Fentanyl derivatives bearing aliphatic alkaneguanidinium moieties: A new series of hybrid molecules with significant binding affinity for μ-opioid receptors and I2-imidazoline binding sites

Article abstract of DOI:10.1016/j.bmcl.2003.10.048

A new series of fentanyl derivatives [i.e., N-[1-(2-phenethyl)-4-piperidyl] -N-(guanidinoalkyl)propanamide] bearing aliphatic alkaneguanidinium moieties were prepared. Their affinities for the μ opioid receptors and for the I ₂-imidazoline binding sites (I₂-IBS) were determined on human post-mortem prefrontal cortex membranes. All of these hybrid compounds had significant and/or very high affinity for both receptors in the nanomolar range, meaning an improvement compared to the prototype N-[1-(2-phenethyl)-4- piperidyl]-N-(guanidinopropyl)propanamide previously reported.

Full text of DOI:10.1016/j.bmcl.2003.10.048

Bioorganic & Medicinal Chemistry Letters 14 (2004) 491–493
Fentanyl derivatives bearing aliphatic alkaneguanidinium moieties:
a new series of hybrid molecules with significant binding affinity
for ꢀ-opioid receptors and I2-imidazoline binding sites
Christophe Dardonville,a,* Nadine Jagerovic,a Luis F. Calladob and J. Javier Meanab
a
´
Instituto de Quımica Medica, CSIC, Juan de la Cierva 3, E-28006 Madrid, Spain
´
Departamento de Farmacologıa, Universidad del Paıs Vasco/Euskal Herriko Unibertsitatea, Leioa, E-48940 Bizkaia, Spain
b
´
´
Received 30 July 2003; revised 30 September 2003; accepted 20 October 2003
Abstract—A new series of fentanyl derivatives [i.e., N-[1-(2-phenethyl)-4-piperidyl]-N-(guanidinoalkyl)propanamide] bearing
aliphatic alkaneguanidinium moieties were prepared. Their affinities for the m opioid receptors and for the I2-imidazoline binding
sites (I2-IBS) were determined on human post-mortem prefrontal cortex membranes. All of these hybrid compounds had significant
and/or very high affinity for both receptors in the nanomolar range, meaning an improvement compared to the prototype N-[1-
(2-phenethyl)-4-piperidyl]-N-(guanidinopropyl)propanamide previously reported.
# 2003 Elsevier Ltd. All rights reserved.
1. Introduction
published earlier, studies on the replacement of the
phenyl ring of fentanyl by a butyl or propyl group
afforded m-opioid molecules that retained some analge-
sic activity of the parent compound.9
Several pharmacological studies revealed the interac-
tions between I2-imidazoline binding site (IBS) and
opioid systems.1À4 Very recently, it has been shown that
2-BFI, an I2-IBS ligand, when chronically admini-
strated, led to an attenuation of both the hyperactivity
of rat locus coeruleus neurons during opiate withdrawal
and the development of tolerance to morphine.5 Other
studies demonstrated the potential of the putative endo-
genous IBS ligand agmatine6 (1-amino-4-guanidinobu-
tane) to attenuate the escalation of intravenous cocaine
and fentanyl self-administration in rats7 and to regulate
morphine tolerance/dependence and withdrawal symp-
toms.8 Thus, I2-IBS ligands might be useful agents for
the management of opioid dependence and tolerance.
A first attempt to obtain hybrid molecules with opioid
activity and I2-IBS affinity led us to identify the proto-
type 5e (Scheme 1).10 This compound displayed high
affinity for the m opioid receptor whereas it only showed
low affinity for the I2-IBS.
In a previous SAR studies of a series of alkane bisgua-
nidinium aliphatic derivatives,11 we found out that
increasing the number of methylene units between two
guanidine moieties led to an increase of the I2-IBS affi-
nity. We rationalised that modulating the chain length
of the methylenic spacer between the fentanyl pharma-
cophore and the guanidine moiety should increase the
affinity of the prototype 5 for I2-IBS. Hence, a new ser-
ies of fentanyl hybrids compounds bearing a guanidine
at the end of a methylene chain of 2, 6, 8 and 12 units
was prepared (Scheme 1) and their binding affinity to
both kinds of receptors was evaluated.
One approach that seems interesting and promising for
pharmaceutical regulation of opioid dependence and
tolerance is the use of hybrid drugs in which opioid
(e.g., fentanyl) and I2-imidazoline agents (e.g., guani-
dine group) are combined in one molecule. Among
structure–activity relationships (SAR) that have been
Keywords: I2-Imidazoline binding site affinity; m-opioid affinity;
Alkane guanidine; Dual acting drug; Opioid tolerance; Opioid with-
drawal; Human brain.
