P. Urbani et al. / Bioorg. Med. Chem. 16 (2008) 7510–7515
7515
as a yellow solid; mp 135 °C; 1H NMR (CDCl3) d 7.25–7.14 (m,
2.3. Binding assay
15H); 6.98 (d, 2H, J = 6.4 Hz); 6.63 (d, 2H, J = 6.4 Hz); 5.57 (br s,
1H); 4.78 (s, 1H); 3.83 (t, 1H, J = 6.0 Hz); 3.22 (q, 2H, J = 5.0 Hz)
2.17 (q, 2H, J = 5.0 Hz). HR m/z 443.2101 correlates with the chem-
ical formula [C29H28N2O+Na]+ within 62 ppm.
For CB1 and CB2 receptor binding assays, the new compounds
were tested using membranes from HEK cells transfected with
either the human CB1 or CB2 receptor and [3H]-(ꢂ)-cis-3-[2-hydro-
xy-4-(1,1-dimethylheptyl)-phenyl]-trans-4-(3-hydroxy-propyl)-
cyclohexanol ([3H]CP-55,940) (Kd = 0.31 nM for CB2 and 0.18 nM
for CB1 receptors) as the high affinity ligand as described by the
manufacturer (Perkin-Elmer, Italia).23 Displacement curves were
generated by incubating drugs with [3H]CP-55,940 (0.084 for CB2
and 0.14 nM for CB1 binding assay). In all cases, Ki values were cal-
culated by applying the Cheng–Prusoff equation to the IC50 values
(obtained by GraphPad) for the displacement of the bound radioli-
gand by increasing concentrations of the test compounds.
2.2.8. N-(3-Phenyl)propyl-2,2-diphenylacetamide (10a)
Starting from 2,2-diphenylacetic acid (400 mg, 1.89 mmol) and
3-phenylpropylamine (383 mg, 2.83 mmol), the title compound
was obtained as reported for 9a as a white solid in a 63% yield;
mp 135 °C; 1H NMR (CDCl3) d 7.39–6.94 (m, 15H), 5.60 (br s, 1H),
4.88 (s, 1H), 3.40 (q, 2H, J = 6.9 Hz), 2.66 (t, 2H, J = 7.0 Hz), 1.89–
1.77 (m, 2H). HR m/z 352.1678 correlates with the chemical for-
mula [C23H23NO+Na]+ within 62 ppm.
2.2.9. N-(3-Phenyl)propyl-2-(4-chlorophenyl)-2-
2.4. cAMP assay
phenylacetamide (10b)
Starting from 2-(4-chlorophenyl)2-phenylacetic acid16 (620 mg,
2.52 mmol), and 3-phenylpropylamine (681 mg, 5.04 mmol) the ti-
tle compound was obtained as reported for 9a as a white solid in a
70% yield; mp 125 °C; 1H NMR (CDCl3) d 7.35–7.05 (m, 14H), 5.57
(br s, 1H), 4.86 (s, 1H), 3.35 (q, 2H, J = 6.7 Hz), 2.62 (t, 2H,
J = 7.6 Hz), 1.89–1.80 (m, 2H). HR m/z 386.1250 correlates with
the chemical formula [C23H22NOCl+Na]+ within 610 ppm.
Cyclic AMP assays were performed on intact confluent N18TG2
cells plated in six-well dishes and stimulated for 10 min at 37°C
with forskolin 1 lM in 400 lL of serum-free Dulbecco’s modified
Eagle’s medium containing 20 mM Hepes, 0.1 mg/mL BSA,
0.1 mM 1-methyl-3-isobutylxanthine.24 Cells were treated with
vehicle (methanol, 0.1%) or compounds (at various concentrations)
or WIN-55,212 (100 nM) or WIN-55,212 plus compound 4b
(1 lM). After incubation, 800 lL of ethanol was added, cells were
2.2.10. N-(3-Phenyl)propyl-2-(4-bromophenylacetamide) (10c)
Starting from 2-(4-bromophenyl)-2-phenylacetic acid16 (1.85 g,
6.35 mmol) and 3-phenylpropylamine (1.72 g, 12.70 mmol), the ti-
tle compound was obtained as reported for 9a as a white solid in a
81% yield; mp 132 °C; 1H NMR (CDCl3) d 7.50–7.10 (m, 14H), 5.57
(br s, 1H), 4.84 (s, 1H), 3.34 (q, 2H, J = 6.7 Hz), 2.62 (t, 2H,
J = 7.6 Hz); 1.96–1.82 (m, 2H). HR m/z 430.0765 correlates with
the chemical formula [C23H22NOBr+Na]+ within 63 ppm.
extracted and cyclic AMP was determined by means of a cyclic
AMP assay kit (Amersham, UK), as advised by the manufacturer.
Acknowledgment
The authors thank Mr. Marco Allarà for technical assistance.
References and notes
1. Pertwee, R. G. Pharmacol. Ther. 1997, 74, 129.
2. Pertwee, R. G. Biochem. Soc. Trans. 1998, 26, 267.
3. Devane, W. A.; Dysarz, F. A.; Johnson, M. R.; Melvin, L. S.; Howlett, A. C. Mol.
Pharmacol. 1988, 34, 605.
4. Matsuda, L. A.; Lolait, S. J.; Brownstein, M. J.; Young, A. C.; Bonner, T. I. Nature
1990, 346, 561.
