L.-W. Hsin et al. / Bioorg. Med. Chem. Lett. 10 (2000) 707±710
709
We found that the high lipophilicity of antalarmin
caused troublesome problems (e.g., solubility, bioavail-
ability). To increase the hydrophilicity of ¯uoro-anta-
larmin derivatives, compound 11e containing an
additional basic piperazine nitrogen was designed and
synthesized. The ¯uoride 11e did possess much better
solubility than antalarmin. However, it had little anity
11. Schulz, D. W.; Mansbach, R. S.; Sprouse, J.; Braselton, J.
P.; Collins, J.; Corman, M.; Dunaiskis, A.; Faraci, S.;
Schmidt, A. W.; Seeger, T.; Seymour, P.; Tingley, F. D. I.;
Winston, E. N.; Chen, Y. L.; Heym, J. Proc. Natl. Acad. Sci.
USA 1996, 93, 10477.
1
2. Griebel, G.; Perrault, G.; Sanger, D. J. Psychopharmacol-
ogy 1998, 138, 55.
3. Deak, T.; Nguyen, K. T.; Ehrlich, A. L.; Watkins, L. R.;
1
for the CRHR (>1 mM). It was also observed in recent
1
0,20,21
Spencer, R. L.; Maier, S. F.; Licinio, J.; Wong, M.-L.;
Chrousos, G. P.; Webster, E.; Gold, P. W. Endocrinology
1999, 140, 79.
1
SAR studies
into a variety of potent CRHR antagonists was detri-
that introducing polar functionalities
1
mental to their CRHR binding anity.
1
14. Baram, T. Z.; Chalmers, D. T.; Chen, C.; Koutsoukos, Y.;
de Souza, E. B. Brain Res. 1997, 770, 89.
1
ent Pharm. Des. 1999, 5, 289.
5. McCarthy, J. R.; Heinrichs, S. C.; Grigoriadis, D. E. Curr-
In summary, we report the synthesis and CRHR bind-
1
ing anity of a novel series of ¯uoro-substituted anta-
larmin derivatives. Among them, compounds 11a and
1
9
1
6. Chen, Y. L. International Publication Number WO
413676-A1.
7. Shimizu, M.; Nakahara, Y.; Yoshioka, H. Tetrahedron
1
1b are high anity CRHR ligands and are potential
1
18
candidates for the development of F-containing PET
tracers for the CRHR . Furthermore, these nonpeptide
Lett. 1985, 26, 4207.
1
18. Experimental procedures: (A) Coupling reaction: A mix-
ture of 4-chloro-2,5,6-trimethyl-7-(2,4,6-trimethylphenyl)-
pyrrolo[2,3-d]pyrimidine (2.00 g, 6.37 mmol) and 6 to 10
equiv of the corresponding amine in DMSO (15 mL) was
¯
uorides may prove to be useful as research tools for
studying the physiological and pathological roles of
CRH system, and in the development of new medications
for CRH-related disorders. In vivo pharmacological
studies of these ¯uoro-antalarmin analogues are in
progress and the results will be reported in due course.
ꢀ
heated at 130 C for 5 h and the crude product was puri®ed
using silica gel column chromatography to aord 10a±f in
9
butylammonium ¯uoride (3.0 mmol) in THF (10 mL) in the
0±98% yield; (B) Fluorination: To a solution of tetra-
Ê
presence of molecular sieves 4 A (3 g) was added a mixture of
p-toluenesulfonyl ¯uoride (2 mmol) and the corresponding
aminoalcohol (1.0 mmol) in THF (5 mL) at room tempera-
ture. The reaction was stirred at re¯ux for 20 h and the ®nal
product was chromatographed to aord 11a±e in 65±85%
yield. All of the intermediates and ®nal products were char-
Acknowledgements
Ling-Wei Hsin thanks NIDDK and the Fogarty Inter-
national Center for a Visiting Fellowship.
1
acterized by H NMR, CIMS, and high-resolution EIMS.
1
9. CRHR binding assay: Frozen whole rat brains, dissected
1
References and Notes
from male Sprague±Dawley rats, 200±300 g, were purchased
from Taconic Farms (Germantown, NY). The brains were
rapidly frozen, shipped to NIH on dry ice and stored at
1
1
2
4
3
4
. Vale, W.; Spiess, J.; Rivier, C.; Rivier, J. Science 1981, 213,
394.
. Owens, M. J.; Nemero, C. B. Pharmacol. Rev. 1991, 43,
25.