* Corresponding author. Tel.: +34-91-562-2900; fax: +34-91-564-
4853; e-mail: dardonville@iqm.csic.es
2. Chemistry
Compounds 1e–4e were synthesised in six steps using a
shorter procedure than the one previously described by
0960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2003.10.048

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C. Dardonville et al. / Bioorg. Med. Chem. Lett. 14 (2004) 491–493
Scheme 1. Reagents and conditions: (i) amine (5 equiv), Boc2O (1 equiv), CHCl3, rt; (ii) NaBH3CN, MS (3 A), MeOH, rt; (iii) (EtCO)2O, DMAP,
pyridine, CH2Cl2, rt; (iv) CF3CO2H, CH2Cl2, 30 min; (v) HgCl2, Et3N, CH2Cl2, rt.
Montero et al.10 The mono Boc-protected diaminoalk-
anes (m=2, 6, 8 and 12) were directly reacted with 1-
phenethyl-4-piperidone in the presence of NaBH3CN
maintaining the pH at ca. 7–8 with AcOH. The resulting
amines (1a–4a) were acylated with propionic anhydride/
DMAPcat./pyridine/CH2Cl2 to afford the corresponding
propanamides 1b–4b which were easily isolated by flash
chromatography.12 Following the removal of the Boc-
protecting group, the guanidines were introduced with
the classical Boc-thiourea reagent.13 Compounds 1e–4e
were finally isolated as their trifluoroacetate salt and
purified by crystallisation.14 The NMR data of these
series of propanamides (1b–e to 4b–e) show character-
istic duplicated signals corresponding to the observation
of the cis–trans isomerism around the N–CO bond as
previously reported for 5e.10
Table 1. Affinity data [Ki (nM)] for I2-IBS and m-opioid receptors
Compd
–(CH2)m–
ma
[3H]-2BFI
I2
[3H]-DAMGO
nb
m
Idazoxan
Fentanyl
—
—
2437
3
28Æ11
5462Æ1343d
Æ228
ndc
5
4
3
3
3
3
3
2.9Æ1.5
433Æ83
23Æ4.5
1.04Æ0.28
Æ9.7
1e
5e
2e
3e
4e
1920Æ996
409Æ238
12 Æ7237
Æ3.0
6
8
126.5
6
477Æ75
a Number of methylene units in the spacer.
b Values expressed as meanÆstandard error mean of n experiments.
c Not determined.
d Ki value for mouse brain membrane (from ref 10).
compared to the prototype 5e (Table 1). Remarkably,
compound 4e, with a 12-methylene-units spacer between
the propanamide and guanidine groups, was a more
potent ligand than idazoxan (Ki=28 nM) for I2-IBS
with Ki=6.5 nM. In this series, we observed a good
correlation between the number of methylene units in
the spacer and the affinity for I2-IBS.
3. Pharmacology
The binding affinity of the new synthesised compounds
(1e–4e) and 5e was evaluated through competition
binding studies against the selective I2-IBS radioligand
[3H]-2-BFI and the selective m-opioid ligand [3H]-
DAMGO following the procedure previously descri-
bed.10,11,15,16 The assays were performed in membranes
from post-mortem human frontal cortex, a brain area
that shows an important density of I2-IBS and m-opioid
receptors. Drug competition studies were performed
with either [3H]-2-BFI (1 nM) or [3H]-DAMGO (2nM)
in the absence or presence of various concentrations of
competing drugs (10À12, 10À10 and 10À3 M concentra-
tions). Non-specific binding was estimated in the pre-
sence of 10À3 M idazoxan in experiments with [3H]-2-
BFI, or with 10À4 M naloxone in [3H]-DAMGO assays.
The opioid agonist fentanyl and the a2-adrenergic/I2-IBS
ligand idazoxan were used as references (Table 1). Com-
pounds 1e–5e were studied as their trifluoroacetate salts.
