Synthesis and analgesic activity of a series of new azaalkane bis-guanidinium and bis(2-aminoimidazolinium) compounds

Article abstract of DOI:10.1016/S0968-0896(02)00637-5

In the present paper, we wish to report the synthesis and antinociceptive activity of a series of new azaalkane bis(2-aminoimidazolinium) compounds from which, N,N′-di(4,5-dihydro-1H-imidazol-2-yl)-3-aza-1,6-hexanediamine 2a has shown the best analgesic p

Full text of DOI:10.1016/S0968-0896(02)00637-5

Bioorganic & Medicinal Chemistry 11 (2003) 1283–1291
Synthesis and Analgesic Activity of a Series of New Azaalkane
Bis-guanidinium and Bis(2-aminoimidazolinium) Compounds
Christophe Dardonville,a,* Isabel Rozas,b Pilar Goya,a Rocıo Giron,c
Carlos Goicoecheac and Ma Isabel Martınc
a
´
´
Instituto de Quımica Medica (CSIC). Juan de la Cierva 3, 28006-Madrid, Spain
bDepartment of Chemistry, Trinity College Dublin, Dublin 2, Ireland
c
´
Unidad de Farmacologıa, Facultad de Ciencias de la Salud, Universidad Rey Juan Carlos,
´
28922-Alcorcon, Madrid, Spain
Received 2 August 2002; accepted 29 November 2002
Abstract—In the present paper, we wish to report the synthesis and antinociceptive activity of a series of new azaalkane bis(2-
aminoimidazolinium) compounds from which, N,N0-di(4,5-dihydro-1H-imidazol-2-yl)-3-aza-1,6-hexanediamine 2a has shown the
best analgesic properties in vivo in two different assays (i.e., acetic acid-induced writhing test and hot-plate test in mice), as well as
oral bioavailability.
# 2003 Elsevier Science Ltd. All rights reserved.
Introduction
of the NSAIDs or respiratory depression, tolerance
and dependence in the case of opioids. The mechan-
Pain is a problem not yet solved by Medicine. Accord-
ingto the World Health Organization, 90% of diseases
are associated with pain. Studies from the International
Association for the Study of Pain (IASP) show that pain
is reported in 50% of cancer patients (all stages) and in
75% of patients with advanced tumours. In addition,
accordingto IASP, in Denmark, researchers found that
approximately 38% of the general population suffer
from chronic pain,1 and the yearly costs of back pain
and sciatica in the UK are E9 billion, with E1 billion
spent each year on direct health care costs.2 Moreover,
there are certain kinds of pain, such as the neuropathic
pain associated to amputations, which at present have
no adequate treatment. Now, if we take into account
the analgesics being used nowadays, those of reference
continue to be acetylsalicylic acid and morphine, both
isolated in the 19th century.
ism of pain transmission is very complex and around
10–15 neuromodulators of the pain response have
been found in different studies. Thanks to these
investigations, new approaches to this problem are
3
beingconsidered.
In our search for new molecules with analgesic activity,
we discovered the lead compound 2a (Scheme 1) show-
ingan interestingantinociceptive activity in the acetic
acid-induced writhingtest in mice (73.9 and 63.9%
inhibition at 100 mg/kg ip and po, respectively) as well
as in the hot-plate test (50% inhibition at 100 mg/kg ip).
We decided to prepare a series of simple analogues of
this lead in order to investigate the structure–activity
relationships (SARs) of this kind of azaalkane deriva-
tives. Thus, the cationic 2-aminoimidazolinium moiety
was changed for a guanidinium cation which is very
similar structurally and electronically4 (molecule 2b),
the alkane chain was lengthened (1a) or shortened (3a)
with one methylene group, the secondary amino group
was replaced by a methylene group (4a) or substituted
by a phenylethyl (5a) or 3(2-ethyl)indole moiety (6a).
Moreover, a conformationally restricted analogue was
prepared (13) in order to investigate the active con-
formation of this kind of molecules and finally, a mono-
2-aminoimidazolinium derivative was synthesised (14)
The classical therapies for pain relief consist mainly in
the use of Non-Steroidal Anti-inflammatory Drugs
(NSAIDs) and opiates. Both families show quite ser-
ious secondary effects such as renal toxicity in the case
*Correspondingauthor at current address: Welsh School of Phar-
macy, Cardiff University, Redwood building, CF10 3XF Cardiff, UK.
Fax: +44-29-2087-4149; e-mail: dardonvillec@cf.ac.uk
0968-0896/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0968-0896(02)00637-5

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C. Dardonville et al. / Bioorg. Med. Chem. 11 (2003) 1283–1291
Scheme 1. (a) 2-Methylmercapto-4,5-dihydroimidazole iodide, MeOH, reflux.
and tested to assess the importance of the two cationic
groups at the extremes of the molecules.
methanol. The compounds were purified either by direct
crystallisation of the hydroiodide salt (3a, 4a7, 14), or by
formation of their picrate salt (1a, 2a, 6a, 13) or oxalate
salt (5a), when direct isolation of the iodide salt from
the reaction mixture was unsuccessful. In the case of the
purification by formation of the picrate salts, regener-
ation of the biologically compatible and water soluble
hydrochloride salts was carried out with a strongly basic
anion exchange resin (IRA400/Clꢀ form), yieldingafter
lyophilisation the hydrochloride salts as highly hygro-
scopic solids.
In the present article, we report the synthesis and anti-
nociceptive activity of the lead compound 2a as well as
the synthesis and analgesic activity (acetic-induced wri-
thingtest in mice) of seven 2-aminoimidazolinium ana-
logues and three guanidinium analogues of this
compound. Moreover, the affinity of the lead compound
(2a) for different receptors was investigated in order to
explore its mechanism of action.
The non-commercial N-substituted amines 5 and 6 were
prepared as described in Scheme 3. The polyamine 2
was selectively protected on the primary amino groups
with benzylcyanoformate,8 giving 8 in good yield. The
free secondary amino group of 8 was alkylated with
2-bromoethylbenzene and 3(2-bromoethyl)indole in
presence of K2CO3 to give compounds 9 and 10,
respectively. Hydrogenolysis of these compounds with
Pd/C 10% in MeOH yielded the correspondingamines
5 and 6.
Chemistry
The guanidinium derivatives 1b and 2b have been
described elsewhere.5 Followingthe same methodology
involvingthe nucleophilic substitution of S-methyli-
sothiourea by the correspondingprimary amines, we
also prepared 3b (Scheme 1). The compounds were
purified by crystallisation and isolated as their sulphate
salts.
