A new class of NO-donor H3-antagonists

Article abstract of DOI:10.1016/j.farmac.2003.12.008

Synthesis and pharmacological characterisation of a series of compounds obtained by joining, through appropriate spacers, NO-donor furoxan and nitrooxy moieties to the imidazole ring, as well as their structurally related analogues devoid of NO-donating properties are described. All the products were studied for their capacity to interact with H₃-receptors present on the guinea-pig ileum and with H₂-receptors present on guinea-pig right atrium. The whole series of products displayed reversible H₃- antagonistic activity. No activity on H₂-receptors was observed when the products were tested at 10 μM concentration. Many of the products were also able to induce partial relaxation when added to the bath after electrical contraction of the guinea-pig ileum during the study of their H ₃-antagonism. This phenomenon seems to be dependent on various factors; for some compounds it proved to be dependent on NO-mediated sGC activation, for other products it could be due to their weak M ₃-antagonism. The investigation of the lipophilic-hydrophilic balance of all the products indicates, for many of them, an ideal value to cross the blood-brain barrier.

Full text of DOI:10.1016/j.farmac.2003.12.008

IL FARMACO 59 (2004) 359–371
www.elsevier.com/locate/farmac
A new class of NO-donor H3-antagonists
Paolo Tosco a, Massimo Bertinaria a, Antonella Di Stilo a,
Elisabetta Marini a, Barbara Rolando a, Giovanni Sorba b,
Roberta Fruttero a,*, Alberto Gasco a
a Dipartimento di Scienza e Tecnologia del Farmaco, Via P. Giuria 9, 10125 Torino, Italy
b Dipartimento di Scienze Chimiche, Alimentari, Farmaceutiche e Farmacologiche, Università degli Studi del Piemonte Orientale,
Viale Ferrucci 33, 28100 Novara, Italy
Received 6 October 2003; accepted 23 December 2003
Abstract
Synthesis and pharmacological characterisation of a series of compounds obtained by joining, through appropriate spacers, NO-donor
furoxan and nitrooxy moieties to the imidazole ring, as well as their structurally related analogues devoid of NO-donating properties are
described. All the products were studied for their capacity to interact with H3-receptors present on the guinea-pig ileum and with H2-receptors
present on guinea-pig right atrium. The whole series of products displayed reversible H3-antagonistic activity. No activity on H2-receptors was
observed when the products were tested at 10 µM concentration. Many of the products were also able to induce partial relaxation when added
to the bath after electrical contraction of the guinea-pig ileum during the study of their H3-antagonism. This phenomenon seems to be
dependent on various factors; for some compounds it proved to be dependent on NO-mediated sGC activation, for other products it could be
due to their weak M3-antagonism. The investigation of the lipophilic-hydrophilic balance of all the products indicates, for many of them, an
ideal value to cross the blood-brain barrier.
© 2004 Elsevier SAS. All rights reserved.
Keywords: Histamine; H3-antagonists; Molecular hybrids; NO-donors; Furoxans
1. Introduction
Since NO too is implicated in learning and memory forma-
tion [6], the simultaneous block of histamine H3-receptors
and NO release in specific areas of the brain could sinergis-
tically contribute to a curative effect in such pathological
conditions. In a previous study we designed a series of
[3-(1H-imidazol-4-yl)propyl]guanidines, analogues of SKF
91486 [7], containing differently substituted NO-donor
furoxan moieties, which displayed selective and quite potent
H3-antagonistic properties [8]. Because of the presence of
the guanidine substructure, all of them are strong bases
(pKa>11) almost fully protonated at physiological pH. Con-
sequently they are typically polar hydrophilic compounds
which do not readily enter the CNS.
As further development of this work, in order to obtain
less polar compounds potentially able to cross the blood-
brain barrier (BBB), we now describe the synthesis and the
preliminary in vitro pharmacological characterisation of a
series of products structurally related to aryloxyalkylimida-
zoles [9] (e.g., 10) and formally obtained from these models
by substituting furoxan or nitrooxy moieties for aryl groups.
Today there is a widespread interest in the design of
“hybrid” drugs in which an appropriate pharmacophoric
group is joined to a nitric oxide (NO) releasing moiety [1,2].
Some of these products have been object of patents and are
now under clinical investigation [1]. For a long time our
laboratory has taken an active interest in this approach, and
we have developed a number of hybrid structures, particu-
larly in the cardiovascular and gastroenteric fields [3,4].
Recently we investigated the possibility of obtaining new
H3-receptor antagonists endowed with NO-donor properties.
A number of potential therapeutic applications for H3-
receptor antagonists in several CNS pathologies, including
memory and learning disorders, have been suggested [5].
* Corresponding author.
E-mail address: roberta.fruttero@unito.it (R. Fruttero).
© 2004 Elsevier SAS. All rights reserved.
doi:10.1016/j.farmac.2003.12.008

360
P. Tosco et al. / IL FARMACO 59 (2004) 359–371
A number of sulphur and sulfonyl analogues, as well as a
number of structurally related furazans devoid of the capac-
ity to release NO, are also considered.
Finally, the dinitrooxy model 9 was obtained according to
the pathway reported in Scheme 2. The reaction of 1 in THF
with allyl bromide and NaH, followed by deprotection with
2 N HCl in EtOH afforded the intermediate 8. This product
was Boc (t-butoxycarbonyl) protected and allowed to react
with a I2/AgNO3 mixture in acetonitrile to give the desired
final compound 9.
O
The synthetic strategy followed to obtain the sulphur-
containing models is reported in Scheme 3. The common
starting material for the preparation of all these products was
the O-ethyl dithiocarbonate S-ester 12. This compound was
obtained by tosylation in pyridine of the imidazole derivative
1, followed by reaction in acetonitrile solution with potas-
sium O-ethyl dithiocarbonate. Treatment of this intermediate
with the appropriate phenylsulfonyl furoxan and furazan
derivatives in CHCl3 or CH3CN solution in the presence of
N,N-dimethylpropane 1,3-diamine gave the expected nucleo-
philic substitution products. Working with 2, 2a, 2b, 4 the
reaction was carried out at different temperatures according
to the reactivity of the substrates, and the resulting protected
products were immediately deprotected using 90% aqueous
TFA to give the final compounds. When 4a was used the
reaction temperature was kept at -15 °C and a mixture of
trityl derivatives of 15a and 15b in a 1:5 ratio was obtained.
Fractional crystallisation gave pure protected 15b, which
once deprotected afforded pure 15b, and a mixture of pro-
tected 15a and 15b in about 1:1 ratio. This mixture was
deprotected with TFA and again protected with the less
lipophilic Boc, then resolved by reverse phase HPLC. Depro-
tection in TFA gave pure 15a. The structures of the two
products were assigned on the basis of their 13C NMR spec-
tra, using appropriate correlation tables previously obtained
using suitable furoxan models [11]. The reference compound
13 was prepared in a similar manner by direct interaction of
thiophenol with 11 in acetonitrile solution at room tempera-
ture followed by deprotection in TFA. Derivatives 16, 16a,
16b were easily prepared by KMnO4 oxidation of the thio-
analogues in acetic acid solution.
HN
N
10
2. Chemistry
The synthetic strategy for the preparation of the ether
models 5, 5a, 6, 6a, 7, 7a is outlined in Scheme 1. Of the two
possible furoxan isomers, only the products bearing the oxy-
gen atom at the 4-position of the ring were prepared, since
they are the thermally stable isomers. The potassium salt of
the trityl (triphenylmethyl) protected imidazole derivative 1
was treated in THF solution with the appropriate phenylsul-
fonyl furoxan and furazan derivatives to give the expected
nucleophilic substitution products. When 3,4-bis(phenyl-
sulfonyl)furoxan (4a) was allowed to react under these con-
ditions, the sole 3-phenylsulfonylfuroxan 7a was obtained as
indicated by its 13C NMR spectrum. This finding is in line
with other reports which claim that alkoxy groups selectively
displace the 4-phenylsulfonyl substituent in 4a [10]. The
products obtained were immediately deprotected by using
90% aqueous trifluoroacetic acid (TFA) to give the final
compounds.
OH
N
N
Ph3C
1
i)
t-BuOK, ∆
R
Ph
Ph
SPh
SPh
SO2Ph
SO2Ph
n
0
1
0
1
0
1
2
2a
3
3a
4
ii)
PhSO2
R
N
N
(
)
O n
O
3. Lipophilic-hydrophilic balance
4a
The ionisation constants (pKa) and the partition coeffi-
cients (log P in n-octanol/water) of the products discussed in
the present work, measured by the pH-metric method using a
Sirius GlpKa instrument, are reported in Table 1. The distri-
bution coefficients at pH 7.4 (log D7.4), calculated from these
figures, are also reported. The pKa values lie in quite a narrow
range (6.80÷7.19) and are in keeping with the presence in the
structures of the basic imidazole ring bearing an oxy or thio
substituted alkyl chain at the 4-position [12]. The log P and
log D values are similar, as a consequence of the low ionisa-
tion degree of the products at pH 7.4 and range about 2 to 3.5.
Hansch has suggested that a parabolic relationship exists for
the passive diffusion of neutral molecules into the CNS, with
an optimum log Poct value of about 2 0.5 [13]. Conse-
quently, the majority of the products should readily enter the
CNS.
(
)
n
O
O
N
N
O
R
N
N
Ph3C
90% aq TFA
R
Ph
Ph
SPh
SPh
SO2Ph
SO2Ph
n
0
1
0
1
0
1
(
)
n
O
O
5
5a
6
6a
7
N
N
O
R
HN
N
7a
Scheme 1. Synthesis of the ether models 5, 5a, 6, 6a, 7, 7a.