The clear improvement in I2-IBS binding affinity
observed when increasing the number of methylene
units from m=3 (5e) to m=12( 4e) was in agreement
with our previous observations on alkane bis-guanidi-
nium compounds in which the affinity for I2-IBS
increased with the chain length. However, in that study,
the presence of both guanidinium at the end of the ali-
phatic chain appeared essential for I2-IBS activity since
monoguanidinium counterparts (e.g., octyl-, nonyl- and
dodecylguanidinium) showed extremely poor I2-IBS
affinity.11 Thus, in the case of the hybrid molecules 1e–
5e, it is interesting to note that the presence of the
piperidyl propanamide pharmacophore proceeding
from the fentanyl structure confers significant I2-IBS
affinity to the alkyl guanidinium moiety. Since fentanyl
alone is a very poor I2-IBS ligand (see Table 1), there is
some evidence for a possible synergic effect of both
pharmacophoric groups for I2-receptor affinity in these
hybrid compounds.
All the new compounds synthesised (1e–4e) showed
submicromolar to nanomolar affinity for I2-IBS which
represented a raise from 4- (1e) to almost 300-fold (4e)

C. Dardonville et al. / Bioorg. Med. Chem. Lett. 14 (2004) 491–493
493
Regarding m-opioid receptor, all the molecules bound
with affinities in the nanomolar range (Table 1). One
compound (3e, Ki=37 nM) was roughly equipotent with
5e (Ki=23 nM), two molecules (1e,4e) showed lower
affinity whereas 2e (Ki=1.04 nM) was an excellent m-
opioid ligand with a binding affinity comparable to that
of fentanyl (2.9 nM).
4. Boronat, M. A.; Olmos, G.; Garcia-Sevilla, J. A. Br. J.
Pharmacol. 1998, 125, 175.
5. Ruiz-Durantez, E.; Torrecilla, M.; Pineda, J.; Ugedo, L.
Br. J. Pharmacol. 2003, 138, 494.
6. Reis, D. J.; Regunathan, S. Trends Pharmacol. Sci. 2000,
21, 187.
7. Morgan, A. D.; Campbell, U. C.; Fons, R. D.; Carroll,
M. E. Pharmacol. Biochem. Behav. 2002, 72, 873.
8. Aricioglu-Kartal, F.; Regunathan, S. Life Sci. 2002, 71,
1695.
9. Essawi, M. Y. H. Bull. Fac. Pharm. (Cairo Univ.) 1990,
28, 11.
10. Montero, A.; Goya, P.; Jagerovic, N.; Callado, L. F.;
Meana, J. J.; Giron, R.; Goicoechea, C.; Martin, M. I.
Bioorg. Med. Chem. 2002, 10, 1009.
With respect to the spacer chain length, these results
showed that the best m-opioid ligand was obtained with
six methylene units (2e, m=6) whereas either increasing
(m=8, 12) or decreasing (m=2, 3) the length of the
spacer affords less potent molecules. This observation
would need confirmation by the assay of more spacers
of different lengths (e.g., m=4, 5, 7, 9).
11. Dardonville, C.; Rozas, I.; Callado, L. F.; Meana, J. J.
Bioorg. Med. Chem. 2002, 10, 1525.
12. Using only 1.5 equiv of propionic anhydride and DMAP
cat. made the purification easier than the method pre-
viously described.
13. Poss, M. A.; Iwanowicz, E.; Reid, J. A.; Lin, J.; Gu, Z.
Tetrahedron Lett. 1992, 33, 5933.
To conclude, we have reported a new series of N-[1-(2-
phenethyl)-4-piperidyl]-N-(guanidinoalkyl)propanamide
hybrid molecules with improved affinity for the I2-IBS
and m-opioid receptor compared to the prototype 5e.