For the 2-aminoimidazolinium derivatives 1a–6a
(Scheme 1) and 14 (Scheme 2), a similar procedure was
followed using2-methylmercapto-4,5-dihydroimidazole
iodide6 for the reaction with diamines 1–6 in refluxing
The synthesis of the conformationally restricted ana-
logue 13 is depicted in Scheme 4. The 1-(4-piperi-
dyl)piperazine skeleton was formed by reductive
amination of N-Boc piperidone with N-Boc piperazine
and sodium cyanoborohydride9 at pH 7, giving 11a in
65% yield. The Boc groups were removed easily with
TFA to give 11b as its trifluoroacetate salt quanti-
tatively. Introduction of the imidazolinium cations into
the molecule 11b with 2-methylmercapto-4,5-dihydro-
imidazole iodide in refluxingethanol as described above
(Scheme 1) was not successful and only startingmaterial
Scheme 2. (a) 2-methylmercapto-4,5-dihydroimidazole iodide, MeOH,
reflux; (b) picric acid, H2O; (c) IRA400 (Clꢀ) basic exchange resin,
H2O–THF; (d) S-methylisothiouronium sulfate, CH3CN, reflux.

C. Dardonville et al. / Bioorg. Med. Chem. 11 (2003) 1283–1291
1285
Scheme 3. (e) benzylcyanoformate, CH2Cl2 (79%); (f) K2CO3, CH3CN, reflux; (g) H2/Pd/C 10%, MeOH.
Scheme 4. (h) NaBH3CN, AcOH, MeOH, pH 7 (65%); (i) TFA, CH2Cl2; (j) THF, rt, 2 days, (50%); (k) EtI, EtOH, 60 ꢂC; (l) NaOEt, EtOH, rt.
was recovered. Hence, another way to generate 2-amino-
imidazolinium moiety was attempted, in particular the
method described by Anslyn et al.10 that involves the five-
member ringintramolecular cyclisation of a S-alkyl-
isothiouronium salt with an amine. Thus, the free base
of 11b reacted with two equivalents of the commercially
available [(tert-butoxycarbonyl)amino]ethyliso thiocya-
nate to yield the thiourea 12. The next three steps were
conducted in a one-pot reaction in the followingman-
ner. The first step consisted in the alkylation of the
thiourea with 2-iodoethanol to form a thioethanol-
leavinggroup. This intermediate was not isolated and
was treated with TFA to remove the Boc protecting
groups and generate an intramolecular nucleophile that
could cyclise in basic conditions, namely with sodium
ethoxide, to yield the free base of compound 13. The
product 13 was purified by formation of its picrate salt
and then isolated as its hydrochloride salt.
2. Morphine was used as reference compound: its anti-
nociceptive effect was evaluated (1 mg/kg: 16ꢁ2.8%,
2.5 mg/kg: 44.2ꢁ7.7%; 3.5 mg/kg: 79.6ꢁ2.8%; 5 mg/
kg: 95.6ꢁ1.7%; 10 mg/kg: 100%) and its ED50 value
was calculated (Table 2, footnote).
Compounds 2a, 2b, 3a, 3b and 4a showed a dose-
dependent inhibition of the acetic acid-induced con-
tractions at 25, 50 and 100 mg/kg, with the lead com-
pound 2a havinga maximum effect of 100% inhibition
at 150 mg/kg. The toxicity of compounds 2b and 4a
appeared at 100 mg/kg with behavioural changes for 2b
and a death rate above 50% for 4a at this dose. In con-
trast, no behavioural changes or visible signs of toxicity
appeared for compounds 2a, 3a and 3b at the doses
tested.
Since the best activity found was that of the lead com-
pound 2a, the oral bio-availability of that compound
Table 1. Analgesic activity screening of azaalkane bis-cationic
derivatives
Pharmacology
The analgesic activity of all the compounds was eval-
uated with the acetic acid-induced writhingtest in
mice.11 The compounds were first screened at an intra-
peritoneal (ip) dose of 50 mg/kg (Table 1). At this dose,
four compounds (1a, 5a, 6a and 13) were toxic (>50%
death), one compound showed no analgesic activity (14)
and five derivatives (2a, 2b, 3a, 3b, 4a) had a fair anti-
nociceptive activity (>25% inhibition at 50 mg/kg).
Compd
Acetic acid-induced writhingtest (mice)
% inhibition at 50 mg/kg ip
1a
2a
2b
3a
3b
4a
5a
6a
13
14
Toxic @ 25
60.9ꢁ2
47.4ꢁ1.5
50.4ꢁ1.3
30ꢁ1.6
24.7ꢁ2.4
Toxic @ 50
Toxic @ 50
Toxic @ 25
0
The active molecules were further tested at different
doses to assess their analgesic profile, and their ED50
values were calculated. The results are reported in Table

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C. Dardonville et al. / Bioorg. Med. Chem. 11 (2003) 1283–1291
Table 2. Analgesic activity profile (ip) and ED50 values of compounds 2a, 2b, 3a, 3b and 4a
c
Compd
% Inhibition of acetic acid-induced writhes in mice
ED50
10 mg/kg
25 mg/kg
50 mg/kg
100 mg/kg
150 mg/kg
100
mg/kg (confidence interval)
2a
2b
3a
3b
4a
30ꢁ1.4
34.8
60.9ꢁ2
73.9ꢁ1.8
82.2 ꢁ1.4
55.3ꢁ1.6
53 ꢁ1.9
30.69 (16.47–57.19)
54.91 (7.897–381.8)
53.14 (19.72-143.20)
110.0 (67.54–179.20)
a
a
—
—
12.8ꢁ4.1
40.2ꢁ1
8.4ꢁ4.2
25.7ꢁ1.7
47.4ꢁ1.5
50.4ꢁ1.3
30ꢁ1.6
—
—
a
a
a
—
ꢀ18.5ꢁ1.8
55ꢁ1.7b
a
24.7ꢁ2.4
15.5 ꢁ2.9
—
aDosis not tested for these compounds.
bDosis of 125 mg/kg.
cReference: morphine: ED50=1.8 mg/kg (0.53–6.25).
was tested. Compound 2a, when orally administered
(po), showed a dose-dependent inhibition of writhes of
14% at 25 mg/kg, 30.9% at 50 mg/kg, and 63.9% at 100
mg/kg, with a similar profile to that of the ip admini-
stration and a maximum effect of 75.5% inhibition at
150 mg/kg.
Regarding the length of the alkane chain, the addition
of one methylene group (1a) provokes a high toxicity
whereas the shorteningin one methylene carbon ( 3a)
reduces slightly the activity without inducing toxicity.