P. Tosco et al. / IL FARMACO 59 (2004) 359–371
361
2 N HCl, EtOH
reflux
THF, reflux
O
1
N
N
Ph3C
, NaH
Br
THF, Boc2O
6 N NaOH
O
O
HN
N
N
N
Boc
8
i)
AgNO3, I2
0 °C
CH3CN
ii)AgNO3, reflux
O
ONO2
HN
N
ONO2
9
Scheme 2. Synthesis of the dinitrooxy derivative 9.
4. Pharmacology
tested at 10 µM concentration. They are more potent H3-
antagonists than reference 10, with the sole exception of
furoxan 7a bearing a sulfonyl group at the ring. The whole
series displays feeble competitive antagonism at the M3-
receptor. The sole furazan 6 appears to be a feeble non-
competitive antagonist. Only two products, namely furoxans
6a and 7a, induced partial relaxation when added to the bath
at 1 µM concentration after electrical contraction of the
tissue. For the derivative 6a this effect was completely re-
versed in the presence of ODQ, and consequently the relax-
ation can be regarded as NO-dependent (Fig. 1). By contrast,
in the case of 7a ODQ only partly inhibits this relaxation that
therefore is in part dependent on other factors. The antago-
nistic properties of the product at the M3-receptor might play
a role in this behaviour.
The H3-antagonistic activity of the products was assessed
by their ability to antagonise the concentration-dependent
inhibitory effect of (R)-a-methylhistamine (MHA) on
electrically-evoked twitches of isolated guinea-pig ileum
segments [14]. pA2 values were calculated using the Gaddum
equation (pA2 = -log[B] + log[CR-1]) and are reported in
Table 2. When necessary, some experiments were carried out
in the presence of 1 µM ODQ (1H-[1,2,4]oxadiazolo[4,3-
a]quinoxalin-1-one), a well known inhibitor of the soluble
guanilate cyclase (sGC). Selectivity for the H3-receptor was
evaluated on the basis of the ability of the compounds to
modify the spontaneous contraction frequency or to antago-
nise the positive chronotropic action of histamine on isolated
guinea-pig atria (H2-receptor), and the ability to antagonise
the carbachol-induced ileum contraction (M3-receptor) at
10 µM concentration. The results are reported in Table 2.
The dinitrooxy derivative 9 was found to be a potent and
selective H3-antagonist. At 1 µM concentration it triggers a
large relaxation of the electrically stimulated tissue, and
again this effect can be ascribed to NO, since it is completely
abolished by ODQ.
5. Results and Discussion
Ether derivatives behave as reversible antagonists at H3-
receptors and did not show affinity for the H2-receptor when
All the compounds containing sulphur in the spacer be-
tween the two heterorings (14, 14a-b, 15, 15a-b, 16, 16a-b)

362
P. Tosco et al. / IL FARMACO 59 (2004) 359–371
1
TsCl
pyridine, 4 °C
S
S
EtO
S- K+
OTs
S
OEt
N
N
N
N
CH3CN
Ph3C
Ph3C
11
12
CH3CN
PhSH, Et3N
90% aq TFA
S
S
N
N
HN
N
Ph3C
13
( )
O
n
O
i) CHCl3 or CH3CN,
Me2N
N
N
NH2
S
R
12
N
N
Ph3C
R
Ph
2a 3-Ph
2b 4-Ph
SO2Ph
4a SO2Ph
n
0
1
1
0
1
ii)
PhSO2
R
2
N
N
90% aq TFA
4
O
( )
O
n
( )
O
n
O
N
S
N
R
n
0
1
1
0
1
1
14 Ph
14a 3-Ph
14b 4-Ph
15 SO2Ph
15a 3-SO2Ph
15b 4-SO2Ph
R
HN
O
N
( )
O
n
N
N
KMnO4
CH3COOH
R
n
16 Ph
16a 3-Ph
16b 4-Ph
0
1
1
14, 14a, 14b
SO2
R
HN
N
Scheme 3. Synthesis of the intermediate 12, of the reference compound 13 and of compounds 14, 14a-b, 15, 15a-b, 16, 16a-b containing sulfur in the side chain.

P. Tosco et al. / IL FARMACO 59 (2004) 359–371
363
Table 1
6. Experimental
Dissociation constants and lipophilicity descriptors in octanol/water of the
compounds under study
6.1. Synthesis of compounds
a
Compd
10
5
5a
6
6a
7
7a
9
pKa
log Poct
2.37
3.16
2.72
3.66
3.03
2.57
2.26
1.50
2.99
3.81
3.21
3.17
3.20
2.70
2.73
2.38
2.03
2.01
log D7.4 b
2.17
2.99
2.55
3.51
2.88
2.42
2.12
1.31
2.78
3.67
3.06
3.02
3.06
2.56
2.60
2.28
1.93
1.90
7.18c
7.07c
7.07c
6.99c
7.00c
7.00c
7.00c
7.13c
7.19d
6.99d
7.02d
7.03d
6.99d
6.97d
6.94d
6.80c
6.81c
6.85c
Melting points were determined with a capillary apparatus
(Büchi B-540) and are uncorrected. All the products were
routinely checked by IR (Shimadzu FT-IR 8101 M). 1H and
13C NMR spectra were obtained on a Bruker Avance 300, at
300 and 75 MHz respectively; d in ppm rel. to SiMe4 as the
internal standard; coupling constants J in Hz. Exchangeable
protons either are not visible or are found at low fields (13-15
ppm). 13C NMR spectra were fully decoupled. Following
abbreviations are used: s: singlet, d: doublet, t: triplet, qt:
quartet, qn: quintet, oc: octet, br: broad; Fx: furoxan, Fz:
furazan. Mass spectra were recorded on a Finnigan-Mat
TSQ-700. Flash chromatography (FC) was performed on
BDH silica gel (particle size 40-63 µm). Reverse phase
HPLC chromatography was performed on a Waters Prep LC
3000 instrument using a Merck column RT 250-25 pre-
packed with LiChrospher® 100 RP-18e. When not otherwise
specified, anhydrous magnesium sulphate (MgSO4) was
used as the drying agent of organic phases. Analysis (C,H,N)
of the new compounds was performed by REDOX (Monza).
Structures 1 [15], 2 [16], 2a and 2b [10], 4 [17], 4a [18], 10
[19] were synthesised according to the reported methods.
13
14
14a
14b
15
15a
15b
16
16a
16b
a log P of the neutral form determined by octanol/water titrations:
SD < 0.04.
b log D at pH = 7.4 calculated from the following equation:
a
1
10pK −pH
D = PN·
+ PI·
pK −pH
pK −pH
ͩ
a
ͪ ͩ
a
ͪ
1+10
1+10
c dissociation constants determined by aqueous titrations: SD < 0.04.
d Dissociation constants determined by aqueous titrations in the presence
of methanol as cosolvent (20-60 wt%) according to Yasuda-Shedlovsky
procedure: SD < 0.05.
6.1.1. 3-(phenylsulfonyl)-4-(phenylthio)furazan (3)
To a stirred solution of 4 (2.00 g, 5.71 mmol) in CH3CN
(60 mL) kept under N2 triethylamine (1.59 mL, 11.4 mmol)
was added. The reaction mixture was heated under reflux and
a solution of thiophenol (0.64 mL, 6.28 mmol) in CH3CN
(90 mL) was added dropwise over 30 min. After cooling the
reaction mixture was poured into water (40 mL) and the
solvent was evaporated under reduced pressure; the residue
was dissolved in CH2Cl2 (40 mL) and washed with 1 N HCl
(3×20 mL), 1 N NaOH (3×20 mL), brine (20 mL), dried and
evaporated under reduced pressure to give an oil. The latter
was dissolved in hot n-hexane with a few drops of iPrOH;
upon cooling white crystals of 3 were collected (1.64 g,
91%). M.p.: 72.5-73 °C (n-hexane/iPrOH); 1H NMR
(CDCl3): d, 8.17-8.14 (m, 2H, CH), 7.80-7.76 (m, 1H, CH),
7.68-7.61 (m, 4H, CH), 7.46-7.43 (m, 3H, CH); 13C NMR
(CDCl3): d, 155.28 (Fz-3-C), 152.28 (Fz-4-C), 137.94
(PhSO2-1-C), 135.58 (PhSO2-4-C), 134.51 (CH), 130.52
(PhS-4-C), 129.88 (CH), 129.78 (CH), 128.88 (CH), 126.13
(PhS-1-C); MS (EI): m/e 318 (M+, 23), 141 (67), 109 (35), 77
(100). Anal. C14H10N2O3S2 (318.36), calcd: C, 52.82; H,
3.17; N, 8.80; found: C, 52.62; H, 3.13; N 8.76.
behave as reversible antagonists at H3-receptors present on
the ileum. As seen for the ether series, also in this case the
furoxans 15a-b, 16a-b bearing a sulfonyl group at the ring
were less potent than reference 13, while all the other prod-
ucts were as potent as or more potent than this compound. In
addition the whole series displays quite good selectivity for
the H3-receptor with respect to the H2. In fact when tested at
10 µM concentration they did not trigger any H2 activity. All
the products when added to the bath at 1 µM concentration,
after electrical contraction of the tissue, induced relaxation.
This effect was particularly evident for the furoxan deriva-
tives 15a-15b, 16a (100%) and 14b (60%). For these prod-
ucts the phenomenon is partly mediated by NO, since its
extent was reduced by ODQ. Since all the substances dis-
played to some degree a non-competitive antagonism with
respect to carbachol at the M3-receptor present on the guinea-
pig ileum this behaviour could be due, at least in part, to their
ability to interfere with this receptor.
In conclusion, a new series of NO-donor H3-antagonists
and related compounds devoid of the ability to release NO
has been described. Many of these products have a
lipophilic-hydrophilic balance appropriate to enter the CNS
and display on the electrically stimulated guinea-pig ileum
both H3-antagonistic and NO-donor properties. These fea-
tures make them interesting candidates for further in vivo
studies.
6.1.2. 4-(phenylsulfonyl)-3-(phenylthio)furoxan (3a)
To a stirred solution of 4a (4.00 g, 10.9 mmol) in CHCl3
(200 mL) kept at -15 °C under N2 triethylamine (3.04 mL,
21.8 mmol) was added. A solution of thiophenol (1.23 mL,
12.0 mmol) in CHCl3 (80 mL) was added dropwise over
45 min. The reaction mixture was poured into cold 1 N HCl
(80 mL). The organic layer was separated and washed with
1 N HCl (2×80 mL), then with 5% NaHCO3 (6×80 mL),

