), under the action of 3-
(1-tritylimidazol-4-yl)propylamine (6). Preparation of 6
is achieved by the two different approaches depicted in
Scheme 1. The first implies synthesis of the phtalimido
derivative 8 by reaction of phtalimide, triphenylphos-
phine, diisopropylazodicarboxylate (DIAD) and 7 in
THF at 0 ꢁC. Product 8 dissolved in ethanol is easily
converted at room temperature into 6 by aqueous 40%
methylamine. The second route starts from trityl-pro-
tected iodoimidazole 2. Transformation of this product
into the aldehyde 3 was previously achieved by action of
n-butylithium at ꢀ78 ꢁC in dry THF followed by rapid
quenching of the reaction mixture with DMF.15 We
modified this procedure using ethylmagnesium bromide
in CH2Cl2 at room temperature. The aldehyde 3 pro-
vides a,b-unsaturated nitrile 4 by means of a modified
Wittig approach using cyanomethyl diethylpho-
sphonate, according to the procedure described in lit-
erature.16 Also subsequent reduction of 4 over 10% Pd/
C to give 5 was obtained according to literature.16
Finally, action on 5 of a mixture of LiAlH4/AlCl3 in 2:1
Et2O/THF solution at room temperature affords in
1
and H NMR spectra of the products 21, 22 indicate
that, in this case too, selective displacement of 4-benze-
nesulfonyl group occurs. Subsequent deprotection of
17–20 in boiling 5N HCl gives the final products 23–26
(Scheme 2). Surprisingly, this procedure does not work
with benzenesulfonyl derivatives 21, 22. In fact, exten-
sive decomposition occurs with formation of a mixture
of products among which we isolated, in the case of 22,
benzenesulfonylmethyl chloride (29) in quantitative
yield. The mechanism of this transformation remains
to be cleared. Final derivatives 27, 28 were obtained
in good yield by treating derivatives 21, 22 with 3 M
p-TsOH at 95 ꢁC for 72 h.
Detection of nitrite
To evaluate the thiol-induced NO-generation the
appropriate furoxan derivative was dissolved in pH 7.4
buffered water and incubated for 1 h at 37 ꢁC in the
presence of a large excess of l-cysteine (1:50). Nitrite,
which is an important oxidation product of NO in
aerobic solution, was determined by the Griess reaction
Scheme 2. (a) 5N HCl, reflux; (b) 3 M p-TsOH, reflux.

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M. Bertinaria et al. / Bioorg. Med. Chem. 11 (2003) 1197–1205
Table 1. NO-release characteristics, H3 receptor antagonism and H2-agonistic properties of synthesised compounds 23–28, reference 1, and hista-
mine
Compd
NOꢀ2 (%)a (+l-cys)
H3-antagonismb
Guinea-pig ileum
H2-agonistic activityc Guinea-pig
papillary muscle
pA2ꢂSEM
(pD2ꢂSEM)
(i.a.)d
1
—
—
7.24ꢂ0.07
8.19ꢂ0.07
7.82ꢂ0.09
8.49ꢂ0.03
8.25ꢂ0.07
7.42ꢂ0.03
7.02ꢂ0.03
7.05ꢂ0.05e
—
4.49ꢂ0.07 (0.52)
4.34ꢂ0.04 (0.41)
4.54ꢂ0.07 (0.61)
5.70ꢂ0.04 (0.40)
5.90ꢂ0.06 (0.59)
5.88ꢂ0.07 (0.53)
5.28ꢂ0.012 (0.63)
0.52
0.41
0.61
0.40
0.59
0.53
0.63
23
24
25
26
27
28
3.8ꢂ0.1
—
4.0ꢂ0.06
—
2.67ꢂ3.4
Histamine
—
6.15ꢂ0.02 (1.00)
1.00d
— not determined.
aYields are reported in% [mol/molꢂstandard error of the mean (SEM)].
bpA2 was calculated only for one surmountable concentration (1 mM) of the antagonist using the Gaddum’s equation pA2=ꢀlog [B]+log [CRꢀ1];
data are the meanꢂSEM of 5–11 observations.
cpD2=ꢀlog EC50; data are the meanꢂSEM of 4–6 observations.
di.a., intrinsic activity; histamine=1.
eDetermined in the presence of ODQ 1 mM.
according to a previously reported procedure.6 The
results expressed as a percentage of NOꢀ2 (mol/mol) are
reported in Table 1. It is worthy of note that nitrite
production by furoxan is strongly dependent on the
medium, the concentration and the nature of the thiol
employed; therefore, it only gives a trend of the NO
production which might occur in the cellular environ-
ment; it does not give information about the NO-redox
form(s) involved in the release.
Pharmacology
The H3-antagonistic activity of the products was asses-
sed by their ability at 1 mM concn to antagonise the
concentration-dependent inhibitory effect of (R)-a-
methylhistamine (MHA) on electrically evoked twitches
of isolated guinea-pig ileum segments.18 pA2 values
were calculated using the Gaddum equation
(pA2=ꢀlog [B]+log [CR-1]) and are reported in Table
1. The H2-agonistic activity of the products was investi-
gated by their ability to induce changes in the contrac-
tion force of electrically stimulated guinea-pig papillary
muscle according to the reported method.19 Potencies
were expressed as pD2 values (pD2=ꢀlog EC50) and
reported in Table 1.
Discussion
All the products display quite good antagonistic activity
at H3 receptor. In fact they are able to inhibit, at 1 mM
concentration, the concentration-dependent inhibitory
effect of (R)-a-methylhistamine (MHA) on electrically
evoked contractile response (acetylcholine release) of
guinea-pig ileum preparations. Analysis of pA2 values
of the furazan derivatives 23, 25, 27 shows that they
display more potent or similar antagonistic activity with
Figure 1. NO-dependent effect of compound 28 on electrically stimu-
lated guinea-pig ileum (panel a); experiment repeated in the presence
of ODQ (panel b); MHA reported as M concentration.

