260
J.-U. Peters et al. / Bioorg. Med. Chem. Lett. 18 (2008) 256–261
Oliver, K. R.; Kinsey, A. M.; Wainwright, A.; Siri-
nathsinghji, D. J. S. Brain Res. 2000, 867, 131.
tori, K.; Dias, S.; Peerschke, E. I. B.; Moore, M. A. S.;
Blanset, D. L.; Lang, P. C.; Petrone, M.; Rafii, S. Exp.
Hematol. 2001, 29, 1417.
6. (a) Schoeffter, P.; Ullmer, C.; Bobirnac, I.; Gabbiani, G.;
Lubbert, H. Br. J. Pharmacol. 1996, 117, 993; (b)
Gustafson, E. L.; Durkin, M. M.; Bard, J. A.; Zgombick,
J.; Branchek, T. A. Br. J. Pharmacol. 1996, 117, 657; (c)
To, Z. P.; Bonhaus, D. W.; Eglen, R. M.; Jakeman, L. B.
Br. J. Pharmacol. 1995, 115, 107; (d) Neumaier, J. F.;
Sexton, T. J.; Yracheta, Y.; Diaz, A. M.; Brownfield, M. J.
Chem. Neuroanat. 2001, 21, 63.
7. Thomas, D. R. Pharmacol. Ther. 2006, 111, 707.
8. Suckling, C. J.; Murphy, J. A.; Khalaf, A. I.; Zhou, S.;
Lizos, D. E.; Van Nhien, A. N.; Yasumatsu, H.; McVie,
A.; Young, L. C.; McCraw, C.; Waterman, P. G.; Morris,
B. J.; Pratt, J. A.; Harvey, A. L. Bioorg. Med. Chem. Lett.
2007, 17, 264.
21. Radioligand binding assays to assess the affinity of
compounds for 5-HT receptors: recombinant human 5-
HT receptors (5-HT1A, 1D, 2A, 2C, 5A, 6 and 7) were
expressed in HEK-293-EBNA cells using transient trans-
fection then homogenised. All radioligand binding assays
were carried out in 96-well plates in the presence of
radioligand ([3H]LSD for 5-HT1D (2 nM), 5-HT5A
(1.3 nM), 5-HT6 (1.6 nM), 5-HT7 (2 nM); [3H]-8-OH-
DPAT for 5-HT1A (1 nM); [3H] ketanserin for 5-HT2A
(1 nM); [3H]-mesulergine for 5-HT2C (1.5 nM)) and 10
concentrations of compound (ranging from 10 lM to
0.03 nM). Non-specific binding was defined using 2 lM
pindolol (5-HT1A), sumatriptan (5-HT1D), spiperone (5-
HT2A), mianserin (5-HT2C), methiothepin (5-HT5A and 5-
HT6) and 10 lM SB269970 (5-HT7). Each well contained
an aliquot of receptor membrane homogenate (varying
concentrations), 0.5 mg of Ysi-poly-l-lysine SPA beads
9. Pittala, V.; Salerno, L.; Modica, M.; Siracusa, M. A.;
Romeo, G. Mini-Rev. Med. Chem. 2007, 7, 945.
10. Lattmann, E.; Merino, I.; Dunn, S.; Parveen, B.; Latt-
mann, P.; Billington, D. C.; Bunprakob, Y.; Sattayasai, J.
Lett. Drug Design Disc. 2006, 3, 49.
(Amersham; for all SPA assays (5-HT1D, 5-HT2A, 5-HT2C
,
11. Hedlund, P. B.; Huitron-Resendiz, S.; Henriksen, S. J.;
Sutcliffe, J. G. Biol. Psychiatry 2005, 58, 831.
5-HT5A), not filtration assays (5-HT1A, 5-HT6, 5-HT7)) in
a final volume of 200 ll of buffer containing 50 mM
Tris,10 mM MgCl2, 1 mM EGTA and 10 lM pargiline
(pH 7.4). All assays were conducted in duplicate and
repeated at least twice. Assay plates were incubated for
varying times at room temperature (5-HT1A 30 min; 5-
HT1D, 5-HT2A 60 min; 5-HT2C 90 min, 5-HT5A120 min)
or at 37 ꢁC (5-HT7 60 min and 5-HT6 90 min) before
centrifugation (SPA) or filtration. For filtration assays
these were terminated by rapid filtration under vacuum
through GF/C filters, presoaked for at least 30 min with
PEI (polyethylenimine; 0.3%), with 5 · 0.4 ml washes of
ice-cold Tris buffer (50 mM, pH 7.4). For both SPA and
filtration plates, bound ligand was determined using a
Packard Topcount scintillation counter. XLfit, was used
to iteratively plot the data and determine IC50 and Hill
coefficient values. Ki was determined using the Cheng-
Prussoff calculation.
12. Guscott, M.; Bristow, L. J.; Hadingham, K.; Rosahl, T.
W.; Beer, M. S.; Stanton, J. A.; Bromidge, F.; Owens, A.
P.; Huscroft, I.; Myers, J.; Rupniak, N. M.; Patel, S.;
Whiting, P. J.; Hutson, P. H.; Fone, K. C.; Biello, S. M.;
Kulagowski, J. J.; McAllister, G. Neuropharmacology
2005, 48, 492.
