Guanidinium and aminoimidazolinium derivatives of N-(4-piperidyl)propanamides as potential ligands for μ opioid and I2-imidazoline receptors: Synthesis and pharmacological screening

Article abstract of DOI:10.1016/S0968-0896(01)00356-X

Derivatives of N-(1-phenethyl-4-piperidyl)propanamides incorporating guanidinium and 2-aminoimidazolinium groups have been prepared by a synthetic approach involving first introduction of a spacer between the piperidine and the functional group by reductive amination of piperidinone. The formation of each of these functional groups was carried out using N-N′ -di(tert-butoxycarbonyl) thiourea and 2-methylthioimidazolinium iodide, respectively. These structures have been designed to incorporate two pharmacologic goals into one entity. Radioligand binding assays ave been used to study their affinity for opioid (μ δ and κ) and I ₂ -imidazoline receptors. Two of them, 10 and 16, showed high affinity for μ opioid receptors and functionally they had moderate analgesic properties in the hot plate and writhing tests. The in vitro studies on guinea pig ileum (GPI) indicated that both compounds are μ opioid agonists. In what concerns I2 -imidazoline receptor activity, these derivatives showed low affinity around 6 to 7 times less than idazoxan. Copyright

Full text of DOI:10.1016/S0968-0896(01)00356-X

Bioorganic & Medicinal Chemistry 10 (2002) 1009–1018
Guanidinium and Aminoimidazolinium Derivatives of
N-(4-Piperidyl)propanamides as Potential Ligands for ꢀ Opioid
and I2-imidazoline Receptors: Synthesis and
Pharmacological Screening
Ana Montero,a Pilar Goya,a Nadine Jagerovic,a,* Luis F. Callado,b J. Javier Meana,b
Rocıo Giron,c Carlos Goicoecheac and Ma Isabel Martınc
a
´
Instituto de Quımica Medica, CSIC, Juan de la Cierva, 3, E-28006 Madrid, Spain
´
Departamento de Farmacologıa, Universidad del Pais Vasco/Euskal Herriko Unibertsitatea, Leioa, E-48940 Bizkaia, Spain
b
´
c
´
Facultad de Ciencias de la Salud, Area de Farmacologıa, Universidad Rey Juan Carlos, Av. de Atenas s/n, E-28922 Madrid, Spain
Received 9 July 2001; accepted 5 October 2001
Abstract—Derivatives of N-(1-phenethyl-4-piperidyl)propanamides incorporating guanidinium and 2-aminoimidazolinium groups
have been prepared by a synthetic approach involving first introduction of a spacer between the piperidine and the functional group
by reductive amination of piperidinone. The formation of each of these functional groups was carried out using N-N0-di(tert-
butoxycarbonyl)thiourea and 2-methylthioimidazolinium iodide, respectively. These structures have been designed to incorporate
two pharmacologic goals into one entity. Radioligand binding assays have been used to study their affinity for opioid (m, d and k)
and I2-imidazoline receptors. Two of them, 10 and 16, showed high affinity for m opioid receptors and functionally they had mod-
erate analgesic properties in the hot plate and writhing tests. The in vitro studies on guinea pig ileum (GPI) indicated that both
compounds are m opioid agonists. In what concerns I2-imidazoline receptor activity, these derivatives showed low affinity around 6
to 7 times less than idazoxan. # 2002 Elsevier Science Ltd. All rights reserved.
Introduction
In this paper, we wish to report a new class of molecular
entities combining the pharmacophoric features of m
opioid binding ligands withthose of imidazoline recep-
tor ligands (I2-imidazoline preferring binding sites).
Recent studies6,7 have shown that imidazoline receptor
agonists combined withsome opioid agonists produce
antinociceptive synergy and it has been recently
demonstrated8 that some I2-imidazoline ligands can
attenuate tolerance to opioid induced antinociception.
The functional interaction between I2-imidazoline and
opioid receptors suggests a new effective therapeutic
strategy to manage pain by administration of I2-opioid
ligands combinations. The rationale behind this combi-
nation in the same molecule would be to obtain com-
pounds withenhanced opioid analgesic properties and
with somewhat reduced side effects. On the basis of
these considerations and within the framework of dual
acting drugs, the structures here proposed combine in
the same molecule a potent opioid moiety with I2-imi-
dazoline receptor features. The opioid moiety would
provide analgesic properties and the imidazoline ligand
moiety would enhance the activity and confer attenuation
‘Dual acting drugs’, which are drugs containing two
pharmacophoric groups combined in a single molecule
is one of the strategies used in drug design.1 These dual
acting drugs can exert their action on two binding sites
of a single receptor or on two different receptors.
Very recent examples include, among others, a new class
of prodrug of an HIV protease inhibitor conjugated
witha reverse transcriptase inhibitor, 2 dual inhibitors of
cyclooxygenase-2 and 5-lipoxygenase as novel anti-
arthritic agents,3 a dual inhibitor of thymidilate syn-
thase and dihydrofolate reductase as antitumor agent4
and a novel compound for treating bronchial asthma
which is a dual antagonist for leukotriene D4 and
thromboxane A2 receptors.5
*Corresponding author. Tel.:+34-91-562-2900; fax:+34-91-564-4853;
e-mail: iqmj307@iqm.csic.es
0968-0896/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(01)00356-X

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A. Montero et al. / Bioorg. Med. Chem. 10 (2002) 1009–1018
Figure 1. Chemical structure of fentanyl, compounds tested biologically included in this work and some known imidazoline ligands.
Results and Discussion
to the opioid induced tolerance, which is one of the
major side effects of opioid therapy.
Chemistry
The general formulae of the compounds described is
depicted in Figure 1 and corresponds to the combina-
tion of an N-[1-(2-phenethyl)-4-piperidyl]propanamide,
present in fentanyl like analgesics9 witha guanidinium
or 2-aminoimidazolinium groups which are found in
imidazoline ligands (see Fig. 1).10 A synthetic method-
ology has been developed for these novel structures
and their binding to both kinds of receptors studied. In
the most promising compounds, in vitro and in vivo
antinociceptive assays have also been carried out.
The syntheses of 10, 11, 16, and 17 were achieved fol-
lowing the sequences described in Schemes 1 and 3 for
the aliphatic series and in Schemes 2 and 3 for the aro-
matic series. The reaction sequences began with reduc-
tive amination of 1-phenethyl-4-piperidone (5). In th e
usual method of preparation of fentanyl derivatives,11
this reductive amination proceeds in two steps with first,
formation of the corresponding imine followed by
LiAlH4 reduction. The formation of aminopiperidines 6
and 12 from the piperidone 5 was carried out in an one
Scheme 1. (a) 4/NaBH3CN/MeOH/3 A sieves; (b) (EtCO)2O; (c) H2/Pd/C/AcOH/35–40 psi.