M. Bertinaria et al. / Bioorg. Med. Chem. 11 (2003) 1197–1205
1201
respect to lead 1 (SKF 91486). Furoxan derivatives 24
and 26, which are feeble NO-donors, behave in a strictly
similar manner as the furazan analogues 23 and 25,
unable to release NO. Different behaviour is presented
by the furoxan 28, a potent NO-donor. In fact, when
the product was added to the bath at 1 mM concen-
tration after the electrical contraction of the tissue, a
partial relaxation (ꢃ20%) of the preparation was
observed (Fig. 1a). This effect was transient and
disappeared after 5min.
antagonists could be an interesting starting point for the
design of further hybrids.
Experimental
Melting points were determined with a capillary appa-
ratus (Buchi 530) and are uncorrected or by differential
scanning calorimetry (Perkin-Elmer DSC 7, heating rate
10 ꢁC/min). All the compounds were routinely checked
1
by IR (Shimadzu FT-IR 8101 M). H and 13C NMR
The phenomenon was absent when the experiment was
repeated in the presence of 1 mM ODQ (1H-[1,2,4]oxa-
diazolo[4,3-a]quinoxalin-1-one), a selective inhibitor of
soluble guanilate cyclase (sGC) (Fig. 1b), thus suggest-
ing the involvement of NO as the final mediator of this
effect. Accordingly, relaxing effect on guinea-pig ileum
induced by NO-donors was earlier observed.20 Morover
other furoxan derivatives tested in our laboratory dis-
play similar behaviour, as observed in preliminary
experiments.21 The calculated pA2 value for derivative
28 was 7.02 (ꢂ0.03) close to those of the parent furazan
27 and of the lead 1.
spectra were obtained on a Bruker AC-200 at 200 and
50 MHz resp. and on a Bruker Avance 300, at 300 and
75MHz resp.; d in ppm rel. to SiMe4 as internal stan-
dard; coupling constants J in Hz; 13C NMR spectra
were fully decoupled. Mass spectra were recorded on
Finnigan-Mat TSQ-700. Flash chromatography (FC):
silica gel (Merck Kieselgel 60) 70–230 mesh ASTM.
Anhydrous magnesium sulfate (MgSO4) was used as the
drying agent of organic phases unless otherwise stated.
Analysis (C, H, N) of the new compounds was per-
formed by REDOX (Monza). Structures 2,15 4, 5,16 7,23
9,24 13,25 14,26 15,17 1627 were synthesised according to
reported methods. Following abbreviations are used:
DMF, N,N-dimethylformamide; Et2O, diethyl ether;
Since lead 1 shows weak agonistic properties at the H2
receptor, we further characterised compounds 23–28 for
their ability to interact with the H2 receptor.
THF,
tetrahydrofuran;
DIAD,
diisopropylazo-
dicarboxylate; PE, petroleum ether 40–60 ꢁC; AcOEt,
ethyl acetate.
The data reported in Table 1 show that all the synthe-
sised compounds induce a concentration-dependent
increase in the contractile force when tested on
electrically stimulated guinea-pig papillary muscle.
Unlike histamine they are unable to produce the max-
imal response evoked by H2 receptor stimulation on this
tissue. The positive inotropic effect of the compounds
23–28, was antagonised by the presence of 1 mM
Famotidine, a well-known H2-antagonist. The pD2
values of compounds 23–28 suggest that the activity at
H2 receptors occurs at concentrations 100–500 times
higher than those required to block H3 receptors.
Therefore all the synthesised products behave, similarly
to the lead 1, as weak partial agonists at H2 receptor,
their efficacy lying between 40 and 63% of that of his-
tamine. Development of the present work will address
structural modulation of this new class of hybrids in
order to obtain products endowed with suitable brain
penetrating properties,22 and to evaluate their ability to
interfere with different H3 receptor subtypes which
occur in central neurons.11
[(1-Triphenylmethyl)imidazol-4-yl]carbaldehyde (3). To a
stirred solution of 2 (1.50 g, 3.44 mmol) in 14 mL of dry
CH2Cl2 a solution of ethylmagnesium bromide 3 M in
Et2O (1.27 mL) was added at room temperature and the
reaction mixture was stirred for 30 min. After this time,
dry DMF (0.29 mL, 3.78 mmol) was added and the
mixture stirred at room temperature for another 30 min.
The mixture was treated with half-saturated NH4Cl
solution and the organic layer washed with water (3ꢄ20
mL), dried and evaporated under reduced pressure to
give a yellow solid which was purified by FC (silica gel;
PE/AcOEt 6:4) to afford 3 (0.96 g, 82%) as white solid.
MS (EI): m/z (M+): 338; mp 199–201 C (iPrOH). H
NMR (CDCl3) d 9.89 (s, 1H); 7.62 (s, 1H); 7.54 (s, 1H);
7.41–7.31 (m, 9H); 7.16–7.08 (m, 6H). 13C NMR
(CDCl3): 186.43; 141.38; 140.71; 140.47; 129.50; 128.24;
127.88; 116.88; 76.75. Analysis for C23H18N2O (338.4),
calcd C, 81.63; H, 5.36; N, 8.28; found: C, 81.36; H,
5.34; N, 8.25.
ꢁ
1
N-{3-[(1-Triphenylmethyl)imidazol-4-yl]propyl}phtalimide
(8). To a stirred solution of 6 (4.00 g, 10.9 mmol) in dry
THF (100 mL) at 0 ꢁC phtalimide (2.39 g, 16.3 mmol)
and triphenylphosphine (4.27 g, 16.3 mmol) were added.