Regarding the nature of the cationic group, the 2-amino-
imidazolinium moiety seems to give slightly better
activity than the guanidinium one (Table 1, 2a vs 2b and
3a vs 3b). The presence of two terminal cationic groups
seems to be indispensable as reflected by the lack of
activity of the mono-2-aminoimidazolinium compound
14. Finally, the presence of a non-substituted secondary
amino group in the alkylic linker appears to be impor-
tant to avoid the toxicity in this family of azaalkane
compounds (see the toxicity of derivatives 4a, 5a, 6a and
13).
The most active compound 2a was also tested usinga
second test: the hot-plate test in mice. The mean per-
centage of the Maximum Possible Effect (M.P.E.; the
formula for the calculation of the M.P.E. is defined in
the Experimental) induced by the ip administration of
2a was: 25ꢁ4.2% at 50 mg/kg and 50ꢁ9.7% at 100
mg/kg. As a comparison, the mean percentage of
M.P.E. for morphine was 41ꢁ3.2% at 5 mg/kg in this
assay.
Concerningthe mechanism underlyingthe analegsic
effect of 2a, functional tests and bindingassays permit
to discard direct interaction with a wide number of
receptors enumerated above. It is interestingto remark
that interactions with opioid receptors could be dis-
carded because m and k activation induces dose-depen-
dent inhibition of the contractile electrically-induced
contractile response in GPI13 and d activation inhibits
the electrically induced contraction in MVD.14 This is of
great importance because activation of the opioid sys-
tem is one of the main and most common analgesic
mechanisms.
In order to investigate the possible mechanism of action
of 2a, the compound was screened accordingto stan-
dard protocols for bindingand inhibition of a range of
targets considered to be involved in pain,12 discarding
any affinity for Cyclooxygenase-2 (COX-2), Nitric
Oxide Synthase inducible (iNOS), Bradykinin B2,
Dopaminergic D4.2, Glutamate NMDA (agonist and
phencyclidine), Muscarinic (M1, M2, and M4), Nicotinic
Acetylcholine (central), Serotonin 5-HT1A and Tachy-
kinin NK1 receptors. Compound 2a was also tested in a
functional assay with guinea pig ileum (GPI) prepa-
rations. This molecule did neither provoke any con-
tractile response of the GPI, nor antagonize the
contractile response induced by acetylcholine, ser-
otonine, histamine, bradykinin or substance P. These
results suggest that the analgesic effect of 2a is not due
to a direct interaction with these receptors. Moreover,
the absence of action mediated by opioid receptors or
by a1-, a2-adrenergic receptors was confirmed by func-
tional tests on isolated mouse vas deferens (MVD) pre-
parations; 2a did not elicit contractile responses in this
tissue and was not able to modify the contraction
induced by electrical stimulation or by noradrenaline.
Conclusion
In this series of bis-guanidinium and bis(2-aminoimi-
dazolinium) azaalkane derivatives synthesised and tested
for their antinociceptive activity, the N,N0-di(4,5-dihy-
dro-1H-imidazol-2-yl)-3-aza-1,6-hexanediamine 2a has
shown to be the most potent compound. One of the
structural elements that seems to be important is the
nature of the cation, because the bis-guanidinium ana-
logue is toxic producing conduct changes in mice.
Moreover, the length of the aliphatic chain is rather
important. Thus, shorteningin one C atom diminishes
the activity, whereas the lengthening in one methylene
unit increases considerably the toxicity. Finally, the
secondary amine group seems to be essential because its
absence produces high toxicity. Besides, the introduc-
tion over such amine group of aromatic groups such as
phenethyl or 3(2-ethylindole) produces very high toxi-
city.
Discussion
From these results, a few conclusions can be drawn
regarding the SAR of compound 2a (Fig. 1). First of all,
in general, it seems that only few changes can be made
to the structure without losingthe analgesic activity or
increasingconsiderably the toxicity of the molecule.

C. Dardonville et al. / Bioorg. Med. Chem. 11 (2003) 1283–1291
1287
Figure 1. SAR studies on compound 2a.
Method A. Preparation of the bis(2-aminoimidazo-
linium) compounds 1a–6a
Concluding, compound 2a has shown the best analgesic
properties in vivo, as well as very good oral bio-avail-
ability. This derivative possesses potent non-opioid
antinociceptive activity the mechanism action of which
still remains to be determined.
A solution of 4.75 mmol of amine (1–6) and 2-methyl-
mercapto-4,5-dihydroimidazole iodide (10 mmol) of in
dry MeOH (15 mL) was heated under reflux for 2.5 h. A
current of nitrogen was bubbled into the solution to
remove CH3SH. The solvent was removed in vacuo and
the product was purified by formation of the picrate
salt. The crude residue dissolved in water (50 mL) was
treated with a hot aqueous solution of picric acid (10
mmol). The yellow picrate salt that precipitated was
washed successively with H2O, hexane, Et2O and crys-
tallized from hot CH3CN. The hydrochloride salts were
regenerated with Amberlyte IRA400 (Clꢀ form)
exchange resin. For this purpose, the picrate salt was
dissolved in H2O/THF (300 mL) and stirred with the
resin (previously washed) overnight. The resin was
removed by filtration and the solution concentrated in
vacuo. The oily residue was taken up in a little water
and washed twice with EtOAc. The product was lyo-
philised to afford the pure chloride salt.
Experimental
Chemistry
Reagents were used as received. 2-methylmercapto-4,5-
dihydroimidazole iodide was synthesised accordingto
the procedure described in ref 6. Reaction solvents were
purchased anhydrous and used as received. Chromato-
graphy was performed either with silica gel 60 PF254
(Merck, particle size 40–63 mm) or with the BiotageTM
TM
‘Flash 40’ system usingKP-Sil
or 40M cartridges
(particle size 32–63 mm, 60 A). CAUTION: the noxious
gas CH3SH is evolved duringthe synthesis and it should
be trapped with a concentrated aqueous solution of
NaOH and then destroyed with sodium hypochlorite.
Other solvents used were reagent grade. 1H and 13C
NMR spectra were recorded at 200 and 50 MHz,
respectively, on a Varian Gemini 200 spectrometer.
1
Chemical shifts of the H NMR spectra were internally
N,N0-Di(4,5-dihydro-1H-imidazol-2-yl)-4-aza-1,7-hepta-
nediamine (1a). Picrate salt: yellow solid (26% yield);
mp 188–189 ꢂC; IR (KBr) 3420, 3320, 3100, 1680, 1640,
referenced to the residual proton resonance in D2O (d
4.6 ppm) and in DMSO (d 2.49 ppm). Chemical shifts of
the 13C NMR spectra were referenced with a capilar of
DMSO-d6 (d 39.5 ppm). IR spectra were recorded on a
Perkin–Elmer 681 FTIR spectrophotometer as KBr
pellets. Meltingpoints were determined with a Reichert-
JungThermovar apparatus and are uncorrected. Mass
spectra were recorded on a Hewlett Packard Series
1100 MSD spectrometer (ES, APCI) and on a VG
Autospec spectrometer (FAB). Elemental analysis was
performed on a Heraeus CHN-O Rapid analyser. Ana-
lytical results were withinꢁ0.4% of the theoretical
values unless otherwise noted.
