4730
U. Funke et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4727–4730
iments as well as HPLC27 (Table 2). Experimentally, for [18F]20c a
logD value of 2.47 0.28 was determined by batch experiments.
For [18F]20t, similar results with logD = 2.36 ( 0.15, extractive)
were obtained. Within the applied pH range of 7.2–7.4, remarkably
higher logD values of 4.1725 and 3.8626 were calculated for 20c and
20t (neutral form), respectively. However, the experimental values
correspond to a logP of 2.31 1.0,25 calculated for the monoionic
form, and could be caused by solvation effects in the aqueous sys-
tem. LogD data collected by HPLC methods24,27b are dependent on
the column systems applied but not significantly on the isomeric
structure of 20. A logD of 3.27 0.52 for 20c and 20t, determined
on the Multisorb C18 column, corresponds to the calculated
logD = 3.86.26 Results of analyses on the SupelcosilTM C18 column
(logD = 2.54 0.42 for 20c, logD = 2.49 0.41 for 20t) are more
consistent with the extractive data and the calculated logD of
the monoionic form. Since logP values within a range of 0.9–2.5
indicate a good brain penetration of tracer compounds, we assume
that [18F]20c and [18F]20t are able to cross the blood–brain barrier
in a sufficient amount.
Determination of the reversible plasma protein binding of the
radioligands resulted in method-dependent data. While acetoni-
trile precipitated only 2.7% of [18F]20c incubated in rat plasma,28
ultrafiltration yielded about 50% binding of [18F]20c or [18F]20t to
rat plasma protein.29 This fraction still contains blood platelets
which are known targets for SERT ligands.2 Hence the free radio-
tracer fraction is about 50%, which is high enough to allow quanti-
tative SERT imaging.30
S.; Kung, H. F.; Shiue, C. Y. J. Nucl. Med. 2003, 44, 1890; (e) Huang, Y.; Narendran,
R.; Bae, S. A.; Erritzoe, D.; Guo, N.; Zhu, Z.; Hwang, D. R.; Laruelle, M. Nucl. Med.
Biol. 2004, 31, 727.
8. (a) Freter, K. J. Org. Chem. 1975, 40, 2525; (b) Guillaume, J.; Dumont, C.; Laurent,
J.; Nédélec, L. Eur. J. Med. Chem. 1987, 22, 33.
9. Wustrow, D. J.; Smith, W. J.; Corbin, A. E.; Davis, M. D.; Georgic, L. M.; Pugsley,
T. A.; Whetzel, S. Z.; Heffner, T. G.; Wise, L. D. J. Med. Chem. 1997, 40, 250.
10. Taeger, C. Diploma Thesis, University of Leipzig, 2006.
11. Gairaud, C. B.; Lappin, G. R. J. Org. Chem. 1953, 18, 1.
12. Anderson, W. K.; McPherson, H. L., Jr.; New, J. S.; Rick, A. C. J. Med. Chem. 1984,
27, 1321.
13. (a) Pictet, A.; Spengler; T. Chem. Ber. 1911, 44, 2030.; (b) Whaley, W. M.;
Govindachari, T. R. Org. React. 1951, 6, 151.
14. (a) Sall, D. J.; Grunewald, G. L. J. Med. Chem. 1987, 30, 2208; (b) Plobeck, N.;
Delorme, D.; Wei, Z.-Y.; Yang, H.; Zhou, F.; Schwarz, P.; Gawell, L.; Gagnon, H.;
Pelcman, B.; Schmidt, R.; Yue, S. Y.; Walpole, C.; Brown, W.; Zhou, E.; Labarre,
M.; Payza, K.; St-Onge, S.; Kamassah, A.; Morin, P.-E.; Projean, D.; Ducharme, J.;
Roberts, E. J. Med. Chem. 2000, 43, 3878.
15. Fisher, M. J.; Gunn, B.; Harms, C. S.; Kline, A. D.; Mullaney, J. T.; Nunes, A.;
Scarborough, R. M.; Arfsten, A. E.; Skelton, M. A.; Um, S. L.; Utterback, B. G.;
Jakubowski, J. A. J. Med. Chem. 1997, 40, 2085.
16. Abdel-Magid, A. F.; Carson, K. G.; Harris, B. D.; Maryanoff, C. A.; Shah, R. D. J.
Org. Chem. 1996, 61, 3849.
17. Cells obtained from: (a) Blakely, R. hSERT-HEK293, Vanderbilt University,
Nashville, USA.; (b) Boenisch, H. hNET-HEK293, University of Bonn, Germany.;
(c) Storch, A. hDAT-HEK293, Technical University Dresden, Germany.
