Synthesis and pharmacological characterization of new H2-antagonists containing NO-donor moieties, endowed with mixed antisecretory and gastroprotective activities

Article abstract of DOI:10.1002/(SICI)1522-2675(20000119)83:1<287::AID-HLCA287>3.0.CO;2-2

Synthesis, structural characterization, and pharmacological profile of a series of H₂-antagonists able to release nitric oxide (NO) are reported. These compounds were obtained by using appropriate spacers to join H₂- antagonistic pharmacophoric groups related to lamtidine and tiotidine to different NO-donor moieties such as esters of HNO₃, nitrosothio groups, and benzenesulfonyl-substituted furoxans. All of the compounds were tested for their NO-donor properties. Furthermore, the hybrid structures synthesized, together with some selected reference compounds, were tested for their H₂- antagonistic properties, both in vitro and in vivo, and for their gastroprotective effects. Only the hybrid compounds were able both to antagonize histamine effects on guinea-pig papillary muscle and to display in vivo antisecretory and gastroprotective action. The best results were obtained with the lamtidine/furoxan hybrid structure.

Full text of DOI:10.1002/(SICI)1522-2675(20000119)83:1<287::AID-HLCA287>3.0.CO;2-2

Helvetica Chimica Acta ± Vol. 83 (2000)
287
Synthesis and Pharmacological Characterization of New H2-Antagonists
Containing NO-Donor Moieties, Endowed with Mixed Antisecretory and
Gastroprotective Activities
by Massimo Bertinariaa), Giovanni Sorbab), Claudio Medanaa), Clara Cenaa), Maristella Adamic),
Giuseppina Morinic), Cristina Pozzolic), Gabriella Coruzzic), and Alberto Gascoa)*
a
Á
) Dipartimento di Scienza e Tecnologia del Farmaco, Universita degli Studi di Torino, Via Pietro Giuria 9,
10125 Torino, Italy
b
Á
) Dipartimento di Scienze Chimiche, Alimentari, Farmaceutiche e Farmacologiche, Universita degli Studi del
Piemonte Orientale, Viale Ferrucci 33, 28100 Novara, Italy
c
Á
) Istituto di Farmacologia, Universita degli Studi di Parma, Via Volturno 39, 43100 Parma, Italy
Synthesis, structural characterization, and pharmacological profile of a series of H2-antagonists able to
release nitric oxide (NO) are reported. These compounds were obtained by using appropriate spacers to join
H2-antagonistic pharmacophoric groups related to lamtidine and tiotidine to different NO-donor moieties such
as esters of HNO3, nitrosothio groups, and benzenesulfonyl-substituted furoxans. All of the compounds were
tested for their NO-donor properties. Furthermore, the hybrid structures synthesized, together with some
selected reference compounds, were tested for their H2-antagonistic properties, both in vitro and in vivo, and for
their gastroprotective effects. Only the hybrid compounds were able both to antagonize histamine effects on
guinea-pig papillary muscle and to display in vivo antisecretory and gastroprotective action. The best results
were obtained with the lamtidine/furoxan hybrid structure.
1. Introduction. ± H2-Antagonists are a well-established class of drugs used in the
management of peptic ulcer and gastric-acid-related disorders [1]. Since H2-receptors
were found to be involved not only in histamine response, but also in those of gastrin
and acetylcholine, blocking of H2-receptors causes an inhibition of acid secretion even
if other stimuli are applied to the parietal cells [2]. Another interesting application of
these drugs is their use for suppressing gastric-acid production during administration of
non-steroidal anti-inflammatory drugs (NSAIDs). In this regard, however, proton-
pump inhibitors are generally preferred, either for their stronger acid-suppressing
capacity or for their additional gastroprotective effects [3]. The presence of the latter
property is controversial in H2-receptor antagonists. Although some studies indicate
that they display gastroprotective effects in a number of ulceration models, they fail to
trigger protection against gastric damage induced by NSAIDs [4][5]. Nitric oxide
(NO), a recently discovered second messenger, displays a number of important roles in
different compartments of the body [6]. At the level of the gastric tract, it is involved in
muscle relaxation, hemodynamic control, and cytoprotection of the gastric lining [7].
Based on these facts, we thought it would be useful to combine the antisecretory
activity of H2-receptor antagonists with the NO-dependent gastroprotective effect in
the same molecule. This can be achieved by joining a H2-antagonistic pharmacophoric
group to an appropriate NO-donor moiety, according to the general block structure I
(Fig. 1).

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Fig. 1. General block structure of H2-antagonists and the reference compounds lamtidine and tiotidine
In this work, the H2-antagonistic substructures were derived from lamtidine and
tiotidine, respectively, two well-known H2-receptor antagonists. As NO-releasing
moieties, we chose phenylsulfonyl-substituted furoxan (ˆ furazan 2-oxide), esters of
HNO3, and nitrosothio functions. All the structures prepared are collected in Fig. 2. As
control, the derivatives lacking the NO-releasing moieties were also studied. Synthesis
and characterization of 29 were discussed in a preliminary report [8]; nevertheless, a
few properties of this compound are also considered in the present paper for
comparison.
2. Results and Discussion. ± 2.1. Synthesis of the Nitrates 8 ± 11. Synthesis of the
hybrid models 8, 9, 10, and 11, containing nitroxy groups, was realized according to the
pathway shown in Scheme 1. Diphenyl N-cyanocarbonimidate (3) was used as starting
material. This compound shows a very high reactivity towards nucleophiles such as
amines [9]. Displacement of the first PhO group occurs under mild conditions, even
with poor nucleophiles. Intermediates 4 and 5 were obtained in good yields simply on
stirring 3, at room temperature in MeCN, with nitric-acid esters 1 and 2. Longer
reaction times were necessary for the displacement of the second PhO group by amines
6 and 7, which led to the formation of 8 ± 11 in high yields. 13C-NMR Data are in
agreement with the proposed structures (see Exper. Part). In particular, signals in the
range 70 ± 79 ppm are attributable to nitroxy-substituted C-atoms.
2.2. Synthesis of the Nitrosothio Derivatives 20 and 21. Unlike the esters of nitric
acid, structures containing nitrosothio groups are characterized by high instability and
can only rarely be isolated and purified [10]. One exception is S-nitroso-N-
acetylpenicillamine (SNAP) (ˆ N-acetyl-3-(nitrosothio)valine), which was chosen
for our experiments. The unusual stability of this compound allowed an X-ray study of
its structure to be carried out and also stimulated the preparation of some analogues
[11][12]. We planned a sequential approach to the final products 20 and 21, in which the
nitrosothio group was introduced in the last step of the reaction sequence (Scheme 2).
At room temperature, the reaction of an excess of ethylenediamine (14) with 13
afforded 16. Compound 15 was similarly obtained from 12 according to the procedure
reported in [13]. These two intermediates, treated with thietan-2-one 17 in MeOH
solution, led to the expected N-acetylpenicillaminyl derivatives 18 and 19 in good