ꢀ
� 70 C until the time of the assay. At the time of assay, brains
were thawed on ice, cerebellar hemispheres were dissected,
weighed, and homogenized in 20 volumes of ice cold homo-
. Chrousos, G. P. New Engl. J. Med. 1995, 332, 1351.
. Bremner, J. D.; Licinio, J.; Darnell, A.; Krystall, J. H.;
genization buer containing PBS, 10 mM MgCl , 2 mM
2
EGTA, and 0.1 mM bacitracin, pH 7.0. The homogenate was
centrifuged for 10 min at 40,000 g. The pellet was resuspended
in cold PBS buer and recentrifuged for 10 min at 40,000 g.
The ®nal pellet was resuspended in homogenization buer at
30 mg tissue wet weight/mL buer and 0.1 mL homogenate
was added to 1.5 mL microfuge tubes containing 0.1 mL of
Owens, M. J.; Southwick, S. M.; Nemero, C. B.; Charney, D.
S. Am. J. Psychiatry 1997, 154, 624.
5. Nemero, C. B.; Widerlov, E.; Bissette, G.; Wallens, H.;
Karlsson, I.; Eklund, K.; Kilts, C. D.; Loosen, P. T.; Vale, W.
Science 1984, 226, 1342.
125
0
6
. Zhou, Y.; Spangler, R.; LaForge, K. S.; Maggos, C. E.; Ho,
A.; Kreek, M. J. J. Pharmacol. Exp. Ther. 1996, 279, 351.
. Chen, Y. L.; Mansbach, R. S.; Winter, S. M.; Brooks, E.;
[
I]-Tyr -sauvagine (New England Nuclear, Boston, MA) in
incubation buer (homogenization buer plus 0.01% BSA),
and 0.1 mL of incubation buer containing increasing con-
7
�
10
� 6
Collins, J.; Corman, M. L.; Dunaiskis, A. R.; Faraci, W. S.;
Gallaschun, R. J.; Schmidt, A.; Schulz, D. W. J. Med. Chem.
centrations (10
to 10 M) of the CRH antagonist analo-
gues. Nonspeci®c binding was de®ned by 1 mM ovine CRH
and 1 mM sauvagine (Peninsula, Belmont, CA) which gave
identical nonspeci®c binding values. Binding reactions were
incubated for 2 h at room temperature. Radioactivity bound
to tissue was separated from free ligand by centrifugation in a
microfuge (Beckman, Palo Alta, CA) for 10 min at 12,000 g at
1
8
997, 40, 1749.
. Webster, E. L.; Lewis, D. B.; Torpy, D. J.; Zachman, E. K.;
Rice, K. C.; Chrousos, G. P. Endocrinology 1996, 137, 5747.
. Chen, C.; Dagnino, R. J.; De Souza, E. B.; Grigoriadis, D.
E.; Huang, C. Q.; Kim, K.-I.; Liu, Z.; Moran, T.; Webb, T.
9
ꢀ
R.; Whitten, J. P.; Xie, Y. F.; McCarthy, J. R. J. Med. Chem.
1
4 C. The supernatant was aspirated and the radioactivity of
996, 39, 4358.
0. Chorvat, R. J.; Bakthavatchalam, R.; Beck, J. P.; Gilligan,
the pellet was measured in a gamma counter at approximately
80% eciency. The computer program GraphPad Prism 2.0a
for Power Macintosh (GraphPad Software Inc., San Diego
1
P. J.; Wilde, R. G.; Cocuzza, A. J.; Hobbs, F. W.; Cheeseman,
R. S.; Curry, M.; Rescinito, J. P.; Krenitsky, P.; Chidester, D.;
Yarem, J. A.; Klaczkiewicz, J. D.; Hodge, C. N.; Aldrich, P.
E.; Wasserman, Z. R.; Fernandez, C. H.; Zaczek, R.; Fitzger-
ald, L. W.; Huang, S.-M.; Shen, H. L.; Wong, Y. N.; Chien, B.
M.; Quon, C. Y.; Arvanitis, A. J. Med. Chem. 1999, 42, 833.
CA) was used to calculate K
i
values from the IC50 of the [1 I]-
25
0
Tyr -sauvagine competitive binding curves for each antagonist
analogue. Unless noted in Table 1, three binding curves con-
ducted in duplicate were generated for each analogue and the
i
K values represent the mean of the three experimentsSEM.