18. For hSERT, hNET, and hDAT, the IC50 values of the test compounds were
determined on homogenates of the respective HEK293 cell line17 versus
[3H]paroxetine (SERT; PerkinElmer Life Sciences; AS = 706 GBq/mmol),
[3H]citalopram (SERT; Amersham GE Healthcare; AS = 3121 GBq/mmol)
[3H]nisoxetine (NET; PerkinElmer Life Sciences; AS = 2960 GBq/mmol), and
[3H]WIN35,428 (DAT; PerkinElmer Life Sciences; AS = 3145 GBq/mmol). r5HT1A
affinity was determined on rat cortical membrane homogenate versus [3H]-8-
OH-DPAT (Amersham GE Healthcare; AS = 8399 GBq/mmol). For binding
experiments, homogenates were diluted with the respective assay buffer
(hSERT and hNET: 50 mM Tris–HCl, pH 7.4, 120 mM NaCl, 5 mM KCl; hDAT:
50 mM Tris–HCl, pH 7.4, 100 mM NaCl; r5HT1A: 50 mM Tris–HCl, pH 7.4, 4 mM
CaCl2, 0.1% ascorbic acid) and incubated with the respective radioligand
(ꢀworking concentrations: 1 nM [3H]paroxetine, 0.5 nM [3H]citalopram,
0.5 nM [3H]nisoxetine, 0.5 nM [3H]WIN35,428, 0.3 nM [3H]-8-OH-DPAT) and
6–12 concentrations ofthe test compoundsat 21 °Cfor60 min(hSERT, hNET, and
r5HT1A) or on ice for 120 min (hDAT). Non-specific binding was determined with
No degradation was observed for [18F]20c and [18F]20t after
120 min incubation under physiological conditions31 as deter-
mined by analytical radio-HPLC and TLC. Also under conditions
more relevant for radiosyntheses such as basic conditions, and a
bath temperature of 80 °C, [18F]20c and [18F]20t remained stable.32
In summary, radiochemical, physicochemical and pharmacolog-
ical in vitro data identified the [18F]fluoropropoxy derivative
300
l
M clomipramin (hSERT), 100
lM protriptyline (hNET), 10 lM GBR12909
(hDAT), or 10
l
M serotonin hydrochloride (r5HT1A). KD values were obtained by
homologous competition according to the described protocols:
[3H]paroxetine = 0.69 nM, [3H]citalopram = 4.46 nM, [3H]nisoxetine = 6.77 nM,
and [3H]WIN35,428 = 24.1 nM. KD of [3H]-8-OH-DPAT (3.1 nM on rat cortex) was
taken from Newman-Tancredi et al.19
[
18F]20c to be a promising radiotracer for the imaging of the SERT.
Subsequently, further evaluation of [18F]20c will be performed to
determine the radiotracer affinity and to investigate the biodistri-
bution and the brain uptake.
19. Newman-Tancredi, A.; Assié, M.-B.; Leduc, N.; Ormière, A.-M.; Danty, N.; Cosi,
C. Int. J. Neuropsychopharmacol. 2005, 8, 341.
20. Henry, L. K.; Field, J. R.; Adkins, E. M.; Parnas, M. L.; Vaughan, R. A.; Zou, M. F.;
Newman, A. H.; Blakely, R. D. J. Biol. Chem. 2006, 281, 2012.
21. Chen, F.; Larsen, M. B.; Neubauer, H. A.; Sanchez, C.; Plenge, P.; Wiborg, O. J.
Neurochem. 2005, 92, 21.
Acknowledgments
22. Block, D.; Coenen, H. H.; Stöcklin, G. J. Labelled Compd. Radiopharm. 1987, 24,
1029.
23. Block, D.; Coenen, H. H.; Stöcklin, G. J. Labelled Compd. Radiopharm. 1988, 25, 201.
24. (a) EU. Guideline 67/548/EWG 1967, A.8.; (b) Hansch, C.; Fujita, T. J. Am. Chem.
Soc. 1964, 86, 1616; (c) Wilson, A. A.; Houle, S. J. Labelled Compd. Radiopharm.
1999, 42, 1277.
This work has been supported by a grant of the Deutsche For-
schungsgesellschaft. We wish to thank the cyclotron operators of
the department of Nuclear Medicine at the University of Leipzig
and collaborators of the Institute of Analytical Chemistry on the
Faculty of Chemistry and Mineralogy at the University of Leipzig.
We are grateful to Tina Ludwig for her excellent performance of
in vitro studies.
25. ACD/LogD. Version 4.56/27, April 2000, Advanced Chemistry Development Inc.,
Toronto, Canada.