Scheme 2. (a) 3-Nitroaniline/NaBH3CN/MeOH/3 A sieves; (b) (EtCO)2O; (c) H2/Pd/C/AcOH/35–40 psi.

A. Montero et al. / Bioorg. Med. Chem. 10 (2002) 1009–1018
1011
Scheme 3. (a) (BocNH)2CS/TEA/HgCl2/CH2Cl2/0 ꢀC; (b) TFA/CH2Cl2; (c) 2-methylthioimidazolinium iodide/MeOH.
Scheme 4. (a) (Boc)2O/CHCl3/0 ꢀC; (b) CbzCl/THF/NaOH(3.3 N); (c) TFA/CH2Cl2.
pot reaction withpropanediamine 4 and 3-nitroaniline
respectively, using NaBH3CN as reductive agent. The
monoprotected propanediamine 4, required for the
reductive amination in the propyl series, was obtained
(Scheme 4) through monoprotection of diaminopropane
1 with tert-butoxycarbonyl group, followed by sub-
stitution of the free amine with benzyloxycarbonyl
group and finally the tert-butoxycarbonyl protecting
group was removed. Acylation of aminopiperidines 6
and 12 with propionic anhydride led to the correspond-
ing propanamides 7 and 13. Catalytic hydrogenation of
7 allowed the deprotection of amine to provide 8 and
catalytic hydrogenation of 13 resulted in reduction of
the nitro group affording 14. The guanidinylation of
Experimental, show (i) in the 1H NMR: duplicated
signals for the methyl and methylenic protons of the
amide group, for piperidine H-4 and for the methylenic
protons of the propyl chain, (ii) in the 13C NMR: two
signals for the carbonyl and for piperidine C-4. These
data indicate that the rotation around the N–CO bond
is slow enoughto observe cis–trans isomerism in NMR
studies. For the propanamides 13–17, corresponding to
the aromatic series, cis–trans conformers were not
observed by NMR studies at room temperature. How-
ever, in the 1H NMR spectra, the methylenic protons of
the amide group form an ABX3 pattern indicating
restricted rotation around N–Cphenyl bond due probably
of the presence of the meta-substituted group.
amines
8
and 14 with N-N0-di(tert-butoxy-
carbonyl)thiourea, a guanidinylation agent which was
prepared as previously reported by Iwanowicz et al.,12
in the presence of mercuric chloride and triethylamine
provided 9 and 15 respectively (Scheme 3). Treatment of
the protected guanidinium derivatives 9 and 15 with
trifluoroacetic acid afforded the corresponding guanidi-
nium derivatives 10 and 16 as trifluoroacetate salts.
Formation of the aminoimidazoline from the amines 8
and 14 was attempted with N,N0-di(tert-butoxy-
carbonyl)imidazolin-2-thione but did not lead to the
desired product. However, aminoimidazoline deriva-
tives 11 and 17 were obtained according to the Aspinall
et al.13 procedure by treatment of the amines 8 and 14
with 2-(methylthioimidazolinium) iodide (Scheme 3).
This reagent was prepared by reacting methyl iodide
with N,N0-ethylenethiourea in refluxing methanol.
The biological activities of compounds 16 and 17 have
been studied on their oxalate salt. For compound 11,
the iodide salt was used. Concerning product 10, bind-
ing assays have been realized on its trifluoracetate salt
and in vivo and in vitro studies have been carried on its
chloride salt.
Pharmacology
Binding assays. Compounds 10, 11, 16 and 17 were
evaluated for their binding affinities at the m, d and k
opioid receptors and at I2-imidazoline receptors following
reported procedures14,15 withminor modifications.
Fentanyl and idazoxan were also tested for comparative
purposes. Mouse brain membranes (P2 fraction) were
3
used with[ H]DAMGO, [3H]DPDPE and [3H]U-69593
ligands for the m, d and k receptors, respectively,
and [3H]2-BFI for I2-imidazoline receptor. Data of
radioligand binding assays are reported in Table 1.
The NMR data of the propanamides 7–11, correspond-
ing to the aliphatic series and which are reported in the

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A. Montero et al. / Bioorg. Med. Chem. 10 (2002) 1009–1018
Table 1. Affinity data [Ki (nM)] for m, d, k and I2-imidazoline receptora
Receptors
[3H]DAMGO (2 nM)
[3H]DPDPE (4 nM)
[3H]U-69593 (2 nM)
[3H]2-BFI (1 nM)
I2
m
d
k
Idazoxan
Fentanyl
10
11
16
17
n.d.
6ꢂ1.5
n.d.
n.d.
307ꢂ183
5462ꢂ1343
2022ꢂ949
2327ꢂ811
1890ꢂ499
9630ꢂ6731
663ꢂ184
298ꢂ40
37ꢂ12
1744ꢂ185
24955ꢂ17612
181ꢂ42
355ꢂ134
5043ꢂ483
841ꢂ282
11551ꢂ4381
1751ꢂ1135
7.8ꢂ2.5
7119ꢂ4089
12078ꢂ9686
aValues are expressed as meanꢂstandard error of the mean of three experiments.
Concerning the opioid receptors, compounds 10 and 16
show high affinity for the m receptor, the latter having a
Ki similar to that of fentanyl, a well-established potent m
opioid used as the reference compound.
the writhes induced by 25 mg/kg of compound 10, was
only of 5ꢂ2.7% (n=10). Non toxic doses (ꢃ 25 mg/kg)
were not able to induce analgesia (25 mg/kgꢁ% MEP
12.3ꢂ3.8%, n=12) when the hot plate test was done.
The affinity values obtained for the I2-imidazoline
receptor are less relevant than for the m opioid receptor,
with the compounds showing low or no affinity. Never-
theless, the best m opioid ligands 16 and 10 show some
affinity for the I2-imidazoline receptor although their
potencies are 6 times lower than idazoxan.
Compound 16 also inhibited the number of writhes (2
5
mg/kgꢁ20ꢂ4.4% (n=6),
mg/kgꢁ55.3ꢂ1.3%
(n=12), 10 mg/kgꢁ84.1ꢂ0.9% (n=13) in mice. In hot
plate test the% MPE induced by compound 16 was: 10
mg/kgꢁ15.8ꢂ8% (n=10) and 20 mg/kgꢁ50.7ꢂ10%
(n=11), naloxone (1 mg/kg) antagonized the analgesia
induced by 16 (% MPE after naloxone 13.2ꢂ11.8
(n=10). Larger doses (40 mg/kg) of this compound were
tested but must be disregarded because of their toxicity.
Functional activity. The low affinity shown in the binding
assays for the I2-imidazoline receptor together with the
fact that functional assays for this kind of receptor are
not very specific, prompted us to performe in vitro and in
vivo tests of the compounds only on the m opioid receptors.