1610, 1340, 1280, 795, 755, 720 cmꢀ1
;
1H NMR
(DMSO-d6) d: 8.59 (s, 2H), 8.4–8.0 (m, 4H), 3.6 (s, 8H),
3.25–3.1 (m, 4H), 3.0–2.8 (m, 4H), 1.76 (quint, 4H).
Anal. calcd for C30H34N16O21: C, 37.74; H, 3.59; N,
23.47. Found: C, 37.97; H, 3.65; N, 23.65. Hydrochlo-
ride salt: slightly orange hygroscopic oily solid (33%);
IR (KBr): 1670, 1605, 1470, 1290, 1050 cmꢀ1; 1H NMR
(D2O) d: 3.5 (s, 8H); 3.11 (t, 4H, J=6.8 Hz); 2.89 (t, 4H,
J=7.6 Hz), 1.77 (quint 4H, J=7.1 Hz); 13C NMR
(D2O) d: 158.9, 44.1, 41.7, 38.5, 24.5; MS (ES+) m/e:
268 (MH+, 100%), 303 (M+HCl).

1288
C. Dardonville et al. / Bioorg. Med. Chem. 11 (2003) 1283–1291
N,N0-Di(4,5-dihydro-1H-imidazol-2-yl)-3-aza-1,6-hexane-
diamine (2a). Picrate salt: yellow solid; mp 210–212 ꢂC;
IR (KBr) 3440, 3360, 3260,1660, 1620, 1575, 1535, 1345,
1315, 1255, 890, 770, 725, 690 cmꢀ1; 1H NMR (DMSO-
d6) d: 8.59 (s, 2H), 8.5–8.0 (m, 8H), 3.6 (d, 8H), 3.4-2.8
(m, 8H), 1.77 (quint, 2H); 13C NMR (DMSO-d6) d:
160.9; 159.3; 159.3; 141.8; 125.3; 124.4; 45.7; 44.6; 42.6;
42.5; 25.5; MS (ES+) 254 (MH+). Anal. calcd for
C29H36N16O21: C, 36.87; H, 3.84; N, 23.72. Found: C,
36.64; 3.55; 23.48. Hydrochloride salt: yellowish hygro-
low oil. The solvent was removed from the flask, Et2O
was added and the residue was triturated with a glass
rod until a solid crystallised. The product was recrys-
tallised from MeOH/Et2O followingthe same proce-
dure, and dried in vacuo (45 ꢂC) to afford 5a as an
hygroscopic yellow solid (50%); Dihydroiodide mono-
oxalate: Pf>90 ꢂC (dec); IR (KBr): 1643, 1575, 1260,
695, 668 cmꢀ1; 1H NMR (D2O) d: 7.5–7.2 (m, 5H); 3.70
(s, 4H); 3.68 (s, 4H); 3.6–3.0 (m, 12H); 2.0 (m, 2H); 13C
NMR (D2O) d: 168.9; 160.3; 136.5; 129.8; 129.5; 128.1;
54.5; 52.3; 51.4; 43.4; 43.3; 40.1; 38.0; 37.1; 30.0; LRMS
(ES+) m/e: 358 (MH+); 486 ([M+HI]). Anal. calcd for
scopic solid (66%); mp 194–197 ꢂC; H NMR (D2O) d:
1
3.59 (s, 4H); 3.56 (s, 4H); 3.48 (t, 2H, J=5.9 Hz), 3.3–
3.1 (m, 4H); 1.88 (quint, 2H, J=7.8 Hz); 13C NMR
(D2O) d: 159.25, 159.13, 45.6, 44.7, 42.2, 42.1, 38.9,
38.3, 24.7; MS (FAB+) m/e: 254 (MH+, 100%), 290.2
(M+HCl, 32%), 322 (M+2HCl). Anal. calcd for
C11H26N7Cl3/H2O: C, 34.70; H, 7.41; N, 25.75. Found:
C, 35.11; H, 7.25; N, 25.72.
.
C19H31N7I2 1/2C2H2O4: C, 36.60; H, 4.76; N, 14.94.
Found: C, 36.53; H, 4.96; N: 12.85.
3-(2-Ethylindol-3-yl)-3-aza-1,6-hexanediamine (6).
A
mixture of 10 (830 mg, 1.57 mmol), Pd/C 10% (360 mg)
in MeOH (50 mL) was shaken under H2 (33 Psi) for 18
h. After filtration and evaporation of the solvent, 6 was
N,N0-Di(4,5-dihydro-1H-imidazol-2-yl)-3-aza-1,5-penta-
nediamine (3a). The product was isolated directly as the
hydroiodide salt from the reaction mixture in the fol-
lowingmanner: the solvent was removed in vacuo and
obtained as a colourless oil (408 mg, 100%); H NMR
1
(D2O) d: 7.5–7.2 (m, 2H); 7.1–6.8 (m, 3H); 2.9–2.1 (m,
12H); 1.56 (quint, 2H); 13C NMR (D2O) d: 154.9; 141.3;
131.9; 128.5; 127.1; 124.4; 123.8; 117.2; 58.3; 55.3; 54.7;
42.98; 41.5; 28.6; 26.3; LRMS (ES+) m/e: 261 (MH+).
i
the resultingthick oily residue dissolved in hot PrOH (5
mL) was triturated with a glass rod. The yellow solid
that formed was recrystallized successively from PrOH
and EtOH to give 3a as a white solid (20%); mp 145–
146 ꢂC. Hydroiodide salt: IR (KBr) 3300–3000, 1640,
N,N0-Di(4,5-dihydro-1H-imidazol-2-yl)-3-(2-ethylindol-3-
yl)-3-aza-1,6-hexanediamine (6a). Method A, starting
from 6 (220 mg, 0.85 mmol), 2-methylmercapto-4,5-di-
hydroimidazole iodide (413 mg, 1.69 mmol) in EtOH (3
mL) for 24 h; Monopicrate monoiodide salt: yellow solid
i
1
1560, 1265 cmꢀ1; H NMR (D2O) d: 3.61 (s, 8H,); 3.24
(t, 4H, J=6.2 Hz); 2.7 (t, 4H, J=6.2 Hz); 13C NMR
(D2O) d: 162.63, 47.35, 43.13, 42.32. Anal. calcd for
C10H23N7I2: C, 24.26; H, 4.68; N, 19.80. Found: C,
24.50; H, 4.69; N, 19.54.