26. MarvinSketch. Version 5.0.3, April 2008, ChemAxon Ltd., Budapest, Hungary.
27. Experimental determination of logD: (a) Extractive method. n = 3 per buffer
system. Phosphate buffer: according to Sørensen, KH2PO4/Na2HPO4Á2H2O,
50 mM, pH 7.2. Dulbecco buffer: Dulbecco’s PBS, Thermo Fischer Scientific,
USA, Product # 1890535, pH 7.2. Tris–HCl buffer: Thermo Fischer Scientific,
USA, Product # BP153-500, 30 mM, pH 7.4.; (b) HPLC-method. n P 3 per
References and notes
1. Pineyro, G.; Blier, P. Pharmacol. Rev. 1999, 51, 533.
standard or sample. Columns: Multisorb RP18-7 (250 Â 4 mm,
7 lm, CS
2. Brust, P.; Hesse, S.; Müller, U.; Szabo, Z. Curr. Psychiatry Rev. 2006, 2, 111.
3. Cipollina, J. A.; Mattson, R. J.; Sloan, C. P. U.S. Patent 5,607,961, 1997.
4. Deskus, J. A.; Epperson, J. R.; Sloan, C. P.; Cipollina, J. A.; Dextraze, P.; Qian-
Cutrone, J.; Gao, Q.; Ma, B.; Beno, B. R.; Mattson, G. K.; Molski, T. F.; Krause, R.
G.; Taber, M. T.; Lodge, N. J.; Mattson, R. J. Bioorg. Med. Chem. Lett. 2007, 17,
3099.
5. Mewshaw, R. E.; Zhou, P.; Zhou, D.; Meagher, K. L.; Asselin, M.; Evrard, D. A.;
Gilbert, A. M. U.S. Patent 6,313,126, 2001.
6. Meagher, K. L.; Mewshaw, R. E.; Evrard, D. A.; Zhou, P.; Smith, D. L.; Scerni, R.;
Spangler, T.; Abulhawa, S.; Shi, X.; Schechter, L. E.; Andree, T. H. Bioorg. Med.
Chem. Lett. 2001, 11, 1885.
7. (a) Wilson, A. A.; Ginovart, N.; Schmidt, M.; Meyer, J. H.; Threlkeld, P. G.; Houle,
S. J. Med. Chem. 2000, 43, 3103; (b) Vercouillie, J.; Tarkiainen, J.; Halldin, C.;
Emond, P.; Chalon, S.; Sandell, J.; Langer, O.; Guilloteau, D. J. Labelled Compd.
Radiopharm. 2001, 44, 113; (c) Huang, Y.; Hwang, D. R.; Narendran, R.; Sudo, Y.;
Chatterjee, R.; Bae, S. A.; Mawlawi, O.; Kegeles, L. S.; Wilson, A. A.; Kung, H. F.;
Laruelle, M. J. Cereb. Blood Flow Metab. 2002, 22, 1377; (d) Shiue, G. G.; Choi, S.
R.; Fang, P.; Hou, C.; Acton, P. D.; Cardi, C.; Saffer, J. R.; Greenberg, J. H.; Karp, J.
Chromatography Service, Germany), SupelcosilTM ABZ+ Plus (250 Â 4 mm,
5 lm, Sigma–Aldrich, Germany). Gradient: eluent A (5% MeCN + 20 mM
ammonium acetate) to eluent B (80% MeCN + 20 mM ammonium acetate) in
55 min; flow rate 1 ml/min.
28. Rat plasma, freshly isolated: protein content 95 mg/ml, V = 75
l
l. n = 3, two
extraction steps of precipitate each, MeCN V = 500
37 °C. Centrifugation: 2000g, 30 min, rt.
29. Rat plasma, freshly isolated: protein content 95 mg/ml, V = 100–450
ll. Incubation: 30 min,
ll. Buffer
system: Dulbecco, V = 125–350 l. Incubation: 30 min, 37 °C. MPS
l
Micropartition Ultrafiltration Device: Mfilter = 30 kDa, Millipore Corporation,
USA. n = 3, one wash each. Centrifugation: 2000g, 30 min, rt.
30. Laruelle, M.; Slifstein, M.; Huang, Y. Mol. Imaging Biol. 2003, 5, 363.
31. Rat cortex homogenate (V = 300
ll, in Tris–HCl), NaCl phys. (V = 300 ll),
Dulbecco (V = 300 l), pH 7.2–7.4, respectively. One milliliter reaction vials,
l
37 °C, aluminum heating plate. Sampling intervals: 30 min.
32. NaOHaq 0.1 and 1.3 M, respectively, one milliliter reaction vials, 80 °C,
aluminum heating plate. Sampling intervals: 30 min.