The mixture was kept at 0 ꢁC and a solution of DIAD
(4.71 g, 23.3 mmol) in dry THF (120 mL) was added
dropwise during 1.5h. The reaction mixture was con-
centrated under reduced pressure to precipitate a solid
which was filtered and washed with THF (2ꢄ20 mL) to
yield 8 (4.97 g, 92%) as a white solid. CIMS (isobutane)
m/z 498 (MH+); mp 217–219 ꢁC (iPrOH); 1H NMR
(CDCl3) d 7.80–7.69 (m, 4H); 7.32–7.26 (m, 10H); 7.17
(s, 1H); 7.15–7.05 (m, 6H); 6.58 (s, 1H); 3.73 (t, J=7.2,
2H); 2.61 (t, J=7.2, 2H); 1.90 (m, 2H). 13C NMR
Conclusion
The results show that conjugation of the substituted
1,2,5-oxadiazole moiety or of its 2-oxide analogue with
the [3-(1H-imidazol-4-yl)propyl]guanidino pharmaco-
phore is not detrimental to the H3-antagonistic activity,
most of the derivatives being more active than lead
compound 1. The response evoked by our compounds
at the H3 receptor is remarkably higher than the
response evoked at the H2 receptor. Compound 28 dis-
plays a dual NO-dependent muscle relaxation and H3-
antagonistic effect. The described class of NO-donor-H3

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M. Bertinaria et al. / Bioorg. Med. Chem. 11 (2003) 1197–1205
(CDCl3): 168.35; 142.57; 140.59; 138.35; 133.78; 132.19;
129.8; 127.98; 127.92; 123.12; 118; 75.07; 37.65; 28.28;
25.96. Analysis for C33H27N3O2 (497.57), calcd C, 79.66;
H, 5.47; N, 8.44; found: C, 79.30; H, 5.49; N, 8.32.
1H NMR (CDCl3) d 10.6 (br s, exchangeable signal not
always observed); 8.18 (m, 2H); 7.43–7.34 (m, 13H);
7.18–7.11 (m, 6H); 6.61 (s, 1H); 3.80–3.67 (m, 4H); 3.41
(m, 2H); 2.62 (t, J=6.2, 2H); 1.95(m, 2H). 13C NMR
(CDCl3) d 178.81; 161.91; 142.10; 140.27; 138.61;
137.81; 130.68; 129.57; 128.58; 128 (two overlapping
carbons); 127.75; 118.43; 72.75; 64.43; 44.19; 39.78;
29.30; 23.13. Analysis for C35H35N5O2 (557.7), calcd C,
75.38; H, 6.33; N, 12.56; found: C, 75.48; H, 6.45; N, 12.33.
3-[(1-Triphenylmethyl)imidazol-4-yl]propylamine (6) and
1-benzoyl-2-{3-[(1-triphenylmethyl)imidazol-4-yl]propyl}-
O-phenylisourea (10). Method A. To a stirred suspen-
sion of 8 (4.00 g, 8.04 mmol) in EtOH (100 mL),
methylamine 40% aq solution (10 mL, 120 mmol) was
added. After 3 h of stirring at room temperature, the
mixture was evaporated under reduced pressure. The
residue was dissolved in CH2Cl2 (30 mL), and 9 (2.55 g,
8.04 mmol) was added. After 30 min at room tempera-
ture, the solvent was evaporated and the crude product
purified by FC (silica gel; CH2Cl2/AcOEt 8.5:1.5) to
obtain 10 as white foam (2.94 g, 62%). The product
showed identical data when compared with a sample
synthesised according to method B. Method B. To a
stirred suspension of LiAlH4 (0.25g, 6.60 mmol) in dry
N-Benzoyl-N0 -(3-hydroxypropyl)-N00 -{3-[(1-triphenyl-
methyl)imidazol-4-yl]propyl}guanidine (12). The reaction
was carried out as described above using 10 (1.41 g, 2.35
mmol) and 3-aminopropan-1-ol (1.89 mL, 24.7 mmol).
(Yield: 61%). White foam; mp 123–124 ꢁC (MeOH/
ꢁ
1
H2O). H NMR (DMSO-d6) recorded at 55 C d 10.25
(s, 1H, observed at 25 ꢁC); 8.07 (m, 2H); 7.40–7.36 (m,
13H); 7.10 (m, 6H); 6.64 (s, 1H); 4.23 (s, 1H); 3.51 (m,
2H); 3.36 (m, 4H); 2.56–2.50 (m, 2H); 1.85 (m, 2H); 1.72
(m, 2H). 13C NMR (DMSO-d6) d 175.03; 160.34;
142.64; 140.71; 139.68; 137.91; 130.44; 129.37; 128.58;
128.26; 128.13; 127.7; 117.68; 74.71; 58.53; 38.03; 32.57;
Et2O (12.5mL) a solution of AlCl (0.88 g, 6.60 mmol)
3
in dry Et2O (12.5mL) was added and, after 10 min of
stirring at room temperature, 5 (2.00 g, 5.50 mmol) in
dry THF (12 mL) was added carefully. Reduction was
completed in 1 h, the reaction mixture was treated with
H2O (1.5mL), NaOH 10% (2.5mL) and again H 2O
(2.5mL). The precipitate was filtered and washed thor-
oughly with THF (200 mL) and CH2Cl2 (300 mL). The
organic phase was dried (K2CO3) and evaporated under
.
30.71; 29; 25.33. Analysis for C36H37N5O2 0.3H2O
(577.13), calcd C, 74.92; H, 6.47; N, 12.14; found: C,
74.94; H, 6.51; N, 12.1.