ꢂ
1
(573 mg; 92%); mp 95–105 C (>118 dec.); H NMR
(DMSO-d6) d: 11.01 (s, 1H): 9.45 (bs, 1H); 8.59 (s, 2H);
8.4–8.0 (m, 3H); 7.56 (d, 1H, J=6.8 Hz); 7.38 (d, 1H,
J=7.4 Hz); 7.26 (s, 1H); 7.07 (q, 2H); 3.62 (bd, 8H);
3.5–2.8 (m, 14H), 1.91 (m, 2H); LRMS (ES+) m/e: 397
(MH+/100%). Anal. calcd for C27H34 I N11O6: C,
44.08; H, 4.66; N, 20.95. Found: C, 43.35; H, 4.31; N,
20.81. Trihydrochloride salt: yellow hygroscopic solid
N,N0 -Di(4,5-dihydro-1H-imidazol-2-yl)-1,6-hexanedia-
mine (4a).7 The product was isolated as the hydro-
iodide salt by crystallisation from MeOH/Et2O: white
solid (26%); mp 197–199 ꢂC; H NMR (D2O) d: 3.48(s,
1
1
8H); 2.99 (t, 4H, J=6.8 Hz); 1.5–1.25 (m, 4H); 1.25–
1.00 (m, 4H); 13C NMR (D2O) d: 158.9, 41.7, 41.5, 27.2,
24.5; MS (APCI+) m/e: 253 (MH+). Anal. calcd for
C12H26N6I2: C, 28.36; H, 5.16; N, 16.54. Found: C,
28.43; H, 5.02; N, 16.64.
(74%); IR (KBr): 1633; 1017; 992; 970 cmꢀ1; H NMR
(D2O) d: 7.61 (d, 1H, J=7.4 Hz); 7.45 (d, 1H, J=7.6
Hz); 7.28 (s, 1H); 7.14 (q, 2H); 3.7–3.0 (m, 20H); 1.92
(br, 2H); 13C NMR (D2O) d: 160.1; 137.0; 126.7; 124.8;
122.9; 120.2; 118.6; 112.7; 108.8; 53.7; 52.4; 51.6; 43.3;
40.0; 37.9; 23.6; 20.6. Anal. calcd for C21H34N8Cl3/
4.5H2O: C, 42.97; H, 7.55; N, 19.09. Found: C, 42.90;
H, 7.27, N, 18.26.
3-(2-Aminoethylamino-2-phenethyl)propylamine (5).
A
mixture of 9 (590 mg, 1.2 mmol) and Pd/C 10% (55 mg)
in MeOH (50 mL) was shaken under H2 (20 Psi) for 16
h. After filtration and evaporation of the solvent, 5 was
N,N00 -Ddi(benzyloxycarbonyl)-3-aza-1,6-hexanediamine
(8). A two-neck flask was charged with 3-(2-amino-
ethylamino)propylamine 2 (1.814 g, 15.48 mmol) dis-
solved in dry CH2Cl2 (40 mL). Caution: the reaction
should be carried out with a positive pressure of nitro-
gen and one neck of the flask connected to a trap con-
taininga concentrated aqueous NaOH solution to trap
cyanhydric acid that is evolved duringthe reaction.
Benzyl cyanoformate (5 g, 31 mmol) dissolved in dry
CH2Cl2 (100 mL) was added dropwise at room tem-
perature over a 7 h period. The reaction was stirred
another 15 h at room temperature. The solvent was
removed in vacuo and the product was purified by flash
chromatography on silica with CHCl3/MeOH (3/1). 8
was dissolved in a little CH2Cl2 and filtered on a path of
Celite to remove traces of silica. White solid (4.59 g,
1
obtained as a colourless oil (272 mg, 100%); H NMR
(CDCl3) d: 7.3–7.1 (m, 5H); 4.19 (br, 4H, NH2); 2.85–
2.40 (m, 12H); 1.65 (quint, 2H); 13C NMR (D2O) d:
141.0; 129.5; 129.4; 127.0; 55.7; 55.3; 51.7; 39.4; 38.1;
32.3; 27.5. Anal. calcd for C13H23N3/1.6H2O: C, 61.94;
H, 10.52; N, 16.67. Found: C, 62.20; H, 9.29; N, 16.67.
N,N0-Di-(4,5-dihydro-1H-imidazol-2-yl)-3-phenethyl-3-
aza-1,6-hexanediamine (5a). Followingmethod A, from
5 (270 mg, 1.2 mmol) and 2-methylmercapto-4,5-di-
hydroimidazole iodide (617 mg, 2.53 mmol) in MeOH
(10 mL) for 3.5 h. The solvent was removed in vacuo
and oxalic acid (108 mg, 1 equiv) was added to the
residue dissolved in a small quantity of MeOH. EtOAc
was added and the product precipitated as a thick yel-

C. Dardonville et al. / Bioorg. Med. Chem. 11 (2003) 1283–1291
1289
79%); mp 96–97 ꢂC (iPrOH); IR (KBr): 3280, 1665,
(s, 4H); 3.24 (t, 2H, J=6.8 Hz); 3.03 (t, 2H, J=7.7
Hz); 2.95 (t, 2H, J=7.5 Hz); 1.91 (quint, 2H, J=7.5
Hz); 1.6 (sext, 2H); 0.89 (t, 3H, J=7.5 Hz); 13C NMR
(D2O) d: 174.2; 162.6; 52.3; 47.7; 45.7; 42.6; 28.3; 22.2;
13.4; LRMS (ES+) m/e: 185 (MH+/100%). Anal.
calcd for C10H22N4O2I: C, 33.61; H, 6.20; N, 15.68.
Found: C, 33.98; H, 5.78; N, 15.26.
1
1515, 1255, 1225 cmꢀ1; H NMR (CDCl3) d: 7.38–7.26
(m, 10H); 5.4 (b, NH); 5.33 (b, NH); 5.08 (s, 2H); 5.07
(s, 2H); 3.25 (bm, 4H); 2.69 (b, 2H); 2.63 (t, 2H, J=6.2
Hz); 1.62 (quint, 2H); 1.54 (b, NH); 13C NMR (CDCl3)
d: 156.6; 156.5; 136.7; 136.6; 128.4; 128.0; 66.6; 66.5;
48.9; 46.8; 40.5; 39.3; 29.8; LRMS (FAB+) m/e: 386
(MH+/100%). Anal. calcd for C21H27N3O4: C, 65.43;
H, 7.06; N, 10.90. Found: C, 65.72; H, 7.14; N,
11.14.