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289
Fig. 2. Summary of synthesized hybrids containing H2-antagonistic and NO-donor moieties
yields. Subsequent nitrosation of these compounds was satisfactorily achieved with
t-BuONO in AcOH, yielding 20 and 21, respectively. The final products were
sufficiently stable to be isolated by lyophilization of the frozen reaction mixture and to
be characterized by routine spectroscopic techniques. 13C-NMR Data were in
agreement with the proposed structures (see Exper. Part). S-Nitrosation led to a
significant downfield-shift (13 ppm) of the resonances of the quaternary C-atoms
linked to the S-atom.
2.3. Synthesis of the Furoxan and Furazan Derivatives. Synthesis of the hybrid
furoxan model compounds 29 and 30, and of the related furazan derivative 32 was
realized by the pathway depicted in Scheme 3. The PhO group present in the
intermediates 12 and 13 was displaced at room temperature by the amino alcohols 22

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Scheme 1
Scheme 2
and 23 to yield H2-antagonistic pharmacophores 24 ± 27 that bear hydroxyalkyl spacers
on the terminal amino group of the cyanoguanidine moiety. Compounds 24 and 25,
dissolved in THF (THF/DMF in the case of 25) in the presence of 50% aqueous NaOH
solution, were able to react with 3,4-bis(phenylsulfonyl)furoxan (28) to yield the final
products 29 and 30. It is known that 28, dissolved in THF, reacts with EtOH under basic
conditions to give 4-ethoxy-3-(phenylsulfonyl)furoxan [14]. Based on this observation,
29 and 30 were expected to be the products of an analogous reaction of 28 with 24 and
25, respectively. This assumption was also confirmed by 13C-NMR data (see Exper.
Part). In particular, the signals at 110.7 and 158.9 ppm, detected for both compounds,
are characteristic for C(3) and C(4), respectively, of a 4-alkoxy-3-(phenylsulfonyl)-
substituted furoxan ring [15]. The furazan analogue 32 was synthesized in a similar
fashion from 25 and 31 in DMF in the presence of NaH.
2.4. Detection of Nitrite and Determination of Initial NO-Release Rate. Nitrite is the
major oxidation products of NO in aqueous systems, and their detection serves as a
basis for the determination of the original NO concentration. However, it is known that

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291
Scheme 3
in the presence of thiols, besides this oxidation, a variety of other reactions can occur.
To examine spontaneous NO release, each hybrid compound was dissolved (10 4 m) in
pH-7.4-buffered H2O and kept at 378 for 1 h. The nitrites formed were detected by the
Griess reaction [16]. Furthermore, the NO2 concentration was determined in the
presence of a large excess of cysteine (Cys) (50 :1) after 1 h of incubation. The results,
expressed as % NO2 (mol/mol), are summarized in Table 1. The initial rates of NO
release in the presence of Cys (5-fold molar excess) and, if necessary, in its absence,
were determined at selected concentrations for each hybrid, employing a spectropho-
tometric technique based on the NO-induced oxidation of oxyhemoglobin (HbO2) to
methemoglobin (MetHb) [17]. The reaction itself was monitored by detecting the
1
increase of absorbance (DA) at 401 nm. The initial rates of NO release (in nmol ml
1
1
min ) were calculated from the measured rates (in DA min ) using a molar extinction
coefficient De ˆ e401(MetHb) e401(HbO2) ˆ 39.9 (Æ1.4) mm cm . All results are
1
1
reported in Table 1.
Hybrids 8 and 10, bearing one nitroxy function, behaved as weak NO-donors only in
the presence of cysteine. The extent of NO release at the applied concentration (10 4 m)
was too small to allow the determination of the initial rate of the process. When two
nitroxy functions were present (structures 9 and 11), the extent of release increased,
but, again, it was impossible to determine the initial rate. However, working with cells,
tissues, or in vivo, not only thiol-induced but also enzymatic production of NO from
nitrates should be considered [18].
NO Release by nitrosothio groups was quite complex to follow. Both 20 and 21 led
to the formation of nitrite to a similar extent. The initial rates of NO release,
determined at 378 in 5 Â 10 4 m solutions, were, within the limits of experimental error,
identical for the two structures. Analogous measurements in the presence of an excess
of cysteine showed a high increase in the amounts of nitrite formed, as well as in the
initial rates of NO release. This behavior might be explained by transnitrosation from

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Table 1. NO-Release Characteristics of Hybrids under Different Conditionsa)
Compound
NO2 [%]b)
NO2 [%]b)
(‡l-cys)
NO2 [%]b)
(‡Cu ) )
NO
NO
d
c
[nmol ml 1 min 1] c)
[nmol ml min 1] )
‡
1
(
l-cys)
(
l-cys)
(‡l-cys)
8
9
0
0
0
0
0.43 Æ 0.11
6.0 Æ 0.3
±
±
±
±
0
0
0
0
0
0
0
0
10
11
20
21
29
30
0.47 Æ 0.11
4.4 Æ 0.1
2.1 Æ 0.4
28.0 Æ 1.0
50.5 Æ 0.6
56.9 Æ 3.4
31.6 Æ 1.5
64.6 Æ 2.0
0.989 Æ 0.012e)
0.346 Æ 0.025
0.556 Æ 0.030
0.596 Æ 0.077
1.248 Æ 0.085
3.8 Æ 0.8
96.8 Æ 1.2
1.015 Æ 0.025e)
0
0
±
±
0
0
a) Yields are reported in % (mol/mol Æ standard error of the mean (SEM), cf. Exper. Part). b) Nitrite was
determined according to the Griess procedure; compound: 100 mm; l-cysteine: 5 mm; cf. Exper. Part. c) The
initial NO-release rate was determined by the oxyhemoglobin method; compound: 10 mm; l-cysteine: 50 mm.
d
e
‡
‡
) [Cu ]: 10 mm. ) With 10 mm Cu , immediate oxyhemoglobin oxidation.
the rather stable SNAP analogues 20 and 21 to very unstable S-nitrosocysteine [19]. An
even more dramatic change in the rate of NO release was observed in the presence of
‡
Cu ions (10 5 m). Within 1 h, the tiotidine hybrid 21 was 100% denitrosated, and the
lamtidine hybrid 20 was about 65% denitrosated. For both compounds, it was
impossible to measure the initial rates of NO production since the reactions were too
fast. Efficient metal-ion catalysis in nitrosothio-group decomposition would explain our
observations [20][21].
NO Release from furoxan derivatives occurred only in the presence of cysteine. The
lamtidine hybrid 29 was a stronger overall NO-donor than the tiotidine hybrid 30, but
the initial rate of release was ca. twofold higher for 30 than for 29. Both the extent and
the initial rate of NO release in the two hybrids were high. With respect to biological
systems, one should consider that NO might also be released enzymatically from
furoxans. However, to the best of our knowledge, this possibility has not been
investigated yet.
2.5. Pharmacological Characterization. All of the hybrid drugs, as well as the related
compounds lacking NO-donor moieties, were screened for their H2-antagonistic
properties on isolated guinea-pig papillary muscle in vitro, and on anesthetized rats
with a lumen-perfused stomach in vivo (i.v. administration). In the former case, the
activity was expressed as pA2 (CL 95%), in the latter as ID50 (in mg/kg) (CL 95%).
Gastroprotective activity was assessed against HCl-induced (0.6n) gastric lesions in
conscious rats (i.g. administration). The results were expressed as %-reduction of the
lesion index compared to a control group. It should be noted that, due to different
routes of administration, gastroprotective effects of H2-receptor antagonists containing
NO-donor moieties were observed at doses 3 ± 10 times higher than those usually
eliciting antisecretory effects. The results are reported in Table 2.
Hybrids 8 and 10, containing one nitroxy function, exhibited 12- and 3-fold lower
potency, respectively, at guinea-pig papillary muscle H2-receptors than the reference
compounds 26 and 27. The ability of 10 to inhibit gastric-acid secretion was almost
comparable to that of reference compound 27. However, a similar comparison between
the two lamtidine derivatives 8 and 26 was not possible. In fact, hybrid 8 reduced