N-[3-(1H-Imidazol-4-yl)propyl]-N0-{2-[(4-phenylfurazan-
3-yl)oxy]ethyl}guanidine dihydrochloride (23). To a stir-
red solution of 11 (1.38 g, 2.47 mmol) and 13 (0.85g,
2.97 mmol) in distilled THF (30 mL), 50% (w/w) aq
NaOH solution (0.8 g, 9.9 mmol) was added maintain-
ing the temperature at 20–25 ꢁC. The reaction mixture
was stirred vigorously at room temperature for 5.5 h,
then diluted with H2O (20 mL) and the organic solvent
was evaporated under reduced pressure (25 ꢁC). The
aqueous residue was extracted with AcOEt (3ꢄ30 mL),
the org. phase dried and evaporated to afford a gummy
solid which was purified by FC (silica gel; CH2Cl2/
AcOEt 7:3) to give the protected derivative 17 (1.24 g,
72%). The intermediate was readily hydrolysed with 68
mL of 5N HCl at reflux for 16 h. After cooling the
mixture was filtered through a sinter, the aqueous phase
extracted with Et2O (3ꢄ30 mL) and then evaporated
under reduced pressure (60 ꢁC). The semisolid residue
was treated with benzene (3ꢄ20 mL), evaporated and
dried in a desiccator over P2O5/NaOH/paraffin under
high vacuum to obtain 23 (0.66 g, 63% from 11) as
white amorphous solid. Mp 192–197 ꢁC (MeOH/Et2O).
1H NMR (DMSO-d6) d 14.61 (br s, 2H); 9.03 (s, 1H);
8.27 (m, 2H); 8.01 (m, 2H); 7.83 (s, 2H); 7.65–7.56 (m,
3H); 7.44 (m, 10H); 4.54 (t, J=4.5, 2H); 3.8 (m, 2H);
3.26–3.17 (m, 2H); 2.71 (t, J=7.4, 2H); 1.83 (m, 2H).
13C NMR (DMSO-d6) d 163.34; 156.24; 145.27; 133.59;
132.44; 131.15; 129.42; 127.55; 124.22; 115.81; 71.20;
40.08; 39.97; 23.42; 21.04. Analysis for C17H21
1
reduced pressure to afford 6 as a yellow oil. H NMR
(CDCl3) d 7.35–7.28 (m, 10H); 7.18–7.10 (m, 6H); 6.6 (s,
1H); 2.71 (t, J=7.0, 2H); 2.58 (t, J=7.5, 2H); 2.02 (br s,
2H); 1.79 (m, 2H). 13C NMR (CDCl3) d 142.36; 141.16;
138.12; 129.58; 127.79 (two overlapping carbons);
117.59; 74.87; 41.54; 33.01; 25.56. The product 6 was
not further characterised but directly converted to 10 by
addition of N-benzoyldiphenylimidocarbonate (9) (2.54
g, 7.94 mmol) to a solution of 6 (2.02 g, 5.49 mmol) in
CH2Cl2 (50 mL). After 30 min of stirring at room tem-
perature the solvent was evaporated under reduced
pressure and the oily residue purified by FC (silica gel;
CH2Cl2/AcOEt 9:1) to yield 10 (2.79 g, 86%) as white
foam. Mp 144–145 ꢁC (cold acetone/H2O). 1H NMR
(CDCl3) d 10.2 (br s, 1H); 7.8 (m, 2H); 7.37–7.14 (m,
24H); 6.6 (s, 1H); 3.59 (br m, 2H); 2.71 (br m, 2H); 2.08
(m, 2H). 13C NMR (CDCl3) d 177.63; 162.61; 151.82;
142.51; 140.35; 138.59; 137.34; 131.55; 129.71; 129.23;
128.99; 127.98; 127.79; 127.79; 125.60; 121.68; 118.05;
75.08; 41.14; 29.34; 25.65. Analysis for C39H34N4O2
(590.69), calcd C, 79.30; H, 5.80; N, 9.48.
N-Benzoyl-N0-(2-hydroxyethyl)-N00-{3-[(1-triphenylmethyl)-
imidazol-4-yl]propyl}guanidine (11). To a stirred solu-
tion of 10 (1.41 g, 2.35mmol) in dry THF (10 mL), 2-
aminoethanol (1.50 mL, 24.7 mmol) was added and the
mixture was refluxed for 40 min. After cooling the mix-
ture was diluted with H2O (10 mL), the solvent was
removed under reduced pressure and the aqueous resi-
due extracted with CH2Cl2 (5ꢄ20 mL). The organic
layer was dried and the solvent evaporated under
reduced pressure to give a solid which was purified by
FC (silica gel; CH2Cl2/MeOH 9.5:0.5) yielding 11 (0.92
g, 70%) as white foam. Mp 164–165 ꢁC (MeOH/H2O).
.
.
.
N7O2 2HCl 0.5H2O 0.2 C4H10O (452.16), calcd C, 47.28;
H, 5.80; N, 21.68; found: C, 47.17; H, 5.76; N, 21.46.