Method B. Preparation of bisguanidinium compounds 1b,
2b, 3b, 5b
N,N00 -Di(benzyloxycarbonyl)-3-phenylethyl-3-aza-1,6-
hexanediamine (9). A solution of 8 (1 g, 2.6 mmol),
bromoethylbenzene (0.5 mL, 3.6 mmol) and K2CO3
(180 mg, 1.3 mmol) in CH3CN (25 mL) was refluxed for
43 h. The reaction mixture was filtered on Celite and the
filter cake was rinsed with CH2Cl2. The solvent was
removed in vacuo and the product was purified by
chromatography on silica using CHCl3/MeOH (gra-
dient 70/1 to 20/1); colourless oil (1.062 g, 83%); IR
Followingthe procedure of Villaroya et al. A solution
5
of 3 mmol of diamine (1–3) and S-methylisothio-
uronium sulfate (3 mmol) of in 10 mL of dry CH3CN
was heated under reflux for 20 h. A white solid pre-
cipitated from the solution. The solid was washed with
cold CH3CN and recrystallised from EtOH/H2O (1/5).
1,7-Diguanidinium-4-azaheptane sulfate (1b). White
amorphous solid (24% yield); mp 275–279 ꢂC (Lit.5
280–283 ꢂC dec). Anal. calcd for C8H26N7O7S1.5: C,
25.25; H, 6.89; N, 25.77; S, 12.63. Found: C, 25.13; H,
6.87; N, 25.06; S, 11.75.
1
(neat): 3300; 1680; 1500; 1432; 1230; 715; 675 cmꢀ1; H
NMR (CDCl3) d: 7.4–7.1 (m, 15H); 5.68 (br, NH); 5.42
(br, NH); 5.14 (s, 4H); 3.21 (m, 4H); 2.71 (s, 4H); 2.55
(m, 4H); 1.61 (quint, 2H); 13C NMR (CDCl3) d: 156.92;
156.86; 140.6; 137.1; 129.1; 128.8; 128.77; 128.4; 126.4;
66.8; 66.7; 55.9; 53.6; 51.9; 39.9; 39.1; 33.8; 27.39. Anal.
calcd for C29H35N3O4: C, 71.14; H, 7.20; N, 8.58.
Found: C, 71.35; H, 7.04; N, 9.02.
1,6-Diguanidinium-3-azahexane sulfate (2b). White
amorphous solid (30% yield); mp: 244–248 ꢂC (Lit.5
192 ꢂC dec). Anal. calcd for C7H21N7O4S: C, 28.09; H,
7.07; N, 32.75; S, 10.71. Found: C, 28.35; H, 7.27; N,
32.77; S, 10.43.
N,N0-Di(benzyloxycarbonyl)-3-(2-ethylindol-3-yl)-3-aza-
1,6-hexanediamine (10). A solution of 8 (1.108 g, 2.88
mmol), 3(2-bromoethyl)indole (537 mg, 2.4 mmol) and
K2CO3 (331 mg, 2.4 mmol) in CH3CN (15 mL) was
refluxed for 21 h. The reaction mixture was filtered on
Celite and the filter cake was rinsed with CH2Cl2. The
solvent was removed in vacuo and flash chromato-
graphy on silica using a 40S cartridge (CH2Cl2/MeOH
5%) afforded 10 as an orange oil (1.035 g, 82%); IR
(neat): 3365; 3300; 3020; 2990; 2910; 1680; 1500; 1235;
1,5-Diguanidinium-3-azapentane sulfate (3b). White solid
(12%); mp: 229–232 ꢂC; H NMR (D2O/TFA) d: 3.09
1
(t, 4H, J=6 Hz); 2.58 (t, 4H, J=6 Hz); 13C NMR
(D2O/TFA) d: 150.8, 46.0, 40.0; MS (ES+) m/e: 286
(M+H2SO4, 100%), 188 (M+H). Anal. calcd for
C6H19N7O4S. H2O: C, 23.75; H, 6.98; N, 32.32; S,
10.57. Found: C, 23.90; H, 7.52; N, 32.21; S, 10.54.
1-(tert-Butoxycarbonyl)-4-[(1-tert-butoxycarbonyl)piperi-
din-4-yl]piperazine (11a). Sodium cyanoborohydride
(880 mg, 14 mmol) was added at once to a stirred solu-
tion of N-Boc-piperidone (1.407 g, 7 mmol) and N-Boc-
piperazine (1.447 g, 7.8 mmol) in MeOH (30 mL). The
pH of the solution was adjusted with CH3CO2H at ca. 7
(and checked duringthe course of the reaction) and the
reaction mixture was stirred at room temperature until
the N-Boc piperidone was consumed (48 h). The solvent
was removed in vacuo and the crude residue was parti-
tioned between CH2Cl2 and water. The aqueous phase
was extracted three times with CH2Cl2. Combined
organic extracts were washed with brine, dried
(Na2SO4) and concentrated in vacuo to afford 11a as a
white solid. The product was crystallised from hexane/
EtOAc at ꢀ20 ꢂC and cold filtered; white solid (1.669 g;
65%); mp 134–139 ꢂC; IR (KBr): 1685; 1670; 1410;
1215; 720; 675 cmꢀ1 1H NMR (CDCl3) d: 8.66 (br,
;
NH); 7.58 (d, 1H, J=7.2 Hz); 7.4–7.0 (d, 1H, J=1.6
Hz); 5.65 (br, NH); 5.47 (br, NH); 5.07 (s, 2H); 5.05 (s;
2H); 3.2–2.9 (m, 4H); 2.9–2.6 (m, 4H); 2.5–2.3 (m, 4H);
1.5 (m, 2H); 13C NMR (CDCl3) d: 156.96; 156.90; 137.0;
136.6; 128.7; 128.2; 127.6; 122.3; 121.9; 119.2; 118.8;
113.8; 111.7; 66.7; 66.6; 54.5; 53.5; 52.0; 39.9; 39.1; 27.1;
23.2; LRMS (ES+) m/e: 529 (MH+/100%). Anal. calcd
for C31H36N4O4/0.5H2O: C, 69.25; H, 6.75; N, 10.42.
Found: C, 69.06; H, 6.73; N, 10.35.