13. (a) Wesolowska, A.; Tatarczynska, E.; Nikiforuk, A.;
Chojnacka-Wojcik, E. Eur. J. Pharmacol. 2007, 555, 43;
(b) Wesolowska, A.; Nikiforuk, A.; Stachowicz, K. Eur. J.
Pharmacol. 2006, 53, 185; (c) Wesolowska, A.; Nikiforuk,
A.; Stachowicz, K.; Tatarczynska, E. Neuropharmacology
2006, 51, 578.
14. Graf, M.; Jakus, R.; Kantor, S.; Levay, G.; Bagdy, G.
Neurosci. Lett. 2004, 359, 45.
15. Sprouse, J.; Reynolds, L.; Li, X.; Braselton, J.; Schmidt,
A. Neuropharmacology 2004, 46, 52.
16. Corbett, David F.; Heightman, Tom D.; Moss, Stephen
F.; Bromidge, Steven M.; Coggon, Sara A.; Longley,
Mark J.; Roa, Ana Maria; Williams, Jennifer A.; Thomas,
David R. Bioorg. Med. Chem. Lett. 2005, 15, 4014.
17. Thomas, D. R.; Soffin, E. M.; Roberts, C.; Kew, J. N. C.;
de la Flor, R. M.; Dawson, L. A.; Fry, V. A.; Coggon, S.
A.; Faedo, S.; Hayes, P. D.; Corbett, D. F.; Davies, C. H.;
Hagan, J. J. Neuropharmacol. 2006, 51, 566.
22. Determination of competitive action of guanidines at the
5-HT5A receptor using [35S]GTPcS Schild analysis: 5-HT
agonist EC50 curves were determined in the presence of
different antagonist concentrations. Experiments for each
compound at different concentrations were repeated three
times and then a pA2 Schild analysis performed. Exper-
iments were conducted in 96-well plates, each well
containing 0.2 nM [35S]GTPcS (25 ll), 100 ll of buffer
(50 mM Tris, 10 mM MgCl2, 50 mM NaCl, 1 mM EGTA,
10lM pargiline and dithiothreitol 100 lM (pH 7.4)) alone
or containing 5-HT (11 concentrations ranging from
100 lM to 0.1 nM) or non-specific definer GTPcS
(100 lM), and GDP (3 lM). Compound (25 ll) was tested
at three different concentrations per experiment (ranging
from 1 lM to 1 nM). Each well contained an aliquot
(50 ll) of receptor membrane homogenate (5-HT5A cell
homogenates prepared for the radioligand binding were
used) premixed with (1.5 mg/well) PVT-WGA SPA beads
(Amersham). Plates were incubated at 30 ꢁC for 30 min,
then centrifuged for 5 min, before rapidly (within 15 min)
counting the radioactivity using a Packard Topcount
scintillation counter.
18. Data presented at the 36th Annu. Meet. Soc. Neurosci.
(Oct 14–Oct 18, Atlanta) 2006, (a) Garcia-Ladona, F.;
Amberg, W.; Kling, A.; Lange, U. E. W.; Hornberger, W.;
Wernet, W.; Netz, A.; Ochse, M.; Mezler, M.; Meyer, A.
H.; Hahn, A.; Hillen, H.; Beyerbach, A.; Bowmik, S.;
Schoemaker, H.; Sullivan, J. P., Abst. 33.1; (b) Drescher,
K. U.; Amberg, W.; Kling, A.; Gross, G.; Schoemaker,
H.; Sullivan, J. P.; Garcia-Ladona F. J., Abst. 33.2; (c)
Rueter, L. E.; Wicke, K.; Basso, A. M.; Jongen-Relo, A.
L.; van Gaalen, M. M.; Gross, G.; Decker, M. W.; Kling,
A.; Schoemaker, H.; Sullivan, J. P.; Amberg, W.; Garcia-
Ladona, F. J., Abst. 33.3; (d) Ebert, U.; Kling, A.;
Amberg, W.; Garcia-Ladona, F. J.; Gross, G.; Schoe-
maker, H.; Sullivan, J. P., Abst. 529.25; (e) Jongen-Relo,
A. L.; Bespalov, A. Y.; Rueter, L. E.; Freeman, A. S.;
Decker, M. W.; Gross, G.; Schoemaker, H.; Sullivan, J.
P.; van Gaalen, M. M.; Wicke, K. M.; Zhang, M.;
Amberg, W.; Garcia-Ladona, F. J., Abst. 529.26.
19. Rusak, B.; Zucker, I. Physiol. Rev. 1979, 59, 449.
20. Interestingly, 2 is a metabolite of the PDE3 inhibitor,
anagrelide, and has been proposed to be responsible for
anagrelide’s thrombocytopenic effect: Lane, W. J.; Hat-
23. Trinka, P.; Slegel, P.; Reiter, J. J. Prakt. Chem. 1996, 338, 675.
24. Stalder, H. Helv. Chim. Acta 1986, 69, 1887.
25. Sikkar, R.; Martinson, P. Acta Chem. Scand. B 1980, B34,
551.
26. Preparation of 12: (a) 1-(2-amino-6-chloro-phenyl)-etha-
none (29): a suspension of 2-amino-6-chlorobenzonitrile
(28, 3.67g, 24 mmol) in diethyl ether (60 ml) was added
slowly to methylmagnesium bromide (56 ml, 3 M in Et2O,