364
P. Tosco et al. / IL FARMACO 59 (2004) 359–371
Table 2
H3-antagonism, M3-antagonism and miorelaxing properties of synthesised compounds
Electrically stimulated guinea-pig ileum (H3-receptor)
Guinea-pig ileum (M3-receptor)
Compd
pA2 SEa
6.64 0.10
7.41 0.12
7.64 0.08
7.31 0.07
6.97 0.09
7.02 0.09d
7.45 0.04
Relaxation % SEb
0
pA2 SEc
NT
Carbachol maximum response % SEc
10
5
5a
6
-
0
4.83 0.13
4.96 0.27
NCA
100
0
100
0
63.9 3.5
100
6a
46.4 3.1
0d
5.51 0.04
7
7a
0
5.12 0.08
5.40 0.07
100
100
e
-
68.8 6.8
19.4 3.7d
83.5 4.4
0d
5.62 0.03d
7.81 0.05
7.82 0.03d
6.89 0.17
8.12 0.10
7.94 0.06
7.89 0.02d
7.73 0.13
7.79 0.11d
7.16 0.11
9
NT
-
13
14
14a
36.0 7.0
21.9 3.0
22.2 2.3
23.7 2.0d
60.3 3.9
38.0 5.4d
24.7 2.4
100
51.8 1.2d
100
47.3 4.9d
28.4 4.2
100
NCA
NCA
NCA
88.1 1.1
28.7 1.0
83.2 2.8
14b
NCA
78.6 3.2
15
15a
NCA
NCA
25.4 3.6
70.3 5.8
e
-
6.10 0.12d
e
15b
-
NCA
26.6 4.6
5.94 0.08d
6.82 0.09
16
NCA
NCA
89.2 1.5
87.4 9.6
e
16a
-
5.94 0.12d
5.50 0.08
5.39 0.08d
23.7 3.5d
28.8 2.3
0d
16b
NCA
80.6 4.0
NT: not tested; NCA: non competitive antagonist.
a pA2 values were estimated at the concentrations reported in the experimental section.
b Determined at 1 µM concentration.
c Determined at 10 µM concentration.
d In the presence of 1 µM ODQ.
e It was impossible to estimate pA2 value in the absence of ODQ.
dried and evaporated under reduced pressure to give a yellow
oil. The latter was dissolved in a mixture of hot
n-hexane/iPrOH; upon cooling pale yellow crystals of 3a
were collected (2.72 g, 75%). M.p.: 111-112 °C (n-
additional 0.17 g potassium tert-butoxide. After 2 h at r.t. the
reaction mixture was poured into brine (20 mL) and extracted
with EtOAc (3×20 mL). The combined organic layers were
washed with brine (15 mL), dried and evaporated under
reduced pressure affording a crude oil which was purified by
FC (CH2Cl2 in gradient from 5% to 20% of EtOAc) to yield
a white solid. The latter was dissolved in 5 mL 90% aqueous
TFA and stirred for 12 h. The reaction mixture was evapo-
rated under reduced pressure, the residue was treated with
1 N HCl (40 mL) and filtered through a celite pad. The filtrate
was washed with Et2O (4×15 mL); the aqueous phase was
made alcaline with Na2CO3 and extracted with EtOAc (3×20
mL). The combined organic layers were washed with brine
(15 mL), dried over K2CO3 and evaporated under reduced
pressure to yield 5 free base (0.26 g, 72%) as white solid. The
latter was converted into the corresponding hydrochloride,
white crystalline solid. M.p.: 151-152 °C (MeOH/Et2O); 1H
NMR (DMSO-d6): d, 9.05 (s, 1H, Im-2-H), 7.94-7.89 (m,
2H, Ph-2,6-H), 7.61-7.59 (m, 3H, Ph-3,4,5-H), 7.51 (s, 1H,
Im-5-H), 4.50 (t, 2H, J = 6.0 Hz, CH2-O), 2.90 (t, 2H,
1
hexane/iPrOH); H NMR (CDCl3): d, 8.16-8.13 (m, 2H,
CH), 7.78-7.76 (m, 1H, CH), 7.67-7.62 (m, 2H, CH), 7.40-
7.30 (m, 5H, CH); 13C NMR (CDCl3): d, 160.07 (Fx-4-C),
137.15 (PhSO2-1-C), 136.15 (PhSO2-4-C), 133.65 (CH),
130.39 (PhS-4-C), 130.21 (CH), 130.17 (CH), 129.99 (CH),
126.93 (PhS-1-C), 108.75 (Fx-3-C); MS (EI): m/e 334 (M+,
7), 274 (71), 141 (73), 77 (100). Anal. C14H10N2O4S2
(334.36), calcd: C, 50.29; H, 3.01; N, 8.38; found: C, 49.98;
H, 3.00; N 8.39.
6.1.3.
3-[3-(1H-imidazol-4-yl)propoxy]-4-phenylfurazan
hydrochloride (5)
To a stirred solution of 1 (0.50 g, 1.36 mmol) in 10 mL dry
THF under N2 potassium tert-butoxide (0.17 g, 1.50 mmol)
was added, then the reaction mixture was heated under re-
flux. After 30 min the reaction was cooled to 40 °C and 2 was
added (0.54 g, 1.90 mmol). The reaction mixture was stirred
at r.t. for 2 h, then heated again to 40 °C and treated with
J = 7.5 Hz, CH2-Im), 2.26 (qn, 2H, J = 6.7 Hz, -CH2-); 13
C

P. Tosco et al. / IL FARMACO 59 (2004) 359–371
365
Fig. 1. Experimental curves for derivative 6a in the absence and in the presence of ODQ.

