Design, synthesis and biological evaluation of a bivalent μ opiate and adenosine A1 receptor antagonist

Article abstract of DOI:10.1016/j.bmcl.2009.09.112

The cross talk between different membrane receptors is the source of increasing research. We designed and synthesized a new hetero-bivalent ligand that has antagonist properties on both A₁ adenosine and μ opiate receptors with a K_i of 0.8 ± 0.05 and 0.7 ± 0.03 μM, respectively. This hybrid molecule increases cAMP production in cells that over express the μ receptor as well as those over expressing the A₁ adenosine receptor and reverses the antalgic effects of μ and A₁ adenosine receptor agonists in animals.

Full text of DOI:10.1016/j.bmcl.2009.09.112

Bioorganic & Medicinal Chemistry Letters 19 (2009) 6736–6739
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis and biological evaluation of a bivalent
and adenosine A1 receptor antagonist
l opiate
Smitha C. Mathew a, Nandita Ghosh a, Youlet By b, Aurélie Berthault a, Marie-Alice Virolleaud a,
Louis Carrega b, Gaëlle Chouraqui a, Laurent Commeiras a, Jocelyne Condo b, Mireille Attolini a,
a,
b,c,
*
Anouk Gaudel-Siri a, Jean Ruf b, Jean-Luc Parrain a, , Jean Rodriguez , Régis Guieu
*
*
a Aix-Marseille Université, Institut des Sciences Moléculaires de Marseille, iSm2-UMR CNRS 6263, Centre Saint Jérôme, Service 531, 13397 Marseille Cedex 20, France
b Université de la Méditerranée, UMR MD2, Faculté de Médecine, Boulevard Jean Moulin, 13005 Marseille, France
c Assistance Publique des Hôpitaux de Marseille, Boulevard Jean Moulin, 13005 Marseille, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 29 July 2009
Revised 28 September 2009
Accepted 29 September 2009
Available online 3 October 2009
The cross talk between different membrane receptors is the source of increasing research. We designed
and synthesized a new hetero-bivalent ligand that has antagonist properties on both A1 adenosine and
opiate receptors with a Ki of 0.8 0.05 and 0.7 0.03 M, respectively. This hybrid molecule increases
cAMP production in cells that over express the receptor as well as those over expressing the A1 aden-
osine receptor and reverses the antalgic effects of
l
l
l
l
and A1 adenosine receptor agonists in animals.
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Adenosine
Fentanyl
Bivalents antagonists
l-Receptors
A1 receptors
The cross talk between different G protein-coupled receptors
evaluate its biological effects. A hetero-bivalent ligand is a single
chemical entity that is composed of two covalently linked pharma-
cophores with a dual mode of action, acting on two different recep-
tor subtypes.1a,f,4
(GPCRs) is the source of increasing research in the area of simulta-
neous targeting of more than one GPCR.1 Most of the cross talk be-
tween different GPCRs concerns cAMP production via the
modulation of adenylyl-cyclase activity. There is a great number
of GPCRs that either stimulate or inhibit adenylyl-cyclase activity,
depending on the nature of the G protein (Gs, Gi or other). Among
Modulating both A1ARs and MORs could have therapeutic appli-
cation in some diseases. For example, the release of adenosine
aggravates the hypotension during severe sepsis or septic shock,
leading to subsequent tissue hypoperfusion and ischaemia. Most
of these effects are secondary to the activation of A1 adenosine
receptors, explaining the absence of response to pressor amines
GPCRs, there is evidence that A1 adenosine receptors (A1ARs) and
l
opioid receptors (MORs) are implicated in such cross talk and their
activation leads to a decrease in cAMP levels in the target cells.