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Table 2. H2-Antagonistic Properties and Gastroprotective Activity of Hybrids 8 ± 11, 20, 21, 29, and 30, and the
Related Reference Compounds 18, 19, 24 ± 27, and 32
H2-antagonistic activity
Guinea-pig papillary Rat lumen-perfused
Gastroprotective activity
Rat stomach
% reduction of lesion index
mean Æ SEM
muscle
stomach
pA2 (CL 95%)
ID50 [mg/kg] (CL 95%)
8
9
5.18 (4.29 ± 6.07)a)b)
±c)
44 Æ 18 (30 mg/kg)d)
56 Æ 7 (100 mg/kg)d)
6.16 (5.99 ± 6.33)a)
0.35 (0.16 ± 0.79)
62 Æ 8 (30 mg/kg)d)
97 Æ 1 (100 mg/kg)e)
10
11
18
19
20
21
24
26
27
5.95 (5.36 ± 6.54)a)
6.59 (5.9 ± 7.28)a)
5.34 (5.09 ± 5.59)a)
5.83 (5.13 ± 6.52)a)
5.43 (5.28 ± 5.59)a)
5.62 (5.04 ± 6.19)a)
6.95 (6.44 ± 7.46)a)
6.25 (5.88 ± 6.62)
6.40 (5.83 ± 6.97)a)
0.60 (0.32 ± 1.18)
1.01 (0.90 ± 1.14)
0.68 (0.28 ± 1.64)
0.26 (0.08 ± 1.00)
0.94 (0.45 ± 1.96)
0.42 (0.18 ± 0.88)
0.02 (0.005 ± 0.12)
0.26 (0.14 ± 0.45)
0.34 (0.20 ± 0.55)
35 Æ 20 (30 mg/kg)
±c)
inactive up to 100 mg/kg
inactive up to 100 mg/kg
36 Æ 12 (3 mg/kg)d)
50 Æ 7 (0.03 mg/kg)d)
inactive up to 100 mg/kg
inactive up to 100 mg/kg
inactive up to 100 mg/kg
29
5.78 (5.65 ± 5.91)b)
0.40 (0.20 ± 0.76)b)
20 Æ 6 (3 mg/kg)
80 Æ 6 (10 mg/kg)e)
90 Æ 2 (30 mg/kg)e)
30
32
6.71 (6.62 ± 6.81)f)
6.30 (4.84 ± 7.76)f)
0.20 (0.15 ± 0.26)
0.11 (0.06 ± 0.20)
65 Æ 8 (30 mg/kg)d)
inactive up to 100 mg/kg
a) pA2 was calculated only for two surmountable concentrations of the antagonist. b) P < 0.05 vs. the related
reference compound (t-test for unpaired data). c) See text. d) P < 0.05 vs. the vehicle-treated group (ANOVA
followed by Newman-Keuls test). e) P < 0.01 vs. the vehicle-treated group (ANOVA followed by Newman-
Keuls test). f) pA2 was calculated only for one surmountable concentration of the antagonist.
histamine response by approximately 50% when tested at a dose of 1.55 mg/kg, but
higher doses were toxic for the animals, thus preventing us from evaluating the ID50
value. Compounds 8 and 10 showed gastroprotective activity, which was not the case for
the related compounds devoid of NO-donor moieties. For 8, the observed effect did not
vary significantly for doses of 30 and 100 mg/kg, respectively, leading in both cases to a
ca. 50% reduction of the lesion index. Due to the low solubility of 10, only 30 mg/kg-
doses could be evaluated; they were found to provide 35% protection. Compound 9,
containing two nitroxy functions, displayed good H2-antagonistic activity both in vitro
and in vivo. In addition, it was a better gastroprotective agent than the mononitroxy
structure 8. This is in keeping with its better NO-donor properties. The tiotidine hybrid
11 was a more potent H2-receptor blocker in vitro, but in vivo, it was less effective than
10. The gastroprotective action of 11 was not evaluated because of its low solubility.
The nitrosothio-substituted hybrids 20 and 21 were as effective as the reference
compounds 18 and 19 on papillary muscle and in inhibiting gastric-acid secretion. In
both tests, the tiotidine structures were more potent than the lamtidine analogues. As
expected, the reference compounds were devoid of any gastroprotective effect, while
the nitrosothio-substituted hybrids showed a rather complex behavior: structure 20

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caused a 36% reduction of the lesion index at a dose of 3 mg/kg, higher doses, however,
were ineffective. Experiments with compound 21 led to very ambiguous results: in
some cases, protective effects were observed, in other cases, 21 led to gastric lesions,
and sometimes it did not trigger any action at all. However, at very low doses (0.03 mg/
kg), we were able to show a statistically significant, partially protective effect (50 Æ
7%). This contradictory behavior might be caused by individual differences in tissue
availability of cysteine and/or of those metal ions which strongly influence NO release
from nitrosothio-substituted compounds.
The tiotidine-furoxan hybrid 30 was a more potent H2-receptor antagonist both in
vitro and in vivo than the corresponding lamtidine hybrid 29, and it showed properties
similar to the furazan analogue 32. Compound 29 was less active than the related
compound 24, but it still displayed a good H2-antagonistic profile. It caused a dose-
dependent reduction of gastric damage, resulting in complete protection at a dose of
30 mg/kg. This compound appears to be a well-balanced hybrid endowed with both H2-
antagonistic activity and gastroprotective effect in vivo. The tiotidine-derived structure
30 was only a partial gastroprotector, causing a 65% reduction of the lesion index at
30 mg/kg, which was, due to problems of solubility, the maximum dose tested. As
expected, neither of the two reference compounds 24 and 32 displayed this kind of
effect as they lack the necessary NO-donor moieties.
In conclusion, all of the hybrids tested are endowed with both antisecretory and
gastroprotective activity the lamtidine-derived structures showing the most promising
properties. In this series, the hybrid behavior was optimised by the presence of a
(phenylsulfonyl)furoxan substructure. These compounds could be the prototypes of a
new class of drugs, which may be useful in the therapy of inflammatory disorders and in
the management of conditions involving gastric hypersecretion.
The financial support from MURST, Rome, and Consiglio Nazionale delle Ricerche, Rome, is gratefully
acknowledged. The authors are grateful to Mrs. Maura Vallaro for her skillful technical assistance.
Experimental Part
General. Compounds 1 [22], 2 [23], 3 [9], 6 [24], 7 [25], 12 [26], 13 [27], 15 [13], 17 [12], 24 [13], 28 [28], 29
[8], and 31 [29] were prepared according to the reported methods. Flash chromatography (FC): silica gel 60
(Merck 230 ± 400 mesh ASTM). M.p.: Büchi 530 apparatus, uncorrected. Dec. temp. was detected by
introducing the sample into the bath approx. 108 below dec. started, heating rate 38/min. Some compounds were
amorphous solids without defined m.p. For some of these structures, the initial point of dec. detected by
differential scanning calorimetry (Perkin-Elmer DSC 7, heating rate 108/min) is reported. All the products were
characterized by routine spectroscopy. IR Spectra: Shimadzu FT-IR8101 M. 1H- and 13C-NMR spectra: Bruker
AC-200, at 200 and 50 MHz, resp.; d in ppm rel. to SiMe4 as internal standard, coupling constants J in Hz;
13C-NMR spectra were fully decoupled. Elemental analyses (C, H, N) were performed by Redox (Cologno M.)
3-{[(Cyanoimino)(phenoxy)methyl]amino}propyl Nitrate (4). To a stirred soln. of 1 (4.00 g, 21.8 mmol)
and Et3N (3.47 ml, 24.9 mmol) in MeCN (22 ml), 3 (5.19 g, 21.8 mmol) was added, and the mixture was stirred
for 30 min at r.t. The solvent was evaporated under reduced pressure, the oily residue was diluted with CH2Cl2
(20 ml) and washed with H2O (3 Â 20 ml). The org. phase was dried (MgSO4) and evaporated under reduced
pressure to give a viscous oil which was purified by FC (silica gel; AcOEt/CH2Cl2 1:40 (300 ml) and AcOEt/
CH2Cl2 1:4 (500 ml)) to afford 4 (5.00 g, 87%). White solid. M.p. 778 (EtOH). 1H-NMR (CDCl3): 8.09 (s, NH);
7.07 ± 7.45 (m, 5 arom. H); 4.57 (t, J ˆ 6.1, CH2ONO2); 3.53 (m, CH2NH); 2.08 (m, CH2). 13C-NMR (CDCl3):
164.49; 151.48; 130.08; 127.15; 121.86; 116.16; 70.73; 39.35; 27.15. Anal. calc. for C11H12N4O4 (264.2): C 50.00,
H 4.58, N 21.20; found: C 50.04, H 4.56, N 21.08.