366
P. Tosco et al. / IL FARMACO 59 (2004) 359–371
NMR (DMSO-d6): d, 163.79 (Fz-3-C), 145.71 (Fz-4-C),
133.98 (Im-2-C), 132.88 (Im-4-C), 131.60 (Ph-4-C), 129.87
(Ph-3,5-C), 127.77 (Ph-2,6-C), 124.87 (Ph-1-C), 116.16 (Im-
5-C), 72.42 (CH2-O), 27.73 (CH2-Im), 21.15 (-CH2-). Anal.
C14H14N4O2·HCl (306.75), calcd: C, 54.82; H, 4.93; N,
18.26; found: C, 54.83; H, 4.97; N, 18.19.
27.00 (CH2-Im), 20.36 (-CH2-). Anal. C14H14N4O2S·HCl
(338.81), calcd: C, 49.63; H, 4.46; N, 16.54; found: C, 49.69;
H, 4.49; N, 16.42.
6.1.6.
4-[3-(1H-imidazol-4-yl)propoxy]-3-(phenylthio)-
furoxan hydrochloride (6a)
To a stirred solution of 1 (0.50 g, 1.36 mmol) in 10 mL dry
THF under N2 potassium tert-butoxide (0.17 g, 1.50 mmol)
was added, then the reaction mixture was heated under re-
flux. After 30 min the reaction was cooled to 0 °C and 3a was
added (0.64 g, 1.90 mmol). After 1 h at 0 °C the mixture was
worked up, purified and hydrolised in 90% aqueous TFA as
described for 6 to yield 6a (0.29 g, 68%) as pale yellow oil.
The latter was converted into the corresponding hydrochlo-
ride, white crystalline solid. M.p.: 118.5-119.5 °C
(MeOH/Et2O); 1H NMR (DMSO-d6): d, 9.08 (s, 1H, Im-2-
H), 7.41 (m, 6H, Im-4-H, Ph), 4.40 (t, 2H, J = 5.9 Hz,
CH2-O), 2.72 (t, 2H, J = 7.5 Hz, CH2-Im), 2.16-2.10 (m, 2H,
-CH2-); 13C NMR (DMSO-d6): d, 163.46 (Fx-4-C), 133.58
(Im-2-C), 132.22 (Im-4-C), 130.29 (Ph-3,5-C), 129.98 (Ph-
2,6-C), 128.79 (Ph-4-C), 128.73 (Ph-1-C), 115.64 (Im-5-C),
104.88 (Fx-3-C), 69.59 (CH2-O), 26.93 (CH2-Im), 20.34
(-CH2-). Anal. C14H14N4O3S·HCl (354.81), calcd: C, 47.39;
H, 4.26; N, 15.79; found: C, 47.34; H, 4.27; N, 15.76.
6.1.4.
4-[3-(1H-imidazol-4-yl)propoxy]-3-phenylfuroxan
hydrochloride (5a)
To a stirred solution of 1 (0.50 g, 1.36 mmol) in 10 mL dry
THF under N2 potassium tert-butoxide (0.17 g, 1.50 mmol)
was added, then the reaction mixture was heated under reflux.
After 30 min the reaction was cooled to 0 °C and 2a was added
(0.57 g, 1.90 mmol). After 1.5 h at r.t., the mixture was worked
up, purified and hydrolised in 90% aqueous TFA as described
for 5 to yield 5a free base (0.27 g, 69%) as white solid. The
latter was converted into the corresponding hydrochloride,
white crystalline solid. M.p.: 157-158 °C (MeOH/Et2O); 1H
NMR (DMSO-d6): d, 9.05 (s, 1H, Im-2-H), 8.04-7.98 (m,
2H, Ph-2,6-H), 7.67-7.56 (m, 3H, Ph-3,4,5-H), 7.52 (s, 1H,
Im-5-H), 4.52 (t, 2H, J = 6.0 Hz, CH2-O), 2.90 (t, 2H,
J = 7.4 Hz, CH2-Im), 2.27 (qn, 2H, J = 6.7 Hz, -CH2-); 13
C
NMR (DMSO-d6): d, 162.26 (Fx-4-C), 133.55 (Im-2-C),
132.39 (Im-4-C), 130.75 (Ph-4-C), 129.16 (Ph-3,5-C),
126.25 (Ph-2,6-C), 122.04 (Ph-1-C), 115.75 (Im-5-C),
107.63 (Fx-3-C), 69.91 (CH2-O), 27.14 (CH2-Im), 20.68
(-CH2-). Anal. C14H14N4O3·HCl (322.75), calcd: C, 52.10;
H, 4.68; N, 17.30; found: C, 52.13; H, 4.73; N, 17.30.
6.1.7. 3-[3-(1H-imidazol-4-yl)propoxy]-4-(phenylsulfonyl)
furazan hydrochloride (7)
To a stirred solution of 1 (0.50 g, 1.36 mmol) in 10 mL dry
THF under N2 potassium tert-butoxide (0.17 g, 1.50 mmol)
was added, then the reaction mixture was heated under re-
flux. After 30 min the reaction was cooled to -15 °C and 4
was added (0.67 g, 1.90 mmol). After 2 h at r.t., the mixture
was worked up, purified and hydrolised in 90% aqueous TFA
as described for 5 to yield 7 free base (0.20 g, 52%) as white
solid. The latter was converted into the corresponding hydro-
6.1.5. 3-[3-(1H-imidazol-4-yl)propoxy]-4-(phenylthio)fura-
zan hydrochloride (6
)
To a stirred solution of 1 (0.50 g, 1.36 mmol) in 10 mL dry
THF under N2 potassium tert-butoxide (0.17 g, 1.50 mmol)
was added, then the reaction mixture was heated under re-
flux. After 30 min the reaction was cooled to 40 °C and 3 was
added (0.60 g, 1.90 mmol).After 30 min at 40 °C the reaction
mixture was poured into brine (20 mL) and extracted with
EtOAc (3×20 mL). The organic layer was washed with brine
(15 mL), dried and evaporated under reduced pressure af-
fording a crude oil which was purified by FC (CH2Cl2 in
gradient from 5% to 20% of EtOAc) to yield a yellow viscous
oil. The latter was dissolved in 5 mL 90% aqueous TFA and
stirred for 12 h. The reaction mixture was poured into 0.1 N
HCl (150 mL) and extracted with CHCl3 (5×30 mL). The
combined organic layers were washed with 5% Na2CO3
(5×20 mL), dried over K2CO3 and evaporated under reduced
pressure to yield a crude product which was purified by FC
(CH2Cl2 in gradient from 1% to 5% of MeOH) to yield 6
(0.35 g, 86%) as pale yellow oil. The latter was converted into
the corresponding hydrochloride, white crystalline solid.
1
chloride. M.p.: 152-153 °C (dec.) (MeOH/Et2O); H NMR
(DMSO-d6): d, 9.08 (s, 1H, Im-2-H), 8.11 (d, 2H, J = 7.4 Hz,
Ph-2,6-H), 7.94 (t, 1H, J = 7.3 Hz, Ph-4-H), 7.80 (t, 2H,
J = 7.4 Hz, Ph-3,5-H), 7.46 (s, 1H, Im-5-H), 4.43 (t, 2H,
J = 5.9 Hz, CH2-O), 2.74 (t, 2H, J = 7.5 Hz, CH2-Im), 2.15
(qn, 2H, J = 6.6 Hz, -CH2-); 13C NMR (DMSO-d6): d, 161.02
(Fz-3-C), 148.64 (Fz-4-C), 136.66 (Ph-1-C), 136.35 (Ph-4-
C), 133.80 (Im-2-C), 132.18 (Im-4-C), 130.42 (Ph-3,5-C),
128.86 (Ph-2,6-C), 115.72 (Im-5-C), 73.04 (CH2-O), 27.00
(CH2-Im), 20.42 (-CH2-). Anal. C14H14N4O4S·HCl (370.81),
calcd: C, 45.35; H, 4.08; N, 15.11; found: C, 45.23; H, 4.06;
N, 15.11.
6.1.8. 4-[3-(1H-imidazol-4-yl)propoxy]-3-(phenylsulfonyl)
furoxan oxalate (7a)
1
M.p.: 111-112 °C (MeOH/Et2O); H NMR (DMSO-d6): d,
To a stirred solution of 1 (0.50 g, 1.36 mmol) in 10 mL dry
THF under N2 potassium tert-butoxide (0.17 g, 1.50 mmol)
was added, then the reaction mixture was heated under re-
flux. After 30 min the reaction was cooled to -15 °C and 4a
was added (0.70 g, 1.90 mmol). After 1 h at r.t. the mixture
was worked up, purified and hydrolised in 90% aqueous TFA
as described for 5 to yield 7a free base (0.20 g, 55%) as white
9.08 (s, 1H, Im-2-H), 7.65-7.42 (m, 6H, Im-5-H, Ph), 4.36 (t,
2H, J = 6.0 Hz, CH2-O), 2.66 (t, 2H, J = 7.5 Hz, CH2-Im),
2.08 (qn, 2H, J = 6.7 Hz, -CH2-); 13C NMR (DMSO-d6): d,
163.16 (Fz-3-C), 144.27 (Fz-4-C), 133.66 (Im-2-C), 132.70
(Ph-3,5-C), 132.25 (Im-4-C), 129.94 (Ph-2,6-C), 129.54 (Ph-
4-C), 127.36 (Ph-1-C), 115.59 (Im-5-C), 71.91 (CH2-O),