5. Munro, S.; Thomas, K. L.; Abu-Shaar, M. Nature 1993, 365, 61.
6. Walker, J. M.; Huang, S. M. Pharmacol. Ther. 2002, 95, 127.
7. Iwamura, H.; Suzuki, H.; Ueda, Y.; Kaya, T.; Inaba, T. J. Pharmacol. Exp. Ther.
2001, 296, 420.
8. Mallat, A.; Teixeira-Clerc, F.; Deveaux, V.; Lotersztajn, S. Expert Opin. Ther.
Targets 2007, 11, 403.
2.2.11. N-(3,3-Diphenyl)propyl-2-phenylacetamide (11a)
Starting from phenylacetic acid (300 mg, 2.21 mmol) and 3,3-
diphenylpropylamine (700 mg, 3.31 mmol), the title compound
was obtained as reported for 9a as a white solid in a 85% yield;
mp 85 °C; 1H NMR (CDCl3) d 8.0 (s, 1H), 7.41–7.06 (m, 15H), 4.10
(t, 1H, J = 7.2 Hz), 3.44 (s, 2H), 3.33 (q, 2H, J = 6.9 Hz), 2.19 (q, 2H,
J = 7.5 Hz). HR m/z 352.1675 correlates with the chemical formula
[C23H23NO+Na]+ within 62 ppm.
9. Matias, I.; Di Marzo, V. Trends Endocrinol. Metab. 2007, 18, 27.
10. Muccioli, G. G. Chem. Biodivers. 2007, 4, 1805.
11. Muccioli, G. G.; Lambert, D. M. Curr. Med. Chem. 2005, 12, 1361.
12. Van Gaal, L. F.; Rissanen, A. M.; Scheen, A. J.; Ziegler, O.; Rossner, S. Lancet 2005,
365, 1389.
13. Després, J. P.; Golay, A.; Sjostrom, L. N. Engl. J. Med. 2005, 353, 2121.
14. Scheen, A. J.; Finer, N.; Hollander, P.; Jensen, M. D.; Van Gaal, L. F. Lancet 2006,
368, 1660.
15. Lin, L. S.; Lanza, T. J., Jr.; Jewell, J. P.; Liu, P.; Shah, S. K.; Qi, H.; Tong, X.; Wang, J.;
Xu, S. S.; Fong, T. M.; Shen, C. P.; Lao, J.; Xiao, J. C.; Shearman, L. P.; Stribling, D.
S.; Rosko, K.; Strack, A.; Marsh, D. J.; Feng, Y.; Kumar, S.; Samuel, K.; Yin, W.;
Van der Ploeg, L. H.; Goulet, M. T.; Hagmann, W. K. J. Med. Chem. 2006, 49, 7584.
16. Halterman, R. L.; McEvoy, M. A. J. Am. Chem. Soc. 1990, 112, 6690.
17. Corey, E. J.; Chaykovsky, M. J. Am. Chem. Soc. 1962, 84, 866.
18. Eisenbraun, E. J. Org. Synth., 1973, Collect Vol. V, 310.
19. Friedrich, E. C.; DeLucca, G. J. Org. Chem. 1983, 48, 1678.
20. Secrist, J. A., III.; Logue, M. J. Org. Chem. 1972, 37, 335.
21. Meschler, J. P.; Kraichely, D. M.; Wilken, G. H.; Howlett, A. C. Biochem.
Pharmacol. 2000, 60, 1315.
2.2.12. N-(3,3-Diphenyl)propyl-2-(4-chlorophenyl)acetamide
(11b)
Starting from 4-chlorophenylacetic acid (500 mg, 2.93 mmol)
and 3,3-diphenylpropylamine (1.2 g, 5.86 mmol), the title com-
pound was obtained as reported for 9a as a white solid in a 71%
yield; mp 107 °C; 1H NMR (CDCl3) d 7.37–7.16 (m, 14H), 5.27 (s,
1H), 3.91 (t, 1H, J = 7.7 Hz), 3.47 (s, 2H), 3.27 (q, 2H, J = 6.6 Hz),
2.25 (q, 2H, J = 7.4 Hz). HR m/z 386.1247 correlates with the chem-
ical formula [C23H22NOCl+Na]+ within 610 ppm.
2.2.13. N-(3,3-Diphenyl)propyl-2-(4-bromophenyl) acetamide
(11c)
Starting from 4-bromophenylacetic acid (500 mg, 2.32 mmol)
and 3,3-diphenylpropylamine (980 mg, 4.64 mmol) the title com-
pound was obtained as reported for 9a as a white solid in a 75%
yield; mp 110 °C; 1H NMR (CDCl3) d 7.50 (d, 2H, J = 7.4 Hz), 7.32–
7.12 (m, 10H), 6.96 (d, 2H, J = 7.5 Hz), 5.33 (br s, 1H), 3.90 (q, 1H,
J = 7.5 Hz), 3.46 (s, 2H); 3.24 (q, 2H, J = 6.6 Hz), 2.25 (q, 2H,
J = 7.2 Hz). HR m/z 430.0776 correlates with the chemical formula
[C23H22NOBr+Na]+ within <1 ppm.
22. Muccioli, G. G.; Wouters, J.; Scriba, G. K.; Poppitz, W.; Poupaert, J. H.; Lambert,
D. M. J. Med. Chem. 2005, 48, 7486.
23. Brizzi, A.; Brizzi, V.; Cascio, M. G.; Bisogno, T.; Sirianni, R.; Di Marzo, V. J. Med.
Chem. 2005, 48, 7343.
24. Melck, D.; Rueda, D.; Galve-Roperh, I.; De Petrocellis, L.; Guzman, M.; Di Marzo,
V. FEBS Lett. 1999, 463, 235.