Helvetica Chimica Acta ± Vol. 83 (2000)
295
3-{[(Cyanoimino)(phenoxy)methyl]amino}propane-1,2-diyl Dinitrate (5). To a stirred soln. of 2 (2.50 g,
10.2 mmol) and Et3N (1.62 ml, 11.6 mmol) in MeCN (10 ml), 3 (2.57 g, 10.7 mmol) was added, and the mixture
was stirred vigorously for 30 min at r.t. The solvent was evaporated under reduced pressure, the residue was
diluted with CH2Cl2 and washed with H2O (1 Â 20 ml) and brine (2 Â 20 ml). The org. phase was dried (MgSO4)
and evaporated under reduced pressure to afford a crude product which was purified by FC (silica gel; CH2Cl2
(300 ml) and AcOEt/CH2Cl2 1 :10 (500 ml)) to yield 5 (2.19 g, 66%). White solid. M.p. 938 (i-PrOH). 1H-NMR
(CDCl3): 8.37 (t, J ˆ 5.8, NH); 7.47 ± 7.08 (m, 5 arom. H); 5.44 (m, CHONO2); 4.74 (m, CH2ONO2); 3.64 ± 3.92
(m, 2 H, CH2NH). 13C-NMR (CDCl3): 164.11; 150.17; 129.59; 126.91; 121.16; 115.25; 76.70; 68.89; 40.48. Anal.
calc. for C11H11N5O7 (325.2): C 40.62, H 3.41, N 21.53; found: C 40.47, H 3.37, N 21.45.
3-(2-Cyano-3-{3-[3-(piperidinomethyl)phenoxy]propyl}guanidino)propyl Nitrate (8). Compound 3 (1.00 g,
3.67 mmol) was added portionwise during 2 h to vigorously stirred 6 (2.46 g, 7.67 mmol). The resulting mixture
was stirred for 24 h at r.t., and the crude product was purified by FC (silica gel; MeOH/CH2Cl2 1:10) to afford 8
(1.54 g, 97%). Pale-yellow oil. The product was characterized as the oxalate (EtOH/Et2O). 1H-NMR
((D6)DMSO): 6.98 ± 7.38 (m, 6 H); 4.53 (t, J ˆ 6.1, 2 H); 4.14 (s, 2 H); 4.02 (m, 2 H); 3.85 (t, J ˆ 5.6, 2 H); 3.21 ±
3.41 (m, 4 H); 2.98 (m, 4 H); 1.82 ± 1.95 (m, 4 H); 1.71 (m, 4 H); 1.52 (m, 2 H). 13C-NMR ((D6)DMSO): 165.04;
159.42; 158.71; 132.59; 129.86; 123.11; 118.11; 116.78; 115.28; 71.59; 65.37; 59.48; 52.04; 39.95; 37.76; 28.64;
26.29; 22.71; 21.80. Anal. calc. for C20H30N6O4 ´ C2H2O4 ´ 0.5 H2O (517.5): C 51.06, H 6.43, N 16.24; found:
C 51.30, H 6.81, N 16.58.
3-(2-Cyano-3-{3-[3-(piperidinomethyl)phenoxy]propyl}guanidino)propane-1,2-diyl Dinitrate (9). To
a
stirred soln. of 6 (2.43 g, 7.56 mmol) in MeCN (3 ml), 5 (1.81 g, 5.56 mmol) was added, and the mixture was
stirred for 20 h at r.t. The solvent was evaporated under reduced pressure, and the residue was purified by FC
(silica gel; MeOH/CH2Cl2 1 :10) to afford 9 (2.59 g, 97%). Pale-yellow oil. The product was characterized as
oxalate. M.p. 130 ± 1328 (DSC) (MeOH/Et2O). 1H-NMR ((D6)DMSO): 6.98 ± 7.48 (m, 6 H); 5.56 (m, 1 H);
4.66 ± 4.98 (m, 2 H); 4.14 (s, 2 H); 4.02 (m, 2 H); 3.29 ± 3.60 (m, 4 H); 3.00 (m, 4 H); 1.95 (m, 2 H); 1.72
(m, 4 H); 1.52 (m, 2 H). 13C-NMR ((D6)DMSO): 164.65; 159.60; 158.68; 132.11; 129.88; 123.19; 117.64; 116.83;
115.38; 78.30; 70.44; 65.30; 59.30; 51.90; 39.78; 38.61; 28.53; 22.54; 21.70. Anal. calc. for C20H29N7O7 ´ C2H2O4
(569.5): C 46.40, H 5.49, N 17.22; found: C 46.62, H 5.56, N 17.06.
3-{2-Cyano-3-[2-({2-[(diaminomethylidene)amino]thiazol-4-yl}methylthio)ethyl]guanidino}propyyl Nitrate
(10). To a stirred suspension of 7 ´ 2 HCl (4.60 g, 15.1 mmol) and Et3N (11.5 ml, 82.5 mmol) in MeCN
(66 ml), 4 (2.00 g, 7.57 mmol) was added, and the mixture was stirred for 72 h at r.t. The liquid was decanted, and
the solid residue was dissolved in 2n NaOH soln. and extracted with AcOEt (3 Â 50 ml). The combined org.
layers were dried (MgSO4) and evaporated under reduced pressure, yielding a white solid. The crude product
was purified by FC (silica gel; MeOH/CH2Cl2 1:10) affording 10 (1.54 g, 54%). Yellow oil. The product was
characterized as the oxalate (MeOH/Et2O). 1H-NMR (MeOD): 7.06 (s, 1 H); 4.57 (t, J ˆ 6.2, 2 H); 3.83 (s, 2 H);
3.33 ± 3.47 (m, 4 H); 2.72 (t, J ˆ 6.9, 2 H); 2.01 (m, 2 H). 13C-NMR (MeOD): 167.06; 162.06; 161.39; 156.55;
150.91; 120.01; 111.18; 72.39; 42.28; 39.57; 32.21; 32.04; 28.10. Anal. calc. for C14H21N9O7S2 (491.5): C 34.21,
H 4.31, N 25.65; found: C 34.36, H 4.34, N 25.74.
3-{2-Cyano-3-[2-({2-[(diaminomethylidene)amino]thiazol-4-yl}methylthio)ethyl]guanidino}propane-1,2-diyl
Dinitrate (11). To a stirred suspension of 7 ´ 2 HCl (3.80 g, 12.5 mmol) and Et3N (9.25 ml, 66.3 mmol) in MeCN
(10 ml), a soln. of 5 (2.04 g, 6.27 mmol) in MeCN (4 ml) was added and the reaction mixture stirred vigorously
for 70 h at r.t. The solvent was evaporated under reduced pressure, the residue dissolved in 2n NaOH soln. and
extracted with AcOEt (3 Â 50 ml). The dried (MgSO4) org. phase was evaporated under reduced pressure and
the residue purified by FC (silica gel, MeOH/CH2Cl2 1:10) to give 11 (1.64 g, 56%) as colorless oil. The product
was characterized as the oxalate. M.p. 2108 (dec.) (DSC) (MeOH). 1H-NMR ((D6)DMSO): 7.82 (m, 4 H); 7.39
(m, 2 H); 6.81 (s, 1 H); 5.54 (m, 1 H); 4.84 (m, 2 H); 3.70 (s, 2 H); 3.31 ± 3.59 (m, 4 H); 2.60 (m, 2 H). 13C-NMR
((D6)DMSO): 168.16; 166.10; 159.45; 155.92; 148.06; 117.55; 107.21; 78.27; 70.44; 40.85; 39.77; 30.85; 30.16.
Anal. calc. for C12H18N10O6S2 ´ 0.5 C2H2O4 (507.5): C 30.77, H 3.77, N 27.60; found: C 31.18, H 3.82, N 27.25.
1-(2-Aminoethyl)-3-[2-({2-[(diaminomethylidene)amino]thiazol-4-yl}methylthio)ethyl]guanidino-2-carbo-
nitrile (16). To powdered 13 (6.00 g, 16.0 mmol), ethane-1,2-diamine (14) (4.8 ml, 71.7 mmol) was added keeping
the temp. at 208. The resulting viscous mixture was stirred for 3 h at r.t., excess 14 was partially removed under
high vacuum, and the oily residue was purified by FC (silica gel; MeOH/CH2Cl2 1:1 (1000 ml) ‡ 32% aq.
NH4OH (13 ml)) to afford 16 (4.78 g, 88%). Highly hygroscopic solid foam. 1H-NMR ((D6)DMSO): 6.88
(m, 4 H); 6.50 (s, 1 H); 3.62 (s, 2 H); 3.31 (t, J ˆ 7.1, 2 H); 3.09 (t, J ˆ 5.8, 2 H); 2.56 ± 2.67 (m, 4 H). 13C-NMR
((D6)DMSO): 175.59; 159.82; 157.10; 147.80; 118.29; 104.58; 44.57; 41.07; 40.67; 31.39; 30.46. The product was
not further characterized, but converted to 19.