P. Tosco et al. / IL FARMACO 59 (2004) 359–371
367
solid. The latter was converted into the corresponding ox-
alate. M.p.: 152-153 °C (dec.) (absolute EtOH/acetone); 1H
NMR (DMSO-d6): d, 8.46 (s, 1H, Im-2-H), 8.03 (d, 2H,
J = 8.6 Hz, Ph-2,6-H), 7.91 (t, 1H, J = 7.4 Hz, Ph-4-H), 7.77
(t, 2H, J = 7.7 Hz, Ph-3,5-H), 7.23 (s, 1H, Im-5-H), 4.44 (t,
2H, J = 6.1 Hz, CH2-O), 2.74 (t, 2H, J = 7.4 Hz, CH2-Im),
2.12 (qn, 2H, J = 6.8 Hz, -CH2-); 13C NMR (DMSO-d6): d,
165.40 (oxalate), 159.71 (Fx-4-C), 137.99 (Ph-1-C), 137.01
(Ph-4-C), 135.09 (Im-2-C), 134.28 (Im-4-C), 130.91 (Ph-
3,5-C), 129.22 (Ph-2,6-C), 116.87 (Im-5-C), 111.39 (Fx-3-
C), 71.32 (CH2-O), 27.95 (CH2-Im), 21.94 (-CH2-). Anal.
C14H14N4O5S·H2C2O4 (440.39), calcd: C, 43.64; H, 3.66; N,
12.72; found: C, 44.03; H, 3.75; N, 13.06.
evaporated under reduced pressure to yield a pale yellow oil.
The latter was promptly dissolved in 15 mL CH3CN and
treated with AgNO3 (0.78 g, 4.60 mmol), then the reaction
mixture was cooled at 0 °C and a solution of I2 (1.07 g,
4.21 mmol) in 80 mL CH3CN was added dropwise over
30 min. The mixture was filtered through a celite pad, and the
filtrate was treated with additional AgNO3 (1.30 g,
7.66 mmol), then refluxed for 12 h. After cooling the solvent
was removed under reduced pressure and the residue was
treated with water (30 mL) and excess KI, then with 1 N HCl
(50 mL) and Et2O (20 mL). The mixture was filtered through
a celite pad, the biphasic filtrate was separated and the aque-
ous phase washed with Et2O (3×20 mL). The aqueous phase
was made alcaline with Na2CO3 and extracted with EtOAc
(4×20 mL). The combined organic layers were washed with
brine (20 mL), dried over K2CO3 and evaporated under
reduced pressure to afford a crude oil which was purified by
FC (CH2Cl2/MeOH 97:3) to yield 9 free base (0.40 g, 28%)
as yellow oil. The latter was readily converted into the corre-
sponding oxalate, white crystalline solid. M.p.: 134-135 °C
(dec.) (iPrOH); 1H NMR (DMSO-d6): d, 8.46 (s, 1H, Im-2-
H), 7.17 (s, 1H, Im-5-H), 5.60-5.57 (m, 1H, CH-ONO2), 4.95
(dd, 1H, J = 2.8/12.9 Hz, CH2-ONO2), 4.78 (qt, 1H,
J = 6.5 Hz, CH2-ONO2), 3.74-3.67 (m, 2H, O-CH2-CH),
3.50-3.42 (m, 2H, CH2-CH2-O), 2.63 (t, 2H, J = 7.6 Hz,
CH2-Im), 1.83 (qn, 2H, J = 6.9 Hz, -CH2-); 13C NMR
(DMSO-d6): d, 165.52 (oxalate), 134.70 (Im-2-C), 134.32
(Im-4-C), 116.68 (Im-5-C), 79.70 (CH-ONO2), 71.07 (CH2-
O), 70.59 (CH2-O), 67.97 (CH2-O), 28.85 (CH2-Im), 21.90
(-CH2-); MS (CI, isobutane): m/e 291 (MH+). Anal.
C9H14N4O7·H2C2O4 (380.27), calcd: C, 34.74; H, 4.24; N,
14.73; found: C, 34.85; H, 4.33; N, 15.10.
6.1.9. 4-[3-(allyloxy)propyl]-1H-imidazole oxalate (8)
To a stirred solution of 1 (1.00 g, 2.71 mmol) in dry THF
(10 mL) under N2 a 60% suspension of NaH in mineral oil
(0.54 g, 13.6 mmol) was added, then the reaction mixture was
heated under reflux. After 30 min a solution of allyl bromide
(1.15 mL, 13.6 mmol) in dry THF (5 mL) was added, and the
reaction mixture was kept refluxing for 1 h; after cooling, the
mixture was cautiously poured into cold brine (30 mL) and
extracted with Et2O (3×15 mL). The organic layer was
washed with brine (10 mL), dried and evaporated under
reduced pressure to afford a yellow oil which was promptly
dissolved in 10 mL EtOH, treated with 2 N HCl (5 mL), then
refluxed for 1 h.After cooling the solvent was evaporated and
the residue treated with water and filtered through a celite
pad. The filtrate was washed with Et2O (3×15 mL), and the
aqueous phase was evaporated under reduced pressure. The
solid residue was mixed with wet K2CO3 and extracted with
EtOAc (3×20 mL); the combined extracts were evaporated
under reduced pressure affording a crude oil which was
purified by FC (CH2Cl2/MeOH 95:5) to yield 8 free base
(0.30 g, 67%) as pale yellow oil. The latter was converted into
the corresponding oxalate, white crystalline solid. M.p.: 153-
6.1.11.
O-ethyl S-[3-(1-trityl-1H-imidazol-4-yl)propyl]
dithiocarbonate (12)
To a stirred solution of 1 (4.00 g, 10.9 mmol) in dry
pyridine (40 mL) at 0 °C p-toluenesulfonyl chloride (4.16 g,
21.8 mmol) was added. After standing at 4 °C for 6 h the
mixture was poured into cold water (200 mL) and cold 85%
H3PO4 (40 mL) was added dropwise under stirring, then it
was extracted with CHCl3 (3×20 mL); the combined organic
layers were washed with 5% citric acid (2×40 mL), then with
10% NaHCO3 (20 mL), dried over K2CO3 and evaporated
under reduced pressure to yield 11 as viscous oil. The latter
was immediately dissolved in CH3CN (40 mL), potassium
O-ethyl dithiocarbonate (2.60 g, 16.2 mmol) was added, and
the reaction mixture was stirred for 60 h at r. t. The solvent
was evaporated under reduced pressure and the residue was
treated with water (80 mL) and extracted with CH2Cl2 (3×20
mL). The combined organic layers were washed with brine
(20 mL), dried and evaporated under reduced pressure to
afford a yellow oil which was dissolved in 200 mL hot
n-hexane. The insoluble white solid was separated by filtra-
tion and the filtrate was concentrated on a hot plate until
cloudy; upon cooling, 12 crystallised (3.03 g, 59%) as white
solid. M.p.: 101-102 °C (n-hexane); 1H NMR (DMSO-d6): d,
1
154 °C (absolute EtOH/acetone); H NMR (DMSO-d6): d,
8.48 (s, 1H, Im-2-H), 7.19 (s, 1H, Im-5-H), 5.87 (oc, 1H,
J = 5.2 Hz, -CH=), 5.22 (dd, 1H, J = 1.6/17.1 Hz, =CH2
trans), 5.12 (d, 1H, J = 10.4 Hz, =CH2 cis), 3.90 (d, 2H,
J = 5.3 Hz, O-CH2-CH), 3.38 (t, 2H, J = 6.3 Hz, CH2-CH2-
O), 2.65 (t, 2H, J = 7.6 Hz, CH2-Im), 1.83 (qn, 2H, J = 6.9 Hz,
-CH2-); 13C NMR (DMSO-d6): d, 165.78 (oxalate), 136.19
(Im-2-C), 134.72 (-CH=), 134.67 (Im-4-C), 117.08 (=CH2),
116.84 (Im-5-C), 71.65 (CH2-O), 69.36 (CH2-O), 29.24
(CH2-Im), 22.35 (-CH2-). Anal. C9H14N2O·0.7 H2C2O4
(229.25), calcd: C, 54.48; H, 6.77; N, 12.22; found: C, 54.40;
H, 6.70; N, 12.08.
6.1.10. 4-{3-[2,3-bis(nitrooxy)propoxy]propyl}-1H-imida-
zole oxalate (9)
To a stirred solution of 8 free base (0.64 g, 3.83 mmol) in
distilled THF (15 mL) at 0 °C 6 N NaOH (0.70 mL) was
added, followed by Boc2O (0.92 g, 4.21 mmol). After 1 h at
r.t. the mixture was poured into brine (15 mL) and the
aqueous phase was extracted with Et2O (3×15 mL). The
organic layer was washed with brine (10 mL), dried and