Binding data show that two of the new compounds (10
and 16) selectively bind to m opioid receptors, however
these data give no information about intrinsic activity and
the functional study is necessary in order to determine
whether they are agonists or antagonists.
Intraperitoneal administration of morphine induced a
dose-dependent antinociceptive effect in bothwrithing
test and hot plate test. In the writhing test, the percen-
tage of inhibition of writhes induced by morphine were:
1.3 mg/kgꢁ19.5ꢂ0.5% (n=10); 2.5 mg/kgꢁ46.6ꢂ1.4%
(n=12); 3.5 mg/kgꢁ80.4ꢂ0.5% (n=11); 5 mg/kgꢁ96ꢂ
0.3% (n=10) and 10 mg/kgꢁ100% (n=10). In hot plate
test the percentage MEP induced by morphine were:
2.5 mg/kgꢁ29.8ꢂ4.8% (n=12), 5 mg/kgꢁ41ꢂ8.9%
(n=12), 7.5 mg/kgꢁ64.1ꢂ7.1% (n=12) and 10 mg/kgꢁ
86.3ꢂ7.5% (n=11).
To obtain this information, the analgesic activity of
these two compounds was tested using the writhing test
and the hot plate test in mice. The writhing test was
used because it is widely employed at the first stages of
the evaluation of antinociceptive drugs16 and it was
used as a first approachto determine tehir anti-
nociceptive potency. Then they were also studied using
the hot plate test,17 to corroborate the analgesia by this
more sensitive test. Reversal of the antinociceptive effect
withnaloxone, a selective opioid antagonist, was used
to verify that the analgesia might be attributed to
interaction withopioid receptors.
To assess the participation of the opioid system on the
effect of these compounds their functional activity on
different subtypes of receptors myenteric plexus—long-
itudinal muscle strips of the guinea pig ileum18ꢁ20—was
analyzed. In guinea pig ileum, 10 and 16, as well as the
reference compound, morphine, induced a dose-depen-
dent inhibition of the electrically-induced contractions
(Fig. 2). Table 2 shows the EC50 values. The in vitro
effect of all the tested drugs was completely reversed by
the in vitro administration of low concentration of
naloxone (5ꢄ10ꢁ8 M) a m opioid antagonist. The inhibition
of the contractile response in this tissue may be mediated by
activation of m or k receptors18,19,21ꢁ23 but considering
When the antinociceptive activity of the new com-
pounds 10 and 16 was determined carrying out the wri-
thing test, they elicited a dose-dependent inhibition of
the number of writhes induced by the intraperitoneal
administration of acetic acid. In the writhing test, the
percentage of inhibition of writhes induced by com-
pound 10 were: 2 mg/kgꢁ19.5ꢂ2.3% (n=12); 10 mg/
kgꢁ20.5ꢂ1.6% (n=12) and 25 mg/kgꢁ47.4ꢂ2.2%
(n=12). The increase of the dosage induced behavioral
impairments and results were disregarded. Naloxone was
able to antagonize the antinociceptive effect of the largest
tested dose of compound 10, after the ip administration
of 1 mg/kg of naloxone, the percentage of inhibition of
Table 2. Inhibitory effect (EC50) of morphine, 10 and 16 on the
electrically-induced contractions in guinea pig ileum
Compound
EC50 (M)
Confidence intervals 95%
Morphine
10
16
2.1ꢄ10ꢁ7
6.61ꢄ10ꢁ6
1.9ꢄ10ꢁ6
8.7ꢄ10ꢁ8 to 5.4ꢄ10ꢁ7
5ꢄ10ꢁ6 to 8.9ꢄ10ꢁ6
4.5ꢄ10ꢁ7 to 7.9ꢄ10ꢁ6

A. Montero et al. / Bioorg. Med. Chem. 10 (2002) 1009–1018
1013
Experimental
Chemistry
The melting points were determined with a Reichert
Jung Thermovar apparatus and are uncorrected. Elec-
trospray mass spectra were recorded on a MSD-Serie
1100 Hewlett Packard apparatus. Elemental analysis
were performed on a Heraeus CHNO Rapid Analyser.
Flashcolumn chromatographies were run on silica gel
60 (230–400 meshASTM, Merck) or on medium pres-
1
sure 40i prepacked KP-Sil cartridge (Biotage). H and
13C NMR spectra were recorded on a Varian Gemini
200 spectrometer and on Varian 300 and 400 unity
spectrometers. Dry MeOH was obtained by distillation
over Mg(OH)2. Anhydrous CH2Cl2 was obtained by
distillation over CaCl2. THF and toluene were distilled
over sodium-benzophenone. TEA and propylamine
were purified by distillation over KOH.
Figure 2. Lines show the mean percentage ꢂSEM of inhibition of the
electrically induced contractile responses induced by cumulative addi-
tion of morphine (open circles) or of 10 (triangles) or 16 (squares).
N-(tert-Butoxycarbonyl)-1,3-propanediamine (2). To 1,3-
propanediamine (550 mL) in CHCl3 (1 L) at 0 ꢀC was
added (Boc)2O (24.0 g, 110 mmol) dissolved in CHCl3
(150 mL) dropwise during 0.5 h. The reaction mixture
was allowed to warm to ambient temperature and stir-
red for 2 h. After filtrating the mixture, the filtrate was
concentrated in vacuo and the oily residue was dissolved
in EtOAc (500 mL). The solution was washed with brine
and dried over MgSO4. Evaporation of the solvent in
vacuo gave 2 (18.8 g) as a white solid. Yield 98%, mp
that their effect was antagonized by low concentrations
of naloxone, it could be suggested that the effect on this
tissue could be mediated by the m opioid receptor. It is
interesting to note that there is a very good correlation
between the antinociceptive potency of tested drugs (10
and 16 and morphine) and their effectiveness on guinea
pig ileum which is an interesting result because it is
characteristic of opioid analgesic drugs.24
54–56 ꢀC. H NMR (CDCl3) d 5.14 (br, 1H), 3.23 (br,
1
2H), 3.16 (m, 2H), 2.75 (t, 2H, J=6.7 Hz), 1.61 (tt, 2H,
J=6.7 and 6.7 Hz), 1.39 (s, 9H); 13C NMR (CDCl3) d
156.2, 79.0, 38.9, 38.1, 32.3, 28.3; MS (ES+) m/z 175
([MH]+, 100).
In summary, although compounds 10 and 16 were not
well tolerated after their in vivo administration and the
behavioral impairment restrained their analgesic utility,
data obtained from the study of the antinociception and
of the naloxone antagonism permits the suggestion that
the activation of opioid receptors plays a role in their
effect. Moreover, when isolated guinea pig ileum, a tis-
sue containing mainly m and k receptors, was used, it
was confirmed that 10 and 16 are m opioid agonists.