N-Propyl-N0-(4,5-dihydro-1H-imidazol-2-yl)-1,3-propa-
nediamine (14). Followingmethod A with N-propyl-
1,3-propanediamine (994 mg, 8.57 mmol) and 2-
methylmercapto-4,5-dihydroimidazole iodide (2.3 g,
9.42 mmol) in MeOH (15 mL). The product was iso-
lated as the oxalate salt. After the reaction finished, the
solvent was removed in vacuo and the crude residue
1
1355; 1230; 1155; 1110 cmꢀ1; H NMR (CDCl3) d: 4.3–
4.1 (br, 2H); 3.48 (br s, 4H); 2.7 (br t, 2H, J=12 Hz);
2.6–2.45 (m, 5H); 1.9–1.7 (m, 2H); 1.46 (br s, 20H); 13C
NMR (CDCl3/100 MHz) d: 154.7; 79.7; 79.5; 62.2;
49.1; 43.9 (br); 43.5 (br); 43.3 (br); 28.6; 28.2; LRMS
(ES+) m/e: 370 (MH+). Anal. calcd for C19H35N3O4:
C, 61.76; H, 9.55; N, 11.37. Found: C, 61.90; H, 9.53;
N, 11.12.
i
dissolved in PrOH was treated with 2 equiv of oxalic
acid. The product was allowed to crystallize in the
i
fridge overnight. The crystals were washed with PrOH
and ether, yielding 14 as a white solid (65%); Mono-
iodide monooxalate salt: mp 184–190 ꢂC; IR (KBr)
1
1650; 1580; 1290; 1270 cmꢀ1; H NMR (D2O) d: 3.63

1290
C. Dardonville et al. / Bioorg. Med. Chem. 11 (2003) 1283–1291
1-(4-Piperidyl)piperazine (11b). A solution of 11a (291
mg, 0.79 mmol) in 50% TFA/CH2Cl2 was stirred 2.5 h
at room temperature. The solvent was evaporated to
dryness and the residue was triturated in ether with a
spatula until a white solid crystallised. The solid was
washed with ether and dried in vacuo (282 mg); Tri-
fluoroacetate salt of 11b: 92%; mp 140–150 ꢂC; 1H
NMR (D2O) d: 3.4–3.1 (m, 2H); 3.0 (br s, 4H); 2.9–2.3
(br m, 7H); 2.1–1.75 (br m, 2H); 1.6–1.25 (br m, 2H);
13C NMR (D2O) d: 162 (q, TFA); 116.2 (q, TFA); 61.8;
46.5; 43.1; 41.5; 23.8; LRMS (ES+) m/e: 170 (MH+).
Anal. calcd for C15H22F9N3O6: C, 35.23; H, 4.36; N,
8.21. Found: C, 35.25; H, 4.79; N, 8.30.
158.8; 158.6; 141.8; 125.1; 124.3; 61.4; 47.1; 44.5; 43.2;
42.8; 42.7; 25.3; Monohydrochloride monoiodide salt:
white hygroscopic solid; 13%; H NMR (D2O) d: 3.8–
3.4 (br m, 5H); 3.65 (s, 4H); 3.61 (s, 4H); 3.4–2.9 (br m,
8H); 2.2–2.0 (br m, 2H); 1.8–1.5 (br m, 2H); 13C NMR
(D2O) d: 159.1 ; 62.4; 48.2; 45.6; 44.4; 43.6; 43.5; 26.4;
LRMS (ES+) m/e: 306 (MH+/100%). Anal. calcd for
C15H30N7ClI/2H2O: C, 33.22; H, 6.32; N, 18.08. Found:
C, 33.39; H, 6.03; N, 17.49.
1
Pharmacology
Functional activity. In vivo assays. CD1 male mice in the
weight range of 27–30 g were used for the study. All the
animals were supplied with food and water ‘ad libitum’
and were housed in a temperature-controlled room at
21 ꢂC. Lighting was on a 12/12-h light/dark cycle. The
mice were housed for a least 1 day in the test-room
before experimentation.
1-[[2-[(1,1-Dimethylethoxy)carbonyl]amino]ethyl]amino]-
thioxomethyl]-amino]-4-[[[2-[(1,1-dimethylethoxy)carbo-
nyl]amino]ethyl]amino]thioxomethyl] - amino]piperidin - 4-
yl]piperazine (12). The trifluoroacetate salt of 11b (2.473
g) was dissolved in water (200 mL) and stirred with 65
mL of IRA 400 (OHꢀ form) to liberate the freebase of
the amine. After 5 h, the pH of the aqueous solution was
about 8–9. The resin was filtered off and the solution
concentrated in vacuo to afford the free base of 11b as a
white solid (648 mg). In a flask flushed with nitrogen, a
solution of [(tert-butoxycarbonyl)amino]ethylisothio-
cyanate (1.244 g, 6.16 mmol) in THF (20 mL) was added
dropwise to a solution of 11b in a mixture of dry THF (25
mL) and DMF (5 mL added to help solubilisation of
11b). The reaction was stirred 2 days at room tempera-
ture and the solvent was removed in vacuo. The yellow
residue was purified by chromatography on silica with
CHCl3/MeOH 5% to yield 12 as a white foam (804 mg,
50%); mp 147–149 ꢂC; 1H NMR (CDCl3) d: 7.7 (br, 1H);
7.1 (br, 2H); 5.77 (br, 2H); 4.45–4.2 (m, 2H); 3.52 (br,
3H); 3.34 (br, 3H); 3.05 (br, 3H); 2.68 (s, 4H); 2.56 (s,
3H); 2.26 (br, 4H); 1.52 (m, 2H); 1.12 (s, 18H); 13C NMR
(CDCl3) d: 181.3; 180.8; 162.6; 158.0; 79.5; 74.2; 61.0;
48.6; 47.3; 46.6; 39.5; 36.5; 31.4; 28.3; 27.8; LRMS (ES+)
m/e: 574 (MH+/100%). Anal. calcd for C25H47N7O4S2:
C, 52.33; H, 8.26; N, 17.09; 11.17. Found: C, 52.04; H,
8.40; N, 17.31; S, 10.98.
To detect antinociceptive activity, several doses (25–100
mg/kg) of the tested compounds or saline solution were
ip administered to separated groups of mice (nꢃ10) 30
min before the analgesic effect was recorded. In
order to compare the antinociception level with that
of a reference compound, the effect of ip adminis-
tration of morphine (3.5 mg/kg) was also tested. Each
mouse was used only once and an observer who was
unaware of the treatment performed the testingand
data recording.
Writhing test. The mice were ip injected with a 2% ace-
tic acid solution to produce the typical writhingreac-
tion15 which is characterised by a wave of contraction of
the abdominal musculature followed by extension of the
hinds limbs. After the acetic acid administration, the
mice were placed in individual transparent containers
and, 5 min later, the number of writhes was counted
duringa 10-min period.
To test the antinociceptive effect, writhingtest was car-
ried out 30 min after the administration of separate doses
of: compound 1a, 2a–b, 3a–b, 4a, 5a, 6a, 13 and 14.
1-(4,5-Dihydro-1H-imidazol-2-yl)-4-[1-(4,5-dihydro-1H-
imidazol-2-yl)piperidin-4-yl] piperazine (13). A solution
of 12 (140 mg, 0.24 mmol) and iodoethane (3 mL) in
EtOH (2 mL) was heated 22 h (bath temperature: 60 ꢂC)
under a nitrogen atmosphere. The solvent was evapor-
ated in vacuo to give an orange foam. The product was
not isolated and was dissolved in 3 mL of a TFA/H2O
solution (2/1). This mixture was stirred 3.5 h at room
temperature and the solvent was removed in vacuo.