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P. Tosco et al. / IL FARMACO 59 (2004) 359–371
7.39 (m, 9H, Ph), 7.28 (s, 1H, Im-2-H), 7.12-7.08 (m, 6H,
Ph), 6.66 (s, Im-5-H), 4.61 (qt, J = 7.1 Hz, CH2-O), 3.11 (t,
J = 7.3 Hz, CH2-S), 2.57-2.51 (m, CH2-Im), 1.93-1.87 (m,
-CH2-), 1.33 (t, J = 7.1 Hz, CH3); 13C NMR (DMSO-d6): d,
214.55 (C=S), 142.44 (Ph-1-C), 139.90 (Im-4-C), 137.90
(Im-2-C), 129.28 (Ph-3,5-C), 128.26 (Ph-2,6-C), 128.03 (Ph-
4-C), 117.93 (Im-5-C), 74.44 (Ph3C-N), 70.15 (CH2-O),
34.71 (CH2-S), 27.80 (CH2-Im), 26.88 (-CH2-), 13.66 (CH3).
Anal. C28H28N2OS2 (472.66), calcd: C, 71.15; H, 5.97; N,
5.93; found: C, 71.30; H, 6.07; N, 5.91.
aqueous CH3COOH (40 mL), 5% citric acid (2×40 mL),
10% NaHCO3 (20 mL), brine (20 mL), dried and evaporated
under reduced pressure affording a crude oil which was
purified by FC (petroleum ether in gradient from 20% to 50%
of EtOAc) to yield a white solid. The latter was dissolved in
10 mL 90% aqueous TFA and stirred for 12 h. The reaction
mixture was evaporated under reduced pressure, the residue
was treated with 1 N HCl (40 mL) and filtered through a
celite pad. The filtrate was washed with Et2O (4×15 mL), the
aqueous phase was made alcaline with Na2CO3 and extracted
with EtOAc (3×20 mL). The combined organic layers were
washed with brine (15 mL), dried over K2CO3 and evapo-
rated under reduced pressure to yield 14 free base (0.48 g,
79%) as clear oil. The latter was converted into the corre-
sponding hydrochloride, white crystalline solid. M.p.: 105-
106 °C (MeOH/Et2O); 1H NMR (DMSO-d6): d, 9.07 (s, 1H,
Im-2-H), 7.84-7.77 (m, 2H, Ph-2,6-H), 7.66-7.59 (m, 3H,
Ph-3,4,5-H), 7.49 (s, 1H, Im-5-H), 3.33 (t, 2H, J = 7.1 Hz,
CH2-S), 2.84 (t, 2H, J = 7.4 Hz, CH2-Im), 2.16 (qn, 2H,
J = 7.3 Hz, -CH2-); 13C NMR (DMSO-d6): d, 152.69 (Fz-4-
C), 151.60 (Fz-3-C), 133.62 (Im-2-C), 132.23 (Im-4-C),
131.34 (Ph-4-C), 129.54 (Ph-3,5-C), 128.07 (Ph-2,6-C),
124.57 (Ph-1-C), 115.79 (Im-5-C), 31.51 (CH2-S), 27.14
(CH2-Im), 22.97 (-CH2-). Anal. C14H14N4OS·HCl (322.81),
calcd: C, 52.09; H, 4.68; N, 17.36; found: C, 51.94; H, 4.78;
N, 17.21.
6.1.12. 4-[3-(phenylthio)propyl]-1H-imidazole hydrochlo-
ride (13)
The intermediate tosylate 11 was prepared as described
above using 1 (1.00 g, 2.71 mmol), p-toluenesulfonyl chlo-
ride (1.03 g, 5.42 mmol) and dry piridine (10 mL). 11 was
immediately dissolved in CH3CN (5 mL), triethylamine
(0.45 mL, 3.25 mmol) and thiophenol (1.39 mL, 13.6 mmol)
were added, and the reaction mixture was stirred under N2 for
60 h. The solvent was evaporated under reduced pressure and
the residue was dissolved in CH2Cl2 (20 mL); the combined
organic layers were washed with 1 N NaOH (2×20 mL), 5%
citric acid (3×20 mL), brine (10 mL), dried and evaporated
under reduced pressure to yield a cream-coloured solid,
which was purified by FC (CH2Cl2 in EtOAc gradient from
5% to 50%) to afford a white solid which was promptly
dissolved in 12 mL 90% aqueous TFA and stirred for 12 h.
The reaction mixture was evaporated under reduced pressure
and the residue was treated with 2 N HCl (20 mL) and filtered
through a celite pad. The filtrate was washed with Et2O (3×15
mL); the aqueous phase was made alcaline with Na2CO3,
then extracted with EtOAc (3×15 mL). The combined or-
ganic layers were washed with brine (15 mL), dried over
K2CO3 and evaporated under reduced pressure to yield 13
free base (0.27 g, 46%) as clear oil. The latter was converted
into the corresponding hydrochloride, white crystalline solid.
6.1.14. 4-{[3-(1H-imidazol-4-yl)propyl]thio}-3-phenylfuro-
xan hydrochloride (14a)
To a stirred solution of 12 (1.00 g, 2.12 mmol) in 10 mL
CHCl3 under N2 at 0 °C N,N-dimethylpropane-1,3-diamine
(10 mL) was added; after 45 min at r.t., it was diluted with
CHCl3 (30 mL), then 2a (0.96 g, 3.18 mmol) was added.
After 2 h the reaction mixture was worked up, purified and
hydrolised in 90% aqueous TFA as described for 14 to yield
14a free base (0.53 g, 82%) as clear oil. The latter was
converted into the corresponding hydrochloride, white crys-
1
M.p.: 138-139 °C (MeOH/Et2O); H NMR (DMSO-d6): d,
1
9.04 (s, 1H, Im-2-H), 7.43 (s, 1H, Im-5-H), 7.35-7.29 (m, 4H,
Ph-2,3,5,6-C), 7.22-7.18 (m, 1H, Ph-4-C), 3.00 (t, 2H,
J = 7.2 Hz, CH2-S), 2.79 (t, 2H, J = 7.5 Hz, CH2-Im), 1.94
(qn, 2H, J = 7.4 Hz, -CH2-); 13C NMR (DMSO-d6): d, 135.85
(Ph-1-C), 133.33 (Im-2-C), 132.29 (Im-4-C), 128.97 (Ph-
3,5-C), 127.96 (Ph-2,6-C), 125.56 (Ph-4-C), 115.40 (Im-5-
C), 31.04 (CH2-S), 27.35 (CH2-Im), 22.80 (-CH2-). Anal.
C12H14N2S·HCl (254.78), calcd: C, 56.57; H, 5.93; N 11.00;
found: C, 56.51; H, 6.08; N, 10.87.
talline solid. M.p.: 130-131 °C (MeOH/Et2O); H NMR
(DMSO-d6): d, 9.07 (s, 1H, Im-2-H), 7.84-7.80 (m, 2H,
Ph-2,6-H), 7.68-7.60 (m, 3H, Ph-3,4,5-H), 7.48 (s, 1H, Im-5-
H), 3.29 (t, 2H, J = 7.0 Hz, CH2-S), 2.83 (t, 2H, J = 7.4 Hz,
CH2-Im), 2.15-2.07 (qn, 2H, J = 7.1 Hz, -CH2-); 13C NMR
(DMSO-d6): d, 154.71 (Fx-4-C), 133.61 (Im-2-C), 132.22
(Im-4-C), 131.08 (Ph-4-C), 129.27 (Ph-3,5-C), 127.73 (Ph-
2,6-C), 122.04 (Ph-1-C), 115.79 (Im-5-C), 114.42 (Fx-3-C),
29.79 (CH2-S), 27.21 (CH2-Im), 22.94 (-CH2-). Anal.
C14H14N4O2S·HCl (338.81), calcd: C, 49.63; H, 4.46; N,
16.54; found: C, 49.42; H, 4.43; N, 16.52.
6.1.13. 3-{[3-(1H-imidazol-4-yl)propyl]thio}-4-phenylfura-
zan hydrochloride (14)
To a stirred solution of 12 (1.00 g, 2.12 mmol) in 10 mL
CH3CN under N2 at 0 °C N,N-dimethylpropane-1,3-diamine
(10 mL) was added; after 45 min at r.t., it was diluted with
CH3CN (20 mL), treated with 2 (0.91 g, 3.18 mmol) and
heated under reflux for 1 h. After cooling, the mixture was
poured into brine (40 mL), the solvent was removed under
reduced pressure and the residue extracted with CHCl3 (3×15
mL). The combined organic layers were washed with 50%
6.1.15. 3-{[3-(1H-imidazol-4-yl)propyl]thio}-4-phenylfuro-
xan hydrochloride (14b)
To a stirred solution of 12 (1.00 g, 2.12 mmol) in 10 mL
CHCl3 under N2 at 0 °C N,N-dimethylpropane-1,3-diamine
(10 mL) was added; after 45 min at r.t., it was diluted with
CHCl3 (70 mL) and cooled to 0 °C, then 2b (0.96 g,
3.18 mmol) was added. After 30 min at 0 °C the reaction
mixture was worked up, purified and hydrolised in 90%