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Helvetica Chimica Acta ± Vol. 83 (2000)
2-Acetamido-N-(2-cyano-3-{3-[3-(piperidinomethyl)phenoxy]propyl}guanidino)-3-mercapto-3-methylbut-
anamide (18). To a soln. of 15 (1.58 g, 4.41 mmol) in MeOH (30 ml) N-(2,2-dimethyl-4-oxothietan-3-
yl)acetamide (17) (0.76 g, 4.41 mmol) was added, and the mixture was stirred for 11h at r.t. The solvent was
evaporated under reduced pressure, and the crude product was purified by FC (silica gel; MeOH/CH2Cl2 1:5)
affording 18 (1.90 g, 81%). White solid. The product was characterized as the oxalate. M.p. 798 (MeOH/Et2O).
1H-NMR ((D6)DMSO): 8.34 (s, 1 H); 8.05 (d, J ˆ 9.3, 1 H); 7.34 (s, 1 H); 6.97 ± 7.19 (m, 4 H); 4.45 (d, J ˆ 9.3,
1 H); 4.07 (s, 2 H); 4.01 (m, 2 H); 3.21 ± 3.37 (m, 6 H); 2.94 (m, 4 H); 2.79 (s, 1 H); 1.93 (m, 3 H); 1.70 (m, 4 H);
1.51 (m, 2 H); 1.36 (s, 3 H); 1.33 (s, 3 H). 13C-NMR ((D6)DMSO): 169.75; 169.45; 165.07; 159.45; 158.68;
132.90; 129.79; 122.98; 118.06; 116.67; 115.12; 65.34; 60.99; 59.65; 52.11; 45.99; 40.70; 38.50; 38.20; 30.01; 29.22;
28.61; 22.85; 22.57; 21.95. Anal. calc. for C26H41N7O3S ´ C2H2O4 ´ 0.2 Et2O (636.5): C 54.34, H 7.13, N 15.40;
found: C 54.34, H 7.49, N 15.06.
2-Acetamido-N-{2-cyano-3-[2-({2-[(diaminomethylidene)amino]thiazol-4-yl}methylthio)ethyl]guanidino}-3-
mercapto-3-methylbutanamide (19). To a soln. of 16 (2.00 g, 5.86 mmol) in MeOH (12 ml), 8 (1.02 g, 5.86 mmol)
was added, and the mixture was stirred for 2 h at r.t. Evaporation of the solvent under reduced pressure and FC
(silica gel; MeOH/CH2Cl2 3 :20 (300 ml) and MeOH/CH2Cl2 3 :10 (400 ml)) afforded 19 (2.72 g, 90%). Pale-
yellow solid. The product was characterized as the oxalate (MeOH/Et2O). 1H-NMR ((D6)DMSO): 8.28
(s, 1 H); 8.00 (d, J ˆ 9.3, 1 H); 7.90 (m, 4 H); 6.99 (s, 1 H); 6.85 (s, 1 H); 4.49 (d, J ˆ 9.3, 1 H); 3.71 (s, 2 H);
3.11 ± 3.45 (m, 4 H); 2.75 (s, 1 H); 2.58 (m,2 H); 1.92 (s, 3 H); 1.36 ± 1.38 (s, 6 H). 13C-NMR ((D6)DMSO):
169.78; 169.52; 167.32; 165.67; 159.33; 155.77; 148.15; 117.96; 107.49; 60.94; 46.05; 40.23; 38.15; 30.74; 30.16;
30.04; 29.18; 22.58. Anal. calc. for C18H30N10O2S3 ´ 0.5 C2H2O4 ´ H2O ´ 0.3 Et2O (600.0): C 40.44, H 6.05, N 23.35;
found: C 40.75, H 5.89, N 23.16.
2-Acetamido-N-(2-cyano-3-{3-[3-(piperidinomethyl)phenoxy]propyl}guanidino)-3-methyl-3-(nitrosothio)bu-
tanamide (20). To a soln. of 18 (1.00 g, 1.88 mmol) in AcOH (50 ml) in a light-protected vessel, a soln. of t-
BuONO (0.22 g, 2.16 mmol) in AcOH (5 ml) was added dropwise. After 30 min at r.t., the mixture was frozen
and the solvent lyophilized to leave a green gum which was triturated at 108 with dry Et2O (2 Â 20 ml),
decanted, and dried (high vacuum) to afford 20 (1.01 g, 86%). Pale-green solid. The product was characterized
as the oxalate (MeOH/Et2O). 1H-NMR ((D6)DMSO): 8.61 (s, 1 H); 8.47 (d, J ˆ 9.6, 1 H); 7.39 ± 7.00 (m, 5 H);
5.18 (d, J ˆ 9.6, 1 H); 4.20 (s, 2 H); 4.02 (m, 2 H); 3.05 (m, 4 H); 3.20 ± 3.47 (m, 6 H); 1.98 (s, 3 H); 1.93 (s, 3 H);
1.86 (s, 3 H); 1.73 (m, 4 H); 1.54 (m, 2 H). 13C-NMR ((D6)DMSO): 169.52; 168.79; 164.86; 132.08; 129.93;
123.24; 118.08; 116.97; 115.38; 65.44; 59.07; 58.91; 51.96; 40.35; 39.10; 28.39; 26.59; 24.90; 22.59; 22.43; 21.73.
Anal. calc. for C26H40N8O4S ´ C2H2O4 (650.8): C 51.68, H 6.51, N 17.22; found: C 52.04, H 7.01, N 16.78.
2-Acetamido-N-{2-cyano-3-[2-({2-[(diaminomethylidene)amino]thiazol-4-yl}methylthio)ethyl]guanidino}-3-
methyl-3-(nitrosothio)butanamide (21). To a soln. of 19 (1.00 g, 1.94 mmol) in AcOH (50 ml) in a light-protected
vessel, t-BuONO (0.25 ml, 2.14 mmol) was added dropwise. After 30 min of stirring at r.t., the mixture was
frozen, and the solvent was lyophilized to leave a green-red gum which was dissolved in 5% aq. AcOH soln.
(15 ml) and extracted with AcOEt (3 Â 10 ml). AcOH (15 ml) was added to the aq. phase, and the mixture was
frozen and lyophilized again. The green-red residue was triturated at 108 with dry Et2O (2 Â 20 ml), decanted,
and dried (high vacuum) to afford 21 (0.78 g, 67%). Cream-colored solid. 1H-NMR ((D6)DMSO): 8.60
(m, 1 H); 8.46 (d, J ˆ 9.6, 1 H); 7.64 (m, 4 H); 7.20 (m, 1 H); 7.06 (m, 1 H); 6.82 (s, 1 H); 5.17 (d, J ˆ 9.6, 1 H);
3.70 (s, 2 H); 3.20 ± 3.31 (m, 6 H); 2.60 (m, 2 H); 1.85 ± 1.98 (s, 12 H). 13C-NMR ((D6)DMSO): 172.21; 169.52;
168.72; 167.81; 159.28; 155.78; 148.13; 131.63; 117.93; 107.28; 59.63; 59.18; 40.75; 40.58; 38.33; 30.74; 30.18; 26.81;
24.87; 22.40; 21.16. Anal. calc. for C18H29N11O3S3 ´ 2 AcOH ´ 3 H2O (717.8): C 36.81, H 6.04, N 21.48; found:
C 36.38, H 5.54, N 21.63.
1-[2-({2-[(Diaminomethylidene)amino]thiazol-4-yl}methylthio)ethyl]-3-(hydroxyethyl)guanidine-2-carboni-
trile (25). To a stirred suspension of 13 (3.00 g, 8.00 mmol) in MeOH (36 ml), 2-aminoethanol (22) (12.5 ml,
207 mmol) was added, and the mixture was stirred for 4 h at r.t. The solvent was evaporated under reduced
pressure, and the crude product was purified by FC (silica gel; CH2Cl2 (300 ml) and MeOH/CH2Cl2 1:10
(500 ml)) to afford 25 (2.41 g, 88%). White solid. The product was characterized as the oxalate. M.p. 130 ± 1318
(dec.) (DSC) (H2O/i-PrOH). 1H-NMR ((D6)DMSO): 8.39 (m, 4 H); 7.16 (m, 1 H); 7.01 (s, 1 H); 6.94 (m, 1 H);
3.76 (s, 2 H); 3.48 (t, J ˆ 5.7, 2 H); 3.14 ± 3.36 (m, 4 H); 2.60 (m, 2 H). 13C-NMR ((D6)DMSO): 165.59; 163.49;
159.62; 155.19; 148.39; 118.11; 108.86; 59.70; 44.00; 40.70; 30.50; 30.30. Anal. calc. for C11H18N8OS2 ´ C2H2O4
(432.5): C 36.10, H 4.66, N 25.91; found: C 35.92, H 4.66, N 25.90.
1-(3-Hydroxypropyl)-3-{3-[3-(piperidinomethyl)phenoxy]propyl}guanidine-2-carbonitrile (26). Finely
powdered 12 (2.00 g, 5.09 mmol) was dissolved in 3-aminopropan-1-ol (23; 12 ml, 157 mmol) and stirred for
1 h at r.t. The mixture was poured into H2O (20 ml) and extracted with AcOEt (4 Â 30 ml). The org. phase was
dried (MgSO4) and evaporated under reduced pressure yielding a brown oil, which was purified by FC (silica