In peripheral nervous system, there is a cross-tolerance and
cross withdrawal between A1ARs and MORs, indicating that these
receptors are localised on the same primary afferent nociceptors
and that A1ARs and MORs cooperate as a multiple receptor com-
plex.2 Our group has also demonstrated that the activation of
MORs increases adenosine concentration in the extra cellular
spaces of the central nervous system, suggesting that most effects
of opioids are due to adenosine release.3
in these patients.5 Furthermore, naloxone, a
l receptor antagonist
has been successfully used against the drop of blood pressure dur-
ing septic shock6 or hypovolemic shock,7 suggesting that the re-
lease of endogenous opioids participates into the severity of
these syndromes. Thus, blocking both A1ARs and MORs may be
of a great interest in these pathologies. Modulating both these
receptors may also be of interest in the area of drug-withdrawal
therapy.8
Based on the A1AR/MOR cross talk, the aim of this study was to
synthesize a potential hetero-bivalent A1AR/MOR ligand and to
Fentanyl [N-phenyl-N-(1-phenethyl-4-piperidinyl)propanamide]
is one of the most powerful opioid analgesics, with a potency approx-
imately eighty times that of morphine and forty times that of
oxycodone.9 Assuch,weplannedtoassociatefentanylwithadenosine
to generate an appropriate hetero-bivalent ligand (Fig. 1). Herein, we
report the synthesis of such a compound 10, its MOR and A1AR
binding affinities and biological properties.
* Corresponding authors. Tel.: +33 4 91 28 89 14; fax: +33 4 91 28 91 87 (J.-L.P.);
tel.: +33 4 32 43 82; fax: +33 4 91 (R.G.).
E-mail addresses: jl.parrain@univ-cezanne.fr (J.-L. Parrain), guieu.regis@numer
icable.fr (R. Guieu).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.09.112

S. C. Mathew et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6736–6739
6737
O
O
R
N
O
a
NH2
N
N
N
N
N
Bn
H2N
6 ; R=H
7; R=C(O)Et
b
N
N
HO
O
O
NH2
OH OH
c
N
N
Bn
8
adenosine
linker
fentanil
Scheme 2. Synthesis of aminofentanyl 8. Reagents and conditions: (a) 1-pheneth-
ylpiperidine-4-one (5), NaBH(OAc)3, AcOH, DCE, rt, 77%; (b) propionyl chloride,
Et3N, DCM, reflux, 95%; (c) NH4OAc, NaBH3CN, MeOH, 50 °C, 55%.
Figure 1. Concept of hetero-bivalent A1AR/MOR ligand.
Considering that the main approach for discovering A1AR ago-
nists has been the modification of adenosine itself and that the
N6-cyclopentyl adenosine (CPA) derivative displays high A1AR
selectivity,10 we first turned our attention to the synthesis of com-
pound 10 containing an adenosine N6-cyclopentyl group and an
amide bond linker at the 50-position.
Our approach for this potential hetero-bivalent ligand would in-
volve amide bond formation between the adenosine and the fenta-
nyl derivatives 4 and 8 respectively.
HN
N
N
O
N
H
N
N
a
b
N
O
9
4
N
O
Bn
10
HO
OH
The synthesis of acid 4 (Scheme 1) commenced with the conver-
sion of inosine (1) into the corresponding chloro-acetal derivative
2. Acetylation of inosine with acetic anhydride in the presence of
triethylamine and DMAP afforded the corresponding triacetylino-
sine in quantitative yield.
Scheme 3. Synthesis of adenosyl fentanyl 10. Reagents and conditions: (a) 7, BOP,
Et3N, THF, rt, 77%; (b) 80% aq TFA, rt, 32%.