P. Tosco et al. / IL FARMACO 59 (2004) 359–371
369
aqueous TFA as described for 14 to yield 14b free base
(0.52 g, 80%) as clear oil. The latter was converted into the
corresponding hydrochloride, white crystalline solid. M.p.:
140-141 °C (MeOH/Et2O); 1H NMR (DMSO-d6): d, 9.03 (s,
1H, Im-2-H), 7.91-7.88 (m, 2H, Ph-2,6-H), 7.65-7.58 (m,
3H, Ph-3,4,5-H), 7.34 (s, 1H, Im-5-H), 3.00 (t, 2H,
J = 7.3 Hz, CH2-S), 2.69 (t, 2H, J = 7.3 Hz, CH2-Im), 1.86
(qn, 2H, J = 7.3 Hz, -CH2-); 13C NMR (DMSO-d6): d, 158.72
(Fx-4-C), 134.28 (Im-2-C), 132.78 (Im-4-C), 132.23 (Ph-4-
C), 129.95 (Ph-3,5-C), 128.75 (Ph-2,6-C), 126.74 (Ph-1-C),
116.38 (Im-5-C), 112.04 (Fx-3-C), 30.84 (CH2-S), 28.92
(CH2-Im), 23.37 (-CH2-). Anal. C14H14N4O2S·HCl (338.81),
calcd: C, 49.63; H, 4.46; N, 16.54; found: C, 49.89; H, 4.53;
N, 16.49.
(t, 1H, J = 7.4 Hz, Ph-4-H), 7.75 (t, 2H, J = 7.7 Hz, Ph-3,5-
H), 7.10 (s, 1H, Im-5-H), 2.93 (t, 2H, J = 7.3 Hz, CH2-S),
2.57 (t, 2H, J = 7.3 Hz, CH2-Im), 1.67 (qn, 2H, J = 7.3 Hz,
-CH2-); 13C NMR (DMSO-d6): d, 164.29 (oxalate), 159.48
(Fx-4-C), 136.05 (Ph-4-C), 135.78 (Ph-1-C), 133.88 (Im-2-
C), 132.56 (Im-4-C), 129.98 (Ph-3,5-C), 129.03 (Ph-2,6-C),
115.89 (Im-5-C), 108.99 (Fx-3-C), 30.05 (CH2-S), 28.23
(CH2-Im), 22.86 (-CH2-). Anal. C14H14N4O4S2·0.8 H2C2O4
(438.44), calcd: C, 42.74; H, 3.59; N, 12.78; found: C, 42.74;
H, 3.62; N, 12.83.
The mother liquor from the fractional recrystallisation
was evaporated under reduced pressure obtaining a mixture
of the two trityl derivatives of 15a and 15b in a 1:1 ratio. This
mixture was hydrolised in 90% aqueous TFA and purified as
for 14 to yield a mixture of 15a and 15b free base as clear oil.
This mixture was dissolved in EtOAc (10 mL) and treated
with excess Boc2O; the solvent was removed under reduced
pressure to yield a crude oil which was purified by FC
(petroleum ether in gradient from 20% to 50% of EtOAc) to
remove excess Boc2O, and then resolved by RP18-HPLC
(CH3CN/H2O 7:3) obtaining pure Boc-protected 15a. The
latter was hydrolised in 90% aqueous TFA and purified as for
14 to yield 15a free base as clear oil. The product was
converted into the corresponding oxalate, white crystalline
solid. M.p.: 122-123 °C (acetone); 1H NMR (DMSO-d6): d,
8.31 (s, 1H, Im-2-H), 8.03 (d, 2H, J = 7.9 Hz, Ph-2,6-H), 7.94
(t, 1H, J = 7.4 Hz, Ph-4-H), 7.79 (t, 2H, J = 7.7 Hz, Ph-3,5-
H), 7.17 (s, 1H, Im-5-H), 3.24 (t, 2H, J = 7.2 Hz, CH2-S),
2.75 (t, 2H, J = 7.4 Hz, CH2-Im), 2.08 (qn, 2H, J = 7.3 Hz,
-CH2-); 13C NMR (DMSO-d6): d, 164.85 (oxalate), 154.20
(Fx-4-C), 137.30 (Ph-4-C), 134.93 (Im-2-C), 134.01 (Im-4-
C), 133.40 (Ph-1-C), 131.04 (Ph-3,5-C), 129.07 (Ph-2,6-C),
117.92 (Fx-3-C), 116.94 (Im-5-C), 30.03 (CH2-S), 27.95
(CH2-Im), 24.50 (-CH2-). Anal. C14H14N4O4S2·0.6 H2C2O4
(420.43), calcd: C, 43.42; H, 3.64; N, 13.32; found: C, 43.47;
H, 3.68; N, 13.01.
6.1.16. 3-{[3-(1H-imidazol-4-yl)propyl]thio}-4-(phenylsul-
fonyl)furazan hydrochloride (15)
To a stirred solution of 12 (1.00 g, 2.12 mmol) in 10 mL
CHCl3 under N2 at 0 °C N,N-dimethylpropane-1,3-diamine
(10 mL) was added; after 45 min at r.t., it was diluted with
CHCl3 (70 mL) and cooled to -15 °C, then 4 (1.16 g,
3.18 mmol) was added. After 30 min at -15 °C the reaction
mixture was worked up, purified and hydrolised in 90%
aqueous TFA as described for 14 to yield 15 free base (0.55 g,
67%) as clear oil . The latter was converted into the corre-
sponding hydrochloride, white crystalline solid. M.p.: 168-
1
169 °C (absolute EtOH/acetone); H NMR (DMSO-d6): d,
9.04 (s, 1H, Im-2-H), 8.09 (d, 2H, J = 8.6 Hz, Ph-2,6-H), 7.92
(t, 1H, J = 7.4 Hz, Ph-4-H), 7.82 (t, 2H, J = 7.3 Hz, Ph-3,5-
H), 7.45 (s, 1H, Im-5-H), 3.26 (t, 2H, J = 7.1 Hz, CH2-S),
2.77 (t, 2H, J = 7.4 Hz, CH2-Im), 2.08 (qn, 2H, J = 7.3 Hz,
-CH2-); 13C NMR (DMSO-d6): d, 156.10 (Fz-4-C), 152.49
(Fz-3-C), 137.62 (Ph-1-C), 137.20 (Ph-4-C), 134.38 (Im-2-
C), 132.85 (Im-4-C), 131.24 (Ph-3,5-C), 129.32 (Ph-2,6-C),
116.54 (Im-5-C), 32.06 (CH2-S), 27.59 (CH2-Im), 23.64
(-CH2-). Anal. C14H14N4O3S2·HCl (386.88), calcd: C, 43.46;
H, 3.91; N, 14.48; found: C, 43.53; H, 4.00; N, 14.51.
6.1.18. 3-{[3-(1H-imidazol-4-yl)propyl]sulfonyl}-4-phenyl-
furazan oxalate (16)
6.1.17.
4-{[3-(1H-imidazol-4-yl)propyl]thio}-3-(phenyl-
sulfonyl)furoxan oxalate (15a) and 3-{[3-(1H-imidazol-4-
yl)propyl]thio}-4-(phenylsulfonyl)furoxan oxalate (15b)
To a stirred solution of 12 (1.00 g, 2.12 mmol) in 10 mL
CHCl3 under N2 at 0 °C N,N-dimethylpropane-1,3-diamine
(10 mL) was added; after 45 min at r.t., it was diluted with
CHCl3 (70 mL) and cooled to -15 °C, then 4a (1.16 g,
3.18 mmol) was added. After 30 min at -15 °C the reaction
mixture was worked up and purified as described for 14 to
afford a white solid (0.90 g, 70%) which by NMR analysis
proved to be constituted by a mixture of isomers, namely the
trityl derivatives of 15a and 15b in a 1:5 ratio. Pure crystal-
line 4-phenylsulfonyl isomer was obtained by fractional re-
crystallisation (MeOH/EtOH 1:4). This crystalline product
was hydrolised in 90% aqueous TFA and purified as for 14 to
yield 15b free base as clear oil. The latter was converted into
the corresponding oxalate, white crystalline solid. M.p.: 135-
To a stirred solution of 14 (0.48 g, 1.68 mmol) in 4.8 mL
CH3COOH at 0 °C potassium permanganate (0.53 g,
3.36 mmol) was added, then the cooling bath was replaced by
a water bath. After 6 h the solvent was evaporated under
reduced pressure, the residue was treated with water (30 mL)
and Na2SO3 was added until decoloration, followed by
Na2CO3 until pH 10 was reached. 20 mL EtOAc were added,
the biphasic mixture was shaken and filtered through a celite
pad, then the aqueous layer was extracted further with EtOAc
(3×15 mL). The combined organic layers were washed with
brine (15 mL), dried over K2CO3, treated with Boc2O
(0.43 g, 1.96 mmol) and evaporated under reduced pressure
to yield a pale yellow oil which was purified by FC (petro-
leum ether in gradient from 30% to 40% of EtOAc) to yield
the Boc-protected derivative of 16 (0.44 g, 63%). The latter
was dissolved in absolute ethanol (10 mL), treated with
oxalic acid (0.25 g) and refluxed for 1 h; upon cooling 16
1
136 °C (absolute EtOH/acetone); H NMR (DMSO-d6): d,
8.41 (s, 1H, Im-2-H), 8.11 (d, 2H, J = 8.6 Hz, Ph-2,6-H), 7.88