N-(tert-Butoxycarbonyl)-N0-(benzyloxycarbonyl)-1,3-
propanediamine (3). To propanediamine 2 (18.7 g, 107
mmol) in THF (600 mL) at 0 ꢀC was added benzyl
chloroformate (23 mL, 161 mmol) in NaOH (3.2 N).
The reaction mixture was allowed to warm to ambient
temperature and stirred for 2 h. The solution was acidi-
fied withHCl (6 N), extracted withEtOAc. The organic
layer was dried over MgSO4. The solvent was removed
in vacuo to give the crude product which was flash
chromatographed on silica gel (CH2Cl2/EtOAc, 9:1) to
provide 3 (30.2 g) as a white solid. Yield 91%, mp 45–
Conclusions
In this paper, we have described compounds with dual
action on two types of receptors, m opioid and I2-imi-
dazoline receptors. A synthetic methodology has been
developed for incorporating in the same molecule the
two pharmacophoric groups required, N-phenylpiperi-
dine and guanidinium and aminoimidazolinium
groups. Concerning the affinity for the two kinds of
receptors, radioligand binding assays indicate that two
compounds (10 and 16) are potent m opioid ligands, one
of them (16) withpotency similar to that of fentanyl,
and they also show moderate analgesic properties in
vivo. The results for the other receptor, the I2-imidazo-
line one, are less significant and the compounds (10 and
16) show only low affinity. This kind of hybrid struc-
tures descrived here for the first time, can be regarded as
a lead for structural modifications mainly aimed at
improving their binding to the I2-imidazoline receptors,
in order to get analgesic compounds withreduced-
tolerance side effects.
46 ꢀC. H NMR (CDCl3) d 7.26–7.33 (m, 5H), 5.42 (br,
1
1H), 5.06 (s, 2H), 4.95 (br, 1H), 3.20 (m, 2H), 3.14 (m,
2H), 1.58 (tt, 2H, J=6.7 and 6.7 Hz), 1.41 (s, 18H); 13C
NMR (CDCl3) d 156.7, 156.4, 136.5, 128.4, 128.0, 127.4,
126.8, 79.2, 66.5, 37.6, 37.0, 30.4, 28.3; MS (ES+) m/z
331 ([MNa]+, 100).
N-(Benzyloxycarbonyl)-1,3-propanediamine (4). A solu-
tion of 3 (30.0 g, 97 mmol) in CH2Cl2 (100 mL) and
TFA (100 mL) was stirred for 4 hat room temperature.
The solvents were removed in vacuo. The residue was
dissolved in CH2Cl2 and extracted withNaOH (2.5 N).
The organic layer was dried over MgSO4. The solvent
was removed in vacuo to give 4 (20.0 g). Yield 98%, mp
83–84 ꢀC. H NMR (CDCl3) d 7.28–7.35 (m, 5H), 5.28
1
(br, 1H), 5.08 (s, 2H), 3.28 (m, 2H), 2.77 (t, 2H, J=6.6
Hz), 1.63 (tt, 2H, J=6.6 and 6.6 Hz); 13C NMR

1014
A. Montero et al. / Bioorg. Med. Chem. 10 (2002) 1009–1018
(CDCl3) d 156.6, 136.6, 128.5, 128.1, 66.6, 39.6, 39.1,
32.6; MS (ES+) m/z 209 ([MH]+, 100).
Hz, 1H), 3.50 (t, J=12.0 Hz, 1H), 3.26 (t, 2H, J=6.7
Hz), 3.18 (t, 2H, J=6.7 Hz), 3.0 (d, 4H, J=12.0 Hz),
2.72 (m, 4H), 2.66 (m, 4H), 2.52 (m, 4H), 2.36 (q, 2H,
J=7.4 Hz), 2.34 (q, 2H, J=7.3 Hz), 2.0 (t, 4H,
J=12.0 Hz), 1.82 (d, 4H, J=12.0 Hz), 1.67 (q, 4H,
J=12.0 Hz), 1.62 (m, 4H), 1.61 (t, 2H, J=7.3 Hz), 1.63
(t, 2H, J=7.3 Hz); 13C NMR (CDCl3) d 173.7, 173.5,
140.0, 128.5, 128.3, 126.0, 60.1, 55.4, 53.1, 39.6, 39.1,
33.6, 32.7, 30.7, 26.7, 9.5; MS (ES+) m/z 318 ([MH]+,
100); MS (ESꢁ) m/z 352 ([MCl]ꢁ, 100).
1-(2-Phenethyl)-4-(N-benzyloxycarbonyl-N0-diaminopro-
pyl)piperidine (6). To 4 (2.2 g, 10.5 mmol) in anhydrous
MeOH (25 mL) was added a solution of concd HClg in
anhydrous MeOH until pH 6. To this solution were
successively added 1-phenethyl-4-piperidone (1.8 g, 8.75
mmol), NaBH3CN (820 mg, 13.1 mmol) and 3 A molec-
ular sieves. The solution was then acidified to pH 6 with
a solution of concd HClg in MeOH and stirred for 24 h
at ambient temperature. The reaction mixture was fil-
tered over a Celite bed which was then washed with
MeOH. Concentration of the combined filtrates in
vacuo gave a brown precipitate which was purified by
medium pressure chromatography on silica gel
(CH2Cl2/NH3(g) in MeOH, 9:1) to provide 6 (1.2 g) as a
N-[(2,3-di-tert-Butoxycarbonyl)guanidinopropyl]-N-[1-(2-
phenethyl)-4-piperidyl]propanamide (9). To a solution of
8 (2.0 g, 6.3 mmol), thiourea (1.7 g, 6.3 mmol) and 2 mL
of TEA (18.9 mmol) in 20 mL of anhydrous CH2Cl2
under N2 and at 0 C was added 2.2 g (8.2 mmol) of
HgCl2. The reaction mixture was stirred at 0ꢀC for 1 h.
The temperature was allowed to warm to ambient tem-
perature and the mixture was stirred for additional 24 h.