Conversion to the chloride (SOCl2 in DMF) was directly followed
by deacetylation using a solution of ammonia in methanol to give
the expected chloroinosine (90% yield over three steps). Selective
protection of the two secondary alcohols as the corresponding ace-
tonide was achieved using dimethoxypropane in presence of p-
TSA. Treatment of 2 with cyclopentylamine in the presence of Hu-
nigs base provided the corresponding secondary amine 3 in 98%
yield. Finally the primary alcohol was oxidised to the correspond-
ing acid 4 under TEMPO-iodobenzene diacetate conditions11 in 65%
yield. Attention was next turned to the fentanyl subunit, as part of
target molecule 8 (Scheme 2). Reductive amination of 4-aminoace-
tophenone with commercially available 1-phenethylpiperidine-4-
one (5) afforded amine 6.12
Completion of the targeted ligand 10 (Scheme 3) was achieved
in two steps by using a BOP-assisted coupling between acid 4 and
amine 813 to give 9 then followed by acetal deprotection using
aqueous trifluoroacetic acid. Using this approach, the potential het-
ero-bivalent ligand 10 was efficiently synthesized on 30 milligram
scale (93% HPLC purity grade) in eight steps.14
This new hetero-bivalent ligand 10 was then submitted to bio-
logical evaluation. Adenosyl fentanyl 10 was first tested using noci-
ceptive tests in mice (n = 12 per group, using intra cerebro
ventricular route (icv)). Ligand 10 alone had no effect on latencies.
However it is worthy of note that DAMGO, a well known synthetic
opioid peptide with
l agonist properties, co-injected with 10
showed shorter latencies than DAMGO alone both in hot plate
This secondary amine was next subjected to propionylation to
provide 7 in 95% yield. Further reductive amination of 7 with
ammonium acetate and sodium cyanoborohydride then furnished
subunit 8 in 55% yield.11
and tail flick tests.
Adenosyl fentanyl 10 also significantly reversed the increase in
latency induced by CPA, an A1 AR agonist (Fig. 2A). Ligand 10 also
reversed the increase in latencies induced by injection of both CPA
and DAMGO (Fig. 2B). Comparable results were obtained using
intraperitoneal (ip) administration (data not shown).
We also evaluated the acute toxicity of 10 in animals: LD 50 va-
Cl
lue was >50 lg/mouse (when icv administered) and >500 mg/kg
(when ip administered).
N
N
a, b
c
We tested the properties of 10 to modulate cAMP production in
cell culture. Compound 10 activated cAMP production in a dose
dependent manner both in CHO K1 cells that over expressed MORs
and in CHO Chem 3 cells that over expressed A1ARs, with a maxi-
mal stimulation of 29 7% and 17 3%, respectively. Compara-
tively, naloxone, a MOR antagonist, increased cAMP production
with a maximal stimulation of 50 16% while 8-cyclopentyl-1,3-
dipropyl-xanthine (DPCPX), a A1AR antagonist, increased cAMP
production with a maximal stimulation of 45 8% (Fig. 3). We also
evaluated the affinities (Ki) of the drugs tested, using binding assay
on cell membranes (Table 1). Compound 10 had substantially low-
er affinities for the A1ARs as compared with A1AR antagonist
DPCPX and/or lower affinities for the MORs as compared with
the MOR antagonist naloxone or with the MOR agonist DAMGO.
However, its affinity was sufficient to produce biological effects
at reasonable concentrations.
inosine 1
N
N
HO
O
2
O
O
HN
N
HN
N
N
N
N
N
N
N
O
d
HO
HO
O
O
4
3
O
O
O
O
Scheme 1. Synthesis of acid 4. Reagents and conditions: (a) (1) Ac2O, DMAP, Et3N,
ACN, rt, quant.; (2) SOCl2, DMF, DCM, 0 °C then reflux, 90%; (3) NH3/MeOH, 0 °C
then ꢀ20 °C, quant.; (b) dimethoxypropane, p-TSA, acetone, rt, 70%; (c) cyclopen-
tylamine, DIEA, EtOH, 80 °C, 98%; (d) BAIB, TEMPO, ACN/H2O, rt, 65%.

6738
S. C. Mathew et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6736–6739
Figure 3. Comparative effects of 10, naloxone and DPCPX on cAMP production.