370
P. Tosco et al. / IL FARMACO 59 (2004) 359–371
precipitated as oxalate, white crystalline solid. M.p.: 154-155
°C (absolute EtOH); H NMR (DMSO-d6): d, 8.61 (s, 1H,
6.2. Lipophilicity studies
1
Potentiometric titrations were performed with GlpKa ap-
paratus (SiriusAnalytical Instruments Ltd, Forrest Row, East
Sussex, UK) [20]. Ionisation constants were determined ac-
cording to ref. 21 by four separate titrations for each com-
pound (ca. 0.5 mM). The low aqueous solubility of the
compounds containing sulfur in the side chain required titra-
tions in the presence of variable amounts of methanol as
cosolvent (20-60 wt%); pKa values were determined by ex-
trapolation to zero content of cosolvent according to the
Yasuda-Shedlovsky procedure [22]. To obtain lipophilicity
data, at least four separate titrations for each compound (ca.
0.5 mM), containing various volumes of octan-1-ol (from
1 mL of organic solvent/15 mL of H2O to 10 mL of organic
solvent/5 mL of H2O), were performed in the pH range 2 to
11.5. Log P data were obtained by the Multiset approach
[21,23]. All titrations were carried out under Ar at
25.0 0.1 °C [23].
Im-2-H), 7.85 (d, 2H, J = 8.1 Hz, Ph-2,6-H), 7.69-7.58 (m,
3H, Ph-3,4,5-H), 7.26 (s, 1H, Im-5-H), 3.83 (t, 2H,
J = 7.7 Hz, CH2-S), 2.80 (t, 2H, J = 7.3 Hz, CH2-Im), 2.13
(qn, 2H, J = 7.5 Hz, -CH2-); 13C NMR (DMSO-d6): d, 164.00
(oxalate), 154.37 (Fz-3-C), 152.46 (Fz-4-C), 134.13 (Im-2-
C), 132.38 (Im-4-C), 131.59 (Ph-4-C), 129.39 (Ph-3,5-C),
128.93 (Ph-2,6-C), 122.68 (Ph-1-C), 115.95 (Im-5-C),
54.31 (CH2-SO2), 22.70 (CH2-Im), 20.95 (-CH2-). Anal.
C14H14N4O3S·0.9 H2C2O4 (399.39), calcd: C, 47.52; H,
3.99; N, 14.03; found: C, 47.52; H, 4.02; N, 14.03.
6.1.19. 4-{[3-(1H-imidazol-4-yl)propyl]sulfonyl}-3-phenyl-
furoxan oxalate (16a)
To a stirred solution of 14a (0.53 g, 1.75 mmol) in 5.3 mL
acetic acid at 0 °C potassium permanganate (0.55 g,
3.50 mmol) was added, then the cooling bath was replaced by
a water bath at r.t. After 6 h the mixture was worked up and
purified as described for 16 to yield the Boc-protected deriva-
tive of 16a (0.20 g, 26%). The latter was dissolved in absolute
ethanol (10 mL), treated with oxalic acid (0.10 g) and re-
fluxed for 1 h; upon cooling 16a precipitated as oxalate,
white crystalline solid. M.p.: 189-190 °C (absolute EtOH);
1H NMR (DMSO-d6): d, 8.62 (s, 1H, Im-2-H), 7.75-7.73 (m,
2H, Ph-2,6-H), 7.59 (m, 3H, Ph-3,4,5-H), 7.24 (s, 1H, Im-5-
H), 3.62 (t, 2H, J = 7.5 Hz, CH2-SO2), 2.75 (t, 2H, J = 7.0 Hz,
CH2-Im), 2.11-2.06 (m, 2H, -CH2-); 13C NMR (DMSO-d6):
d, 164.55 (oxalate), 157.30 (Fx-4-C), 134.58 (Im-2-C),
132.74 (Im-4-C), 131.74 (Ph-4-C), 129.97 (Ph-3,5-C),
129.20 (Ph-2,6-C), 120.98 (Ph-1-C), 116.51 (Im-5-C),
113.60 (Fx-3-C), 53.79 (CH2-SO2), 23.08 (CH2-Im), 21.31
(-CH2-). Anal. C14H14N4O4S·H2C2O4 (424.38), calcd: C,
45.28; H, 3.80; N, 13.20; found: C, 45.27; H, 3.77; N, 13.11.
6.3. Functional Studies
Ileum and heart were isolated from male albino guinea-
pigs weighing 250-350 g which had been anaesthetised with
CO2 and killed by decapitation. All animals were treated
humanely in accordance with recognised guidelines on ex-
perimentation. As few animals as possible were used. The
purposes and the protocols of our studies have been approved
by the Ministero della Salute, Rome, Italy. The tissues were
mounted in organ baths containing 30 mL of Krebs-
bicarbonate buffer of the following composition (mM): NaCl
111.2, KCl 5.0, CaCl2 2.5, MgSO4 1.2, KH2PO4 1.0,
NaHCO3 12, glucose 11.1. The solution was maintained at
37 °C for the ileum and at 31 °C for the heart and continu-
ously gassed with 95% O2-5% CO2 (pH = 7.4).
6.1.20. 3-{[3-(1H-imidazol-4-yl)propyl]sulfonyl}-4-phenyl-
furoxan oxalate (16b)
6.3.1. Electrically stimulated guinea-pig ileum
To a stirred solution of 14b (0.52 g, 1.72 mmol) in 5.2 mL
acetic acid at 0 °C potassium permanganate (0.54 g,
3.44 mmol) was added, then the cooling bath was replaced by
a water bath at room temperature. After 6 h the mixture was
worked up and purified as described for 16 to yield the
Boc-protected derivative of 16b (0.25 g, 33%). The latter was
dissolved in absolute ethanol (10 mL), treated with oxalic
acid (0.10 g) and refluxed for 1 h; upon cooling 16b precipi-
tated as oxalate, white crystalline solid. M.p.: 143-145 °C
(dec.) (absolute EtOH); 1H NMR (DMSO-d6): d, 8.46 (s, 1H,
Im-2-H), 7.70 (d, 2H, J = 8.1 Hz, Ph-2,6-H), 7.66-7.54 (m,
3H, Ph-3,4,5-H), 7.13 (s, 1H, Im-5-H), 3.64 (t, 2H,
J = 7.8 Hz, CH2-SO2), 2.73 (t, 2H, J = 7.3 Hz, CH2-Im), 2.06
(qn, 2H, J = 7.5 Hz, -CH2-); 13C NMR (DMSO-d6): d, 164.39
(oxalate), 155.47 (Fx-4-C), 134.52 (Im-2-C), 133.06 (Im-4-
C), 131.56 (Ph-4-C), 129.82 (Ph-3,5-C), 128.65 (Ph-2,6-C),
125.04 (Ph-1-C), 116.79 (Fx-3-C), 116.33 (Im-5-C),
53.26 (CH2-SO2), 23.25 (CH2-Im), 20.69 (-CH2-). Anal.
C14H14N4O4S·H2C2O4 (424.38), calcd: C, 45.28; H, 3.80; N,
13.20; found: C, 45.30; H, 3.73; N, 13.13.
(H3-receptors)
After equilibration for 2 h, with washings every 20 min,
the muscle segments were stimulated with square wave
pulses (10 V, 0.1 Hz, 0.5 ms) for 30 min and then cumulative
concentration-response curves (half-log increments) to the
histamine H3-agonist (R)-a-methylhistamine (MHA) were
recorded until no change in response was found. To avoid the
desensitisation phenomena, a single curve to the H3-agonist
MHA was carried out in the same preparation. From each
animal four segments of the ileum were prepared: one was
used as a control and the others were incubated with the
antagonist for 20 min before generating concentration-
response curves with MHA.All the experiments were carried
out in the presence of H1 and H2-receptor blockers (1 µM
Pyrilamine and 1 µM Ranitidine) to prevent the activation of
these receptors by the highest concentration of MHA. In the
case of derivatives 6a, 7a, 9, 14a-b, 15a-b, 16a-b we
determined pA2 values either in the absence or in the pres-
ence of 1 µM ODQ which was added to the bath at least
15 min before the addition of the antagonist. pA2 values are

P. Tosco et al. / IL FARMACO 59 (2004) 359–371
371
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10 µM concentration. None of the compounds produced a
modification of the spontaneous contraction frequency of the
atrium. The compounds were then maintained in the bath for
20 min and a second concentration-response curve to the
agonist was obtained.
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