Helvetica Chimica Acta ± Vol. 83 (2000)
297
gel; MeOH/CH2Cl2 1:10) to afford 26 (1.90 g, quant.) as pale-yellow oil. The product was characterized as the
oxalate. M.p. 121 ± 1228 (dec.) (DSC) (MeOH/Et2O). 1H-NMR ((D6)DMSO): 7.63 (s, 1 H); 7.00 ± 7.39 (m, 6 H);
4.21 (s, 2 H); 4.02 (m, 2 H); 3.05 ± 3.47 (m, 8 H); 1.93 (m, 2 H); 1.52 ± 1.73 (m, 8 H). 13C-NMR ((D6)DMSO):
164.48; 159.58; 158.75; 131.52; 129.94; 123.36; 118.33; 116.99; 115.85; 65.44; 59.10; 58.46; 51.81; 38.73; 38.43;
32.02; 28.76; 22.38; 21.56. Anal. calc. for C20H31N5O2 ´ C2H2O4 ´ 0.5 H2O (472.5): C 56.39, H 7.31, N 14.94; found:
C 55.92, H 7.25, N 14.82.
1-[2-({2-[(Diaminomethylidene)amino]thiazol-4-yl}methylthio)ethyl]-3-(hydroxypropyl)guanidine-2-car-
bonitrile (27). Finely powdered 13 (1.02 g, 2.98 mmol) was dissolved in 23 (1.9 ml, 24.9 mmol) and stirred
vigorously for 1 h at r.t. FC (silica gel; MeOH/CH2Cl2 1:10) afforded 27 (0.95 g, 90%). Colorless oil. The
product was characterized as the oxalate (MeOH/Et2O). 1H-NMR ((D6)DMSO): 7.93 (m, 4 H); 7.02 (m, 2 H);
6.83 (s, 1 H); 3.70 (s, 2 H); 3.43 (m, 2 H); 3.32 (m, 2 H); 3.15 (m, 2 H); 2.58 (m, 2 H); 1.82 (m, 2 H). 13C-NMR
((D6)DMSO): 167.41; 165.96 159.38; 155.83; 148.20; 118.17; 107.41; 58.45; 40.86; 39.75; 31.91; 30.78; 30.33. Anal.
calc. for C12H20N8OS2 ´ 0.5 C2H2O4 ´ H2O ´ 0.3 Et2O (441.7): C 38.61, H 5.93, N 25.37; found: C 38.59, H 5.52,
N 25.78.
1-[2-({2-[(Diaminomethylidene)amino]thiazol-4-yl}methylthio)ethyl]-3-{2-[2-oxido-3-(phenylsulfonyl)fur-
azan-4-yloxy]ethyl}guanidine-2-carbonitrile (30). To a stirred soln. of 25 (1.00 g, 2.9 mmol) and 3,4-bis-
(phenylsulfonyl)furazan 2-oxide (28; 1.28 g, 3.5 mmol) in DMF/THF 1:20 (25 ml), 50% (w/w) aq. NaOH soln.
(0.46 g, 5.75 mmol) was added at r.t., keeping the temp. at 20 ± 258. The mixture was reacted for 40 min, the
solvent was removed under reduced pressure, and the residue was treated with sat. NaCl soln. (30 ml) and
extracted with AcOEt (6 Â 40 ml). The org. layers were dried (MgSO4) and evaporated under reduced pressure.
The crude product was purified by FC (silica gel; MeOH/AcOEt 1 :10) yielding an orange oil, which was
immediately converted to the oxalate by treatment with a soln. of oxalic acid in acetone. The collected solid was
washed with boiling EtOH to afford 30 (1.05 g, 60%). Cream-colored solid. M.p. 173 ± 1748 (dec.) (DSC).
1H-NMR ((D6)DMSO): 8.22 (m, 4 H); 7.74 ± 8.13 (m, 5 H); 7.28 (m, 2 H); 6.98 (s, 1 H); 4.47 (m, 2 H); 3.75
(s, 2 H); 3.58 (m, 2 H); 3.33 (m, 2 H); 2.82 (m, 2 H). 13C-NMR ((D6)DMSO): 167.66; 165.52; 159.38; 158.95;
155.77; 148.10; 138.61; 136.28; 117.84; 110.75; 107.40; 69.48; 40.88; 39.80; 30.83; 30.19. Anal. calc. for
C19H22N10O5S3 ´ C2H2O4 (656.7): C 38.41, H 3.68, N 21.31; found: C 38.70, H 3.83, N 21.33.
1-[2-({2-[(Diaminomethylidene)amino]thiazol-4-yl}methylthio)ethyl]-3-{2-[3-(phenylsulfonyl)furazan-4-
yloxy]ethyl}guanidine-2-carbonitrile (32). NaH (80% in mineral oil; 0.10 g, 3.36 mmol) was washed with abs.
Et2O (2 Â 5 ml), decanted, and the solid was dissolved in dry DMF (2 ml). A soln. of 25 (1.00 g, 2.92 mmol) in
dry DMF (4 ml) was slowly added to the NaH soln. and, after evolution of gas ceased, 3,4-bis(phenylsulfo-
nyl)furazan (31) (1.07 g, 3.07 mmol) was added. The mixture was stirred for 3 h at r.t., then poured into crushed
ice and filtered. The deliquescent yellow solid was purified by FC (silica gel; MeOH/CH2Cl2 1:10) to afford 32
(1.01 g, 62%). White foam. The product was characterized as the oxalate. M.p. 1288 (dec.) (DSC) (MeCN).
1H-NMR ((D6)DMSO): 8.05 ± 7.52 (m, 9 H); 7.50 (m, 2 H); 6.83 (s, 1 H); 4.50 (m, 2 H); 3.71 (s, 2 H); 3.60
(m, 2 H); 3.34 (m, 2 H); 2.63 (m, 2 H). 13C-NMR ((D6)DMSO): 164.83; 164.52; 161.07; 159.34; 155.21; 148.86;
148.29; 137.00; 136.26; 117.74; 108.55; 71.99; 40.82; 39.91; 30.60; 30.18. Anal. calc. for C19H22N10O4S3 ´ C2H2O4
(640.7): C 39.37, H 3.78, N 21.86; found: C 39.43, H 3.81, N 21.59.
Quantitative Nitrite Detection. A soln. of the appropriate compound (20 ml) in DMSO was added to 2 ml of
50 mm phosphate buffer (pH 7.4), or to 2 ml of 50 mm phosphate buffer containing 5 mm l-cysteine,
4
respectively. The final concentration of the drug was 10 m. After 1 h at 378, 1 ml of this mixture was treated
with 250 ml of Griess reagent (4 g sulfanilamide, 0.2 g N-naphthylethylenediamine bis(hydrochloride), 10 ml
85% phosphoric acid in distilled H2O (final volume: 100 ml)). After 10 min at r.t., absorbance was measured at
540 nm; 10 ± 80 nmol/ml NaNO2 standard solns. were used for the calibration curve. The nitrite yield was
expressed in % NO2 (mol/mol) Æ standard deviation.
Kinetics of NO Release. The rate of NO release was determined by a spectrophotometric technique based
on the oxidation of oxyhemoglobin (HbO2) to methemoglobin (MetHb). The formation of MetHb was
followed by recording the increase of absorbance (DA) at 401 nm on a Perkin-Elmer-l5 spectrophotometer in a
thermostated (378C) cuvette. The reaction was started by adding the drugs, dissolved in DMSO, to a 4 mm HbO2
soln. in 50 mm phosphate buffer (pH 7.4), containing l-cysteine (5-fold molar excess) and, if necessary, in its
5
absence (final drug concentration 10 m). HbO2 was prepared according to the method previously described
[30]. The increase of absorbance (DA) was recorded over the first 3 min. The initial rates were calculated from
the slope of the straight-line portion of each curve. Every NO-releasing rate is the average of at least three
determinations. The molar extinction coefficient De ˆ e401(MetHb) e401(HbO2) ˆ 39.9 (Æ1.4) mm 1 cm 1 was
used [30].