Table 1
Ki values for drugs at the A1 AR, A2A ARs and the MORs DPCPX: 8-cyclopentyl-1,3-
dipropylxanthine (A1 AR antagonist)
Ki A1ARs (nM)
12.9
>50,000
>100,000
800 57
Ki A2AARs (nM)
Ki MORs (nM)
Figure 2. Results of nociceptive tests in animals.
DPCPX
Naloxone
DAMGO
10
3
>100,000
1.5 0.2
3.8 0.7
728 37
In summary, we have synthesized a hetero-bivalent ligand 10
that has antagonist properties on both A1ARs and MORs. Its affinity
remained low for the two receptors but sufficient to have biological
effects at reasonable concentration. Compound 10 reversed signif-
icantly the antalgic effects of DAMGO or CPA and activated cAMP
production in both cell lines tested. To the best of our knowledge,
compound 10 constitutes the first drug that blocks both MORs and
A1ARs. Multivalent-ligands are also called hybrid molecules, and
are defined as chemical entities having different biological ef-
fects.4c There is evidence that adenosine via A1ARs, is implicated
in the modulation of opiate action in the central nervous system.15
Morphine16 and adenosine17 inhibit Ca2+ dependent neurotrans-
mitter release; this inhibition is blocked by theophiline an adeno-
sine receptors antagonist.18 A1ARs and MORs are both implicated
>40,000
Naloxone: MOR antagonist; DAMGO: ([
D-Ala2, N-Me Phe4, Gly-ol]enkephalin)(MOR
agonist).
in septic or hypovolemic shock,5–7 withdrawal,8,19 mood regula-
tion20 and pain21,22 Thus drugs which modulate both A1ARs and
MORs could prove highly interesting in these therapeutic areas.
Acknowledgements
The authors thank Agence Nationale pour la Recherche (ANR-
05-BLAN-0175-01), CNRS, Université Paul Cézanne, Université de

S. C. Mathew et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6736–6739
7. Faden, A. I.; Holaday, J. W. Science 1979, 205, 317.
6739
la Méditerranée, Faculté de Médecine, Assistance Publique des
Hôpitaux de Marseille, Fondation Mérieux and Conseil Régional
Provence Alpes Côte d’Azur for fundings; Johnson and Johnson lab-
oratories for CHO K1 cells supply and Conseil Général des Bouches
du Rhône are also gratefully acknowledged; Drs T. Meert and van
Hijfte, and Miss De Chabanne for helpful suggestions.
8. Khorasani, M. Z.; Hajizadeh, S.; Fathollahi, Y.; Semnanian, S. Brain Res. 2006,
1084, 61.
9. Fentanyl: Incapacitating Agent. National Institute for Occupational Safety and
Health. Emergency Response Database. August 22, 2008. Retrieved December
31, 2008.
10. Ju, Y.; Kumar, D.; Varma, R. S. J. Org. Chem. 2006, 71, 6697.
11. Boger, D. L.; Patane, M. A.; Zhou, J. J. Am. Chem. Soc. 1994, 116, 8544.
12. Abdel-Magid, A. F.; Carson, K. G.; Harris, B. D.; Maryanoff, C. A.; Shah, R. D. J.
Org. Chem. 1996, 61, 3849.
13. Borch, R. F.; Bernstein, M. D.; Dupont Durst, H. J. Am. Chem. Soc. 1971, 93, 2897.
14. 1H NMR (CDCl3, 300 MHz) d 8.05 (s, 1H), 7.98 (s, 1H), 7.33–7.07 (m, 9H), 6.93
(d, J = 12 Hz, 2H), 5.87 (d, J = 6 Hz, 1H), 5.17 (d, J = 6 Hz, 1H), 4.64–4.58 (m, 2H),
4.49 (s, 1H), 4.42 (br s, 1H), 4.29 (d, J = 6 Hz, 1H), 3.12–3.05 (m, 4H), 2.94–2.82
(m, 4H), 2.01–1.84 (m, 6H), 1.68–1.52 (m, 12H), 0.90 (t, J = 12 Hz, 3H) 13C NMR
(CDCl3, 75 MHz) d 175.1, 170.2, 153.1, 144.6, 136.2, 130.2, 129.4, 129.0, 127.8,
90.3, 85.4, 73.5, 72.8, 58.5, 52.6, 50.4, 49.8, 49.5, 48.4, 33.5, 31.3, 30.8, 28.9,
28.0, 24.1, 21.9, 9.7; HRMS: C39H50N8O5 calcd: [M+H] 711.3904. Found:
711.3919.