14. Spectroscopic data: 1e. (2TFA): NMR (400 MHz, D2O/
CD3OD) 1H d ppm 7.3–7.1 (m, 5H); 4.0–3.8 (m, 1H); 3.55
(m, 2H); 3.35 (m, 1H); 3.2 (br m, 4H); 3.12 (br m, 1H);
3.05–2.85 (m, 4H); 2.33 (m, 2H); 2.25–2.0 (m, 1H); 1.95–
1.75 (m, 3H); 0.88 (m, 3H); 13C d ppm 178.3; 157.2; 136.2;
129.2; 128.9; 127.5; 57.8; 52.5; 52.0; 40.4; 39.7; 30.0; 27.4;
26.7; 9.0; ES+MS m/z 346 [M+H]; 173.6 [(M+2H),
100%]. 2e. (2TFA): NMR (400 MHz, D2O) 1H d ppm
7.63–7.49 (m, 5H); 4.45 (m, 0.6H); 4.30 (m, 0.4H); 3.91 (br
m, 2H); 3.56 (m, 2H); 3.45 (t, 2H); 3.35 (m, 3H); 3.27 (m,
3H); 2.67 (q, 1H, J=7.5 Hz); 2.60 (q, 1H, J=7.5 Hz);
2.46–2.1 (m, 4H); 1.77 (br m, 3H); 1.68 (m, 1H); 1.54 (br
m, 4H); 1.27 (m, 3H); 13C d ppm 178.3 (s); 177.7 (s); 157.7
(s); 137.17 (s); 137.1 (s); 130.0 (d); 129.7 (d); 128.3 (d);
58.6 (d); 53.4 (d); 53.1 (d); 53.0 (d); 52.0 (t); 46.3 (d); 42.8
(d); 42.0 (d); 30.9 (t); 30.8 (t); 29.5 (t); 28.9 (t); 28.8 (t);
28.4 (t); 27.7 (t); 27.6 (t); 27.4 (t); 26.9 (t); 26.6 (t); 10.1 (q);
10.0 (q); ES+MS m/z 402.5 [M+H]; 201.9 [(M+2H),
100%]. 3e. (2TFA): NMR (300 MHz, CD3OD) 1H d ppm
7.3–7.1 (m, 5H); 4.2(br m, 0.5H); 4.0 (br m, 0.5H); 3.61
(br d, 2H); 3.3–2.85 (m, 10H); 2.43–2.0 (m, 4H); 1.85 (br t,
2H); 1.5 (br m, 4H); 1.27 (br m, 8H); 1.01 (br t, 3H,
J=7.3 Hz); 13C d ppm 177.0 (s); 176.3 (s); 163.8 (s); 163.3
(s); 138.0 (s); 130.4 (d); 130.2(d); 128.7 (d); 59.4 (d); 54.0
(d); 53.6 (t); 52.7 (t); 46.9 (t); 43.5 (t); 42.8 (t); 32.4 (t); 31.9
(t); 30.9 (t); 30.65 (t); 30.60 (t); 30.5 (t); 30.2(t); 29.3 (t);
28.5 (t); 28.3 (t); 28.27 (t); 28.05 (t); 27.97 (t); 10.4 (q);
ES+MS m/z 430.5 [M+H]; 215.9 [(M+2H), 100%]. 4e.
This study showed that the incorporation in a same
molecule of m-opioid and I2-IBS pharmacophoric moi-
eties (i.e., fentanyl and alkylguanidine respectively),
linked by a methylene spacer, led to a synergistic effect
for the binding to both receptors. The modulation of
the affinity for both receptors by modification of the
length of the methylene spacer between the two phar-
macophoric moieties afforded two hybrid compounds
(1e and 3e) with balanced I2/m affinity, a 400-fold m-
opioid selective compound (2e) and a 80-fold I2-IBS
selective compound (4e). The in vitro and in vivo func-
tional activity of these new dual acting drugs is cur-
rently being evaluated and will be reported elsewhere.
Finally, these results are relevant in the field of IBS
since they further demonstrate that aliphatic alkane-
guanidines can afford high-affinity ligands for the I2-
IBS.
Acknowledgements
This work was supported by the Spanish Grant SAF00-
0114-C02and the Basque Government (INTEK-
ER02ME04). C.D. is recipient of a postdoctoral fellow-
ship from the Spanish Ministerio de Educacion Cultura
y Deporte (SB2001-0174). We thank the staff members
of the Instituto Vasco de Medicina Legal for their
cooperation in the study.
1
(2TFA): NMR (500 MHz, CD3OD) H d ppm 7.55–7.43
(m, 5H); 4.49 (br t, 0.6H); 4.32(m, 0.4H); 3.91 (br m, 2H);
3.6–3.2(m, 10H); 2.67 (q, 0.8H, J=7.3 Hz); 2.58 (q, 1.2H,
J=7.3 Hz); 2.47 (br q, 1.2H); 2.37 (br q, 0.8H); 2.19 (br d,
0.8H); 2.13 (br d, 1.2H); 1.81–1.65 (m, 4H); 1.6–1.4 (br m,
16H); 1.30 (td, 3H, J=7.3 Hz); 13C d ppm 177.2; 176.5;
158.8; 137.8; 130.2; 130.1; 128.5; 59.3; 53.8; 53.4; 52.5;
46.7; 43.4; 42.7; 32.2; 31.7; 30.9; 30.8; 30.6; 30.53; 30.48;
30.0; 29.1; 28.4; 28.13; 27.97; 27.87; 10.43; 10.38; ES+MS
m/z 486.5 [M+H]; 243.9 [(M+2H), 100%].
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