J. W. Corbett et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6250–6256
6255
Verhoest, P. R. WO 2003/72107; (c) Corbett, J. W.; Ennis,
M. D.; Frank, K. E.; Fu, J. -M.; Hoffman, R. L.;
Verhoest, P. R. WO 2003/91225; (d) Corbett, J. W.; Fu,
J. -M.; Ennis, M. D.; Frank, K. E.; Hoffman, R. L.;
Verhoest, P. R. WO 2004/024719.
incorporating this pyridinone. The beneficial impact on
binding affinity of the pyridyl ring in 18 was confirmed
by preparing 3-methylphenyl derivative 25 (Ki = 35
and 436 nM, respectively). Moving the methyl group
around the pyridyl ring, as found in analogs 34–36, re-
sulted in >13-fold losses in binding affinity compared
to 18 (Ki = 509, 456 and 472 nM, respectively). 4-Ethyl-
pyridyl analog 33 was approximately 1.5-fold less potent
than 18 (Ki = 53 and 35 nM, respectively) and the
pyrimidyl analog 43 was >4-fold less potent than 19
(Ki = 51 and 11 nM, respectively).
7. (a) Altman, R. A.; Buchwald, S. L. Org. Lett. 2007, 9, 643;
(b) Klapars, A.; Huang, X.; Buchwald, S. L. J. Am. Chem.
Soc. 2002, 124, 7421.
8. 2-Chloro-3,6-diethylpyrazine 1 (R1 = R2 = ethyl) was
prepared following a literature procedure in Sato, Nobu-
hiro; Matsuura, Tomoyuki J. Chem Soc., Perkin Trans. 1
1996, 19, 2345.
9. Application of Ullman coupling conditions by heating the
bromopyrazine derivative with 2-hydroxy-4-methylpyri-
dine in DMF at 150 °C in the presence of K2CO3 and
catalytic CuI resulted in complex mixtures containing low
yields (<20%) of the desired coupled products. Slightly
improved yields were achieved by substituting bromopyr-
azine 3a with iodopyrazine 3b.
10. Catalytic use of N,N-dimethylethylenediamine was found
to provide slightly improved yields of product compared
to the use of trans-1,2-diaminocyclohexane. Also, cesium
carbonate provided improved yields over potassium
carbonate.
In conclusion, potent, low nanomolar CRF1 receptor
antagonists were prepared by a multi-step sequence, with
the key steps involving a palladium catalyzed coupling of
cis-1-amino-2-indanols and copper catalyzed coupling of
pyridinones to functionalized pyrazines. Different SAR
was developed depending upon whether an oxygen or
nitrogen atom was the linker between the pyrazine ring
and the pendant pyridyl group. Interactions resulting in
improved activity for 46 versus 44 are unclear, but nitro-
gen-linked derivatives may have a different preferred
mode of binding than oxygen-linked pyridyl compounds.
The most potent analog, 19, derived from (1R,2S)-(+)-
cis-1-amino-2-indanol, had a Ki = 11 1 nM. Com-
pound 19 was not advanced owing to toxicities observed
with derivatives in other series’ associated with the
2-fluoroethyl ether moiety. Alkyl substitution of the
pyridinone ring indicated a preference for 4-methylpyrid-
inone. In an effort to identify CRF1 receptor antagonists
possessing improved drug characteristics, compounds
from the indanylpyrazine chemotype are less lypophilic
than previously reported heteroatom-linked pyrazines
and have improved binding affinities.
11. Yields for the coupling reaction ranged from 20% to 70%.
12. Yin, J.; Zhao, M. M.; Huffman, M.; McNamara, J. M.
Org. Lett. 2002, 4, 3481.
13. Assay results are reported as duplicates. The following is a
description of the preparation of differentiated human
neuroblastoma IMR32 cell membranes for use in the
standard radioligand binding assay, as well as a description
of the binding assay itself. IMR32 cells were maintained in
high glucose DMEM supplemented with 10% fetal bovine
serum, 1% non-essential amino acids, and 10 U/ml penicil-
lin. In order to increase receptor expression, the cells were
differentiated by the addition of 2.5 lM 5-bromo-20-deox-
yuridine to the cell medium. The cells were grown under
differentiation conditions for ten days before harvesting for
radioligand binding. To prepare the membranes, the
differentiated IMR32 cells were grown to confluence and
harvested in ice-cold Dulbecco’s phosphate-buffered saline.
After collection, the cells were pelleted by low speed
centrifugation (2500 rpm), and frozen at ꢂ80 °C until
needed. On the day of the assay, the pellets were thawed
and resuspended in 10 ml of 50 mM Hepes, pH 7.0,
containing 10 mM MgCl2, 2 mM EGTA, 1 lg/ml aprotinin,
1 lg/ml leupeptin, and 1 lg/ml pepstatin. The cell suspen-
sions were then homogenized, using a Brinkmann polytron
(setting 5 for 10 s), and centrifuged for 10 min at 20,000 rpm
at 4 °C. Following centrifugation, the pellets were resus-
pended and assayed for protein concentration. Radioligand
binding assays were conducted in disposable polypropylene
96-well plates. The CRF competition assays were initiated
by the addition of 150 ll membrane homogenate (30 lg/
well) to 150 ll assay buffer (50 mM Hepes, pH 7.0,
containing 10 mM MgCl2, 2 nM EGTA, 1 lg/ml aprotinin,
1 lg/ml leupeptin, 1 lg/ml pepstatin, 0.1% ovalbumin, and
0.15 nM bacitracin) containing [125I]Tyr°-CRF (ovine)
(140 pM) with or without competing ligand. Radioligand
binding was terminated after 2 h at room temperature by
filtration through Packard GF/C unifilter plates (presoaked
with 0.3% polyethyleneimine) using a Packard cell harves-
tor. Filters were washed three times with ice-cold
phosphate-buffered saline, pH 7.0, containing 0.01% Triton
X-100. Filters were then assessed for radioactivity in a
Packard TopCount. Apparent dissociation constants (Ki
values) from the competition experiments were calculated
using an iterative nonlinear regression curve-fitting pro-
gram (Prism; GraphPAD Software, San Diego, CA).
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