Lyophilization of the residue afforded an orange solid
that was dissolved in a mixture of MeOH (5 mL) and
EtOH (10 mL). This solution was added dropwise,
under nitrogen, to a sodium ethoxide solution (25 mg of
sodium in 3 mL of absolute EtOH). The reaction was
stirred 24 h at room temperature. The solvent was
removed in vacuo and the product was isolated by for-
mation of its picrate salt (method B). Picrate salt.
Yellow solid (31 mg); mp 204–207 ꢂC and 220–225 ꢂC;
1H NMR (DMSO) d: 8.54 (s, 2H); 8.49 (s, 1H); 8.33 (s,
1H); 3.62 (s, 4H); 3.60 (s, 4H); 4.0–2.9 (br m, 13H); 2.07
(br d, 2H); 1.59 (br d, 2H); 13C NMR (DMSO) d: 160.7,
The effect of 2a was also tested 30 min after po (intra-
gastric cannula) administration. SS (saline solution) of
one dose (25, 50, 100 and 150 mg/kg) of 2a were given
30 min before testing.
Hot plate test. This test was carried out with a hot plate
at 55 ꢂC as a nociceptive stimulus.16 The control reac-
tion latency of the animals was measured before the
treatment. The time of lickingof the front paws was
taken as an index of nociception. The latency was mea-
sured before drugadministration (control) and 30 min
after treatment. The cut-off time was 30 s and analgesia
was quantified with the formula of the % M.P.E.:
M:P:E: ¼ ðlatency after treatment À control latencyÞ=
ðcut-off time À control latencyÞ
To test the antinociceptive effect, hot plate test was
carried out 30 min after the administration of SS or

C. Dardonville et al. / Bioorg. Med. Chem. 11 (2003) 1283–1291
1291
separated doses of: compound 2a (50 and 100 mg/kg)
and morphine (5 mg/kg).
Marie Curie Research TrainingGrant for which he
thanks the European Commission, Directorate General
XII (Science, Research and Development).
In vitro assays
Isolated tissues. Male guinea-pigs weighing 300–450 g
were used for this study. Myenteric plexus-longitudinal
muscle strips (MP-LM) were isolated from guinea-pig
ileum (GPI) as described by Ambache.17 Tissues were
suspended in a 10 mL organ bath containing Krebs
solution (NaCl 118, KCl 4.75; CaCl2 2.54; KH2PO4
1.19; MgSO4 1.2; NaHCO3 25; glucose 11 mM). This
solution was continuously gassed with 95% O2 and 5%
CO2. Tissues were kept under 1 gof restingtension at
32 ꢂC and were electrically stimulated through two pla-
tinum ringelectrodes with rectangular pulses of 70 V,
0.1 ms duration and 0.3 Hz frequency. The isometric
force was recorded on a Grass model 7A polygraph.
References and Notes
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4. Dardonville, C.; Rozas, I.; Alkorta, I. J. Mol. Graphics
Mod. 1999, 16, 150.
5. Villarroya, M.; Gandıa, L.; Lopez, M. G.; Garcıa, A. G.;
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7. Compound 4a had already been prepared by the author
and evaluated for its bindingaffinity at the I 2-imidazoline
bindingsite in human brain tissues: Dardonville, C.; Rozas, I.;
Callado, L. F.; Meana, J. Bioorg. Med. Chem. 2002, 10, 1525.
8. Murahashi, S.-I.; Naota, T.; Nakajima, N. Chem. Lett.
1987, 879.
In order to detect agonistic or antagonist effects medi-
ated through the receptors presents in GPI, cumulative
administration of increasingconcentrations of 2a were
carried out in a step by step manner, beingthe interval
between applications 15 min. The effect of 2a was tested
in electrically stimulated tissues as well as in non stimu-
lated tissues.
9. Borch, R. F.; Bernstein, M. D.; Durst, H. J. Am. Chem.
Soc. 1971, 93, 2897.
10. Ariga, K.; Anslyn, E. V. J. Org. Chem. 1992, 57, 417.
11. (a) Collier, H. O.; Dinneen, L. C.; Johnson, C. A.;
Schneider, C. Br. J. Pharmac. Chemother. 1968, 32, 295. (b)
Martınez, V.; Thakur, S.; Mogil, J. S.; Tache, Y.; Mayer, E. A.
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12. MDS Pharma Services, 11804 North Creek PKwy. S.
Bothell, WA 98011-8890, USA.
13. Martın, M. I.; Goicoechea, C.; Ormazabal, M. J.; Alfaro,
M. J. Br. J. Pharmacol. 1996, 119, 804.
14. (a) Capasso, A. Neuropharmacology 1999, 38, 871. (b)
Schmidt, R.; Menard, D.; Mrestani-Klaus, C.; Chung, N. N.;
Lemieux, C.; Schiller, P. W. Peptides 1997, 18, 1615. (c) Men-
zies, J. R.; Paterson, S. J.; Duwiejua, M.; Corbett, A. D. Eur.
J. Pharmacol. 1998, 350, 101. (d) Saito, T.; Aoki, F.; Hirai, H.;
Inagaki, T.; Matsunaga, Y.; Sakakibara, T.; Sakemi, S.;
Suzuki, Y.; Watanabe, S.; Suga, O.; Sujaku, T.; Smogowicz,
A. A.; Truesdell, S. J.; Wong, J. W.; Nagahisa, A.; Kojima,
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Finally, to test antagonistic activity, separated doses of
2a, adequate to reach 10ꢀ8, 5ꢄ10ꢀ8, 10ꢀ7 and 5ꢄ10ꢀ6 M
concentration in the organ bath, were administered.
Then, GPI was stimulated by addition of acetylcholine
(5ꢄ10ꢀ8 M), serotonine (2ꢄ10ꢀ7 M), histamine (10ꢀ5
M), bradykinin (5ꢄ10ꢀ7 M) or substance P (10ꢀ6 M), and
MVD with noradrenaline (5ꢄ10ꢀ7 M). The concen-
tration of the agonists was those needed to approximately
reach a contractile response of 1 gin control tissues.
The general receptor and enzyme screenings were per-
formed by MDS Pharmacology Services, Bothell, WA
98011-8890, USA.12
Acknowledgements
16. Martın, M. I.; Goicoechea, C.; Colado, M. I.; Alfaro,
M. J. Eur. J. Pharmacol. 1992, 224, 77.
17. Ambache, N. J. Physiol. 1954, 127, 53.
Financial support was provided by the Spanish DGI-
CYT (Project SAF 97-0044-C02). C.D was a holder of a