N-[3-(1H-Imidazol-4-yl)propyl]-N0-{2-[(3-phenylfuroxan-
4-yl)oxy]ethyl}guanidine dihydrochloride (24). To a stir-
red solution containing 11 (1.00 g, 1.79 mmol) and 14
(0.76 g, 2.50 mmol) in distilled THF (30 mL), 50% (w/
w) aq NaOH solution (0.56 g, 7.02 mmol) was added

M. Bertinaria et al. / Bioorg. Med. Chem. 11 (2003) 1197–1205
1203
maintaining the temperature at 20–25 ꢁC. After 3 h of
vigorous stirring at room temperature, the mixture was
diluted with H2O (20 mL) and the organic solvent was
evaporated under reduced pressure (25 ꢁC). The aqu-
eous residue was extracted with CH2Cl2 (3ꢄ30 mL) and
the extracts dried and evaporated under reduced pres-
sure to afford a pale yellow oil. The crude product was
purified by FC (silica gel; CH2Cl2/AcOEt 6:4) to obtain
the protected intermediate 18 (1.22 g, 95%) as a white
solid. Derivative 18 was immediately hydrolysed by
refluxing in 5N HCl (66 mL) for 16 h. The cooled mix-
ture was filtered through a sinter, extracted with Et2O
(3ꢄ30 mL) and the aqueous layer evaporated under
reduced pressure (60 ꢁC). After drying under high
vacuum over P2O5/NaOH/paraffin 24 (0.64 g, 78%
from 11) was obtained as white amorphous solid. Mp
162–169 ꢁC (dec.) (dry MeOH/Et2O). 1H NMR
(DMSO-d6) d 14.53 (br s, 2H); 9.04 (s, 1H); 8.33 (br m,
2H); 8.09 (m, 2H); 7.87 (br m, 2H); 7.64–7.53 (m, 3H); 7.44
(s, 1H); 4.57 (br s, 2H); 3.81 (m, 2H); 3.23 (m, 2H); 2.71 (t,
J=7, 2H); 1.83 (m, 2H). 13C NMR (DMSO-d6) d 162.25;
156.28; 133.54; 132.47; 130.75; 129.18; 126.46; 121.86;
115.84; 107.87; 69.22; 39.9 (two overlapping carbons);
(0.56 g, 7.02 mmol) was added at room temperature;
after 3 h of stirring the mixture was diluted with H2O
(25mL) and the organic solvent evaporated under
reduced pressure (25 ꢁC). The aqueous residue was
extracted with CH2Cl2 (3ꢄ30 mL), the extracts dried
and evaporated to obtain the crude product which was
purified by FC (silica gel, CH2Cl2/AcOEt 6:4) to obtain
pure 20 (1.00 g; 78%) as a pale yellow foam. The pro-
tected intermediate 20 was refluxed in 5N HCl for 16 h;
after cooling the mixture was filtered and extracted with
Et2O (3ꢄ30 mL) and the aqueous phase evaporated to
dryness under reduced pressure (60 ꢁC). The product
was dried over P2O5/NaOH/paraffin under high vacuum
to yield 26 (0.63 g; 78% from 12) as white amorphous
solid. An analytical sample was obtained as tri-
fluoroacetate. Mp 184 ꢁC (DSC) (MeOH/Et2O). 1H
NMR (DMSO-d6) d a few exch. protons not observed;
9.01 (s, 1H); 8.07 (m, 4H); 7.61 (m, 4H); 7.45(s, 1H);
4.57 (t, J=5.6, 2H); 3.4 (m, 2H); 3.2 (m, 2H); 2.72 (t,
J=7, 2H); 2.11 (m, 2H); 1.87 (m, 2H). 13C NMR
(DMSO-d6) d 162.28; 156.04; 134.20; 133.60; 132.58;
130.75; 129.12; 126.31; 122.04; 115.66; 107.03; 68.43;
39.99; 37.77; 27.91; 27.50; 21.13. Analysis for
.
.
.
.
27.50; 21.08. Analysis for C17H21N7O3 2HCl 0.6H2O
(455.1), calcd C, 44.86; H, 5.36; N, 21.54; found: C,
44.63; H, 5.64; N, 21.35.
C18H23N7O3 1.5CF 3COOH 1.5H2O (583.5), calcd C,
43.23; H, 4.75; N, 16.8; found: C, 43.43; H, 4.94; N,
16.67.
N-[3-(1H-Imidazol-4-yl)propyl]-N0-{3-[(4-phenylfurazan-
3-yl)oxy]propyl}guanidine dihydrochloride (25). To a
stirred solution of 12 (1.00 g, 1.75mmol) and 13 (0.70 g,
2.45mmol) in distilled THF (30 mL), 50% (w/w) aq
NaOH solution (0.56 g, 7.02 mmol) was added at room
temperature and the reaction stirred vigorously for 6 h.
The mixture was diluted with H2O (20 mL) and the
organic solvent evaporated under reduced pressure
(25 ꢁC). The aqueous residue was extracted with CH2Cl2
(4ꢄ30 mL), the organic phase dried and evaporated
under reduced pressure to give a pale yellow oil, which
was purified by FC (silica gel; CH2Cl2/AcOEt 6:4) to
obtain 19 (0.92 g, 74%) as a white solid. This inter-
mediate was deprotected by refluxing with 5N HCl (50
mL) for 16 h. The cooled reaction mixture was filtered,
extracted with Et2O (3ꢄ30 mL), CH2Cl2 (1ꢄ30 mL),
and the aqueous layer was evaporated under reduced
pressure (60ꢁ). The semisolid product was dried in a
desiccator under high vacuum over P2O5/NaOH/paraf-
fin to afford 25 (0.47 g, 61% from 12) as a white amor-
phous solid. Mp 170–174 ꢁC (dec.) (dry MeOH/Et2O).