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Helvetica Chimica Acta ± Vol. 83 (2000)
Guinea-Pig Papillary Muscle. The procedure previously described was followed [31]. After removing the
heart, left papillary muscles were set up at 378 in a 10-ml organ bath filled with oxygenated (95% O2 and 5%
CO2) Ringer soln. of the following composition (mm): NaCl 153.98; NaHCO3 5.95; KCl 5.63; CaCl2 ´ 2 H2O 2.16;
glucose 5.55. Two Pt electrodes, inserted into the ventricular basis of the papillary muscle, were used to drive the
tissue electrically. Square-wave pulses of 2 Hz frequency, 1 ms duration and 20% above-threshold voltage were
applied. Changes in force of contraction were recorded by an isometric transducer connected to a plotter.
Before new compounds were applied, the tissue was allowed to equilibrate for ca. 60 min under a load of 1 g.
Then, cumulative concentration-response curves (CRC) to histamine were constructed by increasing the doses
by 0.5 log units after each response had reached a plateau (3 ± 4 min). For antagonist studies, the first CRC to
histamine was followed by incubation of antagonist for 20 min before a second CRC was obtained. For
antagonists which produced surmountable antagonism, linear regression analysis was applied, and pA2 values
(95% confidence limits) and the slope of the Schild plot were obtained [32]. For this purpose, at least three
antagonist concentrations were used. For compounds which caused surmountable antagonism only at one or two
concentrations, pA2 values were estimated by Gaddumꢀs equation: pA2 ˆ log [B] ‡ log [CR 1], where [B]
represents the concentration of the antagonist and CR is the concentration ratio at the EC50 level. pA2 Values
were reported as geometric means with 95% confidence limits.
Lumen-Perfused Stomachs of Anesthetized Rats. The technique developed by Bertaccini et al. [33] was
followed. Male rats (Wistar, 200 ± 250 g) were used after 18 h fasting. After urethane anesthesia (1.25 g/kg, i.p.
administration), the stomach was perfused at constant volume (60 ml/h) with saline (0.9% NaCl) at 378 through
an esophageal cannula. The perfusion fluid was collected via a duodenal cannula. The acid in the perfusate was
estimated every 10 min by titration to pH 7 with 10 mm NaOH with an automatic titrator system (Radiometer,
Copenhagen). Gastric-acid secretion was stimulated by
a continuous infusion (6 ml/h) of histamine
(20 mmol kg 1 h 1). Inhibitory compounds were administered in separate experiments by bolus injection at
the plateau of acid secretion induced by histamine. Acid-secretory responses were expressed as mean values Æ
SEM in mequiv. HCl kg 1 min 1. The inhibitory effects were expressed as % inhibition in comparison with
plateau levels of acid output considered as 100%. The ID50 values were calculated from inhibitory dose-response
curves and reported as geometric means with 95% confidence limits.
Gastroprotection. Male Wistar rats (180 ± 200 g) were used after 24 h fasting. The new compounds or saline
were administered intragastrically in a 10-ml/kg volume; 30 min later, 0.6n HCl (1 ml/rat), was given
intragastrically. The animals were killed by cervical dislocation 30 min after HCl administration. Stomachs were
opened along the lesser curvature and examined under a stereomicroscope. Each individual hemorrhagic lesion
was measured along its greatest length (<1 mm: rating of 1; 1 ± 2 mm: rating of 2; >2 mm: rating according to
length in mm). The overall sum was designed as ꢁlesion indexꢀ. The results were expressed as % reduction of
lesion index compared to controls.
Statistics. Statistical significance was determined by the Studentꢀs t-test for unpaired data, or ANOVA
followed by the Newman-Keuls test.
REFERENCES
[1] ꢁPharmacology of Histamine Receptorsꢀ, Eds. C. R. Ganellin, M. E. Parsons, Wright PSG, Bristol 1992.
[2] A. Walan, J. P. Bader, C. B. H. W. Lamers, D. W. Piper, K. Rutgersson, S. Eriksson, New Eng. J. Med. 1989,
320, 69.
[3] I. Murakami, H. Satoh, S. Asano, R. Maeda, Jpn. J. Pharmacol. 1996, 72, 137.
[4] A. Robert, C. Lancaster, J. P. Davis, S. O. Field, J. E. Nezamis, Scand. J. Gastroenterol. 1989, 19 (Suppl.
101), 69.
[5] J. M. Scheiman, Drugs of Today 1997, 33, 655.
[6] J. F. Kerwin, Jr., J. R. Lancaster, Jr., P. I. Feldman, J. Med. Chem. 1995, 38, 4343.
[7] J. L. Wallace, D. N. Granger, FASEB J. 1996, 10, 731.
[8] G. Sorba, A. Gasco, G. Coruzzi, M. Adami, C. Pozzoli, G. Morini, G. Bertaccini, Arzneim.-Forsch./Drug
Res. 1997, 47(II), 849.
[9] R. L. Webb, C. S. Labaw, J. Heterocycl. Chem. 1982, 19, 1205.
[10] A. R. Butler, P. Rhodes, Anal. Biochem. 1997, 249, 1.
[11] L. Field, R. V. Dilts, R. Ravichandran, P. G. Lenhert, G. E. Carnahan, J. Chem. Soc., Chem. Commun. 1978,
249.
[12] H. A. Moynihan, S. M. Roberts, J. Chem. Soc., Perkin Trans. 1 1994, 797.