The mixture was diluted with EtOAc, filtered over
Celite. The filtrate was washed with H2O, washed with
brine, and dried over MgSO4. The solvent was evapo-
rated in vacuo. The residue was purified by medium
pressure chromatography on silica gel (EtOAc) to pro-
vide 9 (1.9 g) as a yellowishoil. Yield 51%. 1NMR
(CDCl3) d 12.01 (s, 2H), 11.50 (s, 2H), 7.26–7.21 (m,
4H), 7.17–7.14 (m, 6H), 4.42 (t, 1H, J=12.0 Hz), 3.53
(t, 1H, J=12.0 Hz), 3.40 (t, 2H, J=7.0 Hz), 3.38 (t, 2H,
J=7.0 Hz), 3.24 (m, 4H), 3.08 (d, 4H, J=12.0 Hz), 2.73
(m, 4H), 2.56 (m, 2H), 2.34 (q, 1H, |J|=7.3 Hz), 2.31 (q,
1H, J=7.3 Hz), 2.05 (t, 4H, J=12.0 Hz), 1.73–1.84 (m,
8H), 1.67 (m, 4H), 1.46 (s, 36H), 1.13 (t, 6H, J=7.3 Hz);
13C NMR (CDCl3) d 173.7, 173.5, 163.3, 156.1, 155.9,
140.0, 128.5, 128.3, 126.0, 83.2, 82.7, 60.2, 55.3, 53.1,
39.2, 38.7, 33.7, 30.7, 29.6, 28.2, 27.9, 26.8, 9.5; MS
(ESꢁ) m/z 352 ([MCl]ꢁ, 100).
yellowishsolid. Yield 45%, mp 131–133 ꢀC. H NMR
1
(CDCl3) d 7.13–7.17 (m, 3H), 7.22–7.25 (m, 2H), 7.25–
7.29 (m, 5H), 5.87 (br, 1H), 5.04 (s, 2H), 4.17 (br, 1H),
3.27 (t, 2H, J=6.5 Hz), 3.0 (d, 2H, J=12.0 Hz), 2.83 (t,
2H, J=6.6 Hz), 2.75 (m, 3H), 2.56 (m, 2H), 2.06 (t, 2H,
J=12.0 Hz), 2.02 (d, 2H, J=12.0 Hz), 1.91 (tt, 2H,
J=6.7 and 6.7 Hz), 1.70 (q, 2H, J=12.0 Hz); 13C NMR
(CDCl3) d 158.1, 139.8, 136.6, 128.7, 128.5, 128.4, 128.1,
128.0, 126.2, 67.7, 60.9, 56.1, 52.7, 43.8, 39.5, 34.4, 30.5,
28.8; MS (ES+) m/z 396 ([MH]+, 100).
N-[(N0-Benzyloxycarbonyl)aminopropyl]-N-[1-(2-phenethyl)-
4-piperidyl]propanamide (7). A solution of 6 (6.1 g, 15.4
mmol) in propionic anhydride (50 mL) was stirred and
heated to reflux for 3 h. After cooling, the solution was
poured on ice, basified withNH OH (30%) and extrac-
4
ted withEtOAc. Teh combined organic layers were
dried over MgSO4 and concentrated in vacuo. To the
residue dissolved in EtOAc was added Et2O, the pre-
cipitate was filtered. The filtrate was concentrated to
give the crude product which was purified by flash
chromatography on silica gel (EtOAc/MeOH, 9:1) to
N-[1-(2-Phenethyl)-4-piperidyl]-N-(guanidinopropyl)pro-
panamide (10). A solution of 9 (1.6 g, 2.89 mmol) in a
mixture 1:1 of CH2Cl2/TFA was stirred at room tem-
perature for 4 h. After evaporation of the solvents in
vacuo, the residue was dried in vacuo to give 10 (1.6 g)
1
provide 7 (5.0 g) as an orange oil. Yield 72%. H NMR
(CDCl3) d 7.32–7.25 (m, 10H), 7.23–7.17 (m, 4H), 7.12–
7.10 (m, 6H), 5.0 (s, 4H), 4.32 (t, 1H, J=12.0 Hz), 3.47
(t, 1H, J=12.0 Hz), 3.25 (m, 4H), 3.06 (m, 4H), 2.97 (d,
4H, J=12.0 Hz), 2.69 (m, 4H), 2.51 (m, 4H), 2.27 (q,
2H, J=7.4 Hz), 2.20 (q, 2H, J=7.4 Hz), 1.98 (t, 4H,
J=12.0 Hz), 1.77 (d, 4H, J=12.0 Hz), 1.72–1.60 (m,
8H), 1.06 (t, 3H, J=7.3 Hz), 1.03 (t, 3H, J=7.3 Hz);
13C NMR (CDCl3) d 173.8, 156.4, 139.8, 136.6, 128.4,
128.2, 127.8, 127.7, 127.6, 125.8, 66.1, 60.0, 55.4, 53.0,
38.5, 38.2, 33.5, 30.6, 29.5, 26.6, 9.5; MS (ES+) m/z 452
([MH]+, 100), 474 ([MNa]+); MS (ESꢁ) m/z 486
([MCl]ꢁ).
as a trifluoroacetic salt. Yield 98%, mp 158–160 ꢀC. H
1
NMR (D2O) d 7.31–7.26 (m, 4H), 7.23–7.19 (m, 6H),
4.12 (t, 1H, J=12.0 Hz), 4.0 (t, 1H, J=12.0 Hz), 3.59
(m, 4H), 3.25 (m, 4H), 3.23 (m, 4H), 3.08 (m, 4H), 3.06
(m, 2H), 2.98 (m, 4H), 2.36 (q, 2H, |J|=7.4 Hz), 2.27 (q,
2H, J=7.4 Hz), 2.06 (q, 2H, J=12.0 Hz), 1.96 (d, 4H,
J=1.02 Hz), 1.67 (tt, 2H, J=7.4 and 7.4 Hz), 1.64 (tt,
2H, J=7.4 and 7.4 Hz), 0.95 (t, 3H, J=7.4 Hz), 0.94 (t,
3H, J=7.4 Hz); 13C NMR (D2O) d 177.8, 177.5, 163.0
(d, JCCF=35.2 Hz), 156.9, 136.3, 129.2, 128.9, 127.5,
116.4 (d, JCF=291.6 Hz), 58.0, 52.5, 52.1, 51.3, 43.0,
39.0, 30.0, 28.1, 27.4, 26.5, 9.1. The chloride salt of 10
was formed by treating an aqueous solution of the tri-
fluoroacetic salt withAmberlite IRA 400 resine: MS
(ES+) m/z 180 [(M+2H)/2]2+, 360 ([MH]+, 100%).
N-(Aminopropyl)-N-[1-(2-phenethyl)-4-piperidyl]propana-
mide (8). A mixture of 7 (340 mg, 0.76 mmol), 10% Pd/C
(15 mg) and glacial AcOH (1 mL) in MeOH (30 mL)
was shaken under H2 (35–40 psi) for 6 h. After filtration
and evaporation of the solvent, the residue was dis-
solved in NaOH (2.5 N) and extracted withEtOAc. The
organic layer was dried over MgSO4 and concentrated
in vacuo to give 8 (235 mg) as a lighly yellowish oil.
Yield 98%. 1H NMR (CDCl3) d 7.23–7.18 (m, 4H),
7.12–7.10 (m, 6H), 5.78 (s, 4H, 2H), 4.45 (t, J=12.0
.
Anal.
52.26(52.10); H: 8.35(8.15); N: 15.24(14.96).