1H NMR (DMSO-d6) d 14.7 (br s, 2H); 9.06 (s, 1H); 8.1
(m, 2H); 7.93 (m, 2H); 7.70 (m, 2H); 7.68 (m, 3H); 7.47
(s, 1H); 4.54 (t, J=5.6, 2H); 3.42–3.18 (m, 4H); 2.72 (t,
J=7.2, 2H); 2.10 (m, 2H); 1.84 (m, 2H). 13C NMR
(DMSO-d6) d 163.39; 156.08; 145.30; 133.55; 132.51;
131.18; 129.46; 127,44; 124.46; 115.67; 70.47; 39.95;
N-[3-(1H-Imidazol-4-yl)propyl]-N0-{3-[(4-benzenesulfonyl-
furazan-3-yl)oxy]propyl}guanidine ditosylate (27). To a
solution of 12 (1.17 g, 2.04 mmol) and bis(benzene-
sulfonyl)furazan (15) (1.00 g, 2.85mmol) in distilled
THF (10 mL), 50% (w/w) aq NaOH solution (0.65 g,
8.16 mmol) was added and the mixture stirred vigor-
ously at room temperature for 2 h. The reaction was
diluted with water (10 mL) and the solvent evaporated
under reduced pressure (25 ꢁC). The aqueous residue
was treated with brine (10 mL) and extracted with
CH2Cl2 (3ꢄ30 mL), the organic phase was dried and
evaporated under reduced pressure to give a pale yellow
oil. The product was purified by FC [silica gel, CH2Cl2/
AcOEt 8:2 (300 mL) then CH2Cl2/AcOEt 1:1 (400 mL)]
to obtain pure 21 (1.43 g, 90%) as a white foam. Inter-
mediate 21 was hydrolysed by heating at 95 ꢁC with
p-TsOH 3 M (75mL) for 72 h; after cooling the mixture
was diluted with water (40 mL), extracted with Et2O
(5ꢄ40 mL) and the aqueous phase evaporated to dry-
ness (60 ꢁC). The residue was dried thoroughly in a
desiccator over P2O5/NaOH/paraffin under high
vacuum for 48 h. The obtained cream-coloured solid
was triturated with Et2O (6ꢄ100 mL) to eliminate
excess acid and the residue was crystallised from dry
MeOH/Et2O to obtain 27 (1.09 g, 75% from 12) as a
white amorphous solid. Mp 134 ꢁC (dec.) (DSC). 1H
NMR (DMSO-d6) d 9.01 (s, 1H); 8.10 (m, 2H); 7.96–
7.74 (m, 4H); 7.60–7.44 (m, 10H); 7.13 (m, 4H); 4.42 (t,
J=5.8, 2H); 3.22 (m, 4H); 2.68 (t, J=7.4, 2H); 2.30 (s,
6H); 1.96 (m, 2H); 1.83 (m, 2H). 13C NMR (DMSO-d6)
d 161; 155.66; 148.63; 145.11; 138.18; 136.88; 136.33;
134.01; 132.62; 130.32; 128.78; 128.29; 125.53; 115.73;
71.39; 40.18; 37.46; 27.78; 27.30; 21.20; 20.87. Analysis
37.80;
28.13;
.
27.52;
21.10.
Analysis
for
.
C18H23N7O2 2HCl 0.6H2O (453.15), calcd C, 47.71; H,
5.83; N, 21.64; found: C, 47.67; N, 5.87; N, 21.29.
N-[3-(1H-Imidazol-4-yl)propyl]-N0-{3-[(3-phenylfuroxan-
4-yl)oxy]propyl}guanidine dihydrochloride (26). To a
stirred solution of 12 (1.00 g, 1.75mmol) and 3-phenyl-
4-benzenesulfonylfuroxan (14) (0.74 g, 2.45mmol) in
distilled THF (75mL), 50% (w/w) aq NaOH solution
.
.
.
for C18H23N7O4S 2 C7H8O3 H2O 0.2 C4H10O (810.7),
calcd C, 48.59; H, 5.35; N, 12.09; found: C, 48.8; H,
5.14; N, 12.06.

1204
M. Bertinaria et al. / Bioorg. Med. Chem. 11 (2003) 1197–1205
N-[3-(1H-Imidazol-4-yl)propyl]-N0-{3-[(3-benzenesulfonyl-
furoxan-4-yl)oxy]propyl}guanidine ditosylate (28). To a
solution of 12 (1.00 g, 1.75mmol) and bis(benzene-
sulfonyl)furoxan (16) (0.90 g; 2.45mmol) in dry THF (8
mL), 50% (w/w) aq NaOH solution (0.56 g, 7.0 mmol)
was added dropwise and the reaction mixture stirred
vigorously at room temperature for 1 h. The mixture
was diluted with H2O (10 mL), the organic solvent eva-
porated under reduced pressure (25 ꢁC). The obtained
residue was treated with brine (10 mL) and extracted
with CH2Cl2 (3ꢄ30 mL). The extracts were dried and
evaporated under reduced pressure to afford the crude
product which was purified by FC [silica gel, CH2Cl2/
AcOEt 7:3 (400 mL) then CH2Cl2/AcOEt 1:2 (300 mL)]
to give the protected intermediate 22 as a white foam
(1.23 g, 88%). The protected product 22 (0.60 g, 0.75
mmol) was suspended in 3 M p-TsOH (35mL) and
heated at 95 ꢁC for 72 h. After cooling the mixture was
filtered, extracted with Et2O (5ꢄ20 mL) and the aqu-
eous phase evaporated under reduced pressure (45 ꢁC).
The residue was dried under high vacuum, then washed
with Et2O (7ꢄ50 mL). The semisolid residue was dis-
solved in water (10 mL) and extracted with AcOEt
(3ꢄ20 mL), the aqueous layer was evaporated under
reduced pressure (45 ꢁC) to obtain a white solid which
was crystallised from dry MeOH/Et2O to give 28 (0.48
g, 70% from 12) as a white amorphous solid. Mp 144–
mL)]. After 10 min at room temperature, absorbance
was measured at 540 nm; 10–80 nmol/mL NaNO2
standard solutions were used for the calibration curve.
The nitrite yield was expressed in % NO-2 (mol/
mol)ꢂstandard error.