15. Salem, A.; Hope, W. Pharmacol. Biochem. Behav. 1997, 57, 671.
16. Michaelson, D. M.; McDowall, G.; Sarne, Y. J. Neurochem. 1984, 43, 614.
17. Silinsky, E. M. J. Physiol. 2004, 558, 389.
18. Cahill, C. M.; White, T. D.; Sawynok, J. J. Pharmacol. Exp. Ther. 1995, 275, 84.
19. Hack, S. P.; Christie, M. J. Crit. Rev. Neurobiol. 2003, 15, 235.
20. Kaster, M. P.; Budni, J.; Santos, A. R.; Rodrigues, A. L. Eur. J. Pharmacol. 2007, 576,
91.
References and notes
1. (a) Portoghese, P. S.; Larson, D. L.; Sayre, L. M.; Yim, C. B.; Ronsisvalle, G.; Tam,
S. W.; Takemori, A. E. J. Med. Chem. 1986, 29, 1855; (b) Jacobson, K. A.; Xie, R.;
Young, L.; Chang, L.; Liang, B. T. J. Biol. Chem. 2000, 275, 30272; (c) Daniels, D. J.;
Kulkarni, A.; Xie, Z.; Bhushan, R. G.; Portoghese, P. S. J. Med. Chem. 2005, 48,
1713; (d) Savini, L.; Campiani, G.; Gaeta, A.; Pellerano, C.; Fattorusso, C.;
Chiasserini, L.; Fedorko, J. M.; Saxena, A. Bioorg. Med. Chem. Lett. 2001, 11, 1779;
(e) Peng, X.; Knapp, B. I.; Bidlack, J. M.; Neumeyer, J. L. J. Med. Chem. 2006, 49,
256; (f) Karellas, P.; McNaughton, M.; Baker, S. P.; Scammells, P. J. J. Med. Chem.
2008, 51, 6128.
2. Aley, K. O.; Levine, J. D. J. Neurosci. 1997, 17, 735.
3. Halimi, G.; Devaux, C.; Clot-Faybesse, O.; Sampol, J.; Legof, L.; Rochat, H.; Guieu,
R. Eur. J. Pharmacol. 2000, 398, 217.
4. (a) Perez, M.; Jorand-Lebrun, C.; Pauwels, P. J.; Pallard, I.; Halazy, S. Bioorg. Med.
Chem. Lett. 1998, 8, 1407; (b) Bhushan, R. G.; Sharma, S. K.; Xie, Z.; Daniels, D. J.;
Portoghese, P. S. J. Med. Chem. 2004, 47, 2969; (c) Meunier, B. Acc. Chem. Res.
2008, 41, 69.
5. Martin, C.; Leone, M.; Viviand, X.; Ayem, M. L.; Guieu, R. Crit. Care Med. 2000,
28, 3198.
21. Guieu, R.; Peragut, J. C.; Roussel, P.; Hassani, H.; Sampieri, F.; Bechis, G.; Gola,
R.; Rochat, H. Pain 1996, 68, 271.
22. Pham, T.; Carrega, L.; Sauze, N.; Fund-Saunier, O.; Devaux, C.; Peragut, J. C.;
Saadjian, A.; Guieu, R. Anesthesiology 2003, 98, 459.
6. Peters, W. P.; Johnson, M. W.; Friedman, P. A.; Mitch, W. E. Lancet 1981, 1, 529.