(C20H33N5O.2HCl 1.5H2O)
(found)
C:
N-[1-(2-Phenethyl)-4-piperidyl]-N-[N0-(4,5-dihydro-1H-
imidazol-2-yl)aminopropyl]propanamide (11). A solution
of 8 (248 mg, 0.78 mmol) and 2-(methylthio)imidazoli-

A. Montero et al. / Bioorg. Med. Chem. 10 (2002) 1009–1018
1015
nium iodide (400 mg, 1.64 mmol) in anhydrous MeOH
(3 mL) was stirred and heated to reflux for 48 h. A
stream of N2 was passed through the solution for 15
min then the solution was allowed to cool to room
temperature. A precipitate was formed on adding di-
ethyl ether to the solution. The solid was filtered, washed
with diethyl ether and recrystallized from water at 5 ꢀC.
The crystals were dried in vacuo at 30 ꢀC to give 11 (140
J=8.0 Hz), 7.46 (ddd, 1H, J=8.0, 2.0 and 1.0 Hz),
7.28–7.23 (m, 2H), 7.20–7.13 (m, 3H), 4.72 (tt, 1H,
J=12.0 and 4.0 Hz), 3.01 (dd, 2H, J=12.0 and 4.0 Hz),
2.70 (m, 2H), 2.54 (m, 2H), 2.18 (dt, 2H, J=12.0 and
2.0 Hz), 1.92 (m, 2H), 1.85 (dd, 2H, J=12.0 and 2.0
Hz), 1.38 (dq, 2H, J=12.0 and 4.0 Hz), 1.05 (t, 3H,
J=7.4 Hz). 13C NMR (CDCl3) d 172.8, 148.7, 140.2,
140.0, 136.8, 130.2, 128.6, 128.4, 126.1, 125.5, 123.4,
60.3, 52.9, 52.4, 33.8, 30.6, 28.9, 9.4; MS (ES+) m/z 382
([MH]+, 100).
mg) as a iodide salt. Yield 36%, mp 143–145 ꢀC. H
1
NMR (CD3OD) d 7.30–7.25 (m, 4H), 7.21–7.18 (m,
6H), 4.10 (t, 1H, J=12.0 Hz), 4.0 (t, 1H, J=12.0 Hz),
3.40 (m, 4H), 3.5 (s, 8H), 3.22 (m, 2H), 3.20 (m, 2H),
3.06 (m, 2H), 3.0 (m, 4H), 2.96 (m, 4H), 2.27 (q, 2H,
J=7.4 Hz), 2.36 (q, 2H, J=7.4 Hz), 2.06 (q, 4H,
J=12.0 Hz), 1.88 (d, 4H, J=12.0 Hz), 1.70 (tt, 2H,
J=7.4, 7.4 Hz), 1.64 (tt, 2H, J=7.4 and 7.4 Hz), 1.16 (t,
3H, J=7.4 Hz), 1.12 (t, 3H, J=7.4 Hz); 13C NMR
(CD3OD) d 177.6, 177.4, 158.9, 136.3, 130.2, 130.0,
128.8, 58.5, 52.8, 52.2, 51.4, 44.1, 43.5, 40.0, 30.1, 28.3,
27.6, 26.2, 9.7; MS (ES+) m/z 386 ([MH]+). Anal.
(C22H35N5O.2HI) (found) C: 41.20 (41.11); H: 5.81
(5.37); N: 10.92 (10.49).
N-[1-(2-Phenethyl)-4-piperidyl]-N-(3-aminophenyl)propa-
namide (14). A mixture of oxalate salt of 13 (1.3 g, 2.8
mmol) and 10% Pd/C (57 mg) in MeOH (30 mL) was
shaken under H2 (35–40 psi) for 0.5 h. After filtration
and evaporation of the solvent, the residue was dis-
solved in CH2Cl2. The solution was washed with K2CO3
(0.5 N). The organic layer was dried over MgSO4 and
concentrated in vacuo to give 14 (600 mg) as a lighly
yellowishoil. Yield 61%. 1H NMR (CDCl3) d 7.23–7.06
(m, 6H), 7.05 (t, 1H, J=8.0 Hz), 6.54 (d, 1H, J=7.7
Hz); 6.46 (dd, 1H, J=8.3 and 2.2 Hz), 4.56 (t, 1H,
J=12.0 Hz), 2.93 (d, 2H, J=12.0 Hz), 2.68 (m, 2H),
2.50 (m, 2H), 2.06–1.91 (m, 4H), 1.75 (d, 2H, J=12.0
Hz), 1.37 (q, 2H, J=12.0 Hz), 0.93 (t, 3H, J=7.4 Hz).
13C NMR (CDCl3) d 175.6, 164.6, 149.0, 139.3, 136.7,
130.8, 129.4, 129.2, 127.7, 116.9, 116.6, 113.7, 58.3, 52.8,
50.7, 31.0, 28.7, 28.1, 9.8. MS (ES+) m/z 352 ([MH]+,
100).
1-(2-Phenethyl)-4-(3-nitroanil-1-yl)piperidine (12). To 3-
nitroaniline (1.5 g, 7.4 mmol) in anhydrous MeOH at
pH 6 were successively added 1-phenethyl-4-piperidone
(1.2 g, 8.8 mmol), NaBH3CN (700 mg, 11.1 mmol) and
3 A molecular sieves. During 78 hof stirring of the
reaction mixture at room temperature the solution was
kept acidifying to pH 6 witha solution of concd HCl g in
MeOH. Then the reaction mixture was filtered over a
Celite bed. Concentration of the filtrate in vacuo gave a
crude which was purified by flash chromatography on
silica gel (EtOAc/hexane, 9:1) to provide 12 (3.6 g) as a
yellow solid. Yield 37%, mp 115–119 ꢀC. 1H NMR
(CDCl3) d 1H NMR (acetone-d6) d 7.02 (ddd, 1H,
J=8.0, 2.0 and 1.0 Hz), 7.13–7.18 (m, 1H), 7.20–7.29
(m, 3H), 7.30–7.33 (m, 1H), 7.34–7.38 (m, 1H), 7.41 (t,
1H, J=2.0 Hz), 3.40 (t, 1H, J=12.0 Hz), 2.96 (d, 2H,
J=12.0 Hz), 2.77 (m, 2H), 2.56 (m, 2H), 2.22 (t, 2H,
J=12.0 Hz), 2.01 (d, 2H, J=12.0 Hz), 1.52 (q, 2H,
J=12.0 Hz); 13C NMR (acetone-d6) d 150.4, 150.0,
141.7, 130.7, 129.5, 129.0, 126.6, 119.4, 110.8, 106.8,
61.1, 52.9, 50.5, 34.4, 32.7; MS (ES+) m/z 326 ([MH]+,
100).