Isolated guinea-pig papillary muscle
Left papillary muscles were dissected from guinea pig
hearts and suspended under 0.5–1 g of passive load in
10-mL organ baths, containing Ringer solution, at 37 ꢁC
and gassed with 95% O2 and 5% CO2. Two platinum
electrodes, implanted into the ventricular basis, were
used to drive electrically the tissues. Square wave pulses
(2 Hz, 1 ms, 20% above threshold voltage) were con-
tinuously delivered and changes in contraction force of
papillary muscles were recorded by an isometric trans-
ducer, connected with a pen recorder. After 60–90 min
equilibration period, cumulative concentration–
response curves to histamine and to H2 receptor ago-
nists were constructed by increasing doses by half-log
units after each response had reached a plateau (1–2
min). Up to three reproducible concentration–response
curves could be constructed in the same preparation at
1-h intervals, without any sign of desensitisation. When
H2 receptor antagonists were tested, the curve to hista-
mine or to other agonists was repeated in the same
tissue after a 20-min incubation with the antagonist.
153 ꢁC (dec.) (DSC) (MeOH/Et2O). H NMR (DMSO-
1
d6) d few exch. protons not observed; 9.02 (s, 1H); 8.04
(m, 2H); 7.96–7.72 (m, 4H); 7.62–7.40 (m, 9H); 7.14 (m,
4H); 4.46 (t, J=5.9, 2H); 3.49–3.19 (m, 4H); 2.69 (t,
J=7.4, 2H); 2.30 (s, 6H); 2.02 (m, 2H); 1.84 (m, 2H).
13C NMR (DMSO-d6) d 158.93; 155.67; 145.16; 138.13;
137.11; 136.3; 134; 132.59; 130.16; 128.48; 128.29;
125.54; 115.73; 110.68; 68.82; 40.22; 37.54; 27.66; 27.29;
Isolated guinea-pig ileum
All compounds were tested for H3-antagonistic effects in
vitro using the guinea pig ileum (a portion of the small
intestine 20–50 cm proximal to the ileocaecal valve)18
isolated from male albino guinea-pigs weighing 250–350
g. The tissues were mounted under 1 g tension (isotonic
transducer) and placed in Krebs–bicarbonate buffer
(composition in mM: NaCl 111.2; KCl 5.0; CaCl2 2.5;
MgSO4 1.2; KH2PO4 1.0; NaHCO3 12; glucose 11.1;
pH=7.4) at 37 ꢁC and gassed with 95% O2–5% CO2.
After equilibration of 2 h with washings every 20 min,
the muscle segments were stimulated with square wave
pulses delivered by a Grass Stimulator S-44 (10 V, 0.1
Hz, 0.5ms). After 30 min of stimulation, cumulative
concentration–response curves (half-log increments) of
the histamine H3 agonist (R)-a-methylhistamine (MHA)
were recorded until no change in response was found.
To avoid the desensitisation phenomena, a single con-
centration-response curve to the H3 agonist MHA was
carried out in the same preparation. From each animal
four segments of ileum were prepared: one was used as a
control and the others were incubated with the antago-
nist for 20 min before the generation of concentration–
response curves with MHA. All the experiments were
carried out in the presence of H1 and H2 receptor
blockers (Pyrilamine 1 mM and Ranitidine 1 mM) to
prevent the activation of these receptors by the highest
concentration of MHA.29 In order to check eventual effect
by NO on the concentration–response curve of MHA,
preliminary experiments were carried out. The concen-
tration–response curve for MHA was recorded in the
presence of the furoxan NO-donor CHF 2363 (0.1 mM)30
without observing any significant change in the EC50
.
21.29;
20.87.
.
Analysis
for
C18H23N7O5S 2
.
C7H8O3S H2O 0.2 C4H10O (826.7), calcd C, 47.65; H,
5.24; H, 11.86; found: C, 47.85; H, 5.01; N, 11.51.
Attempted hydrolysis of 22 with 5 NHCl: synthesis of
benzenesulfonylmethyl chloride (29). The protected
intermediate 22 (0.45g, 0.56 mmol) was refluxed with
5N HCl (35 mL) for 16 h. The cooled reaction mixture
was filtered and extracted with Et2O (5ꢄ15mL). The
ethereal phase was washed with 10% NaHCO3 (3ꢄ10
mL), dried and evaporated under reduced pressure to
give a pale orange oil which was purified by FC (silica
gel, PE/CH2Cl2 1:1) to afford the title product 29 (0.10
g, 98%) as white solid. Mp 53–54 ꢁC (EtOH/H2O) (lit.28
52–53 ꢁC). H NMR (CDCl3) d 7.97 (m, 2H); 7.84–7.72
1
(m, 3H); 5.33 (s, 2H). 13C NMR (DMSO-d6) d 136;
134.82; 129.57; 128.87; 57.48. Analysis for C7H7ClO2S
(190.6), calcd C, 44.1; H, 3.70; found: C, 43.94; H,
3.73.
Quantitative nitrite detection. A solution of the appro-
priate compound (20 mL) in DMSO was added to 2 mL
of 50 mM phosphate buffer (pH 7.4) containing 5 mM
l-cysteine. The final concentration of the drug was 10ꢀ4
M. After 1 h at 37 ꢁC, 1 mL of this mixture was treated
with 250 mL of Griess reagent [4 g sulfanilamide, 0.2 g
N-naphtylethylenediamine bis(hydrochloride), 10 mL
85% phosphoric acid in distilled H2O (final volume: 100

M. Bertinaria et al. / Bioorg. Med. Chem. 11 (2003) 1197–1205
1205
value. Similarly no change in the affinity of the reference
H3-antagonist Tioperamide was observed when the
experiment was repeated in the presence of CHF 2363.