Helvetica Chimica Acta ± Vol. 83 (2000)
299
[13] J. Hirschfeld, A. Buschauer, J. Elz, W. Schunack, M. Ruat, E. Traiffort, J. C. Schwartz, J. Med. Chem. 1992,
35, 2231.
[14] G. Sorba, G. Ermondi, R. Fruttero, U. Galli, A. Gasco, J. Heterocycl. Chem. 1996, 33, 327.
[15] R. Fruttero, G. Sorba, G. Ermondi, M. Lolli, A. Gasco, Farmaco 1997, 52, 405.
[16] W. R. Tracey, Neuroprotocols 1992, 1, 125.
[17] E. Noack, D. Kubitzek, G. Kojda, Neuroprotocols 1992, 1, 133.
[18] H. Schröder, Adv. Drug Res. 1996, 28, 253.
[19] D. L. H. Williams, J. Chem. Soc., Chem. Commun. 1996, 1085.
[20] J. McAninly, D. L. H. Williams, S. C. Askew, A. R. Butler, C. Russel, J. Chem. Soc., Chem. Commun. 1993,
1758.
[21] A. P. Dicks, H. R. Swift, D. L. H. Williams, A. R. Butler, H. H. Al Saꢀdoni, B. G. Cox, J. Chem. Soc., Perkin
Trans. 2 1996, 481.
[22] L. B. Romanova, M. E. Ivanova, D. A. Nesterenko, L. T. Ermenenko, Russ. Chem. Bull. 1994, 43, 1207.
[23] J. Barbiere, Bull. Soc. Chim. Fr. 1944, 11, 470.
[24] A. Buschauer, S. Postius, I. Szeleny, W. Schunack, Arzeneim.-Forsch./Drug Res. 1985, 35, 25.
[25] T. O. Yellin, D. J. Gilman, J. D. Fleet, J. M. Wardleworth, Ger. Offen. 2.817.07802, Nov. 1975 (CA: 1979, 90,
87452d).
[26] A. Buschauer, I. Kramer, W. Schunack, Arch. Pharm. (Weinheim) 1986, 319, 434.
[27] F. R. Schulze, R. A. Alisch, A. Buschauer, W. Schunack, Arch. Pharm. (Weinheim) 1994, 327, 455.
[28] W. V. Farrar, J. Chem. Soc. 1964, 604.
[29] D. Boschi, A. Di Stilo, C. Cena, M. Lolli, R. Fruttero, A. Gasco, Pharm. Res. 1997, 14, 1750.
[30] G. Sorba, C. Medana, R. Fruttero, C. Cena, A. Di Stilo, U. Galli, A. Gasco, J. Med. Chem. 1997, 40, 463; ibid.
1997, 40, 2288.
[31] G. Bertaccini, G. Coruzzi, Naunyn-Schmiedebergꢀs Arch. Pharmacol. 1981, 317, 225.
[32] O. Arunlakshana, H. O. Schild, Br. J. Pharmacol. 1959, 14, 48.
[33] G. Bertaccini, R. Endean, V. Ersparmer, M. Impicciatore, Br. J. Pharmacol. 1968, 34, 311.
Received September 1, 1999