N-[m-(2,3-di-tert-Butoxycarbonyl)guanidinophenyl]-N-[1-
(2-phenethyl)-4-piperidyl]propanamide (15). To a solu-
tion 14 (500 mg, 1.42 mmol), N-N0-di(tert-butoxy-
carbonyl)thiourea (392 mg, 1.42 mmol) and TEA (0.6
mL, 4.26 mmol) in anhydrous MeOH (20 mL) under N2
and at 0 ꢀC was added HgCl2 (390 mg, 1.82 mmol). The
reaction mixture was stirred at 0 ꢀC for 1 h. The tem-
perature was allowed to warm to ambient temperature
and the mixture was stirred for additional 20 h. The
mixture was diluted withEtOAc, filtered over Celite.
The filtrate was washed with H2O, washed with brine,
and dried over MgSO4. The solvent was evaporated in
vacuo. The residue was purified by flash chromato-
graphy on silica gel (EtOAc/hexane, 9:1) to provide 15
1
(625 mg) as an oil. Yield 74%. H NMR (D2O) d 7.40–
7.35 (t, 1H, J=8.0 Hz), 7.25–7.13 (m, 6H), 7.10–7.04
(dd, 1H, J=8.0 and 2.0 Hz), 7.0 (t, 1H, J=2.0 Hz), 3.60
(t, 1H, J=12.0 Hz), 3.13 (d, 2H, J=12.0 Hz), 2.83 (m,
2H), 2.63 (m, 2H), 2.16 (t, 2H, J=12.6 Hz), 1.83 (m,
4H), 1.58 (m, 2H), 1.53 (s, 9H), 1.51 (s, 9H), 0.91 (t, 3H,
J=7.4 Hz); 13C NMR (CDCl3) d 173.6, 163.3, 153.4,
149.2, 140.3, 137.7, 129.1, 128.6, 128.4, 126.1, 109.7,
109.4, 107.4, 84.0, 79.6, 60.6, 55.7, 53.6, 33.9, 29.6, 28.1,
28.0, 22.6, 11.3; MS (ES+) m/z 593 ([MH]+, 100).
N-[1-(2-Phenethyl)-4-piperidyl]-N-(3-nitrophenyl)propa-
namide (13). A solution of 12 (538 mg, 1.65 mmol) in
propionic anhydride (80 mL) was stirred and heated to
reflux for 4 h. After allowing to cool to room tempera-
ture, the excess of propionic acid was decomposed by
heating to reflux the solution with water (100 mL). The
solution was then basified with NH4OH (30%) and
extracted withCH 2Cl2. The organic layer was dried
over MgSO4 and concentrated in vacuo. To the residue
dissolved in EtOAc was added an excess of oxalic acid
in diethylether, this mixture was stirred at room tem-
perature for 1 h. The precipitate was filtered, washed
withEtOAc then withEt 2O. The crude was first crys-
tallized from MeOH/Et2O then from H2O at 5 ꢀC to
provide the oxalate salt of 13 (540 mg). Yield 70%, mp
142–143 ꢀC. 1H NMR (CDCl3) d 8.27 (ddd, 1H, J=8.0,
2.0 and 1.0 Hz), 7.98 (t, 1H, J=2.0 Hz), 7.63 (t, 1H,
N-[1-(2-Phenethyl)-4-piperidyl]-N-(m-guanidinophenyl)-
propanamide (16). A solution of 15 (300 mg, 0.84
mmol) in a mixture 1:1 of CH2Cl2/TFA was stirred at
room temperature for 4 h. After evaporation of the sol-
vents in vacuo, the residue was dissolved in CH2Cl2 and
washed with NaOH (2.5 N). The organic layer was dried
over MgSO4 and the solvent was evaporated. To the
residue dissolved in EtOAc was added an excess of

1016
A. Montero et al. / Bioorg. Med. Chem. 10 (2002) 1009–1018
oxalic acid dissolved in Et2O. The white precipitate was
filtered, washed with EtOAc then with Et2O. The white
solid was dried in vacuo at 30 ꢀC to give 16 (303 mg) as
membranes which were incubated with [3H]DAMGO (2
nM), [3H]U-69593 (2 nM), [3H]DPDPE (4 nM) or
[3H]2-BFI (1 nM). Neural membranes were incubated
for 60 min at 25 ꢀC in [3H]DAMGO and [3H]U-69593
assays, 60ꢀmin at 37 ꢀC in th e 3[H]DPDPE assays or 45
min at 25 C for [3H]2-BFI, in the absence or presence
of the competing compounds (10ꢁ12 M to 10ꢁ3 M, 10
concentrations). Total binding was determined and
plotted as a function of the compound concentration.
an oxalate salt. Yield 75%, mp 110–112 ꢀC. H NMR
1
(D2O) d 7.33 (t, 1H, J=8 Hz), 7.25–7.13 (m, 6H), 7.07
(dd, 1H, J=8.0 and 2.0 Hz), 7.0 (t, 1H, J=2.0 Hz), 3.8
(t, 1H, J=12.0 Hz), 3.57 (d, 2H, J=12.0 Hz), 3.23 (m,
2H), 2.95 (td, 2H, J=12.0 and 2.0 Hz), 2.90 (m, 2H),
2.06 (dq, 2H, J=12.0 and 2.0 Hz), 1.80 (dq, 2H, J=12.0
and 4.0 Hz), 1.29 (q, 1H), 1.24 (q, 1H), 0.69 (t, 3H,
J=7.3 Hz); 13C NMR (D2O) d 174.5, 167.7, 157.4,
143.0, 141.0, 136.28, 131.6, 129.2, 128.9, 127.5, 122.2,
119.0, 117.0, 58.3, 57.7, 51.7, 30.0, 26.0, 19.0, 10.3; MS
Incubations were terminated by diluting the samples
with5 mL of ice-cold Tris incubation buffer (4 ꢀC).
Membrane bound was separated by vacuum filtration
through Whatman GF/C glass fibre filters. Then, the
filters were rinsed twice with5 mL of incubation buffer
and transferred to minivials containing 3 mL of Opti-
Phase ‘HiSafe’ II cocktail and counted for
radioactivity by liquid scintillation spectrometry.
(ES+)
m/z
.
394
(MH+,
100).
Anal.
(C23H31N5O H2C2O4) (found) C: 62.10 (61.82); H: 6.88
(6.82); N: 14.48 (14.35).
N-[1-(2-Phenethyl)-4-piperidyl]-N-[m-[N0-(4,5-dihydro-1H-
imidazol-2-yl)aminophenyl]propanamide (17). A solution
of 14 (300 mg, 0.85 mmol) and 2-(methylthio)imidazo-
linium iodide (410 mg, 1.70 mmol) in anhydrous MeOH
(3 mL) was stirred and heated to reflux for 48 h. A
stream of N2 was passed through the solution for 15
min then the solution was allowed to cool to room
temperature. A precipitate was formed on adding die-
thyl ether to the solution. The solid was filtered, washed
with diethyl ether and recrystallized from water at 5 ꢀC.