11. Drutel, G.; Peitsaro, N.; Karlstedt, K.; Wieland, K.; Smit,
M. J.; Timmerman, H; Panula, P.; Leurs, R. Mol. Pharmacol.
2001, 59, 1.
12. Kervin, J. F., Jr.; Lancaster, J. R., Jr.; Feldman, P. L. J.
Med. Chem. 1995, 38, 4343.
13. Gammie, S. C.; Dawson, V. L.; Nelson, R.J. In Nitric
Oxide Biology and Pathobiology; Ignarro, L. J. Ed., Academic:
New York, 2000; p 429.
14. Ganellin, C.R. In Pharmacology of Histamine Receptors,
Ganellin, C. R., Parsons, M. E., Eds., Wright-PSG: London,
1982; p 10.
Acknowledgements
This work was supported by a COFIN grant from
MIUR, Roma and by grants from the University of
Parma (MURST 60%).
15. Kirk, K. L. J. Heterocycl. Chem. 1985, 22, 5 7.
16. Adger, B. M.; Surtees, J. Synth. Commun. 1987, 17, 223.
17. Sorba, G.; Ermondi, G.; Fruttero, R.; Galli, U.; Gasco, A.
J. Heterocycl. Chem. 1996, 33, 327.
References and Notes
1. (a) Wang, P. G.; Xian, M.; Tang, X.; Wu, X.; Wan, Z.; Cai,
T.; Janczuk, A. J. Chem. Rev. 2002, 102, 1109. (b) Janero,
D. R. Free Radic. Biol. Med. 2000, 28, 1495.
18. Vollinga, R. C.; Zuiderveld, O. P.; Scheerens, H.; Bast, D.;
Timmerman, H. Meth. Find. Exp. Clin. Pharmacol. 1992, 105,
747.
2. Freduzzi, S.; Mariucci, G.; Tantucci, M.; del Soldato, P.;
Ambrosini, M. V. Neurosci. Lett. 2001, 302, 121.
19. Bertaccini, G.; Coruzzi, G. Naunyn-Schmiedeberg’s Arch.
Pharmacol. 1981, 317, 225.
3. Tallet, D.; del Soldato, P.; Oudart, N.; Burgaud, J. L. Bio-
chem. Biophys. Res. Commun. 2002, 290, 125.
20. Osthaus, L. E.; Galligan, J. J. J. Pharmacol. Exp. Ther.
1992, 260, 140.
4. Fiorucci, S.; Antonelli, E.; Morelli, O.; Mencarelli, A.;
Casini, A.; Mello, T.; Palazzetti, B.; Tallet, D.; del Soldato, P.;
Morelli, A. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 8897.
5. Schonafinger, K. Farmaco 1999, 54, 316.
21. Di Stilo, A. Personal communication.
22. Stark, H.; Krause, M.; Arrang, J.-M.; Ligneau, X.;
Schwartz, J.-C.; Schunack, W. Bioorg. Med. Chem. Lett. 1994,
4, 2907.
6. Sorba, G.; Medana, C.; Fruttero, R.; Cena, C.; Di Stilo, A.;
Galli, U.; Gasco, A. J. Med. Chem. 1997, 40, 463.
7. Lolli, M. L.; Cena, C.; Medana, C.; Lazzarato, L.; Morini,
G.; Coruzzi, G.; Manarini, S.; Fruttero, R.; Gasco, A. J. Med.
Chem. 2001, 44, 3463.
8. Bertinaria, M.; Sorba, G.; Medana, C.; Cena, C.; Adami,
M.; Morini, G.; Pozzoli, C.; Coruzzi, G.; Gasco, A. Helv.
Chim. Acta 2000, 83, 287.
9. Hill, S. J.; Ganellin, C. R.; Timmerman, H.; Schwartz, J.-
C.; Shankley, N. M.; Young, J. M.; Schunack, W.; Levi, R.;
Haas, H. L. Pharm. Rev. 1997, 49, 253.
10. (a) Leurs, R.; Blandina, P.; Tedford, C.; Timmerman, H.
TiPS 1998, 19, 177. (b) Stark, H.; Arrang, J.-M.; Ligneau, X.;
Garbarg, M.; Ganellin, C. R.; Schwartz, J.-C.; Schunack, W.
Prog. Med. Chem. 2001, 38, 279. (c) Bertaccini, G.; Coruzzi,
G.; Poli, E. In The Histamine H3 Receptor; Leurs, R., Tim-
merman, H., Eds., Elsevier Science: Amsterdam, 1998; p 59.
23. Stark, H.; Purand, K.; Huls, A.; Ligneau, X.; Garbarg,
M.; Schwartz, J.-C.; Schunack, W. J. Med. Chem. 1996, 39,
1220.
24. Buschauer, A. Arch. Pharm. (Weinheim) 1987, 320, 377.
25. Calvino, R.; Mortarini, V.; Gasco, A.; Sanfilippo, A.;
Ricciardi, M. L. Eur. J. Med. Chem. Chim. Ther. 1980, 15,
485.
26. Boschi, D.; Di Stilo, A.; Cena, C.; Lolli, M.; Fruttero, R.;
Gasco, A. Pharm. Res. 1997, 14, 1750.
27. Farrar, W. V. J. Chem. Soc. 1964, 604.
28. Cinquini, M.; Colonna, S. J. Chem. Soc., Perkin Trans. 1
1972, 1883.
29. Poli, E.; Pozzoli, C.; Coruzzi, G.; Bertaccini, G. J. Phar-
macol. Exp. Ther. 1994, 270, 788.
30. Civelli, M.; Giossi, M.; Caruso, P.; Razzetti, R.; Berga-
maschi, M.; Bongrani, S.; Gasco, A. Br. J. Pharmacol. 1996,
118, 923.