To the crystals dissolved in EtOAc was added an excess
of oxalic acid dissolved in Et2O. The precipitate was fil-
tered, washed with EtOAc then with Et2O. The white
solid was dried in vacuo at 30 ꢀC to give 17 (102 mg) as
Analysis of binding data. Analysis of competition
experiments to obtain the inhibition constant (Ki) were
performed by nonlinear regression using the EBDA-
LIGAND program. All experiments were analysed
assuming a one-site model of radioligand binding.
Drugs. [3H]DAMGO (specific activity 50 Ci/mmol)
was purchased from American Radiolabeled Chemicals
Inc., USA. [3H]U69593 (specific activity 41.4 Ci/mmol)
and [3H]DPDPE (specific activity 45 Ci/mmol) were
obtained from NEN Life Science Products Inc., USA.
[3H]2-BFI (specific activity 70 Ci/mmol) was purchased
from Amersham International, UK. Idazoxan HCl was
synthesized by Dr. F. Geijo at S.A. Lasa Laboratories,
Barcelona, Spain. Other reagents were obtained from
Sigma Chemical Co. (St. Louis, MO, USA).
an oxalate salt. Yield 26%, mp 130–132 ꢀC. H NMR
1
(CD3OD) d 7.40 (t, 1H, J=8.0 Hz), 7.26–7.15 (m, 6H),
7.1 (dd, 1H, J=8.0 and 2.0 Hz), 6.9 (t, 1H, J=2.0 Hz),
3.9 (t, 1H, J=12.0 Hz), 3.62 (s, 4H), 3.55 (d, 2H,
J=12.0 Hz), 3.20 (m, 2H), 3.0 (td, 2H, J=12.0 and 2.0
Hz), 2.92 (m, 2H), 2.0 (dq, 2H, J=12.0 and 2.0 Hz),
1.86 (dq, 2H, J=12.0 and 4.0 Hz), 1.32 (m, 2H), 0.94 (t,
3H, J=7.3 Hz); 13C NMR (CD3OD) d 173.7, 167.0,
159.4, 143.0, 141.0, 136.0, 132.4, 128.8, 128.3, 127.7,
119.8, 116.5, 115.6, 57.8, 57.5, 52.8, 43.0, 31.0, 27.0,
20.7, 11.2; MS (ES+) m/z 420 ([MH]+, 100). Anal.
Functional activity. In vivo assays. CD1 male mice
weighing 25–30 g were used. All the animals were sup-
plied withfood and water ‘ad libitum’ and were housed
in a temperature-controlled room at 21 ꢀC. Lighting was
on a 12/12-h light/dark cycle. The mice were housed for
at least 1 day in the test-room before experimentation.
.
(C25H33N5O H2C2O4) (found) C: 63.64 (63.41); H: 6.92
(6.67); N: 13.74 (13.61).
To detect antinociceptive activity, several doses (2–50
mg/kg) of the tested compounds or saline solution were
intraperitoneally (ip) administered to separated groups
of mice (nꢅ10), 30 min before the analgesic effect was
tested. In order to evaluate the antinociception level, the
effect of ip administration of morphine (1.3–10 mg/kg)
was also tested. Eachmouse was used only once and an
observer who was unaware of the treatment performed
the testing and data recording.
Pharmacology
Binding assays. Preparation of membranes. Neural
membranes (P2 fractions) were prepared from the whole
brain of male Swiss Webster mice. Briefly, the tissue
samples were homogenized in 5 mL of ice-cold Tris
sucrose buffer (5 mM Tris–HCl, 250 mM sucrose, pH
7.4). The homogenates were centrifuged at 1100g for 10
min, and the supernatants were then recentrifuged at
40,000g for 10 min. The resulting pellet was incubated
at 25 ꢀC for 30 min to remove endogenous opioids.
After that, the pellet was washed twice and resuspended
in 50 mM Tris–HCl buffer (pH 7.5) to a final protein
Writhing test. The mice were ip injected with a 2% acetic
acid solution to produce the typical writhing reaction which
is characterised by a wave of contraction of the abdominal
musculature followed by extension of the hind limbs. After
the acetic acid administration, mice were placed in indivi-
dual transparent containers and, 5 min later, the number of
writhes was counted during a 10 min period.
content of 0.87ꢂ0.11 mg mLꢁ1
.
Binding assay. Total binding was measured in 0.55
mL-aliquots (50 mM Tris–HCl, pH 7.5) of the neural

A. Montero et al. / Bioorg. Med. Chem. 10 (2002) 1009–1018
1017
To test the antinociceptive effect writhing test was car-
ried out 30 min after the administration of separated
doses of: compound 10 (2, 10, 25 and 50 mg/kg), com-
pound 16 (5 and 10 mg/kg) and morphine (1.3, 2.5, 3.5,
5 and 10 mg/kg).
The effect of the drugs was evaluated 5 min after the
addition of eachdose, as percentage of inhibition, tak-
ing the amplitude of the last contraction before the first
addition of agonist as 100%. The opioid agonists were
added to the organ bath 15 min after the beginning of
electrical stimulation. Eachtissue was employed to
construct only one concentration–response curve.
Hot plate test. This test was carried out with a hot
plate at 55 ꢀC as a nociceptive stimulus. The control
reaction latency of the animals was measured before the
treatment. The time of licking of the front paw was
taken as an index of nociception. The latency was mea-
sured before drug or saline administration (control) and
30 min after treatment. The cut-off time was 30 s and
analgesia was quantified with the formula of the % of
maximum possible effect (M.P.E.):
To corroborate that the inhibitory effect of the selective
opioid agonists or of the new compounds was mediated
through interaction with mu opioid receptors, one low
dose naloxone (5ꢄ10ꢁ8 M) was added to the organ bath
at the end of each experiment.
Acknowledgements
M:P:E: ¼ ðlatency aftertreatment À control latencyÞ=
ðcut-off time À control latencyÞ
This research was supported by Spanish grants SAF 97-
0044-CO2, FIS (95/1731) and SAF 00-0114-C02. LFC is
recipient of a postdoctoral fellowship from the Basque
Government.
To test the antinociceptive effect hot plate test was car-
ried out 30 min after the administration of separated
doses of: compound 10 (2, 10, 25 and 50 mg/kg), com-
pound 16 (5, 10 and 20 mg/kg) and morphine (2.5, 5, 7.5
and 10 mg/kg).
References and Notes
1. Bourguignon, J.-J. In The Practice of Medicinal Chemistry;
Wermouth, C. G., Ed.; Academic: London, 1996; p 261.
2. Matsumoto, H.; Hamawaki, T.; Ota, H.; Kimura, T.;
Goto, T.; Sano, K.; Hayashi, Y.; Kiso, Y. Bioorg. Med. Chem.
Lett. 2000, 10, 1227.
3. Inagaki, M.; Tsuri, T.; Jyoyama, H.; Ono, T.; Yamada, K.;
Kobayashi, M.; Hori, Y.; Arimura, A.; Yasui, K.; Ohno, K.;
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